Discovery of (R)-1-(3-(4-Amino-3-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (CHMFL-EGFR-202) as a Novel Irreversible EGFR Mutant Kinase Inhibitor with a Distinct Binding Mode

Article abstract of DOI:10.1021/acs.jmedchem.6b01907

On the basis of Ibrutinib’s core pharmacophore, which was moderately active to EGFR T790M mutant, we discovered novel epidermal growth factor receptor (EGFR) inhibitor compound 19 (CHMFL-EGFR-202), which potently inhibited EGFR primary mutants (L858R, del

Full text of DOI:10.1021/acs.jmedchem.6b01907

Subscriber access provided by University of Newcastle, Australia
Article
Discovery of (R)-1-(3-(4-Amino-3-(3-chloro-4-(pyridin-2-
ylmethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)prop-2-en-1-one (CHMFL-EGFR-202) as a Novel Irreversible
EGFR Mutants Kinase Inhibitor with a Distinct Binding Mode
Aoli Wang, Xixiang Li, Hong Wu, Fengming Zou, Xiao-E Yan, Cheng Chen, Chen
Hu, Kailin Yu, Wenchao Wang, Peng Zhao, Jiaxin Wu, Ziping Qi, Wei Wang, Beilei
Wang, Li Wang, Tao Ren, Shanchun Zhang, Cai-Hong Yun, Jing Liu, and Qingsong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01907 • Publication Date (Web): 10 Mar 2017
Downloaded from http://pubs.acs.org on March 11, 2017
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 67
Journal of Medicinal Chemistry
1
2
3
4
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

Journal of Medicinal Chemistry
Page 2 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Discovery of (R)ꢀ1ꢀ(3ꢀ(4ꢀAminoꢀ3ꢀ(3ꢀchloroꢀ4ꢀ
(
pyridinꢀ2ꢀylmethoxy)phenyl)ꢀ1Hꢀpyrazolo[3,4ꢀ
d]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)propꢀ2ꢀenꢀ1ꢀone
CHMFLꢀEGFRꢀ202) as a Novel Irreversible
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
EGFR Mutants Kinase Inhibitor with a Distinct
Binding Mode
1,2,7
1,3,7
1,3,7
1,3,7
4,7
Aoli Wang , Xixiang Li , Hong Wu , Fengming Zou , Xiao-E Yan , Cheng
1
,2
1,2
1,2
1,3
4
1,2
Chen , Chen Hu , Kailin Yu , Wenchao Wang , Peng Zhao , Jiaxin Wu , Ziping
1
,3
1,3
1,2
1,2
5
3,6
Qi, Wei Wang , Beilei Wang , Li Wang , Tao Ren , Shanchun Zhang , Cai-Hong
4*
1,3*
1,2,3,5*
Yun , Jing Liu , Qingsong Liu
1. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei, Anhui 230031, P. R. China
2
. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
3. CHMFLꢀHCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei
Anhui 230031, P. R. China
4
. Department of Biophysics and Institute of Systems Biomedicine, School of Basic
Medical Sciences, Peking University Health Science Center, Beijing 100191, China
ACS Paragon Plus Environment

Page 3 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation,
Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui
2
30088, P. R. China
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
6
. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui
230031, P. R. China
7
. These authors contribute equally
ABSTRACT
Based on Ibrutinib’s core pharmacophore, which was moderately active to EGFR T790M
mutant, we discovered a novel EGFR inhibitor compound 19 (CHMFLꢀEGFRꢀ202),
which potently inhibited EGFR primary mutants (L858R, del19) and drug resistant
mutant L858R/T790M. Compound 19 displayed a good selectivity profile among 468
kinases/mutants tested in the KINOMEscan assay (S score (1) = 0.02), especially it did
not exhibit apparent activities against INSR and IGF1R kinases. The Xꢀray crystal
structure revealed that this class of inhibitors formed a covalent bond with Cys797 in a
distinct “DFGꢀinꢀCꢀHelixꢀout” inactive EGFR conformation. Compound 19 displayed
strong antiproliferative effects against EGFRꢀmutants driven NSCLC cell lines such as
H1975, PC9, HCC827 and H3255 but not the EGFR wt expressing cells. In the H1975
and PC9 cells inoculated xenograft mouse model, compound 19 exhibited dose dependent
tumor growth suppression efficacy without obvious toxicity. Compound 19 might be a
potential drug candidate for EGFRꢀmutant driven NSCLC.
INTRODUCTION
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Nonꢀsmall cell lung cancer (NSCLC) accounts for about 85%ꢀ90% lung cancers
1
which remains the leading cause of cancerꢀrelated deaths worldwide. EGFR (epidermal
2
growth factor receptor) kinase dysregulation is detected in 43–89% of NSCLCs, thereby
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
making EGFR inhibitors attractive for antiꢀNSCLC therapies. EGFR, also called
3
ErbB1/HER1, belongs to the ErbB family of receptor tyrosine kinases (RTKs), which
4
also includes ErbB2 (HER2/neu), ErbB3 (HER3) and ErbB4 (HER4). Gainꢀofꢀfunction
primary mutations of EGFR such as L858R or deletions within exon 19, lead to increased
5
6
cell proliferation and survival. The firstꢀgeneration EGFR inhibitors such as erlotinib
7
and gefitinib have achieved dramatic initial clinical responses but usually followed by an
8
acquired drug resistance. The most frequent drug resistant mutation is EGFR gatekeeper
9
T790M mutation, which is observed in approximately 50% of patients with first
generation EGFR inhibitor resistance. This encouraged the development of secondꢀ
10
generation irreversible EGFR inhibitors such as apatinib (BIBW2992, 1) (Figure 1) and
1
1
neratinib (HKI272, 2), which potently inhibit both primary EGFR mutations and the
T790M resistance mutation through formation of a covalent bond with Cys797. However,
due to the narrow selectivity window between EGFR wt and mutants, some adverse
effects were observed in the clinic. In order to improve the safety profile, EGFR mutants
1
2
selective inhibitors, so called thirdꢀgeneration inhibitors such as WZ4002 (3) , CO1686
1
3
14
15
16
(4) , AZD9291 (5) and other structurally related compounds 6 , HM61713 (7) , as
17
well as EGF816 (8) etc were developed. However, by examining the chemical
structures, most of the EGFR irreversible inhibitors bear either quinazoline or
aminopyrimidine core pharmacophores. Given the fact that different pharmacophores can
potentially provide different pharmacological properties (e.g., the effect for the
ACS Paragon Plus Environment

Page 5 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
16
18
hyperglycemia etc) and currently the patent space is becoming more and more
crowded, novel pharmacophore based new inhibitors are always attractive for developing
new antiꢀNSCLC therapies. Recently PCIꢀ32765 (9, Figure 2), an irreversible BTK
kinase inhibitor, has been reported to exhibit moderate activities against EGFR T790M
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
9
mutant in NSCLC. On the basis of this work, here we describe our medicinal chemistry
effort to the discovery of a novel pyrazolopyrimidine scaffold based potent irreversible
EGFR primary mutants (L858R, del19) and drug resistant (T790M) mutant inhibitor
compound 19 (CHMFLꢀEGFRꢀ202), which adopts a distinct “DFGꢀinꢀCꢀHelixꢀout”
inactive binding conformation with EGFR (Figure 2).
Figure 1. Chemical structures of the representative EGFR kinase inhibitors.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Schematic illustration of discovery of compound 19.
RESULTS AND DISCUSSION
Chemistry
Compounds 10ꢀ11 and 13-29 were prepared as described in Scheme 1. The synthesis
started from Oꢀalkylation between substituted 4ꢀbromophenol and substituted methyl
bromide analogues which generated intermediates 31a-p. Then a Suzuki coupling
reaction offered pinacol protected boronic esters 32a-p. Another Suzuki coupling
between intermediates 33-36, which were prepared following previously reported
2
0
procedures, and the corresponding pinacol boronic esters 32a-o yielded Bocꢀprotected
intermediates 37a-o. Finally acidic deprotection of the Boc group followed by Nꢀ
acylation reaction with either acryloyl chloride or (E)ꢀ4ꢀbromobutꢀ2ꢀenoyl chloride
afforded the target molecules.
a
Scheme 1. Synthesis of Compounds 10-11 and 13-29
ACS Paragon Plus Environment

Page 7 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R
O
R
OH
Br
O
R1
Br
a
b
R
R
1
2
R1
+
R
R
3
R
2
31a-32a, 37a, 13, 19; R=2-pyridine, R
=Cl, R =R =H;
1 2 3
R
3
R
3
R
2
B
31b-32b, 37b, 25; R=2-pyridine, R =F, R =R =H;
1
2
3
O
O
Br
31c-32c, 37c, 26; R=2-pyridine, R =Me, R =R =H;
1 2 3
31d-32d, 37d, 28; R=2-pyridine, R
1
=methoxy, R
2 3
=R =H;
3
3
3
3
1e-32e, 37e, 29; R=2-pyridine, R =CN, R =R =H;
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
2
3
3
1a-p
32a-p
1f-32f, 37f, 14; R=benzene, R =R =R =H;
1
2
3
1g-32g, 37g, 15; R=benzene, R =Cl, R =R =H;
1
2
3
1h-32h, 37h, 16; R=4-fluorobenzene, R
1
=Cl, R
=Cl, R
31j-32j, 37j, 20; R=3-(methylsulfonyl)benzeneR =Cl, R =R =H;
2
=R
3
=H;
33, R4=
31i-32i, 37i, 18; R=3,4-difluorobenzene, R
1
2
=R
3
=H;
NH2
NH
N
2
I
NBoc
NBoc
I
1
2
3
c
N
N
3
3
1 2 3
1k-32k, 37k, 21; R=2-methyl-1H-imidazole, R =Cl, R =R =H;
N
N
1l-32l, 37l, 22; R=3-dimethyl-1H-indazole, R =Cl, R =R =H;
1
2
3
N
34
, R =
4
N
H
N
R
4
31m-32m, 37m, 27; 6-dimethylpyridine, R
1
=Me, R
2
3
=R =H;
31n-32n, 37n, 23; R=3-fluorobenzene, R =R =Cl, R =H;
1
3
2
3
3-36
3
1o-32o, 37o, 24; R=3-fluorobenzene, R =R =H, R =Cl;
1 3 2
3
5, R4=
31p-32p, 10, 11, 17; R=3-fluorobenzene, R =Cl, R =R =H;
1 2 3
NHBoc
NBoc
36, R4=
R
R
R
O
O
O
R
1
R1
R
1
R
3
R3
R
3
NH
NH
2
NH2
2
R
2
R
N
d, e
R
2
f
2
N
N
N
N
or
N
32a-o
+
33
N
N
N
N
N
N
N
N
O
O
N
NH
37a-o
13
14-16, 18-29
R
R
O
R
O
O
R
2
1
R
3
R1
R1
R3
NH2
R
NH
3
NH
2
2
R
d, e, f
R
R2
N
2
N
or
N
32p
+
34/35/36
N
or
N
N
N
N
N
N
N
N
N
O
N
O
HN
11
O
10
17
a
Reagents and conditions: (a) DMF, K CO , rt, 2 h; (b) bis(pinacolato)diboron, 1,4ꢀ
2
3
dioxane, KOAc, Pd(dppf)Cl , 100 °C, 12 h; (c) R OH, THF, triphenylphosphine, DIAD,
2
4
0
°C to rt, 12 h; (d) 1,4ꢀdioxane/H O, K CO , Pd(PPh ) , 90 °C, 12 h; (e) 4 N HCl in
2 2 3 3 4
EtOAc, rt, 1 h; (f) for 10-11 and 14-29, acryloyl chloride, DIEA, DCM, 0 °C, 5 min; for
13, (E)ꢀ4ꢀbromobutꢀ2ꢀenoyl chloride, DIEA, DCM, 0 °C, 5 min, and then dimethylamine,
rt, 30 min.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 12 was prepared as depicted in Scheme 2. The synthesis of the compound
started from nucleophilic substitution of 3ꢀiodoꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine
with 1ꢀfluoroꢀ3ꢀnitrobenzene which afforded intermediate 38. Suzuki coupling between
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
8 and the corresponding pinacol boronic ester 32p followed by hydrogenation of the
nitro group provided aniline 39. Finally acylation reaction with acryloyl chloride
furnished the desired product 12.
a
Scheme 2. Synthesis of Compound 12
a
Reagents and conditions: (a) 1ꢀfluoroꢀ3ꢀnitrobenzene, K CO , DMF, 120 °C, 12 h; (b)
2
3
32p, 1,4ꢀdioxane/H O, K CO , Pd(PPh ) , 90 °C, 12 h; (c) SnCl , MeOH, 70 °C for 2 h;
2
2
3
3 4
2
(d) acryloyl chloride, DIEA, DCM, 0 °C, 5 min.
Design Rationale and Structure-Activity Relation (SAR) Exploration
Recently we reported that compound 9 exhibited potent inhibitory activities against
EGFR primary mutant L858R (H3255 cell, GI : 0.11 µM) and del19 (PCꢀ9 cell, GI :
5
0
50
0
.05 µM) but only moderate activities against first generation EGFR inhibitors resistant
1
9
mutant L858R/T790M (H1975 cell, GI : 1.2 µM). In addition, Xꢀray crystal structure
50
revealed that compound 9 bound to EGFR T790M with a distinct “DFGꢀin/CꢀHelixꢀout”
ACS Paragon Plus Environment

Page 9 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
21
inactive conformation. In order to improve the potency of this pharmacophore, we first
analyzed the coꢀcrystal structure of EGFR T790M with compound 9 (PDB 4YNJ) to
direct the chemical modification (Figure 3). Compound 9 formed two hydrogen bonds
between the pyrazolopyrimidine and Met793 and Gln791 residues in the hinge binding
area. The acrylamide in 9 formed a covalent bond with Cys797 to achieve the irreversible
binding. The Oꢀbridged diphenyl moiety directed into the hydrophobic pocket formed by
the DFG motif and αCꢀHelix. Interestingly, the Met790 sideꢀchain formed a beneficial
hydrophobic interaction with the phenyl ring (Figure 3A). Compared to the binding mode
of the typical EGFR irreversible inhibitor 3, the CꢀHelix in compound 9 shifted out which
caused Glu672 to move away from the orientation that usually required for the
coordination with ATP through Mg ions in the active kinase conformation (Figure 3B).
This CꢀHelixꢀout inactive conformation generated a larger hydrophobic pocket that could
accommodate larger groups than the phenyl ring (Figure 3C). Based on this analysis, we
envisioned that there were two directions we could take to improve the binding affinity of
this pharmacophore against EGFR T790M mutant. First, we could modify the Oꢀbridged
diphenyl moiety (R3 in Figure 3D) to more flexible and larger substituents to improve the
potency and selectivity. Second, the piperidine acrylamide moiety (R1 and R2 in Figure
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
3
D) could be modified to achieve better irreversible binding.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Schematic illustration of SAR exploration rationale. (A) Binding mode of
compound 9 with EGFRꢀT790M (PDB 4YNJ). (B) Demonstration of the CꢀHelixꢀout
conformation by overlapping the Xꢀray crystal structures of compounds 9 and 3 (PDB
4YNJ and 3IKA). (C) Demonstration of the hydrophobic pocket generated by the Cꢀ
Helix out conformation. (D) Illustration of the chemical modification strategy from
compound 9.
As the starting point, we first explored the SAR of the covalent bond formation part
(R1 and R2). TEL transfused EGFR kinase with T790M mutant was transformed into
BaF3 cell, whose growth is dependent on the EGFR T790M, and this cell was used to
monitor the EGFRꢀT790M kinase inhibitory activities of the compounds. Meanwhile,
parental BaF3 cell was used to monitor the general selectivity. Replacement of the R1
group from piperidine (9) to pyrrolidine (10) significantly increased the activity to
ACS Paragon Plus Environment

Page 11 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EGFRꢀT790M (GI : 0.16 ꢁM versus 0.013 ꢁM) however started to gain activity against
5
0
parental BaF3 cell (GI : >10 ꢁM versus 4.1 ꢁM) (Table 1). Switching piperidine (9) to
50
cyclohexanamine (11) only slightly increased the EGFRꢀT790M activity (GI : 0.11 ꢁM),
5
0
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
while showing potency against parental BaF3 cell (GI : 1.7 ꢁM). Further change of R1
5
0
to a phenyl group (12) resulted in decreased activity to EGFRꢀT790M (GI : 0.59 ꢁM),
5
0
although it still kept a good selectivity against parental BaF3 cell (GI : >10 µM).
5
0
Attempt to switch the R2 group from the acrylamide (9) to a more hydrophilic group, i.e.,
E)ꢀ5ꢀ(dimethylamino)pentꢀ3ꢀenamide (13), also caused the activity loss to EGFRꢀ
T790M (GI : 0.47 ꢁM), meanwhile narrowing the selectivity window between parental
(
5
0
BaF3 cell (GI : 1.3 ꢁM). Based on these results, we decided to keep the R1 and R2
5
0
groups as they are in compound 9.
a
Table 1. SAR Exploration Focused on the R1/R2 Moieties
BaF3ꢀtelꢀ
EGFRꢀT790M
Parental BaF3
GI : µM)
Compd
R1
R2
(
5
0
(
GI : µM)
50
9
0.16±0.02
＞10
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
1
0
1
2
0.013±0.003
4.1±0.11
1.7±0.1
＞10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.11±0.005
0.59±0.072
1
3
0.47±0.051
1.3±0.29
a
All GI values were obtained by triplet testing.
5
0
We then turned our attention to the R3 and R4 moieties that directed into the
hydrophobic pocket generated by the CꢀHelixꢀout conformation (Table 2). As described
above, the CꢀHelixꢀout conformation generated a relatively larger hydrophobic pocket
which might accommodate larger groups. Therefore, we first kept the R3 group as the
benzene ring and the oxygen atom as the linkage like in compound 9 but added one more
carbon to the R4 moiety (14) to direct the benzyl group into the hydrophobic pocket more
deeply. To our satisfaction, this modification significantly improved 14’s potency against
EGFR T790M mutant (GI : 0.013 µM, 12ꢀfold better than 9), meanwhile keeping a good
5
0
selectivity against parental BaF3 cell (GI : 8.2 µM). Slightly increasing the size of R3 by
5
0
adding one ꢀCl atom at 3ꢀposition of the benzene ring (15) retained the activity against
EGFR T790M (GI : 0.01 µM) but also started to gain activity against parental BaF3 cell
5
0
(GI : 4.8 µM). Increasing the hydrophobicity of R4 moiety by adding the paraꢀF atom
5
0
(
16) retained the activity to EGFR T790M but still did not improve the selectivity against
ACS Paragon Plus Environment

Page 13 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
parental BaF3 cells. Switching the ꢀF atom to meta-position (17) slightly decreased the
activity against EGFR T790M (GI : 0.097 µM) compared to 14. Addition of two ꢀF
5
0
atoms to both para- and meta-positions in R4 (18) gained a GI value of 0.049 µM to
5
0
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
EGFR T790M, which was still less potent than compound 13. However, replacement of
the benzene ring in R4 to the pyridine ring (19) remarkably increased the potency against
EGFR T790M (GI : <0.0003 µM), meanwhile exhibiting great selectivity against
5
0
parental BaF3 cell (GI : >10 µM). Addition of the methyl sulfonyl group to the benzene
5
0
ring (20) significantly decreased the activity to EGFR T790M but also gained potency to
parental BaF3 cells. When changing the pyridine group to the methylimidazole (21), the
compound lost about 100ꢀfold activity compared to 19, although still displayed good
selectivity to parental BaF3 cell. Further increasing the size of R4 moiety with the methyl
indazole (22) also decreased the activity against EGFR T790M (GI : 0.32 µM),
5
0
meanwhile narrowing the selectivity window to parental BaF3 cell (GI : 3.3 µM).
5
0
Addition of one more ꢀCl atom at 5-position of the benzene ring in R3 of 17 (23) slightly
increased the potency to EGFR T790M (GI : 0.071 µM) but also showed moderate
5
0
activity to parental BaF3 cell (GI : 5.5 µM). Similarly, shifting the ꢀCl atom from 3ꢀ to
5
0
2
ꢀposition (24) also significantly lowered the activity against EGFR T790M (GI : 0.17
50
µM). When changing the ꢀCl in 19 to the smaller ꢀF atom (25), it also led to more than
00ꢀfold activity loss (GI : 0.066 µM). Increasing the substituent’s size with the methyl
2
5
0
group (26) further decreased the activity (GI : 0.15 µM). However, adding one methyl
5
0
group to the 6ꢀposition of the pyridine ring in R4 of 19 (27) started to gain back the
potency though still much less potent than 19. Further switching the methyl group in R3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
moiety to larger methoxy (28) and nitrile (29) groups both resulted in remarkable activity
loss to EGFR T790M mutant.
a
Table 2. SAR Exploration Focused on the R3/R4 Moieties
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BaF3ꢀtelꢀ
Parental BaF3
Compd
R3
R4
EGFRꢀT790M
GI : µM)
(GI : ꢁM)
5
0
(
50
9
0.16±0.02
0.013±0.005
0.01±0.006
＞10
1
1
4
5
8.2±0.38
4.8±0.32
1
6
0.016±0.002
0.097±0.004
5.6±0.17
9.4±0.26
17
ACS Paragon Plus Environment

Page 15 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
18
0.049±0.0026
2.9±0.2
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
＜0.0003
1.0±0.06
＞10
5.4±0.036
＞10
2
2
2
0
1
2
0.037±0.028
0.32±0.006
3.3±0.19
Cl
2
2
2
3
4
5
0.071±0.016
0.17±0.021
0.066±0.0072
0.15±0.0058
0.072±0.013
2.6±1.0
5.5±0.021
2.3±0.058
＞10
Cl
26
27
28
＞10
5.6±0.43
＞10
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
9
2.2±0.32
＞10
a
All GI values were obtained by triplet testing.
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Biochemical and Cellular Property Evaluation of 19
Since compound 19 exhibited the best activity against EGFR T790M as well as the
best selectivity window against parental BaF3 cells, we then further tested it in a panel of
EGFR mutants engineered BaF3 cells (Table 3). In order to better understand its binding
mode, we also prepared 19’s reversible version compound 30 by saturation of its
potential electrophile acrylamide for covalent binding to propionyl group for comparison.
The results demonstrated that neither compound 19 nor 30 exhibited apparent potency
against parental BaF3 cells (both GI s: >10 µM), while compound 5 showed moderated
50
activity to BaF3 cells (GI : 1.5 µM). For the wild type EGFR, compound 19 exhibited
5
0
better selectivity than compound 5 (GI : 2.0 µM versus 0.3 µM). Compound 19 was as
5
0
potent as compound 5 for the primary EGFR mutants L858R and del 19. Meanwhile, it
displayed strong potencies against EGFR T790M (GI : <0.0003 µM) and EGFR
50
L858R/T790M (GI : <0.0003 µM) but only moderate activity to EGFR del19 T790M
5
0
mutant (GI : 0.63 µM), which showed similar trends as compound 5. In addition, for all
50
of the EGFR wt/mutants, when the C797 residue that was required for covalent bond
formation was mutated to serine, both compounds 19 and 5 lost activities remarkably,
which indicated that 19 exerted its inhibitory activities through an irreversible binding
mode. Furthermore, for all of the 19 sensitive EGFR mutants, the reversible version 30
exhibited much lower potencies, which again implied the covalent bond formation feature
ACS Paragon Plus Environment

Page 17 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of 19. These data suggested that compound 19 might irreversibly bind to EGFR mutants
and showed proper selectivity compared to the wide type EGFR, meanwhile exhibiting
better general cellular toxicity selectivity window than compound 5.
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
Table 3. Antiproliferative Effects of Compounds 5, 19 and 30 against a Panel of EGFRꢀ
a
BaF3 Isogenic Cell Lines
cell line
19 (GI : ꢁM) 30 (GI : ꢁM) 5 (GI : ꢁM)
5
0
50
50
ParentalꢀBaF3
>10
2.0±0.57
4.6±0.1
>10
1.5±0.058
BaF3ꢀtelꢀEGFR
1.1±0.38
3.7±0.38
1.6±0.26
2.6±0.4
0.3±0.011
0.59±0.055
<0.0003
BaF3ꢀtelꢀEGFRꢀC797S
BaF3ꢀtelꢀEGFRꢀL858R
BaF3ꢀtelꢀEGFRꢀL858RꢀC797S
BaF3ꢀtelꢀEGFRꢀT790M
BaF3ꢀtelꢀEGFRꢀT790MꢀC797S
BaF3ꢀFLꢀEGFRꢀDEL19
BaF3ꢀFLꢀEGFRꢀT790MꢀL858R
BaF3ꢀFLꢀEGFRꢀT790MꢀL858RꢀC797S
<0.0003
5.7±1.2
0.59±0.085
0.013±0.0006
0.55±0.02
0.001±0.0005
＜0.0003
<0.0003
2.3±0.35
0.004±0.0005
<0.0003
7.9±0.38
1.1±0.11
3.7±0.15
6.3±0.2
3.2±0.2
7.7±0.62
0.31±0.01
a
All GI values were obtained by triple testing.
5
0
Selectivity Profile Evaluation of Compound 19
We next examined compound 19’s kinome wide selectivity profile with DiscoveRx’s
2
2
KINOMEScan technology . The results demonstrated that 19 bore a good selectivity (S
score (1) = 0.02 at 1 µM) among 468 kinases and mutants tested (Figure 4A and
Supplemental Table S1). Among EGFR wt and mutants, 19 displayed strong binding
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
affinities against EGFR wt, EGFR (G719C/S), EGFR (L858R), EGFR (L858R/T790M),
EGFR (L861Q) and EGFR (T790M). In addition, 19 also exhibited strong binding
affinities against BLK, BMX, BTK, ERBB2, ERBB4, LOK, MEK1 and MEK5 kinases
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(percent of control score less than 1% at 1 ꢁM of 19). It is not surprising that 19 might
also bind to BLK, BMX, BTK, ERBB2 and ERBB4 since these kinases also bear a
similar cysteine residue as C797 of EGFR at the identical position. However, it is
interesting that 19 bound to LOK, MEK1 and MEK5 kinases strongly. Given the fact that
KINOMEscan is a binding assay and may not really reflect the inhibitory activity of the
compound, we then tested it in the Invitrogen SelectScreen assay and it revealed that 19
displayed strong potencies against EGFR(T790M) and EGFR kinases (IC : 5.3 nM and
50
8.3 nM respectively), and also strongly inhibited BLK (IC : 8.1 nM), BTK (IC : 24.5
5
0
50
nM), ERBB2 (IC : 8.1 nM), ERBB4 (IC : 3.2 nM), and moderately inhibited BMX
5
0
50
(
IC : 111.0 nM) and MEK1 (IC : 161.0 nM) kinases (Figure 4B). In addition, we
50 50
compared the compounds’ inhibitory activities against IGF1R and INSR, which might
induce the hyperglycemia upon inhibition. The results showed that compounds 1ꢀ5 all
bore moderate inhibitory activities against IGF1R and INSR, while compound 19 was
much less active to these kinases compared to them (Figure 4C). Furthermore, compared
to these known inhibitors, compound 19 also exhibited better selectivity to the parental
BaF3 cell, which indicated a better general toxicity profile. To further confirm 19’s
EGFR activity, we examined its effect on the EGFR autoꢀphosphorylation in cells. The
results demonstrated that 19 could potently block the Y1068 phosphorylation of EGFR
with an EC of 7 nM in the EGFR L858R/T790M mutant and an EC of 70 nM in the
5
0
50
ACS Paragon Plus Environment

Page 19 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EGFR wt (Figure 4D), which suggested that there was about 10ꢀfold selectivity between
the EGFR wt and drug resistance mutant EGFR L858R/T790M.
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

Journal of Medicinal Chemistry
Page 20 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 4. Selectivity profiling of compound 19. (A) KINOMEscan profiling of 19 at a
concentration of 1 ꢁM against 468 kinases and mutants. (B) Invitrogen SelectScreen
biochemical characterization of 19 (values = mean±SEM, n = 3). (C) Comparison of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EGFR inhibitors’ inhibitory activities against INSR and IGF1R kinases. (D) 19 and 5’s
inhibitory effects on EGFR wt and EGFR L858R/T790M mutant in cells.
Binding Mode Exploration
In order to better understand the SAR data and binding mode of this series of
compounds, we tried to determine the crystal structure of EGFR T790M in complex with
the inhibitor and we were able to coꢀcrystallize both EGRF T790M/19 and EGFR
T790M/21. However the former turned out to be twining crystals which could not be
used for structure determination. Therefore, we determined the crystal structure of EGFR
T790M in complex with compound 21 (PDB 5GNK), which revealed that EGFR T790M
protein adopted the “DFGꢀinꢀCꢀHelixꢀout” inactive conformation upon 21 binding
(Figure 5A). Compared to the aminopyridimidine scaffold based representative inhibitor
compound 3 (PDB 3IKA), CꢀHelix shifted further out in the complex with compound 21
which provided more space to accommodate the R4 moiety (Figure 5B). The
pyrazolopyrimidine formed two hydrogen bonds with Met793 and Gln791 in the hinge
area, respectively (Figure 5C). As expected, the acrylamide formed a covalent bond with
Cys797 residue. Met790 adopted a conformation in which the sideꢀchain was placed in
parallel to the R3 benzene ring and formed a useful hydrophobic interaction with it. In
addition, the ꢀCl atom was 3.2 Å distant from the backbone carbonyl of Leu788 and
formed a halogen bond there, which could significantly increase the binding affinity. This
explained the results observed in the SAR study that all of the groups at this position
ACS Paragon Plus Environment

Page 21 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
which lacked the halogen bond, e.g., ꢀH, ꢀF, methyl, methoxy and nitrile groups were less
2
3
favored than –Cl. The methyl imidazole occupied the hydrophobic pocket generated by
the CꢀHelixꢀout conformation and formed a hydrogen bond with Asp855. Compound 19
adopted an irreversible binding mode similar to compound 21 when docked into this
conformation (Figure 5D). According to the crystal structure and the docking model, a
pyridine group in compound 19 to replace the methyl imidazole moiety in compound 21
would better fit to the hydrophobic pocket without losing the hydrogen bond with
Asp855, which might explain the higher inhibitory activity of 19 than 21 against EGFR
T790M transformed BaF3 cells.
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 5. Binding mode analysis. (A) Superimposition of the crystal structure of 21 with
EGFR T790M (PDB 5GNK, in magenta) on 3 with EGFR T790M (PDB 3IKA, in cyan).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
B) Illustration of the hydrophobic pocket generated by CꢀHelixꢀout conformation. (C)
Binding mode analysis of 21 with EGFR T790M (PDB 5GNK). (D) Virtual docking of
9 into EGFR T790M (PDB 5GNK).
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Cell Activity Examination
We then tested compounds 5, 19, and 30 against a panel of NSCLC cell lines (Table
4
). In the EGFR L858R/T790M mutant cell H1975, 19 exhibited strong antiproliferative
effect (GI : 0.005 µM), which was as same as 5. Compound 19 also potently inhibited
5
0
the growth of PC9 (del19, GI : 0.004 ꢁM), HCC827 (del19, GI : 0.015 ꢁM) and H3255
5
0
50
(
L858R, GI : 0.075 ꢁM) cells. In addition, it exhibited good selectivity against EGFR wt
50
NSCLC cell lines such as A549, NCIꢀH2122, H1355 and H1703. A similar trend was
observed for compound 5. The reversible version compound 30 lost over 600ꢀfold
activities against those 19 sensitive cell lines and did not show apparent activities against
the 19 insensitive cell lines, indicating that the irreversible covalent binding observed in
the Xꢀray crystal structure was biologically relevant. Furthermore, compound 19 did not
display any apparent activities against normal Chinese hamster ovary (CHO) cells or
CHL cells, indicating no general toxicity.
Table 4. Antiproliferative Effects of Compounds 19, 30 and 5 against a Panel of Intact
a
Cancer Cell Lines
cell line
H1975
19 (GI : ꢁM)
30 (GI : ꢁM)
5 (GI : ꢁM)
5
0
50
50
0.005±0.001
3.0±1.0
4.4±0.25
＞10
0.005±0.001
0.002±0.0001
0.001±0.0001
(
L858R/T790M)
PCꢀ9 (del19)
0.004±0.001
0.015±0.007
HCC827 (del19)
ACS Paragon Plus Environment

Page 23 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
H3255 (L858R)
A549 (wt)
NCIꢀH2122 (wt)
H1355 (wt)
H1703 (wt)
CHO
0.075±0.015
>10
>10
>10
>10
>10
>10
>10
>10
0.033±0.013
3.5±1.5
>10
1.2±0.11
3.0±0.32
3.5±0.81
4.2±0.11
2.9±0.2
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
>10
8.3±0.9
>10
CHL
>10
a
All GI values were obtained by triple testing.
5
0
We next investigated compound 19’s effects on the EGFR mediated signaling
pathways in the established NSCLC cell lines. The results demonstrated that 19 potently
inhibited both EGFR wt/mutants’ autoꢀphosphorylation at Y1068 (Figure 6). In EGFRꢀ
mutant cell lines (H1975, PC9, H3255, HCC827) it also potently inhibited the
phosphorylation of EGFR downstream mediator ERK (EC50 less than 10 nM).
Interestingly, it significantly affected the phosphorylation of pAKT (Thr308/Ser473),
p70S6K (Thr389) in H1975 and HCC827 cells at the concentration of 100 nM. Similar
trends were observed for compounds 4 and 5. In addition, in wt EGFRꢀexpressing
NSCLC cell lines A549 and NCIꢀH2122, none of the downstream mediators were
affected.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Effects of compound 19 on EGFR mediated signaling pathways in EGFR wt
and mutant NSCLC cell lines.
Compound 19 could also doseꢀdependently arrest the cell cycle progression in G0/G1
phase in the EGFRꢀmutant NSCLC cells but not A549 or H2122 cells (Figure 7A). Upon
4
8 h of drug treatment, 19 started to induce doseꢀdependent cell apoptotic death in
ACS Paragon Plus Environment

Page 25 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EGFRꢀmutant cells from the concentration of 30 nM, which exhibited similar trends as
the wellꢀestablished EGFR inhibitors 4 and 5 (Figure 7B).
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

Journal of Medicinal Chemistry
Page 26 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 7. (A) Effects of compound 19 on the cell cycle progression in EGFR wt/mutant
NSCLC cell lines. (B) Effects of compound 19 on the apoptosis in EGFR wt/mutant
NSCLC cell lines.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
PK/PD Evaluation.
We next evaluated compound 19’s PK properties in rats following intravenous and
oral administration (Table 5). Upon intravenous injection compound 19 exhibited a Cmax
value of 2343.41 ng/mL. With oral administration it showed a halfꢀlife about 2 hours and
a moderate bioavailability (F=10.29%).
Table 5. Pharmacokinetic Study of Compound 19 on Sprague Dawley Rats
1
9
iv (1 mg/kg)
403.75±27.66
405.24±27.32
2343.41±284.86
0.017±0
po (10 mg/kg)
415.39±117.53
441.01±132.11
129.58±2.09
0.58±0.38
AUC(0ꢀt) (ng/mL·h)
AUC(0ꢀ∞) (ng/mL·h)
AUC(0ꢀt) (ng/mL·h)
Tmax (h)
T (h)
0.30±0.06
1.07±0.24
2.48±0.17
0.21±0.03
/
1.95±0.43
1
/2
Vz (mL/kg)
CLz (mL/h/kg)
MRT (0ꢀ ∞) (h)
F (%)
67.83±24.39
24.38±8.58
2.55±0.63
10.29
We finally tested the antitumor efficacy of compound 19 in H1975 (EGFRꢀ
L858R/T790M) and PC9 (EGFRꢀDel19) cell lines inoculated xenograft mouse models.
ACS Paragon Plus Environment

Page 27 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Based on the PK data, we chose the oral administration. The results demonstrated that
none of the 25, 50 and 100 mg/kg/day treatment affected the mice body weights (Figure
8
A and Figure 9A). After 21 days of continuous treatment, 19 doseꢀdependently inhibited
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
the growth of the H1975 and PC9 cells mediated tumors. A 50 mg/kg/day dosage almost
completely suppressed the tumor progression and exhibited a tumor growth inhibition
(TGI) efficacy of 45% in H1975 tumor (Figure 8BꢀD) and a 100mg/kg/day dosage
significantly slowed down the PC9 tumor progression (TGI = 50%) (Figure 9BꢀD). The
western blotting analysis of PC9 mediated tumor tissues demonstrated that EGFR Y1068
phosphorylation was effectively inhibited, which indicated the onꢀtarget engagement of
1
9 at those dosages (Figure 9E). In addition, the immunohistochemistry (IHC) stain
showed that 19 doseꢀdependently inhibited the tumor cell proliferation (examined by Kiꢀ
7 staining) and induced apoptosis (examined by TUNEL staining) in both H1975
Figure 8E) and PC9 mediated tumors (Figure 9F).
6
(
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 67
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Compound 19’s antitumor efficacy in H1975 xenograft mouse model. Female
nu/nu mice bearing established H1975 tumor xenografts were treated with 19 at 25.0,
5
0.0 and 100 mg/kg/d dosage or vehicle. Daily oral administration was initiated when
3
H1975 tumors had reached a size of 200 to 400 mm . Each group contained seven
animals. Data, mean ± SEM. (A) Body weight and (B) Tumor size measurements from
ACS Paragon Plus Environment

Page 29 of 67
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
H1975 xenograft mice after 21 days 19 administration. Initial body weight and tumor size
were set as 100%. (C) Representative photographs of tumors in each group after 0, 25.0,
5
0.0, 100 mg/kg/d 19 or vehicle treatment. (D) Comparison of the final tumor weights in
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
each group after 21ꢀday treatment period of 19. Numbers in columns indicate the mean
tumor weight in each group. ns, p>0.05, (∗) p<0.05, (∗∗) p<0.01. (E) Representative
micrographs of hematoxylin and eosin (HE), Kiꢀ67, and TUNEL staining of tumor tissues
with 19 treatment groups in comparison with the vehicle treatment group. Note the
specific nuclear staining of cells with morphology consistent with proliferation and
apoptosis (E, red arrows).
ACS Paragon Plus Environment