Design, synthesis, and biological evaluation of novel aminopyrimidinylisoindolines as AXL kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.10.013

A novel series of aminopyrimidinylisoindoline derivatives 1a-w having an aminopyrimidine scaffold as a hinge region binding motif were designed and synthesized. Among them, six compounds showed potent inhibitory activities against AXL kinase with IC₅₀ values of submicromolar range. Especially, compound 1u possessing (4-acetylpiperazin-1-yl)phenyl moiety exhibited extremely excellent efficacy (IC₅₀ = <0.00050 μM). Their in vitro antiproliferative activities were tested over five cancer cell lines. Most compounds showed good antiproliferative activities against HeLa cell line. The kinase panel profiling of 50 different kinases and the selected inhibitory activities for the representative compound 1u were carried out. The compound 1u exhibited excellent inhibitory activities (IC₅₀ = <0.00050, 0.025, and 0.050 μM for AXL, MER, and TYRO3, respectively) against TAM family, together with potent antiproliferative activity against MV4-11 cell line (GI₅₀ = 0.10 μM) related to acute myeloid leukemia (AML).

Full text of DOI:10.1016/j.bmcl.2018.10.013

Accepted Manuscript
Design, synthesis, and biological evaluation of novel aminopyrimidinylisoin-
dolines as AXL kinase inhibitors
Min Jung Choi, Eun Joo Roh, Wooyoung Hur, So Ha Lee, Taebo Sim, Chang-
Hyun Oh, Sun-Hwa Lee, Jong Seung Kim, Kyung Ho Yoo
PII:
S0960-894X(18)30803-5
https://doi.org/10.1016/j.bmcl.2018.10.013
BMCL 26072
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
24 August 2018
1 October 2018
10 October 2018
Please cite this article as: Choi, M.J., Roh, E.J., Hur, W., Lee, S.H., Sim, T., Oh, C-H., Lee, S-H., Kim, J.S., Yoo,
K.H., Design, synthesis, and biological evaluation of novel aminopyrimidinylisoindolines as AXL kinase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.013
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Design, synthesis, and biological evaluation of novel
aminopyrimidinylisoindolines as AXL kinase inhibitors
a,d
a
a
a
a
Min Jung Choi , Eun Joo Roh , Wooyoung Hur , So Ha Lee , Taebo Sim ,
b
c
d
a,
Chang-Hyun Oh , Sun-Hwa Lee , Jong Seung Kim , Kyung Ho Yoo *
a
Chemical Kinomics Research Center, Korea Institute of Science and Technology, PO Box 131,
Cheongryang, Seoul 02792, Republic of Korea
b
Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131, Cheongryang,
Seoul 02792, Republic of Korea
c
Efficacy Assessment Support Department, New Drug Development Center, 80 Chembok-ro Dong-
gu, Daegu 41061, Republic of Korea
d
Department of Chemistry, Korea University, Seoul 02841, Republic of Korea
*
Corresponding author. Tel.: +82 2 958 5152; fax: +82 2 958 5549.
E-mail address: khyoo@kist.re.kr (K. H. Yoo).
1

ABSTRACT
A
novel series of aminopyrimidinylisoindoline derivatives 1a-w having an
aminopyrimidine scaffold as a hinge region binding motif were designed and synthesized.
Among them, six compounds showed potent inhibitory activities against AXL kinase with
IC50 values of submicromolar range. Especially, compound 1u possessing (4-
acetylpiperazin-1-yl)phenyl moiety exhibited extremely excellent efficacy (IC50 = <
0
.00050 M). Their in vitro antiproliferative activities were tested over five cancer cell
lines. Most compounds showed good antiproliferative activities against HeLa cell line. The
kinase panel profiling of 50 different kinases and the selected inhibitory activities for the
representative compound 1u were carried out. The compound 1u exhibited excellent
inhibitory activities (IC50 = < 0.00050, 0.025, and 0.050 M for AXL, MER, and TYRO3,
respectively) against TAM family, together with potent antiproliferative activity against
MV4-11 cell line (GI50 = 0.10 M) related to acute myeloid leukemia (AML).
Keywords: Aminopyrimidinylisoindolines; Inhibitors; AXL kinase; TAM family; Enzyme
inhibitory activity; Antiproliferative activity
2

AXL is a receptor tyrosine kinase (RTK) involved in the growth, differentiation,
1
survival, and motility of many different cell types. In recent years, AXL kinase has
emerged as a key facilitator of immune escape and drug-resistance by eliminating
intercellular antibodies and regulating the secretion, and release of cytokines. The AXL
protein was latter classified as a RTK belonging to the TAM (TYRO3, AXL, and MER)
2
subfamily. The TAM RTKs are overexpressed in a large number of human cancers
3
,4
including myeloid leukemias, colon, liver and prostate cancer. Elevated expression of one
or more TAM receptors is often associated with cancer progression, resistance to targeted
5
-7
therapies, and metastasis. Overexpression of AXL was shown to correlate with poorer
prognosis, increased invasiveness, and xenograft growth of human cancers, further
8
indicating that AXL has strong oncogenic potential. AXL has been implicated as a cancer
driver and correlated with poor survival in numerous aggressive tumors including
9
10
11
12
glioblastoma multiforme, breast and lung cancers, osteosarcoma, and acute myeloid
1
3
leukemia. Therefore, AXL has recently been proposed as an potential oncology target due
to its overexpression in several types of cancers and a number of small molecule inhibitors
1
4-17
have been developed.
Many kinase inhibitors utilize the pyrimidine scaffold as a hinge anchor with
1
8,19
various substitutions at 2- and 4-positions.
In this work, a novel series of
aminopyrimidinylisoindoline derivatives 1a-w having an aminopyrimidine scaffold as a
1
5
20
hinge region binding motif, based on the structural features of TP-0903, TAE-684, and
2
1
Fedratinib, were designed and synthesized (Fig. 1). Here isoindoline moiety having a rigid
bicyclic structure to enhance efficacy was introduced at 4-position of pyrimidine nucleus.
3

Their in vitro enzyme inhibitory and antiproliferative activities were tested over AXL
kinase and five cancer cell lines. Also the kinase panel profiling of 50 different kinases and
the selected inhibitory activities for the representative compound 1u are reported.
Fig. 1. Structures of aminopyrimidine derivatives as AXL and TAM inhibitors and the target
compounds 1a-w.
Aminopyrimidinylisoindolines 1a-w were synthesized by the pathways illustrated in
Scheme 1. Amination of 2,4-dichloro-5-trifluoromethylpyrimidine (2) as a starting material
with 7N ammonia solution in methanol gave 2-chloro-5-(trifluoromethyl)pyrimidin-4-
2
2
amine (3),
methylmethanesulfonamide (8) in presence of sodium hydride to yield the key intermediate
in good yield. Nucleophilic substitution of the chloro group of compound 4 with the
which was then cyclized with N-(2,3-bis(bromomethyl)phenyl)-N-
4
appropriate aryl amines in presence of p-toluenesulfonic acid led to the corresponding title
4

2
3
compounds 1a–w.
Scheme 1. Reagents and conditions: (a) 7N ammonia solution in MeOH, rt, 2.5 h, 38%; (b) NaH,
o
THF, rt, 24 h, 84%; (c) p-TsOH (2M in dioxane), DMF, 90 C, 24 h, 11-70%.
Synthesis of N-(2,3-bis(bromomethyl)phenyl)-N-methylmethanesulfonamide (8)
was carried out by the sequence of reactions shown in Scheme 2. N-2,3-Trimethylaniline
(
6) was obtained by N-methylation of 2,3-dimethylaniline (5) with tetrabutylammonium
bromide and dimethylsulfate in toluene followed by 15% aqueous sodium hydroxide
solution. Treatment of 6 with methanesulfonyl chloride gave N-(2,3-dimethylphenyl)-N-
methylmethanesulfonamide (7) in good yield, which was then brominated with N-
bromosuccinimide in presence of AIBN under reflux condition to produce the desired
2
4
compound 8.
Scheme 2. Reagents and conditions: (a) tetrabutylammonium bromide, dimethylsulfuric acid,
toluene, 15% aqueous NaOH solution, rt, 24 h, 51%; (b) methanesulfonyl chloride, pyridine, rt, 30
min, 98%; (c) N-bromosuccinimide, AIBN, CH
2
Cl , reflux, 4 h, 73%.
2
The in vitro inhibitory activity of the target compounds was tested against AXL
5

2
5
kinase, together with staurosporine as a reference standard because of its high potency.
The ability of aminopyrimidinylisoindolines 1a-w to inhibit the activity of AXL kinase is
summarized in Table 1. As shown in Table 1, compounds 1f, 1j, 1l, 1r showed good
potencies (IC50 = 1.5 – 9.1 M) and compounds 1a, 1d, 1v exhibited potent inhibitory
activities with IC50 values below submicromolar range (IC50 = 0.080 – 0.89 M). Upon
investigating the effect of substituents onto aryl nucleus, compounds with electron-donating
group were generally found to be more potent than compounds having electron-
withdrawing group. Compounds 1a, 1d, 1f, 1j with methoxy or methyl group showed
superior potencies to other compounds having the different substituents, except for
compound 1l. This may be rationalized that the electronic effect of substituents at receptor
site may affect the affinity and potency. Bicyclic compound 1w exhibited excellent
inhibitory activity with IC50 value of 0.0045 M. Especially, compounds 1t (IC50 = 0.00094

M) and 1u (IC50 = < 0.00050 M) with longer chains at 2-position were remarkably more
potent than other compounds having the chains of relatively short length. This may be
attributed to that the longer chain, piperazinyl moiety, may geometrically permit
appropriate fitting of the molecule at the ATP-binding site of the receptor kinase domain. In
our series, compound 1u possessing (4-acetylpiperazin-1-yl)phenyl moiety was selected as
a representative compound. As compared with parent compound 1u, compound 1v having
additional methoxy group on aryl nucleus displayed the result of much lowered efficacy.
Interestingly, the comparison of compound 1t with p–orientation and compound 1s of o-
orientation showed a very surprising difference (IC50 = 0.00094 and > 20 M, respectively)
in efficacy. Namely, compound 1s did not possess any meaningful activity. It can be
6

concluded that the introduction of solubilizing group at the para position of the phenyl
group greatly improved the potency and that steric and/or electronic properties of the
substituents of the phenyl group play a very important role in the drug-receptor interaction.
Table 1. The in vitro enzyme inhibitory activity of aminopyrimidinylisoindolines 1a-w against
AXL kinase.
Compd. No.
R
IC (M)
Compd. No.
1m
R
IC (M)
50
50
1
a
0.080
> 20
1
1
1
b
c
> 20
> 20
0.89
1n
1o
1p
> 20
> 30
> 20
d
1e
> 20
1q
> 20
1
1
1
f
2.9
1r
1s
2.6
g
h
> 20
> 20
> 20
2
6
1t
0.00094
7

2
6
1
1
i
> 20
9.1
1u
< 0.00050
0.79
j
1v
1
1
k
l
> 20
1.5
1w
0.0045
0.0039
Staurosporine
2
7
Table
2
shows
the
in
vitro
antiproliferative
activity
of
aminopyrimidinylisoindolines 1a-w to inhibit the growth of five cancer cell lines. Most
compounds showed superior antiproliferative activities against HeLa cell line to other cell
lines. As previous, the long-chained compounds 1t-w were found to be more potent than
those of the shorter length against all the tested cell lines. Compound 1t having 4-
morpholinophenyl moiety showed good activities (GI50 = 0.79 - 6.8 M) for all of these. In
particular, the representative compound 1u exhibited potent antiproliferative activities with
GI50 values of submicromolar range (GI50 = 0.73 and 0.97 M, respectively) against HeLa
and OVCAR-3 cell lines.
8

Table 2. The in vitro antiproliferative activity of aminopyrimidinylisoindolines 1a-w against five
cancer cell lines.
GI50 (M)
Compd. No.
A549
24.8
7.3
U87-MG
> 30
6.2
HeLa
7.0
OVCAR-3
0.63
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
21.5
>50
Hep G2
1.8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
16.5
17.2
5.1
25.6
> 30
22.1
> 30
19.1
> 30
> 30
19.6
16.8
> 30
> 30
> 30
10.3
9.9
> 30
> 30
> 30
20.7
> 30
> 30
25.5
> 30
> 30
> 30
> 30
> 30
6.7
> 30
> 30
29.4
> 30
> 30
> 30
28.3
> 30
> 30
> 30
> 30
16.8
5.5
d
e
14.0
10.6
> 30
> 30
17.4
6.5
f
g
h
i
j
k
l
10.9
5.3
m
n
o
p
q
r
> 30
9.2
4.3
15.2
6.2
> 30
29.8
19.5
> 30
6.8
11.1
7.3
16.0
8.6
>50
19.0
> 30
2.5
4.3
24.9
>50
23.0
> 30
1.9
s
23.9
0.79
0.73
1.3
t
2.4
u
v
w
1.3
22.1
4.7
0.97
1.6
1.4
3.3
3.8
12.1
2.1
> 30
3.7
6.3
> 30
1.8
9.8
PF-562271
1.7
1.2
9

Compound 1u selected as a representative compound was tested at a single dose
concentration of 10 μM over a panel of 50 oncogenic kinases at Reaction Biology
Corporation (Malvern, PA) (Table 3). As shown in Table 3, good potencies more than 90%
inhibition were observed against ten kinases at this concentration. Compound 1u exhibited
excellent enzymatic inhibitions above 95% against four kinases (Aurora A, FAK, PLK1,
SYK). The inhibition exerted in twenty-four kinases was below 50%.
Table 3. Percentages of enzymatic inhibitions exerted by the representative compound 1u
over a panel of 50 kinases.
a,b
Kinase
ABL1
% Inhibition
63.1
91.1
-3.6
Kinase
% Inhibition
-2.5
CK1 epsilon
DAPK1
DNA-PK
EGFR
ACK1
42.2
-4.0
AKT1
ALK
71.3
95.3
92.1
6.7
2.0
Aurora A
AXL
EPHA1
FAK/PTK2
FGFR1
FGR
13.1
96.4
26.0
80.6
49.0
62.2
83.8
14.1
89.8
19.3
95.2
-14.6
85.2
6.9
BRAF
c-KIT
17.8
17.9
88.2
20.5
37.2
75.4
45.1
55.8
84.1
69.1
3.6
c-MET
c-SRC
CDK1/cyclin B
CHK1
FLT1/VEGFR1
FYN
HIPK1
IKKa/CHUK
IR
JAK1
JNK1
PKCa
KDR/VEGFR2
LCK
PLK1
RAF1
LYN
RET
MEK1
ROCK1
1
0

MER
90.6
25.3
66.6
-34.8
65.5
16.2
9.8
RON/MST1R
ROS/ROS1
SYK
40.1
92.2
95.3
65.4
92.2
87.7
90.5
MST4
MUSK
P38a/MAPK14
p70S6K/RPS6KB1
PAK4
TIE2/TEK
TRKA
TYRO3/SKY
YES/TES1
PIMI
a
Test compound was used in a single dose concentration of 10 M.
b
%
Inhibition was calculated by subtracting % activity from 100.
The selected eight enzyme inhibitory and fourteen antiproliferative activities for the
representative compound 1u are summarized in Table 4. As shown in Table 4, compound
u exhibited excellent inhibitory activities with IC50 values of < 0.00050 M (AXL), 0.025
M (MER), and 0.050 M (TYRO3) against TAM family. Four kinases such as Aurora A,
1

FLT3, PLK1, and SYK showed 1-digit nanomolar IC50 values. Compound 1u have shown
good antiproliferative activities less than 1-digit micromolar range (GI50 = 0.10 – 5.2 M)
against all the tested cell lines, except U87-MG. It was found to possess potent activities
with GI50 values of submicromolar range for seven cell lines. Among them, MV4-11 cell
line related to acute myeloid leukemia (AML) displayed superior potency with GI50 value
of 0.10 M to other cell lines.
11

Table 4. Determination of the selected enzyme inhibitory and antiproliferative activities for the
representative compound 1u.
Kinase
IC (M)
Cell line
U87-MG
IMR-32
GI (M)
50
50
a
Aurora A
0.0030
< 0.00050
0.025
22.1
a
AXL
0.26
0.50
5.2
MER
FAK
MDA-MB-231
HCC-78
0.011
a
FLT3
PLK1
SYK
0.0046
0.0088
0.0051
0.050
A549
1.3
NOMO-1
MV4-11
3.8
0.10
1.4
a
TYRO3
Hep G2
Panc-1
0.43
0.97
0.73
0.42
1.0
a
OVCAR-3
a
HeLa
HCT 116
A375
HT-1080
1.8
a
These data were used from Tables 1 and 2.
As a continuation of our ongoing anticancer development research project, a novel
series of aminopyrimidinylisoindoline derivatives 1a-w were designed and synthesized.
The target compounds were evaluated for inhibitory activities against AXL kinase and
antiproliferative activities against five cancer cell lines. Among them, compound 1u
possessing (4-acetylpiperazin-1-yl)phenyl moiety exhibited extremely excellent efficacy
with IC50 value of < 0.00050 M against AXL kinase. In our series, a representative
compound 1u exhibited the best combination of enzyme inhibitory activities (IC50 = <
0
.00050, 0.025, and 0.050 M for AXL, MER, and TYRO3, respectively) against TAM
1
2

family and antiproliferative activity (GI50 = 0.10 M for MV4-11). It can be used as a
promising lead for the development of potent AXL and TAM inhibitors.
Acknowledgments
This work was funded by the KIST Institutional Program (Grant No. 2E28010)
from Korea Institute of Science and Technology, and by the Creative Fusion Research
Program through the Creative Allied Project funded by the National Research Council of
Science & Technology (CAP-17-01-KIST).
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5. In vitro kinase assay. The AXL assay is performed using the AXL Kinase Enzyme
TM
System (Promega, USA) and ADP-Glo Kinase Assay Kit (Promega, USA). Because the
AXL reaction utilizes ATP and generates ADP, the kinase reaction is terminated
TM
simultaneously and the remaining ATP is depleted, when the ADP-Glo reagent is added.
After the reaction, the kinase detection reagent is added, and then the ATP newly
synthesized is converted to light, using the luciferase/luciferin reaction. First, the enzyme,
substrate, ATP and inhibitor are diluted in kinase buffer. The mixture is added to the wells
of 384 low volume plate: 1 µL of inhibitor or (5% DMSO), 2 µL of enzyme, and 2 µL of
TM
substrate/ATP mix. After incubated at room temperature for an hour, 5 µL of ADP-Glo
reagent is added. Again, incubate at room temperature for 40 min, and then add 10 µL of
kinase detection reagent. Lastly, incubate at room temperature for 30 min and record
luminescence.
o
1
2
6. Selected data. Compound 1t: Grey solid; mp: 247.5 – 249.5 C; H NMR (400 MHz,
DMSO-d ) δ 9.58 (s, 1H), 8.37 (s, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.49 (q, J = 4.3 Hz, 1H),
.46 (s, 1H), 7.44 (d, J = 1.5 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 5.16 (s, 2H), 5.03 (s, 2H),
6
7
3
.75 (t, J = 4.7 Hz, 4H), 3.24 (s, 3H), 3.08 - 3.35 (m, 7H); HRMS (ESI, positive) calcd for
+
C
25
H
27
F
3
N
6
O
3
SNa [M+Na] 571.1715, found 571.1713. Compound 1u: Pale brown solid;
o
1
mp: 229.5 – 232.0 C; H NMR (400 MHz, DMSO-d ) δ 9.58 (s, 1H), 8.37 (s, 1H), 7.66 (d,
6
J = 8.7 Hz, 2H), 7.50 (q, J = 4.3 Hz, 1H), 7.46 (s, 1H), 7.42 - 7.46 (m, 1H), 6.98 (d, J = 8.9
Hz, 2H), 5.16 (s, 2H), 5.03 (s, 2H), 3.53 - 3.63 (m, 4H), 3.24 (s, 3H), 3.11 (t, J = 4.9 Hz,
2
H), 3.08 (s, 3H), 3.04 (t, J = 5.1 Hz, 2H), 2.05 (s, 3H); HRMS (ESI, positive) calcd for
+
C
27
H
30
F
3
N
7
O
3
SNa [M+Na] 612.1981, found 612.1990.
2
7. Cellular antiproliferation assay. Lung cancer cells (A549), glioblastoma cells (U87-MG),
cervix adenocarcinoma cells (HeLa), ovarian carcinoma cells (OVCAR-3), and
hepatocellular carcinoma cells (Hep G2) were obtained from ATCC (Manassas, VA).
1
5

Stock cultures were grown in 5 mL RPMI 1640 supplemented with 5% fetal bovine serum.
Extracted cells in 96-well plates (100 µL cells/well) were exposed to different sample
o
concentrations in DMSO/RPMI. And then, they are incubated at 37 C with 5% CO
2
for 2
days. Afterwards, cells were fixed with 50% trichloroacetic acid and cell proliferation was
estimated by spectrophotometric quantification (540 nm wavelength) of cellular protein
content. The measurements were obtained three times (time zero, 48 hr post-incubation for
compound free, and tested cells) and concentration-response curve for each cell line was
plotted through an equation. For the concentration-response curve for each cell line, TGI
(
concentration that produces 100% of cell growth inhibition) value was determined.
1
6

Design, synthesis, and biological evaluation of novel
aminopyrimidinylisoindolines as AXL kinase inhibitors
a,d
a
a
a
a
Min Jung Choi , Eun Joo Roh , Wooyoung Hur , So Ha Lee , Taebo Sim ,
b
c
d
a,
Chang-Hyun Oh , Sun-Hwa Lee , Jong Seung Kim , Kyung Ho Yoo *
1
7

Highlights
■
■
■
■
Six compounds showed potent inhibitory activities against AXL kinase
Most compounds showed good antiproliferative activities against HeLa cell line
1u exhibited extremely excellent efficacy (IC50 = < 0.00050 M) against AXL kinase
1u showed the best combination of enzyme inhibitory and antiproliferative activities
1
8