Analogues of neurohypophyseal hormones, oxytocin and arginine vasopressin, conformationally restricted in the N-terminal part of the molecule

Article abstract of DOI:10.1021/jm058038f

It is generally accepted that the conformation of the N-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. In this work, we decided to investigate how the substitution of positions 2 and 3 with the ethyle

Full text of DOI:10.1021/jm058038f

2016
J. Med. Chem. 2006, 49, 2016-2021
Analogues of Neurohypophyseal Hormones, Oxytocin and Arginine Vasopressin,
Conformationally Restricted in the N-Terminal Part of the Molecule
Wioleta Kowalczyk,*,† Adam Prahl,† Izabela Derdowska,† Dariusz Sobolewski,† Jadwiga Olejnik,‡ Janusz Zabrocki,‡
Lenka Borovickova´,§ Jirˇina Slaninova´,§ and Bernard Lammek†
Faculty of Chemistry, UniVersity of Gdan˜sk, Sobieskiego 18, 80-952 Gdan˜sk, Poland, Institute of Organic Chemistry, Technical UniVersity of
Ło´dz´, 90-924 Ło´dz´, Poland, and Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
FlemingoVo sq. 2, 16610 Prague, Czech Republic
ReceiVed July 21, 2005
It is generally accepted that the conformation of the N-terminal part of neurohypophyseal hormones analogues
is important for their pharmacological activity. In this work, we decided to investigate how the substitution
of positions 2 and 3 with the ethylene-bridged dipeptide -Phe-Phe would alter the pharmacological properties
of OT, [Mpa1]OT, and [Cpa1]OT (OT ) oxytocin; Mpa ) 3-mercaptopropionic acid; Cpa ) 1-mercapto-
cyclohexaneacetic acid) and to investigate how a bulky 3,3-diphenyl-L-alanine residue incorporated in position
2 of AVP, [Mpa1]AVP, and [Cpa1]AVP (AVP ) arginine vasopressin) would change the pharmacological
profile of the compounds. The next analogues, [Val4]AVP, [Mpa1,Val4]AVP, and [Cpa1,Val4]AVP, had
N-benzyl-L-alanine introduced at position 3. The last peptide was designed by Cys1 substitution in AVP by
its sterically restricted bulky counterpart, R-hydroxymethylcysteine. All the peptides were tested for their in
vitro uterotonic, pressor, and antidiuretic activities in the rat. The results of these assays showed that the
reduction of conformational freedom of the N-terminal part of the molecule had a significant impact on
pharmacological activities.
Introduction
hormones, may be a reason for the overlap of pharmacological
profiles of OT and AVP that are seen. However, the affinities
of these hormones for receptors are different, as are their
biological capacities to transduce signals and elicit a biological
response.
Oxytocin (OT),a a physiologically important nonapeptide
hormone and neurotransmitter containing a 20-membered ring
and an acyclic tripeptide tail, regulates several physiological
functions, such as milk ejection, uterine contraction, vascular
and cardiac relaxation, etc. Another neurohypophyseal hormone,
arginine vasopressin (AVP), in addition to its well-known
antidiuretic activity, has also complex cardiovascular actions.
The AVP and OT receptor family consists of four subtypes of
receptors. V1a receptors modulate the vasopressor actions of
AVP,1 the adrenocorticotropic hormone (ACTH) releasing
activity of AVP is evoked by V1b receptors,2 and OT receptors
control milk ejection and uterine smooth muscle contractions.
These three receptor subtypes function via the phosphatidyli-
nositol pathway. The last subtype, V2 receptor, present in the
renal tubule and collecting ducts, modulates antidiuretic re-
sponses to AVP and is linked to adenylate cyclase activity.2
The AVP and OT receptor subtypes show a high degree of
homology. This, along with the similar structure of both
In the last few years, there has been an upsurge of interest in
both neurohypophyseal hormones, and new physiological and
pathophysiological roles for these peptides have been indicated.
Most of this work would not have been possible without the
aid of OT and AVP antagonists as pharmacological tools.3,4
However, during the past decade, we noticed that antagonists
of OT, which seemed to be less readily investigated than their
AVP counterparts, became more and more interesting because
of their potential application in the prevention of premature labor
and treatment of dysmenorrhea. There is a substantial body of
evidence pointing to the significance of OT in both premature
and mature parturitions; thus, one alternative to the current
therapy for preterm labor is the use of OT receptor blockers.5
Although in recent years much progress has been made toward
improving the selectivity of OT antagonists,6 the design of
compounds that are both potent and selective as OT blockers
remains a challenge.
A great deal of evidence points to the importance of the
conformation of the N-terminal part of OT and AVP analogues
for their pharmacological activity.3,7-9 This is supported also
by our recent description of some pharmacological properties
of analogues with bulky L- or D-â-(1-naphthyl)-alanine (Nal)
moieties at position 2 of [(Mpa)1]AVP (Mpa ) 3-mercapto-
propionic acid). Both compounds surprisingly exhibited moder-
ate antioxytocic activity in vitro and were very selective.10 We
assumed that the hindering effect caused by the naphthyl ring
has a significant impact on conformation of the analogues and
thus greatly influences their interactions with receptors. In
another study, Prochazka and Slaninova´11 described the synthesis
and pharmacological evaluation of analogues with L- or D-Nal
at position 2 of OT or AVP. This, together with the results of
* To whom correspondence should be addressed. Faculty of Chemistry,
University of Gdan˜sk, Sobieskiego 18, 80-952 Gdan˜sk, Poland. Tel.:
(0 48 58) 345 03 88. Fax: (0 48 58) 341 03 57. E-mail: wiola@
chem.univ.gda.pl.
† University of Gdan˜sk.
‡ Technical University of Ło´dz´, 90-924 Ło´dz´.
§ Academy of Sciences of the Czech Republic.
a Abbreviations: AVP, arginine vasopressin; Cpa, 1-mercaptocyclohexa-
neacetic acid; Dap, diaminopropionic acid; DCM, dichloromethane; DIEA,
diisopropylethylamine; Dpa, 3,3-diphenyl-L-alanine; DMF, dimethylforma-
mide; HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate; R-HmC, R-hydroxymethylcysteine; HOAt, 1-hydroxyben-
zotriazole; HOBt, 1-hydroxy-benzotriazole; MBHA, p-methoxybenzhydryl-
amine; Mob, 4-methoxybenzyl; Mpa, 3-mercaptopropionic acid; Nal, â-(1-
naphthyl)-L-alanine; NMP, 1-methyl-2-pyrrolidone; OT, oxytocin, TBTU,
2-1H-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFA,
trifluoroacetic acid. The symbols of the amino acids and peptides are in
accordance with the 1983 Recommendations of the IUPAC-IUB Joint
Commission on Biochemical Nomenclature and “A Revised Guide to
Abbreviations in Peptide Science” published in J. Pept. Sci. 2003, 9, 1-8.
10.1021/jm058038f CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/25/2006

Analogues of Neurohypophyseal Hormones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2017
our previous study,12 clearly demonstrates new possibilities
opened up by utilizing the bulky Nal residue for modification
of both OT or AVP for designing analogues of neurohypophy-
seal hormones. It has been shown that single substitution with
a Nal residue results in selective and quite potent analogues of
both hormones.11,12 Another approach, which also resulted in
moderately potent and fairly selective antagonists of OT, was
used in Manning’s laboratory and consisted of multiple modi-
fications of the OT molecule, e.g., desGly-NH2, d(CH2)5[D-
Trp2,Thr4,Dap5]OVT, where Dap is diaminopropionic acid, and
d(CH2)5 stands for 1-mercaptocyclohexaneacetic acid.
of conformational constraint. Considerable information exists
concerning outlining of the structural perturbations induced by
this modification: occurrence of the cis isomer for tertiary amide
bonds, steric constraints due to the N-alkyl group, suppression
of a proton-donating N-H group capable of hydrogen bonding,
and increased basicity of the carbonyl group. Conformational
studies indicate that the influence of N-alkylation on a confor-
mation depends to a large extent on the chirality of the residues
surrounding the modified peptide bond.15
Finally, it seemed worthwhile to modify the Cys1 in AVP to
check how its sterically restricted bulky counterpart, R-hy-
droxymethylcysteine (R-HmC), will change the pharmacological
profile of the hormone.
The analogues have the following structure: [-Phe-Phe2,3]-
OT (I), [Mpa1, -Phe-Phe2,3]OT (II), [Cpa1, -Phe-Phe2,3]OT (III),
Biologically active peptides exhibit multiple conformations
in solution. Thus, the synthesis of conformationally restricted
analogues is a valuable approach for determining structure-
activity relationships. Restrictions can be imposed by formation
of cyclic structures within the peptide framework by disulfide
and lactam bridges, or by substitution of chosen amino acid
residues with sterically restricted fragments that limit confor-
mational freedom, forcing peptide backbone and/or side chains
to adopt specific orientations. Another approach to achieve
certain stabilization of the structure, is the preparation of various
types of pseudopeptides through additional, short-range cycliza-
tion. One such constraint consists of the -CH2-CH2- link
bridging two consecutive peptide nitrogens leading to the
formation of a piperazinone ring, which incorporates the relevant
N-C-C′-N′ peptide segment as its inherent part.13 Using
theoretical conformational analysis, the impact of such constraint
(N,N′-ethylene-bridged dipeptide unit) was studied, taking N,N′-
bridged N-acetyl-Phe-Phe-N′-methylamide as an example. The
results indicate equal preferences for assuming either of two
enantiomeric twisted chair/boatlike pucker types for the piper-
azinone ring. The size and/or pucker type of the ring constraint
affects little the peptide backbone, which always tends to exist
in either of two distinct semi-extended forms. They are equally
populated for the piperazinone-constrained peptide. The side-
chain benzyl group shows conformational preferences dependent
on whether it belongs to the first (in the ring) or to the second
(outside the ring) Phe residue. In the first Phe generally two,
Viz. extended-to-N (EN) and extended-to-O (EO) staggered
conformations are preferred over the third folded one (F). The
notable exceptions are the piperazinone-constrained peptides
where the F form tends to be distinctly favored. The benzyl
ring belonging to the second Phe (that outside the rings) typically
prefers the EN and never assumes the F conformation.14
[Dpa2]AVP (IV), [Mpa1,Dpa2]AVP (V), [Cpa1,Dpa2]AVP (VI),
[N-Bzl-Ala3,Val4]AVP (VII), [Mpa1,N-Bzl-Ala3,Val4]AVP (VIII),
[Cpa1,N-Bzl-Ala3,Val4]AVP (IX), [R-HmC1]AVP (X).
Results
The 10 new analogues of AVP and OT (I-X) were
synthesized using Boc-chemistry on a methoxybenzhydryl resin
(I-IX) and on a chloromethylated Merrifield resin (X). For
analogues II, V, VIII, and III, VI, IX, Mpa(Mob) and Cpa-
(Mob) were used in final step, respectively. As we were not
able to couple Boc-N(Bzl)Ala during the synthesis to the
growing peptide chain for peptides VII-IX, Boc-Tyr(Bzl)-
N(Bzl)Ala-Val-OH was used in the fifth coupling step. On
completion of the syntheses, the protected peptidyl or acylpep-
tidyl resin (analogues I-IX) were treated with liquid HF in
the presence of anisole at 0 °C (in the case of analogue X the
protected nonapeptidyl resin was ammonolysed in methanol)
and oxidized with I2 in methanol. The crude products were
desalted on a Sephadex G-15 and purified by RP-HPLC. The
purity and identity of each peptide were determined by HPLC
and FAB mass spectrometry (molecular ion).
Physicochemical properties and pharmacological data of the
new peptides I-X, and those of AVP, OT, [Mpa1]AVP, and
some related peptides, are presented in Tables 1 and 2. The
activities of the analogues were determined in the in vitro rat
uterotonic test in the absence of magnesium ions, in the rat
pressor test, and in the antidiuretic assay on conscious rats as
described in the experimental section. None of these new
analogues exhibited antivasopressor activity. A comparison of
the antidiuretic activities of the new analogues with those
published previously must be done with caution as different
testing procedures were used. To compare the effects of
compounds with different regression lines of the dose response
curve, doses of AVP were compared with such doses of new
analogues that gave the same antidiuretic response, i.e., the doses
that caused rats to excrete half of the water load (t1/2) in 60 and
200 min. For AVP, the activity was arbitrary taken to be 465
IU/mg for both responses. Analogues IV and V have antidiuretic
activity comparable to AVP at the level of t1/2 60 min. However,
their dose-response curves are much steeper being similar to
those of dDAVP. It means that at a t1/2 level of 200 min the
activity is much higher than that of AVP (2000 and 10000% of
that of AVP, respectively). The remaining analogues (I-III and
VI-IX) exhibited negligible antidiuretic activity, while com-
pound X was a moderately potent agonist.
In summary, all these findings prompted us to continue our
studies on the influence of sterically restricted fragments placed
in this part of OT or AVP analogues on their pharmacological
properties. First, we decided to check how substitution of
positions 2 and 3 with an ethylene-bridged dipeptide -Phe-Phe
(a -CH2-CH2- bridge spanning two subsequent peptide
nitrogens, thus forming a piperazinone ring) would alter the
pharmacological properties of OT, [Mpa1]OT, and [Cpa1]OT.
Next, we designed three peptides by substituting position 2
in AVP, [Mpa1]AVP, and [Cpa1]AVP with bulky 3,3-diphenyl-
L-alanine residue. Both modifications restrict the conformational
freedom of the resulting peptides, which should influence their
pharmacological properties. The third group of analogues was
designed by N-benzyl-L-alanine substitution at position 3 of
[Val4]AVP, [Mpa1,Val4]AVP, and [Cpa1,Val4]AVP. The N-
benzyl-L-alanine may be considered as a structural analogue of
phenylalanine, in which the side chain is moved from CR to
nitrogen. At the same time, introduction of such a peptoid unit
into a peptide chain may also be considered as an alkylation of
the peptide bond, which is attractive as a local and subtle mode
Peptides I-VI and X exhibited different antioxytocic activi-
ties ranging from pA2 ) 6.40 to 7.23. Analogues VII and IX
did not show any activity in the uterotonic test, while compound
VIII showed very low agonistic activity.

2018 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Kowalczyk et al.
Table 1. Physicochemical Properties of Peptides I-Xa
HPLC tr
[M + H+]
calculated
yield [%]
analogue
[Phe-Phe2,3]OT
a
b
formula
found
A
B
I
9.89
13.84
18.37
11.98
17.72
23.74
C48H66N12O11S2
C48H65N11O11S2
C52H76N11O11S2
1051.2
1036.2
1104.2
1052.2
1036.1
1104.2
89
65
83
59
24
50
[Mpa1, Phe-Phe2,3]OT
[Cpa1, Phe-Phe2,3]OT
II
III
[Dpa2]AVP
IV
V
11.36
14.00
15.87
9.70
11.63
13.97
7.38
13.18
17.18
19.96
11.75
13.56
16.99
9.82c
C52H69N15O11S2
C52H68N14O11S2
C57H76N14O11S2
C47H68N14O11S2
C47H67N13O11S2
C52H73N13O11S2
C47H66N15O13S2
1143.8
1128.8
1197.4
1069.3
1054.3
1122.3
1113.5
1144.5
1129.7
1197.6
1069.8
1054.3
1122.2
1114.7
86
79
88
71
85
87
71
63
46
59
38
54
66
16
[Mpa1,Dpa2]AVP
[Cpa1,Dpa2]AVP
VI
VII
VIII
IX
X
[N-Bzl-Ala3,Val4]AVP
[Mpa1,N-Bzl-Ala3,Val4]AVP
[Cpa1,N-Bzl-Ala3,Val4]AVP
[R-HmC1]AVP
a Headings: a, Linear gradient from 20 to 80% of [B] for 20 min; b, linear gradient from 20 to 80% of [B] for 30 min; c, linear gradient from 10 to 45%
of [B] for 15 min. A, yields were calculated on the base of the glycine content of the starting resin; B, yields are based on the amount of crude peptide.
Table 2. Pharmacological Properties of the New AVP Analoguesa
activity
analogue
uterotonic in vitro no Mg2+
pressor IU/mg or pA2
antidiuretic IU/mgc
AVPb
17
412
465
[Mpa1]AVPb
[Cpa1]AVPb
OTb
27-63
pA2 ) 8.15
450
803
pA2 ) 7.61
346-370
pA2 ) 8.35
5
1300-1745
0.033
5
[Mpa1]OTb
1.44
19
[Cpa1]OTb
[Val4]AVPb
[Mpa1,Val4]AVPb
[Cpa1,Val4]AVPb
[Phe-Phe2,3]OT
32
51
738
1150
0.32
pA2 ) 7.34
pA2 ) 6.82
pA2 ) 7.97
0
I
<0.04
[Mpa1, Phe-Phe2,3]OT
[Cpa1, Phe-Phe2,3]OT
II
III
pA2 ) 6.50
pA2 ) 6.72
0
0
<0.04
<0.04
[Dpa2]AVP
IV
V
pA2 ) 7.00
pA2 ) 7.23
pA2 ) 6.10
0
very low agonist
0
0
0
0
0
0
0
0.18
450 (9000)
450 (45000)
<0.04
[Mpa1,Dpa2]AVP
[Cpa1,Dpa2]AVP
VI
VII
VIII
IX
X
[N-Bzl-Ala3,Val4]AVP
[Mpa1,N-Bzl-Ala3,Val4]AVP
[Cpa1,N-Bzl-Ala3,Val4]AVP
[R-HmC1]AVP
<0.04
<0.04
<0.04
4.5 (∼22.5)
pA2)6.40
a IU/mg or pA2. b Values taken from ref 7. c The activities obtained by comparing doses of AVP and the analogue resulting in an antidiuresis time of t1/2
) 60 min; in parentheses, the activities obtained by comparing doses of AVP and the analogue resulting in an antidiuresis time of t1/2 ) 200 min.
Discussion
out to be a potent and selective antagonist of the vasopressor
response to AVP.12
The present work is a continuation of our already 10-year
studies aimed at clarifying the impact of steric restrictions and
bulky subtituents in the N-terminal part of the AVP and OT
molecules on pharmacological properties of the resulting
analogues. Previously, we reported that such modifications
influenced the interaction of molecules with V1a, V2, and
oxytocic receptors. Our approach to this problem included the
ethylene-bridged dipeptide fragment -Phe-Phe, at positions 2
and 3.16 One peptide from this series was a highly potent and
selective blocker of V1 receptors. It is noteworthy that OT
analogues substituted at positions 2 and 3 with the ethylene-
bridged dipeptide -D-Phe-D-Phe exhibited either no or very weak
activities.17 We also showed that modification of position 2 of
AVP and some of its analogues with 1-aminocyclohexane-1-
carboxylic acid or 1-aminocyclopentane-1-carboxylic acid re-
sulted in compounds having differently modified activities (high
antidiuretic potency, low and graded pressor activity, and either
no activity or low oxytocin antagonizing activity in the
uterotonic in vitro test), thus selectively altered interaction with
receptors.18-20 Furthermore, we demonstrated that the analogues
of AVP modified at position 2 with bulky L or D â-(1-naphthyl)-
alanine were moderately potent and exceptionally selective
oxytocin antagonists in vitro.21 On the other hand, [D-Arg8]VP
substituted at position 3 with â-(2-naphthyl)-L-alanine turned
This paper describes the synthesis and pharmacological
evaluation of 10 new analogues with various types of modifica-
tions in the N-terminal part of the molecule. First, we substituted
positions 2 and 3 of OT, [Mpa1]OT, and [Cpa1]OT with a
conformationally constrained dipeptide unit Phe-Phe, to obtain
analogues I, II, III. This constraint consists of the -CH2-CH2-
link bridging two consecutive peptide chain nitrogens leading
to the formation of a piperazinone ring that incorporates the
relevant -N-C-C′-N′- peptide segment as its inherent part.
This modification converted OT and its agonist [Mpa1]OT into
rather weak but selective antagonists of oxytocic activity in the
in vitro uterus test. With [Cpa1]OT, this modification was not
beneficial. On the contrary, it significantly reduced the antago-
nistic potency (see analogue III).
Of the next three analogues, IV, V, and VI, peptide IV
designed by only a single modification of the AVP molecule,
i.e., substitution of Tyr2 with 3,3-diphenyl-L-alanine, has strik-
ingly different pharmacological properties in comparison to the
parent hormone. It displays no pressor activity, while its
antidiuretic potency is preserved and even prolonged due to the
much steeper dose-response curve. Moreover, this compound
is a moderately potent blocker of oxytocic uterotonic activity.
This combination of pharmacological properties makes our

Analogues of Neurohypophyseal Hormones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2019
Tyr(Bzl)-N(Bzl)Ala-Val-OH (4), which was used for the solid-phase
peptide synthesis procedure.
peptide interesting as a tool in physiological experiments.
Deamination of compound IV results in peptide V having an
essentially similar profile of activities. However, both anti-
diuretic and antiuterotonic potencies are enhanced. It is interest-
ing to notice that additional modification of compound IV at
position 1 with 1-mercaptocyclohexaneacetic acid (Cpa) (in-
tended to enhance its antagonistic properties), resulted in peptide
VI with substantially reduced antioxytocic potency and practi-
cally devoid of antidiuretic activity. This finding suggests that
two bulky groups at positions 1 and 2 together might not be
compatible with the binding site of the receptors. Previously,
when we combined Cpa1 modification with substitution of
position 2 of sterically restricted amino acids, e.g., 1-aminocy-
clohexane-1-carboxylic acid or 1-aminocyclopentane-1-car-
boxylic acid,18-20 we also observed the reduction of activity.
The condition that Cpa1 (or similar residue) is essential for
substantial antagonistic activity3,4,7 appears not to be valid for
analogues that contain either bulky or effectively reducing
conformational freedom fragments in the N-terminus.
N-Benzyl-L-alanine, a chiral peptoid unit, may be considered
as an analogue of L-phenylalanine, in which the side chain is
moved from CR to nitrogen. In other words, the nitrogen of the
peptide bond is de facto alkylated by benzyl. This may appear
to be a local and subtle conformational change. However, all
three analogues with N-benzyl-L-alanine in position 3 (VII-
IX) showed no activities in the tests; thus, this modification
turned out to be deleterious for the interaction with all V1a, V2,
and oxytocic receptors.
Peptide X prepared by substitution of R-hydroxymethylcys-
teine in position 1 of AVP instead of Cys has an interesting
pharmacological profile, although the activities are not too high,
being comparable to those of any analogue having the modified
amino group. It is a weak antagonist of oxytocin, practically
devoid of pressor activity; however, it still has weak antidiuretic
properties (about 1% of that of AVP). This modification in
combination with single or multiple additional changes in
positions 2, 3, and 8 may result in a compound having interesting
pharmacological properties.
Summing up, our studies resulted in several analogues with
interesting pharmacological properties that may contribute to
mapping the receptor binding sites. We believe that even more
important is the fact that we could once more demonstrate that
the modification of the N-terminal part of the molecule,
especially the reduction of conformational freedom, has a
dramatic impact on the pharmacological activities, which in turn
opens new possibilities for designing new analogues of AVP
and OT with desired activities.
Melting points were uncorrected. Optical rotations were measured
in a 1-dm cell on a Horiba polarimeter (model SEPA-200) at 589
nm (Na D line). NMR spectra were recorded on a Bruker Avance
DPX 250 spectrometer. Analytical HPLC was performed on a
Milton Roy instrument with a SpectroMonitor 3100 detector using
Vydac C18 (250 × 4.6 mm) column, flow rate 1 mL/min, detection
at 220 nm, and solvents [A] 0.05% trifluoroacetic acid in water
and [B] 0.038% trifluoroacetic acid in acetonitrile/water 90:10 in
a gradient application. Thin-layer chromatography (TLC) was
carried out on 250 nm silica gel GF precoated uniplates (Merck).
The chromatograms were visualized with ninhydrin or with chlorine
followed by starch/KI. FAB mass spectra were recorded on an APO
Electron model MI 1200 1E mass spectrometer equipped with a
FAB ion source.
Boc-N(Bzl)Ala-Val-OBzl (1). To the stirred suspension of Boc-
N(Bzl)Ala-OH (1.396 g; 5 mmol), HOBT (0.675 g; 5 mmol) and
TBTU (1.605 g; 5 mmol) in DCM (20 mL), DIPEA (0.856 mL; 5
mmol) were added. After 20 min of stirring, the crystalline
TosOHxNH2Val-OBzl (1.897 g, 5 mmol) and DIPEA (0.856 mL;
5 mmol) were added. The reaction mixture was left with stirring
for 3 days, diluted with ethyl acetate (100 mL), and washed
successively with 1 M aqueous KHSO4, 5% aqueous NaHCO3 and
water. The organic layer was dried over anhydrous MgSO4 and
then evaporated to dryness, yielding 2.17 g of crude oil, which
was purified on “flash” chromatography in the solvent system ethyl
acetate/hexane ) 1:1.5 (A). Yield 2.01 g (85.6%) of pure
homogeneous product as a light yellow oil. HPLC purity 100%, tR
) 15.2 min (gradient 70-90%B in 25min), R2D0 -42.6 (c ) 1,
MeOH), Rf ) 0.6 (A), FAB/MS m/e 469 [M + H]+ Calcd for
1
C27H36O5N2 ) 468. H NMR (CDCl3), δ (ppm): 0.84 (dd, 6H, J
) 7.5; 5 Hz Val γCH3), 1.24 (d, 3H, J ) 7.5 Hz Ala âCH3), 1.44
(s, 9H Boc CH3), 2.06-2.11 (m, 1H, Val âCH), 4.12 (dd, 1H, J )
7.5, 5.0 Hz Val RCH), 4,27 (s, 2H, PhCH2N), 4.33 (s, 2H PhCH2O),
4.71 (1H, d, J ) 8.2 Hz NH), 5.08-5.14 (m, 1H, Ala RCH) 7.21-
7.35 (m, 10H, PhCH2O and PhCH2N).
Boc-Tyr(Bzl)-N(Bzl)Ala-Val-OBzl (3). Removal of Boc Group
from Dipeptide 1. Dipeptide 1 (1.874 g, 4 mmol) was treated with
a 4 M HCl solution in ethyl acetate (15 mL). After 30 min, the
solvent was removed at room temperature under reduced pressure.
The crude waxy product was treated with ether and filtered off to
yield 1.140 g (70%) of 2, which was used for the next step without
further purification.
Coupling with Boc-Tyr(Bzl)-OH. A solution of 1.03 g (2.77
mmol) of Boc-Tyr(Bzl)-OH in 20 mL of dichloromethane was
cooled to -15 °C and treated with 0.304 mL (2.77 mmol) of
N-methylmorpholine (NMM), followed by 0.366 mL (2.77 mmol)
of isobutyl chloroformate. The mixture was stirred for 15 min, then
1.12 g (2.77 mmol) of 2 was added, followed by addition of 0.304
mL (2.77 mmol) of NMM. After being stirred 1 h at -10 °C, the
mixture was warmed slowly to room temperature and stirred
overnight. The solvent was removed under reduced pressure to
dryness. The residue was taken up in ethyl acetate (70 mL) and
washed with 1 M KHSO4 (2 × 25 mL), 1 M NaHCO3 (2 × 25
mL), water (2 × 25 mL), and brine (1 × 25 mL). After the sample
was dried over anhydrous Na2SO4, ethyl acetate was evaporated,
yielding 1.82 g of crude product, which was purified on “flash”
chromatography in the solvent system ethyl acetate/hexane ) 1:1.5
(A). The tripeptide 3 was isolated as an amorphous powder. Yield
0.746 g (37%); HPLC purity 99.5%, tR ) 16.85 min (gradient 70-
90%B in 25 min); Rf (A) ) 0.4; R20 -28 (c ) 1, MeOH); FAB/
MS m/e 722.3 [M + H]+, 744.4 [M D+ Na]+ Calcd. for C43H51N3O7
Experimental Section
Chemistry. Racemic S-benzyl-R-hydroxymethylcysteine [R-
HmC(Bzl)] was synthesized by selective S-benzyl-cysteine R-hy-
droxymethylation and was resolved into its enantiomers by
fractional crystallization of the diastereomeric salts of their N-
benzoyl derivatives with (-)-quinine, using the method previously
described.22 [R]- and [S]-Boc-R-HmC(Bzl)-OH were prepared in
acetonitrile using tetramethylammonium hydrate (TMAH) and
Boc2O according to the published procedure.23
The tripeptide building block [Boc-Tyr(Bzl)-N(Bzl)Ala-Val-OH]
(4) was synthesized separately in solution. Following the routine
strategy, the dipeptide [Boc-N(Bzl)-Ala-Val-OBzl (1) was depro-
tected on the N-terminus with HCl solution in ethyl acetate (4 M),
and the resulting hydrochloride salt of H-N(Bzl)Ala-Val-OBzl (2)
was extended by coupling with Boc-Tyr(Bzl)-OH to yield the
protected tripeptide Boc-Tyr(Bzl)-N(Bzl)Ala-Val-OBzl (3). Re-
moval of the benzyl ester group from 3 by careful hydrogenolysis
in methanol over 10% Pd on charcoal afforded the free acid, Boc-
1
) 721.8; H NMR (CDCl3) δ (ppm): 0.84 (dd, 6H, J ) 6.75, 5
Hz Val γ-CH3), 1.30 (d, 3H J ) 6.75 Hz Ala âCH3), 1.42 (s, 9H
Boc-CH3), 2.00-2.11 (m, 1H, Val âCH), 2.90-2.97 (m, 2H, Tyr
âCH2), 3.88-4.12 (m, 1H Val RCH), 4.30-4.41 (m, 1H, Tyr RCH),
5.04-5.22 (m, 7H, 6H CH2Ph, 1H Ala RCH), 6.05 (d, 1H, J ) 8.5
Hz Val NH), 6.63 (d, 1H, J ) 8.5 Hz Tyr NH), 7.34-7.38 (19H,
4H Tyr Ph, 10H Tyr and Ala OCH2Ph, 5H NCH2Ph);

2020 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Kowalczyk et al.
Boc-Tyr(Bzl)-N(Bzl)Ala-Val-OH (4). A solution of protected
tripeptide 3 (0.516 g, 0.715 mmol) in 5 mL of methanol was
hydrogenated (3 h, monitoring by TLC) over 10% Pd on charcoal.
The filtered solution was evaporated yielding a homogeneous
amorphous powder 0.430 g (95,1%). HPLC purity 98%, tR ) 6.20
min. (gradient 70-90% B in 25 min.), R2D0 -16,7 (c ) 1, MeOH);
FAB/MS m/e 632.1 [M + H]+ Calcd. for C36H46N3O7 ) 631.4; 1H
NMR (CDCl3) δ (ppm): 0.92 (dd, 6H, J ) 6.75; 5.25 Val γCH3),
1.22 (d, 3H, J ) 7.00 Hz, Ala âCH3), 1.40 (s, 9H, Boc CH3), 1.96-
2.00 (m, 1H Val âCH), 2.87-2.92 (m, 1H, Tyr âCH2), 3.85-3.87
(m, 1H, Val RCH), 4.36-4.82 (m, 6H, 1H Ala RCH, 1H Tyr RCH,
2H OCH2Ph, 2H NCH2Ph), 5.51 (d, 1H, J ) 6.90 Hz Val NH),
5.77 (d, 1H, J ) 6.75 Hz Tyr NH), 7.20-7.26 (m, 14H, 4H Tyr
Ph, 5H OCH2Ph and 5H NCH2Ph). 13C NMR (CDCl3) δ(ppm),
17.74 (Ala â C), 18.68, 18.97 (Val γC), 28.11 (Boc CH3), 30.64
(Val âC), 52.82 (Tyr â C), 55.62 (Ala RC), 57.63 (Tyr RC), 58.83
(Val RC), 65.80 (CH2Ph), 80.45 (Boc CdO), 115.6, 126.39, 126.82,
127.96, 130.46, 137.48, (Tyr, OBzl, Ph), 155.10, 155.57, 155.96,
(N-Bzl Ph), 156.2 (Boc CdO), 170.47, 171.28, 174.32 (Ala, Tyr,
Val CdO).
The completeness of each coupling reaction during synthesis was
monitored by the Kaiser test29 or chloranil test.30 Recoupling was
performed when the test was positive. With peptides II, III, V,
VI, VIII, and IX, Mpa(Mob) or Cpa(Mob) were used in the final
coupling step.
After completion of the synthesis, the protected peptidyl or
acylpeptidyl resins were treated with 10 mL of liquid hydrogen
fluoride (HF) containing 1 mL of anisole at -70 °C and stirred for
60 min at 0 °C.31 After removal of HF and anisole in vacuo, the
mixture was washed successively with anhydrous diethyl ether and
acetic acid, and the solution was diluted with methanol.
With analogue X, the protected nonapeptidyl resin was am-
monolysed in methanol.27,28 After evaporation of the solvent, the
product was extracted into hot DMF, precipitated with boiling water,
and left overnight at room temperature. The peptide was collected
by filtration, washed with water, and dried in-vacuo over P2O5.
The product was further purified by dissolving in DMF and
reprecipitating with CH3-OH: Et2O (3:1). Then a solution of the
peptide intermediate (0.15 mmol) in a sodium-dried and redistilled
ammonia was treated at boiling point, and with stirring with sodium
from a stick of the metal contained in a small-bore glass tube until
a light-blue color persisted in the solution for 30 s, NH4Cl was
added to discharge the color. The solution was evaporated, the
residue was dissolved in glacial acetic acid (150 mL), and the
solution was diluted with methanol (1500 mL).
Boc-Phe-Phe was synthesized using a procedure described in
the literature.12
Mpa(Mob) was obtained as described for Mpa(Bzl)24 using
p-methoxybenzyl chloride. Cpa(Mob) was synthesized using a
procedure described in the literature.25
The resulting dithiols were oxidatively cyclized with 0.1 M I2
in methanol using the standard procedure. The solvents were
evaporated under reduced pressure, and the residue was dissolved
in water and lyophilized. The crude products were desalted on a
Sephadex G-15 column and eluted with aqueous acetic acid (30%)
at a flow rate of 3-4 mL/h. 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, λ ) 254 nm. The peptide was eluted as a single peak.
Peptides V, VII-IX were purified by RP-HPLC. The peptides were
eluted as single peaks. The purity and identity of each peptide were
determined by HPLC and FAB mass spectrometry (molecular ion).
Biological Evaluation. Wistar rats were used in all experiments.
Female rats were estrogenized 48 h before the experiment. The
uterotonic test was carried out in vitro in the absence of magnesium
ions.32,33 The vasopressor test was performed using phenoxyben-
zamine-treated male rats.34 Synthetic oxytocin was used as a
standard in uterotonic tests, and synthetic arginine vasopressin was
used in pressor test. Dose-response (single administration) or
cumulative dose-response (measurements without washing steps
between administration of enhanced doses) curves were constructed.
The values reported are averages of three to five separate experi-
ments.
Tests to assess the antidiuretic or diuretic properties were
conducted on conscious male rats in two variations of the modified
Burn test.35,36 In the standard manner with hydrated rats, the animals
having fasted for 16 h were weighed and then given tap water
through a stomach catheter. The water load was 4% of the body
weight. Immediately after the water load, the tested substances (or
physiological saline as control) were administered subcutaneously
at doses of 0.001-100 nmol/kg. The rats were then placed in
individual metabolic cages, and their urine was collected over a 5
h period. The time t1/2 in which the rats excreted half the water
load was determined and then plotted against the dose. For
comparison of the compounds, such doses were chosen as to yield
All amino acid derivatives were purchased from NovaBiochem,
except Boc-Dpa, which was provided by Merck.
Peptide Synthesis. Thin-layer chromatography (TLC) was
carried out on silica plates (Merck), and spots were visualized with
iodine or ninhydrin. The solvent system used was 1-butanol/acetic
acid/water/ethyl acetate (1:1:1:1, v/v). High-performance liquid
chromatography (HPLC) was carried out on a Waters (analytical
and preparative) chromatograph equipped with a UV detector (λ
) 226 nm). The purity of the peptides I-IX was determined on a
Vydac C18 column (5 µm, 100A; 25 × 0.46 cm). The following
solvent systems were used: [A] 0.1% aqueous trifluoroacetic acid
(TFA) and [B] acetonitrile: 0.1% aqueous TFA (80:20 v/v). Linear
gradients from 20 to 80% of solution [B] for 20 min and from 20
to 80% of solution [B] for 30 min were applied for peptides at a
flow rate of 1 mL/min. Preparative HPLC was carried out using a
Kromasil C8 column (5 µm, 25 × 250 mm) in a gradient running
from 10 to 50% of [B] for 120 min at a flow rate of 10 mL/min.
The purity and identity of the peptide X were determined by HPLC
[a Gold System Beckman chromatograph, Vydac C18 column (5
µm, 4.6 × 250 mm) with precolumn Ultrasphere ODS (5 µm, 4.6
× 45 mm), solvent systems were used: [A] 0.1% aqueous
trifluoroacetic acid (TFA), [B] acetonitrile: 0.1%TFA (80:20 v/v)].
A linear gradient from 10 to 45% of solution [B] for 15 min was
applied for peptide at a flow rate of 1 mL/min (λ ) 226 nm). FAB/
MS of the peptides were recorded on a MALDI TOF mass
spectrometer.
All peptides were obtained by solid-phase peptide synthesis.
Analogues I-IX were synthesized manually using Boc-chemistry
on a methoxybenzhydryl resin (MBHA resin, Senn Chemicals AG,
1% DVB, 200-400 mesh, 0.67 mmol/g) on a scale of 150 µmol
according to standard procedures, using in situ neutralization.26 The
protected peptide precursor: Boc-R-HmC(Bzl)-Tyr(Bzl)-Phe-Gln-
Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-NH2 (analogue X) was synthesized
manually by a solid-phase method, i.e., by the stepwise coupling
of Boc-amino acids to the growing peptide chain on a chlorom-
ethylated Merrifield resin (Novabiochem, 200-400 mesh, 0.7
mmol/g) on a scale of 150 µmol. Fully protected peptide resin was
synthesized according to standard procedures involving (i) depro-
tection 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) couplings of Boc-amino acid mediated by TBTU and HOBt
in the presence of DIEA in DMF or in a mixture of DMF, NMP,
and DCM (1:1:1 v/v) containing 1% Triton, in the case of Boc-
(RHm)Cys(Bzl) coupling.27,28
t
1/2 equal to 200 min and the so-called threshold doses yielding t1/2
equal to 60 min (equal to the value of t1/2 obtained with the
physiological solution). On each day of the experiment, 21 rats
divided into 5 groups of 4 or 5 animals to which different doses
and compounds were administered were used; each dose being
tested in two or three independent experiments (different days,
different rats). To test for diuretic effects with nonhydrated rats,
no water load was given to the fasting animals. For details see ref
37.

Analogues of Neurohypophyseal Hormones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2021
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Acknowledgment. Partial funding for this work was pro-
vided by the Polish State Committee for Scientific Research
under Grant No. 0161/T09/2004/26 and 1312/T09/2005/29 and
by research project No. Z4055905 of the Academy of Sciences
of the Czech Republic.
Supporting Information Available: HPLC data for peptides
I-IX. This material is available free of charge via the Internet at
http://pubs.acs.org.
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