Synthesis of a new class of druglike angiotensin II C-terminal mimics with affinity for the AT2 receptor

Article abstract of DOI:10.1021/jm0613469

Four tripeptides corresponding to the C-terminal region of angiotensin II were synthesized. One of these peptides (Ac-His-Pro-Ile) showed moderate binding affinity for the AT₂ receptor. Two aromatic histidine-related scaffolds were synthesized and introduced in the tripeptides to give eight new peptidomimetic structures. Three of the new peptide-derived druglike molecules exhibited selective, nanomolar affinity for the AT₂ receptor. These ligands may become lead compounds in the future development of novel classes of selective AT₂ receptor agonists.

Full text of DOI:10.1021/jm0613469

J. Med. Chem. 2007, 50, 1711-1715
1711
Synthesis of a New Class of Druglike Angiotensin II C-Terminal Mimics with Affinity for the
AT2 Receptor
Jennie Georgsson,† Christian Sko¨ld,† Milad Botros,‡ Gunnar Lindeberg,† Fred Nyberg,‡ Anders Karle´n,† Anders Hallberg,† and
Mats Larhed*,†
Department of Medicinal Chemistry, DiVision of Organic Pharmaceutical Chemistry, BMC, Uppsala UniVersity, P.O. Box 574,
SE-751 23 Uppsala, Sweden, and Department of Pharmaceutical Sciences, DiVision of Biological Research on Drug Dependence, BMC,
Uppsala UniVersity, P.O. Box 591, SE-751 24 Uppsala, Sweden
ReceiVed NoVember 21, 2006
Four tripeptides corresponding to the C-terminal region of angiotensin II were synthesized. One of these
peptides (Ac-His-Pro-Ile) showed moderate binding affinity for the AT2 receptor. Two aromatic histidine-
related scaffolds were synthesized and introduced in the tripeptides to give eight new peptidomimetic
structures. Three of the new peptide-derived druglike molecules exhibited selective, nanomolar affinity for
the AT2 receptor. These ligands may become lead compounds in the future development of novel classes of
selective AT2 receptor agonists.
Introduction
The peptide hormone angiotensin II (Ang II,a Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe) activates two receptors, angiotensin II type
I and angiotensin II type 2 (AT1 and AT2, respectively). The
receptors are both G-protein coupled, but differ considerably
in amino acid sequence and in the physiological effects.
Knowledge of the AT1 receptor has been available for 35 years
and it is mostly recognized for its important role in the regulation
of blood pressure, salt and water retention, and cellular growth.1,2
The AT2 receptor, on the other hand, was not characterized until
the early 1990s.3,4 The AT2 receptor often exerts opposite effects,
as compared to that of the AT1 receptor; its activation results
in vasodilatation and suppressed cellular growth.1,5,6 It appears
to have a regulatory role in the renin-angiotensin system,
balancing the effects of the AT1 receptor especially in patho-
logical conditions. The receptor also promotes antiproliferation,
neurite outgrowth in certain cell types, apoptosis, and cell
differentiation.5,7,8 It has been proposed that the AT2 receptor
could be an important target in the therapeutic area of hyperten-
sion and cardiac remodeling.5,9
Using the endogenous peptide ligand as a starting point for
the development of a new lead series of druglike compounds
is, to us, an appealing approach, and over the years, we have
made some progress in our efforts to identify selective AT2
receptor agonists. For example, we have designed and synthe-
sized a number of bioactive AT2 receptor selective analogues
of similar size to Ang II, where the dipeptide fragment Tyr4-
Ile5 was replaced by various γ-turn mimetic scaffolds.10-12 In
1991, de Gasparo et al. showed that removal of three amino
acids from the N-terminus of Ang II ([Val5]Ang II (4-8) and
[Val,5Ile8]Ang II (4-8)) resulted in AT2 receptor selectivity.13
Inspired by these results, we synthesized and evaluated the
structurally similar compound I (Figure 1) and the corresponding
Figure 1. AT2 receptor agonists with common and potentially
important structural elements indicated.
analogue with Phe as the C-terminal amino acid. Interestingly,
both of them were AT2 receptor agonists.14 In the same study,
we also demonstrated that replacement of the Tyr-Ile unit by a
single aromatic core produced ligand II (Figure 1) with AT2
receptor affinity and agonistic activity comparable to Ang II.14
In parallel, nonpeptide AT2 receptor ligands have been devel-
oped in our group, originating from the first reported nonpeptide
AT1 receptor agonist L-162,313 disclosed by Merck.15,16 L-162,-
313 was found to also exert agonistic properties toward the AT2
receptor,17 and structural modifications have resulted in the first
selective nonpeptide AT2 receptor agonist M024 (Figure 1).18
* To whom correspondence should be sent. Phone: +46-184714667.
Fax: +46-184714474. E-mail: mats@orgfarm.uu.se.
† Department of Medicinal Chemistry.
‡ Department of Pharmaceutical Sciences.
a Abbreviations: Ang II, angiotensin II; AT1, angiotensin II type 1; AT2,
angiotensin II type 2.
10.1021/jm0613469 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/15/2007

1712 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Brief Articles
Chart 1
protecting group was chosen because of the high stability in
basic conditions and the possibility to selectively cleave it using
tetrabutylammonium fluoride (TBAF) in the presence of the
sulfonamide.21 The methyl ester was next reduced with LiAlH4
in dry THF. The resulting alcohol was treated with mesyl
chloride (MsCl) overnight.22 As a result of the prolonged
reaction time, the benzyl chloride 15 was formed instead of the
mesylate via a double substitution reaction. To obtain a better
leaving group, the benzyl iodide 16 was prepared immediately
before usage by classic treatment with NaI in acetone. An ortho-
directed lithiation of the protected imidazole 13 with n-BuLi in
THF at -78 °C followed by the slow addition of benzyl iodide
16 afforded structure 17.23 The two silyl protecting groups were
easily cleaved by treatment with TBAF to furnish free 18 in an
83% yield. A mild two-step oxidation of benzyl alcohol 18 was
utilized to reduce the risk of oxidation at the second benzylic
position. First, the aldehyde was generated by a Swern oxidation
at the alcohol. The intermediate aldehyde was further oxidized
to the benzoic acid by sodium chlorite and sodium dihydrogen
phosphate.24 Benzoic acid 19 was then coupled to the appropri-
ate tert-butyl ester protected amino acid using standard amide
coupling conditions, with HATU as the coupling reagent.
Finally, removal of the remaining protecting groups with HCl
in dioxane generated compounds 5, 9, 6, and 10 as salts.
Chart 2
The synthetic route to scaffold B25 is presented above
(Scheme 3). As for compound 14, the free benzoic acid of
monomethyl isophthalate was selectively reduced by BH3‚THF
to give monoester 20. The alcohol was converted to the benzyl
bromide (21) by the standard reaction with PBr3. Treatment of
21 with imidazole in DMF followed by hydrolysis of the methyl
ester with LiOH gave scaffold B, which was isolated as the
HCl salt. To generate the final products 7, 8, 11, and 12, scaffold
B was coupled to the amino acids using the same conditions as
above. No loss of the stereochemistry was detected.
Binding Assay. Compounds 1-12 were evaluated in a
radioligand binding assay relying on displacement of [125I]Ang
II from AT1 receptors in rat liver membranes26 and AT2
receptors in pig uterus membranes.27 Ang II and [4-NH2-Phe6]-
Ang II were used as reference substances. These results are
presented in Table 1. Tripeptide 2 with an N-terminal Ile residue
binds selectively to the AT2 receptor with a Ki value of 1.1
µM, while peptide 3 was the only analogue that displayed
affinity for the AT1 receptor. Of the evaluated eight pseudopep-
tides (5-12), compounds 9, 11, and 12 had nanomolar affinity
for the AT2 receptor, with Ki values ranging from 16.6 to 40.6
nM. Despite being very good AT2 receptor ligands, the
compounds did not display AT1 receptor binding.
Based on structural comparison, we suggest that the nonpep-
tidic agonist M024 mimics the C-terminal of Ang II and the
truncated analogues I and II, as illustrated in Figure 1. Thus,
the imidazole group of M024 corresponds to the histidine side
chain, the acylsulfonamide functionality provides an acidic
proton similar to the carboxylic acid, and either the isobutyl
chain or the n-butyl chain of M024 shares the same interaction
as the C-terminal Phe/Ile side chain. However, no apparent
structural motif can be found in M024 corresponding to a Tyr
side chain, which is present in Ang II and the truncated
analogues. Thus, the following question arises: Is a Tyr side
chain really necessary in the case of the more Ang II-like
analogues or can it be removed to further reduce the molecular
size and peptide character? Herein, we address this question by
presenting the synthesis of four novel tripeptide analogues (1-
4), the synthesis of two aromatic scaffolds (A and B), and their
incorporation, providing eight peptidomimetic analogs (5-12)
as well as the biological evaluation of all 12 compounds.
Results and Discussion
Discussion
Chemistry. The four peptides 1-4 (Chart 1) were obtained
from commercially available resin-bound protected tripeptides.
Scaffolds A and B (Chart 2) were prepared as described below
and were thereafter coupled to one or two amino acids to
generate eight pseudopeptides, 5-12 (Chart 3). For the synthesis
of scaffold A, a benzoic acid with a histidine side chain, an
orthogonally diprotected imidazole was required. To obtain this,
N,N-dimethylimidazole-1-sulfonamide was TIPS-protected in
position 2 using n-BuLi to deprotonate, followed by addition
of triisopropylsilyl triflate (TIPS-OTf) to afford compound 13
(Scheme 1). The subsequent preparation of scaffold A (as
sulfonamide 19) is outlined in Scheme 2.
We have previously reported on the development of a set of
pentapeptides and pseudopeptides, starting from the octapeptide
Ang II. Most of the ligands synthesized by us displayed high
affinity for the AT2 receptor.14 Four of the compounds were
also tested in a functional assay based on neurite outgrowth
from NG 108-15 cells and proved to be agonists. In parallel
and using a more classical medicinal chemistry approach starting
from the AT1 agonist L-162,313,15,16 the nonpeptidic selective
AT2 receptor agonist M024 was developed in our group.18 As
mentioned above, the nonpeptidic AT2 receptor agonists may
mimic the C-terminal part of Ang II, and if this hypothesis is
correct, removal of the Tyr residue should be possible without
loss of activity. To evaluate this hypothesis, a set of four
tripeptides (1-4) lacking the Tyr residue were synthesized and
tested for AT1 and AT2 receptor affinity (Chart 1). Only
First, the free carboxylic acid of the commercially available
monomethyl isophthalate was selectively reduced using BH3-
THF to yield the benzyl alcohol.19 The alcohol was directly
protected by TIPS-OTf to form compound 14.20 The TIPS

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1713
Chart 3
Scheme 1a
Scheme 3a
a Reagents and conditions: (a) n-BuLi, THF, -78 °C, TIPS-OTf.
Scheme 2a
a Reagents and conditions: (a) (i) BH3-THF, THF; (b) PBr3, diethyl
ether; (c) (i) imidazole, DMF; (ii) LiOH, H2O/MeOH/THF; (iii) aq. HCl;
(d) (i) amino acid, HATU, DIEA, DMF; (ii) HCl in dioxane.
Table 1. Binding Affinities for the AT1 and AT2 Receptors
a
b
AT1
AT2
compound
Ki ( SEM (nM)
Ki ( SEM (nM)
Ang II
0.5
0.3
0.9
[4-NH2-Phe6] Ang II
>10000
>10000
>10000
1598 ( 66
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
1
2
3
4
5
6
7
8
>10000
1110 ( 17
>10000
>10000
>10000
>10000
>10000
>10000
40.6 ( 1.0
>10000
37.5 ( 0.6
16.6 ( 0.5
9
a Reagents and conditions: (a) (i) BH3‚THF, THF; (ii) TIPS-OTf, PPTS,
DMAP, pyridine, DCM; (b) (i) LiAlH4, THF; (ii) NaOH/THF; (iii) MsCl,
NEt3, DCM; (c) NaI, acetone; (d) n-BuLi in hexane, dry THF, -78 °C, 13;
(e) TBAF, THF; (f) (i) oxalyl chloride, NEt3, DMSO, DCM; (ii) NaClO2,
NaH2PO4‚H2O, cyclohexene, tert-butyl alcohol; (g) (i) amino acid, HATU,
DIEA, DMF; (ii) HCl in dioxane.
10
11
12
a Binding assay relying on displacement of [125I] Ang II from rat liver
membranes. b Binding assay relying on displacement of [125I] Ang II from
pig uterus myometrium.
analogue 2, with the Ile residue in the C-terminal, showed
moderate AT2 receptor binding affinity (Ki ) 1110 nM). In fact,
Ile in this position has previously been found to enhance the
AT2 receptor binding affinity when compared to the corre-
sponding Phe analogue.14 Furthermore, it also seemed that the
N-terminal acetyl group was necessary for receptor binding
because primary amine 4 lacked affinity. The reasonable binding
affinity of the N-acetylated tripeptide 2 suggests that it could
serve as a lead compound in producing less peptidic compounds.
With this in mind, a series of analogues were synthesized
based on the 1,3-disubstituted benzene core structure. This
simple unit has previously proved successful as a mimic of Tyr-
Ile when incorporated into Ang II related pseudopeptides.12,14
However, after appropriate functionalizations, this scaffold might
also be worthwhile investigating as a mimic of His or His-Pro.
Thus, compound 19 (protected A) equipped with a His mimick-
ing side chain was synthesized (Scheme 2). In addition, isomer
B containing an N-imidazolemethyl side chain similar to that
found in the nonpeptide AT2 receptor agonist M024 was
prepared (Scheme 3, Figure 1). Because a direct atom-to-atom
comparison in the His region was not possible between 2 and
scaffolds A or B, it was difficult to decide which amino acid

1714 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Brief Articles
should be coupled to the C-terminal end of the scaffold. To
circumvent the problem, two series of compounds were
produced. In the first set of ligands (5-8), scaffolds A and B
substitute His, while in the second series (9-12) they occupy
the position of the dipeptide His-Pro. The resulting compounds
in the first series showed no detectable affinity up to 10 µM
(Table 1). It can be concluded that in the series of monoamides
(9, 11, and 12) both scaffolds A and B effectively replace the
dipeptide fragment His-Pro, as indicated by their reasonably
high AT2 receptor affinities. Compound 12 with an Ile C-
terminal showed slightly better affinity (Ki ) 16.6 nM) than
the corresponding Phe analogue (Ki ) 37.5 nM), which is in
accordance with previous observations.14 No great difference
in binding affinity was observed when scaffolds A and B were
coupled to Phe (compare 9 and 11). Surprisingly, structure 10
showed no detectable binding up to 10 µM. It is remarkable
that substitution of Phe with Ile when having a histidine side
chain (compare 9 and 10) results in loss of AT2 receptor affinity
when the same substitution increases the affinity in the analogues
with an N-imidazolemethyl group (compare 11 and 12).
However, similar sensitivity in affinity to the AT2 receptor has
been found previously, where relatively small structural modi-
fications of the sulfonylcarbamate side chain in a series of M024
analogues resulted in inactive compounds.17 This sensitivity was
also present but not equally profound in compounds having the
same N-imidazolemethyl group as M024.28 In 10, the combina-
tion of the Ile and His side chain is clearly not suitable for a
favorable AT2 receptor interaction, which seems to reflect a
similar sensitivity in this series.
To examine if 12 and M024 could adopt a similar binding
mode at the AT2 receptor, pharmacophore modeling was
performed. As a result of their similarity, it was hypothesized
that the imidazole and the acidic groups, respectively, correspond
to pharmacophore groups and are superimposed upon binding.
A third possible pharmacophore group in 12 corresponds to the
Ile side chain. However, it is not obvious which of the isobutyl
or n-butyl groups of M024 should share the same receptor
interaction as the Ile side chain in that case. To address this
issue, these compounds were modeled using the DISCOtech29
program in Sybyl.30 Because it has been proposed that Lys215
in the AT2 receptor is involved in an ionic interaction with the
C-terminal of Ang II,31 the acidic groups in 12 and M024 were
defined as a pharmacophore group that all models were forced
to contain. Using this approach, DISCOtech found 194 models.
Guided by the superimposed features and the overall structural
overlap of the superimposed structures in each model, two
reasonable models were identified. In these models, the imida-
zole groups and the acidic groups overlay nicely, while the Ile
side chain can be either superimposed on the n-butyl group
(Figure 2a) or in the vicinity of the isobutyl group (Figure 2b).
It is shown in Figure 2b that the Ile side chain is not perfectly
matched to the isobutyl group but is oriented more toward the
thiophene ring (Figure 2b). Based on the present modeling, it
therefore seems that 12 and M024 should be able to adopt
similar binding modes in general when binding to the AT2
receptor. However, M024 will have additional interactions with
the AT2 receptor, considering that 12 only has one hydrophobic
side chain.
Figure 2. Possible pharmacophore models of 12 (white carbons) and
M024 (green carbons), with important structural elements indicated
(Figure 1). The colored spheres correspond to DISCOtech features. (a)
Model in which the Ile side chain of 12 and the n-butyl group of M024
are overlaid. (b) Model in which the Ile side chain of 12 and the isobutyl
group are overlaid.
compounds was prepared, and three of them were found to
possess nanomolar affinity for the AT2 receptor. These ligands
were developed based on the Ang II octapeptide and may serve
as unique lead structures in the development of a novel and
selective class of AT2 receptor agonists.
Experimental Section
Preparation of Peptides 1-4. Fmoc-His(Trt)-Pro-Phe-Wang
resin10 or Fmoc-His(Trt)-Pro-Ile-Wang resin10 was swelled in DMF.
The Fmoc-group was removed by treatment with 20% piperidine
in DMF, and the resin was washed with DMF, DCM, and MeOH
before being dried under vacuum overnight. For the acetylated
analogues, an aliquot of the resin was swelled in DMF. The acetic
acid was dissolved together with PyBOP or HBTU and DIEA in
DMF. The solution was added to the resin and agitated by rotation
overnight. The resin was deprotected, washed, and dried as above.
The peptides were cleaved from the resin using triethylsilane and
95% aqueous TFA. The polymer was filtered off and washed with
TFA. The filtrate was evaporated in a stream of nitrogen, and the
product was precipitated by addition of diethyl ether. The precipitate
was collected by centrifugation, washed with ether, and dried. The
crude peptide was purified by RP-HPLC and isolated at yields of
11-50%.
General Procedure for Amide Coupling and Deprotection.
Scaffold 19 or B, HATU, the appropriate amino acid, and DIEA
were dissolved in DMF. After stirring in ambient temperature
overnight, the reaction mixture was poured into water and extracted
with ethyl acetate. The combined organic phase was washed with
saturated NH4Cl, water, and brine and dried with Na2SO4. The crude
product was purified on preparative RP-LC-MS. The product was
dissolved in 4 M HCl in dioxane and stirred at room temperature
overnight (or compounds with dimethylsulfonamide protecting
group present, at 70 °C for 5 h). The dioxane was evaporated under
vacuum. The crude products were purified by RP-LC-MS and
isolated in yields of 12-31%.
Compound 9. Isolated yield of 9 was 12.5 mg (14%). 1H NMR
(CD3CN + D2O) δ 8.50 (d, J ) 1.5 Hz, 1H), 7.59 (m, 1H), 7.57
(m, 1H), 7.44-7.37 (m, 2H), 7.29-7.18 (m, 5H), 7.16 (m, 1H),
4.79 (dd, J ) 5.1, 9.4 Hz, 1H), 4.07 (s, 2H), 3.29 (dd, J ) 5.1,
14.0 Hz, 1H), 3.08 (dd, J ) 9.4, 14.0 Hz, 1H); 13C NMR (CD3CN
+ D2O) δ 174.3, 168.7, 138.5, 138.4, 135.2, 134.6, 133.6, 133.1,
130.2, 130.1, 129.4, 128.5, 127.8, 126.8, 117.3, 55.1, 37.7, 30.8;
LC/MS (M, 349.1) (350.1; M + H+). HRMS (M + 1) calcd,
Conclusion
350.1505; found, 350.1501. [R]20 ) -20.5 (c 0.44, DMSO-d6)
In summary, two aromatic scaffolds functionalized with either
a histidine side chain or an N-imidazolemethyl group were
successfully synthesized and coupled to C-terminal peptide
fragments of Ang II. Based on these scaffolds, a novel set of
D
Compound 10. Isolated yield of 10 was 18.1 mg (23%).
Diasteromeric mixture of 1:0.11 Ile/allo-Ile determined by amino
acid analysis. 1H NMR (DMSO-d6) δ (major) 8.30 (d, J ) 7.4 Hz,

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1715
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NH), 7.73 (m, 1H), 7.69 (m, 1H), 7.53 (m, 1H), 7.37 (m, 1H),
7.35 (m, 1H), 6.76 (m, 1H), 4.27 (t, J ) 7.4 Hz, 1H), 3.89 (s, 2H),
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1
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NMR (CD3CN + D2O) δ 8.70 (m, 1H), 7.67 (m, 1H), 7.65 (m,
1H), 7.52-7.37 (m, 4H), 7.26-7.16 (m, 5H), 5.37 (s, 2H), 4.75
(dd, J ) 5.0, 9.4 Hz, 1H), 3.28 (dd, J ) 5.0, 14.0 Hz, 1H), 3.05
(dd, J ) 9.4 14.0 Hz, 1H); 13C NMR (CD3CN + D2O) δ 175.1,
168.5, 138.4, 136.0, 135.6, 135.5, 132.8, 130.6, 130.2, 129.4, 128.6,
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B.; Botros, M.; Karlen, A.; Hallberg, M.; Gallo-Payet, N.; Alterman,
M. Selective angiotensin II AT2 receptor agonists: Arylbenzylimi-
dazole structure-activity relationships. J. Med. Chem. 2006, 49,
7160-7168.
NMR (CD3CN + D2O) δ 8.74 (m, 1H), 7.79 (m, 1H), 7.75 (m,
1H), 7.55-7.49 (m, 2H), 7.42 (m, 2H), 5.40 (s, 2H), 4.42 (d, J )
6.4 Hz, 1H), 1.95 (m, 1H), 1.50 (m, 1H), 1.24 (m, 1H), 0.94 (d, J
) 6.9 Hz, 3H), 0.87 (t, J ) 7.4 Hz, 3H); 13C NMR (CD3CN +
D2O) δ 175.3, 169.5, 135.8, 135.41, 135.39, 132.9, 130.6, 128.9,
128.5, 122.8, 121.2, 58.8, 53.1, 37.4, 26.0, 15.9, 11.6; LC/MS (M,
315.2) (316.1; M + H+). HRMS (M + 1) calcd, 316.1661; found,
316.1659. [R]20 ) -10.9 (c 0.46, DMSO-d6).
D
Acknowledgment. We gratefully acknowledge the financial
support from the Swedish Research Council and the Swedish
Foundation for Strategic Research. We also thank Shane
Peterson and Dr. Luke Odell for linguistic revision.
Supporting Information Available: Experimental details,
HPLC tracings and spectroscopic data for compounds 1-21 and
B, procedures for AT1 and AT2 receptor binding assays, and
description of the molecular modeling. This material is available
free of charge via the Internet at http://pubs.acs.org.
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