Identification of 2(1H)-pyrimidinones as potential EGFR T790M inhibitors for the treatment of gefitinib-resistant non-small cell lung cancer

Article abstract of DOI:10.1016/j.bioorg.2019.102994

A new class of 2(1H)-pyrimidinone derivatives was identified as potential EGFR T790M inhibitors against TKI-resistant NSCLC. These novel compounds inhibited the EGFR T790M kinase activity at concentrations in the range of 85.3 to 519.9 nM. In particular,

Full text of DOI:10.1016/j.bioorg.2019.102994

Accepted Manuscript
Identification of 2(1H)-Pyrimidinones as Potential EGFR T790M Inhibitors for
the Treatment of Gefitinib-resistant Non-small Cell Lung Cancer
Jianping Huang, Jiaxin Huang, Ning Wang, Luhong Wang, Lei Li, Changyuan
Wang, Xiuli Sun, Yongming Li, Xiaodong Ma, Guoping Huang
PII:
S0045-2068(18)31323-3
https://doi.org/10.1016/j.bioorg.2019.102994
102994
DOI:
Article Number:
Reference:
YBIOO 102994
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 November 2018
5 May 2019
17 May 2019
Please cite this article as: J. Huang, J. Huang, N. Wang, L. Wang, L. Li, C. Wang, X. Sun, Y. Li, X. Ma, G. Huang,
Identification of 2(1H)-Pyrimidinones as Potential EGFR T790M Inhibitors for the Treatment of Gefitinib-resistant
Non-small Cell Lung Cancer, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102994
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Identification of 2(1H)-Pyrimidinones as Potential EGFR
T790M Inhibitors for the Treatment of Gefitinib-resistant Non-
small Cell Lung Cancer
Jianping Huang,a,e,1 Jiaxin Huang,b,1 Ning Wang,b Luhong Wang,b Lei Li,b Changyuan
Wang,b Xiuli Sun,c Yongming Li,d Xiaodong Mab, and Guoping Huange,
aInstitute of functional materials and agricultural applied chemistry, College of Science, Jiangxi Agricultural
University, Nanchang 330045, P. R. China.
bCollege of Pharmacy, Dalian Medical University, Dalian, 116044, P. R. China.
cDepartment of Hematology, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China.
dInstitute of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, No. 138, Xianlin Avenue,
Qixia District, Nanjing, 210023, PR China.
e Nanchang Jiangteng Pharmaceutical Co., Ltd., Nanchang 330045, P. R. China.
E-mail address: xiaodong.ma@139.com (X.D Ma)
Keywords: NSCLC; EGFR T790M; Resistant; Inhibitor; Pyrimidinone.
Abstract
A new class of 2(1H)-pyrimidinone derivatives was identified as potential EGFR
T790M inhibitors against TKI-resistant NSCLC. These novel compounds inhibited
the EGFR T790M kinase activity at concentrations in the range of 85.3 to 519.9 nM.
In particular, compound 7e exhibited the strongest activity against both EGFRWT
(IC50= 96.9 nM) and EGFRT790M (IC50= 85.3 nM) kinases in the cells. Compared with
inhibitor 7e, compound 7b displayed enhanced antiproliferative activity against
gefitinib-resistant H1975 cells harboring the EGFR T790M mutation. In addition,
compound 7b also has low toxicity against the normal human liver cells LO2, with an
IC50 of 11.1 µM. Moreover, both the AO/EB and DAPI staining assays also
demonstrated the inhibitory efficacy of 7b against the resistant H1975 cells. This
contribution provides a new scaffold 2(1H)-pyrimidinone as potential EGFR T790M
inhibitor against drug-resistant NSCLC.
1. Introduction
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and
accounts for 85% of all lung cancer patients [1]. The epidermal growth factor receptor
(EGFR, also known as ErbB1 and HER1) is expressed in 20–80% of NSCLC tumors
and is recognized as one of the most promising drug targets for the treatment of
1

NSCLC [2]. The first generation of small-molecule EGFR inhibitors include gefitinib
(1, Figure 1) and erlotinib (2, Figure 1), which compete with ATP in a reversible
manner to bind the kinase domain of their targets [3,4]. Unfortunately, all patients
who experience marked improvement on these drugs eventually develop progression
of disease after a median of 12 months due to the acquisition of drug resistance [5,6].
About half of the cases with acquired resistance to first-generation EGFR inhibitors
can be accounted for by a second-site mutation in exon 20 of the EGFR kinase
domain, which results in the substitution of methionine for threonine at position 790
(T790M). The bulkier methionine residue at position 790 sterically hinders binding of
gefitinib and erlotinib to the ATP-binding pocket [7,8]. Additional research has
indicated that the T790M mutation also increases the affinity of EGFR for ATP,
therefore outcompeting ATP-competitive tyrosine-kinase inhibitors (TKIs) and
restoring enzymatic activity in their presence [9].
N
Cl
F
O
F
N
HN
N
Cl
O
HN
N
O
HN
N
O
O
O
O
N
N
HN
O
O
N
N
O
2
3
1
O
N
N
O
N
O
N
O
N
N
HN
HN
N
HN
O
O
O
O
N
NH
N
NH
HN
N
NH
N
Cl
N
F3C
6
4
5
Figure 1. Structures of novel EGFR inhibitors.
To overcome the T790M-mediated acquired resistance to EGFR TKIs, many trials
have studied the intensification of EGFR inhibition through use of second- and third-
generation inhibitors, such as afatinib (3) [10], WZ4002 (4) [11], rociletinib (5)
[12],and osimertinib (6) [13,14] (Figure 1). These inhibitors overcome T790M
resistance mainly through covalent inhibition of EGFR by binding to a cysteine side
2

chain (Cys797) through Michael addition with a suitable electrophile attached to the
inhibitor molecule [15]. Despite of their excellent clinical efficacy in the treatment of
EGFR mutant NSCLC patients, the development of resistance to these new third-
generation EGFR inhibitors still occurs 14-20 months later [16]. Therefore,
identifying of more potent EGFR T790M inhibitors to overcome TKI resistance is
still very urgent.
hydrophilic group
O
Covalent binding section
N
NH
O
U
O
NH
U
N
N
Cl
WZ4002
crystal structure with EGFRT790M
2(1H)-Pyrimidinones
Title molecules 7a-e
Figure 2. Discovery strategy of the desired 2(1H)-pyrimidinone analogues.
The co-crystal structure (PDB code: 3IKA) of the EGFR T790M kinase domain in
complex with WZ4002 (4) [11] indicated that a “U-shaped” configuration of a
pyrimidine core, together with an aniline ring bearing a hydrophilic group and an
acrylamide moiety, is propitious to bind with EGFR target (Figure 2) [5]. Accordingly,
a class of 2(1H)-pyrimidinone derivatives were described as potential EGFR T790M
inhibitors to overcome TKI resistance in this manuscript.
2. Results and Discussion
2.1 Synthesis of the title molecules
3

Cl
O
Cl
O
N
NO2
NO2
b
c
N
NO2
d
a
N
HN
N
Cl
NH
NH2
2`
O
4`
CN
O2N
10
N
R
11a R=
11b R=
H
8
9
N
O
Cl
Cl
O
N
N
R1
O
O
e
N
NH2
N
NH
7a,b R1=
7c R1=
HN
HN
O
2`
2`
4`
4`
12a,b
R
R
e
Cl
N
Br
Cl
O
O
O
N
N
NH
N
NH
X
f
O
HN
O
HN
O
N
N
H
H
7d X= O
7e X= CH2OH
14a
Scheme 1. Synthetic route of the title molecules. Reagents and conditions: (a)K2CO3, KI, CH3CN,
48 h,100 C, 54%; (b)(COCl)2, DCM, r.t, 68%; (c) TFA, 2-BuOH, 100 C, 56-67%; (d)Fe-NH4Cl,
MeOH-H2O, 2 h, 70 C, 68-75%; (e)NaHCO3, CH3CN, r.t., 53-76%; (f)K2CO3, KI, CH3CN, 100
C, 48 h, 66-70%.
The synthetic methods for the preparation of the title molecules 7a-e are depicted
in Scheme 1 [17-19]. 3-Nitroaniline 18 was reacted with bromoacetonitrile to produce
compound 9. By reacting compound 9 with oxalic dichloride, the 2(1H)-pyrimidinone
scaffold (10) was formed. Compounds 11a,b were synthesized by a nucleophilic
substitution reaction between intermediate 10 and anilines. After reducing the nitro
group in compound 11, the aniline derivatives 12a,b were generated. Finally, the title
molecules 7a-c and intermediate 14a were respectively synthesized by an acylation of
compounds 12a,b with acylation reagent, comprised of acetyl chloride , acryloyl
chloride and bromoacetyl bromide. The title molecules 13d,e were also prepared
through an additional nucleophilic substitution reaction between 14a and N-
heterocycle.
4

2.2 Inhibition of Kinase Activity and Cancer Cell Viability
Table 1. Anti-EGFRWT and EGFRT790M Enzymatic activity of the title molecules 7a-e.a
Cl
N
H
N
N
R2
HN
R
O
O
R1
Enzymatic activityb
(IC50, nM)b
Compd.
R
R1
R2
EGFRL858R/T790M
EGFRWT
O
7a
7b
7c
7d
H
230
227.4
N
H
N
O
H
266
504
285
298.1
337.2
519.9
O
H
H
CH3
N
H
O
N
O
N
H
O
N
7e
H
85.3
96.9
15.5
N
H
OH
Gefitinib
832.3
aData represent the mean of at least three separate experiments. bConcentration needed to inhibit the
autophosphorylation of the cytoplasmic domain of EGFR by 50%, as calculated using GraphPad Prim version 5.0
2.2.1 Enzymatic activity
All the title molecules were evaluated for their effect on the enzymatic activity of
wild type EGFR (EGFRWT) and mutant EGFR T790M (EGFRT790M) kinases using the
ADP-GloTM kinase assay system [20,21]. Gefitinib was also evaluated as a positive
control. The assay results, listed in Table 1, indicated that these 2(1H)-pyrimidinone
analogues inhibited the kinase activity at concentrations in the range of 85.3 to 519.9
nM. In particular, among these derivatives, compound 7e exhibited the strongest
inhibitory activity against both EGFRWT(IC50= 96.9 nM) and EGFRT790M kinases
(IC50= 85.3 nM). The results of the kinase-based assay clearly revealed that
compounds (7a and 7b) bearing an acryloyl group are more active than those without
5

this substituent (7c and 7d).
2.2.2 Antiproliferative activity
In addition, all these molecules were also explored their effect on the proliferation
of various cancer cells, including lung cancer cell line (H1975 cells, A549 cells, A431
cells), and lymphoma cell line (Ramos cells and Raji cells). Moreover, normal
hepatocyte cell line (LO2) was also used as normal control to evaluate their
cytotoxicity on non-cancerous cells. The results shown in Table 2 indicated that most
of the prepared compounds remarkably inhibited the proliferation of lung cancer cells
at micromolecular concentrations. On the other hand, these compounds were
ineffective in blocking the proliferation of lymphoma cells (IC50> 22.9 μM). Among
these 2(1H)-pyrimidinones, compound 7e, the strongest EGFRT790M inhibitor, was
also a very potent inhibitor of the proliferation of H1975 cells harboring EGFR
T790M mutation (IC50= 7.69 μM). However, compound 7e had certain cytotoxic
effect on normal LO2 cells (IC50= 1.79 μM). In contrast, another potent EGFRT790M
inhibitor 7b, not only displayed stronger antiproliferative activity against H1975 cells
than 7e, but it also exhibited very low toxicity to LO-2 cells (IC50>11.1μM). Thus,
compound 7b may serve as a new lead compound for the discovery of anti-TKI-
resistant agent..
Table 2. Antiproliferative activity of the title molecules.a
Antiproliferative activityb
(IC50, μM)
Compd.
H1975
8.31
A431
5.51
A549
3.40
6.09
12.1
>20
Raji
42.7
22.9
>40
>40
>40
Ramous
>40
LO2
14.3
11.1
15.4
23.9
1.79
7a
7b
7c
7d
7e
6.60
10.3
>40
>10.0
12.4
4.50
>40
>16.0
24.4
>40
7.69
>20
>40
6

Gefitinib
10.89
3.308
10.07
>40
>40
>10
aData represent the mean of at least three separate experiments. bConcentration needed to inhibit the
autophosphorylation of the cytoplasmic domain of EGFR by 50%, as calculated using GraphPad Prim version 5.0
2.3 Effect on the proliferation of H1975 Cells
2.3.1 Wound Healing and Transwell Assays
A wound healing assay was performed to determine whether 7b can inhibit H1975
cell migration. As shown in Figures 5, compound 7b strongly inhibited migration of
H1975 cells at a drug concentration range from 1 to 20 µM. In addition, the AO/EB
and DAPI staining assays also indicated that compound 7b remarkably induced
apoptosis in H1975 cells with the increase of the treatment time and drug
concentration
2.3.2 AO/EB and DAPI staining
Control
7b(μM)
A)
10
5
Gefitinib(μM)
10
5
Control
7b(μM)
B)
5
10
Gefitinib(μM)
5
10
Figure 4. Morphological changes of H1975 cells (100 ×, final magnification, A): AO/EB (Acridine
7

Orange/Ethidium Bromide) double fluorescent staining of H1975 cells treated with different
concentrations of gefitinib and 7b for 72 h; B): DAPI ( 4′,6′-diamidino-2-phenylindole
hydrochloride) staining of H1975 cells treated with different concentrations of gefitinib and 7b for
72 h.
Control
1μM
10μM
20μM
0h
72h
72h
0726-01
Gefitinib
Figure 3. Representative images of H1975 cells treated with different concentrations of 7b for 0
and 72 h by the wound-healing assay.
2.4 Molecular Simulation
Asp800
Leu799
a)
Cys797
Gly719
Lys722
Leu844
Pro797
Met790
Met793
b)
c)
Met790
Met790
Thr854
Asn842
Asn842
Val726
Thr854
Arg841
Arg841
Ala743
Ala743
Pro794
Met793
Met793
Val726
Pro794
Cys797
Phe795
Cys797
Phe795
Asp800
Asp800
Figure 5. a) Binding sites of WZ4002 within EGFRT790M (PDB code: 4I22) [11]; b)Putative
binding modes of pyrimidinone 7b ; c)Putative binding modes of pyrimidinone 7e
EGFRT790M enzyme.
with
8

To investigate the putative interaction mechanism of pyrimidinones with the
EGFRT790M -mutated kinase (PDB code: 3IKA), two representative molecules, namely
7b and 7e were separately docked into the the ATP-binding pocket. For comparison,
the novel EGFRT790M inhibitor WZ4002 was also analyzed as well. The software
AutoDock 4.2 its default parameters [22,23] was used for docking performance
analysis. Apparently, both compounds 7b (Figure 5a) and 7e (Figure 5b) can come
into tight contact with the EGFRT790M binding pocket. Also, as anticipated, compound
7b formed a very strong covalent bond between its introduced acryloyl group and the
Cys797 reside in the EGFRT790M protein. Instead, compound 7e formed a hydrogen-
bond between the carbonyl group in the C-2 aniline side chain and amino acid
Met793. In contrast, the pyrimidinone core in 7b mainly comes to contact with resides
Asn842 and Val726, while the core in compound 7e mainly interacts with resides
Asp800 and Phe795. It is possible that these contacts produce are responsible for the
inhibitory of these compounds against the kinase activity of the mutated EGFRT790M
enzyme. By contrast, WZ4002 forms several stronger interactions with EGFRT790M
,
including: (1) covalent bond between the acryl amide functionality with the amino
acid Cys797; (2) strong contacts generated from the chlorine atom at the C-5 position
of pyrimidine core with the mutant gatekeeper residue Met790; (3) hydrogen bond
between the N-1 nitrogen atom of pyrimidine core and amino acid Met793; (4) polar
forces formed by the piperazine ring with the outside of the ATP-binding pocket of
EGFRT790M. Accordingly, their binding contacts are in accord with the biological
activity against EGFRT790M kinase.
3
Conclusion
A novel anti-EGFRT790M kinase scaffold, namely 2(1H)-pyrimidinone, was
identified in this work to act against the gefitinib-resistant NSCLC cancer cells. These
2(1H)-pyrimidinone derivatives could significantly inhibited both EGFRWT and
EGFRT790M kinase activity at concentrations in the range of 85.3 to 519.9 nM. Among
these derivatives, compound 7e exhibited the strongest activity against the EGFRWT
(IC50= 96.9 nM) and EGFRT790M (IC50= 85.3 nM) cells, while compound 7b displayed
9

the highest antiproliferative activity against EGFRT790M-mutated H1975 cells.
Noteworthy, compound 7b also had low toxicity against the normal LO2 cells (IC50 >
11.1 nM). Moreover, further analysis with the AO/EB and DAPI staining assays also
indicated its efficiency against gefitinib-resistant H1975 cells. Thus, compound 7b
may serve as a new scaffold to discover more potent EGFR T790M inhibitors.
4
Materials and Methods
4.1 Chemistry
4.1.1 General information
Commercial available solvents and reagents were directly used without further
purifications.
High
resolution
ESI-MS
was
performed
on
an
1
AB Sciex TripleTOF® 4600 LC/MS/MS system. H NMR and 13C NMR spectra,
which were recorded in [d] DMSO, were respectively performed on Brucker AV 100
and 400 MHz spectrometer. Coupling constants (J) are expressed in hertz (Hz).
Chemical shifts (δ scale) are reported in parts per million (ppm) relative to the central
peak of the solvent. All reactions were monitored by TLC, using silica gel plates with
fluorescence GF254 and UV light visualization. Preparative TLC separations were
obtained on Silica Gel (300–400 mesh) using dichloromethane/methanol (20:1) as
developing solvents.
4.1.2. General procedure for the synthesis of 3, 5-dichloropyrazin-2(1H)-one 10.
Oxalyl chloride (3.4 mL, 40 mmol) was added dropwise to the solution of
compound 9 (1.77g, 10 mmol) in dry toluene (20 mL). After stirring for 45 min,
triethylamine hydrochloride (2.1 mL, 15 mmol) and DMF (3d) was added by small
portions to the mixture, and was then stirred for 2 days. The reaction mixture was
concentrated and the residue was purified by silica gel column chromatography (from
10% to 30% EtOAc in petroleum ether) to afford substituted 3, 5-dichloropyrazin-
2(1H)-one 10.
4.1.3. General procedure for the synthesis of the intermediates 11a,b
A flask was charged with 3, 5-dichloropyrazin-2(1H)-one 10 (2.85g, 10 mmol),
anilines (12 mmol), TFA (0.08 mL, 1.05 mmol), and 2-BuOH (10 mL). The slurry
10

was heated to 100 °C and maintained at this temperature for 12 h. Then, the reaction
mixture was allowed to cool to room temperature and was neutralized with a saturated
aqueous sodium bicarbonate solution. The aqueous mixture was extracted with
CH2Cl2 (20 mL) three times and concentrated to obtain the crude product. The crude
product was directly used in the next step without further purification.
4.1.4. General procedure for the synthesis of the intermediates 12a,b.
Compound 11 (10 mmol) was dissolved in methanol (30 mL) and water (30 mL).
Iron powder (1.68 g, 30 mmol) and ammonium chloride (2.67 g, 50 mmol) were
added, and the resulting mixture was heated to 70 °C and stirred for 3 h. The reaction
mixture was cooled to room temperature and filtered through Celite. The methanol
solvent was removed in vacuo, and the resulting residue was basified with sodium
bicarbonate and extracted with dichloromethane (20 mL) three times. The organic
layer was separated, dried using anhydrous sodium sulfate, concentrated, and directly
used in the subsequent step.
4.1.5. General procedure for the synthesis of the title compounds 7a-c and
intermediate 13a.
Acetyl chloride (0.257 g, 12 mmol) or acryloyl chloride (1.09 g, 12 mmol) was
added dropwise to a solution of compound 12a,b (0.814 g, 3.18 mmol) and
triethylamine (2.42 g, 24 mmol) in methylene chloride (20 mL) at 0 °C. The reaction
was stirred for 1 h, and then was concentrated. The residue was purified by silica gel
preparative TLC using dichloromethane/methanol (20:1) as developing solvents to
prepare the title molecules 7a-c.
4.1.6. General procedure for the synthesis of the title compounds 7a,b and 7d,e.
Compound 14a (490 mg, 10 mmol), potassium carbonate (2.76 g, 20 mmol), and
morpholine (1.31 g, 15 mmol) were added to 20 mL of acetone. The resulting mixture
was heated to reflux for 4 h. The reaction mixture was cooled to room temperature
and filtered. The filtrate was collected and concentrated in vacuo. The residue was
purified by silica gel preparative TLC using dichloromethane/methanol (20:1) as
developing solvents to produce the desired compounds 7d,e.
(7a)N-(3-(3-(2-(acetylamino)phenylamino-5-chloro-2-oxo-1(2H)-
11

1
pyrazinyl)phenyl)acrylamide. Yield: 53.1%; off-yellow solid. H NMR (400 MHz,
DMSO-d6): δ 11.84 (s, 1H), 10.14 (s, 1H), 8.51 (d, J = 8.2 Hz, 1H), 8.41 (d, J = 4.4
Hz, 1H), 7.63 (s, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.29 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H),
7.03 (s, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.85 (t, J = 7.4 Hz, 1H), 6.26 – 6.09 (m, 1H),
6.00 (dd, J = 17.0, 1.8 Hz, 1H), 5.50 (dd, J = 10.0, 1.8 Hz, 1H), 3.10 – 3.04 (m, 3H);
13C NMR (400 MHz, DMSO-d6): δ 168.94, 163.87, 150.62, 147.53, 140.13, 139.76,
139.34, 132.30, 132.07, 130.01, 128.62, 127.91, 124.21, 122.56, 121.60, 121.49,
119.84, 119.78, 117.53, 116.84, 26.65; HRMS (ESI) for C21H18ClN5O3, [M+H]+
calcd: 424.1098; found: 424.1114.
(7b)N-(3-(3-(4-(1-morpholino)methyl)phenylamino-5-chloro-2-oxo-1(2H)-pyrazinyl)
1
phenyl)acrylamide. Yield: 68.5%; off-yellow solid. H NMR (400 MHz, DMSO-d6):
δ 10.44 (s, 1H), 9.65 (s, 1H), 8.01 – 7.77 (m, 3H), 7.70 (d, J = 7.6 Hz, 1H), 7.50 (t, J
= 8.0 Hz, 1H), 7.34 – 7.21 (m, 4H), 6.55 – 6.37 (m, 1H), 6.29 (d, J = 16.6 Hz, 1H),
13
5.89 – 5.69 (m, 1H), 3.59 (m, 4H), 2.39 (m, 4H), 1.23 (s, 2H); C NMR (400 MHz,
DMSO-d6): δ 163.88, 150.43, 147.77(2C), 140.24, 139.91, 138.43, 132.08, 130.07,
129.82, 127.94, 124.60, 121.73, 120.53(4C), 119.83, 117.50, 116.14, 66.54, 62.34,
53.49, 29.50; HRMS (ESI) for C24H24ClN5O3, [M+H]+ calcd: 466.1568;
found:466.1713.
(7c)N-(3-(3-(2-(acetylamino)phenylamino-5-chloro-2-oxo-1(2H)-pyrazinyl) phenyl)
1
acetamide. Yield: 75.9%; off-yellow solid. H NMR (400 MHz, DMSO-d6): δ 12.12
(s, 1H), 10.21 (s, 1H), 8.79 (d, J = 8.2 Hz, 1H), 8.70 (d, J = 4.4 Hz, 1H), 7.82 (s, 1H),
7.74 (dd, J = 7.8, 1.2 Hz, 1H), 7.63–7.54 (m, 2H), 7.46 (s, 1H), 7.30 (s, 1H), 7.20–
7.12 (m, 2H), 2.79 (d, J = 4.6 Hz, 3H), 2.07 (s, 3H); 13C NMR (400 MHz, DMSO-d6):
δ 171.93, 169.10, 168.93, 150.61, 147.52, 140.43, 139.72, 139.33, 132.29, 129.90,
128.61, 124.16, 121.49, 121.10, 119.77, 119.46, 117.10, 116.88, 26.65, 22.98; HRMS
(ESI) for C20H18ClN5O3, [M+H]+ calcd:412.1098; found:412.1115.
(7d)N-(3-(3-(2-(acetylamino)phenylamino-5-chloro-2-oxo-1(2H)-pyrazinyl) phenyl)
1
-(1-morpholino)acetamide. Yield: 70.1%; off-yellow solid. H NMR (400 MHz,
DMSO-d6): δ 10.06 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 11.2 Hz, 4H), 7.70
(s, 2H), 7.55–7.40 (m, 2H), 7.27–7.16 (m, 2H), 4.61 (s, 2H), 4.07 (d, J = 4.8 Hz, 3H),
12

3.64 (s, 4H), 1.23 (s, 4H); 13C NMR (400 MHz, DMSO-d6): δ 170.68, 158.73, 154.78,
145.70, 142.30, 139.91, 139.54, 135.74, 135.24, 129.95, 129.18, 128.68, 126.93,
121.76, 121.67, 120.36, 119.76, 117.78, 108.75, 66.45, 60.50, 53.57, 44.58, 42.11;
HRMS (ESI) for C24H25ClN6O4, [M+H]+ calcd:497.1626; found:497.1675.
(7e)N-(3-(3-(2-(acetylamino)phenylamino-5-chloro-2-oxo-1(2H)-pyrazinyl) phenyl)
1
- (4-hydroxy-1-piperidinyl)acetamide. Yield: 65.6%; off-yellow solid. H NMR (400
MHz, DMSO-d6): δ 9.95 (s, 1H), 8.23 (s, 1H), 8.21 (s, 1H), 7.97 (s, 2H), 7.92 (s, 2H),
7.88 (s, 1H), 7.70 (s, 1H), 7.50 (s, 1H), 7.25 (s, 1H), 7.20 (d, J = 7.8 Hz, 2H), 4.02 (d,
J = 5.2 Hz, 2H), 3.54 (s, 4H), 1.75 (s, 4H), 1.23 (s, 3H), 0.84 (d, J = 6.6 Hz, 1H); 13C
NMR (400 MHz, DMSO-d6): δ 158.73, 154.78, 145.70(2C), 142.32, 139.83, 139.56,
135.74(2C), 130.00, 129.19, 128.68, 126.93, 121.67, 120.36, 119.69, 117.74,
108.75(2C), 66.21, 60.64, 51.36, 42.60, 32.53, 29.49; HRMS (ESI) for C25H27ClN6O4,
[M+H]+ calcd:511.1782; found:511.1420.
4.2 Biological evaluation method
4.2.1 Cell culture and reagents
NSCLC cells (H1975, A431, and A549), and human normal hepatocyte cells (LO2)
were obtained from Fuheng Biology Company (Shanghai, China). The MTT was
obtained from Biotool Company (Switzerland). The wild-type EGFR, and mutant
EGFR L858R/T790M kinase assay systems were purchased from Promega
Corporation (USA). 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 LO2 cells were grown in
®
®
DMEM (Gibco , USA) supplemented with 10% FBS (Gibco , USA), 1% penicillin-
streptomycin (Beyotime Company, China). All cells were maintained and propagated
as monolayer cultures at 37 ℃ in humidified 5% CO2 incubator.
4.2.2 Kinase enzymatic assays
The experiments were generally performed according to the instructions of the
manufacturer. Concentrations consisting of suitable levels from 10 to 1000 nM were
used for all of the tested compunds. The more detailed and complete protocols, see the
13

ADP-GloTM kinase Assay Technical Manual #313, and the active kinase datasheet
available
at:
http://www.promega.com/tbs/tm313/tm313/tm313.html
and
(or
http://www.promega.com/KESProtocol
http://www.promega.com/tbs/signaling.htm), respectively. The test was performed in
a 384-well plate, and includes the major steps below: (1) perform a 5 μL kinase
reaction using 1×kinase buffer (e.g., 1×reaction buffer A), (2) incubate at room
temperature for 60 minutes, (3) add 5 μL of ADP-Glo™ Reagent to stop the kinase
reaction and deplete the unconsumed 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 and introduce luciferase and
luciferin to detect ATP, (7) incubate at room temperature for 30 minutes, (8) plate was
measured on TriStar® LB942 Multimode Microplate Reader (BERTHOLA) to detect
the luminescence (Integration time 0.5-1 second). Curve fitting and data presentations
were performed using Graph Pad Prism version 5.0.
4.2.3 Cell Growth Inhibitory Activity
4.2.3.1 MTT assay
The cells were seeded at a density of 1 to 5×103 cells/mL in 96-well plates in growth
medium supplemented with 10% serum at 37 °C with 5% CO2 for one day. After 12 h
of incubation, 100 μL of medium was removed, and 100 μL of sample solution with
different concentrations of inhibitor was added and then the cells were incubated for
48 or 72 h. Subsequently, 10 μL of MTT reagent (Biotool Company, 5.0 mg/mL)
dissolved in phosphate-buffered saline (PBS) was added and the cells were incubated
for another 4 h. The absorbance was read at 570 nm with a microplate reader (Thermo,
USA). The data were calculated using GraphPad Prim version 5.0
4.2.3.32 Wound-healing assay
The cancer cells were cultured in 6-well plates for 48 h at 37 °C. Wounds were
created in the cell monolayer and washed with PBS to remove cell debris, then the
cells were treated with different concentrations of inhibitor for 72 h. After that, the
dead cells were washed away with PBS, and the images were taken by the inverted
microscope (OLYMPUS, Japan).
14

4.2.3.3 AO/EB and DAPI staining assay
Approximately 2×105 cells/well of H1975 cells in 6-well plate were incubated in an
incubator for 12 h, then treated with different concentrations of inhibitors for 72 h.
After incubation, the cells were washed with PBS for two times. Then, total of 20 μL
of the solution containing the AO/EB dye mix (1.0 μg/mL of AO and 1.0 μg/mL of
EB in PBS) was added to the cells. The apoptotic, necrotic and live cells were
observed and counted under the fluorescent inverted microscope (OLYMPUS, Japan).
DAPI staining was performed after being treated as mentioned above. The cells plated
in 6-well plates were washed twice with PBS and fixed with 4% paraformaldehyde
for 10 min, then washed with PBS for three times. Cells were subsequently incubated
in DAPI (1.0 μg/mL) solution at room temperature for 15 min, then examined under
the fluorescent inverted microscope (OLYMPUS, Japan).
4.3 Molecular modeling
AutoDock 4.2 software was used for docking studies. Most of the parameters were
seted as default. 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 binding interaction energy was calculated to include van der Waals, electrostatic,
and torsional energy terms defined in the tripos force field. The WWW site also
includes many resources for use of AutoDock, including detailed Tutorials that guide
users through basic AutoDock usage, docking with flexible rings, and virtual
screening
with
AutoDock.
Tutorials
may
be
found
at:
http://autodock.scripps.edu/faqs-help/tutorial
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 81672944),
Research Fund of Higher Education of Liaoning province (LQ2017008, LQ2017039),
the National Natural Science Foundation of Liaoning province (20180530066), the
Cultivation Project of Youth Techstars, Dalian, China (No. 2016RQ043), the Natural
Science Foundation of Science and Technology Department of Jiangxi Province (No.
15

20171BBE50027, 20171BBG70029), the Natural Science Foundation of Education
Department of Jiangxi Province (No. GJJ170274), and the Natural Science
Foundation of Nanchang City (No. 2018CXTD014) for the financial support of this
research.
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Graphical abstract
hydrophilic group
O
Covalent binding section
N
NH
O
U
O
U
NH
N
N
Cl
Pyrimidine scaffold
2(1H)-Pyrimidinone scaffold
WZ4002
EGFRT790M Inhibitor
crystal structure with EGFRT790M
20

Highlights
 2(1H)-Pyrimidinone as potential EGFR T790M inhibitor against drug-resistant
NSCLC.
 7b,e displayed enhanced antiproliferative activity against gefitinib-resistant
H1975 cells harboring the EGFR T790M mutation.
 7b has low toxicity against the normal LO2 cells ( IC50< 11.1 µM).
21