Synthesis and pharmacological evaluation of 5-(4-biphenyl)-3-methyl-4-phenyl-1,2,4-triazole derivatives as a novel class of selective antagonists for the human vasopressin V1a receptor

Article abstract of DOI:10.1021/jm010544r

A series of 5-(4-biphenyl)-3-methyl-4-phenyl-1,2,4-triazole derivatives were prepared and evaluated as selective antagonists for the human vasopressin V_1A receptor. The compounds were examined for their affinity to the cloned human V_1A receptor (hV_1A) and selectivity vs the cloned human V₂ receptor (hV₂). By utilizing the structure-activity relationship on 4,4-difluoro-5-methylidene-2,3,4,5-tetrahydrobenzazepine derivatives as dual antagonists for the V_1A and V₂ receptors in our previous study, we found that substituting the methoxy group at the 2-position of the 4-phenyl ring with (4-methylpiperazin-1-yl)alkoxy moieties brought about marked improvement of both affinity to hV_1A and selectivity vs hV₂. Further introduction of a methyl group into the 6-position of the 4-phenyl ring resulted in additional improvement of selectivity. One particular compound, 5-(4-biphenyl)-3-methyl-4-{2-[6-(4-methyl-l-piperazinyl)-hexyloxy]phenyl} -1,2,4-triazole (19) showed potent affinity to hV_1A with a K_i value of 1.04 nM and high selectivity with a 1700-fold selectivity vs hV₂. We also found marked differences in the affinity of compounds in this series between the human and the rat receptors. Compound, 19 was further examined for its V_1A receptor antagonist activity in rats. As a result, 19 demonstrated antagonist activities toward an arginine vasopressin-induced increase in diastolic blood pressure after intravenous or oral administration and long-lasting oral activity.

Full text of DOI:10.1021/jm010544r

J . Med. Chem. 2002, 45, 2589-2598
2589
Syn th esis a n d P h a r m a cologica l Eva lu a tion of
5-(4-Bip h en yl)-3-m eth yl-4-p h en yl-1,2,4-tr ia zole Der iva tives a s a Novel Cla ss of
Selective An ta gon ists for th e Hu m a n Va sop r essin V1A Recep tor
Akio Kakefuda,* Takeshi Suzuki, Takahiko Tobe, J unko Tsukada,† Atsuo Tahara, Shuichi Sakamoto, and
Shin-ichi Tsukamoto
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., 21 Miyukigaoka, Tsukuba,
Ibaraki 305-8585, J apan
Received November 30, 2001
A series of 5-(4-biphenyl)-3-methyl-4-phenyl-1,2,4-triazole derivatives were prepared and
evaluated as selective antagonists for the human vasopressin V1A receptor. The compounds
were examined for their affinity to the cloned human V1A receptor (hV1A) and selectivity vs the
cloned human V2 receptor (hV2). By utilizing the structure-activity relationship on 4,4-difluoro-
5-methylidene-2,3,4,5-tetrahydrobenzazepine derivatives as dual antagonists for the V1A and
V2 receptors in our previous study, we found that substituting the methoxy group at the
2-position of the 4-phenyl ring with (4-methylpiperazin-1-yl)alkoxy moieties brought about
marked improvement of both affinity to hV1A and selectivity vs hV2. Further introduction of a
methyl group into the 6-position of the 4-phenyl ring resulted in additional improvement of
selectivity. One particular compound, 5-(4-biphenyl)-3-methyl-4-{2-[6-(4-methyl-1-piperazinyl)-
hexyloxy]phenyl}-1,2,4-triazole (19) showed potent affinity to hV1A with a Ki value of 1.04 nM
and high selectivity with a 1700-fold selectivity vs hV2. We also found marked differences in
the affinity of compounds in this series between the human and the rat receptors. Compound
19 was further examined for its V1A receptor antagonist activity in rats. As a result, 19
demonstrated antagonist activities toward an arginine vasopressin-induced increase in diastolic
blood pressure after intravenous or oral administration and long-lasting oral activity.
In tr od u ction
ylene]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl}carbonyl)-
2-phenylbenzanilide (3), which is a dual antagonist for
the V1A and V2 receptors.14 A subsequent research
program was aimed at the development of subtype
selective antagonists for the AVP receptor, which ap-
pears essential in elucidating the pathophysiological
roles of AVP.
Arginine vasopressin (AVP) is a peptide hormone
secreted by the posterior pituitary and has been known
to play important physiological roles in vasoconstriction
and antidiuresis. AVP exerts its effects through binding
to specific receptors coupled to distinct second mes-
sengers. To date, two peripheral AVP receptor subtypes,
the V1A and V2 receptors, have been identified.1,2
Through the V1A receptors, AVP activates phospholipase
A2, C, and D,3 which results in the mobilization of
intracellular calcium and protein phosphorylation.4 The
V1A receptors are present in vascular smooth muscle
cells, hepatocytes, platelets, and mesangial cells. These
receptors serve to mediate the contraction, proliferation,
and hypertrophy of cells.5-9 In contrast, the V2 receptors
stimulate adenylate cyclase resulting in the production
of cyclic adenosine 3′,5′-phosphate (AMP) and are
present in renal epithelial cell lines (LLC-PK1) where
they control free water and urea reabsorption.10-11
Abnormal secretion of AVP is thought to be involved
in the pathology of several diseases and disorders
including heart failure, hypertension, renal diseases,
and hypernatremia.12 Consequently, antagonists for the
AVP receptors are considered to be beneficial in treating
the above diseases.13
So far, two nonpeptide selective antagonists for the
V1A receptor, OPC-21268 (1)15,16 and SR49059 (2),17 have
been reported (Chart 1). Clinical studies for 2 are un-
derway, and its therapeutic effects have been found.18,19
These facts prompted us to investigate a novel class of
selective antagonists for the V1A receptor.
It has been reported that there are marked differences
in the AVP receptor specificity between different
species.17,20-21 For instance, 1 showed poor affinity to
the cloned human V1A receptor even at 10 µM,22 despite
its high affinity to the rat V1A receptor (Ki ) 23.5 nM).23
Therefore, we employed Chinese hamster ovary (CHO)
cells, which stably express the cloned human V1A or V2
receptor subtypes (hV1A and hV2) for a receptor binding
assay.23 High-throughput screening (HTS) of the Ya-
manouchi chemical library based on a hV1A binding
assay led to the identification of 5-(4-biphenyl)-4-(2-
methoxyphenyl)-3-methyl-1,2,4-triazole (4). Compound
4 was found to possess a novel structure and show
moderate hV1A affinity and selectivity vs hV2 (Ki ) 120
nM, hV2/hV1A ) 21). Blockade of the V2 receptor is
known to cause an increase in urine volume,23 which
may complicate the pharmacological evaluation of com-
pounds. Therefore, the aim of our research was focused
Investigations into the development of nonpeptide
antagonists for the AVP receptors afforded (Z)-4′-({4,4-
difluoro-5-[(4-dimethylamino-1-piperidyl)carbonylmeth-
* To whom correspondence should be addressed. Tel: +81-298-54-
1545. Fax: +81-298-52-5387. E-mail: kakefuda@yamanouchi.co.jp.
† Clinical Development Department.
10.1021/jm010544r CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/11/2002

2590 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Kakefuda et al.
Ch a r t 1
compounds. Furthermore, 5-(4-Biphenyl)-3-methyl-4-{2-
[6-(4-methyl-1-piperazinyl)hexyloxy]phenyl}-1,2,4-tria-
zole (19), which showed high affinity and selectivity to
hV1A, was examined for its V1A antagonist activity in
rats.
Ch em istr y
The synthesis of the novel 5-(4-biphenyl)-3-methyl-
4-phenyl-1,2,4-triazole derivatives is summarized in
Schemes 1 and 2. Compounds 16-32 were prepared
from aminophenols as shown in Scheme 1. The ami-
nophenols were converted to the thioimidates (5-8) in
four steps, and then, compounds 5-8 were subjected to
a cyclization reaction with an acyl hydrazide to give the
1,2,4-triazole derivatives (9-12). Compounds 9-12 were
finally transformed to the target compounds 16-27 and
30 by bromoalkylation and substitution with amines.
The hydrochloride salts of versatile intermediates (13-
15) were prepared by condensation of acyl chlorides with
diamines, which were then subjected to a reaction with
9 or 12 to afford 28, 29, 31, and 32.
The synthesis of 4-piperidyl derivatives (37-39) is
illustrated in Scheme 2. The ethylene and butylene
derivatives (37 and 38) were prepared from the com-
mercially available alcohols (33 and 34) by successive
protection, tosylation, substitution, and deprotection.
The propylene derivative (39) was synthesized from 9
by condensation with alcohol and hydrogenation of the
pyridine ring.
on not only enhancement of hV1A affinity but also
improvement of selectivity vs hV2. Our previous study
on the structure-activity relationships (SAR) of 3 and
its derivatives revealed that the terminal basic moiety
of 3 was important in exerting potent binding affinity,
especially for the V1A receptor.14 Thus, on the basis of
our hypothetical superimposition of 4 and the 1-benzoyl-
2,3,4,5-tetrahydrobenzazepine part of 3 as shown in
Figure 1, we examined the effect of the basic moiety on
hV1A, linked with the 4-phenyl ring of 4 using an
appropriate tether. Subsequently, we modified the
terminal basic part and the tether. Finally, we intro-
duced a methyl moiety into the 6-position of the 4-phen-
yl ring to investigate its steric effect. Representative
compounds were studied for their binding affinity to the
rat V1A and V2 receptors (rV1A and rV2) in order to
determine the species differences in the affinity of
P h a r m a cology
Bin d in g Assa y. Radioligand binding assays for hV1A
and hV2 were performed according to the reported
protocols22 using [3H]AVP on CHO cells stably express-
ing hV1A or hV2, respectively (Table 1). Binding assays
for rV1A and rV2 were also performed using [3H]AVP
on rat liver or [3H]AVP on rat kidney, respectively
(Table 2).23
In Vivo Assa y. In vivo activities of 19 were examined
by measuring the inhibition of the AVP-induced increase
F igu r e 1. Hypothetical superimposition of 3 and 4.

Antagonists for the Human Vasopressin Receptor
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2591
Sch em e 1. Synthesis of
5-(4-Biphenyl)-3-methyl-4-phenyl-1,2,4-triazole
Derivativesa
a
Reagents and conditions: (a) Ac2O, AcOEt; (b) BnBr, K2CO3, CH3CN, reflux; (c) P2S5, toluene, 70 °C; (d) MeI, K2CO3, CH3CN, 50 °C;
(e) ArCONHNH2, DMF, 120 °C; (f) H2, Pd/C, DMF; (g) Br(CH2)nBr, K2CO3, CH3CN, reflux; (h) amine, K2CO3, CH3CN, reflux; (i) 13-15,
K2CO3, CH3CN, reflux; (j) amine, CH3CN.
Sch em e 2. Synthesis of 4-Piperidyl Derivativesa
a
Reagents and conditions: (a) TrCl, Et3N, DMF; (b) TsCl, pyridine; (c) 9, K2CO3, CH3CN, reflux; (d) 4 N HCl(g)/AcOEt; (e) 4-(3-
hydroxypropyl)pyridine, PPh3, DEAD, THF; (f) H2, PtO2, AcOH.
in diastolic blood pressure (DBP) in pithed rats after
intravenous (iv) or in conscious rats after oral (po)
administration (Figures 3 and 4).23 The dose of 19
required to inhibit AVP-induced DBP response by 50%,
the ID50 value, was determined.
basic part. The propoxy derivative (16) showed improved
hV1A affinity (Ki ) 38.3 nM) and selectivity vs hV2 (hV2/
hV1A ) 270), by 3-fold and 13-fold as compared to 4,
respectively. We then examined the effects of changing
the length of the alkyl chain between the 4-methylpi-
perazine moiety and the 4-phenyl ring of 16. Figure 2A
shows the relation between the hV1A affinity and the
alkyl chain number (n) of compounds 16-22. The more
the alkyl chain number from propylene (n ) 3, 16) was,
the higher hV1A affinity of derivatives was until a peak
for hV1A affinity appeared at hexylene (n ) 6, 19).
Further elongation of the alkyl chain from 19 caused a
decrease in the hV1A affinity of compounds (20-22). A
similar tendency was observed between the hV2 affinity
and the alkyl chain number; however, the increase in
range for hV2 affinity was lower than that for hV1A
affinity. These results revealed that a peak for selectiv-
ity also appeared at hexylene (n ) 6, 19) as shown in
Figure 2B. Of the compounds 16-22, 19 had the high-
est hV1A affinity (Ki ) 1.04 nM) and selectivity (hV2/
hV1A ) 1700), which were 120-fold and 81-fold higher
Resu lts a n d Discu ssion
The synthesized compounds were primarily evaluated
for their binding affinity to hV1A and hV2. The results
were expressed as Ki values, and the selectivity for hV1A
vs hV2 (Ki ratio, hV2/hV1A) is presented in Table 1.
Initially, we examined the effects of the tether be-
tween the terminal basic moiety and the 4-phenyl ring.
Although the 4-(dimethylamino)-1-piperidyl moiety of
3 has been shown to be effective in exerting high affinity
to the V1A receptor,14 a variety of metabolism at the
4-dimethylamino part has been observed in our previous
study. In addition, replacement of the 4-dimethylami-
nopiperidine moiety with other basic moieties has been
found to retain the affinity to the V1A receptor.14 We
chose a 4-methyl-1-piperazinyl moiety as the terminal

2592 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Kakefuda et al.
Ta ble 1. Binding Affinities of 5-(4-Biphenyl)-3-methyl-4-phenyl-1,2,4-triazole Derivatives to the Cloned Human V1A and V2 Receptors
a
Ki values were obtained from three to four independent experiments performed in duplicate. Each value indicates a mean ( SEM.
Mean from two experiments. c Not tested.
b

Antagonists for the Human Vasopressin Receptor
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2593
Ta ble 2. Binding Affinities of Representative Compounds to the Rat V1A and V2 Receptors and the Cloned Human V1A and V2
Receptors
Ki (nM)a
selectivity Ki ratio
rV2/rV1A hV2/hV1A
72 1700
100
5.8
compd
rV1A
hV1A
rV2
hV2
19
26
29
30
32
1
17.3 ( 2.3
9.69 ( 2.5
62.7 ( 12
1.04 ( 0.15
1.05 ( 0.11
3.03 ( 0.31
1.46 ( 0.13
1.01 ( 0.15
1240 ( 110
1010 ( 220
364 ( 65
11 700 ( 1700
1840 ( 330
1780 ( 160
979 ( 70
585 ( 32
930
190
102 ( 8.8
14 400 ( 1300
1920 ( 130
>10 000
110
57
9900
1900
32.4 ( 5.7
23.5 ( 4.4
1.43 ( 0.17
0.160 ( 0.040
>10 000
>10 000
285 ( 36
0.770 ( 0.16
>430
200
2
3
0.530 ( 0.080
0.620 ( 0.23
178 ( 41
1.19 ( 0.02
340
4.8
1.9
a
Ki values were obtained from four to six independent experiments performed in duplicate. Each value indicates a mean ( SEM.
F igu r e 4. Time course of inhibitory effects of oral administra-
tion of 19 on AVP (30 mg/kg)-induced increases in DBP in
conscious rats. The inhibition was expressed as a percent
change of control response. Each value indicates a mean (
SEM from five independent experiments.
Subsequently, we studied the effect of substituting the
position of the 6-(4-methyl-1-piperazinyl)hexyloxy moi-
ety on the 4-phenyl ring. The m-substituted (23) and
the p-substituted (24) derivatives showed significantly
decreased or no hV1A affinity. These results suggested
that ortho substitution on the 4-phenyl ring might be
suitable for high hV1A affinity.
The 4-piperidyl derivatives (37-39) were also evalu-
ated. The butylene derivative (38) exhibited high hV1A
affinity and selectivity (Ki ) 2.12 nM, hV2/hV1A ) 2100),
which were comparable to those of 19. This result
F igu r e 2. Relation between hV1A affinities of the alkyl chain
suggested that the 4-(4-piperidyl)butoxy moiety of 38
number (A) and selectivities and the alkyl chain number (B)
might contribute to high hV1A affinity, as in the case
in 16-22.
with the 6-(4-methyl-1-piperazinyl)hexyloxy moiety of
19.
Next, we examined the effects of modifying the
piperazine part of 19 to investigate the role of the
nitrogen atoms in affinity. The 1-piperidyl derivative
(25), the 1-imidazolyl derivative (27), and the amide
derivative (28) showed approximately 1/10 times the
hV1A affinity of 19. However, the 4-(1-piperidyl)-1-
piperidyl derivative (26) exhibited comparable hV1A
affinity (Ki ) 1.05 nM) to 19. It appered that compounds
with two basic nitrogens were superior to compounds
with one basic nitrogen or aromatic nitrogens with
respect to the hV1A affinity. On the other hand, 29 with
the 4-carbonylbenzyloxy moiety as the tether possessed
high hV1A affinity (Ki ) 3.03 nM), despite the fact that
it has only one basic nitrogen. The 4-carbonylbenzyloxy
moiety was found to be one of the preferable tethers.
Finally, we introduced a methyl group into the
6-position of the 4-phenyl ring to investigate the steric
effect of the six substituent. With respect to hV1A
affinity, the 6-hexyloxy derivative (30) and the 4-car-
bonylbenzyloxy derivative (31) were comparable to or
F igu r e 3. Inhibitory effects of intravenous administration of
19 on AVP (30 mU/kg)-induced increases in DBP in pithed
rats. The inhibition was expressed as a percent change of
control response. Each value indicates a mean ( SEM from
four independent experiments. The ID50 value (half-maximal
dose inhibiting an AVP-induced response) was 0.695 ( 0.044
mg/kg iv.
than those of 4, respectively. Moreover, 19 was better
than 2 with respect to selectivity (hV2/hV1A ) 1700 for
19 and 340 for 2, respectively).

2594 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Kakefuda et al.
slightly less than the corresponding unsubstituted
derivatives (19 and 29, respectively). For hV2 affinity,
however, both 30 and 31 were much less than the
unsubstituted derivatives. These resulted in approxi-
mately 6-fold improvement of selectivity in both 30 and
31. In particular, 30 showed the highest selectivity (hV2/
hV1A ) 9900) in this series. Furthermore, the 4-(1-
piperidyl)-1-piperidyl derivative (32) of 31 displayed
high hV1A affinity (Ki ) 1.01 nM) and selectivity (hV2/
hV1A ) 1900), which were comparable to those of 19.
These results indicated that further introduction of a
methyl group into the 6-position of the 4-phenyl ring
afforded an additional improvement in selectivity. This
methyl group may contribute to the improvement in
selectivity by maintaining the 4-phenyl ring in a more
preferable conformation. From these results described
above, we concluded that introduction of a basic moiety
linked with the 4-phenyl ring by an appropriate tether
(i.e., hexyloxy) brought about marked improvement of
both hV1A affinity and selectivity vs hV2.
ring with (4-methyl-1-piperazinyl)alkoxy moieties brought
about marked improvement of both affinity and selec-
tivity for hV1A. Alteration of the length of the alkyl chain
between the 4-methylpiperazine moiety and the 4-phen-
yl ring revealed that peaks appeared at hexylene for
both hV1A affinity and selectivity. It was found that
further introduction of a methyl group into the 6-posi-
tion of the 4-phenyl ring afforded an additional im-
provement of selectivity. One of the synthesized com-
pounds, 19, showed high affinity and selectivity for hV1A
with a Ki value of 1.04 nM and a 1700-fold selectivity
vs hV2, respectively. We also found marked differences
in both the affinity for V1A and the selectivity vs hV2 of
compounds between the human and the rat AVP recep-
tors. In vivo evaluation in rats demonstrated that
compound 19 was a selective and orally active antago-
nist for the V1A receptor and that its activity was long-
lasting. We believe that this study would provide a novel
approach to selective antagonists for the human V1A
receptor.
To investigate differences in the affinity of compounds
to the AVP receptors of different species, we examined
the binding affinity of our representative compounds
together with known AVP antagonists to the rat AVP
receptors, as shown in Table 2. All selected compounds
in this series (19, 26, 29, 30, and 32) showed lower
affinity to rV1A than to hV1A by 9-70-fold. Interestingly,
the series of compounds exhibited similar affinity to
both rV2 and hV2, which resulted in a lower selectivity
of compounds for the rat receptors than those for the
human receptors. For example, 19 showed relatively low
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Yanaco
MP-500D melting point apparatus and are uncorrected. 1H
nuclear magnetic resonance (NMR) spectra were recorded on
a J EOL J MN-LA300 or J EOL J MN-EX400 spectrometer, and
the chemical shifts are expressed in δ (ppm) values with
tetramethylsilane as an internal standard (in NMR descrip-
tion, s ) singlet, d ) doublet, t ) triplet, m ) multiplet, and
br ) broad peak). Mass spectra were recorded on a J EOL J MS-
LX2000 spectrometer. For salts, assignments of ion peaks are
based on the basic component. The elemental analyses were
performed with a Yanaco MT-5 microanalyzer (C, H, N) and
a Yokogawa IC-7000S ion chromatographic analyzer (halogens)
and were within (0.4% of theoretical values. High-perfor-
mance liquid chromatography (HPLC) analyses were per-
formed on a Hitachi L-7400 (UV detector) with a Hitachi
L-7100 (pump); a TSKgel ODS-80TM column was used; eluent
CH3CN/0.01 M KH2PO4(aq) system at a flow rate of 1.0 mL/
min. Drying of organic solutions during workup was done over
anhydrous Na2SO4.
5-(4-Bip h en yl)-4-(2-h yd r oxyp h en yl)-3-m eth yl-1,2,4-tr i-
a zole (9). To a solution of 2-aminophenol (8.12 g, 74.4 mmol)
in AcOEt (80 mL) was added dropwise Ac2O (16.3 mL, 168
mmol) at 0 °C, and the mixture was stirred at room temper-
ature for 3 h. The mixture was concentrated in vacuo, and the
residual solid was recrystallized from AcOEt-hexane to give
2-hydroxyacetanilide (10.9 g, 97%) as a light yellow powder.
To a suspension of the acetanilide obtained above (10.9 g, 72.0
mmol) and K2CO3 (19.9 g, 144 mmol) in CH3CN (200 mL) was
added benzyl bromide (9.72 mL, 79.2 mmol), and the mixture
was stirred at 70 °C for 8 h. After it was cooled at room
temperature, the precipitate was removed by filtration and the
filtrate was concentrated in vacuo. The residual solid was
recrystallized from AcOEt-hexane to give 2-(benzyloxy)acet-
anilide (15.2 g, 86%) as a colorless powder. To a solution of
the anilide obtained above (22.6 g, 93.5 mmol) in toluene (300
mL) was added P2S5 (23.0 g, 104 mmol), and the mixture was
stirred at 70 °C for 2 h. After it was cooled at room temper-
ature, the precipitate was removed by filtration and the filtrate
was concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/AcOEt ) 2/1) to give
2-(benzyloxy)thioacetanilide (19.5 g, 81%) as a brown oil. A
suspension of the thioacetanilide obtained above (19.4 g, 75.4
mmol), K2CO3 (30.0 g, 217 mmol), and MeI (20.0 g, 140 mmol)
in CH3CN (300 mL) were stirred at 50 °C for 3 h. After it was
cooled at room temperature, the mixture was concentrated in
vacuo. The residue was partitioned between AcOEt (500 mL)
and H2O (300 mL). The organic layer was washed with
saturated NaCl(aq) (300 mL), dried, and concentrated in vacuo.
The residue was purified by column chromatography on silica
rV1A affinity (Ki ) 17.3 nM) and selectivity (rV2/rV1A
)
72), which were 17- and 24-fold lower than those to the
human receptors, respectively. In contrast, 2 and 3
exhibited similar binding affinity to both the rat and
the human receptors. Therefore, differences in the
affinity of compounds between the human and the rat
receptors should depend on their structures.
Compounds in this series had good rV1A affinity and
selectivity vs rV2, although their potencies were less
than those for the human receptors. Compound 19 was
chosen and examined further to evaluate its V1A recep-
tor antagonist activity in rats. Intravenous administra-
tion of 19 to pithed rats dose dependently antagonized
an AVP (30 mU/kg)-induced increase in DBP via the
V1A receptor with an ID50 value of 0.695 mg/kg (Figure
3). Furthermore, after oral administration of 19 (1, 10,
and 100 mg/kg) to conscious rats, the time courses of
antagonism effects against AVP-induced increase in
DBP were monitored (Figure 4). Compound 19 dose
dependently antagonized an AVP-induced increase in
DBP, and its effect lasted for at least 8 h at doses of
more than 10 mg/kg. In contrast, 19 did not affect the
urine volume even at a dose of 100 mg/kg. These results
demonstrated that 19 was a selective and orally active
antagonist for the V1A receptor in rats.
Con clu sion
A series of 5-(4-biphenyl)-3-methyl-4-phenyl-1,2,4-
triazole derivatives were prepared and evaluated for
their affinity to hV1A and selectivity vs hV2. By utilizing
the SAR on 4,4-difluoro-5-methylidene-2,3,4,5-tetrahy-
drobenzazepine derivatives, we found that substitution
of the methoxy group at the 2-position of the 4-phenyl

Antagonists for the Human Vasopressin Receptor
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2595
gel (hexane/AcOEt ) 2/1) to give 5 (16.2 g, 79%) as a yellow
oil. A solution of 5 (5.06 g, 18.7 mmol) and 4-biphenylcarboxylic
hydrazide (3.60 g, 17.0 mmol) in N,N-dimethylformamide
(DMF; 50 mL) was stirred at 120 °C for 23 h. After it was
cooled at room temperature, the mixture was concentrated in
vacuo. The residue was purified by column chromatography
on silica gel (CHCl3/MeOH ) 50/1) to give crude 4-(2-benzyl-
oxy)-5-(4-biphenyl)-3-methyl-1,2,4-triazole (5.60 g, 79%) as a
light yellow solid. To a solution of the triazole obtained above
(4.62 g, 11.0 mmol) in DMF (75 mL) was added 10% Pd/C (w/
w, 0.900 g), and the mixture was stirred under hydrogen
pressure (4 kg/cm3) at room temperature for 19 h. The catalyst
was removed by filtration, and the filtrate was concentrated
in vacuo. The residual solid was recrystallized from AcOEt-
hexane to give 2-hydroxyacetanilide (10.9 g, 97%) as a light
yellow powder The syrup was crystallized from AcOEt-hexane
J ) 7.5, 1.5 Hz), 7.31-7.56 (11H, m). MS (FAB): m/z 468
(MH+). Anal. (C29H33N5O) C, H, N.
5-(4-Biph en yl)-3-m eth yl-4-{2-[4-(4-m eth yl-1-piper azin yl)-
bu toxy]p h en yl}-1,2,4-tr ia zole (17). 60%; mp 103-104 °C
(Et2O-hexane). 1H NMR (300 MHz, CDCl3): δ 1.35-1.45 (2H,
m), 1.54-1.63 (2H, m), 2.21-2.40 (10H, m), 2.27 (3H, s), 2.30
(3H, s), 3.82-3.89 (1H, m), 3.93-4.00 (1H, m), 7.03-7.08 (1H,
m), 7.18 (1H, dd, J ) 8.1, 1.8 Hz), 7.31-7.56 (11H, m). MS
(FAB): m/z 482 (MH+). Anal. (C30H35N5O‚0.5H2O) C, H, N.
5-(4-Biph en yl)-3-m eth yl-4-{2-[5-(4-m eth yl-1-piper azin yl)-
p en tyloxy]p h en yl}-1,2,4-tr ia zole (18). 59%; mp 115-116 °C
(AcOEt-hexane). 1H NMR (300 MHz, DMSO-d6): δ 1.01-1.12
(2H, m), 1.16-1.29 (2H, m), 1.37-1.48 (2H, m), 2.04-2.21
(10H, m), 2.10 (3H, s), 2.16 (3H, s), 3.81-4.01 (2H, m), 7.12
(1H, t, J ) 7.7 Hz), 7.27 (1H, d, J ) 8.1 Hz), 7.34-7.56 (7H,
m), 7.63-7.67 (4H, m). MS (FAB): m/z 496 (MH+). Anal.
(C31H37N5O) C, H, N.
1
to give 9 (3.45 g, 95%) as a beige powder. H NMR (400 MHz,
DMSO-d6): δ 2.17 (3H, s), 6.95 (1H, t, J ) 8.0 Hz), 7.07 (1H,
t, J ) 8.0 Hz), 7.30-7.53 (7H, m), 7.60-7.65 (4H, m), 10.33
(1H, s). MS (FAB): m/z 328 (MH+).
5-(4-Biph en yl)-3-m eth yl-4-{2-[7-(4-m eth yl-1-piper azin yl)-
h ep tyloxy]p h en yl}-1,2,4-tr ia zole (20). 67%; mp 67-68 °C
(Et2O-hexane). 1H NMR (300 MHz, CDCl3): δ 1.20 (6H, br
s), 1.36-1.44 (2H, m), 1.50-1.57 (2H, m), 2.24-2.30 (4H, m),
2.28 (3H, s), 2.29 (3H, s), 2.43 (6H, br s), 3.80-3.87 (1H, m),
3.90-3.98 (1H, m), 7.02-7.06 (2H, m), 7.16 (1H, dd, J ) 8.1,
1.8 Hz), 7.31-7.56 (10H, m). MS (FAB): m/z 524 (MH+). Anal.
(C33H41N5O‚0.15H2O) C, H, N.
5-(4-Biph en yl)-3-m eth yl-4-{2-[8-(4-m eth yl-1-piper azin yl)-
octyloxy]p h en yl}-1,2,4-tr ia zole (21). 64%; mp 70-71 °C
(Et2O-hexane). 1H NMR (300 MHz, CDCl3): δ 1.19 (8H, br
s), 1.39-1.46 (2H, m), 1.52-1.57 (2H, m), 2.25-2.30 (6H, m),
2.28 (3H, s), 2.29 (3H, s), 2.45 (4H, br s), 3.79-3.87 (1H, m),
3.90-3.98 (1H, m), 7.02-7.06 (2H, m), 7.16 (1H, dd, J ) 8.0,
1.7 Hz), 7.30-7.56 (10H, m). MS (FAB): m/z 538 (MH+). Anal.
(C34H43N5O‚0.25H2O) C, H, N.
5-(4-Bip h en yl)-3-m et h yl-4-{2-[10-(4-m et h yl-1-p ip er a -
zin yl)d ecyloxy]p h en yl}-1,2,4-tr ia zole (22). 50%; mp 87-
88 °C (Et2O-hexane). 1H NMR (300 MHz, CDCl3): δ 1.22-
1.78 (12H, m), 1.40-1.57 (4H, m), 2.27-2.32 (4H, m), 2.28 (3H,
s), 2.29 (3H, s), 2.45 (6H, br s), 3.80-3.87 (1H, m), 3.90-3.98
(1H, m), 7.02-7.06 (2H, m), 7.16 (1H, dd, J ) 8.1, 1.7 Hz),
7.30-7.56 (10H, m). MS (FAB): m/z 566 (MH+). Anal.
(C36H47N5O) C, H, N.
Compounds 10-12 were prepared by a procedure similar
to that described for 9. Yields referr to the cyclization step to
construct the 1,2,4-triazole ring.
5-(4-Bip h en yl)-4-(3-h yd r oxyp h en yl)-3-m eth yl-1,2,4-tr i-
1
a zole (10). 68%. H NMR (300 MHz, DMSO-d6): δ 2.25 (3H,
s), 6.76 (1H, d, J ) 1.8 Hz), 6.80 (1H, d, J ) 7.8 Hz), 6.95 (1H,
dd, J ) 8.4, 2.4 Hz), 7.33-7.49 (7H, m), 7.66-7.68 (4H, m).
MS (FAB): m/z 328 (MH+).
5-(4-Bip h en yl)-4-(4-h yd r oxyp h en yl)-3-m eth yl-1,2,4-tr i-
1
a zole (11). 66%. H NMR (300 MHz, DMSO-d6): δ 2.22 (3H,
s), 6.90 (2H, d, J ) 8.7 Hz), 7.23 (2H, d, J ) 8.7 Hz), 7.34-
7.46 (7H, m), 7.65-7.68 (4H, m). MS (FAB): m/z 328 (MH+).
5-(4-Bip h en yl)-4-(2-h yd r oxy-6-m eth ylp h en yl)-3-m eth -
yl-1,2,4-tr ia zole (12). 41%. 1H NMR (300 MHz, DMSO-d6):
δ 1.80 (3H, s), 2.13 (3H, s), 6.82 (1H, d, J ) 7.5 Hz), 6.95 (1H,
d, J ) 8.1 Hz), 7.25-7.52 (7H, m), 7.64-7.68 (4H, m), 10.46
(1H, br s). MS (FAB): m/z 328 (MH+).
5-(4-Biph en yl)-3-m eth yl-4-{2-[6-(4-m eth yl-1-piper azin yl)-
h exyloxy]p h en yl}-1,2,4-tr ia zole (19). To a suspension of 9
(1.04 g, 3.18 mmol) and K2CO3 (1.76 g, 12.7 mmol) in CH3CN
(30 mL) was added 1,6-dibromohexane (1.52 mL, 9.54 mmol),
and the mixture was stirred at 70 °C for 3 h. After it was cooled
at room temperature, the mixture was concentrated in vacuo.
The residue was partitioned between CHCl3 (30 mL × 3) and
H2O (50 mL). The organic layer was concentrated in vacuo,
and the residue was purified by column chromatography on
silica gel (CHCl3/MeOH ) 40/1) to give 5-(4-biphenyl)-4-[2-(6-
bromohexyloxy)phenyl]-3-methyl-1,2,4-triazole (0.924 g, 59%)
as a light yellow solid. To a suspension of the bromide obtained
above (0.333 g, 0.679 mmol) and K2CO3 (0.282 g, 2.04 mmol)
in CH3CN (8 mL) was added 1-methylpiperazine (0.452 mL,
4.07 mmol), and the mixture was stirred at 70 °C for 14 h.
After it was cooled at room temperature, the mixture was
concentrated in vacuo. The residue was partitioned between
CHCl3 (15 mL × 3) and H2O (30 mL). The organic layer was
concentrated in vacuo, and the residue was purified by column
chromatography on silica gel (CHCl3/MeOH ) 10/1) to give
crude 19 (0.252 g) as a light yellow foam. The foam was
crystallized from Et2O-hexane to give 19 (0.201 g, 58%) as a
light yellow powder; mp 96-97 °C. 1H NMR (300 MHz,
CDCl3): δ 1.18-1.23 (4H, m), 1.35-1.44 (2H, m), 1.51-1.60
(2H, m), 2.22-2.30 (4H, m), 2.27 (3H, s), 2.29 (3H, s), 2.42 (6H,
br s), 3.80-3.87 (1H, m), 3.91-3.98 (1H, m), 7.02-7.07 (2H,
m), 7.17 (1H, dd, J ) 7.7, 1.7 Hz), 7.31-7.56 (10H, m). MS
(FAB): m/z 510 (MH+). Anal. (C32H39N5O) C, H, N.
5-(4-Bip h en yl)-3-m eth yl-4-{2-[6-(p ip er id in o)h exyloxy]-
p h en yl}-1,2,4-tr ia zole (25). 49%; mp 79-81 °C (Et2O-
hexane). 1H NMR (300 MHz, CDCl3): δ 1.16-1.26 (4H, m),
1.48-1.60 (8H, m), 1.73 (4H, br s), 2.29 (3H, s), 2.39-2.65 (4H,
m), 3.83-3.90 (1H, m), 3.94-4.01 (1H, m), 7.03-7.08 (2H, m),
7.17 (1H, dd, J ) 8.1, 2.1 Hz), 7.31-7.56 (10H, m). MS (FAB):
m/z 495 (MH+). Anal. (C32H38N4O‚2.0H2O) C, H, N.
5-(4-Bip h en yl)-3-m eth yl-4-(2-{6-[(4-p ip er id in o)p ip er i-
d in o]h exyloxy}p h en yl)-1,2,4-tr ia zole (26). 51%; mp 90-
91 °C (Et2O-hexane). 1H NMR (300 MHz, CDCl3): δ 1.16-
1.22 (4H, m), 1.36-1.46 (4H, m), 1.52-1.60 (8H, m), 1.71-
1.85 (4H, m), 2.16-2.21 (3H, m), 2.29 (3H, s), 2.48 (4H, br s),
2.85-2.95 (2H, m), 3.80-3.87 (1H, m), 3.91-3.98 (1H, m),
7.02-7.06 (2H, m), 7.16 (1H, dd, J ) 8.0, 1.7 Hz), 7.31-7.56
(10H, m). MS (FAB): m/z 578 (MH+). Anal. (C37H47N5O)
C, H, N.
5-(4-Bip h en yl)-4-{2-[6-(1-im id a zolyl)h exyloxy]p h en yl}-
3-m eth yl-1,2,4-tr ia zole Dih yd r och lor id e (27). 69%; mp
1
80-82 °C (AcOEt-hexane). H NMR (300 MHz, DMS-d6): δ
1.09-1.20 (4H, m), 1.41-1.50 (2H, m), 1.65-1.74 (2H, m), 2.36
(3H, s), 3.86-3.94 (1H, m), 3.96-4.04 (1H, m), 4.11 (2H, t,
J ) 7.2 Hz), 7.20 (1H, t, J ) 7.7 Hz), 7.33 (1H, d, J ) 8.4 Hz),
7.37-7.77 (15H, m), 9.21 (1H, s). MS (FAB): m/z 478 (MH+).
Anal. (C30H31N5O‚2HCl‚2.25H2O) C, H, N, Cl.
Compounds 16-18, 20-22, and 25-27 were prepared from
9 by a procedure similar to that described for 19. Yields referr
to the final substitution step.
5-(4-Biph en yl)-3-m eth yl-4-{3-[6-(4-m eth yl-1-piper azin yl)-
h exyloxy]p h en yl}-1,2,4-tr ia zole (23). Compound 23 was
prepared from 10 by a procedure similar to that described for
19. Yields refer to the final substitution step; 43%; mp 85-87
°C (AcOEt-hexane). 1H NMR (300 MHz, CDCl3): δ 1.31-1.55
(4H, m), 1.72-1.81 (2H, m), 2.27-2.45 (12H, m), 2.28 (3H, s),
2.38 (3H, s), 3.92 (2H, t. J ) 6.6 Hz), 6.73 (1H, t, J ) 2.0 Hz),
5-(4-Biph en yl)-3-m eth yl-4-{2-[3-(4-m eth yl-1-piper azin yl)-
p r op oxy]p h en yl}-1,2,4-tr ia zole (16). 41%; mp 127-128 °C
(Et2O-hexane). 1H NMR (300 MHz, CDCl3): δ 1.67-1.76 (2H,
m), 2.14-2.34 (10H, m), 2.23 (3H, s), 2.29 (3H, s), 3.86-3.94
(1H, m), 3.97-4.04 (1H, m), 7.04-7.10 (1H, m), 7.19 (1H, dd,

2596 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Kakefuda et al.
6.79-6.81 (1H, m), 7.01-7.05 (1H, m), 7.31-7.57 (10H, m).
MS (FAB): m/z 510 (MH+). Anal. (C32H39N5O‚0.25H2O) C,
H, N.
12.8 Hz), 6.92 (1H, d, J ) 8.1 Hz), 6.97 (1H, d, J ) 7.8 Hz),
7.08 (2H, d, J ) 8.1 Hz), 7.26-7.57 (12H, m). MS (FAB): m/z
558 (MH+). Anal. (C35H35N5O2) C, H, N.
5-(4-Biph en yl)-3-m eth yl-4-{4-[6-(4-m eth yl-1-piper azin yl)-
h exyloxy]p h en yl}-1,2,4-tr ia zole (24). Compound 24 was
prepared from 11 by a procedure similar to that described for
19. Yields refer to the final substitution step; 76%; mp 124-
125 °C (AcOEt-hexane). 1H NMR (300 MHz, CDCl3): δ 1.34-
1.56 (6H, m), 1.78-1.87 (2H, m), 2.21-2.45 (10H, m), 2.29 (3H,
s), 2.35 (3H, s), 4.00 (2H, t. J ) 6.3 Hz), 6.97-7.15 (4H, m),
1-(4-Ch lor om eth ylben zoyl)-4-piper idin opiper idin e Hy-
d r och lor id e (15). Compound 15 was prepared from 4-chlo-
romethylbenzoyl chloride and 4-piperidinopiperidine by a
procedure similar to that described for 13 (96%). This com-
pound was used in the next step without further purification.
5-(4-Biph en yl)-3-m eth yl-4-(6-m eth yl-2-{4-[(4-piper idin o-
piper idin o)car bon yl]ben zyloxy}ph en yl)-1,2,4-tr iazole (32).
Compound 32 was prepared from 12 and 15 by a procedure
similar to that described for 28 (60%); mp 147-150 °C
(AcOEt-hexane). 1H NMR (300 MHz, CDCl3): δ 1.44-1.60
(8H, m), 1.75-1.97 (2H, m), 1.99 (3H, s), 2.26 (3H, s), 2.51 (5H,
br s), 2.66-2.91 (2H, m), 3.70 (1H, br s), 4.71 (1H, br s), 5.02
(1H, d. J ) 12.6 Hz), 5.09 (1H, d. J ) 12.6 Hz), 6.92 (1H, d,
J ) 8.1 Hz), 6.97 (1H, d, J ) 7.2 Hz), 7.08 (2H, d, J ) 8.1 Hz),
7.25-7.57 (12H, m). MS (FAB): m/z 626 (MH+). Anal.
(C40H43N5O2‚0.25H2O) C, H, N.
5-(4-Bip h en yl)-3-m et h yl-4-{2-[2-(4-p ip er id yl)et h oxy]-
p h en yl}-1,2,4-tr ia zole (37). To a suspension of 9 (0.220 g,
0.670 mmol) and K2CO3 (1.00 g, 7.24 mmol) in CH3CN (10 mL)
was added 35 (n ) 2, 0.370 g, 0.700 mmol), and the mixture
was stirred at 80 °C for 4 h. After it was cooled at room
temperature, the mixture was concentrated in vacuo. The
residue was partitioned between CHCl3 (15 mL × 3) and H2O
(30 mL). The organic layer was concentrated in vacuo, and
the residue was purified by column chromatography on silica
gel (CHCl3/MeOH ) 50/1) to give 5-(4-biphenyl)-3-methyl-4-
{2-[2-(1-trityl-4-piperidyl)ethoxy]phenyl}-1,2,4-triazole (0.250
g, 55%) as a colorless foam. To a solution of the piperidine
obtained above (0.250 g, 0.370 mmol) in EtOH (3 mL) was
added 4 N HCl(g)/1,4-dioxane (2 mL), and the mixture was
stirred at 70 °C for 20 min. After it was cooled at room
temperature, the mixture was concentrated in vacuo. The
residue was made alkaline with K2CO3(aq) and extracted with
CHCl3 (15 mL × 3). The combined extract was concentrated
in vacuo, and the residue was purified by column chromatog-
raphy on silica gel (CHCl3/MeOH ) 10/1) to give crude 37 as
a light yellow solid. The solid was recrystallized from AcOEt-
hexane to give 37 (0.092 g, 57%) as a colorless powder; mp
120-122 °C. 1H NMR (300 MHz, CDCl3): δ 0.95-1.55 (7H,
m), 2.29 (3H, s), 2.35-2.55 (2H, m), 2.90-3.00 (2H, m), 3.85-
4.05 (2H, m), 7.07 (1H, t, J ) 7.2 Hz), 7.10-7.60 (12H, m).
MS (FAB): m/z 439 (MH+). Anal. (C28H30N4O‚0. 5H2O) C,
H, N.
5-(4-Bip h en yl)-3-m et h yl-4-{2-[4-(4-p ip er id yl)b u t oxy]-
p h en yl}-1,2,4-tr ia zole Dih yd r och lor id e (38). Compound 38
was prepared from 9 and 36 (n ) 4) by a procedure similar to
that described for 37 (47%). 1H NMR (300 MHz, DMSO-d6): δ
0.95-1.75 (11H, m), 2.30 (3H, s), 2.55-2.85 (2H, m), 3.00-
3.25 (2H, m), 3.80-4.05 (2H, m), 7.10-7.80 (13H, m), 8.81 (1H,
br s), 9.05 (1H, br s). MS (FAB): m/z 467 (MH+). Anal.
(C29H32N4O‚2HCl‚2.5H2O) C, H, N, Cl.
5-(4-Bip h en yl)-3-m eth yl-4-{2-[3-(4-p ip er id yl)p r op oxy]-
p h en yl}-1,2,4-tr ia zole Dih yd r och lor id e (39). To a solution
of 9 (0.220 g, 0.670 mmol), 4-(3-hydroxypropyl)pyridine (0.274
g, 2.00 mmol), and Ph3P (0.520 g, 2.00 mmol) in tetrahydro-
furan (THF, 10 mL) was added dropwise diethyl azodicarbox-
ylate (0.350 g, 2.00 mmol), and the mixture was stirred at room
temperature for 2 h. The mixture was concentrated in vacuo,
and the residue was made alkaline with K2CO3(aq) and
extracted with CHCl3 (15 mL × 3). The combined extract was
concentrated in vacuo, and the residue was purified by column
chromatography on silica gel (CHCl3/MeOH ) 40/1) to give
5-(4-biphenyl)-3-methyl-4-{2-[3-(4-pyridyl)propoxy]phenyl}-
1,2,4-triazole (0.251 g, 84%) as a colorless solid. To a solution
of the pyridine obtained above (0.195 g, 0.430 mmol) in AcOH
(10 mL) was added PtO2 (0.090 g), and the mixture was stirred
under hydrogen atmosphere at room temperature for 7 h. The
catalyst was removed by filtration, and the filtrate was
concentrated in vacuo. The residue was made alkaline with
K2CO3(aq) and extracted with CHCl3 (15 mL × 3). The
combined extract was concentrated in vacuo, and the residue
7.23-7.57 (9H, m). MS (FAB): m/z 510 (MH+). Anal. (C32H39
N5O‚0.25H2O) C, H, N.
-
5-(4-Bip h en yl)-3-m eth yl-4-{6-m eth yl-2-[6-(4-m eth yl-1-
p ip er a zin yl)h exyloxy]p h en yl}-1,2,4-t r ia zole Tr ih yd r o-
ch lor id e (30). Compound 30 was prepared from 12 by a
procedure similar to that described for 19. Yields refer to the
1
final substitution step; 25%. H NMR (300 MHz, DMSO-d6):
δ 1.15-1.20 (6H, m), 1.47-1.67 (2H, m), 1.91-2.00 (2H, m),
1.99 (3H, s), 2.25 (3H, s), 2.83 (3H, s), 3.03 (1H, br s), 3.40-
3.70 (4H, m), 3.98-4.07 (3H, m), 7.06 (1H, d, J ) 7.8 Hz), 7.19
(1H, d, J ) 8.7 Hz), 7.37-7.54 (6H, m), 7.69-7.75 (4H, m).
MS (FAB): m/z 524 (MH+). Anal. (C32H39N5O‚2.75HCl‚4.0H2O)
C, H, N, Cl.
1-(6-Br om oh exa n oyl)-4-m eth ylp ip er a zin e Hyd r och lo-
r id e (13). To a solution of 1-methylpiperazine (0.399 g, 3.98
mmol) in CH3CN (4 mL) was added dropwise a solution of
6-bromohexanoyl chloride (1.02 g, 4.78 mmol) in CH3CN (10
mL) at 0 °C, and the mixture was stirred at room temperature
for 2 h. The mixture was concentrated in vacuo, and the
residual solid was suspended with Et2O and filtered to give
13 (1.22 g, 98%) as a light yellow powder. This compound was
used in the next step without further purification.
5-(4-Bip h en yl)-3-m eth yl-4-(2-{5-[(4-m eth yl-1-p ip er a zin -
yl)ca r bon yl]p en tyloxy}p h en yl)-1,2,4-tr ia zole Dih yd r o-
ch lor id e (28). To a suspension of 9 (0.249 g, 0.770 mmol) and
K2CO3 (0.426 g, 3.08 mmol) in CH3CN (10 mL) was added 13
(0.362 g, 1.16 mmol), and the mixture was stirred at 70 °C for
23 h. After it was cooled at room temperature, the mixture
was concentrated in vacuo. The residue was partitioned
between CHCl3 (15 mL × 3) and H2O (30 mL). The organic
layer was concentrated in vacuo, and the residue was purified
by column chromatography on silica gel (CHCl3/MeOH ) 10/
1) to give the free base of 28 (0.287 g) as a colorless syrup.
This material was converted to its hydrochloride salt by
treating it with 4 N HCl(g)/AcOEt (3.0 mL, 12.0 mmol). The
crude salt was suspended with Et2O and filtered to give 28
(0.282 g, 56%) as a colorless amorphous powder. 1H NMR (300
MHz, DMSO-d6): δ 1.07-1.11 (3H, m), 1.32-1.54 (4H, m),
2.20-2.25 (2H, m), 2.36 (3H, s), 2.73 (3H, s), 2.91-3.02 (3H,
m), 3.30-3.45 (2H, m), 3.86-4.00 (3H, m), 4.38-4.42 (1H, m),
7.19 (1H, t, J ) 7.8 Hz), 7.33 (1H, d, J ) 8.1 Hz), 7.37-7.52
(5H, m), 7.60-7.75 (7H, m), 11.43 (1H, br s). MS (FAB): m/z
524 (MH+). Anal. (C32H37N5O2‚1.75HCl‚4.0H2O) C, H, N, Cl.
1-(4-Ch lor om et h ylb en zoyl)-4-m et h ylp ip er a zin e H y-
d r och lor id e (14). Compound 14 was prepared from 4-chlo-
romethylbenzoyl chloride by a procedure similar to that
described for 13 (95%). This compound was used in the next
step without further purification.
5-(4-Bip h en yl)-3-m eth yl-4-(2-{4-[(4-m eth yl-1-p ip er a zin -
yl)ca r bon yl]ben zyloxy}p h en yl)-1,2,4-tr ia zole (29). Com-
pound 29 was prepared from 9 and 14 by a procedure similar
to that described for 28 (60%); mp 179-180 °C (AcOEt-
hexane). 1H NMR (300 MHz, CDCl3): δ 2.27-2.43 (4H, m),
2.29 (3H, s), 2.32 (3H, s), 3.35 (2H, br s), 3.75 (2H, br s), 4.95
(1H, d. J ) 12.5 Hz), 5.06 (1H, d. J ) 12.5 Hz), 7.02-7.14
(4H, m), 7.24-7.57 (13H, m). MS (FAB): m/z 544 (MH+). Anal.
(C34H33N5O2) C, H, N.
5-(4-Bip h en yl)-3-m eth yl-4-(6-m eth yl-2-{4-[(4-m eth yl-1-
p ip er a zin yl)ca r b on yl]b en zyloxy}p h en yl)-1,2,4-t r ia zole
(31). Compound 31 was prepared from 12 and 14 by a
procedure similar to that described for 28 (60%); mp 161-164
1
°C (AcOEt-hexane). H NMR (300 MHz, CDCl3): δ 1.99 (3H,
s), 2.22-2.32 (4H, m), 2.26 (3H, s), 2.29 (3H, s), 3.36 (2H, br
s), 3.74 (2H, br s), 5.02 (1H, d. J ) 12.8 Hz), 5.09 (1H, d. J )

Antagonists for the Human Vasopressin Receptor
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2597
was purified by column chromatography on silica gel (CHCl3/
MeOH ) 10/1) to give the free base of 39 (0.180 g) as a colorless
syrup. This material was converted to its hydrochloride salt
by treating it with 4 N HCl(g)/AcOEt (1.0 mL, 4.0 mmol). The
crude salt was suspended with AcOEt and filtered to give 39
(0.144 g, 64%) as a colorless amorphous powder. 1H NMR (300
MHz, DMSO-d6): δ 0.95-1.75 (11H, m), 2.30 (3H, s), 2.50-
2.75 (2H, m), 3.00-3.15 (2H, m), 3.80-4.05 (2H, m), 7.10-
7.80 (13H, m), 8.79 (1H, br s), 9.02 (1H, br s). MS (FAB): m/z
453 (MH+). HPLC analysis (CH3CN/0.01 M KH2PO4(aq) (8:2))
tR ) 4.85 min (97.2%).
with a single dose of the 0.5% methylcellulose aqueous solution
of 19 or the vehicle (0.5% methylcellulose aqueous solution),
and any changes in blood pressure were noted.
Ack n ow led gm en t. We thank Dr. J . Ohmori for his
critical reading of the manuscript and the staff of
Analysis and Metabolism Laboratories for performing
spectral measurements.
Refer en ces
P h a r m a cology. Bin d in g Assa y: F or th e Clon ed Hu -
m a n Recep tor s. The cloned human AVP receptor subtypes
were stably expressed in CHO cells and plasma membranes
prepared according to the reported protocols.22 [3H]AVP (0.5-
1.0 nM, 80 Ci mmol-1, DuPont-New England Nuclear) was
added to each membrane preparation, which was then incu-
bated with various concentrations of compounds in 250 µM
assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 0.1%
bovine serum albumin). After an incubation period (60 min,
25 °C), the reaction was terminated by the addition of 3 mL
of ice-cold Tris buffer (50 mM Tris-HCl, pH 7.4, and 10 mM
MgCl2) followed immediately by rapid filtration through 96
well GF/B UniFilter Plates using a MicroMate Cell Harvester
(Packard Instrument Company). The filters were rinsed twice,
and the radioactivity retained on the filters was counted with
a TopCount Microplate Scintillation Counter (Packard Instru-
ment Company). Nonspecific binding was determined using 1
µM unlabeled AVP. The radioligand binding data were ana-
lyzed by GraphPad PRISM (GraphPAD Software, Inc.). The
inhibitory dissociation constant (Ki) was calculated from the
following formula: Ki ) IC50/(1 + [L]/Kd), where [L] is the
concentration of radioligand present in the tube and Kd is the
dissociation constant of radioligand obtained from Scatchard
plot analysis. The Kd values were 0.39 ( 0.13 nM for hV1A and
1.21 ( 0.37 nM for hV2, respectively (n ) 5).
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