Chemical syntheses and biological studies on dimeric chimeras of oxytocin and the V2-antagonist, d(CH2)5[D-Ile2,Ile4]arginine vasopressin

Article abstract of DOI:10.1021/jm9900220

Parallel and antiparallel heterodimers have been synthesized that combine into a single molecule the neurohypophyseal hormone oxytocin and the potent vasopressin V₂-antagonist d(CH₂)₅[D-Ile²,Ile⁴]argi

Full text of DOI:10.1021/jm9900220

5002
J . Med. Chem. 1999, 42, 5002-5009
Ch em ica l Syn th eses a n d Biologica l Stu d ies on Dim er ic Ch im er a s of Oxytocin
a n d th e V2-An ta gon ist, d (CH2)5[D-Ile2,Ile4]a r gin in e Va sop r essin |,∇
Lin Chen,† Regina Golser,†,§ Alena Machova´,‡ J irina Slaninova´,‡ and George Barany*,†
Departments of Chemistry and Laboratory Medicine & Pathology, University of Minnesota, 207 Pleasant Street S.E.,
Minneapolis, Minnesota 55455, and Institute of Organic Chemistry and Biochemistry, Academy of Science of
The Czech Republic, 166 10 Prague 6, Flemingovo na´m. 2, The Czech Republic
Received J anuary 19, 1999
Parallel and antiparallel heterodimers have been synthesized that combine into a single
molecule the neurohypophyseal hormone oxytocin and the potent vasopressin V2-antagonist
d(CH2)5[D-Ile2,Ile4]arginine vasopressin. Solid-phase synthesis with NR-9-fluorenylmethyloxy-
carbonyl (Fmoc) chemistry, featuring appropriate combinations of orthogonal protecting groups
for the thiols [S-(N-methyl-N-phenylcarbamoyl)sulfenyl (Snm); S-acetamidomethyl (Acm);
S-triphenylmethyl (Trt)], was used to assemble the required linear nonapeptide amide monomer
intermediates, which were then brought together in defined ways by solution reactions to
provide the two heterodimers. The first disulfide bridge was formed by a directed approach
involving attack by the free thiol of the 1-â-mercapto-â,â-cyclopentamethylenepropionic acid
(Pmp) residue of one monomer onto the Snm group of a cysteine residue on the other monomer;
the inverse directed strategy failed due to steric hindrance. The second disulfide bridge was
formed by iodine co-oxidation of Cys(Acm) residues on adjacent chains. Biological studies
revealed that both the parallel and antiparallel chimeras lack pressor activity, have low
uterotonic activity, and have diuretic activities comparable to that of the monomeric V2-
antagonist. Sodium excretion depends on experimental conditions. Thus, with a 4% water load,
both chimeras display effects similar to that of an equimolar mixture of oxytocin and
V2-antagonist, i.e., lower sodium excretion than that resulting from administration of oxytocin
alone but higher than that when V2-antagonist was administered alone. However, when no
water load was used, the parallel chimera proved to be more effective in promoting sodium
excretion than either oxytocin alone or an equimolar mixture of oxytocin and V2-antagonist.
In tr od u ction
sized,3,4 and a number of them are used worldwide as
valuable tools in medicine, pharmacology, and physiol-
ogy.5,6 Vasopressin acts primarily as an antidiuretic and
pressor agent, whereas oxytocin has potent uterotonic
and galactogogic actions. In addition, oxytocin plays a
significant physiological role in the regulation of salt
and water balance.7 The optimal doses for the natri-
uretic effect are between 0.1 and 1 µg of oxytocin/kg of
animal weight;8 at higher doses, detection of this effect
is complicated by simultaneous antidiuretic action.
Several oxytocin analogues have been designed and
synthesized which showed enhanced natriuresis, but all
of these were also antidiuretic.8,9 The recent discovery
of oxytocin receptors in the heart10-12 has led McCann
and co-workers to suggest that intracardiac oxytocin
binding to its receptors stimulates the release of atrial
natriuretic factor (ANF), which mediates the natriuretic
effect.
Starting with the initial syntheses of oxytocin and
vasopressin reported by du Vigneaud and co-workers1,2
in 1954, these two neurohypophyseal hormones, as well
as their agonists and antagonists, have been the subject
of extensive pharmacological investigations. Hundreds
of analogues of these hormones have been synthe-
| Taken in part from the diploma thesis of R. Golser, based on
research at the University of Minnesota as a joint study exchange
fellow from the Karl-Franzens Universita¨t Graz, Austria.
* To whom correspondence should be addressed. Phone: (612) 625-
1028. Fax: (612) 626-7541. E-mail: barany@tc.umn.edu.
† University of Minnesota.
‡ Academy of Science of The Czech Republic.
§ Current address: Institute of Pharmacology and Toxicology,
Universita¨tsplatz 2, A-8010 Graz, Austria.
∇ Abbreviations used for amino acids and the designation of peptides
follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J . Biol. Chem. 1972, 247, 977-983. The following
additional abbreviations are used: Acm, S-acetamidomethyl; AVP,
arginine vasopressin; Boc, tert-butyloxycarbonyl; Bzl, benzyl; DIEA,
N,N-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide;
DMF, N,N-dimethylformamide; DTT, dithiothreitol; FABMS, fast atom
bombardment mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbo-
nyl; HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetraethyluronium hexaflu-
orophosphate; HOAc, acetic acid; HOAt, 1-hydroxy-7-azabenzotriazole;
HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chro-
matography; MALDI, matrix-assisted laser desorption ionization; Meb,
methylbenzyl; Mpa, â-mercaptopropionic acid; NMM, N-methylmor-
pholine; PAL, “peptide amide linker” [5-(4-Fmoc-aminomethyl-3,5-
dimethoxyphenoxy)valeric acid]; PEG-PS, poly(ethylene glycol)-
polystyrene (resin support); Pfp, pentafluorophenyl; Pmp, 1-â-mercapto-
â,â-cyclopentamethylenepropionic acid; Scm, S-methyloxycarbonylsul-
fenyl; Snm, (N-methyl-N-phenylcarbamoyl)sulfenyl; tBu, tert-butyl;
TFA, trifluoroacetic acid; Trt, triphenylmethyl. All amino acids used
were of the L-configuration unless indicated otherwise.
Our laboratories have long been interested in eluci-
dating the significance of disulfide bridges in the actions
of peptide hormones.13-21 A major aspect of our studies
has been the directed synthesis and pharmacological
evaluation of dimers of neurohypophyseal hormones.17,19
Dimers of oxytocin were observed originally by Ressler22
and isolated by Yamashiro, Hope, and du Vigneaud as
byproducts from the oxidation step to close the hetero-
detic ring.23 Furthermore, Aanning and Yamashiro
reported an ingenious intentional synthesis of the
parallel dimer.24 Relatedly, dimers were isolated as
10.1021/jm9900220 CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/10/1999

Dimeric Chimeras of Oxytocin and V2-Antagonist
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5003
Sch em e 1a
a
The covalent structures, using standard amino acid abbreviations, are shown on top. A shorthand representation to indicate the
orientations of chains, with the arrowheads representing the N-termini, is shown on the bottom.
byproducts during the syntheses of [2-O-methyltyrosine]-
oxytocin25 and deamino[8-D-arginine]vasopressin.26 Also,
dimers of lysine vasopressin were isolated (inconclusive
as to whether parallel or antiparallel) either from a
hypophysial extract or upon repeated lyophilization of
the monomer from a slightly alkaline solution, as
described by Schally and Guillemin.27 The isolation of
a naturally occurring antiparallel dimeric vasotocin
analogue by Proux and colleagues,28 and confirmation
of its structure by unambiguous chemical synthesis of
both parallel and antiparallel forms, is of further
interest.
We have described previously efficient methods to
synthesize directly parallel and antiparallel homo- and
heterodimers of oxytocin and deaminooxytocin.17,19 These
dimers were evaluated in a series of biological assays17,19
which gave results that were consistent with the
hypothesis that dimers exhibit their biological activities
after dissociation into monomers. Continuing in this
vein, the present paper reports the design and synthesis
of both parallel and antiparallel heterodimers (I and II
in Scheme 1) that combine oxytocin and the potent
vasopressin V2-antagonist d(CH2)5[D-Ile2, Ile4]arginine
vasopressin29 into a single molecule. We hoped that this
combination would preserve the oxytocic activity but
that the interaction of oxytocin with V2-receptors lead-
ing to antidiuresis would be blocked. More specifically,
we assumed that due to dissociation of the chimera into
one molecule of oxytocin and one molecule of an an-
tagonist of the vasopressin V2-receptor, a potent agent
with prolonged diuretic and natriuretic actions could be
obtained. Our experiments reported herein show that
the overall design principle is valid.
diisopropylcarbodiimide (DIPCDI) in the presence of
1-hydroxybenzotriazole (HOBt) in N,N-dimethylforma-
mide (DMF); 4 equiv each of Fmoc-protected amino acid,
coupling reagent, and additive were used with respect
to the initial loading of resin-bound amine functionality.
However, couplings of Gln and Asn (side chain unpro-
tected) were achieved with the respective pentafluo-
rophenyl (Pfp) active esters in the presence of HOBt (4
equiv each), and a mixture of O-(7-azabenzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU), 1-hydroxy-7-azabenzotriazole (HOAt), and N,N-
diisopropylethylamine (DIEA) (4 equiv each) was used
to mediate the manual introduction of the N-terminal
residue. Three S-protecting groups, S-(N-methyl-N-
phenylcarbamoyl)sulfenyl (Snm), S-acetamidomethyl
(Acm), and/or S-triphenylmethyl (Trt), were incorpo-
rated in various combinations at two positions of the
sequences, so that their placement in the orthogonal
protection scheme would define the final orientations
of the two chains in the target heterodimer. Pmp(Snm)-
OH, the required Snm-protected derivative30 of 1-â-
mercapto-â,â-cyclopentamethylenepropionic acid (Pmp),
was prepared in good yield (43-56%) from either Pmp-
(Acm)-OH or Pmp(Meb)-OH by reacting with (chloro-
carbonyl)sulfenyl chloride followed by N-methylaniline.
The remaining building blocks were commercially avail-
able or made by published procedures.20
Syntheses of the heterodimers, both parallel and
antiparallel (I and II), relied on appropriate linear
nonapeptide amide monomer intermediates (Scheme 2).
Three peptide-resins were created: the vasopressin V2-
antagonist sequence was synthesized once with internal
S-Trt and N-terminal Pmp(Acm) and a second time with
internal S-Acm and N-terminal Pmp(Snm), while the
oxytocin was synthesized with internal S-Acm and
N-terminal Boc-Cys(Snm). Upon acidolytic cleavage/
deprotection to release these peptides from the supports,
S-Acm or S-Snm moieties are retained, while S-Trt is
removed to furnish a free thiol function. The V2-
antagonist sequence was required with either the
internal Cys or the N-terminal Pmp group as the free
thiol (see below); the former intermediate was obtained
directly when the S-Trt group was used at the internal
Cys and the latter formed in two stages based on
reductive conversion of an S-Snm group. The key
Resu lts a n d Discu ssion
Syn th esis of Dim er s. A previously described strat-
egy19 for management of peptide thiols and controlled
formation of two disulfide bridges was followed, with
certain modifications (Scheme 2). Automated stepwise
solid-phase chemistry using the base-labile 9-fluorenyl-
methyloxycarbonyl (Fmoc) group for NR-amino protec-
tion, as well as tris(alkoxy)benzylamide (PAL) anchoring
with commercially available poly(ethylene glycol)-
polystyrene (PEG-PS) supports, was used to assemble
the linear sequences of oxytocin and the vasopressin V2-
antagonist. Couplings were mediated primarily by N,N′-

5004 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Chen et al.
Sch em e 2a
a
See Scheme 1 for explanation of nomenclature. Products and intermediates have Roman numerals in the same order as text mention.
dimerization steps were carried out in solution, and they
involved attack by the nucleophilic, liberated thiol of one
peptide onto an electrofugal S-Snm moiety on a separate
chain. The aforementioned directed reactions were
generally rapid and effective in mixed aqueous media
at pH 8 and occurred with minimal formation of
undesired side products. The resultant intermolecular
disulfide-linked chimeric intermediates were then treated
with iodine to co-oxidize Cys(Acm) residues on adjacent
chains.
Initial studies to create the parallel heterodimer (I)
focused on the directed reaction of the free thiol of Cys1
of the oxytocin chain with the Snm group of a Pmp
residue on the vasopressin V2-antagonist chain. Sur-
prisingly, the expected linear disulfide-linked het-
erodimer was not formed; instead, the major observed
product was the disulfide dimer of [Cys6(Acm)]oxytocin,
presumably due to air oxidation of the corresponding
monomeric species. It is plausible that no mixed disul-
fide formed because of steric hindrance from the six-
membered ring of Pmp. This assumption was consistent
with simpler model reactions, carried out under a wide
range of conditions both in solution and on the solid
phase, which revealed that the Snm group on Pmp was
resistant to displacement by thiols (see Supporting
Information). Consequently, the strategy to form the
heterodimer was reversed with respect to the sulfur
protecting groups. Thus, we examined attack by a free
thiol group from a Pmp residue of the vasopressin V2-
antagonist (III) onto an S-Snm group of the N-terminal
cysteine residue of the oxytocin monomer (IV). To create
the required Pmp residue with a free thiol, the corre-
sponding peptide-resin with N-terminal Pmp(Snm) was
reduced with dithiothreitol (DTT, 20 equiv) in the
presence of Et3N (20 equiv) in DMF for 1 h, followed by
cleavage from the support. This method has the advan-
tage that excess DTT can be removed readily by simple
filtration and washing. An alternative way to access the
same species was to cleave the peptide first, then dilute
the TFA solution with methanol, and add Zn metal to
promote reduction (details in Supporting Information).
Given the new protection strategy, the desired direct
reaction was indeed successful (Figure 1A,B), and the
heterodimeric intermediate (V) with one disulfide bridge
between the N-terminal Pmp residue of the V2-antago-
nist and the corresponding N-terminal Cys residue of
oxytocin was isolated by preparative HPLC and oxidized
further with iodine in HOAc-H2O (4:1) to give the

Dimeric Chimeras of Oxytocin and V2-Antagonist
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5005
at the doses applied. These results may be explained
by the antiuterotonic activity of the V2-antagonist. As
expected, neither chimera has measurable pressor
activity.
Effects of these compounds on diuresis and natriuresis
were tested using conscious male rats either without a
water load or with a 4% water load. The dynamics of
urine excretion was followed every 15 min for 5 h. The
quantity of urine excreted during the entire 5-h period
was recorded, and the total sodium and potassium in
the urine was determined. Finally, for experiments with
a starting water load (4%), the diuresis half-time,
defined as the time at which one-half of the original load
is excreted, was calculated. Once the optimal effective
dose for the chimeras was established, i.e., 50 nmol/kg,
the relevant parameters were determined using the
same dose of oxytocin alone, V2-antagonist alone, and
an equimolar mixture of oxytocin and V2-antagonist
(Table 2).
F igu r e 1. Analytical HPLC evaluation of intermediates I-V.
Results from the diuresis test indicate that the
activities of the parallel and antiparallel chimeric
peptides are comparable to the activity of the monomer
V2-antagonist; i.e., in the dosage range 2.5-100 nmol/
kg, the quantity of urine excreted is approximately the
same and the chimeras do not display initial antidiure-
sis as is the case for oxytocin itself. Furthermore, the
effect is somewhat more pronounced for the parallel
chimera over the antiparallel one.
target heterodimer (I) containing two disulfide bridges
(Figure 1C). The overall yield after preparative HPLC
purification was 25% (see Figure 1D for analytical
HPLC evaluation). When intermediate V was oxidized
by iodine without first carrying out a purification, the
yields were much lower.
Synthesis of the antiparallel heterodimer (II) followed
a similar strategy. The directed reaction to form the first
disulfide bridge involved attack of a free thiol group
from the internal Cys6 residue of the vasopressin V2-
antagonist monomer (VI) [which also had N-terminal
Pmp(Acm)] onto the N-terminal Cys(Snm) of oxytocin
(VII) [which also had internal Cys(Acm)]. This step went
very smoothly, in reasonable yield and purity. Next, the
iodine oxidation step was carried out, to provide the
desired dimeric product as the main component detected
by analytical HPLC. Preparative HPLC resulted in the
isolation of the dimer, albeit in a disappointingly low
overall yield of 1.1%.
There can be no doubt that the desired II was indeed
made. This was proven by a doping experiment with I,
which showed that these two heterodimers were distinct
entities upon HPLC analysis. One possibility that was
considered to explain the isolated yield was that the
iodine oxidative reaction had not proceeded for a suf-
ficiently long time. However, no improvements were
observed upon increasing reaction times 2-, 10-, or 20-
fold (data not shown). Steric hindrance of the Pmp
residue presumably inhibits the desired intramolecular
co-oxidation to close the peptide cycle and allows
competing scrambling to occur. Intramolecularly disul-
fide-cyclized monomers of both oxytocin and the vaso-
pressin V2-antagonist were indeed observed, and oligo-
mers (not detected by HPLC) could also form leading
to a reduced overall yield.
Biologica l Stu d ies. Chimeras I and II, prepared as
just described, were tested in the classical assays for
neurohypophyseal hormones, and the measured activi-
ties were compared to values determined for the indi-
vidual components (Table 1). In the uterotonic test,
activities were somewhat lower than was expected from
prior activity data on parallel and antiparallel oxytocin
homodimers. The effect starts immediately after ad-
ministration, and hardly any prolongation was observed
Excretion of Na+ ions is different depending on
whether the rats are hydrated. For rats with a water
load, the level of ion excretion promoted by chimeras is
more or less equal to that of the equimolar mixture of
oxytocin and V2-antagonist, i.e., lower than that of
oxytocin alone and higher than that of V2-antagonist
alone. Oxytocin alone causes much higher excretion of
sodium. Thus, in the water load case, the effects of
oxytocin and V2-antagonist were not additive. These
results could be explained by insufficient selectivity of
the vasopressin analogue just for the V2-receptor.
However, when nonhydrated rats were used, the paral-
lel chimeric dimer showed the best parameters. The
quantity of excreted sodium with the parallel chimera
exceeded that of oxytocin alone, V2-antagonist alone, or
a mixture of oxytocin and V2-antagonist. The quantity
of excreted sodium after application of the antiparallel
dimer was comparable to that of V2-antagonist alone,
oxytocin alone, or the equimolar mixture of both. While
we hypothesize that effects of the dimers are displayed
after dissociation, the observed differences between
parallel and antiparallel dimers could be explained by
different kinetics of dissociation of the dimers. However,
no direct experimental data bearing on this issue are
available. The possibility of direct interaction of the
chimeras with the receptors has not been directly
excluded and is in fact consistent with the immediate
effect and lack of prolongation in the uterotonic test.
P er sp ective a n d Con clu sion s
One of the goals of the present study was to determine
whether one could use disulfide bridging to tailor
chimeric molecules that would combine in a single
dimeric species defined properties of the corresponding
monomers. Based on the hypothesis that the dimers
function after their dissociation into monomers, it

5006 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Ta ble 1. Biological Activities of Compoundsa
Chen et al.
activities
pressor
uterotonic
antidiuretic
peptides
in vitro (no Mg2+
)
in vivo
450
pA2 ) 6.9
13.8
1.1
0.8
anesth rats
conscious rats
oxytocinb
450
4.5
5
100%e
inhibnf
∼10%
V2-antagonistc
pA2 ) 7.47
pA2 ) 6.42
0.5
0
pA2 ) 8.04
oxytocin dimerd
1.37
0.22
0.8
ND
ND
ND
parallel dimer oxytocin/V2-antagonist
antiparallel dimer oxytocin/V2-antagonist
inhibnf
inhibnf
0
a
Assays and data analysis summarized in the Experimental Section. Unless indicated otherwise, the units of activity are IU/mg; ND,
not determined. Literature values from ref 3; used to standardize remaining experiments. c The pA2 values are taken from ref 6 and
represent the negative logarithm (base 10) of the concentration (in M) required for half-inhibition of oxytocin activity. As described in
b
d
ref 19, a mixture of parallel and antiparallel homodimers was isolated as a byproduct from synthetic efforts directed at certain heterodimers.
Activity data are also repeated from ref 19, except for antidiuretic activity in conscious rats (this study). e Relative to the activity of
oxytocin alone, which is defined as 100%. f All three compounds with this label have the same degree of inhibitory effects (antagonism)
at similar doses.
Ta ble 2. Effects of Various Compounds on Diuresis Half-Time, Volume of Excreted Urine, and Monovalent Cation Content in Urinea
diuresis
half-time (min)
vol of excreted
urine in 5 h (mL)
total Na+ (µmol)
water load water load
0% 4%
total K+ (µmol)
water load
4%
water load
0%
water load
4%
water load
water load
4%
peptides
0%
physiological saline control
oxytocin
V2-antagonist
equimolar mixture of oxytocin
and V2-antagonist
65 ( 5
178 ( 5
54 ( 2
41 ( 5
1.8 ( 0.4
4.8 ( 0.3
9.8 ( 0.1
8.1 ( 1.0
8.3 ( 0.5
11.3 ( 0.4
14.2 ( 0.9
14.1 ( 0.7
72 ( 29
563 ( 15
431 ( 49
480 ( 81
71 ( 14
773 ( 78
275 ( 41
476 ( 20
79 ( 21
202 ( 17
220 ( 26
194 ( 17
82 ( 16
287 ( 28
152 ( 24
196 ( 25
parallel chimeric dimer
antiparallel chimeric dimer
antiparallel oxytocin homodimerb
42 ( 2
49 ( 2
208 ( 10
9.4 ( 0.7
6.3 ( 0.6
14.2 ( 0.8
13.1 ( 0.7
6.4 ( 1.3
762 ( 79
358 ( 39
375 ( 71
480 ( 30
1146 ( 181
217 ( 27
200 ( 30
162 ( 29
123 ( 10
231 ( 45
a
All compounds were tested at the same dose, i.e., 50 nmol/kg of animal. Compounds were administered subcutaneously, and values
b
are expressed as average ( standard error of mean. The oxytocin dimer was also used at the 50 nmol/kg dose. Thus, for proper comparison
to other data in the table, it should be noted that after dissociation, this would give 100 nmol of the monomer (i.e., oxytocin).
follows that properties of the various dimer analogues
should depend on properties of the individual compo-
nents. Our previous work with synthetic oxytocin and
deaminooxytocin homo- and heterodimers showed that
these compounds exhibit prolonged in vivo activity in
the uterotonic test. Furthermore, preliminary tests for
antidiuretic activity on conscious rats revealed that the
oxytocin dimer (present as a mixture of parallel and
antiparallel) is active, although the potency is 1 order
of magnitude less than that of oxytocin (Table 1). With
the goal to attain an agent with both natriuretic and
diuretic effects, and without the initial antidiuretic
phase, we decided to synthesize chimeric dimers com-
bining into a single molecule oxytocin and a V2-
antagonist. Indeed, the chimeras showed most of the
predicted effects, which in most cases were quite close
to those of an equimolar mixture of the two individual
components. A major exception, which may be of con-
siderable practical importance, occurred in the case of
the parallel chimeric peptide dimer when administered
to rats without water load. It is known that the diuretic
and natriuretic effect of oxytocin depends on the hydra-
tion state of the animal.7 Humans are usually not in a
state of overhydration, so the results from our studies
are encouraging as a lead toward natriuretic drugs with
favorable Na+/K+ ratios and minimum side effects.
Somewhat puzzling in light of previous results was
the absence of any prolongation effect with the new
dimeric compounds. It is possible that the different ways
in which peptides were applied may play a role, i.e.,
intravenous administration for the uterotonic in vivo
test versus subcutaneous administration for the antidi-
uretic test. The difference in time scalestens of minutes
for the uterotonic in vivo test versus hours in the
antidiuretic testsmay also be significant.
Our concept of using chimeric dimers could be im-
proved by using a more V2-selective antagonist, together
with a more potent natriuretic analogue of oxytocin. The
currently chosen antagonist,29 as a monomer, inhibits
in vitro and in vivo uterotonic activities of oxytocin.29
Hence, this antagonist as a component of the chimera
inhibits not only the undesired interaction of the oxy-
tocin component with the V2-receptors but also the
desired interaction with oxytocin receptors. We expect
that appropriate iterative redesign and testing of chi-
meras along the lines described herein could lead to
useful active and specific compounds that are diuretic
as well as uterotonic and/or natriuretic.
Exp er im en ta l Section
Gen er a l. Materials, solvents, instrumentation, and general
methods were essentially as described in previous publications
from our laboratories.20,31 Fmoc-PAL-PEG-PS supports (initial
loading 0.15-0.18 mmol/g) for peptide synthesis were obtained
from the BioSearch Division of PerSeptive Biosystems (Framing-
ham, MA). Protected Fmoc-amino acid derivatives and cou-
pling reagents were from PerSeptive Bioystems or Advanced
Chemtech (Louisville, KY), while Pmp derivatives were from
Peptide International (Louisville, KY). Boc-Cys(Snm)-OH was
prepared and characterized as described previously.20 TFA,
piperidine, DIEA, and NMM were obtained from Fisher
(Pittsburgh, PA). Peptide chain assembly was carried out
either manually or on a PerSeptive model 9050 continuous-
flow peptide synthesizer. Fmoc removal was achieved with
piperidine-DMF (1:4, 2 + 8 min). DIPCDI/HOBt- or HATU/
HOAt/DIEA-mediated couplings were carried out for 1-2 h
using 4 equiv each of Fmoc-amino acids, reagents, and addi-
tives over resin-bound amine. Washings between chemical

Dimeric Chimeras of Oxytocin and V2-Antagonist
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5007
steps were carried out with DMF and/or CH2Cl2. Upon
completion of on-resin steps, peptide-resins (∼20 mg, except
indicated otherwise) were washed with CH2Cl2 and then
cleaved for 1 h with either reagent B, TFA-CH2Cl2-Et3SiH-
phenol-H2O (92:5:1:1:1) (2 mL), or reagent R, TFA-thioani-
sole-1,2-ethanedithiol-anisole (90:5:3:2) (2 mL). The cleavage
mixtures were then filtered and washed with TFA (2 × 2 mL).
The combined filtrates were evaporated by bubbling with a
stream of N2. The crude peptides were precipitated with Et2O-
CH2Cl2 (10 mL of 20:1 mixture) at 0 °C, collected by low-speed
centrifugation, and dissolved in a suitable medium for analyti-
cal HPLC.
mmol) in CHCl3 (2 mL) was added dropwise at 0-5 °C into an
ice bath-cooled suspension of Pmp(Acm)-OH (0.5 g, 2.0 mmol)
in CHCl3 (8 mL) over 5 min. After an additional 15 min of
stirring, CH3OH (20 mL) was added and the solution was
stirred for a further 30 min. The reaction mixture was next
concentrated in vacuo, and the residue was purified by flash
column chromatography to give the product as an off-white
solid. Yield: 96 mg (18%), mp 77-84 °C, Rf ) 0.44 [CHCl3-
CH3OH-HOAc (30:1:0.1)]. 1H NMR (CDCl3): δ 3.91 (s, 3H),
2.67 (s, 2H), 1.32-2.0 (m, 10H). FABMS: m/z calcd for
C
10H16O4S2 264.0, found 265.0 [(MH)+].
1-â-Mer ca p to-â,â-cyclop en ta m eth ylen ep r op ion ic Acid
Amino acid analysis was performed on a Beckman 6300
analyzer with a sulfated polystyrene cation-exchange column
(0.4 cm × 21 cm). Peptides were hydrolyzed in 6 N aqueous
HCl-propionic acid (1:1 v/v) plus 2 drops of liquefied phenol
to prevent degradation of Tyr, for 1 h at 160 °C. Analytical
HPLC was performed using a Vydac analytical C-18 reversed-
phase column (218TP54; 5 µm, 300 Å; 0.46 × 25 cm) on a
Beckman system using Beckman System Gold software. Pep-
tide samples were chromatographed at 1.2 mL/min flow rate,
using a linear gradient over 20 min of 0.1% aqueous TFA and
acetonitrile (containing 0.1% TFA) from 9:1 in 20 min to 3:2,
detection at 220 nm. Purification of crude peptide products
was carried out on a Waters Deltaprep 3000 semipreparative
HPLC using a Vydac semipreparative C-18 reversed-phase
column (218TP1610; 10 µm, 300 Å; 1.0 × 25 cm), elution at 5
mL/min, with a linear gradient of 0.1% aqueous TFA-CH3-
CN from 9:1 to 1:4 over 65 min with detection at 220 nm.
Fractions with the desired peptide were pooled and lyophilized
to provide white powders. The final isolated yields were
calculated by comparison of amino acid analyses of the purified
peptide to the initial loading of the resin.
(P m p -OH). Pmp(Snm)-OH (0.2 g, 0.59 mmol) was dissolved
in CH3OH (40 mL), and then, TFA (2 mL) and zinc dust (385
mg, 5.9 mmol) were added. The reaction mixture was stirred
and monitored by TLC [CHCl3-CH3OH-HOAc (15:1:0.1)].
After 2 h, the mixture was concentrated in vacuo, and the
residue was dissolved in EtOAc (20 mL), washed with H2O (3
× 20 mL), dried (Na2SO4), and concentrated in vacuo to give
the product as a white powder. Yield: 58 mg (57%), mp 68-
1
71 °C, Rf ) 0.45. H NMR (CDCl3): δ 2.66 (s, 2H), 1.30-1.85
(m, 10H). FABMS: m/z calcd for C8H14O2S 174.3, found 175.1
[(MH)+].
P m p (SH )-D-Ile -P h e -Ile -Asn -Cys(Acm )-P r o-Ar g-Gly-
NH2 (III). A. The peptide-resin, Pmp(Snm)-D-Ile-Phe-Ile-Asn-
Cys(Acm)-Pro-Arg(Pmc)-Gly-PAL-PEG-PS (30 mg, initial load-
ing 0.15 mmol/g), was treated with DTT (14 mg, 90 µmol) in
H2O-CH3CN (1:1, 1 mL) for 1 h, with stirring, under an argon
atmosphere. The peptide-resin was then washed with DMF
and CH2Cl2 and cleaved from the resin with reagent R (2 mL).
After the workup as described in “General”, analytical HPLC
revealed the formation of the title product (tR ) 24.2 min, 73%).
FABMS: m/z calcd for C52H85N14O11S2 1144.4, found 1145.6.
Alternative methods involving acidolytic cleavage of the pep-
tide-resin, followed by solution reductions either with zinc in
the mixture of CH3CN, H2O, and CH3OH or with aqueous
DTT-Et3N, were also studied (see Supporting Information).
1H NMR spectra were observed in the indicated solvents
with a Varian VXR 300 instrument. Exchangeable protons are
not reported. Low-resolution fast atom bombardment mass
spectroscopy (FABMS) was carried out on a VG Analytical
707E-HF low-resolution double-focusing mass spectrometer
P a r a llel Heter od im er of Oxytocin a n d V2-An ta gon ist
(I). The starting peptides for the procedure were obtained upon
cleavage and workup of the corresponding peptide-resins
(Scheme 2), as described in “General”, and quantified by amino
acid analyses and analytical HPLC peak areas. A solution of
Pmp(SH)-D-Ile-Phe-Ile-Asn-Cys(Acm)-Pro-Arg-Gly-NH2 (III; 5
µmol, tR ) 24.2 min) in H2O-CH3CN (1:1; 10 mL) and a
solution of H-Cys(Snm)-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-
Gly-NH2 (IV; 5 µmol, tR ) 17.0 min) in H2O-CH3CN (1:1; 3.1
mL) were combined and mixed with 0.1 M phosphate buffer,
pH 8 (2 mL). The resultant homogeneous mixture was stirred
under an argon atmosphere; formation of the linear single
disulfide heterodimer V (tR ) 19.6 min) was monitored by
analytical HPLC. After 1.5 h reaction, the intermediate was
purified by preparative HPLC and lyophilized. The intermedi-
ate was then dissolved in H2O-CH3CN (1:1; 2 mL) and
combined with a solution of I2 (25 mg, 0.1 mmol, 20 equiv) in
HOAc (47 mL). After stirring for 1.5 h, H2O (50 mL) was added,
and the excess I2 was removed by extraction with CCl4 (4 ×
40 mL). The aqueous phase was lyophilized, and the title
peptide (tR ) 20.0 min) was purified by preparative HPLC (see
“General”). Yield: 5.2 mg (25%, based on initial loading).
MALDI-MS: m/z calcd for C92H143N25O22S4 2077.97, found
2079.0 [(MH)+]. Amino acid composition: Asx, 2.27; Glx, 1.06;
Pro, 2.19; Gly, 2.23; Ile, 2.44; Leu, 1.03; Tyr, 0.86; Phe, 0.89.
An tip a r a llel Heter od im er of Oxytocin a n d V2-An ta go-
n ist (II). Following a similar procedure as above, Pmp(Acm)-
D-Ile-Phe-Ile-Asn-Cys(SH)-Pro-Arg-Gly-NH2 (VI; 19 µmol, tR
) 21.4 min) and H-Cys(Snm)-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-
Leu-Gly-NH2 (IV; 19 µmol, tR ) 17.0 min) were dissolved
separately in H2O-CH3CN (1:1, 3 mL each). The solutions
were combined, diluted with phosphate buffer, pH 8 (3 mL),
and stirred under an argon atmosphere. The resultant linear
single disulfide heterodimer VII (tR ) 20.5 min) was purified
by preparative HPLC and lyophilized. The intermediate was
then dissolved in H2O-CH3CN (1:1, 2 mL), and a solution of
I2 (95 mg, 0.37 mmol) in HOAc (8 mL) was added. After stirring
equipped with
a VG 11/250 data system, operated at a
resolution of 2000 and 4000. MALDI was performed using a
Finnigan FT/MS 2001 instrument. A nitrogen laser was used
as the light source (6 mW power, 377 nm wavelength, 3 ns
duration), and argon was pulsed into the cell to assist in
trapping and cooling of the ions. The matrix used was
dihydroxybenzoic acid. A solution of matrix and sample in
CH3CN-H2O (1:4) was allowed to evaporate on the target.
Poly(propylene glycol) was used as the reference material in
accurate mass determination.
N-Met h yl-N-p h en ylca r b a m oylsu lfen yl-1-â-m er ca p t o-
â,â-cyclop en t a m et h ylen ep r op ion ic Acid [P m p (Sn m )-
OH]. A. A solution of (chlorocarbonyl)sulfenyl chloride32 (0.7
mL, 8.2 mmol) in CHCl3 (8 mL) was added dropwise at 0-5
°C into an ice bath-cooled suspension of Pmp(Acm)-OH (2.0 g,
8.2 mmol; Rf ) 0.29 [CHCl3-HOAc (100:1)]) in CHCl3 (35 mL)
over 10 min. After an additional 15 min of stirring, the reaction
mixture was filtered and the filtrate was added slowly to a
solution of N-methylaniline (8.8 mL, 81 mmol) in CHCl3 (25
mL) at 0 °C over 10 min. The reaction mixture was stirred for
an additional 30 min and then washed with 1 N aqueous HCl
(2 × 80 mL) and H2O (80 mL). The organic phase was dried
(Na2SO4), concentrated in vacuo, and purified by flash column
chromatography [CHCl3-HOAc (100:1)] to provide the product
as a brown crystalline mass. Yield: 1.21 g (43%), mp 151-
152 °C, Rf ) 0.79 [CHCl3-HOAc (100:1)]. 1H NMR (CDCl3): δ
7.19-7.52 (m, 5H), 3.34 (s, 3H), 2.51 (s, 2H), 1.25-1.77 (m,
10H). FABMS: m/z calcd for C16H21NO3S2 339.10, found 340.10
[(MH)+].
B. Title product was obtained in the same way (1.2-mmol
scale), except starting with S-4-methylbenzyl-1-â-mercapto-
â,â-cyclopentamethylenepropionic acid [Pmp(Meb)-OH] in-
stead of Pmp(Acm)-OH. Yield: 0.23 g (56%).
S-(Met h yloxyca r b on yl)su lfen yl-1-â-m er ca p t o-â,â-cy-
clop en ta m eth ylen ep r op ion ic Acid [P m p (Scm )-OH]. A
solution of (chlorocarbonyl)sulfenyl chloride (0.17 mL, 2.0

5008 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Chen et al.
(5) J ost, K. Practical use in human and veterinary medicine. In ref
3, Vol. II, Part 2, pp 87-125.
(6) Manning, M.; Sawyer, W. H. Design, Synthesis and some uses
of receptor-specific agonists and antagonists of vasopressin and
oxytocin. J . Recept. Res. 1993, 13, 195-214.
(7) Hrbas, P. Natriuretic Action. In ref 3, Vol. II, Part 2, pp 41-85.
(8) Hrbas, P.; Barth, T.; Skopkova, J .; Lebl, M.; J ost, K. Effect of
some oxytocin analogues on natriuresis in rats. Endocrinol. Exp.
1980, 14, 151-157.
for 1.5 h, H2O (38 mL) was added, and the excess I2 was
removed by extraction with CCl4 (4 × 40 mL). The aqueous
phase was lyophilized, and the title peptide (tR ) 19.6 min)
was purified by preparative HPLC (see “General”). Yield: 0.85
mg (1.1%). MALDI-MS: m/z calcd for C92H143N25O22S4 2077.97,
found 2079.6 [(MH)+]. Amino acid composition: Asx, 2.15; Glx,
0.93; Pro, 1.95; Gly, 2.21; Ile, 2.75; Leu, 0.94; Tyr, 0.59; Phe,
1.06.
P r oof Th a t P a r a llel a n d An tip a r a llel Dim er s Ar e
Distin ct Sp ecies. Because antiparallel heterodimer II, iso-
lated in low yield as described above, is an isomer of the
parallel heterodimer I, MALDI-MS alone was not considered
to provide conclusive evidence for its structure. Therefore, a
co-injection experiment was carried out. Using a Vydac C4
analytical column, an approximately equimolar mixture of I
and II was chromatographed at 1.0 mL/min flow rate, under
isocratic conditions of 0.1% aqueous TFA and 0.1% TFA in
acetonitrile (7:3) over 25 min, detection at 218 nm; I (tR ) 15.2
min) and II (tR ) 14.5 min) were baseline separated.
(9) Hrbas, P.; Skopkova, J .; Zicha, J .; Barth, T.; Lebl, M.; J ost, K.
Nacartocin - Analogue of oxytocin with enhanced natriuretic
properties: Natriuretic and hemodynamic characteristics. En-
docrinol. Exp. 1984, 18, 117-124.
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J .; McCann, S. M.; Antunes-Rodrigues, J . Oxytocin mediates
atrial natriuretic peptide release and natriuresis after volume
expansion in the rat. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
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C.; Gutkowska, J .; Antunes-Rodrigues, J .; McCann, S. M.
Oxytocin releases Atrial Natriuretic Peptide from Rat Atria In
Vitro that Exerts Negative Inotropic and Chronotropic Action.
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heart. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11704-11709.
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and protracted action of carba-analogues of deamino-oxytocin
with O-methyltyrosine in position 2. Collect. Czech. Chem.
Commun. 1980, 45, 3045-3050.
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the antiparallel dimer of deamino-1-carba-oxytocin. In Peptides
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Bicyclic Tridecapeptide Amide with Two Disulfide Bridges:
Illustration of Novel Protection Schemes and Oxidation Strate-
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Synthesis and Biological Activity of the Parallel Dimer of
Deamino-Oxytocin. Pept. Res. 1993, 6, 155-159.
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Biologica l Eva lu a tion .33 Wistar rats were used in all
experiments. Female rats were estrogenized 24-48 h before
the experiment. The uterotonic test was carried out in vitro
in the absence of magnesium ions34,35 and also in vivo.36 The
vasopressor test was performed using phenoxybenzamine-
treated male rats.37 Synthetic oxytocin was used as a standard
in uterotonic tests, and synthetic arginine vasopressin was
used in pressor tests. Dose-response (single administration)
or cumulative dose-response (measurements without washing
steps between administration of enhanced doses) curves were
constructed. The activities were determined by comparing the
threshold doses of the standard and the analogues. Values
reported (Table 1) are averages of 3-5 separate experiments.
Tests to estimate antidiuretic, diuretic, and natriuretic
properties were conducted on conscious male rats in two
variations of the Burn38 test, as modified.39 In the standard
way with hydrated rats, the animals fasted for 16 h were
weighed and then given tap water by means of stomach
catheter. The water load was 4% of body weight. Immediately
after the water load, the tested substances (or physiological
saline as a control) were applied subcutaneously in doses of
1-100 nmol/kg. The rats were then placed into individual
metabolic cages, and their urine was collected over a 5-h
period. Each day of the experiment, 21 rats were used, divided
into 5 groups of 4 or 5 rats getting different doses and
compounds; each dose was tested in 2 or 3 independent
experiments (different days, different rats). To test for effects
with nonhydrated rats, no water load was given to the fasting
animals. The concentration of sodium and potassium ions in
the rat urine was determined using an Eppendorf model FCM
6341 flame spectrophotometer.
(19) Chen, L.; Bauerova´, H.; Slaninova´, J .; Barany, G. Syntheses and
Biological Activities of Parallel and Antiparallel Homo and
Hetero Dimers of Oxytocin and Deamino-Oxytocin. Pept. Res.
1996, 9, 114-121.
(20) Chen, L.; Zoulikova´, I.; Slaninova´, J .; Barany, G. Synthesis and
Pharmacology of Novel Analogues of Oxytocin and Deamino-
Oxytocin: Directed Methods for the Construction of Disulfide
and Trisulfide Bridges in Peptides. J . Med. Chem. 1997, 40,
864-876.
Ack n ow led gm en t . We thank NIH GM 43552 for
financial support.
(21) Hargittai, B.; Barany, G. Controlled syntheses of natural and
disulfide-mispaired regioisomers of R-conotoxin SI. J . Pept. Res.
1999, 54, 468-479.
(22) Ressler, C. Inactivations of Oxytocin Suggesting Peptide Dena-
turation. Science 1958, 128, 1281-1282.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
scriptions of model studies and routes that failed or gave less-
than-optimal results. This material is available free of charge
via the Internet at http://pubs.acs.org.
(23) Yamashiro, D.; Hope, D. B.; du Vigneaud, V. Isomeric Dimers
of Oxytocin. J . Am. Chem. Soc. 1968, 90, 3857-3860.
(24) Aanning, H. L.; Yamashiro, D. Synthesis of the parallel dimer
of oxytocin. J . Am. Chem. Soc. 1970, 92, 5214-5216.
(25) Flegel, M.; Barth, T.; Fric, I.; Blaha, K.; J ost, K. The dimer of
[2-O-Methyltyrosine]oxytocin: preparation and properties. Col-
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(26) Flegel, M.; Barth, T.; Zaoral, M. Isolation and characterization
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sis. Peptides 1976; Proc. 14th Eur. Pept. Symp., Wepion, 1976;
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(27) Schally, A.; Guillemin, R. Some Biological and Chemical Proper-
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J M9900220