Synthesis and biological evaluation of azole-diphenylpyrimidine derivatives (AzDPPYs) as potent T790M mutant form of epidermal growth factor receptor inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.09.001

A series of novel azole-diphenylpyrimidine derivatives (AzDPPYs) were synthesized and biologically evaluated as potent EGFR^T790Minhibitors. Among these analogues, the most active inhibitor 6e not only displayed high activity against EGFR^T790M/L858Rkinase (IC₅₀?=?3.3?nM), but also was able to repress the replication of H1975 cells harboring EGFR^T790Mmutation at a concentration of 0.118?μmol/L. In contrast to the lead compound rociletinib, 6e slightly reduces the key EGFRT790M-minduced drug resistance. Significantly, inhibitor 6e demonstrates high selectivity (SI?=?299.3) for T790M-containing EGFR mutants over wild type EGFR, hinting that it will cause less side effects.

Full text of DOI:10.1016/j.bmc.2016.09.001

Accepted Manuscript
Synthesis and Biological Evaluation of Azole-diphenylpyrimidine Derivatives
(AzDPPYs) as Potent T790M Mutant Form of Epidermal Growth Factor Re-
ceptor Inhibitors
Zhendong Song, Yue Jin, Yang Ge, Changyuan Wang, Jianbin Zhang, Zeyao
Tang, Jinyong Peng, Kexin Liu, Yanxia Li, Xiaodong Ma
PII:
S0968-0896(16)30688-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.001
BMC 13254
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 August 2016
31 August 2016
1 September 2016
Please cite this article as: Song, Z., Jin, Y., Ge, Y., Wang, C., Zhang, J., Tang, Z., Peng, J., Liu, K., Li, Y., Ma, X.,
Synthesis and Biological Evaluation of Azole-diphenylpyrimidine Derivatives (AzDPPYs) as Potent T790M Mutant
Form of Epidermal Growth Factor Receptor Inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.09.001
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Synthesis and Biological Evaluation of Azole-
diphenylpyrimidine Derivatives (AzDPPYs)
as Potent T790M Mutant Form of Epidermal
Growth Factor Receptor Inhibitors
Zhendong Songa,1, Yue Jina,1, Yang Gea, Changyuan Wanga, Jianbin Zhanga, , Zeyao Tang a, Jinyong Penga,
Kexin Liua, Yanxia Lib, and Xiaodong Maa, 
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
bRespiratory Department, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China.
1These authors contributed equally to this work.
E-mail address: xiaodong.ma@139.com (X.D. Ma). liyanxia001@163.com (Y.X. Li).
Asp800
O
Hydrogen bond
Cys797
N
Multibiological
functions
N
N
O
N
O
HN
HN
O
HN
N
NH
HN
F3C
N
NH
N
Met790
Cl
N
AzDPPY 6e
Rociletinib
IC50(EGFRT790M/L858R) = 3.3 nM
IC50(EGFRWT) = 1000 nM
IC50(EGFRT790M/L858R) = 21.5 nM
IC50(EGFRWT) = 460 nM
SI (EGFRT790M/L858R/EGFRWT)= 299.3
IC50(H1975 cell) = 0.118 μM
SI (EGFRT790M/L858R/EGFRWT)= 21.4
IC50(H1975 cell) = 0.137 μM
2

Bioorganic & Medicinal Chemistry
Synthesis and Biological Evaluation of Azole-diphenylpyrimidine Derivatives
(AzDPPYs) as Potent T790M Mutant Form of Epidermal Growth Factor Receptor
Inhibitors
Zhendong Songa,1, Yue Jina,1, Yang Gea, Changyuan Wanga, Jianbin Zhanga, Zeyao Tang a, Jinyong Penga,
Kexin Liua, Yanxia Lib,, and Xiaodong Maa,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
bRespiratory Department, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China.
1These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel azole-diphenylpyrimidine derivatives (AzDPPYs) were
synthesized and biologically evaluated as potent EGFRT790M inhibitors.
Among these analogues, the most active inhibitor 6e not only displayed high
activity against EGFR T790M/L858R kinase (IC50 = 3.3 nmol), but also was able
to repress the replication of H1975 cells harboring EGFRT790M mutation at a
concentration of 0.118 µmol. In contrast to the lead compound rociletinib, 6e
slightly reduces the key EGFRT790M-minduced drug resistance.
Significantly, inhibitor 6e demonstrates high selectivity (SI = 299.3) for
T790M-containing EGFR mutants over wild type EGFR, hinting that it will
cause less side effects.
Available online
Keywords:
NSCLC
EGFR T790M
Inhibitors
Synthesis
Azole-diphenylpyrimidine
1. Introduction
The epidermal growth factor receptor (EGFR) protein, is over-expressed in up to 80% of non-small cell
lung cancers (NSCLC), and has been a primary therapeutic target for NSCLC.1-3 Inhibitors targeting the
kinase domain of EGFR, such as gefitinib (1, Fig.1)4,5 and erlotinib (2, Fig.1)6, have shown promise in
patients with activating mutations (i.e. in exons 18, 19 or 21) in EGFR. Unfortunately, drug resistance
often occurs as a result of a secondary mutation such as the Thr790 to Met790 (T790M) mutation for most
patients who initially respond to gefitinib and erlotinib.7-9 Irreversible EGFR inhibitors containing a
Michael acceptor functional group have been developed to circumvent the T790M mutation-related
resistance. These inhibitors form a covalent bond with the Cys797 within the EGFR active site and have
shown preclinical activity against T790M-containing mutants of EGFR.10 Unfortunately, the family of the
irreversible inhibitors always have skin rash, diarrhea, and other serious side effects,15,16 with the exception
of a few molecules, such as WZ400211, rociletinib12, and osimertinib13,14, Therefore, it is necessary to
3

develop new molecules for the development of novel drugs with better anti-NSCLC activity and less host
toxicity.
F
O
HN
N
Cl
HN
N
O
O
O
O
N
O
O
N
N
1
2
Gefitinib
Erlotinib
O
N
O
N
N
O
N
O
O
N
N
N
HN
HN
HN
O
O
N
N
NH
HN
F3C
rociletinib
N
NH
HN
Cl
N
O
N
N
4
3
5
WZ4002
osimertinib
Figure 1. Chemical structures of novel EGFR inhibitors.
Amino acid
O
Hydrogen bond
N
Multibiological functions
X = N,C
N
N
X
Cys797
N
O
O
HN
HN
R2
O
HN
R1
N
NH
HN
F3C
N
NH
N
N
Met790
EGFRT790M Inhibitor
Rociletinib
AzDPPY derivatives
6a-j
Figure 2. Designed strategy of the azole-diphenylpyrimidine derivatives.
Generally, a common pyrimidine structure core is essential for these inhibitors to maintain the high anti-
EGFRT790M activity, as shown in figure 1. In addition, an acryloyl functionality is also important in forming
a strong covalent bond with amino acid Cys797 in EGFRT790M.11 Azoles, particularly the bioisosteric 1, 3,
4-oxadiazoles, 1, 2, 4-triazoles, and 1, 3, 4-thiadiazoles, are well-known small heterocyclic templates that
exhibit a broad-spectrum of biological activities such as antimalarial, antitubercular, antitumor,
antibacterial, anticancer, CNS depressant, anticonvulsant, molluscicidal, analgesic, inflammatory, and anti-
HIV. Typically, the azole functionality exhibited successful interactions with particular receptors on
4

enzyme active sites through polar interactions.17-19 Herein, a series of pyrimidine derivatives bearing an
azole group are designed and synthesized to enhance the binding affinity with EGFRT790M enzyme. Indeed,
the molecular simulations shown in figure 5 indicates that the new azole substituent forms a new strong
hydrogen bond with EGFRT790M, suggesting that these molecules can improve the anti-EGFRT790M activity.
Therefore, in this manuscript, a class of azole-diphenylpyrimidine derivatives (AzDPPYs) along with their
activity against NSCLC were described.
2. Results and discussion
2.1. Chemistry
The designed compounds AzDPPYs 6a-j were prepared according to the general strategy outlined in
Scheme 1.20,21 Commercially available reagent 3-nitroaniline (7) was reacted with acryloyl chloride to
produce the nitro-substituted intermediate 8. By reducing the nitro group in compound 8 using Fe-NH4Cl
conditions, the N-acryloyl-4-aminobenzamide (9) was produced in excellent yield (92%). Installation of the
4-aniline substituent 9 of the pyrimidine was achieved by direct regioselective displacement of 4-chloro in
the presence of N,N-diisopropylethylamine (DIPEA). As specified in scheme 2, scheme 3 and scheme 4,
the azole-substituted anilines 11a-g were prepared by applying the reported synthetic methods,22-25
including nucleophilic substitution reaction, acylation, and reaction reduction reaction. The desired azole-
diphenylpyrimidine derivatives 6a-j were conveniently synthesized when coupled by various anilines
containing the azole group with the pyrimidine scaffold 10a-c.
NO2
R1
NH2
N
NO2
Cl
N
Cl
a
b
NH
NH
c
NH2
O
O
8
7
9
R1
NH
R2
R1
N
N
N
N
NH2
Cl
N
NH
HN
N
X
R2
11a-g
NH
d
NH
N
O
O
X
10a-c
N
X = N,C
R1 = H, Cl, F, NO2
R2 = H, Cl
6a-j
Scheme 1. Synthetic route of title compounds 6a-j. Reagents and conditions: (a) acryloyl chloride, NaHCO3, CH3CN, rt,
0.5 h, 95%; (b) Fe-NH4Cl, MeOH-H2O, 2 h, 70 C, 92%; (c) ArNH2, DIPEA, 1,4-dioxane, rt, 2 h, 91%; (d) trifluoroacetic
acid, 4-azole-substituted aniline, 2-BuOH, 100 C, 12 h, 33–42%.
5

NO2
NO2
NH2
R2
R2
R2
b
a
Br
N
N
N
N
R= H or MeO
R= H or MeO
12a,b
13a,b
11a,b
Scheme 2. Synthetic route of intermediates 11a-b. (a) imidazole, K2CO3, CH3CN, reflux, 5 h, 80–92%; (b) Fe-NH4Cl,
MeOH-H2O, 2 h, 70 C, 72–81%.
NO2
NH2
NO2
R2
NO2
a
b
c
O
O
O
N
N
OH
X
Cl
X
N
N
X
X
X = H or N
X = H or N
14
16a,b
11c,d
15
Scheme 3. Synthetic route of title intermediates 11c-d. Reagents and conditions: (a) 1-bromo-3-chloropropane, K2CO3,
CH3CN, reflux, 12 h, 80–91%; (b) imidizole or tetrazole, K2CO3, CH3CN, reflux, 5 h, 55–73%; (c) Fe-NH4Cl, MeOH-
H2O, 2 h, 70 C, 62–81%.
NO2
NH2
NO2
d
c
F
N
N
17
N
N
a
11e
18
NO2
NO2
NH2
NO2
b
c
d
O
O
O
O
MsO
N
X
N
X
HO
19
N
N
12f,g
X = H or N
20
11f,g
Scheme 4. Synthetic route of title intermediates 10a-m. Reagents and conditions: (a) HOCH2CH2OH, K2CO3, 80 C, 2 h,
72–87%; (b) MeSO2Cl, NaHCO3, CH3CN, r.t, 2 h, 67–85%; (c) Azole, K2CO3, CH3CN, reflux, 5 h, 47–71%; (d) Fe-
NH4Cl, MeOH-H2O, 2 h, 70 C, 48–62%.
2.2 Biological activity
These compounds were evaluated by their inhibition ability for the autophosphorylation of wild-type
EGFR and the T790M/L858R-mutated EGFR kinases using ADP-Glo™ Kinase Assay.26-28 The novel
inhibitors rociletinib and gefitinib were also evaluated as references using the same procedure. As seen in
Table 1, the family of these molecules were able to interfer with the activity of EGFR T790M/L858R
kinase within 100 nmol/L concentrations. Among them, inhibitors 6c, 6e, 6f, possessing the IC50 values of
9.1 nmol, 3.3 nmol, and 9.0 nmol, were better than rociletinib (IC50 = 21.5 nmol) within EGFR T790M/
6

L858R kinase inhibition. In particular, the most active inhibitor 6e, that contains an imidazolyl
functionality, displyed approximately 8-fold higher potency against EGFRT790M/L858R than rociletinib.
However, by simply installing a MeO substituent on the phenyl ring of compound 6e, the inhibitor 6d (IC50
= 109 nmol) is reduced and was able to reduce the potency of anti-EGFR T790M/L858R nearly 30-times.
The addition of a long linker between the azole heterocycle and the phenyl ring also was not benefical. The
example compunds 6a and 6i, which have the IC50 values of 106.0 nmol and 40.6 nmol, lost the anti-BTK
capacitcy rapidly. These biological explortaions also demonstrated that addition one or two nitrogen atom
to the imidazole ring was unfavorabe. For instances, molecules 6b, and 6j, featuring a triazole or tetrazole
functionality, exhibited only moderate activity, with IC50 values aroud 100 nmol. Interestingly, the activity
againt the wild-type EGFR found that these comounds are less sensitive to WT EGFR, with IC50 values
being greater than 505.6 nmol. Note, the azo-DPPYs, possess high selectivity index, hinting that they will
produce less side effects particularly the imidizle-substited compunds 6c (SI >109.9) and 6e (SI = 299.3).
Table 1 In vitro EGFR tyrosine kinases (wild type and L858R/T790M mutation) activities of the title compounds 6a-j a
R1
N
HN
R
N
NH
R2
NH
O
6a-j
EGFR (IC50,nM)b
SI
R1
Cl
Cl
F
R2
H
H
H
R
Compd
6a
WT L858R/T790M
(WT:L858R/T790M)
N
>1000
>1000
>1000
506
106.0
103.4
9.1
>9.434
>9.671
>109.9
4.639
N
O
N
N
N
6b
N
N
O
O
6c
N
Cl MeO
109.0
3.3
6d
N
N
N
Cl
F
H
H
H
H
1000
299.3
6e
N
987
9.0
110.2
6f
N
N
N
NO2
NO2
>1000
>1000
118.7
98.2
>8.425
>10.19
6g
N
H
O
6h
N
HN
7

O
O
N
NO2
NO2
H
H
>1000
>1000
460
40.6
100.6
21.5
>24.63
>9.940
21.4
6i
6j
N
N
N
N
N
rociletinib
gefitinib
b
a Data represent the mean of at least three separate experiments. Concentration needed to inhibit the autophosphorylation
of the cytoplasmic domain of EGFR by 50%, as determined from the dose-response curve.
These newly synthesized compounds were also evaluated for their activity against the NSCLC cell lines
(A431WT, H1975L858R/T790M, A549k-ras mutation), as well as the normal lung cells HBE by using the MTT
method. Gefitinib and rociletinib were also compared for cell viability, as seen in Table 2. Notably,
molecule 6e, was the most active inhibitor against EGFRT790M kinase, and also has the strongest capacity
(IC50 = 0.118 µmol) for inhibiting the H1975 cell line harboring EGFRT790M mutation. This molecule
possesses almost 10 times higher potency than gefitinib, and is equivalent to rociletinib in repression of the
H1975 cell line replication. Although molecule 6a, beared an additional carbon atom linker between the
imidazolyl functionality and phenyl ring, has a moderate activity against EGFRT790M/L858R kinase (IC50 =
106.0 nmol), it could strongly inhibit H1975 cells at concentrations of 0.391 nmol. It is possible to form a
new interaction mechanism to interfere with the T790M-mutated cells. In terms of their activity against
A431 cell line harboring wild-type EGFR, compound 6f (IC50 = 0.152 nmol), which has a fluorine atom at
the C-5 position of pyrimidine core, was the most potent inhibitor within this class of inhibitors. When
replacing the fluorine atom in compound 6f with a chlorine substituent, the molecule 6e yielded a 5 times
increased activity, with an IC50 value of 0.733 nmol. It was apparent that the most active inhibitors were 6e
and 6f, which displayed stronger potency than references against the A431 cells. Furthermore, the newly
obtained AzDPPYs were effective in interference with the proliferation of the K-ras mutated A549 cell line
within micromolar concentrations. Compounds 6e and 6f were the most effective inhibitors against A549
cells, having the IC50 values of 2.730 µmol and 3.563 µmol, respectively. Fortunately, most of these
inhibitors were less sensitive to the normal HBE cell line (IC50 > 40µmol), indicating that they are less
cytotoxic. Overall, the biological evaluations of AzDPPY derivatives, eventually led to the discovery of a
promising inhibitor 6e with high anti-EGFRT790M activity and low cytotoxicity.
Table 2 Cellular antiproliferative activities of the title compounds 6a-j.
Cellular antiproliferative activity (IC50, μM)b
Compd
H1975
A431
A549
HBE
8

0.391±0.015
2.366±0.098
1.814±0.056
0.932±0.074
0.118±0.004
1.723±0.067
11.51±0.460
12.62±0.510
8.320±0.312
8.050±0.303
0.137±0.004
10.89±0.960
1.337±0.042
5.950±0.266
2.476±0.102
6.775±0.289
0.733±0.031
0.152±0.008
16.25±0.607
12.35±0.507
6.460±0.277
7.960±0.299
1.290±0.037
3.308±0.106
8.436±1.525
10.93±0.663
8.366±0.554
14.30±1.004
2.730±0.234
3.563±0.205
>40
39.15±3.236
>40
6a
6b
39.95±3.241
>40
6c
6d
>40
6e
>40
6f
>40
6g
6h
>40
>40
27.37±1.986
19.35±0.822
6.502±0.267
29.43±2.506
39.19±3.238
>40
6i
6j
>40
rociletinib
gefitinib
23.76±2.368
a
b
Data represent the mean of at least three separate experiments. Dose-response curves were determined at five
concentrations. The IC50 values are the concentrations in micromolar needed to inhibit cell growth by 50% as determined
from these curves.
H1975
A431
(μmol/L)
(nmol/L)
Figure 3. Various treating time of inhibitor 6e on the affects of the ability against H1975 and A431 cell lines.
Additionally, the effects of treating time (24h, 48h, 72h) on the ability to inhibit H1975 and A431 cell
lines was measured using the most active inhibitor, 6e. Figure 1 confirms that the proliferations of the two
cell lines were inhibited significantly by 6e in dose-and time-dependent manners. Moreover, the effects of
the inhibitor 6e on apoptosis in H1975 cell line were also determined using flow cytometer analysis
(FCMA). The result, shown in figure 4, displayed that the inhibitory effects of 6e obviously
increased with the increasing of drug concentration.
9

50nmol
Blank
)
500nmol
%
200nmol
6e
(
s
e
t
a
r
s
i
s
i
t
o
t
t
o
p
A
5
10
Blank
15
Concentrations (μM/L)
Figure 4. Compound 6e induced H1975 cell apoptosis in vitro. The cells were incubated with the indicated concentrations
of 6e for 48 h, and the cells were stained with a annexin V/FTIC, followed by flow cytometry analysis. One representative
experiment is shown. p <0.05 (n = 3)
2.3. Molecular modelling analysis
To explore the interaction mechanism of AzDPPYs with EGFRT790M enzyme, molecular simulations
were performed by docking the representative compounds, specifically 6d, 6e, 6f, 6g and 6i, into the ATP
binding pocket of EGFRT790M (PDB: 4I22).29 For comparison, the lead compound WZ4002 was also
performed using same procedure (Fig. 5a). The program AutoDock 4.2 with its default parameters was
used.30-32 As shown in figure 5a, WZ4002 forms several strong interactions with EGFRT790M, including: (1)
covalent bond between the acryl amide functionality with the amino acid Cys797; (2) strong contacts
generated from the chlorine atom at the C-5 position of pyrimidine core with the mutant gatekeeper residue
Met790; (3) hydrogen bond between the N-1 nitrogen atom of pyrimidine core and amino acid Met793; (4)
polar forces formed by the piperazine ring with the outside of the ATP-binding pocket of EGFRT790M
.
Evidently, the binding mode of the most active inhibitor 6e (c, Fig.5b) with EGFRT790M is similar to that of
WZ4002. For compound 6e, it produced the excepted strong hydrogen bond between the newly introduced
imidazole ring with the reside Asp803 through a water molecule. However, by only replacing the chlorine
atom at the C-5 position of pyrimidine core with a fluorine atom, the binding sites of the obtained
compound 6f with EGFRT790M are different from those of 6e. In terms of inhibitor 6f, the covalent bond
between the acryl amide and the amino acid Cys797 remains, but the desired hydrogen bond formed by the
azole ring disappeared. Moreover, the C-5 fluorine atom of the pyrimidine core was removed far away from
the mutant reside Met790, making apparent loss of contacts with this reside. While for the less active
10

inhibitors 6d and 6f, the important contacts of covalent bond with Cys797 or hydrogen bond with Met793
still appeared. But the less active molecule 6j not only lost these important interaction forces, but also made
the essential covalent bond disappeared. Overall, all these effects were in accordance with their activity
data.
Asp800
Asp800
Leu799
Asp800
b)
Leu799
c)
a)
Leu799
H2O
Cys797
Gly719
Pro797
Cys797
Cys797
Gly719
Gly719
Lys722
Leu844
Lys722
Pro797
Leu844
Leu844
Pro797
Met790
Met793
Met793
Met790
Met793
Met790
Asp800
Asp800
d)
Leu799
Asp800
Leu799
e)
Leu799
f)
Cys797
Cys797
Gly719
Gly719
Cys797
Gly719
Lys722
Lys722
Leu844
Met790
Leu844
Met790
Lys722
Pro797
Leu844
Met790
Pro797
Pro797
Met793
Met793
Met793
Figure 5. a) Binding site of WZ4002 within EGFRT790M (PDB code: 4I22). b) Putative binding mode of 6e within
EGFRT790M (PDB code: 4I22). c) Putative binding mode of 6d within EGFRT790M (PDB code: 4I22). d) Putative binding
mode of 6d within EGFRT790M (PDB code: 4I22). d) Putative binding mode of 6g within EGFRT790M (PDB code: 4I22). d)
Putative binding mode of 6j within EGFRT790M (PDB code: 4I22).
3. Conclusion
A series of novel azole-diphenylpyrimidine derivatives were designed and synthesized to strengthen the
interactions with EGFRT790M target. Biological evaluation studies in kinase and in NSCLC cell lines
eventually led to the identification of a novel inhibitor 6e, which not only displayed high activity against
EGFRT790M/L858R kinase (IC50 = 3.30 nmol), but also were able to repress the replication of H1975 cell line
harboring EGFRT790M mutation at concentrations of 0.118 µmol. Moreover, this compound also high
selectivity (SI = 299) for T790M-containing EGFR mutants over wild type EGFR, hinting that it will cause
less side effects. Taken together, this exploration provided a promising DPPY derivative 6e featuring a
novel azole functionality with enhanced anticancer activity.
4. Experimential section
4.1. General Methods and Chemistry.
Solvents and reagents were obtained from commercial supplies and were used without further purification.
High resolution ESI-MS were performed on an AB Sciex TripleTOF® 4600 LC/MS/MS system. 1H NMR
11

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 6a–j.20-25
4-Azole-substituted aniline intermediates 11a-g were prepared according to the reported literatures 22 to 25.
While the intermediates N-(3-((2-chloro-5-substitutedpyrimidin-4-yl)oxy)phenyl)acrylamide 10a-b were
synthesized using the reported procedure described in references 20 to 21. All these intermediates were
directly used without any purification and structural characterization. With these intermediates in hand, the
newly obtained compounds were synthesized as described below.
A flask was charged with compound 10a-c (0.70 mmol), 11a-g (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, 1:1) as eluents.
N-[3-[[5-Chloro-2-[4-[2-(1H-imidazol-1-yl)ethoxy]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6a).
Yield 46.1%; off-white solid. 1H NMR (400 MHz, d6-DMSO): δ 4.12 (s, 2H), 4.31 (s, 2H), 5.75 (d, J =
12.0 Hz, 1H), 6.24 (d, J = 16.8 Hz, 1H), 6.44 (dd, J = 12.0, 16.8 Hz, 1H) , 6.70 (s, 2H), 6.90 (s, 1H), 7.22
(s, 1H), 7.29-7.31 (m, 2H), 7.44-7.49 (m, 3H), 7.66 (s, 1H), 7.87 (s, 1H), 8.10 (s, 1H), 8.90 (s, 1H), 9.18 (s,
1H), 10.20 (s, 1H). HRMS (ESI) for C24H22ClN7O2, [M+H]+ calcd: 476.1605, found: 476.1613.
N-[3-[[5-Chloro-2-[4-[2-(1H-triazol-1-yl)ethoxy]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6b).
Yield 40.2%; off-white solid. 1H NMR (400 MHz, d6-DMSO): δ 4.07 (s, 2H), 4.26 (s, 2H), 5.70 (d, J =
12.0 Hz, 1H), 6.19 (d, J = 16.8 Hz, 1H), 6.39 (dd, J = 12.0, 16.8 Hz, 1H) , 6.64 (s, 2H), 6.84 (s, 1H), 7.17
(s, 1H), 7.24-7.29 (m, 2H), 7.44-7.48 (m, 2H), 7.61 (s, 1H), 7.82 (s, 1H), 8.05 (s, 1H), 8.84 (s, 1H), 9.13 (s,
1H), 10.15 (s, 1H). HRMS (ESI) for C23H21ClN9O2, [M+H]+ calcd: 475.1517, found: 475.1563.
N-[3-[[5-Fluoro-2-[4-[2-(1H-imidazol-1-yl)ethoxy]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6c).
Yield 36.7%; off-white solid. 1H NMR (400 MHz, d6-DMSO): δ 4.12 (t, J = 4.0 Hz, 2H)), 4.31 (t, J = 4.0
Hz, 2H), 5.75 (d, J = 12.0 Hz, 1H), 6.24 (d, J = 16.8 Hz, 1H), 6.44 (dd, J = 12.0, 16.8 Hz, 1H) , 6.74 (d, J =
8.0 Hz, 2H), 6.91(s, 1H), 7.24 (d, J = 6.0 Hz,1H), 7.29 (d, J = 12.0 Hz, 2H), 7.42 (d, J = 8.4 Hz, 1H), 7.53
(d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.93 (s, 1H), 8.07 (s, 1H), 9.01 (s, 1H), 9.39 (s, 1H), 10.15 (s, 1H).
HRMS (ESI) for C24H22FN7O2, [M+H]+ calcd: 460.1927, found: 460.1901.
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N-[3-[[5-Chloro-2-[2-methoxyl-4-(1H-imidazol-1-ylmethyl)]phenylamino]-4-
pyrimidinyl]amino]phenyl]-2-propenamide (6d).
Yield 46.5%; off-white solid. 1H NMR (400 MHz, d6-DMSO): δ 3.78 (s, 3H), 5.05 (s, 2H), 5.74 (dd, J = 2.4,
10.0 Hz, 1H), 6.24 (dd, J = 2.0, 16.8 Hz, 1H), 6.42 (dd, J = 10.0, 17.2 Hz, 1H), 6.66 (d, J = 8 Hz, 1H),
6.88 (s, 1H), 6.97 (s, 1H), 7.15 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.25 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H),
7.73 (s, 1H), 7.79 (s, 1H), 7.91 (d, J = 6.0 Hz, 1H), 7.92 (s, 1H), 8.11 (s, 1H), 8.95 (s, 1H), 10.14 (s, 1H).
HRMS (ESI) for C24H22ClN7O2, [M+H]+ calcd: 476.1608, found: 476.1614.
N-[3-[[5-Chloro-2-[4-(1H-imidazol-1-ylmethyl)]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6e).
Yield 56.5%; off-white solid. 1H NMR (400 MHz, d6-DMSO): δ 5.02 (s, 2H), 5.75 (dd, J = 2.4, 10.0 Hz,
1H), 6.25 (dd, J = 2.0, 16.8 Hz, 1H), 6.47 (dd, J = 10.0, 17.2 Hz, 1H), 6.86 (s, 1H), 7.04 (d, J = 8.4 Hz,
2H), 7.10 (s, 1H), 7.30 (d, J = 6.0 Hz, 2H), 7.50 (d, J = 6.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.67 (s, 1H),
7.85 (s, 1H), 8.14 (s, 1H), 8.97 (s, 1H), 9.37 (s, 1H), 10.18 (s, 1H).
HRMS (ESI) for C23H20ClN7O, [M+H]+ calcd: 446.1478, found: 446.1490.
N-[3-[[5-Fluoro-2-[4-(1H-imidazol-1-ylmethyl)]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6f).
Yield 36.7%; off-white solid. 1H NMR (400 MHz, d6-DMSO): δ 5.04 (s, 2H), 5.76 (dd, J = 2.0, 10.0 Hz,
1H), 6.26 (dd, J = 2.0, 16.8 Hz, 1H), 6.50 (dd, J = 10.0, 16.8 Hz, 1H), 6.88 (s, 1H), 7.03 (d, J = 8.4 Hz,
2H), 7.09 (s, 1H), 7.27 (d, J = 6.0 Hz, 1H), 7.43 (d, J = 6.0 Hz, 1H), 7.51 (d, J = 6.0 Hz, 1H), 7.64 (s, 1H),
7.69 (s, 1H), 7.93 (s, 1H), 8.11 (s, 1H), 9.24 (s, 1H), 9.46 (s, 1H), 10.16 (s, 1H).
HRMS (ESI) for C23H20FN7O, [M+H]+ calcd: 430.1768, found: 430.1782.
N-[3-[[5-Nitro-2-[4-[2-(1H-imidazol-1-yl)acetylamino]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6g).
Yield 66.1%; faint yellow solid. 1H NMR (400 MHz, d6-DMSO): δ 5.07 (s, 2H), 5.71 (d, J = 10.0 Hz, 1H),
6.21 (d, J = 16.8 Hz, 1H), 6.40 (dd, J = 10.0, 16.8 Hz, 1H), 6.88 (s, 1H), 7.01 (s, 1H), 7.11 (d, J = 8.4Hz,
2H), 7.27 (d, J = 6.0Hz, 1H), 7.44 (d, J = 6.0Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H), 7.65 (d, J = 8.4Hz, 2H),
7.69 (s, 1H), 7.83 (s, 1H), 7.95 (s, 1H), 8.51(s, 1H), 9.04 (s, 1H), 10.32 (s, 1H).
HRMS (ESI) for C24H21N9O4, [M+H]+ calcd: 500.1722, found: 500.1714.
N-[3-[[5-Nitro-2-[4-(1H-triazol)]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-propenamide (6h).
Yield 60.2%; faint yellow solid. 1H NMR (400 MHz, d6-DMSO): δ 5.67 (d, J = 10.0 Hz, 1H), 6.19 (d, J =
16.8 Hz, 1H), 6.29 (dd, J = 10.0, 16.8 Hz, 1H), 6.39 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H), 6.84 (s, 1H), 7.01 (s,
1H), 7.21 (d, J = 8.4Hz, 1H), 7.28 (d, J = 6.0 Hz, 1H), 7.38 (d, J = 6.0Hz, 1H), 7.41 (s, 1H), 7.43 (s, 1H),
7.86 (s, 1H), 8.02 (s, 1H), 8.96 (s, 1H), 9.23 (s, 1H), 10.29 (s, 1H).
HRMS (ESI) for C22H18N9O3, [M+H]+ calcd: 443.1531, found: 443.1574.
13

N-[3-[[5-Nitro-2-[ 4-[3-(1H-imidazol-1-yl)propoxy]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6i).
Yield 61.0%; faint yellow solid. 1H NMR (400 MHz, d6-DMSO): δ 3.86 (m, 2H), 4.07 (t, J = 4.0 Hz, 2H),
4.57 (t, J = 4.0 Hz, 2H), 5.72 (d, J = 10.0 Hz, 1H), 6.17 (d, J = 16.8 Hz, 1H), 6.21 (dd, J = 10.0, 16.8 Hz,
1H), 6.39 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H), 6.84 (s, 1H), 7.17 (d, J = 8.4Hz, 2H), 7.24 (d, J = 6.0 Hz, 1H),
7.44 (d, J = 8.4Hz, 2H), 7.61 (s, 1H), 7.82 (s, 1H), 8.05 (s, 1H), 9.03 (s, 1H), 9.45 (s, 1H), 10.27 (s, 1H).
HRMS (ESI) for C25H24N9O4, [M+H]+ calcd: 501.1987, found: 501.2006.
N-[3-[[5-Nitro-2-[ 4-[3-(1H-tetrazol-1-yl)propoxy]phenylamino]-4-pyrimidinyl]amino]phenyl]-2-
propenamide (6j).
Yield 56.8%; faint yellow solid. 1H NMR (400 MHz, d6-DMSO): δ 3.87 (m, 2H), 4.10 (t, J = 4.0 Hz, 2H),
4.62 (t, J = 4.0 Hz, 2H), 5.75 (d, J = 10.0 Hz, 1H), 6.18 (d, J = 16.8 Hz, 1H), 6.25 (dd, J = 10.0, 16.8 Hz,
1H), 6.45 (s, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H), 7.20 (d, J = 8.4Hz, 1H), 7.28 (d, J = 6.0 Hz, 1H),
7.49 (d, J = 8.4Hz, 2H), 7.71 (s, 1H), 7.88 (s, 1H), 8.15 (s, 1H), 9.13 (s, 1H), 9.55 (s, 1H), 10.26 (s, 1H).
HRMS (ESI) for C23H22N10O4, [M+Na]+ calcd: 525.1787, found: 525.1739.
4.3. In vitro kinase enzymatic assay
The wild-type EGFR enzyme, mutant EGFR T790M/L858R enzyme, and the ADP-Glo™ Kinase Assay
system that measures ADP formed from a kinase reaction were purchased from Promega Corporation
(USA). Concentrations consisting of suitable levels from 0.1 to 1000 nM were used for all of the tested
compunds. For the ADP-Glo kinase assay, ADP is converted into ATP, which is converted into light by
Ultra-Glo™ Luciferase. The experiments were performed according to the instructions of the
manufacturer.26-29 Plate was measured on TriStar2 LB 942 Multimode Microplate Reader (BERTHOLA) to
detect the Luminescence. Curve fitting and data presentations were performed using Graph Pad Prism
version 5.0.
4.4. Cellular activity assay
H1975 cells were obtained from Fuheng Biology Company (Shanghai, China). Cells were seeded in 96-
well plates at a density of 2,000 to 3,000 cells/well and were maintained at 37 C in a 5% CO2 incubator in
DMEM or RPMI1640 containing 10% fetalbovine serum (FBS, Gibico). Cell viability was then assessed
with MTT reagent (Thiazolyl blue tetrazolium bromide; Sigma, Oakville, ON). Cells were esposed to
treatment for 48 h, and the number of cells used per experiment for each cell lines was adjusted to obtain an
absorbance of 0.5 to 1.2 at 570 nm. Compounds were tested at appropriate concentrations (0.05 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.
4.5. Cell Apoptosis Assay
Cells were treated with solvent control (DMSO), or compound 6e in medium containing 5% FBS for 48
hours. Both non-adherent and adherent cells were collected and pooled, washed twice with PBS and
14

resuspended in 70% ethanol for permeabilization. Cell suspensions were then treated with propidium iodide
solution (48 μg/ml propi-dium iodide, 40 μg/ml RNase A) for 30 minutes at room temperature. A total of 3
× 105 cells were evaluated using the Beckman Coulter Epics XL flow cytometer (Beckman-
Coulter,Mississauga, ON) and the percentage of apoptotic cells was calculated from cells with less than
2NDNA content using FCS Express flow cytometry analysis software (ModFit LT 3.1).
4.6. Molecular docking study
Docking studies were carried out on AutoDock 4.2. The crystal structure (PDB: 4I22) of the kinase
domain of EGFRT790M bound to inhibitor 1 was used in the docking studies. 29 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 gefitinib. By moving
gefinitib and the irrelevant water, molecule 6d, 6e, 6f, 6g, and 6j was placed, respectively 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.30-32
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 81402788), and the PhD Start-
up Fund of Natural Science Foundation of Liaoning Province, China (No. 20141115) for the financial
support of this research.
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