C-2 (E)-4-(Styryl)aniline substituted diphenylpyrimidine derivatives (Sty-DPPYs) as specific kinase inhibitors targeting clinical resistance related EGFRT790M mutant

Article abstract of DOI:10.1016/j.bmc.2017.03.032

With the aim to overcome the drug resistance induced by the EGFR T790M mutation (EGFR^T790M), herein, a family of diphenylpyrimidine derivatives (Sty-DPPYs) bearing a C-2 (E)-4-(styryl)aniline functionality were designed and synthesized as potential EGFR^T790M inhibitors. Among them, the compound 10e displayed strong potency against the EGFR^T790M enzyme, with the IC₅₀ of 11.0?nM. Compound 10e also showed a higher SI value (SI?=?49.0) than rociletinib (SI?=?21.4), indicating its less side effect. In addition, compound 10e could effectively inhibit the proliferation of H1975 cells harboring the EGFR^T790M mutation, within the concentration of 2.91?μM. Significantly, compound 10e has low toxicity against the normal HBE cell (IC₅₀?=?22.48?μM). This work provided new insights into the discovery of potent and selective inhibitor against EGFR^T790M over wild-type (EGFR^WT).

Full text of DOI:10.1016/j.bmc.2017.03.032

Accepted Manuscript
C-2 (E)-4-(Styryl)aniline Substituted Diphenylpyrimidine Derivatives (Sty-
DPPYs) as Specific Kinase Inhibitors Targeting Clinical Resistance related
EGFRT790M Mutant
Anran Song, Jianbin Zhang, Yang Ge, Changyuan Wang, Qiang Meng, Zeyao
Tang, Jinyong Peng, Kexin Liu, Yanxia Li, Xiaodong Ma
PII:
S0968-0896(17)30335-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.032
BMC 13630
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 February 2017
14 March 2017
16 March 2017
Please cite this article as: Song, A., Zhang, J., Ge, Y., Wang, C., Meng, Q., Tang, Z., Peng, J., Liu, K., Li, Y., Ma,
X., C-2 (E)-4-(Styryl)aniline Substituted Diphenylpyrimidine Derivatives (Sty-DPPYs) as Specific Kinase
Inhibitors Targeting Clinical Resistance related EGFRT790M Mutant, Bioorganic & Medicinal Chemistry (2017), doi:
http://dx.doi.org/10.1016/j.bmc.2017.03.032
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Bioorganic & Medicinal Chemistry
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C-2 (E)-4-(Styryl)aniline Substituted Diphenylpyrimidine Derivatives (Sty-DPPYs)
as Specific Kinase Inhibitors Targeting Clinical Resistance related EGFRT790M Mutant
Anran Songa,1, Jianbin Zhanga,1, Yang Gea, Changyuan Wanga, Qiang Menga, Zeyao Tanga, Jinyong Penga, Kexin
Liua, Yanxia Lib and Xiaodong Maa, ∗
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
b Department of Respiratory Medicine, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China.
1These authors contributed equally to this work.
∗ Corresponding author.
Email address: xiaodong.ma@139.com (X. Ma).
ARTICLE INFO
ABSTRACT
Article history:
Received
With the aim to overcome the drug resistance induced by the EGFR T790M
mutation (EGFRT790M), herein, a family of diphenylpyrimidine derivatives
(Sty-DPPYs) bearing a C-2 (E)-4-(styryl)aniline functionality were designed
and synthesized as potential EGFRT790M inhibitors. Among them, the
compound 10e displayed strong potency against the EGFRT790M enzyme, with
the IC50 of 11.0 nM. Compound 10e also showed a higher SI value (SI = 49.0)
than rociletinib (SI = 21.4), indicating its less side effect. In addition,
compound 10e could effectively inhibit the proliferation of H1975 cells
harboring the EGFRT790M mutation, within the concentration of 2.91 µM.
Significantly, compound 10e has low toxicity against the normal HBE cell
(IC50 = 22.48 µM). This work provided new insights into the discovery of
potent and selective inhibitor against EGFRT790M over wild-type (EGFRWT).
Received in revised form
Accepted
Available online
Keywords:
NSCLC
Resistance
EGFRT790M
Inhibitor
Diphenylpyrimidine
1. Introduction
Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, with the 5-
year survival rate of less than 5%.1-3 The standard treatment for advanced NSCLC is a combination of
platinum compounds and taxanes, however, the treatment is associated with severe adverse effects, low
selectivity and high toxicity.4-6 In recent decades, the application of target-specific agents to treat patients
with lung cancer has shown impressive therapeutic effects in clinical trials, especially the agents targeting
to the activating mutations of epidermal growth factor receptor (EGFR). EGFR, existing on the cell surface,
is a member of the ErbB family of receptors. It plays an important role in many signaling processes, that
regulate numerous cellular functions of cell growth, differentiation, and angiogenesis.7,8 The mutations of
EGFR, which lead to its overexpression or overactivity, are associated with various cancers.9

EGFR mutations account for approximately 10-15% of NSCLC cases in North America and Europe, and
30-40% of NSCLCs in Asian countries.10 Gefitinib (1, Fig.1)11,12 and erlotinib (2, Fig.1)13, the first
generation of EGFR tyrosine kinase inhibitors (TKIs), have shown good response rates in NSCLC patients
with activating EGFRDel 19 or EGFRL858R mutations. Compared with platinum-based chemotherapy, they
conferred excellent radiographic overall response rates of 50-75%, improved progression-free survival by
8-14 months and improved the quality of life.14 Unfortunately, the clinical efficacy of gefitinib and erlotinib
is diminished because patients have developed resistance within 10-16 months.15 In particular,
approximately 60% of the patients who have developed resistance are associated with a single second-site
EGFR kinase domain mutation at the gatekeeper position (Thr790 to Met790, T790M).16,17 The second-
generation EGFR-TKIs (such as afatinib, 3, Fig.1) are developed to overcome the acquired resistance after
the failure of the first generation of EGFR-TKIs. Despite the promising cellular potency against the
EGFRT790M mutation, afatinib exhibits insufficient efficacy in patients at clinically achievable
concentrations. The non-selectivity against wild-type EGFR (EGFRWT) and mutant EGFR could lead
serious epithelium-based toxicities such as rash and diarrhea, which have limited the clinical application of
afatinib.18 Hence, there is an urgent need for EGFR-TKIs that can selectively identify and inhibit the
EGFRT790M mutation, while also leaving the wild-type form of EGFR.
N
O
N
O
N
O
N
N
N
O
O
O
O
NH
Cl
NH
NH
NH
O
O
F
N
F
O
Cl
N
2
Erlotinib
1
Gefitinib
3
Afatinib
O
N
N
N
N
N
O
O
O
N
HN
N
HN
HN
O
O
O
N
NH
HN
F3C
Rociletinib
N
NH
N
HN
Cl
O
N
N
N
5
4
WZ4002
6
Osimertinib
Figure 1. Chemical structures of the novel EGFR inhibitors
Recently, the third-generation EGFR-TKIs including WZ-4002 (4)19, rociletinib (CO-1686, 5)20 and
osimertinib (AZD9291, 6)21,22, have been developed with high potency against T790M-containing mutants
and selectivity over the wild-type EGFR (Fig.1). They have demonstrated a high and sustained response in
patients with advanced EGFR-mutant NSCLC who have experienced treatment failure with first- and
second-generation EGFR-TKIs. Osimertinib was approved by the United States Food and Drug

Administration agency in late 2015, and rociletinib is undergoing phase III clinical trial evaluation.
Generally, a common pyrimidine structure core, which could interact with the hinge residue Met793 by
forming a bidentate hydrogen bonding interaction, is essential for the third-generation EGFR inhibitors to
maintain the high anti-EGFRT790M activity. Moreover, a Michael addition receptor moiety, acrylamide, is
also necessary to form a covalent binding with the conserved cysteine residue present in the lip of the
EGFR ATP binding site (Cys797).19
The stilbene group is a novel biological functionality, contained in several molecules, such as resveratrol
(7)23, diethylstilbestrol (8)24, and pterostilbene (9)25 (Fig.2). The typical agent, resveratrol, has been widely
reported to possess bioactivities of anti-inflammatory, anticancer, antiviral, antioxidant and neuroprotective
bioactive effects. As anticancer agents, resveratrol and its analogues inhibit the activity of tyrosinase and
induce the apoptosis and autophagy of NSCLC cells.23 Remarkably, resveratrol has been reported to
overcome the acquired resistance of NSCLC cells when treated in combination with gefitinib. Accordingly,
we hypothesized that EGFR-TKIs combined with the trans-stilbene structure might have a synergistic
effect on inhibiting the EGFR mutation. In this study, a series of pyrimidine derivatives bearing a trans-
stilbene group were designed and synthesized to discover more effective EGFRT790M inhibitors (Fig.2). In
addition, their anti-EGFRT790M activities were also evaluated by in vitro kinase enzymatic assay and cellular
activity assay.
O
N
O
Covalent bond
Cys797
N
HN
R
O
O
HN
N
NH
HN
N
F3C
Rociletinib
EGFRT790M Inhibitor, Phase Ш
HN
R1
N
X
OH
R
N
Met790
3
R2
R1
Sty-DAPYs 10a-h
R
4
X = NH, O
5
R = Me, Cl, MeO, etc
R1 = Cl, F
7 Resveratrol R = H, R1 = 3,5-Di(OH)2, R2 = H
8 Diethylstilbestrol R = Ethyl, R1 = 4-OH, R2 = H
9 Pterostilbene R = H, R1 = 3,5-Di(MeO)2, R2 = OH
Figure 2. Designed strategy of the Sty-DPPY derivatives.
2. Results and discussion
2.1. Chemistry
The designed compounds 10a-h were prepared following the general strategy outlined in Scheme 1.26,27
Briefly, a family of benzyl bromide derivatives 11a-d were reacted with the nucleophilic reagent triethyl
phosphate to generate intermediates 12a-d, which were then reacted with p-nitrobenzaldehyde following

the Wittig-Horner reaction to produce the (E)-4-nitrostilbene analogues 13a-d. Subsequently, the nitro
substituent in compounds 13a-d was reduced by Fe-NH4Cl to form the (E)-4-aminostilbene intermediates
14a-d. Additionally, 3-aminophenol and m-phenylenediamine materials 15a-b were reacted with acryloyl
chloride to prepare the 3-(N-acrylamide)amines 16a-b. After regioselectively coupling with the 4-chloro
group of the 2,4-dichloropyrimidine core, the amino-substituted compounds 16a-b were conveniently
converted to the pyrimidines 17a-d. Finally, the desired aminopyrimidines derivatives containing the (E)-
4-nitrostilbene group 10a-h were synthesized when coupled by flexible construction containing the (E)-4-
aminostilbene group with the pyrimidine framework 17a-d.
R
c
R
R
R
CHO
O2N
R
a
O
b
P
O
Br
O
O
HN
R = 3,5-DiMe2
,
3,5-Di(MeO)2
,
NO2
NH2
2,4-DiMe2 ,
11a-d
3,5-DiCl2
f
14a-d
13a-d
12a-d
X
N
NH
H
N
O
N
X
R1
Cl
X
R1
N
Cl
N
d
R = 3,5-DiMe2
,
3,5-Di(MeO)2
,
O
X
N
Cl
2,4-DiMe2
R1 = Cl, F
, 3,5-DiCl2
e
NH2
N
H
N
R1
X = O or NH
R1 = Cl, F
10a-h
X = OH or NH2
15a-b
X = O or NH
X = OH or NH2
16a-b
17a-d
Scheme 1. Synthetic route of the title compounds 10a-h. Reagents and conditions: (a) P(OEt)3, n-Bu4NBr, 150 °C, reflux, 6 h,
82-91%; (b) p-nitrobenzaldehyde, CH3ONa, DMF, 0 °C to rt., overnight, 68-88%; (c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 78-
92%; (d) acryloyl chloride, NaHCO3, CH3CN, rt., 0.5 h, 93-95%; (e) DIPEA, 1,4-dioxane, rt., 2 h, 88-91%; (f)
trifluoroacetic acid, 2-BuOH, 100 °C, 12 h, 30.5–68.5%.
2.2. Biological activity
2.2.1. Kinase inhibitory activity
All the Sty-DPPY derivatives were evaluated for their activities against the wild-type EGFR and the
T790M/L858R-mutated EGFR applying with the ADP-GloTM kinase assay system.28,29 Two novel agents,
gefitinib and rociletinib, were also tested as positive controls. The results of this evaluation, shown in
Table 1, obviously revealed that Syt-DPPYs have strong inhibitory activity against the mutant
EGFRT790M/L858R, with IC50 values in the range of 6.8 to 26.5 nM. In contrast, this class of inhibitors
displayed the potency to inhibit EGFRT790M/L858R activity 21 to 57 times higher than gefitinib. Among them,
four typical compounds 10e (IC50 = 11.0 nM), 10f (IC50 = 6.8 nM), 10g (IC50 = 8.1 nM), 10h (IC50 = 11.3
nM), still exhibited stronger activity than rociletinib (IC50 = 21.5 nM). Structure and activity relationship
(SAR) analysis showed that substituents installed on the C-2 (E)-4-(styryl)aniline slightly affect their anti-

EGFRT790M/L858R activity. Generally, the 2,4-disubtituted compound 10f was superior than 10d (26.5 nM)
with 3,5-disubstituents. In addition, Sty-DPPYs exhibited moderate activity against the wild-type EGFR
with the IC50 values ranging from 51.3 to 1037 nM. It was evident that compounds 10f and 10h had the
least potency against the wild-type EGFR, with IC50 values of 996.1 nM and 1037 nM, respectively. In
particular, inhibitor 10f, which had a SI value of 146.5, displayed higher selectivity than rociletinib.
Moreover, compounds 10e (SI = 49.0), 10g (SI = 47.7), and 10h (SI = 91.8) also had improved selectivity,
indicating their less side effects.
Table 1
In vitro enzymatic inhibitory activity of compounds 10a-h against EGFRWT and EGFR790M/L858R
.
EGFR IC50 nM) a
（
Compd.
R
R1
X
SI
(WT:T790M/L858R)
WT
T790M/L858R
3,5-DiMe2 Cl
3,5-DiCl2
O
N
N
N
N
N
N
N
87.0
22.4
14.2
15.7
26.5
11.0
6.8
3. 9
10a
F
388.1
216.1
606.5
539.2
996.1
386.7
1037
10.6
27.3
13.8
22. 9
49.0
146.5
47.7
91.8
0.009
21.4
10b
3,5-diMeO2 Cl
3,5-DiMe2 Cl
10c
10d
3,5-DiMe2
2,4-DiMe2 Cl
2,4-DiMe2
3,5-DiCl2 Cl
F
10e
10f
F
8.1
10g
11.3
1202
21.5
10h
_
_
_
gefitinib
rociletinib
_
460.2
a EGFR activity assays were performed using the ADP-Glo™ Kinase Assay according to the manufacturer’s instructions. The compounds were
incubated with the kinase reaction mixture for 1h before measurement. The data were means from at least three independent experiments.
2.2.2. Cellular antiproliferative activities
Table 2
Antiproliferative activity of compounds 10a-h against cells harboring a different status of EGFR.
Cellular antiproliferative activity (IC50, µM) a
Compd.
A431
4.71
1.29
1.78
5.56
10.55
3.16
3.11
H1975
13.73
6.98
A549
10.75
7.01
HCC827
11.66
10.79
3.32
HBE
6.07
10a
10b
10c
10d
10e
10f
5.42
3.76
6.84
9.18
2.32
5.59
4.00
17.01
22.48
15.83
10.44
2.91
12.81
4.94
2.07
4.39
9.71
10g
7.16
1.32
>20

10h
6.56
6.12
1.79
6.98
15.83
36.21
3.24
9.16
35.2
13.13
0.0099
17.64
gefitinib
rociletinib
0.137
0.031
23.80
a The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data were
means from at least three independent experiments.
100
80
60
40
20
0
Control
1
2
3
Figure 3. Compound 10e induced H1975 cell apoptosis in vitro. The cells were incubated with the indicated
concentrations of 10e for 72 h, and the cells were stained with annexin V/FTIC, followed by flow cytometry analysis. One
representative experiment is shown.
The antiproliferative activities of these newly synthesized compounds were also investigated by the MTT
assay in four NSCLC cell lines (A431, H1975, A549 and HCC827), and one normal lung cell line (HBE).
A431 cells have the wild-type EGFR, while H1975, A549 and HCC827 cells harbor different EGFR
mutations (H1975 harboring EGFRL858R/T790M mutation, A549 harboring EGFRK-ras mutation, and HCC827
harboring EGFRdel E746-A750 mutation). The two novel agents, gefitinib and rociletinib, were studied as
references. As seen in Table 2, Sty-DPPYs are able to inhibit H975 cells within micromolar concentrations,
with IC50 values in the range of 2.32 µM to 13.73 µM. Among them, compounds 10d and 10e were the
most potent inhibitors against H1975 cells, with IC50 values of 2.32 µM, 2.91 µM, respectively. While for
the activity against the wild-type A431 cells, compounds 10d and 10e have IC50 values of 5.56 µM and
10.55 µM, revealing that both 10d and 10e have weak selectivity against resistant H1975 cells over wild-
type A431 cells. Notably, inhibitor 10e also displayed the strongest potency for inhibiting the replication of
HCC827 cells among this class of inhibitors, with an IC50 value of 2.07 µM. Interestingly, the 2,4-
dimethyl-substituted analogue 10g could interfere with the K-ras mutated A549 cells at a low concentration

of 1.32 µM. The activity against the normal HBE cells showed that most of Sty-DPPYs have weak
inhibitory capability, indicating that they will produce low cytotoxicity. In particular, the most active
inhibitor 10e still has the lowest activity for inhibiting HBE cells (IC50 = 22.48 µM), exhibiting excellent
biological property for the further development. Additionally, the effects of inhibitor 10e on apoptosis in
the H1975 cell line were also explored using flow cytometry analysis. The inhibitory effects of molecule
10e were clearly increased in a concentration dependent manner, with an apoptosis rate ranging from 40.4
to 79.8%, as shown in Figure 3.
2.3. Molecular modelling analysis
A
B
C
Met790
Met790
Val726
Val726
Leu718
Leu718
Leu792
Leu792
Thr854
Met793
Pro794
Thr854
Met793
Pro794
Asn842
Asn842
Phe795
Phe795
Arg841
Arg841
Cys797
Asp800
Cys797
Asp800
Leu799
Leu799
Figure 4. A) Conformation alignment of WZ4002 and inhibitor 10e; B) binding mode of WZ4002 within EGFRT790M
enzyme (PDB code: 3IKA); C) putative binding mode of 10e within EGFRT790M
.
Additionally, the most active inhibitor 10e was docked into the ATP binding pocket of EGFRT790M
(PDB: 3IKA)19 to investigate the putative interaction mechanism with the EGFR target. The software
AutoDock 4.2 was used with its default parameters.30-31 For comparison, the x-ray crystal conformation of
the lead compound WZ4002 was reserved and analyzed as well. As shown in Figure 4, both the typical Sty-
DPPY 10e and WZ4002 could tightly contact with EGFR kinase in an identical manner, including: (1)
covalent bond forces produce by the acryl amide group with the amino acid Cys797; (2) strong contacts
come from the chlorine atom at the C-5 position of the pyrimidine core with the mutant gatekeeper residue
Met790; (3) hydrogen-bond forces generated from the N-1 nitrogen atom of the pyrimidine core with amino
acid Met793; (4) polar forces formed by the piperazine ring with the outside of the ATP-binding pocket of
EGFRT790M. For inhibitor 10e, the phenyl ring in the C-2 (E)-4-(styryl)aniline moiety formed additional
strong π-π contacts with the amino acid Phe795, which might improve the inhiibitory activity against
theEGFRT790M activity. Overall, this docking model was consistent with the biological activity data.
3. Conclusion
Based on the pyrimidine template, a series of C-2 (E)-4-(styryl)aniline substituted Sty-DPPYs were
synthesized and evaluated as potent EGFRT790M mutant inhibitors. In particular, compound 10e displayed

strong anti-EGFRT790M activity (IC50 =11.0 nM), while its effect on the wild-type EGFR (IC50 = 49.0 nM)
was significantly less potent. Apparently, compound 10e showed higher selectivity and stronger potency
than the novel agent rociletinib. Moreover, inhibitor 10e could interfere with the proliferation of H1975
cells at a concentration of 2.91 µM, and had little effect on the normal HBE cells at this concentration.
Flow cytometry analysis demonstrated that 10e could significantly induce the apoptosis (52.9%) of H1975
cell at a concentration of 4 µM. Overall, the results obtained with compound 10e suggested that it might be
used as a lead compound for further development as a selective EGFR inhibitor to overcome resistance
effects conferred by the EGFR mutations.
4. Experimental section
4.1. General Methods and Chemistry.
All commercial chemicals and solvents were analytical grade and were used without further purification.
High resolution ESI-MS were performed on an AB Sciex TripleTOF® 4600 LC/MS/MS system. 1H NMR
and 13C NMR spectra on a Brucker AV 400 MHz spectrometer were recorded in [d]DMSO. Coupling
constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm)
units relative to internal control (TMS). All reactions were monitored by TLC, using silica gel plates with
fluorescence F254 and UV light visualization. Flash chromatography separations were obtained on Silica
Gel (300–400 mesh) using dichloromethane/methanol as eluents.
4.2. General procedure for the synthesis of 10a–j.26,27
Compounds 14a-d were synthesized according to our previously reported procedure. While compounds
17a-d were prepared using the reported mehthod32,33. All these intermediates were used without any
purification. With these intermediates in hand, the newly obtained compounds were synthesized as
described below. A flask was charged with compounds 14a-d (0.70 mmol), 17a-d (0.70 mmol), TFA (0.08
mL, 1.05 mmol), and 2-BuOH (10 mL).The slurry was heated to 100 °C for 5 h. The reaction mixture was
allowed to cool to room temperature and was neutralized with a saturated aqueous sodium bicarbonate
solution. The aqueous mixture was then extracted with CH2Cl2 (20 mL) three times. The crude product was
purified using flash chromatography with dichloromethane/methanol (v/v, 30:1) as eluents.
N-[3-[[5-Chloro-2-[(E)-4-(3,5-dimethylstyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10a)
Yield 30.5%; deep yellow solid; 1H NMR (DMSO-d6): δ 2.28(s, 6H), 5.76-5.79 (d, J = 12 Hz, 1H), 6.26-
6.30 (d, J = 16 Hz, 1H), 6.43-6.50 (m, 1H), 6.87-7.08 (m, 4H), 7.16 (s, 2H), 7.25-7.51 (m, 5H), 7.65-7.69
(m, 2H), 8.50 (s, 1H), 9.90 (s, 1H), 10.46 (s, 1H); 13C NMR (DMSO-d6): δ 21.43 (2C), 105.27, 113.49,
117.29, 117.53, 119.27, 124.53 (2C), 126.85, 126.91 (2C), 127.88, 128.17, 129.34, 130.54, 131.11, 132.15,

137.63, 138.05 (2C), 139.68, 140.90, 152.79, 157.84, 158.63, 163.90 (2C), 164.38; HRMS (ESI+) for
C29H25ClN4O2, [M+H]+ calcd: 497.1739, found: 497.1749.
N-[3-[[5-Fluoro-2-[(E)-4-(3,5-dichlorostyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10b)
Yield 44.6%; light gray solid; 1H NMR (DMSO-d6): δ 5.73-5.76 (d, J =12 Hz, 1H), 6.24-6.29 (d, J = 20 Hz,
1H), 6.46-6.53 (m, 1H), 7.03-7.07 (d, J = 16 Hz, 1H), 7.33-7.37 (m, 2H), 7.44-7.52 (m, 5H), 7.60-7.63 (d, J
= 12 Hz, 4H), 7.99 (s, 1H), 8.23-8.24 (d, J = 4 Hz, 1H), 9.90 (s, 1H), 10.19 (s, 1H), 10.29 (s, 1H); 13C NMR
(DMSO-d6): δ 114.84, 116.56, 118.88, 120.39 (2C), 124.16, 125.10 (2C), 126.69, 127.51, 127.77 (2C),
129.33, 131.11, 131.94, 132.38, 134.95 (2C), 138.32, 139.21, 139.78, 141.68, 141.77, 151.74, 151.86 (d, J
= 46.8 Hz, 1C), 153.07, 163.74; HRMS (ESI+) for C27H20Cl2FN5O, [M+H]+ calcd: 520.1102, found:
520.1138.
N-[3-[[5-Chloro-2-[(E)-4-(3,5-dimethoxystyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10c)
Yield 61.3%; gray white solid; 1H NMR (DMSO-d6): δ 3.78 (s, 6H), 5.72-5.75 (m, 1H), 6.25-6.53 (m, 3H),
6.72-6.73 (d, J = 4 Hz, 2H), 6.94-6.98 (d, J = 16 Hz, 1H), 7.11-7.15 (d, J = 16 Hz, 1H), 7.28-7.38 (m, 4H),
7.59-7.64 (m, 3H), 7.93 (s, 1H), 8.20 (s, 1H), 9.19 (s, 1H), 9.68 (s, 1H), 10.33 (s, 1H); 13C NMR (DMSO-
d6): δ 55.70 (2C), 100.03, 104.58 (2C), 116.06, 116.38, 119.48 (2C), 119.97, 126.56, 127.19 (2C), 127.39,
129.17, 129.23, 130.64, 132.43, 139.03, 139.67, 139.93, 140.18, 153.72, 153.74, 156.94, 157.03, 161.15
(2C), 163.71; HRMS (ESI+) for C29H26ClN5O3, [M+H]+ calcd: 528.1797, found: 528.1870.
N-[3-[[5-Chloro-2-[(E)-4-(3,5-dimethylstyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10d)
Yield 68.5%; light gray solid; 1H NMR (DMSO-d6): δ 2.28 (s, 6H), 5.73-5.76 (m, 1H), 6.25-6.30 (m, 1H),
6.50-6.57 (m, 1H), 6.89 (s, 1H), 6.96-7.07 (m, 2H), 7.17 (s, 2H), 7.24-7.26 (d, J = 8 Hz, 1H), 7.35-7.41 (m,
3H), 7.53-7.65 (m, 3H), 7.98 (s, 1H), 8.30 (s, 1H), 9.73 (s, 1H), 10.18 (s, 1H), 10.46 (s, 1H); 13C NMR
(DMSO-d6): δ 21.45 (2C), 104.74, 114.29, 116.47, 117.08, 120.34, 124.59 (2C), 127.09 (2C), 127.30,
127.48, 128.08, 129.29, 129.45, 132.05, 132.40, 137.58, 138.08 (2C), 138.33, 138.60, 138.63, 139.81,
157.58, 159.15, 163.78 (2C); HRMS (ESI+)for C29H26ClN5O, [M+H]+ calcd: 496.1899, found: 496.1962.
N-[3-[[5- Fluoro-2-[(E)-4-(3,5-dimethylstyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10e)
Yield 47.0%; light yellow solid; 1H NMR (DMSO-d6): δ 2.28 (s, 6H), 5.73-5.76 (m, 1H), 6.25-6.30 (m,
1H), 6.45-6.52 (m, 1H), 6.87-7.15 (m, 5H), 7.31-7.39 (m, 3H), 7.49-7.51 (d, J = 8 Hz, 2H), 7.69-7.71 (d, J
= 8 Hz, 2H), 7.96 (s, 1H), 8.13-8.14 (d, J = 4 Hz, 1H), 9.35 (s, 1H), 9.49 (s, 1H), 10.20 (s, 1H); 13C NMR
(DMSO-d6): δ 20.95 (2C), 113.69, 115.01, 117.57, 118.36 (2C), 123.95 (2C), 125.69, 126.61 (2C), 126.89,
127.98, 128.73, 129.69, 131.96, 137.31, 137.55 (2C), 138.98, 139.12, 139.37, 140.48, 140.84 (d, J = 78.8

Hz, 1C), 141.82, 149.84 (d, J = 43.6 Hz, 1C), 155.27 (d, J = 11.6 Hz, 1C), 163.15; HRMS (ESI+) for
C29H26FN5O, [M+H]+ calcd: 480.2194, found: 480.2241.
N-[3-[[5-Chloro-2-[(E)-4-(2,4-dimethylstyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10f)
Yield 59.7%; gray white solid; 1H NMR (DMSO-d6): δ 2.26 (s, 3H), 2.36 (s, 3H), 5.70-5.73 (m, 1H), 6.22-
6.27 (m, 1H), 6.42-6.49 (m, 1H), 6.89-6.93 (d, J = 16 Hz, 1H), 7.00 (s, 2H), 7.13-7.17 (d, J = 16 Hz, 1H),
7.32-7.37 (m, 4H), 7.48-7.56 (m, 2H), 7.62-7.64 (d, J = 8 Hz, 2H), 7.89 (s, 1H), 8.17 (s, 1H), 9.01 (s, 1H),
9.46 (s, 1H), 10.21 (s, 1H); 13C NMR (DMSO-d6): δ 20.00, 21.23, 104.44, 116.03, 116.17, 119.24 (2C),
119.86, 123.72, 125.17, 127.05 (2C), 127.35, 127.39, 128.99, 129.16, 130.83, 131.52, 132.37, 133.79,
135.48, 136.73, 139.28, 139.57, 140.38, 155.31, 156.73, 157.96, 163.67; HRMS (ESI+) for C29H26ClN5O,
[M+H]+ calcd: 496.1899, found: 496.1954.
N-[3-[[5-Fluoro-2-[(E)-4-(2,4-dimethylstyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10g)
Yield 31.2%; pale yellow solid; 1H NMR (DMSO-d6): δ 2.26 (s, 3H), 2.36 (s, 3H), 5.70-5.77 (m, 1H), 6.23-
6.29 (m, 1H), 6.93-7.01 (m, 3H), 7.15-7.19 (d, J = 16 Hz, 1H), 7.27-7.34 (m, 1H), 7.40-7.45 (m, 4H), 7.50-
7.52 (d, J = 8 Hz, 1H), 7.58-7.60 (d, J = 8 Hz, 1H), 7.76-7.78 (d, J = 8 Hz, 2H), 8.13-8.14 (d, J = 4 Hz, 1H),
8.25-8.32 (m, 1H), 9.46 (s, 1H), 9.54 (s, 1H), 10.52 (s, 1H); 13C NMR (DMSO-d6): δ 19.53, 20.73, 113.13,
115.65, 117.25, 118.44, 123.01, 124.67, 126.56, 126.64 (2C), 128.66 (2C), 128.90, 130.00, 131.03, 131.93,
132.16, 133.40, 134.96, 136.17, 137.97, 139.17, 139.38, 141.66 (d, J = 81.6 Hz, 1C), 141.86, 149.75 (d, J =
43.6 Hz, 1C), 155.23 (d, J = 11.6 Hz, 1C), 163.23; HRMS (ESI+) for C29H26FN5O, [M+H]+ calcd:
480.2194, found: 480.2229.
N-[3-[[5-Chloro-2-[(E)-4-(3,5-dichlorostyryl)phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
acrylamide (10h)
Yield 60.4%; gray white solid; 1H NMR (DMSO-d6): δ 5.72-5.75 (m, 1H), 6.23-6.28 (m, 1H), 6.43-6.50 (m,
1H), 6.99-7.03 (d, J = 16 Hz, 1H), 7.30-7.44 (m, 6H), 7.57-7.62 (m, 5H), 7.88 (s, 1H), 8.23 (s, 1H), 9.39 (s,
1H), 9.79 (s, 1H), 10.27 (s, 1H); 13C NMR (DMSO-d6): δ 104.80, 116.33, 116.71, 119.80 (2C), 120.36,
123.78, 125.04 (2C), 126.60, 127.46, 127.63 (2C), 129.22, 130.41, 132.03, 132.28, 134.93 (2C), 138.79,
139.70, 140.34, 141.83, 156.18, 157.22, 159.19, 163.73; HRMS (ESI+) for C27H20Cl3N5O, [M+H]+ calcd:
536.0806, found: 536.0841.
4.3. Kinase enzymatic assays
The ADP-GloTM system (EGFRWT, Catalog. V3831, EGFRT790M/L858R Catalog. V5324), purchased from
Promega Corporation (USA) were used to perform the enzymatic assays. The experiments were performed
according to the instructions of the manufacturer. The more detailed and complete protocols, and the active
kinase data were available at: http://www.promega.com/tbs/tm313/tm313/tm313.html and
http://www.promega.com/KES Protocol (or http://www.promega.com/tbs/signaling.htm), respectively. For

all of the tested compunds, concentrations consisting of suitable levels from 0.1 to 1000 nM were used. The
test was performed in a 384-well plate, and includes the major steps below: (1) perform a 5 µL kinase
reaction using 1×kinase buffer (e.g., 1×reactionbuffer A), (2) incubate at room temperature for 60 minutes,
(3) add 5 µL of ADP-Glo™ reagent to stop the kinase reaction and deplete theunconsumed ATP, leaving
only ADP and a very low background of ATP, (4) incubate at room temperature for 40 minutes, (5) add 10
µL of kinase detection, (6) reagent to convert ADP to ATP andintroduce luciferase and luciferin to detect
ATP, (7) incubate at room temperature for 30 minutes, (8) plate was measured on TriStar® LB942
Multimode Microplate Reader (BERTHOLD) to detect the luminescence (Integration time 0.5-1 second).
Curve fitting and data presentations were performed using GraphPad Prism version 5.0.
4.4. Cellular activity assay
H1975, H431, HCC827, and A549 human NSCLC cells were obtained from the American Type
Culture Collection. HBE normal cells were kind gifts from Fuheng Biology Company (Shanghai, China).
H1975, HCC827 and A549 cells were grown in RPMI-1640 (Gibco®, USA) supplemented with 10% FBS
(Gibco®, USA), 1% penicillin-streptomycin (Beyotime Company, China). A431 and HBE cells were grown
in DMEM (Gibco®, USA) supplemented with 10% FBS (Gibco®, USA), 1% penicillin-streptomycin
(Beyotime Company, China). All cells were maintained and propagated as monolayer cultures at 37 °C in
humidified 5% CO2 incubator.
Cell viability was then assessed with MTT reagent (Thiazolyl blue tetrazolium bromide; Sigma, Oakville,
ON). Cells were seeded in 96-well plates at a density of 3,000 to 5,000 cells/well and were maintained at 37
°C in a 5% CO2 incubator in DMEM or RPMI1640 containing 10% fetalbovine serum (FBS, Gibico). Cells
were esposed to treatment for 72 h, and the number of cells used per experiment for each cell line was
adjusted to obtain an absorbance of 0.5 to 1.2 at 570 nm. Compounds were tested at appropriate
concentrations (0.01 to 40 µM), with each concentration duplicated five times. The data were calculated
using GraphPad Prim version 5.0. Dose-response curves were fitted using a nonlinear regression model
with a sigmoidal dose-response.
4.7. Molecular docking study
AutoDock 4.2.6 software was used to carry out the docking studies. Detailed tutorials that guide users
through basic AutoDock usage, docking with flexible rings, and virtual screening with AutoDock may be
found at: http://autodock.scripps.edu/faqs-help/tutorial. Generally, the crystal structure (PDB: 3IKA) of the
kinase domain of EGFRT790M bound to inhibitor 4 was used in the docking studies. The enzyme preparation
and the hydrogen atoms adding was performed in the prepared process. The whole EGFR enzyme was
defined as a receptor and the site sphere was selected on the basis of the binding location of WZ4002. By
moving WZ4002 and the irrelevant water, molecule 10e was placed. The binding interaction energy was
calculated to include Van der Waals, electrostatic, and torsional energy terms defined in the tripos force

field. The structure optimization was performed using a genetic algorithm, and only the best-scoring ligand
protein complexes were kept for analyses.
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 81402788), and the
Cultivation Project of Youth Techstars, Dalian, 2016RQ043) for the financial support of this research.
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