Virtual screening and further development of novel ALK inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.04.008

Anaplastic lymphoma kinase (ALK) has been in the spotlight in recent years as a promising new target for therapy of non-small-cell lung cancer (NSCLC). Since the identification of the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene in some NSCLC patients was reported in 2007, various research groups have been seeking ALK inhibitors. Above all, crizotinib (PF-02341066) has been under clinical trial, and its therapeutic efficacy of inhibiting ALK in NSCLC has been reported. Among anticancer drugs, drug resistance appears frequently necessitating various kinds of inhibitors. We identified novel ALK inhibitors by virtual screening from the public chemical library collected by the Chemical Biology Research Initiative (CBRI) at the University of Tokyo, and inhibitors that are more potent were developed.

Full text of DOI:10.1016/j.bmc.2011.04.008

Bioorganic & Medicinal Chemistry 19 (2011) 3086–3095
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Virtual screening and further development of novel ALK inhibitors
Masako Okamoto a,b, Hirotatsu Kojima b, Nae Saito b, Takayoshi Okabe b, Yoshiaki Masuda a,b
,
Toshio Furuya a,b, Tetsuo Nagano b,c,
⇑
a Drug Discovery Department, Research & Development Division, PharmaDesign, Inc., 2-19-8 Hatchobori, Chuo-ku, Tokyo 104-0032, Japan
b Chemical Biology Research Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
c Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Anaplastic lymphoma kinase (ALK) has been in the spotlight in recent years as a promising new target for
therapy of non-small-cell lung cancer (NSCLC). Since the identification of the echinoderm microtubule-
associated protein-like 4 (EML4)-ALK fusion gene in some NSCLC patients was reported in 2007, various
research groups have been seeking ALK inhibitors. Above all, crizotinib (PF-02341066) has been under
clinical trial, and its therapeutic efficacy of inhibiting ALK in NSCLC has been reported. Among anticancer
drugs, drug resistance appears frequently necessitating various kinds of inhibitors. We identified novel
ALK inhibitors by virtual screening from the public chemical library collected by the Chemical Biology
Research Initiative (CBRI) at the University of Tokyo, and inhibitors that are more potent were developed.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 3 March 2011
Revised 2 April 2011
Accepted 4 April 2011
Available online 7 April 2011
Keywords:
ALK, anaplastic lymphoma kinase
NSCLC, non-small-cell lung cancer
Virtual screening
Chemical library
1. Introduction
anaplastic large cell lymphoma (ALCL) cell line, and its therapeutic
efficacy of inhibiting ALK and c-Met in NSCLC is truly remarkable.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase
and is normally expressed in the central and peripheral nervous
systems. ALK was first identified as a fusion partner of nucleophos-
min (NPM) in anaplastic large-cell lymphomas (ALCLs) with a
t(2;5)(p23;q35) chromosomal rearrangement.1 Hence, ALK has
been expected to be a target for anticancer therapy similar to other
tyrosine kinases. Various research of ALK inhibitors has been con-
ducted to date; for example, a small molecule ALK inhibitor, NVP-
TAE684, was reported by The Genomics Institute of the Novartis
Research Foundation, showing potent and selective inhibitory
activity.2
However, since drug resistance appears frequently for anticancer
drugs, various kinds of inhibitors are still required.
To obtain novel ALK inhibitors having new scaffolds, we
adopted an in silico approach. In the past several years, we have at-
tempted to find various biologically active compounds against each
target protein from a commercially available compound database
using in silico approach and have reported successful results.5,6
Furthermore, a public chemical library has been collected and sup-
plied by the Chemical Biology Research Initiative (CBRI)7 at the
University of Tokyo in Japan since 2007; we also used this chemical
library and virtual screening approach.
Moreover, in 2007, the transforming echinoderm microtubule-
associated protein-like 4 (EML4)-ALK fusion gene in non-small-cell
lung cancer (NSCLC) was reported by Mano et al.3 ALK has since
been in the spotlight as a promising new target for therapy of
non-small-cell lung cancer (NSCLC). Since this report, many re-
search groups have been seeking ALK inhibitors and have reported
some potent inhibitors. Above all, crizotinib (PF-02341066)4 pro-
duced by Pfizer, Inc., has already been under clinical trial; its IC50
value is 24 nM against NPM-ALK oncogenic fusion variant of the
ALK receptor tyrosine kinase expressed by the KARPAS299 human
Notably, we have had many successful experiments finding hit
compounds using structure-based virtual screening (SBVS). We
usually construct protein–ligand complex models using an X-ray
crystal structure of the target protein and known inhibitors before
SBVS for an efficient screen. However, at the time of starting this re-
search, there were no known X-ray crystal structures of the ALK ki-
nase domain. Therefore, we obtained a 3D structure of the ALK
kinase domain by homology modeling using one of the crystal
structures of the activated insulin receptor tyrosine kinase (PDB
code: 1IR38) as a template. Protein–ligand complex models were
then made using this homology model protein structure and known
inhibitors, and docking calculations were conducted using CON-
SENSUS-DOCK.9
⇑
Corresponding author. Tel.: +81 3 5841 4850; fax: +81 3 5841 4855.
E-mail addresses: okamoto@pharmadesign.co.jp (M. Okamoto), kojima@mol.f.
u-tokyo.ac.jp (H. Kojima), nae-saito@mol.f.u-tokyo.ac.jp (N. Saito), tokabe@mol.f.
u-tokyo.ac.jp (T. Okabe), masuda@pharmadesign.co.jp (Y. Masuda), furuya@phar
madesign.co.jp (T. Furuya), tlong@mol.f.u-tokyo.ac.jp (T. Nagano).
As a result, we were successful in the identification of hit com-
pounds as novel ALK inhibitors by SBVS from the public chemical
library collected by CBRI at the University of Tokyo. In addition,
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.008

M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
3087
we succeeded in the organic synthesis of lead compounds from hit
compounds to obtain more potent inhibitors.
structures of activated insulin receptor tyrosine kinase as a
template.
To select the template for homology modeling, we practiced
BLAST12 search against PDB database using ALK kinase domain
sequence (a part of Q9UM73 (Genbank Accession code)). The result
from BLAST search is shown in Table 1, and some crystal structures
of IGF-1R and INSR were high-ranking. In particular, INSR (PDB
code: 1IR3), a ligand-complex crystal structure having high resolu-
tion (1.90 Å), had been used as a template for homology modeling
in other papers.13,14 Therefore we selected 1IR3 as our template.
Sequence alignment and homology modeling were carried out
by MOE15 module. The alignment is shown in Figure 1. In general,
gatekeeper residue is considered important in the role of obtaining
selectivity. In the case of this homology model of ALK, Leu (red let-
ter in Fig. 1) was predicted as the gatekeeper, as found by Gunby
et al.13 After the alignment, we constructed a modeling structure
with 1IR3 as a template using the MOE module.
2. Results and discussion
2.1. Sequence alignment and homology modeling
Protein 3D structures are indispensable for SBVS; however,
X-ray crystal structures had not been included in the PDB data-
base10 until the release of three crystal structures analyzed by sev-
eral groups.11 Therefore, we obtained the 3D structure of ALK
kinase domain by homology modeling using one of the crystal
Table 1
BLAST search results using ALK kinase domain sequence as a query
PDB code
Release
Protein
Ligand
%ID
1M7N
1P4O
1JQH
2OJ9
3D94
1IRK
1P14
1K3A
2Z8C
1IR3
1RQQ
2ZM3
3BKB
3CD3
1I44
2003/1/7
IGF-1R
IGF-1R
IGF-1R
IGF-1R
IGF-1R
INSR
INSR
IGF-1R
INSR
—
—
ANP
BMI
D94
—
46
45
45
45
45
45
45
44
44
44
44
43
42
42
41
40
40
39
39
39
2.2. Protein–ligand complex model
2003/4/29
2002/4/19
2007/5/1
2008/7/29
1995/2/27
2003/7/22
2001/11/28
2008/8/12
1998/1/7
2003/12/30
2008/6/10
2007/12/25
2008/3/25
2001/3/7
2002/9/11
1997/12/3
2008/2/26
2001/4/18
2003/4/8
For more efficient SBVS, we usually construct a protein–ligand
complex model before virtual screening.5 We use an X-ray crystal
structure ordinarily, but in this case, because crystal structures
were lacking at the time of conducting this research, we adopted
the model structure of ALK kinase domain described above. In
the constructed complex model, we used known potent ALK inhib-
itors selected from patent information16 (Table 2). Various ALK
inhibitors such as 3H-pyrazolo[3,4-c]isoquinoline derivatives were
described abundantly in international publication patent informa-
tion,16 and we were able to consider structure–activity relation-
ships from these compounds’ assay data. Therefore we selected
compounds in Table 2 to build protein–ligand complex models.
Then, we tried to construct appropriate protein–ligand complex
models for efficient virtual screening.
—
ACP
S91
ANP
112
575
STU
STU
ACP
—
INSR
INSR
IGF-1R
v-Fes
v-Fes
INSR
MuSK
LCK
FGFR2
c-Abl
c-Abl
1LUF
3LCK
3C4F
1IEP
—
C4F
STI
STI
1OPJ
Figure 1. Sequence alignment of INSR and ALK kinase domain. Numbers are residue UID of INSR. Color bars show actual secondary structure of INSR (PDB code: 1IR3).
Reversed letters indicate conserved residues. The gatekeeper residues are shown in red letters. This figure was prepared by MOE.

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M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
Table 2
Known potent ALK inhibitors16 used for building protein–ligand complex models
Compound
Structure
Inhibitory activity
OH
H
N
N
N
1
IC50 ꢀ 99 nM
O
O
Cl
OH
H
N
N
N
F
2
IC50 ꢀ 99 nM
O
O
N
In this study, we used a molecular dynamics simulation module
in MOE15 by modifying an SVL script to build the complex models.
This SVL script, called MultiCopyMD,5 was devised to enable
several ligand molecules to be positioned in the binding site of
the target protein simultaneously during the simulation so that
the consensus binding conformation of that protein for multiple
ligands can be generated. Water molecules were soaked within
20 Å of the center of the ligands in the complex model with wall
restraints around them, while the side chain atoms within 4.5 Å
of the center of the ligands and the backbone atoms of the gly-
cine-rich loop were unfixed in the MD calculations. The system
was gradually heated to 300 K, and an additional 1 ns simulation
at constant temperature and volume (NVT ensemble, NPA algo-
rithm) was carried out. We then clustered the coordinates in this
trajectory and selected several structures to compare each enrich-
ment of virtual screening.
We compared the enrichments among representative complex
model structures by small-scale (approximately a 1000-compound
test set database) virtual screening using CONSENSUS-DOCK.9
These 1000 compounds were selected randomly from the commer-
cially available compound databases, filtered by drug-likeness and
clustering. The small-scale test set database had these 1000 com-
pounds and known ALK inhibitors, and a complex model structure
having the best enrichment of known ALK inhibitors in small-scale
virtual screening was selected.
Figure 2. The protein–ligand complex model structures of ALK kinase domain. (A)
One known inhibitor, compound 1, is shown in pink; and the other known inhibitor,
compound 2, is shown in light blue. (B) To show detailed interactions, only
compound 2 is drawn. There are four hydrogen bonds between compound 2 and
ALK kinase domain, and a hydrophobic interaction is also predicted with Leu1196.
The most appropriate complex model structure selected is
shown in Figure 2. In this binding mode, there are four hydrogen
bonds between compound 2 and ALK kinase domain including
two hydrogen bonds with hinge region, which seem to be core
interactions for most kinase inhibitors; hydrophobic interaction
is also predicted with Leu1196.
could possibly make hydrogen bonds with the hinge region of
the ATP binding site.
Through SBVS, as shown in Figure 3, we selected 300 com-
pounds from CBRI Library and 591 compounds from the commer-
cially available compound database. Among these compounds, we
performed an ALK inhibition assay. As a result, 13 compounds in
2.3. Structure-based virtual screening
total exhibited greater than 50% inhibition at 10 lM, as shown in
Table 3. To obtain more potent compounds, we conducted a simi-
Using the complex model described above, we conducted struc-
ture-based virtual screening using CONSENSUS-DOCK9 against the
CBRI Library and commercially available compound database. At
the time we carried out these screenings, the CBRI Library
contained 71,558 compounds. To compensate for the numbers of
compounds for structure-based virtual screening, we also screened
against a commercially available compound database. After dock-
ing calculation, we selected compounds by pharmacophore that
larity search using these compounds as queries.
2.4. Second screening (similarity search)
Using the 13 hit compounds as queries, we carried out a similar-
ity search with BIT_MACCS fingerprint and selected 6036
compounds. As a result, we were able to obtain 64 compounds
with equivalent or higher inhibitory activity (% inhibition at

M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
3089
the O of indolinone and NH of pyrrole. Furthermore, hydrophobic
interactions between compound 3 and Leu1196, Leu1256 were
predicted. Based on these points, indolinone was predicted as a
promising scaffold for further development.
2.5. Design and synthesis
For further development, we planned to synthesize designed
compounds. To add more interactions between compound 3 and
adjacent amino acids of the ATP binding site outside of the hinge
region, we synthesized compounds in Table 5 as shown in Schemes
1–3; detail protocols are described in Section 4.
We conducted in vitro assays of the synthesized compounds,
and the results are shown in Table 6. The most potent inhibitor
was compound 25a; its predicted binding mode is shown in Figure
5. Compared to the binding mode of compound 3, two additional
hydrogen bonds were predicted: NH2 on the indolinone ring and
O in the side chain of Asp178, and NH2 on the benzene ring and
O in the side chain of Asp111. These interactions were thought to
increase inhibitory activity of compound 25a. According to the
comparison between 25a and 25b, m-position of amino group on
benzene ring seemed to be better than p-position. We designed
compounds 26a and 26b to obtain additional hydrogen bonds,
but those inhibitory activities decreased. This is due to the expo-
sure of the (2-aminoethyl)amino group to solvent, but hydrogen
bonds were not made with neighboring amino acids.
Figure 3. Structure-based virtual screening protocol.
2.6. Kinase panel assay
10
l
M > 50). IC50 value was determined to find relatively potent
To examine kinase selectivity of synthesized compound 23a, we
conducted kinase panel assay including 21 tyrosine kinases (Table
7). As a result, 23a showed highly selectivity against ABL, CSK,
EGFR, EPHA2, EPHB4, and JAK3. However, against FLT3, IGF-1R,
INSR, TRKA, and TYRO3, inhibitory activities of 23a were equal to
ALK, EML4-ALK, and NPM1-ALK. Especially, selectivity against INSR
is essential for drugs as ALK inhibitors, we have been considering
drug design for obtaining selective compounds.
compounds. Fifteen compounds showed an IC50 value under
10 M (Table 4); the most potent compound’s IC50 value was
0.85 M (compound 3).
The predicted binding mode of compound 3 is shown in Figure
4. In this binding mode, there were two hydrogen bonds between
compound 3 and the hinge region of ALK kinase domain; these
interactions were thought to stabilize the binding of compound
3. Intramolecular hydrogen bonding was also predicted between
l
l
Table 3
Hit compounds from structure-based virtual screening (% inhibition at 10
l
M > 50)
1st hit compounds from SBVS
H
N
O
OH
O
O
O
OH
OH
OH
O
OH
NH2
O
HO
O
HO
H
OH
OH
N
O
OH
HN
N
N
O
OH
HO
OH
H
N
HO
HO
OH
N
OH
OH
N
O
N
OH
Br
HN
HN
HO
N
N
OH
N
OH
O
S
H
O
O
OH
O
OH
N
HO
HO
N
O
N
O
N
O
N
H
O
N
O
O2N

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M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
Table 4
Compounds with high inhibitory activity in 2nd screening (IC50 ꢀ 10
lV)
Compound
Structure
IC50
(l
M)
Compound
Structure
IC50 (lM)
H
N
N
O
N
Br
N
H
SO2
N
3
0.85
10
2.85
N
NH
HO
H
N
H
N
N
N
O
H
O
N
F
N
4
0.91
11
3.47
O
NH
O
Cl
OH
H
N
H
N
O
O
H
N
O
5
1.38
12
3.75
S
N
O
N
HN
H
N
OH
N
N
HN
6
1.73
13
4.24
N
NH
HO
H
OH
N
O
N
H
N
N
7
1.81
14
6.46
NH
N
Cl
N
O
O
N
N
NO2
8
2.40
15
8.66
NH
NH
HO
HO
HO
N
H
N
N
S
N
O
N
9
2.40
16
9.29
N
OH
Cl
O
The results described in this paper indicate that our SBVS ap-
proach is very efficient for finding a novel scaffold on a target pro-
tein’s active compounds. After finding hit compounds, we could
confirm improvements in inhibitory activity through stepwise
structure-based drug design. We conducted a kinase panel study
to inspect kinase selectivity, and we would like to develop our no-
vel ALK inhibitors. We hope our discovery of these promising ALK
inhibitors possessing new scaffolds will be useful for research of
chemotherapy for NSCLC.
3. Conclusion
Using SBVS, we identified novel ALK inhibitors from the CBRI
Library and a commercially available compound database; these
hit compounds had new scaffolds for ALK inhibitor development.
Furthermore, we developed lead compounds from hits by
designing compounds, and we were successful in obtaining potent
ALK inhibitors (the most potent inhibitor: compound 25a, IC50
50.8 nM).
=

M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
3091
Table 5
Synthesized compounds
H
N
O
H
X
N
R1
R2
R3
Compound
X
R1
R2
R3
23a
23b
25a
25b
26a
26b
31a
31b
32a
32b
H
H
NH2
NH2
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
m-NH2–C6H4
p-NH2–C6H4
m-NH2–C6H4
p-NH2–C6H4
m-NH(CH2)2NH2–C6H4
p-NH(CH2)2NH2–C6H4
CH3
CH3
CH3
CH3
m-NH2–C6H4
p-NH2–C6H4
m-NH(CH2)2NH2–C6H4
p-NH(CH2)2NH2–C6H4
H
Library; a similarity search was conducted using MOE15 with com-
pounds in Table 3 as queries. Compounds with similarity were se-
lected by visual inspection for in vitro assay.
Figure 4. Predicted binding mode of compound 3.
4. Experimental
4.1. Compounds
4.3. Kinase assay
Tested compounds in Table 2 and 3 were purchased from sev-
eral suppliers. Their characterization and purity were confirmed
using LC–MS.
Activity was measured by mobility shift assay. The reaction
mixture containing 1.5
l
M FL-Peptide 13, 5-FAM-KKSRGDYMTM-
M ATP,
QIG-CONH2 (Caliper Life Science, Hopkinton, MA), 67.9
l
42 ng/mL ALK (Invitrogen, Carlsbad, CA), 50 mM Hepes (pH 7.4),
10 mM MgCl2, 1 mM DTT, 1% Protease Inhibitor Cocktail Set V
(Merck Darmstat, Germany), 1% Phosphatase Inhibitor Cocktail
Set III (Merck), 0.01% Brij-35 and a test compound (5% DMSO)
was incubated at room temperature for 60 min, and the reaction
4.2. Similarity search
We calculated a fingerprint based on the descriptors of the
BIT_MACCS: MACCS structural keys for all compounds in CBRI
Scheme 1. Synthetic protocol of compounds 23a, 23b, 25a, and 25b in Table 5. Reagents: (a) CH3CO2NH4, AcOH, CH3NO2; (b) ethyl 3-oxobutanoate, MeOH, CH3ONa, NH3/
MeOH; (c) TFA; (d) DMF/POCl3, 1,2-dichloroehtane; (e) indoline-2-one, MeOH, piperidine; (f) Pd/C, THF; (g) 5-nitroindoline-2-one, MeOH, piperidine; (h) Pd/C, THF.

3092
M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
H
N
H
N
O
O
H
Br
N
H
NH2
N
NH
NH2
H2N
23a: m-NH2
23b: p-NH2
26a: m-NH(CH2)2NH2
26b: p-NH(CH2)2NH2
Scheme 2. Synthetic protocol of compounds 26a, 26b in Table 4. Reagents: 2-bromoethylamine hydrobromide, K2CO3, DMF.
H
N
H
N
O
O
O
H
N
a
b
H
H
H
N
N
Br
27
28
29
H
N
H
N
O
O
H
H
c
d
N
N
O2N
H2N
31a: m-NH2
31b: p-NH2
30
H
N
O
e
H
N
32a: m-NH(CH2)2NH2
32b: p-NH(CH2)2NH2
H2N
N
H
Scheme 3. Synthetic protocol of compounds 31a, 31b, 32a, and 32b in Table 5. Reagents: (a) indoline-2-one, MeOH, piperidine; (b) NBS, (C6H5CO)2O2, CCl4; (c) Suzuki
coupling, 4-nitrophenylboronic acid, K2CO3, Pd(dppf)Cl2, H2O, dioxane; (d) Pd/C, THF; (e) 2-bromoethylamine hydrobromide, K2CO3, DMF.
was stopped by adding 140 mM EDTA. The phosphorylated and
unphosphorylated peptides were separated and detected by Lab-
Chip EZ Reader II (Caliper Life Science).
tra were recorded at 300 MHz, and TMS was used as an internal
standard. High resolution mass spectra were obtained using JEOL
AccuTOF JMS-T100LC and an electrospray ionization (ESI) source.
LC–MS was taken on a quadrupole Mass Spectrometer on Agilent
LC/MSD 1200 Series (Column: Welchrom XB-C18 (50 Â 4.6 mm,
4.4. Kinase panel assay
5
l
m) operating in ES (+) or (À) ionization mode; T = 30 °C; flow
This experiment was performed by Carna Biosciences (Kobe,
Japan). Briefly, the reaction mixture containing the enzyme,
1 mM ATP and 1 lM appropriate peptide was analyzed by mobility
rate = 1.5 mL/min. Prep-HPLC was performed under the following
conditions: Welchrom C18 (150 Â 20) column; wavelength
220 nm; mobile phase: A MeCN (0.1% TFA), B water (0.1% TFA);
flow rate: 25 mL/min; injection volume: 2 mL; run time: 30 min;
equilibration: 5 min.
shift assay after incubation.
4.5. Chemistry
4.5.1. General procedure for preparation of compound 18
To a solution of compound 17 (20.0 g) and ammonium acetate
(25.5 g) in AcOH (265 mL) was added CH3NO2 (21 mL). The mixture
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. 1H NMR spec-

M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
3093
Table 6
Table 7
IC50 values of synthesized compounds with high inhibitory activity
Kinase panel assay of compound 23a. Compounds 23a
(4 M) were tested against 21 tyrosine kinases
H
N
l
O
Kinase
% Inhibition at 4 lM
H
X
ALK
97.6
98.7
93.8
5.1
À6.5
À2.3
4.9
N
R1
EML4-ALK
NPM1-ALK
ABL
R2
R2
R3
CSK
Compound
X
R1
R3
IC50
(nM)
EGFR
EPHA2
EPHB4
FGFR1
FLT3
IGF-1R
INSR
ITK
JAK3
KDR
LCK
MET
PDGFR
PYK2
SRC
SYK
TIE2
5.1
25a
23a
25b
26a
NH2 CH3
CH3
NH2 CH3
H
H
H
H
m-NH2–C6H4
m-NH2–C6H4
p-NH2–C6H4
m-NH(CH2)2NH2–
C6H4
p-NH(CH2)2NH2–
C6H4
p-NH2–C6H4
CH3
50.8
61.8
68.3
89.1
62.7
98.1
91.2
97.2
34.2
9.0
81.7
69.8
12.7
56.6
86.5
39.0
58.5
14.0
99.4
100.5
H
H
H
CH3
CH3
CH3
26b
H
149.3
23b
32b
H
H
H
367.6
415.3
CH3 p-NH(CH2)2NH2–
C6H4
a
32a
H
CH3 m-NH(CH2)2NH2–
C6H4
CH3
604.5
TRKA
TYRO3
AcOEt. The combined organic layers were concentrated and puri-
fied by column chromatography to give the product (4.2 g).
4.5.3. General procedure for preparation of compound 20
To compound 19 (1.0 g) was added TFA (10 mL) at 0 °C. The
mixture was then stirred and refluxed overnight. After cooling
down to room temperature, the solvent was removed under re-
duced pressure. The residue was washed with saturated NaHCO3
and extracted with AcOEt. The combined organic layers were con-
centrated to give the crude product. The crude product was recrys-
tallized by ethyl ether and petroleum ether to afford the product
(600 mg) as a red solid.
4.5.4. General procedure for preparation of compound 21
To DMF (2.7 mL) under N2 at 0 °C was added POCl3 (3.2 mL)
drop wise over 5 min. The cooling bath was removed, and after
15 min 1,2-dichloroethane (15 mL) was added. The reaction mix-
ture was again cooled to 0 °C and a solution of compound 20
(3.0 g) in 1,2-dichloroethane (15 mL) was added drop wise over
15 min. The reaction was heated to reflux for 15 min, and then
Figure 5. Predicted binding mode of compound 25a.
was stirred at 115 °C for 2 h. After cooling down to room temper-
ature, the solvent was removed under reduced pressure. The
resulting mixture was washed with saturated NaHCO3 and ex-
tracted with CH2Cl2. The combined organic layers were concen-
trated and purified by column chromatography to give the
product (10.0 g).
cooled to room temperature.
A solution of sodium acetate
(14.4 g) in water (45 mL) was added slowly to the reaction mix-
ture, and the resulting mixture was again heated to reflux for
20 min. After the reaction mixture was cooled to room tempera-
ture, it was diluted with CH2Cl2, and the aqueous phase was
washed with CH2Cl2 (2Â 50 mL). The combined organic fractions
were washed with saturated NaHCO3, dried (Na2SO4), and concen-
trated in vacuo. The crude material was purified by chromatogra-
phy on silica gel to provide 1.1 g of desired compound 21.
4.5.2. General procedure for preparation of compound 19
To a solution of ethyl 3-oxobutanoate (6.2 mL) in methanol
(60 mL) at 0 °C was added sodium methoxide (560 mg). Compound
18 (9.0 g) was then added to the mixture at the same temperature.
The mixture was stirred at room temperature for 1 h. Subse-
quently, 5 M NH3/methanol solution (200 mL) was poured into
the mixture and the resulting mixture was stirred at room temper-
ature overnight. The mixture was poured into AcOEt. After NH3
was given off, the solvent was removed under reduced pressure.
The resulting mixture was washed with water and extracted with
4.5.5. General procedure for preparation of compound 22
To a solution of compound 21 (5.5 g) and indolin-2-one (2.5 g)
in methanol (50 mL) was added piperidine (0.5 mL). The mixture
was stirred at 65 °C overnight. After cooling down to room temper-
ature, the resulting solid gave desired compound 22 (5.6 g) as a
yellow solid.

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M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
4.5.6. 3-[[3-(3-Aminophenyl)-5-methyl-1H-pyrrol-2-
yl]methylene]-1,3-dihydro-2H-indol-2-one (23a)
2.93–2.97 (m, 2H), 2.36 (s, 3H); HRMS: calcd for C22H23N4O
(M+H)+, 359.18719; found: 359.18838.
To a solution of compound 22 (550 mg) in THF (60 mL) was
added 10% Pd/C catalyst (90 mg). The black mixture was then stir-
red overnight at room temperature under an atmosphere of hydro-
gen. After the removal of catalyst, the filtrate was concentrated and
purified by flash silica chromatography to give compound 23a
(260 mg) as a red solid. 1H NMR (DMSO-d6, 300 MHz): d 13.59 (s,
1H), 10.87 (s, 1H), 7.48 (s, 1H), 7.30–7.32 (d, 2H), 7.08–7.15 (m,
2H), 6.87–6.98 (m, 2H), 6.70–6.71 (d, 1H), 6.57–6.62 (m, 1H),
6.23–6.24 (d, 1H), 2.38 (s, 3H); HRMS: calcd for C20H18N3O
(M+H)+, 316.14499; found: 316.14822.
4.5.14. Preparation of compound 28
To a solution of 2-formyl-3,5-dimethylpyrrole (compound 27,
7.17 g) and indolin-2-one (5.0 g) in methanol (100 mL) was added
piperidine (1.0 mL). The mixture was stirred at 65 °C overnight.
After cooling down to room temperature, the resulting solid gave
desired compound 29 (10.4 g) as a yellow solid.
4.5.15. Preparation of compound 29
Compound 28 (1.0 g), NBS (0.785 g) and benzoyl peroxide
(25 mg) were mixed with CCl4 (100 mL). The mixture was stirred
at room temperature overnight. The resulting solid afforded a
crude product. The crude product was recrystallized by methanol
to give the product (1.1 g) as a yellow solid.
4.5.7. 3-[[3-(4-Aminophenyl)-5-methyl-1H-pyrrol-2-
yl]methylene]-1,3-dihydro-2H-indol-2-one (23b)
1H NMR (DMSO-d6, 300 MHz): d 13.51 (s, 1H), 10.79 (s, 1H),
7.39 (s, 1H), 7.28–7.31 (d, 1H), 7.04–7.15 (m, 3H), 6.64–6.67 (d,
2H), 6.87–6.94 (m, 2H), 6.17–6.17 (d, 1H), 5.24 (s, 2H), 2.36 (s,
3H); HRMS: calcd for C20H18N3O (M+H)+, 316.14499; found:
316.14889.
4.5.16. General procedure for preparation of compound 30
A mixture of compound 29 (1.0 g), 4-nitrophenylboronic acid
(0.79 g), K2CO3 (1.31 g), Pd(dppf)Cl2 (258 mg), H2O (4.5 mL), and
dioxane (10 mL) was heated to 105 °C for 1 h. After cooling down
to room temperature, water was added. The mixture was then ex-
tracted with CH2Cl2 and MeOH. The combined organic layer was
dried over anhydrous sodium sulfate and concentrated. The resi-
due was purified by column chromatography to give the product
(850 mg) as a yellow solid.
4.5.8. General procedure for preparation of compound 24
To a solution of compound 21 (320 mg) and 5-nitroindolin-2-
one (200 mg) in methanol (5 mL) was added piperidine
(0.05 mL). The mixture was stirred at 65 °C overnight. After cooling
down to room temperature, the resulting solid gave desired com-
pound 24 (400 mg) as a yellow solid.
4.5.17. 3-[[4-(3-Aminophenyl)-3,5-dimethyl-1H-pyrrol-2-
yl]methylene]-1,3-dihydro-2H-indol-2-one (31a)
4.5.9. 5-Amino-3-[[3-(3-aminophenyl)-5-methyl-1H-pyrrol-2-
yl]methylene]-1,3-dihydro-2H-indol-2-one (25a)
To a solution of compound 30 (700 mg) in THF (80 mL) was
added 10% Pd/C catalyst (120 mg). The black mixture was then stir-
red overnight at room temperature under an atmosphere of hydro-
gen. After the removal of catalyst, the filtrate was concentrated and
purified by flash silica chromatography to give compound 1
(500 mg) as a red solid. 1H NMR (DMSO-d6, 300 MHz): d 13.59 (s,
1H), 10.80 (s, 1H), 7.71–7.73 (d, 1H), 7.62 (s, 1H), 7.04–7.08 (m,
2H), 6.95–6.96 (m, 1H), 6.85–6.87 (m, 1H), 6.40–6.49 (m, 3H),
5.04 (s, 2H), 2.26–2.30 (d, 6H); HRMS: calcd for C21H20N3O
(M+H)+, 330.16064; found: 330.16533.
1H NMR (DMSO-d6, 300 MHz): d 2.39 (s, 3H), 4.70 (s, 2H), 5.22
(s, 2H), 6.19 (d, 1H), 6.37–6.40 (m, 1H), 6.57–6.60 (m, 4H), 6.67
(s, 1H),7.13 (t, 1H), 7.31 (s, 1H), 10.45 (s, 1H), 13.72 (s, 1H); HRMS:
calcd for C20H19N4O (M+H)+, 331.15589; found: 331.15984.
4.5.10. 5-Amino-3-[[3-(4-aminophenyl)-5-methyl-1H-pyrrol-2-
yl]methylene]-1,3-dihydro-2H-indol-2-one (25b)
1H NMR (DMSO-d6, 300 MHz): d 13.57–13.58 (d, 1H), 10.37 (s,
1H), 7.20 (s, 1H), 7.08–7.11 (m, 2H), 6.64–6.66 (d, 2H), 6.53–6.56
(m, 2H), 6.31–6.35 (m, 1H), 6.11–6.12 (d, 1H), 5.26 (s, 2H), 4.66
(s, 2H), 2.34 (s, 3H); HRMS: calcd for
331.15589; found: 331.15814.
C
20H19N4O (M+H)+,
4.5.18. 3-[[4-(4-Aminophenyl)-3,5-dimethyl-1H-pyrrol-2-
yl]methylene]-1,3-dihydro-2H-indol-2-one (31b)
1H NMR (DMSO-d6, 300 MHz): d 13.56 (s, 1H), 10.74 (s, 1H),
7.68–7.71 (d, 1H), 7.59 (s, 1H), 7.04–7.07 (m, 1H), 6.92–6.97 (m,
3H), 6.84–6.86 (d, 1H), 6.59–6.62 (d, 2H), 5.04 (s, 2H), 2.24–2.28
(d, 6H); HRMS: calcd for C21H20N3O (M+H)+, 330.16064; found:
330.15895.
4.5.11. General procedure for preparation of compound 26
Compound 23 (300 mg), 2-bromoethylamine HBr salt (298 mg)
and K2CO3 (66 mg) was mixed with DMF (15 mL). The mixture was
stirred at 100 °C for 3.5 h. After the removal of the solid, the filtrate
was concentrated. The residue was washed with saturated water
and extracted with CH2Cl2. The combined organic layers were con-
centrated and purified by column chromatography to give the
product (60 mg).
4.5.19. 3-[[4-[3-[(2-Aminoethyl)amino]phenyl]]-3,5-dimethyl-
1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (32a)
Compound 31a (470 mg), 2-bromoethylamine HBr salt (3.2 g)
and Et3N (2.16 mL) was mixed with isopropanol (50 mL). The mix-
ture was stirred and refluxed for 24 h. The solvent was removed
under reduced pressure. The residue was purified by column chro-
matography to give the product (410 mg). 1H NMR (DMSO-d6,
300 MHz): d 13.64 (s, 1H), 10.82 (s, 1H), 7.73–7.75 (m, 3H), 7.65
(s, 1H), 7.08–7.20 (m, 2H), 6.98–7.00 (m, 1H), 6.87–6.89 (d, 1H),
6.53–6.57 (m, 3H), 5.75–5.80 (m, 1H), 3.29–3.38 (m, 2H), 2.97–
2.99 (m, 2H), 2.29–2.33 (d, 6H); HRMS: calcd for C23H25N4O
(M+H)+, 373.20284; found: 373.20285.
4.5.12. 3-[[3-[3-[(2-Aminoethyl)amino]phenyl]]-5-methyl-1H-
pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (26a)
1H NMR (DMSO-d6, 300 MHz): d 13.63 (s, 1H), 10.87 (s, 1H),
7.68 (s, 2H), 7.47 (s, 1H), 7.29–7.32 (d, 1H), 7.19–7.25 (m, 1H),
7.07–7.13 (m, 1H), 6.92–6.97 (m, 1H), 6.86–6.89 (d, 1H), 6.68–
6.70 (m, 2H), 6.61–6.64 (m, 1H), 6.25–6.26 (d, 1H), 5.88–5.90 (m,
1H), 3.28–3.33 (m, 2H), 2.95–2.99 (m, 2H), 2.39 (s, 3H); HRMS:
calcd for C22H23N4O (M+H)+, 359.18719; found: 359.186.
4.5.13. 3-[[3-[4-[(2-Aminoethyl)amino]phenyl]]-5-methyl-1H-
pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (26b)
1H NMR (DMSO-d6, 300 MHz): d 13.53 (s, 1H), 7.40 (s, 1H),
7.22–7.29 (m, 3H), 7.04–7.07 (m, 1H), 6.86–6.95 (m, 2H), 6.71–
6.74 (d, 2H), 6.18–6.19 (d,1H), 6.05 (s, 1H), 3.30–3.32 (m,2H),
4.5.20. 3-[[4-[4-[(2-Aminoethyl)amino]phenyl]]-3,5-dimethyl-
1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (32b)
1H NMR (DMSO-d6, 300 MHz): d 13.61 (s, 1H), 10.79 (s, 1H),
7.71–7.80 (m, 4H), 7.62 (s, 1H), 7.06–7.12 (m, 3H), 6.94–6.99 (m,
1H), 6.86–6.89 (d, 1H), 6.67–6.70 (d, 2H), 5.78 (s, 1H), 3.32–3.41

M. Okamoto et al. / Bioorg. Med. Chem. 19 (2011) 3086–3095
3095
6. Okamoto, M.; Saito, N.; Kojima, H.; Okabe, T.; Takeda, K.; Ichijo, H.; Furuya, T.;
Nagano, T. Bioorg. Med. Chem. 2011, 19, 486.
7. Chemical Biology Research Initiative (CBRI). http://www.cbri.u-tokyo.ac.jp/
index_e.html.
(m, 2H), 2.97–3.01 (m, 2H), 2.26–2.30 (d, 6H); HRMS: calcd for
23H25N4O (M+H)+, 373.20284; found: 373.20581.
C
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Acknowledgements
This work was supported by the Target Protein Research Pro-
grams of the Ministry of Education, Culture, Sports, Science, and
Technology of Japan.
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