Synthesis and biological activity of oxytocin analogues containing unnatural amino acids in position 9: Structure activity study

Article abstract of DOI:10.1007/s00726-009-0372-2

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of L- or D-β-(2-thienyl)-alanine [L- or D-Thi], or L- or D-3-Pyridylalanine [L- or D-3-Pal]) were combined with D-tyrosine(OEthyl) [D-Tyr(Et)] or D-1-naphthylalanine [D-1-Nal] in position 2 and β-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having α-aminoisobutyric acid [Aib] or D-1,2,3,4- tetrahydroisoquinoline-3- carboxylic acid (D-Tic) or diethylglycine (Deg) in position 9 and D-Tyr(Et) or D-1-Nal or D-Tic in position 2 and Mpa or Pen (ββ-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and D-Tyr(Et) or D-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa ¹, D-Tyr(Et)², Deg⁹]OT (pA₂ = 8.68 ± 0.26); this analogue was also selective. Springer-Verlag 2009.

Full text of DOI:10.1007/s00726-009-0372-2

Amino Acids (2010) 38:1549–1559
DOI 10.1007/s00726-009-0372-2
ORIGINAL ARTICLE
Synthesis and biological activity of oxytocin analogues containing
unnatural amino acids in position 9: structure activity study
•
Vassiliki Magafa Lenka Borovickova
•
ˇ
Jirina Slaninova Paul Cordopatis
´
•
ˇ
´
Received: 26 June 2009 / Accepted: 12 October 2009 / Published online: 3 November 2009
Ó Springer-Verlag 2009
Abstract We report the solid phase synthesis and some
pharmacological properties of 24 oxytocin (OT) ana-
logues. Basic modifications at position 9 (introduction of
L- or D-b-(2-thienyl)-alanine [L- or D-Thi], or L- or
D-3-Pyridylalanine [L- or D-3-Pal]) were combined with
D-tyrosine(OEthyl) [D-Tyr(Et)] or D-1-naphthylalanine
[D-1-Nal] in position 2 and b-mercaptopropionic acid
(Mpa) in position 1 modifications in altogether 14
analogues. Additionally, 8 analogues having a-aminoiso-
butyric acid [Aib] or D-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (D-Tic) or diethylglycine (Deg) in position
9 and D-Tyr(Et) or D-1-Nal or D-Tic in position 2 and Mpa
or Pen (bb-dimethylcysteine) in position 1 were prepared.
Two of these analogues have one more modification in
position 6, i.e. Pen. Furthermore, two analogues having
Mpa in position 1 and D-Tyr(Et) or D-1-Nal in position 2
were prepared for comparison purposes. The analogues
were tested for rat uterotonic activity in vitro, in the rat
pressor assay and for binding affinity to human OT
receptor. The analogue having the highest anti-oxytocic
activity was [Mpa1, D-Tyr(Et)2, Deg9]OT (pA2 = 8.68
0.26); this analogue was also selective.
Keywords Oxytocin antagonists Á Position 9 Á
Unnatural amino acids Á Biological activity Á
Binding affinity
Abbreviations
OT
Oxytocin
Mpa
b-Mercaptopropionic acid
[Mpa1]OT Deamino-oxytocin
Aib
a-Aminoisobutyric acid
Diethylglycine
Deg
1-Nal
3-Pal
Pen
1-Naphthylalanine
3-Pyridylalanine
bb-Dimethylcysteine
b-(2-Thienyl)-alanine
1,2, 3, 4, -Tetrahydroisoquinoline-3-
carboxylic acid
Thi
Tic
Abbreviations of common amino acids are in accordance with the
recommendations of IUPAC-IUB Joint Commission on Biochemical
Nomenclature: Arch Biochem Biophys 206 (1988) v–xxii, J Biol
Chem 264 (1989) 668–673, J Peptide Sci 12 (2006) 1–8.
Tyr(Et)
Fmoc
But
Tyrosine(OEthyl)
9-Fluorenylmethoxycarbonyl
t-Butyl
Trityl
Trt
V. Magafa (&) Á P. Cordopatis
DIC
Diisopropylcarbodiimide
1-Hydroxybenzotriazole
Dimethylformamide
Laboratory of Pharmacognosy and Chemistry of Natural
Products, Department of Pharmacy, University of Patras,
26500 Patras, Greece
HOBt
DMF
DMSO
TBAF
TFA
e-mail: magafa@upatras.gr
Dimethylsulphoxide
Tetrabutylammonium fluoride
Trifluoroacetic acid
ˇ
´
´
L. Borovickova Á J. Slaninova
Department of Antimicrobial Peptides,
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of Czech Republic,
16610 Prague 6, Czech Republic
HPLC
ESI–MS
High-performance liquid chromatography
Electrospray ionization–mass spectrometry
123

1550
V. Magafa et al.
more effective treatment of preterm labour with a lower
risk of side effects. Moreover, the efficacy of the OT
antagonist Atosiban (trade name Tractocile) to inhibit
premature uterine contractions in humans is indicative of a
role for OT in human labour (Goodwin et al. 1994; Romero
et al. 2000; Moutquin et al. 2001). It should be noted that a
number of peptide oxytocin antagonists with higher affinity
and selectivity for the human oxytocin receptor than
atosiban have been synthesized over the last 20 years.
These analogues are promising new candidates for dis-
covery and design potential tocolytic agents for the pre-
vention of premature labour (Manning et al. 2008).
Furthermore, barusiban is a new OT receptor antagonist
with a high affinity for the human oxytocin receptor, cur-
rently being developed for the treatment of preterm labour,
which is under investigation (Nilsson et al. 2003; Rein-
heimer et al. 2005; Reinheimer et al. 2006; Thornton et al.
2009). Moreover, in a view of the widespread OT-related
actions, OT antagonists may not only be studied as prom-
ising inhibitors of preterm labour but may also prove
useful in the treatment of dysmenorrhoea, benign prostatic
hyperplasia and psychiatric illnesses such as anxiety, sex-
ual dysfunctions, eating disorders, etc. (Borthwick 2006).
Generally, inhibitors of the uterotonic activity of OT
have been produced by the introduction of bulky b-carbon
substituents into position 1 and/or by substitution of
L-tyrosine in position 2 of OT with an aromatic D-amino
acid. Previous structure–activity studies in our laboratory
revealed that minimal structural changes of the OT mole-
cule could provide quite potent antagonists (Lebl 1987;
Assimomytis et al. 1994; Assimomytis et al. 1996). Fur-
thermore, the importance of conformational flexibility for
agonist activity and relative conformational rigidity and
steric constraints for antagonism has been emphasized
(Urry and Walter 1971; Hill et al. 1990; Lebl et al. 1992).
The proper orientation and the sequence of the
C-terminal tripeptide are critical for obtaining high potency
OT analogues (Ting et al. 1980; Hruby 1986; Fragiadaki
et al. 2003). Therefore, structural modifications of the side-
chain moieties in the C-terminal tripeptide might lead to
analogues with variable biological properties at different
OT target tissues. Moreover, synthesis of analogues of
biologically active peptides in which structural modifica-
tions should enhance resistance towards enzymatic cleav-
age of peptide bonds, resulting in prolonged course of
action, is of great importance. In this regard, OT analogues
with varying ring sizes wherein sulphur is replaced
by carbon (1,6-carba analogues) showed significantly
improved metabolic lifetimes and increased biological
stability (Stymiest et al. 2005). It has also been reported
that modification of the C-terminal tripeptide side chain
influences the chymotryptic cleavage of Tyr-Ile peptide
Introduction
Oxytocin (OT), a physiologically important nonapeptide
hormone and neurotransmitter containing a 20-membered
tocin ring (from Cys-1 to Cys-6) and an acyclic tripeptide
tail (from Pro-7 to GlyNH2-9) (see Fig. 1) regulates several
physiological functions, such as milk ejection, uterine
contractions, vascular and cardiac relaxation, interferes
with salt and water balance, and is known to play a role in
social and reproductive behaviour and emotions (Gimpl
and Fahrenholz 2001; Lippert et al. 2003). Subsequent
studies have demonstrated synthesis of OT mRNA and the
peptide in several peripheral tissues, including ovary,
endometrium or deciduas (Lefebvre et al. 1992).
The multiple established and proposed actions of OT are
all mediated by one type of OT receptor (Gimpl and
Fahrenholz 2001), a member of the super-family of seven-
transmembrane G-protein coupled receptors (GPCRs)
(Phaneuf et al. 1993), which has no subtypes but is struc-
turally related to the vasopressin receptors. The effect on
uterine contractions is of major pharmacological impor-
tance because OT is the strongest uterotonic agent known
and is commonly used in obstetrical practice to speed up
labour.
Preterm birth is defined by the World Health Organi-
zation as birth occurring prior to 37 weeks gestation. Pre-
term labour occurs in up to 20% of all human pregnancies
and leads to preterm delivery with a variety of neonatal
health problems, e.g. neurosensory deficits, respiratory
distress syndrome, low body weight, subnormal height,
while can also possibly result in death of the newborn (Hall
et al. 1997; Moster et al. 2008). Drugs with a variety of
pharmacologic actions, but not specific, have been devel-
oped to suppress contractions associated with preterm
labour. These include magnesium sulphate, beta-adrenergic
agonists (terbutaline and ritodrine), prostaglandin synthe-
tase inhibitors (indomethacin and rofecoxib) or calcium
channel blockers (nifedipine) (Gyetvai et al. 1999; Giles
and Bisits 2007). To date, the use of most of these drugs is
compromised by side effects and limited efficacy. Thus,
there is a need for optimization of existing therapeutic
options and discovery of new drugs for the management of
preterm labour.
OT receptor antagonists represent relatively new class of
tocolytics under investigation. They afford greater speci-
ficity and can be expected to exhibit improved efficacy and
˚
risk profiles (Thornton et al. 2001; Lamont 2003; Akerlund
2006; Allen et al. 2006). Such compounds would allow
H Cys1 Tyr2 Ile3 Gln4 Asn5 Cys6 Pro7 Leu8 Gly9 NH2
´ˇ ˇ
bond in the OT molecule (Hlavacek and Fric 1989).
Fig. 1 Amino acid sequence of oxytocin
123

Oxytocin analogues: synthesis and biological activity
1551
Fig. 2 Structure of the
unnatural amino acids used in
the present study
H2N CO2H
C
H3CH2C CH2CH3
H2N CO2H
H2N
CO2H
H
CO2H
CH2
C
H3C CH3
SH
Aib
Deg
Mpa
D-1-Nal
CO2H
H2N
CO2H
H2N
CO2H
H2N CO2H
C
N
H
H3C
CH3
SH
N
N
L-3-Pal
D-3-Pal
Pen
D-Tic
H2N
CO2H
CH2
H2N
CO2H
S
H2N
CO2H
S
OCH2CH3
D-Tyr(Et)
L-Thi
D-Thi
In continuing our work aimed at the design of selective
OT antagonists (Fragiadaki et al. 2003, 2007), we investi-
gated the effectiveness of modifications in position 9 by
unnatural amino acids (Fig. 2). Here, we present the syn-
thesis of twenty-four new OT analogues which contain in
position9 thefollowingresidues: L- orD-b-(2-thienyl)-alanine
(Thi), L- or D-3-pyridylalanine (3-Pal), a-aminoisobutyric
acid (Aib), D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid (D-Tic), and diethylglycine (Deg) alone or in combi-
nation with D-tyrosine(OEthyl) [D-Tyr(Et)], D-1-naphthyl-
alanine [D-1-Nal] and D-Tic in position 2 (Fig. 3, analogues
1–6, 8–17, and 19–24). Two analogues have one more
modification in position 6, i.e. bb-dimethylcysteine [Pen]
(13 and 16). For comparison purposes we have synthesized
peptides [Mpa1, D-Tyr(Et)2] (analogue 7) and [Mpa1,
D-1-Nal2]OT (analogue 18). The structures of the analogues
are summarized in Fig. 3. All 24 analogues were tested for
biological potency in rat uterotonic test in vitro and rat
pressor test and for binding affinity to human OTR.
resin from Nova Biochem; the derivative S-Trityl-b-
mercaptopropionic acid [Mpa(Trt)] was prepared according
to the literature (Photaki et al. 1979). Peptide reagents were
purchased from Bachem AG and Nova Biochem. All sol-
vents and reagents used for solid-phase synthesis were of
analytical quality and used without further purification.
Peptide synthesis and purification
The analogues were synthesized by Fmoc solid phase
methodology (Fields and Noble 1990) utilizing either Rink
Amide MBHA resin (Rink 1987) or 2-chlorotrityl-chloride
resin (Barlos et al. 1991) bearing a Rink-Bernatowitz linker
(Bernatowitz et al. 1989) as solid support to provide the
peptide amide. Fmoc-protected amino acids were used with
the Trityl group (Trt) [Asn, Gln, Cys, Pen] and the t-Butyl
group (But) [Tyr] as side-chain protection groups. Stepwise
synthesis of a peptide analogue was achieved with diiso-
propylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt)
¨
as coupling agents in dimethylformamide (DMF) (Koning
and Geiger 1970; Sarantakis et al. 1976) in 2.0 (Fmoc-amino
acid), 2.2 (DIC) and 3.0 (HOBt) molar excess for 2 h at room
temperature. Completeness of the reaction was monitored by
the Kaiser test (Kaiser et al. 1970), except in the case of Pro
and Tic residues where the end of the reaction was moni-
tored by the Chloranil test (Vojkovsky 1995). The Fmoc
groups were removed by treatment with 20% piperidine in
Materials and methods
Materials
All 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino
acids were obtained from CBL Patras; Rink Amide MBHA
123

1552
V. Magafa et al.
Fig. 3 Structure of oxytocin
analogues
H-X1-Y2-Ile3-Gln4-Asn5-V6-Pro7-Leu8-Z9-NH2
Analogue
X1
Y2
V6
Z9
1
2
L-Thi
L-3-Pal
D-3-Pal
L-Thi
3
4
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Mpa
Pen
5
L-3-Pal
D-3-Pal
6
7
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tyr(Et)
D-Tic
8
L-Thi
D-Thi
L-3-Pal
D-3-Pal
Aib
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Pen
Pen
Aib
Deg
D-Tic
D-Tic
Aib
D-1-Nal
D-1-Nal
L-Thi
D-Thi
L-3-Pal
D-3-Pal
Aib
D-1-Nal
D-1-Nal
D-1-Nal
D-1-Nal
D-1-Nal
Aib
DMF for 30 min. Cleavage of peptide-linker bond and
removal of the side chain protecting groups were accom-
plished using solution of trifluoroacetic acid (TFA)/1,2-
ethanedithiol/triethylsilane/water (94:2.5:2.5:1:1, v/v, 15 ml/g
peptide resin) in 4 h at room temperature. The released
peptides were precipitated upon concentration of solvent
and addition of cold diethyl ether. The formation of the
disulphide bridge (cyclization) was mediated either by the
dimethylsulphoxide (DMSO)/water method in a solution
of 20% (DMSO)/H2O for 18–30 h at room temperature
(Tam et al. 1991) or via the carbon tetrachloride/tetra-
butylammonium fluoride (CCl4/TBAF) (Maruyama et al.
1999) method in a solution of 20% CCl4 in acetonitrile in
the presence of 150 lL of 1 M TBAF in dry tetrahydro-
furan for 30 min. In both methods of cyclization we used
1.5 mL of the oxidative solution per mg of crude linear
peptide. Completeness of the formation of the disulfide
bond was monitored either by the Ellman’s test or by
analytical HPLC.
The products were purified by gel filtration chromatog-
raphy on Sephadex G-15 using 25% acetic acid as the eluent
with the exception of Thi residue containing peptides for
which 40% acetic acid was used. Final purification was
¨
achieved by semi-preparative HPLC (Mod.10 AKTA,
Amersham Biosciences, Piscataway, USA) on reversed-
phase support LICHROSORB C18 (5 lm, 250 9 8 mm)
with a linear gradient from 5 to 85% acetonitrile (0.1% TFA)
for 30 min at a flow rate of 1.5 ml/min and UV detection at
230 and 254 nm. The appropriate fractions were pooled and
lyophilized. All products gave single spots on thin layer
chromatography (Merck pre-coated silica gel plates, type
G60-F254) in the solvent systems: (A) 1-butanol:acetic
acid:water (4:1:5, upper phase), (B) 1-butanol:acetic
acid:water:pyridine (15:3:10:6) and (C) acetonitrile: water
(5:1). Analytical HPLC (Pharmacia LKB-2210) equipped
with a Nucleosil 100 C18 column (5 lm; 250 9 4.6 mm)
produced single peaks with at least 98% of the total peptide
peak integrals. The solvent system used was the same as that
123

Oxytocin analogues: synthesis and biological activity
1553
Fig. 4 a Analytical HPLC chromatograms of pure analogues [Mpa1,
D-Tyr(Et)2, Deg9]OT (A1) and [Mpa1, D-Tyr(Et)2, D-Tic9]OT (A2) [UV
detection at 220 nm]; b Mass spectra of analogues [Mpa1, D-Tyr(Et)2,
Deg9]OT (B1) and [Mpa1, D-Tyr(Et)2, D-Tic9]OT (B2) resulting from
ESI–MS analysis
for the semi-preparative HPLC. The final verification of
the peptide sequence was achieved by Electrospray Ioni-
sation-Mass Spectrometry (ESI–MS, Micromass-Platform
LC instrument). An example of analytical HPLC-chro-
matograms of pure peptides and ESI–MS spectra are
shown in Fig. 4 for analogues 14 and 15. The physico-
chemical properties of the new analogues are summarized
in Table 1.
with the analogue. In cases where the analogues were not
able to reach the same maximal response as oxytocin, the
single dosing procedure was employed. The inhibitory
potencies are expressed as pA2 values. The pA2 values
represent the negative logarithm to the base 10 of the molar
concentration of an antagonist which reduces the response
to 2 x units of the agonist to the response to x units of the
´
agonist (Slaninova 1987). Each analogue was tested on
uteri from four to five different rats. Pressor activity
was determined on phenoxybenzamine-treated male rats
(Dekanski 1952). The responses to standard doses of
oxytocin or vasopressin were stable for several hours,
without problems with tachyphylaxis.
Biological assay methods
The uterotonic activity was determined in vitro on an iso-
lated strip of rat uterus in the absence of magnesium
´
(Holton 1948; Munsick 1960; Slaninova 1987). In princi-
ple, cumulative dosing was applied in most experiments,
i.e. doses of the 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. The agonistic activity was
determined by comparing the threshold doses of oxytocin
Binding affinity determination
Determination of binding affinity to human OTR was
performed basically as described (Fahrenholz et al. 1984)
using tritiated oxytocin from NEN Life Science, Boston,
MA, USA. In brief, a crude membrane fraction of HEK
OTR cells, i.e. HEK cells having stable expressed human
123

1554
V. Magafa et al.
Table 1 Physiochemical properties of oxytocin analogues used in the present study
Analogues
Yielda
HPLCb
TLC, Rcf
MW
[M ? 1]? d
(%)
tR (min)
A
B
C
1
[L-Thi9] OTe
[L-3-Pal9]OT
[D-3-Pal9]OT
[Mpa1, L-Thi9]OT
[Mpa1, L-3-Pal9]OT
[Mpa1, D-3-Pal9]OT
[Mpa1, D-Tyr(Et)2]OT
[Mpa1, D-Tyr(Et)2, L-Thi9]OT
[Mpa1, D-Tyr(Et)2, D-Thi9]OT
[Mpa1, D-Tyr(Et)2, L-3-Pal9]OT
[Mpa1, D-Tyr(Et)2, D-3-Pal9]OT
[Mpa1, D-Tyr(Et)2, Aib9]OT
[Mpa1, D-Tyr(Et)2, Pen6, Aib9]OT
[Mpa1, D-Tyr(Et)2, Deg9]OT
[Mpa1, D-Tyr(Et)2, D-Tic9]OT
[Mpa1, D-Tyr(Et)2, Pen6, D-Tic9]OT
[Mpa1, D-Tic2, Aib9]OT
[Mpa1, D-1-Nal2]OT
[Mpa1, D-1-Nal2, L-Thi9]OT
[Mpa1, D-1-Nal2, D-Thi9]OT
[Mpa1, D-1-Nal2, L-3-Pal9]OT
[Mpa1, D-1-Nal2, D-3-Pal9]OT
[Mpa1, D-1-Nal2, Aib9]OT
[Pen1, D-1-Nal2, Aib9]OT
58
69
67
56
68
66
70
55
57
63
65
66
63
68
63
61
64
69
56
55
65
63
64
62
18.47
14.09
13.71
19.48
17.69
19.89
22.96
25.07
28.94
20.10
22.79
25.12
30.44
24.58
22.47
28.97
23.89
26.68
29.32
31.09
23.74
24.97
24.55
21.59
0.54
0.50
0.51
0.68
0.62
0.60
0.48
0.81
0.87
0.77
0.76
0.77
0.67
0.74
0.73
0.76
0.66
0.68
0.83
0.89
0.29
0.46
0.67
0.64
0.58
0.55
0.56
0.70
0.65
0.63
0.67
0.76
0.73
0.70
0.71
0.71
0.73
0.72
0.70
0.71
0.60
0.76
0.75
0.74
0.61
0.75
0.86
0.60
0.55
0.52
0.50
0.55
0.49
0.50
0.35
0.51
0.52
0.59
0.57
0.50
0.46
0.70
0.48
0.51
0.48
0.55
0.51
0.59
0.68
0.61
0.55
0.34
1,104.32
1,099.28
1,099.28
1,089.31
1,084.27
1,084.27
1,020.23
1,089.31
1,089.31
1,112.32
1,112.32
1,049.26
1,077.32
1,077.32
1,123.34
1,151.40
1,017.22
1,026.23
1,123.37
1,123.37
1,118.33
1,118.33
1,055.27
1,098.34
1,105.15
1,100.13
1,100.25
1,090.15
1,085.02
1,085.18
1,021.30
1,090.20
1,090.25
1,113.28
1,113.24
1,050.18
1,078.30
1,078.10
1,124.17
1,152.16
1,018.20
1,027.20
1,124.31
1,124.18
1,119.15
1,119.15
1,056.13
1,099.23
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
a
Yields were calculated on the basis of the amino acid content of the resin. All peptides were at least 98% pure
b
c
d
For elution conditions, see the Experimental Section
Solvent systems and conditions are given in the Experimental Section
Data obtained by ESI–MS
OT receptor (kindly donated by Dr. G. Gimpl (Gimpl et al.
or the 2-chlorotrityl-chloride resin bearing a Rink-Berna-
towitz linker as solid support using standard coupling
procedures and Fmoc/But strategy. Except for the cases of
the Fmoc-Gln(Trt)-OH coupling to Fmoc-Cys(Trt)-OH or
Fmoc-Pen (Trt)-OH whereby the coupling reaction lasted
1 h longer, couplings were complete within 2.5 h.
1997)), 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 and the buffer used was 50 mM HEPES at pH 7.6
containing 10 mM MnCl2 and 1 mg/ml bovine serum
albumin. The reaction was terminated by quick filtration on
a Brandel cell harvester. Binding affinities were expressed
as IC50 values calculated from the binding curves using
GraphPad Prism 3.02.
The overall yield of the syntheses of the OT analogues
was in the range 55–70% (calculated on the amount of
linker initially coupled to the resin). Higher yields were
obtained using the Rink Amide MBHA as solid support.
The purification of the Thi residue containing analogues
gives lower yields than those of the rest of the analogues
because of their insolubleness.
Results
The formation of the disulfide bond (cyclization) was
carried out according to the following two oxidized
methods: 1: dimethylsulphoxide (DMSO)/water method
and 2: carbon tetrachloride/tetrabutylammonium fluoride
(CCl4/TBAF) method. The cyclization was quantitative by
Peptide synthesis and purification
All analogues shown in Table 1 were synthesized either
manually or automatically on the Rink Amide MBHA resin
123

Oxytocin analogues: synthesis and biological activity
1555
Table 2 Biological activities of oxytocin analogues
Analogues
Biological activity
Uterotonic in vitro
Pressor
Agonistic
(IU/mg)
Antagonistic
(pA2)
Agonisticb
(IU/mg)
Antagonistic
(pA2)
I
Oxytocina
546
3.10
1
[L-Thi9] OT
[L-3-Pal9]OT
[D-3-Pal9]OT
4.3 0.8
0.25
0
0
0
0
0
2
3
*4
*7.0
II
4
[Mpa1]OTa (deamino-oxytocin)
[Mpa1, L-Thi9]OT
[Mpa1, L-3-Pal9]OT
[Mpa1, D-3-Pal9]OT
[D-Tyr(Et)2]OTa
803
5.5 0.9
*1.1
*7.0
0
0
0
\5.8
5
*7.0
0
6
*7.2
III
7
7.36
[Mpa1, D-Tyr(Et)2]OT
7.82 0.07
8.35 0.26
8.61 0.14
7.82 0.08
8.13 0.07
8.22 0.19
8.37 0.21
8.68 0.26
8.37 0.21
8.28 0.22
6.75 0.35
7.92 0.21
7.83 0.17
8.08 0.13
7.02 0.12
7.77 0.15
8.48 0.08
8.36 0.23
8.29 – 0.05
0
0
0
0
0
0
0
0
0
0
0
8
[Mpa1, D-Tyr(Et)2, L-Thi9]OT
[Mpa1, D-Tyr(Et)2, D-Thi9]OT
[Mpa1, D-Tyr(Et)2, L-3-Pal9]OT
[Mpa1, D-Tyr(Et)2, D-3-Pal9]OT
[Mpa1, D-Tyr(Et)2, Aib9]OT
[Mpa1, D-Tyr(Et)2, Pen6, Aib9]OT
[Mpa1, D-Tyr(Et)2, Deg9]OT
[Mpa1, D-Tyr(Et)2, D-Tic9]OT
[Mpa1, D-Tyr(Et)2, Pen6, D-Tic9]OT
[Mpa1, D-Tic2, Aib9]OT
[Mpa1, D-1-Nal2]OT
[Mpa1, D-1-Nal2, L-Thi9]OT
[Mpa1, D-1-Nal2, D-Thi9]OT
[Mpa1, D-1-Nal2, L-3-Pal9]OT
[Mpa1, D-1-Nal2, D-3-Pal9]OT
[Mpa1, D-1-Nal2, Aib9]OT
[Pen1, D-1-Nal2, Aib9]OT
[Mpa1, D-Tyr(Et)2, Thr4, Orn8]OT (Atosiban)a
0
9
6.19 0.33
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
IV
a
0
0
0
0
0
0
0
*5.60
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.02 – 0.02
The biological assays for the oxytocin analogues 1-24 were performed as outlined in the text. The values are averages SEM of at least three
experiments. The biological activities of the other analogues reported here as references are taken from the literature: for oxytocin and deamino-
2
oxytocin, see Hill et al. (1990), for [D-Tyr(Et) ]OT see Jezek et al. (1994) and for Atosiban, see Melin et al. (1986). If no SEM is given for the
ˇ
reference compound, it means that it was not given in the original papers
b
0 means no activity up to the dose 0.16 mg/kg of exp. animal
c
Means no activity or very low inhibitory activity pA2 \ 6
Biological activity (rat)
both applied methods, whereas using the carbon CCl4/
TBAF method the reaction time for achieving completion
of the cyclization was 10 min, the yields and purity of the
crude peptides, according to their chromatographic pro-
files, were lower because of the presence of residual TBAF
left after solvent evaporation.
Biological evaluation of the new analogues (1–24) is
summarized in Tables 2 and 3. In analogues 1–6 amino
acid Gly in position 9 of oxytocin and deaminooxytocin
was replaced by unnatural amino acids with a heterocyclic
ring in side-chain, e.g. L-Thi or L- or D-3-Pal. Analogue 1
([Thi9]OT) and analogue 2 [L-3-Pal9]OT were found to be
agonists in the uterotonic in vitro assay with a potency
approximately 130 and 2,200 times, respectively, lower
Finally, ESI mass spectrometry revealed that the
purified peptides were the desired products and their
purity determined by analytical HPLC was higher than
98%.
123

1556
V. Magafa et al.
of the native hormone and weak anti-oxytocin activity (pA2
value about 7.0). The potency of analogue 3 was not
affected (analogue 6).
Table 3 Binding affinities of oxytocin analogues
Analogues
Binding affinitya
IC50 (nM)
Previously reported studies suggested that the D-con-
figuration and hydrophobicity of the aromatic amino acid
in position 2 and the proper orientation of the C-terminal
glycine carboxamide are critical for obtaining high potency
OT analogues (Flouret et al. 1991; Lebl et al. 1992). We
have thus synthesized the analogues 8–11 and analogues
19–22 having D-Tyr(Et) and D-1-Nal in position 2,
respectively, and the above-mentioned modifications in
positions 1 and 9. Analogues 7 and 18 were synthesized for
comparison reasons.
Oxytocin
[L-Thi9] OT
[L-3-Pal9]OT
[D-3-Pal9]OT
[Mpa1, L-Thi9]OT
[Mpa1, L-3-Pal9]OT
[Mpa1, D-3Pal9]OT
[Mpa1, D-Tyr(Et)2]OT
[Mpa1, D-Tyr(Et)2, L-Thi9]OT
[Mpa1, D-Tyr(Et)2, D-Thi9]OT
[Mpa1, D-Tyr(Et)2, L-3-Pal9]OT
[Mpa1, D-Tyr(Et)2, D-3-Pal9]OT
[Mpa1, D-Tyr(Et)2, Aib9]OT
[Mpa1, D-Tyr(Et)2, Pen6, Aib9]OT
[Mpa1, D-Tyr(Et)2, Deg9]OT
[Mpa1, D-Tyr(Et)2, D-Tic9]OT
[Mpa1, D-Tyr(Et)2, Pen6, D-Tic9]OT
[Mpa1, D-Tic2, Aib9]OT
[Mpa1, D-1-Nal2]OT
[Mpa1, D-1-Nal2, L-Thi9]OT
[Mpa1, D-1-Nal2, D-Thi9]OT
[Mpa1, D-1-Nal2, L-3-Pal9]OT
[Mpa1, D-1-Nal2, D-3-Pal9]OT
[Mpa1, D-1-Nal2, Aib9]OT
[Pen1, D-1-Nal2, Aib9]OT
2.7 0.2
570 149
4,740 287
393 221
145 58
1
2
3
4
5
1,636 128
174 99
6
7
148 26
8
33.6 4.9
9
301
92
3
2
5
Upon substitution of the residue in position 2 by
D-Tyr(Et) or D-1-Nal the analogues 4–6 lost the agonistic
activity and become strong antagonists (analogues 8–11,
pA2 = 7.82–8.61). It is notable that in the pressor test
only analogue 9 showed a weak antagonistic activity
(pA2 = 6.19 0.33).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
a
33
150 34
80
6
98.0 9.2
46 15
As can be seen from the table, most of the analogues
having in position 2 D-1-Nal—a more compact residue than
D-Tyr(Et)—showed slightly lower oxytocin antagonism in
the uterotonic test, ensuring the major role of D-aromatic
amino acid in position 2. Furthermore, all analogues were
selective, showing no pressor activity.
25
3
1,800 150
34.5 2.9
32.4 15.8
16.2 5.7
155 15
In an attempt to further investigate the role of the residue
in position 9, we synthesized analogues 12–16 and 23–24
with residues without a heterocyclic ring in the side chain,
e.g. the conformationally interesting lipophilic residues
Aib, Deg and D-Tic, without a heteroatom in side-chain.
Analogues having combined substitutions in position 1
(Mpa), in position 2 D-Tyr(Et) and in position 9 Aib or Deg
or D-Tic retain the oxytocin antagonistic potency. The
highest one had analogue 14 ([Mpa1, D-Tyr(Et)2, Deg9]OT,
pA2 = 8.68 0.26). It is notable that further modification
with Pen6 (peptides 13 and 16) led to no change in the
inhibitory potency, ensuring the role of the folding of the
tail portion above or below the plane of the ring. Similarly,
further Pen1/Mpa1 exchange (analogues 23 and 24) led to
no change in inhibitory potency (pA2 = 8.48 0.08 and
pA2 = 8.36 0.23, respectively).
37.5 6.5
44 14
142 18
The values given are averages SEM from three experiments
performed in duplicates
than that of the native hormone. D-diastereoisomer con-
taining 3-Pal (analogue 3) was found to be a partial agonist
in the oxytocic assay, with agonistic potency approxi-
mately 140 times lower than that of the native hormone (16
times higher than that of the L-counterpart) and weak
antagonist with pA2 value about 7.0. The analogues 1–3
showed no pressor activity.
It has been known that substitution of the N-terminal
cysteine by a residue without the amino group, i.e.
b-mercaptopropionic acid (Mpa) (Hope et al. 1962),
enhances the biological activity. Literature data indicate
that the neurohypophyseal hormones lacking the N-termi-
nal amino group are inactivated quite slowly (Grzonka
et al. 1983) and that the first amino acid plays a decisive
Finally, D-Tic2 modification in analogue 12 resulted
in an antagonism with substantially lower potency
(pA2 = 6.75 0.35, analogue 17).
Among the analogues having Aib in position 9, the Tic2
analogue had considerably lower anti-oxytocic potency
than the D-1-Nal and D-Tyr(Et) analogues.
´
role in receptor binding (Toth et al. 1999). Therefore, we
synthesized analogues 4–6. Replacement of Cys1 by Mpa1
in analogue 1 did not affect the agonistic activity (analogue
4) in the uterotonic test; negligible antagonistic activity in
the pressor test was detected. In analogue 2, this change led
to partial agonism (analogue 5) in the oxytocic assay, with
agonistic potency approximately 500 times lower than that
Binding affinity (human receptor)
Table 3 summarizes the binding affinities of the analogues
to human OTR permanently expressed on HEK cells.
As can be seen, introduction of the conformationally
123

Oxytocin analogues: synthesis and biological activity
1557
constrained residues with an aromatic heterocyclic ring at
the side chain (L-Thi or L- or D-3-Pal) decreased the binding
affinity significantly. The lowest affinity from these ana-
logues with a single modification had analogue 2 ([L-3-
Pal9]OT, IC50 = 4,740 287 nM), the highest analogue 3
([D-3-Pal9]OT, IC50 = 393 221 nM).
changed susceptibility of the molecules to enzymatic
´ˇ ˇ
cleavage (Hlavacek and Fric 1989).
We have evaluated the potency of the new analogues in
uterotonic in vitro test, pressor test and affinity to human
OTR stable expressed on the surface of HEK cells. The
comparison of the results of biological activity and binding
affinity has to be performed with caution as the tests were
performed with rats and the binding experiments using
human OTR.
Deamination in position 1 of analogues 1–3 increased
the affinity slightly (compare analogues 1 and 4, 2 and 5, 3
and 6).
Further modification using introduction of D-Tyr(Et)2
significantly increased the affinity (compare peptides 4 and
8; 5 and 10; 6 and 11). Replacement of the L-Thi in position
As can be seen from the Table 2, the substitution of
L-Thi9, L-3-Pal9 or D-3-Pal9 for Gly9 in OT or deamino OT
(analogues 1–6) leads to drastic losses in agonistic activity.
The L-diastereoisomers of 3-Pal (analogues 2 and 5)
showed the highest decrease in agonistic activity. Ana-
logues 1 and 4 with L-Thi9 showed no inhibitory potency
compared to analogues with L- or D-3-Pal9, an amino acid
residue with slightly larger (one methylene group) hetero-
cyclic ring. Our findings are consistent with those previ-
ously reported which suggested that the C-terminal
tripeptide sequence and especially the proper orientation of
the C-terminal glycine carboxamide are critical for
obtaining high potency OT agonist analogues (Ting et al.
1980; Hruby 1986; Fragiadaki et al. 2003).
9
with the D-counterpart (analogue 9) surprisingly
decreased the binding affinity to the OT receptor (compare
IC50 = 33.6 4.9 nM and 301 3 nM for 8 and 9,
respectively).
On the other hand, the D-1-Nal2/D-Tyr(Et)2 exchange in
analogues 8–11 had an inconsistent effect; in the case of
analogues 8, 10 and 11 there was almost no change in affinity
(compare with analogues 19, 21 and 22, respectively), in the
case of the D-Thi9 analogue there was strong decrease of
affinity (compare peptides 9 and 20). In analogue 12 it
improved the affinity about 3.5 times (compare IC50 = 150
and 44 nM for analogues 12 and 23, respectively). Further-
more, replacement of Mpa1 with Pen in analogue 23
decreasedtheaffinityabout three times (compare IC50 = 150
and 44 nM for analogues 23 and 24, respectively).
Analogues having combined substitutions in position 1
(Mpa), in position 2 [D-Tyr(Et)] and in position 9 with resi-
dues having more compact side chains than Gly exhibited in
most of the cases slightly higher anti-OT potency in com-
parison to the [Mpa1, D-Tyr(Et)2]OT (analogue 7) which has
no modification in position 9. In the case of analogues with
D-1-Nal2—more rigid and lipophilic side chain than that of
D-Tyr(Et)2—the same modifications in position 9 did not
lead to substantial change in the antagonistic potency in
comparison to analogue 18 having no change in position 9.
Only in the case of analogue 21 having L-3-Pal in position 9,
the antagonistic activity was decreased. If we order the
studied amino acids introduced into position 9 according to
their effect on antagonistic activity, than in the [Mpa1,
D-Tyr(Et)2] series it would be (from the best till the worst):
Deg = D-Thi [L-Thi = Aib = D-Tic [D-3-Pal [L-3-Pal =
Gly and in the [Mpa1, D-1-Nal2] series: Aib [ D-Thi [
Gly = L-Thi = D-3-Pal [ L-3-Pal.
The D-Tic substitution in position 2 seems to have no
advantage in comparison to D-Tyr(Et) or D-1-Nal in pro-
ducing antagonists (both the potency in rat uterotonic test
and affinity to human OTR are substantially lower). This
finding ensures the role of the side chain of the residue in
position 2 for binding to the uterotonic receptor and it is
consistent with data previously observed (Fragiadaki et al.
2007).
Modifications of analogue 7 by the introduction of Aib
or Deg or D-Tic also influenced the binding affinity insig-
nificantly. The values lie in the range of 46–150 nM.
Finally, analogue 17 with the combination of deamina-
tion, D-Tic2 and Aib9 modifications, exhibited significantly
low binding affinity (IC50 = 1,800 nM). This finding is
consistent with that previously observed by Fragiadaki
et al. (2007), ensuring the role of the side chain of the
residue in position 2 for binding to the uterotonic receptor.
Discussion
We have synthesized 24 analogues of OT with the aim to
study the influence of modifications in position 9 on the
activity of OT and some of its analogues as deamino[D-
Tyr(Et)2]OT or deamino[D-1-Nal2]OT. We have introduced
into position 9 amino acids both containing a heterocyclic
ring in the side chain (Thi, 3-Pal) and lipophilic residues
without a heteroatom in the side-chain (Aib, Deg and
D-Tic). Structural modifications of the side-chain moieties
in the C-terminal tripeptide might lead to analogues with
variable biological properties at different OT target tissues.
Such variations in biological activity could result from
conformational changes influencing interactions of the
analogues with the receptor, from steric demands, or from
The binding affinities to human OTR more or less mirror
the biological activities. There seems to be a species dif-
ference in the preference of the receptors for D-1-Nal2
analogues. The D-1-Nal2 modification generally improves
123

1558
V. Magafa et al.
Fields BG, Noble LR (1990) Solid phase peptide synthesis utilizing 9-
fluorenylmethoxycarbonyl-amino acids. Int J Pept Protein Res
35:161–214
Flouret G, Brieher W, Mahan K (1991) Design of potent oxytocin
antagonists featuring D-tryptophan at position 2. J Med Chem
34:642–646
the binding affinity to human OTR in comparison to the
D-Tyr(Et)2 or D-Tic2 substituted analogues.
The fact that almost all the analogues are inactive in the
pressor test shows that receptors mediating the pressor
activity are not at all tolerant to the conformational changes
arising from replacement of the naturally occurring glycyl
residue in position 9.
´
Fragiadaki M, Magafa V, Slaninova J, Cordopatis P (2003) Synthesis
and biological evaluation of oxytocin analogues containing L-a-t-
butylglycine [Gly(But)] in positions 8 or 9. Peptides 24:1425–
1431
Additional substitutions of Cys in position 6 by a residue
with a more restricting and bulky side chain did not sig-
nificantly influence the antagonistic biological activity. The
introduction of Pen6 seems not to change the folding of the
tail portion above or below the plane of the ring.
´
´
Fragiadaki M, Magafa V, Borovickova L, Slaninova J, Cordopatis P
(2007) Synthesis and biological activity of oxytocin analogues
containing conformationally restricted residues in position 7. Eur
J Med Chem 42:799–806
Giles W, Bisits A (2007) The present and future of tocolysis. Best
Pract Res Clin Obstet Gynaecol 21:857–868
Gimpl G, Fahrenholz F (2001) The oxytocin receptor system:
structure, function, and regulation. Physiol Rev 81:629–683
Gimpl G, Burger K, Fahrenholz F (1997) Cholesterol as modulator of
receptor function. Biochemistry 36:10959–10974
Goodwin TM, Paul R, Silver H et al (1994) The effect of the oxytocin
antagonist atosiban on preterm uterine activity in the human. Am
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Grzonka Z, Lammek B, Kasprzykowski F, Gazis D, Schwartz IL (1983)
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Conclusion
These results confirm the fact that the proper topological
arrangement in the C-terminal part of the molecule is
crucial for the agonistic activity and much less important
for the antagonistic activity. Of all the 24 analogues, the
[Mpa1, D-Tyr(Et)2]OT analogues 14 and 23 containing Deg
and Aib in position 9, respectively, are the most promising
leads for the synthesis of potent and selective OT antago-
nists as they show potent anti-OT activity in the rat
uterotonic test in vitro, high affinity to human OTR and at
the same time exhibit no rat pressor or anti-diuretic (data
not shown) activities.
´
Hill PS, Smith D, Slaninova J, Hruby VJ (1990) Bicyclization of a
weak oxytocin agonist produces a highly potent oxytocin
antagonist. J Am Chem Soc 112:3110–3113
Acknowledgments Partial funding for this work was provided by
research project No. Z40550506 of the Academy of Sciences of the
Czech Republic.
´ˇ
ˇ
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Czech Chem Commun 54:2261–2270
Holton P (1948) A modification of the method of Dale and Laidlaw
for standardization of posterior pituitary extract. Br J Pharmacol
Chemother 3:328–334
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