Influence of enantiomers of 1-naphthylalanine in position 2 of VAVP and dVAVP on their pharmacological properties

Article abstract of DOI:10.1016/j.ejmech.2004.09.011

In this study, we described the synthesis and some pharmacological properties of four new analogues of arginine vasopressin (AVP). Two peptides are substituted in position 2 with l-1-naphthylalanine (l-1-Nal) or its d-enantiomer and in position 4 with val

Full text of DOI:10.1016/j.ejmech.2004.09.011

European Journal of Medicinal Chemistry 40 (2005) 63–68
www.elsevier.com/locate/ejmech
Original article
Influence of enantiomers of 1-naphthylalanine in position 2 of VAVP
and dVAVP on their pharmacological properties
Izabela Derdowska a, Adam Prahl a, Wioleta Kowalczyk a,*, Marcin Janecki b, Samed Melhem b,
Henryk I. Trzeciak b, Bernard Lammek a
a Department of Chemistry, University of Gdan´sk, Sobieskiego 18, 80-952 Gdan´sk, Poland
b Department of Pharmacology, Silesian Medical University, Jagiellon´ska 4, 41-200 Sosnowiec, Poland
Received 26 April 2004; accepted 15 September 2004
Available online 05 November 2004
Abstract
In this study, we described the synthesis and some pharmacological properties of four new analogues of arginine vasopressin (AVP). Two
peptides are substituted in position 2 with L-1-naphthylalanine (L-1-Nal) or its D-enantiomer and in position 4 with valine. In the further two
compounds, we combined the above modifications with placement into position 1 of 3-mercaptopropionic acid residue (Mpa). All new
peptides were tested for vasopressor and antidiuretic activities. We also estimated the uterotonic activities of these compounds in vitro. Urine
samples prior and after peptide administration were analyzed for electrolytes excretion. All analogues are potent oxytocin antagonists. One of
them, namely [L-1-Nal2,Val4]AVP, which appears practically not to interact with V1a and V2 receptors, is exceptionally selective. Our results
open new possibilities for the design of very potent and selective oxytocin antagonists in vitro.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Arginine vasopressin; Naphthylalanine; Oxytocin antagonists
1. Introduction
corporation of nonproteinogenic amino acid residues, to in-
troduce bulky groups with the aim being to restrict the con-
formational flexibility of
a peptide. Conformational
Many analogues of the arginine vasopressin (AVP) have
been synthesized in the course of extensive investigations into
their structure and activity [1–4]. However, despite intensive
efforts in many laboratories, attempts to design potent and
truly selective agonists and antagonists ofAVP receptors still
remain a real challenge. To find a solution to the aforemen-
tioned problem, the discovery of new clues to the design of
AVP analogues would be certainly helpful.
restrictions are of particular value for peptide design targeted
towards an increase of receptor selectivity, metabolic stabil-
ity and the development of highly potent agonists and antago-
nists. Also, in our laboratory we have shown that such an
approach could result in analogues with very interesting
pharmacological properties [5,6]. In 1997, we described the
synthesis and some pharmacological properties of analogues
having L-1-naphthylalanine (L-1-Nal) or L-2-naphthylalanine
(L-2-Nal) in position 3 [5]. One of the new peptides, [L-2-
Nal3,D-Arg8]VP, was among the most potent and selective
antagonists of V1a receptors reported to date. Moreover, it
was the first V1a antagonist devoid of antiuterotonic activity,
and its high antipressor potency arises without modification
of position 1, which was previously thought to be essential
for substantial pressor antagonism [5]. Two other peptides,
[Mpa1,L-1-Nal3,D-Arg8]VP and [Mpa1,L-2-Nal3,D-Arg8]VP,
were highly potent V2 agonists. The second peptide was
exceptionally selective. Our results suggested that position
3 in AVP and its analogues is important not only for
The most straightforward approach for peptide modifica-
tion is to introduce changes into the side chains of chosen
amino acids. This strategy allows, among others, by the in-
Abbreviations: AVP, arginine vasopressin; Mpa, 3-mercaptopropionic
acid; Mob, 4-methoxybenzyl; L-1-Nal, L-1-naphthylalanine; D-1-Nal, D-1-
naphthylalanine; L-2-Nal, L-2-naphthylalanine; D-2-Nal, D-2-naph-
thylalanine; NMP, 1-methyl-2-pyrrolidone; TBTU, 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid.
* Corresponding author. Tel.: +48 58 345 03 88; fax: +48 58 341 03 57.
E-mail address: wiola@chem.univ.gda.pl (W. Kowalczyk).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.011

64
I. Derdowska et al. / European Journal of Medicinal Chemistry 40 (2005) 63–68
binding and recognition as previously thought, but also for
pressor, antidiuretic and uterotonic activities [5]. We also
assumed that the hindering effect caused by bulky naphthyl
moiety has a significant impact on the bioactive conforma-
tions of molecules which contain Nal residue, and can thus
influence their interaction withV1,V2 and oxytotic receptors.
Subsequently, knowing that the tyrosine residue in position
2 seems to play a part in initiating the pressor response of
AVP [7] we decided to place there, in AVP and its analogues,
L-1-Nal or its D-enantiomer [6]. These modifications resulted
in the removal of pressor activity, or conversion into moder-
ate antagonists. All new peptides exhibited varying degrees
of antiuterotonic activity. Two compounds, [Mpa1,L-1-
Nal3]AVP and [Mpa1,D-1-Nal3]AVP, were exceptionally se-
lective antagonists of oxytocin in vitro and thus are promis-
ing candidates for the development of potential tocolytic
agents for the prevention of pre-term labour.
Recently, continuing in this direction, we decided to re-
place the residues in positions 2 or 3 of AVP and some of its
analogues with 1-aminocyclohexane-1-carboxylic acid
(Acc) [8]. Again, we selected the Acc in order to reduce the
flexibility of the peptides by imposing a sterically con-
strained residue limiting conformational freedom, thus forc-
ing the peptide backbone and side chains to adopt specific
orientations. Our results clearly demonstrated that Acc3
modification was deleterious to interaction with V1, V2 and
oxytocin receptors. In the case of Acc2 substitution, the
situation was different, as it selectively modified interaction
with the aforementioned receptors. Two of the new ana-
logues, [Acc2]AVP and [Acc2, D-Arg8]VP, are potent antidi-
uretic agonists. The results described also provided useful
information about structure–activity relationships and thus
opened up new possibilities, particularly in the design of
highly potent and selective V2 agonists.
points were determined on a capillary melting point appara-
tus and are uncorrected. Thin layer chromatography (TLC)
was carried out on silica plates (Merck). The spots being
visualized by iodine or ninhydrin. The acetic acid/butan-1-
ol/ethyl acetate/water (1:1:1:1) solvent system was used.
High-performance liquid chromatography (HPLC) for pep-
tides was carried out on a Gold System Beckman analytical
chromatograph equipped with a UV detector. The purity of
the peptides was determined on a Vydac C18 column (5 µm,
10 × 250 mm) with precolumn Ultrasphere ODS (5 µm, 4.6 ×
45 mm). The following solvent systems were used: (1) 0.1%
aqueous trifluoroacetic acid (TFA), (2) acetonitrile: 0.1%
TFA (80:20 v/v); a linear gradient from 30–70% of (2) for
20 min at a flow rate of 1 ml/min (k = 226 nm). FAB MS of
peptides was recorded on a TRIO-3 mass spectrometer at
7 keV with argon as the bombarding gas.
All amino acid derivatives were purchased from Bachem
AG. S-p-methoxybenzyl-3-mercaptopropionic acid, Mpa-
(Mob), was obtained according to Mpa(Bzl) [9] using
p-methoxybenzyl chloride as follows:
200 mmol (4.6 g) of metallic natrium was added to mix-
ture of 100 mmol (10.6 g, 8.7 ml) Mpa in 300 ml of anhy-
drous ethanol; and then 105 mmol (16.4 g, 14.5 ml, (98%)) of
4-methoxybenzoyl chloride (Mob-Cl) were add with stirring.
The reaction mixture was heated and stirred for an additional
30 min, then the solvents were removed under reduced pres-
sure. The oily residue was dissolved in water end extracted
with diethyl ether (2×). The water layer was washed with
ethyl acetate (2 × 30 ml), acidified with 6 N HCl to pH
= 2 and extracted with ethyl acetate (3 × 50 ml). The organic
layer was dried over anhydrous MgSO4 and evaporated to
dryness. The crude residue was crystallized from CHCl3:n-
hexane yielding 82% of white crystalline product. Mp. 73–
75 °C.
Considering all this, as a continuation of our efforts to
better understand the role of Tyr2, we have now designed,
synthesized and determined some pharmacological proper-
ties of two new peptides modified in positions 2 and 4 with
L-1-Nal or D-1-Nal and valine, respectively. In the further
two compounds, we combined the above substitutions with
placement into position 1 of 3-mercaptopropionic acid resi-
due (Mpa). The structures of these peptides are as follows:
2.2. Peptide synthesis
All peptides were synthesized manually by solid phase
method, i.e. by the stepwise coupling of Boc-amino acids to
the growing peptide chain on a 4-methylbenzhydrylamine
resin (MBHA resin, Senn Chemicals AG, 1% DVB, 200–
400 mesh, 0.67 mmol/g). Fully protected peptide resins or
acylopeptide resins were synthesized according to the stan-
dard procedures involving (i) deprotection steps using 33%
TFA in the presence of anisole (1%), 5 and 25 min; (ii)
neutralization with 10% TEA/DCM, 3 and 7 min (iii), cou-
plings in DCM/DMF (1:1, v/v) carried out using DCC and
HOBT. The couplings of Boc-1-Nal, Boc-D-1-Nal, Boc-
Cys(Mob) and Mpa(Mob) were mediated by TBTU and
HOBt in the presence of DIEA in a mixture of DMF, NMP
and DCM (1:1:1 v/v) containing 1% Triton. The complete-
ness of each coupling reaction was monitored by the Kaiser
et al. [10] or chloranil test [11]. Re-coupling was performed
when the test was positive. After completion of the synthesis,
the protected nonapeptidyl resins were treated with 10 ml of
liquid hydrogen fluoride (HF) containing 0.5 ml of anisole at
X-Y-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH2
where:
X = Cys
X = Cys
X = Mpa
X = Mpa
Y = L-1-Nal
Y = D-1-Nal
Y = L-1-Nal
Y = D-1-Nal
(I)
(II)
(III)
(IV)
2. Materials and methods
2.1. General
All solvents were purified by conventional methods.
Evaporation was carried out under reduced pressure. Melting

I. Derdowska et al. / European Journal of Medicinal Chemistry 40 (2005) 63–68
65
–70 °C and stirred for 60 min at 0 °C [12]. After the removal
of HF and anisole in-vacuo, the mixture was washed with
anhydrous diethyl ether, then with acetic acid and the solu-
tion diluted with methanol. The resulting dithiols were oxi-
datively cyclized with 0.1 M I2 in methanol using the normal
procedure. The solvents were evaporated under reduced pres-
sure and the resulting materials dissolved in water and lyo-
philized. The crude products were desalted on a Sephadex
G-15 column eluted with aqueous acetic acid (30%) at a flow
rate of 4.0 ml/h, k = 254 nm. The eluates were fractionated,
and the fractions containing the major peak were pooled and
lyophilized. The residue was then subjected to gel filtration
on a Sephadex LH-20 column eluted with 10% aqueous
acetic acid at a flow rate of 4.0 ml/h, k = 254 nm. The
peptides were eluted as single peaks. The purity and identity
of each peptide were determined by HPLC and FAB mass
spectrometry (molecular ion). The values of the molecular
ions were as expected. The physicochemical properties of
peptides are presented in Table 1.
method [15]. Anaethesia, however, was induced by injecting
150 mg/kg of thiobutabarbital (Inactin, RBI, USA) ip.
[16,17]. Catheters were inserted into the jugular vein for
peptide infusion. After laparatomy, a polyethylene (PE) can-
nula was introduced into the urinary bladder and ligated. The
urine flow was collected directly from the urinary bladder up
to urine flow rate stabilization (0.8–1.2 ml/10 min.).
From the beginning of the experiment, the animals were
connected by PE catheter to a syringe infusion pump (Medi-
pan, type 612, Poland). Water balance was kept constant for
each rat by rehydrating with hypoosmotic water solution
(180 mOsm/kg) containing 0.24% NaCl and 1.8% glucose,
adding arbitrary excess (1–2 ml/h) to compensate for extra-
renal water loss.
Peptides were injected into the jugular vein as a bolus in a
total volume of ~0.3 ml (including wash) after achieving the
urine flow stabilization in three consecutive 10 min periods at
40 min intervals. Anesthesia was maintained throughout the
experiment.
Antidiuretic response was defined as: Vp/Vo × 100, where
Vo is the urine volume collected in the 10 min period preced-
ing the injection of AVP or analogue; Vp is the volume of
urine collected during the 10 min starting period immedi-
ately after injection. Agonistic activity was expressed in
international units per milligram (IU/mg) of peptide through
the comparison of regression lines for OT orAVP and related
peptides [18].
2.3. Bioassay methods
The vasopressor and antidiuretic activity of AVP ana-
logues were tested on male Wistar rats, 250–270 g, bred at
the Central Experimental Animal Farm of the Silesian Medi-
cal University.
The use of animals was reviewed and approved by the
Local Ethics Committee for Animals Experiments in Katow-
ice (Poland).
Antagonistic activity of AVP analogues was measured by
the method of Schild [24] and described as effective dose
(ED) and pA2 value.
2.4. Vasopressor activity
Each peptide was administered in two doses; high, which
reduces the response to 2 × units of agonist to less than the
response to 1 × unit of agonist; and low, which did not fully
reduce the response obtained by 2 × units of agonist. The pA2
values estimated in vivo represent the negative logarithms (to
base 10) of EDs divided by an assumed volume of distribu-
tion in rats (67 ml/kg).
The AVP was used as a standard agonist in all assays. Its
pressor and antidiuretic activities were accepted as
369 IU/mg and 323 IU/mg, respectively [19].
Vasopressor assays of AVP analogues were performed in
vivo according to the method of Dekanski [13] as described
by Stürmer [14]. The rats were given 1.75 g/kg urethane
intraperitoneally (ip.). Blood pressure stabilization was
evoked by phenoxybenzamine (SmithKline Beecham, USA)
in two or three repeated doses, each amounting to 1 mg/kg
intravenously (iv.). Peptides were injected into the cannu-
lated jugular vein in a volume of 0.1 ml (saline) and the
cannula was rinsed with 0.2 ml saline. Analogues were in-
jected at 30–40 min intervals. Systolic blood pressure mea-
surements were taken by pressure transducer (type PL3JD,
Gould-Statham, USA) fixed into the carotid artery.
2.6. Uterotonic and antiuterotonic activity
Determination of uterotonic or antiuterotonic activity was
assayed on isolated rat uterus using the procedure of Holton
[20] as described previously with additional modifications
[6,21]. Briefly, virgin female Wistar rats weighing 220–250 g
were used in the proestrus/oestrus stage artificially induced
24 h earlier by injection of oestradiol benzoate (0.8 mg/kg,
intramuscularly). Oestrus stages were determined by vaginal
smears made 1–2 h before each experiment. The uterine
horns were isolated about 2 cm, close to the ovarian end
under Inactin (RBI, USA) anaesthesia (125 mg/kg ip.) [16]
and immediately mounted in a 20 ml bathing chamber.
The bathing fluid was the Munsick’s modification of van
Dyke–Hasting’s solution, supplemented with Mg2+ [22,23].
2.5. Antidiuretic activity
Antidiuretic activity of peptides was estimated in rats in
vivo according to Stürmer’s modification [14] of Sawyer’s
Table 1
Physicochemical properties of peptides I–IV
Analogue
(M+H+)
RP-HPLC
TR (min)
Calculated
1089.51
1089.51
1074.5
Found
I
1091.0
1090.6
1075.3
1075.4
11.60
11.82
13.63
14.59
II
III
IV
1074.5
The purity of analogues determined on HPLC was between 97% and 99%.

66
I. Derdowska et al. / European Journal of Medicinal Chemistry 40 (2005) 63–68
The solution had the following composition: NaCl 114.0;
KCl 6.2; NaHCO3 30.0; NaH2PO4 1.0; CaCl2 0.5; MgCl2
0.5 and glucose 2.8 (in millimolar concentration). The reser-
voir and the bath were gassed with a mixture of 95% oxygen
and 5% carbon dioxide. The temperature of the bath solution
was 37 °C.
2.7. Urinary electrolytes analysis
During the antidiuretic experiments, the urine volumes
were collected in consecutive 10 min periods before and after
peptide administration. Urinary sodium, potassium, calcium
and chloride concentrations of all samples were determined
using Synchrom EL-ISE Electrolite System (model 4410,
Beckman, Instruments Inc., USA). The electrolytes excre-
tion (mEq/10 min) were expressed as a percentage after vs.
before peptide administration.
The results are presented as mean SEM of n samples.
Statistical significance of differences of base-line values be-
tween experimental groups was determined by unpaired two-
tailed t-test. A P < 0.05 value was considered significant.
Contractions were measured isometrically with
a
K-30 force displacement transducer (type 351), two-channel
bridge amplifier (type 301, Hugo Sachs Electronics, Ger-
many) and recorded on R-50 multi-pen recorder (model 83,
Rikadenki, Japan). The tissue was subjected to a base-line
tension of 1 g; if regular tension development was not main-
tained during the initial 30 min stabilization period, it was
discarded.
The uterotonic activity was measured for 10 min by inte-
gration of the area under the response curve (AUC) after the
addition of different concentrations of OT (10–10 ÷ 10–6 M/L)
or AVP (1 × 10–9 ÷ 3 × 10–6 M/L). The organ bath was rinsed
out three times with bathing solution after each application of
agonists.
3. Results
The four new analogues of AVP (I-IV) were synthesized
by stepwise coupling of Boc-amino acids to the growing
peptide chain on a p-methoxybenzhydrylamine resin. The
couplings were mediated by the DCC/HOBT or
TBTU/HOBT methods. For peptide III and IV, Mpa(Mob)
was used in the final coupling step. On completion of the
syntheses, the protected peptidyl or acylpeptidyl resins were
treated with liquid HF in the presence of anisole at 0° and
oxidized with I2 in methanol. The crude peptides were puri-
fied on Sephadex G-15 and LH-20. Some of the pharmaco-
logical properties of the new analogues I-IV, also those of
AVP, [Mpa1]AVP and some related peptides, are presented in
Table 2. None of these new analogues exhibited pressor,
anti-antidiuretic or uterotonic activity.
The antiuterotonic potency of the AVP analogues was
measured by the same procedure and the OT or AVP was
used as the standards in noncumulative increasing concentra-
tions 1 min after administration of the antagonist (10–7 M/L).
To express the antagonistic activity of AVP analogues as a
pA2 value, a similar procedure was used. The pA2 value is
defined as the negative logarithm (to base 10) of the molar
concentration of an antagonist, which reduces the contractile
response of agonist to the effect produced by one half of the
administered agonist concentration. The mean values of con-
centration ratios for the OT or AVP pair were calculated by
the method of Schild [24] and plotted in a diagram by regres-
sion analysis. The pA2 value was obtained from the intercept
of the regression line with the abcissa using PHARM/PCS
4.0 pharmacological calculation system [25]. All values of
pA2 were presented as the mean SEM.
The vasopressin antagonistic activity of analogues was
measured according to the method of Schild [24] as de-
scribed by Manning and Sawyer [19]. Peptides II-IV with
pA2 values ranging from 7.37–7.88 exhibited a moderate
antivasopressor potency: peptide I was inactive.
Table 2
Pharmacological data on vasopressin analogues
Peptide
Uterotonic
IU/mg
Vasopressor
(IU/mg)
Antivasopressor
pA2
Antidiuretic
IU/mg
Antiuterotonic pA2
AVP
Oxytocin
[L-1-Nal2,Val4]AVP a
I
—
ND (6)
ND (6)
ND (6)
ND (6)
ND (4)
ND (4)
ND (4)
ND (4)
ND (7)
<0.001 (8)
<0.003 (8)
<0.001 (8)
<0.001 (8)
ND (4)
7.82 0.09 (9)
7.71 0.08 (7)
8.02 0.02 (8)
8.29 0.05 (6)
6.90 0.12 (6)
7.46 0.15 (8)
8.02 0.02 (8)
7.69 0.07 (6)
—
7.87 0.01 (4)
8.00 0.05 (4)
8.18 0.08 (4)
8.32 0.12 (4)
7.10 0.06 (4)
7.78 0.09 (4)
8.18 0.08 (4)
7.94 0.23 (4)
—
[D-1-Nal2,Val4]AVP a
II
III
—
7.37 0.2 (8)
7.88 0.78 (8)
7.35 0.59 (8)
ND (4)
[Mpa1,L-1-Nal2,Val4]AVP a
[Mpa1,D-1-Nal2,Val4]AVP a IV
—
—
[L-1-Nal2]AVP b
[D-1-Nal2]AVPb
[Mpa1,L-1-Nal2]AVP b
[Mpa1,D-1-Nal2]AVP b
AVP c
V
—
VI
—
ND (4)
ND (4)
VII
VIII
—
7.1 0.53 (8)
ND (4)
<0.02 (8)
ND (4)
—
25.5
486 15
369
6
—
323 16
OT c
[Mpa1]AVP d
66
3
346 13
32
—
—
1745 385
738
—
—
—
[Val4]AVP e
a Results are expressed as a mean SEM. The numbers of rats or rat uterine horns are shown in parentheses. ND, not detectable. None of the naphthylalanine
analogues exhibited uterotonic activity or anti-antidiuretic activity up to 10–6 mol/l.
b Values taken from Ref. [6].
c Values taken from Ref. [26].
d Values taken from Ref. [30].
e Values taken from Ref. [31], estimated in Brattleboro rats.

I. Derdowska et al. / European Journal of Medicinal Chemistry 40 (2005) 63–68
67
The antidiuretic activity of the analogues was estimated
according to Stürmer [14] and Sawyer [15]. From the results,
it is clear that the modifications proposed resulted in almost
complete removal of interaction of peptides with V2 recep-
tors as all new analogues had negligible antidiuretic activity.
However, after administration of analogue I, we observed
statistically significant decrease of concentration of Ca2+,
while peptide II diminished concentration of Na+ and K+ in
urine (Fig. 1).
Antiuterotonic activity was assayed on an isolated rat
uterus by the procedure of Holton [20]. In this assay, all new
compounds I–IV are highly potent oxytocin antagonists
(pA2 7.82–8.29). They also effectively blocked the contrac-
tion of rat uterus caused by AVP (pA2 7.87–8.32). It is worth
pointing out that peptide I, which appears practically not to
interact with V1a and V2 receptors, is exceptionally selective
oxytocin antagonist in vitro. Moreover, the results showed
that all new analogues of AVP had negligible antidiuretic
activity (Table 2).
were exceptionally selective, however moderately potent an-
tagonists of oxytocin in vitro. Surprising activity of peptides
VII and VIII is even more interesting when compared with
[Mpa1]AVP. This compound, which may be treated as a
parent one for both analogues, is a potent pressor and antidi-
uretic agonist. Peptide VII differs from [Mpa1]AVP only in
the presence of different aromatic residues in position 2. We
believe that, as in previous examples [5], the presence of a
bulky naphthyl residue greatly influences the interaction of
analogue with receptors. These, in our opinion interesting
results, prompted us to search for additional modifications,
which would improve the antioxytocic potency of the above
analogues. Knowing that the valine in position 4 of AVP and
its analogues in many cases improves antidiuretic potency
and selectivity [26], but having also in mind unexpected
results discussed above, in this study we decided to deter-
mine how Val4 substitution of peptides V–VIII would
change their pharmacological properties. Additional stimu-
lus for such measure was another unexpected effect, when
placement of D-Arg into position 8 of [L-1-Nal3]AVP, instead
of enhance antidiuretic activity of analogue resulted in highly
potent and selective V1a antagonist [5].
4. Discussion
The present work is a part of our studies aimed at clarify-
ing the impact of sterical restrictions in the N-terminal part of
the AVP analogues on pharmacological properties. The phar-
macological data presented in Table 2 showed that our modi-
fication, commonly used to improve V2 agonism, leads in all
cases to substantial enhancement of antiuterotonic potency.
This improvement is very impressive especially when com-
paring a pair of peptides: I, V, as it is approximately in the
range of one order of magnitude (~10-fold enhancement).
Passing to antipressor activity, it is clear thatVal4 substitution
converted inactive in this respect compounds VII and VIII
into moderately potent V1a antagonists, thus diminishing
their selectivity. Therefore, it is even more interesting to note
that peptide I which appears practically not to interact with
Previously we reported that the hindering effect caused by
the bulky naphthyl moiety of L-1-Nal or D-1-Nal in position
2 ofAVP or [Mpa1]AVP had a significant impact on bioactive
conformations of molecules and thus influenced their inter-
action with V1a, V2 and oxytocic receptors [6]. The data for
compounds V–VIII, described in the above paper and pre-
sented here in Table 2, demonstrate that the above-mentioned
modifications resulted in the removal of pressor activity, or
conversion into moderate antagonists.As regards antidiuretic
activity, the modifications gave peptides, which were either
devoid of or had only negligible agonistic V2 potency. All
analogues exhibited varying degrees of antiuterotonic po-
tency (pA2 6.9–7.69). Three compounds V, VII, and VIII
V
1a and V2 receptors is a potent and exceptionally selective
blocker of oxytocin receptor in vitro and thus promising
candidate for the development of potential tocolytic agents
for the prevention of pre-term labour.
In recent years, there has been increasing interest in oxy-
tocin antagonists, as it is thought that oxytocin mechanisms
are involved in the initiation of term and pre-term labour. In
1995, Manning described a series of moderately potent and
fairly selective antagonists of oxytocin [27]. More recently,
Chan designed a specific antagonist for the oxytocin recep-
tors [28]. This analogue however, still possesses some anti-
V
1a activity. Other studies described the synthesis and phar-
macological evaluation of analogues with L- or D-Nal in
position 2 of oxytocin or AVP. In the case of oxytocin ana-
logues, the peptides obtained were selective and moderately
potent antagonists of this hormone [29]. This and previous
studies [5,6] clearly demonstrated the new possibilities
opened up by the use of Nal for modification of neurohy-
popheseal hormones. The results of this study seem to
strongly support these findings.
Fig. 1. Changes in electrolyte levels in rats’ urine following administration
of argininevasopressin analogues I–IV (expressed in percent relative to the
levels before administration). Results are expressed as mean S.D. Number
of rats n are shown under the bars. Sampling was made at 10 min intervals.
Electrolytes excretion was expressed in percent relative to the levels before
peptide administration (100%), which were determined for Na+ 19 7.6; K+
18 5.7; Ca2+ 0.59 0.11 and Cl– 44.5 15.8 (µEq/10 min). * P < 0.05;
statistically significant differences, when compared with value before pep-
tide administration.

68
I. Derdowska et al. / European Journal of Medicinal Chemistry 40 (2005) 63–68
[8] B. JastrzVbska, I. Derdowska, W. Kowalczyk, A. Machová, J. Slani-
It is well known that AVP plays a major role in the
regulation of water balance and participates in cardiovascular
homeostasis. The vasopressor effects ofAVP are mediated by
nová, B. Lammek, J. Pept. Res. 62 (2003) 70–77.
[9] D.B. Hope, V.V.S. Murti, V. Du Vigneaud, J. Biol. Chem. 237 (1962)
1563.
[10] E. Kaiser, R. Colescott, C.D. Bossinger, P. Cook, Anal. Biochem. 34
(1970) 595.
[11] T. Christensen, in: E. Gross, J. Meienhofer (Eds.),
Peptides—Structure and Biological Function, Pierce Chemical Com-
pany, Rockford, Illinois, 1979, pp. 385.
[12] J.M. Stewart, Solid Phase Peptide Synthesis, Pierce Chem, Corp,
Rockford, USA, 1984.
V
1a receptors, whereas the V2 type receptors, located almost
exclusively in the renal collecting duct, promote water per-
meability and sodium, potassium, calcium, magnesium and
chloride reabsorption in the ascending limb [32,33]. Al-
though the aquaretic effect is in general linked with V2
antagonistic action [34–36], nevertheless also the effect of
V
1a receptor antagonists on urine and electrolytes excretion
[13] J. Dekanski, Br. J. Pharmacol. 7 (1952) 567–572.
[14] E. Stürmer, in: B. Berde (Ed.), Handbook of Experimental Pharma-
cology, Springer Verlag, Berlin, 1968, pp. 130–189.
[15] W.H. Sawyer, Endocrinology 63 (1958) 694–698.
[16] S. Lundin, A. Broeders, M. Ohlin, K. Hansson, H.I. Bengtsson,
J. Trojnar, et al., J. Pharmacol. Exp. Ther. 264 (1993) 783–788.
[17] L.F.O. Obika, J.F. Laycock, Clin. Sci. 76 (1989) 667–671.
[18] P.I. Feder, D.W. Hobson, C.T. Olson, R.L. Joiner, M.C. Mathews,
Neurosci. Biobehav. Rev. 15 (1991) 109–114.
was investigated [37,38]. Our results (Fig. 1) seem to support
the hypothesis that also V1a and oxytocic blocker could
influence the concentration of Ca2+, Na+ and K+.
5. Conclusion
[19] M. Manning, W.H. Sawyer, in: R.W. Schrier (Ed.), Vasopressin,
Raven Press, New York, 1985, pp. 131–144.
[20] P. Holton, Br. J. Pharmacol. 3 (1948) 328–334.
[21] H.I. Trzeciak, W. Kozik, S. Melhem, A. Kania, D. Dobrowolski,
A. Prahl, et al., Peptides 21 (2000) 829–834.
[22] R.A. Munsick, Endocrinology 66 (1960) 451–457.
[23] H. Van Dyke, A.B. Hastings, Am. J. Physiol. 83 (1928) 563–570.
[24] H.O. Schild, Br. J. Pharmacol. Chemother. 2 (1947) 189–206.
[25] R.J. Tallarida, R.B. Murray, in: Manual of Pharmacological Calcula-
tions, Springer Verlag, Berlin, 1987.
Our studies resulted in four analogues with interesting
pharmacological properties. Peptide I is a potent and excep-
tionally selective oxytocin antagonist which has potential as
a tocolytic agent. All new analogues may constitute valuable
tools for studies on the physiological roles of oxytocin.
Moreover, our results offer new possibilities in the design of
new potent and selective oxytocin blocers.
[26] M. Manning, Z. Grzonka, W.H. Sawyer, in: C. Beardwell, G. Robin-
son (Eds.), The Pituitary, Butterworth, Kent England, 1981, pp. 265–
296.
Acknowledgments
[27] M. Manning, K. Miteva, S. Pancheva, S. Stoev, N.C. Wo, W.Y. Chan,
Int. J. Pept. Protein Res. 46 (1995) 244–252.
[28] W.Y. Chan, N.C. Wo, S. Stoev, L.L. Cheng, M. Manning, Exp.
Physiol. 85S (2000) 7S–18S.
[29] Z. Prochazka, J. Slaninova, Collect. Czech. Chem. Commun. 60
(1995) 2170–2177.
Partial funding for this work was provided by the Polish
State Committee for Scientific Research in the form of grant
No. 0863/T09/2002/22.
[30] M. Manning, L. Balaspiri, J. Moehring, J. Haldar, W.H. Sawyer, J.
Med. Chem. 19 (1976) 842–845.
References
[31] W.H. Sawyer, M. Acosta, L. Balaspiri, J. Judd, M. Manning, Endocri-
nology 94 (1974) 1106–1115.
[32] J.M. Elalouf, A. Di Stefano, C. De Rouffignac, Proc. Natl. Acad. Sci.
83 (1986) 2276–2280.
[1] M. Lebl, K. Jošt, F. Brtník, in: K. Jošt, M. Lebl, F. Brtník (Eds.),
Handbook of Neurohypophyseal Hormone Analogs, Vol. II, Part 2,
CRC Press Inc, Boca Raton, FL, 1987, pp. 127–267.
[2] M. Manning, K. Ban´kowski, W.H. Sawyer, in: D.M. Gash, G.J. Boev
(Eds.),Vasopressin, Plenum Publishing Corporation, NewYork, 1987,
pp. 335–368.
[3] M. Manning, W.H. Sawyer, J. Recept. Res. 13 (1993) 195–214.
[4] V.J. Hruby, C.W. Smith, in: S. Undenfriend, J. Meienhofer (Eds.), The
Peptides, Vol. 8, Academic Press, Orlando, FL, 1987, pp. 77–207.
[5] B. Lammek, M. Czaja, I. Derdowska, P. Rekowski, H.I. Trzeciak,
P. Sikora, et al., J. Pept. Res. 49 (1997) 261–268.
[6] M. Sobocin´ska, E. Łempicka, E. Konieczna, I. Derdowska, B. Lam-
mek, S. Melhem, et al., J. Pharm. Pharmacol. 52 (2000) 1105–1112.
[7] J. Hlavacek, in: K. Jošt, M. Lebl, F. Brtník (Eds.), Handbook of
Neurohypophyseal Hormone Analogs, Vol. I, Part 2, CRC Press Inc,
Boca Raton, FL, 1987, pp. 109–129.
[33] M. Wittner, A. Di Steffano, P. Wangemann, R. Nitschke, R. Greger,
C. Bailly, et al., Pflugers Arch. 412 (1988) 412–423.
[34] Y. Yamamura, S. Nakamura, S. Itoh, T. Hirano, T. Onogawa,
T. Yamashita, et al., J. Pharmacol. Exp. Ther. 287 (1998) 860–867.
[35] W. Jim&#x1EBB;nez, C. Serradeil-Le Gal, J. Ros, C. Cano,
P. Cejudo, M. Morales-Ruiz, et al., J. Rode˙s, J. Pharmacol. Exp. Ther
295 (2000) 83–90.
[36] C. Serradeil-Le Gal, C. Lacour, G. Valette, G. Garcia, L. Foulon,
G. Galindo, et al., J. Clin. Invest. 98 (1996) 2729–2738.
[37] E. Szczepan´ska-Sadowska, K. StVpniakowski, M.M. Skellton,
A.W. Cowley Jr., Am. J. Physiol. 267 (1994) R1217–R1225.
[38] K.G. Franchini, A.W. Cowley Jr., Am. J. Physiol. 270 (1996)
R1257–R1264.