Novel analogues of arginine vasopressin containing α-2-indanylglycine enantiomers in position 2

Article abstract of DOI:10.1002/psc.1189

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α-2-indanylglycine or its D-enantiomer (Igl or D-Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities.We also determined the binding affinity of these compounds to human OT receptor. The Igl² substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA₂ values ranging from 7.19 to 7.98) and practically did not interact with V_1a and V₂ receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D-Igl² substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D-Igl peptides showed a strong antioxytocic potency (pA₂ values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright

Full text of DOI:10.1002/psc.1189

Research Article
Received: 4 March 2009
Revised: 25 July 2009
Accepted: 28 August 2009
Published online in Wiley Interscience: 18 November 2009
(www.interscience.com) DOI 10.1002/psc.1189
Novel analogues of arginine vasopressin
containing α-2-indanylglycine enantiomers
in position 2
a∗
a
a
´
´
Anna Kwiatkowska, Małgorzta Sleszynska, Izabela Derdowska,
a
a
b
b
ˇ
´
ˇ
´
Adam Prahl, Dariusz Sobolewski, Lenka Borovickova, Jirina Slaninova
and Bernard Lammeka
Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated
ten new compounds modified at position 2 with α-2-indanylglycine or its D-enantiomer (Igl or D-Igl, respectively). All the
peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these
compounds to human OT receptor. The Igl2 substitution resulted in a significant change of the pharmacological profile of the
peptides. The new analogues were moderate or potent OT antagonists (pA2 values ranging from 7.19 to 7.98) and practically
did not interact with V1a and V2 receptors. It is worth emphasizing that these new peptides were exceptionally selective. On
the other hand, the D-Igl2 substituted counterparts turned out to be weak antagonists of the pressor response to AVP and
displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro:
the D-Igl peptides showed a strong antioxytocic potency (pA2 values ranging from 7.70 to 8.20) at low concentrations and full
c
agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright ꢀ 2009
European Peptide Society and John Wiley & Sons, Ltd.
Keywords: arginine vasopressin (AVP) analogues; antidiuretic hormone; α-2-indanylglycine; conformational restriction; affinity to OT
receptor
Introduction
replacement of Tyr2 with bulky or sterically restricted substituents
is generally favorable for generation of effective antagonistic
analogues. This hypothesis is also supported by our already longer
than 12-years research focused on the design of potent and
selectiveanaloguesofAVP.In2004,wedescribedthesynthesisand
some pharmacological properties of four new analogues having
β-(1-naphthyl)-L-alanine (L-1-Nal) or its D-enantiomer in position
2 [12]. All of the new peptides were potent OT antagonists.
Additionally, one of them, namely [L-1-Nal2, Val4] AVP, was
exceptionally selective, as it practically did not interact with V1a
and V2 receptors. Recently, advancing in this direction, we have
replaced the residue in position 2 of AVP with 3,3-diphenyl-L-
alanine (Dip) or its D-enantiomer (D-Dip) [13]. Both the L- and
AVP, otherwise known as antidiuretic hormone, is a cyclic
nonapeptide (Figure 1) with multiple functions. The well-known
peripheral roles of AVP are the regulation of water balance, the
control of blood pressure, and the release of ACTH. In addition to
these actions, AVP is involved in several CNS-related processes,
including the regulation of memory, body temperature, social
behaviour, stress, and reproduction [1–3].
AVP acts both as a hormone and as a neurotransmitter or
neuromodulator.Itexertsitseffectsuponbindingtothreereceptor
subtypes termed: V1A, V1B (V3), and V2 [4–6]. Furthermore, AVP can
interact to some extent with the OT receptor [5]. The AVP and OT
receptor subtypes show a high degree of homology. This, along
with the similar structure of both hormones, may be the reason for
the overlap of pharmacological profiles of OT and AVP. All these
receptors belong to a large family of G protein-coupled receptors
(GPCRs) with seven transmembrane spanning domains [6].
Numerous studies have been carried out on the SAR of
AVP during the last four decades, trying to cast light on the
mechanism of its action [7–9]. It has been demonstrated that
conformation of the N-terminal part of AVP analogues is crucial
for their pharmacological activity [8,9]. It is well known that
antidiuretic properties of the analogues may be enhanced by
deaminationofposition1[10].Forexample,1-deamino-AVPand1-
deamino-8-D-arginine vasopressin (also known as Desmopressin,
dDAVP) are analogues of AVP with enhanced and prolonged
antidiuretic activity. In addition, dDAVP is selective and suitable
for the treatment of diabetes insipidus [11]. On the other hand,
∗
Correspondence to: Anna Kwiatkowska, Faculty of Chemistry, University of
´
´
Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland.
E-mail: aniak@chem.univ.gda.pl
´
´
a
Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk,
Poland
b
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the
Czech Republic, Flemingovo sq. 2, 166-10 Prague, Czech Republic
Abbreviations used:Aic, 2-aminoindane-2-carboxylicacid;AVP, arginineva-
sopressin;Dip,3,3-diphenyl-L-alanine;D-Dip,3,3-diphenyl-D-alanine;DMTMM,
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Mob, 4-methoxybenzyl;
NMM, N-methyl-morpholine; NMP, 1-methyl-2-pyrrolidone; L-1-Nal, β-(1-
naphthyl)-L-alanine; D-1-Nal, β-(1-naphthyl)-D-alanine; OT, oxytocin.
c
J. Pept. Sci. 2010; 16: 15–20
Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.

KWIATKOWSKA ET AL.
resistanceoftheresultingpeptidestoenzymes.Wehavedesigned,
synthesized, and determined some pharmacological properties
of ten new analogues of AVP where the above mentioned
modification was combined with Mpa1, and/or Val4 and/or
D-Arg8 substitutions. The structure of the new peptides is shown
in Table 1.
H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
Figure 1. Amino acid sequence of arginine vasopressin.
(a)
(b)
NH2
O
Materials and Methods
O
OH
NH2
General
HO
TLC was carried out on silica plates (Merck) and the spots were
visualized with iodine. The solvent system used was butan-1-
ol/aceticacid/water/ethylacetate(1 : 1 : 1 : 1, v/v). HPLCwascarried
out on a Waters (analytical and preparative) chromatograph
equipped with a UV detector (λ = 226 nm). The following solvent
systems were used: [A] 0.1% aqueous TFA and [B] acetonitrile/0.1%
aqueous TFA (80 : 20 v/v). Preparative HPLC was carried out using
Waters C18 column (15 µm, 100 ´Å, 7.8 × 300 mm) in gradients
running from 20 to 50% [B] for 90 min for analogues I, VI, VIII, and
X, from 25 to 55% [B] for 90 min for analogues II–V and VII, and
from 15 to 45% [B] for 80 min for analogue IX, all at a flow rate
of 2.5 ml/min. The mass spectra of the peptides were recorded on
a MALDI TOF mass spectrometer (Biflex III Bruker, λ = 337 nm).
The purity of the peptides was determined on a Vydac C18 column
(5 µm, 4.6×250 mm) or Hypersil ODS C18 column (5 µm, 4.6×250
mm). A linear gradient from 20 to 80% of [B] was applied for 20 min
or 30 min at a flow rate of 1 ml/min.
Figure 2. (a) α-2-indanylglycine (Igl). (b) 2-aminoindane-2-carboxylic acid
(Aic).
D-Dip modifications were sufficient to dramatically change the
propertiesoftheanalogues:theantidiureticactivitywaspreserved
and prolonged, the uterotonic activity was transformed into
antioxytocic one and the effect on blood pressure was eliminated.
Moreover, four compounds, [Mpa1, D-Dip2]AVP, [Mpa1, D-Dip2,
Val4]AVP, [Mpa1, D-Dip2, D-Arg8]VP, and [Mpa1, D-Dip2, Val4,
D-Arg8]VP, were exceptionally potent antidiuretic agents with
significantly prolonged activities. Our approach also included the
2-aminoindane-2-carboxylic acid (Aic) modification in position 2
or 3 [14]. One peptide of this series, namely [Mpa1, Aic2, Val4,
D-Arg8]VP, had antidiuretic activity similar to that of dDAVP, thus
being one of the most potent antidiuretic peptides reported to
date.
These observations backed up by our earlier investigations
prompted us to examine the influence of other sterically restricted
moieties placed in position 2 on pharmacological properties of the
resulting peptides.
All the amino acid derivatives were purchased from Nov-
aBiochem, except for Fmoc-Igl and Fmoc-D-Igl, which were
provided by ChemImpex Int., Inc. Mpa (Trt) was obtained as
described for Cys(Trt) [16] using 3-mercaptopropionic acid instead
of L-cysteine hydrochloride.
We decided to check how substitution of position 2 with bulky
α-2-indanylglycine (Igl) enantiomers (L- and D-, see Figure 2a)
would affect biological potency of the analogues. The α-2-
indanylglycine was earlier successfully applied for the synthesis
of potent and totally enzyme-resistant bradykinin antagonists in
Stewart’s laboratory [15]. This modification resembles previously
used Aic (Figure 2b); however, the α-carbon is not incorporated
into the ring. It makes the molecule less compact. Peptides
modifiedwiththisresidueareconformationallylessrestrictedthan
their Aic2 counterparts. This modification should also enhance the
Peptide synthesis
All the peptides were obtained manually by solid-phase method,
i.e. by stepwise coupling of Fmoc-amino acids to the grow-
ing peptide chain on a polystyrene resin (TentaGel S Ram-
resin, capacity 0.23 mmol/g) on a 150 µmol scale. Fmoc-
protected amino acids were used with the Trt and the 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf) as side
Table 1. Physicochemical properties of peptides I–X
Analogue
Formula
HPLC TR (min)a
MW [M + H+]
[Igl2]AVP
I
II
C48H67N15O11S2
C48H67N15O11S2
C48H66N14O11S2
C48H66N14O11S2
C48H68N14O10S2
C48H68N14O10S2
C48H67N13O10S2
C48H67N13O10S2
C48H67N15O11S2
C48H66N14O11S2
12.84
1093.4
1093.4
1078.4
1078.4
1064.4
1064.4
1049.4
1049.4
1093.4
1078.4
1094.1
1094.3
1079.0
1079.4
1065.0
1065.1
1050.1
1050.3
1095.0
1079.2
[D-Igl2]AVP
22.44b,c
[Mpa1, Igl2]AVP
[Mpa1, D-Igl2]AVP
[Igl2, Val4]AVP
[D-Igl2, Val4]AVP
[Mpa1, Igl2, Val4]AVP
[Mpa1, D-Igl2, Val4]AVP
[Igl2, D-Arg8]VP
[Mpa1, Igl2, D-Arg8]VP
III
IV
V
15.23
21.97b,c
14.78
VI
VII
VIII
IX
X
17.11b
16.90
19.53b
15.81b
19.21b
a Linear gradient from 20 to 80% of [B] for 20 min, Vydac C18 column.
b Linear gradient from 20 to 80% [B] for 30 min, Vydac C18 column.
c Linear gradient from 20 to 80% [B] for 30 min, Hypersil ODS C18 column.
The following solvent systems were used: [A] 0.1 aqueous trifluoroacetic acid (TFA); [B] acetonitrile: 0.1% aqueous TFA (80 : 20 v/v).
c
Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.
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J. Pept. Sci. 2010; 16: 15–20

ANALOGUES OF AVP SUBSTITUTED WITH A-2-INDANYLGLYCINE ENANTIOMERS
chain protecting groups for Asn, Gln, Cys, and Arg, respectively.
Full protected peptide resins were synthesized according to stan-
dard procedures [17] involving (i) deprotection steps using a 20%
solution of piperidine in DMF, 5 and 10 min. The couplings in a
mixture of DMF/1-methyl-2-pyrrolidone (NMP) (1 : 1 v/v) contain-
ing 1% Triton were carried out using TBTU, HOBt in the presence of
NMM. In most cases, the amino acids were coupled at a threefold
excess. ThecouplingsafterFmoc-Igl, Fmoc-d-Iglweremediatedby
HATU, HOAt in the presence of NMM in the mixture of DMF/NMP
(1 : 1 v/v) containing 1% Triton or DMTMM in the presence of
DIPEA in the NMP. The completeness of each coupling reaction
was monitored by the Kaiser [18] or chloranil test [19]. Recou-
pling was performed when the test was positive. After completion
of the synthesis, the protected nonapeptidyl resins were treated
with TFA : H2O : TIS : PhOH (92.5 : 2.5 : 2.5 : 2.5) and stirred for 3 h.
Solutions of the cleaved peptides were filtered and evaporated in
vacuo to a volume of about 1 ml. Then the peptides were precip-
itated with diethyl ether to afford crude products. The resulting
dithiols were oxidatively cyclized with a 0.1 m I2 in methanol using
a standard procedure [16]. The solvents were evaporated under
reduced pressure and the resulting materials were dissolved in
water, frozen, and lyophilized to give the final crude oxidized
products. The crude products were desalted on a Sephadex G-15
column, and eluted with aqueous acetic acid (30%) at a flow
rate of 3 ml/h. After freeze-drying, the fractions comprising the
major peak were purified by RP-HPLC as described above. The pu-
rity of the peptides was controlled using analytical HPLC. MALDI
TOF mass spectroscopy (molecular ion) was used to confirm the
identity of the pure products.
logarithm to the base 10 of the average molar concentration of an
antagonistwhichreducestheresponseto2x unitsoftheagonistto
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
values reported are averages of 3 to 5 separate experiments. The
responses to standard doses of OT or vasopressin were stable for
several hours, without problems with tachyphylaxis. For details
see Ref. 23.
Tests to assess the antidiuretic property were conducted on
conscious male rats in a modified Burn test [24,25]. In the standard
mannerwithhydratedrats, theanimalshaving fastedfor16 hwere
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 comparison of the
compound’s potency yielding t1/2 equal to 200 min and the so-
called threshold doses yielding t1/2 equal to 60 min (equal to the
valueof t1/2 obtainedwiththephysiologicalsolution). Oneachday
of the experiment, 21 rats divided into 5 groups of 4 or 5 animals
each were administered different doses of different compounds;
each dose being tested in 2 or 3 independent experiments
(different days, different rats). As standards, synthetic AVP and
dDAVP were used. For details see Ref. 23. The results were thus
expressed in IU/mg in comparison to AVP (the value 465 IU/mg
was taken for AVP for both t1/2 60 min and t1/2 200 min)
Binding affinities to the human OT receptor were determined as
described in Refs 26,27, using tritiated OT from NEN Life Science,
Boston, MA, USA. In brief, a membrane fraction from HEK OTR cells
was incubated with [3H]OT (2 nM) and various concentrations of
peptides (0.1–10 000 nM) for 30 min at 35 ◦C. The total volume of
the reaction mixture was 0.25 ml. Incubation medium consisted
in HEPES (50 mM, pH 7.6), MnCl2 (10 mM), and BSA (1 mg/ml).
The reaction was terminated by quick filtration on a Brandel cell
harvester. OT was used as a control and for determination of
nonspecific binding. Binding affinities were expressed as Ki values
calculatedaccordingtotheexpressionKi = IC50/[(c3HOT/KdOT)+1],
where KdOT is taken as 1.8 nM [28].
Biological Evaluation
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 §23 of the
law of the Czech Republic no. 246/1992.
The uterotonic test was carried out in vitro on the strips of rat
uterus in the absence of magnesium ions [20,21]. Rats in induced
estrus by the injection of estrogen 48 h before the experiments
were used and the height of the single isometric contraction
of a uterine strip was measured. In principle, cumulative 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
concentrations and at 1 min intervals without the fluid being
changed until the maximal response was obtained. Synthetic OT
was used as standard. Each analogue was tested on uteri excised
from 4 to 5 different rats.
The pressor activity was determined on phenoxybenzamine
treated rats [22]. In short, male rats (220–260 g) were anesthetized
using urethane and their vena femoralis and arteria carotis were
canulated for drug administration and blood pressure determi-
nation, respectively. Then phenoxybenzamine was administered
in two doses. After pressure stabilization, usually after 30 min,
bolus doses of the standard or tested substance were adminis-
tered in random order. When antagonistic activity was expected,
a standard dose of vasopressin was administered 1 min after the
administration of the test substance. Each analogue was tested on
3–4 animals. A more detailed description of these protocols can
be found in Ref. 23.
Results
The ten new analogues of AVP (I–X) were obtained as crude
products in about 70–85% yields. After HPLC purification, their
purity was higher than 98% as determined by analytical HPLC.
The MALDI TOF mass spectrometry confirmed that the purified
peptides were the desired products. Physicochemical data of the
new peptides are presented in Table 1.
Pharmacologicaldataofthenewanaloguestogetherwiththose
of AVP and some related peptides are summarized in Table 2.
PeptideshavingIglinposition2weremoderate(I,IX)orhigh(III,
V, VII, X) oxytocic antagonists in the uterus in vitro test. Analogues
with D-Igl at position 2 (II, IV, VI, VIII) exhibited dual activity. At low
concentrations, they were strong antioxytocic agents, whereas at
high concentrations they were full agonists.
Regarding the pressor activity, all the new analogues modified
at position 2 with Igl (I, III, V, VII, IX, X) were devoid of any activity,
while their counterparts with D-Igl were weak antagonists of AVP.
With agonists, the activity was expressed in IU/mg, with
antagonists as pA2. The pA2 values represent the negative
c
J. Pept. Sci. 2010; 16: 15–20
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KWIATKOWSKA ET AL.
Table 2. Pharmacological properties of the new AVP analogues
Analogue
Activity
Pressorc
Oxytocic uterus
Antidiuretic IU/mg
60 min (200 min)
Affinity to human
in vitro test no Mg2+
IU/mg or pA2
OTR Ki (nM)
AVPa
–
–
–
–
–
–
I
17
–
412
465
738d
114–257d
1300–1745d
800 – 50 000d
–
–
[Val4]AVPa
32
[D-Arg8]VPa
[Mpa1]AVPa
[Mpa1, D-Arg8]VPa
[Aic2]AVPb
[Igl2]AVP
[D-Igl2]AVP
[Mpa1, Aic2]AVPb
[Mpa1, Igl2]AVP
[Mpa1, D-Igl2]AVP
[Aic2, Val4]AVPb
[Igl2, Val4]AVP
[D-Igl2, Val4]AVP
[Mpa1, Aic2, Val4]AVPb
[Mpa1, Igl2, Val4]AVP
[Mpa1, D-Igl2, Val4]AVP
[Igl2, D-Arg8]VP
[Mpa1, Igl2, D-Arg8]VP
0.4
4.1
–
27–63
346–370
–
1.5–5.1
∼0.39
–
pA2 = 7.27 0.22
pA2 = 7.19 0.10
pA2 ∼ 8.20 and agonist ∼1.1 IU/mg
pA2 = 7.49 0.16
pA2 = 7.94 0.22
pA2 ∼ 8.20 and agonist ∼5.5 IU/mg
pA2 = 7.93 0.17
pA2 = 7.68 0.16
pA2 ∼ 7.88 and agonist ∼0.9 IU/mg
pA2 = 8.06 0.11
pA2 = 7.75 0.10
pA2 ∼ 7.70 and agonist ∼0.8 IU/mg
pA2 = 7.40 0.15
pA2 = 7.98 0.24
9.4 2.8
∼450 (45 000)
740
e
0
1005 282
209 59
215
e
II
pA2 ∼ 5.7
5.3 2.5
∼450 (45 000)
e
III
IV
0
202 16
35 59
1800
e
pA2 ∼ 5.7
0
∼450 (45 000)
e
V
0
745 175
138 38
1550
e
VI
pA2 ∼ 5.7
pA2 = 6.25 0.12
∼4500 (450 000)
e
VII
VIII
IX
0
658 57
70 30
540 10
188 22
e
pA2 ∼ 7.0
0
0
∼0.45 (∼20)
∼4.5 (∼4.5)
X
a Values taken from Ref. 8.
b Values taken from Ref. 14.
c 0 means no activity up to the dose of 0.15 mg/kg of experimental animal.
d Values taken from Ref. 23.
e Negligible antidiuretic activity; about 1000 times lower than AVP at the threshold level (60 min) and about ten times or more lower at the effect
level (200 min); steeper dose response curve.
All the new compounds exhibited only negligible antidiuretic
activity, about 1000 times lower than that of AVP at the threshold
level (60 min) and about ten times or more lower at the effect
level of 200 min. Analogue X, where Igl2 modification was
combined with Mpa1 and D-Arg8 substitutions, showed about
1% of vasopressin antidiuretic activity at both activity levels.
Binding properties of new analogues to human OT receptors
are also summarized in Table 2.
crucial position, which thus results in a more compact structure
and a decreased conformational freedom of the analogues. Both
noncoded amino acids have a bulky planar side chain, however
in the case of Igl in comparison to Aic, the α-carbon is not
incorporated into the ring which results in a higher flexibility of
the side chain.
In regard to the pressor test, all the analogues having L-
enantiomer at position 2 (I, III, V, VII, IX, X) were devoid of
the pressor potency, while their counterparts with D-Igl were
moderately (VIII) or weakly (II, IV, VI) potent antagonists of AVP.
The data presented in Table 2 show that the combination of D-Igl2
and deamination of position 1 and Val4 substitution results in an
increase of the pressor antagonism (peptide VIII). Also in the case
of the pressor activity, the structural difference between Aic and
Igl plays an important role as the [Aic2]AVP and deamino [Aic2]AVP
peptides are pressor agonists, while those containing Igl have no
pressor activity.
As far as the oxytocic test is concerned, single substitution of
Tyr in the AVP molecule with Igl, resulted in a moderately potent
and selective antagonist of OT (compound I). Deamination, Val4
substitution, a change of the configuration of Arg at position 8, or a
combination of these changes significantly increased antioxytocic
potency of the resulting analogues (III, V, VII, IX, X). The most
potentantioxytocicpeptideswereobtainedeitherbycombination
of Igl2 modification with deamination of position 1 (analogue III,
pA2 = 7.94) or by deamination and the change of configuration
of Arg at position 8 (analogue X, pA2 = 8.00). [Mpa1, Igl2]AVP
is thus a very potent and selective OT antagonist, while its Aic2
counterpart is medium potent and not a selective blocker of OT
Discussion
One of the main topics of our research is investigation of the role
of Tyr2 of AVP and its analogues in pharmacological properties.
This paper describes the synthesis and pharmacological
evaluation of ten new analogues (I–X) designed by substitution
of position 2 of AVP and of some of its analogues (having
different combinations of changes at position 1, 4, and 8) with
Igl enantiomers. The results presented in Table 2 show that the
Igl2 and D-Igl2 peptides have strikingly different pharmacological
properties as compared to that of the parent hormone.
The antidiuretic potency of all the new analogues is negligible
with exception of peptides IX and X, where Igl modification
is combined with D-Arg substitution and deamination, which
are nevertheless only weak agonists. This finding is extremely
interesting especially when compared with antidiuretic activity
of Aic2 analogues. The Aic2 peptides, which were considered as
models of the new compounds I–VI, exhibited potent and long
lasting antidiuretic effect. Aic differs from Igl only by lacking
one –CH–group (Figure 2a and b); however, the group is in its
c
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J. Pept. Sci. 2010; 16: 15–20

ANALOGUES OF AVP SUBSTITUTED WITH A-2-INDANYLGLYCINE ENANTIOMERS
receptors. AnalogueshavingD-Iglinposition2arealsoexceptional
as all of them show dual activity in this test – they showed high
antioxytocic potency (pA2 values ranging from 7.70 to 8.20) at
low concentrations and full agonism at high concentrations. This
phenomenon is difficult to explain.
[30]. Morerecently, Flouretet al. designednovelbicyclicanalogues
derived from potent OT antagonists [31]. Other studies described
the synthesis and biological activity of OT analogues containing
conformationally restricted residues in position 7 [32]. All these
studies resulted in several potent and selective antiuterotonic
agents. From this point of view our finding that only a single
modification of position 2 of AVP with Igl is sufficient to obtain
selective and potent antagonists of OT is important.
On the basis of these results, we are undertaking further SAR
studies on our best Igl2 modified AVP analogues using NMR, CD,
and theoretical molecular modelling methods. These methods
may contribute to the explanation of the relations between the
backbone structure and the functional group orientation and
biological activity.
The data of pharmacological tests on rats were supplemented
bydeterminationoftheaffinitiesoftheanaloguestothehumanOT
receptors stably expressed on the HEK cells [27] using tritiated OT.
The data are given in Table 2. The most potent oxytocic antagonist
of the pure antagonist (compounds I, III, V, VII, IX, X), [Mpa1, Igl2,
D-Arg8]VP (pA2 = 8.00), showed also the highest affinity to the
OT receptors (affinity constant 188 nM). However, the affinity of
compounds with D-Igl, which exhibited dual uterotonic activity
displayed even higher binding affinity (values ranging from 35 to
209 nM). The substances with D-Igl have mostly by one order of
magnitude higher Ki than their Igl containing counterparts. The
high binding to human OTR points to the fact that the analogues
should be active not only in rats but also in humans. The question
is however whether they would be antagonists or agonists. The
binding data to human OTR are not sufficient to explain the dual
activity in the rat uterotonic test. More light for the explanation
of this paradox might be shed by binding data using vasopressin
ligands and vasopressin receptors. We plan to carry out such
investigation in the future.
Acknowledgement
Partial funding for this work was provided by Polish State Commit-
teeforScientificResearchunderthegrantNo.0230/B/H03/2008/35
and 2385/B/HO3/2008/34 and by research project No. Z4055905
of the Academy of Sciences of the Czech Republic.
References
These, in our opinion interesting findings, demonstrate the
usefulness of our approach to the SAR study and for the
design of new highly active and selective analogues of AVP
with desired pharmacological properties. These results are even
more interesting when we recall our previous findings claiming
that the presence of other aromatic amino acid residues in
position 2 (3,3-diphenylalanine enantiomers, L-1-Nal enantiomers,
2-aminoindane-2-carboxylic acid [12–14]) has strong influence
on pharmacological properties of the analogues, and when
combined with additional substitution of positions 1, 4, and 8,
may give peptides with favorable antidiuretic or antioxytocic
activities. One of the examples is [Mpa1, D-Dip2]AVP which is
an exceptionally potent and selective antidiuretic agent with
significantly prolonged activity. On the other hand, the results
presented in this paper are very similar to those obtained
by modification of position 2 of AVP by L-1-Nal enantiomers.
Analogues modified with L- or β-(1-naphthyl)-D-alanine (D-1-
Nal) were potent or moderately potent OT antagonists in vitro,
and displayed either no or low antidiuretic activity while their
pressor potency was removed or had become converted into
moderate antagonistic [12,29]. It is clear that introduction of
Igl or Nal enantiomers into position 2 of AVP forces peptide
backbone and side chains to adopt specific orientations that
resulted in compounds having high antioxytocin potency. These
results are encouraging to perform the structural analysis and
molecular modelling of these peptides in order to explain similar
pharmacological profile. Moreover, comparison of Aic2 and Igl2
substituted analogues of AVP has once more demonstrated that a
slight difference in the structure could cause significant changes
in biological activity. Our new Igl2-substituted peptides proved
that the presence of a less sterically restricted but bulky and
aromatic amino acid residue may result in highly active and
selective antioxytocic agents. It should be emphasized that one
of the new peptides, [Mpa1, Igl2]AVP, has a very high antioxytocic
potency. In the recent years, there has been increasing interest in
OT antagonists, as it is thought that OT is involved in the initiation
of the term and preterm labour. In 1995, Manning described a
series of moderately potent and fairly selective antagonists of OT
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