Design and synthesis of Lapatinib derivatives containing a branched side chain as HER1/HER2 targeting antitumor drug candidates

Article abstract of DOI:10.1016/j.ejmech.2014.10.006

A series of Lapatinib derivatives were designed and prepared by changing the straight alkyl side chain of Lapatinib into a branched one. ELISA assay and western blot analysis showed that these derivatives can significantly inhibit HER1/HER2 as well as their downstream signal transduction proteins. In vitro cytotoxicity assay revealed that these compounds had potent cytotoxic effect against the HER1/HER2-overexpressing cancer cells. A representive compound, 2i, showed potent in vivo antitumor activity comparable to Lapatinib, which was found to block the cell-cycle progression of BT474 cells in the G1 phase causing tumor cell apoptosis in the flow cytometry study. Moreover, the pharmacokinetic investigation on 2i also indicated it had a good performance on both absorption and elimination profiles.

Full text of DOI:10.1016/j.ejmech.2014.10.006

European Journal of Medicinal Chemistry 87 (2014) 631e642
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of Lapatinib derivatives containing a branched
side chain as HER1/HER2 targeting antitumor drug candidates
,
b
,
,
, *
Aifeng Lyu a 1, Lei Fang a 1, Shaohua Gou a
a Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research and School of Chemistry and Chemical Engineering, Southeast University, Nanjing
211189, China
b Jiangsu Hansoh Pharmceutical Corporation, Lianyungang 222000, 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 Lapatinib derivatives were designed and prepared by changing the straight alkyl side chain of
Lapatinib into a branched one. ELISA assay and western blot analysis showed that these derivatives can
significantly inhibit HER1/HER2 as well as their downstream signal transduction proteins. In vitro
cytotoxicity assay revealed that these compounds had potent cytotoxic effect against the HER1/HER2-
overexpressing cancer cells. A representive compound, 2i, showed potent in vivo antitumor activity
comparable to Lapatinib, which was found to block the cell-cycle progression of BT474 cells in the G1
phase causing tumor cell apoptosis in the flow cytometry study. Moreover, the pharmacokinetic inves-
tigation on 2i also indicated it had a good performance on both absorption and elimination profiles.
© 2014 Elsevier Masson SAS. All rights reserved.
Received 1 June 2014
Received in revised form
29 September 2014
Accepted 2 October 2014
Available online 5 October 2014
Keywords:
Lapatinib derivatives
Tyrosine kinase inhibitors
HER1/HER2 targeting
Antitumor
1. Introduction
bind to any growth factor so that physiological HER2 homodimer
formation is unlikely. However, unphysiological overexpression of
Human epidermal growth factor receptor (EGFR) family, also
called HER family, is composed of four tyrosine kinase receptors,
which are known as erbB-1/HER1, erbB2/HER2, erbB3/HER3 and
erbB4/HER4. The family members such as HER1 and HER2 play a
key role in catalyzing phosphorylation reactions in signaling cas-
cades that affect every aspect of cell growth, differentiation and
metabolism. The deregulation of HER1 and/or HER2 by gene over-
expression, frame deletions or activated somatic mutations has
been proven prevalent in many types of cancers including non-
small cell lung cancer, head and neck, breast, colon and several
other cancers with poor clinical outcome [1]. Thus, they have
emerged in recent years as key and validated targets of therapies
for solid tumors [2,3]. These receptors consist of an extracellular
domain, a single hydrophobic transmembrane segment, and an
intracellular portion with a juxtamembrane segment, a protein
kinase domain, and a carboxyterminaltail. Like all protein-tyrosine
kinase receptors, the HER family members function as homo and
heterodimers. But HER-3 has a non-functional kinase, whose
induced homodimer formation is unable to stimulate protein ki-
nase activity and downstream signaling. In addition, HER2 fails to
HER2 leads to the formation of a functional homodimer, which is
important in the pathogenesis of many breast cancers [4]. It is
believed that the growth factor binding to the receptors can induce
a large conformational change in the extracellular domain. In the
way to find small molecules as HER protein-tyrosine kinase in-
hibitors, it is necessary to identify and characterize the catalytic
kinase domain. However, the results turned out that the family
members share a significant sequence homology with many
members of other human kinase. This brings out a unique challenge
in the development of selective inhibitors. One way to overcome
the problem of selectivity in inhibition is to design ligands that
specifically interact with pockets within the kinase active site.
These key pockets are localized nearby the adenosine triphosphate
(ATP) binding site, which is the natural ligand of HER protein-
tyrosine kinase domain. There are two different conformations,
namely the active and the inactive, of protein kinases at the ATP
binding site. It is known that high specificity can be achieved while
targeting the inactive conformation, but while targeting the active
conformation the treatment of the diseased state arising from
activating mutations is favorable, and the second type of inhibitors
has no specificity since they generally act as ATP competitors to
inhibit several protein kinases [5]. So far, most of the approved HER
protein-tyrosine kinase inhibitors, including Gefitinib, Erlotinib,
and Lapatinib (1, Fig. 1), target the ATP binding site of the kinase.
* Corresponding author.
E-mail address: sgou@seu.edu.cn (S. Gou).
A. Lyu and L. Fang contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.10.006
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
Fig. 1. Structures of Lapatinib and its derivatives 2aen.
However, Gefitinib and Erlotinib are HER1 inhibitors, whereas
Lapatinib is a mixed HER1/HER2 inhibitor. Interestingly, Lapatinib
preferentially binds the inactive conformation of HER1 differenti-
ating it from other HER tyrosine kinase inhibitors, such as Erlotinib
or Gefitinib. From the structure-interaction point of view, Wood
et al. analyzed the complex of HER1 kinase with Lapatinib and
found that there are several key interactions with the ATP binding
pocket of HER1: the ATP pocket is enlarged in the inactive confor-
mation by rotation and translation of the C-helix, which is neces-
sary to accommodate a 3-fluorobenzyloxy group and explains why
Lapatinib can only bind to an inactive conformation; the quinazo-
line moiety forms the critical H-bond with the hinge region which
is similar to those observed for Gefitinib and Erlotinib; the large 30-
chloro-40-[(3-fluorobenzyl)oxy]aniline group goes into the gate-
keeper pocket making mostly hydrophobic contacts [6]. However,
the methylsulfonylethylaminomethylfuryl group of Lapatinib,
which was found to point into the solvent region, makes no sig-
nificant contacts with the protein. It is believed that the solvent
region generally tolerates a variety of polar functional groups, and
the modulation of the side chain in the solvent region may alter the
physico-chemical properties of the inhibitors and improve the
binding affinity [5,7]. The success of Icotinib that employs a crown
ether moiety instead of the two ether chains in Erlotinib is a good
example of this strategy [8]. Before long, Dong et al. also reported
that four-membered heterocycles-containing Lapatinib analogs
showed higher HER protein-tyrosine kinase inhibitory activities
and similar antitumor potency, indicating the potency of Lapatinib
is still possible to be improved by structural modifications [7].
Based on the above reports, we were encouraged to design and
prepare new derivatives by making structural modifications on the
side chain. Our strategy is to shorten the length of the side chain of
Lapatinib and at the same time change the straight nitrogen-
containing chain into
a branched one by introducing a
substituted amino group, resulting in a series of Lapatinib de-
rivatives containing a branched side chain (Fig. 1). Our preliminary
molecular modeling study suggested that the derivative could
effectively enter the ATP binding pocket of HER1 and maintain the
binding mode of Lapatinib, meanwhile, the side chain moiety ex-
tends into the solvent region and makes contacts with residues
Asp855 and Thr854 (Fig. 2, A). These preliminarily results sup-
ported our rationale that the designed derivatives may retain or
even improve the HER1 inhibitory activity of Lapatinib.
2. Results and discussion
2.1. Chemistry
For the synthesis of the derivatives, a similar synthetic proce-
dure previously used for Lapatinib was employed [9]. Generally, the
quinazoline compound was coupled to the boronic acid (4) via a

A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
633
Fig. 2. A) Docking of compound 2i (shown in red) within the HER1 kinase domain (PDB code: 1XKK) in comparison with Lapatinib (shown in blue). B) The illustration of the
interaction mode of 2i with the corresponding active sites of HER1. Note the new H-bonds formed between Asp855 and Thr854 residues and the amino group of 2i. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Suzuki coupling reaction to form the key intermediate 5. For the
synthesis of the racemic products, compound 5 was treated with
dimethyl sulfone in the presence of n-butyllithium and then
refluxed in methanol/40% H2SO4 to give compound 6, which was
further reacted with the corresponding alkyl amines to give the
final products 2aeg, respectively (Scheme 1). For the synthesis of
the stereoisomers 2hen, the t-butanesulfinyl group was used as a
powerful chiral directing group [10]. In detail, compound 5 was
firstly aminated by (R) or (S)-t-butylsulfinamide to give the corre-
sponding (R) or (S)-Schiff’s base (7 or 8) which was further treated
with dimethyl sulfone in the presence of n-butyllithium and fol-
lowed by removing of the sulfinyl group to offer the enantiomers
2h and 2m, respectively. Products 2i and 2n were obtained by
treating 2h or 2m with formic acid and 40% formaldehyde aqueous
solution in DMSO. For the preparation of the amide products,
compound 2h was directly acylated by the corresponding acyl
chloride to offer 2jel, respectively (Scheme 2).
2.2. Tyrosine kinase inhibition
To examine the potential of the newly synthesized derivatives
for use as tyrosine kinase inhibitors, compounds 2aen were sub-
jected to an ELISA assay [11]. Table 1 shows the inhibitory activity of
the derivatives against HER1, HER2, c-Kit, PDGFR and KDR,
respectively. It was found that the derivatives significantly inhibi-
ted the autophosphorylation of HER1 and HER2 kinases. The IC50
values of the derivatives (except the amide products 2jel) are
below 60 nM for HER2 inhibition and within the range of
4.1e22.2 nM for HER1 inhibition. Particularly, compound 2c
showed potent inhibitory activity towards both HER2 and HER1

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A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
Scheme 1. The synthetic route of compounds 2aeg. Reagents and conditions: i) (Ph3P)2PdCl2, NEt3, MeOH, reflux, 12 h; ii) dimethyl sulfone, n-butyllithium, THF, r.t., 4 h; then
methanol/40% H2SO4, reflux, 4 h; iii) corresponding amine, MeOH, reflux, 24 h.
kinases with IC50 values of 3.6 and 6.8 nM, respectively, which is in
magnitude at the same order as Lapatinib with IC50 value of 2.7 and
7.9 nM, respectively. Considering there is a chiral center at the side
chain of 2c, we managed to prepare two enantiomers 2i and 2n via
chiral synthesis and investigated whether there is any difference of
the activities between the two enantiomers. It turned out that the
(S)-isomer 2i showed higher potency than the (R)-isomer 2n,
indicating that the steric configuration of the side chain has an
influence on the activity of the enantiomers. In contrast, com-
pounds 2jel, which possess amide substituents on the side chain,
showed only a weak inhibitory activity with IC50 values around
100 nM. All derivatives did not show significant inhibitory effects
on c-Kit, PDGFR and KDR kinases, suggesting that the derivatives
could be considered as HER1/HER2 selective inhibitors, which are
similar to the action manner of the lead compound Lapatinib.
2.4. In vitro cytotoxicity
Given the positive results of the HER1/HER2 inhibition test, we
further investigated the in vitro cytotoxicity of the derivatives
against HER2-overexpressing cancer cell lines (e.g. SK-BR-3 and
BT474), HER1-overexpressing cancer cell line (e.g. A431), as well as
normal cancer cell line MCF-7 without HER1/HER2 expression us-
ing Sulforhodamine B (SRB) assay. Lapatinib was used as the posi-
tive control. Table 2 revealed that most of the newly synthesized
derivatives showed comparable cytotoxicity to Lapatinib against
the HER1/HER2 overexpressing cancer cell lines with IC50 values
below 0.1 mM. Consistent with the order of the HER1/HER2 inhib-
itory activity, compound 2c as well as its enantiomers 2i and 2n
showed higher potency than the other compounds. Particularly, the
cytotoxicity of 2i (IC50 28.8 nM) against BT474 cell line is even over
2-fold higher than that of Lapatinib (IC50 63.9 nM). However,
compounds 2jel that bear amide substituents on the side chain
showed only a weak cytotoxic activity. As for the MCF-7 cell line
which has no HER1/HER2 expression, the derivatives did not show
any cytotoxicity, which is consistent with the performance of
Lapatinib as well as with the kinase inhibition results. Upon
analyzing the structureeactivity relationship, the alkylamine sub-
stituents on the side chain seem beneficial for the HER1/HER2
inhibitory and cytotoxic activity; however, converting the amine
groups to the amide groups (e.g. 2jel) remarkably decreases the
activity. The size of the substituents also has an influence on the
activity. Bulky groups such as cyclohexylamine (2e) and 2-(meth-
ylsulfonyl)ethylamine (2g) seem to have negative effects on the
activity, while small groups such as dimethylamine appear optimal
for the activity. The steric configuration of the chiral carbon also
plays an important role in the activity. It is noted that the (S)-iso-
mers (e.g. compounds 2h and 2i) generally showed higher potency
than the (R)-isomers (e.g. compounds 2m and 2n). Docking study
revealed that the amino group on the side chain of 2i can interact
with the Asp855 and Thr854 residues via H-bonds (Fig. 2, B). In
contrast, such interaction was not observed in the case of the (R)-
isomer. We assumed that this might be the reason for the activity
difference of the isomers. These results have also somehow
confirmed that the structure of the side chain can not only influ-
ence the physico-chemical properties but also affect the kinases
inhibitory activity and consequently the cytotoxic activity.
2.3. Western blot analysis
Since compound 2i showed the best activity in the ELISA assay, it
was selected to perform a western blot assay to investigate its effect
on the phosphorylation of tyrosine kinases as well as the activation
of the downstream signal transduction pathways. It is well known
that growth factors (e.g. VEGF165, EGF, PDGFBB and SCF-1) binding
to the corresponding tyrosine kinase receptors can induce the
phosphorylation of the tyrosine kinases, and cause a dose depen-
dent inhibition of biomarker phosphorylation, resulting in a series
of biological effects. Though different types of tyrosine kinase re-
ceptors share a great degree of commonality, they also have their
own specificity [5]. In order to determine the inhibitory effect of 2i
on the different downstream signal transduction pathways, five
different cell lines, including NCI-H526 (overexpressing c-Kit),
U87MG (overexpressing PDGFR), HUVEC (overexpressing KDR),
A431 (overexpressing HER1) and BT474 (overexpressing HER2),
were employed in the assay. Consistent with the former tests, 2i
significantly inhibited the phosphorylation of HER1 (in A431) and
HER2 (in BT474) as well as the activation of the biomarkers within
the downstream signal transduction pathways in a dose dependent
manner (Figs. 3 and 4). It is noted that the inhibitory potency of 2i
towards HER2 is even higher than that of Lapatinib. However, both
2i and Lapatinib had no effect on c-Kit, PDGFR, KDR and their
downstream signal transduction proteins (Figs. 5e7).
Considering that compounds 2h and 2i showed good activity in
the preliminary assays, we further investigated the cytotoxicity of

A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
635
Scheme 2. The synthetic route of compounds 2hen. Reagents and conditions: i) (R)-t-butylsulfinamide, titanium(IV) isopropoxide, THF, reflux, 4 h; ii) (S)-t-butylsulfinamide,
titanium(IV) isopropoxide, THF, reflux, 4 h; iii) dimethyl sulfone, n-butyllithium, THF, ꢀ40 ꢁC, 1 h; iv) HCl/ethanol (1:10, v/v), r.t., 30 min; v) HCOOH, 40% HCHO, DMSO, 50 ꢁC, 12 h;
vi) corresponding acyl chloride, NEt3, THF, r.t., 2 h.
these two compounds against other six cell lines, including HER2
Table 1
overexpressing Calu-3, MDA-MB-453, SK-OV-3 cancer cell lines,
and MDA-MB-231, NCI-H460 cancer cell lines without HER1/HER2
expression as well as normal human lung cell MRC-5 (Table 3).
Similar to the former results, both 2h and 2i exhibited quite high
activity towards the HER1/HER2 expressing tumor cells, while only
low or none toxicity to the cells without the expression of HER1/
HER2 was observed, indicating a selective cytotoxicity. This result
also indicated that HER1 and HER2 are the main action targets of
the Lapatinib derivatives.
The tyrosine kinase inhibitory activity of the derivatives 2aen expressed as IC50
values (nM).
Compounds
IC50 (nM)a
HER2
HER1
PDGFR
b
KDR
c-Kit
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
Lapatinib
3.4
1.0
3.6
15.5
58.6
48.3
52.2
2.9
17.6
34.8
6.8
14.7
77.2
39.2
28.7
10.8
4.1
89.6
92.8
110.2
13.5
30.4
7.9
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
>200,000
2.5. Flow cytometry study
0.5
152.0
163.8
158.2
10.6
26.1
2.7
Furthermore, a flow cytometry study was performed with 2i to
examine the effect on BT474 cell-cycle progression and the mech-
anism of cell death (necrosis or apoptosis). The resulting diagrams
are shown in Fig. 8. It was observed that, after being co-incubated
with BT474 cells for 24 h, both 2i and Lapatinib could cause accu-
mulation of cells in the G1 phase and produce a decreased
a
Data are the mean values of at least three determinations.

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A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
Fig. 6. The effect of 2i and Lapatinib on PDGFR (in U87MG cell line).
Fig. 3. The effect of 2i and Lapatinib on HER1 (in A431 cell line) and its downstream
signal transduction pathways.
2i
Lapatinib
0
0.001 0.01 0.1
1
10 0.001 0.01 0.1
1
10
μM
Fig. 7. The effect of 2i and Lapatinib on KDR (in HUVEC cell line).
P-HER2
P-ERK1/2
ERK1/2
P-Akt
Table 2
In vitro cytotoxicity of 2aen and Lapatinib.
Compd.
IC50 (nM)a
SK-BR-3
BT474
A431
MCF-7
6621
2a
2b
26.6
30.1
75.4
76.8
682.6
648.0
604.4
884.1
773.2
623.2
683.9
662.9
634.1
884.0
1089.0
1201.0
632.2
767.7
643.1
Akt
6521
6121
8731
9129
6112
7012
6832
5669
8934
9210
>10,000
9336
6666
8468
2c
36.2
48.6
Fig. 4. The effect of 2i and Lapatinib on HER2 (in BT474 cell line) and its downstream
2d
2e
2f
2g
33.9
108.7
174.0
94.4
104.2
69.2
signal transduction pathways.
162.2
101.2
122.8
21.0
2h
population of cells in S phase relative to the negative control,
indicating that the cell-cycle progression is blocked in the G1 phase
by 2i. In addition, at a dose of 10 mM both 2i (apoptosis: 11%) and
Lapatinib (apoptosis: 13%) showed positive effects on tumor
apoptosis.
2i
18.4
28.8
2j
2k
2l
2m
2n
106.0
701.5
1054.0
84.1
119.7
1005.0
1919.0
116.5
83.2
27.8
Lapatinib
30.1
63.9
2.6. In vivo antitumor activity
a
Data are the mean values of at least three determinations (IC50 is the drug
concentration effective in inhibiting 50% of the cell growth measured by SRB assays
after 72 h drug exposure).
In view of the good results of the HER1/HER2 inhibition test and
the in vitro cytotoxicity assay, compounds 2c (200 mg/kg, po) and
2i (100 and 200 mg/kg, po) were selected for further in vivo efficacy
studies in a HER2-overexpressing Calu-3 tumor xenograft mouse
model (Fig. 9). Lapatinib (100 and 200 mg/kg, po) was used as the
positive control and the vehicle (0.5% CMC aqueous solution con-
taining 0.1% Tween-80) group was used as the negative control. It
was found that 2i exhibited significant in vivo efficacy. At a high
dose of 200 mg/kg, the tumor inhibiting rate of 2i reached 83%,
much higher than that of Lapatinib (70%), while the tumor inhib-
iting rate of 2c (58%) is lower than that of Lapatinib. At a low dose of
100 mg/kg, 2i still exhibited a good tumor inhibiting activity with
an inhibiting rate 68%, superior to the activity of Lapatinib (56%) at
the same dose. Furthermore, no body weight loss was observed
after the administration of the tested compounds (data not shown),
indicating that the compounds possess a good safety. Thus, on the
basis of its potent in vivo efficacy and good in vitro profiles, 2i was
selected as a candidate for further pharmacokinetic investigation.
2.7. Pharmacokinetic investigation
It is well known that the structural modification of the side
chain could change the pharmacokinetic properties. For example,
the propylmorpholino side chain of the Gefitinib helps to achieve
Table 3
In vitro cytotoxicity of 2h, 2i and Lapatinib against HER2 overexpressing Calu-3,
MDA-MB-453, SK-OV-3 cancer cell line, and MDA-MB-231, NCI-H460 cancer cell
line without HER1/HER2 expression and normal human lung cell MRC-5.
Compd.
IC50
Calu-3 MDA-MB-453 SK-OV-3 MDA-MB-231 NCI-H460 MRC-5
(m
M)a
2h
2i
0.22
0.23
0.24
0.26
0.19
0.41
0.44
0.59
>100
95.31
>100
>100
>100
>100
>100
>100
>100
Lapatinib 0.23
a
Data are the mean values of at least three determinations (IC50 is the drug
concentration effective in inhibiting 50% of the cell growth measured by SRB assays
after 72 h drug exposure).
Fig. 5. The effect of 2i and Lapatinib on c-Kit (in NCI-H526 cell line).

A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
637
Fig. 8. The effect of 2i and Lapatinib on BT474 cell-cycle distribution tested by flow cytometry.
high and sustained blood levels in vivo over a 24-h period [5]. In
order to investigate whether the branched side chain of the newly
synthesized derivatives has an effect on the pharmacokinetic
properties or not, compounds 2h and 2i were selected for single
dose study on the pharmacokinetics property. Beagle dogs were
treated with 2h or 2i at a dose of 240 mg/kg by gavage, and after
24 h the pharmacokinetic parameters of maximum plasma con-
centration (Cmax), time to reach Cmax (Tmax), mean residence time
(MRT) and area under the plasma concentrationetime curve for
0ꢀ24 h after dosing (AUC0e24 h) were determined (Table 4). It was
noticed that both 2h and 2i exhibited good performance in the
accumulate with multiple doses [13], multiple dose study was
further performed for 2i (Table 5). After administration of doses of
240 mg/kg for 14 days in 6 beagle dogs, oral absorption was delayed
by ~30 min as indicated by Tmax prolonged from 4.0 h to 4.5 h. The
Cmax, AUC0ꢀ24h and AUC0e∞ (area under the plasma concen-
trationetime curve from time zero to infinity after dosing) of 2i
reached 2.9 mg/ml, 30.1 mg h/ml and 30.6 mg h/ml, respectively. As
for the elimination profile of 2i, the elimination half life t1/2, the
total body clearance CL and MRT of 2i were also determined. The
results are shown in Table 4. Judging from both the absorption and
elimination profiles of 2i, it can be concluded that after oral
administration 2i could be effectively absorbed, achieve the ther-
apeutic concentration and maintain the concentration for a long
while in vivo. This good pharmacokinetic property may support the
potent in vivo efficacy of 2i.
terms of absorption. The Cmax and AUC0ꢀ24h of 2i were 3.1
mg/ml
and 29.8 g h/ml, respectively, which is comparable to Lapatinib
m
[12]. Considering single dose study may not accurately predict
systemic exposure (AUC) or the half life t1/2 since concentrations
3. Conclusion
HER protein-tyrosine kinases have emerged in recent years as
key and validated targets of targeted therapies for solid tumors,
thus HER protein-tyrosine kinases inhibitors thereby have attracted
Table 4
Pharmacokinetic parameters of 2h and 2i after single dose administration in beagle
dogs subjects (n ¼ 6).
Compd. Pharmacokinetic parameters
Cmax (CV%a) (
m
g/ml) Tmax (h) AUC0ꢀ24 h (CV%a) MRT (CV%a) (h)
g$h/ml)
(
m
2h
2i
2.1 (37%)
3.1 (45%)
5.0
4.0
20.3 (31%)
29.8 (42%)
6.8 (28%)
6.1 (38%)
Fig. 9. In vivo anti-tumor effect of different dose of 2c, 2i and Lapatinib in Calu-3
tumor-bearing mice models (n ¼ 6).
a
CV% means coefficient of variation.

638
A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
Table 5
The resulted solution was stirred at room temperature for 1 h, then
was added to a solution of compound 5 (3.0 g, 6.0 mmol) [9] dis-
solved in 250 ml of THF and stirred for another 3 h. Concentrated
sulfuric acid (5 ml) was then added and the mixed solution was
refluxed for 1 h. Thereafter, the reaction solution was neutralized by
saturated NaHCO3 aqueous solution and extracted with CH2Cl2
(300 ml ꢂ 3). The organic phase was then dried with anhydrous
Na2SO4, and the solvent was removed under vacuum to give the
crude product which was further purified by column chromatog-
raphy (CH2Cl2/MeOH ¼ 60:1) to give the desired product 6 (1.7 g) in
50% yield as a yellow solid. m.p. 94e96 ꢁC. 1H NMR (400 MHz,
Pharmacokinetic parameters of 2i after multiple dose administration in beagle dogs
subjects (n ¼ 6).
Compd. Pharmacokinetic parameters
Cmax (CV%a) Tmax (h) AUC0ꢀ24 h AUC0ꢀ∞ (CV%a) CL
t1/2 MRT
(m
g/ml)
(CV%a)
g$h/ml)
(
mg$h/ml)
(L/h) (h) (CV%a)
(h)
(m
2i
2.9 (47%)
4.5
29.8 (29%) 30.6 (36%)
25.9 4.8 6.6 (36%)
a
CV% means coefficient of variation.
much attention in the field of searching for novel antitumor drug
candidates. Our current work is focused on the structural modifi-
cation of the side chain of HER1/HER2 inhibitor Lapatinib by
changing the straight nitrogen-containing alkyl chain into a
branched one. It was found that most of the newly synthesized
derivatives retained or even improved the inhibitory activity of
Lapatinib against HER1/HER2 as well as the downstream signal
transduction proteins. In vitro cytotoxicity assay revealed that the
target compounds could significantly inhibit the proliferation of the
HER1/HER2-overexpressing cancer cells. Particularly, the in vitro
cytotoxicity and the kinases inhibition potency of compound 2i is
even 2e5 folds higher than that of Lapatinib. SAR analysis revealed
that the alkylamine substituents on the side chain seem beneficial
for the HER1/HER2 inhibitory and cytotoxic activity. However,
converting the amine groups to the amide groups significantly
decreased the activity. Besides, bulky groups on the side chain have
negative effects on the activity, while small groups such as dime-
thylamine group appear optimal for the activity. The steric config-
uration of the chiral carbon also plays an important role in the
activity. It is noted that the (S)-isomers generally showed higher
potency than the (R)-isomers.
For the further pharmacological evaluations, the most active
compound 2i was selected for flow cytometry study. It was found
that 2i could block the cell-cycle progression of BT474 cells in the
G1 phase and cause tumor cells apoptosis. In vivo assay also
showed that 2i possessed a comparable antitumor activity to
Lapatinib. Further pharmacokinetics investigation on 2i revealed
that it had a good performance on both absorption and elimination
profiles. Consequently, these derivatives, especially 2i, are expected
to be promising antitumor candidates for further evaluations.
DMSO-d6)
d
: 10.01 (s, 1H, NH), 8.82 (d, J ¼ 4.4 Hz, 1H, ArH), 8.51 (s,
1H, ArH), 8.20 (dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.06 (d, J ¼ 2.0 Hz, 1H,
ArH), 7.80 (d, J ¼ 8.4 Hz, 2H, ArH), 7.50e7.44 (m, 1H, ArH), 7.31e7.26
(m, 3H, ArH), 7.20e7.17 (m, 1H, ArH), 7.11e7.09 (m, 1H, ArH), 6.53 (d,
J ¼ 3.2 Hz,1H, ArH), 6.20 (d, J ¼ 14.2 Hz,1H, ArH), 6.15 (d, J ¼ 14.2 Hz,
1H, ArH), 5.27 (s, 2H, OCH2), 3.11 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6)d: 162.6,158.1, 157.9, 154.7, 151.9, 150.2, 149.4, 140.1, 133.6,
130.9, 129.1, 128.8, 128.6, 128.1, 124.9, 123.7, 123.1, 121.5, 120.5, 117.2,
115.8, 115.1, 114.8, 114.4, 108.5, 108.3, 69.9, 41.6. MS-ESI (m/z): 550.0
[MþH]þ.
4.1.3. General procedure for the synthesis of 2aeg
Compound 6 (1.0 mmol) was added to a solution of the corre-
sponding amine (8.0 mmol) dissolved in absolute ethanol and then
refluxed for 14 h. The resulted solution was cooled to the room
temperature, and then the solvent was removed under vacuum to
give the crude product which was furtehr purified by column
chromatography (CH2Cl2/MeOH ¼ 50:1) to give 2aeg, respectively.
4.1.3.1. 6-(5-(1-Amino-2-(methylsulfonyl)ethyl)furan-2-yl)-N-(3-
chloro-4-((3-fluorobenzyl)oxy)phenyl) quinazolin-4-amine (2a).
Yield 66%. m.p. 121e123 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d: 10.03
(s, 1H, NH), 8.88 (d, J ¼ 4.4 Hz, 1H, ArH), 8.56 (s, 1H, ArH), 8.20 (dd,
J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.07 (d, J ¼ 2.0 Hz, 1H, ArH), 7.80 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.51e7.45 (m, 1H, ArH), 7.36e7.28 (m, 3H, ArH),
7.21e7.16 (m,1H, ArH), 7.11e7.10 (m,1H, ArH), 6.57 (d, J ¼ 3.2 Hz,1H,
ArH), 5.27 (s, 2H, OCH2), 4.52e4.49 (m, 1H, CH), 3.63e3.61 (m, 2H,
CH2), 3.12 (s, 3H, CH3), 2.38 (br, 2H, NH2). 13C NMR (100 MHz,
DMSO-d6) d: 162.6, 158.1, 157.9, 154.7, 151.9, 150.2, 149.4, 140.1,
133.6, 130.9, 129.1, 128.8, 128.6, 124.9,123.7, 123.1, 121.5, 117.2, 115.8,
115.1, 114.8, 114.4, 108.5, 108.3, 69.9, 59.6, 46.2, 43.0. HPLC purity:
tR ¼ 9.00, 99.44%. MS-ESI (m/z): 567.2 [MþH]þ.
4. Experimental protocols
4.1. Chemistry
4.1.3.2. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-(meth-
ylamino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-amine
4.1.1. Materials and instruments
(2b). Yield 69%. m.p. 134e135 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d:
All chemicals and reagents were of analytical reagent grade and
used without further purification. All the solvents were dried by the
standard methods wherever needed. Melting points were determined
in open capillary tubes on a Meltemp melting point apparatus and
uncorrected. 1H NMR and 13C NMR were recorded using a Bruker 400/
100 MHz spectrometer and referenced to the residue CHCl3 7.26 ppm
or DMSO-d6 2.5 ppm. Mass spectra were measured on Finnigan MAT
SSQ 710. The purity of the tested compounds was >95% based on the
HPLC analysis (Agilent 1200 and the mobile phase was water:
acetonitrile ¼ 55:45; the detection wavelength was set at 254 nm).
Diastereoselectivity was determined by chiral HPLC analysis
9.87 (s, 1H, NH), 8.75 (d, J ¼ 1.2 Hz, 1H, ArH), 8.57 (s, 1H, ArH), 8.21
(dd, J ¼ 8.8, 2.0 Hz, 1H, ArH), 8.03 (d, J ¼ 2.4 Hz, 1H, ArH), 7.82 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.75 (dd, J ¼ 8.8, 2.4 Hz, 1H, ArH), 7.51e7.46
(m, 1H, ArH), 7.36e7.29 (m, 3H, ArH), 7.21e7.17 (m, 1H, ArH), 7.08
(d, J ¼ 3.2 Hz, 1H, ArH), 6.63 (d, J ¼ 3.2 Hz, 1H, ArH), 5.28 (s, 2H,
OCH2), 4.28e4.25 (m, 1H, CH), 3.77e3.71 (m, 1H, CH2-a), 3.60e3.55
(m, 1H, CH2-b), 3.38 (br, 1H, NH), 3.07 (s, 3H, CH3), 2.29 (s, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6)
d: 162.7, 158.1, 154.9, 154.7,
152.3, 150.3, 149.4, 140.1, 133.5, 130.9, 129.1, 128.9, 128.6, 124.9,
123.7, 123.1, 121.6, 117.1, 115.7, 115.1, 114.8, 114.4, 110.4, 108.1, 69.9,
57.6, 53.3, 42.7, 33.4. HPLC purity: tR ¼ 10.10, 96.59%. MS-ESI (m/z):
581.3 [MþH]þ.
(25 cm ꢂ 5
mm ID Microsorb Si normal phase columnwith UVdetection
at 280 nm and at 254 nm). Retention times (tR) were given in minutes.
4.1.3.3. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-(dime-
4.1.2. (E)-N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(2-
(methylsulfonyl)vinyl)furan-2-yl)quinazolin-4-amine (6)
thylamino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-amine
(2c). Yield 65%. m.p. 90e92 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d:
To a solution of methyl sulfone (3.0 g, 32.0 mmol) in 50 ml of
THF was added n-butyllithium-hexane solution (2.5 M，11.4 ml).
9.86 (s, 1H, NH), 8.73 (d, J ¼ 1.2 Hz, 1H, ArH), 8.57 (s, 1H, ArH), 8.19
(dd, J ¼ 1.6, 8.8 Hz, 1H, ArH), 8.00 (d, J ¼ 2.8 Hz, 1H, ArH), 7.83 (d,

A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
639
J ¼ 8.8 Hz, 1H, ArH), 7.73 (dd, J ¼ 2.4, 8.8 Hz, 1H, ArH), 7.48e7.46 (m,
1H, ArH), 7.32e7.30 (m, 3H, ArH), 7.19 (td, J ¼ 2.0, 8.8 Hz, 1H, ArH),
7.09 (d, J ¼ 3.6 Hz, 1H, ArH), 6.63 (d, J ¼ 3.2 Hz, 1H, ArH)，5.28 (s,
2H, OCH2), 4.34e4.32 (m, 1H, CH), 3.94 (dd, J ¼ 8.8, 14.8 Hz,1H, CH2-
a), 3.72 (dd, J ¼ 5.6, 14.8 Hz,1H, CH2-b), 3.04 (s, 3H, CH3), 2.23 (s, 6H,
7.55e7.51 (m, 1H, ArH), 7.36e7.29 (m, 3H, ArH), 7.21e7.18 (m, 1H,
ArH), 7.02 (d, J ¼ 3.2 Hz, 1H, ArH), 6.62 (d, J ¼ 3.2 Hz, 1H, ArH), 5.28
(s, 2H, OCH2), 4.39e4.36 (m, 1H, CH), 3.79e3.59 (m, 4H, SO2CH2,
SO2CH2CH2), 3.45 (br, 1H, NH), 3.07 (s, 3H, CH3), 3.02 (s, 3H, CH3),
2.51e2.48 (m, 2H, CH2), 0.99 (d, 6H, J ¼ 7.2 Hz, (CH3)2). 13C NMR
N(CH3)2)；13C NMR (DMSO-d6, 100 Hz)
d: 162.6, 158.1, 154.8, 152.4,
(100 MHz, DMSO-d6) d: 162.7, 157.9, 157.6, 154.0, 152.1, 150.9, 149.1,
151.6, 150.4, 149.4, 140.1, 133.4, 130.9, 129.1, 128.9, 128.6, 125.0,
123.7, 123.2, 121.6, 117.2, 115.7, 115.1, 114.8, 114.4, 111.9, 107.9, 69.9,
57.4, 55.3, 54.6, 42.4, 41.2. HPLC purity: tR ¼ 17.19, 99.08%. ESI-MS
(m/z)595.3 [MþH]þ.
140.0, 135.1, 130.9, 129.0, 128.2, 128.0, 124.2, 123.8, 122.9, 121.0,
117.3, 115.3, 115.0, 114.9, 114.2, 109.8, 108.7, 69.9, 60.1, 51.2, 50.3,
43.9, 43.1, 34.5. HPLC purity: tR ¼ 11.21, 97.01%. MS-ESI (m/z): 673.2
[MþH]þ.
4.1.3.4. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-(ethyl-
amino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-amine (2d).
4.1.4. Gerneal procedure for the synthesis of 9 and 10
A mixture of compound 5 (1.0 mmol), (R) or (S)-t-butylsulfi-
namide (1.2 mmol), titanium(IV) isopropoxide (3 mmol) in THF
(15 ml) was refluxed for 4 h. After cooled to the room tempera-
ture, the mixture was treated with saturated NaCl aqueous so-
lution and a great amount of deposit was formed. The deposit
was filtrated off. The filtrate was then concentrated, diluted by
CH2Cl2, and washed by water. Then, the organic phase was
separated, dried with anhydrous Na2SO4, and the solvent was
removed under vacuum to give the desired product (7 or 8) as
yellow oil which was applied in the next reaction without
purification.
Yield 57%. m.p. 97e99 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d: 9.86 (s,
1H, NH), 8.73 (d, J ¼ 1.6 Hz, 1H, ArH), 8.56 (s, 1H, ArH), 8.20 (dd,
J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.02 (d, J ¼ 2.4 Hz, 1H, ArH), 7.82 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.74 (dd, J ¼ 8.8, 2.4 Hz, 1H, ArH), 7.51e7.45 (m,
1H, ArH), 7.36e7.29 (m, 3H, ArH), 7.21e7.16 (m, 1H, ArH), 7.06 (d,
J ¼ 3.2 Hz, 1H, ArH), 6.61 (d, J ¼ 3.2 Hz, 1H, ArH), 5.28 (s, 2H, OCH2),
4.35e4.32 (m, 1H, CH), 3.74e3.51 (m, 2H, CH2), 3.31 (br, 1H, NH),
3.07 (s, 3H, CH3), 2.63e2.45 (m, 2H, CH2),1.03 (t, J ¼ 7.2 Hz, 3H, CH3).
13C NMR (100 MHz, DMSO-d6)
d: 162.6, 158.1, 155.3, 154.7, 152.2,
150.3, 149.4, 140.1, 133.5, 130.9, 129.1, 128.9, 128.6, 124.9, 123.7,
123.1, 121.6, 117.0, 115.7, 115.1, 114.8, 114.4, 110.1, 108.1, 69.9, 58.0,
51.6, 42.9, 40.9, 15.4. HPLC purity: tR ¼ 13.53, 95.12%. MS-ESI (m/z):
595.3 [MþH]þ.
To a ꢀ40 ꢁC solution of methyl sulfone (6.0 mmol) in THF
(15 ml) was slowly added a solution of n-butyllithium (4.0 mmol)
in n-hexane, and the resulted solution was stirred at ꢀ40 ꢁC for
0.5 h. Compound 7 or 8 obtained from the last step (1.0 mmol) in
15 ml of THF was then quickly added, and the resulted solution
was stirred at ꢀ40 ꢁC for another 0.5 h. Thereafter, the reaction
solution was poured into 200 ml of saturated NaCl aqueous so-
lution. The organic phase was separated, dried with anhydrous
Na2SO4, and the solvent was removed under vacuum to give the
crude product which was further purified by column chroma-
tography (CH2Cl2/MeOH ¼ 60:1) to give the desired product (9 or
10).
4.1.3.5. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-(cyclo-
hexylamino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-amine
(2e). Yield 73%. m.p. 80e81 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d:
9.86 (s, 1H, NH), 8.75 (d, J ¼ 1.6 Hz, 1H, ArH), 8.56 (s, 1H, ArH), 8.20
(dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.00 (d, J ¼ 2.4 Hz, 1H, ArH), 7.83 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.74 (dd, J ¼ 8.8, 2.4 Hz, 1H, ArH), 7.50e7.45 (m,
1H, ArH), 7.34e7.28 (m, 3H, ArH), 7.20e7.17 (m, 1H, ArH), 7.04 (d,
J ¼ 3.2 Hz, 1H, ArH), 6.63 (d, J ¼ 3.2 Hz, 1H, ArH), 5.28 (s, 2H, OCH2),
4.37e4.35 (m, 1H, CH), 3.76e3.64 (m, 2H, CH2), 3.41 (br, 1H, NH),
3.07 (s, 3H, CH3), 2.64e2.55 (m, 1H, CH), 1.61e1.17 (m, 10H, (CH2)5).
4.1.4.1. (S)-N-(1-(5-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)quinazolin-6-yl)furan-2-yl)-2-(methylsulfonyl)ethyl)-2-
methylpropane-2-sulfinamide (9). Yield 57%. m.p. 103e105 ꢁC.
½aꢃ2D0 ¼ 15.4ꢁ (c 1, DMSO). e.e. 96% (major enantiomer, tR ¼ 6.10;
13C NMR (100 MHz, DMSO-d6)
d: 162.5, 158.0, 155.3, 154.9, 152.0,
150.3, 149.2, 140.1, 133.5, 130.9, 129.1, 128.9, 128.2, 124.8, 123.3,
122.7, 121.1, 117.9, 115.8, 115.6, 114.6, 113.5, 110.0, 108.7, 69.6, 59.9,
53.0, 43.2, 42.0, 33.9, 26.8, 25.4. HPLC purity: tR ¼ 11.26, 96.25%. MS-
ESI (m/z): 649.2 [MþH]þ.
minor enantiomer, tR ¼ 9.03). 1H NMR (400 MHz, DMSO-d6)
d: 10.01
(s, 1H, NH), 8.87 (d, J ¼ 4.4 Hz, 1H, ArH), 8.56 (s, 1H, ArH), 8.21 (dd,
J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.07 (d, J ¼ 2.0 Hz, 1H, ArH), 7.80 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.52e7.48 (m, 1H, ArH), 7.36e7.29 (m, 3H, ArH),
7.21e7.16 (m,1H, ArH), 7.11e7.10 (m,1H, ArH), 6.57 (d, J ¼ 3.2 Hz,1H,
ArH), 5.65 (br, 1 h, NH), 5.27 (s, 2H, OCH2), 4.59e4.55 (m, 1H, CH),
4.1.3.6. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-(iso-
propylamino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-
amine (2f). Yield 79%. m.p. 86e88 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d: 9.86 (s,1H, NH), 8.77 (d, J ¼ 1.6 Hz,1H, ArH), 8.56 (s,1H, ArH), 8.22
3.65e3.61 (m, 2H, CH2), 3.12 (s, 3H, CH3), 1.36 (s, 9H, C(CH3)3). 13
C
(dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.01 (d, J ¼ 2.4 Hz, 1H, ArH), 7.83 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.72 (dd, J ¼ 8.8, 2.4 Hz, 1H, ArH), 7.51e7.46 (m,
1H, ArH), 7.34e7.28 (m, 3H, ArH), 7.20e7.16 (m, 1H, ArH), 7.02 (d,
J ¼ 3.2 Hz, 1H, ArH), 6.62 (d, J ¼ 3.2 Hz, 1H, ArH), 5.28 (s, 2H, OCH2),
4.37e4.35 (m, 1H, CH), 3.76e3.64 (m, 2H, CH2), 3.35 (br, 1H, NH),
3.07 (s, 3H, CH3), 2.62e2.59 (m, 1H, CH), 0.99 (d, 6H, J ¼ 7.2 Hz,
NMR (100 MHz, DMSO-d6) d: 162.5, 158.0, 157.9, 154.7, 151.9, 150.6,
149.4, 140.1, 133.2, 130.1,129.0,128.3,128.1,124.4, 123.5,123.1, 121.0,
117.7, 115.2, 115.0, 114.1, 113.8, 108.9, 108.6, 69.9, 62.1, 59.9, 46.6,
43.4, 21.5. HPLC purity: tR ¼ 12.09, 97.14%. MS-ESI (m/z): 671.2
[MþH]þ.
(CH3)2). 13C NMR (100 MHz, DMSO-d6)
d
: 162.4, 158.1, 157.9, 154.7,
4.1.4.2. (R)-N-(1-(5-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)quinazolin-6-yl)furan-2-yl)-2-(methylsulfonyl)ethyl)-2-
methylpropane-2-sulfinamide (10). Yield 50%. m.p. 103e105 ꢁC.
½aꢃ2D0 ¼ ꢀ16.0ꢁ (c 1, DMSO). e.e. 95% (major enantiomer, tR ¼ 9.03;
151.5, 150.2, 149.2, 140.1, 133.6, 130.2, 129.0, 128.8, 128.6, 124.9,
123.7, 122.9, 121.5, 117.2, 115.8, 115.1, 114.8, 114.4, 108.5, 108.3, 69.9,
59.9, 50.7, 46.2, 43.0, 22.5. HPLC purity: tR ¼ 9.29, 98.71%. MS-ESI
(m/z): 609.1 [MþH]þ.
minor enantiomer, tR ¼ 6.11).1H NMR (400 MHz, DMSO-d6)
d: 10.01
(s, 1H, NH), 8.87 (d, J ¼ 4.4 Hz, 1H, ArH), 8.56 (s, 1H, ArH), 8.21 (dd,
J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.07 (d, J ¼ 2.0 Hz, 1H, ArH), 7.80 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.52e7.48 (m, 1H, ArH), 7.36e7.29 (m, 3H, ArH),
7.21e7.16 (m,1H, ArH), 7.11e7.10 (m,1H, ArH), 6.57 (d, J ¼ 3.2 Hz,1H,
ArH), 5.65 (br, 1 h, NH), 5.27 (s, 2H, OCH2), 4.59e4.55 (m, 1H, CH),
4.1.3.7. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(2-(meth-
ylsulfonyl)-1-((2-(methylsulfonyl)ethyl) amino)ethyl)furan-2-yl)qui-
nazolin-4-amine (2g). Yield 42%. m.p. 79e80 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 9.87 (s, 1H, NH), 8.75 (d, J ¼ 1.6 Hz, 1H, ArH), 8.55 (s,
1H, ArH), 8.22 (dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.01 (d, J ¼ 2.4 Hz, 1H,
3.65e3.61 (m, 2H, CH2), 3.12 (s, 3H, CH3), 1.36 (s, 9H, C(CH3)3). 13
C
ArH), 7.83 (d, J ¼ 8.8 Hz, 1H, ArH), 7.72 (dd, J ¼ 8.8, 2.4 Hz, 1H, ArH),
NMR (100 MHz, DMSO-d6) d: 162.5, 158.0, 157.9, 154.7, 151.9, 150.6,

640
A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
149.4,140.1, 133.2, 130.1, 129.0,128.3,128.1, 124.4, 123.5,123.1, 121.0,
117.7, 115.2, 115.0, 114.1, 113.8, 108.9, 108.6, 69.9, 62.1, 59.9, 46.6,
43.4, 21.5. HPLC purity: tR ¼ 12.21, 98.41%. MS-ESI (m/z): 671.2
[MþH]þ.
(m, 1H, CH), 3.94 (dd, J ¼ 8.8, 14.8 Hz, 1H, CH2-a), 3.72 (dd, J ¼ 5.6,
14.8 Hz, 1H, CH2-b), 3.04 (s, 3H, CH3), 2.23 (s, 6H, N(CH3)2). 13C NMR
(DMSO-d6, 100 Hz)d: 162.6, 158.1, 154.8, 152.4, 151.6, 150.4, 149.4,
140.1,133.4, 130.9,129.1,128.9,128.6,125.0,123.7, 123.2,121.6, 117.2,
115.7, 115.1, 114.8, 114.4, 111.9, 107.9, 69.9, 57.4, 55.3, 54.6, 42.4, 41.2.
HPLC purity: tR ¼ 17.12, 96.42%. ESI-MS (m/z)595.3 [MþH]þ.
4.1.5. General procedure for the synthesis of 2h and 2m
Compound 9 or 10 (1.0 mmol) was added into 10 ml of HCl-
ethanol solution (1:10, v/v) and the resulted solution was stirred
at room temperature for 0.5 h. The reaction solution was then
neutralized by 25% aqueous ammonia and extracted by AcOEt
(30 ml ꢂ 3). The organic phase was dried with anhydrous Na2SO4,
and the solvent was removed under vacuum to give the crude
product which was furtehr purified by column chromatography
(CH2Cl2/MeOH ¼ 60:1) to give the desired product (2h or 2m).
4.1.7. (R)-N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-
(dimethylamino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-
amine (2n)
Yellow solid, yield 75%. m.p. 90e92 ꢁC. ½aꢃD20 ¼ 15.7ꢁ (c 1,
DMSO). e.e. 97% (major enantiomer, tR ¼ 10.07; minor enantiomer,
tR ¼ 7.08). 1H NMR (DMSO-d6, 400 MHz)
d: 9.86 (s, 1H, NH), 8.73
(d, J ¼ 1.2 Hz, 1H, ArH), 8.57 (s, 1H, ArH), 8.19 (dd, J ¼ 1.6, 8.8 Hz,
1H, ArH), 8.00 (d, J ¼ 2.8 Hz, 1H, ArH), 7.83 (d, J ¼ 8.8 Hz, 1H, ArH),
7.73 (dd, J ¼ 2.4, 8.8 Hz, 1H, ArH), 7.48e7.46 (m, 1H, ArH),
7.32e7.30 (m, 3H, ArH), 7.19 (td, J ¼ 2.0, 8.8 Hz, 1H, ArH), 7.09 (d,
J ¼ 3.6 Hz, 1H, ArH), 6.63 (d, J ¼ 3.2 Hz, 1H, ArH), 5.28 (s, 2H,
OCH2), 4.34e4.32 (m, 1H, CH), 3.94 (dd, J ¼ 8.8, 14.8 Hz, 1H, CH2-a),
3.72 (dd, J ¼ 5.6, 14.8 Hz, 1H, CH2-b), 3.04 (s, 3H, CH3), 2.23 (s, 6H,
4.1.5.1. (S)-6-(5-(1-amino-2-(methylsulfonyl)ethyl)furan-2-yl)-N-(3-
chloro-4-((3-fluorobenzyl)oxy)phenyl) quinazolin-4-amine (2h).
Yield 64%. m.p. 121e123 ꢁC. ½aꢃ2D0 ¼ þ16.6ꢁ (c 1, DMSO). e.e. 94%
(major enantiomer, tR ¼ 7.17; minor enantiomer, tR ¼ 9.38).1H NMR
(400 MHz, DMSO-d6)
d
: 10.03 (s, 1H, NH), 8.88 (d, J ¼ 4.4 Hz, 1H,
ArH), 8.56 (s, 1H, ArH), 8.20 (dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.07 (d,
J ¼ 2.0 Hz, 1H, ArH), 7.80 (d, J ¼ 8.4 Hz, 2H, ArH), 7.51e7.45 (m, 1H,
ArH), 7.36e7.28 (m, 3H, ArH), 7.21e7.16 (m, 1H, ArH), 7.11e7.10 (m,
1H, ArH), 6.57 (d, J ¼ 3.2 Hz, 1H, ArH), 5.27 (s, 2H, OCH2), 4.52e4.49
(m, 1H, CH), 3.63e3.61 (m, 2H, CH2), 3.12 (s, 3H, CH3), 2.38 (br, 2H,
N(CH3)2). 13C NMR (DMSO-d6, 100 Hz)
d: 162.6, 158.1, 154.8, 152.4,
151.6, 150.4, 149.4, 140.1, 133.4, 130.9, 129.1, 128.9, 128.6, 125.0,
123.7, 123.2, 121.6, 117.2, 115.7, 115.1, 114.8, 114.4, 111.9, 107.9, 69.9,
57.4, 55.3, 54.6, 42.4, 41.2. HPLC purity: tR ¼ 17.31, 97.38%. ESI-MS
(m/z)595.3 [MþH]þ.
NH2). 13C NMR (100 MHz, DMSO-d6)
d: 162.6, 158.1, 157.9, 154.7,
151.9, 150.2, 149.4, 140.1, 133.6, 130.9, 129.1, 128.8, 128.6, 124.9,
123.7, 123.1, 121.5, 117.2, 115.8, 115.1, 114.8, 114.4, 108.5, 108.3, 69.9,
59.6, 46.2, 43.0. HPLC purity: tR ¼ 9.03, 99.13%. MS-ESI (m/z): 567.2
[MþH]þ.
4.1.8. General procedure for the synthesis of 2jel
A mixture of 2i (2.0 g, 3.3 mmol), triethylamine (0.4 g, 4.0 mmol)
in THF (20 ml) was stirred for 5 min at room temperature. Then
corresponding acyl chloride (3.0 mmol) was added and the resulted
solution was stirred at room temperature for 2 h. The deposit was
filtrated off and the filtrate was concentrated to give the crude
product which was further purified by column chromatography
4.1.5.2. (R)-6-(5-(1-amino-2-(methylsulfonyl)ethyl)furan-2-yl)-N-
(3-chloro-4-((3-fluorobenzyl)oxy)phenyl) quinazolin-4-amine (2m).
Yield 49%. m.p. 120e123 ꢁC. ½aꢃ2D0 ¼ ꢀ16.2ꢁ (c 1, DMSO). e.e. 97%
(major enantiomer, tR ¼ 9.38; minor enantiomer, tR ¼ 7.17). 1H NMR
(CH2Cl2/MeOH
respectively.
¼
80:1) to give the desired product 2jel,
(400 MHz, DMSO-d6)
d
: 10.03 (s, 1H, NH), 8.88 (d, J ¼ 4.4 Hz, 1H,
ArH), 8.56 (s, 1H, ArH), 8.20 (dd, J ¼ 8.8, 1.6 Hz, 1H, ArH), 8.07 (d,
J ¼ 2.0 Hz, 1H, ArH), 7.80 (d, J ¼ 8.4 Hz, 2H, ArH), 7.51e7.45 (m, 1H,
ArH), 7.36e7.28 (m, 3H, ArH), 7.21e7.16 (m, 1H, ArH), 7.11e7.10 (m,
1H, ArH), 6.57 (d, J ¼ 3.2 Hz, 1H, ArH), 5.27 (s, 2H, OCH2), 4.52e4.49
(m, 1H, CH), 3.63e3.61 (m, 2H, CH2), 3.12 (s, 3H, CH3), 2.38 (br, 2H,
4.1.8.1. (S)-ethyl (1-(5-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)quinazolin-6-yl)furan-2-yl)-2-(methylsulfonyl)ethyl)carba-
mate (2j). Yellow solid, yield 82%. m.p. 78e80 ꢁC. ½aꢃD20 ¼ 33.2ꢁ(c 1,
THF). 1H NMR (DMSO-d6, 400 MHz)
d: 9.88 (s, 1H, NH), 8.75 (d,
J ¼ 1.2 Hz, 1H, ArH), 8.58 (s, 1H, ArH), 8.20 (dd, J ¼ 1.6, 8.8 Hz, 1H,
ArH), 8.03 (d, J ¼ 2.4 Hz, 1H, ArH), 7.99 (br, 1H, NH), 7.83 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.75 (dd, J ¼ 2.4, 8.8 Hz, 1H, ArH), 7.51e7.46
(m, 1H, ArH), 7.36e7.29 (m, 3H, ArH), 7.19 (td, J ¼ 2.0, 8.8 Hz, 1H,
ArH), 7.06 (d, J ¼ 3.2 Hz, 1H, ArH), 6.59 (d, J ¼ 3.2 Hz, 1H, ArH),
5.35e5.32 (m, 1H, CH), 5.28 (s, 2H, OCH2), 4.07 (q, J ¼ 7.2 Hz, 2H,
OCH2CH3), 3.86e3.81 (m, 1H, CH2-a), 3.73e3.67 (m, 1H, CH2-b),
3.04 (s, 3H, CH3), 1.20 (t, J ¼ 7.2 Hz, 3H, OCH2CH3). 13C NMR
NH2). 13C NMR (100 MHz, DMSO-d6)
d: 162.6, 158.1, 157.9, 154.7,
151.9, 150.2, 149.4, 140.1, 133.6, 130.9, 129.1, 128.8, 128.6, 124.9,
123.7, 123.1, 121.5, 117.2, 115.8, 115.1, 114.8, 114.4, 108.5, 108.3, 69.9,
59.6, 46.2, 43.0. HPLC purity: tR ¼ 9.12, 99.21%. MS-ESI (m/z): 567.2
[MþH]þ.
4.1.6. (S)eN-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-(5-(1-
(dimethylamino)-2-(methylsulfonyl)ethyl)furan-2-yl)quinazolin-4-
amine (2i)
(DMSO-d6, 100 Hz)d: 163.9, 161.4, 158.1, 156.0, 154.8, 153.8, 152.4,
150.3, 149.5, 140.1, 133.5, 131.0, 129.1, 128.9, 128.4, 124.8, 123.8,
123.0, 121.6, 117.3, 115.7, 115.2, 115.0, 114.8, 114.6, 114.4, 109.9, 108.2,
69.9, 60.7, 56.7, 44.9, 15.0. HPLC purity: tR ¼ 16.55, 98.37%. MS-ESI
(m/z): 639.1 [MþH]þ.
A mixture of compound 2h (11.5 g, 20.0 mmol), dimethyl sulf-
oxide (110 ml), 40% formaldehyde aqueous solution (21 ml), and
formic acid (10.6 ml) was stirred at 50 ꢁC for 12 h. Then the solution
was diluted by 500 ml of water and the pH value was adjusted to 9
by adding 25% aqueous ammonia dropwise. The formed yellow
deposit was collected by filtration and purified by column chro-
matography (CH2Cl2/MeOH ¼ 60:1) to give the desired product 2i
in 78% yield as a yellow solid. m.p. 90e92 ꢁC. ½aꢃD20 ¼ ꢀ15.5ꢁ (c 1,
DMSO). e.e. 98% (major enantiomer, tR ¼ 7.08; minor enantiomer,
4.1.8.2. (S)-butyl (1-(5-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)quinazolin-6-yl)furan-2-yl)-2-(methylsulfonyl)ethyl)carba-
mate (2k). Yellow solid, yield 85%. m.p. 88e89 ꢁC. ½aꢃD20 ¼ 25.5ꢁ (c 1,
THF). 1H NMR (DMSO-d6, 400 MHz)
d: 9.88 (s, 1H, NH), 8.76 (d,
J ¼ 1.2 Hz, 1H, ArH), 8.58 (s, 1H, ArH), 8.20 (dd, J ¼ 1.6, 8.8 Hz, 1H,
ArH), 8.03 (d, J ¼ 2.4 Hz, 1H, ArH), 8.00 (br, 1H, NH), 7.83 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.76 (dd, J ¼ 2.4, 8.8 Hz, 1H, ArH), 7.51e7.46 (m,
1H, ArH), 7.36e7.29 (m, 3H, ArH), 7.19 (td, J ¼ 2.0, 8.8 Hz, 1H, ArH),
7.06 (d, J ¼ 3.2 Hz, 1H, ArH), 6.59 (d, J ¼ 3.2 Hz, 1H, ArH), 5.34e5.31
(m, 1H, CH), 5.28 (s, 2H, OCH2), 4.04 (m, 2H, CH2O), 3.85 (dd, J ¼ 4.8,
14.4 Hz, 1H, CH2-a), 3.72 (dd, J ¼ 8.8, 14.4 Hz, 1H, CH2-b), 3.05 (s, 3H,
tR ¼ 10.07). 1H NMR (DMSO-d6, 400 MHz)
d: 9.86 (s, 1H, NH), 8.73 (d,
J ¼ 1.2 Hz, 1H, ArH), 8.57 (s, 1H, ArH), 8.19 (dd, J ¼ 1.6, 8.8 Hz, 1H,
ArH), 8.00 (d, J ¼ 2.8 Hz, 1H, ArH), 7.83 (d, J ¼ 8.8 Hz, 1H, ArH), 7.73
(dd, J ¼ 2.4, 8.8 Hz, 1H, ArH), 7.48e7.46 (m, 1H, ArH), 7.32e7.30 (m,
3H, ArH), 7.19 (td, J ¼ 2.0, 8.8 Hz, 1H, ArH), 7.09 (d, J ¼ 3.6 Hz, 1H,
ArH), 6.63 (d, J ¼ 3.2 Hz, 1H, ArH)，5.28 (s, 2H, OCH2), 4.34e4.32

A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
641
CH3), 1.58e1.55 (m, 2H, CH2CH2O), 1.36e1.34 (m, 2H, CH2CH3), 0.89
phenylenediamine in 0.1 M citrate buffer, pH 5.5, was added and
samples were incubated at room temperature until color emerged.
The reaction was terminated by the addition of 50 mL of 2 M
H2SO4, and the plate was read using a multiwell spectrophotom-
eter (SPECTRAMAX 190) at 490 nm. The inhibition rate (%) was
(t, J ¼ 7.2 Hz, 3H, CH2CH3). 13C NMR (DMSO-d6, 100 Hz)
d: 162.6,
158.1, 156.1,154.8,153.8,152.4,150.3,149.5,140.1,133.5,130.9,129.1,
128.9, 128.4, 124.8, 123.7, 123.0, 121.6, 117.3, 115.7, 115.1, 114.8, 114.4,
109.8,108.22, 69.9, 64.5, 56.7, 45.0, 42.1, 31.1,19.0,14.0. HPLC purity:
tR ¼ 23.25, 99.22%. MS-ESI (m/z): 667.4 [MþH]þ.
calculated using the following equation: [1
ꢀ
(A490/A490
control)] ꢂ 100%. IC50 values were obtained by Logit method and
were determined from the results of at least three independent
tests. The results were analyzed by Graphpad Prism 5.0 software.
4.1.8.3. (S)-hexyl (1-(5-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)quinazolin-6-yl)furan-2-yl)-2-(methylsulfonyl)ethyl)carba-
mate (2l). Yellow solid, yield 79%. m.p. 101e103 ꢁC. ½aꢃD20 ¼ 22.2ꢁ (c
1, THF). 1H NMR (DMSO-d6, 400 MHz)
d
: 9.88 (s, 1H, NH), 8.75 (d,
4.2.3. Western blot analysis
J ¼ 1.2 Hz, 1H, ArH), 8.58 (s, 1H, ArH), 8.20 (dd, J ¼ 1.6, 8.8 Hz, 1H,
ArH), 8.03 (d, J ¼ 2.4 Hz, 1H, ArH), 8.00 (br, 1H, NH), 7.83 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.75 (dd, J ¼ 2.4, 8.8 Hz, 1H, ArH), 7.49e7.45 (m,
1H, ArH), 7.36e7.29 (m, 3H, ArH), 7.19 (td, J ¼ 2.0, 8.8 Hz, 1H, ArH),
7.06 (d, J ¼ 3.6 Hz, 1H, ArH), 6.59 (d, J ¼ 3.2 Hz, 1H, ArH), 5.34e5.31
(m, 1H, CH), 5.28 (s, 2H, OCH2), 4.03e4.00 (m, 2H, CH2O), 3.85e3.80
(m, 1H, CH2-a), 3.72e3.66 (m, 1H, CH2-b), 3.04 (s, 3H, CH3),
1.58e1.22 (m, 8H, (CH2)4), 1.20 (t, J ¼ 7.2 Hz, 3H, CH3CH2). 13C NMR
NCI-H526, U87MG, HUVEC, A431 and BT474 cells were grown to
half-confluence in six-well plates, starved in serum-free medium
for 24 h, and then exposed to the tested compound at concentration
of 0.001, 0.01, 0.1 and 1 mM for 2 h. For analysis of receptor tyrosine
kinase phosphorylation and downstream signal transduction
pathways, cells were stimulated with 20 ng/ml EGF or SCF-1, or
PDGFBB or VEGF (all from R&D Systems, Minneapolis, MN) at 37 ꢁC
after the tested compound treatment. Western blot analyses were
subsequently performed using a standard procedure [14]. Anti-
bodies against the following were used: phospho-ErbB2 and ErbB2,
phospho-ERK1/2 and ERK1/2, phospho-AKT and AKT (all from Cell
(DMSO-d6, 100 Hz) d: 162.7, 158.1, 156.1, 154.8, 153.8, 152.4, 150.3,
149.4,140.1,133.5,131.0,129.1,128.9,128.4,124.8,123.7,123.0,121.6,
117.3,115.7,115.1,114.8,114.4,109.8,108.2, 69.9, 64.7, 56.7, 45.0, 42.1,
31.3, 29.0, 25.4, 22.4, 14.2. HPLC purity: tR ¼ 30.70, 97.15%. MS-ESI
(m/z): 695.3 [MþH]þ.
Signaling Technology, Beverly, MA) and b-tubulin (from Sigma).
4.2.4. Cell proliferation assay
4.2. Biological studies
SK-BR-3, BT474, A431, and MCF-7 cell lines were used for the
cell proliferation assays. All cell lines were obtained from the
American Type Culture Collection. Cells were maintained in RPMI-
1640 medium supplemented with 5% fetal bovine serum. Cells
were plated in 96-well plates at the densities of 5.0 ꢂ 104/ml. On
the next day, the tested compounds were dosed at 0.5, 5, 50, 500,
1000, and 5000 ng/ml concentrations and the cells were cultured
for 3 days. At the end of incubation, cell survival was determined
by the sulforhodamine B assay as previously described. The IC50
values were obtained from the growth curves using GraphPad
Prism 5.0.
4.2.1. Molecular modeling
Homology modeling was carried out using the MOE (Molecular
Operating Environment) software (Chemical Computing Group
Inc.). Initial minimization was performed within the homology
modeling function of MOE. The model from MOE was minimized
with a few thousand cycles of minimization using the ABNR
(adopted-basis NewtoneRaphson) method. Ligands were modeled
by positioning them in the active site in accordance with the
published crystal structure of the HER1 kinase domain complexed
with Lapatinib (PDB code: 1XKK). A 7.5 Å sphere of water mole-
cules was added around the ligand. The entire complex was then
subjected to alternate cycles of minimization and dynamics. Each
dynamics run was short, about 3 ps. The intent was to get a
satisfactory structure for the complex that was consistent with the
published crystal structure.
4.2.5. Flow cytometry study
1 ꢂ 106 BT474 cells were seeded in a six-well plate and incu-
bated at 37 ꢁC under 5% CO2 overnight. Then cells were treated
with the tested compounds for 12 h. Cells were harvested with
trypsin and washed twice with PBS solution. Afterward, cells were
fixed in cold 70% ethanol and stored at 4 ꢁC. On the day of analysis,
ethanol was removed by centrifugation (5 min, 25 ꢁC, 3000 rpm),
and cells were washed twice with PBS, and then treated with
RNase (75 kU mlꢀ1) for 30 min at 37 ꢁC. Propidium iodide (PI) was
finally added (50 mg/ml), and samples were processed by a flow
cytometer (FACScan, Becton Dickson, USA). Cells (1 ꢂ 104) were
acquired for each sample and recording of PI signal in the FL2
channel. The cell-cycle results were analyzed by FCSExpress
software.
4.2.2. Tyrosine kinase assays
ELISA assay was conducted with the help of the Beckman
Coulter (Fullerton, CA) Biomek FX robotic instrument or auto-
mated microtiter plate washer for most manipulations. In a typical
ELISA assay, 1 mL of 1 mg/ml test compounds or reference standard
in DMSO was added to plate wells by the Beckman Coulter Biomek
FX robotic instrument. The final DMSO concentration did not
exceed 1%, and DMSO controls were employed. Then, 80 mL 5 mM
ATP solution diluted in kinase reaction buffer (50 mM HEPES pH
7.4, 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was
added to each well. Subsequently, the kinase reaction was initiated
by the addition of purified tyrosine kinase proteins (20 ng) diluted
4.2.6. In vivo antitumor activity study
Calu-3 tumor cell line was grown in culture, and the cells were
washed with PBS, dispersed in a 0.05% (w/v) trypsin solution, and
resuspended. After centrifugation for 5 min, the cell pellets were
resuspended in PBS and adjusted to a concentration of 1 ꢂ107 cells/
ml. BALB/cA nude female mice, 6e7 weeks old, were implanted
subcutaneously (s.c.) on the midright side with 2 ꢂ 106 Calu-3 tu-
mor cells in 0.2 ml PBS. After the mean Calu-3 tumor volume
reached 60e150 mm3, mice were randomly divided into six groups
with six mice per treatment group and twelve mice in the negative
control group. Tumors were measured individually with a vernier
caliper. Volumes were determined using the formula: tumor
volume ¼ length ꢂ width2 ꢂ 0.5. Relative tumor volumes (RTV)
in 10
at 37 ꢁC, the plate was washed three times with phosphate buff-
ered saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 L of
mL of kinase reaction buffer solution. After incubation for 1 h
m
antiphosphotyrosine (PY99) antibody (1:500 dilution) diluted in T-
PBS containing 5 mg/ml BSA was added. After 30 min incubation
at 37 ꢁC, the plate was washed three times as before. Horseradish
peroxidase-conjugated goat antimouse IgG (100
mL) diluted
1:2000 in T-PBS containing 5 mg/ml BSA was added. The plate was
reincubated at 37 ꢁC for 30 min and washed as before. Finally,
100
mL of a solution containing 0.03% H2O2 and 2 mg/ml O-

642
A. Lyu et al. / European Journal of Medicinal Chemistry 87 (2014) 631e642
were determined using the formula: RTV
¼
Vt/V0, where
Acknowledgment
V0 ¼ tumor volume measured at the beginning day of the treat-
ment; Vt ¼ tumor volume measured in the course of the treatment.
Therapeutic effects on tumor growth were expressed as mean tu-
mor volumes versus time, calculated as (1 ꢀ T/C) ꢂ 100%, where
T ¼ treated relative tumor volume and C ¼ control relative tumor
volume. At the same time, the weight of each mouse was also
measured every other day for 21 d.
This work is supported by Jiangsu Province Hi-Tech Key Labo-
ratory for Bio-medical Research and Jiangsu Hansoh Pharmceutical
Corporation. The authors are thankful to the software support of
State Key Laboratory of Natural Medicines, China Pharmaceutical
University.
4.2.7. Pharmacokinetic study
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Blood samples (~1 ml) from Beagle dogs were collected on ice
until centrifugation was performed with in one hour (1000ꢂ g
and 4 ꢁC for 15 min). All plasma samples were stored at ꢀ20 ꢁC
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versus time curve within the 24-h dosing intervals (AUC0e24
)
were determined by standard model independent methods
based on the plasma concentration-time data. Cmax, MRT, and
Tmax were determined directly from the plasma concentration-
time data.