Discovery of JND3229 as a New EGFRC797S Mutant Inhibitor with in Vivo Monodrug Efficacy

Article abstract of DOI:10.1021/acsmedchemlett.8b00373

EGFR^C797S mutation inducing resistance against third generation EGFR inhibitor drugs is an emerging unmet clinical need for nonsmall cell lung cancer patients. The pyrimidopyrimidinone derivative JND3229 was identified as a new highly potent EGFR^C797S inhibitor with single digit nM potency. It also exhibited good in vitro and in vivo monodrug anticancer efficacy in a xenograft mouse model of BaF3/EGFR^{19D/T790M/C797S} cells. A high-resolution X-ray crystallographic structure was also determined to elucidate the interactions between JND3229 and EGFR^T790M/C797S. Our study provides an important structural and chemical basis for future development of new generation EGFR^C797S inhibitors as anticancer drugs.

Full text of DOI:10.1021/acsmedchemlett.8b00373

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Letter
Discovery of JND3229 as a New EGFRC797S
Mutant Inhibitor with In Vivo Mono-drug Efficacy
Xiaoyun Lu, Tao Zhang, Su-jie Zhu, Qiuju Xun, Linjiang Tong, Xianglong Hu, Yan Li,
Shingpan Chan, Yi Su, Yiming Sun, Yi Chen, Jian Ding, Cai-Hong Yun, Hua Xie, and Ke Ding
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00373 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 9, 2018
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ACS Medicinal Chemistry Letters
Discovery of JND3229 as a New EGFRC797S Mutant
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Inhibitor with In Vivo Mono-drug Efficacy
Xiaoyun Lu#*a, Tao Zhang#b,e,f, Su-Jie Zhu#c, Qiuju Xund, Lingjiang Tongb, Xianglong Hua, Yan Lib,
Shingpan Chana, Yi Sub, Yiming Sunb, Yi Chenb, Jian Dingb, Cai-Hong Yun*c, Hua Xie*b, Ke Ding*a
a
International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development
of Chinese Ministry of Education (MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou
510632, China.
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b
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
Department of Biophysics and Peking University Institute of Systems Biomedicine, Peking University Health Science
c
Center, Beijing 100191, China
d
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou
510530, China
e School of Life Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, China
f School of Pharmacy, University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
KEYWORDS: EGFRC797S, Clinical resistance, Fourth-generation inhibitors, Mono-drug efficacy.
ABSTRACT: EGFRC797S mutation induced resistance against the 3rd generation EGFR inhibitor drugs is an emerging “unmet
clinical need” for NSCLC patients. A pyrimidopyrimidinone derivative JND3229 was identified as a new highly potent EGFRC797S
inhibitor with single digit nM potency. It also exhibited good in vitro and in vivo mono-drug anticancer efficacy in a xenograft
mouse model of BaF3/EGFR19D/T790M/C797S cells. A high resolution X-ray crystallographic structure was also determined to elucidate
the interactions between JND3229 and EGFRT790M/C797S. Our study provides important structural and chemical basis for future
development of new generation EGFRC797S inhibitors as anticancer drugs.
The epidermal growth factor receptor (EGFR, ErbB1, HER1)
is one of the most validated molecular targets for anticancer
drug discovery.1 Three generations of EGFR inhibitors, e.g.
gefitinib, 2 erlotinib, 3 afatinib 4 and osimertinib (AZD9291), 5
have been approved by US FDA and achieved significantly
clinical benefit in non-small cell lung cancer (NSCLC)
patients. Particularly, the wild-type sparing 3rd generation
EGFR threonine790 to methionine790 mutant (T790M) inhibitor
osimertinib demonstrates approximately 75% overall response
rate (ORR) in EGFRT790M mutation-positive NSCLC patients
and was approved as the first-line treatment for metastatic
MGH121-res2 NSCLC cancer cells with EGFRC797S mutations
by combinations with EGFR antibody cetuximab or
pantitumumab.17 Nevertheless, it is still highly valuable to
discover novel EGFRC797S inhibitors with mono-drug efficacy
for overcoming the required resistance against the 3rd
generation EGFR inhibitors.18-20 Herein, we report the
identification of JND3229 (6, Figure 1) as a new reversible
EGFRC797S mutant inhibitor demonstrating both in vitro and in
vivo mono-drug efficacy against the proliferation of
EGFRC797S mutated cancer cells.
In order to discover new EGFRC797S inhibitors, a random
screening was initially conducted by utilizing our in-house
7
NSCLC patients in 2018,6, representing one of the most
advanced progress in human cancer therapy.
kinase inhibitor library containing approximate 3000
21-26
However, a tertiary Cys797 to Ser797 (C797S) point mutation,
disturbing covalent bond formation with the irreversible 3rd
generation EGFR inhibitors, becomes a leading mechanism of
clinically acquired resistance in approximately 40% of patients
treated with osimertinib.8-10 An allosteric inhibitor, EAI045 (1,
Figure 1),11 has been discovered to exhibit low nM IC50 values
against EGFRT790M and EGFRC797S mutants. However, the
combination with an EGFR antibody cetuximab was required
for EAI045 to demonstrate in vivo therapeutic efficacy
because of asymmetric dimerization of the receptor.11 (2-
Hydroxy)phenyl-4-substitututed quinazoline derivatives (2a
and 2b),12 tri-substituted imidazole (3),13-15 and a pan kinase
inhibitor Gö6976 (4)16 were also discovered to display strong
inhibition against EGFRC797S kinase, but neither cell-based
activity nor in vivo efficacy of these molecules was disclosed.
Encouragingly, brigatinib (5),17 an FDA approved ALK kinase
inhibitor, was recently reported to strongly suppress the kinase
activity of EGFRC797S and inhibit the proliferation of NSCLC
cancer cells harboring EGFRC797S mutation with IC50 values in
nM ranges. Moreover, brigatinib also exhibited significant
antitumor efficacy in a xenograft mouse model of PC9 and
compounds.
The effort helped us to identify JND3229
(Figure 1), a pyrimidopyrimidinone derivative, as a new
EGFRC797S inhibitor. The molecule was readily synthesized by
using a protocol outlined in Scheme 1. Briefly, a commercially
available
ethyl
4-chloro-2-(methylthio)-pyrimidine-5-
carboxylate (7) reacted with 1-Boc-1, 4-cyclohexanediamine
(8), followed by reduction, oxidation, Borch reductive
amination and cyclization to yield key pyrimidopyrimidinone
14. Oxidation of 14 with 3-chloroperbenzoic acid (m-CPBA)
yielded the sulfone 15, which was consequently subjected to
nucleophilic, deprotection and acylation reaction to give the
titled compound JND3299.
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F
HO
N
N
S
O
CN
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OH
N
N
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H
H
N
O
F
HN
N
HO
HN
Y
2a
: Y=C
2b
: Y=N
EAI045, 1
2
3
Cl
N
HN
N
N
NH
N
N
H
N
O
O
P
Cl
O
HN
N
N
O
O
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HN
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N
N
CN
JND3229, 6
GÖ6976, 4
Brigatinib, 5
Figure 1. Chemical structures of the representative EGFRC797S
inhibitors 1-6.
O
O
O
N
H
NH2
N
OH
NH
N
O
c
a
b
N
O
S
N
NH
S
N
+
S
N
NH
S
N
Cl
NHBoc
NHBoc
11
NHBoc
10
NHBoc
9
8
7
Cl
Cl
Cl
N
N
e
N
N
N
N
f
d
H
O
Cl
S
N
NH
S
N
N
O
S
N
N
O
O
H2
N
12
Figure 2 JND3229 potently inhibits the kinase activities of
EGFRL858R/T790M and EGFRL858R/T790M/C797S. The kinase inhibitory
efficacy of JND3229 (A), AZD9291 (B), staurosporine (C),
inhibitor 2b (D) and brigatinib (E) against EGFRL858R/T790M (blue
mark), EGFRL858R/T790M/C797S (red mark) and EGFRWT (Purple
mark) were tested by ELISA assay and the IC50 values were
calculated bases on 3 independent repeats.
NHBoc
13
NHBoc
15
NHBoc
14
Cl
N
Cl
Cl
N
N
N
N
N
g
h
i
HN
N
N
O
HN
N
N
O
HN
N
N
O
N
N
NH2
16
N
N
HN
N
N
NH2
N
N
NHBoc
The strong kinase inhibition of JND3229 was further validated
by investigating its potential suppression on activity of EGFR
O
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6
17
signals
in
BaF3
cells
stably
transfected
with
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 80 ℃ ,
85%; (b) LiAlH4,THF, −40 to 0 ℃, 50%; (c) MnO2, DCM, room
rt, 92%; (d) AcOH, NaBH4, PhMe, 110 ℃ to rt, 80%; (e)
triphosgene, Et3N, DCM, 0 ℃ to rt, 94%; (f) m-CPBA, DCM, rt,
88%; (g) F3CCOOH, 2-BuOH, 110 ℃; (h) F3CCOOH, DCM, rt,
50% (for two steps); (i) propanoic acid, HATU, DIPEA, DCM, rt,
90%.
EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S (Figure 3). It was
shown that JND3229 potently inhibited the phosphorylation of
EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S in
a dose-
dependent manner. Consistent to their kinase activity,
staurosporine also demonstrated strong inhibition on the
EGFRC797S activation in the BaF3 cells, but the effect of
osimertinib was minor.
JND3229 exhibited strong inhibition against the kinase
activity of EGFRL858R/T790M/C797S with an IC50 value of 5.8 nM
under the conditions of an enzyme-linked-immunosorbent
assay (ELISA) assay (Figure 2A). Additionally, the compound
also potently suppressed EGFRL858R/T790M and EGFRWT with
IC50 values of 30.5 and 6.8 nM (Figure 2A). Similar to the
previous observation, osimertinib exhibited an approximately
70-folds less potency against the EGFRC797S mutant comparing
with its strong inhibition against EGFRL858R/T790M (Figure 2B).
A pan-kinase inhibitor staurosporine (Figure 2C), EGFRC797S
inhibitor 2b (Figure 2D) and brigatinib (Figure 2E), used as
positive drugs, also displayed strong inhibition against
EGFRL858R/T790M/C797S kinase.
Figure 3. JND3229 potently inhibits the phosphorylation of
EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S in engineering
BaF3 Cells. BaF3 cells that overexpressed EGFRL858R/T790M/C797S
(A) or EGFR19D/T790M/C797S (B) were treated with the indicated
concentrations of JND3229, AZD9291 and staurosporine for 2 h
and stimulated by EGF for 15 min. Cell lysates were harvested for
western blot analysis for EGFR phosphorylation.
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ACS Medicinal Chemistry Letters
indicated by yellow hatched lines to key amino acids. Water is
represented as red dots. (B) The X-ray crystal structure with
interaction surface is shown.
The anti-proliferative activities of JND3229 were also
investigated against a panel of cells with different EGFR
status. It was shown that JND3229 potently inhibited the
proliferation of BaF3 cells harboring the EGFRL858R/T790M/C797S
and EGFR19D/T790M/C797S mutations with IC50 values of 0.51 and
0.32 µM, respectively (Table 1), which are comparable to
those of brigatinib. It also potently suppressed the growth of
NCI-H1975 NSCLC cells with EGFRT790M mutation with an
IC50 value of 0.31 µM. Although osimertinib exhibited strong
anti-proliferative effect on NCI-H1975 cells with an IC50 value
of 0.13 µM, its inhibition against BaF3 cells harboring
EGFRC797S mutation was significantly less potent (IC50 > 4
µM). Consistent to its non-selective inhibition against
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The in vivo anticancer efficacy of JND3229 was also
examined by using a xenograft mouse model. BALB/c mice
bearing established BaF3-EGFR19D/T790M/C797S mouse xenograft
tumors were treated with JND3229 twice daily at dosage of 10
mg/kg by intraperitoneal injection or by vehicle control for 10
days. EAI045 (60 mg/kg, once daily by oral gavage)
combination with cetuximab (1 mg/kg, once other day by
intraperitoneal injection) were used as positive control. The
data showed that administration of JND3229 caused an
obvious suppression of tumor growth with a Tumor Growth
Inhibition (TGI) value of 42.2% (Figure 5A), which was more
potent than that of EAI045/ cetuximab combination (TGI =
22.3%, Figure 5B). In addition, JND3229 was well tolerated
and there is no obvious body weight loss or other obvious
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EGFRWT
,
JND3229 also obviously suppressed the
proliferation of A431 cancer cells overexpressing EGFRWT
with an IC50 value of 0.27 µM.
Table 1. The anti-proliferative activities of JND3229 against the
cells with different mutant EGFR.
toxic
sign
in
the
treated
animals.
Further
immunohistochemistry analysis demonstrated that JND3229
treatment significantly decreased the level of phosphorylated
EGFR (p-EGFR) tin the tumor tissues, confirming in vivo
target inhibition of the compound (Figure 5C).
Anti-proliferation IC50 (µM)
JND3229 AZD9291
EGFR
Status
Cells
brigatinib
0.42±0.08
L858R/T79
0M/C797S
0.51±0.08 5.15±1.57
0.32±0.11 4.61±2.34
BaF3
19D/T790M
/C797S
L858R/T79
0M
0.26±0.02
NCI-
H1975
0.31±0.01 0.13±0.04
0.27±0.18 1.24±0.37
1.09±0.24
-
A431
WT
A 2.65 Å resolution X-ray crystallographic structure of
JND3229-EGFRT790M/C797S complex was also determined to
elucidate detailed interactions between the inhibitor and the
kinase (Figure 4 and ESI Table S1). It was shown that
JND3229 was accommodated in the ATP binding site of the
C797S mutated EGFR with
a reversible “U-shaped”
configuration. The pyrido[2,3-d]pyrimidine-7-one core formed
a bidentate hydrogen bond interactions with the “hinge”
residue Met793 of the protein. The 2-chlorophenyl group was
directed toward the hydrophobic back pocket composed by
Lys745, Glu762, Leu788, Met766 and Met790, and the
carbonyl of pyrido[2,3-d]pyrimidine-7-one formed a hydrogen
bond with the nitrogen of Lys745 mediated by a water
molecule. The propionamide group was located in a solvent-
exposing region and the NH moiety formed a hydrogen bond
with Leu718 mediated by another water molecule. The left-
hand methyl substituted phenyl group interacted with Gly796
by Van der Waals and the hydrophilic methyl piperazine
moiety was extended directly to the solvent.
Figure 5. JND3229 shows in vivo antitumor efficacy in
EGFR19D/T790M/C797S mouse xenograft model. Mice bearing BaF3-
EGFR19D/T790M/C797S tumor xenografts were treated with JND3229
(10 mg/kgꢀin 0.5% HPMC, ip, Bid), or treated with EAI045 (60
mg/kg in 10% NMP/90% PEG300, po, qd) combined with
cetuximab (1 mg/kg in 0.9% w/v NaCl/water, ip, qod). Relative
tumor volume (RTV) (A) and body weight (B) of
EGFR19D/T790M/C797S mouse xenograft model were measured (n =
6) every 2-3 days. The tumor growth inhibition was measured at
the final day of the treatment for the drug-treated group versus the
control (*P < 0.05 vs. control, Student’s t test) compared to the
control group. (C) JND3229 suppressed the expression of p-
EGFR in EGFR19D/T790M/C797S xenograft model. Tumor tissue
sections were stained by hematoxylin, eosin (H&E) (blue) and p-
EGFR antibody (brown), and Relative Integrated Optical Density
(IOD) of p-EGFR labeling was presented by quantitative analysis
(*P < 0.05 vs. control, Student’s t test), IOD Integrated Optical
Density. All values represent mean ± SD or mean ± SEM.
Figure
4 The X-ray crystal structure of JND3229 with
In summary, JND3229 was identified as a new EGFRC797S
inhibitor. The compound potently inhibited EGFRC797S
mutated kinase with an IC50 value of 5.8 nM, and strongly
EGFRL858R/T790M/C797S (PDB ID: 5ZTO). (A)The EGFR kinase is
shown in green and blue stick and ribbon representation.
JND3229 is shown in yellow stick structure. Hydrogen bonds are
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suppressed the proliferation of BaF3 cells harboring the
EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S mutations with
IC50 values of 0.51 and 0.32 µM, respectively. Moreover, the
compound also demonstrated in vivo mono-drug anticancer
efficacy in a xenograft mouse model of BaF3 cells with
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EGFR19D/T790M/C797S mutation.
A high resolution X-ray
crystallographic structure was also determined to elucidate the
interactions between JND3229 and EGFRT790M/C797S protein.
Although the relatively low target selectivity (ESI Table S2)
may raise some concern about the potential off-target toxicity
of this molecule, our study might provide useful structural and
chemical basis for further development of the 4th generation
EGFRC797S inhibitors to overcome the acquired resistance
against osimertinib.
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ASSOCIATED CONTENT
Electronic supplementary information (ESI) available:
Chemistry, biological assay and X-ray crystallography data.
See DOI:
AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: luxy2016@jnu.edu.cn (X. L.),
yunch@hsc.pku.edu.cn (C-H. Y.), hxie@simm.ac.cn (H. X.),
dingke @jnu.edu.cn (K. D.).
9. 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 mediated resistance to AZD9291 in non-
small cell lung cancer harboring EGFR T790M. Nat. Med.
2015, 21, 560-562.
Author Contributions
# These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
10. 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.
11. 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.
12. Park, H.; Jung, H. Y.; Mah S.; Hong, S. Discovery of EGF
receptor inhibitors that are selective for the d746-
750/T790M/C797S mutant through structure-based de novo
design. Angew. Chem. Int. Edit. 2017, 56, 7634-7638.
13. 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. Edit. 2016, 55, 10890-10894.
14. Juchum, M.; Gunther, M.; Doring, E.; Sievers-Engler, A.;
Lammerhofer, M.; Laufer, S. Trisubstituted imidazoles with
a rigidized hinge binding motif act as single digit nM
inhibitors of clinically relevant EGFR L858R/T790M and
L858R/T790M/C797S mutants: an example of target
hopping. J. Med. Chem. 2017, 60, 4636-4656.
ACKNOWLEDGMENT
The authors appreciate the financial support from Guangdong
Natural Science Funds (2015A030306042, 2105A030312014),
National Natural Science Foundation of China (21572230,
81425021, 21702075, 81673285 and 31270769), Guangdong
Nanyue-Baijie Award, Guangzhou City Key Laboratory of
Precision Chemical Drug Development (201805010007),
Institutes for Drug Discovery and Development of Chinese
Academy of Science (CASIMM0120185006) and Jinan
University.
ABBREVIATIONS
EGFR, epidermal growth factor receptor; NSCLC, non-small cell
lung cancer; ORR, overall response rate; C797S, Cys797 to
Ser797; m-CPBA, 3-chloroperbenzoic acid; ELISA, enzyme-
linked-immunosorbent assay; TGI, Tumor Growth Inhibition;
RTV, Relative tumor volume; IOD, Integrated Optical Density.
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