2-Oxo-3,4-dihydropyrimido[4,5-d] pyrimidines as new reversible inhibitors of EGFR C797S (Cys797 to Ser797) mutant

Article abstract of DOI:10.1016/j.cclet.2019.09.044

Extensive structure-activity relationships (SARs) study of JND3229 was conducted to yield a series of new reversible 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidine privileged scaffold as EGFR^C797S inhibitors. One of the most potent compound 6i potently suppressed EGFR^{L858R/T790M/C797S} kinase with an IC₅₀ value of 3.1 nmol/L, and inhibited the proliferation of BaF3 cells harboring EGFR^{L858R/T790M/C797S} and EGFR^{19D/T790M/C797S} mutants with IC₅₀ values of 290 nmol/L and 316 nmol/L, respectively. Further, 6i dose-dependently induced suppression of the phosphorylation of EGFR^{L858R/T790M/C797S} and EGFR^{19D/T790M/C797S} in BaF3 cells. Compound 6i may serve as a promising lead compound for further drug discovery overcoming the acquired resistance of non-small cell lung cancer (NSCLC) patients.

Full text of DOI:10.1016/j.cclet.2019.09.044

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CCLET 5250 No. of Pages 7
Chinese Chemical Letters xxx (2019) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
2
-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible
inhibitors of EGFR C797S (Cys797 to Ser797) mutant
a,1
a,1
b,1
c,1
a
b
Xianglong Hu , Qiuju Xun , Tao Zhang , Su-Jie Zhu , Qian Li , Linjiang Tong ,
b
a
d,
b,
a,
a,
Mengzhen Lai , Tao Huang , Cai-Hong Yun *, Hua Xie *, Ke Ding *, Xiaoyun Lu *
a
International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education
(
MOE), School of Pharmacy, Jinan University, Guangzhou 510632, China
Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
b
2
01203, China
c
Institute for Translational Medicine, College of Medicine, Qingdao University, Qingdao 266021, China
Department of Biochemistry and Biophysics, Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center,
d
Beijing 100191, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Available online xxx
Extensive structure-activity relationships (SARs) study of JND3229 was conducted to yield a series of new
C797S
reversible 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidine privileged scaffold as EGFR
inhibitors. One
L858R/T790M/C797S
of the most potent compound 6i potently suppressed EGFR
kinase with an IC50 value of
L858R/T790M/C797S
3
.1 nmol/L, and inhibited the proliferation of BaF3 cells harboring EGFR
and
Keywords:
19D/T790M/C797S
EGFR
mutants with IC50 values of 290 nmol/L and 316 nmol/L, respectively. Further, 6i
C797S
EGFR
SARs
mutant
L858R/T790M/C797S
dose-dependently induced suppression of the phosphorylation of EGFR
and
19D/T790M/C797S
EGFR
in BaF3 cells. Compound 6i may serve as a promising lead compound for further
2
-Oxo-3,4-dihydropyrimido[4,5-d]
drug discovery overcoming the acquired resistance of non-small cell lung cancer (NSCLC) patients.
pyrimidine
Clinical resistance
Fourth-generation inhibitors
©
2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Epidermal growth factor receptor-tyrosine kinase inhibitors
EGFR-TKIs) have brought great benefits in the clinic for the
it is highly urgent to develop the fourth-generation EGFR
C797S
(
inhibitors to overcome EGFR
mutation [9].
treatment of non-small cell lung cancer (NSCLC) patients harbor-
ing EGFR-mutation. To date, three generations of EGFR inhibitors
Several fourth-generation inhibitors of EGFR with different
binding types have been reported [1,10], including the allosteric
inhibitors EAI045 (1a) and JBJ-04-125-02 (1b) [11], and the ATP-
competitive inhibitors 2-4 [12–15] (Fig. 1). Among them, the
[
1,2], e.g., gefitinib, erlotinib, afatinib, dacomitinib and osimertinib
(
AZD9291), have been approved by Food and Drug Administra-
tion (FDA). Particularly, the third generation EGFR Thr790 to
Met790 mutant (T790 M) inhibitor osimertinib, covalently
(2-hydroxy)pheyl-4-substitututed quinazoline 2 and tri-substitut-
C797S
ed imidazole 3 displayed strong potency against EGFR
kinase
binding to Cys797, demonstrated approximately 75% overall
in biochemical assay, but neither cell-based activity nor in vivo
efficacy of these molecules was disclosed. EAI045 (1a) and
brigatinib (4) were both required to combine with EGFR antibody
cetuximab or pantitumumab to demonstrate in vivo therapeutic
efficacy [11,15]. Most recently, we have identified a pyrimidopyr-
T790M
response rate (ORR) in EGFR
mutation-positive NSCLC
patients and was approved as the first-line treatment for
metastatic NSCLC patients in 2018 [3–5]. However, EGFR tertiary
Cys797 to Ser797 (C797S) point mutation arises rapidly after
treatment of osimertinib in clinic, due to the fact that the less
imidinone-based derivative JND3229 (5) as a new reversible
C797S
reactive serine797 fails to form a covalent bond with osimerti-
EGFR
mutant inhibitor demonstrating both in vitro and in vivo
C797S
C797S
nib [6,7]. EGFR
mutation has become the most common
mono-drug efficacy against the proliferation of EGFR
cancer cells [16]. Structure analysis indicated that JND3229 bound
to the ATP binding site of EGFR
mutated
acquired resistance to the third-generation EGFR-TKIs [8]. Thus,
C797S
with a reversible “U-shaped”
configuration. The 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidine
privileged core formed a bidentate hydrogen bond interactions
with Met793, the 2-chlorophenyl group was directed toward the
hydrophobic back pocket, and the carbonyl in pyrido[4,5-d]
pyrimidine formed a hydrogen bond with Lys745 mediated by a
*
Corresponding authors.
E-mail addresses: yunch@hsc.pku.edu.cn (C.-H. Yun), hxie@simm.ac.cn (H. Xie),
dingke@jnu.edu.cn (K. Ding), luxy2016@jnu.edu.cn (X. Lu).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2019.09.044
1001-8417/© 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
(

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X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
Fig. 1. The reported fourth generation EGFR inhibitors overcoming EGFRC797S mutation.
EGFRL858R/T790M/C797S. When exocyclic N atom in the linker was
incorporated into the cycloalkyl group, the resulting compounds
(
6c–6h)
showed
greatly
decreased
potency
on
L858R/T790M/C797S
EGFR
. Compound 6i with cis-cyclohexanamine
instead of the trans-cyclohexanamine exhibited an IC50 value of
L858R/T790M/C797S
3
.1 nmol/L against EGFR
, which is comparable to
that of JND3229. Moreover, this compound is 7-fold more potent
L858R/T790M
than JND3229with the inhibitoryactivityof EGFR
double
mutant. These results indicated that the cyclohexanamine group in
the linker fraction was preferable for the high inhibitory activity.
After determined that the cyclohexanamine was the optimal
substituent for the L part, we then turned to optimize the R1
substitution (Table 1). It was revealed that the ethyl in JND3229
could be replaced with a variety of relative small substituents (e.g.,
methyl (6j), isopropyl (6k), tert-butyl (6l)), without obviously
affecting the EGFR inhibitory potency. However, when the ethyl was
replaced with larger hydrophobic groups, including cyclopentyl
(6m), or cyclohexyl (6n), and phenyl (6o), the resulting molecules
displayed about 3- to 7-folds potency loss. For instance, 6o with the
Fig. 2. The designed 2-oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines privileged
scaffold based on JND3229.
water molecule. Herein, we would like to describe extensive
structure-activity relationships (SARs) of this 2-oxo-3,4-dihydro-
pyrimido[4,5-d]pyrimidine privileged scaffold based compounds
phenyl substituent exhibited an IC50 value of 14.4 nmol/L against
L858R/T790M/C797S
(
Fig. 2).
The synthetic routes of the designed2-oxo-3, 4-dihydropyrimido
4, 5-d]pyrimidinyl analogues were outlined in Scheme 1. Briefly,
EGFR
. Interestingly, introducing an N atom into this
phenyl group in 6o further reduced the compound’s activity (6p,
IC50 = 35.2 nmol/L). It was worthy to note that when the propionyl
moiety in JND3229 was replaced with a propyl group, the resulting
compound (6x) dramatically decreased the kinase inhibitory
[
reduction of 4-chloro-2-(methylthio)-pyrimidine-5-carboxylate
(
(
7) with diisobutyl aluminium hydride (DIBAL) gave (4-chloro-2-
methyl thio)pyrimidin-5-yl)methanol, which were subsequently
potency with an IC50 value of 139.7 nmol/L against
L858R/T790M/C797S
oxidized byMnO
2
toyieldaldehyde 9. Forcompounds6a-6uand6x,
EGFR
. The investigation suggested that a critical
9
then reacted with BOC-protected aliphatic amines, followed by
interaction mightexist betweenthe carbonylgroup andthe protein.
T790M/C797S
Borch reductive amination, cyclization and oxidation to yield key
pyrimidopyrimidinone 14. For compounds 6v and 6w, 9 went
through Grignard reaction to give alcohol 15, which followed by
chlorination, nucleophilic substitution, cyclization and oxidation to
yield key pyrimidopyrimidinone 20. The intermediates 14 and 20
were respectively subjected to nucleophilic, deprotection and
acylation reaction to give the title compounds 6a-6w.
The kinase inhibitory activities of the title compounds were
evaluated by the enzyme-linked-immunosorbent assay (ELISA)
assay (Table 1). Osimertinib, staurosporine and JND3229 were used
The X-ray structure analysis of EGFR
and JND3229
complex revealed that the methyl substituted phenylpiperazine
moiety of JND3229 was exposed to a solvent accessible surface
0
[16]. We then explored the preliminary SARs of 3 -methyl
0
substituent. When the 3 -methyl group in JND3229 was replaced
0
0
with 3 -CF
3
(6r) or changed to 2 -position (6u), the resulting
compounds exhibited comparable activities, while replacement of
0
0
3 -methyl group with the other substituents, e.g., 3 -F (6r), 3ꢀOCH
3
(6s), or removal of it (6t), yielded compounds with potency loss
ranging from 2- to 3-folds.
as positive controls to validate the screening conditions. As shown
3
At last, we investigated the potential steric impact of R position
L858R/T790M
in Table 1, osimertinib potently inhibits EGFR
, while is
by substitution with methyl (6v) and ethyl (6w). The compound 6v
exhibited the equal activity compared to the parental compound
JND3229, while the ethyl substituted compound 6w displayed
about 4-folds potency loss, suggesting that the bulky moieties
were not tolerated at this site.
L858R/T790M/C797S
less potent against EGFR
. However, staurosporine,
a broad spectrum of multi-kinase inhibitor, suppresses the
L858R/T790M
L858R/T790M/C797S
enzymatic activity of the EGFR
and EGFR
mutants with low nanomolar IC50 values.
We firstlyinvestigatedthe SARs on the linker L(Fig. 2)andtheresults
were summarized in Table 1. Change of trans-cyclohexanamine in
JND3229withacis-cyclopyrrilidinamine(6a)andlinearethylamine
In order to get insight into the binding mode of cys-cyclo-
T790M/C797S
hexanamine with EGFR
, a co-crystal structure of 6i
T790M/C797S
complexed with EGFR
was determined (Fig. 3A and
(
6b) led to 14-fold and complete potency loss against
Table S1 in Supporting information). It was indicated that 6i bound
Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
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X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
3
ꢁ
Scheme 1. General procedures for synthesis of compounds 6a-6 u, 6x, 6v and 6 w. Reagents and conditions: (a) DIBAL, THF, ꢀ78 C, 3–4 h, 88%; (b) MnO
2
, DCM, r.t., overnight,
ꢁ
ꢁ
ꢁ
8
6%; (c) CH
3
CN, r.t., overnight, 70%–82%. (d) i) 2-chloroaniline, AcOH, PhCH
3
, 110 C. ii) NaBH
4
, THF, 70 C, 50%–60%; (e) Triphosgene, DIPEA, DCM, 0 C, 70%–80%; (f) m-CPBA,
, 0 C, 2 h, 70%–80%. (i) 2-chloroaniline, Acetonitrile, r.t., overnight, 80%–85%; (j) 10, CH
overnight, 80%–90%; (k) 3-methyl-4-(4-methylpiperazin-1-yl)aniline, TFA, 2-butyl alcohol, 110 C, overnight, 40%–50%; (l) i) TFA, DCM, r.t.; ii) propionic acid, HATU, DIPEA,
ꢁ
ꢁ
DCM, r.t., 70%–80%; (g) R
3
MgCl, THF, -40 C, 80%–90%; (h) DIPEA, POCl
3
3
CN, r.t.,
ꢁ
ꢁ
DCM, overnight, 30%–50%. (n) i) TFA, DCM, r.t.; ii) iodine propane, EtOH, DCM, 80 C, 20 h.28%–35%.
to EGFR T790M/C797S with a reversible “U-shaped” configuration
with the “hinge” residue Met793. The 2-chlorophenyl group was
directed toward the hydrophobic back pocket composed by Lys745,
Glu762, Leu788, Met766 and Met790. Different conformations of
cyclohexylamine caused the different binding patterns to protein.
The carbonyl of amide in 6i formed a hydrogen bond with Ser720
mediated by a water molecule, while the NH of amide in JND3229
formed a hydrogen bond with Leu718 mediated by another water
molecule.
Further, the kinase inhibition of the most potent compounds 6i
and 6s (both showed sub-micromolar activities against BaF3-
L858R/T790M/C797S
19D/T790M/C797S
EGFR
and BaF3-EGFR
) was further
validated by investigating its suppressive functions on the
L858R/T790M/C797S
activation
BaF3 EGFR
of
EGFR
in
BaF3-EGFR
and
ꢀ
19D/T790M/C797S
mutants. As shown in Fig. 4, compounds
6i and 6s caused a dose-dependent suppression of the phosphor-
L858R/T790M/C797S
19D/T790M/C797S
ylation of EGFR
and EGFR
starting
from the concentration of 100 nmol/L.
The antiproliferative activities of compounds 6a-6x were also
In summary, an extensive SAR investigation was conducted
L858R/T790M
investigated against H1975 (EGFR
) and BaF3 cells stably
based on our recently disclosed 2-oxo-3,4-dihydropyrimido[4,5-d]
L858R/T790M/C797S
19D/T790M/C797S
C797S
transfected with EGFR
and EGFR
pyrimidine privileged scaffold-based EGFR
inhibitor JND3229.
mutants (Table 1). It was showed that 6a-6x exhibited moderate
antiproliferative activities against H1975 and BaF3 cells harboring
One of the most potent compound 6i potently suppressed
L858R/T790M/C797S
EGFR
kinase and inhibited the proliferation of
L858R/T790M/C797S
19D/T790M/C797S
L858R/T790M/C797S
19D/T790M/C797S
EGFR
values of 0.12–3.27
mol/L, respectively. Compound 6i displayed the most potent
and EGFR
m
mutants, with IC50
BaF3 cells harboring EGFR
and EGFR
mol/L, 0.29ꢀ>10
m
mol/L and 0.31ꢀ>10
mutants with IC50 values of 3.1 nmol/L, 290 nmol/L and 316 nmol/L,
m
respectively. Compound 6i dose-dependently induced suppression
L858R/T790M/C797S
L858R/T790M/C797S
antiproliferative activities against BaF3-EGFR
and
mol/L and
mol/L, respectively, which is corresponding to that of
biochemical kinase assay.
of
EGFR
the
phosphorylation
of
EGFR
and
19D/T790M/C797S
19D/T790M/C797S
BaF3-EGFR
.31
with IC50 values of 0.29
m
in BaF3 cells starting from the concentration
0
m
of 100 nmol/L. Compound 6i may serve as a new generation of
EGFR inhibitors for anticancer drug discovery.
Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
(

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X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
Table 1
In vitro EGFR inhibition and anti-proliferation data of compounds 6a-6x.
Cpds
L
R
1
2
R
2
R
3
Kinase IC50 (nmol/L)
Cellar IC50
TM1H1975
0.31 ꢂ 0.01
(m
mol/L)
BaF3-EGFRL858R/TM1
EGFRL858R/DM
EGFRL858R/TM1
BaF3-EGFR19D/TM2
JND3229
C
H
5
3'-CH
3
H
30.5 ꢂ 3.3
5.8 ꢂ 2.5
0.51 ꢂ 0.08
0.32 ꢂ 0.11
6
a
C
2
H
5
3'-CH
3
H
248.1 ꢂ 90.4
45.1 ꢂ 32.3
0.61 ꢂ 0.08
1.29 ꢂ 0.04
1.82 ꢂ 0.67
6
b
c
C
2
2
H
5
5
3'-CH
3'-CH
3
3
H
H
>1000
>1000
1.76 ꢂ 0.33
0.38 ꢂ 0.12
>10
>10
6
C
H
103.2 ꢂ 20.6
66.7 ꢂ 27.9
1.41 ꢂ 0.19
1.66 ꢂ 0.49
6
d
C
2
H
5
3'-CH
3
H
>1000
>1000
1.59 ꢂ 0.52
9.04 ꢂ 0.85
9.64 ꢂ 0.03
6
e
f
C
2
2
H
5
5
3'-CH
3'-CH
3
3
H
H
511.5 ꢂ 103.9
869.4 ꢂ 117.5
>1000
>1000
2.00 ꢂ 0.15
1.43 ꢂ 0.53
8.80 ꢂ 0.75
7.90 ꢂ 1.51
7.10 ꢂ 2.14
9.44 ꢂ 0.21
6
C
H
6g
6h
6i
C
2
C
2
C
2
H
5
H
5
H
5
3'-CH
3'-CH
3'-CH
3
3
3
H
H
H
711.0 ꢂ 153.7
232.6 ꢂ 5.7
5.1 ꢂ 3.0
>1000
1.89 ꢂ 0.59
3.27 ꢂ 0.94
0.12 ꢂ 0.09
9.45 ꢂ 0.31
4.35 ꢂ 1.89
596.0 ꢂ 344.5
3.1 ꢂ 0.9
>10
>10
0.29 ꢂ 0.17
0.31 ꢂ 0.12
6
j
CH
3
3'-CH
3
H
5.1 ꢂ 0.8
4.2 ꢂ 1.2
0.68 ꢂ 0.07
1.55 ꢂ 0.24
1.32 ꢂ 0.49
Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
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X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
5
Table 1 (Continued)
Cpds
L
R
1
R
2
R
3
Kinase IC50 (nmol/L)
Cellar IC50
(
mmol/L)
EGFRL858R/DM
EGFRL858R/TM1
BaF3-EGFRL858R/TM1
BaF3-EGFR19D/TM2
TM1H1975
6
k
3'-CH
3
H
19.1 ꢂ 2.0
6.7 ꢂ 2.0
0.51 ꢂ 0.09
1.09 ꢂ 0.47
1.36 ꢂ 0.53
1.75 ꢂ 0.08
6
l
3'-CH
3
H
27.6 ꢂ 7.0
7.9 ꢂ 1.6
0.65 ꢂ 0.04
2.11 ꢂ 0.14
6
m
n
3'-CH
3'-CH
3
H
H
38.0 ꢂ 9.8
15.8 ꢂ 2.9
43.1 ꢂ 9.3
0.84 ꢂ 0.15
1.11 ꢂ 0.13
1.43 ꢂ 0.42
0.99 ꢂ 0.01
2.04 ꢂ 0.28
6
3
65.8 ꢂ 12.0
1.03 ꢂ 0.004
6o
6p
6q
3'-CH
3'-CH
3'-F
3
H
H
H
17.4 ꢂ 3.3
29.2 ꢂ 4.2
19.9 ꢂ 5.4
14.4 ꢂ 2.5
35.2 ꢂ 5.8
11.0 ꢂ 1.9
1.55 ꢂ 0.31
2.38 ꢂ 0.78
0.31 ꢂ 0.13
1.75 ꢂ 0.24
2.37 ꢂ 0.36
0.66 ꢂ 0.06
1.92 ꢂ 0.14
2.74 ꢂ 0.34
1.28 ꢂ 0.50
3
C
2
H
5
6
r
s
C
2
H
5
3'-CF
3
H
H
4.0 ꢂ 2.5
8.8 ꢂ 6.0
1.80 ꢂ 0.25
0.38 ꢂ 0.16
1.20 ꢂ 0.48
0.47 ꢂ 0.15
0.89 ꢂ 0.49
0.52 ꢂ 0.18
6
C
2
H
5
3'-OCH
3
25.7 ꢂ 9.2
13.2 ꢂ 3.2
6
t
C
2
H
5
3'-H
H
H
24.4 ꢂ 12.0
5.8 ꢂ 2.2
18.3 ꢂ 3.3
9.1 ꢂ 2.6
0.55 ꢂ 0.17
1.76 ꢂ 0.30
0.58 ꢂ 0.28
0.58 ꢂ 0.28
1.13 ꢂ 0.44
1.13 ꢂ 0.84
6
u
C
2
H
5
2'-CH
3
Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
(

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X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
Table 1 (Continued)
Cpds
L
R
1
2
R
2
R
3
Kinase IC50 (nmol/L)
Cellar IC50
(m
mol/L)
BaF3-EGFRL858R/TM1
EGFRL858R/DM
EGFRL858R/TM1
BaF3-EGFR19D/TM2
TM1H1975
6
6
6
v
C
H
5
3'-CH
3
CH
3
39.8 ꢂ 19.0
6.2 ꢂ 1.7
1.48 ꢂ 0.55
2.46 ꢂ 0.61
2.29 ꢂ 0.36
1.49 ꢂ 0.65
7.20 ꢂ 4.46
w
x
C
2
H
5
3'-CH
3
C
2
H
5
23.3 ꢂ 4.2
22.8 ꢂ 2.4
139.7 ꢂ 35.1
3.15 ꢂ 0.91
2.24 ꢂ 0.20
9.67 ꢂ 0.11
2.47 ꢂ 0.64
196.9 ꢂ 71.9
AZD9291
Staurosporine
–
–
2.5 ꢂ 0.7
297.7 ꢂ 125.6
4.6 ꢂ 3.0
<0.016
0.03 ꢂ 0.01
3.39 ꢂ 0.42
0.01 ꢂ 0.01
9.27 ꢂ 0.37
0.05 ꢂ 0.01
26.7 ꢂ 4.7
Note: DM: L858R/T790M, TM1: L858R/T790M/C797S, TM2: 19D/T790M/C797S.
Fig. 3. (A) The X-ray crystal structure of 6i with EGFRT790M/C797S (PDB ID: 6JRX). (B) Overlay the 6i and JND3229 in EGFRT790M/C797S (PDB ID: 5ZTO). The EGFR kinase is shown in
green and blue stick and ribbon representation. 6i and JND3229 are shown in purple and yellow stick. Hydrogen bonds are indicated by yellow and black hatched lines to key
amino acids. Water is represented as red dots.
Fig. 4. Compounds 6i and 6 s potently suppressed the phosphorylation of EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S in BaF3 cells. Cells were treated with or without
compounds 6i and 6 s for 4 h at indicated concentration, respectively. Cells were then stimulated by 50 ng/ml EGF for 10 min and harvested for western blot analysis.
Declaration of competing interest
Acknowledgments
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
The authors appreciate the financial support from National
Natural Science Foundation of China (Nos. 81922062, 81874285
and 81673285), Guangdong International Science and Technology
Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
(

G Model
CCLET 5250 No. of Pages 7
X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
7
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Please cite this article in press as: X. Hu, et al., 2-Oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines as new reversible inhibitors of EGFR C797S
Cys797 to Ser797) mutant, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.09.044
(