Discovery of N-(5-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-methyl-1,4-diazepan-1-yl)phenyl)acrylamide (CHMFL-ALK/EGFR-050) as a potent ALK/EGFR dual kinase inhibitor capable of overcoming a variety of ALK/EGFR associated drug resistant mutants in NSCLC

Article abstract of DOI:10.1016/j.ejmech.2017.08.035

Recently, more and more concomitant EGFR mutations and ALK rearrangement are observed from the clinic, which still lacks effective single-agent therapy. Starting from ALK inhibitor 14 (TAE684), we have developed a highly potent EGFR/ALK dual kinase inhibitor compound 18 (CHMFL-ALK/EGFR-050), which potently inhibited EGFR L858R, del 19 and T790M mutants as well as EML4-ALK, R1275Q, L1196M, F1174L and C1156Y mutants biochemically. Compound 18 significantly inhibited the proliferation of EGFR mutant and EML4-ALK driven NSCLC cell lines. In the cellular context it strongly affected EGFR and ALK mediated signaling pathways, induced apoptosis and arrested cell cycle at G0/G1 phase. In the in vivo studies, 18 significantly suppressed the tumor growth in H1975 cell inoculated xenograft model (40 mg/kg/d, TGI: 99%) and H3122 cell inoculated xenograft model (40 mg/kg/d, TGI: 78%). Compound 18 might be a potential drug candidate for EGFR- or ALK-individual as well as concomitant EGFR/ALK NSCLC.

Full text of DOI:10.1016/j.ejmech.2017.08.035

Accepted Manuscript
Discovery of N-(5-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-
yl)amino)-4-methoxy-2-(4-methyl-1,4-diazepan-1-yl)phenyl)acrylamide (CHMFL-ALK/
EGFR-050) as a potent ALK/EGFR dual kinase inhibitor capable of overcoming a
variety of ALK/EGFR associated drug resistant mutants in NSCLC
Yongfei Chen, Jiaxin Wu, Aoli Wang, Ziping Qi, Taoshan Jiang, Cheng Chen,
Fengming Zou, Chen Hu, Wei Wang, Hong Wu, Zhenquan Hu, Wenchao Wang,
Beilei Wang, Li Wang, Tao Ren, Shanchun Zhang, Qingsong Liu, Jing Liu
PII:
S0223-5234(17)30638-4
10.1016/j.ejmech.2017.08.035
EJMECH 9679
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2017
Revised Date: 13 August 2017
Accepted Date: 15 August 2017
Please cite this article as: Y. Chen, J. Wu, A. Wang, Z. Qi, T. Jiang, C. Chen, F. Zou, C. Hu, W.
Wang, H. Wu, Z. Hu, W. Wang, B. Wang, L. Wang, T. Ren, S. Zhang, Q. Liu, J. Liu, Discovery of N-
(5-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-methyl-1,4-
diazepan-1-yl)phenyl)acrylamide (CHMFL-ALK/EGFR-050) as a potent ALK/EGFR dual kinase inhibitor
capable of overcoming a variety of ALK/EGFR associated drug resistant mutants in NSCLC, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.035.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery
of
N-(5-((5-Chloro-4-((2-
(
isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-
4-methyl-1,4-diazepan-1-yl)phenyl)acrylamide (CHMFL-ALK/EGFR-
(
0
50) as a Potent ALK/EGFR Dual Kinase Inhibitor Capable of
Overcoming a Variety of ALK/EGFR Associated Drug Resistant
Mutants in NSCLC
1
,2,7
1,3,7
1,3,7
1,2,7
1,4, 7
Yongfei Chen , Jiaxin Wu , Aoli Wang , Ziping Qi , Taoshan Jiang
, Cheng
1
,3
1,2
1,3
1,2
1,2
1,2
Chen , Fengming Zou , Chen Hu , Wei Wang , Hong Wu , Zhenquan Hu ,
1
,2
1,3
1,3
4
5
Wenchao Wang , Beilei Wang , Li Wang , Tao Ren , Shanchun Zhang , Qingsong
1
,2,3,5
1,2
Liu
* , Jing Liu *
1
. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei, Anhui 230031, P. R. China
2
. CHMFL-HCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei
Anhui 230031, P. R. China
3
4
. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
. School of Life Sciences, Anhui Agricultural University, Hefei, Anhui 230036, P. R.
China
5
6
. 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
. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui
30031, P. R. China
2

ACCEPTED MANUSCRIPT
7
. These authors contribute equally
AUTHOR INFORMATION
Corresponding Authors
*
E-mail address: qsliu97@hmfl.ac.cn (Q. Liu), E-mail address: jingliu@hmfl.ac.cn (J.
Liu).
Author contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
ABSTRACT
Recently, more and more concomitant EGFR mutations and ALK rearrangement are
observed from the clinic, which still lacks effective single-agent therapy. Starting from
ALK inhibitor 14 (TAE684), we have developed a highly potent EGFR/ALK dual kinase
inhibitor compound 18 (CHMFL-ALK/EGFR-050), which potently inhibited EGFR
L858R, del 19 and T790M mutants as well as EML4-ALK, R1275Q, L1196M, F1174L
and C1156Y mutants biochemically. Compound 18 significantly inhibited the
proliferation of EGFR mutant and EML4-ALK driven NSCLC cell lines. In the cellular
context it strongly affected EGFR and ALK mediated signaling pathways, induced
apoptosis and arrested cell cycle at G0/G1 phase. In the in vivo studies, 18 significantly
suppressed the tumor growth in H1975 cell inoculated xenograft model (40 mg/kg/d,
TGI: 99%) and H3122 cell inoculated xenograft model (40 mg/kg/d, TGI: 78%).
Compound 18 might be a potential drug candidate for EGFR- or ALK-individual as well
as concomitant EGFR/ALK NSCLC.

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Keywords
EGFR; ALK; Dual kinase inhibitor; Non-small cell lung cell cancer
Abbreviations used
NSCLC, non-small cell lung cell cancer; EGFR, epidermal growth factor receptor; ALK,
anaplastic lymphoma kinase; EML4, echinoderm microtubule-associated protein like 4;
NPM, nucleophosmin; SAR, structure-activity relationship; CHL, Chinese hamster Lung;
CHO, Chinese hamster ovary; eIF4E, eukaryotic initiation factor 4E; 4EBP1, 4E-binding
protein 1; P70S6k, p70S6 kinase 1; STAT3, signal transducer and activator of
transcription 3; ERK, extracellular regulated protein kinases; PARP, poly ADP-ribose
polymerase; PK, pharmacokinetics; IHC, immunohistochemistry; HE, hematoxylin-
eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; TGI,
tumor growth inhibition.
1
. INTRODUCTION
Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell
lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Currently a
number of driving oncogenes are identified in NSCLC and among them EGFR activating
mutations and ALK rearrangements are two prevalent ones. EGFR activating mutations
are found in approximately 10–30% of the patients with NSCLC [1-3]. Two frequent and
mutually exclusive primary mutations occur either in the activation loop as a point
mutation (EGFR L858R) or by short deletion in exon 19 (EGFR-del19), which together
4
account for approximately 85% of all cases. First generation EGFR tyrosine kinase
inhibitors (TKIs) such as compounds 1 (Erlotinib) [1,5] and 2 (Gefitinib) [6] are now

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established therapies for NSCLC patients with oncogenic EGFR primary mutations
(
Figure 1). However, upon continuous treatment, patients become resistant and
approximate 60% cases will develop a secondary T790M mutation at the gatekeeper
position of EGFR [7-9]. To overcome the drug resistance caused by T790M mutation,
several second generation irreversible EGFR inhibitors such as compounds 3 (Afatinib
10], 4 (Dacomitinib) [11] and more selective third generation inhibitors such as
compounds 5 (WZ4002) [12], 6 (Rociletinib) [13], 7 (Osimertinib) [14,15] and 8
)
[
(Olmutinib) [16] have been developed.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase of the insulin
receptor superfamily. Various ALK-fused oncogenes such as echinoderm microtubule
associated protein like 4 (EML4)-ALK have been identified in 2-7% NSCLC [17].
Validation of ALK as an important therapeutic target has led to the development of ALK
inhibitors such as compounds 9 (Crizotinib) [18], 10 (Ceritinib) [19], 11 (Alectinib) [20], 12
(Brigatinib) [21] and 13 (ASP3026) [22], which have been approved for clinical use
except 13. It is noteworthy is that compound 12 has been reported to be able to inhibit
both ALK and EGFR kinases [21].

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O
N
O
N
N
O
O
N
O
O
N
O
HN
O
O
N
HN
Cl
F
N
N
HN
O
N
HN
Cl
F
O
HN
Cl
F
O
HN
N
N
1
(Erlotinib)
2 (Gefitinib)
3 (Afatinib)
4 (Dacomitinib)
CF3
NH
Cl
N
N
N
HN
N
HN
N
HN
N
NH
H
O
O
N
N
O
N
O
N
S
NH
O
N
H
N
NH
H
N
N
N
N
O
N
N
N
O
O
N
O
5
(WZ4002)
6 (Rociletinib)
7 (Osimertinib)
8 (Olmutinib)
N
Cl
HN
N
N
N
O
S
HN
N
NH
O
P
N
NH
F
O
O
NH
Cl
O
Cl
N
NH2
O
O
S
HN
N
NH
O
N
N
N
Cl
O
N
N
N
N
N
N
N
N
H
HN
O
9
(Crizotinib)
10 (Ceritinib)
11 (Alectinib)
12 (Brigatinib)
13 (ASP3026)
Figure 1. Chemical structures of representative EGFR and ALK kinase inhibitors.
EGFR mutants and ALK rearrangements have been once considered as mutually
exclusive [23,24]. However, with the development of high throughput sequencing and
deep sequencing technologies, more and more concomitant EGFR mutants and ALK
rearrangements are identified from the single colony of the tumor tissue in patients [25-
3
0], in which case single EGFR or ALK inhibitor could not display a good therapeutic
effect. In addition, in ALK driven NSCLC cells H3122, an EGFR reactivation adaptive
drug resistance mechanism has been identified [31]. Although theoretically combination
of EGFR and ALK inhibitors would enhance the antitumor efficacy against the
EGFR/ALK co-expression or co-activation, drug-drug interaction problems would make

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the dual-target-single-agent strategy more preferred. Based on this concept, via structure
based drug design approach, we have discovered a potent ALK/EGFR dual kinase
inhibitor compound 18 (CHMFL-ALK/EGFR-050), which displayed strong activities
against EGFR mutants such as L858R, del 19, T790M and ALK mutants such as F1174L,
C1156Y, L1196M, etc (Figure 2). During the preparation of this manuscript, Gray team
has also reported a similar compound developed from compound 10 (Ceritinib) which
could inhibit both ALK and EGFR T790M mutant [32].
Figure 2. Schematic illustration of discovery of compound 18 (CHMFL-ALK/EGFR-
0
50).
2
2
. Results and discussion
.1 Design and synthesis
In order to seek the proper core pharmacophore for medicinal chemistry exploration,
we started from screening a panel of kinase inhibitors that are clinically approved or
under clinical investiation against H1975 cells. We found that compound 14 (TAE-684)
[33], which was a well-established ALK inhibitor, exhibited moderate antiproliferative
efficacy (GI = 0.72 ꢀM). Further testing in EGFR transformed BaF3 cells showed that
5
0
1
4 could moderately inhibit the proliferation of BaF3-TEL-EGFR (GI : 0.56 ꢀM), BaF3-
50
TEL-EGFR-T790M (GI : 0.42 ꢀM) and displayed proper selectivity window against
5
0

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parental BaF3 cells (GI50: 1.1 ꢀM), which indicated that it did bear the on-target activity
against EGFR. We then docked 14 into the EGFR T790M X-ray structure (PDB ID:
3
IKA) and found that the distance from R position to Cys797 is about 5 Å and it would
1
be approachable for an electrophilic group. Therefore we postulated that this scaffold
might be amenable for developing an irreversible inhibitor to enhance the inhibitory
effect (Figure 3A). In addition, installment of the irreversible warhead would probably
not affect the ALK kinase binding (PDB ID: 2XB7) because there is no amino acid
residue that can block the binding around this position (Figure 3B). On the basis of the
analysis, we decided to introduce an acrylamide at the R
1 2 3
position and explore the R , R ,
R , R and R positions to obtain a highly potent ALK/EGFR dual kinase inhibitor
4
5
6
(
Figure 3C).

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Figure 3. Design rationale based on compound 14’s core pharmacophore. (A) Docking of
1
4 into EGFR T790M mutant protein (PDB ID: 3IKA). (B) Docking of 14’s into ALK
kinase (PDB ID: 2XB7). (C) Schematic illustration of SAR exploration starting from 14’s
core pharmacophore.
Compounds 15-49 were prepared according to the general synthetic route depicted in
scheme 1. Substitution reaction between substituted amines and 2,4-dichloropyrimidine
analogues afforded intermediates 51a-n. Another substitution with 4-fluoro-3-
nitroaniline analogues under acidic condition provided corresponding products 52a-p.
'
Compounds 53a-i were generated by substitution with piperazine or piperidine
derivatives, or morpholine. The nitro group was then reduced with Fe or SnCl to yield
2
'
intermediates 54a-i . Finally acylation reaction with acryloyl chloride furnished the
desired compounds 15-49. Compound 50, which was compound 18’s reversible version,
was prepared following the same synthetic route except that propionyl chloride was used
instead in the last acylation step to react with compound 54d (Scheme 2).
a
Scheme 1. Synthesis of compounds 15-49

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R4
R6
N
R5
R4
R4
N
NO2
F
R6
N
R5
b
a
R5
R6
N
N
N
H
+
Cl
N
HN
N
N
Cl
Cl
R2
52a-p
5
1a-n
5
5
5
5
5
5
5
5
5
5
5
5
5
5
1a, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H
52a, R2=OMe, R4=Cl, R5=2-(isopropylsulfonyl)phenyl, R6=H
52b, R2=OMe, R4=H, R5=2-(isopropylsulfonyl)phenyl, R6=H
52c, R2=OMe, R4=CH3, R5=2-(isopropylsulfonyl)phenyl, R6=H
4
5
6
1b, R =H, R =2-(isopropylsulfonyl)phenyl, R =H
4
5
6
1c, R =CH , R =2-(isopropylsulfonyl)phenyl, R =H
4
3
5
6
5
2d, R =H,R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H
1d, R =Cl, R =3-(trifluoromethyl)phenyl, R =H
2
4
5
6
4
5
6
1e, R =Cl, R =3-chloro-4-fluorophenyll, R =H
52e, R2=OCH(CH3)2, R4=CH3, R5=2-(isopropylsulfonyl)phenyl, R6=H
52f, R2=OMe, R4=Cl, R5=3-(trifluoromethyl)phenyl, R6=H
52g, R2=OMe, R4=Cl, R5=3-chloro-4-fluorophenyll, R6=H
4
5
6
1f, R =Cl, R =2,6-difluorophenyl, R =H
4
5
6
1g, R =Cl, R =thiophen-2-ylmethyl, R =H
4
5
6
5
2h, R =OMe, R =Cl, R =2,6-difluorophenyl, R =H
1h, R =Cl, R =cyclopropyl, R =H
2 4 5 6
4
5
6
1i, R =Cl, R =2-pivalamidophenyl, R =H
52i, R2=OMe, R4=Cl, R5=thiophen-2-ylmethyl, R6=H
52j, R2=OMe, R4=Cl, R5=cyclopropyl, R6=H
4
5
6
1j, R =Cl, R =3-(trifluoromethyl)phenyl, R =CH
4
5
6
3
1k, R =Cl, R =2-(N,N-dimethylsulfamoyl)phenyl, R =H 52k, R2=OMe, R4=Cl, R5=2-pivalamidophenyl, R6=H
4
5
6
5
2l, R =OMe, R =Cl, R =3-(trifluoromethyl)phenyl, R =CH
1l, R =Cl, R =2-(dimethylphosphoryl, R =H
2 4 5 6 3
4
5
6
1m, R =Cl, R =2-(methylcarbamoyl, R =H
52m, R2=OMe, R4=Cl, R5=2-(N,N-dimethylsulfamoyl)phenyl, R6=H
52n, R2=OMe, R4=Cl, R5=2-(dimethylphosphoryl, R6=H
4
5
6
1n, R =Cl, R =2-cyanophenyl, R =H
4
5
6
52o, R =OMe, R =Cl, R =2-(methylcarbamoyl, R =H
2 4 5 6
5
2p, R =OMe, R =Cl, R =2-cyanophenyl, R =H
2
4
5
6
R4
R4
R4
O
R6
R6
R6
N
R5
N
NO2
N
R5
N
NH2
e
N
R5
N
NH
c
d
N
N
N
HN
R3
HN
R3
HN
R3
R2
3a-i'
R2
R2
54a-i'
15-49
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
3a-54a,15; R =OCH , R =4-(4-methylpiperazin-1-yl)piperidin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3b-54b,16; R =OCH , R =4-(methylsulfonyl)piperazin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3c-54c,17; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3d-54d,18; R =OCH , R =4-methyl-1,4-diazepan-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3e-54e,19; R =OCH , R =4-isopropylpiperazin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3f-54f, 20; R =OCH , R =4-(2-methoxyethyl)piperazin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3g-54g, 21; R =OCH , R =4-cyclohexylpiperazin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3h-54h, 22; R =OCH , R =4-morpholino, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3i-54i, 23; R =OCH , R =piperidin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3j-54j, 24; R =H, R =4-(4-methylpiperazin-1-yl)piperidin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
4
5
6
3k-54k, 25; R =OCH(CH ) , R =4-ethylpiperazin-1-yl, R =Cl, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3 2
3
4
5
6
3l-54l, 26; R =OCH , R =4-(4-methylpiperazin-1-yl)piperidin-1-yl, R =CH , R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
3
5
6
3m-54m, 27; R =OCH , R =4-ethylpiperazin-1-yl, R =CH , R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
3
5
6
3n-54n, 28; R =OCH , R =4-ethylpiperazin-1-yl, R =H, R =2-(isopropylsulfonyl)phenyl, R =H;
2
3
3
4
5
6
3o-54o, 29; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =3-(trifluoromethyl)phenyl, R =H;
2
3
3
4
5
6
3p-54p, 30; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =3-chloro-4-fluorophenyl, R =H;
2
3
3
4
5
6
3q-54q, 31; R 2=OCH , R =4-methylpiperazin-1-yl, R =Cl, R =2,6-difluorophenyl, R =H;
3
3
4
5
6
3r-54r, 32; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =thiophen-2-ylmethyl, R =H;
2
3
3
4
5
6
3s-54s, 33; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =cyclopropyl, R =H;
2
3
3
4
5
6
3t-54t, 34; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =2-pivalamidophenyl, R =H;
2
3
3
4
5
6
3u-54u, 35; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =3-(trifluoromethyl)phenyl, R =CH ;
2
3
3
4
5
6
3
3v-54v, 36; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =2-(N,N-dimethylsulfamoyl)phenyl, R =H;
2
3
3
4
5
6
3w-54w, 37; R =OCH , R =4-ethylpiperazin-1-yl, R =Cl, R =2-(dimethylphosphoryl, R =H;
2
3
3
4
5
6
3x-54x, 38; R =OCH , R =4-4-(dimethylamino)piperidin-1-yl, R =Cl, R =2-(dimethylphosphoryl, R =H;
2
3
3
4
5
6
3y-54y, 39; R =OCH , R =4-morpholino, R =Cl, R =2-(dimethylphosphoryl, R =H;
2
3
3
4
5
6
3z-54z, 40; R =OCH , R =4-(methylsulfonyl)piperazin-1-yl, R =Cl, R =2-(dimethylphosphoryl, R =H;
2
3
3
4
5
6
'
3a -54a', 41; R =OCH , R =1,4'-bipiperidin]-1'-yl, R =Cl, R =2-(dimethylphosphoryl, R =H;
2
3
3
4
5
6
3b'-54b', 42; R =OCH , R =4-methylpiperazin-1-yl, R =Cl, R =2-(methylcarbamoyl, R =H;
2
3
3
4
5
6
3c'-54c', 43; R =OCH , R =4-isopropylpiperazin-1-yl, R =Cl, R =2-(methylcarbamoyl, R =H;
2
3
3
4
5
6
'
3d -54d', 44; R =OCH , R =4-acetylpiperidin-1-yl, R =Cl, R =2-(methylcarbamoyl, R =H;
2
3
3
4
5
6
3e'-54e', 45; R =OCH , R =4-(1-methylpiperidin-4-yl)piperazin-1-yl, R =Cl, R =2-(methylcarbamoyl R =H;
2
3
3
4
5
6
3f'-54f', 46; R =OCH , R =4-ethylpiperazin-1-yl, R =Cl, R =2-cyanophenyl, R =H;
2
3
3
4
5
6
3g'-54g', 47; R =OCH , R =4-isopropylpiperazin-1-yl, R =Cl, R =2-cyanophenyl, R =H;
2
3
3
4
5
6
3h'-54h', 48; R =OCH , R =4-morpholino, R =Cl, R =2-cyanophenyl, R =H;
2
3
3
4
5
6
3i'-54i', 49; R =OCH , R =4-(4-methylpiperazin-1-yl)piperidin-1-yl, R =Cl, R =2-cyanophenyl, R =H;
2
3
3
4
5
6

ACCEPTED MANUSCRIPT
a
o
Reagents and conditions: (a) for 51a, NaH, DMF, 0 C-rt, 12 h; for 51b-n, DIPEA,
propan-2-ol, reflux, 12 h; (b) for 52a-c and 52f-p, 4-fluoro-2-methoxy-5-nitroaniline; for
5
2d, 4-fluoro-3-nitroaniline; for 52e, 4-fluoro-2-isopropoxy-5-nitroaniline, PTSA, 2-
o
pentanol, 115 C, 5 h; (c) for 53a, 53i-j, 53l, 53x, 53a', 53i', piperidine derivatives; for
5
3b-c, 53e-g, 53k, 53m-w, 53z, 53b'-g', piperazine derivatives; for 53d, 1-methyl-1,4-
o
diazepane; for 53h, 53y, 53h', morpholine, DMF, 140 C, 2 h; (d) for 54a-v, Fe, NH Cl,
4
EtOH/H
2
2
O, reflux, 1 h; for 54w-i', SnCl , EtOH, reflux, 2 h; (e) acryloyl chloride,
o
DIPEA, DMF, 0 C, 0.5 h.
a
Scheme 2. Synthesis of compound 50
2
2
.2 Biological evaluation
.2.1 Structure-activity relationship (SAR) investigation
To test our design rationale, we first prepared compound 15 which bears an
acrylamide moiety at R to explore the antiproliferative potency against EGFR mutant
1
T790M driven H1975 cells. Compared with compound 14, 15 significantly increased the
activity against H1975 cells (GI50 = 0.023 µM) (Table 1). Moreover, 15 retained the
activity against EML4-ALK rearrangement driven NSCLC cell line H3122 (GI <0.0003
5
0
µM), which indicated that the molecular scaffold of 14 was suitable for developing ALK/
EGFR dual targets inhibitor.

ACCEPTED MANUSCRIPT
We then prepared a series of modifications at different positions of skeleton of 14 to
explore the SAR based on the anti-proliferative activity against EGFR and ALK driven
cell lines. We first focused on the R R and R moieties by fixing the R and R moieties.
2
,
3
4
5
6
Inhibitory activities (GI ) of these compounds against H1975 and H3122 cells were
5
0
shown in Table 1. The results indicated that when keeping R as methoxy group and R4
2
as –Cl, variation of R group as different piperazine-based derivatives (15-21) all led to
3
potent inhibitory activities against H1975 (GI50: 0.015-0.077 µM) and retained high
potency against H3122 cells (GI s <0.0003 µM except 0.033 µM for 17). In particular,
5
0
the methylated 1,4-diazepane at R
3
(18) exhibited the best activity to H1975 and H3122
cells (GI : 0.015 µM and <0.0003 µM respectively). Interestingly, installment of
5
0
morpholine (22) or piperidine (23) at R resulted in significant activity loss to H1975
3
cells. Keeping compound 14’s R group and removing the methoxy group at R (24) led
3
2
to significant activity loss against H3122 cells (GI50: 4.9 µM). Switching R
2
to a much
larger group (isopropoxyl) (25) also caused obvious activity loss to H1975 (GI = 0.30
5
0
µM) although it retained the inhibitory activity against H3122 (GI50 < 0.0003 µM).
Replacement of the -Cl atom with a methyl group (26, 27) remained good inhibitory
activities to both H1975 (GI = 0.027 µM, 0.043 µM) and H3122 (GI < 0.0003 µM,
5
0
50
0
.002 µM). However, removal of the -Cl atom (28) resulted in activity loss to both
H1975 (GI = 0.19 µM) and H3122 cells (GI = 0.041 µM).
5
0
50
a
Table 1. SAR Exploration Focused on the R
2
3 4
/R /R Moieties

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H1975
H3122
Compd.
R2
R3
R4
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-CH
-CH
-H
(
GI : ꢀM)
(GI : ꢀM)
50
50
1
1
1
1
1
2
2
2
2
2
2
2
2
2
5
6
7
8
9
0
1
2
3
4
5
6
7
8
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-H
3
3
3
3
3
3
3
3
3
0.023±0.0036
0.040±0.0012
0.043±0.0013
0.015±0.0004
0.025±0.0003
0.041±0.04
0.077±0.0047
1.2±0.03
＜0.0003
＜0.0003
0.033±0.0052
＜0.0003
＜0.0003
＜0.0003
＜0.0003
＜0.0003
0.24±0.0053
7.1±0.63
0.029±0.01
4.9±0.072
＜0.0003
-OCH(CH
)
3
0.30±0.011
2
-OCH
-OCH
-OCH
3
3
3
3
3
0.027±0.0021
0.043±0.0053
0.19±0.0058
＜0.0003
0.0020±0.0001
0.041±0.0043

ACCEPTED MANUSCRIPT
a
All GI50 values were obtained by triplet testing.
On the basis of these results, we decided to keep the R
and explore the R , R and R moieties (Table 2). When R
and R was the -H atom or -CH group, replacement of the isopropylsulfonyl benzene
with other aromatic rings or cyclopropane which lack the hydrogen bond acceptor (29-
5) all led to significant activity loss against both H1975 and H3122 cells. However,
2
3 4
(-CH O) and R (-Cl) moieties
3
5
6
3
was set as methyl piperazine
6
3
3
when switching the R moiety back to isopropylsulfonyl benzene (36) started to gain
5
back the activity against H1975 (GI50: 0.031 µM) and H3122 cells (GI50: 0.011 µM),
although compared to compound 15, it still lost 30-fold activity to H3122 cells. This
indicated that the hydrogen bond acceptor at R position is required for the EGFR and
5
ALK kinase activity. We then replaced the isopropylsulfonyl substituent with dimethyl
phosphine oxide and kept the –H atom at R to explore the R moiety. The results
6
3
demonstrated that with ethyl piperazine at R , 37 exhibited good anti-proliferative effect
3
against H1975 (GI50: 0.026 µM) and H3122 (GI50: 0.037 µM). However, switching this
moiety
to
N,N-dimethylpiperidin-4-amine
(38),
morpholine
(39)
or
methylsulfonylpiperazine (40) all led to significant activity loss to both H1975 and
H3122 cells. Interestingly, with the 1,4'-bipiperidine at R , 41 started to gain back the
3
activity to H1975 (GI50: 0.041 µM) and H3122 (GI50: 0.078 µM). We then changed the
isopropylsulfonyl substituent to N-methylacetamide to further explore the R moiety. The
3
data demonstrated that methyl piperazine (42) and isopropyl piperazine (43) exhibited
good activity to H1975 and H3122 cells. But acyl piperazine (44) and 1-(1-
methylpiperidin-4-yl)piperazine (45) both caused significant activity loss compared to

ACCEPTED MANUSCRIPT
compound 15. In addition, the efforts to switch the isopropylsulfonyl substituent to nitrile
group at R position (46-49) all resulted in obvious activity loss to both cell lines.
5
a
Table 2. SAR Exploration Focused on the R /R /R Moieties
3
5
6
Cl
R5
O
N
N
HN
R6
N
HN
R3
O
H1975
H3122
Compd.
R3
R5
R6
H
H
H
H
H
(
GI : ꢀM)
(GI : ꢀM)
50
50
2
3
3
3
3
9
0
1
2
3
0.26
±
0.0012
0.77±0.0006
0.10±0.0058
0.13±0.02
0.22±0.025
>10
0.30±0.017
2.9±0.052
0.27±0.02
3.2±0.0023
3
3
4
5
H
0.95
±
0.032
6.5±0.75
3.6±0.24
CH
1.8±0.11
3

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3
3
3
3
4
4
4
4
4
4
4
6
7
8
9
0
1
2
3
4
5
6
H
H
H
H
H
H
H
H
H
H
H
0.031
0.026
±
0.0012
0.0004
0.011±0.0078
0.037±0.0023
0.14±0.052
0.36±0.032
±
0.22±0.03
0.78
0.89
±
±
0.0051
0.0022
0.80
±
±
0.034
0.041
±0.0016
0.078
0.0031
0.050±0.006
0.0035±0.0001
0.0045±0.0003
0.079±0.006
0.25
±
0.0093
0.012±0.0023
0.12±0.003
0.44±0.07
0.018±0.0003
0.11±0.019

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CN
4
4
4
7
8
9
H
H
H
0.43±0.052
0.11±0.029
0.37±0.021
1.2±0.056
0.20
±
0.0024
0.098±0.0004
a
All GI50 values were obtained by triplet testing.
2
.2.2 EGFR and ALK on-target activity examination of compound 18
Since compound 18 exhibited the best anti-proliferative effects against H1975 and
H3122 cells, we then tested it in the enzymatic assays against purified proteins with
Invitrogen’s SelectScreen technology to confirm its on-target effect (Table 3). The results
showed that 18 was highly potent against EGFR drug resistant mutants T790M (IC : 3.9
5
0
nM) and L858R/T790M (IC : 3.6 nM) meanwhile displayed 20-30 fold selectivity over
5
0
EGFR gain-of-function mutant L858R (IC : 82.5 nM) and EGFR wt (IC : 108 nM). In
5
0
50
addition, it exhibited IC values less than 1 nM against ALK mutants including ALK
5
0
R1275Q, L1196M, F1174L and C1156Y and an IC of 9.8 nM against ALK wt. In order
50
to further confirm these on-target effects, we then tested 18 in a panel of EGFR/ALK
transformed BaF3 cells which grow dependently on the EGFR or ALK mutants. It
showed that 18 was highly potent to EGFR wt (GI : 0.016 µM) and exhibited about 15-
5
0
fold selectivity against EGFR-wt-C797S (GI50: 0.23 µM) (Table 4). Similar trends were
observed for EGFR L858R/L858R-T790M/L858R-T790M-C797S and EGFR del19
transformed BaF3 cells. In addition, compound 50 in which the acrylamide at R was
1

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replaced with propyl amide that lacks the covalent bond formation capability lost the
activities against EGFR wt, L858R and del19 significantly compared to compound 18.
These results indicated that 18 inhibited EGFR and mutants with an irreversible binding
mode through formation of a covalent bond with C797 residue, like the canonical EGFR
irreversible inhibitor compound 5. Furthermore, 18 could inhibit the proliferation of
EGFR T790M and L858R-T790M mutants transformed BaF3 cells, which further
confirmed its capability to overcome the drug-resistant gatekeeper mutations as the
strong anti-proliferative efficacy observed in H1975 cells. Compound 18 also displayed
potent inhibitory activities against NPM and EML4 rearranged ALK transformed BaF3
cells (Table 5). More importantly, it could overcome compound 9’s drug-resistant
mutants including ALK F1174L, C1156Y and L1196M. However, for the ALK G1202R
mutant, neither 18 nor 9 exhibited good inhibitory activity.
Table 3. Compound 18’s Enzymatic Activity against EGFR/ALK and Associated
a
Mutants
Target
Compd. 18 (IC50: nM)
108±1.8
EGFR wt
EGFR L858R
EGFR T790M
EGFR L858R/T790M
ALK wt
82.5±1.5
3.9±0.2
3.6±0.2
9.8±0.3
ALK R1275Q
ALKL 1196M
ALK F1174L
0.82±0.04
0.59±0.02
0.92±0.22

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ALK C1156Y
1.0±0.04
a
All GI values were obtained by triple testing.
5
0
Table 4. Compound 18’s Antiproliferative Effect against a Panel of EGFR/mutants
a
Transformed BaF3 Cell Lines
Compd. 18
µM
Compd. 50
µM
Compd. 5
µM)
Cell Line
(
GI50
:
)
(GI50
:
)
(GI50
:
Parental BaF3
1.2
0.016
0.23
±
0.056
0.0003
0.026
7.1
1.5
±
±
0.077
2.2
1.3
±
±
0.047
0.034
BaF3-TEL-EGFR
±
0.033
BaF3-TEL-EGFR-C797S
BaF3-TEL-EGFR-T790M
BaF3-FL-EGFR-del19
BaF3-FL-EGFR-L858R
BaF3-FL-EGFR-L858R-T790M
±
0.36
±
±
±
±
±
0.0057 0.89
±
0.032
0.0080
0.0085
±
±
0.0004 0.80
0.0005 0.27
0.37
0.049
0.0042
0.04
0.0021
±
0.0003
0.0003
0.0003
0.0002
＜
＜
＜
＜
0.0003
0.0003
＜
0.29
0.042
BaF3-FL-EGFR-L858R-T790M-C797S 1.1
±
0.048
1.7
±0.12
2.2±0.12
a
All GI50 values were obtained by triple testing.
Table 5. Compound 18’s Antiproliferative Effect against a Panel of ALK/mutants
a
Transformed BaF3 Cell Lines
Compd. 18
Compd. 9
Cell Line
(
GI : µM
)
(GI : µM
)
5
0
50
Parental BaF3
1.2
±
0.056
1.1±0.05
BaF3-NPM-ALK
BaF3-EML4-ALK
0.001
±
0.0001
＜
＜
0.0003
0.0003
＜
0.0003

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BaF3-TEL-ALK
＜
0.0003
0.029 0.0031
0.0069 0.0009
0.0003
<0.0003
BaF3-FL-ALK-F1174L
BaF3-TEL-ALK-C1156Y
BaF3-TEL-ALK-L1196M
±
0.32
0.15
0.59
0.43
±
±
±
±
0.026
0.0086
0.042
0.03
±
＜
BaF3-TEL-ALK-G1202R
0.46±0.04
a
All GI values were obtained by triple testing.
5
0
2
.2.3 Compound 18’s binding mode examination
In order to better understand compound 18’s binding mechanism we then docked it
into EGFR T790M (PDB ID: 3IKA) and ALK (PDB ID: 2XB7). The modeling results
showed that in EGFR kinase, 18 formed two hydrogen bonds in the hinge binding area
with Met793 (Figure 4A). The acrylamide at R formed a covalent bond with Cys797
1
residue, and the diazepane at R formed a hydrogen bond with Asp800 residue adjacent
3
to the hinge binding area. Meanwhile, the –Cl at R formed a halogen bond with Met790,
4
which strengthened the binding. This could explain that 18 was much more potent against
EGFR T790M mutant than EGFR wt and EGFR L858R in the biochemical enzymatic
examination (IC ) because the latter two kinases lacked this halogen bond. This is
5
0
12
similar to the canonical EGFR T790M mutant selective inhibitor 7. In addition, the
isopropylsulfonyl group formed a hydrogen bond with Lys745, which could explain why
compounds 29-35 lost activities significantly and the hydrogen bond acceptor at this
position was required. Similarly, in ALK kinase 18 formed two hydrogen bonds with
Met1199 in the hinge binding area (Figure 4B). The acrylamide at R
1
formed a hydrogen
bond with Asp1203, which further strengthened the binding. Also, the isopropylsulfonyl

ACCEPTED MANUSCRIPT
group formed a hydrogen bond with Lys1150, which explained why compounds 29-35
lost activities to ALK.
In addition, we performed the washing out experiment in H1975 and H3122 cells to
examine the phosphorylation of EGFR and ALK kinases. The results showed that 18
exhibited time- and dose-dependent inhibition of the pEGFR Y1068 site (Figure 4C). At
the concentration of 300 nM, even after 24 h washing out the inhibitor, the
phosphorylation level still remained inhibited, which indicated that 18 indeed worked
with irreversible binding mode in the cellular context. However, in H3122 cell, after the
washing out, pALK Y1604 recovered immediately, which indicated that it adopted a
reversible binding mode (Figure 4D). This is consistent with the structure analysis
because there is no approachable cysteine residue by the acrylamide.
Figure 4. Binding mode characterization of compound 18 on EGFR and ALK kinases.
(A) Docking of 18 into X-ray crystal structure of EGFR T790M (PDB ID: 3IKA). (B)

ACCEPTED MANUSCRIPT
Docking of 18 into X-ray crystal structure of ALK kinase (PDB ID: 2XB7). (C) Washing
out experiment of 18 in H1975 cell for the phosphorylation of EGFR kinase at different
concentrations and time points. (D) Washing out experiment of 18 in H3122 cell for the
phosphorylation of ALK kinase at different concentrations and time points.
2
.2.4 Compound 18’s antiproliferative effects on a panel of EGFR and ALK driven
NSCLC cell lines
We next evaluated compound 18’s anti-proliferative effects in the EGFR and ALK
driven NSCLC cell lines (Table 6). The data showed that besides H1975 cell (EGFR
L858R/T790M) and H3122 cell (EML4-ALK), 18 also potently inhibited the
proliferation of PC-9 (EGFR del 19) (GI : 0.015 µM), HCC827 (EGFR del 19) (GI :
5
0
50
0
.0062 µM), H3255 (EGFR L858R) (GI : 0.031 µM) as well as H2228 (EML4-ALK)
50
cells (GI : 0.028 µM). It displayed much less anti-proliferative inhibitory activities
5
0
against EGFR wt expressing cells including A549, H2122 and H1355 cells. In addition,
8 exhibited good selectivity against Chinese hamster ovarian cells CHO (GI : 1.8 µM)
1
5
0
and CHL (GI50: 3.5 µM), which indicated no apparent general cytotoxicity. EGFR
inhibitor compound 5 and ALK inhibitor compound 9 displayed similar trends in the
EGFR driven and ALK driven cell lines respectively. Furthermore, 18 exhibited better
antiproliferative efficacy than 5 in the EGFR mutant driven cell lines and 9 in the ALK
rearrangement driven cell lines. Meanwhile, in the EGFR mutant driven cell lines,
compound 18 was much more potent than its reversible version compound 50, which
further confirmed that the irreversible binding effect of 18 was biologically relevant.
Table 6. Compound 18’s Antiproliferative Effects against a Panel of EGFR/ALK
a
mutants Driven Cell Lines

ACCEPTED MANUSCRIPT
Compd. 18
Compd. 50
Compd. 5
M)
Compd. 9
Cell Line
H1975
(
GI :
µM)
(GI : M) (GI50:
µ
µ
(GI :
µM)
5
0
50
50
0
.015±0.0003 0.7±0.0055 0.056±0.003
0.34±0.042 0.027±0.001
/
/
(
EGFR L858R/T790M)
PC-9 (EGFR del19)
HCC827 (EGFR del19)
H3255 (EGFR L858R)
A549 (EGFR wt)
0.015±0.003
0.0062±0.0005 2.6±0.51
0.0074±0.002 /
0.031±0.0059 0.83±0.0072 0.39±0.034
/
/
/
/
5.6±0.59
1.8±0.02
1.4±0.035
1.2±0.024
3.3±0.28
>10
H2122 (EGFR wt)
5.7±0.35
H1355 (EGFR wt)
2.2±0.0064 5.3±0.19
H3122(EML4-ALK varian1)
＜
0.0003
＜
0.0003
/
0.037±0.003
0.073±0.002
3.2±0.065
H2228 (EML4-ALK varian3) 0.028±0.005
0.1±0.0065
3.6±0.19
1.3±0.1
/
CHO
CHL
1.8±0.032
3.5±0.41
3.9±0.0041
4.4±0.0046
0.45±0.019
a
All GI values were obtained by triple testing.
5
0
2
.2.5 Compound 18’s cellular effects on the EGFR/ALK mutants driven NSCLC cell
lines
We next examined the effects of compound 18 on the EGFR/ALK mediated signaling
pathways. In H1975 and PC9 cells, at the concentration of 30 nM compound 18 could
completely inhibit the phosphorylation of EGFR Y1068 and subsequently block the
downstream mediator ERK Thr202/204 phosphorylation (Figure 5A). As expected, in the
EGFR wt expressing NSCLC cell line A549, only pEGFR started to be affected at the
concentration of 300 nM and none of the downstream mediators was inhibited. In EML4-
ALK rearranged H3122 cells, 18 almost completely blocked the phosphorylation of ALK

ACCEPTED MANUSCRIPT
Y1064 at the concentration of 100 nM, and also affected the phosphorylation of
downstream mediators such as AKT and ERK. In addition, compound 18 could
effectively arrest H1975, PC9 and H3122 cell cycle into G0/G1 phase at 24 h but did not
affect A549 cell cycle progression (Figure 5B). In H1975, PC9 and H3122 cells,
compound 18 could start to induce the apoptosis by examining the cleaved PARP and
Caspase-3 from 300 nM concentration upon 24 h treatment. But in A549 cells, it did not
exhibited apoptosis induction effect up to 1 µM concentration (Figure 5C). These results
indicated that 18’s biological effect was indeed from EGFR/ALK on-target inhibition.

ACCEPTED MANUSCRIPT
Figure 5. Compound 18’s effects in the cellular context in the EGFR/ALK driven
NSCLC cell lines. (A) 18’s effects on the EGFR and ALK mediated signaling pathways
in H1975, PC9, A549 and H3122 cells. (B) 18’s effects on cell cycle progression in
H1975, PC9, A549 and H3122 cells. (C) 18’s effects on apoptosis in H1975, PC9, A549
and H3122 cells.
2
.2.6 In vivo pharmacokinetic properties of compound 18
We then evaluated compound 18’s PK properties in rats following intravenous
injection (I.V.) and oral administration (P.O.) (Table 7). The half-life of 18 was about 1 h
through I.V. and 1.75 h through P.O.. The AUC0-t was about 1140 ng/mL*h via P.O..
These data indicated that 18 was acceptable for oral treatment in the animal model study.
Table 7. In Vivo PK Parameters of Compound 18
Parameter
I.V. (1 mg/kg)
707.336±49.93
720.424±42.218
0.947±0.143
1488.219±377.84
0.017±0
P.O. (10 mg/kg)
1141.84±146.199
1296.403±223.56
4.118±0.33
AUC0-t (ng/mL*h)
AUC0-∞ (ng/mL*h)
MRT(0-t) (h)
Cmax (ng/mL)
Tmax (h)
282.805±28.356
4±0
T
1/2 (h)
0.991±0.14
—
1.747±0.211
16.14%
F (%)
2
.2.7 In vivo antitumor efficacy of compound 18

ACCEPTED MANUSCRIPT
We next tested compound 18’s antitumor efficacy in EGFR mutant driven H1975
cell inoculated mouse xenograft model. The results showed that at dosages of 10
mg/kg/day, 20 mg/kg/day and 40 mg/kg/day, 18 did not affect the body weights which
indicated that it has no obvious cytotoxicity (Figure 6A). 18 could dose dependently
suppress tumor progression and almost completely suppress tumor growth at 20 mg/kg
QD oral dosage with the tumor growth inhibition (TGI) of 96% (Figure 6B-D). At 40
mg/kg/day dosage a TGI of 99% was achieved. Immunohistochemistry (IHC) stain
showed that 18 could dose dependently inhibit the proliferation of tumor cells (Ki-67
stain) and induce tumor cell apoptosis (TUNEL stain) in tumor tissues (Figure 6E).
Similarly, in the ALK rearrangement driven H3122 cell inoculated mouse xenograft
model, 18 did not affect the animal body weights and it could dose dependently suppress
tumor progression (Figure 7A-D). At 40 mg/kg/day oral dosage 18 achieved the TGI of
7
8.4%. IHC stain also demonstrated the dose dependent anti-proliferation and apoptosis
induction in tumor tissues (Figure 7E).

ACCEPTED MANUSCRIPT
Figure 6. Compound 18’s antitumor efficacy in H1975 xenograft mouse model. Female
nu/nu mice bearing established H1975 tumor xenografts were treated with 18 at 10.0,
2
0.0 and 40.0 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

ACCEPTED MANUSCRIPT
H1975 xenograft mice after 21 days 18 administration. Initial body weight and tumor size
were set as 100%. (C) Representative photographs of tumors in each group after 0, 10.0,
2
0.0, 40.0 mg/kg/d 18 or vehicle treatment. (D) Comparison of the final tumor weights in
each group after 21-day treatment period of 18. 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), Ki-67, and TUNEL staining of tumor tissues
with 18 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).

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Figure 7. Compound 18’s antitumor efficacy in H3122 xenograft mouse model. Female
nu/nu mice bearing established H3122 tumor xenografts were treated with 18 at 10.0,
2
0.0 and 40.0 mg/kg/d dosage or vehicle. Daily oral administration was initiated when
3
H3122 tumors had reached a size of 200 to 400 mm . Each group contained seven