Design and synthesis of 4,6-substituted-(diaphenylamino)quinazolines as potent EGFR inhibitors with antitumor activity

Article abstract of DOI:10.1016/j.bmc.2011.10.085

A type of novel 4,6-substituted-(diaphenylamino)quinazolines, which designed based on the 4-(phenylamino)quinazoline moiety, have been discovered as potential EGFR inhibitors. These compounds displayed good antiproliferative activity and EGFR-TK inhibitory activity. Especially, 4-((4-(3-bromophenylamino) quinazolin-6-ylamino)methyl)phenol (5b), showed the most potent inhibitory activity (IC₅₀ = 0.28 μM for Hep G2, IC₅₀ = 0.59 μM for A16-F10 and IC₅₀ = 0.87 μM for EGFR) and effectively induces apoptosis in a dose-dependent manner in the Hep G2 cell line. Molecular docking of 5b into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.

Full text of DOI:10.1016/j.bmc.2011.10.085

Bioorganic & Medicinal Chemistry 20 (2012) 317–323
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 4,6-substituted-(diaphenylamino)quinazolines
as potent EGFR inhibitors with antitumor activity
Huan-Qiu Li a, , Dong-Dong Li b, Xiang Lu b, Yun-Yun Xu a, Hai-Liang Zhu b,
⇑
⇑
a College of Pharmaceutical Science, Soochow University, Suzhou 215123, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A type of novel 4,6-substituted-(diaphenylamino)quinazolines, which designed based on the 4-(phenyla-
mino)quinazoline moiety, have been discovered as potential EGFR inhibitors. These compounds displayed
good antiproliferative activity and EGFR-TK inhibitory activity. Especially, 4-((4-(3-bromophenylami-
Received 13 September 2011
Revised 26 October 2011
Accepted 29 October 2011
Available online 6 November 2011
no)quinazolin-6-ylamino)methyl)phenol (5b), showed the most potent inhibitory activity (IC50 = 0.28
lM
for Hep G2, IC50 = 0.59 M for A16-F10 and IC50 = 0.87 M for EGFR) and effectively induces apoptosis in a
l
l
dose-dependent manner in the Hep G2 cell line. Molecular docking of 5b into EGFR TK active site was also
performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
EGFR-TK inhibitors
4-(Phenylamino)quinazoline
Antitumor
Molecular docking
1. Introduction
Molecular modeling studies suggested, and experimental results
confirmed, that the Michael acceptor side chain of these inhibitors
Receptor protein tyrosine kinases play a key role in signal trans-
duction pathways that regulate cell division and differentiation.
Among the growth factor receptor kinases that have been identified
as being important in cancer is epidermal growth factor receptor
(EGFR) kinase. Activation of EGFR may be because of overexpres-
sion, mutations resulting in constitutive activation, or autocrine
expression of ligand.1,2 Expression of a dominant negative Ras mu-
tant in EGFR overexpressing cells also results in a significant poten-
tiation of EGFR induced apoptosis suggesting that Ras activation is a
key survival signal generated by the EGFR. The role of EGFR has
been most thoroughly studied in breast cancer,3 lung cancer (espe-
cially lung adenocarcinomas)4–6 and in hormone-refractory pros-
tate cancer.7 Compounds that inhibit the kinase activity of EGFR
after binding of its cognate ligand are of potential interest as new
therapeutic antitumor agents.8,9
The 6,7-dialkoxy-4-(phenylamino)quinazolines are a class of
potent, selective, ATP-competitive inhibitors of EGFR tyrosine ki-
nase.10,11 Tosu group and Parke-Davis group developed irreversible
inhibitors the butynamide-substituted 4-anilinoquinazoline A,12 as
well as the acrylamide-substituted 4-anilinoquinazoline B,13 based
on the 4-(phenylamino)quinazoline core structure that have an at-
tached Michael acceptor functional group at the C-6 position.
forms a covalent linkage with the sulfhydryl group of the Cys 773
of EGFR (Scheme 1). These compounds proved to be potent inhib-
itors of tumor growth in a human epidermoid carcinoma xenograft
(A431) model that overexpresses EGFR-TK.13 However, these com-
pounds exhibit poor bioavailability and the induction of apoptosis
by these EGFR-TK inhibitors in solid tumors have not been re-
searched. In continuation of our earlier studies focus on EGFR
inhibitors as antitumor agents,14,15 we now discovery a series of
4,6-substituted-(diaphenylamino)quinazolines as new EGFR-TK
inhibitors. As discussed above, molecular modeling suggests that
a favorable site to attach new phenylamino groups based on the
4-(phenylamino)quinazoline core structure would be at the C-6
position, since these positions point toward the outside of the pro-
tein. These compounds are potent in inhibition of cell growth in
two cancer cell lines (Hep G2 and A16-F10) and with high levels
of EGFR proteins and effectively induces apoptosis in a dose-
dependent manner in the Hep G2 cell line.
2. Results and discussion
2.1. Chemsitry
Other workers have prepared 4-anilinoquinazolines by reacting
substituted anilines with 4-chloroquinazolines.16 We designed a
more efficient synthesis that allowed ring cyclization and incorpo-
ration of the 4-anilino group in a single step, as shown in Scheme 2.
Commercially available 5-nitroanthranilonitrile 1 was converted
⇑
Corresponding authors. Tel./fax: +86 512 65882092 (H.-Q.L.); tel./fax: +86 258
3592572 (H.-L.Z.).
E-mail addresses:
(H.-L. Zhu).
huanqiuli@suda.edu.cn (H.-Q. Li),
zhuhl@nju.edu.cn
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.085

318
H.-Q. Li et al. / Bioorg. Med. Chem. 20 (2012) 317–323
HN
N
Br
HN
N
Br
H
N
H
N
N
N
O
O
B
A
Scheme 1.
O2N
H2N
X
CN
O2N
CN
i
CH3
NH2
N
N
2
1
CH3
ii
HN
N
X
HN
X
O2N
iii
N
N
N
X=Br
3
4
X=Br
X=Cl
5
iv/v
X=Cl
6
N
N
N
H
R1
NH
5a X=Br,R1=2-OH
5b X=Br,R1=4-OH
5c X=Br,R1=2-OCH3
5d X=Br,R1=4-F
6a X=Cl,R1=2-OH
6b X=Cl,R1=4-OH
6c X=Cl,R1=2-OCH3
6d X=Cl,R1=4-F
5e X=Br,R1=4-Cl
5f X=Br,R1=4-Br
5g X=Br,R1=2-Cl
5h X=Br,R1=2-Br
5i X=Br,R1=4-OCH3
6e X=Cl,R1=4-Cl
6f X=Cl,R1=4-Br
6g X=Cl,R1=2-Cl
6h X=Cl,R1=2-Br
6i X=Cl,R1=4-OCH3
Scheme 2. Synthetic routes of 4-(phenylamino)quinazolines. Reagents and conditions: (i) dimethylformamide dimethyl acetal, 70–75 °C; (ii) ArNH2/AcOH, 70–75 °C; (iii) Fe/
AcOH/EtOH/H2O, 70–80 °C; (iv) benzaldehyde/ethanol, 40 °C; (v) sodium borohydride/ethanol, 40–50 °C.
into the corresponding formamidine 2 using DMF acetal. Heating a
solution of formamidine 2 and 3-X-aniline in HOAc gave 6-nitro-4-
(3-X-phenylamino)quinazolines 3 and 4. Reduction of the nitro
group of 3 and 4 with iron in HOAc yielded the intermediate 6-
aminoquinazolines 5 and 6.
characterized by spectroscopic methods and the elemental analysis
together with a crystal structure (5d Fig. 1).
2.2. Antiproliferative activity
Schiff bases of 6-aminoquinazolines were obtained in high
yields using various substituted benzaldehyde and EtOH as the car-
bonyl activator. Then schiff bases of 6-aminoquinazolines were
submitted to the Zn/HCOOH (aq) reductive conditions, leading to
the corresponding, 6-substituted-(diaphenylamino)quinazolines
5a–5i and 6a–6i with moderate to high overall yields (Scheme 2).
All new 4,6-substituted-(diaphenylamino)quinazolines were fully
In former reports, the N1, N3 of the 4-(phenylamino)quinazo-
line core structure would interact with active centre probably by
hydrogen bond, water bridge or else. So we saved the quinazoline
skeleton in the new designed compunds.17 The meta substituent of
the aniline group is oriented toward the kinase pocket. This mode
of binding is in line with the structure–activity relationship ob-
served in the quinazoline inhibition series. This mode of binding

H.-Q. Li et al. / Bioorg. Med. Chem. 20 (2012) 317–323
319
Structure–activity relationships in these new compounds dem-
onstrated that compounds containing 30-X = Br showed better
activity than 30-X = Cl, indicating little tolerance of bulkiness in
the hydrophobic pocket of the ATP site of EGFR of the latter com-
pounds. Compounds with R1 substitution at the para position
(5b, 5d, 5e and 5f) showed better activities than those with substi-
tution at the meta position (5a, 5c, 5g and 5h) based both on cyto-
toxicity and enzyme inhibitory activity, respectively. Hydroxyl
derivatives at R1 position are more potent then corresponding
methoxy derivatives, so the increased lipophilicity of methoxy
derivatives at R1 position may be detrimental to the inhibitory
activity.
2.3. EGFR inhibitory activity and molecular docking
To evaluate the EGFR inhibitory potency of new compounds,
their ability to block EGFR TK was tested in an EGFR TK assay.
We chose ten compounds behaved well for antiproliferation and
their EGFR inhibitory activities were displayed at Table 2. This
could indicate that 4,6-substituted-(diaphenylamino)quinazolines
basically follow the EGFR inhibition path against cancer cell. As
shown in Table 2, compound 5b and 5e displayed the most potent
Figure 1. Crystal structure of compounds 5d.
is in line with the structure–activity relationship observed in the
quinazoline inhibition series. Small substituents are tolerated at
the 30-aniline moiety, preferentially halogens and electron-donat-
ing group at the 6- and/or 7-positions that enhance the binding
of N1 and N3. Thus, in this study, we choose halogen atom (Br
and Cl) at the 30-X-aniline moiety to develop the novel 4,6-substi-
tuted-(diaphenylamino)quinazolines.
The synthesized 4,6-substituted-(diaphenylamino)quinazolines
were evaluated for their antiproliferative activities against Hep G2
and A16-F10 cells by applying the MTT colorimetric assay. The re-
sults were summarized in Table 1. Compounds were tested over a
inhibitory activity (IC50 = 0.87 lM and 1.64 lM for EGFR), less
comparable to the positive control erlotinib (IC50 = 0.03
lM for
EGFR).
To help understand the SARs observed at the EGFR and guide
further SAR studies, molecular docking of the most potent inhibitor
5b into ATP binding site of EGFR kinase was performed on the
binding model based on the EGFR complex structure (1M17.pdb).
The binding model of compound 5b and EGFR was depicted in Fig-
ure 2A and Figure 2B. In the binding model, compound 5b is nicely
bound to the region of EGFR, the quinazoline ring be inserted ni-
cely inside the pocket. The R1 substituted phenyl group in 5b pro-
jects into a hydrophobic region, which is comprised of the side
chains of Pro 717, Leu 768 and Ile 716, that was important for
the potent inhibitory activity of 5b. The modeling also suggested
range of concentrations from 0.01 to 100
lg/ml, and the calculated
IC50 values, that is, the concentration ( g/mL) of a compound that
l
was able to cause 50% cell death with respect to the control culture,
were reported differently according to different cancer cells. As ex-
pected, these compounds containing 30-X = Br or Cl all exhibited re-
marked effects on antiproliferative activities, and generally the
results showed which those applied to Hep G2 cells would
performed better than that in A16-F10 cells, was normal and accept-
able. Especially, 4-((4-(3-bromophenylamino)quinazolin-6-ylami-
no)methyl)phenol (5b), showed the most potent inhibitory
that there is a
p-cation interaction between quinazoline ring of
compound 5b and Lys828,
p-cation interaction energies are of
the same order of magnitude as hydrogen bonds or salt bridges
and play an important role in stabilizing the three dimensional
structure of a protein.18
activity (IC50 = 0.28
lM for Hep G2 and IC50 = 0.59 lM for A16-
F10), and comparable to the positive control erlotinib
(IC50 = 0.12 lM for Hep G2 and IC50 = 0.02 lM for A16-F10).
2.4. Analysis of apoptosis induced by compounds 5b
We evaluated compound 5b for their ability to induce apoptosis
in the Hep G2 cell line using Annexin-V and propium iodide (PI) dou-
ble staining by flow cytometry. The results are shown in Figure 3. As
can be seen, compound 5b is very effective in induction of apoptosis
in a dose-dependent manner. Treatment of the Hep G2 cells by 1 and
Table 1
Structure of 4,6-substituted-(diaphenylamino)quinazolines and the antiproliferative
effects
Compd
X
R1
IC50 (lM)
Hep G2
A16-F10
2 lM of 5b for 3 days results in 20.8% and 54.2% of apoptotic cells
(early + late), as compared to 0.98% of apoptotic cells in an untreated
5a
5b
5c
5d
5e
5f
5g
5h
5j
6a
6b
6c
6d
6e
6f
6g
6h
6j
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2-OH
4-OH
2-OMe
4-F
4-Cl
4-Br
1.26 0.04
0.28 0.15
8.82 1.14
1.77 0.16
0.92 0.16
1.41 0.06
1.82 0.13
2.76 0.07
5.43 0.76
2.08 0.16
1.52 0.07
9.85 2.12
2.69 0.21
1.78 0.21
3.81 0.32
5.33 0.67
4.17 0.48
5.26 0.18
0.12
1.87 0.36
0.59 0.15
12.5 2.2
2.09 0.43
1.82 0.36
2.21 1.05
3.74 0.83
4.38 0.61
10.4 2.7
3.18 0.54
1.58 0.28
14.8 3.1
2.95 0.28
2.67 0.27
4.01 0.14
7.12 0.79
5.83 0.51
8.43 1.26
0.02
Table 2
Inhibition (IC50) of EGFR kinase
2-Cl
2-Br
Compd
EGFR inhibition IC50 (lM)
4-OMe
2-OH
4-OH
2-OMe
4-F
4-Cl
4-Br
2-Cl
2-Br
5a
5b
5c
5e
5f
6a
6b
6c
3.22
0.87
11.22
1.64
3.13
3.54
1.87
13.88
4.41
5.67
0.03
6e
6f
Erlotinib
4-OMe
Erlotinib

320
H.-Q. Li et al. / Bioorg. Med. Chem. 20 (2012) 317–323
4. Experimental
4.1. Chemistry general
All chemicals (reagent grade) used were purchased from Sig-
ma–Aldrich (USA) and Sinopharm Chemical Reagent Co. , Ltd.(Chi-
na).1H NMR spectra were measured on a Bruker AV-300 or AV-500
spectrometer at 25 °C and referenced to Me4Si. Chemical shifts are
reported in ppm (d) using the residual solvent line as internal stan-
dard. Splitting patterns are designed as s, singlet; d, doublet; t, trip-
let; m, multiplet. ESI-MS spectra were recorded on a Mariner
System 5304 Mass spectrometer. Elemental analyses were per-
formed on a CHN–O-Rapid instrument and were within 0.4% of
the theoretical values. Melting points were determined on a XT4
MP apparatus (Taike Corp., Beijing, China) and are as read. Analyt-
ical thin-layer chromatography (TLC) was performed on the glass-
backed silica gel sheets (silica gel 60 Å GF254). All compounds
were detected using UV light (254 or 365 nm).
Figure 2A. Molecular docking modeling of compounds 5b with EGFR kinase: The R1
substituted phenyl group in 5b projects into
a hydrophobic region, which is
comprised of the side chains of Pro 717, Leu 768 and Ile 716, that was important for
4.2. General procedure for the preparation of compounds
the potent inhibitory activity of 5b. The modeling also suggested that there is a
cation interaction between quinazoline ring of compound 5b and Lys828.
p-
4.2.1. General procedure for the preparation of compounds 5
and 6
5-Nitroanthranilonitrile 1 (4.89 g, 30 mmol, 1.0 equiv) was sus-
pended in dimethylformamide dimethyl acetal (10 mL) and the
mixture was refluxed for 2 h. The resulting mixture was cooled
to room temperature for 2–3 h. The yellow precipitate that formed
was filtered, washed with ethyl ether, and dried to give 2 (Yield:
80–90%). A mixture of 2 (2.18 g, 10 mmol, 1.0 equiv) and 3-chloro-
aniline (1.1 equiv) or 3-bromoaniline (1.1 equiv) was heated and
stirred at reflux in acetic acid (20 mL) for 2 h. Aniline and acetic
acid would be added into the reaction flask first and then slowly
added 2 using the medicine spoon within one minute. This treat-
ment is to prevent the formation of lumps between them. The yel-
low precipitate that formed was filtered hot, washed with hot
acetic acid, diethyl ether, and dried to give the desired nitroquinaz-
oline 3 or 4 (Yield: 80–90%).
6-Nitroquinazoline 4 (2.0 g, 6.65 mmol, 1.0 equiv), iron (2.5 g,
45 mmol, 6.7 equiv) and acetic acid (6 mL, 90 mmol, 13.5 equiv)
were suspended in aqueous ethanol (180 mL, 77.8% v/v) and
heated at reflux about 70–80 °C for 5–6 h. Meantime this mixture
would be stirred for one minute every half hour with a glass rod
and the yellow solution would become reddish-brown slowly.
The reaction mixture was cooled to room temperature and alkalin-
ized by addition of concentrated ammonia (40 mL). Insoluble
material was removed by filtration through celite, and the filtrate
was evaporated under reduced pressure. The resulting solid was
extracted with ethyl acetate for column chromatography. Column
chromatography was performed using silica gel (200–300 mesh)
eluting with ethyl acetate and petroleum ether (3:1, v/v) to give
amine 6.
Figure 2B. 3D model of the interaction between compound 5b and the ATP binding
site, which binding energy is equal to ꢀ11.0.
control. This is consistent with its nice binding affinity to EGFR TK
and its potent activity in inhibition of cell growth.
3. Conclusion
In summary, eighteen novel 4,6-substituted-(diaphenylami-
no)quinazolines that may function as inhibitors of EGFR kinases
have been designed and prepared. As expected, these compounds
containing 30-X = Br or Cl all exhibited remarked effects on antipro-
liferative activity and EGFR TK inhibitory activity. Especially,
4-((4-(3-bromophenylamino)quinazolin-6-ylamino)methyl)phe-
4.2.2. General Procedure for synthesis of compounds 5a–5i and
6a–6i
Equimolar amount of amine 5 or 6 (0.5 mmol) and various
substituted benzaldehyde (1.0 mmol) were dissolved in ethanol
(20 mL), and stirred 30–40 °C for 5–6 h. The next step was to add
sodium borohydride (5 mmol) into the reaction mixture and this
solution continued to stirred 40–50 °C for 3–4 h. The reaction mix-
ture was concentrated. The product (5a–5i, 6a–6i) was obtained by
column chromatography.
nol (5b), showed the most potent inhibitory activity (IC50 = 0.28
lM
for Hep G2, IC50 = 0.59 M for A16-F10 and IC50 = 0.87 M for EGFR).
l
l
The EGFR molecular docking model suggested that compound 5b is
nicely bound to the region of EGFR, the quinazoline ring be inserted
nicely inside the pocket, p-cation interaction between N1, N3-qui-
nazoline ring and Lys828 may an important role in stabilizing the
three dimensional structure of a protein. Thus, compound 5b is po-
tent EGFR TK inhibitor and effectively induces apoptosis in a dose-
dependent manner in the Hep G2 cell line as a potential anticancer
agent.
4.2.2.1.
no)methyl)phenol (5a).
DMSO-d6, ppm): 4.38 (d, J = 5.49 Hz, 2H, CH2), 6.40 (t,
2-((4-(3-Bromophenylamino)quinazolin-6-ylami-
Mp 235–236 °C; 1H NMR (300 MHz,
d

H.-Q. Li et al. / Bioorg. Med. Chem. 20 (2012) 317–323
321
Figure 3. Analysis of apoptosis induced by compounds 5b in the Hep G2 liver cancer cell line. Data represent the percentage of apoptotic cells. (a) control, (b) 0. 5
lM , (c)
1.0 lM, (d) 2.0 lM.
J = 5.58 Hz, 1H), 6.76 (t, J = 6.96 Hz, 1H), 6.86 (d, J = 8.07 Hz, 1H),
7.04–7.11 (m, 1H), 7.24–7.40 (m, 5H), 7.55 (d, J = 8.94 Hz, 1H),
7.89 (d, J = 8.04 Hz, 1H), 8.16 (s, 1H, NH), 8.39 (s, 1H), 9.44 (s, 1H,
NHCH2), 9.60 (s, 1H, OH). ESI-MS: 421.1 C21H18BrN4O, [M+H]+).
Anal. Calcd for C21H17BrN4O: C, 59.87%; H, 4.07%; N, 13.30%. Found:
C, 59.53%; H, 4.36%; N, 13.57%.
NHCH2). ESI-MS: 423.1 (C21H17BrN4O, [M+H]+). Anal. Calcd for
21H16BrN4O: C, 59.59%; H, 3.81%; N, 13.24%. Found: C, 59.83%; H,
4.26%; N, 12.87%.
C
4.2.2.5. N6-(4-Chlorobenzyl)-N4-(3-bromophenyl)quinazoline-
4,6-diamine (5e).
DMSO-d6, ppm): 4.46 (d, J = 5.85 Hz, 2H, CH2), 6.78 (t,
Mp 119–121 °C; 1H NMR (300 MHz,
d
4.2.2.2.
methyl)phenol (5b).
4-((4-(3-Bromophenylamino)quinazolin-6-ylamino)
Mp 239–240 °C; 1H NMR (300 MHz,
J = 5.85 Hz, 1H), 7.25–7.37 (m, 4H), 7.41 (d, J = 8.58 Hz, 2H), 7.48
(d, J = 8.61 Hz, 2H), 7.57 (d, J = 8.79 Hz, 1H), 7.88 (d, J = 8.04 Hz,
1H), 8.16 (s, 1H, NH), 8.39 (s, 1H), 9.36 (s, 1H, NHCH2). ESI-MS:
439.0 (C21H17BrClN4, [M+H]+). Anal. Calcd for C21H16BrClN4: C,
57.36%; H, 3.67%; N, 12.74%. Found: C, 59.83%; H, 4.06%; N,
12.57%.
DMSO-d6, d ppm): 4.30 (d, J = 5.31 Hz, 2H, CH2), 6.56 (s, 1H), 6.74
(t, J = 8.4 Hz, 1H), 7.24–7.37 (m, 6H), 7.54 (d, J = 8.97 Hz, 1H),
7.91 (d, J = 8.22 Hz, 1H), 8.18 (s, 1H, NH), 8.38 (s, 1H), 9.30 (s, 1H,
OH), 9.43 (s, 1H, NHCH2). ESI-MS: 421.1 (C21H18BrN4O, [M+H]+).
Anal Calcd for C21H17BrN4O: C, 59.87%; H, 4.07%; N, 13.30%. Found:
C, 59.72%; H, 4.45%; N, 13.11%.
4.2.2.6. N6-(4-Bromobenzyl)-N4-(3-bromophenyl)quinazoline-
4,6-diamine(5f).
DMSO-d6, ppm): 4.44 (d, J = 6.03 Hz, 2H, CH2), 6.78 (t,
Mp 125–127 °C; 1H NMR (300 MHz,
4.2.2.3.
line-4,6-diamine(5c).
N6-(2-Methoxybenzyl)-N4-(3-bromophenyl)quinazo-
Mp 105–107 °C; 1H NMR (300 MHz,
d
J = 5.94 Hz, 1H), 7.24–7.36 (m, 4H), 7.41 (d, J = 8.4 Hz, 2H), 7.53–
7.58 (m, 3H), 7.88 (d, J = 8.07 Hz, 1H), 8.16 (s, 1H, NH), 8.39 (s,
1H), 9.35 (s, 1H, NHCH2). ESI-MS: 482.9 (C21H17Br2N4, [M+H]+).
Anal. Calcd for C21H16Br2N4: C, 52.09; H, 3.33%; N, 11.57%. Found:
C, 52.34%; H, 3.36%; N, 11.27%.
DMSO-d6, d ppm): 3.85 (s, 3H, OCH3), 4.41 (d, J = 5.67 Hz, 2H,
CH2), 6.47 (s, 1H), 6.92 (t, J = 7.40 Hz, 1H), 7.04 (d, J = 8.04 Hz, 1H),
7.24–7.39 (m, 6H), 7.56 (d, J = 8.94 Hz, 1H), 7.84 (d, J = 8.04 Hz,
1H), 8.15 (s, 1H, NH), 8.38 (s, 1H), 9.42 (s, 1H, NHCH2). ESI-MS:
435.1 (C22H20BrN4O, [M+H]+). Anal. Calcd for C22H19BrN4O: C,
60.70%; H, 4.40%; N, 12.87%. Found: C, 60.21%; H, 4.36%; N, 13.07%.
4.2.2.7. N6-(2-Chlorobenzyl)-N4-(3-bromophenyl)quinazoline-
4,6-diamine(5g).
DMSO-d6, ppm): 4.53 (d, J = 5.67 Hz, 2H, CH2), 6.69 (t,
Mp 199–200 °C; 1H NMR (300 MHz,
4.2.2.4. N6-(4-Fluorobenzyl)-N4-(3-bromophenyl)quinazoline-
4,6-diamine(5d).
DMSO-d6, ppm): 4.44 (d, J = 5.85 Hz, 2H, CH2), 6.73 (t,
J = 5.58 Hz, 1H), 7.18 (t, J = 8.88 Hz, 2H), 7.26 (d, J = 8.58 Hz, 1H),
7.34(t, J = 7.95 Hz ,3H), 7.47–7.52 (m, 2H), 7.56(d, J = 8.94 Hz, 1H),
7.89 (d, J = 8.07 Hz, 1H), 8.17 (s, 1H, NH), 8.39 (s, 1H), 9.37 (s, 1H,
d
Mp 122–124 °C; 1H NMR (300 MHz,
J = 5.76 Hz, 1H), 6.76 (t, J = 6.96 Hz, 1H), 7.24–7.40 (m, 6H), 7.48–
7.53 (m, 2H), 7.59 (d, J = 7.71 Hz, 1H), 7.87 (d, J = 8.22 Hz, 1H),
8.16 (s, 1H, NH), 8.40 (s, 1H), 9.41 (s, 1H, NHCH2). ESI-MS: 439.0
(C21H17BrClN4, [M+H]+). Anal. Calcd for C21H16BrClN4: C, 57.36%;
H, 3.67%; N, 12.74%. Found: C, 56.97%; H, 3.36%; N, 13.09%.
d

322
H.-Q. Li et al. / Bioorg. Med. Chem. 20 (2012) 317–323
4.2.2.8. N6-(2-Bromobenzyl)-N4-(3-bromophenyl)quinazoline-
4,6-diamine (5h).
Mp 213–214 °C; 1H NMR (300 MHz,
DMSO-d6, ppm): 4.49 (d, J = 5.49 Hz, 2H, CH2), 6.68 (t,
J = 5.76 Hz, 1H), 7.23–7.40 (m, 6H), 7.50 (d, J = 6.95 Hz, 1H), 7.59
(d, J = 8.97 Hz, 1H), 7.67 (dd, J1 = 7.86 Hz, J2 = 8.04 Hz, 1H), 7.87
(d, J = 8.04 Hz, 1H), 8.16 (s, 1H, NH), 8.39 (s, 1H), 9.44 (s, 1H,
NHCH2). ESI-MS: 482.9 (C21H17Br2N4, [M+H]+). Anal. Calcd for
4.2.2.15. N6-(4-Bromobenzyl)-N4-(3-chlorophenyl)quinazoline-
4,6-diamine (6f).
Mp 126–128 °C; 1H NMR (500 MHz, DMSO-
d
d6, d ppm): 4.41 (d, J = 6 Hz, 2H, CH2), 6.76 (t, J = 5.75 Hz, 1H), 7.07
(dd, J1 = 7.5 Hz, J2 = 8 Hz, 1H), 7.29–7.38 (m, 6H), 7.49 (d, J = 8.2 Hz,
2H), 7.56 (d, J = 9 Hz, 1H), 7.79 (d, J = 8 Hz, 1H), 8.04 (s, 1H, NH),
8.39 (s, 1H), 9.38 (s, 1H, NHCH2). ESI-MS: 439.0 (C21H17BrClN4,
[M+H]+). Anal. Calcd for C21H16BrClN4: C, 57.36; H, 3.67%; N,
12.74%. Found: C, 57.24%; H, 3.39%; N, 13.05%.
C21H16Br2N4: C, 52.09%; H, 3.33%; N, 11.57%. Found: C, 51.89%; H,
3.46%; N, 11.76%.
4.2.2.16. N6-(2-Chlorobenzyl)-N4-(3-chlorophenyl)quinazoline-
4.2.2.9.
line-4,6-diamine (5i).
N6-(4-Methoxybenzyl)-N4-(3-bromophenyl)quinazo-
4,6-diamine (6g).
Mp 195–197 °C; 1H NMR (500 MHz, DMSO-
Mp 115–117 °C; 1H NMR (300 MHz,
d6, d ppm): 4.54 (d, J = 5.5 Hz, 2H), 6.70 (t, J = 5.5 Hz, 2H), 7.12 (dd,
J1 = 6.5 Hz, J2 = 6.5 Hz, 1H), 7.31–7.33 (m, 3H), 7.37–7.40 (m, 2H),
7.48–7.52 (m, 2H), 7.60 (d, J = 9 Hz, 1H), 7.81 (d, J = 8 Hz, 1H),
8.05 (s, 1H, NH), 8.41 (s, 1H), 9.43 (s, 1H, NHCH2). ESI-MS: 394.1
(C21H17Cl2N4, [M+H]+). Anal. Calcd for C21H16Cl2N4: C, 63.81%; H,
4.08%; N, 14.17%. Found: C, 64.07%; H, 3.95%; N, 14.09%.
DMSO-d6, d ppm): 3.37 (s, 3H, OCH3), 4.37 (d, J = 5.49 Hz, 2H,
CH2), 6.64 (t, J = 5.58 Hz, 1H), 6.93 (d, J = 8.61 Hz, 2H), 7.25–7.40
(m, 6H), 7.56 (d, J = 8.97 Hz, 1H), 7.91 (d, J = 8.04 Hz, 1H), 8.18 (s,
1H, NH), 8.39 (s, 1H), 9.39 (s, 1H, NHCH2). ESI-MS: 435.1
(C22H20BrN4O, [M+H]+). Anal. Calcd for C22H19BrN4O: C, 60.70%;
H, 4.40%; N, 12.87%. Found: C, 60.43%; H, 4.34%; N, 13.07%.
4.2.2.17. N6-(2-Bromobenzyl)-N4-(3-chlorophenyl)quinazoline-
4.2.2.10.
methyl)phenol (6a).
2-((4-(3-Chlorophenylamino)quinazolin-6-ylamino)
Mp 232–234 °C; 1H NMR (300 MHz,
4,6-diamine (6h).
Mp 203–205 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.50 (d, J = 5.67 Hz, 2H, CH2), 6.70 (s, 1H), 7.14
(d, J = 7.68 Hz, 1H), 7.25 (t, J = 6.95 Hz, 1H), 7.33–7.43 (m, 4H),
7.59 (d, J = 8.97 Hz, 1H), 7.50 (dd, J1 = 7.86 Hz, J2 = 7.5 Hz, 1H),
7.59 (d, J = 9.15 Hz, 1H), 7.67 (d, J = 7.86 Hz, 1H), 7.80 (d,
J = 8.97 Hz, 1H), 8.04 (s, 1H, NH), 8.41 (s, 1H), 9.46 (s, 1H, NHCH2).
ESI-MS: 439.0 (C21H17BrClN4, [M+H]+). Anal. Calcd for
DMSO-d6, d ppm): 4.38 (d, J = 5.49 Hz, 2H, CH2), 6.41 (t, J = 5.4 Hz,
1H), 6.77 (t, J = 7.41 Hz, 1H), 6.86 (d, J = 8.04 Hz, 1H), 7.07–7.14
(m, 2H), 7.30 (t, J = 6.95 Hz, 2H), 7.40 (t, J = 8.15 Hz, 2H), 7.55 (d,
J = 8.97 Hz, 1H), 7.83 (d, J = 8.22 Hz, 1H), 8.06 (s, 1H, NH), 8.40 (s,
1H), 9.45 (s, 1H, NHCH2), 9.61 (s, 1H, OH). ESI-MS: 377.1
(C21H18ClN4O, [M+H]+). Anal. Calcd for C21H17ClN4O: C, 66.93%; H,
4.55%; N, 14.87%. Found: C, 67.03%; H, 4.36%; N, 14.59%.
C21H16BrClN4: C, 57.36%; H, 3.67%; N, 12.74%. Found: C, 56.99%;
H, 3.42%; N, 13.06%.
4.2.2.11.
methyl)phenol (6b).
4-((4-(3-Chlorophenylamino)quinazolin-6-ylamino)
Mp 228–230 °C; 1H NMR (300 MHz,
4.2.2.18. N6-(4-Methoxybenzyl)-N4-(3-chlorophenyl)quinazo-
line-4,6-diamine (6i).
Mp 103–104 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.31 (d, J = 5.49 Hz, 2H, CH2), 6.56 (s, 1H), 6.75
(d, J = 8.61 Hz, 2H), 7.13 (dd, J1 = 7.86 Hz, J2 = 7.86 Hz, 1H), 7.25–
7.43 (m, 5H), 7.30 (t, J = 6.95 Hz, 2H), 7.40 (t, J = 8.15 Hz, 2H),
7.55 (d, J = 8.97 Hz, 1H), 7.84 (d, J = 8.22 Hz, 1H), 8.07 (s, 1H, NH),
8.39 (s, 1H), 9.32 (s, 1H, OH), 9.40 (s, 1H, NHCH2). ESI-MS: 377.1
(C21H18ClN4O, [M+H]+). Anal. Calcd for C21H17ClN4O: C, 66.93%; H,
4.55%; N, 14.87%. Found: C, 67.14%; H, 4.63%; N, 14.49%.
DMSO-d6, d ppm): 3.73 (s, 3H, OCH3), 4.38 (d, J = 5.49 Hz, 2H,
CH2), 6.64 (t, J = 5.48 Hz, 1H), 6.93 (d, J = 8.61 Hz, 2H), 7.13 (dd,
J1 = 8.07 Hz, J2 = 7.86 Hz, 1H), 7.33–7.43 (m, 5H), 7.56 (d,
J = 8.97 Hz, 1H), 7.85 (d, J = 8.25 Hz, 1H), 8.07 (s, 1H, NH), 8.40 (s,
1H), 9.40 (s, 1H, NHCH2). ESI-MS: 391.1 (C22H20lN4O, [M+H]+).
Anal. Calcd for C22H19ClN4O: C, 67.60%; H, 4.90%; N, 14.33%. Found:
C, 67.13%; H, 5.12%; N, 14.36%.
4.2.2.12. N7-(2-Methoxybenzyl)-N2-(3-chlorophenyl)quinazo-
line-2,7-diamine (6c).
4.3. Cell proliferation assay
Mp 99–101 °C; 1H NMR (500 MHz,
DMSO-d6, d ppm): 3.86 (s, 3H, OCH3), 4.43 (d, J = 6 Hz, 2H, CH2),
6.50 (t, J = 5.75 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.5 Hz,
1H), 7.13 (dd, J1 = 8 Hz, J2 = 8 Hz, 1H), 7.26–7.31 (m, 2H), 7.35–
7.42 (m, 3H), 7.57 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 8.05
(s, 1H, NH), 8.40 (s, 1H), 9.44 (s, 1H, NHCH2). ESI-MS: 391.1
(C22H20ClN4O, [M+H]+). Anal. Calcd for C22H19ClN4O: C, 67.60%; H,
4.90%; N, 14.33%. Found: C, 67.26%; H, 5.24%; N, 14.65%.
The antiproliferative activities of chalcone thiosemicarbazide
derivatives were determined using a standard (MTT)-based colori-
metric assay (Sigma). Briefly, cell lines were seeded at a density of
7 ꢁ 103 cells/well in 96-well microtiter plates (Costar). After 24 h,
exponentially growing cells were exposed to the indicated com-
pounds at final concentrations ranging from 0.1 to 40 mg/mL. After
48 h, cell survival was determined by the addition of an MTT solu-
tion (20 lL of 5 mg/mL MTT in PBS). After 6 h, 100 mL of 10% SDS in
4.2.2.13. N7-(4-Fluorobenzyl)-N2-(3-chlorophenyl)quinazoline-
0.01 N HCl was added, and the plates were incubated at 37 °C for a
further 4 h; optical absorbance was measured at 570 nm on an
LX300 Epson Diagnostic microplate reader. Survival ratios are ex-
pressed in percentages with respect to untreated cells. IC50 values
were determined from replicates of 6 wells from at least two inde-
pendent experiments.
2,7-diamine (6d).
DMSO-d6, ppm): 4.44 (d, J = 5.67 Hz, 2H, CH2), 6.74 (t,
Mp 115–117 °C; 1H NMR (300 MHz,
d
J = 5.85 Hz, 1H), 7.12–7.21 (m, 3H), 7.33–7.43 (m, 3H), 7.47–7.58
(m, 3H), 7.82 (d, J = 8.22 Hz, 1H), 8.06 (s, 1H, NH), 8.40 (s, 1H),
9.39 (s, 1H, NHCH2). ESI-MS: 379.1 (C21H17ClFN4, [M+H]+). Anal.
Calcd for C21H16ClFN4: C, 66.58%; H, 4.26%; N, 14.79%. Found: C,
66.83%; H, 4.62%; N, 14.41%.
4.4. General procedure for preparation, purification of EGFR
inhibitory assay
4.2.2.14. N6-(4-Chlorobenzyl)-N4-(3-chlorophenyl)quinazoline-
4,6-diamine (6e).
Mp 121–123 °C; 1H NMR (300 MHz, DMSO-
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain
(EGFR-CD, amino acids 645-1186) were cloned into baculoviral
expression vectors pBlueBacHis2B and pFASTBacHTc (Huakang
Company China), separately. A sequence that encodes (His)6 was
located at the 50 upstream to the EGFR sequences. Sf-9 cells were
infected for 3 days for protein expression. Sf-9 cell pellets were sol-
ubilized at 0 °C in a buffer at pH 7.4 containing 50 mM HEPES,
d6, d ppm): 4.46 (d, J = 5.85 Hz, 2H, CH2), 6.79 (s, 1H), 7.13 (d,
J = 9.15 Hz, 1H), 7.31–7.49 (m, 6H), 7.57 (d, J = 8.94 Hz, 1H), 7.81
(d, J = 9.15 Hz, 1H), 8.05 (s, 1H, NH), 8.39 (s, 1H), 9.38 (s, 1H,
NHCH2). ESI-MS: 395.0 (C21H17Cl2N4, [M+H]+). Anal. Calcd for
C21H16Cl2N4: C, 63.81%; H, 4.08%; N, 14.17%. Found: C, 64.03%; H,
4.21%; N, 13.84%.

H.-Q. Li et al. / Bioorg. Med. Chem. 20 (2012) 317–323
323
10 mM NaCl, 1% Triton, 10
dium vanadate, 10 g/mL aprotinin, 10
pepstatin, and 16 g/mL benzamidine HCl for 20 min followed by
l
M ammonium molybdate, 100
l
M so-
try. Early apoptotic cells were defined as Annexin-V positive/PI-
negative, late apoptotic cells as Annexin-V/PI-double positive and
necrotic cells as Annexin-V positive/PI positive.
l
l
l
g/mL leupeptin, 10
lg/mL
20 min centrifugation. Crude extract supernatant was passed
through an equilibrated Ni-NTA superflow packed column and
washed with 10 mM and then 100 mM imidazole to remove non-
specifically bound material. Histidinetagged proteins were eluted
with 250 and 500 mM imidazole and dialyzed against 50 mM NaCl,
Acknowledgments
The work was supported by National Natural Science Founda-
tion of China (Grant No.81102316); the Natural Science Foundation
of the Jiangsu Higher Education Institutions of China (Grant
No.11KJD350002) and PAPD (A Project Funded by the Priority Aca-
demic Program Development of Jiangsu Higher Education
Institutions).
20 mM HEPES, 10% glycerol, and 1 lg/mL each of aprotinin, leu-
peptin, and pepstatin for 2 h. The entire purification procedure
was performed at 4 °C or on ice.19
The EGFR kinase assay was set up to assess the level of autophos-
phorylation based on DELFIA/Time-Resolved Fluorometry. Com-
pounds 20–42 were dissolved in 100% DMSO and diluted to the
appropriate concentrations with 25 mM HEPES at pH 7.4. In each
Supplementary data
well, 10
combinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at
room temperature. Then, 10
L of 5 ꢁ buffer (containing 20 mM
HEPES, 2 mM MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20
lL compound was incubated with 10 lL (5 ng for EGFR) re-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.085.
l
l
lL
References and notes
of 0.1 mM ATP-50 mM MgCl2 were added for 1 h. Positive and neg-
ative controls were included in each plate by incubation of enzyme
with or without ATP-MgCl2. At the end of incubation, liquid was
aspirated, and plates were washed three times with wash buffer.
1. Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.; McGuire, W. L.
Science 1987, 235, 177.
2. Slamon, D. J.; Godolphin, W.; Jones, L. A. Science (Wash. DC) 1989, 244, 707.
3. Scheurle, D.; Jahanzeb, M.; Aronsohn, R. S.; Watzek, L.; Narayanan, R. H.
Anticancer Res. 2000, 20, 2091.
4. Cox, G.; Vyberg, M.; Melgaard, B.; Askaa, J.; Oster, A.; O’Byrne, K. J. Int. J. Cancer
2001, 92, 480.
5. Gullick, W. J. Br. Med. Bull. 1991, 47, 87.
6. Moscatello, D. K.; Holgado-Mudruga, M.; Godwin, A. K.; Ramirez, G.; Gunn, G.;
Zoltick, P. W.; Biegel, J. A.; Hayes, R. L.; Wong, A. J. Cancer Res. 1995, 55, 5536.
7. Wikstrand, C. J.; McLendon, R. E.; Friedman, A.; Bigner, D. D. Cancer Res. 1997,
57, 4130.
8. Bridges, A. J. Curr. Med. Chem. 1999, 6, 825.
9. Boschelli, D. H. Drugs Future 1999, 24, 515.
10. Fry, D. W.; Kraker, A. J.; McMichael, A.; Ambroso, L. A.; Nelson, J. M.; Leopold,
W. R.; Connors, R. W.; Bridges, A. J. Science 1994, 265, 1093.
11. Ward, W. H. J.; Cook, P. N.; Slater, A. M.; Davies, D. H.; Holdgate, G. A.; Green, L.
R. Biochem. Pharmacol. 1994, 48, 659.
12. Discafani, C. M.; Carroll, M. L.; Floyd, M. B., Jr.; Hollander, I. J.; Husain, Z.;
Johnson, B. D.; Kitchen, D.; May, M. K.; Malo, M. S.; Minnick, A. A., Jr.;
Nilakantan, R.; Shen, R.; Wang, Y.-F.; Wissner, A.; Greenberger, L. M. Biochem.
Pharmacol. 1999, 57, 917.
A 75 lL (400 ng) sample of europiumlabeled anti-phosphotyrosine
antibody was added to each well for another 1 h of incubation. After
washing, enhancement solution was added and the signal was de-
tected by Victor (Wallac Inc.) with excitation at 340 nm and emis-
sion at 615 nm. The percentage of autophosphorylation inhibition
by the compounds was calculated using the following equation:
100% ꢀ [(negative control)/(positive control ꢀ negative control)].
The IC50 was obtained from curves of percentage inhibition with
eight concentrations of compound. As the contaminants in the en-
zyme preparation are fairly low, the majority of the signal detected
by the anti-phosphotyrosine antibody is from EGFR.
4.5. Molecular docking modeling
13. Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Gries, K. D.; Hicks, J. L.;
Hook, K. E.; Keller, P. R.; Leopold, W. R.; Loo, J. B.; Trumpp-Kallmeyer, S.;
Dobrusin, E. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12022.
14. Lv, P. C.; Li, H. Q.; Sun, J.; Zhou, Y.; Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 4606.
15. Li, H. Q.; Yan, T.; Yang, Y.; Shi, L.; Zhou, C. F.; Zhu, H. L. Bioorg. Med. Chem. 2010,
18, 305.
16. Rewcastle, G. W.; Palmer, B. D.; Bridges, A. J.; Showalter, H. D. H.; Sun, L.;
Nelson, J.; McMichael, A.; Kraker, A. J.; Fry, D. W.; Denny, W. A. J. Med. Chem.
1996, 39, 918.
Molecular docking of compound 5b into the three-dimensional
EGFR complex structure (1M17.pdb, downloaded from the PDB)
was carried out using the AutoDock software package (version
4.0) as implemented through the graphical user interface Auto-
Dock Tool Kit (ADT 1.4.6).
4.6. Analysis of apoptosis induced in cells
17. Rachid, Z.; Brahimi, F.; Katsoulas, A.; Teoh, N.; Jean-Claude, B. J. J. Med. Chem.
2003, 46, 4313.
18. Jeffrey, D. S.; Christopher, G. V. S.; Caldwell, W. S. J. Med. Chem. 1999, 42, 3066.
19. Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y.-F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.
The Hep G2 cells were seeded in 6-well plates at a seeding den-
sity of 105 cells/ml. When all the cells were adhered, various con-
centrations of compound 5b added. Cells were treated with
compounds 5b for 3 days and apoptosis was analyzed using An-
nexin-V and propium iodide (PI) double staining by flow cytome-