Vinyl sulfide cyclized analogues of angiotensin II with high affinity and full agonist activity at the AT1 receptor

Article abstract of DOI:10.1021/jm011063a

Vinyl sulfide cyclized analogues of the octapeptide angiotensin II that are structurally related to the cyclic disulfide agonist c[Hcy^3,5 Ang II have been prepared. The synthesis relies on the reaction of the mercapto group of a cysteine residu

Full text of DOI:10.1021/jm011063a

J . Med. Chem. 2002, 45, 1767-1777
1767
Vin yl Su lfid e Cyclized An a logu es of An gioten sin II w ith High Affin ity a n d F u ll
Agon ist Activity a t th e AT1 Recep tor
Petra J ohannesson,† Gunnar Lindeberg,† Anja J ohansson,† Gregory V. Nikiforovich,‡ Adolf Gogoll,§
Barbro Synnergren,| Madeleine Le Gre`ves,| Fred Nyberg,| Anders Karle´n,† and Anders Hallberg*,†
Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, Box 574,
SE-751 23 Uppsala, Sweden, Department of Biochemistry and Molecular Biophysics, Washington University,
P.O. Box 8036, St. Louis, Missouri 63110, Department of Organic Chemistry, Uppsala University, Box 531,
SE-751 21 Uppsala, Sweden, and Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence,
Uppsala University, Box 591, SE-751 24 Uppsala, Sweden
Received October 17, 2001
Vinyl sulfide cyclized analogues of the octapeptide angiotensin II that are structurally related
to the cyclic disulfide agonist c[Hcy3,5]Ang II have been prepared. The synthesis relies on the
reaction of the mercapto group of a cysteine residue in position 3 with the formyl group of
allysine incorporated in position 5 of angiotensin II. A mixture of the cis and the trans isomers
was formed, and these were separated and isolated by RP-HPLC. Thus, the three-atom CH2-
S-S element of the AT1 receptor agonist c[Hcy3,5]Ang II has been displaced by a bioisosteric
three-atom S-CHdCH element. A comparative conformational analysis of the 13-membered
ring systems of c[Hcy3,5]Ang II and the 13-membered cyclic vinyl sulfides with cis and trans
configuration, respectively, suggested that all three systems adopted very similar low-energy
conformations. This similarity was also reflected in the bioactivity. Both of the compounds
that contained the ring systems encompassing the cis or trans vinyl sulfide elements between
positions 3 and 5 exhibited Ki values less than 2 nM and exerted full agonism at the AT1
receptor. In contrast, vinyl sulfide cyclization involving the amino acid residues 5 and 7 rendered
inactive compounds. The cyclic vinyl sulfides that have agonist activity were both shown to
possess low-energy conformers compatible with the previously proposed 3D model for the
bioactive conformation of Ang II.
In tr od u ction
fully applied. Importantly, for obtaining receptor activa-
tion, it is critical that the cyclization procedure em-
ployed enforces orientation of the important side chain
elements into the correct regions of the receptor protein.
Access to a variety of cyclization methods that allow for
conformational fine-tuning should therefore be highly
desirable.
Knowledge of the bioactive conformations of biologi-
cally active peptides is invaluable for the understanding
of receptor activation and for the stepwise conversion
of target peptides into less peptidic analogues. Because
of the inherent flexibility of linear peptides in solution
and since preferred solution conformations do not neces-
sarily correspond to those adopted when activating the
receptor, the direct determination of bioactive conforma-
tions is still a formidable endeavor. Thus, in the wait
for 3D structural data of peptide/G-protein coupled
receptor complexes to become generally available, al-
ternative strategies must be explored. Constrained
peptide analogues may provide indirect information
about the topological requirements within the peptide-
receptor complex. The principle of reducing flexibility
and thereby limiting the unfavorable entropy loss upon
binding has been widely used.1 Cyclization is a powerful
tool for imposing conformational constraints, and a
variety of methods have been employed for the prepara-
tion of cyclic peptides.1-3 Cyclizations by disulfide and
amide bond formation are most common, but more
elaborate processes, e.g., ring-closing metathesis,4-10
and the use of thioether,11-15 dithioether,16-21 ureido,22
and saturated aliphatic23 bridges has also been success-
Several cyclic analogues of the hypertensive octapep-
tide angiotensin II (Ang II, 1, Chart 1) have been
prepared.20,24-33 Among these, the monocyclic disulfides
c[Cys3,5]Ang II, c[Cys3Hcy5]Ang II, c[Hcy3Cys5]Ang II,
and c[Hcy3,5]Ang II (2) (Chart 1), with ring sizes from
11 to 13 atoms, all showed high affinity for the AT1
receptor. While the 11- and 12-membered ring ana-
logues exerted less than 2% of the activity of Ang II,
the 13-membered c[Hcy3,5]Ang II (2) was found to be a
full agonist, only 2 times less potent than Ang II itself
(pD2 ) 8.48 versus 8.81 for Ang II).25
We wanted access to alternative monocyclization
methods that would deliver active Ang II analogues
encompassing ring systems with slightly different but
overall similar conformational properties compared to
the disulfides but that are devoid of the redox-sensitive
disulfide bridge. Such constrained and active analogues
would serve as valuable tools in the ongoing32-40 search
for the bioactive conformation of Ang II. We therefore
prepared the 13-membered ring analogues 13 and 14
(Scheme 2) of Ang II, encompassing cis and trans vinyl
sulfide bridges between residues 3 and 5. The cyclization
was also performed between residues 5 and 7 to give
* To whom correspondence should be addressed. Phone: +46 18 471
4284. Fax: +46 18 471 4976. E-mail: Anders.Hallberg@bmc.uu.se.
† Department of Medicinal Chemistry, Uppsala University.
‡ Washington University.
§ Department of Organic Chemistry, Uppsala University.
| Department of Pharmaceutical Biosciences, Uppsala University.
10.1021/jm011063a CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/30/2002

1768 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
J ohannesson et al.
Ch a r t 1
Sch em e 1a
the Ang II analogues 16 and 17 (Scheme 3). The cyclized
vinyl sulfides are attractive targets because they can
also serve as precursors for further modifications. They
were prepared by a novel cyclization reaction using a
protected derivative of the ω-formyl-R-amino acid all-
ysine41 as building block.
a
Reagents: (a) Z-Cl, Na2CO3, H2O/dioxane; (b) paraformalde-
hyde, TsOH, benzene, 67%, over two steps from 3; (c) Me2S‚BH3,
THF; (d) PCC, NaHCO3, CH2Cl2; (e) TsOH, MeOH, 61%, over three
steps from 5; (f) NaOMe, MeOH; (g) H2, Pd/C, EtOH, 62%, over
two steps from 8; (h) (i) KOH(aq), MeOH, (ii) Fmoc-Cl, Na2CO3,
H2O/dioxane, 70%.
Resu lts
1. Syn th esis. The vinyl sulfide synthesis relies on the
reaction of a thiol with the formyl group of allysine. The
synthetic route to the dimethylacetal-protected allysine
derivative 11, which is used as a building block for
peptide synthesis, is outlined in Scheme 1. This syn-
thesis is analogous to the one we previously reported31
for the lower homologue, derived from L-glutamic acid.
The commercially available L-2-aminoadipic acid (L-Aad)
(3) was employed as the starting material. The synthesis
requires diprotection of the nitrogen to avoid the
spontaneous cyclization of the nitrogen onto the alde-
hyde function.42-44 Therefore, the nitrogen was pro-
tected both with a benzyloxycarbonyl (Z) group and with
an oxazolidinone. The latter group was used for the
simultaneous protection of the nitrogen as well as of the
R-carboxyl group.45 The resulting compound 5 was then
ready for the transformation of the side chain carboxyl
function into a formyl group. The conversion of carboxy-
lic acid 5 to aldehyde 7 was performed via reduction
with borane dimethyl sulfide to the alcohol 6 and
subsequent oxidation with PCC. The resulting aldehyde
7 was protected as the dimethyl acetal 8. Compound 8
was converted to the free amine 10 through cleavage of
the oxazolidinone with sodium methoxide in methanol
to give the methyl ester 9, followed by removal of the
benzyloxycarbonyl group by catalytic hydrogenation.
The methyl ester of compound 10 was hydrolyzed with
potassium hydroxide, and the zwitterion formed was
directly treated with Fmoc-Cl and aqueous sodium
carbonate in dioxane to give the Fmoc-protected allysine
derivative 11.
deprotection and cleavage from the resin, cyclization
was achieved and the Ang II analogues 13 and 14, which
encompass a vinyl sulfide bridge between positions 3
and 5, were obtained. The cis (13) and trans (14) isomers
were formed in an approximate ratio of 1:2, and the total
yield after purification by RP-HPLC was 10%.
The precursor peptide 15, which incorporates the
masked allysine in position 5 and a cysteine now in
position 7, was also prepared in order to furnish the 5-7
vinyl sulfide bridged analogues 16 and 17 (Scheme 3).
We were not able to separate the cis (16) and trans (17)
isomers by RP-HPLC, but the ratio cis/ trans was
determined from NMR to be approximately 3:2 and the
total isolated yield was 8%.
2. Str u ctu r a l Ch a r a cter iza tion . Proton NMR sig-
nals of the Ang II analogues 13 and 14 were assigned
from results from primitive exclusive correlation spec-
troscopy (PE-COSY),46 total correlation spectroscopy
(TOCSY),47 and rotating-frame Overhauser enhance-
ment spectroscopy (ROESY)48 as described previ-
ously.30,49 Connectivity between CH2 and S-CHdCH in
the CH2-S-CHdCH- segment was established by
nuclear Overhauser effect (NOE) from Hꢀ of allysine to
Hâ of cysteine. The cis geometry of the vinyl sulfide
double bond (Ang II analogue 13) was established from
the coupling constant 3J Hδ-Hꢀ ) 9.2 Hz, and the trans
geometry (Ang II analogue 14) was established from the
coupling constant 3J Hδ-Hꢀ ) 15 Hz. PE-COSY, TOCSY,
and ROESY spectra were also recorded for the mixture
of Ang II analogues 16 and 17. Comparison with the
spectra from compounds 13 and 14 together with the
The building block 11 was incorporated by solid-phase
peptide synthesis (SPPS) into position 5 of the precursor
peptide 12, which also contains a trityl-protected cys-
teine residue in position 3 (Scheme 2). Upon acidic

Analogues of Angiotensin II
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1769
Sch em e 2a
Sch em e 3a
a
Reagents: (a) (i) His(Trt)-Cys(Trt)-Phe-Wang resin, HBTU,
NMM, DMF, (ii) piperidine, DMF; (b) (i) Fmoc-Tyr(tBu), HBTU,
NMM, DMF, (ii) piperidine, DMF; (c) (i) Fmoc-Val, HBTU, NMM,
DMF, (ii) piperidine, DMF; (d) (i) Fmoc-Arg(Pbf), HBTU, NMM,
DMF, (ii) piperidine, DMF; (e) (i) Fmoc-Asp(OtBu), HBTU, NMM,
DMF, (ii) piperidine, DMF; (f) 95% aqueous TFA.
a
Reagents: (a) (i) His(Trt)-Pro-Phe-Wang resin, 2-(1H-benzo-
triazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU), N-methylmorpholine (NMM), DMF, (ii) piperidine, DMF;
(b) (i) Fmoc-Tyr(tBu), HBTU, NMM, DMF, (ii) piperidine, DMF;
(c) (i) Fmoc-Cys(Trt), HBTU, NMM, DMF, (ii) piperidine, DMF;
(d) (i) Fmoc-Arg(Pbf), HBTU, NMM, DMF, (ii) piperidine, DMF;
(e) (i) Fmoc-Asp(OtBu), HBTU, NMM, DMF, (ii) piperidine, DMF;
(f) 95% aqueous TFA.
octapeptides 13 and 14, respectively (Chart 2). Ac-Ala-
Ala-Ala-NMe (1m ) and Ac-c[Hcy-Ala-Hcy]-NMe (2m ),
which were used as model compounds for Ang II and
for the 13-membered ring analogue c[Hcy3,5]Ang II (2),
were included for comparison. The conformational prop-
erties of 1m and 2m have recently been described.20 The
computational method that was used was the same as
in the present study. We used the Amber force field and
the GB/SA water solvation model50 within Macromodel
(version 6.5),51 and all conformations within 5 kcal/mol
of the lowest energy minimum were studied. In addition
to these conformations, we also analyzed the conforma-
tions between 5 and 10 kcal/mol in order to evaluate
whether new conformational families would appear. The
number of conformations within 5 (10) kcal/mol of the
assignment of certain key signals allowed the identifica-
tion of vinyl sulfides 16 and 17.
3. Con for m a tion a l Ch a r a cter iza tion . 3.a . Th eo-
r etica l Ca lcu la tion s on Mod el Tr ip ep tid es. The aim
of this part of the study was to characterize the new
vinyl sulfide ring systems of Ang II analogues 13 and
14 and to compare them to the ring system of c[Hcy3,5]-
Ang II (2) as well as to a linear peptide such as Ang II.
The conformational analysis was performed on the
blocked tripeptide model compounds 13m and 14m of

1770 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
J ohannesson et al.
constraints of the cyclic compounds. Almost all confor-
mations of the cyclic analogues have X1 values between
0° and 180° and X2 values between 0° and -180°. This
is most probably related to the stereochemistry of the
CR-carbon in residues 3-5.
The cis and trans vinyl sulfide cyclized 13m and 14m
adopt fewer low-energy conformations compared to the
disulfide 2m . However, as deduced from the conforma-
tional analysis, the overall conformational preferences
of the tripeptides are comparable. The topographical
similarity between the vinyl sulfides and the disulfide
is also illustrated in Figure 3, where the lowest energy
conformations of 13m , 14m , and 2m are superimposed.
3.b. Mod elin g of Octa p ep tid es. The conformational
analysis of the agonistic octapeptides 13 and 14 will be
described elsewhere.52 It has been performed using the
previously described buildup strategy35 and the ECEPP/2
force field.53,54 Some of the low-energy conformers that
were found for the octapeptides 13 and 14 display a good
overlap with one of the possible bioactive conformations
of Ang II proposed by Nikiforovich and Marshall ear-
lier.35 This particular bioactive conformation of Ang II
has been supported recently by results obtained with a
series of potent cyclic Ang II agonists.52 The conformers
of compounds 13 (green) and 14 (magenta) with the best
fit to this bioactive conformation of Ang II35 (white) are
shown in Figure 4. As is evident from Figure 4, the
general spatial positions of the pharmacophore side
chains of Tyr4, His6, and Phe8 as well as the C-terminal
carboxyl group overlap very well indeed. The main
difference can be found in the CR-Câ bond vector of the
Tyr4 residue. (Note that the buildup procedure em-
ployed52 does not specify low-energy conformations
within the accuracy required to distinguish side chain
rotamers; however, the particular conformations of
compounds 13 and 14 depicted in Figure 4 are of low
energy.)
4. P h a r m a cology. 4.a . In Vitr o Bin d in g Affin ity.
Compounds 13 and 14 and compounds 16 and 17 were
evaluated in a radioligand binding assay based on
displacement of [125I]Ang II from AT1 receptors in rat
liver membranes55 (Table 1). Ang II, c[Hcy3,5]Ang II, and
the non-peptide AT1 antagonist DuP 753 (Losartan)
were used as reference substances. Analogues 13 and
14 were both found to bind with high affinity, 1.7 nM,
to the AT1 receptor. Compounds 16 and 17, which were
tested as a mixture, did not show any affinity to the
receptor.
4.b. F u n ction a l Da ta . Ang II analogues 13 and 14
were evaluated for possible agonistic properties in a
vascular contractility study using rabbit aorta (Table
1). Both analogues 13 and 14 behaved as full agonists
(Figure 5) and were about 20 and 10 times less active,
respectively, than Ang II itself. For both of the com-
pounds the concentration-response curves were poten-
tiated with a maximum effect more than 15% higher
than Ang II. The agonistic properties of 13 and 14 were
completely blocked upon addition of the non-peptide AT1
antagonist DuP 753. Compounds 16 and 17 were not
tested in the functional assay because they lacked
affinity for the AT1 receptor.
F igu r e 1. Parameters used to characterize the model com-
pounds, here exemplified for model compound 13m .
Ch a r t 2
global energy minimum found for model compounds
13m , 14m , 1m , and 2m , were 36 (139), 22 (109), 20
(148), and 50 (303), respectively. To study whether the
cis or trans vinyl sulfide changed the conformational
preference of the peptide backbone within the cycle, the
Ψ2, Φ3, Ψ3, and Φ4 torsion angles (Figure 1) were
compared. We also investigated the overall effect of the
monocyclizations by analyzing the values of the virtual
torsion angles X1 ) (N2-CR2-CR3-Câ3), X2 ) (Câ3-
CR3-CR4-C(4)O), and X3 ) (N2-CR2-CR4-C(4)O) (see
Figure 1 for notation of the atoms). X1, X2, and X3
describe the relative directions of the incoming back-
bone, the side chain attached to CR3, and the outgoing
backbone with respect to each other.
The results are shown in Figure 2, where the torsion
angles of all conformations within 5 kcal/mol of the
lowest energy minimum are plotted as red triangles and
the conformations between 5 and 10 kcal/mol as blue
circles. The Ψ3, Φ3 and Ψ2, Φ4 plots within 5 and 10
kcal/mol of the lowest energy minimum are very similar
for 13m and 14m . This indicates that the cis and trans
vinyl sulfide groups induce similar backbone conforma-
tional properties. When the same plots are compared
to those of the 13-membered disulfide 2m , there is also
an overall resemblance. When 13m and 14m are
compared to the linear 1m , the picture is also the same
except that conformers possessing dihedral angle values
around Φ3 ) 60° and Φ4 ) 60° seem to be energetically
less favorable for the vinyl sulfides and to some extent
also for the disulfide 2m . It thus appears that for the
13-membered cycles in this study, the backbone torsion
angles within the ring are similar.
The X1-X3 plots within 5 and 10 kcal/mol of the
lowest energy minimum are also similar for 13m and
14m . These plots also resemble those of the disulfide
analogue 2m . However, when the X1, X2, and X3 torsion
angles of the cyclic analogues are compared to those of
the linear 1m , it becomes evident that the linear 1m
can adopt many additional conformers not available to
the cyclic 13m and 14m because of the added ring
Discu ssion
ω-Formyl-R-amino acids have frequently been used
for the construction of bicyclic thiazabicycloalkane

Analogues of Angiotensin II
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1771
F igu r e 2. Scatter plots of torsion angles for all conformations below 5 kcal/mol (red triangles) and between 5 and 10 kcal/mol
(blue circles) for model compounds 1m , 2m , 13m , and 14m .
F igu r e 3. Stereoimage of the rmsd best fit of the lowest energy conformations of 13m and 14m to 2m . N2, CR2, CR3, Câ3, CR4,
and C(4)O were included in the fitting procedure. The rmsd values were 0.15 Å for 13m and 0.17 Å for 14m .
dipeptide units.56-60 The thiazabicycloalkanes are most
often synthesized through reaction of the formyl func-
tion of the ω-formyl-R-amino acid with a neighboring
nitrogen and sulfur atom, followed by intramolecular
N-acylation to give bicyclization and to provide a thia-
zolidine in the final step. We previously reported a
spontaneous bicyclization, which delivered tripeptide
mimetic thiazabicycloalkanes upon deprotection of oc-
tapeptides encompassing masked ω-formyl-R-amino
acids.30,31 The regioselectivity of this cyclization could
be directed toward either the C-terminal or the N-
terminal end of the peptide by simply altering the chain
length of the ω-formyl-R-amino acid from two to three
carbons, as illustrated in Scheme 4. We attributed the
driving force for this regioselectivity to the ready
formation of five-membered rings in favor of other ring
sizes. Robl et al.57 have used the ω-formyl-R-amino acid
allysine coupled to homocysteine to accomplish a similar
bicyclization via a six-membered N-acyliminium ion.
They used this to prepare 7,6-fused thiazabicycloalkane
dipeptide units. In contrast, when precursor peptides
12 and 15 are deprotected, cyclization to nitrogen to

1772 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
J ohannesson et al.
Sch em e 4a
F igu r e 4. Low-energy conformers (relative energy less than
10 kcal/mol) of compounds 13 (green) and 14 (magenta), with
the best fit to the suggested bioactive conformation number
II of Ang II35 (white). Geometrical similarity was assessed by
overlapping the CR and Câ atoms for the Val3-Phe8 fragment
of Ang II. The rmsd values were 1.1 and 1.0 Å for 13 and 14,
respectively. Only fragments 3-8 are depicted. All hydrogens
are omitted for clarity.
a
Previously reported bicyclization procedure.30,31 The regiose-
lectivity can be directed either toward the C-terminal or N-
terminal end of the peptide by altering the chain length of the
incorporated masked ω-formyl-R-amino acid from two to three
carbons.
Ta ble 1. In Vitro Rat Liver AT1 Receptor Binding Affinities
and Agonistic Activities in Vascular Contractility Studies on
Rabbit Aorta
have reported the formation of vinyl sulfides through
elimination-addition of enol triflates that occurs via an
intermediate allene in their synthesis of monocyclic
seven-membered dipeptide units. Besides their work,
the formation of vinyl sulfides as a method for the
cyclization of peptides has, to the best of our knowledge,
not been used previously.
AT1 receptor
binding affinities
agonistic activities
compound
Ki (nM) ( SEM
EC50 (nM) ( SEM
Ang II
0.31 ( 0.08
0.23 ( 0.14
25 ( 4.7
1.7 ( 0.26
1.7 ( 0.32
no affinitya
no affinitya
1.40 ( 0.28
c[Hcy3,5]Ang II
We envisioned that the 13-membered vinyl sulfide
ring systems would exhibit overall similar properties
but with slightly different conformational properties
compared to the corresponding disulfide-based ring
systems. The vinyl sulfides should therefore exhibit the
proper requirements and the potential to be exploited
for conformational fine-tuning of structure activity
relationships (SARs) of cyclic peptide analogues. From
a pharmacokinetic viewpoint it would also be of value
to assess whether compounds containing the vinyl
sulfide ring systems display higher metabolic stability
than the disulfides. Encouraged by the high affinity and
agonistic properties reported for the 13-membered Ang
II analogue c[Hcy3,5]Ang II (2),25 we therefore prepared
Ang II analogues 13 and 14 with the cis and trans vinyl
sulfide bridges between residues 3 and 5. Indeed, the
pharmacological evaluation showed both of these ana-
logues to display high affinities and full agonist proper-
ties at the AT1 receptor. As an additional example of
the cyclization method, Ang II analogues 16 and 17,
cyclized between residues 5 and 7, were prepared. The
5-7 region of Ang II has not been as extensively studied
as the 3-5 region. With the exception of the bicyclic
c[Sar1, Hcy5, Mpc7]Ang II, which was shown to be a
weak partial agonist with 10 times lower affinity than
Ang II,29 cyclization between residues 5 and 7 has only
rendered inactive compounds.29-31 In line with this, the
5-7 cyclized vinyl sulfides 16 and 17 showed no
affinities for the rat AT1 receptor.
DuP 753
13
14
16
17
26.0 ( 6.79
16.1 ( 3.52
a
Compounds 16 and 17 were tested for binding as a mixture
(approximate ratio 3:2). This mixture did not show any affinity
below 1 µM.
F igu r e 5. Cumulative concentration-response curves for the
contractile effects of Ang II (b), 13 (2), and 14 (9) in isolated
rabbit aorta strips. Values represent the mean ( SEM (n )
11 for Ang II; n ) 4 for 13 and 14).
We were initially surprised that the conformational
differences between the cis and trans vinyl sulfide ring
systems were so small, as is shown in the conforma-
tional analysis on the tripeptide model compounds.
These similarities are, however, further supported by
the modeling of octapeptides 13 and 14 as well as by
their similar pharmacological profiles. The very similar
binding affinities of the cis and trans vinyl sulfide
generate a six-membered ring does not seem to occur,
but instead, the formyl function cyclizes to the sulfur
atom only. We speculate that the reaction occurs via the
formation of hemithioacetal-like compounds that un-
dergo elimination of water to form both the monocyclic
cis and trans vinyl sulfide compounds. Crescenza et al.61

Analogues of Angiotensin II
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1773
Centre, Uppsala, Sweden, on 24 h hydrolyzates with an LKB
4151 alpha plus analyzer, using ninhydrin detection.
cyclized octapeptides 13 and 14 indicate that there are
no specific interactions between the ring-closed loops
and the receptor.
Solid -P h a se P ep tid e Syn th esis (SP P S). The peptides
were synthesized on an 80 µmol scale with a Symphony
instrument (Protein Technologies, Inc., Tucson, AZ) using
Fmoc/tert-butyl protection. The starting polymer was Fmoc-
Phe-Wang resin (0.51 mmol/g), and the side chain protect-
ing groups were as follows: Asp(OtBu), Arg(Pbf), Cys(Trt), Tyr-
(tBu), and His(Trt). The Fmoc group was removed by 20%
piperidine/DMF using a two-step treatment, 5 + 10 min.
Coupling of the amino acids (125 µmol) was done in DMF (2.5
mL) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HBTU) (125 µmol) in the presence
of N-methylmorpholine (NMM) (0.5 mmol). Double couplings
(2 × 30 min) were used except for the introduction of compound
11 (single coupling, 1 h). At the end of each coupling cycle,
the remaining amino groups were capped by addition of 20%
acetic anhydride/DMF (1.25 mL) to the coupling mixture and
allowing the reaction to proceed for 5 min. After completion
of the synthesis, the Fmoc group was removed and the
partially protected peptide resin was washed with several
portions of DMF and CH2Cl2 and dried in a stream of nitrogen
and in vacuo. Yields for the purified Ang II analogues were
corrected for peptide content.
Initially, we also expected that the vinyl sulfides
would restrict the conformational freedom to a further
extent than is suggested by the conformational analysis.
The expected overall topographical similarities of the
cis and trans vinyl sulfide ring systems to the corre-
sponding 13-membered disulfide ring system in c[Hcy3,5]-
Ang II were, however, confirmed. Hence, it appears that
vinyl sulfides can be considered as methylene disulfide
bioisosteres, at least in 13-membered cyclized peptide
systems. To determine whether vinyl sulfides provide
proper surrogates for methylene disulfides in other ring
systems, further conformational analyses are required.
Both the agonistic octapeptides 13 and 14 can present
the previously proposed bioactive conformation of Ang
II,35 as is shown in Figure 4. Note that the absolute
orientation of the aromatic rings of the pharmacophore
groups that is displayed in this figure is not confirmed
by experimental data. One of the possible ways to
address this issue is by the use of analogues with the
conformationally restricted side chains, as the â-methyl-
Phe62 and â-methyl-Tyr. The two new Ang II agonists
13 and 14 have also served as valuable tools in compu-
tational modeling experiments. This has resulted in a
refined proposed model of the bioactive conformation of
Ang II.52
L-4-(3-(Ben zyloxyca r bon yl)-5-oxo-1,3-oxa zolid in -4-yl)-
bu ta n oic Acid (5).63-65 Commercially available L-2-aminoa-
dipic acid (L-Aad) (3) (7.00 g, 43.4 mmol) was dissolved in a
mixture of 10% aqueous Na2CO3 (200 mL) and dioxane (200
mL) and cooled to 0 °C. Benzylchloroformate (Z-Cl) (95%) (7.2
mL, 48 mmol) was added dropwise over 10 min, and 10%
aqueous Na2CO3 was added until pH 9 was attained. The
reaction mixture was stirred at 0 °C for 1 h and thereafter
was washed with diethyl ether (3 × 400 mL). Diethyl ether
(400 mL) was added to the water phase, which was then
acidified with 1 M aqueous HCl until pH 2 was attained. The
phases were separated, and the water phase was further
extracted with diethyl ether (3 × 400 mL). The combined
organic layers (4 × 400 mL) were dried (MgSO4) and evapo-
rated to give crude Z-L-Aad (4)66,67 as white crystals (12.1 g,
40.9 mmol). Crude Z-L-Aad (4) (6.00 g, 20.3 mmol) was
dissolved in benzene (200 mL), and paraformaldehyde (95%)
(1.22 g, 38.6 mmol) and p-toluenesulfonic acid (monohydrate,
98.5%) (0.230 g, 1.19 mmol) were added. The reaction mixture
was refluxed with azeotropic removal of water, using a Dean-
Stark apparatus, for 18 h. Most of the benzene was evaporated,
and the residue was dissolved in EtOAc (200 mL) and washed
with saturated aqueous NaHCO3 (10 mL). The organic phase
was dried (MgSO4) and evaporated. The residue was purified
by flash column chromatography (gradient system, CH2Cl2 to
2% MeOH in CH2Cl2) to give product 5 (4.45 g, 67% over two
steps, from compound 3), as an oil: [R]D +84.2° (c 1.0, CHCl3);
1H NMR (CDCl3) δ 1.56-2.18 (m, 4H, CH2CH2), 2.29-2.47 (m,
Con clu sion
In summary a new 1-3 cyclization method that
delivers cis and trans vinyl sulfides and that provides
a complement to the common disulfide cyclizations has
been developed. The 13-membered ring systems that are
formed adopt low-energy conformations very similar to
those from the 1-3 disulfide cyclizations with homocys-
teine residues, as deduced from conformational analysis.
Incorporation of the 13-membered vinyl sulfide ring
systems into Ang II produced agonists that were almost
as potent as c[Hcy3,5]Ang II. Thus, the vinyl sulfide
cyclization should serve as a valuable tool in the fine-
tuning of models of bioactive conformations. Although
only two examples of 1-3 vinyl sulfide cyclizations are
given herein, we believe that the method should be
applicable to the cyclization of other short target pep-
tides. The fact that the double bond of vinyl sulfides can
in general be functionalized provides a special advan-
tage of the cyclization procedure.
2H, CH2), 4.30-4.40 (m, 1H, CH), 5.13-5.27 (m, 3H, OCH2a
N
and OCH2Ar), 5.55 (br s, 1H, OCH2bN), 7.34-7.41 (m, 5H, Ar);
13C NMR (CDCl3) δ 19.4, 29.9, 33.1 (CH2CH2CH2), 54.5 (CH),
68.0 (OCH2Ar), 77.9 (OCH2N), 128.2, 128.5, 128.6 (CH Ar),
135.1 (ipso Ar), 152.9 (CO Z), 172.0, 178.5 (C-1, C-5); IR (neat)
3200, 1802, 1724. Anal. (C15H17NO6) C, H, N.
Exp er im en ta l Section
Ch em istr y. Gen er a l Com m en ts. 1H and 13C NMR spectra
were recorded on a J EOL J NM-EX270 at 270 (67.8) MHz or
on a J EOL J NM-EX400 or a Varian Unity 400 spectrometer
at 400 (100.6) MHz. Spectra were recorded at ambient tem-
perature unless otherwise noted. Chemical shifts are reported
as δ values (ppm) referenced to Me4Si. IR spectra were
recorded on a Perkin-Elmer model 1605 FT-IR instrument and
are reported as vmax (cm-1). Optical rotations were measured
at ambient temperature on a Perkin-Elmer model 241 pola-
rimeter. Elemental analyses were performed by Mikro Kemi
AB, Uppsala, Sweden. Flash column chromatography was done
using Riedel-de Hae¨n silica gel S (32-63µm). Mass spectros-
copy was carried out on an Applied Biosystems (Uppsala,
Sweden) BIOION 20 plasma desorption mass spectrometer.
Amino acid analyses and peptide content determinations were
performed at the Department of Biochemistry, Biomedical
L-3-(Ben zyloxyca r bon yl)-4-(4-h yd r oxybu tyl)-1,3-oxa zo-
lid in -5-on e (6).63 Compound 5 (4.11 g, 13.4 mmol) was
dissolved in dry THF (160 mL) under N2 atmosphere and was
cooled to 0 °C. Borane-methyl sulfide complex (2.0 M in THF,
7.0 mL, 14 mmol) was added dropwise over 15 min, whereafter
the reaction mixture was slowly allowed to reach room
temperature and was then stirred for 18 h. Concentration
afforded crude 6 (4.31 g) as a white foam that could be used
in the next step without further purification. An analytical
sample was prepared by purification by flash column chroma-
tography (gradient system, CH2Cl2 to 1% MeOH in CH2Cl2):
1
[R]D +86.3° (c 1.0, CHCl3); H NMR (CDCl3) δ 1.23-1.75 (m,
5H, CH2CH2 and OH), 1.82-2.17 (m, 2H, CH2), 3.61 (br s, 2H,
CH2OH), 4.25-4.40 (m, 1H, CH), 5.12-5.28 (m, 3H, OCH2a
N
and OCH2Ar), 5.51 (br s, 1H, OCH2bN), 7.34-7.41 (m, 5H, Ar);
13C NMR (CDCl3) δ 20.4, 30.2, 31.8 (CH2CH2CH2), 54.7 (CH),

1774 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
J ohannesson et al.
61.9 (CH2OH), 67.7 (OCH2Ar), 77.8 (OCH2N), 128.1, 128.4,
128.5 (CH Ar), 135.2 (ipso Ar), 152.7 (CO Z), 172.3 (C-5); IR
(neat) 3448, 1798, 1715 cm-1. Anal. (C15H19NO5) C, H, N.
residue was purified by flash column chromatography (gradi-
ent system, CH2Cl2 to 3.5% MeOH in CH2Cl2) to give the
product 10 (1.38 g, 62% over two steps, from compound 8) as
1
a colorless oil: [R]D +16.4° (c 1.0, CHCl3); H NMR (CDCl3) δ
L-4-(3-(Ben zyloxyca r bon yl)-5-oxo-1,3-oxa zolid in -4-yl)-
bu ta n a l (7).65 To a solution of alcohol 6 (860 mg, 2.93 mmol)
in CH2Cl2, Celite (1.80 g), PCC (1.84 g, 8.37 mmol), and solid
NaHCO3 (0.29 g, 3.45 mmol) were added. The reaction mixture
was stirred at room temperature for 5 h and thereafter filtered
through a plug of SiO2, using EtOAc/petroleum diethyl ether
1:1 as eluent. Concentration afforded crude aldehyde 7 (660
mg, 2.27 mmol) as a colorless oil that could be used in the next
step without further purification. An analytical sample was
prepared by purification by flash column chromatography
(gradient system, CH2Cl2 to 2% MeOH in CH2Cl2): [R]D +94.0°
(c 1.0, CHCl3); 1H NMR (CDCl3) δ 1.54-2.17 (m, 4H, CH2CH2),
2.38-2.59 (m, 2H, CH2), 4.30-4.40 (m, 1H, CH), 5.13-5.30
(m, 3H, OCH2aN and OCH2Ar), 5.55 (br s, 1H, OCH2bN), 7.34-
7.42 (m, 5H, Ar), 9.72 (br s, 1H, CHO); 13C NMR (CDCl3) δ
16.7, 29.7, 42.8 (CH2CH2CH2), 54.4 (CH), 67.8 (OCH2Ar), 77.8
(OCH2N), 128.1, 128.4, 128.5 (CH Ar), 135.1 (ipso Ar), 152.7
(CO Z), 171.9 (C-5), 201.0 (CHO); IR (neat) 2728, 1799, 1715
cm-1. Anal. (C15H17NO5) C, H, N.
L-3-(Ben zyloxyca r b on yl)-4-(4,4-d im et h oxyb u t yl)-1,3-
oxa zolid in -5-on e (8). Aldehyde 7 (460 mg, 1.58 mmol) and
p-toluenesulfonic acid (monohydrate, 98.5%) (15 mg, 78 µmol)
were dissolved in MeOH (30 mL). After the mixture was stirred
at room temperature for 2.5 h, most of the solvent was
evaporated and the residue was partitioned between EtOAc
(50 mL) and saturated aqueous NaHCO3 (20 mL). The organic
phase was washed with brine (20 mL), dried (MgSO4), and
concentrated. Purification by flash column chromatography
(gradient system, CH2Cl2 to 2% MeOH in CH2Cl2) gave the
product 8 as a colorless oil (383 mg, 1.14 mmol, 61% over
three steps, from compound 5): [R]D +81.4° (c 1.0, CHCl3); 1H
NMR (CDCl3) δ 1.22-1.70 (m, 4H, CH2CH2), 1.80-2.13 (m,
2H, CH2), 3.29 (s, 6H, CH(OCH3)2), 4.24-4.38 (m, 2H, CH and
CH(OCH3)2), 5.13-5.27 (m, 3H, OCH2aN and OCH2Ar), 5.54
(br s, 1H, OCH2bN), 7.32-7.40 (m, 5H, Ar); 13C NMR (CDCl3)
δ 19.3, 30.3, 31.9 (CH2CH2CH2), 52.6, 52.8 (CH(OCH3)2), 54.7
(CH), 67.8 (OCH2Ar), 77.8 (OCH2N), 103.9 (CH(OCH3)2), 128.2,
128.5, 128.6 (CH Ar), 135.2 (ipso Ar), 152.8 (CO Z), 172.1 (C-
5); IR (neat) 1802, 1716 cm-1. Anal. (C17H23NO6) C, H, N.
1.36-1.82 (m, 8H, CH2CH2CH2 and NH2), 3.30 (s, 6H, CH-
(OCH3)2), 3.44 (dd, J ) 5.4, 7.3 Hz, 1H, H-2), 3.72 (s, 3H,
COOCH3), 4.35 (dd, J ) 5.4, 5.4 Hz, 1H, H-6); 13C NMR (CDCl3)
δ 20.6, 32.0, 34.5 (CH2CH2CH2), 51.8 (COOCH3), 52.6 (2C, CH-
(OCH3)2), 54.2 (C-2), 104.1 (C-6), 176.3 (C-1); IR (neat) 3375,
1732 cm-1. Anal. (C9H19NO4) C, H, N.
L -2 -((9 -F l u o r e n y l m e t h o x y c a r b o n y l )a m i n o )-6 ,6 -
d im eth oxyh exa n oic Acid (11). To compound 10 (100 mg,
487 µmol) dissolved in MeOH (6 mL) was added 1 M aqueous
KOH (490 µL, 490 µmol), and the mixture was stirred at room
temperature for 15 h. Then 10% aqueous citric acid was added
until pH 6-7 was attained, and the reaction mixture was
concentrated to give a solid residue. This residue was dissolved
in a mixture of 10% aqueous Na2CO3 (10 mL) and dioxane (5
mL) and cooled to 0 °C. Fmoc-Cl (97%) (190 mg, 712 µmol)
dissolved in dioxane (5 mL) was added dropwise, whereafter
the reaction mixture was allowed to reach room temperature.
The pH was kept around 10-11. Stirring at room temperature
was continued for 48 h. Then 10% aqueous citric acid was
added until pH 8 was attained, and the reaction mixture was
washed with diethyl ether (4 × 50 mL). Diethyl ether (150
mL) was added to the water phase, which was then acidified
to pH 3 with 10% aqueous citric acid, under vigorous stirring.
The phases were separated, and the water phase was further
extracted with diethyl ether (2 × 150 mL). The combined
organic layers (3 × 150 mL) were washed with water (2 × 200
mL), dried (MgSO4), and concentrated to give the building
block 11 (141 mg, 70%) as a white foam: [R]D +8.7° (c 0.38,
1
CHCl3); H NMR (CDCl3) δ 1.35-2.01 (m, 6H, CH2CH2CH2),
3.32 (s, 6H, CH(OCH3)2), 4.23 (dd, J ) 6.9, 6.9 Hz, 1H, CH
Fmoc), 4.35-4.53 (m, 4H, H-2, H-6 and CH2 Fmoc), 5.42 (br
d, J ) 8.1 Hz, 1H, NH), 7.32 (m, 2H, Ar Fmoc), 7.41 (m, 2H,
Ar Fmoc), 7.58-7.63 (m, 2H, Ar Fmoc), 7.77 (m, 2H, Ar Fmoc);
13C NMR (CDCl3) δ 20.3, 31.8, 31.9 (CH2CH2CH2), 47.0 (CH
Fmoc), 52.6, 52.7 (CH(OCH3)2), 53.5 (C-2), 66.9 (CH2 Fmoc),
104.1 (C-6), 119.9, 125.0, 127.0, 127.6 (CH Ar Fmoc), 141.2,
143.6, 143.7 (ipso Fmoc), 156.1 (CO Z), 176.0 (C-1); IR (neat)
3320, 1715. Anal. (C23H27NO6‚1.5H2O) C, H, N.
L-2-(Ben zyloxyca r bon yla m in o)-6,6-d im eth oxyh exa n o-
ic Acid Meth yl Ester (9). Dimethylacetal 8 (4.70 g, 13.9
mmol) was dissolved in dry MeOH (300 mL) under N2
atmosphere and cooled to -12 °C. Sodium methoxide (95%)
(790 mg, 13.9 mmol) was suspended in MeOH (300 mL) and
added dropwise over 1 h, whereafter the reaction mixture was
slowly allowed to reach room temperature. After the mixture
was stirred for 6 h, 10% aqueous citric acid was added until
pH 7 was attained and then half of the solvent was evaporated.
The residue was poured into EtOAc (400 mL) and washed with
20% aqueous NaCl (2 × 250 mL). The water phases were
further extracted with EtOAc (2 × 250 mL). The combined
organic layers were dried (MgSO4) and concentrated to give 9
as a colorless oil (4.23 g, 12.5 mmol). Compound 9 was used
in the next step without further purification. An analytical
sample was prepared through purification by flash column
chromatography (gradient system, CH2Cl2 to 1.5% MeOH in
An g II An a logu es 13 a n d 14. Automated SPPS according
to the general procedure, starting with 159 mg (81.1 µmol)
Fmoc-Phe-Wang resin, produced 285 mg of the partially
protected peptide polymer (weight increase corresponding to
97% yield). Part of the resin (256 mg, 72.0 µmol) was cleaved
and deprotected with 95% aqueous TFA (4 mL) for 1.5 h. The
resin was filtered off and washed with 5% triethylsilane/TFA
(3 × 400 µL). An additional portion of triethylsilane (50 µL)
was added in order to decolorize the filtrate. After 20 min the
peptide was precipitated with cold diethyl ether (40 mL),
collected by centrifugation, washed with diethyl ether (4 × 15
mL), and dried to yield 81.0 mg. The crude product was divided
into five aliquots, each dissolved in 0.1% aqueous TFA
containing 7% MeCN (4.3 mL), and chromatographed on a
Vydac 10 µm C18 column (1 cm × 25 cm) using a 60 min
gradient of 15-45% MeCN in 0.1% aqueous TFA at a flow rate
of 3 mL/min. The separation was monitored at 230 nm and by
plasma desorption mass spectrometry (PDMS). The fractions
corresponding to the two major peaks both contained material
of the expected mass. 13: 2.6 mg (3.4%); PDMS (MW 1046.1)
1047.9 (M + H+); amino acid analysis Asp, 1.02; Arg, 0.96;
Tyr, 0.97; His, 1.01; Pro, 1.02; Phe, 1.01; 1H NMR (DMSO-d6,
30 °C, 400 MHz) δ 1.46 (m, 2H, Hγ′/ Hγ-Arg), 1.49 (m, 1H,
Hâ′-Arg), 1.58 (m, 1H, Hâ-Arg), 1.66 (m, 1H, Hâ′-allysine), 1.77
(m, 1H, Hâ-allysine), 1.79 (m, 1H, Hâ′-Pro), 1.81 (m, 2H, Hγ′/
Hγ-Pro), 1.89 (m, 1H, Hγ′-allysine), 2.04 (m, 1H, Hâ-Pro), 2.21
(m, 1H, Hγ-allysine), 2.64 (m, 1H, Hâ′-Tyr), 2.66 (m, 1H, Hâ′-
Asp), 2.82 (m, 1H, Hâ-Asp), 2.86 (m, 1H, Hâ′-His), 2.88 (m,
1H, Hâ-Tyr), 2.90 (m, 2H, Hâ/Hâ′-Cys), 2.94 (m, 1H, Hâ′-Phe),
3.03 (m, 1H, Hâ-His), 3.04 (m, 1H, Hâ-Phe), 3.09 (m, 2H, Hδ/
Hδ′-Arg), 3.45 (m, 1H, Hδ′-Pro), 3.56 (m, 1H, Hδ-Pro), 4.11
(m, 1H, HR-Asp), 4.25 (m, 1H, HR-allysine), 4.33 (m, 1H, HR-
1
CH2Cl2): [R]D +7.8° (c 1.0, CHCl3); H NMR (CDCl3) δ 1.25-
1.45 (m, 2H, CH2), 1.50-1.92 (m, 4H, CH2 and CH2), 3.29 (s,
6H, CH(OCH3)2), 3.73 (s, 3H, COOCH3), 4.32 (dd, J ) 5.7, 5.7
Hz, 1H, H-6), 4.33-4.43 (m, 1H, H-2), 5.10 (s, 2H, OCH2Ar),
5.39 (br d, J ) 8.1 Hz, 1H, NH), 7.28-7.40 (m, 5H, Ar); 13C
NMR (CDCl3) δ 20.2, 31.7, 32.1 (CH2CH2CH2), 52.1 (COOCH3),
52.5, 52.6 (CH(OCH3)2), 54.2 (C-2), 66.7 (OCH2Ar), 103.9 (C-
6), 127.9, 128.0, 128.3 (CH Ar), 136.1, (ipso Ar), 155.7 (CO Z),
172.7 (C-1); IR (neat) 3375, 1732 cm-1. Anal. (C17H25NO6) C,
H, N.
L-2-Am in o-6,6-d im eth oxyh exa n oic Acid Meth yl Ester
(10).57,68-72 Compound 9 (3.32 g, 9.78 mmol) and 10% Pd/C (550
mg, 517 µmol) were mixed in absolute EtOH (170 mL) and
stirred under H2 (1 atm) at room temperature for 2.5 h. The
mixture was filtered through Celite and concentrated. The

Analogues of Angiotensin II
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1775
Arg), 4.38 (m, 1H, HR-Pro), 4.42 (m, 1H, HR-Phe), 4.51 (m,
1H, HR-Cys), 4.51 (m, 1H, HR-Tyr), 4.76 (m, 1H, HR-His), 5.46
(m, 1H, Hδ-allysine), 5.89 (d, J ) 9.2 Hz, 1H, Hꢀ-allysine),
6.61 (m, 2H, meta-Tyr), 6.96 (m, 2H, ortho-Tyr), 7.17-7.27 (m,
5H, Phe), 7.32 (m, 1H, H4-His), 7.63 (d, J ) 7.3 Hz, 1H, NH-
allysine), 7.64 (m, 1H, NHꢀ-Arg), 7.97 (d, J ) 7.7 Hz, 1H, NH-
His), 8.02 (d, J ) 7.3 Hz, 1H, NH-Cys), 8.28 (d, J ) 7.6 Hz,
1H, NH-Phe), 8.52 (d, J ) 8.9 Hz, 1H, NH-Tyr), 8.58 (d, J )
7.8 Hz, 1H, NH-Arg), 8.85 (m, 1H, H2-His). 14: 4.9 mg (6.5%);
PDMS 1047.2 (M + H+); amino acid analysis Asp, 1.04; Arg,
1.01; Tyr, 0.98; His, 1.00; Pro, 0.98; Phe, 0.99; 1H NMR
(DMSO-d6, 30 °C, 400 MHz) δ 1.47 (m, 3H, Hâ′-Arg, Hγ′/ Hγ-
Arg), 1.58 (m, 2H, Hâ/ Hâ′-allysine), 1.59 (m, 1H, Hâ-Arg), 1.78
(m, 3H, Hâ′-Pro, Hγ′/ Hγ-Pro), 2.02 (m, 1H, Hâ-Pro), 2.08 (m,
2H, Hγ′/ Hγ-allysine), 2.64 (m, 1H, Hâ′-Tyr), 2.66 (m, 1H, Hâ′-
Asp), 2.82 (m, 1H, Hâ-Asp), 2.84 (m, 1H, Hâ′-Cys), 2.86 (m,
1H, Hâ-Tyr), 2.88 (m, 1H, Hâ′-His), 2.90 (m, 1H, Hâ-Cys), 2.93
(m, 1H, Hâ′-Phe), 3.03 (m, 1H, Hâ-Phe), 3.04 (m, 1H, Hâ-His),
3.07 (m, 2H, Hδ/ Hδ′-Arg), 3.48 (m, 1H, Hδ′-Pro), 3.57 (m, 1H,
Hδ-Pro), 4.11 (m, 1H, HR-Asp), 4.24 (m, 1H, HR-allysine), 4.33
(m, 1H, HR-Arg), 4.39 (m, 1H, HR-Pro), 4.43 (m, 1H, HR-Phe),
4.44 (m, 1H, HR-Cys), 4.48 (m, 1H, HR-Tyr), 4.75 (m, 1H, HR-
His), 5.41 (ddd, J ) 7.3, 7.3, 15.0 Hz, 1H, Hδ-allysine), 5.75
(d, J ) 15.0 Hz, 1H, Hꢀ-allysine), 6.60 (m, 2H, meta-Tyr), 6.97
(m, 2H, ortho-Tyr), 7.17-7.27 (m, 5H, Phe), 7.33 (m, 1H, H4-
His), 7.64 (m, 1H, NHꢀ-Arg), 7.64 (d, J ) 7.8 Hz, 1H, NH-
allysine), 7.93 (d, J ) 7.4 Hz, 1H, NH-Cys), 8.16 (d, J ) 7.7
Hz, 1H, NH-His), 8.27 (d, J ) 7.7 Hz, 1H, NH-Phe), 8.58 (d, J
) 7.7 Hz, 1H, NH-Arg), 8.66 (d, J ) 8.6 Hz, 1H, NH-Tyr), 8.87
(m, 1H, H2-His).
mounted in water-jacketed organ baths (3 mL) containing
Kreb’s bicarbonate buffer, maintained at 38 °C and oxygenated
with a gas mixture of 93.5% O2 and 6.5% CO2. One end of the
aorta strips was anchored to a stationary support; the other
end was connected to an isometric force transducer (Radnoti
Glass Technology, Inc.), and the isometric contractions were
recorded on an ink-writing recorder. The aorta strips were
stretched stepwise to a resting tension of 2 g, which was
maintained throughout the experiment, and were then allowed
to equilibrate for 60 min. After equilibration a control cumula-
tive concentration-contractile response curve for Ang II (3 ×
10-10 to 10-7 M) was recorded. Thereafter, the aorta strips were
repeatedly washed and allowed to return to baseline tension.
When stabilized, the contractile response of the test com-
pounds was recorded at concentrations of 3 × 10-10 to 10-6
M
to produce a cumulative concentration-response curve in
analogy with that of Ang II. The results of the experiments
are expressed as a percentage of maximum control contractile
force obtained from the first cumulative concentration-
response curve for Ang II.
Con for m a tion a l En er gy Ca lcu la tion s of Mod el Tr ip -
ep tid es. The calculations of 13m and 14m were performed
using the Amber* all atom force field as implemented in the
program Macromodel 6.5.51 The general Born solvent-acces-
sible surface area (GB/SA) method for water developed by
Still50 was used in all calculations. The number of torsion
angles allowed to vary simultaneously during each Monte
Carlo step ranged from 1 to n - 1, where n equals the total
number of rotatable bonds (n ) 8 for 13m and 14m ). Amide
bonds were fixed in the trans configuration. Conformational
searches were conducted by use of the systematic unbound
multiple minimum search (SUMM) method73 in the batchmin
program (command SPMC); 20 000-step runs were performed,
and those conformations within 50 kJ /mol of the global
minimum were kept. The ring-closure bond was defined as the
bond between the Câ and Cγ atoms of the side chain of allysine.
Torsional memory and geometrical preoptimization were used.
PR conjugate gradient (PRCG) minimization with a maximum
of 5000 iterations was used in the conformational search with
the derivative convergence set to 0.05 (kJ /mol)/Å. In the
subsequent minimization to fully converged structures, a
maximum of 5000 steps of PRCG minimization was followed
by a maximum of 5000 steps of TNCG minimization with the
convergence criteria set to 0.001 (kJ /mol)/Å in both runs.
An g II An a logu es 16 a n d 17. The partially protected
peptide resin was recovered in 98% yield according to the
measured increase in mass. Part of the resin (281 mg, 79.1
µmol) was cleaved and deprotected as described for 13 and 14
to yield 88.3 mg of the crude product. The peptide was divided
into five aliquots and purified as above. The peaks correspond-
ing to products 16 and 17 were not resolved, and their fractions
were therefore collected together. (16 + 17): 7.0 mg (8.4%);
PDMS (MW 1048.0) 1049.8 (M + H+); amino acid analysis Asp,
1.03; Arg, 0.99; Val, 0.98; Tyr, 0.97; His, 1.02; Phe, 1.00. 1H
NMR of the mixture of 16 + 17 indicated the formation of vinyl
sulfides by analogy with 13 + 14, by observation of the
following signals. 1H NMR (DMSO-d6, 30 °C, 400 MHz) δ 5.34
(ddd, J ) 7.1, 7.1, 15.1 Hz, 1H, Hδ-allysine, trans isomer), 5.55
(m, 1H, Hδ-allysine, cis isomer), 5.97 (d, J ) 15.1 Hz, 1H, Hꢀ-
allysine, trans isomer), 5.97 (d, J ) 9.2 Hz, 1H, Hꢀ-allysine,
cis isomer); cis/trans ratio ) 63:37.
Ack n ow led gm en t. We thank the Swedish Founda-
tion for Strategic Research for financial support.
Ra t Liver Mem br a n e AT1-Recep tor Bin d in g Assa y. Rat
liver membranes were prepared according to the method of
Dudley et al.55 Binding of [125I]Ang II to membranes was
conducted in a final volume of 0.5 mL of 50 mM Tris-HCl (pH
7.4), supplemented with 100 mM NaCl, 10 mM MgCl2, 1 mM
EDTA, 0.025% bacitracin, and 0.2% BSA and containing rat
liver homogenate corresponding to 5 mg of the original tissue
weight, [125I]Ang II (0.036 nM), and variable concentrations
of the test substance. Samples were incubated at 25 °C for 1
h, and binding was terminated by filtration through Whatman
GF/B glass-fiber filter sheets, using a Brandel cell harvester.
The filters were washed with 4 × 2 mL of Tris-HCl (pH 7.4)
and transferred to tubes. The radioactivity was measured in
a γ counter. Nonspecific binding was determined in the
presence of 1 µM Ang II. All experiments were performed in
triplicate except for Ang II, which was performed in quadru-
plicate. Ki values were calculated using the Cheng-Prusoff
equation (Kd ) 1.1 ( 0.08 nM, [L] ) 0.036 nM).
F u n ction a l Assa y, Va scu la r Con tr a ctility Stu d ies on
Ra bbit Aor ta . Male New Zealand white rabbits weighing
2.5-3.5 kg were killed by a blow to the head. The thoracic
aorta was excised immediately and placed in 38 °C oxygenated
Kreb’s bicarbonate buffer of the following composition: NaCl
120 mM, KCl 4.75 mM, CaCl2 2.54 mM, MgSO4 1.2 mM, KH2-
PO4 1.19 mM, NaHCO3 25 mM, and D-(+)-glucose 11 mM. The
aorta was rinsed of blood, and the connective tissue was
removed before the aorta was cut into 3 mm segments and
Refer en ces
(1) Hruby, V. J . Conformational Restrictions of Biologically Active
Peptides via Amino Acid Side Chain Groups. Life Sci. 1982, 31,
189-199.
(2) Toniolo, C. Conformationally Restricted Peptides through Short-
Range Cyclizations. Int. J . Pept. Protein Res. 1990, 35, 287-
300.
(3) Lambert, J . N.; Mitchell, J . P.; Roberts, K. D. The Synthesis of
Cyclic Peptides. J . Chem. Soc., Perkin Trans. 1 2001, 471-484.
(4) Miller, S. J .; Blackwell, H. E.; Grubbs, R. H. Application of Ring-
Closing Metathesis to the Synthesis of Rigidified Amino Acids
and Peptides. J . Am. Chem. Soc. 1996, 118, 9606-9614 and
references therein.
(5) Ripka, A. S.; Bohacek, R. S.; Rich, D. H. Synthesis of Novel Cyclic
Protease Inhibitors Using Grubbs Olefin Metathesis. Bioorg.
Med. Chem. Lett. 1998, 8, 357-360.
(6) Piscopio, A. D.; Miller, J . F.; Koch, K. Ring Closing Metathesis
in Organic Synthesis: Evolution of a High Speed, Solid Phase
Method for the Preparation of â-Turn Mimetics. Tetrahedron
1999, 55, 8189-8198.
(7) Fink, B. E.; Kym, P. R.; Katzenellenbogen, J . A. Design,
Synthesis, and Conformational Analysis of a Proposed Type I
â-Turn Mimic. J . Am. Chem. Soc. 1998, 120, 4334-4344.
(8) Reichwein, J . F.; Versluis, C.; Liskamp, R. M. J . Synthesis of
Cyclic Peptides by Ring-Closing Metathesis. J . Org. Chem. 2000,
65, 6187-6195 and references therein.
(9) Prabhakaran, E. N.; Rajesh, V.; Dubey, S.; Iqbal, J . Synthesis
of Cyclic Peptides as Mimics for the Constrained Conformation
of Structural Analogues of HIV Protease Inhibitors. Tetrahedron
Lett. 2001, 42, 339-342.

1776 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
J ohannesson et al.
(10) Creighton, C. J .; Reitz, A. B. Synthesis of an Eight-Membered
Cyclic Pseudo-Dipeptide Using Ring Closing Metathesis. Org.
Lett. 2001, 3, 893-895.
(11) Rudinger, J .; J osˇt, K. A Biologically Active Analogue of Oxytocin
Not Containing a Disulfide Group. Experientia 1964, 20, 570-
571.
(12) Yu, L.; Lai, Y.; Wade, J . V.; Coutts, S. M. A Simple and Efficient
Method for the Syntheses of Thioether Cyclic Peptides. Tetra-
hedron Lett. 1998, 39, 6633-6636 and references therein.
(13) Feng, Y.; Pattarawarapan, M.; Wang, Z.; Burgess, K. Solid-Phase
SN2 Macrocyclization Reactions To Form â-Turn Mimics. Org.
Lett. 1999, 1, 121-124.
(14) Li, H.; J iang, X.; Howell, S. B.; Goodman, M. Synthesis,
Conformational Analysis and Bioactivity of Lan-7, a Lanthionine
Analog of TT-232. J . Pept. Sci. 2000, 6, 26-35.
(15) Li, H.; J iang, X.; Goodman, M. Synthesis, Conformational
Analysis and Biological Activities of Lanthionine Analogs of a
Cell Adhesion Modulator. J . Pept. Sci. 2001, 7, 82-91.
(16) Mosberg, H. I.; Omnaas, J . R. Dithioether-Containing Cyclic
Peptides. J . Am. Chem. Soc. 1985, 107, 2986-2987.
(17) Szewczuk, Z.; Rebholz, K. L.; Rich, D. H. Synthesis and Biological
Activity of New Conformationally Restricted Analogues of Pep-
statin. Int. J . Pept. Protein Res. 1992, 40, 233-242.
(18) Ueki, M.; Ikeo, T.; Iwadate, M.; Asakura, T.; Williamson, M. P.;
Slaninova´, J . Solid Phase Synthesis and Biological Activities of
[Arg8]-Vasopressin Methylenedithioether. Bioorg. Med. Chem.
Lett. 1999, 9, 1767-1772.
(19) Ueki, M.; Ikeo, T.; Hokari, K.; Nakamura, K.; Saeki, A.;
Komatsu, H. A New Efficient Method for S-CH2-S Bond
Formation and Its Application to a Djenkolic Acid-Containing
Cyclic Enkephalin Analog. Bull. Chem. Soc. J pn. 1999, 72, 829-
838 and references therein.
(20) Lindman, S.; Lindeberg, G.; Gogoll, A.; Nyberg, F.; Karle´n, A.;
Hallberg, A. Synthesis, Receptor Binding Affinities and Confor-
mational Properties of Cyclic Methylenedithioether Analogues
of Angiotensin II. Bioorg. Med. Chem. 2001, 9, 763-772.
(21) Walker, M. A.; J ohnson, T. General Method for the Synthesis of
Cyclic Peptidomimetic Compounds. Tetrahedron Lett. 2001, 42,
5801-5804.
(22) Pawlak, D.; Oleszczuk, M.; Wo´jcik, J .; Pachulska, M.; Chung,
N. N.; Schiller, P. W.; Izdebski, J . Highly Potent Side-Chain to
Side-Chain Cyclized Enkephalin Analogues Containing a Car-
bonyl Bridge: Synthesis, Biology and Conformation. J . Pept. Sci.
2001, 7, 128-140.
(23) Alexander McNamara, L. M.; Andrews, M. J . I.; Mitzel, F.;
Siligardi, G.; Tabor, A. B. Peptides Constrained by an Aliphatic
Linkage between Two CR Sites: Design, Synthesis, and Unex-
pected Conformational Properties of an i,(i + 4)-Linked Peptide.
J . Org. Chem. 2001, 66, 4585-4594.
(24) Sugg, E. E.; Dolan, C. A.; Patchett, A. A.; Chang, R. S. L.; Faust,
K. A.; Lotti, V. J . Cyclic Disulfide Analogs of [Sar1,Ile8]-
Angiotensin II. Pept.: Chem., Struct. Biol., Proc. Am. Pept.
Symp., 11th 1990, 305-306.
(25) Spear, K. L.; Brown, M. S.; Reinhard, E. J .; McMahon, E. G.;
Olins, G. M.; Palomo, M. A.; Patton, D. R. Conformational
Restriction of Angiotensin II: Cyclic Analogs Having High
Potency. J . Med. Chem. 1990, 33, 1935-1940.
(26) Plucinska, K.; Kataoka, T.; Yodo, M.; Cody, W. L.; He, J . X.;
Humblet, C.; Lu, G. H.; Lunney, E.; Major, T. C.; Panek, R. L.;
Schelkun, P.; Skeean, R.; Marshall, G. R. Multiple Binding
Modes for the Receptor-Bound Conformations of Cyclic AII
Agonists. J . Med. Chem. 1993, 36, 1902-1913.
(27) Matsoukas, J . M.; Hondrelis, J .; Agelis, G.; Barlos, K.; Gatos,
D.; Ganter, R.; Moore, D.; Moore, G. J . Novel Synthesis of Cyclic
Amide-Linked Analogues of Angiotensins II and III. J . Med.
Chem. 1994, 37, 2958-2969.
(28) Vlahakos, D.; Matsoukas, J . M.; Ancans, J .; Moore, G. J .;
Iliodromitis, E. K.; Marathias, K. P.; Kremastinos, D. T. Biologi-
cal Activity of the Novel Cyclic Angiotensin II Analog [Sar1,Lys3-
Glu5]ANG II. Lett. Pept. Sci. 1996, 3, 191-194.
(33) Polevaya, L.; Mavromoustakos, T.; Zoumboulakis, P.; Grdado-
lnik, S. G.; Roumelioti, P.; Giatas, N.; Mutule, I.; Keivish, T.;
Vlahakos, D. V.; Iliodromitis, E. K.; Kremastinos, D. T.; Mat-
soukas, J . Synthesis and Study of a Cyclic Angiotensin II
Antagonist Analogue Reveals the Role of π*-π* Interactions in
the C-Terminal Aromatic Residue for Agonist Activity and Its
Structure Resemblance with AT1 Non-peptide Antagonists.
Bioorg. Med. Chem. 2001, 9, 1639-1647.
(34) Samanen, J . M.; Peishoff, C. E.; Keenan, R. M.; Weinstock, J .
Refinement of a Molecular Model of Angiotensin II (AII) Em-
ployed in the Discovery of Potent Nonpeptide Antagonists.
Bioorg. Med. Chem. Lett. 1993, 3, 909-914.
(35) Nikiforovich, G. V.; Marshall, G. R. Three-Dimensional Recogni-
tion Requirements for Angiotensin Agonists: A Novel Solution
for an Old Problem. Biochem. Biophys. Res. Commun. 1993, 195,
222-228.
(36) Nikiforovich, G. V.; Kao, J . L.-F.; Plucinska, K.; Zhang, W. J .;
Marshall, G. R. Conformational Analysis of Two Cyclic Analogs
of Angiotensin: Implications for the Biologically Active Confor-
mation. Biochemistry 1994, 33, 3591-3598.
(37) J oseph, M.-P.; Maigret, B.; Scheraga, H. A. Proposals for the
Angiotensin II Receptor-Bound Conformation by Comparative
Computer Modeling of AII and Cyclic Analogs. Int. J . Pept.
Protein Res. 1995, 46, 514-526.
(38) Balodis, J .; Golbraikh, A. Conformational Analysis of Cyclic
Angiotensin II Analogs. Lett. Pept. Sci. 1996, 3, 195-199.
(39) Carpenter, K. A.; Wilkes, B. C.; Schiller, P. W. The Octapeptide
Angiotensin II Adopts a Well-Defined Structure in a Phospho-
lipid Environment. Eur. J . Biochem. 1998, 251, 448-453.
(40) Boucard, A. A.; Wilkes, B. C.; Laporte, S. A.; Escher, E.;
Guillemette, G.; Leduc, R. Photolabeling Identifies Position 172
of the Human AT1 Receptor as a Ligand Contact Point: Recep-
tor-Bound Angiotensin II Adopts an Extended Structure. Bio-
chemistry 2000, 39, 9662-9670.
(41) Eyre, D. R.; Paz, M. A.; Gallop, P. M. Cross-Linking in Collagen
and Elastin. Annu. Rev. Biochem. 1984, 53, 717-748.
(42) Do¨lz, R.; Heidemann, E. Allysine Peptides and Derivatives. Int.
J . Pept. Protein Res. 1988, 32, 307-320.
(43) Do¨lz, R.; Heidemann, E. Reactivity of the Allysine Aldehyde
Group. Connect. Tissue Res. 1989, 18, 255-268.
(44) Ojima, I.; Tzamarioudaki, M.; Eguchi, M. New and Efficient
Route to Pipecolic Acid Derivatives by Means of Rh-Catalyzed
Intramolecular Cyclohydrocarbonylation. J . Org. Chem. 1995,
60, 7078-7079.
(45) Ben-Ishai, D. Reaction of Acylamino Acids with Paraformalde-
hyde. J . Am. Chem. Soc. 1957, 79, 5736-5738.
(46) Mueller, L. P.E.COSY, a Simple Alternative to E.COSY. J . Magn.
Reson. 1987, 72, 191-196.
(47) Braunschweiler, L.; Ernst, R. R. Coherence Transfer by Isotropic
Mixing: Application to Proton Correlation Spectroscopy. J .
Magn. Reson. 1983, 53, 521-528.
(48) Bothner-By, A. A.; Stephens, R. L.; Lee, J .-m.; Warren, C. D.;
J eanloz, R. W. Structure Determination of a Tetrasaccharide:
Transient Nuclear Overhauser Effects in the Rotating Frame.
J . Am. Chem. Soc. 1984, 106, 811-813.
(49) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; J ohn Wiley
& Sons: New York, 1986.
(50) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T.
Semianalytical Treatment of Solvation for Molecular Mechanics
and Dynamics. J . Am. Chem. Soc. 1990, 112, 6127-6129.
(51) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MacroModelsAn Integrated Software System for Modeling
Organic and Bioorganic Molecules Using Molecular Mechanics.
J . Comput. Chem. 1990, 11, 440-467.
(52) Nikiforovich, G. V.; Karle´n, A.; Hallberg, A. 3D Model for
“Receptor-Bound” Conformation at the AT-1 Receptors of An-
giotensin II Analogs Cyclized in Position 3 and 5. Manuscript
in preparation.
(53) Nemethy, G.; Pottle, M. S.; Scheraga, H. A. Energy Parameters
in Polypeptides. 9. Updating of Geometrical Parameters, Non-
bonded Interactions, and Hydrogen Bond Interactions for the
Naturally Occurring Amino Acids. J . Phys. Chem. 1983, 87,
1883-1887.
(54) Dunfield, L. G.; Burgess, A. W.; Scheraga, H. A. Energy
Parameters in Polypeptides. 8. Empirical Potential Energy
Algorithm for the Conformational Analysis of Large Molecules.
J . Phys. Chem. 1978, 82, 2609-2616.
(29) Zhang, W.-J .; Nikiforovich, G. V.; Pe´rodin, J .; Richard, D. E.;
Escher, E.; Marshall, G. R. Novel Cyclic Analogs of Angiotensin
II with Cyclization between Positions 5 and 7: Conformational
and Biological Implications. J . Med. Chem. 1996, 39, 2738-2744.
(30) J ohannesson, P.; Lindeberg, G.; Tong, W.; Gogoll, A.; Karle´n,
A.; Hallberg, A. Bicyclic Tripeptide Mimetics with Reverse Turn
Inducing Properties. J . Med. Chem. 1999, 42, 601-608.
(31) J ohannesson, P.; Lindeberg, G.; Tong, W.; Gogoll, A.; Synner-
gren, B.; Nyberg, F.; Karle´n, A.; Hallberg, A. Angiotensin II
Analogues Encompassing 5,9- and 5,10-Fused Thiazabicycloal-
kane Tripeptide Mimetics. J . Med. Chem. 1999, 42, 4524-4537.
(32) Matsoukas, J . M.; Polevaya, L.; Ancans, J .; Mavromoustakos,
T.; Kolocouris, A.; Roumelioti, P.; Vlahakos, D. V.; Yamdagni,
R.; Wu, Q.; Moore, G. J . The Design and Synthesis of a Potent
Angiotensin II Cyclic Analogue Confirms the Ring Cluster
Receptor Conformation of the Hormone Angiotensin II. Bioorg.
Med. Chem. 2000, 8, 1-10.
(55) Dudley, D. T.; Panek, R. L.; Major, T. C.; Lu, G. H.; Bruns, R.
F.; Klinkefus, B. A.; Hodges, J . C.; Weishaar, R. E. Subclasses
of Angiotensin II Binding Sites and Their Functional Signifi-
cance. Mol. Pharmacol. 1990, 38, 370-377.
(56) 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 and references therein.

Analogues of Angiotensin II
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1777
(57) Robl, J . A.; Sun, C.-Q.; Stevenson, J .; Ryono, D. E.; Simpkins,
L. M.; Cimarusti, M. P.; Dejneka, T.; Slusarchyk, W. A.; Chao,
S.; Stratton, L.; Misra, R. N.; Bednarz, M. S.; Asaad, M. M.;
Cheung, H. S.; Abboa-Offei, B. E.; Smith, P. L.; Mathers, P. D.;
Fox, M.; Schaeffer, T. R.; Seymour, A. A.; Trippodo, N. C. Dual
Metalloprotease Inhibitors: Mercaptoacetyl-Based Fused Het-
erocyclic Dipeptide Mimetics as Inhibitors of Angiotensin-
Converting Enzyme and Neutral Endopeptidase. J . Med. Chem.
1997, 40, 1570-1577.
(58) Siddiqui, M. A.; Pre´ville, P.; Tarazi, M.; Warder, S. E.; Eby, P.;
Gorseth, E.; Puumala, K.; DiMaio, J . Synthesis of Constrained
Bicyclic Dipeptide Mimetics. Tetrahedron Lett. 1997, 38, 8807-
8810.
(59) Witter, D. J .; Famiglietti, S. J .; Cambier, J . C.; Castelhano, A.
L. Design and Synthesis of SH3 Domain Binding Ligands:
Modifications of the Consensus Sequence XPpXP. Bioorg. Med.
Chem. Lett. 1998, 8, 3137-3142.
(60) Khalil, E. M.; Ojala, W. H.; Pradhan, A.; Nair, V. D.; Gleason,
W. B.; Mishra, R. K.; J ohnson, R. L. Design, Synthesis, and
Dopamine Receptor Modulating Activity of Spiro Bicyclic Pep-
tidomimetics of L-Prolyl-L-leucyl-glycinamide. J . Med. Chem.
1999, 42, 628-637.
(61) Crescenza, A.; Botta, M.; Corelli, F.; Santini, A.; Tafi, A. Cyclic
Dipeptides. 3. Synthesis of Methyl (R)-6-[(tert-Butoxycarbonyl)-
amino]-4,5,6,7-tetrahydro-2-methyl-5-oxo-1,4-thiazepine-3-car-
boxylate and Its Hexahydro Analogues: Elaboration of a Novel
Dual ACE/NEP Inhibitor. J . Org. Chem. 1999, 64, 3019-3025.
(62) Shenderovich, M. D.; Ko¨ve´r, K. E.; Nikiforovich, G. V.; J iao, D.;
Hruby, V. J . Conformational Analysis of â-Methyl-para-nitro-
phenylalanine Stereoisomers of cyclo[D-Pen2, D-Pen5]Enkephalin
by NMR Spectroscopy and Conformational Energy Calculations.
Biopolymers 1996, 38, 141-156.
(63) Shiosaki, K.; Nadzan, A. M.; Kopecka, H.; Shue, Y.-K.; Holladay,
M. W.; Lin, C. W.; Nellans, H. N. Patent WO 9119733, 1991.
(64) Nishikawa, M. Patent J P 04193871, 1992.
(65) Nishimura, K.; Fujimura, K.-i.; Matsumoto, J .; Kobayashi, T.
Patent WO 0006594, 2000.
(66) Claesen, M.; Vlietinck, A.; Vanderhaeghe, H. Preparation of the
Enantiomers of 2-Carbobenzyloxyaminoadipic Acid and Their
1-Benzylesters. Bull. Soc. Chim. Belg. 1968, 77, 587-596.
(67) Szirtes, T.; Kisfaludy, L.; Pa´losi, EÄ .; Szporny, L. Synthesis of
Thyrotropin-Releasing Hormone Analogs. 2. Tripeptides Struc-
turally Greatly Different from TRH with High Central Nervous
System Activity. J . Med. Chem. 1986, 29, 1654-1658.
(68) Robl, J . A.; Kronenthal, D. R.; Goderey, J . D., J r. Patent EP
629627, 1994.
(69) Robl, J . A. U.S. Patent 5508272, 1996.
(70) Godfrey, J . D., J r.; Kronenthal, D. R.; Schwinden, M. D.;
Srivastava, S. K.; Ramig, K.; Venit, J . J .; J ass, P. A.; Racha, S.;
Dillon, J . L., J r.; Soundararajan, N.; Powers, G. L.; Kotnis, A.
S. Patent WO 0003981, 2000.
(71) Donovan, M. J .; Goldberg, S.; Hanson, R. L.; J ass, P. A.; Li, W.-
S.; Patel, R. N.; Ramig, K.; Szarka, L. J .; Venit, J . J . Patent WO
0004179, 2000.
(72) Moniot, J . L.; Srivastava, S. K.; Winter, W. J .; Venit, J . J .;
Swaminathan, S.; Ramig, K.; J ass, P. A.; Schwinden, M. D.;
Dillon, J . L., J r.; Racha, S.; Simpson, J .; Chen, C.-k.; Pack, S.
K. U.S. Patent 6162913, 2000.
(73) Goodman, J . M.; Still, W. C. An Unbounded Systematic Search
of Conformational Space. J . Comput. Chem. 1991, 12, 1110-1117.
J M011063A