Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell lines

Article abstract of DOI:10.1016/j.bioorg.2019.103408

A new class of pyrimidine derivatives were designed and synthesized as potential dual FAK and EGFR^T790M inhibitors using a fragment-based drug design strategy. This effort led to the identification of the two most active inhibitors, namely 9a and 9f, against both FAK (IC₅₀ = 1.03 and 3.05 nM, respectively) and EGFR^T790M (IC₅₀ = 3.89 and 7.13 nM, respectively) kinase activity. Moreover, most of these compounds also exhibited strong antiproliferative activity against the three evaluated FAK-overexpressing pancreatic cancer (PC) cells (AsPC-1, BxPC-3, Panc-1) and two drug-resistant cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells) at concentrations lower than 6.936 μM. In addition, 9a was also effective in the in vivo assessment conducted in a FAK-driven human AsPC-1 cell xenograft mouse model. Overall, this study offers a new insight into the treatment of hard to treat cancers.

Full text of DOI:10.1016/j.bioorg.2019.103408

Journal Pre-proofs
Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential
dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell
lines
Min Ai, Changyuan Wang, Zeyao Tang, Kexin Liu, Xiuli Sun, Tengyue Ma,
Yanxia Li, Xiaodong Ma, Lei Li, Lixue Chen
PII:
S0045-2068(19)31065-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103408
YBIOO 103408
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
5 July 2019
28 September 2019
28 October 2019
Please cite this article as: M. Ai, C. Wang, Z. Tang, K. Liu, X. Sun, T. Ma, Y. Li, X. Ma, L. Li, L. Chen, Design
and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential dual EGFR T790M and FAK inhibitors
against a diverse range of cancer cell lines, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103408
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Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as
potential dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell lines
Min Ai a,1, Changyuan Wang a,1, Zeyao Tanga, Kexin Liua, Xiuli Sunb, Tengyue Mac, Yanxia Lib, Xiaodong Maa,
Lei Lia, and Lixue Chena,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
b Department of Hematology, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China.
c Dalian Buyun Biological Technology Co., Ltd., 116085, PR China.
1 These authors contributed equally to this work.
E-mail address: lilei0332@126.com.
Abstract
A new class of pyrimidine derivatives were designed and synthesized as potential dual FAK and
EGFRT790M inhibitors using a fragment-based drug design strategy. This effort led to the
identification of the two most active inhibitors, namely 9a and 9f, against both FAK (IC50 = 1.03
and 3.05 nM, respectively) and EGFRT790M (IC50 = 3.89 and 7.13 nM, respectively) kinase activity.
Moreover, most of these compounds also exhibited strong antiproliferative activity against the three
evaluated FAK-overexpressing pancreatic cancer (PC) cells (AsPC-1, BxPC-3, Panc-1) and two
drug-resistant cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells) at
concentrations lower than 6.936 μM. In addition, 9a was also effective in the in vivo assessment
conducted in a FAK-driven human AsPC-1 cell xenograft mouse model. Overall, this study offers a
new insight into the treatment of hard to treat cancers.
Key Words: FAK; EGFR T790M; Inhibitor; Dual-target; Fragment-based drug design
1

1. Introduction
Cancer is one of the major diseases that endangers human health and even lives worldwide
today. There were 4.3 million new cancer cases and more than 2.8 million cancer deaths in China in
2015, with lung cancer having the highest incidence, and pancreatic cancer (PC) being the leading
cause of cancer death [1,2]. Receptor tyrosine kinases (RTKs) and non-RTKs play important roles
in cancer cell proliferation, survival and migration in response to extracellular signals.
Small-molecule drugs targeting dysregulated RTKs have various clinical benefits, especially for
genetically defined patient populations [3-5]. Focal adhesion kinase (FAK), one of the important
non-receptor protein tyrosine kinases, is highly overexpressed in several cancers, including thyroid,
brain, liver, prostate, breast, colon, head and neck. The triggering of FAK is initiated by the binding
of integrin to the extracellular matrix (ECM), followed by FAK autophosphorylation at Tyr-397,
which is crucial for most cell functions [6-9]. Until now, a number of small-molecule FAK
inhibitors have been identified and shown to be potent inhibitors interfering tumor growth and
metastasis in several preclinical and clinical models [10-13]. TAE-226 (1) is a typical FAK
inhibitor (IC50=5.5 nM), with potent antiproliferative and antitumor effects in vitro and in vivo in
several types of cancers [10]. Another novel FAK inhibitor, CEP-37440 (2, phase I, NCT01922752),
is currently in the clinical testing stage for cancer treatment [11]. Also, VS-4718 (3, phase II,
NCT02465060), significantly impacts cell viability, decreases anchorage-independent growth and
motility in selected PC cells by targeting the FAK kinase activity and inhibiting Y397
phosphorylation [12,13] (Figure 1). Undoubtedly, searching for effective FAK inhibitor offers new
tools to gain further understanding of the antitumor mechanisms underlying these inhibitors.
2

OH
N
A
O
N
N
MeHN
O
N
O
O
N
N
S
MeHN
MeHN
N
Me
OMe
OMe
Me
O
HN
Cl
N
NH
O
HN
Cl
N
NH
HN
F3C
N
NH
N
N
1
2
3
TAE-226
CEP-37440
VS-4718
R
N
B
NMe2
N
O
N
O
O
N
MeN
HN
HN
HN
OMe
OMe
OMe
MeN
X
N
NH
HN
F3C
N
NH
N
NH
N
N
N
R1
4 WZ-4002 X=O, R1= Cl, R= Me
5 CO-1686 X=NH, R1= CF3, R= MeCO-
7
6
Az-DPPY
AZD-9291
Fig. 1. Chemical structures of the novel pyrimidine derivatives as potent non-RTK (A) or RTK (B) inhibitors.
The epidermal growth factor receptor (EGFR) tyrosine kinase is another valuable clinically
validated target for anticancer therapies, especially for non-small-cell lung cancer (NSCLC)
treatment [14,15]. However, the T790M mutation (threonine to methionine) within the ATP site of
the EGFR, occurs frequently (in approximately 60% of the patients) in NSCLC patients who
develop resistance to drugs targeting EGFR, and thus leads to failure of EGFR-targeted therapy
[16,17]. The pyrimidine derivatives WZ-4002 (4) [18], CO-1686 (5, Phase III) [19], and AZD-9291
(6, approved in 2015) [20,21] are novel EGFRT790M inhibitors against resistant NSCLC disease. Our
considerable efforts on the structural modification of the pyrimidine scaffold also led to the
identification of several promising EGFRT790M inhibitors, including Az-DPPYs (7, Figure 1) [22]
and Mor-DPPYs (8, Figure 2) [23]. All these works indicated that the pyrimidine core along with
the C-2,4 dianiline side chains were essential for the high anticancer activity of the inhibitors
3

[18,24,25]. Using
a
fragment-based drug design strategy, herein,
a
class of
N-alkylbenzamide-substituted diphenylpyrimidine (DPPYs) were synthesized based on the
TAE-226 and Mor-DPPY scaffold (Figure 2). Fortunately, most of these compounds not only
displayed strong anti-FAK activity, but also displayed high EGFRT790M inhibitory potency. These
dual FAK and EGFRT790M inhibitors, their synthetic method and the characterization of their
anticancer activity are described in this study.
O
Me
O
Glu506/Gl
N
N
N
O
O
y
O
429
HN
O
N
HN
HN
Improved activity
Asp546
HN
O
R1
OMe
HN
N
NH
HN
F3C
N
NH
H
N
N
N
R
Cl
Mor-DPPY (8)
EGFRT790M inhibitor
Fragment-based
drug design
O
HN
Cl
N
NH
Rociletinib (5)
EGFRT790M inhibitor
Phase III
N
Cys502
O
N
DPPYs 9a-m
FAK and EGFRT790M dual-inhibitor
MeHN
OMe
O
HN
Cl
N
NH
N
TAE-226 (1)
FAK inhibitor
Fig. 2. Designed strategy of the title molecules DPPYs.
2. Results and discussion
2.1 Chemistry
4

Cl
N
NO2
O
NH2 O
NO2
O
Cl
N
NH
O
R
R
R
c
a,b
N
N
OH
d
N
H
H
H
12a-d
13a-d
10
11a-d
NO2
Cl
NH2
NH
N
NO2
HN
N
NH
O
h
f,g
e
R
R1
R1
N
H
R1
NH
N
Br
R1
9a-m
NH2
R = Me, Et, i-Pr,
Cyclopropyl
O
O
O
NH
N
14a-d
16a-d
15a-d
O
O
R1 = H, CH3, OCH3, Cl
Scheme 1. Synthetic route of title compounds 9a-m. Reagents and conditions: a) SOCl2, 1 h, 60 C; b)
methylamine hydrochloride/ethylamine hydrochloride/isopropylamine hydrochloride/cyclopropylamine
hydrochloride, NaHCO3, CH3CN, 4 h, 0 C, 91-97%; c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 C, 65-78%; d)
2,4,5-trichloropyrimidine, N,N-diisopropylethylamine (DIPEA), isopropanol, 6 h, 80 C, 72-85%; e) bromoacetyl
bromide, NaHCO3, CH3CN, 2 h, 0 C, 85-91%; f) morpholine, K2CO3, KI, CH3CN, 4 h, 60 C, 75-80%; g)
Fe-NH4Cl, MeOH-H2O, 2 h, 70 C, 72-81%; h) 13a-d,trifluoroacetic acid, 2-BuOH, 100 C, 12 h, 11-22%.
All the newly designed DPPYs were synthesized applying the similar synthetic method
illustrated in our previous work (Scheme 1) [22,26,27]. Commercially available 2-nitrobenzene acid
(10) was reacted with SOCl2, and then directly reacted with alkylamine in the presence of the base
NaHCO3 to produce 11a-d. The 11a-d was then reduced to give 12a-d using the Fe-NH4Cl
condition. After region-selective substitution of the C-4 chlorine atom in the
2,4,5-trichloropyrmidine reagent with arylamine, the 2,5-dichloropyrimidine intermediate 13a-d
was synthesized. In addition, various C-2 aniline side chains were prepared starting from the
material nitroanilines 14a-d which were reacted with bromoacetyl bromide to yield 15a-d.
Compounds 15a-d were treated with morpholine, and then reduced by Fe-NH4Cl reagent to prepare
the anilines 16a-d. Ultimately, the title molecules 9a-m were conveniently synthesized via the
coupling reaction of anilines 16a-d with chloro-substituted pyrimidines 13a-d in the presence of
trifluoroacetic acid (TFA).
2.2 Biological activity
5

Table 1. Inhibitory activity of the newly synthesized compounds against FAK and EGFRT790M kinases.
Cl
N
HN
N
NH
O
R
N
H
R1
NH
N
O
O
Enzymatic activity(IC50, nM)a
Compound
R
R1
CH3
FAK
EGFRT790M
3.89±0.12
20.7±2.31
32.7±1.99
6.14±0.62
41.5±1.43
7.13±1.26
99.1±4.92
22.3±1.35
44.8±2.71
37.6±1.20
89.2±2.86
66.5±1.25
48.4±2.68
>100
9a
9b
Me
1.03±0.14
106.7±2.98
11.8±2.21
101.0±0.87
125.7±3.35
3.05±0.53
117.7±3.39
3.54±0.93
150.0±2.16
24.9±0.73
9.89±2.70
30.6±2.95
40.0±1.34
6.8±0.79
Me
OCH3
Cl
9c
Me
9d
Cyclopropyl
CH3
OCH3
H
9e
Cyclopropyl
9f
Et
Et
9g
CH3
OCH3
Cl
9h
Et
9i
Et
9j
i-Pr
i-Pr
i-Pr
i-Pr
H
9k
CH3
OCH3
Cl
9l
9m
TAE-226
a
Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in
nanomolar needed to inhibit cell growth by 50% as determined from these curves.
Table 2. Antiproliferative activity of the title molecules against various human cancer cell lines.
Antiproliferative activity(IC50, μM)a
Compound
AsPC-1
BxPC-3
Panc-1
Hpde6-c7
1.347±0.15
2.603±0.25
2.738±0.27
MCF/Adr
0.892±0.10
0.741±0.09
2.143±0.17
H1975
9a
9b
9c
0.909±0.18 0.761±0.14 1.218±0.22
0.814±0.19 1.210±0.14 2.007±0.07
0.909±0.18 1.510±0.19 2.084±0.06
0.333±0.02
0.460±0.05
0.882±0.07
6

9d
2.554±0.28 1.672±0.23 1.767±0.14
4.134±0.32 2.972±0.19 1.695±0.09
1.426±0.23 1.873±0.05 0.994±0.16
1.882±0.13 1.947±0.21 1.787±0.21
1.757±0.24 3.090±0.34 1.996±0.17
2.436±0.38 1.699±0.24 0.856±0.20
2.917±0.12 2.309±0.14 1.299±0.10
3.848±0.14 2.324±0.14 1.990±0.20
6.936±0.41 3.630±0.28 4.020±0.08
3.795±0.29 2.477±0.12 2.134±0.08
1.696±0.17
3.561±0.28
1.367±0.09
2.066±0.20
4.405±0.25
2.447±0.18
5.445±0.25
3.938±0.19
5.967±0.31
4.985±0.27
11.380±0.33
1.835±0.15
1.874±0.03
1.972±0.09
0.716±0.03
2.544±0.15
1.383±0.27
2.796±0.19
3.613±0.23
--
0.602±0.09
0.950±0.09
0.478±0.04
0.513±0.09
0.922±0.13
0.983±0.14
0.892±0.08
0.918±0.10
--
9e
9f
9g
9h
9i
9j
9k
9l
9m
--
--
TAE-226
6.730±0.31 12.330±0.42
>20.00
>8.00
6.79±0.17
a
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.
All the title molecules were evaluated for their inhibitory activity against FAK kinase with the
ADP-GloTM kinase assay, as shown in Table 1[28,29]. Clearly, these newly synthesized compounds
displayed strong inhibitory activity against the FAK enzyme within the drug concentrations ranging
from 1.03 to 150.0 nM. Four typical molecules 9a (IC50=1.03 nM), 9f (IC50=3.05 nM), 9h
(IC50=3.54 nM), and 9k (IC50=9.89 nM) remarkably interfered with the FAK enzymatic activity
with IC50 values lower than 10 nM. The most active inhibitor, 9a, even displayed 6-fold higher
activity than the representative FAK inhibitor TAE-226 (6.79 nM). Moreover, their activity against
the EGFRT790M also showed that these molecules can exert strong inhibitory effect on this mutant
kinase with the drug concentrations in the range of 3.89 to 99.1 nM. In particular, two potent FAK
inhibitory compounds, 9a and 9f, also displayed strong anti-EGFRT790M activity, with IC50 values of
3.89 and 7.13 nM, respectively. Noteworthy, compound 9d significantly enhanced the inhibitory
7

activity against EGFRT790M (IC50 = 6.14 nM) despite its moderate inhibitory activity against FAK
kinase (IC50 = 101 nM). These kinase-based test results revealed that the steric cyclopropyl group
(R) in the C-1 aniline side chain is unfavorable to inhibit FAK enzymatic activity. The exemplary
compounds 9d and 9e only exhibited moderate inhibitory activity, with IC50 values of 101.0 and
125.7 nM, respectively. In addition, compounds bearing an isopropyl group (R) also do not favor to
enhance their anti-FAK and EGFR enzymatic activity, indicating that large substituent (R) is
unbeneficial for these inhibitors. While a relatively small group, such as methyl or ethyl substituent
is suitable. The representative compounds 9a and 9f displayed strong inhibitory effects on activity
of the two kinases. The result that 9h showed strong inhibitory potency to FAK kinase, but is weak
to inhibit the activity of EGFRT790M kinase, suggested that methoxy substitution (R1) is
disadvantageous for identification of dual-target inhibitors. Overall, the activity in the kinase level
indicated that this structural modification leads to the formation of two compounds, namely 9a and
9f, which are potent dual FAK-EGFRT790M inhibitors.
Additionally, in this study, all the title molecules were also evaluated for their antiproliferative
activity towards tumor cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay [30]. Three FAK-overexpressed PC cells (AsPC-1, BxPC-3, Panc-1) and two
drug-resistant cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells) were
used for the evaluation of the title molecules. TAE-226, a typical FAK inhibitor was also
concurrently evaluated as a reference. The test results, shown in Table 2, revealed that these
molecules generally have strong inhibitory activity toward the evaluated tumor cells, with IC50
values less than 6.936 μM (compound 9l). The representative compound 9a strongly inhibited the
8

proliferation of AsPC-1 and BxPC-3 cells at concentrations lower than 1 μM, with IC50 values of
0.909 and 0.761 μM, respectively. Moreover, compound 9a also showed antiproliferative activity
against Panc-1 cells, at such low drug concentration of 1.218 μM. Evidently, the detected activity of
inhibitor 9a against cancer cell is consistent with its strong anti-FAK enzymatic activity. Excitingly,
these molecules also have strong anti-proliferative activity against Gefitinib-resistant H1975 cells
(NSCLC), with IC50 values ranging from 0.333 to 0.918 μM. Among these molecules, the most
active inhibitor 9a still showed the strongest inhibitory effect (IC50=0.333 μM) on this mutant cell
line. Furthermore, compounds 9a, 9b, and 9g were also able to significantly inhibit the
drug-resistant breast cancer cells (MCF-7/adr) within the concentrations of 1 μM. Notably, 9b
exhibited strong anti-proliferative activity against Aspc-1 cells (IC50=0.814 μM), but weak
anti-FAK activity, indicating that it might block the proliferation of cancer cells through other
signaling pathway independent of FAK or had weak capability to penetrate the cell membrane.
Overall, these results showed that compound 9a not only possesses strong inhibitory activity against
the kinase and tumor cells, but also showed weak cytotoxicity activity to the normal human
pancreatic ductal cell (Hpde6-c7) within concentration of 1.347 μM, which warrants further
biological evaluation.
a)
b)
Fig. 3. The effects of treating time and concentrations of inhibitor 9a on cell viability, a) AsPC-1, b) Panc-1.
9

The data shown in Figure 3 indicate that the most active inhibitor, 9a, remarkably decreased the
cell viability of both AsPC-1 and Panc-1 cells with the increase of the treatment time and drug
concentration. Indeed, the AsPC-1 and Panc-1 cells were almost completely inhibited after
treatment with 8 μM of 9a for 24 h, with cell viability rate of 3.4 and 3.7%, respectively.
Fig. 4. Compound 9a induced AsPC-1 cell apoptosis in vitro. The cells were incubated with the indicated
concentrations of 9a for 72 h, and the cells were stained with annexin V/FTIC, followed by flow cytometry
analysis. One representative experiment is shown.
10

0.25μM
0.5μM
Blank
G0/G1: 57.38%
S: 30.80%
G2/M: 15.42%
G0/G1: 66.16%
S: 28.14%
G2/M : 5.7 %
G0/G1: 63.89%
S: 30.39%
G2/M: 5.72%
a)
1.0μM
80
G0/G1
S
60
40
20
0
G2/M
G0/G1: 39.90%
S: 32.91%
G2/M: 27.19%
k
M
M
M
n
μ
μ
μ
a
l
5
5
0
.
.
2
B
.
0
1
0
Drug concentration
Fig. 5. Effects of molecule 9a on AsPC-1 cells cycle arrest detected by flow cytometry assay. Cells were treated
with different concentrations of 9a for 72 h, collected and fixed with 70% ethanol at 4 °C overnight. Then, the
cells were stained by the mixture containing 5 mL propidium iodide for 10 min at 37 °C, and the cell cycle was
analyzed by a flow cytometer.
In addition, the effects of compound 9a on apoptosis and cell-cycle progression in AsPC-1 cells
were also explored by flow cytometry analysis [31]. As shown in Figure 4, compound 9a induced
apoptosis in AsPC-1 cells in a dose- and time-dependent manner, increasing the percentages of
apoptotic cells from 52.9 to 90.4% after treatment with various concentrations of 9a (from 0.25 μM
to 1.0 μM) for 72 h compared with the control group. In addition, the data presented in Figure 5
revealed that the percentage of cells in the G2/M phase increased from 5.72 to 27.19%, and those in
the G0/M phase decreased from 63.89 to 39.90% after treatment with 9a at concentrations from
0.25 to 1.0 μM for 72 h. In comparison, the percentage of cells in the S phase only showed minor
changes. Accordingly, 9a arrested AsPC-1 cells in G2/M phase.
11

a)
Control
9a (30mg/kg)
9a (60mg/kg)
c)
b)
2.0
1.5
1.0
0.5
2500
2000
1500
1000
500
control
9a
(30mg/kg)
9a
6
2
4
8
10
(60mg/kg)
Days post initial treatment (d)
Drug concentration
Fig. 6. In vivo effects of 9a on the tumor growth of AsPC-1 cell Xenografts in nude mice. (A) Images of the tumor
collected from the mice; (B) Tumor weight; (C) Tumor volume.
To further investigate the biological activity of the most active inhibitor 9a, this compound was
also evaluated in vivo using a FAK-driven human AsPC-1 cell xenograft mouse model. The results
shown in Figure 6 indicated that tumor growth was dose-dependently inhibited by repeated oral
administration of 9a (qd) for 10 days. Specifically, the tumor growth inhibition (TGI) rates for
compound 9a were 24.45 and 36.6% for the dosages of 30 and 60 mg/kg/day, respectively.
3. Conclusion
A family of pyrimidine derivatives comprising potent dual FAK and EGFRT790M inhibitors was
identified using a fragment-based drug design strategy. Among these inhibitors, two typical
molecules, namely 9a and 9f, not only displayed strong inhibitory activity against FAK kinase (IC50
= 1.03, 3.05 nM), but also inhibited EGFRT790M mutants at concentrations of 3.89 and 7.13 nM,
respectively. Additionally, these compounds also exhibited strong inhibitory activity toward the
three evaluated FAK-overexpressing PC cells (AsPC-1, BxPC-3, Panc-1) and two drug-resistant
12

cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells), at concentrations
below 6.936 μM. Moreover, the xenograft model study also showed the efficiency of 9a in vivo.
Overall, this work identified and characterized a series of potent dual FAK and EGFRT790M
inhibitors which showed great potential for the treatment of hard to treat cancers.
4. Experimental section
4.1 General methods and chemistry
All solvents and chemicals were analytical reagent (AR) and used as purchased without further
1
13
purification. H NMR and C NMR spectra were respectively recorded on a Bruker AV 400 MHz
and AV 101 MHz spectrometer. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts
(δ) of NMR are reported in parts per million (ppm) units relative to the residual solvent peak
1
(d6-DMSO: 40.0, TMS: 0.0). H NMR spectra are reported in the following order: multiplicity,
approximate coupling constant (J value) in hertz, and number of protons; signals are characterized
as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), and br (broad signal).
High resolution ESI-MS was performed on an AB Sciex TripleTOF® 4600 LC/MS/MS system. All
reactions were monitored by TLC, using silica gel plates with fluorescence F254 (TLC Silica gel 60
F254, Merck) and UV light visualization. Flash chromatography separations were obtained on
Silica Gel (300–400 mesh) using dichloromethane/methanol as eluents.
4.2 Preparation of the title molecules 9a-m
2,5-dichloropyrimidine intermediates 13a-d were conventionally prepared as described in our
previous reported method in literatures 23, 27 in addition to the adjustment of temperature. While
the anilines 16a-d were generally synthesized by using nucleophilic substitution and reduction
13

reactions. as reported in literature 23 but addition of KI to promote reaction in condition f. All these
intermediates were not purified and used indirectly in the following step. The prepared
2,5-dichloropyrimidine intermediate 13a-d (1.14g, 10.0 mmol) was added in one portion to 16a-d
(12 mmol) and TFA (1.29 g, 10.0 mmol) in 2-butanol (20 mL). The resulting mixture was stirred at
100 °C for 12 h. Then the mixture was evaporated, and MeOH was added. The solution was poured
into saturated sodium bicarbonate solution. The residue was purified by flash chromatography
separations using dichloromethane/methanol as eluents MeOH/CH2Cl2 (1:30) to yield the title
molecules below.
(9a)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-meth
o
1
ylbenzamide (Off-white power, m.p.:119.5-121.3 C), H NMR (400 MHz, DMSO-d6) δ 11.61 (s,
1H), 9.39 (s, 1H), 9.27 (s, 1H), 8.78–8.73 (m, 2H), 8.22 (s, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H),
7.61–7.51 (m, 2H), 7.48 (td, J = 7.6, 1.5 Hz, 1H), 7.42 (dd, J = 8.6, 2.5 Hz, 1H), 7.15 (td, J = 7.6,
1.2 Hz, 1H), 3.67 (t, J = 4.5 Hz, 4H), 3.13 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 2.56 (t, J = 4.5 Hz, 4H),
13
2.18 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 169.39, 168.09, 158.21, 155.42, 155.13, 139.80,
137.39, 131.99, 130.69, 130.68, 128.47, 123.83, 122.34, 121.69 (2C), 121.19, 118.07, 105.35, 66.77,
62.26, 53.77, 26.78, 18.37.
HRMS (ESI) for C25H28ClN7O3, [M+Na]+ calcd: 532.1834; found: 532.1849.
(9b)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-m
ethylbenzamide (Off-white power, m.p.:137.3-139.2 oC),
14

1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.79‒8.75 (m, 2H), 8.23
(s, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.76 (dd, J = 7.9, 1.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.38 (s,
1H), 7.28 (dd, J = 8.7, 2.2 Hz, 1H), 7.14 (td, J = 7.6, 1.2 Hz, 1H), 3.78 (s, 3H), 3.66 (t, J = 4.6 Hz,
4H), 3.12 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H), 2.54 (t, J = 4.6 Hz, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 169.39, 167.60, 158.13, 155.38, 155.03, 148.89, 139.84, 137.01, 131.90, 128.48, 122.35, 121.87,
121.62, 121.04, 119.55, 111.89, 105.55, 103.76, 66.95, 62.13, 56.29, 53.65, 26.79.
HRMS (ESI) for C25H28ClN7O4, [M+H]+ calcd: 526.1891; found: 526.1978.
(9c)2-[[5-chloro-2-(3-chloro-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-meth
ylbenzamide (Off-white power, m.p.:230.2-231.9 oC),
1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.73 (s, 1H), 9.66 (s, 1H), 8.80 (q, J = 4.5 Hz, 1H),
8.71 (d, J = 8.4 Hz, 1H), 8.27 (s, 1H), 8.03–8.00 (m, 2H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.55–7.50
(m, 2H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 3.68 (t, J = 4.6 Hz, 4H), 3.17 (s, 2H), 2.82 (d, J = 4.5 Hz,
13
3H), 2.58 (t, J = 4.6 Hz, 4H). C NMR (101 MHz, DMSO-d6): δ 169.37, 168.33, 157.78, 155.48,
155.07, 139.63, 137.92, 132.18, 128.65, 128.54, 124.07, 122.75, 122.55, 121.63, 121.34, 119.49,
118.91, 106.11, 66.88(2C), 62.02, 53.65(2C), 26.79. HRMS (ESI) for C24H25Cl2N7O3, [M+H]+
calcd:
530.1396;
found:530.1476.
(9d)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-cycl
o
1
opropylbenzamide (Off-white power, m.p.:209.0-210.3 C), H NMR (400 MHz, DMSO-d6) δ
11.50 (s, 1H), 9.42 (s, 1H), 9.29 (s, 1H), 8.76 (d, J = 4.3 Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.23 (s,
1H), 7.72 (dd, J = 7.9, 1.6 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.54–7.46 (m, 2H), 7.42 (dd, J = 8.7,
15

2.4 Hz, 1H), 7.14 (td, J = 7.6, 1.2 Hz, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.13 (s, 2H), 3.00–2.72 (m, 1H),
2.56 (t, J = 4.6 Hz, 4H), 2.18 (s, 3H), 0.89–0.68 (m, 2H), 0.66–0.53 (m, 2H). 13C NMR (101 MHz,
DMSO-d6): δ 170.31, 168.11, 158.20, 155.42, 155.17, 139.61, 137.40, 132.06, 130.72, 130.66,
128.81, 123.86, 122.32, 121.72, 121.62, 121.32, 118.00, 105.32, 66.77(2C), 62.24, 53.77(2C),
23.58, 18.39, 6.20(2C). HRMS (ESI) for C27H30ClN7O3, [M+H]+ calcd: 536.2099; found: 536.2191.
(9e)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-cy
o
1
clopropylbenzamide (Off-white power, m.p.:223.1-224.6 C), H NMR (400 MHz, DMSO-d6) δ
11.54 (s, 1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.86–8.59 (m, 2H), 8.24 (s, 1H), 8.04 (d, J = 8.7 Hz, 1H),
7.73 (dd, J = 7.9, 1.6 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.29 (dd, J = 8.7, 2.2 Hz, 1H),
7.13 (td, J = 7.6, 1.2 Hz, 1H), 3.78 (s, 3H), 3.67 (t, J = 4.6 Hz, 4H), 3.12 (s, 2H), 2.98–2.81 (m, 1H),
2.55 (t, J = 4.7 Hz, 4H), 0.76–0.71 (m, 2H), 0.66–0.52 (m, 2H). 13C NMR (101 MHz, DMSO-d6):
δ 170.33, 167.61, 158.14, 155.40, 155.06, 148.90, 139.67, 137.02, 131.95, 128.82, 122.31, 121.87,
121.65, 121.18, 119.56, 111.87, 105.55, 103.73, 66.95(2C), 62.14, 56.30, 53.65(2C), 23.58,
6.19(2C). HRMS (ESI) for C27H30ClN7O4, [M+H]+ calcd: 552.2048; found: 552.2136.
(9f)2-[[5-chloro-2-(4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-ethylbenzamid
e (Off-white power, m.p.:198.6-199.4 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 9.65 (s,
1H), 9.43 (s, 1H), 8.80 (t, J = 5.6 Hz, 1H), 8.75–8.69 (m, 1H), 8.21 (s, 1H), 7.77 (dd, J = 7.9, 1.6 Hz,
1H), 7.59 (d, J = 9.1 Hz, 2H), 7.53 (d, J = 9.1 Hz, 2H), 7.51–7.45 (m, 1H), 7.16 (td, J = 7.6, 1.2 Hz,
1H), 3.65 (t, J = 4.6 Hz, 4H), 3.34–3.28 (m, 2H), 3.12 (s, 2H), 2.55–2.51 (m, 4H), 1.15 (t, J = 7.2
Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.70, 168.06, 158.20, 155.42, 155.09, 139.71, 136.34,
133.34, 131.79, 128.54, 122.37, 121.90, 121.57, 120.40 (2C), 120.32 (2C), 105.27, 66.55 (2C),
16

62.53, 53.69 (2C), 34.58, 14.97. HRMS (ESI) for C25H28ClN7O3, [M+H]+ calcd: 510.1942; found:
510.2042.
(9g)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-ethyl
o
1
benzamide (Off-white power, m.p.:209.0-211.0 C), H NMR (400 MHz, DMSO-d6) δ 11.54 (s,
1H), 9.42 (s, 1H), 9.29 (s, 1H), 8.80 (t, J = 5.5 Hz, 1H), 8.73 (d, J = 8.5 Hz, 1H), 8.22 (s, 1H), 7.77
(dd, J = 7.9, 1.6 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.56–7.44 (m, 2H), 7.42 (dd, J = 8.7, 2.5 Hz, 1H),
7.16 (td, J = 7.6, 1.2 Hz, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.31 (td, J = 7.3, 5.5 Hz, 2H), 3.13 (s, 2H),
2.56 (t, J = 4.6 Hz, 4H), 2.18 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ
168.74, 168.11, 158.20, 155.41, 155.15, 139.72, 137.41, 131.96, 130.71, 130.67, 128.58, 123.86,
122.37, 121.70, 121.64, 121.58, 118.01, 105.31, 66.77(2C), 62.25, 53.77(2C), 34.59, 18.39, 14.97.
HRMS (ESI) for C26H30ClN7O3, [M+H]+ calcd: 524.2099; found: 524.2190.
(9h)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-et
hylbenzamide (Off-white power, m.p.:128.9-130.1 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.58 (s,
1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.81 (t, J = 5.5 Hz, 1H), 8.76 (d, J = 7.5 Hz, 1H), 8.23 (s, 1H), 8.04
(d, J = 8.7 Hz, 1H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H), 7.45 (t, 1H), 7.41–7.37 (m, 1H), 7.29 (dd, J = 8.7,
2.2 Hz, 1H), 7.15 (td, J = 7.6, 1.2 Hz, 1H), 3.78 (s, 3H), 3.67 (t, J = 4.5 Hz, 1H), 3.36–3.23 (m, 1H),
13
3.12 (s, 2H), 2.55 (t, J = 4.6 Hz, 4H), 1.15 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ
168.73, 167.60, 158.13, 155.38, 155.03, 148.89, 139.77, 137.01, 131.84, 128.58, 122.35, 121.87,
121.63, 121.42, 119.55, 111.88, 105.53, 103.74, 66.95 (2C), 62.13, 56.30, 53.65 (2C), 34.59, 14.96.
HRMS (ESI) for C26H30ClN7O4, [M+H]+ calcd: 540.2048; found: 540.2149.
17

(9i)2-[[5-chloro-2-(3-chloro-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-ethyl
o
1
benzamide (Off-white power, m.p.:234.4-235.6 C), H NMR (400 MHz, DMSO-d6) δ 11.56 (s,
1H), 9.73 (s, 1H), 9.66 (s, 1H), 8.82 (t, J = 5.5 Hz, 1H), 8.76–8.62 (m, 1H), 8.27 (s, 1H), 8.11–7.89
(m, 2H), 7.78 (dd, J = 7.9, 1.6 Hz, 1H), 7.70–7.33 (m, 2H), 7.17 (td, J = 7.6, 1.1 Hz, 1H), 3.68 (t, J
= 4.5 Hz, 4H), 3.37–3.24 (m, 2H), 3.17 (s, 2H), 2.58 (t, J = 4.5 Hz, 4H), 1.15 (t, J = 7.2 Hz, 3H).
13C NMR (101 MHz, DMSO-d6) δ 168.69, 168.30, 157.77, 155.47, 155.06, 139.53, 137.91, 132.09,
128.62 (2C), 124.05, 122.73, 122.54, 121.75, 121.65, 119.46, 118.88, 106.07, 66.87 (2C), 62.01,
53.64 (2C), 34.59, 14.96. HRMS (ESI) for C25H27Cl2N7O3, [M+H]+ calcd: 544.1552; found:
544.1640.
(9j)2-[[5-chloro-2-(4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-isopropylbenz
o
1
amide (Off-white power, m.p.:222.4-224.1 C), H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H),
9.65 (s, 1H), 9.43 (s, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.56 (d, J = 7.7 Hz, 1H), 8.21 (s, 1H), 7.77 (dd,
J = 7.8, 1.6 Hz, 1H), 7.59 (d, J = 9.1 Hz, 2H), 7.53 (d, J = 9.1 Hz, 2H), 7.51–7.45 (m, 1H), 7.16 (td,
J = 7.5, 1.2 Hz, 1H), 4.38–3.95 (m, 1H), 3.65 (t, J = 4.6 Hz, 4H), 3.12 (s, 2H), 2.58–2.51 (m, 4H),
1.19 (s, 3H), 1.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.07, 158.22, 155.45, 155.10,
139.51, 136.36, 133.34, 131.68, 128.76, 122.40, 122.15, 121.97, 120.34 (2C), 120.33(2C), 105.23,
66.56(2C), 62.53, 53.69(3C), 41.59, 22.55. HRMS (ESI) for C26H30ClN7O3, [M+H]+ calcd:
524.2099; found: 524.2189.
(9k)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-isopr
opylbenzamide (Off-white power, m.p.:220.0-222.0 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s,
18

1H), 9.42 (s, 1H), 9.29 (s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.57 (d, J = 7.7 Hz, 1H), 8.22 (s, 1H), 7.77
(dd, J = 7.9, 1.6 Hz, 1H), 7.57 (d, J = 2.4 Hz, 1H), 7.54–7.45 (m, 2H), 7.42 (dd, J = 8.7, 2.4 Hz, 1H),
7.16 (td, J = 7.5, 1.2 Hz, 1H), 4.43–3.95 (m, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.13 (s, 2H), 2.56 (t, J =
4.7 Hz, 4H), 2.17 (s, 3H), 1.19 (s, 3H), 1.18 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 168.09,
158.19, 155.41, 155.13, 139.50, 137.40, 131.83, 130.70, 130.63, 128.79, 123.85, 122.37, 122.13,
121.74, 121.58, 117.96, 105.25, 66.76(2C), 62.24, 53.76(2C), 41.58, 22.55(2C). HRMS (ESI) for
C27H32ClN7O3, [M+H]+ calcd: 538.2255; found:538.2348.
(9l)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-iso
propylbenzamide (Off-white power, m.p.:197.1-199.0 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.45
(s, 1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.57 (d, J = 7.7 Hz, 1H), 8.23 (s, 1H),
8.03 (d, J = 8.7 Hz, 1H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.29
(dd, J = 8.7, 2.2 Hz, 1H), 7.15 (td, J = 7.5, 1.2 Hz, 1H), 4.33–4.08 (m, 1H), 3.78 (s, 3H), 3.67 (t, J =
4.5 Hz, 4H), 3.12 (s, 2H), 2.55 (t, J = 4.6 Hz, 4H), 1.19 (s, 3H), 1.18 (s, 3H). 13C NMR (101 MHz,
DMSO-d6): δ 168.11, 167.62, 158.16, 155.41, 155.05, 148.91, 139.59, 137.04, 131.75, 128.81,
122.36, 121.97, 121.87, 121.67, 119.56, 111.86, 105.49, 103.73, 66.96(2C), 62.14, 56.31,
53.66(2C), 41.59, 22.55(2C). HRMS (ESI) for C27H32ClN7O4, [M+H]+ calcd: 554.2204; found:
554.2285.
(9m)2-[[5-chloro-2-(3-chloro-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-isop
o
1
ropylbenzamide (Off-white power, m.p.:258.2-260.0 C), H NMR (400 MHz, DMSO-d6) δ 11.42
(s, 1H), 9.72 (s, 1H), 9.66 (s, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.58 (d, J = 7.7 Hz, 1H), 8.27 (s, 1H),
19

8.09–7.87 (m, 2H), 7.77 (dd, J = 7.9, 1.5 Hz, 1H), 7.62–7.40 (m, 2H), 7.18 (t, J = 7.7 Hz, 1H),
4.17–4.08 (m, 1H), 3.68 (t, J = 4.5 Hz, 4H), 3.17 (s, 2H), 2.58 (t, J = 4.6 Hz, 4H), 1.19 (s, 3H), 1.17
(s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.30, 168.04, 157.77, 155.49, 155.06, 139.31, 137.91,
131.98, 128.83, 128.61, 124.05, 122.73, 122.56, 122.31, 121.71, 119.43, 118.85, 106.01, 66.87(2C),
62.01, 53.64(2C), 41.59, 22.54(2C). HRMS (ESI) for C26H29Cl2N7O3, [M+H]+ calcd: 558.1709;
found: 558.1799.
4.3 In vitro kinase enzymatic assay
The ADP-GloTM Kinase enzyme system (FAK Catalog. V9301; EGFRT790M/L858R Catalog.
V5324 Promega), purchased from Promega Corporation (USA), was used for in vitro enzymatic
assay. Concentrations consisting of suitable levels from 1 nM, 10 nM, 100 nM, 500 nM to 1 μM
were used for all of the tested compunds. The test was performed in a 384-well plate. Detailed and
complete protocols can be founded in the ADP-GloTM kinase Assay Technical Manual available at:
https://cn.promega.com/resources/protocols/product-information-sheets/n/fak-kinase-enzyme-syste
m-protocol/. Briefly, procedures include: (1) perform a 5 μL kinase reaction using 1×kinase buffer
(e.g., 1×reactionbuffer A), (2) incubate at room temperature for 60 min, (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.
20

4.4 Cellular activity assay
All the cancer cell lines were purchased from Fuheng Biology Company (Shanghai, China).
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) reagent was purchased from Biotool
Company (Switzerland). The Annexin V-FITC Apoptosis Detection Kit and Cell Cycle Assay were
purchased from Beyotime Company (China). All cell lines were grown in DMEM (Gibco®, USA)
supplemented with 10% FBS (Gibco®, USA), 1% penicillin-streptomycin (Beyotime Company,
China). The cells were maintained and propagated as monolayer cultures at 37 °C in humidified 5%
CO2 incubator.
Cells were seeded in a 96-well plate at a density of 3,000 to 5,000 cells/well and were
maintained at 37 C in a 5% CO2 incubator in DMEM containing 10% FBS for one day. Cells were
then exposed to the synthesiaed inhibitors 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 with a microplate reader
(Thermo, USA). Compounds were tested at appropriate concentrations (1.25 to 40 μmol/L), with
each concentration duplicated five times. The IC50 values were calculated using GraphPad Prim
version 5.0.
4.5 Cell apoptosis assay
AsPC-1 cells (3 to 5×105 cells/well) incubated in 6-well plates were treated with solvent control
(DMSO), or compound 9a (1, 1.2 or 5 μM) in medium containing 5% FBS for 72 h. Then, collected
and fixed with 70% ethanol at 4 °C overnight. After being fixed with 75% ethanol at 4 °C for 24 h,
the cells were stained with Annexin V-FITC (5 μL)/propidium iodide (5 μL), and analyzed by flow
cytometry (Becton-Dickinson, USA).
21

4.6 Mouse tumour xenograft efficacy study
The female BALB/cJNju-Foxn1nu/Nju mice weighing 20–25 g in 5–6 week-old were purchased
from the Model Animal Research Center of Nanjing University, Nanjing, China. All animals were
housed in a controlled environment at 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. AsPC-1 cells (3×106) were suspended in 0.1mL
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
9a at the doses of 30 and 60 mg/kg for 10 days. In the process, the side length of the tumor was
measured to calculate the tumor volume based on the formula: V = A×B2/2, 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.
Acknowledgement
We are grateful to the National Natural Science Foundation of China (No. 81603186), National
Natural Science Foundation of Liaoning (No. 3512942), Research Fund of Higher Education of
Liaoning province（LQ2017039, LQ2017008）for the financial support of this research.
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Graphical Abstract
Glu506/Gl
O
N
EGFRT790M
y
O
429
O
binding sites
N
HN
O
HN
Asp546
HN
O
R1
H
N
HN
Cl
N
NH
R
Fregment-based
drug design
N
O
HN
Cl
N
NH
Mor-DPPY EGFRT790M inhibitor
N
Cys502
O
DPPYs 9a-m
FAK and EGFRT790M dual inhibitor
N
FAK
binding sites
MeHN
OMe
O
HN
Cl
N
NH
N
TAE226 FAK inhibitor
27

Highlights
 Novel Pyrimidines as potential dual FAK and EGFRT790M inhibitors.
 9a, 9f were the strongest FAK and EGFRT790M inhibitors (IC50 < 7.13 nM)
 9a exhibited strong antiproliferative activity against various cancer cells.
 9a was effective in the in vivo assessment conducted in AsPC-1 cell xenograft mouse
model.
28

We declare that we do not have any commercial or associative interest that represents a conflict of interest
in connection with the work submitted.
29