Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide

Article abstract of DOI:10.1002/jlcr.1933

Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S-oxidations. The ultimate toxic metabolite thioacetamide S,S-dioxide, or its tautomer acetimidoyl sulfinic acid CH ₃C(NH)SO₂H, then acylates lysine side chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the extent of their modification and elucidate the structural details of their modification, we required both radio-labeled and stable-labeled forms of TA and its intermediate metabolite thioacetamide S-oxide (TASO). The latter is stable when purified but can be difficult to isolate. Considering currently available isotopic precursors, we devised and report here methods for the synthesis and isolation of TA and TASO labeled with C-14, C-13, and/or deuterium. The methods are straightforward, utilize readily available precursors, and are amenable to small scale.

Full text of DOI:10.1002/jlcr.1933

Research Article
Received 2 June 2011,
Revised 21 July 2011,
Accepted 17 August 2011
Published online 17 October 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1933
Synthesis of carbon-14, carbon-13 and
deuterium labeled forms of thioacetamide and
thioacetamide S-oxide
*
Diganta Sarma and Robert P. Hanzlik
Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S-oxidations. The ultimate
toxic metabolite thioacetamide S,S-dioxide, or its tautomer acetimidoyl sulfinic acid CH3C(NH)SO2H, then acylates lysine side
chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the
extent of their modification and elucidate the structural details of their modification, we required both radio-labeled and
stable-labeled forms of TA and its intermediate metabolite thioacetamide S-oxide (TASO). The latter is stable when purified
but can be difficult to isolate. Considering currently available isotopic precursors, we devised and report here methods for
the synthesis and isolation of TA and TASO labeled with C-14, C-13, and/or deuterium. The methods are straightforward, uti-
lize readily available precursors, and are amenable to small scale.
Keywords: acetate; thioacetamide; thioacetamide S-oxide; deuterium exchange; pyrolysis
obtained from Cambridge Isotopes (Andover, MA, USA). All other
chemicals and solvents were obtained from Sigma Aldrich (St. Louis,
Introduction
Thioacetamide (TA, 1), a prototype thiono-sulfur (>C = S) com-
pound, has been used industrially in the leather, textile, and paper
MO, USA). Hydrogen peroxide was titrated iodometrically before
use. 1H and 13C NMR spectra were recorded on Bruker AV III
industries, as an accelerator in the vulcanization of buna rubber, as
500 MHz spectrometer, and the spectra were referenced to the
a stabilizer in motor fuels, and as a replacement for hydrogen sul-
residual NMR solvent peak. Electrospray ionization spectra were
fide in qualitative analysis.1 TA was also once used as a fungicide
acquired on a LCT Premier time of flight mass spectrometer.
on oranges, but it was reported that a single dose of this toxin in
Gas chromatography/mass spectrometry (GC/MS) analyses were
animals can produce centrilobular necrosis and that chronic
conducted with an Agilent 6890 N GC (Agilent Technologies, Santa
administration leads to liver cirrhosis and even hepatocarcinoma.2
Clara, CA, USA) interfaced with a quadrupole mass analyzer using
an injector temperature of 240ꢀC and an initial column tempera-
ture of 50 ꢀC for 1 min followed by a gradient of 50–300 ꢀC over
13min. Ionization was by electron impact at 70eV. Thin-layer chro-
matography was performed on Silica Gel GF plates (2.5Â 10cm,
0.25 mm) with fluorescence detection under ultraviolet light at
254 nm for TA and TASO or by spraying the plate with 0.05%
Rhodamine 6 G in 95% ethanol to detect acetamide under ultravio-
let or ambient light. After eluting with 10% methanol in ethyl
acetate (v/v), the Rf values for TA, acetamide, and TASO were
0.80, 0.55, and 0.35, respectively. Column chromatography was
performed using silica gel (63–200 particle size). Radioactivity
was measured using a Packard Tri-Carb liquid scintillation counter.
More recently, TA has been used to generate useful animal models of
3
hepatic fibrosis, hepatic cirrhosis,4 and human cholangiocarci-
noma.5 TA6,7 and its congener thiobenzamide (TB, PhCSNH2)8,9 are
also widely used to model acute hepatotoxicity. The cytotoxicity of
both thioamides is believed to result from a two-step bioactivation
sequence leading first to a thioamide S-oxide such as TASO (2) and
then to a reactive S,S-dioxide such as TASO2 (3) (Scheme 1).10,11 The
iminosulfinic acid tautomers of TASO2 and TBSO2 are highly reactive
acylating agents toward lysine residues in proteins and may initiate
necrosis by covalently binding to liver macromolecules.8,12
Whereas numerous specific cellular protein targets have been
identified for TB8 and a number of other metabolically activated pro-
toxins,13 no specific cellular targets for TA metabolites have thus far
been identified. Detection of proteins adducted by reactive metabo-
lites commonly depends on treating animals or cells with isotopically
labeled protoxins, followed by 2D gel electrophoresis and identifica-
tion of targets by mass spectrometric analysis. Herein, we report sim-
ple and efficient methods for the synthesis of TA and TASO from
sodium acetate labeled with either 14C or 13C and deuterium.
Thioacetamide S-oxide (TASO, 2)
Thioacetamide, 1 (0.050 g, 0.666 mmol) was dissolved in 1 mL
methanol in a 50 mL screw-capped Corex tube. The solution
was cooled to 0 ꢀC, and hydrogen peroxide (30% w/w, 10.9 M
Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045,
USA
Experimental
[1-14C]-acetic acid, sodium salt (59.6 mCi/mmol and radiochemi-
*Correspondence to: Robert P. Hanzlik, Department of Medicinal Chemistry,
University of Kansas, Lawrence, KS 66045, USA.
cal purity >98%) was obtained from Moravek Biochemical (San
Diego, CA, USA). [1,2-13C]-acetic acid-d3, sodium salt was E-mail: rhanzlik@ku.edu
J. Label Compd. Radiopharm 2011, 54 795–798
Copyright © 2011 John Wiley & Sons, Ltd.

D. Sarma and R. P. Hanzlik
Scheme 1. Metabolic activation of thioamides.
by iodometric titration, 0.046 mL, 0.501 mmol) was added. After formed during pyrolysis was removed using a rotovap yielding
stirring for 2 h at 0 ꢀC, the solvent was removed (rotovap, bath crude acetamide (6, 0.074 g, 1.254 mmol, quantitative yield) as
temperature ≤37 ꢀC). The residue was dissolved in a small quan- a highly viscous pale yellow liquid. Tetrahydrofuran (THF, 4 mL)
tity of methanol and transferred to a column packed with 20 g and Lawesson’s reagent (0.510 g, 1.254 mmol) were added
silica gel in ethyl acetate. Elution was started with MeOH/ethyl directly to the crude product, and the mixture was stirred at
acetate (v/v= 1:9), and compound 2 was eluted with MeOH/ethyl room temperature for 2 h. With no work-up, the reaction mixture
acetate (v/v= 1:4). Removal of the solvent provided 2 as a crystal- was transferred to a column packed with 20 g silica gel in ethyl
line white solid (0.054 g, 0.593 mmol, 89% yield). 1H NMR (DMSO- acetate/hexane (1:9 v/v); TA (7) was eluted using ethyl acetate/
d6): d 1.94 (s, 3H), 8.19 (s, 1H), 9.01 (s, 1H). 13C NMR: d 13.83 hexane (1:2 v/v). Removal of the solvent provided 7 as crystalline
(–CH3), 189.97 (>C = S). TOF MS calcd for C2H5OSN [M + H]+ white solid (0.014 g, 0.187 mmol, specific activity 5.65 mCi/
92.0170, found 92.0159. The purified material was quite stable for mmol). Radiochemical purity as assessed by TLC was approxi-
several months when stored as a crystalline solid in a freezer (ca. mately 98%.
À20 ꢀC), as judged by TLC and by retention of its well-defined col-
orless crystalline appearance as tiny plates and pincushions.
[1-14C]-thioacetamide S-oxide (8)
Compound 7 (0.004 g, 0.054 mmol, 5.65 mCi/mmol) was com-
bined with ordinary TA (0.008 g, 0.110 mmol) in 2 mL methanol
[1-14C]-thioacetamide (7)
[1-14C]-acetic acid, .sodium salt (4, 0.017 g, 0.126 mmol, 7.5 mCi)
was dissolved in 1 mL methanol and transferred to a 12 mL
in a culture tube containing a magnetic stirring bar (Scheme 2).
The reaction mixture was cooled to 0 ꢀC, H2O2 (10.9 M by iodo-
metric tiration, 0.012 mL, 0.130 mmol) was added, and the reac-
tion was stirred at 0 ꢀC for 2 h. After removal of solvent (rotovap,
bath temperature ≤37 ꢀC), the residue was redissolved in a small
volume of methanol and transferred to a column of silica gel
(20 g) packed in ethyl acetate. Elution began with MeOH/ethyl
acetate (1:9 v/v) and compound 8 was eluted with MeOH/ethyl
acetate (1:4 v/v). Removal of the solvent provided 8 as a crystal-
line white solid (0.012 g, 0.132 mmol, 1.90 mCi/mmol)
screw-capped culture tube, and solid ammonium acetate (5,
0.087 g, 1.134 mmol) was added. After dissolution, the homoge-
neous mixture was evaporated to dryness (rotovap). The tube
was capped tightly and placed in a 6 in. length of galvanized iron
pipe closed at both ends with threaded pipe caps. The bottom
1.5 in. of the upright assembly was immersed in a sand bath
heated electrically to approximately 240 ꢀC overnight (ca. 24 h)
(Scheme 3). After cooling to room temperature, the water
Scheme 2. Synthetic routes to isotopically-labeled thioacetamide and thioacetamide S-oxide.
Scheme 3. Synthesis of [1-14C]-thioacetamide S-oxide. (a) pyrolysis in sealed screw-cap tube, 240 ꢀC, 24 h; (b) Lawesson’s reagent (p-MeOC6H4PS2)2, THF, r.t. 2 h; (c) 0.95
eq. H2O2, MeOH, 0 ꢀC, 2 h.
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J. Label Compd. Radiopharm 2011, 54 795–798

D. Sarma and R. P. Hanzlik
[1,2-13C]-Acetic acid-d4 (10)
packed with 20 g silica gel in ethyl acetate/hexane (1:9 v/v). Com-
pound 14 was eluted with ethyl acetate/hexane (1:2 v/v).
Removal of the solvent provided 14 as crystalline white solid
(0.012 g, 0.150 mmol). GC/MS examination of a methanol solu-
tion of 14 showed the expected molecular ion at 80 (vs. 75 for
unlabeled TA) and indicated complete retention of deuterium.
A 50 mL pear-shaped flask (19/22 joint) was charged with 1.0 mL
of D3PO4 (prepared by adding 2 vol of D2O to 85% H3PO4 and
then removing excess water using a rotary evaporator at ca.
70 ꢀC for a total of three cycles of D-exchange). The use of
D3PO4 rather than H3PO4 was precautionary to prevent loss of
deuterium by exchange. [1,2-13C]-acetic acid-d3, sodium salt tri-
hydrate (9, 0.508 g, 3.603 mmol) was then added as a solid pow-
der on top of the D3PO4; the flask was rotated several turns to mix
and spread out the viscous mass on the walls. It was immediately
affixed to a 135˚ bend connecting tube leading to a vacuum dis-
tillation adapter that terminated in a 50 mL round bottom receiv-
ing flask. After cooling the receiver flask to À78 ꢀC (dry ice/acet-
one), the system was evacuated to approximately 1 torr, and
the distillation flask was heated gently with a hair dryer until
the acetic acid that was generated all collected in the receiver
as a frozen white solid. Compound 10 was thus obtained as color-
less liquid at room temperature (0.258 g, 3.91 mmol). Identity,
purity, and complete retention of deuterium were confirmed by
GC/MS analysis of a tiny aliquot dissolved in methanol to wash
out the single easily exchangeable proton of the acid group.
[1,2-13C, 2,2,2-2H3]-thioacetamide S-oxide (15)
Compound 14 (0.010 g, 0.125 mmol) was dissolved in 1 mL metha-
nol and oxidized with standardized hydrogen peroxide solution
and isolated by chromatography as described for 2. Removal of
solvent provided 15 as tiny white crystals (0.006 g, 0.062 mmol).
TOF MS calcd for 13C2H2D3OSN [M + H]+ 97.0425, found 97.0439.
Results and Discussion
Thioacetamide S-oxide is a stable compound when pure, but its
synthesis and isolation are complicated by its extreme polarity
and high water solubility. It was first synthesized by Walter
who treated TA with aqueous hydrogen peroxide saturated with
ammonium sulfate.14 Later Porter and Neal prepared TASO on a
40 g scale by oxidizing TA with hydrogen peroxide in DMF and
isolating the product by precipitation with carbon disulfide.15
They also prepared [1-14C]-TASO by oxidizing [1-14C]-TA
[1,2-13C, 2,2,2-2H3]-Thioacetamide (14)
Compound 10 (0.240 g, 3.636 mmol) was cooled to À78 ꢀC in a (obtained commercially) with excess hydrogen peroxide in acet-
50 mL round bottom receiver flask, anhydrous ammonia (2 M one and isolating the product by low temperature crystallization.
solution in ethanol, 2.2 mL, 4.4 mmol) was added, and the reac-
As a precursor for the synthesis of isotopically labeled TASO,
tion mixture was allowed to warm to room temperature. Succes- labeled TA has been prepared by addition of H2S to acetonitrile
sive portions of the solution were transferred to a 16 Â 100 mm and by thionation of acetamide (Scheme 2). Anthoni et al.16 pre-
2
screw-cap culture tube, and the solvent was removed by rotary pared H, 13C, and 15N variants of acetonitrile by reacting CH3I
evaporation. Once all the material was pooled and evaporated, with KCN in glycerol (94–98% yield) and converting the latter
methanol-O-d (1.5 mL) was added to the residue (11) and then to TA using Ph2PS2H in toluene (55% yield after sublimation).
removed by rotary evaporation to wash out all exchangeable Porter et al.11 synthesized [3H]-TA by treating [3H]-MeCN with
hydrogens; this step was repeated one more time. Final evapora- (EtO)2PS2H in 40–70% overall yield. Benincori et al.17 prepared
tion of the solvent afforded 12 as colorless viscous liquid [13C2]-TA at 5% enrichment in 30% overall yield by refluxing a
(0.200 g, 2.326 mmol). The tube was then capped and placed in mixture of [13C2]-acetic acid with urea and thionating the result-
a metal pipe and heated at 240 ꢀC for 24 h as described for 7. ing acetamide product using P2S5 and K2S in toluene.
After cooling, the water formed during pyrolysis was removed
For the identification of hepatocyte proteins adducted by TA
(rotovap) to give 13 as colorless viscous liquid (0.040 g, metabolites, we first needed [14C]-TA to quantitate the overall
0.625 mmol). Complete retention of deuterium was confirmed extent of protein adduction and to locate adducted protein
by GC/MS analysis of a tiny aliquot dissolved in methanol to spots on 2D gels by phosphor imaging. To facilitate the mass
wash out the two exchangeable protons of the amide group. spectral analysis of tryptic peptides derived from the radioactive
Compound 13 (0.035 g, 0.547 mmol) was dissolved in 2 mL THF spots, we also wanted a stable isotope signature having a mass
in a culture tube with a magnetic stirring bar. Lawesson’s reagent shift of at least 5 AMU8,18 in the TA and its metabolites. Consid-
(0.221 g, 0.547 mmol) was added, and the reaction mixture was ering the potential isotopic precursors currently available com-
stirred at room temperature for 2 h. Solvent was then removed mercially, we chose to begin with sodium [1-14C]-acetate and
(rotovap), the crude product was redissolved in small amount sodium [1,2-13C2-2,2,2-2H3]-acetate (Schemes
of ethyl acetate, and the solution was transferred to a column respectively).
3
and 4,
Scheme 4. Synthesis of stable labeled TASO.
J. Label Compd. Radiopharm 2011, 54 795–798
Copyright © 2011 John Wiley & Sons, Ltd.
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D. Sarma and R. P. Hanzlik
Our syntheses of [14C]-TA were predicated on an early report the methyl group. The latter was then thionated with Lawesson’s
by Hofmann19 indicating that acetamide could be prepared sim- reagent and carefully oxidized to TASO using 1.0 eq. of hydrogen
ply by pyrolyzing ammonium acetate. We therefore combined peroxide.
sodium [1-14C]-acetate at high specific activity with an excess
of ammonium acetate in methanol (to ensure isotopic mixing
and to achieve the desired final specific activity), evaporated
the solvent, and pyrolyzed the resulting material overnight in a
Acknowledgements
Funding from the National Institutes of Health (GM-21784) is
gratefully acknowledged.
sealed screw-cap culture tube (protected inside a metal pipe).
The acetamide product, formed in essentially quantitative yield,
was thionated with Lawesson’s reagent in THF to give [14C]-TA,
and the latter was oxidized to [14C]-TASO in ca. 30% overall yield. References
We discovered that it was quite critical to control the amount of
[1] Thioacetamide, in Report on Carcinogens, Eleventh Edition, U.S.
H2O2 added to just below 1.0 mol-equivalent to prevent over-
oxidation of the TA and to avoid having unoxidized TA as an
impurity in the material intended for metabolic studies. When
stored as a crystalline solid at À20 ꢀC, TASO was found to be
stable for several months.
Department of Health and Human Services, Public Health Service,
National Toxicology Program, 2005.
[2] O. G. Fitzhugh and A. A. Nelson, Science. 1948, 108, 626–628.
[3] H. Okuyama, N. H. Shimamura, N. Uyama, Y. W. Kwon, N. Kwada, Y.
Yamaoka and J. Yodoi, J. Hepatol. 2005, 42, 117–123.
[4] X. Li, I. S. Benjamin and B. Alexander, J. Hepatol. 2002, 36, 488–493.
[5] C.-N. Yeh, A. Maitra, K.-F. Lee, Y.-Y. Jan and M.-C. Chen, Carcinogen-
esis. 2004, 25, 631–636.
[6] S. K. Ramaiah, U. Apte and H. Mehendale, Drug metab Dispos. 2001,
29, 1088–1095.
[7] T. Wang, K. Shankar, M. J. J. Ronis and H. M. Mehendale, J. Pharmacol.
Exp. Therap. 2000, 294, 473–479.
[8] K. Ikehata, T. G. Duzhak, N. A. Galeva, T. Ji, Y. M. Koen and R. P. Hanzlik,
Chem. Res. Toxicol. 2008, 21, 1432–1442.
[9] T. Ji, K. Ikehata, Y. M. Koen, S. W. Esch, T. D. Williams and R. P. Hanzlik,
Chem. Res. Toxicol. 2007, 20, 701–708.
[10] J. R. Cashman and R. P. Hanzlik, J. Org Chem. 1982, 47, 4745–4650.
[11] W. R. Porter, M. J. Gudzinowicz and R. A. Neal, J. Pharmacol. Exp.
Therap. 1979, 208, 386–391.
[12] M. C. Dyroff and R.A. Neal, Mol. Pharmacol. 1983, 233, 219–227.
[13] J. Fang, Y. M. Koen and R. P. Hanzlik, BMC Chemical Biology. 2009, 9.
[14] W. Walter, Ann. Chem. 1960, 633, 35–49.
We expected the synthesis of [1,2-13C2-2,2,2-2H3]-TA to pro-
ceed almost as easily but were surprised by the ease with which
the deuterium atoms could be lost during the pyrolysis step,
apparently by exchange with the amide NH2 protons. Therefore,
a slightly more elaborate route was devised (Scheme 4). Sodium
[1,2-13C2-2,2,2-2H3]-acetate (9) was converted to acetic acid-d4
using 85% phosphoric acid (i.e., H3PO4•H2O) that had been deut-
erated by several cycles of adding and evaporating D2O to give
(D3PO4•D2O). This completely avoided the partial loss of deuter-
ium that occurred when nondeuterated 85% H3PO4 was used.
The labeled acetic acid (10) was isolated by bulb-to-bulb transfer
under vacuum in essentially quantitative yield and then neutra-
lized with anhydrous ammonia in ethanol to give ammonium
acetate 11 with full deuterium retention as verified by mass
spectrometry. Because pyrolysis of this material consistently lead
to the loss of most of the deuterium from the methyl group, we
surmised that the protons of the ammonium group of salt 11
were the source of hydrogens. Prior exchange of these with
methanol-O-d afforded labeled ammonium acetate 12, pyrolysis
[15] W. R. Porter and R. A. Neal, Drug metab Dispos. 1978, 6, 379–388.
[16] U. Anthoni and P. H. Nielsen, J. Label. Compd. Radiopharm. 1984, 21,
375–380.
[17] T. Benincori, T. Pilati, S. S. Rizzo M. and F. Sannicolo, Eur. J. Org. Chem.
2003, 2480–2487.
[18] W. Yue, Y. M. Koen, T. D. Williams and R. P. Hanzlik, Chem. Res. Toxicol.
2005, 18, 1748–1754.
of which produced acetamide 13 with no loss of deuterium from [19] A. W. Hofmann. Ber. 1882, 15, 977–984.
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