Structure-based design of 5-Methylpyrimidopyridone derivatives as new wild-type sparing inhibitors of the epidermal growth factor receptor triple mutant (EGFRL858R/T790M/C797S)

Article abstract of DOI:10.1021/acs.jmedchem.9b00576

Tertiary EGFR^C797S mutation induced resistance against osimertinib (1) is an emerging "unmet clinical need" for non-small-cell lung cancer (NSCLC) patients. A series of 5-methylpyrimidopyridone derivatives were designed and synthesized as new selective EGFR^{L858R/T790M/C797S} inhibitors. A representative compound, 8r-B, exhibited an IC₅₀ of 27.5 nM against the EGFR^{L858R/T790M/C797S} mutant, while being a significantly less potent for EGFR^WT (IC₅₀ > 1.0 μM). Cocrystallographic structure determination and computational investigation were conducted to elucidate its target selectivity.

Full text of DOI:10.1021/acs.jmedchem.9b00576

Subscriber access provided by BUFFALO STATE
Brief Article
Structure-Based Design of 5-Methylpyrimidopyridone Derivatives
as New Wild-Type Sparing Inhibitors of Epidermal Growth
Factor Receptor Triple Mutant (EGFRL858R/T790M/C797S)
Jiayi shen, Tao Zhang, Shu-jie Zhu, Min Sun, Linjiang Tong, Mengzhen Lai, Rong
zhang, Wei Xu, Ruibo Wu, Jian Ding, Cai-Hong Yun, Hua Xie, Xiaoyun Lu, and Ke Ding
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00576 • Publication Date (Web): 12 Jul 2019
Downloaded from pubs.acs.org on July 12, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Structure-Based Design of 5-Methylpyrimidopyridone Derivatives as
New Wild-Type Sparing Inhibitors of the Epidermal Growth Factor
Receptor Triple Mutant (EGFRL858R/T790M/C797S
)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jiayi Shen, †, # Tao Zhang, ‡ , # Shu-jie Zhu,, # Min Sun,  Linjiang Tong, ‡ Mengzhen lai, ‡ Rong
Zhang,  Wei Xu, † Ruibo Wu,  Jian Ding, ‡ Cai-Hong Yun, *,  Hua Xie, *, ‡ Xiaoyun Lu*, † and Ke
Ding*, †
†
International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug
Discovery of Chinese Ministry of Education (MOE), Guangzhou City Key Laboratory of Precision Chemical Drug
Development, School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
‡
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
 Institute for Translational Medicine, College of Medicine, Qingdao University, Qingdao 266021, China
 Jiangsu Aosaikang Pharmacceutical Co. Ltd., #699 Kejian Road, Jiangsu Science park, Nanjing 211112, China
 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
 Department of Biophysics and Peking University Institute of Systems Biomedicine, Peking University Health
Science Center, Beijing 100191, China
KEYWORDS: Epidermal Growth Factor Receptor (EGFR), C797S mutation, resistance, Non-small cell lung cancer
(NSCLC)
ABSTRACT: Tertiary EGFRC797S mutation induced resistance against osimertinib (1) is an emerging “unmet clinical need”
for non-small cell lung cancer (NSCLC) patients. A series of 5-methylpyrimidopyridone derivatives were designed and
synthesized as new selective EGFRL858R/T790M/C797S inhibitors. A representative compound, 8r-B, exhibited an IC50 value of
27.5 nM against the EGFRL858R/T790M/C797S mutant, while being a significantly less potent for EGFRWT (IC50 > 1.0 µM). Co-
crystallographic structure determination and computational investigation were conducted to elucidate its target
selectivity.
INTRODUCTION
residence time and restore inhibitory activity against the
10, 11, 12
EGFRT790M mutants.
However, a tertiary Cys797 to
The epidermal growth factor receptor (EGFR) has
been well validated as a therapeutic target for anti-cancer
Ser797 (C797S) point mutation becomes
a leading
1
mechanism of clinically acquired resistance in ∼40% of
drug 1 treated NSCLC patients, by disturbing the
corresponding covalent bond formation.13 Accordingly,
development of selective EGFRC797S inhibitors is
considered as a new potential strategy to overcome
acquired resistance against the 3rd-generation EGFR
inhibitors. 14, 15, 16
drug discovery. Three generations of EGFR inhibitors,
2
3
e.g., reversible gefitinib
and erlotinib,
irreversible
afatinib 4 and dacomitinib,
and the wild-type sparing
5, 6
osimertinib (1, AZD9291), 7 have been approved by the US
FDA and have achieved significant clinical benefit for
NSCLC patients with EGFR activating mutations (i.e.,
L858R and delE746-A750) and/or secondary threonine790
to methionine790 (T790M) mutation, respectively. In
particular, drug 1 has become the first globally accessible
3rd-generation EGFR inhibitor, having been recently
approved as a first-line treatment for metastatic NSCLC
Recently, an allosteric inhibitor EAI045 (2a, Figure 1)
was discovered that selectively exhibits low nM IC50
potency against the EGFRC797S mutant. However,
combination with an EGFR antibody, e.g. cetuximab, was
required for the compound to demonstrate in vivo
therapeutic efficacy, because of asymmetric dimerization
8,
9
patients,
representing one of the most advanced
developments in NSCLC therapy.
17
of the EGFR receptor. Brigatinib (3), and compounds 4
Mode of Action (MOA) investigation revealed that
the acrylamide moiety in drug 1 could undergo a Michael
addition reaction with the side chain of the Cys797 residue
under physiological conditions to increase the target
and 5 were also reported to display strong ATP-
competitive inhibition against EGFRC797S. Similar to the
allosteric inhibitor 2a, drug 3 also required combination
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 8
with cetuximab to display in vivo efficacy in xenograft
18
mouse models with EGFRC797S mutations. Compound 5
1
2
3
4
represents one of the most selective ATP-competitive
inhibitors of EGFRC797S reported, however no in vitro or in
19, 20
vivo efficacy data has been disclosed to date.
Most
5
6
7
8
recently, JBJ-04-125-02 (2b) was disclosed as a new
allosteric inhibitor of the EGFRL858R/T790M/C797S mutant with
promising in vitro and in vivo efficacy. However, the
molecule is inactive against the clinically important
EGFRDel-19 variants (e.g., EGFRdelE746-A750
,
EGFRdelE746-
9
A750/T790M
and EGFRdelE746-A750/T790M/C797S 21. We have also
)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
identified a pyrimidopyrimidinone derivative, JND3229
(6), as a new reversible EGFRC797S mutant inhibitor with in
vitro and in vivo single-agent efficacy, but its relatively
low EGFRL858R/T790M/C797S target specificity over the wild-
type kinase may raise some concern about potential EGFR
mechanism-related toxicity. 22
Figure
2.
Structure-based
design
of
new
EGFRL858R/T790M/C797S inhibitors. A) Chemical structure of 6
and its complex with EGFRT790M/C797S (PDB: 5ZTO); B)
Chemical structure of 7 and its complex with EGFRT790M
(PDB: 5GMP); C) Superimposition of 7 with inhibitor 6 in
EGFRT790M/C797S (PDB: 5GMP); D) Designed new
EGFRL858R/T790M/C797S inhibitors.
In this paper, we describe the design and synthesis of
a series of 5-methylpyrimidopyridone derivatives as new
wild-type sparing EGFRL858R/T790M/C797S inhibitors. X-ray co-
crystallographic
structure
determination
and
computational investigation were employed to rationalize
the target selectivity of the molecules.
CHEMISTRY
N
S
R
O
The designed molecules 8a-8r were readily
synthesized by using a protocol outlined in Scheme 1.
Briefly,
dichloropyrimidine 9 was reacted with protected aliphatic
amines 10 to obtain 11. Compounds 11 were condensed
with trans-but-2-enoic acid under Heck coupling
HN
N
N
N
N
MeO
N
H
O
O
HO
N
H
commercially
available
5-bromo-2,
4-
F
N
2a
EAI045( ): R=H
N
NH
N
2b
JBJ-04-125-02 ( ): R=
1
osimertinib ( , AZD9291)
F
N
24
O
H
N
conditions
in
the
presence
of
N
N
N
bis(benzonitrile)palladium(II) dichloride, before being
treated with acetic anhydride to produce intermediates 12.
Selective bromination of intermediates 12 with liquid
bromine in acetic acid gave 13. Compounds 13 were
coupled with 3-methyl-4-(4-methylpiperazin-1-yl)aniline
to produce intermediates 14. Intermediates 14 underwent
Suzuki coupling with a range of phenylboronic acids to
give 15 which were deprotected under acidic conditions to
yield the substituted amines 16. With 16 in hand,
designed molecules 8a, 8c-8r were easily prepared by a
standard acylation reaction. Alternatively, synthetic
procedures for 8b and 8l are provided in the Supporting
Information.
HN
N
Cl
N
N
H
N
NH
P
N
OH
N
CN
H
N
N
HN
N
O
HO
O
O
3
)
Brigatinib (
5
4
Figure 1. Chemical structures of osimertinib and the
reported EGFRC797S inhibitors.
MOLECULAR DESIGN
Structural feature analysis of the 6-EGFRT790M/C797S
complex (PDB: 5ZTO) revealed that the compound bound
to the ATP-binding pocket with a similar “U-shaped”
configuration to that of our previously reported
22,
EGFRT790M selective inhibitor 7 (PDB: 5GMP, Figure 2).
23
Scheme 1. Synthesis of compounds 8a, 8c-8r.
Previous studies also demonstrated that the 5-methyl
substituent of compound 7 was directed towards the
Br
Br
Br
NH2
R1
N
N
N
N
“gatekeeper” residue Met790, contributing greatly to
d
+
c
a
b
Cl
N
NH
R1
Cl
N
N
O
Cl
N
Cl
Cl
N
N
O
Boc
23
R1
EGFRT790M selectivity over the wild-type protein.
A
Boc
R1
Boc
Boc
12
9
11
13
10
preliminary superimposition further suggested that the 5-
R2
R2
R2
Br
O
methyl pyrido[2, 3-d]pyrimidin-7-one scaffold of the
N
N
N
N
22, 23
HN
N
N
O
O
HN
N
N
O
g
HN
N
N
HN
N
N
O
H
e
f
irreversible inhibitor
7
overlapped with the
R1
R1
R1
R1
Boc
NH
Boc
pyrimido[4, 5-d]pyrimidine-7-one core of
6 in the
EGFRT790M/C797S complex. Based on these observations, a
N
N
N
N
N
N
N
N
8
15
16
14
series of 5-methylpyrimidopyridone derivatives (8) were
designed
and
synthesized
as
new
potential
Reagents and conditions: a) K2CO3, acetonitrile (AN), 0
°C to r.t., 80-95%; b) (i) (E)-but-2-enoic acid,
bis(benzonitrile) palladium(II) dichloride, DIPEA, tri(o-
tolyl)phosphine, THF, 80 °C, argon, 16 hrs, (ii) acetic
anhydride, 90 °C, 24 hrs, 40-50%; c) Br2, AcONa, AcOH,
EGFRL858R/T790M/C797S inhibitors, with the aim of improving
the target selectivity of this class of derivatives over wild-
type EGFR (Figure 2).
ACS Paragon Plus Environment

Page 3 of 8
Journal of Medicinal Chemistry
50 °C, 30 hrs, 60-75%; d) 3-methyl-4-(4-methylpiperazin-
against EGFRWT and EGFRL858R/T790M/C797S, respectively,
which was 27-90 fold less potent than the original lead
1
2
3
4
5
6
7
8
1-yl) aniline, TFA, 2-butanol, 110 °C, 18 hrs, 50-65%; e)
[(C6H5)3P]2PdCl2, Na2CO3, substituted phenylboronic acid,
1, 4-dioxane, H2O, 110 °C, 45-70%; f) TFA, DCM, r.t., 85-
90%; g) propionyl chloride, Et3N, DCM, 0 °C to r.t., 80-
90%.
molecule
6
(Table 1). Preliminary computational
investigation suggested that the 5-methyl group failed to
extend to the gatekeeper Met790 residue and form a
favorable hydrophobic interaction, but caused a steric
collision with this residue. Removal of the 5-methyl group
(8b) indeed improved the inhibitory potency against
EGFRL858R/T790M/C797S by a factor of 13.6-fold. However,
eliminating the methyl group resulted in a greater
RESULTS AND DISCUSSION
Compound 8a, in which the original pyrimido[4, 5-
d]pyrimidine-7-one core of 6 was simply replaced by a 5-
methyl pyrido[2, 3-d]pyrimidin-7-one moiety, was first
designed and synthesized. Its kinase inhibition activities
against EGFRWT and EGFRL858R/T790M/C797S were determined
by utilizing an enzyme-linked immunosorbent assay
9
potency improvement against EGFRWT
,
becoming
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
detrimental to the target selectivity of the molecule. The
resulting compound 8b exhibited IC50 values of 3.8 and
21.
38.1 nM against EGFRWT and EGFRL858R/T790M/C797S
,
(ELISA). Four previously reported EGFRC797S inhibitors,
respectively. These results implied that introduction of a
5-methyl group could be beneficial for the design of wild-
type sparing EGFRL858R/T790M/C797S inhibitors, although it
might have a negative impact on the EGFRC797S inhibitory
potency.
i.e. 2, 3, 5 and 6, were included as the reference
compounds. All of the positive control compounds
exhibited comparable IC50 values to the reported data. 15-19
Disappointingly, compound 8a displayed obviously
decreased potency with IC50 values of 180.9 and 519.9 nM
Table 1. In Vitro Kinase Inhibition of 8a−8r a
3'
4'
5'
R3
2'
N
1'
R2
6'
HN
N
N
O
O
R1
R1=
N



NH
NH
N
N
N
N
N
V
IV
VI
Kinase Inhibition (IC50, nM)
Kinase Inhibition (IC50, nM)
Cpds
8a
R1
R2
R3
Cpds
8l-A
R1
R2
R3
EGFRWT
EGFRTM
EGFRWT
>1000
EGFRTM
2’or 6’-Cl Me 180.9±80.8
(mixture )
519.9±83.4
2’-Br
Me
498.1±126.0
Ⅰ
Ⅴ
8b
8c
2’or 6’-Cl
H
3.8±2.1
38.1±10.1
35.1±6.8
8l-B
6’-Br
Me
Me
>1000
>1000
56.1±9.5
Ⅰ
Ⅱ
Ⅴ
Ⅴ
2’or 6’-Cl Me 176.6±80.6
(mixture )
8m-A
2’-Me
587.6±111.1
8c-A
8c-B
2’-Cl
6’-Cl
Me
>1000
518.8±78.1
27.7±10.3
8m-B
8n
6’-Me
Me
>1000
>1000
227.9±9.5
>1000
Ⅱ
Ⅱ
Ⅴ
Ⅴ
Me 168.8±63.4
2’or 6’-OMe Me
(mixture )
8d
2’or 6’-Cl Me 280.5±39.5 866.9±146.4
(mixture )
8o
2’or 6’-OH
(mixture )
2’-Cl, 5’-F
Me
>1000
>1000
Ⅲ
Ⅴ
8e-A
8e-B
8f-A
2’-Cl
6’-Cl
2’-Cl
Me
Me
Me
>1000
>1000
>1000
>1000
>1000
>1000
8p-A
8p-B
8q-A
Me
Me
Me
>1000
>1000
>1000
207.0±135.0
37.1±1.2
Ⅳ
Ⅳ
Ⅴ
Ⅴ
Ⅴ
Ⅴ
3’-F, 6’-Cl
2’-Cl,4’-F
224.1±6.7
8f-B
8g-A
8g-B
8h
6’-Cl
2’-Cl
6’-Cl
3’-Cl
4’-Cl
H
Me
Me
Me
Me
Me
Me
Me
>1000
>1000
>1000
>1000
>1000
>1000
>1000
65.1±22.7
>1000
8q-B
8r-A
8r-B
2
4’-F, 6’-Cl
Me
Me
Me
-
>1000
>1000
88.6±13.3
>1000
Ⅴ
Ⅵ
Ⅵ
Ⅴ
Ⅴ
Ⅴ
Ⅴ
Ⅴ
Ⅴ
Ⅴ
-
2’-Cl, 3’-F
81.7±23.3
>1000
5’-F, 6’-Cl
>1000
27.5±11.6
73.1±12.9
3.0±1.4
-
-
-
>1000
8i
>1000
3
-
-
76.6±20.2
>1000
8j
>1000
5
-
-
38.7±16.6
5.8±2.5
8k
2’or 6’-F
>1000
6
6.8±3.8
a EGFRL858R/T790M/C797S (EGFRTM) and EGFRWT kinase inhibition was were tested by ELISA assay. The data are mean values from at least three
independent experiments.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Our next investigation mainly focused on
Page 4 of 8
In order to validate the potential contribution of the 2’-
chloro group of 8f-B, compounds 8h and 8i, in which the
chloro-substituent was moved to the 3’-position or 4’-
position, respectively, were design and synthesized. Both
8h and 8i were totally inactive with respect to inhibition
1
2
3
4
5
6
7
8
identification of an optimal R1-linker group, which could
potentially facilitate the 5-methyl substituent to adopt a
suitable orientation avoiding steric collision with Met790
.
The propionamide was kept unchanged because the 6-
EGFRT790M/C797S complex structure (PDB: 5ZTO) suggested
that this moiety was solvent-surface accessible and might
not make much contribution to interaction with the
protein. Interestingly, a replacement of the trans-
of EGFRL858R/T790M/C797S and EGFRWT
, supporting our
hypothesis that a 2’-substituent is optimal for inhibition
of the EGFR kinase. Not surprisingly, removal of the
critical 2’-chloro group also caused a total abolishment of
inhibitory activity against EGFRWT and the C797S mutant
(8j). Further investigation revealed that this position is
highly sensitive to the size and physicochemical
properties of the substituted group. When the 2’-chloro
substituent was replaced with 2’-fluoro (8k), 2’-methoxyl
(8n), or 2’-hydroxyl (8o) groups, all of the resulting
molecules were found to be completely inactive against
both EGFRL858R/T790M/C797S and EGFRWT. The 2’-methyl
analogs (i.e., 8m-A and 8m-B) were also notably less
active than 8f-B. Not surprisingly, the 2’-bromo derivative
(8l-B) exhibited similar EGFRL858R/T790M/C797S inhibitory
potency to that of 8f-B, with an IC50 value of 56.1 nM.
Interestingly, introduction of a second fluoro-substituent
at each available vacant position of the pendent
chlorophenyl ring had varying influence on the biological
activity of the molecules. For instance, the 4’-F, 6’-Cl
disubstituted analogue (8q-B) exhibited almost identical
potency and selectivity to that of 8f-B, but the 3’-F, 6’-Cl
(8q-B) and 5’-F, 6’-Cl (8r-B) were 1.8~2.4 fold more potent
than the lead molecule. Compounds 8q-B and 8r-B
exhibited IC50 values of 37.1 and 27.5 nM, respectively
against the EGFRL858R/T790M/C797S mutant. It was also
noteworthy that both compound 8r-B and 8q-B were
equally potent and selective to the previously reported
EGFRC797S inhibitor 5 in a parallel comparison. Different to
the allosteric inhibitor 2b, 8r-B exhibited similar
inhibition against the EGFRdel746-750/T790M/C797S mutant with
an IC50 value of 49.9 nM.
cyclohexanediamine linker in 8a with
a
cis-
9
cyclohexanediamine moiety (8c) resulted in a significant
improvement in EGFRL858R/T790M/C797S inhibitory potency.
The resulting compound 8c exhibited an IC50 value of 35.1
nM against EGFRL858R/T790M/C797S, which is 14.8-fold more
potent than the parental compound 8a. However, the
modification barely affected the inhibitory activity against
EGFRWT. The steric hindrance between the 5-methyl and
7-carbonyl moieties of the scaffold and the 2’-Cl group of
the pendent phenyl ring of 8c results in hindered rotation
of the corresponding C-C bond, inducing an axial chirality
in the molecule. Two atropisomers present in the mixture
8c (i.e. 8c-A and 8c-B) were separated by chiral High
Performance Liquid Chromatography (HPLC). The
absolute stereochemistry of both compounds was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
determined
by
using
small
molecule
X-ray
crystallographic analysis (Figure 3). Further biological
evaluation revealed that the compounds exhibited IC50
values of 518.8 and 27.7 nM against EGFRL858R/T790M/C797S
,
respectively. Thus, 8c-B represents the preferred
configuration to bind with the triple mutant.
Several other aliphatic heterocyclic linkers were also
explored (Table 1). Although introduction of 4-piperidine
(8d) or (S)-3-piperidine (8e-A & 8e-B) linkers caused total
abolishment of the EGFRL858R/T790M/C797S inhibitory potency,
the (R)-3-piperidine substituted molecule 8f-B and the (R)-
3-pyrrolidine derivative 8g-B exhibited IC50 values of 65.1
and 81.7 nM, respectively, against the EGFRL858R/T790M/C797S
mutant. Notably, compounds 8f-B and 8g-B were almost
equally potent to 8b and 8c, but their inhibition of the
wild-type kinase was abolished. Similar to the above
observation, the alternate stereo-enantiomers 8f-A and
8g-A were totally inactive. Consequently, compound 8f-B
was selected as a new starting point for further structural
optimization because of its relatively potent
EGFRL858R/T790M/C797S inhibition and promising target
selectivity.
X-ray co-crystallographic structures of 8r-B with
EGFRT790M/C797S and EGFRWT were further determined to
elucidate detailed interactions and the structural basis of
the observed target selectivity (Figure 3). These confirmed
that compound 8r-B bound at the ATP binding sites of
EGFRT790M/C797S and EGFRWT with similar “U-shaped”
configurations. The 5-methylpyrimido-pyrimidinone
scaffold formed the classical hydrogen bond donor-
acceptor interactions with the “hinge” residue Met793
while the 6-(2’-chloro-3’-fluoro)phenyl group occupies a
hydrophobic pocket bounded by Lys745, Glu762, Leu788
,
,
Met766 and Met790. Both the 4-methylpiperazinylphenyl
group and the propionyl piperidine were directly
accessible to the solvent surface, which would provide
feasible positions for future optimization of the
physicochemical properties of the compounds. Notably,
the 5-methyl group approaches Met790 within a distance of
3.5 Å in EGFRT790M/C797S to form a favorable hydrophobic
interaction. Alternately, its lipophilic character appears to
be incompatible with the hydrophilic Thr790 in EGFRWT. In
addition, the lipophobic property of Thr790 could also
weaken the hydrophobic interactions of the 6-(2’-chloro-
Figure 3. X-ray structures of compound 8c-A and 8c-B.
Our previous structural investigation implied that a
2’-chloro group in 6 might be critical to force the pendent
phenyl moiety to adopt an appropriate orientation for it
to be accommodated in a hydrophobic pocket bounded
by Lys745, Glu762, Leu788, Met766 and Met790 in EGFRC797S
.
22
ACS Paragon Plus Environment

Page 5 of 8
Journal of Medicinal Chemistry
3’-fluro)phenyl group with wild-type EGFR. This
structural information could provide rational
explanation for the inhibitor’s selectivity between
EGFRT790M/C797S and EGFRWT
obvious (Supporting Information). Investigation also
1
2
3
4
a
revealed that the compound exhibited moderate aqueous
solubility and cellular permeability (Supporting
Information).
.
5
6
CONCLUSION
In summary, a series of 5-methylpyrimidopyridone
derivatives were designed and synthesized as novel
selective wild-type sparing EGFRL858R/T790M/C797S inhibitors.
One of the representative compounds, 8r-B exhibited an
IC50 value of 27.5 nM against the EGFRL858R/T790M/C797S triple
mutant, while being almost totally inactive against the
EGFRWT kinase, identifying 8r-B as one of the most
selective 4th generation EGFR inhbitors reported to date.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. X-ray structures of co-crystals of 8r-B-
EGFRT790M/C797S and 8r-B-EGFRWT complexes. (A) A 2.943
Å
Co-crystallographic
structure
determination
and
computational investigation further validated that target
selectivity of the inhibitors mainly relied on the
hydrophobic nature of the Met790 residue in the
T790M/C797S mutant protein. Although the relatively
low kinome-selectivity and metabolic liability (Supporting
Information) of 8r-B may limit its future development,
our study provides a useful lead compound and some
fundmental structural information for the rational
development of new selective EGFRC797S inhibitor drugs.
Further pharmacokinetics-oriented optimization of 8r-B
is on-going and the results will be disclosed in due course.
crystallographic structure of 8r-B-EGFRT790M/C797S
complex (PDB ID: 6JRJ). (B) A 2.796 Å crystallographic
structure of 8r-B-EGFRTWT complex (PDB ID: 6JRK).
The binding free energy of compound 8r-B in
EGFRWT and the EGFRT790M/C797S mutant were estimated to
be -47.8±2.7 kcal/mol and -57.5±4.0 kcal/mol respectively,
by utilizing classical MD simulations and MM-GBSA
calculations (Supporting Information). These results
suggest that binding of compound 8r-B to EGFRT790M/C797S
is thermodynamically more favorable than binding to
EGFRWT, which is consistent with the experimental results.
Moreover, contribution of the stabilization energy of each
residue to the overall binding affinity of 8r-B was also
evaluated and the difference between the EGFRWT and
EGFRT790M/C797S mutant were calculated using the equation
ΔΔG = ΔGWT-ΔGTMCS. The top ten significant residues
to enhance the binding affinity are summarized in Figure
EXPERIMENTAL SECTION
General Chemistry. Reagents and solvents were
obtained from commercial suppliers and used without
further purification. Flash chromatography was
performed using silica gel (200−300 mesh). All reactions
were monitored by TLC, using silica gel plates with
1
fluorescence F254 and UV light visualization. H and 13C
5. The binding energy difference (ΔΔG =ΔGWT
-
NMR spectra were recorded on a Bruker AV-400
spectrometer at 400 MHz and Bruker AV-500
spectrometer at 125 MHz, respectively. Coupling
constants (J) are expressed in hertz (Hz). Chemical shifts
(δ) of NMR are reported in parts per million (ppm) units
relative to internal control (TMS). The first-order peak
patterns are indicated as s (singlet), d (doublet), t (triplet),
q (quadruplet). Complex non-first order signals are
indicated as m (multiplet). The low- or high- resolution
ESI-MS results were recorded on an Agilent 1200 HPLC-
MSD mass spectrometer or Applied Biosystems Q-STAR
Elite ESILC−MS/MS mass spectrometer, respectively. The
purity of compounds was determined by reverse-phase
high-performance liquid chromatography (HPLC)
analysis confirming to be >95%. Analytical HPLC analyses
were conducted using an Agilent 1260 system (G1310B Iso
pump and G1365D MWD VL detector) with an YMC-
Triart C18 reversed-phase column (250 mm × 4.6 mm, 5
μm) at 254 nm. Elution was MeOH in water, and flow rate
was 1.0 mL/min. Preparative HPLC (PHPLC) purifications
were performed using Agilent 218 solvent delivery module
and an Agilent 325 dual wavelength UV−vis detector with
an YMC-Triart C18 reversed-phase column (250 mm × 20
mm, 5 μm) at 254 nm. The LC column was maintained at
room temperature.
ΔGT790M/C797S) of compounds 3 and 6 were also calculated
to be -1.5 kcal/mol and 2.2 kcal/mol, respectively, which
might give a reasonable explanation for their relatively
low triple mutant selectivity (Supporting Information).
Figure 5 (A) The hydrophobic amino acid residues
are marked in dark green, while the polar amino acid
residues are marked in red. (B) The top ten hot-spot
residues predicited computationally.
Further biological investigation validated that
compound 8r-B potently supressed the activation of
EGFR signaling in BaF3 cells stably transfected with
EGFRL858R/T790M/C797S (Supporting Information) and
inhibited proliferation of this transfected cell line model
with an IC50 value of 0.662 (±0.045) M. However, its
effect on wild-type EGFR signaling was significantly less
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 8
N-((1R,4R)-4-(6-(2-chlorophenyl)-5-methyl-2-((3-
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-7-
oxopyrido[2,3-d]pyrimidin-8(7H)-yl)cyclohexyl)
propionamide (8a). Propionyl chloride (25 L ， 0.28
mmol) was added dropwise to a mixture of 16a which was
The authors appreciate the financial support from National
Natural Science Foundation of China (81820108029, 21572230,
81673285, 81425021, 21702075), Guangdong Province
(2015A030312014,
2015A030306042,
2016A050502041,
readily prepared by
a protocol described in the
2018A050506043, and 2017A030310253), Guangzhou city
(201805010007) and Jinan University. We also thank Profs.
Jeff Smaill and Adam Patterson from the University of
Auckland, New Zealand, for proofreading our manuscript.
Supporting Information (150 mg ， 0.26 mmol) and Et3N
（0.11 mL，0.78 mmol）in dry DCM (10 mL) at 0 oC. The
reaction mixture was stirred at 0 oC for 30 min and
concentrated under reduced pressure. The resulting crude
product was purified by silica gel chromatography to
9
ABBREVIATIONS USED
EGFR, epidermal growth factor receptor; NSCLC, non-small
cell lung cancer; C797S, Cys797 to Ser797; MOA, mode of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
afford 8a as a yellow solid (135 mg，92%). H NMR (400
MHz, DMSO-d6) δ 9.87 (s, 1H), 8.89 (s, 1H), 7.60 (d, J =
2.3 Hz, 1H), 7.57 – 7.53 (m, 1H), 7.49 (dd, J = 8.6, 2.4 Hz,
1H), 7.41 (dd, J = 4.6, 2.3 Hz, 2H), 7.31 – 7.24 (m, 1H), 7.02
(d, J = 8.7 Hz, 1H), 5.42 (s, 1H), 3.86 (s, 1H), 2.81 (s, 5H),
2.47 (s, 4H), 2.28 (s, 3H), 2.23 (s, 3H), 2.15 (d, J = 7.4 Hz,
1H), 2.12 (s, 3H), 1.91 (d, J = 11.1 Hz, 2H), 1.55 (d, J = 9.1 Hz,
2H), 1.42 (s, 2H), 1.24 (d, J = 11.1 Hz, 3H), 1.01 – 0.92 (t, J =
action;
WT,
wild-type;
ELISA,
enzyme
linked
immunosorbent assay; AN, acetonitrile; DIPEA, N, N-
diisopropylethylamine; TFA, trifluoroacetic acid; DCM,
dichloromethane; THF, tetrahydrofuran; HPLC, High
Performance Liquid Chromatography.
REFERENCES
13
7.5 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 172.5, 162.8
(1) Mok, T. S.; Wu, L. Y.; Thongprasert, S.; Yang, C. H.; Chu,
D. T.; Saijo, N.; Sunpaweravong, P.; Han, H.; Margono, B.;
Ichinose, Y.; Nishiwaki, Y.; Ohe, Y.; Yang, J. J.;
Chewaskulyong, B.; Jiang, H.; Duffield, E. L.; Watkins, C. L.;
Armour, A. A.; Fukuoka, M. Gefitinib or carboplatin-
paclitaxel in pulmonary adenocarcinoma. New. Engl. J. Med.
2009, 361, 947 – 957.
(2C), 161.9, 159.2, 157.8, 155.2, 135.5, 134.0 (2C), 132.7, 132.5
(2C), 129.9, 129.5 (2C), 127.5, 119.4, 119.1, 106.7, 106.6, 55.7
(2C), 51.8, 46.2, 36.3, 32.7, 31.2, 29.5, 29.4, 29.0, 28.5, 27.1,
18.4, 15.1, 10.5. HRMS (ESI) for C35H42ClN7O2 [M+H]+, calcd:
628.3089; found, 628.3218. HPLC analysis: MeOH-H2O (85:
15), 11.86 min, 97.3% purity.
The other designed compounds were synthesized by
following a similar procedure (Supporting Information).
(2) Paez, J. G.; Janne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.;
Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T.
J.; Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.; Sellers, W. R.;
Johnson, B. E.; Meyerson, M. EGFR mutations in lung cancer:
correlation with clinical response to gefitinib therapy.
Science. 2004, 304, 1497−1500.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures, in vitro ELISA assay, cell proliferation
inhibition assay, western blot analysis, experimental data for
the X-ray structures of 8c-A and 8c-B, computational study,
solubility assay, metabolic stability, Caco-2 permeability
(3) Dowell, J.; Minna, J. D.; Kirkpatrick, P. Erlotinib
hydrochloride. Nat. Rev. Drug Discovery. 2005, 4, 13−14.
(4) Dungo, R. T.; Keating, G. M. Afatinib: first global
approval. Drugs. 2013, 73, 1503−1515.
1
assay, the kinase selectivity profiling study of 8r-B, H NMR,
13C NMR spectra of compounds 8a-8r (PDF), HPLC purity
analysis of 8a-8r (PDF) and Molecular Formula Strings (CSV).
The material is available free of charge via the Internet at
http://pubs.acs.org.
(5) Wu, Y. L.; Cheng, Y.; Zhou, X.; Lee, K. H.; Nakagawa, K.;
Niho. S.; Tsuji, F.; Linke, R.; Rosell, R.; Corral, J.; Migliorino,
M. R.; Pluzanski, A.; Sbar, E. I.; Wang, T.; White, J. L.;
Nadanaciva, S.; Sandin, R.; Mok, T. S. Dacomitinib versus
gefitinib as first-line treatment for patients with EGFR-
mutation-positive non-small-cell lung cancer. Lancet Oncol.
2017, 18, 1454-1466.
Atomic coordinates and experimental data for the co-crystal
structures of 8r-B with EGFRT790M/C797S and EGFRWT will be
released upon article publication.
(6) Smaill, J. B.; Gonzales, A. J.; Spicer, J. A.; Lee, H.; Reed, J.
E.; Sexton, K.; Althaus, I. W.; Zhu, T.; Black, S. L.; Blaser, A.;
Denny, W. A.; Ellis, P. A.; Fakhoury. S.; Harvey, P. J.; Hook,
K.; McCarthy, F. O.; Palmer, B. D.; Rivault, F.; Schlosser, K.;
Ellis, T.; Thompson, A. M.; Trachet, E.; Winters, R. T.; Tecle,
H.; Bridges, A. Tyrosine kinase inhibitors. 20. Optimization
of substituted quinazoline and pyrido3,4-dpyrimidine
derivatives as orally active, irreversible inhibitors of the
epidermal growth factor receptor family. J. Med. Chem. 2016,
59, 8103-8124.
AUTHOR INFORMATION
Corresponding Authors
*For H.X.: E-mail, hxie@simm.ac.cn
*For C.Y.: E-mail, yunch@bjmu.edu.cn
*For X.L.: E-mail, luxy2016@jnu.edu.cn
*For K.D.: E-mail, dingke@jnu.edu.cn, +86 20 85223764
Author Contributions
(7) Kim, E. S. Olmutinib: first global approval. Drugs. 2016,
76, 1153−1157.
# contributed equally to this work.
Notes
(8) Soria, J. C.; Ohe, Y.; Vansteenkiste, J.; Reungwetwattana,
T.; Chewaskulyong, B.; Lee, K. H.; Dechaphunkul, A.;
Imamura, F.; Nogami, N.; Kurata, T.; Okamoto, I.; Zhou, C.;
The authors declare no competing financial interest.
ACS Paragon Plus Environment

Page 7 of 8
Journal of Medicinal Chemistry
Cho, B. C.; Cheng, Y.; Cho, E. K.; Voon, P. J.; Planchard, D.;
(16) Ayeni, D.; Politi, K.; Goldberg, S. D. Emerging agents and
new mutations in EGFR-mutant lung cancer. Clin. Cancer Res.
2015, 21, 3818-3820.
(17) Jia, Y.; Yun, C. H.; Park, E.; Ercan, D.; Manuia, M.; Juarez,
J.; Xu, C.; Rhee, K.; Chen, T.; Zhang, H.; Palakurthi, S.; Jang,
J.; Lelais, G.; DiDonato, M.; Bursulaya, B.; Michellys, P. Y.;
Epple, R.; Marsilje, T. H.; McNeill, M.; Lu, W.; Harris, J.;
Bender, S.; Wong, K. K.; Janne, P. A.; Eck, M. J. Overcoming
EGFR(T790M) and EGFR(C797S) resistance with mutant-
selective allosteric inhibitors. Nature. 2016, 534, 129−132.
1
2
3
4
5
6
7
8
Su, W. C.; Gray, J. E.; Lee, S. M.; Hodge, R.; Marotti, M.;
Rukazenkov, Y.; Ramalingam, S. S. Osimetinib in untreated
EGFR-mutated advanced non-small-cell lung cancer. N. Engl.
J. Med. 2018, 378, 113−125.
(9) FDA Center for Drug Evaluation and Research.
www.accessdata.fda.gov/drugsatfda_docs/label/2018/208065s
008lbl.pdf (accessed April 19, 2018).
(10) Piotrowska, Z.; Niederst, M. J.; Karlovich, C. A.; Wakelee,
H. A.; Neal, J. W.; Mino-Kenudson, M.; Fulton, L.; Hata, A.
N.; Lockerman, E. L.; Kalsy, A.; Digumarthy, S.; Muzikansky,
A.; Raponi, M.; Garcia, A. R.; Mulvey, H. E.; Parks, M. K.;
DiCecca, R. H.; Dias-Santagata, D.; Iafrate, A. J.; Shaw, A. T.;
Allen, A. R.; Engelman, J. A.; Sequist, L. V. Heterogeneity
underlies the emergence of EGFRT790 wild-type clones
following treatment of T790M-positive cancers with a third-
generation EGFR inhibitor. Cancer Discov. 2015, 5, 713−722.
9
(18) Uchibori, K.; Inase, N.; Araki, M.; Kamada, M.; Sato, S.;
Okuno, Y.; Fujita, N.; Katayama, R. Brigatinib combined with
anti-EGFR antibody overcomes osimertinib resistance in
EGFR-mutated non-small-cell lung cancer. Nat. Commun.
2017, 8, 14768-14784.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Gunther, M.; Juchum, M.; Kelter, G.; Fiebig, H.; Laufer, S.
Lung cancer: EGFR inhibitors with low nanomolar activity
against a therapy-resistant L858R/T790M/C797S mutant.
Angew. Chem. Int. Ed. 2016, 55, 10890−10894.
(11) Engel, J.; Lategahn, J.; Rauh, D. Hope and
disappointment: covalent inhibitors to overcome drug
resistance in non-small cell lung cancer. ACS Med. Chem.
Lett. 2016, 7, 2−5.
(20) Park, H.; Jung, H. Y.; Mah, S.; Hong, S. Discovery of
EGFR (d746-750/T790M/C797S) mutant-selective inhibitors
via structure-based de novo design. Angew. Chem. Int. Ed.
2017, 56, 7634−7638.
(12) Niederst, M. J.; Hu, H.; Mulvey, H. E.; Lockerman, E. L.;
Garcia, A. R.; Piotrowska, Z.; Sequist, L. V.; Engelman, J. A.
The allelic context of the C797S mutation acquired upon
treatment with third-generation EGFR inhibitors impacts
sensitivity to subsequent treatment strategies. Clin. Cancer
Res. 2015, 21, 3924−3933.
(21) To, C.; Jang, J.; Chen, T.; Park, E.; Mushajiang, M.; Clercq,
D. J. H. D.; Xu, M.; Wang, S.; Cameron, M. D.; Heppner, D. E.;
Shin, B. H.; Gero, T. W.; Yang, A.; Dahlberg, S. E.; Wong, K. K;
Eck, M. J.; Gray , N. S.; Jänne, P. A. Single and dual targeting
of mutant EGFR with an allosteric inhibitor. Cancer Discov.
2019, DOI: 10.1158/2159-8290.
(13)Thress, K. S.; Paweletz, C. P.; Felip, E.; Cho, B. C.; Stetson,
D.;Dougherty, B.; Lai, Z.; Markovets, A.; Vivancos, A.; Kuang,
Y.; Ercan,D.; Matthews, S. E.; Cantarini, M.; Barrett, J. C.;
Janne, P. A.; Oxnard,G. R. Acquired EGFR C797S mutation
mediates resistance to AZD9291 in non-small cell lung cancer
harboring EGFR T790M. Nat. Med. 2015, 21, 560−562.
(22) Lu, X.; Zhang, T.; Zhu, S.-J.; Xun, Q.; Tong, L.; Hu, X.; Li,
Y.; Chan, S.; Su, Y.; Sun, Y.; Chen, Y.; Ding, J.; Yun, C.-H.; Xie,
H. and Ding, K. Discovery of JND3229 as a new EGFRC797S
mutant inhibitor with in vivo monodrug efficacy. ACS Med.
Chem. Lett. 2018, 9, 1123-1127.
(14)Cross, D. A.; Ashton, S.E.; Ghiorghiu, S.; Eberlein, C.;
Nebhan, C. A.; Spitzler, P. J.; Orme, J.P.; Finlay, M. R. V.;
Ward, R. A.; Mellor, M. J.; Hughes, G.; Rahi, Amar.; Jacobs, V.
N.; Brewer, M. R.; Ichihara, E.; Sun, J.; Jin, H.; Ballard, P.; Al-
Kadhimi, K.; Rowlinson, R. Klinowska, Teresa.; Richmond, G.
H. P.; Cantarini, M.; Kim, D. M.; Ranson, M. R.; Pao, W.
AZD9291, an irreversible EGFR TKI, overcomes T790M-
mediated resistance to EGFR inhibitors in lung cancer.
Cancer Discov. 2014, 4, 1046-1061.
(15) Wang, S.; Tsui, S. T.; Liu, C.; Song, Y.; Liu, D. EGFR c797s
mutation mediates resistance to third-generation inhibitors
in T790M-positive non-small cell lung cancer. J Hematol
Oncol. 2016, 9, 59-64.
(23) Yu, L.; Huang, M. H.; Xu, T. F.; Tong, L. J.; Yan, X. Y.;
Zhang, Z.; Xu, Y.; Yun, C. H.; Xie, H.; Ding, K.; lu, X. Y. A
structure-guided optimization of pyrido[2,3-d]pyrimidin-7-
ones as selective inhibitors of EGFRL858R/T790M mutant with
improved pharmacokinetic properties. Eur. J. Med. Chem.
2016, 126, 1107-1117.
(24) Xu, T. F.; Peng, T.; Ren, X. M.; Zhang, L. W.; Yu, L.; Luo,
J. F.; Zhang, Z.; Tu, Z. C.; Tong, L. J.; Huang, Z R.; Lu, X .Y.;
Geng, M. Y.; Xie, H.; Ding, J. Ding ,K. C5-substituted
pyrido[2,3-d]pyrimidin-7-ones as highly specific kinase
inhibitors targeting the clinical resistance related EGFR
T790M mutant. Med. Chem. Commun. 2015, 6, 1693-1697.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 8
1
2
3
4
TOC
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment