Synthesis and characterization of hydroxylated mesocarb metabolites for doping control

Article abstract of DOI:10.1002/ardp.200800144

The synthesis and method of analysis of hydroxylated mesocarb metabolites are described. Six potential hydroxylated mesocarb metabolites were prepared, characterized, and compared with the mesocarb metabolites synthesized enzymatically in vitro using human liver proteins and also compared with metabolites extracted from human urine after oral administration of mesocarb. p-Hydroxymesocarb was the most prevalent metabolite (conjugated and non-conjugated) observed. With respect to doping analysis, synthesis of p-hydroxymesocarb, the main urinary metabolite of mesocarb, and its availability as a reference material is important.

Full text of DOI:10.1002/ardp.200800144

Arch. Pharm. Chem. Life Sci. 2009, 342, 201–209
M. Vahermo et al.
201
Full Paper
Synthesis and Characterization of Hydroxylated Mesocarb
Metabolites for Doping Control
Mikko Vahermo1, Tina Suominen2, Antti Leinonen2, and Jari Yli-Kauhaluoma1
1 Faculty of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, Helsinki, Finland
2 United Laboratories Ltd., Doping Control Laboratory, Helsinki, Finland
The synthesis and method of analysis of hydroxylated mesocarb metabolites are described. Six
potential hydroxylated mesocarb metabolites were prepared, characterized, and compared with
the mesocarb metabolites synthesized enzymatically in vitro using human liver proteins and
also compared with metabolites extracted from human urine after oral administration of meso-
carb. p-Hydroxymesocarb was the most prevalent metabolite (conjugated and non-conjugated)
observed. With respect to doping analysis, synthesis of p-hydroxymesocarb, the main urinary
metabolite of mesocarb, and its availability as a reference material is important.
Keywords: Doping analysis / Mesocarb / Metabolite / Sydnone imine /
Received: August 6, 2008; accepted: December 12, 2008
DOI 10.1002/ardp.200800144
Introduction
Mesocarb (Sydnocarb), 3-(1-methyl-2-phenylethyl)-5-[[(phe-
nylamino)carbonyl]amino]-1,2,3-oxadiazolium (Fig. 1) is
a psychostimulant that is used in Russia as a drug for
treating psychiatric disorders such as schizophrenia and
depression [1]. The pharmacological profile of mesocarb
is similar to other stimulants but evidence suggests that
Figure 1. Structure of mesocarb.
it has a different mode of action [2]. When compared to
amphetamines, the effects build up more gradually and
extracted in minute quantities from either rat [6, 9, 10]
or human urine [8]. Several anti-doping laboratories have
studied the metabolism of mesocarb in man [8, 11–16].
Phase I metabolism of mesocarb introduces one or more
hydroxy groups to the phenylpropyl, phenylcarbamoyl
moiety or to both. The sulphate conjugate of p-hydroxy-
mesocarb 6a is likely to be the main phase II metabolite,
and a small amount of b-D-glucuronide conjugate has
also been detected [5]. In addition, Appolonova et al. have
found several mono-, di-, and trihydroxylated mesocarb
metabolites [8]. However, the precise regiochemistry of
these metabolites has remained unknown.
In this paper, we describe a convenient route to synthe-
size six potential mesocarb metabolites 6a–f (Fig. 2). Syn-
thesis methods of mesocarb have been published [17–
19], but to our knowledge no attempts to synthesize its
hydroxylated metabolites have been reported in the liter-
last longer. Mesocarb does not cause euphoria or locomo-
tor excitation, and it has weak addictive liability [3].
Mesocarb was added to the list of banned compounds
in sports by the Medical Commission of the International
Olympic Committee in 1991 [4]. Several analytical meth-
ods for detecting mesocarb or its metabolites have been
developed [5–8]. However, method development has
been difficult, because, until now, no pure metabolites
have been available [5] as reference substances, and
researchers have been dependent on the metabolites
Correspondence: Jari Yli-Kauhaluoma, Faculty of Pharmacy, Division of
Pharmaceutical Chemistry, P. O. Box 56 (Viikinkaari 5 E), FI-00014 Uni-
versity of Helsinki, Finland.
E-mail: Jari.Yli-Kauhaluoma@helsinki.fi
Fax: +358 9 191 59556
Abbreviation: multiple reaction monitoring (MRM)
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Arch. Pharm. Chem. Life Sci. 2009, 342, 201–209
ward approach to prepare metabolites with correct regio-
chemistries was to use appropriately substituted starting
materials; methoxyphenylacetones 1b–d and p-methoxy-
phenylisocyanate 4f, where the methoxy groups are in
the correct positions. Additionally, the demethylation
reaction could be carried out as a last step of the synthe-
sis route (Scheme 1).
Phenylacetones 1a–d were converted to the corre-
sponding amphetamines 2a–d via reductive amination
using ammonium formate as a source for hydrogen and
nitrogen [20]. The amphetamines 2a–d were treated with
KCN and formaldehyde at pH 2-3 to yield alkyl acetoni-
triles 3a–d [17]. These alkyl acetonitriles were subse-
quently nitrosated with NaNO2 in dilute HCl [21]. The
ring closure to the sydnone imines was achieved by
refluxing N-nitroso-a-aminoacetonitriles in concentrated
HCl. The obtained crude oily products were dissolved in
ethanol and precipitated with diethyl ether to give the
corresponding hydrochloride salts and were used with-
out further purification. Sydnone imine hydrochlorides
4a–d were then reacted with phenylisocyanates 4e–f in
anhydrous pyridine to yield the corresponding methoxy-
substituted mesocarbs 5a–f. Demethylation of the phe-
nolic methyl ether was successfully carried out with an
aluminium chloride-ethanethiol system in dichlorome-
thane [22]. Other demethylation reagents, such as BBr3
[23] and thiophenol [24] were also tried but neither of
them worked as well as the AlCl3-EtSH system.
Figure 2. Synthesized mesocarb metabolites.
ature. The synthesized metabolites were characterized by
means of 1H-NMR, 13C-NMR, FTIR spectrometry, and
liquid chromatography – mass spectrometry (LC-MS). As
a part of the characterization protocol, the synthesized
metabolites were compared to those purified from urine
samples collected after oral administration of mesocarb,
and to those synthesized enzymatically in vitro using
human liver proteins.
Results and discussion
Synthetic chemistry
Six hydroxylated mesocarb metabolites were selected for
the synthesis. We found out that the most straightfor-
Reagents and conditions: (a) Pd/C, HCOONH4, MeOH, H2O, r.t., overnight, 56 – 95%; (b) KCN, HCHO, H2O, pH 2 – 3, 08C fi r.t., overnight,
80 – 91%; (ci) NaNO2, THF, 1 M aqueous HCl, 08C fi r.t., 3 h; (cii) conc. HCl, reflux, 40 min, 75 – 93%; (d) pyridine, r.t., overnight, 49 – 92%;
(e) AlCl3, EtSH, CH2Cl2, r.t., 1 h, 20 – 74%.
Scheme 1. Synthesis of the metabolites.
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Mesocarb Metabolites
203
Figure 3. The MS/MS spectra obtained from the compounds 6a–f
using a collision energy of 10 V.
1
The purities of the final products 6a–f were deter- HMBC and H spectra, and the corresponding carbons
mined by LC/UV-VIS and were within the range of 94.9–
from the HSQC spectrum (Table 1).
99.6%. All the metabolites were structurally character-
1
ized by NMR spectroscopy. In addition to the H and 13
C
LC-MS analytics
spectra, the structure of the main metabolite 6a was con- The hydroxylated mesocarb metabolites were also ana-
firmed by the HSQC (Heteronuclear Single Quantum lyzed using LC-MS and LC-MS/MS. [M + H]+ was the domi-
Coherence) and HMBC (Heteronuclear Multiple Bond Cor- nating ion for all the metabolites, and was thus used as
relation) spectra. 1H signals were assigned from the the precursor ion in the tandem mass spectrometric
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Table 1. 1H- and 13C-NMR chemical shifts of 6a in ppm (d).
interfering peaks. The recovery of the extraction proce-
dure was between 82 and 93% at a concentration level of
500 ng/mL (n = 4). Ion suppression of the sample matrix
was studied using the approach of Dams et al. [25] and did
not indicate any ion suppression at the retention time
range where the analytes eluted.
d
d
H1–3
H5
H6
H7
H8
7.19–7.32 (5H)
3.28 (CH2)
5.10
1.63 (CH3)
8.14
C(1)
C(2)
C(3)
C(4)
127.1
128.5 (2C)
128.9 (2C)
136.1
C(5)
40.6
H12
H13
H(NH)
H(OH)
7.39 (2H)
6.62 (2H)
9.01
C(6)
C(7)
C(8)
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
62.5
19.5
102.1
172.0
158.7
132.8
119.4 (2C)
114.9 (2C)
151.9
Enzymatic synthesis
As a final part of the characterization, the chemical struc-
tures of the synthesized metabolites were compared both
to the metabolites synthesized enzymatically in vitro,
using human liver microsomal protein and fraction S9 of
human liver protein, and to an authentic human urine
sample, which was collected during 0 to 48 hours after
p.o. administration of 5 mg of mesocarb (donated from
the Antidoping Centre Moscow).
8.92
For numbering of the atoms, see Fig. 1.
In-vitro enzymatic synthesis yielded only p-hydroxyme-
socarb 6a (Fig. 2) which was also the dominating metabo-
lite found in human urine collected after oral adminis-
tration of mesocarb. Most of the p-hydroxymesocarb in
urine was sulphated (76%), but also some glucuronidated
(22%) and free (2%) metabolite was detected. Traces of the
dihydroxylated metabolite 6f (Fig. 2) were also found in
urine. The results agree with the literature, where p-
hydroxymesocarb has been considered to be the main
metabolite. Appolonova et al. also detected several di- and
trihydroxylated mesocarb metabolites in human urine
[8, 26], but in this experiment those metabolites could
not be detected. A possible explanation for this might be
the administered single low dose and the individual dif-
ferences in the metabolism of mesocarb.
Table 2. Compound-specific MRM parameters and retention
times of the mesocarb metabolites.
Compound
Precursor ion Product
Collision RT
[M + H]+ (m/z) ions (m/z) energy (V) (min)
Mesocarb
323
339
339
339
371
355
355
119
91
193
91
24
44
10
48
40
18
40
18
36
20
38
20
38
20
36
36
6.05
5.37
5.49
5.84
4.64
5.16
4.81
6.61
6a
6b
6c
6d
6e
6f
107
135
107
135
123
151
107
135
107
135
109
97
17a-Methyltestosterone 303
(internal standard)
Conclusion
We have developed a method of synthesis and analysis
for different hydroxy-substituted mesocarb metabolites.
The synthesized metabolites were compared to the
metabolites obtained from in-vitro and in-vivo experi-
ments. LC-MS/MS analysis of the metabolites from the
experiments agreed with the literature that p-hydroxy-
mesocarb is the main metabolite of mesocarb. Thus, with
respect to doping analysis, synthesis of this metabolite
and its availability as a reference material, is naturally of
great importance.
experiments. The fragmentation of the compounds
(Fig. 3) was efficient producing several specific ions,
which is essential for the reliable verification of suspi-
cious samples in doping control. The two most abundant
and specific product ions were selected for multiple reac-
tion monitoring (MRM). The optimized MRM parameters
used are presented in Table 2. The performance of the LC-
MS/MS method was examined for the identification of
mesocarb metabolites in urine. Each metabolite could be
detected at a concentration level of 50 ng/mL (n = 4) with
signal-to-noise ratios higher than 150. Intra-day precision
determined at concentration levels of 50 and 500 ng/mL
(n = 4) varied between 2.7 and 10.4%. Four aliquots of
drug-free urine were analyzed to check the selectivity of
the assay. The biological background was low with no
The authors thank Mrs. Teija Moisander for expert technical
assistance and the Antidoping Centre Moscow for an authentic
human urine mesocarb sample. The project was funded by the
World Anti-Doping Agency WADA (Grant 05D4JY/2005).
The authors have declared no conflict of interest.
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Mesocarb Metabolites
205
2-(2-Methoxyphenyl)-1-methylethylamine 2c
Experimental
1
A pale orange oil. Yield: 1.32 g (65%). H-NMR (300 MHz, DMSO-
d6) d: 7.17 (td, J = 1.8, J = 8.4 Hz, 1H), 7.09 (dd, J = 1.5, 7.2 Hz, 1H),
6.93 (dd, J = 0.6, 7.2 Hz, 1H), 6.85 (td, J = 0.9, 7.2 Hz, 1H), 3.75 (s,
3H), 3.17 (s, 2H), 3.02 (sextet, J = 6.6 Hz, 1H), 2.51 (d, J = 6.6 Hz,
1H), 0.92 (d, J = 6.6 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d: 157.3,
130.7, 128.0, 127.2, 120.2, 110.6, 55.2, 48.6, 46.7, 23.5; FTIR (KBr,
cm– 1): 3400–2800 (br), 2055, 1600.
General
The reagents and materials for synthetic purposes were obtained
from commercial suppliers and were used without further puri-
fication. Pyridine and dichloromethane were distilled and dried
prior to use. Reactions were monitored by thin-layer chromatog-
raphy on silica gel 60-F256 plates acquired from Merck (Darm-
stadt, Germany). Column chromatography was carried out man-
ually by using silica gel 60 (230–400 mesh) from E. Merck or by
Biotage SP1 purification system (Charlottesville, VA, USA) using
Flash 25+M silica cartridges. FTIR spectra were measured on a
Bruker Vertex 70 FT-IR spectrometer (Bruker, Ettlingen, Ger-
many) with KBr technique. 1H-NMR and 13C-NMR were recorded
on Varian Mercury Plus 300 MHz spectrometer (Varian, Palo
Alto, CA., USA) using DMSO-d6 as a solvent. Chemical shifts are
reported relative to TMS. J values are given in Hz. In order to
2-(2,4-Dimethoxyphenyl)-1-methylethylamine 2d
A pale orange oil. Yield: 1.1 g (56%). 1H-NMR (300 MHz, DMSO-d6)
d: 7.01 (d, J = 8.4 Hz, 1H), 6.52 (d, J = 2.7 Hz, 1H), 6.45 (dd, J = 2.4,
8.4 Hz, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.07 (sextet, J = 6.3 Hz, 1H),
2.58 (dd, J = 6.6, 12.6 Hz, 1H), 2.49 (dd, J = 7.2, 12.9 Hz, 1H), 0.96
(d, J = 6.3 Hz, 1H); 13C-NMR (75 MHz, DMSO-d6) d: 159.2, 158.1,
131.1, 119.0, 104.4, 98.4, 55.3, 55.1, 46.8, 38.2, 21.5; FTIR (KBr,
cm– 1): 3359, 2836, 2065, 1612.
1
observe H-13C correlation between the mesoionic proton 8 and
carbon 8 in the HSQC spectrum, the value of one bond coupling
constant had to be altered from the default setting of 140 to
180 MHz. Purity of the synthesized metabolites was checked
using an HP1100 LC/UV-VIS system (Hewlett-Packard, Wald-
bronn, Germany) with a Zorbax Eclipse XDB-C18 (2.1650 mm,
3.5 lm; Agilent, Germany) column using the same chromato-
graphic conditions, which were used in LC-MS/MS detection of
the metabolites.
(1-Methyl-2-phenylethylamino)acetonitrile 3a
2a (2.50 g, 14.6 mmol) was suspended in water (35 mL). The pH
of the solution was adjusted to 2–3 with concentrated HCl. The
reaction mixture was cooled with an ice bath, and formaldehyde
(1.10 mL, 14.6 mmol) and potassium cyanide (0.95 g, 14.6 mmol)
were added. After stirring the reaction mixture overnight at
room temperature, it was extracted with Et2O (4615 mL). The
combined extracts were dried with Na2SO4 and evaporated to
give light yellow oil.
Chemistry
Yield: 2.12 g (83%). Rf = 0.79 (EtOAc / MeOH 4 : 1); 1H-NMR
(300 MHz, CDCl3) d: 7.25 (m, 5H), 3.54 (s, 2H), 3.17 (m, 1H), 2.67
(m, 2H), 1.11 (d, J = 6.3 Hz, 3H); 13C-NMR (75 MHz, CDCl3) d: 138.4,
129.4, 128.9, 126.8, 117.8, 53.1, 43.5, 35.0, 19.6; FTIR (KBr, cm– 1):
3333, 3063, 2234, 1952, 1603.
1-Methyl-2-phenylethylamine 2a
The substance was synthesized under the permission by the
National Agency for Medicines (3/2005, Helsinki, Finland).
Ammonium formate (49 g, 780 mmol) was dissolved in a mix-
ture of water (18 mL) and MeOH (160 mL). Phenylacetone (10 g,
75 mmol) and palladium (2.4 g, 3.0 mmol) on activated charcoal
(10 w/w%) were added. The resulting mixture was stirred over-
night at room temperature. The reaction mixture was filtered
through a pad of Celite to remove the palladium catalyst. The
Celite was washed with MeOH, the filtrate was evaporated and
concentrated HCl (14 mL) was added to the residue. This mixture
was extracted with Et2O (2615 mL). The aqueous phase was
made alkaline with solid KOH, and the resulting amphetamine
base separated. It was removed, and the aqueous phase was
extracted with Et2O (15 mL). The ethereal phase was combined
with the amphetamine layer and the solvent was evaporated.
Amphetamine was dissolved in MeOH and filtered through a
pad of silica. Silica was washed with methanol. Evaporation of
the solvent afforded a pale orange oil.
[2-(4-Methoxyphenyl)-1-methylethylamino]acetonitrile 3b
A colorless oil. Yield: 570 mg (86%). Rf = 0.39 (EtOAc / Hex 1 : 1);
1H-NMR (300 MHz, DMSO-d6) d: 7.08 (td, J = 3.0, 8.4 Hz, 2H), 6.83
(td, J = 2.7, 9.0 Hz, 2H), 3.72 (s, 3H), 3.65 (d, J = 3.9 Hz, 2H), 2.86
(sextet, J = 5.7 Hz, 1H), 2.68 (dd, J = 5.7, 13.5 Hz, 1H), 2.39 (bs, 1H),
2.38 (dd, J = 7.2, 13.2 Hz, 1H), 0.90 (d, 3H, 6.3 Hz); 13C-NMR
(75 MHz, DMSO-d6) d: 157.5, 131.9, 130.0, 113.5, 54.9, 48.4, 45.3,
23.2; FTIR (KBr, cm– 1): 3332, 3061, 2234, 2058, 1614.
[2-(2-Methoxyphenyl)-1-methylethylamino]acetonitrile 3c
A colorless oil. Yield: 660 mg (91%). Rf = 0.30 (EtOAc / Hex 1 : 1);
1H-NMR (300 MHz, DMSO-d6) d: 7.20 (td, J = 1.8, 7.8 Hz, 1H), 7.10
(dd, J = 1.8, 7.2 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.86 (td, J = 0.9,
7.2 Hz, 1H), 3.77 (s, 3H), 3.64 (bd, J = 5.1 Hz, 2H), 2.95 (m, sextet, J
= 6.6 Hz, 1H), 2.77 (dd, J = 5.1, 12.9 Hz, 1H), 2.40 (bs, 1H), 2.36 (dd,
J = 8.1, 12.9 Hz, 1H), 0.89 (d, J = 6.6 Hz, 3H); 13C-NMR (75 MHz,
DMSO-d6) d: 157.3, 130.8, 127.5, 127.1, 120.1, 119.3, 110.7, 55.2,
51.7, 36.7, 34.4, 19.3; FTIR (KBr, cm– 1): 3333, 3065, 2234, 2051,
1601.
Yield: 9.5 g (95%); 1H-NMR (300 MHz, DMSO-d6) d: 7.25 (m, 5H),
3.09 (sextet, J = 6.6 Hz, 1H), 2.66 (dd, J = 6.6, 13.2 Hz, 1H), 2.52 (dd,
J = 6.9, 13.2 Hz, 1H), 0.97 (d, J = 6.3 Hz, 3H); 13C-NMR (75 MHz,
DMSO-d6) d: 139.2, 129.2, 128.2, 126.0, 48.2, 44.7.0, 21.8; FTIR
(KBr, cm– 1): 3365, 3281, 3027, 1947, 1604.
[2-(2,4-Dimethoxyphenyl)-1-
methylethylamino]acetonitrile 3d
A pale yellow oil. Yield: 590 mg (91%). Rf = 0.31 (EtOAc / Hex 1 : 1);
1H-NMR (300 MHz, DMSO-d6) d: 6.99 (d, J = 8.4 Hz, 1H), 6.52 (d, J =
2.1 Hz, 1H), 6.44 (dd, J = 2.1, 8.4 Hz, 1H), 3.75 (s, 3H), 3.73 (s, 3H),
2.88 (sextet, J = 6.6 Hz, 1H), 2.67 (dd, J = 5.1, 13.2 Hz, 1H), 2.34 (bs,
1H), 2.29 (dd, J = 7.5, 13.2 Hz, 1H), 0.88 (d, J = 6.3 Hz, 3H);13C-NMR
2-(4-Methoxyphenyl)-1-methylethylamine 2b
A pale orange oil. Yield: 1.67 g (55%). 1H-NMR (300 MHz, DMSO-
d6) d: 7.08 (td, J = 3.0, 8.4 Hz, 2H), 6.83 (td, J = 3.3, 8.4 Hz, 2H), 3.71
(s, 3H), 2.93 (sextet, J = 6.3 Hz, 1H), 2.60 (d, J = 7.2 Hz, 2H), 0.92 (d,
J = 6.6 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d: 157.5, 131.9, 130.0,
113.5, 54.9, 48.4, 45.3, 23.2; FTIR (KBr, cm– 1): 3359, 3271, 3030,
2059, 1612.
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(75 MHz, DMSO-d6) d: 159.1, 158.1, 131.1, 119.3, 119.2, 104.3,
98.3, 55.3, 55.1, 51.8, 36.1, 34.4, 19.2; FTIR (KBr, cm– 1): 3333,
3078, 2234, 2060, 1614.
(4615 mL). The extracts were combined and washed with aque-
ous CuSO4 solution and water. The organic phase was then dried
with Na2SO4 and evaporated. The crude product was purified by
silica gel column chromatography using EtOAc / toluene (2 : 1)
as an eluent to give a yellow solid.
3-(1-Methyl-2-phenylethyl)sydnone imine 4a
Yield: 1.4 g (92%). Rf = 0.50 (EtOAc / Hex 4 : 1); 1H-NMR
(300 MHz, DMSO-d6) d: 9.14 (s, 1H), 8.17 (s, 1H), 7.53 (d, J = 8.7 Hz,
2H), 7.26 (m, 5H), 6.80 (d, J = 9 Hz, 2H), 5.11 (sextet, J = 6.6 Hz, 1H),
3.69 (s, 3H), 3.28 (m, 2H), 1.64 (d, J = 6.6 Hz, 3H);13C-NMR (75 MHz,
DMSO-d6) d: 172.1, 158.7, 153.9, 136.1, 134.3, 128.9, 128.5, 127.0,
119.1, 113.6, 102.2, 62.5, 55.1, 40.5, 19.4; FTIR (KBr, cm– 1): 3259,
3032, 1635, 1601.
3a (2.12 g, 12.2 mmol) was dissolved in THF (11 mL). The solution
was cooled to 08C and 1 M HCl (8.6 mL) was added. NaNO2
(0.84 g, 12.2 mmol) was dissolved in water (3 mL) and added
dropwise over 20 min. The reaction mixture was stirred at room
temperature for 3 hours after which it was evaporated to dry-
ness. The residue was suspended to concentrated HCl (13 mL)
and refluxed for 40 min. The reaction mixture was evaporated
to dryness, and the residue was dissolved in a small amount of
ethanol and cooled. Et2O was added. The mixture was stirred vig-
orously on an ice bath for 20 min after which the off-white syd-
none imine hydrochloride was filtered.
3-(1-Methyl-2-(4-methoxyphenyl)ethyl)-5-
[[(phenylamino)carbonyl]amino]-1,2,3-oxadiazolium 5b
A pale yellow solid. Yield: 482 mg (59%). Rf = 0.59 (EtOAc / Hex
3 : 1);1H-NMR (300 MHz, DMSO-d6) d: 9.29 (s, 1H), 8.20 (s, 1H), 7.62
(d, J = 7.2 Hz, 2H), 7.20 (t, J = 7.5 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H),
6.86 (m, 3H), 5.06 (sextet, J = 6.6 Hz, 1H), 3.70 (s, 3H), 3.22 (m, 2H),
1.62 (d, J = 6.6 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d: 172.3,
158.9, 158.2, 141.1, 129.9, 128.4, 127.8, 121.1, 117.8, 113.9,
102.3, 62.8, 54.9, 39.9, 19.3; FTIR (KBr, cm– 1): 3228, 3035, 1638,
1593.
Yield: 760 mg (68%); 1H-NMR (300 MHz, DMSO-d6) d: 9.96 (s,
2H), 8.30 (s, 1H), 7.27 (m, 5H), 5.32 (sextet, J = 6.9 Hz, 1H), 3.31 (d, J
= 7.2 Hz, 2H), 1.63 (d, J = 6.6 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6)
d: 169.0, 135.6, 129.2, 129.0, 128.6, 127.2, 101.2, 63.8, 40.5, 18.9;
FTIR (KBr, cm– 1): 3373, 3091, 1684.
3-[1-Methyl-2-(4-methoxyphenyl)ethyl]sydnone imine 4b
An off-white solid. Yield: 558 mg (93%). 1H-NMR (300 MHz,
DMSO-d6) d: 9.95 (s, 2H), 8.27 (s, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.85
(d, J = 8.4 Hz, 2H), 5.25 (sextet, J = 6.9 Hz, 1H), 3.71 (s, 3H), 3.23 (d,
J = 7.8 Hz, 2H), 1.61 (d, J = 6.9 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6)
d: 168.9, 158.3, 130.1, 127.3, 114.0, 101.1, 64.0, 55.0, 39.8, 18.8;
FTIR (KBr, cm– 1): 3334, 3151, 1697.
3-(1-Methyl-2-(2-methoxyphenyl)ethyl)-5-
[[(phenylamino)carbonyl]amino]-1,2,3-oxadiazolium 5c
A yellow solid. Yield: 369 mg (57%). Rf = 0.45 (EtOAc / Hex 3 : 1);
1H-NMR (300 MHz, DMSO-d6) d: 9.27 (s, 1H), 8.13 (s, 1H), 7.62 (d, J =
7.8 Hz, 2H), 7.22 (m, 3H), 7.08 (dd, J = 1.8, 7.8 Hz, 1H), 6.97 (d, J =
1.2, 8.4 Hz, 1H), 6.86 (m, 2H), 5.06 (sextet, J = 6.9 Hz, 1H), 3.74 (s,
3H), 3.24 (d, J = 7.5 Hz, 2H), 1.64 (d, J = 6.9 Hz, 3H); 13C-NMR
(75 MHz, DMSO-d6) d: 172.3, 158.9, 157.3, 141.1, 130.5, 128.8,
128.4, 121.9, 120.3, 117.8, 110.8, 102.3, 61.2, 55.3, 36.1, 19.2;
FTIR (KBr, cm– 1): 3231, 3023, 1639, 1586.
3-[1-Methyl-2-(2-methoxyphenyl)ethyl]sydnone imine 4c
A light brown solid. Yield: 1.3 g (85%). 1H-NMR (300 MHz, DMSO-
d6) d: 9.91 (s, 2H), 8.2 (s, 1H), 7.25 (td, J = 1.8, 8.4 Hz, 1H), 7.11 (dd, J
= 1.8, 7.2 Hz, 1H), 6.97 (dd, J = 0.6, 8.4 Hz, 1H), 6.87 (td, J = 0.9,
7.2 Hz, 1H), 5.20 (sextet, J = 6.9 Hz, 1H), 3.73 (s, 3H), 3.26 (d, J =
6.9 Hz, 2H), 1.64 (d, J = 6.9 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d:
168.9, 157.3, 130.8, 129.0, 123.1, 120.4, 110.8, 101.2, 62.6, 55.3,
36.0, 18.7; FTIR (KBr, cm– 1): 3345, 3039, 1677.
3-(1-Methyl-2-(2,4-dimethoxyphenyl)ethyl)-5-[[(4-
methoxyphenylamino)carbonyl]amino]-1,2,3-
oxadiazolium 5d
3-[1-Methyl-2-(2,4-dimethoxyphenyl)ethyl]sydnone imine
A yellow solid. Yield: 350 mg (49%). Rf = 0.47 (EtOAc / toluene
1 : 1); 1H-NMR (300 MHz, DMSO-d6) d: 9.13 (s, 1H), 8.08 (s, 1H), 7.53
(d, J = 9.3 Hz, 2H), 6.96 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.7 Hz, 2H),
6.52 (d, J = 2.1 Hz, 1H), 6.42 (dd, J = 2.1, 8.7 Hz, 1H), 4.98 (sextet, J =
6.6 Hz, 1H), 3.73 (s, 3H), 3.72 (s, 3H), 3.69 (s, 3H), 3.15 (d, J = 7.5 Hz,
2H), 1.61 (d, J = 6.3 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d: 172.1,
159.9, 158.8, 158.3, 153.8, 134.4, 131.0, 119.1, 115.7, 113.6,
104.6, 102.0, 98.3, 61.3, 55.4, 55.1, 35.6, 19.2; FTIR (KBr, cm– 1):
3228, 3001, 1637, 1589.
4d
4d failed to precipitate from EtOH / Et2O. Water was evaporated
and the residue dried in vacuo before the next reaction step.
1
Yield: 521 mg (75%). H-NMR (300 MHz, DMSO-d6) d: 9.73 (s,
2H), 8.14 (s, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H),
6.45 (dd, J = 2.4, 8.1 Hz, 1H), 5.13 (sextet, J = 7.5 Hz, 1H), 3.73 (s,
3H), 3.71 (s, 3H), 3.16 (d, J = 6.9 Hz, 2H), 1.62 (d, J = 7.2 Hz, 3H); 13C-
NMR (75 MHz, DMSO-d6) d: 168.9, 160.1, 158.3, 131.3, 115.1,
104.8, 101.1, 98.3, 62.9, 55.4, 55.1, 35.6, 18.6; FTIR (KBr, cm– 1):
3335, 2999, 1670.
3-(1-Methyl-2-(2-methoxyphenyl)ethyl)-5-[[(4-
methoxyphenylamino)carbonyl]amino]-1,2,3-
oxadiazolium 5e
A pale yellow solid. Yield: 337 mg (49%). Rf = 0.47 (EtOAc / Hex
3 : 1);1H-NMR (300 MHz, DMSO-d6) d: 9.13 (s, 1H), 8.09 (s, 1H), 7.53
(d, J = 9 Hz, 2H), 7.23 (td, J = 1.8, 8.4 Hz,1H), 7.07 (dd, J = 1.5,
7.5 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 6.82 (m, 3H), 5.05 (sextet, J =
6.9 Hz, 1H), 3.75 (s, 3H), 3.69 (s, 3H), 3.23 (d, J = 6.9 Hz, 2H), 1.64
(d, J = 6.9 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d: 172.1, 158.8,
157.3, 153.9, 134.3, 130.5, 128.8, 123.6, 120.3, 119.1, 113.6,
3-(1-Methyl-2-phenylethyl)-5-[[(4-
methoxyphenylamino)carbonyl]amino]-1,2,3-
oxadiazolium 5a
4a (1.11 g, 4.61 mmol) was dissolved in anhydrous pyridine
(25 mL) under argon atmosphere. The solution was cooled on an
ice bath and 4-methoxyphenyl isocyanate (1.20 mL, 9.22 mmol)
was added. The reaction mixture was stirred overnight at room
temperature. The reaction was quenched by pouring the mix-
ture to ice water. The aqueous phase was extracted with EtOAc
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Arch. Pharm. Chem. Life Sci. 2009, 342, 201–209
Mesocarb Metabolites
207
110.8, 102.1, 61.1, 55.3, 55.1, 36.1, 19.3; FTIR (KBr, cm– 1): 3242,
3002, 1643, 1594.
118.9, 117.8, 115.0, 102.2, 61.1, 36.2, 19.3; FTIR (KBr, cm– 1): 3228,
3186, 1638, 1593, 1313.
3-(1-Methyl-2-(4-methoxyphenyl)ethyl)-5-[[(4-
methoxyphenylamino)carbonyl]amino]-1,2,3-
3-[1-Methyl-2-(2,4-dihydroxyphenyl)ethyl]-5-[[(4-
hydroxyphenylamino)carbonyl]amino]-1,2,3-
oxadiazolium 5f
oxadiazolium 6d
A yellow solid. Yield: 468 mg (59%). Rf = 0.16 (EtOAc / Hex 2 : 1);
1H-NMR (300 MHz, DMSO-d6) d: 9.14 (s, 1H), 8.16 (s, 1H), 7.53 (d, J =
9.0 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.80 (d,
J = 8.7Hz, 2H), 5.04 (sextet, J = 6.6 Hz, 1H), 3.70 (s, 3H ), 3.69 (s,
3H), 3.21 (m, 2H), 1.62 (d, J = 6.6 Hz, 3H);13C-NMR (75 MHz, DMSO-
d6) d: 172.1, 158.7, 158.2, 153.9, 134.3, 129.9, 127.8, 119.1, 113.9,
113.6, 102.1, 62.8, 55.1, 54.9, 19.3; FTIR (KBr, cm– 1): 3225, 3029,
1637, 1592.
A yellow solid. Yield: 204 mg (20%). LC-UV/VIS purity 94.9%; Rf =
0.20 (EtOAc / Hex 4 : 1); H-NMR (300 MHz, DMSO-d6) d: 9.42 (s,
1
1H), 9.13 (s, H), 8.98 (s,1H), 8.91 (s, 1H), 8.03 (s, 1H), 7.39 (d, J =
9 Hz, 2H), 6.71 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 8.7 Hz, 2H), 6.27 (d, J =
2.1 Hz, 1H), 6.09 (dd, J = 2.4, 8.4 Hz, 1H), 4.97 (sextet, J = 7.5 Hz,
1H), 3.06 (d, J = 7.2 Hz, 2H), 1.60 (d, J = 7.2 Hz, 3H); 13C-NMR
(75 MHz, DMSO-d6) d: 172.1, 158.7, 157.5, 156.3, 151.8, 132.8,
130.9, 119.4, 114.9, 112.4, 106.2, 102.4, 101.8, 61.3, 35.8, 19.2;
FTIR (KBr, cm– 1): 3314 (br), 3191, 1620, 1513, 1309.
3-(1-Methyl-2-phenylethyl)-5-[[(4-
3-[1-Methyl-2-(2-hydroxyphenyl)ethyl]-5-[[(4-
hydroxyphenylamino)carbonyl]amino]-1,2,3-
hydroxyphenylamino)carbonyl]amino]-1,2,3-
oxadiazolium 6a
5a (0.515 g, 1.45 mmol) was dissolved in dry dichloromethane
(5 mL). Ethanethiol (5 mL) was added. The solution was cooled to
08C and aluminium chloride (0.66 g, 4.95 mmol) was added. The
reaction mixture was stirred at room temperature for 1 hour.
The reaction was quenched by adding water (10 mL). The prod-
uct precipitated, and the resulting mixture was stirred for
5 min, after which the supernatant was decanted, concentrated,
and extracted with dichloromethane. The organic layer was
combined with the precipitated product and evaporated to dry-
ness. The crude product was purified by silica gel column chro-
matography, and subsequently recrystallized from ethanol to
give light orange crystals.
Yield: 290 mg (59%). LC-UV/VIS purity 99.3%; Rf = 0.35 (EtOAc /
Hex 4 : 1);1H-NMR (300 MHz, DMSO-d6) d: 9.01 (s, 1H), 8.92 (s, 1H),
8.14 (s, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.25 (m, 5H), 6.62 (d, J = 9 Hz,
2H), 5.1 (sextet, J = 6.6 Hz, 1H), 3.28 (m, 2H), 1.63 (d, J = 6.6 Hz,
3H); 13C-NMR (75 MHz, DMSO-d6) d: 172.0, 158.7, 151.9, 136.1,
132.8, 128.9, 128.5, 127.1, 119.4, 114.9, 102.1, 62.5, 40.6, 19.5;
FTIR (KBr, cm– 1): 3155, 1601, 1542, 1433, 1312.
oxadiazolium 6e
A yellow solid. Yield: 290 mg (65%). LC-UV/VIS purity 99.3%; Rf =
1
0.40 (EtOAc / Hex 4 : 1); H-NMR (300 MHz, DMSO-d6) d: 9.61 (s,
1H), 8.99 (s, 1H), 8.93 (s, 1H), 8.07 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H),
7.05 (td, J = 1.5, 9.3 Hz, 1H), 6.97 (dd, J = 1.5, 7.2 Hz, 1H), 6.80 (d, J
= 7.2 Hz, 1H), 6.63 (m, 3H), 5.07 (sextet, J = 7.2 Hz, 1H), 3.19 (d, J =
7.2 Hz, 2H), 1.63 (d, J = 6.9 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d:
172.1, 158.8, 155.5, 151.9, 132.8, 130.6, 128.5, 122.0, 119.5,
119.0, 115.1, 114.9, 101.9, 61.0, 36.3, 19.4; FTIR (KBr, cm– 1): 3221,
1627, 1514, 1310.
3-[1-Methyl-2-(4-hydroxyphenyl)ethyl]-5-[[(4-
hydroxyphenylamino)carbonyl]amino]-1,2,3-
oxadiazolium 6f
A yellow solid. Yield: 275 mg (74%). LC-UV/VIS purity 99.6%; Rf =
1
0.30 (EtOAc / Hex 4 : 1); H-NMR (300 MHz, DMSO-d6) d: 9.31 (s,
1H), 9.01 (bs, 1H), 8.91 (s, 1H), 8.10 (s, 1H), 7.39 (d, J = 8.7 Hz, 2H),
6.97 (d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.4 Hz, 2H), 6.62 (d, J = 9 Hz,
2H), 4.98 (sextet, J = 6.9 Hz, 1H), 3.14 (m, 2H), 1.60 (d, J = 6.6 Hz,
3H); 13C-NMR (75 MHz, DMSO-d6) d: 172.0, 158.7, 156.3, 151.8,
132.8, 129.8, 126.0, 119.4, 115.3, 114.9, 102.0, 62.8, 40.0, 19.3;
FTIR (KBr, cm– 1): 3185, 1637, 1600, 1514.
3-[1-Methyl-2-(4-hydroxyphenyl)ethyl]-5-
[[(phenylamino)carbonyl]amino]-1,2,3-oxadiazolium 6b
A pale yellow solid. Yield: 595 mg (58%). LC-UV/VIS purity 96.6%;
1
Rf = 0.54 (EtOAc / Hex 4 : 1); H-NMR (300 MHz, DMSO-d6) d: 9.31
In-vitro enzymatic synthesis of mesocarb metabolites
In-vitro synthesis of the metabolites was based on the procedure
described by Keski–Hynnilꢀ et al. [27]. The reaction mixture for
enzyme-assisted synthesis of phase I metabolites of mesocarb
consisted of 50 lM of mesocarb in methanol, 5 mM NADPH in
buffer, human liver microsomal protein (0.5 mg/mL) and frac-
tion S9 of human liver protein (0.5 mg/mL). 50 mM phosphate
solution (pH 7.4, 5 mM MgCl2) was used as an incubation buffer.
The total reaction volume was 100 lL.
(s, 1H), 9.28 (s, 1H), 8.17 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.20 (t, J =
8.1 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.88 (t, J = 7.2 Hz, 1H), 6.66 (d, J
= 8.4 Hz, 2H), 5.01 (sextet, J = 6.6 Hz, 1H), 3.15 (m, 2H), 1.61 (d, J =
6.6 Hz, 3H); 13C-NMR (75 MHz, DMSO-d6) d: 172.3, 158.9, 156.3,
141.1, 129.9, 128.4, 126.0, 121.1, 117.8, 115.3, 102.3, 62.9, 39.9,
19.2; FTIR (KBr, cm– 1): 3265, 3184, 1639, 1592, 1517, 1313.
3-[1-Methyl-2-(2-hydroxyphenyl)ethyl]-5-
[[(phenylamino)carbonyl]amino]-1,2,3-oxadiazolium 6c
A yellow solid. Yield: 190 mg (57%). LC-UV/VIS purity 98.9%; Rf =
0.63 (EtOAc / Hex 4 : 1); 1H-NMR (300 MHz, DMSO-d6) d: 9.62 (s,
1H), 9.27 (s, 1H), 8.14 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.21 (d, J =
7.8 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.06 (td, J = 1.8, 7.5 Hz, 1H),
6.97 (dd, J = 1.2, 7.5 Hz, 1H), 6.88 (t, J = 7.8 Hz, 1H), 6.8 (d, J =
7.8 Hz, 1H), 6.68 (td, J = 0.9, 7.2 Hz, 1H), 5.1 (sextet, J = 7.2 Hz, 1H),
3.21 (d, J = 7.2 Hz, 2H), 1.65 (d, J = 6.6 Hz, 3H); 13C-NMR (75 MHz,
DMSO-d6) d: 172.4, 158.9, 155.5, 141.1, 130.6, 128.4, 121.9, 121.1,
The phase I reaction was started by addition of NADPH to the
reaction mixture. The reaction was carried out at 378C in Eppen-
dorf tubes in a dry bath. The incubation was stopped after
4 hours by addition of ice-cold 4 M perchloric acid. After centri-
fugation, the supernatant was moved to a glass vial and its pH
was adjusted to 6–7 with 1 M NaOH. The aqueous phase was
extracted with ethyl acetate (460.5 mL), and the combined
EtOAc extracts were evaporated. The analytical recovery of the
used extraction procedure was 92–95% depending on the
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208
M. Vahermo et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 201–209
metabolite. The mesocarb metabolites were thermally stable
during incubation at 378C (data not shown).
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LC-MS/MS detection of metabolites
The LC-MS/MS-experiments were performed on a ThermoFinni-
gan Surveyor LC from ThermoFinnigan, and a TSQuantum triple
quadrupole mass spectrometer, also from ThermoFinnigan (Ger-
many).
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The examination of the analytes' mass spectrometric proper-
ties was conducted using an electrospray ionization (ESI) in the
positive ion mode. The MS- and MS/MS-spectra were measured in
the scan range m/z 50–500 and two-ion transitions were chosen
per each compound for MRM. Argon was used as the collision
gas at a pressure of 1.5 mTorr. Nitrogen was used both as sheath
and auxiliary gas (49 and 5 arbitrary units). The spray voltage
was 4000 V and the capillary temperature 2708C.
The chromatographic separation was obtained at ambient
temperature on a Zorbax 300SB-CN (2.16150 mm, 5 lm) col-
umn from Agilent, equipped with a guard column of the same
packing material (2.1612.5 mm). The mobile phase consisted of
A: 2.5 mM ammonium acetate with 0.1% acetic acid, pH 4, and B:
methanol. A linear gradient was run with a flow rate of 200 lL/
min from 40% B to 100% B in 6 min, followed by an equilibration
step of 2 min.
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Urine sample pre-treatment
17a-Methyltestosterone was used as an internal standard
(500 ng/mL). A 3 mL-aliquot of urine was applied to a Sep-Pak
C18 cartridge (Waters, Milford, MA, USA), which was previously
conditioned with 2.5 mL of MeOH and 5 mL of water. The sample
was first rinsed with 5 mL of water, and then eluted with 3 mL of
MeOH. The eluate was divided into three parts, which were
evaporated to dryness and dissolved in 1 mL of an appropriate
buffer for the isolation of the free and conjugated metabolites.
Enzymatic hydrolysis of the b-D-glucuronide conjugates was per-
formed in a 0.1 M phosphate buffer (pH 7) with 25 lL of b-glucur-
onidase from Escherichia coli K12 (Boehringer Mannheim, Mann-
heim, Germany) at 508C for 1 h. Sulphate conjugates were enzy-
matically hydrolyzed in 1 mL of 0.1 M acetate buffer (pH 5) con-
taining 5 lM saccharic acid lactone with 10 lL of arylsulfatase
from Helix pomatia (Sigma-Aldrich, St. Louis, MO, USA) at 378C
for 12 h. For isolation of the free metabolites no hydrolyzing
enzyme was added. All three aliquots were finally extracted
twice with 3 mL of ethyl acetate at pH 8 on a vortex-mixer for
30 s. After centrifugation, the organic phase was separated,
evaporated to dryness under a nitrogen stream at 408C, and
finally dissolved in 100 lL of the initial LC mobile phase prior to
injection to the LC-MS/MS. The analytical performance of the
method was evaluated with respect to detection capability, pre-
cision, specificity, extraction recovery, and ion suppression of
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