Design, synthesis, and incorporation of a β-turn mimetic in angiotensin II forming novel pseudopeptides with affinity for AT1 and AT2 receptors

Article abstract of DOI:10.1021/jm051222g

A benzodiazepine-based β-turn mimetic has been designed, synthesized, and incorporated into angiotensin II. Comparison of the mimetic with β-turns in crystallized proteins showed that it most closely resembles a type II β-turn. The compounds exhibited hig

Full text of DOI:10.1021/jm051222g

J. Med. Chem. 2006, 49, 6133-6137
6133
Design, Synthesis, and Incorporation of a â-Turn Mimetic in Angiotensin II Forming Novel
Pseudopeptides with Affinity for AT1 and AT2 Receptors
Ulrika Rosenstro¨m,† Christian Sko¨ld,† Gunnar Lindeberg,† Milad Botros,‡ Fred Nyberg,‡ Anders Karle´n,† and Anders Hallberg*,†
Department of Medicinal Chemistry, BMC, Uppsala UniVersity, P.O. Box 574, SE-751 23 Uppsala, Sweden, and Department of Biological
Research on Drug Dependence, BMC, Uppsala UniVersity, P.O. Box 591, SE-751 24 Uppsala, Sweden
ReceiVed December 6, 2005
A benzodiazepine-based â-turn mimetic has been designed, synthesized, and incorporated into angiotensin
II. Comparison of the mimetic with â-turns in crystallized proteins showed that it most closely resembles
a type II â-turn. The compounds exhibited high to moderate binding affinity for the AT2 receptor, and one
also displayed high affinity for the AT1 receptor. Molecular modeling showed that the high-affinity compounds
could be incorporated into a previously derived model of AT2 receptor ligands.
Introduction
The potent vasoactive octapeptide hormone angiotensin II
(Ang II) acts through the AT1 and the AT2 receptors, both of
which belong to the G-protein-coupled receptor superfamily.
The AT1 receptor mediates most of the known actions of Ang
II, such as vasoconstriction, aldosterone release, and sodium
and water retention.1-3 Recently it has been established that
the AT2 receptor has a physiologic role in the brain and in
cardiovascular and renal functions in adults, as well as in the
modulation of various processes associated with cell differentia-
tion and tissue repair.2 In some cells, AT2 receptor activation
inhibits proliferation and also mediates differentiation in neural
cell lines.4 Often the effects of AT2 receptor activation oppose
those mediated by the AT1 receptor, and it is therefore not
surprising that the AT2 receptor, in recent years, has attracted
interest as a new target for potential therapeutics.
Despite only 32-34% sequence homology,5,6 AT1 and AT2
receptors bind Ang II with high affinity. As deduced from
conformational analyses of conformationally constrained ana-
logues, there is supporting evidence that Ang II adopts a turn
centered at Tyr4 when activating the AT1 receptor.7-16 While
less research has been devoted to molecular recognition involv-
ing the interaction of Ang II with the AT2 receptor, binding
data and conformational analyses of linear and cyclic Ang II
analogues suggest that the AT1 and AT2 receptors accommodate
Ang II differently.9,17-28 We have previously prepared a series
of scaffolds,10,26-28 for examples see Figure 1, designed to mimic
γ-turn secondary structure motifs. Of the pseudopeptides
obtained by incorporation of these motifs into Ang II to
substitute the backbone centered around Tyr4, only that with
scaffold A exhibited high affinity for the AT1 receptor and acted
as an AT1 agonist, inducing contraction of blood vessels in
vitro.10 In contrast, several of the scaffolds were tolerated with
respect to AT2 receptor recognition.26,27 It was proposed that
the turn region around Tyr4, the guanidino group of the Arg2
residue, the N-terminal end, and their relative orientations in
space are critical for favorable interaction with the AT2
receptor.26,27
Figure 1. Some γ-turn mimetics (A-C) previously incorporated in
Ang II.10,26,28
scaffolds mimicking â-turns have been reported in recent
years.30-33 We felt encouraged to elaborate on the benzodi-
azepine structure and to synthesize â-turn mimetics with the
benzodiazepine core as template.
We herein report the synthesis and preliminary pharmacologi-
cal evaluation of Ang II analogues 1-3 (Chart 1) comprising a
benzodiazepine-based type II â-turn mimetic.
Results
Chemistry. The benzodiazepine-based Fmoc-protected â-turn
mimetic 18 was synthesized in solution and incorporated into
Ang II using solid-phase methodology to deliver the pseudopep-
tides 1-3. The bicyclic core structure was utilized as a turn
template to replace the peptide fragments Val3-Tyr4-Ile5-His6
(1), Val3-Tyr4-Ile5 (2), and Tyr4-Ile5 (3) in Ang II.
The synthesis started with a benzylic oxidation of 2-fluoro-
m-xylene to give the diacid 5 (Scheme 1). The diacid was
transformed into the monoester 6 in one step or via the diester.
Nitration of the monoester 6 gave 7, which was transformed to
the acid chloride and further reacted in a Friedel-Crafts
acylation. The substituted benzophenone was obtained as a 2:5
mixture of the ortho- and para-substituted isomers.
The substituted benzophenone 8a was first reduced to the
diphenylmethane derivative 9 to decrease the potential for the
fluorine ipso-carbon to undergo nucleophilic aromatic substitu-
tion (Scheme 2). In the next step, the methyl ester was reduced34
to the alcohol, and Swern oxidation was employed to reoxidize
the alcohol 10 to the aldehyde. Reductive amination of 11 with
2-aminoethanol produced a primary alcohol that was protected
with a tert-butyldimethylsilyl group (TBDMS). Fmoc-L-Ile-OH
was coupled with the secondary amine 12, and the Fmoc group
was removed. Subsequent internal nucleophilic aromatic sub-
stitution delivered the benzodiazepine core structure as a 1:3
mixture of the TBDMS-protected form and the free alcohol 14.
â-Turn secondary structures are much more common than
γ-turns in proteins, as shown by Rose et al.,29 and several
* To whom correspondence should be addressed. Phone: +46-18-
4714284. Fax: +46-18-4714474. E-mail: anders.hallberg@orgfarm.uu.se.
† Department of Medicinal Chemistry.
‡ Department of Biological Research on Drug Dependence.
10.1021/jm051222g CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/01/2006

6134 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Brief Articles
(PDB).35,36 A complete atom-to-atom-based comparison between
the â-turn mimetic and the protein â-turns was not possible
because there is no exact match between the first two amino
acid residues in the turn and the mimetic. For the compari-
son, we choose to include the CR and Câ atoms of the i + 1
and i + 2 residues in the protein â-turns to represent the
direction of the side chains. To allow for a fit of the peptide
backbone, the CR atoms of i - 1, i + 3, and i + 4 and the
carbonyl oxygen atoms of i - 1, i + 2, and i + 3 were also
included. Atoms in the i residue were not considered because
of the lack of correspondence in the turn mimetic. The
corresponding atoms in model compound 19 used for compari-
son are shown in Figure 2.
Chart 1
The investigated conformations were superimposed with the
lowest root-mean-square (rms) atomic pair distances (0.75 Å
cutoff) on type II and type IV â-turns. One example of 19
superimposed on a type II â-turn is shown in Figure 3. By
increase of the rms distance criterion to include superimpositions
up to 1 Å, other turns, such as types I, II′, and VIII, could also
fit the mimetic.
(B) Common Binding Mode for Ligands at the AT2
Receptor. Conformational analysis of model structures of 2 and
3 resulted in 2320 and 1562 conformations, respectively. When
these conformations were superimposed on a previously derived
AT2 receptor binding model,28 conformations with a common
binding mode could be identified (Figure 4).
In Vitro Binding Affinity. Ang II analogues 1-3 were
evaluated in radioligand binding assays using displacement of
[
125I]Ang II from AT1 receptors in rat liver membranes37 and
from AT2 receptors in pig uterus membranes38 (Table 1). The
natural ligand Ang II and the highly AT2 selective analogue
[4-NH2-Phe6]Ang II22 were used as reference substances.
Compound 1, in which the â-turn mimetic is substituted for
the Val3-Tyr4-Ile5-His6 segment, has moderate binding affinity
for the AT2 receptor and only weak binding affinity for the AT1
receptor. In 2, which contains the His6 residue, the AT2 receptor
binding affinity was increased, while the AT1 receptor affinity
remained weak. Compound 3, which contains both Val3 and
His6, exhibited considerable binding affinity for the AT1 and
AT2 receptors.
Scheme 1a
Rabbit Thoracic Aorta String. AT1 Functional Assay.
Compound 3, which has AT1 and AT2 receptor affinity, was
also evaluated for agonistic activity at the AT1 receptor in
thoracic aorta, according to a procedure published by Pendleton
et al.39 The agonist Ang II that was used as a reference
compound induced a concentration-dependent contraction of the
rabbit thoracic aorta, which was inhibited by the AT1 receptor
antagonist saralasin. Compound 3 was tested at seven concen-
trations from 3.0 nM to 3.0 µM but did not produce any
contraction of this tissue.
a Reagents: (a) KMnO4, KOH, H2O, 58%; (b) SOCl2; (c) MeOH, 99%;
(d) LiOH, THF/MeOH/H2O, 47%; (e) MeOH, H2SO4, 40%; (f) HNO3,
H2SO4, 97%; (g) SOCl2; (h) AlCl3, anisole, CH2Cl2, 37% (8a), 15% (8b).
Treatment of the product mixture with tetrabutylammonium
fluoride (TBAF) gave benzodiazepine 14.
Mild conditions were required for the conversion of the
alcohol function in 14 to a carboxylic acid.26 The protecting
group on the phenolic hydroxy group in 15 was changed to a
Boc group to facilitate the solid-phase peptide synthesis. Finally,
the nitro group in 17 was reduced and the aniline nitrogen was
protected with FmocCl. Standard Fmoc/t-Bu SPPS methodology
was used to incorporate the protected benzodiazepine-based turn
mimetic 18 into Ang II.
Molecular Modeling. (A) Comparison of the â-Turn
Mimetic Moiety and Protein â-Turns. Conformational analysis
was performed on model compound 19 (Figure 2). This resulted
in 30 low-energy conformations (within 5 kcal/mol of the lowest
energy minimum). The five conformations within 1 kcal/mol
of the lowest energy conformation were evaluated by comparing
them with an in-house database of 13 784 extracted â-turns from
protein X-ray structures obtained from the Protein Data Bank
Discussion
It has previously been shown that several cyclic analogues
of Ang II have good AT1 and AT2 receptor affinity.9,23,40
Furthermore, a number of Ang II analogues containing a
benzodiazepine-based γ-turn mimetic or a thiazabicycloalkane
dipeptide mimetic have shown considerable binding affinity for
the AT2 receptor and high AT2/AT1 selectivity.24,26,27 Also, an
Ang II pseudopeptide containing a small aromatic ring as a
γ-turn mimicking moiety was recently found to have high
affinity for the AT1 and AT2 receptors. Although several γ-turn
mimetics have been introduced in Ang II,10,14,26-28 â-turn
mimetics have rarely been utilized, even though it has been
suggested that Ang II adopts a â-turn around Tyr4 during

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6135
Scheme 2a
a Reagents: (a) Et3SiH, CF3SO2H, TFA, CH2Cl2, 98%; (b) NaBH4, H2O, dioxane, 65%; (c) ClCOCOCl, DMSO, Et3N, CH2Cl2, 100%; (d) 2-aminoethanol,
NaCNBH3, HOAc, MeOH; (e) TBDMSCl, DBU, THF, 71%; (f) Fmoc-L-Ile-OH, HATU, DIEA, CH2Cl2; (g) DBU, THF, 92%; (h) Et3N, DMSO; (i) TBAF,
THF, 90%; (j) ClCOCOCl, DMSO, Et3N, CH2Cl2; (k) NaClO2, NaH2PO4, cyclohexene, t-BuOH, H2O, 70%; (l) BF3‚Me2S, CH2Cl2, 78%; (m) Boc2O, Et3N,
DMAP, THF/H2O, 74%; (n) H2, Pd/C, MeOH; (o) FmocCl, Na2CO3 (aq), dioxane, 32%.
Figure 2. A â-turn (left) and a model compound of the investigated
â-turn mimetic (19) (right). Atoms used in the â-turn mimetic for
superimposing onto crystallized â-turns are marked by an asterisk.
Figure 4. Conformations of the model structures of 2 (green carbons)
and 3 (orange carbons) together with two of the compounds from the
previously derived AT2 receptor binding model (gray carbons).
Table 1. Binding Affinities for the AT1 and AT2 Receptors
Ki ( SEM (nM)
AT1
AT2
compd
(rat liver membranes) (pig uterus myometrium)
Figure 3. Minimized conformation of the synthesized benzodiazepine-
based â-turn mimetic superimposed onto a type II â-turn (rms distance
) 0.67 Å). The PDB code for the protein is 1H2C (chain A, turn region
Gly141-Pro146). All torsion angles are within (20° from the ideal torsion
angles of a type II â-turn. For clarity, only the backbone and Câ atoms
are shown.
Ang II
0.1
0.6
[4-NH2Phe6]Ang II
>10000
2108 ( 33
1668 ( 20
14.9 ( 0.4
0.7
1
2
3
53.1 ( 1.7
4.7 ( 0.3
1.8 ( 0.04
receptor interaction.13,16 Two locations for a â-turn centered at
Tyr4 are possible in Ang II: the 2-5 and the 3-6 regions. We
decided to synthesize a â-turn mimetic with a tyrosine side chain
in the i + 1 position and an isoleucine side chain in the i + 2
position and introduce this turn mimetic into the 3-6 region of
Ang II.
A â-turn is most often defined as any tetrapeptide unit
occurring in a nonhelical region that causes a reversal of the
direction of the peptide chain (Figure 2).29 Further, the distance
between CR of the first (i) residue and the fourth (i + 3) residue
in the peptide sequence should be less than 7 Å.41 â-Turns
comprise a rather diverse group of structures, and they have
therefore been divided into a number of turn types (I, I′, II, II′,
IV, VIa, VIb, VIII) according to the backbone dihedral angles
φ and ψ of the i + 1 and i + 2 residues of the turn.42 In addition,
a number of slightly different turn types have been presented
in the literature.29,41,43 To classify the turn type mimicked by
19, we focused on comparing the CR-Câ bond vectors of amino
acids i + 1 and i + 2 and the CR atoms that have corresponding
atoms in the turn mimetic (shown in Figure 2). The five
conformations of the â-turn mimetic with the lowest energy
(<1 kcal/mol) were compared with 13 784 â-turns extracted
from X-ray structures of proteins from the PDB.35,36 On the
basis of this comparison, the investigated â-turns with the best
fit to the mimetic were found to belong to the type II and the
type IV classes. Since â-turn mimetics are primarily designed
to mimic the classical turn types and since type IV turns
comprise a diverse collection of nondefined turns, these were
not considered further. Thus, of the classical â-turn types,
scaffold 19 seems to best mimic a type II â-turn.
The synthesized â-turn mimetic was introduced into Ang II,
and the three obtained pseudopeptides 1-3 showed binding

6136 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Brief Articles
affinity for the AT2 receptor. Compound 1, which lacks the
histidine side chain, displayed a moderate binding affinity for
the AT2 receptor (Ki ) 53.1 nM). It has previously been shown
that the binding affinity of Ang II for the AT2 receptor drops
by a factor of 10 when the histidine is replaced by an alanine17
or a glycine.25 In 2, the histidine residue was present and the
binding affinity for the AT2 receptor was increased 10-fold (Ki
) 4.7 nM). A slight gain in affinity was observed when an extra
valine residue was also present, 3 (Ki ) 1.8 nM).
Interestingly, 2 and 3 were capable of adopting binding modes
similar to those found in a previously identified model of
common binding modes of high-affinity AT2 receptor ligands
(Figure 4).28 In this model, all the compounds contained a
γ-turn-like mimetic scaffold in the same region as the investi-
gated â-turn mimetic in 2 and 3. Thus, both of these types of
turn mimetics seem to be able to direct the recognition motifs
in such a way that ligands with high affinity for the AT2 receptor
are obtained. However, although there are geometric similarities
between the mimetics, there are also visible differences. For
example, the isoleucine side chain in the â-turn mimetic does
not have any equivalent in the compounds containing a γ-turn
mimetic when superimposed as shown in Figure 4. Nonetheless,
despite this extra volume, the compounds seem to be tolerated
by the AT2 receptor.
Experimental Section
General Synthesis. The pseudopeptides were synthesized manu-
ally from Phe-2-chlorotrityl resin or His(Trt)-Pro-Phe-2-chlorotrityl
resin44 in 2 mL disposable syringes equipped with a porous
polyethylene filter. Standard Fmoc/t-Bu conditions were used, and
the Fmoc protecting group was removed by treatment with 20%
piperidine/DMF for 5 + 10 min. The resin was washed with DMF
after every coupling or deprotection step. The natural amino acids
were coupled using the appropriate amino acid (5 equiv) in the
presence of PyBOP (5 equiv) and DIEA (10 equiv) in DMF (0.5
mL). The turn templates (1.2 equiv) were reacted with the polymer
in the presence of PyBOP (1.2 equiv) and DIEA (3.2 equiv) in
DMF (0.5 mL). The coupling of the amino acid attached to the
turn templates was repeated using first the conditions described
above, and thereafter the resin was washed and recoupled with the
same amino acid (10 equiv) using HATU (5 equiv) and DIEA (10
equiv) in 0.5 mL of DMF. After the final coupling step the Fmoc
group was removed and the resin was washed with with DMF,
CH2Cl2, and MeOH and dried in vacuo.
Cleavage. The partially protected peptide resin was treated with
triethylsilane (50 µL) and 95% aqueous TFA (1.5 mL) for 1.5 h.
The polymer was removed by filtration and washed with TFA (2
× 0.3 mL). The combined filtrates were evaporated in a stream of
nitrogen to 1.5 mL, and the product was precipitated with diethyl
ether (12 mL). It was collected by centrifugation, washed with
diethyl ether, and dried in vacuo.
The benzodiazepine structure used in this study as a â-turn
mimetic has also previously been used as a γ-turn mimetic (C
in Figure 1). There is, however, a noticeable difference in the
side chains, since the tyrosine side chain was moved from the
benzodiazepine ring in the γ-turn mimetic to the aromatic ring
in the â-turn mimetic. Both of these turn mimetic scaffolds have
been incorporated in Ang II, replacing Val-Tyr-Ile. The
pseudopeptide incorporating scaffold C lacked affinity for the
AT2 receptor (Ki > 10,000 nM), while 2 had excellent affinity
(Ki ) 4.7 nM). This indicates that the relative arrangement of
the Arg and Tyr side chains is important for AT2 receptor
binding in the pseudopeptides, which has also been shown
previously.26
Purification. The crude material was dissolved in a mixture of
H2O (2 mL) and CH3CN (0.5 mL) and purified by RP-HPLC on a
5 µm ACE phenyl column (21.2 mm × 150 mm) using a 60 min
gradient of 15-45% MeCN in 0.1% aqueous TFA at a flow rate
of 5 mL/min. The separation was monitored by UV absorption at
220 nm and by LC/MS and/or analytical RP-HPLC of selected
fractions.
Ang II Analogue 1. Fmoc-Pro-OH was coupled to Phe-2-
chlorotrityl resin (27.9 mg, 23.7 µmol) according to the general
procedure for 3 h. The Fmoc group was removed, and the turn
template 18 was reacted with the polymer for 20 h. Fmoc-Arg-
(Pbf)-OH was coupled using PyBOP for 20 h and HATU for 4 h.
After deprotection the polymer was coupled with Fmoc-Asp(Ot-
Bu)-OH for 3 h. The yield of the deprotected, cleaved, purified,
and lyophilized peptide was 6.6 mg (26%). LC/MS (Mabs 912.45):
913.6 (M + H+), 457.5 ([M + 2H+]/2). Amino acid analysis: Asp,
1.00; Arg, 1.01; Pro, 0.88; Phe, 0.99.
Ang II Analogue 2. Compound 18 was coupled to His(Trt)-
Pro-Phe-2-chlorotrityl resin (60.0 mg, 36.0 µmol) (0.75 mL DMF)
as outlined above for 20 h. The Fmoc group was removed, and the
resin was washed with with DMF, CH2Cl2, and MeOH and dried
in vacuo. Half of the material (38.7 mg, 18.0 µmol) was transferred
to a second syringe and reacted with Fmoc-Arg(Pbf)-OH for 20 h.
The coupling was repeated using HATU for 4 h. Coupling of Fmoc-
Asp(Ot-Bu)-OH for 10 h followed by deprotection, washing,
cleavage, and purification produced the desired pseudopeptide in
8.5 mg (45%) yield. LC/MS (Mabs 1049.51): 1050.8 (M + H+),
526.0 ([M + 2H+]/2), 351.0 ([M + 3H+]/3). Amino acid analysis:
Asp, 1.00; Arg, 1.01; His, 0.97; Pro, 1.00; Phe, 1.02.
Ang II Analogue 3. Fmoc-Val-OH was coupled for 20 h with
the second half (38.7 mg, 18.0 µmol) polymer collected after
incorporation and deprotection of 18. The coupling was repeated
using HATU for 4 h. Successive couplings of Fmoc-Arg(Pbf)-OH
and Fmoc-Asp(Ot-Bu)-OH for 3 h produced the desired pseudopep-
tide. Deprotection, washing, cleavage, and purification gave the
product in 9.1 mg (44%) yield. LC/MS (Mabs 1148.58): 1149.9
(M + H+), 575.5 ([M + 2H+]/2). Amino acid analysis: Asp, 1.00;
Arg, 1.00; Val, 1.00; His, 0.96; Pro, 1.01; Phe, 1.02.
Both pseudopeptides 1 and 2 exhibited very weak binding
affinity for the AT1 receptor, Ki ) 2108 nM and Ki ) 1668
nM, respectively. Interestingly, 3, in which the Tyr4-Ile5 segment
has been replaced by the â-turn mimetic, showed good binding
affinity for the AT1 receptor (Ki ) 14.9 nM). The side chains
of residues 2, 4, 6, and 8 in Ang II are considered key elements
for binding to the AT1 receptor.18 In both 2 and 3, all these
side chains are present, but only 3 shows high binding affinity.
This may be because the distance between the arginine side
chain and the tyrosine side chain is more favorable for a correct
AT1 receptor interaction in 3, which is similar to the results
found for the AT2 receptor ligands (see above).
Conclusion
A â-turn mimetic based on the benzodiazepine core structure
has been synthesized and incorporated into Ang II. The â-turn
mimetic was shown to best mimic a type II â-turn when com-
pared with peptide â-turns in crystallized protein structures.
Two of the three resulting pseudopeptides exhibited high
AT2 receptor affinity. One also showed good AT1 receptor
affinity but displayed no agonistic effect in an AT1 receptor
functional assay. Inclusion of the two compounds with the
highest affinity in a previously published model of Ang II
pseudopeptides showed that, despite the incorporation of dif-
ferent turn mimetics, the pseudopeptides were capable of similar
binding modes.
Acknowledgment. We gratefully acknowledge support from
the Swedish Research Council and the Swedish Foundation for
Strategic Research. We also thank Dr. Ingemar Ander for fruitful
discussions.

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6137
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Supporting Information Available: Experimental procedures
including the synthesis of 5-18, biological assays, molecular
modeling, and 1H, 13C, and elemental analysis data of 6-18. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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