Synthesis and biological activity of novel 4,4-difluorobenzazepine derivatives as non-peptide antagonists of the arginine vasopressin V 1A receptor

Article abstract of DOI:10.1016/j.bmc.2005.10.035

To find potent and selective antagonists of the arginine vasopressin (AVP) V_1A receptor, optimization studies of compounds structurally related to (Z)-N-{4′-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahydro-1H- 1-benzazepin-1-yl)carbonyl

Full text of DOI:10.1016/j.bmc.2005.10.035

Bioorganic & Medicinal Chemistry 14 (2006) 1827–1837
Synthesis and biological activity of novel
4,4-difluorobenzazepine derivatives as non-peptide antagonists
of the arginine vasopressin V1A receptor
Yoshiaki Shimada,a,* Nobuaki Taniguchi,b Akira Matsuhisa,b Hiroaki Akane,c
Noriyuki Kawano,b Takeshi Suzuki,b Takahiko Tobe,b Akio Kakefuda,b Takeyuki Yatsu,b
Atsuo Tahara,a Yuichi Tomura,b Toshiyuki Kusayama,b Koh-ichi Wada,b
Junko Tsukada,c Masaya Orita,b Takashi Tsunodad and Akihiro Tanakae
aDrug Discovery Research, Astellas Pharma Inc., 5-2-3, Tokodai, Tsukuba, Ibaraki 300-2698, Japan
bDrug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
cDevelopment, Astellas Pharma Inc., 3-17-1, Hasune, Itabashi-Ku, Tokyo 174-8612, Japan
dTechnology, Astellas Pharma Inc., 160-2, Akahama, Takahagi, Ibaraki 318-0001, Japan
eCorporate Administration, Astellas Pharma Inc., 2-3-11, Nihonbashi-honcho, Chuo-Ku, Tokyo 103-8411, Japan
Received 2 September 2005; revised 19 October 2005; accepted 20 October 2005
Available online 11 November 2005
Abstract—To find potent and selective antagonists of the arginine vasopressin (AVP) V1A receptor, optimization studies of com-
pounds structurally related to (Z)-N-{40-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl)carbon-
yl]phenyl}carboxamide were performed. The synthesis and pharmacological properties of these compounds are described. We
first investigated the effect of the carboxamide moiety, and found that a 2-methylfuran-3-carbonyl group at this position increased
V1A binding affinity and selectivity for the V1A receptor versus the V2 receptor. The amino group of the 5-carbamoylmethylidene
moiety was also examined, and a 4-piperidinopiperidino group was found to be optimal at this position. The hemifumarate of
compound 12l (YM218) was shown to exhibit potent binding affinity, V1A receptor selectivity, and in vivo antagonist activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
to the V1A receptor,6 and therefore V1A receptor antag-
onists may be useful for treatment of these diseases.
Arginine vasopressin (AVP) is a 9-amino acid poly-
peptide hormone that is released by the posterior pitui-
tary gland and regulated by an intricate homeostatic
mechanism that controls water balance.1,2 AVP recep-
tors are classified into three types, V1A, V1B, and V2
receptors,3–5 and the V1A receptor is found in blood ves-
sels, platelets, the liver, and renal mesangial cells. AVP
plays a role in various physiological effects, such as
vasoconstriction, platelet aggregation, glycogenolysis,
and the contraction of mesangial cells, all of which are
mediated by the V1A receptor. Overproduction of AVP
is thought to cause congestive heart failure, hyperten-
sion, liver disease, and kidney disease through binding
Non-peptide V1A receptor-selective antagonists OPC-
21268 (1), SR49059 (2), and triazole derivatives (3) have
been reported (Fig. 1),7–12 and we have previously
reported V1A receptor antagonists (4–6) containing the
(Z)-4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahy-
dro-1H-1-benzazepine moiety (Fig. 2).13,14 However, the
selectivity of the 2-ethoxybenzoyl derivatives (4, 5) for
binding to the V1A receptor versus the V2 receptor was
insufficient, and the 2-(2-ethyl-1H-imidazol-1-yl)benzoyl
derivative (6) showed decreased V1A receptor selectivity
for the cloned human receptor, compared to the rat
receptor. Conversion of the R1 substituents into groups
containing hetero atoms, such as 2-alkoxybenzoyl- or
2-(2-ethyl-1H-imidazol-1-yl)benzoyl, gave compounds
with potent binding affinity and selectivity for the V1A
receptor, and we found that the substituent at the 2-po-
sition of the benzoyl group at R1 is more important for
Keywords: YM218; Arginine vasopressin; V1A receptor selective antag-
onist; 2-Methylfuran-3-carbonyl group.
*
Corresponding author. Tel.: +81 0 29 847 8611; fax: +81 0 29 847
1536; e-mail: yoshiaki.shimada@jp.astellas.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.035

1828
Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
N N
N
Cl
Me
OR
HO
Cl
N
N
O
N
N
O
Me
CONH2
SO2
N
N
MeO
MeO
R: Me
(CH2)6
O
O
N
O(CH2)3NHAc
1
2
3
Figure 1. Structure of known non-peptide arginine vasopressin V1A receptor selective antagonist.
OEt
OEt
O
O
R2:
R1:
N
N
NMe2
4
5
O
R2
F
F
O
N
N
N
O
R1
Et
O
N
H
6
N
H
Figure 2. Structure of 4,4-difluorobenzazepine derivatives.
potent V1A antagonist activity, compared to those at the
3- or 4-positions of the benzoyl group.15 These results
suggest that introduction of substituents at the R1 posi-
tion containing both a hetero atom and a substituent
ortho to the carbonyl group might give compounds with
potent binding affinity and selectivity for the V1A recep-
tor. Based on this hypothesis, we chose a five-membered
heterocyclic ring with a methyl group ortho to the car-
bonyl group. Moreover, we optimized the R2 position
of the 5-carbonylmethylidene moiety and found a series
of compounds that were highly potent and selective V1A
receptor antagonists. In this paper, we describe the
structure–activity relationships (SAR) and antagonist
activities of this series of compounds.
pyl)carbodiimide hydrochloride (WSC•HCl) and
1-hydroxybenzotriazole (HOBt) gave the target amide
derivatives 12a–n. Among these compounds 12a–d, g,
j–l, and n were treated with a 4 M solution of HCl in eth-
yl acetate and obtained as HCl salts.
3. Results and discussion
The influence of the R1 position on V1A selectivity in
a
series of five-membered heterocyclic carbonyl
derivatives is shown in Table 1. The 1-methyl-1H-pyr-
role-2-carbonyl derivative (12a) and 3-methylthiophen-
2-carbonyl derivative (12b) exhibited a similar or more
potent V1A binding affinity, compared to that of the
2-ethoxybenzoyl derivative (4). The 3-methylfuran-2-
carbonyl derivative (12c) exhibited potent V1A recep-
tor binding affinity and 120-fold V1A selectivity
against the V2 receptor. These results suggest that
the oxygen atom is important for high V1A receptor
selectivity in both five-membered heterocyclic carbonyl
derivatives and 2-alkoxybenzoyl derivatives. The 2-
methylfuran-3-carbonyl derivative (12d) exhibited the
most potent affinity among the heterocyclic carbonyl
derivatives and showed a 331-fold selectivity for the
V1A versus the V2 receptor. These results suggest that
V1A binding affinity and selectivity are influenced by
both an electronic effect and the position of the
oxygen atom. However, the 5-methyl-1,3-oxazole-4-
carbonyl derivative (12e), which has an oxygen atom
in the same position as 12d, showed decreased binding
affinity compared to 12d.
2. Chemistry
The general synthetic pathways for the compounds list-
ed in Tables 1 and 2 are shown in Scheme 1. The key
intermediate, (Z)-methyl [1-(4-aminobenzoyl)-4,4-diflu-
oro-2,3,4,5-tetrahydro-1H-1-benzazepine-5-ylidene]ac-
etate (7), was synthesized by condensation of 816 with
4-nitrobenzoyl chloride to give the nitrobenzoyl deriva-
tive (9). The nitro group of 9 was reduced with tin(II)
chloride17 to obtain the aminobenzoyl derivative
without reduction of the methylidene group. The
heterocyclic acid chlorides were reacted with compound
7 to give methyl ester derivatives 10a–e, which were
treated with 1 M NaOH to give the acetic acid
derivatives (11a–e). Condensation of 11a–e and various
amines in the presence of 1-ethyl-3-(dimethylaminopro-

Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
1829
Table 1. Receptor binding affinities of the five-membered heterocyclic
carbonyl derivatives
Table 2. Receptor binding affinities of the 2-methylfuran-3-carbonyl
derivatives with various amino groups
O
O
NMe2
N
R2
F
F
F
F
N
N
O
O
O
R1
N
N
O
H
H
Compound R1
Binding
affinity Ki
(nM)
Selectivity Ki ratio
Compound R2
Binding
affinity Ki
(nM)
Selectivity Ki ratio
a
b
V2
c
V1A
V2/V1A
a
b
V2
c
V1A
V2/V1A
OEt
O
12d
0.102 33.9
331
N
N
NMe2
4
0.603 22.4
0.398 17.8
37
45
12
12f
1.41
7.94
6
O
O
Me
12aÆHCl
12bÆHCl
N
12gÆHCl
12h
0.562
3.02
5
-NH(CH2)2N
-NH(CH2)3N
-NH(CH2)4N
O
O
O
O
O
Me
0.562
6.92
0.331 10.5
0.145 17.0
32
118
39
28
380
28
24
S
Me
Me
12i
12cÆHCl
12dÆHCl
12e
0.263 31.6
0.102 33.9
0.661 91.2
120
331
138
O
O
O
12jÆHCl
12kÆHCl
12lÆHCl
12m
1.41
1.51
55.0
41.7
N
N
N
N
N i-Pr
N
O
O
Me
O
N
0.112 42.7
a Rat liver membranes were used. Ki values obtained from two inde-
pendent experiments performed in duplicate.
b Rat kidney membranes were used. Ki values obtained from two
independent experiments performed in duplicate.
c V2/V1A shows the selectivity for binding to the V1A receptor versus
the V2 receptor.
0.214
0.339
6.03
-NH(CH2)3N
-NH(CH2)4N
12nÆHCl
8.13
a Refer to Table 1.
b Refer to Table 1.
c Refer to Table 1.
Since five-membered heterocyclic carbonyl substituents
increased V1A selectivity, compared to the 2-alkoxyben-
zoic acid moiety, a 2-methylfuran-3-carbonyl substitu-
ent was chosen for the R1 position. Further
optimization of the amino group at R2 on the 5-carbonylme-
thylidene moiety was then performed, as shown in Table
2. In a preceding paper,13 the introduction of morpholi-
no and 4-dimethylaminopiperidino groups at this posi-
tion gave compounds with potent binding affinity and
selectivity for the V1A receptor. Therefore, we first syn-
thesized and evaluated the morpholino derivative (12f),
but this compound showed only a 6-fold V1A selectivity
against V2. Introduction of an alkylene chain between
the carboxamide and morpholino groups gave com-
pounds 12g–i, which showed increased V1A receptor
binding affinity with increasing alkylene chain length:
C4 (12i) > C3 (12h) > C2 (12g) > none (12f). The V1A
versus V2 receptor selectivity also increased with an in-
creased alkylene chain length: C4 (12i) > C3 (12h) > C2
(12g). The cyclic derivative (C3, 4-morpholinopiperidi-
no, 12j) showed similar V1A selectivity to the open chain
derivative (C3, 12h). These results suggest that a basic
nitrogen atom and a suitable alkylene chain length be-
tween the carboxamide and the basic nitrogen atom
are required at the R2 position to obtain the desired
properties.
Among compounds with 4-dimethylaminopiperidino-
type substituents, the 4-isopropyl-1-piperazinyl deriva-

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Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
O
O
O
OMe
OMe
OMe
F
F
F
F
a
b
c
F
F
N
N
N
O
O
H
O2N
H2N
8
9
7
O
O
O
OMe
OH
R2
F
F
F
F
F
F
e
d
N
N
N
O
O
O
R1
R1
R1
N
H
N
H
N
H
10a-e
11a-e
12a-n
12a-d, g, j-l, n
free base
HCl salt
4M HCl
/AcOEt
O
O
O
O
Me
Me
R2:
N
NMe2
(a-e)
N
N
N
N
O
(j)
(k)
(l)
N
R1:
(a)
(b)
(c)
(d, f-n)
(e)
O
O
N
O
(f)
(g)
(h)
(i)
N
i-Pr
Me
Me
Me
O
S
N
-NH(CH2)2N
-NH(CH2)3N
-NH(CH2)4N
O
O
O
N
-NH(CH2)3N
-NH(CH2)4N
(m)
(n)
O
Scheme 1. Reagents and conditions: (a) 4-nitrobenzoylchloride, Et3N, CH2Cl2; (b) SnCl2, AcOEt; (c) 1—heterocyclic carboxylic acid, (COCl)2,
DMF, THF; 2—R1-Cl, pyridine, acetonitrile; (d) 1—1 M NaOH, MeOH; 2—1 M HCl; (e) amine, HOBt, WSCÆHCl, CH2Cl2, acetonitrile.
tive (12k) showed decreased V1A selectivity compared to
12d. In contrast, the 4-piperidinopiperidino derivative
(12l) exhibited one of the highest affinities and showed
380-fold selectivity for the V1A receptor versus the V2
receptor. In comparing 12l with 12d and 12j, it appears
that the electronegative effect of the oxygen atom in the
morpholino group (12j) decreased the V1A receptor
binding affinity and selectivity. Based on the SAR study
of derivatives 12i and 12l, open chain derivatives with
piperidine (12m, 12n) were chosen for synthesis and
evaluation. However, although compounds 12m and
12n showed high affinities for the V1A receptor, their
V1A selectivity was insufficient.
most selective V1A receptor antagonist activity encoun-
tered in studies of 4,4-difluorobenzazepine derivatives.
The in vivo V1A receptor antagonist activity was evaluat-
edfollowing intravenous administration ofcompound 12l
to pithed rats. The compound showed dose-dependent
antagonism of an AVP (30 mU/kg)-induced increase in
diastolic blood pressure (DBP) via the V1A receptor, with
an ID50 value of 0.0035 mg/kg. The time course of
antagonism of the AVP-induced increase in DBP was also
monitored after oral administration of 12l (0.3, 1, and
3 mg/kg) to conscious rats (Fig. 3). Compound 12l dose-
dependently antagonized the AVP-induced increase in
DBP, and its effect lasted for at least 8 h at doses of
3 mg/kg. In contrast, oral administration (0.3–3 mg/kg)
of 12l did not increase urine excretion in conscious rats.
These results show that 12l is a selective and orally active
antagonist for the V1A receptor.
The binding of compound 12l with cloned human V1A
(hV1A) and V2 (hV2) receptors was examined (Table 3),
since this compound showed potent binding affinity
and high selectivity for the rat V1A receptor. Compound
12l exhibited similarly high binding affinity and selectiv-
ity for the human V1A receptor, showed increased V1A
selectivity compared to 4, 5, and 6, and exhibited the
To investigate the basis of the potent binding affinity
and selectivity for the V1A receptor and the strong V1A

Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
1831
Table 3. AVP binding affinities with cloned human V1A (hV1A) and V2 (hV2) receptors of compounds 12l, 4, 5, and 6
O
R2
F
F
N
O
R1
N
H
Compound
R1
R2
Binding affinity Ki (nM)
Selectivity Ki ratio
a
b
c
hV1A
hV2
hV2/hV1A
O
Me
O
12lÆHCl
0.224
79.4
45.7
354
OEt
O
4ÆHCl
0.891
0.158
51
44
N
NMe2
5
7.08
N
O
N
Et
N
O
6
1.00
141
140
N
H
a The cloned human V1A receptor, which was stably expressed in CHO cells, was used. Ki values obtained from two independent experiments
performed in duplicate.
b The cloned human V2 receptor, which was stably expressed in CHO cells, was used. Ki values obtained from two independent experiments
performed in duplicate.
c hV2/hV1A shows the selectivity for binding to the cloned human V1A receptor versus the cloned human V2 receptor.
140
120
100
80
0.5%MC
0.3 mg/kg
1.0 mg/kg
3.0 mg/kg
60
40
20
0
0
2
4
6
8
Time (hr)
Figure 3. Inhibition of the peak pressor responses induced by AVP (30 mU/kg, iv) in conscious rats by oral administration of 12l. Values represent
means SEM of six independent experiments.
antagonist activity exhibited by compound 12l, the
stereochemical configuration of 12l was examined.
According to Hassner and Amit18 N-acylbenzazepine,
which forms the central skeleton of 12l, has the structure
shown in Figure 4 (13) at room temperature. The
electrons on the nitrogen atom of the amide bond are
located largely on the carbonyl oxygen atom, giving a
double-bond character to the C–N amide bond of
N-acylbenzazepine, and the carbonyl oxygen atom and
the equatorial hydrogen (He) are in a cis-configuration.
1H NMR spectroscopy showed that the carbonyl oxygen
atom was coplanar with He, and He showed a downfield
shift (ca. d 4.8–5.0 ppm) relative to the position of the
axial hydrogen (Ha) (ca. d 2.7–3.0 ppm). Correspon-
dence of the 1H NMR spectrum of N-acylbenzazepine18
with that for compound 12l (He: d 4.86 ppm, Ha: d
3.17 ppm) was observed, and therefore it was concluded
that compound 12l has the same stereochemical config-
uration as 13.
Superimposition of 12l with a model AVP structure was
then performed. The X-ray crystal structure of AVP has

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Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
Figure 4. Stereochemical configuration of N-acylbenzazepine.
not been reported, and therefore the X-ray structure of
oxytocin,19 which has a similar cyclic structure to AVP
and is also secreted from the posterior pituitary gland,
was used as a 3D template incorporating the amino-acid
sequence of AVP. The SYBYL20 program was used to
examine the 3D structures and to match AVP to 12l.
Superimposition was performed using the GASP21 pro-
gram, which permits overlap of hydrogen bonds and
hydrophobic structures. The two terminal groups of
12l, the 2-methylfuran-3-carbonyl moiety and the 4-pip-
eridinopiperidino moiety, were superimposable with Tyr
and Arg residues of AVP, respectively (Figs. 5 and 6).
Based on these results, overlap between the oxygen atom
of the 2-methylfuran-3-carbonyl moiety and the
hydroxyl group of the Tyr residue, and between the
basic nitrogen atom of the 4-piperidinopiperidino moie-
ty and the guanidino group of the Arg residue was
thought to contribute to the potent V1A binding affinity
and selectivity. A superimposition study of a V1A/V2
dual antagonist and AVP has been reported by Cho
et al.,22 and overlap of the antagonist with the AVP
Tyr, Phe, and Arg residues was proposed. Our results
suggest that potent binding affinity and selectivity for
the V1A receptor are dependent on overlap with the
Tyr and Arg residues of AVP for compounds with
N-acylbenzazepine in the central skeleton.
Figure 5. Superimposition of compound 12l with a model AVP
structure.
O
NH
NH2
H
O
N
O
NH2
NH2
H
O
N
N
H
N
N
O
HN
N
O
NH
F
F
O
O
NH2
O
O
S
S
HN
O
N
H
N
O
O
N
H
NH2
O
N
O
H
HO
AVP
Figure 6. Correlation of functional groups between AVP and compound 12l.
12l

Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
1833
4. Conclusion
ture, and the mixture was refluxed for 5 h. After being
cooled at 0 ꢁC, the resulting mixture was made alkaline
with 1 M NaOH and extracted with AcOEt. The organic
layer was dried over anhydrous K2CO3 and concentrated
in vacuo. The residue was recrystallized from hexane–
CHCl3 to give 0.782 g (2.10 mmol, 84%) of 7 as a colorless
powder. 1H NMR (CDCl3) d: 2.66 (2H, m), 3.81 (3 H, s),
4.24 (1H, m), 4.95 (1H, br), 6.45 (2H, m), 6.76 (1H, m),
7.17 (2H, m), 7.24 (2H, m), 7.42 (3H, m), 7.95 (1H, m).
EI MS m/z (M)+ 372.
In this report, we have described the synthesis of (Z)-N-
{40-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tet-
rahydro-1H-1-benzazepin-1-yl)carbonyl]phenyl}carbox-
amide derivatives and evaluated their pharmacological
properties, in order to obtain a potent and selective
antagonist for the V1A receptor. The introduction of
furancarbonyl groups on the carboxamide moiety (R1
position) enhanced V1A binding affinity and selectivity,
and basic substituents with a suitable spacer on the 5-car-
bonylmethylidene moiety (R2 position) were essential for
V1A selectivity. In particular, (Z)-N-[40-(4,4-difluoro-5-
[2-oxoethylidene-2-({4-piperidinopiperidino)]-2,3,4,5-tetra-
hydro-11-benzazepin-1-yl}carbonyl)phenyl]-2-methylfu-
ran-3-carboxamide monohydrochloride (12l) exhibited
potent binding affinity and V1A receptor selectivity across
species, and also showed potent in vivo activities. The hemi-
fumarate of 12l is undergoing clinical trials as YM21823,24
and is expected to be clinically applicable to diseases such
as congestive heart failure and hypertension.
5.4. General procedure for the preparation of compounds
10a–e
5.4.1. (Z)-Methyl {4,4-difluoro-1-[4-(1-methylpyrrole-2-
carbonylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzaze-
pine-5-ylidene}acetate (10a). To an ice-cooled mixture of
1-methylpyrrole-2-carboxylic acid (2.75 g, 22.0 mmol)
and a catalytic amount of dimethylformamide (DMF)
in tetrahydrofuran (30 mL) was added oxalyl chloride
(3.05 g, 24.0 mmol). The mixture was stirred at room tem-
perature for 3 h, diluted with CHCl3, and concentrated in
vacuo to give a corresponding acid chloride as a colorless
powder. The acid chloride was used in the next reaction
without further purification. To an ice-cooled mixture
of 7 (7.45 g, 20.0 mmol) and pyridine (2 mL) in CH3CN
(80 mL) was added a solution of acid chloride in CH3CN
(20 mL). The mixture was stirred at room temperature for
8 h and then concentrated in vacuo. The residue was
diluted with AcOEt. The organic layer was washed with
a saturated aqueous solution of Na2CO3, 1 M HCl, and
brine, and then dried over anhydrous MgSO4 and concen-
trated in vacuo. The crude product was chromatographed
on silica gel and eluted with hexane–AcOEt (60:40) to give
5.93 g (12.4 mmol, 62%) of 10a as a colorless powder. 1H
NMR (CDCl3) d: 2.28–2.82 (2H, m), 3.24 (1H, m), 3.83
(3H, s), 3.93 (3H, s), 5.04 (1H, m), 6.11 (1H, m), 6.19
(1H, s), 6.66 (1H, m), 6.72 (1H, d, J = 8.3 Hz), 6.77 (1H,
m), 7.08–7.18 (3H, m), 7.23 (1H, t, J = 7.8 Hz), 7.36–
7.40 (3H, m), 7.65 (1H, s). EI MS m/z (M)+ 479.
5. Experimental
5.1. General
1H NMR spectra were obtained on a JNM-400 spectro-
meter, and the chemical shifts are expressed in d (ppm)
values with tetramethylsilane as an internal standard.
Abbreviations of 1H NMR signal patterns are as follows:
s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad
peak. Mass spectra were obtained on a JEOL JMS-
DX300 mass spectrometer (low-resolution mass spec-
trometry) and JEOL JMS-700T mass spectrometer
(high-resolution mass spectrometry). Elemental analysis
was performed with a Yanaco MT-5. Column chroma-
tography on silica gel was performed with Merck KGaA
Silica gel 60 (0.040–0.063 mm).
5.2. (Z)-Methyl [4,4-difluoro-1-(4-nitrobenzoyl)-2,3,4,5-
tetrahydro-1H-1-benzazepine-5-ylidene]acetate (9).
5.4.2. (Z)-Methyl {4,4-difluoro-1-[4-(3-methylthiophene-2-
carbonylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzaze-
pine-5-ylidene}acetate (10b). 90% yield. 1H NMR (CDCl3)
d: 2.42 (1H, m), 2.55 (3H, s), 2.68 (1H, m), 3.26 (1H, m), 3.83
(3H, s), 5.05 (1H, m), 6.19 (1H, s), 6.72 (1H, d, J = 7.9 Hz),
6.93 (1H, d, J = 4.9 Hz), 7.11 (1H, t, J = 7.9 Hz), 7.16 (2H,
d, J = 8.5 Hz), 7.24 (1H, t, J = 7.3 Hz), 7.32 (1H, d,
J = 4.9 Hz), 7.40 (2H, d, J = 7.3 Hz), 7.49 (1H, d,
J = 8.5 Hz). FAB MS m/z (M+H)+ 497.
To an ice-cooled mixture of 816 (2.15 g, 8.50 mmol) and
triethylamine (1.13 g, 11.1 mmol) in CH2Cl2 (20 mL)
was added 4-nitrobenzoyl chloride (1.90 g, 10.2 mmol),
and the mixture was stirred for 12 h at room temperature
and then extracted withethyl acetate (AcOEt). The organ-
ic layer was washed with 1 M HCl, 1 M NaOH, and brine,
and then dried over anhydrous MgSO4 and concentrated
in vacuo. The residue was chromatographed on a silica gel
column using CHCl3 as the eluent and recrystallized from
hexane–AcOEt to give 2.67 g (6.64 mmol, 78%) of 9 as a
5.4.3. (Z)-Methyl {4,4-difluoro-1-[4-(3-methylfuran-2-car-
bonylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-
ylidene}acetate (10c). 86% yield. 1H NMR (CDCl3) d: 2.22–
2.80 (2H, m), 2.41 (3H, s), 3.23 (1H, m), 3.83 (3H, s), 5.05
(1H, m), 6.19 (1H, s), 6.38 (1H, s), 6.71 (1H, d, J = 7.3 Hz),
7.10 (1H, dd, J = 7.3, 7.8 Hz), 7.15 (2H, d, J = 8.8 Hz), 7.23
(1H, dd, J = 7.3, 7.8 Hz), 7.34–7.38 (3H, m), 7.44 (1H, s),
7.46 (1H, s), 8.02 (1H, s). FAB MS m/z (M+H)+ 481.
1
colorless powder. H NMR (CDCl3) d: 2.73 (2H, m),
3.81 (3H, s), 4.35 (1H, m), 4.95 (1H, br), 6.62 (1H, m),
7.25 (1H, m), 7.33 (1H, m), 7.48 (3H, m), 8.00 (1H, m),
8.10 (2H, m). EI MS m/z (M)+ 402.
5.3. (Z)-Methyl [1-(4-aminobenzoyl)-4,4-difluoro-2,3,4,5-
tetrahydro-1H-1-benzazepine-5-ylidene]acetate (7)
5.4.4. (Z)-Methyl {4,4-difluoro-1-[4-(2-methylfuran-3-car-
bonylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzaze-
pine-5-ylidene}acetate (10d). 87% yield. 1H NMR
To a solution of 9 (1.00 g, 2.49 mmol) in AcOEt (20 mL)
was added SnCl2 (3.07 g, 16.2 mmol) at room tempera-

1834
Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
(CDCl3) d: 2.20–2.72 (2H, m), 2.61 (3H, s), 3.24 (1H, m),
3.83 (3H, s), 5.05 (1H, m), 6.18 (1H, s), 6.49 (1H, s), 6.70
(1H, d, J = 7.3 Hz), 7.07–7.12 (4H, m), 7.22 (1H, dd,
J = 7.3, 7.8 Hz), 7.27 (1H, dd, J = 7.3, 7.8 Hz), 7.35–
7.45 (3H, m). FAB MS m/z (M+H)+ 481.
(1H, dd, J = 7.3, 7.8 Hz), 7.54–7.58 (3H, m), 9.74 (1H,
s), 13.19 (1H, br). FAB MS m/z (M+H)+ 467.
5.5.5. (Z)-{4,4-Difluoro-1-[4-(5-methyloxazole-4-carbon-
ylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-
ylidene}acetic acid (11e). 87% yield. H NMR (DMSO-
1
d6) d: 2.25–2.80 (2H, m), 2.60 (3H, s), 3.23 (1H, m),
5.05 (1H, m), 6.33 (1H, s), 6.70 (1H, d, J = 7.3 Hz),
6.85 (1H, m), 6.95–7.40 (3H, m), 7.45 (2H, d,
J = 7.8 Hz), 7.66 (2H, d, J = 7.8 Hz), 8.42 (1H, s),
10.05 (1H, s). FAB MS m/z (M+H)+ 468.
5.4.5. (Z)-Methyl {4,4-difluoro-1-[4-(5-methyloxazole-4-
carbonylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzaze-
pine-5-ylidene}acetate (10e). 71% yield. H NMR (CDCl3)
d: 2.25–2.80 (2H, m), 2.67 (3H, s), 3.84 (3H, s), 5.05 (1H,
m), 6.38 (1H, s), 6.71 (1H, d, J = 7.3 Hz), 6.95–7.40 (4H,
m), 7.45 (2H, d, J = 7.8 Hz), 7.50 (2H, d, J = 7.8 Hz),
7.71 (1H, s), 8.77 (1H, s). FAB MS m/z (M+H)+ 482.
1
5.6. General procedure for the preparation of compounds
12a–n
5.5. General procedure for the preparation of compounds
11a–e
5.6.1. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-dimethylaminopi-
peridino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzaze-
pin-1-yl}carbonyl)phenyl]-1-methylpyrrole-2-carboxam-
ide monohydrochloride (12a). To an ice-cooled mixture
of 11a (0.592 mmol) and 1-hydroxybenzotriazole
(0.710 mmol) in CH2Cl2 (10 mL) and CH3CN (10 mL)
was added 1-ethyl-3-(dimethylaminopropyl)carbodii-
mide hydrochloride (0.710 mmol) in CH2Cl2 (10 mL),
and the mixture was stirred at room temperature for
1 h. After being cooled at 0 ꢁC, 4-dimethylaminopiperi-
zine (0.710 mmol) was added, and the mixture was stirred
overnight at room temperature. To the mixture was added
1 M NaOH, which was then extracted with CHCl3. The
organic layer was dried over anhydrous K2CO3 and con-
centrated in vacuo. The residue was chromatographed on
a silica gel column using CHCl3–MeOH (95:5) as the elu-
ent to give a free amine. This resulting amine was diluted
with MeOH, and the solution was cooled at 0 ꢁC. A 4 M
solution of HCl in AcOEt (0.2 mL) was added to this solu-
tion, and the mixture was concentrated in vacuo. It was
then recrystallized from CHCl3-diethyl ether to give 12a
(0.349 mmol, 59%) as a colorless amorphous substance.
1H NMR (DMSO-d6) d: 1.40–1.80 (2H, m), 2.42 (2H,
m), 2.67 (1H, m), 2.72 (6H, s), 2.96–3.23 (2H, m), 3.43
(1H, m), 3.83 (3H, s), 4.06 (1H, m), 4.53 (1H, m), 4.87
(1H, m), 6.07 (1H, m), 6.79 (1H, s), 6.83 (1H, d,
J = 7.8 Hz), 7.00 (2H, d, J = 2.9 Hz), 7.05 (2H, d,
J = 7.8 Hz), 7.18 (1H, dd, J = 7.3, 7.8 Hz), 7.30 (1H, t,
J = 7.3 Hz), 7.51 (1H, d, J = 7.3 Hz), 7.55 (3H, m), 9.82
(1H, s), 10.42 (1H, m). FAB MS m/z (M+H)+ 576. Anal.
Calcd for C32H35N5O3F2ÆHClÆ3H2O: C, 57.70; H, 6.35;
N, 10.51; Cl, 5.32; F, 5.70. Found: C, 57.51; H, 6.30; N,
10.46; Cl, 5.33; F, 5.74.
5.5.1. (Z)-{4,4-Difluoro-1-[4-(1-methylpyrrole-2-carbon-
ylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-
ylidene}acetic acid (11a). The mixture of compounds 11a
(0.96 g, 2.00 mmol) in methanol (MeOH, 10 mL) and
1 M NaOH (4 mL) was stirred at room temperature for
7 h and then concentrated in vacuo. The residue was acid-
ified with 1 M HCl (10 mL) under cooling conditions, and
the solution was extracted with CHCl3. The organic layer
was dried over anhydrous MgSO4 and concentrated in
vacuo. The residue was recrystallized from hexane–
AcOEt to give 0.72 g (1.54 mmol, 77%) of 11a as a color-
less powder. 1H NMR (DMSO-d6) d: 2.35–2.82 (2H, m),
3.21 (1H, m), 3.94 (3H, s), 5.04 (1H, m), 6.10 (1H, m),
6.23 (1H, s), 6.70 (1H, d, J = 8.7 Hz), 6.76 (1H, s), 6.87
(1H, m), 7.07–7.17 (3H, m), 7.22 (1H, t, J = 7.8 Hz),
7.37 (2H, d, J = 7.8 Hz), 7.50 (2H, d, J = 8.7 Hz), 8.72
(1H, s). FAB MS m/z (M+H)+ 466.
5.5.2. (Z)-{4,4-Difluoro-1-[4-(3-methylthiophene-2-carbon-
ylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl-
idene}acetic acid (11b). 96% yield. 1H NMR (DMSO-d6) d:
2.25–2.89 (2H, m), 2.53 (3H, s), 3.24 (1H, m), 5.04 (1H, m),
6.23 (1H, s), 6.71 (1H, d, J = 7.8 Hz), 6.92 (1H, d,
J = 4.9 Hz), 7.12 (1H, t, J = 7.8 Hz), 7.15 (2H, d,
J = 8.7 Hz), 7.23 (1H, t, J = 7.3 Hz), 7.33 (1H, d,
J = 4.9 Hz), 7.38 (1H, d, J = 7.3 Hz), 7.47 (2H, d,
J = 8.7 Hz), 8.40 (1H, s). FAB MS m/z (M+H)+ 483.
5.5.3. (Z)-{4,4-Difluoro-1-[4-(3-methylfuran-2-carbonyl-
amino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-
ylidene}acetic acid (11c). 81% yield. 1H NMR (DMSO-
d6) d: 2.28–2.84 (2H, m), 2.41 (3H, s), 3.23 (1H, m), 5.06
(1H, m), 6.23 (1H, s), 6.39 (1H, s), 6.70 (1H, d,
J = 7.3 Hz), 7.07 (1H, dd, J = 7.3, 7.8 Hz), 7.15 (2H, d,
J = 8.8 Hz), 7.22 (1H, dd, J = 7.3, 7.8 Hz), 7.34–7.39
(3H, m), 7.48 (1H, s), 7.50 (1H, s), 8.33 (1H, s). FAB
MS m/z (M+H)+ 467.
5.6.2. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-dimethylaminopiperi-
dino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzazepin-
1-yl}carbonyl)phenyl]-3-methylthiophene-2-carboxamide
monohydrochloride (12b). 61% yield. 1H NMR
(DMSO-d6) d: 1.38–1.79 (2H, m), 2.10 (2H, m),
2.40 (3H, s), 2.42 (2H, m), 2.68 (1H, m), 2.70 (3H,
s), 2.71 (3H, s), 2.98-3.23 (2H, m), 3.43 (1H, m),
4.06 (1H, m), 4.53 (1H, m), 4.87 (1H, m), 6.33 (1H,
s), 6.71 (1H, d, J = 7.8 Hz), 6.93 (1H, d,
J = 4.9 Hz), 7.10 (2H, d, J = 7.8 Hz), 7.19 (1H, t,
J = 8.7 Hz), 7.23 (1H, t, J = 7.3 Hz), 7.33 (1H, d,
J = 4.9 Hz), 7.40 (2H, m), 7.47 (1H, d, J = 8.7 Hz),
10.07 (1H, s). FAB MS m/z (M+H)+ 593. Anal.
Calcd for C32H34N4O3F2SÆHClÆ1.8 H2O: C, 58.09;
5.5.4. (Z)-{4,4-Difluoro-1-[4-(2-methylfuran-3-carbonyl-
amino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-
ylidene}acetic acid (11d). 68% yield. 1H NMR (DMSO-
d6) d: 2.34–2.67 (2H, m), 2.50 (3H, s), 3.33 (1H, m), 4.87
(1H, m), 6.65 (1H, s), 6.81 (1H, d, J = 7.3 Hz), 7.00 (1H,
s), 7.05–7.15 (3H, m), 7.27 (1H, dd, J = 7.3, 7.8 Hz), 7.37

Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
1835
H, 5.88; N, 8.47; Cl, 5.57; F, 5.74; S, 4.96. Found: C,
58.11; H, 6.12; N, 8.37; Cl, 5.57; F, 5.74; S, 4.96.
3.98 (2H, m), 4.89 (1H, br), 6.58 (1H, s), 6.78 (1H,
d, J = 7.3 Hz), 7.06–7.10 (3H, m), 7.15 (1H, dd,
J = 7.3, 7.8 Hz), 7.28 (1H, dd, J = 7.3, 7.8 Hz), 7.36
(1H, d, J = 6.8 Hz), 7.55–7.58 (3H, m), 9.79 (1H, s).
FAB MS m/z (M+H)+ 579. Anal. Calcd for
C31H32N4O5F2ÆHClÆ2H2O: C, 57.19; H, 5.73; N, 8.60;
Cl, 5.45; F, 5.84. Found: C, 57.19; H, 5.70; N, 8.55;
Cl, 5.39; F, 5.78.
5.6.3. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-dimethylaminopiperi-
dino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzaze-
pin-1-yl}carbonyl)phenyl]-3-methylfuran-2-carboxamide
monohydrochloride (12c). 42% yield. 1H NMR (DMSO-
d6) d: 1.42–1.82 (2H, m), 2.10 (2H, m), 2.42 (2H, m),
2.51 (6H, s), 2.67 (1H, m), 2.70 (3H, s), 2.98–3.30 (2H,
m), 3.44 (1H, m), 4.06 (1H, m), 4.53 (1H, m), 4.87
(1H, m), 6.33 (1H, s), 6.38 (1H, s), 6.70 (1H, d,
J = 7.3 Hz), 7.08 (1H, t, J = 7.3 Hz), 7.19–7.23 (3H, m),
7.35–7.40 (2H, m), 7.43 (1H, s), 8.02 (1H, s), 10.81
(1H, m). FAB MS m/z (M+H)+ 577. FABHRMS Calcd
for C32H34N4O4F2 (M+H)+, 577.2626. Found: 577.2635.
5.6.8. (Z)-N-(40-{[4,4-Difluoro-5-(2-{[3-(4-morpholinyl)pro-
pyl]amino}-2-oxoethylidene)-2,3,4,5-tetrahydro-1H-1-ben-
zazepin-1-yl]carbonyl}phenyl)-2-methylfuran-3-carboxam-
1
ide (12h). 78% yield. H NMR (DMSO-d6) d: 1.60–1.66
(2H, m), 1.89 (2H, m), 2.31 (6H, s), 2.43 (3H, m), 2.68
(3H, s), 2.74 (1H, m), 3.16 (1H, m), 3.97 (1H, d,
J = 13 Hz), 4.62 (1H, d, J = 13 Hz), 5.03 (1H, m), 6.33
(1H, s), 6.76 (1H, d, J = 7.3 Hz), 7.00 (1H, s), 7.07–7.14
(3H, m), 7.26 (1H, dd, J = 7.3, 7.8 Hz), 7.34 (1H, d,
J = 7.8 Hz), 7.52–7.57 (2H, m), 8.33 (1H, m), 9.72 (1H,
s). FAB MS m/z (M+H)+ 593. Anal. Calcd for
C32H34N4O5F2: C, 64.85; H, 5.78; N, 9.45; F, 6.41. Found:
C, 64.42; H, 5.70; N, 9.41; F, 6.43.
5.6.4. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-dimethylaminopi-
peridino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benza
zepin-1-yl}carbonyl)phenyl]-2-methylfuran-3-carboxam-
ide monohydrochloride (12d). 61% yield. 1H NMR
(DMSO-d6) d: 1.40–1.80 (2H, m), 2.09 (2H, br), 2.45
(2H, m), 2.50 (6H, s), 2.67 (1H, m), 2.70 (3H, s), 3.00-
3.25 (2H, m), 3.37 (1H, m), 4.05 (1H, m), 4.53 (1H,
m), 4.90 (1H, br), 6.78 (1H, s), 6.83 (1H, d,
J = 7.3 Hz), 7.06–7.10 (3H, m), 7.18 (1H, dd, J = 7.3,
7.8 Hz), 7.30 (1H, dd, J = 7.3, 7.8 Hz), 7.51 (1H, d,
J = 7.3 Hz), 7.57–7.61 (3H, m), 9.85 (1H, s). FAB MS
m/z (M+H)+ 577. Anal. Calcd for C32H34N4O4F2ÆH-
ClÆ0.5H2O: C, 61.78; H, 5.83; N, 9.01; Cl, 5.70; F,
6.11. Found: C, 61.52; H, 5.86; N, 8.99; Cl, 5.58; F, 5.78.
5.6.9. (Z)-N-(40-{[4,4-Difluoro-5-(2-{[4-(4-morpholinyl)bu-
tyl]amino}-2-oxoethylidene)-2,3,4,5-tetrahydro-1H-1-ben-
zazepin-1-yl]carbonyl}phenyl)-2-methylfuran-3-carboxamide
1
(12i). 72% yield. H NMR (DMSO-d6) d: 1.40 (4H, br),
2.18–2.32 (6H, m), 2.51 (3H, s), 2.52–2.67 (3H, m), 3.14
(3H, m), 3.54 (4H, m), 4.91 (1H, br), 6.45 (1H, s), 6.75
(1H, d, J = 7.3 Hz), 7.01 (1H, s), 7.10–7.14 (3H, m), 7.26
(1H, dd, J = 7.3, 7.8 Hz), 7.36 (1H, d, J = 7.8 Hz), 7.54
(2H, m), 8.32 (1H, m), 9.72 (1H, s). FAB MS m/z
(M+H)+ 607. Anal. Calcd for C33H36N4O5F2: C, 65.33;
H, 5.98; N, 9.24; F, 6.26. Found: C, 65.07; H, 6.00; N,
9.22; F, 6.28.
5.6.5. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-dimethylaminopiperi-
dino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzaze-
pin-1-yl}carbonyl)phenyl]-5-methyloxazole-4-carboxam-
ide (12e). 65% yield. 1H NMR (CDCl3) d: 1.42–1.58 (2H,
m), 1.89 (2H, m), 2.31 (6H, s), 2.43 (2H, m), 2.68 (3H, s),
2.74 (2H, m), 3.16 (2H, m), 3.97 (1H, d, J = 13 Hz), 4.62
(1H, d, J = 13 Hz), 5.03 (1H, m), 6.33 (1H, s), 6.70 (1H,
d, J = 7.3 Hz), 7.08 (1H, dd, J = 7.3, 7.8 Hz), 7.15–7.30
(4H, m), 7.37 (1H, d, J = 7.3 Hz), 7.48 (1H, d,
J = 7.3 Hz), 7.71 (1H, s), 8.72 (1H, s). FAB MS m/z
(M+H)+ 578. Anal. Calcd for C31H33N5O4F2ÆH2O: C,
62.51; H, 5.92; N, 11.76; F, 6.38. Found: C, 62.12; H,
5.79; N, 11.80; F, 6.33.
5.6.10. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-morpholinopiperi-
dino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzazepin-
1-yl}carbonyl)phenyl]-2-methylfuran-3-carboxamide mono-
1
hydrochloride (12j). 76% yield. H NMR (DMSO-d6) d:
1.38–1.92 (6H, m), 2.16–2.23 (4H, m), 2.28–2.45 (2H,
m), 2.50 (3H, s), 2.68 (1H, t, J = 8.8 Hz), 2.90 (4H,
m), 3.17 (1H, m), 4.05 (1H, d, J = 6.7 Hz), 4.52 (1H,
d, J = 6.7 Hz), 4.86 (1H, br), 6.44 (1H, s), 6.78 (1H,
s), 7.08 (2H, m), 7.18 (1H, dd, J = 7.3, 7.8 Hz), 7.30
(1H, dd, J = 7.3, 7.8 Hz), 7.51 (1H, d, J = 7.8 Hz), 7.60
(3H, m), 8.33 (1H, m), 9.85 (1H, s). FAB MS m/z
(M+H)+ 619. Anal. Calcd for C34H36N4O5F2ÆHClÆ3H2O:
C, 57.58; H, 6.11; N, 7.90; Cl, 5.00; F, 5.36. Found: C,
57.68; H, 6.03; N, 7.87; Cl, 5.30; F, 5.37.
5.6.6. (Z)-N-(40-{[4,4-Difluoro-5-(2-morpholino-2-oxoethy-
lidene)-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]carbon-
yl}phenyl)-2-methylfuran-3-carboxamide (12f). 66%
1
yield. H NMR (DMSO-d6) d: 2.34–2.49 (3H, m), 2.51
(3H, s), 3.10 (1H, br), 3.51–3.62 (7H, m), 4.87 (1H, br),
6.77 (1H, s), 6.82 (1H, d, J = 7.3 Hz), 7.00 (1H, s), 7.07
(1H, d, J = 8.7 Hz), 7.16 (1H, dd, J = 7.3, 7.8 Hz), 7.28
(1H, dd, J = 7.3, 7.8 Hz), 7.50 (1H, d, J = 8.7 Hz), 7.57
(4H, m), 9.74 (1H, s). FAB MS m/z (M+H)+ 536. Anal.
Calcd for C29H27N3O5F2Æ0.2H2O: C, 64.61; H, 5.12; N,
7.79; F, 7.05. Found: C, 64.55; H, 5.15; N, 7.79; F, 7.05.
5.6.11. (Z)-N-[40-({4,4-Difluoro-5-[2-(4-isopropyl-1-piperaz-
inyl)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzazepin-
1-yl}carbonyl)phenyl]-2-methylfuran-3-carboxamide mono-
1
hydrochloride (12k). 40% yield. H NMR (DMSO-d6) d:
1.30 (6H, m), 2.42–2.48 (4H, m), 2.90–3.25 (3H, m),
3.42–3.60 (5H, m), 3.72 (1H, m), 4.13 (1H, m), 4.50
(1H, d, J = 6.8 Hz), 4.85 (1H, br), 6.77 (1H, s), 6.84
(1H, d, J = 7.3 Hz), 7.02 (1H, s), 7.08 (2H, d,
J = 7.8 Hz), 7.20 (1H, dd, J = 7.3, 7.8 Hz), 7.31 (1H,
dd, J = 7.3, 7.8 Hz), 7.55 (3H, m), 8.31 (1H, m), 9.80
(1H, s). FAB MS m/z (M+H)+ 577. Anal. Calcd for
C32H34N4O4F2ÆHClÆH2O: C, 60.90; H, 5.91; N, 8.88;
5.6.7.
(Z)-N-(40-{[4,4-Difluoro-5-(2-{[2-(4-morpholi-
nyl)ethyl]amino}-2-oxoethylidene)-2,3,4,5-tetrahydro-1H-
1-benzazepin-1-yl]carbonyl}phenyl)-2-methylfuran-3-car-
boxamide monohydrochloride (12g). 38% yield. H NMR
1
(DMSO-d6) d: 1.03–1.10 (2H, m), 2.40 (2H, m), 2.50
(3H, s), 3.05–3.21 (4H, m), 3.35–3.60 (6H, m), 3.81–

1836
Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
Cl, 5.62; F, 6.02. Found: C, 61.13; H, 5.98; N, 8.84; Cl,
5.74; F, 6.23.
with Tris buffer and radioactivity retained on them
was counted with a liquid scintillation counter. Specific
binding was calculated as the total binding minus non-
specific binding, which was determined using 1 lM
unlabeled AVP. The concentration of test compound
that caused 50% inhibition (IC50) of the specific binding
of [3H]AVP was determined by regression analysis of the
displacement curve. Inhibitory dissociation constant (Ki)
was calculated from the following formula: Ki = IC50/
(1 + [L]/Kd), where [L] is the concentration of radioli-
gand present in the tubes and Kd is the dissociation con-
stant of radioligand obtained from the Scatchard plot.
5.6.12. (Z)-N-[40-({4,4-Difluoro-5-[2-oxoethylidene-2-(4-
piperidinopiperidino)]-2,3,4,5-tetrahydro-1H-1-benzazepin-
1-yl}carbonyl)phenyl]-2-methylfuran-3-carboxamide mono-
1
hydrochloride (12l). 35% yield. H NMR (DMSO-d6) d:
1.38–1.98 (8H, m), 2.16–2.25 (4H, m), 2.28–2.48 (2H,
m), 2.50 (3H, s), 2.68 (1H, t, J = 6.8 Hz), 2.91 (4H, m),
3.17 (1H, m), 4.05 (1H, d, J = 7.6 Hz), 4.52 (1H, d,
J = 7.6 Hz), 4.86 (1H, br), 6.78 (1H, s), 6.84 (1H, d,
J = 7.3 Hz), 7.08 (2H, m), 7.18 (1H, dd, J = 7.3, 7.8 Hz),
7.30 (1H, dd, J = 7.3, 7.8 Hz), 7.51 (1H, d, J = 7.8 Hz),
7.60 (3H, m), 8.33 (1H, m), 9.85 (1H, s). FAB MS m/z
(M+H)+ 617. Anal. Calcd for C35H38N4O4F2ÆHClÆ0.5-
H2O: C, 63.49; H, 6.09; N, 8.46; F, 5.74; Cl, 5.35. Found:
C, 63.30; H, 5.96; N, 8.35; F, 5.34; Cl, 5.19.
5.7.2. Receptor binding assay: for the cloned human
receptors.25,26 The cloned human AVP receptor subtypes
were stably expressed in CHO cells and plasma mem-
branes were prepared according to the reported
protocols.
5.6.13. (Z)-N-(40-{[4,4-Difluoro-5-(2-oxo-2-{[3-(1-piperidi-
nyl)propyl]amino}ethylidene)-2,3,4,5-tetrahydro-1H-1-ben-
zazepin-1-yl]carbonyl}phenyl)-2-methylfuran-3-carboxam-
ide (12m). 87% yield. 1H NMR (DMSO-d6) d: 1.23
(2H, br), 1.45 (6H, br), 1.64 (2H, br), 2.35 (2H, br),
2.51 (3H, s), 2.67 (4H, br), 3.18 (4H, br), 4.89 (1H,
s), 6.51 (1H, s), 6.76 (1H, d, J = 7.3 Hz), 7.06–7.15
(4H, m), 7.27 (1H, dd, J = 7.3, 7.8 Hz), 7.35 (1H, d,
J = 7.8 Hz), 7.57 (2H, d, J = 7.8 Hz), 8.45 (1H, s),
9.79 (1H, s). FAB MS m/z (M+H)+ 591. Anal. Calcd
for C33H36N4O4F2Æ0.2CHCl3ÆH2O: C, 63.04; H, 6.09;
N, 8.86; Cl, 3.36; F, 6.01. Found: C, 62.64; H, 6.10;
N, 8.94; Cl, 3.55; F, 5.95.
5.7.3. V1A receptor antagonistic activity: in pithed rats
(iv).25,26 Pithed rats were maintained at 37 ꢁC by means
of a thermostat-controlled heating board. For iv injec-
tion, compounds were dissolved in DMF. After the stabil-
ization ofblood pressure, compounds orvehicle was given
(0.5 ml/kg iv) 5 min before the injection of AVP (30 mU/
kg, iv). The dose of compound causing a 50% inhibition of
the pressor response to AVP (ID50) was calculated.
5.7.4. V1A receptor antagonistic activity: in conscious rats
(po).23 Male Wistar rats were anesthetized with pen-
tobarbital (50 mg/kg ip). The left carotid artery and
ipsilateral jugular vein were cannulated with a poly-
ethylene tube (PE-50) for determination of blood
pressure and heart rate, and for intravenous adminis-
tration of AVP (30 mU/kg). The animals were al-
lowed to recover for 2 days after operation, during
which time they were housed in individual cages with
free access to rat chow and water. Blood pressure
was measured with a pressure transducer (AP-200T)
coupled to the carotid arterial cannula and continu-
ously recorded via a polygraph system. Heart rate
was measured from blood pressure pulse waves. After
stabilization of both parameters was achieved, AVP
(30 mU/kg) was administered through the venous can-
nula. The injection of AVP was repeated at intervals
of about 15 min, each injection being given as soon
as blood pressure returned to a preinjection level.
Responses to two consecutive doses of AVP showing
approximately constant amplitudes of blood pressure
elevation were averaged to be taken as a control re-
sponse. Each rat was then treated orally with a single
dose of the 0.5% methylcellulose aqueous solution of
12l or the vehicle (0.5% methylcellulose aqueous solu-
tion), and any changes in blood pressure were noted.
5.6.14. (Z)-N-(40-{[4,4-Difluoro-5-(2-oxo-2-{[4-(1-piperid-
inyl)butyl]amino}ethylidene)-2,3,4,5-tetrahydro-1H-1-ben-
zazepin-1-yl]carbonyl}phenyl)-2-methylfuran-3-carboxam-
ide monohydrochloride (12n). 54% yield. 1H NMR
(DMSO-d6) d: 1.37 (2H, m), 1.49 (3H, m), 1.68–1.77
(5H, m), 2.51 (3H, s), 2.82 (2H, m), 3.01 (2H, m), 3.16
(2H, m), 3.33 (3H, m), 3.38 (3H, m), 4.91 (1H, br),
6.51 (1H, s), 6.77 (1H, d, J = 7.3 Hz), 7.06–7.09 (2H,
m), 7.13 (1H, dd, J = 7.3, 7.8 Hz), 7.27 (1H, dd,
J = 7.3, 7.8 Hz), 7.36 (1H, d, J = 7.8 Hz), 7.55–7.58
(3H, m), 8.42 (1H, m), 9.79 (1H, s). FAB MS m/z 605.
Anal. Calcd for C34H38N4O4F2ÆHClÆ2H2O: C, 60.30;
H, 6.40; N, 8.27; F, 5.61; Cl, 5.24. Found: C, 60.45;
H, 6.21; N, 8.30; F, 5.48; Cl, 5.39.
5.7. Pharmacology
5.7.1. Receptor binding assay: for the rat receptors.25,26
Binding assays were performed using [3H]AVP on plas-
ma membranes prepared from rat liver or kidney. Plas-
ma membrane preparations were incubated with
various concentrations of [3H]AVP (0.1–3.0 nM).
Radioligands (0.5 nM) were added to each membrane
preparation and the mixture was incubated with vari-
ous concentrations of the compounds in 250 ll of assay
buffer (50 mM Tris–HCl, pH 7.5, 5 mM MgCl2 and
0.1% bovine serum albumin). After incubation
(60 min at 25 ꢁC), the reaction was terminated by addi-
tion of 3 ml of ice-cooled Tris buffer (50 mM Tris–HCl,
pH 7.5, 5 mM MgCl2), followed immediately by filtra-
tion using glass filters. The filters were rinsed twice
Acknowledgments
The authors are grateful to Dr. Chikashi Saitoh and Ms.
Chika Kitada for performing binding assay of cloned
human receptors and to the staff of the Division of Anal-
ysis Research Laboratories for the spectral
measurements.

Y. Shimada et al. / Bioorg. Med. Chem. 14 (2006) 1827–1837
1837
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16. Shimada, Y.; Akane, H.; Taniguchi, N.; Matsuhisa, A.;
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