Interconversion of functional activity by minor structural alterations in nonpeptide AT2 receptor ligands

Article abstract of DOI:10.1021/ml500427r

Migration of the methylene imidazole side chain in the first reported selective drug-like AT₂ receptor agonist C21/M024 (1) delivered the AT₂ receptor antagonist C38/M132 (2). We now report that the AT₂ receptor antagonist compound 4, a biphenyl derivative that is structurally related to 2, is transformed to the agonist 6 by migration of the isobutyl group. The importance of the relative position of the methylene imidazole and the isobutyl substituent is highlighted herein.

Full text of DOI:10.1021/ml500427r

Letter
pubs.acs.org/acsmedchemlett
Interconversion of Functional Activity by Minor Structural
Alterations in Nonpeptide AT2 Receptor Ligands
Charlotta Wallinder,† Christian Skold,† Milad Botros,‡ Marie-Odile Guimond,§ Mathias Hallberg,‡
̈
Nicole Gallo-Payet,§ Anders Karlen,† and Mathias Alterman*,†
́
†Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, BMC, Uppsala University, SE-751 23 Uppsala, Sweden
‡Beijer Laboratory, Department of Pharmaceutical Biosciences, BMC, Uppsala University SE-751 23 Uppsala, Sweden
§Service of Endocrinology, Faculty of Medicine and Heath Sciences, University of Sherbrooke, Sherbrooke J1H 5N4, Quebec, Canada
S
* Supporting Information
ABSTRACT: Migration of the methylene imidazole side chain
in the first reported selective drug-like AT2 receptor agonist
C21/M024 (1) delivered the AT2 receptor antagonist C38/M132
(2). We now report that the AT2 receptor antagonist compound 4,
a biphenyl derivative that is structurally related to 2, is transformed
to the agonist 6 by migration of the isobutyl group. The impor-
tance of the relative position of the methylene imidazole and the
isobutyl substituent is highlighted herein.
KEYWORDS: AT2 receptor, nonpeptide ligands, agonist, antagonist, C21/M024, C38/M132
he sartans (angiotensin II receptor blockers, ABRs) have
Tbeen on the market as antihypertensives for more than
two decades and act as selective antagonists at the angiotensin
II AT1 receptor. More recently, the angiotensin AT2 receptor
has emerged as a new target for drug intervention.1−3 In 2004,
the first selective and drug-like AT2 receptor agonist C21/
M024 (1) was disclosed,4 and thereafter, a series of structural
analogues with agonistic properties have been reported.5,6 The
activation of the AT2 receptor is known to render a large
number of diverse biological responses.7,8 For example, recep-
tor activation stimulates neurite outgrowth in neuronal cells,
which is one of the first steps in neuronal differentiation.9
In healthy adults the AT2 receptor expression is low and
mainly found in renal, cardiovascular, and brain tissues.7,8
Notably, during certain pathological conditions, such as myo-
cardial infarction, vascular injury, brain ischemia, renal failure,
Figure 1. Structures of compounds 1 and 2.
and cutaneous wounds, the AT2 receptor expression is
significantly up-regulated.7,8
C38/M132 (2) (Figure 1).15,16 Thus, a minor structural alter-
Recently, several studies conducted in experimental disease
ation resulted in the interconversion of a selective AT2 receptor
agonist to a selective AT2 receptor antagonist.
models have revealed that the selective potent AT2 receptor
agonist 1 acts as an anti-inflammatory agent and exerts pro-
nounced tissue protective effects in particular in cardiovascular
and renal disease. Hence, the compound has had a large impact
on the view of the AT2 receptor as a drug target.1,9−14 In addition,
the AT2 receptor is a promising drug target for antagonists. Thus,
the AT2 receptor antagonist EMA401 has demonstrated con-
vincing efficacy data in patients suffering from neuropathic pain
(postherpetic neuralgia) in a phase II study.3
Herein, we report the synthesis and biological evaluation of four
AT2 receptor ligands, the biphenyl derivatives, and regioisomers, 3,
4, 5, and 6 (Figure 2), and that the relative position of the imidazole
and isobutyl substituent determines the functional outcome.
The nonselective Ang II receptor ligand 7 (L-162,782) acts
as an AT1 receptor agonist and 8 (L-162,389) acts as an AT1
We discovered that migration of the methylene imidazole
substituent of 1 from the para to the meta substitution pattern
delivered a selective AT2 receptor antagonist, compound
Received: October 21, 2014
Accepted: December 8, 2014
Published: December 8, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Figure 2. Structures of the regioisomers 3, 4, 5, and 6.
Figure 3. Structures of compounds 7 and 8.
receptor antagonist (Figure 3).17 Notably, these two structures
differ only by a methyl group; the isobutyl group gives rise to
agonism, while the n-propyl group renders antagonism.17
Since the only structural difference between the agonist 1
and the antagonist 2 can be found in the position of the
methylene imidazole substituent, it can be hypothesized that
the change in functionality could be attributed to a reduced
ability to stabilize the activated receptor conformation due to a
differently positioned imidazole group in 2. However, as an
alternative hypothesis the imidazole rings could have the same
receptor interaction point, while the isobutyl groups are forced
to be positioned differently and cause the difference in fun-
ctionality (Figure 4A). If so, the topography and location of the
lipophilic side chain of ligands binding to the AT2 receptor is
the primary determinant of functionality as was the case at the
AT1 receptor (cf. 7 and 8).
Figure 4. (A) Possible binding mode of two model compounds corre-
sponding to structures 1 (white) and 2 (green). (B) Corresponding
hypothesized binding mode for the two model compounds
corresponding to the biphenyl analogues, 3 (purple) and 4 (orange),
and postulated functions at the AT2 receptor. (C) Comparison of the
suggested binding modes of 1 and 2 with the analogues of 3 and 4,
where the isobutyl have been moved to the meta-position, the two
model compounds corresponding to compounds 5 (red) and 6 (blue),
respectively.
In order to explore this alternative hypothesis and to identify
a potential binding mode, a computational analysis was perfor-
med. Potential pharmacophore groups, including the imidazole
rings, of 1 and 2 were superimposed using the pharmacophore
modeling program DISCOtech18 in Sybyl19 (see Supporting
Information for details). In order to reduce the number of
conformations, model structures with methyl instead of butyl
substituents on the sulfonylcarbamate group were used in the
modeling. From the suggested binding mode model identifide,
shown in Figure 4A, the isobutyl groups are oriented differently
in the two thiophene compounds, which could be of impor-
tance for their ability to induce either the agonistic or antago-
nistic response. On the basis of this model, we hypothesized
that it should be possible to convert the meta-substituted
antagonist 2 to an agonist if the isobutyl chain could adopt
a similar orientation as in the para-substituted agonist 1,
illustrated in Figure 4A. To investigate this hypothesis we
performed further modeling where we replaced the thiophene
ring with a phenyl group to obtain biphenyl analogues allowing
better assessment of the impact of relative positions of the
isobutyl side chain (i.e., 3 and 4, see Figure 2). Accordingly,
superimposition of 3 and 4 produced a model, depicted in
Figure 4B, which was very similar to that shown in Figure 4A.
The corresponding analogues with an isobutyl group meta to
the sulfonylcarbamate, compounds 5 and 6 (Figure 2), were
also modeled and compared with the suggested binding modes
of 1 and 2 as seen in Figure 4C. Although the position of the
isobutyl group in 6 did not superimpose optimally to the
corresponding group in 1, it was clearly closer to this binding
mode than that of 2, where the imidazole and the sulfon-
ylcarbamate also could reach the same interactions (Figure 4C).
According to the model in Figure 4C, compound 5, although
having a meta-methylene imidazole substitution, could adopt a
conformation that allows a similar binding mode as 6 and was
therefore also hypothesized to act as an agonist. In order to
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Letter
explore this binding mode hypothesis we set out to synthesize
regioisomers 3−6 and determine their AT2 receptor binding
affinity and, in particular, their functional activity at the AT2
receptor.
Table 1. Biological Activities at the AT2 Receptor
compd
Ki SD (nM)
function (10 nM)
3
4
5
6
0.7 0.1
12.3 0.4
10.1 0.4
8.2 0.2
agoinst
The synthesis of 3 has been previously described.5 The
synthesis of compounds 4−6 are outlined in Scheme 1 and
started with the benzenesulfonamides 9 and 10, prepared as
previously described by Wallinder et al.20 Compound 13 and
14 were prepared by N-alkylation of imidazole by 3- and
4-bromobenzyl bromide in DMF. The crude boronic acids
(11 and 12) were coupled with compounds 13 and 14 under
Suzuki coupling conditions with Pd(PPh3)4 as catalyst and
Na2CO3 as base using microwave irradiation (150 °C, 5 min)
to form compounds 15−17 in good yields (75−87%).
Compounds 4−6 were afforded after deprotection of the
sulfonamide with BCl3 and subsequent acylation with n-butyl
chloroformate in DCM, water, and Na2CO3 to yield the final
products in 27−51% yield, after purification by preparative
HPLC.
antagonist
agonist
agonist
All compounds were evaluated for functionality in a neurite
outgrowth assay with NG108-15 cells. The NG108-15 cells
express mainly the AT2 receptor in their undifferentiated state,
and a three-day treatment with Ang II or the selective peptidic
AT2 receptor agonist CGP-42112 (Nα-nicotinoyl-Tyr-(Nα-Cbz-
Arg)-Lys-His-Pro-Ile)23,24 induce neurite outgrowth.16,25 The
signaling pathways involve a sustained increase in Rap1/B-Raf/
p42/p44mapk activity and activation of the nitric oxide/guanylyl
cyclase/cGMP pathway.26,27 Cells were treated in the absence
or presence of Ang II, 3, 4, 5, and 6. After 3 days of treatment,
cells were examined under a phase-contrast microscope and
micrographs were taken. To establish an adequate test concen-
tration, the compounds were tested at various concentrations
ranging from 0.1 to 100 nM. No evidence of cell death was
observed. All compounds were tested at a concentration of
10 nM, compound 4 was also tested at 100 nM to verify that
the lack of neurite outgrowth was not due to too low concen-
tration (data not shown). Coincubation with the selective AT2
receptor antagonist PD 123,319 reduced neurite outgrowth,
verifying that the effect was mediated through the AT2 receptor.
Treatment with PD 123,319 alone did not alter the morphology
compared to untreated cells (data not shown), as we have shown
previously.16 The antagonistic effect was verified through coin-
cubation with Ang II resulting in reduced neurite outgrowth.
Compounds 3−6 were evaluated for affinity in a radioligand-
binding assay by displacement of [125I]Ang II from AT2
receptors in pig uterus membranes as described previously by
Nielsen et al.21 The natural substrate Ang II and the selective
AT1 receptor antagonist losartan were used as reference
substances.22 The results of the binding study are presented
in Table 1. Only AT2 receptor affinity was evaluated since no
AT1 receptor binding have been seen for these methylene
imidazoles even upon modifications of the lower part of
the structures. This is also verified by compound 3, which
previously has been tested for AT1 receptor affinity (Ki >
10 000 nM).5
a
Scheme 1
a
Reagents: (a) n-BuLi, THF, triisopropyl borate, HCl (aq); (b) Pd(PPh3)4, Na2CO3, toluene, ethanol, H2O; (c) BCl3, DCM; (d) n-butyl
chloroformate, Na2CO3, DCM, H2O.
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and Figure 5). When the substitution pattern for the iso-
butyl chain of 3 was changed to give 5, the affinity dropped
approximately 10 times, but the agonistic property was retained
(Table 1 and Figure 5). When the same structural modification
was performed with 4 to deliver compound 6 the affinity was
slightly increased, but more interestingly, the antagonistic
property of 4 was interconverted to agonism (Table 1 and
Figure 5). Thus, independent of the methylene imidazole
position, both an agonist and an antagonist could be obtained
by moving the isobutyl side chain. This suggests that the
structural feature responsible for functionality is the spatial
relationship between the imidazole ring and the isobutyl side
chain.
When looking at the peptide-activated GPCRs there seems
to be a trend that minor structural alterations in small non-
peptidic ligands can interconvert the functional activity, cf. 7
and 8.17 Small changes in the effector domain of peptides can
frequently also have large impact on functionality, as
exemplified by [Ile8]AngII, which acts as an antagonist at the
AT1 receptor (or partial agonist), while the native [Phe8]AngII
acts as an agonist.28 Likely it is the same effects on the equilib-
rium between the active and inactive receptor conformation
that we see from these small nonpeptidic ligands.
The presented investigation highlights the importance of the
relative position of the methylene imidazole and the isobutyl
substituent for functional activity in this class of small molecule
AT2 receptor ligands. Thus, regardless of the position of the
methylene imidazole group that previously was proposed to
serve as an important determinant for agonism, both agonists
and antagonists can be obtained by a short distance migration
of the isobutyl group attached to the biphenyl scaffold. This
suggests that the structural feature responsible for functionality
is the spatial relationship between the imidazole ring and the
isobutyl side chain. The data gained from molecular modeling
provides a good foundation in the future design of selective
AT2 receptor agonists and antagonists.
Figure 5. Effect of compounds 3−6 on neurite outgrowth in NG108-
15 cells. The cells were plated at a cell density of 3.6 × 104 cells/Petri
dish (35 mm) and were cultured for 3 days in the absence or presence
of 100 nM Ang II, 10 nM 3, 10 nM 4, 10 nM 5, or 10 nM 6 alone or in
combination with 10 μM PD 123,319 or 100 nM Ang II. Cells with at
least one neurite longer than a cell body were counted as positive for
neurite outgrowth. The number of cells with neurites was expressed as
the percentage of the total number in the micrographs (at least 400
cells according to the experiment). The results are significant
according to two-way ANOVA: ****, p < 0.0001; ***, p < 0.001;
**, p < 0.01; ns = not significant.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedure and analytical data of the synthesized
compounds, biological evaluation methods, and computational
methods. This material is available free of charge via the
Internet at http://pubs.acs.org.
The functionalities are summarized in Table 1 and the neurite
outgrowth results are shown in Figure 5.
AUTHOR INFORMATION
■
The biphenyl analogues of 1 and 2, compounds 3 and 4,
respectively, exhibited a similar biological response both in
affinity to and functional activity at the AT2 receptor, indicating
that the biphenyl scaffold is a bioisoster to the thienylphenyl
scaffold in this series of compounds. It is worth noting that the
antagonist 4 exhibited a better inhibition of Ang II-induced
neurite outgrowth compared to PD 123,319, even thought the
concentration of 4 was a 1000-fold lower compared to PD
123,319 (10 nM vs 10 μM), as also seen with 2.15,16 Ang II
stimulation after pretreatment with 4 resulted in neurite out-
growth no longer significantly different compared to untreated
cells, while Ang II stimulation after pretreatment with PD
123,319 showed a neurite outgrowth significantly (****, p <
0.0001) higher compared to untreated cells. This indicates that
compound 4 is a more efficient AT2 receptor antagonist than
PD 123,319 in the cell assay.
Corresponding Author
*Tel: +46-18-4714283. Fax: +46-18-4714474. E-mail: mathias.
alterman@orgfarm.uu.se.
Author Contributions
The manuscript was written through contributions of all
authors.
Funding
We gratefully acknowledge support from the Swedish Research
Council (to V.R.), the Swedish Foundation for Strategic
Research to (S.S.F.), Kjell and Marta Beijer Foundation, Knut
̈
and Alice Wallenberg Foundation, the Canadian Institutes of
Health Research to N.G.P., M.D. Payet (MOP 27912), and
Vicore Pharma AB. N.G.P. is a holder of a Canadian Research
Chair in Endocrinology of the Adrenal Gland.
Notes
Compound 3 that has been previously synthesized and
assessed as an AT2 receptor ligand5 acts as an agonist (Table 1
The authors declare no competing financial interest.
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Letter
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ACKNOWLEDGMENTS
■
We thank Dr. Luke R. Odell for linguistic advice.
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