Development of erlotinib derivatives as CIP2A-ablating agents independent of EGFR activity

Article abstract of DOI:10.1016/j.bmc.2012.08.039

Cancerous inhibitor of PP2A (CIP2A) is a novel human oncoprotein that inhibits PP2A, contributing to tumor aggressiveness in various cancers. Several studies have shown that downregulation of CIP2A by small molecules reduces PP2A-dependent phosphorylation of Akt and induces cell death. Here, a series of mono- and di-substituted quinazoline and pyrimidine derivatives based on the skeleton of erlotinib (an EGFR inhibitor) were synthesized and their bioactivities against hepatocellular carcinoma were evaluated. The di-substituted quinazoline and pyrimidine derivatives were more potent inhibitors of cancer-cell proliferation than the mono-substituted derivatives. In particular, compound 1 with chloride at position 2 of quinazoline was as potent as erlotinib in inducing cell death but no inhibition for EGFR activity. Further assays confirmed a correlation between cell death, and CIP2A and Akt inhibition by these derivatives. Among all the derivatives, compounds 19 and 22 showed the most potent antiproliferative activities and the strongest inhibition of CIP2A and p-Akt expression.

Full text of DOI:10.1016/j.bmc.2012.08.039

Bioorganic & Medicinal Chemistry 20 (2012) 6144–6153
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of erlotinib derivatives as CIP2A-ablating agents independent
of EGFR activity
Kuen-Feng Chen b,c, , Kuan-Chuan Pao a, , Jung-Chen Su a, Yi-Chieh Chou b,c, Chun-Yu Liu a,d
,
Hui-Ju Chen b,c, Jui-Wen Huang e, InKi Kim f, Chung-Wai Shiau a,
⇑
a Institute of Biopharmaceutical Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei 112, Taiwan, ROC
b Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan, ROC
c National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan, ROC
d Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
e Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan, ROC
f ASAN Institute for Life Science, ASAN Medical Center, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancerous inhibitor of PP2A (CIP2A) is a novel human oncoprotein that inhibits PP2A, contributing to
tumor aggressiveness in various cancers. Several studies have shown that downregulation of CIP2A by
small molecules reduces PP2A-dependent phosphorylation of Akt and induces cell death. Here, a series
of mono- and di-substituted quinazoline and pyrimidine derivatives based on the skeleton of erlotinib
(an EGFR inhibitor) were synthesized and their bioactivities against hepatocellular carcinoma were
evaluated. The di-substituted quinazoline and pyrimidine derivatives were more potent inhibitors of
cancer-cell proliferation than the mono-substituted derivatives. In particular, compound 1 with chloride
at position 2 of quinazoline was as potent as erlotinib in inducing cell death but no inhibition for EGFR
activity. Further assays confirmed a correlation between cell death, and CIP2A and Akt inhibition by these
derivatives. Among all the derivatives, compounds 19 and 22 showed the most potent antiproliferative
activities and the strongest inhibition of CIP2A and p-Akt expression.
Received 30 May 2012
Revised 8 August 2012
Accepted 9 August 2012
Available online 30 August 2012
Keywords:
Erlotinib
Quinazoline
Pyrimidine
CIP2A
HCC
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
phoma. Bortezomib contains dipeptidyl boronic acid which is the
binding site of the 26S proteasome and further inhibits the protea-
Overexpression of cancerous inhibitor of PP2A (CIP2A) has been
found in several common human cancers including acute leukemia,
prostate cancer, non-small cell lung cancer, gastric cancer, head and
neck cancer, colon cancer and breast cancer and has been linked to
clinical aggressiveness in tumors and promotion of the malignant
growth of cancer cells.1–7 CIP2A interacts directly with the tran-
scription factor c-Myc and inhibits PP2A dephosphorylation of c-
Myc, thereby stabilizing the oncogenic c-Myc from degradation.7
PP2A, a protein phosphatase, is a crucial regulator of cell prolifera-
tion by dephosphorylation of protein kinases on serine or threonine
residues.8 PP2A is composed of three subunits which regulate sub-
strate specificity, cellular localization and enzymatic activity. For
example, PP2A dephosphorylates p-Akt at serine 473 and reduces
the cell growth. Hence, the CIP2A-PP2A-Akt signaling cascade is
thought to be an important survival regulator in cancers.5
some activity. Interestingly, bortezomib has also been reported to
repress CIP2A expression and subsequently reduce P-Akt level
and induce apoptosis in hepatocellular carcinoma (HCC).1,5 There-
fore, development of small molecules that target CIP2A and trigger
its downregulation might be a promising strategy for anti-cancer
therapy.
Quinazolines have been used as a scaffold for synthesizing a
variety of pharmacological compounds. For example, antagonists
of human adenosine A3 receptor,9 inhibitors of histone lysine
methyltransferase G9a,10 inhibitors of poly(ADP-ribose)polymer-
ase,11 an inhibitor of protein kinase c isotypes,12 agonists of hista-
mine H4 receptor13 and inhibitors of thymidylate synthase
inhibitors.14 Amino substitutes at position 4 of quinazoline have
been demonstrated to be inhibitors of epidermal growth factor
receptor (EGFR) which is a receptor tyrosine kinase regulating cell
proliferation.15–18 These agents, gefitinib, erlotinb, and lapatinib,
have been approved for clinical use in cancer patients19 (Fig. 1).
In addition, a combination of hydroxyheptanamide and quinazo-
line has been synthesized as a dual function inhibitor of histone
acyltransferase and EGFR.20 It exhibited good activity against
HDAC and EGFR in vitro. For the current project, we started with
Recently, bortezomib, a novel proteasome inhibitor, has been
approved for treatment of multiple myeloma and mantel-cell lym-
⇑
Corresponding author. Tel.: +886 2 28267930; fax: +886 2 28201866.
E-mail address: cwshiau@ym.edu.tw (C.-W. Shiau).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.039

K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
6145
Figure 1. Inhibitors with quinazoline skeleton.
typical quinazoline-based EGFR inhibitors containing one phenyla-
mine at position 4. Aiming to expand the structural diversity of the
quinazoline substituents, we added phenylamines at position 2 of
quinazoline. The inhibitory potency of these disubstituent agents
against EGFR was disappointing in comparison with erlotinib, but
we also found that the disubstitute-quinazoline derivatives exhibit
an apoptotic effect on HCC and reduce p-Akt through repressing
the expression of CIP2A. This exciting result prompted us to devel-
op CIP2A-ablating agents.
As the disubstitute-quinazoline derivatives reduced CIP2A level,
we examined their ability to induce growth inhibition in HCC cells.
We investigated the structure–activity relationship (SAR) and
found that these agents mediate apoptosis in association with
downregulation of CIP2A in HCC cells. Western blot analyses of
downstream signaling in the human HCC cell line SK-Hep-1 are
also presented.
quinazolines with various phenylamines yielding compounds 18–
24 (Table 3). These compounds were synthesized based on the gen-
eral procedures described in Scheme 1. The inhibition of CIP2A by
these compounds was analyzed by western blot assay at 20
each compound.
lM of
3. Biological evaluation
3.1. Development of erlotinib derivative lacking inhibition of
EGFR kinase activation
An erlotinib derivative (compound 1) was synthesized by intro-
ducing a chloride atom at the 2-position of the quinazoline ring to
prevent the formation of hydrogen bonds between nitrogen of
compound 1 and T790 and M793 of EGFR. A comparison of the
EGFR kinase activity of erlotinib and compound 1 in PC9 cells
showed that erlotinib was able to inhibit the phsphorylation of
2. Chemistry
EGFR with IC50 at 1.36 lM but that compound 1 was devoid of
EGFR kinase inhibition (Fig. 2). The result suggested that functional
group connected to 2-position of quinazoline ring impeded nitro-
gen atom of quinazoline to act as a hydrogen acceptor and break
the binding with EGFR. These findings prompt us to develop novel
anticancer agents by simple introducing substituents in 2 position
of quinazoline skeleton.
A series of quinazoline derivatives were designed and synthe-
sized by the general procedure illustrated in Scheme 1 and the
SAR of the downregulation of CIP2A by these agents was explored.
Based on its core quinazoline structure, we chose commercially
available dichloro-quinazoline as a starting material. A series of
mono-quinazoline derivatives (compounds 1–10) were generated
with phenylamines by replacement of the chloride in the
quinazoline (Table 1). We further replaced the quinazoline ring
with pyrimidine and used it as a platform to carry out structural
modification, which generated a series of compounds 11–17 (Table
2). Then, we replaced the other chloride from the mono-substitute
3.2. The structure–activity relationship of erlotinib derivative
All the new quinazoline derivatives (compounds 1–24) were
screened against a panel of SK-Hep-1 cell lines for growth-inhibitory
activities. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-

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K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
Scheme 1. General synthesis of mono- and di-substitued quinazoline and pyrimidine derivatives. Reagents and conditions: (a) phenyl amine, HCl, i-PrOH; (b) phenyl amine,
HCl, i-PrOH; (c) phenyl amine, DIPEA, i-PrOH; (d) phenyl amine, DIPEA, i-PrOH.
Table 1
Table 2
Cell growth inhibitory activity of mono-substituted quinazoline derivatives as
measured by MTT assay
Cell growth inhibitory activity of synthesized pyrimidine derivatives as measured by
MTT assay
Compd
R1
R2
Cl
IC50 of cell death (
lM)
Compd
R1
R2
H
IC50 of cell death (
7.5 0.5
lM)
11
>40
1
12
13
Cl
Cl
>40
>40
2
Me
12.3 0.6
3
4
5
H
H
H
16.5 0.8
>40
14
15
Cl
Cl
>40
>40
>40
16
17
3.7 0.2
6
H
8.8 0.4
7
8
H
H
15.3 0.6
>40
13.8 0.4
the phenyl ring as in compounds 4 and 8, no activity against
SK-Hep1 cells was seen suggesting that hydrophobic interaction
is required in this area. Compound 7 bearing a phenyloxy group
exhibited higher activity than compound 9 bearing a 4-cyano-phe-
nyloxy group revealing that an electron-withdrawing group is not
favored for inducing cell death. Among the modified analogues (Ta-
ble 1), compound 1 exhibited the most potent growth inhibitory
activity. Interestingly, as the quinazoline was replaced with a
pyrimidine group to generate mono and di-substituted compounds
(11–17) (Table 2), no inhibitions with mono substituted at position
4 of pyrimidine derivatives 11–15 were detected in cell growth
assays. However, compounds 16 and 17 with phenylamine di-
substituents at positions 2, 4 in pyrimidine showed more potent
anti-tumor activity than mono-substituted pyrimidine in cell
growth assays. This result suggests that a phenylamine group con-
nected to position 2 of pyrimidine plays a crucial role in cancer-cell
growth inhibitory activity. Therefore, we tried to add a second
phenylamine group to the quinazoline. As shown in Scheme 1,
replacement of the chloride in compound 1 with a phenylamino
9
H
H
>40
10
19.8 0.8
lium bromide) assay was used to measure growth inhibition. The
compound concentrations causing 50% cell growth inhibition (IC50
values) are summarized in Tables 1–3.21 The IC50 was determined
by interpolation from dose–response curves.
Compound 2, with methylation at the amine group, was equally
as potent as compound 1 in inducing cell death. This implies that
hydrogen donor ability is not necessary for the induction of cell
death. In addition, as methylation at the amino position is known
to dramatically reduce the binding ability of quinazoline with
EGFR,22 compound 2 further provides a proof of concept that
induction of cell death of quinazoline derivatives is independent
of EGFR inhibition. When a hydroxyl group was introduced into

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6147
Table 3
CIP2A inhibitory activity than the hydrophilic cyanophenyl groups.
In addition, compound 23 showed much better inhibition than
compound 24, suggesting that the connection position of cyano-
phenyl group to phenyl ring plays an important role in CIP2A
inhibition.
Cell growth inhibitory activity of di-substituted quinazoline derivatives as measured
by MTT assay
3.3. Mechanistic validation of the mode of action of quinazoline
derivatives
Quinazoline derivatives have previously been evaluated as
EGFR inhibitors. However, the quinazoline derivatives we gener-
ated had very low potency against EGFR because of the second
substituted group. We did, however, find that quinazoline deriva-
tives repressed oncoprotein CIP2A expression and induced cell
death. Therefore, we hypothesized that quinazoline derivatives
downregulate CIP2A and p-Akt, and consequently enhance cell
apoptosis. We, therefore, screened SK-Hep1 cells treated with the
Compd
R1
Cell growth inhibition IC50
6.8 0.3
(lM)
18
19
20
2.8 0.1
7.1 0.3
quinazoline derivatives at 20 lM by western blot for expression
of CIP2A. As shown in Figure 3, mono-substituted pyrimidine com-
pounds resulted in no appreciable change in CIP2A expression. Di-
substituted pyrimidine and quinazoline compounds on the other
hand showed a high degree of repression of CIP2A. In addition,
we applied quantitative polymerase chain reaction (qPCR) assay
to demonstrate that the correlation coefficient (R2) between the
IC50 of CIP2A inhibition and the IC50 of cell growth is 0.9519, indi-
cating that the decreased level of CIP2A induced by these deriva-
tives is well correlated with cell toxicity. Next, we applied potent
compounds, 19 and 22, to study whether down-regulation CIP2A
and p-Akt is correlated to EGFR phosphorylation. As shown in
Figure 4A, compound 19 and 22 have no inhibitory effect on EGFR.
This data confirms that the second substituted-quinazoline deriva-
tives significantly reduce the binding affinity to ATP bind domain
of EGFR. We assessed CIP2A expression in response to compound
19 or erlotinib treatment in SK-Hep1 cell line. 19 reduced CIP2A
expression and cell viability with a dose dependent manner and
was more potent in its action than erlotinib. These result suggest
that CIP2A plays an important role in regulating cell viability
(Fig. 4B). Next we used three compounds, 4, 19, and 22, to explore
whether downregulation of CIP2A lead to suppression of p-Akt. As
shown in Figure 5A, compounds 19 and 22 that showed CIP2A
21
22
23
4.5 0.2
2.8 0.1
3.9 0.2
24
14.5 0.5
group resulted in di-substitute quinazoline derivatives. These
derivatives showed more potent activity than mono-substituted
derivatives against HCC cells (Table 3), suggesting that the second
substituted component originating from quinazoline plays a signif-
icant role in the activity. In addition, compounds 19 and 22 exhib-
ited higher potency with low IC50 values (2.8 and 2.8 lM,
respectively) against HCC cells whereas compound 24 only showed
moderate activity, indicating that substitutions with hydrophobic
properties, such as phenyloxy and benzyl groups exhibited higher
Figure 2. EGFR phosphorylation activity of erlotinib and compound 1. PC9 cells were exposed to erlotinib or compound 1 at 0.5, 1, 2, 4, and 8 lM for 24 h and cell lysates
were analyzed for EGFR phosphorylation.

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K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
A
B
Figure 3. (A) Western blot analysis of ablative effects of CIP2A versus Actin in SK-Hep-1 cells after treatment for 24 h with compounds 1–24 at a concentration of 20 lM.
(B) The IC50 of CIP2A inhibition versus IC50 of cell growth inhibition of quinazoline derivatives in SK-Hep-1 cells.
inhibitory activity, reduced p-Akt level, induced PARP cleavage, but
compound 4 which showed no CIP2A inhibitory activity, had no ef-
fect on p-Akt and PARP. Analysis of DNA fragmentation and flow
cytometry data showed that compounds 19 and 22 induced cell
apoptosis, a result that is consistent with their inhibition of CIP2A
expression. (Fig. 5B and C).
study we focused on the structural modification of quinazoline and
pyrimidine to develop a novel class of CIP2A ablative agents. Fur-
ther, these quinazoline and pyrimidine-based derivatives with di-
substituted phenylamines not only show great inhibition of CIP2A
but also have no EGFR inhibitory activity. Presumably, the func-
tional group at position 2 of compound 1 reduces its interaction
with EGFR. Subsequent modifications of quinazoline and pyrimi-
dine with a series of phenylamine in the 2 position resulted in
decreasing CIP2A expression.
4. Discussion
Recent data suggest that CIP2A serves as a major regulator of
cell survival by disrupting PP2A activity and subsequently increas-
ing Akt phosphorylation. CIP2A has been shown to be protective in
clinical therapy rendering it a promising drug development target.5
For example, a recent study demonstrated that the effect of bort-
ezomib, a proteasome inhibitor, in HCC cells was associated with
expression of CIP2A. HCC cells which have high levels of expression
of CIP2A were more resistant to borzetomib treatment than HCC
cells with low level of CIP2A. Therefore, agents that downregulate
CIP2A can be expected to act as sensitizing drugs in patients. In this
5. Conclusions
In summary, here, a series of quinazoline and pyrimidine-de-
rived bases were synthesized and their cytotoxicity was explored
with interesting SAR results. Structural modifications indicated
that di-phenylamine derivatives with quinazoline and pyrimidine
skeletons are required for activity. According to MTT assay, most
of these derivatives had micromolar level potency against SK-
Hep-1 cells. Compounds 19 and 22 showed the most potent inhibi-

K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
6149
Figure 4. (A) The effect of erlotinib and compound 19 and 22 on EGFR phosphorylation activity. PC9 cells were treated with erlotinib and compound 19 and 22 at 2 lM for
24 h. (B) Compound 19 repressed CIP2A expression and inhibited cell growth as measured by MTT assay. SK-Hep-1 cells were treated with erlotinib and Compound 19 at 2.5
and 5 M for 24 h. The cells were analyzed with western blot assay and MTT assay.
l
Figure 5. (A) Western blot analysis of the effects of compounds 4, 19, and 22, (each at 5
lM) on CIP2A, phosphorylation of Akt, PARP and Actin, in SK-Hep-1 cells after 30 h of
treatment. (B) Flow cytometry analysis of cell death induced by compounds 4, 19, and 22, at 5
l
M, after 24 h of treatment in SK-Hep-1 cells. Apoptotic cells were stained in
potassium iodide (PI) solution and determined by flow cytometry. Columns, mean (n = 3); bars, SD; ⁄P <0.05. (C) Elisa analysis of cell death. Effects of compounds 4, 19, and 22
on DNA fragmentation in SK-Hep-1 cells. Cells were treated with compounds 4, 19, and 22 with indicated concentration for 24 h and DNA fragmentation was analyzed by
using a cell death ELISA kit. Columns, mean (n = 3); bars, SD; ⁄P <0.05.
tion of CIP2A expression and cell survival activity, whereas com-
pound 4 had no activity in either assay. Furthermore, compounds
19 and 22 reduced Akt phosphorylation after repressing CIP2A,
whereas compound 4 had no activity against p-Akt and CIP2A.
These results suggest selective sensitivity in response to the differ-
ent substituted functional groups in quinazoline. Moreover inhibi-
tion of CIP2A expression correlated with cytotoxicity in SK-Hep-1
cells upon drug treatment. The SARs obtained from both the

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K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
in vitro growth inhibition data and the CIP2A-Akt pathway warrant
further detailed study. Testing of compounds 19 and 22 in an
in vivo HCC model is currently being pursued.
6.2.4. 2-Chloro-N-(3-chlorophenyl)-6,7-dimethoxyquinazolin-
4-amine (3)
1H NMR (400 MHz, DMSO-d6): d 3.90 (s, 3H), 3.95 (s, 3H), 7.18
(s, 1H), 7.20 (d, 1H, J = 8.0 Hz), 7.44 (t, J = 8.0 Hz, 1H), 7.79 (d,
J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.97 (s, 1H), 10.08 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d 56.5, 57.1, 103.3, 106.6, 107.8, 121.3,
122.4, 124.1, 130.5, 133.1, 140.7, 148.2, 149.6, 154.1, 155.6,
158.2; HRMS Calcd for C16H13Cl2N3O2 (M+H): 350.0463. Found:
350.0466. Yield: 75%.
6. Experimental section
6.1. Materials
Proton and carbon nuclear magnetic resonance (1H and 13C NMR)
spectra were recorded on Bruker DPX400 (400 MHz) instruments.
Chemical shifts (d) are reported in ppm relative to the TMS peak.
Peak multiplicities are expressed as follows: s, singlet; d, doublet;
t, triplet; q, quartet; br s, broad singlet; m, multiplet. Reaction pro-
gress was determined by thin layer chromatography (TLC) analysis
on silica gel 60 F254 plate (Merck). Chromatographic purification
was carried on silica gel columns 60 (0.063–0.200 mm or 0.040–
0.063 mm, Merck), basic silica gel. Commercial reagents and sol-
vents were used without additional purification. The following
abbreviations are used: CDCl3, deuterated chloroform; DMSO-d6, di-
methylsulfoxide-d6; i-PrOH, isopropylalcohol; EtOAc, ethyl acetate;
DMF, N,N-dimethylformamide; MeOH, methanol; THF, tetrahydro-
furan; EtOH, ethanol; DMSO, dimethyl sulfoxide; DIPEA, diisopro-
pylethylamine; DCM, dichloromethane. High resolution mass
spectra were recorded on a Finnigan MAT 95S mass spectrometer.
6.2.5. 3-(2-Chloro-6,7-dimethoxyquinazolin-4-ylamino)phenol
(4)
1H NMR (400 MHz, DMSO-d6): d 3.15 (s, 1H), 3.91 (s, 3H), 3.94
(s, 3H), 6.59 (d, J = 6.8 Hz, 1H), 7.13–7.21 (m, 4H), 7.98 (s, 1H),
9.96 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 56.4, 56.9, 58.5,
103.3, 106.4, 107.7, 110.6, 112.1, 114.2, 129.5, 139.8, 147.6,
149.5, 154.2, 155.4, 158.0; HRMS Calcd for C16H14ClN3O3 (M+H):
332.0802. Found: 332.0810. Yield: 60%.
6.2.6. 2-Chloro-N-(2-fluoro-5-methylophenyl)-6,7-
dimethoxyquinazolin-4-amine (5)
1H NMR (400 MHz, DMSO-d6): d 2.32 (s, 3H), 3.87 (s, 3H), 3.92
(s, 3H), 7.12–7.16 (m, 2H), 7.22 (d, J = 10.4 Hz, 1H), 7.29 (d,
J = 7.6 Hz, 1H), 7.90 (s, 1H), 10.05 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d 20.1, 56.0, 56.3, 102.8, 105.7, 106.8, 115.61, 115.8,
124.9, 125.0, 128.1, 128.2, 128.5, 133.6, 133.7, 146.9, 149.1,
153.9, 153.9, 155.1, 156.3, 159.1; HRMS Calcd for C17H15ClFN3O2
(M+H): 348.0915. Found: 348.0911. Yield: 60%.
6.2. Chemical synthesis
6.2.1. General procedure for the synthesis of compounds 1–10
Aniline derivative (0.8 mmol) was added to a solution of 2,4-di-
chloro-6,7-dimethoxyquinazoline (1.0 mmol) in isopropyl alcohol
(5 ml), followed by the addition of a drop of concentrated HCl
6.2.7. 2-Chloro-N-(4-chloro-3-(trifluoromethyl)phenyl)-6,
7-dimethoxyquinazolin-4-a mine (6)
1H NMR (400 MHz, DMSO-d6): d 3.92 (s, 3H), 3.96 (s, 3H), 7.19
(s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.99 (s, 1H), 8.19 (d, J = 8.8 Hz,
1H), 8.42 (s, 1H), 10.32 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d
56.0, 56.8, 102.8, 105.9, 107.3, 118.7, 120.9, 120.9, 121.0, 121.0,
121.5, 124.2, 124.5, 125.9, 126.2, 126.5, 126.9, 131.6, 138.3,
147.5, 149.2, 153.1, 155.2, 157.2; HRMS Calcd for C17H12Cl2F3N3O2
(M+H): 418.0337. Found: 418.0340. Yield: 86%.
(100 ll). The resulting mixture was stirred at 60 °C for 2 h. The
mixture was filtered, and the solid was washed with isopropyl
alcohol then dried under vacuum to give compounds 1–10. This
procedure afforded the expected coupling product as a white or
yellow solid (yield: 21–95%).
6.2.2. 2-Chloro-N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-
4-amine (1)
6.2.8. 2-Chloro-6,7-dimethoxy-N-(4-phenoxyphenyl)
quinazolin-4-amine (7)
1H NMR (400 MHz, DMSO-d6): d 3.93 (s, 3H), 3.99 (s, 3H), 4.21
(s, 1H), 7.18 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz,
1H), 7.85 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.94 (s, 1H), 10.00 (s,
1H); 13C NMR (100 MHz, DMSO-d6): d 56.4, 57.2, 81.1, 83.8,
103.9, 106.6, 107.9, 122.2, 123.8, 126.0, 127.6, 129.2, 139.5,
148.1, 149.5, 154.1, 155.5, 158.4; HRMS Calcd for C18H14ClN3O2
(M+H): 340.0853. Found: 340.0850. Yield: 94%.
1H NMR (400 MHz, DMSO-d6): d 3.91 (s, 3H), 3.94 (s, 3H), 7.03
(d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 7.11–7.16 (m, 2H), 7.40
(t, J = 6.0 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.90 (s, 1H), 9.93 (s,
1H); 13C NMR (100 MHz, DMSO-d6): d 56.5, 57.0, 103.4, 105.9,
107.5, 118.8, 119.5, 123.8, 125.3, 130.5, 134.3, 146.9, 149.5,
153.6, 153.9, 155.5, 157.4, 158.4; HRMS Calcd for C22H18ClN3O3
(M+H): 408.1115. Found: 408.1121. Yield: 55%.
6.2.3. 2-Chloro-N-(3-ethynylphenyl)-6,7-dimethoxy-
N-methylquinazolin-4-amine (2)
6.2.9. 4-(2-Chloro-6,7-dimethoxyquinazolin-4-ylamino)-
3-methylphenol (8)
Methyl iodide (56 ll, 0.90 mmol) was added to a solution of 1
1H NMR (400 MHz, DMSO-d6): d 2.70 (s, 3H), 3.89 (s, 3H), 3.90
(s, 3H), 6.64 (d, J = 8.4 Hz, 1H), 6.71 (s, 1H), 7.04 (d, J = 8.4 Hz,
1H), 7.11 (s, 1H), 7.83 (s, 1H), 9.67 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d 18.5, 56.4, 56.5, 102.8, 107.0, 107.3, 113.5, 117.3,
128.0, 129.2, 136.7, 148.1, 149.2, 155.1, 155.5, 156.5, 160.1; HRMS
Calcd for C17H16ClN3O3 (M+H): 346.0958. Found: 346.0951. Yield:
23%.
(61.0 mg, 0.18 mmol) and sodium hydride (60% oil suspension,
8.63 mg, 0.36 mmol) in 2 ml of DMF cooled to 0 °C. The mixture
was stirred at 0 °C for 1 h, then allowed to warm to room temper-
ature and stirred for another 1 h. The reaction mixture was washed
with water, and then extracted with EtOAc. The organic phase was
dried over MgSO4, filtered and concentrated under reduced pres-
sure. The crude residue was purified by chromatography on a silica
gel column using EtOAc/hexane as eluent (0–40%) to give com-
pound 2 (yield: 60%). 1H NMR (400 MHz, CDCl3): d 3.07 (s, 1H),
3.28 (s, 3H), 3.57 (s, 3H), 3.88 (s, 3H), 6.21 (s, 1H), 7.05 (s, 1H),
7.15 (d, J = 7.2 Hz, 1H), 7.31–7.37 (m, 3H); 13C NMR (100 MHz,
CDCl3): d 42.2, 55.2, 56.1, 78.8, 82.0, 104.8, 106.7, 108.6, 124.2,
126.7, 129.5, 130.0, 130.2, 147.5, 147.8, 150.4, 154.4, 155.0,
161.3; HRMS Calcd for C19H16ClN3O2 (M+H): 354.1009. Found:
354.1016.
6.2.10. 4-(3-(2-Chloro-6,7-dimethoxyquinazolin-
4-ylamino)phenoxy)benzonitrile (9)
1H NMR (400 MHz, DMSO-d6): d 3.91 (s, 3H), 3.94 (s, 3H), 6.94
(d, J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.21 (d, J = 8.8 Hz, 2H), 7.50 (t,
J = 8.0 Hz, 1H), 7.65 (m, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.90 (s, 1H),
10.00 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 56.0, 56.2, 102.1,
105.3, 106.6, 107.2, 113.8, 115.4, 118.4, 118.6, 118.7, 130.2,

K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
6151
134.6, 140.4, 148.2, 149.0, 153.9, 154.6, 155.0, 157.6, 160.7; HRMS
Calcd for C23H17ClN4O3 (M - H): 431.0911. Found: 431.0909. Yield:
74%.
6.3.7. N2,N4-Bis(3-ethynylphenyl)pyrimidine-2,4-diamine (16)
1H NMR (400 MHz, CDCl3): d 3.04 (s, 1H), 3.10 (s, 1H), 6.16 (d,
J = 6.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.19–7.31 (m, 4H), 7.37 (d,
J = 8.0 Hz, 1H), 7.45 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H),
7.92 (br s, 1H), 8.06 (d, J = 6.0 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d 77.0, 77.8, 83.0, 83.8, 97.2, 120.5, 122.4, 122.6, 123.0, 123.1,
125.2, 126.1, 128.0, 128.8, 129.3, 138.5, 139.7, 157.2, 159.8,
6.2.11. N-Benzyl-2-chloro-6,7-dimethoxyquinazolin-4-amine
(10)
1H NMR (400 MHz, MeOH-d4): d 3.90 (s, 3H), 3.92 (s, 3H), 4.79
(s, 2H), 6.96 (s, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.31 (t, J = 7.2 Hz, 2H),
7.39 (d, J = 7.2 Hz, 2H), 7.47 (s, 1H); 13C NMR (100 MHz, DMSO-
d6): d 43.4, 55.8, 56.0, 102.2, 106.5, 106.8, 126.9, 127.4, 128.3,
138.9, 147.2, 148.5, 154.4, 155.0, 159.9; HRMS Calcd for
161.0; HRMS Calcd for
311.1291. Yield: 10%.
C20H14N4 (M+H): 311.1297. Found:
6.3.8. N2,N4-Bis(4-chloro-3-(trifluoromethyl)phenyl)
C
17H16ClN3O2 (M+H): 330.1009. Found: 330.1007. Yield: 21%.
pyrimidine-2,4-diamine (17)
1H NMR (400 MHz, CDCl3): d 6.16 (d, J = 5.6 Hz, 1H), 6.60 (s, 1H),
7.10 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.60 (d,
J = 8.4 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.72 (s, 1H), 7.92 (s, 1H), 8.12
(d, J = 5.6 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): d 99.6, 118.0 (q),
118.4 (q), 118.7, 118.9, 121.4, 121.6, 123.0, 123.3, 123.9, 124.1,
124.2, 124.3, 126.91–128.30 (m), 131.2, 131.4, 139.0, 139.6,
6.3. General procedure for the synthesis of compounds 11–17
6.3.1. General procedure for the synthesis of compounds 11–17
To a solution of 2,4-dichloropyrimidine (1.0 mmol) and DIPEA
(100 ll) in isopropyl alcohol was added 0.7 mmol aniline deriva-
tives and the mixtures stirred under ice-bath condition for
30 min. The resulting mixture was stirred at room temperature
for 8 h. After the reaction was completed, the reaction mixture
was washed with water, extracted with EtOAc, and the organic
layer was dried over MgSO4. After removal of MgSO4 by filtration
and evaporation of solvents, the crude residue was purified by
chromatography on a silica gel column using MeOH/DCM as eluent
(0% to 2%) to give compounds 11–17 (yield: 3–27%).
155.8, 159.1, 160.6; HRMS Calcd for
467.0265. Found: 467.0254. Yield: 5%.
C18H10Cl2F6N4 (M+H):
6.4. General procedure for synthesis of compounds 18 and 24
6.4.1. General procedure for synthesis of compounds 18–24
Aniline derivatives (0.5 mmol) were added to a solution of com-
pound 1 (0.2 mmol) in isopropyl alcohol (3 ml), followed by the
addition of a drop of concentrated HCl (100 ll). The resulting solu-
6.3.2. 2-Chloro-N-(3-ethynylphenyl)pyrimidin-4-amine (11)
1H NMR (400 MHz, MeOH-d4): d 3.48 (s, 1H), 6.66 (d, J = 6.0 Hz,
1H), 7.18 (d, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.61 (d,
J = 8.4 Hz, 1H), 7.72 (s, 1H), 8.05 (d, J = 6.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 78.6, 82.8, 102.9, 123.7, 123.9, 126.5, 129.8,
130.0, 137.5, 158.5, 161.1, 162.6; HRMS Calcd for C12H8ClN3
(M+H): 230.0485. Found: 230.0478. Yield: 5%.
tion was heated by using microwave irradiation to 150 °C for
30 min. After cooling, the mixture was filtered, and the solid was
washed with isopropyl alcohol. The crude solid was dissolved in
DCM and washed with saturated NaHCO3 solution. The organic
phase was dried over MgSO4, filtered and concentrated under re-
duced pressure. The crude residue was purified by chromatogra-
phy on a silica gel column using MeOH/DCM as eluent (0–5%) to
give compounds 18–24 (yield: 20–80%).
6.3.3. 2-Chloro-N-(3-chlorophenyl)pyrimidin-4-amine (12)
1H NMR (400 MHz, CDCl3): d 6.59 (d, J = 5.6 Hz, 1H), 7.18 (d,
J = 8.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.36
(s, 1H), 8.15 (d, J = 5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): d
102.9, 120.6, 122.6, 125.8, 130.6, 135.2, 138.4, 158.2, 160.8,
162.0; HRMS Calcd for C10H7Cl2N3 (M+H): 240.0095. Found:
240.0101. Yield: 6%.
6.4.2. N2-(4-Chloro-3-(trifluoromethyl)phenyl)-N4-
(3-ethynylphenyl)-6,7-dimethoxy-quinazoline-2,4-diamine (18)
1H NMR (400 MHz, MeOH-d4): d 3.47 (s, 1H), 3.91 (s, 3H), 3.93
(s, 3H), 6.88 (s, 1H), 7.22 (d, J = 7.6 Hz, 2H), 7.31 (t, J = 8.0 Hz,
1H), 7.34 (d, J = 9.2 Hz, 1H), 7.53 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H),
7.83 (s, 1H), 8.01–8.04 (m, 2H); 13C NMR (100 MHz, MeOH-d4): d
54.9, 55.4, 77.1, 77.2, 83.1, 101.9, 105.0, 105.1, 117.1–117.2 (q),
121.5, 121.7, 121.9 (d), 122.5, 122.7, 122.9, 123.3, 124.4, 125.5,
126.9, 127.3, 127.6, 128.3, 128.5, 131.17, 139.5, 140.3, 147.0,
6.3.4. 2-Chloro-N-(4-chloro-3-(trifluoromethyl)phenyl)
pyrimidin-4-amine (13)
1H NMR (400 MHz, CDCl3): d 6.55 (d, J = 5.6 Hz, 1H), 7.11 (s, 1H),
7.51 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 8.20 (d,
J = 5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 103.6, 120.8 (q),
123.7, 125.8, 128.1, 129.2, 129.5, 132.5, 136.2, 158.3, 160.9,
161.4; HRMS Calcd for C11H6Cl2F3N3 (M+H): 307.9969. Found:
307.9969. Yield: 3%.
148.3, 155.1, 155.5, 157.8; HRMS Calcd for
(M+H): 499.1149. Found: 499.1142. Yield: 33%.
C25H18ClF3N4O2
6.4.3. N4-(3-Ethynylphenyl)-6,7-dimethoxy-N2-
(4-phenoxyphenyl)quinazoline-2,4-diamine (19)
1H NMR (400 MHz, DMSO-d6): d 3.93 (s, 3H), 3.94 (s, 3H), 4.18
(s, 1H), 6.98 (d, 4H, J = 8.8 Hz), 7.13 (s, 1H), 7.15 (d, 1H,
J = 7.6 Hz), 7.33–7.45 (m, 6H), 7.69 (d, 1H, J = 7.6 Hz), 7.75 (s, 1H),
8.10 (s, 1H), 10.33 (s, 1H), 10.90 (s, 1H); 13C NMR (100 MHz,
CDCl3): d 56.1, 56.2, 77.6, 83.2, 100.0, 104.4, 106.3, 117.9, 120.0,
120.8, 122.4, 122.5, 122.7, 125.2, 127.7, 128.8, 129.5, 135.9,
138.6, 146.7, 149.2, 151.3, 155.1, 155.9, 156.9, 158.2; HRMS Calcd
for C30H24N4O3 (M+H): 489.1927. Found: 489.1925. Yield: 40%.
6.3.5. N-Benzyl-2-chloropyrimidin-4-amine (14)
1H NMR (400 MHz, MeOH-d4): d 4.55 (s, 2H), 6.60 (d, J = 5.2 Hz,
1H), 7.19–7.22 (m, 1H), 7.26–7.31 (m, 4H), 8.12 (d, J = 5.2 Hz, 1H);
13C NMR (100 MHz, MeOH-d4): d 43.8, 104.4, 126.9, 127.4, 128.2,
138.3, 154.3, 160.2, 163.7; HRMS Calcd for C11H10ClN3 (M+H):
220.0642. Found: 220.0640. Yield: 27%.
6.3.6. 2-Chloro-N-(4-phenoxyphenyl)pyrimidin-4-amine (15)
1H NMR (400 MHz, MeOH-d4): d 6.63 (d, J = 6.0 Hz, 1H), 6.96–
7.00 (m, 4H), 7.08 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 8.0 Hz, 2H), 7.54
(d, J = 8.0 Hz, 2H), 8.01 (d, J = 6.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d 102.1, 119.0, 119.6, 123.7, 125.5, 129.8, 131.7, 155.6,
156.7, 158.0, 160.8, 163.0; HRMS Calcd for C16H12ClN3O (MꢀH):
296.0591. Found: 296.0583. Yield: 14%.
6.4.4. N2-(3-Chlorophenyl)-N4-(3-ethynylphenyl)-6,
7-dimethoxyquinazoline-2,4-diamine (20)
1H NMR (400 MHz, MeOH-d4): d 3.47 (s, 1H), 3.93 (s, 3H), 3.94
(s, 3H), 6.88 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 7.17 (t, J = 8.0 Hz,
1H), 7.22 (d, J = 7.6 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.53 (d,
J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.80 (s, 2H), 7.87 (d, J = 8.4 Hz, 1H);
13C NMR (100 MHz, MeOH-d4): d 54.9, 55.4, 77.1, 83.1, 102.0,

6152
K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
104.9, 104.9, 117.0, 118.4, 120.5, 122.6, 123.0, 125.5, 126.9, 128.5,
129.3, 133.7, 139.6, 142.3, 146.9, 148.5, 155.1, 155.8, 157.9; HRMS
Calcd for C24H19ClN4O2 (M+H): 431.1275. Found: 431.1280. Yield:
34%.
EGFR, p-EGFR, PARP and p-Akt (Ser473) were from Cell Signaling
(Danvers, MA).
6.5.3. Cell death detection ELISA
The effect of compounds 4, 19 and 22 on cell viability was as-
sessed by a cell death detection ELISA kit (Roche Applied Science,
Mannheim, Germany). SK-Hep-1 cells were treated with com-
pounds 4 and 19 at 22 at 2.5 and 5 lM for 24 h. The cells were col-
lected and assayed according to the standard protocol provided by
6.4.5. N4-(3-Ethynylphenyl)-N2-(2-fluoro-5-methylphenyl)-
6,7-dimethoxyquinazoline-2,4-diamine (21)
1H NMR (400 MHz, MeOH-d4): d 2.18 (s, 3H), 3.45 (s, 1H), 3.95
(s, 3H), 3.96 (s, 3H), 6.75 (br s, 1H), 6.91–6.98 (m, 2H), 7.23 (d,
J = 7.6 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.75–7.77 (m,
2H), 7.91 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 21.2,
56.1, 56.1, 75.5, 100.3, 104.4, 105.5, 114.0, 114.2, 121.4, 122.2,
122.3, 122.7, 122.7, 125.3, 127.6, 127.7, 127.8, 128.9, 133.7,
133.7, 138.6, 146.9, 148.1, 149.7, 152.1, 155.2, 155.2, 157.1; HRMS
Calcd for C25H21FN4O2 (M+H): 429.1727. Found: 429.1721. Yield:
20%.
the manufacturer.
6.5.4. Apoptosis analysis
The apoptotic cells were assessed by flow cytometry (sub-G1).
After Sk-Hep-1 cells were treated with compounds 4, 19, and 22,
cells were trypsinized, collected by centrifugation and resus-
pended in PBS. After centrifugation, the cells were washed in PBS
and resuspended in propidium iodide (PI) staining solution. (Spec-
imens were incubated in the dark for 30 min at 37 °C and then ana-
lyzed with an EPICS Profile II flow cytometer (Coulter Corp.,
Hialeah, FL). All experiments were performed in triplicate
6.4.6. N2-Benzyl-N4-(3-ethynylphenyl)-6,7-
dimethoxyquinazoline-2,4-diamine (22)
1H NMR (400 MHz, DMSO-d6): d 3.84 (s, 3H), 3.85 (s, 3H), 4.15
(s, 1H), 4.53 (d, J = 6.4 Hz, 2H), 6.75 (s, 1H), 7.10–7.19 (m, 3H),
7.27 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.33 (d, J = 7.2 Hz,
2H), 7.63 (s, 1H), 7.90 (s, 1H), 9.14 (s, 1H); 13C NMR (100 MHz,
CDCl3): d 29.3, 45.2, 55.7, 55.9, 76.8, 83.0, 100.1, 103.4, 105.2,
121.6, 122.2, 124.2, 126.7, 126.9, 127.1, 128.1, 128.4, 138.5,
139.3, 145.7, 154.7, 156.5, 158.0; HRMS Calcd for C25H22N4O2
(M+H): 411.1821. Found: 411.1826. Yield: 15%.
6.5.5. MTT assay
The effect of individual test agents on cell viability was assessed
by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay in 6 replicates. Sk-Hep-1 cells were seeded
and incubated in 96-well, flat-bottomed plates in for 24 hours
and were exposed to various concentrations of test agents dissolved
in DMSO (final concentration, 0.1%) for 48 h. Controls received
DMSO vehicle at a concentration equal to that in drug-treated cells.
6.4.7. 4-(3-(4-(3-Ethynylphenylamino)-6,7-
dimethoxyquinazolin-2-ylamino)phenoxy-)benzonitrile (23)
1H NMR (400 MHz, CDCl3): d 3.05 (s, 1H), 3.93 (s, 3H), 3.95 (s,
3H), 6.61 (d, J = 7.6 Hz, 1H), 6.92 (s, 1H), 6.94 (s, 1H), 7.01 (d,
J = 9.2 Hz, 2H), 7.20 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 8.0 Hz, 2H), 7.35
(d, J = 7.2 Hz, 2H), 7.54 (d, J = 9.2 Hz, 2H), 7.64 (s, 1H), 7.66 (d,
J = 8.0 Hz, 1H), 7.73 (s, 1H); 13C NMR (100 MHz, CDCl3): d 56.1,
56.2, 77.6, 83.2, 100.1, 104.6, 105.3, 106.3, 110.5, 112.8, 115.2,
118.0, 119.0, 122.7, 122.8, 125.4, 127.9, 128.8, 130.1, 133.9, 138.5,
142.1, 147.0, 148.9, 155.1, 155.1, 155.3, 157.0, 161.6; HRMS Calcd
for C31H23N5O3 (M+H): 514.1879. Found: 514.1888. Yield: 80%.
The medium was removed, replaced by 200 lL of 0.5 mg/mL MTT in
10% fetal bovine serum containing DMEM, and cells were incubated
in the carbon dioxide incubator at 37 °C for 2 h. Supernatants were
removed from the wells and the reduced MTT dye was solubilized
in 100 lL/well DMSO. Absorbance at 570 nm was determined on
a plate reader.
6.5.6. Quantitative real time polymerase chain reaction (qPCR)
assay
Total RNA was isolated from SK-Hep1 cell line with TRIzol
(Invitrogen). An aliquot of 2.5 lg/12.1 lL of total RNA was used
6.4.8. 4-(4-(4-(3-Ethynylphenylamino)-6,7-
as the template in the synthesis of first-strand cDNA using an oli-
go(dT) primer and the AMV reverse transcriptase system (Roche
Diagnostics) by Thermal Cycler (RTC-200, MJ Reaserch). The meth-
od of qPCR was followed according to the method described by
Ponchel et al.23 qPCR was performed using a Roche LightCycler
480 sequence detection system (RocheApplued Science,). Thermo-
dimethoxyquinazolin-2-ylamino)phenoxy)-benzonitrile (24)
1H NMR (400 MHz, DMSO-d6): d 3.92 (s, 3H), 3.94 (s, 3H), 4.22
(s, 1H), 7.06 (d, J = 8.4 Hz, 2H), 7.10–7.15 (m, 3H), 7.36 (d,
J = 7.6 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.53 (d, J = 7.6 Hz, 2H), 7.68
(d, J = 7.6 Hz, 1H), 7.74 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 8.03 (s,
1H); 13C NMR (100 MHz, CDCl3): d 56.1, 56.2, 77.6, 83.2, 100.2,
104.6, 105.0, 106.3, 117.3, 119.0, 120.6, 121.0, 122.6, 122.7,
125.5, 127.8, 128.8, 134.0, 137.6, 138.6, 146.8, 148.6, 149.2,
155.1, 155.7, 157.0, 162.4; HRMS Calcd for C31H23N5O3 (M+H):
514.1879. Found: 514.1876. Yield: 75%.
cycling was performed in a final volume of 20
l
l containing 2.5
ll
of cDNA sample, 200 nM of each of the primers, and 6.5
lL of SYBR
Green I master mix (Roche). The relative differences in expression
levels between genes were expressed using cycle time (Ct) values
as follows: the Ct value of the gene of CIP2A was first normalized
to that for GAPDH in the same sample, then the difference between
the treatment and control group was calculated and expressed as
an increase or decrease in cycle numbers compared with the con-
trol. Oligonucleotide sequences were as follows: CIP2A, 50-TGG
CAA GAT TGA CCT GGG ATT TGG A-30(sense) and 50-AGG AGT
AAT CAA ACG TGG GTC CTG A-30(antisense); GAPDH, 50-CGA CCA
CTT TGT CAA GCT CA-30(sense) and 50-AGG GGT CTA CAT GGC
AAC TG-30(antisense). The following PCR conditions were used:
denaturation at 95 °C for 10 min followed by 40 cycles of 94 °C
for 1 min, annealing for 1 min at 60 °C, and elongation for 1 min
at 72 °C, and a final elongation step at 72 °C for 10 min.
6.5. Biological assays
6.5.1. Cell culture
SK-Hep-1, PC9 cells were maintained in DMEM supplemented
with 10% FBS, 100 units/mL penicillin G, 100
lg/mL streptomycin
sulfate and 25 g/mL amphotericin B in a 37 °C humidified incuba-
l
tor in an atmosphere of 5% CO2 in air.
6.5.2. Western blot
SK-Hep-1 and PC9 cells (3 ꢁ 105 cells) were treated with com-
pounds at various doses in 60 mm dish. 40 lg/per lane of cell ly-
sates were analyzed by western blot. Antibodies for
immunoblotting such as anti-Akt1 and CIP2A were purchased from
Santa Cruz Biotechnology (San Diego, CA). Other antibodies such as
6.5.7. Clonogenic assay
For colony formation, SK-Hep1 cells transfected with scramble
siRNA or CIP2A-specific siRNA were seeded in triplicate onto

K.-F. Chen et al. / Bioorg. Med. Chem. 20 (2012) 6144–6153
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Dobrusin, E. M.; Bridges, A. J.; Zhou, H.; Showalter, H. D.; Winters, R. T.;
Leopold, W. R.; Fry, D. W.; Nelson, J. M.; Slintak, V.; Elliot, W. L.; Roberts, B. J.;
Vincent, P. W.; Patmore, S. J. J. Med. Chem. 1803, 1999, 42.
6 cm plates (10,000 cells per plate). After 7 days of culturing, cells
were stained with crystal violet and colony containing more than
50 cells were counted.
Acknowledgments
We thank the National Science Council, Taiwan (NSC98-2320-B-
010-005-My3 and NSC-100-2325-B-010-007) and National Yang
Ming University, Taiwan for financial support. C.W.S. also thanks
the National Research Institute of Chinese Medicine for NMR
support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.039.
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