Synthesis and Structure-Activity Relationships of 5,6,7,8-Tetrahydro-4H-thieno[3,2-b]azepine Derivatives: Novel Arginine Vasopressin Antagonists

Article abstract of DOI:10.1021/jm030287l

A variety of novel heterocyclic compounds having thienoazepine, pyrroloazepine, furoazepine, and thienodiazepine skeletons were synthesized, most of which exhibited potent antagonism of [³H]-AVP specific binding in assays using rat liver (V1), rat kidney (V2), human platelet plasma membranes, and recombinant human CHO cells (V2), as well as antagonizing AVP-induced hypertension in rats (V1, intravenous) and showing a diuretic effect in rats (V2, oral). By detailed studies of the structure-activity relationships of these compounds, the thienoazepine derivative 1 was found to be a very potent combined V1 and V2 antagonist. After further pharmacological and toxicological evaluation as well as physical properties, the hydrochloride 2 (JTV-605) of compound 1 was selected for clinical studies as a potent AVP antagonist with a long duration of action.

Full text of DOI:10.1021/jm030287l

J . Med. Chem. 2004, 47, 101-109
101
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of
5,6,7,8-Tetr a h yd r o-4H-th ien o[3,2-b]a zep in e Der iva tives: Novel Ar gin in e
Va sop r essin An ta gon ists
Hidetsura Cho,* Kengo Murakami, Hiroyuki Nakanishi, Akitaka Fujisawa, Hirotaka Isoshima, Misako Niwa,
Kazuhide Hayakawa, Yasunori Hase, Itsuo Uchida, Hidenori Watanabe, Korekiyo Wakitani, and Kazuo Aisaka
Central Pharmaceutical Research Institute, J apan Tobacco, Inc., 1-1, Murasaki-cho, Takatsuki, Osaka, 569-1125, J apan
Received J une 13, 2003
A variety of novel heterocyclic compounds having thienoazepine, pyrroloazepine, furoazepine,
and thienodiazepine skeletons were synthesized, most of which exhibited potent antagonism
of [3H]-AVP specific binding in assays using rat liver (V1), rat kidney (V2), human platelet
plasma membranes, and recombinant human CHO cells (V2), as well as antagonizing AVP-
induced hypertension in rats (V1, intravenous) and showing a diuretic effect in rats (V2, oral).
By detailed studies of the structure-activity relationships of these compounds, the thienoazepine
derivative 1 was found to be a very potent combined V1 and V2 antagonist. After further
pharmacological and toxicological evaluation as well as physical properties, the hydrochloride
2 (J TV-605) of compound 1 was selected for clinical studies as a potent AVP antagonist with
a long duration of action.
In tr od u ction
Arginine vasopressin (AVP) is a neurohypophyseal
nonapeptide that has a wide variety of physiological
activities in mammals. It plays an important role in
regulating water and solute excretion by the kidneys
and also participates in other physiological functions
such as blood pressure control, regulation of platelet
aggregation, and ACTH secretion. Therefore, AVP may
be involved in various conditions such as heart failure,
hypertension, angina, hyponatremia, and dysmenor-
rhea.1 After development of the early peptide AVP
antagonists that lacked oral bioavailability and had a
short half-life,2 the orally effective nonpeptide antago-
nists OPC-21268 (a selective V1a antagonist), OPC-
31260 (compound 3) (a selective V2 antagonist),3 SR-
49059 (a selective V1a antagonist), SR-121463 (a V2
antagonist),4 YM-087 (V1 and V2 dual antagonists),5
and VP-343 (a V2 antagonist)6 were synthesized. We
began synthetic studies searching for more potent
combined V1 and V2 antagonists as well as selective
V2 antagonists. We constructed several basic heterocy-
clic skeletons, including thienoazepine,7 pyrroloazepine,
furoazepine, and thienodiazepine8 and discovered de-
F igu r e 1. Novel thienoazepines and AVP antagonist 3.
rivatives that exhibited potent AVP antagonism.9 Com-
pound 4, one of the compounds we were studying, was
Evaluation of the pharmacological and toxicological
properties of compound 1 led us to choose its hydro-
chloride 2 for clinical evaluation. In this report we
independently patented by American Cyanamid.10 How-
ever, this compound did not show good oral absorption
in rats. Therefore, we continued investigations to im-
disclose synthetic methods and bioactivity data of
prove oral absorption as well as potency and duration.
compound 1 and analogues.
Using molecular modeling, we found compound 1 among
a set of compounds which showed satisfactory bioavail-
ability and extremely potent activities as AVP antago-
nists. The discovery of a series of potent AVP antagonist
analogues containing a central benzene ring substituted
with a nitrogen-containing side chain was new to AVP
research.
Ch em istr y
We developed new heterocyclic fused azepines and
diazepines for the basic skeleton. As we reported previ-
ously,7 DIBAH (diisobutylaluminum hydride) reduction
of heterocyclic fused cyclohexanone oxime a gave regi-
oselectively heterocyclic fused azepine b as the sole
compound. Benzoylation of compound b afforded com-
pound c which was successively treated with hydrogen
* To whom correspondence should be addressed. Phone: +81-72-
681-9700. Fax: +81-72-681-9725. E-mail: hidetsura.cho@ims.jti.co.jp.
10.1021/jm030287l CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/27/2003

102 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Sch em e 1. Synthesis of Compounds 4-9, 13-15
Cho et al.
Sch em e 2. Synthesis of Compounds 10-12, 16a
a
Reaction conditions: (1) DIBAH, (2) 4-nitrobenzoyl chloride, 3-methoxy-4-nitrobenzoyl chloride, or 2-methoxy-4-nitrobenzoyl chloride/
Et3N, (3) H2/Pd-C, (4) 2-phenylbenzoyl chloride, 2-methylbenzoyl chloride, or 2-chlorobenzoyl chloride/Et3N, (5) NaBH4, (6) MsCl/pyridine,
(7) amine derivatives, (8) LiAlH4.
Sch em e 3a
a
Reaction conditions: (9) MsCl, pyridine, (10) amine deriva-
tives, K2CO3, KI, (11) H2, PtO2, (12) 2-phenylbenzoyl chloride,
Et3N, (13) 3-pyridinylmethyl chloride or benzyl chloride, K2CO3,
KI, (14) chloroacetyl chloride, Et3N, (15) amine derivative, KI.
(see Scheme 1 and Figure 2). On the other hand,
azepines having a dialkylamino group were synthesized
F igu r e 2. A list of thienoazepines, furoazepines, pyr-
as shown in Scheme 2. Namely, reduction of 5,6,7,8-
roloazepines, and thienodiazepines.
tetrahydro-4H-thieno[3, 2-b]azepine-5, 8-dione d 11 with
on Pd-C and substituted benzoyl chloride to give
5,6,7,8-tetrahydrothieno[3,2-b]azepines 4-9 and 13-15
NaBH4 yielded the alcohol, which was treated with
mesyl chloride to give compound e. Nucleophilic sub-

Novel Arginine Vasopressin Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 103
Ta ble 1. Data of Specific Binding of Thienoazepines 4, 8-16, Furoazepine 5, Pyrroloazepines 6 and 7, and Thienodiazepines 17-22
to AVP Receptor, Antagonistic Effect on AVP-Induced Hypertension, and Diuretic Effect
[3H]-AVP binding IC50 (nM)a
compound
no.
blood pressure:b
ID50 (mg/kg, iv)
urine volume:b
(mL/4 h) [dose mg/kg, po]
V1 rat
V2 rat
V1 human
V2 human
3
4
5
6
7
5000 ( 140
290 ( 20
2100 ( 150
390 ( 28
300 ( 22
>10000
300 ( 30
310 ( 25
300 ( 30
200 ( 15
380 ( 36
5100 ( 450
200 ( 15
20 ( 2
2,000 ( 90
300 ( 20
2900 ( 280
2100 ( 120
210 ( 16
4000 ( 250
290 ( 25
2000 ( 90
780 ( 100
2200 ( 160
490 ( 35
50 ( 3
500 ( 15
5.0 ( 0.4
>10000
>10000
60 ( 6
>10000
>10000
30 ( 3
<10
>0.3
9.3 ( 0.7 [30], 2.9 ( 0.1 [10]
0.4 ( 0.2 [30]
n.t.
0.11 ( 0.05
n.t.c
0.07 ( 0.01
0.15 ( 0.07
>0.3
n.t.
0.8 ( 0.4 [30]
n.t.
n.t.
8
9
1900 ( 160
480 ( 14
2000 ( 30
3200 ( 300
960 ( 100
110 ( 10
200 ( 20
2000 ( 140
200 ( 15
300 ( 27
500 ( 45
>10000
>0.3
10
11
12
13
14
15
16
17
18
19
20
21
22
>0.3
9.9 ( 0.8 [30], 7.6 ( 1.3 [10]
2.9 ( 0.4 [10]
3.8 ( 0.4 [10]
3.5 ( 0.2 [30]
4.5 ( 0.5 [30], 2.5 ( 0.2 [10]
2.3 ( 0.1 [10]
12.0 ( 1.1 [30], 9.3 ( 1.6 [10]
6.3 ( 0.4 [10]
6.7 ( 0.5 [10]
1.2 ( 0.2 [10]
n.t.
100 ( 15
50 ( 5
n.t.
50 ( 3
30 ( 3
<10
n.t.
>0.3
30 ( 2
<10
0.11 ( 0.02
0.04 ( 0.01
>0.3
<10
60 ( 4
<10
10 ( 3
3000 ( 250
800 ( 55
400 ( 35
700 ( 50
>10000
400 ( 35
1000 ( 90
20 ( 2
200 ( 20
80 ( 10
60 ( 5
>10000
80 ( 15
50 ( 3
10 ( 3
>0.3
20 ( 2
0.31 ( 0.06
0.14 ( 0.05
n.t.
30 ( 3
100 ( 10
70 ( 5
500 ( 50
200 ( 20
>0.3
>0.3
1.2 ( 0.4 [10]
7.4 ( 0.7 [10]
10 ( 3
a
V1 rat; liver, V1 human; platelet, V2 rat; adrenal medulla, V2 human; recombinant, CHO cells, n ) 3, mean ( SEM (standard
errors). Rat; n ) 3, mean ( SEM. c Not tested.
b
Sch em e 4a
a
Reaction conditions: (16) BBr3 or AlCl3/NaI, (17) BrCH2COOEt or Br(CH2)3COOEt/KI, K2CO3, (18) KOH-H2O, (19) Br(CH2)3Cl or
Br(CH2)4Cl, K2CO3, (20) ClCOOiBu, Et3N, amine derivatives, (21) amine derivatives, KI.
stitution of compound e with several amines and suc-
cessive reduction of the carbonyl group with LiAlH4
yielded compound g, which was converted into com-
pounds 10-12, and 16 by the same procedures as shown
in Scheme 1. As for 5,6,7,8-tetrahydro-4H-thieno[2,3-
b][1,4]diazepine derivatives, compounds 17-22 were
prepared by the procedure reported previously (see
Scheme 3 and Figure 2).8
Next, introduction of new substituents at position A
or B on the benzene ring was performed in order to find
more potent thienoazepine derivatives. As described
later (Table 1), by addition of a suitable nitrogen-
containing chain, good bioavailability, and very potent
V1 and V2 antagonistic activity were obtained. Thus,
compound 14 was converted into compound 15 using
BBr3 or AlCl3/NaI (see Scheme 4). Treatment of com-
pound 15 with ethyl 4-bromobutyrate in the presence
of KI and K2CO3, followed by hydrolysis with KOH, gave
a carboxylic acid 35, which was converted to compound
1 by successive treatment with isobutyl chloroformate
(IBCF) and 4-dimethylaminopiperidine/triethylamine.
Successively, compounds 23-27 were obtained using
either ethyl bromoacetate or ethyl 4-bromobutyrate and
the procedure mentioned above. Compounds 28-30

104 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Cho et al.
methoxy group to the middle benzene ring increased the
inhibition of [3H]-AVP binding. Thus, compounds 14 and
15 exhibited potent V2 (rat and human) as well as V1
(human) antagonistic activity. As for the position of
methoxy group substitution, it was found that the
2-position (B) was preferable to the 3-position (A) for
stronger activity by comparing compounds 14 and 13.
Despite improved potency in these studies, compounds
13-15 had an unsatisfactory effect on urine output in
vivo, compared to compound 3. To achieve potent V2
antagonistic activity in vivo, several substituted or
unsubstituted dialkylamino groups were introduced at
position-8 of the azepine ring. Compounds 10 and 12
were thus obtained, which showed increased oral ab-
sorption and strong V2 antagonism. In particular,
compound 10 exhibited satisfactory V2 selectivity.
Introduction of a substitution at position-8 seemed to
be crucial for V2 selectivity. When a methoxy group was
added to position-2 on the benzene ring, compound 16
was obtained, which was superior to compound 3 in both
potency and V2 selectivity in vitro and in vivo (effect
on urine output) (see Table 1).
Next, we studied the AVP antagonistic activity of
compounds with a thienodiazepine skeleton. Compounds
17-22, synthesized as in our previous report,8 showed
fairly potent V2 receptor binding, but did not always
have good V1 binding. The diuretic effect in vivo
depended upon substitution of the alkyl group on a
nitrogen atom at position-8; a 2-(pyrrolidin-1-yl)ethyl,
a 2-(dimethylamino)ethyl, and a 2-(4-methylpiperazin-
1-yl)ethyl group on the nitrogen atom increased the
diuretic effect, while a benzyl or a pyridin-3-ylmethyl
group reduced it. Thus, compounds 17, 18, and 22 were
comparable in activity to compound 3, but were unfor-
tunately chemically unstable.
F igu r e 3. A list of thienoazepines with a nitrogen-containing
side chain.
were synthesized by alkylation of 15 with ω-haloalkyl
bromide and substitution of the resulting alkyl halide
with the corresponding amines (see Scheme 4 and
Figure 3).
Based on the structure-activity studies mentioned
above, thienoazepines 10 and 16, which were more
potent and more V2-selective than compound 3, were
obtained by introduction of a suitable group at position-8
of the tetrahydrothieno[3,2-b]azepine ring. The finding
that compounds 14 and 15, with a methoxy or a hydroxy
group on the benzene ring and without any substitution
at position-8 of the thienoazepine ring, showed combined
V1 and V2 antagonism prompted us to search for more
potent combined antagonists. In our overlap studies
using the Discover program from Accelrys (see molec-
ular modeling experiments.), suitable overlaps were
observed between the thiophene moiety and the phenyl
group of tyrosine, between the 2-phenyl group on the
biphenyl group and the phenyl group of phenylalanine,
between the carbonyl group near the azepine ring and
that of cysteine-1, and between the carbonyl group near
the biphenyl group and that of phenylalanine (see
Figure 4a and 4b). The N-Me nitrogen of the side chain
of compound 1 seemed to fit the arginine nitrogen of
the AVP molecule. Thus, we supposed that the nitrogen
containing side chain on the middle benzene ring might
contribute more potent activity. Therefore, we first
synthesized compound 1 designed in this way. Addition
of a side chain at position-B on the middle benzene ring
resulted in good bioavailability and extremely strong
specific binding to the AVP receptor (V1; human plate-
lets and V2; recombinant human CHO cells), as well as
Resu lts a n d Discu ssion
Initially, we performed [3H]-AVP binding assays using
rat liver membranes (for V1), rat kidney membranes (for
V2), human platelet plasma membranes (for V1), and
recombinant human CHO cell membranes (for V2) and
successively studied the hypotensive effect (V1, iv) in
rats with AVP-induced hypertension and the diuretic
effect (V2, po) in rats (see Experimental Section). First,
we compared three types of heterocyclic azepines,
thienoazepine, furoazepine, and pyrroloazepine deriva-
tives 4-7. Among them, thienoazepine 4 was very
potent in the human V2 binding assay (IC50 ) 5 nM) as
N-methyl pyrroloazepine 7 (IC50 ) 60 nM). Compounds
4 and 7 were more potent than compound 3, except in
the rat V2 binding assay, but they did not increase 4 h
urine output even at 30 mg/kg. Presumably, this was
due to the nonpolarity of these compounds. Since furo-
azepine 5 and pyrroloazepine 6 were very weak in the
human V2 binding assay, we selected the thienoazepine
skeleton for synthesis of other derivatives. A variety of
R group substituents on the benzene ring were studied
and it was found that the 2-phenyl group was essential
for potent antagonistic activity by comparing this group
with the 2-chloro and 2-methyl groups (see Figure 2).
Substitution at the 2-position was found to be crucial,
because a compound with 4-phenyl substitution on the
benzene ring showed weaker activity (IC50 ) >10 µM)
in the V1 and V2 binding assays. Introduction of a

Novel Arginine Vasopressin Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 105
F igu r e 4. (a) Correlation of functional groups between AVP and 1. (b) Overlay of 1 (white) and AVP (green).
Ta ble 2. Data of Specific Binding of Thienoazepines 1, 23-30 to AVP Receptor, Antagonistic Effect on AVP-induced Hypertension,
and Diuretic Effect
[3H]-AVP binding IC50 (nM)a
compound
no.
blood pressure:b
ID50 (mg/kg, iv)
urine volume:b
(mL/4 h) [dose mg/kg, po]
V1 rat
V2 rat
V1 human
V2 human
1
23
24
25
26
27
28
29
30
6.0 ( 0.4
20 ( 1.2
20 ( 1.1
4.0 ( 0.3
20 ( 1.6
20 ( 1.9
20 ( 1.5
2.0 ( 0.1
3.0 ( 0.2
3.0 ( 0.2
3.0 ( 0.3
1.0 ( 0.1
6.0 ( 0.5
20 ( 1.8
4.0 ( 0.3
8.0 ( 0.8
<1
3.0 ( 0.3
50 ( 4.0
10 ( 0.9
4.0 ( 0.3
10 ( 0.1
50 ( 4.5
5.0 ( 0.1
<1
20 ( 1.0
10 ( 1.0
6.0 ( 0.3
9.0 ( 0.8
30 ( 2.8
8.0 ( 0.7
80 ( 4.3
2.0 ( 0.1
1.0 ( 0.1
0.005 ( 0.002
0.008 ( 0.001
0.03 ( 0.01
0.01 ( 0.005
0.03 ( 0.01
0.02 ( 0.01
0.02 ( 0.01
0.03( 0.01
<0.01
22.0 ( 1.1 [10]
19.1 ( 2.4 [10]
17.6 ( 1.2 [10]
18.0 ( 1.0 [10]
8.0 ( 0.6 [10]
15.0 ( 1.8 [10]
10.2 ( 1.0 [10]
18.3 ( 2.2 [10]
16.9 ( 1.4 [10]
7.0 ( 0.5
5.0 ( 0.1
a
V1 rat; liver, V1 human; platelet, V2 rat; adrenal medulla, V2 human; recombinant, CHO cells, n ) 3, mean ( SEM (standard
b
errors). Rat; n ) 3, mean ( SEM.
potent antagonism of AVP-induced hypertension and a
marked diuretic (Table 2). Compound 1 showed antago-
nism of AVP-induced hypertension and had a potent
diuretic effect as well as showing very strong V1 and
V2 receptor binding. We then modified the side chain
in various ways. The piperidine ring of compound 1 was
replaced with an N-methylhomopiperazine ring (com-
pound 23) or a 4-N-methylpiperazine ring (compound
24), and the N-Me2 group of 1 was replaced with an
N-Et2 group (compound 25) or a piperidine ring (com-
pound 26). The carbonyl group of 1 was changed into a
methylene group and the piperidine ring of 1 was
converted to a 4-N-methylpiperazine group to give
compound 30. Compound 28 with a cleaved piperazine
ring was also prepared, as well as compound 27 with a
shortened methylene chain and compound 29 without
the carbonyl group of compound 23. Although com-
pounds 1, 23-25, 29-30 showed the desired potency
in every assay, compound 1 exhibited the most potent
diuretic effect and hypotensive effect as well as the
strong specific binding to V1 and V2 receptors. In
toxicological evaluations in rats, compounds 24 and 30
were excluded because of hepatic toxicity. The mecha-
nism of action of hydrochloride salt 2 of compound 1
proved to be antagonistic on V2 receptor of human
platelet. Compound 2 was obtained as a fine crystalline
form, while the salts of 23-25 and 29-30 were not
obtained as crystals but as amorphous powders. Com-
pound 2 was found to be the most desirable compound
because of its favorable physical properties (crystalliza-
tion, pharmaceutical stability, etc.), oral absorption,
toxicological profile, and ease of production. Therefore,

106 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Cho et al.
The mixture was washed with water and brine, dried over
MgSO4, and evaporated to give a residue, which was purified
by SiO2 column chromatography (n-hexane:EtOAc ) 2:1) to
afford compound 37 (884 mg, 96.6%). NMR (CDCl3) δ ppm.
1.80-2.05 (4H, m), 2.17-2.40 (1H, m), 2.37 (6H, s), 3.50-3.60
(1H, m), 3.86 (3H, s), 6.17 (1H, d, J ) 3.7 Hz), 6.87 (1H, d, J
) 3.7 Hz), 6.98 (1H, d, J ) 6.8 Hz), 7.13 (1H, brs), 7.65 (1H, d,
J ) 6.8 Hz). MS (FAB, m/z) ) 376 (M+ + 1). Anal. (C18H21N3O4S)
C, H, N.
compound 1 (2) was chosen for further pharmacological
evaluations.
In conclusion, by detailed studies of the structure-
activity relationships of heterocyclic compounds having
thienoazepine, pyrroloazepine, furoazepine, and thieno-
diazepine skeletons, compound 1 (2) was found to be a
very potent combined V1 and V2 antagonist, while
compound 16 was superior to compound 3 (selective V2
antagonist) with respect to potency and V2 selectivity
both in vitro and in vivo. After further pharmacological
and toxicological evaluation (data will be reported
elsewhere) as well as the physical properties (crystal-
lization and stability), the hydrochloride 2 of compound
1 was finally selected for clinical studies as a potent dual
V1 and V2 AVP antagonist.
N-[4-[[8-Dim eth yla m in o-5,6,7,8-tetr a h yd r o-4H-th ien o-
[3,2-b]a zep in -4-yl]ca r bon yl]-2-m et h oxyp h en yl]-1, 1′-b i-
p h en yl-2-ca r boxa m id e (16). To a stirred solution of com-
pound 37 (417 mg, 1.11 mmol) in acetic acid (4.0 mL) were
added Fe powder (248 mg, 4.43 mmol) and water (0.06 mL).
After stirring at 50 °C for 4 h, the reaction was quenched with
CHCl3 (20 mL) and the mixture was filtered through Celite.
The filtrate was made basic with an aqueous solution of 1 N
NaOH (150 mL) and extracted with AcOEt. The organic layer
was washed with brine, dried over MgSO4, and evaporated to
give a residue, which was dissolved in CHCl3 (15 mL). To the
resulting solution were added 2-phenylbenzoyl chloride and
Et3N (0.46 mL, 3.31 mmol), and the mixture was stirred for 3
h at room temperature. [Preparation8,9 of 2-phenylbenzoyl
chloride: To 2-phenylbenzoic acid (264 mg, 1.33 mmol) in
CHCl3 (3 mL) was added thionyl chloride (1.75 mL, 24.0 mmol).
The mixture was refluxed for 4 h. The mixture was evaporated
under reduced pressure to give the residue, which was dis-
solved in toluene. Evaporation in vacuo gave 2-phenylbenzoyl
chloride. Without purification, the chloride was used in the
reaction.] The mixture was washed with saturated NaHCO3
aqueous solution and brine, dried over anhydrous MgSO4, and
evaporated to give a residue, which was purified by SiO2
column chromatography (n-hexane:AcOEt ) 1:1) to afford
compound 16 (374 mg, 64.2%), mp 168.2-168.8 °C (from Et2O).
IR (KBr) 1665, 1635 cm-1, NMR (CDCl3) δ ppm. 1.70-2.03
(4H, m), 2.14-2.30 (1H, m), 2.37 (6H, s), 3.44 (3H, s), 3.64 (1H,
s), 4.05 (1H, m), 6.15 (1H, brs), 6.78 (1H, d, J ) 5.0 Hz), 6.82
(1H, s), 6.90 (1H, d, J ) 8.3 Hz), 7.29-7.53 (8H, m), 7.69 (1H,
s), 7.81 (1H, d, J ) 7.6 Hz), 8.26 (1H, d, J ) 8.3 Hz). MS (FAB,
Exp er im en ta l Section
Gen er a l Meth od s. Ch em istr y. Melting points were de-
termined with a Yanagimoto (Yanako) micro melting point
1
apparatus (HK-10D) and are uncorrected. H NMR spectra
(δ, ppm) were recorded using a J EOL J NMA 300 (300 MHz)
spectrometer with tetramethylsilane as the internal standard,
and structure analysis of AVP was carried out using a Bruker
AMX-500 (500 MHz) spectrometer. IR spectra (cm-1) were
obtained with a Perkin-Elmer FT 1650 infrared spectrometer.
Mass spectra (FAB+) were recorded with a Finnigan TSQ 700
instrument. HPLC was performed with a Shimazu LC-6A
instrument, and thin-layer chromatography (TLC) was carried
out on Merck silica gel plates (60F-254). Column chromatog-
raphy was performed with Merck silica gel (70-230 mesh).
8-H yd r oxy-4,6,7,8-t e t r a h yd r ot h ie n o[3,2-b]a ze p in -5-
on e (35) (see Scheme 2). To a stirred suspension of 6,7-
dihydrothieno[3,2-b]azepin-5,8-dione (1.58 g, 8.78 mmol) in
MeOH (30 mL) was added NaBH4 (330 mg, 8.68 mmol) at room
temperature and stirring was continued for 3 h. The reaction
was quenched with water, and the mixture was extracted with
CHCl3. The organic layer was washed with brine, dried over
anhydrous MgSO4, and evaporated to give compound 35 (996
mg, 61.9%). NMR (DMSO-d6) δ ppm. 1.95-2. 25 (2H, m), 2.30-
2.45 (1H, m), 2.50-2.65 (1H, m), 4.60 (1H, t, J ) 4.5 Hz), 6.70
(1H, d, J ) 5.4 Hz), 7.39 (1H, d, J ) 5.4 Hz), 9.84 (1H, brs).
MS (FAB, m/z) ) 184 (M+ + 1). Anal. (C8H9NO2S) C, H, N.
8-Dim eth ylam in o-4,6,7,8-tetr ah ydr oth ien o[3,2-b]azepin -
5-on e (36). To a stirred solution of compound 35 (500 mg, 2.75
mmol) in pyridine (20 mL) was added methanesulfonyl chloride
(473 mg, 4.13 mmol) at 0 °C. After 4 h, the solvent was
removed in vacuo and the residue was dissolved in DMF (30
mL). To the solution were added K2CO3 (3.8 g, 27.5 mmol) and
dimethylamine hydrochloride (2.2 g, 27.0 mmol). Stirring was
continued for 18 h, and the solvent was removed in vacuo. To
the residue was added a saturated aqueous NaHCO3 solution,
and the mixture was extracted with CHCl3. The organic layer
was washed with water and brine, dried over MgSO4, and
evaporated to give compound 36 (270 mg, 47.0%). NMR
(DMSO-d6) δ ppm. 1.60-1.85 (1H, m), 1.95-2.15 (1H, m), 2.20
(6H, s), 2.30-2.60 (2H, m), 3.92 (1H, dd, J ) 12.0, 6.0 Hz),
6.65 (1H, d, J ) 6.0 Hz), 7.29 (1H, d, J ) 6.0 Hz), 9.69 (1H,
brs). MS (FAB, m/z) ) 211 (M+ + 1). Anal. (C10H14N2OS) C,
H, N.
+
m/z) ) 526 (M + 1). Anal. (C31H31N3O3S) C, H, N.
Eth yl 4-[2-[[[1,1′-Biph en yl]-2-car bon yl]am in o]-5-[(5,6,7,8-
tetr ah ydr o-4H-th ien o[3,2-b]azepin -4-yl)car bon yl]ph en oxy]-
bu tyr a te (31). To a stirred solution of compound 15 (1.0 g,
2.14 mmol) in acetone (85 mL) were added ethyl 4-bromobu-
tyrate (1.2 g, 6.15 mmol), anhydrous K2CO3 (0.87 g, 6.28
mmol), and KI (71 mg, 0.43 mmol). The mixture was refluxed
for 12 h, diluted with water, and extracted with CHCl3. The
organic layer was washed with brine, dried over anhydrous
Na2SO4, and evaporated to give a residue, which was purified
by SiO2 column chromatography (AcOEt:n-hexane ) 1:3) to
give compound 31 (921 mg, 73.8%). NMR (CDCl3) δ ppm. 1.24
(3H, t, J ) 7.2 Hz), 1.70-2.05 (6H, m), 2.27 (2H, t, J ) 7.2
Hz), 2.90-3.00 (2H, m), 3.60-4.00 (4H, m), 4.08 (2H, q, J )
7.2 Hz), 6.20 (1H, brs), 6.68 (1H, d, J ) 5.4 Hz), 6.75-6.80
(1H, m), 6.82 (1H, d, J ) 9.0 Hz), 7.25-7.60 (8H, m), 7.65 (1H,
s), 7.75-7.80 (1H, m), 8.30 (1H, d, J ) 8.0 Hz). MS (FAB, m/z)
+
) 583 (M + 1). Anal. (C34H34N2O5S) C, H, N.
4-[2-[[[1,1′-Bip h en yl]-2-ca r bon yl]a m in o]-5-[(5,6,7,8-tet-
r a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)ca r bon yl]p h en oxy]-
bu tyr ic Acid (32). To a stirred solution of compound 31 (820
mg, 1.41 mmol) in MeOH (100 mL) was added KOH (140 mg,
2.5 mmol). The reaction mixture was refluxed for 2 h, and the
solvent was removed under reduced pressure to give a residue,
which was dissolved in CHCl3 and an aqueous solution of 1 N
HCl (10 mL). The organic layer was separated and washed
with brine, dried over anhydrous Na2SO4, and evaporated to
yield the solid (32) (614 mg, 78.6%). IR(KBr): 1718, 1643, 1528
cm-1, NMR (DMSO-d6) δ ppm. 1.50-2.00 (6H, m), 2.25-2.40
(2H, m), 2.75-3.00 (2H, m), 3.50-4.00 (2H, m), 6.30 (1H, brs),
6.70-7.00 (3H, m), 7.25-7.70 (9H, m), 7.70-7.85 (1H, m), 9.03
(1H, s), 12.2 (1H, s). MS (FAB, m/z) ) 555 (M+ + 1). Anal.
(C32H30N2O5S) C, H, N.
8-Dim eth yla m in o-4-(3-m eth oxy-4-n itr oben zoyl)-5,6,7,8-
tetr a h yd r o-4H-th ien o[3,2-b]a zep in e (37). To a stirred sus-
pension of LiAlH4 (184 mg, 4.84 mmol) in anhydrous THF (40
mL) was added compound 36 (510 mg, 2.44 mmol), and the
mixture was refluxed for 3 h. After cooling, the reaction was
quenched with Et2O (80 mL), and the excess reagent was
carefully inactivated with the minimum amount of water. The
organic layer was decanted, dried over MgSO4, and evaporated
to give a residue, which was dissolved in CHCl3. To the
resulting solution were added Et3N (1.0 mL) and 3-methoxy-
4-nitrobenzoyl chloride [prepared from 3-methoxy-4-nitroben-
zoic acid (575 mg, 2.92 mmol) and thionyl chloride (3.0 mL,
41 mmol)9]. The mixture was stirred 14 h at room temperature.
N-[2-[4-(4-Dim eth yla m in op ip er id in o)-4-oxobu toxy]-4-
[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)ca r bon -
yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e (1). To a stirred

Novel Arginine Vasopressin Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 107
solution of compound 32 (100 mg, 0.18 mmol) in DMF (1.5 mL)
was added Et3N (96 µL, 0.69 mmol) under argon gas. Then
isobutyl chloroformate (29 µL, 0.22 mmol) was added at 0 °C.
After stirring for 30 min, a suspension of 4-(dimethylamino)-
piperidine dihydrochloride (46 mg, 0.23 mmol) and Et3N (64
µL, 0.46 mmol) in DMF (1.5 mL) was added. Stirring was
continued at 0 °C for 2 h and then at room temperature for 1
h. DMF was removed in vacuo (50 °C, 4-6 mmHg) to give a
residue, which was dissolved in AcOEt, and the resulting
mixture was washed with saturated aqueous solution of
NaHCO3. The organic layer was separated, dried over anhy-
drous MgSO4, and evaporated under reduced pressure to give
the solid (1) (120 mg). IR (KBr): 1640 cm-1. NMR (CDCl3) δ
ppm. 1.20-1.50 (2H, m), 1.50-2.10 (11H, m), 2.20-2.40 (8H,
m), 2.45-2.60 (1H, m), 2.80-3.00 (2H, m), 3.60-4.20 (4H, m),
4.50 (1H, d, J ) 12.0 Hz), 6.20 (1H, brs), 6.67 (1H, d, J ) 5.0
Hz), 6.70-6.85 (2H, m), 7.25-7.60 (8H, m), 7.70-7.80 (2H,
m), 8.26 (1H, d, J ) 8.0 Hz). MS (FAB, m/z) ) 665 (M+ + 1).
Anal. (C39H44N4O4S) C, H, N.
d, J ) 8.1 Hz). MS (FAB, m/z) ) 693 (M+ + 1). Anal.
(C41H48N4O4S) C, H, N.
N -[2-[4-(4-P ip e r id in o p ip e r id in o )-4-o x o b u t o x y ]-4-
[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)ca r bon -
yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e (26). Compound
26 (612 mg, 86.9%) was prepared using compound 32 (554 mg,
1.00 mmol) and 4-piperidinopiperidine (336 mg, 2.00 mmol)
as described for compound 1. IR (KBr) 1638, 1522 cm-1. NMR
(CDCl3) δ ppm. 1.25-1.60 (7H, m), 1.77 (4H, m), 1.91 (2H, t,
J ) 5.9 Hz), 1.95-2.05 (2H, m), 2.27 (2H, t, J ) 6.2 Hz), 2.40-
2.60 (6H, m), 2.80-2.85 (3H, m), 3.60-3.80 (6H, m), 4.53 (1H,
d, J ) 10.8 Hz), 6.19 (1H, brs), 6.67 (1H, d, J ) 4.6 Hz), 6.78
(1H, brs), 6.80 (1H, brs), 7.26-7.60 (7H, m), 7.75-7.77 (2H,
+
m), 8.26 (1H, d, J ) 7.0 Hz). MS (FAB, m/z) 705 (M + 1).
Anal. (C42H48N4O4S) C, H, N.
N-[2-[2-(4-Dim eth yla m in op ip er id in o)-2-oxoeth oxy]-4-
[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in 4-yl)ca r bon -
yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e (27). To a stirred
solution of compound 15 (374 mg, 0.80 mmol) in acetone (10
mL) and DMF (10 mL) were added ethyl bromoacetate (200
mg, 1.20 mmol), K2CO3 (112 mg, 0.81 mmol), and KI (120 mg,
0.73 mmol). The mixture was refluxed for 5 h, diluted with
water, and extracted with CHCl3. The organic layer was dried
over MgSO4 and evaporated to leave the residue (480 mg).
Without purification, the residue was dissolved in a solution
of KOH (121 mg, 2.1 mmol) in MeOH. The mixture was
refluxed for 2 h, diluted with water, and extracted with CHCl3.
The organic layer was dried over MgSO4 and evaporated to
leave the residue (300 mg), which was dissolved in CHCl3 (8
mL). To the stirred solution were added 4-dimethylaminopi-
peridine (150 mg, 1.17 mmol), bis(2-oxo-3-oxazolidinyl)phos-
phinic chloride (BOP-Cl) (216 mg, 0.85 mmol), and Et3N (0.24
mL, 1.72 mmol). The mixture was stirred at room temperature
for 12 h. The reaction mixture was diluted with CHCl3, washed
with saturated NaHCO3 aqueous solution, dried over MgSO4,
and evaporated to leave the residue, which was purified by
SiO2 column chromatography (CHCl3:MeOH ) 19:1) to provide
compound 27 (280 mg, 55.0% from compound 15).
N-[2-[4-(4-Dim eth yla m in op ip er id in o)-4-oxobu toxy]-4-
[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)ca r bon -
yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e Hyd r och lor id e
(2) (J TV-605). To a stirred solution of compound 1 (120 mg,
0.18 mmol) in AcOEt (1 mL) was added dropwise 4 N HCl in
AcOEt (0.25 mL). After 30 min, the precipitate was collected,
and recrystallization from acetone (2 mL) yielded colorless
crystals, which were dried at 40 °C and 0.3-0.5 mmHg for 4
h (2) (J TV-605) (102 mg, 82%). mp 211.5-213.5 °C. IR (KBr)
1635, 1525 cm-1. NMR (CDCl3) δ ppm. 1.39-1.59 (2H, m),
1.67-1.87 (4H, brs), 1.90-2.05 (5H, m), 2.25-2.30 (3H, m),
2.46 (1H, t, J ) 12.3 Hz), 2.63 (3H, s), 2.73 (3H, s), 2.89-3.00
(3H, m), 3.24 (1H, t, J ) 12.2 Hz), 3.85 (2H, d, J ) 13.2 Hz),
3.87 (2H, brs), 4.67 (2H, d, J ) 13.2 Hz), 6.18 (1H, brs), 6.72
(1H, d, J ) 5.7 Hz), 7.24-7.35 (4H, m), 7.42-7.58 (4H, m),
7.74-7.78 (2H, m), 8.24 (1H, d, J ) 7.8 Hz), 12.70 (1H, brs).
MS (FAB, m/z) ) 665 (M + + 1). Anal. (C39H45ClN4O4S) C, H,
N.
N-[2-[4-(4-Meth ylp er h yd r o-1,4-d ia zep in -1-yl)-4-oxobu -
toxy]-4-[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)-
car bon yl]ph en yl]-(1,1′-biph en yl)-2-car boxam ide (23). Com-
pound 23 (171 mg, 81%) was prepared using compound 32 (180
mg, 0.33 mmol) and 1-methylhomopiperazine (48 µL, 0.39
mmol) as described for compound 1 (see Figure 3). IR (KBr)
1638 cm-1. NMR (CDCl3) δ ppm. 1.70-2.00 (8H, m), 2.20-
2.40 (5H, m), 2.40-2.60 (4H, m), 2.80-3.00 (2H, m), 3.30-
3.50 (2H, m), 3.50-4.10 (6H, m), 6.20 (1H, brs), 6.60-6.70 (1H,
m), 6.70-6.90 (2H, m), 7.20-7.60 (8H, m), 7.70-7.80 (2H, m),
8.27 (1H, d, J ) 9.0 Hz). MS (FAB, m/z) ) 651 (M+ + 1). Anal.
(C38H42N4O4S) C, H, N.
IR (KBr) 1654 cm-1. NMR (CDCl3) δ ppm. 1.40-1.50 (4H,
m), 1.72-1.82 (2H, m), 1.90-2.00 (2H, m), 2.49 (6H, s), 2.50-
2.60 (2H, m), 2.90-3.00 (4H, m), 3.70-3.90 (2H, m), 4.20-
4.30 (2H, m), 4.54 (1H, m), 6.19 (1H, brs), 6.66 (1H, brs), 6.80
(1H, brs), 6.90 (1H, m), 7.25-7.60 (7H, m), 7.76-7.90 (2H, m),
+
8.20 (1H, m), 8.41 (1H, m). MS (FAB, m/z) ) 637 (M + 1).
Anal. (C37H40N4O4S) C, H, N.
N-[2-(3-Ch lor opr opoxy)-4-[(5,6,7,8-tetr ah ydr o-4H-th ien o-
[3,2-b]a zep in -4-yl)ca r bon yl]p h en yl]-(1,1′-bip h en yl)-2-ca r -
boxa m id e (33). To a stirred solution of compound 15 (600
mg, 1.28 mmol) in acetone were added K2CO3 (531 mg, 3.85
mmol), KI (43 mg, 0.26 mmol), and 1-bromo-3-chloropropane
(0.19 mL, 1.92 mmol). The mixture was stirred with refluxing
for 15 h, concentrated under reduced pressure, and dissolved
in AcOEt. The organic layer was washed with water and brine,
dried over anhydrous Na2SO4, and evaporated to give a
residue, which was purified by SiO2 column chromatography
(AcOEt:n-hexane ) 1:3) to yield compound 33 (581 mg, 83.4%)
as colorless crystals, mp 178.6-179.2 °C (from Et2O). IR (KBr)
1621, 1523 cm-1. NMR (CDCl3) δ ppm. 1.69-1.81 (2H, m),
1.90-2.02 (4H, m), 2.87-2.91 (2H, m), 3.45 (2H, t, J ) 6.8
Hz), 3.73-3.77 (2H, m), 3.92 (2H, brs), 6.18 (1H, brs), 6.65
(1H, d, J ) 5.1 Hz), 6.75 (1H, s), 6.83 (1H, d, J ) 8.4 Hz),
7.27-7.57 (9H, m), 7.75 (1H, d, J ) 7.5 Hz), 8.30 (1H, d, J )
8.4 Hz). MS (FAB, m/z) ) 545 (M+ + 1). Anal. (C31H29ClN2O3S)
C, H, N.
N-[2-(4-Ch lor obu toxy)-4-[(5,6,7,8-tetr ah ydr o-4H-th ien o-
[3,2-b]a zep in -4-yl)ca r bon yl]p h en yl]-(1,1′-bip h en yl)-2-ca r -
boxa m id e (34). Compound 34 (320 mg, 89.4%) was synthe-
sized using compound 15 (300 mg, 0.64 mmol) and 1-bromo-
4-chlorobutane (0.11 mL, 0.96 mmol) as described for compound
33. IR (KBr) 1671, 1637 cm-1. NMR (CDCl3) δ ppm. 1.65-
1.85 (6H, m), 1.90-2.10 (2H, m), 2.85-2.95 (2H, m), 3.45-
4.15 (6H, m), 6.10-6.25 (1H, m), 6.60-6.85 (3H, m), 7.25-
7.80 (10H, m), 8.25-8.40 (1H, m). MS (FAB, m/z) ) 559 (M+
+ 1). Anal. (C32H31ClN2O3S) C, H, N.
N-[2-[4-(4-Meth ylpiper azin -1-yl)-4-oxobu toxy]-4-[5,6,7,8-
tetr ah ydr o-4H-th ien o[3,2-b]azepin -4-yl]car bon yl]ph en yl]-
(1,1′-bip h en yl)-2-ca r boxa m id e (24). Compound 24 (131 mg,
63.4%) was prepared using compound 32 (180 mg, 0.33 mmol)
and 1-methylpiperazine (43 µL, 0.39 mmol) as described for
compound 1. mp 188-190°C (from 2-propanol). IR (KBr) 1641
cm-1. NMR (CDCl3) δ ppm. 1.70-1.85 (2H, m), 1.85-2.05 (4H,
m), 2.20-2.40 (9H, m), 2.85-3.00 (2H, m), 3.35 (2H, t, J ) 5.0
Hz), 3.55 (2H, t, J ) 5.0 Hz), 3.69 (2H, t, J ) 6.0 Hz), 3.70-
4.10 (2H, m), 6.20 (1H, brs), 6.70-6.90 (4H, m), 7.25-7.40 (3H,
m), 7.40-7.60 (5H, m), 7.70-7.80 (2H, m), 8.26 (1H, d, J )
8.0 Hz). MS (FAB, m/z) ) 637 (M + + 1). Anal. (C37H40N4O4S)
C, H, N
N-[2-[4-(4-Diet h yla m in op ip er id in o)-4-oxob u t oxy]-4-
[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)ca r bon -
yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e (25). Compound
25 (741 mg, 63.0%) was prepared using compound 32 (941 mg,
1.70 mmol) and 4-diethylaminopiperidine dihydrochloride (428
mg, 1.87 mmol) as described for compound 1. IR (KBr) 1640,
1522 cm-1. NMR (CDCl3) δ ppm. 1.00 (6H, t, J ) 7.2 Hz), 1.20-
1.40 (2H, m), 1.65-2.00 (8H, m), 2.21 (2H, t, J ) 7.1 Hz), 2.35-
2.60 (5H, m), 2.60-2.90 (4H, m), 3.55-3.95 (5H, m), 4.48 (1H,
d, J ) 12.6 Hz), 6.13 (1H, brs), 6.60 (1H, J ) 4.8 Hz), 6.65-
6.80 (2H, m), 7.20-7.50 (8H, m), 7.65-7.70 (2H, m), 8.19 (1H,

108 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Cho et al.
N-[2-[3-(4-Meth ylp er h yd r o-1,4-d ia zep in -1-yl)p r op oxy]-
4-[(5,6,7,8-tetr a h yd r o-4H-th ien o[3,2-b]a zep in -4-yl)ca r bo-
n yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e (29). To a so-
lution of compound 33 (280 mg, 0.51 mmol) in CH3CN (1.5
mL) and DMF (1.5 mL) were added KI (8.5 mg, 0.051 mmol)
and 1-methylhomopiperazine (95.7 µL, 0.77 mmol). The mix-
ture was refluxed for 4 h and concentrated under reduced
pressure to give a residue, which was dissolved in AcOEt. Then
the mixture was washed with saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and evaporated to give a
residue, which was purified by SiO2 column chromatography
(CHCl3:MeOH ) 40:1 then 10:1) to afford compound 29 (244
mg, 76.3%). IR (neat) 1639, 1522 cm-1. NMR (CDCl3) δ ppm.
1.69-2.02 (8H, m), 2.37-2.50 (2H, m), 2.42 (3H, s), 2.65-2.74
(8H, m), 2.89-2.93 (2H, m), 3.65-3.71 (2H, m), 3.82 (2H, brs),
6.20 (1H, brs), 6.67 (1H, d, J ) 5.1 Hz), 6.75-6.82 (2H, m),
7.30-7.58 (8H, m), 7.66 (1H, s), 7.75 (1H, d, J ) 7.8 Hz), 8.26
(1H, d, J ) 7.5 Hz). MS (FAB, m/z) ) 623 (M+ + 1). Anal.
(C37H42N4O3S) C, H, N.
given from the Insight II molecular modeling system from
Accelrys (see Figure 4b).
P h a r m a cologica l Exp er im en ts. Ma ter ia ls. The radioli-
gand [3H]AVP with a specific activity of 80 Ci/mmol was
obtained from DuPont-New England Nuclear (Boston, MA),
and AVP was obtained from Peptide Institute Inc. (Suita,
J apan). Synthesized compounds were initially dissolved in
dimethyl sulfoxide (DMSO) at 10-2 M and then diluted to the
desired concentration with the assay buffer. The final concen-
tration of DMSO in the buffer did not exceed 1% at which level
[3H]AVP binding was not affected. BSA was purchased from
Sigma Chemical Co. (St. Louis, MO). Kits for the protein assay
were purchased from Bio-Rad Laboratories (Richmond, CA).
An im a ls. Male Sprague Dawley (S.D.) rats weighing 250-
350 g were used. All animals were maintained under a 12 h
light/dark cycle with food and water available ad libitum.
R a d ioliga n d Bin d in g St u d ies. Rats were sacrificed by
decapitation, and their liver and kidneys were quickly re-
moved. All subsequent steps were carried out at 4 °C. The
hepatic plasma membrane fraction was prepared by a modi-
fication of the method of Nakamura et al.17 The renal medul-
lary plasma membrane fraction was prepared by the method
of Cambell et al.18 Human platelet plasma membranes were
obtained from healthy volunteers by the method of Vittet et
al.19 Human V2 receptor plasma membranes were prepared
from human V2 receptor-expressing CHO cells by a modifica-
tion of the method of Morel et al.20 Protein concentrations were
determined with a DC protein assay kit (Bio-Rad Laboratories)
using BSA as the standard. For competition studies, the
radioligand (1-2 nM) was added to each membrane prepara-
tion, and the mixture was incubated with various concentra-
tions of test compounds in 250 µL of assay buffer containing
100 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM EDTA, and
0.1% BSA. Binding was initiated by addition of the plasma
membrane preparations, and incubation was done for 20 min
at 37 °C to allow equilibrium to be established. After incuba-
tion, the reaction was terminated by addition of 2.5 mL of ice-
cold Tris buffer (100 mM Tris-HCl, pH 8.0), followed imme-
diately by rapid filtration through a GF/B UniFilter. After
rinsing three times, the radioactivity retained on the filters
N-[2-[4-[N′-(2-Dim eth yla m in oeth yl)-N′-m eth yla m in o]-
b u t oxy]-4-[5,6,7,8-t et r a h yd r o-4H -t h ien o[3,2-b]a zep in -4-
yl]ca r bon yl]p h en yl]-(1,1′-bip h en yl)-2-ca r boxa m id e (28).
Compound 28 (103 mg, 30.7%) was obtained from compound
34 (300 mg, 0.54 mmol) and N, N, N′-trimethylethylenedi-
amine (110 µL, 0.87 mmol) as described for compound 29. IR
(CHCl3) 1672, 1640 cm-1. NMR (CDCl3) δ ppm. 1.35-1.65 (4H,
m), 1.80-2.10 (4H, m), 2.15-2.55 (15H, m), 2.80-2.95 (2H,
m), 3.50-4.10 (4H, m), 6.10-6.30 (1H, m), 6.60-6.90 (3H, m),
7.20-7.60 (8H, m), 7.67 (1H, brs), 7.70-7.80 (1H, m), 8.20-
+
8.35 (1H, m). MS (FAB, m/z) ) 625 (M
(C37H44N4O3S) C, H, N.
+ 1). Anal.
N-[2-[4-(4-Meth ylp ip er a zin -1-yl)bu toxy]-4-[(5,6,7,8-tet-
r a h yd r o-4H-t h ien o[3,2-b]a zep in -4-yl)ca r bon yl]p h en yl]-
(1,1′-bip h en yl)-2-ca r boxa m id e (30). Compound 30 (150 mg,
44.9%) was obtained as crystals from compound 34 (300 mg,
0.54 mmol) and 1-methylpiperazine (80 µL, 0.72 mmol) as
described for compound 29. mp 134.7-135.4°C (from AcOEt).
IR (CHCl3) 1672, 1639 cm-1. NMR (CDCl3) δ ppm. 1.40-2.05
(10H, m), 2.20-2.65 (11H, m), 2.85-3.00(2H, m), 3.50-4.10
(4H, m) 6.10-6.30 (1H, m), 6.60-6.90 (3H, m), 7.20-7.60 (8H,
m), 7.65 (1H, brs), 7.70-7.80 (1H, m), 8.20-8.35 (1H, m). MS
was counted with
a liquid-scintillation counter (Packard
Instrument Company). Nonspecific binding was determined
with 1 µM unlabeled AVP, and specific binding was calculated
as the total binding minus nonspecific binding. The concentra-
+
(FAB, m/z) ) 623 (M + 1). Anal. (C37H42N4O3S) C, H, N.
Str u ctu r a l An a lysis of AVP . Structural analysis of AVP
in solution was performed following the methodology by
Wuthrich and co-workers.12 All NMR (500 MHz) experiments
were performed with a 5 mM sample of AVP dissolved in
tion of each test compound that caused 50% inhibition (IC50
)
of specific binding of [3H]AVP was determined by regression
analysis of displacement curves.
1
DMSO-d6. H NMR signals of amino acids were assigned at
Assessm en t of th e Effect on Blood P r essu r e Male
Sprague Dawley rats weighing 250-350 g were anesthetized
with urethane (1.3 g/kg ip). Catheters were inserted into the
femoral artery and vein for recording of arterial blood pressure
and intravenous drug administration, respectively. The ani-
mals were kept at 37 °C using a thermostat-controlled heating
board. After stabilization of the blood pressure, each test
compound or vehicle was given (1 mL/kg. iv) at 2 min before
injection of AVP (30 mU/kg. iv). The dose of a test compound
causing 50% inhibition of the maximal pressor response to
AVP (ID50) was calculated.
30 °C using 2D DQF-COSY,13 TOCSY,14 and NOESY15
experiments. Interproton distances were derived from esti-
mates of NOE cross-peak volumes in NOESY experiments
acquired with mixing-times ranging from 50 to 300 ms.
Coupling constants were measured from a E-COSY experi-
ment.16 Amide protection from DMSO-d6 was deduced from
chemical shift temperature coefficients measured using NOE-
SY experiments. The set of restraints derived from these data
included 73 interproton distances and one hydrogen bond
between Asn5 NH and Tyr2 CO.
Molecu la r Mod elin g Exp er im en ts. The overlaid struc-
tures of AVP and 1 were created by Discover program from
Accelrys using CVFF force fields. Molecular dynamics and
minimization were carried out with consensus restrains
between the corresponding groups of AVP and 1 shown by
Figure 4a, taking no account of intermolecular interactions
between two molecules. The distances between centroids were
forced to zero for overlapping the aromatic rings. The distances
of the corresponding atoms of the carbonyl groups or the amino
groups were forced to zero for the studies. Hydrogen atom
protonated to the amino group was also used for the studies
as well as the nitrogen atom of the amino group. The final
minimization was continued until the maximum derivative
was less than 0.01 kcal/Å with 1.0 kcal/mol/Å2 as force
constants for each overlapping. During this calculation, NOE
and hydrogen bond restraints of AVP by proton NMR study
mentioned above were also used. Graphical displays were
Assessm en t of th e Effect on Ur in e Ou tp u t. Rats were
fasted for 16-20 h before the experiment, and some animals
were also deprived of drinking water for 16 to 20 h to stimulate
endogenous AVP secretion. Test compounds or vehicle were
administered orally and spontaneously voided urine was
collected for 4 h using a metabolic cage.
Refer en ces
(1) Laszlo. F. A.; Laszlo, F., J r.; Wied, D. De. Pharmacology and
Clinical Perspectives of Vasopressin Antagonists. Pharmacol.
Rev. 1991, 43, 73-108.
(2) Manning, M.; Przybylski. J . P.; Olma, A.; Klis, W. A.; Kruszynski,
M.; Wo, N. C.; Pelton, G. H.; Sawyer, W. H. No Requirement of
Cyclic Conformation of Antagonists in Binding to Vasopressin
Receptors. Nature 1987, 329, 839-840.
(3) Ogawa, H.; Yamamura, Y.; Miyamoto, H.; Kondo, K.; Yamashita,
H.; Nakaya, K.; Chihara, T.; Mori, T.; Tominaga, M.; Yabuuchi,
Y. Orally Active, Nonpeptide Vasopressin V1 Antagonists. A

Novel Arginine Vasopressin Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 109
Novel Series of 1-(1-Substituted 4-piperidyl)-3,4-dihydro-2(1H)-
quinolinone. J . Med. Chem. 1993, 36, 2011-2017. Ogawa, H.;
Yamashita, H.; Kondo, K.; Yamamura, Y.; Miyamoto, H.; Kan,
K.; Kitano, K.; Tanaka, M.; Nakaya, K.; Nakamura, S.; Mori,
T.; Tominaga, M.; Yabuuchi, Y. Orally Active, Nonpeptide
Vasopressin V2 Receptor Antagonists: A Novel Series of 1-[4(Ben-
zoylamino)benzoyl]-2, 3, 4, 5-tetrahydro-1H-benzazepines and
Related Compounds. J . Med. Chem. 1996, 39, 3547-3555.
(4) Serradeil-Le Gal, C.; Wagnon, J .; Garcia, C.; Lacour, C.; Guirau-
dou, P.; Christophe, B.; Villanova, G.; Nisato, D.; Maffrand, J .
P.; Le Fur, G.; Guillon, G.; Cantau, B.; Barberis, C.; Trueba, M.;
Ala, Y.; J ard, S. Biochemical and Pharmacological Properties of
SR 49059, a New, Potent, Non-peptide Antagonist of Rat and
Human Vasopressin V1a. Receptors. J . Clin. Invest. 1993, 92,
224-231. Serradeil-Le Gal, C.; Bourrie, B.; Ranfaste, D.; Car-
ayon, P.; Garcia, C.; Maffrand, J . P.; Le Fur, G.; Casellas, P.
Effect of a New, Potent, Non-peptide V1a Vasopressin Antagonist,
SR 49059. On the Binding and the Mitogenic Activity of
Vasopressin on Swiss 3T3 Cells. Biochemical Pharmacology
1994, 47, 633-641. Hermecz, I.; Santa-Csutor, A.; Gonczi, C.;
Heja, G.; Csikos, E.; Simon, K.; Smelko-Esek, A.; Podanyi, B.
Chemical Development of the Vasopressin Receptor 2 Antagonist
SR-121463. Pure Appl. Chem. 2001, 73, 1401-1409.
(5) Matsuhisa, A.; Taniguchi N.; Koshio, H.; Yatsu, T.; Tanaka, A.
Nonpeptide Arginine Vasopressin Antagonists for Both V1A and
V2 Receptors: Synthesis and Pharmacological Properties of 4′-
(1,4,5,6-Tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbonyl)-
benzanilide Derivatives and 4′-(5,6-Dihydro-4H-thiazolo[5,4-
d][1]benzoazepine-6-carbonyl)benzanilide Derivatives. Chem.
Pharm. Bull. 2000, 48, 21-31.
(6) Ohtake, Y.; Naito, A.; Hasegawa, H.; Kawano, K.; Morizono, D.;
Taniguchi, M.; Tanaka, Y.; Matsukawa, H.; Naito, K.; Oguma,
T.; Ezure, Y.; Tsuriya, Y. Novel Vasopressin V2 Receptor-
selective Antagonists, Pyrrolo[2,1-a]quinoxaline and Pyrrolo[2,1-
c][1,4]benzodiazepine Derivatives. Bioorg. Med. Chem. 1999, 7,
1247-1254.
(9) Cho, H.; Wakitani, K. J apan Patent Application, H7-291540.
(Nov 9, 1995), WO97/17349.
(10) American Cyanamid Company, Albright, J . D. PCT/US96/01096,
WO96/22294, 1996.
(11) Kunick C. Synthese [b]-Kondensierter Azepindione durch
Dealkoxy-carbonylierung. Arch. Pharm. (Weinheim) 1991, 324,
579-581.
(12) Wuthrich, K. NMR of Proteins and Nucleic Acids; J ohn Wiley
and Sons: New York, 1986.
(13) Rance, M.; Sørensen, O. W.; Bodenhausen, G.; Wagner, G.; Ernst,
R. R.; Wuthrich, K. Improved spectral resolution in cosy 1H
NMR spectra of proteins via double quantum filtering. Biochem.
Biophys. Res. Commun. 1983, 117, 479-485.
(14) Bax, A.; Davis, D. G. MLEV-17-based two-dimensional homo-
nuclear magnetization transfer spectroscopy. J . Magn. Reson.
1985, 65, 355-360.
(15) Kumar, A.; Ernst, R. R.; Wuthrich, K. A two-dimensional nuclear
Overhauser enhancement (2D NOE) experiment for the elucida-
tion of complete proton-proton cross-relaxation networks in
biological macromolecules. Biochem. Biophys. Res. Commun.
1980, 95, 1-6.
(16) Griesinger, C.; Sørensen, O. W.; Ernst, R. R. Practical aspects
of the E. COSY technique. Measurement of scalar spin-spin
coupling constants in peptides. J . Magn. Reson. 1987, 75, 474-
492.
(17) Nakamura, T.; Tomomura A.; Noda, C.; Shimoji, M.; Ichihara,
A. Acquisition of a Beta-Adrenergic Response by Adult Rat
Hepatocytes During Primary Culture. J . Biol. Chem. 1983, 258,
9283-9289.
(18) Campbell, B. J .; Woodward, G.; Borberg, V. Calcium-mediated
Interactions between the Antidiuretic Hormone and Renal
Plasma Membranes, J . Biol. Chem. 1972, 247, 6167-6175.
(19) Vittet, D.; Rondot, A.; Cantau, B.; Launay, J . M.; Chevillard, C.
Nature and Properties of Human Platelet Vasopressin Receptors.
Biochem. J . 1986, 233, 631-636.
(7) Cho, H.; Murakami, K.; Nakanishi, H.; Isoshima, H.; Hayakawa,
K.; Uchida, I. Regioselective Synthesis of Several Heterocyclic
Fused Azepines Using Diisobutylaluminum Hydride. Hetero-
cycles 1998, 48, 919-927.
(8) Cho. H.; Murakami, K.; Fujisawa, A.; Niwa, M.; Nakanishi, H.;
Uchida, I. Synthesis of Novel 5,6,7,8-Tetrahydro-4H-thieno[2,3-
b][1,4]diazepine Derivatives. Heterocycles 1998, 48, 1555-1566.
(20) Morel, A.; O’Carroll, A.-M.; Brownstein, M. J .; Lolait, S. J .
Molecular Cloning and Expression of
a Rat Vla Arginine
Vasopressin Receptor. Nature 1992, 356, 523-526.
J M030287L