AT2-selective angiotensin II analogues containing tyrosine-functionalized 5,5-bicyclic thiazabicycloalkane dipeptide mimetics

Article abstract of DOI:10.1021/jm049651m

This paper reports the synthesis of two angiotensin II analogues with tyrosine-functionalized 5,5-bicyclic thiazabicycloalkane dipeptide mimetics replacing the Tyr⁴-Ile⁵ residues. The preparation of these analogues relies on the synt

Full text of DOI:10.1021/jm049651m

J. Med. Chem. 2004, 47, 6009-6019
6009
AT2-Selective Angiotensin II Analogues Containing Tyrosine-Functionalized
5,5-Bicyclic Thiazabicycloalkane Dipeptide Mimetics
Petra Johannesson,† Ma´te´ Erde´lyi,†,‡ Gunnar Lindeberg,† Per-Anders Fra¨ndberg,§ Fred Nyberg,§
Anders Karle´n,† and Anders Hallberg*,†
Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden, Department of Organic
Chemistry, Uppsala University, BMC, Box 599, SE-751 24 Uppsala, Sweden, and Department of Biological Research on Drug
Dependence, Uppsala University, BMC, Box 591, SE-751 24 Uppsala, Sweden
Received May 10, 2004
This paper reports the synthesis of two angiotensin II analogues with tyrosine-functionalized
5,5-bicyclic thiazabicycloalkane dipeptide mimetics replacing the Tyr4-Ile5 residues. The
preparation of these analogues relies on the synthesis and incorporation of an R,R-disubstituted
chimeric amino acid derivative and on-resin bicyclization to a cysteine residue. The synthesized
analogues both displayed high angiotensin AT2/AT1 receptor binding preferences and had AT2
receptor affinities in the same low nanomolar range as angiotensin II itself. Conformational
analysis, using experimental constraints derived from NMR studies, indicated that the Tyr4
and His6 residues in one of the angiotensin II analogues were in close proximity to each other.
Chart 1. Structure of Angiotensin II
Introduction
The linear octapeptide angiotensin II (Ang II, Chart
1) mediates its actions through binding to two pharma-
cologically distinct receptor subtypes, designated AT1
and AT2. Most of the well-known actions of Ang II, such
as vasoconstriction, aldosterone release, sympathetic
activation and cellular growth are mediated through the
AT1 receptor.1-3 The function of the AT2 receptor is less
clear, but growing evidence supports its role in the
regulation of antiproliferation processes, cellular dif-
ferentiation, apoptosis and vascular relaxation.4-8 The
AT2 receptor has recently attracted considerable interest
as a new therapeutic target.
Chart 2. Structure of Thiazabicycloalkane Dipeptide
Mimetics
Although several different models of Ang II binding
to the AT1 receptor have been proposed,9-18 it seems
likely that the peptide adopts a reverse turn at the
central Tyr4 residue.9-11,19-25 Understanding of the
requirements for molecular recognition at the AT2
receptor is more limited.18,22,26-29 The introduction of
monocyclic or bicyclic amino acid sequences into the 3-5
region of Ang II has been reported to result in analogues
with retained AT2 affinity, and it was postulated that
Ang II binding at the AT2 receptor might also involve a
reverse turn in the Tyr4 region of the molecule.22,29
Recently an Ang II analogue with high AT2 affinity and
encompassing a γ-turn-like mimetic replacing amino
acid residues 4-5 was also reported.30
It has been suggested that a reverse turn in a peptide
could act as a recognition trigger in peptide-receptor
interactions.31 As a consequence, much time has been
dedicated to the synthesis of structures that mimic or
induce reverse turns.32-41 The first generation of turn
mimetics often suffered from the lack of side-chains, but
during recent years many side-chain functionalized
reverse turn mimetics have also been reported. One of
the most successful examples of turn mimetics is seen
in the family of thiazabicycloalkane dipeptide
mimetics,42-52 as represented here by structures 1-5
(Chart 2). The turn mimetics (1-3) were reported by
Hruby and co-workers.51,52 Johnson et al. prepared the
type II′ â-turn mimetic 446 and also compound 5,53 which
* Corresponding author. Telephone: +46 18 471 4284. Fax: +46
18 471 4976. E-mail: Anders.Hallberg@farmkem.uu.se.
† Department of Medicinal Chemistry.
‡ Department of Organic Chemistry.
§ Department of Biological Research on Drug Dependence.
10.1021/jm049651m CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/15/2004

6010 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Johannesson et al.
Scheme 1. Synthetic Strategy Applied for the Preparation of the R,R-Disubstituted Building Block 13a
a Reagents and conditions: (a) Paraformaldehyde, benzene, reflux, 38% (95% based on consumed starting material); (b) KHMDS, allyl
iodide, THF, -78 °C, 65%; (c) OsO4, NaIO4, H2O/dioxane; (d) TsOH, MeOH, 61% over two steps, from compound 8; (e) NaOMe, MeOH,
reflux, 86%; (f) H2, Pd/C, EtOH, 65 °C; (g) (i) KOH (aq), MeOH, reflux (ii) Fmoc-Cl, Na2CO3, H2O/dioxane, 25% over two steps, from
compound 11.
was designed as a mimetic that could not achieve a
â-turn conformation.
outlined by Baldwin et al.56 in their synthesis of the
corresponding aldehyde derivative which was used to
produce phenylalanine side-chain functionalized thiaz-
abicycloalkanes, designated as γ-lactam analogues of
â-lactam antibiotics.
We have previously described a procedure for induc-
ing spontaneous bicyclization which delivers tripeptide
mimetic thiazabicycloalkanes after incorporation of a
masked aldehyde building block and a cysteine residue
into Ang II.54,55 Upon liberation of the aldehydes by
TFA, thiazabicycloalkanes spontaneously formed via
connection of the side chains and a backbone nitrogen
in the final step. This is in contrast to the most
commonly used method for synthesis of thiazabicyclo-
alkanes, which relies on intramolecular N-acylation as
the final step.42
This paper reports the synthesis of Ang II analogues
16A and 16B (Scheme 2) which encompass tyrosine
side-chain functionalized thiazabicycloalkane dipeptide
mimetics replacing the Tyr4 and Ile5 residues, where a
turn has been proposed to be important both for AT1
and AT2 receptor recognition. The preparation of these
analogues relies on incorporation of the R,R-disubsti-
tuted chimeric amino acid derivative 13 (Scheme 1), and
on-resin cyclization, employing methodology similar to
that previously used by our group for formation of the
corresponding tripeptide mimetics.54,55 Both of the Ang
II analogues exhibit high AT2/AT1 selectivity and a high
affinity for the AT2 receptor. We also present a confor-
mational analysis, using experimental constraints de-
rived from NMR studies of the two Ang II analogues in
DMSO solution.
The synthesis started with L-Z-Tyr(OtBu)-OH (6). The
oxazolidinone formation is usually achieved through
reaction with paraformaldehyde and a catalytic amount
of acid, such as p-toluenesulfonic acid, under azeotropic
removal of water.55,57 In this case, however, it was not
possible to use acidic catalysis with p-toluenesulfonic
acid, because of the presence of the acid-labile tert-butyl
protecting group. The conversion to oxazolidinone 7 was
sluggish in the absence of acid and resulted in a low
yield of 38%. Fortunately, the starting material could
be recovered and, based on its consumption, 95% yield
was achieved.
Oxazolidinone 7 was deprotonated with potassium
hexamethyldisilylamide (KHMDS), and the resulting
enolate was alkylated with allyl iodide to form the
racemic, quaternary-substituted amino acid derivative
8. Oxidative cleavage of the double bond with sodium
periodate and catalytic amounts of osmium tetroxide58
afforded the aldehyde 9, which was directly protected
as the dimethyl acetal to give compound 10.
The NMR spectra for compounds 7, 8 and 10 were
recorded at elevated temperatures in order to achieve
signal coalescence of the rotameric mixtures. Nonethe-
less, it was necessary to report the NMR data for
compound 10 as for a mixture of two rotamers (see
Experimental Section).
Results
Conversion to the primary amine 12 was performed
by first cleaving the oxazolidinone with sodium meth-
oxide in methanol and then removing the benzyloxy-
carbonyl group by catalytic hydrogenation. Cleavage of
the oxazolidinone and benzyloxycarbonyl groups from
compounds 10 and 11, respectively, required higher
temperatures than for deprotection of the corresponding
monosubstituted compounds.55 Reflux was also needed
to effect hydrolysis of the methyl ester 12. Finally, the
amino function of the resulting zwitterion was directly
Synthesis. The synthetic route to the R,R-disubsti-
tuted building block 13 is outlined in Scheme 1. Since
the stereochemical outcome of the bridgehead carbon
formed in the cyclization step was not obvious, and since
we were interested in several of the subsequent poten-
tial diastereomeric target peptides, we desired access
to both enantiomers of 13. We therefore chose to use a
racemic process of synthesis. The synthesis of the
intermediate aldehyde 9 was based on a strategy

AT2-Selective Angiotensin II Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6011
Scheme 2. Incorporation of 13 into a Pseudopeptide
reacted with Fmoc-Cl to give the R,R-disubstituted
building block 13, which was incorporated into the
pseudopeptide by standard Fmoc/tert-butyl solid-phase
peptide synthesis (SPPS).
Mimicking Angiotensin II and Its On-Resin Cyclizationa
The SPPS incorporation and cyclization using the
methods successfully employed in our earlier studies54,55
met with limited success. Coupling of the quaternary
substituted building block 13 was slow, as expected, but
could be achieved by prolonging the reaction using
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexaflu-
orophosphate (PyBOP) activation. However, the next
step, coupling of Fmoc-Val to the partially protected R,R-
disubstituted amino acid residue, was unsuccessful.
Several methods for proceeding were then evaluated,
using small-scale experiments followed by cleavage and
MS analysis. Activation with O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) gave exclusively the tetramethylguanidine-
modified starting peptide. Reaction with diisopropyl
carbodiimide (DIC) and 1-hydroxy-7-azabenzotriazole
(HOAt) produced an unidentified product as well as the
starting material. Activation with PyBOP or use of
Fmoc-Val-F or Fmoc-Val-OPfp resulted in only the
starting peptide, although it was largely modified by
monotritylation.
Steric hindrance, due to side-chain interactions and
limited flexibility of the R,R-disubstituted residue, seemed
to be the most likely explanation for the inaccessibility
of the R-amino group for coupling with the carboxyl
component, although a direct transfer of a trityl group
to the R-amino function was also possible. In both cases,
deprotection and on-resin cyclization before incorpora-
tion of Fmoc-Val was expected to facilitate the coupling
reaction. Indeed, upon pretreatment of the peptide resin
with 2% TFA in dichloromethane for 1.5 h, followed by
coupling with Fmoc-Val-F59,60 and removal of the Fmoc
protecting group, the expected hexapeptide analogue
was found to be the main product after cleavage.
However, the reaction was still incomplete and the acid
incubation of the Wang resin, because of the limited
stability of the peptide-linker bond, was accompanied
by an unacceptable loss of starting material. We there-
fore deemed it necessary to consider resins of higher acid
stability.
a Reagents and conditions: (a) Cys(Trt)-His(Trt)-Pro-Phe-P-
linker-2-TentaGel, PyBOP, DIEA, DMF, overnight; (b) 95% TFA
(aq); (c) piperidine, DMF; (d) (i) Fmoc-Val-F, DIEA, DMF, (ii)
piperidine, DMF; (e) (i) Fmoc-Arg(Pbf), PyBOP, DIEA, DMF, (ii)
piperidine, DMF; (f) (i) Fmoc-Asp(OtBu), PyBOP, DIEA, DMF, (ii)
piperidine, DMF; (g) 95% TFA (aq), (h) hν (350 nm), (i) purification
by RP-HPLC.
Preliminary experiments with the acid stable-
HYCRAM resin61,62 were promising. We found, however,
that Pd(0)-induced cleavage led to concomitant oxidation
of methionine to the corresponding sulfoxide. Since our
target peptide would also contain a thioaminal entity,
the risk of oxidation forced us to abandon this approach.
tion of Ang II analogues encompassing four different
diastereomeric core structures is therefore possible.
However, it seems that only two of them were formed
in significant amounts.
Instead, we turned our attention to photolabile link-
ers. The use of the 4-ethyl-2-methoxy-5-nitrophenoxy-
acetyl based P-linker-263,64 attached to TentaGel S NH2,
although of low capacity, finally allowed synthesis, on-
resin cyclization, and cleavage to proceed satisfactorily
(Scheme 2).
The synthesis outlined in Scheme 2 afforded two main
compounds with the expected mass and amino acid
ratios. These two Ang II analogues, referred to as 16A
and 16B, were each isolated in 5% yield after purifica-
tion by RP-HPLC. Building block 13 was incorporated
into the peptides as the racemate, and an additional
asymmetric center of unknown configuration was cre-
ated at the ring junction of the bicyclic system. Forma-
Structural Characterization. The structural as-
signment of Ang II analogues 16Aand 16B (Scheme 2),
as well as determination of the stereochemistry of the
bicyclic part of the molecules, was performed using
data obtained from phase sensitive DQF-COSY,65-67
gNOESY,68,69 ROESY,70 gTOCSY,71 gHMQC72,73 and
gHMBC74 NMR experiments. The observed NMR data
was in good agreement with assignments of previous
angiotensin analogues.54,55 Selected 1H NMR signals for
16A and 16B are given in Table 1 (for full spectral data
and assignments, see Experimental Section). The di-
astereomeric Ang II analogues 16A and 16B both had
an R configuration at the tyrosine side-chain function-
alized carbon atom. 16B also had an R configuration at

6012 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Johannesson et al.
Table 1. Selected 1H NMR Chemical Shifts for Ang II Analogues 16A and 16B (DMSO-d6 solution, 25 °C)
compd
residue
∆δ/∆Ta
NH
H-R
H-â
H-â′
H-γ
H-γ′
other
16A
Asp
Arg
Val
“Tyr”
Xb
Cys
His
Pro
Phe
Asp
Arg
Val
“Tyr”
Xb
4.11
4.36
4.17
-
2.78
1.64
1.88
2.95
2.67
3.19
3.07
2.08
3.04
2.79
1.67
1.96
2.95
2.84
2.41
2.99
2.04
3.02
2.63
1.51
-
-
-
2.9
5.4
6.7
8.58
7.90
8.29
1.51
1.51
NHꢀ: 7.50 (∆δ/∆Ta ) 2.6)
0.84
0.90
2.90
2.33
3.21
2.85
1.83
2.95
2.63
1.52
-
2.90
2.82
2.22
2.83
1.81
2.87
-
-
ortho: 7.02; para: 6.70
-
3.95
-
-
4.79
4.83
4.41
4.47
4.12
4.39
4.26
-
-
-
-
1.0
4.3
7.58
-
-
H2: 9.30; H4: 7.28
Hδ: 3.58; Hδ′: 3.45
Ar: 7.16-7.30
1.83
1.83
8.37
-
-
-
-
16B
3.6
5.9
6.1
8.68
7.95
8.32
1.52
1.43
NHꢀ: 7.59 (∆δ/∆Ta) 6.1)
0.81
0.83
-
-
ortho: 6.99; para: 6.65
-
4.72
-
Cys
His
Pro
Phe
-
5.00
4.76
4.41
4.47
-
-
-
6.4
6.5
8.15
-
-
H2: 9.30; H4: 7.28
Hδ: 3.47; Hδ′: 3.58
Ar: 7.16-7.30
1.78
-
1.81
-
8.36
a NMR temperature coefficients of NH chemical shifts (ppb/K). b X is used to denote the part derived from the aldehyde side chain of
building block 13, see Figure 1.
Figure 2. Model compounds used for conformational char-
acterization.
Figure 1) observed in the ROESY spectra collected in
DMSO-d6 solution. Monte Carlo conformational searches
combined with molecular mechanics energy minimiza-
Figure 1. NOE information from Ang II analogues 16A and
16B.
tion using the OPLS all atom force field were applied,
and the GB/SA water solvation model75 as implemented
in the program Macromodel (version 7.0) was used.76
The calculations resulted in 1154 low energy conforma-
tions for m16A and 695 conformations for m16B within
5 kcal/mol of the lowest energy minimum. The 20 lowest
energy conformations of m16A and m16B are super-
imposed in Figure 3. The different stereochemistry in
the bicyclic ring system enforces different ring geom-
etries for m16A and m16B. This is also indicated by
the computational analysis which shows that the side
chains of Tyr4 and His6 in m16B are in close proximity.
In contrast, this geometric arrangement is not preferred
in the diastereomeric m16A. However, a few of the
conformations identified for m16A adopt similar side-
chain geometries to those seen with m16B. Further-
more, as indicated by Figure 3, both isomers of the
thiazabicycloalkane dipeptide mimetics appear to mimic
an extended backbone conformation.
the newly formed ring-junction carbon, while this ring-
junction carbon had an S configuration in the epimeric
16A. NOE information of Ang II analogues 16A and 16B
is illustrated in Figure 1. For Ang II analogue 16B,
several ROESY cross-peaks indicated that the tyrosine
and histidine side chains are within 5 Å proximity to
each other in DMSO solution at room temperature. Low-
temperature coefficients (Table 1) indicate that the
histidine amide proton in Ang II analogue 16A is
hydrogen bonded and the arginine amide proton is in
equilibrium between hydrogen bonded and non-hydro-
gen bonded states in DMSO solution at room temper-
ature. For Ang II analogue 16B, none of the amide
protons seems to be hydrogen bonded under these
conditions.
Molecular Modeling. To model the preferred con-
formations of Ang II analogues 16A and 16B, calcula-
tions for the blocked bicyclic tetrapeptides m16A and
m16B (Figure 2) were performed using the experimen-
tal constraints derived from ROE cross-peaks (see
In Vitro Binding Affinity. Ang II analogues 16A
and 16B were evaluated in radioligand binding assays
relying on displacement of [125I]Ang II from AT1 recep-
tors in rat liver membranes77 and from AT2 receptors

AT2-Selective Angiotensin II Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6013
Our SPPS and on-resin cyclization, however, delivered
epimer 16A with an S configuration at the newly formed
bridgehead carbon and epimer 16B with the corre-
sponding R configuration. It should be emphasized
though that we cannot exclude that all four of the
diastereomers had been formed in the reaction mixture,
but that two of them escaped detection.
The thiazabicycloalkane 5 (Chart 2), with stereo-
chemistry corresponding to that of 16A, was designed
as a dipeptide mimetic unable to adopt a â-turn con-
formation.53 The crystal structure and conformational
analysis of 3 (Chart 2), with the same stereochemistry
as 16A, indicated that this compound was also unlikely
to adopt a turn conformation, with respect to reversal
of the peptide backbone.51 5,5-Bicyclic thiazabicyclo-
alkanes with stereochemistry corresponding to that of
16B have to the best of our knowledge not yet been
reported. Conformational analysis of carba-analogues
with a carbon replacing the sulfur atom, however,
indicated that these structures have poor reverse turn-
inducing properties.79 Although thiazabicycloalkanes in
general have been successfully used as turn-inducing
fragments, it seems that the 5,5-bicyclic dipeptide
mimetics with the particular stereochemistry reported
here, and with the incoming and outgoing peptide chain
anti to each other, do not have reverse turn-inducing
properties. This was also indicated by our conforma-
tional analysis of model tetrapeptides m16A and m16B,
as can be seen from Figure 3.
Ang II analogues 16A and 16B both had high AT2/
AT1 selectivity. They both displayed high affinity for the
AT2 receptor with Ki values of 1.0 and 3.7 nM, respec-
tively, which is in the same range as that of Ang II itself
(1.2 nM). In contrast to the current situation for the AT1
receptor, relatively few investigations of the structure-
activity relationships and conformation of Ang II bind-
ing to the AT2 receptor have been reported.2,18,22,27-30,80-82
Most of the binding affinity studies performed to date
show that modifications of the linear peptides Ang II
and [Sar1]Ang II are well tolerated by the AT2 receptor.
Miura et al. reported that replacement with Ala at most
positions in Ang II led to only minor decreases in AT2
receptor affinity. The Arg2, Tyr4 and His6 residues were
most sensitive to substitution.28 A recent Gly scan of
Ang II showed that only substitution of Arg2 resulted
in significant reductions in AT2 affinity.83 Conforma-
tional analysis of AT2 binding mono- and bicyclic Ang
II analogues suggested the importance of a turn in the
central Tyr4 region of Ang II in its AT2 binding confor-
mation.22,29 However, contrary to all previous sugges-
tions, and as a result of photoaffinity labeling and site-
directed mutagenesis experiments, Derae¨t et al. proposed
that Ang II binds in an extended â-strand-like confor-
mation at both the AT1 and AT2 receptors.18 Our
conformational analysis of Ang II analogues 16A and
16B indicates an extended backbone conformation in
the Tyr4-Ile5 region of Ang II in solution. This is in
accordance with the proposed model of Derae¨t et al.
Furthermore the conformational analysis indicated that
in analogue 16B the Tyr4 and His6 side chains are in
close proximity to each other.
Figure 3. Overlaid backbones of the 20 lowest energy
conformations of m16A (top) and m16B (bottom) from molec-
ular modeling.
Table 2. In Vitro AT1 and AT2 Receptor Binding Affinities
AT1
AT2
(rat liver embranes) (pig uterus myometrium)
compd
Ki (nM) ( SEM
Ki (nM) ( SEM
Ang II
1.1 ( 0.4
>10000
n.d.a
1.9 ( 0.6
>10000
>10000
1.2 ( 0.4
1.4 ( 0.1
2.1 ( 0.1
-
1.0 ( 0.1
3.7 ( 0.3
[4-NH2-Phe6]AngII
c[Hcy3,5]Ang II
losartan
16A
16B
a n.d. ) not determined.
in pig uterus membranes78 (Table 2). Ang II, c[Hcy3,5]-
Ang II,20 the highly AT2 selective [4-NH2-Phe6]Ang II
and the non-peptide AT1-selective antagonist losartan
were used as reference substances. Ang II analogues
16A and 16B both displayed high AT2/AT1 selectivity
and bound with high affinities to the AT2 receptor
(affinity constants Ki 1.0 nM for 16A and 3.7 nM for
16B). Neither 16A nor 16B had any binding affinity to
the AT1 receptor; no Ki values below 10 µM were found.
Discussion
Building block 13 was incorporated into the peptides
as the racemate. After SPPS and cyclization, two of the
four possible diastereomeric target peptides were iso-
lated. Both of the bicyclic Ang II analogues had the same
stereochemistry as the naturally occurring L-tyrosine
with respect to the tyrosine side-chain functionality.
This may have been due to less effective SPPS incor-
poration of the S-enantiomer of 13 or to a lower
tendency for appropriate folding and cyclization. Ac-
cording to the literature, the ring-junction hydrogen and
the cysteine-derived carbonyl group in the diastereomer
that is usually formed are on the same side of the
thiazolidine ring.42 Similar results were also obtained
by Baldwin et al.56 Their enantiomerically pure L-
phenylalanine side-chain functionalized building block
was reported to deliver the 5,5-bicyclic thiazabicyclo-
alkane with stereochemistry corresponding only to 16A.
For the AT1 receptor, the Ang II aromatic side chains
of the Tyr4, His6 and Phe8 residues, as well as the
C-terminal carboxylate, have been identified as the

6014 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Johannesson et al.
pharmacophore groups.2,84 The pharmacological evalu-
ation showed that Ang II analogues 16A and 16B lacked
affinity for the AT1 receptor. Apparently these analogues
cannot adopt a conformation that enables binding to this
receptor, which fits with their inability to make a
reverse turn around the central Tyr4 residue, as has
been proposed for Ang II in its AT1-binding conforma-
tion.9-11,19-25 Also the close proximity of the Tyr4 and
His6 side chains in solution conformations of Ang II
analogue 16B do not fit proposed models of bioactive
conformations such as that suggested by Nikiforovich
et al.10,11 Interestingly, the close spatial arrangement
of the Tyr4 and His6 aromatic side chains does fit with
the bioactive conformation of Ang II at the AT1 receptor
proposed by Moore and co-workers.15 This model in-
volves a charge relay system that requires clustering
of the Tyr4, His6 and Phe8 side chains. As is the case
for our conformational analysis of Ang II analogues 16A
and 16B, the model of Moore et al. is based on biophysi-
cal studies of Ang II and conformationally constrained
Ang II analogues in solution.
Merck). Chromatographic spots were visualized by UV and/
or spraying with an ethanolic solution of ninhydrin (2%),
followed by heating. Mass spectroscopy was carried out on an
Applied Biosystems (Uppsala, Sweden) BIOION 20 plasma
desorption mass spectrometer. Amino acid analyses and pep-
tide content determinations were performed at the Department
of Biochemistry, Biomedical center, Uppsala, Sweden, on 24
h hydrolyzates with an LKB 4151 alpha plus analyzer, using
ninhydrin detection.
Materials. Z-L-Tyr(tBu)-OH (6) was isolated from the
corresponding commercially available dicyclohexylamine salt
by extraction. Fmoc-Val-F was prepared from Fmoc-Val-OH
and cyanuric fluoride according to the literature.59,60 Fmoc-
Phe-Wang resin was obtained from Calbiochem-Novabiochem
(La¨ufelfingen, Switzerland), and Boc-Phe-HYCRAM resin from
Orpegen Pharma (Heidelberg, Germany). P-linker-2-Tenta-
Gel63,64 was a gift from Doc. Eva Åkerblom, Department of
Medicinal Chemistry, Uppsala University, BMC, Uppsala,
Sweden. Amino acid derivatives and 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were
from Alexis Corp. (La¨ufelfingen, Switzerland), (benzotriazol-
1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (Py-
BOP) from Calbiochem-Novabiochem. DMF (purum) was
purchased from Fluka Chemie GmbH (Buchs, Switzerland)
and was used without further purification.
Conclusion
(4S)-3-(Benzyloxycarbonyl)-4-(4-tert-butoxyphenyl-
methyl)-1,3-oxazolidin-5-one (7). Z-L-Tyr(tBu)-OH (6) (28.3
g, 76.2 mmol) was dissolved in benzene (500 mL), and
paraformaldehyde (95%) (6.86 g, 217 mmol) was added. The
reaction mixture was immersed into a prewarmed oil bath and
refluxed with azeotropic removal of water using a Dean-Stark
apparatus for 24 h. Additional amounts of paraformaldehyde
were added after 17 h (6.86 g, 217 mmol) and 20 h (6.86 g,
217 mmol). Most of the benzene was evaporated, and the
residue was dissolved in EtOAc (500 mL) and washed with
brine (150 mL). The organic phase was dried (MgSO4) and
evaporated. The residue was purified by flash column chro-
matography (gradient system: isohexane to isohexane/EtOAc
3:2) to afford recovered starting material (6) (17.0 g, 60%) and
product (7) (11.0 g, 38% (95% based on consumed starting
material)) as an oil: TLC Rf ) 0.4 (isohexane/EtOAc 3:2); [R]D
The tyrosine derivative 13, R-substituted with a
masked aldehyde side chain, has been synthesized in
seven steps from Z-Tyr(OtBu)-OH (6). Compound 13 was
used in SPPS with the photolabile P-linker-2-TentaGel
as solid support.63,64 Upon acidic liberation of the
aldehyde, on-resin cyclization occurred spontaneously,
leading to formation of 5,5-bicyclic thiazabicycloalkane
dipeptide mimetics substituted with a tyrosine side-
chain.
The cyclization procedure was applied for the syn-
thesis of two Ang II analogues containing the 5,5-bicyclic
conformational constraint located in the important Tyr4-
Ile5 region. The two constrained Ang II analogues
exhibited high AT2/AT1 selectivity and an AT2 binding
affinity in the same range as for Ang II itself. Confor-
mational analysis using experimental constraints de-
rived from NMR studies supported the hypothesis of an
extended backbone conformation in the Tyr4-Ile5 region.
Furthermore, our conformational analysis indicated
close proximity of the Tyr4 and His6 side chains in one
of the AT2-selective angiotensin II analogues.
1
) +189° (c ) 0.99, 99% EtOH, 22 °C); H NMR (acetone-d6,
50 °C, 270.2 MHz) δ 1.26 (s, 9H, C(CH3)3), 3.04 (dd, J ) 3.0,
13.2 Hz, 1H, CH2a-Tyr), 3.27 (br dd, 1H, CH2b-Tyr), 4.39 (br d,
1H, NCH2aO), 4.54 (br dd, 1H, CH2CH(N)-CO), 5.17 (br d, 1H,
CH2a-Z), 5.23 (br d, 1H, CH2b-Z), 5.28 (d, J ) 4.0 Hz, 1H,
NCH2bO), 6.82 (dm, J ) 8.5 Hz, 2H, Ar-Tyr), 6.95 (dm, J )
8.5 Hz, 2H, Ar-Tyr), 7.26-7.44 (m, 5H, Ar-Z). 13C NMR
(acetone-d6, 50 °C, 67.8 MHz) δ 29.7 (C(CH3)3), 36.2 (CH2-Tyr),
57.7 (CH2CH(N)-CO), 68.5 (CH2-Z), 79.16 (C(CH3)3), 79.23
(NCH2O), 125.2 (CH-Ar Tyr), 129.5, 129.6, 129.9 (CH-Ar Z),
131.2 (ipso Ar), 131.5 (CH Ar Tyr), 137.9 (ipso Ar), 153.7, 156.5
(ipso COC(CH3)3, CO Z), 173.0 (CH(N)CO); IR (KBr) 1795,
1685. Anal. (C22H25NO5) C, H, N.
(4R,S)-3-(Benzyloxycarbonyl)-4-(4-tert-butoxyphenyl-
methyl)-4-[1-(2-propenyl)]-1,3-oxazolidin-5-one (8). Com-
pound 7 (14.4 g, 37.5 mmol) was dissolved in dry THF (60 mL)
in a dried three-necked flask fitted with a septum, a ther-
mometer and a gas outlet for flushing with N2. The solution
was cooled to -78 °C. Potassium hexamethyldisilyl amide
(KHMDS) as a 0.5 M toluene solution (187 mL, 93.5 mmol)
was added slowly via a syringe, over 2 h, keeping the
temperature below -70 °C. The temperature was monitored
by a thermometer inside the flask. Thereafter allyl iodide (98%)
(9.7 mL, 104 mmol) was added over 20 min, also via a syringe
and keeping the temperature below -70 °C. The reaction
mixture was stirred at -78 °C for 15 h and thereafter
quenched by the addition of saturated aqueous NH4Cl (250
mL). The quenched mixture was partitioned between diethyl
ether (400 mL) and water (100 mL). The water phase was
further extracted with diethyl ether (500 mL). The combined
organic phases were dried (MgSO4) and concentrated. The
residue was purified by flash column chromatography (CH2-
Cl2) to give pure 8 (10.3 g, 65%) as a solid: mp 81-83 °C; TLC
Experimental Section
Chemistry. General Comments. 1H and 13C NMR spectra
were recorded on a JEOL JNM-EX270 (1H: 270.2 MHz, 13C:
67.8 MHz), on a JEOL JNM-EX400 (1H: 399.8 MHz, 13C:
100.5 MHz), and on a Varian Inova 800 MHz (1H: 799.9 MHz)
spectrometer. The gNOESY spectrum (tmix ) 0.4 s) of the
octapeptide 16A was recorded on a Varian Unity 400 spec-
trometer operating at 400 MHz. Chemical shifts are reported
as δ values (ppm), referenced indirectly to Me4Si via the
solvent residual signal. IR spectra were recorded on a Perkin-
Elmer model 1605 FT-IR instrument, and for solids, a mounted
Microfocus Beam Condenser with ZnSe lenses in a Diasqueeze
Plus Diamond Compressor Cell (Graseby Specac Inc., Smyrna,
U.S.A.) was used unless otherwise noted. IR values are
reported as vmax (cm-1). Optical rotation was measured on a
Perkin-Elmer Model 241 polarimeter. Melting points (uncor-
rected) were determined in open glass capillaries using a
melting point microscope. Elemental analyses were performed
by Mikro Kemi AB, Uppsala, Sweden. Flash column chroma-
tography was performed using Merck silica gel 60 (40-63 µm).
Thin-layer chromatography (TLC) was performed using alu-
minum sheets precoated with silica gel 60 F254 (0.2 mm, E.

AT2-Selective Angiotensin II Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6015
1
Rf ) 0.7 (1% MeOH in CH2Cl2); H NMR (DMSO-d6, 120 °C,
(OMe)2), 3.28 (s, 3H, CH(OMe)2), 3.51 (d, J ) 13.5 Hz, 1H,
CH2b-Tyr), 3.70 (s, 3H, COOMe), 4.30 (dd, J ) 3.0, 8.0 Hz, 1H,
CH(OMe)2), 5.07 (d, 1H, J ) 12 Hz, CH2a-Z), 5.21 (d, 1H, J )
12 Hz, CH2b-Z), 5.82 (s, 1H, NH), 6.74-6.81 (m, 4H, CH Ar
Tyr), 7.31-7.43 (m, 5H, CH Ar Z); 13C NMR (acetone, 50 °C,
67.8 MHz) δ 28.7 (C(CH3)3), 38.9 (CH2CH(OMe)2), 40.3 (CH2-
Tyr), 52.3 (COOMe), 53.4 (CH(OMe)2), 55.0 (CH(OMe)2), 62.5
(C(NH)COOMe), 66.2 (CH2-Z), 78.1 (C(CH3)3), 102.5 (CH-
(OMe)2), 123.6 (CH Ar Tyr), 128.0, 128.1, 128.4 (CH-Ar Z),
130.1 (CH-Ar Tyr), 130.2, 136.7 (ipso Ar), 154.17, 154.21 (CO
Z and ipso COC(CH3)3), 172.7 (COOMe); IR (solid) 3394, 1741,
1709. Anal. (C26H35NO7) C, H, N.
(2R,S)-2-Amino-2-(4-tert-butoxyphenylmethyl)-4,4-di-
methoxybutanoic Acid Methyl Ester (12). Compound 11
(2.55 g, 5.38 mmol) and 10% Pd/C (284 mg, 267 µmol) were
mixed in absolute EtOH (100 mL) and stirred under H2 (1
atm), at 65 °C for 40 min. The mixture was filtered through
Celite, and the Celite was washed with additional EtOH (300
mL). Concentration gave crude 12 (1.89 g) which could be used
in the next step, without further purification. An analytical
sample was prepared through purification by flash column
chromatography (CH2Cl2/MeOH 9:1). White solid material: mp
55-56 °C; TLC Rf ) 0.6 (5% MeOH in CH2Cl2); 1H NMR
(CDCl3, 25 °C, 270.2 MHz) δ 1.32 (s, 9H, C(CH3)3), 1.96 (dd, J
) 14.0, 5.5 Hz, 1H, CH2aCH(OMe)2), 2.05 (br s, 2H, NH2), 2.29
(dd, J ) 14.0, 5.5 Hz, 1H, CH2bCH(OMe)2), 2.77 (d, J ) 13.5
Hz, 1H, CH2a-Tyr), 3.09 (d, J ) 13.5 Hz, 1H, CH2b-Tyr), 3.30
(s, 3H, CH(OMe)2), 3.32 (s, 3H, CH(OMe)2), 3.67 (s, 3H,
COOMe), 4.48 (t, J ) 5.5 Hz, 1H, CH(OMe)2), 6.89 (dm, J )
8.5 Hz, 2H, CH-Ar Tyr), 7.03 (dm, J ) 8.5 Hz, 2H, CH-Ar Tyr);
13C NMR (CDCl3, 25 °C, 67.8 MHz) δ 28.8 (C(CH3)3), 42.4 (CH2-
CH(OMe)2), 46.2 (CH2-Tyr), 51.8 (COOMe), 53.3 (CH(OMe)2),
53.4 (CH(OMe)2), 60.1 (C(NH2)COOMe), 78.2 (C(CH3)3), 102.4
(CH(OMe)2), 123.9 (CH Ar), 130.4 (ipso Ar and CH-Ar, 2 C
overlapping), 154.4 (ipso COC(CH3)3), 176.4 (COOMe); IR
(solid) 3384, 3321, 1732. Anal. (C18H29NO5) C, H, N.
(2R,S)-2-(4-tert-butoxyphenylmethyl)-2-[(9-fluorenyl-
methoxycarbonyl)- amino]-4,4-dimethoxybutanoic Acid
(13). To compound 12 (1.00 g, 2.95 mmol) dissolved in MeOH
(40 mL) was added 1 M aqueous KOH (7.4 mL, 7.4 mmol),
and the mixture was refluxed for 3 h. 10% aqueous citric acid
was added to pH 9, and the reaction mixture was concentrated
to give a solid residue. This residue was dissolved in a mixture
of 10% aqueous Na2CO3 (50 mL) and dioxane (25 mL) and
cooled to 0 °C. Fmoc-Cl (97%, 1.91 g, 2.43 mmol) dissolved in
dioxane (25 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 con-
tinued for 68 h. 10% aqueous citric acid was added to pH 8-9,
and the reaction mixture was washed with hexane (3 × 50
mL and 2 × 100 mL). Diethyl ether (200 mL) was added to
the water phase, which was then acidified to pH 4 with 10%
aqueous citric acid, under vigorous stirring. The phases were
separated, and the water phase was further extracted with
diethyl ether (2 × 200 mL). The combined organic layers were
washed with water (2 × 100 mL), dried (MgSO4) and concen-
trated to give the building block 13 (398 mg, 25% over two
steps, from compound 11) as a white foam; TLC Rf ) 0.35 (5%
MeOH in CH2Cl2); 1H NMR (CDCl3, 25 °C, 270.2 MHz) δ 1.31
(s, 9H, C(CH3)3), 2.33 (dd, J ) 14.0, 7.6 Hz, 1H, CH2aCH-
(OMe)2), 2.70 (dd, J ) 14.0, 3.8 Hz, 1H, CH2bCH(OMe)2), 3.12
(d, J ) 13.5 Hz, 1H, CH2a-Tyr), 3.28 (s, 3H, CH(OMe)2), 3.33
(s, 3H, CH(OMe)2), 3.49 (d, J ) 13.5 Hz, 1H, CH2b-Tyr), 4.26
(t, J ) 6.5 Hz, 1H, CH-Fmoc), 4.36-4.47 (m, 2H, CH2a-Fmoc
and CH(OMe)2), 4.56 (dd, J ) 10.3, 6.5 Hz, 1H, CH2b-Fmoc),
5.89 (s, 1H, NH), 6.83 (d, J ) 7.6 Hz, 2H, CH-Ar), 6.94 (d, J )
7.6 Hz, 2H, CH-Ar), 7.28-7.46 (m, 4H, CH-Ar), 7.58-7.65 (m,
2H, CH-Ar), 7.78 (d, J ) 7.6 Hz, 2H, CH-Ar); 13C NMR (CDCl3,
25 °C, 67.8 MHz) δ 28.8 (C(CH3)3), 38.7 (CH2CH(OMe)2), 40.2
(CH2-Tyr), 47.3 (CH-Fmoc), 53.4 CH(OMe)2), 54.7 CH(OMe)2),
62.2 (C(NH)COOH), 66.3 (CH2-Fmoc), 78.5 (C(CH3)3), 102.5
(CH(OMe)2), 119.9, 123.8, 125.0, 127.1, 127.7 (CH-Ar), 130.1
(ipso Ar), 130.2 (CH-Ar), 141.3, 143.7, 143.8 (ipso Ar), 154.1,
399.8 MHz) δ 1.29 (s, 9H, C(CH3)3), 2.61 (dd, J ) 6.9, 14.0 Hz,
1H, CH2aCHdCH2), 2.98 (d, J ) 13.8 Hz, CH2a-Tyr), 3.06 (br
dd, 1H, CH2bCHdCH2), 3.35 (br d, 1H, CH2b-Tyr), 4.28 (d, J )
3.8 Hz, 1H, NCH2aO), 5.05-5.33 (m, 5H, NCH2bO, CH2-Z, CH2-
CHdCH2), 5.57-5.70 (m, 1H, CH2CH)CH2), 6.81 (m, 2H, Ar
Tyr), 6.88 (m, 2H, Ar Tyr), 7.34-7.46 (m, 5H, Ar Z); 13C NMR
(DMSO-d6, 120 °C, 67.8 MHz) δ 28.1 (C(CH3)3), 38.4, 39.0 (CH2-
CHdCH2, CH2-Tyr), 65.7, 66.3 (C(N)CO, CH2-Z), 76.6 (NCH2O),
77.3 (C(CH3)3), 119.3 (CH2CH)CH2), 122.5 (CH-Ar Tyr), 127.4,
127.5, 127.8 (CH-Ar Z), 128.5 (ipso Ar), 129.2 (CH-Ar Tyr),
130.5 (CH2CHdCH2), 135.5 (ipso Ar), 150.6, 154.2 (ipso COC-
(CH3)3, CO Z,), 172.5 (C(N)CO); IR (solid) 1796, 1711. Anal.
(C25H29NO5) C, H, N.
(4R,S)-3-(Benzyloxycarbonyl)-4-(4-tert-butoxyphenyl-
methyl)-4-(2,2-dimethoxyethyl)-1,3-oxazolidin-5-one (10).
To a solution of compound 8 (4.10 g, 9.68 mmol) in dioxane
(23 mL) and water (4.8 mL) was added OsO4 (4% in H2O) (3.1
mL, 0.51 mmol). To this mixture was added NaIO4 (6.21 mg,
29.0 mmol) in small portions, over 1.5 h, and the reaction
mixture was stirred at room temperature for 22 h. Thereafter,
the mixture was cooled to 0 °C, and the reaction was quenched
by slowly adding saturated aqueous NaHSO3 (70 mL). Stirring
was continued at room temperature for 15 min. The reaction
mixture was partitioned between diethyl ether (250 mL) and
saturated aqueous NaHSO3 (40 mL). The water phase was
further extracted with diethyl ether (2 × 250 mL). The organic
phases were combined and washed with saturated aqueous
NaHSO3 (130 mL). The organic phase was then dried (MgSO4)
and concentrated. The resulting crude aldehyde 9 was dis-
solved in MeOH (200 mL), p-toluenesulfonic acid (monohy-
drate, 98.5%) (1.31 g, 6.68 mmol) was added, and the mixture
was stirred at room temperature for 15 h. Most of the solvent
was evaporated, and the residue was partitioned between
EtOAc (400 mL) and saturated aqueous NaHCO3 (200 mL).
The water phase was further extracted with diethyl ether (400
mL). The combined organic layers were dried (MgSO4) and
concentrated. Purification of the residue by flash column
chromatography (isohexane/EtOAc 4:1) gave acetal 10 as an
oil (2.78 g, 61%): TLC Rf ) 0.55 (isohexane/EtOAc 7:3); NMR
data are reported on a mixture of two conformers:1H NMR
(acetone-d6, 50 °C, 270.2 MHz) δ 1.25 and 1.26 (2 × s, 9H,
C(CH3)3), 2.19-2.31 (m, 1H, CH2aCH(OMe)2), 2.38-2.47 (m,
0.4H, CH2bCH(OMe)2), 2.56-2.65 (m, 0.6H, CH2bCH(OMe)2),
2.81-2.91 (m, 7.3 H, CH(OMe)2 and CH2-Tyr), 3.37-3.47 (m,
0.7H, CH2-Tyr), 4.25-4.36 (m, 2H, CH(OMe)2 and NCH2aO),
4.92-5.02 (m, 1H, NCH2bO), 5.07-5.16 (m, 0.7H, CH2-Z),
5.26-5.35 (m, 1.3H, CH2-Z), 6.70-6.90 (m, 4H, CH Ar Tyr),
7.28-7.56 (m, 5H, CH Ar Z); 13C NMR (acetone-d6, 50 °C, 67.8
MHz) δ 29.7 (C(CH3)3), 38.6, 39.9 (CH2CH(OMe)2), 41.4, 42.7
(CH2-Tyr), 54.2, 54.5, 54.6, 54.7 (2 × OMe), 64.66, 64.74 (C(N)-
CO), 68.1, 68.7 (CH2-Z), 78.2, 78.5 (NCH2O), 79.15, 79.17
(C(CH3)3), 103.2, 103.4 (CH(OMe)2), 125.0 (CH-Ar Tyr), 129.57,
129.62, 129.9, 130.0, 130.4, 130.5, 130.7 (C ipso Ar and CH-
Ar), 131.6 (CH-Ar Tyr), 137.6, 138.1 (C ipso Ar), 152.7, 153.4,
156.5, 156.6 (CO Z and ipso COC(CH3)3), 174.7, 175.1 (5); IR
(neat) 1798, 1715; Anal. (C26H33NO7) C, H, N.
(2R,S)-2-(Benzyloxycarbonylamino)-2-(4-tert-bu-
toxyphenylmethyl)-4,4-dimethoxybutanoic Acid Methyl
Ester (11). Compound 10 (1.15 g, 2.43 mmol) was dissolved
in MeOH (40 mL) under N2 atmosphere. Molecular sieves and
NaOMe (95%) (395 mg, 6.95 mmol) were added. The reaction
was refluxed for 2.5 h. 10% aqueous citric acid was added to
pH 7, and most of the solvent was evaporated. The residue
was partitioned between EtOAc (200 mL) and H2O (100 mL).
The water phase was further extracted with EtOAc (200 mL).
The organic layers were combined, dried (MgSO4) and evapo-
rated. Purification of the residue by flash column chromatog-
raphy (gradient system isohexane/EtOAc 17:3 to 3:1) afforded
pure 11 (0.99 g, 86%) as a white solid: mp 74-76 °C; TLC Rf
1
) 0.65 (isohexane/EtOAc 3:2); H NMR (CDCl3, 25 °C, 399.8
MHz) δ 1.31 (s, 9H, C(CH3)3), 2.26 (dd, J ) 8.0, 14.0 Hz, 1H,
CH2aCH(OMe)2), 2.79 (dd, J ) 3.0, 14.0 Hz, 1H, CH2bCH-
(OMe)2), 3.01 (d, J ) 13.5 Hz, 1H, CH2a-Tyr), 3.23 (s, 3H, CH-

6016 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Johannesson et al.
154.6 (CO Fmoc and ipso COC(CH3)3), 176.4 (COOH); IR (solid)
3408, 1713. Anal. (C32H37NO7 ‚ 1.75 H2O) C, H, N.
(1, 3 and 10 min, respectively) and the polymer washed with
DMF (6 × 2 mL). The resin above was reacted with Fmoc-
Val-F (54.3 mg, 159 µmol) and DIEA (27.7 µL, 159 µmol) in 1
mL of DMF for 17.5 h and then washed and deprotected as
described before. Cleavage and LC-MS (3.0 mg sample)
confirmed that the coupling was complete.
Coupling of Fmoc-Arg(Pbf)-OH and Fmoc-Asp(OtBu)-
OH. The peptide chain was further elongated by coupling with
Fmoc-Arg(Pbf)-OH‚diisopropyl ether (52.6 mg, 70.0 µmol),
PyBOP (36.4 mg, 70.0 µmol) and DIEA (24.4 µL, 140.0 µmol)
in 0.75 mL of DMF for 3 h. The resin was then washed and
deprotected as described above. Fmoc-Asp(OtBu)-OH (28.8 mg,
70.0 µmol) was incorporated by the same procedure. After
washing and removal of the Fmoc group, the resin was further
washed with DMF (6 × 2 mL) and MeOH (6 × 2 mL) and dried
to yield 84.8 mg. Side-chain deprotection was accomplished
with TFA-H2O-triethylsilane (90:5:5; 2 × 1.5 mL, 2 × 30
min). The resin was then washed with CH2Cl2 (6 × 2 mL),
DMF (6 × 2 mL) and MeOH (6 × 2 mL) and dried. Yield: 82.2
mg.
H-Phe-P-linker-2-TentaGel. P-linker-2 resin (508 mg, ca.
90 µmol) was allowed to swell in 2.6 mL of dry CH2Cl2 in a 15
mL centrifuge tube. Fmoc-Phe-OH (132 mg, 340 µmol) was
added and washed down with 2 × 2 mL CH2Cl2 followed by
diisopropylcarbodiimide (53 µL, 340 µmol) and 4-pyrrolidi-
nopyridine (7.1 mg, 48 µmol). The tube was capped, covered
with Al-foil, and rotated at room temperature for 3 h. The resin
was filtered off and washed with CH2Cl2 (5 × 7 mL) and DMF
(5 × 7 mL). Ca. 10 mL 20% piperidine in DMF was then
allowed to slowly pass through the polymer. Final removal of
the Fmoc group was accomplished by treatment with a second
10-mL portion of the deprotection mixture in the capped
filtration tube under rotation for 10 min. The resin was
filtered, washed with DMF (5 × 7 mL), CH2Cl2 (5 × 7 mL)
and MeOH (5 × 10 mL). The yield of the dried resin was 516
mg. The degree of substitution, as determined by amino acid
analysis, was 0.18 mmol/g.
H-Cys(Trt)-His(Trt)-Pro-Phe-P-linker-2-TentaGel. To
H-Phe-P-linker-2 resin (508 mg, 91 µmol) was added a solution
of Fmoc-Pro-OH‚H2O (151 mg, 425 µmol) in DMF (2 mL)
followed by HBTU (161 mg, 425 µmol) dissolved in DMF (2
mL), and DIEA (148 µl, 850 µmol). The contents of the covered
filtration tube were mixed by rotation for 2h and then washed
and deprotected as described above for the H-Phe-P-linker-2-
TentaGel. The same procedure was used for the coupling of
Fmoc-His(Trt)-OH (263 mg, 425 µmol) to yield 548 mg of the
partially protected tripeptide resin. Part of the polymer (252
mg, 39.8 µmol) was transferred to a 5-mL disposable syringe
equipped with a 20 µm polyethylene filter and reacted with
Fmoc-Cys(Trt)-OH (116 mg, 198 µmol) and HBTU (75 mg, 198
µmol) in the presence of DIEA (69 µL, 396 µmol) essentially
as described above for the other amino acids but with a
coupling volume of 2 mL and with 3-mL washings. The dry,
partially protected tetrapeptide resin weighed 267 mg. To a
sample (3.6 mg) weighed into a transparent polypropylene
Eppendorf tube was added MeOH (0.3 mL). The tube was
capped and flushed with nitrogen and the suspension irradi-
ated at 350 nm under stirring for 2 h.63,64 LC-MS analysis
showed the expected m/z values for the major component. No
deletion peptides were detected.
Ang II Analogues 16A and 16B. Coupling of Building
Block 13. In a 2-mL disposable syringe equipped with a
polyethylene filter H-Cys(Trt)-His(Trt)-Pro-Phe-P-linker-2 resin
(95.8 mg, 14.3 µmol) was reacted with building block 13 (9.2
mg, 16.8 µmol), PyBOP (8.7 mg, 16.8 µmol) and DIEA (7.3 µL,
42.0 µmol) in a total volume of 0.5 mL of DMF. The reaction
was allowed to proceed under rotation in the dark for 18 h.
The resin was filtered and washed with DMF (6 × 2 mL),
MeOH (3 × 2 mL), CH2Cl2 (3 × 2 mL) and MeOH (6 × 2 mL)
and dried in vacuo to yield 98.0 mg of compound 14. A sample
(4.1 mg) was cyclized and deprotected by treatment with 95%
aq TFA for 1 h and then with 95% TFA containing 3%
triethylsilane for 10 min in order to scavenge the trityl cations.
After washing with ether, DMF and MeOH, the sample was
cleaved by UV irradiation as described above. LC-MS analysis
showed two isomers of the expected mass. However, since the
incorporation was incomplete, the coupling was repeated with
compound 13 (14.0 mg, 25.6 µmol), PyBOP (13.3 mg, 25.6 µmol)
and DIEA (13.4 µL, 76.6 µmol) for 17 h. The resin was washed
with DMF (6 × 2 mL), CH2Cl2 (6 × 2 mL) and MeOH (6 × 2
mL) and dried in vacuo. Yield of compound 14: 95.9 mg. UV
cleavage of an analytical sample (6.2 mg) now showed the
reaction to be complete.
Cleavage. The fully deprotected peptide resin was divided
into three equal portions and transferred to 5-mL quartz tubes.
After addition of 1.5 mL of MeOH, the tubes were capped with
rubber septa and flushed with nitrogen for 2 min. The resin
suspensions were then irradiated with UV light at 350 nm
under stirring for 2 h. The polymer was removed by filtration
through 0.45 µm centrifuge filters and further extracted with
MeOH (0.5 mL) and 50% aqueous MeOH (2 × 0.5 mL). The
combined filtrates were then evaporated to dryness in vacuo.
Purification. The residue after evaporation was dissolved
in 2.5 mL of 0.1% aqueous TFA containing 10% MeCN and
purified by two runs on a Vydac C18 (218TP) column (10 µm,
22 × 250 mm) using a gradient of MeCN in 0.1% aq TFA (5-
55% MeCN in 75 min) at a flow rate of 3 mL/min and with
detection by UV absorbance at 230 nm. The fractions were
further analyzed by LC-MS using a Chromolith Performance
RP-18e column (4.6 × 100 mm). Two major components were
isolated, both showing the expected m/z values. 16A: 0.65 mg
(5.1%); LC-MS (MW 1059.7) 1060.6 (M + H+), 531.0 (M + 2H+)/
2; amino acid analysis Asp, 0.98; Arg, 0.97; Val, 0.62; His, 1.03;
Pro(+Cys), 1.22; Phe, 1.03 (81% peptide); 1H NMR (DMSO-
d6, 25 °C, 400 MHz) δ 0.84 (d, J ) 6.6 Hz, 3H, CH3-Val), 0.90
(d, J ) 7.0 Hz, 3H, CH3-Val), 1.51 (m, 3H, Hâ′-Arg, Hγ-Arg,
Hγ′-Arg), 1.64 (m, 1H, Hâ-Arg), 1.75-1.90 (m, 4H, Hâ-Val, Hâ′-
Pro, Hγ-Pro, Hγ′-Pro), 2.08 (m, 1H, Hâ-Pro), 2.33 (m, 1H, Hâ′-
X), 2.57-2.67 (m, 2H, Hâ-X, Hâ′-Asp), 2.77-2.98 (m, 5H, Hâ-
Asp, Hâ′-His, Hâ-Phe, Hâ-Tyr, Hâ′-Tyr), 3.00-3.12 (m, 4H, Hâ′-
Phe, Hδ-Arg, Hδ′-Arg, Hâ-His), 3.16-3.25 (m, 2H, Hâ-Cys, Hâ′-
Cys), 3.45 (m, 1H, Hδ′-Pro), 3.58 (m, 1H, Hδ-Pro), 3.95 (t, J )
7.1 Hz, Hγ-X), 4.11 (dd, J ) 3.8, 8.6 Hz, 1H, HR-Asp), 4.17 (t,
J ) 8.2 Hz, 1H, HR-Val), 4.36 (m, 1H, HR-Arg), 4.40-4.50 (m,
2H, HR-Pro, HR-Phe), 4.79 (dd, 1H, J ) 3.8, 6.6 Hz, HR-Cys),
4.83 (m, 1H, HR-His), 6.70 (AA′ part of AA′XX′, 2H, Hortho-Tyr),
7.02 (XX′ part of AA′XX′, 2H, Hmeta-Tyr), 7.28 (m, 6H, Phe,
H-4 His), 7.50 (dd, J ) 6.4 Hz, 1H, NHꢀ-Arg), 7.58 (d, J ) 8.4
Hz, 1H, NH-His), 7.90 (d, J ) 8.4 Hz, 1H, NH-Val), 8.29 (br s,
1H, NH-Tyr), 8.37 (d, J ) 7.7 Hz, 1H, NH-Phe), 8.58 (d, J )
8.2 Hz, 1H, NH-Arg), 9.30 (br s, 1H, H-2 His). The histidine
amide NH is hydrogen bonded, the Arg-NH is in exchange
between H-bonded and non H-bonded states in DMSO solution
at 25 °C. 16B: 0.67 mg (5.3%); LC-MS 1060.6 (M + H+), 531.0
(M + 2H+)/2; amino acid analysis Asp, 0.97; Arg, 0.97; Val,
1
0.90; His, 1.03; Pro(+Cys), 1.12; Phe, 1.03 (84% peptide); H
NMR (DMSO-d6, 25 °C, 400 MHz): δ 0.81 (d, J ) 7.0 Hz, 3H,
CH3-Val), 0.83 (d, J ) 7.0 Hz, 3H, CH3-Val), 1.43 (m, 1H, Hγ′-
Arg), 1.52 (m, 2H, Hâ′-Arg, Hγ-Arg), 1.67 (m, 1H, Hâ-Arg), 1.78
(m, 1H, Hγ-Pro), 1.81 (m, 2H, Hâ′-Pro, Hγ′-Pro), 1.96 (dh, J )
7.0, 7.3 Hz, 1H, Hâ-Val), 2.04 (m, 1H, Hâ-Pro), 2.22 (dd, J )
4.0, 14.3 Hz, 1H, Hâ′-Cys), 2.41 (dd, J ) 7.0, 14.3 Hz, 1H, Hâ-
Cys), 2.63 (dd, J ) 9.0, 18.1 Hz, 1H, Hâ′-Asp), 2.79 (dd, J )
3.3, 18.1 Hz, 1H, Hâ-Asp), 2.83-2.90 (m, 6H, Hâ-X, Hâ′-His,
Hâ′-X, Hâ′-Phe, Hâ-His, Hâ′-Tyr), 2.98-3.10 (4H, m, Hâ-Tyr, Hâ-
Phe, Hδ-Arg, Hδ′-Arg), 3.47 (m, 1H, Hδ′-Pro), 3.58 (m, 1H, Hδ-
Pro), 4.12 (dd, J ) 3.7, 8.4 Hz, 1H, HR-Asp), 4.26 (dd, J ) 7.3,
Cyclization Affording Compound 15. To remove the
side-chain protecting groups and to induce the desired bicy-
clization, the remaining resin (14) (89.8 mg, 12.4 µmol) was
treated with 95% aqueous TFA (2 × 2 mL, 2 × 30 min) and
then with 95% aqueous TFA containing 50 µL of triethylsilane
(2 mL, 10 min). The resin was filtered off and washed with
CH2Cl2 (5 × 2 mL) and DMF (5 × 2 mL).
Coupling of Fmoc-Val-F. The Fmoc group was removed
by reaction with three 2-mL portions of 20% piperidine in DMF

AT2-Selective Angiotensin II Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6017
8.8 Hz, 1H, HR-Val), 4.37-4.48 (m, 3H, HR-Arg, HR-Pro, HR-
Phe), 4.72 (d, J ) 3.2, 8.0 Hz, 1H, Hγ-X), 4.76 (dd, J ) 5.6, 8.0
Hz, 1H, HR-His), 5.00 (dd, J ) 4.0, 7.0 Hz, 1H, HR-Cys), 6.65
(AA′XX′, 2H, Hortho-Tyr), 6.99 (AA′XX′, 2H, Hmeta-Tyr), 7.16-
7.30 (m, 5H, Phe), 7.28 (br s, 1H, H-4 His), 7.59 (t, J ) 5.8 Hz,
1H, NHꢀ-Arg), 7.95 (d, J ) 8.8 Hz, 1H, NH-Val), 8.15 (d, J )
9.5 Hz, 1H, NH-His), 8.32 (br s, 1H, NH-Tyr), 8.36 (d, J ) 7.3
Hz, 1H, NH-Phe), 8.68 (d, J ) 7.7 Hz, 1H, NH-Arg), 9.30 (br
s, 1H, H-2 His). None of the amide NH’s are hydrogen bonded
in the DMSO solution of this diastereomer at 25 °C. X is used
to denote the part derived from the aldehyde side-chain of
building block 13, see Figure 1. The yields are corrected for
peptide content.
Molecular Modeling. The conformational predictions of
m16A and m16B were performed using the OPLS all atom
force field as implemented in the program Macromodel 7.0.76
The General Born Solvent Accessible (GB/SA) surface area
method developed by Still75 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. Amide bonds
where fixed in the trans configuration. Conformational con-
straints derived from ROESY cross-peaks were introduced
using the CDIS command (5 Å ( 1.5 Å) as implemented in
Macromodel 7.0. For the conformational search 10000 Monte
Carlo step runs were performed, and those conformations
within 5 kcal/mol of the lowest energy minimum were kept.
In the subsequent minimization a maximum of 40000 steps
of PR conjugate gradient (PRCG) minimization resulted in
1154 and 695 fully converged structures for m16A and m16B,
respectively.
teristics of the Ang II binding AT2 receptor was determined
by using six different concentrations (0.03-5 nM) of the labeled
[
125I]Ang II. Nonspecific binding was determined in the pres-
ence of 1 µM Ang II. The specific binding was determined by
subtracting the nonspecific binding from the total bound [125I]-
Ang II. The dissociation constant (Kd ) 1.6 ( 0.2 nM) was
determined by Scatchard analysis of data obtained with Ang
II using GraFit (Erithacus Software, UK). The binding data
were best fitted with a one-site fit. All experiments were
performed in triplicates and were repeated three times except
for 16B that were performed in duplicates. Ki values were
calculated using the Cheng-Prusoff equation.
Acknowledgment. We thank Adolf Gogoll, Depart-
ment of Organic Chemistry, Uppsala University, Upp-
sala, Sweden, for valuable advice concerning the NMR
spectra of Ang II analogues 16A and 16B. The Swedish
NMR Centre (Patrik Jarvoll), at Gothenburg University,
Sweden, is gratefully acknowledged for use of a Varian
Unity 800 spectrometer. We also thank Milad Botros,
Department of Biological Research on Drug Depen-
dence, Uppsala University, Sweden, for skillful in vitro
binding experiments, and the Swedish Foundation for
Strategic Research (SSF) for financial support.
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C. Macromodel - an Integrated Software System for Modeling
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