Preparation of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives as novel arginine vasopressin V2 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2008.09.039

The present work describes the discovery of novel series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives as arginine vasopressin (AVP) V₂ receptor agonists. By replacing the amide juncture in YM-35278 with a direct ring connection gave compound 10a, which acts as a V₂ receptor agonist. These studies provided the potent, orally active non-peptidic V₂ receptor agonists 10a and 10j.

Full text of DOI:10.1016/j.bmc.2008.09.039

Bioorganic & Medicinal Chemistry 16 (2008) 9524–9535
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preparation of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)
acetamide derivatives as novel arginine vasopressin V2 receptor agonists
a
a
a
a
Issei Tsukamoto a, , Hiroyuki Koshio , Seijiro Akamatsu , Takahiro Kuramochi , Chikashi Saitoh ,
Takeyuki Yatsu a, Hiroko Yanai-Inamura a, Chika Kitada a, Eisaku Yamamoto a, Shuichi Sakamoto b,
Shin-ichi Tsukamoto a
*
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
b Technology Astellas Pharma Inc., 3-17-1 Hasune, Itabashi, Tokyo 174-8612, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present work describes the discovery of novel series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-ben-
zazepine-5-ylidene)acetamide derivatives as arginine vasopressin (AVP) V2 receptor agonists. By replac-
ing the amide juncture in YM-35278 with a direct ring connection gave compound 10a, which acts as a V2
receptor agonist. These studies provided the potent, orally active non-peptidic V2 receptor agonists 10a
and 10j.
Received 7 August 2008
Revised 11 September 2008
Accepted 12 September 2008
Available online 17 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
V2 receptor agonist
V2 receptor antagonist
(4,4-Difluoro-1,2,3,4-tetrahydro-5H-1-
benzazepin-5-ylidene)acetamide
derivatives
Urine volume
1. Introduction
51803 and VNA932, have been reported2–5; however, none of them
have been launched into market so far (Fig. 1).
Arginine vasopressin (AVP) is a cyclic nonapeptide that is pro-
duced and secreted by the hypothalamo-neurohypophysial sys-
Here, we describe the preparation of the novel V2 receptor ago-
nist 10a via structural conversion from YM-35278,6 a V2 receptor
antagonist found in a previous study (Fig. 2). Further research of
structural requirements for agonist activity to provide another
potent, orally active, non-peptidic V2 receptor agonist, (2Z)-2-
{1-[2-chloro-4-(3-methylpyrrolidin-1-yl)benzoyl]-4,4-difluoro-
1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene}-N-(pyridin-
2-ylmethyl)acetamide hydrochloride (10j), is also reported.
Compounds 10a and 10j were both found to decrease urine volume
in water-loaded rats.
tem. Vasopressin receptors are classified by three subtypes, V1a
V1b, and V2. The V1a receptor, which exists in vascular smooth mus-
cle and platelets, is involved in blood pressure control, and V1b
,
,
which is distributed mainly in the hypophysis pituitary gland, reg-
ulates adrenocorticotropic hormone (ACTH) secretion. The pres-
ence of the V2 receptor, which is involved in water reabsorption,
has been identified in the kidney.1 Stimulation of the V2 receptor
with AVP, for which production is triggered by increased blood
pressure, causes water reabsorption in the kidney by the process
of increasing cyclic adenosine monophosphate (cAMP) with subse-
quent activation of aquaporin-2 water channel. This implies that a
V2 receptor agonist could reduce urine volume, and, thus poten-
tially treat diseases such as central diabetes insipidus and nocturia.
2. Chemistry
All compounds evaluated for V2 agonist activity were prepared
as described in Schemes 1–3. Commercially available 2-chloro-4-
fluorobenzoic acid (1) was esterified under acid conditions to af-
ford 2. Treatment of 2 with cyclic amines following hydrolysis pro-
vided 4-substituted 2-chlorobenzoic acid-derived intermediates
(4a–4f, 4h–4o). 2-Chloro-4-(5-methyl-1H-pyrazol-1-yl)benzoic
acid (4g) was synthesized from 2-chloro-4-fluorobenzonitrile (5)
and 3-methylpyrazole, which was then hydrolyzed7,8 via a benz-
amide derivative (7), as shown in Scheme 1.
Currently, desmopressin (1-desamino-8-D-Arg vasopressin), a
peptide analogue of AVP, is the only V2 receptor agonist on the
market, and is used for the treatment of central diabetes insipidus.
Several other non-peptidic V2 receptor agonists, such as OPC-
* Corresponding author. Tel.: +81 29 863 6682; fax: +81 29 852 5387.
E-mail address: issei.tsukamoto@jp.astellas.com (I. Tsukamoto).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.039

I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
9525
NH2
O
O
NH2
O
O
NH2
NH
H
N
NH2
NH
HN
H
N
HN
NH2
NH2
N
H
O
N
H
O
O
S
HO
HN
O
O
S
HO
HN
O
O
S
NH
O
S
NH
O
O
N
O
H
N
N
O
NH
H
N
NH
NH2
NH2
O
N
O
O
N
O
H
O
H
O
NH2
AVP
Desmopressin
O
N
H
N
N
N
O
N
O
N
Cl
N
Cl
OPC-51803
VNA932
Figure 1. Chemical structures of AVP and well-known arginine vasopressin V2 receptor agonists.
O
O
N
H
N
N
H
N
F
F
F
F
N
N
O
O
O
N
H
N
Cl
YM-35278
V2 receptor antagonist
V2 receptor agonist
Figure 2. Chemical structures of YM-35278 and designs for V2 receptor agonists.
Transformation of 4a–4k, 4n, and 4o into the corresponding
benzoyl chlorides, and subsequent amidation with methyl (4,4-
difluoro-1,2,3,4-tetrahydro-5H-1-benzoazepin-5-ylidene)acetate9
yielded compounds 8a–8k, 8n, and 8o, which were treated with
aqueous sodium hydroxide to afford 9a–9k, 9n, and 9o. Condensa-
tion of those carboxylic acids with 2-aminomethylpyridine in the
presence of N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride and 1-hydroxybenzotriazole yielded the target
derivatives 10a–10k, 10n, and 10o (Scheme 2).
agonist activity. Compound 10a displayed high binding affinity
for the human V2 receptor with a Ki value of 4.8 nM and potent
cAMP accumulation activity (EC50 = 1.8 nM).
This observation agrees with the reports by Kondo et al.2 and
Venkatesan et al.,4 which show that compounds that link to V2
antagonists via an amino group instead of an amide-linkage exhibit
V2 agonist activity.
Table 2 illustrates the exploration of substitutions in the pyra-
zole ring of compound 10a. Compound 10b, in which the 3-methyl
group in the pyrazole ring is absent, showed intense affinity for the
V2 receptor, with a Ki value of 9.2 nM. However, the cAMP accumu-
lation activity of this compound was moderate, with an ED50 value
of 192 nM. More bulky substitutions, such as replacing the methyl
group of 10a with an ethyl group (10c), isopropyl group (10d), or
phenyl group (10e), also resulted in decreased agonist potency,
while binding affinity was maintained. Compounds 10f, 10g, and
10h, which have a 3,5-dimethyl group, 5-methyl group, and an
indazole ring, respectively, showed a similar tendency to decrease
cAMP production.
Compounds 10l and 10m were synthesized from 4l and 4m via
condensation with another benzazepin derivative (13), as shown in
Scheme 3.
3. Result and discussion
The Ki values of compounds 10a–10o for human V2 receptor
were evaluated in a radioligand binding study. The activity of com-
pounds 10a–10o for cAMP production was also determined as EC50
value and intrinsic activity.
As described above, although all compounds substituted into
the pyrazole ring exhibited good V2 receptor binding affinity, most
of them showed less potent cAMP accumulation activity. This indi-
As shown in Table 1, replacing the biphenylamide moiety of
YM-35278, a V2 receptor antagonist found previously, with the
3-methylpyrazol-1-yl group yielded compound 10a, which has V2

9526
I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
CO2H
Cl
CO2Me
Cl
CO2Me
Cl
CO2H
Cl
a
c
b
F
F
R
R
1
2
3a R= (3-Methyl-1H-pyrazol)-1-yl
3b R= Pyrazolyl
4a R= (3-Methyl-1H-pyrazol)-1-yl
4b R= Pyrazolyl
3c R= 3-Ethylpyrazol-1-yl
3d R= 3-Isopropylpyrazol-1-yl
3e R= 3-Phenylpyrazol-1-yl
3f R= 3,5-Dimethylpyrazol-1-yl
3h R= 2H-Indazol-2-yl
4c R= 3-Ethylpyrazol-1-yl
4d R= 3-Isopropylpyrazol-1-yl
4e R= 3-Phenylpyrazol-1-yl
4f R= 3,5-Dimethylpyrazol-1-yl
4h R= 2H-Indazol-2-yl
3i R= Pyrrolidin-1-yl
4i R= Pyrrolidin-1-yl
3j R= 3-Methylpyrrolidin-1-yl
3k R= 2-Methylpyrrolidin-1-yl
3l R= Piperidin-1-yl
4j R= 3-Methylpyrrolidin-1-yl
4k R= 2-Methylpyrrolidin-1-yl
4l R= Piperidin-1-yl
3m R= Azepan-1-yl
4m R= Azepan-1-yl
3n R= 3-(Dimethylamino)pyrrolidin-1-y 4n R= 3-(Dimethylamino)pyrrolidin-1-yl
3o R= 4-Methypiperazin-1-yl
4o R= 4-Methypiperazin-1-yl
CN
Cl
CONH2
CO2H
Cl
CN
Cl
d
e
f
N
N
N
N
N
Cl
N
F
4g
7
6
5
Scheme 1. Reagents and conditions: (a) H2SO4, MeOH; (b) amine, K2CO3, NMP; (c) aq HCl or aq NaOH; (d) NaH, 3-methylpyrazole, DMF; (e) H2O2, K2CO3, DMSO; (f) NaNO2,
H2SO4, H2O.
cates that the steric requirement for producing intrinsic activity is
extremely strict around this region. Among these compounds, the
original 3-methylpyrazole derivative 10a was the only one pos-
sessing both good binding affinity and potent agonist activity. In
the cAMP accumulation assay, the maximum response to 10a
was quite comparable to that of AVP itself.
Table 3 shows the activity of compounds that have aliphatic
rings in place of the pyrazole ring in compound 10a. Compounds
substituted with five- to seven-membered hydrophobic rings
(10i–10m) maintained binding affinity to the V2 receptor. With re-
gard to intrinsic activity, pyrrolidine-substituted 10i and 3-meth-
ylpyrrolidine-substituted 10j exhibited activity comparable to
that of 10a in the cAMP production assay, with ED50 values of
3.13 nM and 1.25 nM, respectively. However, the 2-methyl pyrrol-
idine derivative 10k showed a substantial decrease in cAMP accu-
mulation. Substitution with piperidine (10l) was less effective,
yielding an ED50 value of 104 nM, which is 33-fold less potent than
compound 10i. Substitution of a seven-membered ring (10m) also
caused a decrease (206-fold), with an ED50 value of 646 nM. Amine
functional groups, such as the 3-(dimethylamino)pyrrolidine (10n)
and 4-methylpiperazine-1-yl groups (10o), led to a decrease in V2
receptor binding affinity. Compounds 10n and 10o also showed lit-
tle cAMP production activity.
These results indicate that steric bulkiness does not hinder V2
receptor binding, but it has a severely negative impact on agonist
activity, as is also observed in Table 2. Sterically-unhindered nearly
O
CO2Me
F
CO2H
F
N
N
H
F
F
F
F
CO2H
a
b
c
N
N
(HCl)
R
Cl
N
O
O
O
R
R
Cl
Cl
R
Cl
8a R= (3-Methyl-1H-pyrazol)-1-yl
8b R= Pyrazolyl
9a R= (3-Methyl-1H-pyrazol)-1-yl
9b R= Pyrazolyl
4a-4k, 4n, 4o
10a R= (3-Methyl-1H-pyrazol)-1-yl
10b R= Pyrazolyl
8c R= 3-Ethylpyrazol-1-yl
8d R= 3-Isopropylpyrazol-1-yl
8e R= 3-Phenylpyrazol-1-yl
8f R= 3,5-Dimethylpyrazol-1-yl
8g R= (5-Methyl-1H-pyrazol)-1-yl
8h R= 2H-Indazol-2-yl
9c R= 3-Ethylpyrazol-1-yl
9d R= 3-Isopropylpyrazol-1-yl
9e R= 3-Phenylpyrazol-1-yl
9f R= 3,5-Dimethylpyrazol-1-yl
9g R= (5-Methyl-1H-pyrazol)-1-yl
9h R= 2H-Indazol-2-yl
10c R= 3-Ethylpyrazol-1-yl
10d R= 3-Isopropylpyrazol-1-yl
10e R= 3-Phenylpyrazol-1-yl
10f R= 3,5-Dimethylpyrazol-1-yl
10g R= (5-Methyl-1H-pyrazol)-1-yl
10h R= 2H-Indazol-2-yl
8i R= Pyrrolidin-1-yl
9i R= Pyrrolidin-1-yl
9j R= 3-Methylpyrrolidin-1-yl
9k R= 2-Methylpyrrolidin-1-yl
10i R= Pyrrolidin-1-yl
8j R= 3-Methylpyrrolidin-1-yl
8k R= 2-Methylpyrrolidin-1-yl
10j R= 3-Methylpyrrolidin-1-yl
10k R= 2-Methylpyrrolidin-1-yl
10n R= 3-(Dimethylamino)pyrrolidin-1-yl
10o R= 4-Methypiperazin-1-yl
8n R= 3-(Dimethylamino)pyrrolidin-1-y 9n R= 3-(Dimethylamino)pyrrolidin-1-y
8o R= 4-Methypiperazin-1-yl
9o R= 4-Methypiperazin-1-yl
Scheme 2. Reagents and conditions: (a) SOCl2, cat. DMF, THF then methyl (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetate9 (11), pyridine; (b) aq NaOH;
(c) 2-(aminomethyl)pyridine, WSCD, HOBt, DMF, then HCl.

I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
9527
O
N
H
F
F
N
O
CO2H
F
CO2Me
N
H
F
F
N
F
F
c
b
a
F
N
.HCl
N
H
N
H
N
H
O
11
12
13
R
Cl
10l R= Piperidin-1-yl
10m R= Azepan-1-yl
Scheme 3. Reagents and conditions: (a) aq NaOH; (b) 2-(aminomethyl)pyridine, WSCD, HOBt, DMF; (c) 4l or 4m, SOCl2, cat. DMF, THF, then HCl.
Table 1
modification of the previously found V2 receptor antagonist YM-
35278. The novel V2 agonist 10a was discovered as a result of
replacing the amide linkage of YM-35278 with a direct pyrazolyl
group. Further exploration of structure–activity relationships indi-
cated that the structural requirements for agonist activity are
highly specific, while receptor binding tolerates steric bulkiness.
Among the compounds reported here, 10a and 10j were identified
as potent V2 receptor agonists with high binding affinity. Further-
more, oral administration of 10a or 10j was found to significantly
decrease urine volume in water-loaded rats.
Binding affinity and cAMP accumulation activity of YM-35278 and 10a
Binding
cAMP
affinity for accumulation
V2 Kia (nM) EC50 (nM)
b
O
N
H
F
F
N
YM-35278
0.40 (rat) Not effective
N
5. Experiment
5.1. Chemistry
O
O
N
H
In general, reagents and solvents were used as purchased with-
out further purification. Melting points were determined with a
Yanaco MP-500D melting point apparatus and left uncorrected.
1H NMR spectra were recorded on a JEOL JNM-LA300 or a JEOL
JNM-EX400 spectrometer. Chemical shifts were expressed in d
(ppm) values with tetramethylsilane as an internal standard
(NMR descriptions; s, singlet; d, doublet; t, triplet; q, quartet; dt,
double triplet; m, multiplet, and br, broad peak). Mass spectra
were recorded on a JEOL JMS-LX2000 spectrometer. The elemental
analyses were performed with a Yanaco MT-5 microanalyzer (C, H,
and N) and Yokogawa IC-7000S ion chromatographic analyzer (hal-
ogens), and were within 0.4% of theoretical values.
19 (rat)
4.8
(human)
1.8
O
N
H
F
F
N
10a
N
O
N
N
Cl
5.1.1. Methyl 2-chloro-4-fluorobenzoate (2)
a
Human or rat V2 receptors expressed on CHO cells were used. All assays were
performed in triplicate.
To
a
mixture of 2-chloro-4-fluorobenzoic acid (20.0 g,
115 mmol) in methanol (200 ml) was added sulfuric acid (10 ml),
and the mixture was heated at 60 °C for 17 h. The reaction was
then allowed to cool to room temperature, the solvent was reduced
by evaporation, the residue was diluted in cold water and neutral-
ized with potassium carbonate. The aqueous solution was ex-
tracted with ethyl acetate. The organic phase was washed with
water and brine, dried over MgSO4, filtered, and concentrated in
vacuo to give the title compound 2 (20.9 g, 96%) as pale yellow
oil. 1H NMR (400 MHz, CDCl3) d 3.92 (3H, s), 7.03 (1H, td, J = 8.8,
2.4 Hz), 7.20 (1H, dd, J = 8.4, 2.4 Hz), 7.90 (1H, dd, J = 8.8, 6.0 Hz).
MS (EI) m/z = 188 [M].
b
EC50 values were determined as the concentration of the test compound
required to increase the cAMP level to 50% of the maximum response to AVP. All
assays were performed in triplicate.
neutral structures, such as pyrrolidine-1-yl and 3-methylpyrroli-
dine-1-yl group, maintain effective V2 binding and intrinsic activity
here as observed in Table 3.
The oral antidiuretic effects of compounds 10a and 10j, which
possess good binding affinity and excellent cAMP production activ-
ity, as mentioned above, were evaluated. As shown in Figure 3,
both 10a and 10j reduced urine volume in water-loaded rats with
ED50 values of 0.26 mg/kg po and 0.31 mg/kg po, respectively.
5.1.2. Methyl 2-chloro-4-(3-methyl-1H-pyrazol-1-yl)benzoate
(3a)
4. Conclusion
A mixture of methyl 2-chloro-4-fluorobenzoate (2; 20.9 g,
111 mmol), 3-methylpyrazole (9.38 ml, 117 mmol) and potassium
carbonate (30.7 g, 222 mmol) in 1-methyl-2-pyrrolidinone (NMP)
(150 ml) was heated at 120 °C for 3 h. The reaction was then al-
lowed to cool to room temperature and partitioned between water
In an attempt to discover novel V2 receptor agonists, a series of
(4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acet-
amide derivatives were designed and synthesized via structural

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I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
Table 2
Table 3
Binding affinity and cAMP accumulation activity of substituted pyrazole derivatives
Binding affinity and cAMP accumulation activity of aliphatic ring-substituted
derivatives
O
O
N
H
F
F
N
N
H
F
F
N
N
N
O
O
R
*
Cl
R
Cl
Compound
R
Binding
affinity for
hV2 Kia (nM) EC50 (nM)
cAMP
AMP
Compound
R
Binding
cAMP
AMP
accumulation accumulation
Max activityc
b
affinity for hV2 accumulation
accumulation
Kia (nM)
EC50 (nM)
Max activityc
b
N
10a
10b
10c
10d
10e
10f
4.8
9.2
21
192
95.9
N
N
*
*
10a
10I
4.8
1.8
95.9
N
N
*
61.9
73.4
86.8
4.9
N
*
N
22
14
3.13
1.25
107.1
N
N
*
*
N
>10,000
>10,000
>10,000
>10,000
>10,000
N
10J
90.4
59.3
(racemate)
18
N
*
N
10K
16
1100
N
11
4.9
(racemate)
*
N
*
N
N
10l
14
11
104
646
75.3
62.8
*
N
9.1
6.0
15.9
5.7
*
N
N
10m
*
N
10g
N
N
*
*
N
10n
200
170
>10,000
>10,000
15.9
35.5
N
10h
20
>10,000
8.7
(racemate)
a
Human V2 receptors expressed on CHO cells were used. All assays were per-
formed in triplicate.
*
N
10o
b
N
EC50 values were determined as the concentration of the test compound
required to increase the cAMP level to 50% of the maximum response to AVP. All
assays were performed in triplicate.
Intrinsic activity was calculated as the percentage (%) of the maximum response
to the test compound compared to the maximum response (100%) to AVP. All assays
were performed in triplicate.
a
Human V2, receptors expressed on CHO cells were used. All assays were per-
formed in triplicate.
c
b
EC50 values were determined as the concentration of the test compound
required to increase the cAMP level to 50% of the maximum response to AVP. All
assays were performed in triplicate.
c
Intrinsic activity was calculated as the percentage (%) of the maximum response
to the test compound compared to the maximum response (100%) to AVP. All assays
were performed in triplicate.
and ethyl acetate. The organic phase was washed with water and
brine, dried over MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(n-hexane/ethyl acetate = 20/1–10/1) to give the title compound
3a (9.25 g, 33%) as white solid. 1H NMR (400 MHz, CDCl3) d 2.38
(3H, s), 3.94 (3H, s), 6.30 (1H, d, J = 2.4 Hz), 7.60 (1H, dd, J = 8.8,
2.4 Hz), 7.82 (1H, d, J = 2.4 Hz), 7.85 (1H, d, J = 2.4 Hz), 7.96 (1H,
d, J = 8.8 Hz). MS (FAB) m/z 251 [M+H]+.
5.1.4. Methyl 2-chloro-4-(3-ethyl-1H-pyrazol-1-yl)benzoate
(3c)
Compound 3c was prepared according to the procedure de-
scribed for 3a from 2 and 3-ethylpyrazole. The title compound 3c
(692 mg, 32%) was obtained as colorless oil. 1H NMR (300 MHz,
CDCl3) d 1.31 (3H, t, J = 7.7 Hz), 2.75 (2H, q, J = 7.7 Hz), 3.94 (3H,
s), 6.33 (1H, d, J = 2.6 Hz), 7.60 (1H, dd, J = 8.6, 2.2 Hz), 7.83 (1H,
d, J = 2.2 Hz), 7.86 (1H, d, J = 2.6 Hz), 7.96 (1H, d, J = 8.6 Hz). MS
(FAB) m/z 265 [M+H]+.
5.1.3. Methyl 2-chloro-4-(1H-pyrazol-1-yl)benzoate (3b)
Compound 3b was prepared according to the procedure de-
scribed for 3a from 2 and pyrazole. The title compound 3b
(830 mg, 55%) was obtained as white solid. 1H NMR (300 MHz,
CDCl3) d 3.95 (3H, s), 6.52 (1H, dd, J = 2.6, 1.9 Hz), 7.66 (1H, dd,
J = 8.4, 2.2 Hz), 7.76 (1H, d, J = 1.5 Hz), 7.87 (1H, d, J = 2.2 Hz),
7.98 (1H, d, J = 1.5 Hz) 7.99 (1H, d, J = 8.6 Hz). MS (FAB) m/z 237
[M+H]+.
5.1.5. Methyl 2-chloro-4-(3-isopropyl-1H-pyrazol-1-
yl)benzoate (3d)
Compound 3d was prepared according to the procedure de-
scribed for 3a from 2 and 3-isopropylpyrazole. The title compound

I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
9529
as white solid. 1H NMR (300 MHz, DMSO- d6) d 3.90 (3H, s), 7.13
(1H, t, J = 7.3 Hz), 7.36 (1H, t, J = 8.8 Hz), 7.72 (1H, d, J = 8.8 Hz),
7.78 (1H, d, J = 8.4 Hz), 8.06 (1H, dd, J = 8.4, 1.1 Hz), 8.23 (1H, d,
J = 8.6 Hz), 8.37 (1H, s), 9.29 (1H, s). MS (FAB) m/z 287 [M+H]+.
120
100
80
60
40
20
0
compound10a
5.1.9. Methyl 2-chloro-4-(pyrrolidin-1-yl)benzoate (3i)
Compound 3i was prepared according to the procedure de-
scribed for 3a from 2 and pyrrolidine. The title compound 3i
(6.04 g, 88%) was obtained as pink solid. 1H NMR (300 MHz, CDCl3)
d
2.00–2.06 (4H, m), 3.29–3.35 (4H, m), 3.85 (3H, s), 6.39 (1H, dd,
J = 8.8, 2.4 Hz), 6.54 (1H, d, J = 2.4 Hz), 7.84 (1H, d, J = 8.8 Hz). MS
(FAB) m/z 240 [M+H]+.
Vehicle 0.1mg/kg 0.3mg/kg 1mg/kg
5.1.10. Methyl 2-chloro-4-(3-methylpyrrolidin-1-yl)benzoate
(3j)
po
po
po
Compound 3j was prepared according to the procedure de-
scribed for 3a from 2 and 3-methylpyrrolidine. The title compound
3j (1.69 g, 84%) was obtained as cream solid. 1H NMR (300 MHz,
CDCl3) d 1.13 (3H, d, J = 6.6 Hz), 1.58–1.66 (1H, m), 2.10–2.21
(1H, m), 2.32–2.48 (1H, m), 2.89 (1H, dd, J = 9.5, 7.9 Hz), 3.27–
3.51 (3H, m), 3.85 (3H, s), 6.36 (1H, dd, J = 8.8, 2.4 Hz), 6.51 (1H,
d, J = 2.4 Hz), 7.84 (1H, d, J = 8.8 Hz). MS (FAB) m/z 254 [M+H]+.
120
100
80
60
40
20
0
compound 10j
5.1.11. Methyl 2-chloro-4-(2-methylpyrrolidin-1-yl)benzoate
(3k)
Compound 3k was prepared according to the procedure de-
scribed for 3a from 2 and 2-methylpyrrolidine. The title compound
3k (2.08 g, 78%) was obtained as colorless oil. 1H NMR (300 MHz,
CDCl3) d 1.18 (3H, d, J = 6.2 Hz), 1.69–1.80 (1H, m), 1.98–2.16
(3H, m), 3.16–3.27 (1H, m), 3.40–3.48 (1H, m), 3.85 (3H, s), 3.87–
3.97 (1H, m), 6.41 (1H, dd, J = 8.8, 2.4 Hz), 6.55 (1H, d, J = 2.6 Hz),
7.83 (1H, d, J = 8.8 Hz). MS (FAB) m/z 254 [M+H]+.
Vehicle 0.1mg/kg 0.3mg/kg 1mg/kg
po po po
Figure 3. Effects of the oral administration of compounds 10a and 10j on urinary
excretion rate in water-loaded rats. The urinary excretion rate indicates the
percentage of urine volume to loaded water volume. All assays were performed in
quadruplicate.
5.1.12. Methyl 2-chloro-4-(piperidin-1-yl)benzoate (3l)
Compound 3l was prepared according to the procedure de-
scribed for 3a from 2 and piperidine. The title compound 3l
(1.27 g, 94%) was obtained as colorless oil. 1H NMR (300 MHz,
CDCl3) d 1.63–1.70 (6H, m), 3.28–3.35 (4H, m), 3.86 (3H, s), 6.71
(1H, dd, J = 9.0, 2.6 Hz), 6.85 (1H, d, J = 2.6 Hz), 7.82 (1H, d,
J = 9.0 Hz). MS (APCI) m/z 254 [M+H]+.
3d (982 mg, 35%) was obtained as white solid. 1H NMR (300 MHz,
CDCl3) d 1.32 (6H, d, J = 6.9 Hz), 3.00–3.13 (1H, m), 3.94 (3H, s),
6.34 (1H, d, J = 2.6 Hz), 7.60 (1H, dd, J = 8.6, 2.2 Hz), 7.84 (1H, d,
J = 2.2 Hz), 7.86 (1H, d, J = 2.6 Hz), 7.96 (1H, d, J = 8.6 Hz). MS
(FAB) m/z 279 [M+H]+.
5.1.13. Methyl 4-(azepan-1-yl)-2-chlorobenzoate (3m)
5.1.6. Methyl 2-chloro-4-(3-phenyl-1H-pyrazol-1-yl)benzoate
(3e)
Compound 3m was prepared according to the procedure de-
scribed for 3a from 2 and azepane. The title compound 3m
(794 mg, 56%) was obtained as colorless oil. 1H NMR (300 MHz,
CDCl3) d 1.51–1.60 (4H, m), 1.74–1.84 (4H, m), 3.47 (4H, t,
J = 6.0 Hz), 3.85 (3H, s), 6.53 (1H, dd, J = 9.0, 2.8 Hz), 6.66 (1H, d,
J = 2.6 Hz), 7.82 (1H, d, J = 9.0 Hz). MS (EI) m/z 267 [M].
Compound 3e was prepared according to the procedure de-
scribed for 3a from 2 (1.90 g, 10.1 mmol) and 3-phenylpyrazole
(1.40 g, 10.0 mmol). The title compound 3e (2.00 g, 64%) was ob-
tained as white solid. 1H NMR (400 MHz, DMSO-d6) d 3.89 (3H,
s), 7.15 (1H, d, J = 2.7 Hz), 7.37–7.42 (1H, m), 7.45–7.51 (2H, m),
7.95–8.07 (4H, m), 8.18 (1H, d, J = 1.9 Hz), 8.77 (1H, d, J = 2.7 Hz).
MS (FAB) m/z 313 [M+H]+.
5.1.14. 2-Chloro-4-(3-methyl-1H-pyrazol-1-yl)benzoic acid (4a)
A
mixture of methyl 2-chloro-4-(3-methyl-1H-pyrazol-1-
yl)benzoate (3a; 9.25 g, 36.9 mmol) and aqueous hydrochloric acid
(6 M, 10 ml, 60 mmol) in acetic acid (10 ml) was refluxed for 13 h.
The reaction was then allowed to cool to room temperature and
subsequently poured into cold water. Resulting precipitates were
collected by filtration and dried in vacuo to give the title com-
pound 4a (8.56 g, 98%) as white solid. 1H NMR (400 MHz, CDCl3)
d 2.39 (3H, s), 6.32 (1H, d, J = 2.4 Hz), 7.63 (1H, dd, J = 8.8,
2.4 Hz), 7.86 (1H, d, J = 2.4 Hz), 7.88 (1H, d, J = 2.8 Hz), 8.12 (1H,
d, J = 8.8 Hz). MS (FAB) m/z 235 [MꢀH]ꢀ.
5.1.7. Methyl 2-chloro-4-(3,5-dimethyl-1H-pyrazol-1-
yl)benzoate (3f)
Compound 3f was prepared according to the procedure de-
scribed for 3a from 2 (1.90 g, 10.1 mmol) and 3,5-dimethylpyrazole
(960 mg, 10.0 mmol). The title compound 3f (500 mg, 19%) was ob-
tained as colorless oil. 1H NMR (300 MHz, DMSO- d6) d 2.19 (3H, s),
2.40 (3H, s), 3.88 (3H, s), 6.15 (1H, s), 7.65 (1H, dd, J = 8.4, 2.0 Hz),
7.76 (1H, d, J = 2.0 Hz), 7.95 (1H, d, J = 8.4 Hz). MS (FAB) m/z 265
[M+H]+.
5.1.15. 2-Chloro-4-(1H-pyrazol-1-yl)benzoic acid (4b)
5.1.8. Methyl 2-chloro-4-(2H-indazol-2-yl)benzoate (3h)
Compound 3h was prepared according to the procedure de-
scribed for 3a from 2 (1.90 g, 10.1 mmol) and indazole (1.20 g,
10.0 mmol). The title compound 3h (600 mg, 21%) was obtained
A mixture of methyl 2-chloro-4-(1H-pyrazol-1-yl)benzoate (3b;
1.75 g, 7.39 mmol) and aqueous sodium hydroxide (5 M, 5 ml,
25 mmol) in methanol (20 ml) was refluxed for 45 min. The reac-
tion was then allowed to cool to room temperature and subse-

9530
I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
quently poured into aqueous hydrochloride. Resulting precipitates
were collected by filtration and dried in vacuo to give the title com-
pound 4b (1.59 g, 96%) as white solid. 1H NMR (300 MHz, DMSO-
d6) d 6.61–6.64 (1H, m), 7.84 (1H, d, J = 1.5 Hz), 7.93–7.97 (2H,
m), 8.06 (1H, d, J = 1.8 Hz), 8.68 (1H, d, J = 2.8 Hz). MS (FAB) m/z
223 [M+H]+.
(1H, dd, J = 8.6, 2.4 Hz), 6.51 (1H, d, J = 2.2 Hz), 7.76 (1H, d,
J = 8.8 Hz), 12.16–12.54 (1H, br). MS (FAB) m/z 240 [M+H]+.
5.1.23. 2-Chloro-4-(2-methylpyrrolidin-1-yl)benzoic acid (4k)
Compound 4k was prepared according to the procedure de-
scribed for 4b from 3k. The title compound 4k (1.69 g, 89%) was
obtained as white solid. 1H NMR (300 MHz, DMSO-d6) d 1.10 (3H,
d, J = 6.2 Hz), 1.63–1.75 (1H, m), 1.91–2.11 (3H, m), 3.11–3.21
(1H, m), 3.35–3.44 (1H, m), 3.92–4.02 (1H, m), 6.50–6.56 (2H,
m), 7.73–7.79 (1H, m), 12.24–12.40 (1H, br). MS (FAB) m/z 240
[M+H]+.
5.1.16. 2-Chloro-4-(3-ethyl-1H-pyrazol-1-yl)benzoic acid (4c)
Compound 4c was prepared according to the procedure de-
scribed for 4b from 3c. The title compound 4c (589 mg, 90%) was
obtained as cream solid. 1H NMR (300 MHz, DMSO-d6) d 1.23
(3H, t, J = 7.7 Hz), 2.66 (2H, q, J = 7.7 Hz), 6.46 (1H, d, J = 2.2 Hz),
7.87 (1H, dd, J = 8.8, 1.5 Hz), 7.94 (1H, d, J = 8.6 Hz), 7.99 (1H, d,
J = 1.7 Hz), 8.55 (1H, d, J = 2.4 Hz). MS (FAB) m/z 251 [M+H]+.
5.1.24. 2-Chloro-4-(piperidin-1-yl)benzoic acid (4l)
Compound 4l was prepared according to the procedure de-
scribed for 4b from 3l. The title compound 4l (997 mg, 88%) was
obtained as white solid. 1H NMR (300 MHz, DMSO-d6) d 1.53–
1.62 (6H, m), 3.30–3.36 (4H, m), 6.84–6.94 (2H, m), 7.74 (1H, d,
J = 8.8 Hz), 12.45–12.52 (1H, br).
5.1.17. 2-Chloro-4-(3-isopropyl-1H-pyrazol-1-yl)benzoic acid
(4d)
Compound 4d was prepared according to the procedure de-
scribed for 4b from 3d. The title compound 4d (873 mg, 94%)
was obtained as white solid. 1H NMR (300 MHz, DMSO-d6) d 1.26
(6H, d, J = 6.8 Hz), 2.93–3.04 (1H, m), 6.48 (1H, s), 7.87 (1H, d,
J = 8.6 Hz), 7.92–8.01 (2H, m), 8.54 (1H, s), 13.20–13.40 (1H, br).
MS (FAB) m/z 265 [M+H]+.
5.1.25. 4-(Azepan-1-yl)-2-chlorobenzoic acid (4m)
Compound 4m was prepared according to the procedure de-
scribed for 4b from 3m. The title compound 4m (676 mg, 90%)
was obtained as white solid. 1H NMR (300 MHz, DMSO-d6) d
1.42–1.51 (4H, m), 1.64–1.75 (4H, m), 3.49 (4H, t, J = 5.9 Hz),
6.64–6.70 (2H, m), 7.74 (1H, d, J = 9.3 Hz), 12.27–12.38 (1H,
br).
5.1.18. 2-Chloro-4-(3-phenyl-1H-pyrazol-1-yl)benzoic acid (4e)
Compound 4e was prepared according to the procedure de-
scribed for 4b from 3e (1.90 g, 6.08 mmol). The title compound
4e (1.80 g, 99%) was obtained as white solid. 1H NMR (300 MHz,
DMSO-d6) d 7.14 (1H, d, J = 2.8 Hz), 7.36–7.52 (3H, m), 7.94–8.03
(4H, m), 8.14 (1H, s), 8.76 (1H, d, J = 2.8 Hz), 13.30–13.44 (1H,
br). MS (FAB) m/z 297 [MꢀH]ꢀ.
5.1.26. 2-Chloro-4-(5-methyl-1H-pyrazol-1-yl)benzonitrile (6)
To a suspension of sodium hydride (60% dispersion in mineral
oil, 1.54 g, 38.5 mmol) in N,N-dimethylformamide (DMF) (30 ml)
was added 3-methylpyrazole (3.06 ml, 38.0 mmol), and the mix-
ture was stirred at -20 °C for 1 h. Then, a solution of 2-chloro-4-
fluorobenzonitrile (5; 5.00 g, 32.1 mmol) in DMF (5 ml) was slowly
added to the reaction mixture, and that was stirred overnight at
room temperature. Water was added to the mixture, and resulting
precipitates were collected by filtration. The crude product was
purified by silica gel column chromatography (n-hexane/ethyl ace-
tate = 10/1–9/1) and subsequent recrystallization from Et2O/n-
hexane to give the title compound 6 (690 mg, 10%) as white solid.
1H NMR (300 MHz, DMSO-d6) d 2.46 (3H, s), 6.38 (1H, s), 7.69 (1H,
s), 7.78 (1H, dd, J = 8.4, 2.0 Hz), 7.99 (1H, d, J = 1.8 Hz), 8.12 (1H, d,
J = 8.4 Hz). MS (FAB) m/z 218 [M+H]+.
5.1.19. 2-Chloro-4-(3,5-dimethyl-1H-pyrazol-1-yl)benzoic acid
(4f)
Compound 4f was prepared according to the procedure de-
scribed for 4b from 3f (500 mg, 1.89 mmol). The title compound
4f (440 g, 93%) was obtained as white solid. 1H NMR (300 MHz,
DMSO-d6) d 2.19 (3H, s), 2.39 (3H, s), 6.14 (1H, s), 7.43 (1H, dd,
J = 8.4, 2.2 Hz), 7.72 (1H, d, J = 2.0 Hz), 7.93 (1H, d, J = 8.4 Hz),
13.36–13.54 (1H, br). MS (FAB) m/z 249 [MꢀH]ꢀ.
5.1.20. 2-Chloro-4-(2H-indazol-2-yl)benzoic acid (4h)
Compound 4h was prepared according to the procedure de-
scribed for 4b from 3h (600 mg, 2.09 mmol). The title compound
4 h (580 mg, quantitative yield) was obtained as white solid. 1H
NMR (300 MHz, DMSO-d6) d 7.13 (1H, ddd, J = 8.4, 6.6, 0.8 Hz),
7.35 (1H, ddd, J = 8.8, 6.6, 1.1 Hz), 7.72 (1H, dd, J = 8.8, 1.1 Hz),
7.78 (1H, d, J = 8.4 Hz), 8.03 (1H, d, J = 8.6 Hz), 8.20 (1H, dd,
J = 8.6, 2.2 Hz), 8.33 (1H, d, J = 2.2 Hz) 9.28 (1H, d, J = 0.9 Hz),
13.50–13.62 (1H, br). MS (FAB) m/z 271 [MꢀH]ꢀ.
5.1.27. 2-Chloro-4-(5-methyl-1H-pyrazol-1-yl)benzamide (7)
To a mixture of 2-chloro-4-(5-methyl-1H-pyrazol-1-yl)benzo-
nitrile (6; 650 mg, 2.99 mmol) and potassium carbonate (69 mg,
0.50 mmol) in DMSO (5 ml) was added hydrogen peroxide (30%,
0.42 ml, 3.75 mmol), and the mixture was stirred at room temper-
ature for 30 min. Ice-cold water was added to the mixture, and
resulting precipitates were collected by filtration, and subse-
quently dried in vacuo to give the title compound 7 (520 mg,
74%) as white solid. 1H NMR (300 MHz, DMSO-d6) d 2.39 (3H, s),
6.32 (1H, s), 7.58 (2H, s), 7.61 (1H, d, J = 1.5 Hz), 7.65–7.70 (2H,
m), 7.94–8.00 (1H, br). MS (FAB) m/z 236 [M+H]+.
5.1.21. 2-Chloro-4-(pyrrolidin-1-yl)benzoic acid (4i)
Compound 4i was prepared according to the procedure de-
scribed for 4b from 3i. The title compound 4i (3.39 g, 60%) was ob-
tained as white solid. 1H NMR (300 MHz, DMSO-d6) d 1.92–1.99
(4H, m), 3.25–3.31 (4H, m), 6.50 (1H, dd, J = 8.6, 2.4 Hz),
6.53 (1H, d, J = 2.4 Hz), 7.76 (1H, d, J = 8.6 Hz). MS (FAB) m/z 226
[M+H]+.
5.1.28. 2-Chloro-4-(5-methyl-1H-pyrazol-1-yl)benzoic acid (4g)
To a flask with water (4 ml) at 0 °C were added sulfuric acid
(10 ml), 2-chloro-4-(5-methyl-1H-pyrazol-1-yl)benzamide (7;
500 mg, 2.12 mmol), and sodium nitrite (732 mg, 10.6 mmol)
sequentially, and the mixture was stirred at room temperature
for 3 h. Ice-cold water was added to the mixture, and resulting pre-
cipitates were collected by filtration, and subsequently dried in va-
cuo to give the title compound 4g (450 mg, 90%) as white solid. 1H
5.1.22. 2-Chloro-4-(3-methylpyrrolidin-1-yl)benzoic acid (4j)
Compound 4j was prepared according to the procedure de-
scribed for 4b from 3j. The title compound 4j (1.53 g, quantitative
yield) was obtained as white solid. 1H NMR (300 MHz, DMSO-d6) d
1.07 (3H, d, J = 6.6 Hz), 1.51–1.66 (1H, m), 2.04–2.15 (1H, m), 2.28–
2.42 (1H, m), 2.84 (1H, dd, J = 9.7, 7.7 Hz), 3.22–3.50 (3H, m), 6.47

I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
9531
NMR (300 MHz, DMSO-d6) d 2.43 (3H, s), 6.34 (1H, s), 7.62–7.67
(2H, m), 7.76 (1H, d, J = 2.0 Hz), 7.95 (1H, d, J = 8.4 Hz). MS (FAB)
m/z 235 [MꢀH]ꢀ.
5.1.33. Methyl (2Z)-{1-[2-chloro-4-(3-phenyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8e)
Compound 8e was prepared according to the procedure de-
scribed for 8a from 4e. The title compound 8e (200 mg, 37%) was
obtained as white solid. 1H NMR (300 MHz, DMSO-d6): 3.34 (3H,
s), 6.62 (1H, s), 7.04–7.11 (2H, m), 7.19–7.3 (2H, m), 7.33–7.48
(5H, m), 7.75 (1H, d, J = 7.5 Hz), 7.87–7.93 (2H, m), 7.96 (1H, s),
8.54–8.62 (1H, m). MS (FAB) m/z 534 [M+H]+.
5.1.29. Methyl (2Z)-{1-[2-chloro-4-(3-methyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8a)
A
mixture of 2-chloro-4-(3-methyl-1H-pyrazol-1-yl)benzoic
acid (4a; 8.56 g, 36.2 mmol), DMF (several drops), and thionyl chlo-
ride (3.98 ml, 54.6 mmol) in tetrahydrofuran (THF) (100 ml) was
stirred at room temperature for 1 h. The reaction mixture was
evaporated and azeotroped with toluene. To a solution of this res-
idue in pyridine (50 ml) was added a solution of methyl (2Z)-(4,4-
difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetate9
(9.17 g, 36.2 mmol) in THF (25 ml), and the mixture was stirred at
room temperature for 1 h. The solvent was evaporated, and the res-
idue was partitioned between water and ethyl acetate. The organic
phase was washed with water and brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (CHCl3/methanol = 20:1) to
give the title compound 8a (10.0 g, 59%) as pale yellow amorphous
solid. 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.40–2.60 (2H,
m), 3.20–3.30 (1H, br), 3.78 (3H, s), 4.75–4.85 (1H, br), 6.33 (1H,
d, J = 2.4 Hz), 6.59 (1H, s), 6.90–7.10 (1H, br), 7.06 (1H, d, J = 7.3
Hz), 7.22 (1H, t, J = 7.3 Hz), 7.27 (1H, t, J = 7.3 Hz), 7.35 (1H, d,
J = 7.3 Hz), 7.59 (1H, d, J = 8.3 Hz), 7.81 (1H, s), 8.38 (1H, d, J = 1.9
Hz). MS (FAB) m/z 472 [M+H]+.
5.1.34. Methyl (2Z)-{1-[2-chloro-4-(3,5-dimethyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8f)
Compound 8f was prepared according to the procedure de-
scribed for 8a from 4f. The title compound 8f (400 mg, 47%) was
obtained as white solid. 1H NMR (300 MHz, CDCl3): 2.24 (3H, s),
2.25 (3H, s), 2.10–2.80 (2H, m), 3.25–3.45 (1H, br), 3.85 (3H, s),
4.85–5.15 (1H, br), 5.97 (1H, s), 6.12 (1H, s), 6.90–7.31 (6H, m),
7.43 (1H, s). MS (FAB) m/z 486 [M+H]+.
5.1.35. Methyl (2Z)-{1-[2-chloro-4-(5-methyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8g)
Compound 8g was prepared according to the procedure de-
scribed for 8a from 4g. The title compound 8g (683 mg, 85%) was
obtained as colorless amorphous solid. 1H NMR (300 MHz,
DMSO-d6): 2.29 (3H, s), 2.45–2.65 (2H, m), 3.30–3.40 (1H, m),
3.77 (3H, s), 4.70–4.90 (1H, m), 6.27 (1H, s), 6.58 (1H, s), 7.06–
7.13 (1H, m), 7.20–7.39 (5H, m), 7.54–7.61 (2H, m). MS (FAB) m/z
472 [M+H]+.
5.1.30. Methyl (2Z)-{1-[2-chloro-4-(1H-pyrazol-1-yl)benzoyl]-
4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8b)
Compound 8b was prepared according to the procedure de-
scribed for 8a from 4b. The title compound 8b (570 mg, 63%) was
obtained as pale yellow amorphous solid. 1H NMR (400 MHz,
CDCl3) d 2.20–2.80 (2H, m), 3.20–3.40 (1H, br), 3.86 (3H, s), 4.90–
5.15 (1H, br), 6.13 (1H, s), 6.45 (1H, t, J = 2.8 Hz), 6.95–7.01 (1H,
m), 7.04 (1H, d, J = 7.6 Hz), 7.12 (1H, td, J = 7.6, 1.6 Hz), 7.20 (1H,
t, J = 7.6 Hz), 7.30 (1H, dd, J = 7.6, 1.6 Hz), 7.32–7.37 (1H, m),
7.66–7.70 (2H, m), 7.82 (1H, d, J = 2.8 Hz). MS (FAB) m/z 458
[M+H]+.
5.1.36. Methyl (2Z)-{1-[2-chloro-4-(2H-indazol-2-yl)benzoyl]-
4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8h)
Compound 8h was prepared according to the procedure de-
scribed for 8a from 4h. The title compound 8h (300 mg, 29%)
was obtained as pale yellow amorphous solid. 1H NMR (300 MHz,
CDCl3): 2.30–2.80 (2H, m), 3.20–3.45 (1H, m), 3.87 (3H, s), 4.85–
5.20 (1H, m), 6.15 (1H, s), 7.03–7.23 (5H, m), 7.28–7.34 (2H, m),
7.59 (1H, d, J = 8.3 Hz), 7.65 (1H, d, J = 8.4 Hz), 7.70 (1H, d,
J = 9.0 Hz), 7.91 (1H, d, J = 1.8 Hz), 8.31 (1H, s). MS (FAB) m/z 508
[M+H]+.
5.1.31. Methyl (2Z)-{1-[2-chloro-4-(3-ethyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8c)
Compound 8c was prepared according to the procedure de-
scribed for 8a from 4c. The title compound 8c (785 mg, 83%) was
obtained as colorless amorphous solid. 1H NMR (300 MHz, CDCl3):
1.26 (3H, t, J = 7.6 Hz), 2.25–2.80 (2H, m), 2.69 (2H, q, J = 7.6 Hz),
3.20–3.50 (1H, br), 3.86 (3H, s), 4.80–5.20 (1H, br), 6.12 (1H, s),
6.26 (1H, d, J = 2.6 Hz), 6.92–6.97 (1H, m), 7.03 (1H, d, J = 7.5 Hz),
7.11 (1H, dt, J = 1.5, 7.6 Hz), 7.19 (1H, dt, J = 1.3, 7.4 Hz), 7.27–
7.33 (2H, m), 7.63 (1H, d, J = 2.0 Hz), 7.72 (1H, d, J = 2.6 Hz). MS
(FAB) m/z 486 [M+H]+.
5.1.37. Methyl (2Z)-{1-[2-chloro-4-(pyrrolidin-1-yl)benzoyl]-
4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8i)
Compound 8i was prepared according to the procedure de-
scribed for 8a from 4i. The title compound 8i (920 mg, 92%) was
obtained as yellow amorphous solid. 1H NMR (300 MHz, CDCl3):
1.93–2.07 (6H, m), 2.54–2.61 (2H, br), 3.13–3.23 (4H, br), 3.84
(3H, s), 6.10–6.16 (2H, m), 6.36–6.44 (2H, m), 6.62–6.71 (1H, m),
6.93–7.02 (1H, m), 7.12–7.23 (2H, m),. MS (FAB) m/z 461 [M+H]+.
5.1.32. Methyl (2Z)-{1-[2-chloro-4-(3-isopropyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8d)
5.1.38. Methyl (2Z)-{1-[2-chloro-4-(3-methylpyrrolidin-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8j)
Compound 8d was prepared according to the procedure de-
scribed for 8a from 4d. The title compound 8d (971 mg, 77%)
was obtained as colorless amorphous solid. 1H NMR (300 MHz,
CDCl3): 1.28 (6H, d, J = 7.0 Hz), 2.30–2.80 (2H, m), 2.95–3.10 (1H,
m), 3.20–3.50 (1H, br), 3.86 (3H, s), 4.80–5.15 (1H, br), 6.13 (1H,
s), 6.27 (1H, d, J = 2.6 Hz), 6.9–6.97 (1H, m), 7.04 (1H, d,
J = 7.7 Hz), 7.11 (1H, dt, J = 1.5 ,7.7 Hz), 7.19 (1H, dt, J = 1.3
,7.3 Hz), 7.27–7.34 (2H, m), 7.64 (1H, d, J = 2.0 Hz), 7.71 (1H, d,
J = 2.6 Hz). MS (FAB) m/z 500 [M+H]+.
Compound 8j was prepared according to the procedure de-
scribed for 8a from 4j. The title compound 8j (414 mg, 44%) was
obtained as yellow amorphous solid. 1H NMR (400 MHz, CDCl3):
1.09 (3H, d, J = 6.8 Hz), 1.50–1.70 (3H, m), 2.02–2.15 (1H, m),
2.28–2.80 (4H, m), 3.20–3.35 (3H, m), 3.84 (3H, s), 6.00–6.08 (1H,
m), 6.12 (1H, s), 6.33–6.39 (1H, m), 6.61–6.68 (1H, m), 6.94–7.01
(1H, m), 7.10–7.21 (2H, m), 7.26–7.31 (1H, m). MS (FAB) m/z 475
[M+H]+.

9532
I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
5.1.39. Methyl (2Z)-{1-[2-chloro-4-(2-methylpyrrolidin-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8k)
form. The organic phase was washed with water and brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
crystallized (n-hexane/ethyl acetate) and subsequently recrystal-
lized (n-hexane/ethyl acetate) to give the title compound 9a
(9.00 g, 99%) as colorless crystals. Mp: 252–254 °C; 1H NMR
(400 MHz, DMSO-d6) d 2.22 (3H, s), 2.40–2.65 (2H, br), 3.10–3.25
(1H, br), 4.73–4.91 (1H, br), 6.33 (1H, d, J = 2.4 Hz), 6.50 (1H, s),
7.04 (1H, d, J = 7.3 Hz), 6.93–7.10 (1H, br), 7.20 (1H, dt, J = 1.5,
7.3 Hz), 7.26 (1H, t, J = 7.3 Hz), 7.32 (1H, d, J = 7.3 Hz), 7.60 (1H,
d, J = 8.3 Hz), 7.81 (1H, s), 8.38 (1H, d, J = 2.4 Hz), 13.15–13.30
(1H, br). MS (FAB) m/z 458 [M+H]+.
Compound 8k was prepared according to the procedure de-
scribed for 8a from 4k. The title compound 8k (413 mg, 40%)
was obtained as yellow amorphous solid. 1H NMR (400 MHz,
CDCl3): 1.06–1.12 (3H, m), 1.53–1.72 (3H, m), 1.91–2.09 (4H, m),
2.40–2.58 (1H, m), 3.00–3.11 (1H, m), 3.23–3.34 (1H, m), 3.71–
3.81 (1H, m), 3.84 (3H, s), 6.08–6.17 (2H, m), 6.37–6.45 (1H, m),
6.58–6.69 (1H, m), 6.94–7.03 (1H, m), 7.09–7.23 (2H, m), 7.26–
7.33 (1H, m). MS (FAB) m/z 475 [M+H]+.
5.1.40. Methyl (2Z)-(1-{2-chloro-4-[3-(dimethylamino)pyrro-
lidin-1-yl]benzoyl}-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-ben-
zazepin-5-ylidene)acetate (8n)
5.1.43. (2Z)-2-{1-[2-Chloro-4-(3-methyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10a)
A mixture of methyl 2-chloro-4-fluorobenzoate (2; 1.20 g,
6.36 mmol), 3-(dimethylamino)pyrrolidine (880 mg, 7.68 mmol),
and potassium carbonate (1.30 g, 9.24 mmol) in NMP (8 ml) was
heated overnight at 120 °C. The reaction was then allowed to cool
to room temperature and partitioned between water and ethyl
acetate. The organic phase was washed with water and brine, dried
over sodium sulfate, filtered, and concentrated in vacuo. A mixture
of this residue and aqueous sodium hydroxide (5 M, 4 ml,
20 mmol) in methanol (20 ml) was refluxed for 1 h. The reaction
was then allowed to cool to room temperature and subsequently
poured into aqueous hydrochloride. Resulting precipitates were
collected by filtration and dried in vacuo to give a hydrochloride
salt of compound 4n (1.95 g, quantitative yield) as brown solid. A
mixture of 4n (730 mg, 2.40 mmol) and thionyl chloride (5 ml)
was stirred at 80 °C for 45 min. The reaction mixture was evapo-
rated and azeotroped with toluene. To a solution of this residue
in pyridine (10 ml) was added methyl (2Z)-(4,4-difluoro-1,2,3,4-
A
mixture of (2Z)-{1-[2-chloro-4-(3-methyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetic acid (9a; 770 mg, 1.68 mmol), 2-(aminomethyl)pyr-
idine (0.19 ml, 1.85 mmol), 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (WSCD) (490 mg, 2.52 mmol),
and 1-hydroxy-1H-benzotriazole (HOBt) (310 mg, 2.52 mmol) in
DMF was stirred overnight at room temperature. The reaction mix-
ture was partitioned between water and ethyl acetate, then the or-
ganic phase was washed with water and brine, dried over MgSO4,
filtered, and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (CHCl3/methanol = 25:1) and
subsequently treated with hydrochloride in ethyl acetate, then
evaporated. The residue was crystallized from ethanol and col-
lected by filtration to give the title compound 10a (687 mg, 70%)
as colorless crystals. Mp: 214–217 °C; 1H NMR (400 MHz, DMSO-
d6) d 2.22 (3H, s), 2.40–2.50 (1H, br), 2.67–2.89 (1H, br), 3.11–
3.23 (1H, br), 4.73 (2H, d, J = 5.4 Hz), 4.76–4.90 (1H, br), 6.34 (1H,
d, J = 2.5 Hz), 6.41(1H, s), 6.99 (1H, d, J = 7.8 Hz), 7.19(1H, t,
J = 7.8 Hz), 7.26(1H, t, J = 7.8 Hz), 7.33(1H, d, J = 6.8 Hz), 7.61 (1H,
d, J = 8.3 Hz), 7.79–7.88 (3H, m) ,8.35–8.45 (2H, m) ,8.79 (1H, d,
J = 4.8 Hz), 9.30 (1H, s). MS (FAB) m/z 548 [M+H]+. Anal. Calcd for
C29H24ClF2N5O2ꢁHClꢁ0.1H2O: C, 59.41; H, 4.33; N, 11.95; Cl, 12.09;
F, 6.48. Found: C, 59.19; H, 4.40; N, 11.85; Cl, 12.06; F, 6.40.
tetrahydro-5H-1-benzazepin-5-ylidene)acetate9
(500 mg,
1.97 mmol) and the mixture was stirred at 60 °C for 3 h. The sol-
vent was evaporated, and the residue was partitioned between
aqueous citric acid and CHCl3. The organic phase was washed with
water and brine, dried over sodium sulfate, filtered, and concen-
trated in vacuo. The crude product was purified by silica gel col-
umn chromatography (CHCl3/methanol = 30:1) to give the title
compound 8n (791 mg, 80%) as brown amorphous solid. 1H NMR
(400 MHz, CDCl3): 1.83–1.89 (1H, m), 2.12–2.29 (9H, m), 2.35–
2.65 (1H, m), 2.72–2.83 (1H, m), 2.98–3.07 (1H, m), 3.16–3.27
(1H, m), 3.28–3.41 (2H, m), 3.80–3.86 (4H, m), 6.02–6.13 (2H,
m), 6.35–6.39 (1H, m), 6.63–6.70 (1H, m), 6.92–6.70 (1H, m),
7.11–7.32 (3H, m). MS (FAB) m/z 504 [M+H]+.
5.1.44. (2Z)-2-{1-[2-Chloro-4-(1H-pyrazol-1-yl)benzoyl]-4,4-
difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene}-N-
(pyridin-2-ylmethyl)acetamide hydrochloride (10b)
Methyl (2Z)-{1-[2-chloro-4-(1H-pyrazol-1-yl)benzoyl]-4,4-di-
fluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene}acetate (8b;
560 mg, 1.22 mmol) in THF and methanol was treated with sodium
hydroxide (1.0 M, 3.0 ml, 3.0 mmol) at room temperature for 4 h.
The reaction was partitioned between aqueous hydrochloride
and chloroform. The organic phase was washed with water and
brine, dried over Na2SO4, filtered, and concentrated in vacuo to
give the crude product of 9b. A mixture of foregoing crude product
of 9b, 2-(aminomethyl)pyridine (0.18 ml, 1.93 mmol), WSCD
(390 mg, 1.93 mmol), and HOBt (240 mg, 1.93 mmol) in DMF
(10 ml) was stirred overnight at room temperature. The reaction
mixture was partitioned between water and ethyl acetate, then
the organic phase was washed with water and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (CHCl3/methanol = 60:1)
and subsequently treated with hydrochloride in ethyl acetate, then
evaporated. Resulting solid was crystallized from ethanol and col-
lected by filtration to give the title compound 10b (547 mg, 79%) as
colorless crystals. Mp: 218–220 °C. 1H NMR (400 MHz, DMSO-d6):
d 2.31–2.48 (1H, br), 2.64–2.90 (1H, br), 3.05–3.24 (1H, br), 4.73
(2H, d, J = 5.3 Hz), 4.76–4.90 (1H, br), 6.55 (1H, t, J = 1.5 Hz), 6.56
(1H, s), 7.01 (1H, d, J = 7.3 Hz), 7.05–7.17 (1H, br), 7.20 (1H, dt,
J = 1.5, 7.3 Hz), 7.26 (1H, t, J = 7.3 Hz), 7.32–7.37 (1H, m), 7.68
(1H, dd, J = 1.5, 8.3 Hz), 7.74–7.77 (1H, m), 7.79–7.87 (2H, m),
5.1.41. Methyl (2Z)-{1-[2-chloro-4-(4-methylpiperazin-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetate (8o)
Compound 8o was prepared according to the procedure de-
scribed for 8n. The title compound 8o (614 mg, 57%) was obtained
as pale brown amorphous solid. 1H NMR (400 MHz, CDCl3): 2.28–
2.33 (4H, m), 2.45–2.51 (5H, m), 3.10–3.30 (5H, m), 3.81–3.87
(4H, m), 6.10 (1H, s), 6.46 (1H, d, J = 7.2 Hz), 6.68–6.75 (2H, m),
6.98 (1H, d, J = 8.0 Hz), 7.12 (1H, t, J = 7.2 Hz), 7.18 (1H, t,
J = 7.8 Hz), 7.26–7.29 (1H, m). MS (FAB) m/z 490 [M+H]+.
5.1.42. (2Z)-{1-[2-Chloro-4-(3-methyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}acetic acid (9a)
Methyl
(2Z)-{1-[2-chloro-4-(3-methyl-1H-pyrazol-1-yl)ben-
zoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene}-
acetate (8a; 9.37 g, 19.9 mmol) in THF (50 ml) and methanol
(20 ml) was treated with sodium hydroxide (1.0 M, 20.0 ml,
20.0 mmol) at 50 °C for 12 h. The reaction was then allowed to cool
to room temperature and partitioned between water and chloro-

I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
9533
7.91 (1H, s), 8.43 (1H, t, J = 7.9 Hz), 8.52 (1H, d, J = 2.4 Hz), 8.80 (1H,
d, J = 5.4 Hz), 9.31 (1H, s). MS (FAB) m/z 534 [M+H]+. Anal. Calcd for
C28H22ClF2N5O2ꢁHCl: C, 58.96; H, 4.06; N, 12.28; Cl, 12.43; F, 6.66.
Found: C, 58.95; H, 4.02; N, 12.28; Cl, 12.28; F, 6.85.
5.1.49. (2Z)-2-{1-[2-Chloro-4-(5-methyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10g)
Compound 10g was prepared according to the procedure de-
scribed for 10b. The title compound 10g (153 mg, 79%) was ob-
tained as colorless crystals. Mp: 220–222 °C. 1H NMR (400 MHz,
DMSO-d6): d 2.28 (3H, s), 2.20–2.42 (1H, br), 2.69–2.89 (1H, br),
3.08–3.24 (1H, br), 4.64 (2H, d, J = 4.9 Hz), 4.75–4.97 (1H, br),
6.27 (1H, d, J = 1.2 Hz), 6.34 (1H, s), 7.02 (1H, d, J = 7.3 Hz), 7.05–
7.16 (1H, br), 7.20 (1H, t, J = 7.3 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.31–
7.37 (2H, m), 7.56 (1H, d, J = 1.2 Hz), 7.60 (1H, s), 7.63–7.74 (2H,
m), 8.19–8.31 (1H, m), 7.73 (1H, d, J = 4.9 Hz), 9.19 (1H, s). MS
(FAB) m/z 548 [M+H]+. Anal. Calcd for C29H24ClF2N5O2ꢁHClꢁ0.2H2O:
C, 59.23; H, 4.35; N, 11.91; Cl, 12.06; F, 6.46. Found: C, 59.19; H,
4.36; N, 11.86; Cl, 12.03; F, 6.20.
5.1.45. (2Z)-2-{1-[2-Chloro-4-(3-ethyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10c)
Compound 10c was prepared according to the procedure de-
scribed for 10b. The title compound 10c (363 mg, 71%) was ob-
tained as colorless crystals. Mp: 199–202 °C. 1H NMR (400 MHz,
DMSO-d6): d 1.19 (3H, t, J = 7.6 Hz), 2.25–2.40 (1H, br), 2.60 (2H,
q, J = 7.6 Hz), 2.70–2.85 (1H, br), 3.10–3.25 (1H, br), 4.74 (2H, d,
J = 5.6 Hz), 4.75–4.90 (1H, br), 6.38 (1H, d, J = 2.4 Hz), 6.57 (1H, s),
6.99 (1H, d, J = 7.6 Hz), 7.01–7.15 (1H, br), 7.19 (1H, dt, J = 1.3,
8.0 Hz), 7.26 (1H, t, J = 7.4 Hz), 7.33 (1H, dd, J = 1.2, 8.0 Hz), 7.62
(1H, d, J = 8.8 Hz), 7.79 - 7.88 (3H, m), 8.39 (1H, d, J = 2.4 Hz),
8.43 (1H, t, J = 7.8 Hz), 8.80 (1H, d, J = 5.6 Hz), 9.30 (1H, s). MS
(FAB) m/z 562 [M]+. Anal. Calcd for C30H26ClF2N5O2.HCl: C, 60.21;
H, 4.55; N, 11.70; Cl, 11.85; F, 6.35. Found: C, 60.07; H, 4.41; N,
11.67; Cl, 11.77; F, 6.31.
5.1.50. (2Z)-2-{1-[2-Chloro-4-(2H-indazol-2-yl)benzoyl]-4,4-
difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene}-N-
(pyridin-2-ylmethyl)acetamide hydrochloride (10h)
Compound 10h was prepared according to the procedure de-
scribed for 10b. The title compound 10h (220 mg, 75%) was ob-
tained as colorless crystals. Mp: 158–160 °C. 1H NMR (300 MHz,
DMSO-d6): d 2.25–2.55 (1H, m), 3.10–3.30 (1H, m), 3.95–4.05
(1H, m), 4.69 (2H, d, J = 5.5 Hz), 4.70–4.95 (1H, m), 6.58 (1H,
s), 7.02–7.38 (7H, m), 7.57–7.81 (4H, m), 7.94 (1H, d,
J = 8.6 Hz), 8.19 (1H, s), 8.33 (1H, t, J = 7.8 Hz), 8.76 (1H, d,
J = 5.5 Hz), 9.12 (1H, s), 9.20–9.31 (1H, m). MS (FAB) m/z 584
[M]+. Anal. Calcd for C32H24ClF2N5O2ꢁ0.85HClꢁH2O: C, 60.72; H,
4.28; N, 11.06; Cl, 10.36; F, 6.00. Found: C, 60.62; H, 4.25; N,
10.78; Cl, 10.20; F, 5.75.
5.1.46. (2Z)-2-{1-[2-Chloro-4-(3-isopropyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10d)
Compound 10d was prepared according to the procedure de-
scribed for 10b. The title compound 10d (424 mg, 74%) was ob-
tained as colorless crystals. Mp: 205–208 °C. 1H NMR (400 MHz,
DMSO-d6): d 1.21 (6H, d, J = 6.8 Hz), 2.25–2.40 (1H, br), 2.65–2.80
(1H, br), 2.85–3.02 (1H, m), 3.10–3.25 (1H, br), 4.74 (2H, d,
J = 5.2 Hz), 4.75–4.90 (1H, br), 6.41 (1H, d, J = 2.4 Hz), 6.58 (1H, s),
6.99 (1H, d, J = 7.6 Hz), 7.00–7.15 (1H, br), 7.19 (1H, t, J = 7.6 Hz),
7.26 (1H, t, J = 7.4 Hz), 7.34 (1H, d, J = 8.0 Hz), 7.62 (1H, d,
J = 8.4 Hz), 7.79–7.88 (3H, m), 8.39 (1H, d, J = 2.4 Hz), 8.44 (1H, t,
J = 7.6 Hz), 8.81 (1H, d, J = 5.2 Hz), 9.31 (1H, s). MS (FAB) m/z 576
[M]+. Anal. Calcd for C31H28ClF2N5O2ꢁHCl: C, 60.79; H, 4.77; N,
11.43; Cl, 11.58; F, 6.20. Found: C, 60.50; H, 4.61; N, 11.73; Cl,
11.60; F, 6.04.
5.1.51. (2Z)-2-[1-(2-Chloro-4-pyrrolidin-1-ylbenzoyl)-4,4-
difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene]-N-
(pyridin-2-ylmethyl)acetamide hydrochloride (10i)
Compound 10i was prepared according to the procedure de-
scribed for 10b. The title compound 10i (456 mg, 40%) was ob-
tained as pale yellow crystals. Mp: 194–196 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.82–1.69 (4H, br), 2.41–2.48 (1H, br),
2.53–2.60 (1H, br), 3.08–3.17 (5H, br), 4.72 (2H, d, J = 5.4 Hz),
4.75–4.81 (1H, br), 6.17–6.25 (1H, br), 6.38–6.44 (1H, br), 6.48
(1H, s), 6.67–6.75 (1H, br), 6.84–6.91 (1H, br), 7.15–7.35 (3H, m),
7.79–7.86 (2H, m), 8.43 (1H, t, J = 7.8 Hz), 8.79 (1H, d, J = 4.9 Hz),
9.25 (1H, s). MS (FAB) m/z 537 [M]+. Anal. Calcd for
C29H27ClF2N4O2ꢁHCl: C, 60.74; H, 4.92; N, 9.77; Cl, 12.36; F, 6.63.
Found: C, 60.42; H, 4.78; N, 9.81; Cl, 12.34; F, 6.36.
5.1.47. (2Z)-2-{1-[2-Chloro-4-(3-phenyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide (10e)
Compound 10e was prepared according to the procedure de-
scribed for 10b. The title compound 10e (160 mg, 70%) was ob-
tained as colorless crystals. Mp: 164–166 °C. 1H NMR (300 MHz,
DMSO-d6): d 2.25–2.60 (1H, br), 2.65–2.95 (1H, br), 3.10–3.35
(1H, br), 4.50 (2H, d, J = 5.7 Hz), 4.75–4.95 (1H, br), 6.51 (1H, s),
6.99–7.47 (11H, m), 7.71–7.99 (5H, m), 8.53–8.56 (1H, m) ,8.59
(1H, d, J = 2.8 Hz), 9.05 (1H, s). MS (FAB) m/z 610 [M]+. Anal. Calcd
for C34H26ClF2N5O2: C, 66.94; H, 4.30; N, 11.48; Cl, 5.81; F, 6.23.
Found: C, 66.61; H, 4.53; N, 11.19; Cl, 5.68; F, 6.13.
5.1.52. rac-(2Z)-2-{1-[2-Chloro-4-(3-methylpyrrolidin-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10j)
Compound 10j was prepared according to the procedure de-
scribed for 10b. The title compound 10j (298 mg, 59%) was ob-
tained as colorless crystals. Mp: 200–204 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.02 (3H, d, J = 6.9 Hz), 1.45–1.59 (1H,
m), 1.94–2.08 (1H, m), 2.23–2.33 (1H, m), 2.35–2.47 (1H, br),
2.57–2.64 (1H, br), 2.67–2.78 (1H, m), 2.92–3.05 (1H, br), 3.08–
3.17 (1H, m), 3.19–3.25 (1H, m), 3.28–3.37 (1H, m), 4.74 (2H,
d, J = 5.3 Hz), 4.76–4.93 (1H, br), 6.17–6.26 (1H, br), 6.35–6.43
(1H, br), 6.49 (1H, s), 6.65–6.77 (1H, br), 6.84–6.93 (1H, br),
7.16–7.34 (3H, m), 7.82–7.90 (2H, m), 8.45 (1H, t, J = 7.8 Hz),
8.80 (1H, d, J = 5.3 Hz), 9.27 (1H, t, J = 5.3 Hz). MS (FAB) m/z
551 [M]+. Anal. Calcd for C30H29ClF2N4O2ꢁHClꢁ0.3H2O: C, 60.77;
H, 5.20; N, 9.45; Cl, 11.96; F, 6.41. Found: C, 60.87; H, 5.10; N,
9.50; Cl, 12.15; F, 6.55.
5.1.48. (2Z)-2-{1-[2-Chloro-4-(3,5-dimethyl-1H-pyrazol-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10f)
Compound 10f was prepared according to the procedure de-
scribed for 10b. The title compound 10f (180 mg, 59%) was ob-
tained as colorless crystals. Mp: 221–222 °C. 1H NMR (300 MHz,
DMSO-d6): d 2.13 (3H, s), 2.25 (3H, s), 2.30–2.55 (1H, br), 2.65–
2.90 (1H, br), 3.15–3.30 (1H, br), 4.67 (2H, d, J = 5.7 Hz), 4.75–
4.85 (1H, br), 6.07 (1H, s), 6.54 (1H, s), 6.98–7.35 (6H, m), 7.55
(1H, s), 7.70–7.79 (2H, m), 8.32 (1H, d, J = 7.9 Hz), 8.75 (1H, d,
J = 5.1 Hz), 9.23 (1H, s). MS (FAB) m/z 562 [M]+. Anal. Calcd for
C30H26ClF2N5O2ꢁHClꢁ0.7H2O: C, 58.96; H, 4.68; N, 11.46; Cl, 11.60;
F, 6.22. Found: C, 59.23; H, 4.73; N, 11.18; Cl, 11.24; F, 6.17.

9534
I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
5.1.53. rac-(2Z)-2-{1-[2-Chloro-4-(2-methylpyrrolidin-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10k)
Compound 10k was prepared according to the procedure
described for 10b. The title compound 10k (220 mg, 44%) was
obtained as colorless crystals. Mp: 182–191 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.00 (3H, d, J = 5.4 Hz), 1.56–1.68 (1H,
m), 1.83–2.04 (3H, m), 2.22–2.47 (1H, br), 2.54–2.78 (1H, br),
2.96–3.05 (1H, br), 3.12–3.35 (2H, br), 3.75–3.84 (1H, br), 4.73
(2H, d, J = 5.3 Hz), 4.78–4.87 (1H, br), 6.23–6.32 (1H, br), 6.38–
6.46 (1H, br), 6.50 (1H, s), 6.66–6.75 (1H, br), 6.84–6.93 (1H,
br),7.15–7.29 (2H, m), 7.30–7.38 (1H, m), 7.83–7.89 (2H, m), 8.45
(1H, t, J = 7.8 Hz), 8.80 (1H, d, J = 5.3 Hz), 9.27 (1H, t, J = 5.3 Hz).
MS (FAB) m/z 551 [M]+. Anal. Calcd for C30H29ClF2N4O2ꢁHClꢁ0.3H2O:
C, 60.77; H, 5.20; N, 9.45; Cl, 11.96; F, 6.41. Found: C, 60.77; H,
5.00; N, 9.57; Cl, 12.08; F, 6.28.
5.1.57. (2Z)-2-(4,4-Difluoro-1,2,3,4-tetrahydro-5H-1-
benzazepin-5-ylidene)-N-(pyridin-2-ylmethyl)acetamide (13)
Methyl (2Z)-2-(4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzaze-
pin-5-ylidene)-N-(pyridin-2-ylmethyl)acetate
(12;
4.57 g,
19.1 mmol), 2-(aminomethyl)pyridine (2.22 ml, 21.0 mmol),
WSCD (5.60 g, 29.0 mmol), and HOBt (3.60 mg, 29.0 mmol) in
DMF (45 ml) were stirred overnight at room temperature. The
reaction mixture was partitioned between water and ethyl acetate,
then the organic phase was washed with water and brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The crude prod-
uct was purified by silica gel column chromatography (CHCl3/
methanol = 25:1) to give the title compound 13 (6.29 g, quantita-
tive yield) as green amorphous solid. 1H NMR (300 MHz, CDCl3) d
2.40–2.65 (2H, m), 3.41 (2H, t, J = 5.9 Hz), 4.66 (2H, d, J = 5.3 Hz),
4.75–5.20 (1H, br), 6.30 (1H, s), 6.59 (1H, dd, J = 0.8, 8.1 Hz), 6.79
(1H, dt, J = 0.9, 7.4 Hz), 7.09–7.26 (4H, m), 7.37 (1H, d, J = 7.9 Hz),
7.71 (1H, dt, J = 1.6, 7.8 Hz), 8.52 (1H, d, J = 4.6 Hz). MS (FAB) m/z
330 [M+H]+.
5.1.54. rac-(2Z)-2-(1-{2-Chloro-4-[3-(dimethylamino)pyrro-
lidin-1-yl]benzoyl}-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-
benzazepin-5-ylidene)-N-(pyridin-2-ylmethyl)acetamide
hydrochloride (10n)
5.1.58. (2Z)-2-[1-(2-Chloro-4-piperidin-1-ylbenzoyl)-4,4-
difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene]-N-
(pyridin-2-ylmethyl)acetamide hydrochloride (10l)
Compound 10n was prepared according to the procedure de-
scribed for 10b. The title compound 10n (659 mg, 66%) was ob-
tained as colorless crystals. Mp: >250 °C. 1H NMR (400 MHz,
DMSO-d6): d 2.21–2.49 (4H, m), 2.75 (6H, s), 3.00–3.22 (2H,
m), 3.35–3.64 (3H, m), 3.83–3.97 (1H, m), 4.69 (2H, d,
J = 6.0 Hz), 4.70–5.00 (1H, br), 6.31 (1H, d, J = 7.2 Hz), 6.46 (1H,
s), 6.53 (1 H, s), 6.75–6.77 (1H, m), 6.88 (1H, d, J = 6.8 Hz),
7.15–7.34 (3H, m), 7.71–7.79 (2H, m), 8.33 (1H, t, J = 7.8 Hz),
8.75 (1H, d, J = 5.2 Hz), 9.27 (1H, s), 11.61 (1H, s). MS (FAB) m/
A
mixture of 2-chloro-4-(piperidin-1-yl)benzoic acid (4l;
350 mg, 1.46 mmol), DMF (several drops), and thionyl chloride
(0.22 ml, 2.20 mmol) in THF (10 ml) was stirred at room tempera-
ture for 2.5 h. The reaction mixture was evaporated and azeotr-
oped with toluene. To a solution of this residue in acetonitrile
(20 ml) were added (2Z)-2-(4,4-difluoro-1,2,3,4-tetrahydro-5H-1-
benzazepin-5-ylidene)-N-(pyridin-2-ylmethyl)acetamide
(13,
400 mg, 1.21 mmol) and pyridine (0.40 ml, 4.90 mmol), and the
mixture was stirred overnight at 80 °C. The reaction was then al-
lowed to cool to room temperature and evaporated. The residue
was partitioned between aqueous citric acid and chloroform. The
organic phase was washed with water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (CHCl3/metha-
nol/aq NH3 = 25:1:0.1) and subsequently treated with hydrochlo-
ride in ethyl acetate, then evaporated. Resulting solid was
recrystallized from ethanol and collected by filtration to give
the title compound 10l (176 mg, 24%) as colorless crystals. Mp:
196–200 °C. 1H NMR (400 MHz, DMSO-d6) d 1.46–1.60 (6H, br),
2.44–2.49 (1H, br), 2.55–2.61 (1H, br), 3.11–3.17 (4H, br),
3.22–3.25 (1H, br), 4.68 (2H, d, J = 5.4 Hz), 4.74–4.88 (1H, br),
6.46 (1H, s), 6.64–6.73 (1H, br), 6.75–6.81 (1H, br), 6.83–6.93
(2H, m), 7.15–7.36 (3H, m), 7.73–7.82 (2H, m), 8.34 (1H, t,
J = 7.4 Hz), 8.77 (1H, d, J = 4.9 Hz), 9.21 (1H, s). MS (FAB) m/z 551
[M]+. Anal. Calcd for C30H29ClF2N4O2ꢁHClꢁ0.5H2O: C, 60.41; H,
5.24; N, 9.39; Cl, 11.89; F, 6.37. Found: C, 60.71; H, 5.17; N, 9.38;
Cl, 11.96; F, 6.31.
z
580 [M]+. Anal. Calcd for C31H32ClF2N5O2ꢁ1.8HClꢁ2H2O: C,
54.62; H, 5.59; N, 10.27; Cl, 14.56; F, 5.57. Found: C, 54.77; H,
5.51; N, 10.24; Cl, 14.43; F, 5.34.
5.1.55. (2Z)-2-{1-[2-Chloro-4-(4-methylpiperazin-1-
yl)benzoyl]-4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-
ylidene}-N-(pyridin-2-ylmethyl)acetamide hydrochloride (10o)
Compound 10o was prepared according to the procedure de-
scribed for 10b. The title compound 10o (497 mg, 62%) was ob-
tained as colorless crystals. Mp: 206–208 °C (decomp.). 1H NMR
(400 MHz, DMSO-d6): d 2.25–2.60 (1H, m), 2.65–2.80 (4H, m),
2.95–3.15 (5H, m), 3.37–3.42 (2H, m), 3.77–3.83 (2H, m), 4.75
(2H, d, J = 5.2 Hz), 4.76–4.90 (1H, br), 6.48 (1H, s), 6.72–6.77 (1H,
m), 6.81–6.98 (3H, m), 7.19 (1H, t, J = 7.2 Hz), 7.26 (1H, t,
J = 7.4 Hz), 7.32 (1H, d, J = 6.8 Hz), 7.80–7.88 (2H, m), 8.45 (1H, t,
J = 7.8 Hz), 8.80 (1H, d, J = 5.2 Hz), 9.33 (1H, s), 11.39 (1H, s),. MS
(FAB) m/z 566 [M]+. Anal. Calcd for C30H30ClF2N5O2ꢁ2HClꢁ4H2O: C,
50.68; H, 5.67; N, 9.85; Cl, 14.96; F, 5.34. Found: C, 50.29; H,
5.50; N, 9.82; Cl, 14.78; F, 5.18.
5.1.56. (2Z)-(4,4-Difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-
5-ylidene)acetic acid (12)
5.1.59. (2Z)-2-[1-(4-Azepan-1-yl-2-chlorobenzoyl)-4,4-difluoro-
1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene]-N-(pyridin-2-
ylmethyl)acetamide hydrochloride (10m)
Methyl (2Z)-(4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-
5-ylidene)acetate (11; 8.00 g, 31.6 mmol) in THF (20 ml) and
methanol (20 ml) was treated with sodium hydroxide (1.0 M,
45.0 ml, 45.0 mmol) overnight at room temperature. After the
pH value was adjusted around 5.0–6.0 by addition of aqueous
hydrochloride, the mixture was partitioned between water and
chloroform. The organic phase was washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo to give the title com-
pound 12 (4.57 g, 60%) as yellow solid. 1H NMR (300 MHz, CDCl3)
d 2.54–2.69 (2H, m), 3.42 (2H, t, J = 6.0 Hz), 6.20 (1H, s), 6.61 (1H,
dd, J = 0.9, 8.1 Hz), 6.82 (1H, dt, J = 1.0, 7.4 Hz), 7.16 (1H, dt,
J = 1.3, 8.0 Hz), 7.22 (1H, dd, J = 1.5, 7.7 Hz). MS (FAB) m/z 240
[M+H]+.
Compound 10m was prepared according to the procedure de-
scribed for 10l from 4m and 13. The title compound 10m
(165 mg, 22%) was obtained as colorless crystals. Mp: 194–
197 °C. 1H NMR (400 MHz, DMSO-d6): d 1.33–1.45 (4H, br), 1.55–
1.67 (4H, br), 2.41–2.47 (1H, br), 2.54–2.59 (1H, br), 3.02–3.21
(1H, br), 3.28–3.44 (4H, br), 4.67 (2H, d, J = 5.4 Hz), 4.72–4.93
(1H, br), 6.33–6.42 (1H, br), 6.46 (1H, s), 6.53–6.58 (1H, br),
6.62–6.71 (1H, br), 6.83–6.90 (1H, br), 7.13–7.34 (3H, m), 7.71–
7.80 (2H, m), 8.34 (1H, t, J = 7.3 Hz), 8.76 (1H, d, J = 4.9 Hz), 9.18
(1H, s). MS (FAB) m/z 565 [M]+. Anal. Calcd for
C31H31ClF2N4O2ꢁHClꢁ0.5H2O: C, 60.99; H, 5.45; N, 9.18; Cl, 11.61;
F, 6.22. Found: C, 61.18; H, 5.34; N, 9.19; Cl, 11.68; F, 6.17.

I. Tsukamoto et al. / Bioorg. Med. Chem. 16 (2008) 9524–9535
9535
5.2. Biology
to induce a reaction. The cells were then dissolved in phosphate-
buffered saline (PBS, Invitrogen Japan K.K.) containing 0.2% Triton
X-100. The cAMP level in the cell lysate was determined using
the homogenous time-resolved fluorescence (HTRF) assay with a
cyclic AMP kit (Nihon Schering K.K.).13
Intrinsic activity was calculated as the percentage (%) of the
maximum response to the test compound compared to the maxi-
mum response (100%) to AVP. All data analyses were performed
using SAS. The results are expressed as the means standard error.
The activities of test compounds for cAMP production were calcu-
lated by logistic regression as EC50 values.
5.2.1. Binding assay for human V2 receptor
Chinese hamster ovary (CHO) cells stably expressing human V2
receptors, which were established by Tahara et al.,10 were used.
Cells were washed with phosphate buffered saline, and then col-
lected in ice-cold hypotonic buffer (10 mmol/L Tris–HCl, 5 mmol/
L EDTA, pH 7.4). Subsequently, cells were collected using a cell
scraper and then homogenized using POLYTRONÒ followed by cen-
trifugation (1000g, 10 min) at 4 °C. The supernatant was centri-
fuged (35,000g, 30 min) at 4 °C, and the pellet was suspended in
Tris buffer. Membrane fractions were stored at ꢀ80 °C until used
for binding assay. The concentration of membrane protein was
determined by the Coomassie blue method using BSA as a
standard.
The affinities of test compounds for human V2 receptor were
evaluated by the radioligand binding study. For the competitive
binding study, 50
(final concentration of 0.91 nmol/L) were mixed with 150
membrane suspension in 50 mmol/L Tris–HCl (pH 7.4) buffer con-
5.2.3. Antidiuretic effect in water-loaded rats
A test to determine which rats would be selected for use was
performed at least a week before the beginning of the study. In this
test, male Wistar rats (SLC, 200–300 g) were given distilled water
(30 mL/kg) orally. Afterwards, the animals were kept in metabolic
cages, and urine was collected for 4 h after water loading. Animals
whose urinary excretion rate was at least 70% of the volume of
water loaded (which was regarded as 100%) were used. While
the animals were deprived of feed and water, they orally received
the test compounds without anesthesia. Distilled water (30 mL/kg,
po) was loaded 15 min after administration, and the animals were
kept in a metabolic cage. Urine was collected every hour up to 4 h
after water loading. The urinary excretion rate (%) was calculated
by regarding the volume of loaded water as 100%. Linear regression
was performed to obtain the doses of the test compounds required
to decrease the urinary excretion rate to 50% (ED50). All analyses
were performed using SAS (version 8.2, SAS Institute Japan). The
results are expressed as the mean (95% confidence interval) or
the means standard error.
l
L of drug solution and 50
l
L of [3H]vasopressin
L of
l
taining 10 mmol/L MgCl2 and 0.1% bovine serum albumin in a final
volume of 250 lL. This mixture was incubated at room tempera-
ture for 60 min. Reactions were terminated by filtration through
UniFilterÒ GF/B (Perkin-Elmer) using a MicroMate Cell Harvester
(Packard Instrument Company, Meriden, CT, USA) and the filter
was washed with ice-cold Tris buffer. The radioactivity retained
TM
on the filter was counted by TopCount microplate scintillation
counter (Perkin-Elmer) using the scintillation cocktail (MicroScin-
TM
ti-40 , Perkin-Elmer). Nonspecific binding or total binding was
determined by including 1 lmol/L AVP or without test compounds
in the reaction mixture, respectively. The number of concentra-
tions of compounds was 11, appropriately chosen from 1 ꢂ 10ꢀ11
to 1 ꢂ 10ꢀ5mol/L, using a common ratio of approximately 3. We
also performed the saturation binding study to yield the dissocia-
tion constants (Kd values) of [3H]vasopressin for each human V2
receptors. A membrane suspension was incubated with various
concentrations of [3H]vasopressin (0.1–3.2 nmol/L) in the absence
Acknowledgments
The authors deeply acknowledge Dr. Mitsuaki Ohta and Dr.
Minoru Okada for their helpful support to this research. The
authors are also grateful to the staff of the Division of Analytical
Science Laboratories for elemental analysis and spectral
measurement.
or presence of 1 lmol/L AVP. Assay conditions were the same as
those described for the competitive binding assay.
All values were determined by four separate experiments per-
formed in triplicate and represented as means SEM. Statistical
analysis was performed using a SAS software (SAS Institute,
USA). Specific binding was calculated as total binding minus non-
specific binding. The concentration of each compound required to
reduce specific binding of [3H]vasopressin by 50% (IC50 value)
was obtained by nonlinear regression analysis. A Kd value of
[3H]vasopressin for each vasopressin receptor was yielded by Scat-
chard plot analysis. The affinity constants (Ki values) were calcu-
lated from the following equation,12 using the Kd values yielded
from each separate experiment, Ki = IC50/(1 + [[3H]vasopressin con-
centration]/Kd).
References and notes
1. (a) Hardman, J. G.; Limbird, L. E. Goodman & Gilman’s The Pharmacological Basis
of Therapeutics, 10th ed.; McGraw-Hill: New York, 2001. pp 789–944; (b)
Guyton, A. C.; Hall, J. E. Textbook of Medical Physiology, 9th ed.; W.B. Saunders:
Medina, 1999. pp 933–944; (c) Michel, R. H.; Kirk, C. J.; Billah, M. M. Biochem.
Soc. Trans. 1979, 7, 861; (d) Laszlo, F. A.; Laszlo, F.; De Weid, D. Pharmacol. Rev.
1991, 43, 73.
2. Kondo, K.; Ogawa, H.; Shinohara, T.; Kurimura, M.; Tanada, Y.; Kan, K.;
Yamashita, H.; Nakamura, S.; Hirano, T.; Yamamura, Y.; Mori, T.; Tominaga, M.;
Itai, A. J. Med. Chem. 2000, 43, 4388.
3. Kondo, K.; Kan, K.; Tanada, Y.; Bando, M.; Shinohara, T.; Kurimura, M.; Ogawa,
H.; Nakamura, S.; Hirano, T.; Yamamura, Y.; Kido, M.; Mori, T.; Tominaga, M. J.
Med. Chem. 2002, 45, 3805.
4. Venkatesan, A. M.; Grosu, G. T.; Failli, A. A.; Chan, P. S.; Coupet, J.; Saunders, T.;
Mazandarani, H.; Ru, X. Bioorg. Med. Chem. Lett 2005, 15, 5003.
5. Failli, A. A.; Shumsky, J. S.; Steffan, R. J.; Caggiano, T. J.; Williams, D. K.;
Trybulsky, E. J.; Ning, X.; Lock, Y.; Tanikella, T.; Hartmann, D.; Chan, P. S.; Park,
C. H. Bioorg. Med. Chem. Lett. 2006, 16, 954.
6. Taniguchi, N.; Tanaka, A.; Matsuhisa, A.; Sakamoto, K.; Koshio, H.; Yatsu, T.
Patent WO 9506035, 1995; Chem. Abstr. 1995, 123, 228010.
7. Sudborough, J. J. J. Chem. Soc. 1895, 67, 601.
8. Katritzky, A. R.; Pilarski, B.; Urogdi, L. Synthesis 1989, 949.
9. Shimada, Y.; Akane, H.; Taniguchi, N.; Matsuhisa, A.; Kawano, N.; Tanaka, A.
Chem. Pharm. Bull. 2005, 53, 589.
10. Tahara, A.; Saito, M.; Sugimoto, T.; Tomura, Y.; Wada, K.; Kusayama, T. Br. J.
Pharmacol. 1998, 125, 1463.
11. Tahara, A.; Saito, M.; Sugimoto, T.; Tomura, Y.; Wada, K.; Kusayama, T.;
Tsukada, J.; Ishii, N.; Yatsu, T.; Uchida, W.; Tanaka, A. Br. J. Pharmacol. 1998,
125(7), 1463.
12. Lazareno, S.; Bridsall, N. J. Br. J. Pharamacol. 1993, 109, 1110.
13. Takasaki, J.; Kamohara, M.; Saito, T.; Matsumoto, M.; Matsumoto, S.; Ohishi, T.;
Soga, T.; Matsushime, H.; Furuichi, K. Mol. Pharmacol. 2001, 60(3), 432.
5.2.2. Stimulatory effect on the production of intracellular
cAMP in human vasopressin V2 receptor
CHO cells stably expressing human V2 receptors, prepared by
Tahara et al., were used.11 The cells were incubated in
a-MEM, con-
taining 10% fetal bovine serum (FBS, Invitrogen Japan K.K.), 1% pen-
icillin/streptomycin (Invitrogen Japan K.K.), and 0.1% amethopterin
(dihydrofolate reductase inhibitor), in the absence of nucleic acid,
at 37 °C, in an atmosphere of 95% air/5% CO2.
CHO cells expressing human V2 receptors were grown to sub-
confluence on a 96-well plate, and then incubated in serum-free
medium for 24 h before assay. The medium was replaced with a-
MEM containing 1 mmol/L 3-isobutyl-1-methylxantine (IBMX, Sig-
ma) and 0.1% bovine serum albumin (BSA, Sigma), then the test
compound was added and incubated at 37 °C for 10 min in order