Structural optimization of diphenylpyrimidine scaffold as potent and selective epidermal growth factor receptor inhibitors against L858R/T790M resistance mutation in nonsmall cell lung cancer

Article abstract of DOI:10.1111/cbdd.13370

A new class of thiodiphenylpyrimidine analogs (Thio-DPPY) were synthesized as potent and selective EGFR T790M inhibitors to overcome gefitinib resistance in nonsmall cell lung cancer (NSCLC). This structural optimization led to the identification of two p

Full text of DOI:10.1111/cbdd.13370

PROFESSOR XIAODONG MA (Orcid ID : 0000-0002-5189-6287)
Article type : Research Article
Structural Optimization of Diphenylpyrimidine Scaffold as Potent and
Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors against
L858R/T790M Resistance Mutation in Non-small Cell Lung Cancer
a,1
c,1
c
c
c
b,c
c
Yuanyuan Yi , Luhong Wang , Dan Zhao , Shanshan Huang , Changyuan Wang , Zhihao Liu , Huijun Sun ,
Kexin Liu , Xiaodong Mac, and Yanxia Lia, 
c
a
Department of Respiratory Medicine, the First Affiliated Hospital of Dalian Medical University, Dalian
1
16011, PR China
b
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P. R. China
c
College of Pharmacy, Dalian Medical University, Dalian 116044, PR China
1
These authors contributed equally to this work

Corresponding author
E-mail address: xiaodong.ma@139.com (X. Ma), liyanxia001@163.com (X. Li)
Abstract
A new class of thiodiphenylpyrimidine analogues (Thio-DPPY) were synthesized as
potent and selective EGFR T790M inhibitors to overcome gefitinib resistance in non-small
cell lung cancer (NSCLC). This structural optimization led to the identification of two potent
T790M/L858R
EGFR
inhibitors, 14a and 14e, which possess IC50 values of 27.5 and 9.1 nM,
respectively. Moreover, compounds 14a (SI > 36.4) and 14e (SI > 109.9) exhibited high
selectivity and low activity against the wild type EGFR (IC > 1000 nM). In particular,
5
0
T790M
compound 14a also displayed strong potency against EGFR
-mutated H1975 cells (IC50
=
0.074 μM), but weak activity toward normal cells HBE (IC > 40 μM) and LO-2 (IC =
5
0
50
9
.891 μM). Importantly, compound 14a (SI = 52.6) significantly improved the selectivity
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doi: 10.1111/cbdd.13370
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against mutant H1975 cells over wild-type A431 cells than rociletinib (SI = 6.0), thus
revealing its slight cell cytotoxicity. This study provides a promising Thio-DPPY derivative
as enhanced EGFR T790M inhibitor, and also revealed valuable clues for further
optimization of DPPY scaffold to overcome NSCLC resistance.
Keywords: NSCLC, EGFR T790M, Resistance, Inhibitors, Pyrimidine.
1
. Introduction
The epidermal growth factor receptor (EGFR) tyrosine kinase is one of the most
important clinically validated targets for non-small-cell lung cancer (NSCLC) treatment [1,
]. First-generation EGFR inhibitors, gefitinib (1) and erlotinib (2), were approved by United
States Food and Drug Administration (FDA) for treatment of NSCLC patients in 2003 and
004, respectively (Fig.1) [3, 4]. Both of them achieved excellent improvements in overall
2
2
response rates (ORR) and median progression-free survival (PFS) for NSCLC patients
harboring with EGFR activating mutations (exon 19 deletion or exon 21 L858R substitution
mutation) [5, 6]. Unfortunately, the secondary T790M (substitution of threonine to
methionine) gatekeeper point mutation in exon 20, which accounts for approximately 60% of
the drug resistances, often occurs for most NSCLC patients who are exhibiting activating
mutations that are invariably developed acquired resistance to EGFR tyrosine kinase inhibitor
(
TKIs) therapy within months. The replacement of a threonine by the more sterically
demanding methionine increases the affinity to ATP and provokes a steric repulsion of the
-aminoquinazoline-based inhibitors erlotinib and gefitinib, resulting in a different binding
4
mode and significant loss of inhibitory activity [7, 8].
Second-generation EGFR TKIs which incorporate a Michael acceptor to covalently
target a rare cysteine (Cys797) in EGFR at the lip of the ATP-binding site were discovered to
overcome EGFR T790M drug resistance. In these inhibitors, the electrophile represents the
only distinctive feature as compared to first-generation EGFR inhibitors, and thus, the
potential of these drugs to overcome T790M drug resistance is directly correlated with
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covalent modification of the target protein [9, 10]. Afatinib (3) was the representative
second-generation EGFR inhibitor, showing excellent clinical results to overcome EGFR
T790M drug resistance. But for the dose limiting toxicities related to the inhibition of
wild-type (WT) EGFR, the second EGFR inhibitor displayed limited clinical benefit for
NSCLC patients who have developed T790M acquired resistance [11,12]. Therefore,
third-generation covalent EGFR inhibitors, such as compounds 4 (WZ4002) [13], 5
(CO-1686, rociletinib) [14], and 6 (AZD9291, osimertinib) [15, 16], have been developed as
mutant-selective EGFR inhibitors that specifically target EGFRL858R/T790M mutation
while being less selective for WT EGFR (Fig.1). Generally, most of these third-generation
EGFR T790M inhibitors feature a pyrimidine template along with an acrylamide functional
substituent.
Sulfur, as the core chemical elements needed for biochemical functioning, is an
elemental macronutrient for all life. A large number of sulphur-substituted organic molecules
have been advanced to market for treatment of various disease, including Fudosteine (7) as a
novel mucoactive agent [17], Omeprazole (8) as a medication used in the treatment of
gastroesophageal reflux disease, peptic ulcer disease, and Zollinger–Ellison syndrome [18],
Montelukast (9) as a leukotriene receptor antagonist (LTRA) used for the maintenance
treatment of asthma and to relieve symptoms of seasonal allergies [19], and Modafinil (10) as
a wakefulness-promoting agent (or eugeroic) used for treatment of disorders such as
narcolepsy, shift work sleep disorder, and excessive daytime sleepiness associated
with obstructive sleep apnea [20]. Notably, sulphor-submitted molecules also displayed
excellent biological propriety for treatment of cancers. For example, Compound 11 is a
typical JNK3 inhibitor [21], compound 12 is a EGFR inhibitor [22], and compound 13 is
Bcl-2 inhibitor [23] (Fig.2). Based on the novel pyrimidine biological template [24], we
synthesized a new class of thiopyrimidine derivatives to improve the activity and selectivity
against EGFR T790M enzyme (Fig.3). Moreover, their activity against wild type EGFR and
EGFR T790M-mutated enzymes, as well as their activity against NSCLC cell lines were also
described in this manuscript.
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2
. Results and Discussion
2
.1. Chemistry
Cl
X
N
Cl
b
N
Cl
X
Cl
NH2
O
N
N
NH
O
a
HN
S
N
HN
S
N
NH
X
X
N
H
f
g
1
1
5a X = OH
5b X = NH2
16a X = OH
16b X = NH2
17a X = O
17b X = NH
NH
NH
NO2
NO2
NH2
O
O
O
NO2
c
d
e
N
O
N
O
S
S
S
O
O
14a X = NH
4b X = O
SH
1
14c
Cl
N
N
18
19
20
21
Scheme 1. Synthetic route of the title compounds 14a-c. Reagents and conditions: (a) acryloyl chloride,
NaHCO , CH CN, rt., 0.5 h, 60-80%; (b) X=OH, K CO , DMF, 60 C; X=NH , DIPEA, 1,4-dioxane, rt., 2
3
3
2
3
2
h, 91%; (c) 1-chloro-3-bromopropane, K CO , CH CN, 70 C, 12 h, 95%; (d) morpholine, K CO , KI,
2
3
3
2
3
DMF, 80 C, 12 h, 81%; (e) Fe-NH Cl, MeOH-H O, 2 h, 60 C, 72%; (f) TFA, 2-BuOH, 100 C, 12 h,
4
2
2
2-37%; (g) m-CPBA,CH Cl , rt., 31%.
2
2
Cl
X
Cl
X
N
N
NO2
NO2
NH2
HN
N
N
HN
S
N
N
NO2
a,b
c,d
e
f
g
NH
NH
S
S
S
S
O
O
F
O
O
OH
O
N
O
N
O
O
O
O
O
O
1
4d X = NH
2
2
23
24
25
14f X = NH
1
4e X = O
Scheme 2. Synthetic route of the title compounds 14d-f. Reagents and conditions: (a) ethyl
-mercaptoacetate, K CO , KI, DMF, 100 C, 12 h, 95%; (b) KOH, H O, 50 C, 24 h, 76%; (c) SOCl , 80
2
2
3
2
2

C, 2 h; (d) morpholine, NaHCO , CH CN, rt, 0.5 h, 95%; (e) Fe-NH Cl, MeOH-H O, 2 h, 60 C, 72%; (f)
3
3
4
2
pyrimidines 17a-b, TFA, 2-BuOH, 100 C, 12 h, 16-19%, (g) m-CPBA,CH Cl , rt., 25%.
2
2
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The synthetic routes of the title molecules 14a-f are presented in Schemes 1 and 2
25-28]. Commercially available anilines 15a,b were reacted with acryloyl chloride to
[
prepare the 3-acrylamide-substituted compounds 16a,b in almost quantitative yields. By
regioselectively coupling anilines 16a,b with the 2,4,5-tricholrepyrimidine reagent, the key
C-2 chloro-pyrimidine intermediates 17a,b were synthesized. On the other hand,
4
-nitrothiophenol 18 was reacted with 1-chloro-3-bromopropane to yield the
chloro-substituted compound 19, which was then converted to compound 20 characterized by
a morpholine substituent. Next, the nitro-substituted compound 20 was reduced to the aniline
derivative 21 by using the Fe-NH Cl condition. After completing the reaction between
4
pyrimidines 17a,b and aniline 21 under the action of trifluoroacetic acid (TFA), the title
molecules 14a,b were synthesized. Finally, oxidizing the sulfur atom in molecule 14a with
the m-chloroperbenzoic acid (m-CPBA) reagent prepared the sulfoxide-substituted compound
1
4c. Similarly, the title molecules 14d-f were also prepared as shown in Scheme 2. Starting
from the reaction of 1-fluoro-4-nitrobenzene with ethyl 2-mercaptoacetate, the intermediate
3 was prepared, which was converted to nitro-substituted intermediate 24 via subsequent
2
hydrolysis and acylation reactions. Compound 24 was then reduced to aniline 25 by using
Fe-NH Cl reduction condition. By regionselectvie nucleophilic substitution and oxidation
4
reactions, the desired molecules 14d-f were conventionally synthesized.
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2
2
.2. Biological evaluation of 14a-f
.2.1. Kinase inhibitory activity
Table 1 In vitro EGFR tyrosine kinases (wild type and L858R/T790M mutation) activities of the title
a
compounds 14a-f
Cl
N
HN 2 N 4
X
1
'
2
3
'
'
NH
4
'
R
O
14a-f
Enzymatic activity
b
SI
(
IC , nM)
50
Compound
X
R
(WT: L858R/T790M)
L858R/T790M
WT
EGFR
EGFR
S
N
1
4a
4b
4c
4d
4e
4f
NH
O
27.5±3.3
>1000
1012±101.7
1035±81.5
980.0±72.4
>1000
>36.4
<10.1
10.4
O
S
S
N
N
1
>100.0
98.2±12.1
>100.0
O
O
1
NH
NH
O
O
S
1
<9.8
N
N
N
O
O
O
O
O
O
S
S
1
9.1±1.3
>109.9
12.5
1
NH
79.3±9.2
20.0±4.3
823.3±101.1
992.9±102.1
500.0±67.5
15.5±2.6
O
Rociletinib
Gefitinib
25.0
0.019
a
Data represent the mean of at least three separate experiments.
b
Concentration needed to inhibit the autophosphorylation of the cytoplasmic domain of EGFR by 50%, as
calculated using software GraphPad Prim version 5.0.
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The kinase-based activity against the wild type EGFR and the T790M/L858R-mutated
EGFR was evaluated by using the ADP-Glo™ Kinase Assay system [29, 30]. Two novel
EGFR inhibitors, gefitinib and rociletinib, were also tested as positive controls. The test
results presented in table 1 clearly indicated that compounds 14a and 14e displayed the
T790M/L858R
strongest anti-EGFR
activity among Thio-DPPYs, with IC values of of 27.5 and
50
9
.1 nM, respectively. While the remaining compounds could effectviely inhibit
T790M/L858R
EGFR
activity at concentrations of more than 79.3 nM. Structure and activity
relationship analysis suggested that the oxidation of the sulfur atom to the sulfoxide group is
unfavorable. For example, the sulfur-substituted analogue 14a (IC = 27.5 nM) is markedly
5
0
stronger than the analogue 14c (IC50 = 98.2 nM) which bears a sulfoxide substituent.
Fortunately, most of these compounds are inactive against the wild type EGFR (IC > 980
5
0
nM), indicating their high selectivity against the EGFR T790M mutation over the wild type
EGFR. In particular, the potent inhibitors 14a (SI > 36.4) and 14e (SI > 110.2) possess
greatly higher selectivity than rociletinib (SI = 25), which suggested that they will produce
less side effects.
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a
Table 2 Cellular antiproliferative activities of the title compounds 14a-f.
b
SI
Antiproliferative activity(IC , μM)
5
0
Compd.
(A431/
H1975
HCC827
A431
A549
LO2
HBE
H1975)
1
4a
4b
0.074±0.011 0.152±0.088 3.893±0.708 5.794±1.022 9.891±1.236
>40
52.6
1
0.373±0.091 0.204±0.091 5.762±1.089 5.842±0.931 16.50±2.090 38.5±3.1
0.255±0.078 0.308±0.079 0.573±0.103 3.336±0.926 5.059±0.858 >40
0.464±0.099 0.050±0.019 1.192±0.213 3.576±0.759 9.296±1.086 25.3±4.1
15.4
2.2
2.6
7.2
3.4
6.0
0.7
1
4c
4d
4e
4f
1
1
1.050±0.125 0.198±0.087 7.557±0.834 8.493±2.010 20.90±3.147
0.667±0.101 0.062±0.011 2.247±0.947 33.97±5.78 14.77±2.014
>40
>40
>40
1
Rociletinib 0.299±0.092 0.031±0.009 1.794±0.965 3.242±0.945 12.75±2.084
Gefitinib 4.691±1.050 0.006±0.001 3.300±0.817 10.07±1.03 21.86±4.152 23.8±3.2
a
Data represent the mean of at least three separate experiments.
b
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.
2
.2.2. Antiproliferative activity against NSCLC cell lines
In addition, all these compounds were evaluated for their activity against NSCLC cell
wild type EGFR
lines using the MTT assay method. Four typical NSCLC cell lines (A431
, H1975
EGFR T790M mutation
wild type EGFR and k-ras EGFR mutation
EGFR del E746-A750 mutation
,
A549
and HCC827
), and
two normal cell lines (human bronchial epithelial cells(HBE), and liver cells(LO-2)) were
used to evaluate the cell viability. As shown in Table 2, Thio-DPPY derivatives could
actively interfere with the proliferation of gefitinib-resistant H1975 cells within the 0.074
(compound 14a) to 1.050 μM (compound 14e) concentrations range; however, they are less
potent against the A431 cells harboring wild type EGFR ((compound 14c, IC > 0.573 μM).
5
0
In spite of the strong inhibitory potency toward the T790M-mutated EGFR enzyme,
compound 14e only displayed moderate capability for inhibiting the H1975 cells, with an
T790M
IC value of 1.05 μM. Fortunately, compound 14a, another potent EGFR
inhibitor,
5
0
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exhibited strong potency against H1975 cells at a concentration of 0.074 μM. Compound 14a
also showed moderate activity to inhibit the proliferation of A431 cells (IC = 3.893 μM),
5
0
indicating its high selectivity (SI = 52.6) toward mutant H1975 cells over wild type A431
cells. The activity against H1975 cells also showed that compounds 14d-f (IC =0.464~1.05
5
0
μM) bearing a N-morpholine formamide functional group in the C-2 aniline side chain is
slightly less active than the compounds 14a-c (IC50 = 0.074~0.373 μM) without this
substituent. Furthermore, all these compounds are highly sensitive to the HCC827 cell
harboring EGFR del E746-A750 mutation, possessing IC50 values lower than 0.308 μM.
Compound 14d, the most active inhibitor against HCC827 cells in this series of compounds,
particularly has an IC50 value of 0.050 μM. Additionally, all these compounds displayed
moderate capability for inhibiting the A549 cells bearing the K-ras mutation. In terms of their
activity against the normal cell lines HBE and LO-2, most of these Thio-DPPY derivatives
are inactive, and the typical inhibitor 14a (with IC values of 9.89 and 40 μM, respectively,)
50
displayed considerably low cell cytotoxicity. Figure 4 showed that the most active inhibitor
1
4a significantly blocked the proliferation of H1975 cells with increasing time (24, 48, and
2 h) and drug concentrations (0.2, 0.5, and 1.0 μM). When treatment of 1.0 μM
7
concentration of 14a for 72 h, the cell viability rate (26.8%) was approximately 2-flod lower
than that of rociletinib (44.8%), indicating enhanced anti-gefitinib resistance. These
biological evaluations suggested that compound 14a can be potentially used as a promising
T790M
EGFR
inhibitor toward NSCLC.
2
.2.3. Flow cytometry
The effect of the most potent inhibitor 14a on the apoptosis in EGFR
T790M
-mutated
H1975 cells was also explored. As shown in Figure 5, compound 14a could induce apoptosis
in H1975 cells in a dose- and time-dependent manner. The apoptosis rates apparently
increased from 45.6% to 96.8% in H1975 cells treated with compound 14a (0.2, 0.5, and 1.0
μM) for 72 h.
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2
.3. Binding model analysis
To further elucidate the binding mode of the Thio-DPPY derivatives with the EGFR
T790M enzyme, a docking study was performed using Autodock 4.2 software [31, 32]. The
representative structures of 14a, 14b, 14c and 14e were docked into the crystal structure of
the EGFR T790M binding pocket (PDB: 3IKA), respectively [13]. The autodock default
parameters were used during the docking performance. For comparison, the lead compound
WZ4002 was also docked into the binding pocket by using the same procedure. The
simulation result showed in Figure 6 indicated that compounds 14a and 14b, which possess
strong inhibitory enzymatic activity, tightly bind with the EGFR T790M enzyme similarly to
WZ4002. Typically, all these potent inhibitors formed several important contacts with
T790M
EGFR
kinase, including (1) covalent-bond forces between the acrylamide group with the
amino acid Cys797; (2) a strong contact generated from the chlorine atom at the C-5 position
of the pyrimidine core with the mutant gatekeeper residue Met790; (3) strong hydrogen
bonds between the N-1 nitrogen atom of the pyrimidine core and the amino acid Leu792.
T790M
However, the less potent inhibitors 14b and 14c bind with EGFR
in a quite different
manner from the active compounds, and almost lose these important interaction forces. These
binding modes are accorded with their biological activity data.
3
. Conclusion
To overcome gefitinib resistance in NSCLC, a class of Thio-DPPY derivatives were
synthesized as potent and selective EGFR T790M inhibitors. Among these newly obtained
T790M
compounds, two active EGFR
inhibitors 14a and 14e were identified, possessing IC50
values of 27.5 and 9.1 nM, respectively. Moreover, compounds 14a (SI > 36.4) and 14e (SI >
09.9) displayed less potency against the wild type EGFR (IC50 > 980 nM), but higher
1
selectivity than rociletinib (SI = 25.0). In particular, compound 14a exhibited strong potency
against mutant H1975 cells (IC = 0.074 μM), and weak inhibitory ability toward the two
5
0
normal cells HBE and LO-2 (with IC of 40 and 9.89 μM, respectively). All these results
5
0
proved that inhibitor 14a may be served as a potential EGFR T790M inhibitor to overcome
gefitinib resistance toward NSCLC.
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4
. Experimental section
4
.1. General Methods and Chemistry
All solvents and reagents were obtained from commercial supplies and were used without
further purifications. Solvents were purified and stored according to standard procedures.
®
High resolution ESI-MS was performed on an AB Sciex TripleTOF 4600 LC/MS/MS
1
13
system. H NMR and C NMR spectra on a Brucker AV 400 MHz spectrometer were
recorded in [d] DMSO. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ
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. Flash chromatography separations were obtained on Silica Gel (300–400
mesh) using dichloromethane/methanol as eluents.
4
.2. General procedure for the synthesis of the title compounds 14a,b and 14d,e.
-Chloro-pyrimidine derivatives 17a,b and the morpholine-substituted anilines
2
intermediates 21, 25 were synthesized according to our reported procedures [25-27]. All
these intermediates were directly used without any purification and structural characterization.
With these intermediates in hand, the title molecules 14a, b and 14d, e were synthesized as
described below.
A flask was charged with intermediates 14a,b (0.10 mmol), anilines 21, 25 (0.10 mmol),
TFA (0.08 mL, 1.05 mmol), and 2-BuOH (10 mL). The slurry was heated to 100 °C for 12 h.
The reaction mixture was allowed to cool to room temperature and was neutralized with a
saturated aqueous sodium bicarbonate solution. The aqueous mixture was then extracted with
CH Cl (20 mL) three times. The crude product was purified using flash chromatography
2
2
with dichloromethane/methanol (v/v, 30:1) as eluents.
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4
.2.1.N-[3-[[5-Chloro-2-[4-((1-morpholino)propylthio)phenylamino]-4-pyrimidinyl]amino]
phenyl]-2-propenamide (14a)
Yield: 36.52%; off-yellow solid; H NMR (400 MHz, DMSO-d ): δ 1.60-1.63 (m, 2H),
1
6
2
.28-2.32 (m, 6H), 2.82 (t, J = 8.0 Hz, 2H), 3.53 (t, J = 8.0 Hz, 4H), 5.76 (dd, J = 4.0 Hz, 8.0
Hz, 1H), 6.26 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.44 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 7.11 (d, J =
8
9
5
1
.0 Hz, 2H), 7.18-7.35 (m, 2H), 7.52-7.57 (m, 3H), 7.89 (s, 1H), 8.15 (s, 1H), 9.00 (s, 1H),
1
3
.41 (s, 1H), 10.22 (s, 1H); C NMR (100 MHz, DMSO-d ): δ 26.37, 32.42, 53.98 (2C),
6
7.39, 66.86 (2C), 104.60, 116.17, 116.24, 120.03 (2C), 120.14, 127.35, 127.64, 129.32,
31.03 (2C), 132.53, 139.46, 139.76, 139.83, 155.48, 156.96, 158.18, 163.84; HRMS (ESI)
+
m/z calcd for C H ClN O S, [M+H] 525.1834, found: 525.1843.
2
6
29
6
4
4
.2.2.N-[3-[[5-Chloro-2-[4-((1-morpholino)propylthio)phenylamino]-4-pyrimidinyl]oxygen
phenyl]-2-propenamide (14b)
Yield: 22.14%; off-yellow solid; H NMR (400 MHz, DMSO-d ): δ 1.64-1.69 (m, 2H),
]
1
6
2
.32-2.36 (m, 6H), 2.87 (t, J = 8.0 Hz, 2H), 3.58 (t, J = 8.0 Hz, 4H), 5.83 (dd, J = 4.0 Hz, 8.0
Hz, 1H), 6.32 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.49 (dd, J = 12.0 Hz, 16.0 Hz, 1H), 7.06-7.10
(m, 3H), 7.40 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.76 (s, 1H), 8.54 (s, 1H), 9.89 (s,
1
3
1
H), 10.46 (s, 1H); C NMR (100 MHz, DMSO-d ): δ 26.01, 31.83, 53.67 (2C), 57.04, 66.56
6
(2C), 105.09, 113.30, 117.02, 117.39, 119.75 (2C), 127.79, 128.01, 130.31 (2C), 130.39,
1
31.97, 138.56, 140.78, 152.66, 157.75, 158.51, 163.73, 164.26; HRMS (ESI) m/z calcd for
+
C H ClN O S, [M+H] 526.1674, found: 526.1678.
26
28
5
3
4
.2.3.N-[3-[[5-Chloro-2-[4-((1-morpholino)ethanethioate)phenylamino]-4-pyrimidinyl]ami
no]phenyl]-2-propenamide (14d)
1
Yield: 16.04%; off-white solid; H NMR (400 MHz, DMSO-d ): δ 3.41-3.54 (m, 8H), 3.80
6
(s, 2H), 5.76-5.79 (m, 1H), 6.28 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.48 (dd, J = 8.0 Hz,16.0 Hz,
1
1
4
H), 7.20 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.45-57 (m, 3H), 7.91 (s, 1H), 8.18 (s,
1
3
H), 9.03 (s, 1H), 9.46 (s, 1H), 10.24 (s, 1H); C NMR (100 MHz, DMSO-d ): δ 37.70,
6
2.74, 47.14, 67.04 (2C), 105.06, 116.42, 116.59, 120.18 (2C), 120.48, 126.44, 127.95,
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1
1
29.62, 132.30 (2C), 132.84, 139.75, 140.12, 140.71, 155.75, 157.25, 158.43, 164.15,
+
67.63; HRMS (ESI) m/z calcd for C H ClN O S, [M+H] 525.1470, found: 525.1484.
2
5
25
6
3
4
.2.4.N-[3-[[5-Chloro-2-[4-((1-morpholino)ethanethioate)phenylamino]-4-pyrimidinyl]oxy
gen]phenyl]-2-propenamide (14e)
1
Yield: 19.01%; off-white solid; H NMR (400 MHz, DMSO-d ): δ 3.39 (t, J = 4.0 Hz, 4H),
6
3
1
7
.50 (t, J = 4.0 Hz, 4H), 3.77 (s, 2H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.26 (dd, J = 4.0 Hz,
6.0 Hz, 1H), 6.45 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 7.00-7.03 (m, 1H), 7.11 (d, J = 8.0 Hz, 2H),
.35 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.70 (s, 1H), 8.48
1
3
(
s, 1H), 9.85 (s, 1H), 10.39 (s, 1H); C NMR (100 MHz, DMSO-d ): δ 36.89, 42.14, 46.51,
6
6
6.43 (2C), 105.30, 113.26, 117.09, 117.43, 119.60 (2C), 126.84, 127.81, 130.40, 131.36
(2C), 131.98, 139.24, 140.78, 152.67, 157.74, 158.50, 163.77, 164.28, 166.95; HRMS (ESI)
+
m/z calcd for C H ClN O S, [M+H] 526.1310, found: 526.1323.
2
5
24
5
4
4
.3. General procedure for the synthesis of the title compounds 14c,f.
m-CPBA (75%) (0.24 g, 1.03 mmol) was added to a solution of 14a or 14d (0.25 g, 0.52
mmol) in anhydrous CH Cl (10 mL) at 0 °C. The reaction mixture was allowed to warm to
2
2
room temperature and then stirred for 1 h. The solution was diluted with CH Cl and then
2
2
treated with 50% Na S O /NaHCO solution. The organic layer was separated, washed with
2
2
3
3
brine, dried with Na SO , filtered, and concentrated in vacuo. The resulting crude material
2
4
was subjected to flash column chromatography (CH Cl /MeOH stepwise elution, 30:1) to
2
2
give the title molecules 14c,f .
4
.3.1.N-[3-[[5-Chloro-2-[4-((1-morpholino)propylsulfinyl)phenylamino]-4-pyrimidinyl]ami
no]phenyl]-2-propenamide (14c)
1
Yield: 31.0%; off-brown solid; H NMR (400 MHz, DMSO-d ): δ 1.56-1.68 (m, 2H),
6
2
1
5
.26-2.32 (m, 6H), 2.74-2.86 (m, 2H), 3.52 (t, J = 8.0 Hz, 4H), 5.76 (dd, J = 4.0 Hz, 8.0 Hz,
H), 6.27 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.48 (dd, J = 12.0 Hz, 16.0 Hz, 1H), 7.30-7.40 (m,
H), 7.53-7.58 (m, 1H), 7.83 (d, J = 8.0 Hz, 2H), 8.22 (s, 1H), 9.10 (s, 1H), 9.73 (s, 1H),
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1
3
1
0.28 (s, 1H); C NMR (100 MHz, DMSO-d ): δ 19.14, 19.47, 53.68 (2C), 57.13, 66.78
6
(2C), 105.28, 114.31, 116.17, 116.35, 119.33 (2C), 125.25 (2C), 126.64, 127.63, 129.53,
1
32.56, 135.60, 139.91, 143.62, 155.47, 157.03, 158.01, 163.86; HRMS (ESI) m/z calcd for
+
C H ClN O S, [M+H] 541.1783, found: 541.1788.
26
29
6
3
4
.3.2.N-[3-[[5-Chloro-2-[4-((1-morpholino)acetylsulfinyl)phenylamino]-4-pyrimidinyl]ami
no]phenyl]-2-propenamide (14f)
1
Yield: 25.0%; off-brown solid; H NMR (400 MHz, DMSO-d ): δ 3.37-3.52 (m, 8H), 3.93 (d,
6
J = 12.0 Hz, 1H), 4.11 (d, J = 12.0 Hz, 1H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.27 (dd, J =
4
8
.0 Hz, 16.0 Hz, 1H), 6.47 (dd, J = 8.0 Hz,16.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.49 (d, J =
.0 Hz, 2H), 7.82-7.94 (m, 4H), 8.22 (s, 1H), 9.11 (s, 1H), 9.72 (s, 1H), 10.25 (s, 1H); 13C
NMR (100 MHz, DMSO-d ): δ 42.39, 46.71, 61.09, 66.61, 66.78, 105.36, 116.23, 116.43,
6
1
1
5
19.23 (2C), 120.43, 125.78 (2C), 127.71, 129.36, 132.53, 135.41, 139.40, 139.91, 143.96,
+
55.48, 157.09, 158.01, 163.89, 163.91; HRMS (ESI) m/z calcd for C H ClN O S, [M+H] ]
2
5
25
6
4
41.1419, found: 541.1441.
4
.4. Kinase enzymatic assays
TM
WT
T790M/L858R
ADP-Glo
assay systems (EGFR : Catalog. V3831, EGFR
: Catalog.
V5324), were purchased from Promega Corporation (USA). The experiments were performed
according to the instructions of the manufacturer. The detailed and complete protocols, and
the
active
kinase
data
were
available
at:
https://cn.promega.com/resources/protocols/product-information-sheets/n/egfr-kinase-enzym
e-system-protocol/
and
https://cn.promega.com/resources/protocols/product-information-sheets/n/egfr-t790m-l858r-k
inase-assay-protocol/, respectively. For all of the tested compunds, concentrations consisting
of suitable levels from 0.1 to 1000 nM were used. The test was performed in a 384-well plate,
including the main steps below: (1) perform a 5 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 theunconsumed ATP, leaving
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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.
4
.5. Cellular activity assay
H1975, H431, HCC827, and A549 human NSCLC cells were obtained from the
American Type Culture Collection. HBE cells and LO-2 cells were kind gifts from Fuheng
Biology Company (Shanghai, China). H1975, HCC827 and A549 cells were grown in
®
®
RPMI-1640 (Gibco , USA) supplemented with 10% FBS (Gibco , USA), 1%
penicillin-streptomycin (Beyotime Company, China). A431 , LO-2 and HBE 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 °C in humidified 5% CO incubator.
2
Cell viability was then assessed with MTT reagent (Thiazolyl blue tetrazolium bromide;
Sigma, Oakville, ON). Cells were seeded in 96-well plates at a density of 3,000 to 5,000
cells/well and were maintained at 37 C in a 5% CO incubator in DMEM or RPMI1640
2
®
containing 10% fetalbovine serum (FBS, Gibco ). Cells were esposed to treatment for 72 h,
and the number of cells used per experiment for each cell lines was adjusted to obtain an
absorbance of 0.5 to 1.2 at 570 nm. Compounds were tested at appropriate concentrations
(0.01 to 40 μM), with each concentration duplicated five times. The data were calculated
using GraphPad Prim version 5.0. Dose-response curves were fitted using a nonlinear
regression model with a sigmoidal dose-response.
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4
.6. Molecular docking study
AutoDock 4.2 software and the default parameter was used to perform the docking
studies. Detailed tutorials that guide users through basic AutoDock usage, docking with
flexible rings, and virtual screening with AutoDock may be found at:
http://autodock.scripps.edu/faqs-help/tutorial. Generally, the crystal structure (PDB: 3IKA) of
T790M
the kinase domain of EGFR
bound to inhibitor 4 was used in the docking studies. The
enzyme preparation and the hydrogen atoms adding was performed in the prepared process.
The whole EGFR enzyme was defined as a receptor and the site sphere was selected on the
basis of the binding location of WZ4002. By moving WZ4002 and the irrelevant water,
molecules 14a, b, c and e were placed, respectively. When doing the molecular simulation,
the covalent bond produced by the acryloyl group and the amino acid Cys797 is set as default
if the distance between them is low than 3.82 Å (Within this distance, WZ4002 could form
covalent bond with Cys797). The binding interaction energy was calculated to include Van
der Waals, electrostatic, and torsional energy terms defined in the tripos force field. The
structure optimization was performed using a genetic algorithm, and only the best-scoring
ligand protein complexes were kept for analyses.
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 81672945,
8
1603186), and Research Fund of Higher Education of liaoning province（LQ2017039）for
the financial support of this research.
Figure legends
Figure 1. Novel EGFR inhibitors for treatment of NSCLC.
Figure 2. Chemical structures of the typical organosulfur compounds.
Figure 3. Designed strategy of the Thio-DPPY derivatives.
Figure 4. The effects of treating time and drug concentrations of compound 14a and rociletinib on H1975
cell viability, (A) inhibitor 14a; (B) rociletinib.
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Figure 5. Compound 14a induced H1975 cell apoptosis in vitro. The cells were incubated with the
indicated concentrations of 14a for 72 h, and the cells were stained with annexinV/FTIC, followed by flow
cytometry analysis. One representative experiment is shown.
T790M
Figure 6. A: Binding mode of WZ4002 with EGFR
enzyme (PDB code: 3IKA); B,C,D,E: Proposed
T790M
binding modes of the typical inhibitors with EGFR
enzyme (B: inhibitor 14a, C: inhibitor 14e, D:
inhibitor 14b, E: inhibitor 14c).
Conflict of interest
All these authors were employed by Dalian Medical University, and all of them have no
financial and personal relationships with other people or organizations that can
inappropriately influence this work.
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