Synthesis of [2H3]-labelled sulfamethoxazole and its main urinary metabolites

Article abstract of DOI:10.1002/jlcr.1375

Sulfamethoxazole was labelled at positions 3, 5, and 4′ by H/D exchange in a mixture of 5% ²H₂SO₄ and 95% ²H₂O (v/v) under reflux for 72 h with good isotope incorporation and acceptable yield. Subsequ

Full text of DOI:10.1002/jlcr.1375

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 656–659.
Published online 4 June 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1375
JLCR
Research Article
Synthesis of [2H3]-labelled sulfamethoxazole and its main
urinary metabolites
1,2,
GEORG HEINKELE1,2 and THOMAS E. MURDTER
*
¨
1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany
2 University Tu¨bingen, Germany
Received 23 October 2006; Revised 15 March 2007; Accepted 16 March 2007
Abstract: Sulfamethoxazole was labelled at positions 3, 5, and 40 by H/D exchange in a mixture of 5% 2H2SO4 and
95% 2H2O (v/v) under reflux for 72 h with good isotope incorporation and acceptable yield. Subsequently, [2H3]-
sulfamethoxazole-N1-glucuronide and [2H3]-N4-acetyl-sulfamethoxazole were synthesized. Copyright # 2007 John
Wiley & Sons, Ltd.
Keywords: sulfamethoxazole; deuterium label; sulfamethoxazole-N1-glucuronide; N4-acetyl-sulfamethoxazole
Introduction
catalyzed hydrogen–deuterium exchange reactions4
followed by acetylation and glucuronidation, respec-
tively (Figure 1). In an initial attempt we treated (1) with
a mixture of 20% 2H2SO4 and 80% 2H2O (v/v) under
reflux. H/D exchange and the stability of (1) was
monitored by LC-MS (Figure 2). Whilst H/D exchange
was rapid and almost exhaustive within 24 h, massive
degradation took place and we were not able to isolate
any product from the reaction mixture. To minimize
this degradation the concentration of 2H2SO4 was
Sulfamethoxazole (4-amino-N-(5-methylisoxazol-3-yl)-
benzenesulfonamide) (1) is a chemotherapeutic widely
used in the treatment of urinary and respiratory
infections in humans as well as in agriculture. In the
year 2003 in Germany more than 48 000 kg were
prescribed for human medicine alone.1 In humans (1)
is extensively metabolized and excreted in the urine
mainly as N4-acetyl-sulfamethoxazole (N-(4-{[(5-methy-
lisoxazol-3-yl)amino]sulfonyl}phenyl)-acetamide) (45–
70%) and sulfamethoxazol-N1-glucuronide (N-[(4-ami-
nophenyl)- sulfonyl]-N-(5-methylisoxazol-3-yl)hexapyr-
anuronosylamine) (5–15%).2,3
2
2
reduced to 5% H2SO4 in 95% H2O (v/v). Under these
conditions, after a reaction time of 72 h 38% of 3,5,40-
[2H3]-sulfamethoxazole (2) was isolated with an isotope
incorporation of >93%. Prolonged reaction time even
improved isotope incorporation. However, this resulted
in a dramatic decrease in yield (Table 1). As microwave
irradiation can be very useful in accelerating H/D
exchange,5 in an additional experiment, the H/D
exchange reaction was carried out under microwave-
enhanced conditions. After 1 h in a domestic micro-
wave, at low energy to avoid release of vapor through
the pressure control valve, the isotopic distribution was
comparable to conventional heating under reflux for
50 h (Table 1). However, the reproducibility of results
was unsatisfactory, possibly because of the inhomo-
geneity of the field in our domestic system.
Highly sensitive mass spectrometric methods are
used to investigate the environmental load of these
substances and these assays require stable isotope
internal standards.
Results and discussion
In principle, a de novo synthesis approach using
deuterium-labelled acetanilide can be applied to pre-
pare deuterium-labelled (1). However, this requires a
multi-step synthesis starting with an expensive pre-
cursor. Therefore, we decided to investigate acid
(2) was converted to 3,5,40-[2H3]-N4-acetyl-sulfa-
methoxazole (3) by acetylation in pyridine according
to Fujimoto and Okabe.6 Glucuronidation was per-
formed using 2,3,4-tri-O-acetyl-1-bromo-1-desoxy-a-d-
glucopyranosiduronic acid methyl ester (4) and LiOH in
*Correspondence to: Thomas E. Mu¨rdter, Dr. Margarete Fischer-Bosch
Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376
Stuttgart, Germany. E-mail: thomas.muerdter@ikp-stuttgart.de
Contract/grant sponsor: Robert Bosch Foundation
Copyright # 2007 John Wiley & Sons, Ltd.

LABELLED SULFAMETHOXAZOLE BY H/D EXCHANGE 657
2H
3
H
2''
6'
1'' N
4
2H
2
1
5'
O
4'
2H5
O
O
S
6
N
N
3'
O
2H
AcCl in py
H
3
6'
H2N
H2N
4
2H
3
2
1
5'
2H2O
O
O
4'
2H5
2H
3
O
O
S
S
2H2SO4
6
N
N
N
3'
N
6'
O
O
H2N
4
2H
2
5'
H
H
O
4'
1
2H5
O
1
1. LiOH + 4
S
2
6
N
N
3'
in meOH
O
6''
HOOC
2. LiOH in H2O
O
H
H
5''
H
4''
HO
1''
OH
H
2''
3''
H
OH
5
Figure 1 Synthesis of [2H3]-sulfamethoxazole, [2H3]-sulfamethoxazole-N1-glucuronide and [2H3]-N4-acetyl-sulfamethoxazole.
Figure 2 H/D exchange of sulfamethoxazole in 20% 2H2SO4 in 80% 2H2O (v/v). Reaction was performed as given in text. Samples
were taken from the reaction mixture and immediately frozen and stored at ꢀ288C until isotope distribution was determined by LC-
MS. Quantification of total sulfamethoxazole in the reaction mixture was done by HPLC-UV at 254 nm. m: d0; .: d1; &: d2; *: d3; &:
amount relative to t¼ 0 h.
methanol followed by deprotection with aqueous LiOH
as described previously.7 However, to achieve accep-
table yields of 3,5,40-[2H3]-sulfamethoxazol-N1-glucur-
onide (5) elevated temperature and prolonged reaction
times were necessary.
nologies, Waldbronn, Germany) and calculated by
comparison to the unlabelled compound. Identification
of the localization of deuterium labels was done by
comparison of 1H-NMR spectra of labelled and un-
labelled compounds at 250 or 500 MHz (Bruker,
Karlsruhe, Germany).
Experimental
3,5,40-[2H3]-sulfamethoxazole (2)
Reactions requiring water-free conditions were per-
formed in glass ware which was dried by heating under
a stream of dry argon prior to use. All reactions were
performed under an argon atmosphere. The degree
of isotope labelling was determined by electrospray
ionization-mass spectrometry (HP 1100, Agilent Tech-
Two gram of (1) was dissolved in 5 ml 2H2SO4 and 95 ml
2H2O and heated under reflux for 72 h. At the end of the
reaction time the slightly yellow reaction mixture was
cooled in an ice bath and the pH was adjusted to 5 with
5 M NaOH. During the adjustment of pH a white solid
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 656–659
DOI: 10.1002.jlcr

¨
658 G. HEINKELE AND T. E. MURDTER
Table 1 H/D exchange of sulfamethoxazole in 5% 2H2SO4 in 95% 2H2O (v/v)
Time (h)
Isotope distribution (%)
d2
Isolated yield (%)
d0
d1
d3
d4
48
72
96
60 mina
0.00
0.00
0.00
0.00
0.83
0.09
0.00
0.61
30.2
6.45
3.1
69.0
93.32
96.7
73.4
0.06
0.10
0.23
0.2
62
38
13
45
25.8
Note: Reactions were performed as given in text but terminated at the times indicated in the table.a Microwave.
precipitated. The reaction mixture was extracted three
times with 100 ml ethyl acetate in which the precipitate
dissolved immediately. The organic phase was dried
over sodium sulfate and evaporated to dryness to
obtain 753 mg (38%) of (2) as a white amorphous solid.
Recrystallization from ethanol gave colorless cubic
crystals. Yield: 659 mg (33% of theory). For isotope
distribution see Table 1. Anal.: Calculated for
chloric acid and water and dried to obtain 250 mg (84%
of theory) of (3) as a slightly brown solid of a purity
>99% (HPLC-UV at 254 nm). Anal.: Calculated for
C12H210H3N3O4S: C, 48.31; Hþ2H, 4.40; N, 14.09; O,
21.45; S, 10.75. Found: C, 48.34; Hþ2H, 4.41; N,
14.10; S, 10.94. Isotope distribution: d0: 0.00, d1: 0.07,
d2: 7.03, d3: 93.1, d4: 0.2. 13C-NMR (in 2H6-DMSO):
170.2 (C-50); 169.1 (C-100); 157.5 (C-30); 143.4 (C-4);
132.8 (C-1); 127.9 (C-2, C-6); 118.4 (low intensity)
(C-3, C-5); 95.3 (low intensity) (C-40); 24.1 (C-200); 12.0
C
10H82H3N3O3S: C, 46.86; Hþ2H 4.33; N, 16.40; O,
18.73; S, 12.51. Found: C, 46.83; Hþ2H 4.36; N, 16.43;
13
2
S, 12.68. C-NMR (in C2H3O2H): 171.7 (C-50); 159.6
(C-60). 1H-NMR (in H6-DMSO): 7.79 2H (C-2, C-6); 6.1
(C-30); 154.7 (C-4); 130.2 (C-2, C-6); 126.4 (C-1);
113.6–114.4 (m, low intensity, C-3, C-5); 95.8–96.7
(m, low intensity, C-40); 12.3 (C-60). 1H-NMR (in
C2H3O2H): 7.58 (s 2H, C-2, C-6); 6.13 (s 0.04H, C-40);
2.32 (s 3H, C-60). (Assignment was done according
to COSY experiments and in agreement with Saito
et al.8)
0.05H (C-40); 2.30 s 3H (C-60); 2.10 s 3H (C-200).
(4-amino-N-(b-D-glucopyranuronosyl)-N-(5-methy-
loxazol-3-yl)-benzenesulfonamide) (5)
To a hot solution of 550 mg of (2) in 7.5 ml methanol,
90.2 mg of LiOH and 854 mg of (4) were added and
stirred under reflux for 90 min. After the reaction
mixture was cooled in an ice bath, 496 mg of LiOH in
7.5 ml water was added and the solution was stirred for
additional 30 min at room temperature. The pH of the
reaction mixture was adjusted to pH 4 by the addition
of glacial acetic acid and unchanged (2) was extracted
three times with 15 ml chloroform. The organic phase
was dried over sodium sulfate and evaporated to
dryness to obtain 377 mg (68.5%) of (2) as a yellow
solid which was identified as pure (2) by TLC and
HPLC. The aqueous phase was evaporated to dryness
under reduced pressure. The foamy residue was stored
over potassium hydroxide under reduced pressure to
remove traces of acetic acid. The majority of hydrophilic
by-products were removed by a column chromatogra-
phy on silica gel 60 with acetone:methanol 50:50 (v/v).
Final purification was carried out by chromatography
on a Lobar B column packed with Lichroprep RP 18
(40–64 mm) (Merck, Darmstadt, Germany) with a two
step gradient. Firstly, some brown by-products were
eluted with water:methanol 85:15 (v/v) then (5) was
separated from an unidentified glucuronide with
water:methanol 2:1 (v/v). Freeze drying of the pooled
fractions containing (5) resulted in 123 mg (13.2% of
theory) of a white solid. Isotope distribution: d0: 0.00,
3,5,40-[2H3]-sulfamethoxazole (2) microwave assisted
In a closed PTFE-reaction vessel equipped with a
pressure control valve (approximately 7 bar) 500 mg of
(1) was dissolved in 1.5 ml 2H2SO4 and 28.5 ml 2H2O.
The reaction vessel was irradiated in a domestic
microwave oven at a nominal energy of 80 W (1/10 of
time with a fixed energy of 800 W) for 60 min. After
cooling down to room temperature, the reaction vessel
was opened, cooled in an ice bath and the product was
separated as described above, to obtain 224 mg (45% of
theory) as a white solid.
3,5,40-[2H3]-N4-acetyl-sulfamethoxazole (3)
To a solution of 256 mg (1 mmol) (2) in 5 ml dry
pyridine, 142 ml (2 mmol) acetyl chloride was added.
Spontaneously, a white solid precipitated. This pre-
cipitate re-dissolved while the reaction mixture was
stirred at 1008C. After 30 min the yellow solution was
cooled in an ice bath and diluted with 25 ml of ice-cold
1 M NaOH. Acidifying of the reaction mixture with
chilled 10% hydrochloric acid resulted in precipitation
of (3). The precipitate was washed with diluted hydro-
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 656–659
DOI: 10.1002.jlcr

LABELLED SULFAMETHOXAZOLE BY H/D EXCHANGE 659
d1: 0.08, d2: 6.12, d3: 93.7, d4: 0.04. 13C-NMR (in
2H2O): 175.1 (C-600); 173.2 (C-50); 156.6 (C-30); 153.0
(C-4); 130.2 (C-2, C-6); 123.8 (C-1); 114.3 (m, low
intensity, C-3, C-5); 102.5 (m, low intensity, C-40); 86.6
(C-100); 78.8 (C-500); 76.7 (C-300); 71.4 (C-400); 69.6 (C-200);
11.9 (C60). 1H-NMR (in 2H2O): 7.45 (s, 2H, C-2, C-6);
6.13 (s, 0.05H, C-40); 5.21 (d, 1H, C-100); 3.69 (d, 1H,
C-500); 3.49 (t, 1H, C-300); 3.30 (t, 1H, C-400); 3.14 (t, 1H,
C-200); 2.29 (s, 3H, C-60).
REFERENCES
¨
1. Kummerer K, Henninger A. Clin Microbiol Infect
2003; 9: 1203–1214. DOI: 10.1111/j.1469-0691.
2003.00739.x
2. van der Ven AJ, Vree TB, van Ewijk-Beneken Kolmer
EW, Koopmans PP, van der Meer JW. Br J Clin
Pharmacol 1995; 39: 621–625.
3. Brackett CC, Singh H, Block JH. Pharmacoth-
erapy 2004; 24: 856–870. DOI: 10.1592/phco.24.9.
856.36106
4. Werstiuk NH. Hydrogen isotope labelling of aromatic
compounds in aqueous medium at elevated tem-
perature. In Synthesis and Application of Isotopically
Labelled Compounds 1991, Buncel E, Kabalka GW
(eds). Elsevier Science Publishers BV: Amsterdam,
1992; 303–308.
Conclusion
Acid catalyzed H/D exchange by heating under atmo-
spheric pressure or microwave assisted under elevated
pressure is a suitable method to obtain deuterium-
labelled sulfamethoxazole with high isotope incorpora-
tion and acceptable yield. Labelling proved to be stable
during subsequent conjugation reactions to 3,5,40-
[2H3]-N4-acetyl-sulfamethoxazole and 3,5,40-[2H3]- sul-
famethoxazole-N1-glucuronide, respectively.
5. Barthez JM, Filikov AV, Frederiksen LB, Huguet
M-L, Jones JR, Lu S-Y. Can J Chem 1998; 76:
726–728.
6. Fujimoto M, Okabe K. Chem Pharm Bull 1962; 10:
572–575.
¨
¨
7. Heinkele G, Schanzle G, Murdter TE. J Label Compd
Radiopharm 2002; 45: 1153–1158. DOI: 10.1002/
jlcr.640
Acknowledgements
We are grateful to Dr M. Letzel at the Bavarian
Environment Agency, Department of Analytical Labora-
tories and Evaluation of Chemicals, Munich, for the
initiation of this work. We thank Mr J. Rebell at the
University of Stuttgart, Institute of Organic Chemistry, for
the acquisition of NMR spectra. This work was supported
by the Robert Bosch Foundation, Stuttgart, Germany.
8. Saito T, Hayamizu K, Yanagisawa M, Yamamoto O.
Spectral Database for Organic Compounds (SDBS),
National Institute of Advanced Industrial Science
and Technology, Tokyo. URL: http://www.aist.
go.jp/RIODB/SDBS/cgi-bin/direct frame top.cgi?
lang=eng. access: 2006-09-15
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 656–659
DOI: 10.1002.jlcr