Preparation of non-peptide, highly potent and selective antagonists of arginine vasopressin V1a receptor by introduction of alkoxy groups

Article abstract of DOI:10.1248/cpb.51.1075

A series of compounds structurally related to 4′-[(4,4-difluoro-5- methylidene-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl)carbonyl]benzanilide were synthesized and evaluated for arginine vasopressin (AVP) antagonistic activity. Compounds with alkoxy groups (especially ethoxy group) at the 2′-position of benzanilide possessed potent affinity and selectivity for the V_1A receptor versus V₂ receptor. Further study has shown that the introduction of 4,4-dimethylaminopiperidino and morpholino groups at carbonylmethylene exhibited more potent affinity and selectivity for V _1A receptors. Consequently, we found that the (Z)-4′-({4,4- Difluoro-5-[(4-dimethylaminopiperidino) carbonylmethylene]-2,3,4,5-tetrahydro- 1H-1-benzoazepin-1-yl}carbonyl)-2-ethoxybenzanilide monohydrochloride (8d) and the (Z)-4′-[(4,4-Difluoro-5-morpholinocarbamoylethylene-2,3,4,5- tetrahydro-1H-1-benzoazepin-1-yl)carbonyl]-2-ethoxybenzanilide (8q) exhibited potent and selective V_1A receptor antagonist activity. The synthesis and pharmacological properties of these compounds are detailed in this paper.

Full text of DOI:10.1248/cpb.51.1075

September 2003
Chem. Pharm. Bull. 51(9) 1075—1080 (2003)
1075
Preparation of Non-peptide, Highly Potent and Selective Antagonists of
Arginine Vasopressin V1A Receptor by Introduction of Alkoxy Groups
Yoshiaki SHIMADA,a Nobuaki TANIGUCHI,a Akira MATSUHISA,a Takeyuki YATSU,a Atsuo TAHARA,a and
,b
*
Akihiro TANAKA
a Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd.; 21 Miyukigaoka, Tsukuba, Ibaraki
305–8585, Japan: and b Corporate Communications Department, Yamanouchi Pharmaceutical Co., Ltd.; 2–3–11
Nihonbashi-Honcho, Chuo-ku, Tokyo 103–8411, Japan. Received May 28, 2003; accepted July 15, 2003
A series of compounds structurally related to 4؅-[(4,4-difluoro-5-methylidene-2,3,4,5-tetrahydro-1H-1-ben-
zoazepin-1-yl)carbonyl]benzanilide were synthesized and evaluated for arginine vasopressin (AVP) antagonistic
activity. Compounds with alkoxy groups (especially ethoxy group) at the 2؅-position of benzanilide possessed po-
tent affinity and selectivity for the V1A receptor versus V2 receptor. Further study has shown that the introduction
of 4,4-dimethylaminopiperidino and morpholino groups at carbonylmethylene exhibited more potent affinity
and selectivity for V1A receptors. Consequently, we found that the (Z)-4؅-({4,4-Difluoro-5-[(4-dimethylamino-
piperidino)carbonylmethylene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-ethoxybenzanilide mono-
hydrochloride (8d) and the (Z)-4؅-[(4,4-Difluoro-5-morpholinocarbamoylethylene-2,3,4,5-tetrahydro-1H-1-ben-
zoazepin-1-yl)carbonyl]-2-ethoxybenzanilide (8q) exhibited potent and selective V1A receptor antagonist activity.
The synthesis and pharmacological properties of these compounds are detailed in this paper.
Key words arginine vasopressin antagonist; V1A receptor selective; alkoxy group
Arginine vasopressin (AVP) is a nonapeptide secreted by cal roles of AVP and could lead to new therapeutic tools, we
the posterior pituitary gland. AVP is known as an anti-di- then decided to study selective non-peptide antagonists of the
uretic hormone (ADH) and demonstrates potent anti-diuretic V1A receptor as a next target.
and vasoconstrictor activities. The subtypes of the AVP re-
Selective non-peptide antagonists of the V1A receptor re-
ceptor have been identified as V1A, V1B and V2 in the periph- ported to date include OPC-21268 (3)10) and SR49059 (4)11)
ery.1—3) Among these receptors, the V1A receptor is found in (Fig. 2). We had already discovered compounds possessing
vascular smooth muscle, liver, platelets and renal mesangial high binding affinities for both V1A and V2 receptors as de-
cells. The V2 receptor is found only in the kidney.
scribed above, and chose 2 as the lead compound because it
Considering the physiological properties of AVP, abnormal showed the most potent V1A receptor binding affinity among
secretion of AVP is thought to be associated with high blood our compounds. On modification of 2, we presumed that
pressure, cardiac disease, kidney disease and hyperna- modification of biphenyl moiety, shown in the box in Fig. 3,
tremia.4) Compounds which act as antagonists of AVP recep- would show V1A selectivity versus V2 receptor on the basis of
tors are therefore expected to be highly effective drugs for our investigations of AVP antagonists.5,8)
the treatment of these conditions.
Therefore, we first modified R1 and R2 positions of 2, then
To this end, we have conducted initial investigations into optimized the R3 position.
the discovery of non-peptide antagonists of both V1A and V2
In this paper, we described the SAR within a series of
receptors. In the course of investigations of AVP antagonists, these derivatives that led to the discovery of a highly potent
we have discovered and reported several compounds that ex- and selective V1A receptor antagonist.
hibited similar highly potent affinity for these receptor sub-
Chemistry The synthesis pathways of the compounds
types.5—7) In particular, 4Ј-(2-methyl-1,4,5,6-tetrahydroimi- listed in Table 1—4 are shown in Chart 1. The methyl ester
dazo[4,5-d][1]-benzoazepine-6-carbonyl)-2-phenyl-ben- derivatives (6a—h) were obtained by reacting substituted
zanilide monohydrochloride (1, YM087) and (Z)-4Ј-({4,4-di- benzoyl chloride with methyl (Z)-[4,4-difluoro-1-(4-
fluoro-5-[(4-dimethylaminopiperidino)carbonylmethylene]- aminobenzoyl)-2,3,4,5-tetrahydro-1H-1-benzoazepin-5-yli-
2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2- dene] acetate (5).9) Each derivative was hydrolyzed to acetic
phenylbenzanilide monohydrochloride (2, YM-35471) pos- acid derivative (7a—h) under basic condition. Condensation
sessed high binding affinities for both V1A and V2 receptors of 7 and various amines in the presence of 1-ethyl-3-(di-
and potent AVP antagonist activity (Fig. 1).8,9)
methylaminopropyl)carbodiimide hydrochloride (WSC.HCl)
Considering that subtype selective AVP receptor antago- and 1-hydroxybenzotriazole (HOBt) gave the target amide
nists may be useful for investigation of the pathophysiologi- derivatives (2, 8b—r).
Results and Discussion
Binding Affinities Methods for determining the in vitro
AVP receptor binding affinities are described in Experimen-
tal. The results of the binding assay of compounds are shown
in Tables 3 and 4.
Initially, we investigated the influence of various sub-
stituent groups at the R1 and R2 position of 4-(N,N-dimethyl-
Fig. 1
amino) piperidine derivatives, as shown in Table 3. The
To whom correspondence should be addressed. e-mail: shimada@yamanouchi.co.jp
© 2003 Pharmaceutical Society of Japan

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Vol. 51, No. 9
Chart 1
Table 1. Physical and Spectral Data of Methyl Ester Derivatives
Yield
(%)
1H-NMR
(CDCl3) d
FAB-MS m/z
(Mϩϩ1)
No.
R1
Ph
R2
H
6a
95
2.30—2.80 (2H, m), 3.21 (1H, m), 3.83 (3H, s), 5.03 (1H, m), 6.17 (1H, s), 6.66 (1H, m),
6.91 (3H, m), 7.01 (2H, m), 7.09 (1H, t, Jϭ7 Hz), 7.24 (1H, t, Jϭ8 Hz), 7.33—7.49 (8H, m),
7.50—7.60 (1H, m), 7.83 (1H, d, Jϭ7 Hz)
2.02—2.82 (2H, m), 2.47 (3H, s), 3.21 (1H, m), 3.83 (3H, s), 5.06 (1H, m), 6.21 (1H,
s), 6.73 (1H, d, Jϭ8 Hz), 7.12—7.50 (12H, m)
2.26—2.84 (2H, m), 3.26 (1H, m), 3.83 (3H, s), 4.03 (3H, s), 5.03 (1H, m), 6.21 (1H, s),
6.73 (1H, m), 7.02 (1H, d, Jϭ7 Hz), 7.09—7.28 (5H, m), 7.37 (1H, d, Jϭ8 Hz), 7.50 (3H,
m), 8.23 (1H, m), 9.84 (1H, m)
1.61 (3H, t, Jϭ7 Hz), 2.32—2.82 (2H, m), 3.24 (1H, m), 3.84 (3H, s), 4.26 (2H, q, Jϭ
7 Hz), 5.03 (1H, m), 6.22 (1H, s), 6.73 (1H, m), 7.02 (1H, d, Jϭ7 Hz), 7.06—7.18 (4H,
m), 7.24 (1H, t, Jϭ8 Hz), 7.37 (1H, d, Jϭ8 Hz), 7.47 (3H, m), 8.24 (1H, m), 10.12 (1H, m)
1.50 (6H, d, Jϭ6 Hz), 2.35—2.82 (2H, m), 3.23 (1H, m), 3.84 (3H, s), 4.82 (1H, m), 5.07
(1H, m), 6.22 (1H, s), 6.73 (1H, m), 7.00 (1H, d, Jϭ7 Hz), 7.08—7.18 (4H, m), 7.24 (1H,
t, Jϭ8 Hz), 7.38 (1H, d, Jϭ8 Hz), 7.47 (3H, m), 8.24 (1H, m), 10.25 (1H, m)
2.27—2.82 (2H, m), 3.26 (1H, m), 3.83 (3H, s), 5.04 (1H, m), 6.21 (1H, s), 6.73 (1H, m),
7.10—7.20 (3H, m), 7.26 (1H, t, Jϭ8 Hz), 7.34—7.50 (6H, m), 7.72 (1H, d, Jϭ8 Hz),
7.98 (1H, s)
2.25—2.82 (2H, m), 3.24 (1H, m), 3.83 (3H, s), 5.04 (1H, m), 6.24 (1H, s), 6.75 (1H, m),
7.10—7.20 (3H, m), 7.14 (1H, t, Jϭ8 Hz), 7.38 (1H, m), 7.52 (2H, d, Jϭ8 Hz), 7.57—7.67
(2H, m), 7.72 (1H, t, Jϭ7 Hz), 8.08 (1H, d, Jϭ7 Hz), 10.09 (1H, s)a)
2.30—2.80 (2H, m), 3.24 (1H, m), 3.81 (6H, s), 3.83 (3H, s), 5.07 (1H, m), 6.20 (1H,
s), 6.58 (2H, d, Jϭ8 Hz), 6.74 (1H, d, Jϭ8 Hz), 7.11—7.18 (3H, m), 7.26 (1H, d, Jϭ
7 Hz), 7.31 (1H, t, Jϭ7 Hz), 7.38 (1H, d, Jϭ7 Hz), 7.40—7.52 (4H, m)
552b)
490b)
6b
6c
Me
H
H
98
95
OMe
506b)
520b)
6d
6e
6f
OEt
O-i-Pr
Cl
H
H
99
97
99
89
94
535
H
510, 512b)
522
6g
6h
NO2
OMe
H
OMe
537
a) 1H-NMR spectra were measured in DMSO-d6. b) EI-MS (Mϩ).

September 2003
1077
Table 2. Physical and Spectral Data of Acetic Acid Derivatives
Yield
(%)
1H-NMR
(CDCl3) d
FAB-MS m/z
No.
R1
Ph
R2
H
(Mϩϩ1)
7a
76
2.41 (1H, m), 2.67 (1H, m), 3.24 (1H, m), 3.68 (1H, m), 5.00 (1H, m), 6.19 (1H, s),
6.67 (1H, m), 6.92 (2H, m), 6.98 (3H, m), 7.10 (1H, t, Jϭ8 Hz), 7.24 (1H, d, Jϭ7 Hz),
7.30—7.50 (8H, m), 7.53 (1H, m), 7.80 (1H, d, Jϭ8 Hz)
2.15—3.00 (2H, m), 2.43 (3H, s), 3.35 (1H, m), 5.02 (1H, m), 6.20 (1H, s), 6.75 (1H, m),
7.08—7.49 (12H, m), 7.67 (1H, br)
2.20—2.80 (2H, m), 3.26 (1H, m), 4.03 (3H, s), 5.03 (1H, m), 6.21 (1H, s), 6.73 (1H,
m), 7.02 (1H, d, Jϭ7 Hz), 7.09—7.28 (5H, m), 7.37 (1H, d, Jϭ8 Hz), 7.50 (3H, m), 8.23
(1H, m), 9.84 (1H, m)
1.59 (3H, t, Jϭ7 Hz), 2.28—2.90 (2H, m), 3.28 (1H, m), 4.22 (2H, q, Jϭ7 Hz), 5.03 (1H,
m), 6.27 (1H, s), 6.73 (1H, m), 6.93 (1H, d, Jϭ8 Hz), 7.06—7.18 (4H, m), 7.24 (1H, t, Jϭ
8 Hz), 7.26—7.30 (4H, m), 8.22 (1H, d, Jϭ7 Hz), 10.16 (1H, m)
538c)
476c)
7b
7c
Me
H
H
90
89
OMe
492c)
507
7d
7e
OEt
H
H
99
75
O-i-Pr
1.47 (6H, d, Jϭ6 Hz), 2.28—2.86 (2H, m), 3.28 (1H, m), 4.79 (1H, m), 5.07 (1H, m), 6.27
(1H, s), 6.73 (1H, m), 6.98 (1H, d, Jϭ8 Hz), 7.06—7.20 (4H, m), 7.24 (1H, t, Jϭ8 Hz), 7.39
(1H, d, Jϭ8 Hz), 7.41—7.50 (4H, m), 8.21 (1H, m), 10.26 (1H, m)
521
7f
Cl
H
H
90
55
2.15—2.82 (2H, m), 3.22 (1H, m), 5.03 (1H, m), 6.24 (1H, s), 6.72 (1H, m), 7.09—7.20 (3H,
m), 7.25 (1H, t, Jϭ8 Hz), 7.32—7.48 (4H, m), 7.50—7.65 (3H, m), 9.85 (1H, s)a)
2.46 (2H, m), 3.11 (1H, m), 4.88 (1H, m), 6.68 (1H, s), 6.84 (1H, m), 7.09 (2H, d, Jϭ
8 Hz), 7.21 (1H, t, Jϭ8 Hz), 7.31 (1H, t, Jϭ8 Hz), 7.38 (1H, d, Jϭ8 Hz), 7.50 (2H, d, Jϭ
7 Hz), 7.74—7.80 (2H, m), 7.86 (1H, t, Jϭ7 Hz), 8.14 (1H, d, Jϭ7 Hz), 10.74 (1H, s), 13.18
(1H, br)a)
497, 499
7g
NO2
508
523
7h
OMe
OMe
88
2.50 (2H, m), 3.10 (1H, m), 3.73 (6H, s), 4.89 (1H, m), 6.66 (1H, s), 6.71 (2H, d, Jϭ8 Hz),
6.83 (1H, m), 7.04 (2H, d, Jϭ8 Hz), 7.20 (1H, t, Jϭ8 Hz), 7.26—7.41 (3H, m), 7.52 (2H, d,
Jϭ7 Hz), 10.30 (1H, s), 13.21 (1H, m)a)
a) 1H-NMR spectra were measured in DMSO-d6. b) EI-MS (Mϩ).
Table 3. Receptor-Binding Affinities for 4-(N,N-dimethylamino)piperidine
Table 4. Receptor-Binding Affinities for 2-Ethoxybenzanilide Derivatives
Derivatives
Binding affinity (pKi)
c)
No.
R3
V1A/V2
37
a)
b)
Binding affinity (pKi)
V1A
V2
c)
No.
R1
R2
V1A/V2
a)
b)
V1A
V2
8d
9.22
7.65
8i
8j
NH2
NHMe
NHEt
NH-n-Pr
NH-i-Pr
NH-c-Pr
NMe2
8.81
9.26
9.04
8.95
9.20
9.39
8.95
8.59
7.86
8.18
8.29
8.11
8.21
8.14
8.28
7.83
9
12
6
2
Ph
Me
OMe
OEt
O-i-Pr
Cl
H
H
H
H
H
H
H
OMe
10.1
8.76
9.38
8.12
7.62
7.65
8.10
7.67
7.44
6.30
5
4
8b
8c
8d
8e
8f
8k
8l
8m
8n
8o
8p
8.89
9.22
9.19
8.99
8.78
8.08
19
37
12
21
22
60
7
10
18
5
8g
8h
NO2
OMe
NEt2
6
8q
8r
9.80
8.75
8.19
8.28
41
3
a) pKi of [3H]vasopressin binding to rat liver membranes. b) pKi of [3H]vaso-
pressin binding to rat kidney membranes. c) V1A/V2 showed the selectivity of binding
affinity for V1A versus V2 receptor.
a—c) See footnotes for Table 3.

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Vol. 51, No. 9
Table 5. Binding Affinities of Cloned Human V1A (hV1A) and V2 (hV2) Receptors and AVP-Antagonist Activities
Binding affinity (pKi)
Antagonist activities
V1Ad) ID50 (mg/kg)
c)
No.
R3
V1A/V2
a)
b)
hV1A
hV2
8d
8q
9.05
9.80
8.37
7.34
8.15
8.72
51
44
0.0091
0.019
0.013
YM0878)
0.45
a—c) See footnotes for Table 3. d) ID50 represents the drug concentration (mg/kg) required to inhibit the AVP-induced pressor response in pithed rats by 50% on intra-
venous administration.
methyl-substituted derivative (8b) had lower binding affinity binding affinity was observed.
for both V1A and V2 receptors than 2. In contrast, alkoxy-sub-
Antagonist Activity V1A receptor antagonist activity
stituted derivatives (8c—e) showed potent V1A receptor bind- was determined by measuring inhibition of the AVP-induced
ing affinity and improvement of selectivity compared with 2. diastolic blood pressure (DBP) response in pithed rats after
In particular, the ethoxy-substituted derivative (8d) exhibited intravenous (i.v.) administration. The dose of compound
most potent affinity among alkoxy derivatives and 37-fold se- causing a 50% inhibition of pressor response to AVP (ID50)
lectivity for V1A versus V2 receptor.
was calculated. The experimental method used to determine
Additionally, chloro derivative (8f) and nitro derivative AVP antagonist activity is described in Experimental. Com-
(8g) exhibited potent affinity and selectivity for V1A receptor pounds 8d and 8q were tested (Table 5). Both compounds
compared with 2. These results indicated that introduction of significantly suppressed the AVP-induced blood pressure in-
an alkoxy group was effective to showing potent affinity and crease. The V1A antagonist activity of these compounds was
selectivity for the V1A receptor. We reasoned that introduc- more effective than that of YM087. In particular, the 4-(N,N-
tion of two alkoxy groups to the R1 and R2 (symmetry posi- dimethylamino) piperidine derivative (8d) showed 1.4-fold
tion of R1) positions might produce more potent and selective more potent V1A antagonist activity than YM087.
compounds by synergistic effect, and therefore prepared 2,6-
dimethoxy derivative (8h). Results showed that compound 8h Conclusion
exhibited 60-fold selectivity for V1A versus V2 receptor, but
As part of efforts to discover new selective V1A receptor
that it had lower binding affinity for V1A receptors than the antagonists, we synthesized several 4Ј-(4,4-difluoro-5-
alkoxy derivatives (8c—e). Consequently, to obtain a com- methylidene-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl)car-
pound with favorable V1A receptor binding affinity and selec- bonyl derivatives, and evaluated their pharmacological prop-
tivity, introduction of one alkoxy (especially ethoxy) group at erties. Results showed that introduction of an alkoxy (espe-
R1 position would be effective.
cially ethoxy) group at the 2Ј-position of benzanilide was ef-
For the next modification, we investigated the influence of fective to obtaining high potency and selectivity for the V1A
various amine substituent groups at the R3 position of 2- receptor. Furthermore, introduction of 4,4-dimethyl-
ethoxybenzanilide derivatives, as shown in Table 4. The car- piperidino (8d) and morpholino (8q) groups at the 1-position
bamoyl-substituted derivative (8i) had lower binding affinity of carbonylmethylene exhibited more potent affinity and se-
for V1A and V2 receptors than 8d. The alkyl amine substi- lectivity for V1A receptors.
tuted derivatives (8j—p) showed potent V1A receptor binding
These compounds possessed powerful antagonist activity
affinity, but their selectivity was not sufficient. In the investi- for the V1A receptor and are expected to prove valuable as
gation of 4Ј-[(4,4-difluoro-5-methylidene-2,3,4,5-tetrahydro- therapeutic tools in the investigation of abnormal secretion of
1H-1-benzoazepin-1-yl)carbonyl]-2-phenylbenzanilide deriv- AVP.
atives,9) we found that introduction of a morpholino group
showed high V1A receptor selectivity. We, therefore, at-
tempted to introduce a morpholino group at the R3 position.
The morpholino-substituted derivative (8q) exhibited most
potent V1A receptor binding affinity and 41-fold selectivity in
Experimental
1H-NMR spectra were obtained on a JEOL JNM-EX90 or JNM-A500
spectrometer and the chemical shifts are expressed in d (ppm) values with
1
tetramethylsilane as an internal standard. Abbreviations of H-NMR signal
patterns are as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; q,
quartet; m, multiplet; br, broad peak. Mass spectra were obtained on a JEOL
JMS-DX300 spectrometer. Elemental analysis was performed with a Yanaco
MT-5. Melting points were determined on a Yanaco MP-500D micro melting
point apparatus without correction. Column chromatography on silica gel
was performed with Merck KGaA Silica gel 60 (0.040—0.063 mm).
General Procedure for Synthesis of Methyl Ester Derivatives (6a—h)
To an ice-cooled mixture of benzoic acid (2.00 mmol) and catalytic N,N-di-
methylformamide (DMF) in CH2Cl2 (10 ml) was added oxalyl chloride
(3.32 mmol). The mixture was stirred at room temperature for 3 h, diluted
with benzene and concentrated in vacuo to give a crude acid chloride. To an
ice-cooled mixture of 5 (620 mg, 1.67 mmol) and pyridine (5 ml) in CH2Cl2
(10 ml) was added a solution of the above acid chloride in CH2Cl2 (10 ml).
The mixture was stirred at room temperature for 1 h, and then concentrated
V1A versus V2 receptor. However, the thiomorpholino-substi-
tuted derivative (8r) showed lower binding affinity and selec-
tivity than 8q. These results suggested that introduction of
moieties including an oxygen or nitrogen atom at both the R1
and R3 positions was important to the manifestation of potent
and selective V1A receptor antagonist activity.
Subsequently, the compounds (8d, 8q) that showed potent
binding affinity and selectivity for the V1A receptor were
tested for their binding affinities for cloned human V1A
(hV1A) and V2 (hV2) receptors (Table 5). Both compounds
showed the same potent binding affinity and selectivity for
the V1A receptor as in the rat, and no species difference in in vacuo. The residue was diluted with ethyl acetate (AcOEt). The organic

September 2003
1079
zanilide Monohydrochloride (8f): Colorless amorphous. Yield; 17%. 1H-
NMR (DMSO-d6) d: 1.40—1.80 (2H, m), 2.09 (2H, m), 2.43 (2H, m), 2.67
(1H, m), 2.71 (6H, s), 2.95—3.22 (2H, m), 3.43 (1H, m), 4.06 (1H, m), 4.53
(1H, m), 4.87 (1H, m), 6.82 (1H, s), 6.85 (1H, m), 7.10 (2H, m), 7.20 (1H, t,
Jϭ8 Hz), 7.31 (1H, t, Jϭ7 Hz), 7.41—7.63 (7H, m), 10.61 (1H, m). FAB-
MS m/z: 607, 609 (Mϩϩ1).
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-nitroben-
zanilide Monohydrochloride (8g): Colorless amorphous. Yield; 42%. 1H-
NMR (DMSO-d6) d: 1.40—1.80 (2H, m), 2.10 (2H, m), 2.43 (2H, m), 2.67
(1H, m), 2.70 (3H, s), 2.71 (3H, s), 3.02—3.24 (2H, m), 3.40 (1H, m), 4.05
(2H, m), 4.53 (1H, m), 4.88 (1H, m), 6.83 (1H, s), 6.86 (1H, m), 7.11 (2H,
m), 7.22 (1H, m), 7.32 (1H, t, Jϭ6 Hz), 7.52 (2H, m) 7.74—7.78 (2H, m),
7.86 (1H, t, Jϭ7 Hz), 8.14 (1H, d, Jϭ6 Hz), 10.76 (1H, m), 10.80 (1H, s).
FAB-MS m/z: 618 (Mϩϩ1).
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2,6-dimethoxy-
benzanilide Monohydrochloride (8h): Colorless amorphous. Yield; 57%. 1H-
NMR (DMSO-d6) d: 1.40—1.80 (2H, m), 2.10 (2H, m), 2.42 (2H, m), 2.68
(1H, m), 2.70 (3H, s), 2.71 (3H, s), 2.95—3.23 (2H, m), 3.45 (1H, m), 3.73
(6H, s), 4.05 (1H, m), 4.53 (1H, m), 4.87 (1H, m), 6.71 (2H, d, Jϭ8 Hz),
6.80 (1H, s), 6.84 (1H, m), 7.06 (1H, m), 7.21 (1H, m), 7.36—7.38 (2H, m),
7.53 (3H, m), 10.31 (1H, s), 10.83 (1H, m). FAB-MS m/z: 633 (Mϩϩ1).
(Z)-4؅-[(5-Carbamoylmethylene-4,4-difluoro-2,3,4,5-tetrahydro-1H-1-
benzoazepin-1-yl)-carbonyl]-2-ethoxybenzanilide (8i) To an ice-cooled
mixture of 7d (1.00 mmol) and HOBt (1.10 mmol) in CH2Cl2 (10 ml) and
CH3CN (10 ml) was added WSC.HCl (211 mg, 1.10 mmol) in CH2Cl2
(10 ml), and the mixture was stirred at room temperature for 1 h. After being
cooled at 0 °C, 28% NH4OH (0.3 ml) was added, and the mixture was stirred
at room temperature overnight. To the mixture was added 1 M NaOH and ex-
tracted with CHCl3. The organic layer was dried over anhydrous K2CO3 and
concentrated in vacuo. The residue was chromatographed on a silica gel col-
umn using CHCl3–MeOH (95 : 5) as eluent, then recrystallized from
AcOEt–Et2O to give 100 mg (0.197 mmol, 20%) of 8i as a colorless powder.
mp 134—139 °C. 1H-NMR (CDCl3) d: 1.61 (3H, t, Jϭ8 Hz), 2.40—2.80
(2H, m), 3.34 (1H, m), 4.23 (2H, d, Jϭ8 Hz), 4.86 (1H, d, Jϭ8 Hz), 5.66
(1H, s), 6.14 (1H, s), 6.35 (1H, s), 6.72 (1H, d, Jϭ8 Hz), 6.94 (1H, d,
Jϭ8 Hz), 7.00—7.30 (5H, m), 7.38—7.50 (4H, m), 8.24 (1H, d, Jϭ8 Hz),
10.16 (1H, s). FAB-MS m/z: 506 (Mϩϩ1). Anal. Calcd for
C28H25N3O4F2·0.5H2O: C, 65.36; H, 5.09; N, 8.17; F, 7.38. Found: C, 65.24;
H, 5.13; N, 8.12; F, 7.22.
layer was washed with a saturated aqueous solution of Na2CO3, 1 M HCl and
brine, dried over anhydrous MgSO4 and concentrated in vacuo. The crude
product was chromatographed on silica gel and eluted with n-hexane–AcOEt
to give a colorless amorphous substance. All physical and spectral data of
methyl ester derivatives are shown in Table 1.
General Procedure for Synthesis of Acetic Acid Derivatives (7a—h)
To an ice-cooled solution of 6 (1.55 mmol) in methanol (MeOH) (10 ml) was
added lithium hydroxide monohydrate (4.65 mmol) in water (2 ml). The mix-
ture was stirred at room temperature for 7 h, and then concentrated in vacuo.
The residue was dissolved in CHCl3 and 1 M HCl and extracted with CHCl3.
The organic layer was dried over anhydrous MgSO4 and concentrated in
vacuo. The product was purified by recrystallization. All physical and spec-
tral data of acetic acid derivatives are shown in Table 2.
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmeth-
ylene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-phenyl-
benzanilide monohydrochloride (2) To an ice-cooled mixture of 7a
(0.364 mmol) and 1-hydroxybenzotriazole hydrate (HOBt) (0.437 mmol) in
CH2Cl2 (10 ml) and acetonitrile (CH3CN) (10 ml) was added 1-ethyl-3-(di-
methylaminopropyl) carbodiimide hydrochloride (WSC.HCl) (0.437 mmol)
in CH2Cl2 (10 ml), and the mixture was stirred at room temperature for 1 h.
After being cooled at 0 °C, 4-N,N-dimethylamino piperidine (0.437 mmol)
was added, and the mixture was stirred at room temperature overnight. To
the mixture was added 1 M NaOH followed by extraction with CHCl3. The
organic layer was dried over anhydrous K2CO3 and concentrated in vacuo.
The residue was chromatographed on
a silica gel column using
CHCl3–MeOH (95 : 5) as eluent to yield a free amine. The resulting amine
was diluted with MeOH, and the solution was cooled at 0 °C. To the solu-
tion, 4 N-HCl/AcOEt was added and the mixture was concentrated in vacuo,
and recrystallized from CHCl3–Et2O to give 160 mg (0.233 mmol, 72%) of 2
as a colorless powder. mp 240 °C (dec.). 1H-NMR (CDCl3) d: 1.39—1.80
(2H, m), 2.07 (2H, m), 2.41 (2H, m), 2.66 (1H, m), 2.72 (6H, s), 2.95—3.20
(2H, m), 3.43 (1H, m), 4.04 (1H, m), 4.52 (1H, m), 4.86 (1H, m), 6.78 (1H,
s), 6.81 (1H, m), 7.01 (2H, m), 7.19 (1H, m), 7.26—7.43 (8H, m), 7.44—
7.60 (5H, m), 10.35 (1H, s). EI-MS m/z: 649 (Mϩϩ1). Anal. Calcd for
C39H38N4O3F2·HCl: C, 68.36; H, 5.74; N, 8.18; Cl, 5.17; F, 5.55. Found: C,
68.44; H, 5.87; N, 8.09; Cl, 5.07; F, 5.41.
Compounds 8b—h were synthesized in the same manner.
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-methylben-
zanilide Monohydrochloride (8b): Colorless powder. Yield; 62%. mp 204—
206 °C. 1H-NMR (DMSO-d6) d: 1.40—1.80 (2H, m), 2.09 (2H, m), 2.42
(2H, m), 2.67 (1H, m), 2.71 (6H, s), 2.98—3.21 (2H, m), 4.06 (1H, d,
Jϭ13 Hz), 4.53 (1H, d, Jϭ13 Hz), 4.89 (1H, m), 6.81 (1H, s), 6.85 (1H, d,
Jϭ8 Hz), 7.09 (2H, d, Jϭ8 Hz), 7.20 (1H, t, Jϭ8 Hz), 7.25—7.44 (5H, m),
7.52 (1H, d, Jϭ8 Hz), 7.58 (2H, d, Jϭ8 Hz), 10.39 (1H, s), 10.72 (1H, m).
EI-MS m/z: 586 (Mϩ). Anal. Calcd for C34H36N4O3F2·HCl·2H2O: C, 61.95;
H, 6.27; N, 8.50; Cl, 5.38; F, 5.76. Found: C, 61.99; H, 6.34; N, 8.21; Cl,
5.40; F, 5.68.
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-methoxyben-
zanilide Monohydrochloride (8c): Colorless amorphous. Yield; 69%. 1H-
NMR (DMSO-d6) d: 1.42—1.78 (2H, m), 2.08 (2H, m), 2.42 (2H, m), 2.67
(1H, m), 2.72 (6H, s), 2.99—3.22 (2H, m), 3.43 (1H, m), 3.85 (3H, s), 4.04
(1H, m), 4.52 (1H, m), 4.83 (1H, m), 6.79 (1H, s), 6.83 (1H, m), 7.02—7.21
(5H, m), 7.31 (1H, t, Jϭ8 Hz), 7.44—7.62 (5H, m), 10.19 (1H, s), 10.56
(1H, m). FAB-MS m/z: 603 (Mϩϩ1).
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-ethoxyben-
zanilide Monohydrochloride (8d): Colorless amorphous. Yield; 51%. 1H-
NMR (DMSO-d6) d: 1.35 (3H, s), 1.40—1.80 (2H, m), 2.09 (2H, m), 2.47
(2H, m), 2.68 (1H, m), 2.70 (3H, s), 2.71 (3H, s), 2.95—3.54 (3H, m), 4.06
(1H, m), 4.15 (2H, q, Jϭ7 Hz), 4.53 (1H, m), 4.86 (1H, m), 6.81 (1H, s),
6.84 (1H, m), 7.02—7.21 (4H, m), 7.31 (1H, t, Jϭ8 Hz), 7.44—7.62 (4H,
m), 10.20 (1H, s), 10.76 (1H, m). FAB-MS m/z: 617 (Mϩϩ1).
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-isopropoxyben-
zanilide Monohydrochloride (8e): Colorless amorphous. Yield; 62%. 1H-
NMR (DMSO-d6) d: 1.31 (6H, d, Jϭ6 Hz), 1.40—1.80 (2H, m), 2.08 (2H,
m), 2.42 (2H, m), 2.67 (1H, m), 2.72 (6H, s), 2.99—3.22 (2H, m), 3.43 (1H,
m), 3.85 (3H, s), 4.04 (1H, m), 4.53 (1H, m), 4.73 (1H, m), 4.86 (1H, m),
6.81 (1H, s), 6.84 (1H, m), 6.98—7.36 (5H, m), 7.31 (1H, m), 7.42—7.70
(5H, m), 10.19 (1H, s), 10.77 (1H, m). FAB-MS m/z: 631 (Mϩϩ1).
(Z)-4Ј-({4,4-Difluoro-5-[(4,4-dimethylaminopiperidino)carbonylmethyl-
ene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-chloroben-
Compounds 8j—r were synthesized in the same manner.
(Z)-4Ј-{[4,4-Difluoro-5-(N-methylcarbamoylmethylene)-2,3,4,5-tetrahy-
dro-1H-1-benzoazepin-1-yl]carbonyl}-2-ethoxybenzanilide (8j): Colorless
powder. Yield; 69%. mp 213—215 °C. 1H-NMR (CDCl3) d: 1.60 (3H, t,
Jϭ7 Hz), 2.38 (1H, m), 2.65 (1H, m), 2.96 (3H, d, Jϭ5 Hz), 3.31 (1H, s),
4.19 (2H, q, Jϭ7 Hz), 4.87 (1H, Br), 6.27 (1H, m), 6.35 (1H, s), 6.69 (1H, d,
Jϭ8 Hz), 6.91 (1H, d, Jϭ8 Hz), 7.00—7.10 (4H, m), 7.23 (1H, t, Jϭ7 Hz),
7.40—7.50 (4H, m), 8.22 (1H, d, Jϭ6 Hz), 10.15 (1H, s). FAB-MS m/z: 520
(Mϩϩ1). Anal. Calcd for C29H27N3O4F2: C, 67.04; H, 5.24; N, 8.09; F, 7.31.
Found: C, 66.82; H, 5.33; N, 8.10; F, 7.16.
(Z)-2-Ethoxy-4Ј-{[5-(N-ethylcarbamoylmethylene)-4,4-difluoro-2,3,4,5-
tetrahydro-1H-1-benzoazepin-1-yl]carbonyl}benzanilide (8k): Colorless
powder. Yield; 77%. mp 197—199 °C. 1H-NMR (CDCl3) d: 1.24 (3H, t,
Jϭ7 Hz), 1.60 (3H, t, Jϭ7 Hz), 2.40 (1H, m), 2.70 (1H, m), 3.34 (1H, m),
3.44 (1H, m), 4.17 (2H, q, Jϭ7 Hz), 4.88 (1H, m), 6.36 (2H, m), 6.67 (1H, d,
Jϭ8 Hz), 6.68 (1H, d, Jϭ8 Hz), 7.00—7.10 (4H, m), 7.23 (1H, t, Jϭ7 Hz),
7.40—7.50 (4H, m), 8.22 (1H, d, Jϭ6 Hz), 10.14 (1H, s). FAB-MS m/z: 534
(Mϩϩ1). Anal. Calcd for C30H29N3O4F2·0.1H2O: C, 67.30; H, 5.50; N, 7.85;
F, 7.10. Found: C, 67.01; H, 5.64; N, 7.93; F, 6.83.
(Z)-4Ј-({4,4-Difluoro-5-[N-(1-propyl)-carbamoylmethylene]-2,3,4,5-
tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-ethoxybenzanilide (8l): Col-
orless powder. Yield; 79%. mp 214—216 °C. 1H-NMR (CDCl3) d: 0.98 (3H,
t, Jϭ7 Hz), 1.60—1.70 (5H, m), 2.40 (1H, m), 2.66 (1H, m), 3.30—3.40
(3H, m), 4.18 (2H, q, Jϭ7 Hz), 4.88 (1H, m), 6.36 (2H, br), 6.68 (1H, d,
Jϭ8 Hz), 6.89 (1H, d, Jϭ8 Hz), 7.00—7.10 (4H, m), 7.23 (1H, t, Jϭ7 Hz),
7.40—7.50 (4H, m), 8.22 (1H, d, Jϭ6 Hz), 10.15 (1H, s). FAB-MS m/z: 548
(Mϩϩ1). Anal. Calcd for C31H31N3O4F2·0.2H2O: C, 67.55; H, 5.74; N, 7.62;
F, 6.89. Found: C, 67.74; H, 6.13; N, 7.65; F, 6.65.
(Z)-4Ј-({4,4-Difluoro-5-[N-(2-propyl)carbamoylmethylene]-2,3,4,5-
tetrahydro-1H-1-benzoazepin-1-yl}carbonyl)-2-ethoxybenzanilide
(8m):
1
Colorless powder. Yield; 82%. mpϾ230 °C. H-NMR (CDCl3) d: 1.25 (6H,
t, Jϭ7 Hz), 1.60 (3H, t, Jϭ7 Hz), 2.37 (1H, m), 2.67 (1H, m), 3.33 (1H, m),
4.18 (2H, q, Jϭ7 Hz), 4.25 (1H, m), 4.88 (1H, s), 6.11 (1H, m), 6.35 (1H, s),

1080
Vol. 51, No. 9
diately by filtration using glass filters. The filters were rinsed twice with Tris
buffer and radioactivity retained on them was counted with a liquid scintilla-
tion counter. Specific binding was calculated as the total binding minus non-
specific binding, which was determined using 1 mM unlabeled AVP. The con-
centration of test compound that caused 50% inhibition (IC50) of the specific
binding of [3H]AVP was determined by regression analysis of the displace-
ment curve. Inhibitory dissociation constant (Ki) was calculated from the
following formula: KiϭIC50/(1ϩ[L]/Kd), where [L] is concentration of radi-
oligand present in the tubes and Kd is the dissociation constant of radioli-
gand obtained from the Scatchard plot.
6.67 (1H, d, Jϭ8 Hz), 6.89 (1H, d, Jϭ8 Hz), 7.00—7.10 (4H, m), 7.23 (1H,
t, Jϭ7 Hz), 7.40—7.50 (4H, m), 8.22 (1H, d, Jϭ6 Hz), 10.14 (1H, s). FAB-
MS m/z: 548 (Mϩϩ1). Anal. Calcd for C31H31N3O4F2·0.1H2O: C, 67.72; H,
5.73; N, 7.65; F, 6.91. Found: C, 67.56; H, 5.77; N, 7.59; F, 6.71.
(Z)-4Ј-{[5-(N-Cyclopropylcarbamoylmethylene)-4,4-difluoro-2,3,4,5-
tetrahydro-1H-1-benzoazepin-1-yl]carbonyl}-2-ethoxybenzanilide
(8n):
Colorless powder. Yield; 80%. mpϾ230 °C. 1H-NMR (CDCl3) d: 0.60—
0.70 (2H, m), 0.80—0.90 (2H, m), 1.59 (3H, t, Jϭ7 Hz), 2.38 (1H, s), 2.68
(1H, m), 2.85 (1H, m), 3.28 (1H, m), 4.14 (2H, q, Jϭ7 Hz), 4.87 (1H, s),
6.33 (1H, s), 6.60—6.70 (2H, m), 6.83 (1H, d, Jϭ8 Hz), 7.00—7.10 (4H,
m), 7.22 (1H, t, Jϭ7 Hz), 7.30—7.40 (4H, m), 8.20 (1H, d, Jϭ6 Hz), 10.13
(1H, s). FAB-MS m/z: 546 (Mϩϩ1). Anal. Calcd for
C31H29N3O4F2·0.25H2O: C, 67.69; H, 5.41; N, 7.64; F, 6.91. Found: C,
67.42; H, 5.42; N, 7.96; F, 6.77.
Receptor Binding Assay: For the Cloned Human Receptors12,13) The
cloned human AVP receptor subtypes were stably expressed in CHO cells
and plasma membranes prepared according to the reported protocols.
V1A Receptor Antagonistic Activity12,13) Pithed rats were maintained at
37 °C by means of a thermostat-controlled heating board. For i.v. injection,
compounds were dissolved in DMF. After the stabilization of blood pressure,
compounds or vehicle was given (0.5 ml/kg i.v.) 5 min before the injection of
AVP (30 mU/kg i.v.). The dose of compound causing a 50% inhibition of the
pressor response to AVP (ID50) was calculated.
(Z)-4Ј-{[4,4-Difluoro-5-(N,N-dimethylcarbamoylmethylene)-2,3,4,5-
tetrahydro-1H-1-benzoazepin-1-yl]carbonyl}-2-ethoxybenzanilide (8o): Col-
orless powder. Yield; 84%. mp 195—198 °C. 1H-NMR (CDCl3) d: 1.61 (3H,
t, Jϭ7 Hz), 2.30—2.80 (2H, m), 3.04 (3H, s), 3.09 (3H, s), 3.28 (1H, m),
4.26 (2H, q, Jϭ7 Hz), 5.04 (1H, m), 6.34 (1H, s), 6.72 (1H, d, Jϭ8 Hz), 6.99
(1H, d, Jϭ8 Hz), 7.10—7.50 (9H, m), 8.25 (1H, d, Jϭ6 Hz), 10.18 (1H, s).
FAB-MS m/z: 534 (Mϩϩ1). Anal. Calcd for C30H29N3O4F2·0.5H2O: C,
66.41; H, 5.57; N, 7.74; F, 7.00. Found: C, 66.37; H, 5.84; N, 7.73; F, 6.70.
(Z)-4Ј-{[5-(N,N-Diethylcarbamoylmethylene)-4,4-difluoro-2,3,4,5-
Acknowledgements The authors are grateful to Dr. Yuichi Tomura, Mr.
Koh-ichi Wada, Ms. Junko Tsukada, Dr. Chikashi Saitoh, Mr. Masanao
Sanagi, Ms. Hiroko Yanai and Mr. Eisaku Yamamoto for the biological eval-
uation and to the staff of the Division of Analysis Research Laboratories for
the spectral measurements.
tetrahydro-1H-1-benzoazepin-1-yl]carbonyl}-2-ethoxybenzanilide
(8p):
1
Colorless powder. Yield; 73%. mp 164—165 °C. H-NMR (CDCl3) d: 1.21
(6H, t, Jϭ8 Hz), 1.60 (3H, t, Jϭ7 Hz), 2.40—2.80 (2H, m), 3.30 (1H, m),
3.67 (4H, q, Jϭ8 Hz), 4.26 (2H, q, Jϭ7 Hz), 5.05 (1H, m), 6.38 (1H, s), 6.71
(1H, d, Jϭ8 Hz), 6.98 (1H, d, Jϭ8 Hz), 7.00—7.30 (5H, m), 7.36 (1H, t,
Jϭ7 Hz), 7.40—7.50 (3H, m), 8.24 (1H, d, Jϭ6 Hz), 10.17 (1H, s). FAB-MS
m/z: 562 (Mϩϩ1). Anal. Calcd for C32H33N3O4F2·0.5H2O: C, 67.35; H,
6.01; N, 7.37; F, 6.66. Found: C, 67.56; H, 5.98; N, 7.43; F, 6.63.
References
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(Z)-4Ј-[(4,4-Difluoro-5-morpholinocarbamoylmethylene-2,3,4,5-tetrahy-
dro-1H-1-benzoazepin-1-yl)carbonyl]-2-ethoxybenzanilide (8q): Colorless
powder. Yield; 82%. mp 144—147 °C. 1H-NMR (CDCl3) d: 1.61 (3H, t,
Jϭ7 Hz), 2.40—2.80 (2H, m), 3.30 (1H, m), 3.58 (2H, m), 3.74 (6H, m),
4.26 (2H, q, Jϭ7 Hz), 5.05 (1H, m), 6.33 (1H, s), 6.74 (1H, d, Jϭ8 Hz), 6.99
(1H, d, Jϭ8 Hz), 7.10—7.60 (9H, m), 8.25 (1H, d, Jϭ6 Hz), 10.17 (1H, s).
FAB-MS m/z: 576 (Mϩϩ1). Anal. Calcd for C32H31N3O5F2: C, 66.77; H,
5.43; N, 7.30; F, 6.60. Found: C, 66.81; H, 5.35; N, 7.33; F, 6.63.
(Z)-4Ј-[(4,4-Difluoro-5-thiomorpholinocarbamoylmethylene-2,3,4,5-
tetrahydro-1H-1-benzoazepin-1-yl)carbonyl]-2-ethoxybenzanilide (8r): Col-
orless powder. Yield; 42%. mp 156—158 °C. 1H-NMR (CDCl3) d: 1.61 (3H,
t, Jϭ7 Hz), 2.27—2.86 (6H, m), 3.26 (1H, m), 3.70—4.14 (4H, m), 4.26
(2H, q, Jϭ7 Hz), 5.02 (1H, m), 6.33 (1H, s), 6.74 (1H, m), 6.99 (1H, t,
Jϭ8 Hz), 7.07—7.29 (6H, m), 7.36 (1H, m), 7.53 (3H, m), 8.24 (1H, m),
10.17 (1H, s). FAB-MS m/z: 592 (Mϩϩ1). Anal. Calcd for C32H31N3O4SF2:
C, 64.96; H, 5.28; N, 7.10; S, 5.42; F, 6.42. Found: C, 63.07; H, 5.26; N,
6.86; S, 5.26; F, 6.25.
Receptor Binding Assay: For the Rat Receptors12,13) Binding assays
were performed using [3H]AVP on plasma membranes prepared from rat
liver or kidney. Plasma membrane preparations were incubated with various
concentrations of [3H]AVP (0.1—3.0 nM). Radioligands (0.5 nM) were added
to each membrane preparation and the mixture was incubated with various
concentrations of the compounds in 250 ml of assay buffer (50 mM Tris–HCl,
pH 7.5, 5 mM MgCl2 and 0.1% bovine serum albumin). After incubation
(60 min at 25 °C), the reaction was terminated by addition of 3 ml of ice-
cooled Tris buffer (50 mM Tris–HCl, pH 7.5, 5 mM MgCl2), followed imme-