Short pseudopeptides containing turn scaffolds with high AT2 receptor affinity

Article abstract of DOI:10.1016/j.bmc.2006.05.019

Two pentapeptides, Ac-Tyr-Ile-His-Pro-Phe/Ile, were synthesized and shown to have angiotensin II AT₂ receptor affinity and agonistic activity. Based on these peptides, a new series of 13 pseudopeptides was synthesized via introduction of five d

Full text of DOI:10.1016/j.bmc.2006.05.019

Bioorganic & Medicinal Chemistry 14 (2006) 5963–5972
Short pseudopeptides containing turn scaffolds with
high AT2 receptor affinity
a
a
a
b
´ `
Jennie Georgsson, Ulrika Rosenstro¨m, Charlotta Wallinder, Helene Beaudry,
b
b
c
c
a
´
Bianca Plouffe, Gunnar Lindeberg, Milad Botros, Fred Nyberg, Anders Karlen,
Nicole Gallo-Payetb,* and Anders Hallberga,*
aDepartment of Medicinal Chemistry, Division of Organic Pharmaceutical Chemistry, Uppsala University,
PO Box 574, SE-751 23 Uppsala, Sweden
bService of Endocrinology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que., Canada J1H 5N4
cDepartment of Pharmaceutical Sciences, Division of Biological Research on Drug Dependence, Uppsala University,
PO Box 591, SE-751 24 Uppsala, Sweden
Received 19 December 2005; revised 4 May 2006; accepted 15 May 2006
Available online 5 June 2006
Abstract—Two pentapeptides, Ac-Tyr-Ile-His-Pro-Phe/Ile, were synthesized and shown to have angiotensin II AT2 receptor affinity
and agonistic activity. Based on these peptides, a new series of 13 pseudopeptides was synthesized via introduction of five different
turn scaffolds replacing the Tyr-Ile amino acid residues. Pharmacological evaluation disclosed subnanomolar affinities for some of
these compounds at the AT2 receptor. Substitution of Phe by Ile in this series of ligands enhanced the AT2 receptor affinity of all
compounds. These results suggest that the C-terminal amino acid residues can be elaborated on to enhance the AT2 receptor affinity
in truncated Ang II analogues.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
pathological conditions such as heart failure, myocardi-
al infarction, brain lesions, vascular injury, and wound
healing.3,4,7,8 It has also been demonstrated that AT2
receptor activation mediates antiproliferation, cellular
differentiation, programmed cell death (apoptosis), and
even vasodilation.3,4,7 Furthermore, agonistic activation
of the AT2 receptor in neuronal cells induces neurite
outgrowth and elongation, promotes cellular migration,
and modulates neuronal excitability.9 The mechanism of
action involves activation of the nitric oxide/guanyl cy-
clase/cGMP pathway10 and a sustained increase in
p42/p22mapk activity.11
The octapeptide angiotensin II (Ang II, Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe) is the endogenous ligand for the
AT1- and AT2-receptors. Both of these receptors are
G-protein coupled. They differ considerably by sharing
only 32–34% sequence homology,1,2 and by having dif-
ferent signaling pathways as well as exerting different
physiological effects.3,4 Vasoconstriction, aldosterone
release, and stimulation of sympathetic transmission,
and cellular growth are generally attributed to AT1
receptor activation.3–5 The role of the AT2 receptor is
less clear, largely because of its low level of expression
in adults. The AT2 receptor is the predominant angio-
tensin receptor in fetal tissues, but is rapidly down-
regulated after birth, suggesting an involvement in fetal
development.6 Importantly, it is up-regulated in certain
Losartan was the first selective non-peptide AT1 receptor
antagonist on the market, launched in 1994 as an anti-hy-
pertensive drug and developed from a non-peptide
lead.12,13 Recently, our group reported the first selective
non-peptide AT2 receptor agonist.14 It has been proposed
that such AT2 receptor agonists might find a role as a ther-
apeutic agent in the cardiovascular area since it has been
demonstrated that AT2 receptor activation affects cardiac
remodeling and leads to reduced blood pressure.7,15 The
selective AT2 receptor agonists originate from an agonist
(L-162,313) that is structurally related to the sartans and
Keywords: Angiotensin II; AT1 receptor; AT2 receptor; AT2 receptor
agonist; Pseudopeptides; Turn scaffolds.
*
Corresponding authors. Tel.: +46 18 4714284; fax: +46 18 4714474
(A.H.); tel.: +1 819 564 5243; fax: +1 819 564 5292 (N.G.-P.); e-mail
addresses: nicole.gallo-payet@usherbrooke.ca; anders.hallberg@
orgfarm.uu.se
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.019

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J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
that activates both the AT1 receptor16,17 and the AT2
receptor18. Hence, the receptor-selective AT2 agonists14
and the AT1 antagonists13 were developed by classical
medicinal chemistry approaches from non-peptide leads.
An alternative way to produce novel lead series would be
to start from the bioactive peptide and stepwise remove
structural elements less crucial for receptor activation
and at the same time strengthen important ligand receptor
interactions.19 To make peptidomimetics by such an iter-
ative procedure is a challenging task. Nevertheless, in re-
cent years a number of elegant examples have been
disclosed.20–22 We have been intrigued by and are address-
ing the question whether Ang II can be converted to low-
molecular weight drug-like AT2 receptor agonists via a
stepwise process.
Table 1. Binding affinities for the AT1- and AT2-receptors
Compound
Ki (nM) SEM
AT1 (rat liver
membranes)
AT2 (pig uterus
zmyometrium)
Ang II
[4-NH2-Phe6]Ang II
0.95
0.2
0.7
>10,000
>10,000
>10,000
31.0 1.7
>10,000
>10,000
>10,000
>10,000
9.4 0.7
>10,000
195.9 5.1
>10,000
566.6 17.4
27.7 1.8
>10,000
>10,000
>10,000
>10,000
1
2
37.9 2.5
3.0 0.2
11.0 1.3
4.2 0.2
23.9 1.3
114.6 2.7
12.1 0.8
9.2 0.2
12.3 1.5
24 2.2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
38.7 1.2
102.7 1.1
0.8 0.02
2.5 0.1
0.5 0.03
21.8 0.7
0.7 0.04
The ligand binding interactions with the AT1- and AT2-
receptors seem to be different. The AT1 receptor is sen-
sitive to fairly small changes in the amino acid sequence
of Ang II. For example, substitution of Phe8 by Ile8
results in an AT1 receptor antagonist.23 The AT2 recep-
tor, on the contrary, seems to be more tolerant both to-
ward structural alterations in Ang II24,25 and also with
regard to truncation of the peptide as de Gasparo
et al. reported in a study from 1991.26 In that study, it
was found that the acetylated pentapeptides Ac-Tyr-
Val-His-Pro-Phe and Ac-Tyr-Val-His-Pro-Ile both
bound to the AT2 receptor with high selectivity and with
Ki values in the nanomolar range.26 Unfortunately, no
data were disclosed concerning the agonistic and/or
antagonistic properties of these compounds.
AT2 receptor based on their ability to induce neurite
outgrowth. Furthermore, we disclose that highly potent
AT2 receptor ligands can be obtained starting from
these pentapeptides by substitution of the dipeptide
fragment Tyr-Ile of 1–4 with properly substituted benzo-
diazepine or diphenylmethane scaffolds, that is, A, B, C,
D, or E (Chart 2). Two of these pseudopeptides were
also shown to be AT2 receptor agonists.
We herein report that pentapeptide derivatives of Ang
II, with either an N-terminal comprising an acetyl group
(1) or a free amine (3), are high-affinity AT2 receptor
ligands (Chart 1 and Table 1). Substitution of the C-ter-
minal Phe by Ile in these pentapeptides results in 2 and
4, which both have higher AT2 receptor affinities, as
compared to 1 and 3, respectively. We also report that
compounds 1 and 2 exerted agonistic effects at the
2. Results
2.1. Chemistry
The turn scaffolds A, B, C, and E either N-acetylated or
with a free N-terminal were synthesized and introduced
OH
OH
N
N
NH
NH
O
O
O
O
H
N
H
N
N
N
H
H
N
N
O
N
N
O
N
N
H
H
H
H
O
O
O
O
O
O
OH
OH
1
2
OH
OH
N
O
N
O
NH
O
NH
H
N
N
O
H
H
H2N
N
O
N
N
N
H
H
O
H2N
N
O
O
N
H
OH
O
O
OH
3
4
Chart 1.

J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
OH
5965
outlined in Scheme 2. The subsequent Fmoc-protected
amino acids were coupled using either HBTU or PyBOP
as coupling reagent. Piperidine in DMF was used for
deprotection of the N-terminal. Scaffolds A, B, C, D,
and E (A, B, C, and E were N-terminal Fmoc-protected)
were introduced with the identical coupling conditions
as above but with a prolonged reaction time. The
pseudopeptides containing an acetylated N-terminal
were finally coupled to acetic acid, again using standard
conditions. The final cleavage of the products from the
resin was performed using 95% aq TFA.
OH
H
H
N
O
O
H2N
OH
N
O
O
N
N
OH
OH
H2N
B
A
OH
OH
2.2. Receptor affinity studies
OH
OH
H2N
OH
H2N
O
O
O
Compounds 1–17 were evaluated in radioligand binding
assays relaying on displacement of [125I]Ang II from
AT1 receptors in rat liver membranes33 and from AT2
receptors in pig uterus membranes34 (Table 1). Ang II
and [4-NH2-Phe6]Ang II were used as reference sub-
stances. The results are given as Ki values in Table 1.
C
D
E
Chart 2.
instead of Tyr-Ile in 5–17 (Chart 3). The synthetic routes
to the benzodiazepine c-turn scaffolds A27 and B,28 and
the aromatic c-turn scaffolds C,29 and E29 were previ-
ously published. The synthesis of scaffold D was per-
formed as outlined in Scheme 1. Compound 18 was
prepared by a Friedel–Crafts acylation of anisole with
the acid chloride of the commercially available monom-
ethyl isophthalate. The acylation gave ortho and para
regioisomers where the major product was found to be
the para substituted 18.30 Separation of the two regioi-
somers was conducted with column chromatography
and 58% of 18 was isolated. The subsequent reduction
of the carbonyl group of 18, to afford 19, was performed
under acidic conditions with triethylsilane as hydride
source.31 The deprotection of the methoxy group was
accomplished with boron trifluoride dimethylsulfide
complex in DCM.32 Without any further purification
the mild hydrolysis was conducted with LiOH in a
mixture of THF/H2O/MeOH to yield compound D.
The pentapeptides 1–4 all exhibit nanomolar affinity for
the AT2 receptor. In addition, peptide 3 exerts affinity
for the AT1 receptor. All of the pseudopeptide ligands
5–17 displayed nanomolar affinity for the AT2 receptor
and exhibited Ki values ranging from 0.5 nM to
114.6 nM. Four of the compounds (8, 10, 12, and 13)
are non-selective and their AT1 receptor affinities range
from Ki = 9.4 nM to 567 nM.
2.3. In vitro morphological effects induced by compounds
1, 2, 8, and 15 in NG 108-15 cells
To study the effects of compounds 1, 2, 8, and 15 on cell
differentiation, NG108-15 cells were used. In their undif-
ferentiated state, neuroblastoma · glioma hybrid
NG108-15 cells have a rounded shape and divide actively.
It has previously been shown by us that these cells express
only the AT2 receptor35,36 and that a three-day treatment
with Ang II or the selective peptidic AT2 receptor agonist
CGP 42112A induces neurite outgrowth.36
The peptides 1–4 and the pseudopeptides 5–17 were pre-
pared using standard Fmoc-strategy solid-phase peptide
synthesis (SPPS) with Phe-Wang or Ile-Wang resins as
Compounds 1, 2, 8, and 15 were first tested at various
concentrations ranging from 1 pM to 1 lM and none
of the doses induced cell death. When the cells were
stimulated for three days with each of the compounds,
they induced neurite outgrowth. The 3-day treatment in-
creased the number of cells with neurites longer than one
cell body from 8.32 0.29% in control cells to
17.29 0.13%, 17.85 0.55%, 18.21 0.12%, and
16.28 0.69% in the cells treated with compound 1
(0.01 lM), 2 (0.01 lM), 8 (0.1 lM), and 15 (0.01 lM),
respectively (Fig. 1). This effect was mediated through
the AT2 receptor, since co-incubation of 1, 2, 8, and
15 with the AT2 receptor antagonist, PD123,319
(10 lM), virtually halted neurite elongation. We have
previously shown that PD123,319 administered alone
does not alter the morphology of the untreated cells.11,36
O
O
OH
O
a
b
O
O
O
O
18 (58%)
OH
O
c
OH
O
O
O
19 (96%)
D (74%)
2.4. Rabbit thoracic aorta strips, AT1 functional assay
Scheme 1. Reagents and conditions: (a) i—SOCl2, reflux; ii—anisole,
AlCl3, DCM; (b) (H3CH2C)3SiH, F3CSO3H, TFA; (c) i—BF3ÆS(CH3)2,
DCM; ii—LiOH, THF/MeOH/H2O.
The effect of compound 8 was tested at six different con-
centrations ranging from 10 nM to 3 lM at the AT1

5966
J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
HO
HO
HO
O
N
O
N
O
N
O
NH
NH
NH
H
N
H
N
H
N
H
H2N
N
O
O
O
N
N
N
N
N
N
H
H
H
N
N
N
H
O
O
O
N
N
N
H
H
H2N
O
O
O
O
O
O
OH
OH
OH
5
6
7
OH
OH
OH
N
N
N
O
NH
NH
NH
O
O
N
N
N
H
H
H
N
N
H
N
H
N
H
O
O
O
N
N
O
O
O
O
O
O
OH
OH
OH
NH2
NH
O
8
9
10
OH
OH
N
O
N
NH
NH
O
N
N
H
N
H
N
H
N
H
O
O
N
O
O
O
O
O
OH
OH
H2N
OH
N
H
11
12
OH
OH
N
O
NH
N
O
N
O
NH
NH
N
H
N
N
H
O
N
N
H
N
H
N
N
H
O
N
H
O
O
O
O
O
OH
O
O
OH
OH
NH2
NH
13
14
15
O
OH
OH
N
O
N
O
NH
NH
N
N
H
N
H
N
N
H
O
N
H
O
O
O
O
O
O
OH
OH
H2N
N
H
16
17
Chart 3.
receptor using the rabbit aorta strips according to the
literature procedure.37 At these experimental settings
compound 8 did neither induce contraction nor inhibit
Ang II induced contraction at concentrations as high
as 3 lM.
A
B
C
D
E
5
6, 7
8, 9, 13, 14
10, 15
A
B
C
D
E
a
11, 12, 16, 17
Scheme 2. Reagents: (a) i—His-Pro-Phe-Wang or His-Pro-Ile-Wang
resins, HBTU or PyBOP, DIEA, DMF; ii—20% piperidine in DMF;
iii—95% aq TFA.
3. Discussion
the peptide and then their 3D-relationship. Some of
the tools used to determine the pharmacophore elements
are truncation of the peptide and Ala-scans. The 3D-
relationship can be studied using, for example,
To develop a bioactive and less peptide-like molecule
affecting a certain peptide receptor utilizing the endoge-
nous ligand as lead structure is an intricate task. One ap-
proach is to determine the pharmacophore elements of

J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
5967
E on the other hand are easily accessible and position
the N- and C-terminal in a correct position relative to
each other as compared to a c-turn.29,40 Since previous
studies have shown that all of these scaffolds can substi-
tute Tyr-Ile in Ang II with retained AT2 receptor affin-
ities, we planned to further reduce the peptide
character of the pentapeptides by introducing these scaf-
folds in this position.
Scaffolds A, B, C, and E were introduced in the penta-
peptides to give compounds 5, 6, 8, and 11 which all
showed nanomolar affinity for the AT2 receptor (Table
1). Interestingly, the basicity of the N-terminal does
not appear to be critical for good affinity since, for
example, the free benzyl amine 11 (calcd pKa ꢀ 9.2)
has a Ki of 38.7 nM and the aniline 8 (calcd pKa ꢀ 3.5)
a Ki of 9.2 nM. The corresponding acetylated com-
pounds (7, 9, and 12) were also synthesized for compar-
ison (except for scaffold A). Compound 7 gained 10-fold
in affinity (Ki = 12.1 nM) compared to its non-acetylated
equivalent 6. On the other hand, 9 exhibits a Ki of
12.3 nM, which is comparable to the free aniline 8.
The affinity of 12 (Ki = 102.7) dropped by a factor of 3
as compared to that of 11 (Ki = 38.7). Apparently, the
N-terminal end can be both neutral and charged, and
still good affinities are obtained. We therefore incorpo-
rated scaffold D, where the nitrogen is absent, in the
Tyr-Ile position. Interestingly, compound 10 displayed
a Ki value of 24 nM showing that the N-terminal nitro-
gen is not necessary for binding. The eight pseudopep-
tides (5–12) discussed so far all showed good affinity
to the AT2 receptor with Ki values ranging from
9.2 nM to 114.6 nM.
Figure 1. The effect on neurite outgrowth in NG108-15 cells. White,
untreated cells; grey, cells treated with Ang II, 1, 2, 8 or 15; black, cells
treated with the same compounds in combination with the AT2
receptor antagonist, PD 123,319. Cells with at least one neurite longer
than a cell body were counted as positive for neurite outgrowth. The
number of cells with neurites represents the percentage of the total
number of cells in the micrographs (at least 290 cells according to the
experiment). Concentrations used in the assay: 0.1 lM of Ang II,
0.01 lM of 1, 0.01 lM of 2, 0.1 lM of 8, 0.1 lM of 15, and 10.0 lM of
PD 123,319.
cyclization of the peptide followed by replacement of
these cyclic fragments for well-defined secondary
structure mimics.19
De Gasparo et al. synthesized the truncated pentapep-
tide analogues Ac-Tyr-Val-His-Pro-Phe and Ac-Tyr-
Val-His-Pro-Ile of Ang II and showed that they had
binding affinity to AT2 receptors.26 We were intrigued
by this finding and decided to resynthesize these com-
pounds, although using Ile (compounds 1 and 2 as in
Ang II) instead of Val. We also synthesized the com-
pounds with a free N-terminal group (compounds 3
and 4) to investigate the importance of the acetyl group
and the charge in this position. Compounds 1–4 were
evaluated in the AT1 and AT2 receptor binding assays
and compounds 1 and 2 in the agonist assay using NG
108-15 cells. All four compounds showed affinity to
the AT2 receptor with Ki values ranging between
3.0 nM and 37.9 nM (Table 1). Peptides 2 and 4 contain-
ing the C-terminal Ile residue had somewhat higher
affinity as compared to the Phe analogues, which was
also observed by de Gasparo et al. Furthermore, the
charged and acetylated N-terminal groups had about
the same affinity. Finally, both compounds 1 and 2 were
shown to be AT2 receptor agonists. It was therefore
foreseen that these truncated analogues could be an
excellent starting point in the search for less peptidic
AT2 receptor agonists.
The results of peptides 1–4 revealed that substitution of
Phe in the C-terminal for Ile afforded ligands with up to
10-fold higher affinities. Therefore, a series of pseudo-
peptides encompassing Ile in the C-terminal were syn-
thesized. Because of their simplicity and synthetic
accessibility the aromatic scaffolds C, D, and E were
chosen for incorporation in the Tyr-Ile part. As expect-
ed, compounds 13–17 exhibited better affinity for the
AT2 receptor than their Phe equivalents. It is interesting
to note that regardless of which scaffolds were used and
whether the N-terminal is free, acetylated or not present,
the AT2 receptor affinities were enhanced by 2–145 times
for the series of Ile analogues as compared to the corre-
sponding Phe analogues. Thus, the present data suggest
that the C-terminal isoleucine side chain is a key residue
that promotes enhanced AT2 receptor affinity and selec-
tivity toward the AT1 receptor. However, the impor-
tance of the N-terminal appears to be of less
significance. In fact, the Ile analogues 13, 15, and 17
are all equipotent with Ang II.
Previous studies including 3–5 disulfide monocycliza-
tions and other types of cyclizations have provided sup-
port for the hypothesis that Ang II adopts a c-turn-like
conformation in the Val3-Tyr4-Ile5 region when binding
to AT2 receptors.27,39 A series of c-turn mimicking scaf-
folds (A, B, C, and E) have therefore been incorporated
into Ang II to replace the Val-Tyr-Ile and Tyr-Ile resi-
dues.27,29,38,40 The benzodiazepine based scaffolds A
and B mimic a c-turn well but they are synthetically very
demanding to prepare.27,38 The aromatic scaffolds C and
Compound 8 was selected for functional studies using
both the AT2 receptor agonist assay and the rabbit aorta
strip AT1 receptor assay because it had affinity to both
these receptors. A compound with a pharmacological
profile corresponding to both an AT1 receptor antago-
nist, and AT2 receptor agonist should, for example, be
interesting as a therapeutic agent in the cardiovascular
area (see Section 1).7,15 The functional assays revealed

5968
J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
that compound 8 had AT2 receptor agonistic properties
but neither agonistic nor antagonistic activities were
detected at the AT1 receptor. Compound 15 was also
evaluated as an AT2 receptor agonist because of its high
affinity and peptidomimetic character and was also
shown to be an agonist. Taken together, the identifica-
tion of compounds 8 and 15 has led to the discovery
of less peptidic AT2 receptor agonists and opens up
for further developments of drug-like compounds.
on a Thermo Hypersil HyPurity C4, 5 lm, 4.6 mm ·
50 mm column and a ACE 5 C18, 4.6 mm · 50 mm col-
umn. MikroKemi AB, Uppsala, Sweden or Analytische
Laboratorien, Industriepark Kaiserau, Lindlar, Germa-
ny performed elemental analyses. Molecular masses
(HRMS) were determined on a Micromass Q-Tof2 mass
spectrometer equipped with an electrospray ion source.
Column chromatography was performed using commer-
cially available silica gel 60 (particle size: 0.040–
0.063 mm Merck). Thin–layer chromatography was per-
formed with aluminum sheets coated with silica gel 60
F254 (0.2 mm, E. Merck) and the analytes were visual-
ized with UV-light. Dichloromethane used in the reac-
tions was distilled over calcium hydride. Other
chemicals were used without further purification. The
AT1 functional assay was performed at Cerep, France.
Concerning the AT1 receptor very few truncated peptide
analogues of Ang II have shown affinity to this receptor.41
It was therefore very surprising that the pentapeptide 3
had a Ki of 31 nM. It has also been very difficult to intro-
duce secondary structure mimetics in Ang II and preserve
the affinity. The AT1 receptor binding affinities obtained
with 8, 10, 12, and 13 were therefore much unexpected.
These compounds are anticipated to further elucidate
the recognition requirements for AT1 receptor ligands.
5.2. 3-(4-Methoxybenzoyl)-methyl benzoate (18)
Monomethyl isophthalate (4.78 g, 26.5 mmol) was
refluxed in 35 mL thionyl chloride for 1 h. The thionyl
chloride was evaporated under reduced pressure. An ali-
quot of the crude product (1.35 g, 6.80 mmol) was dis-
solved in 15 ml of dry DCM. AlCl3 (2.72 g,
20.4 mmol) was suspended in 10 ml of dry DCM and
cooled to 0 ꢁC. The acid chloride solution was added
slowly to the cooled reaction mixture and was stirred
at 0 ꢁC for 45 min. Anisole (2.2 mL, 20.4 mmol) was
added dropwise and the reaction mixture was stirred
at room temperature over night. The reaction mixture
was poured into a mixture of 100 g ice and 100 mL of
1 M HCl and extracted with DCM. The organic phase
was washed with NaHCO3, H2O and brine, dried over
Na2SO4 and evaporated. After subsequent purification
by column chromatography (DCM/isohexane 3:2), 18
(1.06 g, 58%) was isolated as a clear oil. 1H NMR
(CDCl3) d: 8.39 (ddd, J = 1.8, 1.7, 0.7 Hz, 1H, CH),
8.24 (ddd, J = 7.9, 1.7, 1.3 Hz, 1H, CH), 7.96 (ddd,
J = 7.7, 1.8, 1.3 Hz, 1H, CH), 7.82 (m, 2H, 2· CH),
7.58 (ddd, J = 7.9, 7.7, 0.7 Hz, 1H, CH), 6.98 (m, 2H,
2· CH), 3.94 (s, 3H, CH3), 3.90 (s, 3H, CH3). 13C
NMR (CDCl3) d: 194.6 (CO), 166.4 (CO), 163.5 (C),
138.6 (C), 133.8 (CH), 132.7 (CH), 132.6 (2· CH),
130.7 (CH), 130.2 (C), 129.6 (C), 128.5 (CH), 113.8
(2· CH), 55.5 (CH3), 52.4 (CH3). MS m/z (70 eV) 270
(M+). Anal. (C16H14O4) C, H, N.
4. Conclusion
In summary, a new class of ligands for the Ang II AT2
receptor is presented. Initially, four pentapeptides were
synthesized of which two were confirmed to be agonists.
Subsequently, five tyrosine-based scaffolds encompass-
ing a benzodiazepine or a benzene ring were introduced
N-terminally to generate a range of pseudopeptides with
high affinity to the AT2 receptor. C-terminal substitu-
tion of Phe by Ile in this series of ligands enhanced the
AT2 receptor affinity of all compounds. Three of these
pseudopeptides (13, 15, and 17) were equipotent to the
endogenous ligand Ang II. Furthermore, it was shown
that the N-terminal nitrogen was not needed obtaining
high AT2 receptor affinity. Four of the compounds 1,
2, 8 and 15 were selected for examination in a functional
assay and all were found to act as AT2 receptor agonists.
5. Experimental
5.1. General
1H NMR and 13C NMR were obtained on a JEOL
JNM-400 spectrometer or on a Varian Mercury plus
spectrometer (1H at 400 MHz, 13C at 100.6 MHz). Spec-
tra were recorded at ambient temperature unless other-
wise stated. Chemical shifts are reported as d values
(ppm) referenced to d 7.26 ppm for CHCl3, d 77.0 ppm
for CDCl3, d 2.05 ppm and d 29.84 ppm for acetone-d6
and d 1.94 and 1.32 ppm for MeCN-d3. GC–MS was
performed with an electron impact (70 eV) mass–selec-
tive detector, and an HP-1 capillary column using a
70–305 ꢁC temperature gradient. The preparative RP-
HPLC column used was a 10 lm Vydac C18 column
(22 mm · 250 mm) with a mobile phase of MeCN in
0.1% aqueous TFA at a flow rate of 5 ml/min. Analyti-
cal RP-LC/MS was performed on a Gilson HPLC sys-
5.3. 3-(4-Methoxy-benzyl)-methyl benzoate (19)
A solution of 18 (1.8 g, 6.66 mmol) in 40 mL of dry
DCM was cooled to 0 ꢁC and TFA (5.1 mL, 66.6 mmol)
and triflic acid (15 lL, 0.17 mmol) was added. Trieth-
ylsilane (3.2 mL, 20.0 mmol) was added dropwise from
an addition funnel to the solution. The ice bath was re-
moved and the reaction mixture was stirred at ambient
temperature over night. The reaction mixture was
washed with 1 M NaOH and brine, dried over MgSO4
and evaporated. Compound 19 (1.64 g, 96%) was isolat-
ed as a clear oil. 1H NMR (CDCl3) d: 7.89–7.86 (m, 2H,
2· CH), 7.36 (m, 1H, CH), 7.35 (m, 1H, CH), 7.10 (m,
2H, 2· CH), 6.84 (m, 2H, 2· CH), 3.97 (m, 2H, CH2),
3.90 (s, 3H, CH3), 3.78 (s, 3H, CH3). 13C NMR (CDCl3)
d: 167.2 (CO), 158.1 (C), 141.9 (C), 133.4 (CH), 132.6
tem with
a
Finnigan AQA quadropole mass
spectrometer using a Chromolith Performance RP–18
4.6 mm · 100 mm column (Merck) at a flow rate of
4 mL/min. The pseudopeptide purities were determined

J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
5969
(C), 130.3 (C), 129.9 (CH), 129.8 (2· CH), 128.5 (CH),
127.3 (CH), 114.0 (2· CH), 55.2 (CH3), 52.1 (CH3),
40.8 (CH2). MS m/z (70 eV) 256 (M+). Anal.
(C16H16O3) C, H, N.
([M+2H+]/2), 359.4 ([M+MeCN+2H+]/2). Amino acid
analysis: Tyr, 1.00; Ile, 0.91; His, 0.92; Pro, 1.01; Phe,
0.99. HRMS (M+1) calcd: 676.3459. Found: 676.3467.
5.5.4. Peptide 4. Part of the crude material (33.0 mg) was
purified to give 15.8 mg (49%) of the desired peptide.
LC/MS (M: 641.4): 642.2 (M+H+), 321.7 ([M+2H+]/2),
342.3 ([M+MeCN+2H+]/2). Amino acid analysis: Tyr,
1.00; Ile, 1.87; His, 0.90; Pro, 1.00. HRMS (M+1) calcd:
642.3615. Found: 642.3602.
5.4. 3-(4-Hydroxybenzyl)-benzoic acid (D)
Compound 19 (1.3 g, 5.1 mmol) was dissolved in dry
DMC (100 mL). The reaction vessel was fitted with a
septum, cooled in an ice bath, and BF3ÆS(CH3)2
(1.4 mL, 12.7 mmol) was added slowly. The reaction
mixture was allowed to reach ambient temperature
and was left stirring over night. It was poured into
1 M HCl aqueous (100 mL) and extracted with DCM.
The organic phase was washed with H2O and brine,
dried with MgSO4 and evaporated. The crude product
(5.1 mmol) was dissolved in THF (100 mL) and MeOH
(12.5 mL) and H2O (25 mL) was added. An 1 M aque-
ous solution of LiOH (25 mL) was added dropwise
and the reaction was stirred over night. The reaction
mixture was concentrated and then portioned between
5 N NaOH and ethyl acetate. The aqueous phase was
acidified with concd HCl and extracted with ethyl ace-
tate. The organic phase was washed with H2O and brine,
dried with MgSO4 and evaporated to give D (864 mg,
74%) as an off-white solid. 1H NMR (acetone-d6) d:
11.22 (br s, 1H), 8.20 (br s, 1H), 7.89–7.86 (m, 2H, 2·
CH), 7.36 (m, 1H, CH), 7.35 (m, 1H, CH), 7.10 (m,
2H, 2· CH), 6.84 (m, 2H, 2· CH), 3.97 (m, 2H, CH2),
3.90 (s, 3H, CH3), 3.78 (s, 3H, CH3). 13C NMR (ace-
tone-d6) d: 167.2 (CO), 158.1 (C), 141.9 (C), 133.4
(CH), 132.6 (C), 130.3 (C), 129.9 (CH), 129.8 (2· CH),
128.5 (CH), 127.3 (CH), 114.0 (2· CH), 55.2 (CH3),
52.1 (CH3), 40.8 (CH2). LC/MS (M: 228): 457
(2M+H). Anal. (C14H12O3) C, H, N.
5.6. General synthesis of pseudopeptides
5.6.1. Coupling. Fmoc-His(Trt)-Pro-Phe-Wang resin27
or Fmoc-His(Trt)-Pro-Ile-Wang resin27 was weighed
into a 2 mL disposable syringe equipped with a porous
polyethylene filter and allowed to swell in DMF
(1.5 mL) for 1 h. The Fmoc-group was removed by a
three-step treatment with 20% piperidine in DMF (3·
1.5 mL, 1 + 3 + 10 min) and the polymer was washed
with DMF (6· 1.5 mL, 6· 1 min). The turn scaffold,
Fmoc-protected A, Fmoc-protected B, Fmoc-protected
C, D or Fmoc-protected E (1.25 equiv), and PyBOP
(1.25 equiv) (or HBTU (1.25 equiv) for couplings to
the Fmoc-His(Trt)-Pro-Ile-Wang resin) were dissolved
in DMF (1.5 mL) in the presence of DIEA (2.5 equiv)
and allowed to react with the resin over night (18 h).
LC/MS analysis after cleavage of an analytical sample
showed complete reaction. The rest of the resin was
washed with DMF (6· 2 mL, 6· 1 min). The Fmoc-
group was cleaved as described above and the resin
was washed with DMF (6· 2 mL, 6· 1 min), DCM
(5· 2 mL, 5· 1 min) and MeOH (5· 2 mL, 5· 1 min) be-
fore being dried in vacuum over night. For the acetylat-
ed analogues, an aliquot of the resin was swelled in
DMF (2 mL). The acetic acid (5 equiv) was dissolved
together with PyBOP (5 equiv) or HBTU (5 equiv) and
DIEA (10 equiv) in DMF (0.5 mL). The solution was
added to the resin and agitated by rotation over night.
The resin was deprotected, washed and dried as above.
5.5. General synthesis of peptides
The peptides 1–4 were synthesized with a Protein Tech-
nologies Symphony instrument using standard Fmoc/t-
Bu chemistry as earlier described.24 Acetylation was
accomplished by allowing the resin to react with 20%
acetic anhydride in DMF for 30 min directly after the
last Fmoc deprotection, that is, without intermediate
washing. Cleavage and purification was carried out as
described below.
5.6.2. Cleavage. To cleave the peptide from the resin, tri-
ethylsilane (100 lL) and 95% aqueous TFA (1.5 mL)
were added and the mixture was agitated for 1 h. The
polymer was filtered off and washed with TFA (2·
0.3 mL). The filtrate was evaporated in a stream of
nitrogen and the product was precipitated by addition
of diethyl ether (13 mL). The precipitate was collected
by centrifugation, washed with ether (3· 7 mL) and
dried.
5.5.1. Peptide 1. Purification of 10.3 mg of the crude
cleavage product gave 6.4 mg (57% based on amount
of starting resin) of the target peptide. LC/MS (M:
717.3): 718.3 (M+H+). Amino acid analysis: Tyr, 1.00;
Ile, 0.86; His, 0.89; Pro, 1.00; Phe, 1.01. HRMS (M+1)
calcd: 718.3564. Found: 718.3555.
5.6.3. Purification. The crude peptide was purified by
RP-HPLC. The separation was monitored at 230 nm
and by analytical RP-HPLC and/or LC/MS of selected
fractions.
5.5.2. Peptide 2. An aliquot (13.5 mg) of the crude prod-
uct was purified to yield 10.0 mg (80%). LC/MS (M:
683.4): 684.3 (M+H+). Amino acid analysis: Tyr, 1.00;
Ile, 1.86; His, 0.89; Pro, 1.00. HRMS (M+1): Calcd:
684.3721. Found: 684.3713.
5.6.4. Analogue 5. The synthesis was conducted as
described above. An aliquot of the Fmoc-His(Trt)-
Pro-Phe-Wang resin was deprotected, and coupled to
Fmoc-protected A and after appropriate washing and
deprotection, the resin was subjected to the standard
cleavage and purification procedures to yield 6.0 mg,
8.2 lmol (30%), LC/MS (M: 722.3): 723.2 (M+H+),
5.5.3. Peptide 3. Preparative RP-HPLC of 22.4 mg of
the crude material afforded 11.1 mg (57%) of the pure
peptide. LC/MS (M: 675.3): 676.4 (M+H+), 338.9

5970
J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
362.2 ([M+2H+]/2), 382.8 ([M+MeCN+2H+]/2), Amino
acid analysis: His, 1.02; Pro, 0.99; Phe, 0.99. HRMS
(M+1) calcd: 723.3255. Found: 723.3242.
washed, deprotected again, and then cleaved according
to the general procedure. After purification on the
ACE Phenyl column instead of the standard, 11.6 mg,
18.1 lmol (20%) were recovered. LC/MS (M: 638.29):
639.6 (M+H+), 320.4 ([M+2H+]/2), 340.9 ([M+MeCN+
2H+]/2), 361.4 ([M+2MeCN+2H+]/2). Amino acid anal-
ysis: His, 1.00; Pro, 1.00; Phe, 1.01. HRMS (M+1) calcd:
639.2931. Found: 639.2938.
5.6.5. Analogue 6. The synthesis was conducted as de-
scribed above. An aliquot of the Fmoc-His(Trt)-Pro-
Phe-Wang resin was deprotected, and coupled to
Fmoc-protected B and after appropriate washing and
deprotection, the resin was subjected to the standard
cleavage and purification procedures to yield 11.2 mg,
15.5 lmol (54%), LC/MS (M: 722.3): 723.1 (M+H+),
362.2 ([M+2H+]/2), 382.8 ([M+MeCN+2H+]/2), Amino
acid analysis: His, 1.00; Pro, 1.00; Phe, 1.00. HRMS
(M+1) calcd: 723.3255. Found: 723.3242.
5.6.11. Analogue 12. The Fmoc-His(Trt)-Pro-Phe-Wang
resin (58 mg, 30 lmol) was deprotected coupled to
Fmoc-protected E. After the Fmoc-deprotection the res-
in was reacted with acetic anhydride (760 lmol) and
DIEA (760 lmol) in DMF (0.5 mL) for 1 h. Standard
cleavage gave 20.0 mg crude product which was purified
on the ACE Phenyl column instead of the standard col-
umn. Yield: 3.4 mg, 5.0 lmol (16%); LC/MS (M:
680.30): 681.5 (M+H+), 341.3 ([M+2H+]/2), 361.9
([M+MeCN+2H+]/2). Amino acid analysis: His, 0.99;
Pro, 1.00; Phe, 1.01. HRMS (M+1): Calcd: 681.3037.
Found: 681.3032.
5.6.6. Analogue 7. The synthesis was conducted as
described above. An aliquot of the Fmoc-His(Trt)-
Pro-Phe-Wang resin was deprotected, and coupled to
Fmoc-protected B and after appropriate washing and
deprotection, subsequent coupling of acetic acid. The
resin was subjected to the standard cleavage and purifi-
cation procedures to yield 9.9 mg, 12.9 lmol (59%), LC/
MS (M: 764.3): 765.4 (M+H+), 383.3 ([M+2H+]/2),
Amino acid analysis: His, 1.00; Pro, 1.00; Phe, 1.00.
HRMS (M+1) calcd: 765.3360. Found: 765.3374.
5.6.12. Analogue 13. An aliquot of the Fmoc-His(Trt)-
Pro-Ile-Wang resin (80 mg, 50 lmol) was deprotected
and coupled to Fmoc-protected C. The resin was sub-
jected Fmoc-deprotection and cleavage. Purification
was conducted according to the standard procedure.
Yield 5.2 mg (18%). LC/MS (M: 590.3): 591.2
(M+H+), 296.2 ([M+2H+]/2), 316.8 ([M+MeCN+
2H+]/2). Amino acid analysis: His, 1.00; Pro, 1.00;
Ile, 0.99. HRMS (M+1): Calcd: 591.2931. Found:
591.2935.
5.6.7. Analogue 8. The synthesis was conducted as de-
scribed above. An aliquot of the Fmoc-His(Trt)-Pro-
Phe-Wang resin (70 mg, 42 lmol) was deprotected, and
coupled to Fmoc-protected C and after appropriate
washing and deprotection, the resin was subjected to
the standard cleavage and purification procedures to
yield 14.2 mg (54%). LC/MS (M: 624.3): 625.4
(M+H+),
333.9
([M+MeCN+2H+]/2),
354.4
5.6.13. Analogue 14. A portion of the Fmoc-His(Trt)-
Pro-Phe-Wang resin (80 mg, 50 lmol) deprotected and
coupled to Fmoc-protected C followed by deprotection
and subsequent coupling of acetic acid (200 lmol) as
outlined above. After cleavage, the crude product was
purified and 7.2 mg (23%) was isolated. LC/MS (M:
632.3): 633.3 (M+H+). Amino acid analysis: His, 1.00;
Pro, 1.01; Ile, 0.99. HRMS (M+1) calcd: 633.3037.
Found: 633.3050.
([M+2MeCN+2H+]/2). Amino acid analysis: His, 1.00;
Pro, 0.99; Phe, 1.00. HRMS (M+1) calcd: 625.2775.
Found: 625.2777.
5.6.8. Analogue 9. A portion of the Fmoc-His(Trt)-Pro-
Phe-Wang resin (75 mg, 45 lmol) was deprotected and
subjected to coupling of Fmoc-protected C, and subse-
quently with acetic acid (200 lmol) as outlined above.
After cleavage, the crude product was purified according
to the standard procedure. Yield 6.4 mg (23%). LC/MS
(M: 666.3): 667.4 (M+H+). Amino acid analysis: His,
1.00; Pro, 1.00; Phe, 1.00. HRMS (M+1) calcd:
667.2880. Found: 667.2885.
5.6.14. Analogue 15. A part of the Fmoc-His(Trt)-Pro-
Ile-Wang resin (80 mg, 50 lmol) was deprotected and
further reacted with D, washed, deprotected, and
cleaved according to the general method. After purifica-
tion 4.6 mg (16%) were recovered. LC/MS (M: 575.3):
576.2 (M+H+). Amino acid analysis: His, 1.00; Pro,
1.01; Ile, 1.00. HRMS (M+1): Calcd: 576.2822. Found:
576.2818.
5.6.9. Analogue 10. The synthesis was conducted as out-
lined above, starting from Fmoc-His(Trt)-Pro-Phe-Wang
resin (83 mg, 50 lmol). After the initial deprotection, the
resin was coupled to D followed by deprotection, washing
and drying. The standard procedure to cleave the product
from the resin was used. The purification of the peptide
differed slightly from the standard procedure. After puri-
fication on an ACE phenyl column instead of the standard
11.0 mg (36%) was recovered. LC/MS (M: 609.3): 610.2
(M+H+). Amino acid analysis: His, 1.01; Pro, 0.99; Phe,
1.00. HRMS (M+1) calcd: 610.2666. Found: 610.2656.
5.6.15. Analogue 16. The Fmoc-His(Trt)-Pro-Ile-Wang
resin (80 mg, 50 lmol) was subjected Fmoc-deprotection
and coupling to Fmoc-protected E. The crude product
was cleaved and purified according to the standard pro-
cedure. Yield 5.2 mg (17%). LC/MS (M: 604.3): 605.3
(M+H+), 303.2 ([M+2H+]/2), 323.7 ([M+MeCN+2H+]/
2). Amino acid analysis: His, 0.99; Pro, 1.01; Ile, 0.99.
HRMS (M+1) calcd: 605.3088. Found: 605.3098.
5.6.10. Analogue 11. An aliquot of the Fmoc-His(Trt)-
Pro-Phe-Wang resin (55 mg, 29 lmol) was deprotected,
and coupled to Fmoc-protected E. The resin was
5.6.16. Analogue 17. The Fmoc-His(Trt)-Pro-Ile-Wang
resin (80 mg, 50 lmol) was subjected Fmoc-deprotection

J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
5971
and coupling to Fmoc-protected E. After deprotection it
was subjected to coupling with acetic acid (200 lmol) as
outlined above. After cleavage, the crude product was
purified using the standard system. Yield 6.4 mg
(20%). LC/MS (M: 646.3): 647.4 (M+H+), 344.8
([M+MeCN+2H+]/2). Amino acid analysis: His, 1.00;
Pro, 1.01; Ile, 0.99. HRMS (M+1) calcd: 647.3193.
Found: 647.3191.
Cheng–Prusoff equation42 (Kd = 0.7 0.1 nM, [L] =
0.057 nM). The binding data were best fitted with a
one-site fit. All experiments were performed in triplicate.
5.8. In vitro morphological effects
To study the in vitro morphological effects NG108-15
cells (provided by Drs M. Emerit and M. Hamon; IN-
SERM, U. 238, Paris, France) were used as well as
transfected NG108-15/pcDNA3 cells. The transfected
cell line has previously been shown to have the same
behavior as the native cell line.43 Both cell lines were
cultured (NG108-15 passage 18–28, NG108-15/
pcDNA3 passage 12–18) in Dulbecco’s modified Ea-
gle’s medium (DMEM, Gibco BRL, Burlington,
ONT, Canada) with 10% fetal bovine serum (FBS,
Gibco), HAT supplement (Hypoxanthine, Aminopter-
in, Thymidine from Gibco) and 50 mg/l gentamycin
(Gibco) at 37 ꢁC in 75 cm2 Nunclon Delta flasks in
a humidified atmosphere of 93% air and 7% CO2, as
previously described.35 The transfected cell line was
kept stable by addition of Geneticin (G-418, 200 lg/
ml) to the media. Subcultures were performed at sub-
confluency. Under these conditions, cells express only
the AT2 receptor subtype.35,36 Cells were stimulated
during three days, once a day (first stimulation 24 h
after plating). Cells were cultured for three subsequent
days under these conditions. For all experiments, cells
were plated at the same initial density of 3.6 · 104
cells/35 mm Petri dish. Cells were treated without
(control cells), with [Val5]-angiotensin II from Bachem
5.7. Rat liver membrane AT1 receptor binding assay
Rat liver membranes were prepared according to the
method of Dudley et al.33 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 lM bacitracin,
10 lM pepstatin A, 10 lM bestatin, 10 lM 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 concen-
trations (0.01 nM–1.0 lM) of test substance. Samples
were incubated at 25 ꢁC for 2 h, and binding was termi-
nated 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 c-counter. Non-specific binding was determined in
the presence of 1 lM Ang II. The specific binding was
determined by subtracting the non-specific binding from
the total bound [125I]Ang II. IC50 was determined by
Scatchard analysis of data obtained with Ang II by
using GraFit (Erithacus Software, UK). The apparent
dissociation constants Ki were calculated from IC50 val-
ues using the Cheng–Prusoff equation42 (Kd = 1.7
0.1 nM, [L] = 0.057 nM). The binding data were best
fitted with a one-site fit. All experiments were performed
in triplicate.
(Marina Delphen, CA, USA) (100 nM) or 1,
2
(10 nM), 8 or 15 (100 nM), in the absence or in the
presence of PD 123,319 (RBI Natick, MA, USA)
(10 lM), an AT2 receptor antagonist introduced daily
30 min prior to Ang II, 1, 2, 8, or 15. During the three
day treatment the transfected cell line was cultured
without Geneticin.
5.7.1. Pig myometrial membrane AT2 receptor binding
assay. Myometrial membranes were prepared from por-
cine uteri according to the method of Nielsen et al.34
Potential interference by binding to AT1 receptors was
blocked by addition of 1 lM losartan. Binding of
5.9. Determination of cells with neurites
Cells were examined under a phase contrast microscope
and micrographs were taken after three days under the
various experimental conditions. Cells with at least
one neurite longer than a cell body were counted as po-
sitive for neurite outgrowth. At least 290 cells were
counted in three independent experiments.10
[
125I]Ang II to membranes was conducted in a final vol-
ume of 0.5 mL containing 50 mM Tris–HCl (pH 7.4),
100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10 lM
bacitracin, 10 lM pepstatin A, 10 lM bestatin, 10 lM
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 lM) of test substance. Samples were incu-
bated 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 trans-
ferred to tubes. The radioactivity was measured in a
c-counter. Nonspecific binding was determined in the
presence of 1 lM Ang II. The specific binding was deter-
mined by subtracting the nonspecific binding from the
total bound [125I]Ang II. IC50 was determined by Scat-
chard analysis of data obtained with Ang II by using
GraFit (Erithacus Software, UK). The apparent dissoci-
ation constants Ki were calculated using the
5.10. Vascular contractility studies
This study was performed at Cerep (France) according
to literature.37 Seven concentrations (3, 10, 30, 100,
300, 1000 nM) of 8 were tested. Ang II was used as ref-
erence agonist and saralasin (0.03 lM) as reference
antagonist.
Acknowledgments
We gratefully acknowledge the financial support from
the Swedish Research Council and the Swedish Founda-
tion for Strategic Research. We also thank Kristofer
Olofsson for linguistic revision.

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J. Georgsson et al. / Bioorg. Med. Chem. 14 (2006) 5963–5972
Butler, B. S.; Foor, F.; Pasternak, A.; Pan, Y.; Silva, M.;
Supplementary data
Freidinger, R. M.; Smith, R. G.; Chapman, K.; Schaeffer,
J. M.; Patchett, A. A. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 10836–10841.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2006.05.019.
22. Gouin, S. G.; Murphy, P. V. J. Org. Chem. 2005, 70,
8527–8532.
23. Khosla, M. C.; Leese, R. A.; Maloy, W. L.; Ferreira, A.
T.; Smeby, R. R.; Bumpus, F. M. J. Med. Chem. 1972, 15,
792–795.
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