The synthesis of 13C6-labeled L-thyronine, 3,5-diiodothyronine, 3,3′,5-triiodothyroacetic acid and 3,3′,5,5′-tetraiodothyroacetic acid

Article abstract of DOI:10.1016/j.tet.2020.131352

The effects of thyroid hormone metabolites (THMs) other than T3, rT3 and T4 are largely unknown, partially due to the lack of adequate methods. For adequate analysis, internal standards for all THMs are essential, but unfortunately not commercially available. Reported approaches for the synthesis of T0, 3,5-T2, TA3 and TA4 lack sensitivity and/or are not adaptable for ¹³C₆-labeled analogues. In this paper, we describe the synthesis of four ¹³C₆-labeled THMs, T0-¹³C₆, 3,5-T2-¹³C₆, TA3-¹³C₆, TA4-¹³C₆. Starting with ¹³C₆-bromo-benzene, a short and versatile synthesis route was developed in which the formation of the diphenyl ether by a Chan-Lam coupling reaction was fundamental.

Full text of DOI:10.1016/j.tet.2020.131352

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13
The synthesis of C -labeled l-thyronine, 3,5-diiodothyronine, 3,3′,5-
6
triiodothyroacetic acid and 3,3′,5,5′-tetraiodothyroacetic acid
Gregor S. Pilzak, Rutchanna Jongejan, Toine van den Bergh, Robin P. Peeters,
Tommi Meulemans, Yolanda B. de Rijke
PII:
S0040-4020(20)30507-X
https://doi.org/10.1016/j.tet.2020.131352
TET 131352
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 23 April 2020
Revised Date: 17 June 2020
Accepted Date: 19 June 2020
Please cite this article as: Pilzak GS, Jongejan R, van den Bergh T, Peeters RP, Meulemans T, de Rijke
13
YB, The synthesis of C -labeled l-thyronine, 3,5-diiodothyronine, 3,3′,5-triiodothyroacetic acid and
6
3,3′,5,5′-tetraiodothyroacetic acid, Tetrahedron (2020), doi: https://doi.org/10.1016/j.tet.2020.131352.
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The synthesis of 13C6-labeled L-thyronine,
3,5-diiodothyronine, 3,3',5-triiodothyroacetic
acid and 3,3',5,5'-tetraiodothyroacetic acid
Leave this area blank for abstract info.
Gregor S. Pilzaka , Rutchanna M.S. Jongejanb, Toine van den Bergha, Robin P. Peetersc,d, Tommi
Meulemansa and Yolanda B. de Rijkeb,d
aMercachem B.V. Kerkenbos 1013, 6503 GE Nijmegen, The Netherlands
bDepartment of Clinical Chemistry, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
cDepartment of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
dAcademic Centre for Thyroid Diseases, Erasmus MC University Medical Center, Rotterdam, Dr. Molewaterplein 40 3015 GD Rotterdam, The
Netherlands

1
Tetrahedron
journal homepage: www.elsevier.com
The synthesis of 13C6-labeled L-thyronine, 3,5-diiodothyronine, 3,3',5-
triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid
Gregor S. Pilzaka , Rutchanna M.S. Jongejanb, Toine van den Bergha, Robin P. Peetersc,d, Tommi
Meulemansa and Yolanda B. de Rijkeb,d
aMercachem B.V. Kerkenbos 1013, 6503 GE Nijmegen, The Netherlands
bDepartment of Clinical Chemistry, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
cDepartment of Internal Medicine, Erasmus MC University Medical Center, Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
dAcademic Centre for Thyroid Diseases, Erasmus MC University Medical Center, Rotterdam, Dr. Molewaterplein 40 3015 GD Rotterdam, The Netherlands
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The effects of thyroid hormone metabolites (THMs) other than T3, rT3 and T4 are largely
unknown, partially due to the lack of adequate methods. For adequate analysis, internal
standards for all THMs are essential, but unfortunately not commercially available. Reported
approaches for the synthesis of T0, 3,5-T2, TA3 and TA4 lack sensitivity and/or are not
adaptable for 13C6-labeled analogues. In this paper, we describe the synthesis of four 13C6-
labelled THMs, T0-13C6, 3,5-T2-13C6, TA3-13C6, TA4-13C6. Starting with 13C6-bromo-benzene, a
short and versatile synthesis route was developed in which the formation of the diphenyl ether
by a Chan-Lam coupling reaction was fundamental.
Available online
Keywords:
Thyroid hormone
Triac
2009 Elsevier Ltd. All rights reserved
.
Tetrac
3,5-diiodothyronine
L-thyronine
———
Corresponding author. Address: Erasmus MC University Medical Center; Department of Clinical Chemistry; Postbus 2040; 3000 CA
Rotterdam, The Netherlands; Tel.: +31-10-7033543; e-mail: y.derijke@erasmusmc.nl

2
Tetrahedron
brominated using selectfluor and sodium bromide.21 The boronic
1. Introduction
acid BA1 was prepared via lithium exchange with the use of
triisopropyl borate. This procedure22 resulted in a higher yield of
BA1 compared to borylation with trimethoxy borate23 or Grignard
chemistry.24 The formation of BA1 was further optimized
performing cryogenic quench with Glauber’s salt instead of HCl
in water to yield BA1 quantitively.
Thyroid hormones (THs) have diverse effects in several
important organs such as bone, heart, fat, liver, pituitary, muscle
and brain regulating differentiation, growth and metabolism.1 The
thyroid gland is regulated by thyrotropin-releasing hormone
(TRH) in the hypothalamus and thyroid-stimulating hormone
(TSH) in the pituitary. This pathway is known as the
hypothalamic-pituitary-thyroid (HPT) axis. Predominantly the
prohormone 3,3',5,5'-tetraiodothyronine (T4) and approximately
20% of the active metabolite 3,3',5-triiodothyronine (T3) are
produced by the thyroid gland. A negative feedback loop
maintains the equilibrium of THs in the bloodstream as increased
T4 and T3 in the bloodstream inhibits the secretion of TRH in the
hypothalamus. TH metabolism produces 3,5-diiodothyronine
(3,5-T2) and L-thyronine (T0) via deiodination or 3,3',5-
triiodothyroacetic acid (TA3) and 3,3',5,5'-tetraiodothyroacetic
acid (TA4) via decarboxylation and oxidative deamination
(Figure 1).2 Their role in (patho)physiology during health, disease
and comorbidity remains unknown due to the lack of adequate
methods to measure these thyroid hormone metabolites (THMs).
Potential roles are described for 3,5-T2 in energy metabolism, for
TA3 in brain, liver, muscle and heart and for TA4 in heart and
pituitary, also elevated levels are observed in Graves' disease
patients.3-8 To study the role of THMs in health, disease and
comorbidity, selective and sensitive LC-MS/MS methods
measuring multiple THMs in a single analysis are needed. In
In the next step, the ether bond was prepared by a Chan-Lam
coupling reaction of BA1 with N-Acetyl-L-tyrosine ethyl ester
using copper (II) acetate, triethylamine and pyridine in
dichloromethane at room temperature.25 One-pot removal of the
protective groups using hydroiodic acid or hydrobromic acid in
boiling acetic acid was previously reported for T0-13C6
analogues.26 Unfortunately, this procedure was not robust and
often led to decomposition of the intermediate 4. Therefore, the
protective groups were removed stepwise instead of one-pot
removal. First, boron tribromide was used to remove the methoxy
ether followed by saponification of the methyl ester with lithium
hydroxide and deacetylation of the amide group with hydrochloric
acid in boiling acetic acid. With this synthesis route, T0-13C6 was
obtained as a HCl salt in a good yield and high purity.
recent years, several LC-MS/MS methods have been developed to
9-14
measure single and multiple THMs.5,
Despite the apparent
chemical and physical differences of these THMs, internal
standards of other THMs have predominantly been used with the
exception of 13C9-15N-labeled 3,5-T2 in the most recent studies.11-
12, 15
This internal standard is however reported to be
contaminated with 3,5-T2 itself.12 Using the correct internal
standard in LC-MS/MS panels is essential, as sample preparations
and liquid chromatography and mass spectrometry settings are
less optimal. For the synthesis of T0, 3,5-T2, TA3 and TA4
several approaches are previously reported.16-19 These approaches
either lack sensitivity and/or are not adaptable for the synthesis of
13C6-labeled analogues. In this paper, we describe the synthesis of
four 13C6-labeled THM, T0-13C6, 3,5-T2-13C6, TA3-13C6, TA4-
13C6, which were used to develop a nine-THM panel with liquid
chromatography-tandem mass spectrometry (LC-MS/MS).14
Scheme 1. Synthesis of T0-13C6.
Reagents and conditions: a) NaOMe, Cu(I)Br, anh. DMF, 120 °C, 5
hours, 91%, b) selectfluor, NaBr, MeCN, 16 hours, room temp., 98%,
c) n-butyllithium , B(OiPr)3, anh. THF/toluene (1:4), -78 ˚C, 2 hours,
Glauber’s salt, quant., d) N-Acetyl-L-tyrosine ethyl ester
monohydrate, Cu(OAc)2, pyridine, powdered molsieves, DCM, 16
hours, room temp., 58%. e). i. BBr3, anh. DCM, -78 ˚C, 1 hour, ii.
THF, LiOH, 3 hours, iii. HCl/AcOH (1/2), reflux temp., 6 hours,
87%.
This strategy was also used for the synthesis of TA3-13C6 and
TA4-13C6 (Scheme 2). The precursor 5 was obtained via a Chan-
Lam coupling reaction of BA1 with commercially available
methyl 2-(4-hydroxy-3,5-diiodophenyl)acetate. Direct iodination
of 5 using iodide in ammonia to form TA3-13C6 and TA4-13C6
analogues was not successful and led to over iodination and
decomposition. Therefore, both protective groups were removed
stepwise and intermediate 7 was halogenated using iodide in
ammonia. With this synthesis route, a 2:1 mixture of TA3-13C6
and TA4-13C6 was formed. TA3-13C6 and TA4-13C6 were separated
Figure 1. Chemical structures of thyroid hormone metabolites T0,
3,5-T2, TA3 and TA4.
2. Results/Discussion
We developed a new synthesis route for four 13C6-labeled
THM analogues starting from readily available 13C6-bromo-
benzene which was first converted to 13C6-anisole 2 using sodium
methoxide and copper(I) bromide in DMF under Dean-Stark
conditions (Scheme 1).20 Subsequently, 2 was selectively

3
by preparative HPLC, which yielded 10% of TA3-13C6 and 30%
of TA4-13C6 in a high purity.
Scheme 2. Synthesis of TA3-13C6 and TA4-13C6.
Reagents and conditions: a) Cu(OAc)2, pyridine, powdered
molsieves, DCM, 16 hours, room temp., 57%, b) BBr3, anh. DCM, -
78 °C, 79% c) THF, LiOH, 16 hours, 82% d) NH3, I2, MeOH, 0 °C, 1
hour, 10% TA3-13C6, 30% TA4-13C6.
Scheme 3. Synthesis of 3,5-T2-13C6.
Reagents and conditions: a) BBr3, anh. DCM, -78 °C, 87%, b)
Metabolite 3,5-T2-13C6 was synthesized via a Chan-Lam
coupling reaction using triisopropyl silyl ether protected boronic
acid BA2 and Boc-protected 3,5-diiodo-L-tyrosine methyl ester
(Scheme 3). The previously used conditions to remove O-
methoxy and N-acetyl groups in the synthesis of T0-13C6 (Scheme
1) were considered to be too harsh for 3,5-T2-13C6 and would
cause decomposition of the product. To prepare BA2,
bromoanisole 3 was first deprotected followed by protection as its
triisopropyl silyl ether. Next the bromide was converted into its
boronic acid by lithium exchange with the use of triisopropyl
TIPSCl, imidazole, 0 °C, DCM, 1 hour, 62%, c) n-butyllithium
,
B(OiPr)3, anh. THF, -70 °C, 2 hours, 66%, d) Cu(OAc)2, pyridine,
powdered molsieves, DCM, 16 hours, room temp., 49%, e) TBAF,
THF, room temp., 10 minutes, 76%, f) THF/water (4/1), LiOH, 0 °C,
3 hours, 99%, g) HCl, dioxane, 3 hours, 54%.
4. Experimental Section
NMR spectra were recorded on a Bruker AMX 400 NMR (400
MHz) spectrometer. Signal positions are recorded as chemical
shifts (δ) in parts per million (ppm) and referenced to the residual
solvent peaks (CDCl3 or DMSO-d6) and TMS as an internal
standard. The multiplicities are reported as follows br = broad, s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet and
combination of these. Doublet of multiplets (dm) is used to
describe the complex splitting patterns in 13C-labeled compounds,
27
borate. The Chan-Lam coupling reaction with Boc-3,5-diiodo-
L-tyrosine methyl ester and boronic acid BA2, gave the diphenyl
ether in moderate yield. The silyl ether was cleaved using TBAF
in good yield and the ester group hydrolysed using lithium
hydroxide. After deprotection of the amine by hydrochloric acid
in dioxane, 3,5-T2-13C6 was obtained as HCl salt in a high purity.
1
3. Conclusion
and the reported JH,C coupling constant is measured as peak-to-
peak distance between the middle of each multiplet group. HRMS
spectra were recorded on a LC-MS Q Exactive Focus HRMS
spectrometer, which was calibrated with the Pierce calibration
solution both in positive and negative mode.
Bromobenzene-13C6 (99 atom% 13C) was purchased from Sigma-
Aldrich (Darmstadt, Germany).
In this paper, we described a new synthesis route for the
preparation and isolation of pure T0-13C6, TA3-13C6, TA4-13C6 and
3,5-T2-13C6. The Chan-Lam coupling reaction can be used to
synthesize 13C6-labeled thyroid hormone analogues.
4.1
anisole-1,2,3,4,5,6-13C6
2
In a one neck-flask equipped with Dean-Stark apparatus,
bromobenzene-1,2,3,4,5,6-13C6 (1.9 mL, 18.4 mmol) was

4
Tetrahedron
dissolved in N,N-dimethylformamide (dry) (5 mL). A 5.4 M as eluents to yield the title compound (42 mg, 0.116 mmol, 58%
solution of sodium methoxide in methanol (5.1 mL, 27.6 mmol) yield).
was added dropwise with a syringe. The mixture was refluxed at
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.02 (d, J=8.5 Hz, 2H);
120 °C for ~1.5 hours until ~3 mL of methanol was collected.
Then, copper (I) bromide (0.264 g, 1.841 mmol) was added and
the reaction was continued for ~2 hours. The mixture was diluted
with dichloromethane (25 mL) and filtered through a plug of
kieselguhr. Subsequently, the filtrate was mixed with
water/dichloromethane (1:1; 100 mL) and the layers were
separated. Additionally, the dichloromethane layer was washed
four times with 50 mL of water, separated and dried with sodium
sulphate. After concentration under reduced pressure, the title
compound was obtained in 91% yield.
7.00 (dm, JHC=160 Hz, 2H); 6.90 (dm, JHC=160 Hz, 2H); 6.85 (d,
J=8.5 Hz, 2H); 5.93 (d, J=7.6 Hz, 1H); 4.84 (q, J=7.2 Hz, 1H);
4.16 (q, J=7.2 Hz, 2H); 3.81 (d, J=4.2 Hz, 3H); 3.11 (dd, J=14.0,
5.8 Hz, 1H); 3.06 (dd, J=14.0, 5.8 Hz, 1H); 2.0 (s, 3H), 1.26 (t,
J=7.1 Hz, 3H).
4.5.
(S)-2-amino-3-(4-(4-hydroxyphenoxy-1,2,3,4,5,6-
13C6)phenyl)propanoic acid HCl-salt T0-13C6
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.30 (dm, JHC=132 Hz,
2H); 6.94 (dm, JHC=168 Hz, 3H); 3.81 (d, J=4.1 Hz, 3H).
To a solution of 4 (45 mg, 0.124 mmol) in anhydrous
dichloromethane (10 mL) at -78 °C under nitrogen atmosphere, a
1 M solution of boron tribromide in dichloromethane (0.8 mL,
0.800 mmol) was dropwise added and stirred for 1 hour at -78 °C.
The mixture was then quenched with methanol and concentrated
under reduced pressure. The crude material was dissolved in
4.2
1-bromo-4-methoxybenzene-1,2,3,4,5,6-13C6
3
To a solution of 2 (0.95 g, 5.00 mmol) in anhydrous tetrahydrofuran (10 mL) and lithium hydroxide (1 M) (0.619 mL,
acetonitrile (20 mL) was added sodium bromide (0.51 g, 5.0 0.619 mmol) was added. The reaction was stirred at room
mmol) and selectfluor (1.77 g, 5.0 mmol). The reaction mixture temperature for 3 hours. Subsequently, the reaction mixture was
was stirred overnight at room temperature under nitrogen
diluted with 30 mL ethyl acetate and 30 mL of 1 M HCl solution
atmosphere. The suspension was diluted with ethyl acetate (50 in water. The layers were separated and the water layer was
mL) and filtered through a silicagel plug. The filtrate was extracted twice with 50 mL ethyl acetate. The organic layers were
concentrated under reduced pressure to yield 3 (1.05 g, 4.90 dried and concentrated under reduced pressure. The crude mixture
mmol) in a 98% yield.
was dissolved in a solution of hydrochloric acid (10 mL, 329
mmol) and acetic acid (20 mL, 349 mmol) and refluxed for 6
hours. The slightly yellow solution was concentrated under
reduced pressure and the obtained solids were triturated with
dichloromethane (10 mL). The title compound was obtained as a
HCl-salt (30 mg, 0.107 mmol) in 87% yield.
1H-NMR (400 MHz, DMSO-d6) δ: 13.71 (s, 1H); 9.40 (s, 1H);
8.24 (bs, 2H); 7.21 (d, J = 8.5 Hz, 2H); 6.86 (dm, JHC=161 Hz,
2H); 6.85 (d, J = 8.5 Hz, 2 H); 6.78 (dm, JHC=162 Hz, 2H); 4.13
(t, J = 6.0 Hz, 1H); 3.08 (dd, J = 14.4, 6.1 Hz, 1H); 3.02 (dd, J =
14.5, 6.9 Hz, 1H).
13C-NMR (DMSO-d6, 100 MHz) δ (ppm): 153.9 (td, J =
65.3, 9.0 Hz), 147.8 (td, J = 68.4, 9.0 Hz), 121.7–119.9 (m),
117.1–115.3 (m).
HRMS 280.127513 ([M+H]+, calculated), 280.12558 ([M+H]+,
found), ∆ = -6.91 ppm.
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.38 (dm, JH,C=156 Hz,
2H); 6.80 (dm, JH,C=176 Hz, 2H); 3.78 (d, 3H, J=4.2 Hz).
4.3
(4-methoxyphenyl-1,2,3,4,5,6-13C6)boronic acid BA1
In a three neck flask equipped with nitrogen in and outlet, 3
(0.6 g, 3.11 mmol) was dissolved in a mixture of anhydrous
tetrahydrofuran (2 mL) and anhydrous toluene (8 mL) and cooled
down to -75 °C. Then, a 2.5 M solution of n-butyllithium in
hexanes (1.37 mL, 3.42 mmol) was added dropwise over the
course of 15 minutes, maintaining the temperature below -70 °C.
After stirring for 1 hour at -75 °C, triisopropyl borate (0.79 mL,
3.42 mmol) was added dropwise with the temperature not
exceeding -60 °C. The reaction was continued for 30 minutes and
quenched with 10 grams of Glauber’s salt at -50 °C. After 15
minutes at -50 °C, 50 mL of methanol was added and the mixture
was left to warm up to room temperature. Subsequently, the
mixture was filtered and concentrated under reduced pressure.
The crude material was co-evaporated three times with 20 mL of
toluene and three times with a 1:1 solution of dichloromethane
and pentane to yield the title compound (490 mg, 3.11 mmol)
quantitively as a white solid.
4.6.
methyl 2-(3,5-diiodo-4-(4-methoxyphenoxy-1,2,3,4,5,6-
13C6)phenyl)acetate
5
To BA1 (200 mg, 1.267 mmol), methyl 2-(4-hydroxy-3,5-
diiodophenyl)acetate (265 mg, 0.633 mmol), copper (II) acetate
(345 mg, 1.900 mmol), molecular sieves (1 g) in anhydrous
dichloromethane (10 mL) was added pyridine (0.102 mL, 1.267
mmol). The brown/green mixture was stirred at room temperature
overnight. Subsequently, BA1 (100 mg, 0.633 mmol) was added
and the reaction was stirred for 1 hour. The reaction mixture was
diluted with dichloromethane (20 mL) and concentrated in vacuo.
The product was purified using flash column chromatography
with 360% ethyl acetate in heptane as eluents to yield the title
compound (192 mg, 0.362 mmol, 57% yield) as a colourless oil.
1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.80 (dm, JH,C=164
Hz, 2H); 6.87 (dm, JH,C=160 Hz, 2H); 3.78 (d, 3H, J=4.2 Hz).
4.4.
ethyl
(S)-2-acetamido-3-(4-(4-methoxyphenoxy-
1,2,3,4,5,6-13C6)phenyl)propanoate
4
BA1 (62.8 mg, 0.398 mmol), ethyl acetyl-L-tyrosinate (50 mg,
0.199 mmol) and copper (II) acetate (108 mg, 0.597 mmol) were
mixed in dichloromethane (1 mL) and powdered 4Å molsieves in
8 mL screw cap reaction vial. Then pyridine (0.161 mL, 1.990
mmol) was added and the mixture was stirred overnight. The
crude mixture was concentrated under reduced pressure and
purified on silica gel column using 0-20% ethyl acetate in heptane
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.78 (s, 2H); 6.84 (dm,
JH,C=156 Hz, 2H); 6.72 (dm, JH,C=156 Hz, 2H); 3.78 (s, 3H), 3.75
(s, 3 H); 3.57 (s, 2H).

5
4.7.
methyl
2-(4-(4-hydroxyphenoxy-1,2,3,4,5,6-13C6)-3,5-
HRMS 626.776342 ([M-H]-, calculated), 626.77685 ([M-H]-,
diiodophenyl)acetate
6
found), ∆ = 2.56 ppm.
To a solution of 5 (220 mg, 0.394 mmol) in anhydrous
dichloromethane (30 mL) at -78 °C under nitrogen atmosphere
was added a 1 M solution of boron tribromide in dichloromethane
(1 mL, 1.000 mmol) and the mixture was allowed to reach room
temperature. After 2.5 hours, the mixture was cooled to -78 °C
under nitrogen atmosphere and a 1 M solution of boron
tribromide in dichloromethane (1 mL, 1.000 mmol) was added
dropwise. The cold mixture was quenched with 60 mL of
methanol and concentrated under reduced pressure. The crude
product was purified on a silica gel column with 0-40% ethyl
acetate in heptane as eluents to yield the title compound (170 mg,
0.310 mmol, 79 % yield).
TA4-13C6
1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 12.50 (bs, 1H); 9.20
(bs, 1H); 7.84 (s, 2H); 7.06 (dm, JH,C=165 Hz, 2H); 3.58 (s, 2H).
13C-NMR (DMSO-d6, 100 MHz) δ (ppm): 152.6–148.5
(m), 125.9–124.1 (m), 88.6–86.9 (m).
HRMS 752.672985 ([M-H]-, calculated), 752.67334 ([M-H]-,
found), ∆ = 1.93 ppm.
4.10.
4-bromophenol-13C6
8
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.77 (s, 2H); 6.76 (dm,
JH,C=161 Hz, 2H); 6.67 (dm, JH,C=156 Hz, 2H); 4.48 (s, 1H); 3.75
(s, 3H); 3.57 (s, 2H).
To a solution of 3 (2.3 g, 8.34 mmol) in dichloromethane (50
mL) was added boron tribromide (0.868 mL, 9.18 mmol) and left
stirring overnight at room temperature. As the reaction was not
completed two more portions of a 1 M solution of boron
tribromide in dichloromethane were added over a period of 24
hours (8.34 mL and 4.17 mL). The mixture was quenched with
100 mL water. The organic layer was evaporated at 35 °C and
300 mbar reduced pressure. The residue was dissolved in diethyl
ether (100 mL) and washed with saturated aqueous solution of
sodium bicarbonate, brine and dried over sodium sulphate,
filtered and concentrated at 35 °C and 300 mbar reduced pressure
to afford the title compound (2.46 g, 7.29 mmol, 87% yield) as a
brown oil containing 13% of unreacted starting material. The title
compound was used as such in step 4.11.
4.8.
diiodophenyl)acetic acid
2-(4-(4-hydroxyphenoxy-1,2,3,4,5,6-13C6)-3,5-
7
To a solution of 6 (50 mg, 0.097 mmol) in tetrahydrofuran (10
mL) was added lithium hydroxide (0.6 mL, 0.600 mmol)
dropwise during 2 minutes. The reaction was stirred overnight at
room temperature. Additional lithium hydroxide (0.1 mL, 0.100
mmol) was added and reaction was continued until completion (2
hours). The mixture was partitioned between ethyl acetate (50
mL) and brine (20 mL). The water layer was extracted two
additional times with ethyl acetate (50 mL). The combined
organic layers were dried on sodium sulphate and concentrated
under reduce pressure. The solids were triturated with
dichloromethane and pentane, and dried to yield the title
compound (40 mg, 0.080 mmol, 82% yield).
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.31 (dm, JH,C=166
Hz, 2H); 6.72 (dm, JH,C=160 Hz, 2H); 6.55-6.45 (m, 1H); 5.90 (s,
1H).
1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 12.22 (bs, 1H); 9.13
(s, 1H); 7.85 (s, 2H); 6.69 (dm, JHC=156 Hz, 2H); 6.52 (dm,
JHC=156 Hz, 2H); 3.51 (s, 2H).
4.11.
(4-bromophenoxy-1,2,3,4,5,6-13C6)triisopropylsilane 9
Triisopropylsilyl chloride (1.698 mL, 8.01 mmol) was added
to a solution of 8 (2.46 g, 7.29 mmol) in dichloromethane (50
mL). The reaction mixture was cooled with an ice bath and
imidazole (1.240 g, 18.21 mmol) was added. Subsequently, the
reaction was stirred for 30 minutes at 0 °C and slowly warmed up
to room temperature and stirred overnight. The reaction mixture
was diluted with diethyl ether and washed two times with 0.5 M
hydrochloric solution in water and saturated aqueous sodium
bicarbonate and brine. Then, the mixture was dried over sodium
sulphate, filtered and concentrated in vacuo. The crude product
was purified by flash chromatography with 0-35% solution of
ethyl acetate in heptane as eluents to obtain the title compound
(1.52 g, 4.53 mmol, 62.2 % yield).
4.9.
3,3',5-triiodo thyroacetic acid-13C6 TA3-13C6 and
3,5,3',5'-tetraiodo thyroacetic acid-13C6 TA4-13C6
To a solution of 7 (30 mg, 0.060 mmol) in methanol (0.6 mL)
was added a solution of 32% ammonia in water (0.5 mL, 7.32
mmol) at 0 °C, followed by the dropwise addition of 0.1 M iodine
in methanol (0.149 mL, 0.015 mmol). The light yellow mixture
was reacted at 0 °C for 1 hour, followed by a second portion of
0.1 M iodine in methanol (0.447 mL, 0.045 mmol). After 1 hour
the reaction mixture was poured out in 10% solution of sodium
metabisulfite in water (10 mL) and extracted with ethyl acetate
(3x10 mL). The combined organic layers were dried on Na2SO4
and concentrated in vacuo. The crude mixture was purified by
preparative LC-MS and lyophilised to yield pure TA3-13C6 (3.71
mg, 0.006 mmol, 10% yield) and pure TA4-13C6 (13.59 mg, 0.018
mmol, 30% yield).
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.30 (dm, JH,C=160 Hz,
2H); 6.75 (dm, JH,C=156 Hz, 2H); 1.35-1.15 (m, 3H); 1.11 (s,
9H); 1.09 (s, 9H).
4.12.
acid
(4-((triisopropylsilyl)oxy)phenyl-1,2,3,4,5,6-13C6)boronic
BA2
TA3-13C6
1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 12.54 (bs, 1H);
10.06 (s, 1 H); 7.82 (s, 1H); 6.99 (dm, JH,C=163 Hz, 1H); 6.83
(dm, JH,C=160 Hz, 1H); 6.58 (dm, JH,C=164 Hz, 2H); 3.57 (s,
2H).
13C-NMR (DMSO-d6, 100 MHz) δ (ppm): 152.7–151.1
(m), 149.9–148.1 (m), 124.7 (t, J = 68.0 Hz), 117.0–114.2 (m),
85.3–83.6 (m).
Under nitrogen atmosphere, 9 (1.52 g, 4.53 mmol) was
dissolved in tetrahydrofuran (dry) (50 mL). The reaction mixture
was cooled to -78 °C and 2.5 M n-butyllithium solution in
hexanes (2.176 mL, 5.44 mmol) was added. The reaction mixture
was stirred for 1 hour followed by dropwise addition of
triisopropyl borate (1.421 mL, 6.12 mmol). The reaction mixture
was stirred for one hour at -78 °C and was then allowed to warm

6
Tetrahedron
up to room temperature. After stirring overnight at room
11 (134 mg, 0.208 mmol) was dissolved in a mixture of
temperature the reaction mixture was cooled to 0 °C and tetrahydrofuran (4 mL) and water (1 mL) and cooled down with
quenched with 3 N solution of HCl in water (3 mL) and stirred for an ice bath. Then, lithium hydroxide monohydrate (43.6 mg,
1 hour at 0 °C. Then, the aqueous layer was extracted with ethyl 1.038 mmol) was added and the reaction mixture was conducted
acetate. The combined organic layers were washed with brine, for 3 hours at 0 °C. Reaction mixture was acidified with 0.5 M
dried over sodium sulphate, filtered and concentrated in vacuo. solution of HCl in water and extracted two times with ethyl
The crude product was purified by flash chromatography with 0- acetate. The combined organic layers were washed with brine,
60% solution of ethyl acetate in heptane as eluents to yield BA2 dried over sodium sulphate, filtered and concentrated to yield the
(905 mg, 3.01 mmol, 66.5 % yield).
title compound (130 mg, 0.206 mmol, 99% yield).
1H-NMR (CDCl3, 400 MHz) δ (ppm): 8.08 (dm, JH,C=163 Hz,
2H); 6.98 (dm, JH,C=160 Hz, 2H); 1.35-1.20 (m, 3H); 1.14 (s,
9H); 1.12 (s, 9H).
4.16.
3,5-diiodo-L-thyronine-13C6 HCl-salt
3,5-T2-13C6
12 (130 mg, 0.206 mmol) was dissolved in 4 M hydrochloric
acid in 1,4-dioxane (2 mL, 57.6 mmol) and reacted for 3 hours at
4.13.
diiodo-4-(4-((triisopropylsilyl)oxy)phenoxy-1,2,3,4,5,6-
13C6)phenyl)propanoate
10
methyl
(S)-2-((tert-butoxycarbonyl)amino)-3-(3,5- room temperature. Then, the reaction mixture was concentrated
under reduced pressure and the resulting solids triturated in
methyl tert-butyl ether (2 mL), filtered and dried to yield a white
solid. This material was dissolved in acetonitrile/water and
lyophilized to yield the title compound (63 mg, 0.111 mmol, 53.9
% yield) as a white solid.
Molecular sieves (4 Å, powdered, 700 mg, 0.555 mmol), Boc-
3,5-diiodo-L-tyrosine methyl ester (304 mg, 0.555 mmol), BA2
(250 mg, 0.833 mmol) and copper (II) acetate (302 mg, 1.665
mmol) were mixed in anhydrous dichloromethane (8 mL).
1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 9.11 (s, 1H); 8.09
Pyridine (0.090 mL, 1.110 mmol) was added and the reaction (bs, 2H); 7.82 (s, 2H); 6.68 (dm, JH,C=161 Hz, 2H); 6.55 (dm,
mixture was stirred overnight. Additional pyridine (0.045 mL, JH,C=156 Hz, 2H); 4.03 (s, 1H); 3.13 (dd, J = 14.3, 5.3 Hz, 1H),
0.555 mmol) was added and the reaction was continued at room 3.02 (dd, J = 14.3 Hz, 7.7 Hz, 1H).
temperature for 24 hours. The reaction mixture was quenched
13C-NMR (DMSO-d6, 100 MHz) δ (ppm): 153.0–151.1 (m),
149.7–147.9 (m), 116.8–114.8 (m).
HRMS 531.920800 ([M+H]+, calculated), 531.91812 ([M+H]+,
found), ∆ = 5.04 ppm.
with 0.5 M solution of HCl in water. The aqueous layer was
extracted two times with ethyl acetate and the combined organic
layers were washed with brine, dried over sodium sulphate,
filtered and concentrated in vacuo. The crude product was
purified by flash chromatography with 0-50% solution of ethyl
acetate in heptane as eluent to yield the title compound (220 mg,
0.274 mmol, 49% yield)
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.62 (s, 1 H); 6.79 (dm,
JH,C=162 Hz, 2H); 6.61 (dm, JH,C=160 Hz, 2H); 5.09 (d, J=8.1
Acknowledgments
Hz, 1H); 4.54 (d, J=7.2 Hz, 1H); 4.50 (bs, 1H); 3.76 (s, 3H); 3.10 This research did not receive any specific grant from funding
agencies in public, commercial or not-for-profit sectors.
(dd, J = 13.7 Hz, 5.6 Hz, 1H); 2.92 (dd, 13.9 Hz, 6.5 Hz, 1H);
1.45 (s, 9H); 1.15-1.30 (m, 3H); 1.09 (s, 12H); 1.07 (s, 6H).
References
4.14.
Methyl
(S)-2-((tert-butoxycarbonyl)amino)-3-(4-(4-
hydroxyphenoxy-1,2,3,4,5,6-13C6)-3,5-diiodophenyl)propanoate
1.
Yen, P. M., Physiological Reviews 2001, 81 (3), 1097-
1142.
2.
11
Bianco, A. C.; Salvatore, D.; Gereben, B.; Berry, M. J.;
To a solution of 10 (220 mg, 0.274 mmol) in anhydrous
tetrahydrofuran (5 mL) was dropwise added a 1 M solution of
tetra-n-butylammonium fluoride in tetrahydrofuran (0.412 mL,
0.412 mmol). After ten minutes the reaction mixture was
quenched with 0.5 M solution of HCl in water. The reaction
mixture was diluted with water and extracted three times with
ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulphate, filtered and concentrated in
vacuo. The crude product was purified by flash chromatography
with 0-60% solution of ethyl acetate in heptane as eluent to yield
the title compound (134 mg, 0.208 mmol, 76 % yield) as a white
solid.
Larsen, P. R., Endocrine Reviews 2002, 23 (1), 38-89.
3.
945-50.
4.
Endocrinol 1994, 99 (1), 89-94.
5.
Schwartz, S.; Trierweiler, A.; Niziol, L. M.; Atkins, S.; Scanlan, T.
S.; Smith, T. J., J Clin Endocrinol Metab 2016, jc20162762.
Horst, C.; Rokos, H.; Seitz, H. J., Biochem J 1989, 261 (3),
Lanni, A.; Moreno, M.; Lombardi, A.; Goglia, F., Mol Cell
Fernando, R.; Placzek, E.; Reese, E. A.; Placzek, A. T.;
6.
Sherman, S. I.; Ringel, M. D.; Smith, M. J.; Kopelen, H.
A.; Zoghbi, W. A.; Ladenson, P. W., J Clin Endocrinol Metab 1997,
82 (7), 2153-8.
7.
A., Eur J Endocrinol 2001, 144 (2), 145-54.
8. Horn, S.; Kersseboom, S.; Mayerl, S.; Muller, J.; Groba, C.;
Trajkovic-Arsic, M.; Ackermann, T.; Visser, T. J.; Heuer, H.,
Endocrinology 2013, 154 (2), 968-79.
9.
Lameloise, N.; Siegrist-Kaiser, C.; O'Connell, M.; Burger,
1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.63 (s, 2H); 6.76 (dm,
JH,C=159 Hz, 2H); 6.65 (dm, JH,C=159 Hz, 2H); 5.09 (d, J=8.1
Hz, 1H); 4.55 (bs, 1H); 4.50 (bs, 1H); 3.76 (s, 3H); 3.10 (d, J =
13.0 Hz, 1H); 2.90 (dd, 13.6 Hz, 6.6 Hz, 1H); 1.45 (s, 9H).
Wang, D.; Stapleton, H. M., Anal Bioanal Chem 2010, 397
(5), 1831-9.
10.
Hansen, M.; Luong, X.; Sedlak, D. L.; Helbing, C. C.;
Hayes, T., Anal Bioanal Chem 2016.
4.15.
hydroxyphenoxy-1,2,3,4,5,6-13C6)-3,5-diiodophenyl)propanoic
acid 12
(S)-2-((tert-butoxycarbonyl)amino)-3-(4-(4-
11.
Lorenzini, L.; Nguyen, N. M.; Sacripanti, G.; Serni, E.;
Borso, M.; Saponaro, F.; Cecchi, E.; Simoncini, T.; Ghelardoni, S.;
Zucchi, R.; Saba, A., Front Endocrinol (Lausanne) 2019, 10, 88.

7
12.
Rijntjes, E.; Kohrle, J., Anal Bioanal Chem 2019.
13. Piehl, S.; Heberer, T.; Balizs, G.; Scanlan, T. S.; Kohrle, J.,
Rapid Commun Mass Spectrom 2008, 22 (20), 3286-96.
14. Jongejan, R. M. S.; Klein, T.; Meima, M. E.; Visser, W. E.;
Richards, K. H.; Monk, R.; Renko, K.; Rathmann, D.;
van Heerebeek, R. E. A.; Luider, T. M.; Peeters, R. P.; de Rijke, Y.
B., Clin Chem 2020, in press.
15.
Hackenmueller, S. A.; Scanlan, T. S., Synth Commun 2013,
43 (10), 1439-1446.
16.
Raja, K.; Mugesh, G., Angew Chem Int Ed Engl 2015, 54
(26), 7674-8.
17.
Canzanelli, A.; Harington, C. R.; Randall, S. S., Biochem J
1934, 28 (1), 68-72.
18.
19.
Wilkinson, J. H., Biochem J 1956, 63 (4), 601-5.
Bridoux, A.; Khan, R. A.; Chen, C.; Cheve, G.; Cui, H.;
Dyskin, E.; Yasri, A.; Mousa, S. A., J Enzyme Inhib Med Chem 2011,
26 (6), 871-82.
20.
Aalten, H. L.; van Koten, G.; Grove, D. M.; Kuilman, T.;
Piekstra, O. G.; Hulshof, L. A.; Sheldon, R. A., Tetrahedron 1989, 45
(17), 5565-5578.
21.
Rieth, R. D., J Org Chem 2002, 67 (13), 4487-93.
22. Li, W.; Nelson, D. P.; Jensen, M. S.; Hoerrner, R. S.; Cai,
Syvret, R. G.; Butt, K. M.; Nguyen, T. P.; Bulleck, V. L.;
D.; Larsen, R. D.; Reider, P. J., The Journal of Organic Chemistry
2002, 67 (15), 5394-5397.
23.
Rosen, B. M.; Wilson, D. A.; Wilson, C. J.; Peterca, M.;
Won, B. C.; Huang, C.; Lipski, L. R.; Zeng, X.; Ungar, G.; Heiney, P.
A.; Percec, V., J Am Chem Soc 2009, 131 (47), 17500-21.
24.
Leermann, T.; Leroux, F. R.; Colobert, F., Org Lett 2011,
13 (17), 4479-81.
25.
26.
Qiao, J. X.; Lam, P. Y. S., Synthesis 2011, 06, 829-856.
Leeson, P. D.; Ellis, D.; Emmett, J. C.; Shah, V. P.;
Showell, G. A.; Underwood, A. H., J Med Chem 1988, 31 (1), 37-54.
27. Hart, M. E.; Suchland, K. L.; Miyakawa, M.; Bunzow, J.
R.; Grandy, D. K.; Scanlan, T. S., J Med Chem 2006, 49 (3), 1101-
12.

Highlights
•
13C6-labeled thyroid hormone metabolites analogues were synthesized from readily
available13C6-bromo-benzene
•
•
Preparation of corresponding boronic acids was optimized
Chan-Lam coupling reaction was used as key step