A selective AT2 receptor ligand with a gamma-turn-like mimetic replacing the amino acid residues 4-5 of angiotensin II.

Article abstract of DOI:10.1021/jm030921v

Three angiotensin II (Ang II) analogues encompassing a benzodiazepine-based gamma-turn-like scaffold have been synthesized. Evaluation of the compounds in a radioligand binding assay showed that they had no affinity to the rat liver AT(1) receptor. Howeve

Full text of DOI:10.1021/jm030921v

J . Med. Chem. 2004, 47, 859-870
859
A Selective AT2 Recep tor Liga n d w ith a γ-Tu r n -Lik e Mim etic Rep la cin g th e
Am in o Acid Resid u es 4-5 of An gioten sin II
Ulrika Rosenstro¨m,† Christian Sko¨ld,† Gunnar Lindeberg,† Milad Botros,‡ Fred Nyberg,‡ Anders Karle´n,† and
Anders Hallberg†,*
Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, BMC, Box 574, Uppsala University,
SE-751 23 Uppsala, Sweden, and Department of Biological Research on Drug Dependence, BMC, Box 591, Uppsala
University, SE-751 24 Uppsala, Sweden
Received J une 10, 2003
Three angiotensin II (Ang II) analogues encompassing a benzodiazepine-based γ-turn-like
scaffold have been synthesized. Evaluation of the compounds in a radioligand binding assay
showed that they had no affinity to the rat liver AT1 receptor. However, one of the compounds
displayed considerable affinity to the pig uterus AT2 receptor (Ki ) 3.0 nM) while the other
two lacked affinity to this receptor. It was hypothesized that the reason for the inactivity of
one of these analogues to the AT2 receptor was that the guanidino group of the Arg2 residue
and/or the N-terminal end of the pseudopeptide could not interact optimally with the receptor.
To investigate this hypothesis, a conformational analysis was performed and a comparison
was carried out with the monocyclic methylenedithioether analogue cyclo(S-CH2-S)[Cys3,5]-
Ang II which is known to bind with high affinity to the AT2 receptor (Ki ) 0.62 nM). This
comparison showed that, in the compounds with high AT2 receptor affinity, the guanidino group
of the Arg2 residue and the N-terminal end could access common regions of space that were
not accessible to the inactive compound. To examine the importance of the guanidino group
for binding, the Arg side chain was removed by substituting Arg2 for Ala2 in the analogue
having the high affinity. This analogue lacked affinity to AT2 receptors, which supports the
role of the guanidino group in receptor binding.
In tr od u ction
receptor-peptide complexes, strategies relying on con-
strained analogues should provide valuable informa-
tion.16 Cyclization can be a powerful tool for imposing
conformational constraints. In addition, properly se-
lected cyclization methodologies could serve to facilitate
the stepwise transformation of peptide leads into pep-
tidomimetic molecules.17,18 Models of Ang II bound to
the AT1 receptor have been proposed and there is
growing evidence that the peptide adopts a turn cen-
tered at the Tyr4 residue.19-27 Considerably less is
known about the structural requirements for AT2 recep-
tor affinity, but there was strong early support, based
on studies of linear and cyclic [Sar1]Ang II analogues,
for significant differences from those for AT1 receptor
affinity.21,28-30 However, recently, as a result of photo-
affinity labeling experiments combined with site-
directed mutagenesis, Derae¨t et al. proposed that Ang
II binds in an almost identical fashion and adopts an
extended â-strand-like conformation at both the AT1 and
the AT2 receptors,31 contrary to previous predictions.
The octapeptide angiotensin II (Ang II) exerts its
actions by binding to two pharmacologically distinct
receptor subtypes designated AT1 and AT2. The AT1
receptor mediates the well-known physiological effects
of Ang II, such as vasoconstriction, aldosterone release,
stimulation of sympathetic transmission, and cellular
growth.1-3 AT1 receptor antagonists (the sartans) are
currently used as effective clinical antihypertensives.
The function of the AT2 receptor subtype, which was
cloned more recently and found to share only 32-34%
sequence identity with the AT1 receptor,4,5 remains
elusive and somewhat controversial. It has been sug-
gested that it plays a role in mediating antiproliferation,
cellular differentiation, apoptosis, and vasodilation.6-9
Importantly, the receptor is expressed at low densities
in adult tissues but is up-regulated in pathological
conditions such as heart failure, renal failure, myocar-
dial infarction, brain lesions, vascular injury, and wound
healing.10-15 The AT2 receptor has recently attracted
considerable interest as a new potential therapeutic
target.
Our long-term objective is to enable transformation
of peptides to nonpeptides by iterative incorporation of
well-defined secondary structure mimetics in the target
peptides with a special focus on AT1 and AT2 receptor
ligands. This paper reports the design, synthesis, and
binding data of the four Ang II analogues 1-4, encom-
passing γ-turn-like mimicking scaffolds (Chart 1). One
of these pseudopeptides, compound 2, exhibits very high
AT2 receptor selectivity. We have also evaluated the
previously synthesized Ang II analogues 5a and 5b,
which contains a similar γ-turn scaffold, for AT2 receptor
affinity. Finally, we present molecular modeling studies
Knowledge of the conformation of Ang II when bound
to the two receptor subtypes is invaluable for the
understanding of the topological requirements within
the AT1 and AT2 receptor-peptide complexes. Since the
preferred solution conformations of the flexible linear
octapeptide do not necessarily correspond to those in
* To whom correspondence should be addressed. Tel +46 18 471
4284. Fax +46 18 471 4976. E-mail: Anders.Hallberg@orgfarm.uu.se.
† Department of Medicinal Chemistry.
‡ Department of Biological Research on Drug Dependence.
10.1021/jm030921v CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/17/2004

860 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Rosenstro¨m et al.
Ch a r t 1
of 1, 2, and 5a and a comparison with compound 6,
which has been shown to have high affinity to the AT2
receptor.32
ethylamine. Glycine or its methyl ester could not be used
as building blocks because of the facile formation of a
diketopiperazine in a later step. The subsequent cou-
pling of the secondary amine 8 with Fmoc-L-Tyr(t-Bu)-
OH was performed using O-(7-azabenzothiazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) as activating reagent. The Fmoc group was
removed with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
rather than with the more commonly used piperidine,
since the latter was found to undergo nucleophilic
aromatic substitution at the fluorine ipso-carbon. The
deprotected amine 9 underwent an internal nucleophilic
aromatic substitution under the basic conditions used.
A significantly improved yield of the benzodiazepine
scaffold was achieved in the cyclization if the depro-
tected amine was first purified through column chro-
matography to remove the deprotection side products
and thereafter immediately treated with an aqueous
triethylamine/DMSO solution. Treatment of 10 with
TFA/CH2Cl2 removed both the acetal protection and the
Resu lts
Ch em istr y. The bicyclic core structure of 1-4, pre-
pared by solution chemistry, was incorporated into Ang
II using solid-phase methodology. The synthesis of
pseudopeptide 5, encompassing a γ -turn mimetic region
centered at Tyr4 has been previously reported.20 Inser-
tion of NH into this γ-turn mimic gives the benzodi-
azepine core structure that has previously been recog-
nized and successfully applied as a turn mimic.33 The
benzodiazepine system was used as a turn template for
substituting Val3-Tyr4-Ile5 (1), Tyr4-Ile5 (2 and 3), or Ile5-
His6-Pro7 (4) segments of Ang II.
The synthesis of 12, outlined in Scheme 1, was based
on the strategy described by Keenan at al.,33 starting
with a reductive amination of the substituted benzal-
dehyde 7 with the glycine equivalent 2,2-dimethoxy-

Selective AT2 Receptor Ligand
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 861
Sch em e 1a
a
Reagents: (a) 2,2-dimethoxyethylamine, HOAc, NaCNBH3, MeOH, 72%; (b) Fmoc-L-Tyr(t-Bu)-OH, HATU, DIEA, CH2Cl2, 90%; (c) (i)
DBU, THF, (ii) Et3N, DMSO, H2O, 64%; (d) (i) TFA, CH2Cl2, (ii) NaClO2, NaH2PO4, cyclohexene, t-BuOH, THF, H2O, 77%; (e) (i) HCOONH4,
Pd/C, MeOH, (ii) FmocCl, Na2CO3 (aq), dioxane, 47%.
Sch em e 2a
a
Reagents: (a) 2-aminoethanol, HOAc, NaCNBH3, MeOH, 85%; (b) TBDPSCl, DBU, CH3CN, 98%; (c) Fmoc-L-His(Boc)-OH, HATU,
DIEA, CH2Cl2, 86%; (d) (i) DBU, THF, (ii) Et3N, DMSO, H2O, 81%; (e) (i) ClCOCOCl, DMSO, Et3N, CH2Cl2, (ii) NaClO2, NaH2PO4,
cyclohexene, t-BuOH, H2O, 55%; (f) (i) HCOONH4, Pd/C, MeOH, (ii) FmocCl, Na2CO3 (aq), dioxane, 33%.
tert-butyl group. The unstable aldehyde was immedi-
ately converted to the carboxylic acid 11 using mild
oxidation by sodium chlorite in a mixture of tert-butyl
alcohol, THF, and sodium dihydrogenphosphate solution
at 0 °C.34 At higher temperatures or with other oxidizing
agents (e.g. J ones reagent), the carboxylic acid 11 was
not detected. Instead, J ones reagent produced a mixture
of products where benzylic oxidation followed by cleav-
age of the tyrosine side chain was the predominant
reaction. Reduction of the nitro group was performed
with ammonium formiate and Pd/C. The zwitterion
obtained was not isolated but directly reacted with
FmocCl.35 Compound 12 decomposed during column
chromatography and was therefore purified by repeated
extractions.
The peptides 1, 2, and 3 were obtained by incor-
poration of the Fmoc derivative 12 into Ang II using
standard Fmoc/t-Bu SPPS methodology. Some of
the couplings, particularly the acylation of the ani-
linic nitrogen, were expected to proceed slowly and
require long reaction times. Therefore, (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP) rather than 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) was
used as activating agent in order to eliminate the risk
of formation of tetramethylguanidino derivatives. No
protection was used for the phenolic hydroxyl group.
According to MS analysis, 2 equiv of the Fmoc-amino
acid were incorporated in each of the following coupling
steps. However, the presumed phenyl ester was cleaved
smoothly in the ensuing deprotection reaction. The
target peptides 1, 2, and 3 were liberated from the resin
by treatment with TFA in the presence of water and
triethylsilane, isolated by precipitation with ether, and
purified by RP-HPLC on a C18 column.
The histidine analogue 18 was synthesized employing
a similar route, outlined in Scheme 2. Aminoethanol was
now used instead of amino acetal as glycine ester
equivalent, to avoid the need for acidic deprotection. The
benzaldehyde 7 underwent a reductive amination with
unprotected 2-aminoethanol using 5 equiv of amine and
with NaCNBH3 as the reducing agent. A longer reaction
time or higher temperature lowered the yield. The
alcohol 13 was protected with tert-butyldiphenylchlo-
rosilane (TBDPSCl) to circumvent side reactions, such
as Boc migration, and to simplify the purification.
Coupling of 14 with Fmoc-L-His(Boc)-OH, deprotection
of the Fmoc-group, and the subsequent cyclization
proceeded smoothly. The TBDPS-protecting group was
spontaneously cleaved during the cyclization, probably

862 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ta ble 1. Binding Affinities for the AT1 and AT2 Receptors
Rosenstro¨m et al.
AT1 (rat liver
membranes)
Ki (nM) ( SEM
AT2 (pig uterus
myometrium)
Ki (nM) ( SEM
compound
Ang II
0.24 ( 0.07
>10000
>10000
>10000
>10000
>10000
>10000
>10000
44 ( 1
0.23 ( 0.01
2.1 ( 0.5
>10000
[4-NH2-Phe6]Ang II
1
2
3
4
5a
5b
632
3.0 ( 1.1
>10000
>10000
61 ( 2
>10000
0.62 ( 0.04
assisted by the fluoride ions liberated. Since the direct
oxidation of the alcohol to carboxylic acid with TEMPO
and sodium hypochlorite36 failed, the oxidation was
performed in two steps. Swern oxidation gave the
aldehyde in high yield. However, the aldehyde was
found to decompose on silica and during storage and
was therefore directly oxidized to the carboxylic acid 17
using sodium chlorite at 0 °C. In contrast to the
observations for the tyrosine derivative, THF had to be
excluded from the reaction mixture to produce the acid
17 in a satisfactory yield. Reduction of the nitro group
with ammonium formiate and Pd/C gave the zwitterion,
which was subsequently protected using FmocCl. It was
necessary to keep the reaction time short and to add
FmocCl before the base in order to prevent migration
of the Boc group from the imidazole to the aniline
nitrogen. The building block 18 was incorporated into
Ang II, using the SPPS procedure described for 12
above, to yield the analog 4. However, in this case a
combination of ion-exchange chromatography and RP-
HPLC was required for purification of the final product.
Biologica l Resu lts. Compounds 1-5 were evaluated
in radioligand binding assays relying on displacement
of [125I]Ang II from AT1 receptors in rat liver mem-
branes37 and from AT2 receptors in pig uterus mem-
branes38 (Table 1). Ang II and the highly AT2 receptor-
selective [4-NH2-Phe6]Ang II were used as reference
substances. Compound 2 and compound 5a exhibited
high AT2/AT1 selectivities, and the former compound (2)
displayed a considerable binding affinity to the AT2
receptor (Ki ) 3.0 nM). Compounds 1, 3, 4, and 5b were
devoid of binding affinity to either receptor subtype.
Con for m ation al P r efer en ces. Conformational analy-
sis was performed on the model compounds 1m , 2m ,
and 6m (see Figure 1) of the pseudopeptides 1, 2, and 6
using the MMFFs force field and the General Born
Solvent Accessible (GB/SA)39 water solvation in Mac-
roModel 7.1.40 We previously used the AMBER* force
field and the GB/SA water solvation model in Macro-
Model to calculate the conformational preferences of
turn mimetics and peptide analogues (see, for example,
20, 41, 42). However, when using the AMBER* force
field and the GB/SA water solvation model for the
compounds in this study, there seemed to be a strong
bias toward conformations in which the guanidino group
formed intramolecular hydrogen bonds to the backbone
carbonyl groups. Because of these hydrogen bonds and/
or the lack of high quality force field parameters for the
benzamide moiety, the amide bond near the aromatic
ring became distorted in 1m and 2m . Since this distor-
tion was not observed with MMFFs, this force field was
used in the further analysis. The number of conforma-
F igu r e 1. Model compounds used in conformational analysis
and molecular modeling. A star indicates those atoms that
were superimposed in order to reduce the number of identified
conformations.
tions within 5 kcal/mol of the global energy minimum
found for 1m , 2m , and 6m were 4342, 2901, and 1273,
respectively.43 Three different ring conformations of the
diazepine system in 1m and 2m were identified; two of
these were very similar to the inverse γ-turn and one
was similar to the classic γ-turn.44
To reduce the number of identified conformations and
facilitate the analysis, the minimization convergence
criterion was increased, and only key atoms that
describe important structural features were superim-
posed (Figure 1). This reduced the number of conforma-
tions to 1726, 1217, and 803 for 1m , 2m , and 6m ,
respectively. Inspection of the results showed that in
many of the conformations, the guanidino group still
formed a hydrogen bond with one of the carbonyl
oxygens of the peptide backbone. The hydrogen bond
was especially pronounced in 2m , since this molecule
is more flexible and has an extra carbonyl group as
compared to 1m . This probably does not apply to the
case when 2m binds to the receptor since the Arg2 side
chain in this case is more likely to participate in receptor
interactions45 rather than in intramolecular hydrogen
bonds. Even though the conformational distribution was
biased toward these conformations, we believe that the
general conformational properties of the pseudopeptides
are relevant for our purposes.
Discu ssion
In comparison to the current situation for the AT1
receptor, few investigations of the structure-activity
relationships of the AT2 receptor have been re-
ported.2,21,30,32,45-47 Also, little information about the
functional properties of AT2 receptor ligands, except for
a few compounds (e.g. PD123,319, CGP-42112 and

Selective AT2 Receptor Ligand
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 863
[4-NH2-Phe6]Ang II), is available. Most of the binding
affinity studies performed to date show that modifica-
tion of the linear peptides Ang II or [Sar1]Ang II is well
tolerated by the AT2 receptor. The amino acid residue
Asp1 is not important for affinity to the receptor since
Ang III (Ang II(2-8)) and Ang II have similar affinity
and since many modifications in position 1 are al-
lowed.47 Ang IV (Ang II(3-8)) has been reported to have
a very low affinity to the AT2 receptor, indicating a role
for Arg2 in receptor binding.47 The importance of the
charge and the length of the amino acid side chain in
position two in Ang II has also been shown from
mutagenesis studies of the AT2 receptor.45 Furthermore,
Miura et al. recently introduced Ala into several posi-
tions in Ang II and only observed a minor decrease in
affinity to the AT2 receptor for most of the synthesized
Ala analogues.30 The side chains of Arg2, Tyr4 and His6
seemed to be sensitive to this modification, implying
their possible role as pharmacophore groups.
F igu r e 2. A γ-turn with the i, i+1, and i+2 residues indicated.
F igu r e 3. Stereopresentation (relaxed) of the best fit of the
γ-turn mimetic moiety of 1m /2m (blue) and a model compound
of 5a (orange) to 6m (green). The atoms in the i+1 residue
(except the carbonyl oxygen) and the N atom and CR atom of
the i+2 residue were superimposed in 1m /2m and 6m . The
corresponding atoms in 5a (with Csp2 in the i+1 residue
instead of N) were fitted to 6m . Only the γ-turn/γ-turn mimetic
region is shown, with hydrogens omitted for clarity. The global
minimum was used for 1m , the lowest energy conformation
with an equatorial Tyr substituent for 5a and the lowest
energy conformation with a γ-turn conformation for 6m .
Several cyclic analogues of Ang II have also been
synthesized and evaluated for AT2 receptor affinity.
Marshall’s group performed disulfide monocyclizations
in the 3-5 region of [Sar1]Ang II that resulted in a more
than 10-fold reduction of the affinity to the AT2 receptor,
while affinities to the AT1 receptor subtype were es-
sentially unaffected.21 Notably, disulfide-based bicy-
clizations in the same region delivered [Sar1]Ang II
analogues with high affinities for the AT2 receptor but
with lower AT1 receptor affinities in general.21 We
recently observed that monocyclic methylenedithioether
analogues with Cys and Hcy in the 3 and 5 positions of
Ang II had binding preference for the AT2 receptor. The
most potent analogue (6) in the series, encompassing a
12-membered Cys3,5 cyclized ring system, exhibited a
Ki of 0.62 nM. It preferentially adopted low energy
inverse γ-turn conformations centered at Tyr4, as de-
duced from conformational analysis of model tripep-
tides.32 These results suggested that incorporation of
rigid bicyclic γ-turn mimicking scaffolds in this region
could be a relevant first approach toward less peptide-
like AT2 receptor ligands derived from the native
octapeptide.
at which it leaves. An atom-to-atom comparison of the
backbone atoms of compound 1 (or 2) and 6 shows that
an exact match can be obtained from the i+1 residue
and onward to the C-terminus (Figure 1). However, the
i residue of the γ-turn-like mimetic region in 1 cannot
be mapped onto 6. This difference in geometry will affect
the relative position of the Asp-Arg residues as com-
pared to the rest of the peptide and will thus affect the
ability to adopt similar conformations. Preliminary
conformational analysis of the γ-turn/γ-turn mimetic
region in 1, 5a , and 6, followed by superimposition of
the i+1 and i+2 residue, indicated that the residues
entering the γ-turn region are directed quite differently
(Figure 3). Because of the geometric differences, we
synthesized not only compound 1, which is more similar
to 5a , but also compound 2 with the Val3 residue. The
lipophilic Ile5 side chain was omitted in 1 and 2 for
synthetic reasons and also because it is considered only
to have a conformational stabilizing role and to induce
a turn motif.19,48
Both compound 1 and compound 2 lacked affinity (Ki
> 10 000 nM) to the AT1 receptor. Compound 1 also
lacked affinity (Ki > 10 000 nM) to the AT2, receptor
while, interestingly, compound 2 bound with high
affinity to the same receptor (Ki ) 3.0 nM). We hypoth-
esized that the reason for the lack of affinity of 1 was
not that Val3 was missing per se but rather that the
guanidino group of the Arg2 residue and/or the N-
terminal end of the pseudopeptide could not interact
optimally with the receptor due to the geometric differ-
ences between 1 and 6 (Figure 3). In comparison, Val3
could function as a spacer in compound 2 and allow the
Arg residues and the N-terminal end in the two active
compounds 2 and 6 to interact in similar positions
despite the geometric difference. To test this hypothesis,
we performed conformational analysis of the model
compounds of 1, 2, and 6 as shown in Figure 1. As a
basis for the modeling we omitted the Asp residue, since
it is known not to be important for binding to the AT2
In this work, two different γ-turn mimicing bicyclic
scaffolds, a benzodiazepine and a isoquinoline, differing
only in the presence (1-4) or absence (5a and 5b) of an
NH group have been studied (Chart 1). The isoquinoline
scaffold, which was previously introduced in the 3-5
position of Ang II and evaluated for AT1 receptor
affinity, delivered two diastereoisomers 5a and 5b (a
and b denote single diastereoisomers of unassigned
absolute stereochemistry).20 It was confirmed in this
study that both of the diastereoisomers lacked affinity
to the AT1 receptor. However, when evaluated for AT2
receptor affinity, it was shown that one of the diaster-
oisomers bound fairly well to this receptor (Ki ) 61 nM).
We hypothesized that the stereochemistry of this dias-
tereoisomer corresponds to the natural L-configuration
(5a ).
The other γ-turn-like mimetic incorporated in Ang II
in this study, to give 1-4, contains the benzodiazepine
structure (Chart 1). The benzodiazepine scaffold is more
bulky than the peptide γ-turn, and the angle at which
the peptide chain enters the turn is different from that

864 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Rosenstro¨m et al.
F igu r e 5. Superimposition of 2m (yellow carbons) and 6m
(green carbons) showing the similar positioning of the Arg side-
chain, the N-terminal backbone direction and γ-turn/γ-turn
mimetic moiety. When the γ-turn mimetic moiety is used as
the basis for superimposition, the guanidino group in com-
pound 1m (violet carbons) and the N-terminal acetyl group
cannot reach the same binding site.
regions of space as compared to the guanidino carbon
in 1m , and that the N-terminal end of the peptide
backbone (CH3-CO bond vector) also seems to be
oriented quite differently.
To examine whether 1m and/or 2m could position the
carbon of the guanidino group and the N-terminal
methyl carbon in the same region of space as the
corresponding groups in 6m when the γ-turn/γ-turn
mimetic regions are superimposed, we searched for
conformations that had an root-mean-square (RMS)
distance between these atoms below 3 Å (Figure 4b).
Inspection of Figure 4b shows that the common spatial
positions of these atoms in 1m /6m and 2m /6m is
different and that there are more conformations of
compounds 1m /6m (blue dots) having these atoms in a
similar position than for compounds 2m /6m (yellow
dots). This is most likely because compounds 1m and
6m more closely resemble each other. However, accord-
ing to the above reasoning, none of the sets of conforma-
tions of 1m /6m should correspond to the “bioactive”
conformation. In contrast, there are two areas in Figure
4b that are unique regarding the positioning of the Arg
side chain in 2m and 6m and which cannot be adopted
by 1m /6m . In Figure 5 one of these sets of conformations
from the area behind the azepine ring is shown along
with the conformation of 1m with the guanidino carbon
closest to this set. It can be seen that the direction of
the N-terminal group in 2m and 6m is similar but
slightly separated in distance. The direction of the
outgoing C-terminal group (C(O)-NHCH3) is also dif-
ferent, but the energy penalty for 2m to adopt the same
direction as in 6m is only about 1 kcal/mol (MMFFs
force field). 2m and 6m also have more of an extended
backbone conformation in accordance with the hypoth-
esis of Derae¨t et al.31 To rationalize the above results,
we therefore suggest that the extra Val residue in 2
enables the guanidino group and the N-terminal chain
to adopt optimal conformation(s) for binding (as defined
by the strong binder 6) when the γ-turn/γ-turn mimetic
regions are superimposed. This conformation(s) cannot
be adopted by the inactive analogue 1, assuming the
overlay shown in Figure 5. To challenge the hypotheses
regarding the importance of the Arg side chain, we
F igu r e 4. (a) RMS best fit of 1m /2m to 6m using the atoms
defined in Figure 3. Only the bicyclic moiety, the carbon in
the guanidino group in 1m (blue) and in 2m (yellow), and the
N-terminal CH3-CO bond vector in 1m (methyl carbon red,
carbonyl carbon white) and in 2m (methyl carbon green,
carbonyl carbon white) are shown. The benzodiazepine scaffold
that does not seem to overlap corresponds to the classic γ-turn
conformation. (b) Only those conformations where 1m and/or
2m could position the carbon of the guanidino group and the
N-terminal methyl carbon near those of the corresponding
groups in 6m are shown.
receptor. The His-Pro-Phe residues were also omitted,
since we assumed that they occupy similar conforma-
tional space in the three compounds.
On the basis of the results from the conformational
analysis, the cyclic moieties were superimposed and the
position of the carbon in the guanidino group in 1m
(blue) and in 2m (yellow) were plotted (Figure 4a) for
all identified conformations. We also plotted the N-
terminal CH3-CO bond vector in 1m (methyl carbon
red, carbonyl carbon white) and in 2m (methyl carbon
green, carbonyl carbon white). It can be seen that the
carbon in the guanidino group in 2m reaches further
out from the bicyclic scaffold and occupies different

Selective AT2 Receptor Ligand
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 865
(ca. 20 h) were allowed for incorporation of compounds 12 and
18 as well as for the following amino acid. The Fmoc amino
acids were protected as follows: Asp(Ot-Bu), Arg(Pbf), Tyr(t-
Bu), His(Trt).
Completeness of the coupling reactions was ascertained by
PDMS analysis of cleaved resin samples (ca 1 mg) before and/
or after deprotection.
synthesized compound 3 in which Arg has been substi-
tuted for Ala. This compound lacked affinity for AT2
receptors thus supporting a role of the guanidino group
of the Arg2 residue in receptor binding.
The γ-turn-like scaffold in 1 and 2 was also introduced
in the 5-7 region of Ang II since previous studies have
shown that the AT1 receptor can accommodate peptides
cyclized in this position.49 In that study, the bicyclic
[Sar1,Hcy3,Mpt5]Ang II analogue had an affinity of 1.3
nM to the AT1 receptor and the [Sar1,Hcy5,Mpc7]Ang II
analogue exhibited an affinity of 20 nM. These data
suggest that Ang II might adopt a turn also centered
at His6 when binding to the AT2 receptor. Notably,
compound 4 was inactive at both the AT1 and AT2
receptors. With regard to AT2 receptor binding, support
for the presence of a turn motif located in the 5-7 region
of Ang II has to the best of our knowledge so far not
been reported.
(2,2-Dim eth oxyeth yl)(2-flu or o-5-n itr oben zyl)am in e (8).
HOAc (2.00 mL, 35.0 mmol) and 2,2-dimethoxyethylamine
(24.97 g, 47.3 mmol) were added to the aldehyde 7 (4.01 g,
23.7 mmol) dissolved in 50 mL of MeOH. The reaction mixture
was stirred overnight before NaCNBH3 (3.01 g, 47.9 mmol)
was added in portions. After another night, water was added
and the MeOH was evaporated. The remaining aqueous
solution was extracted with EtOAc, dried over Na2SO4, and
evaporated. Purification by column chromatography (gradient
system CH2Cl2 to 2% MeOH in CH2Cl2) gave 8 as a yellow oil
1
(4.39 g, 72%). H NMR (CDCl3) δ: 8.34 (m, 1H, CH), 8.12 (m,
1H, CH), 7.15 (m, 1H, CH), 4.47 (t, J ) 5.3 Hz, 1H, CH), 3.91
(s, 2H, CH2), 3.36 (s, 6H, CH3), 2.74 (d, J ) 5.3 Hz, 2H, CH2),
2.02 (s, 1H, NH). 13C NMR (CDCl3) δ: 164.3 (d, J ) 257.5 Hz,
CF), 144.3 (C), 129.9 (d, J ) 16.9 Hz, C), 125.9 (d, J ) 6.9 Hz,
CH), 124.5 (d, J ) 10.7 Hz, CH), 116.1 (d, J ) 24.6 Hz, CH),
103.6 (CH), 54.1 (CH3), 50.4 (CH2), 46.2 (CH2). GC-MS (M+):
258. Anal. (C11H15FN2O4) C, H, N.
Con clu sion
In summary, four analogues of angiotensin II encom-
passing a benzodiazepine-based γ-turn-like peptidomi-
metic have been synthesized. One of these analogues
(2) binds with high affinity to the Ang II AT2 receptor.
Our conformational analysis suggested that the guani-
dino group of the Arg2 residue and the N-terminal end
of the active analogues 2 and 6 could access common
regions of space inaccessible to the compound that
lacked affinity (1). To challenge this hypothesis, com-
pound 3, which lacks the Arg side chain, was synthe-
sized. The latter compound had no affinity to the AT2
receptor, which supports the hypothesis and emphasizes
the importance of the Arg side chain for binding to the
receptor.
(2S)-3-(4-ter t-Bu t oxyp h en yl)-N-(2,2-d im et h oxyet h yl)-
2-[(9H-flu or en -9-ylm eth oxylcar bon yl)am in o]-N-(2-flu or o-
5-n itr oben zyl)p r op ion a m id e (9). Fmoc-Tyr(t-Bu)-OH (5.12
g, 11.1 mmol), HATU (4.25 g, 11.2 mmol) and DIEA (5.00 mL,
28.7 mmol) were added to a solution of the amine 8 (2.71 g,
10.5 mmol) in 50 mL of CH2Cl2. The reaction mixture was
stirred overnight. After addition of water, the mixture was
extracted with EtOAc. The organic phase was washed with 1
M KHSO4, water, saturated NaHCO3 solution, and brine, dried
(Na2SO4), and evaporated. The residue was purified by column
chromatography (pentane:EtOAc 3:1) to give 9 as a yellow
foam (6.65 g, 90%). [R]22 ) -13° (c ) 1.02, MeOH). The
D
product was observed as rotamers at 20 °C in NMR, increasing
the temperature to 120 °C results in one set of peaks, but they
1
are still very broad in the aliphatic region. H NMR (DMSO-
Exp er im en ta l Section
d6) δ: 8.20 (m, 1H, CH), 8.08 (m, 1H, CH), 8.02 and 7.95
(minor) (m, 1H, NH), 7.87 (m, 2H, 2 × CH), 7.67 and 7.62
(minor) (m, 2H, 2 × CH), 7.49 (m, 1H, CH), 7.40 (m, 2H, 2 ×
CH), 7.30 (m, 2H, 2 × CH), 7.18 and 7.05 (minor) (m, 2H, 2 ×
CH), 6.81 and 6.74 (minor) (m, 2H, 2 × CH), 4.73 and 4.50
(minor) (m, 1H, CH), 4.71 (m, 1H, CH2), 4.58 (m, 1H, CH2),
4.38 (m, 1H, CH), 4.20 (m, 1H, CH2), 4,14 (m, 1H, CH2), 3.83
Gen er a l Meth od s. 1H and 13C NMR spectra were recorded
on a J EOL J NM-EX400 at 400 and 100.5 MHz, respectively.
Spectra were recorded at ambient temperature unless other-
wise noted. Chemical shifts are reported as δ values (ppm)
referenced to δ 7.26 ppm CHCl3, δ 77.0 ppm CDCl3, δ 2.50
ppm, δ 39.5 (DMSO-d6), δ 1.98 CH3CN, δ 117.7 CD3CN, CH3-
OH δ 3.30 ppm and CD3OD δ 49.15 ppm. Optical rotations
were measured on a Perkin-Elmer model 241 polarimeter.
Elemental analyses were performed by Mikro Kemi AB,
Uppsala Sweden. Mass spectra were recorded on LC-MS
and 3.57 (minor) (m, 1H, CH2), 3.30-3.18 (m, 7H, 2 × CH3
+
CH2), 2.95-2.81 (m, 2H, CH2), 1.19 and 1.15 (s, 9H, 3 × CH3).
13C NMR (DMSO-d6) δ (major peaks): 173.0 (C), 163.6 (d, J )
253 Hz, CF), 156.0 (C), 155.6 (C), 153.6 (C), 144.0 (C), 143.7
(C), 140.7 (C), 132.3 (C), 129.8 (CH), 127.6 (CH), 127.2 (CH),
125.3 (CH), 124.84 (CH), 124.81 (CH), 123.5 (CH), 120.1 (CH),
116.8 (d, 24.5, CH), 103.3 (CH), 77.6 (C), 65.9 (CH), 55.3 (CH3),
54.3 (CH3), 52.3 (CH), 50.0 (CH2), 46.6 (CH2), 44.3 (CH2), 36.5
utilizing electron-spray ionization (ESI) or on
a GC-MS,
equipped with a nonpolar capillary column, utilizing electron
impact (EI) at an ionizing energy of 70 eV.
Thin-layer chromatography (TLC) was performed using
aluminum sheets precoated with silica gel 60 F254 (0.2 mm, E.
Merck). The spots were identified using UV-detection and/or
by spraying with 3% methanolic solution of ninhydrin followed
by heating. Flash column chromatography was performed
using Merck silica gel 60 (40-63 µm). The starting material
7 was synthesized according to a previously published proce-
dure.50
Solid -P h a se P ep tid e Syn th esis (SP P S). The starting
Fmoc-His(Trt)-Pro-Phe-Wang resin (0.25 mmol) was synthe-
sized manually, in a 5 mL disposable syringe equipped with a
porous polyethylene filter, using standard Fmoc/t-Bu condi-
tions. Removal of the Fmoc group was achieved by treatment
with 20% piperidine/DMF for 2 + 10 min. The Fmoc amino
acids (0.75 mmol) were coupled by reaction with HBTU (0.75
mmol) and diisopropylethylamine (DIEA) (1.50 mmol) in DMF
(1.2 mL) for 45 min.
(CH2), 28.5 (CH3). LC-MS (M + H+): 700.0. Anal. (C39H42
FN3O8) C, H, N.
-
(2S)-2-(4-ter t-Bu toxyben zyl)-4-(2,2-d im eth oxyeth yl)-7-
n itr o-1,2,4,5-tetr ah ydr oben zo[1,4]diazepin -3-on e (10). DBU
(1.41 mL, 9.43 mmol) was added to 9 (6.06 g, 8.66 mmol) in
dry THF (15 mL). After 2 h, the reaction was complete
according to TLC (2% MeOH in CH2Cl2). The reaction mixture
was evaporated, and the residue was purified by column
chromatography (CH2Cl2 to elute Fmoc-related side products,
then 2% MeOH in CH2Cl2). The free amine was obtained in
3.98 g. The amine (3.98 g, 8.34 mmol) was dissolved in 170
mL of DMSO and 1.7 mL of water and 3.4 mL of Et3N were
added. After 3 days at room temperature, EtOAc and saturated
NaHCO3 (aq) were added. The layers were separated and the
aqueous layer was extracted with EtOAc. The combined
organic layers were washed with water and brine, dried
(Na2SO4) and evaporated. The crude residue was purified by
column chromatography (gradient CH2Cl2 to 1% MeOH in CH2-
Cl2) to give 10 as a yellow foam (2.55 g, 64% over two steps).
The angiotensin II analogues were synthesized in 2 mL
syringe vessels using a similar procedure. However, PyBOP/
DIEA were used for activation, and prolonged reaction times

866 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Rosenstro¨m et al.
1
[R]22 ) -327° (c ) 1.03, MeOH). H NMR (CDCl3) δ: 7.89-
Hz, 1H, CH2). 13C NMR (CD3OD) δ: 173.9 (C), 173.5 (C), 157.2
(C), 156.4 (C), 145.4 (C), 143.7 (C), 142.8 (C), 131.6 (C), 131.5
(CH), 130.3 (C), 128.9 (CH), 128.3 (CH), 126.3 (CH), 122.3
(CH), 122.0 (CH), 121.3 (C), 121.1 (CH), 118.5 (CH), 116.7
(CH), 67.7 (CH2), 57.7 (CH), 53.9 (CH2), 50.3 (CH2), 48.6 (CH),
37.4 (CH2). LC-MS (M + H+): 564.1. Anal. (C33H29N3O6‚H2O)
C, H, N.
D
7.40 (m, 2H, CH and CH), 7.19 (m, 2H, 2 × CH), 6.97 (m, 2H,
2 × CH), 6.35 (m, 1H, CH), 5.37 (d, J ) 16.6 Hz, 1H, CH2),
5.00 (dd, J ) 5.3, 9.0 Hz, 1H, CH), 4.42 (bs, 1H, NH), 4.34
(dd, J ) 4.8, 5.4 Hz, 1H, CH), 4.06 (d, J ) 16.6 Hz, 1H, CH2),
3.71 (dd, J ) 4.8, 14.0 Hz, 1H, CH2), 3.54 (dd, J ) 5.4, 14.0
Hz, 1H, CH2), 3.37 (s, 3H, CH3), 3.36 (dd, J ) 5.3, 14.7 Hz,
1H, CH2), 3.21 (s, 3H, CH3), 2.89 (dd, J ) 9.0, 14.7 Hz, 1H,
CH2), 1.34 (s, 9H, 3 × CH3). 13C NMR (CDCl3) δ: 169.1 (C),
154.5 (C), 151.0 (C), 137.8 (C), 130.9 (C), 129.6 (CH), 126.6
(CH), 125.3 (CH), 124.6 (CH), 118.4 (C), 115.3 (CH), 103.3
(CH), 78.6 (C), 54.9 (CH and CH3), 54.8 (CH3), 52.1 (CH2), 49.5
(CH2), 36.1 (CH2), 28.8(CH3). LC-MS (M + H+): 457.9. Anal.
(C24H31N3O6) C, H, N.
2-[(2-Flu or o-5-n itr oben zyl)am in o]eth an ol (13). The ben-
zaldehyde 7 (1.00 g, 5.92 mmol) was dissolved in 60 mL of
MeOH. HOAc (0.50 mL, 8.75 mmol), 2-aminoethanol (1.80 mL,
29.9 mmol), and NaCNBH3 (0.370 g, 5.89 mmol) were added,
and the reaction mixture was heated to reflux. The conversion
was complete after 1.5 h according to TLC (10% MeOH in CH2-
Cl2). The mixture was cooled to room temperature, acidified
to pH 1 by addition of 2 M HCl, and partly evaporated. The
residue was washed with ether and 6 M NaOH was added to
adjust the pH to 11. The water layer was extracted with diethyl
ether, and the combined organic layers were dried over K2-
CO3, evaporated, and purified by column chromatography (3%
MeOH in CH2Cl2) to give 13 as a yellow solid (1.07 g, 85%).
1H NMR (CDCl3) δ: 8.33 (m, 1H, CH), 8.17 (m, 1H, CH), 7.17
(m, 1H, CH), 3.91 (s, 2H, Ar-CH2), 3.69 (m, 2H, CH2), 2.80
(m, 2H, CH2), 1.99 (br s, 2H, OH and NH). 13C NMR (CDCl3)
δ: 164.4 (d, J ) 256 Hz, CF), 144.0 (C), 129.0 (d, J ) 17.1 Hz,
C), 125.9 (d, J ) 6.1 Hz, CH), 124.8 (d, J ) 9.8 Hz, CH), 116.3
(d, J ) 24.4 Hz, CH), 61.0 (CH2), 50.5 (CH2), 46.2 (CH2). LC-
MS (M + H+): 214.8. Anal. (C9H11FN2O3) C, H, N.
[(2S )-2-(4-H y d r o x y b e n z y l)-7-n i t r o -3-o x o -1,2,3,5-
tetr a h yd r oben zo[1,4]d ia zep in -4-yl]a cetic Acid (11). Com-
pound 10 (400 mg, 0.87 mmol) was dissolved in 16 mL of TFA/
CH2Cl2 (1:1). After 1 h, the aldehyde was deprotected according
to TLC (10% MeOH in CH2Cl2), and the reaction mixture was
evaporated. The residue was dissolved in EtOAc and water,
and the layers were separated. The organic layer was washed
with water, dried over Na2SO4, and evaporated. The obtained
aldehyde was dissolved in 3 mL of THF and 4.5 mL of t-BuOH.
The solution was cooled on an ice-bath, and cyclohexene (1.43
mL, 14.1 mmol) was added. A freshly prepared ice-cold solution
of NaClO2 (752 mg, 80%, 6.65 mmol) and NaH2PO4‚H2O (760
mg, 5.53 mmol) in 3.5 mL of water was added dropwise. After
45 min at 0 °C, the reaction was complete according to TLC
(20% MeOH in CH2Cl2). EtOAc and water were added, and
the layers were separated. The organic layer was washed with
brine, dried (Na2SO4), and evaporated. After purification by
column chromatography (gradient 2% MeOH in CH2Cl2 to 30%
MeOH in CH2Cl2), the product was redissolved in EtOAc. The
EtOAc solution was washed with 1 M KHSO4, dried over Na2-
[2-(ter t-Bu tyld ip h en ylsila n yloxy)eth yl]-(2-flu or o-5-n i-
tr oben zyl)a m in e (14). DBU (1.40 mL, 9.36 mmol) and
TBDPSCl (2.40 mL, 9.38 mmol) were added to a solution of
13 (1.45 g, 6.78 mmol) in 35 mL of CH3CN. The reaction
mixture was stirred overnight. After evaporation, the residue
was dissolved in CH2Cl2, washed with water and brine, dried
over Na2SO4, and evaporated. Purification by column chro-
matography (2% MeOH in CH2Cl2) gave 14 as a yellow oil (3.02
g, 98%).
SO4, and evaporated to give 11 as a yellow foam (249 mg, 77%).
1
[R]22 ) -399° (c ) 0.54, MeOH). H NMR (CD3CN) δ: 7.92
1H NMR (CDCl3) δ: 8.39 (m, 1H, CH), 8.17 (m, 1H,
D
(m, 1H, CH), 7.88 (m, 1H, CH), 7.24 (m, 2H, 2 × CH), 6.79 (m,
2H, 2 × CH), 6.59 (m, 1H, CH), 6.37 (br s, 2H, 2 × OH), 5.48
(d, J ) 17.2 Hz, 1H, CH2), 5.46 (bs, 1H, NH), 5.12 (dd, J )
6.2, 7.7 Hz, 1H, CH), 4.29 (d, J ) 17.6 Hz, 1H, CH2), 4.14 (d,
J ) 17.6 Hz, 1H, CH2), 4.03 (d, J ) 17.2 Hz, 1H, CH2), 3.24
(dd, J ) 6.2, 14.3 Hz, 1H, CH2), 2.81 (dd, J ) 7.7, 14.3 Hz,
1H, CH2). 13C NMR (CD3CN) δ: 170.2 (C), 170.1 (C), 156.0
(C), 152.6 (C), 137.4 (C), 130.9 (CH), 128.8 (C), 126.5 (CH),
125.3 (CH), 118.9 (C), 115.5 (CH), 115.4 (CH), 55.4 (CH), 51.6
(CH2), 48.5 (CH2), 35.5 (CH2). LC-MS (M + H+): 371.8. Anal.
(C18H17N3O6‚1/2 H2O) C, H, N.
[(2S)-7-(9H-F lu or en -9-ylm eth oxyca r bon yla m in o)-2-(4-
h y d r o x y b e n zy l)-3-o x o -1,2,3,5-t e t r a h y d r o b e n zo [1,4]-
d ia zep in -4-yl]a cetic Acid (12). HCO2NH4 (300 mg, 4.76
mmol) was added to 11 (80 mg, 0.22 mmol) and Pd/C (23 mg
10%, 0.02 mmol) in 2.2 mL of MeOH and stirred under
nitrogen in a sealed tube. After 3 h, the reaction was completed
according to TLC (20% MeOH in CH2Cl2). Pd/C was removed
by filtration. The crude product obtained after evaporation was
dissolved in 4 mL of 10% Na2CO3 (aq) and 2 mL of dioxane
and cooled to 0 °C. FmocCl (112 mg, 0.43 mmol) dissolved in
2 mL of dioxane was added dropwise, and the reaction mixture
was allowed to reach room temperature. After 24 h, 10%
aqueous citric acid was added until pH 8-9. The mixture was
washed several times with ether. New ether was added to the
water phase, which was acidified to pH 3 by addition of 10%
aqueous citric acid. The layers were separated, and the water
layer was extracted with ether and EtOAc. The organic layers
were combined and washed with water, dried over Na2SO4,
and evaporated. The product 12 was obtained as white foam
(57 mg, 47%). [R]22D ) -107° (c ) 0.42, MeOH). 1H NMR (CD3-
OD) δ: 7.76 (m, 2H, 2 × CH), 7.65 (m, 2H, 2 × CH), 7.36 (m,
2H, 2 × CH), 7.28 (m, 2H, 2 × CH), 7.14 (m, 2H, 2 × CH),
7.08-6.90 (m, 2H, 2 × CH), 6.71 (m, 2H, 2 × CH), 6.46 (m,
1H, CH), 5.35 (d, J ) 16.3 Hz, 1H, CH2), 4.79 (dd, J ) 5.6, 8.1
Hz, 1H, CH), 4.48 (d, J ) 17.4 Hz, 1H, CH2), 4.39 (d, J ) 6.2
Hz, 2H, CH2), 4.20 (m, 1H, CH), 3.86 (m, 2H, CH2 and CH2),
3.15 (dd, J ) 5.6, 14.3 Hz, 1H, CH2), 2.72 (dd, J ) 8.1, 14.3
CH), 7.66 (m, 4H, CH), 7.48-7.36 (m, 6H, CH), 7.18 (m, 1H,
CH), 3.96 (s, 2H, CH2), 3.83 (t, J ) 5.1 Hz, 2H, CH2), 2.80 (t,
J ) 5.1 Hz, 2H, CH2), 2.70 (br s, 1H, NH), 1.07 (s, 9H, 3 ×
CH3). 13C NMR (CDCl3) δ: 164.7 (d, J ) 256.8 Hz, CF), 144.3
(d, J ) 1.5 Hz, C), 135.5 (C), 133.3 (C), 129.7 (CH), 127.9, 127.7
(CH), 126.0 (d, J ) 6.9 Hz, CH), 124.6 (d, J ) 10.0 Hz, CH),
116.2 (d, J ) 24.6 Hz, CH), 62.8 (CH2), 50.7 (CH2), 46.1 (CH2),
26.8 (CH3), 19.1 (C). LC-MS (M + H+): 454.2. Anal. (C25H29
-
FN2O3Si) C, H, N.
1-(ter t-Bu toxyca r bon yl)-5-[(2S)-2-[[2-(ter t-bu tyld ip h e-
n ylsila n yloxy)eth yl]-(2-flu or o-5-n itr oben zyl)ca r ba m oyl]-
2-(9H -flu or e n -9-ylm e t h oxyca r b on yla m in o)e t h yl]im -
id a zole (15). The amine 14 (4.60 g, 10.2 mmol) was dissolved
in 80 mL of CH2Cl2. HATU (3.89 g, 10.2 mmol), Fmoc-L-His-
(Boc)-OH (4.89 g, 10.2 mmol), and DIEA (5.30 mL, 30.4 mmol)
were added. The reaction mixture was stirred at room-
temperature overnight. Water and EtOAc were added, and the
layers were separated. The aqueous layer was extracted with
EtOAc, and the combined organic layers were washed with 1
M KHSO4, water, saturated NaHCO3, and brine, dried over
Na2SO4, and evaporated. The residue was purified by column
chromatography (CH2Cl2 to 2% MeOH in CH2Cl2) to give 15
as white foam (7.98 g, 86%). [R]23 ) -19° (c ) 1.04, MeOH).
D
Rotamers in NMR. An attempt to run NMR at 120 °C in
DMSO-d6 failed since the substance was unstable at these
conditions. 1H NMR (CDCl3) δ: 8.21 and 8.18 (minor) (m, 1H,
CH), 8.13 and 8.10 (minor) (m, 1H, CH), 7.99 and 7.94 (minor)
(m, 1H, CH), 7.76 (m, 2H, 2 × CH), 7.70-7.53 (m, 6H, 6 ×
CH), 7.47-7.28 (m, 10H, 10 × CH), 7.22-7.08 (m, 2H, 2 ×
CH), 6.01 and 5.87 (minor) (d, J ) 8.2 Hz, 1H, NH), 5.06 (m,
1H, CH), 4.93 (d, J ) 15.9 Hz, 1H, CH2), 4.57 (d, J ) 15.9 Hz,
1H, CH2), 4.44-4.13 (m, 3H, CH2 and CH), 3.89 (m, 2H, CH2),
3.67 (m, 2H, CH2), 3.05 (dd, J ) 6.2, 14.8 Hz, 1H, CH2), 2.93
(dd, J ) 6.6, 14.8 Hz, 1H, CH2), 1.56 (s, 9H, 3x CH3), 1.02 (s,
9H, 3 × CH3). 13C NMR (CDCl3) δ (major peaks): 172.3 (C),
164.0 (d, J ) 256.8 Hz, CF), 155.6 (C), 146.6 (C), 144.4 (d, J )
1.5 Hz, C), 143.8 (C), 141.1 (C), 137.7 (C), 136.6 (CH), 135.4
(CH), 132.6 (C), 129.8 (CH), 127.7 (CH), 127.6 (CH), 127.0

Selective AT2 Receptor Ligand
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 867
(CH), 126.2 (C), 125.6 (d, J ) 6.2 Hz, CH), 125.2 (CH), 124.7
(d, J ) 10.0 Hz, CH), 119.8 (CH), 116.0 (d, J ) 24.6 Hz, CH),
114.7 (CH), 85.6 (C), 67.0 (CH2), 62.6 (CH2), 50.2 (CH), 49.8
(CH2), 47.0 (CH), 44.1 (CH2), 31.3 (CH2), 27.7 (CH3), 26.7 (CH3),
18.9 (C). LC-MS (M + H+): 912.2. Anal. (C51H54FN5O8Si‚H2O)
C, H, N.
dissolved in EtOAc and 0.5 M KHSO4, the layers were
separated and the aqueous layer was extracted with EtOAc.
The organic layer was washed with brine, dried (Na2SO4), and
evaporated. The product 17 (123 mg, 59%) was obtained as
yellow foam. [R]22 ) -316° (c ) 0.53, MeOH). 1H NMR (CD3-
D
OD) δ: 8.17 (m, 1H, CH), 7.94 (m, 1H, CH), 7.85 (m, 1H, CH),
7.40 (m, 1H, CH), 6.60 (m, 1H, CH), 5.59 (d, J ) 16.6 Hz, 1H,
CH2), 5.29 (m, 1H, CH), 4.39 (d, J ) 17.5 Hz, 1H, CH2), 4.15
(d, J ) 17.5 Hz, 1H, CH2), 4.08 (d, J ) 16.6 Hz, 1H, CH2),
3.20 (dd, J ) 6.5, 15.0 Hz, 1H, CH2), 2.89 (dd, J ) 7.6, 15.0
Hz, CH2), 1.61 (s, 9H, 3 × CH3). 13C NMR (CD3OD) δ: 172.4
(C), 172.2 (C), 153.9 (C), 148.2 (C), 140.1 (C), 138.5 (CH), 138.4
(C), 127.5 (CH), 126.2 (CH), 119.5 (C), 116.8 (CH), 116.5 (CH),
87.3 (C), 54.5 (CH), 53.1 (CH2), 49.8 (CH2), 29.8 (CH2), 28.1
(CH3). LC-MS (M + H+): 446.0. Anal. (C20H23N5O7‚H2O) C,
H, N.
1-(ter t-Bu t oxyca r b on yl)-5-[(2S)-4-(2-h yd r oxyet h yl)-7-
n itr o-3-oxo-2,3,4,5-tetr a h yd r o-1H-ben zo[1,4]d ia zep in -2-
ylm eth yl]im id a zole (16). DBU (0.910 mL, 5.98 mmol) was
added to a solution of 15 (5.318 g, 5.83 mmol) in 30 mL of
THF. After 1 h at room temperature, the reaction was
completed according to TLC (10% MeOH in CH2Cl2). The
MeOH was evaporated and the product purified by column
chromatography (gradient CH2Cl2 to 2% MeOH in CH2Cl2) to
yield 3.70 g of the deprotected amine. The amine was dissolved
in 90 mL of DMSO, and 0.90 mL of water and 2.60 mL of Et3N
were added. The reaction mixture was stirred for 72 h at room
temperature. EtOAc and NaHCO3 (aq) were added, and the
layers were separated. The aqueous layer was extracted with
EtOAc, and the combined organic layers were washed with
water and brine, dried over Na2SO4, and evaporated. The
residue was purified through column chromatography (gradi-
ent CH2Cl2 to 3% MeOH in CH2Cl2), and the product 16 was
obtained as yellow foam (2.05 g, 81%). [R]23D ) -300° (c ) 1.05,
MeOH). 1H NMR (CDCl3) δ: 8.02 (d, J ) 1.4 Hz, 1H, CH),
7.89 (m, 1H, CH), 7.86 (m, 1H, CH), 7.28 (d, J ) 1.4 Hz, 1H,
CH), 6.50 (m, 1H, CH), 6.07 (d, J ) 2.2 Hz, 1H, NH), 5.40 (d,
J ) 16.9 Hz, 1H, CH2), 5.05 (ddd, J ) 2.2, 4.5, 9.0 Hz, 1H,
CH), 3.98 (d, J ) 16.9 Hz, 1H, CH2), 3.77 (m, 3H, CH2 and
CH2), 3.60 (m, 1H, CH2), 3.13 (dd, J ) 4.5, 15.2 Hz, 1H, CH2),
1-(ter t-Bu toxycar bon yl)-5-[(2S)-4-car boxym eth yl-7-(9H-
flu or en -9-ylm eth oxycar bon ylam in o)-3-oxo-2,3,4,5-tetr ah y-
d r o-1H -b en zo[1,4]d ia zep in -2-ylm et h yl]im id a zole (18).
HCO2NH4 (128 mg, 2.03 mmol) was added to a mixture of Pd/C
(7 mg 10%, 7 µmol) and 17 (30 mg, 0.07 mmol) in 1 mL of
MeOH. The reaction, was performed under nitrogen in a sealed
tube, and was complete within 1.5 h according to TLC (20%
MeOH in CH2Cl2). More MeOH was added, and the Pd/C was
removed by filtration. After evaporation, the crude product was
immediately suspended in 2 mL of dioxane and cooled to 0
°C. FmocCl (26 mg, 0.10 mmol) was added, followed by
dropwise addition of 1 mL of 10% aqueous Na2CO3, pH 8-9.
After 1.5 h, ether and water were added. The layers were
separated, and the aqueous layer was washed with ether until
TLC showed that all impurities with Rf values higher than
that of the product had been removed. EtOAc was added to
the aqueous layer, which was acidified to pH 3 using 10% citric
acid. The layers were separated, and the aqueous layer was
extracted with EtOAc. The combined organic layers were
washed with water, dried (Na2SO4), and evaporated to give
18 as white foam (14 mg, 33%). 1H NMR (CD3OD) δ: 8.12 (m,
1H, CH), 7.78 (m, 2H, 2 × CH), 7.67 (m, 2H, 2 × CH), 7.43-
7.33 (m, 3H, 2 × CH and CH), 7.30 (m, 2 × CH), 7.11-6.92
(m, 2H, 2 × CH), 6.52 (m, 1H, CH), 5.49 (d, J ) 16.4 Hz, 1H,
CH2), 4.97 (dd, J ) 6.0, 7.7 Hz, 1H, CH), 4.51 (d, J ) 17.4,
1H, CH2), 4.41 (m, 2H, CH2), 4.22 (m, 1H, CH), 3.90 (d, J )
17.4 Hz, 1H, CH2), 3.86 (d, J ) 16.4 Hz, 1H, CH2), 3.13 (dd, J
) 6.0, 14.8 Hz, 1H, CH2), 2.83 (dd, J ) 7.7, 14.8 Hz, 1H, CH2),
1.61 (s, 9H, 3 × CH3). 13C NMR (CD3OD) δ: 173.4 (C), 173.3
(C), 156.5 (C), 148.4 (C), 145.5 (C), 145.4 (C), 143.7 (C), 142.8
(C), 140.9 (C), 138.2 (CH), 130.3 (C), 129.0 (CH), 128.3 (CH),
126.3 (CH), 122.4 (CH), 122.1 (CH), 121.1 (CH), 118.5 (CH),
116.5 (CH), 87.2 (C), 67.8 (CH2), 55.4 (CH), 53.9 (CH2), 50.2
(CH2), 48.6 (CH), 30.5 (CH2), 28.9 (CH3). Accurate mass
(C35H35N5O7) calculated 637.2536, found 637.2519.
2.98 (dd, J ) 9.0, 15.2 Hz, 1H, CH2), 1.60 (s, 9H, 3 × CH3). 13
C
NMR (CDCl3) δ: 169.9 (C), 151.7 (C), 146.7 (C), 138.9 (C), 137.4
(C), 136.9 (CH), 126.1 (CH), 125.6 (CH), 117.6 (C), 115.5 (CH),
115.1 (CH), 85.9 (C), 61.4 (CH2), 54.4 (CH), 52.1 (CH2), 50.5
(CH2), 29.0 (CH2), 27.9 (CH3). LC-MS (M + H+): 432.1. Anal.
(C20H25N5O6‚H2O) C, H, N.
1-(ter t-Bu toxyca r bon yl)-5-((2S)-4-ca r boxym eth yl-7-n i-
tr o-3-oxo-2,3,4,5-tetr a h yd r o-1H-ben zo[1,4]d ia zep in -2-yl-
m eth yl)im id a zole (17). A solution of oxalyl chloride (58 µL,
0.68 mmol) in 3 mL of CH2Cl2 was cooled to -78 °C. DMSO
(100 µL, 1.41 mmol) was added dropwise, and the mixture was
stirred 15 min before a solution of 16 (250 mg, 0.58 mmol) in
2.5 mL of CH2Cl2 was added dropwise over 10 min. After
another 30 min at -78 °C, Et3N (275 µL, 2.01 mmol) was added
and the reaction mixture was allowed to warm to -30 °C over
50 min. Water (2 mL) was added to quench the reaction. The
layers were separated, and the aqueous layer was extracted
with CH2Cl2. The combined organic layers were washed with
1 M KHSO4, water, saturated NaHCO3 solution, and brine,
dried (Na2SO4), and evaporated. The aldehyde was obtained
as yellow foam (233 mg, 94%). [R]22 ) -266° (c ) 1.03, CH2-
D
An g II An a logu e 1. Fmoc-His(Trt)-Pro-Phe-Wang resin
(143.7 mg, 58.9 µmol) was weighed into a 2 mL reaction vessel
and allowed to swell in DMF (1.5 mL) for 20 min. The Fmoc
group was removed by a three-step treatment with 20%
piperidine in DMF (3 × 1.5 mL, 1 + 3 + 10 min), and the
polymer was then washed with DMF (6 × 1.5 mL, 6 × 1 min).
Compound 12 (41.0 mg, 72.8 µmol; contaminated with the NO2
derivative 11) and PyBOP (37.9 mg, 72.8 µmol) were dissolved
in DMF (1.0 mL) in the presence of DIEA (30.4 µL, 175 µmol)
and allowed to react with the resin for 20 h. LC/MS analysis
after cleavage of an analytical sample showed that the coupling
was incomplete. The resin was washed with DMF (6 × 1.5 mL,
6 × 1 min) and recoupled with 12 (25.0 mg, 44.4 µmol), PyBOP
(23.1 mg, 44.4 µmol) and DIEA (15.5 µL, 88.8 µmol) in DMF
(1 mL) for 17.5 h. After washing with DMF (6 × 1.5 mL, 6 ×
1 min), the resin was deprotected and washed as described
above. Fmoc-Arg(Pbf)-OH (130.7 mg, 174 µmol) was then
coupled for 17 h using PyBOP (90.5 mg, 174 µmol) and DIEA
(60.6 µL, 348 µmol) in DMF (0.7 mL). Fmoc-Asp(Ot-Bu)-OH
was introduced in the same way, using a coupling time of 4 h.
The peptide resin was then deprotected, washed with DMF (6
× 1.5 mL, 6 × 1 min) and CH2Cl2 (6 × 2 mL, open column),
and dried to yield 156.2 mg.
Cl2), 1H NMR (CDCl3) δ: 9.59 (s, 1H, CH), 8.13 (d, J ) 1.3 Hz,
1H, CH), 7.90 (m, 1H, CH), 7.79 (m, 1H, CH), 7.34 (d, J ) 1.3
Hz, 1H, CH), 6.60 (m, 1H, CH), 6.34 (br s, 1H, NH), 5.50 (d, J
) 17.0 Hz, 1H, CH2), 5.12 (m, 1H, CH), 4.67 (d, J ) 18.5 Hz,
1H, CH2), 4.15 (d, J ) 18.5 Hz, 1H, CH2), 3.73 (d, J ) 17.0
Hz, 1H, CH2), 3.17 (dd, J ) 4.7, 15.1 Hz, 1H, CH2), 3.06 (dd,
J ) 8.6, 15.1 Hz, 1H, CH2), 1.61 (s, 9H, 3 × CH3). 13C NMR
(CDCl3) δ: 196.2 (CH), 169.6 (C), 151.7 (C), 146.5 (C), 138.4
(C), 137.4 (C), 136.6 (CH), 125.9 (CH), 125.6 (CH), 117.0 (C),
115.7 (CH), 115.3 (CH), 86.2 (C), 57.4 (CH2), 54.0 (CH), 52.1
(CH2), 27.9 (CH2), 27.8 (CH3).
A suspension of the aldehyde (220 mg, 0.47 mmol) in 13 mL
of t-BuOH and 2.65 mL of cyclohexene was cooled to 0 °C, and
a freshly prepared ice-cold solution of NaClO2 (590 mg (80%),
5.22 mmol) and NaH2PO4‚H2O (560 mg, 4.08 mmol) in 5.5 mL
of water was added dropwise over 10 min. The reaction
mixture was stirred for 1.5 h at 0 °C. Water and EtOAc were
added, and the layers were separated. The aqueous layer was
extracted with EtOAc, and the combined organic layers were
washed with water and brine, dried (Na2SO4), and evaporated.
After purification by column chromatography (gradient 2%
MeOH in CH2Cl2 to 20% MeOH in CH2Cl2), the product was

868 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Rosenstro¨m et al.
At all stages, unless otherwise indicated, PDMS analysis
of deprotected samples showed the expected m/z value for the
major peak. Incomplete couplings were not observed. However,
after the introduction of 12, the C-terminal tripeptide acylated
by the nitro derivative 11 was detected as an impurity in all
samples. Analysis of the Fmoc protected resin after coupling
of Arg and Asp showed that 2 equiv of the amino acid were
initially incorporated.
Triethylsilane (100 µL) and 95% aq TFA (4 mL) were added
to the partially protected peptide resin (147.2 mg), and the
mixture was agitated by rotation for 2 h. The polymer was
filtered off and washed with TFA (3 × 0.7 mL). The yellow
filtrate was evaporated in a stream of nitrogen to ca. 4 mL
and the product precipitated by the addition of ether (25 mL).
After cooling in an ice-bath, the precipitate was collected by
centrifugation, washed with ether (4 × 15 mL), and dried to
give 53.5 mg.
The crude peptide was dissolved in a mixture of H2O (7.6
mL) and MeCN (0.4 mL) and purified in two runs by RP-HPLC
on a 10 µm Vydac C18 column (2.2 × 25 cm) with an 80 min
gradient of 5-45% MeCN in 0.1% aq TFA at a flow rate of 3
mL/min. The separation was monitored at 230 nm and by
PDMS and/or analytical RP-HPLC of selected fractions. The
yield of the purified and lyophilized peptide was 10.7 mg (19%).
PDMS (Mw 994.1): 995.4 (M + H+); amino acid analysis: Asp,
0.99; Arg, 1.00; His, 1.03; Pro, 0.98; Phe, 0.99.
An g II An a logu e 2. Reaction of the starting Fmoc-His(Trt)-
Pro-Phe-Wang resin (95.5 mg, 39.2 µmol) essentially as
described above for 1, although including coupling of Val,
produced 109.8 mg of the partially protected peptide resin.
Cleavage (103.2 mg) with TFA-H2O-triethylsilane gave 38.7
mg of the crude peptide, which was purified according to the
above procedure. Yield: 9.1 mg (23%); PDMS (Mw 1093.2):
1095.2 (M + H+); amino acid analysis: Asp, 0.99; Arg, 0.99;
Val, 0.98; His, 1.01; Pro, 1.01; Phe, 1.00.
An g II An a logu e 3. This compound was similarly prepared
from the same starting resin (31.9 mg, 25.6 µmol) by consecu-
tive coupling of Val, Ala, and Asp. Cleavage of the partially
protected peptide resin (42.1 mg) and purification by RP-HPLC
produced 10.1 mg (39.1%) of the desired product. PDMS (Mw
1008.1): 1008.8 (M + H+); amino acid analysis: Asp, 1.00; Ala,
0.99; Val, 1.00; His, 1.02; Pro, 1.00; Phe, 0.99.
An g II An a logu e 4. Fmoc-Phe-Wang resin (35.5 mg, 35.5
µmol) was deprotected and allowed to react with 18 (27.0 mg,
42.3 µmol), PyBOP (22.0 mg, 42.3 µmol), and DIEA (18.4 µL,
106 µmol) in DMF (0.9 mL) for 26 h. Fmoc-Tyr(t-Bu)-OH (64.3
mg, 140 µmol) was coupled with the aid of PyBOP (72.8 mg,
140 µmol) and DIEA (48.8 µL, 280 µmol) in DMF (0.7 mL) for
22 h. The same conditions were used for the remaining amino
acids, but with reaction times of 2-2.5 h. The deprotected,
washed, and dried peptide resin weighed 53.3 mg. TFA
cleavage (48.1 mg) in the presence of H2O and triethylsilane
for 2 h gave 23.3 mg of the crude product.
PDMS analysis showed the expected m/z value for the major
peak and, in addition, the presence of an impurity correspond-
ing to the deletion peptide Asp-Arg-Val-Tyr-Phe. Since the two
peptides seemed to coelute on the C18 column, a two-stage
purification procedure was applied. The crude product (12.0
mg) was dissolved in 0.1% aq TFA containing 25% MeCN (2
mL) and applied onto a cation exchange column (Dyno NeoBar
CS4, 0.65 × 6 cm, sodium form). The product was eluted using
a gradient of NaCl (0-0.4 M in 30 min) in the same buffer at
a flow rate of 3 mL/min and with UV detection (254 nm). The
fractions corresponding to the major peak were pooled and
evaporated to dryness in a vacuum. The residue was redis-
solved in 0.1% aq TFA (2.5 mL) and further purified by RP-
HPLC on a Vydac C18 column (1 × 25 cm) using a flow rate
of 3 mL/min and an 80-min gradient of 10-50% MeCN in 0.1%
TFA. The separation was monitored at 230 nm and by PDMS
and RP-HPLC of selected fractions. Yield of pure 4: 1.9 mg
(12%); PDMS (Mw 996.1): 997.7 (M + H+); amino acid
analysis: Asp, 1.02; Arg, 0.99; Val, 0.99; Tyr, 1.01; Phe, 0.99.
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.37 Binding of [125I]Ang II to membranes was
conducted in a final volume of 0.5 mL containing 50 mM Tris-
HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA,
0.025% bacitracin, 0.2% BSA (bovine serum albumin), liver
homogenate corresponding to 5 mg of the original tissue
weight, [125I]Ang II (80 000 cpm, 0.03 nM) and variable
concentrations of test substance. Samples were incubated at
25 °C for 2 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 3 × 3 mL of Tris-HCl
(pH 7.4) and transferred to tubes. The radioactivity was
measured in a γ-counter. The characteristics of the Ang II
binding AT1 receptor were determined using six different
concentrations (0.03-5 nmol/L) of labeled [125I]Ang II. Non-
specific binding was determined in the presence 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 ) 1.7 ( 0.1 nM, [L] ) 0.057 nM) was
determined by Scatchard analysis of data obtained with Ang
II by using GraFit (Erithacus Software, UK). The binding data
were best fitted with a one-site fit. All experiments were
performed in triplicate. Ki values were calculated using the
Cheng and Prusoff equation (Kd ) 1.7432).
P or cin e (P ig) Myom etr ia l Mem br a n e AT2 Recep tor
Bin d in g Assa y. Myometrial membranes were prepared from
porcine uteri according to the method of Nielsen et al.38
Potential interference by binding to AT1 receptors was blocked
by addition of 1 µM losartan. Binding of [125I]Ang II to
membranes was conducted in a final volume of 0.5 mL
containing 50mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM
MgCl2, 1 mM EDTA, 0.025% bacitracin, 0.2% BSA, homoge-
nate corresponding to 10 mg of the original tissue weight, [125I]-
Ang II (80 000 cpm, 0.03 mM), and variable concentrations of
test substance. Samples were incubated at 25 °C for 1.5 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 3 × 3 mL of Tris-HCl (pH 7.4) and
transferred to tubes. The radioactivity was measured in a
γ-counter. The characteristics of the Ang II binding AT2
receptor were determined using six different concentrations
(0.03-5 nmol/L) of labeled [125I]Ang II. Nonspecific binding was
determined in the presence 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
)
0.7 ( 0.1 nM, [L] ) 0.057 nM) was determined by Scatchard
analysis of data obtained with Ang II by using GraFit
(Erithacus Software, UK). The binding data were best fitted
with a one-site fit. All experiments were performed in tripli-
cate. Ki values were calculated using the Cheng-Prusoff
equation (Kd ) 0.7292).
Con for m a t ion a l An a lysis a n d Molecu la r Mod elin g.
The conformational search was performed on structures 1m ,
2m , and 6m using the MMFFs force field as implemented in
the program MacroModel 7.1.40 The General Born Solvent
Accessible (GB/SA) surface area method for water developed
by Still et al.39 was used in all calculations. Amide bonds were
fixed in the trans configuration and 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 ) 17 for 6m , n ) 13 for 1m , and
n ) 16 for 2m ). Conformational searches were conducted using
the Systematic Unbound Multiple Minimum (SUMM) search
method51 in the batchmin program. 5000 steps per rotatable
bond were used. Conformations within 5 kcal/mol of the lowest
found minimum were kept. The ring closure bond was defined
as the bond between Câ and the sulfur atom of the i residue of
the γ-turn in 6m and the bond between N and CR of the Ala
residue. Torsional memory and geometric preoptimization
were used. Truncated Newton Conjugate Gradient (TNCG)
minimization with a maximum of 100 iterations was used in
the conformational search, with derivative convergence set to
0.05 kJ /mol/Å. A maximum of 500 iterations of TNCG was used
in the subsequent refinement minimization, with the conver-
gence criteria set to 0.001 kJ /mol/Å and only the key atoms

Selective AT2 Receptor Ligand
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