Synthesis of tritium labelled thiorphan, an enkephalinase inhibitor

Article abstract of DOI:10.1002/jlcr.1566

Tritium labelling of the enkephalinase inhibitor, thiorphan, is complicated by the presence of mercapto functional group. Reactions often used in aromatic tritiation, such as halogination and catalytic halogen/tritium displacement, are adversely affected

Full text of DOI:10.1002/jlcr.1566

Research Article
Received 21 July 2008,
Revised 13 October 2008,
Accepted 17 October 2008
Published online 17 November 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1566
Synthesis of tritium labelled thiorphan,
an enkephalinase inhibitor
Ã
Shao-Yong Wu, and Mohammad R. Masjedizadeh
Tritium labelling of the enkephalinase inhibitor, thiorphan, is complicated by the presence of mercapto functional group.
Reactions often used in aromatic tritiation, such as halogination and catalytic halogen/tritium displacement, are adversely
affected by the presence of the divalent sulfur moeity. By protecting the SH group with t-butyl group, the tritiation reaction
proceeded smoothly without catalyst poisoning. The mercapto functionality was re-generated with great ease and
efficiency using 2-nitrobenzenesulfenyl chloride and dithiothreitol (DTT). [3H]-Thiorphan, thus, obtained had
a
radiochemical purity of 499% by AN-HPLC and a specific activity of 18.42 Ci/mmol. [3H]-Thiorphan showed good stability
when stored at 41C in an aqueous solution containing 10% methanol and 0.2% DTT in the dark.
Keywords: tritium labelled thiorphan; enkephalinase; neutral endopeptidase; NEP; radioligand; tritiation; thiorphan stability
Two bromine containing thioesters
8 and 9 were then
Introduction
synthesized (Scheme 1).10 Methyl 3-(dimethylamino)propionate
3 was coupled individually with 3- and 4-bromobenzyl bromides
4 to form aminoesters 5 in 36 and 32% yield, respectively. The
low yields from these reactions were mostly due to competing
N-benzylation followed by elimination leading to N,N-dimethyl-
bromobenzylamine by-products. Compounds 5 were further
derivatized into the ammonium iodides 6 (85 and 82% yield),
which underwent base-catalyzed elimination to afford vinyl
acids 7 (85 and 93% yield). Sulfur was introduced by Michael
addition of thioacetic acid to give thioesters 8 in 95% and 9 in
87% yield.
Following an analogous literature procedure9 compound 9
was converted into disulfide 12 through three transformations
(Scheme 2). Amide formation under standard peptide coupling
conditions afforded 10 in 91% yield. The protecting groups in
compound 10 were then removed by treatment with NaOH in
methanol to give mercapto acid 11, which underwent oxidation
by iodine to form the disulfide 12 in 81% yield in two steps.
Catalytic debromination by deuterium gas for compound 12, as
well as for simpler analog 8, was examined under various
conditions (catalysts: Pd/C, Pd/BaSO4, Pd(OH)2/C, Pd(OAc)2,
PdCl2(dppf); reductants: D2, NaBD4; solvents: THF, dioxane,
AcOEt, DMF, EtOH, MeOH, H2O; bases: TEA, EDTA). Most of the
conditions showed either no reaction or undesired side
reactions (S–C and S–S cleavage). In the best cases where
reductions were conducted in H2O with 20% Pd(OH)2/C as
catalyst (catalyst/substrate ꢀ2:1 or more, by weight) and in the
presence of triethylamine, only trace amounts (o5%) of
the desired product 13 from 12 and up to 20% of
Thiorphan is a thiol containing drug, which has been clinically
administered as its S-acetyl O-benzyl prodrug racecadotril (also
known as acetorphan) for the treatment of diarrhea.1,2 Thiorphan
shows low nanomolar inhibitory activity against neutral en-
dopeptidase (NEP or enkephalinase), a zinc-metallopeptidase
widely distributed in peripheral tissues and in the brain whose
biological functions include catabolism of the opioid peptides.3,4
Enkephalinase inhibitors have received comprehensive studies
for their potential therapeutic applications namely in CNS and
digestive tract diseases.5 Tritium labelled thiorphan6,7 and
acetorphan8 have been used as radioactive probes for char-
acterization of enkephalinase (Figure 1).
There are two types of tritium labelled thiorphan known in
the literature, one with the labelling on glycine methylene (1)
utilizing commercially available [3H]-glycine as tritium source,6,7
and another labelled on phenyl ring (2) through tritium–bro-
mine exchange reaction, which required preparation of the
corresponding halogenated precursor.9 However, the yield and
specific activity in both cases were very low, encountering either
catalyst poisoning due to the presence of the divalent sulfur
group or somewhat lengthy experimental preparation. These
problems are the predominant factors that have limited the
availability and use of [3H]-thiorphan. Therefore, a new and
robust synthesis of [3H]-thiorphan is needed.
Results and discussion
Initial experiments indicated that direct halogenations of
thiorphan or acetorphan suffered from S-oxidation, forming
either disulfide in aromatic iodination with bis(pyridine)iodonium
tetrafluoroborate or sulfonic acid in bromination with bromine.
In the latter experiment the phenyl group was brominated.
It became clear that bromine had to be introduced prior to
Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
*Correspondence to: Shao-Yong Wu, Roche Palo Alto, 3431 Hillview Avenue,
Palo Alto, CA 94304, USA.
generation of free thiol group in order to avoid the S-oxidation. E-mail: shao-yong.wu@roche.com
J. Label Compd. Radiopharm 2008, 52 23–28
Copyright r 2008 John Wiley & Sons, Ltd.

S.-Y. Wu and M. R. Masjedizadeh
bromine/deuterium exchange product from 8 were observed reaction between 16 and glycine benzyl ester gave the amide 17 in
along with the starting materials and some S–C and S–S 79% yield. Compound 17 was used as the precursor for tritium
cleavage by-products. Such poor conversions were mostly due incorporation. It was expected that under palladium-catalyzed
to catalysts poisoning caused by divalent sulfur groups in the tritiation reaction, the bromo and benzyl groups would be reduced
molecules.
Activity of the sulfur groups in these molecules (SAc in 8 and
simultaneously to afford tritium labelled S-t-butyl thiorphan.
Indeed, in a cold run using deuterium gas (Scheme 4),
SS in 12) needed to be attenuated in order to increase its bromine/deuterium exchange and de-benzylation occurred
stability and minimize the observed catalyst poisoning effects. under the optimized catalytic reduction conditions (D2,
Among SH protecting groups, the more bulky and stable S-t- Pd(OH)2/C, TEA, H2O) to afford the D-labelled 18 quantitatively.
butyl group is considered a better choice for preventing the MS analysis of 18 (D) revealed the deuterium distribution as D0
interaction between sulfur and the active sites of catalyst as well 30%, D1 47%, and D2 22%, respectively. The effectiveness of S-t-
as reducing the chance to release the free SH group or undergo butyl protection was also observed when compound 16 was
S–C cleavage.
subjected to a similar bromine/deuterium exchange conditions
The acetyl group in compound 8 was removed by basic affording the reduction product exclusively with D-incorpora-
hydrolysis to form mercapto acid 15 (Scheme 3), which was then tion as D0 10%, D1 60%, D2 29%. In contrast, when 17 was
heated with t-butanol in the presence of aq. HCl to form 16 with the deuterated to form 18 in ethyl acetate with Pd/C as catalyst,
desired S-t-butyl group11 in 51% yield in two steps. The coupling product 18 (D) had a more specific D1 labelling (D0 3%, D1 94%,
D2 3%) even though the reaction was slower and not
quantitative (79% yield). The removal of t-butyl group in 18
O
O
T
T
(D) was accomplished by using 2-nitrobenzenesulfenyl chlor-
ide,11 which effectively displaced the t-butyl group with
nitrobenzenethio group giving the disulfide 19 (D) via a
sulfonium ion intermediate.12 Upon treatment with DTT13 and
triethylamine in methylene chloride, the S–S bond in 19 (D) was
easily reduced to thiols yielding deuterium-labelled thiorphan
20 quantitatively.
OH
T
OH
N
N
H
O
H
O
SH
SH
T
1
2
Figure 1. [3H]-Thiorphan in the literature.
O
O
LDA
MeI
IPA
Br
O
Br
Br
+
N
O
THF
N
3
4
5
O
S
O
O
1N NaOH
AcSH
OH
O
Br
Br
OH
Br
N
I
O
6
7
8 (3-Br), 9 (4-Br)
Scheme 1. Preparation of 2-(acetylsulfanylmethyl)-3-(3- or 4-bromophenyl) propionic acid 8 and 9.
O
S
O
S
O
O
Gly-Bn-ester
OH
N
H
1N NaOH
MeOH
OH
N
H
O
Br
Br
DCC/HOBT
O
SH
Br
TEA/THF/DCM
O
O
9
10
11
O
O
OH
OH
O
N
H
N
H
O
O
O
I2
MeOH
D2
catalyst
S
S
S
S
Br
Br
D
D
OH
N
H
O
O
H
N
H
N
SH
D
OH
OH
O
O
12
13
14
Scheme 2. Preparation of brominated disulfide 12 as labelling precursor.
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 52 23–28

S.-Y. Wu and M. R. Masjedizadeh
O
S
O
O
Br
Br
OH
OH
0.2N NaOH
t-BuOH
Br
OH
SH
S
37%aq. HCl
O
8
15
16
O
Gly-Bn-ester
Br
O
N
HOBT / DCC
H
O
THF / DCM
Et3N
S
17
Scheme 3. Preparation of 2-[3-(3-bromophenyl)-2-(S-t-butylsulfanylmethyl)propionylamino] acetic acid benzyl ester 17 as labelling precursor.
O
O
OH
(D or T)
O
Br
N
D2 or T2, Pd(OH)2
Et3N, H2O
N
H
H
O
O
S
S
18
17
NO2
O
S
OH
(D or T)
Cl
N
O
H
O
DTT
Et3N, CH2Cl2
OH
(D or T)
S
S
N
H
AcOH
O
SH
NO2
19
20
Scheme 4. Preparation of [2H] and [3H]-thiorphan.
It is well known that thiorphan is unstable in solutions and easily
undergoes oxidation to form the corresponding disulfide. According
to some reports,14,15 aqueous solutions with slightly acidic pH seem
essential for prolonging shelf-life. An initial study indicated that when
unlabelled thiorphan was kept as a 0.005% ethanol solution at 41C
under Ar with or without addition of acetic acid (0.01%), its purity
degraded to 61 or 64% in 30 days, respectively. The major
degradation product was the expected diastereomeric pair of
thiorphan disulfides, which showed characteristic 1:1 peaks on HPLC
and identical MS profiles. In search of a suitable solvent system to
store the synthesized [3H]-thiorphan, stability studies were conducted
on non-labeled thiorphan in H2O and EtOH as the primary solvents
and DTT or b-mercaptoethanol (BME) as an antioxidant. Some of the
solutions contained a cosolvent (MeOH, H2O) and/or an acid additive
(TFA, HCl, AcOH). The solutions were kept at 41C under N2 in the dark
and were analyzed by HPLC at 3, 15, 30 and 90 days after initial
preparations. Thiorphan was more stable in solutions containing DTT
than BME. Acid additives had adverse effects in most cases. Solutions
in the water–DTT series showed the highest level of purity retention
across all the time points. Among them, the one containing 10%
methanol and 0.2% DTT had the least degradation (4% in 90 days).
The deuterium labelling conditions were then applied for
tritiation reaction (Scheme 4). Compound 17 (6 mg) was
reduced with 5 Ci of carrier-free tritium gas affording 219 mCi
of crude product 18 (T) at 87% radiochemical purity. A portion
of the crude 18 (T, 44 mCi) underwent consecutive de-t-
butylation and S–S bond reduction reaction in one-pot. These
two reactions proceeded well at ambient temperature and
reached completion within 1 h for de-t-butylation and 30 min for
DTT-induced S–S reduction. The HPLC radiochemical purities of
starting material 18 (T, 87%), the crude products 19 (T, 88%) and
20 (T, 86%) were remarkably consistent, indicating highly
specific transformations affording [3H]-thiorphan in 71% isolated
yield. From 17 to 20 (T), only one purification (prep-HPLC) was
needed. The radiochemical purity was 499%. The specific
activity was determined by MS as 18.4 Ci/mmol. [3H]-thiorphan
showed good stability when stored at 41C in an aqueous
solution containing 10% methanol and 0.2% DTT in the dark.
Experimental
Proton NMR spectra were recorded on Bruker 300 MHz spectro-
meters. LC/MS analyses were carried out on a Finnigan LCQ-
Solutions in the other series showed moderate to severe degradation Advantage ion trap mass spectrometer equipped with an
electrospray ionization source. Analytic HPLC was run on a
Waters 2695 System with a Waters 2996 Photodiode Array
Detector, and preparative HPLC on a Beckman System 32 Karat
Gold using a 125 Solvent Module and a 166 detector. Column
and thirophan purity dropped 20–90% in 90 days. In those that had
the most degradation, the major impurity was the disulfide arising
from cross coupling of thiorphan and anti-oxidant BME. The disulfide
of thiorphan itself was consistently less than 1% in all solutions.
J. Label Compd. Radiopharm 2008, 52 23–28
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

S.-Y. Wu and M. R. Masjedizadeh
chromatography was run on a Teledyne Isco Combiflash 2-(Acetylsulfanylmethyl)-3-(3- or 4-bromophenyl)propionic acid (8
or 9)
Companion System with Thompson prepacked E-Merck silica
gel cartridges. Thin layer chromatography used Analtech Silica
Gel GF 250 micro plates. UV detection was at 220 or 256 nm and
radio detection was on an IN/US System b-Ram (HPLC) and a
Bioscan System 200 Imaging Scanner (TLC).
To a 5 ml pear-shaped flask containing 2-(3-bromobenzyl)acrylic
acid 7 (0.44 g, 1.83 mmol) was added thioacetic acid (1 ml,
1.065 g, 13.99 mmol). The mixture was heated at 501C for 2.5 h
and after HPLC confirmation of completion, excess thioacetic
acid was removed by rotary evaporation. The residue was co-
evaporated with toluene twice and purified with silica gel
column chromatography eluting with 0–2% MeOH in CH2Cl2 to
3-(3- or 4-Bromophenyl)-2-dimethylaminomethylpropionic acid
methyl ester (5)
To a 100 ml round bottom flask under N2 was added anhydrous give 8 as a colorless thick oil (0.55 g) in 95% yield. HPLC purity:
THF (20 ml) and lithium diisopropylamide (1.8 M in THF/ 97%. The 4-bromo isomer 9 was prepared similarly from 4-
heptane/ethylbenzene, 4 ml, 7.2 mmol) and the mixture was bromo 7 in 87% yield. 1H NMR (CDCl3)d2.35 (s, 3H), 2.98 (m, 5H),
cooled to À301C with a dry ice/MeOH bath. To this, a solution of 7.07 (d, J = 8.30 Hz, 2H), 7.43 (d, J = 8.50 Hz, 2H); MS (ESÀ)
methyl 3-(dimethylamino)propionate 3 (0.92 g, 7 mmol) in THF [MÀCH3CO+]: 273, 275; [2M+Na+À2H+]: 653, 655, 657.
(5 ml) was added dropwise; the initial yellowish solution turned
into a yellowish suspension. The mixture was aged at À301C for 2-[3-(4-Bromophenyl)-2-(S-acetylsulfanylmethyl)propionylamino]a-
cetic acid benzyl ester (10)
15 min, a solution of 3-bromobenzyl bromide 4 (1.75 g, 7 mmol)
in THF (5 ml) was added dropwise causing the suspension to
become clear. Stirring continued at –30 ꢀ À151C for 3 h. The
mixture was quenched with saturated NH4Cl solution to pH ꢀ8,
and extracted with ether three times. Organic layers were
combined, washed with water and dried over sodium sulfate.
The crude product was purified by silica gel flash column
chromatography eluting with 0–5% MeOH in CH2Cl2 to give the
desired 3-bromo 5 as a light yellow oil (0.762 g) in 36% yield. 1H
NMR (CDCl3)d2.24 (s, 6H), 2.28, 2.65(2H, m), 2.83 (m, 3H), 3.62 (s,
3H), 7.13 (m, 2H), 7.36 (s, d, 2H); MS (ES+) [M+H+]: 300, 302. The
4-bromo isomer of 5 was prepared similarly from 4-bromobenzyl
bromide 4 in 32% yield. 1H NMR (CDCl3)d2.23 (s, 6H), 2.28,
2.64(2H, 2 m), 2.82 (m, 3H), 3.61 (s, 3H), 7.03 (d, J = 8.42 Hz, 2H),
7.39 (d, J = 8.42 Hz, 2H); MS (ES+) [M+H+]: 300, 302. As a by-
product, N,N-dimethyl-4-bromobenzylamine was isolated in 13%
yield. 1H NMR (CDCl3)d2.22 (s, 6H), 3.37 (s, 2H), 7.18 (d,
J = 8.32 Hz, 2H), 7.44 (d, J = 8.36 Hz, 2H); MS (CI) [M+H+]: 214, 216.
To a 100 ml round bottom flask containing the thioester 9
(320 mg, 1 mmol) was added THF (10 ml) and the mixture was
cooled at 01C. A solution of glycine benzyl ester hydrochloride
(201 mg, 1 mmol) and triethylamine (101 mg, 1 mmol) in CH2Cl2
(10 ml) was added dropwise, followed by additions of a solution
of HOBT (153 mg, 1 mmol) in THF (7 ml) and a solution of DCC
(247 mg, 1.2 mmol) in CH2Cl2 (8 ml). The cooling bath was
removed and the reaction mixture was stirred at ambient
temperature overnight. Both HPLC and TLC analysis confirmed
completion of the reaction. The mixture was concentrated and
the residue purified by silica gel flash column chromatography
eluting with 0–2% ethyl acetate in CH2Cl2 to give 10 as a white
solid (420 mg) in 91% yield. 1H NMR (CDCl3)d2.33 (s, 3H), 2.61 (m,
1H), 2.92 (m, 4H), 3.95 (m, 2H), 5.15 (d, J = 2.3 Hz, 2H), 5.84 (m,
1H), 7.05 (d, J = 8.20 Hz, 2H), 7.36 (m, 9H); MS (ES+) [M+H+]: 264,
266; (ESÀ) [MÀCH3CO+]: 420, 422.
2-[3-(4-Bromophenyl)-2-(sulfanylmethyl)propionylamino]acetic
acid (11)
2-(3- or 4-Bromobenzyl)-3-methoxy-N,N,N-trimethyl-3-oxo-1-pro-
panaminium iodide (6)
To a 100 ml round bottom flask containing the benzyl ester 10
(420 mg, 0.9 mmol) was added MeOH (15 ml). The resulting
solution was cooled at 01C and 1 N NaOH solution (2 ml) was
added via a syringe pump over 1 h. The mixture was stirred at
01C for 2.5 h and then at room temperature for 3 h. HPLC
analysis showed 75% of desired product 11 and 10% of disulfide
12. The remainder consisted of partially hydrolyzed products.
MS of 11: (ES+) [M+H+]: 332, 334. This reaction mixture was used
without workup for the preparation of the disulfide 12.
To a 10 ml pear-shaped flask containing 3-(3-bromophenyl)-2-
dimethylaminomethylpropionic acid methyl ester 5 (0.762 g,
2.54 mmol) was added isopropanol (5 ml) and iodomethane
(480 ml, 1.09 g, 7.68 mmol). The mixture was stirred at ambient
temperature overnight. The resulting white suspension was
filtered, washed with i-PrOH and air-dried to give the 3-bromo
ammonium iodide 6, (0.95 g) in 85% yield. The 4-bromo isomer 6
was prepared similarly from 4-bromo 5 in 82% yield. MS (ES+)
[MÀIÀ]: 314, 316. HPLC purity was 95%.
2-[3-(4-Bromophenyl)-2-(sulfanylmethyl)propionylamino]acetic
acid disulfide (12)
2-(3- or 4-Bromobenzyl)acrylic acid (7)
To a 10 ml round bottom flask containing 2-(3-bromobenzyl)-3-
methoxy-N,N,N-trimethyl-3-oxo-1-propanaminium iodide
The crude mixture of 11 was cooled to 01C, a solution of I2 (127 mg,
0.5 mmol) in MeOH (1.0 ml) was added slowly over 5 min and the
resulting mixture was stirred for additional 30 min. A few drops of
sat. Na2SO3 solution was added to de-colorize the mixture followed
by careful addition of 0.2 N HCl to adjust pH to ꢀ2. The mixture
was extracted with ethyl acetate twice, organic phases were
combined, washed with water, brine and dried over sodium sulfate.
After filtration and solvent evaporation, the residue was passed
through a short silica gel cartridge eluting with 5–10% MeOH in
CH2Cl2 to afford 244 mg of 12 in 81% yield from 9. HPLC purity was
490%. MS of 12: (ES+) [M+H+]: 661, 663, 665.
6
(0.95 g, 2.15 mmol) was added 1 N NaOH (4.3 ml), the mixture
was heated at 1051C for 2 h. After cooling to room temperature,
it was acidified with 0.5 N HCl to pH ꢀ1, the white precipitate
was isolated by filtration, washed with water and air-dried to
give 3-bromo 7 (0.44 g) in 85% yield. AN-HPLC purity was 93%.
The 4-bromo isomer 7 was prepared similarly from 4-bromo 6 in
1
93% yield. HPLC purity was 94%. H NMR (CDCl3)d3.58 (s, 2H),
5.61 (s, 1H), 6.38 (s, 1H), 7.08 (d, J = 8.27 Hz, 2H), 7.42 (d,
J = 8.35 Hz, 2H); MS (ESÀ) [MÀH+]: 239, 241; [2M+Na+À2H+]: 501,
503, 505.
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 52 23–28

S.-Y. Wu and M. R. Masjedizadeh
Catalytic hydrodebromination of 2-[3-(4-bromophenyl)-2-(sulfanyl- filled up with D2 gas). The mixture was stirred for 2 h at ambient
methyl)propionylamino]acetic acid disulfide (12)
temperature. The catalyst was removed by filtration and the
solvent was evaporated. The residue was analyzed by HPLC,
which showed a clean and complete conversion. MS of [2H]-18
(ES+) [M+H+]: 254, 255, 256. The amount of deuterium
incorporation was determined by LC/MS as 30% D0, 47% D1,
22% D2 and 3% D3.
The tritiation was performed by ViTrax Co. based on a slightly
modified reaction scale (17/20%Pd(OH)2/H2O/TEA/reaction
time = 6 mg : 18 mg : 2.5 ml : 25 ml : 5 h). This resulted in a total
activity of 219 mCi of crude product [3H]-18, which showed
radiochemical purity of ꢀ90% by TLC and a 87% purity by
HPLC. Analytical HPLC conditions: Zorbax SB-C8, 4.6 Â 150 mm,
5 mm. A: 0.1% TFA in H2O, B: acetonitrile, gradient from 30 to
90% B 0–20 min; hold 90% B 5 min; post time 10 min; flow rate:
1 ml/min; temp. 301C, radiodetector. Retention time for [3H]-18:
8.28 min.
A typical reaction is as follows: to a 25 ml two-neck flask were
added the substrate 12 (12 mg), water (4 ml), 20% Pd(OH)2 on
carbon (24 mg), and triethylamine (20 ml). The mixture was
stirred under D2 (balloon) and monitored by HPLC. Sampling
was done at 2, 5 and 16 h. The reaction gave o5% of the desired
product 13.
2-Sulfanylmethyl-3-(3-bromophenyl)propionic acid (15)
To a two-neck 25 ml round bottom flask containing the thioester
8 (0.25 g, 0.79 mmol) was added 0.2 N NaOH (5 ml, 1 mmol). The
mixture was stirred at ambient temperature for 17 h. Additional
base (1.9 N NaOH, 0.7 ml) was added and the mixture was stirred
for additional 15 min. HPLC analysis showed completion of
reaction. HPLC purity: 81% (14% disulfide), LC–MS was used to
identify the products. MS of 15: (ESÀ) [M–H+]: 273, 275. This was
used for the next reaction without further purification.
[3H]-S-(2-Nitrophenylsulfanyl) thiorphan ([3H]-19)
To a 25 ml pear-shaped flask was added a solution of [3H]-18
(43.8 mCi) in MeOH (4 ml). Solvent was evaporated and to the
residue was added acetic acid (6 ml) followed by addition of a
solution of 2-nitrobenzenesulfenyl chloride (3.0 mg) in acetic
acid (3 ml). The mixture was stirred for 1 h. AcOH was
evaporated at reduced pressure and the residue was used for
the next reaction without further purification. Analytical HPLC
conditions: Zorbax SB-C8, 4.6 Â 150 mm, 5 mm. A: 0.1% TFA in
H2O, B: acetonitrile, gradient from 30 to 90% B 0–20 min; hold
90% B 5 min; post time 10 min; flow rate: 1 ml/min; temp. 301C,
adiodetector. Retention time for [3H]-19: 10.20 min.
2-(S-t-butylsulfanylmethyl)-3-(3-bromophenyl)propionic acid (16)
To the crude reaction mixture of 15 under N2 was injected a
mixture of t-BuOH (3 ml) and 37% HCl (2.5 ml). It was heated at
reflux for 4 h, cooled to room temperature and partitioned
between water and CH2Cl2. Aqueous phase was re-extracted
with CH2Cl2, the combined CH2Cl2 extracts were washed with
brine twice and dried over sodium sulfate. The crude product
was purified by silica gel chromatography eluting with 0–5%
EtOH in CH2Cl2 to give 16 as a colorless thick oil (0.133 g) in 51%
yield in two steps. HPLC purity: 95%, MS of 16: (ESÀ) [M–H+]:
329, 331.
[3H]-Thiorphan (20)
2-[3-(3-Bromophenyl)-2-(S-t-butylsulfanylmethyl)propionylami-
no]acetic acid benzyl ester (17)
To the crude [3H]-19 were added CH2Cl2 (4 ml), DTT (2.9 mg) and
triethylamine (10 ml). The mixture was stirred under N2 for
To a 25 ml pear-shaped flask containing acid 16 (0.22 g, 30 min, concentrated to dryness, re-dissolved in 200 ml of 0.2%
0.665 mmol) and THF (7 ml) at 01C was dropwise added a DTT in MeOH/H2O (1:1) and purified by preparative HPLC to give
solution of glycine benzyl ester hydrochloride (0.134 g, 26.97 mCi [3H]-thiorphan with a radiochemical purity of 99.8%
0.665 mmol) and triethylamine (93 ml, 67.3 mg, 0.665 mmol) in by AN-HPLC. Specific activity was 18.42 Ci/mmol. It was stored as
CH2Cl2 (7 ml), followed by a dropwise addition of a solution of 0.2375 mCi/ml aqueous solution containing 10% MeOH and
HOBT (0.102 g, 0.665 mmol) in THF (5 ml) and a solution of 1,3- 0.2% DTT. Analytical HPLC conditions: Zorbax SB-C8,
dicyclohexyl carbodiimide (0.164 g, 0.796 mmol) in CH2Cl2 (6 ml). 4.6 Â 150 mm, 5 mm. A: 0.1% TFA in H2O, B: 0.1% TFA in ACN,
The ice/water cooling bath was removed and the mixture was gradient from 10 to 90% B 0–20 min; hold 90% B 5 min; post
stirred at ambient temperature overnight. After concentration, time 10 min; flow rate: 1 ml/min; temp. 301C, radiodetector.
the residue was partitioned between water and CH2Cl2 and the Rentention time for [3H]-thiorphan: 9.16 min.
aqueous layer was extracted twice with CH2Cl2. The combined
extract was washed with brine, dried over sodium sulfate and Thiorphan stability in solutions
purified by silica gel flash column chromatography eluting with
Four series of 0.01% unlabelled thiorphan solutions were
prepared by a combination of a primary solvent and 0.2% of
an anti-oxidant (water–DTT, water–BME, ethanol–DTT, and
0–50% ether in hexane to give 17 as a colorless thick oil (0.25 g)
1
in 79% yield. HPLC purity: 100%. H NMR (CDCl3)d1.29 (s, 9H),
2.51 (m, 1H), 2.64 (m, 1H), 2.85 (m, 3H), 4.02 (m, 2H), 5.16 (s, 2H),
ethanol–BME, respectively) with or without additives such as
5.95 (m, 1H), 7.12 (m, 2H), 7.35 (m, 7H); MS (ES+) [M+H+]: 478,
co-solvent (MeOH, H2O) or various acids. The five solutions in
480; (ESÀ) [M–H+]: 476, 478.
each of the two aqueous series included (in addition to
[2H]- or [3H]-S-t-Butyl thiorphan ([2H]- or [3H]-18)
water–DTT or water–BME) (1) 10% methanol, (2) 40% methanol,
(3) 10% methanol+0.005% TFA, (4) 10% methanol+0.005% conc.
Procedure for reduction with deuterium gas: to a 10 ml pear-
shaped flask containing a magnetic stirrer bar was added the
ester 17 (2.5 mg, 0.0052 mmol), 20% Pd(OH)2/C (7.2 mg), H2O
(1 ml), and triethylamine (10 ml). The air inside the flask was
removed by reduced pressure and it was refilled with D2 gas
from a balloon (about 10 ml of open space inside the flask was
HCl, (5) 10% methanol+0.01% acetic acid. The five solutions in
each of the two ethanol series included (in addition to
ethanol–DTT or ethanol–BME) (1) no other additives, (2) 40%
water, (3) 0.005% TFA, (4) 0.005% conc. HCl, (5) 0.01% acetic
acid. These solutions were stored at 41C under N2 in dark, and
HPLC samplings were done at 3, 15, 30 and 90 days after initial
J. Label Compd. Radiopharm 2008, 52 23–28
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

S.-Y. Wu and M. R. Masjedizadeh
preparations. Based on the findings, the labelled [3H]-thiorphan Catalytic tritium/bromine exchange reaction was carried out by
with 99.8% radiochemical purity was stored under optimal ViTrax Co.
conditions (aqueous solution containing 10% methanol and
0.2% DTT at 0.24 mCi/ml, at 41C under N2 in dark) and re-
analyzed by HPLC after 130 days; only 6% radiochemical
degradation was found.
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Conclusion
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Acknowledgement
The authors thank Tatyana Voronin and Rong Teng for
purification and analysis of [3H]-thiorphan with HPLC and MS.
www.jlcr.org
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J. Label Compd. Radiopharm 2008, 52 23–28