Discovery of N-[4-(Quinolin-4-yloxy)phenyl]benzenesulfonamides as Novel AXL Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00672

The overexpression of AXL kinase has been described in many types of cancer. Due to its role in proliferation, survival, migration, and resistance, AXL represents a promising target in the treatment of the disease. In this study we present a novel compound family that successfully targets the AXL kinase. Through optimization and detailed SAR studies we developed low nanomolar inhibitors, and after further biological characterization we identified a potent AXL kinase inhibitor with favorable pharmacokinetic profile. The antitumor activity was determined in xenograft models, and the lead compounds reduced the tumor size by 40% with no observed toxicity as well as lung metastasis formation by 66% when compared to vehicle control.

Full text of DOI:10.1021/acs.jmedchem.8b00672

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES
Article
Discovery of N-[4-(quinolin-4-yloxy)-phenyl]-
benzenesulfonamides as novel AXL kinase inhibitors
István Szabadkai, Robert Torka, Rita Garamvölgyi, Ferenc Baska, Pál Gyulavári,
Sándor Boros, Eszter Illyés, Axel Choidas, Axel Ullrich, and László #rfi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00672 • Publication Date (Web): 21 Jun 2018
Downloaded from http://pubs.acs.org on June 21, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of N-[4-(quinolin-4-yloxy)-phenyl]-benzenesulfonamides as novel
AXL kinase inhibitors
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
†
,┴
#,┴
†,∞
†
#
István Szabadkai , Robert Torka , Rita Garamvölgyi , Ferenc Baska , Pál Gyulavári ,
†
†
∫
‡
*,†,∞
Sándor Boros , Eszter Illyés , Axel Choidas , Axel Ullrich , László Őrfi
†
Vichem Chemie Research Ltd., Budapest, Hungary
#
Institute of Physiological Chemistry, University HalleꢀWittenberg, Halle (Saale), Germany
Department of Molecular Biology, Max Planck Institute of Biochemistry, Martinsried,
‡
Germany
∫
Lead Discovery Center GmbH, Dortmund, Germany
∞
Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary
ABSTRACT
The overexpression of AXL kinase has been described in many types of cancer. Due to its
role in proliferation, survival, migration and resistance AXL represents a promising target in
the treatment of the disease. In this study we present a novel compound family which
successfully targets the AXL kinase. Through optimization and detailed SAR studies we
developed low nanomolar inhibitors and after further biological characterization we identified
a potent AXL kinase inhibitor with favorable pharmacokinetic profile. The antitumor activity
was determined in xenograft models and the lead compounds reduced the tumor size by 40%
with no observed toxicity as well as lung metastasis formation by 66% when compared to
vehicle control.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 55
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
KEYWORDS
AXL kinase, inhibitor, metastasis, antitumor agents, cancer, Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀ
benzenesulfonamides.
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
INTRODUCTION
Receptor tyrosine kinases (RTKs) are transmembrane proteins, linking the extraꢀ and
intracellular environment. As mediators of signal transduction, they play a major role in
normal cellular processes, including differentiation, proliferation, adhesion, migration,
apoptosis and metabolism.
A subfamily of mammalian RTKs is called TAM family, including AXL, MER and TYROꢀ3.
1
AXL was originally identified in chronic myelogenous leukemia (CML) and chronic
2
myeloproliferative disorder. Subsequently, its overexpression has been reported in many
human cancers, comprising breast, lung, prostate, colon, esophageal, liver and other, which
3
have been reviewed by Paccez et al.
4
AXL can primarily be stimulated by Gas6 (Growth arrestꢀspecific 6). As a result of the
activation of AXL kinase, various signaling pathways can be activated, such as
5
6
phosphoinositide 3ꢀkinase (PI3K), including AKT, S6K or NFꢀκB as downstream targets,
7
mitogenꢀactivated protein kinase (MAPK) pathway or signal transducer and activator of
8
transcription (STAT). Aberrant regulation of Gas6/AXL signaling contribute to cell
9
proliferation, increased cell survival, migration and angiogenesis, therefore they play an
important role in oncogenic transformation, metastasis and tumor growth. AXL may be a
10
biomarker of resistance in targeted therapyꢀresistant cancers.
According to its central role in neoplastic processes, the AXL signaling pathway represents a
notable molecular therapeutic target in the fight against cancer. A number of agents inhibiting
2
ACS Paragon Plus Environment

Page 3 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the biological activity of AXL has been described in the literature (Figure 1), e.g. amuvatinib
1
1
12
13
14
(MPꢀ470) (1), bosutinib (SKIꢀ606) (2), foretinib (XL880) (3), BMSꢀ777607 (4),
1
5
16
cabozantinib (XL184) (5) or S49076 (6). However, most of these small molecule kinase
inhibitors were developed against other targets, such as cKIT, SRC/ABL, cꢀMET or VEGFRꢀ
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
2. The first selective inhibitor developed purposely to target AXL kinase was compound R428
17
(7).
O
O
H
N
H
N
O
O
Cl
Cl
O
F
H
N
F
N
O
O
HN
S
HN
O
F
N
N
O
N
N
O
O
O
O
O
F
N
Cl
O
N
N
N
N
N
O
H2N
N
1 Amuvatinib (MPꢀ470)
2 Bosutinib (SKIꢀ606)
3 Foretinib (XL880)
4 BMSꢀ777607
O
N
H
N
H
N
O
O
O
F
N
O
N
O
N
H
N
NH2
N
O
N
N
N
S
O
N
O
N
H
NH
5
Cabozatinib (XLꢀ184)
6 S49076
7 R428 (BGB 324)
Figure 1. Structures of reported smallꢀmolecule tyrosine kinase inhibitors exhibiting activity
against AXL kinase
TM
18
We have applied Vichem's Nested Chemical Library
technology to discover hit
compounds against AXL kinase. As we previously reported, bosutinib (2) was found to inhibit
1
2
the AXL kinase activity in a dose dependent manner, with an IC50 of 0.56 µM. The 4ꢀ
19
phenoxyquinoline derivative (Figure 2) Ki8751 (8) was originally developed by Kubo et al.
as a VEGFRꢀ2 tyrosine kinase inhibitor. Since structural similarity can be found between
bosutinib (2) and Ki8751 (8), it was practical to determine the AXL kinase inhibitory effect of
Ki8751 (8), which was found to be even better (IC50 = 0.30 µM) than that of bosutinib (2).
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 55
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
F
F
H
N
H
N
O
O
N
F
O
O
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
8
Ki8751
Figure 2. Structure of an NꢀphenylꢀN’ꢀ{4ꢀ(4ꢀquinolyloxy)phenyl}urea derivative
In this manuscript, we present a novel Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamide
series we have designed to target AXL kinase, describe their synthesis, biological activity and
structureꢀactivity relationships.
Chemical Synthesis
(All chemical synthesis procedures are described in detail in the Supporting Information.)
The Nꢀ[4ꢀ(6,7ꢀdimethoxyꢀquinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamide derivatives (12aꢀ
1
2u) were prepared according to the general synthetic route shown in Scheme 1.
Scheme 1. Synthesis of Nꢀ[4ꢀ(6,7ꢀdimethoxyꢀquinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamide
a
derivatives
R1
R3
O
R3
R3
R4
N+
R4
H
N
NH
2
Oꢀ
R4
O
S
R2
O
O
Cl
N
O
O
O
O
O
O
O
O
a
b
c
O
O
N
N
N
9
10a-10g
11a-11g
12a-12u
a
Reagents and conditions: (a) 4ꢀnitrophenols, chlorobenzene, DIPEA, reflux, 2 days; (b) Zn,
NH
4
Cl, EtOH/H
2
O, reflux, 3 hours; (c) benzenesulfonyl chlorides, pyridine, rt, 2 days.
4
ACS Paragon Plus Environment

Page 5 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The starting material (4ꢀchloroꢀ6,7ꢀdimethoxyꢀquinoline, 9) was commercially available. This
compound was coupled with various 4ꢀnitrophenols by heating them in chlorobenzene, in the
presence of N,Nꢀdiisopropylethylamine (DIPEA). The isolated 4ꢀ(4ꢀnitroꢀphenoxy)ꢀquinoline
compounds (10aꢀ10g) were reduced by zinc powder and ammonium chloride in ethanol/water
mixture at reflux temperature. The coupling reaction of the anilines (11aꢀ11g) with
benzenesulfonyl chlorides in pyridine provided the corresponding sulfonamide derivatives
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
(12aꢀ12u).
In order to introduce alkoxy side chains containing aminoꢀ or ester groups into the positions 6
and 7 of the quinoline ring, required the protection of the hydroxyl group. The general
synthetic route for the preparation of these compounds is shown in Scheme 2.
Scheme 2. Synthesis of Nꢀ[4ꢀ(substitutedꢀquinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamide
7
a
derivatives bearing a side chain (R )
O
N+
O
N+
O
N+
F
F
Oꢀ
F
Oꢀ
R5 = H or OCH
R5
Oꢀ
3
Cl
N
O
O
O
R5
R5
R5
a
b
c
O
O
HO
O
R7
N
N
N
1
3a-d
14a-14d
15a-15d
16a-16o
R1
R2
f
R1
H
H
N
F
NH
2
F
N
F
S
S
R2
O
O
O
O
O
R5
O
O
d
e
R5
O
O
R7
O
R7
N
N
2
H N
O
N
17a-17o
18a-18d, 19a-19n
HCl
20a-20k, 21a-21k
20l, 21l
a
Reagents and conditions: (a) 4ꢀnitrophenols, chlorobenzene, reflux, 2 days; (b) 33% HBr
solution, rt, 2 hours; (c) alkyl halogenides, acetonitrile, Cs CO , KI, rt, 45 min, then reflux 3
hours; (d) Zn, NH Cl, EtOH/H O, reflux, 3 hours; (e) benzenesulfonyl chlorides, pyridine, 60
2
3
4
2
°C, 3 days, (f) transformation of 20k and 21k, HCl, ethyl acetate, rt, 3 hours.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 55
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
The starting materials (4ꢀchloroꢀbenzyloxyꢀquinoline derivatives, 13a-13d) were available
from commercial sources. These benzyloxyꢀ4ꢀchloroquinoline derivatives (13a-13d) were
coupled with 2ꢀfluoroꢀ4ꢀnitrophenol by heating them in chlorobenzene in the presence of N,Nꢀ
diisopropylethylamine. The benzyloxy protective group was removed from the isolated 4ꢀ(4ꢀ
nitroꢀphenoxy)ꢀquinoline compounds (14aꢀ14d) by hydrobromic acid in acetic acid. The
obtained hydroxyꢀquinolines (15a-15d) were alkylated by the proper alkyl halogenides
resulting in the appropriate alkoxy derivatives (16a-16o). The reduction of the nitro groups of
these compounds was carried out by zinc powder and ammonium chloride in ethanol/water
mixture at reflux temperature, whereas the coupling of (17aꢀ17o) with benzenesulfonyl
chlorides in pyridine gave the desired sulfonamides (18aꢀ18d, 19aꢀ19n, 20aꢀ20k and 21aꢀ
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
21k) as final products. In the case of 20k and 21k, after removing the tertꢀbutoxycarbonyl
(BOC) protecting group we isolated the aminopropoxy derivatives as hydrochloride salt (20l
and 21l).
RESULTS AND DISCUSSION
The compounds prepared for this study were primarily evaluated by in vitro AXL kinase
assay. In order to determine the structureꢀactivity relationships, the following moieties were
1
2
changed: substituents of the benzenesulfonamide ring (R , R ), substituents of the pꢀ
3
4
5
6
aminophenol ring (R , R ) and substituents of the quinoline ring (R , R ) as shown in Figure
3.
6
ACS Paragon Plus Environment

Page 7 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R1
R3
H
R4
O
N
S
R2
O
O
R5
R6
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
N
Figure 3. General structure of the prepared Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀ
benzenesulfonamide series
1
2
First, the effect of the benzenesulfonamide substituents (R , R ) was evaluated as shown in
Table 1, while the substituents of the quinoline and the pꢀaminophenol ring remained
unchanged, identical to those in Ki8751 (8) (Figure 2). The analogous 2,4ꢀdifluoro derivative
(12h) (IC50 = 6.400 µM) showed significantly weaker inhibitory potency against AXL than
Ki8751 (8) (IC50 = 0.300 µM). However, we have discovered a number of compounds having
submicromolar IC50, from which 2ꢀCF
3 3
(12c) and 2ꢀOCF (12f) groups showed the best
results in this set (0.360 µM and 0.300 µM, respectively).
Table 1. Inhibitory activities of Nꢀ[4ꢀ(6,7ꢀdimethoxyꢀquinolinꢀ4ꢀyloxy)ꢀ2ꢀfluoroꢀphenyl]ꢀ
benzenesulfonamide derivatives
R1
F
H
N
S
R2
O
O
O
N
O
O
1
2
a
No
R
R
AXL IC50 (µM)
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 55
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
2a
12b
2c
2d
2e
2f
2g
12h
H
H
H
2.600
2.350
0.360
6.000
>3.000
0.300
>1.000
6.400
0.840
>1.000
3.010
>1.000
1.610
1.770
>1.000
2ꢀCH
3
1
2ꢀCF
3
H
1
3ꢀCF
3
H
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
1
2ꢀOCH
3
H
1
2ꢀOCF
3
H
1
2ꢀF
H
2ꢀF
4ꢀF
5ꢀF
6ꢀF
H
1
2i
2j
2ꢀF
1
2ꢀF
1
2k
2l
2m
3ꢀF
1
2ꢀCl
2ꢀCl
2ꢀCl
3ꢀCl
H
1
5ꢀCl
6ꢀCl
4ꢀF
12n
12o
a
IC values are average of at least two independent experiments
5
0
3
4
Optimization of substituents of the pꢀaminophenol ring revealed, that the R and R
substituents (see Fig.3) can strongly influence the kinase inhibitory activity (Table 2.).
Introducing a methyl group in either one of these positions (12q, 12r) significantly reduced
the AXL inhibitory effect. Surprisingly, in the case of methoxy and fluoro moieties we
3
observed a diverse effect. In R position these substituents did not cause considerable changes
4
(
12s, 0.289 µM; 12c, 0.360 µM), however, in R position the methoxy group reduced the
effect (12t, 1.960 µM), while the fluorine substituent unexpectedly increased the AXL
inhibitory effect (12u, 0.077 µM).
8
ACS Paragon Plus Environment

Page 9 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 2. Inhibitory activities of Nꢀ[4ꢀ(6,7ꢀdimethoxyꢀquinolinꢀ4ꢀyloxy)ꢀ2ꢀsubtitutedꢀphenyl]ꢀ
2ꢀtrifluoromethylꢀbenzenesulfonamide derivatives
R3
H
N
R4
S
O
O
F
F
F
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
O
R5
R6
N
3
4
a
No.
R
R
AXL IC50 (µM)
1
2p
2q
H
H
H
0.350
>3.000
2.250
0.289
1.960
0.360
0.077
1
CH
3
1
2r
2s
H
CH
3
1
CH
3
O
H
12t
12c
H
CH
3
O
F
H
12u
H
F
a
IC50 values are average of at least two independent experiments
5
6
In the next step we explored the effect of R and R substituents and how interchangeable are
they (Table 3). To evaluate the effect of bosutinib’s (2) side chain, we introduced the 3ꢀ(4ꢀ
5
6
methylꢀpiperazinꢀ1ꢀyl)ꢀpropoxy moiety into R or R position. We have discovered that
6
compounds featuring this side chain in R position (18b, 18d) were significantly more potent
5
than those in R position (18a, 18c). We have also found, that a methoxy substituent next to
the 3ꢀ(4ꢀmethylꢀpiperazinꢀ1ꢀyl)ꢀpropoxy moiety (18a and 18c; 18b and 18d) increased the
inhibitory potency against AXL kinase. As a result, 18d was identified as the most potent
compound in this set with an IC50 of 0.021 µM.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 55
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
Table 3. Inhibitory activities of Nꢀ(3ꢀfluoroꢀ4ꢀ{6,7ꢀsubstitutedꢀquinolinꢀ4ꢀyloxy}ꢀphenyl)ꢀ2ꢀ
trifluoromethylꢀbenzenesulfonamide derivatives
H
N
F
S
O
O
F
F
F
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
O
R5
R6
N
5
3
6
3
a
No.
R
R
AXL IC50 (µM)
1
2u
8a
CH
O
CH
O
O
0.077
0.910
N
O
1
H
N
N
O
18b
8c
H
0.093
0.101
0.021
N
N
O
1
CH
3
N
N
O
18d
CH
3
O
N
a
IC50 values are average of at least two independent experiments
5
6
Having selected the optimal substituent combinations on the quinoline (R , R ) and pꢀ
3
4
1
2
aminophenol ring (R , R ), we aimed to reꢀevaluate the role of the R , R substituents as
shown in Table 4. Based on this comparison, in the group the 2ꢀCF moiety bearing (18d)
remained to be the most active with 0.021 µM IC50. Furthermore, the following substituents
were confirmed to express similar efficacy against AXL kinase: 2ꢀOCF (19e), 2,5ꢀdiF (19h),
,6ꢀdiF (19i).
3
3
2
Table 4. Inhibitory activities of Nꢀ(3ꢀfluoroꢀ4ꢀ{6ꢀmethoxyꢀ7ꢀ[3ꢀ(4ꢀmethylꢀpiperazinꢀ1ꢀyl)ꢀ
propoxy]ꢀquinolinꢀ4ꢀyloxy}ꢀphenyl)ꢀbenzenesulfonamide derivatives
1
0
ACS Paragon Plus Environment

Page 11 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R1
H
N
S
F
R2
O
O
O
O
O
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
N
N
N
1
2
a
No.
18d
9a
9b
9c
9d
9e
19f
9g
9h
R
R
AXL IC50 (µM)
2ꢀCF
3
H
H
0.021
0.218
0.430
0.354
1.434
0.051
0.121
0.380
0.027
0.035
0.388
0.184
0.201
0.068
0.202
1
H
1
2ꢀCH
3ꢀCF
2ꢀOCH
3
H
1
3
H
1
3
H
1
2ꢀOCF
3
H
2ꢀF
H
1
2ꢀF
4ꢀF
5ꢀF
6ꢀF
H
1
2ꢀF
1
9i
2ꢀF
1
9j
3ꢀF
1
9k
19l
9m
9n
2ꢀCl
2ꢀCl
2ꢀCl
3ꢀCl
H
5ꢀCl
6ꢀCl
4ꢀF
1
1
a
IC50 values are average of at least two independent experiments
6
Finally, our attention was focused on the R substituent. Two series of derivatives were
prepared bearing 2ꢀtrifluoromethylbenzenesulfonamide (18d, 20aꢀ20l) or 2,5ꢀ
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 55
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
difluorobenzenesulfonamide (19h, 21aꢀ21l) moiety to validate the effect of the side chain.
The results are summarized in Table 5 and Table 6. As a result of our optimization process we
were able to identify numerous submicromolar AXL kinase inhibitors in this compound class.
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
Table 5. Inhibitory activities of Nꢀ(3ꢀfluoroꢀ4ꢀ{6ꢀmethoxyꢀquinolinꢀ4ꢀyloxy}ꢀphenyl)ꢀ2ꢀ
trifluoromethylꢀbenzenesulfonamide derivatives
H
N
F
S
O
O
F
F
F
O
O
R7
O
N
7
a
No.
R
AXL IC50 (µM)
18d
20a
3ꢀ(4ꢀmethylꢀpiperazinꢀ1ꢀyl)ꢀpropylꢀ
2ꢀ(morpholinꢀ4ꢀyl)ꢀethylꢀ
0.021
0.054
0.021
0.021
0.018
0.070
0.031
0.046
0.046
0.796
0.027
0.170
0.238
2
0b
0c
0d
0e
0f
20g
3ꢀ(morpholinꢀ4ꢀyl)ꢀpropylꢀ
4ꢀ(morpholinꢀ4ꢀyl)ꢀbutylꢀ
2
2
3ꢀ(piperidinꢀ1ꢀyl)ꢀpropylꢀ
2
3ꢀ(2ꢀmethylꢀpiperidinꢀ1ꢀyl)ꢀpropylꢀ
3ꢀ(3ꢀmethylꢀpiperidinꢀ1ꢀyl)ꢀpropylꢀ
3ꢀ(4ꢀmethylꢀpiperidinꢀ1ꢀyl)ꢀpropylꢀ
4ꢀacetoxyꢀbutylꢀ
2
20h
20i
dimethylcarbamoylmethylꢀ
3ꢀ(dimethylamino)ꢀpropylꢀ
3ꢀ(tertꢀbutoxycarbonylamino)ꢀpropylꢀ
3ꢀaminoꢀpropylꢀ
20j
20k
20l*
a
IC values are average of at least two independent experiments, *HCl salt
50
12
ACS Paragon Plus Environment

Page 13 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 6. Inhibitory activities of Nꢀ(3ꢀfluoroꢀ4ꢀ{6ꢀmethoxyꢀquinolinꢀ4ꢀyloxy}ꢀphenyl)ꢀ2,5ꢀ
difluoroꢀbenzenesulfonamide derivatives
F
H
N
F
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
S
F
O
O
O
O
O
R7
N
7
a
No.
R
AXL IC50 (µM)
1
9h
1a
1b
1c
1d
21e
1f
1g
1h
3ꢀ(4ꢀmethylꢀpiperazinꢀ1ꢀyl)ꢀpropylꢀ
2ꢀ(morpholinꢀ4ꢀyl)ꢀethylꢀ
0.053
0.052
0.035
0.027
0.072
0.050
0.028
0.016
0.091
>1.000
0.036
0.120
0.636
2
2
3ꢀ(morpholinꢀ4ꢀyl)ꢀpropylꢀ
4ꢀ(morpholinꢀ4ꢀyl)ꢀbutylꢀ
2
2
3ꢀ(piperidinꢀ1ꢀyl)ꢀpropylꢀ
3ꢀ(2ꢀmethylꢀpiperidinꢀ1ꢀyl)ꢀpropylꢀ
3ꢀ(3ꢀmethylꢀpiperidinꢀ1ꢀyl)ꢀpropylꢀ
3ꢀ(4ꢀmethylꢀpiperidinꢀ1ꢀyl)ꢀpropylꢀ
4ꢀacetoxyꢀbutylꢀ
2
2
2
2
2
1i
dimethylcarbamoylmethylꢀ
3ꢀ(dimethylamino)ꢀpropylꢀ
3ꢀ(tertꢀbutoxycarbonylamino)ꢀpropylꢀ
3ꢀaminoꢀpropylꢀ
1j
21k
1l*
2
a
IC values are average of at least two independent experiments, *HCl salt
50
A detailed proteinꢀinhibitor coꢀcrystal structure of AXL kinase has not been published yet. To
identify the important pharmacophore features for the binding we used the Phase module of
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 55
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
Schrödinger Suite 2009 and determined a five point pharmacophore (AAAHR, survival score:
3.037) for the active molecules (Figure 4).
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 4. The five point pharmacophore of AXL kinase inhibitors. Compound 18d (orange),
19h (grey) and 21f (green) are shown on the image.
The marked motifs (A1, A2, A6, H8, R17) are analogous in the best and the moderate active
compounds, and according to other kinase inhibitors these points are necessary for the
interactions with the hinge region (A1), DFG motif (A2) and gatekeeper residue (A2, R17).
The in vitro AXL kinase assay revealed that the most active compounds differ in two motifs
6
from less active ones. The flexible side chains in R position enhance not only the inhibitory
effect (Table 3) but the solubility as well. The position and substitution of the aromatic ring
R17 is also crucial, because this phenyl ring can be involved in hydrophobic interactions with
the gatekeeper residue (Leu620 of the AXL kinase). Although a small gatekeeper residue, like
Leu620, could allow larger and more hydrophobic substituents, only small substituents are
3
4
well tolerated, especially in R and R position (Table 2). Due to the size of the molecules the
14
ACS Paragon Plus Environment

Page 15 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
whole large back cavity can be occupied by the sulfonamide group and the substituted phenyl
ring.
Having identified the biochemical potency of our compounds against AXL kinase, cellular
assays were performed. 18d, 19e, 19h, 19i and 21f, all displaying low µM IC50 values of AXL
phosphorylation, were selected for further characterization of kinase selectivity profiles and in
migration and invasion related cell based assays.
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
First we determined the kinase selectivity profile of 18d, 19h, 19i and 21f using three
different types of assays, namely IMAP, binding and HTRF assay. The kinase profile of 18d,
19h, 19i and 21f displayed common target profile by inhibition of AXL as well as cꢀMET,
SRC, ABL, cKIT, TIE2, PDGFRbeta, AurA and VEGFRꢀ2 with efficacy of 83ꢀ100% when
used at 10 µM concentration (Figure 5). The comparison of the kinase selectivity profiles
identified additional targets with lower affinity especially to compound 18d such as DDR1,
FLT3, RET, FGFR3, CSK, BꢀRAF, ERBB2, SYK, INSR, and JAK3. 50% of the tested
kinases have not been targeted by any of the compounds with notable affinity.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 55
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 5. The kinase selectivity profile against a panel of 36 human kinases proves AXL as a
common target of Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamides derivates. The
selectivity profiling was performed in triplicates at a compound concentration of 10 ꢁmol/L.
The plots indicate the percentages of inhibition for each individual kinase.
We compared the molecular targets of Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamides
derivates using the kinomeꢀwide DiscoveRx KinomeScan assay. The kinaseꢀbinding
selectivity of 20b and 20g was determined on a panel of 442 human wild type and mutated
kinases (Kinomscan) (Supporting Information Table S1.). At 5 µmol/L, apart from the
primary target AXL, 12 kinases (8 wild type kinases and 4 mutants) were identified as hits of
20b and 23 of 20g (17 wild type kinases and 6 mutants), exhibiting equal or lower percentage
control values than of AXL (Supporting Information Table S2.). Lower numbers indicate
20
stronger binding . Additional common targets are ABL, LOK, cKIT, BRK, SRC, BLK, LCK,
RIPK2, PDGFRA and B, CSF1R, cꢀMET, DDR1 and EGFR. ABL, cKIT; SRC; PDGFRbeta,
cꢀMET and DDR1 were verified by chemical binding assays as displayed in Figure 5.
16
ACS Paragon Plus Environment

Page 17 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Increasing the threshold of percentage control to ten times the percentage control value of
AXL we identified 45 out of 442 tested kinases for 20b and 71 out of 442 for 20g (Supporting
Information Table S2.). This corresponds to 10.2% and 16.0% of the kinome being analyzed.
Wilson et al, published the selectivity profiles of R428/BGB324 at a concentration of 1
µmol/L using a similar KinomeScan panel. This most selective AXL inhibitor published until
now targets 24 kinases and mutants more potently than AXL and hits 128 out of 452 kinases
and mutants within a range of ten times the percentage control value of AXL. This represents
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
20
27.8% of the tested kinome . This data indicate a higher selectivity of Nꢀ[4ꢀ(quinolinꢀ4ꢀ
yloxy)ꢀphenyl]ꢀbenzenesulfonamides 20b and 20g for AXL compared to R428/BGB324.
More in vivo relevant targets were analyzed by KinAffinity technology to determine the target
profiles of the small molecule kinase inhibitors 19h and 19i in lysates from the human breast
cancer cell line MDAꢀMBꢀ231 (Table 7 and 8). The KinAffinity analysis of MDAꢀMBꢀ231
lysates demonstrated a broad selectivity of compounds 19h and 19i for protein kinases. Target
spectra for both compounds were fairly similar and revealed high affinities in the low
nanomolar range for protein kinases AXL, cꢀMET, TNK1, MAP4K5, LYN, RIPK2, EGFR,
SRC and several others.
The receptor tyrosine kinase AXL exhibited the highest affinity with a Kd value of 0.015 µM
for compound 19h. Additional target kinases with Kd values < 0.150 µM (10 fold the Kd of
AXL) were TNK1, MAP4K5, LYN, cꢀMET, RIPK2, ACVR1, PLK4, FYN, AURKB, ABL2,
EGFR and LIMK1. In summary, 13 protein kinases were identified with Kd values < 0.150
µM for compound 19h. In total, 168 kinase targets were enriched from MDAꢀMBꢀ231 cells,
48 of which were identified as target proteins of compound 19h within a Kd range between
0.015 and 7.650 ꢁM. This corresponds to 7.7% and 25% of the kinome being detected in
MDAꢀMB231 cells. The remaining 126 kinases bound to the KinAffinity matrix but were not
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 55
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
competed by compound 19h. Consequently, these kinases showed no binding characteristics
expected for target proteins of compound 19h. Table 7 shows the target protein kinases of
compound 19h in MDAꢀMBꢀ231 cells. Targets were ranked according to their affinities and
targets with low Kd values ranging from 0.015 to 7.650 µM.
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
Table 7. The target protein kinases of compound 19h in MDAꢀMBꢀ231 cells (analyzed by
KinAffinity technology)
Uniprot Protein Name
Gene
Name
AXL
KD_free
[µM]
1
2
3
4
5
6
7
8
9
1
P30530
Q13470
AXL oncogene
0.015
Nonꢀreceptor tyrosineꢀprotein kinase TNK1
TNK1
0.024
Q9Y4K4 Mitogenꢀactivated protein kinase kinase kinase kinase 5
MAP4K5 0.024
P07948
P08581
O43353
Q04771
O00444
P06241
Tyrosineꢀprotein kinase Lyn
Tyrosineꢀprotein kinase cꢀMET
Receptorꢀinteracting serine/threonineꢀprotein kinase 2
Activin receptor type I
LYN
0.029
0.041
0.043
0.052
0.053
0.064
>0.069
0.071
0.122
0.130
0.178
0.254
0.270
cꢀMET
RIPK2
ACVR1
PLK4
FYN
Poloꢀlike kinase 4
Tyrosineꢀprotein kinase Fyn
0
Q96GD4 Aurora kinase B
AURKB
ABL2
EGFR
LIMK1
YES
11 P42684
12 P00533
Tyrosineꢀprotein kinase ABL2
Epidermal growth factor receptor
LIM domain kinase 1
13
14
15
16
P53667
P07947
P12931
P41240
Protoꢀoncogene tyrosineꢀprotein kinase Yes
Protoꢀoncogene tyrosineꢀprotein kinase Src
Tyrosineꢀprotein kinase CSK
SRC
CSK
18
ACS Paragon Plus Environment

Page 19 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
7
Q14164
Inhibitor of nuclear factor kappaꢀB kinase subunit epsilon IKKE
0.282
18 Q13131
5ꢀAMPꢀactivated protein kinase catalytic subunit alphaꢀ1 PRKAA1 0.616
19
20
21
22
23
P16591
O75460
P54619
O14965
O43741
Tyrosineꢀprotein kinase Fer
FER
0.679
0.723
Serine/threonineꢀprotein kinase/endoribonuclease IRE1
5ꢀAMPꢀactivated protein kinase subunit gammaꢀ1
Aurora kinase A
IRE1
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
PRKAG1 0.825
AURKA 0.847
5ꢀAMPꢀactivated protein kinase subunit betaꢀ2
PRKAB2 0.969
PRKAG2 1.078
24 Q9UGJ0 5ꢀAMPꢀactivated protein kinase subunit gammaꢀ2
25 Q9NYL2 Mitogenꢀactivated protein kinase kinase kinase MLT
ZAK
1.180
26
27
28
29
Q9Y478 5ꢀAMPꢀactivated protein kinase subunit betaꢀ1
P37173 Transforming growth factorꢀbeta receptor type II
Q8IVH8 Mitogenꢀactivated protein kinase kinase kinase kinase 3
PRKAB1 1.257
TGFBR2 1.860
MAP4K3 2.080
Q13043
Mammalian STE20ꢀlike protein kinase 1
CyclinꢀGꢀassociated kinase
MST1
GAK
2.433
2.448
>2.514
2.857
30 O14976
31 Q6PHR2 Serine/threonineꢀprotein kinase ULK3
ULK3
ECH1
32
33
34
35
Q13011
Q99759
Q15418
Q92844
Delta(3,5)ꢀDelta(2,4)ꢀdienoylꢀCoA isomerase
Mitogenꢀactivated protein kinase kinase kinase 3
90 kDa ribosomal protein S6 kinase 1
MAP3K3 3.133
RPS6KA1 3.152
TRAF family memberꢀassociated NFꢀkappaꢀB activator
Mitogenꢀactivated protein kinase kinase kinase kinase 4
Lymphocyteꢀoriented kinase
TANK
3.418
36 O95819
37 O94804
MAP4K4 3.75
LOK
3.78
38
39
40
41
P27448
MAP/microtubule affinityꢀregulating kinase 3
MARK3
3.854
Q9Y2U5 Mitogenꢀactivated protein kinase kinase kinase 2
Q05397 Focal adhesion kinase 1
MAP3K2 4.396
FAK
4.445
4.455
Q7KZI7 MAP/microtubule affinityꢀregulating kinase 2
MARK2
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 55
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
4
2
Q9H0K1 Qinꢀinduced kinase
QIK
4.518
5.146
43 Q96PY6 Never in mitosis Aꢀrelated kinase 1
NEK1
MARK4
PCTK3
MST2
44
45
46
47
48
Q96L34 MAP/microtubule affinityꢀregulating kinase 4
5.975
Q07002
Q13188
P36507
Q00537
Serine/threonineꢀprotein kinase PCTAIREꢀ3
Mammalian STE20ꢀlike protein kinase 2
>6.194
>6.454
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
Dual specificity mitogenꢀactivated protein kinase kinase 2 MAP2K2 6.917
Serine/threonineꢀprotein kinase PCTAIREꢀ2 PCTK2 >7.651
The receptor tyrosine kinase AXL and the receptor tyrosineꢀprotein kinase cꢀMET exhibited
the highest affinities each with a Kd value of 0.006 µM for compound 19i. Additional high
affinity target kinases with Kd values < 0.060 µM (10 fold the Kd of AXL) were MAP4K5,
LYN, TNK1, ABL2, EGFR, RIPK2, CSK, FYN, YES, and PLK4. In summary, 13 protein
kinases were identified with Kd values < 0.060 µM for compound 19i. In total, 168 kinase
targets were enriched from MDAꢀMBꢀ231 cells, 52 of which were identified as target proteins
of compound 19i within a Kd range between 0.006 and 9.230 ꢁM. This corresponds to 7.7%
and 25.6% of the kinome being detected in MDAꢀMB231 cells. The remaining 125 kinases
bound to the KinAffinity matrix but were not competed by compound 19i. Consequently,
these kinases showed no binding characteristics expected for target proteins of compound 19i.
Table 8 shows the target protein kinases of compound 19i in MDAꢀMBꢀ231 cells. Targets are
ranked according to their affinities with Kd values ranging from 0.006 to 9.230 ꢁM.
Table 8. The target protein kinases of compound 19i in MDAꢀMBꢀ231 cells (analyzed by
KinAffinity technology)
Uniprot Protein Name
Gene
KD_free
20
ACS Paragon Plus Environment

Page 21 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Name
[µM]
1
2
3
4
5
6
7
8
9
1
P30530 AXL oncogene
AXL
MET
0.006
0.006
P08581 Tyrosineꢀprotein kinase cꢀMet
Q9Y4K4 Mitogenꢀactivated protein kinase kinase kinase kinase 5
P07948 Tyrosineꢀprotein kinase Lyn
MAP4K5 0.008
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
LYN
0.011
0.014
0.016
0.016
0.017
0.030
0.034
0.038
0.040
0.048
>0.070
0.093
0.096
0.122
0.146
Q13470 Nonꢀreceptor tyrosineꢀprotein kinase TNK1
P42684 Tyrosineꢀprotein kinase ABL2
TNK1
ABL2
EGFR
RIPK2
CSK
P00533 Epidermal growth factor receptor
O43353 Receptorꢀinteracting serine/threonineꢀprotein kinase 2
P41240 Tyrosineꢀprotein kinase CSK
0
Q96GD4 Aurora kinase B
AURKB
FYN
11 P06241 Tyrosineꢀprotein kinase Fyn
12
13
14
15
16
P07947 Protoꢀoncogene tyrosineꢀprotein kinase Yes
O00444 Poloꢀlike kinase 4
YES
PLK4
ACVR1
LIMK1
SRC
Q04771 Activin receptor type I
P53667 LIM domain kinase 1
P12931 Protoꢀoncogene tyrosineꢀprotein kinase Src
17 Q14164 Inhibitor of nuclear factor kappaꢀB kinase subunit epsilon IKKE
18
19
20
21
22
O14965 Aurora kinase A
AURKA
TGFBR1 0.205
P36897 Transforming growth factorꢀbeta receptor type I
O75460 Serine/threonineꢀprotein kinase/endoribonuclease IRE1
P16591 Tyrosineꢀprotein kinase Fer
IRE1
FER
0.243
0.304
Q13163 Dual specificity mitogenꢀactivated protein kinase kinase 5 MAP2K5 0.539
23 Q13131 5ꢀAMPꢀactivated protein kinase catalytic subunit alphaꢀ1 PRKAA1 0.786
24
Q8IVH8 Mitogenꢀactivated protein kinase kinase kinase kinase 3
MAP4K3 1.367
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 55
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
5
Q92844 TRAFꢀinteracting protein
TANK
1.567
26 P54619 5ꢀAMPꢀactivated protein kinase subunit gammaꢀ1
PRKAG1 1.871
PRKAB2 1.907
27
28
29
30
31
O43741 5ꢀAMPꢀactivated protein kinase subunit betaꢀ2
Q13043 Mammalian STE20ꢀlike protein kinase 1
TBKBP1 TANKꢀbinding kinase 1ꢀbinding protein 1
Q05397 Focal adhesion kinase 1
MST1
TBKBP1 2.090
FAK 2.212
2.080
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
Q9Y478 5ꢀAMPꢀactivated protein kinase subunit betaꢀ1
PRKAB1 2.279
PRKAG2 2.296
32 Q9UGJ0 5ꢀAMPꢀactivated protein kinase subunit gammaꢀ2
33 Q6PHR2 Serine/threonineꢀprotein kinase ULK3
ULK3
LIMK2
AZI2
LOK
>2.513
2.784
2.982
3.039
3.210
3.273
3.545
>3.674
4.166
5.043
5.223
34
35
36
37
P53671 LIM domain kinase 2
Q9H6S1 5ꢀazacytidineꢀinduced protein 2
O94804 Lymphocyteꢀoriented kinase
Q9NYL2 Mitogenꢀactivated protein kinase kinase kinase MLT
ZAK
38 Q9UHD2 TANKꢀbinding kinase 1
TBK1
QIK
39 Q9H0K1 Qinꢀinduced kinase SNF1ꢀlike kinase 2
40
41
42
43
P42680 Tyrosineꢀprotein kinase Tec
TEC
O14976 CyclinꢀGꢀassociated kinase
GAK
NEK1
MST2
Q96PY6 Never in mitosis Aꢀrelated kinase 1
Q13188 Mammalian STE20ꢀlike protein kinase 2
44 Q15418 90 kDa ribosomal protein S6 kinase 1
RPS6KA1 5.474
MAP3K2 5.518
45 Q9Y2U5 Mitogenꢀactivated protein kinase kinase kinase 2
46
47
48
49
P27448 MAP/microtubule affinityꢀregulating kinase 3
Q14289 Focal adhesion kinase 2
MARK3
PYK2
5.622
6.928
7.086
Q7KZI7 MAP/microtubule affinityꢀregulating kinase 2;
P37173 Transforming growth factorꢀbeta receptor type II
MARK2
TGFBR2 7.385
22
ACS Paragon Plus Environment

Page 23 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
0
Q9Y2K2 Serine/threonineꢀprotein kinase QSK
51 P36507 Dual specificity mitogenꢀactivated protein kinase kinase 2 MAP2K2 8.852
P30084 EnoylꢀCoA hydratase 1 ECHS1 9.237
QSK
7.630
5
2
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
Complete lists of all kinases that bound to the KinAffinity matrix and of all identified target
kinases of compound 19h and 19i are provided in Supporting Information Table S3. This gave
us the opportunity to discriminate between Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀ
benzenesulfonamides derivates with higher and lower target selectivity profiles to AXL.
To elucidate the affinities of examples 18d, 19e, 19h, 19i, 20b, 20g and 21f to four migration
related kinases in more detail, we determined IC50 values for AXL, SRC, cꢀMET and EGFR
kinases (Table 9). All seven examples exhibit low IC values for AXL confirming the high
5
0
affinity of these Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamides as AXL inhibitors.
The affinity to SRC, cꢀMET and EGFR was more variable for the tested examples. (Table 9.)
The IC50 values especially for cꢀMET and EGFR were increasing from 0.127 ꢁM for cꢀMET
and 0.178 ꢁM for EGFR using example 19i, to 1.535 ꢁM for cꢀMET and 3.081 ꢁM for EGFR
in the case of example 19e. The IC values were increasing in general from 19i < 19h <18d <
5
0
20b < 20g < 21f < 19e identifying 19e as the most selective compound for AXL. Finally we
were able to categorize compound 19i and 19h into a lower selective group, 18d, 20b, 20g as
an intermediate group and 21f and 19e as compounds with higher selectivity for AXL.
Table 9. The IC50 values of Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamide derivates
on AXL, SRC, cꢀMET and wild type EGFR. The IC50 determinations were performed in
triplicates.
No.
AXL IC50 (ꢁM)
SRC IC50 (ꢁM)
cꢀMET IC50 (ꢁM)
EGFR wt IC50 (ꢁM)
19i
19h
0.035
0.027
0.044
0.088
0.127
0.358
0.178
0.149
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 55
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
18d
20b
0.021
0.021
0.027
0.028
0.051
0.544
0.995
1.723
0.113
1.951
0.488
0.338
0.609
1.307
1.535
0.281
0.650
0.592
1.128
3.081
2
0g
1f
9e
2
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
Overexpression of the RTK AXL and its implication in the migration and invasion of
2
1, 22
mammary carcinoma has already been demonstrated in various publications.
Therefore
we studied the impact of Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀphenyl]ꢀbenzenesulfonamides derivates on
AXL mediated migration and invasion of an AXLꢀoverexpressing human breast cancer cell
line, namely MDAꢀMB231.
First, we determined the inhibitory effect of 18d, 19e, 19h, 19i and 21f on cell migration
using Boyden chamber assays. The IC50 value was 1.5 ꢁM for 19i, 2.5 µM for 19h, 3.3 for
21f, 4.6 µM for 18d and 4.3 ꢁM for 19e in MDAꢀMB231 cells (Figure 6 and 7).
Figure 6. Migration inhibition (%) of the compounds
24
ACS Paragon Plus Environment

Page 25 of 55
Journal of Medicinal Chemistry
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 7. Representative images of MDAꢀMB231 transꢀmigrating the porous membrane.
MDAꢀMB231 cells were incubated with indicated compound concentrations relative to
DMSO control for 3.5 hours. Scale bars indicate 100ꢀꢁm.
Comparing the IC50 values we discovered that migration inhibition was correlating with the
selectivity profiles of the compounds. Thereby it was evident that less selective compounds,
namely 19h and 19i, were superior in blocking migration of MDAꢀMBꢀ231 cells in contrast to
more AXL selective derivates, in terms of migration related kinases, like SRC, cꢀMET and
EGFR as displayed in Table 9. A similar conclusion were drawn by Pénzes et al, 2014, that
inhibition of MDAꢀMB231 cell migration by AXL inhibitors is dependent on additional
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 55
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
targets like a SRC family kinase LYN and the migration related CRKꢀassociated substrate
2
3
p130Cas .
In summary, the inhibition values of migration were significantly (more than 50ꢀfold) higher
than those determined for inhibition of AXL phosphorylation, suggesting that migration of
MDAꢀMB231 does not solely depend on AXL phosphorylation, but might be regulated by
multiple additional kinases such as SRC, cꢀMET and EGFR.
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
Secondly, we determined the inhibitory effect of 18d, 19e, 19h, 19i and 21f on invasion of
MDAꢀMB231 cells (Figure 8). MDAꢀMB231 cell have the ability for invasion of semisolid
Matrigel. We used this capacity of MDAꢀMB231 cells and seeded a single cell suspension on
top of a solid Matrigel. Then we allowed the single cells to migrate towards each other within
5
hours, resulting in the formation of large cell aggregates (Figure 9). Compounds blocking
invasion are able to arrest MDAꢀMB231 cells in a single cell status prohibiting mesenchymal
cell invasion and subsequent aggregate formation. The percentage of the large multicellular
aggregates was subsequently quantified.
Figure 8. Invasion inhibition (%) of the molecules
26
ACS Paragon Plus Environment

Page 27 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The IC value for aggregate formation was 156 nM for 19i, 158 nM for 19h, 185 nM for 21f,
50
158 nM for 18d and 103 nM for 19e in MDAꢀMB231 cells. The comparison of IC values of
5
0
invasion and aggregate formation correlated with the IC50 values of AXL phosphorylation
inhibition. Thereby it was evident that all compounds exhibit very similar IC50 blocking
invasion of MDAꢀMBꢀ231 cells in nM range.
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 9. Representative images of MDAꢀMB231 aggregates. MDAꢀMB231 cells were
incubated with indicated compound concentrations relative to DMSO control for 5 hours.
Scale bars indicate 100 ꢁm.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 55
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
In summary, the inhibition values of invasion were twoꢀ to sixꢀfold higher than those
determined for inhibition of AXL phosphorylation, suggesting that invasion inhibition of
MDAꢀMB231 is closely correlated to AXL phosphorylation inhibition in contrast to
migration. This is in line with the results that invasion is more affected than migration by
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
24
AXL specific siRNA in colorectal cancer cell lines , AXL specific blocking antibody 20G7ꢀ
2
5
D9 in tripleꢀnegative breast cancer cell line HBLꢀ100 and selective RNAꢀbased aptamer,
GL21.T that binds the extracellular domain of AXL at high affinityAXL specific Aptamer in
26
GBM cell line U87MG .
Migration inhibition correlates positively with lower selectivity of Nꢀ[4ꢀ(quinolinꢀ4ꢀyloxy)ꢀ
phenyl]ꢀbenzenesulfonamides towards AXL kinase and highlights the importance of
additional migration related kinases, as EGFR, cꢀMET, SRC family kinases, LIMK1 or ABL.
These migration related kinases were validated as target proteins with < 1µM Kdꢀvalues in
MDAꢀMB231 cells using KinAffinity. Nevertheless, we cannot exclude other factors being
2
7
important for metastasis inhibition as shown for NKꢀcell activation in vivo .
Two compounds 19h and 21f exhibiting good selectivity profiles and low nanomolar AXL
inhibition values were selected for further ADME and pharmacokinetic studies. The obtained
parameters for compound 19h and 21f are summarized in Table 10.
Table 10. ADME and pharmacokinetic parameters obtained for compound 19h and 21f
Parameter
19h
68.8
24.3
7.4
21f
18.0
46.1
2.4
SolRank pH 7.4 rel. Conc. [µM]
PAMPA pH 7.4 %Flux [%]
ꢀ6
Cacoꢀ2 Papp Aꢀ>B [10 cm/s]
28
ACS Paragon Plus Environment

Page 29 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ꢀ
6
Cacoꢀ2 Papp Bꢀ>A [10 cm/s]
17.0
14.6
6.6
Cacoꢀ2 RatioBꢀ>A:Aꢀ>B
2.3
60.5
143.3
96.3
108.0
99.1
97.2
Microsomal Stab. Phase I human Clint [µL/min/mg]
Microsomal Stab. Phase I murine Clint [µL/min/mg]
Plasma Stab. Human %Remain
18.3
19.1
99.1
98.8
98.5
95.9
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
Plasma Stab. Murine %Remain
Plasma Protein Binding human %Bound
Plasma Protein Binding murine %Bound
PK mouse IV AUC(0ꢀINF) [ng*h/mL]
PK mouse IV AUC(0ꢀTz) [ng*h/mL]
PK mouse IV CL [l/h/kg]
2646.0 1395.0
1433.0
0.4
669.0
0.7
PK mouse IV C0 [ng/mL]
310.0
18.7
216.0
8.5
PK mouse IV t1/2z [h]
PK mouse IV Vz [l/kg]
10.2
8.8
Compound 21f and 19h displayed a good solubility at pH 7.4 (18.0 or 68.8 µM, respectively)
and exhibited a good permeability in PAMPA and Cacoꢀ2 assays. The intrinsic clearance had
been assessed by microsomal stability assays displaying sufficient stability of 18.3
µL/min/mg in human microsomes and 19.1 µL/min/mg in mouse microsomes for compound
21f, and 60.5 µL/min/mg in human microsomes and 143.3 µL/min/mg in mouse microsomes
for compound 19h. Both compounds feature very high human and murine plasma stability
and are characterized by low plasma protein binding especially to murine plasma proteins
with 95.9% and 97.2 % (Table 10).
29
ACS Paragon Plus Environment