Novel paramagnetic AT1 receptor antagonists

Article abstract of DOI:10.1039/c1cc14920b

Novel paramagnetic selective angiotensin AT₁ receptor antagonists (sartans) bearing nitroxides (3, 4) have been prepared and their pharmacology evaluated in vitro as well as in vivo. Compounds 3, 4 proved to be effective sartans with pK_B estimates in the range 6.2-9.1. In addition, the sodium salt (11) of 4 (R = Bu) is able to protect against vascular injury in hypertensive rats as determined by its ability to attenuate the development of intimal thickening caused by balloon injury of the carotid artery. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc14920b

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COMMUNICATION
Novel paramagnetic AT1 receptor antagonistsw
ab
Nichole P. H. Tan,ab Michelle K. Taylor,z Steven E. Bottle,ac Christine E. Wright,ad
James Ziogas,ad Jonathan M. White,b Carl H. Schiesser*ab and Nitya V. Jani*ad
Received 9th August 2011, Accepted 21st September 2011
DOI: 10.1039/c1cc14920b
Novel paramagnetic selective angiotensin AT1 receptor antagonists
(sartans) bearing nitroxides (3, 4) have been prepared and their
pharmacology evaluated in vitro as well as in vivo. Compounds 3, 4
proved to be effective sartans with pKB estimates in the range
6.2–9.1. In addition, the sodium salt (11) of 4 (R = Bu) is able to
protect against vascular injury in hypertensive rats as determined
by its ability to attenuate the development of intimal thickening
caused by balloon injury of the carotid artery.
superoxide dismutase to reduce superoxide. Moreover,
nitroxides in addition to directly scavenging free radicals
may also enter into reductive cycles to form hydroxylamines
or oxoammonium ions that can interrupt biological redox
processes.6 The high antagonist affinity and slow dissociation
of the sartan pharmacophore would be expected to target and
retain these hybrid nitroxide antioxidants to AT1 receptors.
AT1 receptors are upregulated at sites of oxidative stress in
cardiovascular diseases exacerbating free radical production.7
In atherosclerotic plaques, cholesterol laden macrophages
produce an excess of extracellular superoxide that cause
further oxidative damage.8 Thus combining a sartan and an
antioxidant moiety may enable a targeted decrease in radical-
mediated damage and inflammation by reducing AT1 receptor-
dependent and -independent free radicals.
Dual action drugs describe agents with different pharmacophore
groups combined within the one molecule. Such drugs have
been designed to improve the pharmacological profile of
individual pharmacophores, particularly in the treatment of
multifactorial diseases in which complex pathologies arise.1,2
One such disease is hypertension which involves a range of
mediators and co-morbidities.3 Angiotensin II acting on AT1
receptors is a key mediator in the pathogenesis of hyper-
tension.4 AT1 receptor antagonists (sartans) possess antihy-
pertensive actions by antagonising angiotensin II-induced
vasoconstriction as well as anti-inflammatory actions by
inhibiting angiotensin II-derived free radicals which are also
AT1 receptor-dependent.5 Free radicals have also been
strongly implicated in the maladaptive changes in vascular
function that has been observed in hypertensive patients.3
Consequently, a dual action drug combining a free radical
antioxidant moiety (nitroxide) and a sartan may target the
angiotensin II and the oxidative stress component of hyper-
tension using one compound. The nitroxide moiety was chosen
as it is a free radical antioxidant that can mimic the enzyme
As a consequence, we chose to prepare and examine nitroxide-
containing sartans. As part of on-going work aimed at the
development of novel compounds for the treatment of hyper-
tension, we prepared analogues of existing sartans with the aim
of exploring the effect of heteroatom substitution and substituent
a ARC Centre of Excellence for Free Radical Chemistry and
Biotechnology, Victoria, Australia
b School of Chemistry and Bio21 Molecular Science and Biotechnology
Institute, The University of Melbourne, Victoria, 3010, Australia.
E-mail: carlhs@unimelb.edu.au; Fax: +61 3 9347 8189;
Tel: +61 3 8344 2432
c School of Physical and Chemical Sciences, Queensland University of
Technology, GPO Box 2432, Brisbane, QLD 4001, Australia
d Department of Pharmacology, The University of Melbourne,
Victoria, 3010, Australia
w Electronic supplementary information (ESI) available: Experimental
details for the preparation of new compounds; HPLC traces for compounds
3, 4; EPR spectrum of 4 (R = Bu); 1H and 13C NMR spectra for compound
9; CIF for 4 (R = Me). CCDC 833015. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c1cc14920b
z Current address: Division of Chemistry, School of Science and
Technology, The University of New England, Armidale, NSW,
2351, Australia.
c
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Chem. Commun., 2011, 47, 12083–12085 12083

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Fig. 1 Effects of NADPH (100 mM; Control) on luminescent super-
oxide counts detected using lucigenin (5 mM) in the presence of 1–10 mM
compound 10 or compound 11 in rat isolated aorta. Values are mean
(ꢀSEM) superoxide luminescent counts per milligram of dry tissue
weight (counts mgꢁ1). Error bars not visible are contained within bars.
*P o 0.05 vs. Control using one-way ANOVA with Dunnett’s post-test.
n = 5–7 vessel segments, taken from separate rats.
trityl-protected drugs 8 in moderate yield (Scheme 1). Subsequent
reflux in methanol afforded the deprotected compounds 3 and 4
in 18–56% yield following flash chromatography.
Scheme 1 Reagents and conditions: (a) NaH, LiBr, DMF, 6 or 7,
0 1C - rt; (b) MeOH, reflux, 18–56% (2 steps).
As these novel compounds are paramagnetic, it was not
possible to obtain 1H or 13C NMR spectra, although each
compound exhibited the ‘‘classic’’ triplet expected for nitroxides
(g factor 2.005; aN 13.875 G);13 an example is provided in the
ESI.w The purity of compounds 3, 4 was assessed by HPLC
analysis (see ESIw). In addition, 3 and 4 were characterised by 1H
and 13C NMR spectroscopy of their (diamagnetic) methoxyamine
derivatives 9, prepared as per the example shown in Scheme 2.
Fortunately nitroxide 4 (R = Me) proved to be a crystalline
solid and its structure was established by X-ray crystallography
and is displayed in Fig. S1 (ESIw).
Chinese hamster ovary (CHO) cells stably expressing the rat
AT1A receptor (CHO-AT1A cells) were used in a preliminary
study to assess AT1 receptor antagonist potency of the sartan
analogues prepared in this study.14 In these experiments, the
ability of 30 nM of compounds 3, 4 to inhibit angiotensin
II-induced calcium release was determined. The pKB estimates
of antagonist potency are summarized in Table 1 together with
those of milfasartan and eprosartan (standards). Clearly
compounds 3 and 4 are effective AT1 receptor antagonists in
this assay, with the propyl and butyl substituted systems
(entries 5, 6, 9, and 10) being more potent in this assay.
On the basis of the pKB data provided in Table 1, we chose
to examine the antioxidant capacity of the most potent AT1
receptor antagonist, namely 3 and 4 (R = Bu); these were first
converted to their sodium salts 10 and 11 (for solubility
reasons) by treatment with sodium hydride in THF
(Scheme 2).
Scheme 2 Reagents and conditions: (a) FeSO4, DMSO, H2O2, 89%;
(b) NaH, THF, 85%.
size on AT1 receptor activity of sartans. We reported recently
that a selenium substitution has a minimal negative effect; for
example selenomilfasartan (1) proved to have a pKB value within
one order of magnitude of the parent compound;9 while benzo
analogues (e.g. 2) also proved to be effective sartans.10 As a
consequence, we examined whether nitroxide-containing com-
pounds that include 3 and 4 would be effective AT1 receptor
antagonists with added therapeutic benefits.
Compounds 3, 4 were readily prepared by the coupling of
the ‘‘biphenyl core’’ (5)11 with the appropriate halide (6, 7),
themselves prepared following literature procedures,12 to give the
Table 1 pKB Estimates of the sartans (3, 4) prepared in this study as
compared with milfasartan and eprosartan
Table 2 Effects of 7 day treatment with compounds on mean arterial
pressure (MAP) in conscious unrestrained SHR using telemetry.
Values are mean ꢀ SEM MAP (mmHg) on day 0 prior to treatment
and on day 7 of treatment. Treatment was delivered over 7 days via an
osmotic mini-pump implanted s.c. *P o 0.05 on DMAP over 7 days
compared to rats treated with DMSO. There were 4–6 rats in each
treatment group
Entry
Sartan
pKB estimatea
1
2
3
4
5
6
7
8
9
10
Milfasartan
Eprosartan
3 (R = Me)
(R = Et)
9.5 ꢀ 0.3
8.4 ꢀ 0.5
6.6 ꢀ 0.3
6.2 ꢀ 0.3
8.2 ꢀ 0.2
9.1 ꢀ 0.4
7.1 ꢀ 0.3
7.2 ꢀ 0.3
8.4 ꢀ 0.4
8.4 ꢀ 0.5
(R = Pr)
(R = Bu)
4 (R = Me)
(R = Et)
(R = Pr)
(R = Bu)
Day 0
(mmHg) (mmHg) mmHg
Day 7
DMAP/
Treatment
50% DMSO (240 ml per day)
132 ꢀ 3 138 ꢀ 3
6
Compound 11 (30 mg kgꢁ1 per day) 135 ꢀ 4 112 ꢀ 4 ꢁ23*
a
Eprosartan (30 mg kgꢁ1 per day)
128 ꢀ 3 113 ꢀ 4 ꢁ15*
See the text.
c
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average intimal thickness was 1.8 ꢀ 0.2 mm (n = 6). This was
27-fold thicker in the injured vessels from the same rats with a
change in the intimal thickness of 46 ꢀ 10 mm (Fig. 2A).
Treatment with milfasartan (30 mg kgꢁ1 per day) had no effect
on intimal thickening (Fig. 2B). However, treatment with 11
(30 mg kgꢁ1 per day) significantly attenuated intimal thickening
in injured vessels observed as a change in intimal thickness of
only 12 ꢀ 4 mm. Indeed in 2 of the 6 rats treated with 11, intimal
thickness was completely prevented (Fig. 2C). This was
associated with a reduction in superoxide counts detected using
lucigenin-enhanced chemiluminescence in abdominal aorta of
rats treated with 11. It is interesting to note that treatment with
sodium 2,2,5,5-tetramethyl-2,5-dihydropyrrole-1-oxyl-3-carboxylate
(12), a representative nitroxide, also decreased superoxide
production, however this was insufficient to significantly
attenuate intimal thickening (Fig. 2D).
Fig. 2 Representative pictures of uninjured (top panels) and injured
(bottom panels) carotid artery cross-sections (5 mm thick; H & E
staining) taken from SHR allocated to different treatment groups: (A)
saline (120 ml per day), (B) milfasartan, (C) compound 11 or (D)
compound 12 (30 mg kgꢁ1 per day, each) in saline s.c. via an osmotic
mini-pump for 14 days. Lumen (L), intima (I), media (M) and
adventitia (A) are marked on the bottom left panel. Injury was caused
by passing an inflated balloon catheter (Fogarty 2F) through the left
common carotid artery 3 times, with the right, uninjured vessel serving
as a control.
Nitroxide 11 is a water soluble dual action drug. Its dual
actions have been determined in a number of different assays
both in vitro and in vivo. In vivo, 11 possessed anti-hypertensive
actions and decreased free radical production and vascular
injury in SHR. This may result in a novel therapeutic agent
that targets both the hypertension and oxidative stress
components of cardiovascular diseases using one drug.
We thank the Australian Research Council through the
Centres of Excellence Scheme for generous financial support.
NADPH was used to stimulate superoxide production in rat
isolated aorta segments in the presence or absence of compounds
(10, 11) which was detected using lucigenin-enhanced chemi-
luminescence as described previously.15,16 Fig. 1 represents
NADPH-derived superoxide per mg of dry tissue weight
(counts mgꢁ1; details provided in the ESIw) which for control
tissues was 2009 ꢀ 197 counts mgꢁ1. Compound 11 (3 or 10 mM
only) significantly reduced superoxide counts to 535 ꢀ 98 or
221 ꢀ 65 counts mgꢁ1, respectively. All three concentrations
of 10 concentration-dependently decreased superoxide counts
to 991 ꢀ 162, 310 ꢀ 39 or 115 ꢀ 14 counts mgꢁ1, respectively,
indicating that both of these compounds act as antioxidants,
with 11 (R = Bu) performing slightly better in this assay. For
this reason, we chose to progress 11 (R = Bu) to preliminary
in vivo assays.
Notes and references
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Mean arterial pressure (MAP) was measured in conscious,
unrestrained male spontaneously hypertensive rats (SHR)
using telemetry.17 As shown in Table 2, subcutaneous 50%
DMSO had no significant effect on MAP. Importantly, SHR
receiving eprosartan or 11 had significant decreases in MAP
over 7 days, confirming that 11 is an effective antihypertensive.
Finally, we examined the ability of 11 to attenuate the develop-
ment of intimal thickening caused by balloon injury of the carotid
artery, a model for restenosis and atherosclerosis.18 Carotid artery
rubbing in SHR results in a thickening of the intimal layer due to
local production of free radicals, growth factors and cytokines at
the site of damage.18,19 There is also an upregulation of AT1
receptors at the site of injury, which contributes to smooth muscle
cell hypertrophy and hyperplasia.20
Fig. 2 shows representative histological sections from injured
vessels and their respective contralateral uninjured controls. In
the uninjured vessels from rats treated with saline for 2 weeks the
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12083–12085 12085