Design, synthesis and structure-activity relationships of novel diaryl urea derivatives as potential EGFR inhibitors

Article abstract of DOI:10.3390/molecules21111572

Two novel series of diaryl urea derivatives 5a-i and 13a-l were synthesized and evaluated for their cytotoxicity against H-460, HT-29, A549, and MDA-MB-231 cancer cell lines in vitro. Therein, 4-aminoquinazolinyl-diaryl urea derivatives 5a-i demonstrated significant activity, and seven of them are more active than sorafenib, with IC₅₀ values ranging from 0.089 to 5.46 μM. Especially, compound 5a exhibited the most active potency both in cellular (IC₅₀ = 0.15, 0.089, 0.36, and 0.75 μM, respectively) and enzymatic assay (IC₅₀ = 56 nM against EGFR), representing a promising lead for further optimization.

Full text of DOI:10.3390/molecules21111572

molecules
Article
Design, Synthesis and Structure-Activity
Relationships of Novel Diaryl Urea Derivatives as
Potential EGFR Inhibitors
Nan Jiang 1, Yanxin Bu 1, Yu Wang 1, Minhua Nie 1, Dajun Zhang 2,* and Xin Zhai 1,
*
1
Key Lab. of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China;
jiangnan@syphu.edu.cn (N.J.); yanxinbu@163.com (Y.B.); wangyubobby@163.com (Y.W.);
18604019493@163.com (M.N.)
Department of Chemsitry, Shenyang Medical College, 146 Huanghe North Street, Huanggu District,
Shenyang 110034, China
2
*
Correspondence: zhangdajun2008@126.com (D.Z.); leixinjia@126.com (X.Z.);
Tel./Fax: +86-24-2398-6429 (X.Z.)
Academic Editor: Derek J. McPhee
Received: 19 October 2016; Accepted: 15 November 2016; Published: 18 November 2016
Abstract: Two novel series of diaryl urea derivatives 5a
for their cytotoxicity against H-460, HT-29, A549, and MDA-MB-231 cancer cell lines in vitro. Therein,
4-aminoquinazolinyl-diaryl urea derivatives 5a demonstrated significant activity, and seven of
them are more active than sorafenib, with IC50 values ranging from 0.089 to 5.46 M. Especially,
compound 5a exhibited the most active potency both in cellular (IC50 = 0.15, 0.089, 0.36, and 0.75 M,
–i and 13a–l were synthesized and evaluated
–
i
µ
µ
respectively) and enzymatic assay (IC50 = 56 nM against EGFR), representing a promising lead for
further optimization.
Keywords: diaryl urea; 4-aminoquinazolinyl; synthesis; cytotoxicity; EGFR inhibitors
1. Introduction
Recently, small molecular multiple targeted drugs have played a crucial role in cancer therapy due
to their high efficiency and low toxicity. Diaryl urea derivative sorafenib [
inhibitor targeted Raf and receptor tyrosine kinases (RTKs), has been applied for the treatment of
advanced renal cell carcinoma (RCC) [ ], unresectable hepatocellular carcinoma (HCC) [ ], and
differentiated thyroid carcinoma (DTC) [ ]. It is reported that the lipophilic diaryl urea moiety served
as a key structural fragment for binding with the hydrophobic pocket of the kinase domain through
hydrogen bonds and hydrophobic interactions [ ]. Subsequently, diverse diaryl urea derivatives
were developed successively in the past decades, such as linifanib [ ], tivozanib [ ], and ki8751 [8]
1], the first oral multikinase
2
3
4
5
6
7
(Figure 1). Therein, thieno[3,2-d]pyrimidine derivative S1 bearing diaryl urea moieties at the C-2
positon exhibited significant inhibition of tyrosine kinases, including c-Kit, orphan receptor tyrosine
kinase (RET), and FLT3 for its prominent framework [9]. Similarly, the 4-aminoquinazolinyl skeleton,
which competitively binds to the ATP binding pocket of intracellular kinase domains and blocks the
conduction of downstream signaling networks mediated by tyrosine kinase, is also extensively used in
the design of RTKs inhibitors (e.g., gefitinib, erlotinib and S2) [10,11].
In light of the abovementioned considerations, and as ongoing efforts to identify new
potent antitumor agents, a novel series of 4-aminoquinazolinyl-diaryl urea derivatives 5a–i were
designed according to bioisosterism theory to achieve synergistic antitumor effects (Figure 2).
In addition, a variety of substituents and aliphatic amino were introduced into the terminal
phenyl group and C-4 position on the quinazoline ring to explore the influence of electronic and
Molecules 2016, 21, 1572; doi:10.3390/molecules21111572
www.mdpi.com/journal/molecules

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steric hindrance effect. Furthermore, another novel series of diaryl urea derivatives 13a
–
l
bearing
a 5-pyridyl-4-aminopyrimidinyl motif were designed based on scaffold hopping principle, in the
hope of attaining structural diversity and optimal cellular potency via improving water solubility.
All compounds were synthesized and evaluated for their cytotoxicity against HT-29, H-460, A549, and
MDA-MB-231 cancer cell lines. Additionally, enzymatic assay of the most active compound 5a was
also presented in this study.
Figure 1. Structures of diaryl urea derivatives.
Figure 2. Design of target compounds.
2. Results and Discussion
2.1. Chemistry
The synthesis of target compounds 5a
have been described in detail in our previous work [12], so the synthetic method is not
listed here. [12]. A Suzuki reaction of 3a with commercially available 4-aminophenylboronic
acid pinacol ester under nitrogen protection provided the key intermediates 4a 13]. A series
of substituted aromatic isocyanates and isothiocynates were subsequently prepared by treating the
corresponding arylamine with triphosgene (BTC) or 1,4-diazabicyclo[2.2.2]octane (DABCO) without
further purification [14 15]. Eventually, target compounds 5a were successfully obtained via the
reaction of 4a,b with corresponding 6 or 7 in THF at 30 ◦C, respectively.
–i is depicted in Scheme 1. The synthesis of intermediates
3a–b
,
b
,b [
6
7
,
–i

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◦
Scheme 1. Synthesis of target compounds 5a–i. Reagents and conditions: (a) urea, 160 C, 12 h; (b) POCl3,
◦
◦
DIPEA, 90 C, 6 h; (
c
) NH2(CH2)nR1, THF, TEA, 30 C, 15 min; (
d
) 4-aminophenylboronic acid pinacol
◦
ester, Na2CO3, Pd(PPh3)2Cl2, THF, N2, r.f., 5 h; (
e)
6a or 7a
–d
–b, THF, 30 C, 6 h; (
f
) BTC, 1,4-dioxane,
r.f., 8–12 h; (g) (i) DABCO, CS2, toluene, r.t., 12 h; (ii) BTC, CHCl3, r.f., 1 h.
The synthetic route of target compounds 13a
the mixture of 4-aminobenzonitrile in EtOH and 1,4-dioxane in an ice bath to generate compound
which was further replaced with dry NH3 to afford intermediate 16]. 10 was prepared via reaction of
2-(pyridin-2-yl)acetonitrile and excess N,N-dimethylformamide dimethyl acetal (DMF-DMA) at 30 C
for 24 h [17]. Condensation reaction of and 10 in MeOH-H2O solution in the presence of Na2CO3
gave rise to intermediate 11 18]. Subsequently, 11 condensed with corresponding aromatic isocyanate
6c in a similar manner as described for compounds 5a to furnish 12a . Finally, 12a were treated
with 20%–30% hydrochloride ethanol solution to provide target compounds 13a–l.
–l is outlined in Scheme 2. Dry HCl was bubbled into
8,
9
[
◦
9
[
–
n
–
i
–
l
–l
Scheme 2. Synthesis of target comp◦ounds 5a
–
i
. Reagents and conditions: (
a
) HCl, EtOH-1,4-dioxane,
◦
◦
0 C, 6 h, r.t., 48 h; (
b
) NH3, EtOH, 0 C, 6 h, r.t., 24 h; (
c) DMF-DMA, MeOH, 30 C, 24 h; (
d) Na2CO3,
◦
◦
MeOH-H2O, 70 C, 24 h; (e) 6c–n, THF, 30 C, 6 h; and (f) HCl-EtOH, CHCl3, 1 h.

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2.2. Biological Results and Discussion
For summarizing structure-activity relationships (SARs), all target compounds (5a–i and
13a–l) were evaluated for their cytotoxicity against HT-29 (human colon cancer), H-460 (human
lung cancer), A549 (human lung cancer), and MDA-MB-231 (human breast cancer) cell lines by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as the
positive control. The results expressed as IC50 values (µM) are presented in Tables 1 and 2.
Table 1. Structures and cytotoxicity of compounds 5a
–i
against HT-29, H-460, A-549, and MDA-MB-231
cells in vitro.
IC50 (µM) a
R2
R1
Compd.
n
X
HT-29
H-460
A-549
MDA-MB-231
5a
5b
5c
5d
5e
5f
O
O
O
O
O
S
2
2
3
3
3
2
2
3
3
2-Cl, 6-CH3
3,4-(CH3)2
2-Cl, 6-CH3
3,4-(CH3)2
4-F
0.15 ± 0.14
1.09 ± 0.21
0.36 ± 0.72
1.72 ± 0.37
2.64 ± 0.53
0.24 ± 0.17
1.05 ± 0.012
0.65 ± 0.33
0.089 ± 0.11 0.36 ± 0.27
0.75 ± 0.65
2.42 ± 0.28
1.08 ± 0.15
3.92 ± 1.24
2.72 ± 0.42
2.59 ± 0.36
3.02 ± 0.51
3.13 ± 0.85
1.50 ± 0.13
0.27 ± 0.12
2.93 ± 0.62
1.19 ± 0.96
0.77 ± 0.22
1.28 ± 0.68
1.02 ± 0.74
2.33 ± 1.52
0.82 ± 0.43
5.46 ± 0.38
3.41 ± 1.27
2.30 ± 0.18
2.81 ± 0.73
1.44 ± 0.39
4-Cl
5g
5h
S
4-OCF3
4-Cl
S
5i
S
4-OCF3
2.41 ± 0.21
3.27 ± 0.32
2.58 ± 0.74
2.15 ± 0.43
4.86 ± 0.56
4.47± 0.28
4.07 ± 1.02
3.81± 0.50
Sorafenib
a
Results are expressed as means ± SD (standard deviation) of three independent experiments.
Table 2. Structures and cytotoxicity of compounds 13a–l against HT-29, H-460, A-549, and
MDA-MB-231 cells in vitro.
IC50 (µM) a
R1
Compd.
HT-29
H-460
A549
MDA-MB-231
13a
13b
13c
13d
13e
13f
13g
13h
H
2,4-diCH3
2-Cl, 6-CH3
2,6-diCH3
2,6-diF
3-Cl, 4-F
3-Cl
30.52 ± 2.56
45.08 ± 1.37
12.95 ± 0.68
18.37 ± 0.45
35.10 ± 2.81
6.33 ± 0.93
10.07 ± 0.32
ND
28.64 ± 1.08
44.83 ± 2.42
32.16 ± 1.59
NA
NA
NA
24.66 ± 0.80
24.62 ± 2.75
ND
15.28 ± 1.19
5.86 ± 1.34
ND
30.71 ± 1.73
15.94 ± 0.95
NA
10.49 ± 2.26
15.43 ± 1.02
ND
25.42 ± 1.14
28.12 ± 2.44
20.94 ± 1.63
12.75 ± 2.97
21.95 ± 2.58
ND
3-CF3
13i
13j
13k
13l
4-OCF3
4-F
3-F
26.58 ± 1.27
17.95 ± 0.94
30.50 ± 3.16
24.18 ± 1.35
3.27 ± 0.32
23.46 ± 2.29
32.28 ± 2.87
ND
16.54 ± 0.70
30.65 ± 3.06
ND
NA
NA
NA
4.47 ± 0.28
NA
2-F
32.29 ± 2.63
35.56 ± 2.85
Sorafenib
2.15 ± 0.43
3.81 ± 0.50
a
Results are expressed as means
showing IC50 value > 50 µM. ND: Not determined.
±
SD (standard deviation) of three independent experiments. NA: compound
As shown in Table 1, compounds 5a
lines with IC50 values ranging from 0.089 to 5.46
–
i
demonstrated excellent activity towards four tested cell
M, and seven of them were more active against
µ
tested cell lines than the positive control sorafenib. It was noticeable that all compounds exhibited
prominent cytotoxicity against HT-29 superior to sorafenib, reflecting good selectivity for colon cancer.
In general, antitumor potency was related to the amino group R1 on side chain, and dimethylamino
was more effective than morpholinyl (5a vs. 5c
, 5b vs. 5d, 5g vs. 5i), which might be due to its smaller
bulk and stronger hydrophilia. Additionally, the substituent R2 on the terminal phenyl ring exerted

Molecules 2016, 21, 1572
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a major influence on pharmacological activity. Electrophilic groups are beneficial to the improvement
of antitumor potency, as might be the reason of compounds 5a 5c 5f, and 5h with the Cl group
,
,
showing eminent cytotoxicity. Conversely, a certain decrease in activity was observed once 3,4-(CH3)2
were introduced, such as compounds 5b and 5d. Interestingly, 2-CH3 and 6-Cl analogue 5a showed
best activity, with IC50 values of 0.15, 0.089, 0.36, and 0.75 µM, which were five- to 24-fold more potent
than sorafenib respectively. The results indicated that steric hindrance in ortho-position was preferred.
To further verify the optimized structural skeleton and explore the SARs, pharmacological data
of compounds 13a–l bearing 4-aminopyrimidinyl moiety are illustrated in Table 2. Unexpectedly,
the results were unsatisfied although multiple hydrophilic modifications were performed by
introducing pyridine group and further salifying with hydrochloride. The contrast of antitumor
potency between 4-aminoquinazoline and 4-aminopyrimidine derivatives suggested that further
structural modification might have broken the binding affinity to intracellular kinase domains or
switched the binding affinity to other targets, while the 4-aminoquinazolinyl moiety was an excellent
skeleton for EGFR inhibitors. The activity toward EGFR kinase of compound 5a was surprised with
the IC50 value of 56 nM, indicating that 4-aminoquinazolinyl-diaryl urea derivatives 5a–i were potent
EGFR inhibitors (Table 3). Efforts to identify its mechanisms of action and further optimization are
ongoing and will be reported in due course.
Table 3. EGFR and VEGFR2/KDR kinases inhibitory activity of compound 5a in vitro.
IC50 (nM)
Compd.
VEGFR2/KDR
EGFR
5a
>3000
93
56
-
Sorafenib
3. Experimental Section
3.1. Chemistry
Melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in electrospray ionization
(ESI) mode on an Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear magnetic
resonance spectroscopy were performed using a Bruker ARX-300, 300 or 400 MHz spectrometers
(Bruker Bioscience, Billerica, MA, USA) with tetramethylsilane (TMS) as an internal standard.
Thin-layer chromatography (TLC) analysis was carried out on silica gel plates GF254 (Qingdao
Haiyang Chemical, Qingdao, China). Unless otherwise noted, all common reagents and solvents were
used as obtained from commercial suppliers without further purification.
3.2. General Procedure for Preparation of 2-(4-Aminophenyl)-4-aminoquinazolines (4a–b)
Dioxane (100 mL) was added to a solution of sodium carbonate (3.2 g, 0.03 mol) in water (25 mL).
Under argon, 2-chloro-4-aminoquinazoline 3 (0.01 mol), 4-aminophenylboronic acid pinacol ester (2.4 g,
0.011 mol) and Pd(PPh3)2Cl2 (0.7 g, 1 mmol) were added to the mixture successively. After refluxing for
6 h, water (50 mL) was added to the reaction mixture. The mixture was extracted by dichloromethane
(50 mL
×
3). The organic phase was washed by brine (50 mL
×
1), dried, and evaporated to yield 4a–b.
N1-(2-(4-aminophenyl)quinazolin-4-yl)-N2,N2-dimethylethane-1,2-diamine (4a). Yield: 73.5%;
MS (ESI) m/z: 308.2 [M + H+]. 2-(4-Aminophenyl)-N-(3-morpholinopropyl)quinazolin-4-amine (4b).
Yield: 78.1%; MS (ESI) m/z: 364.5 [M + H+].
3.3. General Procedure for Preparation of Componds 5a–i
A mixture of intermediate
4 (1 mmmol) and corresponding aromatic isocyanate 6 or isothiocyanate 7
(1.1 mmol) in dry THF (15 mL) was stirred at 30 ◦C for 6 h and monitored by TLC. The precipitate was

Molecules 2016, 21, 1572
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collected by filtration, washed with ether, and purified by silica gel chromatography (MeOH:CH2Cl2 = 20:1)
to afford target compounds 5a–i.
1-(2-Chloro-6-methylphenyl)-3-(4-(4-((2-(dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)urea (5a). Yield:
◦
62%; m.p.: 144.5–147.0 C; MS (ESI) m/z: 474.9 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 9.08
(s, 1H), 8.2.4 (d, J = 8.8 Hz, 2H ), 8.12 (t, J = 5.2, 4.8 Hz, H), 8.04 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.56 (m,
2H), 7.45 (d, J = 8.4 Hz, 2H), 7.29 (m, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.05 (m, 1H)
,
3.17 (q, 2H), 2.47 (t, J = 8.0 Hz, 2H), 2.38 (s, 3H), 2.21 (s, 6H). Anal. Calcd for C26H27ClN6O (%): C,
65.74; H, 5.73; N, 17.69; Found (%): C, 65.71; H, 5.76; N, 17.68.
1-(4-(4-((2-(Dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)-3-(3,4-dimethylphenyl)urea (5b). Yield: 68%;
m.p.: 132.0–134.0 ◦C; MS (ESI) m/z: 455.2 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 10.04
(s, 1H), 8.39 (t, J = 7.6 Hz, 1H), 8.24 (d, J = 7.2 Hz, 3H), 8.10 (d, J = 8.4 Hz, 1H), 7.55 (m, 3H), 7.46 (d,
J = 8.4 Hz, 2H), 7.28 (t, J = 7.2 Hz, 1H), 6.89 (d, J = 7.2 Hz, 1H), 6.60 (d, J = 7.2 Hz, 1H), 3.17 (q, 2H), 2.47
(t, J = 8.0 Hz, 2H), 2.23 (s, 3H), 2.22 (s, 3H), 2.19 (s, 6H). Anal. Calcd for C27H30N6O (%): C, 71.34; H,
6.65; N, 18.49; Found (%): C, 71.36; H, 6.62; N, 18.47.
1-(2-Chloro-6-methylphenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)urea (5c). Yield:
◦
70%; m.p.: 152.5–154.5 C; MS (ESI) m/z: 531.1 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 9.20
(s, 1H), 8.38 (d, J = 8.4 Hz, 2H ), 8.28 (t, J = 4.8, 5.2 Hz, H), 8.18 (d, J = 8.4 Hz, 1H), 8.07 (s, 1H), 7.71
(m, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.42 (m, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H), 7.19
(m, 1H), 3.70 (m, 2H), 3.57 (t, J = 8.4 Hz, 4H), 2.40 (m, 6H), 2.27 (s, 3H), 1.87(m, 2H). Anal. Calcd for
C29H31ClN6O2 (%): C, 65.59; H, 5.88; N, 15.83; Found (%): C, 65.61; H, 5.85; N, 15.88.
1-(3,4-Dimethylphenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)urea (5d). Yield: 56%;
m.p.: 140.7–143.0 ◦C; MS (ESI) m/z: 510.9 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 10.18
(s, 1H), 8.54 (t, J = 7.2 Hz, 1H), 8.37 (d, J = 7.2 Hz, 3H), 8.23 (d, J = 8.0 Hz, 1H), 7.69 (m, 3H), 7.59 (d,
J = 8.0 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.73 (d, J = 7.2 Hz, 1H), 3.70 (m, 2H),
3.57 (t, J = 7.2 Hz, 4H), 2.42 (t, J = 7.2 Hz, 2H), 2.37 (t, J = 7.2 Hz, 4H), 2.24 (s, 3H), 2.23 (s, 3H), 1.86 (m,
2H). Anal. Calcd for C30H34N6O2 (%): C, 70.56; H, 6.71; N, 16.46; Found (%): C, 70.55; H, 6.76; N, 16.42.
1-(4-Fluorophenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)urea (5e). Yield: 52%; m.p.:
137.0–139.5 ◦C; MS (ESI) m/z: 501.2 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 9.91 (s, 2H),
8.51 (t, J = 5.2 Hz, 1H), 8.39 (d, J = 8.8 Hz, 2H), 8.30 (d, J = 8.4 Hz, 1H), 7.72 (m, 4H), 7.60 (d, J = 8.8 Hz
,
2H), 7.43 (m, 1H), 7.36 (d, J = 8.4 Hz, 2H), 4.15 (t, J = 6.8 Hz, 2H ), 3.72 (t, J = 6.4 Hz, 4H), 2.43 (t,
J = 6.8 Hz, 2H), 2.38 (t, J = 6.4 Hz, 4H), 1.86 (m, 2H). Anal. Calcd for C28H29FN6O2 (%): C, 67.18; H,
5.84; N, 16.79; Found (%): C, 67.15; H, 5.88; N, 16.75.
1-(4-Chlorophenyl)-3-(4-(4-((2-(dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)thiourea (5f). Yield: 65%;
m.p.: 131.5–134.0 ◦C; MS (ESI) m/z: 477.1 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 10.75 (s,
2H), 8.31 (d, J = 8.4 Hz, 3H), 8.07 (d, J = 8.1 Hz, 1H), 7.68–7.59 (m, 4H), 7.45 (d, J = 8.4 Hz, 2H), 7.37–7.26
(m, 3H), 3.86–3.77 (q, 2H), 2.73 (t, J = 6.4 Hz, 2H), 2.33 (s, 6H). Anal. Calcd for C25H25ClN6S (%): C,
62.95; H, 5.28; N, 17.62; Found (%): C, 62.94; H, 5.27; N, 17.64.
1-(4-(4-((2-(Dimethylamino)ethyl◦)amino)quinazolin-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)thiourea (5g).
1
Yield: 60%; m.p.: 150.8–153.0 C; MS (ESI) m/z: 527.2 [M + H+]; H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 10.12 (s, 1H), 10.03 (s, 1H), 8.25 (d, J = 8.4 Hz, 2H), 8.18 (t, J = 6.8 Hz, 1H), 8.05 (d, J = 8.0 Hz,
1H), 7.58 (m, 3H), 7.47 (d, J = 8.8 Hz, 2H), 7.31 (m, 3H), 6.96 (d, J = 8.4 Hz, 1H), 3.38 (q, 2H), 2.56 (t,
J = 7.2 Hz, 2H), 2.23 (s, 6H). Anal. Calcd for C26H25F3N6OS (%): C, 59.30; H, 4.79; N, 15.96; Found (%):
C, 59.32; H, 4.76; N, 15.94.
1-(4-Chlorophenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)thiourea (5h). Yield: 63%;
m.p.: 145.5–148.0 ◦C; MS (ESI) m/z: 533.8 [M + H+]; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 10.90 (s,
2H), 8.42 (d, J = 8.8 Hz, 3H), 8.24 (d, J = 8.4 Hz, 1H), 7.73 (m, 4H), 7.65 (d, J = 8.4 Hz, 2H), 7.44 (m, 1H),
7.38 (d, J = 8.8 Hz, 2H), 3.71 (m, 2H), 3.58 (t, J = 8.4 Hz, 4H), 2.43 (t, J = 7.2 Hz, 2H), 2.38 (t, J = 8.4 Hz,

Molecules 2016, 21, 1572
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4H), 1.88 (m, 2H). Anal. Calcd for C28H29ClN6OS (%): C, 63.09; H, 5.48; N, 15.76; Found (%): C, 63.05;
H, 5.47; N, 15.79.
1-(4-(4-((3-Morpholinopropyl)amino)quinazolin-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)thiourea
(
5i).
Yield: 59%; m.p.: 164.5–166.5 C; MS (ESI) m/z: 583.9 [M + H+]; H-NMR (400 MHz, DMSO-d6)
(ppm): 10.25 (s, 1H), 10.16 (s, 1H), 8.45 (d, J = 8.8 Hz, 2H), 8.35 (t, J = 7.2 Hz, 1H), 8.22 (d, J = 8.4 Hz
◦
1
δ
,
1H), 7.74 (m, 3H), 7.64 (d, J = 8.8 Hz, 2H), 7.48 (m, 3H), 7.11 (d, J = 8.0 Hz, 1H), 3.73 (m, 2H), 3.59
(t, J = 4.4 Hz, 4H), 2.44 (t, J = 7.2 Hz, 2H), 2.39 (t, J = 4.4 Hz, 4H), 1.89 (m, 2H). Anal. Calcd for
C29H29F3N6O2S (%): C, 59.78; H, 5.02; N, 14.42; Found (%): C, 59.75; H, 5.01; N, 14.47.
3.4. General Procedure for Preparation of Aromatic Isocyanates (6a–n)
Appropriate aromatic amine (0.02 mol) was added slowly to a stirred solution of BTC (3.0 g,
0.01 mol) in 1,4-dioxane (30 mL) at room temperature. After refluxing for 8-12 h, excess 1,4-dioxane
was evaporated. The residue was distilled under reduced pressure to afford compounds 6a–n.
1-Chloro-4-isocyanatobenzene (6a). Purity: 78.2%; b.p.: 110–114 ◦C (30–40 mmHg).
4-Isocyanato-1,2-dimethylbenzene (6b). Purity: 80.5%; b.p.: 59–65 ◦C (30–40 mmHg).
1-Chloro-2-isocyanato-3-methylbenzene (6c). Purity: 80.8%; b.p.: 116–118 ◦C (30–40 mmHg).
1-Fluoro-4-isocyanatobenzene (6d). Purity: 84.2%; b.p.: 94–95 ◦C (30–40 mmHg).
1-Fluoro-3-isocyanatobenzene (6e). Purity: 83.6%; b.p.: 85–91 ◦C (30–40 mmHg).
1-Fluoro-2-isocyanatobenzene (6f). Purity: 84.3%; b.p.: 76–80 ◦C (30–40 mmHg).
1-Chloro-3-isocyanatobenzene (6g). Purity: 80.1%; b.p.: 125–128 ◦C (30–40 mmHg).
2-Chloro-1-fluoro-4-isocyanatobenzene (6h). Purity: 85.2%; b.p.: 93–98 ◦C (30–40 mmHg).
1,3-Difluoro-2-isocyanatobenzene (6i). Purity: 80.9%; b.p.: 67–71 ◦C (30–40 mmHg).
2-Isocyanato-1,3-dimethylbenzene (6j). Purity: 76.9%; b.p.: 67–71 ◦C (30–40 mmHg).
1-Isocyanato-2,4-dimethylbenzene (6k). Purity: 77.5%; b.p.: 71–77 ◦C (30–40 mmHg).
1-Isocyanato-3-(trifluoromethyl)benzene (6l). Purity: 83.2%; b.p.: 105–111 ◦C (30–40 mmHg).
1-Isocyanato-4-(trifluoromethoxy)benzene (6m). Purity: 81.0%; b.p.: 113–116 ◦C (30–40 mmHg).
Isocyanatobenzene (6n). Purity: 80.2%; b.p.: 45–50 ◦C (30–40 mmHg).
3.5. General Procedure for Preparation of Aromatic Isothiocyanates (7a,b)
Appropriate aromatic amine (0.02 mol) and DABCO (6.7 g, 0.06 mol) were dissolved in toluene
(25 mL) at room temperature. Under stirring, CS2 (4.6 g, 0.06 mol) was added dropwise to the solution
within 20 min. After stirring at room temperature for 12 h, the precipitate was collected by filtration
and washed with toluene. The residue was dried and suspended in CHCl3 (25 mL), and BTC (19.6 g,
0.066 mol) in CHCl3 (25 mL) was added dropwise to the mixture slowly in an ice bath. Then the
mixture was stirred for 1 h at room temperature and refluxed for 1 h. The mixture was filtered and
evaporated to give compounds 7a,b.
1-Chloro-4-isothiocyanatobenzene (7a). Purity: 77.5%.
1-Isothiocyanato-4-(trifluoromethoxy)benzene (7b). Purity: 79.8%.
3.6. Ethyl 4-aminobenzimidate Dihydrochloride (8)
Dry hydrogen chloride was bubbled into the mixture of 4-aminobenzonitrile (15 g, 0.127 mol),
1,4-dioxane (100 mL) and anhydrous ethanol (100 mL) for 6 h in an ice bath. Then the mixture was

Molecules 2016, 21, 1572
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sealed and stirred at room temperature for 48 h. After completion, the solution was concentrated under
reduced pressure and the semisolid residue was diluted with anhydrous ether (200 mL). The suspension
was filtered, washed with anhydrous ether and dried to give the title compound
(29.3 g, 97.7%). MS (ESI) m/z: 164.9 [M + H+].
8 as a white solid
3.7. 4-Aminobenzimidamide Hydrochloride (9)
Dry ammonia was bubbled into the mixture of compound 8 (29.3 g, 0.123 mol) and anhydrous
ethanol (200 mL) for 6 h in an ice bath. Then the mixture was sealed and stirred at room temperature
for 24 h. Then the solution was concentrated under a reduced pressure and the semisolid residue was
diluted with anhydrous ether (200 mL). The suspension was filtered, washed with anhydrous ether,
and dried to give the title compound 9 as a white solid (20.2 g, 95.3%). MS (ESI) m/z: 135.9 [M + H+].
3.8. 3-(Dimethylamino)-2-(pyridin-2-yl)acrylonitrile (10)
A mixture of 2-(pyridin-2-yl)acetonitrile (15.0 g, 0.127 mol), DMF-DMA (30 mL, 0.229 mol) and
methanol (36 mL) was stirred at 30 ◦C for 24 h. The solvent was evaporated, moderate methanol and
ethyl acetate was added in. The solution was concentrated under a reduced pressure to remove excess
DMF-DMA completely. The residue was poured into petroleum ether (100 mL) and stirred for 6 h.
The suspension was separated by filtration and washed with ether to give compound 10 as a dark red
solid (18.3 g, 83.2%). MS (ESI) m/z: 174.0 [M + H+].
3.9. 2-(4-Aminophenyl)-5-(pyridin-2-yl)pyrimidin-4-amine (11)
A mixture of compound
9 (20.2 g, 0.118 mol ), compound 10 (17.0 g, 0.098 mol), sodium carbonate
(12.5 g, 0.0118 mol), methnol (200 mL) and water (60 mL) was heated to 70 ◦C for 24 h. The solution
was concentrated and added to water (200 mL). The precipitate was collected by filtration, washed
with ether, and recrystallized with ethnol to afford compound 11 as a pale white solid (13.9 g, 53.8%).
MS (ESI) m/z: 264.1 [M + H+].
3.10. General Procedure for Preparation of Compounds 12a–l and 13a–l
A mixture of intermediate 11 (1 mmmol) and corresponding aromatic isocyanate
dry THF (15 mL) was stirred at 30 ◦C for 6 h and monitored by TLC. The precipitate was collected
by filtration, washed with ether, and dried to afford compounds 12a without further purification.
6 (1.1 mmol) in
–
l
At room temperature, 20%–30% hydrochloride ethanol solution (1 mL) was added dropwise to a stirred
solution of compound 12 dissolved in CHCl3 (10 mL). The suspension was stirred for 1 h, filtered and
dried to give the corresponding compounds 13a–l.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-phenylurea dihydrochloride (13a). Yield: 79%; m.p.:
303.0–306.0 ◦C; MS (ESI) m/z: 383.1 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 9.41 (s, 1H),
9.11 (s, 1H), 8.93 (s, 2H), 8.77 (s, 1H), 8.68 (d, J = 4.4 Hz, 1H), 8.32 (d, J = 8.4 Hz, 2H), 8.10 (d, J = 8.0 Hz
,
1H), 7.94 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.4 Hz), 7.38–7.32 (m, 1H), 7.30 (t,
J = 7.7 Hz, 2H), 7.00 (t, J = 6.9 Hz). Anal. Calcd for C22H20Cl2N6O (%): C, 58.03; H, 4.43; N, 18.46;
Found (%): C, 58.07; H, 4.46; N, 18.42.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2,4-dimethylphenyl)urea dihydrochloride (13b).
◦
1
Yield: 65%; m.p.: 286.0–288.5 C; MS (ESI) m/z: 411.3 [M + H+]; H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 9.77 (s, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.6 Hz, 1H), 8.25(d, J = 8.6 Hz, 2H), 8.22 (d, J = 7.8 Hz,
1H), 8.05 (t, J = 7.9 Hz, 1H), 7.72(d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.52 (t, J = 4.8 Hz, 1H), 7.02
(s, 1H), 6.98 (d, J = 8.2 Hz, 2H), 2.24 (s, 6H). Anal. Calcd for C24H24Cl2N6O (%): C, 59.63; H, 5.00; N,
17.39; Found (%): C, 59.60; H, 5.06; N, 17.34.
1-(4-(4-Amino-5-(pyridin-2-yl)py◦rimidin-2-yl)phenyl)-3-(6-chloro-2-methylphenyl)urea dihydrochloride (13c).
1
Yield: 72%; m.p.: 252.0–254.0 C; MS (ESI) m/z: 431.0 [M + H+]; H-NMR (300 MHz, DMSO-d6)
δ

Molecules 2016, 21, 1572
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(ppm): 9.78 (s, 1H), 8.85 (s, 1H), 8.74 (d, J = 4.2 Hz, 1H), 8.56 (s, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.22 (d,
J = 8.1 Hz, 1H), 8.04 (t, J = 6.7 Hz, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.54–7.50 (m, 1H), 7.36 (d, J = 5.4 Hz,
1H), 7.27–7.18 (m, 2H), 2.28 (s, 3H). Anal. Calcd for C23H21Cl3N6O (%): C, 54.83; H, 4.20; N, 16.68;
Found (%): C, 54.84; H, 4.23; N, 16.65.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2,6-dimethylphenyl)urea dihydrochloride (13d).
◦
1
Yield: 76%; m.p.: 271.0–274.0 C; MS (ESI) m/z: 411.2 [M + H+]; H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 9.83 (s, 1H), 8.85 (s, 1H), 8.74(d, J = 4.2 Hz, 1H), 8.34 (s, 1H), 8.23 (d, J = 9.3 Hz, 2H), 8.21 (d,
J = 8.1 Hz, 1H), 8.05(t, J = 7.8 Hz, 1H), 7.72 (d, J = 9.0 Hz, 2H), 7.54–7.50 (m, 1H), 7.08 (s, 3H), 2.23 (s, 6H).
Anal. Calcd for C24H24Cl2N6O (%): C, 59.63; H, 5.00; N, 17.39; Found (%): C, 59.67; H, 5.02; N, 17.36.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2,6-difluorophenyl)urea dihydrochloride (13e). Yield:
◦
68%; m.p.: 279.5–281.0 C; MS (ESI) m/z: 419.1 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 9.78
(s, 1H), 8.88 (s, 1H), 8.73 (d, J = 4.7 Hz, 1H), 8.53 (s, 1H), 8.40 (s, 2H), 8.24 (d, J = 8.8 Hz, 2H), 8.20 (d,
J = 9.3 Hz, 1H), 8.04 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.54–7.49 (m, 1H), 7.32–7.25 (m, 3H).
Anal. Calcd for C22H18Cl2F2N6O (%): C, 53.78; H, 3.69; N, 17.10; Found (%): C, 53.75; H, 3.72; N, 17.08.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(3-chloro-4-fluorophenyl)urea dihydrochloride (13f).
◦
1
Yield: 80%; m.p.: 289.5–291.5 C; MS (ESI) m/z: 435.0 [M + H+]; H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 9.69 (s, 1H), 9.55 (s, 1H), 8.88 (s, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.21 (d,
J = 8.2 Hz, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.85–7.82 (m, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.54–7.49 (m, 1H),
7.40–7.34 (m, 2H). Anal. Calcd for C22H18Cl3FN6O (%): C, 52.04; H, 3.57; N, 16.55; Found (%): C, 52.05;
H, 3.52; N, 16.56.
1-(4-(4-Amino-5-(pyridi◦n-2-yl)pyrimidin-2-yl)phenyl)-3-(3-chlorophenyl)urea dihydrochloride (13g). Yield:
71%; m.p.: 270.0–272.5 C; MS (ESI) m/z: 417.0 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 9.89
(s, 1H), 9.71 (s, 1H), 8.86 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.25 (d, J = 8.7 Hz, 2H), 8.21 (d, J = 8.2 Hz, 1H),
8.05 (t, J = 7.9 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.72 (s, 1H), 7.55–7.51 (m, 1H), 7.33–7.30 (m, 2H), 7.04 (d,
J = 5.8 Hz, 1H). Anal. Calcd for C22H19Cl3N6O (%): C, 53.95; H, 3.91; N, 17.16; Found (%): C, 53.94; H,
3.92; N, 17.14.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea dihydrochloride (13h).
◦
1
Yield: 65%; m.p.: 267.0–269.5 C; MS (ESI) m/z: 451.1 [M + H+]; H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 9.78 (s, 1H), 9.74 (s, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 8.21 (m,
2H), 8.05 (s, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.61–7.50 (m, 4H), 7.35 (d, J = 7.6 Hz, 1H). Anal. Calcd for
C23H19Cl2F3N6O (%): C, 52.79; H, 3.66; N, 16.06; Found (%): C, 52.75; H, 3.69; N, 16.05.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea dihydrochloride
◦
13i). Yield: 78%; m.p.: 308.5–310.5 C; MS (ESI) m/z: 467.1 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ
(
(ppm): 9.50 (s, 1H), 9.35 (s, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.24 (d, J = 8.7 Hz, 2H), 8.19 (d,
J = 8.4 Hz, 1H), 8.04 (t, J = 6.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.59 (d, 2H), 7.60–7.51 (m, 1H), 7.32 (d,
J = 8.1 Hz, 2H). Anal. Calcd for C23H19Cl2F3N6O2 (%): C, 51.22; H, 3.55; N, 15.58; Found (%): C, 51.24;
H, 3.59; N, 15.54.
1-(4-(4-Amino-5-(pyridi◦n-2-yl)pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea dihydrochloride (13j). Yield:
75%; m.p.: 264.0–267.0 C; MS (ESI) m/z: 401.0 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 9.80
(s, 1H), 9.47 (s, 1H), 8.87 (s, 1H), 8.75 (d, J = 4.6 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.22 (d, J = 7.8 Hz,
1H), 8.07 (t, J = 7.9 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.56–7.52 (m, 2H), 7.49 (t, J = 4.8 Hz, 1H), 7.15 (t,
J = 8.8 Hz, 2H). Anal. Calcd for C22H19Cl2FN6O (%): C, 55.82; H, 4.05; N, 17.76; Found (%): C, 55.84; H,
4.01; N, 17.79.
1-(4-(4-Amino-5-(pyridi◦n-2-yl)pyrimidin-2-yl)phenyl)-3-(3-fluorophenyl)urea dihydrochloride (13k). Yield:
67%; m.p.: 228.5–231.0 C; MS (ESI) m/z: 401.1 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 9.89
(s, 1H), 9.73 (s, 1H), 8.86 (s, 1H), 8.74 (d, J = 4.5 Hz), 8.26 (d, J = 8.9 Hz, 2H), 8.22 (d, J = 8.4 Hz, 1H), 8.05
(t, J = 7.8 Hz, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.55 (s, 1H), 7.53–7.50 (m, 1H), 7.37–7.29 (m, 1H), 7.14 (d,

Molecules 2016, 21, 1572
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J = 8.1 Hz, 1H), 6.82 (t, J = 8.4 Hz, 1H). Anal. Calcd for C22H19Cl2FN6O (%): C, 55.82; H, 4.05; N, 17.76;
Found (%): C, 55.81; H, 4.06; N, 17.72.
1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2-fluorophenyl)urea dihydrochloride (13l). Yield:
73%; m.p.: 258.5–261.5 ◦C; MS (ESI) m/z: 401.0 [M + H+]; 1H-NMR (300 MHz, DMSO-d6)
δ (ppm):
10.01 (s, 1H), 8.94 (s, 1H), 8.87 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.22 (d, J = 8.1 Hz,
1H), 8.16–8.03 (m, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.55–7.50 (m, 1H), 7.29–7.03 (m, 3H). Anal. Calcd for
C22H19Cl2FN6O (%): C, 55.82; H, 4.05; N, 17.76; Found (%): C, 55.86; H, 4.03; N, 17.75.
3.11. Evaluation of the Biological Activity
The antitumor activity of compounds 5a–i and 13a–l was evaluated with HT-29, H-460, A549, and
MDA-MB-231 by the MTT method in vitro, with sorafenib as positive control. The cancer cells were
cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS).
Approximately 4
×
103 cells, suspended in MEM medium, were plated onto each well of a 96-well
◦
plate and incubated in 5% CO2 at 37 C for 24 h. The test compounds were added to the culture
medium at the indicated final concentrations and the cell cultures were continued for 72 h. Fresh MTT
was added to each well at a final concentration of 5
The formazan crystals were dissolved in 100 L DMSO per each well, and the absorbency at 492 nm
µ
g/mL and incubated with cells at 37 ◦C for 4 h.
µ
(for the absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured
with the ELISA reader. All of the compounds were tested twice in each of the cell lines. The results
expressed as IC50 (inhibitory concentration of 50%) were the averages of two determinations and were
calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
3.12. EGFR and VEGFR2/KDR Kinases Assay In Vitro
The target compound 5a was tested for its activity against EGFR and VEGFR2/KDR kinases
through the mobility shift assay. All kinase assays were performed in 96-well plates in a 50 µL reaction
volume. The kinase buffer contains 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM
DTT. The stop buffer contains 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, and
50 mM ethylene diamine tetraacetic acid (EDTA). Compounds were diluted to 500
then 10 L of the compounds were transfered to a new 96-well plate as the intermediate plate, and
90 L kinase buffer was added to each well. Five microliters of each well of the intermediate plate was
µM by 100% DMSO,
µ
µ
transferred to a 384-well plate. The following amounts of enzyme and substrate were used per well:
kinase base buffer, FAM-labeled peptide, ATP, and enzyme solution. Wells containing the substrate,
enzyme, and DMSO without compound were used as the DMSO control. Wells containing just the
substrate without enzyme were used as the low control. The compounds were incubated at room
temperature for 10 min. Ten microliters of peptide solution was added to each well and incubated at
◦
28 C for a specified period of time and the reaction stopped by 25
µL of stop buffer. Finally, data was
collected using the Caliper program, which converted conversion values to inhibition values.
Percent inhibition = (max − conversion)/(max − min) × 100,
(1)
where “max” stands for DMSO control; “min” stands for low control.
4. Conclusions
Taken as a whole, two novel series of diaryl urea derivatives were synthesized and evaluated
for their cytotoxicity against four human cancer cell lines (H-460, HT-29, A549, and MDA-MB-231).
Our strategy that 4-aminoquinazolinyl moiety was incorporated into the diaryl urea scaffold conveyed
cellular potency and resulted in analogues 5a
–i demonstrating excellent activity in the single-digit
µM range, superior to sorafenib. The preliminary SARs showed that electrophilic groups on terminal
phenyl ring and steric hindrance in ortho-position were beneficial to improved antitumor potency.
The exploration of detailed SARs led to the identification of EGFR inhibitor 5a as a valuable lead,

Molecules 2016, 21, 1572
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which possessed prominent activity both in cellular (IC50 = 0.15, 0.089, 0.36, and 0.75
µM, respectively)
and enzymatic assay (IC50 = 56 nM against EGFR). Further optimization and investigation based on
these structures are in progress.
Acknowledgments: This work was supported by National Natural Science Foundation of China (21002065),
Project of Education Department of Liaoning (L2013382), Development Project of Ministry of Education Innovation
Team (IRT1073) and Science and Technology Program of Shenyang (No. F15-139-9-02).
Author Contributions: N.J. and D.Z. conceived and designed the experiments; Y.B. and M.N. performed the
experiments; Y.W. analyzed the data; X.Z. contributed reagents/materials/analysis tools; N.J. wrote the paper.
Conflicts of Interest: The authors declare no conflict interest.
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Sample Availability: Samples of the compounds are available from the authors.
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