New selective AT2 receptor ligands encompassing a γ-turn mimetic replacing the amino acid residues 4-5 of angiotensin II act as agonists

Article abstract of DOI:10.1021/jm0491492

New benzodiazepine-based γ-turn mimetics with one or two amino acid side chains were synthesized. The γ-turn mimetics were incorporated into angiotensin II (Ang II) replacing the Val³-Tyr⁴-Ile ⁵ or Tyr⁴-Ile

Full text of DOI:10.1021/jm0491492

J. Med. Chem. 2005, 48, 4009-4024
4009
New Selective AT2 Receptor Ligands Encompassing a γ-Turn Mimetic Replacing
the Amino Acid Residues 4-5 of Angiotensin II Act as Agonists
Ulrika Rosenstro¨m,† Christian Sko¨ld,† Bianca Plouffe,‡ He´le`ne Beaudry,‡ Gunnar Lindeberg,† Milad Botros,§
Fred Nyberg,§ Gunter Wolf,| Anders Karle´n,† Nicole Gallo-Payet,*,‡ and Anders Hallberg*,†
Department of Medicinal Chemistry, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden, Service of
Endocrinology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada, Department of Biological
Research on Drug Dependence, BMC, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, Sweden, and Department of
Medicine, Division of Nephrology and Osteology, University of Hamburg, University Hospital Eppendorf, Martinistrasse 52,
D-20246 Hamburg, FRG
Received October 25, 2004
New benzodiazepine-based γ-turn mimetics with one or two amino acid side chains were
synthesized. The γ-turn mimetics were incorporated into angiotensin II (Ang II) replacing the
Val3-Tyr4-Ile5 or Tyr4-Ile5 peptide segments. All of the resulting pseudopeptides displayed high
AT2/AT1 receptor selectivity and exhibited AT2 receptor affinity in the low nanomolar range.
Molecular modeling was used to investigate whether the compounds binding to the AT2 receptor
could position important structural elements in common areas. A previously described
benzodiazepine-based γ-turn mimetic with high affinity for the AT2 receptor was also included
in the modeling. It was found that the molecules, although being structurally quite different,
could adopt the same binding mode/interaction pattern in agreement with the model hypothesis.
The pseudopeptides selected for agonist studies were shown to act as AT2 receptor agonists
being able to induce outgrowth of neurite cells, stimulate p42/p44mapk, and suppress proliferation
of PC12 cells.
Introduction
The signaling pathways activated by the AT2 receptor
are still controversial. The AT2 receptor is not coupled
to any of the classical, well-established, second mes-
sengers for G-protein-coupled receptors, such as cAMP
or inositol phosphatases, and its coupling to a GRi
protein, reported by several authors is not viewed in
consensus (for reviews see refs 4, 8, and 19-22).
However, various mediators, which could individually
exert opposite effects, such as cGMP, tyrosine or serine/
threonine phosphatases, and the extracellular signal
regulated kinases ERK1/ERK2 (p42/p44mapk), have been
associated with the activation of the AT2 receptor,
depending on the cell types and experimental conditions
used. More precisely, a sustained increase in p42/
p44mapk activity is associated with neuronal differentia-
tion.23,24
Information of the bioactive conformation of Ang II
when bound to its receptors is invaluable for the
understanding of the topological requirements within
the peptide-receptor complex. In this context, con-
strained analogues are important research tools and
various cyclization strategies have been employed to
introduce conformational constrains in Ang II.25-34 An
important driving force for the research is to enable the
conversion of peptides into more druglike compounds.
Cyclization can be a powerful tool and transform peptide
leads into peptidomimetic compounds.35,36 Several mod-
els of the bioactive conformation of Ang II when
interacting with the AT1 receptor have been pro-
posed.32,37-44 Thus, it has been suggested by several
groups that Ang II adopts a turn conformation around
the Tyr4 residue25,26,29,30,37-39,45-47 but also extended
conformations of Ang II have been considered.48 Much
less is reported on the structural requirements for AT2
The octapeptide angiotensin II (Ang II, Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe) mediates its biological actions by
activating at least two distinct receptor subtypes,
designated AT1 and AT2. Both receptors are seven-
transmembrane G-protein coupled receptors with 32-
34% sequence homology.1,2 Most of the more well-known
physiological effects of Ang II, including vasoconstric-
tion, aldosterone release, stimulation of sympathetic
transmission, and cellular growth, are generally at-
tributed to AT1 receptor activation.3-5 The role of the
AT2 receptor has been less clear, largely because of its
often low level of expression in adults. However, roles
of the AT2 receptor in mediating antiproliferation,
cellular differentiation, programmed cell death (apop-
tosis), and even vasodilation have been proposed.6-10
The AT2 receptor is the predominant angiotensin recep-
tor in fetal tissues but is rapidly downregulated after
birth, suggesting its involvement in fetal develop-
ment.11,12 Importantly in adults it is upregulated in
certain pathological conditions such as heart failure,
myocardial infarction, brain lesions, vascular injury, and
wound healing.13-18 It has also been shown that activa-
tion of the AT2 receptor in cells of neuronal origin induce
neurite outgrowth and elongation, modulate neuronal
exitability, and promote cellular migration in adults.8
* Corresponding
authors:
e-mail
anders.hallberg@
orgfarm.uu.se, tel +46-18-4714284, fax +46-18-4714474; e-mail
nicole.gallo-payet@usherbrooke.ca, tel +1-819-5645243, fax +1-819-
5645292.
† Department of Medicinal Chemistry, Uppsala University.
‡ Service of Endocrinology, University of Sherbrooke.
§ Department of Biological Research on Drug Dependence, Uppsala
University.
| Department of Medicine, University of Hamburg.
10.1021/jm0491492 CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/13/2005

4010 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
Chart 1
Results
Chemistry. Two Fmoc-protected benzodiazepine-
based γ-turn mimics 18a and 18b were synthesized in
solution and thereafter incorporated into Ang II using
solid-phase peptide synthesis (SPPS) to deliver the
pseudopeptides 3-8. The benzodiazepine core structure
was used as a turn template to replace Val3Tyr4Ile5 (4,
6, and 8) or Tyr4Ile5 (3, 5, and 7) in Ang II. The
pseudopeptides 7 and 8 comprise the isoleucine side
chain that previously has been proposed to be important
for stabilizing a turn conformation at Tyr4 in Ang II.25,59
The synthetic routes to the γ-turn mimetics 18a and
18b are outlined in Scheme 1 and start with a reduction
of 2-chloro-3-nitrobenzoic acid to the alcohol followed by
a Swern oxidation to afford aldehyde 11 in 91% yield.
The aldehyde was reacted with either the glycine
equivalent 2-aminoethanol or the isoleucine equivalent
(S,S)-2-amino-3-methyl-1-pentanol in reductive amina-
tion. An amino acid or its methyl ester was not employed
as a building block because of the tentative formation
of a diketopiperazine in a later step. Protection of the
alcohol group of 12a with tert-butyldiphenylchlorosilane
(TBDPSCl) was performed to improve the yield and to
achieve cleaner reactions. A much lower yield (20%
compared to 64% over two steps) was obtained when
the TBDPS-protected 2-aminoethanol was used in the
reductive amination. The isoleucine derivative 12b could
be protected using the same conditions but the subse-
quent reaction with the TBDPS-protected alcohol 13b,
that is, the amide formation, was very slow and provided
a mixture of two diastereoisomers in a low yield.
Therefore, the TBDPS-protected glycine derivative 13a
and the unprotected 12b were used in the amide
coupling with Fmoc-L-Tyr(t-Bu)-OH using O-(7-azaben-
zothiazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HATU) as the activating reagent. To
further improve the yield and to considerably shorten
the reaction time in the coupling of the sterically
hindered 12b with Fmoc-L-Tyr(t-Bu)-OH, the reaction
mixture was heated to 100 °C for 15 min using con-
trolled microwave irradiation. When these conditions
are used, the Fmoc-protected product was obtained in
56% yield and only one diastereoisomer was observed
in NMR. The less sterically hindered amine 13a was
reacted at room temperature overnight, and the amide
was obtained in excellent yield.
Deprotection of the Fmoc group was performed using
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Intramolecu-
lar nucleophilic aromatic substitution at the chlorine
ipso carbon in 14a proceeded smoothly in DMSO/Et3N
at 100 °C. Reacting 14b using the same conditions gave
a ∼7:3 mixture of two diastereoisomers. The diastere-
oisomers were separated using column chromatography,
and the structural assignment and the determination
of the stereochemistry of the molecules were performed
using correlation spectroscopy, heteronuclear correlation
spectroscopy, and rotating-frame Overhauser enhance-
ment spectroscopy NMR experiments. The nuclear
Overhauser effect (NOE) information of the intermedi-
ate 16b and its isomer 16c are illustrated in Figure 1.
The major product in the cyclization step (16b) was
confirmed to have S configuration in the ring, which is
the stereochemistry of the natural amino acid used as
the starting material. The minor diastereoisomer 16c
receptor affinity,49-56 although it is known that mono-
and bicyclizations in the 3-5 region of Ang II may give
analogues with retained affinity.27,29,57
Recently, we described the Ang II derivatives (1 and
2) as both possessing a benzodiazepine scaffold intended
to mimic a γ-turn backbone replacing either the amino
acid residues 4-5 or 3-4-5 in the target peptide.58
None of these compounds bound to the AT1 receptor.
However, compound 1, but not compound 2, the latter
lacking the Val3 residue, exhibited a high AT2 receptor
affinity (Ki ) 3.0 nM), Chart 1. It was concluded that
the position of the guanidino group of the Arg2 residue
in space, in relation to the Tyr side chain and the
N-terminal end, was critical for AT2 receptor affinity.
Molecular modeling suggested that a more favorable
positioning of the Arg2-Tyr4 side chains could be ob-
tained with a benzodiazepine γ-turn mimicking scaffold,
where the 9-position rather than the 7-position of the
benzodiazepine serves as a handle for attachment of the
N-terminal residues. We herein report the design,
synthesis, and binding data of the pseudopeptides 3-8,
comprising a new γ-turn mimic replacing the Val3-Tyr4-
Ile5 and Tyr4-Ile5 segments of Ang II (Chart 2). These
compounds act as high-affinity AT2 receptor ligands but
do not bind to the AT1 receptor. We also report that 1,
3, and 4 are full agonists and thus induce cell outgrowth
of neurite cells, an effect that was suppressed by the
selective AT2 receptor antagonist PD123,319. Further-
more, compound 1 suppresses proliferation of PC12 cells
expressing only AT2 receptors. This is to the best of our
knowledge the first report on AT2 receptor stimulation
mediated by constrained Ang II analogues.

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4011
Chart 2
seems to have the stereochemistry outlined in Figure
1. The TBDPS-protecting group was removed from the
glycine derivative 15a using tetrabutylammonium fluo-
ride (TBAF) in THF, and the alcohol 16a was obtained
in a good yield. Oxidation of the alcohols 16a and 16b
was performed in two steps, first a Swern oxidation to
the aldehyde and thereafter a mild oxidation using
sodium chlorite at 0 °C60 to deliver the carboxylic acids
17a and 17b in 90% and 76% yield, respectively. The
aldehydes were not stable and were purified by extrac-
tions but could not be stored. Reduction of the nitro
groups in 17a and 17b was performed using ammonium
formate and Pd/C. The zwitterions obtained were iso-
lated by extractions and thereafter reacted with FmocCl
in dioxane and aqueous Na2CO3. The pure compound
18a was obtained in 68% yield using repeated extraction
while the reduction and/or protection of 17b produced
an ∼8:2 mixture of diastereoisomers. After purification
by reversed-phase high-performance liquid chromatog-
raphy (RP-HPLC), the major component 18b was ob-
tained in 35% yield.
tively. The protected γ-turn mimetics 18a or 18b were
manually coupled to the His(Trt)-Pro-Phe-Wang-resin
in DMF using PyBOP as an activating agent and
diisopropylethylamine (DIEA) as a base. Prolonged
reaction times were used for the incorporation of the
turn templates and the adjacent amino acids where the
couplings were expected to proceed slowly. After comple-
tion of the synthesis, TFA in the presence of water and
triethylsilane was used to liberate the target peptides
from the resin. The products were precipitated with
diethyl ether and purified by RP-HPLC. Compound 18a
gave the single pseudopeptides 3-6, while compound
18b produced two diastereoisomers of the pseudopep-
tides 7 and 8 in ∼5:1 and ∼2:1 ratios, respectively. The
diastereoisomers could be separated in the final puri-
fication step to give 7a, 7b, 8a, and 8b (a and b denote
single diastereoisomers of unassigned absolute stereo-
chemistry). When the intermediate 16b corresponded
to the natural L-configuration, we hypothesize that the
stereochemistry of the major products (7a and 8a) also
corresponds to the natural L-configuration.
Incorporation of the Fmoc derivatives 18a and 18b
into Ang II using standard Fmoc/t-Bu SPPS methodol-
ogy produced the pseudopeptides 3-6 and 7-8, respec-
Binding Assays. Radioligand binding assays relying
on the displacement of [125I]Ang II from AT1 receptors
in rat liver membranes61 and from AT2 receptors in pig

4012 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
Scheme 1a
a Reagents: (a) (i) NaBH4, BF3‚Et2O, THF, (ii) ClCOCOCl, DMSO, Et3N, CH2Cl2, 91%; (b) 2-aminoethanol or (S,S)-2-amino-3-methyl-
1-pentanol, HOAc, NaCNBH3, MeOH, 56-69%; (c) tert-butylchlorodiphenylsilane, DBU, THF, 85-93%; (d) (i) Fmoc-L-Tyr(t-Bu)-OH, HATU,
DIEA, CH2Cl2, (ii) DBU, THF, 46-90%; (e) Et3N, DMSO, 41-82%; (f) TBAF, THF, 89%; (g) (i) oxalyl chloride, DMSO, Et3N, CH2Cl2, (ii)
NaClO2, NaH2PO4, cyclohexene, t-BuOH, H2O, 76-86%; (h) (i) HCOONH4, Pd/C, MeOH, (ii) FmocCl, Na2CO3 (aq), dioxane, 35-68%.
Table 1. Binding Affinities for the AT1 and AT2 Receptors
AT1 (rat liver
membranes)
Ki (nM) ( SEM
AT2 (pig uterus
myometrium)
Ki (nM) ( SEM
compound
Ang II
0.24 ( 0.07
>10000
>1000058
>1000058
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
0.23 ( 0.03
0.9 ( 0.3
[4-NH2-Phe6]Ang II
1
3.0 ( 1.158
>1000058
2
3
0.8 ( 0.1
4
2.8 ( 0.2
5
9.3 ( 1.4
6
37.4 ( 2.1
0.3 ( 0.01
0.08 ( 0.003
2.6 ( 0.2
Figure 1. NOE information from the major diastereoisomer
16b and the minor diastereoisomer 16c.
7a
7b
8a
8b
uterus membranes62 were used to evaluate compounds
3-8 (Table 1). The natural ligand Ang II and the AT2
receptor selective agonist [4-NH2-Phe6]Ang II53 were
used as reference substances. Compound 1 has previ-
ously been reported to have a high affinity to the AT2
receptor (Ki ) 3.0 nM) while compound 2, without the
extra Val residue, lacks affinity to the AT2 receptor (Ki
> 10 µM).58 In contrast, the pseudopeptides 3 and 4
encompassing the new γ-turn mimetic 18a, exhibit both
good binding affinity and high AT2/AT1 receptor selec-
tivity (Table 1). Compound 3, incorporating a Val
residue, has a slightly better binding affinity (Ki ) 0.8
nM) than compound 4 (Ki ) 2.8 nM). Replacement of
the Arg2 residue in compounds 3 and 4 with Ala gave
compounds 5 and 6, respectively. In both of these
compounds the binding affinity was reduced by a factor
117.4 ( 7.7
of 10. The pseudopeptides 7a and 8a encompassing the
γ-turn mimetic 18b, with an isoleucine side chain in the
i + 2 position, both have good binding affinity for the
AT2 receptor. Compound 7a, with a Val residue, exhibits
a Ki of 0.3 nM and 8a, without the Val residue, a Ki of
2.6 nM. Interestingly, 7b, the diastereoisomer of 7a,
exhibits an even better binding affinity for the AT2
receptor (Ki ) 0.08 nM) while 8b, the diastereoisomer
of 8a, has a moderate binding affinity (Ki ) 117.4 nM).
None of the pseudopeptides displayed any affinity for
the AT1 receptor. Compounds 1, 3, and 4, with defined
stereochemistry, were chosen for further more advanced
in vitro studies.

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4013
Figure 3. Various inhibitors effect on neurite outgrowth in
NG108-15 cells induced by compound 1. NG108-15 cells were
plated at a density of 4 × 104 cells per dish in 35 mm Petri
dishes and were cultured for 3 days in the absence (control
(A)) or in the presence of 0.1 µM 1 (B) or in the presence of
compound 1 (0.1 µM) and 1 µM PD123,319 (C), compound 1
(0.1 µM) and 10 µM PD98,059 (D), compound 1 (0.1 µM) and
0.5 µM LY-83,583 (E), or compound 1 (0.1 µM) and 1 µM KT
5823 (F). All panels are seen at the same magnification.
Figure 2. Comparative effects of the three compounds 1, 3,
and 4 and angiotensin II (Ang II) on neurite outgrowth in
NG108-15 cells. NG108-15 cells were plated at a density of 4
× 104 cells per dish in 35 mm Petri dishes and were cultured
for 3 days in the absence (control (A)), or in the presence of
0.1 µM 1 (B), 0.1 µM 3 (C), 0.1 µM 4 (D), or 0.1 µM Ang II (E).
Neurite outgrowth was quantified as explained in the Experi-
mental Section (F). All panels are seen at the same magnifica-
tion.
0.41%, and 9.35 ( 0.35% in the cells treated with
compounds 1, 3, and 4, respectively (Figure 2F). This
effect was mediated through the AT2 receptor, since
coincubation of 1, 3, or 4 with the AT2 receptor antago-
nist, PD123,319 (1 µM), virtually halted neurite elonga-
tion. An illustration is shown in Figure 3C for compound
1. Similar observations were made with compounds 3
and 4. We have previously shown that PD123,319 alone
did not alter the morphology of the untreated cells.23,64
To further explore if compounds 1, 3, and 4 share the
same signaling transduction as Ang II,23,65 cells were
preincubated for 3 days with 10 µM PD98,059: a dose
that abolished MAPK activity. When coincubated with
1, 3, or 4, PD98,059 decreased the length and number
of neurites (Figure 3D). Cells were also preincubated
with LY-83,583 (0.5 µM), an inhibitor of soluble guanylyl
cyclase (sGC) or with KT 5823 (1 µM), an inhibitor of
cGMP-dependent protein kinases. PD98,089, LY-83,583,
and KT 5823 treated cells had the same morphological
appearance alone as the control, untreated cells. How-
ever, cells coincubated with 1, 3, or 4 and LY-83,583 or
KT 5823 have only one or two thin processes (parts E
and F of Figure 3). Treatment with Ang II increased
the number of cells by 9.74 ( 0.51%. This effect was
abolished in cells coincubated with PD123,319, PD98,-
059, LY-83,583, and KT 5823 (Figure 4).
In Vitro Morphological Effects Induced by Com-
pounds 1, 3, and 4 in NG 108-15 Cells. To study the
effects of compounds 1, 3, and 4 on differentiation,
NG108-15 cells were used. In their undifferentiated
state, neuroblastoma × glioma hybrid NG108-15 cells
have a rounded shape and divide actively. We have
shown previously that these cells express only the AT2
receptor63,64 and that a 3 day treatment with Ang II or
the selective peptidic AT2 receptor agonist CGP 42112
induces neurite outgrowth.64 The mechanisms involve
a sustained increase in p42/p44mapk activity23,24 and
activation of the nitric oxide/guanylyl cyclase/cGMP
pathway65 (for review see ref 8).
For all experimental conditions, cells were plated at
the same initial density (4 × 104 cells/35 mm Petri dish)
and were treated with Ang II or one of the pseudo-
peptides 1, 3, or 4. After 3 days of culture, the cells were
examined under a phase-contrast microscope and mi-
crographs were taken. The pseudopeptides were first
tested at various concentrations ranging from 1 pM to
1 µM. Except for the higher concentration of 1 µM, none
of the other doses induced cell death. In addition, in cells
stimulated for 3 days with concentrations higher than
0.1 nM, the three tested pseudopeptides induced neurite
outgrowth. As shown by phase-contrast microscopy,
nontreated control cells had rounded cell bodies, without
or with some thin processes (Figure 2A). After a 3 day
treatment with 1 (Figure 2B), 3 (Figure 2C), or 4 (Figure
2D) (0.1 µM), most cells extended one or two neurite
processes exhibiting a growth cone at the tip,64 while
the cell body retained a rounded appearance. In com-
parison to Ang II (0.1 µM) (Figure 2E), the tested
compounds induced the same morphological changes.64
Quantification of these results indicated that a 3 day
treatment with 1, 3, or 4 increased the number of
cells with neurites longer than one cell body from
2.26 ( 0.10% in control cells to 11.07 ( 0.46%, 9.88 (
The pseudopeptides 1, 3, and 4 also induced phos-
phorylation of p42/p44mapk, as determined by using an
antibody directed against the phosphorylated form of
p42/p44mapk. Thirty minutes of application (optimum
time period for maximal effect of Ang II)23,65 of com-
pound 1, 3, or 4 stimulated phosphorylation of p42/
p44mapk with a (2.2 ( 0.2)-, (2.8 ( 0.9)-, and (2.9 ( 0.4)-
fold increase over control, respectively. The stimulation
was abolished in cells preincubated with 10 µM PD123,-
319 or 10 µM PD98,059 (Figure 5).

4014 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
Figure 4. Various inhibitors effects on neurite outgrowth in
NG108-15 cells induced by compounds 1, 3, and 4. Cells with
at least one neurite longer than a cell body were counted as
positive for neurite outgrowth The number of cells with
neurites represent the percentage of the total number of cells
in the micrographs (from 50 to 100 cells according to the
experiment). The control bar in each experiment corresponds
to an experiment with only the reference compound.
Figure 6. A γ-turn with the i, i + 1, and i + 2 residues
indicated and scaffolds that have been used as γ-turn mimet-
ics.
Figure 5. The effect of compounds 1, 3, and 4 on p42/p44mapk
activation. NG108-15 cells were stimulated without or with
0.1 µM of the Ang II analogues 1, 3, or 4 for 30 min alone or
in the presence of 10 µM PD123,319 or 10 µM PD98,059.
Figure 7. A comparison of scaffolds 20 and 21 with respect
to distances and angles between the CR atoms. The measured
distances are given outside, and the angles inside, the triangle.
The aromatic carbon connected to the N-terminal handle is
selected as CRi. The top triangle shows distances and angles
in a minimized ideal inverse γ-turn.
Table 2. Proliferation of PC12 Cells as Measured with
[3H]thymidine Incorporation
counts per minute
10% FCS
62585 ( 3853
48887 ( 3737a
68707 ( 3483
45421 ( 2883a
10% FCS + 1.0 µM Ang II
10% FCS + 1.0 µM PD123,319
10% FCS + 1.0 µM compound 1
a p < 0.05 vs 10% FCS only, n ) 10-12.
Proliferation of PC12 Cells. It was also investi-
gated whether compound 1 could inhibit proliferation
of PC12 cells only expressing AT2 receptors.66 As shown
in Table 2, compound 1 inhibited proliferation of cells
grown in 10% FCS to the same extent as Ang II at 1.0
µM.
Figure 8. The γ-turn mimetic 21 (green) and a γ-turn from
a crystallized protein (gray) superimposed.
Molecular Modeling. A conformational analysis was
performed on 20 and 21, Figure 6, but with Ala
replacing Tyr, which resulted in 10 and 11 conforma-
tions, respectively, within 5 kcal/mol of the lowest
energy minimum. The lowest energy conformations
were used in the geometrical comparison shown in
Figure 7. The distances and angles in the other confor-
mations of each structure were within 0.1 Å and 5°,
respectively. A minimized peptide structure of an ideal
inverse γ-turn was also included for comparison.
lowest energy minimum (three conformations) were
evaluated by comparing the torsion angles Φi, Ψi, Φi+1
,
Ψi+1, Φi+2, and Ψi+2 in 21 with the corresponding torsion
angles in γ-turns from an in-house database of 2101
extracted inverse γ-turns from protein X-ray structures
from the protein data bank (PDB).67 The third lowest
energy conformation compared well to an inverse γ-turn
in the database (PDB ID: 1NNF, chain A, sequence
Thr82-Ala83-Gln84). A superimposition of these structures
can be seen in Figure 8.
Scaffold 21 was further studied to see if a correspond-
ing peptide γ-turn could be identified in a crystallized
protein. The conformations within 1 kcal/mol of the
Using the pharmacophore modeling program DIS-
COtech68, we investigated if the active compounds 1, 3,

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4015
Figure 9. Model compounds used in the conformational
analysis and molecular modeling. Asterisks indicate atoms
used for comparison between conformations in the conforma-
tional analysis. These atoms also correspond to the DISCOtech
features (the guanidino carbon only indirectly).
Figure 10. The model of superimposed structures obtained
by DISCOtech that has the highest structural overlap. 1m is
shown with white carbons, 3m with green carbons, and 4m
with yellow carbons. The cyan sphere indicates the hydrogen
bonding acceptor site in the receptor.
and 4 could position important structural elements
(features) in common regions of space not accessible to
the inactive compound 2. In this study model com-
pounds 1m, 2m, 3m, and 4m were used, and the
structural elements included were, see Figure 9, (a) the
Ala side chain (corresponding to Câ in the Tyr residue
in the full-length pseudopeptides), (b) the C-terminal
benzodiazepine anchor point (corresponding to CR in
Gly), (c) the binding site in the receptor corresponding
to the Arg guanidino group (as defined by DISCOtech),
and (d) the N-terminal end (corresponding to CR in Asp).
DISCOtech requires as input a set of conformations
(max 300) of each compound. These were generated with
a Monte Carlo search in MacroModel 8.569 using the
MMFFs force field and the generalized Born solvent
accessible surface area (GB/SA)70 water solvation model.
To reduce the number of conformations and to identify
a representative set of conformations (<300) for each
compound, we removed those where the guanidino
group participates in an intramolecular hydrogen bond
to a carbonyl oxygen. In our hypothesis of the receptor-
bound conformation of Ang II, the Arg side chain is
interacting with Asp297 (see refs 71 and 72) and should
therefore not be intramolecularly hydrogen bonded. The
number of conformations found within 5 kcal/mol of the
energy minimum for 1m, 3m, and 4m (Figure 9) were
3704, 620, and 3207, respectively. By removing very
similar conformations, we were able to reduce the
number of conformations further. This was accom-
plished by minimization with (a) increased convergence
criterion, (b) a reduced number of atoms for identifying
unique conformations (only the atoms that represent a
feature in the later DISCOtech run), and (c) the distance
between atoms compared for defining unique conforma-
tions (BatchMin CRMS command) set to 0.75 Å for 3m
and to 1.5 Å for 1m and 4m (the 1.5 Å value for 1m
and 4m was used to arrive at less than 300 conforma-
tions). The number of conformations found with this
protocol for 1m, 3m, and 4m was 212, 167, and 158,
respectively. DISCOtech identified 82 different models
with superimposed conformations where the important
elements were positioned in the same space. However,
many of the models had very low overall structural
overlap and were therefore not convincing. To select the
most relevant model(s), the total van der Waals volume
of each model was calculated. A small total volume
indicates a high structural overlap, and a large total
volume, when the structures are not matched, indicates
a low structural overlap. The model with the lowest total
volume is shown in Figure 10. This model corresponds
to a plausible binding mode for the active compounds
with each of the four investigated structural elements
confined within 2.5 Å for all three structures.
An additional DISCOtech analysis including 2m was
performed to investigate if the inactive compound could
fit this model as well. With the same protocol as above
(with CRMS set to 0.75 Å), the number of conformations
identified for 2m was initially 687 and after refinement
237. The same DISCOtech protocol was applied, and 57
models were found. However, none of these models had
a convincing alignment, and none of the conformations
included in the previous model (Figure 10) were present
in any of these new models. This indicates that the
active compounds may have a common binding mode
not accessible to the inactive compound.
Discussion
Until recently only a small set of receptor selective
compounds have been available for the functional stud-
ies of the AT2 receptor, for example, PD123,319,73 CGP-
42112,74 and (4-NH2-Phe6)Ang II,53 the latter being used
as a reference compound in the present study. We
recently disclosed the first selective nonpeptidic AT2
receptor agonist,75 developed from a nonselective AT1/
AT2 receptor agonist76-78 structurally related to the
“sartans”. However, our major research interest and
long-term objective is to enable transformation of pep-
tides to druglike non-peptides via incorporation of well-
defined mimetics of secondary structure motifs in the

4016 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
backbones as illustrated in Figure 8, where 21 is
compared to a γ-turn found in a crystallized protein.
Taken together, this shows that scaffold 21 resembles
more the native γ-turn than 20. When 21 is used as a
γ-turn mimetic, it could also allow the pseudopeptide
without the extra Val residue to reach the common
regions of space and possibly pick up affinity. This
prompted us to synthesize both compound 3 with the
extra Val residue, and compound 4 and evaluate the AT2
receptor binding affinities.
Both pseudopeptides 3 (Ki ) 0.8 nM) and 4 (Ki ) 2.8
nM) exhibited high affinity to AT2 receptors. It seems
that the extra valine residue is not required for optimal
interaction with the receptor when scaffold 21 is used,
which supports our hypothesis. When the arginine
residue in pseudopeptide 1 was replaced with an ala-
nine, all of the AT2 receptor affinity was lost.58 Interest-
ingly, when the same substitution was made in pseudo-
peptides 3 and 4, the affinity for the obtained com-
pounds, 5 and 6, only dropped 10 times. This is more in
line with the decrease in affinity that is observed when
arginine2 is substituted by glycine56 or glutamine49 in
Ang II. It therefore seems that 3, 4, and Ang II interact
similarly with the AT2 receptor. The reason for the large
decrease in affinity for 1 is most likely because of the
removal of the Arg side chain in combination with a
nonoptimal interaction with the receptor.
To study if compounds 1, 3, and 4, which all have high
affinity to the AT2 receptor, could adopt similar confor-
mations, we superimposed key features in the molecules
using the pharmacophore search program DISCOtech.
Among the identified models, one held all the included
groups in the same areas and had a reasonable scaffold
alignment (see Figure 10). This shows that although the
three active molecules encompass quite different scaf-
folds, they can still adopt the same binding mode/
interaction pattern. This model had no equivalent
among the models found when the inactive molecule (2)
was included in the analysis.
Another interesting observation that can be made
from the analysis is that the valine side chains of 1 and
3 are located in the same region in the model (see Figure
10). This could imply that the valine not only allowed
for a better receptor alignment due to the extra flex-
ibility but also may be of some importance for direct
receptor interaction. This interaction may be the reason
for the moderate increase in affinity when the extra
valine is inserted in 4 to give 3, since a reasonable
receptor alignment already seems to have been obtained
for both of these compounds.
The lipophilic side chains Val3 and Ile5 in Ang II are
considered to have only conformational stabilizing roles
and to induce a turn motif when Ang II interacts with
the AT1 receptor.25,59 It has also been indicated that
these side chains are less important for Ang II binding
to the AT2 receptor.49,56 Therefore, to study the impact
of the lipophilic side chain Ile5 for affinity in the
pseudopeptide 3, we synthesized and evaluated com-
pounds 7a and 8a (Chart 2). Compound 7a had ap-
proximately the same binding affinity to the AT2
receptor as the analogue without the Ile side chain (3)
Ki ) 0.3 nM and Ki ) 0.8 nM, respectively. The two
analogues that did not contain the extra valine residue
(4 and 8a) also had very similar binding affinities to
Figure 11. Superimposition of the γ-turn moieties of 9 and
20. The 7- and 9-position in the benzodiazepine skeleton are
indicated by arrows.
target peptides. For example, several cyclic analogues
of Ang II have been synthesized and assessed as ligands
to the AT2 receptor.27,29 One of the most potent ligands
(Ki ) 0.62 nM), a monocyclic methylenedithioether
analogue of Ang II (9), was found to preferentially adopt
low-energy inverse γ-turn conformations centered at
Tyr4, as deduced from the conformational analysis.27 We
reasoned that incorporation of suitable rigid scaffolds
mimicking this inverse γ-turn should constitute a
relevant first approach in the search for the bioactive
conformation(s) of Ang II when binding and activating
the AT2 receptor.
The isoquinoline 19 and the benzodiazepine 20 have
previously been employed as scaffolds mimicking the
presumed γ-turn centered at Tyr4 in Ang II (Figure
6).45,58 Substitution of Val3-Tyr4-Ile5 of Ang II by the
isoquinoline 19 renders an AT2 receptor ligand with a
Ki of 61 nM, while the same substitution with the
benzodiazepine 20 provides an inactive pseudopeptide
(2).58 When a Val3 residue was introduced in 2 and only
the Tyr-Ile peptide segment was substituted, a highly
potent analogue (1) was obtained with a Ki of 3 nM.58
Using 9 as a reference peptide, we hypothesized that
the reason for the lack of affinity of the shorter peptide
2 was not that the Val3 was missing per se but rather
that the guanidino group of the Arg2 residue and/or the
N-terminal part of the pseudopeptide could not interact
optimally with the receptor due to the geometric differ-
ence between 2 and 9. The main reason for this is that
the γ-turn moiety in 1 and 2 is not optimal from a
geometrical point of view (Figure 7). However in peptide
1, Val3 could function as a spacer and allow the Arg
residues and the N-terminal part in 1 and 9 to interact
in similar positions despite the geometric difference.
When the γ-turn moieties of 20 and 9 are superim-
posed (Figure 11), it can be seen that by moving the
entry point of the peptide chain in the benzodiazepine
scaffold from the 7-position to the 9-position a geo-
metrically better γ-turn mimetic could be obtained (21
in Figure 6). This is shown in more detail in Figure 7
where a γ-turn in a reference peptide and the γ-turn
mimetics 20 and 21 and some key distances and angles
are compared. In this comparison, we used the inverse
γ-turn conformation of the peptide and focused on the
distances and angles between the three CR atoms within
the γ-turn moiety. It can be seen that the reference
γ-turn and the mimetic 21 are very similar. We also
compared the benzodiazepine scaffold with peptide

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4017
the AT2 receptor. Compound 8a exhibited a Ki of 2.6
nM compared to a Ki of 2.8 nM for pseudopeptide 4
without the Ile side chain. These results indicate that
the Ile side chain can be omitted in the turn templates
without affecting the binding affinities. While com-
pounds 7a and 8a were synthesized, partial epimeriza-
tion took place, and two additional compounds (7b and
8b) could be isolated and evaluated. Interestingly,
pseudopeptide 7b, a diastereoisomer to compound 7a
exhibited a very good binding affinity to the AT2
receptor (Ki ) 0.08 nM). In contrast, the diastereoisomer
of compound 8a, pseudopeptide 8b, had only a moderate
binding affinity (Ki ) 117.4 nM) to the AT2 receptor.
Notably, the absolute stereochemistry of neither 7b nor
8b has been determined. All the synthesized pseudopep-
tides lacked affinity (Ki > 10 000 nM) to the AT1
receptor.
To establish if these compounds are agonists or
antagonists, we selected compounds 1, 3, and 4 and
evaluated their potential for inducing neurite outgrowth
and antiproliferation. The present results demonstrate
that the three tested pseudopeptides (1, 3, and 4) induce
neurite outgrowth, one of the first steps of neuronal
differentiation, as does Ang II and CGP-42112.64 In
addition, 1, 3, and 4 as well as Ang II stimulate MAPK
activity, and as previously shown,23 this activation of
the p42/p44mapk pathway is essential to promote neurite
outgrowth by cells stimulated with 1, 3, or 4. These
effects are attributabed to the AT2 receptor, since
coincubation with PD123,319 abolishes the effect. More-
over, inhibition of cGMP-dependent kinases with KT
5823 or sGC with LY-83,583 indicate that cGMP par-
ticipates in the early steps of neurite elongation sug-
gesting that compounds 1, 3, and 4 induce neurite
outgrowth through MEK/p42/p44mapk and the nitric
oxide/guanylyl cyclase/cGMP pathway. All of these
results corroborate previous observations made with
Ang II or the peptide agonist CGP-42112,23,64,65 indicat-
ing that the pseudopeptides 1, 3, and 4 act as agonists
at the AT2 receptor. Compound 1 also inhibits prolifera-
tion of PC12 cells only expressing AT2 receptors, a
finding which further supports the view that pseudopep-
tide 1 acts as an agonist.
receptor. Furthermore, it has been demonstrated by
molecular modeling that, for the bioactive compounds,
key structural elements can reach common regions in
space and therefore probably bind to the AT2 receptor
in a similar fashion. A successful design of organic
scaffolds mimicking the secondary structure adopted by
Ang II when interacting with the AT1 receptor remains
a challenge.
Experimental Section
General Methods. 1H and 13C NMR spectra were recorded
on a JEOL JNM-EX400 at 400 and 100.5 MHz, respectively,
or a Varian MERCURY+ 400 spectrometer (400 MHz). Spectra
were recorded at ambient temperature unless otherwise noted.
Chemical shifts are reported as δ values (ppm) referenced to
δ 7.26 CHCl3, δ 77.0 CDCl3, δ 3.30 CH3OH, δ 49.15 CD3OD, δ
2.50, and δ 39.5 DMSO-d6. Optical rotations were measured
on a Perkin-Elmer model 241 polarimeter. Analytical Labo-
ratories, Lindlar, Germany, performed the elemental analyses.
Analytical reversed-phase liqiud chromatography-mass spec-
trometry (RP LC-MS) was performed with a Gilson HPLC
system combined with a Finnigan AQA quadropole mass
spectrometer using a Chromolith Performance RP-18e column
at a flow rate of 4 mL/min. Preparative RP LC-MS was
performed using an identical LC-MS system but with a Zorbax
SB-C8, 5 µm column (21.2 mm × 150 mm) at a flow rate of 15
mL/min.
Thin-layer chromatography (TLC) was performed using
aluminum sheets precoated with silica gel 60 F254 (0.2 mm, E.
Merck). The spots were identified by UV detection and/or by
spraying with a 3% methanolic solution of ninhydrin followed
by heating. Flash column chromatography was performed
using Merck silica gel 60 (40-63 µm). The microwave reactions
were conducted in heavy-walled glass Smith process vials (5
mL) sealed with aluminum crimp caps that were fitted with a
silicon septum. The microwave heating was performed in a
Smith synthesizer single-mode microwave cavity (Biotage,
Uppsala, Sweden).
2-Chloro-3-nitrobenzaldehyde,79,80 11. 2-Chloro-3-nitrob-
ezoic acid (10.00 g, 49.6 mmol) in THF (50 mL) was added
dropwise over 50 min to an ice-cooled suspension of sodium
borohydride (3.60 g, 94.7 mmol) in THF (160 mL). The solution
was treated dropwise with boron trifluoride etherate (18 mL,
142 mmol) over 30 min. The reaction mixture was stirred for
5 h at room temperature, cooled on an ice bath, and acidified
with 1 M hydrochloric acid. EtOAc and water were added, the
layers were separated, and the aqueous layer was extracted
with EtOAc. The combined organic layers were washed with
water, saturated NaHCO3, water, 1 M hydrochloric acid, and
brine, dried (MgSO4), and evaporated to give (2-chloro-3-
nitrophenyl)methanol79 (8.90 g, 96%) as a white solid. Anal.
(C7H6ClNO3) C, H, N. A solution of oxalyl chloride (2.74 mL,
32.0 mmol) in CH2Cl2 (140 mL) was cooled to -78 °C. DMSO
(4.80 mL, 67.7 mmol) was added dropwise, and the mixture
was stirred for 15 min before a solution of (2-chloro-3-
nitrophenyl)methanol (4.00 g, 21.3 mmol) in CH2Cl2 (40 mL)
was added dropwise during 20 min. The reaction mixture was
stirred for 1 h at -78 °C before Et3N (15.0 mL, 108 mmol)
was added, and the reaction temperature was allowed to reach
-30 °C in 40 min. Water and more CH2Cl2 were added, and
the layers were separated. The aqueous layer was extracted
with CH2Cl2, and the combined organic layers were washed
with 1 M KHSO4, water, saturated NaHCO3, and brine, dried
over Na2SO4, and evaporated. The benzaldehyde 11 was
obtained as a beige solid (3.76 g, 95%). Anal. (C7H4ClNO3) C,
H, N.
The nonpeptidic AT2 selective agonist that we previ-
ously reported75 lacks structural elements that are likely
to mimic the N-terminal (Asp-Arg-Val-Tyr-Ile) of Ang
II, but the compound is still serving as a full AT2
receptor agonist. Thus, Ang II and the pseudopeptide
agonists 1, 3, and 4 reported herein and the nonpeptidic
agonist seem to bind very differently to the AT2 receptor,
which is essentially in accordance with the similar
findings by Perlman et al. regarding AT1 receptor
activation.77
Conclusion
A new, improved γ-turn mimetic with one or two
amino acid side chains has been synthesized and
incorporated into Ang II. The resulting pseudopeptides
showed high AT2 receptor affinity and selectivity. The
three compounds selected for agonist evaluation were
shown to induce neurite outgrowth and stimulate p42/
p44mapk, and furthermore, one was shown to suppress
proliferation of PC12 cells. It can therefore be concluded
that these pseudopeptides act as agonists at the AT2
2-(2-Chloro-3-nitro-benzylamino)ethanol, 12a. HOAc
(0.39 mL, 6.83 mmol) and 2-aminoethanol (1.26 mL, 20.9
mmol) were added to a solution of 11 (773 mg, 4.16 mmol) in
MeOH (45 mL). After 10 min, NaCNBH3 (290 mg, 4.62 mmol)
was added and the reaction mixture was heated to reflux for
3 h. The mixture was cooled to room temperature, acidified to
pH 1 by addition of 2 M HCl, and partly evaporated. The

4018 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
residue was washed with diethyl 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 Na2SO4 and evaporated to give the alcohol 12a as a yellow
solid (665 mg, 69%). Anal. (C9H11N2O3) C, H, N.
[2-(tert-Butyldiphenylsilanyloxy)ethyl]-(2-chloro-3-ni-
trobenzyl)amine, 13a. DBU (1.00 mL, 6.69 mmol) and
TBDPSCl (1.70 mL, 6.64 mmol) were added to an ice-cooled
solution of the alcohol 12a (1.30 g, 5.64 mmol) in CH3CN (30
mL). The reaction mixture was stirred for 5 h at room
temperature. After evaporation the residue was dissolved in
water and EtOAc and extracted with EtOAc. The combined
organic layers were washed with water and brine, dried (Na2-
SO4), and evaporated. Purification by column chromatography
(EtOAc/pentane, 1:4) gave 13a as a pale yellow oil (2.46 g,
93%). Anal. (C25H29ClN2O3Si) C, H, N.
added dropwise, and the mixture was stirred for 15 min before
a solution of 16a (1.148 g, 2.78 mmol) in 5.0 mL of CH2Cl2
was added dropwise over 15 min. After another 2 h at -78
°C, Et3N (1.60 mL, 11.48 mmol) was added, and the reaction
mixture was allowed to warm to -30 °C over 1 h. 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 an orange foam
(1.03 g, 90%). [R]23 ) -170° (c ) 1.02, MeOH). 1H NMR
D
(CDCl3) δ: 9.60 (s, 1H, CH), 8.43 (d, J ) 2.9 Hz, 1H, NH),
8.05 (dd, J ) 8.7, 1.7 Hz, 1H, CH), 7.27 (m, 2H, 2 × CH), 7.07
(dd, J ) 7.1, 1.7 Hz, 1H, CH), 6.98 (m, 1H, 2 × CH), 6.54 (dd,
J ) 8.7, 7.1 Hz, 1H, CH), 5.58 (d, J ) 16.5 Hz, 1H, CH2), 5.17
(m, 1H, CH), 4.67 (d, J ) 18.5 Hz, 1H, CH2), 4.15 (d, J ) 18.5
Hz, 1H, CH2), 3.75 (d, J ) 16.5 Hz, 1H, CH2), 3.40 (dd, J )
14.6, 4.5 Hz, 1H, CH2), 2.96 (dd, J ) 14.6, 9.6 Hz, 1H, CH2),
1.33 (s, 9H, 3 × CH3). 13C NMR (CDCl3) δ: 196.3 (C), 169.5
(C), 154.6 (C), 143.9 (C), 135.7 (CH), 133.2 (C), 130.9 (C), 129.9
(CH), 127.5 (CH), 124.5 (CH), 121.9 (C), 115.4 (CH), 78.5 (C),
56.8 (CH2), 56.0 (CH), 52.9 (CH2), 36.5 (CH2), 28.8 (CH3). Anal.
(C22H25N3O5‚0.5H2O) C, H, N.
(2S)-2-Amino-3-(4-tert-butoxy-phenyl)-N-[2-(tert-butyl-
diphenylsilanyloxy)ethyl]-N-(2-chloro-3-nitrobenzyl)-
propionamide, 14a. The amine 13a (2.30 g, 4.9 mmol) was
dissolved in CH2Cl2 (30 mL). HATU (2.00 g, 5.26 mmol), Fmoc-
L-Tyr(t-Bu)-OH (2.48 g, 5.40 mmol), and DIEA (1.80 mL, 10.35
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 (EtOAc/pentane, 1:4) to give the Fmoc-
protected amine as a white foam (4.23 g, 95%). Anal. (C53H56-
ClN3O7Si) C, H, N. DBU (0.770 mL, 5.15 mmol) was added to
a solution of the Fmoc-protected amine (3.90 g, 4.28 mmol) in
25 mL of THF. After 2 h at room temperature, the reaction
was completed according to TLC (2% MeOH in CH2Cl2). The
MeOH was evaporated, and the crude product purified by
column chromatography (gradient CH2Cl2 to 2% MeOH in CH2-
The obtained aldehyde was dissolved in 25 mL of t-BuOH
and cyclohexene (4.70 mL, 46.2 mmol). The solution was cooled
on an ice bath, and a freshly prepared ice-cold solution of
NaClO2 (2.62 g, 80%, 23.2 mmol) and NaH2PO4‚H2O (2.55 g,
18.6 mmol) in 15 mL of water was added dropwise over 10
min. After 2 h 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 CH2Cl2 to 30% MeOH
in CH2Cl2) the product was redissolved in EtOAc. The EtOAc
solution was washed with 0.5 M KHSO4, dried over Na2SO4,
Cl2) to give the deprotected amine 14a (2.81 g, 95%). [R]23
+19° (c ) 1.00, MeOH). Anal. (C38H46ClN3O5Si) C, H, N.
)
D
and evaporated to give 17a as a yellow foam (947 mg, 96%).
1
[R]23 ) -173° (c ) 1.02, MeOH). H NMR (CD3OD) δ: 7.91
D
(dd, J ) 8.7, 1.6 Hz, 1H, CH), 7.32 (m, 2H, 2 × CH), 7.23 (dd,
J ) 7.1, 1.7 Hz, 1H, CH), 6.96 (m, 2H, 2 × CH), 6.51 (dd, J )
8.7, 7.1 Hz, 1H, CH), 5.62 (d, J ) 16.6 Hz, 1H, CH2), 5.38 (dd,
J ) 9.9, 4.6 Hz, 1H, CH), 4.40 (d, J ) 17.5 Hz, 1H, CH2), 4.13
(d, J ) 17.5 Hz, 1H, CH2), 4.08 (d, J ) 16.6 Hz, 1H, CH2),
3.32 (dd, J ) 14.5, 4.6 Hz, 1H, CH2), 2.90 (dd, J ) 14.5, 9.9
Hz, 1H, CH2), 1.30 (s, 9H, 3 × CH3). 13C NMR (CD3OD) δ:
172.5 (C), 172.2 (C), 155.7 (C), 145.3 (C), 137.5 (CH), 134.2
(C), 133.2 (C), 131.3 (CH), 127.8 (CH), 125.7 (CH), 124.6 (C),
116.7 (CH), 79.8 (C), 56.8 (CH), 53.5 (CH2), 49.6 (CH2), 37.4
(CH2), 29.3 (CH3). Anal. (C22H25N3O6‚0.6H2O) C, H, N.
(2S)-2-(4-tert-Butoxybenzyl)-4-[2-(tert-butyldiphenyl-
silanyloxy)ethyl]-9-nitro-1,2,4,5-tetrahydrobenzo[1,4]-
diazepin-3-one, 15a. The amine 14a (2.60 g, 3.78 mmol) was
dissolved in 30 mL of DMSO, and Et3N (0.77 mL, 5.5 mmol)
was added. The reaction mixture was heated to 100 °C and
stirred for 24 h. EtOAc, water, and NaHCO3 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 (CH2-
Cl2), and the product 15a was obtained as a yellow foam (2.03
g, 82%). [R]23D ) -109° (c ) 1.03, MeOH). Anal. (C38H45N3O5-
Si) C, H, N.
[(2S)-2-(4-tert-Butoxybenzyl)-9-[(9H-fluoren-9-ylmeth-
oxycarbonyl)amino]-3-oxo-1,2,3,5-tetrahydrobenzo[1,4]-
diazepin-4-yl]-acetic Acid, 18a. HCO2NH4 (450 mg, 7.14
mmol) was added to a mixture of Pd/C (45 mg 10%, 42 µmol)
and 17a (203 mg, 0.47 mmol) in MeOH (7 mL). The reaction
was performed under nitrogen in a sealed tube and was
complete within 2.5 h. The Pd/C was removed by filtration.
After evaporation, the crude product was dissolved in EtOAc
and washed with water and brine. The organic layer was dried
over Na2SO4 and evaporated. The residue was suspended in
dioxane (5 mL) and cooled to 0 °C. FmocCl (182 mg, 0.70 mmol)
was added, followed by the dropwise addition of 4.7 mL of 10%
aqueous Na2CO3 at a pH of 8-9. After 48 h, diethyl ether and
water were added. The layers were separated, and the aqueous
layer (with a precipitate) was washed with diethyl ether until
TLC showed that all impurities with Rf values higher than
that of the product had been removed. More diethyl ether 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 diethyl ether. The combined organic
layers were washed with water, dried (Na2SO4), and evapo-
(2S)-2-(4-tert-Butoxybenzyl)-4-[2-hydroxyethyl]-9-nitro-
1,2,4,5-tetrahydrobenzo[1,4]diazepin-3-one, 16a. TBAF (1
M solution in THF, 3.40 mL, 3.4 mmol) was added to 15a (1.85
g, 2.84 mmol) in THF (40 mL). After 1.5 h water and EtOAc
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 over Na2SO4, and evapo-
rated. Column chromatography (1% MeOH in CH2Cl2) gave
15a as a yellow foam (1.05 g, 89%). [R]23 ) -201° (c ) 1.06,
D
MeOH). 1H NMR (CDCl3) δ: 8.41 (d, J ) 2.8 Hz, 1H, NH),
8.04 (m, 1H, CH), 7.27 (m, 2H, 2 × CH), 7.12 (m, 1H, CH),
6.98 (m, 2H, 2 × CH), 6.52 (m, 1H, CH), 5.44 (d, J ) 16.7 Hz,
1H, CH2), 5.10 (m, 1H, CH), 3.96 (d, J ) 16.7 Hz, 1H, CH2),
3.88-3.73 (m, 3H, CH2 and CH2), 3.62-3.50 (m, 1H, CH2), 3.40
(dd, J ) 4.4, 14.6 Hz, 1H, CH2), 2.94 (dd, J ) 9.6, 14.6 Hz,
1H, CH2), 2.26 (br s, 1H, OH), 1.33 (s, 9H, 3 × CH3). 13C NMR
(CDCl3) δ: 169.8 (C), 154.5 (C), 144.0 (C), 135.8 (CH), 133.0
(C), 131.1 (C), 129.9 (CH), 127.3 (CH), 124.5 (CH), 122.6 (C),
115.3 (CH), 78.5 (C), 61.7 (CH2), 56.4 (CH), 53.0 (CH2), 50.2
(CH2), 36.7 (CH2), 28.8 (CH3). Anal. (C22H27N3O5) C, H, N.
rated to give 18a as a yellow foam (197 mg, 68%). [R]23
)
D
1
-28° (c ) 1.01, MeOH). H NMR (CD3OD) δ: 7.90-7.60 (m,
4H, 4 × CH), 7.48-7.21 (m, 4H, 4 × CH), 7.10 (m, 2H, 2 ×
CH), 7.02 (m, 1H, CH), 6.86 (m, 1H, CH), 6.59 (m, 1H, CH),
5.44 (d, J ) 16.5 Hz, 1H, CH2), 4.91 (m, 1H, CH), 4.48 (d, J )
[(2S)-2-(4-tert-Butoxybenzyl)-9-nitro-3-oxo-1,2,3,5-
tetrahydrobenzo[1,4]diazepin-4-yl]-acetic Acid, 17a. A
solution of oxalyl chloride (300 µL, 3.50 mmol) in 16 mL of
CH2Cl2 was cooled to -78 °C. DMSO (525 µL, 7.40 mmol) was

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4019
17.4 Hz, 1H, CH2), 4.44 (m, 1H, CH), 4.19 (m, 2H, CH2), 4.03
(d, J ) 16.5 Hz, 1H, CH2), 3.97 (d, J ) 17.4 Hz, 1H, CH2),
3.29 (dd, J ) 14.3, 4.0 Hz, 1H, CH2), 2.79 (dd, J ) 14.3, 10.0
Hz, 1H, CH2), 1.13 (s, 9H, 3 × CH3). 13C NMR (CD3OD) δ:
173.9 (C), 172.8 (C), 157.3 (C), 155.3 (C), 145.6 (C), 145.2 (C),
142.8 (C), 133.9 (C), 130.9 (CH), 129.0 (CH), 128.6 (CH), 128.3
(CH), 127.7 (C), 126.7 (CH), 126.4 (CH), 125.6 (CH), 123.0 (C),
121.1 (CH), 119.0 (CH), 79.4 (C), 68.5 (CH2), 57.0 (CH), 53.6
(CH2), 50.1 (CH2), 48.3 (CH), 37.7 (CH2), 29.3 (CH3). Anal.
(C37H37N3O6‚1.5H2O) C, H, N.
(2S,3S)-2-(2-Chloro-3-nitrobenzylamino)-3-methylpen-
tan-1-ol, 12b. Compound 12b was prepared from 11 (3.90 g,
21.0 mmol) and (S,S)-2-amino-3-methyl-1-pentanol (2.50 g,
21.3 mmol) as described above in the synthesis of 12a. The
reaction mixture was heated overnight before being worked
up to give 12b as a pale yellow oil (3.29 g, 55%). [R]23D ) +20°
(c ) 1.03, MeOH). Anal. (C13H19ClN2O3) C, H, N.
1.92 (m, 1H, CH), 1.34 (m, 1H, CH2), 1.32 (s, 9H, 3 × CH3),
1.05 (m, 1H, CH2), 0.99 (d, J ) 6.6 Hz, 3H, CH3), 0.84 (t, J )
7.3 Hz, 3H, CH3). 13C NMR (CDCl3) δ: 170.2 (C), 154.5 (C),
144.1 (C), 136.0 (CH), 133.0 (C), 131.4 (C), 129.9 (2 × CH),
127.2 (CH), 124.5 (2 × CH), 123.2 (C), 115.3 (CH), 78.5 (C),
62.8 (CH2), 61.3 (CH), 56.4 (CH), 48.0 (CH2), 36.6 (CH2), 32.1
(CH), 28.8 (3 × CH3), 25.8 (CH2), 15.7 (CH3), 10.5 (CH3).
(2S,3S)-2-[(2S)-2-(4-tert-Butoxybenzyl)-9-nitro-3-oxo-
1,2,3,5-tetrahydrobenzo[1,4]diazepin-4-yl]-3-methylpen-
tanoic Acid, 17b. Compound 17b was obtained after a two-
step oxidation of 16b (300 mg, 0.64 mmol) as described above
for 17a. Column chromatography (gradient CH2Cl2 to 10%
MeOH in CH2Cl2) gave 17b as a yellow foam (236 mg, 76%).
1
[R]23 ) -70° (c ) 1.03, MeOH). H NMR (CDCl3) δ: 8.40 (d,
D
J ) 2.9 Hz, 1H, NH), 8.03 (dd, J ) 8.7, 1.6 Hz, 1H, CH), 7.25
(m, 2H, 2 × CH), 7.16 (dd, J ) 7.2, 1.6 Hz, 1H, CH), 6.97 (m,
1H, 2 × CH), 6.54 (dd, J ) 8.7, 7.2 Hz, 1H, CH), 5.32 (d, J )
17.6 Hz, 1H, CH2), 5.16-5.08 (m, 2H, 2 × CH), 4.48 (d, J )
17.6 Hz, 1H, CH2), 3.40 (dd, J ) 14.8, 4.2 Hz, 1H, CH2), 2.96
(dd, J ) 14.8, 10.1 Hz, 1H, CH2), 1.84 (m, 1H, CH), 1.31 (s,
9H, 3 × CH3), 1.08 (m, 1H, CH2), 0.96 (d, J ) 6.6 Hz, 3H, CH3),
0.85 (m, 1H, CH2), 0.52 (t, J ) 7.4 Hz, 3H, CH3). 13C NMR
(CDCl3) δ: 174.9 (C), 170.9 (C), 154.4 (C), 143.8 (C), 135.9 (CH),
133.1 (C), 131.1 (C), 129.9 (2 × CH) 127.3 (CH), 124.5 (2 ×
CH), 122.9 (C), 115.6 (CH), 78.6 (C), 60.8 (CH), 56.3 (CH), 48.1
(CH2), 36.5 (CH2), 35.1 (CH), 28.8 (3 × CH3), 25.4 (CH2), 15.5
(CH3), 10.4 (CH3). Anal. (C26H33N3O6) C, H, N.
[(1S,2S)-1-(tert-Butyldiphenylsilanyloxymethyl)-2-
methylbutyl]-(2-chloro-3-nitrobenzyl)amine, 13b. Com-
pound 13b was prepared from 12b (1.40 g, 4.88 mmol) as
described above for 13a. The reaction mixture was stirred for
7 h at room temperature before the work up. Purification by
column chromatography (gradient CH2Cl2/pentane (3:1) to
CH2Cl2) gave 13b as a pale yellow oil (2.17 g, 85%). [R]19
+20° (c ) 1.01, MeOH). Anal. (C29H37ClN2O3Si) C, H, N.
)
D
(2S)-2-Amino-3-(4-tert-butoxyphenyl)-N-(2-chloro-3-ni-
tro-benzyl)-N-[(1S,2S)-(1-hydroxymethyl-2-methylbutyl-
)]propionamide, 14b. The amine 12b (1.75 g, 6.10 mmol) was
dissolved in CH2Cl2 (35 mL). The solution was divided into
seven process vials (5 mL each). To each vial HATU (365 mg,
0.96 mmol), Fmoc-L-Tyr(t-Bu)-OH (440 mg, 0.96 mmol), and
DIEA (320 µL, 1.84 mmol) were added. The reaction mixtures
were irradiated with microwaves at 100 °C for 15 min and
were then combined and diluted with water and EtOAc. 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 (gradient CH2-
Cl2 to 2% MeOH in CH2Cl2) to give the Fmoc-protected amine
as a white foam (2.47 g, 56%). DBU (160 µL, 1.07 mmol) was
added to a solution of the Fmoc-protected amine (650 mg, 0.89
mmol) in 6 mL of THF. After 2 h at room temperature, the
MeOH was evaporated, and the product purified by column
chromatography (gradient CH2Cl2 to 2% MeOH in CH2Cl2) to
give the deprotected amine 14b (370 mg, 82%). [R]23D ) +2.5°
(c ) 1.03, MeOH). Anal. (C26H36ClN3O5) C, H, N.
(2S,3S)-2-[(2S)-2-(4-tert-Butoxybenzyl)-9-[(9H-fluoren-
9-ylmethoxycarbonyl)amino]-3-oxo-1,2,3,5-tetrahydro-
benzo[1,4]diazepin-4-yl]]-3-methylpentanoic Acid, 18b.
Compound 18b was prepared from 17b (200 mg, 0.414 mmol)
as described above in the synthesis of 18a, using Pd/C (40 mg
10%, 37 µmol) and HCO2NH4 (520 mg, 8.25 mmol). Dioxane
(4.5 mL), FmocCl (320 mg, 1.24 mmol), and 10% Na2CO3 (aq)
(3.6 mL) were added as described above, and the reaction was
stirred at room temperature for 15 h. Extraction gave 18b and
a diastereoisomeric mixture in a ratio of ∼8:2. The crude
product was purified by RP-HPLC (30 min gradient of 60-
85% CH3CN in 0.05% aqueous formic acid) to give 18b (97 mg,
35%) as a white solid. 1H NMR (CD3OD) δ: 7.90-7.53 (m, 4H,
4 × CH), 7.50-7.17 (m, 4H, 4 × CH), 7.10 (m, 2H, 2 × CH),
7.03 (m, 1H, CH), 6.89 (m, 1H, CH), 6.65 (m, 2H, 2 × CH),
6.58 (t, J ) 7.7 Hz, 1H, CH), 5.35 (m, 1H, CH2), 4.99 (d, J )
10.7, 1H, CH), 4.45 (m, 1H, CH2), 4.42 (m, 1H, CH), 4.32-
4.00 (m, 2H, CH2), 3.19 (dd, J ) 4.3, 14.4 Hz, 1H, CH2), 2.81
(dd, J ) 10.3, 14.4 Hz, 1H, CH2), 1.86 (m, 1H, CH), 1.16 (m,
1H, CH2), 1.14 (s, 9H, 3 × CH3), 0.93 (d, J ) 6.6 Hz, 3H, CH3),
0.79 (m, 1H, CH2), 0.48 (t, J ) 7.5 Hz, 3H, CH3). 13C NMR
(CD3OD) δ: 174.6 (C), 174.2 (C), 157.3 (C), 155.3 (C), 145.6
(C), 142.8 (C), 134.0 (C), 130.9 (CH), 129.0 (CH), 128.3 (CH),
127.7 (C), 126.8 (CH), 125.9 (CH), 125.6 (CH), 123.1 (C), 121.1
(CH), 119.0 (CH), 79.4 (C), 68.6 (CH2), 62.2 (CH), 56.7 (CH),
49.9 (CH2), 48.3 (CH), 37.6 (CH2), 36.4 (CH), 29.3 (CH3), 26.6
(CH2), 16.2 (CH3), 11.0 (CH3). Anal. (C41H45N3O6‚H2O) C, H,
N.
Solid-Phase Peptide Synthesis (SPPS). The peptides
were synthesized manually, in 2 mL disposable syringes
equipped with a porous polyetehylene filter, from His(Trt)-
Pro-Phe-Wang resin58 using standard Fmoc/t-Bu conditions.
The Fmoc group was removed by treatment with 20% piperi-
dine/DMF for 5 + 10 min. Coupling was achieved with PyBOP
in the presence of DIEA. When templates and adjacent amino
acids were coupled, prolonged reaction times were used and
the completeness of the reaction was checked by PDMS or LC/
MS analysis of cleaved resin samples. The Fmoc amino acids
were protected as follows, Asp(Ot-Bu), Arg(Pbf), Tyr(t-Bu), and
His(Trt).
(2S)-2-(4-tert-Butoxybenzyl)-4-[(1S,2S)-(1-hydroxy-
methyl-2-methyl-butyl)]-9-nitro-1,2,4,5-tetrahydrobenzo-
[1,4]diazepin-3-one, 16b. Compound 16b was prepared from
14b (857 mg, 1.69 mmol) as described above in the synthesis
of 15a. Column chromatography (gradient CH2Cl2 to 2% MeOH
in CH2Cl2) gave 16b as a yellow foam (326 mg, 41%) and the
diastereoisomer 16c (126 mg, 16%). 16b: [R]23 ) -51° (c )
D
0.55, MeOH). 1H NMR (CDCl3) δ: 8.42 (d, J ) 2.9 Hz, 1H,
NH), 8.02 (dd, J ) 8.7, 1.6 Hz, 1H, CH), 7.25 (m, 2H, 2 × CH),
7.13 (dd, J ) 7.2, 1.6 Hz, 1H, CH), 6.96 (m, 2H, 2 × CH), 6.52
(dd, J ) 8.7, 7.2 Hz, 1H, CH), 5.32 (d, J ) 17.2 Hz, 1H, CH2),
5.15 (m, 1H, CH), 4.38 (m, 1H, CH), 4.14 (d, J ) 17.2 Hz, 1H,
CH2), 3.92 (dd, J ) 11.8, 3.6 Hz, 1H, CH2), 3.75 (dd, J ) 11.8,
8.5 Hz, 1H, CH2), 3.40 (dd, J ) 14.6, 4.3 Hz, 1H, CH2), 2.96
(dd, J ) 14.6, 9.7 Hz, 1H, CH2), 2.17 (br s, 1H, OH), 1.56 (m,
1H, CH), 1.30 (s, 9H, 3 × CH3), 1.10 (m, 1H, CH2), 0.90 (d, J
) 6.6 Hz, 3H, CH3), 0.86 (m, 1H, CH2), 0.49 (t, J ) 7.4 Hz,
3H, CH3). 13C NMR (CDCl3) δ: 171.3 (C), 154.4 (C), 144.0 (C),
135.6 (CH), 132.9 (C), 131.4 (C), 129.9 (2 × CH), 127.1 (CH),
124.5 (2 × CH), 123.3 (C), 115.3 (CH), 78.5 (C), 62.1 (CH2),
61.0 (CH), 56.5 (CH), 47.8 (CH2), 36.6 (CH2), 34.4 (CH), 28.8
(3 × CH3), 26.0 (CH2), 15.5 (CH3), 10.4 (CH3). Anal. (C26H35N3O5‚
Ang II Analogue 3. Compound 18a (40.0 mg, 64.5 µmol)
and PyBOP (33.6 mg, 64.5 µmol) were dissolved in DMF (0.8
mL) together with DIEA (22.5 µL, 129 µmol) and allowed to
react with His(Trt)-Pro-Phe-Wang resin (67.2 mg, 53.8 µmol)
in a 2 mL syringe vessel for 20.5 h. PDMS analysis of a cleaved
analytical sample confirmed that the coupling was complete.
The resin was washed with DMF (6 × 1.5 mL, 6 × 1 min) and
1
H2O) C, H, N. 16c: H NMR (CDCl3) δ: 8.42 (d, J ) 2.9 Hz,
1H, NH), 8.04 (m, 1H, CH), 7.27 (m, 2H, 2 × CH), 7.17 (m,
1H, CH), 6.96 (m, 2H, 2 × CH), 6.52 (m, 1H, CH), 5.27 (d, J )
16.7 Hz, 1H, CH2), 5.10 (m, 1H, CH), 4.30 (m, 1h, CH), 4.10
(d, J ) 16.7 Hz, 1H, CH2), 3.72 (m, 2H, CH2), 3.41 (dd, J )
14.4, 5.0 Hz, 1H, CH2), 2.98 (dd, J ) 14.4, 9.3 Hz, 1H, CH2),

4020 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
the Fmoc group removed by treatment with 20% piperidine/
DMF (2 × 1.5 mL, 5 + 10 min). After the sample was washed
with DMF (6 × 1.5 mL, 6 × 1 min), CH2Cl2 (6 × 2 mL, open
column), and MeOH (6 × 2 mL), the resin was dried in vacuo
to give 83.3 mg.
mg, 45.1 µmol) was reacted with Fmoc-Val-OH (76.5 mg, 226
µmol), PyBOP (117 mg, 226 µmol), and DIEA (78.6 µL, 452
µmol) in DMF (0.75 mL) for 16.5 h, and the coupling was
repeated under the same conditions for 4 h. The polymer
weighed 72.0 mg after deprotection, washing, and drying.
Fmoc-Ala-OH‚H2O (36.6 mg, 111 µmol) and Fmoc-Asp(Ot-Bu)-
OH (45.7 mg, 111 µmol) were then coupled successively to an
aliquot (35.5 mg, 22.2 µmol) of the resin using PyBOP/DIEA
activation for 3 h. The desired, partially protected resin
weighed 40.1 mg. The pseudopeptide was cleaved and depro-
tected by treatment with triethylsilane (50 µL) and 95% aq
TFA (1.5 mL) for 2 h to give 21.2 mg of crude material. The
product was dissolved in 10% MeCN (2.2 mL) and purified on
a 5 µm ACE phenyl column (21.2 mm × 150 mm) using a 45
min gradient of 10-40% MeCN in 0.1% aq TFA at a flow rate
of 5 mL/min. Yield: 8.0 mg (35.8%). LC/MS (Mabs 1007.45):
1008.7 (M + H+), 505.1 ([M + 2H+]/2). Amino acid analysis:
Asp, 1.01; Ala, 0.98; Val, 0.52; His, 1.00; Pro, 1.02; Phe, 0.99.
Ang II Analogue 6. A second aliquot (36.9 mg, 23.6 µmol)
of the resin obtained after coupling of compound 18a (analogue
3 above) was reacted with Fmoc-Ala-OH‚H2O (38.9 mg, 118
µmol), PyBOP (61.4 mg, 118 µmol), and DIEA (41.2 µL, 236
µmol) in DMF (0.5 mL) for 22 h. The product was washed with
DMF and then the coupling was repeated for 4 h under the
same conditions. The resin was deprotected and Fmoc-Asp-
(Ot-Bu)-OH (48.6 mg, 118 µmol) was coupled in the same way
using a reaction time of 2.5 h. The Fmoc group was removed,
and the polymer was washed and dried to give 41.0 mg.
Cleavage with 95% TFA in the presence of triethylsilane
afforded 23.7 mg of the crude product, which was purified on
the semipreparative phenyl column as described above but
with a slightly modified gradient: 5-35% MeCN in 45 min.
Yield: 8.9 mg (26.6%). LC-MS (Mabs 908.38): 909.2 (M + H+),
455.4 ([M + 2H+]/2). Amino acid analysis: Asp, 0.98; Ala, 0.28;
His, 1.01; Pro, 1.02; Phe, 1.00.
Part of the polymer (39.6 mg, 24.3 µmol) was transferred to
a second syringe and coupled in DMF (0.5 mL) with Fmoc-
Val-OH (41.2 mg, 122 µmol) and PyBOP (63.2 mg, 122 µmol)
in the presence of DIEA (42.3 µL, 244 µmol). PDMS analysis
after 19 h showed that some unreacted material was still
present on the resin. Therefore, the resin was washed with
DMF (3 × 2 mL, 3 × 1 min) and the coupling was repeated in
the same way for 3.5 h but with O-(1H-6-chlorobenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU)
(43.2 mg, 122 µmol) as the activation agent. There was little
improvement according to the PDMS analysis; a small amount
of unreacted material still remained. The resin was washed
and deprotected as described above, then washed with DMF
(6 × 1.5 mL, 6 × 1 min). The desired pseudopeptide resin was
produced (47.3 mg) after successive couplings of Fmoc-Arg-
(Pbf)-OH (78.8 mg, 122 µmol) and Fmoc-Asp(Ot-Bu)-OH (50.0
mg, 122 µmol) using PyBOP/DIEA activation for 2.5 h and
followed by deprotection and washing with DMF, CH2Cl2, and
MeOH.
Triethylsilane (75 µL) and 95% aq TFA (1.5 mL) were added
to the partially protected peptide resin in a 3.5 mL centrifuge
tube, and the mixture was agitated by rotation for 1.5 h. The
polymer was removed by filtration through a small plug of
glass wool in a Pasteur pipet and washed with TFA (2 × 0.3
mL). The combined filtrates were evaporated in a stream of
nitrogen to 1.5 mL, and the product was precipitated with
diethyl ether (12 mL). It was collected by centrifugation,
washed with diethyl ether (4 × 8 mL), and dried in air and in
vacuo to give 32.2 mg. LC-MS analysis showed the expected
m/z value for the major component.
The crude material was dissolved in a mixture of H2O (4
mL) and MeCN (0.2 mL) and purified in two runs by RP-HPLC
on a 10 µm Vydac C18 column (2.2 × 25 cm) using a 60 min
gradient of 5-45% MeCN in 0.1% aq TFA at a flow rate of 5
mL/min. The separation was monitored by UV absorption at
230 nm and by LC/MS and/or analytical RP-HPLC of selected
fractions. The yield of the purified and lyophilized peptide was
8.0 mg (30.1%). LC/MS (Mabs 1092.51): 1093.7 (M + H+), 547.6
([M + 2H+]/2), 365.4 ([M + 3H+]/3). Amino acid analysis: Asp,
1.00; Arg, 0.95; Val, 0.48; His, 1.04; Pro, 0.98; Phe, 1.03.
Ang II Analogue 4. The second half (42.2 mg, 25.9 µmol)
of the peptide resin obtained after coupling and deprotection
of compound 18a was reacted with Fmoc-Arg(Pbf)-OH (84.0
mg, 130 µmol), PyBOP (67.4 mg, 130 µmol), and DIEA (45.1
µL, 260 µmol) in DMF (0.5 mL) for 22.5 h. PDMS analysis
indicated that some unreacted material still remained on the
resin. The coupling, however, was not repeated since the
corresponding recoupling of Fmoc-Val-OH (see above for
analogue 3) was essentially without effect. Instead, the resin
was deprotected and coupled with Fmoc-Asp(Ot-Bu)-OH (53.3
mg, 130 µmol), PyBOP (67.4 mg, 130 µmol), and DIEA (45.1
µL, 260 µmol) in DMF (0.5 mL) for 2.5 h. The Fmoc group was
removed, and the polymer was washed with DMF, CH2Cl2, and
MeOH and dried as described above. The resin (47.7 mg, 25.6
µmol) was treated with triethylsilane and 95% aq TFA for 1.5
h, and the product was precipitated and washed with diethyl
ether and dried, giving 32.0 mg. LC/MS analysis showed that
the material contained a considerable amount (ca 30%) of the
des-Arg analogue.
Ang II Analogues 7a and 7b. Compound 18b (43.0 mg,
63.6 µmol) was coupled to His(Trt)-Pro-Phe-2-Cl-Trityl resin
(95.4 mg, 57.2 µmol) with the aid of PyBOP (33.1 mg, 63.6
µmol) and DIEA (22.2 µL, 127µmol) in DMF (1.0 mL) for 18 h.
LC/MS analysis after cleavage of an analytical sample showed
the reaction to be complete. The resin was washed, depro-
tected, and dried as above to yield 119 mg. An aliquot of the
polymer (58.1 mg, 27.9 µmol) was coupled in DMF (0.5 mL)
with Fmoc-Val-OH (47.5 mg, 140 µmol) using PyBOP (72.8 mg,
140 µmol) and DIEA (48.8 µL, 280 µmol) for 20 h. The reaction
was incomplete according to LC-MS analysis. The coupling
was repeated using the same conditions but with HATU (53.2
mg, 140 µmol) as the activating agent. The pseudo-peptide
chain was further elongated by consecutive couplings with
Fmoc-Arg(Pbf)-OH (90.8 mg, 140 µmol) and Fmoc-Asp(Ot-Bu)-
OH (57.6 mg, 140 µmol) using PyBOP activation for 2 h. After
deprotection, the resin was washed and dried and weighed 73.5
mg. Part of the material (57.9 mg) was treated with triethyl-
silane (50 µL) and 95% aq TFA (1.5 mL) for 1.5 h to yield 32.6
mg of the crude product. This was dissolved in 10% MeCN
(2.2 mL) and purified on the ACE phenyl column using a 60
min gradient of 15-45% MeCN in 0.1% aq TFA. Two isomers
were collected. The one with the lowest retention was further
purified by RP-HPLC on a 5 µm ACE CN column (21.2 mm ×
50 mm) with a 30 min gradient of 5-35% MeCN in 0.1% aq
TFA at a flow rate of 3 mL/min followed by rechromatography
on the phenyl column. 7a: yield, 8.4 mg (33.2%). LC/MS (Mabs
1148.58): 1149.3 (M + H+), 575.3 ([M + 2H+]/2), 384.0 ([M +
3H+]/3). Amino acid analysis: Asp, 0.99; Arg, 0.99; Val, 0.49;
His, 0.88; Pro, 1.01; Phe, 1.01. 7b: yield, 1.5 mg (5.9%). LC/
MS (Mabs 1148.58): 1149.3 (M + H+), 575.4 ([M + 2H+]/2),
384.0 ([M + 3H+]/3). Amino acid analysis: Asp, 1.00; Arg, 0.98;
Val, 0.42; His, 0.93; Pro, 1.01; Phe, 1.01.
The crude pseudopeptide was chromatographed in two runs
on the Vydac C18 column using the same conditions as for
analogue 3 above. The yield of pure lyophilized product was
5.6 mg (22.0%). LC/MS (Mabs 993.45): 994.6 (M + H+), 497.9
([M + 2H+]/2), 332.4 ([M + 3H+]/3). Amino acid analysis: Asp,
1.00; Arg, 0.33; His, 1.00; Pro, 1.00; Phe, 0.99.
Ang II Analogue 5. Compound 18a (84.7 mg, 114 µmol)
was coupled to His(Trt)-Pro-Phe-Wang resin (142 mg, 114
µmol), deprotected, washed, and dried as described above for
analogue 3 to give 178 mg of product. Part of the resin (70.5
Ang II Analogues 8a and 8b. The second half of the
product obtained after coupling compound 18b to the tripeptide
resin (61.3 mg, 29.4 µmol) was reacted with Fmoc-Arg(Pbf)-
OH (95.4 mg, 147 µmol), PyBOP (76.5 mg, 147 µmol), and
DIEA (51.2 µL, 294 µmol) in DMF (0.5 mL) for 20 h. LC/MS
analysis showed the reaction to be incomplete, and the

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4021
coupling was repeated using the same conditions but with
HATU as the activating agent. Although a small amount of
unreacted material still remained, the resin was deprotected
and coupled with Fmoc-Asp(Ot-Bu)-OH (60.5 mg, 147 µmol)
using PyBOP/DIEA activation as above for 5 h. After the
removal of the Fmoc group, the polymer was washed and dried
to yield 71.1 mg. Part of the material (59.0 mg) was treated
with triethylsilane (50 µL) and 95% aq TFA (1.5 mL) for 1.5 h
to give 32.4 mg of the crude product. Purification was achieved
by RP-HPLC on the ACE phenyl column as described for
compound 7 above. 8a: yield, 9.6 mg (37.5%). LC-MS (Mabs
1049.50): 1050.4 (M + H+), 526.0 ([M + 2H+]/2), 351.0 ([M +
3H+]/3). Amino acid analysis: Asp, 0.99; Arg, 0.42; His, 0.88;
Pro, 1.01; Phe, 1.00. Two isomers were isolated. 8b: yield, 4.8
mg (18.7%). LC-MS (Mabs 1049.50): 1050.4 (M + H+), 525.9
([M + 2H+]/2), 350.9 ([M + 3H+]/3). Amino acid analysis: Asp,
0.95; Arg, 0.39; His, 0.77; Pro, 1.02; Phe, 1.03.
Rat Liver Membrane AT1 Receptor Binding Assay. Rat
liver membranes were prepared according to the method of
Dudley et al.61 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, 10
µM bacitracin, 10 µM pepstatin A, 10 µM bestatin, 10 µM
captopril, 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 (0.01
nM-1.0 µM) of test substance. The 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. 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. IC50 was determined by a
Scatchard analysis of data obtained with Ang II by using
GraFit (Erithacus Software, U.K.). The apparent dissociation
constants Ki were calculated from IC50 values using the
Cheng-Prusoff equation81 (Kd ) 1.7 ( 0.1 nM, [L] ) 0.057
nM). The binding data were best fitted with a one-site fit. All
of the experiments were performed in triplicate.
Pig Myometrial Membrane AT2 Receptor Binding
Assay. Myometrial membranes were prepared from porcine
uteri according to the method of Nielsen et al.62 Potential
interference by binding to AT1 receptors was blocked by the
addition of 1 µM losartan. 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,
10 µM bacitracin, 10 µM pepstatin A, 10 µM bestatin, 10 µM
captopril, 0.2% BSA, homogenate corresponding to 10 mg of
the original tissue weight, [125I]Ang II (80 000 cpm, 0.03 mM),
and variable concentrations (0.01 nM-1.0 µM) of the 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. 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. IC50 was determined by a Scatchard analysis of data
obtained with Ang II by using GraFit (Erithacus Software,
U.K.). The apparent dissociation constants Ki were calculated
using the Cheng-Prusoff equation81 (Kd ) 0.7 ( 0.1 nM, [L]
) 0.057 nM). The binding data were best fitted with a one-
site fit. All of the experiments were performed in triplicate.
CA); PD98,059, anti-phosphorylated p42/p44mapk, and total p42/
p44mapk antibodies from the New England Biolabs (Beverly,
MA); horseradish peroxidase-conjugated anti-mouse and anti-
rabbit antibodies from Amersham Pharmacia Biotech Inc.
(NJ); complete protease inhibitor, poly(vinylidene difluoride)
(PVDF) membranes, and enhanced chemiluminescence (ECL)
detection system from Roche Laboratories (Montreal, QC,
Canada). All other chemicals were of grade-A purity.
(B) Cell Culture. NG108-15 cells (provided by Drs M.
Emerit and M. Hamon, INSERM, U. 238, Paris, France) were
cultured (passage 7 to 30) in DMEM with 10% fetal bovine
serum (FBS, Gibco BRL, Burlington, ON, Canada), HAT
supplement, and 50 mg/L gentamycin at 37 °C in 75 cm2
Nunclon Delta flasks in a humidified atmosphere of 93% air
and 7% CO2, as previously described.63 Subcultures were
performed at subconfluency. Under these conditions, cells
expressed only the AT2 receptor subtype.63,64 Cells were
stimulated for periods ranging from minutes (MAPK) to 3 days
(neurite elongation) (first stimulation 24 h after plating)
according to the experiments. Cells were cultured for three
subsequent days under these conditions. For all experiments,
cells were plated at the same initial density of 4 × 104 cells/
35 mm Petri dish. Cells were treated without (control cells)
or with Ang II (100 nM) or one of the test compounds (100
nM), in the absence or in the presence of the various inhibitors
to be tested: PD123,319 (1 µM), an AT2 receptor antagonist,
PD98,059 (10 µM), an inhibitor of MEK, LY-83,583 (0.5 µM),
an inhibitor of soluble guanylyl cyclase or KT-5823 (1 µM),
and an inhibitor of cGMP-dependent protein kinases (each
introduced daily with inhibitors applied 30 min prior to Ang
II or the tested pseudopeptide).
(C) Determination of Cells with Neurites. Cells were
examined daily under a phase contrast microscope, and
micrographs were taken after 3 days under the various
experimental conditions. Cells with at least one neurite longer
than a cell body were counted as positive for neurite outgrowth.
At least 64 cells were counted in three independent experi-
ments.65
(D) Western Blotting for p42/p44mapk. After 3 days of
culture, cells were washed with Hanks buffered saline (HBS)
(HBS: 130 mM NaCl; 3.5 mM KCl; 2.3 mM CaCl2‚2H2O; 0.98
mM MgCl2‚6H2O; 5 mM HEPES; 0.5 mM EGTA) and then
incubated for 10 min with 800 µL of stabilization buffer (100
nM staurosporine, 1 mM Na3VO4 in HBS) and finally lysed in
25 µL of lysis buffer (50 mM HEPES pH 7.8, 100 nM
staurosporine, 1 mM Na3VO4, 1% Triton-X100, protease
inhibitors). Samples were separated on 10% SDS-polyacryl-
amide gels. Proteins were transferred electrophoretically to
PVDF membranes. Membranes were blocked with 1% gelatin,
and 0.05% Tween 20 in TBS buffer (pH 7.5). After samples
were washed with TBS-Tween 20 (0.05%), membranes were
incubated overnight at 4 °C with antiphosphorylated p42/
p44mapk (1:1000) or anti-p42/p44mapk (1:1000) and diluted in
TBS-Tween 20 (0.05%) plus BSA (0.1%). After samples were
washed with TBS-Tween 20, detection was accomplished using
horseradish peroxidase-conjugated anti-mouse or anti-rabbit
IgG (1:2000) and an enhanced chemiluminescence (ECL)
detection system, as described previously.23
(E) Data Analysis. The data were presented as a mean (
standard error of mean (SEM) of the number of experiments
indicated in the text, each performed in duplicate or triplicate.
Statistical analyses of the data were performed using the one-
way analysis of variance (ANOVA) test. Homogeneity of
variance was assessed by Bartlett’s test, and p values were
obtained from Dunnett’s tables.
Proliferation of PC12 Cells. PC12 cells, a well-character-
ized rat adrenal pheochromocytoma cell line, were obtained
from the European Collection of Animal Cell Cultures (ECACC,
Salisbury, United Kingdom). Cells were grown in suspension
cultures in DMEM (Gibco-BRL, Eggenstein, Germany) with
10% fetal calf serum (FCS) in 5% CO2 at 37 °C. PC12 cells
expressed only AT2 receptors.
In Vitro Morphological Effects. (A) General. The chemi-
cals used in the present study were obtained from the following
sources: Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), HAT supplement (Hypoxanthine, Ami-
nopterin, Thymidine), and gentamycin from Gibco BRL (Bur-
lington, ON, Canada); [Val5]-angiotensin II from Bachem
(Marina Delphen, CA); PD123,319 from RBI (Natick, MA); LY-
83,583 and KT 5823 from Calbiochem-Novabiochem (La Jolla,
Proliferation was assessed by incorporation of [3H]thymidine
into DNA. Cells (104 cells per well) were transferred to a 96-

4022 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
well microtiter plate and made quiescent by incubation in
serum-free medium for 12 h. Cells were then stimulated as
indicated for 24 h in 10% FCS in the presence of 10-6 M Ang
II (Sigma), 10-6 M PD123,319 (Sigma), or 10-6 M compound 1
and were pulsed with 1 µCi [3H]thymidine (5 Ci/mmol,
Amersham) during the last 6 h of culture. At the end of the
incubation period, cells were collected on glass-fiber paper with
an automatic cell harvester. Radioactivity of dry filters was
measured by liquid scintillation spectroscopy. All experiments
were independently performed with 10-12 replicates. All of
the values are presented as means ( SEM. Statistical signifi-
cance among multiple groups was first tested with the non-
parametric Kruskal-Wallis test. Individual groups were then
tested using the Wilcoxon-Mann-Whitney test. A p value of
<0.05 was considered significant.
Conformational Analysis and Molecular Modeling. (A)
General Protocol for Conformational Analysis. Confor-
mational analysis was performed in MacroModel 8.569 using
the MMFFs force field and the general Born solvent accessible
(GB/SA) surface area method for water developed by Still et
al.70 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. The
ring closure bond was defined as the bond between N and CR
of the Ala residue in the model compounds. The conformational
search was conducted using the systematic unbound multiple
minimum (SUMM) search method82 in the BatchMin program.
Truncated Newton conjugate gradient (TNCG) minimization
with a maximum of 500 iterations was used in the conforma-
tional search. Torsional memory and geometric preoptimiza-
tion were used. Conformations within 5 kcal/mol of the lowest
energy minimum were kept.
valine and to 1.5 Å for the analogues with valine. Conforma-
tions within 5 kcal/mol of the lowest energy minimum were
saved.
The DISCOtech68 module in Sybyl 6.985 for Linux was used
to find an alignment of interesting features among the
conformations. Extra features were added to the molecules in
DISCOtech by transforming relevant atoms of the molecules
into DISCOtech recognizable features. Câ of Ala (corresponding
to Câ in Tyr) was transformed into a quaternary nitrogen
(DISCOtech NP feature), and the CH3 carbon of the acetyl
capping group (corresponding to CR in Asp) and the carbon of
the N-CH3 group (corresponding to CR in Gly) were trans-
formed into tert-butyl groups (DISCOtech HY features). These
features plus the acceptor sites (DISCOtech AS features)
originating from the guanidino group were used in the
analysis. To simplify the analysis, all atoms not included in a
feature were transformed into hydrogen atoms. In this trans-
formation, only the atom types were changed but not the
geometry. The conformations from the conformational analysis
of 1m (212), 3m (158), and 4m (167) were imported to
DISCOtech. The DISCOtech setting “Features by Class” was
used to search for models containing more than zero acceptor
sites (receptor points corresponding to the guanidino group),
exactly two hydrophobic features (CR of the Gly and Asp
residue), and exactly one positive nitrogen (Câ of the Tyr
residue). The tolerance for a feature match between the three
molecules was set to start at 0.25 Å and increased in incre-
ments of 0.25 Å up to 2.5 Å if necessary. Compound 3m, with
the lowest number of identified conformations, was used as
the reference molecule. The total volume of the models was
calculated using the spreadsheet function Autofill (molprop_vol-
ume) in Sybyl. The same setup was used in the DISCOtech
analysis when the inactive 2m was also included (237 confor-
mations of 2m was used).
(B) CR Distance Comparisons in γ-Turn Mimetics.
Conformational analysis using the general protocol was per-
formed on model compounds of scaffold 20 and 21 where
tyrosine was substituted for alanine. One thousand search
steps were used, and for the minimization the derivative
Acknowledgment. We gratefully acknowledge sup-
port from the Swedish Research Council, the Swedish
Foundation for Strategic Research, and the Canadian
Institutes of Health Research to N.G.P. (MOP 37891).
N.G.P. is a holder of a Canada Research Chair in
Endocrinology of the Adrenal Gland.
convergence criterion was set to 0.001 kJ mol-1 Å-1
.
The distances and angles between the CR atoms in the
identified conformations were measured and compared to those
in an inverse γ-turn. In this comparison, the aromatic carbon
of the benzene ring in 20 and 21 that is bound to methylamide
was defined as CRi. The ideal inverse γ-turn was generated by
minimizing (MMFFs) a trialanine sequence with Φi+1 adjusted
to -77° and Ψi+1 adjusted to 65° prior to the minimization.
Supporting Information Available: 1H and 13C NMR
spectral data of (2-chloro-3-nitrophenyl)methanol and com-
pounds 11, 12a, 13a, 14a, 15a, 12b, 13b, and 14b and
elemental analysis for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(C) Comparison of 21 to γ-Turns in Protein Crystal
Structures. The three conformations identified within 1 kcal/
mol of the lowest found minimum of the model compound of
21 were compared to 2101 inverse γ-turns (defined as having
torsion angles (30° from the ideal values of residue i + 1),
extracted from a representative set of crystallized proteins
selected using the PDB_SELECT list from December 2003.83,84
The comparison was performed by calculating the root mean
References
(1) Kambayashi, Y.; Bardhan, S.; Takahashi, K.; Tsuzuki, S.; Inui,
H.; Hamakubo, T.; Inagami, T. Molecular cloning of a novel
angiotensin II receptor isoform involved in phosphotyrosine
phosphatase inhibition. J. Biol. Chem. 1993, 268, 24543-24546.
(2) Mukoyama, M.; Nakajima, M.; Horiuchi, M.; Sasamura, H.;
Pratt, R. E.; Dzau, V. J. Expression cloning of type 2 angiotensin
II receptor reveals a unique class of seven-transmembrane
receptors. J. Biol. Chem. 1993, 268, 24539-24542.
(3) Timmermans, P. B. M. W. M.; Wong, P. C.; Chiu, A. T.; Herblin,
W. F.; Benfield, P.; Carini, D. J.; Lee, R. J.; Wexler, R. R.; Saye,
J. A. M.; Smith, R. D. Angiotensin II receptors and angiotensin
II receptor antagonists. Pharm. Rev. 1993, 45, 205-251.
(4) de Gasparo, M.; Catt, K. J.; Inagami, T.; Wright, J. W.; Unger,
T. International union of pharmacology. XXIII. The angiotensin
II receptors. Pharm. Rev. 2000, 52, 415-472.
(5) Kaschina, E.; Unger, T. Angiotensin AT1/AT2 receptors: regula-
tion, signaling and function. Blood Pressure 2003, 12, 70-88.
(6) Horiuchi, M. Functional aspects of angiotensin type 2 receptor.
Adv. Exp. Med. Biol. 1996, 396, 217-224.
(7) Csikos, T.; Chung, O.; Unger, T. Receptors and their classifica-
tion: focus on angiotensin II and the AT2 receptor. J. Hum.
Hypertens. 1998, 12, 311-318.
square distance between the torsion angles Φi, Ψi, Φi+1, Ψi+1
,
Φi+2, and Ψi+2 in the protein turns and the corresponding
torsion angles in 21.
(D) DISCOtech Analysis. The conformational search was
performed on structures 1m, 2m, 3m, and 4m using the
general protocol. For the minimization the derivative conver-
gence criterion was set to 0.05 kJ mol-1 Å-1 and 5000 search
steps per rotatable bond were used. To reduce the number of
identified conformations, all folded conformations which have
intramolecular hydrogen bonds with a distance of 4 Å or less
between the carbon in the guanidino group and any of the
carbonyl oxygens were removed. The number of conformations
was reduced further in a subsequent minimization of the found
conformations by increasing the convergence criteria to 0.001
kJ mol-1 Å-1 and using only four key atoms (see Figure 9) as
comparison atoms (COMP). In this minimization, the criterion
for a unique conformation was changed from the default
maximum distance of 0.25 Å between the compared atoms
after superposition (CRMS) to 0.75 Å for the analogues without
(8) Gendron, L.; Payet, M. D.; Gallo-Payet, N. The angiotensin type
2 receptor of angiotensin II and neuronal differentiation: from
observations to mechanisms. J. Mol. Endocrinol. 2003, 31, 359-
372.
(9) Widdop, R. E.; Jones, E. S.; Hannan, R. E.; Gaspari, T. A.
Angiotensin AT2 receptors: cardiovascular hope or hype? Br.
J. Pharmacol. 2003, 140, 809-824.

New Selective AT2 Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4023
(10) Li, X. C.; Widdop, R. E. AT2 receptor-mediated vasodilatation
is unmasked by AT1 receptor blockade in conscious SHR. Br. J.
Pharmacol. 2004, 142, 821-830.
(11) Grady, E. F.; Sechi, L. A.; Griffin, C. A.; Schambelan, M.;
Kalinyak, J. E. Expression of AT2 receptors in the developing
rat fetus. J. Clin. Invest. 1991, 88, 921-933.
(32) Matsoukas, J. M.; Polevaya, L.; Ancans, J.; Mavromoustakos,
T.; Kolocouris, A.; Roumelioti, P.; Vlahakos, D. V.; Yamdagni,
R.; Wu, Q.; Moore, G. J. The design and synthesis of a potent
angiotensin II cyclic analogue confirms the ring cluster receptor
conformation of the hormone angiotensin II. Bioorg. Med. Chem.
2000, 8, 1-10.
(12) Tsutsumi, K.; Saavedra, J. M. Characterization and development
of angiotensin II receptor subtypes (AT1 and AT2) in rat brain.
Am. J. Physiol. 1991, 261, R209-R216.
(13) Ohkubo, N.; Matsubara, H.; Nozawa, Y.; Mori, Y.; Murasawa,
S.; Kijima, K.; Maruyama, K.; Masaki, H.; Tsutumi, Y.; Shiba-
zaki, Y.; Iwasaka, T.; Inada, M. Angiotensin type 2 receptors
are reexpressed by cardiac fibroblasts from failing myopathic
hamster hearts and inhibit cell growth and fibrillar collagen
metabolism. Circulation 1997, 96, 3954-3962.
(33) Johannesson, P.; Lindeberg, G.; Tong, W.; Gogoll, A.; Synner-
gren, B.; Nyberg, F.; Karle´n, A.; Hallberg, A. Angiotensin II
analogues encompassing 5,9- and 5,10-fused thiazabicycloalkane
tripeptide mimetics. J. Med. Chem. 1999, 42, 4524-4537.
(34) Johannesson, P.; Lindeberg, G.; Tong, W.; Gogoll, A.; Karle´n,
A.; Hallberg, A. Bicyclic tripeptide mimetics with reverse turn
inducing properties. J. Med. Chem. 1999, 42, 601-608.
(35) Hruby, V. J.; Balse, P. M. Conformational and topographical
considerations in designing agonist peptidomimetics from pep-
tide leads. Curr. Med. Chem. 2000, 7, 945-970.
(14) Chung, O.; Unger, T. Unopposed stimulation of the angiotensin
AT2 receptor in the kidney. Nephrol., Dial., Transplant. 1998,
13, 537-540.
(36) Hruby, V. J. Designing peptide receptor agonists and antago-
nists. Nat. Rev. Drug Discovery 2002, 1, 847-858.
(15) Kimura, B.; Sumners, C.; Phillips, M. I. Changes in skin
angiotensin II receptors in rats during wound healing. Biochem.
Biophys. Res. Commun. 1992, 187, 1083-1090.
(16) Nio, Y.; Matsubara, H.; Murasawa, S.; Kanasaki, M.; Inada, M.
Regulation of gene transcription of angiotensin II receptor
subtypes in myocardial infarction. J. Clin. Invest. 1995, 95, 46-
54.
(17) Viswanathan, M.; Saavedra, J. M. Expression of angiotensin II
AT2 receptors in the rat skin during experimental wound
healing. Peptides 1992, 13, 783-786.
(18) Nakajima, M.; Hutchinson, H. G.; Fujinaga, M.; Hayashida, W.;
Morishita, R.; Zhang, L.; Horiuchi, M.; Pratt, R. E.; Dzau, V. J.
The angiotensin II type 2 (AT2) receptor antagonizes the growth
effects of the AT1 receptor: gain-of-function study using gene
transfer. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10663-10667.
(19) Horiuchi, M.; Akishita, M.; Dzau, V. J. Recent progress in
angiotensin II type 2 receptor research in the cardiovascular
system. Hypertension 1999, 33, 613-621.
(37) Samanen, J. M.; Peishoff, C. E.; Keenan, R. M.; Weinstock, J.
Refinement of a molecular model of angiotensin II (AII) employed
in the discovery of potent nonpeptide antagonists. Bioorg. Med.
Chem. Lett. 1993, 3, 909-914.
(38) Nikiforovich, G. V.; Kao, J. L. F.; Plucinska, K.; Zhang, W. J.;
Marshall, G. R. Conformational analysis of two cyclic analogues
of angiotensin: implications for the biologically active conforma-
tion. Biochemistry 1994, 33, 3591-3598.
(39) Nikiforovich, G. V.; Marshall, G. R. Three-dimensional recogni-
tion requirements for angiotensin agonists: a novel solution for
an old problem. Biochem. Biophys. Res. Commun. 1993, 195,
222-228.
(40) Joseph, M. P.; Maigret, B.; Scheraga, H. A. Proposals for the
angiotensin II receptor-bound conformation by comparative
computer modeling of AII and cyclic analogues. Int. J. Pept.
Protein Res. 1995, 46, 514-526.
(41) Balodis, J.; Golbraikh, A. Conformational analysis of cyclic
angiotensin II analogues. Lett. Pept. Sci. 1996, 3, 195-199.
(42) Carpenter, K. A.; Wilkes, B. C.; Schiller, P. W. The octapeptide
angiotensin II adopts a well-defined structure in a phospholipid
environment. Eur. J. Biochem. 1998, 251, 448-453.
(20) Nouet, S. S.; Nahmias, C. C. Signal transduction from the
angiotensin II AT2 receptor. Trends Endocrin. Metab. 2000, 11,
1-6.
(21) Inagami, T.; Kambayashi, Y.; Ichiki, T.; Tsuzuki, S.; Eguchi, S.;
Yamakawa, T. Angiotensin receptors: molecular biology and
signaling. Clin. Exp. Pharmacol. Physiol. 1999, 26, 544-549.
(22) Stoll, M.; Unger, T. Angiotensin and its AT2 receptor: new
insights into an old system. Regul. Pept. 2001, 99, 175-182.
(23) Gendron, L.; Laflamme, L.; Rivard, N.; Asselin, C.; Payet, M.
D.; Gallo-Payet, N. Signals from the AT2 (angiotensin type 2)
receptor of angiotensin II inhibit p21ras and activate MAPK
(mitogen-activated protein kinase) to induce morphological
neuronal differentiation in NG108-15 cells. Mol. Endocrinol.
1999, 13, 1615-1626.
(24) Stroth, U.; Blume, A.; Mielke, K.; Unger, T. Angiotensin AT2
receptor stimulates ERK1 and ERK2 in quiescent but inhibits
ERK in NGF-stimulated PC12W cells. Mol. Brain Res. 2000, 78,
175-180.
(25) Spear, K. L.; Brown, M. S.; Reinhard, E. J.; McMahon, E. G.;
Olins, G. M.; Palomo, M. A.; Patton, D. R. Conformational
restriction of angiotensin II: cyclic analogues having high
potency. J. Med. Chem. 1990, 33, 1935-1940.
(43) Polevaya, L.; Mavromoustakos, T.; Zoumboulakis, P.; Grdado-
lnik, S. G.; Roumelioti, P.; Giatas, N.; Mutule, I.; Keivish, T.;
Vlahakos, D. V.; Iliodromitis, E. K.; Kremastinos, D. T.; Mat-
soukas, J. Synthesis and study of a cyclic angiotensin II
antagonist analogue reveals the role of π*-π* interactions in
the C-terminal aromatic residue for agonist activity and its
structure resemblance with AT1 non-peptide antagonists. Bioorg.
Med. Chem. 2001, 9, 1639-1647.
(44) Boucard, A. A.; Wilkes, B. C.; Laporte, S. A.; Escher, E.;
Guillemette, G.; Leduc, R. Photolabeling identifies position 172
of the human AT1 receptor as a ligand contact point: receptor-
bound angiotensin II adopts an extended structure. Biochemistry
2000, 39, 9662-9670.
(45) Schmidt, B.; Lindman, S.; Tong, W.; Lindeberg, G.; Gogoll, A.;
Lai, Z.; Thornwall, M.; Synnergren, B.; Nilsson, A.; Welch, C.
J.; Sohtell, M.; Westerlund, C.; Nyberg, F.; Karlen, A.; Hallberg,
A. Design, synthesis, and biological activities of four angiotensin
II receptor ligands with γ-turn mimetics replacing amino acid
residues 3-5. J. Med. Chem. 1997, 40, 903-919.
(26) Johannesson, P.; Lindeberg, G.; Johansson, A.; Nikiforovich, G.
V.; Gogoll, A.; Synnergren, B.; Le Greves, M.; Nyberg, F.; Karle´n,
A.; Hallberg, A. Vinyl sulfide cyclized analogues of angiotensin
II with high affinity and full agonist activity at the AT1 receptor.
J. Med. Chem. 2002, 45, 1767-1777.
(27) Lindman, S.; Lindeberg, G.; Fra¨ndeberg, P.-A.; Nyberg, F.;
Karle´n, A.; Hallberg, A. Effect of 3-5 monocyclizations of
angiotensin II and 4-AminoPhe6-Ang II on AT2 receptor affinity.
Bioorg. Med. Chem. 2003, 11, 2947-2954.
(28) Lindman, S.; Lindeberg, G.; Gogoll, A.; Nyberg, F.; Karlen, A.;
Hallberg, A. Synthesis, receptor binding affinities and confor-
mational properties of cyclic methylenedithioether analogues of
angiotensin II. Bioorg. Med. Chem. 2001, 9, 763-772.
(29) Plucinska, K.; Kataoka, T.; Yodo, M.; Cody, W. L.; He, J. X.;
Humblet, C.; Lu, G. H.; Lunney, E.; Major, T. C.; Panek, R. L.;
Schelkum, P.; Skeean, R.; Marshall, G. R. Multiple binding
modes for the receptor-bound conformations of cyclic AII ago-
nists. J. Med. Chem. 1993, 36, 1902-1913.
(30) Sugg, E. E.; Dolan, C. A.; Patchett, A. A.; Chang, R. S. L.; Faust,
K. A.; Lotti, V. J. Cyclic disulfide analogues of [Sar1,Ile8]-
angiotensin II. In Peptides, Chemistry, Structure Biology, Pro-
ceedings of the Eleventh American Peptide Symposium; Rivier,
J. E., Marshall, G. R., Eds.; ESCOM: Leiden, The Netherlands,
1990; pp 305-306.
(46) Printz, M. P.; Nemethy, G.; Bleich, H. Proposed models for
angiotensin II in aqueous solution and conclusions about recep-
tor topography. Nature (London) 1972, 237, 135-140.
(47) Lindman, S.; Lindeberg, G.; Nyberg, F.; Karle´n, A.; Hallberg,
A. Comparison of three γ-turn mimetic scaffolds incorporated
into angiotensin II. Bioorg. Med. Chem. 2000, 8, 2375-2383.
(48) Derae¨t, M.; Rihakova, L.; Boucard, A.; Perodin, J.; Sauve, S.;
Mathieu, A. P.; Guillemette, G.; Leduc, R.; Lavigne, P.; Escher,
E. Angiotensin II is bound to both receptors AT1 and AT2,
parallel to the transmembrane domains and in an extended
form. Can. J. Physiol. Pharmacol. 2002, 80, 418-425.
(49) Miura, S.-I.; Karnik, S. S. Angiotensin II type 1 and type 2
receptors bind angiotensin II through different types of epitope
recognition. J. Hypertens. 1999, 17, 397-404.
(50) Samanen, J.; Regoli, D. Structure-activity relationships of
peptide angiotensin II receptor agonists and antagonists. In
Angiotensin II Receptors, Volume I: Molecular Biology, Bio-
chemistry, Pharmacology and Clinical Perspectives; Ruffolo, R.
R., Jr., Ed.; CRC Press: Boca Raton, FL, 1994; pp 11-97.
(51) Bouley, R.; Perodin, J.; Plante, H.; Rihakova, L.; Bernier, S. G.;
Maletinska, L.; Guillemette, G.; Escher, E. N- and C-terminal
structure-activity study of angiotensin II on the angiotensin
AT2 receptor. Eur. J. Pharmacol. 1998, 343, 323-331.
(52) Cody, W. L.; He, J. X.; Lunney, E. A.; Humblet, C. C.; Lu, G. H.;
Panek, R. L.; Dudley, D. T. Modification of the C-terminal
dipeptide of angiotensin II yielded a novel series of analogues
with II (AT2) receptor selectivity. Protein Pept. Lett. 1996, 3,
107-112.
(31) Zhang, W.-J.; Nikiforovich, G. V.; Perodin, J.; Richard, D. E.;
Escher, E.; Marshall, G. R. Novel cyclic analogues of angiotensin
II with cyclization between positions 5 and 7: conformational
and biological implications. J. Med. Chem. 1996, 39, 2738-2744.

4024 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Rosenstro¨m et al.
(53) Speth, R. C.; Kim, K. H. Discrimination of two angiotensin II
receptor subtypes with a selective agonist analogue of angio-
tensin II, p-aminophenylalanine6 angiotensin II. Biochem. Bio-
phys. Res. Commun. 1990, 169, 997-1006.
(54) Aiyar, N.; Griffin, E.; Edwards, R.; Weinstock, J.; Samanen, J.;
Nambi, P. Characterization of bovine ovary angiotensin II
receptors using subtype-selective antagonists. Pharmacology
1993, 46, 1-8.
(55) Samanen, J.; Aiyar, N.; Edwards, R. Modifications to angiotensin
II (AII) peptide analogues that reduce AT1 receptor affinity have
little to no effect upon AT2 receptor affinity. In Peptides,
Proceedings of the Thirteenth American Peptide Symposium;
Hodges, R. S., Smith, J. A., Eds.; ESCOM: Leiden, The Neth-
erlands, 1994; pp 640-642.
(56) Rosenstro¨m, U.; Sko¨ld, C.; Lindeberg, G.; Botros, M.; Nyberg,
F.; Hallberg, A.; Karle´n, A. Synthesis and AT2 receptor binding
properties of angiotensin II analogues. J. Pept. Res. 2004, 64,
194-201.
(57) Johannesson, P.; Lindeberg, G.; Erde´lyi, M.; Fra¨ndeberg, P.-A.;
Nyberg, F.; Karle´n, A.; Hallberg, A. AT2-selective angiotensin
II analogues encompassing tyrosine functionalized 5,5-bicyclic
thiazabicycloalkane dipeptide mimetics. J. Med. Chem. 2004,
47, 6009-6019.
(58) Rosenstro¨m, U.; Sko¨ld, C.; Lindeberg, G.; Botros, M.; Nyberg,
F.; Karle´n, A.; Hallberg, A. A selective AT2 receptor ligand with
a γ-turn-like mimetic replacing the amino acid residues 4-5 of
angiotensin II. J. Med. Chem. 2004, 47, 859-870.
(59) Marshall, G. R. Determination of the receptor bound conforma-
tion of angiotensin. Dtsch. Apoth. Ztg. 1986, 126, 2783-2786.
(60) Uenishi, J. i.; Kobayashi, N.; Komine, S.; Okadai, T.; Yonemitsu,
O.; Sasaki, T.; Yamada, Y. Total synthesis of (()-acetomycin and
design of esterase-resistant analogues. Chem. Pharm. Bull. 1999,
47, 517-523.
(61) Dudley, D. T.; Panek, R. L.; Major, T. C.; Lu, G. H.; Bruns, R.
F.; Klinkefus, B. A.; Hodges, J. C.; Weishaar, R. E. Subclasses
of angiotensin II binding sites and their functional significance.
Mol. Pharmacol. 1990, 38, 370-377.
(62) Nielsen, A. H.; Schauser, K.; Winther, H.; Dantzer, V.; Poulsen,
K. Angiotensin II receptors and renin in the porcine uterus:
myometrial AT2 and endometrial AT1 receptors are down-
regulated during gestation. Clin. Exp. Pharmacol. Physiol. 1997,
24, 309-314.
(63) Buisson, B.; Bottari, S. P.; De Gasparo, M.; Gallo-Payet, N.;
Payet, M. D. The angiotensin AT2 receptor modulates T-type
calcium current in nondifferentiated NG108-15 cells. FEBS Lett.
1992, 309, 161-164.
(64) Laflamme, L.; de Gasparo, M.; Gallo, J.-M.; Payet, M. D.; Gallo-
Payet, N. Angiotensin II induction of neurite outgrowth by AT2
receptors in NG108-15 cells. Effect counteracted by the AT1
receptors. J. Biol. Chem. 1996, 271, 22729-22735.
(65) Gendron, L.; Cote, F.; Payet, M. D.; Gallo-Payet, N. Nitric oxide
and cyclic GMP are involved in angiotensin II AT2 receptor
effects on neurite outgrowth in NG108-15 cells. Neuroendocri-
nology 2002, 75, 70-81.
(66) Wolf, G.; Harendza, S.; Schroeder, R.; Wenzel, U.; Zahner, G.;
Butzmann, U.; Freeman, R. S.; Stahl, R. A. K. Angiotensin II’s
antiproliferative effects mediated through AT2-receptors depend
on down-regulation of SM-20. Lab. Invest. 2002, 82, 1305-1317.
(67) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.
N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The protein data
bank. Nucleic Acids Res. [Online] 2000, 28, 235-242. http://
www.pdb.org/.
(68) Martin, Y. C.; Bures, M. G.; Danaher, E. A.; DeLazzer, J.; Lico,
I.; Pavlik, P. A. A fast new approach to pharmacophore mapping
and its application to dopaminergic and benzodiazepine agonists.
J. Comput.-Aided Mol. Des. 1993, 7, 83-102.
(69) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MacroModel - an integrated software system for modeling
organic and bioorganic molecules using molecular mechanics.
J. Comput. Chem. 1990, 11, 440-467.
(70) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T.
Semianalytical treatment of solvation for molecular mechanics
and dynamics. J. Am. Chem. Soc. 1990, 112, 6127-6129.
(71) Knowle, D.; Kurfis, J.; Gavini, N.; Pulakat, L. Role of Asp297 of
the AT2 receptor in high-affinity binding to different peptide
ligands. Peptides 2001, 22, 2145-2149.
(72) Heerding, J. N.; Yee, D. K.; Jacobs, S. L.; Fluharty, S. J.
Mutational analysis of the angiotensin II type 2 receptor:
contribution of conserved extracellular amino acids. Regul. Pept.
1997, 72, 97-103.
(73) Blankley, C. J.; Hodges, J. C.; Klutchko, S. R.; Himmelsbach,
R. J.; Chucholowski, A.; Connolly, C. J.; Neergaard, S. J.; Van
Nieuwenhze, M. S.; Sebastian, A.; Quin, J., III.; Essenburg, A.
D.; Cohen, D. M. Synthesis and structure-activity relationships
of a novel series of non-peptide angiotensin II receptor binding
inhibitors specific for the AT2 subtype. J. Med. Chem. 1991, 34,
3248-3260.
(74) Whitebread, S.; Mele, M.; Kamber, B.; de Gasparo, M. Prelimi-
nary biochemical characterization of two angiotensin II receptor
subtypes. Biochem. Biophys. Res. Commun. 1989, 163, 284-291.
(75) Wan, Y.; Wallinder, C.; Plouffe, B.; Beaudry, H.; Kannan, M.
A.; Wu, X.; Johansson, B.; Holm, M.; Botros, M.; Karle´n, A.;
Pettersson, A.; Nyberg, F.; Fa¨ndriks, L.; Gallo-Payet, N.; Hall-
berg, A.; Alterman, M. Design, synthesis and biological evalu-
ation of the first selective nonpeptide AT2 receptor agonist. J.
Med. Chem. 2004, 47, 5995-6008.
(76) Kivlighn, S. D.; Huckle, W. R.; Zingaro, G. J.; Rivero, R. A.; Lotti,
V. J.; Chang, R. S. L.; Schorn, T. W.; Kevin, N.; Johnson, R. G.,
Jr. Discovery of L-162,313: a nonpeptide that mimics the
biological actions of angiotensin II. Am. J. Physiol. 1995, 268,
R820-R823.
(77) Perlman, S.; Schambye, H. T.; Rivero, R. A.; Greenlee, W. J.;
Hjorth, S. A.; Schwartz, T. W. Non-peptide angiotensin agonist.
Functional and molecular interaction with the AT1 receptor. J.
Biol. Chem. 1995, 270, 1493-1496.
(78) Wan, Y.; Wallinder, C.; Johansson, B.; Holm, M.; Mahalingam,
A. K.; Wu, X.; Botros, M.; Karlen, A.; Pettersson, A.; Nyberg,
F.; Fa¨endriks, L.; Hallberg, A.; Alterman, M. First reported
nonpeptide AT1 receptor agonist (L-162,313) acts as an AT2
receptor agonist in vivo. J. Med. Chem. 2004, 47, 1536-1546.
(79) Rovnyak, G.; Andersen, N.; Gougoutas, J.; Hedberg, A.; Kimball,
S. D.; Malley, M.; Moreland, S.; Porubcan, M.; Pudzianowski,
A. Studies directed toward ascertaining the active conformation
of 1,4-dihydropyridine calcium entry blockers. J. Med. Chem.
1988, 31, 936-944.
(80) Ricci, A.; Martani, A.; Graziani, O.; Oliva, M. L. Benzisothiazoles.
II. Oxidation and nitration. Ann. Chim. (Rome) 1963, 53, 1860-
1868.
(81) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (I50) of an enzymic reaction. Biochem. Pharmacol.
1973, 22, 3099-3108.
(82) Goodman, J. M.; Still, W. C. An unbounded systematic search
of conformational space. J. Comput. Chem. 1991, 12, 1110-1117.
(83) Hobohm, U.; Scharf, M.; Schneider, R.; Sander, C. Selection of
representative protein data sets. Protein Sci. 1992, 1, 409-417.
(84) Hobohm, U.; Sander, C. Enlarged representative set of protein
structures. Protein Sci. 1994, 3, 522-524.
(85) SYBYL Molecular Modeling Software, version 6.9; Tripos Inc.:
St. Louis, MO.
JM0491492