Design and synthesis of pyrrole-5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones with C-3′ side chains as potent Met kinase inhibitors

Article abstract of DOI:10.1039/c4ra08720h

Pyrrole-5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones of scaffold 4 with various C-3′ side chains were designed as potent Met kinase inhibitors. Structural optimization led to compounds 10, 20, and 22-24 which demonstrated subnanomolar IC₅₀ values in the biochemical assay. The potent compound 20 inhibited Met with IC₅₀ value of 0.37 nM and the proliferation of MKN45 cells with IC₅₀ of 0.22 μM. It suppressed Met autophosphorylation with the downstream signaling through Gab-1, PLC-γ, FAK, Akt, STAT3, and ERK in cell. Complete inhibition of STAT3 and ERK phosphorylation was observed in MKN45 cells treated with 20 at the concentration of 100 nM. A computation simulation study was performed to reveal the interaction of 20 with Met.

Full text of DOI:10.1039/c4ra08720h

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Design and synthesis of pyrrole–5-(2,6-
dichlorobenzyl)sulfonylindolin-2-ones with C-30
Cite this: RSC Adv., 2014, 4, 58990
side chains as potent Met kinase inhibitors†
Chia-Wei Liu,a Chun-Liang Lai,a Yu-Hsiang Lin,a Li-Wei Teng,a Sheng-chuan Yang,a
Win-Yin Wei,a Shu Fu Lin,a Ju-Ying Yang,a Hung-Jyun Huang,a Ru-Wen Wang,a
Chao-Cheng Chiang,a Mei-Hui Lee,a Yu-Chuan Wang,b Shih-Hsien Chuang,a
a
a
ab
*
Jia-Ming Chang, Ying-Shuan E. Lee and Jiann-Jyh Huang
Pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones of scaffold 4 with various C-30 side chains were
designed as potent Met kinase inhibitors. Structural optimization led to compounds 10, 20, and 22–24
which demonstrated subnanomolar IC50 values in the biochemical assay. The potent compound 20
inhibited Met with IC50 value of 0.37 nM and the proliferation of MKN45 cells with IC50 of 0.22 mM. It
suppressed Met autophosphorylation with the downstream signaling through Gab-1, PLC-g, FAK, Akt,
STAT3, and ERK in cell. Complete inhibition of STAT3 and ERK phosphorylation was observed in MKN45
cells treated with 20 at the concentration of 100 nM. A computation simulation study was performed to
reveal the interaction of 20 with Met.
Received 15th August 2014
Accepted 30th October 2014
DOI: 10.1039/c4ra08720h
www.rsc.org/advances
subsequent scaffold hopping based on the structure of 2 led to
PF-2341066 (3),13 the most advanced Met and ALK kinase
Introduction
inhibitor recently approved by FDA for the treatment of NSCLC
(see Fig. 1).
Met is a transmembrane receptor tyrosine kinase expressed in
normal and malignant cells. Upon binding to its natural
ligand hepatocyte growth factor/scatter factor (HGF/SF), Met
induces several signaling pathways responsible for cell
proliferation, motility, migration, and survival.1–3 The cellular
events play an important role in normal embryonic develop-
ment and wound healing4 but nevertheless, overexpression of
HGF and/or Met or activating mutation of Met can lead to
cancer.5 As a result, Met is considered as a potential target for
cancer therapy.6–8
C-5 substituted pyrrole–indolin-2-ones SU11274 (1)9 and
PHA665752 (2)10 are the prototype Met kinase inhibitors from in
silico design before the three dimensional structure of Met was
characterized (Fig. 1). They are obtained using the homology
model of Met built from the closely related FGFR1 kinase.11 The
C-40 amino–amido side chains of 1 and 2 were designed to
modulate the pharmacokinetic properties12 and would provide
additional interactions with the enzyme to enhance the activi-
ties. Though 1 and 2 are only used as the biology tool,
Based on the literatures reporting the discovery of pyrrole–
indolin-2-ones as multikinase inhibitors for VEGFR-2, FGFR1,
and PDGFR-b kinases, different inhibition proles are observed
with different positions of the side chains.14,15 For FGFR1 inhi-
bition, moving the C-40 side chain in a pyrrole–indolin-2-one to
the C-30 position provided compounds with 5- to 10-fold
increased potency (see the structures of 1 and 2 in Fig. 1). As
compound 2 was designed from the Met homology model of
FGFR1, we envisaged that pyrrole–indolin-2-ones with C-30 side
chains would also be potent Met inhibitors. Currently, the role
of C-30 side chain for Met inhibition has not been studied in
detail.
In this study, we used pyrrole–5-(2,6-dichlorobenzyl)sul-
fonylindolin-2-one of scaffold 4 as the template to study the
role of C-30 substituents for the inhibition of Met kinase.
Except for the C-30 substituents, scaffold 4 possesses a pyrrole-
fused cyclohexanone moiety for patent purposes.16 Subse-
quent optimization led to compounds 10, 20, and 22–24 that
demonstrated subnanomolar IC50 values for Met inhibition in
the biochemical assay. A computation modeling was used to
reveal the different interactions of the C-30 side chain of 4 with
Met in comparison with the C-40 side chain of 2. We expected
the structural information would contribute to the further
design of new scaffolds against Met, as in the case of
designing 3 from 1 and 2.
aDevelopment Center for Biotechnology, No. 101, Lane 169, Kangning St., Xizhi
District, New Taipei City 22180, Taiwan. E-mail: lukehuang@mail.ncyu.edu.tw;
Fax: +886 5 271 7901; Tel: +886 5 271 7959
bDepartment of Applied Chemistry, National Chiayi University, No. 300, Syuefu Rd.,
Chiayi City 60004, Taiwan
† Electronic supplementary information (ESI) available: Ligplot diagrams of the
ATP binding site of Met complexed with compounds 2 and 20, and the kinase
proling data of 20. See DOI: 10.1039/c4ra08720h
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Fig. 1 Structures of SU11274 (1), PHA665752 (2), PF-2341066 (3), and pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-one of scaffold 4.
pyrroles 13a–e served as the starting material to react with
Results and discussion
Chemistry
phenylsulfonyl chloride (PhSO2Cl) in the presence of NaOH to
provide the N1-protected pyrrole 14a–e in 86–94% yields
(Scheme 2). The carbonyl groups in 14a–e could be reacted
with ethane-1,2-dithiol to give the dithiane 15a–e in 87–99%
yields. Removal of phenylsulfonyl group in 15a–e under basic
condition provided 16a–e in 96% to >99% yields, which was
treated with LiAlH4 in THF at room temperature to afford
amine 17a–e in 60–96% yields. Introduction of a formyl group
Scheme 1 presents the synthesis of pyrrole–5-(2,6-dichlor-
obenzyl)sulfonylindolin-2-ones 7–12 of scaffold 4 bearing C-30
carboxyl-, ethoxycarbonyl-, and amido-ethyl side chains (X ¼
CO2H, CO2Et, and CONR2R3 in 4, Fig. 1). 2-Formylpyrrole 5,
prepared according to our published procedures,17 was
condensed with 5-[(2,6-dichlorobenzyl)sulfonyl]indolin-2-one
(6) to provide compound 7 bearing a C-30 carboxylethyl group in
89% yield. Esterication of 7 in EtOH in the presence of SOCl2
provided 8 that possessed a C-30 ethoxycarbonylethyl group in
92% yield. Amidation of the carboxyl group in 7 by various
amines using CDI as the coupling agent provided pyrrole–5-(2,6-
dichlorobenzyl)sulfonylindolin-2-ones 9–12 bearing
amido-ethyl group in 70–85% yields.
a
C-30
For the synthesis of pyrrole–indolin-2-ones 20–24 bearing
C-30 aminopropyl side chains (X ¼ CH2NR2R3 in 4, Fig. 1),
Scheme 2 Synthesis of pyrrole–5-(2,6-dichlorobenzyl)sulfonylindo-
lin-2-ones 20–24 equipped with C-30 aminopropyl side chain.
Reagents and conditions: (a) PhSO2Cl, NaOH, CH2Cl2, 86–94%; (b)
Scheme 1 Synthesis of pyrrole–5-(2,6-dichlorobenzyl)sulfonylindo- ethane-1,2-dithiol, BF3$OEt2, MeOH, 87–99%; (c) NaOH, H2O, MeOH,
lin-2-ones 7–12 bearing C-30 carboxyl-, ethoxycarbonyl-, or amido- reflux, 96% to >99%; (d) LiAlH4, THF, r.t., 60–96%; (e) POCl3, DMF, 71–
ethyl side chains as Met kinase inhibitors. Reagents and conditions: (a) 97%; (f) HgCl2, CaCO3, CH3CN, H2O, 66–93%; and (g) 5-(2,6-
piperidine, EtOH, reflux, 12 h, 89%; (b) SOCl2, EtOH, 92%; and (c) 7, dichlorobenzylsulfonyl)indolin-2-one (6), piperidine, EtOH, reflux, 60–
amine, CDI, CH2Cl2, 70–85%.
78%.
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to the C-2 position of 17a–e was accomplished by use of 5-(2,6-dichlorobenzylsulfonyl)indolin-2-one (6) to provide the
Vilsmeier–Haack reaction to generate 18a–e in 71–97% yields. corresponding 20–24 with aminopropyl groups at their C-30
Deprotection of the dithianyl group using HgCl2 afforded positions in 60–78% yields.
19a–e in 66–93% yields, which was eventually condensed with
Table 1 The enzymatic and cellular activities of pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones 7–12 and 20–24 with different C-30 side
chains as Met kinase inhibitors
% inhibition at 1.0 mM
Compd
X
Met IC50a (nM)
30.40
MKN45 IC50a,b (mM)
Aurora A
65.5%c
Aurora B
7
>10
9.4%
8
9
20.20
4.22
0.15
3.25 ꢀ 0.41
>10
22.7%
13.0%
10.8%
16.3%
6.9%
9.2%
10
2.34 ꢀ 0.13
11
12
7.17
2.94
9.25 ꢀ 0.32
0.99 ꢀ 0.10
2.2%
8.7%
5.5%
1.3%
20
21
0.37
7.70
0.22 ꢀ 0.03
0.40 ꢀ 0.05
2.7%
1.9%
34.3%
6.2%
22
23
0.31
0.43
0.82 ꢀ 0.08
0.47 ꢀ 0.05
4.2%
22.3%
35.4%
23.7%
24
0.26
9d
0.24 ꢀ 0.02
0.10 ꢀ 0.03
6.0%
15.3%
2
—
—
—
a
b
c
d
The IC50 values were averaged from two independent dose–response curves. A 72 h cell proliferation assay. IC50: 855 nM. Obtained from
literature.19
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In vitro potency
Western blotting
The IC50 values of pyrrole–5-(2,6-dichlorobenzyl)sulfonylindo- As shown in Table 1, the great enhancement for the Met inhi-
lin-2-ones 7–12 and 20–24 for the inhibition of Met kinase and bition by compounds 20 and 22–24 did not reect on their
the proliferation of MKN45 cells (gastric carcinoma) that carry antiproliferative activities (MKN45 IC50: 0.22–0.82 mM). We then
an amplied Met proto-oncogene18 are presented in Table 1. We characterized the effect of potent compound 20 (MKN45 IC50:
also tested the potencies of 7–12 and 20–24 for the inhibition of 0.22 mM) on Met signal transduction in MKN45 cells by Western
Aurora kinases for comparison due to pyrrole–indolin-2-ones blot, and the results are presented in Fig. 2. Strong inhibition of
possessing a C-5 bulky group and a C-3 carboxylethyl side chain tyrosine phosphorylation in the Met kinase domain (pY1234/
were found to show Aurora kinase inhibitory activities in our 1235) was observed with 20 at a concentration of 1.1 mM and the
previous study.17 As shown in Table 1, the prototype pyrrole–5- reduced level of phosphorylated Met (IC50 ꢁ 0.1–1.1 mM)
(2,6-dichlorobenzyl)sulfonylindolin-2-one 7 bearing a C-30 car- correlated to the antiproliferative IC50 value of 20 (0.22 mM). The
boxyethyl group displayed an IC50 value of 30.40 nM for Met phosphorylation of Met docking protein Gab-1 was completely
inhibition. However, it did not show signicant anti- inhibited at the concentration of 0.4 mM. Compound 20 also
proliferative activity for MKN45 cells (IC50 > 10 mM). Compound potently inhibited the constitutive downstream signaling
8, the ethyl ester analog to 7, showed a slightly improved through PLC-g, FAK, Akt, STAT-3, and Erk, which provided the
potency (IC50: 20.20 nM) as well as enhanced antiproliferative evidence of Met inhibition by 20 in cell. Regarded as the
activity for MKN45 (IC50: 3.25 mM). These two compounds did downstream cellular effectors of Met inhibition, the phos-
not signicantly inhibit Aurora kinases expect 7 inhibited phorylation of STAT3 and ERK in MKN45 cells was completely
Aurora A with an IC50 value of 855 nM.
abolished by 20 at the concentration below 100 nM, showing
For further improving the Met and MKN45 inhibitory better correlation to the results from biochemical assay than the
activities, we evaluated compounds 9–12 with the C-30 ami- antiproliferative activity.
doethyl substituents. These compounds showed remarkable
improvements for Met inhibition with IC50 values less than 10
nM (IC50 for 2: 9 nM).19 The most potent amido compound 10
Kinase proling
with
a
N,N-diethylaminoethylamino (Et2NCH2CH2NH–)
Since compound 20 shared a similar scaffold with the multi-
kinase inhibitor sunitinib,14 we explored its inhibition prole
against a panel of 451 kinases at the concentration of 1.0 mM on
DiscoveRx Kinomescan scanMax platform.20 The results
revealed that CAMKK1, CDKL2, CSF1R, DCAMKL3, FLT3
(D835Y), FLT3 (ITD), GRK1, GRK7, IRAK4, JAK2 (JH1domain-
catalytic), MAP4K2, MAP4K3, MAP4K4, MAP4K5, MET,
MET(M1250T), MET(Y1235D), MST2, PDGFRB, RSK3 (Kin.-
Dom.1-N-terminal), TAOK1, TAOK2, TAOK3, TNIK, TRKB, and
YSK4 kinases were inhibited signicantly by 20 with a
percentage of control of <10% (see the table in the ESI†).
substituent inhibited Met at subnanomolar concentration
(IC50: 0.15 nM) and showed antiproliferative activity for
MKN45 cells at low micromolar concentration (IC50: 2.34 mM).
The N-methylpiperazinyl analog 12 displayed the most potent
antiproliferative activity (MKN45 IC50: 0.99 mM) among the
amido compounds 9–12. Respective comparison of kinase
and cell activities of 9 with 11 and 10 with 12 suggested that
the NH group in the amido moiety would prot the enzymatic
but not the cell activity. The difference in the cell activities of
these compounds might come from their different cell
permeability or inhibition proles against other kinases, e.g.,
VEGFR-2, PDGFR, and FGFR1.15
Pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones 20–24
bound with C-30 aminopropyl groups showed enhanced
activities both for Met kinase and MKN45 cells (Table 1).
Except for 21 with a diethylamino group, these compounds
inhibited Met at subnanomolar concentrations (IC50: 0.26–
0.43 nM) and demonstrated better antiproliferative activities
than their carboxyl, ester, and amido analogs (i.e. 7–12) on
MKN45 cells with IC50 values less than 1.0 mM. The promising
results from the biochemical assay shown in Table 1 indicated
that pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones of
scaffold 4 with C-30 side chains also showed promising Met
kinase inhibitory activity as their C-40 analog 2. Furthermore,
the use of the C-30 ethyl ester, amido, and amino side chains
also diminished the activity of the corresponding compounds
against Aurora A and Aurora B kinases (IC50 > 1.0 mM, Table 1)
in comparison with the use of a C-30 carboxyl side chain
(compound 7). A great selectivity (ꢁ100- to 1000-fold) was thus
achieved for Met over Aurora kinases for the compounds in
Table 1.
Fig. 2 Effect of 20 on Met phosphorylation and signal transduction in
MKN45 cells.
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through Gab-1, PLC-g, FAK, Akt, STAT-3, and ERK were found
on MKN45 cells treated with 20. Among the proteins, STAT3 and
ERK were completely inhibited by 20 at the concentration of
100 nM.
Experimental
Chemistry
Reagents were used as purchased without further purication.
5-(2,6-Dichlorobenzyl)sulfonylindolin-2-one (6) was prepared
according to literature procedures.22,23 Proton NMR spectra were
recorded on a Bruker (500 MHz) spectrometer with CDCl3 or
DMSO-d6 as solvent. Multiplicities are abbreviated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling
constant (hertz). Carbon-13 NMR spectra were obtained from a
Bruker spectrometer (125 MHz) by use of CDCl3 or DMSO-d6 as
solvent. Carbon-13 chemical shis are referenced to the center
peak of CDCl3 (d 77.0 ppm) or DMSO-d6 (d 39.5 ppm). ESI-MS
spectra were recorded with an Applied Biosystems API 300 mass
spectrometer. The purities of the compounds were greater than
95% as determined from reversed-phase HPLC. The synthesis of
compounds 5, 13a–e, 17a–e, 18a–e, and 19a–e was followed our
previously reported procedures.17
Fig. 3 Overlapping of the docked binding modes of compounds 2 and
20 in the ATP binding site of Met kinase. Compound 2 is shown as light
blue sticks, 20 is shown as yellow sticks, hydrogen bonds are shown as
dashed gray line, and the oxygen, nitrogen, and sulfur atoms are
colored in red, blue, and orange, respectively. The C-30 side chain of
20 faced the region around Ile1084 and the C-40 side chain of 2 faced
the region around Tyr1159 and His1094.
General procedure for the synthesis of N-phenysulfonyl-
pyrroles 14a–e. A CH2Cl2 solution (250 mL) containing NaOH
suspension (0.10 mol, 5.0 equiv.) was added with compound
13a–e (ꢁ0.020 mol, 1.0 equiv.). The mixture was stirred at 0 ꢂC
for 30 min. A solution of phenylsulfonyl chloride (0.030 mol, 1.5
equiv.) in CH2Cl2 (50 mL) was slowly added to the reaction
mixture in a period of 30 min. Aer the addition was completed,
Molecular modeling
To explore the interaction of pyrrole–5-(2,6-dichlorobenzyl)sul-
fonylindolin-2-ones of scaffold 4 with Met, we modeled the
binding mode of the potent compound 20 as well as reference
compound 2 in Met (PDB code 2RFS)12 using docking simula-
tions. Aer energy minimization, the binding poses of 20 and 2
were overlapped as illustrated in Fig. 3. Obviously, 20 shared
ꢂ
the reaction mixture was stirred for additional 30 min at 0 C
similar binding pose to 2 in Met. Both compounds formed two
and for 12 h at room temperature. The solution was added with
H2O (100 mL) and the organic layer was separated. The aqueous
layer was re-extracted with CH2Cl2 (3 ꢃ 50 mL), and the
combined organic layer was dried over MgSO4(s) and concen-
trated under reduced pressure to give the desired 14a–e in 86–
94% yields.
N,N-Dimethyl-3-[4-oxo-1-(phenylsulfonyl)-4,5,6,7-tetrahydro-
1H-indol-3-yl]propanamide (14a). Yield: 94%; 1H NMR (CDCl3) d
7.85 (d, J ¼ 7.6 Hz, 2H), 7.66 (t, J ¼ 7.5 Hz, 1H), 7.55–7.58 (m,
2H), 7.06 (s, 1H), 3.02 (s, 3H), 2.89–2.98 (m, 7H), 2.56–2.61 (m,
2H), 2.36–2.41 (m, 2H), 2.02–2.09 (m, 2H); ESI-MS m/z 374.7
(M + H)+.
0
H-bonds from N1–H and N1 –H with Pro1158 and Met1160,
respectively, and an H-bond from C2]O with Met1160 in the
hinge region. The oxygen atom of the C-5 sulfonyl group formed
an H-bond with Asp1222, and the 2,6-dichlorophenyl tted the
hydrophobic pocket formed by Val1092, Tyr1230, and Gly1085
from the analysis of Ligplot.21 However, the side chain at the C-
30 position of 20 headed to the different region from the C-40
amino–amido group in 2, and thus interacted with different
residues in Met other than 2: 20 interacted with Ile1084 and 2
interacted with Tyr1159 and His1094 (Fig. 3). The hydrophilic
interaction of the C-30 dimethylamino group might contribute
the promising activity of 20.
N,N-Diethyl-3-[4-oxo-1-(phenylsulfonyl)-4,5,6,7-tetrahydro-1H-
indol-3-yl]propanamide (14b). Yield: 93%; 1H NMR (CDCl3) d
7.85 (d, J ¼ 7.6 Hz, 2H), 7.66 (t, J ¼ 7.5 Hz, 1H), 7.54–7.57 (m,
2H), 7.06 (s, 1H), 3.29–3.38 (m, 4H), 2.96 (t, J ¼ 6.8 Hz, 4H),
Conclusions
Pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones of scaffold 2.54–2.59 (m, 2H), 2.37–2.43 (m, 2H), 2.03–2.10 (m, 2H), 1.07–
4 with a C-30 side chain was designed as potent Met kinase 1.13 (m, 6H); ESI-MS m/z 402.9 (M + H)+.
inhibitors. Structural optimization led to compounds 10, 20,
3-[3-Oxo-3-(pyrrolidin-1-yl)propyl]-1-(phenylsulfonyl)-6,7-dihy-
and 22–24 that showed subnanomolar IC50 values in the dro-1H-indol-4(5H)-one (14c). Yield: 89%; 1H NMR (CDCl3) d
biochemical assay. Computation modeling revealed that the C- 7.85 (d, J ¼ 7.5 Hz, 2H), 7.65 (t, J ¼ 7.5 Hz, 1H), 7.53–7.56 (m,
30 side chain of 4 interacted with Ile1084 and would contribute 2H), 7.06 (s, 1H), 3.38–3.45 (m, 4H), 2.92–2.97 (m, 4H), 2.50–
to its promising activity. The potent compound 20 possessed an 2.55 (m, 2H), 2.34–2.41 (m, 2H), 2.02–2.09 (m, 2H), 1.85–1.92
IC50 value of 0.37 nM also inhibited the proliferation of MKN45 (m, 2H), 1.77–1.84 (m, 2H); ESI-MS m/z 400.8 (M + H)+.
cells (IC50: 0.22 mM). Cellular events including the inhibition of
Met autophosphorylation and the downstream signaling 1H-indol-4(5H)-one (14d). Yield: 89%; H NMR (CDCl3) d (d, J
3-(3-Morpholino-3-oxopropyl)-1-(phenylsulfonyl)-6,7-dihydro-
1
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¼ 7.6 Hz, 2H), 7.67 (t, J ¼ 7.5 Hz, 1H), 7.56–7.59 (m, 2H), 7.07 70% H2O were added with NaOH (5.0 equiv.). The reaction
(s, 1H), 3.55–3.65 (m, 8H), 2.90–2.99 (m, 4H), 2.57–2.62 (m, mixture was heated at reux for 3.0 h. The solution was
2H), 2.37–2.42 (m, 2H), 2.04–2.11 (m, 2H); ESI-MS m/z 417.0 neutralized with 3.0 N HCl and the resulting precipitate was
(M + H)+.
collected to provide the desired 16a–e in 96% to >99% yields.
3-[3-(4-Methylpiperazin-1-yl)-3-oxopropyl]-1-(phenylsulfonyl)- The spectroscopic data of were consistence with our previous
6,7-dihydro-1H-indol-4(5H)-one (14e). Yield: 86%; 1H NMR report.17
(CDCl3) d 7.86 (d, J ¼ 7.5 Hz, 2H), 7.67 (t, J ¼ 7.5 Hz, 1H), 7.56–
7.59 (m, 2H), 7.06 (s, 1H), 3.50–3.64 (m, 4H), 2.90–2.98 (m, dichlorobenzyl)sulfonylindolin-2-ones
4H), 2.57–2.62 (m, 2H), 2.33–2.41 (m, 6H), 2.28 (s, 3H), 2.04– mylpyrroles 5 or 19a–e (ꢁ0.50 mmol, 1.0 equiv.) was mixed with
2.10 (m, 2H); ESI-MS: m/z 430.0 (M + H)+.
5-(2,6-dichlorobenzyl)sulfonylindolin-2-one (6, 1.0 equiv.) and
General procedure for the synthesis of pyrrole–5-(2,6-
7
and 20–24. 2-For-
General procedure for the synthesis of dithianes 15a–e. To a piperidine (0.10 mL) in EtOH (5.0 mL). The reaction mixture
methanol solution containing compound 14a–e (ꢁ0.20 mol, 1.0 was heated at reux for 12 h. The resulting precipitate was
equiv.) and ethane-1,2-dithiol (1.1 equiv.) was added BF3$OEt2 ltered and washed with EtOH to provide the corresponding
(0.25 equiv.). The reaction mixture was stirred at room pyrrole–5-(2,6-dichlorobenzyl)sulfonylindolin-2-ones 7 and 20–24
temperature for 45 min. The solution was mixed with 10% in 60–89% yields.
aqueous NaOH (50 mL) and concentrated under reduced pres-
(Z)-3-(2-{[5-(2,6-Dichlorobenzylsulfonyl)-2-oxoindolin-3-ylidene]-
sure to remove the methanol. The solution was extracted with methyl}-4-oxo-4,5,6,7-tetrahydro-1H-indol-3-yl)propanoic acid (7).
CH2Cl2 (3 ꢃ 200 mL), and the organic layer was washed with Yield: 89%; 1H NMR (DMSO-d6) d 13.65 (s, 1H), 12.11 (brs, 1H),
H2O, dried over MgSO4(s), and concentrated under reduced 11.52 (s, 1H), 8.21 (d, J ¼ 1.6 Hz, 1H), 7.94 (s, 1H), 7.46–7.52 (m,
pressure to give the desired 15a–e in 87–99% yields.
3H), 7.39 (dd, J ¼ 8.7, 7.4 Hz, 1H), 7.06 (d, J ¼ 8.2 Hz, 1H), 4.88
N,N-Dimethyl-3-[10-(phenylsulfonyl)-10,50,60,70-tetrahydrospiro (s, 2H), 3.20 (t, J ¼ 7.6 Hz, 2H), 2.93 (t, J ¼ 6.1 Hz, 2H), 2.52 (t, J ¼
[[1,3]dithiolane-2,40-indole]-30-yl]propanamide (15a). Yield: 87%; 7.6 Hz, 2H), 2.40–2.44 (m, 2H), 2.02–2.10 (m, 2H); 13C NMR
1H NMR (CDCl3) d 7.77 (d, J ¼ 7.5 Hz, 2H), 7.62 (t, J ¼ 7.5 Hz, (DMSO-d6) d 19.28, 21.83, 21.91, 33.87, 37.50, 56.89, 108.78,
1H), 7.51–7.54 (m, 2H), 6.99 (s, 1H), 3.42–3.50 (m, 2H), 3.30–3.36 114.70, 118.27, 118.77, 124.91, 124.99, 125.43, 126.16, 126.53,
(m, 2H), 3.06–3.11 (m, 2H), 3.02 (s, 3H), 2.97 (s, 3H), 2.61–2.70 127.81, 130.16, 131.02, 133.03, 135.51, 142.09, 147.59, 168.70,
(m, 4H), 2.15–2.19 (m, 2H), 1.80–1.89 (m, 2H); ESI-MS m/z 450.8 172.97, 192.83; ESI-MS m/z 573.0 (M + H)+.
(M + H)+.
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-({3-[3-(dimethylamino)propyl]-
N,N-Diethyl-3-[10-(phenylsulfonyl)-10,50,60,70-tetrahydrospiro 4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl}methylene)indolin-2-one (20).
1
[[1,3]dithiolane-2,40-indole]-30-yl]propanamide (15b). Yield: 92%; Yield: 70%; H NMR (DMSO-d6) d 13.51 (s, 1H), 11.46 (brs, 1H),
1H NMR (CDCl3) d 7.77 (d, J ¼ 7.6 Hz, 2H), 7.61 (t, J ¼ 7.4 Hz, 7.97 (s, 1H), 7.42–7.57 (m, 3H), 7.34–7.42 (m, 2H), 7.07 (d, J ¼ 6.9
1H), 7.50–7.54 (m, 2H), 6.99 (s, 1H), 3.44–3.51 (m, 2H), 3.37–3.43 Hz, 1H), 4.87 (s, 2H), 2.92–3.05 (m, 4H), 2.35–2.45 (m, 4H), 1.97–
(m, 2H), 3.30–3.36 (m, 4H), 3.07–3.13 (m, 2H), 2.61–2.69 (m, 2.19 (m, 8H), 1.57–1.72 (m, 2H); 13C NMR (DMSO-d6) d 19.73,
4H), 2.15–2.19 (m, 2H), 1.82–1.88 (m, 2H), 1.10–1.20 (m, 6H); 21.84, 21.96, 26.58, 37.56, 43.98, 56.37, 56.86, 108.88, 114.08,
ESI-MS m/z 478.8 (M + H)+.
117.69, 118.80, 124.90, 124.95, 125.04, 125.77, 126.62, 127.83,
3-[10-(Phenylsulfonyl)-10,50,60,70-tetrahydrospiro[[1,3]dithiolane- 130.17, 130.82, 134.51, 135.52, 142.03, 147.73, 168.67, 192.72; ESI-
2,40-indole]-30-yl]-1-(pyrrolidin-1-yl)propan-1-one (15c). Yield: MS m/z 586.0 (M + H)+.
1
90%; H NMR (CDCl3) d 7.74–7.80 (m, 2H), 7.62 (t, J ¼ 7.5 Hz,
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-({3-[3-(diethylamino)propyl]-
1H), 7.51–7.54 (m, 2H), 6.99 (s, 1H), 3.39–3.53 (m, 6H), 3.28–3.38 4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl}methylene)indolin-2-one
(m, 2H), 3.05–3.15 (m, 2H), 2.57–2.68 (m, 4H), 2.12–2.22 (m, (21). Yield: 78%; 1H NMR (DMSO-d6) d 13.62 (s, 1H), 8.11 (s, 1H),
2H), 1.92–2.00 (m, 2H), 1.79–1.90 (m, 4H); ESI-MS m/z 477.0 (M 7.89 (s, 1H), 7.43–7.56 (m, 3H), 7.37–7.42 (m, 1H), 7.07 (d, J ¼
+ H)+.
8.10 Hz, 1H), 4.87 (s, 2H), 2.90–3.02 (m, 4H), 2.32–2.47 (m, 8H),
1-Morpholino-3-[10-(phenylsulfonyl)-10,50,60,70-tetrahydrospiro
2.01–2.12 (m, 2H), 1.65–1.71 (m, 2H), 0.91 (t, J ¼ 7.1 Hz, 6H); 13
C
[[1,3]dithiolane-2,40-indole]-30-yl]propan-1-one (15d). Yield: 99%; NMR (DMSO-d6) d 10.36, 21.02, 21.85, 21.98, 26.59, 37.59, 44.85,
1H NMR (CDCl3) d 7.77 (d, J ¼ 7.4 Hz, 2H), 7.62 (t, J ¼ 7.4 Hz, 50.24, 56.88, 108.86, 114.10, 117.77, 118.83, 124.97, 125.12,
1H), 7.51–7.54 (m, 2H), 6.99 (s, 1H), 3.61–3.69 (m, 6H), 3.42–3.52 125.48, 126.33, 126.51, 127.81, 130.15, 130.95, 134.42, 135.50,
(m, 4H), 3.31–3.38 (m, 2H), 3.07–3.12 (m, 2H), 2.61–2.71 (m, 142.04, 147.72, 168.70, 192.67; ESI-MS m/z 614.0 (M + H)+.
4H), 2.15–2.19 (m, 2H), 1.83–1.87 (m, 2H); ESI-MS m/z 492.9 (M
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-({4-oxo-3-[3-(pyrrolidin-1-
yl)propyl]-4,5,6,7-tetrahydro-1H-indol-2-yl}methylene)indolin-2-
+ H)+.
1-(4-Methylpiperazin-1-yl)-3-[10-(phenylsulfonyl)-10,50,60,70-tetra- one (22). Yield: 66%; 1H NMR (DMSO-d6) d 13.62 (s, 1H), 11.52
hydrospiro[[1,3]dithiolane-2,40-indole]-30-yl]propan-1-one
(15e). (s, 1H), 8.05 (s, 1H), 7.86 (s, 1H), 7.45–7.50 (m, 3H), 7.34–7.41
Yield: 96%; 1H NMR (CDCl3) d 7.77 (d, J ¼ 7.5 Hz, 2H), 7.62 (t, J ¼ (m, 1H), 7.05 (d, J ¼ 8.2 Hz, 1H), 4.85 (s, 2H), 3.03 (t, J ¼ 6.8 Hz,
7.5 Hz, 1H), 7.50–7.53 (m, 2H), 6.99 (s, 1H), 3.64–3.68 (m, 2H), 2H), 2.93 (t, J ¼ 5.9 Hz, 2H), 2.27–2.42 (m, 8H), 2.01–2.09 (m,
3.50–3.54 (m, 2H), 3.42–3.49 (m, 2H), 3.30–3.37 (m, 2H), 3.07– 2H), 1.63–1.73 (m, 6H); 13C NMR (DMSO-d6) d 20.92, 21.84,
3.12 (m, 2H), 2.61–2.72 (m, 4H), 2.36–2.40 (m, 4H), 2.31 (s, 3H), 21.97, 22.17, 28.05, 37.58, 52.45, 53.26, 56.87, 108.87, 114.18,
2.16–2.20 (m, 2H), 1.82–1.88 (m, 2H); ESI-MS m/z 506.0 (M + H)+. 117.66, 118.78, 124.93, 124.99, 125.04, 126.40, 126.51, 127.81,
General procedure for the synthesis of pyrroles 16a–e. 130.17, 130.93, 134.71, 135.52, 142.03, 147.70, 168.70, 192.70;
Compounds 15a–e (1.0 equiv.) in a solution of 30% MeOH and ESI-MS m/z 612.0 (M + H)+.
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(dd, J ¼ 4.2, 4.2 Hz, 1H), 7.46–7.53 (m, 3H), 7.39 (dd, J ¼ 8.7, 7.4
Hz, 1H), 7.06 (d, J ¼ 8.2 Hz, 1H), 4.88 (s, 2H), 3.22 (t, J ¼ 7.3 Hz,
2H), 2.94 (t, J ¼ 6.1 Hz, 2H), 2.48 (s, 3H), 2.45–2.40 (m, 2H), 2.37
(t, J ¼ 7.3 Hz, 2H), 2.03–2.10 (m, 2H); 13C NMR (DMSO-d6) d
19.67, 21.85, 21.93, 24.44, 35.34, 37.54, 56.90, 108.80, 114.46,
118.06, 118.74, 125.00, 125.37, 126.23, 126.55, 127.81, 130.18,
131.10, 133.66, 135.51, 142.06, 147.57, 168.67, 170.75, 192.81;
ESI-MS m/z 586.0 (M + H)+.
(Z)-3-(2-{[5-(2,6-Dichlorobenzylsulfonyl)-2-oxoindolin-3-ylidene]
methyl}-4-oxo-4,5,6,7-tetrahydro-1H-indol-3-yl)-N-[2-(dieth-
ylamino)ethyl]propanamide (10). Yield: 72%; 1H NMR (DMSO-d6)
d 13.60 (s, 1H), 11.49 (brs, 1H), 8.19 (s, 1H), 7.89 (s, 1H), 7.64 (d, J
¼ 5.5 Hz, 1H), 7.46–7.54 (m, 3H), 7.35–7.43 (m, 1H), 7.06 (d, J ¼
8.2 Hz, 1H), 4.87 (s, 2H), 3.18–3.27 (m, 2H), 2.89–3.03 (m, 4H),
2.40–2.45 (m, 2H), 2.37 (t, J ¼ 7.2 Hz, 2H), 2.28 (q, J ¼ 7.0 Hz,
4H), 2.22 (t, J ¼ 7.2 Hz, 2H), 2.01–2.12 (m, 2H), 0.77 (t, J ¼ 7.0 Hz,
6H); 13C NMR (DMSO-d6) d 10.56, 19.74, 21.85, 21.93, 35.47,
35.93, 37.55, 45.51, 50.64, 56.89, 108.76, 114.41, 117.94, 118.73,
124.93, 125.05, 125.49, 126.34, 126.47, 127.80, 130.17, 131.13,
133.50, 135.52, 142.07, 147.50, 168.69, 170.21, 192.79; ESI-MS
m/z 671.0 (M + H)+.
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-{[3-(3-morpholinopropyl)-4-
oxo-4,5,6,7-tetrahydro-1H-indol-2-yl]methylene}indolin-2-one (23).
Yield: 72%; 1H NMR (DMSO-d6) d 13.64 (s, 1H), 11.48 (brs, 1H),
8.21 (s, 1H), 7.88 (s, 1H), 7.46–7.50 (m, 3H), 7.33–7.41 (m, 1H),
7.05 (d, J ¼ 8.2 Hz, 1H), 4.86 (s, 2H), 3.51–3.57 (m, 4H), 3.02 (t, J
¼ 7.1 Hz, 2H), 2.93 (t, J ¼ 6.0 Hz, 2H), 2.21–2.42 (m, 8H), 1.99–
2.09 (m, 2H), 1.67–1.74 (m, 2H); 13C NMR (DMSO-d6) d 21.12,
21.84, 21.98, 26.07, 37.58, 52.34, 56.35, 56.90, 65.22, 108.81,
114.20, 118.05, 118.84, 124.94, 125.02, 125.10, 126.34, 126.46,
127.81, 130.16, 131.03, 134.94, 135.52, 142.02, 147.70, 168.72,
192.70; ESI-MS m/z 628.0 (M + H)+.
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-({3-[3-(4-methylpiperazin-1-
yl)propyl]-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl}methylene)indolin-2-
one (24). Yield: 60%; 1H NMR (DMSO-d6) d 13.67 (s, 1H), 11.52 (brs,
1H), 8.20 (s, 1H), 7.85 (s, 1H), 7.46–7.51 (m, 3H), 7.37–7.42 (m, 1H),
7.06 (d, J ¼ 8.2 Hz, 1H), 4.88 (s, 2H), 2.91–3.03 (m, 4H), 2.22–2.44
(m, 10H), 2.01–2.14 (m, 7H), 1.70 (d, J ¼ 6.8 Hz, 2H); 13C NMR
(DMSO-d6) d 21.22, 21.84, 21.97, 26.59, 37.59, 44.66, 51.70, 53.72,
56.01, 56.95, 108.78, 114.22, 118.13, 118.83, 124.91, 125.06, 126.31,
126.48, 127.81, 130.16, 130.96, 135.03, 135.51, 142.00, 143.92,
147.67, 168.72, 192.68; ESI-MS m/z 641.0 (M + H)+.
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-({4-oxo-3-[3-oxo-3-(pyrroli-
din-1-yl)propyl]-4,5,6,7-tetrahydro-1H-indol-2-yl}methylene)indo-
lin-2-one (11). Yield: 82%; H NMR (DMSO-d6) d 13.64 (s, 1H),
(Z)-Ethyl-3-(2-{[5-(2,6-dichlorobenzylsulfonyl)-2-oxoindolin-3-
ylidene]methyl}-4-oxo-4,5,6,7-tetrahydro-1H-indol-3-yl)propanoate
(8). To a solution of 7 (105.5 mg, 0.1840 mmol) in EtOH (2.0 mL)
was added with SOCl2 (0.10 mL). The reaction mixture was
heated at reux for 12 h. The solution was concentrated under
reduced pressure. The residue was added with H2O (5.0 mL) and
heated. The solution was cooled to room temperature and the
resultant solids were collected to give the desired 8 (101.8 mg,
0.1692 mmol) in 92% yield: 1H NMR (DMSO-d6) d 13.68 (s, 1H),
11.53 (brs, 1H), 8.27 (d, J ¼ 1.1 Hz, 1H), 7.95 (s, 1H), 7.46–7.53
(m, 3H), 7.34–7.42 (m, 1H), 7.06 (d, J ¼ 8.2 Hz, 1H), 4.88 (s, 2H),
4.02 (q, J ¼ 7.1 Hz, 2H), 3.25 (t, J ¼ 7.4 Hz, 2H), 2.94 (t, J ¼ 6.0 Hz,
2H), 2.61 (t, J ¼ 7.4 Hz, 2H), 2.38–2.45 (m, 2H), 2.01–2.11 (m,
2H), 1.13 (t, J ¼ 7.1 Hz, 3H); 13C NMR (DMSO-d6) d 13.13, 19.12,
21.84, 21.88, 33.49, 37.47, 56.90, 58.83, 108.81, 114.87, 118.32,
118.77, 124.93, 124.99, 125.34, 126.23, 126.63, 127.81, 130.14,
131.08, 132.42, 135.50, 142.11, 147.61, 168.68, 171.27, 192.87;
ESI-MS m/z 601.0 (M + H)+.
General procedure for the synthesis of pyrrole–5-(2,6-
dichlorobenzyl)sulfonylindolin-2-ones 9–12. Compound 7 (ꢁ1.0
g, 1.0 equiv.) was suspended in CH2Cl2 (20 mL) and mixed with
CDI (1.2 equiv.). The reaction mixture was stirred at room
temperature for 3.0 h. The solution was added with methyl-
amine, N,N-diethylethane-1,2-diamine, pyrrolidine, or 1-meth-
ylpiperazine (2.0 equiv.), and the reaction mixture was stirred at
room temperature for 12 h. The solution was diluted with
CH2Cl2 (200 mL), and washed with 0.1 N HCl (50 mL), saturated
Na2CO3 (50 mL), and brine (50 mL). The organic layer was dried
over anhydrous MgSO4(s) and concentrated under reduced
pressure to provide the desired 9–12 as yellow solids in 70–85%
yields.
1
11.44 (s, 1H), 8.22 (d, J ¼ 1.3 Hz, 1H), 7.94 (s, 1H), 7.46–7.51 (m,
3H), 7.35–7.42 (m, 1H), 7.05 (d, J ¼ 8.2 Hz, 1H), 4.88 (s, 2H),
3.19–3.39 (m, 6H), 2.94 (t, J ¼ 5.9 Hz, 2H), 2.52–2.57 (m, 2H),
2.40–2.45 (m, 2H), 2.03–2.09 (m, 2H), 1.79–1.86 (m, 2H), 1.68–
1.76 (m, 2H); 13C NMR (DMSO-d6) d 19.35, 21.85, 21.93, 22.97,
24.65, 34.28, 37.54, 44.32, 44.83, 56.86, 108.75, 114.49, 118.19,
118.85, 124.99, 125.06, 125.41, 126.19, 126.48, 127.82, 130.15,
131.04, 133.81, 135.51, 142.04, 147.58, 168.70, 168.75, 192.86;
ESI-MS m/z 626.0 (M + H)+.
(Z)-5-(2,6-Dichlorobenzylsulfonyl)-3-({3-[3-(4-methylpiperazin-1-
yl)-3-oxopropyl]-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl}methylene)
indolin-2-one (12). Yield: 70%; 1H NMR (DMSO-d6) d 13.66 (s,
1H), 11.51 (brs, 1H), 8.21 (d, J ¼ 0.93 Hz, 1H), 7.91 (s, 1H), 7.47–
7.50 (m, 3H), 7.35–7.42 (m, 1H), 7.05 (d, J ¼ 8.2 Hz, 1H), 4.88 (s,
2H), 3.37–3.45 (m, 4H), 3.20 (t, J ¼ 7.6 Hz, 2H), 2.94 (t, J ¼ 6.0 Hz,
2H), 2.57 (t, J ¼ 7.6 Hz, 2H), 2.43 (t, J ¼ 6.0 Hz, 2H), 2.14–2.17 (m,
4H), 2.12 (s, 3H), 2.04–2.09 (m, 2H); 13C NMR (DMSO-d6) d 19.85,
21.86, 21.93, 32.63, 37.53, 39.90, 43.75, 44.65, 53.31, 53.76,
56.89, 108.76, 114.63, 118.27, 118.89, 124.99, 125.39, 126.23,
126.54, 127.83, 130.17, 131.05, 133.42, 135.51, 142.06, 147.62,
168.70, 169.02, 192.98; ESI-MS m/z 655.0 (M + H)+.
Biological and computational methods
Met and Aurora kinase assays. Inhibition of kinase activity
by the test compound was measured by quantifying the amount
of 33P incorporation into the substrate in the presence of a test
compound. Standard assay conditions utilized
5 ng of
recombinant Met kinase (Upstate, cat. no. 14-526), 1 mg poly
Glu-Tyr (Sigma, Product number P0275), 100 mM ATP, 0.2 mCi
[33P]ATP (specic activity 3000 Ci mmolꢄ1, Perkin Elmer), 8 mM
MOPS–NaOH (pH 7.0), and 1 mM EDTA in a total volume of 25
(Z)-3-(2-{[5-(2,6-Dichlorobenzylsulfonyl)-2-oxoindolin-3-ylidene]
methyl}-4-oxo-4,5,6,7-tetrahydro-1H-indol-3-yl)-N-methyl-
propanamide (9). Yield: 85%; 1H NMR (DMSO-d6) d 13.61 (s, 1H),
11.49 (brs, 1H), 8.22 (d, J ¼ 1.6 Hz, 1H), 7.90 (s, 1H), 7.72
ꢂ
mL. Reaction mixtures were incubated at 30 C for 30 min and
58996 | RSC Adv., 2014, 4, 58990–58998
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reactions stopped by addition of 3% phosphoric acid; mixtures Two hundred genetic algorithm (GA) runs were performed for
were harvested onto a 96-well GF/B Unilter (Perkin Elmer) docking compounds 2 and 20. GOLD was run to save up to best
using a Unilter harvester (Perkin Elmer), and counted with a docking solutions for each ligands. The results were visually
TopCount microplate scintillation counter (PerkinElmer). The analyzed using PyMOL program (http://www.pymol.org/) and
IC50 values of inhibitors were determined aer assays were Ligplot.21
conducted at 3-fold serially diluted concentrations of each
compound in duplicate. The results were analyzed using linear
regression soware (GraphPad Prism version 4; GraphPad
Acknowledgements
Soware Inc., San Diego, CA). For Aurora assay, 1 ng of
We are grateful to the Ministry of Economic Affairs of the
recombinant Aurora A or Aurora B kinase (Upstate Biotech-
Republic of China and Ministry of Science and Technology of
nology), 2 mg peptide substrate (tetra-LRRLSLG, synthesized by
Republic of China (102-2113-M-415-005-MY2) for nancial
Genesis Biotech Inc.), 10 mM ATP (for Aurora A) or 40 mM ATP
support.
(for Aurora B), 0.2 mCi [33P]ATP (specic activity 3000 Ci
mmolꢄ1, Perkin Elmer), 8 mM MOPS–NaOH (pH 7.0), and 1 mM
EDTA in a total volume of 25 mL were utilized.
Notes and references
Cell proliferation assay. The antiproliferative activity of the
compounds with respect to human gastric carcinoma cell,
MKN45 (Japanese Collection of Research Bioresources), was
measured using the CellTiter 96 assay kit (Promega) following
the manufacturer's instruction. In brief, the cells were main-
tained in DMEM containing 0.2% FBS and incubated at 37 ꢂC in
5% CO2 atmosphere. Cells were plated at a density of 2500 cells
per well of a 96-well plate for 24 h, then treated with different
concentrations of the compounds, and incubated for another 72
hours. At the end of the incubation, CellTiter 96 Aqueous One
Solution Reagent (Promega) was added and incubated for
another 3.0 hours. Cell viability was determined by measuring
absorbance at 490 nm using EMax® microplate reader (Molec-
ular Devices). Data were processed and analyzed using Graph-
Pad Prism version 4.
1 R. Haddad, K. E. Lipson and C. P. Webb, Anticancer Res.,
2001, 21, 4243–4252.
2 P. Longati, P. M. Comoglio and A. Bardelli, Curr. Drug
Targets, 2001, 2, 41–55.
3 G. Maulik, A. Shrikhande, T. Kijima, P. C. Ma, P. T. Morrison
and R. Salgia, Cytokine Growth Factor Rev., 2002, 13, 41–59.
4 S. Giordano, C. Ponzetto, M. F. Di Renzo, C. S. Cooper and
P. M. Comoglio, Nature, 1989, 339, 155–156.
5 C. Birchmeier, W. Birchmeier, E. Gherardi and G. F. Vande
Woude, Nat. Rev. Mol. Cell Biol., 2003, 4, 915–925.
6 C. M. Stellrecht and V. Gandhi, Cancer Lett., 2009, 280, 1–14.
7 X. Liu, R. C. Newton and P. A. Scherle, Trends Mol. Med.,
2009, 16, 37–45.
8 P. M. Comoglio, S. Giordano and L. Trusolino, Nat. Rev. Drug
Discovery, 2008, 7, 504–516.
9 M. Sattler, Y. B. Pride, P. Ma, J. L. Gramlich, S. C. Chu,
L. A. Quinnan, S. Shirazian, C. Liang, K. Podar,
J. G. Christensen and R. Salgia, Cancer Res., 2003, 63,
5462–5469.
Western blot analysis. The effect of Met inhibitors on
cellular protein phosphorylation was evaluated by Western blot
analysis on MKN45 cells. Cells grown to subconuency were
grown overnight in 0.5% (FBS) and DMEM. Cells in low serum
were treated with compound 20 in DMSO (20 mM stock) for 30
minutes. Drug-treated cells were then harvested and washed 10 J. G. Christensen, R. Schreck, J. Burrows, P. Kuruganti,
twice with PBS, and the cell pellets were boiled in 2ꢃ SDS
sample buffer and subjected to SDS-PAGE and immunoblotting
analysis. Western blots were done using standard procedures
E. Chan, P. Le, J. Chen, X. Wang, L. Ruslim, R. Blake,
K. F. Lipson, J. Ramphal, S. Do, J. J. Cui, J. M. Cherrington
and D. B. Mendel, Cancer Res., 2003, 63, 7345–7355.
and probed with the following antibodies: anti-Met (Cell 11 X. Wang, P. Le, C. Liang, J. Chan, D. Kiewlich, T. Miller,
Signaling Technology), anti-phospho-Met (Tyr1234/1235), anti-
phospho-Gab-1 (Tyr627, Thermo Scientic), anti-phospho-FAK
(Tyr861, Thermo Scientic), anti-phospho-PLC-g (Tyr783), anti-
D. Harris, L. Sun, A. Rice, S. Vasile, R. A. Blake,
A. R. Howlett, N. Patel, G. McMahon and K. E. Lipson, Mol.
Cancer Ther., 2003, 2, 1085–1092.
phospho-Akt (Ser473), anti-phospho-STAT3 (Tyr705), anti- 12 S. F. Bellon, P. Kaplan-Leo, Y. Yang, Y. Zhang, J. Moriguchi,
phospho-p44/42 ERK mitogen-activated protein kinase (Thr202/
Tyr204), and horseradish peroxidase-conjugated secondary
antibodies (Cell Signaling).
K. Rex, C. W. Johnson, P. E. Rose, A. M. Long, A. B. O'Connor,
Y. Gu, A. Coxon, T. S. Kim, A. Tasker, T. L. Burgess and
I. Dussault, J. Biol. Chem., 2008, 283, 2675–2683.
Computational method. The three dimensional structure of 13 H. Y. Zou, Q. Li, J. H. Lee, M. E. Arango, S. R. McDonnell,
Met complexed with 1 was obtained from the Protein Data Bank
(entry: 2RFS).12 Hydrogen atoms were added, and water mole-
cules cocrystallized with the protein were removed from the
original structure using Accelrys Discovery Studio 2.5 (Accelrys,
S. Yamazaki, T. B. Koudriakova, G. Alton, J. J. Cui,
P.-P. Kung, M. D. Nambu, G. Los, S. L. Bender,
B. Mroczkowski and J. G. Christensen, Cancer Res., 2007,
67, 4408–4417.
Inc.). The modied crystal structure of Met was docked by a 14 M. Krug and A. Hilgeroth, Mini-Rev. Med. Chem., 2008, 8,
genetic algorithm based program (GOLD) using the Chem 1312–1327.
scoring (CCDC Soware Limited, Cambridge, U.K.). The genetic 15 L. Sun, N. Tran, C. Liang, F. Tang, A. Rice, R. Schreck,
algorithm was executed at the default settings, and the active
K. Waltz, L. K. Shawver, G. McMahon and C. Tang, J. Med.
Chem., 1999, 42, 5120–5130.
˚
site radius is 10 A from inhibitor complex for the docking study.
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16 J.-J. Huang, C.-C. Chiang, C. Liu, C.-L. Lai, S. F. Lin, Y.-H. Lin, 20 M. A. Fabian, W. H. Biggs III, D. K. Treiber, C. E. Atteridge,
R.-W. Wang, S.-c. Yang, J.-M. Chang, S.-H. Chuang and
P. Chen, US Pat., 7897602, March 1, 2011.
M. D. Azimioara, M. G. Benedetti, T. A. Carter, P. Ciceri,
P. T. Edeen, M. Floyd, J. M. Ford, M. Galvin, J. L. Gerlack,
R. M. Grotzfeld, S. Herrgard, D. E. Insko, M. A. Insko,
A. G. Lai, J.-M. Lelias, S. A. Mehta, Z. V. Milanov,
A. M. Velasco, L. M. Wodicka, H. K. Patel, P. P. Zarrinkar
and D. J. Lockhart, Nat. Biotechnol., 2005, 23, 329–336.
21 A. C. Wallace, R. A. Laskowski and J. M. Thornton, Protein
Eng., 1995, 8, 127–134.
17 C.-C. Chiang, Y.-H. Lin, S. F. Lin, C.-L. Lai, C. Liu, W.-Y. Wei,
S.-c. Yang, R.-W. Wang, L.-W. Teng, S.-H. Chuang,
J.-M. Chang, T.-T. Yuan, Y.-S. Lee, P. Chen, W.-K. Chi,
J.-Y. Yang, H.-J. Huang, C.-B. Liao and J.-J. Huang, J. Med.
Chem., 2010, 53, 5929–5941.
18 G. Gambarotta, C. Boccaccio, S. Giordano, M. Andŏ,
M. C. Stella and P. M. Comoglio, Oncogene, 1996, 13, 1911– 22 L. Sun, N. Tran, F. Tang, H. App, P. Hirth, G. McMahon and
1917. C. Tang, J. Med. Chem., 1998, 41, 2588–2603.
19 G. A. Smolen, R. Sordella, B. Muir, G. Mohapatra, 23 H. Guan, A. D. Laird, R. A. Blake, C. Tang and C. Liang,
A. Barmettler, H. Archibald, W. J. Kim, R. A. Okimoto,
D. W. Bell, D. C. Sgroi, J. G. Christensen, J. Settleman and
D. A. Haber, Proc. Natl. Acad. Sci., 2006, 103, 2316–2321.
Bioorg. Med. Chem. Lett., 2004, 14, 187–190.
58998 | RSC Adv., 2014, 4, 58990–58998
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