Synthesis of [125I]-, [2H]-, and [ 3H]-labeled 3-iodothyronamine (T1AM)

Article abstract of DOI:10.1080/00397910500466074

3-Iodothyronamine (T₁AM) is a novel metabolite of thyroid hormone. In HEK-293 cells expressing an orphan G-protein coupled receptor, the trace amine receptor, T₁AM, potently increased cAMP accumulation. In mice, T₁AM rapid

Full text of DOI:10.1080/00397910500466074

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Synthesis of [125I]‐, [2H]‐, and [3H]‐Labeled
3‐Iodothyronamine (T1AM)
Motonori Miyakawa a & Thomas S. Scanlan a
a Departments of Pharmaceutical Chemistry and Cellular and Molecular
Pharmacology , University of California at San Francisco , San Francisco,
California, USA
Published online: 16 Aug 2006.
To cite this article: Motonori Miyakawa & Thomas S. Scanlan (2006) Synthesis of [125I]‐, [2H]‐, and
[3H]‐Labeled 3‐Iodothyronamine (T1AM), Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 36:7, 891-902, DOI: 10.1080/00397910500466074
To link to this article: http://dx.doi.org/10.1080/00397910500466074
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Synthetic Communicationsw, 36: 891–902, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910500466074
Synthesis of [125I]-, [2H]-, and [3H]-Labeled
3-Iodothyronamine (T1AM)
Motonori Miyakawa and Thomas S. Scanlan
Departments of Pharmaceutical Chemistry and Cellular and
Molecular Pharmacology, University of California at San Francisco,
San Francisco, California, USA
Abstract: 3-Iodothyronamine (T1AM) is a novel metabolite of thyroid hormone. In
HEK-293 cells expressing an orphan G-protein coupled receptor, the trace amine
receptor, T1AM, potently increased cAMP accumulation. In mice, T1AM rapidly
induced hypothermia and bradycardia within minutes of administration. These
results suggest the existence of a new signaling pathway, the stimulation of which
leads to rapid physiological and behavioral consequences. Isotope-labeled T1AM
derivatives would be useful to study the biology and pharmacology of T1AM.
Herein we describe efficient syntheses of [125I]-, [2H]-, and [3H]-T1AM.
Keywords: 3-Iodothyronamine (T1AM), [125I]-T1AM, [2H]-T1AM, [3H]-T1AM
INTRODUCTION
3,5,30-Triiodothyronine (T3, Figure 1) is a high-affinity ligand for the nuclear
thyroid hormone (TH) receptors TRa and TRb, whose activation controls
normal vertebrate development and physiology.[1] T3-modulated transcription
of target genes via activation of TRa and TRb is a slow process taking hours
to days to manifest. However, there are many rapid effects associated with TH
that occur in seconds to minutes, precluding TR-mediated transactivation as a
mechanism of action.[2,3] Some examples of these nongenomic effects include
Received in the USA September 15, 2005
Address correspondence to Thomas S. Scanlan, Departments of Pharmaceutical
Chemistry and Cellular and Molecular Pharmacology, University of California at
San Francisco, 600 16th St., San Francisco, CA 94143-2280, USA. Fax: þ1-415-
4763620; E-mail: scanlan@cgl.ucsf.edu
891

892
M. Miyakawa and T. S. Scanlan
Figure 1. The predominant thyroid hormone secreted from the thyroid gland,
triiodothyronine (T3), and novel metabolite, 3-iodothyronamine (T1AM).
sodium channel activation[4] and increased isolated cardiac myocyte contrac-
tile function.[5] Additionally, there is evidence that rapid effects of TH have
therapeutic potential.[6] Patients suffering from congestive heart failure
experienced increased cardiac performance upon a bolus injection of T3.
We previously reported the discovery of 3-iodothyronamine (T1AM,
Figure 1), a novel metabolite of TH.[7] In HEK-293 cells expressing an orphan
G-protein coupled receptor, the trace amine associated receptor[8,9] (TAAR1),
T1AM potently increased cAMP accumulation. In mice, T1AM rapidly
induced hypothermia and bradycardia within minutes of an i.p. injection. In
isolated rat hearts, T1AM rapidly decreased cardiac output. These results
suggest the existence of a new signaling pathway, the stimulation of which
leads to rapid physiological and behavioral consequences. To study the biology
and pharmacology of T1AM, isotope-labeled T1AM derivatives would be very
helpful. Herein we describe efficient syntheses of [125I]-, [2H]-, and [3H]-T1AM.
RESULTS AND DISCUSSION
Synthesis of [125I]-T1AM
[
125I]-T1AM was synthesized by the route outlined in Scheme 1. 4-(Methoxy-
methoxy)phenylboronic acid 3[10] was prepared from 4-bromophenol 1 in two
steps in good yield. Boronic acid 3 was coupled with N-t-Boc-3-iodotyramine
4[11] utilizing stochiomeric copper(II) acetate[12–14] to give biaryl ether 5 in
48% yield. Refluxing 5 with hexamethyldistannane in toluene in the presence
of the catalytic amount of tetrakis(triphenylphoshine)palladium(0) provided
the trimethylstannyl-thyronamine derivative 6[15] in 92% yield, which was the
precursor needed to synthesize [125I]-T1AM. The radioiododestannylation of 6
with [125I]-sodium iodine (1 mCi, carrier free, sp act. 2200Ci/mmol Perkin
Elmer Inc.) was completed within 30min at ambient temperature using Chlora-
mine-T as oxidant.[16] The [125I]-intermediate was deprotected in the presence of
3 N HCl in anhydrous ethyl acetate to give the desired [125I]-T1AM (7) as the
hydrochloride salt. The radioactive purity of the final compound was checked
by exposing the TLC plate to X-ray film. The total radiochemical yield of
[
125I]-T1AM after silica-gel flash chromatography purification was 20%.

Synthesis of [125I]-, [2H]-, and [3H]-Labeled 3-Iodothyronamine (T1AM)
893
Scheme 1. Reagents and conditions: a, MOMCl, NaH, DMF, RT, 92%; b, i) nBuLi,
THF, 2788C, ii) (iPrO)3B, 2788C to RT; iii) HCl, 08C, 71%; c, 3, Cu(OAc)2, iPr2NEt,
Py, CH2Cl2, RT, 48%; d, (SnMe3)2, Pd(PPh3)4, PhMe, reflux, 92%; e, i) 125INa (1 mCi),
Chloramine-T, EtOH, H2O, RT; ii) 3 N HCl-AcOEt, RT.
Synthesis of [2H]-T1AM
The synthesis of [2H]-T1AM was begun with the protection of commercially
available 4-hydroxybenzyl cyanide 8 as the triisopropylsilyl ether 9
(Scheme 2). The benzyl protons of 9 were exchanged with deuterium by
treatment with sodium methoxide in methanol-d4. The deuterium exchange
Scheme 2. Reagents and conditions: a, TIPSCl, imidazole, DMF, RT, 84%; b, i)
NaOMe, CD3OD, RT; ii) LiAl2H4-AlCl3, THF, RT; iii) Boc2O, K2CO3, THF, H2O,
RT, 67%; c, TBAF, THF, RT, 97%; d, NaI, NaOCl, KOH, MeOH, 2208C, 82%; e,
i
3, Cu(OAc)2, Pr2NEt, Py, CH2Cl2, RT, 73%; f, 3 N HCl-AcOEt, RT, 94%.

894
M. Miyakawa and T. S. Scanlan
efficiency was checked by NMR. The di-deuterated intermediate was suffi-
ciently pure and used without any further purification. Reduction of the
di-deuterated intermediate with a lithium aluminum deuteride/aluminum
chloride complex[17] followed by protection of the resulting primary amine
gave the tetra-deuterated tyramine derivative 10 in respectable yield. The
tyramine derivative 10 was treated with tetrabutyl ammonium fluoride, and
the resulting phenol 11 was iodinated with sodium iodide and sodium hypo-
chlorite,[18] providing the mono-iodinated product 12 in good yield.
Copper(II)-mediated coupling of 12 with boronic acid 3 gave biaryl ether
13. Concomitant hydroxyl and amine deprotection in the presence of 3 N
HCl in anhydrous ethyl acetate gave the desired [2H]-T1AM (14) as the hydro-
chloride salt in excellent yield.
Synthesis of [3H]-T1AM
The synthesis of [3H]-T1AM was achieved by reductive amination utilizing
sodium cyanoborotritiride (Scheme 3). Mono-iodinated phenol 16 was syn-
thesized from methyl 2-(4-hydroxyphenyl)acetate 15 with iodine monochloride
and butylamine in moderate yield. Compound 16 was coupled with boronic
acid 3 utilizing the copper(II)-mediated procedure outlined previously to
give biaryl ether 17. Reduction of 17 proceeded satisfactorily by utilizing
DIBAL-H at 2788C to give aldehyde 18. Treatment of 18 with ammonium
acetate in methanol followed by treatment with [3H] sodium cyanoboro-
hydride (10mCi, 3–10 mCi/mg, American Radiolabeled Chemicals Inc.)
gave the [3H]-intermediate. The [3H]-intermediate was deprotected in the
presence of 3 N HCl in anhydrous ethyl acetate to give the desired [3H]-
T1AM (19) as hydrochloride salt. The radioactive purity of final compound
was checked by exposing the TLC plate to X-ray film.
In summary, we have described a synthesis of [125I]-, [2H]-, and [3H]-
T1AM, employing readily available materials and simple reaction conditions.
These compounds should be the valuable tools to study the biology and
pharmacology of T1AM.
Scheme 3. Reagents and conditions: a, ICl, BuNH2, THF, 2788C, 29%; b, 3,
i
Cu(OAc)2, Pr2NEt, Py, CH2Cl2, RT, 32%; c, DBAL-H, THF, 2788C, 58%; d, i)
NH4OAc, NaB3H3CN, MeOH, THF, RT; ii) 3 N HCl-AcOEt, RT.

Synthesis of [125I]-, [2H]-, and [3H]-Labeled 3-Iodothyronamine (T1AM)
895
EXPERIMENTAL
General
1H and 13C NMR spectra were taken on a Varian 400 (400 MHz and 100 MHz
respectively). High-resolution mass spectra were obtained from the depart-
mental mass spectrometry facility. High-resolution mass spectrometry
(HRMS) using electrospray ionization was performed by the National
Bio-organic, Biomedical Mass Spectrometry Resource at UCSF. Anhydrous
THF, DCM, diethyl ether, pyridine, and diisopropyl ethyl amine were
filtered through two columns of activated basic alumina and transferred
under an atmosphere of argon gas in a solvent purification system designed
and manufactured by J. C. Meyer (University of California, Irvine).
Anhydrous DMF was obtained by passing through two columns of activated
molecular sieves. All other anhydrous solvents and reagents were purchased
from Aldrich, Sigma-Aldrich, Fluka, or Acros and were used without any
further purification unless otherwise stated.
1-Bromo-4-(methoxymethoxy)benzene (2)
4-Bromophenol (1) (10.0 g, 57.8 mmol) in DMF (10 mL) was added dropwise
to a stirring slurry of sodium hydride pellets (2.5 g, 61.7 mmol) in DMF
(40 mL) at ambient temperature. The reaction mixture was stirred until the
evolution of hydrogen ceased (30 min). The choloromethyl methyl ether
(4.3 mL, 56.4 mmol) was added during 30 min. The reaction was stirred for
an additional 30 min after which excess sodium hydride was destroyed by
cautious addition of methanol (5 mL). The reaction mixture was diluted
with ether, washed with water and brine, and dried over MgSO4. The crude
product was purified via SiO2 flash chromatography (eluted hexane/ethyl
acetate 20:1) to give the pure product 2 (11.4g, 92% yield). 1H NMR
(400 MHz, chloroform-d) d 7.38 (d, J ¼ 9.0 Hz, 2 H), 6.92 (d, J ¼ 9.0 Hz, 2 H),
5.14 (s, 2 H), 3.46 (s, 2 H); 13C NMR (100 MHz, chloroform-d) d 156.3,
132.3, 118.1, 114.2, 94.5, 56.0; HRMS (EIþ) for C8H9BrO2 calcd.
217.9786, found 217.9764.
4-(Methoxymethoxy)phenylboronic Acid (3)
n-Butyl lithium (11.2 mL, 2.5 M in hexane, 27.9 mmol) dropwise was added to
a stirring solution of 1-bromo-4-(methoxymethoxy)benzene (2) (5.0 g,
23.3 mmol) in THF (120 mL) at 2788C. The reaction mixture was stirred
for 30 min, then triisopropyl borate (7 mL, 30.2 mmol) was added in one
portion. The reaction was stirred at 2788C for 1 h, allowed to warm to
ambient temperature over 4 h, quenched with 1 N HCl (20 mL), and stirred

896
M. Miyakawa and T. S. Scanlan
for 30 min at 08C. The aqueous layer was extracted with ethyl acetate, and
combined organic layers were dried over MgSO4. The crude product was
purified via SiO2 flash chromatography (eluted hexane/ethyl acetate 10:1
1
to 1:1) to give the pure product 3 (3.0 g, 71% yield). H NMR (400 MHz,
chloroform-d) d 8.16 (d, J ¼ 8.6 Hz, 2 H), 7.11 (d, J ¼ 8.6 Hz, 2 H), 5.21
(s, 2 H), 3.52 (s, 2 H); 13C NMR (100 MHz, chloroform-d) d 160.81,
137.45, 115.53, 94.04, 56.16; HRMS (EIþ) for C8H11BO4 [M+H+Na]
calcd. 205.0648, found 205.0650.
tert-Butyl 4-(40-Methoxymethoxyphenoxy)-3-
iodophenethylcarbamate (5)
4-(Methoxymethoxy)phenylboronic acid (3) (365 mg, 2 mmol) and tert-butyl
4-hydroxy-3-iodophenethylcarbamate (4) (363 mg, 1 mmol) were dissolved in
dichloromethane (12 mL) at ambient temperature in a flask flushed with dry
˚
air. A large excess of 4 A powdered molecular sieves were added, and the
mixture was allowed to stir for 10 min with a dry tube attached. Copper(II)
acetate (185 mg, 1 mmol), N,N-diisopropylethylamine (0.9 mL, 5 mmol), and
pyridine (0.4 mL, 5 mmol) were added in succession, and the reaction was
stirred at ambient temperature for overnight. The reaction mixture was
diluted with ether and filtered through celite, and the filtrate was sequentially
washed with 0.5 N HCl, water, and brine, then dried over MgSO4. The crude
product was purified via SiO2 flash chromatography (eluted hexane/ethyl
acetate 5:1) to give the pure product 5 (240 mg, 48% yield). 1H NMR
(400MHz, chloroform-d) d 7.67 (d, J ¼ 1.7 Hz, 1 H), 7.07 (d, J ¼ 8.2 Hz, 1 H),
7.02 (d, J ¼ 9.2 Hz, 2 H), 6.92 (d, J ¼ 9.2 Hz, 2 H), 6.78 (d, J ¼ 8.4 Hz, 1
H), 5.14 (s, 2 H), 3.49 (s, 3 H), 7.07 (d, J ¼ 8.2 Hz, 1 H), 3.34 (m, 2 H),
2.73 (brd t, J ¼ 6.8 Hz, 2 H), 1.44 (s, 9 H); HRMS (EIþ) for C21H26 INO5
[M þ H] calcd. 499.0856, found 499.0847.
tert-Butyl 4-(40-Methoxymethoxy)phenoxy-3-
(trimethylstannyl)phenethylcarbamate (6)
Hexamethylditin (170 mg, 0.52 mmol) and tetrakis(triphenylphoshine)palla-
dium(0) (15 mg, 0.013 mmol) were added to a solution of tert-butyl 4-(40-meth-
oxymethoxyphenoxy)-3-iodophenethyl-carbamate (5) (130 mg, 0.26 mmol) in
toluene (4 mL). The reaction was refluxed for 2 h under argon atmosphere.
After cooling to ambient temperature, the reaction mixture was diluted with
ether, filtered through celite, and concentrated in vacuo. The crude product
was purified via SiO2 flash chromatography (eluted hexane/ethyl acetate
1
10:1) to give the pure product 6 (130 mg, 92% yield). H NMR (400 MHz,
chloroform-d) d7.25 (d, J ¼ 2.2 Hz, 1 H), 7.06 (dd, J ¼ 2.2, 8.4 Hz, 1 H),
7.00 (d, J ¼ 9.2 Hz, 2 H), 6.90 (d, J ¼ 9.2 Hz, 1 H), 6.69 (d, J ¼ 8.4 Hz,

Synthesis of [125I]-, [2H]-, and [3H]-Labeled 3-Iodothyronamine (T1AM)
897
1 H), 5.14 (s, 2 H), 4.58 (brd s, 1H), 3.49 (s, 3H), 3.36 (m, 1 H), 2.76
(t, J ¼ 7.0 Hz, 1 H), 1.44 (s, 9 H), 0.26 (s, 9 H); 13C NMR (100 MHz, chloro-
form-d) d 161.21, 155.84, 153.12, 151.91, 137.12, 133.33, 132.47, 130.32,
120.00, 117.55, 115.93, 95.03, 79.18, 55.94, 41.99, 35.48, 28.42, 29.17;
HRMS (EIþ) for C24H35NO5Sn calcd. 537.1537, found 537.1487.
4-(40-(2-Aminoethyl)-2-[125I]iodophenoxy)phenol Hydrochloride (7)
[
125I]-T1AM
Chloramine-T (20 mL, 4 mg/mL in water, 0.21 mmol), 5% HCl (5 mL), and
125I] sodium iodine (1 mC, carrier free, sp act. 2200Ci/mmol, Perkin Elmer
[
Inc.) was added to a solution of tert-butyl 4-(40-methoxymethoxy)phenoxy-
3-(trimethylstannyl)-phenethylcarbamate (6) (100 mg, 0.19 mmol) in ethanol
(10 mL). in vial. The reaction was allowed to proceed at ambient temperature
for 30 min then concentrated. The reaction mixture was diluted with brine and
extracted with ether. The combined organic layer was passed through a
MgSO4 column and concentrated in vacuo. The mixture was dissolved in a
3 N HCl solution in ethyl acetate (200 mL, anhydrous), and the reaction
mixture was allowed to proceed at ambient temperature for 3 h and concen-
trated in vacuo. The crude product was purified via SiO2 flash chromatography
(eluted ethyl acetate/methanol 1:0 to 2:1 to give the pure product (7)
(20% radioactive yield).
2-(4-(Triisopropyl)silyloxyphenyl)acetonitrile (9)
Triisopropylsilyl chloride (2.1 mL, 10 mmol) was added to a stirred solution of
4-hydroxybenzyl cyanide (8) (1.3 g, 10 mmol) in DMF (5 mL). The reaction
mixture was cooled to 08C, imidazole (1.7 g, 25 mmol) was added, and the
mixture was stirred at 08C for 30 min then allowed to warm to ambient temp-
erature over 24 h. The reaction mixture was diluted with ether and sequentially
washed with 1 N HCl, sat. aq. NaHCO3, and brine, then dried over MgSO4.
The crude product was purified via SiO2 flash chromatography (eluted
1
hexane/ethyl acetate 20:1) to give the pure product 9 (2.4 g, 84% yield). H
NMR (400MHz, chloroform-d) d 7.16 (d, J ¼ 8.4 Hz,
2 H), 6.87
(d, J ¼ 8.4 Hz, 2 H), 3.67 (s, 2 H), 1.25 (m, 3 H), 1.10 (d, J ¼ 8.4 Hz, 18 H);
HRMS (EIþ) for C17H27NOSi calcd. 289.1862, found 298.1859.
tert-Butyl 2-[4-(Triisopropylsilyloxy)phenyl]-
[1,1,2,2,-2H4]ethylcarbamate (10)
Sodium methoxide (3.2 mg, 0.06 mmol) was added to a stirred solution of
2-[4-(Triisopropyl)silyloxyphenyl]acetonitrile (9) (350 mg, 1.2 mmol) in

898
M. Miyakawa and T. S. Scanlan
CD3OD (3 mL) and stirred for 2 h at ambient temperature. The reaction
mixture was concentrated in vacuo and dried under high vacuum pressure.
The residue was dissolved to THF (3 mL) and added to the suspension of
lithium aluminum deuteride (125 mg, 3 mmol) and aluminum chloride
(400 mg, 3 mmol) in THF (7 mL) dropwise. The reaction was stirred at
ambient temperature for 1 h and quenched with water. The reaction mixture
was diluted with ether and filtered through celite, and filtrate was sequentially
washed with brine and then dried over MgSO4. The reaction mixture was con-
centrated in vacuo and dried under high vacuum pressure. Di-tret-butyl dicar-
bonate (290 mg, 1.3 mmol) was added to a stirred solution of the crude mixture
in THF (5 mL) and a solution of potassium carbonate (182 mg, 1.3 mmol) in
water (2.5 mL). After 3 h the reaction mixture was diluted with ether and
sequentially washed with 1 N HCl, water, and brine, then dried over
MgSO4. The crude product was purified via SiO2 flash chromatography
(eluted hexane/ethyl acetate 15:1) to give the pure product 10
(320 mg, 67% yield). 1H NMR (400 MHz, chloroform-d) d 7.02
(d, J ¼ 8.4 Hz, 2 H), 6.81 (d, J ¼ 8.4 Hz, 2 H), 4.47 (brd s, 1 H), 1.41
(s, 9 H), 1.24 (m, 3 H), 1.09 (d, J ¼ 8.4 Hz, 18 H); HRMS (EIþ) for
C22H235H4NO3Si calcd. 397.295, found 397.294.
tert-Butyl 2-(4-Hydroxyphenyl)-[1,1,2,2,-2H4]ethylcarbamate (11)
TBAF (0.89 mL, 1 M in THF, 0.89 mmol) was added dropwise to a stirred
solution of tert-butyl 2-[4-(triisopropylsilyloxy)phenyl]-[1,1,2,2,-2H4] ethyl-
carbamate (10) (320 mg, 0.81 mmol) in THF (10 mL). The reaction mixture
was stirred for 1 h, then diluted with ether. The mixture was washed with
water and brine, then dried over MgSO4. The crude product was purified
via SiO2 flash chromatography (eluted hexane/ethyl acetate 3:1) to give the
1
pure product 11 (190 mg, 97% yield). H NMR (400 MHz, chloroform-d)
d 7.04 (d, J ¼ 8.1 Hz, 2 H), 6.77 (d, J ¼ 8.1 Hz, 2 H), 4.99 (brd s, 1 H),
4.52 (brd s, 1 H), 1.44 (s, 9 H); HRMS (EIþ) for C13H152H4NO3 calcd.
241.1616, found 241.1618.
tert-Butyl 2-(4-Hydroxy-3-iodophenyl)-
[1,1,2,2,-2H4]ethylcarbamate (12)
A stirring solution of tert-butyl 2-(4-hydroxyphenyl)-[1,1,2,2,-2H4] ethyl-
carbamate (11) (110 mg, 0.46 mmol), sodium iodine (72.5 mg, 0.48 mmol),
and 5 N potassium hydroxide (0.1 mL) in methanol (5 mL) was cooled to
2208C. Sodium hypochlorite (0.9 mL, 0.48 mmol) was added to the
mixture over 30 min and the reaction was stirred at 2208C for 1 h. The
reaction mixture was diluted with ether, sequentially washed with 0.1 M
Na2S2O3, water, and brine; and dried over MgSO4. The crude product was

Synthesis of [125I]-, [2H]-, and [3H]-Labeled 3-Iodothyronamine (T1AM)
899
purified via SiO2 flash chromatography (eluted dichloromethane/ethyl acetate
1
20:1) to give the pure product 12 (139 mg, 82% yield). H NMR (400 MHz,
chloroform-d) d 7.49 (s,
1
H), 7.07 (d, J ¼ 8.4 Hz,
1 H), 6.92
(d, J ¼ 8.4 Hz, 1 H), 5.26 (s, 1 H), 4.51 (brd s, 1 H), 1.44 (s, 9 H); HRMS
(EIþ) for C13H214H4INO3[M+H] calcd. 367.0582, found 367.0565.
tert-Butyl 2-[3-Iodo -4-(40-methoxymethoxyphenoxy)phenyl]-
[1,1,2,2,-2H4]ethylcarbamate (13)
4-(Methoxymethoxy)phenylboronic acid (3) (127 mg, 0.7 mmol) and tert-butyl
2-(4-hydroxy-3-iodophenyl)-[1,1,2,2,-2H4]ethylcarbamate (12) (135 mg,
0.37 mmol) were dissolved in dichloromethane (5 mL) at ambient temperature
˚
in a flask flushed with dry air. A large excess of 4 A powdered molecular sieves
was added, and the mixture was allowed to stir for 10 min with a dry tube
attached. Copper (II) acetate (67 mg, 0.37 mmol), N,N-diisopropylethylamine
(0.32 mL, 1.84 mmol), and pyridine (0.15 mL, 1.84 mmol) were added in suc-
cession, and the reaction was stirred at ambient temperature overnight. The
reaction mixture was diluted with ether and filtered through celite, and the
filtrate was sequentially washed with 0.5 N HCl, water, and brine, then dried
over MgSO4. The crude product was purified via SiO2 flash chromatography
(eluted hexane/ethyl acetate 5:1) to give the pure product 13 (135 mg, 73%
yield). 1H NMR (400 MHz, chloroform-d) d 7.67 (brd s, 1 H), 7.07
(d, J ¼ 8.4 Hz, 1 H), 7.02 (d, J ¼ 9.2 Hz, 2 H), 6.92 (d, J ¼ 9.2 Hz, 2 H),
6.74 (d, J ¼ 8.4 Hz, 2 H), 5.15 (brd s, 1 H), 3.49 (s, 3 H), 1.44 (s, 9 H);
HRMS (EIþ) for C21H222H4INO5 [MþH] calcd. 503.1107, found 503.1096.
4-[40-(2-Amino[1,1,2,2,-2H4]ethyl)-20-iodophenoxy]phenol
Hydrochloride (14) [2H]-T1AM
tert-Butyl 2-[3-iodo-4-(40-methoxymethoxyphenoxy)phenyl]-[1,1,2,2,-2H4]
ethyl-carbamate (13) (135 mg, 0.27 mmol) was dissolved in 3 N HCl
solution in ethyl acetate (2 mL, anhydrous), and the reaction mixture was
stirred at ambient temperature over night. The crude mixture was concentrated
in vacuo and dried under high vacuum pressure to give the pure product 14
1
(100 mg, 94% yield). H NMR (400 MHz, DMSO-d6) d 7.83 (brd s, 1 H),
7.77 (d, J ¼ 2.1 Hz, 1 H), 7.21 (dd, J ¼ 2.1, 8.4 Hz, 1 H), 6.82
(d, J ¼ 8.7 Hz, 2 H), 6.77 (d, J ¼ 8.7 Hz, 2 H), 6.71 (d, J ¼ 8.4 Hz, 1 H);
HRMS (EIþ) for C14H210H4INO2 calcd. 359.032, found 359.031.
Methyl 2-(4-Hydroxy-3-iodophenyl)acetate (16)
A stirring solution of methyl 2-(4-hydroxyphenyl)acetate (15) (1.0 g,
6.0 mmol) and n-butylamine (3 mL, 30 mmol) in THF (30 mL) was cooled

900
M. Miyakawa and T. S. Scanlan
to 2788C. Iodine monochloride (6.6 mL, 1 M in dicholoromethane, 6.6 mmol)
was added to the mixture over 1 h and the reaction was stirred at 2788C for
2 h. The reaction mixture was diluted with ether and washed with 0.5 M HCl.
The aqueous layer was extracted with ether, and then the combined organic
layers were sequentially washed with 0.1 M Na2S2O3, water, and brine and
dried over MgSO4. The crude product was purified via SiO2 flash chromato-
graphy (eluted dichloromethane/ethyl acetate 50:1 to 10:1) to give the pure
1
product 16 (735 mg, 29% yield). H NMR (400 MHz, chloroform-d) d 7.58
(d, J ¼ 2.2 Hz, 1 H), 7.14 (dd, J ¼ 2.2, 8.2 Hz, 1 H), 6.90 (d, J ¼ 8.2 Hz, 1
H), 5.51 (brd s, 1 H), 3.70 (s, 3 H), 3.52 (s, 2 H); 13C NMR (100 MHz, chloro-
form-d) d 172.0, 154.1, 138.8, 131.1, 127.8, 115.0, 85.4, 52.2, 39.6; HRMS
(EIþ) for C9H9IO3 [MþH] calcd. 291.9596, found 291.9600.
Methyl 2-[3-Iodo-4-(40-methoxymethoxyphenoxy)phenyl]acetate (17)
4-(Methoxymethoxy)phenylboronic acid (3) (2 g, 11.0 mmol) and methyl
2-(4-hydroxy-3-iodophenyl)acetate (16) (1.7 g, 5.8 mmol) were dissolved in
dichloromethane (70 mL) at ambient temperature in a flask flushed with dry
˚
air. A large excess of 4 A powdered molecular sieves was added, and the
mixture was allowed to stir for 10 min with a dry tube attached. Copper(II)
acetate (1 g, 5.8 mmol), N,N-diisopropylethylamine (5 mL, 29 mmol), and
pyridine (2.4 mL, 29 mmol) were added in succession, and the reaction was
stirred at ambient temperature for overnight. The reaction mixture was
diluted with ether and filtered through celite. The filtrate was sequentially
washed with 0.5 N HCl, water, and brine, then dried over MgSO4. The
crude product was purified via SiO2 flash chromatography (eluted hexane/
1
ethyl acetate 5:1) to give the pure product 17 (1.5 g, 32% yield). H NMR
(400 MHz, chloroform-d) d 7.76 (d, J ¼ 2.2 Hz, 1 H), 7.20 (d, J ¼ 8.2 Hz, 1
H), 7.01 (d, J ¼ 8.7 Hz, 2 H), 6.93 (d, J ¼ 8.7 Hz, 2 H), 6.73 (d, J ¼ 8.2 Hz,
1 H), 5.15 (s, 2 H), 3.70 (s, 3 H), 3.55 (s, 3 H), 3.49 (s, 3 H); 13C NMR
(100 MHz, chloroform-d) d 171.5, 157.3, 156.5, 153.6, 151.2, 140.3, 130.4,
120.6, 120.1, 117.6, 95.0, 87.8, 56.0, 52.0, 40.3; HRMS (EIþ) for
C17H17IO5 [M+H] calcd. 428.0121, found 428.0123.
2-[3-Iodo-4-(40-methoxymethoxyphenoxy)phenyl]acetaldehyde (18)
Diisobutylaluminum hydride (1.15 mL, 1 M in toluene, 1.15 mmol) dropwise
was added to a stirring solution of methyl 2-[3-iodo-4-(40-methoxymethoxy-
phenoxy)phenyl]acetate (17) (214 mg, 0.5 mmol) in THF (5 mL) at 2788C.
The reaction was stirred at 2788C for 1 h and quenched with sat. NH4Cl.
The reaction mixture was diluted with ether and filtered through celite. The
filtrate was sequentially washed with brine, then dried over MgSO4.
The crude product was purified via SiO2 flash chromatography (eluted

Synthesis of [125I]-, [2H]-, and [3H]-Labeled 3-Iodothyronamine (T1AM)
901
hexane/ethyl acetate 4:1) to give the pure product 18 (90 mg, 58% yield). 1H
H), 7.70
NMR (400 MHz, chloroform-d) d 9.74 (t, J ¼ 2.0 Hz,
2
(d, J ¼ 2.0 Hz, 1 H), 7.08 (dd, J ¼ 2.0, 8.0 Hz, 1 H), 7.03 (d, J ¼ 8.8 Hz, 2
H), 6.94 (d, J ¼ 8.8 Hz, 2 H), 6.77 (d, J ¼ 8.0 Hz, 1 H), 5.15 (s, 2 H), 3.64
(d, J ¼ 2.0 Hz, 2 H), 3.50 (s, 3 H); 13C NMR (100 MHz, chloroform-d)
d 198.55, 156.90, 153.73, 150.86, 140.61, 130.72, 128.15, 120.67, 120.26,
117.64, 94.93, 88.21, 55.98, 48.99; HRMS (EIþ) for C16H15IO4[M+H]
calcd. found 398.0012.
4-[4-(2-Amino[2-3H]ethyl]-2-iodophenoxy)phenol Hydrochloride
(19) [3H]-T1AM
Ammonium acetate (100 mg, 1.29 mmol) in methanol (5 mL) in vial was added
a
solution of 2-(3-iodo-4-(40-methoxymethoxyphenoxy)phenyl)acet-
to
aldehyde (20) (100 mg, 0.25 mmol) in THF (5 mL). The reaction was
allowed to proceed at ambient temperature for 3 h, then [3H] sodium cyano-
borohydride (10 mCi, 3–10 mCi/mg, American Radiolabeled Chemicals
Inc.) in methanol (200 mL) was added. After 2 h the reaction was added to
3 N HCl solution in ethyl acetate (200 mL, anhydrous), and the reaction
mixture was allowed to proceed at ambient temperature for 1 h and concen-
trated in vacuo. The crude product was purified via SiO2 flash chromatography
(eluted ethyl acetate/methanol 1:0 to 2:1 to give the pure product 19
(0.4% radioactive yield).
ACKNOWLEDGMENT
This work was supported by a grant from the National Institutes of Health
(DK-52798).
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