Design and synthesis of novel Gefitinib analogues with improved anti-tumor activity

Article abstract of DOI:10.1016/j.bmc.2010.04.046

There is an urgent need to design and develop new and more potent EGFR inhibitors with improved anti-tumor activity. Here we describe the design and synthesis of two series of 4-benzothienyl amino quinazolines as new analogues of the EGFR inhibitor Gefiti

Full text of DOI:10.1016/j.bmc.2010.04.046

Bioorganic & Medicinal Chemistry 18 (2010) 3812–3822
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel Gefitinib analogues with improved
anti-tumor activity
Xiaoqing Wu a,b, Mingdong Li a, Yang Qu b, Wenhua Tang b, Youguang Zheng a, Jiqin Lian b,
b,
Min Ji a, , Liang Xu
*
*
a School of Chemistry and Chemical Engineering, Southeast University, 87 Dingjiaqiao, Nanjing 210009, Jiangsu, China
b Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan Medical School and Comprehensive Cancer Center,
4424E Med Sci I, 1301 Catherine St., Ann Arbor, MI 48109-5637, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
There is an urgent need to design and develop new and more potent EGFR inhibitors with improved anti-
tumor activity. Here we describe the design and synthesis of two series of 4-benzothienyl amino quina-
zolines as new analogues of the EGFR inhibitor Gefitinib. The anti-tumor activity of these novel Gefitinib
analogues in 6 human cancer cell lines was examined. Compared with the parental Gefitinib, most of the
new compounds show a markedly increased cytotoxicity to cancer cells. Furthermore, several of the ser-
ies B compounds that side chains at position 7 contain either a methyl or ethyl group are potent pan-RTK
inhibitors. Two representative compounds in this class, 15 and 17, have an enhanced capability to inhibit
cancer cell growth and induce apoptosis in vitro and inhibit tumor formation in vivo in human cancer
cells with high HER-2, as compared with the parental Gefitinib. Thus they may be promising lead com-
pounds to be developed as an alternative for current Gefitinib therapy or for Gefitinb-resistant patients,
potentially via simultaneously blocking multiple RTK signaling pathways.
Received 14 January 2010
Revised 11 April 2010
Accepted 16 April 2010
Available online 21 April 2010
Keywords:
Gefitinib, 4-Benzothienyl amino
quinazolines
Cancer cells
Apoptosis
Receptor tyrosine kinase
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
nib) (Fig. 1), are two selective EGFR inhibitors approved by the FDA
in 2003 and 2004 for locally advanced or metastatic non-small-cell
The epidermal growth factor receptor (EGFR) belongs to the
ErbB family of receptor tyrosine kinases (RTK), which includes four
members: ErbB-1/EGFR, ErbB-2/HER-2/neu, ErbB-3/HER-3, and
ErbB-4/HER-4.1,2 Activation of EGFR (ligand-dependent or ligand-
independent) leads to the phosphorylation at cytoplasmic tails of
the receptor and subsequent recruitment of adapter moleculars,
which results in pro-growth/survival signal transduction via sev-
eral pathways. The major pathways implicated in EGFR signaling
include RAS–RAF–MAPK (mitogen-activated protein kinase), PI3K
(phosphatidylinositol 3-kinase)–AKT, and JAK–STAT (signal trans-
ducers and activators of transcription).2–4
EGFR overexpression occurs frequently in human epithelial
malignancies and its activation plays a significant role in the devel-
opment and progression of human cancers, since EGFR signaling
pathways are associated with cell proliferation, survival promotion
and apoptosis inhibition.5,6 Therefore, EGFR is a very attractive
molecular target for cancer therapy. Over the past 20 years, numer-
ous small molecular inhibitors and monoclonal antibodies targeting
EGFR have been successfully developed. The 4-anilino quinazolines
derivatives, Iressa (ZD1839, Gefitinib) and Tarceva (OSI-774, Erloti-
lung cancer (NSCLC) therapy, and are currently under evaluation in
clinical trials for other tumor types.7,8 Clinical data show that
10–20% of all NSCLC patients partially respond to these two EGFR
inhibitors,9,10 but only Erlotinib prolongs the survival of patients
with recurrent NSCLC.11 Moreover, most of the patients who
responded to initial treatment eventually developed resistance to
the EGFR inhibitors.12 Thus there is an urgent need to design and
develop new and more potent EGFR inhibitors with improved
anti-tumor activity.
Numerous novel derivatives have been synthesized using Gefiti-
nib as a leading compound. Most modifications of the structure of
Gefitinib are focused on substitution of the benzene ring, but its
replacement with another aromatic ring is rarely reported. In this
study, we designed and synthesized two series of 4-benzothienyl
amino quinazoline derivatives that show significantly improved
anti-tumor activity. New compounds that side chains at position 7
contain either a methyl or ethyl group were identified as potential
pan-RTK inhibitors with enhanced apoptosis-inducing capability.
2. Chemistry
* Corresponding authors. Tel.: +86 025 83272078 (M.J.); tel.: +1 734 615 7017;
fax: +1 734 615 3422 (L.X.).
Based on the structure–activity relationships (SAR) and quanti-
tative structure–activity relationships (QSAR) of the 4-anilinoqui-
E-mail addresses: jimin@seu.edu.cn (M. Ji), liangxu@umich.edu (L. Xu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.046

X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
3813
F
Cl
HN
N
O
HN
O
N
O
O
O
O
N
N
O
N
S
Erlotinib
Gefitinib
S
COOC2H5
COOC2H5
HN
N
HN
N
MeO
R
R
N
N
MeO
series A
series B
Figure 1. Structure of Gefitinib, Erlotinib and core structure of analogues.
nazolines reported previously,13–21 and using Gefitinib as a leading
compound, we designed two series of novel analogues: series A,
replacement of the benzene ring with benzothiophene, secondary
amino-substituted propoxy side chain at position 6 and methoxy
group at position 7 of the quinazoline nucleus; series B, replace-
ment of the benzene ring with benzothiophene, methoxy group
at position 6 and secondary amino-substituted propoxy side chain
at position 7 of the quinazoline nucleus. Our objective was to
determine whether these compounds favor greater inhibition of
cell proliferation and higher induction of cell death.
As summarized in Table 1, up to 20 compounds in series A
(1–10) and series B (11–20) were synthesized by two main routes,
as illustrated in Schemes 1 and 2, respectively. Methyl 3-hydroxy-
4-methoxy benzoate as starting material was alkylated with 1-bro-
mo-3-chloropropane to give 21 in 95% yield. Nitration of 21 with
nitric acid in acetic acid afforded 22, which was then reduced by
powdered iron in acetic acid to give 23 in satisfactory yield
(77%). In contrast, catalytic hydrogenation using Raney/Ni or 5%
Pd/C gave incomplete conversions, even after a long reaction time.
Cyclization of 23 with formamidine acetate and chlorination with
Table 1
Structures and yields of compounds 1–20
S
S
COOC2H5
COOC2H5
HN
HN
R
MeO
N
N
MeO
N
R
N
series A
series B
Compd
R
Yield (%)
Compd
11
R
Yield (%)
1
2
3
4
77
68
58
71
68
62
51
68
O
O
N
N
O
N
O
N
O
12
CH3
O
N
N
CH3
13
O
O
N
N
O
N
14
O
N
5
6
O
O
N
N
69
64
15
16
O
N
72
65
CH3
O
N
O
CH3
H3C
H3C
7
59
17
60
O
O
N
O
N
8
9
N
61
65
18
19
N
CH2CH3
52
67
O
N
N
N
CH2CH3
O
O
N
N
10
59
20
N
N
59
O
N

3814
X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
Cl
O
O
COOCH3
Cl
O
O
COOCH3
HO
O
COOCH3
c
a
b
NO2
21
22
Cl
O
Cl
O
O
Cl
O
O
S
Cl
O
COOCH3
NH2
N
NH
e
d
N
N
O
25
24
23
S
S
COOC2H5
COOC2H5
COOC2H5
Cl
HN
g
HN
H2N
O
O
R
O
N
N
f
N
N
26
1-10
Scheme 1. Reagents and conditions: (a) ClCH2CH2CH2Br, K2CO3, 70 °C; (b) HNO3, AcOH, Ac2O, 0–5 °C; (c) Fe, AcOH, MeOH, N2, 50 °C; (d) formamidine acetate, EtOH, reflux;
(e) SOCl2, DMF, reflux; (f) i-PrOH, reflux; (g) aliphatic amine, KI, 70 °C.
O
O
COOCH3
O
O
COOCH3
O
COOCH3
c
a
b
Cl
NO2
Cl
Cl
HO
O
N
O
O
O
O
O
COOCH3
NH2
e
N
NH
d
Cl
N
Cl
O
S
Cl
S
S
COOC2H5
COOC2H5
COOC2H5
HN
N
H2N
HN
g
O
O
O
R
N
N
f
Cl
N
27
11-20
Scheme 2. Reagents and conditions: (a) ClCH2CH2CH2Br, K2CO3, 70 °C; (b) HNO3, AcOH, Ac2O, 0–5 °C; (c) Fe, AcOH, MeOH, N2, 50 °C; (d) formamidine acetate, EtOH, reflux;
(e) SOCl2, DMF, reflux; (f) i-PrOH, reflux; (g) aliphatic amine, KI, 70 °C.
thionyl chloride gave 25. Aminolysis of 25 was performed using
excess ethyl 5-aminobenzo[b]thiophene-2-carboxylate to afford
26 in 93% yield. The final step was nucleophilic displacement of
the chlorine atom with different aliphatic amines to yield the cor-
responding target compounds series A 1–10.
Starting from methyl 4-hydroxy-3-methoxybenzoate (Scheme
2), the compounds 11–20 could be obtained via the same synthetic
methods as described above. This is the first study to replace the
benzene ring of Gefitinib with benzothiophene to design com-
pletely new compounds. All the target compounds are new chem-
ical entities and obtained in satisfactory yields (Table 1).
3.2. Series B compounds that side chains at position 7 contain
either a methyl or ethyl group are pan-RTK inhibitors
As Gefitinib was a selective EGFR inhibitor, we determined the
EGFR inhibitory activity of novel Gefitinib analogues using an EGFR
Kinase Assay Kit. Unexpectedly, the inhibitory activity of the new
compounds on EGFR tyrosine kinase was partially reduced
(Table 2). The compounds were then tested for their ability to inhibit
two other EGFR-related receptor tyrosine kinases, HER-2 and MET.
As also shown in Table 2, most series B compounds maintained or in-
creased HER-2 inhibitory ability while increasing MET inhibitory
ability compared to Gefitinib and Erlotinib, while series A com-
pounds appeared to have less inhibitory potency in both kinases.
Our result is consistent with an earlier report22 that Gefitinib ana-
logues with the secondary amino-substituted propoxy side chain
at position 7 were more potent RTK inhibitors than the compounds
substituted at position 6. However, we replaced the benzene ring
with benzothiophene to generate 4-benzothienyl amino quinazo-
line derivatives, which are totally new class of analogues.
3. Results and discussion
3.1. New compounds showed higher cytotoxicity in different
cancer cell lines compared with Gefitinib and Erlotinib
We first examined the cytotoxicity of the 20 new compounds, as
well as the parental compounds Gefitinib and Erlotinib, in 6 human
cancer cell lines by the MTT-based cytotoxicity assay (Table 2). The
IC50 values of most of the compounds in those 6 cell lines were re-
duced compared to the two parental drugs, indicating that the
anti-tumor activity of these new compounds was significantly
improved.
Based on the above results, we tried to summarize preliminary
SAR: replacement of the benzene ring with benzothiophene in-
creased cytotoxicity but decreased EGFR inhibitory activity. Shift-
ing the secondary amino-substituted propoxy side chain at
position 6 (series A compounds 1–10) to position 7 (series B com-

X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
3815
Table 2
Cytotoxicity in different cell lines and enzyme inhibition activity of Gefitinib, Erlotinib and compounds 1–20
Compd
Cytotoxicity in different cell lines (IC50
,
l
M)a
A549
Enzyme inhibitiona,b (%)
HER-2 MET
Miapaca2
Panc1
DU145
PC3
NCI-H661
EGFR
Gefitinib
Erlotinib
1
2
3
4
5
6
7
50.62
82.47
14.11
5.208
3.090
6.813
3.295
2.511
2.272
4.640
2.331
3.561
31.84
4.096
7.556
2.273
3.694
87.19
2.630
7.105
2.892
3.044
40.42
>100
42.17
19.40
19.69
5.756
7.780
27.20
7.132
8.64
20.00
3.035
8.529
29.99
>100
6.669
61.85
24.84
6.552
>100
5.320
27.30
8.977
30.67
22.86
36.76
18.17
6.216
2.538
7.375
2.453
6.810
7.703
3.051
6.238
2.278
32.46
3.872
33.91
6.002
6.685
>100
37.93
33.03
19.23
8.026
5.828
21.12
7.695
6.812
10.94
3.929
9.043
9.120
62.98
5.826
64.79
8.976
8.518
>100
31.06
12.71
17.70
9.670
9.353
7.746
7.066
8.841
7.719
6.966
7.013
10.58
36.79
15.76
20.53
6.745
13.52
>100
98.37
96.95
22.43
45.38
41.02
18.07
22.69
30.47
33.73
40.28
37.61
25.40
8.27
72.91
13.26
11.33
62.74
1.52
70.47
67.88
60.14
19.59
81.11
77.94
46.02
67.61
62.35
47.32
47.82
57.01
54.74
63.20
77.45
59.54
71.22
83.39
69.33
74.46
86.85
19.52
86.08
74.82
82.81
73.01
28.18
39.76
24.11
32.27
38.65
21.09
21.98
12.80
25.16
21.62
44.64
35.21
61.51
51.02
44.86
52.25
57.26
14.14
69.80
44.96
51.62
44.20
27.72
15.66
18.75
18.52
13.17
10.86
17.26
4.220
5.948
12.25
53.28
6.756
39.70
6.694
7.276
>100
8
9
10
11
12
13
14
15
16c
17
18
19
20
7.502
18.45
4.029
11.11
2.716
16.16
2.342
5.621
4.147
21.47
5.680
10.26
6.533
26.28
5.744
7.030
Miapaca2, Panc1: human pancreatic cancer cell line; DU145, PC3: human prostate cancer cell line; A549, NCI-H661: human lung cancer cell line.
a
Values are averages of at least two independent experiments, SD <10%.
b
Compounds tested at a concentration of 10
Low solubility in DMSO stock.
lM.
c
pounds 11–20) increased HER-2 and MET inhibitory ability but de-
creased EGFR inhibitory potency with an exception: the side chains
at position 7 contain either a methyl or ethyl group (compounds
12, 15, 17, 18 and 19; compound 16 was an exception due to its
low solubility). Thus compounds 12, 15, 17, 18 and 19 might be
pan-RTK inhibitors.
shown). Taken together, these results strongly support that HER-
2 is one of the targets of 15 and 17.
3.4. Compounds 15 and 17 significantly suppressed pancreatic
cancer Miapaca2 cell proliferation
Since crosstalk between EGFR and HER-2/MET was one of the
mechanisms of resistance to Gefitinnib,23,24 using these com-
pounds as pan-RTK inhibitors might overcome this problem, allow-
ing compounds 12, 15, 17, 18 and 19 to serve as potential
alternatives to Gefitinib. Based on IC50 and RTK inhibitory activity,
the compounds 15 and 17 were chosen for further characterization
and mechanism studies.
To examine the effects of 15 and 17 on cancer cells, we first car-
ried out the MTT-based cytotoxicity (Fig. 3A), the cell growth
curves (Fig. 3B) and colony formation assays (Fig. 3C and D) with
human pancreatic cancer Miapaca2 cells. After treatment with 15
or 17, Miapaca2 cell growth was significantly suppressed and col-
ony formation was reduced in a dose-dependent manner, while
Gefitinib had very limited effects. These results suggested that 15
and 17 markedly suppress the proliferation of Miapaca2 cells that
are HER-2 positive and low cMET.
3.3. Compounds 15 and 17 exhibit inhibitory effect on HER-2
kinase and selectively inhibit the growth of breast cancer cells
transfected with HER-2
3.5. Compounds 15 and 17 potently induced apoptosis in
Miapaca2 cells
The compounds 15 and 17 were examined for their effects on
the HER-2 kinase activity. As shown inFigure 2A and B, the IC50 val-
ues are 2.29 lM and 1.32 lM for 15 and 17, respectively. The
We next examined the mechanism of 15 and 17 in cell growth
inhibition. As shown in Figure 4A and B, 15 or 17 potently induced
apoptosis in a dose- and time-dependent manner. In contrast,
Gefitinib did not induce any significant apoptosis comparing with
DMSO control. Cleaved PARP (Fig. 4C) provided further evidence of
15 and 17-induced apoptosis. Furthermore, as PARP was one of the
substrates of caspase-3, our functional caspase-3 activation assay
showed a similar result. Caspase-3 activity increased more than
parental compounds showed much lower activity. To further vali-
date their inhibitory effect on the proliferation function of HER-2,
we compared the cytotoxicity of 15 and 17 in HER-2 negative hu-
man breast cancer cell line MCF-7 and that with HER-2-transfected
MCF-7 clone that is highly tumorigenic and dependent on HER-2
(MCF-HER2, Fig. 2C). Representative results of two independent
experiments are shown in Figure 2D and E. MCF-HER2 cells were
more sensitive to 15 and 17 than HER-2-negative MCF-7 cells. 17
appears to be more potent and selective than 15, in terms of cyto-
toxicity to MCF-HER2 versus MCF-7 (the IC50 of 15 and 17 are 5.9
eight-fold after treatment with 20
lM 15 or 17, whereas Gefitinib
had only a minor effect (Fig. 4D). The above data demonstrate that
15 and 17 are potent apoptosis inducers. Further studies are ongoing
to delineate the exact mechanism(s) of action and determine
whether these compounds have other molecular targets.
and 2.7 lM for MCF-HER2 cells, 7.3 and 6.3 lM for MCF-7 cells,
respectively). This apparent selectivity is correlated with their
HER-2 kinase inhibitory activity shown above. The IC50 of the
3.6. Compounds 15 and 17 inhibited Miapaca2 tumor formation
in vivo
parental compounds were all >30
showed limited cytotoxicity to normal breast epithelial cell line
MCF-10A (IC50 >30 M). The responses of MCF-7-vector cells to
these compounds were similar to that of MCF-7 cells (data not
lM in these cells. 15 and 17
l
To further confirm the anti-tumor activity of 15 and 17, Mia-
paca2 cells were treated by 2.5 lM Gefitinib, 15 or 17 for 48 h

3816
X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
A
B
C
100
100
80
60
40
20
0
80
IC50=2.29μM
IC50 =1.32μM
HER-2
Actin
60
40
20
0
MCF-
HER2
MCF-7
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
Cpd.15 (logM)
Cpd.17 (logM)
IC50 (μM)
D
E
IC50 (μM)
120
120
100
80
60
40
20
0
MCF-7
7.377
MCF-7
6.380
100
80
60
40
20
0
MCF-HER2 5.963
MCF-HER2 2.762
1
10
100
1
10
100
Cpd. 15(μM)
Cpd. 17(μM)
Figure 2. Compounds 15 and 17 exhibit inhibitory effect on HER-2 kinase and selectively inhibits the growth of cancer cells with high HER-2. (A, B) Representative inhibition
curve of HER-2 kinase by 15 and 17 by a HER-2 Kinase Assay Kit (repeated three times). (C) Western blot analysis of HER-2 protein expression in MCF-7 and HER-2 transfected
stable clone of MCF-7 (MCF-HER2) cells. Actin is shown as a loading control. (D, E) The MTT-based cytotoxicity assay in MCF-7 and MCF-HER-2 cells was carried out as
described in Section 5. MCF-HER2 cells were more sensitive to 15 and 17 than HER-2 negative MCF-7 cells.
and then implanted into nude mice subcutaneously. Tumor inci-
dence and tumor sizes were measured. Notably, pre-treatment
with 15 and 17 effectively delayed Miapaca2 tumor formation
in vivo (Fig. 5A and B) and inhibited tumor growth (Fig. 5C and
D). The data demonstrate the improved anti-tumor activity as
compared with the parent compound Gefitinb. Compound 17
showed even better activity than 15, which is correlated with its
better inhibitory activity on HER-2.
open capillaries and are uncorrected. 1H NMR and 13C NMR spectra
were recorded in CDCl3 on a Bruker Avance 500 spectrometer.
Chemical shifts (d) are reported in parts per million (ppm) relative
to tetramethylsilane (TMS), used as an internal standard. Mass
spectra (MS) were obtained from Agilent 1100LC/MS. IR spectra
were run on FI-IR Spectrometer (Perkin–Elmer). Element analysis
was run on Elementa Vario ELIII element analyzer. All compounds
were routinely checked by TLC with silica gel GF-254 glass plates
and viewed under UV light at 254 nm.
4. Conclusion
5.1.1. Methyl 3-(3-chloropropoxy)-4-methoxybenzoate (21)
A mixture of methyl 3-hydroxy-4-methoxybenzoate (84.6 g,
0.47 mol), 1-bromo-3-chloropropane (101.6 g, 0.65 mol), and
potassium carbonate (138.1 g, 1.0 mol) in DMF (500 mL) was
heated at 70 °C for 4 h. The reaction mixture was cooled to room
temperature, then poured slowly into ice water (3 L) while stirring
constantly. The solid formed was filtered off and washed with cold
water. The off-white product was recrystallized from ethyl acetate
(200 mL) to give 113.9 g of 21 in 95% yield. Mp: 111–113 °C; 1H
NMR d: 2.02–2.22 (tt, 2H, –CH2CH2CH2–), 3.65 (t, 2H, –CH2Cl),
3.79 (s, 3H, –OCH3), 3.88 (s, 3H, –OCH3), 4.10 (t, 2H, –CH2O), 6.84
(d, 1H, HAr), 7.49 (s, 1H, HAr), 7.71 (d, 1H, HAr).
In our study, two series of 20 novel analogues of Gefitinb were
designed and synthesized, and certain structure–activity relation-
ships were proposed. Most of the new compounds show improved
anti-tumor activity in the 6 human cancer cell lines tested. More
significantly, the series B compounds that side chains at position
7 contain either a methyl or ethyl group were identified to be po-
tent pan-RTK inhibitors. Two representative compounds, 15 and
17, showed more potent and selective apoptosis-inducing capabil-
ity and improved anti-tumor activity in cancer cells with HER-
overexpression, as compared with the parental Gefitinb. The novel
pan-RTK inhibitors discovered in our study hold promise as alter-
natives to current Gefitinib therapy or for treatment of Gefitinib-
resistant patients, potentially via simultaneously blocking multiple
RTK signaling pathways.
5.1.2. Methyl 5-(3-chloropropoxy)-4-methoxy-2-nitrobenzoate
(22)
Nitric acid (84.5 ml, 66%) was added dropwise at 0–5 °C to a
solution of methyl 3-(3-chloropropoxy)-4-methoxybenzoate (21,
93.0 g, 0.36 mol) in a mixture of acetic acid (300 mL) and acetic
anhydride (100 mL). This mixture was stirred at room temperature
for 6 h, then slowly poured into ice water (2 L) and extracted with
ethyl acetate (4 Â 200 mL). The combined organic layer was
washed with saturated sodium bicarbonate (2 Â 200 mL) and brine
5. Experimental
5.1. Synthesis
All reagents were purchased from commercial sources and used
without further purification. Melting points were measured in

X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
3817
IC50 (μM)
A
B
Gefitinib 45.80
120
100
80
60
40
20
0
DMSO
300
250
200
150
100
50
Cpd. 15 3.160
Cpd. 17
2.369
Gefitinib
Cpd. 15
Cpd. 17
**
***
***
0
0
24h
48h
72h
1
10
100
Drug (μM)
C
D
DMSO
200
DMSO
Gefitinib (1μM)
0.25 μM
0.5 μM
1.0 μM
*
150
100
50
***
***
***
***
***
***
Cpd. 15 (1μM)
Cpd. 17 (1μM)
0
Gefitinib
Cpd. 15
Cpd. 17
Figure 3. Compounds 15 and 17 significantly suppress HER-2(+) human pancreatic cancer Miapaca2 cell proliferation as compared with Gefitinib. (A) The MTT-based
cytotoxicity assay in Miapaca2 cells was carried out as described in Section 5. (B) Growth curve of Miapaca2 cells after treatment with 2.5 M Gefitinib, 15 or 17. Miapaca2
l
cells were seeded in 6-well plates and, after treatment for the indicated time, trypsinized and counted. Mean SD (n = 3). **P <0.01, ***P <0.001, versus DMSO control,
two-way ANOVA. (C) Miapaca2 cells were seeded in 6-well plates and treated with Gefitinib, 15 or 17 at indicated doses. After 9–10 days incubation, the plates were gently
washed with PBS and stained with 0.1% crystal violet. Colonies with over 50 cells were counted. Mean SD (n = 3). *P <0.05, ***P <0.001, versus DMSO control, two-way
ANOVA. (D) Representative pictures of the colonies.
(2 Â 100 mL) and dried (Na2SO4). The ethyl acetate was evaporated
to give a yellow oil that solidified after standing in a refrigerator for
12 h and was then recrystallized from ethyl acetate/petroleum
ether to afford the product 22 as light yellow crystals (97.1 g,
89% yield). Mp: 54–56 °C; 1H NMR d: 2.03–2.24 (tt, 2H,
–CH2CH2CH2–), 3.66 (t, 2H, –CH2Cl), 3.78 (s, 3H, –OCH3), 3.89 (s,
3H, –OCH3), 4.12 (t, 2H, –CH2O), 7.82 (s, 1H, HAr), 8.01 (d, 1H, HAr).
0.51 mol) in ethanol (800 mL) was heated at reflux for 6 h with
overhead stirring. The mixture was allowed to stand in the refrig-
erator overnight. The precipitate was then collected by filtration,
washed with ethanol and air dried to give 24 as a white power
(88.7 g, 92% yield). Mp: 218–219 °C; 1H NMR d: 2.10–2.31 (tt, 2H,
–CH2CH2CH2–), 3.72 (t, 2H, –CH2Cl), 3.83 (s, 3H, –OCH3), 4.02 (t,
2H, –CH2O), 6.98 (d, 1H, HAr), 7.89 (s, 1H, HAr), 8.02 (d, 1H,
HAr), 9.03–9.42 (br, 1H, –NH–).
5.1.3. Methyl 5-(3-chloropropoxy)-2-amino-4-methoxybenzoate
(23)
5.1.5. 4-Chloro-6-(3-Chloropropoxy)-7-methoxyquinazoline (25)
6-(3-Chloropropoxy)-7-methoxyquinazolin-4(3H)-one (24, 102 g,
0.38 mol) was added to thionyl chloride (500 mL) with magnetic
stirring. DMF (20 mL) was then slowly added dropwise and the
mixture was heated to reflux for 4 h. Most of the excess of thionyl
chloride was then removed under reduced pressure and the yellow
residue was dissolved in chloroform (500 mL), washed with a sat-
urated solution of sodium carbonate (2 Â 100 mL) and water
(2 Â 100 mL), and dried (Na2SO4). The chloroform was then re-
moved under reduced pressure to give an off-white power, which
was recrystalized from ethyl acetate to give the product 25 (93.5 g,
86% yield). Mp: 150–152 °C; 1H NMR d: 2.43 (tt, 2H, –CH2CH2CH2–),
3.85 (t, 2H, –CH2Cl), 4.09 (s, 3H, –OCH3), 4.39 (t, 2H, –OCH2), 7.43
(s, 1H, HAr), 7.47 (s, 1H, HAr), 8.91 (s, 1H, HAr).
Powdered iron (50 g, 0.89 mol) was added to acetic acid
(500 mL). The resulting suspension was stirred for 15 min at
50 °C under an atmosphere of N2, and a solution of methyl 5-(3-
chloropropoxy)-4-methoxy-2-nitrobenzoate (22, 90.0 g, 0.30 mol)
in methanol (300 mL) was added dropwise. The mixture was stir-
red for another 30 min at 50–60 °C. The catalyst was filtered, and
the filtrate was slowly poured into water (4 L) and extracted with
ethyl acetate (4 Â 200 mL). The organic phase was washed with a
saturated solution of sodium carbonate (2 Â 100 mL) and brine
(2 Â 100 mL) and then dried (Na2SO4). The solvent was removed
under vacuum and the brown solid residue was recrystallized from
ethyl acetate/petroleum ether to give the product 23 as light
brown crystals (63.1 g, 77% yield). Mp: 96–98 °C; 1H NMR d:
1.98–2.20 (tt, 2H, –CH2CH2CH2–), 3.62 (t, 2H, –CH2Cl), 3.76 (s,
3H, –OCH3), 3.85 (s, 3H, –OCH3), 4.07 (t, 2H, –CH2O), 5.10–5.35
(br, 2H, –NH2), 6.09 (d, 1H, HAr), 7.21 (s, 1H, HAr).
5.1.6. Ethyl 5-(6-(3-chloropropoxy)-7-methoxyquinazolin-4-yl-
amino)benzo[b]thiophene-2-carboxylate (26)
Ethyl 5-aminobenzo[b]thiophene-2-carboxylate (16.1 g, 73 mmol)
was added to a solution of 4-chloro-6-(3-chloropropoxy)-7-meth-
oxyquinazoline (25, 14.9 g, 52 mmol) in isopropanol (300 mL).
The mixture was heated to reflux for 3 h, and then left standing
5.1.4. 6-(3-Chloropropoxy)-7-methoxyquinazolin-4(3H)-one (24)
A solution of methyl 5-(3-chloropropoxy)-2-amino-4-methoxy-
benzoate (23, 98.2 g, 0.36 mol) and formamidine acetate (52.6 g,

3818
X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
A
B
24h
48h
30
25
20
15
10
5
DMSO
***
***
DMSO
5.0 μM
10.0 μM
***
5.0 μM
10.0 μM
***
***
***
20
10
0
***
***
0
Gefitinib
Cpd. 15
Cpd. 17
Gefitinib
Cpd. 15
Cpd. 17
D
C
10
8
***
DMSO
Gefitinib Cpd.15 Cpd.17
10 20 10 20
***
5.0 μM
10.0 μM
20.0 μM
10 20 μM
***
6
PARP
Cleaved
PARP
***
4
***
2
β-Actin
* *
*
0
Gefitinib
Cpd. 15
Cpd. 17
Figure 4. Compounds 15 and 17 potently induced cell apoptosis. Miapaca2 cells were seeded in 6-well plates and incubated with Gefitinib, 15 or 17 at doses of 5, 10, or 20
lM,
respectively. 24 h or 48 h after incubation, cells were harvested and processed for further detection. (A, B) Apoptotic cell population after 24 h and 48 h treatment. Miapaca2 cells
were stained with propidium iodide after ethanol fixation and analyzed by flow cytometry. Mean SD (n = 2). ***P <0.001, two-way ANOVA versus DMSO control. (C) Western
blot analysis of apoptosis-related protein PARP after 24 h treatment. Actin is shown as a loading control. (D) Caspase-3 activation after 24 h treatment. Whole-cell lysates (20
were reacted with fluorogenic substrate DEVD-AFC. After incubation at 37 °C for 2 h, released AFC was monitored by a microplate reader (BMG LABTECH). Fold increase of
***
lg)
*
fluorescence signal was expressed by normalizing activity to DMSO control. Mean SD (n = 2). P <0.05,
P <0.001, two-way ANOVA versus DMSO control.
in the refrigerator overnight; the precipitate was collected by fil-
tration, washed with chilled isopropanol (2 Â 150 mL), and recrys-
talized from ethyl acetate to give an off-white power (22.8 g, 93%
yield). Mp: >270 °C.
(CDCl3) d: 1.42 (t, 3H), 2.10 (q, 2H), 2.27 (s, 3H), 2.45–2.54 (br,
8H), 2.55 (t, 2H), 3.99 (s, 3H), 4.22 (t, 2H), 4.42 (q, 2H), 7.23 (s,
1H), 7.34 (s, 1H), 7.73 (dd, 1H), 7.82 (d, 1H), 7.95 (br, 1H), 8.02
(s, 1H, HAr), 8.31 (d, 1H), 8.65 (s, 1H); 13C NMR (CDCl3) d: 14.29,
26.43, 45.98, 53.20, 54.87, 55.05, 56.16, 61.62, 67.84, 100.90,
108.04, 118.07, 122.76, 123.06, 130.22, 136.08, 149.10, 153.59; IR
(cmÀ1): 3406, 3235, 3209, 2941, 2806, 2149, 1708, 1624, 1593,
1522, 1510, 1428, 1237, 1147, 1070, 1008, 962, 866, 756; M/z:
536.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 62.78; H, 6.21; N,
13.07. Found: C, 62.09; H, 6.25; N, 13.25.
5.1.7. Ethyl 5-(7-methoxy-6-(3-morpholinopropoxy)quinazolin-
4-ylamino)benzo[b]thiophene-2-carboxylate (1)
Ethyl 5-(6-(3-chloropropoxy)-7-methoxyquinazolin-4-ylamino)-
benzo[b]thiophene-2-carboxylate (26, 0.80 g, 2 mmol) and potas-
sium iodide (0.2 g) were added to the solution of morpholine
(5 mL) in DMF (15 mL).The solution was stirred at 70 °C for
30 min. The excess morpholine was then removed under reduced
pressure and the residue dissolved in chloroform (60 mL), washed
with water (2 Â 20 mL), and then dried (Na2SO4). The solvent was
removed under vacuum. The crude product was purified by column
chromatography on silica gel, eluting with ethyl acetate/triethyl-
amine (20:1) to afford white powder (1, 0.81 g, 77% yield). Mp:
192–194 °C; 1H NMR (CDCl3) d: 1.43 (t, 3H), 2.12 (dd, 2H), 2.49 (t,
4H), 2.58 (t, 2H), 3.72 (t, 4H), 4.00 (s, 3H), 4.19 (t, 2H), 4.42 (q, 2H),
7.13 (s, 1H), 7.37 (s, 1H), 7.70 (d, 1H), 7.85 (d, 1H), 8.04 (s, 1H,
HAr), 8.30 (d, 1H), 8.67 (s, 1H); 13C NMR (CDCl3) d: 14.21, 26.23,
35.79, 55.40, 56.22, 60.39, 66.96, 67.76, 100.79, 108.19, 109.04,
118.07, 122.71, 123.14, 130.22, 135.19, 136.07, 138.23, 139.48,
147.67, 149.15, 153.63, 155.29, 156.47, 162.74; IR (cmÀ1): 3445,
3367, 3027, 2976, 2870, 2109, 1710, 1621, 1592, 1526, 1503, 1424,
1391, 1240, 1141, 1110, 996, 957, 857, 754; M/z: 523.2 ([M+H]+,
100%). Elem. Anal. Calcd: C, 62.05; H, 5.79; N, 10.72. Found: C,
61.57; H, 6.04; N, 10.09.
5.1.9. Ethyl 5-(7-methoxy-6-(3-(pyrrolidin-1-yl)propoxy)quina-
zolin-4-ylamino)benzo[b] thiophene-2-carboxylate (3))
As in the procedure described for 1, compound 3 was prepared
as light yellow powder (0.52 g, 58% yield). Mp: 162–164 °C; 1H
NMR (CDCl3) d: 1.41 (t, 3H), 1.81 (m, 4H), 2.12 (p, 2H), 2.54 (br,
4H), 2.65 (t, 2H), 3.98 (s, 3H), 4.17 (t, 2H), 4.40 (q, 2H), 7.18 (s,
1H), 7.53 (s, 1H), 7.71 (dd, 1H), 7.84 (d, 1H), 8.02 (s, 1H), 8.30 (d,
1H), 8.67 (s, 1H); 13C NMR (CDCl3) d: 14.30, 23.45, 28.28, 52.90,
54.25, 56.16, 61.60, 67.91, 101.12, 107.91, 109.16, 118.04, 122.77,
122.96, 130.27, 134.92, 136.23, 138.04, 139.39, 147.43, 148.97,
153.54, 155.09, 156.59, 162.76; IR (cmÀ1): 3452, 3313, 3280,
2871, 2186, 1705, 1624, 1587, 1513, 1438, 1413, 1288, 1225,
1140, 921, 855; M/z: 507.2 ([M+H]+, 100%). Elem. Anal. Calcd: C,
64.01; H, 5.97; N, 11.06. Found: C, 63.59; H, 6.05; N, 10.85.
5.1.10. Ethyl 5-(7-methoxy-6-(3-(piperidin-1-yl)propoxy)quina-
zolin-4-ylamino)benzo[b] thiophene-2-carboxylate (4)
As in the procedure described for 1, compound 4 was prepared
as white powder (0.67 g, 71% yield). Mp: 153–155 °C; 1H NMR
(CDCl3) d: 1.40–1.46 (m, 5H), 1.57–1.65 (m, 4H), 2.12 (t, 2H),
2.44 (br, 4H), 2.53 (t, 2H), 4.00 (s, 3H), 4.19 (t, 2H), 4.43 (q, 2H),
7.20 (s, 1H), 7.51 (s, 1H), 7.73 (dd, 1H), 7.85 (d, 1H), 8.04 (s, 1H),
5.1.8. Ethyl 5-(7-methoxy-6-(3-(4-methylpiperazin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (2)
As in the procedure described for 1, compound 2 was prepared
as white powder (0.72 g, 68% yield). Mp: 169–171 °C; 1H NMR

X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
3819
A
B
1.0 Million
Gefitinib
Cpd. 15
0.5 million
Gefitinib
Cpd. 15
Cpd. 17
100
80
60
40
20
0
100
Cpd. 17
80
*
60
*
40
20
0
0
10
20
30
40
0
10
20
30
40
Days after implantation
Days after inoculation
C
D
0.5 million
1.0 million
100
80
60
40
20
0
120
100
80
60
40
20
0
Gefitinib
Cpd. 15
Cpd. 17
Gefitinib
Cpd. 15
Cpd. 17
*
**
***
***
0
12
19
26
33
40
0
12
19
26
33
40
Days after implantation
Days after implantation
Figure 5. Compounds 15 and 17 inhibited tumor formation and tumor growth. Miapaca2 cells were treated with 2.5 lM Gefitinib, compound 15 or 17 for 48 h. Cells were
collected, counted for live cells and inoculated into nude mice subcutaneously (sc) on both flanks, 0.5 Â 106 cells or 1 Â 106 cells/0.2 ml, respectively. (A, B) Kaplan–Meier
curves show effects of Gefitinib, compound 15 or 17 pre-treatment on tumor formation. *P <0.05 versus Gefitinib, Mantel-Cox test. (C, D) The tumor sizes were measured
using a caliper. Tumor volume was calculated using the formula: (length  width2)/2. Mean SEM (n = 5). P <0.05, P <0.01,
*
**
***
P <0.001 versus Gefitinib, two-way ANOVA.
8.32 (d, 1H), 8.67 (s, 1H); 13C NMR (CDCl3): 14.35, 24.35, 25.90,
26.52, 54.68, 55.67, 56.21, 61.64, 68.13, 100.98, 108.13, 109.10,
118.08, 122.78, 123.09, 130.28, 135.09, 136.14, 138.18, 139.47,
147.62, 149.15, 153.60, 155.29, 156.49, 162.76; IR (cmÀ1): 3466,
2930, 2850, 2071, 1706, 1622, 1594, 1525, 1507, 1427, 1390,
1238, 1194, 1147, 1065, 1010, 951, 862, 756; M/z: 521.3 ([M+H]+,
100%). Elem. Anal. Calcd: C, 64.59; H, 6.20; N, 10.76. Found: C,
64.24; H, 6.17; N, 10.91.
1H), 7.70 (dd, 1H), 7.74 (s, 1H), 7.80 (d, 1H), 7.99 (d, 1H), 8.27 (d,
1H); 13C NMR (CDCl3) d: 14.29, 21.79, 26.63, 30.75, 34.30, 54.04,
55.27, 61.60, 68.00, 100.92, 107.88, 109.10, 118.12, 122.79,
122.97, 130.20, 13.95, 136.09, 138.09, 139.36, 147.44, 149.07,
153.30, 155.18, 156.54, 162.73; IR (cmÀ1): 3282, 3094, 2944,
2913, 1713, 1624, 1577, 1527, 1516, 1451, 1428, 1393, 1295,
1241, 1202, 1146, 1066, 1003, 920, 886, 857, 752; M/z: 535.2
([M+H]+, 100%). Elem. Anal. Calcd: C, 65.14; H, 6.41; N, 10.48.
Found: C, 65.02; H, 6.39; N, 10.79.
5.1.11. Ethyl 5-(6-(3-(diethylamino)propoxy)-7-methoxyquina-
zolin-4-ylamino)benzo[b] thiophene-2-carboxylate (5)
5.1.13. Ethyl 5-(7-methoxy-6-(3-(2-methylpiperidin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (7)
As in the procedure described for 1, compound 7 was prepared
as white powder (0.56 g, 59% yield). Mp: 177–178 °C; 1H NMR
(CDCl3) d: 1.05 (d, 3H), 1.25–1.30 (m, 2H), 1.41 (t, 3H), 1.60–1.66
(m, 2H), 2.02 (t, 2H), 2.13 (t, 1H), 2.42 (br, 1H), 2.44 (p, 1H), 2.54
(s, 2H), 2.85 (p, 2H), 3.95 (s, 3H), 4.06–4.14 (m, 2H), 4.40 (q, 2H),
7.22 (s, 1H), 7.32 (s, 1H), 7.73 (dd, 1H), 7.99 (s, 1H), 8.06 (d, 1H),
8.28 (d, 1H), 8.65 (d, 1H); 13C NMR (CDCl3) d: 14.28, 23.76, 25.35,
25.96, 34.38, 50.20, 52.11, 56.11, 61.59, 68.00, 101.30, 107.76,
109.21, 118.09, 122.82, 122.91, 130.24, 136.24, 138.01, 139.34,
147.37, 148.92, 153.51, 156.67, 162.76; IR (cmÀ1): 3331, 2977,
2931, 2849, 2156, 1704, 1626, 1595, 1577, 1524, 1453, 1427,
1392, 1290, 1235, 1221, 1191, 1142, 1077, 1014, 942, 887, 862,
754; M/z: 535.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 65.14; H,
6.41; N, 10.48. Found: C, 65.17; H, 6.60; N, 10.34.
As in the procedure described for 1, compound 5 was prepared
as white powder (0.56 g, 69% yield). Mp: 144–146 °C; 1H NMR
(CDCl3) d: 1.01 (t, 6H), 1.41 (t, 3H), 2.02 (p, 2H), 2.57 (hex, 6H),
3.97 (s, 3H), 4.14 (t, 2H), 4.40 (q, 2H), 7.24 (s, 1H), 7.67 (s, 1H),
7.70 (dd, 1H), 7.82 (d, 1H), 8.00 (s, 1H), 8.26 (d, 1H), 8.67 (s, 1H);
13C NMR (CDCl3) d: 11.62, 14.28, 26.82, 46.99, 49.07, 56.12,
61.60, 67.79, 100.81, 107.90, 109.09, 118.06, 122.74, 122.99,
130.20, 134.97, 136.09, 138.09, 139.37, 147.44, 149.14, 153.49,
155.20, 156.51, 162.73; IR (cmÀ1): 2950, 2795, 1711, 1623, 1574,
1521, 1445, 1425, 1389, 1232, 1192, 1142, 1065, 1009, 954, 867,
759; M/z: 509.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 63.76; H,
6.34; N, 11.02. Found: C, 63.89; H, 6.34; N, 11.61.
5.1.12. Ethyl 5-(7-methoxy-6-(3-(4-methylpiperidin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (6)
As in the procedure described for 1, compound 6 was prepared
as white powder (0.61 g, 64% yield). Mp: 170–172 °C; 1H NMR
(CDCl3) d: 0.90 (d, 3H), 1.20–1.23 (m, 2H), 1.34 (m, 1H), 1.41 (t,
3H), 1.60 (d, 2H), 1.90 (t, 2H), 2.06 (p, 2H), 2.46 (t, 2H), 2.86 (d,
2H), 3.96 (s, 3H), 4.12 (t, 2H), 4.40 (q, 2H), 7.22 (s, 1H), 7.23 (s,
5.1.14. Ethyl 5-(6-(3-(dimethylamino)propoxy)-7-methoxyqui-
nazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (8)
As in the procedure described for 1, compound 8 was prepared
as white powder (0.47 g, 61% yield). Mp: 201–203 °C; 1H NMR

3820
X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
(CDCl3) d: 1.14 (t, 3H), 2.07 (t, 2H), 2.25 (s, 6H), 2.45 (t, 3H), 3.98 (s,
3H), 4.17 (t, 2H), 4.40 (q, 2H), 7.24 (s, 1H), 7.67 (s, 1H), 7.70 (dd,
1H), 7.82 (d, 1H), 8.01 (s, 1H), 8.29 (d, 1H), 8.67 (s, 1H); 13C NMR
(CDCl3) d: 14.21, 27.22, 45.56, 56.16, 56,21, 61.65, 67.74, 100.85,
108.05, 109.15, 118.04, 122.71, 123.06, 130.28, 136.14, 138.12,
139.45, 147.49, 149.17, 153.58, 155.16, 156.50, 162.78; IR (cm
À1): 3452, 3281, 3089, 2962, 2753, 2099, 1702, 1625, 1579, 1517,
1473, 1430, 1395, 1292, 1233, 1148, 1067, 993, 888, 862, 754; M/
z: 509.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 62.48; H, 5.87; N,
11.66. Found: C, 62.42; H, 5.93; N, 10.92.
ethylamine (20:1) to afford white powder (11, 0.71 g, 68%). Mp:
155–157 °C; 1H NMR (CDCl3) d: 1.41 (t, 3H), 2.09 (t, 2H), 2.48 (t,
4H), 2.54 (t, 2H), 3.72 (t, 4H), 3.97 (s, 3H), 4.21 (t, 2H), 4.40 (q,
2H), 7.13 (s, 1H), 7.55 (s, 1H), 7.69 (d, 1H), 7.83 (d, 1H), 8.01 (s,
1H), 8.26 (d, 1H), 8.6 (s, 1H); 13C NMR (CDCl3) d: 14.27, 25.92,
53.67, 55.24, 56.32, 61.62, 66.93, 67.38, 99.60, 108.67, 108.88,
118.07, 122.73, 123.03, 130.16, 135.04, 136.01, 138.15, 139.37,
147.47, 149.84, 153.48, 154.34, 156.46, 162.71; IR (cmÀ1): 3780,
3456, 2953, 2853, 2064, 1706, 1679, 1629, 1521, 1453, 1416,
1228, 1114, 1069, 1010, 858; M/z: 523.2 ([M+H]+, 100%). Elem.
Anal. Calcd: C, 62.05; H, 5.79; N, 10.72. Found: C, 61.77; H, 6.05;
N, 9.99.
5.1.15. Ethyl 5-(6-(3-(4-ethylpiperazin-1-yl)propoxy)-7-methoxy-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (9)
As in the procedure described for 1, compound 9 was prepared
as white powder (0.54 g, 65% yield). Mp: 174–175 °C; 1H NMR
(CDCl3) d: 1.06 (t, 3H), 1.41 (t, 3H), 2.08 (t, 3H), 2.35–2.53 (m,
12H), 3.97 (s, 3H), 4.15 (t, 2H), 4.41 (q, 2H), 7.19 (s, 1H), 7.24 (s,
1H), 7.61 (br, 1H), 7.70 (d, 1H), 7.83 (d, 1H), 8.01 (s, 1H), 8.28 (s,
1H), 8.67 (s, 1H); 13C NMR (CDCl3) d: 11.94, 14.30, 26.56, 52.29,
52.83, 53.32, 54.95, 56.15, 61.61, 67.96, 100.99, 108.13, 109.08,
118.06, 122.73, 123.04, 130.18, 136.14, 138.17, 139.45, 147.63,
149.19, 153.58, 155.35, 156.50, 162.71; IR (cmÀ1): 3296, 3181,
3117, 2965, 2934, 2810, 1710, 1625, 1578, 1522, 1506, 1472,
1450, 1428, 1393, 1332, 1233, 1199, 1144, 1066, 994, 923, 845,
752, 639; M/z: 550.3 ([M+H]+, 100%). Elem. Anal. Calcd: C, 63.37;
H, 6.42; N, 12.74. Found: C, 62.95; H, 6.32; N, 12.84.
5.1.19. Ethyl 5-(6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (12)
As in the procedure described for 11, compound 12 was pre-
pared as white powder (0.60 g, 62% yield). Mp: 173–174 °C; 1H
NMR (CDCl3) d: 1.42 (t, 3H), 2.10 (p, 2H), 2.31 (s, 3H), 2.25 (br,
8H), 2.57 (t, 2H), 4.01 (s, 3H), 4.24 (t, 2H), 4.42 (q, 2H), 7.07 (s,
1H), 7.33 (s, 1H), 7.70 (dd, 1H), 7.86 (d, 1H), 8.05 (d, 1H), 8.31 (d,
1H), 8.67 (s, 1H); 13C NMR (CDCl3) d: 14.35, 26.35, 46.00, 53.13,
54.85, 55.16, 56.45, 61.67, 67.61, 99.45, 108.89, 108.95, 118.02,
122.69, 123.14, 130.23, 136.08, 138.22, 139.49, 147.71, 149.98,
153.59, 154.49, 156.40, 162.75; IR (cmÀ1): 3302, 3181, 3103,
2940, 2838, 2241, 1712, 1624, 1587, 1521, 1454, 1417, 1245,
1229, 1147, 1057, 1010, 922, 854, 755; M/z: 536.2 ([M+H]+,
100%). Elem. Anal. Calcd: C, 62.78; H, 6.21; N, 13.07. Found: C,
62.72; H, 6.15; N, 12.85.
5.1.16. Ethyl 5-(7-methoxy-6-(3-(4-phenylpiperazin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (10)
As in the procedure described for 1, compound 10 was prepared
as white powder (0.61 g, 59% yield). Mp: 204–206 °C; 1H NMR
(CDCl3) d: 1.41 (t, 3H), 1.97 (br, 2H), 2.13 (p, 2H), 2.59–2.65 (m,
6H), 3.20 (t, 4H), 3.99 (s, 3H), 4.20 (t, 2H), 4.41 (q, 2H), 6.82–6.89
(m, 3H), 7.20–7.23 (m, 3H), 7.58 (s, 1H), 7.68 (dd, 1H), 7.80 (d,
1H), 8.00 (s, 1H), 8.27 (d, 1H), 8.67 (s, 1H); 13C NMR (CDCl3) d:
14.30, 26.51, 49.13, 53.33, 54.90, 56.17, 61.60, 67.90, 101.22,
108.20, 109.13, 116.04, 118.06, 119.80, 122.72, 123.05, 129.10,
130.20, 136.16, 139.46, 147.69, 149.16, 151.22, 153.62, 155.38,
162.70; IR (cmÀ1): 3454, 3347, 2968, 2815, 1697, 1678, 1623,
1597, 1574, 1528, 1508, 1473, 1434, 1392, 1240, 1219, 1142,
1076, 1008, 916, 866, 758, 692; M/z: 598.2 ([M+H]+, 100%). Elem.
Anal. Calcd: C, 66.31; H, 5.90; N, 11.72. Found: C, 65.32; H, 5.79;
N, 11.81.
5.1.20. Ethyl 5-(6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)qui-
nazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (13)
As in the procedure described for 11, compound 13 was pre-
pared as yellow powder (0.45 g, 51% yield). Mp: 205–207 °C; 1H
NMR (CDCl3) d: 1.48 (t, 3H), 1.80 (m, 4H), 2.14 (s, 2H), 2.56 (br,
4H), 2.68 (q, 2H), 3.73 (t, 2H), 4.02 (s, 3H), 4.23 (t, 2H), 7.24 (s,
1H), 7.40 (s, 1H), 7.51 (dd, 1H), 7.68 (d, 1H), 8.01 (d, 1H), 8.16 (d,
1H), 8.60 (s, 1H); 13C NMR (CDCl3) d: 14.29, 23.54, 24.17, 26.62,
47.44, 49.28, 52.93, 54.25, 56.57, 67.62, 100.73, 108.59, 109.20,
118.56, 122.43, 122.66, 125.79, 136.07, 136.47, 139.56, 136.89,
139.56, 139.89, 147.50, 149.80, 153.65, 154.29, 157.05, 162.74; IR
(cmÀ1): 3422, 3332, 3139, 2869, 2178, 1706, 1625, 1588, 1511,
1450, 1417, 1296, 1224, 1140, 913, 852; M/z: 507.2 ([M+H]+,
100%). Elem. Anal. Calcd: C, 64.01; H, 5.97; N, 11.06. Found: C,
63.59; H, 6.34; N, 10.58.
5.1.17. Ethyl 5-(7-(3-chloropropoxy)-6-methoxyquinazolin-4-
ylamino)benzo[b]thiophene-2-carboxylate (27)
5.1.21. Ethyl 5-(6-methoxy-7-(3-(piperidin-1-yl)propoxy)quina-
zolin-4-ylamino)benzo[b] thiophene-2-carboxylate (14)
Ethyl 5-aminobenzo[b]thiophene-2-carboxylate (15.0 g, 68 mmol)
was added to a solution of 4-chloro-7-(3-chloropropoxy)-6-meth-
oxyquinazoline (15.2 g, 52 mmol) in isopropanol (300 mL). The
mixture was heated to reflux for 3 h, and then left standing in
the refrigerator overnight. The precipitate was then collected by
filtration, washed with chilled isopropanol (2 Â 150 mL), and
recrystalized from ethanol to give an off-white power (21.5 g,
86% yield). Mp: >270 °C.
As in the procedure described for 11, compound 14 was pre-
pared as off-white powder (0.64 g, 68% yield). Mp: 166–167 °C;
1H NMR (CDCl3) d: 1.39–1.46 (m, 5H), 1.57–1.65 (m, 4H), 2.12 (t,
2H), 2.44 (br, 4H), 2.54 (t, 2H), 4.00 (s, 3H), 4.21 (t, 2H), 4.41 (q,
2H), 7.10 (s, 1H), 7.41 (s, 1H), 7.70 (dd, 1H), 7.85 (d, 1H), 8.04 (s,
1H), 8.30 (d, 1H), 8.66 (s, 1H); 13C NMR (CDCl3) d: 14.33, 24.33,
25.84, 26.23, 54.58, 55.65, 56.43, 61.66, 67.73, 99.49, 108.84,
108.88, 118.04, 122.75, 122.80, 123.10, 130.24, 136.07, 138.19,
139.44, 147.62, 149.90, 153.54, 154.40, 156.41, 162.76; IR (cm
À1): 3466, 3352, 3110, 2930, 2856, 2107, 1708, 1681, 1628, 1578,
1526, 1451, 1419, 1234, 1219, 1140, 1067, 1012, 944, 856, 756;
M/z: 521.3 ([M+H]+, 100%). Elem. Anal. Calcd: C, 64.59; H, 6.20;
N, 10.76. Found: C, 64.67; H, 6.34; N, 10.18.
5.1.18. Ethyl 5-(6-methoxy-6-(7-morpholinopropoxy)quinazolin-
4-ylamino)benzo[b] thiophene-2-carboxylate (11)
Ethyl 5-(7-(3-chloropropoxy)-6-methoxyquinazolin-4-ylami-
no)benzo[b] thiophene-2-carboxylate (27, 0.78 g, 2 mmol). and
potassium iodide (0.2 g) were added to the solution of morpholine
(5 mL) in DMF (15 mL). The solution was stirred at 70 °C for
30 min, the excess morpholine was removed under reduced pres-
sure, and the residue was dissolved in chloroform (60 mL), washed
with water (2 Â 20 mL), and then dried (Na2SO4). The solvent was
removed under vacuum. The crude product was purified by col-
umn chromatography on silica gel, eluting with ethyl acetate/tri-
5.1.22. Ethyl 5-(7-(3-(diethylamino)propoxy)-6-methoxyquina-
zolin-4-ylamino)benzo[b] thiophene-2-carboxylate (15)
As in the procedure described for 11, compound 15 was pre-
pared as white powder (0.71 g, 72% yield). Mp: 181–183 °C; 1H
NMR (CDCl3) d: 1.03 (t, 6H), 1.42 (t, 3H), 2.05 (m, 2H), 2.55 (q,

X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
3821
4H), 2.65 (t, 3H), 3.99 (s, 3H), 4.21 (t, 2H), 4.42 (q, 2H), 7.12 (s, 1H),
7.50 (s, 1H), 7.70 (dd, 1H), 7.84 (d, 1H), 8.02 (d, 1H), 8.28 (d, 1H),
8.67 (s, 1H); M/z: 509.2, ([M+H]+, 100%); 13C NMR (CDCl3) d:
11.86, 14.22, 14.34, 26.61, 47.09, 47.15, 49.17, 56.43, 61.67,
67.68, 99.57, 108.86, 108.90, 118.06, 122.76, 123.10, 130.24,
135.11, 136.13, 138.18, 139.46, 147.70, 149.98, 153.57, 154.54,
156.47, 162.77; IR (cmÀ1): 3453, 3281, 3198, 3093, 2957, 2165,
1705, 1625, 1577, 1522, 1450, 1417, 1234, 1194, 1147, 1065,
1018, 955, 869, 753; M/z: 509.2 ([M+H]+, 100%). Elem. Anal. Calcd:
C, 63.76; H, 6.34; N, 11.02. Found: C, 63.64; H, 6.34; N, 10.60.
7.17 (s, 1H), 7.24 (s, 1H), 7.67 (dd, 1H), 7.71 (s, 1H), 7.80 (d, 1H),
7.97 (s, 1H), 8.23 (d, 1H), 8.64 (s, 1H); 13C NMR (CDCl3) d: 11.87,
14.26, 26.31, 52.25, 52.77, 53.15, 54.84, 56.34, 61.60, 67.57,
99.86, 108.72, 108.95, 118.09, 122.77, 122.97, 130.17, 135.01,
136.14, 138.12, 139.37, 147.57, 149.89, 153.49, 154.44, 156.54,
162.73; IR (cmÀ1): 3420, 3335, 2975, 2948, 2808, 1719, 1697,
1626, 1596, 1579, 1527, 1511, 1453, 1419, 1395, 1237, 1220,
1201, 1162, 1145, 1061, 1012, 923, 866, 756, 649; M/z: 550.3
([M+H]+, 100%). Elem. Anal. Calcd: C, 63.37; H, 6.42; N, 12.74.
Found: C, 63.33; H, 6.32; N, 12.40.
5.1.23. Ethyl 5-(6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (16)
As in the procedure described for 11, compound 16 was pre-
pared as white powder (0.62 g, 65% yield). Mp: 166–168 °C; 1H
NMR (CDCl3) d: 0.91 (d, 3H), 1.23–1.26 (m, 2H), 1.35 (m, 1H),
1.41 (t, 3H), 1.61 (d, 2H), 1.95 (t, 2H), 2.09 (p, 2H), 2.51 (t, 2H),
2.89 (d, 2H), 3.97 (s, 3H), 4.19 (t, 2H), 4.41 (q, 2H), 7.11 (s, 1H),
7.50 (s, 1H), 7.69 (dd, 1H), 7.83 (s, 1H), 8.01 (s, 1H), 8.27 (d, 1H),
8.75 (s 1H); 13C NMR (CDCl3) d: 14.30, 21.83, 26.48, 30.80, 34.32,
54.02, 55.29, 56.37, 61.63, 67.80, 99.59, 108.76, 108.87, 118.06,
122.74, 123.05, 130.21, 136.08, 139.41, 153.51, 156.45; IR (cm
À1): 3431, 3359, 2947, 2918, 2861, 2171, 1709, 1680, 1627, 1579,
1525, 1453, 1419, 1236, 1143, 1067, 1015, 980, 849, 756; M/z:
535.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 65.14; H, 6.41; N,
10.48. Found: C, 65.04; H, 6.67; N, 10.00.
5.1.27. Ethyl 5-(6-methoxy-7-(3-(4-phenylpiperazin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b]thiophene-2-carboxylate (20)
As in the procedure described for 12, compound 20 was pre-
pared as white powder (0.48 g, 59% yield). Mp: 170–172 °C; 1H
NMR (CDCl3) d: 1.43 (t, 3H), 1.69 (br, 2H), 2.16 (p, 2H), 2.61–2.67
(m, 6H), 3.22 (t, 4H), 4.02 (s, 3H), 4.27 (t, 2H), 4.42 (q, 2H), 6.86
(t, 31),6.92 (s, 1H), 6.95 (s, 1H), 7.09 (s, 1H), 7.24–7.30 (m, 2H),
7.34 (s, 1H), 7.70 (dd, 1H), 7.85 (d, 1H), 8.04 (s, 1H), 8.30 (d, 1H),
8.67 (s, 1H); 13C NMR (CDCl3) d: 14.30, 26.37, 49.20, 53.30, 54.94,
56.45, 61.61, 67.58, 70.47, 99.64, 109.04, 116.08, 119.65, 122.64,
123.09, 129. 09, 130,17, 136.12, 138.21, 139.49, 147.73, 150.01,
151.39, 153.58, 154.50, 156.42, 162.70; IR (cmÀ1): 3428, 3329,
3180, 2939, 2820, 1693, 1624, 1599, 1557, 1494, 1453, 1395,
1384, 1313, 1278, 1238, 1212, 1139, 1042, 1011, 927, 898, 783,
694; M/z: 598.3 ([M+H]+, 100%). Elem. Anal. Calcd: C, 66.31; H,
5.90; N, 11.72. Found: C, 65.35; H, 5.99; N, 12.50.
5.1.24. Ethyl 5-(6-methoxy-7-(3-(2-methylpiperidin-1-yl)propoxy)-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (17)
As in the procedure described for 11, compound 17 was pre-
pared as off-white powder (0.51 g, 60% yield). Mp: 160–161 °C;
1H NMR (CDCl3) d: 1.06 (d, 3H), 1.26–1.32 (m, 2H), 1.42 (t, 3H),
1.51–1.67 (m, 4H), 2.01–2.21 (m, 4H), 2.54 (p, 1H), 2.90 (p, 2H),
3.97 (s, 3H), 4.10–4.20 (m, 2H), 4.41 (q, 2H), 7.12 (s, 1H), 7.55 (s,
1H), 7.69 (dd, 1H), 7.84 (d, 1H), 8.02 (s, 1H), 8.27 (s, 1H), 8.67 (s,
1H); 13C NMR (CDCl3) d: 14.34, 19.02, 23.98, 25.31, 26.20, 34.66,
50.23, 52.25, 54.66, 55.96, 56.42, 61.67, 67.84, 99.66, 108.82,
108.92, 118.09, 122.77, 123.09, 130.24, 135.10, 136.13, 138.19,
139.45, 147.67, 149.98, 153.57, 154.53, 156.60, 162.78; IR (cm
À1): 3450, 3253, 3076, 2928, 2166, 1725, 1702, 1621, 1576, 1525,
1506, 1452, 1418, 1242, 1222, 1146, 1067, 1008, 958, 852, 752;
M/z: 535.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 65.14; H, 6.41;
N, 10.48. Found: C, 64.48; H, 6.40; N, 10.17.
5.2. Biological evaluation
5.2.1. In vitro kinase assays
In vitro kinase inhibitory ability was determined using the
HTScan EGFR Kinase Assay Kit, HTScan HER-2 Kinase Assay Kit,
and HTScan MET Kinase Assay Kit (Cell Signaling Technology), fol-
lowing the manufacturer’s instructions.
5.2.2. Cell culture and reagents
Human pancreatic cancer cell lines, prostate cancer cell lines,
lung cancer cell lines and human breast cell line MCF-7 were pur-
chased from American Type Culture Collection and cultured in
high-glucose DMEM (HyClone) supplemented with 10% fetal bo-
vine serum (FBS; HyClone) in a 5% CO2 humidified incubator at
37 °C. All media were also supplemented with 100 units/mL peni-
cillin and 100 lg/mL streptomycin. MCF-7-HER2 cells were ob-
5.1.25. Ethyl 5-(7-(3-(dimethylamino)propoxy)-6-methoxyquina-
zolin-4-ylamino)benzo[b] thiophene-2-carboxylate (18)
tained by transfecting MCF-7 cells with plasmid containing
human HER-2 cDNA under CMV promoter and selected for highly
proliferating and tumorigenic clone.
As in the procedure described for 11, compound 18 was pre-
pared as white powder (0.47 g, 52% yield). Mp: 176–178 °C; 1H
NMR (CDCl3) d: 1.43 (t, 3H), 2.37 (t, 2H), 2.76 (s, 6H), 3.15 (d,
3H), 4.05 (s, 3H), 4.26 (t, 2H), 4.41 (q, 2H), 7.18 (s, 1H), 7.60 (s,
1H), 7.84–7.91 (m, 2H), 8.05 (s, 1H), 8.43 (s, 1H), 8.54 (s, 1H); 13C
NMR (CDCl3) d: 13.70, 24.31, 38.96, 39.51, 35.79, 42.97, 54.90,
56.04, 60.88, 65.57, 101.83, 107.50, 109.19, 118.04, 121.92,
123.05, 129.63, 133.94, 136.27, 136.97, 138.55, 146.06, 148.74,
152.84, 156.47, 162.00; IR (cmÀ1): 3487, 2825, 2662, 1709, 1622,
1587, 1513, 1453, 1418, 1230, 1146, 1065, 1015, 958, 859, 756;
M/z: 509.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 62.48; H, 5.87;
N, 11.66. Found: C, 62.02; H, 5.51; N, 11.92.
5.2.3. Cell cytotoxicity assay
Cells were seeded in 96-well culture plates (5000 cells/well)
and treated with serially diluted testing compounds in triplicates.
After 4–5 days culture, cell growth medium was removed, prolifer-
ation reagent WST-8 (Sigma) was added to each well and incu-
bated at 37 °C for 1–3 h. Absorbance was measured with a plate
reader at 450 nm with correction at 650 nm. The results were
expressed as the percentage of absorbance of treated wells versus
that of vehicle control. IC50, the drug concentration causing 50%
growth inhibition, was calculated via sigmoid curve fitting using
GRAPHPAD PRISM 5.0, as we described previously.25
5.1.26. Ethyl 5-(7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxy-
quinazolin-4-ylamino)benzo[b] thiophene-2-carboxylate (19)
As in the procedure described for 11, compound 19 was pre-
pared as white powder (0.56 g, 67% yield). Mp: 155–156 °C; 1H
NMR (CDCl3) d: 1.07 (t, 2H), 1.38 (t, 2H), 2.06 (p, 2H), 2.20 (t,
2H), 2.37–2.55 (m, 12H), 3.93 (s, 3H), 4.17 (t, 2H), 4.39 (q, 2H),
5.2.4. Colony formation assay
For colony formation assay, Miapaca2 cells were seeded in
6-well plates (200 cells/well, in triplicate) and treated with Gefiti-
nib, compound 15 or 17 at different doses. 0.5 ml FBS was added
per well on Day 5. After 9–10 days incubation, plates were gently

3822
X. Wu et al. / Bioorg. Med. Chem. 18 (2010) 3812–3822
washed with PBS and stained with 0.1% of crystal violet. Colonies
with over 50 cells were manually counted. Plating efficiency was
calculated by dividing the number of colonies formed in the trea-
ted group by that in the control, as we previously described.26–28
Acknowledgments
Grant support: This study was supported in part by China
Scholarship Council and NIH grants R01 CA121830, R21 CA128220
and R01 CA134655 (L.X.), and by NIH through the University of
Michigan’s Cancer Center Support Grant (P30 CA46592).
We thank Dr. Susan Harris for help with the manuscript; the
University of Michigan Comprehensive Cancer Center (UMCCC)
Flow Cytometry Core for flow cytometry analysis, and UMCCC Unit
of Laboratory Animal Medicine (ULAM) for help with animal
experiments.
5.2.5. Apoptosis analysis
For apoptosis analysis by flow cytometry, MiaPaCa2 cells were
treated with Gefitinib, compound 15 or 17 at different doses for
24 h or 48 h, then trypsinized and washed with phosphate-buf-
fered saline and fixed in 70% ethanol on ice. After centrifugation,
cells were stained with 50 lg/ml propidium iodide and 0.1 lg/ml
RNase A, and analyzed by flow cytometry using a FACStar Plus™.
Each histogram was constructed with the data from at least 5000
events. Data were analyzed to calculate the percentage of cell pop-
ulation in each phase using the CellQuest software, as we described
previously.25
References and notes
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5.2.6. Western blot analysis
Cells were washed with PBS and lysed in an ice-cold RIPA lysis
buffer (1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, and Com-
plete Protease Inhibitor cocktail 100 lg/ml, in PBS) for 15 min on
ice. The cell lysates were then cleared by centrifugation at
13,000g for 10 min at 4 °C. The supernatants were collected. Pro-
tein concentrations were determined with the Bradford method
9. Kris, M. G.; Natale, R. B.; Herbst, R. S.; Lynch, T. J., Jr.; Prager, D.; Belani, C. P.;
Schiller, J. H.; Kelly, K.; Spiridonidis, H.; Sandler, A.; Albain, K. S.; Cella, D.; Wolf,
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(Bio-Rad); 40 lg of protein were electrophoresed by 10% SDS–
PAGE. Separated proteins were transferred to polyvinylidene
difluoride membranes (Bio-Rad). After blocking with 5% milk, blots
were probed with anti-HER-2, anti-PARP (Cell Signaling Technol-
ogy) and anti-b-Actin (Sigma), followed by horseradish peroxi-
dase-conjugated secondary antibody (Pierce), and detected with
the SuperSignal West Pico chemiluminescence substrate (Pierce)
as we described previously.27
5.2.7. Caspase-3 activation assay
Caspase activation in MiaPaCa2 cells treated with Gefitinib,
compound 15 or 17 at different doses was determined following
the instructions of a Caspase-3 activation assay kit (BioVision).
Twenty-four hours after treatment, cells were lysed and the whole
16. Smaill, J. B.; Rewcastle, G. W.; Bridges, A. J.; Zhou, H. R.; Showalter, H. D. H.; Fry,
D. W.; Nelson, J. M.; Sherwood, V.; Elliott, W. L.; Vincent, P. W.; DeJohn, D. E.;
Loo, J. A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Denny, W. A. J. Med.
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in the DMSO control, as we described previously.27,29
5.2.8. Animal studies
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purchased from NCI. MiaPaCa2 cells were treated with 2.5 lM
Gefitinib, compound 15 or 17 for 48 h. Cells were collected and
inoculated into nude mice subcutaneously (sc) on both flanks after
alcohol preparation of the skin, using a sterile 22-gauge needle with
0.2 ml cell suspension of 0.5 Â 106 or 1 Â 106 cells, with manual
restraint. Tumor sizes were measured using a caliper. Tumor volume
was calculated using the formula: (length  width2)/2, as we de-
scribed previously.29 All animal experiments were done according
to the protocol approved by the University of Michigan Guidelines
for Use and Care of Animals.
5.2.9. Statistical analysis
Two-way ANOVA was employed to analyze the cell assay data
using PRISM 5.0 software (GRAPHPAD). Kaplan–Meier analysis and
Mantel–Cox test were used for animal studies. P <0.05 was defined
as statistically significant.