Design, synthesis and biological evaluation of potent EGFR kinase inhibitors against 19D/T790M/C797S mutation

Article abstract of DOI:10.1016/j.bmcl.2020.127327

The efficacy of EGFR inhibitors is frequently affected by acquired resistance. EGFR^{19D/T790M/C797S} mutation is one of the primary reasons for the emergence of resistance after treatment with the third-generation EGFR inhibitors such as AZD9291, CO1686 and Olmutinib. To overcome the resistance mutation 19D/T790M/C797S, we designed and prepared a series of indole derivatives with the terminal hydroxyl of alkyl chain to increase extra interaction with the Asp855 in the conservative DFG site. Activity evaluation, structure-activity relationship and docking analysis were also carried out. Among them, compound 12e displayed significant inhibitory activity against EGFR^{19D/T790M/C797S} (IC₅₀ = 15.3 nM) and good selectivity over EGFR WT (IC₅₀ > 1000 nM), L858R/T790M (IC₅₀, 156.6 nM) and L858R/T790M/C797S (IC₅₀, 218.3 nM) respectively. Furthermore, 12e exhibited good growth inhibition activity, induced G1 phase cell cycle arrest and apoptosis in BaF3/EGFR^{19D/T790M/C797S} cells by suppressing EGFR phosphorylation signaling pathway. In all, our study might provide a novel structural design method and lay the solid foundation for the development of the 4th generation EGFR^{19D/T790M/C797S} inhibitors.

Full text of DOI:10.1016/j.bmcl.2020.127327

Journal Pre-proofs
Design, synthesis and biological evaluation of potent EGFR kinase inhibitors
against 19D/T790M/C797S mutation
Zhicheng Su, Tingyuan Yang, Jie Wang, Mengzhen Lai, Linjiang Tong,
Gulinuer Wumaier, Zhuo Chen, Shengqing Li, Honglin Li, Hua Xie,
Zhenjiang Zhao
PII:
S0960-894X(20)30438-8
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127327
BMCL 127327
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 April 2020
26 May 2020
4 June 2020
Please cite this article as: Su, Z., Yang, T., Wang, J., Lai, M., Tong, L., Wumaier, G., Chen, Z., Li, S., Li, H.,
Xie, H., Zhao, Z., Design, synthesis and biological evaluation of potent EGFR kinase inhibitors against 19D/
T790M/C797S mutation, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2
020.127327
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Ltd.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
A novel indole derivative 12e was designed as a
Design, synthesis and biological evaluation of potent
EGFR kinase inhibitors against 19D/T790M/C797S
mutation
potential fourth generation EGFR19D/T790M/C797S
#
,a
#,a
#,a
b,d
b
c
Zhicheng Su , Tingyuan Yang , Jie Wang , Mengzhen Lai , Linjiang Tong , Gulinuer·Wumaier , Zhuo
a,*
c
a
b,*
a,*
Chen , Shengqing Li , Honglin Li , Hua Xie , Zhenjiang Zhao
N
HN
N
O
N
IC50 (nM)
15.3
EGFR19D/T790M/C797S
EGFR L858R/T790M/C797S 218.3
OH
EGFR L858R/T790M
EGFR WT
156.6
N
N
>1000
12e

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and biological evaluation of potent EGFR kinase inhibitors
against 19D/T790M/C797S mutation
#
,a
#,a
#,a
b,d
b
c
Zhicheng Su , Tingyuan Yang , Jie Wang , Mengzhen Lai , Linjiang Tong , Gulinuer·Wumaier ,
a,*
c
a
b,*
a,*
Zhuo Chen , Shengqing Li , Honglin Li , Hua Xie , Zhenjiang Zhao
a
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science & Technology, Shanghai 200237, China
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
b
2
c
01203, China.
Department of Pulmonary and Critical Care Medicine, Huashan Hospital, Fudan University, Shanghai, China.
School of Pharmacy, Fudan University, Shanghai 201203, China
Authors contributed equally to this work.
d
#
ARTICLE INFO
ABSTRACT
Article history:
Received
The efficacy of EGFR inhibitors is frequently affected by acquired resistance.
1
9D/T790M/C797S
EGFR
mutation is one of the primary reasons for the emergence of resistance after
Revised
Accepted
Available online
treatment with the third-generation EGFR inhibitors such as AZD9291, CO1686 and Olmutinib.
To overcome the resistance mutation 19D/T790M/C797S, we designed and prepared a series of
indole derivatives with the terminal hydroxyl of alkyl chain to increase extra interaction with the
Asp855 in the conservative DFG site. Activity evaluation, structure-activity relationship and
docking analysis were also carried out. Among them, compound 12e displayed significant
1
9D/T790M/C797S
inhibitory activity against EGFR
(IC50 =15.3 nM) and good selectivity over EGFR
WT (IC50 > 1000 nM), L858R/T790M (IC50, 156.6 nM) and L858R/T790M/C797S (IC50, 218.3
Keywords:
EGFR
NSCLC
C797S
nM) respectively. Furthermore, 12e exhibited good growth inhibition activity, induced G1 phase
1
9D/T790M/C797S
cell cycle arrest and apoptosis in BaF3/EGFR
cells by suppressing EGFR
phosphorylation signaling pathway. In all, our study might provide a novel structural design
method and lay the solid foundation for the development of the 4th generation
1
9D/T790M/C797S
EGFR
inhibitors.
2
019 Elsevier Ltd. All rights reserved.
Lung cancer is the leading cause of cancer-related deaths in
second-generation EGFR inhibitors cause serious side effects7.
The development of third-generation EGFR inhibitors (Olmutinib
and AZD9291) with aminopyrimidine as a novel scaffold
male or female worldwide, in which non-small cell lung cancer
1
(
NSCLC) accounts for 85% of lung cancer . EGFR is a member
8
,9
of the ErbB receptor tyrosine family and plays a key role in cell
successfully avoid these problems .
2
proliferation, differentiation and apoptosis . Meanwhile, EGFR
However, acquired resistance reappeared during the period of
clinical treatment with the third-generation EGFR inhibitors such
as AZD9291 and Olmutinib. At present, some studies have found
that the tertiary EGFR exon 20 C797S mutation appeared to be
the most common in a variety of drug resistance mechanisms.
For example, Thress et al first exposed that C797S mutation was
mutations have an important impact on cell growth and other
physiological processes. Thus, EGFR is a promising drug target
in the treatment of NSCLC. First-generation EGFR inhibitors
(
Gefitinib and Erlotinib) show high inhibitory activity for the
patients with sensitizing EGFR mutations, namely L858R
3
,4
mutation and exon 19 deletion . However, acquired drug
resistance occurs on patients who gain benefits from the first
generation EGFR inhibitors. Approximately 60% of the acquired
mutation is the EGFRT790M point mutation, which reduces the
binding affinity towards drugs and reinforces interaction with
1
0
one of the causes of AZD9291 resistance . Subsequently, Yu et
1
9D/T790M
al reported that a patient bearing EGFR
mutations was
detected C797S mutation after treated with AZD9291 for 9
1
1
months . Meanwhile, one study also revealed that C797S
1
2
mutation appeared in patients treated with Olmutinib . It was the
C797S mutation that disrupted the covalent bond between
inhibitors and Cys797 residue and caused the loss of inhibitor
activity.
5
ATP . To overcome T790M resistance mutation, second-
generation EGFR inhibitors (Afatinib and Dacomitinib) have
been developed by introducing a Michael acceptor to form a
6
covalent bond with Cys797 residue . Nevertheless, due to the
lack of selectivity between WT EGFR and mutant EGFR, the
*
Corresponding author. Tel.: +86-21-64253962; email: zhjzhao@ecust.edu.cn, hxie@simm.ac.cn, chenzhuo@ecust.edu.cn

Thus, the development of EGFR C797S mutation inhibitor is
an emergent issue, but so far, few researches have been done.
Recently, some potential EGFR inhibitors for the
By analyzing the binding patterns of the small molecule
inhibitors compound 7 and AZD9291 in Figure 2A, we found
that two molecules exhibited similar binding modes. However,
the additional hydrogen bond of compound 7 with the Asp855
side chain of the DFG motif stabilizes the flexible linker chain
into an orientation directed away from Cys797, so we inferred
that the alkyl chain terminal hydroxyl group of compound 7
extending to kinase phosphate binding site played a key role for
the tertiary mutation inhibitory activity through a polar contact
(H-O polar bond) with the Asp855 side chain in DFG motif.
Therefore, with the help of molecular docking, the alkyl chain
with terminal hydroxyl structural unit was introduced into the
AZD9291 scaffold to increase interaction with the Asp855. So a
series of indole derivatives were designed and prepared.
L858R/T790M/C797S
EGFR
mutation have been reported with high
inhibitory activity1
3,14,15,16
. Meanwhile, Park et al found some
inhibitors against EGFR1
9D/T790M/C797S
mutation by virtual
research found that
9D/T790M/C797S mutation is more general phenomenon in
1
4
screening .
Since
previous
1
1
7
patients after the use of third-generation drug AZD9291 , we
herein reported the development of a series of EGFR inhibitors
1
9D/T790M/C797S
against EGFR
mutation based on Osimertinib
(
AZD9291) and compound 7 in Figure 2 reported with single-
L858R/T790M/C797S
digit nanomolar IC50 values against EGFR
1
8
mutation , some compounds displayed significant inhibitory
activity against EGFR1
9D/T790M/C797S
and good selectivity over
EGFR WT, L858R/T790M and L858R/T790M/C797S
respectively.
HO
A
B
First-generation
Third-generation
F
S
N
O
N
NH
O
HN
N
Cl
HN
N
HN
N
O
HN
N
O
O
O
O
N
H3CO
H3CO
N
F
NH
N
N
O
N
N
1
(Gefitinib)
2 (Erlotinib)
H
5
(Olmutinib)
O
Second-generation
N
7
F
F
HN
N
HN
Cl
O
H
N
O
N
Figure 2. (A) Overlay poses between AZD9291 (yellow sticks, co complexed
crystal conformation, PDB 4ZAU) and compound 7 docked with EGFR
T790M (purple sticks, PDB 2JIU); (B) the structure of compound 7.
HN
N
Cl
N
N
H
N
N
N
N
O
H
O
N
N
N
O
O
O
3
(Afatinib)
4 (Dacomitinib)
6 (AZD9291)
Compound 8-12 were synthesized starting from 1H-indole
derivatives as presented in Scheme 1. A commercially available
Figure 1. Structures of representative first-, second-, and third-generation
EGFR inhibitors
NO2
NO2
NH2
O
O
e
f
O
N
F
N
N
N
HN
N
O
N
CHO
N
H
N
a
g
OH
R2
N
N
R1
R2
N
R1
R3
H
N
b or c
N
d
12e
R3
N
a : R1 = H, R = (CH2)2OH, R = H
2
3
8
8
N
Cl
b : R1 = (CH2)2OH, R = H, R = H
2
3
H
N
8c-e, 8g : R1 = (CH2)3OH, R = H, R = H
2
3
9a-g
c
12a : R1 = H, R =(CH2)2OH, R =H, R = propionylamino
2
3
4
f : R1 = (CH2)3OH, R = H, R = F
2
3
8
2b : R1 = (CH2)2OH, R =H, R =H, R = propionylamino
2
3
4
1
1
1
1
F
2c : R1 = (CH2)3OH, R =H, R =H, R = propionylamino
2
3
4
2f : R1 = (CH2)3OH, R =H, R =F, R = propionylamino
2
3
4
g
2g : R1 = (CH2)3OH, R =H, R =H, R = acryloylamino
2
3
4
N
N
R3
R3
R3
HN
N
N
N
HN
N
N
O
N
O
O
N
N
N
HN
F
HN
F
HN
F
N
R2
R2
j
R2
N
H
O
O
OH
i+j
O
N
R1
N
R1
h
N
R1
NO2
i
N
N
N
R4
NH2
HO
NO2
N
12d
11d
10a-d, 10f-g
11a-c, 11f-g
12a-c, 12f-g
Scheme 1. aReagents and conditions: (a) (1)P(Ph)
CH
I, KHMDS, THF, 25 oC, 2h, (2) BH
· THF, 0 oC, NaOH, H
O
2
, reflux, 1h; (b) (1) (2-
, 0
, MeOH, 25 C, 5 h; (g)
3
2
3
2
o
o
o
bromoethanoxy)(tert-butyl)dimethylsilane, DMF, NaH, 100 C, 4h;, (2) TBAF, THF, 25 C, 4h; (c) DMSO, KOH, 25 C, 8 h; (d) (1) DCE, anhydrous AlCl
3
o
o
o
o
C, (2) 2,4-dichloropyrimidine, 55 C, 1.5 h; (e) N,N,N’- trimethylethane -1,2-diamine , DMF, K
2
CO
3
, 110 C, 3 h; (f) 10%Pd/C, H
2

o
o
TsOH, 2-pentanol, 4-fluoro-2-methoxy-5- nitroaniline, 105 C, 2.5 h; (h) N,N,N’- trimethylethane -1,2-diamine, DMAC, DIPEA, 85 C, 3 h; (i) Fe, MeOH/DCM,
o
o
AcOH, 55 C, 4 h; (j) acryloyl chloride, DCM, DIPEA, 0 C, 1.5 h.
1
H-indole derivatives were carried out with Friedel-Crafts
Furthermore, in order to explore whether the substituent on
alkylation reaction after 2,4-dichloropyrimidine addition to afford
compound 9a-g. Subsequently, compound 10a-d&10f-g were
synthesized by the nucleophilic substitution of 9a-d&f-g with 4-
fluoro-2-methoxy-5-nitroaniline. The nitro group of compound
indole benzene ring had any effects on the bioactivity, 12f with F
substitution in R -postion was synthesized. As a result, 12f
3
displayed similar bioactivities to that of 12c in EGFR wild-type
1
9D/T790M/C797S
and mutations. For EGFR
mutation, 12f (IC50 = 40.4
1
0a-d&10f-g was performed reduction catalyzed by Fe powder
nM) showed 2 times more potent in contrast to 12c (IC50 = 72.5
nM), in fact the substitution effect on indole benzene ring was
not obvious.
in glacial acetic acid condition to yield corresponding amine,
which was used in the next step without further purification.
Finally, the target compounds 12a-d&f-g were prepared by an
acylation reaction with either acryloyl chloride or propionyl
chloride. Compound 12e was also synthesized by the
To our delighted, we obtained 12g with a high inhibitory
L858R/T790M
activity against EGFR
EGFR
(IC50
=
2.2 nM) and
1
9D/T790M/C797S
(IC50 = 17.9 nM) and high selectivity over
1
nucleophilic substitution of 9e with N -(2-(dimethylamino)ethyl)-
3
L858R/T790M/C797S
EGFR wild-type and EGFR
mutation. 12g with
1
-methoxy-N -methylbenzene-1,4-diamine.
4
Michael receptor on R -position represented typical third
generation EGFR inhibitor. To verify the effects of substituents
Considering the possibility for polar interactions with Asp855
2
in phosphate-binding site, we first obtained compounds bearing
the hydroxyethyl group in different positions at the indole
acrylamide, we could compare 12c with propionamide on R -
position 12g, its inhibitory activity against EGFR
L858R/T790M
and
1
19D/T790M/C797S
moiety. 12b (IC50 = 80.5 nM) with hydroxyethyl in R -postion
EGFR
was both weaker than 12g, especially the
showed better inhibitory activity against EGFR1
9D/T790M/C797S
inhibitory activity against EGFR
L858R/T790M
(2.2 nM vs 126.8 nM),
2
mutation than 12a (IC50 = 663.4 nM) with hydroxyethyl in R -
which demonstrated that acrylamide was the key Michael
receptor for a third generation EGFR inhibitor and not too critical
postion. Then, we tested the effects of length of the terminal
hydroxyl alkyl group on inhibitory activity. 12c (IC50 = 72.5 nM)
with 1'-hydroxypropyl had slight advantage over 1'-hydroxyethyl
1
9D/T790M/C797S
for EGFR
.
To prove this inference above, compound 12e (Figure S1) with
R =H was obtained at last. The results were exactly as expected,
1
2b.
4
In the solvent-exposed region, the binding mode in Figure 2A
12e showed the best inhibitory activity (IC50 = 15.3 nM) against
1
9D/T790M/C797S
revealed that hydrophilic tail was oriented to solvent-exposed
region. Therefore, we introduced hydrophilic group
ethylenediamine in an attempt to increase inhibitory activity. 12d
EGFR
and excellent selectivity over EGFR wild-
a
type and other mutations. We could conclude that 12e was a
promising fourth generation drug lead compound for
1
9D/T790M/C797S
19D/T790M/C797S
(
EGFR
IC50 = 73.0 nM) displayed about same
EGFR
.
inhibitory activity comparable to 12c (IC50 = 72.5 nM), which
suggested that the structure modification of solvent region had
little effect on the molecular level activity.
.
Table 1. Determination of Inhibitory Activities against EGFR Wild-Type (WT), L858R/T790M (LR/TM), L858R/T790M/C797S (LR/TM/CS) and
a
1
9D/T790M/C797S (19D/TM/CS) .
EGFR IC50[nM]
LR/TM/CS
Compound
WT
LR/TM
19D/TM/CS
N
HN
F
N
1
2a
O
N
> 1000
208.0 ± 16.2
>1000
663.4 ± 154.5
O
H
OH
N
H
N
HN
F
N
1
2b
O
N
> 1000
170.1 ± 9.5
> 1000
80.5 ± 21.6
O
N
H
OH
N
1
2c
HN
F
N
> 1000
126.8 ± 10.5
422.3 ± 211.5
72.5 ± 2.4
O
N
O
N
H
OH

N
HN
N
O
N
O
1
2d
> 1000
169.3 ± 16.5
638.4 ± 129.4
73.0 ± 14.9
N
H
OH
N
N
N
HN
N
O
N
1
2e
> 1000
156.6 ± 2.8
218.3 ± 29.8
15.3 ± 1.4
OH
N
N
F
N
HN
N
O
N
O
1
2f
> 1000
142.0 ± 3.1
332.2 ± 194.6
40.4 ± 4.5
N
H
OH
F
N
1
2g
HN
F
N
282.5 ± 54.5
2.2 ± 0.5
>1000
17.9 ± 2.7
O
N
O
N
H
OH
Brigatinib
AZD9291
48.3 ± 8.7
1.5 ± 0.4
7.2 ± 3.3
2.5 ± 0.2
1.5 ± 0.1
332.6 ± 185.3
167.1 ± 40.0
/
aKinase activity assays were examined by using the ELISA-based EGFR-TK assay. Data are averages of at least two independent determinations and reported as
the means ± SDs.
To further clarify the structure-activity relationship, molecular
docking was adopted to explain the binding modes of compound
exon 20 C797S mutation, a tertiary Cys797 to Ser797 point
mutation disturbed the corresponding covalent bond formation,
1
2a, 12b and 12c. We inferred that the difference of the activities
thus the activity difference between 12c and 12g was not as
L858R/T790M
between 12a and 12b may be attributed to the fact that 1'-
hydroxyethyl substitution could lead to a better orientation
towards Asp855. Indeed, a polar contact (H-O polar bond)
between the terminal hydroxyl group of 12b with the Asp855
side chain could be seen, which could not be seen in 7'-
hydroxyethyl 12a. In addition, 12c with 1'-hydroxypropyl formed
an additional hydrogen bond with Lys745 led it to a slight
advantage over 1'-hydroxyethyl 12b (Figure 3A), thus 12c
showed the best activity among them.
significant as that in EGFR
, which demonstrated that
acrylamide was the key Michael receptor for a third generation
1
9D/T790M/C797S
EGFR inhibitor and not too critical for EGFR
.
4
Furthermore, the representative compound 12e with R =H bound
T790M/C797S
in ATP-binding cleft of the EGFR
mutants, maintaining
the hydrogen bonds with Met793 by the structure of
aminopyrimidine. Besides, the terminal hydroxyl group formed
two hydrogen bonds with Asp855 and Lys745 respectively while
the hydrophilic group ethylenediamine formed salt bridge
interaction with Asp800 (Figure 3C). In brief, compared with
solvent region and Michael receptor group, we thought that the
introduction of the alkyl chain with terminal hydroxyl structural
unit was considerable for the improvement of activity against
Moreover, both 12c and 12g could form hydrogen bond
interaction with Asp855 and Lys745 side chain through the
terminal hydroxyl alkyl group. However, 12g with Michael
2
receptor on R -position formed a covalent bond with Cys797
1
9D/TM/CS, which could explain the reason that the compounds
(Figure 3B), which could explain its high inhibitory activity
1
2g with key Michael receptor still showed strong activities
L858R/T790M
against EGFR
(IC50 = 2.2 nM). Compared with 12g, 12c
against 19D/TM/CS. That is, when an appropriate alkyl chain
with terminal hydroxyl structural unit was added to the AZD9291
scaffold, the indole derivatives (such as 12e) could form a
2
with propionamide on R -position could not form a covalent
bond with Cys797 result in a weaker inhibitory activity against
EGFR
L858R/T790M
(IC50 = 126.8 nM), As for the tertiary EGFR

hydrogen bond with the Asp855 in the conservative DFG site to
cells, EGFR-L858R/T790M mutant H1975 cells and
L858R/T790M/C797S
19D/T790M/C797S
enhance the inhibitory activity against EGFR
which coincided with our original design idea.
,
BaF3/EGFR
cells. It was shown that 12e potently
inhibited the proliferation of BaF3 cells harboring the
1
9D/T790M/C797S
EGFR
mutant with IC50 value at 8.51 μM (Figure 4,
Table 2) which was comparable with Brigatinib. 12e also
inhibited the growth of A431 and H1975 cells with an IC50
values of 20.48 and 16.18 μM, respectively.
A
B
The mechanism of cell death was evaluated by two-
dimensional cell sorting by staining DNA with PI and staining
phosphatidylserine (PS) with a fluorescent annexin V derivative.
We evaluated the effects of compound 12e after 24 h treatments
1
9D/T790M/C797S
in BaF3/EGFR
cells. As shown in Figure 5,
1
9D/T790M/C797S
C
compound 12e induced apoptosis of BaF3/EGFR
cells in a concentration-dependent manner. It induced 24.18%
early-stage apoptosis and 14.53% late-stage apoptosis at 10 μM
concentration, and 74.01% early-stage apoptosis and 23.81%
late-stage apoptosis at 50 μM concentration. The percentage of
apoptosis in the 12e-treated group was higher than that in the
control group.
Figure 3. (A) The docked binding modes of 12c in EGFRT790M/C797S mutant
blue sticks, PDB 5ZTO); (B) The overlaid docked binding modes of 12g in
EGFRL858R/T790M mutant (white sticks, PDB 5Y25) and EGFRT790M/C797S mutant
pink sticks, PDB 5ZTO) (C) The docked binding modes of 12e in
(
(
EGFRT790M/C797S mutant (green sticks, PDB 5ZTO).
The antiproliferative activities of 12e were investigated against
a panel of cancer cells including EGFR-overexpressing A431
Table 2. Antiproliferative Activities of 12e against Cells with Different Mutant EGFR
a
IC50, μM.
Comp.
A431 / H1975
A431 / BaF3/EGFR19D/TM/CS
A431
20.48 ± 1.15
1.51 ± 0.35
H1975
BaF3/EGFR19D/TM/CS
8.51 ± 0.78
1
2e
16.18 ± 0.82
0.74 ± 0.11
1.27
2.04
2.41
0.28
Brigatinib
5.47 ± 0.56
Figure 4. The in vitro cell inhibitory efficacy of 12e against A431(A), H1975(B), and BaF3/EGFR19D/T790M/C797S (C) were tested by CCK-8 assay.

Figure 5. Apoptotic effect of 12e on BaF3/EGFR19D/T790M/C797S cells after treatment for 24 h. The lower left quadrant represents live cells, the lower right is for
early/primary apoptotic cells, the upper right is for late/secondary apoptotic cells, and the upper left represents cells damaged during the procedure.
To demonstrate the effect of compound 12e on the cell cycle,
we used flow cytometry for analysis. BaF3/EGFR19D/T790M/C797S
cells were treated with compound 12e (1, 10, and 20 μM) and
brigatinib (1 μM). As shown in Figure 6A, the percentage of cells
in the G1, S and G2 phase was obtained with flow cytometry, and
then summarized in Figure 6B. Compound 12e caused
accumulation of cells in the G1 phase of the cell cycle in a dose-
dependent manner. The percentage of G1 phase cells from
3
9.62% to 53.37% from absence to the presence of 20 μM 12e.
Figure 7. (A) BaF3 cells that overexpressed EGFR19D/T790M/C797S were treated
with the indicated concentrations of 12e, and brigatinib for 3 h and stimulated
by EGF for 10 min. Cell lysates were harvested for Western blot analysis for
EGFR, Akt and ERK phosphorylation. (B) Protein bands were quantified
using ImageJ and GraphPad Prism software.
In conclusion, to overcome this resistance mutation
1
9D/T790M/C797S, a series of indole derivatives bearing alkyl
chains with the terminal hydroxyl group which formed the
hydrogen bond with Asp855 and intended to increase the binding
affinities of the AZD9291 were synthesized and evaluated as
1
9D/T790M/C797S
potent EGFR
inhibitors. The results were exactly as
expected through structure-activity relationship and docking
analysis. The representative compound 12e strongly inhibited the
1
9D/T790M/C797S
kinase activity of EGFR
with IC50 value of 15.3 nM
WT
and exhibited highly selectivity over EGFR and
Meanwhile, 12e could significantly
L858R/T790M/C797S
EGFR
.
suppress the EGFR phosphorylation and its downstream
signaling molecules Akt and ERK, induce the apoptosis, arrest
cell cycle at G1, and inhibit proliferation with IC50 values of 8.5
1
9D/T790M/C797S
μM in BaF3/EGFR
cells in a concentration-
dependent manner. Our study provided a novel structural design
concept which is promising for the development of the fourth-
Figure 6. Effect of 12e on cell cycle progression in BaF3/EGFR19D/T790M/C797S
cells. (A) Cells were treated with different concentrations of 12e and
brigatinib for 24 h, then the cell cycle distribution was analyzed by flow
cytometry. (B) Graph reporting percentile cell-cycle phase distributions. G1,
S and G2 phase distributions were determined by FlowJo analysis.
1
9D/T790M/C797S
generation EGFR
inhibitors.
Acknowledgments
We evaluated 12e of its ability to interfere with EGFR and
downstream signaling. To this end, BaF3/EGFR19D/T790M/C797S
cells were treated with different doses of 12e and analyzed the
phosphorylation of EGFR and downstream molecules Akt and
ERK. As shown in Figure 7, 12e significantly induced reduction
of phosphorylated EGFR and ERK in a dose-dependent manner,
and phosphorylated Akt signal decreased slightly.
The research is supported in part by the National Key
Research and Development Program (Grant 2016YFA0502304),
the National Natural Science Foundation of China (grant
81825020), the National Science & Technology Major Project
“Key New Drug Creation and Manufacturing Program”, China
(No. 2018ZX09711002), the Special Program for Applied
Research on Super Computation of the NSFC-Guangdong Joint
Fund (the second phase) under Grant No. U1501501. Honglin Li
is also sponsored by National Program for Special Supports of
Eminent Professionals and National Program for Support of Top-
Notch Young Professionals.

References and notes
1
2
3
.
.
.
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.;
Jemal, A. CA: a cancer journal for clinicians 2018, 68, 394.
Appert-Collin, A.; Hubert, P.; Cremel, G.; Bennasroune, A.
Frontiers in pharmacology 2015, 6, 283.
Cohen, M. H.; Williams, G. A.; Rajeshwari, S.; Gang, C.; W
David, M.; David, M.; Sophia, A.; Atiqur, R.; Chenyi, L.; Richard,
L. Clinical Cancer Research An Official Journal of the American
Association for Cancer Research 2004, 10, 1212.
Declaration of interests
The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
☒
4
.
Xu,
Y.;
Liu,
H.;
Chen
,
J.;
Zhou
Q.
,
Canc
er
biolo
gy &
thera
py
2
010, 9, 572.
the work reported in this paper.
5
.
Bean, J.; Riely, G. J.; Balak, M.; Marks, J. L.; Ladanyi, M.; Miller,
V. A.; Pao, W. Clinical cancer research : an official journal of the
American Association for Cancer Research 2008, 14, 7519.
Singh, M.; Jadhav, H. R. Drug discovery today 2018, 23, 745.
Specenier, P.; Vermorken, J. Expert opinion on pharmacotherapy
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
6
7
.
.
2
016, 17, 1295.
8
.
.
Soria, J. C.; Sequist, L. V.; Gadgeel, S.; Goldman, J.; Wakelee, H.;
Varga, A.; Fidias, P.; Wozniak, A. J.; Neal, J. W.; Doebele, R. C.
Journal of Thoracic Oncology 2014, 32, S141.
9
1
Jiang, T.; Zhou, C. Translational Lung Cancer Research 2014, 3,
3
70.
0. 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. Nature medicine 2015, 21, 560.
1
1
1. Song, H. N.; Jung, K. S.; Yoo, K. H.; Cho, J.; Lee, J. Y.; Lim, S.
H.; Kim, H. S.; Sun, J. M.; Lee, S. H.; Ahn, J. S.; Park, K.; Choi,
Y. L.; Park, W.; Ahn, M. J. Journal of thoracic oncology : official
publication of the International Association for the Study of Lung
Cancer 2016, 11, e45.
2. Chabon, J. J.; Simmons, A. D.; Lovejoy, A. F.; Esfahani, M. S.;
Newman, A. M.; Haringsma, H. J.; Kurtz, D. M.; Stehr, H.;
Scherer, F.; Karlovich, C. A.; Harding, T. C.; Durkin, K. A.;
Otterson, G. A.; Thomas Purcell, W.; Ross Camidge, D.;
Goldman, J. W.; Sequist, L. V.; Piotrowska, Z.; Wakelee, H. A.;
Neal, J. W.; Alizadeh, A. A.; Diehn, M. Nature communications
2
016, 7, 13513.
3. Grabe, T.; Lategahn, J.; Rauh, D. ACS medicinal chemistry letters
018, 9, 779.
4. Park, H.; Jung, H. Y.; Mah, S.; Hong, S. Angewandte Chemie
017, 56, 7634.
1
1
1
2
2
5. Gunther, M.; Lategahn, J.; Juchum, M.; Doring, E.; Keul, M.;
Engel, J.; Tumbrink, H. L.; Rauh, D.; Laufer, S. J Med Chem
2
017, 60, 5613.
1
6. Li, Q.; Zhang, T.; Li, S.; Tong, L.; Li, J.; Su, Z.; Feng, F.; Sun, D.;
Tong, Y.; Wang, X.; Zhao, Z.; Zhu, L.; Ding, J.; Li, H.; Xie, H.;
Xu, Y. ACS medicinal chemistry letters 2019, 10, 869.
1
1
7. Wang, S.; Tsui, S. T.; Liu, C.; Song, Y.; Liu, D. Journal of
hematology & oncology 2016, 9, 59.
8. Chen, L.; Fu, W.; Zheng, L.; Liu, Z.; Liang, G. J Med Chem 2017,
6
0, 5613.
Supplementary Material
Supplementary material that may be helpful in the review
process should be prepared and provided as a separate electronic
file. That file can then be transformed into PDF format and
submitted along with the manuscript and graphic files to the
appropriate editorial office.