Design and synthesis of the first selective agonists for the rat vasopressin V1b receptor: Based on modifications of deamino-[cys 1]arginine vasopressin at positions 4 and 8

Article abstract of DOI:10.1021/jm060928n

The neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) mediate a wide variety of peripheral and central physiological and behavioral effects by acting on four different G-protein coupled receptors, termed V _1a (vascular), V<

Full text of DOI:10.1021/jm060928n

J. Med. Chem. 2007, 50, 835-847
835
Design and Synthesis of the First Selective Agonists for the Rat Vasopressin V1b Receptor:
Based on Modifications of Deamino-[Cys1]arginine Vasopressin at Positions 4 and 8†
Ana Pena,‡ Brigitte Murat,‡ Miguel Trueba,‡ Maria A. Ventura,§ Nga C. Wo,| Hazel H. Szeto,| Ling Ling Cheng,
Stoytcho Stoev, Gilles Guillon,‡ and Maurice Manning*,
Institut de Ge´nomique Fonctionnelle, CNRS UMR 5203-INSERM U.661, UniVersite´ Montpellier I et II, 34094 Montpellier Cedex 05, France,
Department of Pharmacology, Weill Medical College of Cornell UniVersity, New York, New York 10021, Department of Biochemistry and
Cancer Biology, UniVersity of Toledo College of Medicine, Toledo, Ohio (formerly The Medical UniVersity of Ohio), and Institut Cochin,
INSERM U.567, Paris, France
ReceiVed August 1, 2006
The neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) mediate a wide variety of
peripheral and central physiological and behavioral effects by acting on four different G-protein coupled
receptors, termed V1a (vascular), V1b (pituitary), V2 (renal), and OT (uterine). We recently reported that
d[Cha4]AVP (A), d[Leu4]AVP (B), d[Orn4]AVP (C), and d[Arg4]AVP (D) have high affinity and are selective
agonists for the human V1b receptor. However, peptides A-D were subsequently shown to be potent
antidiuretic agonists in the rat and are, thus, not selective V1b agonists in the rat. Peptides A-D served as
leads for the studies reported here. They were modified at position 8 by Lys, ornithine (Orn), diaminobutyric
acid (Dab), and diaminopropionic acid (Dap) to give d[Cha4,Lys8]VP (1), d[Cha4,Orn8]VP (2), d[Cha4,-
Dab8]VP (3), d[Cha4,Dap8]VP (4), d[Leu4,Lys8]VP (5), d[Leu4,Orn8]VP (6), d[Leu4,Dab8]VP (7), d[Leu4,-
Dap8]VP (8), d[Orn4,Lys8]VP (9), d[Orn4,Orn8]VP (10), d[Arg4,Lys8]VP (11), d[Arg4,Orn8]VP (12), and
d[Arg4,Dab8]VP (13). All peptides were synthesized by the Merrifield solid-phase method. Their binding
and functional properties were evaluated in rat AVP V1a, V1b, and V2 receptors and on the rat OT receptor
expressed either in native tissues or in stably transfected cells. They were also examined in rat vasopressor,
antidiuretic, and in in vitro (no Mg++) oxytocic assays. Functional studies performed on chinese hamster
ovary cells expressing the different AVP/OT receptors confirm that d[Cha4,Lys8]VP (1), d[Cha4,Dab8]VP
(3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8) are the first selective agonists for the rat V1b receptor.
These selective V1b agonists are promising new tools for studies of the role of the V1b receptor in the rat.
Introduction
and glucagon release by the pancreas,5-8 steroid, and catechola-
mine secretion by the adrenals,9 glycogenolysis in the liver,2
and the release of atrial natriuretic factor from the heart,10 and
paradoxically, it also elicits a vasodilatory response.11,12 Cen-
trally, AVP has multiple effects, including the regulation of
memory, synaptic transmission, body temperature, pain, anxiety,
and depression in rats and in humans (for reviews, see refs 13
and 14). The central effects of vasopressin and those of its
closely related neurohypophyseal peptide, oxytocin (OT), have
received intense investigative scrutiny in recent years.15-38 The
diverse peripheral and central effects of AVP and OT are
mediated by four different 7-transmembrane G-protein-coupled
receptors termed: V2, V1a, V1b (V3), and OT receptors.1-4,39
V2 receptors, present in the kidney, mediate the antidiuretic
effects of AVP via the adenylate cyclase pathway.1-4 V1a
receptors, present in many tissues, mediate the vascular effects
of AVP by causing vasoconstriction of the vascular smooth
muscle cells.1-4 The OT receptor mediates the uterine contract-
ing and milk let-down effects of OT.1-4 In the central nervous
system, V1a receptors mediate the effects of AVP on anxi-
ety.14,24,38 V1b receptors, present in the pituitary, pancreas,
adrenals, and in the CNS, mediate the release of ACTH from
the anterior pituitary,1,2 steroids from the adrenal medulla,9 and
insulin and glucagon from the pancreas.5-8 Recently, the V1b
receptor has also been shown to mediate anxiety and depression
in rats13,14,24,33,38,40 and also in humans.13,22,34 By contrast, OT
has been shown to relieve anxiety in rats,41-43 mice,28,44
monkeys,45 and in humans.46 The V1a, V1b, and OT receptors
act via the phosphoinositide pathway.1-4 The V2, V1a, V1b, and
OT receptors from a variety of species, including rat and human,
Arginine vasopressin (AVPa) exerts three well-known pe-
ripheral physiological effects: antidiuresis, vasoconstriction, and
the release of adrenocorticotropin hormone (ACTH; for reviews,
see refs 1-4). In addition to these well-established functions,
AVP is also involved peripherally in the regulation of insulin
† Dedicated to the memory of Professor Bruce Merrifield.
* To whom correspondence should be addressed. Tel.: (419) 383-4131.
Fax: (419) 383-6228. E-mail: maurice.manning@utoledo.edu.
‡ Institut de Ge´nomique Fonctionnelle.
§ Institut Cochin.
| Weill Medical College of Cornell University.
University of Toledo College of Medicine.
a Abbreviations: Symbols and abbreviations are in accordance with the
recommended IUB Commission on Biochemical Nomenclature (Eur. J.
Biochem. 1989, 180, A9-A11) and IUPHAR (Trends Pharmacol. Sci. 2001).
All amino acids are in the L-configuration unless otherwise noted. AcOH,
acetic acid; ACTH, adrenocorticotropin hormone, human; AVP, arginine8
vasopressin; dAVP, deamino-[Cys1]arginine vasopressin; Boc, tert-buty-
loxycarbonyl; Bzl, benzyl; cAMP, cyclic adenosine monophosphate; Cha,
1-amino-cyclopentane-1-carboxylic acid (cyclohexylalanine); CHO cells,
chinese hamster ovary cells; Dab, L-2,4-diaminobutyric acid; Dap, L-2,3-
diaminopropionic acid; DCC, dicyclohexylcarbodiimide; DMF, dimethyl-
formamide; DMEM, Dubbelco’s modified Eagles medium; E, maximal
efficiency; Et3N, triethylamine; Et2O, ethyl ether; HBS, Hank’s buffered
saline; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid
chromatography; hVx-R, human VP receptors; InsPs, total inositol phos-
phates; Kact, concentration of agonist leading to half-maximal activity; Kd,
concentration of peptide leading to half-maximal specific binding deduced
from saturation experiments; Ki, concentration of peptide leading to half-
maximal specific binding deduced from competition experiments; Meb,
p-methylbenzyl; Mob, p-methoxybenzyl; Mpr, 3-mercaptopropionyl; ONp,
p-nitrophenylester; OT, oxytocin; rVx-R, rat VP receptors; SI, selectivity
index; TLC, thin-layer chromatography; Tos, tosyl; VP, vasopressin; VT,
vasotocin; V1a, vascular; V1b, pituitary; V2, renal; Z, benzyloxycarbonyl.
10.1021/jm060928n CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/20/2007

836 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pena et al.
Table 1. Pharmacological Activities in Rat Bioassays of Lys8, Orn8, Dab8, and Dap8 Analogues of dAVP, d[X4]AVP Analogues A-D (where X )
Cha, Leu, Orn, and Arg) and Related Analogues
antidiuretic activity
(units/mg)
vasopressor activity
(units/mg)
oxytocic activity (in vitro)
no Mg++ (units/mg)
no.
peptide
AVPa
323 ( 16
369 ( 6
13.9 ( 0.5
63 ( 4
12 ( 0.5
15 ( 2
25 ( 7
0.9
dAVPb
1745 ( 385
346 ( 13
d[Lys8]VPc
d[Orn8]VPd
d[Dab8]VPe
d[Dap8]VPf
dDAVPg
550 ( 1.7k (301 ( 11)
486 ( 17k (202 ( 23)
463 ( 24k (1373 ( 128)
165 ( 12k (1079)
1200 ( 126
145 ( 7k (126 ( 2)
355 ( 11k (355 ( 30)
385 ( 12k (450 ( 30)
8.7 ( 0.7k (14.0)
0.39 ( 0.02
1.5
26
d[Val4]AVPh
dVDAVPh
d[D-3-Pal2]AVPi
1150 ( 110
51 ( 2
1230 ( 170
<0.01
∼8
∼1
antagonist
NDo
pA2 ) 6.22n
A
1
d[Cha4]AVPj
133.6 ( 5.61
0.82 ( 0.01
0.067 ( 0.005
0.043 ( 0.008
NDo
d[Cha4,Lys8]VPk
pA2 ) 6.48 ( 0.03p
M ) 3.41-7 ( 0.2
pA2 ) 6.55 ( 0.05p
M ) 3.04-7 ( 0.36
pA2 ) 6.35 ( 0.3
M ) 4.67-7 ( 0.34
pA2 ) 6.32 ( 0.03p
M ) 4.87-7 ( 0.3
0.67 ( 0.12
2
3
4
d[Cha4,Orn8]VPk
d[Cha4,Dab8]VPk
d[Cha4,Dap8]VPk
1.0 ( 0.2
1.0 ( 0.1
0.3 ( 0.1
0.22 ( 0.02
0.18 ( 0.02
∼0.002
B
5
d[Leu4]AVPl
378 ( 23.67
10.5 ( 0.9k
5-6m
3.11 ( 0.11
0.9 ( 0.1k
0.55m
d[Leu4,Lys8]VPk,m
0.054 ( 0.002k
6
d[Leu4,Orn8]VPk
5.7 ( 0.9
3.1 ( 0.1
mixed pA2 ∼< 4.89
agonistic activity, ∼0.04
0.27 ( 0.03
0.046
7
8
C
9
10
D
11
12
13
d[Leu4,Dab8]VPk
d[Leu4,Dap8]VPk
d[Orn4]AVPl
5.7 ( 0.5
0.7 ( 0.1
3.5 ( 0.2
0.05 ( 0.01
1.54 ( 0.13
0.23 ( 0.02
1.9 ( 0.1
159.93 ( 6.5
83 ( 4
260.32 ( 22.4
7.8 ( 0.4
6.49 ( 0.63
d[Orn4,Lys8]VPk
d[Orn4,Orn8]VPk
d[Arg4]AVPl
3.1 ( 0.1
7.9 ( 0.5
2.8 ( 0.1
748.32 ( 12.96
784 ( 54
0.027 ( 0.003
0.15 ( 0.02
0.06 ( 0.01
0.14 ( 0.02
d[Arg4,Lys8]VPk
d[Arg4,Orn8]VPk
d[Arg4,Dab8]VPk
823 ( 133
431 ( 58
173 ( 3
190 ( 5
a Original synthesis reported in ref 57. Data from ref 87. b Original synthesis reported in ref 72. Repeated synthesis and data from ref 86. c Original
synthesis and data reported in ref 73. d Original synthesis and data reported in ref 74. e Original synthesis and data reported in ref 75. f Original synthesis and
data reported in ref 76. g Original synthesis reported in ref 95a. Repeated synthesis and data from ref 86. h Original synthesis reported in ref 96a. Data from
ref 86. i Original synthesis and data reported in ref 97. j Original synthesis and data reported in ref 65. k This publication. l Original synthesis reported in ref
67. Data reported in ref 68. m Original synthesis and data reported in ref 81. n In vivo pA2 values are estimates since the [M] for the in vivo pA2 is estimated
by dividing ED (effective dose) by the estimated volume of distribution of 67 mL/kg. ED is defined as the dose (nmol/kg intravenously) of the antagonist
that reduces the response to 2x units of agonist to the response with x units of agonist administered in the absence of the antagonist. o ND ) not determined.
p In vitro pA2 values represent the negative logarithm to the base 10 of the average molar concentration [M] of the antagonist that reduces the response to
2x units of agonist to equal the response seen with x units of agonist administered in the absence of the antagonist.
have been cloned during the past decade or so.47-54 To date,
the receptor that mediates the vasodilating effects of AVP has
not been characterized.55 Following the original syntheses of
both OT56 and AVP,57 structure-activity studies, based on rat
bioassays, which measure antidiuretic, vasopressor, and in vitro
oxytocic activities,58-61 uncovered structural modifications of
these two peptides, which led to the design of agonists and
antagonists that possess potent and selective antidiuretic, pressor,
and oxytocic activities in the standard rat bioassays (for reviews,
see refs 1 and 58-61). In fact, these findings led to the
delineation of the V2, V1a, and OT receptors long before these
receptors were cloned.1,2 Although it was known that AVP could
cause the release of ACTH (see ref 62a,b and references therein),
it was not until the mid-eighties that the receptor that mediates
this response was characterized pharmacologically and shown
to be different than the V1a, V2, and OT receptors.63,64 Because
it was found to utilize the same second messenger system as
the V1a receptor, this AVP receptor was termed the V1b
receptor.64 There is no routine bioassay for the ACTH releasing
effect of AVP. Consequently, with the exception of those VP
analogues examined in corticotropin-releasing factor in vitro
and in vivo assays prior to the early eighties (see ref 62a,b and
references therein), virtually all of the many hundreds of VP
analogues reported to date58-61 are lacking quantitative data on
their ACTH-releasing activities. Thus, the design of selective
agonists and antagonists for the V1b receptor has, until recently,
been a very daunting challenge. It was not until the V1b receptor
was cloned in the mid-nineties48,52,53 that it became possible,
through the use of V1b receptor binding and functional assays,
to design selective agonists and an antagonist for the AVP V1b
receptor.65-68Werecentlyreportedthatdeamino-[cyclohexylalanine4]-
arginine vasopressin (d[Cha4]AVP, A) is the first selective
agonist for the human V1b receptor.65 This peptide has since
become a valuable pharmacological tool in a variety of
studies.25,31,69,70a-d A selective nonpeptide V1b antagonist, shown
to be effective in humans, was also recently reported.66 This
nonpeptide hV1bR antagonist was suggested to have beneficial
effects for treating depression in humans.71 We subsequently
reported67 the synthesis of 21 position-4 analogues of deamino-
[Cys1]-arginine vasopressin (dAVP).72 Virtually all of these
analogues exhibit very high affinities for the human V1b
receptor.67 Furthermore, a number of these analogues, most
notably, d[Leu4]AVP, d[Orn4]AVP and d[Arg4]AVP, with high
affinities for the human V1b receptor, have very low affinities
for the human V1a, V2, and OT receptors.67 In functional assays,
these three peptides are also highly selective for the human V1b
receptor with respect to the human V1a, V2, and OT receptors.67
Thus, they and d[Cha4]AVP65 are the first high affinity selective

Agonists for the Rat Vasopressin V1b Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 837
Table 2. Binding Properties of Lys8,Orn8, Dab8, and Dap8 Analogues of d[X4]AVP Analogues A-D (where X ) Cha, Leu, Orn, and Arg) for Rat
Vasopressin and Oxytocin Receptorsa
affinity, Ki (nM)
rV1b-R SI
V1a/V1b
no.
peptide
rV1b-R
rV2-R
rV1a-R
rOT-R
V2/V1b
OT/V1b
AVPb,c
0.29 ( .05
0.45 ( 0.1
0.8 ( 0.2d
0.3f
1.54 ( 0.97
10.8 ( 2.6 b
100g
1.17 ( 0.11
1.5
3.8
5.4
54
5.9
4.8
dAVPd (d[Gln4]AVP)
dDAVPe
0.20 ( 0.03b
0.97 ( 0.16 b
d[Val4]AVPh
0.25 ( 0.06
0.3 ( 0.1
60 ( 15
826 ( 150
1.2
240
3304
dVDAVPh
0.26f
316g
d[D-3-Pal2]AVPj
d[Cha4]AVPk
112 ( 30
69 ( 17
450 ( 22
0.6
9
4.0
1641
4837
967
5542
175
31 300
23 662
5523
4112
8603
2000
6227
1412
99
A
1
2
3
4
B
5
6
7
8
1.4 ( 0.1b
1.9 ( 0.4
12.7 ( 2.8k
600 ( 100
450 ( 150
450 ( 90
1050 ( 300
3.1 ( 0.8
100 ( 25
425 ( 100
210 ( 20
240 ( 50
3.4 ( 0.3
51 ( 18
2300 ( 600b
9100 ( 700
2900 ( 40
4400 ( 200
1800 ( 500
1252 ( 214
3800 ( 600
1400 ( 500
1000 ( 200
3300 ( 500
900 ( 245
2800 ( 1000
1300 ( 400
12.9 ( 0.4
130 ( 7
1430 ( 330k
590 ( 90
550 ( 20
430 ( 60
1200 ( 200
481 ( 49
64 ( 10
1021
312
182
547
122
12 025
400
213
164
352
2215
49
41
27 323
219
218
d[Cha4, Lys8]VPb
d[Cha4, Orn8]VPb
d[Cha4, Dab8]VPb
d[Cha4, Dap8]VPb
d[Leu4]AVPl
317
148
566
101
77
631
1638
828
624
7.6
113
83
1.5
6.7
5.1
3.0 ( 0.6
0.8 ( 0.2
10 ( 2
0.04 ( 0.01
0.16 ( 0.1
0.26 ( 0.02
0.25 ( 0.02
0.38 ( 0.03
0.45 ( 0.12
0.45 ( 0.04
0.91 ( 0.10
0.13 ( 0.12
0.6 ( 0.2
d[Leu4, Lys8]VPb,m
d[Leu4, Orn8]VPb
d[Leu4, Dab8]VPb
d[Leu4, Dap8 ]VPb
d[Orn4]AVPl
55 ( 6
41 ( 10
134 ( 22
997 ( 196
22 ( 6
C
9
d[Orn4, Lys8]VPb
d[Orn4, Orn8]VPb
d[Arg4 ]AVPl
10
D
11
12
13
76 ( 17
37 ( 9
0.20 ( 0.02
3.8 ( 0.9
3.1 ( 1.5
1.8 ( 0.3
3550 ( 790
125 ( 7
d[Arg4, Lys8]VPb
d[Arg4, Orn8 ]VPb
d[Arg4, Dab8 ]VPb
228
112
129
0.6 ( 0.2
0.19 ( 0.04
67 ( 16
25 ( 10
130 ( 30
195 ( 30
9.3
1026
a Bindings assays were performed on crude plasma membranes as described in the legend of Figure 1 and in the Experimental Section. Ki values are the
mean ( SEM of at least three independent experiments, each performed in triplicate. For each analogue, the rV1b-R SI was calculated as follows: SI ) (Ki
analogue for rVx-R)/(Ki analogue for rV1b-R), where rVx-R is the rV1a, rV2, or rOT receptor. b This publication. c Original synthesis reported in ref 57.
Repeat synthesis reported in ref 87. d Original synthesis reported in ref 72. Data from ref 65. e Original synthesis reported in ref 95a. f Data from ref 95b.
g Data from ref 96b. h Original synthesis reported in ref 96a; data from ref 65, unless otherwise noted. j Original synthesis reported in ref 97. Data from ref
65. k Original synthesis reported in ref 65. Data from ref 65. l Original synthesis reported in ref 67. Data from ref 68. m Original synthesis reported in ref 81.
agonists for the human V1b receptor. Due to species differences
between the human and the rat V2 receptors, these four peptides
were subsequently shown not to be selective V1b agonists in
the rat.68 While they were shown to exhibit high affinities for
the rat V1b receptor, they were also found to possess potent
where X4 ) Cha (A); Leu (B); Orn (C), and Arg (D).
antidiuretic activities in the rat bioassay and to have high
All four amino acids, Lys, Orn, Dab, and Dap, were
affinities for the rat V2 receptor.68 In searching for clues to the
incorporated at position 8 in peptides A and B to give peptides
design of selective agonists for the rat V1b receptor, we selected
1-8. The Lys8 and Orn8 substituents were incorporated in
the four peptides that exhibited the highest selectivity for the
peptide C to give peptides 9 and 10. The Lys8, Orn8, and Dab8
human V1b receptor, namely, d[Cha4]AVP (A), d[Leu4]AVP (B),
substituents were incorporated in peptide D to give peptides
d[Orn4]AVP (C), and d[Arg4]AVP (D).67 Using these four
11-13. The 13 peptides designed according to this rationale
are as follows:
peptides (A-D) as leads, we were confronted with the intriguing
challenge of where and how they could be modified to give
peptides that retain the V1b receptor agonism and diminished
V1a and OT receptor agonism of peptides A-D, while eliminat-
peptides 1-13
side chain at position 8
ing the potent rat V2 receptor agonism of peptides A-D.67,68
Because previous studies had shown that a basic L-amino acid
at position 8 is a requirement for the ACTH-releasing activity
of AVP analogues in the rat,62a,b we focused on finding basic
amino acid replacements for the Arg8 residue in peptides A-D.
We decided to replace the Arg8 residue in peptides A-D with
basic amino acids that have incremental differences in the sizes
of their side chains. We thus selected the basic amino acids,
Lys, ornithine (Orn), diaminobutyric acid (Dab), and diamino-
propionic acid (Dap), which have, respectively, 4, 3, 2, and 1
methylene groups in their side chains. These four amino acids
had previously been incorporated in dAVP72 to give, respec-
tively, d[Lys8]VP,73 d[Orn8]VP,74 d[Dab8]VP,75 and d[Dap8]-
VP.76 These four position 8 analogues of dAVP were resyn-
thesized for this study. In rat antidiuretic assays carried out on
the resynthesized peptides, all four analogues exhibit substantial
reductions in antidiuretic activity relative to that exhibited by
dAVP72 (Table 1). The greatest reduction is exhibited by d[Dap8]-
VP, the analogue with the shortest side-chain at position 8.
Peptides A-D67,68 have the following general structure:
1
2
3
4
5
6
7
8
9
10
11
12
13
d[Cha4,Lys8]VP
d[Cha4,Orn8]VP
d[Cha4,Dab8]VP
d[Cha4,Dap8]VP
d[Leu4,Lys8]VP
d[Leu4,Orn8]VP
d[Leu4,Dab8]VP
d[Leu4,Dap8]VP
d[Orn4,Lys8]VP
d[Orn4,Orn8]VP
d[Arg4,Lys8]VP
d[Arg4,Orn8]VP
d[Arg4,Dab8]VP
-CH2-CH2-CH2-CH2-NH2
-CH2-CH2-CH2-NH2
-CH2-CH2-NH2
-CH2-NH2
-CH2-CH2-CH2-CH2-NH2
-CH2-CH2-CH2-NH2
-CH2-CH2-NH2
-CH2-NH2
-CH2-CH2-CH2-CH2-NH2
-CH2-CH2-CH2-NH2
-CH2-CH2-CH2-CH2-NH2
-CH2-CH2-CH2-NH2
-CH2-CH2-NH2
We now report the synthesis and the binding affinities in rat
V1b, V1a, V2, and OT receptor assays of peptides 1-13 (Table
2). We also report their antidiuretic, vasopressor, and oxytocic
activities in rat bioassays77-80 (Table 1). It may be recalled that
one of these peptides, d[Leu4,Lys8]VP (5), had been previously
reported in 1973.81 This was at a time when the existence of
the AVP V1b receptor was neither hypothesized or known. So,

838 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pena et al.
Table 3. Functional Properties of V1b Specific Analogues d[Cha4,Lys8]VP (1), d[Cha4,Dab8]VP (3), d[Leu4,Lys8]VP (5) and d[Leu4,Dap8]VP (8) and
Related Peptides (A) and (B) for Rat Vasopressin V2 and V1b Receptors
V1b receptor
V2 receptor (rat kidney)
(At-T20 cells transfected with rat V1b-R)
adenylate
cyclase (AC)
activity
maximal AC
activity
(% of max
maximal
PLC activity
(% of max
antidiuretic
activity
(units/mg)
binding [3H]
AVP Kd
(nM)
phospholipase
C activation
Kact (nM)
binding [3H]
AVP Kd (nM)
no.
peptides
K
act(nM)
AVP response)
AVP response)
AVPa
323 ( 16b
0.45 ( 0.10
12.7 ( 2.8
596 ( 123
447 ( 91
3.1 ( 0.8
101 ( 25
237 ( 47
0.32 ( 0.08c
117 ( 15c
NDe
100
0.29 ( 0.05
1.40 ( 0.09
1.88 ( 0.39
0.79 ( 0.17
0.04 ( 0.01
0.16 ( 0.09
0.38 ( 0.03
2.3 ( 0.3
0.6 ( 0.1
2.7 ( 0.4
1.4 ( 0.2
0.4 ( 0.1
1.5 ( 0.4
1.6 ( 0.1
100
A
1
3
B
5
8
d[Cha4]AVPa
133 ( 6c
86
110 ( 3
73 ( 6
97 ( 4
128 ( 6
124 ( 4
108 ( 5
d[Cha4, Lys8]VPa
d[Cha4, Dab8]VPa
d[Leu4]AVPa
0.82 ( 0.01
1.0 ( 0.1
378 ( 24d
10.5 ( 0.9
0.7 ( 0.1
NDe
174 ( 25
75 ( 4
NDe
NDe
d[Leu4, Lys8]VPa
d[Leu4, Dap8]VPa
196 ( 71
300 ( 49
76 ( 5
97 ( 3
a This publication, unless otherwise noted. b Data from ref 87. c Data from ref 65. d Data from ref 68. e ND ) not determined.
Table 4. Physicochemical Properties of Free Peptides 1-13
TLC,b Rf
no.
peptide
yielda (%)
a
b
c
d
e
HPLC tR (min)
formula
MW
MW found
1
2
3
4
5
6
7
8
9
10
11
12
13
d[Cha4,Lys8]VP
d[Cha4,Orn8]VP
d[Cha4,Dab8]VP
d[Cha4,Dap8]VP
d[Leu4,Lys8]VP
d[Leu4,Orn8]VP
d[Leu4,Dab8]VP
d[Leu4,Dap8]VP
d[Orn4,Lys8]VP
d[Orn4,Orn8] VP
d[Arg4,Lys8]VP
d[Arg4,Orn8] VP
d[Arg4,Dab8]VP
33.0
48.0
28.1
17.8
59.9
44.6
46.6
46.3
48.9
37.5
68.9
49.5
56.5
0.32
0.33
0.32
0.36
0.30
0.31
0.31
0.32
0.16
0.12
0.19
0.18
0.20
0.24
0.10
0.54
0.43
0.50
0.42
0.49
0.50
0.41
0.39
0.28
0.27
0.32
0.29
0.35
0.31
0.25
0.17
0.28
0.29
0.28
0.23
0.24
0.06
0.13
0.06
0.12
0.03
23.5
20.1
20.1
20.2
17.6
17.8
17.5
17.1
14.0
14.0
13.7
13.9
17.4
C50H71O11N11S2
C49H69O11N11S2
C48H67O11N11S2
C47H65O11N11S2
C47H67O11N11S2
C46H65O11N11S2
C45H63O11N11S2
C44H61O11N11S2
C46H66O11N12S2
C45H64O11N12S2
C47H68O11N14S2
C46H66O11N14S2
C45H64O11N14S2
1066.3
1052.3
1038.3
1024.3
1026.3
1012.2
998.2
1065.9
1051.0
1038.2
1024.2
1026.0
1014.5
998.5
0.41
0.51
0.21
0.21
0.51
0.45
0.10
984.2
984.3
1027.3
1013.2
1069.3
1055.3
1041.3
1027.3
1013.6
1070.0
1056.1
1041.2
0.10
0.05
0.18
0.04
a Peptides 1-13 were obtained from the corresponding protected peptides I-XIII, Table 5. Yields are based on the protected peptide in the reduction-
reoxidation step in each case and are uncorrected for AcOH and water content. b Solvent systems and conditions are given in the Experimental Section.c All
peptides were at least 95% pure. For elution, a linear gradient from 90:10 to 30:70 (0.05% aqueous TFA/0.05%TFA in CH3CN) over 30 min with a flow
rate of 1.0 mL/min, as detailed in the Experimental Section, was applied.
Table 5. Physicochemical Properties of Protected Peptides I-XIIIa
TLC,c Rf
no.
peptidea
yieldb (%)
mp °C
a
b
c
d
e
I
II
Mpr(Meb)-Tyr(Bzl)-Phe-Cha-Asn-Cys(Bzl)-Pro-Lys(2Cl-Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Cha-Asn-Cys(Bzl)-Pro-Orn(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Cha-Asn-Cys(Bzl)-Pro-Dab(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Cha-Asn-Cys(Bzl)-Pro-Dap(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Leu-Asn-Cys(Bzl)-Pro-Lys(2Cl-Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Leu-Asn-Cys(Mob)-Pro-Orn(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Leu-Asn-Cys(Bzl)-Pro-Dab(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Leu-Asn-Cys(Bzl)-Pro-Dap(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Orn(Z)-Asn-Cys(Mob)-Pro-Lys(2Cl-Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Orn(Z)-Asn-Cys(Mob)-Pro-Orn(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Arg(Tos)-Asn-Cys(Bzl)-Pro-Lys(2Cl-Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Arg(Tos)-Asn-Cys(Bzl)-Pro-Orn(Z)-Gly-NH2
Mpr(Meb)-Tyr(Bzl)-Phe-Arg(Tos)-Asn-Cys(Bzl)-Pro-Dab(Z)-Gly-NH2
90.3
91.5
84.3
84.4
91.1
94.7
73.0
77.8
80.1
92.9
96.6
89.7
80.0
225-226
230-231
239-241
244-246
230-231
227-228
235-237
247-249
226-228
222-224
179-180
179-180
183-185
0.88
0.89
0.89
0.78
0.88
0.80
0.83
0.82
0.77
0.81
0.85
0.80
0.76
0.70
0.67
0.87
0.88
0.65
0.65
0.87
0.86
0.96
0.87
0.89
0.86
0.83
0.85
0.83
0.73
0.77
0.80
0.75
0.72
0.74
0.76
0.72
0.71
0.74
0.74
0.76
0.72
0.87
0.86
0.87
0.87
0.86
0.74
0.81
0.76
0.81
0.86
0.85
0.77
0.78
III
IV
V
VI
VII
VIII
IX
X
0.67
0.66
0.66
0.66
0.68
XI
XII
XIII
a The protected peptides I-XIII, synthesized as described in the Experimental Section, are the immediate protected precursors for the peptides 1-13
given in Table 4. b Yields are based on the amino acid content of the resin. c Solvent systems are described in the Experimental Section.
the ACTH-releasing potency of this peptide was not investigated
at that time. This peptide had been shown to exhibit negligible
vasopressor activity and low antidiuretic activity in rat bioas-
says81 (Table 1). It thus possessed the desired pharmacological
properties needed for a selective V1b agonist in the rat. The
question now to be answered was, would this peptide, which
possessed low bioactivities in the rat antidiuretic and vasopressor
bioassays, retain the potent rat V1b receptor agonism of its parent
d[Leu4]AVP?68 We also report the functional properties for the
rat V1b and V2 receptors of those peptides that exhibit high
selectivity for the rat V1b receptors with respect to the rat V2,
V1a, and OT receptors in binding assays and that also exhibit
very low activities in all three rat bioassays (Table 3).
Peptide Synthesis
The synthesis of the free peptides 1-13 (Table 4) was carried
out utilizing the Merrifield solid-phase method82-84 with the
modifications previously described.85-87 Starting from Boc-Gly
resin, the protected precursors I-XIII (Table 5) were synthe-
sized. HCl (1M)/acetic acid (AcOH) was used in all the
deprotection steps. Neutralizations were carried out with 10%
Et3N/CH2Cl2 in all the neutralization steps. Coupling reactions
were mediated mainly by DCC/HOBt88 in CH2Cl2/DMF except
for Boc-Asn, which was incorporated as its p-nitrophenyl
ester89,90 in DMF. Cleavage from the acylpeptide resin was by
ammonolysis in methanol with DMF extraction85,90 to give the

Agonists for the Rat Vasopressin V1b Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 839
56
protected peptide amides I-XIII (Table 5). Na in liquid NH3
was used to deblock each protected precursor, as previously
described.85-87 The resulting disulphydryl compounds were
oxidatively cyclized with K3[Fe(CN)6]91 using the modified
reverse procedure.92 The free peptides were desalted and purified
by gel filtration on Sephadex G-15 and Sephadex LH-20, mainly
in a two-step procedure93 using 50% AcOH and 2 M AcOH as
eluents, respectively, as previously described.85-87 When neces-
sary, an additional purification on Sephadex G-15 or Sephadex
LH-20 with 0.2 M AcOH as eluent was carried out. The purity
of the free peptides 1-13 (Table 4) was checked by thin-layer
chromatography (TLC), high performance liquid chromatogra-
phy (HPLC), and mass spectrometry. The four previously
published position 8 analogues of dAVP, d[Lys8]VP,73 d[Orn8]-
VP,74 d[Dab8]VP,75 and d[Dap8]VP76 (Table 2), were resynthe-
sized by the solid-phase method as described for dAVP.86 They
were purified and analyzed as described for peptides 1-13.
Bioassays. Peptides were assayed for agonistic activity in
the rat antidiuretic assay, in the rat vasopressor assay, and in
the in vitro rat oxytocic assay using the four-point assay
design.77-80 All experimental procedures were approved by the
Institutional Committee for the Care and Use of Animals at
Weill Medical College of Cornell University. Synthetic AVP
and OT, which had been standardized in vasopressor and
oxytocic units against the USP Posterior Pituitary Reference
Standard, were used as working standards in all bioassays.
Antidiuretic assays were on water-loaded rats under ethanol
anesthesia, as described by Sawyer.77 Vasopressor assays were
performed on urethane-anesthetized and phenoxybenzamine-
treated rats, as described by Dekanski.78 Oxytocic assays were
performed on isolated uteri from diethylstilbestrol-primed rats
in a Mg2+-free van Dyke-Hasting’s solution.79 When standard
errors are presented in the tables, the means reflect results from
at least four independent assay groups. A preliminary report
on the bioassay activities and rat receptor affinities of some of
these peptides has been reported.94
Figure 1. Binding properties of d[Leu4,Lys8]VP for rat AVP/OT
receptors. The binding properties of d[Leu4,Lys8]VP were determined
by binding competition experiments using [3H]AVP as radioligand and
membranes from At-T20 and CHO transfected cells (rV1b-R and rOT-
R, respectively) or from native tissues (rV1a-R and rV2-R), as described
in the Experimental Section. Specific binding was plotted against the
concentration of the unlabeled analogue used in the assay and expressed
as percent of the specific binding measured in the presence of vehicle
(C). Results are the mean ( SEM of at least three independent
experiments, each performed in triplicate.
The functional properties in rat V1b and V2 receptor assays of
the peptides, which are the most selective in the rat receptor
binding assays, namely, d[Cha4,Dab8]VP (3), d[Leu4,Lys8]VP81
(5), and d[Leu4,Dap8]VP (8) together with those of A, B, and
d[Cha4,Lys8]VP (1), are given in Table 3 and Figure 2.
Examination of the rat V1b receptor affinities of peptides 1-13
in Table 2 shows that, with the exception of peptide 4, d[Cha4,-
Dab8]VP, all of these peptides exhibit high affinities for the rat
V1b receptor. Ten peptides (3, 5-13) exhibit subnanomolar
affinities for the rat V1b receptor. The Lys8, Orn8, Dab8, and
Dap8 analogues of d[Cha4]AVP and d[Leu4]AVP exhibit low
affinities for the rat V2, V1a, and OT receptors. By contrast, the
position 8 analogues of C and D retain high affinities for the
rat V2 receptors and, thus, do not exhibit the desired V1b/V2
selectivity. Thus, in binding assays, only peptides 1-8 exhibit
the desired high selectivity for the rat V1b receptor with respect
to the rat V2, V1a, and OT receptors. We selected four of these,
1, 3, 5, and 8, for examination in functional assays (see below).
Examination of the rat bioassay data for peptides 1-13,
presented in Table 1, shows that the four position 8 analogues
of d[Cha4]AVP (A; peptides 1-4) all exhibit the desired
reduction in antidiuretic activity compared to that exhibited by
A (133.6 units/mg). The four peptides 1-4 exhibit antidiuretic
activities in the range of 0.3 to 1.0 unit/mg. These four peptides
also exhibit very weak vasopressor activities (0.002 to 0.22 unit/
mg). Peptides 1-4 exhibit weak OT antagonism in in vitro (no
Mg++) assays. As noted earlier, peptide 5, d[Leu4,Lys8]VP, had
previously been reported to exhibit 5-6 units/mg of antidiuretic
activity.81 In this study, we report a value of 10.5 units/mg for
the resynthesized peptide. These values show that replacing the
Arg8 residue by a Lys8 residue in the recently reported parent
peptide d[Leu4]AVP,67 which has an antidiuretic activity ) 378
units/mg,68 led to a drastic reduction in its antidiuretic activity.
The vasopressor activity of d[Leu4,Lys8]VP was originally
reported to be 0.55 unit/mg.81 We report here vasopressor
potency of 0.9 unit/mg for d[Leu4,Lys8]VP. We also report that
d[Leu4,Lys8]VP exhibits very weak in vitro (no Mg++) oxytocic
activity (0.054 unit/mg). The three remaining analogues of
d[Leu4]AVP (B), peptides 6-8, also exhibit reductions in
antidiuretic potencies relative to B. With an antidiuretic potency
of 0.75 unit/mg, the d[Leu4,Dap8]VP analogue (8) exhibits the
greatest reduction. With vasopressor potencies of 3.1 units/mg
and 3.5 units/mg, the Orn8 and Dab8 analogues of B do not
show reductions in vasopressor potencies relative to that
Receptor Binding and Functional Assays. These are
described in the Experimental Section. They were carried out
as previously described.65,68,95b,96b,99,100
Results
The rat V1b, V2, V1a, and OT receptor affinities for the
position 8 modified peptides 1-13, together with those of their
parent peptides d[Cha4]AVP (A), d[Leu4]AVP (B), d[Orn4]AVP
(C), and d[Arg4]AVP (D)67,68 and the related peptides, AVP,57,87
dAVP,72,86 dDAVP,86,95a dVDAVP,96a and d[D-3-Pal2]AVP97 are
given in Table 2 and Figure 1. Their antidiuretic, vasopressor,
and oxytocic (in vitro, no Mg++) activities in rat bioassays
together with those of the related analogues above and of d[Lys8]-
VP,73 d[Orn8]VP,74 d[Dab8]VP,75 and d[Dap8]VP76 (resynthe-
sized and original values) are given in Table 1. It should be
noted that we obtained different values for the antidiuretic
activities of these four resynthesized position 8 modified
analogues compared to those from the original syntheses73-76
(Table 1). In units/mg, with the original values in brackets, these
values are as follows: d[Lys8]VP, 550 (301);73 d[Orn8]VP, 486
(202);74 d[Dab8]VP, 463 (1373);75 and d[Dap8]VP, 165 (1079).76
Thus, for the Lys8 and Orn8 analogues, our values are higher
than those originally reported.73,74 For the Dab8 and Dap8
analogues, the values are strikingly lower than the original
values.75,76 We have no explanation for these striking differences
in antidiuretic activities. The vasopressor activities of the
resynthesized position 8 analogues of dAVP are in general much
closer to the values in the original publications73-76 (Table 1).

840 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pena et al.
mg. Peptides 11 and 12 exhibit antidiuretic activity of 784 and
823 units/mg, respectively. Replacement of the Arg8 residue in
D by a Dab8 residue to give peptide 13 reduced the antidiuretic
activity. However, with an antidiuretic activity of 431 units/
mg, peptide 13 is more potent than AVP (antidiuretic activity
) 332 unit/mg). All three analogues of D (peptides 11-13)
exhibit substantial vasopressor potencies of 83, 173, and 190
units/mg, respectively, with one (peptide 11) being lower and
two (peptides 12 and 13) being higher than D (vasopressor
potency ) 160 U/mg). Thus, in reviewing the rat receptor
affinity data in Table 2 and the rat bioassay data in Table 1, it
became clear that although the majority of these 13 peptides
have high affinities for the rat V1b receptor, only the two position
8 analogues of d[Cha4]AVP (A), namely, d[Cha4,Lys8]VP (1)
and d[Cha4,Dab8]VP (3), and two of the analogues of d[Leu4]-
AVP (B), namely, d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8),
exhibit the desired reductions in rat antidiuretic and vasopressor
activities to merit further scrutiny in functional assays.
Functional Properties of d[Cha4,Lys8]VP (1), d[Cha4,Dab8]-
VP (3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8; Table
3, Figure 2). To further analyze the V1b and V2 agonist
properties in the rat of the four analogues that exhibit the best
affinities and selectivities for the rat V1b receptor with respect
to the rat V2 receptor, namely, d[Cha4,Lys8]VP (1), d[Cha4,-
Dab8]VP (3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8), we
tested their abilities to stimulate phospholipase C(PLC) (inositol
phosphates accumulation) on At-T20 cells transfected with the
V1b receptor and adenylate cyclase (cyclic AMP accumulation)
on rat kidney membranes expressing the V2 receptor. In the
PLC functional assays, there is a good correlation between their
binding affinities for the rat V1b receptor and their Kact
(concentration of agonist leading to half-maximal activity)
values (Table 3). Similarly, in the adenylate cyclase assay,
peptides 3, 5, and 8 exhibit a good correlation between their
Kact values and their binding affinities for the rat V2 receptor.
Thus, in contrast to their parent peptides, d[Cha4]AVP (A) and
d[Leu4]AVP (B), peptides 1, 3, 5, and 8 are excellent high
affinity selective agonists for the rat V1b receptor.
Figure 2. Agonist properties of some d[X4,Y8]VP analogues for the
rat V1b and V2 receptors. Upper panel: [3H] myo inositol prelabeled
At-T20 cells stably transfected with the rV1b-R were incubated 15 min
at 37 °C in an HBS medium supplemented with 15 mM LiCl with or
without (C) increasing amounts of AVP or d[X4,Y8]VP analogues. Total
InsP, which accumulated, was determined in each experimental
condition. Results, expressed as % of maximal InsP response obtained
with 100 nM AVP, are the mean ( SEM of at least three independent
experiments, each performed in triplicate. Lower panel: Rat kidney
membranes naturally expressing the rV2-R were incubated at 37 °C
with or without (C) increasing amounts of AVP or d[X4,Y8]VP
analogues in the presence of R[32P]-ATP. [32P]-cAMP produced was
determined as described in the Experimental Section and expressed as
% of maximal cAMP response obtained with 100 nM AVP. Results
are the mean ( SEM of at least three distinct experiments, each
performed in triplicate.
Discussion
Effects of Position 8 Modification in Peptides A-D on
Rat V1b Receptor Affinities and Selectivities. In searching for
clues to the design of selective agonists for the rat V1b receptor,
we selected as leads four peptides, d[Cha4]AVP (A), d[Leu4]-
AVP (B), d[Orn4]AVP (C), and d[Arg4]AVP (D), which are
highly selective for the human V1b receptor with respect to the
human V2, V1a, and OT receptors.67 Although these four peptides
were subsequently found to exhibit high affinities for the rat
V1b receptor,68 they were also found to exhibit high affinities
for the rat V2 receptor and to exhibit potent antidiuretic activities
in rat bioassays.68 Consequently, they are not selective for the
exhibited by B (3.11 units/mg). With a vasopressor potency of
0.05 unit/mg, the d[Leu4,Dap8]VP analogue (peptide 8) exhibits
a striking reduction in vasopressor potency relative to B
(vasopressor activity ) 3.11 units/mg). All three peptides 6-8
exhibit very weak oxytocic activities; 0.04, 0.27, and 0.046 unit/
mg, respectively. With antidiuretic activities of 7.8 units/mg
and 7.9 units/mg, peptides 9 and 10, the Lys8 and Orn8 analogues
of d[Orn4]AVP (C), exhibit reductions in antidiuretic potencies
relative to C (antidiuretic activity ) 260 units/mg). These two
V1b versus the V2 receptor in the rat. To use these four peptides
as leads for the design of ligands that are selective for the rat
V1b receptor, we needed to retain their affinities for the V1b
receptor while diminishing their affinities for the rat V2 receptor.
In the study reported here, the Arg8 residue in peptides A-D
was replaced with one or more of the following basic amino
acids: Lys, Orn, Dab, and Dap. The resulting peptides 1-13
(Table 2) exhibit varying degrees of high V1b receptor affinity
and selectivity. With the exception of peptides 11-13, all
peptides exhibit very low affinities for the rat V1a receptor. Thus,
peptides 1-10 are highly selective for the rat V1b receptor with
respect to the rat V1a receptor. All 13 peptides exhibit diminished
affinities for the rat OT receptor, especially in relationship to
analogues also exhibit ∼50% reductions in in vitro (no Mg++
)
oxytocic activities relative to C (oxytocic activity ) 6.49 units/
mg). The Lys8 analogue (9) exhibits diminished vasopressor
potency (0.23 units/mg) relative to C (vasopressor activity )
1.54 units/mg), whereas the Orn8 analog exhibits slightly higher
vasopressor potency (1.9 units/mg). Replacement of the Arg8
residue in d[Arg4]AVP (D) by Lys and Orn to give peptides 11
and 12 led in each instance to enhancements of antidiuretic
activity. Peptide D exhibits antidiuretic activity ) 748 units/

Agonists for the Rat Vasopressin V1b Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 841
four peptides 5-8 exhibit rat V1b receptor affinities equivalent
to that of AVP (Ki ) 0.29 nM). The high affinity of the Dap8
analogue of B, peptide 8, is in striking contrast to the relatively
low affinity of the Dap8 analogue of A, peptide 4. These findings
demonstrate that the effects of the Dap8 modification in d[X4]-
AVP analogues on rat V1b receptor affinity is very dependent
on the structure of the amino acid at position 4 and on the
number of aliphatic carbons of the residue at position 8 (Figure
3, upper panel). These findings show that when position 4 in
dAVP is occupied by Leu, position 8 can be occupied with a
wide variety of basic amino acids with retention of high affinity
for the rat V1b receptor. These findings are in contrast to those
observed when position 4 in dAVP is occupied by Cha (see
above).
Effects on Rat V1b Receptor Affinity of Position 8
Modifications in d[Orn4]AVP (C; Peptides 9 and 10). d[Orn4]-
AVP (C) exhibits subnamolar affinity for the rat V1b receptor
(Ki ) 0.45 nM).68 Replacement of the Arg8 residue in C by
Lys and by Orn to give peptides 9 and 10 resulted in retention
of subnanomolar affinities for the rat V1b receptor. The affinity
of peptide 9 for the rat V1b receptor is equivalent to that of C.
The affinity of peptide 10 for the rat V1b receptor is about half
that of C. These finding show that Arg8, Lys8, and Orn8
substitutions in combination with an Orn4 substitution in dAVP
are well tolerated by the rat V1b receptor.
Effects on Rat V1b Receptor Affinity of Position 8
Modifications in d[Arg4]AVP (D; Peptides 11-13, Table 2).
d[Arg4]AVP (D) exhibits subnamolar affinity for the rat V1b
receptor (Ki ) 0.13 nM).68 Replacement of the Arg8 residue in
D by Lys, Orn, and Dab to give peptides 11-13 was very well
tolerated. Peptides 11-13 all exhibit subnamolar affinities for
the rat V1b receptor. These findings show that when position 4
in dAVP is occupied by Arg, position 8 can be occupied by
Arg, Lys, Orn, and Dab with retention of high affinity for the
rat V1b receptor.
Figure 3. Influence of the number of aliphatic carbons in the side-
chain at position-8 of d[Leu4,Y8]VP analogues on rat V1b receptor
properties. The affinity (upper panel) and the V1b receptor SI relative
to other VP/OT receptor isoforms (lower panel) for given d[Leu4,Y8]-
VP analogues were plotted as a function of the number of aliphatic
carbons present on the side chain of their eight residue. Ki values and
V1b SI were from Table 2.
dAVP (Ki ) 0.97 nM). They thus exhibit varying degrees of
selectivity for the rat V1b receptor with respect to the rat OT
receptor. Peptides 1-8, the position 8 analogues of d[Cha4]AVP
(A) and d[Leu4]AVP (B), all exhibit diminished affinities for
the rat V2 receptor and, in binding assays, are consequently
highly selective for the rat V1b receptor with respect to the rat
V2 receptor. These are the first such peptides to exhibit high
affinity and selectivity for the rat V1b receptor with respect to
the rat V1a, V2, and OT receptors.
Effects on Rat V1b Receptor Affinity of Position 8
Modifications in d[Cha4]AVP (A; Peptides 1-4, Table 2).
d[Cha4]AVP (A) exhibits a Ki value for the rat V1b receptor )
1.40 nM.68 Replacement of the Arg8 residue in d[Cha4]AVP
by Lys, Orn, Dab, and Dap to give peptides 1-4 led to varying
degrees of retention of rat V1b receptor affinity (Table 2). The
Lys8 and Dab8 substituents are clearly superior to the Orn8 and
Dap8 substituents in retaining or enhancing rat V1b receptor
affinity. d[Cha4,Lys8]VP (1) and d[Cha4,Dab8]VP (3) are the
most promising peptides to emerge from this series of position
8 analogues of A. These findings show that when the position
4 residue in dAVP is occupied by Cha, the affinity for the rat
V1b receptor is highly dependent on the structure of the basic
amino acid at position 8.
These data prove conclusively that the four lead peptides
A-D could be modified at position 8 to give a series of peptides
that, with the exception of three peptides, the Lys8, Orn8, and
Dap8 analogues of A (peptides 1, 2, and 4), exhibit subnano-
molar affinity for the rat V1b receptor.
Effects of Position 8 Modifications in Peptides A-D on
Rat V2 Receptor Affinities (Table 2; Figure 3). As is clear
from the rat V2 receptor affinity data in Table 2, the effects of
the Lys8, Orn8, Dab8, and Dap8 replacements for Arg8 in peptides
A-D depends very much on the nature of the amino acid at
position 4. When these substitutions were carried out in the Cha4
and Leu4 analogues (peptides A and B), the resulting peptides
1-8 exhibited significant reductions in rat V2 receptor affinities,
whatever the size of the position 8 residue (1-4 aliphatic
carbons; Figure 3, lower panel). By contrast, when these
substitutions were carried out in the Orn4 and Arg4 analogues
(peptides C and D), the resulting peptides 9-13 exhibit only
very modest reductions in rat V2 receptor affinities relative to
those exhibited by the parent peptides C and D.
Effects on Rat V2 Receptor Affinities and Selectivities of
Position 8 Modifications in d[Cha4]AVP (A; Peptides 1-4,
Table 2). d[Cha4]AVP (A) exhibits a Ki value for the rat V2
receptor ) 12.7 nM.65 Replacement of the Arg8 residue in A
by Lys, Orn, Dab, and Dap to give peptides 1-4 brought about
striking reductions in affinities for the rat V2 receptor. These
reductions combined with the high affinities for the rat V1b
receptor lead to strikingly high rat V1b/V2 receptor selectivities
exhibited by peptides 1-4 relative to that of A
Effects on Rat V1b Receptor Affinity of Position 8
Modifications in d[Leu4]AVP (B; Peptides 5-8, Table 2;
Figure 3). d[Leu4]AVP (B) exhibits a very high Ki value for
the rat V1b receptor ) 0.04 nM.68 Replacement of the Arg8
residue in B by Lys, Orn, Dab, and Dap led to excellent retention
of rat V1b receptor affinities by the resultant peptides 5-8. All

842 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pena et al.
.
Effects on Rat V2 Receptor Affinities and Selectivities of
Position 8 Modifications in d[Leu4]AVP (Peptides 4-8, Table
2). d[Leu4]AVP (B) exhibits a Ki value for the rat V2 receptor
) 3.1 nM.68 Peptides 5-8 exhibit reductions in rat V2 receptor
affinities relative to B. Thus, the replacement of the Arg8 residue
in B with Lys, Orn, Dab, and Dap resulted in the desired
reductions in affinities for the rat V2 receptor. These reductions
combined with the high affinities for the rat V1b receptor led to
striking enhancements in rat V1b/V2 receptor selectivities of
peptides 5-8 relative to B.
Effects on Rat V2 Receptor Affinities and Selectivities of
Position 8 Modifications in d[Orn4]AVP (C; Peptides 9 and
10) and in d[Arg4]AVP (D; Peptides 11-13, Table 2). With
Ki values of 3.4 nM and 0.2 nM, peptides C and D exhibit high
and very high affinities, respectively, for the rat V2 receptor.
Replacement of the Arg8 residue in d[Orn4]AVP (C)68 by Lys
and Orn resulted in only modest reductions in rat V2 receptor
affinities relative to that exhibited by C (Table 2). Consequently,
peptides 9 and 10 do not exhibit the same gains in rat V1b/V2
receptor selectivity as peptides 1-8. The affinities of peptides
11-13 for the rat V2 receptor, while reduced in comparison
with the very high affinity of the parent peptide D, nonetheless,
are in the same high range as peptides B and C. Thus, peptides
11-13 exhibit very low gains in rat V1b/V2 receptor selectivities
(Table 2).
It is clear from the rat V2 receptor affinities and rat V1b/V2
receptor selectivities of the 13 position 8 analogues of A-D in
Table 2 that the neutral lipophilic substitutions, Cha and Leu,
are superior to the basic amino acids, Orn and Arg, at position
4 in leading to peptides that exhibit reduced rat V2 receptor
affinities and high rat V1b/V2 receptor selectivities.
Figure 4. Correlation between antidiuretic and vasopressor activities
of d[X4,Y8]VP analogues and their corresponding binding properties
(Ki.). Data from this figure are from Tables 1 and 2. The correspondence
between peptides (A-D and 1-13) is given in Table 2.
same substitutions in peptides A-C, (d[Cha4]AVP, d[Leu4]-
AVP, and d[Orn4]AVP), illustrate the need for caution in making
generalizations on the effects of a series of substitutions based
on making these changes in only one peptide.
Effects of Position 8 Modifications of Peptides A-D on
Antidiuretic Activities: Comparisons with Rat V2 Receptor
Affinities (Peptides 1-13, Tables 1 and 2; Figure 4). As noted
earlier, all four parent peptides, d[Cha4]AVP (A), d[Leu4]AVP
(B), d[Orn4]AVP (C), and d[Arg4]AVP (D),68 although pos-
sessing excellent affinities for the rat V1b receptor, are clearly
not selective agonists for the rat V1b receptor, because they also
exhibit potent antidiuretic activities. Replacement of the Arg8
residue in d[Cha4]AVP (A) with Lys, Orn, Dab, and Dap
resulted in each case in drastic reductions in antidiuretic
potencies (Table 1). These findings all correlate very well with
the low affinities for peptides 1-4 in the rat V2 receptor binding
assays (Table 2, Figure 4). Replacement of the Arg8 residue in
d[Leu4]AVP (B) by Lys, Orn, Dab, and Dap also brought about
marked reductions in antidiuretic activities. The original syn-
thesis of d[Leu4, Lys8]VP (peptide 5) reported antidiuretic
activity of 5-6 units/mg.81 In peptides 4-8, with the exception
of d[Leu4, Dap8]VP (peptide 8), the low rat receptor affinities
of peptides 5-7 do not correlate well with their rat antidiuretic
activities. These findings are illustrated in Figure 4, upper panel,
showing that receptor affinities may not always accurately reflect
agonistic properties. In striking contrast to their effects in A,
B, and C, replacement of the Arg8 residue in d[Arg4]AVP (D)
by Lys and Orn to give peptides 11 and 12 effected unexpected
increases in antidiuretic activities. With antidiuretic activities
of 784 and 823 units/mg, peptides 11 and 12 are among the
most potent antidiuretic agonists reported to date. These two
peptides and indeed peptide D are clearly far more potent
antidiuretic agonists than AVP (antidiuretic activity ) 332 units/
mg).86 These findings on the contrasting effects on antidiuretic
activities of the Lys8, Orn8, Dab8, and Dap8 substitutions in
dAVP73-76 and in d[Arg4]AVP (D) with those obtained for those
Effects of Position 8 Modifications of Peptides A and B
on Vasopressor Activities (Peptides 1-8, Table 1, Figure 4).
d[Cha4]AVP (A) exhibits very weak vasopressor activity.
Replacement of the Arg8 residue in A by Lys, Orn, Dab, and
Dap resulted in two peptides (1 and 4) with reductions of
vasopressor potencies and two peptides (2 and 3) with very
modest increases in vasopressor potencies. This pattern was also
observed with d[Leu4]AVP (B). The Lys8 and Dap8 analogues
of B (peptides 5 and 8) exhibit diminished vasopressor potencies.
Whereas, the Orn8 and Dab8 analogues (peptides 7 and 8) exhibit
vasopressor potencies that are virtually equipotent with that of
B. The greatest reductions in vasopressor potencies in peptides
A and B were clearly brought about by the Dap8 modification,
which has a single carbon in its side chain. The Lys8 residue
with four carbons also brought about a marked reduction in
vasopressor potency in peptide B. By contrast, the Orn8 and
Dab8 residues, with three and four carbons in their side chains,
led to slight enhancements in vasopressor potency in A and good
retention of vasopressor potency in B. These patterns of reduced
and retained or enhanced vasopressor activities brought about
by the Lys, Orn, Dab, and Dap substituents at position 8 in
d[Cha4]AVP (A) and in d[Leu4]AVP (B), closely mirror the
effects on vasopressor activities of these four substituents at
position 8 in dAVP73-76 (Table 1). Thus, there is no correlation
between the number of carbons in the side chain at position 8
and the vasopressor potency of the resultant analogue. As noted
above for the rat V2 receptor, there is not always a good
correlation between binding data for the rat V1a receptor and
for rat vasopressor activities (see Figure 4, lower panel).

Agonists for the Rat Vasopressin V1b Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 843
HPLC was performed on a Waters 810 instrument under the
following conditions: 90:10 to 30:70 0.05% aqueous TFA/0.05%
TFA in CH3CN, linear gradient over 60 min at 1.0 mL/min (λ )
210 nm), on a Microsorb C18 column (Rainin Instrument Co., Inc.).
All peptides were at least 95% pure. Mass spectra were done by
the Tuft’s Core Facility, Physiology Department (Boston, MA) on
a MALDI-TOF Voyager mass spectrometer (Perspective Biosys-
tems/Applied Biosystems) using dihydrobenzoic acid as the matrix.
Solid-Phase Synthesis Procedures. Peptides 1-13 (Table 4)
were synthesized using the Merrifield method82-84 with the
modifications previously described.85-87 Starting with 0.25 nM of
Boc-Gly resin, eight cycles of deprotection, neutralization, and
coupling were carried out by the DCC/HOBt88 or the active
ester89-90 procedure. The side chain protection of the Boc-amino
acids incorporated was as follows: Mpr(Meb), Tyr(Bzl), Orn(Z),
Arg(Tos), Cys(Mob) or Cys(Bzl), Lyz(2-Cl-Z), Dab(Z), and Dap-
(Z). Ammonolysis in MeOH85,90 was used to cleave the protected
peptides from the resin. The protected precursors obtained by
ammonolysis were extracted with hot DMF and isolated by
precipitation with hot water, as previously described.85-87 They were
purified by reprecipitations with DMF/MeOH/Et2O until adjudged
pure by TLC85 to give the required protected peptides I-XIII (Table
5). All protected precursors I-XIII were deblocked using sodium
in liquid ammonia,56 as previously described.85-87 The resulting
disulphydryl compounds were oxidatively cyclized with K3[Fe-
(CN)6]91 using the modified reverse procedure.92 The free peptides
were purified by a two-step gel filtration procedure93 on Sephadex
G-15 (eluent 50% AcOH) and Sephadex LH-20 (eluent 2 M AcOH).
For some peptides, an additional purification by gel filtration on
Sephadex G-15 and/or LH-20 was used. The physicochemical data
for the free peptides 1-13 are given in Table 4.
Mpr(Meb)-Tyr(Bzl)-Phe-Leu-Asn-Cys(Bzl)-Pro-Dap(Z)-Gly-
NH2 (VIII, Table 5). Boc-Gly resin (0.5 g, 0.35 mmol) was
subjected to eight cycles of deprotection, neutralization, and
coupling with Boc-Dap(Z), Boc-Pro, Boc-Cys(Bzl), Boc-Asn-ONp,
Boc-Leu, Boc-Phe, Boc-Tyr(Bzl), and Mpr(Meb), respectively. The
resulting protected peptidyl resin was cleaved by ammonolysis, as
previously described.85-87 The protected peptide was extracted with
hot DMF (30 mL), and the product was precipitated by the addition
of hot water (ca. 300 mL). After cooling, the product was collected,
dried in vacuo over P2O5, and reprecipitated from methanol (30
mL) and ether (ca. 200 mL). Collection and drying in vacuo over
P2O5 gave the required nonapeptide amide 490 mg, yield 77.8%
(VIII, Table 5). The same procedure was used for the synthesis
and purification of all other protected nonapeptide amides I-VII,
IX-XIII (Table 5). The physicochemical properties of the protected
peptides I-XIII are given in Table 5.
Conclusion
We report here the first analogues of AVP, which are selective
agonists for the rat V1b receptor with respect to the rat V1a, V2,
and OT receptors. These peptide analogues were designed by
using as leads the four peptides, d[Cha4]AVP (A), d[Leu4]AVP
(B), d[Orn4]AVP (C), and d[Arg4]AVP (D), which were the
first high affinity selective agonists for the human V1b receptor.67
Peptides A-D exhibit properties very similar to those of the
potent selective antidiuretic agonist dDAVP,86,95(a) widely used
clinically for the treatment of diabetes insipidus.1 Recently,
dDAVP was shown to be a full V1b agonist in humans and a
partial V1b agonist in rats.98 Replacement of the Arg8 residue
in peptides A-D by Lys, Orn, Dab, and Dap resulted in peptides
1-13 (Table 1). With the exception of peptides 1, 2, and 4, all
of these peptides exhibit subnanomolar affinities for the rat V1b
receptor (Table 2). Examination of these 13 peptides in rat V1a,
V2, and OT receptor binding assays and in rat antidiuretic,
vasopressor, and oxytocic (in vitro; no Mg++) bioassays
uncovered four peptides: d[Cha4,Lys8]VP (1), d[Cha4,Dab8]VP
(3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8), which exhibit
very high affinities for the rat V1b receptor and very low
affinities for the rat V1a, V2, and OT receptors. These four
peptides exhibit high selectivities for the rat V1b receptor with
respect to the rat V2, V1a, and OT receptors. In functional assays,
these four peptides are potent agonists for the rat V1b receptor
and weak agonists for the rat antidiuretic, vasopressor, and OT
receptors. One of these peptides, d[Leu4,Lys8]VP, was first
reported in 1973.81 Long before the discovery of the AVP V1b
receptor,63,64 it was shown to be a weak antidiuretic and
vasopressor agonist in rat bioassays.81 We now report that it is
also a weak oxytocic agonist and that it is a potent and a highly
selective agonist for the rat V1b receptor. The selective agonists
for the rat V1b receptor reported here are potentially useful new
tools for studies on the role of the V1b receptor in modulating
anxiety in the rat. They also offer promising new leads to the
design of peptide antagonists for the rat V1b receptor and of
radiolabeled and fluorescent ligands for the rat V1b receptor.
Experimental Section
Materials. All reagents used were analytical grade. Most standard
chemicals were purchased from Sigma (St. Louis, MO), Roche
Molecular Biochemicals (Mannheim, Germany), or Merck & Co.,
Inc. (Darmstadt, Germany), unless otherwise indicated. AVP came
from Bachem (Bubendorf, Switzerland). [3H]AVP (60-80 Ci/
mmol) was from Perkin-Elmer Life Sciences (Coutaboeuf, France).
DMEM (Dubbelco’s modified Eagles medium)-F12 was from
Sigma (St. Louis, MO), geneticine and Lipofectine Plus Reagent
were from Gibco, L-glutamine was purchased from In vitrogen
(Cergy Pontoise, France), Nu-serum was from Becton Dickinson
Biosciences (Ozyme, Saint Quentin en Yvelines, France), and
Fetalclone III was from Hyclone (Perbio, Bre´vie`res France). Dowex
AG1-X8 formate form 200-400 mesh was purchased from Bio-
Rad Laboratories, Inc. (Munchen, Germany). The Merrifield resin
was purchased from Calbiochem-Novabiochem Corp. (San Diego,
CA). The Boc-Asn-ONp was provided by Bachem Bioscience, Inc.
(King of Prussia, PA). The Boc-Phe, Boc-Leu, and Boc-Pro
derivatives were provided by Bachem (Torrance, CA). All other
amino acid derivatives were purchased from Chem-Impex Inter-
national, Inc. (Wood Dale, IL). TLC was run on precoated silica
gel plates (60F-254, E. Merck) with the following solvent sys-
tems: (a) 1-butanol/AcOH/H2O (4:1:5, upper phase); (b) 1-butanol/
AcOH/H2O (4:1:1); (c) 1-butanol/AcOH/H2O (4:1:2); (d) 1-butanol/
AcOH/H2O/pyridine (15:3:3:10); and (e) 1-butanol/AcOH/H2O (2:
1:1). Loads of 10-15 µg were applied and chromatograms were
developed at a minimal length of 10 cm. The chlorine gas procedure
for the KI-starch reagent was used for detection.84 Analytical
d[Leu4,Dap8] Vasopressin (8, Table 4). The Na/liquid NH3
procedure56 was used for the deprotection of all protected non-
apeptide amides I-XIII, as described here for peptide VIII. A
solution of protected nonapeptide amide (VIII, Table 5) (120 mg)
in sodium-dried ammonia (ca. 400 mL) was treated at the boiling
point and with stirring with sodium from a stick of metal contained
in a small-bore glass tube until a light-blue color persisted in the
solution for about 30 s.85-87 NH4Cl was added to discharge the
color. Reoxidation of the deblocked disulphydryl peptide 8 was
performed with K3[Fe(CN6)]91 by the modified reverse procedure92
as follows. The resulting disulphydryl peptide residue was dissolved
in 25 mL of 50% AcOH, and the solution was diluted with 50 mL
of H2O. The peptide solution was added dropwise with stirring over
a period of 15-30 min to a 600 mL aqueous solution that contained
20 mL of a 0.01 M solution of potassium ferricyanide. Meanwhile,
the pH was adjusted to approximately 7.0 with concentrated
ammonium hydroxide. Following oxidation, the free peptide 8 was
isolated and purified as follows: after acidification with AcOH to
pH 4.5 and stirring for 20 min with an anion exchange resin (Bio-
Rad, AG 3 × 4, Cl- form, 5 g damp weight), the suspension was
slowly filtered and washed with 0.2 M AcOH (3 × 30 mL). The
combined filtrate and washings were lyophilized. The resulting
powder was desalted on a Sephadex G-15 column (110 × 2.7 cm),
eluting with aqueous AcOH (50%), with a flow rate of 5 mL/h93.

844 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pena et al.
The eluate was fractionated and monitored for absorbance at 254
nm. The fractions making up the major peak were checked by TLC,
pooled, and lyophilized. The residue was further subjected to two
consecutive gel filtrations on Sephadex LH-20 (100 × 1.5 cm),
eluting with aqueous AcOH (2 M and 0.2 M, respectively), with a
flow rate of 4 mL/min. The peptide was eluted in a single peak
(absorbance at 254 nm). Lyophilization of the pertinent fractions
gave the desired vasopressin analogue 8 (48.7 mg, 46.3%; Table
4). The deprotection, reoxidation, and purification of peptides 1-7
and 9-13 (Table 4) were carried out in essentially the same manner.
Cell Transfection. Mouse corticotroph At-T20 cells (ATCC,
CRL-1795) were transfected using the cDNA coding for the rat
V1b-R.53 This cDNA called rV3 was amplified by polymerase chain
reaction, inserted into the HindIII-Xbal sites of the expression vector
pcDNA3, and verified by restriction enzyme digestion and DNA
sequencing. Transfection was achieved using the Lipofectamine Plus
reagent according to the instruction of the manufacturer. Cells stably
expressing pcDNA-rV3 were selected using the geneticine added
in the cell culture medium. The clones able to bind [3H]AVP were
purified by the limited dilution techniques and screened again by
the [3H]AVP assay.
Data analysis were analyzed by GraphPad Software, Inc., Prism
(GraphPad Software, Inc., San Diego, CA), as previously de-
scribed.65 The inhibitory dissociation constants (Ki) for unlabeled
AVP analogues were calculated from binding competition experi-
ments according to the Cheng and Prusoff equation: Ki ) IC50/(1
+ [L]/Kd),101 where IC50 is the concentration of unlabeled analogue
leading to half-maximal inhibition of specific binding, [L] is the
concentration of the radioligand present in the assay, and Kd is its
affinity for the VP receptor studied. Concentrations of VP analogue
leading to half-maximal stimulation of second messenger accumula-
tions (Kact) were calculated from functional studies using GraphPad
Software, Inc., Prism. Results are expressed as the mean ( SEM
of the number of distinct experiments indicated.
Acknowledgment. The rat V1b cDNA was provided by Dr
M. Saito (Japan), to whom we are greatly indebted, as well as
F. Letourneur from the sequencing facility of the Institut Cochin.
We want to thank the NIH for its financial support (Grant
GM25280 to M.M.), the Basque Country University (Bilbao,
Spain; Grant 9/UPV-00042.310-15852/2004), and “Secretar´ıa
de Estado de Educacio´n y Universidades” (Madrid, Spain) for
the fellowship to M.T. We thank Ms. Ann Chlebowski for her
expert assistance in the preparation of this manuscript and
Mireille Passama for preparing the illustrations.
Cell Culture. Chinese hamster ovary (CHO) cells stably
expressing the rat isoforms of the vasopressin V1a, V1b, V2, and
OT receptors were cultured in DMEM supplemented with 10% of
fetal calf serum, 2 mM L-glutamine, 500 units/mL penicillin and
streptomycin, and 1× nonessential amino acids in an atmosphere
of 95% air and 5% CO2 at 37 °C, as previously described (65).
References
Mouse corticotroph At-T20 cells stably expressing the rat V1b
receptor isoform were cultured in the same conditions as the CHO
cells but in a DMEM/F12 medium supplemented with 7.5%
Fetalclone II and 7.5% Nu-serum, 0.5 mM L-glutamine and 200
µg/mL geneticine.
(1) Barberis, C.; Morin, D.; Durroux, T.; Mouillac, B.; Guillon, G.; Seyer,
R.: Hibert, M.; Tribollet, E.; Manning, M. Molecular pharmacology
of AVP and OT receptors and therapeutic potential. Drug News
Perspect. 1999, 12, 279-292.
(2) Jard, S. Vasopressin receptors. A historical survey. In AdVances in
Experimental Medicine and Biology; Zingg, H. H., Bourque, C. W.,
Bichet, D. G., Eds.; Plenum Press: New York, 1998; pp 1-13.
(3) Holmes, C. L.; Landry, D. W.; Granton, J. T. Science review:
Vasopressin and the cardiovascular system part 1sreceptor physiol-
ogy. Crit. Care 2003, 7, 427-434.
(4) Holmes, C. L.; Landry, D. W.; Granton, J. T. Science Review:
Vasopressin and the cardiovascular system part 2sclinical physiol-
ogy. Crit. Care 2004, 8, 15-23.
(5) Lee, B.; Yang, C.; Chen, T. H.; al-Azawi, N.; Hsu, W. H. Effect of
AVP and oxytocin on insulin release: involvement of V1b receptors.
Am. J. Physiol. Endocrinol. Metab. 1995, 269, E1095-E1100.
(6) Yibchok-Anun, S.; Cheng, H.; Heine, P. A.; Hsu, W. H. Character-
ization of receptors mediating AVP- and OT-induced glucagon release
from the rat pancreas. Am. J. Physiol. Endocrinol. Metab. 1999, 277,
E56-E62.
(7) Oshikawa, S.; Tanoue, A.; Koshimizu, T.; Kitagawa, Y.; Tsujimoto,
G. Vasopressin stimulates insulin release from islet cells through V1b
receptors: A combined pharmacological/knockout approach. Mol.
Pharmacol. 2004, 65, 623-629.
(8) Yibchok-anun, S.; Abu-Basha, E. A.; Yao, C. Y.; Panichkriangkrai,
W.; Hsu, W. H. The role of arginine vasopressin in diabetes-
associated increase in glucagon secretion. Regul. Pept. 2004, 122,
157-162.
(9) Guillon, G.; Trueba, M.; Joubert, D.; Grazzini, E.; Chouinard, L.;
Cote, M.; Payet, M. D.; Manzoni, O.; Barberis, C.; Robert, M.; Gallot-
Payet, N. Vasopressin stimulates steroid secretion in human adrenal
glands: Comparison with angiotensin-II effect. Endocrinology 1995,
136, 1285-1295.
(10) Manning, P. T.; Schwartz, D.; Katsube, N. C.; Holmberg, S. W.;
Needleman, P. Vasopressin-stimulated release of atriopeptin: En-
docrine antagonists in fluid homeostasis. Science 1985, 229, 395-
397.
(11) Hirsch, A. T.; Dzau, V.; Majzoub, J. A.; Creager, M. A. Vasopressin-
mediated forearm vasodilation in normal humans. Evidence for a
vascular vasopressin V2 receptor. J. Clin. InVest. 1989, 84, 418-
426.
(12) Chan, W. Y.; Levi, R.; Wo, N. C.; Koyama, M.; Stoev, S.; Cheng,
L. L.; Manning, M. Novel selective hypotensive vasopressin pep-
tides: Cardiovascular and structure-activity-relationship studies. Eur.
J. Pharmacol. 2001, 419, 65-72.
(13) Ring, R. H. The central vasopressinergic system: Examining the
opportunities for psychiatric drug development. Curr. Pharm. Des.
2005, 11, 205-225.
(14) Landgraf, R. The involvement of the vasopressin system in stress-
related disorders. CNS Neurol. Disord. Drug Targets 2006, 5, 167-
179.
Binding Experiments. CHO and At-T20 cells, stably transfected
with vasopressin or OT receptors, were used to prepare crude
plasma membrane, as previously described (65). Rat kidney (inner
medulla) and liver membrane preparations naturally expressing V2
and V1a receptor isoforms were obtained as previously described.99
Membrane binding assays were performed as previously described99
using [3H]AVP as radioligand. Membranes (5-50 µg protein) were
incubated 60 min at 30 °C (membranes from transfected cells) or
at 37 °C (membranes from native tissues) in a medium containing
50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 1 mg/mL bovine serum
albumin (BSA), and 0.01 mg/mL leupeptine. The binding properties
of the unlabeled analogues were determined by competition
experiments. Briefly, 0.5 to 3 nM of [3H]AVP was added in the
incubation medium with (nonspecific binding) or without (total
binding) 1 µM of unlabeled AVP and increasing amounts of the
unlabeled analogues to be tested or with vehicle (control).
Radioactivity found associated to plasma membranes was deter-
mined by filtration through GF/C filters. Specific binding was
calculated as the difference between total and nonspecific binding
and expressed as percent of control specific binding.
Functional Assays. Total inositol phosphate (InsP) accumulation
was determined as previously described.9 Briefly, At-T20 cells
expressing the rat vasopressin V1b receptor were cultured in 24-
well plates at 100 000 cells/well. Cells were grown for 48 h in
DMEM/F12 medium supplemented with 7.5% Fetalclone II, 7.5%
Nu-serum, and 0.5 mM L-glutamine and with 1 µCi/mL myo-[2-
3H]-inositol. Cells were then washed twice with a Hank’s buffered
saline medium (HBS), equilibrated at 37° in HBS for 30 min and
incubated for 15 min in HBS supplemented with 15 mM LiCl, 1
mg/mL glucose, 1 mg/mL BSA, and 2.1 g/L NaHCO3. Cells were
then stimulated for 15 min with increasing concentrations of the
analogues to be tested. The reaction was stopped with 5% (v/v)
perchloric acid. InsP accumulated were extracted and purified on
Dowex AG1-X8 anion exchange chromatography column and
counted.
Adenylate cyclase activity was assessed on plasma membranes
from rat kidney and was determined by measuring the conversion
of R[32P]-ATP to [32P]-cAMP, as previously described.95b,102

Agonists for the Rat Vasopressin V1b Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 845
(15) Legros, J.-J. Inhibitory effect of oxytocin on corticotrope function
in humans: Are vasopressin and oxytocin ying-yang neurohormones?
Psychoneuroendocrinology 2001, 26, 649-655.
(40) Overstreet, D. H.; Griebel, G. Antidepressant-like effects of the
vasopressin V1b receptor antagonist SSR149415 in the Flinders
Sensitive Line rat. Pharmacol. Biochem. BehaV. 2005, 82, 223-227.
(41) Windle, R. J.; Shanks, N.; Lightman, S. L.; Ingram, C. D. Central
oxytocin administration reduces stress-induced corticosterone release
and anxiety behavior in rats. Endocrinology 1997, 138, 2829-2834.
(42) Neumann, I. D.; Wigger, A.; Tomer, L.; Holsboer, F.; Landgraf, R.
Brain oxytocin inhibits basal and stress-induced activity of the
hypothalamoEnDash-pituitary-adrenal axis in male and female
rats: Partial action within the paraventricular nucleus. J. Neuroen-
docrinol. 2000, 12, 235-243.
(43) Windle, R. J.; Gamble, L. E.; Kershaw, Y. M.; Wood, S. A.;
Lightman, S. L.; Ingram, C. D. Gonadal steroid modulation of stress-
induced hypothalamo-pituitary-adrenal activity and anxiety behav-
ior: Role of central oxytocin. Endocrinology 2006, 147, 2423-2431.
(44) Ring, R. H.; Malberg, J. E.; Potestio, L.; Ping, J.; Boikess, S.; Luo,
B.; Schechter, L. E.; Rizzo, S.; Rahman, Z.; Rosenzweig-Lipson, S.
Anxiolytic-like activity of oxytocin in male mice: behavioral and
autonomic evidence, therapeutic implications. Psychopharmacology
(Berlin) 2006, 185, 218-225.
(45) Parker, K. J.; Buckmaster, C. L.; Schatzberg, A. F.; Lyons, D. M.
Intranasal oxytocin administration attenuates the ACTH stress
response in monkeys. Psychoneuroendocrinology 2005, 30, 924-
929.
(46) Kirsch, P.; Esslinger, C.; Chen, Q.; Mier, D.; Lis, S.; Siddhanti, S.;
Gruppe, H.; Mattay, V. S.; Gallhofer, B.; Meyer-Lindenberg, A.
Oxytocin modulates neural circuitry for social cognition and fear in
humans. J. Neurosci. 2005, 25, 11489-11493.
(47) Birnbaumer, M.; Selbold, A.; Gilbert, S.; Ishido, M.; Barberis, C.;
Antaramian, A.; Brabet, P.; Rosenthal, W. Molecular cloning of the
receptor for human antidiuretic hormone. Nature 1992, 357, 333-
335.
(48) de Keyzer, Y.; Auzan, C.; Lenne, F.; Beldjord, C.; Thibonnier, M.;
Bertagna, X.; Clauser, E. Cloning and characterization of the human
V3 pituitary vasopressin receptor. FEBS Lett. 1994, 356, 215-220.
(49) Gorbulev, V.; Buchner, H.; Akhundova, A.; Fahrenholz, F. Molecular
cloning and functional characterization of V2 [8-lysine] vasopressin
and oxytocin receptors from a pig kidney cell line. Eur. J. Biochem.
1993, 215, 1-7.
(50) Kimura, T.; Tanizawa, O.; Mori, K.; Brownstein, M. J.; Okayama,
H. Structure and expression of a human oxytocin receptor. Nature
1992, 356, 526-529.
(51) Morel, A.; O’Carroll, A. M.; Brownstein, M. J.; Lolait, S. J. Molecular
cloning and expression of a rat V1a arginine vasopressin receptor.
Nature 1992, 356, 523-526.
(52) Sugimoto, T.; Saito, M.; Mochizuki, S.; Watanabe, Y.; Hashimoto,
S.; Kawashima, H. Molecular cloning and functional expression of
a cDNA encoding the human V1b vasopressin receptor. J. Biol. Chem.
1994, 269, 27088-27092.
(53) Saito, M.; Sugimoto, T.; Tahara, A.; Kawashima, H. Molecular
cloning and characterization of rat V1b receptor: Evidence for its
expression in extra-pituitary tissues. Biochem. Biophys. Res. Commun.
1995, 212, 751-757.
(54) Thibonnier, M.; Auzan, C.; Madhun, Z.; Wilkins, P.; Berti-Mattera,
L.; Clauser, E. Molecular cloning, sequencing, and functional
expression of a cDNA encoding the human V1a vasopressin receptor.
J. Biol. Chem. 1994, 269, 3304-3310.
(55) Stoev, S.; Cheng, L. L.; Manning, M.; Wo, N. C.; Szeto, H. H. Design
and synthesis of potent, highly selective vasopressin hypotensive
agonists. J. Pept. Sci. 2006, 12, 592-604.
(56) du Vigneaud, V.; Ressler, C.; Swan, J. M.; Roberts, C. W.;
Katsoyannis, P. G. The synthesis of oxytocin. J. Am. Chem. Soc.
1954, 76, 3115-3121.
(57) du Vigneaud, V.; Gish, D. T.; Katsoyannis, P. G. A synthetic
preparation possessing biological properties associated with arginine-
vasopressin. J. Am. Chem. Soc. 1954, 76, 4751-4752.
(58) Manning, M.; Sawyer, W. H. Design, synthesis, and some uses of
receptor-specific agonists and antagonists of vasopressin and oxytocin.
J. Recept. Res. 1993, 13, 195-214.
(59) Chan, W. Y.; Wo, N. C.; Stoev, S.; Cheng, L. L.; Manning, M.
Discovery and design of novel and selective vasopressin and oxytocin
agonists and antagonists: The role of bioassays. Exp. Physiol. 2000,
85, 7S-18S.
(60) Hruby, V. J.; Smith, C. W. Structure-activity relationships of
neurohypophyseal peptides. In Chemistry, Biology, and Medicine of
Neurohypophyseal Hormones and Their Analogs; Smith, C. W., Ed.;
Undenfriend, S., Meienhofer, J., Eds.; Academic Press: Orlando, FL,
1987; Vol. 8 (The Peptides), pp 77-207.
(16) Insel, T. R.; Young, L. J. The neurobiology of attachment. Nat. ReV.
Neurosci. 2001, 2, 129-136.
(17) Francis, D. D.; Young, L. J.; Meaney, M. J.; Insel, T. R. Naturally
occurring differences in maternal care are associated with the
expression of oxytocin and vasopressin (V1a) receptors: gender
differences. J. Neuroendocrinol. 2002, 14, 349-353.
(18) Scott, L. V.; Dinan, T. G. Vasopressin as a target for antidepressant
development: An assessment of the available evidence. J. AffectiVe
Disord. 2002, 72, 113-124.
(19) Bielsky, I. F.; Hu, S. B.; Szegda, K. L.; Westphal, H.; Young, L. J.
Profound impairment in social recognition and reduction in anxiety-
like behavior in vasopressin V1a receptor knockout mice. Neurop-
sychopharmacology 2004, 29, 483-493.
(20) Amico, J. A.; Mantella, R. C.; Vollmer, R. R.; Li, X. Anxiety and
stress responses in female oxytocin deficient mice. J. Neuroendo-
crinol. 2004, 16, 319-324.
(21) Winslow, J. T.; Insel, T. R. Neuroendocrine basis of social recogni-
tion. Curr. Opin. Neurobiol. 2004, 14, 248-253.
(22) Dinan, T. G.; O’Brien, S.; Lavelle, E.; Scott, L. V. Further
neuroendocrine evidence of enhanced vasopressin V3 receptor
responses in melancholic depression. Psychol. Med. 2004, 34, 169-
172.
(23) Theodosis, D. T.; Schachner, M.; Neumann, I. D. Oxytocin neuron
activation in NCAM-deficient mice: Anatomical and functional
consequences. Eur. J. Neurosci. 2004, 20, 3270-3280.
(24) Landgraf, R. Neuropeptides in anxiety modulation. Handb. Exp.
Pharmacol. 2005, 335-369.
(25) Huber, D.; Veinante, P.; Stoop, R. Vasopressin and oxytocin excite
distinct neuronal populations in the central amygdala. Science 2005,
308, 245-248.
(26) Pittman, Q. J.; Spencer, S. J. Neurohypophysial peptides: Gatekeepers
in the amygdala. Trends Endocrinol. Metab. 2005, 16, 343-344.
(27) Bosch, O. J.; Meddle, S. L.; Beiderbeck, D. I.; Douglas, A. J.;
Neumann, I. D. Brain oxytocin correlates with maternal aggression:
link to anxiety. J. Neurosci. 2005, 25, 6807-6815.
(28) Takayanagi, Y.; Yoshida, M.; Bielski, I. F.; Ross, H. E.; Kawamata,
M.; Onaka, T.; Yanagisawa, T.; Kimura, T.; Matzuk, M. M.; Young,
L. J.; Nishimori, K. Pervasive social deficits, but normal parturition,
in oxytocin receptor-deficient mice. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 16096-16101.
(29) Nair, H. P.; Gutman, A. R.; Davis, M.; Young, L. J. Central oxytocin,
vasopressin, and corticotrophin-releasing factor densities in the basal
forebrain predict isolation potentiated startle in rats. J. Neurosci. 2005,
25, 11479-11488.
(30) Wismer Fries, A. B.; Ziegler, T. E.; Kurian, J. R.; Jacoris, S.; Pollak,
S. D. Early experience in humans is associated with changes in
neuropeptides critical for regulating social behavior. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 17237-17240.
(31) Reymond-Marron, I.; Raggenbass, M.; Zaninetti, M. Vasopressin
facilitates glycinergic and GABAergic synaptic transmission in
developing hypoglossal motoneurons. Eur. J. Neurosci. 2005, 21,
1601-1609.
(32) Debiec, J. Peptides of love and fear: Vasopressin and oxytocin
modulate the integration of information in the amygdala. Bioessays
2005, 27, 869-873.
(33) Ma, S.; Shipston, M. J.; Morilak, D.; Russell, J. A. Reduced
hypothalamic vasopressin secretion underlies attenuated adrenocor-
ticotropin stress responses in pregnant rats. Endocrinology 2005, 146,
1626-1637.
(34) Dinan, T. G.; Scott, L. V. Anatomy of melancholia: Focus on
hypothalamic-pituitary-adrenal axis overactivity and the role of
vasopressin. J. Anat. 2005, 207, 259-264.
(35) Kramer, K. M.; Choe, C.; Carter, C. S.; Cushing, B. S. Developmental
effects of oxytocin on neural activation and neuropeptide release in
response to social stimuli. Horm. BehaV. 2006, 49, 206-214.
(36) Pedersen, C. A.; Boccia, M. L. Vasopressin interactions with oxytocin
in the control of female sexual behavior. Neuroscience 2006, 139,
843-851.
(37) Goekoop, J. G.; de Winter, R. P.; de Rijk, R.; Zwinderman, K. H.;
Frankhuijzen-Sierevogel, A.; Wiegant, V. M. Depression with above-
normal plasma vasopressin: validation by relations with family
history of depression and mixed anxiety and retardation. Psychiatry
Res. 2006, 141, 201-211.
(38) Bosch, O. J.; Kromer, S. A.; Neumann, I. D. Prenatal stress: Opposite
effects on anxiety and hypothalamic expression of vasopressin and
corticotrophin-releasing hormone in rats selectivity bred for high and
low anxiety. Eur. J. Neurosci. 2006, 23, 541-551.
(39) Barberis, C.; Tribollet, E. Vasopressin and oxytocin receptors in the
central nervous system. Crit. ReV. Neurobiol. 1996, 10, 119-154.
(61) Lebl, M. In Handbook of Neurohypophyseal Hormone Analogs; Jost,
K., Lebl, M., Brtnik, F., Eds.; CRC Press: Boca Raton, FL, 1988;
Vol. 2, Part 1, pp 17-74; Part 2, pp 127-267.

846 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pena et al.
(62) (a) Aizawa, T.; Yasuda, N.; Greer, M. A.; Sawyer, W. H. In vitro
adrenocorticotropin-releasing activity of neurohypophyseal hormones
and their analogs. Endocrinology 1982, 110, 98-104. (b) Gaillard,
R. C.; Schoenenberg, P.; Favrod-Coune, C. A.; Muller, A. F.; Marie,
J.; Bockaert, J.; Jard, S. Properties of rat anterior pituitary vasopressin
receptors: Relation to adenylate cyclase and the effect of corticotro-
phin-releasing factors. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2907-
2911.
(63) Antoni, F. A. Novel ligand specificity of pituitary vasopressin
receptors in the rat. Neuroendocrinology 1984, 39, 186-188.
(64) Jard, S.; Gaillard, R. C.; Guillon, G.; Marle, J.; Schoenenberg, P.;
Muller, A. F.; Manning, M.; Sawyer, W. H. Vasopressin antagonists
allow demonstration of a novel type of vasopressin receptor in the
rat adenohypophysis. Mol. Pharmacol. 1986, 30, 171-177.
(65) Derick, S.; Cheng, L. L.; Voirol, M. J.; Stoev, S.; Giacomini, M.;
Wo, N. C.; Szeto, H. H.; Ben Mimoun, M.; Andres, M.; Gaillard, R.
C.; Guillon, G.; Manning, M. [1-Deamino-4-cyclohexylalanine]-
arginine vasopressin: A potent and specific agonist for vasopressin
V1b receptors. Endocrinology 2002, 143, 4655-4664.
(66) Serradeil-Le Gal, C.; Wagnon, J.; Simiand, J.; Griebel, G.; Lacour,
C.; Guillon, G.; Barberis, C.; Brossard, G.; Soubrie, P.; Nisato, D.;
Pascal, M.; Pruss, R.; Scatton, B.; Maffrand, J. P.; Le Fur, G.
Characterization of (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)-
sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-
hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), a
selective and orally active vasopressin V1b receptor antagonist. J.
Pharmacol. Exp. Ther. 2002, 300, 1122-1130.
(67) Cheng, L. L.; Stoev, S.; Manning, M.; Derick, S.; Pena, A.; Ben
Mimoun, M.; Guillon, G. Design of potent and selective agonists
for the human vasopressin V1b receptor based on modifications of
[deamino-cys1]arginine vasopressin at position 4. J. Med. Chem. 2004,
47, 2375-2388.
(68) Guillon, G.; Pena, A.; Murat, B.; Derick, S.; Trueba, M.; Ventura,
M. A.; Szeto, H. H.; Wo, N.; Stoev, S.; Cheng, L. L.; Manning, M.
Position 4 analogues of [deamino-Cys(1)]arginine vasopressin exhibit
striking species differences for human and rat V(2)/V(1b) receptor
selectivity. J. Pept. Sci. 2006, 3, 190-198.
(69) Griffante, C.; Green, A.; Curcuruto, O.; Haslam, C. P.; Dickinson,
B. A.; Arban, R. Selectivity of d[Cha4]AVP and SSR149415 at human
vasopressin and oxytocin receptors: evidence that SSR149415 is a
mixed vasopressin V1b/oxytocin receptor antagonist. Br. J. Pharma-
col. 2005, 146, 744-751.
(70) (a) Andres, M.; Pena, A.; Derick, S.; Raufaste, D.; Trojnar, J.;
Wisniewski, K.; Trueba, M.; Serradeil-Le-Gal, C.; Guillon, G.
Comparative pharmacology of bovine, human and rat vasopressin
receptor isoforms. Eur. J. Pharmacol. 2004, 501, 59-69. (b) Derick,
S.; Pena, A.; Durroux, T.; Wagnon, J.; Serradeil-Le Gal, C.; Hibert,
M.; Rognan, D.; Guillon, G. Key amino acids located within the
transmembrane domains 5 and 7 account for the pharmacological
specificity of the human V1b vasopressin receptor. Mol. Endocrinol.
2004, 18, 2777-2789. (c) Volpi, S.; Liu, Y.; Aguilera, G. Vasopressin
increases GAGA binding activity to the V1b receptor promoter
through transactivation of the MAP kinase pathway. J. Mol. Endo-
crinol. 2006, 36, 581-590. (d) Rodrigo, J.; Pena, A.; Murat, B.;
Trueba, M.; Durroux, T.; Guillon, G.; Rognan, D. Mapping the
Binding Site of Arginine-Vasopressin to V1a and V1b Vasopressin
Receptors. Mol. Endocrinol. 2006, epublication No. 12.
(71) Griebel, G.; Simiand, J.; Serradeil-Le Gal, C.; Wagnon, J.; Pascal,
M.; Scattton, B.; Maffrand, J. P.; Soubrie, P. Anxiolytic- and
antidepressant-like effects of the nonpeptide vasopressin V1b receptor
antagonist, SSR149415, suggest an innovative approach for the
treatment of stress-related disorders. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 6370-6375.
.
(75) Zaoral, M.; Brtnik, F.; Barth, T.; Machova, A. [1-â-mercaptopropionic
acid, 8-R,γ-diaminobutyric acid]vasopressin and [1-â-mercaptopro-
pionic acid, 8-D-ornitine]-vasopressin, synthesis and biological effects.
Collect. Czech. Chem. Commun. 1978, 41, 2088-2095.
(76) Krchnak, V.; Zaoral, M.; Machova, A. [1-â-mercaptopropionic acid,
8-R,â-diaminopropionic acid]vasopressin and [1-â-mercaptopropionic
acid, 8-D-R,â-diaminopropionic acid]vasopressin, two lysine-vaso-
pressin analogs with considerable antidiuteric effect. Collect. Czech.
Chem. Commun. 1979, 44, 2161-2164.
(77) Sawyer, W. H. Biologic assays for oxytocin and vasopressin. Methods
Med. Res. 1961, 9, 210-219.
(78) Dekanski, J. The quantitative assay of vasopressin. Br. J. Pharmacol.
1952, 7, 567-572.
(79) Munsick, R. A. Effect of magnesium ion on the response of the rat
uterus to neurohypophysial hormones and analogues. Endocrinology
1960, 66, 451-457.
(80) Holton, P. A. A modification of the method of Dale and Laidlaw for
standardization of posterior pituitary extract. Br. J. Pharmacol. 1948,
3, 328-334.
(81) Dyckes, D. F.; Ferger, M. F.; du Vigneaud, V. Synthesis and some
of the pharmacological properties of [4-leucine]-8-lysine-vasopressin
and [1-deamino, 4-leucine]-8-lysine-vasopressin. J. Med. Chem. 1973,
16, 843-847.
(82) Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of
a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149-2154.
(83) Merrifield, R. B. Solid phase synthesis. Science 1986, 232, 341-
347.
(84) Stewart, J. M.; Young, J. D. Solid phase synthesis; Pierce Chemical
Company: Rockford, IL, 1984.
(85) Manning, M. Synthesis by the Merrifield method of a protected
nonapeptide amide with the amino acid sequence of oxytocin. J. Am.
Chem. Soc. 1968, 90, 1348-1349.
(86) Manning, M., Balaspiri, L.; Moehring, J.; Haldar, J.; Sawyer, W. H.
Synthesis and some pharmacological properties of deamino [4-threo-
nine, 8-D-arginine]vasopressin and deamino [8-D-arginine]vasopressin,
highly potent and specific antidiuretic peptides, and [8-D-arginine]-
vasopressin and deamino-arginine-vasopressin. J. Med. Chem. 1976,
19, 842-845.
(87) Manning, M.; Coy, E. J.; Sawyer, W. H.; Acosta, M. Solid-phase
synthesis and some pharmacological properties of 4-threonine analogs
of vasopressins and vasotocin and of arginine-vasopressin and
arginine-vasotocin. J. Med. Chem. 1973, 16, 463-466.
(88) Konig, W.; Geiger, R. New method for synthesis of peptides:
activation of the carboxyl group with dicyclohexylcarbodiimide using
1-hydroxybenzotriazoles as additives. Chem. Ber. 1970, 103, 788-
798.
(89) Bodanszky, M.; du Vigneaud, V. A method of synthesis of long
peptide chains using a synthesis of oxytocin as an example. J. Am.
Chem. Soc. 1959, 81, 5688-5691.
(90) Bodanszky, M.; Sheehan, J. T. Active esters and resins in peptide
synthesis. Chem. Ind. 1964, 1423-1424.
(91) Hope, D. B.; Murti, V. V.; du Vigneaud, V. A highly potent analogue
of oxytocin, desamino-oxytocin. J. Biol. Chem. 1962, 237, 1563-
1566.
(92) Rivier, J.; Keiser, R.; Galyean, R. Solid phase synthesis of soma-
tostatin and glucagon-selective analogs in gram quantities. Biopoly-
mers 1978, 17, 1927-1938.
(93) Manning, M.; Wuu, T. C.; Baxter, J. W. M. The purification of
synthetic oxytocin and analogues by gel filtration on Sephadex G-15.
J. Chromatogr. 1968, 38, 396-398.
(94) Manning, M.; Cheng, L. L.; Stoev, S. B.; Wo, N.; Szeto, H. H.; Pena,
A.; Murat, B.; Trueba, M.; Ventura, M. A.; Guillon, G. A Surprise
End to 20 Year Search for Selective Agonists for Rat Vasopressin
V
1b Receptor. In Understanding Biology Using Peptides, Proceedings
(72) Huguenin, R. L.; Boissonnas, R. A. Synthese de la [desamino1-Arg8]-
vasopressine et de la [desamino1-Phe2-Arg8]-vasopressine, deux
analogues possedant une activite antidiuretique plus elevee et plus
selective que celle des vasopressines naturelles [Synthesis of the
[deamino1-Arg8]-vasopressin and the [deamino1-Phe2-Arg8]-vaso-
pressin, two analogues possessing an antidiuretic activity more
elevated and selective than that of the natural vasopressins). HelV.
Chim. Acta 1966, 49, 695-705.
(73) Kimbrough, R. D., Jr.; Cash, W. D.; Branda, L. A.; Chan, W. Y.; du
Vigneaud, V. Synthesis and biological properties of 1-desamino-8-
lysine-vasopressin. J. Biol. Chem. 1963, 238, 1311-1414.
(74) Huguenin, R. L. Synthese de la desamino1-Orn8-vasopressine, de la
desamino1-Phe2-Orn8-vasopressine, de la desamino1-Ile3-Orn8-vaso-
pressine (-desamino1-Orn8-oxytocine) et de la desamino1-Phe2-Ile3-
Orn8-vasopressine (-desamino1-Phe2-Orn8-oxytocine). HelV. Chim.
Acta 1966, 49, 711-755
of the 19th American Peptide Society Symposium, San Diego, CA,
2005; Blondelle, S. E., Ed.; Springer: New Jersey, 2005.
(95) (a) Zaoral, M.; Kolc, J.; Sorm, F. Amino acids and peptides. LXXI.
Synthesis of 1-deamino-8-D-γ-aminobutyrine-vasopressin, 1-deamino-
8-D-lysine-vasopressin and 1-deamino-8-D-arginine-vasopressin. Col-
lect. Czech. Chem. Commun. 1967, 32, 1250-1257. (b) Bulten, B.;
Guillon, G.; Rajerison, R. M.; Jard, S.; Sawyer, W. H.; Manning,
M. Structural requirements for activation of vasopressin-sensitive
adenylate cyclase, hormone binding, and antidiuretic actions: effects
of highly potent analogues and competitive inhibitors. Mol. Phar-
macol. 1978, 14, 1006-1017.
(96) (a) Sawyer, W. H.; Acosta, M.; Balaspiri, L.; Judd, J.; Manning, M.
Structural changes in the arginine vasopressin molecule that enhance
antidiuretic activity and specificity. Endocrinology 1974, 94, 1106-
1115. (b) Cantau, B.; Keppens, S.; De, Wulf, H.; Jard, S. (3H)-
vasopressin binding to isolated rat hepatocytes and liver mem

Agonists for the Rat Vasopressin V1b Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 847
branes: regulation by GTP and relation to glycogen phosphorylase
activation. J. Recept. Res. 1980, 1, 137-168.
(100) Bradford, M. M. A. rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein-
dye binding. Anal. Biochem. 1976, 72, 248-254.
(101) Cheng, Y.; Prusoff, W. H. Relation between the inhibition constant
(Ki) and the concentration of inhibitor which causes 50 percent
inhibition (IC50) of an enzymatic reaction. Biochem. Pharmacol. 1973,
22, 3099-3108.
(102) Salomon, Y.; Londos, C.; Rodbell, M. A highly-sensitive adenylate
cyclase assay. Anal. Biochem. 1974, 58, 541-548.
(97) Schwartz, J.; Derdowska, I.; Sobocinska, M.; Kupryszewski, G. A
potent new synthetic analog of vasopressin with relative agonist
specificity for the pituitary. Endocrinology 1991, 129, 1107-1109.
(98) Saito, M.; Tahara, A.; Sugimoto, T. 1-Desamino-8-D-arginine vaso-
pressin (dDAVP) as an agonist on V1b vasopressin receptor. Biochem.
Pharmacol. 1997, 53, 1711-1717.
(99) Andres, M.; Trueba, M.; Guillon, G. Pharmacological characterization
of F-180: A selective human V(1a) vasopressin receptor agonist of
high affinity. Br. J. Pharmacol. 2002, 135, 1828-1836.
JM060928N