Suppression of store overload-induced calcium release by hydroxylated metabolites of carvedilol

Article abstract of DOI:10.1016/j.bmcl.2015.11.008

Carvedilol is a drug widely used in the treatment of heart failure and associated cardiac arrhythmias. A unique action of carvedilol is its suppression of store overload-induced calcium release (SOICR) through the cardiac ryanodine receptor (RyR2), which can trigger ventricular arrhythmias. Since the effects of carvedilol metabolites on SOICR have not yet been investigated, three carvedilol metabolites hydroxylated at the 3-, 4′ and 5′-positions were synthesized and assayed for SOICR inhibition in mutant HEK 293 cells expressing the RyR2 mutant R4496C. This cell line is especially prone to SOICR and calcium release through the defective RyR2 channel was measured with a calcium-sensitive fluorescent dye. These results revealed that the 3- and 4′-hydroxy derivatives are slightly more effective than carvedilol in suppressing SOICR, while the 5′-analog proved slightly less active. Metabolic deactivation of carvedilol via these hydroxylation pathways is therefore insignificant.

Full text of DOI:10.1016/j.bmcl.2015.11.008

Bioorganic & Medicinal Chemistry Letters 26 (2016) 149–153
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Suppression of store overload-induced calcium release by
hydroxylated metabolites of carvedilol
a,
Thomas Malig a, Zhichao Xiao b, S. R. Wayne Chen b, , Thomas G. Back
⇑
⇑
a Department of Chemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada
b Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Carvedilol is a drug widely used in the treatment of heart failure and associated cardiac arrhythmias. A
unique action of carvedilol is its suppression of store overload-induced calcium release (SOICR) through
the cardiac ryanodine receptor (RyR2), which can trigger ventricular arrhythmias. Since the effects of car-
vedilol metabolites on SOICR have not yet been investigated, three carvedilol metabolites hydroxylated at
the 3-, 40 and 50-positions were synthesized and assayed for SOICR inhibition in mutant HEK 293 cells
expressing the RyR2 mutant R4496C. This cell line is especially prone to SOICR and calcium release
through the defective RyR2 channel was measured with a calcium-sensitive fluorescent dye. These
results revealed that the 3- and 40-hydroxy derivatives are slightly more effective than carvedilol in sup-
pressing SOICR, while the 50-analog proved slightly less active. Metabolic deactivation of carvedilol via
these hydroxylation pathways is therefore insignificant.
Received 25 September 2015
Revised 31 October 2015
Accepted 4 November 2015
Available online 5 November 2015
Keywords:
Carvedilol metabolites
Store overload-induced calcium release
Ryanodine receptor
HEK 293 mutant cell line (R4496C)
Ó 2015 Elsevier Ltd. All rights reserved.
Heart failure patients are at high risk of sudden death from ven-
tricular arrhythmias (VAs). As a result, considerable effort has been
directed toward the development of new antiarrhythmic drug
therapies, but several clinical trials have demonstrated limited
survival benefits to the patients.1–3 Excessive stimulation of
b-adrenergic receptors has been implicated in VAs and special
attention has been focused on the use of antagonists of b-receptors
as antiarrhythmic agents.4–6 Carvedilol (1) is a nonselective inhibi-
ing overload of Ca2+ in the SR.18–25 This store overload-induced cal-
cium release (SOICR) then triggers delayed after depolarizations
(DADs),26–32 leading to catecholaminergic polymorphic ventricular
tachycardias (CPVTs) as well as to ventricular tachyarrhythmias
and sudden death.4,5,33,34 The inhibition of SOICR by various drugs
can be conveniently measured with a human embryonic kidney
cell line (HEK 293) that expresses
a mutant RyR2 channel
(R4496C) which makes it especially prone to SOICR. The sponta-
neous release of Ca2+ through this channel can then be observed
by means of a fluorescent indicator (fura 2/AM) that responds to
the presence of Ca2+. This cell line has been used extensively for
studying the mechanism of SOICR and for screening inhibitors of
SOICR.16,17
It is noteworthy that carvedilol is unique among the family of b-
blockers in its ability to suppress SOICR16 both in the mutant HEK
293 cell line and in a mouse model20–22 characterized by the same
R4496C mutation of the RyR2 receptor. Moreover, several carvedi-
lol analogs have been prepared, which display a 1000-fold attenu-
ated inhibition of the b-adrenergic receptor, but show comparable
SOICR inhibition relative to carvedilol itself. These analogs are
therefore free of the side effects associated with the excessive b-
blockade resulting from therapeutically effective doses of carvedi-
lol. Furthermore, they are equally or better capable of protecting
mice with the defective RyR2 from VAs induced by the administra-
tion of stimulants (caffeine and epinephrine).16
tor of both
a- and b-adrenergic receptors, with exceptionally
potent (nanomolar) b-blocking properties.7–10 It has also been sug-
gested that its antioxidant properties may contribute to its salutary
effects.11–15 Consequently, carvedilol has frequently served as the
drug therapy of choice for the treatment of heart failure and in
the prevention of associated VAs.
However, other b-blockers fail to produce the antiarrhythmic
effects of carvedilol, suggesting that b-blockade alone is not the
principal source of its salutary effects. More recently, it has been
demonstrated that a major benefit of carvedilol stems from its abil-
ity to regulate the passage of Ca2+ ions from the sarcoplasmic retic-
ulum (SR) into the cytosol of cardiomyocytes via the cardiac
ryanodine receptor (RyR2).16,17 Certain defects in RyR2 result in
spontaneous and sudden release of Ca2+ through this channel dur-
⇑
Corresponding authors. Tel.: +1 403 220 4235 (S.R.W.C.), +1 403 220 6256
(T.G.B.).
The metabolism and pharmacokinetics of carvedilol have been
E-mail addresses: swchen@ucalgary.ca (S.R.W. Chen), tgback@ucalgary.ca
(T.G. Back).
extensively studied in dogs, rats and mice,35 as well as in man.36–40
http://dx.doi.org/10.1016/j.bmcl.2015.11.008
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

150
T. Malig et al. / Bioorg. Med. Chem. Lett. 26 (2016) 149–153
β-amino alcohol linker
6'
O
O
O
N
H
5'
4'
O
N
H
5
8
OH
OH
4
OH
MeO
3
2
6
7
MeO
2'
3'
N
H
2
N
H
1
catechol
carbazole
Carvedilol (1)
O
O
OH
O
N
O
N
H
H
OH
OH
MeO
OH
MeO
N
H
N
3
4
H
Figure 1. Structures of carvedilol and three of its hydroxylated metabolites.
O
O
OH
O
OH
O
Cl
89%
MCPBA
30%
O
N
H
N
H
N
H
5
7
O
6
53%
H2N
2
MeO
8
1. NH4OH
via ref. 52
O
HO
6
Br
2.
3
10%
MeO
OH
OH
MeO
CHO
10
9
1. NH4OH
via ref. 52
O
HO
CHO
2. 6
Br
4
12%
MeO
MeO
12
11
Scheme 1. Synthesis of hydroxycarvedilol derivatives 2–4.
Only a small portion of administered carvedilol is excreted intact.
Among the principal metabolites are a series of hydroxylated deriva-
tives, including the 3-, 40- and 50-derivatives 2–4, respectively, pro-
duced by cytochrome P450-mediated oxidation of either the
carbazole moiety or the catechol ring. The formation of glucuronides,
demethylated products and oxidative cleavage products has also
been observed. The pharmacology of hydroxylated carvedilol
metabolites has been investigated and they are reported to inhibit
the migration and proliferation of smooth muscle cells41 and to act
as antioxidants. In particular, the antioxidant properties of the 3-
hydroxy carvedilol derivative 2 (also known as BM-910228 or SB
211475) are associated with infarct-limiting and antiapoptotic activ-
ity toward cardiomyocytes,42 cardioprotective effects in the preven-
tion of mitochondrial damage from oxidative stress and ischemic
reperfusion injury,43–47 the suppression of radical-induced contrac-
tile dysfunction,48 neuroprotective activity49 and attenuation of
endothelin-1 production.50 Thus, these metabolites may contribute
to the therapeutic properties of carvedilol. However, their ability to
inhibit SOICR has not yet been reported. Since SOICR is a key process
in triggering cardiac arrhythmias, it was of interest to determine
whether the hydroxylated metabolites of carvedilol could suppress
SOICR as effectively as their parent compound 1. We now report the
synthesis of 2–4 (Fig. 1) and their inhibitory effects upon SOICR.
The syntheses of 2–4 were performed as illustrated in Scheme 1
and procedures, along with characterization data for all three
metabolites, are provided in Supporting information. Thus,
4-hydroxycarbazole
5 was converted into the epoxide 6 as
reported previously.16,17 Oxidation of 6 with m-chloroperbenzoic
acid (MCPBA) provided the 3-hydroxy derivative 7 directly, albeit
in low yield.51 Ring-opening of the epoxide moiety of 7 with the
amine
8
then afforded 3-hydroxycarvedilol 2. The 40- and
50-hydroxy derivatives 3 and 4 were obtained by minor variations
of the procedure reported by Senthilkumar et al.52 Thus, vanillin
(9) and isovanillin (11) were converted into phenols 10 and 12,
respectively, by alkylation with 1,2-dibromoethane, followed by
Baeyer–Villiger oxidation and hydrolysis of the corresponding
formate esters, as described previously.52 Substitution of the bro-
mide moieties of 10 and 12 with ammonium hydroxide, followed
by reaction of the resulting primary amines with epoxide 6 then
afforded the desired products in low yields.53
All products subjected to bioassay had purities >95% based
on HPLC analysis (Novapak C18 reversed phase column,

T. Malig et al. / Bioorg. Med. Chem. Lett. 26 (2016) 149–153
151
Figure 4. Inhibition of SOICR by 50-hydroxycarvedilol (4).
Figure 2. Inhibition of SOICR by 3-hydroxycarvedilol (2).
very similar to that of carvedilol (1), with an IC50 of 15.9
l
mol.17
Figures 2a, 3a and 4a, where the decrease in the percentage of cells
displaying SOICR is plotted against the dose of the metabolite, indi-
cate that all of the metabolites caused noticeable inhibition of
SOICR at concentrations of ca. 10
inhibited SOICR by ca. 70% at a concentration of 10
effected complete suppression at 30 mol. Metabolites 3 and 4
showed lower inhibition of only ca. 10–30% at 10 mol, but 3 com-
pletely inhibited SOICR at the 30 mol concentration, while 4 only
suppressed ca. 80% of SOICR at the concentration of 30 mol. Thus,
lmol and higher. Compound 2
lmol and
l
l
l
l
2 appears to be slightly more potent than 3 or 4, while 4 is the least
effective of the three compounds.
Figures 2b, 3b, and 4b show representative traces of fura-2
ratios of single cells before and after the addition of increasing con-
centrations of metabolites 2–4, respectively. It can be seen that all
three metabolites 2–4 markedly reduced the amplitude of sponta-
neous Ca2+ oscillations at the 10
l
mol concentration. Spontaneous
Ca2+ oscillations were completely abolished by all of the metabo-
lites 2–4 at the 30 mol concentration. Furthermore, cells pre-
l
treated with metabolites 2–4 remained responsive to caffeine, an
agonist of RyR2, indicating that these metabolites do not com-
pletely block the RyR2 channel in HEK293 cells. Interestingly,
metabolite 2, but not metabolites 3 or 4, also caused an elevation
Figure 3. Inhibition of SOICR by 40-hydroxycarvedilol (3).
in cytosolic Ca2+ at the 30
lmol concentration and reduced the
amplitude of caffeine-induced Ca2+ release.
We have previously shown that carvedilol suppresses
RyR2-mediated SOICR by directly modulating the gating of the
RyR2 channel.16 We found that carvedilol reduces the open duration
and increases the open frequency of single RyR2 channels.16 This
suppressive effect of carvedilol on SOICR is unlikely to be due to
the depletion of intracellular Ca2+ stores as a result of carvedilol’s
inhibitionof the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA),
as the addition of caffeine can still trigger a robust Ca2+ release from
carvedilol-treated cells. Furthermore, carvedilol treatment does not
induce elevation in cytosolic Ca2+. However, the effect of 3-hydrox-
ycarvedilol differs from those of carvedilol and those of 40 and 50
hydroxycarvedilol. 3-Hydroxycarvedilol induced an elevation of
the cytosolic Ca2+ level and diminished the caffeine-induced Ca2+
release, suggesting that 3-hydroxycarvedilol may cause Ca2+ leakage
or inhibit SERCA activity, leading to the elevation of cytosolic Ca2+
3.9 ꢀ 150 mm, with isocratic acetonitrile–aqueous buffer (0.02 M
NH4OAc and 0.012 M formic acid) 70:30 as the solvent, with a flow
rate of 0.8 mL/min; UV detector at 254 nm).
The bioassays of metabolites 2–4 were conducted by the
method described previously,16,17 employing the HEK293 cell line
that expresses the RyR2 mutant (R4496C). Spontaneous Ca2+ oscil-
lations (SOICR) occurred readily in these cells. The suppression of
SOICR caused by carvedilol metabolites was determined by mea-
suring the percentage of HEK293 cells that displayed spontaneous
Ca2+ oscillations in the presence of increasing concentrations (0, 3,
10, and 30 lM) of carvedilol metabolites using the fluorescence
Ca2+ indicator, fura-2 AM, and single cell Ca2+ imaging.
The results of the SOICR bioassays are shown in Figures 2–4 and
Table 1. The table indicates that the IC50 values of 9.4 0.3,
10.4 0.0, and 19.4 3.6 for compounds 2–4, respectively, are all

152
T. Malig et al. / Bioorg. Med. Chem. Lett. 26 (2016) 149–153
Table 1
SOICR inhibition by carvedilol metabolites
Compound
Structure
IC50
(
l
M) SEM
Repeats (n)
No. of cells
375
O
O
O
N
H
OH
15.9 2.517
6
MeO
Carvedilol 1
N
H
O
N
H
OH
OH
MeO
2
9.4 0.3
3
262
N
H
O
O
O
N
H
OH
OH
MeO
OH
OH
3
4
10.4 0.0
3
3
237
264
N
H
O
N
H
MeO
19.4 3.6
N
H
and depletion of intracellular Ca2+stores. On the other hand, the
effects of 40- and 50-hydroxycarvedilol are similar to those of
carvedilol.
Supplementary data
Supplementary data (NMR spectra of 2–4) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmcl.2015.11.008.
In the HEK293 cell experiments, we intended to show whether
acutely-applied carvedilol metabolites are capable of suppressing
spontaneous Ca2+ oscillations (SOICR) in a concentration-depen-
dent manner. HEK293 cells were perfused continuously with
increasing concentrations of these drugs. We did not pre-incubate
the cells with carvedilol derivatives. Hence, relatively high concen-
trations of the drugs were used to suppress spontaneous Ca2+
release. It should be noted that, however, carvedilol has a high
degree of lipophilicity and a large volume of distribution (132 L),7
indicating that it accumulates over time in extravascular tissues
such as cardiac muscle.8 In line with this view, the concentrations
of carvedilol in the heart are five to sevenfold higher than those in
the plasma.9,10 It is therefore possible that carvedilol metabolites
may accumulate in tissues during long-term treatment and con-
tribute to the observed beneficial effects of carvedilol.
In conclusion, these results indicate that CYP-450-mediated
hydroxylation of the 3-, 40- or 50-position of carvedilol has a rela-
tively minor effect on the inhibitory action of the parent carvedilol
on SOICR. While the 3- and 40-hydroxy derivatives 2 and 3 are
slightly more potent than the parent drug 1, the 50-analog 4 reveals
slightly diminished activity. Metabolic deactivation of carvedilol
via these hydroxylation pathways is therefore minimal, although
clearance of the drug may be increased through formation of more
polar and more water-soluble metabolites.
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Acknowledgments
T.G.B. thanks the Natural Sciences and Engineering Research
Council of Canada for financial support. T.M. was the recipient of
a Department of Chemistry Entrance Award and a Queen Elizabeth
II Scholarship from the University of Calgary. S.R.W.C. acknowl-
edges financial support from the Heart and Stroke Foundation of
Canada, the Canadian Foundation for Innovation, and the Heart
and Stroke Foundation/Libin Cardiovascular Institute of Alberta
Professorship in Cardiovascular Research. None of the sponsors
played a role in the design, execution or interpretation of the
results reported herein, or in the writing of this communication.

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