Design, synthesis and structure-activity relationship of novel tricyclic benzimidazolone derivatives as potent 18 kDa translocator protein (TSPO) ligands

Article abstract of DOI:10.1016/j.bmc.2012.12.024

The 18 kDa translocator protein (TSPO) was identified as a discrete receptor for diazepam (1). Since TSPO in the central nervous system (CNS) is believed to regulate neurosteroids biosynthesis, selective TSPO ligands are expected to be useful in the treatment of psychiatric disorders. We synthesized three novel tricyclic benzimidazolone derivatives, and selected the dihydroimidazoquinolinone derivative 27 as a lead TSPO ligand. Study of the structure-activity relationship (SAR) of dihydroimidazoquinolinone derivatives revealed compounds with potent affinity for TSPO (subnanomolar K_i values), but poor metabolic stability. The optimization of these compounds led to compound 48 with potent affinity for TSPO and good in vitro PK profile.

Full text of DOI:10.1016/j.bmc.2012.12.024

Bioorganic & Medicinal Chemistry 21 (2013) 1257–1267
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationship of novel
tricyclic benzimidazolone derivatives as potent 18 kDa translocator
protein (TSPO) ligands
b
a
c
a
a
Takayuki Fukaya a, , Toru Kodo , Takeo Ishiyama , Hiroyuki Nishikawa , Satoko Baba , Shuji Masumoto
⇑
a Dainippon Sumitomo Pharma Co. Ltd, Enoki 33-94, Suita, Osaka 564-0053, Japan
b Dainippon Sumitomo Pharma Co. Ltd, Doshomachi 2-6-8, Chuo-ku, Osaka 541-0045, Japan
c Dainippon Sumitomo Pharma Co. Ltd, Kasugade Naka 3-1-98, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The 18 kDa translocator protein (TSPO) was identified as a discrete receptor for diazepam (1). Since TSPO
in the central nervous system (CNS) is believed to regulate neurosteroids biosynthesis, selective TSPO
ligands are expected to be useful in the treatment of psychiatric disorders. We synthesized three novel
tricyclic benzimidazolone derivatives, and selected the dihydroimidazoquinolinone derivative 27 as a
lead TSPO ligand. Study of the structure–activity relationship (SAR) of dihydroimidazoquinolinone deriv-
atives revealed compounds with potent affinity for TSPO (subnanomolar Ki values), but poor metabolic
stability. The optimization of these compounds led to compound 48 with potent affinity for TSPO and
good in vitro PK profile.
Received 27 November 2012
Revised 17 December 2012
Accepted 18 December 2012
Available online 27 December 2012
Keywords:
18 kDa translocator protein (TSPO)
Peripheral benzodiazepine receptor (PBR)
Tricyclic benzimidazolone
Dihydroimidazoquinolinone
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
activity relationship (SAR) studies of different substitutents at
the benzoxazolone ring revealed that the 7-Ph derivative 7 exhibits
The 18 kDa translocator protein (TSPO) was identified in 1977 as
a discrete receptor for diazepam (1).1 TSPO was previously called
the peripheral benzodiazepine receptor (PBR), but was renamed
by Papadopoulous and colleagues in 2006 as TSPO.2 TSPO, which
forms a heterotrimeric complex with the 32 kDa voltage-dependent
anion channel (VDAC) and the 30 kDa adenine nucleotide trans-
porter (ANT)3 is mainly distributed on the outer mitochondrial
membrane in peripheral tissues and the central nervous system
(CNS).4 Although the physiological role of TSPO in CNS is not clear,
a study on the function of TSPO suggested that TSPO regulates cho-
lesterol transport from the outer to the inner mitochondrial mem-
brane,5 which is known as the rate-determining step of
neurosteroids biosynthesis.6 As evidence has shown that neuroster-
oids play a role in psychiatric disorders,7 it is believed that TSPO li-
gands might be useful in the treatment of these disorders.
moderate affinity for TSPO (Ki = 9.0 nM). As part of our search for
compounds with various heteroaryls, we obtained interesting re-
sults in the benzimidazolone derivatives.13 Compound 8 exhibited
moderate affinity for TSPO (86% inhibition at 100 nM), and intro-
duction of a methyl group at the N3 position (9) increased this
affinity (100% inhibition at 100 nM). Considering the results
O
Me
Me
N
Me
N
O
O
Me
N
Me
N
N
N
Cl
Cl
5
5
Cl
Cl
1
3
)
Diazepam (
)
Ro5-4864 (2)
PK11195 (
NMe2
As shown in Figure 1, in addition to the classical TSPO ligands
Ro5-4864 (2)8 and PK11195 (3),9 several other TSPO ligands have
been reported, including the 8-oxopurine derivative AC-5216
(4)10 and indoleacetamide derivative SSR180575 (5).11 We have re-
cently reported a series of benzoxazolone derivatives, including
compound 6 as potent and selective TSPO ligands.12 Structure–
O
Me
N
N
N
O
Cl
N
N
N
Et
N
N
Me
O
O
AC-5216 (4)
SSR180575 (5)
⇑
Corresponding author. Tel.: +81 6 6337 5865; fax: +81 6 6337 6010.
E-mail address: takayuki-fukaya@ds-pharma.co.jp (T. Fukaya).
Figure 1. Chemical structures of selected TSPO ligands.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.024

1258
T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
O
O
O
NR1R2
N
X
N
N
N
7
4
4
7
Me
6
Me
5
6
N
O
N
O
O
O
5
N
R
10
6
7
5-Ph
7-Ph
8
R = H
9 R = Me
Figure 2. Design of novel TSPO ligands.
obtained with compounds 7 and 9, introduction of other substi-
tutes at the C-4 position and the N3 position of the benzimidazo-
lone ring was expected to be tolerated in relation to TSPO
activity. As the benzoxazolone ring in compound 6 plays an impor-
tant role as a planar aromatic region for the interaction with
TSPO,14 conversion of the benzoxazolone skeleton to a tricyclic
benzimidazolone ring which expands the planar region was
considered as favorable for enhanced interaction with TSPO.
Based on these exploratory findings, we have designed tricyclic
benzimidazolone derivatives as novel TSPO ligands (10). Here
we present the SAR of a series of dihydroimidazoquinolinone,
dihydroimidazobenzazepinone and dihydroimidazobenzoxazinone
derivatives as novel TSPO ligands (see Fig. 2).
O
O
OH
N
Me
Br
Br
N
N
N
a
b
O
21
O
N
39
40
c, d or e
f
O
O
N
N
Me
Me
X
N
N
N
O
O
N
2. Chemistry
41-48
49
The compounds in this study were synthesized as illustrated
in Schemes 1–3. Scheme 1 shows the preparation of 8-phenyl
dihydroimidazoquinolinone and 9-phenyl dihydroimidazobe-
zazepinone derivatives. Methoxycarbonylation of 1,2,3,4-tetra-
hydroquinoline (11) with methyl chloroformate afforded the
N-carbamate-protected 1,2,3,4-tetrahydroquinoline 13. Bromina-
tion of 13 with N-bromosuccinimide (NBS) in DMF proceeded to
give the expected 6-bromo derivative 15. Subsequent regioselec-
tive nitration of 15 was effected by action of NO2BF4 in CH3CN to
give compound 17. Reductive cyclization of 17 was achieved by
treatment with iron powder in AcOH at 80 °C to provide compound
19. Introduction of an acetate moiety was achieved by N-alkylation
of 19 with t-butyl bromoacetate to afford acetate 21, which was
subjected to Suzuki–Miyaura coupling with phenylboronic acid
to give compound 23. Deprotection of compound 23 with HCl
provided the key intermediary acid derivative 25. Condensation
of 25 with appropriate amines was carried out by combination of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotriazole (HOBt) or by the standard acid
chloride method using oxalyl chloride to afford the corresponding
Scheme 2. Synthesis of the dihydroimidazoquinolinone derivatives 41–49.
Reagents and conditions: (a) HCl/1,4-dioxane, AcOH, 50 °C; (b) N-methylaniline,
EDCI, HOBt, DMF, rt; (c) ArB(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux; (d)
ArSnBu3, Pd(PPh3)4, toluene, reflux; (e) 3-aminopyridine, Pd2(dba)3, Xantphos,
Cs2CO3, toluene, reflux; (f) H2, 10% Pd-C, MeOH, THF, rt.
amide derivatives 27–37. The 9-Ph dihydroimidazobenzazepinone
derivative 38 was obtained from 2,3,4,5-tetrahydro-1H-1-benzaze-
pine (12) in the same way as that for synthesis of 27. The chemical
structures of compounds 27–38 are shown in Tables 1 and 2.
Scheme 2 depicts the synthesis of a series of dihydroimidazo-
quinolinone derivatives with various substituents at the C-8 posi-
tion. Deprotection of 21 with HCl followed by condensation of 39
with N-methylaniline in the presence of EDCI and HOBt in DMF
gave compound 40. The target compounds 41–47 were prepared
via Suzuki–Miyaura coupling or Stille coupling of 40 with boronic
acid or an organotin reagent. Compound 48 was obtained by cou-
pling reaction with 3-aminopyridine in the presence of Pd2(dba)3
and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos)
using Cs2CO3 as a base in toluene.15 The bromine atom in compound
H
N
Br
Br
NO2
Br
O
a
c
e
b
d
N
NH
NCOOMe
n
NCOOMe
n
NCOOMe
n
n
n
11
13
15 n=1
16 n=2
17
19
n=1
20 n=2
n=1
n=1
n=1
12 n=2
14 n=2
18 n=2
O
O
O
O
OtBu
NR1R2
OtBu
OH
O
Br
N
N
f
N
N
g
N
N
N
N
h or i
O
O
O
n
n
n
n
21
23
25
27 37
n=1
n=1
n=1
-
n=1
n=2
22 n=2
24 n=2
26 n=2
38
Scheme 1. Synthesis of 27–38. Reagents and conditions: (a) methyl chloroformate, K2CO3, DMF, 50 °C; (b) NBS, DMF, rt; (c) NO2BF4, CH3CN, 0 °C; (d) Fe, AcOH, 80 °C; (e) t-
butyl bromoacetate, K2CO3, DMF, 50 °C; (f) PhB(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux; (g) HCl/1,4-dioxane, AcOH, 55 °C; (h) amine, EDCI, HOBt, DMF, rt; (i) (COCl)2,
DMF, CH2Cl2, then amine, Et3N, THF, rt.

T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
1259
NO2
NH2
Br
NO2
NH2
Br
NO2
NO2
NO2
NH2
a
d, e
b
c
f
O
O
NH
O
NH
O
O
O
O
O
OH
50
tBuO
tBuO
51
52
53
54
O
O
O
OtBu
OH
N
H
N
Me
NH2
NH
N
N
N
N
N
N
g
i
j
h
O
O
O
O
N
O
O
O
O
O
55
56
57
58
59
Scheme 3. Synthesis of the dihydroimidazobenzoxazinone derivative 59. Reagents and conditions: (a) t-butyl bromoacetate, K2CO3, DMF, rt; (b) NBS, DMF, rt; (c) p-
TsOHꢀH2O, toluene, 80 °C; (d) PhB(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, reflux; (e) p-TsOHꢀH2O, toluene, 80 °C; (f) LiAlH4, THF, rt; (g) CDI, THF, reflux; (h) t-butyl
bromoacetate, K2CO3, DMF, 70 °C; (i) HCl/1,4-dioxane, AcOH, 65 °C; (j) N-methylaniline, EDCI, HOBt, DMF, rt.
Table 1
Table 2
TSPO and CBR binding affinity, and metabolic stability of dihydroimidazoquinolinone,
dihydroimidazobenzazepinone and dihydroimidazobenzoxazinone derivatives
TSPO and CBR binding affinity, and metabolic stability of dihydroimidazoquinolinone
derivatives with various amide moieties
O
O
N
NR1R2
Me
N
O
N
N
O
N
Y
n
Compd
R1
R2
TSPO Kia
(nM)
CBR inhibitionb
(%)
Metabolic
stabilityc
(remaining %)
Compd
n
Y
TSPO Kia
(nM)
CBR inhibitionb
(%)
Metabolic stabilityc
(remaining %)
PK11195 (3)
6
27
38
59
1.7
1.6
0.94
0.32
0.21
27
28
29
30
31
32
33
34
35
36
37
Me
n-Pr
Me
Me
Me
Me
Me
Me
Me
Me
H
Ph
n-Pr
Bn
m-MeO-Ph
p-MeO-Ph
m-Cl-Ph
p-Cl-Ph
2-Py
0.94
1.7
14
11
72
6
3
3
12
1
1
5
14
30
68
1
12
1
1
2
1
CH2
CH2
O
0.23
0.28
0.73
0.44
0.13
2.5
2.1
15
33
0
1
0d
0
a
Ki Values represent the means of 1–3 separate experiments run in duplicate
4
0
0
0
0
using four concentrations of each compound.
Percent inhibition of [3H]-flumazenil specific binding at 10
lM of the
b
34
36
24
27
3-Py
4-Py
Ph
compound.
c
Metabolic stability data refer to percent of compound remaining after incuba-
tion with rat liver S-9 fraction (ca. 2.0 mgprotein/ml) and NADPH (ca. 3.0 mM) for
62
30 min at 37 °C. The initial concentration of each compound was 1.0 lM.
a
Ki Values represent the means of 1–3 separate experiments run in duplicate
using four concentrations of each compound.
b
Percent inhibition of [3H]-flumazenil specific binding at 10
lM of the
40 was removed by standard hydrogenation over Pd-C to give com-
pound 49. The chemical structures of compounds 41–48 are shown
in Table 3.
compound.
c
Metabolic stability data refer to percent of compound remaining after incuba-
tion with rat liver S-9 fraction (ca. 2.0 mgprotein/ml) and NADPH (ca. 3.0 mM) for
30 min at 37 °C. The initial concentration of each compound was 1.0 lM.
Synthesis of the dihydroimidazobenzoxazinone derivative 59
was achieved as illustrated in Scheme 3. O-Alkylation of 2-ami-
no-3-nitrophenol (50) with t-butyl bromoacetate using K2CO3 as
a base in DMF followed by bromination of 51 with NBS in DMF
afforded compound 52. Ring closure in 52 was realized with p-tol-
uenesulfonic acid in toluene at 80 °C to give compound 53. Open-
ing of the oxazinone ring was occurred under basic condition by
Suzuki–Miyaura coupling of 53 with phenylboronic acid to give
[(4-amino-5-nitrobiphenyl-3-yl)oxy]acetic acid, which was again
subjected to acidic condition with p-toluenesulfonic acid in tolu-
ene at 80 °C to afford the desired compound 54. Reduction of the
nitro group and the amide moiety in 54 was simultaneously
achieved with LiAlH4 to give compound 55. Cyclization of 55 by
1,10-carbonyldiimidazole (CDI) generated the dihydroimidazoquin-
olinone scaffold 56. The target compound 59 was obtained from 56
by a method similar to that for the synthesis of 40.
d
Metabolic stability determined at 10 lM of the compound.
binding to membranes prepared from rat kidney and rat cerebral
cortex, respectively. Most of the synthesized compounds showed
high selectivity for TSPO over CBR with only few compounds
exhibiting more than 50% inhibition of CBR at 10 lM.
As shown in Table 1, all three tricyclic benzimidazolone deriva-
tives were very potent TSPO ligands, showing subnanomolar Ki val-
ues. The dihydroimidazoquinolinone derivative 27 exhibited
stronger TSPO activity than that of the benzoxazolone derivative
6. As expected, this finding indicates that the expansion of the pla-
nar region strengthens interaction with the TSPO recognition site.
Further expansion of the ring to a seven-membered ring, as in
the dihydroimidazobenzazepinone derivative 38, led to a threefold
increase in TSPO affinity compared to that of 27, albeit with poor
metabolic stability. In an attempt to improve metabolic stability,
we prepared the dihydroimidazobenzoxazinone derivative 59 by
replacing methylene chain at the C-6 position of 27 by an oxygen
atom. Although compound 59 showed the highest affinity for TSPO
among all three tricyclic derivatives (Ki = 0.21 nM), it had low met-
abolic stability. Taking all these results into account, compound 27,
3. Results and discussion
Affinity of the prepared compounds for TSPO and the central
benzodiazepine receptor (CBR) was evaluated by measuring each
compound’s ability to displace [3H]-PK11195 and [3H]-flumazenil

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T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
Table 3
Next, we examined the effect of a substitution at the C-8 posi-
tion. As shown in Table 3, most compounds showed excellent affin-
ity for TSPO. Compound 40, an intermediate of the C-8 substituted
derivatives, exhibited very potent affinity for TSPO, albeit with
poor metabolic stability. Introduction of a methoxy or trifluoro-
methyl group onto the phenyl ring at the C-8 position of 27 in-
creased TSPO binding affinity. In particular, compounds 41 and
44 were very potent TSPO ligands with Ki values of 0.11 and
0.16 nM, respectively. Based on the results obtained from com-
pounds 41–44, it was predicted that the electron density of the
phenyl ring at the C-8 position does not affect the binding affinity
for TSPO. Compounds bearing a pyridyl moiety, that is, 45–47, also
exhibited high affinity and selectivity for TSPO. Especially, replace-
ment of the phenyl ring at the C-8 position with a 4-pyridyine ring
(47) increased the affinity for TSPO with a Ki value of 0.55 nM.
However, contrary to our expectation, introduction of a para-elec-
tron withdrawing substituent (44) or replacement of the phenyl
group with a pyridine (45–47) did not improve metabolic stability.
Finally we prepared compound 48 to examine the effect of insert-
ing a linker between the conjugated two rings. Fortunately, intro-
TSPO and CBR binding affinity, and metabolic stability of dihydroimidazoquinolinone
derivatives with various substituents at the C-8 position
O
N
Me
X
N
O
N
Compd
X
TSPO Kia
(nM)
CBR inhibitionb
(%)
Metabolic stabilityc
(remaining %)
49
40
27
41
42
43
44
45
46
47
48
H
Br
Ph
2.5
0
5
14
9
5
3
0d
0d
12
1
0.057
0.94
0.11
0.30
0.23
0.16
2.7
3.3
0.55
2.0
m-MeO-Ph
p-MeO-Ph
m-CF3-Ph
p-CF3-Ph
2-Py
3-Py
4-Py
3-PyNH
2d
0
0
0
48
47
41
70
0
0d
3
41
duction of an aminopyridyl moiety led to
a remarkable
a
Ki Values represent the means of 1–3 separate experiments run in duplicate
improvement of metabolic stability (41%). Although not reported
here, other compounds with an amino or ether linker between
the conjugated two rings exhibited low metabolic stability. As
compound 48 showed strong affinity for TSPO and acceptable
aqueous solubility (0.004 mg/mL, at pH 7.4 and >1.0 mg/mL, at
pH 2.5), further biological evaluation of this compound is in
progress.
using four concentrations of each compound.
b
Percent inhibition of [3H]-flumazenil specific binding at 10
lM of the
compound.
c
Metabolic stability data refer to percent of compound remaining after incuba-
tion with rat liver S-9 fraction (ca. 2.0 mg protein/ml) and NADPH (ca. 3.0 mM) for
30 min at 37 °C. The initial concentration of each compound was 1.0 lM.
d
Metabolic stability determined at 10 lM of the compound.
4. Conclusion
which showed high selectivity for TSPO over CBR and moderate
metabolic stability, was selected as a lead compound for further
evaluation.
We have synthesized and evaluated three novel tricyclic benz-
imidazolone derivatives as potent ligands, and selected the dihy-
droimidazoquinolinone derivative 27 as a lead compound. SAR of
dihydroimidazoquinolinone derivatives with various substituents
at the amide part and C-8 position revealed very potent TSPO li-
gands with subnanomolar Ki values, but poor metabolic stability.
Optimization of these compounds led to compound 48, which
showed potent affinity for TSPO with good in vitro PK profiles.
Further biological evaluation of compound 48 is under
investigation.
In our investigation of the amide part of the dihydroimidazo-
quinolinone derivatives, we kept the phenyl ring at the C-8 posi-
tion in the dihydroimidazoquinolinone core and explored various
substituents on the acetamide nitrogen. TSPO affinity and meta-
bolic stability of the tested compounds are shown in Table 2. First
of all, we converted R1 and R2 from an alkyl–phenyl to an alkyl–al-
kyl and alkyl–benzyl, respectively. Compounds 28 (R1 = R2 = nPr)
and 29 (R1 = Me, R2 = Bn) exhibited potent affinity for TSPO. In par-
ticular, compound 29 showed fourfold more potent activity than
that of 27. Although compounds 28 and 29 showed excellent activ-
ity, their metabolic stability was low. We therefore decided to
examine the effect of a substitution at the phenyl ring (R2) of 27.
Introduction of a methoxy group (30, 31) or a chlorine atom (32,
33) onto the phenyl ring resulted in better activity. In particular,
incorporation of a methoxy group into the meta-position and a
chlorine atom into the para-position provided significant increase
in TSPO affinity with compounds 30 and 33 showing Ki values of
0.28 and 0.13 nM, respectively. Next we examined the effect of
replacing the phenyl ring with a pyridine analogue. Although com-
pound 36 was found to be 16-fold less active than compound 27,
compounds 34 and 35 exhibited potent affinity for TSPO with Ki
values of 2.5 and 2.1 nM, respectively. These results surprised us,
since replacing the phenyl group with a pyridine in the benzoxazo-
lone derivatives led to at least 10-fold reduction in TSPO affinity.12
Although we were able to obtain a series of compounds with excel-
lent TSPO affinity and selectivity, these potent compounds suffered
from low metabolic stability. In general, compounds with second-
ary amides have low affinity for TSPO,16 however compound 37
showed moderate affinity for TSPO (Ki = 33 nM) with remarkable
high metabolic stability (62%). This result encouraged us to explore
dihydroimidazoquinolinone derivatives having a secondary amide
moiety in the side chain, however, our efforts led to no further in-
crease in TSPO affinity (data not shown).
5. Experimental section
5.1. Chemistry
Melting points were determined on Stanford Research Systems
OptiMelt MPA100 without correction. NMR spectra were recorded
at ambient temperature on a JEOL JNM-AL400 FT NMR spectrome-
ter. Chemical shifts are expressed in d values (ppm) relative to tet-
ramethylsilane as an internal standard, and signals are expressed
as s (singlet), d (doublet), t (triplet), m (multiplet), or br (broad).
IR spectra were recorded on a JEOL JIR-SPX60 spectrometer as
ATR. High-resolution mass spectra (HRMS) were recorded on a
Thermo Fisher Scientific LTQ orbitrap Discovery MS equipment.
Elemental analysis was performed on a CE Instrument EA1110
and a Yokokawa analytical system IC7000. In general, reagents
and solvents were used as obtained from commercial suppliers
without further purification. Reaction progress was determined
by thin layer chromatography (TLC) analysis on a Merck silica gel
60 F254 precoated glass plate. Visualization was done with UV
light (254 nm) or iodine. Flash column chromatography was con-
ducted using Merck silica gel 60 (70–230 mesh). All reactions were
carried out under
mentioned.
a nitrogen atmosphere unless otherwise

T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
1261
5.1.1. Methyl 3,4-dihydroquinoline-1(2H)-carboxylate (13)
To a suspension of tetrahydroquinoline (18.0 mL, 143 mmol)
and K2CO3 (79.3 g, 574 mmol) in DMF (100 mL) was added methyl-
chloroformate (33.2 mL, 430 mmol) with cooling in an ice bath.
The reaction mixture was stirred at 50 °C for 6 h and cooled to
room temperature. Water was then added, and the mixture was
extracted with a mixture of toluene and EtOAc (1:1). The organic
layer was washed with H2O and brine, and dried over anhydrous
sodium sulfate. After filtration, the solvent was removed in vacuo
to give 13 (27.2 g, 99%) as a yellow oil. This product was used in
the following reaction without further purification: 1H NMR
(400 MHz, CDCl3) d 7.66 (1H, d, J = 7.6 Hz), 7.16 (1H, dd, J = 7.7,
7.7 Hz), 7.09 (1H, d, J = 7.3 Hz), 7.01 (1H, dd, J = 7.3, 7.3 Hz), 3.79
(3H, s), 3.76 (2H, t, J = 6.1 Hz), 2.77 (2H, t, J = 6.6 Hz), 1.99–1.90
(2H, m); IR (ATR) 1701, 1697, 1493, 1439, 1327 cmꢁ1; HRMS
(ESI) m/z calcd for C11H14NO2 [M+H]+ 192.1019; found 192.1015.
a yellow solid: mp 99–101 °C; 1H NMR (400 MHz, CDCl3) d 7.88
(1H, s), 7.49 (1H, s), 3.81 (3H, br s), 3.64–3.62 (2H, br m), 2.85–
2.71 (2H, br m), 2.07–1.92 (2H, br m); IR (ATR) 1701, 1483, 1439,
1321, 1190 cmꢁ1; HRMS (ESI) m/z calcd for C11H12BrN2O4 [M+H]+
314.9975; found 314.9974.
5.1.6. Methyl 7-bromo-9-nitro-2,3,4,5-tetrahydro-1H-1-
benzazepine-1-carboxylate (18)
Compound 18 was prepared from 16 (6.09 g, 21.4 mmol) in a
manner similar to that described for compound 17 as a yellow solid
(4.89 g, 72%): mp 73–75 °C; 1H NMR (400 MHz, CDCl3) d 7.91–7.86
(1H, m), 7.63–7.60 (1H, m), 4.53–4.27 (1H, m), 3.77 and 3.57 (3H,
each s), 3.00–2.71 (3H, m), 2.12–1.78 (3H, m), 1.52–1.32 (1H, m);
IR (ATR) 1713, 1522, 1303, 1169, 1032 cmꢁ1; HRMS (ESI) m/z calcd
for C12H14BrN2O4 [M+H]+ 329.0131; found 329.0129.
5.1.7. 8-Bromo-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-
2(1H)-one (19)
5.1.2. Methyl 2,3,4,5-tetrahydro-1H-1-benzazepine-1-
carboxylate (14)
To a solution of reduced iron (24.1 g, 431 mmol) in AcOH
(250 mL) was added dropwise a solution of 17 (19.4 g, 61.6 mmol)
in AcOH (200 mL) at 80 °C. The reaction mixture was stirred at
80 °C for 2 h and cooled to room temperature. The reaction mixture
was filtered through Celite, and the filtrate was concentrated. The
residue was diluted with EtOAc and H2O. The organic layer was
separated, washed with brine, and dried over anhydrous sodium
sulfate. After filtration, the solvent was removed in vacuo. The
resulting solid was triturated with hexane to give 19 (14.6 g,
94%) as a brown solid: mp 235–236 °C; 1H NMR (400 MHz,
DMSO-d6) d 10.81 (1H, s), 6.99 (1H, s), 6.94 (1H, s), 3.68 (2H, t,
J = 5.7 Hz), 2.76 (2H, t, J = 6.0 Hz), 2.04–1.94 (2H, m); IR (ATR)
3143, 1707, 1657, 1641, 1491 cmꢁ1; HRMS (ESI) m/z calcd for
Compound 14 was prepared from 2,3,4,5-tetrahydro-1H-
benzo[b]azepine (800 mg, 5.43 mmol) in a manner similar to that
described for compound 14 as a white solid (706 mg, 63%): mp
113–115 °C; 1H NMR (400 MHz, CDCl3) d 7.25–7.10 (4H, m),
4.58–4.32 (1H, m), 3.79 and 3.65 (3H, each s), 2.87–2.58 (3H, m),
2.04–1.75 (3H, m), 1.55–1.27 (1H, m); IR (ATR) 1689, 1495, 1439,
1385, 1307 cmꢁ1; HRMS (ESI) m/z calcd for C12H16NO2 [M+H]+
206.1176; found 206.1173.
5.1.3. Methyl 6-bromo-3,4-dihydroquinoline-1(2H)-carboxylate
(15)
To a solution of 13 (26.3 g, 138 mmol) in DMF (140 mL) was
added N-bromosuccinimide (26.9 g, 151 mmol) with cooling in
an ice bath, and the mixture was stirred at room temperature for
2.5 h. Water was then added, and the mixture was extracted with
a mixture of toluene and EtOAc (1:1). The organic layer was
washed with H2O and brine, and dried over anhydrous sodium sul-
fate. After filtration, the solvent was removed in vacuo to give 15
(37.1 g, 100%) as a brown oil. This product was used in the follow-
ing reaction without further purification: 1H NMR (400 MHz,
CDCl3) d 7.59 (1H, d, J = 8.5 Hz), 7.28–7.21 (2H, m), 3.79 (3H, s),
3.74 (2H, t, J = 6.1 Hz), 2.74 (2H, t, J = 6.6 Hz), 1.96–1.89 (2H, m);
IR (ATR) 1701, 1483, 1441, 1321, 727 cmꢁ1; HRMS (ESI) m/z calcd
for C11H13BrNO2 [M+H]+ 270.0124; found 270.0124.
C
10H10BrN2O [M+H]+ 252.9971; found 252.9967.
5.1.8. 9-Bromo-4,5,6,7-tetrahydroimidazo[4,5,1-
jk][1]benzazepin-2(1H)-one (20)
Compound 20 was prepared from 18 (2.53 g, 7.69 mmol) in a
manner similar to that described for compound 19 as a white solid
(1.73 g, 84%): mp 147–149 °C; 1H NMR (400 MHz, DMSO-d6) d
11.01 (1H, br s), 6.99 (1H, d, J = 1.5 Hz), 6.94 (1H, d, J = 2.0 Hz),
3.75 (2H, t, J = 5.2 Hz), 2.93 (2H, t, J = 5.6 Hz), 2.53–2.48 (4H, m);
IR (ATR) 2864, 1686, 1610, 1471, 1149 cmꢁ1; HRMS (ESI) m/z calcd
for C11H12BrN2O [M+H]+ 267.0128; found 267.0126.
5.1.9. tert-Butyl (8-bromo-2-oxo-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)acetate (21)
5.1.4. Methyl 7-bromo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-
carboxylate (16)
To a suspension of 19 (28.9 g, 114 mmol) and K2CO3 (23.7 g,
171 mmol) in DMF (400 mL) was added tert-butyl bromoacetate
(18.5 mL, 126 mmol) with cooling in an ice bath. The reaction mix-
ture was stirred at 50 °C for 3 h and cooled to room temperature.
Water was then added, and the mixture was extracted with a mix-
ture of toluene and EtOAc (1:1). The organic layer was washed with
H2O and brine, and dried over anhydrous sodium sulfate. After fil-
tration, the solvent was removed in vacuo, and the residue was
triturated with hexane to give 21 (41.5 g, 99%) as a beige solid:
Compound 16 was prepared from 14 (521 mg, 2.54 mmol) in a
manner similar to that described for compound 15 as a colorless oil
(590 mg, 82%): 1H NMR (400 MHz, CDCl3) d 7.37–7.27 (2H, m),
7.21–6.96 (1H, m), 4.61–4.23 (1H, m), 3.79 and 3.65 (3H, each s),
2.83–2.53 (3H, m), 2.04–1.19 (4H, m); IR (ATR) 1701, 1487, 1441,
1383, 1300 cmꢁ1; HRMS (ESI) m/z calcd for C12H15BrNO2 [M+H]+
284.0281; found 284.0280.
5.1.5. Methyl 6-bromo-8-nitro-3,4-dihydroquinoline-1(2H)-
carboxylate (17)
mp 178–180 °C; 1H NMR (400 MHz, CDCl3)
d 7.02 (1H, d,
J = 1.7 Hz), 6.87 (1H, d, J = 1.7 Hz), 4.47 (2H, s), 3.85 (2H, t,
J = 5.7 Hz), 2.82 (2H, t, J = 6.1 Hz), 2.15–2.07 (2H, m), 1.48 (9H, s);
IR (ATR) 1741, 1697, 1498, 1421, 1232 cmꢁ1; HRMS (ESI) m/z calcd
for C16H19BrN2O3Na [M+Na]+ 389.0471; found 389.0473; Anal.
Calcd for C16H19BrN2O3ꢀ0.10H2O: C, 52.07; H, 5.24; N, 7.59; Br,
21.65. Found: C, 52.43; H, 5.29; N, 7.45; Br, 21.25.
To a solution of nitronium tetrafluoroborate (5.17 g, 38.9 mmol)
in CH3CN (150 mL) was added dropwise a solution of 15 (7.51 g,
27.8 mmol) in CH3CN (150 mL) with cooling in an ice bath, and
the mixture was stirred with cooling in an ice bath for 1 h. Water
was then added, and the mixture was extracted with a mixture
of toluene and EtOAc (1:1). The organic layer was washed with
aqueous saturated NaHCO3 and brine, and dried over anhydrous
sodium sulfate. After filtration, the solvent was removed in vacuo,
and the residue was purified by silica gel column chromatography
using hexane/EtOAc (5:1, v/v) as eluent to give 175 (5.77 g, 66%) as
5.1.10. tert-Butyl (9-bromo-2-oxo-4,5,6,7-
tetrahydroimidazo[4,5,1-jk][1]benzazepin-1(2H)-yl)acetate (22)
Compound 22 was prepared from 20 (1.71 g, 6.40 mmol) in a
manner similar to that described for compound 21 as a white solid

1262
T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
(2.42 g, 99%): mp 172–174 °C; 1H NMR (400 MHz, CDCl3) d 7.02
(1H, s), 6.84 (1H, s), 4.48 (2H, s), 3.95 (2H, t, J = 5.4 Hz), 2.98 (2H,
t, J = 4.8 Hz), 2.06–1.93 (4H, m), 1.48 (9H, s); IR (ATR) 1741,
zole (203 g, 1.50 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (431 mg, 2.25 mmol) at room temper-
ature. The reaction mixture was stirred at room temperature for
7 h. Water was then added, and the mixture was extracted with
a mixture of toluene and EtOAc (1:1). The organic layer was
washed with H2O and brine, and dried over anhydrous sodium sul-
fate. After filtration, the solvent was removed in vacuo, and the res-
idue was purified by silica gel column chromatography using
CHCl3/EtOAc (4:1, v/v) as eluent to give 27 (460 mg, 77%) as a
white solid: mp 126–127 °C (MeOH); 1H NMR (400 MHz, CDCl3)
d 7.56–7.51 (2H, m), 7.51–7.45 (2H, m), 7.45–7.38 (3H, m), 7.37–
7.27 (3H, m), 7.06 (1H, s), 6.89 (1H, s), 4.41 (2H, s), 3.84 (2H, t,
J = 5.7 Hz), 3.30 (3H, s), 2.88 (2H, t, J = 6.0 Hz), 2.18–2.08 (2H, m);
IR (ATR) 1713, 1666, 1493, 1423, 762 cmꢁ1; HRMS (ESI) m/z calcd
for C25H24N3O2 [M+H]+ 398.1863; found 398.1855; Anal. Calcd for
1693, 1425, 1230, 1149 cmꢁ1
;
HRMS (ESI) m/z calcd for
C
17H22BrN2O3 [M+H]+ 381.0808; found 381.0804.
5.1.11. tert-Butyl (2-oxo-8-phenyl-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl)acetate (23)
To a suspension of 21 (1.50 g, 4.09 mmol) and phenylboronic acid
(0.647 g, 5.31 mmol) in 2 M K2CO3 solution (6.14 mL, 12.3 mmol)
and 1,4-dioxane (30 mL) was added Pd(PPh3)4 (236 mg, 0.204 mmol)
in room temperature. The reaction mixture was stirred at reflux for
2.5 h and cooled to room temperature. Water was then added, and
the mixture was extracted with EtOAc. The organic layer was
washed with brine, and dried over anhydrous sodium sulfate. After
filtration, the solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography using hexane/EtOAc
(1:1, v/v) as eluent to give 23 (0.929 g, 62%) as a beige solid: mp
202–203 °C; 1H NMR (400 MHz, CDCl3) d 7.52 (2H, d, J = 8.0 Hz),
7.41 (2H, m), 7.31 (1H, t, J = 7.1 Hz), 7.09 (1H, s), 6.91 (1H, s), 4.54
(2H, s), 3.90 (2H, t, J = 5.7 Hz), 2.90 (2H, t, J = 6.0 Hz), 2.20–2.12
C
25H23N3O2ꢀ0.25H2O: C, 74.70; H, 5.89; N, 10.45. Found: C, 74.65;
H, 5.83; N, 10.51.
5.1.16. 2-(2-Oxo-8-phenyl-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)-N,N-dipropylacetamide (28)
Compound 28 was prepared from 25 (308 mg, 1.00 mmol) and
dipropylamine (165 lL, 1.20 mmol) in a manner similar to that de-
scribed for compound 27 as a white solid (244 mg, 62%): mp 129–
131 °C (iPrOH); 1H NMR (400 MHz, CDCl3) d 7.53 (2H, dd, J = 8.2,
1.3 Hz), 7.42–7.37 (2H, m), 7.32–7.27 (1H, m), 7.08 (1H, d,
J = 1.2 Hz), 7.02 (1H, d, J = 1.2 Hz), 4.69 (2H, s), 3.90 (2H, t,
J = 5.9 Hz), 3.39–3.24 (4H, m), 2.90 (2H, t, J = 6.0 Hz), 2.21–2.10
(2H, m), 1.72–1.50 (4H, m), 0.99 (3H, t, J = 7.4 Hz), 0.86 (3H, t,
J = 7.4 Hz); IR (ATR) 1705, 1649, 1230, 764, 702 cmꢁ1; HRMS (ESI)
m/z calcd for C24H30N3O2 [M+H]+ 392.2333; found 392.2324; Anal.
Calcd for C24H29N3O2: C, 73.63; H, 7.47; N, 10.73. Found: C, 73.27;
H, 7.44; N, 10.71.
(2H, m), 1.47 (9H, s); IR (ATR) 1741, 1713, 1701, 1228, 1155 cmꢁ1
;
HRMS (ESI) m/z calcd for C22H25N2O3 [M+H]+ 365.1860; found
365.1855; Anal. Calcd for C22H24N2O3ꢀ0.25H2O: C, 71.62; H, 6.69;
N, 7.59. Found: C, 71.50; H, 6.64; N, 7.71.
5.1.12. tert-Butyl (2-oxo-9-phenyl-4,5,6,7-
tetrahydroimidazo[4,5,1-jk][1]benzazepin-1(2H)-yl)acetate (24)
Compound 24 was prepared from 22 (1.84 g, 4.83 mmol) in a
manner similar to that described for compound 23 as a white solid
(1.74 g, 95%): mp 141–143 °C; 1H NMR (400 MHz, CDCl3) d 7.54
(2H, d, J = 7.3 Hz), 7.45–7.38 (2H, m), 7.32 (1H, t, J = 7.1 Hz), 7.10
(1H, s), 6.89 (1H, s), 4.56 (2H, s), 4.03–3.94 (2H, m), 3.11–3.04
(2H, m), 2.09–1.97 (4H, m), 1.47 (9H, s); IR (ATR) 1741, 1697,
1234, 1153, 754 cmꢁ1; HRMS (ESI) m/z calcd for C23H27N2O3
[M+H]+ 379.2016; found 379.2009.
5.1.17. N-Benzyl-N-methyl-2-(2-oxo-8-phenyl-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl)acetamide (29)
Compound 29 was prepared from 25 (462 mg, 1.50 mmol) and
benzylmethylamine (290 lL, 2.25 mmol) in a manner similar to
5.1.13. (2-Oxo-8-phenyl-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)acetic acid (25)
that described for compound 27 as a white solid (473 mg, 77%):
mp 173–175 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 7.55 (2H, d,
J = 8.0 Hz), 7.46–7.38 (2H, m), 7.34–7.21 (5H, m), 7.13–7.06 (2H,
m), 7.03 (1H, s), 4.77–4.58 (4H, m), 3.91 (1H, t, J = 5.7 Hz), 3.73
(1H, t, J = 5.7 Hz), 3.05 and 3.01 (3H, each s), 2.91 (1H, t,
J = 6.0 Hz), 2.84 (1H, t, J = 6.0 Hz), 2.21–2.13 (1H, m), 2.11–2.03
(1H, m); IR (ATR) 1693, 1653, 1493, 756, 704 cmꢁ1; HRMS (ESI)
m/z calcd for C26H26N3O2 [M+H]+ 412.2020; found 412.2007; Anal.
Calcd for C26H25N3O2: C, 75.89; H, 6.12; N, 10.21. Found: C, 75.66;
H, 6.02; N, 10.26.
To a solution of 23 (19.6 g, 53.8 mmol) in AcOH (100 mL) was added
4 N HCl in 1,4-dioxane (80.7 mL). The reaction mixture was stirred at
55 °C for3 h andcooled to room temperature. The solvent was removed
in vacuo, and the resulting solid was triturated with toluene to give 25
(13.6 g, 84%) as a brown solid: mp 216–218 °C; 1H NMR (400 MHz,
DMSO-d6) d 13.03 (1H, br s), 7.64–7.60 (2H, m), 7.45–7.40 (2H, m),
7.34–7.27 (2H, m), 7.17 (1H, s), 4.64 (2H, s), 3.78 (2H, t, J = 5.6 Hz),
2.87 (2H, t, J = 5.9 Hz), 2.11–2.01 (2H, m); IR (ATR) 2364, 1728, 1668,
1659, 1643 cmꢁ1; HRMS (ESI) m/z calcd for C18H16N2O3Na [M+Na]+
331.1053; found 331.1046; Anal. Calcd for C18H16N2O3ꢀ0.50H2O: C,
68.13; H, 5.40; N, 8.83. Found: C, 67.76; H, 5.19; N, 8.78.
5.1.18. N-(3-Methoxyphenyl)-N-methyl-2-(2-oxo-8-phenyl-5,6-
dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl)acetamide (30)
To a suspension of 25 (308 mg, 1.00 mmol) in CH2Cl2 (3.0 mL)
5.1.14. (2-Oxo-9-phenyl-4,5,6,7-tetrahydroimidazo[4,5,1-
jk][1]benzazepin-1(2H)-yl)acetic acid (26)
were added oxalyl choride (96.0
lM, 1.10 mmol) and DMF
(5.0 L) with cooling in an ice bath, and then the mixture was stir-
l
Compound 26 was prepared from 24 (1.57 g, 4.15 mmol) in a
manner similar to that described for compound 25 as a yellow so-
lid (1.30 g, 97%): mp 207–209 °C; 1H NMR (400 MHz, DMSO-d6) d
13.05 (1H, br s), 7.69–7.64 (2H, m), 7.46–7.40 (2H, m), 7.37 (1H,
d, J = 1.2 Hz), 7.31 (1H, t, J = 7.3 Hz), 7.20 (1H, s), 4.68 (2H, s),
3.89–3.81 (2H, m), 3.09–3.00 (2H, m), 2.03–1.88 (4H, m); IR
(ATR) 2931, 1732, 1662, 1655, 1227 cmꢁ1; HRMS (ESI) m/z calcd
for C19H19N2O3 [M+H]+ 323.1390; found 323.1385.
red at room temperature for 1 h. The solvent was removed in va-
cuo, and then the residue was azeotropied with toluene. A
solution of acid chloride thus obtained in THF (3.0 mL) was added
to a solution of 3-methoxy-N-methylaniline (165 mg, 1.20 mmol)
and triethylamine (209 lM, 1.50 mmol) in THF (3.0 mL) at room
temperature, and then the mixture was stirred at room tempera-
ture for 1 h. The reaction was quenched by adding aqueous satu-
rated NaHCO3, and then the mixture was extracted with EtOAc.
The organic layer was washed with H2O and brine and dried over
anhydrous sodium sulfate. After filtration, the solvent was re-
moved in vacuo, and the residue was purified by silica gel column
chromatography using EtOAc as eluent to give 30 (220 mg, 52%) as
a yellow solid: mp 166–168 °C (MeOH); 1H NMR (400 MHz, CDCl3)
5.1.15. N-Methyl-2-(2-oxo-8-phenyl-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl)-N-phenylacetamide (27)
To a solution of 25 (462 mg, 1.50 mmol) in DMF (3.0 mL) were
added N-methylaniline (244 lL, 2.25 mmol), 1-hydroxybenzotria-

T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
1263
d 7.56–7.50 (2H, m), 7.46–7.28 (4H, m), 7.06 (1H, s), 6.96–6.88 (3H,
m), 6.84–6.80 (1H, m), 4.46 (2H, s), 3.89–3.79 (5H, m), 3.29 (3H, s),
2.88 (2H, t, J = 6.0 Hz), 2.18–2.08 (2H, m); IR (ATR) 1705, 1674,
1489, 1421, 754 cmꢁ1; HRMS (ESI) m/z calcd for C26H26N3O3
[M+H]+ 428.1969; found 428.1962; Anal. Calcd for C26H25N3O3: C,
73.05; H, 5.89; N, 9.83. Found: C, 72.72; H, 5.94; N, 9.75.
to that described for compound 27 as a white solid (302 mg, 76%):
mp 165–167 °C (iPrOH); 1H NMR (400 MHz, CDCl3) d 8.65 (1H, br
s), 8.58 (1H, d, J = 2.7 Hz), 7.73 (1H, d, J = 8.0 Hz), 7.56–7.50 (2H,
m), 7.47–7.37 (3H, m), 7.31 (1H, t, J = 7.3 Hz), 7.08 (1H, s), 6.93
(1H, s), 4.41 (2H, s), 3.86–3.78 (2H, m), 3.32 (3H, s), 2.88 (2H, t,
J = 6.0 Hz), 2.19–2.08 (2H, m); IR (ATR) 1709, 1666, 1493, 1423,
;
764 cmꢁ1 HRMS (ESI) m/z calcd for C24H23N4O2 [M+H]+
5.1.19. N-(4-Methoxyphenyl)-N-methyl-2-(2-oxo-8-phenyl-5,6-
dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl)acetamide (31)
Compound 31 was prepared from 25 (308 mg, 1.00 mmol) and
N-methyl-p-anisidine (165 mg, 1.20 mmol) in a manner similar
to that described for compound 30 as a brown solid (348 mg,
81%): mp 172–174 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 7.53
(2H, d, J = 7.3 Hz), 7.44–7.38 (2H, m), 7.31 (1H, t, J = 7.4 Hz),
7.28–7.24 (2H, m), 7.05 (1H, s), 6.97 (2H, d, J = 8.8 Hz), 6.88 (1H,
s), 4.39 (2H, s), 3.89–3.80 (5H, m), 3.27 (3H, s), 2.88 (2H, t,
J = 5.9 Hz), 2.18–2.09 (2H, m); IR (ATR) 1691, 1662, 1504, 1425,
399.1816; found 399.1810; Anal. Calcd for C24H22N4O2ꢀ0.25H2O:
C, 71.53; H, 5.63; N, 13.90. Found: C, 71.43; H, 5.64; N, 13.87.
5.1.24. N-Methyl-2-(2-oxo-8-phenyl-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl)-N-(pyridin-4-yl)acetamide
(36)
Compound 36 was prepared from 25 (308 mg, 1.00 mmol) and
4-(methylamino)pyridine (108 mg, 1.00 mmol) in a manner similar
to that described for compound 27 as a white solid (225 mg, 56%):
mp 141–143 °C (iPrOH); 1H NMR (400 MHz, CDCl3) d 8.68 (2H, d,
J = 5.9 Hz), 7.53 (2H, d, J = 7.1 Hz), 7.45–7.38 (2H, m), 7.34–7.23
(3H, m), 7.09 (1H, s), 6.92 (1H, s), 4.60 (2H, s), 3.83 (2H, t,
J = 5.7 Hz), 3.37 (3H, s), 2.89 (2H, t, J = 6.0 Hz), 2.19–2.07 (2H, m);
IR (ATR) 1693, 1672, 1587, 1491, 1429 cmꢁ1; HRMS (ESI) m/z calcd
for C24H23N4O2 [M+H]+ 399.1816; found 399.1807; Anal. Calcd for
C24H22N4O2ꢀ0.25H2O: C, 71.53; H, 5.63; N, 13.90. Found: C, 71.80;
H, 5.56; N, 13.94.
;
1238 cmꢁ1 HRMS (ESI) m/z calcd for C26H26N3O3 [M+H]+
428.1969; found 428.1965; Anal. Calcd for C26H25N3O3ꢀ0.25H2O:
C, 72.29; H, 5.95; N, 9.73. Found: C, 72.60; H, 5.87; N, 9.79.
5.1.20. N-(3-Chlorophenyl)-N-methyl-2-(2-oxo-8-phenyl-5,6-
dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl)acetamide (32)
Compound 32 was prepared from 25 (308 mg, 1.00 mmol) and 3-
chloro-N-methylaniline (170 mg, 1.20 mmol) in a manner similar to
that described for compound 27 as a white solid (135 mg, 31%): mp
161–162 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 7.54 (2H, d,
J = 7.8 Hz), 7.46–7.23 (7H, m), 7.08 (1H, s), 6.91 (1H, s), 4.45 (2H,
s), 3.83 (2H, t, J = 5.4 Hz), 3.28 (3H, s), 2.89 (2H, t, J = 6.0 Hz), 2.19–
2.09 (2H, m); IR (ATR) 1697, 1686, 1674, 760, 694 cmꢁ1; HRMS
5.1.25. 2-(2-Oxo-8-phenyl-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)-N-phenylacetamide (37)
Compound 37 was prepared from 25 (462 mg, 1.50 mmol) and
aniline (164 lL, 1.80 mmol) in a manner similar to that described
for compound 27 as a white solid (479 mg, 83%): mp 223–224 °C
(MeOH); 1H NMR (400 MHz, CDCl3) d 8.49 (1H, br s), 7.58–7.47
(4H, m), 7.45–7.38 (2H, m), 7.36–7.23 (3H, m), 7.17 (2H, d,
J = 6.1 Hz), 7.09 (1H, t, J = 7.4 Hz), 4.67 (2H, s), 3.95 (2H, t,
J = 5.7 Hz), 2.94 (2H, t, J = 6.0 Hz), 2.24–2.16 (2H, m); IR (ATR) 1687,
1558, 1497, 1238, 700 cmꢁ1; HRMS (ESI) m/z calcd for C24H22N3O2
[M+H]+ 384.1707; found 384.1700; Anal. Calcd for C24H21N3O2: C,
75.18; H, 5.52; N, 10.96. Found: C, 75.07; H, 5.61; N, 11.00.
(ESI) m/z calcd for
C
25H23ClN3O2 [M+H]+ 432.1473; found
432.1467; Anal. Calcd for C25H22ClN3O2: C, 69.52; H, 5.13; N, 9.73;
Cl, 8.21. Found: C, 69.15; H, 5.15; N, 9.80; Cl, 8.12.
5.1.21. N-(4-Chlorophenyl)-N-methyl-2-(2-oxo-8-phenyl-5,6-
dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl)acetamide (33)
Compound 33 was prepared from 25 (308 mg, 1.00 mmol) and
4-chloro-N-methylaniline (145 lL, 1.20 mmol) in a manner similar
to that described for compound 30 as a pale yellow solid (306 mg,
71%): mp 178–180 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 7.53
(2H, d, J = 7.6 Hz), 7.46–7.38 (4H, m), 7.31 (1H, t, J = 7.1 Hz),
7.28–7.22 (2H, m), 7.07 (1H, s), 6.89 (1H, s), 4.41 (2H, s), 3.83
(2H, t, J = 5.6 Hz), 3.27 (3H, s), 2.88 (2H, t, J = 6.0 Hz), 2.19–2.07
(2H, m); IR (ATR) 1695, 1670, 1489, 1425, 754 cmꢁ1 cmꢁ1; HRMS
(ESI) m/z calcd for
432.1466; Anal. Calcd for C25H22ClN3O2: C, 69.52; H, 5.13; N,
9.73; Cl, 8.21. Found: C, 69.23; H, 5.15; N, 9.75; Cl, 8.22.
5.1.26. N-Methyl-2-(2-oxo-9-phenyl-4,5,6,7-
tetrahydroimidazo[4,5,1-jk][1]benzazepin-1(2H)-yl)-N-
phenylacetamide (38)
Compound 38 was prepared from 26 (129 mg, 0.400 mmol) and
N-methylaniline (43.3 lL, 0.400 mmol) in a manner similar to that
described for compound 27 as a beige solid (164 mg, 100%): mp
157–159 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 7.54 (2H, d,
J = 7.1 Hz), 7.51–7.46 (2H, m), 7.46–7.29 (6H, m), 7.07 (1H, s),
6.88 (1H, s), 4.42 (2H, s), 3.96–3.87 (2H, m), 3.30 (3H, s), 3.09–
3.01 (2H, m), 2.07–1.95 (4H, m); IR (ATR) 1707, 1662, 1425, 758,
C
25H23ClN3O2 [M+H]+ 432.1473; found
5.1.22. N-Methyl-2-(2-oxo-8-phenyl-5,6-dihydro-4H-
700 cmꢁ1 HRMS (ESI) m/z calcd for C26H26N3O2 [M+H]+
;
imidazo[4,5,1-ij]quinolin-1(2H)-yl)-N-(pyridin-2-yl)acetamide (34)
Compound 34 was prepared from 25 (462 mg, 1.50 mmol) and 2-
412.2020; found 412.2013; Anal. Calcd for C26H25N3O2: C, 75.89;
H, 6.12; N, 10.21. Found: C, 75.78; H, 6.16; N, 10.23.
(methylamino)pyridine (185 lL, 1.80 mmol) in a manner similar to
that described for compound 27 as a white solid (331 mg, 55%): mp
171–172 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 8.50 (1H, d,
J = 2.9 Hz), 7.79 (1H, t, J = 7.0 Hz), 7.54 (2H, d, J = 8.0 Hz), 7.44–7.19
(5H, m), 7.08 (1H, s), 7.01 (1H, s), 4.82 (2H, s), 3.87 (2H, t, J = 5.7 Hz),
3.44 (3H, s), 2.89 (2H, t, J = 6.0 Hz), 2.19–2.10 (2H, m); IR (ATR) 1697,
1660, 1587, 1419, 1313 cmꢁ1; HRMS (ESI) m/z calcd for C24H23N4O2
[M+H]+ 399.1816; found 399.1810; Anal. Calcd for C24H22N4O2ꢀ
0.25H2O: C, 71.53; H, 5.63; N, 13.90. Found: C, 71.80; H, 5.65; N, 13.94.
5.1.27. (8-Bromo-2-oxo-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)acetic acid (39)
Compound 39 was prepared from 21 (16.7 g, 45.5 mmol) in a
manner similar to that described for compound 25 as a yellow so-
lid (14.1 g, 100%): mp 213–215 °C; 1H NMR (400 MHz, DMSO-d6) d
13.08 (1H, br s), 7.28 (1H, d, J = 1.7 Hz), 7.05 (1H, d, J = 1.7 Hz), 4.58
(2H, s), 3.74 (2H, t, J = 5.7 Hz), 2.79 (2H, t, J = 6.0 Hz), 2.05–1.95 (2H,
m); IR (ATR) 1718, 1653, 1635, 1624, 1427 cmꢁ1; HRMS (ESI) m/z
calcd for C12H11BrN2O3Na [M+Na]+ 332.9845; found 332.9841.
5.1.23. N-Methyl-2-(2-oxo-8-phenyl-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl)-N-(pyridin-3-yl)acetamide
(35)
Compound 35 was prepared from 25 (308 mg, 1.00 mmol) and
N-methy-3-pyridinamine (108 mg, 1.00 mmol) in a manner similar
5.1.28. 2-(8-Bromo-2-oxo-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)-N-methyl-N-phenylacetamide (40)
Compound 40 was prepared from 39 (11.2 g, 36.0 mmol) and N-
methylaniline (4.68 mL, 43.2 mmol) in a manner similar to that de-

1264
T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
scribed for compound 27 as a yellow solid (11.3 g, 78%): mp 195–
197 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 7.53–7.46 (2H, m),
7.42 (1H, t, J = 7.1 Hz), 7.35 (2H, d, J = 7.8 Hz), 6.98 (1H, s), 6.84
(1H, s), 4.32 (2H, s), 3.80 (2H, t, J = 5.9 Hz), 3.31 (3H, s), 2.79 (2H,
t, J = 6.0 Hz), 2.13–2.03 (2H, m); IR (ATR) 1705, 1662, 1497, 1421,
C
C
26H23F3N3O2 [M+H]+ 466.1737; found 466.1724; Anal. Calcd for
26H22F3N3O2ꢀ0.50H2O: C, 65.82; H, 4.89; N, 8.86; F, 12.01. Found:
C, 65.69; H, 4.81; N, 8.87; F, 11.95.
5.1.33. N-Methyl-2-[2-oxo-8-(pyridin-2-yl)-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl]-N-phenylacetamide (45)
To a solution of 40 (200 mg, 0.500 mmol) and 2-(tributylstan-
nyl)pyridine (0.192 mL, 0.600 mmol) in toluene (3.0 mL) was added
Pd(PPh3)4 (28.9 mg, 0.025 mmol) at room temperature. The reaction
mixture was stirred at reflux for 14.5 h and cooled to room temper-
ature. Water was then added, and the mixture was extracted with
EtOAc. The organic layer was washed with brine, and dried over
anhydrous sodium sulfate. After filtration, the solvent was removed
in vacuo, and the residue was purified by silica gel column chroma-
tography using EtOAc/MeOH (10:1, v/v) as eluent to give 45
(42.9 mg, 22%) as a white solid: mp 200–202 °C (MeOH); 1H NMR
(400 MHz, CDCl3) d 8.65 (1H, d, J = 4.9 Hz), 7.74–7.65 (2H, m),
7.53–7.45 (3H, m), 7.44–7.33 (4H, m), 7.20–7.15 (1H, m), 4.44 (2H,
s), 3.85 (2H, t, J = 5.7 Hz), 3.30 (3H, s), 2.90 (2H, t, J = 6.0 Hz), 2.18–
2.09 (2H, m); IR (ATR) 1704, 1666, 1425, 781, 706 cmꢁ1; HRMS
(ESI) m/z calcd for C24H23N4O2 [M+H]+ 399.1816; found 399.1805;
Anal. Calcd for C24H22N4O2ꢀ0.50H2O: C, 70.74; H, 5.69; N, 13.75.
Found: C, 70.55; H, 5.49; N, 13.72.
1406 cmꢁ1
;
HRMS (ESI) m/z calcd for C19H19BrN3O2 [M+H]+
400.0655; found 400.0652; Anal. Calcd for C19H18BrN3O2ꢀ0.25H2O:
C, 56.38; H, 4.61; N, 10.38; Br, 19.74. Found: C, 56.11; H, 4.51; N,
10.25; Br, 20.03.
5.1.29. 2-[8-(3-Methoxyphenyl)-2-oxo-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl]-N-methyl-N-
phenylacetamide (41)
Compound 41 was prepared from 40 (200 mg, 0.500 mmol) and
3-methoxyphenylboronic acid (98.8 mg, 0.650 mmol) in a manner
similar to that described for compound 23 as a white solid
(105 mg, 49%): mp 221–223 °C (MeOH); 1H NMR (CDCl3) d 7.52–
7.46 (2H, m), 7.41 (1H, t, J = 7.2 Hz), 7.37–7.30 (3H, m), 7.12 (1H,
d, J = 7.8 Hz), 7.08–7.04 (2H, m), 6.90–6.84 (2H, m), 4.40 (2H, s),
3.87 (3H, s), 3.84 (2H, t, J = 5.9 Hz), 3.30 (3H, s), 2.88 (2H, t,
J = 6.0 Hz), 2.18–2.08 (2H, m); IR (ATR) 1705, 1660, 1491, 1429,
;
1236 cmꢁ1 HRMS (ESI) m/z calcd for C26H26N3O3 [M+H]+
428.1969; found 428.1960; Anal. Calcd for C26H25N3O3ꢀ0.25H2O:
C, 72.29; H, 5.95; N, 9.73. Found: C, 72.09; H, 6.02; N, 9.39.
5.1.34. N-Methyl-2-[2-oxo-8-(pyridin-3-yl)-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl]-N-phenylacetamide (46)
Compound 46 was prepared from 40 (200 mg, 0.500 mmol) and
3-pyridineboronic acid (79.9 mg, 0.650 mmol) in a manner similar
to that described for compound 23 as a white solid (199 mg, 100%):
mp 130–132 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 8.79 (1H, s),
8.55 (1H, d, J = 4.9 Hz), 7.87–7.79 (1H, m), 7.54–7.31 (6H, m), 7.05
(1H, s), 6.88 (1H, s), 4.42 (2H, s), 3.86 (2H, t, J = 5.7 Hz), 3.31 (3H, s),
2.90 (2H, t, J = 6.0 Hz), 2.20–2.10 (2H, m); IR (ATR) 1691, 1659,
1643, 1491, 1431 cmꢁ1; HRMS (ESI) m/z calcd for C24H23N4O2
5.1.30. 2-[8-(4-Methoxyphenyl)-2-oxo-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl]-N-methyl-N-
phenylacetamide (42)
Compound 42 was prepared from 40 (200 mg, 0.500 mmol) and
4-methoxyphenylboronic acid (98.8 mg, 0.650 mmol) in a manner
similar to that described for compound 23 as a white solid
(165 mg, 77%): mp 138–140 °C (MeOH); 1H NMR (400 MHz, CDCl3)
d 7.51–7.38 (5H, m), 7.34 (2H, d, J = 7.3 Hz), 7.01 (1H, s), 6.96 (2H,
d, J = 8.8 Hz), 6.84 (1H, s), 4.40 (2H, s), 3.85 (3H, s), 3.83 (2H, t,
J = 5.9 Hz), 3.30 (3H, s), 2.87 (2H, t, J = 6.0 Hz), 2.16–2.08 (2H, m);
IR (ATR) 1695, 1668, 1497, 1242, 825 cmꢁ1; HRMS (ESI) m/z calcd
for C26H26N3O3 [M+H]+ 428.1969; found 428.1960; Anal. Calcd for
[M+H]+
24H22N4O2ꢀ1.50H2O: C, 67.75; H, 5.92; N, 13.17. Found: C, 67.41;
H, 5.84; N, 13.02.
399.1816;
found
399.1810;
Anal.
Calcd
for
C
C
26H25N3O3ꢀ0.75H2O: C, 70.81; H, 6.06; N, 9.53. Found: C, 70.59; H,
5.91; N, 9.55.
5.1.35. N-Methyl-2-[2-oxo-8-(pyridin-4-yl)-5,6-dihydro-4H-
imidazo[4,5,1-ij]quinolin-1(2H)-yl]-N-phenylacetamide (47)
5.1.31. N-Methyl-2-{2-oxo-8-[3-(trifluoromethyl)phenyl]-5,6-
dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl}-N-
phenylacetamide (43)
Compound 47 was prepared from 40 (100 mg, 0.250 mmol) and
4-pyridineboronic acid (36.9 mg, 0.300 mmol) in a manner similar
to that described for compound 23 as a white solid (39.7 mg, 40%):
mp 238–239 °C (MeOH); 1H NMR (400 MHz, CDCl3) d 8.62 (2H, d,
J = 6.3 Hz), 7.57 (2H, d, J = 6.3 Hz), 7.54–7.48 (2H, m), 7.43 (1H, t,
J = 7.1 Hz), 7.37 (2H, d, J = 7.6 Hz), 7.16 (1H, s), 6.99 (1H, s), 4.42
(2H, s), 3.86 (2H, t, J = 5.7 Hz), 3.32 (3H, s), 2.91 (2H, t, J = 6.0 Hz),
Compound 43 was prepared from 40 (200 mg, 0.500 mmol) and
3-(trifluoromethyl)phenylboronic acid (123 mg, 0.650 mmol) in a
manner similar to that described for compound 23 as a white solid
(207 mg, 89%): mp 259–260 °C (MeOH); 1H NMR (400 MHz, CDCl3)
d 7.77 (1H, s), 7.71 (1H, d, J = 7.3 Hz), 7.58–7.47 (4H, m), 7.42 (1H, t,
J = 7.1 Hz), 7.36 (2H, d, J = 7.3 Hz), 7.06 (1H, s), 6.89 (1H, s), 4.42
(2H, s), 3.85 (2H, t, J = 5.6 Hz), 3.31 (3H, s), 2.89 (2H, t, J = 6.0 Hz),
2.21–2.08 (2H, m); IR (ATR) 1709, 1695, 1662, 1497, 1429 cmꢁ1
;
HRMS (ESI) m/z calcd for C24H23N4O2 [M+H]+ 399.1816; found
399.1803; Anal. Calcd for C24H22N4O2ꢀ1.75H2O: C, 67.04; H, 5.98;
N, 13.03. Found: C, 66.77; H, 5.75; N, 12.83.
2.20–2.09 (2H, m); IR (ATR) 1716, 1660, 1421, 1329, 1117 cmꢁ1
;
HRMS (ESI) m/z calcd for C26H23F3N3O2 [M+H]+ 466.1737; found
466.1726; Anal. Calcd for C26H22F3N3O2: C, 67.09; H, 4.76; N,
9.03; F, 12.24. Found: C, 66.97; H, 4.78; N, 9.03; F, 12.26.
5.1.36. N-Methyl-2-[2-oxo-8-(pyridin-3-ylamino)-5,6-dihydro-
4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl]-N-phenylacetamide (48)
A
mixture of 40 (200 mg, 0.500 mmol), 3-aminopyridine
5.1.32. N-Methyl-2-{2-oxo-8-[4-(trifluoromethyl)phenyl]-5,6-
dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl}-N-
phenylacetamide (44)
(70.6 mg, 0.750 mmol), Pd2(dba)3 (22.9 mg, 0.0250 mmol), Xant-
phos (43.4 mg, 0.0750 mmol) and Cs2CO3 (228 mg, 0.700 mmol)
in toluene (4.0 mL) was heated at reflux for 11 h and cooled to
room temperature. The reaction was quenched by adding aqueous
saturated NaHCO3, and then the mixture was extracted with
CH2Cl2. The organic layer was washed with brine and dried over
anhydrous sodium sulfate. After filtration, the solvent was re-
moved in vacuo, and the residue was purified by silica gel column
chromatography using EtOAc/MeOH (20:1, v/v) as eluent to give 48
(110 mg, 53%) as a yellow solid: mp 194–196 °C (MeOH); 1H NMR
(400 MHz, CDCl3) d 8.25 (1H, d, J = 2.7 Hz), 8.06 (1H, dd, J = 4.4,
Compound 44 was prepared from 40 (200 mg, 0.500 mmol) and
4-(trifluoromethyl)phenylboronic acid (123 mg, 0.650 mmol) in a
manner similar to that described for compound 23 as a white solid
(196 mg, 84%): mp 207–209 °C (MeOH); 1H NMR (400 MHz, CDCl3)
d 7.68–7.61 (4H, m), 7.53–7.46 (2H, m), 7.42 (1H, t, J = 7.1 Hz), 7.36
(2H, d, J = 7.3 Hz), 7.07 (1H, s), 6.90 (1H, s), 4.41 (2H, s), 3.85 (2H, t,
J = 5.7 Hz), 3.31 (3H, s), 2.89 (2H, t, J = 6.0 Hz), 2.20–2.10 (2H, m); IR
(ATR) 1708, 1660, 1323, 1111, 1065 cmꢁ1; HRMS (ESI) m/z calcd for

T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
1265
1.2 Hz), 7.50–7.44 (2H, m), 7.40 (1H, t, J = 6.7 Hz), 7.32 (2H, d,
J = 7.3 Hz), 7.20–7.15 (1H, m), 7.12–7.06 (1H, m), 6.65 (1H, s),
6.54 (1H, s), 4.32 (2H, s), 3.80 (2H, t, J = 5.7 Hz), 3.29 (3H, s), 2.77
(2H, t, J = 6.0 Hz), 2.14–2.04 (2H, m); IR (ATR) 1695, 1662, 1506,
1429, 694 cmꢁ1; HRMS (ESI) m/z calcd for C24H24N5O2 [M+H]+
414.1925; found 414.1911; Anal. Calcd for C24H23N5O2ꢀ0.25H2O:
C, 68.96; H, 5.67; N, 16.76. Found: C, 69.21; H, 5.63; N, 16.47.
lid: mp 179–180 °C; 1H NMR (400 MHz, DMSO-d6) d 10.50 (1H, s),
7.91 (1H, d, J = 2.2 Hz), 7.68 (1H, d, J = 2.2 Hz), 4.78 (2H, s); IR (ATR)
2343, 1699, 1684, 1338, 1286 cmꢁ1; HRMS (ESI) m/z calcd for
C8H4BrN2O4 [M-H]- 270.9360; found 270.9352.
5.1.41. 5-Nitro-7-phenyl-2H-1,4-benzoxazin-3(4H)-one (54)
To a suspension of 53 (1.30 g, 4.76 mmol) and phenylboronic
acid (0.697 g, 5.71 mmol) in 2 M K2CO3 solution (7.14 mL,
14.3 mmol) and 1,4-dioxane (15 mL) was added Pd(PPh3)4
(275 mg, 0.238 mmol) at room temperature. The reaction mixture
was stirred at reflux for 3 h and cooled to room temperature. 1 M
HCl solution was then added, and the separated solid was collected
by filtration to give [(4-amino-5-nitrobiphenyl-3-yl)oxy]acetic
acid (921 mg, 67%) as a brown solid. To a suspension of [(4-ami-
no-5-nitrobiphenyl-3-yl)oxy]acetic acid (900 mg, 3.12 mmol) in
toluene (15 mL) was added p-toluenesulfonic acid monohydrate
(0.119 g, 0.624 mmol) at room temperature The reaction mixture
was stirred at reflux for 1.5 h, and cooled to room temperature.
The reaction mixture was concentrated, and the residue was di-
luted with CH2Cl2 and aqueous saturated NaHCO3. The organic
layer was separated, washed with brine, and dried over anhydrous
sodium sulfate. After filtration, the solvent was removed in vacuo.
The resulting solid was triturated with hexane to give 54 (718 mg,
85%) as a yellow solid: mp 163–165 °C; 1H NMR (400 MHz, DMSO-
d6) d 10.47 (1H, s), 8.01 (1H, d, J = 2.2 Hz), 7.78–7.71 (3H, m), 7.52–
7.45 (2H, m), 7.42 (1H, t, J = 7.1 Hz), 4.80 (2H, s); IR (ATR) 3261,
5.1.37. N-Methyl-2-(2-oxo-5,6-dihydro-4H-imidazo[4,5,1-
ij]quinolin-1(2H)-yl)-N-phenylacetamide (49)
To a solution of 40 (100 mg, 0.250 mmol) in MeOH (5.0 mL) and
THF (5.0 mL) was added 10% Pd/C (50% wet, 10.0 mg), and stirred
at room temperature for 1 h under hydrogen atmosphere. The
reaction mixture was filtered through Celite, and the filtrate was
concentrated. The residue was recrystallized from iPrOH to give
49 (27.6 mg, 34%) as a white solid: mp 173–174 °C; 1H NMR
(400 MHz, CDCl3) d 7.51–7.44 (2H, m), 7.40 (1H, t, J = 7.7 Hz),
7.32 (2H, d, J = 7.1 Hz), 6.95 (1H, dd, J = 7.7, 7.7 Hz), 6.83 (1H, d,
J = 7.7 Hz), 6.71 (1H, d, J = 7.7 Hz), 4.37 (2H, s), 3.81 (2H, t,
J = 5.9 Hz), 3.30 (3H, s), 2.83 (2H, t, J = 6.1 Hz), 2.14–2.06 (2H, m);
IR (ATR) 1699, 1660, 1497, 1421, 739 cmꢁ1; HRMS (ESI) m/z calcd
for C19H20N3O2 [M+H]+ 322.1550; found 322.1548; Anal. Calcd
for C19H19N3O2ꢀ0.25H2O: C, 70.03; H, 6.03; N, 12.89. Found: C,
69.90; H, 5.94; N, 12.82.
5.1.38. tert-Butyl (2-amino-3-nitrophenoxy)acetate (51)
To a suspension of 2-amino-3-nitrophenol (2.51 g, 16.3 mmol)
and K2CO3 (3.15 g, 22.8 mmol) in DMF (15 mL) was added tert-bu-
tyl bromoacetate (2.55 mL, 17.3 mmol) with cooling in an ice bath.
The reaction mixture was stirred at room temperature for 2.5 h.
Water was then added, and the mixture was extracted with tolu-
ene. The organic layer was washed with H2O and brine, and dried
over anhydrous sodium sulfate. After filtration, the solvent was re-
moved in vacuo, and the residue was purified by silica gel column
chromatography using Hexane/EtOAc (10:1, v/v) as eluent to give
51 (3.59 g, 82%) as an orange solid: mp 103–105 °C; 1H NMR
(400 MHz, CDCl3) d 7.79 (1H, dd, J = 8.8, 1.2 Hz), 6.82 (1H, d,
J = 7.8 Hz), 6.62–6.52 (3H, m), 4.58 (2H, s), 1.50 (9H, s); IR (ATR)
3479, 1736, 1628, 1522, 1151 cmꢁ1; HRMS (ESI) m/z calcd for
1716, 1699, 1338, 1176 cmꢁ1
14H9N2O4 [M-H]- 269.0568; found 269.0561.
; HRMS (ESI) m/z calcd for
C
5.1.42. 7-Phenyl-3,4-dihydro-2H-1,4-benzoxazin-5-amine (55)
To a solution of LiAlH4 (387 mg, 10.2 mmol) in THF (15 mL) was
added dropwise a solution of 54 (689 mg, 2.55 mmol) in THF
(25 mL) at 50 °C. The reaction mixture was stirred at 50 °C for 3 h
and cooled to room temperature. The reaction was then quenched
by dropwise addition of H2O (0.400 mL), 15% NaOH solution
(0.400 mL) and H2O (1.20 mL) with cooling in an ice bath. The reac-
tion mixture was filtered through Celite, and the filtrate was con-
centrated. The residue was purified by silica gel column
chromatography using hexane/EtOAc (1:4, v/v) as eluent to give
55 (279 mg, 48%) as a brown oil: 1H NMR (400 MHz, CDCl3) d
7.55–7.47 (2H, m), 7.41–7.34 (2H, m), 7.30–7.23 (1H, m), 6.65 (1H,
d, J = 2.0 Hz), 6.60 (1H, d, J = 2.0 Hz), 4.23 (2H, t, J = 4.4 Hz), 3.46
(2H, t, J = 4.4 Hz); IR (ATR) 3334, 2870, 1487, 1340, 754 cmꢁ1; HRMS
(ESI) m/z calcd for C14H15N2O [M+H]+ 227.1179; found 227.1176.
C
12H17N2O5 [M+H]+ 269.1132; found 269.1127.
5.1.39. tert-Butyl (2-amino-5-bromo-3-nitrophenoxy)acetate
(52)
To a solution of 51 (2.54 g, 9.47 mmol) in DMF (15 mL) was
added N-bromosuccinimide (1.77 g, 9.94 mmol) with cooling in
an ice bath, and the mixture was stirred at room temperature for
3 h. Water was then added, and the mixture was extracted with
Et2O. The organic layer was washed with H2O and brine, and dried
over anhydrous sodium sulfate. After filtration, the solvent was re-
moved in vacuo. The resulting solid was triturated with hexane to
give 52 (3.26 g, 99%) as a brown solid: mp 73–74 °C; 1H NMR
(400 MHz, CDCl3) d 7.95 (1H, d, J = 2.2 Hz), 6.89 (1H, s), 6.61 (2H,
br s), 4.58 (2H, s), 1.51 (9H, s); IR (ATR) 3369, 1743, 1516, 1209,
5.1.43. 8-Phenyl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-
2(1H)-one (56)
To a solution of 55 (263 mg, 1.16 mmol) in THF (5.0 mL) was
added 1,10-carbonyldiimidazole (226 mg, 1.39 mmol) at room tem-
perature. The mixture was stirred at reflux for 1 h and cooled to
room temperature. The reaction was then quenched by adding
2 M HCl solution, and the mixture was extracted with EtOAc. The
organic layer was washed with brine and dried over anhydrous so-
dium sulfate. After filtration, the solvent was removed in vacuo.
The residue was purified by silica gel column chromatography
using EtOAc as eluent to give 56 (234 mg, 80%) as a brown solid:
mp 195–197 °C; 1H NMR (400 MHz, DMSO-d6) d 10.84 (1H, s),
7.60–7.53 (2H, m), 7.45–7.37 (2H, m), 7.30 (1H, t, J = 7.3 Hz), 6.83
(2H, s), 4.41 (2H, t, J = 4.5 Hz), 3.92 (2H, t, J = 4.5 Hz); IR (ATR)
3000, 1686, 1660, 1490, 1410 cmꢁ1; HRMS (ESI) m/z calcd for
;
1151 cmꢁ1 HRMS (ESI) m/z calcd for C12H16BrN2O5 [M+H]+
347.0237; found 347.0236.
5.1.40. 7-Bromo-5-nitro-2H-1,4-benzoxazin-3(4H)-one (53)
To a solution of 52 (2.33 g, 6.71 mmol) in toluene (10 mL) was
added p-toluenesulfonic acid monohydrate (0.100 g, 0.526 mmol)
at room temperature The reaction mixture was stirred at 80 °C
for 2 h, and cooled to room temperature. The reaction mixture
was concentrated, and the residue was diluted with CH2Cl2 and
aqueous saturated NaHCO3. The organic layer was separated,
washed with brine, and dried over anhydrous sodium sulfate. After
filtration, the solvent was removed in vacuo. The resulting solid
was triturated with hexane to give 53 (1.82 g, 99%) as a yellow so-
C
15H13N2O2 [M+H]+ 253.0972; found 253.0965.
5.1.44. tert-Butyl (2-oxo-8-phenyl-4,5-dihydroimidazo[1,5,4-
de][1,4]benzoxazin-1(2H)-yl)acetate (57)
Compound 57 was prepared from 56 (228 mg, 0.904 mmol) in a
manner similar to that described for compound 21 as a beige solid

1266
T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
(259 mg, 78%): mp 160–162 °C; 1H NMR (400 MHz, CDCl3) d 7.51
(2H, d, J = 7.6 Hz), 7.44–7.38 (2H, m), 7.32 (1H, t, J = 7.6 Hz), 6.88
(1H, s), 6.73 (1H, s), 4.54 (2H, s), 4.44 (2H, t, J = 4.6 Hz), 4.05 (2H,
t, J = 4.6 Hz), 1.48 (9H, s); IR (ATR) 1741, 1707, 1653, 1234,
nized in 10 volumes of ice-cold potassium phosphate buffer
(200 mM KCl, 20 mM KOH, 20 mM KH2PO4, pH 7.4) with a micro-
homogenizer (Physcotron, Niti-on Inc.). The homogenate was cen-
trifuged at 32,500ꢂg and 4 °C for 15 min, and the resulting pellet
was resuspended in the same volume of fresh buffer and recentri-
fuged. These resuspension and centrifugation procedures were re-
peated once more, and the obtained pellet was resuspended in
potassium phosphate buffer (200 mM KCl, 20 mM KOH, 20 mM
KH2PO4, pH 7.4) at a protein concentration of 2.63 mg/mL and
stored frozen at ꢁ80 °C until use. The crude cerebral cortex prepa-
ration (0.895 mL) was incubated with [3H]-flumazenil (final con-
1157 cmꢁ1
;
HRMS (ESI) m/z calcd for C21H23N2O4 [M+H]+
367.1652; found 367.1648.
5.1.45. (2-Oxo-8-phenyl-4,5-dihydroimidazo[1,5,4-
de][1,4]benzoxazin-1(2H)-yl)acetic acid (58)
Compound 58 was prepared from 57 (239 mg, 0.652 mmol) in a
manner similar to that described for compound 25 as a beige solid
(202 mg, 100%): mp 226–228 °C; 1H NMR (400 MHz, DMSO-d6) d
13.08 (1H, br s), 7.61 (2H, t, J = 4.3 Hz), 7.46–7.39 (2H, m), 7.31
(1H, t, J = 7.3 Hz), 7.16 (1H, d, J = 1.0 Hz), 6.91 (1H, d, J = 1.0 Hz),
4.64 (2H, s), 4.44 (2H, t, J = 4.6 Hz), 3.98 (2H, t, J = 4.6 Hz); IR
(ATR) 2875, 1718, 1647, 1201, 1034 cmꢁ1; HRMS (ESI) m/z calcd
for C17H15N2O4 [M+H]+ 311.1026; found 311.1022.
centration 1.0 nM) and 10 lM of each test compound in a total
volume of 1.0 mL for 1 h at 25 °C. The reaction was terminated
by rapid fitration through a GF/B glass filter presoaked with 0.3%
polyethyleneimine. The filters were immediately washed with
ice-cold potassium phosphate buffer (200 mM KCl, 20 mM KOH,
20 mM KH2PO4, pH 7.4), and the filter-bound radioactivity was
quantified using a liquid scintillation analyzer (Tri Carb 2700TR,
Packard). Nonspecific binding was determined in the presence of
5.1.46. N-Methyl-2-(2-oxo-8-phenyl-4,5-dihydroimidazo[1,5,4-
de][1,4]benzoxazin-1(2H)-yl)-N-phenylacetamide (59)
Compound 59 was prepared from 58 (55.0 mg, 0.177 mmol) and N-
10 l
M [3H]-flumazenil. All assays were done in duplicate. Specific
binding was determined by subtracting nonspecific binding from
methylaniline (19.2 lL, 0.177 mmol) in a manner similar to that de-
total binding.
scribed for compound 27 as a white solid (50.1 mg, 71%): mp 166–
167 °C (iPrOH); 1H NMR (400 MHz, CDCl3) d 7.55–7.46 (4H, m), 7.45–
7.38 (3H, m), 7.37–7.29 (3H, m), 6.85 (1H, s), 6.71 (1H, s), 4.46–4.35
(4H, m), 3.99 (2H, t, J = 4.6 Hz), 3.31 (3H, s); IR (ATR) 1718, 1662,
1497, 1284, 1192 cmꢁ1; HRMS (ESI) m/z calcd for C24H22N3O3 [M+H]+
400.1656; found 400.1648; Anal. Calcd for C24H21N3O3: C, 72.16; H,
5.30; N, 10.52. Found: C, 71.80; H, 5.43; N, 10.37.
Acknowledgments
We thank Ms. K. Bando for performing the elemental analysis,
and Mr. T. Ueda and Ms. M. Homma for recording high-resolution
MS spectra.
Supplementary data
5.2. Biology
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.024.
5.2.1. TSPO-binding assay
Male SD rats (Japan Charles River) were decapitated, and the
kidney was dissected. The kidney was homogenized in five vol-
umes of ice-cold 50 mM Tris–HCl buffer (pH 7.6) with a microho-
mogenizer (Physcotron, Niti-on Inc.). The homogenate was
centrifuged at 20,000ꢂg and 4 °C for 10 min, and the resulting pel-
let was resuspended in the same volume of fresh buffer and recen-
trifuged. These resuspension and centrifugation procedures were
repeated once more, and the obtained pellet was resuspended in
50 mM Tris–HCl buffer (pH 7.6) at ae protein concentration of
2.63 mg/mL and stored frozen at ꢁ80 °C until use. The crude mito-
chondrial preparation (0.895 mL) was incubated with [3H]-
PK11195 (final concentration 1.0 nM) and various concentrations
of test compounds in a total volume of 1.0 mL for 1 h at 4 °C. The
reaction was terminated by rapid fitration through a GF/B glass fil-
ter presoaked with 0.3% polyethyleneimine. The filters were imme-
diately washed with ice-cold 50 mM Tris–HCl buffer (pH 7.6), and
the filter-bound radioactivity was quantified using a liquid scintil-
lation analyzer (Tri Carb 2700TR, Packard). Nonspecific binding
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cerebral cortex was dissected. The cerebral cortex was homoge-

T. Fukaya et al. / Bioorg. Med. Chem. 21 (2013) 1257–1267
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