Trace amine-associated receptor 1 (TAAR1) is activated by amiodarone metabolites

Article abstract of DOI:10.1016/j.bmcl.2008.08.013

Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabolites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR₁). This result demonstrates a novel molecular target for amiodarone derivatives with potential clinical significance.

Full text of DOI:10.1016/j.bmcl.2008.08.013

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5920–5922
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Trace amine-associated receptor 1 (TAAR1) is activated
by amiodarone metabolites
Aaron N. Snead a, Motonori Miyakawa b, Edwin S. Tan a, Thomas S. Scanlan b,c,
*
a Graduate Program in Chemistry and Chemical Biology, University of California at San Francisco, 600 16th Street, San Francisco, CA 94143, USA
b Departments of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, University of California at San Francisco, 600 16th Street, San Francisco, CA 94143, USA
c Department of Physiology & Pharmacology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide
variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential
amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabo-
lites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G pro-
tein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR1). This result demonstrates a
novel molecular target for amiodarone derivatives with potential clinical significance.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 June 2008
Revised 4 August 2008
Accepted 5 August 2008
Available online 9 August 2008
Keywords:
Amiodarone
Trace amine-associated receptor 1
TAAR1
G protein-coupled receptors
Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) has
been available in the United States since 1985 and its biological
activity and pharmacokinetics have been well studied. It blocks
myocardial potassium channels and inhibits adrenergic receptors1
as well as type-1 and type-2 50deiodinases.2 Despite what is
known, there are numerous side effects of amiodarone use in hu-
mans that suggest additional activities beyond the identified tar-
gets of amiodarone. Amiodarone is known to be a substrate of
the cytochrome P450 enzyme group. One major observed metabo-
lite of amiodarone, desethylamiodarone, has antiarrhythmic prop-
erties of its own3 as well as distinct antagonistic effects on the
thyroid hormone receptor.4
By comparison to the biosynthesis and metabolism of thyrox-
ine, thyroid hormone (TH), we hypothesized that amiodarone
treatment may result in multiple other amiodarone metabolites
with potential biological activity. The hypothetical amiodarone
metabolites would result as products of known deiodination and
desethylation pathways in the body.3,4 As iodinated benzofuran
derivatives, these amiodarones contain significant structural simi-
larity to TH, and a recently described class of thyroid hormone
metabolites, iodothyronamines.5
matic and rapid pharmacological properties in rodents when
administered exogenously,5 including significant effects on cardiac
performance. While no direct link has been made between T1AM
action with TAAR1 and the observed pharmacology, a correlation
between the potency of several T1AM derivatives against TAAR1
and their ability to induce T1AMs pharmacological effects has been
noted.6 This suggests T1AM activity with TAAR1 may result in the
observed pharmacology.
Given that both amiodarone and thyronamines are both known
to have a variety of cardiac effects and both share significant struc-
tural similarities we sought to understand whether this clinically
used TH derivative, amiodarone or its potential metabolites, may
also target TAAR1. To test this idea, a panel of eight potential ami-
odarone metabolites was synthesized (Scheme 1 and Supplemental
Information). These derivatives include all possible permutations
of amiodarone desethylation and deiodination (Table 1). The total
panel of eight amiodarone derivatives (compounds 2–9) plus the
parent compound amiodarone 1 was then screened against several
mammalian homologs of TAAR1. Specifically, we tested these com-
pounds for both agonist and antagonist activity against mouse, rat
and a chimeric human TAAR1. This screen evaluated the amount of
intracellular cAMP levels induced by the amiodarone analogs by
stimulation of TAAR1, a GPCR coupled to a stimulatory G-protein
(GS).
Iodothyronamines rapidly and potently activate a novel family
of orphan G protein-coupled receptors (GPCRs) the trace amine-
associated receptors (TAARs). The most thoroughly characterized
of the thyronamines, T1AM, is an endogenous compound with dra-
When screened against rat TAAR1 (rTAAR1) four of the potential
amiodarone metabolites, compounds 6, 7, 8, and 9 demonstrated
significant activity against rat TAAR1 (rTAAR1) (Fig. 1a). These com-
* Corresponding author. Tel.: +1 503 494 9292; fax: +1 503 494 9275.
pounds demonstrated specific agonistic activity at doses of 10 lM
E-mail address: scanlant@ohsu.edu (T.S. Scanlan).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.013

A. N. Snead et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5920–5922
5921
R1
R1
R1
O
OH
O
O
O
O
O
a
d
N
H
O
b
NH2
R2
O
O
10
7-9
13
O
c
f
R1
R1
OH
R2
O
O
O
O
O
Br
N3
e
R2
R2
O
O
O
10-12
14,15
16,17
g
i
R1
R1
O
O
O
O
O
O
N
N
N
H
R2
R2
R2
h
O
O
O
4-6
1-3
18-20
Scheme 1. Synthesis of amiodarone panel. For compounds 1, 4, 7, 12, 15, 17, and 20, R1 = R2 = I. For compounds 2, 5, 8, 11, 14, 16, and 19, R1 = H and R2 = I. For compounds 3,
6, 9, 10, and 18, R1 = R2 = H. Reagents and conditions: (a) Cs2CO3, BocNHCH2CH2OMs, DMF, 50 °C; (b) 3 N anhydrous HCl in EtOAc; (c) NaI, NaOCl, 5M KOH, MeOH; (d) EtBr2,
K2CO3, DMF; (e) NaN3, DMF; (f) PPh3, THF/H2O; (g) N-(2-chloroethyl)-N-ethylbenzylamine HCl, NaI, K2CO3, DMF; (h) i—5, 1-chloroethyl carbonochloride, EtCl2, reflux, 2 h; ii—
MeOH, reflux, 1 h, 3 N HCl/EtOAc; (i) NaI, Et2NCH2CH2Cl.HCl, K2CO3, DMF, RT.10
Table 1
Amiodarone panel
Lastly, the amiodarone panel was screened against a chimeric
rat–human TAAR1 (r-hTAAR1). This chimera, as described previ-
ously,7 was generated by exchanging portions of the N terminus
(residues 1–20), C terminus (residues 305–340), and third intracel-
lular loop (residues 204–258) of the wild-type human sequence for
that of rTAAR1 to enhance expression and plasma membrane
trafficking of the receptor.7 Importantly, all of the transmembrane
domains from the human TAAR1 were retained. The chimeric
r-hTAAR1 receptor has been shown to respond similarly to the wild
type human TAAR1 (hTAAR1) in regard to ligand specificity and
potency.7b,8 No published reports, however, have verified the
complete ligand profile of this chimeric receptor as compared with
the reported activity of wild-type hTAAR1. In our screen of the
r-hTAAR1 receptor none of the amiodarones demonstrate any
significant agonistic or antagonistic activity at any of the doses
tested (Fig. 1c). Despite the lack of amiodarone activity against
the r-hTAAR1 chimera follow-up studies with the WT receptor
are warranted because of the wide ranging ligand profiles between
the TAAR homologs.6,9 Despite our observations amiodarones
could have activity with the WT hTAAR1.
R1
O
O
R3
N
R2
R4
O
Compound
R1
R2
R3
R4
Ami
dE-Ami
ddE-Ami
dI-Ami
dEdI-Ami
ddEdI-Ami
ddI-Ami
dEddI-Ami
ddEddI-Ami
1
4
7
2
5
8
3
6
9
I
I
I
I
I
I
H
H
H
Et
I
I
H
H
H
H
H
H
Et
Et
H
Et
Et
H
Et
Et
H
H
H
Et
H
H
Et
H
H
or below with the most potent analog 7, ddE-Ami, giving an ob-
served half maximal effective concentration (EC50) of 1 M (data
not shown). The other five amiodarone derivatives (Ami, dE-Ami,
dI-Ami, dEdI-Ami, and ddI-Ami) did not display any antagonistic
activity when tested at 10 lM in competition with the reported
l
In summary, we synthesized several potential amiodarone
metabolites, products of desethylation and/or deiodination, and
found that several act as agonists against both mouse and rat
TAAR1 and several also weakly antagonize mTAAR1. By looking at
the specific compounds that demonstrated specific activity with
either the rat or mouse TAAR a striking structure–activity relation-
ship was observed. As iodine content increases and ethylation state
decreases compounds gain efficacy and potency with rTAAR1 and
amiodarones with no iodines, independent of ethylation state,
are weak agonists of mTAAR1. However, none of these compounds
had activity against the r-hTAAR1 chimera. Given that the usual
therapeutic serum level of amiodarone is low micromolar, with
significant accumulation in adipose and other tissues, our data
potentially have clinical relevance. These results demonstrate a no-
vel molecular target for amiodarone derivatives, at least in rodents,
agonist T1AM at its EC50 concentration of 33 nM.
To extend the conclusions of amiodarone activity, the amioda-
rone panel was tested against another TAAR1 homolog the mouse
TAAR1 (mTAAR1). As was seen for rTAAR1 several of the amiodaro-
nes showed significant agonist activity at 10 lM concentration. A
different subset of four compounds (compounds 3, 6, 8, and 9)
demonstrated significant but partial or weak agonistic activity
against m TAAR1 (Fig. 1b). Additionally, distinct from the amioda-
rone activity with rTAAR1, several compounds in the amiodarone
panel appear to also demonstrate partial or weak antagonistic
activity (>10 lM IC50 against mTAAR1) (data not shown).

5922
A. N. Snead et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5920–5922
125
100
75
50
25
0
100 nM
1 μM
10
M
μ
rTAAR1
125
100
75
50
25
0
mTAAR1
125
100
75
50
25
0
r-hTAAR1ch
Figure 1. HEK293 cells stably transfected with either (a) rTAAR1, (b) mTAAR1, (c) r-hTAAR1, or empty pcDNA3 vector were harvested in Krebs–Ringer–Hepes buffer (KRH) and
incubated in KRH with 133 M IBMX and 3 L of the test compound, forskolin (10 M), or vehicle (dimethly sulfoxide, DMSO) for 1 h at 37 °C. The cells were boiled and the
l
l
l
cell lysate was analyzed for cAMP content by use of the Hithunter cAMP XS kit (DiscoveRX, Fremont, CA). Data were reported as percent maximal stimulation of reported
agonists of each TAAR1 homolog (T1AM for rat and mouse TAAR1 and phenethylamine (PEA) for r-hTAAR1). Data were plotted and analyzed with Prism software (Graphpad,
San Diego, CA). Standard error of the mean was calculated for at least three separate experiments performed in triplicate.10
2. Basaria, S.; Cooper, D. S. Am. J. Med. 2005, 118, 706.
3. (a) Plomp, T. A.; van Rossum, J. M.; Robles de Medina, E. O.; van Lier, T.;
and suggest another family of receptors amiodarone may target in
vivo.
Maes, R. A. Arzneim. Forsch. 1984, 34, 513; (b) Wiersinga, W. M.; Trip, M. D.
Postgrad. Med. J. 1986, 62, 909; (c) Wiersinga, W. M. Eur. J. Endocrinol. 1997,
137, 15.
Acknowledgments
4. (a) Latham, K. R.; Sellitti, D. F.; Goldstein, R. E. J. Am. Coll. Cardiol. 1987, 9, 872;
(b) Bakker, O.; van Beeren, H. C.; Wiersinga, W. M. Endocrinology 1994, 134,
We thank the David K. Grandy Lab for the donation of the r-
hTAAR1 cell line. This work was supported by fellowship from
the Ford Foundation (A.N.S.), and a grant from the National Insti-
tutes of Health (DK52798, T.S.S.).
1665; (c) van Beeren, H. C.; Bakker, O.; Wiersinga, W. M. Mol. Cell. Endocrinol.
1995, 112.
5. Scanlan, T. S.; Suchland, K. L.; Hart, M. E.; Chiellini, G.; Huang, Y.; Kruzich, P. J.;
Frascarelli, S.; Crossley, D. A.; Bunzow, J. R.; Ronca-Testoni, S.; Lin, E. T.; Hatton,
D.; Zucchi, R.; Grandy, D. K. Nat. Med. 2004, 10, 638.
6. Hart, M. E.; Suchland, K. L.; Miyakawa, M.; Bunzow, J. R.; Grandy, D. K.; Scanlan,
T. S. J. Med. Chem. 2006, 49, 1101.
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E. A.; Bunzow, J. R.; Arttamangkul, S.; Sonders, M. S.; Grandy, D. K. J. Pharmacol.
Exp. Ther. 2007, 321, 178.
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Wainscott, D. B.; Little, S. P.; Yin, T.; Tu, Y.; Rocco, V. P.; He, J. X.; Nelson, D. L. J.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.013.
9. Tan, E. S.; Miyakawa, M.; Bunzow, J. R.; Grandy, D. K.; Scanlan, T. S. J. Med. Chem.
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References and notes
10. Complete Experimental and Synthetic Methods can be found in the
Supplementary data.
1. Hartong, R.; Wiersinga, W. M.; Plomp, T. A. Horm. Metab. Res. 1990, 22,
85.