Discovery of a novel B-cell lymphoma 6 (BCL6)–corepressor interaction inhibitor by utilizing structure-based drug design

Article abstract of DOI:10.1016/j.bmc.2017.07.037

B-cell lymphoma 6 (BCL6) is a transcriptional repressor that can form complexes with corepressors via protein–protein interactions (PPIs). The complexes of BCL6 and corepressors play an important role in the formation of germinal centers (GCs), and differentiation and proliferation of lymphocytes. Therefore, BCL6–corepressor interaction inhibitors would be drug candidates for managing autoimmune diseases and cancer. Starting from high-throughput screening hits 1a and 2a, we identified a novel BCL6–corepressor interaction inhibitor 8c (cell-free enzyme-linked immunosorbent assay [ELISA] IC₅₀ = 0.10 μM, cell-based mammalian two-hybrid [M2H] assay IC₅₀ = 0.72 μM) by utilizing structure-based drug design (SBDD) based on an X-ray crystal structure of 1a bound to BCL6. Compound 8c also showed a good pharmacokinetic profile, which was acceptable for both in vitro and in vivo studies.

Full text of DOI:10.1016/j.bmc.2017.07.037

Accepted Manuscript
Discovery of a novel B-cell lymphoma 6 (BCL6)–corepressor interaction in-
hibitor by utilizing structure-based drug design
Takeshi Yasui, Takeshi Yamamoto, Nozomu Sakai, Kouhei Asano, Takafumi
Takai, Yayoi Yoshitomi, Melinda Davis, Terufumi Takagi, Kotaro Sakamoto,
Satoshi Sogabe, Yusuke Kamada, Weston Lane, Gyorgy Snell, Masashi Iwata,
Masayuki Goto, Hiroshi Inooka, Jun-ichi Sakamoto, Yoshihisa Nakada,
Yasuhiro Imaeda
PII:
S0968-0896(17)31203-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.037
BMC 13874
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 June 2017
18 July 2017
19 July 2017
Please cite this article as: Yasui, T., Yamamoto, T., Sakai, N., Asano, K., Takai, T., Yoshitomi, Y., Davis, M.,
Takagi, T., Sakamoto, K., Sogabe, S., Kamada, Y., Lane, W., Snell, G., Iwata, M., Goto, M., Inooka, H., Sakamoto,
J-i., Nakada, Y., Imaeda, Y., Discovery of a novel B-cell lymphoma 6 (BCL6)–corepressor interaction inhibitor by
utilizing structure-based drug design, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.07.037
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Discovery of a novel B-cell lymphoma 6 (BCL6)–
corepressor interaction inhibitor by utilizing
structure-based drug design
Takeshi Yasui*, †, Takeshi Yamamoto†, Nozomu Sakai†, Kouhei Asano†, Takafumi Takai†, Yayoi
Yoshitomi†, Melinda Davis‡, Terufumi Takagi†, Kotaro Sakamoto†, Satoshi Sogabe†, Yusuke
Kamada†, Weston Lane‡, Gyorgy Snell‡, Masashi Iwata†, Masayuki Goto†, Hiroshi Inooka†, Jun-
ichi Sakamoto†, Yoshihisa Nakada†, Yasuhiro Imaeda*, †
†Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-chome, Fujisawa,
Kanagawa 251-8555, Japan
‡Takeda California Inc. 10410 Science Center Dr. San Diego CA 92121, USA
Corresponding Authors
*For T. Y.: phone, +81-466-32-4261; E-mail, takeshi.yasui@takeda.com
*For Y. I.: phone, +81-466-32-4112; E-mail, yasuhiro.imaeda@takeda.com
1

Abstract: B-Cell lymphoma 6 (BCL6) is a transcriptional repressor that can form complexes
with corepressors via protein–protein interactions (PPIs). The complexes of BCL6 and
corepressors play an important role in the formation of germinal centers (GCs), and
differentiation and proliferation of lymphocytes. Therefore, BCL6–corepressor interaction
inhibitors would be drug candidates for managing autoimmune diseases and cancer. Starting
from high-throughput screening hits 1a and 2a, we identified a novel BCL6–corepressor
interaction inhibitor 8c (cell-free enzyme-linked immunosorbent assay [ELISA] IC50 = 0.10 µM,
cell-based mammalian two-hybrid [M2H] assay IC50 = 0.72 µM) by utilizing structure-based drug
design (SBDD) based on an X-ray crystal structure of 1a bound to BCL6. Compound 8c also
showed a good pharmacokinetic profile, which was acceptable for both in vitro and in vivo
studies.
Keywords: B cell lymphoma 6 (BCL6), protein–protein interaction (PPI), structure-based drug
design (SBDD), diphenylamine
2

1. Introduction
B-Cell lymphoma 6 (BCL6) protein is known as a transcriptional repressor; it is composed of
three domains: broad-complex, tramtrack and bric-a-brac (BTB) domain; repression domain 2
(RD2); and zinc finger (ZF) domain.1, 2 A specific groove constructed by BCL6 BTB domain
(BCL6BTB) homodimers is involved in a protein–protein interaction (PPI) with a corepressor such
as BCL6 corepressor (BCoR), silencing mediator for retinoid and thyroid receptors (SMRT), and
nuclear receptor corepressor (NCoR).3–5 BCL6 is involved in proliferation and survival of
germinal center (GC) B cells, from which diffuse large B-cell lymphomas (DLBCL) originate,
by recruiting the SMRT, BCoR, and NCoR corepressors to the lateral groove of the BCL6BTB
homodimers.6–12 Accordingly, disruption of the PPI between BCL6 and corepressors by BCL6
inhibitors may serve as a therapeutic strategy for managing autoimmune diseases and cancer.13, 14
To date, several BCL6 inhibitors have been reported. In 2008, Evans et al. discovered rifamycin
SV, a member of the ansamycin family, based on a screening of a natural product library.15 In
2014, Leandro et al. identified a small molecular BCL6 inhibitor 79-6, which is functionally
active in vivo and induces apoptosis of BCL6-dependent lymphoma cell lines, by a combination
of computer-aided drug design and screens of small molecule libraries.16 Further, they confirmed
the binding of 79-6 to the corepressor-binding groove of BCL6BTB homodimers by X-ray
crystallography and nuclear magnetic resonance (NMR) analysis. In 2016, the same group
reported a 79-6 derivative, FX1, which showed improved potency compared to that of 79-6.17
However, even the most active compound, FX1, among the BCL6 inhibitors reported so far has a
binding affinity of only around 7 µM as a KD value. In general, development of small molecular
PPI inhibitors is relatively difficult mainly because of following reasons18–20: (1) Contact surfaces
involved in PPIs are relatively large compared to those of conventional targets such as enzymes
or G-protein-coupled receptors (GPCRs); and (2) regions of residues that contribute to high-
3

affinity binding, called hotspots, are generally small on the protein–protein interface.
Accordingly, PPI inhibitors tend to have low ligand efficiency and high molecular weights.
Very recently, we identified
a
BCL6 inhibiting tridecapeptide, F1324 (Ac-
LWYTDIRMSWRVP-OH), which binds to the corepressor-binding groove of the BCL6BTB
homodimers and inhibits PPI between BCL6BTB and BCoR with strong KD and IC50 values (0.57
nM and 1 nM, respectively).21, 22 Interestingly, an evaluation of truncated tetrapeptides of F1324
revealed that the C-terminus tetrapeptide region of F1324 (WRVP-OH) plays an important role
in potentiation. In addition, according to the X-ray co-crystal structure of 79-6 with BCL6BTB, the
binding site of 79-6 is the same as the C-terminus tetrapeptide-binding region.16 On the basis of
these findings, it was suggested that the corepressor-binding groove of the BCL6BTB homodimers
could have a hotspot for small molecular inhibitors to bind with high affinity. Herein, we report
the identification of diphenylamine derivatives as BCL6 inhibitors by high-throughput screening
(HTS) and their subsequent structure-based optimization into a cellular potent BCL6 inhibitor
with a good pharmacokinetic profile.
2. Chemistry
Synthesis of diphenylamine derivatives 1b–h is shown in Scheme 1. SNAr displacement
reaction of the commercially available aryl fluorides 10b–f with 5-amino-2-benzimidazolinone 9
gave the corresponding diphenylamine derivatives 1b–f. Carboxylic acid 1g was prepared from
ester 1f by hydrolysis. Subsequent amidation of 1g with HOBt·NH3 afforded carboxamide 1h.
Hereinafter, the left-hand side aryl group and the right-hand side aryl group of the diphenylamine
compounds are referred to as "ring A" and "ring B," respectively, as described in Scheme 1.
4

Scheme 1. Synthesis of 1b–ha
a Reagents and conditions: (a) TEA or DIPEA, DMSO, rt–160 °C, 2 h–3 d, 1–80%; (b) 1 M
NaOH, MeOH, THF, 50 °C, 3 h, 57%; (c) HOBt·NH3, EDC, DMF, rt, 4 d, 70%.
Introduction of substituents to the 5-position of ring A of 1a was achieved in two steps
(Scheme 2). Intermediates 13a–d were obtained by the SNAr displacement reaction of aryl
fluoride 11 with the corresponding commercially available amines 12a–d. Ether 13e was
prepared from 11 and 4-hydroxytetrahydropyran 12e in the presence of sodium hydride.
Treatment of 13a and 13b with aniline 9 under microwave irradiation provided alcohol 3 and
ester 4, respectively. Subsequent hydrolysis of 4 provided carboxylic acid 5. Intermediates 13c–e
were employed for syntheses of 6, 7, and 8a via a palladium (Pd)-mediated cross-coupling
reaction, Buchwald-Hartwig amination, with aniline 9.
Scheme 2. Synthesis of 3–8aa
5

a Reagents and conditions: (a) 12a–d, TEA or DIPEA, DMA, or DMSO, rt, 3–16 h, 85–89%; (b)
12e, NaH, DMF, 0 °C, 20 min, 58%; (c) 9, DIPEA, DMA, or NMP, MW, 150–200 °C, 2–3 h, 5–
31%; (d) 9, Pd2(dba)3, BINAP, Cs2CO3, DME, 100 °C, 4–16 h, 5–47%; (e) 2 M NaOH, THF,
MeOH, rt, 16 h, 47%.
Installation of an oxindole scaffold as ring B by the Buchwald-Hartwig amination of 13e with
5-aminoindolin-2-one gave compound 8b (Scheme 3). In the same manner, 3,4-
dihydroquinolinones 8c were prepared from 13e with 6-amino-3,4-dihydroquinoline-2(1H)-one.
Scheme 3. Synthesis of 8b–ca
6

a Reagents and conditions: (a) 5-aminoindolin-2-one or 6-amino-3,4-dihydroquinolin-2(1H)-one,
Pd2(dba)3, BINAP, Cs2CO3, DME, 100 °C, 4 h, 17–56%.
3. Results and discussion
3. 1. PPI inhibitory activities
The synthesized compounds were evaluated by an enzyme-linked immunosorbent assay
(ELISA) as the primary cell-free assay to determine the PPI inhibitory activities (IC50) between
BCL6BTB and BCoR. HTS of around 130,000 compounds using the ELISA monitoring the
interaction between the BCL6BTB and the BCoR peptide provided diphenylamine derivatives 1a
and 2a as chemical starting points for BCL6BTB-BCoR PPI inhibition (Fig. 1). The hit compounds
1a and 2a displayed more potent inhibitory activities (IC50 = 4.5 and 14 µM, respectively) than
that of the reported compound 79-6 (IC50 = 200 µM).16 The IC50 value of 79-6 in our ELISA
system was comparable to the reported data (IC50 = 212 µM). The binding of 1a or 2a to BCL6
protein was confirmed by surface plasmon resonance (SPR) and their affinities (KD values)
against BCL6BTB were similar to their IC50 values (1a, KD = 5.0 µM; 2a, KD = 24 µM). The
reported KD value of FX1 is 7 µM determined by microscale thermophoresis.17
7

a
Fig 1. Known BCL6 inhibitors 79-6 and FX1, and hit compounds 1a and 2a from HTS.
Displacement of a SMRT-BBD peptide from BCL6BTB corepressor complex was measured by a
fluorescence polarization competitive binding assay.16
thermophoresis.17
Determined by microscale
b
With the aim of improving the PPI inhibitory activity and exploring the structure-activity
relationship (SAR), effects of substituents at the 2- and 4-positions of ring A of 1a were
investigated. The results are summarized in Table 1. Replacement of the chlorine atom of 1a
with a bromine atom (1b) or trifluoromethyl group (1c) led to a reduction of the inhibitory
activity (IC50 = 83 and > 300 µM, respectively). On the other hand, the cyano derivative 1d
showed potency similar to that of 1a (IC50 = 4.3 µM). Regarding the replacement of the nitro
group at the 4-position of 1a, the cyanide (1e), ester (1f), carboxylic acid (1g), and carboxamide
(1h) derivatives resulted in decreased inhibitory activities.
Table 1. Effects of the 2-, 4-substituents of ring A on cell-free PPI inhibition
8

ELISA IC50 (µM)a, b
4.5 (3.5–5.7)
83 (22–310)
>300
Compounds
X
Cl
Y
NO2
1a
1b
1c
1d
1e
1f
Br
CF3
CN
Cl
NO2
NO2
NO2
4.3 (3.9–4.9)
110 (23–490)
8.6 (4.7–16)
62 (50–75)
CN
Cl
CO2Me
CO2H
CONH2
Cl
1g
1h
Cl
43 (37–51)
a
b
Inhibition of BCL6-corepressor (BCoR) interaction was measured. 95% confidence intervals
are shown in parentheses.
3. 2. X-ray crystal structure
To elucidate and further investigate the binding mode of 1a to BCL6BTB, an X-ray
crystallographic analysis was conducted. It revealed that 1a binds to a part of the corepressor-
binding groove, which is the same site as that of 79-6 in the crystal structure of a BCL6BTB/79-6
complex16 and/or tetrapeptide Ac-WRVP-OH in the crystal structure of a BCL6BTB/F1324(10–13)
complex21 (Fig. 2). In the crystal structure of 1a bound to BCL6BTB, the ring A moiety is
positioned in a hydrophobic pocket composed of side chains of Tyr58, Asn21, Arg24, and Arg28
(Fig. 2a). A hydrogen bond between the NH group of the diphenylamino moiety and the
carbonyl group of the backbone of Met51 is observed (Fig. 2b). The chlorine atom at the 2-
position of 1a fully occupies the relatively small hydrophobic pocket surrounded by Met51,
Leu25, and Ala52, as shown in Fig. 2a. This binding mode information is consistent with the fact
9

that the relatively large substituents, such as the bromine atom and trifluoromethyl group, were
not tolerated as substituents at the 2-position of 1a.
Fig. 2. X-ray crystal structure of 1a with BCL6 (PDB code: 5X9O). (a) Amino acid residues of
BCL6 and 1a are shown in light green and yellow, respectively. (b) Schematic diagram of the
ligand binding site.
3. 3. Docking simulation
Regarding the benzoxadiazole derivative 2a identified from HTS, removal of the
hydroxypropyl group (2b) resulted in a 5-fold decrease in potency (IC50 = 69 µM, Fig. 3a). This
data suggested that the hydroxypropyl group formed an interaction with some residues on
BCL6BTB, and contributed to the improvement of the potency. To estimate the binding mode, a
docking model of 2a with BCL6 was constructed by MOE using the BCL6/1a complex
structure.23 The docked pose of 2a overlaid well with the binding mode of the 1a-bound BCL6
10

crystal structure (Fig. 3b). In this docking model, the alcohol moiety of the hydroxypropyl group
of 2a was located in the vicinity of Arg28, and the interaction between the alcohol moiety and
the guanidyl side chain of Arg28 was indicated. Based on this docking study, the
hydroxypropylamino group was introduced into the 5-position of ring A of 1a. As a result, the
hybridized compound 3 showed a 20-fold improvement in BCL6BTB–BCoR PPI inhibitory
potency (IC50 = 0.24 µM) as compared with that of 1a.
Fig. 3. Presumed effect of hydroxypropylamino group of 2a by docking study. (a) Introduction
of the hydroxypropylamino group led to improvement of the potency. (b) Superposition of
BCL6-bound 1a (yellow) and the docking structure of 2a (magenta) constructed by MOE. A
hydrogen bonding between the hydroxy group of 2a and the guanidyl side chain of Arg28 is
suggested. Some amino acid residues were omitted for clarification.
3. 4. Physicochemical properties
11

Although inhibitory activity was improved, compound 3 displayed insufficient aqueous
solubility (0.09 µg/mL) and parallel artificial membrane permeability assay (PAMPA)
permeability (undetected) under neutral conditions (at pH 6.8 for aqueous solubility and 7.4 for
PAMPA). To improve these physicochemical properties that could affect its cellular activity,
conversion of the substituent at the 5-position of ring A was explored, and the results are
summarized in Table 2. Ester 4 exhibited improved PAMPA permeability (151 nm/s) with good
PPI inhibitory activity (IC50 = 0.43 µM), although its aqueous solubility remained low (0.42
µg/mL). On the other hand, carboxylic acid 5 showed high aqueous solubility (> 72 µg/mL)
while its PAMPA permeability was poor. Introduction of a 3-pyridylmethylamino group (6) or 4-
tetrahydropyranylamino group (7) at the 5-position resulted in enhancement of the potencies
(IC50 = 0.15 and 0.13 µM, respectively), although their aqueous solubilities were still insufficient
(< 0.23 and 0.61 µg/mL, respectively). Interestingly, the 4-tetrahydropyranyloxy derivative 8a
exhibited significantly improved aqueous solubility (18 µg/mL) while maintaining high potency
(IC50 = 0.22 µM) and moderate PAMPA permeability (45 nm/s). The significantly improved
aqueous solubility of 8a might be attributed to its more flexible conformation through disruption
of the planar conformation due to its intramolecular hydrogen bond between the oxygen atom of
the nitro group and the NH group of the 4-tetrahydropyranylamino moiety of 7a.24, 25
Table 2. Effects of the 5-substituent of ring A on cell-free PPI inhibition, solubility, and
membrane permeability
12

ELISA IC50 (µM)a, b
0.24 (0.18–0.33)
0.43 (0.37–0.50)
PAMPA (nm/s)d
undetected
151
Compound
Aqueous
solubility
(µg/mL)c
R
0.09
3
4
0.42
0.13 (0.10–0.15)
>72
undetected
5
6
0.15 (0.12–0.19)
0.13 (0.11–0.15)
<0.23
0.61
147
101
7
0.22 (0.18–0.27)
18
45
8a
a
b
Inhibition of BCL6-corepressor (BCoR) interaction was measured. 95% confidence intervals
are shown in parentheses. c Measured at pH 6.8. d Measured at pH 7.4.
Compounds 3–8a, whose PPI inhibitory activities were improved by introducing side chains
with a hydrogen bond acceptor (HBA) into 1a, were crystallized in complex with BCL6 to
clarify binding modes. An X-ray crystal structure of carboxylic acid 5 bound to BCL6 was
obtained. It elucidated that 5 binds to the same site as 1a in the same manner (Fig. 4). However,
13

the electron density map showed that the carboxylate moiety of 5 is exposed to solvent and
mostly disordered (data not shown). No clear interactions were observed between the
carboxylate moiety and the guanidyl side chain of Arg28, and the effect of the carboxylate
moiety for the improved potency remains unclear because no specific structural features are
observed.
Fig. 4. X-ray crystal structure of 5 bound to BCL6 (PDB code: 5X9P)
Our next efforts were focused on modification of ring B for further improvement of
membrane permeability. The benzimidazolone scaffold (8a) with two hydrogen bond donors
(HBDs) was considered to be one of the reasons for the low PAMPA permeability. Therefore,
we synthesized oxindole 8b and tetrahydroquinolinone 8c to improve PAMPA permeability
while maintaining potency. As expected, the PAMPA permeabilities of 8b and 8c (288 and 206
nm/s, respectively) were dramatically improved compared with that of 8a (45 nm/s). The
14

potency and aqueous solubility of 8b (IC50 = 0.13 µM, aqueous solubility = 12 µg/mL) and 8c
(IC50 = 0.10 µM, aqueous solubility = 12 µg/mL) were comparable to those of 8a (IC50 = 0.22
µM, aqueous solubility = 18 µg/mL). In addition, 8b and 8c did not exhibit significant
cytotoxicity even at 30 µM (Table 3).
Table 3. In vitro profiles of diphenylamine derivatives having modified ring B
X = NH, n = 0
X = CH2, n = 0
X = CH2, n = 1
Compound
8a
8b
8c
ELISA IC50 (µM)a, b
0.22 (0.18–0.27)
0.13 (0.12–0.15)
0.10 (0.091–0.12)
Aqueous solubility
(µg/mL)c
18
45
12
288
99
12
206
83
PAMPA (nm/s)d
Cytotoxicity (ATP, %
at 30 µM)
100
a
b
Inhibition of BCL6-corepressor (BCoR) interaction was measured. 95% confidence intervals
are shown in parentheses. c Measured at pH 6.8. d Measured at pH 7.4.
3. 5. Cell-based M2H assay
15

Compounds 8b and 8c, having improved physicochemical properties, were further evaluated
in a cell-based assay using a mammalian two-hybrid (M2H) system (Table 4). Cellular PPI
inhibition between BCL6BTB and BCoR by 8b was observed with an IC50 value of 1.2 µM.
Compound 8c also exhibited good cellular PPI inhibitory activity in the submicromolar range
(M2H IC50 = 0.72 µM). Although the cell-free PPI inhibitory activity of compound 3 was
comparable to those of 8b and 8c, no cellular activity was observed even at 100 µM of 3,
indicating that its physicochemical properties were inadequate.
Table 4. Cell-based PPI inhibition of diphenylamine derivatives using M2H method
Compound
8b (n = 1)
8c (n = 2)
3
M2H IC50 (µM)a
>100
1.2
0.72
a Inhibition of BCL6-corepressor (BCoR) interaction was measured.
3. 6. PK profiles
The pharmacokinetic profile of compound 8c was evaluated by a mouse cassette-dosing study
(0.1 mg/kg iv; 1 mg/kg po). As summarized in Fig. 5, compound 8c exhibited a good
16

pharmacokinetic profile (MRT = 3.3 h, AUC = 1.27 µg•h/mL, F = 79.9%). These data indicate
that compound 8c could be utilized as a tool compound for in vitro and in vivo biological studies.
Fig. 5. Pharmacokinetic parameters for 8c by mouse cassette-dosing (0.1 mg/kg iv; 1 mg/kg po).
Cmax = maximal plasma concentration reached after oral administration. Tmax = time to reach the
maximal plasma concentration after oral administration. MRT = mean residence time after oral
administration. F = oral bioavailability. AUC = exposure (total area under the plasma
concentration–time curve) after oral administration. Cltotal = total clearance. VDss = volume of
distribution.
4. Conclusion
Novel BCL6BTB-BCoR PPI inhibitors were identified by a structure-based drug design
(SBDD) approach starting from the HTS hit compounds 1a and 2a. On the basis of the
superposition of the X-ray crystal structure of 1a bound to BCL6 and the docking model of 2a,
we designed diphenylamine 3 by introducing a hydroxypropylamino group, and it showed the
significantly improved PPI inhibitory activity compared to that of 1a. Further exploration of the
substituents on rings A and B of 3 led to the identification of oxindole 8b and
17

tetrahydroquinolinone 8c with approximately 40-fold greater PPI inhibitory activities than that of
1a. They also exhibited sufficient membrane permeabilities and aqueous solubilities to give
measurable activity in the M2H assay. Evaluation of 8b and 8c by the M2H assay demonstrated
good cellular PPI inhibitory activities with IC50 values of 1.2 µM and 0.72 µM, respectively. In
addition, the good pharmacokinetic profile of 8c in mice indicated that 8c could be a suitable
chemical probe as a BCL6 inhibitor for both in vivo and in vitro experiments. Although PPI
inhibitors tend to be larger molecules (MW ≥ 500) than enzyme inhibitors in general,26 the
molecular weights of our compounds remained around 400. We believe this research is a good
example of how an SBDD approach can work efficiently against PPI targets.
5. Experimental section
5. 1. Chemistry
Melting points were determined on a Büchi melting point apparatus B-545 and were
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker
AVANCE-300 (300 MHz) instruments in CDCl3 or DMSO-d6 solution. Chemical shifts are given
in parts per million (ppm) with tetramethylsilane as an internal standard. Abbreviations are used
as follows: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, dd =
doublet of doublets, dt = doublet of triplets, brs = broad singlet. Coupling constants (J values) are
given in hertz (Hz). Elemental analyses were carried out by Elemental Microanalysis Team in
Medicinal Chemistry Research Laboratories and were within 0.4% of the theoretical values
unless otherwise noted. Low-resolution mass spectra (MS) were acquired using an Agilent
LC/MS system (Agilent1200SL/Agilent6130MS or Agilent1200SL/Agilent1956MS or
18

Agilent1200SL/Agilent6110MS), Shimadzu UFLC/MS (Prominence UFLC high pressure
gradient system/LCMS-2020) operating in electron spray ionization mode (ESI+). The column
used was an L-column 2 ODS (3.0 x 50 mm I.D., 3 µm, CERI, Japan) with a temperature of 40
°C and a flow rate of 1.2 or 1.5 ml/min. Condition 1: Mobile phases A and B under an acidic
condition were 0.05% TFA in water and 0.05% TFA in MeCN, respectively. The ratio of mobile
phase B was increased linearly from 5% to 90% over 0.9 min, 90% over the next 1.1 min.
Condition 2: Mobile phases A and B under a neutral condition were a mixture of 5 mmol/L
AcONH4 and MeCN (9:1, v/v) and a mixture of 5 mmol/L AcONH4 and MeCN (1:9, v/v),
respectively. The ratio of mobile phase B was increased linearly from 5% to 90% over 0.9 min,
90% over the next 1.1 min.
The purities of all compounds tested in biological systems were assessed as being > 95%
using elemental analysis or analytical HPLC. Purity data were collected by HPLC with NQAD
(Nano Quality Analyte Detector) or Corona CAD (Charged Aerosol Detector). The column was
an L-column 2 ODS (30 x 2.1 mm I.D., CERI, Japan) or a Capcell Pak C18AQ (50 mm x 3.0
mm I.D., Shiseido, Japan) with a temperature of 50 °C and a flow rate of 0.5 ml/min. Mobile
phases A and B under a neutral condition were a mixture of 50 mmol/L ammonium acetate,
water and acetonitrile (1:8:1, v/v/v) and a mixture of 50 mmol/L ammonium acetate and MeCN
(1:9, v/v), respectively. The ratio of mobile phase B was increased linearly from 5% to 95% over
3 min, 95% over the next 1 min. Reagents and solvents were obtained from commercial sources
and used without further purification.
Reaction progress was determined by thin layer chromatography (TLC) analysis on Merck
Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic purification was carried out
on silica gel columns ((Merck Kieselgel 60, 70‒230 mesh or 230‒400 mesh, Merck),
19

(Chromatorex NH-DM 1020, 100‒200 mesh, Fuji Silysia Chemical, Ltd.) or (Inject column and
Universal column, YAMAZEN Co.)) or on Purif-Pack (Si or NH, Shoko Scientific Co., Ltd.).
Preparative HPLC were acquired using a Gilson Preparative HPLC System (GX-D) with UV
detector (220 and 254 nm). The column used was a Symmetrix ODS-R (150 x 30 mm I.D., 5 µm,
Boston Analytics, Inc. China) and a flow rate of 25 ml/min. Condition 1: Mobile phases A and B
under an acidic condition were 0.225% formic acid in water and MeCN, respectively. Condition
2: Mobile phases A and B under a basic condition were 0.05% ammonium hydroxide in water
and MeCN, respectively. The ratio of mobile phase B was increased linearly over between 8 and
15 min. All commercially available solvents and reagents were used without further purification.
Yields were not optimized. The following abbreviations are used. CDCl3, deuterated chloroform;
DMA, N,N'-dimethylacetamide; DME, 1,2-dimethoxyethane; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; DMSO-d6, dimethyl sulfoxide-d6; DIPEA, N,N-
diisopropylethylamine; EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; EtOAc, ethyl
acetate; EtOH, ethanol; HOBt·NH3, 1-hydroxybenzotriazole ammonium salt; IPE, diisopropyl
ether; MeCN, acetonitrile; MeOH, methanol; NMP, 1-methyl-2-pyrrolidone; Pd2(dba)3,
Tris(dibenzylideneacetone)dipalladium(0); BINAP, racemic-2,2'-bis(diphenylphosphino)-1,1'-
binaphthyl; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
5-((2-Bromo-4-nitrophenyl)amino)-1H-benzo[d]imidazol-2(3H)-one (1b)
To a solution of compound 9 (149 mg, 1.0 mmol) and TEA (0.21 mL, 1.5 mmol) in DMSO (4
mL) was added compound 10b (242 mg, 1.1 mmol) at rt. The mixture was stirred at rt for 3 d
and at 50 °C for 1 d. The reaction mixture was cooled to rt and diluted with MeCN (4 mL) and
water. The precipitated solid was collected by filtration, washed with MeCN–water (1:1) and
20

1
water, and dried to give compound 1b (145 mg, 0.42 mmol, 42%) as an orange solid. H NMR
(DMSO-d6) 6.79 (1H, d, J = 9.3 Hz), 6.83–6.89 (2H, m), 6.95–7.00 (1H, m), 8.00 (1H, dd, J =
9.3, 2.6 Hz), 8.35 (1H, d, J = 2.6 Hz), 8.38 (1H, brs), 10.70 (2H, brs). MS m/z 351 (M+H)+. Mp
299–301 ºC. Anal. Calcd for C13H9BrN4O3·2.7H2O: C, 39.25; H, 3.65; N, 14.08. Found: C, 39.08;
H, 3.46; N, 14.02.
5-((4-Nitro-2-(trifluoromethyl)phenyl)amino)-1H-benzo[d]imidazol-2(3H)-one (1c)
To a solution of compound 9 (149 mg, 1.0 mmol) and TEA (0.21 mL, 1.5 mmol) in DMSO (4
mL) was added compound 10c (0.15 mL, 1.1 mmol) at rt. The mixture was stirred at rt for 3 d
and diluted with EtOH (2 mL) and water (6 mL). The precipitated solid was collected by
filtration, washed with MeCN–water (1:1), water and MeCN, and dried to give compound 1c
1
(157 mg, 0.46 mmol, 46%) as a yellow solid. H NMR (DMSO-d6) 6.78–6.89 (3H, m), 6.99
(1H, d, J = 8.1 Hz), 8.15 (1H, dd, J = 9.4, 2.6 Hz), 8.30 (1H, d, J = 2.6 Hz), 8.65 (1H, s), 10.74
(2H, s). MS m/z 339 (M+H)+. Mp 279–281 ºC. Anal. Calcd for C14H9F3N4O3·0.25H2O: C, 49.06;
H, 2.79; N, 16.35. Found: C, 49.15; H, 2.67; N, 16.18.
5-Nitro-2-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)benzonitrile (1d)
To a solution of compound 9 (149 mg, 1.0 mmol) and TEA (0.21 mL, 7.5 mmol) in DMSO (4
mL) was added compound 10d (183 mg, 1.1 mmol) at rt. The mixture was stirred at rt for 3 d,
diluted with EtOH (2 mL) and water (4 mL). The precipitated solid was collected by filtration
and washed with MeCN–water (1:1). The solid was suspended in MeCN, collected by filtration,
washed with MeCN, and dried to give compound 1d (237 mg, 0.80 mmol, 80%) as an orange
1
solid. H NMR (DMSO-d6) 6.85–6.99 (4H, m), 8.15 (1H, dd, J = 9.5, 2.7 Hz), 8.51 (1H, d, J =
21

2.7 Hz), 9.36 (1H, s), 10.72 (2H, s). MS m/z 296 (M+H)+. Mp 342–344 ºC. Anal. Calcd for
C14H9N5O3·0.4H2O: C, 55.59; H, 3.27; N, 23.15. Found: C, 55.77; H, 3.18; N, 22.97.
3-Chloro-4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)benzonitrile (1e)
To a solution of compound 9 (179 mg, 1.2 mmol) and DIEPA (0.35 mL, 2.0 mmol) in DMSO
(4 mL) was added compound 10e (156 mg, 1.0 mmol) at rt. The mixture was stirred at 100 °C
overnight and at 140 °C overnight. The reaction mixture was cooled to rt and diluted with water.
The precipitated solid was collected by filtration, washed with water, and dried. The residue was
suspended in EtOAc–MeOH and purified by silica gel column chromatography (EtOAc:MeOH =
10:0 to 9:1). The fractions were combined and evaporated. The residue was suspended in EtOAc,
collected by filtration, washed with EtOAc, and then dried to give compound 1e (105 mg, 0.37
1
mmol, 37%) as a pale purple solid. H NMR (DMSO-d6) 6.78–6.87 (3H, m), 6.91–6.98 (1H,
m), 7.47 (1H, dd, J = 8.6, 1.9 Hz), 7.85 (1H, d, J = 2.0 Hz), 8.19 (1H, s), 10.64 (2H, s). MS m/z
285 (M+H)+. Mp 324–326 ºC. Anal. Calcd for C14H9ClN4O·0.25H2O: C, 58.14; H, 3.31; N, 19.37.
Found: C, 58.01; H, 3.18; N, 19.24.
Methyl 3-chloro-4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)benzoate (1f)
To a solution of compound 10f (834 mg, 4.4 mmol) and compound 9 (550 mg, 3.7 mmol) in
DMSO (5 mL) was added DIPEA (1.3 mL, 7.4 mmol) at rt. The mixture was stirred at 100 °C
for 1 d and heated under microwave irradiation at 160 °C for 2 h. The reaction mixture was
cooled to rt, diluted with EtOAc and water, and extracted with EtOAc. The extract was washed
with water and brine, dried over Na2SO4, and then evaporated. The residual solid was suspended
in EtOAc and insoluble materials were removed by filtration. The filtrate was purified by silica
22

gel column chromatography (EtOAc:MeOH = 10:0 to 9:1). The fractions were combined and
evaporated. The residual solid was suspended in EtOAc, collected by filtration, washed with
1
EtOAc, and dried to give compound 1f (17 mg, 0.05 mmol, 1%) as an off-white solid. H NMR
(DMSO-d6) 33.78 (3HH, s), 66..80–6.95 (4H, m), 7.66 (1H, dd, J = 8.7, 2.0 Hz), 7.85 (1H, d, J = 2.0
Hz), 8.03 (1H, s), 10.61 (1H, brs), 10.62 (1H, brs). MS m/z 318 (M+H)+. Mp 304–306 ºC. Anal.
Calcd for C15H12ClN3O3·0.3H2O: C, 55.75; H, 3.93; N, 13.00. Found: C, 55.93; H, 3.86; N, 12.75.
3-Chloro-4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)benzoic acid (1g)
To a solution of compound 1f (230 mg, 0.72 mmol) in MeOH (5 mL) and THF (5 mL) was
added 1 M NaOH (2.9 mL, 2.9 mmol) at rt. The mixture was stirred at 50 °C for 3 h, cooled to rt,
and acidified with 5% aqueous citric acid solution. The precipitated solid was collected by
filtration and washed with water. The residue was dissolved in MeOH and MeCN, and
evaporated. The resulting solid was suspended in MeCN–MeOH, collected by filtration, washed
with MeCN, and dried to give compound 1g (125 mg, 0.41 mmol, 57%) as a pale brown solid. 1H
NMR (DMSO-d6) 6.788–6.94 (4H, m), 7.64 (1H, dd, J = 8.6, 1.9 Hz), 7.83 (1H, d, J = 2.0 Hz),
7.94 (1H, s), 10.59 (1H, brs), 10.60 (1H, brs), 12.55 (1H, brs). MS m/z 304 (M+H)+. Mp 319–321
ºC. Anal. Calcd for C14H10ClN3O3·1.6H2O: C, 50.57; H, 4.00; N, 12.64. Found: C, 50.57; H, 3.86;
N, 12.78.
3-Chloro-4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)benzamide (1h)
To a solution of compound 1g (100 mg, 0.33 mmol) and HOBt·NH3 (65 mg, 0.43 mmol) in
DMF (5 mL) was added EDC (67 mg, 0.43 mmol) at rt. The mixture was stirred at rt for 4 d,
cooled to 0 °C, and diluted with water. The precipitated solid was collected by filtration, washed
23

with water, and dried. The resulting solid was suspended in MeOH–EtOAc, collected by
filtration, washed with EtOAc, and dried to give compound 1h (70 mg, 0.23 mmol, 70%) as a
pale brown solid. 1H NMR (DMSO-d6) 6.78–6.93 (4H, m), 7.13 (1H, brs), 7.62 (1H, dd, J = 8.6,
2.0 Hz), 7.71–7.81 (2H, m), 7.89 (1H, d, J = 2.0 Hz), 10.55 (1H, s), 10.57 (1H, s). MS m/z 303
(M+H)+. Mp 289–291 ºC. Anal. Calcd for C14H11ClN4O2·0.4H2O: C, 54.26; H, 3.84; N, 18.08.
Found: C, 54.37; H, 3.67; N, 17.80.
3-((4,5-Dichloro-2-nitrophenyl)amino)propan-1-ol (13a)
Compound 11 (0.40 mL, 3.1 mmol) was added to a solution of DIPEA (0.80 mL, 4.6 mmol)
and compound 12a (0.23 mL, 3.1 mmol) in DMA (5 mL) at 0 °C. The mixture was stirred at rt
overnight. The reaction mixture was poured into water and extracted with EtOAc. The organic
layer was separated, washed with water and brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography (hexane:EtOAc = 4:1 to 1:1) to
give compound 13a (723 mg, 2.73 mmol, 89 %) as an orange solid. 1H NMR (DMSO-d6) 1.70–
1.80 (2H, m), 3.38–3.48 (2H, m), 3.49–3.58 (2H, m), 4.74 (1H, t, J = 5.0 Hz), 7.35 (1H, s), 8.24
(1H, s), 8.41 (1H, t, J = 5.4 Hz). MS m/z 266 (M+H)+.
5-((2-Chloro-5-((3-hydroxypropyl)amino)-4-nitrophenyl)amino)-1H-benzo[d]imidazol-2(3H)-
one (3)
A mixture of compound 13a (100 mg, 0.38 mmol), compound 9 (68 mg, 0.45 mmol) and
DIPEA (0.10 mL, 0.57 mmol) in DMA (1 mL) was heated at 150 °C for 30 min and then heated
under microwave irradiation at 200 °C for 2 h. The mixture was poured into water and extracted
with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4,
24

and concentrated in vacuo. The precipitate was crystallized from EtOAc to give an orange solid.
The solid was purified by preparative HPLC (L-Column 2 ODS, eluted with H2O in MeCN
containing 0.1% TFA). The desired product was crystallized in the fraction, collected by
filtration, and then washed with water, IPE and hexane to give compound 3 (7.2 mg, 0.019
1
mmol, 5 %) as an orange solid. H NMR (DMSO-d6) 1.48–1.75 (2H, m), 2.99–3.14 (2H, m),
3.38–3.47 (2H, m), 4.42–4.70 (1H, m), 6.03 (1H, s), 6.75–7.06 (3H, m), 8.06 (1H, s), 8.24–8.54
(2H, m), 10.22–10.96 (2H, m). MS m/z 378 (M+H)+. Mp 294–296 ºC. Anal. Calcd for
C16H16ClN5O4·0.1H2O: C, 50.63; H, 4.30; N, 18.45. Found: C, 50.61; H, 4.22; N, 18.29.
Ethyl 3-((4,5-dichloro-2-nitrophenyl)amino)propanoate (13b)
Compound 13b was prepared from ethyl 3-aminopropanoate hydrochloride in a similar
manner to that described for compound 13a and obtained in 85% yield as an orange solid. 1H
NMR (DMSO-d6) 1.18 ((3H, t,, J = 7.1 Hz), 2.67 (2H, t, J = 6.6 Hz), 3.59–3.68 (2H, m), 4.08
(2H, q, J = 7.1 Hz), 7.44 (1H, s), 8.24 (1H, s), 8.25–8.30 (1H, m). MS m/z 307 (M+H)+.
Ethyl 3-((4-chloro-2-nitro-5-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-
yl)amino)phenyl)amino)propanoate (4)
A mixture of compound 13b (685 mg, 2.2 mmol), compound 9 (400 mg, 2.7 mmol) and
DIPEA (0.58 mL, 3.4 mmol) in NMP (3 mL) was heated under microwave irradiation at 200 °C
for 3 h. The mixture was directly charged on silica gel and purified by silica gel column
chromatography (hexane:EtOAc = 3:1 to 0:10 and then EtOAc:MeOH = 10:0 to 20:1) to give a
yellow solid. The solid was crystallized from EtOAc to give compound 4 (287 mg, 0.68 mmol,
1
31 %) as a yellow solid. H NMR (DMSO-d6) 1.12 (3H, t, J = 7.1 Hz), 2.52–2.58 (2H, m),
25

3.21–3.30 (2H, m), 4.01 (2H, q, J = 7.1 Hz), 6.04 (1H, s), 6.86–6.97 (3H, m), 8.06 (1H, s), 8.33
(1H, t, J = 5.2 Hz), 8.43 (1H, brs), 10.68 (2H, brs). MS m/z 420 (M+H)+. Mp 221–223 ºC. Anal.
Calcd for C18H18ClN5O5·H2O: C, 49.38; H, 4.60; N, 16.00. Found: C, 49.12; H, 4.48; N, 15.86.
3-((4-Chloro-2-nitro-5-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-
yl)amino)phenyl)amino)propanoic acid (5)
A mixture of compound 4 (50 mg, 0.12 mmol) and 2 M NaOH in H2O (1 mL, 2.0 mmol) in
THF (1 mL) and MeOH (1 mL) was stirred at rt overnight. The mixture was neutralized with 1
M HCl at 0 °C and extracted with EtOAc. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. The residue was crystallized from EtOAc–hexane
to give compound 5 (22 mg, 0.056 mmol, 47 %) as a yellow solid. 1H NMR (DMSO-d6) 2.39–
2.48 (2H, m), 3.16–3.28 (2H, m), 6.08 (1H, s), 6.88–6.97 (3H, m), 8.06 (1H, s), 8.33 (1H, t, J =
5.2 Hz), 8.42 (1H, s), 10.67 (2H, d, J = 3.6 Hz), 12.40 (1H, brs). MS m/z 392 (M+H)+. Mp 282–
284 ºC. Anal. Calcd for C16H14ClN5O5·0.1EtOAc·0.3H2O: C, 48.52; H, 3.82; N, 17.25. Found: C,
48.69; H, 3.70; N, 17.01.
4,5-Dichloro-2-nitro-N-(pyridin-3-ylmethyl)aniline (13c)
A mixture of compound 11 (2.5 g, 12 mmol), compound 12c (1.3 g, 12 mmol) and TEA (1.7
mL, 12 mmol) in DMSO (30 mL) was stirred at rt for 3 h. The mixture was diluted with EtOAc,
washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The residue was
1
washed with IPE to give compound 13c (3.0 g, 10 mmol, 85 %) as a yellow solid. H NMR
(CDCl3) 4.55 (2H, d, J = 5.7 Hz), 6.93 (1H, s), 7.34 (1H, dd, J = 7.7, 4.7 Hz), 7.62–7.70 (1H,
m), 8.23–8.36 (2H, m), 8.56–8.69 (2H, m). MS m/z 298 (M+H)+.
26

5-((2-Chloro-4-nitro-5-((pyridin-3-ylmethyl)amino)phenyl)amino)-1H-benzo[d]imidazol-2(3H)-
one (6)
Pd2(dba)3 (31 mg, 0.030 mmol) was added to a solution of compound 13c (200 mg, 0.67
mmol), compound 9 (100 mg, 0.67 mmol), cesium carbonate (328 mg, 1.0 mmol) and BINAP
(42 mg, 0.070 mmol) in DME (5 mL). The mixture was stirred at 100 °C under N2 for 4 h and
then diluted with EtOAc and water. The precipitate was collected by filtration. The solid was
purified by preparative HPLC (L-Column 2 ODS, eluted with H2O in MeCN containing 0.1%
TFA). To the desired fraction was added aqueous NaHCO3 solution and concentrated into half
volume in vacuo. The precipitate was collected by filtration to give compound 6 (15 mg, 0.037
mmol, 5 %) as a light brown solid.1H NMR (DMSO-d6) 4.37 (2H, d, J = 5.7 Hz), 5.87 (1H, s),
6.42 (1H, d, J = 8.2 Hz), 6.68 (1H, s), 6.80 (1H, d, J = 8.2 Hz), 7.22–7.27 (1H, m), 7.46 (1H, d, J
= 7.6 Hz), 8.06 (1H, s), 8.30 (2H, brs), 8.45 (1H, d, J = 4.4 Hz), 8.85 (1H, t, J = 6.0 Hz), 10.65
(2H, d, J = 12.7 Hz). MS m/z 411 (M+H)+. Mp 298–300 ºC. Anal. Calcd for
C19H15ClN6O3·0.1H2O: C, 55.31; H, 3.71; N, 20.37. Found: C, 55.22; H, 3.59; N, 20.24.
N-(4,5-Dichloro-2-nitrophenyl)tetrahydro-2H-pyran-4-amine (13d)
Compound 11 (0.40 mL, 3.1 mmol) was added to a solution of DIPEA (0.80 mL, 4.6 mmol)
and compound 12d (310 mg, 3.1 mmol) in DMSO (5 mL) at rt. The mixture was stirred at rt
overnight. The mixture was poured into water and extracted with EtOAc. The organic layer was
separated, washed with aqueous citric acid solution and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel column chromatography
(hexane:EtOAc = 3:1 to 3:2) to give compound 13d (784 mg, 2.7 mmol, 88 %) as an orange
27

1
solid. H NMR (CDCl3) 1.59–1.78 (2H, m), 2.01–2.11 (2H, m), 3.53–3.73 (3H, m), 4.03 (2H,
dt, J = 12.0, 3.8 Hz), 6.98 (1H, s), 8.01 (1H, d, J = 6.5 Hz), 8.30 (1H, s). MS m/z 291 (M+H)+.
5-((2-Chloro-4-nitro-5-((tetrahydro-2H-pyran-4-yl)amino)phenyl)amino)-1H-benzo[d]imidazol-
2(3H)-one (7)
A mixture of compound 13d (300 mg, 1.0 mmol), compound 9 (184 mg, 1.2 mmol) and
DIPEA (0.22 mL, 1.2 mmol) in NMP (1 mL) was heated under microwave irradiation at 200 °C
for 3 h. The mixture was directly charged on silica gel and purified by silica gel column
chromatography (hexane:EtOAc = 3:1 to 0:10 and then EtOAc:MeOH = 10:0 to 9:1) and
following preparative HPLC (L-Column 2 ODS, eluted with H2O in MeCN containing 0.1%
TFA). The desired fraction was concentrated in vacuo. The residue was washed with water, IPE
and hexane to give compound 7 (55 mg, 0.14 mmol, 13 %) as an orange solid.1H NMR (DMSO-
d6) 1.40–1.55 (2H, m), 1.74–1.87 (2H, m), 3.19–3.30 (3H, m), 3.74–3.84 (2H, m), 6.12 (1H, s),
6.87–6.98 (3H, m), 8.07 (1H, s), 8.12 (1H, d, J = 7.0 Hz), 8.48 (1H, brs), 10.69 (2H, brs). MS
m/z 404 (M+H)+. Mp 305–307 ºC. Anal. Calcd for C18H18ClN5O4·0.25H2O: C, 52.95; H, 4.57; N,
17.15. Found: C, 53.07; H, 4.37; N, 16.98.
4-(4,5-Dichloro-2-nitrophenoxy)tetrahydro-2H-pyran (13e)
Compound 12e (1.5 mL, 15 mmol) was added to a suspension of sodium hydride (60% wt,
0.67 g, 17 mmol) in DMF (30 mL) at 0 °C. After being stirred at 0 °C for 20 min, compound 11
(2.0 mL, 15 mmol) was added to the reaction mixture. The mixture was stirred at rt for 20 min.
The mixture was quenched with water at rt and extracted with EtOAc. The organic layer was
separated, washed with water and brine, dried over MgSO4, and concentrated in vacuo. The
28

residue was purified by silica gel column chromatography (hexane:EtOAc = 10:0 to 3:1) to give
1
compound 13e (2.6 g, 8.9 mmol, 58 %) as a pale yellow solid. H NMR (CDCl3) 1.78–1.96
(2H, m), 1.97–2.16 (2H, m), 3.51–3.75 (2H, m), 3.89–4.06 (2H, m), 4.46–4.80 (1H, m), 7.18
(1H, s), 7.98 (1H, s).
5-((2-Chloro-4-nitro-5-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)amino)-1H-benzo[d]imidazol-
2(3H)-one (8a)
Compound 8a was prepared from compound 13e in a similar manner to that described for
compound 6 and obtained in 47% yield as a yellow solid. 1H NMR (DMSO-d6) 1.52–1.65 (2H,
m), 1.78–1.91 (2H, m), 3.37–3.51 (2H, m), 3.70–3.82 (2H, m), 4.41–4.50 (1H, m), 6.51 (1H, s),
6.85–6.97 (3H, m), 8.07 (1H, s), 8.45 (1H, s), 10.66 (1H, s), 10.69 (1H, s). MS m/z 405 (M+H)+.
Mp 309–311 ºC. Anal. Calcd for C18H17ClN4O5·0.25H2O: C, 52.82; H, 4.31; N, 13.69. Found: C,
53.00; H, 4.33; N, 13.46.
5-((2-Chloro-4-nitro-5-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)amino)indolin-2-one (8b)
Compound 8b was prepared from compound 13e and 5-aminoindolin-2-one in a similar
manner to that described for compound 6 and obtained in 17% yield as a yellow solid.1H NMR
(DMSO-d6) 1.53–1.65 (2H, m), 1.80–1.91 (2H, m), 3.40–3.53 (4H, m), 3.70–3.82 (2H, m),
4.44–4.53 (1H, m), 6.46 (1H, s), 6.85 (1H, d, J = 8.1 Hz), 7.09–7.19 (2H, m), 8.07 (1H, s), 8.45
(1H, s), 10.43 (1H, s). MS m/z 404 (M+H)+. Mp 222–224 ºC. Anal. Calcd for
C19H18ClN3O5·0.25H2O: C, 55.89; H, 4.57; N, 10.29. Found: C, 55.87; H, 4.59; N, 10.19.
29