Novel vasopressin V2 receptor-selective antagonists, pyrrolo[2,1-a]quinoxaline and pyrrolo[2,1-c][1,4]benzodiazepine derivatives

Article abstract of DOI:10.1016/S0968-0896(99)00049-8

The intent of the work was to study the structure-activity relationships of AVP receptor antagonists bearing a chiral ring as a partial structure since such studies had been reported for only achiral compounds. In the present paper, we deal with compounds consisting of the chiral tricyclic hetero ring (1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline and 1,2,3,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine) and 2-phenylbenzanilide analogues. These compounds exhibited a highly selective affinity for V2 receptor, and their stereochemical configuration had a great influence on V2 receptor binding. VP-343 (N-[4-[[(2S,3aR)-2-hydroxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl]carbonyl]phenyl]-4'-methyl[1,1'-biphenyl]-2-carboxamide), VP-365 (N-[4-[[(11aS)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-yl]carbonyl]phenyl][1,1'-biphenyl]-2-carboxamide) and VP-339 (N-[4-[[(11aS)-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-yl]carbonyl]phenyl][1,1'-biphenyl]-2-carboxamide) were the most potent compounds in vitro and in vivo. The IC₅₀ values of VP-343, VP-365 and VP-339 against V2 receptor were 0.772, 1.18 and 0.216nM, respectively. The ED₃₀₀ values (dose required to increase three times the urine volume of the control rats; oral administration) of VP-343, VP-365 and VP-339 were 0.22, 0.31 and 0.78mg/kg, respectively. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00049-8

Bioorganic & Medicinal Chemistry 7 (1999) 1247±1254
Novel Vasopressin V2 Receptor-selective Antagonists,
Pyrrolo[2,1-a]quinoxaline and Pyrrolo[2,1-c][1,4]benzodiazepine
Derivatives
Yasuhiro Ohtake,* Akira Naito, Hisashi Hasegawa, Katsuhiro Kawano,
Daisuke Morizono, Makoto Taniguchi, Yoko Tanaka, Hidehiko Matsukawa,
Kenji Naito, Touru Oguma, Yohji Ezure and Yoshihiro Tsuriya
Sagami Research Laboratories, Wakamoto Pharmaceutical Co., Ltd., 378, Kanade, Ohimachi, Ashigarakami-gun, Kanagawa,
258-0018, Japan
Received 18 December 1998; accepted 29 January 1999
AbstractÐThe intent of the work was to study the structure±activity relationships of AVP receptor antagonists bearing a chiral ring
as a partial structure since such studies had been reported for only achiral compounds. In the present paper, we deal with com-
pounds consisting of the chiral tricyclic hetero ring (1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline and 1,2,3,10,11,11a-hexahy-
dro-1H-pyrrolo[2,1-c][1,4]benzodiazepine) and 2-phenylbenzanilide analogues. These compounds exhibited a highly selective
anity for V2 receptor, and their stereochemical con®guration had a great in¯uence on V2 receptor binding. VP-343 (N-[4-
[[(2S,3aR)-2-hydroxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl]carbonyl]phenyl]-40-methyl[1,10-biphenyl]-2-carboxamide),
VP-365 (N-[4-[[(11aS)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-yl]carbonyl]phenyl][1,10-biphenyl]-2-carb-
oxamide) and VP-339 (N-[4-[[(11aS)-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-yl]carbonyl]phenyl]-
[1,10-biphenyl]-2-carboxamide) were the most potent compounds in vitro and in vivo. The IC50 values of VP-343, VP-365 and VP-
339 against V2 receptor were 0.772, 1.18 and 0.216 nM, respectively. The ED300 values (dose required to increase three times the
urine volume of the control rats; oral administration) of VP-343, VP-365 and VP-339 were 0.22, 0.31 and 0.78 mg/kg, respectively.
# 1999 Elsevier Science Ltd. All rights reserved.
Introduction
where they controlled free water and urea reabsorption.
The receptor stimulates adenylate cyclase, which results
in the production of cyclic AMP.
Arginine vasopressin (AVP) is a well-known peptide
hormone with a variety of biological actions on distinct
tissues. Recently, three receptor subtypes (V1a, V1b and
V2) have been cloned and found to belong to the seven
transmembrane G protein-coupled receptors super-
family.1±3 The V1a receptors are mainly located in vas-
cular smooth muscle4 and liver cells.5 Manning et al.6
reported that the vasoconstriction activity by AVP was
due to the interaction between AVP and the V1a recep-
tor. Jard et al.7 reported that the V1b receptors were
located in the anterior pituitary where they stimulated
corticotropin release. AVP activates phospholipase C-
mediated hydrolysis of polyphosphoinositides through
the V1a and V1b receptors to generate two second
messengers, inositol-1,4,5-triphosphate, which induces
an increase of free intracellular calcium from the endo-
plasmic reticulum, and 1,2-diacylglycerol, which acti-
vates protein kinase C.8 As for the V2 receptor, Butlen
et al.9 reported that they were located in the kidney
AVP may play a role in several disease conditions,
including heart failure, hypertension, hyponatremia,
and syndrome of inappropriate antidiuretic hormone
secretion. The study on selective AVP antagonists is
essential for the elucidation of the pathophysiological
role of AVP.
Until recently, all potent AVP receptor antagonists
reported have been peptide analogues of AVP10±14 and
have not had good oral bioavailability, with some exhi-
biting partial agonistic activity.15,16 Recently, orally
e€ective nonpeptide V1a-selective (OPC-2126817) and
V2-selective (OPC-3126018) antagonists have been
reported (Fig. 1).
The study on structure±activity relationships (SAR) for
OPC-31260 indicated several important facts. They can
be summarized as follows: (1) the benzene-fused seven-
membered ring system was signi®cant for V2 (and V1a)
antagonists; (2) the ortho substituents on the terminal
Key words: Antagonists; benzodiazepines; hormones; receptors.
*Corresponding author.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00049-8

1248
Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
Figure 1.
benzoyl ring seemed to be important both for a high
anity for the V2 receptor and good oral activity.
(Fig. 2). These compounds were synthesized from the
standpoint of introducing chirality into the six and
seven-membered ring system such as OPC-21268, OPC-
31260 and YM-087. In the present paper, we deal with
the synthesis and biological properties of these novel
AVP antagonists.
Tahara et al.19 reported the biochemical and pharma-
cological properties of YM-087, which was a nonpep-
tide antagonist of both V1a and V2 receptors. The study
on the systematically modi®ed compounds relevant to
YM-087 clari®ed the structure features required for the
binding anity for vasopressin receptors. These studies
revealed that the introduction of a lipophilic group such
as 2-phenyl or 2-(4-substituted phenyl) on the terminal
benzoyl ring exhibited more potent antagonistic activ-
ities than that of 2-methyl group in the in vivo tests
towards both V1a and V2 receptors.20
Results and Discussion
Chemistry
The synthesis of Q type compounds (1a±1d) is shown in
Schemes 1 and 2. As the starting material, trans-4-hy-
droxy-l-proline (or cis-4-hydroxy-d-proline) methyl
ester hydrochloride was used for the synthesis of 4a and
4c. The stereochemical inversion of the hydroxy group
of 4a,c that used the improved Mitsunobu condition,24
gave 4b and 4d, then the resulting esters 5a and 5c were
hydrolyzed with aqueous NaOH to give 4b,d. Recrys-
tallization in AcOEt/CHCl3 gave optically pure 4b,d.
Because the amides 6a±d were very labile, the two con-
secutive reductions of the nitro group (4a±d) and the
resulting amide group (6a±d) were successively done
without isolating 6a±d. The amines 7a±d were puri®ed
as the hydrochloride. The compounds 1a±d were pre-
pared by acylating 7a±d with 4-(40-methyl-2-biphenyl-
carboxamido)benzoyl chloride. Overall yields of 1a±d
were 21±37% in general.
The study on the SAR of the compound bearing chiral
ring as a partial structure has not been reported on the
AVP receptor antagonists. As for the several peptide
hormones such as cholecystokinin,21 substance P22 and
Progesterone,23 the importance of the chirality of
receptor antagonists has been elucidated.
We intended to perform the SAR studies on AVP
receptor antagonists with chirality. Therefore, our study
was focused on the SAR of the compounds consisting of
the chiral tricyclic hetero ring and the benzanilide moi-
ety bearing 2-phenyl or 2-(4-substituted phenyl) group.
The two di€erent types of compounds were studied with
respect to the six and seven-membered ring system.
One was 1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxa-
line derivatives (Q type, compound 1). The other was
1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzo-
diazepine derivatives (B type, compounds 2 and 3)
The synthesis of B type compounds (2, 3) is shown in
Scheme 3. Reduction of the carbonyl groups in the
dilactams 8a25 and 8b25 with lithium aluminium hydride
gave the diamines 9a26 and 9b in 87% and 90% yields,
Figure 2.

Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
1249
Scheme 1. i) 1-¯uoro-2-nitrobenzene, Et3N, CH3CN, 60 ꢀC, 16 h; ii) Ph3P, DIPAD, AcOH, toluene, rt, 2 h; iii) 1. aq. NaOH, rt, 20 h 2.HCl
Scheme 2. i) 10% Pd-C, Et3N, THF, 45 ꢀC, 4 h; ii) NaBH4, THF, ref., 18 h; iii) HCl; iv) 4-(40-methyl-2-biphenylcarboxamido)benzoyl chloride,
pyridine, CH2Cl2, rt, 18 h.
respectively. Selective reduction of the carbonyl group
at C-11 position in 8a and 8b in re¯uxing glyme con-
taining 2 equiv NaBH4 and 1.3 equiv tri¯uoroacetic acid
(TFA)27 for 4 h gave quantitatively the monoamines
10a28 and 10b. The yield, however, was generally poor
when the acid instead of TFA (e.g. AcOH or MsOH)
was used. To achieve the quantitative yield, the above
experimental condition could be recommended. The
compounds 2a and 2b were prepared by acylating 9a, 9b
with 4-[(2-phenylbenzoyl)amino]benzoyl chloride29 in
81% and 79% yields, respectively. The compounds 3a
and 3b were prepared by acylating 10a and 10b with the
above acid chloride in 73% and 70% yields, respectively.
Binding study
The binding studies were done by a slightly modi®ed
method of Thibonnier et al.30 with rat liver (V1a recep-
tor) and kidney (V2 receptor) plasma membranes. The
anity of compounds was determined by competition
studies with [3H]d(CH2)5Tyr(Me)AVP ([3H]-V1 anta-
gonists) and [3H]desGly(NH2)d(CH2)5[d-Ile2,Ile4]AVP
([3H]-V2 antagonists) as a radioligand.
As shown in Tables 1 and 2, all the test compounds
showed the strong anity and the high selectivity for
V2 receptor binding. The results of the binding study on

1250
Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
Scheme 3. i) LiAlH4, THF, ref., 3 h; ii) NaBH4, TFA, glyme, ref., 4 h; iii) 4-[(2-phenylbenzoyl)amino]benzoyl chloride, pyridine, CH2Cl2, rt, 18 h
Table 1. Vasopressin receptor binding anities of Q type
stereochemical con®guration at C-11a position of
1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzo-
diazepine ring was not important, because the binding
anity for V2 receptor of 2a and 2b was similar. On the
contrary, as for compound 3, in which a lactam moiety
is contained, the con®guration at C-11a position
strongly a€ected the binding anity for V2 receptor (3a
versus 3b).
Compound
C-3a
position
H
C-2
position
X
IC50 (nM) UV (mL/kg)
V1a V2
i.v.
p.o.
1a
1b
(S)
(S)
(R)
(R)
H
H
H
H
(R)
(S)
(R)
(S)
OH 8130 27.6 15.3
OH 2600 7.30 28.3
OH 780 9.77 60.6
OH 110 0.772 79.2
N.T.
N.T.
90.3
96.3
1c
1d (VP-343)
N.T., not tested.
Diuretic activity
The diuretic activity was determined by measuring the
urine volume (UV in Tables 1 and 2) collected for 4 h
after administration. The dose of the test compounds
was 3 mg/kg for intravenous administration (i.v.) and
30 mg/kg for oral administration (p.o.), and 4 rats were
evaluated. The mean values of UV for control rats after
i.v. (n=21) and p.o. (n=8) were 12.5 and 6.9 mL/kg,
respectively. All the compounds, except for the com-
pound 3b, exhibited the antagonistic activities for V2
receptor. The compounds 1c, 1d, 2b and 3a remarkably
increased the urine volume in comparison with that of
the control rats. The ED300 values (oral dose required to
increase three times the urine volume of the control rats)
of 1d (VP-343), 2b (VP-365) and 3a (VP-339) were 0.22,
0.31 and 0.78 mg/kg, respectively.
Table 2. Vasopressin receptor binding anities of B type
Compound
C-11a
position
H
IC50 (nM)
UV (mL/kg)
V1a
V2
i.v.
p.o.
2a
(S)
(R)
(S)
(R)
H
H
H
H
21.5%a
127
3.07
1.18
29.6
77.5
58.6
9.9
73.8
121.6
102.7
N.T.
2b (VP-365)
3a (VP-339)
3b
15.8%a 0.216
0%a
116
N.T., not tested.
aThe values are the inhibition percent at the concentration of 10 M.
6
Q type (1 in Fig. 2) are shown in Table 1. It is of interest
that the stereochemical con®guration at C-3a position
of 1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline ring
had an in¯uential e€ect on the binding anity for V2
(and V1a) receptor (1a versus 1c, 1b versus 1d). It is also
noted that the binding anity for the V1a and V2
greatly changed depending on the stereochemical con-
®guration of C-2 position, i.e. (S)-isomers were more
potent than (R)-isomers (1a versus 1b, 1c versus 1d).
Conclusion
The data presented here revealed, for the ®rst time, that
the stereochemical con®guration of the chiral com-
pounds had a great in¯uence on V2 receptor binding.
These results have important implications for the
development of V2 receptor antagonists and could be
useful to develop more potent V2 receptor antagonists.
The results of the binding study on B type (2, 3 in Fig.
2) are shown in Table 2. In the case of compound 2, the

Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
1251
Since the compounds of pyrrolo[1,2-a]quinoxaline and
pyrrolo[2,1-c][1,4]benzodiazepine with di€erent chirality
bind to V2 receptor in greatly di€erent anity, it seems
that such chromophores could be the pharmacophores
for V2 receptor. Through the present studies, VP-339,
VP-343 and VP-365 could be candidates for diuretics
and the more detailed biological activities of these
compounds will be reported elsewhere.
(2S,4S)-4-Hydroxy-1-(2-nitrophenyl)proline (4b). Diiso-
propyl azodicarboxylate (DIPAD, 3.27 mL, 16.62 mM)
was added dropwise to a solution of 4a (2.95 g,
11.08 mM), triphenylphosphine (4.36 g, 16.62 mM) and
acetic acid (1.27 mL, 22.16 mM) in dry toluene (45 mL)
in an ice-bath. After being stirred at room temperature
for 2 h, 2 N NaOH (33.2 mL) was added to the reaction
mixture, and then the reaction mixture was stirred at
room temperature for 20 h. The water layer was sepa-
rated, acidi®ed with 4N HCl (25 mL) and then extracted
with AcOEt. The organic layer was washed with brine,
dried over Na2SO4, ®ltered and then concentrated under
reduced pressure. Recrystallization from AcOEt/CHCl3
gave 4b (2.01 g, 63%) as light yellow crystals: mp 245±
250 ꢀC (dec.); 1H NMR (DMSO-d6) d 2.06 (1H, dt,
J=11.7, 4.9 Hz), 2.42±2.55 (1H, m), 2.99 (1H, dd,
J=10.7, 2.9 Hz), 3.36 (1H, dd, J=10.7, 4.9 Hz), 4.19
(1H, dd, J=8.8, 4.9 Hz), 4.37 (1H, bs), 6.78±6.88 (2H,
m), 7.22 (1H, d, J=7.8 Hz), 7.47±7.55 (1H, m), 7.77±
7.82 (1H, m); IR (KBr) 3440, 1700, 1605, 1565, 1505,
1485, 1440, 1275.
Experimental
General procedures
Melting points were determined on a Yanoco MP-S3
apparatus and are uncorrected. IR spectra (cm 1) were
1
recorded on a JASCO IR-810 spectrometer. H NMR
spectra were measured with a JEOL JMN-GSX 270
spectrometer. Chemical shifts are recorded in the d scale
relative to tetramethylsilane as an internal standard.
Mass spectra (MS) were measured on a JEOL JMS-AX
505W instrument. Optical rotations were determined
with a JASCO DIP-140 polarimeter. In general, reac-
tions were carried out in dry solvents under N2, unless
otherwise mentioned. All commercially available che-
micals were used as supplied by the manufacturer. Col-
umn chromatography was performed on silica gel 60
(70±230 mesh) from Merck. The radioligands [3H]d
(CH2)5Tyr(Me)AVP ([3H]-V1 antagonists; speci®c
activity, 1813 GBq/mM) and [3H]desGly(NH2)d(CH2)5
[d-Ile2,Ile4]AVP ([3H]-V2 antagonists; speci®c activity,
2116.4 GBq/mM) were obtained from DuPont.
(2R,4S)-4-Hydroxy-1-(2-nitrophenyl)proline (4d). In the
similar manner as described above, 4d (63%) was pre-
pared from 4c. Recrystallization from AcOEt/CHCl3
gave light yellow crystals: mp 225±230 ꢀC (dec.); 1H
NMR (DMSO-d6) d 2.07±2.20 (1H, m), 2.39 (1H, dd,
J=12.7, 6.8 Hz), 2.57±2.64 (1H, m), 3.67 (1H, dd, J=
10.8, 3.4 Hz), 4.45 (1H, s), 4.59 (1H, dd, J=10.3, 6.8 Hz),
5.13 (1H, bs), 6.86±6.94 (1H, m), 7.00 (1H, d, J=8.1 Hz),
7.51±7.59 (1H, m), 7.83 (1H, dd, J=8.1, 1.5 Hz); IR (KBr)
3380, 1700, 1605, 1575, 1515, 1445, 1350, 1280, 1080.
(2S,4R)-4-Hydroxy-1-(2-nitrophenyl)proline methyl ester
(4a). A solution of trans-4-hydroxy-l-proline methyl
ester hydrochloride (7.26 g, 40 mM), 1-¯uoro-2-nitro-
benzene (6.0 g, 42.5 mM) and triethylamine (7.7 mL,
54.7 mM) in acetonitrile (100 mL) was stirred for 16 h at
60 ꢀC. The reaction mixture was cooled to room tem-
perature, poured into 1 N HCl (100 mL) cooled in an
ice-bath and then extracted with Et2O. The organic
layer was washed with brine, dried over Na2SO4, ®ltered
and then concentrated under reduced pressure. The
residue was chromatographed on silica gel column
(mobile phase; CHCl3, and then CHCl3:MeOH=99:1)
4b: 100% e.e., 4d: 100% e.e. [The e.e. values were deter-
mined by HPLC with Mightysil1 RP-18 (0.1% AcOH,
30% acetonitrile/water, 1 mL/min). The retention times
were 10 and 7 min for 4b and 4d, respectively.]
(2R,3aS)-2-Hydroxy-1,2,3,3a,4,5-hexahydro-pyrrolo[1,2-
a]quinoxaline hydrochloride (7a). A solution of 4a
(30.7 g, 115 mM) and Et3N (16.1 mL, 115 mM) in THF
(310 mL) was stirred at 45 ꢀC in the presence of 10%
palladium carbon (3.1 g) under H2 at 1 atm pressure for
4 h. The catalyst was removed by ®ltration and washed
with THF (200 mL). To the combined THF solution
was added NaBH4 (17.4 g, 461 mM), and the reaction
mixture was stirred while re¯uxing for 18 h under nitro-
gen atmosphere. The reaction mixture was cooled in an
ice bath and diluted with water (500 mL). The resulting
mixture was carefully neutralized with 4N HCl (100 mL)
in an ice bath, extracted with AcOEt (2Â400 mL). The
organic layer was washed with brine, dried over Na2SO4
and concentrated to the half volume (ab. 600 mL). To
the ice-cooled extract (ab. 600 mL) was added dropwise
4N HCl/AcOEt (57.7 mL, 0.231 M) and the mixture was
stirred for 1 h in an ice bath. The precipitate was col-
lected by ®ltration and recrystallized from MeOH to
giveꢀ 7a (10.68 g, 41%) as pale red needles: mp 215±
1
to give 4a as a red oil (9.9 g, 93%): H NMR (CDCl3) d
1.74 (1H, d, J=4.0 Hz), 2.20±2.35 (1H, m), 2.43±2.55
(1H, m) 2.82±2.92 (1H, m), 3.70 (3H, s), 3.86 (1H, dd,
J=11.0, 3.7 Hz), 4.55±4.77 (2H, m), 6.81±6.99 (2H, m),
7.34±7.47 (1H, m), 7.77 (1H, dd, J=8.4, 1.8 Hz); IR
(neat) 3480, 1735, 1605, 1510, 1360, 1275, 1205,
1
1175 cm
.
(2R,4R)-4-Hydroxy-1-(2-nitrophenyl)proline methyl ester
(4c). cis-4-Hydroxy-l-proline methyl ester hydrochlo-
ride and 1-¯uoro-2-nitrobenzene were subjected to the
same reaction described for the synthesis of 4a. The
compound 4c (97%) was obtained as a red oil: 1H NMR
(CDCl3) d 2.16±2.26 (1H, m), 2.50±2.62 (1H, m), 2.91 (1H,
d, J=9.0 Hz) 3.41±3.48 (1H, m), 3.57 (1H, dd, J=10.5,
5.4 Hz), 3.75 (3H, s), 4.40±4.55 (2H, m), 6.81±6.89 (2H,
1
221 C (dec.); H NMR (DMSO-d6) d 1.70±1.80 (1H,
m), 2.14 (1H, dd, J=12.5, 5.3 Hz), 2.84 (1H, t, J=
11.5 Hz), 3.34±3.47 (2H, m), 3.81 (1H, dd, J=11.5,
3.2 Hz), 4.01±4.12 (1H, m), 4.54 (1H, t, J=3.9 Hz),
6.67±6.74 (2H, m), 7.23±7.30 (2H, m), 11.51 (2H, br); IR
m), 7.35±7.43 (1H, m), 7.64±7.79 (1H, m); IR (neat)
1
3450, 1740, 1605, 1510, 1350, 1280, 1210, 1180 cm
.

1252
Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
(KBr) 3450, 2920, 2890, 2850, 2740, 2675, 2640, 1620,
1
(1H, d, J=4.8 Hz), 6.33±6.39 (1H, m), 6.56 (1H, dd,
J=8.1, 1.5 Hz), 6.73 (1H, dd, J=8.1, 1.5 Hz), 6.93 (1H,
ddd, J=8.1, 7.3, 1.5 Hz), 7.16 (2H, dd, J=8.4, 0.7 Hz),
7.27±7.35 (4H, m), 7.41±7.57 (6H, m), 10.02 (1H, s); IR
(KBr) 3400, 1630, 1605, 1515, 1505, 1410, 1375, 1320,
1250 cm 1; HRMS m/e 503.2238 (C12H16N2 requires
503.2209).
1515, 1360, 1350, 1200, 755 cm
.
(2S,3aS)-2-Hydroxy-1,2,3,3a,4,5-hexahydro-pyrrolo[1,2-
a]quinoxaline hydrochloride (7b). In a similar manner as
described above, 7b (48%) was prepared from 4b.
Recrystallization from MeOH gave pale red needles: mp
206±212 ꢀC (dec.); 1H NMR (DMSO-d6) d 1.61 (1H, dt,
J=12.2, 7.6 Hz), 2.32 (1H, quint, J=6.1 Hz), 2.88 (1H,
t, J=11.2 Hz), 3.04 (1H, dd, J=9.8, 5.4 Hz), 3.54±3.78
(3H, m), 4.44 (1H, quint, J=6.1 Hz), 6.62±6.67 (2H, m),
N-[4-[[(2R,3aR)-2-Hydroxy-2,3,3a,4-tetrahydro-pyrrolo-
[1,2-a]quinoxalin-5(1H)-yl]carbonyl]-phenyl]-40 -methyl-
[1,10-biphenyl]-2-carboxamide (1c). In a similar manner
as described above, 1c (60%) was prepared from 7c as a
yellowish powder. [a]d27=+341.55 (MeOH, c=1.042).
1H NMR and IR were identical to those of 1b; HRMS
m/e 503.2189 (C12H16N2 requires 503.2209).
7.14±7.19 (2H, m), 11.28 (2H, br); IR (KBr) 3450, 2850,
1
2740, 2670, 2640, 1615, 1510, 1365, 1355, 750 cm
.
(2R,3aR)-2-Hydroxy-1,2,3,3a,4,5-hexahydro-pyrrolo[1,2-
a]quinoxaline hydrochloride (7c). In a similar manner as
described above, 7c (49%) was prepared from 4c.
Recrystallization from MeOH gave pale red needles. 1H
NMR, mp and IR were identical to those of 7b.
N-[4-[[(2S,3aR)-2-Hydroxy-2,3,3a,4-tetrahydro-pyrrolo-
[1,2-a]quinoxalin-5(1H)-yl]carbonyl]-phenyl]-40 -methyl-
[1,10-biphenyl]-2-carboxamide (1d). In a similar manner
as described above, 1d (79%) was prepared from 7d as a
yellowish amorphous powder. [a]2d7=+355.36 (MeOH,
(2S,3aR)-2-Hydroxy-1,2,3,3a,4,5-hexahydro-pyrrolo[1,2-
a]quinoxaline hydrochloride (7d). In a similar manner as
described above, 7d (76%) was prepared from 4d.
Recrystallization from MeOH gave pale red needles. 1H
NMR, mp and IR were identical to those of 7a.
1
c=1.082). H NMR and IR were identical to those of
1a; HRMS m/e 503.2207 (C12H16N2 requires 503.2209).
1a: 99% e.e., 1b: 99% e.e., 1c: 99% e.e. and 1d: 99 %
e.e. [The e.e. values were determined by HPLC with
Chiralcel OD-RH1 (60% acetonitrile/water, 0.5 mL/
min). The retention times were 35, 18, 12 and 11 min for
1a, 1b, 1c and 1d, respectively.
N-[4-[[(2R,3aS)-2-Hydroxy-2,3,3a,4-tetrahydro-pyrrolo-
[1,2-a]quinoxalin-5(1H)-yl]carbonyl]-phenyl]-40 -methyl-
[1,10-biphenyl]-2-carboxamide (1a). To an ice-cold solu-
tion of 7a (1.50 g, 6.62 mM) and pyridine (1.1 mL,
13.23 mM) in CH2Cl2 (30 mL) was added dropwise
while stirring a solution of 4-(40-methyl-2-biphenyl-
carboxamido)benzoyl chloride (2.31 g, 6.62 mM) in
CH2Cl2 (46 mL) over a period of 0.5 h. The reaction
mixture was stirred at room temperature for 18 h and
diluted with water (100 mL). The resulting solution was
extracted with AcOEt (100 mL) and the organic layer
was washed with 0.8 N HCl (100 mL), saturated aqu-
eous NaHCO3 (100 mL) and brine, dried over Na2SO4
and then concentrated under reduced pressure. The
residue was chromatographed on silica gel (mobile
phase; CHCl3:MeOH=49:1) to give 1a (2.18 g, 65%) as
a yellowish powder: [a]2d7= 333.28 (MeOH, c=1.013);
1H NMR (DMSO-d6, 90 ꢀC) d 1.69 (1H, ddd, J=12.5,
10.5, 4.4 Hz), 2.14 (1H, dd, J=12.5, 5.4 Hz), 2.39 (3H,
s), 2.66 (1H, dd, J=12.2, 10.5 Hz), 3.46 (2H, d,
J=2.4 Hz), 3.88±3.99 (1H, m), 4.55 (1H, bs), 4.76 (1H,
dd, J=12.5, 3.9 Hz), 4.88 (1H, d, J=3.7 Hz), 6.41±6.47
(1H, m), 6.63 (1H, dd, J=8.1, 1.5 Hz), 6.81 (1H, dd,
J=8.1, 1.5 Hz), 7.02 (1H, ddd, J=8.1, 7.3, 1.5 Hz), 7.25
(2H, d, J=7.8 Hz), 7.37±7.44 (4H, m), 7.50±7.66 (6H,
m), 10.12 (1H, s); IR (KBr) 3400, 2920, 2850, 1620,
1600, 1515, 1505, 1410, 1320 cm 1; HRMS m/e 503.2177
(C12H16N2 requires 503.2209).
(11aS)-1,2,3,10,11,11a-Hexahydro-5H-pyrrolo-[2,1-c][1,4]-
benzodiazepine (9a). A solution of 8a (3 g, 13.9 mM) in
dry THF (100 mL) was added dropwise to the suspen-
sion of LiAlH4 (2.1 g, 55.2 mM) in dry THF (50 mL) at
0 ꢀC. The mixture was re¯uxed for 3 h. It was cooled to
room temperature, and the excess of LiAlH4 was
decomposed by adding AcOEt and then water. The
mixture was diluted with ether. The organic layer was
separated, and the aqueous layer was extracted with ether.
The combined organic layer was washed with brine,
dried over MgSO4 and then concentrated under reduced
pressure. Recrystallization from AcOEt gave 9a (2.3 g,
ꢀ
1
87%) as colorless crystals: mp 108±109 C; H NMR
(CDCl3) d 1.39±1.54 (1H, m), 1.69±1.98 (3H, m), 2.40±
2.52 (2H, m), 2.74 (1H, dd, J=13.1, 12.7 Hz), 3.12±3.19
(1H, m), 3.29±3.46 (1H, m), 3.50 (1H, d, J=13.4 Hz),
3.81 (1H, d, J=13.4 Hz), 6.71 (1H, d, J=7.6 Hz), 6.81
(1H, dd, J=7.6, 7.2 Hz), 7.04±7.13 (2H, m); IR (KBr)
2960, 1600, 1580, 1480, 1370, 1290, 1090 cm 1; HRMS
m/e 188.1291 (C12H16N2 requires 188.1313).
(11aR)-1,2,3,10,11,11a-Hexahydro-5H-pyrrolo-[2,1-c][1,4]-
benzodiazepine (9b). In a similar manner as described
above, 9b (90%) was prepared from 8b. Recrystalliza-
1
tion from AcOEt gave a yellowish powder. H NMR,
mp and IR were identical to those of 9a. 9a: 98% e.e.
and 9b: 99% e.e. (The e.e. values were determined by
HPLC with Chiralcel OD-RH1, 25% acetonitrile/0.1M
aqueous KPF6, 0.5 mL/min. The retention times were
14 and 17 min for 9a and 9b, respectively.).
N-[4-[[(2S,3aS)-2-Hydroxy-2,3,3a,4-tetrahydro-pyrrolo-
[1,2-a]quinoxalin-5(1H)-yl]carbonyl]-phenyl]-40 -methyl-
[1,10-biphenyl]-2-carboxamide (1b). In a similar manner
as described above, 1b (77%) was prepared from 7b as a
yellowish powder: [a]2d7= 334.12 (MeOH, c=1.014);
1H NMR (DMSO-d6, 90 ꢀC) d 1.49±1.60 (1H, m), 2.26±
2.35 (4H, m), 2.74 (1H, dd, J=12.5, 10.3 Hz), 3.01±3.08
(1H, m), 3.56±3.69 (2H, m), 4.44±4.60 (2H, m), 4.89
(11aS)-1,2,3,10,11,11a-Hexahydro-5H-pyrrolo-[2,1-c][1,4]-
benzodiazepin-5-one (10a). To a solution of 8a (5.05 g,
23.4 mM) in glyme (50 mL) was added NaBH4 (1.76 g,

Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
1253
46.6 mM) at room temperature. Tri¯uoroacetic acid
(3.43 g, 30.1 mM) in glyme (50 mL) was added dropwise
to the reaction mixture over a period of 0.5 h at the
room temperature. The mixture was re¯uxed for 4 h.
The reaction mixture was cooled, carefully quenched
with brine, dried over MgSO4, ®ltered and then con-
centrated under reduced pressure. Recrystallization
from AcOEt gave 10a (4.52 g, 96%) as colorless crystals:
mp 181±182 ꢀC; 1H NMR (CDCl3) d 1.64±1.97 (3H, m),
2.17±2.32 (1H, m), 3.33 (1H, dd, J=12.5, 8.5 Hz), 3.50±
3.73 (2H, m), 3.77±3.94 (2H, m), 6.55 (1H, dd, J=8.1,
1.1 Hz), 6.74±6.81 (1H, m), 7.15±7.22 (1H, m), 8.01 (1H,
dd, J=8.1, 1.5 Hz); IR (KBr) 3330, 2950, 1620, 1595,
1495, 1450, 1365, 1260 cm 1; HRMS m/e 202.1057
(C12H14N2O requires 202.1106).
solution of 4-[(2-phenylbenzoyl)amino]benzoyl chloride
(1.53 g, 4.4 mM) in CH2Cl2 (15 mL) over a period of
0.5 h. The reaction mixture was stirred at room tem-
perature for 18 h and diluted with water (100 mL). The
resulting solution was extracted with CH2Cl2 and the
organic layer was washed with 0.8 N HCl (50 mL),
saturated aqueous NaHCO3 (50 mL) and brine, dried
over Na2SO4 and then concentrated under reduced
pressure. The residue was chromatographed on silica gel
(mobile phase; CHCl3:MeOH=49:1), followed by crys-
tallization from AcOEt:hexane (1:3) to give 3a (1.64 g,
73%) as a white powder: mp 233±235 ꢀC; [a]d27=
1
+531.50 (MeOH, c=1.042); H NMR (CDCl3) d 1.87±
1.96 (1H, m), 2.03±2.19 (3H, m), 3.58±3.69 (2H, m),
3.79±3.93 (2H, m), 4.29 (1H, t, J=12.8 Hz), 6.64 (1H, d,
J=6.7 Hz), 6.79±6.91 (3H, m), 7.07 (2H, d, J=8.4 Hz),
7.13 (1H, td, J=7.7, 1.8 Hz), 7.23±7.31 (1H, m), 7.32±
7.57 (8H, m), 7.76 (1H, dd, J=7.3, 1.5 Hz), 7.86 (1H,
d, J=7.3 Hz); IR (KBr) 1630, 1520, 1410, 1360,
1320 cm 1; FABHRMS m/e 502.2101 [M+H]+ (C32H27
N3O3 requires 502.2131).
(11aR)-1,2,3,10,11,11a-Hexahydro-5H-pyrrolo-[2,1-c][1,4]-
benzodiazepin-5-one (10b). In a similar manner as
described above, 10b (95%) was prepared from 8b.
Recrystallization from AcOEt gave a yellowish powder.
1H NMR, mp and IR were identical to those of 10a.
N-[4-[[(11aS)-2,3,11,11a-Tetrahydro-1H-pyrrolo-[2,1-c]-
[1,4]benzodiazepin-10(5H)-yl]carbonyl]-phenyl][1,10 -bi-
phenyl]-2-carboxamide (2a). To an ice-cold solution of
9a (1.50 g, 6.62 mM) and pyridine (1.1 mL, 13.23 mM) in
CH2Cl2 (30 mL) was added dropwise while stirring a
solution of 4-[(2-phenylbenzoyl)amino]benzoyl chloride
(2.31 g, 6.62 mM) in CH2Cl2 (45 mL) over a period of
0.5 h. The reaction mixture was stirred at room tem-
perature for 18 h and diluted with water (100 mL). The
resulting mixture was extracted with CH2Cl2 and the
organic layer was washed with 0.8 N HCl (100 mL),
saturated aqueous NaHCO3 (100 mL) and brine, dried
over Na2SO4 and then concentrated under reduced
pressure. The residue was chromatographed on silica gel
(mobile phase; CHCl3:MeOH=49:1), followed by crys-
tallization from AcOEt:hexane (1:3) to give 2a (2.70 g,
81%) as a white powder: mp 178±179.5 ꢀC; [a]d27=
N-[4-[[(11aR)-5-Oxo-2,3,11,11a-tetrahydro-1H-pyrrolo-
[2,1-c][1,4]benzodiazepin-10(5H)-yl]-carbonyl]phenyl][1,10-
biphenyl]-2-carboxamide (3b). In a similar manner
as described above, 3b (70%) was prepared from 10b.
Recrystallization from AcOEt:hexane gave a white
powder. 1H NMR, mp and IR were identical to those of
3a: mp 233±235 ꢀC; [a]d27= 518.94 (MeOH, c=1.018);
FABHRMS m/e 502.2104 [M+H]+ (C32H27N3O3
requires 502.2131).
3a: 100% e.e. and 3b: 100% e.e. (The e.e. values were
determined by HPLC with Chiralcel OD-RH1, 40%
acetonitrile/water, 0.5 mL/min. The retention times were
36 and 29 min for 3a and 3b, respectively.).
Biological methods
1
297.18 (MeOH, c=1.003); H NMR (CDCl3) d 1.42±
1. Receptor binding studies. For the V1a receptor bind-
5
12
1.58 (1H, m), 1.71±1.93 (2H, m), 1.95±2.10 (1H, m),
2.48±2.71 (3H, m), 3.10±3.21 (1H, m), 3.72±3.93 (2H,
m), 5.05±5.20 (1H, m), 6.61 (1H, d, J=8.1 Hz), 6.80±
6.99 (4H, m), 7.02±7.14 (3H, m), 7.26±7.29 (1H, m),
7.33±7.57 (8H, m), 7.85 (1H, d, J=6.6 Hz); IR (KBr)
1640, 1600, 1520, 1400, 1320, 1270 cm 1; HRMS m/e
487.2284 (C32H29N3O2 requires 487.2260).
ing assay, the test compounds (3Â10
10 M) and
2.12 nM [3H]-V1 antagonist were incubated with 5 mg
(protein equivalent) of liver membranes in 1 mL of
50 mM Tris±HCl (pH 7.4) containing 10 mM KCl,
10 mM MgCl2 and 0.1% bovine serum albumin (BSA).
For the V2 receptor binding assay, the test compounds
10 5 M) and 1.8 nM [3H]-V2 antagonist were
12
(3Â10
incubated with 5 mg (protein equivalent) of kidney
membranes in 1 mL of 50 mM Tris±HCl (pH 7.4) con-
taining 2 mM KCl, 1 mM MgCl2 and 0.1% BSA. After
incubation for 60 min at 25 ꢀC, the free ligands were
separated by ®ltration with a cell harvester, and then the
®lters were washed three times with 50 mM Tris±HCl
(pH 7.4). The radioactivity trapped on the ®lters was
counted with a liquid scintillation counter. Speci®c
N-[4-[[(11aR)-2,3,11,11a-Tetrahydro-1H-pyrrolo-[2,1-c]-
[1,4]benzodiazepin-10(5H)-yl]carbonyl]-phenyl][1,10 -bi-
phenyl]-2-carboxamide (2b). In a similar manner as
described above, 2b (79.3%) was prepared from 9b.
Recrystallization from AcOEt:hexane gave a white
powder. 1H NMR, mp and IR were identical to those of
2a: mp 178±179.5 ꢀC; [a]2d7=+298.40 (MeOH, c=1.009);
FABHRMS m/e 488.2301 [M+H]+ (C32H29N3O2
requires 488.2338).
binding was calculated as a total binding minus non-
5
speci®c binding, which was determined using 10
M
unlabeled AVP. The concentration of test compounds
that caused 50% inhibition (IC50; nM) of the speci®c
binding of [3H]-V1 or [3H]-V2 antagonist was deter-
mined by regression analysis of displacement curves.
The values are the means obtained from 1±5 independent
experiments performed in duplicate.
N-[4-[[(11aS)-5-Oxo-2,3,11,11a-tetrahydro-1H-pyrrolo-
[2,1-c][1,4]benzodiazepin-10(5H)-yl]-carbonyl]phenyl][1,10-
biphenyl]-2-carboxamide (3a). To an ice-cold solution of
10a (0.81 g, 4.0 mM) and pyridine (0.66 mL, 8.0 mM) in
CH2Cl2 (20 mL) was added dropwise while stirring, a

1254
Y. Ohtake et al. / Bioorg. Med. Chem. 7 (1999) 1247±1254
2. Diuretic activity. Rats (weighting 240±320 g) were
fasted for 16±20 h. Immediately after the test com-
pounds or vehicle were administered intravenously
(3 mg/kg) or orally (30 mg/kg) to the rats, saline (25 mL/
kg) was administered orally. As a vehicle, N,N-di-
methylformamide and 5% gum arabic were used for
intravenous and oral administration, respectively.
Spontaneously voided urine was collected for 4 h. The
mean values of the urine volume for control rats after
intravenous (n=21) and oral (n=8) administration
were 12.5 and 6.9 mL/kg, respectively. The oral dose
(ED300; mg/kg) required to increase three times the
urine volume of the control rats, to which the vehicle
was administered, was determined with respect to sev-
eral compounds.
13. Manning, M.; Sawyer, W. H. J. Lab. Clin. Med., 1989,
114, 617±632.
14. Manning, M.; Chan. W. Y. Regul. Pept., 1993, 45, 279±283.
15. Mah, S. C.; Hofbauer, K. G. J. Pharmacol. Exp. Ther.,
1988, 245, 1028±1032.
16. Hu€man, W. F.; Albrightson-Winslow, C.; Brickson, B.;
Bryan, H. G.; Caldwell, N.; Dytko, G.; Eggleston, D. S.;
Kinter, L. B.; Moore, M. L.; Newlander, K. A.; Schmidt, D.
B.; Silvestri, J. S.; Stassen, F. L.; Yim, N. C. F. J. Med. Chem.,
1989, 32, 880±884.
17. (a) Yamamura, Y.; Ogawa, H.; Chihara, T.; Kondo, K.;
Onogawa, T.; Nakamura, S.; Mori, T.; Tominaga, M.;
Yabuuchi, Y. Science, 1991, 252, 572±574. (b) Ogawa, H.;
Yamamura, Y.; Yamamoto, H.; Kondo, K.; Yamashita, H.;
Nakaya, K.; Chihara, T.; Mori, T.; Tominaga, M.; Yabuuchi,
Y. J. Med. Chem., 1993, 36, 2011±2017.
18. 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. J. Med.
Chem., 1996, 39, 3547±3555.
References
19. Tahara, A.; Tamura, Y.; Wada, K.; Kusayama, T.; Tsu-
kada, J.; Takanashi, M.; Yatsu, T.; Uchida, W.; Tanaka, A. J.
Pharmacol. Exp. Ther., 1997, 282, 301±308.
20. Matsuhisa, A.; Tanaka, A.; Kikuchi, K.; Shimada, Y.;
Yatsu, T.; Yanagisawa, I. Chem. Pharm. Bull., 1997, 45, 1870±
1874.
21. Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.;
Whitter, W. L.; Veber, D. F.; Anderson, P. S.; Freidinger, R.
M. J. Med. Chem., 1989, 32, 13±16.
1. Thibonnier, M.; Auzan, C.; Madhun, Z.; Wilkins, P.;
Berti-Mattera, L.; Clauser, E. J. Biol. Chem., 1994, 269,
3304±3310.
2. Sugimoto, T.; Saito, M.; Mochizuki, S.; Watanabe, Y.;
Hashimoto, S.; Kawashima, H. ibid., 1994, 269, 27088±27092.
3. Birnbaumer, M.; Seibold, A.; Gilbert, S.; Ishido, M.; Bar-
beris, C.; Antaramian, A.; Bradet, P.; Rosenthal, W. Nature,
1992, 357, 333±335.
4. Serradeil-Le-Gal, C.; Herbert, J. M.; Delisee, C.; Schae€er,
P.; Raufaste, D.; Garcia, C.; Dol, F.; Marty, E.; Ma€rand, J.
P.; Le Fur, G. Am. J. Physiol., 1995, 268, H404±H410.
5. Howl, J.; Ismail, T.; Strain, A. J.; Kirk, C. J.; Anderson,
D.; Wheatley, M. Biochem., 1991, 276, 189±195.
6. Manning, M.; Sawyer, W. H. J. Recept. Res., 1993, 13,
195±214.
7. Jard, S.; Gaillard, R. C.; Guillon, G.; Marie, J.; Schoenen-
berg, P.; Muller, A. F.; Manning, M.; Sawyer, W. H. Mol.
Pharmacol., 1986, 30, 171±177.
22. Desai, M. C.; Lefkowitz, S. L.; Thadeio, P. F.; Longo, K.
P.; Snider, R. M. J. Med. Chem., 1992, 35, 4911±4913.
23. Hamann, L. G.; Farmer, L. J.; Johnson, M. G.; Bender, S.
L.; Mais, D. E.; Wang, M. W.; Crombie, D.; Goldman, M. E.;
Jones, T. K. J. Med. Chem., 1996, 39, 1778±1789.
24. Anderson, N. G.; Lust, D. A.; Colapret, K. A.; Simpson,
J. H.; Malley, M. F.; Gougoutas, J. Z. J. Org. Chem., 1996, 61,
7955±7958.
25. Fryer, R. I.; Zhang, P.; Lin, Kuei-Ying; Upasani, R. B.;
Wong, G.; Skolnick, P. Med. Chem. Res., 1993, 3, 183±191.
26. Edwin, V.; Namkyu, L. J. Am. Chem. Soc., 1995, 117,
891±900.
8. Michell, R. H.; Kirk, C. J.; Billah, M. M. Biochem. Soc.
Trans., 1979, 7, 861±865.
9. Butlen, D.; Guillon, G.; Rajerison, R. M.; Jard, S.; Sawyer,
W. H.; Manning, M. ibid., 1978, 14, 1006±1017.
10. Manning, M.; Sawyer, W. H. J. Recept. Res., 1993, 13,
195±214.
11. Kinter, L. B.; Hu€man, W. F.; Stassen, F. L. Am. J. Phi-
siol., 1988, 254, F165±F177.
27. Umino, N.; Iwamura, T.; Itoh, N. Tetrahedron Letters,
1976, 763±766.
28. Kamal, A.; Rao, N. V. Chem. Commun., 1996, 385±386.
29. Jay, D. A.; Aranapakam, M. V.; John, P. D.; Fun-Wah
Sum. US Patent, 5700796. Chem. Abstr. 1997, 128, 88934.
30. Thibonnier, M; Bayer, A. L.; Simonson, M. S.; Kester, M.
Endocrinology, 1991, 129, 2845±2856.
12. Laszlo, F. A.; Laszlo Jr, F.; De Wied, D. Pharmacol.
Rev., 1991, 43, 73±108.