Arginine vasopressin and its analogues - The influence of position 2 modification with 3,3-diphenylalanine enantiomers. Highly potent V2 agonists

Article abstract of DOI:10.1016/j.ejmech.2008.12.010

Eleven new analogues of arginine vasopressin (AVP) modified in position 2 by 3,3-diphenyl-l-alanine or its d-enantiomer (Dip or d-Dip) were synthesized and pharmacologically evaluated for their pressor, antidiuretic and in vitro uterotonic activities. Bot

Full text of DOI:10.1016/j.ejmech.2008.12.010

European Journal of Medicinal Chemistry 44 (2009) 2862–2867
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Arginine vasopressin and its analogues – The influence of position
2 modification with 3,3-diphenylalanine enantiomers.
Highly potent V2 agonists
,
a
a
b
Anna Kwiatkowska a , Dariusz Sobolewski , Adam Prahl , Lenka Borovickova ,
*
ˇ
´
b
a
ˇ
´
Jirina Slaninova , Bernard Lammek
a Faculty of Chemistry, University of Gdan´sk, Sobieskiego 18, 80-952 Gdan´sk, Poland
b Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo sq. 2, 166 10 Prague, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven new analogues of arginine vasopressin (AVP) modified in position 2 by 3,3-diphenyl-L-alanine or
Received 25 August 2008
Received in revised form
7 December 2008
Accepted 9 December 2008
Available online 24 December 2008
its
D
-enantiomer (Dip or
D
-Dip) were synthesized and pharmacologically evaluated for their pressor,
antidiuretic and in vitro uterotonic activities. Both the Dip and
D-Dip modifications at position 2 of AVP
are sufficient to completely change the pharmacological profile of the peptides. They preserve or increase
antidiuretic activity, cause its prolongation, transform uterotonic property in antagonistic one and cancel
the effect on blood pressure. Four of the new peptides ([Mpa1, -Dip2]AVP, [Mpa1, -Dip2,Val4]AVP,
D D
[Mpa1,
D
-Dip2,
D
-Arg8]VP, [Mpa1,
D
-Dip2,Val4,
D
-Arg8]VP) are exceptionally potent antidiuretic agents with
Keywords:
significantly prolonged activities.
Arginine vasopressin (AVP) analogues
3,3-Diphenylalanine enantiomers
V2 agonists
Ó 2008 Elsevier Masson SAS. All rights reserved.
Antidiuretic activity
1. Introduction
natriuretic factor from the heart [8]. Vasopressin exerts its biolog-
ical functions upon binding to four different 7-transmembrane G-
protein-coupled receptors (GPCRs) termed: V1a, V1b, V2 and OT
receptors [1–3]. V1a receptors, present in many tissues including
brain, mediate the vasopressor actions of AVP [1]. V1b receptors
present in the pituitary, pancreas and adrenals mediate the ACTH-
release from the anterior pituitary gland [3], steroid secretion from
the adrenal medulla [6] and the insulin and glucagon release from
the pancreas [7]. OT receptors control milk ejection and uterine
smooth muscle contractions. These three receptor subtypes func-
tion via the phosphatidylinositol pathway. The antidiuretic activity
of AVP is evoked by V2 receptors present in the renal tubule and
particularly the collecting ducts. They are linked to adenylate
cyclase signaling [3].
Vasopressin plays an important role in water metabolism and
impairment of its synthesis, secretion or metabolism induces some
clinical disorders such as inappropriate antidiuretic hormone
secretion syndrome (SIADH) or diabetes insipidus. Elevated AVP
secretion leads to renal water retention and extracellular fluid
expansion that is compensated for by enhanced urinary Naþ
excretion. The combination of water retention and Naþ excretion
leads to hyponatremia. Criteria for SIADH were first described by
Bartter and Schwartz [9] and are still applicable today. These are:
hypotonic hyponatremia, urine osmolality higher than appropriate
Arginine vasopressin (AVP), a neurohypophyseal hormone and
neuromodulator, is a cyclic nonapeptide with a disulfide bridge
between Cys residues at positions 1 and 6 [1]. This results in a six-
amino-acid cyclic part and a C-terminal amidated three-residue
tail.
Main physiological roles played by AVP are the regulation of
water balance, the control of blood pressure, and the secretion of
adrenocorticotropin hormone (ACTH) [1–3]. Moreover, AVP also
exhibits to some extent typical oxytocin (OT, a closely related
neurohypophyseal peptide) activities such as the galactogogic and
the uterotonic effects [4]. Apart from these well-known functions,
AVP is also involved in glycogenolysis in the liver [5], in the steroid
and catecholamine secretion by the adrenals [6], release of insulin
and glucagon by the pancreas [7], and in the secretion of atrial
Abbreviations: Acc, 1-Aminocyclohexane-1-carboxylic acid; Aic, 2-Amino-
indane-2-carboxylic acid; Apc, 1-Aminocyclopentane-1-carboxylic acid; AVP,
Arginine vasopressin; Cpa, 1-Mercaptocyclohexaneacetic acid; Dip, 3,3-Diphenyl-
L-
alanine; -Dip, 3,3-Diphenyl- -alanine; MBHA, p-Methoxybenzhydrylamine; Mob,
D
D
4-Methoxybenzyl; NMP, 1-Methyl-2-pyrrolidone; OT, Oxytocin.
* Corresponding author. Tel.: þ48 58 523 53 16.
E-mail address: aniak@chem.univ.gda.pl (A. Kwiatkowska).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.12.010

A. Kwiatkowska et al. / European Journal of Medicinal Chemistry 44 (2009) 2862–2867
2863
for the concomitant plasma osmolality, increased natriuresis,
absence of edema or volume deletion, and normal renal and
adrenal functions [10]. On the other hand, diabetes insipidus is
a heterogeneous condition characterized by polyuria and poly-
dipsia caused by lack of vasopressin secretion (physiological
suppression following excessive water intake) or kidney resistance
to its action [11].
(a steep dose–response curve). Both were moderately potent
blockers of the oxytocin uterotonic activity.
In this study we further checked the influence of the bulky Dip
isomers (L- and D, see Fig. 1) at position 2 of the AVP and some of its
analogues on the pharmacological properties. The synthesized
analogues I–XI are listed in Table 1.
Since 1954, when du Vigneaud et al. characterized and
synthesized AVP [12], many analogues of this hormone have been
obtained and pharmacologically evaluated. A synthetic vasopressin
2. Chemistry
2.1. General
analogue, V2 agonist, 1-deamino-8-D-arginine vasopressin (dDAVP,
commercial names Minirin, Minrin, Desmopressin, Adiuretin-SD)
has been a standard drug for the treatment of diabetes insipidus for
over 30 years [13]. On the other hand, V2 antagonists have potential
therapeutic value in the treatment of SIADH [14]. The OT antago-
nists have been useful for suppressing premature labor [15]. The
design of analogues that selectively interact with appropriate AVP
receptors is still a matter of interest.
Thin-layer chromatography (TLC) was carried out on silica plates
(Merck), and spots were visualized with iodine. The solvent system
used was butan-1-ol/acetic acid/water/ethyl acetate (1:1:1:1, v/v).
High-performance liquid chromatography was carried out on
a Waters (analytical and preparative) chromatograph equipped
with a UV detector (
determined on Vydac C18 column (5
ODS C18 column (5
m, 4.6 ꢀ 250 mm) or Hypersil BDS C18 column
(5
l
¼ 226 nm). The purity of the peptides was
m
m, 4.6 ꢀ 250 mm) or Hypersil
Parallel to the development of selective peptide agonist and
antagonists, the research of non-peptide agonists and antagonists
was carried out. As a result, a number of interesting compounds of
varied structure have been synthesized and tested for their activity.
At present there are available non-peptide antagonists for all 4
types of neurohypophyseal hormone receptors [16,17], e.g. orally
active V2 receptor antagonists (Vaptans), notably, Tolvaptan, Lix-
ivaptan and Satavaptan [16] and the mixed V1a/V2 receptor antag-
onist Conivaptan (YM087) [18]. To date, only Conivaptan (also
known as Vaprisol) has been approved by the US FDA for clinical
use (by i.v. administration) for the treatment of euvolemic and
hypervolemic hyponatremia in hospitalized patients [16]. Espe-
cially interesting is the recent development of orally active specific
V1b receptor antagonist (SSR149415) which opened new possibili-
ties for the study and treatment of stress and depression [19].
Biological activity of peptides is determined by their structure
and conformation. Conformational restriction is therefore a well-
established strategy to change their pharmacological profile.
Peptide flexibility can be restricted by a local constraint imposed,
e.g. by introducing amino acids with limited conformational
freedom that has an impact on specific orientations of the other
amino acid side chains and peptide backbone.
m
m
m, 4.6 ꢀ 250 mm). The following solvent systems were used:
[A] 0.1% aqueous TFA, [B] acetonitrile/0.1% aqueous TFA (80:20 v/v).
Preparative HPLC was carried out using a Waters C18 column
(15
m
m, 100 Å, 7.8 ꢀ 300 mm). The mass spectra of the peptides
were recorded on a MALDI TOF mass spectrometer.
Mpa(Mob) was obtained as described for Mpa(Bzl) [28] using
p-methoxybenzyl chloride. Cpa(Mob) was synthesized using a
procedure described in literature [29].
All the amino acid derivatives were purchased from Nova-
Biochem, except Boc-Dip and Boc-
ChemImpex.
D-Dip, which were provided by
2.2. Peptide synthesis and purification
All peptides were obtained manually using Boc-chemistry on
methoxybenzhydryl resin (MBHA resin, Senn Chemicals AG, 1%
DVB, 200–400 mesh, 0.67 mmol/g) on scale of 150 mmol according
to standard procedures, using in situ neutralization [30]. Fully
protected peptide resin was synthesized according to standard
procedures involving (i) deprotection steps 33% TFA/DCM in the
presence of anisole (1%), 5 and 10 min; (ii) neutralization with 10%
TEA/DCM, 5 and 10 min; (iii) couplings of Boc-amino acid; 3 h at
room temperature. In most cases, the amino acids were coupled at
3-fold excess using TBTU/HOBt/NMM in the mixture of DMF/NMP
(1:1 v/v) containing 1% Triton, and if necessary amino acid/HATU/
HOAt/NMM (1:1:1:2) in mixture of DMF/NMP (1:1 v/v) containing
1% Triton for recoupling steps. After 3 h coupling time, the Kaiser
test [31] or chloranil test [32] was performed to estimate the
completeness of the reaction. After completion of the synthesis, the
protected peptidyl or acylpeptidyl resins were treated with 10 mL
of liquid hydrogen fluoride (HF) containing 1 mL of anisole at
ꢁ70 ꢂC and stirred for 60 min at 0 ꢂC [33]. After removal of HF and
It is generally accepted that the conformation of the N-
terminal part of neurohypophyseal hormone analogues is crucial
for their pharmacological activity [20,21]. This has also been
supported by our already 12-years research focused on the impact
of steric restriction and bulky substituent in the N-terminal part
of AVP molecule on biological properties of the resulting
analogues. We demonstrated that the arginine vasopressin
analogues modified at position 2 with either
thyl)-alanine were moderately potent and selective oxytocin
antagonists in vitro [22], while [
-Arg8]VP substituted at position
3 with -(2-naphthyl)-alanine turned out to be a potent and
L or D b-(1-naph-
D
b
selective antagonist of the pressor response to AVP [23]. We have
also shown that introduction of either 1-aminocyclohexane-1-
carboxylic acid (Acc, also known as Ac6c) or 1-amino-
cyclopentane-1-carboxylic acid (Apc, also known as Ac5c) into
O
position
2 of AVP and some of its analogues resulted in
compounds having high antidiuretic potency, low and graded
pressor activity, and either no activity or low oxytocin antago-
nizing activity in the uterotonic in vitro tests [24–26]. Recently,
we described some pharmacological activities of three analogues
OH
NH2
having bulky 3,3-diphenyl-L-alanine (Dip) in position 2 [27]. The
new peptides had strikingly different biological properties in
comparison with those of the parent hormone. Two of them,
[Dip2]AVP and [Mpa1,Dip2]AVP, displayed no pressor activity
while their antidiuretic potency was preserved and prolonged
Fig. 1. Structure of 3,3-diphenylalanine.

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A. Kwiatkowska et al. / European Journal of Medicinal Chemistry 44 (2009) 2862–2867
Table 1
Physicochemical properties of peptides I–XI.
Analogue
Formula
HPLC TR (min)
[M þ Hþ]
aa
bb
Calculated
Found
[Mpa1,Dip2,Val4]AVP
[Dip2, -Arg8]VP
[Mpa1,Dip2, -Arg8]VP
[Mpa1,Dip2,Val4, D-Arg8]VP
-Dip2]AVP
I
II
C52H69N13O10S2
C52H69N15O11S2
C52H68N14O11S2
C52H69N13O10S2
C52H69N15O11S2
C52H68N14O11S2
C57H76N14O11S2
C52H69N13O10S2
C52H69N15O11S2
C52H68N14O11S2
C52H69N13O10S2
15.85
11.41
14.19
40.06d
12.74
14.46
16.16
16.52
12.95
14.80
16.64
19.06c
15.62
17.99
18.68
14.49c
18.21
21.13
22.08
14.74c
18.49
22.45
1100.3
1144.3
1128.5
1100.3
1144.3
1129.3
1197.4
1100.3
1144.3
1129.3
1100.3
1100.5
1144.3
1129.2
1100.2
1144.2
1129.2
1197.4
1100.2
1144.2
1129.5
1100.3
D
D
III
IV
V
VI
VII
VIII
IX
X
D
[D
[Mpa1,
D
-Dip2]AVP
-Dip2]AVP
-Dip2,Val4]AVP
-Arg8]VP
-Dip2, -Arg8]VP
-Dip2,Val4, -Arg8]VP
[Cpa1,
D
[Mpa1,
D
[D D
-Dip2,
[Mpa1,
[Mpa1,
D
D
D
D
XI
a
Linear gradient from 20 to 80% of [B] in [A] for 20 min, Vydac C18 column.
Linear gradient from 30 to 90% of [B] in [A] for 30 min, Hypersil ODS C18 column.
Linear gradient from 30 to 90% of [B] in [A] for 30 min, Hypersil BDS C18 column.
b
c
d
Linear gradient from 1 to 60% of [B] in [A] for 60 min, Vydac C18 column; [A] 0.1% aqueous trifluoroacetic acid (TFA), [B] acetonitrile: 0.1% aqueous TFA (80:20 v/v).
anisole in vacuo, the mixture was washed successively with
anhydrous diethyl ether, then with acetic acid and the solution was
diluted with methanol. The resulting dithiols were oxidatively
cyclized with a 0.1 M I2 in methanol using a standard procedure
[34]. The solvents were evaporated under reduced pressure and the
residue was dissolved in water and lyophilized.
All the products were purified by gel filtration chromatography
on Sephadex G-15 using 30% acetic acid as the eluent. After freeze-
drying, the fractions comprising the major peak were finally puri-
fied by RP-HPLC Waters chromatograph equipped with a UV
concentrations and at 1 min intervals without the fluid being
changed until the maximal response (the highest contraction) was
obtained. The dose–response curves were constructed. Synthetic
oxytocin was used as standard. Each new analogue was tested using
uteri from 3 to 5 different animals; the values in Table 2 are
averages ꢃ SEM.
3.3. Vasopressor activity
The vasopressor test was performed using phenoxybenzamine-
treated male rats [37] under urethane anesthesia. AVP was used as
a standard. Changes of the arterial blood pressure were registered.
Dose (single administration into vena femoralis)–response curves
were constructed in the presence and absence of an antagonist. The
antagonist was administered 1 min prior to the administration of
the standard. Each analogue was tested in 3–5 independent
experiments, the values in Table 2 are averages ꢃ SEM.
detector
(
l
¼ 226 nm), Waters C18 column (15
mm, 100 Å,
7.8 ꢀ 300 mm) in the linear gradient running from 25 to 55% of [B]
for 90 min for analogues I, III, IV, VI–VII, X, XI, and from 15 to 45%
for 90 min for analogues II, V, IX, at a flow rate of 2.5 mL/min; [A]
0.1% aqueous trifluoroacetic acid (TFA), [B] acetonitrile/0.1%
aqueous TFA (80:20, v/v). The appropriate fractions were pooled
and lyophilized. The analytical HPLC (Waters, equipped with Vydac
C18 column (5
(5
m
m, 4.6 ꢀ 250 mm) or Hypersil ODS C18 column
With agonists, the activity was expressed in IU/mg, whereas
with antagonists as pA2. The pA2 values represent the negative
logarithm to the base 10 of the average molar concentration of an
antagonist that reduces the response to 2x units of the agonist to
the response to x units of the agonist. The volume of distribution in
the in vivo experiments is arbitrarily taken as 67 ml/kg. The
responses to standard doses of oxytocin or vasopressin were stable
for several hours, without problems with tachyphylaxis. For details
see Ref. [38].
m
m, 4.6 ꢀ 250 mm) or Hypersil BDS C18 column (5
mm,
4.6 ꢀ 250 mm)) produced single peaks with at least 98% of the total
peptide peak integrals. The final verification of the peptide
sequence and purity were achieved by MALDI TOF mass spectros-
copy (we did not detect unexpected molecular ions). The physico-
chemical properties of new analogues are summarized in Table 1.
3. Pharmacology
3.1. Bioassay methods
3.4. Antidiuretic activity
Wistar rats were used in all experiments. Handling of the
experimental animals was done under supervision of the Ethics
Committee of the Academy of Sciences according to x 23 of the Law
of the Czech Republic No. 246/1992.
Tests to assess the antidiuretic properties were conducted on
conscious male rats using the modified Burn test [39,40]. In the
standard manner with hydrated rats, the animals having fasted for
16 h were weighed and then given tap water through a stomach
catheter. The water load was 4% of the body weight. Immediately
after the water load, the tested substances (or physiological saline
as control) were administered subcutaneously at doses of 0.001–
100 nmol/kg. The rats were then placed in individual metabolic
cages, and their urine was collected over a 5 h period. The time t1/2
in which the rats excreted half the water load was determined and
then plotted against the dose. As the dose–response curves were
not parallel, such doses were chosen for calculating the com-
pound’s potency which yield t1/2 equal to 60 min (the so-called
threshold doses equal to the value of t1/2 obtained with the phys-
iological solution) and t1/2 equal to 200 min. On each day of the
experiment, 21 rats divided into 5 groups of 4 or 5 animals were
administered different doses of different compounds; in an average
3.2. Uterotonic activity
The uterotonic test was carried out in vitro on the strips of rat
uterus in the absence of magnesium ions [35,36]. Rats in induced
estrus by the injection of estrogen 48 h before the experiments
were used. After decapitation, the uterine horns were excised,
longitudinally cut, placed into a bathing chamber and hooked up to
recorder of contractions. The height of the single isometric
contraction of a uterine strip was measured. In principle, cumula-
tive dosing was applied in the experiments, i.e. doses of standard
(in the presence or absence of analogues) or of the analogue were
added successively to the uterus in the organ bath in doubling

A. Kwiatkowska et al. / European Journal of Medicinal Chemistry 44 (2009) 2862–2867
2865
Table 2
Pharmacological properties of the new AVP analogues together with the values for AVP and some related analogues.
Activityf
Analogue
Uterotonic in vitro no
Mg2þ IU/mg or pA2
Pressorg IU/mg or pA2
Antidiuretich
IU/mg
% of activity of dDAVP
t1/2 ¼ 60
450
t1/2 ¼ 200
450
–
–
–
–
–
w9300
9000
45 000
45 000
20 000
3000
20 000
<4.5
4500
45 000
200 000
400
t1/2 ¼ 60
10
100
23.2
73.9
–
–
–
10
10
t1/2 ¼ 200
AVPa
17
1.5–5.1
0.4
27–63
412
w0.39
4.1
346–370
0.125e
100e
1.7e
97.3e
–
[Mpa1,
D
-Arg8]VPa (dDAVP)
800–50 000
111–257
1300–1745
758
0.033
750–900
450
450
w450
100
30
30
<4.5
100
1000
4500
4.5
4500
1000
9000
9000
[
D
-Arg8]VPa (DAVP)
[Mpa1]AVPa (dAVP)
[Val4]AVPa
–
32
[Cpa1]AVPa
pA2 ¼ 8.15
pA2 ¼ 8.35
–
–
3.5
18
18
8
1.2
8
<0.1
1.8
[Acc2]AVPb
pA2w5.6
56.6
[Dip2]AVPc
pA2 ¼ 7.00 ꢃ 0.20
pA2 ¼ 7.20 ꢃ 0.08
pA2 ¼ 7.27
0
[Mpa1,Dip2]AVPc
[Aic2]AVPd
pA2 ¼ 6.20
9.4 ꢃ 2.8
10
[Mpa1,Dip2,Val4]AVP (I)
1.7 ꢃ 0,2
0
0
0
0
0
0
0
2.2
0.7
0.7
<0.1
2.2
22
100
0.1
100
22
[Dip2,
D
-Arg8]VP (II)
0.44 ꢃ 0,11
0.04 ꢃ 0 01
pA2 ¼ 6.23 ꢃ 0.14
0.24 ꢃ 0,12
pA2 ¼ 7.82 ꢃ 0.39
pA2 ¼ 7.80 ꢃ 0.14
pA2 ¼ 6.83 ꢃ 0.32
7.6 ꢃ 4.3
[Mpa1,Dip2,
[Cpa1,Dip2]AVPc (IV)
[Mpa1,Dip2,Val4, -Arg8]VP (IV)
-Dip2]AVP (V)
D
-Arg8]VP (III)
D
[D
18
[Mpa1,
D
-Dip2]AVP (VI)
-Dip2]AVP (VII)
-Dip2,Val4]AVP (VIII)
-Arg8]VP (IX)
-Dip2, -Arg8]VP (X)
-Dip2,Val4, -Arg8]VP (XI)
w80
0.5
w150
8
w100
w150
[Cpa1,
D
pA2 ¼ 7.4
pA2 < 5.8
0
[Mpa1,
D
450 000
20 000
225 000
450 000
[
D
-Dip2,
D
pA2 ¼ 7.83 ꢃ 0.08
pA2 ¼ 7.79 ꢃ 0.09
pA2 ¼ 7.78 ꢃ 0.08
[Mpa1,
[Mpa1,
D
D
pA2 ¼ 5.8
200
200
D
D
pA2 < 5.8
a
Values taken from Ref. [17].
Values taken from Ref. [20].
b
c
Values taken from Ref. [21]; in that paper 3,3-diphenylalanine was abbreviated as Dpa.
Values taken from Ref. [41].
Values taken from Ref. [45].
d
e
f
The values are given as averages ꢃ SEM, n ¼ 3–5.
g
h
0 means no activity up to the dose of 0.15 mg/kg of experimental animal.
Activity was estimated on the threshold level of activity 60 min and on the level of the 200 min activity; activity of AVP taken as 450 IU/mg at both levels.
each compound was tested in 3–5 different doses, each dose being
tested in 2 or 3 independent experiments (different days, different
rats). The results were thus expressed in IU/mg in comparison to
AVP (the value 450 IU/mg was taken for AVP for both t1/2 60 min
and t1/2 200 min). All peptides showed some degree of antidiuretic
activity and no peptide enhanced the quantity of excreted urine or
speed-up the excretion in the course of any experiment (in
comparison to controls). We have thus not expected a diuretic
effect and not performed the diuretic test using non-hydrated rats.
300
250
200
150
100
50
4. Results
The 11 new analogues of AVP (I–XI) were synthesized by Boc
strategy, purified by HPLC and characterized. The purity of all the
analogues was higher than 98%. Physicochemical properties of the
new peptides and their pharmacological data are presented in
Tables 1 and 2, respectively.
The activities of the analogues were determined in the in vitro rat
uterotonic test in the absence of magnesium ions, in the rat pressor
test, and in the antidiuretic assayusing conscious ratsas described in
the experimental section. The comparison of the antidiuretic activ-
ities of the new analogues with those published previously in the
literature is complicated by the fact that different methods were
used for the activity determination and that the dose–response
curves of the analogues and that of standard AVP have different
slopes (Fig. 2). It is therefore necessary to give two values of potency,
the firstone resulting fromcomparison of the threshold doses of AVP
with those of the analogues (antidiuresis time t1/2 60 min) and the
second one originating from comparison of doses giving an anti-
diuresis time of 200 min. The antidiuresis time (t1/2) corresponds to
XI
X
VIII
IX
dDAVP
AVP
0
8
7
6
5
4
3
2
1
-log d [mg/kg]
Fig. 2. Dose–response curves of the antidiuretic effect of analogues deamino[D-Dip2,
Val4,D-Arg8]VP (XI, -), deamino[D-Dip2,D-Arg8]VP (X, O), [D-Dip2,D-Arg8]VP (IX, B),
[Cpa1,D-Dip2]AVP (VIII, :) and standards deamino[D-Arg8]VP (dDAVP, ꢄ) and AVP (A).

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A. Kwiatkowska et al. / European Journal of Medicinal Chemistry 44 (2009) 2862–2867
the time in which the rat excretes half of the water load. For AVP, the
activity has arbitrarily been set to 450 IU/mg for both responses.
Peptides I–IV with Dip at position 2 have antidiuretic activity on
the threshold level 4–15 times lower than that of AVP, at the 200-
min level they are however 7–45 fold more effective than AVP.
increases the selectivity [42,43], did not further increase the anti-
diuretic activity substantially. All the Dip2 substituted analogues
are very selective as they do not show any pressor activity and
exhibit either very low or negligible uterotonic activity.
As far as analogues V–XI modified in position 2 with
concerned, it can be seen that all the new peptides, with the
exception of [Cpa1, -Dip2]AVP, are very potent and selective agonists
D-Dip are
Analogues V, VI, VIII–XI with D-Dip at position 2 exhibit much
higher activity than AVP already at the level of t1/2 60 min (2–20
times), and as their dose–response curves are also much steeper in
comparison with that of AVP (comparable to dDAVP), the activity
at the t1/2 level of 200 min is much higher than that of AVP (up to
1000 times). Analogue VII showed only low antidiuretic activity –
Cpa1 modification decreased both antioxytocic and antidiuretic
activities.
Peptides I–VI and IX did not exhibit pressor or antipressor
activities while the remaining analogues were either moderate
(VII) or week (VIII, X, XI) pressor antagonists. Analogues I–IV and
VIII showed low agonistic activity in the uterotonic test in vitro,
while peptides V–VI and IX–XI exhibited high antioxytocic potency
(pA2 ¼ 7.78–7.83), only analogue VII displayed one order of
magnitude lower antioxytocic activity (pA2 ¼ 6.83).
D
of V2 receptors. Their activity is about 2–20 fold higher than that of
AVP as calculated on the basis of the threshold doses. They are,
however, 44–1000fold moreactivethan AVP whencomparingdoses
giving the 200 min antidiuresis. This means that up to 1000 times
less of the compound is necessary to evoke the same effect
(t1/2 ¼ 200 min). Of course we do not know whether the prolonga-
tion of the activity is due to better binding to the receptor, or better
signal transformation or slower elimination from the organism.
It is very interesting to note that only a single modification of
position 2 with either Dip or
D-Dip changes dramatically the
properties of the parent hormone: preservation of the antidiuretic
activity and its prolongation, transformation of uterotonic into
antiuterotonic properties and elimination of any effect on blood
pressure. It is also worth noting that the effect is more pronounced
5. Discussion
with the
D-Dip than with Dip (compare the activities of analogue V
with that of its Dip counterpart). In the
D
-series, the impact of
We have previously demonstrated significant impact of steric
restrictions and bulkiness of the substituents in the N-terminal part
of the AVP and OT molecules on pharmacological properties of the
resulting analogues. Our studies included among others the
modification of AVP and some of its analogues with Acc and Apc,
which resulted in compounds having high antidiuretic potency,
decreased pressor activity and either no activity or low oxytocin
antagonizing activity in the uterotonic in vitro tests [24–26].
Recently we presented the synthesis and some pharmacological
properties of the AVP analogues substituted in position 2 with 2-
aminoindane-2-carboxylic acid (Aic) [41]. This modification resul-
ted in similar changes of pharmacological properties as that which
caused modification with Apc and Acc. Enlargement of the cyclic
side chain and restoring its aromatic character, placing the aromatic
ring closer to the backbone in comparison to Tyr, led either to
preservation or a slight decrease in antidiuretic activity as calcu-
lated from the threshold doses, as well as enhancement of activity
when comparing doses eliciting a 200 min antidiuresis. One of the
deamination of position 1 (Mpa1) was as expected – the antidiuretic
potency of the resulting compound VI was enhanced. Additional
substitution of this analogue in position 4 with Val resulted in
further prolongation of the activity (peptide VIII) and appearance
of a very weak antipressor activity.
In regard to the uterotonic test, the new peptides V–XI have
high antioxytocic potency with the exception of analogue VII which
displays weak antioxytocic properties and of analogue VIII which
exhibits low agonistic activity. High antioxytocic activity of most of
our analogues is very interesting, especially that of peptide V that
differs from AVP in position 2 only. It is also worth emphasizing that
analogue VII having a bulky and lipophilic substituent in position 1
is a relatively weak antagonist of oxytocin in the uterus in vitro test,
which is in contrast with the previous knowledge [42,43]. Another
surprising finding is the low uterotonic agonistic activity of peptide
VIII which differs from the potent antagonist of oxytocin (peptide
VI) by the presence of Val at position 4 only. The Val4 modification
was thought to increase antidiuretic activity. In the case of
dAVP [21] introduction of Val in position 4 also enhanced the ute-
rotonic activity. These observations support the hypothesis that
in the case of analogues having conformational restriction in the
N-terminal part of the molecule accumulation of modifications
does not result in enhancement of the desired activity. Single
modifications leading to the same effect, e.g. enhancement of
antidiuretic activity, may not necessarily have an additive effect if
introduced together.
peptides, namely [Mpa1,Aic2,Val4, -Arg8]VP, had an antidiuretic
D
activity similar to that of dDAVP, thus being one of the most potent
antidiuretic peptides reported to date [41]. These, in our opinion
interesting findings, promoted us to further investigate the impact
of reducing the conformational freedom of the N-terminal part of
the AVP analogues by placing in position 2 other bulky residue 3,3-
diphenyl-L-alanine (Dip). The results were encouraging [27]. Single
modification of the AVP or dAVP molecules, i.e. substitution of Tyr2
with Dip resulted in peptides with strikingly different pharmaco-
logical properties in comparison with the parent compounds (Table
2). The two analogues were moderately potent uterotonic antago-
nists, displayed no activity or low antagonism in the pressor test
while their antidiuretic activity was preserved and prolonged.
In this study we have introduced the same non-proteinogenic
amino acid Dip into other 4 analogues. We have also introduced its
As far as the pressor test is concerned, it is clear that in
comparison to Tyr2 the -Dip2 analogues are either devoid of the
D
pressor activity or become converted into very weak vasopressin
antagonists. In the case of [Cpa1]AVP, both the Dip2 [27] and -Dip2
D
modifications (analogue VII) strongly reduced its antipressor
potency. Probably two bulky substituents in the vicinity of one
another (positions 1 and 2) cannot be tolerated by the receptors.
Usually, replacement of Tyr2 with proteinogenic amino acids
leads to the decrease of all biological activities. An exception
D-enantiomer (D-Dip) into position 2 of the same 7 analogues. The
results presented in Table 2 show that antidiuretic potency of the
analogues modified at position 2 with Dip and additionally having
different combinations of changes at positions 1 and 4 is lower than
that of AVP as calculated on the basis of the threshold doses. On the
other hand, the peptides exhibit significantly prolonged activity.
The Dip modification itself results in prolongation of the effect.
Mpa1 and Val4 substitutions commonly used to increase anti-
presents [Phe2]AVP and [
[21]. They have decreased uterotonic activity, slightly reduced
pressor activity and preserved or enhanced antidiuretic activity.
D
-Tyr2]AVP and their deamino analogues
D
-
Dip with its 2 bulky aromatic phenyl groups may disturb the so
called stacking of aromatic side chains in positions 2 and 3 and this
way reduce markedly the pressor effect. The so called ‘‘stacking’’ is
not important for the antidiuretic activity.
diuretic potency and
D-Arg introduction in position 8 which

A. Kwiatkowska et al. / European Journal of Medicinal Chemistry 44 (2009) 2862–2867
2867
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In general, the presence of
with the
activity of the peptides and in the case of pressor activity it triggers
a weak vasopressin V1a antagonism.
D
-Dip at position 2, in comparison
L
-enantiomer, more distinctly enhances antidiuretic
On the basic of these results, we are undertaking further
investigations on our Dip2 and -Dip2 modified AVP analogues to
D
determine binding affinity to V1a, V1b, V2 and OT receptors. Parallely
we are performing the structural analysis and molecular modeling
of the peptides that may help to reveal processes underlying these
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6. Conclusion
ˇ
ˇ
[21] M. Lebl, K. Jost, F. Brtnik, in: K. Jost, M. Lebl, F. Brtnik (Eds.), Tables of Analogs
in Handbook of Neurohypophyseal Hormone Analogs, vol. II, CRC Press Inc.,
Boca Raton, FL, 1987, pp. 127–267 Part 2.
In summary, our studies have shown once more that the
modification of position 2 which changes conformation of the N-
terminal part of the analogue has a dramatic impact on the phar-
macological profile of the compound (in this case it resulted in
a high and prolonged V2 agonistic activity). This finding supports
our previous results showing that single substitution with some
bulky residues may result in quite potent antagonists of OT
receptors [44]. Such substitution is clearly disadvantageous when
applied to antagonists of V1a receptors, as has been documented in
our previous paper [25]. It should be emphasized that four of the
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´
new peptides ([Mpa1,
D
-Dip2]AVP, [Mpa1,
D
-Dip2,Val4]AVP, [Mpa1,
[27] W. Kowalczyk, A. Prahl, I. Derdowska, D. Sobolewski, J. Olejnik, J. Zabrocki,
D
-Dip2, -Arg8]VP, [Mpa1,
D
D
-Dip2,Val4, -Arg8]VP) are exceptionally
D
´
´
L. Borovickova, J. Slaninova, B. Lammek, J. Med. Chem. 49 (2006) 2016–2021.
[28] M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis, Springer-
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Partial funding for this work was provided by grant 0230/B/H03/
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