Synthesis and structure-activity relationships of amide derivatives of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetic acid as selective arginine vasopressin V2 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2009.03.001

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives was synthesized, and their structure-activity relationships were examined in order to identify potent and selective arginine vasopressin V₂ receptor agonists. Attempts to substitute other chemical groups in place of the 2-pyridilmethyl moiety of 1a led to the discovery that potent V₂ binding affinity could be obtained with a wide range of functional groups. This structural tolerance allowed for the manipulation of other attributes, such as selectivity against V_1a receptor affinity or avoidance of the undesirable inhibition of cytochrome P450 (CYP), without losing potent affinity for the V₂ receptor. Some representative compounds obtained in this study were also found to decrease urine volume in awake rats.

Full text of DOI:10.1016/j.bmc.2009.03.001

Bioorganic & Medicinal Chemistry 17 (2009) 3130–3141
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of amide derivatives of
(4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetic acid
as selective arginine vasopressin V2 receptor agonists
a
a
a
a
Issei Tsukamoto a, , Hiroyuki Koshio , Takahiro Kuramochi , Chikashi Saitoh , Hiroko Yanai-Inamura ,
Chika Kitada-Nozawa a, Eisaku Yamamoto a, Takeyuki Yatsu a, Yoshiaki Shimada 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:
A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives was syn-
thesized, and their structure–activity relationships were examined in order to identify potent and selec-
tive arginine vasopressin V2 receptor agonists. Attempts to substitute other chemical groups in place of
the 2-pyridilmethyl moiety of 1a led to the discovery that potent V2 binding affinity could be obtained
with a wide range of functional groups. This structural tolerance allowed for the manipulation of other
attributes, such as selectivity against V1a receptor affinity or avoidance of the undesirable inhibition of
cytochrome P450 (CYP), without losing potent affinity for the V2 receptor. Some representative com-
pounds obtained in this study were also found to decrease urine volume in awake rats.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 24 December 2008
Revised 2 March 2009
Accepted 3 March 2009
Available online 9 March 2009
Keywords:
(4,4-Difluoro-1,2,3,4-tetrahydro-5H-1-
benzazepin-5-ylidene)acetamide
derivatives
Vasopressin V2 receptor
Vasopressin V1a receptor
CYP inhibitory activity
1. Introduction
we recently reported that (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-
benzazepin-5-ylidene)acetamide derivatives are also novel V2
Arginine vasopressin (AVP) is a cyclic nonapeptide that is pro-
duced and secreted by the hypothalamo-neurohypophysical sys-
tem. The vasopressin receptor family consists of three subtypes,
V1a, V1b, and V2.1–3 The V1a receptor, which exists mainly in vascu-
lar smooth muscle and platelets, is involved in blood pressure con-
trol. The V1b receptor, which is found in the hypophysis pituitary
gland, regulates adrenocorticotropic hormone (ACTH) secretion.
The V2 receptor, which has been identified in the kidney, stimu-
lates coupled adenylate cyclase, increases cyclic adenosine mono-
phosphate (cAMP), and plays an important role in water
reabsorption. In other words, stimulation of the V2 receptor with
AVP causes water reabsorption in the kidney by increasing cAMP
with subsequent activation of the aquaporin-2 water channel,
which results in reducing urine volume. This implies that a V2
receptor agonist can be used to treat diseases such as central
diabetes insipidus and nocturia.4 Desmopressin (dDAVP),5 OPC-
51803,6 and VNA9327 are established in the literature as
compounds with this mechanism of action (Fig. 1). In addition,
receptor agonists (Fig. 2).8
As we reported previously, the chemical structure of the ‘tail’-
moiety (Fig. 2) in our compound strictly regulates agonist-antago-
nist switching for the vasopressin V2 receptor.8 In this paper, we
describe the synthesis, biological activity, and structure–activity
relationships (SARs) of the ‘head’-moiety of 1a as investigated by
replacing the 2-pyridilmethyl group with various other chemical
groups. In addition to the binding affinities for the V2 and V1a
receptors, the cAMP accumulation activity, in vivo action, and
CYP inhibitory activity of some representative compounds are also
shown.
2. Chemistry
The syntheses of test compounds (1a–1w) are shown in Scheme
1–8. Carboxylic acid (1q), which is the common intermediate for
preparation of various amide derivatives, was synthesized from 2
in the same manner as reported in the previous paper.8 Condensa-
tion of 1q with amines in the presence of N-ethyl-N0-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (WSCD) and 1-
hydroxybenzotriazole (HOBt) yielded the target compounds
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.001

I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
3131
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
O
NH
H
NH
N
NH2
N
NH2
O
N
O
O
N
O
H
O
H
O
NH2
AVP
Desmopressin
O
N
N
H
N
N
O
N
O
N
Cl
N
Cl
OPC-51803
VNA932
Figure 1. Chemical structure of AVP and well-known arginine vasopressin V2 receptor agonists.
(1a–1p, 1s–1u, and 1w;Scheme 1). The carboxamide form (1r) was
prepared from 1q and aqueous ammonia via activation by thionyl
chloride (Scheme 2). Compound 1g, which is an amino pyridine
derivative, and 1v, which has a glycine unit, were synthesized in
a stepwise manner as shown in Schemes 3 and 4, respectively.
Schemes 5–8 show the preparation of the benzylamine parts,
which are used for the amidation shown in Scheme 1. Methoxy-
substituted 9a and dimethylamino-substituted 9b were synthe-
sized from 2,6-dibromopyridine (6). ipso-Substitution reactions
with 6 and corresponding nucleophiles afforded 7a and 7b. The
other bromo group was converted to a formyl group via lithiation
by n-BuLi and quenching by dimethylformamide. The resulting
aldehydes (8a and 8b) were treated with hydroxylamine and sub-
O
N
H
N
CO2H
CONH2
F
F
F
F
F
F
'head' moiety
a
N
N
N
'tail' moiety
O
O
O
N
N
N
Cl
N
N
Cl
N
Cl
1a
1q
1r
Figure 2. Chemical structure of arginine vasopressin V2 agonist recently reported.
Scheme 2. Reagents and conditions: (a) SOCl2, cat. DMF, THF then aq NH3.
O
CO2H
R
F
F
F
F
CO2H
CO2H
d, e
f
a, b, c
N
N
N
N
Cl
F
Cl
O
O
2
3
N
N
N
Cl
N
Cl
1q
1a-1p, 1s-1u, 1w
Scheme 1. Reagents and conditions: (a) H2SO4, MeOH; (b) 3-methylpyrazole, K2CO3, NMP; (c) aq HCl, acetic acid; (d) SOCl2, cat. DMF, THF then methyl (4,4-difluoro-1,2,3,4-
tetrahydro-5H-1-benzazepin-5-ylidene)acetate9, pyridine; (e) aq NaOH, MeOH; (f) amine, WSCD, HOBt, DMF, (then HCl).

3132
I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
O
O
N
H
N
N
N
H
NHBoc
b
NH2
HCl
F
F
.
F
F
a
1q
N
N
O
O
N
N
N
N
Cl
Cl
1g
4
Scheme 3. Reagents and conditions: (a) 11, WSCD, HOBt, DMF; (b) HCl, ethyl acetate.
CO2Me
CO2H
O
O
N
H
N
H
F
F
F
F
a
b
1q
N
N
O
O
N
N
N
N
Cl
Cl
1v
5
Scheme 4. Reagents and conditions: (a) glycine methyl ester, WSCD, HOBt, DMF; (b) aq NaOH, MeOH.
c, d
a
b
H2N
Br
N
Br
Br
N
R
OHC
N
R
N
R
6
7a: R = OMe
7b: R = N(CH3)2
8a: R = OMe
8b: R = N(CH3)2
9a: R = OMe
9b: R = N(CH3)2
Scheme 5. Reagents and conditions: (a) NaOMe, MeOH (for 7a) or dimethylamine, DMF, H2O (for 7b); (b) n-BuLi, THF then DMF; (c) hydroxylamine hydrochloride, K2CO3,
EtOH; (d) H2, Raney-Ni, EtOH.
sequently hydrogenated in the presence of Raney-Ni to yield the
(aminomethyl)pyridine derivatives 9a and 9b (Scheme 5). tert-Bu-
tyl [6-(aminomethyl)pyridin-2-yl]carbamate (11) was synthesized
from a mesylate intermediate already reported (10)10,11 via an azi-
dation and a reduction (Scheme 6). 6-(Aminomethyl)pyridine-2-
carbonitrile (16) was prepared as shown in Scheme 7. A cyano
group was introduced at the 6-position of the 2-chloropyridine in
accordance with an established method12 using a commercially
available N-oxide material (12). The chloro-substituted compound
(13) underwent a Stille-coupling13 reaction to afford 14. The vinyl
group in 14 was transformed into a hydroxymethyl group via
ozonolysis, and subsequently reduced by NaBH4 to yield an alcohol
(15). Lastly, 15 was converted into the targeted amine (16) via a
phthalimide intermediate. Scheme 8 illustrates the synthesis of
3-(aminomethyl)pyridazine (20). Compound 18 was synthesized
by condensation of 3-methylpyridazine (17) with benzaldehyde.14
The olefin compound (18) was oxidatively cleaved to afford the
aldehyde (19), which was then converted into the amine com-
pound (20) in a manner similar to that described in Scheme 5.
a, b
OMs
NH2
BocHN
N
10
BocHN
N
11
Scheme 6. Reagents and conditions: (a) NaN3, DMF; (b) H2, Pd–C, EtOH.
a, b, c
d
e, f
g, h
+
N
Cl
OH
NH2
HCl
NC
N
Cl
NC
N
NC
NC
N
N
O
12
13
14
15
16
Scheme 7. Reagents and conditions: (a) KOMe, MeOH; (b) dimethyl sulfate; (c) NaCN, H2O; (d) tributyl(vinyl)tin, Pd(PPh3)4, toluene; (e) O3, MeOH then Me2S; (f) NaBH4,
MeOH; (g) phthalimide, DEAD, PPh3, THF; (h) hydrazine hydrate, MeOH, CHCl3.

I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
3133
Table 2
Ph
Binding affinity of substituted-pyridyl derivatives for V2 and V1a receptors
NH2
CHO
a
b
c, d
N
N
N
N
N
N
O
N
N
N
N
H
F
R2
18
17
19
20
F
Scheme 8. Reagents and conditions: (a) benzaldehyde, ZnCl2; (b) NaIO4, cat. OsO4,
t-BuOH, acetone, H2O; (c) hydroxylamine hydrochloride, K2CO3, EtOH; (d) H2, Pd–C,
EtOH.
N
O
N
Cl
N
3. Results and discussion
Compound
R2
Binding affinity: Ki (nM)
Selectivity
V1a/V2
Synthesized compounds 1a–1u were evaluated for their human
V2 and V1a receptor binding affinity, which was quantified in terms
of Ki in a radioligand binding assay.
a
b
V2
V1a
1a
1e
1f
1g
1h
1i
H
4.8
5.6
16
26
30
47
38
48
22
5.4
5.4
2.9
3.8
4.4
3.0
Compound 1a, which was reported previously, showed V28 and
V1a receptor affinity with Ki values of 4.8 nM and 26 nM, respec-
tively. The replacement of the pyridin-2-yl group in 1a with a pyri-
din-3-yl group (1b), a pyridin-4-yl group (1c), and a phenyl group
(1d) were examined first. As shown in Table 1, both 1b and 1c had
a binding affinity for the V2 receptor about fivefold lower than 1a.
Phenyl substitution (1d) also caused affinity for the V2 receptor to
decrease. In contrast, even with all the structural conversions de-
scribed above, no obvious fluctuation in the affinity for the V1a
receptor was observed. This indicates that polarity at some posi-
tions, such as the pyridin-2-yl group, is preferable because it in-
CH3
OCH3
NH2
N(CH3)2
CN
10
11
7.4
a
Binding affinity for human V2 receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
b
Binding affinity for human V1a receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
suggest that the region near the pyridin-2-yl group gives wide lat-
itude to structural conversion, both electronically and sterically,
while maintaining high affinity for the V2 receptor.
creases affinity for V2 receptor binding. Therefore, such
a
The SARs of compounds in which various hetero aromatic rings
were introduced instead of a pyridin-2-yl group are summarized in
Table 3. All compounds from 1j to 1p possess a hetero atom at a
position similar to that of the nitrogen atom on the pyridine ring
of 1a. Among those compounds, the pyridazin-3-yl substituted
1k showed higher V2 binding affinity than 1a, with a Ki value of
3.4 nM. The binding affinity of 1k for the V1a receptor was slightly
lower than that of 1a, and the V2/V1a ratio was 13:1, which was sig-
nificantly better than that for 1a. Other compounds with nitrogen-
containing hetero aromatic rings (1j, 1l, 1m, 1n, and 1o) main-
tained moderate affinity to the V2 receptor. Meanwhile the intro-
duction of a furan ring (1p), which is less polar than pyridine or
pyridazine, resulted in reduced binding affinity for the V2 receptor.
This tendency agrees with the SARs in Table 1.
modification is expected to generate better selectivity against V1a
binding affinity, which is not affected by the polar atom.
Table 2 shows the binding affinity values for compounds 1e to
1i, which have various functional groups near the nitrogen atom
on the pyridine ring of 1a (Table 2). Compound 1e which possesses
an electronically neutral methyl group, and each compound in Ta-
ble 2, including 1f to 1h, to which an electron donating substituent
was introduced, and 1i, which has electron deficient cyano group,
maintained both a binding affinity for V2 and selectivity against the
V1a receptor comparable to those for compound 1a. Sterically hin-
dered substitution of a dimethylamino group (1h) did not cause
obvious changes in the binding affinity profile either. These results
Table 1
Table 4 exhibits the SARs of compounds containing aliphatic
groups in the ‘head’ moiety. Interestingly, the carboxylic acid
derivative (1q) and carboxamide form (1r), each of which have a
structurally simple ‘head’ moiety, had modest binding affinity for
the V2 receptor, although the values were lower than that for 1a.
More noteworthy is that the binding affinity of 1q for the V1a
receptor almost disappeared (Ki = 6300 nM), and the V2/V1a selec-
tivity ratio increased to 252:1. The effect of the introduction of sev-
eral alkyl chains were also examined (1s–1w). While compound 1s,
which has a hydrophobic n-propyl group, had lower affinity for the
V2 receptor than 1a, hydroxyethyl-substituted 1t recovered its
comparably high binding affinity to 1a. This observation is similar
the case of aromatic substituents, where a polar functional group
at the appropriate position is preferable to V2 binding affinity (Ta-
ble 1 and Table 3). However, (dimethylamino)ethylamide (1u) had
about 10-fold lower affinity to the V2 receptor. The introduction of
a glycine unit maintained moderate binding affinity for the V2
receptor, with a Ki value of 22 nM, and showed low affinity for
the V1a receptor, with a Ki value of 230 nM (1v). The results for
1q and 1v clearly indicate that an acidic function could decrease
binding affinity for the V1a receptor. An amide derivative of 1v
had high affinity for the V2 receptor (1w, Ki = 11 nM), but high
selectivity was not retained.
Binding affinity of pyridyl and benzyl-substituted derivatives for V2 and V1a receptors
R1
O
N
H
F
F
N
O
N
N
Cl
Compound
R1
Binding affinity: Ki (nM)
Selectivity
V1a/V2
a
b
V2
4.8
21
V1a
1a
1b
1c
1d
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
Phenyl
26
40
15
43
5.4
1.9
0.75
2.3
20
19
a
Binding affinity for human V2 receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
b
Binding affinity for human V1a receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.

3134
I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
Table 3
Table 4
Binding affinity of hetero aromatic ring-substituted derivatives for V2 and V1a
receptors
Binding affinity of aliphatic group-substituted derivatives for V2 and V1a receptors
O
O
R4
R3
F
F
F
F
N
N
O
O
N
N
Cl
N
Cl
N
Compound
R4
Binding affinity: Ki (nM)
Selectivity
V1a/V2
Compound
R3
Binding affinity: Ki (nM)
Selectivity
V1a/V2
a
b
V2
V1a
a
b
V2
V1a
N
1a
1q
1r
1s
4.8
26
6300
190
13
5.4
252
N
H
H
N
1a
1j
4.8
26
5.4
4.1
N
N
25
OH
N
H
N
14
57
33
18
5.8
NH
2
N
H
Me
OH
N
H
1k
1l
3.4
43
44
43
13
0.72
N
N
N
N
H
N
H
1t
9.7
28
59
2.9
1.1
11
4.0
3.9
N
NMe
2
S
S
N
H
1u
1v
53
N
H
1m
11
11
N
N
N
H
CO H
22
11
230
40
10
2
N
H
1n
1o
55
72
72
5.0
6.0
2.7
N
H
CONH
1w
3.6
2
H
N
a
N
H
12
27
Binding affinity for human V2 receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
Binding affinity for human V1a receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
N
b
N
H
1p
O
a
Binding affinity for human V2 receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
Lastly, the CYP inhibitory activity of 1a, 1k, 1q, and 1t were
examined (Table 6). While compounds with a nitrogen-containing
hetero aromatic ring (1a, 1k) did not have a preferable overall CYP
inhibitory activity profile, compounds with an aliphatic group sub-
stitution (1q, 1t) were found to avoid potent inhibitory activity
against almost all of the CYP species described in Table 6.
b
Binding affinity for human V1a receptors. The receptors expressed on CHO cells
were used. All assays were performed in triplicate.
Some representative compounds identified in the binding as-
says were evaluated for their agonistic activity to V2 receptor and
in vivo activity. Their agonistic activity was assessed using the
cAMP accumulation test to determine EC50 value and maximal pro-
duction. Pyridazine-substituted 1k exhibited potent cAMP accu-
mulation activity (EC50 = 16 nM, Emax = 86%), although it was
slightly less intense than that of 1a. The agonistic activity of 1q
and 1t, in which an aliphatic group is substituted in the ‘head’ moi-
ety, was found to be extremely potent and comparable to that of 1a
(Table 5).
In vivo activity was assessed as total antidiuretic effect after 4-h
in water-loaded rats (Fig. 3). Compounds 1k and 1t suppressed the
urine excretion rate considerably within the 4 h immediately fol-
lowing intravenous administration. However, the carboxylic acid
derivative 1q did not decrease urine volume. The particularly high
polarity of its carboxyl group was considered to have a negative
impact on pharmacokinetic parameters such as metabolism and
elimination.
4. Conclusion
A series of amide derivatives of (4,4-difluoro-1,2,3,4-tetrahy-
dro-5H-1-benzazepin-5-ylidene)acetic acid were synthesized and
evaluated with respect to following biological profiles: binding
affinity for the V2 and V1a receptors, cAMP production activity,
in vivo antidiuretic effect, and CYP inhibitory activity. As a result
of this exploration and the SAR study, the new insights described
below were gained.
(1) The ‘head’ moiety (Fig. 2) of this derivative tolerates a wide
range of structural conversions, including aromatic and aliphatic
parts with various functional groups, which maintain high affinity
for the V2 receptor. In particular, the presence of a polar hetero
atom at a certain position often increases the affinity for V2
receptor.

I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
3135
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,
N) and Yokogawa IC-7000S ion chromatographic analyzer (halo-
gens) and were within 0.4% of theoretical values.
2-Chloro-4-(3-methyl-1H-pyrazol-1-yl)benzoic acid (3), (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 (1q) and
(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-(pyri-
din-2-ylmethyl)acetamide hydrochloride (1a) were prepared from
2-chloro-4-fluorobenzoic acid (2) according to the procedure de-
scribed in our previous paper.8
Table 5
cAMP accumulation activity of compound 1a, 1k, 1q and 1t
Compound
cAMP accumulation
a
b
EC50 (nM)
Emax (%)
1a
1k
1q
1t
1.8
16
4.2
6.3
95.9
86.4
92.4
93.9
a
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.
b
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.
5.1.1. 2-Bromo-6-methoxypyridine (7a)
To a mixture of 2,6-dibromopyridine (3.00 g) in methanol
(30 ml) was added sodium methoxide (28% in methanol,
25.3 ml), and the mixture was stirred overnight at 85 °C. The sol-
vent was evaporated, and the residue was partitioned between
water and ethyl acetate. The organic phase was washed with brine,
dried over sodium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(n-hexane/ethyl acetate = 2/1) to give the title compound 7a
(2.10 g, 88%) as colorless oil. 1H NMR (400 MHz, DMSO-d6) d 3.85
(3H, s), 6.87 (1H, d, J = 10.0 Hz), 7.23 (1H, d, J = 10.0 Hz), 7.66
(1H, t, J = 10.0 Hz). MS (GC) m/z 188 [M].
Figure 3. Effects of the iv administration of compounds 1a, 1k, 1q and 1t on urinary
excretion rate in water-loaded rats. The urinary excretion rate is the ratio of urine
volume to the volume of water loaded. All assays were performed in quadruplicate.
Table 6
Inhibitory activity against cytochrome P450
5.1.2. 2-Bromo-6-dimethylaminopyridine (7b)
A solution of 2,6-dibromopyridine (3.00 g) and aqueous dimeth-
ylamine (50%, 40 ml) in DMF (40 ml) was refluxed for 3 h. The reac-
tion mixture was partitioned between water and ethyl acetate. The
organic phase was washed with brine, dried over magnesium sul-
fate, filtered, and concentrated in vacuo. The crude product was
purified by silica gel column chromatography (n-hexane/ethyl ace-
tate = 10/1) to give the title compound 7b (2.37 g, 93%) as colorless
oil. 1H NMR (400 MHz, DMSO-d6) d 2.99 (6H, s), 6.60 (1H, d,
J = 8.4 Hz), 6.70 (1H, d, J = 7.6 Hz), 7.39 (1H, dd, J = 8.4, 7.6 Hz).
MS (EI) m/z 200 [M-1], 202 [M+1].
Compound
IC50
CYP 2C19b
(lM)
CYP 2C9a
CYP 2D6c
CYP 3A4d
1a
1k
1q
1t
9.1
13
>20
>20
5.1
7.0
>20
5.5
14
8.9
11
7.9
>100
47
>20
>20
a
IC50 values against CYP2C9 were determined in fluorescent P450 inhibition
assays. All assays were performed in duplicate.
b
IC50 values against CYP2C19 were determined in fluorescent P450 inhibition
assays. All assays were performed in duplicate.
c
IC50 values against CYP2D6 were determined in fluorescent P450 inhibition
assays. All assays were performed in duplicate.
5.1.3. 2-Methoxypyridine-6-carboxaldehyde (8a)
d
IC50 values against CYP3A4 were determined in competitive inhibition assays
To a solution of 7a (2.00 g) in THF (20 ml) was added n-butyl-
lithium (1.52 mol/l in n-hexane, 8.40 ml) at ꢀ78 °C. After stirring
for 1 h, DMF (949 mg) was slowly added, and the reaction mixture
was stirred for 30 min with the maintenance of ꢀ78 °C for the
whole process. The reaction was quenched by aqueous sodium
hydrogen carbonate and extracted with ethyl acetate. The organic
phase was washed with brine, dried over magnesium sulfate, fil-
tered, and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (n-hexane/ethyl ace-
tate = 20/1) to give the title compound 8a (1.02 g, 70%) as colorless
oil. 1H NMR (400 MHz, DMSO-d6) d 3.96 (3H, s), 7.16 (1H, dd,
J = 11.2, 1.2 Hz), 7.59 (1H, dd, J = 9.6, 1.2 Hz), 7.95 (1H, ddd,
J = 11.2, 9.6, 0.8 Hz), 9.90 (1H, d, J = 0.8 Hz). MS (FAB) m/z 138
[M+1]+.
using Midazolam. All assays were performed in duplicate.
(2) The introduction of an acidic functional group in the ‘head’
region effectively decreases the affinity for the V1a receptor.
(3) Some compounds with
a substituted aliphatic group
avoided the undesirable inhibition of CYP without losing potent
affinity for the V2 receptor.
These findings eventually led to the representative compound
1t, which is a practical vasopressin V2 receptor agonist that could
be used for the treatment of disorders such as central diabetes
insipidus and nocturia.
5. Experiment
5.1. Chemistry
5.1.4. 2-Dimethylaminopyridin-6-carboxaldehyde (8b)
Compound 8b was prepared according to the procedure de-
scribed for 8a from 7b. The title compound 8b (1.55 g, 87%) was
obtained as colorless oil. 1H NMR (400 MHz, DMSO-d6) d 3.10
(6H, s), 6.95 (1H, d, J = 8.8 Hz), 7.14 (1H, d, J = 6.8 Hz), 7.71 (1H,
dd, J = 8.8, 6.8 Hz), 9.81 (1H, s). MS (FAB) m/z 151 [M+1]+.
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.

3136
I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
5.1.5. 1-(6-Methoxypyridin-2-yl)methanamine (9a)
5.1.9. 6-Vinylpyridine-2-carbonitrile (14)
A mixture of 8a (1.00 g), hydroxylamine hydrochloride (1.04 g)
and potassium carbonate (2.05 g) in ethanol (20 ml) was stirred at
80 °C for 2 h. After evaporation, the residue was partitioned be-
tween water and ethyl acetate. The organic phase was washed
with brine, dried over magnesium sulfate, filtered, and concen-
trated in vacuo to give 6-methoxypyridine-2-carbaldehyde oxime
(1.02 g, 91%) as yellow solid. 1H NMR (400 MHz, DMSO-d6) d 3.86
(3H, s), 6.81 (1H, d, J = 10.0 Hz), 7.35 (1H, d, J = 10.0 Hz), 7.73
(1H, t, J = 10.0 Hz), 7.99 (1H, s), 11.62 (1H, s). MS(FAB) m/z 153
[M+1]+. 6-Methoxypyridine-2-carbaldehyde oxime (720 mg) was
hydrogenated at the pressure of 2.0 kgf/cm2 in the presence of Ra-
ney-Ni in ethanol (20 ml) for 3.5 h. The catalyst was removed by
filtration, and the filtrate was concentrated in vacuo. This residue
was purified by silica gel column chromatography (chloroform/
methanol = 25/1) to give the title compound 9a (101 mg, 15%) as
colorless oil. 1H NMR (400 MHz, DMSO-d6) d 3.25–3.45 (2H, br),
3.70 (2H, s), 3.83 (3H, s), 6.62 (1H, d, J = 10.8 Hz), 7.00 (1H, d,
J = 10.8 Hz), 7.64 (1H, t, J = 10.8 Hz). MS (EI) m/z 138 [M].
A mixture of 13 (3.00 g), tributyl(vinyl)tin (6.54 ml) and tetra-
kis(triphenylphosphine)palladium (627 mg) in toluene (30 ml)
was refluxed for 5 h. After the reaction was cooled to room temper-
ature, aqueous potassium fluoride was added, then resulting insol-
uble matter was removed by filtration. The filtrate was extracted
with ethyl acetate, and the organic phase was washed with brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The crude product was purified by silica gel column chromatogra-
phy (n-hexane/ethyl acetate = 10/1) to give the title compound 14
(2.63 g, 93%) as colorless oil. 1H NMR (400 MHz, CDCl3) d (1H, d,
J = 10.8 Hz), 6.33 (1H, d, J = 17.6 Hz), 6.81 (1H, dd, J = 17.6,
10.8 Hz), 7.53 (1H, d, J = 7.6 Hz), 7.55 (1H, d, J = 6.8 Hz), 7.78 (1H,
t, J = 8.0 Hz). MS (EI) m/z 130 [M].
5.1.10. 6-Hydroxymethylpyridine-2-carbonitrile (15)
A mixture of 14 (2.11 g) in methanol (20 ml) was stirred at
ꢀ78 °C for 30 min with ozone-bubbling. Dimethyl sulfide
(5.95 ml) was added to the mixture at ꢀ78 °C, and the reaction
was then allowed to be warmed to room temperature. The solvent
was evaporated, and the residue was partitioned between water
and chloroform. The organic phase was washed with brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(n-hexane/ethyl acetate = 10/1) to give 6-formylpyridine-2-carbo-
nitrile (1.72 g, 80%) as white solid. 1H NMR (400 MHz, CDCl3) d
7.93 (1H, dd, J = 7.8, 1.5 Hz), 8.07 (1H, t, J = 7.8 Hz), 8.17 (1H, dd,
J = 7.8, 1.5 Hz), 10.07 (1H, d, J = 1.0 Hz). MS (GC) 132 [M]. To a solu-
tion of 6-formylpyridine-2-carbonitrile (600 mg) in methanol
(12 ml) was added sodium borohydride (172 mg), and the mixture
was stirred at room temperature for 30 min. The solvent was evap-
orated, and the residue was purified by silica gel column chroma-
tography (chloroform/methanol = 50/1) to give the title compound
15 (460 mg, 76%) as white solid. 1H NMR (400 MHz, CDCl3) d 3.18
(1H, t, J = 5.2 Hz), 4.83 (2H, d, J = 5.2 Hz), 7.56 (1H, dd, J = 8.0,
0.4 Hz), 7.63 (1H, dd, J = 8.0, 0.4 Hz), 7.86 (1H, t, J = 8.0 Hz). MS
(FAB) 135 m/z [M+1]+.
5.1.6. 6-(Aminomethyl)-N,N-dimethylpyridin-2-amine (9b)
Compound 9b was prepared according to the procedure de-
scribed for 9a from 8b. The title compound 9b (994 mg, 64% in
two steps) was obtained as colorless oil. 1H NMR (400 MHz,
DMSO-d6) d 1.65–1.85 (2H, br), 3.00 (6H, s), 3,60 (2H, s), 6.44
(1H, d, J = 8.4 Hz), 6,57 (1H, d, J = 7.2 Hz), 7.41–7.45 (1H, m). MS
(FAB) m/z 152 [M+1]+.
5.1.7. tert-Butyl [6-(aminomethyl)pyridin-2-yl]carbamate (11)
To a mixture of {6-[(tert-butoxycarbonyl)amino]pyridin-2-yl}-
methyl methanesulfonate11 (2.82 g) in DMF (30 ml) was added so-
dium azide (910 mg) at ice-chilled temperature, and the mixture
was stirred at room temperature for 4 h. The reaction mixture
was partitioned between water and ethyl acetate, and the organic
phase was washed with brine, dried over magnesium sulfate, fil-
tered, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (n-hexane/ethyl acetate = 5/1) to give
tert-butyl [6-(azidomethyl)pyridin-2-yl]carbamate (2.29 g, 98%) as
colorless oil. 1H NMR (400 MHz, DMSO-d6) d 1.47 (9H, s), 4.41 (2H,
s), 7.06 (1H, dd, J = 6.0, 2.4 Hz), 7.75–7.78 (2H, m), 9.77 (1H, s). MS
(FAB) 250 [M+1]+. tert-Butyl [6-(azidomethyl)pyridin-2-yl]carba-
mate (1.55 g) was hydrogenated in the presence of Pd–C (10%,
300 mg) in ethanol (20 ml) for 2 h. The catalyst was removed by fil-
tration, and the filtrate was concentrated in vacuo to give the title
compound 11 (1.38 g, quantitative yield) as colorless oil. 1H NMR
(400 MHz, DMSO-d6) d 1.46 (9H, s), 3.69 (2H, s), 4.30–4.40 (2H,
br), 7.06 (1H, d, J = 7.2 Hz), 7.58–7.69 (2H, m), 9.57 (1H, s) MS
(FAB) m/z 224 [M+1]+.
5.1.11. 6-Aminomethylpyridine-2-carbonitrile (16)
To a solution of 15 (460 mg), phthalimide (505 mg) and triphen-
ylphosphine (900 mg) in THF (10 ml) was added DEAD (40% in tol-
uene, 1.58 ml), and the mixture was stirred overnight at room
temperature. Resulting precipitate was collected by filtration,
washed by THF, and dried in vacuo to give 6-[(1,3-dioxo-1,3-dihy-
dro-2H-isoindol-2-yl)methyl]pyridine-2-carbonitrile (366 mg, 41%)
as pale yellow solid. 1H NMR (400 MHz, CDCl3) d 5.05 (2H, s),
7.50 (1H, d, J = 7.6 Hz), 7.59 (1H, d, J = 7.6 Hz), 7.76–7.82 (3H, m),
7.89–7.92 (2H, m). MS (FAB) m/z 264 [M+1]+. A mixture of 6-
[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]pyridine-2-car-
bonitrile (365 mg) and hydrazine hydrate (0.074 ml) in methanol
(5 ml) and chloroform (10 ml) was stirred overnight at room tem-
perature. The solvent was evaporated, and the residue was purified
by silica gel column chromatography (chloroform/methanol = 10/
1) to give the title compound 16 (40 mg, 22%) as colorless amor-
phous solid. 1H NMR (400 MHz, CDCl3) d 1.85–1.95 (2H, br), 3.96
(2H, s), 7.49–7.55 (2H, m), 7.75 (1H, t, J = 8.0 Hz). MS (GC) m/z
133 [M].
5.1.8. 6-Chloropyridine-2-carbonitrile (13)12
To a solution of 2-chloropyridin-N-oxide (11.2 g) hydrochloride
in methanol (100 ml) was added potassium methoxide (4.59 g) at
ice-chilled temperature, and the mixture was stirred for 10 min.
After evaporating the solvent, ethanol was added to the residue
and insoluble matter was removed by filtration. The filtrate was
concentrated in vacuo, and to the residue was added dimethyl sul-
fate (6.20 ml), then the mixture was stirred at room temperature
for 3 h. The mixture was washed by diethyl ether using decanta-
tion manner, and to the residue were added water (140 ml) and so-
dium cyanide (12.8 g). The mixture was stirred at 0 °C, and
resulting brown solid was collected by filtration. The crude solid
was purified by silica gel column chromatography (chloroform/
methanol = 50/1) to give the title compound 13 (4.08 g, 45%) as
white solid. 1H NMR (400 MHz, CDCl3) d 7.58 (1H, dd, J = 8.4,
0.8 Hz), 7.65 (1H, dd, J = 7.6, 0.8 Hz), 7.82 (1H, dd, J = 8.4, 7.6 Hz).
MS (EI) m/z 138 [M].
5.1.12. 3-(2-Phenylvinyl)pyridazine (18)14
A
mixture of 3-methylpyridazine (1.83 ml), benzaldehyde
(4.07 ml) and zinc chloride (545 mg) was heated at 150 °C for 2 h.
After the reaction mixture was cooled to room temperature, that
was partitioned between aqueous sodium hydroxide and chloroform.
The organic phase was washed with water and brine, dried over mag-
nesium sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (chloroform/metha-

I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
3137
nol = 20/1) to give the title compound 18 (2.19 g, 60%) as pale brown
solid. 1H NMR (400 MHz, CDCl3) d 7.32–7.47 (5H, m), 7.59–7.73 (4H,
m), 9.06 (1H, dd, J = 4.8, 1.6 Hz). MS (FAB) m/z 183 [M+1]+.
(2H, d, J = 6.0 Hz), 4.74–4.90 (1H, br), 6.33 (1H, d, J = 2.4 Hz),
6.50 (1H, s), 6.98 (1H, d, J = 7.8 Hz), 7.00–7.08 (1H, br), 7.18
(1H, td, J = 7.8, 1.4 Hz), 7.26 (1H, t, J = 7.8 Hz), 7.30–7.36 (3H,
m), 7.58 (1H, d, J = 6.9 Hz), 7.84 (1H, s), 8.38 (1H, d, J = 2.4 Hz),
8.53–8.56 (2H, m), 8.99 (1H, s). MS (FAB) m/z 548 [M+1]+. Anal.
Calcd for C29H24ClF2N5O2ꢁHClꢁ0.5H2O: C, 58.69; H, 4.42; N,
11.80; Cl, 11.95; F, 6.40. Found: C, 58.48; H, 4.27; N, 11.71; Cl,
11.92; F, 6.19.
5.1.13. Pyridazin-3-carboxaldehyde (19)
A mixture of 18 (1.57 g), sodium periodate (3.57 g), osmium tet-
raoxide (2.5 wt.% in t-BuOH, 11.0 ml), acetone (15 ml), t-BuOH
(15 ml) and water (15 ml) was stirred at room temperature for
2 days. Insoluble matter was removed by filtration, and the filtrate
was concentrated in vacuo. The residue was purified by silica gel
column chromatography (chloroform/methanol = 50/1) to give
the title compound 19 (730 mg, 81%) as colorless oil. 1H NMR
(400 MHz, CDCl3) d 7.71 (1H, dd, J = 8.4, 5.2 Hz), 8.08 (1H, dd,
J = 8.4, 2.0 Hz), 9.40 (1H, dd, J = 5.2, 2.0 Hz), 10.41–10.42 (1H, m).
MS (GC) m/z 108 [M].
5.1.17. (Z)-N-Benzyl-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}acetamide (1d)
A
mixture of 1q (220 mg), benzylamine (56 mg), WSCD
(150 mg) and HOBt (104 mg) in DMF (20 ml) was stirred overnight
at room temperature. The reaction mixture was partitioned be-
tween aqueous sodium hydrogen carbonate and ethyl acetate, then
the organic phase was washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The residue was crys-
tallized and recrystallized (ethyl acetate/diethyl ether) to give
the title compound 1d (229 mg, 87%) as colorless crystals. Mp:
168–169 °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), 4.40 (2H, d,
J = 5.9 Hz), 4.76–4.90 (1H, br), 6.33 (1H, d, J = 2.5 Hz), 6.41 (1H, s),
6.97 (1H, d, J = 7.8 Hz), 6.97–7.07 (1H, br), 7.17 (1H, t, J = 7.8 Hz),
7.25 (1H, t, J = 7.8 Hz), 7.32 (1H, d, J = 7.3 Hz), 7.35–7.37 (5H, m),
7.55 (1H, d, J = 8.3 Hz), 7.83 (1H, s), 8.37 (1H, d, J = 2.5 Hz),8.89
(1H, s). MS (FAB) m/z 547 [M+1]+. Anal. Calcd for C30H25ClF2N4O2:
C, 65.87; H, 4.61; N, 10.24; Cl, 6.48; F, 6.95. Found: C, 65.75; H,
4.52; N, 10.23; Cl, 6.42; F, 6.90.
5.1.14. Pyridazin-3-ylmethylamine (20)
Pyridazin-3-carbaldehyde oxime (64 mg, 52%), which is the
intermediate for the title compound 20, was prepared according
to the procedure described for 8a from 19 (108 mg). 1H NMR
(400 MHz, CDCl3) d 3.50 (1H, s), 7.50 (1H, dd, J = 8.8, 4.8 Hz), 8.01
(1H, dd, J = 8.8, 2.0 Hz), 8.52 (1H, s), 9.16 (1H, dd, J = 4.8, 2.0 Hz).
MS (EI) 123 [M]. Pyridazin-3-carbaldehyde oxime (60 mg) was
hydrogenated in the presence of Pd–C (10%, 60 mg) in ethanol
(10 ml) for 1 h. The catalyst was removed by filtration, and the fil-
trate was concentrated in vacuo to give the title compound 20
(55 mg, quantitative yield) as pale yellow oil. 1H NMR (400 MHz,
CDCl3) d 1.60–1.80 (2H, br), 4.21 (2H, s), 7.46 (1H, dd, J = 8.4,
4.8 Hz), 7.53 (1H, dd, J = 8.4, 1.6 Hz), 9.10 (1H, dd, J = 4.8, 1.6 Hz).
MS (EI) m/z 109 [M].
5.1.18. (Z)-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-[(6-methyl-2-pyridyl)methyl]acetamide
hydrochloride (1e)
5.1.15. (Z)-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-3-ylmethyl)acetamide hydrochloride (1b)
A mixture of 1q (300 mg), 3-(aminomethyl)pyridine (71 mg), 1-
Compound 1e was prepared according to the procedure de-
scribed for 1b from 1q and 1-(6-methylpyridin-2-yl)methan-
amine.15 The title compound 1e (343 mg, 87%) was obtained as
colorless crystals. Mp: 185–188 °C; 1H NMR (400 MHz, DMSO-d6)
d 2.22 (3H, s), 2.35–2.55 (1H, br), 2.70 (3H, s), 2.70–2.85 (1H, br),
3.12–3.30 (1H, br), 4.67 (2H, br s), 4.75–4.90 (1H, br), 6.34 (1H,
d, J = 2.5 Hz), 6.53 (1H, s), 6.99 (1H, d, J = 7.8 Hz), 7.00–7.12 (1H,
br), 7.19 (1H, td, J = 7.8, 1.5 Hz), 7.26 (1H, d, J = 7.8 Hz), 7.33 (1H,
d, J = 7.8 Hz), 7.55–7.70 (3H, m), 7.84 (1H, s), 8.23–8.33 (1H, br),
8.39 (1H, d, J = 2.5 Hz), 9.23 (1H, br s). MS (FAB) m/z 562 [M]+. Anal.
Calcd for C30H26ClF2N5O2ꢁHClꢁ1.3H2O: C, 57.94; H, 4.80; N, 11.26;
Cl, 11.40; F, 6.11. Found: C, 58.02; H, 4.41; N, 11.27; Cl, 11.07; F,
6.23.
ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(WSCD) (192 mg) and 1-hydroxy-1H-benzotriazole (HOBt)
(135 mg) in DMF (15 ml) was stirred at room temperature for
3 h. The reaction mixture was partitioned between aqueous so-
dium hydrogen carbonate and ethyl acetate, then the organic
phase was washed with brine, dried over magnesium sulfate, fil-
tered, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (chloroform/methanol = 50/1) to yield
the salt-free form of 1b, which was dissolved in chloroform and
subsequently treated with hydrochloride (4 M in ethyl acetate).
Resulting precipitate was collected by filtration and dried in vacuo
to give the title compound 1b (221 mg, 57%) as colorless amor-
phous solid. 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), 4.57 (2H, s),
4.76–4.90 (1H, br), 6.34 (1H, d, J = 2.4 Hz), 6.52 (1H, s), 6.98 (1H,
d, J = 7.3 Hz), 6.96–7.14 (1H, br), 7.18 (1H, t, J = 7.3 Hz), 7.25 (1H,
t, J = 7.3 Hz), 7.32 (1H, d, J = 7.4 Hz), 7.58 (1H, d, J = 8.8 Hz), 7.83
(1H, s), 7.95–8.02 (1H, m), 8.38 (1H, d, J = 2.4 Hz), 8.40 (1H, s),
8.81 (1H, s), 8.82 (1H, s), 9.15 (1H, s). MS (FAB) m/z 548 [M+1]+.
Anal. Calcd for C29H24ClF2N5O2ꢁ0.3C4H8Oꢁ0.2C2H6Oꢁ1.3HClꢁ1.3H2O:
C, 56.57; H, 4.89; N, 10.78; Cl, 12.55; F, 5.85. Found: C, 56.37; H,
4.64; N, 10.91; Cl, 12.65; F, 5.66.
5.1.19. (Z)-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-[(6-methoxy-2-pyridyl)methyl]acetamide
hydrochloride (1f)
Compound 1f was prepared according to the procedure de-
scribed for 1b from 1q and 9a. The title compound 1f (289 mg,
71%) was obtained as colorless crystals. Mp: 165–167 °C; 1H
NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.35–2.55 (1H, br),
2.70–2.90 (1H, br), 3.05–3.30 (1H, br), 3.87 (3H, s), 4.40 (2H, d,
J = 4.9 Hz), 4.75–5.00 (1H, br), 6.34 (1H, d, J = 2.4 Hz), 6.48 (1H, s),
6.72 (1H, d, J = 8.3 Hz), 6.95–6.97 (2H, m), 7.00–7.10 (1H, br),
7.18 (1H, td, J = 7.8, 1.5 Hz), 7.26 (1H, td, J = 7.8, 1.5 Hz), 7.33 (1H,
dd, J = 7.8, 1.5 Hz), 7.57 (1H, d, J = 8.3 Hz), 7.72 (1H, dd, J = 8.3,
7.3 Hz), 7.83 (1H, s), 8.38 (1H, d, J = 2.4 Hz), 8.98 (1H, br s). MS
(FAB) m/z 578 [M]+. Anal. Calcd for C30H26ClF2N5O3ꢁHClꢁ0.2H2O:
C, 58.30; H, 4.47; N, 11.33; Cl, 11.47; F, 6.15. Found: C, 58.15; H,
4.46; N, 11.28; Cl, 11.14; F, 6.26.
5.1.16. (Z)-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-4-ylmethyl)acetamide hydrochloride (1c)
Compound 1c was prepared according to the procedure de-
scribed for 1b from 1q and 4-(aminomethyl)pyridine. The title
compound 1c (247 mg, 64%) was obtained as colorless crystals.
Mp: 272–274 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s),
2.30–2.55 (1H, br), 2.57–2.90 (1H, br), 3.10–3.31 (1H, br), 4.43

3138
I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
5.1.20. (Z)-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-[(6-dimethylamino-2-pyridyl)methyl]acetamide
hydrochloride (1h)
MS (FAB) m/z 549 [M+1]+. Anal. Calcd for C28H23ClF2N6O2ꢁ0.2H2O:
C, 60.86; H, 4.27; N, 15.21; Cl, 6.42; F, 6.88. Found: C, 60.75; H,
4.26; N, 15.09; Cl, 6.30; F, 6.89.
Compound 1h was prepared according to the procedure de-
scribed for 1b from 1q and 9b. The title compound 1h (199 mg,
73%) was obtained as white solid. Mp: 113–116 °C (decomposed);
1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.35–2.55 (1H, br),
2.70–2.85 (1H, br), 3.10–3.35 (1H, br), 3.24 (6H, s), 4.55 (2H, br
s), 4.73–4.90 (1H, br), 6.34 (1H, d, J = 2.4 Hz), 6.53 (1H, s), 6.80
(1H, d, J = 6.3 Hz), 6.99 (1H, d, J = 7.3 Hz), 6.99–7.12 (1H, br), 7.19
(1H, t, J = 7.3 Hz), 7.27 (1H, t, J = 7.3 Hz), 7.34 (1H, d, J = 7.3 Hz),
7.58 (1H, d, J = 8.3 Hz), 7.84(1H, s), 7.86–7.94 (1H, br), 7.96 (1H,
s), 8.38 (1H, d, J = 2.4 Hz), 9.08–9.30 (1H, br). MS (FAB) m/z 591
[M+1]+. Anal. Calcd for C31H29ClF2N6O2ꢁHClꢁ2H2O: C, 56.11; H,
5.16; N, 12.67; Cl, 10.69; F, 5.73. Found: C, 56.06; H, 5.28; N,
12.62; Cl, 10.67; F, 5.46.
5.1.24. (Z)-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-(pyrimidin-4-ylmethyl)acetamide (1l)
Compound 1l was prepared according to the procedure de-
scribed for 1d from 1q and 1-(pyrimidin-4-yl)methanamine. The
title compound 1l (246 mg, 22%) was obtained as colorless crystals.
Mp: 208–210 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.35–
2.55 (1H, br), 2.65–2.90 (1H, br), 3.15–3.30 (1H, br), 4.48 (2H, d,
J = 5.8 Hz), 4.75–4.92 (1H, br), 6.34 (1H, d, J = 2.4 Hz), 6.52 (1H, s),
6.98 (1H, d, J = 7.8 Hz), 7.00–7.12 (1H, br), 7.18 (1H, td, J = 7.8,
1.5 Hz), 7.26 (1H, t, J = 7.8 Hz), 7.34 (1H, d, J = 7.8 Hz), 7.50 (1H,
d, J = 4.9 Hz), 7.58 (1H, d, J = 6.8 Hz), 7.84 (1H, s), 8.38 (1H, d,
J = 2.5 Hz), 8.81 (1H, d, J = 4.9 Hz), 9.08–9.16 (2H, m). MS (FAB)
m/z 549 [M+1]+. Anal. Calcd for C28H23ClF2N6O2: C, 61.26; H,
4.22; N, 15.31; Cl, 6.46; F, 6.92. Found: C, 61.22; H, 4.21; N,
14.99; Cl, 6.37; F, 6.97.
5.1.21. (Z)-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-[(6-cyano-2-pyridyl)methyl]acetamide (1i)
Compound 1i was prepared according to the procedure de-
scribed for 1d from 1q and 16. The title compound 1i (31 mg,
18%) was obtained as colorless crystals. Mp: 197–199 °C; 1H
NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.25–2.55 (1H, br),
2.60–2.90 (1H, br), 3.05–3.30 (1H, br), 4.53 (2H, br s), 4.47–4.57
(1H, br), 6.34 (1H, d, J = 2.4 Hz), 6.50 (1H, s), 6.98 (1H, d,
J = 7.8 Hz), 6.99–7.08 (1H, br), 7.18 (1H, td, J = 7.8, 1.5 Hz), 7.25
(1H, t, J = 7.8 Hz), 7.32 (1H, dd, J = 7.8, 1.5 Hz), 7.57 (1H, d,
J = 8.8 Hz), 7.73 (1H, d, J = 8.3 Hz), 7.84(1H, s), 7.97 (1H, d,
J = 8.3 Hz), 8.10 (1H, d, J = 8.3 Hz), 8.38 (1H, d, J = 2.4 Hz), 9.14
(1H, br s). MS (FAB) m/z 573 [M+1]+. Anal. Calcd for
C30H23ClF2N6O2ꢁ0.25C3H8Oꢁ0.1HClꢁ0.5H2O: C, 61.49; H, 4.38; N,
13.99; Cl, 6.49; F, 6.33. Found: C, 61.69; H, 4.49; N, 13.67; Cl,
6.59; F, 6.15.
5.1.25. (Z)-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-(thiazol-2-ylmethyl)acetamide (1m)
Compound 1m was prepared according to the procedure de-
scribed for 1d from 1q and 1-(1,3-thiazol-2-yl)methanamine.17
The title compound 1m (271 mg, 74%) was obtained as colorless
crystals. Mp: 218–219 °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),
4.69 (2H, d, J = 4.3 Hz), 4.74–4.90 (1H, br), 6.33 (1H, d, J = 2.5 Hz),
6.45 (1H, s), 6.98 (1H, d, J = 7.5 Hz), 6.98–7.10 (1H, br), 7.18 (1H,
t, J = 7.5 Hz), 7.25 (1H, t, J = 7.5 Hz), 7.32 (1H, d, J = 7.5 Hz), 7.56
(1H, d, J = 8.6 Hz), 7.68 (1H, d, J = 3.3 Hz), 7.76 (1H, d, J = 3.3 Hz),
7.83 (1H, s), 8.38 (1H, d, J = 2.5 Hz), 9.28 (1H, s). MS (FAB) m/z
554 [M]+. Anal. Calcd for C27H22ClF2N5O2Sꢁ0.5H2O: C, 57.60; H,
4.12; N, 12.44; S, 5.70; Cl, 6.30; F, 6.75. Found: C, 57.78; H, 4.02;
N, 12.33; S, 6.10; Cl, 6.01; F, 6.43.
5.1.22. (Z)-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-(pyrazin-2-ylmethyl)acetamide hydrochloride (1j)
Compound 1j was prepared according to the procedure de-
scribed for 1b from 1q and 1-pyrazin-2-ylmethanamine.16 The title
compound 1j (324 mg, 84%) was obtained as white amorphous so-
lid. 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), 4.61 (2H, d, J = 5.8 Hz), 4.76–
4.90 (1H, br), 6.33 (1H, d, J = 2.4 Hz), 6.49 (1H, s), 6.97 (1H, d,
J = 7.3 Hz), 6.97–7.10 (1H, br), 7.17 (1H, t, J = 7.3 Hz), 7.25 (1H, t,
J = 7.3 Hz), 7.32 (1H, d, J = 7.2 Hz), 7.57 (1H, d, J = 8.3 Hz), 7.83
(1H, s), 8.37 (1H, d, J = 2.4 Hz), 8.57 (1H, d, J = 2.4 Hz), 8.61–8.63
(1H, m), 8.68 (1H, s), 9.13 (1H, s). MS (FAB) m/z 549 [M+1]+. Anal.
Calcd for C28H23ClF2N6O2ꢁ0.9HClꢁ1.5H2O: C, 55.49; H, 4.42; N,
13.87; Cl, 11.11; F, 6.27. Found: C, 55.54; H, 4.28; N, 13.45; Cl,
10.97; F, 5.73.
5.1.26. (Z)-N-(1,3-Benzothiazol-2-ylmethyl)-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}acetamide (1n)
Compound 1n was prepared according to the procedure de-
scribed for 1d from 1q and 1-(1,3-benzothiazol-2-yl)methan-
amine.18 The title compound 1n (281 mg, 70%) was obtained as
colorless crystals. Mp: 204–205 °C; 1H NMR (400 MHz, DMSO-d6)
d 2.22 (3H, s), 2.30–2.55 (1H, br), 2.67–2.90 (1H, br), 3.10–3.24
(1H, br), 4.76–4.94 (3H, m), 6.33 (1H, d, J = 2.5 Hz), 6.48 (1H, s),
6.99 (1H, d, J = 7.8 Hz), 7.00–7.12 (1H, br), 7.18 (1H, t, J = 7.6 Hz),
7.26 (1H, t, J = 7.3 Hz), 7.34 (1H, d, J = 7.8 Hz), 7.45 (1H, t,
J = 7.6 Hz), 7.49–7.58 (2H, m),7.84 (1H, s), 7.98 (1H, d, J = 7.9 Hz),
8.12 (1H, d, J = 7.9 Hz), 8.38 (1H, d, J = 2.4 Hz), 9.39 (1H, s). MS
(FAB) m/z 604 [M]+. Anal. Calcd for C31H24ClF2N5O2S: C, 61.64; H,
4.00; N, 11.59; S, 5.31; Cl, 5.87; F, 6.29. Found: C, 61.46; H, 4.09;
N, 11.50; S, 5.24; Cl, 5.87; F, 6.31.
5.1.23. (Z)-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-(pyridazin-3-ylmethyl)acetamide (1k)
5.1.27. (Z)-N-(1H-Benzoimidazol-2-ylmethyl)-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}acetamide
Compound 1k was prepared according to the procedure de-
scribed for 1d from 1q and 20. The title compound 1k (105 mg,
38%) was obtained as pale yellow crystals. Mp: 211–213 °C; 1H
NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.27–2.55 (1H, br),
2.65–2.85 (1H, br), 3.10–3.30 (1H, br), 4.63–4.73 (2H, br), 4.74–
4.88 (1H, br), 6.33 (1H, d, J = 2.4 Hz), 6.49 (1H, s), 6.98 (1H, d,
J = 7.3 Hz), 6.96–7.12 (1H, br), 7.18 (1H, t, J = 7.3 Hz), 7.25 (1H, t,
J = 7.3 Hz), 7.32 (1H, d, J = 7.3 Hz), 7.57 (1H, d, J = 8.3 Hz), 7.66
(1H, d, J = 8.3 Hz), 7.74 (1H, dd, J = 4.9 Hz, 8.3 Hz), 7.83 (1H, s),
8.38 (1H, d, J = 2.4 Hz), 9.12–9.19 (1H, br), 9.17 (1H, d, J = 4.9 Hz).
hydrochloride (1o)
Compound 1o was prepared according to the procedure de-
scribed for 1b from 1q and 1-(1H-benzimidazol-2-yl)methan-
amine. The title compound 1o (67 mg, 16%) was obtained as
white solid. Mp: 199–201 °C; 1H NMR (400 MHz, DMSO-d6) d
2.23 (3H, s), 2.40–2.50 (1H, br), 2.67–2.89 (1H, br), 3.11–3.23
(1H, br), 4.75–4.90 (1H, br), 4.83 (2H, s), 6.35 (1H, d, J = 2.4 Hz),
6.53 (1H, s), 7.00 (1H, d, J = 7.8 Hz), 7.02–7.15 (1H, br), 7.20 (1H,

I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
3139
t, J = 7.8 Hz), 7.27 (1H, t, J = 7.8 Hz), 7.32 (1H, d, J = 7.8 Hz), 7.50
(2H, dd, J = 6.1,3.2 Hz), 7.61 (1H, d, J = 8.7 Hz), 7.79 (2H, dd,
J = 6.1,3.2 Hz), 7.83 (1H, s), 8.38 (1H, d, J = 2.4 Hz), 9.39 (1H, s).
MS (FAB) m/z 587 [M]+. Anal. Calcd for C31H25ClF2N6O2ꢁHClꢁ1.5H2O:
C, 57.24; H, 4.49; N, 12.92; Cl, 10.90; F, 5.84. Found: C, 57.45; H,
4.58; N, 12.86; Cl, 10.71; F, 5.63.
(1H, br), 2.65–2.90 (1H, br), 3.15–3.35 (1H, br), 3.23 (1H, q,
J = 5.8 Hz), 3.48 (1H, m), 4.73 (1H, t, J = 5.3 Hz), 4.76–4.88 (1H,
br), 6.33 (1H, d, J = 2.5 Hz), 6.36 (1H, s), 6.97 (1H, d, J = 7.3 Hz),
6.98–7.07 (1H, br), 7.16 (1H, dt, J = 1.5, 7.3 Hz), 7.24 (1H, t,
J = 7.3 Hz), 7.31 (1H, dd, J = 1.5, 7.3 Hz), 7.55 (1H, d, J = 8.3 Hz),
7.83 (1H, s), 8.37 (1H, d, J = 2.5 Hz), 8.39–8.47 (1H, br). MS (FAB)
m/z 501 [M+1]+. Anal. Calcd for C25H23ClF2N4O3: C, 59.94; H,
4.63; N, 11.18; Cl, 7.08; F, 7.59. Found: C, 59.68; H, 4.68; N,
11.09; Cl, 6.89; F, 7.63.
5.1.28. (Z)-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-(2-furylmethyl)acetamide (1p)
Compound 1p was prepared according to the procedure de-
scribed for 1d from 1q and 1-(2-furyl)methanamine. The title
compound 1p (158 mg, 45%) was obtained as colorless crystals.
Mp: 187–188 °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), 4.38
(2H, d, J = 5.4 Hz), 4.75–4.90 (1H, br), 6.30–6.35 (2H, m), 6.41
(1H, s), 6.41–6.45 (1H, m), 6.96 (1H, d, J = 7.8 Hz), 6.96–7.10
(1H, br), 7.17 (1H, t, J = 7.3 Hz), 7.24 (1H, t, J = 7.3 Hz), 7.30
(1H, d, J = 7.3 Hz), 7.55 (1H, d, J = 6.9 Hz), 7.61 (1H, s), 7.83 (1H,
s), 8.36 (1H, d, J = 2.4 Hz), 8.89 (1H, s). MS (FAB) m/z 537
[M+1]+. Anal. Calcd for C28H23ClF2N4O3: C, 62.63; H, 4.32; N,
10.43; Cl, 6.60; F, 7.08. Found: C, 62.59; H, 4.40; N, 10.39; Cl,
6.56; F, 6.93.
5.1.32. (Z)-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-(2-dimethylaminoethyl)acetamide hydrochloride
(1u)
Compound 1u was prepared according to the procedure de-
scribed for 1b from 1q and N,N-dimethylethane-1,2-diamine. The
title compound 1u (50 mg, 19%) was obtained as white solid.
Mp: 172–174 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s),
2.30–2.55 (1H, br), 2.70–2.90 (7H, br), 3.05–3.20 (3H, br), 3.48–
3.60 (2H, br), 4.75–4.90 (1H, br), 6.34 (1H, d, J = 2.5 Hz), 6.44 (1H,
s), 6.99 (1H, d, J = 7.3 Hz), 6.99–7.11 (1H, br), 7.19 (1H, td, J = 7.3,
2.0 Hz), 7.25 (1H, td, J = 7.3, 2.0 Hz), 7.30 (1H, dd, J = 7.3, 2.0 Hz),
7.57 (1H, d, J = 8.3 Hz), 7.83 (1H, s), 8.38 (1H, d, J = 2.5 Hz) 8.58–
8.67 (1H, br),. MS (FAB) m/z 528 [M+1]+. Anal. Calcd for
C27H28ClF2N5O2ꢁHClꢁ3H2O: C, 52.43; H, 5.70; N, 11.32; Cl, 11.46;
F, 6.14. Found: C, 52.08; H, 5.70; N, 11.17; Cl, 11.36; F, 6.00.
5.1.29. (Z)-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}acetamide (1r)
To a mixture of 1q (210 mg) in 1,2-dichloroethane (20 ml) was
added thionyl chloride (2.0 ml), and the mixture was stirred at
room temperature for 30 min. Reaction mixture was evaporated
and subsequently azeotroped with toluene. A solution of the resi-
due in acetonitrile was poured into aqueous ammonia, then result-
ing precipitate was collected by filtration and dried in vacuo to give
the title compound 1r (259 mg, quantitative yield) as white solid.
Mp: 235–236 °C; 1H NMR (400 MHz, DMSO-d6) d 2.20 (3H, s),
2.30–2.55 (1H, br), 2.60–2.85 (1H, br), 3.11–3.23 (1H, br), 4.75–
4.90 (1H, br), 6.33 (1H, d, J = 2.4 Hz), 6.34 (1H, s), 6.95 (1H, d,
J = 7.3 Hz), 6.94–7.10 (1H, br), 7.17 (1H, t, J = 7.3 Hz), 7.21–7.31
(2H, m), 7.34 (1H, s), 7.57 (1H, d, J = 8.1 Hz), 7.83 (1H, s), 7.85
(1H, s), 8.37 (1H, d, J = 2.4 Hz). MS (FAB) m/z 457 [M+1]+. Anal.
Calcd for C23H19ClF2N4O2ꢁ0.4NH3: C, 59.58; H, 4.39; N, 13.29; Cl,
7.65; F, 8.19. Found: C, 59.80; H, 4.31; N, 13.55; Cl, 7.58; F, 8.08.
5.1.33. (Z)-N-Carbamoylmethyl-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}acetamide (1w)
Compound 1w was prepared according to the procedure de-
scribed for 1d from 1q and glycinamide. The title compound
1w (158 mg, 61%) was obtained as colorless crystals. Mp: 157–
159 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.35–2.52
(1H, br), 2.68–2.85 (1H, br), 3.10–3.30 (1H, br), 3.71–3.81 (2H,
m), 4.60–5.00 (1H, br), 6.33 (1H, d, J = 2.5 Hz), 6.42 (1H, s), 6.98
(1H, d, J = 7.3 Hz), 6.95–7.13 (1H, br), 7.10–7.17 (1H, br), 7.17
(1H, t, J = 7.3 Hz), 7.25 (1H, t, J = 7.3 Hz), 7.25–7.33 (1H, br),
7.34 (1H, d, J = 7.3 Hz), 7.57 (1H, d, J = 6.8 Hz), 7.84 (1H, s), 8.38
(1H, d, J = 2.5 Hz), 8.59–8.70 (1H, br). MS (FAB) m/z 513 [M]+.
Anal. Calcd for C25H22ClF2N5O3ꢁ0.2H2O: C, 58.02; H, 4.36; N,
13.53; Cl, 6.85; F, 7.34. Found: C, 57.98; H, 4.31; N, 13.46; Cl,
6.84; F, 7.61.
5.1.30. (Z)-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-propylacetamide (1s)
5.1.34. (Z)-N-[(6-Aminopyridin-2-yl)methyl]-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}acetamide
Compound 1s was prepared according to the procedure de-
scribed for 1d from 1q and propan-1-amine. The title compound
1s (20 mg, 8%) was obtained as colorless crystals. Mp: 196–
198 °C; 1H NMR (400 MHz, DMSO-d6) d 0.91 (3H, t, J = 7.3 Hz),
1.50 (2H, sextet, J = 7.3 Hz), 2.22 (3H, s), 2.30–2.50 (1H, br), 2.65–
2.85 (1H, br), 3.00–3.25 (3H, br), 4.75–4.90 (1H, br), 6.33 (1H, d,
J = 2.0 Hz), 6.36 (1H, s), 6.97 (1H, d, J = 7.8 Hz), 6.98–7.08 (1H, br),
7.16 (1H, td, J = 7.8, 1.5 Hz), 7.24 (1H, t, J = 7.8 Hz), 7.30 (1H, d,
J = 7.8 Hz), 7.56 (1H, d, J = 8.8 Hz), 7.83 (1H, s), 8.33–8.39 (1H, br),
8.37 (1H, d, J = 2.0 Hz). MS (FAB) m/z 499 [M+1]+. Anal. Calcd for
C26H25ClF2N4O2ꢁ0.2H2O: C, 62.14; H, 5.09; N, 11.15; Cl, 7.05; F,
7.56. Found: C, 62.07; H, 5.26; N, 11.01; Cl, 6.98; F, 7.39.
hydrochloride (1g)
Compound 4 (270 mg, 62%) was prepared according to the pro-
cedure described for 1d from 1q and 11 as colorless oil. MS (FAB)
m/z 663 [M]+. Compound 4 (270 mg) was treated with 4 M hydro-
chloride in ethyl acetate (20 ml) at room temperature for 12 h. Eth-
anol (0.5 ml) and ethyl acetate (20 ml) were added to the mixture,
then the resulting precipitate was collected and dried in vacuo to
give the title compound 1g (74 mg, 30%) as white solid. Mp:
185–189 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.30–
2.50 (1H, br), 2.65–2.85 (1H, br), 3.10–3.30 (1H, br), 4.47 (2H, d,
J = 7.3 Hz), 4.65–5.05 (1H, br), 6.34 (1H, d, J = 2.5 Hz), 6.57 (1H, s),
6.78 (1H, d, J = 7.3 Hz), 6.90 (1H, d, J = 9.3 Hz), 6.99 (1H, d,
J = 7.8 Hz), 7.03–7.14 (1H, br), 7.19 (1H, dt, J = 1.9, 7.8 Hz), 7.26
(1H, t, J = 7.8 Hz), 7.32 (1H, d, J = 2.0 Hz), 7.62 (1H, d, J = 7.3 Hz),
7.83 (1H, s), 7.92 (1H, t, J = 7.8 Hz), 8.03–8.16 (2H, br), 8.37 (1H,
d, J = 2.4 Hz), 9.15 (1H, s), 13.79–14.20 (1H, br). MS (FAB) m/z
563 [M]+. Anal. Calcd for C29H25ClF2N6O2ꢁHClꢁ1.3H2O: C, 55.92; H,
4.63; N, 13.49; Cl, 11.38; F, 6.10. Found: C, 56.01; H, 4.57; N,
13.11; Cl, 11.47; F, 6.08.
5.1.31. (Z)-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-(2-hydroxyethyl)acetamide (1t)
Compound 1t was prepared according to the procedure de-
scribed for 1d from 1q and 2-aminoethanol. The title compound
1t (425 mg, 85%) was obtained as colorless crystals. Mp: 181–
183 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.25–2.52

3140
I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
5.1.35. Methyl (Z)-(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}acetylamino)acetate (5)
to 1 ꢂ 10ꢀ5 mol/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
Compound 5 was prepared according to the procedure de-
scribed for 1d from 1q and glycine methyl ester. The title com-
pound 5 (420 mg, 53%) was obtained as colorless crystals. 1H
NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.30–2.55 (1H, br),
2.65–2.80 (1H, br), 3.15–3.30 (1H, br), 3.67 (3H, s), 3.98 (2H, d,
J = 5.9 Hz), 4.75–4.90 (1H, br), 6.33 (1H, d, J = 2.5 Hz), 6.41 (1H, s),
6.98 (1H, d, J = 7.8 Hz), 6.99–7.09 (1H, br), 7.17 (1H, td, J = 7.8,
1.4 Hz), 7.25 (1H, t, J = 7.6 Hz), 7.32 (1H, d, J = 7.3 Hz), 7.57 (1H,
d, J = 8.3 Hz), 7.83 (1H, s), 8.38 (1H, d, J = 2.4 Hz), 8.87 (1H, br s).
MS (FAB) m/z 529 [M+1]+.
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 the mean SEM. Statistical
analysis was performed using a SAS software (SAS Institute, USA).
Specific binding was calculated as total binding minus nonspecific
binding. The concentration of each compound required to reduce
specific binding of [3H]vasopressin by 50% (IC50 value) was ob-
tained by non-linear regression analysis. A Kd value of [3H]vaso-
pressin for each vasopressin receptor was yielded by Scatchard
plot analysis. The affinity constants (Ki values) were calculated
from the following equation,20 using the Kd values yielded from
each separate experiment. Ki = IC50/(1 + [[3H]vasopressin concen-
tration]/Kd).
5.1.36. (Z)-(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}acetylamino)acetic acid (1v)
A mixture of 5, 1 M aqueous sodium hydroxide in methanol was
stirred overnight at room temperature. The mixture was neutralized
with 1 M aqueous hydrochloride, and that was extracted with chlo-
roform. The organic phase was washed with brine, dried over mag-
nesium sulfate, filtered, and concentrated in vacuo. The residue
was crystallized and recrystallized (n-hexane/ethyl acetate = 3/2) to
give the title compound 1v (116 mg, 48%) as colorless crystals. Mp:
167–169 °C; 1H NMR (400 MHz, DMSO-d6) d 2.22 (3H, s), 2.30–2.55
(1H, br), 2.60–2.85 (1H, br), 3.10–3.25 (1H, br), 3.89 (2H, br s),
4.75–4.95 (1H, br), 6.33 (1H, d, J = 2.5 Hz), 6.39 (1H, s), 6.97 (1H, d,
J = 7.9 Hz), 6.95–7.11 (1H, br), 7.17 (1H, td, J = 7.9, 1.4 Hz), 7.25
(1H, t, J = 7.9 Hz), 7.33 (1H, d, J = 7.9 Hz), 7.57 (1H, d, J = 8.3 Hz),
7.83 (1H, s), 8.38 (1H, d, J = 2.5 Hz), 8.76 (1H, br s), 12.64 (1H, br
s). MS (FAB) m/z 515 [M+1]+. Anal. Calcd for C25H21ClF2N4O4: C,
58.31; H, 4.11; N, 10.88; Cl, 6.89; F, 7.38. Found: C, 58.02; H, 4.10;
N, 10.81; Cl, 6.73; F, 7.13.
5.2.2. Binding assay for human V1a receptor
The binding assay for human V1a receptor was performed in a
manner similar to that for human V2 receptor.
5.2.3. 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.21 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
5.2. Biology
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
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.).22
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 mean 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.19 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
5.2.4. Antidiuretic effect in water-loaded rats
binding study, 50
lL of drug solution and 50
l
L of [3H]vasopressin
L of
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
(final concentration of 0.91 nmol/L) were mixed with 150
l
membrane suspension in 50 mmol/L Tris–HCl (pH 7.4) buffer con-
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 (PerkinElmer) 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 (PerkinElmer) using the scintillation cocktail (MicroScin-
TM
ti-40 , PerkinElmer). Nonspecific binding or total binding were
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

I. Tsukamoto et al. / Bioorg. Med. Chem. 17 (2009) 3130–3141
3141
were performed using SAS (version 8.2, SAS Institute Japan). The re-
sults are expressed as the mean (95% confidence interval) or the
mean standard error.
study, verapamil, a potent and specific CYP3A4 inhibitor, was
used as the positive control.
Acknowledgments
5.3. CYP inhibitory activity
The authors deeply acknowledge Dr. Mitsuaki Ohta, Dr. Minoru
Okada and Dr. Seijiro Akamatsu for their helpful support to this re-
search. The authors are also grateful to the staff of the Division of
Analytical Science Laboratories for elemental analysis and spectral
measurement.
5.3.1. Fluorescent P450 inhibition assay (CYP2C9)23
Using a 96-well plate and 7-methoxy-4-trifluoromethylcouma-
rin (75
20 M) was incubated with rCYP2C9 bactsome (3.1 pmol, pur-
chased from Cypex Ltd). These incubations were conducted for
30 min at 37 °C in 200 L (total volume) of 25 mM phosphate buf-
fer (pH 7.4) and in the presence of a NADPH-generating system
(8.2
M NADP+, 0.41 mM glucose-6-phosphate, 0.41 mM MgCl2,
lM) as a substrate, each test compound (from 0.31 to
l
References and notes
l
1. (a) Hardman, J. G.; Limbird, L. E. Goodman & Gilman’s The Pharmacological Basis
of Therapeutics, 10th ed.; McGraw-Hill: New York, 2001; (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.
2. Thibonnier, M.; Roberts, J. M. J. Clin. Invest. 1985, 76, 1857.
3. (a) Laszlo, F. A.; Laszlo, F.; De Weid, D. Pharmacol. Rev. 1991, 43, 73; (b) Bouby,
N.; Bachmann, S.; Bichet, D.; Bankir, L. Am. J. Physiol. 1990, 258, F973.
4. (a) Asplund, R.; Sundberg, B.; Bengtsson, P. BJU Int. 1999, 83, 591; (b) Weiss, J.
P.; Blaivas, J. G. J. Urol. 2000, 163, 5.
l
and 0.4 units/mL glucose-6-phosphate dehydrogenase). Sulfaphen-
azole, a potent and specific CYP2C9 inhibitor, was used as the po-
sitive control in each study. The reaction was terminated by adding
0.5 M 2-amino-2-hydroxymethyl-1,3-propanediol aqueous solu-
tion containing 80% acetonitrile, and the fluorescence intensity
(excitation wavelength: 409 nm, fluorescence wavelength:
530 nm) was measured using a fluorescence plate reader.
5. (a) Dimson, S. B. Lancet 1977, 1, 1260; (b) Tuvemo, T. Acta Pediatr. Scand. 1978,
67, 753.
6. (a) 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; (b) 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.
7. (a) 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; (b) 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.
5.3.2. Fluorescent P450 inhibition assays (CYP2C19)23
The CYP2C19 inhibition assay was performed in a manner sim-
ilar to that for CYP2C9. The positive control (tranylcypromine) was
incubated for 20 min with substrate (3-cyano-7-ethoxycoumarin,
25 lM), enzyme (2.4 pmol), and phosphate buffer (50 mM). The
excitation and fluorescence wavelengths used were 409 nm and
460 nm, respectively.
8. Tsukamoto, I.; Koshio, H.; Kuramochi, T.; Akamatsu, S.; Saitoh, C.; Yanai-
Inamura, H.; Kitada, C.; Yamamoto, E.; Yatsu, T.; Sakamoto, S.; Tsukamoto, S.
Bioorg. Med. Chem. 2008, 16, 9524.
9. Shimada, Y.; Akane, H.; Taniguchi, N.; Matsuhisa, A.; Kawano, N.; Tanaka, A.
Chem. Pharm. Bull. 2005, 53, 589.
5.3.3. Fluorescent P450 inhibition assays (CYP2D6)23
The CYP2D6 inhibition assay was performed in a manner similar
to that for CYP2C9. The positive control (quinidine) was incubated
for 15 min with substrate (3-[2-(N,N-diethyl-N-methylamino)-
ethyl]-7-methoxy-4-methylcoumarin, 1.5 lM), enzyme (5.0 pmol),
and phosphate buffer (100 mM). The excitation and fluorescence
10. Ager, D. J.; Prakash, I. Synth. Commun. 1995, 25, 739.
11. Mitsuya, M.; Kobayashi, K.; Kawakami, K.; Satoh, A.; Ogino, Y.; Kakikawa, T.;
Ohtake, N.; Kimura, T.; Hirose, H.; Sato, A.; Numazawa, T.; Hasegawa, T.;
Noguchi, K.; Mase, T. J. Med. Chem. 2000, 43, 5017.
12. Elman, B. Tetrahedron 1985, 41, 4941.
13. Stille, J. K. Angew. Chem. 1986, 98, 504.
wavelengths used were 390 nm and 460 nm, respectively.
14. Bunnage, M.E.; Mathias, J.P.; Street, S.D.A.; Wood, A. Patent U.S. 6,251,904.
15. Catherine, B.; Catherine Bougault; Marie-Therese, A.; Andre, D.; Jean-Louis, P.;
Jean-Marc, L.; Laurent Le, P. J. Am. Chem. Soc. 2001, 123, 8053.
16. Abushanab, E.; Bindra, A. P.; Goodman, L.; Petersen, H. J. Org. Chem. 1973, 38,
2049.
5.3.4. Competitive inhibition of CYP3A4 in human liver
microsomes
Midazolam 10-hydroxylation was used to monitor CYP3A4
activity. Each test compound (0.3ꢀ100
l
M) was co-incubated
17. Alessandro, D.; Francisxo, M.; Pedro, M.; Isabel, R.; Tomas, T. Synthesis 1996, 5,
641.
18. Ozawa, T.; Kitagawa, H.; Yamamoto, Y.; Takahata, S.; Iida, M.; Osaki, Y.;
Yamada, K. Bioorg. Med. Chem. Lett. 2007, 15, 7325.
19. Tahara, A.; Saito, M.; Sugimoto, T.; Tomura, Y.; Wada, K.; Kusayama, T. Br. J.
Pharmacol. 1998, 125, 1463.
20. Lazareno, S.; Bridsall, N. Br. J. Pharmacol. 1993, 109, 1110.
21. 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,
1463.
with midazolam (2 M) in the presence of human liver micro-
l
somes (0.1 mg/mL) and NADPH (1.0 mM) at 37 °C for 20 min. At
the end of the incubation, the reaction was terminated by adding
an aqueous solution containing 80% acetonitrile. The concentra-
tion of 10-hydroxymidazolam was determined using LC–MS/MS
analysis. The inhibition of CYP3A4 activity was assessed by com-
paring the amount of 10-hydroxymidazolam formed in the pres-
ence of varying concentrations of inhibitor to the amount of
10-hydroxymidazolam formed in the solvent control. In each
22. Takasaki, J.; Kamohara, M.; Saito, T.; Matsumoto, M.; Matsumoto, S.; Ohishi, T.;
Soga, T.; Matsushime, H.; Furuichi, K. Mol. Pharmacol. 2001, 60, 432.
23. Crespi, C. L.; Miller, V. P.; Penman, B. W. Analyt. Biochem. 1997, 248, 188.