Angiotensin II pseudopeptides containing 1,3,5-trisubstituted benzene scaffolds with high AT2 receptor affinity

Article abstract of DOI:10.1021/jm050280z

Two 1,3,5-trisubstituted aromatic scaffolds intended to serve as γ-turn mimetics have been synthesized and incorporated in five pseudopeptide analogues of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), replacing Val-Tyr-Ile, Val-Tyr, or Tyr-Ile. All the tested compounds exhibited nanomolar affinity for the AT₂ receptor with the best compound (3) having a K_i of 1.85 nM. Four pseudopeptides were AT₂ selective, while one (5) also exhibited good affinity for the AT₁ receptor (K_i = 30.3 nM). This pseudopeptide exerted full agonistic activity in an AT₂ receptor induced neurite outgrowth assay but displayed no agonistic effect in an AT₁ receptor functional assay. Molecular modeling, using the program DISCOtech, showed that the high-affinity ligands could interact similarly with the AT₂ receptor as other ligands with high affinity for this receptor. A tentative agonist model is proposed for AT₂ receptor activation by angiotensin II analogues. We conclude that the 1,3,5-trisubstituted benzene rings can be conveniently prepared and are suitable as γ-turn mimics.

Full text of DOI:10.1021/jm050280z

6620
J. Med. Chem. 2005, 48, 6620-6631
Angiotensin II Pseudopeptides Containing 1,3,5-Trisubstituted Benzene
Scaffolds with High AT2 Receptor Affinity
Jennie Georgsson,† Christian Sko¨ld,† Bianca Plouffe,‡ Gunnar Lindeberg,† Milad Botros,§ Mats Larhed,†
Fred Nyberg,§ Nicole Gallo-Payet,*,‡ Adolf Gogoll,# Anders Karle´n,† and Anders Hallberg*,†
Department of Medicinal Chemistry, Division of Organic Pharmaceutical Chemistry, Uppsala University,
P.O. Box 574, SE-751 23 Uppsala, Sweden, Service of Endocrinology, Faculty of Medicine, University of Sherbrooke,
Sherbrooke J1H 5N4, Quebec, Canada, Department of Pharmaceutical Sciences, Division of Biological Research
on Drug Dependence, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, Sweden, and Department of Chemistry,
Organic Chemistry, Uppsala University, P.O. Box 599, SE-751 24 Uppsala, Sweden
Received March 29, 2005
Two 1,3,5-trisubstituted aromatic scaffolds intended to serve as γ-turn mimetics have been
synthesized and incorporated in five pseudopeptide analogues of angiotensin II (Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe), replacing Val-Tyr-Ile, Val-Tyr, or Tyr-Ile. All the tested compounds
exhibited nanomolar affinity for the AT2 receptor with the best compound (3) having a Ki of
1.85 nM. Four pseudopeptides were AT2 selective, while one (5) also exhibited good affinity for
the AT1 receptor (Ki ) 30.3 nM). This pseudopeptide exerted full agonistic activity in an AT2
receptor induced neurite outgrowth assay but displayed no agonistic effect in an AT1 receptor
functional assay. Molecular modeling, using the program DISCOtech, showed that the high-
affinity ligands could interact similarly with the AT2 receptor as other ligands with high affinity
for this receptor. A tentative agonist model is proposed for AT2 receptor activation by angiotensin
II analogues. We conclude that the 1,3,5-trisubstituted benzene rings can be conveniently
prepared and are suitable as γ-turn mimics.
Introduction
The natural abundance of AT2 receptors is very high
in the fetus but low in adults, compared to the AT1
receptor. Notably, the AT2 receptor is up-regulated in
several pathological conditions, e.g., congestive heart
failure, renal failure, myocardial infarction, vascular
injury, and wound healing.1,2,6,11
The octapeptide angiotensin II (Ang II, Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe), an important hormone in the
renin-angiotensin system, exerts its action by activa-
tion of AT1 and AT2 receptors. The two receptor types
are both G-protein coupled receptors, but they differ
considerably by exhibiting a sequence homology of only
32-34% by having distinguished signaling pathways
and by having different physiological effects.1,2 The
physiological effects that are most commonly associated
with Ang II, e.g., vasoconstriction and aldosteron secre-
tion, are caused by an activation of the AT1 receptor.1-3
The AT2 receptor was cloned in 1993,4,5 but there are
still uncertainties regarding the effects of its activation.
Recent studies suggest “anti-AT1” effects such as va-
sodilation and hypotension in animal models.1,6 The AT2
receptor is also involved in apoptosis, cell differentiation,
and tissue repair and in the increase of duodenal
mucosal alkalization.1,2,6-8 In neuronal cells, agonistic
activation of the AT2 receptor induces neurite elongation
and outgrowth, modulates neuronal excitability, and
promotes cellular migration in adults.8 The mechanisms
involve an activation of the nitric oxide/guanyl cyclase/
cGMP pathway9 and a sustained increase in p42/p22mapk
activity.10
AT2 receptor agonists may find a role as therapeutics,
e.g., in treatment of hypertension and cardiovascular
remodeling.6,12 The bioactive conformation of Ang II,
especially regarding binding to the AT1 receptor, has
been thoroughly studied.13-18 There are indications that
Ang II adopts a γ-turn-like conformation centered at
Tyr4 when binding to and activating the AT1 receptor
based on calculations and rigidifications of Ang II.19-21
The bioactive conformation of Ang II when binding the
AT2 receptor have so far not been fully investigated.
However, photoaffinity labeling experiments suggests
an extended â-strand conformation of Ang II when
binding to the AT2 receptor (and also to the AT1
receptor).22 Other investigations based on, for instance,
computational modeling and binding affinities for the
AT2 receptor of both linear and rigidified Ang II
analogues have also been performed that suggest that
a turnlike structure may be present in the 3-5 region
of Ang II.23-25 Even less is known regarding the
conformations mediating the stimulation of the AT2
receptor, primarily because of the lack of robust in vitro
agonist assays. Cyclizations involving the amino acid
residues 3 and 5 in Ang II, e.g., the cyclization delivering
the methylenedithioether in A25,26 that induces γ-turn
conformations, as well as incorporation of the γ-turn
mimicking benzodiazepine scaffolds B23 and C23,24 and
the dihydroisoquinolinone scaffold D21 in Ang II, have
provided ligands with high affinity for the AT2 receptor
* To whom correspondence should be addressed. For N.G.-P.: phone,
+1-819-564-5243; fax, +1-819-564-5292; e-mail, nicole.gallo-payet@usher
brooke.ca. For A.H.: phone, +46-18-4714284; fax, +46-18-4714474;
e-mail, anders.hallberg@orgfarm.uu.se.
† Division of Organic Pharmaceutical Chemistry, Uppsala Univer-
sity.
‡ University of Sherbrooke.
§ Division of Biological Research on Drug Dependence, Uppsala
University.
# Department of Chemistry, Organic Chemistry, Uppsala University.
10.1021/jm050280z CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/23/2005

Angiotensin II Pseudopeptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6621
Chart 1
γ-turn scaffold in the 3-5 region of Ang II, was found
to lack all AT1 receptor affinity.27 It seems that the AT1
receptor responds to small variations of the turn geom-
etry in the 3-5 region of Ang II while the AT2 receptor
is less sensitive. Accordingly, we speculated that
pseudopeptides encompassing the synthetically easily
accessible γ-turn scaffolds F and the even more compact
G might serve as good AT2 receptor ligands.
Herein, we report the synthesis of the two γ-turn
mimetic scaffolds F and G, each with an aromatic ring
as a common core structure, and the synthesis of five
peptide analogues of Ang II (Chart 2) containing these
two scaffolds. We disclose that pseudopeptides compris-
ing the aromatic scaffold G can exhibit high AT1 and
AT2 receptor affinity while those containing F demon-
strate high AT2 receptor selectivity. One of the Ang II
analogues was found to act as a full AT2 receptor
agonist.
Results
Chemistry. Compound 12 was previously synthe-
sized by us, although with the phenolic hydroxy group
Fmoc-protected and by a procedure delivering a consid-
erably lower total yield.27 The synthetic route to inter-
mediates 8a,b is outlined in Scheme 1, and the prepa-
ration of templates 12 and 15 is in Schemes 2 and 3,
respectively.
(Chart 1). Some of these ligands were recently shown
to exert agonistic activity at the AT2 receptor.24
Compounds 7a,b were prepared by Friedel-Crafts
acylation of anisole using the acid chlorides of the
commercially available benzoic acid derivatives 6a,b. In
the acylation step, the ortho/para ratios were found to
be highly substrate-dependent, providing high para
selectivity from 6a but not from 6b.28 The benzophenone
7a was isolated as the pure para derivative in 73% yield
by simple crystallization. The same synthetic transfor-
Whereas the pseudopeptides incorporating B, C, or
D in the 3-5 region of Ang II did not bind to the AT1
receptor, the analogues containing A and the azepine
γ-turn mimic E showed affinity for the AT1 recep-
tor.21,25,26 Furthermore, a pseudopeptide with the 1,3,5-
trisubstituted benzene F, representing a more compact
Chart 2

6622 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Georgsson et al.
Scheme 1a
phy, and 7b was isolated in 42% yield. The subsequent
high-yielding reduction of ketones 7a,b to form 8a,b was
conducted under acidic conditions in DCM, employing
triethylsilane as the hydride source.29-31 It was essential
to conduct the carbonyl reduction at this stage, since it
was found to be remarkably difficult to perform subse-
quent palladium(0)-catalyzed coupling reactions with an
intact acyl substituent.
The ester functionality in 9 was introduced by a
microwave-assisted palladium-catalyzed alkoxy carbo-
nylation (Scheme 2).32 In this protocol, Mo(CO)6 was
utilized as a solid carbon monoxide source and pal-
ladium on charcoal was used as a catalyst without extra
addition of phosphine ligands. With substrate 8a, the
reactivity of the iodide was considerably higher than for
the bromide and thus allowed chemoselective introduc-
tion of the ester-protected carboxylic acid function
despite the high temperature (130 °C). The major side
product, as deduced from GC-MS, was the dehalogen-
ated starting material. To obtain compound 10, a
palladium-catalyzed cyanation was performed using a
microwave-accelerated procedure.33,34 Pd2(dba)3 and tri-
o-tolylphosphine provided the catalytic system in situ
and Zn(CN)2 served as the cyanide source. To attain
reproducible results, it was crucial to keep the amount
a Reagents: (a) (i) SOCl2, reflux; (ii) anisole, AlCl3, DCM; (b)
(CH3CH2)3SiH, F3CSO3H, TFA, DCM.
Scheme 2a
of Zn(CN)2 low to avoid poisoning of the catalyst.35
A
standard hydrogenation of nitrile 10 under an atmo-
spheric pressure of hydrogen gas employing palladium
on charcoal in absolute ethanol smoothly generated the
benzylamine, which was subsequently reacted with
Fmoc chloride in a solution of aqueous Na2CO3 and
dioxane to produce 11.36 Simultaneous cleavage of the
TMSE ester and the methoxy group was accomplished
with a boron trifluoride dimethyl sulfide complex in
DCM to afford the Fmoc-protected γ-turn scaffold 12.37
Compound 13 was synthesized by hydrolysis of com-
pound 8b with LiOH in THF/H2O/MeOH. The condi-
tions described above for the preparation of compound
11 were thereafter used for reduction of the nitro group
of 13 and the subsequent Fmoc protection to produce
compound 14. The final deprotection of the hydroxyl
function to produce the Fmoc-protected γ-turn template
15 was conducted as described above for the preparation
of 12. The scaffolds F and G were introduced in the
pseudopeptides as their Fmoc-protected equivalents 12
and 15. The pseudopeptides 1-5 were synthesized by
standard Fmoc strategy solid-phase peptide synthesis
(SPPS) using standard SPPS with PyBOP as the
coupling reagent. The introduced γ-turn mimetic scaf-
folds F and G replaced Val-Tyr-Ile in compounds 1 and
4, Tyr-Ile in 3 and 5, and Val-Tyr in 2.
a Reagents: (a) Mo(CO)6, Pd/C, DMAP, DIEA, HOCH2CH2Si-
(CH3)3,dioxane, microwave heating, 130 °C, 15 min; (b) Zn(CN)2,
Pd2(dba)3, P(o-tolyl)3, DMF, microwave heating, 180 °C, 10 min;
(c) (i) H2, Pd/C, EtOH; (ii) Fmoc-Cl, aqueous Na2CO3, dioxane; (d)
BF3‚S(CH3)2, DCM.
Scheme 3a
NMR signal assignment was obtained following rou-
tine methodology, revealing connectivity within amino
acid residues by primitive exclusive correlation spec-
troscopy (PE COSY)38 and total correlation spectroscopy
(TOCSY)39 experiments and sequential assignments
from rotating-frame Overhauser enhancement spectros-
copy (ROESY)40 experiments. These experiments were
combined with solvent suppression by the wet tech-
nique.41 Chemical shift data are available in Supporting
Information.
a Reagents: (a) LiOH, THF/MeOH/H2O; (b) (i) H2, Pd/C, EtOH;
(ii) Fmoc-Cl, aqueous Na2CO3, dioxane; (c) BF3‚S(CH3)2, DCM.
mation to obtain 7b required additional optimization,
and a preactivation of the acid chloride with the AlCl3
was found to be beneficial. Despite this improvement,
only approximately 65% conversion was achieved after
5 days at ambient temperature. Thus, the ortho and
para isomers were separated by column chromatogra-
At room temperature, the spectra of the peptidomi-
metics 3 and 4 indicate the presence of two conformers
due to cis-trans isomerism of the proline peptide bond.

Angiotensin II Pseudopeptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6623
Table 1. Binding Affinities for the AT1 and AT2 Receptors
AT1 (rat liver
membranes)
Ki ( SEM (nM)
AT2 (pig uterus
myometrium)
Ki ( SEM (nM)
compd
Ang II
0.95 ( 0.08
0.16 ( 0.03
>10000
0.2
0.18 ( 0.01
0.7
10.1 ( 0.6
463.7 ( 10.9
1.85 ( 0.1
41.3 ( 0.8
9.8 ( 0.3
[Sar1]Ang II
[4-NH2-Phe6]Ang II
1
2
3
4
5
>10000
>10000
>10000
>10000
30.3 ( 1.5
Figure 1. Effect of the AT2 receptor antagonist PD 123,319
on pseudopeptide 5 induced neurite outgrowth in NG108-15
cells. Cells with at least one neurite longer than the 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 (from 67 to 186 cells
according to the experiment): control, with Ang II (0.1 µM)
stimulation, Ang II (0.1 µM) in the presence of PD 123,319
(1.0 µM), 5 (0.1 µM), and 5 (0.1 µM) in the presence of PD
123,319 (1.0 µM).
As expected, the number of signals is reduced when the
temperature is increased (to 60 °C) because of the faster
exchange between isomers at elevated temperature.
Binding Assay. Compounds 1-5 were evaluated in
radioligand binding assays relying on displacement of
[
125I]Ang II from AT1 receptors in rat liver membranes42
and from AT2 receptors in pig uterus membranes43
(Table 1). Ang II, [4-NH2-Phe6]Ang II, and [Sar1]Ang II
were used as reference substances. The results are
summarized in Table 1. All compounds showed affinity
for the AT2 receptor, and one compound also showed
affinity for the AT1 receptor. The pseudopeptide 1 with
the scaffold F replacing Val-Tyr-Ile in Ang II displayed
a Ki of 10.1 nM for the AT2 receptor. When F replaced
the two amino acids Val-Tyr (2), a 40-fold drop in
affinity was observed compared to 1. The highest affinity
for the AT2 receptor was encountered with compound 3
(Ki ) 1.85 nM) where F replaces Tyr-Ile. Scaffold G was
introduced in compounds 4 and 5 as a substitute for Val-
Tyr-Ile and Tyr-Ile, respectively. Compound 4 had
moderate affinity for the AT2 receptor (Ki ) 41.3 nM).
Notably, compound 5 exhibited affinity for both the AT1
and the AT2 receptors. This substance was further
evaluated in functional AT1 and AT2 assays to elucidate
whether it serves as an agonist to these receptors.
the AT1 receptor in the rabbit thoracic aorta with the
agonist angiotensin II as reference according to litera-
ture procedure.46 Angiotensin II induced a concentra-
tion-dependent contraction of the rabbit thoracic aorta,
which was inhibited by the AT1 receptor antagonist
saralasin. Unlike Ang II, compound 5 did not produce
any contraction of this tissue at a concentration as high
as 3.0 µM.
Comparison of Scaffolds F and G to Peptide
γ-Turns. Conformational analysis of compounds F and
G modified with an N-terminal acetyl cap, C-terminal
N-methylamide cap, and an alanine side chain instead
of the tyrosine side chain resulted in eight and four
conformations, respectively, within 5 kcal/mol of the
lowest energy minimum. The identified conformations
were evaluated by least-squares alignment of the scaf-
folds to γ-turns from an in-house database of 2101
extracted inverse γ-turns from crystallized protein
structures from the PDB.47 Conformations from both
scaffolds were superimposed with a low root-mean-
square (rms) value onto many of the inverse γ-turns in
the database; 586 and 347 inverse γ-turns could be
superimposed onto a conformation of F and G, respec-
tively, with an rms value less than 0.5 Å. One example
is shown as a stereo image in Figure 2 (rms ) 0.11 and
0.29 Å for F and G, respectively, superimposed onto a
In Vitro Morphological Effects Induced by Com-
pound 5 in NG 108-15 Cells. To study the effect of
pseudopeptide 5 on morphological differentiation, i.e.,
neurite outgrowth, NG108-15 cells were used. In their
undifferentiated state, neuroblastoma-glioma hybrid
NG108-15 cells have a rounded shape and divide
actively. We have previously shown that these cells
exclusively express the AT2 receptor44,45 and exhibit long
neurites after a 3-day treatment with Ang II or the
selective peptide agonist CGP 421112.45 After a 3-day
treatment, pseudopeptide 5 induced neurite outgrowth
and most cells extended one or two neurite processes,
exhibiting a growth cone at the tip, while the cell body
retained a rounded appearance. Compared to Ang II at
the same concentration, pseudopeptide 5 induced the
same morphological changes as previously shown.45
Quantification indicated an increase in the number
of cells with neurites longer than the cell body from 3.8%
( 0.2% in control cells to 14.3% ( 0.2% in cells treated
with compound 5 (Figure 1). Incubation of 5 with the
AT2 receptor antagonist PD 123,319 (1.0 µM) halted the
neurite elongation. We have previously shown that PD
123,319 did not alter the morphology of the untreated
cells.10,45
protein γ-turn from PDB 1EUV, chain A, Pro596-Leu597
Asp598, Leu597, as amino acid i + 1).
-
DISCOtech Analysis. To study if the compounds
with high affinity for the AT2 receptor could adopt
similar binding modes, the pharmacophore modeling
program DISCOtech48 was used. Compounds 1m, 3m,
and 5m (Chart 3) served as models for 1, 3, and 5. The
model compounds 16m, 17m, and 18m were also
included in the analysis (see ref 24 for the structures of
the parent compounds, which have high affinity for AT2
receptors). DISCOtech identified 316 different models
with superimposed conformations where the selected
structural elements were positioned in the same regions
in space. Some of the models had a low overall struc-
tural overlap and were therefore not considered further.
Many of the models were also redundant because they
had very similar alignments. DISCOtech was set to
produce models with all or all but one compound
included. It seems that it was difficult to make models
Rabbit Thoracic Aorta String, AT1 Functional
Assay. Compound 5 was tested for agonist activity at
seven concentrations ranging from 3.0 nM to 3.0 µM at

6624 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Georgsson et al.
Figure 2. Stereo image of models of scaffolds F (green carbons) and G (yellow carbons) superimposed onto an inverse γ-turn
found in a crystallized protein.
Figure 3. (a) DISCOtech model including all compounds. (b) Same model but with only 3m (yellow carbons) and 17m (white
carbons) displayed. Spheres indicate the DISCOtech features: yellow, N-terminal; red, C-terminal; orange, tyrosine phenolic
oxygen; green, proposed receptor interaction point for the arginine guanidino group. The proposed interaction point located in
the area occupied by the tyrosine side chain should be ignored.
Chart 3
that included 16m, since it was omitted from most
models. One of the few models with all compounds
reasonably aligned is shown in Figure 3 where each of
the four investigated structural elements are confined
within 2.0 Å from each other. Since DISCOtech does not
consider structural information, the proposed interac-
tion point of the guanidino group found in the area
occupied by the tyrosine side chain should therefore be
ignored. The other proposed interaction point is more
realistic. A DISCOtech analysis was also made using
only the AT2 receptor agonist model compounds 5m,
16m, 17m, and 18m. DISCOtech found 145 models.
Interestingly only one of these included all four agonists.
This model was very similar to the one presented in
Figure 3. In the remaining models only three of the four
compounds were included.
Discussion
The first selective non-peptide AT1 receptor agonist
was discovered in 1994.49 Recently, we reported the first

Angiotensin II Pseudopeptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6625
nonpeptidic selective AT2 receptor agonist.50 Both these
agonists, which are structurally similar to the “sartans”,
originate from a nonselective AT1/AT2 agonist.51 The
synthetic optimization to afford the druglike AT2 recep-
tor ligand was performed by classical medicinal chem-
istry methodology. In parallel, we are addressing the
challenging task of systematically transforming Ang II
to a more druglike molecule by a process where the
determination and examination of essential recognition
elements in the bioactive conformation are key compo-
nents. An important tool is amino acid scans that can
early reveal valuable information on essential elements.
The Arg2 residue seems to contribute to the binding
affinity to the AT2 receptor, but considering this recep-
tor, no particular amino acid is crucial for binding.52,53
Furthermore, replacing parts in the backbone of the
natural peptide by well-defined secondary structure
motifs constitutes a basic strategy in such an iterative
process.
There is support that Ang II adopts a γ-turn-like
conformation in the 3-5 region when binding to the AT1
receptor. In the binding of Ang II to the AT2 receptor,
an extended â-strand conformation has been suggested
in addition to turnlike motifs. These two hypotheses do
not necessarily have to be in conflict with each other,
since a reasonable extended conformation might be
adopted even with a turn-mimicking scaffold present in
the central part of the pseudopeptide. We have therefore
probed this region in Ang II and introduced the slightly
geometrically different monocyclic and bicyclic γ-turn
mimetic scaffolds shown in Chart 1 and measured the
affinity of the pseudopeptides. Only one of the synthe-
sized analogues (built around scaffold E) showed affinity
(Ki ) 2.8 nM) and agonist activity at the AT1 receptor.
In contrast, several pseudopeptides prepared displayed
high affinity for the AT2 receptor.
We previously showed that the 1,3,5-trisubstituted
benzene γ-turn scaffold, when introduced in Ang II to
give 1 and 2, delivered pseudopeptides inactive at the
AT1 receptor.27 We have now resynthesized these com-
pounds using a modified and more efficient route to the
core structure. In the design of these compounds the
side chain of Val3 was deleted. It is assumed that Val3
has only a conformationally stabilizing role in Ang II
and thus can be removed in the γ-turn mimetics.
Furthermore, since it is unclear what to introduce at
the C-terminal end of this scaffold, we coupled both His-
Pro-Phe and Ile-His-Pro-Phe to give 1 and 2.27 Com-
pounds 1 and 2 were reevaluated in the binding assay,
and the lack of affinity for the AT1 receptor was
confirmed. In contrast, in the AT2 receptor-binding
assay these compounds exhibited Ki values of 10.1 and
463.7 nM, respectively. Apparently, the introduction of
the isoleucine residue between scaffold F and the
C-terminal provided a pseudopeptide (2) with 2300
times weaker affinity than Ang II for the AT2 receptor.
In contrast, compound 1 lacking the isoleucine residue
is only 10 times less potent than Ang II. Thus, the 1,3,5-
trisubstituted scaffold seems to be a better mimic of Val-
Tyr-Ile than Val-Tyr. With knowledge about the pre-
ferred C-terminal of these peptides for binding to the
AT2 receptor, we set out to optimize the N-terminal part.
Our previous SAR studies of pseudopeptides interacting
with the AT2 receptor have shown that a valine residue
inserted between the arginine and the γ-turn mimetic
can improve receptor interaction.23,24 Compound 3 was
therefore synthesized and was found to provide a 5
times higher affinity for the AT2 receptor compared to
1.
In the benzodiazepine γ-turn mimetics B and C, the
nitrogen is linked directly to the benzene ring, while in
the trisubstituted benzene γ-turn mimetic F, a meth-
ylene group separates the nitrogen from the aromatic
ring. To study how G, where the 1,3,5-trisubstituted
benzene scaffold with the nitrogen attached directly to
the benzene ring, compares to γ-turns in proteins, we
searched an in-house database of γ-turns derived from
the PDB and superimposed these onto low-energy
conformations of a model structure of the scaffold. A
model structure of F was also included in the compari-
son. Even though the aromatic ring is smaller compared
to the peptide γ-turn, the CR of the i - 1 residue, the
CR of the i + 3 residue, and the side chain of the i + 1
residue align reasonably well. This indicates that both
F and G may replace the inverse γ-turn structure. Since
the core structure G is also accessible via a synthetic
route similar to the one used for F, we synthesized
pseudopeptides 4 and 5. In the AT2 receptor-binding
assay, these pseudopeptides displayed only a somewhat
lower affinity than 3, which further supports the finding
that the AT2 receptor is quite tolerant toward changes
in the 3-5 region of Ang II.
In pseudopeptides 1 and 4 the core structures replace
the amino acid residues Val-Tyr-Ile. These two com-
pounds bind to the AT2 receptor with a somewhat lower
affinity compared to Ang II. However, when Val3 is also
introduced (compounds 3 and 5), the affinity is improved
by 4-5 times. The extra valine residue may have
introduced extra flexibility and enabled more favorable
binding interactions of the pseudopeptides with the AT2
receptor. The same impact of an incorporation of Val3
was also observed for the corresponding benzodiazepine-
based γ-turn mimetics introduced in the same region
in Ang II.24
Several of the models identified by DISCOtech had
all of the key groups in the same region and also had a
reasonable overall alignment of the scaffolds (Figure 3a).
In Figure 3b only 3m and 17m are shown for clarity.
This model is very similar to the one previously pub-
lished, which was based on only 16m, 17m, and 18m,
which are all based on a benzodiazepine scaffold. It thus
seems possible to find a common alignment of key
groups in the pseudopeptides incorporating a 1,3,5-
trisubstituted benzene scaffold and a 7- or 9-substituted
benzodiazepine. DISCOtech models based on only the
four AT2 receptor agonists (5m 16m, 17m, and 18m)
were also produced. Interestingly only one model could
be identified that included all four agonists. This model
was geometrically very similar to the one based on the
six compounds with high affinity (Figure 3). However,
more AT2 receptor agonists are needed to validate and
explore this model further.
We found it surprising that compound 5 (lacking Ile5)
showed affinity for the AT1 receptor (AT1 Ki ) 30.3 nM).
In fact, out of the many γ-turn-like mimetics synthe-
sized and introduced in Ang II, only one has resulted
in affinity for the AT1 receptor. It is not straightforward
to rationalize the present findings because, for example,

6626 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Georgsson et al.
tions was distilled over calcium hydride. Other chemicals were
used without further purification. The AT1 functional assay
was performed at Cerep, France.
the only difference between 5 and 3 (devoid of AT1
receptor affinity) is the methylene group between the
nitrogen and the aromatic ring. Thus, subtle differences
in the ligands seem to be of key importance for obtaining
high AT1 receptor affinity.
3-Bromo-5-iodo-4′-methoxybenzophenone (7a). The com-
mercially available 3-bromo-5-iodobenzoic acid (9.9 g, 30.4
mmol) was refluxed in 60 mL of thionyl chloride for 1 h. The
thionyl chloride was evaporated under reduced pressure. The
crude product was dissolved in 160 mL of dry DCM, and
anisole (9.9 mL, 91.2 mmol) was added. AlCl3 (8.0 g, 60.8
mmol) was added in portions over 30 min. The reaction
mixture was stirred at room temperature overnight and was
then poured into a mixture of 100 mL of ice and 150 mL of 1
M HCl and extracted with DCM. The organic phase was
washed with water and brine, dried over MgSO4, and evapo-
rated. Crystallization from ethanol afforded the para product
7a as off-white crystals (9.23 g, 73%). Anal. (C14H10BrIO2) C,
H.
1-Bromo-3-iodo-5-(4-methoxybenzyl)benzene (8a). A
solution of 7a (9.0 g, 21.6 mmol) in 150 mL of dry DCM was
cooled to 0 °C, and TFA (16.6 mL, 216 mmol) and triflic acid
(50 µL, 0.52 mmol) were added. Triethylsilane (10.3 mL, 64.7
mmol) was added dropwise with an addition funnel to the
solution. The ice bath was removed, and the reaction mixture
was refluxed for 6 h, then washed with 1 M NaOH and brine,
dried over MgSO4, and evaporated. Compound 8a was isolated
as an off-white solid (8.22 g, 94%). Anal. (C14H12BrIO) C, H.
(2-Trimethylsilanylethyl)-3-bromo-5-(4-methoxybenzyl)-
benzoate (9). Each of 13 microwave 2-5 mL vials was
charged with a stirring bar, palladium on charcoal (10%, 80
mg, 0.08 mmol), and Mo(CO)6 (411 mg, 1.55 mmol). A solution
of 8a (8.16 g, 20.2 mmol) and DMAP (2.5 g, 20.5 mmol) in 44
mL of dioxane was divided into 13 aliquots and added to the
microwave vials. DIEA (0.77 mL, 4.4 mmol) and 2-(trimeth-
ylsilyl)ethanol (1.0 mL, 7.0 mmol) were added to each vial,
which was sealed under air. The samples were agitated and
heated sequentially by microwave irradiation to 130 °C for 15
min. After cooling, the reaction mixtures were filtered, pooled,
and concentrated in vacuo. The crude product was diluted with
EtOAc, washed with 5% citric acid, water, and brine, dried
over MgSO4, and evaporated. The residue was purified by
column chromatography on silica with 40% isohexane in DCM
as the mobile phase to give 9 as a clear oil (4.73 g, 55%). Anal.
(C20H25BrO3Si) C, H, N.
(2-Trimethylsilanylethyl)-3-cyano-5-(4-methoxybenzyl)-
benzoate (10). Each of 10 microwave 2-5 mL vials was
charged with a stirring bar and Zn(CN)2 (36 mg, 0.31 mmol).
A 25 mL stock solution of 9 (2.12 g, 5.0 mmol) in DMF was
prepared, and aliquots of 2.5 mL were added to each of 10
microwave vials precharged with Pd2(dba)3 (10.5 mg, 12 nmol)
and P(o-tolyl)3 (15 mg, 48 nmol) in DMF (2 mL). After 15 min,
the reaction mixture was added to a microwave vial, sealed,
and immediately heated to 180 °C for 10 min. After cooling,
all 10 reaction mixtures were combined and poured into 2 M
NH4OH (250 mL). The product was subsequently extracted
into EtOAc. The combined organic phases were washed with
water and brine, dried with Na2SO4, and evaporated under
reduced pressure. The crude product was purified on a silica
column with DCM/isohexane (1:1) as the mobile phase to give
compound 10 (1.57 g, 85%). Anal. (C21H25NO3Si) C, H, N.
Conclusion
Herein, we described straightforward synthetic routes
to the two small aromatic scaffolds F and G, both of
which proved to be useful peptidomimetic core struc-
tures. Molecular modeling supports the view that 1,3,5-
trisubstituted aromatic scaffolds may serve as relevant
γ-turn mimetics. The scaffolds F and G were used as
substitutes for the tripeptide Val-Tyr-Ile or for the
dipeptides Tyr-Ile and Val-Tyr in the proposed turn in
order to find the optimal position of the scaffolds for this
class of compounds. All five pseudopeptides exhibit
nanomolar affinities for the AT2 receptor, confirming
that considerable differences in the central part of Ang
II are tolerated at this receptor. In contrast, minor
changes have a great impact on the AT1 receptor affinity
and thereby the selectivity. Functional assays estab-
lished that the nonselective AT1/AT2 receptor ligand 5
is a full agonist at the AT2 receptor but lacks agonistic
activity at the AT1 receptor. Molecular modeling con-
firms that the model based on only the confirmed
agonists is very similar to the one with all six high-
affinity ligands included. Thus, a tentative model is
proposed for activation of the AT2 receptor by angio-
tensin II analogues.
Experimental Section
General. 1H NMR and 13C NMR were obtained on a JEOL
JNM-400 spectrometer (1H at 400 MHz, 13C at 100.6 MHz) or
on a Varian Mercury plus spectrometer (1H at 400 MHz, 13C
at 100.6 MHz). Spectra were recorded at ambient temperature
unless otherwise stated. Chemical shifts are reported as δ
values (ppm) referenced to δ 7.26 ppm for CHCl3, δ 77.0 ppm
for CDCl3, δ 2.05 ppm and δ 29.84 ppm for acetone-d6, and δ
1.94 and 1.32 ppm for MeCN-d3. NMR spectra for the
pseudopeptides were recorded on a Varian UNITY INOVA (1H
at 499.9 MHz) spectrometer. Signal assignment was made
using PE COSY,38 TOCSY,39 and ROESY40 experiments, using
solvent suppression with the wet41 technique. A mixing time
of 0.4 s was used for the ROESY experiments. IR spectra were
generated on a Fourier transform IR spectrometer. GC-MS
was performed with an electron impact (70 eV) mass-selective
detector and a HP-1 capillary column using a 70-305 °C
temperature gradient. Analytical RP-LC-MS was performed
on a Gilson HPLC system with a Finnigan AQA quadropole
mass spectrometer using a Chromolith Performance RP-18 100
mm × 4.6 mm column (Merck) at a flow rate of 4 mL/min.
Preparative RP-HPLC was performed with a Zorbax SB-C8,
5 µm, 150 mm × 21.2 mm column (Agilent Technologies) at a
flow rate of 12 mL/min. The pseudopeptide purities were
determined on a Thermo Hypersil HyPurity C4, 5 µm, 4.6 mm
× 50 mm column and a ACE 5 C18, 4.6 mm × 50 mm column.
MikroKemi AB, Uppsala, Sweden, or Analytische Laborato-
rien, Industriepark Kaiserau, Lindlar, Germany, performed
elemental analyses. Molecular masses (HRMS) were deter-
mined on a Micromass Q-Tof2 mass spectrometer equipped
with an electrospray ion source. The microwave heating was
conducted in a Smith synthesizer system at a frequency of
2450 MHz under noninert conditions. Column chromatography
was performed using commercially available silica gel 60
(particle size: 0.040-0.063 nm, Merck). Thin-layer chroma-
tography performed with aluminum sheets coated with silica
gel 60 F254 (0.2 mm, E. Merck), and the analytes were
visualized with UV light. Dichloromethane used in the reac-
(2-Trimethylsilanylethyl)-3-[(9H-fluoren-9-ylmethoxy-
carbonylamino)methyl]-5-(4-methoxybenzyl)benzoate
(11). A round-bottom flask was charged with a stirring bar
and palladium on charcoal (10%, 404mg, 0.38 mmol). A
solution of 10 (1.4 g, 3.8 mmol) in absolute ethanol (70 mL)
was added, and the flask was sealed with a septum and
purged, first with nitrogen and then with hydrogen gas. The
reaction mixture was stirred under hydrogen at atmospheric
pressure for 80 min and was then filtered and evaporated. The
crude product was analyzed by GC-MS and found to be
adequate for further reaction. The generated amine (3.8 mmol)
was dissolved in dioxane (50 mL) and cooled in an ice bath. A
10% aqueous solution of Na2CO3 (100 mL) was added slowly.
Fmoc chloride (1.47 g, 5.7 mmol), dissolved in dioxane (50 mL),
was added dropwise to the reaction mixture, which was

Angiotensin II Pseudopeptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6627
allowed to reach ambient temperature under stirring for 4 h.
Water (100 mL) was added, and the mixture was extracted
with ethyl acetate. The organic phase was washed with water
and brine, dried over MgSO4, and evaporated. The product was
purified on a silica column using 5% ethyl acetate in toluene.
Compound 11 was isolated as a clear oil (1.17 g, 52%). Anal.
(C36H39NO5Si) H, N. C: calcd, 72.82; found, 73.6.
3-[(9H-Fluoren-9-ylmethoxycarbonylamino)methyl]-5-
(4-hydroxybenzyl)benzoic Acid (12). Compound 11 (591
mg, 1.0 mmol) was dissolved in dry DCM (15 mL). The reaction
vessel was fitted with a septum and cooled in an ice bath. BF3‚
S(CH3)2 (1.1 mL, 10.0 mmol) was added slowly to the solution.
The reaction mixture was allowed to reach ambient temper-
ature and was left stirring overnight. It was then poured into
water and extracted with ethyl acetate. The organic phase was
extracted with 1 M K2CO3. The aqueous phase was acidified
with 6 M HCl and extracted with ethyl acetate. The organic
phase was then dried over MgSO4 and evaporated to give 12
(376 mg, 78.4 mmol) as a clear oil in 78% yield. Anal. (C30H25-
NO5) C, H, N.
phases were pooled, washed with water and brine, dried with
MgSO4, and evaporated. The product was purified by silica
column chromatography using ethyl acetate/isohexane/formic
acid (49:50:1) and was eluated from the column with methanol.
The eluate was evaporated and then distributed between ethyl
acetate and water. The organic phase was dried and evapo-
rated to yield 14 (204 mg, 49%). Anal. (C30H25NO5‚1/3H2O) C,
N. H: calcd, 5.33; found 4.8.
3-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-(4-hy-
droxybenzyl)benzoic Acid (15). Compound 15 was prepared
from 14 (500 mg, 1.04 mmol) according to the procedure
outlined for compound 12 except for the workup and purifica-
tion, which were modified. The reaction mixture was poured
into 1 M HCl (100 mL) and extracted into ethyl acetate. The
organic phase was washed with NaHCO3, water, and brine,
dried with Na2SO4, and evaporated. The product was purified
by RP-HPLC using a 40 min gradient of 40-85% CH3CN in
0.05% aqueous formic acid and gave, after freeze-drying, 15
as a white solid (288 mg, 59%). Anal. (C29H23NO5‚H2O) C, H,
N.
General Synthesis of Angiotensin Analogues. Coup-
ling. Fmoc-His(Trt)-Pro-Phe-Wang resin (1 equiv) 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, 12 or 15 (1.25 equiv), and PyBOP (1.25
equiv) were dissolved in DMF (1.5 mL) in the presence of DIEA
(2.5 equiv) and allowed to react with the resin overnight (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) before it was dried in a vacuum overnight.
An aliquot of the resin was swelled in DMF (2 mL). The
appropriate acid (5 equiv) was dissolved with PyBOP (5 equiv)
and DIEA (10 equiv) in DMF (0.5 mL). The solution was added
to the resin and agitated by rotation overnight. In all cases,
LC-MS analysis of a cleaved sample showed complete reac-
tion. The resin was washed, and the Fmoc group was cleaved
as described above. Coupling of additional amino acids was
conducted as described above, but the reaction time was
shortened to 2 h. The resin was deprotected, washed, and dried
as above.
3-(4-Methoxybenzoyl)-5-nitromethylbenzoate (7b). 5-Ni-
troisophthalic acid monomethyl ester (3.0g, 13.3 mmol) was
refluxed in 20 mL of thionyl chloride for 1 h. The thionyl
chloride was evaporated under reduced pressure and kept
under vacuum overnight. The crude product was dissolved in
25 mL of dry DCM. AlCl3 (5.32 g, 39.9 mmol) was suspended
in 15 mL of DCM and cooled to 0 °C. The solution of the acid
chloride was added slowly to the suspension, and the reaction
mixture was stirred at 0 °C for 30 min. Anisole (1.9 mL, 17.5
mmol) was added dropwise, and the temperature was raised
to room temperature. After 4 h another portion of anisole (2.45
mL, 22.5 mmol) was added, and the mixture was stirred for 5
days. The reaction mixture was poured into a mixture of 100
mL of ice and 150 mL of 1 M HCl and extracted with DCM.
The organic phase was washed with saturated NaHCO3
solution, water, and brine, dried over MgSO4, and evaporated.
The product was purified with column chromatography (mobile
phase: isohexane 15% in DCM). The para product, 7b (1.78
g, 42%), was isolated as a yellow oil. Anal. (C16H13NO6) C, H,
N.
3-(4-Methoxybenzyl)-5-nitromethylbenzoate (8b). Start-
ing from compound 7b (2.37 g, 7.53 mmol), compound 8b was
prepared as described above for 8a except that the reaction
mixture was stirred at room temperature overnight instead
of being refluxed for 6 h. The product 8b was isolated as an
off-white solid (1.62 g, 71%). Anal. (C16H15NO5) C, H, N.
Cleavage. To cleave the peptide from the resin, triethylsi-
lane (100 µL) 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 evapo-
rated 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.
Purification. The crude peptide, unless otherwise stated,
was dissolved in MeCN (0.4 mL) and H2O (3.6 mL) and purified
in two runs by RP-HPLC on a 10 µm Vydac C18 column (2.2
cm × 25 cm) with a 90 min gradient of 15-45% MeCN in 0.1%
aqueous TFA at a flow rate of 5 mL/min. The separation was
monitored at 230 nm by analytical RP-HPLC and/or LC-MS
of selected fractions.
3-(4-Methoxybenzyl)-5-nitrobenzoic Acid (13). A solu-
tion of 8b (1.62 g, 5.37 mmol) was prepared in THF (95 mL),
H2O (27 mL), and MeOH (13.5 mL). A sample of 1 M LiOH
(27 mL) was added slowly. The reaction mixture was left
stirring at room temperature overnight, then concentrated,
diluted with water, and acidified with 6 M HCl. The aqueous
solution was extracted with ethyl acetate. The organic phase
was washed with water and brine, dried with MgSO4, and
evaporated. Product 13 was isolated as a clear oil (1.38 g, 89%).
Anal. (C15H13NO5) C, H, N.
3-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-(4-meth-
oxybenzyl)benzoic Acid (14). A round-bottom flask was
charged with a solution of 13 (250 mg, 0.87 mmol) in methanol
(15 mL). Palladium on charcoal (10%, 90 mg, 0.087 mmol) was
added to the flask, which was sealed with a septum and purged
with nitrogen and then with hydrogen gas. The reaction
mixture was stirred under hydrogen at atmospheric pressure
for 60 min and was then filtered and evaporated. The raw
product was analyzed by LC-MS and used without further
purification. The generated aniline (0.87 mmol) was dissolved
in dioxane (10 mL) and cooled in an ice bath. An aqueous
solution of Na2CO3 (0.3 M, 10 mL) was added slowly. Fmoc
chloride (248 mg, 0.96 mmol) was dissolved in dioxane (10 mL)
and added dropwise to the reaction mixture, which was
allowed to attain ambient temperature under stirring for 40
h. The reaction mixture was acidified slowly with 6 M HCl
and partitioned between water and ethyl acetate. The water
phase was extracted further with ethyl acetate. All organic
Analogues 1 and 2. The synthesis of these compounds has
been described earlier.27
Analogue 3. An aliquot (53 mg, 28 µmol) of the resin
obtained after introduction of template 12 was coupled with
Fmoc-Val-OH (140 µmol) and deprotected according to the
general procedure. The peptide chain was further elongated
by successive couplings of Fmoc-Arg(Pbf)-OH and Fmoc-Asp-
(Ot-Bu)-OH in the same manner but using reaction times of
2.5 h. After removal of the Fmoc group and after being washed
and dried, the product was cleaved from the resin by treatment
with triethylsilane and 95% aqueous TFA as described above.
The crude product, dissolved in 2.2 mL of 10% MeCN, was
purified by RP-HPLC on a 5 µm ACE phenyl column (21.2 mm

6628 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Georgsson et al.
× 150 mm) using a 60 min gradient of 10-40% MeCN in 0.1%
aqueous TFA and a flow rate of 5 mL/min. Yield: 6.6 mg (17%).
LC-MS (Mabs ) 1008.48): 1009.3 (M + H+), 505.4 ([M + 2H+]/
2), 337.3 ([M + 3H+]/3), 350.9 ([M + MeCN + 3H+]/3). Amino
acid analysis: Asp, 0.99; Arg, 1.01; Val, 0.70; His, 0.98; Pro,
1.01; Phe, 0.98.
Analogue 4. After incorporation of scaffold 15, an aliquot
of the resin (75 mg, 40 µmol) was subjected to successive
couplings with Fmoc-Arg(Pbf)-OH (200 µmol) and Fmoc-Asp-
(Ot-Bu)-OH (200 µmol) as outlined above. The product was
cleaved and purified according to the standard procedures to
yield 14.9 mg (26%). LC-MS (Mabs ) 895.4): 448.9 ([M + 2H+]/
2). Amino acid analysis: Asp, 0.97; Arg, 0.99; His, 1.01; Pro,
1.03; Phe, 1.00.
Analogue 5. A second aliquot of the same resin (75 mg, 40
µmol) was coupled seqentially with Fmoc-Val-OH (200 µmol),
Fmoc-Arg(Pbf)-OH (200 µmol), and Fmoc-Asp(Ot-Bu)-OH (200
µmol) as described above. Standard cleavage and purification
yielded 6.9 mg (11%). LC-MS (Mabs ) 994.6): 498.6 ([M +
2H+]/2), 346.4 ([M + MeCN + 3H+]/3). Amino acid analysis:
Asp, 0.99; Arg, 0.99; Val, 0.98; His, 1.01; Pro, 1.02; Phe, 1.01.
Canada) with 10% fetal bovine serum (FBS, Gibco), HAT
supplement (hypoxanthine, aminopterin, thymidine from Gib-
co), 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.44 Subcultures were
formed at subconfluency. Under these conditions, cells express
only the AT2 receptor subtype.44,45 Cells were stimulated for 3
days, once a day (first stimulation 24 h after plating). Cells
were cultured for 3 subsequent days under these conditions.
For all experiments, cells were plated at the same initial
density of 6 × 104 cells /35 mm Petri dish. Cells were treated
without (control cells) and with [Val5]angiotensin II from
Bachem (Marina Delphen, CA) (100 nM) or 5 (100 nM) and in
the absence or in the presence of PD 123,319 (RBI Natick, MA)
(1 µM), an AT2 receptor antagonist introduced daily 30 min
prior to Ang II or 5.
Determination of Cells with Neurites. Cells were ex-
amined daily under a phase contrast microscope, and micro-
graphs 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
140 cells were counted in three independent experiments.9
Rat Liver Membrane AT1 Receptor Binding Assay. Rat
liver membranes were prepared according to the method of
Dudley et al.42 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 to 1.0 µM) of test substance. Samples were incubated at
25 °C for 2 h, and binding was terminated by filtration through
Whatman GF/B glass-fiber filter sheets using a Brandel cell
harvester. The filters were washed with 3 × 3 mL of Tris-HCl
(pH 7.4) and transferred to tubes. The radioactivity was
measured with a γ-counter. Nonspecific binding was deter-
mined 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 Scatchard
analysis of data obtained with Ang II by using GraFit
(Erithacus Software, U.K.). The apparent dissociation con-
stants Ki were calculated from IC50 values using the Cheng-
Prusoff equation54 (Kd ) 1.7 ( 0.1 nM, [L] ) 0.057 nM). The
binding data were best fitted with a one-site fit. All experi-
ments 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.43 Potential
interference by binding to AT1 receptors was blocked by
addition of 1 µM losartan. Binding of [125I]Ang II to membranes
was conducted in a final volume of 0.5 mL containing 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 to 1.0 µM) of test
substance. Samples were incubated at 25 °C for 1.5 h, and
binding was terminated by filtration through Whatman GF/B
glass-fiber filter sheets using a Brandel cell harvester. The
filters were washed with 3 × 3 mL of Tris-HCl (pH 7.4) and
transferred to tubes. The radioactivity was measured with 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 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 equation54 (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.
Vascular Contractility Studies. This study was per-
formed at Cerep (France) according to literature.46 Seven
concentrations (3, 10, 30, 100, 300, 1000, 3000 nM) of 5 were
tested. Ang II was used as a reference agonist, and saralasin
(0.03 µM) was used as a reference antagonist.
Conformational Analysis and Molecular Modeling.
General Protocol for Conformational Analysis. The con-
formational search was conducted using the systematic un-
bound multiple minimum (SUMM) search method55 in the
BatchMin program. The OPLS-AA force field and the general
Born solvent accessible (GB/SA) surface area method for water
developed by Still et al.,56 as implemented in the program
MacroModel 8.5,57 were used in the calculations. The number
of torsion angles allowed to vary simultaneously during each
Monte Carlo step ranged from 1 to n - 1. Amide bonds were
fixed in the trans configuration. Truncated Newton conjugate
gradient (TNCG) minimization with a maximum of 500
iterations was used in the conformational search, with the
derivative convergence set to 0.05 kJ mol-1 Å-1. Torsional
memory was used. All heavy atoms were used in the compari-
son for unique conformations, if nothing else is stated.
Conformations within 5 kcal/mol of the lowest energy mini-
mum were kept. The obtained conformations were subse-
quently minimized with derivative convergence set to 0.001
kJ mol-1 Å-1 using TNCG minimization with a maximum of
500 iterations.
Comparison of Scaffolds to γ-Turns in Protein Crystal
Structures. Conformational analysis of compounds of F and
G modified with an N-terminal acetyl cap, C-terminal N-
methylamide cap, and an alanine side chain instead of the
tyrosine side chain was performed according to the general
protocol above with 1000 search steps per rotatable bond. The
conformations identified within 5 kcal/mol of the lowest found
minimum of the model compound of F (eight conformations)
and G (four conformations) were compared to 2101 inverse
γ-turns (defined as ideal angles (30°), extracted from a
representative set of crystallized proteins selected using the
PDB_SELECT list58,59 from December 2003. The comparison
was performed by least-squares alignment of CRi-1, CRi+1, Câi+1
,
and CRi+3 in the protein turns and the corresponding atoms in
the model compounds.
DISCOtech Analysis. The conformational search of struc-
tures 1m, 3m, 5m, 16m, 17m, and 18m was performed
according to the general protocol above. The number of steps
was set to 2000 per rotatable bond. The low-energy inverse
γ-turn-like conformation was used for the ring system of the
benzodiazepine-based scaffolds and was not searched. To
reduce the number of identified conformations, all conforma-
tions with a distance of 4 Å or less between the carbon in the
guanidino group and any oxygen atom were removed because
there is support for the interaction of the Arg2 side chain with
Asp297 in the AT2 receptor.60 The criterion for unique confor-
In Vitro Morphological Effects. NG108-15 cells (provided
by Drs. M. Emerit and M. Hamon; INSERM, U. 238, Paris,
France) were cultured (passage 18-28) in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco BRL, Burlington, Ontario,

Angiotensin II Pseudopeptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6629
mations was also changed from the default maximum distance
of 0.25 Å to 1.0 Å between the compared atoms after super-
position (CRMS). The compared atoms were (1) the methyl
carbon of the N-terminal cap, (2) the arginine CR, (3) the carbon
in the guanidino group, (4) the tyrosine CR (the aromatic
carbon in 1m, 3m and 5m), (5) the tyrosine phenolic oxygen
atom, and (6) the methyl carbon of the C-terminal cap. The
number of conformations found within 5 kcal/mol of the energy
minimum for 1m, 3m, 5m, 16m, 17m, and 18m were 590,
1159, 2865, 1963, 893, and 772, respectively. Owing to the limit
in the number of conformations that DISCOtech48 can handle
(e300 conformations per molecule), only conformations of a
compound that could superimpose selected atoms with a low
rms atomic distance to the conformations of the other com-
pounds were used. The superimposed atoms were the same
as the atoms used for comparison in the conformational
analysis. In our previous DISCOtech analysis,24 the conforma-
tions selected and used were based on conformational diver-
sity. In the present study, intermolecular conformational
similarity was used to select conformations for the DISCOtech
analysis. We believe that this approach of selecting conforma-
tions makes it easier to obtain good alignments in DISCOtech.
One-hundred-thirty-four conformations of 3m that all could
be superimposed with an rms atomic pair distance below 1.5
Å onto any conformation of 17m and 18m were used as
reference conformations. These conformations were aligned to
all the conformations of the other compounds to guide the
selection of conformations to include in DISCOtech. For 18m,
360 conformations were superimposed with an rms distance
below 1.5 Å onto the reference set. Of these, the 300 conforma-
tions with the lowest energy were selected. For 5m, 807
conformations matched but were reduced to 265 by decreasing
the rms distance criterion to 1.1 Å. Owing to a very low number
of matched conformations of 16m, the rms distance criterion
was set to 2.2 Å for this compound, giving 100 conformations.
The number of conformations that matched the reference set
for 1m (159) and 17m (268) was within the DISCOtech limit
using the 1.5 Å criterion.
The DISCOtech module in SYBYL,version 6.9,61 was used
to find an alignment of selected structural elements. The
selected structural elements were (1) the methyl carbon of the
N-terminal cap (corresponding to CR in the aspartic acid in
the full-length pseudopeptide), (2) the receptor acceptor sites
originating from the guanidino group (as defined by DIS-
COtech), (3) the tyrosine phenolic oxygen atom, and (4) the
methyl carbon of the C-terminal cap (corresponding to CR in
the histidine in the full-length pseudopeptide). Different
DISCOtech features were created for the tyrosine phenolic
oxygen atom and the capped ends by transforming relevant
atoms into DISCOtech recognizable features. The hydrogen
atoms of the acetyl capping group were transformed into
carbons, thus creating a hydrophobic tert-butyl feature. The
methyl group of the C-terminal cap was transformed into a
quaternary nitrogen group, thus creating a positively charged
feature. The nitrogen atoms in the guanidino group were
transformed into oxygen atoms, thus creating a unique feature
from where only DISCOtech acceptor site features can be
obtained. The tyrosine phenolic oxygen atom was transformed
into a quaternary nitrogen atom and defined as a unique
feature, thus removing the potential DISCOtech acceptor site
feature. To simplify the procedure, all atoms not included in
a feature were transformed into hydrogen atoms. In the
transformations only the atom types were changed, not the
geometry. The DISCOtech setting “features by class” was used
to search for models containing (1) more than zero acceptor
sites (receptor points corresponding to the guanidino group),
(2) one hydrophobic feature (the transformed feature corre-
sponding to the CR of the aspartic acid residue in the full-length
pseudopeptides), (3) one tyrosine phenolic oxygen atom, and
(4) one positively charged nitrogen atom (the transformed
feature corresponding to CR of the histidine residue). The
tolerance for a feature match between the molecules was set
from 0.25 to 2.5 Å in increments of 0.25 Å if no models were
found at the lower tolerance. DISCOtech was set to obtain
models where all or all but one compound were included. 17m,
corresponding to the pseudopeptide with the highest affinity,
was used as the reference molecule.
The DISCOtech analysis with only the AT2 receptor agonist
model compounds 5m, 16m, 17m, and 18m included was made
using the same DISCOtech protocol. The reference set for
selecting similar conformations among the different com-
pounds consisted of the conformations of 17m that could be
superimposed with an rms atomic pair distance below 1.5 Å
onto any conformation of 5m and 18m and below 2.2 Å onto
any conformations of 16m. Included conformations of 5m were
those that could be superimposed onto the reference set with
an rms atomic pair distance below 1.25 Å. For 16m and 18m
the rms distance criterion was set to 2.2 and 1.5 Å, respec-
tively.
Acknowledgment. We gratefully acknowledge the
financial support from the Swedish Research Council
and the Swedish Foundation for Strategic Research. We
also thank Biotage AB for providing the Smith micro-
wave synthesizer.
Supporting Information Available: 1H and 13C NMR
spectral data of compounds 7a,b, 8a,b, and 9-15, MS, LC-
MS, and IR data, elemental analysis results of all new
compounds, purity determination and HRMS data for the
pseudopeptides 1-5, and 1H NMR data for 3 and 4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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