New, potent, selective, and short-acting peptidic V1a receptor agonists

Article abstract of DOI:10.1021/jm200278m

[Arg⁸]vasopressin (AVP) produces vasoconstriction via V _1a receptor (V_1aR)-mediated vascular smooth muscle cell contraction and is being used to increase blood pressure in septic shock, a form of vasodilatory hypotension.

Full text of DOI:10.1021/jm200278m

ARTICLE
pubs.acs.org/jmc
New, Potent, Selective, and Short-Acting Peptidic V1a
Receptor Agonists
Kazimierz Wiꢀsniewski,* Robert Galyean, Hiroe Tariga, Sudarkodi Alagarsamy, Glenn Croston,
Joshua Heitzmann, Arash Kohan, Halina Wiꢀsniewska, Rꢀegent Laporte, Pierre J-M. Riviꢁere, and
Claudio D. Schteingart
Ferring Research Institute Inc., 4245 Sorrento Valley Boulevard, San Diego, California 92121, United States
S Supporting Information
b
ABSTRACT: [Arg8]vasopressin (AVP) produces vasoconstriction via
V1a receptor (V1aR)-mediated vascular smooth muscle cell contraction
and is being used to increase blood pressure in septic shock, a form of
vasodilatory hypotension. However, AVP also induces V2 receptor
(V2R)-mediated antidiuresis, vasodilation, and coagulation factor
release, all deleterious in septic shock. The V1aR agonist terlipressin
(H-Gly3[Lys8]VP) also lacks selectivity vs the V2R and has sizably
longer duration of action than AVP, preventing rapid titration of its
vasopressor effect in the clinic. We designed and synthesized new short
acting V1aR selective analogues of general structure [Xaa2,Ile3,Yaa4,
Zaa8]VP. The most potent and selective compounds in in vitro functional assays (e.g., [Phe2,Ile3,Asn(Me2)4,Orn8]VP (31), [Phe2,
Ile3,Asn((CH2)3OH)4,Orn8]VP (34), [Phe2,Ile3,Hgn4,Orn(iPr)8]VP (45), [Phe2,Ile3,Asn(Et)4,Dab8]VP (49), [Thi2,Ile3,Orn-
(iPr)8]VP (59), [Cha2,Ile3,Asn4,Orn(iPr)8]VP (68)) were tested by intravenous bolus in rats for duration of vasopressive action.
Analogues 31, 34, 45, and 49 were as short-acting as AVP. Compound 45, FE 202158, is currently undergoing clinical trials in
septic shock.
’ INTRODUCTION
Recently, the first large-scale multicenter study, the Vasopressin
in Septic Shock Trial (VASST), has suggested that such therapy
may reduce mortality in less severe septic shock.10
Arginine vasopressin, 1 (AVP), is the endogenous ligand for
the three known subtypes of vasopressin receptors, V1aR, V1bR
(V3R), and V2R, and also acts on the oxytocin receptor (OTR) to
regulate a variety of physiological processes both centrally and
peripherally (Figure 1).1ꢀ4 1 is involved in the continuous main-
tenance of plasma osmolality, and in this role it is also known as
the antidiuretic hormone (ADH). In response to elevations of
plasma osmolality above a physiological set point, 1 is secreted
from the posterior pituitary gland and activates renal V2R to
promote water conservation. 1 also controls the secretion of
adrenocorticotropic hormone (ACTH), mediated by both per-
ipherally and centrally located V1bR, and plays an important role
in stress/anxiety/depression management by activation of cen-
trally located V1bR.4 It has been recently suggested that 1
stimulated release of insulin from Islet cells is mediated by the
pancreatic V1bR.5 In sitof severe hypotension, 1 is acutely
secreted by the posterior pituitary to produce arterial vasocon-
striction by activation of the V1a vasopressin receptor subtype
(V1aR) located in vascular smooth muscle cells, with consequent
increase in blood pressure.6,7
Terlipressin (H-Gly3-LVP), a synthetic analogue of lysine
vasopressin (LVP), believed to behave as a prodrug of LVP, is
approved in some European and Asian countries for the treat-
ment of bleeding esophageal varices and in France for hepator-
enal syndrome.11 This analogue was shown to be effective in
raising mean arterial pressure in septic shock patients after high-
dose intravenous bolus administration,12 which was confirmed
by multiple exploratory clinical trials.13,14
The lack of V1aR selectivity of 1 could possibly limit its use in
vasodilatory hypotension states such as septic shock due to V2R-
mediated unwanted side effects such as vasodilation and coagula-
tion factor release.15 Likewise, the use of terlipressin in such critical
care conditions may be disadvantageous due to the long duration
of vasopressive action,16 which would prevent the desirable rapid
titration of effect to optimize safety and clinical efficacy. Further-
more, the lack of selectivity vs the V2R of its putative active metabolite
LVP16 could also lead to the same V2R-mediated adverse effects.
There has been substantial progress in discovering selective small-
molecule agonists and antagonists for the family of vasopressin/
oxytocin receptors with different degrees of receptor selectivity.4,17,18
AVP deficiency has been observed in patients with septic
shock, a form of vasodilatory hypotension, and low-dose con-
tinuous intravenous infusion of 1 effectively increased blood
pressure in these patients.7,8 Numerous small pilot studies us-
ing such an “add-back” AVP therapy confirmed these results.9
Received: December 22, 2010
Published: June 20, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/jm200278m J. Med. Chem. 2011, 54, 4388–4398
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structures of 1 (AVP), terlipressin, and 2 (F-180).
Table 1. Structure and in Vitro Pharmacological Profile of Reference Compounds 1, 3ꢀ7
in vitro pharmacological profile
EC50 receptor [nM] (pEC50 ( SEM)
selectivity vs receptora
analogue
structure
AVP19
[Orn8]VP, OVP28
[Phe2]AVP27
[Phe2]OVP22
[Ile3]OVP28
hV1a
hV2
hV1b
hOT
hV2
0.20
hV1b
hOT
62
1
3
4
5
6
7
0.24 (9.63 ( 0.02)
0.69 (9.16 ( 0.17)
0.15 (9.81 ( 0.18)
0.14 (9.85 ( 0.06)
0.23 (9.63 ( 0.08)
0.27 (9.57 ( 0.16)
0.05 (10.3 ( 0.04)
0.45 (9.35 ( 0.19)
1.4 (8.85 ( 0.15)
9.2 (8.04 ( 0.08)
4.1 (8.39 ( 0.22)
155 (6.81 ( 0.10)
4.3 (8.37 ( 0.02)
7.5 (8.12 ( 0.13)
16 (7.80 ( 0.04)
15 (7.82 ( 0.20)
56 (7.25 ( 0.19)
25 (7.60 ( 0.16)
15 (7.82 ( 0.07)
71 (7.15 ( 0.07)
>10000b (<5.00)
>10000b (<5.00)
3.3 (8.48 ( 0.06)
>10000b (<5.00)
17
10
0.65
9.3
100
>66000
>71000
14
100
100
240
92
65
17
[Phe2,Ile3]OVP22
570
>37000
a EC50 (receptor)/EC50 (V1aR) ratio. If no significant agonism was observed at the highest concentration tested, selectivity is >highest conc tested/EC50
(hV1aR). b No significant agonism at the highest concentration tested: 10000 nM.
However, for the treatment of patients with vasodilatory hypo-
tension in a hospital setting, a peptide-based drug appears as the
most suitable approach to provide very high selectivity for the
V1aR coupled with short half-life in vivo. This is expected to allow
fast and accurate up and down titration of the infusion rate of the
compound to regulate its vasopressor effect in response to the
changing demands of critically ill patients, without off target effects
due to activation of the V2, V1b, or OT receptors in the periphery
or at these and the V1aR in the CNS.
The structure of 1 was elucidated by du Vigneaud in the early
1950s,19 and it was shown to be a nonapeptide consisting of a
20-membered ring, closed by a disulfide bridge between the Cys1
and Cys6 residues, and a C-terminal tripeptide amide. The ring is
essential for preserving agonistic activity of AVP analogues at the
V1aR, but its presence is not required for binding to the V1aR, as
potent linear peptidic V1aR and mixed V1aR/V2R antagonists
were discovered by Manning’s group.20 Interestingly, the same
group reported linear mixed V1aR antagonists/V2R agonists with
low/moderate antidiuretic potency in a rat in vivo model.21
Analogues of 1 showing improved V1aR selectivity were first
described by Huguenin: replacements of the Arg8 residue with
Orn and of the Tyr2 residue with Phe resulted in analogues showing
reduced antidiuretic activity in rat.22 A vasopressin analogue
[Hmp1,Phe2,Ile3,Hgn4,Dab(Abu)8]VP, 2 (F-180), was discovered
by Ferring scientists and found to be a long-acting vasopressor
in vivo23 and very selective for the human V1aR in vitro.24
We report here the synthesis and pharmacological evaluation
of a series of analogues of 1 modified in positions 2, 3, 4, and/or 8
designed to provide highly V1aR selective and short-acting pep-
tides for the treatment of vasodilatory hypotension states.
’ RESULTS AND DISCUSSION
1 and its analogues 3ꢀ7 were synthesized in our laboratory as
reference compounds for this study and tested in vitro in reporter
gene assays (RGA) at the hV1a and related receptors (Table 1).
In these assays, 1 turned out to be a potent and fully efficacious
agonist at all receptors with the lowest potency (EC50 = 15 nM,
selectivity ratio = 62) at the hOTR. It is noteworthy that 1 has
been reported a partial agonist in other in vitro systems (e.g.,
Wootten et al. demonstrated partial agonism of 1 at the OTR in
an inositol triphosphate accumulation assay).25,26 The improved
V1aR selectivity of analogues 3ꢀ7 as compared to 1 in a rat in vivo
model was reported earlier by Huguenin.22,27,28 This enhanced
selectivity was indeed confirmed in our in vitro assays, suggesting
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Table 2. Structures and in Vitro Pharmacological Profile of Compounds 8ꢀ44
in vitro pharmacological profile
EC50 receptor (nM) (pEC50 ( SEM)
hV2 hV1b
structurea
Yaa
selectivity vs receptorb
analogue
Xaa
Zaa
Dab
hV1a
hOT
hV2
140
hV1b
52
hOT
18
8
Phe
His
Gln
Gln
Gln
0.50
73
26
9.1c
(9.31 ( 0.10)
510c
(7.14 ( 0.04) (7.58 ( 0.20)
(8.04 ( 0.04)
>10000d
(<5.00)
9
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Dab
Orn
Orn
Orn
Orn
Orn
Orn
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
2500c
>10000d
(<5.00)
200
>19 >19
>19
>330
naf
(6.29 ( 0.16)
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
3-Pal
>10000d
>330
naf
6.6
(7.53 ( 0.12)
>1000e
(6.71 ( 0.20)
360
(<5.00)
Ala(BzThi) Gln
Ala(Thz) Gln
>10000d
(<5.00)
1200c
naf
22
(<6.00)
(6.44 ( 0.71)
430
19
130
63
(7.73 ( 0.21)
0.54
(5.60 ( 0.07) (6.36 ( 0.27)
120 150
(6.94 ( 0.05) (6.82 ( 0.65)
200
50c
(6.69 ( 0.14) (7.30 ( 0.56)
330 210
(6.48 ( 0.09) (6.68 ( 0.36)
(5.91 ( 0.30)
29 c
Thi
Gln
Gln
220 270
53
(9.27 ( 0.01)
0.61
(7.53 ( 0.09)
30c
3-Thi
320
150
naf
81
95
naf
49
(9.21 ( 0.04)
2.2
(7.53 ( 0.04)
105
Ala(2-Fur) Gln
Ala Gln
Ala(tBu) Gln
Leu Gln
Ala(cPe) Gln
47
(8.65 ( 0.18)
>1000e
(6.98 ( 0.03)
>10000d
(<5.00)
>10000d
(<5.00)
470
>10000d
(<5.00)
44
naf
(<6.00)
11
>10000d
42
4.0
>900
>760
>12000
>40000
190
(7.97 ( 0.09)
13
(6.33 ( 0.08) (7.36 ( 0.07)
1200 250
(5.91 ( 0.03) (6.60 ( 0.47)
1200 20
(5.93 ( 0.07) (7.71 ( 0.21)
760 8.0
(6.12 ( 0.10) (8.10 ( 0.17)
380 64
(6.42 ( 0.08) (7.20 ( 0.15)
210 59
(6.68 ( 0.09) (7.23 ( 0.21)
2600c
97
(5.58 ( 0.05) (7.01 ( 0.10)
3000c
150
(5.52 ( 0.12) (6.83 ( 0.33)
1700 28
(5.77 ( 0.03) (7.55 ( 0.04)
(<5.00)
>10000d
(<5.00)
>10000d
92
19
25
32
68
(7.90 ( 0.28)
0.80
1500
3000
400
35
(9.10 ( 0.13)
0.25
(<5.00)
Cha
Ac4cg
Phe
Phe
Phe
Phe
Phe
Phe
Gln
>10000d
(<5.00)
180c
(9.60 ( 0.23)
0.94
Gln
(9.03 ( 0.15)
6.0
(6.76 ( 0.20)
>10000d
(<5.00)
Gln(Me)
Gln(Et)
Gln(iBu)
9.8
>1600
>250
>470
>9000
>140
>830
(8.22 ( 0.20)
39
>10000d
66
2.4
7.1
(7.41 ( 0.33)
21
(<5.00)
>10000d
(<5.00)
>10000d
140
1500
17
(7.68 ( 0.22)
1.1
Gln(OMe)
Gln(Me,OMe)
Gln(OH)
25
(8.94 ( 0.39)
69
(<5.00)
1200
540 (6.27 ( 0.17) >10000d (<5.00)
7.8
5.8
(7.16 ( 0.07)
12
(5.91 ( 0.05)
170
70
>10000d
(<5.00)
14
(7.93 ( 0.14)
(6.78 ( 0.10) (7.15 ( 0.59)
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ARTICLE
Table 2. Continued
in vitro pharmacological profile
EC50 receptor (nM) (pEC50 ( SEM)
structurea
selectivity vs receptorb
analogue
Xaa
Yaa
Zaa
Orn
hV1a
hV2
hV1b
hOT
>10000d
hV2
hV1b
20
hOT
28
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asn
1.4
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
2600
29
>7100
>7100
(8.84 ( 0.08)
3.6
(7.54 ( 0.34)
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
33
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Asn(Me)
Asn(Et)
Asn(Me2)
Asn(OH)
Orn
Orn
Orn
Orn
Orn
Orn
Orn
39
>2700
>1700
>5800
630
10
>2700
>1700
>5800
>2400
>1200
>4700
>620
>1900
>2000
>11000
>330
86
(8.45 ( 0.16)
5.7
(7.41 ( 0.13)
30
5.2
36
(8.24 ( 0.22)
1.7
(7.53 ( 0.05)
62
(8.78 ( 0.19)
4.1
(7.20 ( 0.14)
56
13
(8.39 ( 0.09)
8.0
(5.58 ( 0.05) (7.25 ( 0.25)
>10000d
Asn(CH2)2OH)
Asn(CH2)3OH)
Asn(CH(CH2OH)2)
26
>1200
>4700
>620
>1900
>2000
1700
3.2
9.0
3.8
5.3
8.5
51
(8.10 ( 0.18)
2.1 (8.68 ( 0.14) >10000d
(<5.00)
(7.59 ( 0.15)
19
(<5.00)
(7.71 ( 0.15)
16c
>10000d
(<5.00)
>10000d
(<5.00)
>10000d
(<5.00)
1600c
62c
(7.79 ( 0.09)
5.2
(7.21 ( 0.22)
Asn(CH2CH(OH)CH3) Orn
Asn((CH2)2O(CH2)2OH) Orn
28
(8.29 ( 0.16)
4.8
(7.56 ( 0.32)
41
(8.32 ( 0.08)
0.90c
(7.38 ( 0.24)
Hgn
Gln
Gln
Gln
Gln
Gln
Gln
Orn
Dap
46
(9.05 ( 0.12)
30
(5.78 ( 0.23) (7.34 ( 0.15)
720 210
(6.14 ( 0.08) (6.68 ( 0.17)
290c
44
(6.55 ( 0.11) (7.35 ( 0.12)
130 74
(6.88 ( 0.21) (7.13 ( 0.44)
140 38
(6.86 ( 0.15) (7.42 ( 0.20)
260c 32c
(6.59 ( 1.02) (7.50 ( 0.16)
190 100
(6.73 ( 1.23) (7.00 ( 0.23)
24
7.0
(7.53 ( 0.16)
Orn(iPr) 0.38c
760 110
(9.42 ( 0.12)
Orn(Me) 0.26
(9.58 ( 0.11)
Orn(Pr) 0.21
(9.68 ( 0.12)
Orn(Et) 0.57
(9.24 ( 0.20)
Orn(iAm) 0.82
(9.09 ( 0.33)
(7.48 ( 0.06)
16c
500 280
660 180
61
(7.81 ( 0.07)
32
150
(7.49 ( 0.10)
32
450
56
56
(7.50 ( 0.17)
44
230 120
53
(7.36 ( 0.13)
a See the list of abbreviations for unnatural amino acid residues definitions. b EC50 (receptor)/EC50 (hV1aR) ratio. If no significant agonism was obser-
ved at the highest concentration tested selectivity is >highest conc. tested/EC50 (hV1aR). c Partial agonist, efficacy <70%. d No significant agonism at the
highest concentration tested: 10000 nM. e No significant agonism at the highest concentration tested; 1000 nM. Nonapplicable, EC50 at the hV1aR not
f
calculated due to low efficacy. g In the case of Ac4c R1 = R2 = ꢀ(CH2)3ꢀ, in all other cases R2 is H.
positions 2, 3, and 8 of the AVP molecule are important to attain
V1aR specificity. The presence of the N-terminal amino group
appears to be beneficial for V1aR selectivity, as the desamino
analogue dDAVP is a V2R selective compound29 with some
residual V1b agonistic activity.30 In addition, the replacement of
the Gln4 residue with homoglutamine in F-180 confers enhanced
V1aR selectivity.23 Therefore, we decided to explore positions 2,
3, 4, and/or 8 for designing new, selective and short-acting V1aR
agonists. A total of 63 new compounds (8ꢀ70) were prepared by
either Fmoc or Boc strategy. The peptides with alkylated side
chains in position 8 were assembled by Fmoc chemistry. The dia-
mino acid residue in position 8 was introduced with the N-δ-Mmt
(methoxytrityl) protecting group.31 The Mmt group was re-
moved and the N-ω-isopropyl analogues were synthesized by
reductive alkylation of the resin bound peptides with acetone/
NaBH(OAc)3. To prepare peptides with straight N-alkyl groups,
the side chain amino function was derivatized with o-NBS-Cl
(2-nitrobenzenesulfonyl chloride)32 and the resulting resin-
bound sulfonamide was alkylated with an appropriate alcohol
under Mitsunobu reaction conditions.33 The peptides N-alky-
lated in position 4 were assembled by Boc chemistry, and the
position 4 residue was introduced in the sequence as Boc-
Asp(OFm)-OH or Boc-Glu(OFm)-OH.34,35 After the complete
assembly of the peptide, the side chain protection was removed
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Table 3. Structures and in Vitro Pharmacological Profile of Compounds 45ꢀ70
in vitro pharmacological profile
EC50 receptor (nM) (pEC50 ( SEM)
hV2 hV1b
structurea
Yaa
selectivity vs receptorb
analogue
Xaa
Phe
Zaa
hV1a
hOT
hV2
hV1b
hOT
450
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
Hgn
Hgn
Asn
Orn(iPr) 2.4 (8.62 ( 0.07) 2700c (5.58 ( 0.05) 340 (6.47 ( 0.16) 1100c (8.62 ( 0.07)
Dab(iPr) 18 (7.74 ( 0.24) 1100 (5.95 ( 0.09) 960 (6.02 ( 0.25) >10000d (<5.00)
1100 140
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
61
>3400
1300
590
53
12
>550
>3400
>2500
>3700
78
Dab
2.9 (8.54 ( 0.23) >10000d (<5.00)
37 (7.43 ( 0.29)
>10000d (<5.00)
>10000d (<5.00)
>10000d (<5.00)
Asn(Me) Dab
Asn(Et) Dab
3.9 (8.41 ( 0.13) 5300c (5.28 ( 0.10) 8.7 (8.06 ( 0.43)
2.7 (8.57 ( 0.10) 1600c (5.79 ( 0.05) 21 (7.68 ( 0.17)
2.2
7.7
Asn
Asn
Asn
Asn
Orn(iPr) 1.4 (8.87 ( 0.10) 3700c (5.43 ( 0.11) 250 (6.60 ( 0.16) 110c (6.98 ( 0.12)
2600 170
>4100 27
>10000 120
>6600 20
360 210
Orn(Me) 2.4 (8.62 ( 0.30) >10000d (<5.00)
Orn(Et) 0.99 (9.00 ( 0.13) >10000d (<5.00)
Orn(Pr) 1.5 (8.82 ( 0.40) >10000d (<5.00)
Orn(iPr) 2.6 (8.58 ( 0.22) 950 (6.02 ( 0.10)
65 (7.19 ( 0.01)
>10000d (<5.00)
>4100
>10000
>6600
100
120 (6.93 ( 0.04) >10000d (<5.00)
31c (7.51 ( 0.19) >10000d (<5.00)
570 (6.24 ( 0.06) 260 (6.59 ( 0.07)
530 (6.28 ( 0.28) >10000d (<5.00)
1800 (5.75 ( 0.19) 970 (6.01 ( 0.09)
460 (6.34 ( 0.16) 780c (6.11 ( 0.12)
220 (6.65 ( 0.26) >10000d (<5.00)
Ala(2-Fur) Gln
Ala(2-Fur) Asn
Ala(2-Fur) Asn
Ala(2-Fur) Asn
Orn
10 (8.00 ( 0.24) >10000d (<5.00)
>1000
53
>1000
64
Orn(iPr) 15 (7.81 ( 0.42) >10000d (<5.00)
>660 120
Dab
14 (7.84 ( 0.24) >10000d (<5.00)
15 (7.84 ( 0.28) >10000d (<5.00)
>710
>660
32
14
55
Thi
Asn
Gln
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Orn
>660
150
Thi
Orn(iPr) 0.69 (9.16 ( 0.15) 390c (6.41 ( 0.20) 370 (6.44 ( 0.24) 110 (6.96 ( 0.06)
560 530
Thi
Dab
Orn(iPr) 14 (7.86 ( 0.18) >10000d (<5.00)
12 (7.94 ( 0.13) >10000d (<5.00)
510 (6.29 ( 0.52) >10000d (<5.00)
260 (6.58 ( 0.09) 860 (6.06 ( 0.04)
350 (6.46 ( 0.26) >10000d (<5.00)
380 (6.42 ( 0.44) >10000d (<5.00)
110 (6.98 ( 0.11) >10000d (<5.00)
120 (6.93 ( 0.21) >10000d (<5.00)
120 (6.92 ( 0.17) >10000d (<5.00)
>830
42
18
47
51
13
10
15
>830
61
Thi
>710
>1300
>1300
>1200
>830
3-Thi
3-Thi
Ac4ce
Ac4ce
Ala(cPe)
Ala(cPe)
Cha
Orn
Dab
Orn
Dab
Orn
Dab
7.3 (8.14 ( 0.11) >10000d (<5.00)
7.4 (8.13 ( 0.12) >10000d (<5.00)
7.9 (8.10 ( 0.11) >10000d (<5.00)
12 (7.91 ( 0.11) >10000d (<5.00)
7.7 (8.11 ( 0.23) >10000d (<5.00)
11 (7.98 ( 0.25) >10000d (<5.00)
>1300
>1300
>1200
>830
>1200
>900
>7100
>3100
>6200
>1200
>900
76 (7.12 ( 0.17)
>10000d (<5.00)
6.9
Orn(iPr) 1.4 (8.85 ( 0.14) 970 (6.01 ( 0.04)
110 (6.96 ( 0.11) >10000d (<5.00)
140 (6.84 ( 0.30) >10000d (<5.00)
690
78
43
11
Cha
Orn
3.2 (8.49 ( 0.57) >10000d (<5.00)
1.6 (8.80 ( 0.16) >10000d (<5.00)
>3100
Cha
Dab
18 (7.75 ( 0.05)
>10000d (<5.00)
>6200
a See Table 2 for general structure and the list of abbreviations for unnatural amino acid residues definitions. b EC50 (receptor)/EC50 (hV1aR) ratio. If no
significant agonism was observed at the highest concentration tested selectivity is >highest conc tested/EC50 (hV1aR). c Partial agonist, efficacy <70%.
d No significant agonism at the highest concentration tested: 10000 nM. e In the case of Ac4c R1 = R2 = ꢀ(CH2)3ꢀ, in all other cases R2 is H.
Figure 2. Agonist activity of 1 (AVP) and compound 45 in reporter gene assays at the hV1aR and related receptors.
with 30% piperidine/DMF. The resulting free carboxylic group
was converted to the desired amide by coupling with an appro-
priate amine mediated by PyBOP or BOP/DIPEA. The struc-
tures and the results of in vitro evaluation of the new analogues
8ꢀ70 are reported in Tables 2 and 3. Compounds for which a full
dose response curve could be completed were found to have
efficacy greater than 70% at the hV1aR and rV1aR by comparison
with the maximum efficacy of 1, unless noted otherwise in the
tables.
Replacement of the Tyr2 residue with Phe in reference
compounds 1 and 6 results in equipotent V1a agonists 4 and 7,
respectively. Analogues 4 and 7 show better overall selectivity
profile than their parent compounds. Therefore, we prepared a
series of analogues 8ꢀ15, where position 2 Xaa was a residue
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Table 4. Duration of Action of Selected Analogues in Vivo
structurea
Yaa
in vitro potency at rV1aR
relative duration of action
equieffective dose (nmol/kg) mean ( SEM no. of rats tested
analogue
Xaa
Tyr
Zaa
Arg
EC50 (nM) (pEC50 ( SEM)
1a
2a
3a
4a
Gln
Hgn
Gln
Gln
Gln
Gln
Gln
Asn
0.07 (10.2 ( 0.02)
0.54 (9.27 ( 0.39)
0.35 (9.46 ( 0.21)
0.07 (10.1 ( 0.12)
0.12 (9.91 ( 0.34)
4.2 (8.38 ( 0.24)
2.1 (8.68 ( 0.22)
8.2 (8.09 ( 0.15)
1.0 (9.00 ( 0.15)
2.7 (8.56 ( 0.16)
0.34 (9.47 ( 0.14)
0.13 (9.90 ( 0.14)
0.29 (9.54 ( 0.15)
0.10 (9.98 ( 0.06)
0.16 (9.79 ( 0.19)
0.55 (9.26 ( 0.05)
2.0 (8.71 ( 0.19)
4.8 (8.32 ( 0.09)
8.0 (8.10 ( 0.40)
0.62 (9.21 ( 0.15)
0.32 (9.49 ( 0.10)
57 (7.24 ( 0.18)
0.1
1.0
0.1
0.3
0.3
3.0
2.0
3.0
1.0
2.0
2.0
0.2
0.3
0.3
0.2
0.3
6.0
3.0
6.0
1.0
0.6
1.0 ( 0.1b
5.0 ( 0.6c
1.1 ( 0.1b
1.4 ( 0.2b
2.3 ( 0.2c
2.3 ( 0.4b,c
3.1 ( 0.5c
1.4 ( 0.1b,c
1.6 ( 0.1b,c
1.7 ( 0.3b,c
3.4 ( 0.3c
2.3 ( 0.2c
2.6 ( 0.5c
1.9 ( 0.1c
2.1 ( 0.2c
1.4 ( 0.1b
2.0 ( 0.2b,c
1.3 ( 0.1b
2.4 ( 0.3c
1.5 ( 0.1b,c
2.7 ( 0.5c
4.1 ( 0.8c
9
7
3
3
3
5
5
6
3
3
6
3
4
3
3
3
4
3
3
3
3
6
Phe
Tyr
Dab(Abu)
Orn
Phe
Phe
Ala(cPe)
Cha
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Arg
7
Orn
19
20
28
31
34
38
40
41
42
43
45
47
49
52
54
59
68
Dab
Dab
Orn
Asn(Me2)
Orn
Asn((CH2)3OH) Orn
Hgn
Gln
Orn
Orn(iPr)
Orn(Me)
Orn(Pr)
Orn(Et)
Orn(iPr)
Dab
Gln
Gln
Gln
Hgn
Asn
Asn(Et)
Asn
Dab
Orn(Et)
Orn(iPr)
Orn(iPr)
Orn(iPr)
Ala(2-Fur) Gln
Thi
Gln
Cha
Asn
60
a Compounds 1, 3, and 4 contain Phe and all other analogues contain Ile in position 3. Compound 2 is an Hmp1 analogue. See abbreviations list for
definitions of unnatural amino acid residues. b Significantly different from 2. c Significantly different from 1.
containing an unsubstituted aromatic or heteroaromatic ring and
the diaminobutyric acid (Dab) residue in position 8 as the
starting point for a systematic exploration of the effect of the
side chain length in this position. The compounds containing
nitrogen heterocycles were generally less potent at the V1aR than
the Phe2 analogue 8, with the loss of potency apparently correlated
to the basicity of the aromatic ring. The compound containing an
imidazole ring (9, Xaa = His) was the least potent in this series,
and the compounds with pyridine (10) and thiazol (12) rings
were markedly less active as V1aR agonists than 8. The introduc-
tion of the bulky heterocyclic β-(3-benzothiazolyl)alanine in
position 2 resulted in an inactive analogue 11. The Thi2 (13) and
the 3-Thi2 (14) compounds were equipotent with 8 as V1aR
agonists but showed a better selectivity profile. The replacement
of the phenyl ring with the oxygen-containing furan ring resulted
in slightly less potent but more selective compound 15.
Next, we explored the possibility of replacing the aromatic
residue in position 2 with an aliphatic one. As in the case of
previously reported compound [Ala2]AVP,36 the Ala2 analogue
16 turned out to be completely inactive (no significant V1aR
agonist activity up to 1000 nM). Surprisingly, the compounds
with bulkier acyclic side chains 17 (R1 = neopentyl, EC50 = 11
nM) and 18 (R1 = isobutyl, EC50 = 13 nM) were found to be
moderately potent as V1a agonists. Analogues 19ꢀ21 with cyclic
aliphatic residues in position 2 were equipotent with the Phe2
analogue 8, but their selectivity versus other related receptors
(i.e., OTR) was substantially improved. Although AVP analo-
gues with cyclic aliphatic residues in position 2 have been pre-
viously reported,37ꢀ39 and a few fully nonaromatic naturally
occurring vasopressin/conopressin analogues have been described,40
compounds 19ꢀ21 appear to be the first examples of fully nona-
romatic V1aR agonists.
Position 4 (Yaa) seems to be essential for tuning the receptor
specificity in the family of vasopressin/oxytocin receptors. Com-
pounds with improved V2 selectivity have been obtained by
replacing the Gln4 residue with hydrophobic amino acids such as
Val.41 V1bR selective compounds have been discovered by
replacing the Gln4 residue in the dAVP molecule with a variety
of basic (Lys, Orn) or hydrophobic (Cha, Leu) amino acids.42ꢀ45
Consequently, position 4 of compound 7 was modified with a
variety of polar amino acids (analogues 22ꢀ38). Shortening the
position 4 side chain length resulted in analogue 28 showing
similar in vitro profile as 7, whereas analogue 38 with the longer
side chain of homoglutamine, Hgn, exhibited high selectivity
versus both hV2R and hOTR. The N-substitutions on the gluta-
mine primary amide in most cases led to compounds with
decreased potency (23, 24, 26, and 27). Only the N-methyl
(22) and the N-methoxy (25) substituents were well tolerated
but selectivity versus related receptors was not improved. Inter-
estingly, N-substitutions on the primary amide of an asparagine
residue with alkyl, hydroxyalkyl, or hydroxyl groups resulted in a
series of relatively potent compounds 29ꢀ34, 36, 37, with the N,
N-dimethyl analogue 31 and the N-3-hydroxypropyl analogue 34
showing remarkable selectivity versus both V2R and OTR.
The length of the side chain in position 8 has a significant
impact on potency and selectivity of V1aR agonists. The diami-
nopropionic acid Dap8 compound (39) is a substantially less
potent V1aR agonist than the Dab (8) and Orn (7) analogues.
Compound 8 is equipotent with but slightly less selective than
compound 7. N-Alkylation of the Orn8 side chain with small alkyl
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Figure 3. Typical traces of diastolic (DAP) arterial pressure time course (mmHg over baseline DAP) showing the decay of vasopressor effect of
analogues 1, 45, and 2 after an intravenous bolus injection of equieffective doses to the reference dose of 1. Traces in a given graph were obtained in the
same animal.
groups (analogues 40ꢀ44) was well tolerated and resulted in
potent V1aR agonists with improved selectivity versus both V2R
and V1bR. It should be pointed out that the enhanced V1b selec-
tivity could not be achieved by any other modifications described
in this paper.
no obvious SAR rules for the duration of action in vivo could be
identified, compounds 20 and 68, which contain a cyclic aliphatic
Cha residue in position 2, had considerably long duration of
action. Compounds 31, 34, 45, and 49 displayed the most
favorable overall pharmacological profile, and analogue 45 was
selected for further evaluation as a potential treatment in critical
care medicine. The differences in vasopressive duration of action
in vivo for compounds 1, 2, and 45 are presented in Figure 3.
Analogues 45ꢀ70 combine two or more of the single mod-
ifications in positions 2, 4, and/or 8 employed in compounds
8ꢀ44. Although a number of potent/selective V1aR agonists
were found in this set (e.g., compounds 45, 47, 49ꢀ52, 54, 59,
68, 70), the in vitro data suggests that the effects of the structural
changes introduced in compounds 8ꢀ44 are not fully additive.
For example, the combination of aromatic residues containing
five-membered rings (Thi, Ala(2-Fur)) in position 2 with the
Asn4 residue (55ꢀ58, 60ꢀ63) yielded analogues significantly
less potent at the V1aR. Nonaromatic residues in position 2 in
combination with other modifications (64ꢀ67) led to markedly
less potent V1aR agonists with the notable exception of the Cha2
compounds 68ꢀ70.
’ CONCLUSION
We have designed, synthesized, and pharmacologically char-
acterized a series of vasopressin analogues modified in position 2,
3, 4, and/or 8. We identified compounds with high potency as
V1aR agonists, with high selectivity versus related receptors, in
particular V2R, and short duration of action in vivo. Several pep-
tides, such as [Phe2,Ile3,Asn(Me2)4,Orn8]VP (31), [Phe2,Ile3,
Asn((CH2)3OH)4,Orn8]VP (34), [Phe2,Ile3,Hgn4,Orn(iPr)8]
VP (45), and [Phe2,Ile3,Asn(Et)4,Dab8]VP (49), showed the
desired pharmacological profile. Compound 45 (FE 202158)47 is
currently undergoing clinical trials for the treatment of vasodi-
latory hypotension conditions such as septic shock. The full
pharmacological characterization of peptide 45 has just been
published.46
The analogues with the best in vitro pharmacological profile
(potent and V1aR-selective) contain the Phe or the Thi residues
inposition 2, the N-alkylated Asn(R3), the Gln or the Hgnresidues
in position 4, and/or the Dab, the Orn or the N-alkylated
Orn(R5) residues in position 8 (e.g., 31, 34, 38, 45, 49, 59,
and 68). A graphical illustration of in vitro receptor selectivity of
compound 45 as compared to 1 is shown in Figure 2. Although
maximum efficacy of 45 in the RGA in vitro assay at the hV1aR
appears to be slightly lower than that of 1, we believe the dif-
ference is insignificant as the analogue has elicited full agonistic
response in various in vitro, ex vivo and in vivo assays.46
The analogues showing a favorable in vitro profile at the human
receptors were tested in vivo in a rat vasopressor model for their
duration of action. Differences in intrinsic potency at the rat V1aR
(see Table 4 for in vitro EC50 values) and interanimal variation in
V1aR agonist responsiveness were compensated for by experi-
mentally selecting the dose of each compound that produced an
elevation of diastolic blood pressure equivalent to that of a
0.1 nmol/kg dose (ED80) of 1. All compounds were effective
in raising arterial blood pressure at the equieffective doses but
showed considerable differences in the rates of disappearance of
the vasopressor effect (Table 4).
’ EXPERIMENTAL SECTION
Synthesis. General. Amino acid derivatives and resins were pur-
chased from Novabiochem, Bachem Peptide International, and Pep-
Tech Corporation. Fmoc-Hgn-OH was synthesized according to the
published procedure.48 Other chemicals and solvents were purchased
from Sigma-Aldrich, Fisher Scientific, and VWR.
Mass spectra were recorded on a Finnigan MAT spectrometer.
Preparative HPLC was performed on a Waters 2000 liquid chromato-
graph using a 15 μm PrePak 47 mm ꢁ 300 mm cartridge at a flow rate of
100 mL/min. Final purity of analogues was assessed on a 1100 Agilent
liquid chromatograph using the following analytical method: column,
Vydac C18, 5 μm, 2.1 mm ꢁ 250 mm; column temperature ꢀ40 °C;
flow rate ꢀ0.3 mL/min; solvent A, 0.01% TFA; solvent B, 70% CH3CN,
0.01% TFA; gradient, 0ꢀ20% B in 1 min, then 20ꢀ40% B in 20 min,
then held at 100% B for 5 min; when necessary, the first two segments of
the gradient were adjusted for compound lipophilicity; UV detection at
214 nm. The purity of analogues exceeded 95% unless stated otherwise
in the Supporting Information.
The analogues were considered short-acting when the rate of
the decay of the vasopressor effect was less than 2 times of that of
1. For comparison, the relative duration of action of the pre-
viously described long-acting23 analogue 2 used as a reference
compound in this assay was 5 times longer than that of 1. While
HPLC retention times (tR) were determined on a 1200rr Agilent
liquid chromatograph using the following analytical method: column,
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Zorbax SB-C18, 1.8 μm, 50 mm ꢁ 4.6 mm; column temperature ꢀ40 °C;
flow rate ꢀ1.5 mL/min; solvent A, 0.05% TFA; solvent B, 90% CH3CN,
0.05% TFA; gradient, 10% B in 1 min then 10ꢀ50% B in 9 min; UV
detection at 214 nm. HPLC capacity factors (k0) were calculated using
the equation k0 = (tR ꢀ t0)/t0 where t0 is retention time of the unretained
species (actual value was t0 = 0.425 ( 0.002 min). The purities, tR, and
k0 values are reported in the online Supporting Information.
Synthesis of Peptides with Alkylated Side Chain in Position 8 (40ꢀ
46, 50ꢀ54, 56, 59, 61, 68). The peptides were assembled by Fmoc
chemistry. The diamino acid residue in position 8 was introduced
with the N-δ-Mmt (methoxytrityl) protecting group31 and the N-terminal
Cys residue was coupled as Boc-Cys(Trt)-OH. The resin bound peptide
was treated withthe DCM/TIS/TFA 93/5/2(v/v/v) cocktail (2 ꢁ 1.5 h)
to remove the Mmt group. The N-ω-isopropyl analogues (40, 45, 46, 50,
54, 56, 59, 61, and 68) were synthesized by reductive alkylation of the
resin bound peptides with acetone/NaBH(OAc)3 in DCE/TMOF.
To prepare other N-alkyl peptides (41ꢀ44, 51ꢀ53), the side chain
amino group was derivatized with o-NBS-Cl (2-nitrobenzenesulfonyl
chloride)32 and the resulting resin bound sulfonamide was alkylated with
an appropriate alcohol under Mitsunobu reaction conditions (TPP/
DIAD in 1,2-dimethoxyethane).33 The o-NBS group was then removed
with 5% potassium thiophenolate in DMF,32 and finally the peptides
were cleaved from the resin.
Synthesis of Peptides with N-Hydroxy (27, 32) or N-Alkylated Side
Chain in Position 4 (22ꢀ26, 29ꢀ37, 48, 49). The peptides were
assembled by Boc chemistry. The residue in position 4 was introduced in
the sequence as Boc-Asp(OFm)-OH or Boc-Glu(OFm)-OH.34,35 After
complete assembly, the side chain protection was removed with 30%
piperidine/DMF. The resulting free carboxylic group was converted to
the desired amide by coupling with an appropriate amine mediated by
PyBOP or BOP/DIPEA. The N-terminal Boc group was removed prior
to the HF cleavage.
Cleavage and Purification. The peptides synthesized by Fmoc
chemistry were cleaved with the TFA/TIS/H2O 96/2/2 cocktail and
the ones synthesized by Boc chemistry were cleaved with 90% HF/10%
anisole. The ring formation was achieved by oxidation of linear peptides
dissolved in 10% aqueous TFA with iodine. The peptides were purified
by preparative HPLC in triethylammonium phosphate buffers. Finally,
the compounds were converted to their acetates by a modification of a
published HPLC method.49 The fractions with purity exceeding 97%
were pooled and lyophilized.
model from Xlfit (IDBS) and used to estimate EC50 and efficacy values.
Agonist potency is presented as geometric means in nanomol/L (nM).
Selectivity values are given as EC50 ratios of the test compound at a
receptor vs V1aR.
In Vivo Pharmacological Methods. The duration of the vasopressive
effect (i.e., the increase in arterial pressure) induced by intravenous
bolus administration of test compound was determined and compared
to the duration of action of equieffective doses of the short-acting 1 and
the long-acting and selective V1aR agonist 2.23,24
Specifically, 91 male Wistar rats (206ꢀ340 g) were obtained from
Harlan (Indianapolis, IN) for this assay. Upon delivery, rats were housed
two per microisolator cage at Ferring Research Institute Animal Care
Facility under controlled environmental conditions (12 h/12 h light
ꢀdark cycle, lights on at 7 a.m.; 22ꢀ25 °C) in a small animal housing
cabinet (Scantainer Classic, Scanbur A/S, Karlslunde, Denmark) or in a
positive-pressure clean room (BioBubble, BioBubble, Fort Collins, CO)
with free access to standard rodent chow (5001 Rodent Diet, PMI
Nutrition International, Richmond, IN, or 18% Protein Rodent Diet,
product no. 2018, Harlan Teklad, Madison, WI) and acidified tap water
(pH 2.5ꢀ3.0). Housing conditions were in accordance with the Guide
for the Care and Use of Laboratory Animals published by the National
Research Council. The rats were allowed to acclimate for at least 2 days
prior to study use. The in vivo portion of all study protocols was
approved by the Institutional Animal Care and Use Committee.
After overnight fasting, rats were anesthetized with isoflurane (2.0ꢀ2.5%
in oxygen), and anesthesia was maintained with thiobutabarbital sodium
(150 mg/kg intraperitoneally, redosed at 50 mg/kg if needed), mechani-
cally ventilated with 36% oxygen compressed air (HSE-HA Multiple-
Channel Ventilator, Harvard Apparatus, Holliston, MA), and their body
temperature maintained at 37ꢀ38 °C using a water circulating heating
pad (MUL-T-PAD, Gaymar Industries, Orchard Park, NY). A carotid
artery was catheterized to monitor arterial blood pressure (disposable
pressure transducer sets “DTX Plus TNF-R, Becton Dickinson Critical
Care Systems, Franklin Lakes, NJ), and the opposite jugular vein was
catheterized for vehicle and drug administration. Arterial pressure data
was continuously acquired with Notocord-hem (Notocord Systems
SAS, Croissy sur Seine, France). Prior to agonist administration, rats
were pretreated intravenously with the R-adrenergic receptor antagonist
dibenamine (1.8 mg/rat administered divided into 7 boluses spread over
30 min) to stabilize arterial blood pressure and enhance vasopressor
responsiveness to V1aR agonists.52ꢀ54
After stable hemodynamic recordings were established (∼45 min),
animals were administered intravenous boluses at a volume of 0.5 mL/kg
of: (i) heparinized vehicle solution (20 unit/mL heparin in 2.5% w/v
dextrose and 0.45% w/v NaCl) to assess any nonspecific hemodynamic
effects, followed by (ii) 1 (0.1 nmol/kg; ∼ED80) to assess vasopressive
integrity of the animal preparation, (iii) a second administration of 1,
also at 0.1 nmol/kg, to induce an internal (control) reference response,
and (iv) two doses of test compound to identify the equieffective dose to
the second administration of the 0.1 nmol/kg dose of 1 in terms of
diastolic arterial pressure (DAP) rise. For each compound, these two
doses were estimated based on the in vitro potency of the compound at
the rV1aR, relative to 1 (Table 4). The first potentially equieffective dose
Biological Methods. In Vitro Receptor Assays. Agonist activity of
compounds at the human or rat vasopressin V1aR were determined in a
reporter gene assays (RGA) in HEK293 cells by transiently cotransfect-
ing recombinant vasopressin V1aR expression vectors and the reporter
plasmid containing a luciferase gene under the control of NFAT
response elements (NFAT-luciferase).50,51 Assays for human vasopressin
V2R were performed in HEK293 cells cotransfected with recombinant
human vasopressin V2R expression vector and the reporter plasmid contain-
ing a luciferase gene under the control of cAMP responsive elements.
Assays for V1b receptor activity were performed in a Flp-In-293 cell
line (Invitrogen) stably expressing recombinant human vasopressin V1b
receptor and transiently transfected with the NFAT-luciferase reporter
construct.50,51 Assays for OT receptor activity were performed in CHO-
K1 cells stably expressing human OT receptor and transiently trans-
fected with the NFAT-luciferase reporter construct.50,51 Two days
following transfection, cells were treated with appropriate doses of
peptides, incubated at 37 °C for 5 h, lysed in the presence of luciferin,
and total luminescence measured. 1 was used as an internal control for
the vasopressin V1a and V1b receptors, dDAVP was used as an internal
control for the vasopressin V2 receptor, and carbetocin, a selective OT
analogue, was used as an internal control for the OTR assay.
was calculated as 0.1 nmol/kg EC50(Compound)/EC50(1). The sec-
3
ond dose was set to three times this value. The actual equieffective dose
was selected as the one that produced the elevation of diastolic arterial
pressure closer to that produced by the 0.1 nmol/kg dose of 1. In time
control rats, the test compound administration was replaced by repeated
administration of the 0.1 nmol/kg dose of 1. An additional control group
received an equieffective dose (1 nmol/kg) of the long-acting selective
V1aR agonist 2 instead of test compound. Dosing intervals were set as the
time required for the DAP to decrease to a stable baseline (∼20 min for
1 to ∼120 min for 2). The vehicle solution did not produce any measur-
able hemodynamic effect in any of the animals.
Compounds were tested in at least three independent experiments.
Doseꢀresponse curves were generated using a one-site four-parameter
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For each rat, the absolute duration of action of the vasopressor effect
of either test compound, 1, or 2 was calculated as the average of the DAP
decay rate, measured over 10 s intervals, from the time of peak DAP
increase to the time where 80% of this increase had dissipated. To
normalize for interindividual variation in V1aR agonist responsiveness,
the relative duration of action of the vasopressor effect for the dose of
test compound equieffective to the 0.1 nmol/kg dose of 1 inducing the
internal (control) reference response was calculated for each rat as the
ratio of the average decay rate for 1 divided by the average decay rate for
test compound or 2 such that a ratio greater than 1 is indicating a longer
duration of action and a ratio smaller than 1 a shorter duration of action.
Results are expressed as mean ( SEM and are presented in Table 4.
Statistical comparison was done using a one-way ANOVA after trans-
forming the relative duration of action into its reciprocal to ensure that
the assumptions of normal distribution and equal variance were not
violated. The significant ANOVA (P < 0.05) was followed by all pairwise
multiple comparison procedures using the Holm Sidak method (Sigma-
Stat v. 3.11, Aspire Software International, Ashburn, VA).
LVP, 8-lysine vasopressin; Mmt, methoxytrityl; MPE, maximum
possible effect; Orn(iPr), Nδ-isopropylornithine; Orn(Me), Nδ-
methylornithine; Orn(Pr), Nδ-propylornithine; Orn(Et), Nδ-ethyl-
onithine; Orn(iAm), Nδ-isoamylornithine (Nδ-(3-methylbutyl)-
ornithine); OT, oxytocin; OTR, oxytocin receptor; OVP, 8-or-
nithine vasopressin; 2-Pal, β-(2-pyridyl)alanine; 3-Pal, β-(3-pyr-
idyl)alanine; 4-Pal, β-(4-pyridyl)alanine; PyBOP, benzotriazol-
1-yloxy trispyrrolidinephosphonium hexafluorophosphate; RGA,
reporter gene assay; TFA, trifluoroacetic acid; Thi, β-(2-thie-
nyl)alanine; 3-Thi, β-(3-thienyl)alanine; TIS, triisopropylsilane;
TMOF, trimethyl orthoformate; TPP, triphenylphosphine;
V1aR, vasopressin V1a receptor; VP, vasopressin; V1bR (V3R),
vasopressin V1b receptor; V2R, vasopressin V2 receptor; Xaa, any
amino acid residue in position 2; Yaa, any amino acid residue in
position 4; Zaa, any amino acid residue in position 8
’ REFERENCES
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pressin/Oxytocin Receptor Function. J. Endocrinol. 1998, 156, 223–229.
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Med. Biol. 1998, 449, 1–13.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed synthetic procedures
b
and physicochemical properties of compounds 1ꢀ7 and 8ꢀ70.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(4) Ring, R. H. The Central Vasopressinergic System: Examining the
Opportunities for Psychiatric Drug Development. Curr. Pharm. Des.
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moto, G. Vasopressin Stimulates Insulin Release from Islet Cells through
V1b Receptors: A Combined Pharmacological/Knockout Approach.
Mol. Pharmacol. 2004, 65, 623–629.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 1-858-657-1597. E-mail: Kazimierz.Wisniewski@ferring.com.
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’ ACKNOWLEDGMENT
We thank Marlene Brown, John Kraus, and Brian Ly for their
excellent technical assistance. We also thank Denise Riedl and
Michael Dunn for auditing the data and critical reading of the
manuscript.
’ ABBREVIATIONS USED
Ac4c, 1-aminocyclobutane-1-carboxylic acid; ACTH, adrenocor-
ticotropic hormone; ADH, antidiuretic hormone; Ala(BzThi), β-
3-benzothienylalanine; Ala(cPe), β-cyclopentylalanine; Ala(2-Fur),
β-(2-furyl)alanine; Ala(t-Bu), β-(tert-butyl)alanine; Ala(Thz),
β-4-(1,3-thiazolyl)alanine; Asn(CH(CH2OH)2), Nγ-((1,1-dihy-
droxymethyl)methyl)asparagine; Asn(CH2CH(OH)CH3), Nγ-
(2-hydroxypropyl)asparagine; Asn((CH2)2O(CH2)2OH), Nγ-
(5-hydroxy-3-oxapentyl)asparagine; Asn((CH2)2OH), Nγ-(2-
hydroxyethyl)asparagine; Asn(CH2)3OH), Nγ-(3-hydroxypropyl)
asparagine; Asn(Et), Nγ-ethylasparagine; Asn(Me), Nγ-methyla-
sparagine; Asn(Me2), Nγ,Nγ-dimethylasparagine; Asn(OH), Nγ-
hydroxyasparagine; AVP, 8-arginine vasopressin; BOP, benzo-
triazol-1-yloxy trisdimethylaminophosphonium hexafluoropho-
sphate; Cha, β-cyclohexylalanine;Dab, 2,4-diaminobutyric acid; Dap,
2,3-diaminopropionic acid; DAP, diastolic arterial pressure;
dAVP, desamino-8-arginine vasopressin; DCE, 1,2-dichloroethane;
dDAVP, desamino-8-D-arginine vasopressin; DCM, dichloro-
methane; DIAD, diisopropyl azodicarboxylate; DIPEA, N,N-
diisopropyl-N-ethylamine; Gln(Et), Nδ-ethylglutamine; Gln(iBu),
Nδ-isobutylglutamine (Nδ-(2-methylpropyl)glutamine); Gln-
(Me), Nδ-methylglutamine; Gln(Me,OMe), Nδ-methoxy-Nδ-
methylglutamine; Gln(OMe), Nδ-methoxyglutamine; Gln(OH),
Nδ-hydroxyglutamine; HF, hydrogen fluoride; Hgn, homoglutamine;
(16) Wiꢀsniewski, K.; Alagarsamy, S.; Taki, H.; Miampamba, M.;
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