Synthesis and pharmacological evaluation of an analogue of the peptide hormone oxytocin that contains a mimetic of an inverse γ-turn

Article abstract of DOI:10.1021/jm0110744

Oxytocin is a neurohypophyseal peptide hormone that induces labor and lactation in mammals. An inverse γ-turn mimetic corresponding to the tripeptide Ile-Val-Asn has been synthesized and incorporated instead of residues 3-5 of oxytocin to probe the hypoth

Full text of DOI:10.1021/jm0110744

2512
J . Med. Chem. 2002, 45, 2512-2519
Syn th esis a n d P h a r m a cologica l Eva lu a tion of a n An a logu e of th e P ep tid e
Hor m on e Oxytocin Th a t Con ta in s a Mim etic of a n In ver se γ-Tu r n
ZhongQing Yuan,† David Blomberg,† Ingmar Sethson,† Kay Brickmann,†,‡ Kjell Ekholm,§ Birgitta J ohansson,§
Anders Nilsson,§ and J an Kihlberg*,†
Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden, and Ferring AB, P.O. Box 30047,
SE-200 61 Limhamn, Sweden
Received October 26, 2001
Oxytocin is a neurohypophyseal peptide hormone that induces labor and lactation in mammals.
An inverse γ-turn mimetic corresponding to the tripeptide Ile-Val-Asn has been synthesized
and incorporated instead of residues 3-5 of oxytocin to probe the hypothesis that a γ-turn
involving these residues is found in the receptor bound conformation of oxytocin. In the turn
mimetic, residues i and i + 1 are connected by a ψ[CH2O] isostere while a covalent methylene
bridge replaces the hydrogen bond that is often found between residues i and i + 2 in γ-turns.
The turn mimetic was assembled from three types of building blocks: an azido epoxide, an
R-bromo acid, and a protected â-amino alcohol. The oxytocin analogue did not induce
contractions of the uterus nor did it inhibit oxytocin-induced contractions. It is suggested that
the loss of bioactivity is mainly due to the presence of a ψ[CH2O] isostere instead of an amide
bond between residues i and i + 1 in the turn mimetic.
In tr od u ction
Peptides display a multitude of diverse and important
biomedicinal activities, but use of peptides as therapeu-
tic agents is often limited by poor pharmacokinetic
properties, i.e., low uptake on oral administration, rapid
enzymatic degradation, and facile excretion.1 In addi-
tion, conformational flexibility may reduce the biological
F igu r e 1. Closely related structures of the hormones oxytocin
(1) and vasopressin (2).
activity and receptor selectivity of peptides, whereas low
solubility can impose restrictions on their use under
physiological conditions. Substantial efforts have been
pressin,8,9 Leu-enkephalin,10-12 angiotensin II,13 and
devoted to the design and synthesis of mimetics of
bradykinin14 have been suggested to populate γ-turn
peptides in order to circumvent the above problems.2-5
conformations.
Since peptidomimetics are often conformationally re-
Oxytocin (1, Figure 1) is a neurohypophyseal nonamer
stricted, they may also be used in attempts to establish
peptide hormone, the physiological function of which is
the bioactive conformation of peptides.
to regulate milk ejection and uterine contractions in
Turns are defined as regions where a peptide chain
mammals.15,16 Previous conformational studies have
reverses its overall direction.6,7 In a γ-turn, this occurs
suggested that [Pen1]oxytocin populates an inverse
over three residues and a hydrogen bond is often formed
γ-turn centered at Gln4.17-19 Gly-[Lys8]vasopressin, Gly-
between residues i and i + 2 so that a pseudo-seven-
Gly-Gly-[Lys8]vasopressin,20 and the drug desmopressin
membered ring is formed. Depending on if the side chain
([Mpr1,DArg8]vasopressin),21 which have closely related
of residue i + 1 is in an equatorial or axial orientation
structures [cf. vasopressin (2), Figure 1], were also found
on the pseudo-seven-membered ring, γ-turns are clas-
to adopt inverse γ-turns at this position. In addition,
sified as inverse or classical, respectively. In the related
the conformation of desmopressin showed large simi-
â-turns, chain reversal instead involves four residues
larities to the solution structure of neurophysin-bound
and a hydrogen bond may then be formed between
oxytocin22 for residue Tyr2-Cys6.21 These observations
residues i and i + 3. Structural studies have revealed
suggest that it should be interesting to probe the
that a large number of naturally occurring small pep-
biologically active conformation of oxytocin by replacing
tides that function as hormones or neurotransmitters,
the Ile3-Gln4-Asn5 fragment with a mimetic of an
or have other regulatory roles in organisms, may adopt
inverse γ-turn.
â- or γ-turn conformations. â-Turns are more frequent
than γ-turns in biologically active peptides, but vaso-
Resu lts a n d Discu ssion
We recently reported the design of inverse γ-turn
* To whom correspondence should be addressed. Phone: +46-90-
mimetics 3 (Figure 2) and incorporation of such a
mimetic into the drug desmopressin.23 In 3 the amide
bond between residue i and i + 1 of the γ-turn has been
replaced by a methylene ether isostere. In addition, a
786 68 90. Fax: +46-90-13 88 85. E-mail: jan.kihlberg@chem.umu.se.
† Umeå University.
‡ Present address: Medicinal Chemistry, AstraZeneca R&D Mo¨ln-
dal, SE-431 83 Mo¨lndal, Sweden.
§ Ferring AB.
10.1021/jm0110744 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/04/2002

Analogue of Oxytocin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2513
Sch em e 2a
F igu r e 2. Classical and inverse γ-turns have the CdO of
residue i close to the NH of residue i + 2 and are often
stabilized by a hydrogen bond between these groups. In inverse
γ-turn mimetic 3, the hydrogen bond has been replaced by a
methylene bridge that provides conformational restriction.
a
(a) D-(-)-Diisopropyl tartrate, Ti(OiPr)4, tBuOOH in toluene,
methylene bridge ensures the close spatial location of
residues i and i + 2 by forming a six-membered
morpholin-3-one ring. Ab initio calculations revealed
that this covalent linkage effectively restricts the tor-
sional angles of the i + 1 residue to values close to those
of an inverse γ-turn.23,24 We have now focused on
preparation of inverse γ-turn mimetic 4 (Scheme 1),
which has side chains corresponding to those of the
tripeptide Ile-Val-Asn, and on incorporation of 4 in place
of residues 3-5 in oxytocin. In oxytocin, Gln4 can be
replaced by Val with retention of >25% of the agonistic
activity at the uterine receptor.25,26 Val was therefore
used instead of Gln at position i + 1 of turn mimetic in
order to simplify the synthesis. A retrosynthetic analysis
suggested that azido epoxide 5, (R)-2-bromo-3-methyl-
butanoic acid 6, and protected asparaginol 7 are suitable
building blocks for synthesis of mimetic 4. The azido
group in 5 serves as a precursor of the amino group of
residue i in the turn, while the tert-butyldiphenylsilyl-
protected alcohol in 7 corresponds to the carboxyl group
of residue i + 2 of the turn.
CH2Cl2, -20 °C, 82%; (b) Ti(OiPr)2(N3)2, benzene, reflux, 92%; (c)
collidine, BzCl, -10 °C f room temp, then MsCl, CH2Cl2, 0 °C f
room temp, 66%; (d) NaOEt in EtOH, THF, room temp, 83%.
Sch em e 3a
Sch em e 1a
a
Retrosynthetic analysis showing that the target mimetic 4 may
be assembled from three building blocks: azido epoxide 5, R-bromo
acid 6, and protected â-amino alcohol 7.
Synthesis of azido epoxide 5 began with a Katsuki-
Sharpless asymmetric epoxidation27 of allylic alcohol 828
to furnish epoxy alcohol 9 (Scheme 2) (82%, 88% de
according to 1H NMR analysis). Regioselective opening
of the epoxide in 9 by Ti(OiPr)2(N3)229 then yielded azido
diol 10. Benzoylation of the primary hydroxyl group of
10 directly followed by mesylation of the secondary
hydroxyl group gave 11. Finally, treatment of 11 with
sodium ethoxide resulted in removal of the benzoyl
group and intramolecular ring closure to give building
block 5 (41% overall yield from 8, 90% de based on H
NMR analysis).
Assembly of inverse γ-turn mimetic 4 began by
nucleophilic attack at the primary position of azido
epoxide 5 with silylated asparaginol 723 (Scheme 3). At
a
(a) EtOH, reflux, 68%; (b) Et3SiCl, imidazole, CH2Cl2, 0 °C f
room temp, 86%; (c) (R)-(+)-2-bromo-3-methylbutanoic acid (6),
diisopropylcarbodiimide, CH2Cl2, 0 °C f room temp; (d) 2 M
HCl(aq)/THF (3:2), room temp, 56% from 13; (e) KH, THF/DMF
(2.4:1), 0 °C, 89%; (f) TBAF‚H2O, THF, room temp, 97%; (g) NaIO4,
RuCl3‚H2O, CCl4/CH3CN/H2O (2:2:3), room temp, 61%; (h) solid-
phase synthesis, 29%.
this stage, the minor diastereomer originating from the
synthesis of 5 could be removed by flash column
chromatography so that amino alcohol 12 was obtained
in enantiomerically pure form as determined by 1H
NMR spectroscopy (68% yield). Attempts to acylate the
amino group of 12, without acylation of the secondary
hydroxyl group, were not successful. Therefore, the
1

2514 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Yuan et al.
Ta ble 1. 1H NMR Data (δ, ppm) for Peptidomimetic 18 in Aqueous Solutiona
residue
NH
H-R
4.03
H-â
H-γ
others
6.84 and 7.16 (arom)
Cys1
3.13, 3.28
3.03, 3.07
Tyr2
9.28
7.55
4.75
mimetic
Ile3
3.70
4.14
5.32
1.63
2.22
2.74, 2.91
0.91, 1.02
0.76 (δ-CH3), 0.92 (γ-CH3), 3.86 (CHO)
0.87 and 1.01 (γ-CH3)
7.01 and 7.78 (CONH2)
Val4
Asn5
bridge
Cys6
Pro7
3.26 and 3.60 (CH2N)
8.11
5.05
4.40
4.28
3.84, 3.91
2.78, 3.26
1.90, 2.27
1.60, 1.65
2.01b
1.63
3.65 and 3.74 (H-δ)
0.87 and 0.92 (δ-CH3)
7.16 and 7.47 (CONH2)
Leu8
Gly9
8.66
8.51
a
Obtained at 400 MHz, 278 K, and pH ) 6.2 (phosphate buffer) for a ∼14.3 mM solution of 18 in water containing 10% D2O [HDO (δ
b
) 4.98) as internal standard]. Degeneracy has been assumed.
hydroxyl group of 12 was first protected by treatment
with triethylsilyl chloride to furnish 13. Despite this,
coupling of R-bromo acid 6 with secondary amine 13
turned out to be difficult, most likely because of steric
hindrance. Conversion of 6 into the corresponding acid
chloride allowed rapid and almost complete acylation
of 13, but the product 14 was found to be a mixture of
diastereomers, most likely due to epimerization of the
stereogenic center of 6. Initially the yield was low when
N,N′-diisopropylcarbodiimide (DIC) was used as cou-
pling reagent, but 14 was obtained as a single diastereo-
mer according to 1H NMR spectroscopy. To improve the
yield, the influence of the ratio of 6 to DIC, the
concentrations of the reactants, and the reaction time
were investigated carefully. By use of an excess of
R-bromo acid 6 (8 equiv), a 2:1 ratio of 6 to DIC, a high
concentration of 13 (0.09 M), and a prolonged reaction
time (2 days), a satisfactory yield of 14 could be
obtained. Despite substantial efforts, 14 could not be
obtained completely free from diisopropylurea, the side
product of the coupling. However, removal of the tri-
ethylsilyl group by treatment with aqueous HCl allowed
the remaining contaminants of diisopropylurea to be
removed by flash column chromatography and 15 was
isolated in 56% yield based on 13. The key cyclization
of 15 was achieved by conversion of 15 into the corre-
sponding alkoxide by treatment with potassium hydride
in a mixture of THF and DMF, which induced intra-
molecular ring closure to give morpholin-3-one 16 in
high yield (89%). Formation of side products due to
â-elimination or epimerization of the stereocenter ad-
jacent to the carbonyl group in 16 was not detected in
the ring-closure step. Finally, deprotection of the pri-
mary hydroxyl group in 16 with tetrabutylammonium
fluoride and subsequent oxidation of 17 with the bipha-
esters.33 In the first attempt to prepare oxytocin ana-
logue 18, mimetic 4 was activated as an azabenzotria-
zolyl ester by treatment with HOAt and DIC in DMF.34
Only a slight excess (∼30%) of activated 4 relative to
the capacity of the resin was used in the coupling. After
attachment of 4 to the solid phase, the azido group was
reduced by treatment with tin(II) chloride in the pres-
ence of thiophenol and triethylamine.35,36 The reduction
could be monitored conveniently by the disappearance
of the N3 stretch in the IR spectrum obtained from a
few resin beads.37 After reduction of the azido group had
reached completion, it was found that it is essential to
wash the resin with 20% piperidine in DMF before
proceeding with the synthesis. If washing with piperi-
dine was omitted, incorporation of the following amino
acid did not reach completion. This was assumed to be
due to complexation of tin salts to the liberated amino
group in the attached mimetic. The azido group thus
served as a masked amino group throughout the syn-
thesis of 4 and during attachment to the peptide resin.
After solid-phase synthesis had been completed, the
resin was treated with trifluoroacetic acid containing
water, thioanisole, and ethanedithiol as scavengers to
liberate the peptide analogue from the solid support and
to deprotect the amino acid side chains.38 Disulfide bond
formation was effected by oxidation with iodine in
methanol,39 and purification by reversed-phase HPLC
furnished peptide analogue 18 (33% yield based on the
capacity of the resin). The product was homogeneous
when analyzed by reversed-phase HPLC and had the
correct molecular weight according to FAB mass spec-
trometry. However, 1H NMR spectroscopy revealed two
sets of resonances, both of which were compatible with
the structure of 18. N-Alkylated amino acids, such as
turn mimetic 4, are more susceptible to epimerization
during activation and coupling than ordinary amino
acids.40,41 We therefore assumed that the heterogeneity
of the product could be due to epimerization of the
stereocenter adjacent to the carboxyl group of 4. Indirect
support for this assumption was provided by the obser-
vation that the chirality of the Tyr, Cys, Pro, and Leu
residues in the product was intact, as determined by
chiral amino acid analysis.42 Use of HATU43,44 as
coupling reagent, in combination with collidine as base
and dichloromethane as solvent, has been shown to
suppress epimerization in coupling of peptide seg-
ments.45 When mimetic 4 was incorporated using these
conditions, 18 was obtained in 29% yield based on the
capacity of the resin. In this case, 1H NMR spectroscopy
revealed only one set of signals, which was in complete
30
sic RuCl3-NaIO4 system gave the desired inverse
γ-turn mimetic 4 (61%) after purification by reversed-
phase HPLC. The structure of mimetic 4 was confirmed
by 1H NMR spectroscopy, which revealed a strong NOE
between hydrogen atoms H-2 and H-6 in the morpholin-
3-one ring. This observation is consistent with the
expectation that the inverse γ-turn mimetic should
adopt a half-chair conformation (cf. 4, Scheme 3), with
H-2 and H-6 in axial orientations.
The target oxytocin analogue 18 was prepared on a
polystyrene resin grafted with polyethylene glycol chains
that were functionalized with the Rink linker.31,32 The
synthesis was performed by coupling of NR-Fmoc amino
acids, which carried standard side chain protecting
groups and had been preactivated as benzotriazolyl

Analogue of Oxytocin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2515
agreement with the structure of 18 (Table 1). The
structure of 18 was further confirmed by means of fast-
atom bombardment mass spectrometry and amino acid
analysis.
as an inhibitor of oxytocin-induced contractions. This
may be due to the structural features of the γ-turn
mimetic, in particular the presence of a methylene ether
isostere instead of an amide bond between residues 3
and 4 in 18.
Oxytocin (1) is a potent inducer of uterine contrac-
tions, and the ability of analogue 18 to induce contrac-
tions of rat uterine tissue segments, or inhibit oxytocin-
induced contractions, was investigated in an organ
bath.46 It was found that 18 did not act as an oxytocin
agonist even at the highest concentration (2.0 µM)
evaluated in this assay. Furthermore, 18 did not display
any antagonistic effect at concentrations ranging from
0.49 to 7.8 µM when oxytocin was used as agonist. These
findings could be viewed in the context of early observa-
tions, which showed that modifications in the macro-
cyclic ring of oxytocin, for instance by replacement of
Gln4 by isoglutamine, resulted in loss of activity at the
uterine receptor.47-49
Exp er im en ta l Section
Gen er a l Meth od s a n d Ma ter ia ls. All reactions were
carried out under a nitrogen atmosphere with dry solvents
under anhydrous conditions unless otherwise stated. CH2Cl2
and THF were distilled from calcium hydride and potassium
benzophenone, respectively. DMF was distilled and then dried
over 3 Å molecular sieves. TLC was performed on silica gel 60
F254 (Merck) with detection by UV light and charring with
phosphomolybdic acid in EtOH. Flash column chromatography
(eluents given in brackets) was performed on silica gel (Matrex,
60 Å, 35-70 µm, Grace Amicon).
1H NMR spectra for compounds 4-17 were recorded at 400
or 500 MHz, whereas 13C NMR spectra were obtained at 100
MHz, for solutions in CDCl3 [residual CHCl3 (δH 7.26 ppm) or
CHCl3 (δC 77.0 ppm) as internal standard] at 298 K. The 1H
NMR spectrum of oxytocin analogue 18 was recorded at 400
MHz and 278 K in a mixture of phosphate buffer (500 µL, 40
mM, pH ) 6.2, ∼15 mM in NaN3) and D2O (50 µL) using the
resonance of H2O (δH 4.98 ppm) as internal standard. Proton
resonances were assigned from appropriate combinations of
COSY,52 TOCSY,53 HSQC,54 and NOESY55-57 experiments.
Diastereomeric excesses and ratios of the rotameric mixtures
The lack of activity of 18 may be due to incorporation
of turn mimetic 4 having a drastic effect on the
conformation of 18 compared to oxytocin. It is also
possible that the structural modifications introduced by
mimetic 4 prevent 18 from binding to receptors in the
uterus. As outlined in the preceding paper,50 conforma-
tional studies of an analogue of the drug desmopressin
([Mpr1,DArg8]vasopressin) in which a γ-turn mimetic
similar to 4 has been incorporated revealed that the
desmopressin analogue was able to populate similar
conformations as desmopressin itself. In view of the
close structural and conformational21 similarities be-
tween oxytocin and desmopressin, it therefore appears
unlikely that the lack of activity of 18 is due only to
conformational influences from the γ-turn mimetic. It
should, however, be kept in mind that 18 is expected to
be more rigid than oxytocin and that this could contrib-
ute to the low activity of 18. It may therefore be
proposed that structural features of the γ-turn mimetic
also are important when accounting for the loss of
activity of 18. For instance, the methylene bridge might
provide steric hindrance for binding of 18 to the uterine
receptor. However, replacement of the amide bond
between residues 3 and 4 in desmopressin with a
methylene ether isostere was found to result in a
substantial loss of activity at the vasopressin V2 recep-
tor.50 Accordingly, it is possible that the lack of an amide
bond between residues 3 and 4 of 18 significantly
contributes to the loss of bioactivity. This, in turn, would
suggest that the corresponding amide bond in oxytocin
is involved in hydrogen bonding with the uterine G-
protein-coupled receptor, a situation that is regarded
to be uncommon for peptide hormones that interact with
transmembrane receptors.51 Finally, these results may
also suggest that the receptor bound conformation of
oxytocin does not contain an inverse γ-turn at residues
3-5.
1
were derived from the H NMR spectra. Ions for positive fast-
atom bombardment mass spectra were produced by a beam of
xenon atoms (6 keV) from a matrix of glycerol and thioglycerol.
Melting points are uncorrected.
The purity of oxytocin analogue 18 and building block 4 was
analyzed by reversed-phase HPLC using a Kromasil C-8
column (100 Å, 5 µm, 4.6 × 250 mm). Linear gradients of 0%
to 100% of solvent B in solvent A over 60 min and 40% to 100%
of solvent B in solvent A over 30 min were used for 17 and 4,
respectively, with a flow rate of 1.5 mL/min and detection at
214 nm (solvent systems: A, 0.1% aqueous trifluoroacetic acid;
B, 0.1% trifluroacetic acid in CH3CN). Purification of crude
18 and 4 by HPLC was performed on a Kromasil C-8 column
(100 Å, 5 µm, 20 mm × 250 mm) using the same eluent and a
flow rate of 11 mL/min.
(2R,3R,4S)-2,3-Ep oxy-4-m eth ylh exa n -1-ol (9). D-(-)-Di-
isopropyl tartrate (30.3 µL, 0.145 mmol) and Ti(OiPr)4 (28.4
µL, 96.4 µmol) were added sequentially to a mixture of CH2-
Cl2 (7 mL) and crushed 4 Å molecular sieves (55 mg) at -20
°C. After the mixture was stirred for 15 min at -20 °C,
tBuOOH in toluene (3.55 M, 1.19 mL, 4.24 mmol) was added
slowly. After a further 30 min, allylic alcohol 828 (0.220 g, 1.93
mmol) was added dropwise. The reaction was allowed to run
overnight at -20 °C. A cooled (0 °C) solution of FeSO4‚7H2O
(0.64 g) and tartaric acid (0.193 g) in water (5 mL) was
subsequently added, and the mixture was stirred for 10 min.
The phases were separated, and the aqueous phase was
extracted twice with CH2Cl2. The combined organic phases
were cooled to 0 °C and treated with aqueous 30% NaOH
saturated with NaCl (1.2 mL) and water (8.8 mL), and the
mixture was stirred vigorously for 1 h until two clear phases
were formed. The aqueous phase was extracted with CH2Cl2
(2 × 8 mL), and the combined organic phases were dried with
Na2SO4, filtered, and concentrated. Flash chromatography
(pentane/diethyl ether, 10:1 f 1:1) of the residue gave epoxy
alcohol 9 as a colorless oil (0.205 g, 82%, 88% de as determined
In conclusion, an inverse γ-turn mimetic, 4, which has
side chains functionalities corresponding to an Ile-Val-
Asn tripeptide, has been synthesized from three build-
ing blocks: an azido epoxide, an O-silylated amino
alcohol, and an R-bromo acid. Mimetic 4 was then
incorporated in place of residues 3-5 in the peptide
hormone oxytocin by Fmoc solid-phase synthesis to give
oxytocin analogue 18. Analogue 18 did not induce
contractions of uterine tissue segments nor did it act
1
by integration of the H NMR resonances for the CH3CHCHO
protons): 1H NMR (400 MHz, CDCl3) δ 3.89 (1H, brd, J ) 12.6
Hz, CH2OH), 3.58 (1H, brd, J ) 12.4 Hz, CH2OH), 2.96 (1H,
ddd, J ) 4.8, 2.4, and 2.4 Hz, OCHCH2OH), 2.69 (1H, dd, J )
7.3, 2.4 Hz, CH3CHCHO), 2.38 (1H, s, OH), 1.47-1.37 (1H,
m, CH2CH3), 1.36-1.23 (2H, m, CHCH3 and CH2CH3), 0.99
(3H, d, J ) 6.5 Hz, CHCH3), 0.90 (3H, t, J ) 7.3 Hz, CH2CH3);
13C NMR (100 MHz, CDCl3) δ 61.9, 60.5, 58.3, 37.1, 26.4, 16.6,
11.6; HRMS (FAB) calcd for C7H15O2 131.1072 (M + H+), found
131.1071.

2516 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Yuan et al.
(2S,3S,4S)-3-Azid o-4-m eth ylh exa n -1,2-d iol (10). Epoxy
alcohol 9 (3.19 g, 24.5 mmol) in benzene (32 mL) was added
to a suspension of Ti(OiPr)2(N3)2 (7.56 g, 30.2 mmol) in benzene
(200 mL) at 85 °C. After 30 min, the mixture was concentrated,
then diethyl ether (400 mL) and 5% aqueous H2SO4 (200 mL)
were added, and the mixture was stirred for 10 min until two
clear phases were formed. The aqueous phase was extracted
with CH2Cl2 (4 × 20 mL), and the combined organic phases
were dried with Na2SO4, filtered, and concentrated. The
residue was washed with pentane and then purified by flash
chromatography (pentane/Et2O, 5:1 f 1:3) to give azido diol
10 as a colorless oil (3.93 g, 92%, 88% ∼de): 1H NMR (400
MHz, CDCl3) δ 3.85-3.76 (2H, m, CHOH and CH2OH), 3.71
(1H, dd, J ) 11.3, 6.6 Hz, CH2OH), 3.39 (1H, t, J ) 6.3 Hz,
CHN3), 2.34 (2H, brs, OH), 1.75-1.64 (1H, m, CHCH3), 1.63-
1.56 (1H, m, CH2CH3), 1.33-1.17 (1H, m, CH2CH3), 1.00 (3H,
7.70-7.60 (4H, m, Ph), 7.49-7.33 (6H, m, Ph), 7.31-7.19 (15H,
m, Ph), 3.77 (1H, ABX-type dd, J ) 10.4, 4.2 Hz, CH2O), 3.63
(1H, ABX-type dd, J ) 10.4, 4.9 Hz, CH2O), 3.59-3.52 (1H,
m, CHO), 3.02-2.93 (1H, m, CHNH), 2.80 (1H, dd, J ) 7.1,
3.9 Hz, CHN3), 2.65 (1H, ABX-type dd, J ) 11.6, 9.6 Hz, CH2-
CO), 2.56-2.40 (3H, m, CH2CO and CH2NH), 2.30 (2H, bs,
OH and NH), 1.79-1.67 (1H, m, CHCH3), 1.50-1.39 (1H, m,
CH2CH3), 1.26-1.13 (1H, m, CH2CH3), 1.09 (9H, s, C(CH3)3),
0.94 (3H, d, J ) 6.8 Hz, CHCH3), 0.90 (3H, t, J ) 7.4 Hz,
CH2CH3); 13C NMR (100 MHz, CDCl3) δ 170.8, 144.9, 135.5,
133.0, 129.9, 128.6, 127.8, 127.8, 126.8, 70.4, 70.3, 67.0, 64.0,
56.4, 50.4, 38.9, 35.6, 26.9, 24.9, 19.2, 16.2, 11.1; IR (neat) 3319,
2958, 2929, 2100, 1657, 1537, 1109, 698 cm-1; HRMS (FAB)
calcd for C46H56N5O3Si 754.4152 (M + H+), found 754.4163.
N-Tr ip h en ylm eth yl-(3S)-3-[(2S,3S,4S)-3-a zid o-4-m eth -
yl-2-t r iet h ylsilyloxyh exyla m in o]-4-(ter t-b u t yld ip h en yl-
silyloxy)bu tyr a m id e (13). Imidazole (0.155 g, 2.28 mmol)
was added to a solution of azido alcohol 12 (0.735 g, 0.990
mmol) in CH2Cl2 (50 mL) at room temperature. The solution
was cooled to 0 °C, and chlorotriethylsilane (0.366 mL, 2.18
mmol) was added. After 20 h, the mixture was poured into
saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 ×
20 mL). The combined extracts were dried with Na2SO4, and
the solvents were evaporated. Flash chromatography (heptane/
ethyl acetate, 15:1 f 5:1) of the residue furnished silylated
d, J ) 6.9 Hz, CHCH3), 0.92 (3H, t, J ) 7.4 Hz, CH2CH3); 13
C
NMR (100 MHz, CDCl3) δ 71.4, 70.1, 63.2, 36.0, 24.7, 16.2,
11.2; IR (neat) 3365, 2969, 2100, 1462, 1269 cm-1; HRMS
(FAB) calcd for C7H16N3O2 174.1243 (M + H+), found 174.1245.
(2S,3S,4S)-3-Azido-2-m eth an esu lfon yloxy-4-m eth ylh ex-
1-yl ben zoa te (11). 2,4,6-Collidine (2.03 mL, 15.4 mmol) and
benzoyl chloride (1.51 mL, 13.0 mmol) were added sequentially
to a solution of azido diol 10 (2.05 g, 11.8 mmol) in CH2Cl2 (55
mL) at -10 °C. The solution was allowed to attain room
temperature overnight, then it was cooled again to 0 °C, and
methanesulfonyl chloride (1.00 mL, 12.8 mmol) was added.
After being allowed to attain room temperature overnight, the
mixture was poured into saturated aqueous NaCl (50 mL). The
aqueous phase was extracted with EtOAc (4 × 20 mL), and
the combined organic phases were dried with Na2SO4, filtered,
and concentrated. Flash chromatography (heptane/ethyl ac-
etate, 15:1 f 5:1) of the residue gave benzoate 11 as a colorless
oil (2.53 g, 66%, 88% de as determined by integration of the
1H NMR resonances for the CH2O protons): 1H NMR (400
MHz, CDCl3) δ 8.09-8.05 (2H, m, Ph), 7.62-7.57 (1H, m, Ph),
7.50-7.43 (2H, m, Ph), 5.22-5.17 (1H, m, CHOMs), 4.71 (1H,
ABX-type dd, J ) 12.7, 2.3 Hz, CH2O), 4.49 (1H, ABX-type
dd, J ) 12.7, 7.8 Hz, CH2O), 3.69 (1H, dd, J ) 8.3, 4.4 Hz,
CHN3), 3.06 (3H, s, SCH3), 1.78-1.67 (1H, m, CH2CH3), 1.66-
1.55 (1H, m, CHCH3), 1.33-1.21 (1H, m, CH2CH3), 1.08 (3H,
alcohol 13 as a white solid (0.712 g, 86%): mp 49-52 °C; [R
20
D
]
+1.4° (c 0.9, CHCl3); 1H NMR (400 MHz, CDCl3) δ 9.02
(1H, s, NHTrt), 7.66-7.60 (4H, m, Ph), 7.47-7.31 (6H, m, Ph),
7.29-7.18 (15H, m, Ph), 3.82-3.74 (2H, m, CHOSi and CH2-
OSi), 3.55 (1H, ABX-type dd, J ) 10.4, 5.2 Hz, CH2OSi), 3.04-
2.97 (1H, m, CHN), 2.75 (1H, dd, J ) 7.3, 4.4 Hz, CHN3), 2.71
(1H, ABX-type dd, J ) 11.4, 6.8 Hz, CH2N), 2.64 (1H, ABX-
type dd, J ) 11.6, 5.7 Hz, CH2N), 2.46-2.42 (2H, m, CH2CO),
1.94 (1H, s, NH), 1.74-1.65 (1H, m, CHCH3), 1.49-1.39 (1H,
m, CH2CH3), 1.17-1.08 (1H, m, CH2CH3), 1.06 (9H, s, C(CH3)3),
0.96-0.84 (15H, m, CHCH3, CH2CH3, and OSi(CH2CH3)3), 0.56
(6H, q, J ) 7.9 Hz, OSi(CH2CH3)3); 13C NMR (100 MHz, CDCl3)
δ 170.8, 144.9, 135.5, 133.0, 132.8, 129.9, 128.6, 127.8, 126.8,
73.0, 70.1, 69.8, 64.1, 56.7, 49.5, 38.8, 34.5, 26.9, 24.9, 19.2,
16.5, 11.1, 6.9, 5.2; IR (neat) 3319, 2958, 2102, 1668, 1489,
1105 cm-1; HRMS (FAB) calcd for C52H70N5O3Si2 868.5017 (M
+ H+), found 868.5022.
d, J ) 6.8 Hz, CHCH3), 0.94 (3H, t, J ) 7.4 Hz, CH2CH3); 13
C
NMR (100 MHz, CDCl3) δ 166.1, 133.5, 129.7, 129.2, 128.6,
79.2, 69.4, 62.4, 38.8, 36.2, 25.4, 15.5, 10.7; IR (neat) 2966,
2104, 1724, 1344, 1269, 1176, 916 cm-1; HRMS (FAB) calcd
for C15H22N3O5S 356.1580 (M + H+), found 356.1293.
N-Tr ip h en ylm eth yl-(3S)-3-{N′-[(2S,3S,4S)-3-a zid o-2-h y-
d r oxy-4-m eth ylh exyl]-N′-[(1R)-1-br om o-2-m eth ylp r op yl-
ca r b on yl]a m in o}-4-(ter t-b u t yld ip h en ylsilyloxy)b u t yr -
a m id e (15). 1,3-Diisopropylcarbodiimide (DIC, 86.0 µL, 0.432
mmol) was added to a solution of (R)-(+)-2-bromo-3-methyl-
butanoic acid (6, 157 mg, 0.865 mmol, Aldrich) in CH2Cl2 (0.6
mL) at 0 °C. The mixture was stirred at 0 °C for 30 min, after
which amine 13 (90.8 mg, 0.108 mmol) in CH2Cl2 (0.6 mL) was
added. After a further 28 h at 0 °C, the diisopropylurea was
filtered off and the filtrate was extracted with saturated
aqueous NaHCO3 (2 × 5 mL), 10% aqueous citric acid (2 × 5
mL), and brine (5 mL). The organic solution was dried over
Na2SO4 and concentrated. Flash chromatography (heptane/
ethyl acetate, 25:1 f 15:1) of the residue gave somewhat
impure triethylsilylated amide 14 (84.4 mg). Compound 14
(84.4 mg, ∼81.8 µmol) was then dissolved in a mixture of 2 M
aqueous HCl (3 mL) and THF (2 mL), and the resulting
mixture was stirred at room temperature for 5 days. The
mixture was poured into saturated aqueous NaHCO3 (5 mL)
and was extracted with CH2Cl2 (5 × 10 mL). The combined
organic solutions were dried over Na2SO4 and concentrated.
Flash chromatography (heptane/ethyl acetate, 15:1 f 5:1) of
the residue yielded 15 as a white solid (54.6 mg, 56% over two
steps): mp 65-68 °C; [R]2D0 -45° (c 0.7, CHCl3); 1H NMR (400
MHz, CDCl3, 4:1 mixture of rotamers) δ (major) 7.66-7.60
(3.2H, m, Ph), 7.41-7.34 (4.8H, m, Ph), 7.30-7.20 (7.2H, m,
Ph), 7.20-7.16 (4.8H, m, Ph), 6.69 (0.8H, s, NHTrt), 4.30-
4.20 (1.6H, m, CHBr and CH2OSi), 4.04-3.95 (1.6H, m, CHN
and CHOH), 3.66 (0.8H, ABX-type dd, J ) 10.2, 5.5 Hz, CH2-
OSi), 3.55 (0.8H, ABX-type dd, J ) 15.3, 10.0 Hz, CH2N), 3.44-
3.24 (1.6H, m, CH2N and CH2CO), 2.67 (0.8H, dd, J ) 7.4, 3.7
Hz, CHN3), 2.37 (0.8H, ABX-type dd, J ) 15.7, 3.2 Hz, CH2-
(2R,3S,4S)-3-Azid o-1,2-ep oxy-4-m eth ylh exa n e (5). NaO-
Et in EtOH (2 M, 2.36 mL, 4.72 mmol) was added dropwise to
benzoate 11 (1.39 g, 4.29 mmol) in THF (28 mL). After being
stirred for 30 min at room temperature, the solution was
poured into saturated aqueous NH4Cl (15 mL). The mixture
was extracted with CH2Cl2 (2 × 10 mL), and the combined
organic phases were dried with Na2SO4, filtered, and concen-
trated. Flash chromatography (pentane/Et2O, 60:1 f 40:1) of
the residue gave epoxide 5 as a colorless oil (0.56 g, 83%, 90%
de as determined by integration of the 1H NMR resonances
for the CH2O protons): 1H NMR (400 MHz, CDCl3) δ 3.08 (1H,
ddd, J ) 6.6, 4.0, and 2.6 Hz, CHO), 2.92-2.84 (2H, m, CH2O
and CHN3), 2.66 (1H, dd, J ) 4.8, 2.6 Hz, CH2O), 1.72-1.64
(2H, m, CHCH3 and CH2CH3), 1.33-1.20 (1H, m, CH2CH3),
0.99 (3H, d, J ) 6.9 Hz, CHCH3), 0.92 (3H, t, J ) 7.3 Hz,
CH2CH3); 13C NMR (100 MHz, CDCl3) δ 69.3, 53.4, 45.6, 37.3,
25.8, 15.3, 11.1; IR (c ) 0.7 in CH2Cl2) 2968, 2099 1461, 1252
cm-1; HRMS (FAB) calcd for C7H17N4O 173.1483 (M + NH4+),
found 173.1413.
N -Tr ip h e n ylm e t h yl-(3S )-3-[(2S ,3S ,4S )-3-a zid o-2-h y-
d r oxy-4-m et h ylh exyla m in o]-4-(ter t-b u t yld ip h en ylsilyl-
oxy)bu tyr a m id e (12). Azido epoxide 5 (0.225 g, 1.45 mmol)
and amine 723 (0.87 g, 1.45 mmol) were dissolved in EtOH (35
mL), and the solution was refluxed for 96 h. The solvent was
evaporated, and flash chromatography (heptane/ethyl acetate,
10:1 f 1:1) of the residue yielded azido alcohol 12 as a white
solid (0.74 g, 68%): mp 145-148 °C; [R]2D0 -6.8° (c 0.7,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.85 (1H, s, NHTrt),

Analogue of Oxytocin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2517
CO), 2.33-2.23 (0.8H, m, CH(CH3)2), 1.84-1.73 (0.8H, m,
CHCH3), 1.61-1.50 (0.8H, m, CH2CH3), 1.31-1.17 (0.8H, m,
CH2CH3), 1.15 (2.4H, d, J ) 6.6 Hz, CH(CH3)2), 1.05 (7.2H, s,
C(CH3)3), 1.00 (2.4H, d, J ) 6.6 Hz, CH(CH3)2), 0.94 (2.4H, t,
J ) 7.4 Hz, CH2CH3), 0.91-0.81 (2.4H, m, CHCH3); 1H NMR
(400 MHz, CDCl3, 4:1 mixture of rotamers) δ (minor) 7.60-
7.54 (0.8H, m, Ph), 7.46-7.42 (1.2H, m, Ph), 7.30-7.20 (1.8H,
m, Ph), 7.16-7.12 (1.2H, m, Ph), 6.83 (0.2H, s, NHTrt), 4.64
(0.2H, d, J ) 6.9 Hz, CHBr), 4.62-4.54 (0.2H, m, CHOH),
4.16-4.10 (0.4H, m, CHN and CH2OSi), 3.72 (0.2H, ABX-type
dd, J ) 10.7, 4.3 Hz, CH2N), 3.60-3.50 (0.2H, m, CH2N), 3.44-
3.24 (0.2H, m, CH2OH), 2.88-2.78 (0.4H, m, CHN3 and CH2-
CO), 2.50 (0.2H, ABX-type dd, J ) 15.5, 6.7 Hz, CH2CO), 2.33-
2.23 (0.2H, m, CH(CH3)2), 1.84-1.73 (0.2H, m, CHCH3), 1.67-
1.62 (0.2H, m, CH2CH3), 1.31-1.17 (0.2H, m, CH2CH3), 1.05
(1.8H, s, C(CH3)3), 0.91-0.81 (1.8H, m, CH2CH3, CH(CH3)2),
0.83 (0.6H, d, J ) 6.8 Hz, CHCH3); IR (neat) 3359, 2962, 2102,
1633, 1491, 1449, 1036 cm-1; HRMS (FAB) calcd for C35H44N5O4
598.3393 (M + H+), found 598.3409.
(2S)-2-{(2S,6S)-6-[(1S,2S)-1-Azid o-2-m eth ylbu tyl]-2-iso-
p r op ylm or p h olin -3-on e-4-yl}-3-(N-t r ip h en ylm et h ylca r -
ba m oyl)p r op ion ic Acid (4). A catalytic amount (2.2 mol %)
of ruthenium trichloride hydrate was added to a mixture of
alcohol 17 (72 mg, 0.12 mmol), CCl4 (0.3 mL), acetonitrile (0.3
mL), H2O (0.45 mL), and sodium metaperiodate (80 mg, 0.37
mmol). The black mixture was stirred vigorously for 3.5 h at
room temperature, then CH2Cl2 (5 mL) and H2O (3 mL) were
added, and the phases were separated. The aqueous phase was
extracted with CH2Cl2 (3 × 5 mL), and the combined organic
extracts were dried with MgSO4 and concentrated. The residue
was diluted with diethyl ether (5 mL), filtered through a Celite
pad, and concentrated. Flash chromatography (toluene/etha-
nol, 10:1 f 1:1) followed by purification by reversed-phase
HPLC (linear gradient 40% f 100% solvent B in solvent A
during 30 min) furnished acid 4 as a white solid (45 mg,
61%): mp 105-107 °C; [R]2D0 -90° (c 0.4, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 7.27-7.14 (15H, m, Ph), 4.21-4.13 (1H,
m, CHN), 4.03 (1H, d, J ) 1.5 Hz, COCHO), 3.76-3.68 (1H,
m, CHN3CHO), 3.68 (1H, t, J ) 10.8 Hz, CH2N), 3.39 (1H, bd,
J ) 9.5 Hz, CH2N), 3.05 (1H, ABX-type dd, J ) 16.5, 5.4 Hz,
CH2CO), 2.96 (1H, ABX-type dd, J ) 16.8, 8.3 Hz, CH2CO),
2.87 (1H, dd, J ) 6.5, 4.7 Hz, CHN3), 2.47-2.34 (1H, m,
CH(CH3)2), 1.78-1.66 (1H, m, CHCH3), 1.46-1.36 (1H, m, CH2-
CH3), 1.22-1.10 (1H, m, CH2CH3), 1.13 (3H, d, J ) 7.0 Hz,
CH(CH3)2), 1.02 (3H, d, J ) 6.7 Hz, CHCH3), 0.96 (3H, d, J )
6.8 Hz, CH(CH3)2), 0.89 (3H, t, J ) 7.4 Hz, CH2CH3); 13C NMR
(100 MHz, CDCl3) δ 172.8, 170.1, 169.4, 144.6, 128.8, 127.8,
127.0, 81.6, 73.6, 70.6, 67.3, 58.7, 51.5, 36.4, 34.2, 30.6, 24.5,
19.3, 16.3, 15.7, 10.9; IR (neat) 3323, 2962, 2104, 1732, 1687,
1643, 1491, 1184, 1024 cm-1; HRMS (FAB) calcd for C35H41N5O5-
Na 634.3006 (M + Na+), found 634.3074.
P r oced u r e for Solid -P h a se Syn th esis of 18. Peptide 18
was synthesized in a mechanically agitated reactor on a
polystyrene resin grafted with aminated poly(ethylene glycol)
chains (TentaGel-S-NH2, Rapp Polymere, Germany; 0.276 g,
0.25 mmol/g, 69 µmol). The resin was functionalized with the
linker p-[R-[(9-fluorenylmethoxy)formamido]-2,4-dimethoxy-
benzyl]phenoxyacetic acid (Novabiochem, Switzerland). Re-
agent solutions and DMF for washing were added manually
to the reactor. NR-Fmoc amino acids (Bachem, Switzerland)
with the following side chain protective groups were used:
triphenylmethyl (Trt) for Cys and tert-butyl (tBu) for Tyr.
The linker and the NR-Fmoc amino acids were activated as
1-benzotriazolyl esters and then added to the resin. Activation
was performed by reaction of the appropriate NR-Fmoc amino
acid (0.275 mmol), 1-hydroxybenzotriazole (HOBt, 55.8 mg,
0.414 mmol), and 1,3-diisopropylcarbodiimide (41.6 µL, 0.269
mmol) in DMF (1.0 mL) for 30 min. Acylation was performed
for 1-2 h and monitored by addition of bromophenol blue
(0.05% of the resin capacity).58 Fmoc deprotection was per-
formed by treatment with 20% piperidine in DMF (3 + 7 min).
The inverse γ-turn mimetic 4 (55.0 mg, 90 µmol) was activated
using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate43,44 (HATU; 40 mg, 0.105 mmol) and
collidine (24.0 µL, 0.173 mmol) in CH2Cl2 (1.0 mL). Activated
4 was then coupled for 60 h, and the coupling was monitored
by bromophenol blue. Before proceeding further, the resin was
capped with acetic anhydride (0.5 mL) in CH2Cl2 for 45 min.
After incorporation of 4 into the peptide, reduction of the azido
group35,36 was achieved by sequential addition of triethylamine
(240 µL, 1.73 mmol), thiophenol (145 µL, 1.38 mmol), and
SnCl2 (66 mg, 0.345 mmol) to the suspended resin in THF (1
mL). After 19 h at room temperature, the resin was washed
with THF (5 × 1 mL), 20% piperidine (2 × 1 mL), DMF (6 ×
1 mL), and CH2Cl2 (5 × 1 mL) and was dried under vacuum.
The disappearance of the N3 stretch at 2100 cm-1 (IR spectrum
of a few resin beads37) indicated successful reduction. The resin
was then swelled in DMF (1 mL), and synthesis of 18 was
continued as described above.
1685, 1649, 1489, 1111 cm-1; HRMS (FAB) calcd for C51H63
-
BrN5O4Si 916.3833 (M + H+), found 916.3845.
N-Tr ip h en ylm eth yl-(3S)-3-{(2S,6S)-6-[(1S,2S)-1-a zid o-
2-m eth ylbu tyl]-2-isop r op ylm or p h olin -3-on e-4-yl}-4-(ter t-
bu tyld ip h en ylsilyloxy)bu tyr a m id e (16). KH (4.7 mg, 0.12
mmol) was suspended at 0 °C in a mixture of THF (2.4 mL)
and DMF (1.0 mL). Bromo alcohol 15 (70 mg, 76 µmol) in THF
(3.8 mL) was added dropwise to this suspension. After 90 min
at 0 °C, the mixture was poured into saturated aqueous
NaHCO3 (5 mL) and extracted with CH2Cl2 (6 × 10 mL). The
combined extracts were dried over Na2SO4, and the solvents
were evaporated under reduced pressure. Flash chromatog-
raphy (heptane/ethyl acetate, 10:1 f 3:1) of the residue gave
the cyclic morpholinone derivative 16 as a white solid (57 mg,
89%): mp 84-88 °C; [R]D20 -43° (c 0.6, CHCl3); H NMR (500
1
MHz, CDCl3) δ 7.68-7.60 (4H, m, Ph), 7.48-7.34 (6H, m, Ph),
7.32-7.21 (9H, m, Ph), 7.21-7.15 (6H, m, Ph), 6.78 (1H, s,
NHTrt), 4.14 (1H, ABX-type dd, J ) 9.7, 8.0 Hz, CH2O), 3.84
(1H, d, J ) 2.3 Hz, COCHO), 3.64-3.75 (4H, m, CHN3CHO,
CH2N, CHN, and CH2O), 3.33 (1H, ABX-type dd, J ) 15.3,
10.3 Hz, COCH2), 3.22 (1H, ABX-type dd, J ) 17.5, 9.4 Hz,
CH2N), 2.75 (1H, ABX-type dd, J ) 7.1, 3.5 Hz, CHN3), 2.54
(1H, ABX-type dd, J ) 15.3, 4.5 Hz, COCH2), 2.48-2.35 (1H,
m, CH(CH3)2), 1.83-1.71 (1H, m, CHCH3), 1.55-1.42 (1H, m,
CH2CH3), 1.25-1.13 (1H, m, CH2CH3), 1.08 (3H, d, J ) 7.2
Hz, CH(CH3)2), 1.05 (9H, s, C(CH3)3), 0.95 (3H, d, J ) 6.7 Hz,
CHCH3), 0.9 (3H, d, J ) 6.8 Hz, CH(CH3)2), 0.87 (3H, t, J )
7.4 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 169.7, 169.4,
144.6, 135.5, 133.1, 129.8, 128.6, 127.9, 127.8, 127.0, 81.8, 73.3,
70.4, 67.1, 63.3, 61.2, 52.2, 37.1, 34.2, 30.6, 26.8, 24.8, 19.2,
19.1, 16.2, 15.9, 10.9; IR (neat) 3313, 2962, 2102, 1684, 1633,
1489, 1107 cm-1; HRMS (FAB) calcd for C51H62N5O4Si 836.4571
(M + H+), found 836.4561.
N-Tr ip h en ylm eth yl-(3S)-3-{(2S,6S)-6-[(1S,2S)-1-a zid o-
2-m et h ylb u t yl]-2-isop r op ylm or p h olin -3-on e-4-yl}-4-h y-
d r oxybu tyr a m id e (17). Tetrabutylammonium fluoride (19
mg, 74 µmol) was added to a solution of protected alcohol 16
(56 mg, 67 µmol) in THF (2.0 mL) at room temperature. After
the mixture was stirred for 2.5 h, the solvent was evaporated.
Flash chromatography (heptane/ethyl acetate, 1:1 f 1:5) of
the residue furnished alcohol 17 as a white solid (39 mg, 97%):
mp 85-87 °C; [R]2D0 -72° (c 0.4, CHCl3); H NMR (400 MHz,
1
CDCl3) δ 7.31-7.21 (9H, m, Ph), 7.21-7.17 (6H, m, Ph), 7.07
(1H, s, NHTrt), 4.08 (1H, bs, OH), 3.90 (1H, d, J ) 2.3 Hz,
COCHO), 3.81-3.67 (4H, m, CH2OH, CHN3CHO, and CHN),
3.52 (1H, t, J ) 11.1 Hz, CH2N), 3.30 (1H, ABX-type dd, J )
11.6, 2.6 Hz, CH2N), 3.15 (1H, ABX-type dd, J ) 15.2, 8.1 Hz,
CH2CO), 2.83 (1H, ABX-type dd, J ) 15.0, 6.0 Hz, CH2CO),
2.80 (1H, dd, J ) 6.7, 4.5 Hz, CHN3), 2.47-2.34 (1H, m,
CH(CH3)2), 1.80-1.69 (1H, m, CHCH3), 1.52-1.40 (1H, m, CH2-
CH3), 1.24-1.10 (1H, m, CH2CH3), 1.06 (3H, d, J ) 7.0 Hz,
CH(CH3)2), 0.95 (3H, d, J ) 6.7 Hz, CHCH3), 0.88 (3H, d, J )
6.6 Hz, CH(CH3)2), 0.85 (3H, t, J ) 7.4 Hz, CH2CH3); 13C NMR
(100 MHz, CDCl3) δ 170.9, 169.5, 144.6, 128.7, 128.1, 127.2,
81.7, 73.4, 70.7, 67.2, 64.1, 60.7, 51.3, 36.3, 34.3, 31.0, 24.8,
19.2, 16.4, 15.7, 10.9; IR (neat) 3319, 2962, 2106, 1684, 1668,
After completion of the synthesis, the resin carrying the
protected peptide was washed with CH2Cl2 (5 × 1 mL) and

2518 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Yuan et al.
dried under vacuum. The peptide-resin was then cleaved, and
the amino acid side chains were deprotected by treatment with
trifluoroacetic acid/water/thioanisole/ethanedithiol [87.5:5:5:
2.5, 30 mL] for 2.5 h, followed by filtration. Acetic acid was
added to the filtrate, the solution was concentrated, and acetic
acid was added twice more followed by concentration. The
residue was triturated with diethyl ether, which gave a solid
crude peptide that was dissolved in a mixture of acetic acid
and water and was freeze-dried. Cyclization39 was performed
immediately by alternating additions of portions of the crude
peptide in acetic acid and a 0.1 M solution of I2 in methanol
to a 10% solution of acetic acid in methanol (2 mL/mg cleaved
resin). After the final addition of I2, a light-brown solution was
obtained that was neutralized and decolorized by stirring with
Dowex 2 × 8-100 anion-exchange resin (Fluka, Cl- form, 50-
100 mesh; converted into acetate form by washing with 1 M
aqueous NaOH, water, acetic acid, water, and methanol),
filtered, and concentrated. The residue was then dissolved in
a mixture of acetic acid and water and freeze-dried. Purifica-
tion by preparative reversed-phase HPLC using a linear
gradient of 0 f 100% of solvent B in solvent A over 60 min
gave 18 as a white solid after free-drying (20 mg, 29%):
1H NMR data are given in Table 1; MS (FAB) calcd for
C44H69N10O11S2 978 (M + H+), found 977. Amino acid analysis
gave the following: Cys 2.00 (2), Gly 1.01 (1), Leu 1.01 (1),
Pro 0.99 (1), Tyr 0.99 (1).
P h a r m a cologica l Eva lu a tion of 18.46 Sprague-Dawley
rats (body weight ∼250 g, M & B A/S, Denmark) were selected
at natural estrus by investigation of vaginal smears. The
selected animals were sacrificed, and the whole uterus was
removed and dissected free from fat and blood vessels. Four
equal segments from each uterus were cut longitudinally, and
two or three of these were then used in the evaluation. The
uterine segments were mounted in organ baths containing 10
mL of de J alon’s nutrient solution aerated with carbogen gas.
All segments were allowed to stabilize for 60 min at a resting
tension of 1.0 g (10 mN). The isometric contractions were
measured using a Grass force displacement transducer (model
FT03) connected to an amplifier and recorded on a Grass
polygraph (model 7D). The net maximal increase in uterine
contraction was chosen as the index of response. The Grass
system was calibrated so that 1.0 g equaled 20 mm (10 mN).
An oxytocin house standard was used as agonist and control
substance. The oxytocin house standard (616.5 IU/mg) and
analogue 18 were both dissolved in aqueous 0.9% NaCl
solution.
Fund at Umeå University, and the Go¨ran Gustafsson
Foundation for Research in Natural Sciences and Medi-
cine.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectra for compounds 4, 5, 9-13, and 15-17. This material
is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) Plattner, J . J .; Norbeck, D. W. Obstacles to drug development
from peptide leads. In Drug discovery technologies; Clark, C. R.,
Moos, W. R., Eds.; Ellis Harwood Ltd.-Halstead Press: Chi-
chester, England, 1989; pp 92-126.
(2) Ball, J . B.; Alewood, P. F. Conformational constraints: Nonpep-
tide â-turn mimics. J . Mol. Recognit. 1990, 3, 55-64.
(3) Giannis, A.; Kolter, T. Peptidomimetics for receptor ligandss
discovery, development, and medical perspectives. Angew. Chem.,
Int. Ed. Engl. 1993, 32, 1244-1267.
(4) Kahn, M. Peptide Secondary Structure Mimetics: Recent Ad-
vances and Future Challenges. Synlett 1993, 821-826.
(5) Hanessian, S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell,
W. D. Design and Synthesis of Conformationally Constrained
Amino Acids as Versatile Scaffolds and Peptide Mimetics.
Tetrahedron 1997, 53, 12789-12854.
(6) Smith, J . A.; Pease, L. G. Reverse turns in peptides and proteins.
CRC Crit. Rev. Biochem. 1980, 8, 315-399.
(7) Rose, G. D.; Gierasch, L. M.; Smith, J . A. Turns in peptides and
proteins. In Advances in Protein Chemistry; Anfinsen, C. B.,
Edsall, J . T., Richards, F. M., Eds.; Academic Press: Orlando,
FL, 1985; pp 1-109.
(8) Langs, D. A.; Smith, G. D.; Stezowski, J . J .; Hughes, R. E.
Structure of pressinoic acid: The cyclic moiety of vasopressin.
Science 1986, 232, 1240-1242.
(9) Schmidt, J . M.; Ohlenschla¨ger, O.; Ru¨terjans, H.; Grzonka, Z.;
Kojro, E.; Pavo, I.; Fahrenholz, F. Conformation of [8-arginine]-
vasopressin and V1 antagonists in dimethyl sulfoxide solution
derived from two-dimensional NMR spectroscopy and molecular
dynamics simulation. Eur. J . Biochem. 1991, 201, 355-371.
(10) Smith, G. D.; Griffin, J . F. Conformation of [Leu5]enkephalin
from X-ray diffraction: Features important for recognition at
opiate receptor. Science 1978, 199, 1214-1216.
(11) Amodeo, P.; Naider, F.; Picone, D.; Tancredi, T.; Temussi, P. A.
Conformational sampling of bioactive conformers: A low-tem-
perature NMR study of 15N-Leu-enkephalin. J . Pept. Sci. 1998,
4, 253-265.
(12) Aubry, A.; Birlirakis, N.; Sakarellos-Daitsiotis, M.; Sakarellos,
C.; Marraud, M. A crystal molecular conformation of Leucine-
enkephalin related to the morphine molecule. Biopolymers 1989,
28, 27-40.
(13) Schmidt, B.; Lindman, S.; Tong, W.; Lindeberg, G.; Gogoll, A.;
Lai, Z.; Tho¨rnwall, M.; Synnergren, B.; Nilsson, A.; Welch, C.
J .; Sohtell, M.; Westerlund, C.; Nyberg, F.; Karle´n, A.; Hallberg,
A. Design, synthesis, and biological activities of four angiotensin
II receptor ligands with γ-turn mimetics replacing amino acid
residues 3-5. J . Med. Chem. 1997, 40, 903-919.
(14) Sato, M.; Lee, J . Y. H.; Nakanishi, H.; J ohnson, M. E.; Chrusciel,
R. A.; Kahn, M. Design, synthesis and conformational analysis
of γ-turn peptide mimetics of bradykinin. Biochem. Biophys. Res.
Commun. 1992, 187, 999-1006.
(15) Gimpl, G.; Fahrenholz, F. The oxytocin receptor system: struc-
ture function and regulation. Physiol. Rev. 2001, 81, 629-683.
(16) Shojo, H.; Kaneko, Y. Characterization and expression of oxy-
tocin and the oxytocin receptor. Mol. Genet. Metab. 2000, 71,
552-558.
(17) Meraldi, J .-P.; Hruby, V. J .; Brewster, A. I. R. Relative confor-
mational rigidity in oxytocin and [1-penicillamine]-oxytocin: A
proposal for relationship of conformational flexibility to peptide
hormone agonism and antagonism. Proc. Natl. Acad. Sci. U.S.A.
1977, 74, 1373-1377.
(18) Mosberg, H. I.; Hruby, V. J .; Meraldi, J .-P. Conformational study
of the potent peptide hormone antagonist [1-penicillamine,2-
leucine]oxytocin in aqueous solution. Biochemistry 1981, 20,
2822-2828.
(19) Hruby, V. J .; Lebl, M. I. Conformational properties of neurohy-
pophyseal hormone analogs in solution as determined by NMR
and laser Raman spectroscopies. In CRC Handbook of neuro-
hypophyseal hormone analogs; J ost, K., Lebl, M., Brtnik, F., Eds.;
CRC Press: Boca Raton, FL, 1987; pp 105-155.
Evalution of 18 as an oxytocin agonist was performed
against the oxytocin house standard using a 2 + 2 test. At
least one group of doses consisting of standardlow, standardhigh
,
testlow, and testhigh was given in randomized order to each
uterine segment. Analogue 18 was evaluated on two different
occasions in a total of nine experiments but did not show any
agonistic effect even at the highest concentration evaluated
in the organ bath (2.0 µM).
Each evaluation of 18 as an antagonist of oxytocin was
started by administration of an exercise series of oxytocin
consisting of six different concentrations. This series was not
used for calculations. Six new oxytocin concentrations ranging
from 3.2 nM to 1.6 µM were then administered at constant
time intervals (45 s), and a cumulative dose-response curve
was constructed. The uterine segments were rinsed only after
the whole series had been completed. Then analogue 18 was
administered, and after 60 s the oxytocin series was repeated
and the uterine segment was rinsed again. The procedure was
repeated with concentrations of 18 ranging from 0.49 to 7.84
µM in the organ bath. Analogue 18 was evaluated at three
different occasions in a total of 16 experiments but did not
show any antagonistic activity at any of the evaluated con-
centrations.
(20) Zieger, G.; Andreae, F.; Sterk, H. Assignment of proton NMR
resonances and conformational analysis of [Lys 8]-vasopressin
homologues. Magn. Reson. Chem. 1991, 29, 580-586.
(21) Walse, B.; Kihlberg, J .; Drakenberg, T. Conformation of DDAVP,
an analogue of the peptide hormone vasopressin, in aqueous
solution as determined by NMR spectroscopy. Eur. J . Biochem.
1998, 252, 428-440.
Ack n ow led gm en t. This work was funded by grants
from the Swedish Research Council, the Biotechnology

Analogue of Oxytocin
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2519
(22) Lippens, G.; Hallenga, K.; van Belle, D.; Wodak, S. J .; Nirmala,
N. R.; Hill, P.; Russell, K. C.; Smith, D. D.; Hruby, V. J . Transfer
nuclear Overhauser effect study of the conformation of oxytocin
bound to bovine neurophysin I. Biochemistry 1993, 32, 9423-
9434.
(23) Brickmann, K.; Yuan, Z.; Sethson, I.; Somfai, P.; Kihlberg, J .
Synthesis of Conformationally Restricted Mimetics of γ-Turns
and Incorporation into Desmopressin, an Analogue of the Peptide
Hormone Vasopressin. Chem. Eur. J . 1999, 5, 2241-2253.
(24) Brickmann, K.; Somfai, P.; Kihlberg, J . An approach to Enan-
tiomerically Pure Inverse γ-Turn Mimetics for Use in Solid-
Phase Synthesis. Tetrahedron Lett. 1997, 38, 3651-3654.
(25) du Vigneaud, V.; Flouret, G.; Walter, R. Synthesis and some
biological properties of 4-valine-oxytocin and 1-deamino-4-valine-
oxytocin. J . Biol. Chem. 1965, 241, 2093-2096.
(26) Hruby, V. J .; Smith, C. W. Structure-Activity Relationships of
Oxytocin Agonists. In Chemistry, biology, and medicine of
neurohypophyseal hormones and their analogs; Smith, C. W.,
Ed.; Academic Press: Orlando, FL, 1987; pp 82-170.
(27) Gao, Y.; Hanson, R. M.; Klunder, J . M.; Ko, S. Y.; Masamune,
H.; Sharpless, K. B. Catalytic asymmetric epoxidation and
kinetic resolution: Modified procedures including in situ de-
rivatization. J . Am. Chem. Soc. 1987, 109, 5765-5780.
(28) Castejo´n, P.; Moyano, A.; Perica`s, M. A.; Riera, A. A straight-
forward, highly stereoselective synthesis of protected isostatine
derivatives. Chem. Eur. J . 1996, 2, 1001-1006.
(29) Caron, M.; Carlier, P. R.; Sharpless, K. B. Regioselective azide
opening of 2,3-epoxy alcohols by (Ti(OiPr)2(N3)2): Synthesis of
R-amino acids. J . Org. Chem. 1988, 53, 5185-5187.
(30) Carlsen, P. H. J .; Katsuki, T.; Martin, V. S.; Sharpless, K. B. A
greatly improved procedure for ruthenium tetraoxide catalyzed
oxidations of organic compounds. J . Org. Chem. 1981, 46, 3936-
3938.
(31) Rink, H. Solid-phase synthesis of protected peptide fragments
using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett.
1987, 28, 3787-3790.
(32) Bernatowicz, M. S.; Daniels, S. B.; Ko¨ster, H. A comparison of
acid labile linkage agents for the synthesis of peptide C-terminal
amides. Tetrahedron Lett. 1989, 30, 4645-4648.
(41) Davies, J . S.; Mohammed, A. K. Assessment of racemisation in
N-alkylated amnio-acid derivatives during peptide coupling in
a dipeptide system. J . Chem. Soc., Perkin Trans. 1 1981, 2982-
2990.
(42) Performed by C.A.T. GmbH & Co. Chromatographie und Analy-
sentechenik KG (Germany).
(43) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F.
Advantageous applications of azabenzotriazole (triazolopyridine)-
based coupling reagents to solid-phase peptide synthesis. J .
Chem. Soc., Chem. Commun. 1994, 201-203.
(44) Carpino, L. A.; El-Faham, A.; Albericio, F. Racemization studies
during solid-phase peptide synthesis using azabenzotriazole-
based coupling reagents. Tetrahedron Lett. 1994, 35, 2279-2282.
(45) Carpino, L. A.; El-Faham, A. The diisopropylcarbodiimide/1-
hydroxy-7-azabenzotriazole system: Segment coupling and step-
wise peptide assembly. Tetrahedron 1999, 55, 6813-6830.
(46) Melin, P.; Vilhardt, H.; Lindeberg, G.; Larsson, L.-E.; A° kerlund,
M. Inhibitory effect of O-alkylated analogues of oxytocin and
vasopressin on human and rat myometrial activity. J . Endo-
crinol. 1981, 88, 173-180.
(47) Ressler, C.; du Vigneaud, V. The isoglutamine isomer of oxyto-
cin: Its synthesis and comparison with oxytocin. J . Am. Chem.
Soc. 1957, 79, 4511-4515.
(48) Lutz, W. B.; Ressler, C.; Nettleton, D. E., J r.; du Vigneaud, V.
Isoasparagine-oxytocin: The isoasparagine isomer of oxytocin.
J . Am. Chem. Soc. 1959, 81, 167-173.
(49) J arvis, D.; Bodanszky, M.; du Vigneaud, V. The synthesis of
1-(hemi-homocystine)-oxytocin and a study of some of its phar-
macological properties. J . Am. Chem. Soc. 1961, 83, 4780-4784.
(50) Hedenstro¨m, M.; Yuan, Z.; Brickmann, K.; Carlsson, J .; Ekholm,
K.; J ohansson, B.; Kreutz, E.; Nilsson, A.; Sethson, I.; Kihlberg,
J . Conformations and receptor activity of desmopressin ana-
logues which contain γ-turn mimetics or a Ψ[CH2O] isostere. J .
Med. Chem. 2002, 45, 2501-2511.
(51) Marshall, G. R. Peptide interactions with G-protein coupled
receptors. Biopolymers 2001, 60, 246-277.
(52) Derome, A. E.; Williamson, M. P. Rapid-pulsing artifacts in
double-quantum-filtered COSY. J . Magn. Reson. 1990, 88, 177-
185.
(53) Bax, A.; Davis, D. G. MLEV-17-based two-dimensional homo-
nuclear magnetization transfer spectroscopy. J . Magn. Reson.
1985, 65, 355-360.
(54) Kay, L. E.; Keifer, P.; Saarinen, T. Pure absorption gradient
enhanced heteronuclear single quantum correlation spectroscopy
with improved sensitivity. J . Am. Chem. Soc. 1992, 114, 10663-
10665.
(55) J eener, J .; Meier, B. H.; Bachmann, P.; Ernst, R. R. Investigation
of exchange processes by two-dimensional NMR spectroscopy.
J . Chem. Phys. 1979, 71, 4546-4553.
(56) Kumar, A.; Ernst, R. R.; Wu¨thrich, K. A two-dimensional nuclear
Overhauser enhancement (2D NOE) experiment for the elucida-
tion of complete proton-proton cross-relaxation networks in
biological macromolecules. Biochem. Biophys. Res. Commun.
1980, 95, 1-6.
(57) Macura, S.; Ernst, R. R. Elucidation of cross relaxation in liquids
by two-dimensional NMR spectroscopy. Mol. Phys. 1980, 41, 95-
117.
(58) Flegel, M.; Sheppard, R. C. A sensitive general method for
quantitative monitoring of continuous flow solid phase peptide
synthesis. J . Chem. Soc., Chem. Commun. 1990, 536-538.
(33) Ko¨nig, W.; Geiger, R. A new method for synthesis of peptides:
Activation of the carboxyl group with dicyclohexylcarbodiimide
using 1-hydroxybenzotriazoles as additives. Chem. Ber. 1970,
103, 788-798.
(34) Carpino, L. A. 1-Hydroxy-7-azabenzotriazole. An efficient peptide
coupling additive. J . Am. Chem. Soc. 1993, 115, 4397-4398.
(35) Bartra, M.; Romea, P.; Urpi, F.; Vilarrasa, J . A fast procedure
for the reduction of azides and nitro compounds based on the
reducing ability of Sn(SR)3- species. Tetrahedron 1990, 46, 587-
594.
(36) Kim, J .-M.; Bi, Y.; Paikoff, S. J .; Schultz, P. G. The solid phase
synthesis of oligoureas. Tetrahedron Lett. 1996, 37, 5305-5308.
(37) Kick, E. K.; Ellman, J . A. Expedient method for the solid-phase
synthesis of aspartic acid protease inhibitors directed toward
the generation of libraries. J . Med. Chem. 1995, 38, 1427-1430.
(38) King, D. S.; Fields, C. G.; Fields, G. B. A cleavage method which
minimizes side reactions following Fmoc solid phase peptide
synthesis. Int. J . Pept. Protein Res. 1990, 36, 255-266.
(39) Kihlberg, J .; A° hman, J .; Walse, B.; Drakenberg, T.; Nilsson, A.;
So¨derberg-Ahlm, C.; Bengtsson, B.; Olsson, H. Glycosylated
peptide hormones: Pharmacological properties and conforma-
tional studies of analogues of [1-Desamino,8-D-arginine]vaso-
pressin. J . Med. Chem. 1995, 38, 161-169.
(40) McDermott, J . R.; Benoiton, N. L. N-Methylamino Acids in
Peptide Synthesis. IV. Racemization and Yields in Peptide-Bond
Formation. Can. J . Chem. 1973, 51, 2562-2570.
J M0110744