Synthesis and biological evaluation of morpholine-substituted diphenylpyrimidine derivatives (Mor-DPPYs) as potent EGFR T790M inhibitors with improved activity toward the gefitinib-resistant non-small cell lung cancers (NSCLC)

Article abstract of DOI:10.1016/j.ejmech.2017.03.083

Potential new EGFR^T790M inhibitors comprised of structurally modified diphenylpyrimidine derivatives bearing a morpholine functionality (Mor-DPPYs) were used to improve the activity and selectivity of gefitinib-resistant non-small cell lung cancer (NSCLC) treatment. This led to the identification of inhibitor 10c, which displayed high activity against EGFR^T790M/L858R kinase (IC₅₀?=?0.71?nM) and repressed H1975?cell replication harboring EGFR^T790M mutations at a concentration of 0.037?μM. Inhibitor 10c demonstrated high selectivity (SI?=?631.9) for T790M-containing EGFR mutants over wild type EGFR, suggesting that it will cause less side effects. Moreover, this compound also shows promising antitumor efficacy in a murine EGFR^T790M/L858R-driven H1975 xenograft model without affecting body weight. This study provides new potential lead compounds for further development of anti-NSCLC drugs.

Full text of DOI:10.1016/j.ejmech.2017.03.083

Accepted Manuscript
Synthesis and biological evaluation of morpholine-substituted diphenylpyrimidine
derivatives (Mor-DPPYs) as potent EGFR T790M inhibitors with improved activity
toward the gefitinib-resistant non-small cell lung cancers (NSCLC)
Zhendong Song, Shanshan Huang, Haiqing Yu, Yu Jiang, Changyuan Wang, Qiang
Meng, Xiaohong Shu, Hunjun Sun, Kexin Liu, Yanxia Li, Xiaodong Ma
PII:
S0223-5234(17)30251-9
10.1016/j.ejmech.2017.03.083
EJMECH 9343
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 January 2017
Revised Date: 29 March 2017
Accepted Date: 31 March 2017
Please cite this article as: Z. Song, S. Huang, H. Yu, Y. Jiang, C. Wang, Q. Meng, X. Shu, H. Sun,
K. Liu, Y. Li, X. Ma, Synthesis and biological evaluation of morpholine-substituted diphenylpyrimidine
derivatives (Mor-DPPYs) as potent EGFR T790M inhibitors with improved activity toward the gefitinib-
resistant non-small cell lung cancers (NSCLC), European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.03.083.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Asp800
O
Cys797
O
covalent bond
N
N
N
O
L
Flexiable linkers
R1
O
HN
HN
O
HN
R
N
NH
HN
F3C
N
NH
N
N
Met790
Rociletinib
EGFRT790M Inhibitor
10a-l
Improved activity and selectivity
Phase Ш

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of
Morpholine-substituted Diphenylpyrimidine Derivatives
(Mor-DPPYs) as Potent EGFR T790M Inhibitors with
Improved Activity toward the Gefitinib-resistant Non-small
Cell Lung Cancers (NSCLC)
Zhendong Song a,1, Shanshan Huang a,1, Haiqing Yu a, Yu Jiang a, Changyuan Wang a,
Qiang Meng a, Xiaohong Shu a, Hunjun Sun a, Kexin Liu a, Yanxia Li b, and Xiaodong
Ma a,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
bDepartment 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).
Abstract
Potential new EGFRT790M inhibitors comprised of structurally modified
diphenylpyrimidine derivatives bearing a morpholine functionality (Mor-DPPYs)
were used to improve the activity and selectivity of gefitinib-resistant non-small cell
lung cancer (NSCLC) treatment. This led to the identification of inhibitor 10c, which
displayed high activity against EGFRT790M/L858R kinase (IC50 = 0.71 nM) and repressed
H1975 cell replication harboring EGFRT790M mutations at a concentration of 0.037
µM. Inhibitor 10c demonstrated high selectivity (SI = 631.9) for T790M-containing
EGFR mutants over wild type EGFR, suggesting that it will cause less side effects.
Moreover, this compound also shows promising antitumor efficacy in a murine
EGFRT790M/L858R-driven H1975 xenograft model without affecting body weight. This
study provides new potential lead compounds for further development of anti-NSCLC
drugs.
Keywords: NSCLC, EGFR T790M, Resistance, Inhibitors, Pyrimidine.
1

ACCEPTED MANUSCRIPT
1. Introduction
First-generation epidermal growth factor receptor (EGFR) tyrosine kinase
inhibitors (TKIs), gefitinib (1, Fig.1) [1,2] and erlotinib (2, Fig.1) [3] provide
significant clinical benefits in non-small-cell lung cancer (NSCLC) patients by
harboring active mutations in the EGFR tyrosine kinase domain (L858R and del
E746-A750). However, most patients who respond to therapy ultimately develop
disease progression after 9 to 14 months of treatment. The “gatekeeper” EGFR
T790M mutation (threonine790 to methionine790, EGFRT790M) is one of the primary
resistance mechanisms, affecting nearly 50% of NSCLC patients who acquire clinical
resistance by binding ATP with kinase. This EGFRT790M mutation restores ATP
affinity similarly to wild-type (WT) EGFR by preventing the reversible inhibitors
from binding at higher ATP concentrations [4–6].
N
O
O
N
O
O
N
O
N
N
N
O
O
NH
Cl
NH
NH
NH
O
O
F
N
F
O
Cl
N
2
1
3
Erlotinib
Gefitinib
Afatinib
O
N
N
N
N
N
O
N
O
O
N
HN
N
HN
HN
O
O
O
N
NH
HN
F3C
Rociletinib
N
NH
HN
Cl
WZ4002
N
O
N
N
5
4
6
Osimertinib
Figure 1. Novel EGFR inhibitors for treatment of NSCLC.
Second-generation irreversible EGFR inhibitors containing a Michael acceptor
functional group have been developed to overcome EGFRT790M mutation-related
resistance. Afatinib (3, Fig.1) [7], a novel exemplary second-generation inhibitor, was
approved by the US FDA in 2013 to treat late-stage NSCLC patients with actively
mutated EGFR. Unfortunately, these inhibitors produced a relatively low
maximal-tolerated-dose (MTD) because of the non-selective inhibition against
wild-type EGFR and/or other kinases, resulting in poor clinical patient outcomes [8–
2

ACCEPTED MANUSCRIPT
10]. Development of selective EGFRT790M inhibitors is thus urgent to treat NSCLC
patients with acquired resistance.
More recently, third-generation EGFR inhibitors, including WZ4002 (4) [11],
rociletinib (5) [12] and osimertinib (6) [13,14] combine the advantageous properties
of covalent inhibition with an improved selectivity profile since they spare the
inhibition of wild type EGFR (Fig.1). Most of these third-generation inhibitors exhibit
beneficial properties in terms of residence times and toxicity profiles. Both rociletinib
and osimertinib were designated as breakthrough therapies to treat mutant NSCLC by
the US FDA in 2014. A year later, osimertinib was approved, but more phase III
clinical trials were required for rociletinib due to the serious hyperglycemia problem
in NSCLC patients [15].
F
O
O
O
N
N
O
N
O
N
HN
HN
F
O
O
NH2
N
H
N
O
N
O
O
N
O
HN
NH
F
N
O
N
N
H
F
O
N
F
7
8
9
Copanlisib
Foretinib
VS-4718
PI3K inhibitor
Phase
c-Met Inhibitor
Phase
FAK Inhibitor
Phase
Figure 2. Typical anticancer agents bearing a morpholine substituent.
Morpholine group is an important pharmacophore, presented in a number of
anticancer agents, including the novel EGFR inhibitor gefitinib, PI3K inhibitor
copanlisib (7) [16, 17], c-Met inhibitor foretinib (8) [18], and FAK inhibitor VS-4718
(9) [19, 20] (Fig.2). As our continuous efforts to develop small molecule EGFR
inhibitors with better anti-NSCLC activity and less host toxicity [21–23], in this study,
we synthesized a series of morpholine-substituted pyrimidine derivatives
(Mor-DPPYs), and obtained several promising EGFRT790M inhibitors possessing
strong activity and high selectivity. Structure and activity relationship (SAR) studies
of DPPY template indicated that conformational constraint strategy is effective to
improve the selectivity against EGFRT790M over wild-type EGFR [24–26]. In this
manuscript, we also found that installing a flexible morpholine-substituted aniline at
3

ACCEPTED MANUSCRIPT
the C-2 position of the pyrimidine core also could improve the selectivity property
(Fig.3). Herein, these structural modifications of Mor-DPPY analogues and their
anti-NSCLC biological explorations are explained.
Asp800
O
O
Cys797
covalent bond
N
N
O
Flexiable Linkers
L
N
O
HN
R1
HN
O
HN
R
N
NH
HN
N
NH
N
N
F3C
Met790
Rociletinib
10a-l
EGFRT790M Inhibitor
Phase
Ш
Figure 3. Designed strategy of the Mor-DPPY derivatives.
2. Results and Discussion
2.1. Chemistry
The title molecules 10a-l were prepared according to the general strategy [26, 27],
as shown in Schemes 1-5. Commercially available material anilines 11a-c were
converted to acrylamide-substituted phenols and acrylamide-substituted anilines
12a-c by reacting with acryloyl chloride in almost quantitative yields. Anilines 12a-c
regioselectively reacted with the 4-chloro atom of the 2,4-dichloropyrimidine reagent
in the presence of N,N-diisopropylethylamine (DIPEA) to produce the key pyrimidine
intermediates 13a-d. Finally, the desired diphenylpyrimidine derivatives 10a-c, f, g, h
were conveniently synthesized by coupling various morpholine-substituted anilines
with pyrimidines 13a-d. The key intermediates morpholine-substituted anilines 18, 23,
27, 31 were prepared as specified in Schemes 2-4 through classical
nucleophilic substitution, acylation, and reduction reactions [28–31]. After coupling
various morpholine-substituted anilines with the 2-chloropyrimidines 13a-d utilizing
trifluoroacetic acid (TFA) reagent at reflux temperature, the desired molecules 10d-e
and 10i-l were synthesized in the yield of 21.0% to 40.8%.
4

ACCEPTED MANUSCRIPT
R1
X
5
N
2
4
HN
N
O
2''
2'
3'
R2
R2
NH
NH2
3''
O
N
O
R1
X
c
N
N
NH2
NH
O
Cl
N
R1 = Cl, F; R2 = H, MeO(C-2')
X = O, NH
10a-c,h
O
2'
3'
2'
X
a
b
X
3'
NH
Cl
N
H2N
O
N
R1 = Cl, F
X = O, NH
13a-d
X = OH,
NH2(m- or o-)
11a-c
X = OH, NH2
12a-c
HN
N
X
c
O
O
O
N
H
N
O
X = O, NH
10f,g
Scheme 1. Synthetic route of the title compounds 10a-c, f, g, h. Reagents and conditions: (a)
acryloyl chloride, NaHCO3, CH3CN, rt., 0.5 h, 91-96%; (b) 2,4-dichloropyrimidines, DIPEA,
1,4-dioxane, rt., 2 h, 85-92%; (c) TAF, 2-BuOH, 100 °C, 12 h, 26.1–40.8%.
Cl
N
NH2
NO2
NO2
NO2
HN
O
N
R
N
NH
NO2
2''
3''
NH
e
R
d
R
R
a
c
b
R
O
N
O
N
O
O
R
O
F
OMs
OH
O
O
O
R = H, Cl
14a-b
R = H, Cl
10d,e,i
17a-b
15a-b
16a-b
18a-b
Scheme 2. Synthetic route of the title compounds 10d, e, i. Reagents and conditions: (a)
HOCH2CH2OH, K2CO3, 80 °C, 2 h, 80–85%; (b) MeSO2Cl, NaHCO3, CH3CN, rt., 2 h,78–89%;
(c) morpholine, K2CO3, CH3CN, reflux, 5 h, 63–81%; (d) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 60–
82%; (e) TFA, 2-chloropyrimidines 13a-d, 2-BuOH, 100 °C, 12 h, 21.0–36.5%.
Cl
N
NO2
NO2
NO2
NH2
HN
N
NH
NO2
H
N
O
b
a
c
e
f
O
O
O
O
O
O
O
O
d
O
O
OH
OH
OMe
N
N
N
O
O
O
10k
19
20
21
22
23
5

ACCEPTED MANUSCRIPT
Scheme 3. Synthetic route of the title compound 10k. Reagents and conditions: (a) ethyl
bromoacetate, K2CO3, CH3CN, reflux, 5 h, 91%; (b) NaOH, MeOH-H2O, 2 h, 70 °C; (c) (COCl)2,
DMF; (d) morpholine, NaHCO3, CH3CN, reflux, 5 h, 62–77%; (e) Fe-NH4Cl, MeOH-H2O, 2 h, 70
°C, 71–83%; (f) TFA, 2-chloropyrimidine 13, 2-BuOH, 100°C, 12 h, 30.5%.
Cl
N
NH2
NO2
NO2
HN
O
N
NH
H
NO2
OH
N
O
a
b
c
d
O
O
O
N
N
Cl
N
O
O
O
10j
24
26
27
25
Scheme 4. Synthetic route of the title compound 10j. Reagents and conditions: (a)
1-bromo-3-chloropropane, K2CO3, CH3CN, reflux, 12 h, 92%; (b) morpholine, K2CO3, CH3CN,
reflux, 5 h, 88%; (c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 85%; (d) TFA, 2-chloropyrimidine 13,
2-BuOH, 100°C, 12 h, 26.8%.
Cl
N
NO2
NO2
NH2
HN
HN
N
NH
NO2
H
N
O
a
c
b
d
HN
O
HN
O
HN
O
N
O
N
NH2
N
Br
O
O
O
10l
29
28
30
31
Scheme 5. Synthetic route of the title compound 10l. Reagents and conditions: (a) bromoacetyl
bromide, NaHCO3, CH3CN, rt., 0.5 h, 97%; (b) morpholine, K2CO3, CH3CN, reflux, 5 h, 81%; (c)
Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 82%; (d) TFA, 2-chloropyrimidine 13, 2-BuOH, 100°C, 12 h,
40.5%.
2.2 Biological activity
2.2.1 Kinase inhibitory activity
Applying with the ADP-Glo™ Kinase Assay method, all the newly synthesized
compounds were evaluated according to their inhibition ability for
autophosphorylation of the wild-type EGFR and the T790M/L858R-mutated EGFR
kinases [32, 33]. For comparison, two representative drugs rociletinib and gefitinib
were also tested using the same procedure. As shown in Table 1, most of these
Mor-DPPYs could interfer with the EGFRT790M/L858R kinase activity within 100
nmol/L concentrations, and nearly two thirds of them (with IC50 values of lower than
24.8 nM) displayed equivalent or stronger actvity than rociletinb. The most active
6

ACCEPTED MANUSCRIPT
inhibitor was 10c (IC50 = 0.71 nM), bearing a methylene linker between the phenyl
group of the C-2 aniline side chain and the morpholine ring. This inhibitor exhibited
approximately 28-fold higher potency for inhibiting the EGFRT790M/L858R activity than
rociletinib (IC50 = 20.0 nM). However, by simply replacing the chlorine atom with a
fluorine substitutent at the C-5 position of the pyrimidine core in compound 10c, the
yielded molecule 10b (IC50 = 80.8 nM) remarkably lost the anti-EGFRT790M/L858R
capacity (approximately 113-times). The addition of a long linker (two carbon)
between the phenyl group in the C-2 aniline side chain and the morpholine ring was
unbenefical. The exemplary compounds 10d and 10e, are clearly inactive against the
EGFRT790M mutant, with IC50 values of more than 94.7 nM. The kinase-based test
result also indicated that installing a methoxy group at the C-2
' position of the C-2
aniline side chain (compound 10a with an IC50 of 17.0 nM), or a chloride atom at C-3
'
position (compound 10e with an IC50 of more than 100 nM) are uneffective. When the
methylene linker was replaced by an acetamide linker, the yielded compounds 10f and
10g only exhibited moderate activity against the EGFRT790M/L858R kinase, with IC50
values of 96.6 nM and 100.5 nM, respectively. Notably, the class of compounds 10h-l,
which bear an acryl amide functional group at the C-2'' position of the C-4 aniline side
chain, were also potent to inhibit the EGFRT790M activity, possessing the IC50 values in
the range of 13.4 nM to 24.8 nM. Compared with the C-3'' acryl amide substituted
analogues 7a-g, these molecules slightly reduced the anti-EGFRT790M/L858R activity.
Whereas, with the exception of inhibitor 10k (IC50 = 142.9 nM), most of the
Mor-DPPY analogues are not active against the wild type EGFR kinase at
concentrations of more than 448.7 nM, indicating that these inhibitors have high
selectivity against EGFRT790M/L858R over wild type EGFR. Among them, five
compounds 10a (SI > 29.5), 10c (SI = 631.9), 10h (SI > 52.3), 10i (SI = 36.6), and
10j (SI = 35.7) possess higher SI values than rociletinib (SI = 25.0), and the most
active EGFRT790M inhibitor 10c, displayed 25 times higher selectivity than rociletinib.
2.2.2 Antiproliferative activity against NSCLC cell lines
7

ACCEPTED MANUSCRIPT
Additionally, all the new Mor-DPPY analogues were also evaluated for their
activity against the NSCLC cell lines (A431WT, H1975L858R/T790M, HCC827del E746_A750
,
A549WT and k-ras mutation). Evaluation of cytotoxicity was also performed by interfering
with cell viability of the normal human bronchial epithelial cell line (HBE cells) with
MTT method. Gefitinib and rociletinib were also tested as positive controls. The test
result shown in Table 2 indicated that Mor-DPPYs are more active than rociletinib
(IC50 = 0.299 µM) to interfere with the proliferation of the EGFR T790M-mutated
H1975 cells, with IC50 values ranging from 0.037 µM to 0.296 µM. Molecule 10c is
the most potent EGFRT790M inhibitor, exhibiting the strongest capacity (IC50 = 0.037
µM) for inhibiting H1975 cells harboring the EGFRT790M mutation. In contrast with
rociletinib (IC50 = 0.299 µM), compound 10c showed approximately 8 times higher
inhibitory potency against H1975 cells. When it comes to the activity against the wild
type A431 cells, most of these molecules displayed stronger inhibitory capability than
rociletinib (IC50 = 1.794 µM), with IC50 values of lower than 0.0870 µM.
Unfortunately, this result indicated that Mor-DPPY analogues possess lower
selectivity against the mutant H1975 cells over the wild type A431 cells compared
with rociletinib. In addition, Mor-DPPY analogues also displayed nanomolar activity
against HCC827del E746_A750 cell line, which is equivalent to rociletinib. As for the
activity against HCC827 cells, the C-3'' acryl amide substituted compounds, including
10c (IC50 = 0.044 µM) and 10d (IC50 = 0.043 µM) displayed 10 times higher
inhibitory potency than those (10h, 10i) with a C-2'' substituent. Interestingly,
Mor-DPPYs exhibited moderate activity against A549 cells harboring the nearly
insurmountable K-ras mutation. Among them, compound 10f, which bears an amide
liner between the phenyl group in the C-2 aniline side chain and the morpholine ring
was the most effective inhibitor to block the replication of A549 cells, with an IC50
value of 1.662 µM. Moreover, all Mor-DPPY analogues were also tested the cytotoxic
activity against the normal HBE cells, which showed that these inhibitors are not
sensitive to the normal HBE cells with concentrations up to 40 µM, proving their low
cytotoxicity at the cell level. In summary, these biological evaluations eventually led
to the discovery of the promising inhibitor 10c, which possesses high anti-EGFRT790M
activity and low cytotoxicity.
Table 1 In vitro EGFR tyrosine kinases (wild type and L858R/T790M mutation)
activities of the title compounds 10a-l a
8

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R1
5
N
2
4
HN
N
X
2'
3'
2''
3''
R2
N
O
H
4'
R
Enzymatic activity
(IC50, nM) b
O
R2
(C-2' or
C-3')
SI(WT:
L858R/T
790M)
N
Compd.
X
R
R1
H
(C-2'' or
C-3'')
EGFR
EGFR
L858R/T790M
WT
OMe
(C-2')
N
N
N
NH
NH
NH
Cl
F
C-3''
C-3''
C-3''
17.0
80.8
0.71
>500.0
500.0
448.7
10a
10b
10c
>29.5
6.19
O
O
O
H
H
Cl
631.9
O
NH
NH
NH
Cl
Cl
Cl
H
C-3''
C-3''
C-3''
94.7
>100.0
96.6
961.3
>1000
<500.0
10d
10e
10f
9.6
N
O
O
O
Cl
(C-3')
10.0
<5.7
N
O
N
H
O
O
O
N
O
Cl
Cl
Cl
H
H
H
C-3''
C-2''
C-2''
100.5
15.3
13.4
996.0
>800.0
491.0
10g
10h
10i
9.97
>52.3
36.6
N
NH
NH
O
O
O
N
O
N
NH
Cl
H
C-2''
21.5
24.8
766.6
10j
35.7
5.8
O
O
O
NH
NH
Cl
Cl
H
H
C-2''
C-2''
142.9
592.1
10k
N
O
O
HN
21.7
20.0
10l
27.3
25.0
N
O
Rociletinib
Gefitinib
500.0
15.5
823.3
0.019
b
a 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 nanomolar needed to
inhibit cell growth by 50%, as calculated using GraphPad Prism version 5.0.
Table 2 Cellular antiproliferative activities of the title compounds 10a-l a
Cellular antiproliferative activity (IC50, ꢀM) b
Compd.
9

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H1975
0.287
0.609
0.037
0.215
0.500
0.256
0.296
0.104
0.240
0.217
0.132
0.128
0.299
4.691
A431
2.055
0.444
0.382
0.385
0.870
0.760
2.191
0.112
0.478
0.584
0.042
0.104
1.794
3.300
HCC827
0.052
0.041
0.044
0.043
0.057
0.058
0.048
0.995
0.524
0.587
0.418
0.491
0.031
0.006
A549
16.60
4.457
4.450
5.707
13.95
1.662
2.944
2.642
4.702
4.739
2.649
5.113
3.242
10.07
HBE
28.76
22.68
>40
10a
10b
10c
10d
10e
31.46
22.43
22.04
>40
10f
10g
10h
10i
>40
>40
10j
>40
10k
10l
39.54
21.58
>40
Rociletinib
Gefitinib
23.8
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 calculated using GraphPad Prism version 5.0.
2.3 Western blot analysis
10

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B
A
control
(µM)
1
control
(µM)
1
0.2
0.04 0.2
1
1
1
1
1
1
5
EGFR
EGFR
p-EGFR
AKT
p-EGFR
AKT
p-AKT
ERK
p-AKT
ERK
p-ERK
Actin
p-ERK
Actin
Figure 4. The effects of 10c on the activation of EGFR and downstream signaling in A431 (A)
and H1975 cells (B).
To gain insight into the mechanisms underlying 10c activity, the effects on EGFR
activation and downstream signaling in H1975 and A431 cells were investigated.
Expression and activation of proteins involved in EGFR mediated AKT pathways
were assessed via immunoblotting and the results are presented in Fig.4. Assessment
in H1975 cells demonstrated that 10c treatment significantly repressed EGFR
phosphorylation and downstream signaling proteins of the AKT pathway in a
dose-dependent manner. Complete inhibition was achieved at a concentration of 5.0
µM, while complete inhibition was achieved at a concentration of 1.0 µM for A431
cells.
11

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B
5
A
Control
10c
(50mg/kg)
4
3
2
10c
(20mg/kg)
10c
(10mg/kg)
1
Rociletinib
(50mg/kg)
control
Rociletinib
(50mg/kg)
10c
10c
(10mg/kg)
10c
(20mg/kg)
(50mg/kg)
Effect on Body Weight
in Xenograft Model of H1975
D
Effect on Tumor Volume
in Xenograft Model of H1975
C
Days post initial treatment (d)
Days post initial treatment (d)
Figure 5. In vivo effects of 10c on the tumor growth of H1975 cell xenografts in nude mice. (A)
Images of the tumor collected from the mice; (B) Tumor weight; (C) Tumor volume; (D) Body
weight. Data are presented as mean ±SD ( n = 5). ∗p <0.05 and ∗∗p <0.01 compared with control
group.
2.4 In vivo antitumor activity
Given the strong observed inhibition of EGFRT790M kinase and on NSCLC cell
growth in combination with its promising cytotoxicity, the in vivo antitumor efficacy
of 10c was evaluated via an EGFRT790M-driven human H1975 xenograft mouse model
(Fig.5). Repeated oral administration of 10c (qd) for 14 days displayed
dose-dependent inhibition of tumor growth. Tumor growth inhibition (TGI) values
were 64.6%, 45.3%, and 38.97% at dosages of 50, 20, and 10 mg/kg/day, respectively
(Fig.5A). In addition, all doses of 10c were well tolerated, without mortality, or
significant loss of body weight (<5% relative to vehicle-matched controls) during
treatment (Fig.5D). This exploration suggests that 10c could inhibit cancer growth as
potently as rociletinb (70.15%) at a dose of 50 mg, however without posing a major
improvement compared to osimertinib (20 mg/kg/day, 98.75%).
12

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2.5 Molecular modeling analysis
Several typical inhibitors, including 10c (the most active), 10b, d, f (the least
active), and 10h (the moderately active) were docked into the ATP binding pocket of
EGFRT790M (PDB: 3IKA) to investgate the putative interaction mechanism of the
DPPYs with the EGFRT790M enzyme, respectively [11]. The program AutoDock 4.2
with its default parameters were used [34, 35]. For comparison, the lead compound
WZ4002 was also analyzed using the same procedure (Fig. 6A).
As shown in Figs.6A and 6B, both inhibitors 7c and WZ4002 could tightly contact
with EGFRT790M through several important binding forces, including: (3) A hydrogen
bond formed by the N-1 nitrogen atom in pyrimidine core with the amino acid
Leu792; (2) A covalent bond between the acryl amide and the amino acid Cys797; (2)
van der Walls force produced by the C-5 chlorine atom in pyrimidine core with the
mutant gatekeeper amino acid Met790. The major difference in EGFRT790M binding
models is that there is less distance between the C-5 chlorine atom of 10c (3.74Å) and
Met790 than in WZ4002 (3.82Å). Possibly, a shorter distance may strengthen the
interaction force with EGFRT790M, thus improving activity and overcoming gefitinib
resistance. Yet, the chlorine atom at the C-5 position of the pyrimidine scaffold
moved away from the key mutant reside Met790 in less potent inhibitors, including
10b (4.15Å, Fig. 6C) with a C-5 fluoro substituent, 10d (4.42Å, Fig. 6D) bearing an
ethyl linker between the morpholine ring and the C-2 aniline, and 10f (5.82Å, Fig. 6E)
with an acetamide liner at the same position. The movement at this position also led to
the disappearance of the important covalent bond between the acryl group and the
amide acid Cys797. Thus, molecules 10b, 10f, and 10g have nearly 100 times lower
inhibitory potency than inhibitor 10c. In the case of the inhibitor 10h (Fig. 6F) with a
C-2'' acrylamide group, the C-5 chloro substituent in pyrimidine core greatly reached
the amino acid Met790 (3.33Å). In addition, compound 10h also retained a strong
hydrogen bond between the N-1 atom in pyrimidine core and the amino acid Leu792.
However, the acrylamide functional group of the C-2 aniline side chain at the C-2''
position moved far away from the amide acid Cys797, eventually resulting in the loss
13

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of the significant covalent bond. These docking observations reasonably explain their
activity data.
A
B
C
Met790
Met790
Met790
Avl743
Leu792
Avl743
Leu792
Avl743
Leu792
Val726
。
3.82A
Val726
Val726
。
3.74A
。
4.15A
Phe795
Thr654
Thr654
Asn842
Phe795
Phe795
Thr654
Asn842
Asn842
Cys797
Cys797
Cys797
Leu799
Val726
Asp800
Leu799
Leu799
Asp800
Asp800
F
D
E
Met790
Met790
Met790
Avl743
Leu792
Avl743
Avl743
。
Val726
Val726
。
4.42A
3.33A
。
5.82A
Leu792
Leu792
Phe795
Phe795
Thr654
Asn842
Phe795
Thr654
Asn842
Thr654
Asn842
Cys797
Cys797
Cys797
Leu799
Leu799
Leu799
Asp800
Asp800
Asp800
Figure 6. Proposed binding models of the typical inhibitors with EGFRT790M enzyme (PDB code:
3IKA), A: WZ4002, B: inhibitor 10c, C: inhibitor 10b, D: inhibitor 10d, E: inhibitor 10f, F:
inhibitor 10h.
3. Conclusion
Structural optimization of the Mor-DPPY template led to a series of potential
EGFRT790M inhibitors with improved activity and selectivity for treatment of the
gefitinib-resistant NSCLC. The most promising inhibitor 10c, not only displayed high
activity against EGFRT790M/L858R kinase (IC50 = 0.71 nM), but also was able to repress
the replication of H1975 cells harboring EGFRT790M mutation at a concentration of
0.037 µM. Inhibitor 7c showed high selectivity (SI = 631.9) for T790M-containing
EGFR mutants over wild type EGFR, and was lower toxic to normal HBE cells,
suggesting its less side effects. Furthermore, it exhibited high antitumor efficacy in
vivo in an EGFRT790M/L858R-driven H1975 xenograft mouse model without obvious
toxic signs during treatment. Overall, this discovery provides new promising lead
compounds for the further development of anti-NSCLC drugs.
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4. Experimental section
4.1. General Methods and Chemistry.
Unless otherwise noted, solvents and reagents were obtained from commercial
supplies and were used without further purifications. High resolution ESI-MS was
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–l [26–31].
4-Morpholine-substituted aniline intermediates 18, 23, 27, 31 were generally
prepared using the procedures showed in literatures 28 to 31. While the intermediates
N-(3-((2-chloropyrimidin-4-yl)oxy)phenyl)acrylamides 13a-d were synthesized based
on the reported method [26, 27]. All these intermediates were directly used without
any purification and structural characterization. With these intermediates in hand, the
desired compounds were synthesized as described below.
A flask was charged with compounds 13a-d (0.70 mmol), anilines 18, 23, 27, 31
(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-[2-methoxyl-4-((1-morpholino)methyl)phenylamino]-4-pyrimidi
nyl]amino]phenyl]acrylamide (10a).
15

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Yield 40.1%; off-white solid.1H NMR (DMSO-d6): δ 10.16 (s, 1H), 8.96 (s, 1H), 8.13
(s, 1H), 7.98 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.77 (s, 1H), 7.44 (s, 1H), 7.26 (d, J =
4.8 Hz, 2H), 6.90 (s, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.44 (dd, J = 16.8, 9.6 Hz, 1H),
6.27 (dd, J = 16.8, 2.0 Hz, 1H), 5.74 (dd, J = 12.0, 2.0 Hz, 1H), 3.81 (s, 3H), 3.56 (s,
6H), 2.32 (s, 4H). HRMS (ESI) for C25H27ClN6O3, [M+H]+ calcd: 495.1906, found:
495.1858.
N-[3-[[5-Fluoro-2-[4-((1-morpholino)methyl)phenylamino]-4-pyrimidinyl]amino]p
henyl]acrylamide (10b).
Yield 38.2%; off-white solid. 1H NMR (DMSO-d6): δ 10.15 (s, 1H), 9.43 (s, 1H), 9.16
(s, 1H), 8.10 (s, 1H), 7.95 (s, 1H), 7.60 (d, J = 6.8 Hz, 2H), 7.47 (s, 2H), 7.28 (s, 1H),
7.08 (s, 2H), 6.45 (dd, J = 16.0, 9.6 Hz, 1H), 6.26 (d, J = 16.0 Hz, 1H), 5.76 (d, J =
8.8 Hz, 1H), 3.55 (s, 3H), 3.34 (s, 5H), 2.31 (s, 2H). HRMS (ESI) for C24H25FN6O2,
[M+H]+ calcd: 449.2096, found: 449.2015.
N-[3-[[5-Chloro-2-[4-((1-morpholino)methyl)phenylamino]-4-pyrimidinyl]amino]
phenyl]acrylamide (10c).
Yield 40.8%; off-white solid. 1H NMR (DMSO-d6): δ 10.20 (s, 1H), 9.32 (s, 1H), 8.96
(s, 1H), 8.15 (s, 1H), 7.91 (s, 1H), 7.56 (d, J = 8.4 Hz, 3H), 7.31 (d, J = 6.0 Hz, 2H),
7.04 (d, J = 8.4 Hz, 2H), 6.48 (dd, J = 17.2, 10.4 Hz, 1H), 6.27 (dd, J = 16.8, 2.0 Hz,
13
1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.55 (br, 4H), 3.32 (s, 2H), 2.29 (s, 4H); C
NMR (DMSO-d6) δ 163.6 , 158.2, 156.7, 155.3, 139.8, 139.7, 139.6, 139.3, 132.4,
130.6, 129.4 (2C), 129.0, 127.3, 119.8, 119.3 (2C), 115.8, 104.2, 66.7 (2C), 62.6, 53.6
(2C). HRMS (ESI) for C24H25ClN6O2, [M+H]+ calcd: 465.1800, found: 465.1808.
N-[3-[[5-Chloro-2-[4-((1-morpholino)ethotxy)phenylamino]-4-pyrimidinyl]amino]
phenyl]acrylamide (10d).
Yield 32.5%; off-white solid. 1H NMR (DMSO-d6): δ 10.18 (s, 1H), 9.14 (s, 1H), 8.88
(s, 1H), 8.08 (s, 1H), 7.87 (s, 1H), 7.47 (d, J = 8.8 Hz, 3H), 7.29 (d, J = 6.8 Hz, 2H),
6.68 (d, J = 8.8 Hz, 2H), 6.44 (dd, J = 16.8, 10.0 Hz, 1H), 6.24 (dd, J = 16.8, 2.0 Hz,
1H), 5.74 (dd, J = 10.0, 2.0 Hz, 1H), 3.94 (t, J = 6.0 Hz, 2H), 3.66-3.44 (m, 4H), 3.35
16

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13
(s, 2H), 2.62 (t, J = 5.6 Hz, 2H), 2.49 (s, 2H); C NMR (DMSO-d6) δ 163.6, 158.2,
156.6, 155.3, 153.6, 139.5 (2C), 139.3, 134.0, 132.3, 129.1, 127.4, 121.1 (2C), 119.6,
115.9, 114.6 (2C), 103.7, 66.79 (2C), 65.8, 57.6, 54.19 (2C).
HRMS (ESI) for C25H27ClN6O3, [M+H]+ calcd: 495.1906, found: 495.1813.
N-[3-[[5-Chloro-2-[3-chloro-4-((1-morpholino)ethotxy)phenylamino]-4-pyrimidiny
l]amino]phenyl]acrylamide (10e).
Yield 36.5%; off-white solid. 1H NMR (DMSO-d6): δ 10.23 (s, 1H), 9.60 (s, 1H), 9.31
(s, 1H), 8.21 (s, 1H), 7.86 (s, 1H), 7.75 (s, 1H), 7.44 (s, 2H), 7.31 (s, 2H), 7.00 (s,
1H), 6.47 (s, 1H), 6.24 (d, J = 16.4 Hz, 1H), 5.75 (s, 1H), 4.33 (s, 2H), 3.99 (s, 2H),
3.27 (s, 3H), 2.28 (s, 6H). HRMS (ESI) for C25H26Cl2N6O3, [M+H]+ calcd: 529.1516,
found: 529.1476.
N-[3-[[5-Chloro-2-[4-((1-morpholino)acetyl)phenylamino]-4-pyrimidinyl]amino]p
henyl]acrylamide (10f).
Yield 29.7%; off-white solid. 1H NMR (DMSO-d6): δ 10.19 (s, 1H), 9.30 (s, 1H), 8.95
(s, 1H), 8.14 (s, 1H), 7.87 (s, 1H), 7.50-7.55 (m, 3H), 7.32 (d, J = 7.6 Hz, 2H), 6.97 (d,
J = 8.4 Hz, 2H), 6.46 (dd, J = 16.8, 10.0 Hz, 1H), 6.26 (dd, J = 16.8, 1.6 Hz, 1H), 5.76
13
(dd, J = 10.4, 2.0 Hz, 1H), 3.57 (s, 2H), 3.51 (d, J = 4.4 Hz, 2H), 3.44 (s, 6H); C
NMR (DMSO-d6) δ 169.7, 163.6, 158.1, 156.6, 155.2, 139.6, 139.3, 139.2, 132.4,
129.2 (2C), 129.0, 128.6, 127.4, 119.8, 119.3 (2C), 115.9, 115.7, 104.2, 66.5 (2C),
46.4,
42.1
(2C).
HRMS (ESI) for C25H25ClN6O3,
[M+H]+ calcd: 493.1749, found: 493.1766.
N-[3-[[5-Chloro-2-[4-((1-morpholino)acetyl)phenylamino]-4-pyrimidinyl]oxygen]p
henyl]acrylamide (10g).
Yield 26.1%; off-white solid. 1H NMR (DMSO-d6): δ 10.39 (s, 1H), 9.75 (s, 1H), 8.48
(s, 1H), 7.70 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.44-7.48 (m,1H), 7.33 (d, J = 6.8 Hz,
2H), 7.02 (dd, J = 7.6, 1.6 Hz, 1H), 6.90 (d, J = 8.0 Hz, 2H), 6.44 (dd, J = 17.2, 10.4
Hz, 1H), 6.27 (dd, J = 16.8, 2.0 Hz, 1H), 5.78 (dd, J = 10.0, 1.6 Hz, 1H), 3.56 (s, 2H),
13
3.54-3.47 (m, 2H), 3.43 (d, J = 4.4 Hz, 6H); C NMR (DMSO-d6) δ 169.6, 164.3,
17

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163.8, 158.5, 158.0, 152.8, 140.8, 138.5, 132.1, 130.4, 129.3, 129.2 (2C), 127.8, 119.2
(2C),
117.5,
117.0,
113.2,
66.5
(2C),
46.4,
42.1
(2C).
HRMS (ESI) for C25H24ClN5O4, [M+H]+
calcd: 494.1590, found: 494.1604.
N-[2-[[5-Chloro-2-[4-((1-morpholino)methyl)phenylamino]-4-pyrimidinyl]amino]
phenyl]acrylamide (10h).
Yield 30.5%; off-white solid. 1H NMR (DMSO-d6): δ 10.21 (s, 1H), 9.60 (s, 1H), 8.72
(s, 1H), 8.18 (s, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.56 (d, J =
7.6 Hz, 1H), 7.32 (dd, J = 11.2, 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 6.53 (dd, J =
16.8, 10.0 Hz, 1H), 6.31 (d, J = 16.8 Hz, 1H), 5.80 (d, J = 10.0 Hz, 1H), 4.23 (s, 2H),
13
3.94 (d, J = 12.0 Hz, 2H), 3.21 (d, J = 12.0 Hz, 3H), 3.14 -2.99 (m, 3H); C NMR
(DMSO-d6) δ 164.7, 157.6, 157.1, 142.0, 138.3 (2C), 132.3, 132.1, 131.7, 131.5,
128.6 (2C), 128.3, 128.1, 126.0 (2C), 121.7, 119.0, 105.1, 63.7, 59.7, 51.0, 43.3, 21.3.
HRMS (ESI) for C24H25ClN6O2, [M+H]+ calcd: 465.1800, found: 465.1835.
N-[2-[[5-Chloro-2-[4-((1-morpholino)ethotxy)phenylamino]-4-pyrimidinyl]amino]
phenyl]acrylamide (10i).
Yield 21.0%; off-white solid. 1H NMR (DMSO-d6): δ 10.19 (s, 1H), 9.87 (s, 1H), 9.48
(s, 1H), 8.91 (s, 1H), 8.17 (s, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 8.8 Hz, 2H),
7.30 (dd, J = 16.4, 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.53 (dd, J = 16.8, 10.0 Hz,
1H), 6.32 (d, J = 16.8 Hz, 1H), 5.81 (d, J = 10.0 Hz, 1H), 4.35-4.20 (m, 2H), 3.99 (d,
J = 12.4 Hz, 2H), 3.71 (t, J = 12.0 Hz, 4H), 3.50 (d, J = 12.4 Hz, 2H), 3.19 (d, J =
13
18.0 Hz, 2H); C NMR (DMSO-d6) δ 164.7, 157.1, 153.2, 138.3 (2C), 132.2, 131.5,
128.6 (3C), 128.3, 128.0, 126.5, 126.0 (2C), 124.8, 121.5, 115.1, 104.5, 63.7, 62.6,
55.6, 52.2, 21.3 (2C). HRMS (ESI) for C25H27ClN6O3, [M+H]+ calcd: 495.1906,
found: 495.1950.
N-[2-[[5-Chloro-2-[4-((1-morpholino)propoxy)phenylamino]-4-pyrimidinyl]amino
]phenyl]acrylamide (10j).
18

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Yield 26.8%; off-white solid. 1H NMR (DMSO-d6): δ 10.20 (s, 1H), 9.74 (s, 1H), 9.44
(s, 1H), 8.88 (s, 1H), 8.16 (s, 1H), 7.70 (d, J = 6.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H),
7.31 (dd, J = 9.2, 1.6 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 6.53 (dd, J = 17.2, 10.4 Hz,
1H), 6.32 (dd, J = 16.8, 1.6 Hz, 1H), 5.80 (d, J = 10,0 Hz, 1H), 3.96-4.01 (m, 4H),
3.67 (t, J = 12.0 Hz, 3H), 3.28 (d, J = 5.2 Hz, 3H), 3.16-3.02 (m, 2H), 2.11 (dd, J =
10.0, 6.0 Hz, 2H); 13C NMR (DMSO-d6) δ 164.7, 157.0, 153.9, 145.9, 138.3 (2C),
132.2, 131.5, 128.6 (3C), 128.3, 128.0, 126.0 (2C), 124.8, 121.5, 114.7, 104.4, 65.4,
63.9,
54.1,
51.7,
21.7,
21.3
(2C).
HRMS (ESI) for C23H22ClN10O4,
[M+H]+ calcd: 509.2062, found: 509.2110.
N-[2-[[5-Chloro-2-[4-((1-morpholino)acetoxyl)phenylamino]-4-pyrimidinyl]amino
]phenyl]acrylamide (10k).
Yield 30.5%; off-white solid. 1H NMR (DMSO-d6): δ 10.23 (s, 1H), 9.15 (s, 1H), 8.49
(s, 1H), 8.09 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.42 (d, J =
9.2 Hz, 2H), 7.32-7.35 (m,1H), 7.27 (dd, J = 10.8, 7.2 Hz, 1H), 6.72 (d, J = 8.8 Hz,
2H), 6.52 (dd, J = 17.2, 10.4 Hz, 1H), 6.33 (dd, J = 16.8, 1.6 Hz, 1H), 5.81 (dd, J =
10.4, 1.6 Hz, 1H), 4.73 (s, 2H), 3.69-3.50 (m, 4H), 3.47 (s, 4H); 13C NMR
(DMSO-d6) δ 166.7, 164.7, 158.3, 156.5, 155.0, 153.1, 134.4, 132.1, 131.8, 131.4,
128.2, 127.9, 126.0, 125.9, 124.9, 120.8 (2C), 114.7 (2C), 104.0, 66.7, 66.6 (2C), 45.3,
40.6. HRMS (ESI) for C25H25ClN6O4, [M+H]+ calcd: 509.1699, found: 509.1748.
N-[2-[[5-Chloro-2-[4-((1-morpholino)acetylamino)phenylamino]-4-pyrimidinyl]am
ino]phenyl]acrylamide (10l).
1
Yield 40.5%; faint yellow solid. H NMR (DMSO-d6): δ10.23 (s, 1H), 9.58 (s, 1H),
9.28 (s, 1H), 8.53 (s, 1H), 8.12 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.38-7.48 (m, 3H),
7.37 (d, J = 8.4 Hz, 2H), 7.30 (dd, J = 15.6, 7.6 Hz, 2H), 6.53 (dd, J = 16.8, 10.4 Hz,
1H), 6.33 (d, J = 17.2 Hz, 1H), 5.81 (d, J = 10.4 Hz, 1H), 3.64 (d, J = 4.0 Hz, 4H),
13
3.37 (s, 4H), 3.09 (s, 2H); C NMR (DMSO-d6) δ 168.0, 164.7, 158.1, 156.6, 155.0,
136.6, 132.8, 132.0, 131.5, 128.2, 128.0, 126.0, 125.0, 120.2 (2C), 120.0, 119.3 (2C),
66.6 (2C), 62.5, 53.7 (2C). HRMS (ESI) for C25H26ClN7O3, [M+H]+ calcd: 508.1858,
19

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found: 508.1900.
4.3. Kinase enzymatic assays
All the enzymatic assays were tested applying with the ADP-GloTM assay system
(EGFRWT: Catalog. V3831, EGFRT790M/L858R: Catalog. V5324), purchased from
Promega Corporation (USA). The experiments were performed according to the
instructions of the manufacturer. The 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, including the main steps below: (1)
perform a 5 ꢀL kinase reaction using 1× kinase buffer (e.g., 1× reaction buffer A), (2)
incubate at room temperature for 60 minutes, (3) add 5 ꢀL 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 ꢀL 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%
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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 lines 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 ꢀM), 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. Western blotting assay
The lysates from H1975 and A431 cells in different groups were extracted and
centrifuged at 12,000 g for 15 min at 4 °C, then the total proteins were obtained. An
aliquot (50 ꢀg protein) was loaded onto a 8%–12% SDS-PAGE gels and separated
electrophoretically. Then the target proteins were transferred to a PVDF membrane
(Millipore, USA). After blocking the PVDF membrane in 5% dried skim milk (Boster
Biological Technology, China) for 3 h at room temperature, the membrane was
incubated overnight at 4 °C with primary antibodies for 3 h at room temperature.
Protein detection was performed based on an enhanced chemiluminescence (ECL)
method and photographed by using a BioSpectrum Gel Imaging System (HR410,
UVP, USA). In order to eliminate the variations, data were adjusted to Actin
expression: IOD of objective protein versus IOD of Actin expression
4.6. Mouse tumor xenograft efficacy study
The STOCK-Foxn1nu/Nju nude mice weighing 20–25 g in 5–6 week-old were
provided by the Model Animal Research Center of Nanjing University, Nanjing,
China (SCXK: 2015-0001). All animals were housed in a controlled environment at
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23 ±2 °C under a 12-h dark/light cycle with free access to food and water. The animal
maintenance and experiments were performed in accordance with the guidelines of
the Animal Care and Use Committee. H1975 cells (3× 106) were suspended in 0.1 mL
of PBS and injected subcutaneously into the right oxter region of the mice. Three days
after implantation, the mice were randomly divided into seven groups in which the
mice in control group were received by oral 25% PEG-400, and the mice in other six
groups were oral administrated with 10c at the doses of 10, 20 and 50 mg/kg,
rociletinib at the doses of 50 mg/kg, osimertinib at the doses of 10, 20 mg/kg for 14
days. In the process, the side length of the tumor was measured to calculate the tumor
2
volume based on the formula: V = A ×B /2, where A means the lager perpendicular
diameters and B is the smaller perpendicular diameters. At the end of the test, the
animals were sacrificed and the tumors were obtained, photographed and weighted.
4.7. Molecular docking study
AutoDock 4.2 software was used to carried 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, molecules 10b-d, f and 10h
were 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.
Acknowledgements
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We are grateful to the National Natural Science Foundation of China (No.
81402788), and the Cultivation Project of Youth Techstars, Dalian, China (No.
2016RQ043) for the financial support of this research.
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