Discovery of novel 2,4-diarylaminopyrimidine derivatives as potent and selective epidermal growth factor receptor (EGFR) inhibitors against L858R/T790M resistance mutation

Article abstract of DOI:10.1016/j.ejmech.2018.04.052

A series of novel 2,4-diarylaminopyrimidine derivatives of AZD9291 were discovered as L858R/T790M mutant selective epidermal growth factor receptor (EGFR) inhibitors. The majority of these compounds exhibited moderate to excellent EGFR T790 M/L858R inhibitory activities and comparable anti-proliferative activities against double mutant over-expressed NCI-H1975 cells to that of AZD9291. The most promising compounds 8a displayed an IC₅₀ of 4.1 nM against EGFR L858R/T790M mutants. 8a also showed excellent cytotoxic effect against NCI-H1975 cells with an IC₅₀ of 59 nM and 100-fold selectivity over wide-type EGFR over-expressed A431 cells. Compound 8a significantly inhibited tumor growth in NCI-H1975 xenograft models at a non-toxic dose. Docking study performed for 8a with ATP binding site of EGFR-TK showed the similar binding mode to that of AZD9291. All these results suggested that compound 8a was a potential mutant-selective EGFR inhibitor.

Full text of DOI:10.1016/j.ejmech.2018.04.052

Accepted Manuscript
Discovery of novel 2,4-diarylaminopyrimidine derivatives as potent and selective
epidermal growth factor receptor (EGFR) inhibitors against L858R/T790M resistance
mutation
Qi Yan, Yuzhe Chen, Baiyou Tang, Qiang Xiao, Rong Qu, Linjiang Tong, Jian Liu,
Jian Ding, Yi Chen, Ning Ding, Wenfu Tan, Hua Xie, Yingxia Li
PII:
S0223-5234(18)30394-5
10.1016/j.ejmech.2018.04.052
EJMECH 10401
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2018
Revised Date: 25 April 2018
Accepted Date: 25 April 2018
Please cite this article as: Q. Yan, Y. Chen, B. Tang, Q. Xiao, R. Qu, L. Tong, J. Liu, J. Ding, Y. Chen,
N. Ding, W. Tan, H. Xie, Y. Li, Discovery of novel 2,4-diarylaminopyrimidine derivatives as potent and
selective epidermal growth factor receptor (EGFR) inhibitors against L858R/T790M resistance mutation,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.052.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of Novel 2,4-Diarylaminopyrimidine Derivatives as Potent and
Selective Epidermal Growth Factor Receptor (EGFR) inhibitors against
L858R/T790M Resistance Mutation
Qi Yan1,#, Yuzhe Chen2,3,#, Baiyou Tang2,4, Qiang Xiao1, Rong Qu2,3, Linjiang Tong2,
Jian Liu1, Jian Ding2, Yi Chen2, Ning Ding1, Wenfu Tan1*, Hua Xie2,*, Yingxia Li1,*
1School of Pharmacy, Fudan University, Shanghai 201203, China
2Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
3University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
4Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha
410008, Hunan, China
Abstract
A series of novel 2,4-diarylaminopyrimidine derivatives of AZD9291 were discovered
as L858R/T790M mutant selective epidermal growth factor receptor (EGFR)
inhibitors. The majority of these compounds exhibited moderate to excellent EGFR
T790M/L858R inhibitory activities and comparable anti-proliferative activities
against double mutant over-expressed NCI-H1975 cells to that of AZD9291. The most
promising compounds 8a displayed an IC50 of 4.1 nM against EGFR L858R/T790M
mutants. 8a also showed excellent cytotoxic effect against NCI-H1975 cells with an
IC50 of 59 nM and 100-fold selectivity over wide-type EGFR over-expressed A431
*
Corresponding author. Tel./Fax.: +86 21 51980039 (W.T.), Tel.: +86 21 50805897; fax: +86 21 50806722
(H.X.), Tel/Fax.: +86 21 51980127 (Y.L.)
# These authors contributed equally to this work.
E-mail address: wftan@fudan.edu.cn (W. Tan), hxie@simm.ac.cn (H. Xie), liyx417@fudan.edu.cn (Y. Li).
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cells. Compound 8a significantly inhibited tumor growth in NCI-H1975 xenograft
models at a non-toxic dose. Docking study performed for 8a with ATP binding site of
EGFR-TK showed the similar binding mode to that of AZD9291. All these results
suggested that compound 8a was a potential mutant-selective EGFR inhibitor.
Keywords
EGFR inhibitors, Pyrimidine derivatives, Mutant selective inhibitors, AZD9291,
T790M
1. Introduction
The epidermal growth factor receptor (EGFR) protein belongs to the ErbB family of
receptor tyrosine kinases (RTKs), which play an important role in the regulation of
cell growth, differentiation, and survival [1]. EGFR over-expression is observed in
many human malignancies, including non-small cell lung cancer (NSCLC), ovarian
cancer, and colon cancer [2]. Therefore, EGFR has become one of the targets in
anticancer drug research. In recent years, several EGFR inhibitors have been
approved for the treatment of advanced NSCLC by FDA, such as gefitinib (1) and
erlotinib (2) (Fig. 1). EGFR tyrosine kinase inhibitors (TKIs) have been emerging as
one of the effective and targetable treatment strategies for NSCLC [3].
F
O
HN
Cl
HN
N
N
O
O
O
O
N
O
O
N
N
1
2
N
N
F
F
O
O
HN
Cl
HN
N
Cl
HN
O
HN
O
N
N
O
N
3
4
Fig. 1. First- and second- generation EGFR inhibitors
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First-generation EGFR TKIs provide significant clinical benefit in patients with
activating EGFR mutations [4, 5]. However, patients ultimately develop disease
progression, often accompanied with the EGFR T790M mutation [6]. To overcome
the T790M mutation-related resistance, subsequent identification of irreversible
EGFR inhibitors (second-generation EGFR inhibitors) containing a Michael acceptor
functional group such as dacomitinib (3) and afatinib (4) have been developed. These
agents inhibit mutant EGFR as well as the wild type EGFR (EGFR wt), offering a
potential therapeutic option for T790M positive patients [7, 8]. However, less
selectivity between EGFR wt and EGFR T790M mutation eventually limits their
utility due to the severe toxicities such as diarrhea and skin rash [9]. Therefore,
discovery of mutant selective third-generation EGFR inhibitors, has become the focus
of more attention recently [3].
AZD9291 (5) (Fig. 2) is a novel oral, potent and selective third-generation
irreversible inhibitor, developed for the treatment of advanced non-small cell lung
cancer. It has been designed to target the EGFR T790M mutation, while sparing
wild-type EGFR [10]. This compound is structurally distinct from other
third-generation EGFR TKIs, such as WZ4002 (6) and CO-1686 (7) [11], which
offers a pharmacologically differentiated profile from earlier generation EGFR TKIs
[12]. In November 2015, AZD9291 was granted accelerated approval in the USA for
the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC
who have progressed on or after EGFR TKI therapy [13].
Fig. 2. Third generation EGFR inhibitors
2. Design
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As depicted in the modeled structure for AZD9291 with EGFR (Fig. 3), the
orientation of the indole group is adjacent to the gatekeeper residue (Met790),
indicating the selectivity over the wild-type form of the receptor [12].
Fig. 3. AZD9291 binding mode
To exploit novel irreversible EGFR inhibitors to overcome the problem of drug
resistance with mutant selectivity, we focused on the replacement of the indole
residue of AZD9291 with a o-heterocycle substituted aniline as shown in compound
8a. The more flexible and bulky motif was excepted to occupy the binding pocket
better than indole residue, therefore higher selectivity and potency might be achieved
(Fig. 4). A similar ring expansion strategy was utilized by Zhu et al [14]. Based on
this strategy, a series of novel 2,4-diarylaminopyrimidine analogues (8a-m) with
different heterocycle substituents and polar fragments were designed and synthesized.
Their EGFR kinase inhibitory activity, in vitro antiproliferative effects and in vivo
antitumor efficacy were evaluated. Some of these compounds were identified with
greater potency.
N
Het
N
N
Hets are five-
member
heterocycles
such as pyzole,
trizole etc. R are
polar fragments
O
Design
O
NH
O
NH
NH
O
Modification
NH
O
NH
O
N
N
R
N
N
N
N
N
N
N
N
N
N
N
H
H
H
5
8a
8
Fig. 4. Design strategy of pseudo-ring replacement
3. Chemistry
The synthetic routine of target compounds 8a-m was displayed in Scheme 1.
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Compounds 13a-d were synthesized as reported in the literature [15, 16].
Condensation of commercially available 2,4-dichloropyrimidine with 13a-d in 2-butyl
alcohol
containing
DIPEA
provided
intermediates
15a-d.
Adding
4-methylbenzenesulfonic acid hydrate in one portion to the mixture of 15 and
4-fluoro-2-methoxy-5-nitroaniline [10] in 2-pentanol at a high temperature gave
compounds 16a-d. 17a-m were synthesized through nucleophilic reaction of 16a-d
with different amines in moderate to good yields. The target compounds were
generated from compounds 17a-m via reduction and the subsequent acylation.
Scheme 1. Reagent and conditions: (a) DMF-DMA, 110 oC, 100%; (b) NH2NH2,
AcOH (for 11a) or EtOH (for 11b), 100 oC, 87%; (c) MeI, NaH, THF, r.t., 63% (12a),
54% (12b); (d) Fe, NH4Cl, EtOH/H2O, 80 oC, 93% (13a), 95% (13b); (e) pyrazole
(for 12c) or 1,2,3-trizole (for 12d), CuI, DMF, 110 oC, 87% (12c), 75% (12d); (f)
2,4-dichloropyrimidine, 2-BuOH, DIPEA, 110 oC, 76-89%; (g)
4-fluoro-2-methoxy-5-nitroaniline, 2-pentanol, 110 oC, 56-77%; (h) amine, DMF, 110
oC, 78-90%; (i) H2, Pd/C, MeOH, 70-95%; (j) acryloyl chloride, DIPEA, CH2Cl2, r.t.,
35-45%.
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4. Results and discussion
4.1 Kinase inhibitory activity
All the synthesized compounds were evaluated in terms of in vitro inhibitory
activities on EGFR wt and EGFR T790M/L858R mutants (Table 1). As shown in
Table 1, compounds 8a, 8b, 8f, and 8m showed good or modest inhibition against
EGFR wt and EGFR T790M/L858R. 1-Methyl-1H-pyrazole and pyrazole derivative
(8a and 8f) showed the best activity (IC50 = 4.1 nM), while other heterocycle such as
1-methyl-1H-1,2,4-triazole was well tolerated (8h and 8m). The hydrophilic motif R
showed a great impact on activities. The ether linked analogue 8b was a replacement
that was tolerated which resulted in a very similar profile with a slight erosion in
kinase inhibition potency (EGFR T790M/L858R IC50 = 16.4 nM) and a loss in
selectivity. When the R was replaced with other amines such as piperazine and
morpholine, the inhibitory activities against EGFR wt and EGFR T790M/L858R were
diminished significantly (8c, 8g), in some cases resulted in a complete loss of potency
(8d, 8e and 8h). As mentioned in a previous study [17], they employed a glutathione
(GSH) reaction assay to determine the relative reactivity of inhibtiors, results showed
a dependence of GSH half-life on the pKa of the basic group. The intramolecular base
catalysis would be a explanation for the observed loss of activity in some cases.
Table 1. EGFR kinase inhibition (IC50, nM) by the pseudo-ring compounds
Compd.
8a
EGFR wt
349.9±252.6
316.8±295.3
>1000
EGFR T790M/L858R
4.1±2.2
Enzyme selectivitya
85
19
16.4±5.8
8b
38.5±16.5
>1000
NDb
ND
ND
158
8c
>1000
8d
>1000
>1000
8e
648.2±208.7
4.1±0.2
8f
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>1000
142.7±59.2
>1000
ND
ND
8
8g
>1000
280.6±160.4
>1000
8h
33.1±26.0
202.3±84.7
174.5±78.6
838.5±98.4
7.7±2.5
8i
ND
ND
ND
86
8j
8k
>1000
>1000
8l
660.0±165.3
270.4±53.5
8m
12.4±4.3
22
AZD9291
a WT:DM; bNot determined
4.2 In vitro anti-proliferative activity
Compounds 8a, 8b, 8f, 8j and 8m were chosen to further evaluate their
anti-proliferative activities against the A431 epidermoid carcinoma cell line and the
NCI-H1975 non-small cell lung cancer cell line, which expressed high levels of
EGFR wt or EGFR T790M/L858R, respectively. As shown in Table 2, all compounds
demonstrated comparable anti-proliferative activities against NCI-H1975 cells to that
of AZD9291, and displayed less inhibition of the growth of A431 cells. Intriguingly,
compounds 8a and 8f showed 124-fold and 148-fold selectivity over A431 cells,
which were much better than AZD9291.
Table 2. NCI-H1975 and A431 cancer cell proliferation inhibition (IC50, µM)a
Cellular
Compd.
A431
NCI-H1975
selectivityb
7.368±2.491
1.583±0.492
10.071±6.103
0.059±0.007
0.085±0.015
0.068±0.018
124
18
8a
8b
8f
148
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2.760±0.607
1.499±1.112
1.260±0.032
0.070±0.015
39
20
30
8j
8m
0.074±0.016
0.041±0.031
AZD9291
aIC50 data are averages of at least three independent experiments; bA431:H1975
4.3. Compound 8a blocks the phosphorylation of EGFR and downstream
signaling transduction in human cancer cells
We further examined the ability of the representative compound 8a to inhibit the
phosphorylation of EGFR T790M/L858R and EGFR wt in NCI-H1975 cells and
A431 cells, respectively. The results demonstrated that compound 8a potently and
dose-dependently inhibited the phosphorylation of EGFR T790M/L858R and
downstream molecules indicated by p-Akt and p-Erk in NCI-H1975 cells (Fig. 5A),
whilst showing less effective inhibition against the phosphorylation of EGFR wt in
A431 cells (Fig. 5B).
Fig. 5. Effect of compound 8a on the phosphorylation of EGFR T790M/L858R and
EGFR wt in cancer cells induced by EGF. Representative Western blot analysis for the
EGFR T790M/L858R inhibition by 8a in the NCI-H1975 cells (A) and the EGFR wt
inhibition in A431 cells (B).
4.4. In vivo anti-tumor activity of 8a
In vivo anti-tumor activity of 8a
We further evaluated the in vitro antitumor activity of 8a in xenograft models of
NCI-H1975 and A431, respectively. 8a was administrated orally twice daily (10
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mg/kg or 20 mg/kg) and AZD9291 (10 mg/kg) was used for comparison. As
illustrated in Fig. 6A, 8a significantly and dose-dependently suppressed the tumor
growth in NCI-H1975 xenograft model, which was comparable with AZD9291.
Whereas, in A431 xenograft mouse model (Fig. 6C), 8a only exhibited moderate
tumor growth inhibition, with TGI less than 20% even at 20 mg/kg dose treatment.
During the course of experiment, no obvious body weight loss of mice was observed
in 8a treatment group both in NCI-H1975 model (Fig. 6B) and in A431 models (Fig.
6D), indicating this compound was well tolerated.
The target inhibition of 8a in NCI-H1975 and A431 tumor was investigated by
Western blot analysis. As shown in Fig. 6E and 6F, 8a (10 mg/kg) treatment
significantly blocked the phosphorylation of EGFR T790M/L858R and the activation
of downstream molecule Akt in NCI-H1975 cells (*p < 0.05). While in A431
xenograft tumor, the inhibition of phosphorylation about EGFR and Akt was not
significant by 8a which was consistent with the weak inhibition of 8a on A431 tumor
growth（Fig. 6G and 6H）. These results clearly indicated that 8a might serve as a
promising third-generation EGFR–TKI for further development to overcome EGFR
T790M mutation-related lung cancer.
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Fig. 6. In vivo antitumor activity of compound 8a against NCI-H1975 and A431
xenograft mouse models. NCI-H1975 tumor growth (A) and A431 tumor growth (C)
were inhibited by 8a. Body weight changes of control and 8a treatment group were
monitored in NCI-H1975 model (B) and A431model (D). The phosphorylation of
EGFR and Akt in NCI-H1975 tumor was inhibited by 8a treatment compared with
vehicles control group (E). (F) Qutification data of Fig. 6E. In 8a treatment tumor, the
phosphorylation EGFR T790M/L858R and the activation of Akt was significantly less
than that of vehicle control group (*p < 0.05).
4.5 Binding mode of compound 8a
In order to provide deeper insights into the structure-activity relationship of the
new series, compound 8a was selected based on the potency and scaffold type and
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docked with the ATP-binding pocket of EGFR (PDB code: 3IKA) [18]. As illustrated
in Fig. 7, compound 8a irreversibly binds to the T790M mutant EGFR by targeting
the Cys797 residue in the ATP binding site via the formation of a covalent bond. The
pyrimidine core forms an expected bidentate hydrogen-bonded interaction with the
hinge Met793 residue, the orientation of the heterocycle is adjacent to the gatekeeper
residue Met790; the amine motif positions in the solvent exposed region. (Fig. 7).
Fig. 7. Docking model for the binding of 8a in the ATP-pocket of EGFR T790M. The
result was calculated by Maestro 10.1 (Schrödinger Release 2015-3). The figure was
generated using Pymol.
Conclusions
We have described the structural optimization of AZD9291aimed at discovery of
mutant-selective EGFR inhibitors. A novel series of 2,4-diarylaminopyrimidine
derivatives based on the pseudo-ring replacement strategy was designed, synthesized
and evaluated as EGFR inhibitors. Compounds 8a and 8f showed good inhibitory
activities against EGFR T790M/L858R in vitro, with IC50 values of 4.1 nM and
comparable antiproliferation potency to AZD9291 in H1975 cells (59 nM for 8a and
68 nM for 8f). Meanwhile, 8a and 8f exhibited 124-fold and 148-fold selectivity over
A431 cells, which are better than AZD9291. 8a also effectively blocked the
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phosphorylation of EGFR and downstream signaling pathway. In NCI-H1975
xenograft model, oral dosing of compound 8a at 10 and 20mg/kg/day led to
significant tumor growth inhibition with no observed loss in body weight. The
improved biological activities of 8a indicate that the design strategy could be useful
optimization for better activity and selectivity. This inhibition results in
antiproliferative effects in cancer cell lines harboring mutant forms of EGFR, and
translates into pronounced antitumor activity in corresponding in vivo xenograft
models. These compounds show much less activities against wild-type EGFR. In
conclusion, the findings presented herein show that compounds 8a has potential as
promising anticancer agents.
5. Experimental section
5.1 Chemistry
All commercially available starting materials, reagents and solvents were used
without further purification. All the solid compounds are recrystallized from methanol.
Melting points were determined with an Electro thermal melting point apparatus, and
are uncorrected. High resolution mass spectra (HRMS) were recorded on QSTAR XL
1
13
Hybrid MS/MS mass spectrometer. H NMR and C NMR spectra on a Bruker AV
400 or 600 MHz spectrometer were recorded in CDCl3. Chemical shifts are reported
in δ (ppm) units relative to the internal standard tetramethylsilane (TMS).
5.1.1. General method to synthesize compounds
General method to synthesize compounds 16a-d: Compounds 13a-d were
synthesized as reported in the literature [14-15]. A solution of commercially available
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2-nitroacetophenone and N,N-dimethylformamide dimethyl acetal in DMF was heated
o
at 100 C for 2 h. The reaction mixture was concentrated and the resultant solid was
washed with Et2O and collected by filtration to afford 10a. 10a and hydrazine
monohydrate in ethanol was refluxed for 10 h. The mixture was concentrated and the
resultant was subjected to column chlomatography on silica gel to give 11a.
2-nitrobenzamide was dissolved in dimethylformamide dimethylacetal and heated to
100 oC for 1 h. The reaction mixture was cooled and a white precipitate was collected
by filtration and rinsed with hexane to provide 10b. 10b in acetic acid and n-butanol
was treated with hydrazine monohydrate, and heated to reflux for 2 h. The solvent
was removed in vacuo, and the residue was subjected to column chlomatography on
silica gel to give 11b. 13a-b could be obtained through methylation and reduction. A
cooper-catalized coupling was used to synthesize 12c and 12d and followed by a
reduction, 13c and 13d were obtained in good yields. Condensation of commercially
available 2,4-dichloropyrimidine with 13a-d in 2-BuOH containing DIPEA provided
intermediates with structure 15a-d. 4-methylbenzenesulfonic acid hydrate was added
in one portion to 15 and 4-fluoro-2-methoxy-5-nitroaniline in 2-pentanol. The
resulting mixture was stirred at 105 °C for 2.5 h. The mixture was cooled to room
temperature. The precipitate was collected by filtration, washed with 2-pentanol, and
dried under vacuum to afford 16a-16d as a yellow solid, which were used in the
subsequent reaction without purification.
General method to synthesize compounds 17a-m: different amines (0.1 mmol)
was added to a solution of 16a-d (0.9 mmol) and DIPEA (0.1 mmol) in DMF. The
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mixture was heated at 110 oC for 2 hours. The solvent was removed in vacuo, and the
crude product was purified by flash silica gel chromatography (4% 7 N NH3/MeOH in
CH2Cl2) to obtain the title product.
General method to synthesize compounds 18a-m: A solution of compound
17a-m (0.10 mmol) in enthanol (20 mL) was treated with 10% palladium on charcoal
(10 wt %) and hydrogenated at 30 psi for 12 h. The catalyst was filtered through
Celite, and the filtrate was concentrated under reduced pressure. The resulting crude
product was purified by flash silica gel chromatography (dichloromethane/methanol =
15:1, v/v) to obtain the title product.
General method to synthesize compounds 8a-m: Acryloyl chloride (34.5 mg,
0.38 mmol) in CH2Cl2 (1 mL) was added dropwise to a stirred solution of 18a-m (170
mg, 0.38 mmol) and DIPEA (0.073 mL, 0.42 mmol) in DCM (5 mL), cooled in an
ice/water bath. The mixture was stirred for 90 min and then diluted with CH2Cl2 (25
mL) and washed with saturated aqueous NaHCO3 solution (50 mL). The organics
were removed, and the aqueous portion was further extracted with CH2Cl2 (2 × 25
mL). The combined organics were dried over MgSO4, filtered, and concentrated onto
silica for purification. The crude product was purified by flash silica chromatography,
with elution gradient 0 −4% 7 N NH3/MeOH in DCM to afford 8a-m.
5.1.2.
N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((2-(1-methyl-1
H-pyrazol-3-yl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide (8a)
According to general method to synthesize compounds 8a-m, compound 8a was
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synthesized in 45% yield as a yellow solid. mp: 63-65 oC; 1H NMR (400 MHz, CDCl3)
δ 10.37 (s, 1H), 10.07 (s, 1H), 9.49 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 8.11 (d, J = 5.7
Hz, 1H), 7.62–7.59 (m, 1H), 7.39 (s, 1H), 7.35 (s, 1H), 7.26-7.23 (m, 2H), 7.02 (t, J =
7.6 Hz, 1H), 6.76 (s, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.37–6.31 (m, 3H), 6.22 (d, J = 5.8
Hz, 1H), 5.66 (dd, J = 9.5, 2.4 Hz, 1H), 3.99 (s, 3H), 3.85 (s, 3H), 2.88 (t, J = 5.6 Hz,
2H), 2.69 (s, 3H), 2.27 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 163.24, 161.09,
159.92, 156.61, 151.11, 145.25, 137.22, 135.68, 132.57, 130.95, 129.47, 128.10,
128.05, 127.15, 125.92, 122.20, 121.71, 121.63, 112.36, 104.68, 104.48, 99.48, 57.51,
56.16, 45.56, 43.80, 39.27, 29.84; HRMS (ESI) (m/z): [M+H]+ calcd for C29H36N9O2
542.2992; found 542.2990.
5.1.3.
N-(2-(2-(Dimethylamino)ethoxy)-4-methoxy-5-((4-((2-(1-methyl-1H-pyrazol-3-yl)
phenyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide (8b)
According to general method to synthesize compounds 8a-m, compound 8b was
o
synthesized in 35% yield as a solid. mp:125-128 C; 1H NMR (400 MHz, CDCl3) δ
10.39 (s, 1H), 9.78 (s, 1H), 9.34 (s, 1H), 8.41 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 5.8 Hz,
1H), 7.61 (dd, J = 7.8, 1.6 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.24 (t, J = 4.0 Hz, 2H),
7.02 (t, J = 7.6 Hz, 1H), 6.61 (s, 1H), 6.57 (d, J = 2.3 Hz, 1H), 6.42 (d, J = 1.8 Hz,
1H), 6.38 (d, J = 1.8 Hz, 1H), 6.29 (d, J = 10.1 Hz, 1H), 6.24 (d, J = 10.0 Hz, 1H),
6.20 (d, J = 5.8 Hz, 1H), 5.68 (dd, J = 10.0, 1.8 Hz, 1H), 4.11 (t, J = 5.2 Hz, 2H), 4.00
(s, 3H), 3.85 (s, 3H), 2.62–2.50 (m, 2H), 2.36 (s, 6H); 13C NMR (150 MHz, CDCl3) δ
163.3, 161.1, 160.0, 156.6, 151.1, 145.7, 142.8, 137.2, 132.1, 130.9, 128.1, 128.0,
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126.3, 125.0, 124.40, 122.1, 121.3, 113.9, 104.4, 101.8, 99.4, 70.3, 58.1, 56.2, 45.3,
39.2, 29.8; HRMS (ESI) (m/z): [M + H]+ calcd for C28H31N8O3 529.2676; found
529.2680
5.1.4.
N-(2-(4-(Dimethylamino)piperidin-1-yl)-4-methoxy-5-((4-((2-(1-methyl-1H-pyraz
ol-3-yl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide (8c)
According to general method to synthesize compounds 8a-m, compound 8c was
o
synthesized in 50% yield as a solid. mp:80-83 C; 1H NMR (400 MHz, CDCl3) δ
10.37 (s, 1H), 9.41 (s, 1H), 8.51 (s, 1H), 8.38 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 5.8 Hz,
1H), 7.62 (dd, J = 7.8, 1.6 Hz, 1H), 7.40 (d, J = 2.3 Hz, 1H), 7.32 (s, 1H), 7.25-7.23
(m, 2H), 7.04 (t, J = 7.6 Hz, 1H), 6.72 (s, 1H), 6.58 (d, J = 2.3 Hz, 1H), 6.38–6.25 (m,
2H), 6.23 (d, J = 6.0 Hz, 1H), 5.72 (d, J = 9.8 Hz, 1H), 3.99 (s, 3H), 3.86 (s, 3H), 3.04
(d, J = 11.6 Hz, 2H), 2.71 (t, J = 11.4 Hz, 2H), 2.38 (s, 6H), 2.30-2.28 (m, 2H),
13
2.20-2.15 (m, 1H); C NMR (150 MHz, CDCl3) δ 162.8, 161.1, 159.8, 156.5, 151.0,
145.2, 137.1, 135.9, 132.3, 130.9, 128.1, 128.0, 126.7, 126.6, 126.4, 122.3, 121.7,
112.1, 104.4, 103.2, 99.5, 62.1, 56.1, 52.4, 42.0, 39.2, 30.0; HRMS (ESI) (m/z):
[M+H]+ calcd for C31H38N9O2, 568.3134, found 568.3137.
5.1.5.
N-(4-Methoxy-5-((4-((2-(1-methyl-1H-pyrazol-3-yl)phenyl)amino)pyrimidin-2-yl)
amino)-2-(4-methylpiperazin-1-yl)phenyl)acrylamide (8d)
According to general method to synthesize compounds 8a-m, compound 8d was
o
1
synthesized in 60% yield as a solid. mp:66-70 C; H NMR (400 MHz, CDCl3) δ
16

ACCEPTED MANUSCRIPT
10.70 (s, 1H), 9.21 (s, 1H), 8.21-8.18 (m, 2H), 8.07 (d, J = 8.2 Hz, 1H), 7.41-7.37 (m,
1H), 7.14-7.09 (m, 1H), 6.60 (s, 1H), 6.41 (d, J = 5.8 Hz, 1H), 4.01 (s, 3H), 3.85 (s,
3H), 2.95-2.89 (m, 4H), 2.60-2.58 (m, 4H), 2.38 (s, 3H); 13C NMR (150 MHz, CDCl3)
δ 162.1, 160.7, 159.8, 156.6, 143.3, 141.3, 138.4, 135.4, 132.2, 129.7, 128.5, 126.8,
122.1, 121.7, 118.4, 107.3, 104.3, 99.4, 56.9, 56.0, 51.3, 46.2, 36.5; HRMS (ESI)
(m/z): [M+H]+ calcd for C29H34N9O2, 540.2830; found 540.2835.
5.1.6.
N-(4-Methoxy-5-((4-((2-(1-methyl-1H-pyrazol-3-yl)phenyl)amino)pyrimidin-2-yl)
amino)-2-morpholinophenyl)acrylamide (8e)
According to general method to synthesize compounds 8a-m, compound 8e was
o
1
synthesized in 44% yield as a solid. mp:98-101 C; H NMR (400 MHz, CDCl3) δ
10.40 (s, 1H), 9.46 (s, 1H), 8.53 (s, 1H), 8.39 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 5.7 Hz,
1H), 8.06 (s, 1H), 7.64–7.61 (m, 1H), 7.40 (d, J = 2.5 Hz, 2H), 7.25 (d, J = 7.4 Hz,
1H), 7.04 (t, J = 7.5 Hz, 1H), 6.74 (s, 1H), 6.58 (d, J = 2.3 Hz, 1H), 6.41–6.26 (m,
2H), 6.23 (d, J = 5.9 Hz, 1H), 5.74 (d, J = 9.9 Hz, 1H), 4.00 (s, 3H), 3.88-3.85 (m,
7H), 2.88 (t, J = 4.5 Hz, 4H); 13C NMR (150 MHz, CDCl3) δ 162.7, 161.0, 160.9,
159.8, 156.4, 151.0, 145.3, 137.1, 135.0, 132.2, 130.9, 128.1, 128.0, 127.2, 126.8,
126.6, 122.3, 121.6, 112.2, 104.4, 103.8, 99.6, 67.8, 67.3, 66.5, 56.2, 52.9, 45.5, 40.7,
39.2; HRMS (ESI) (m/z): [M + H]+ calcd for C28H31N8O3, 527.2514, found 527.2516.
5.1.7.
N-(5-((4-((2-(1H-pyrazol-1-yl)phenyl)amino)pyrimidin-2-yl)amino)-2-((2-(dimeth
ylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (8f)
17

ACCEPTED MANUSCRIPT
According to general method to synthesize compounds 8a-m, compound 8f was
o
1
synthesized in 43% yield as a solid. mp:90-94 C; H NMR (400 MHz, CDCl3) δ
10.07 (s, 1H), 9.42 (s, 1H), 9.20 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.09 (d, J = 5.7 Hz,
1H), 7.2 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 2.5 Hz, 1H), 7.32 (q, J = 3.5, 3.0 Hz, 3H),
7.1 –7.06 (m, 1H), 6.75 (s, 1H), 6.50–6.42 (m, 1H), 6.42–6.28 (m, 2H), 6.11 (d, J =
5.8 Hz, 1H), 5.68 (dd, J = 9.2, 2.6 Hz, 1H), 3.84 (s, 3H), 2.88 (t, J = 5.6 Hz, 2H), 2.69
(s, 3H), 2.29 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 163.2, 160.6, 159.8, 157.2, 145.2,
141.2, 135.8, 132.9, 132.5, 130.6, 130.3, 129.3, 127.9, 126.8, 126.0, 123.8, 123.7,
123.2, 112.3, 107.1, 104.6, 98.6, 57.4, 56.4, 56.1, 45.5, 43.7; HRMS (ESI) (m/z):
[M+H]+ calcd for C28H34N9O2, 528.2830; found 528.2828.
5.1.8.
N-(5-((4-((2-(1H-pyrazol-1-yl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-
(4-methylpiperazin-1-yl)phenyl)acrylamide (8g)
According to general method to synthesize compounds 8a-m, compound 8g was
synthesized in 66% yield as a solid. mp:93-95 oC; 1H NMR (400 MHz, CDCl3) δ 9.69
(s, 1H), 9.43 (s, 1H), 8.49 (s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.25–8.12 (m, 4H), 8.04 (s,
1H), 7.37 (d, J = 8.0 Hz, 2H), 7.08 (t, J = 7.4 Hz, 1H), 6.79 (s, 1H), 6.39–6.29 (m,
2H), 5.73 (d, J = 9.7 Hz, 1H), 3.85 (s, 3H), 2.94 (s, 4H), 2.46 (s, 3H); 13C NMR (150
MHz, CDCl3) δ 162.7, 160.6, 159.8, 157.2, 145.1, 141.2, 135.3, 132.9, 132.3, 130.5,
130.2, 127.9, 126.8, 126.7, 126.4, 123.6, 123.3, 111.9, 107.1, 107.1, 103.5, 98.7, 56.1,
56.1, 52.5, 46.2; HRMS (ESI) (m/z): [M+H]+ calcd for C28H32N9O2, 526.2673; found
526.2669.
18

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5.1.10.
N-(5-((4-((2-(1H-pyrazol-1-yl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-
morpholinophenyl)acrylamide (8h)
According to general method to synthesize compounds 8a-m, compound 8h was
o
1
synthesized in 45% yield as a solid. mp:123-126 C; H NMR (400 MHz, CDCl3) δ
9.41 (s, 1H), 9.27 (s, 1H), 8.50 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 5.8 Hz,
1H), 7.82 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.38–7.27 (m, 3H), 7.10 (t, J =
7.6 Hz, 1H), 6.72 (s, 1H), 6.46 (t, J = 2.2 Hz, 1H), 6.42 – 6.24 (m, 2H), 6.14 (d, J =
5.8 Hz, 1H), 5.80–5.71 (m, 1H), 3.86-3.79 (m, 7H), 2.86 (t, J = 4.5 Hz, 4H); 13C
NMR (150 MHz, CDCl3) δ 162.7, 160.6, 159.7, 157.1, 145.1, 141.2, 135.0, 132.8,
132.2, 130.5, 130.2, 127.8, 126.9, 126.7, 126.5, 123.6, 123.3, 112.1, 107.1, 103.4,
98.8, 67.8, 56.1, 52.9; HRMS (ESI) (m/z): [M + H] + calcd for C27H29N8O3, 513.2363;
found 513.2360.
5.1.11.
N-(5-((4-((2-(2H-1,2,3-triazol-2-yl)phenyl)amino)pyrimidin-2-yl)amino)-2-((2-(di
methylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (8i)
According to general method to synthesize compounds 8a-m, compound 8i was
o
synthesized in 33% yield as a solid. mp:66-68 C; 1H NMR (400 MHz, CDCl3) δ
10.09 (s, 1H), 9.67 (s, 1H), 9.48 (s, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.13 (d, J = 5.6 Hz,
1H), 7.95 (dd, J = 8.2, 1.6 Hz, 1H), 7.91 (s, 2H), 7.39–7.31 (m, 2H), 7.17–7.11 (m,
1H), 6.76 (s, 1H), 6.26 (d, J = 5.8 Hz, 1H), 5.71–5.66 (m, 1H), 3.85 (s, 3H), 2.88 (t, J
= 5.6 Hz, 2H), 2.69 (s, 3H), 2.33 – 2.26 (m, 9H); 13C NMR (150 MHz, CDCl3) δ
19

ACCEPTED MANUSCRIPT
163.2, 160.6, 159.9, 157.5, 145.1, 135.8, 135.0, 134.9, 132.6, 131.4, 129.9, 129.4,
128.4, 126.8, 125.9, 123.3, 123.3, 123.1, 112.3, 104.6, 98.4, 57.5, 56.1, 45.5, 43.7;
HRMS (ESI) (m/z): [M+H]+ calcd for C27H33N10O2, 529.2782; found 529.2780.
5.1.12.
N-(5-((4-((2-(2H-1,2,3-triazol-2-yl)phenyl)amino)pyrimidin-2-yl)amino)-2-(4-(di
methylamino)piperidin-1-yl)-4-methoxyphenyl)acrylamide (8j)
According to general method to synthesize compounds 8a-m, compound 8j was
synthesized in 40% yield as a solid. mp:69-73 oC; 1H NMR (400 MHz, CDCl3) δ 9.67
(s, 1H), 9.41 (s, 1H), 8.49 (s, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 5.7 Hz, 1H),
7.96 (d, J = 9.3 Hz, 1H), 7.91 (s, 2H), 7.38–7.31 (m, 2H), 7.16 (t, J = 7.7 Hz, 1H),
6.71 (s, 1H), 6.39–6.29 (m, 1H), 6.27 (d, J = 5.7 Hz, 1H), 5.77–5.72 (m, 1H), 3.85 (s,
3H), 3.05 (d, J = 11.4 Hz, 2H), 2.78 – 2.68 (m, 2H), 2.43 (s, 6H), 2.39-2.35 (m, 2H),
13
2.09-2.01 (m, 2H), 1.79–1.65 (m, 2H); C NMR (150 MHz, CDCl3) δ 162.8, 160.6,
159.9, 157.4, 135.1, 132.3, 131.4, 129.9, 128.2, 126.7, 126.4, 123.4, 123.3, 112.0,
103.2, 98.4, 62.2, 56.2, 52.3, 41.8, 29.8; HRMS (ESI) (m/z): [M+H]+ calcd for
C29H35N10O2, 555.2939; found 555.2928.
5.1.13.
N-(5-((4-((2-(2H-1,2,3-triazol-2-yl)phenyl)amino)pyrimidin-2-yl)amino)-4-metho
xy-2-(4-methylpiperazin-1-yl)phenyl)acrylamide (8k)
According to general method to synthesize compounds 8a-m, compound 8k was
synthesized in 51% yield as a solid. mp:84-86 oC; 1H NMR (400 MHz, CDCl3) δ 9.69
(s, 1H), 9.43 (s, 1H), 8.50 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 5.8 Hz, 1H),
20

ACCEPTED MANUSCRIPT
7.96 (dd, J = 8.1, 1.5 Hz, 1H), 7.91 (s, 2H), 7.45 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.16
(t, J = 7.7 Hz, 1H), 6.76 (s, 1H), 6.37 (d, J = 1.7 Hz, 1H), 6.32 (d, J = 9.9 Hz, 1H),
6.27 (d, J = 5.8 Hz, 1H), 5.75 (d, J = 10.0 Hz, 1H), 3.85 (s, 3H), 2.95-2.90 (m, 4H),
13
2.74-2.63 (m, 4H)2.46 (s, 3H); C NMR (150 MHz, CDCl3) δ 162.7, 160.7, 159.7,
157.1, 135.1, 132.3, 131.3, 129.9, 128.4, 126.8, 126.5, 123.4, 123.3, 112.0, 103.6,
98.5, 56.1, 55.9, 52.2, 45.9; HRMS (ESI) (m/z): [M+H]+ calcd for C27H31N10O2,
527.2626; found 527.2626.
5.1.14.
N-(5-((4-((2-(2H-1,2,3-triazol-2-yl)phenyl)amino)pyrimidin-2-yl)amino)-4-metho
xy-2-morpholinophenyl)acrylamide (8l)
According to general method to synthesize compounds 8a-m, compound 8l was
o
synthesized in 45% yield as a solid. mp:117-170 C; 1H NMR (400 MHz, CDCl3) δ
9.69 (s, 1H), 9.47 (s, 1H), 8.53 (s, 1H), 8.21 (d, J = 8.4 Hz, 11H), 8.13 (d, J = 5.8 Hz,
11H), 7.97 (dd, J = 8.1, 1.4 Hz, 10H), 7.92 (d, J = 0.9 Hz, 19H), 7.34 (t, J = 7.6 Hz,
9H), 7.16 (t, J = 7.7 Hz, 11H), 6.74 (s, 11H), 6.43–6.29 (m, 18H), 6.28 (d, J = 6.0 Hz,
13
14H), 5.76 (d, J = 10.0 Hz, 10H), 5.30 (s, 10H), 2.88 (t, J = 4.5 Hz, 47H); C NMR
(150 MHz, CDCl3) δ 160.7, 135.1, 132.3, 128.4, 126.5, 123.4, 103.5, 98.5, 67.8, 56.2,
53.5, 53.0, 29.8, 29.4; HRMS (ESI) (m/z): [M+H]+ calcd for C26H28N9O3, 514.2310;
found 514.2314.
5.1.15.
N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((2-(1-methyl-1
H-1,2,4-triazol-3-yl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide (8m)
21

ACCEPTED MANUSCRIPT
According to general method to synthesize compounds 8a-m, compound 8m was
1
synthesized in 33% yield as a solid. H NMR (400 MHz, CDCl3) δ 10.52 (s, 1H),
10.03 (s, 1H), 9.45 (s, 1H), 8.44 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.13 (s,
1H), 8.13 (s, 2H), 7.54 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 12.3 Hz, 2H), 7.33 (t, J = 7.8
Hz, 1H), 7.14–7.12 (m, 1H), 7.05 (t, J = 7.6 Hz, 1H), 6.76 (s, 1H), 4.00 (s, 3H), 3.86
(s, 3H), 2.89 (t, J = 5.6 Hz, 2H), 2.69 (s, 3H), 2.32 (t, J = 5.8 Hz, 2H), 2.28 (s, 6H);
13C NMR (150 MHz, CDCl3) δ 163.3, 162.1, 160.9, 159.9, 156.8, 145.3, 143.4, 138.3,
135.8, 132.6, 129.8, 129.3, 128.4, 127.0, 125.9, 124.8, 121.9, 121.1, 118.3, 104.6,
99.5, 56.1, 45.4, 43.8, 36.5; HRMS (ESI) (m/z): [M+H]+ calcd for C28H35N10O2
543.2944; found 543.2950.
6.1. Pharmacology
6.1.1. In vitro enzymatic activity assay
The kinases domain of EGFR wt and EGFR T790M/L858R was expressed using
the Bac-to-Bac™ baculovirus expression system (Invitrogen, Carlsbad, CA, USA)
and purified on Ni-NTA columns (QIAGEN Inc., Valencia, CA, USA). The screening
of tyrosine kinase inhibitors was based on enzyme-linked-immunosorbent assay
(ELISA). Briefly, 20 µg/mL Poly (Glu, Tyr)4:1 (Sigma, St. Louis, MO) was precoated
in 96-well ELISA plates as substrate. Kinases were incubated with indicated test
compounds or reference compound in kinase reaction buffer (50 mM HEPES pH 7.4,
20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) containing 5 µmol/L
o
ATP at 37 C for 1 h. After incubation, the wells were washed with PBS and then
incubated with an anti-phosphotyrosine (PY99) antibody (1:1000, Santa Cruz
22

ACCEPTED MANUSCRIPT
Biotechnology, Santa Cruz, CA) followed by a horseradish peroxidase
(HRP)-conjugated secondary antibody. The wells were visualized using
o-phenylenediamine (OPD) and read with a multiwell spectrophotometer
(VERSAmaxTM, Molecular Devices, Sunnyvale, CA, USA) at 492 nM.
6.1.2. Cytotoxicity assay
The cytotoxic activity in vitro was measured using sulforhodamine B (SRB)
assay. All the compounds were dissolved in DMSO at the concentrations 10.0 mg/ml
and were then diluted to appropriate concentrations. Cells were plated in 96-well
plates (5*103 per well) for 24 h and subsequently treated with different concentrations
of all tested compounds for 72 h. Cells were then fixed with 10% (v/v) trichloroacetic
o
acid at 4 C for 1 h. After washing, the cells were stained for 15 min with 4 mg/mL
SRB (Sigma) dissolved in 1% acetic acid. Before measured, 100 µL Tris base was
added per well. The absorbance was measured at 515 nm using a multiscan spectrum
(VERSAmax, Molecular Devices, Sunnyvale, CA, USA). The inhibition rate was
calculated as [1–(A515 treated/A515 control)]×100%. The IC50 value was obtained using
the logit method.
6.1.3. In vitro EGFR phosphorylation assays
NCI-H1975 cells or A431 cells were treated with each compound or without for
2h and then stimulated with 50 ng/ml of EGF for 10 minutes. Cell samples were lysed
in SDS lysis buffer and the Western blot analysis was subsequently performed using
standard procedures. Antibodies directed against the following proteins were used:
p-EGFR, EGFR, p-ERK, ERK, p-AKT, AKT, which were obtained from Cell
23

ACCEPTED MANUSCRIPT
Signaling Technologies (Cambridge, MA, USA), and tubulin, which was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6.1.4. Molecular modeling
A published structure of the EGFR T790M mutant (PDB code 3IKA ) was used
as template. Crystal structures were prepared using the protein preparation wizard in
Maestro (Schrödinger Release 2015-3) which optimizes hydrogen placements. The
active site was defined by using the bound ligand, and the covalent docking protocol
was used to model potential binding modes. These were ranked using the assigned
scores and manually inspected for the retention of the key hinge interactions to the
hinge region residue M793.
6.1.5. In vivo antitumor activity assay
Pathogen-free, 6-8 week-old, female BALB/c nude mice (Shanghai Institute of
Materia Medica, Chinese Academy of Sciences) were maintained under clean-room
conditions in sterile filter-top cages with Aspen Chip bedding. NCI-H1975 xenograft
model was established by inoculated 5×106 cancer cells subcutaneous in the right
flanks of athymic mice. When the tumor reached a volume of 100-300 mm3, the mice
were randomly assigned into control and treatment groups (n = 6 per group). Control
groups were given vehicle alone, and treatment groups received different drugs.
Health conditions of mice were observed daily. The size of tumor and body weight
was measured on the indicated days during the treatment. Tumor volume (V) was
calculated as V = (length×width2)/2. The body weight change (%) was calculated as
follows: body weight change (%)= (Wt/W0 - 1)×100, where Wt represented the body
24

ACCEPTED MANUSCRIPT
weight measured on the indicated days and W0 represented the predosing body weight.
Results are presented as mean for each group. Error bars represent the standard error
of the mean (SEM). Animal experiments were carried out in compliance with the
Animal Management Rules of the Ministy of Health of the People’s Republic of
China (document no. 55, 2001) and in the guidelines for the Care and Use of
Laboratory Animals of Shanghai Institute of Materia Medica.
Acknowledgments
We gratefully acknowledge financial support from Shanghai Science and
Technology Innovation Fund (17431902400) for N.D. and YX.L. We are also grateful for
“Personalized Medicines-Molecular Signature-based Drug Discovery and
Development”, Strategic Priority Research Program of the Chinese Academy of
Sciences (Grant XDA12020209) for H.X.
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Highlights
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A series of novel 2,4-diarylaminopyrimidine derivatives were designed as EGFR-TKIs.
Most of the compounds displayed moderate to good inhibition toward EGFR
L858R/T790M kinase and low anti-proliferative potencies against NCI-H1975 cells.
Compound 8a significantly inhibited tumor growth in NCI-H1975 xenograft models at a
non-toxic dose.
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