Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives: Potent inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase

Article abstract of DOI:10.3109/14756361003671060

A series of salicylanilide derivatives (compounds 1-32) were synthesised by reacting substituted salicylic acids and anilines. The chemical structures of these compounds were determined by ¹H-NMR, electrospray ionisation mass spectrometry (ESI-MS) and elemental analysis. The compounds were assayed for their antiproliferative activities against the Hep-G2 cell line by the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Among the compounds tested, 22 and 28 showed the most favouable antiproliferative activities with 50% inhibitory concentration (IC₅₀) values of 1.7 and 1.3 μM, respectively, which were comparable to the positive control of 5-fluorouracil (IC₅₀ = 1.8 μM). A solid-phase ELISA assay was also performed to evaluate the ability of compounds 1-32 to inhibit the autophosphorylation of the epidermal growth factor receptor tyrosine kinase (EGFR TK). Docking simulations of 22 and 28 were carried out to illustrate the binding mode of the molecule into the EGFR active site, and the result suggested that both compounds 22 and 28 could bind the EGFR kinase well.

Full text of DOI:10.3109/14756361003671060

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(1): 37–45
Copyright © 2011 Informa UK Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756361003671060
RESEARCH ARTICLE
Synthesis and antiproliferative activities against Hep-G2 of
salicylanide derivatives: potent inhibitors of the epidermal
growth factor receptor (EGFR) tyrosine kinase
Zhen-Wei Zhu, Lei Shi, Xiao-Ming Ruan, Ying Yang, Huan-Qiu Li, Suo-Ping Xu, and Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
Abstract
A series of salicylanilide derivatives (compounds 1–32) were synthesised by reacting substituted salicylic acids and
1
anilines. The chemical structures of these compounds were determined by H-NMR, electrospray ionisation mass
spectrometry (ESI-MS) and elemental analysis. The compounds were assayed for their antiproliferative activities
against the Hep-G2 cell line by the 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.
Among the compounds tested, 22 and 28 showed the most favouable antiproliferative activities with 50% inhibitory
concentration (IC50) values of 1.7 and 1.3 μM, respectively, which were comparable to the positive control of
5-fluorouracil (IC50 =1.8 μM). A solid-phase ELISA assay was also performed to evaluate the ability of compounds
1–32 to inhibit the autophosphorylation of the epidermal growth factor receptor tyrosine kinase (EGFR TK). Docking
simulations of 22 and 28 were carried out to illustrate the binding mode of the molecule into the EGFR active site,
and the result suggested that both compounds 22 and 28 could bind the EGFR kinase well.
Keywords: Salicylanilide derivatives; antiproliferative activities; EGFR TK; structure-activity relationship;
docking simulations
Introduction
a type of liver cancer cell often selected for research
Liver cancer is one of the most threatening diseases
in the world today. A study made by the International
Agency for Research on Cancer (IARC) indicated that
liver cancer was the third common cancer and caused
more than 620,000 deaths per year all over the world.
There are several drugs for the treatment of liver can-
cer such as doxorubicin, fluorouracil, cisplatin, and
α-interferon, but all of them have serious side effects
and therefore limit their clinical applications. Due to the
shortage of effective drugs, the discovery of new com-
pounds with potent anti-hepatoma activities is a very
important task. Here, we synthesised a series of salicy-
lanilide derivatives (compounds 1–32). Salicylanilides
are usually synthesised from substituted salicylic acids
and anilines. Salicylanilide derivatives isolated from
natural plants have been reported to possess anti-
proliferative activities against the Hep-G2 cell line,
to test compounds that may possess potent activity
for liver cancer treatment [1–3]. Pei-Wen Hsieh et al.
reported the isolation of 4-methoxydianthramide B, a
salicylanilide derivative, from the traditional Chinese
medicinal plant Dianthus superbus. The compound
4-methoxydianthramide B showed antiproliferative
activities against the Hep-G2 cancer cell line with a
50% inhibitory concentration (IC50) value of 4.08 μg/mL
[4]. This investigation led to the idea that salicylanilide
derivatives could possess potential antiproliferative
activities against the Hep-G2 cell line, and by structure
activity relationship (SAR), the results may be useful
to gain more understanding about the antiproliferative
activities of salicylanilide derivatives. In this paper, we
report the synthesis of some salicylanilide compounds
and their antiproliferative activities against the Hep-G2
cell line.
Address for Correspondence: Hai-Liang Zhu, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
People’s Republic of China. Tel.: +86-25-8359 2572; Fax: +86-25-8359 2672; E-mail zhuhl@nju.edu.cn
(Received 29 October 2009; revised 28 December 2009; accepted 07 January 2010)
37

38
Hai-Liang Zhu et al.
Docking simulations of compounds 22 and 28
humanlivercancercelllineHep-G2byapplyingthe3-(4,5-
dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) colorimetric assay. e compounds were
tested over a range of concentrations from 0.5 to 300 μM,
and the calculated IC50 values i.e. the concentration
(μM) of compounds which were able to cause 50% of cell
death with respect to the control culture, are reported in
Table 2.
Compounds 1–32 could be classed into 4 series: a)
salicylic acid series; b) 4-methyl salicylic acid series; c)
5-iodo salicylic acid series; d) 3,5-dibromo salicylic acid
series (Table 1). Among these salicylanilide derivatives
tested, compounds 22 and 28 were found to show the
most potent activities with IC50 values of 1.8 and 1.3 μM,
respectively. Compounds 21, 24, 25, 26, 27, 30, 31 and
32 showed the best antiproliferative activities with IC50
values ranging from 5-50 μM. e remaining compounds
showed weak antiproliferative activities with IC50 values
ranging from 50–300 μM (compounds 2–7, 9–12, 14,
15, 18, 19 and 29), or not active with IC50 values over
300 μM (compounds 1, 8, 13, 16, 17, 20, 23).
Compounds 1–32 were evaluated for their ability to
inhibit the autophosphorylation of EGFR kinases using
a solid-phase ELISA assay. A number of the synthesised
compounds showed potent EGFR inhibitory activities.
e results showed the same trends for antiprolifera-
tive activities against Hep-G2 as EGFR was an important
factor in liver cancer. (Table 2). Here, again, compounds
22 and 28 showed the most potent inhibitory activi-
ties (IC50 = 1.7 μM and 1.3 μM, respectively), and these
were comparable to the positive control 5-fluorouracil
(IC50 = 1.8 μM).
Among the four salicylic acid series, SAR studies dem-
onstrated that almost all the compounds belonging to
the 5-iodo salicylic acid series showed the best activity
both in the antiproliferative activities against Hep-G2 cell
line and for EGFR inhibition. e results indicated that
the iodine atom substituent at the 5-position could sig-
nificantly increase the activity. Accordingly, compounds
22 and 28, which contained an iodinated substituent at
the 5-position, showed the best activity.
In every salicylic acid series, for instance a) salicylic
acid series and c) 5-iodo salicylic acid series, substituents
at the 2’-position could significantly increase the activity
compared with substituents at the 4’-position [Table 1].
Furthermore, among these compounds where
substituents took place at the 4’-position, electron-
donating groups such as Me and iPr groups, showed
better activity than electron-withdrawing groups such as
OMe, F, Cl and Br.
However, the SAR result summarised above was a brief
overview of the whole 32 compounds synthesised, and
for compound 28, was a little different and seemed not
to obey the SAR rule stated above. Docking simulations
suggested that compound 28 had a good binding activ-
ity with EGFR kinase, and due to its chemical structure
28 was also a good EGFR inhibitor, and thus had good
antiproliferative activity against Hep-G2.
were carried out to give structural insights into the
binding mode with epidermal growth factor recep-
tor tyrosine kinase (EGFR TK) [5–8], to illustrate the
antiproliferative activities against the Hep-G2 cancer
cell line. Molecules designed to block EGFR TK, a
class of potent, selective, ATP-competitive inhibi-
tors of EGFR TK, induced signaling on their own and
further degrade to a DNA damaging species, should
induce significant cell-killing in tumours [9,10]. The
first molecular probe designed to verify the combi-
targeting postulates, was shown to strongly block
the EGFR TK activity on its own in a short exposure
enzyme assay [11,12]. The receptor protein tyrosine
kinases played a key role in signal transduction path-
ways that regulate cell division and differentiation.
The interaction of growth factors with these recep-
tors was a necessary event in the normal regulation
of cell growth. However, under certain conditions, as
a result of overexpression, mutation, or coexpression
of the ligand and the receptor, these receptors could
become hyperactivated and induce uncontrolled cell
proliferation [13]. Among the growth factor receptor
kinases, EGFR kinase (also known as erb-B1 or HER-1)
was important in cancer deregulation of growth-factor
signaling due to that hyperactivation of EGFR was seen
in several cancer types [14,15]. Activation of EGFR
might be a result of overexpression, mutations result-
ing in constitutive activation, or autocrine expression
of the ligand. EGFR overexpression was often seen in
various cancers [16–18]. Compounds that inhibit the
kinase activity of EGFR after binding of its cognate
ligand were of potential interest as new therapeutic
antitumor agents [19,20].
Chemistry
A series of salicylanilides were synthesised by reacting
four kinds of substituted salicylic acids and several kinds
of substituted anilines (Scheme 1). All the compounds
gave satisfactory chemical analysis. e chemical struc-
tures of these compounds were determined by 1H-NMR,
electrospray ionisation mass spectrometry (ESI-MS)
spectra and elemental analyses.
Results and discussion
Biological activity
e in vitro antiproliferative activities of the synthesised
salicylanilide derivatives 1–32 were studied using the
R5
O
R3
R2
R5
COOH
OH
R3
R2
a
N
H
+
H2N
R4
OH
R4
R1
R1
1–32
Scheme 1. Synthesis of salicylanilide derivatives 1–32. (a) EDC,
CH2Cl2, reflux, 8h, 70–86% yield.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives
39
Table 1. Physical Properties of salicylanilide derivatives 1–32.
R3
R2
COOH
OH
+
H2N
R1
Compound R1
R2
H
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
R4
R5
H
Formula
Mp °C
Yield, %
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
H
F
C13H11NO2
127–129
120–122
161–162
155–157
157–158
157–159
159–161
183–185
191–193
145–147
156–157
90–92
74
78
77
85
79
83
71
75
70
81
86
72
77
83
81
80
71
76
73
82
85
70
81
77
74
86
79
75
85
73
71
82
2
3
4
5
6
7
8
9
H
H
C13H10FNO2
C13H10ClNO2
C13H10BrNO2
C13H10FNO2
C13H10ClNO2
C13H10BrNO2
C13H9F2NO2
C13H9Cl2NO2
C14H13NO2
H
Cl
Br
H
H
H
F
H
H
H
H
F
H
Cl
Br
F
H
H
H
Cl
H
H
H
H
Br
H
H
F
Cl
CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
H
H
OCH3
C14H13NO3
H
CH(CH3)2
C16H17NO2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
C14H13NO2
193–195
165–167
135–137
158–160
196–198
171–173
154–156
173–175
194–195
181–182
230–232
238–240
236–237
209–211
241–243
232–234
204–205
200–202
166–168
153–155
H
C14H12BrNO2
C14H12FNO2
C14H12ClNO2
C14H11F2NO2
C14H11Cl2NO2
C15H15NO2
F
Cl
F
Cl
H
H
Cl
Br
H
H
H
Cl
H
H
H
H
H
H
Cl
CH3
CH(CH3)2
C17H19NO2
H
C13H9ClINO2
C13H9BrINO2
C13H9FINO2
C13H9ClINO2
C13H9BrINO2
C13H8Cl2INO2
C14H12INO2
C14H12INO3
C13H8Br2FNO2
C13H8Br2ClNO2
C13H8Br3NO2
C14H11Br2NO3
H
I
H
H
I
F
H
I
Cl
H
I
Br
H
I
Cl
H
I
CH3
OCH3
F
H
I
H
Br
Br
Br
Br
H
Cl
H
Br
H
OCH3
Cation-π interaction is a noncovalent molecular interac-
tion between the face of an electron-rich π system (e.g.
benzene, ethylene) with an adjacent cation. is unusual
interaction is an example of noncovalent bonding between
amonopole(cation)andaquadrupole(πsystem). Cation-π
interaction energies are of the same order of magnitude as
hydrogen bonds or salt bridges and play an important role
in molecular recognition [24]. Cation-π interaction made
the 22/EGFR kinase complex more stable.
In the binding model of compound 28 and EGFR,
there was also a cation-π interaction between the
Lys828 side chain and the salicylic ring (C1, C2, C3, C4,
C5 and C6 shown in Table 1) of 28. Unexpectedly, there
were two H-bonds observable for compound 28: one
between the N-H of 28 and the carboxylate of Gln767,
and the other between the phenolic hydroxyl of 28 and
the carboxylate of Gln767.
Binding Mode of 22 and 28 into EGFR Kinase
To give an structural insight into the ligand/enzyme
interactions, and to give an explanation and understand-
ing of good activity observed, molecular docking of the
most active compounds 22 and 28 into the ATP binding
site of EGFR kinase, the binding model based on the
EGFR complex structure (PDB code: 1M17, [10]) was
performed using the automated docking tools AutoDock
(version 4.0) [21–23]. e binding models of compounds
22 and 28 into EGFR were depicted in Figures 1 and 2.
Docking studies of both compounds 22 and 28 into the
active site of EGFR provided well clustered solutions. In
the binding model of compound 22 and EGFR, there was a
hydrogen bond between the carbonyl oxygen of 22 and the
N-H of the Lys828 side chain. Moreover, a cation-π inter-
action between the Lys828 side chain and the aniline ring
(C1,’ C2,’ C3,’ C4,’ C5,’ C6’ [Table 1]) of 22 was also observed.
© 2011 Informa UK Ltd.

40
Hai-Liang Zhu et al.
In both cases, the binding was further stabilised by
hydrophobic interactions between the phenyl group and
a hydrophobic region, comprised of the side chains of
Leu754, r766 and the Met769 side chains.
Table 2. Antiproliferative activity data and inhibition of EGFR
kinase for the salicylanilide derivatives 1–32.a
Antiproliferative effects against
EGFR
inhibitionIC50
(μM)
Hep-G2
IC50 (μM)
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
NA
>50
24.83
14.18
21.92
>50
Conclusions
80 28
52 17
61 10
246 69
137 37
140 38
NA
A series of salicylanilide derivatives were synthesised by
reacting substituted salicylic acids and anilines then eval-
uated for antiproliferative activities against the human
cancer cell line Hep-G2. Compounds 22 and 28 showed
the most potent antiproliferative activities with IC50 val-
ues of 1.7 and 1.3 μM. e EGFR inhibitory ability of these
synthesised salicylanilide derivatives were also evaluated
using a solid-phase ELISA assay, which had almost the
same trend as the antiproliferative activities assay. SAR
results suggested that the substituents at the 5-position
and 2’-position could increase the activity significantly.
Docking simulations were performed to give the prob-
able binding modes of compounds 22 and 28 into the
ATP binding site of EGFR kinase. Compounds 22 and 28
both formed a cation-π interaction with Lys828 through
their phenyl rings. However, compound 28 formed two
H-bonds with Gln767 through its N-H and phenolic
hydroxyl into the EGFR binding site, and compound 22
formed only one H-bond with Lys828 through its carbo-
nyl oxygen. Still, both 22 and 28 could bind the EGFR
kinase well. e result indicated that compounds 22 and
28, in particular, had significant Hep-G2 antiproliferative
activities and EGFR inhibitory activity, and are promising
potential agents for the treatment of liver cancer.
41.76
>50
>50
114 28
84 26
111 21
82 31
NA
45.29
25.74
39.98
29.62
>50
56 13
110 49
NA
22.19
40.17
>50
NA
>50
78 17
104 33
NA
31.09
34.67
>50
5.9 1.3
1.7 0.28
NA
4.37
0.92
>50
24 3.5
24 7.1
19 2.5
5.1 0.85
1.3 0.2
154 44
22 5.2
47 16
13.04
13.7
9.66
2.68
0.63
>50
Experimental Section
12.19
28.53
22.39
0.32
Chemistry
All chemicals (reagent grade) used in the experiment were
purchased from Aldrich (St. Louis, USA) and Sinopharm
37
1
5-fluorouracil
1.8 0.23
aNA, not active, the IC50 is greater than the maximal
concentration tested (300 μM).
Figure 1. Binding mode of compound 22 with EGFR kinase. For
clarity only the interacting residues were displayed. Ligand (green)
and interacting key residues (white) were represented as stick models,
while the proteins (white) were represented as ribbons. e H-bond
was displayed as spherical surface, and the cation-π interaction was
displayed as coniform surface.
Figure 2. Binding mode of compound 28 with EGFR kinase. For
clarity only the interacting residues were displayed. Ligand (green)
and interacting key residues (white) were represented as stick models,
while the proteins (white) were represented as ribbons. e H-bond
was displayed as spherical surface, and the cation-π interaction was
displayed as coniform surface.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives
41
Chemical Reagent (Shanghai, China). Melting points
(uncorrected) were determined on a XT4 MP apparatus
(Taike, Beijing). TLC was run on the silica gel coated
aluminum sheets (silica gel 60 GF254,E. Merk, Darmstadt,
Germany) and visualised in UV light (254nm). EI spec-
tra were obtained on a Waters GCT mass spectrometer
(Illinois, USA), and 1H NMR spectra were recorded
on a Bruker DPX-300, AV-300 or AV-500 spectrometer
(Rheinstetten, Germany) at 25 °C with TMS and solvent
signals allotted as internal standards. Chemical shifts were
reported in ppm (δ). Elemental analyses were performed
on a CHN-O-Rapid instrument (Hanau, Germany) and
were within 0.4 % of the theoretical values.
(m, 2H); 7.71 (dd, J1 = 1.6 Hz, J2 = 8.3 Hz, 1H); 8.05 (dd,
J1 = 1.9 Hz, J2 = 8 Hz, 1H); 8.3 (dd, J1 = 1.6 Hz, J2 = 8.3 Hz);
10.76 (s, 1H); 11.91 (s, 1H). MS (ESI): 293.9 (C13H11BrNO2,
[M+H]+). Anal. Calcd for C13H10BrNO2: C, 53.45; H, 3.45;
Br, 27.35; N, 4.79%; Found: C, 53.52; H, 3.42; Br, 27.36; N,
4.75%.
N-(4-fluorophenyl)-2-hydroxybenzamide (5)
White powder, mp 157–158°C 1H NMR (500 MHz,
d6-DMSO): 6.95–6.99 (m, 2H); 7.19–7.23 (m, 2H);
7.42–7.46 (m, 1H); 7.71–7.74 (m, 2H); 7.95 (dd, J1 = 1.5
Hz, J2 = 7.8 Hz, 1H); 10.39 (s, 1H); 11.75 (s, 1H). MS
(ESI): 232.1 (C13H11FNO2, [M+H]+). Anal. Calcd for
C13H10FNO2: C, 67.53; H, 4.36; F, 8.22; N, 6.06%; Found:
C, 67.61; H, 4.32; F, 8.24; N, 6.01%.
General method of synthesis 1–32
Equimolar quantities (1 mmol) of substituted salicylic
acid and aniline were dissolved in CH2Cl2 (5 mL), and
1 mmol EDC (1-(3-Dimethylaminopropyl)-3-ethylcar-
bodiimide) were also added to the solution as cata-
lyst. e solution was then stirred under refluxing for
approximately 8 hours. e solvent was evaporated and
the residue purified by column chromatography on
silica gel, eluting with petroleum ether/EtOAc (3:1) to
give a white, light yellow or brown solid of high purity
with a good to high yield (70–86%).
N-(4-chlorophenyl)-2-hydroxybenzamide (6)
Light yellow powder, mp 157–159°C H NMR (300 MHz,
1
d6-DMSO): 6.99 (d, J= 8.4 Hz, 2H); 7.43 (dd, J1 = 1.8 Hz,
J2 = 7 Hz, 3H); 7.76 (dd, J1 = 2 Hz, J2 = 6.9 Hz, 2H); 7.93 (dd,
J1= 1.7 Hz, J2 = 7.9 Hz, 1H); 10.44 (s, 1H); 11.63 (s, 1H).
MS (ESI): 248.7 (C13H11ClNO2, [M+H]+). Anal. Calcd for
C13H10ClNO2: C, 63.04; H, 4.07; Cl, 14.31; N, 5.66%; Found:
C, 62.98; H, 4.1; Cl, 14.29; N, 5.7%.
N-(4-bromophenyl)-2-hydroxybenzamide (7)
White powder, mp 159–161°C 1H NMR (300 MHz, d6-
DMSO): 6.94–7 (m, 2H); 7.41–7.47 (m, 1H); 7.56 (d, J=8.8
Hz, 2H); 7.71 (d, J=8.8 Hz, 2H); 7.93 (d, J=7.9 Hz, 1H);
10.45 (s, 1H); 11.63 (s, 1H). MS (ESI): 293.1 (C13H11BrNO2,
[M+H]+). Anal. Calcd for C13H10BrNO2: C, 53.45; H, 3.45; Br,
27.35; N, 4.79%; Found: C, 53.5; H, 3.48; Br, 27.28; N, 4.77%.
2-Hydroxy-N-phenylbenzamide (1)
Light yellow powder, mp 127–129°C 1H NMR
(300 MHz, d6-DMSO): 6.97 (t, J = 8.3 Hz, 2H);
7.15 (t, J = 7.5 Hz, 1H); 7.35–7.47 (m, 3H); 7.71 (d,
J = 7.7 Hz, 2H); 7.97 (dd, J1 = 1.3 Hz, J2 = 7.7 Hz,
1H); 10.37 (s, 1H); 11.79 (s, 1H). MS (ESI): 214.1
(C13H12NO2, [M+H]+). Anal. Calcd for C13H11NO2:
C, 73.23; H, 5.2; N, 6.57%; Found: C, 73.31; H, 5.17; N,
6.54%.
N-(2,4-difluorophenyl)-2-hydroxybenzamide (8)
Brown powder, mp 183–185°C 1H NMR (500 MHz,
d6-DMSO): 6.98–7.02 (m, 2H); 7.12–7.15 (m, 1H); 7.37–
7.42 (m, 1H); 7.44–7.48 (m, 1H); 8.01 (dd, J1 = 1.8 Hz, J2 = 8.0
Hz, 1H); 8.09–8.13 (m, 1H); 10.58 (s, 1H); 11.89 (s, 1H).
MS (ESI): 250.2 (C13H10F2NO2, [M+H]+). Anal. Calcd for
C13H9F2NO2: C, 62.65; H, 3.64; F, 15.25; N, 5.62%; Found:
C, 62.58; H, 3.66; F, 15.19; N, 5.64%.
N-(2-fluorophenyl)-2-hydroxybenzamide (2)
White powder, mp 120–122°C 1H NMR (300 MHz, d6-
DMSO): 7.03 (d, J= 8.8 Hz, 2H); 7.16–7.36 (m, 3H); 7.43–
7.48 (m, 1H); 8.03 (d, J= 7.9 Hz, 1H); 8.18–8.24 (m, 1H);
10.69 (s, 1H); 11.91 (s, 1H). MS (ESI): 232.1 (C13H11FNO2,
[M+H]+). Anal. Calcd for C13H10FNO2: C, 67.53; H, 4.36; F,
8.22; N, 6.06%; Found: C, 67.42; H, 4.34; F, 8.26; N, 6.08%.
N-(2,4-dichlorophenyl)-2-hydroxybenzamide (9)
1
Light yellow powder, mp 191–193°C H NMR (300 MHz,
d6-DMSO): 6.99–7.06 (m, 2H); 7.44–7.5 (m, 2H); 7.74
(d, J= 2.4 Hz, 1H); 8.04 (d, J= 7.9 Hz, 1H); 8.46 (d, J= 8.8
Hz, 1H); 10.94 (s, 1H); 11.95 (s, 1H). MS (ESI): 283.1
(C13H10Cl2NO2, [M+H]+). Anal. Calcd for C13H9Cl2NO2: C,
55.34; H, 3.22; Cl, 25.13; N, 4.96%; Found: C, 55.24; H,
3.23; Cl, 25.19; N, 4.99%.
N-(2-chlorophenyl)-2-hydroxybenzamide (3)
White powder, mp 161–162°C H NMR (500 MHz, d6-
1
DMSO): 7–7.05 (m, 2H); 7.17–7.2 (m, 1H); 7.37–7.41 (m,
1H); 7.44–7.48 (m, 1H); 7.56 (dd, J1 = 1.5 Hz, J2 = 8 Hz, 1H);
8.05 (dd, J1 = 1.8 Hz, J2 = 8 Hz, 1H); 8.39 (dd, J1 = 1.5 Hz,
J2 = 8.2 Hz, 1H); 10.88 (s, 1H); 11.91 (s, 1H). MS (ESI): 248
(C13H11ClNO2, [M+H]+). Anal. Calcd for C13H10ClNO2: C,
63.04; H, 4.07; Cl, 14.31; N, 5.66%; Found: C, 63.13; H,
4.02; Cl, 14.28; N, 5.69%.
2-Hydroxy-N-p-tolylbenzamide (10)
1
Light yellow powder, mp 145–147°C H NMR (300 MHz,
d6-DMSO): 2.29 (s, 3H); 6.98 (d, J= 7.7 Hz, 2H); 7.18
(d, J= 8.2 Hz, 2H); 7.41-7.47 (m, 1H); 7.59 (d, J= 8.4 Hz,
2H); 7.97 (d, J= 8.2 Hz, 1H); 10.32 (s, 1H); 11.89 (s, 1H).
MS (ESI): 228.3 (C14H14NO2, [M+H]+). Anal. Calcd for
N-(2-bromophenyl)-2-hydroxybenzamide (4)
White powder, mp 155–157°C 1H NMR (500 MHz, d6-
DMSO): 6.99–7.05 (m, 2H); 7.11–7.14 (m, 1H); 7.41–7.48
© 2011 Informa UK Ltd.

42
Hai-Liang Zhu et al.
C14H13NO2: C, 73.99; H, 5.77; N, 6.16%; Found: C, 73.87;
H, 5.79; N, 6.21%.
J = 8.6 Hz, 1H); 7.35-7.43 (m, 1H); 7.91 (d, J = 8.4 Hz, 1H);
8.05-8.13 (m, 1H); 10.52 (s, 1H); 11.87 (s, 1H). MS (ESI):
264.2 (C14H12F2NO2, [M+H]+). Anal. Calcd for C14H11F2NO2:
C, 63.88; H, 4.21; F, 14.43; N, 5.32%; Found: C, 63.97; H,
4.18; F, 14.38; N, 5.29%.
2-Hydroxy-N-(4-methoxyphenyl)benzamide (11)
1
White powder, mp 156–157°C H NMR (500 MHz, d6-
DMSO): 3.76 (s, 3H); 6.94–6.97 (m, 4H); 7.42–7.45 (m,
1H); 7.6 (d, J= 9.2 Hz, 2H); 7.98 (d, J= 7.7 Hz, 1H); 10.27 (s,
1H); 11.99 (s, 1H). MS (ESI): 244.3 (C14H14NO3, [M+H]+).
Anal. Calcd for C14H13NO3: C, 69.12; H, 5.39; N, 5.76%;
Found: C, 69.24; H, 5.34; N, 5.80%.
N-(2,4-dichlorophenyl)-2-hydroxy-4-
methylbenzamide (18)
White powder, mp 171–173°C 1H NMR (500 MHz, d6-
DMSO): 2.31 (s, 3H); 6.85–6.87 (m, 2H); 7.2 (s, 1H); 7.26
(d, J= 8.0 Hz, 1H); 7.88–7.90 (m, 2H); 10.1 (s, 1H); 12.88
(br s, 1H). MS (ESI): 297.2 (C14H12Cl2NO2, [M+H]+). Anal.
Calcd for C14H11Cl2NO2: C, 56.78; H, 3.74; Cl, 23.94; N,
4.73%; Found: C, 56.7; H, 3.78; Cl, 23.99; N, 4.75%.
2-Hydroxy-N-(4-isopropylphenyl)benzamide (12)
White powder, mp 90–92°C 1H NMR (300 MHz, d6-
DMSO): 1.21 (d, J= 7.0 Hz, 6H); 2.84–2.93 (m, 1H); 6.97 (d,
J= 7.9Hz, 2H); 7.24 (d, J= 8.4 Hz, 2H); 7.41–7.46 (m, 1H);
7.61 (d, J= 8.4 Hz, 2H); 7.98 (d, J= 8.2 Hz, 1H); 10.31 (s,
1H); 11.89 (s, 1H). MS (ESI): 256.3 (C16H18NO2, [M+H]+).
Anal. Calcd for C16H17NO2: C, 75.27; H, 6.71; N, 5.49%;
Found: C, 75.35; H, 6.68; N, 5.46%.
2-Hydroxy-4-methyl-N-p-tolylbenzamide (19)
Light yellow powder, mp 154–156°C H NMR (300 MHz,
1
d6-DMSO): 2.29 (s, 3H); 2.3 (s, 3H); 6.78 (d, J= 4.8 Hz, 2H);
7.18 (d, J= 8.3 Hz, 2H); 7.57 (d, J= 8.4 Hz, 2H); 7.91 (d,
J= 8.4 Hz, 1H); 10.24 (s, 1H); 12.02 (s, 1H). MS (ESI): 242.3
(C15H16NO2, [M+H]+). Anal. Calcd for C15H15NO2: C, 74.67;
H, 6.27; N, 5.81%; Found: C, 74.8 H, 6.22; N, 5.78%.
2-Hydroxy-4-methyl-N-phenylbenzamide (13)
1
White powder, mp 193–195°C H NMR (300 MHz, d6-
DMSO): 2.31 (s, 3H); 6.79 (d, J = 4.9 Hz, 2H); 7.14 (t,
J = 7.5 Hz, 1H); 7.37 (t, J = 8 Hz, 2H); 7.69 (d, J = 7.5 Hz,
2H); 7.91 (d, J = 8.4 Hz, 1H); 10.31 (s, 1H); 11.93 (s, 1H).
MS (ESI): 228.3 (C14H14NO2, [M+H]+). Anal. Calcd for
C14H13NO2: C, 73.99; H, 5.77; N, 6.16%; Found: C, 73.87;
H, 5.81; N, 6.19%.
2-Hydroxy-N-(4-isopropylphenyl)-4-
methylbenzamide (20)
White powder, mp 173–175°C 1H NMR (300 MHz, d6-
DMSO): 1.21 (d, J= 7 Hz, 6H); 2.3 (s, 3H); 2.86–2.9 (m,
1H); 6.77–6.79 (m, 2H); 7.24 (d, J= 8.4 Hz, 2H); 7.6 (d,
J= 8.6 Hz, 2H); 7.91 (d, J= 8.4 Hz, 1H); 10.28 (s, 1H); 12.04
(s, 1H). MS (ESI): 270.3 (C17H20NO2, [M+H]+). Anal. Calcd
for C17H19NO2: C, 75.81; H, 7.11; N, 5.20%; Found: C, 75.7;
H, 7.16; N, 5.23%.
N-(2-bromophenyl)-2-hydroxy-4-methylbenzamide (14)
White powder, mp 165–167°C 1H NMR (500 MHz,
d6-DMSO): 2.31 (s, 3H); 6.81–6.84 (m, 2H); 7.1–7.13
(m, 1H); 7.4–7.43 (m, 1H); 7.70 (dd, J1 = 1.5 Hz, J2 = 8.3
Hz, 1H); 7.93 (dd, J1 = 1.5 Hz, J2 = 8 Hz, 1H); 8.29 (dd,
J1 = 1.5 Hz, J2 = 8.3 Hz, 1H); 10.68 (s, 1H); 11.83 (s, 1H).
MS (ESI): 307.2 (C14H13BrNO2, [M+H]+). Anal. Calcd
for C14H12BrNO2: C, 54.92; H, 3.95; Br, 26.1; N, 4.58%;
Found: C, 55.01; H, 3.94; Br, 26.07; N, 4.59%.
N-(2-chlorophenyl)-2-hydroxy-5-iodobenzamide (21)
Brown powder, mp 194–195°C 1H NMR (500 MHz,
d6-DMSO): 7.02 (d, J = 8.5 Hz, 1H); 7.2 (t, J = 7.7 Hz, 1H);
7.4 (t, J = 7.7 Hz, 1H); 7.57 (d, J = 8 Hz, 1H); 7.62 (dd, J1 = 2.5
Hz, J2 = 8.5 Hz, 1H); 8.13 (d, J = 2.5 Hz, 1H); 8.38 (d, J = 8.2
Hz, 1H); 10.85 (s, 1H); 12.25 (s, 1H). MS (ESI): 374.6
(C13H10ClINO2, [M+H]+). Anal. Calcd for C13H9ClINO2: C,
41.8; H, 2.43; Cl, 9.49; I, 33.97; N, 3.75%; Found: C, 41.72;
H, 2.45; Cl, 9.53; I, 33.92; N, 3.77%.
N-(4-fluorophenyl)-2-hydroxy-4-methylbenzamide (15)
White powder, mp 135–137°C 1H NMR (300 MHz, d6-
DMSO): 2.30 (s, 3H); 6.79 (d, J= 4.4 Hz, 2H); 7.21 (t, J= 9
Hz, 2H); 7.69–7.73 (m, 2H); 7.89 (d, J= 8.2 Hz, 1H); 10.34
(s, 1H); 11.9 (s, 1H). MS (ESI): 246.3 (C14H13FNO2, [M+H]+).
Anal. Calcd for C14H12FNO2: C, 68.56; H, 4.93; F, 7.75; N,
5.71%; Found: C, 68.69; H, 4.89; F, 7.72; N, 5.69%.
N-(2-bromophenyl)-2-hydroxy-5-iodobenzamide (22)
1
Brown powder, mp 181–182°C H NMR (500 MHz, d6-
DMSO): 7.02 (d, J= 8.8 Hz, 1H); 7.15 (t, J= 7.7 Hz, 1H);
7.44 (t, J= 7.7 Hz, 1H); 7.62 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz,
1H); 7.72 (d, J= 8 Hz, 1H); 8.13 (d, J= 2.7 Hz, 1H); 8.29 (d,
J= 8.2 Hz, 1H); 10.74 (s, 1H); 12.24 (s, 1H). MS (ESI): 419
(C13H10BrINO2, [M+H]+). Anal. Calcd for C13H9BrINO2:
C, 37.35; H, 2.17; Br, 19.11; I, 30.36; N, 3.35%; Found: C,
37.44; H, 2.16; Br, 19.08; I, 30.31; N, 3.37%.
N-(4-chlorophenyl)-2-hydroxy-4-methylbenzamide (16)
White powder, mp 158–160°C 1H NMR (300 MHz,
d6-DMSO): 2.3 (s, 3H); 6.79 (d, J = 6.4 Hz, 2H); 7.43 (dd,
J1 = 2 Hz, J2 = 6.8 Hz, 2H); 7.75 (dd, J1 = 2 Hz, J2 = 6.8 Hz,
2H); 7.87 (d, J = 8.4 Hz); 10.4 (s, 1H); 11.79 (s, 1H). MS
(ESI): 262.7 (C14H13ClNO2, [M+H]+). Anal. Calcd for
C14H12ClNO2: C, 64.25; H, 4.62; Cl, 13.55; N, 5.35%; Found:
C, 64.31; H, 4.64; Cl, 13.49; N, 5.37%.
N-(4-fluorophenyl)-2-hydroxy-5-iodobenzamide (23)
White powder, mp 230–232°C 1H NMR (500 MHz, d6-
DMSO): 6.96 (d, J= 8.8 Hz, 1H); 7.22 (t, J= 8.8 Hz, 2H); 7.58
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H); 7.7–7.73 (m, 2H); 8.07 (d,
J= 2.5 Hz, 1H); 10.42 (s, 1H); 11.81 (s, 1H). MS (ESI): 358.1
N-(2,4-difluorophenyl)-2-hydroxy-4-methylbenzamide (17)
1
Light yellow powder, mp 196–198°C H NMR (300 MHz,
d6-DMSO): 2.3 (s, 3H); 6.81 (d, J = 5 Hz, 2H); 7.13 (t,
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives
43
(C13H10FINO2, [M+H]+). Anal. Calcd for C13H9FINO2: C,
43.72; H, 2.54; F, 5.32; I, 35.54; N, 3.92%; Found: C, 43.83;
H, 2.52; F, 5.31; I, 35.59; N, 3.9%.
3,5-Dibromo-N-(4-chlorophenyl)-2-
hydroxybenzamide (30)
1
Brown powder, mp 200–202°C H NMR (500 MHz, d6-
DMSO): 7.47 (dd, J1 = 2.1 Hz, J2 = 6.8 Hz, 2H); 7.72 (dd,
J1 = 2.2 Hz, J2 = 6.7 Hz, 2H); 8.02 (d, J= 2.4 Hz, 1H); 8.26 (d,
J= 2.1Hz,1H);10.72(s,1H);12.72(brs,1H).MS(ESI):406.5
(C13H9Br2ClNO2, [M+H]+). Anal. Calcd for C13H8Br2ClNO2:
C, 38.51; H, 1.99; Br, 39.41; Cl, 8.74; N, 3.45%; Found: C,
38.6; H, 1.99; Br, 39.37; Cl, 8.72; N, 3.44%.
N-(4-chlorophenyl)-2-hydroxy-5-iodobenzamide (24)
White powder, mp 238–240°C 1H NMR (500 MHz,
d6-DMSO): 6.96 (d, J= 8.8 Hz, 1H); 7.43 (d, J= 8.8 Hz,
2H); 7.58 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H); 7.74 (d, J= 8.8
Hz, 2H); 8.03 (d, J= 2.5 Hz, 1H); 10.46 (s, 1H); 11.7 (s, 1H).
MS (ESI): 374.6 (C13H10ClINO2, [M+H]+). Anal. Calcd for
C13H9ClINO2: C, 41.8; H, 2.43; Cl, 9.49; I, 33.97; N, 3.75%;
Found: C, 41.72; H, 2.43; Cl, 9.52; I, 34.03; N, 3.78%.
3,5-Dibromo-N-(4-bromophenyl)-2-
hydroxybenzamide (31)
Brown powder, mp 166–168°C 1H NMR (500 MHz,
d6-DMSO): 7.46 (d, J = 8.9 Hz, 2H); 7.71 (d, J = 8.6 Hz, 2H);
8.24 (d, J = 1.8 Hz, 1H); 8.37 (d, J = 1.8 Hz, 1H); 10.71 (s,
1H); 13 (br s, 1H). MS (ESI): 450.9 (C13H9Br3NO2, [M+H]+).
Anal. Calcd for C13H8Br3NO2: C, 34.7; H, 1.79; Br, 53.28; N,
3.11%; Found: C, 34.81; H, 1.78; Br, 53.24; N, 3.09%.
N-(4-bromophenyl)-2-hydroxy-5-iodobenzamide (25)
White powder, mp 236–237°C 1H NMR (500 MHz, d6-
DMSO): 6.97 (d, J=8.5 Hz, 1H); 7.55–7.59 (m, 3H); 7.69 (d,
J=8.8 Hz, 2H); 8.03 (d, J=2.5 Hz, 1H); 10.45 (s, 1H); 11.7 (s,
1H). MS (ESI): 419 (C13H10BrINO2, [M+H]+). Anal. Calcd for
C13H9BrINO2: C, 37.35; H, 2.17; Br, 19.11; I, 30.36; N, 3.35%;
Found: C, 37.49; H, 2.16; Br, 19.07; I, 30.4; N, 3.32%.
3,5-Dibromo-2-hydroxy-N-(4-methoxyphenyl)benzamide (32)
1
Brown powder, mp 153–155°C H NMR (500 MHz, d6-
N-(2,4-dichlorophenyl)-2-hydroxy-5-
iodobenzamide (26)
DMSO): 3.77 (s, 3H); 6.98 (dd, J1 = 2.2 Hz, J2 = 6.8 Hz,
2H); 7.57 (dd, J1 = 2.2 Hz, J2 = 6.8 Hz, 2H); 8.01 (d, J= 2.5
Hz, 1H); 8.31 (d, J= 2.5 Hz, 1H); 10.56 (s, 1H); 13.25 (br s,
1H). MS (ESI): 402.1 (C14H12Br2NO3, [M+H]+). Anal. Calcd
for C14H11Br2NO3: C, 41.93; H, 2.76; Br, 39.85; N, 3.49%;
Found: C, 42.01; H, 2.75; Br, 39.81; N, 3.47%.
Brown powder, mp 209–211°C 1H NMR (300 MHz,
d6-DMSO): 7.3 (d, J = 8.8 Hz, 1H); 7.49 (dd, J1 = 2.4 Hz,
J2 = 8.8 Hz, 1H); 7.63 (dd, J1 = 2.7 Hz, J2 = 8.6 Hz, 1H);
7.75 (d, J = 2.4 Hz, 1H); 8.11 (d, J = 2.6 Hz, 1H); 8.43 (d,
J = 8.8 Hz, 1H); 10.91 (s, 1H); 12.3 (s, 1H). MS (ESI): 409
(C13H9Cl2INO2, [M+H]+). Anal. Calcd for C13H8Cl2INO2:
C, 38.27; H, 1.98; Cl, 17.38; I, 31.1; N, 3.43%; Found: C,
38.41; H, 1.96; Cl, 17.35; I, 31.07; N, 3.44%.
Antiproliferative activities assay
e antiproliferative activities of the salicylanilide
derivatives 1–32 were determined using a standard
3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) colorimetric assay (Sigma, St.
Louis, USA). Briefly, cell lines were seeded at a density
of 7 × 103 cells/well in 96-well microtiter plates (Costar,
Bethesda, USA). After 24 h, exponentially growing cells
were exposed to the indicated compounds at final con-
centrations ranging from 0.5 to 300 μΜ, 5-fluorouracil
was used as a positive control [25]. After 48 h, cell sur-
vival was determined by the addition of an MTT solu-
tion (10 μL of 5 mg/mL MTT in PBS). After 4 h, 100 μL 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 Epson Diagnostic
microplate reader (Offenburg, Germany). Survival ratios
were expressed as a percentage with respect to untreated
cells. IC50 values were determined from replicates of six
wells from at least two independent experiments.
2-Hydroxy-5-iodo-N-p-tolylbenzamide (27)
1
Light yellow powder, mp 241–243°C H NMR (300 MHz,
d6-DMSO): 2.29 (s, 3H); 6.96 (d, J = 8.8 Hz, 1H); 7.18 (d,
J = 8.2 Hz, 2H); 7.58 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 3H); 8.1
(d, J = 2.4 Hz, 1H); 10.34 (s, 1H); 11.95 (s, 1H). MS (ESI):
354.2 (C14H13INO2, [M+H]+). Anal. Calcd for C14H12INO2:
C, 47.61; H, 3.42; I, 35.93; N, 3.97%; Found: C, 47.72; H,
3.41; I, 35.87; N, 3.95%.
2-Hydroxy-5-iodo-N-(4-methoxyphenyl)benzamide (28)
White powder, mp 232–234°C 1H NMR (300 MHz, d6-DMSO):
3.76 (s, 3H); 6.95 (d, J=9 Hz, 3H); 7.56–7.61 (m, 3H); 8.12 (d,
J=2.6 Hz, 1H); 10.31 (s, 1H); 12.04 (s, 1H). MS (ESI): 370.2
(C14H13INO3, [M+H]+). Anal. Calcd for C14H12INO3: C, 45.55;
H, 3.28; I, 34.38; N, 3.79%; Found: C, 45.43; H, 3.3; I, 34.45; N,
3.82%.
3,5-Dibromo-N-(4-fluorophenyl)-2-hydroxybenzamide (29)
Brown powder, mp 204–205°C H NMR (500 MHz, d6-
EGFR Inhibitory Assay
1
A 1.6kb cDNA encoded for the EGFR cytoplasmic domain
(EGFR-CD, amino acids 645–1186) were cloned into the
baculoviral expression vector pFASTBacHTc. A sequence
that encodes (His)6 was located at the 5’ upstream to the
EGFR sequence and Sf-9 cells were infected for three days
for protein expression. e Sf-9 cell pellets were solubi-
lised at 0°C in a buffer at pH 7.4 containing 50mM HEPES,
DMSO): 7.25 (t, J = 7.9 Hz, 2H); 7.68 (dd, J1 = 5.2 Hz,
J2 = 7.9 Hz, 2H); 8.23 (s, 1H); 8.39 (s, 1H); 10.66 (s, 1H);
13.16 (br s, 1H). MS (ESI): 390 (C13H9Br2FNO2, [M+H]+).
Anal. Calcd for C13H8Br2FNO2: C, 40.14; H, 2.07; Br, 41.08;
F, 4.88; N, 3.6%; Found: C, 40.22; H, 2.06; Br, 41.01; F, 4.86;
N, 3.59%.
© 2011 Informa UK Ltd.

44
Hai-Liang Zhu et al.
2. Ksander GM, de Jesus R, Yuan A, Fink C, Moskal M, Carlson E,
Kukkola P, Bilci N, Wallace E, Neubert A, Feldman D, Mogelesky
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Webb R, Navarrete A, Lee W, Gibson J, Alexander N, Sharif H,
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New cytotoxic cyclic peptides and dianthramide from Dianthus
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BC, Ye F, Nilakantan R, Shen R, Discafani C, DeBlanc
R, Davis R, Koehn FE, Greenberger LM, Wang YF,
10mM NaCl, 1% Triton, 10 μM ammonium molybdate,
100 μM sodium vanadate, 10 μg/mL aprotinin, 10 μg/mL
leupeptin, 10 μg/mL pepstatin, and 16 μg/mL benzami-
dine HCl for 20min followed by 20min centrifugation.
e crude extract supernatant was passed through an
equilibrated Ni-NTA superflow packed column (Qiagen,
Hilden, Germany) and washed with 10mM and then
100mM imidazole to remove any nonspecifically bound
material. e histidine tagged proteins were eluted with
250 and 500mM imidazole and dialysed against 50mM
NaCl, 20mM HEPES, 10% glycerol, and 1 μg/mL each of
aprotinin, leupeptin, and pepstatin for 2h. e entire puri-
fication procedure was performed at 4°C or on ice [26].
e EGFR kinase assay was set up to assess the level of
autophosphorylation based on DELFIA/Time-Resolved
Fluorometry. Compounds 1–32 were dissolved in 100%
DMSO and diluted to the appropriate concentrations with
25mM HEPES at pH 7.4. In each well, 10 μL of compound
was incubated with 10 μL (12.5ng for HER-2 or 5ng for
EGFR) of recombinant enzyme (1:80 dilution in 100mM
HEPES) for 10min at room temperature. en, 10 μL of
5mM buffer (containing 20mM HEPES, 2mM MnCl2,
100 μM Na3VO4, and 1mM DTT) and 20 μL of 0.1mM
ATP-50mM MgCl2 was added for 1h. Positive and nega-
tive controls were included in each plate by incubation
of enzyme with or without ATP-MgCl2. At the end of the
incubation period, the liquid was aspirated, and plates
were washed three times with wash buffer. A 75 μL (400ng)
sample of europium labeled anti-phosphotyrosine anti-
body was added to each well for a further 1h of incuba-
tion. After washing, enhancement solution was added and
the signal was detected by Victor (Wallac, Massachusetts,
USA) with excitation at 340nm and emission at 615nm.
e percentage of autophosphorylation inhibition by the
compounds was calculated using the following equation:
100% - [(negative control)/(positive control- negative
control)]. e IC50 was obtained from the percentage inhi-
bition curves with eight concentrations of the compound.
As the contaminants in the enzyme preparation are fairly
low, the majority of the signal detected by the anti-phos-
photyrosine antibody was from EGFR.
Wissner
A.
6-Substituted-4-(3-bromophenylamino)
quinazolines as putative irreversible inhibitors of the epidermal
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8. Bridges AJ, Zhou H, Cody DR, Rewcastle GW, McMichael A,
Showalter HDH, Fry DW, Kraker AJ, Denny W. Tyrosine kinase
inhibitors 8. An unusually steep structure-activity relationship
for analogues of 4-(3-bromoanilino)-6,7-dimethoxyquinazoline
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Molecular docking of compounds 22 and 28 into the
three-dimensional EGFR complex structure (download
from the Protein data Bank (PDB) PDB code: 1M17) was
carried out using the AutoDock software package (version
4.0) as implemented through the graphical user interface
AutoDockTools (ADT 1.4.6).Declaration of Interest
is work was supported by the Jiangsu National Science
Foundation (No. BK2009239) and Anhui National Science
Foundation (No. 070416274X).
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