Synthesis of N-acetyl-S-(3-coumarinyl)-cysteine methyl ester and HPLC analysis of urinary coumarin metabolites

Article abstract of DOI:10.1016/S0300-483X(03)00204-X

N-Acetyl-S-(3-coumarinyl)cysteine, a metabolite of coumarin in rodents, has been synthesized as methyl ester. A new synthetic route to prepare N-acetyl-S-(3-coumarinyl)-D,L-cysteine methyl ester (1) comprises reaction of 3-mercaptocoumarin (3) with N-acetyl-3-chloro-D,L-alanine methyl ester (4). N-acetyl-S-(4-coumarinyl)-L-cysteine (10) was obtained by reaction of 3-bromocoumarin (12) and N-acetyl-L-cysteine (13). A method for the determination of N-acetyl-S-(3-coumarinyl)cysteine as its methyl ester in urine by HPLC has been developed.

Full text of DOI:10.1016/S0300-483X(03)00204-X

Toxicology 190 (2003) 249ꢂ258
/
www.elsevier.com/locate/toxicol
Synthesis of N-acetyl-S-(3-coumarinyl)-cysteine methyl ester
and HPLC analysis of urinary coumarin metabolites
Gerhard Eisenbrand *, Michael Otteneder, Weici Tang
Division of Food Chemistry and Environmental Toxicology, Department of Chemistry, University of Kaiserslautern,
D-67663 Kaiserslautern, Germany
Received 5 March 2003; received in revised form 6 May 2003; accepted 6 May 2003
Abstract
N-Acetyl-S-(3-coumarinyl)cysteine, a metabolite of coumarin in rodents, has been synthesized as methyl ester. A new
synthetic route to prepare N-acetyl-S-(3-coumarinyl)-
mercaptocoumarin (3) with N-acetyl-3-chloro- -alanine methyl ester (4). N-acetyl-S-(4-coumarinyl)-
was obtained by reaction of 3-bromocoumarin (12) and N-acetyl- -cysteine (13). A method for the determination of N-
D
,
L-cysteine methyl ester (1) comprises reaction of 3-
D,
L
L-cysteine (10)
L
acetyl-S-(3-coumarinyl)cysteine as its methyl ester in urine by HPLC has been developed.
# 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Coumarin; N-Acetyl-S-(3-coumarinyl)cysteine; Metabolism; Synthesis; HPLC-determination
1. Introduction
body weight from cosmetic products (Lake, 1999).
Coumarin has been reported to be used clinically
for treatment of lymphedemas, mainly in breast
cancer patients (Burgos et al., 1999; Loprinzi et al.,
1999); for treatment of renal cell carcinoma
(Sagaster et al., 1995; Marshall et al., 1994); and
for prophylaxis of thromboembolism in gynecolo-
gic oncology (Graf et al., 1998).
The biotransformation of coumarin is known to
be species-dependent (Pearce et al., 1992; Bogan et
al., 1996). It appears that 7-hydroxylation of
coumarin is the major phase I pathway in the
majority of human subjects (Rautio et al., 1992;
Bogan et al., 1995), catalyzed predominantly by
cytochrome P450 isoenzyme CYP2A6 (Li et al.,
1997). In phase II biotransformation, 7-hydroxy-
coumarin is converted into glucuronide or sulfate
Coumarin (2H-1-benzopyran-2-one (11)), is a
widely occurring secondary plant metabolite that
has been used as a fragrance in food and cosmetic
products. After rapid absorption from the gastro-
intestinal tract coumarin is distributed throughout
the body (Cohen, 1979; Fentem and Fry, 1993).
Coumarin has also been found to be well absorbed
through the skin (Beckley-Kartey et al., 1997;
Yourick and Bronaugh, 1997). The daily intake
of coumarin has been estimated to reach 0.02 mg
kgꢀ1 body weight via food and 0.04 mg kgꢀ1
* Corresponding author. Tel.: ꢁ
631-205-3085.
E-mail address: eisenbra@rhrk.uni-kl.de (G. Eisenbrand).
/
49-631-205-2974; fax: ꢁ49-
/
0300-483X/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0300-483X(03)00204-X

250
G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ258
/
conjugates (Price et al., 1995; Duffy and O’Ken-
nedy, 1998). 7-Hydroxycoumarin and its glucur-
onide or sulfate represent about 80% of human
urinary coumarin metabolites following oral ad-
ministration (Shilling et al., 1969). However, hu-
mans also convert coumarin to coumarin-3,4-
epoxide as a minor metabolic pathway, resulting
in the formation of 2-hydroxyphenylacetic acid. In
24 h urine, 2-hydroxyphenylacetic acid has been
reported to account for about 4% of the coumarin
dose administered orally (200 mg per person)
(Shilling et al., 1969). CYP2A6 has been found
to be expressed polymorphically. About 15% of
Chinese subjects bear a defective CYP2A6 allele as
be excreted in the urine of rats given [benze-
ne-14C]coumarin, obviously as a result of cou-
marin-3,4-epoxide conjugation with glutathione
(Huwer et al., 1991). Glutathione conjugation
appears as an important detoxification reaction,
since the formation of 2-hydroxyphenylacetalde-
hyde can be inhibited by glutathione (Zhuo et al.,
1999).
It thus appeared of interest to quantify the
metabolic pathway encompassing formation of
coumarin 3,4-epoxide (Ford et al., 2001), with
subsequent urinary excretion of o-hydroxypheny-
lacetic acid or, after glutathione conjugation, of
N-acetyl-S-(3-coumarinyl)cysteine. N-Acetyl-S-
(3-coumarinyl)cysteine until now has not been
synthesized. Its identity as a urinary metabolite
compared with 1ꢂ3% of European populations
/
(Oscarson et al., 1998, 1999). After oral adminis-
tration of coumarin to subjects (2 mg) with a
defective CYP2A6 allele, no 7-hydroxycoumarin
1
was established earlier by comparison of the H-
NMR and mass spectra of N-acetyl-S-(3-coumar-
inyl)cysteine isolated from rat urine with those
of synthetic N-acetyl-S-(4-coumarinyl)cysteine
(Huwer et al., 1991). In the present study, we
report the synthesis of N-acetyl-S-(3-coumarinyl)-
was detected in the urine (0ꢂ8 h). However, about
/
50% of the dose was eliminated as 2-hydroxy-
phenylacetic acid (Hadidi et al., 1997). Single
amino acid substitution (Leu160His) in CYP2A6
has been found to cause switching from 7- to 3-
hydroxylation, resulting in predominant excretion
of 2-hydroxyphenylacetic acid after oral coumarin
(2 mg) (Hadidi et al., 1997). Among a population
of two hundred, two individuals were deficient for
7-hydroxycoumarin formation after an oral dose
of 5 mg coumarin (Oscarson et al., 1998). Thus, as
a result of CYP2A6 polymorphism, biotransfor-
mation of coumarin via coumarin 3,4-epoxide
might represent a major pathway in a very minor
human population.
D,
L
-cysteine methyl ester (1) from 3-mercaptocou-
marin (3) and N-acetyl-3-chloro- -alanine meth-
yl ester (4). N-Acetyl-S-(4-coumarinyl)- -cysteine
(10) was synthesized by reaction of 3-bromocou-
marin (12) with N-acetyl- -cysteine (13).
D,L
L
L
A
method for the determination of N-acetyl-S-(3-
coumarinyl)cysteine in urine by HPLC as its
methyl ester is described.
2. Chemistry
In contrast to the normal human situation, 3,4-
epoxidation represents the major metabolic route
in rat and mouse, resulting in the formation of an
unstable coumarin 3,4-epoxide which degrades
spontaneously to form 2-hydroxyphenylacetalde-
hyde that may be subsequently converted to 2-
hydroxyphenylethanol and 2-hydroxyphenylacetic
acid (Lake, 1999; Lake et al., 1995; Born et al.,
1997; Lovell et al., 1999). Recently, von Weymarn
and Murphy have reported that the major cou-
marin metabolites formed by rat esophageal
microsomes are 8-hydroxycoumarin, o-hydroxy-
phenylacetaldehyde, and o-hydroxyphenylacetic
acid (von Weymarn and Murphy, 2001) N-Ace-
tyl-S-(3-coumarinyl)cysteine has been reported to
N-Acetyl-S-(3-coumarinyl)-
ester (1) was prepared by reaction of 3-mercapto-
coumarin (3) with N-acetyl-3-chloro- -alanine
methyl ester (4) (Scheme 1). 3-Mercaptocoumarin
(3) was obtained from 2-hydroxybenzylidenerho-
danine (5) by refluxing in diluted ethanolic sodium
hydroxide solution (Xing et al., 1987). Compound
5 was prepared by condensation of 2-hydroxyben-
zaldehyde (6) with rhodanine (7) (Mackie and
D,L-cysteine methyl
D,L
Misra, 1954).
ride (8) was used as starting material for prepara-
tion of 4. Chlorination of -serine methyl ester
hydrochloride in acetyl chloride with PCl5 yielded
3-chloro- -alanine methyl ester hydrochloride
D,L-Serine methyl ester hydrochlo-
D,L
D,L

G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ
/
258
251
Scheme 1. Synthetic route of N-acetyl-S-(3-coumarinyl)-D,L-cysteine methyl ester (1).
(9), which was N-acetylated by refluxing with
acetyl chloride in benzene.
After elution with methanol/buffer pH 2.5, it was
converted to the methylester as described before,
using borone trifluoride in methanol. Recoveries
N-Acetyl-S-(4-coumarinyl)-
prepared by the reaction of 3-bromocoumarin (12)
with N-acetyl- -cysteine (13). 3-Bromocoumarin
L-cysteine (10) was
reached 65ꢂ
/
90%.
N-Acetyl-S-(3-coumarinyl)-
L
-cysteine methyl
L
ester (1) and its 4-coumarinyl counterpart 2 can
be clearly separated under the HPLC conditions
used (Fig. 2a). The separation profile of 11, 14, 15
and 1 is demonstrated in Fig. 2b.
(12) can be easily obtained by bromination of
coumarin (11) in chloroform in the presence of
triethylamine (Brogden et al., 1974). Methylation
of 10 to 2 was carried out using borone trifluoride
in methanol (Hallas, 1965). The synthetic route of
N-acetyl-S-(4-coumarinyl)-L-cysteine methyl ester
(2) is demonstrated in Scheme 2.
3. Discussion
Compounds 1 and 2 were characterized by
elemental analysis; NMR-spectroscopy, IR-spec-
troscopy, UV-spectroscopy and mass spectrome-
N-Acetyl-S-(3-coumarinyl)-D,L-cysteine methyl
ester (1) (coumarin 3-mercapturic acid methyles-
ter) is easily accessible by reaction of 3-mercapto-
coumarin (3) with N-acetyl-3-chloro-D,L-alanine
methyl ester (4). Formation of N-acetyl-S-(3-
coumarinyl)cysteine has been suggested to result
from conjugation of phase I metabolite coumarin-
3,4-epoxide with glutathione.
The 4-mercapturic acid isomer 10 was prepared
by the reaction of 3-bromocoumarin (12) with N-
1
try. The H-NMR-spectra of 1 and 2 are given in
1
Fig. 1. The H-NMR-spectrum of 1 matched with
1
the H-NMR-spectrum of N-acetyl-S-(3-coumar-
inyl)cysteine isolated from urine of rats given
coumarin orally (Huwer et al., 1991).
N-Acetyl-S-(4-coumarinyl)cysteine (10) in urine
was concentrated using solid phase extraction on a
Merck Lichrolut Extraction Cartridge, RP 18.

252
G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ258
/
Scheme 2. Synthetic route of N-acetyl-S-(4-coumarinyl)-L-cysteine methyl ester (2).
acetyl-
L
-cysteine (13). This reaction was found to
al., 1994; Hadidi et al., 1998; Meineke et al., 1998).
In the case of a defective CYP2A6 allele, higher
formation rates of coumarin 3,4-epoxide are to be
expected. The urinary excretion of N-acetyl-S-(3-
coumarinyl)cysteine in such subjects is expected to
be quantifiable on the basis of the above HPLC
method.
proceed more smoothly and at higher yields than
by using 4-chlorocoumarin, as described before.
As can be seen from the HPLC chromatograms,
coumarin 3-mercapturic acid can be easily deter-
mined in urine as its methyl ester, in the presence
of parent compound and further hydroxylated
metabolites, with a limit of determination around
1ꢂ
of 100ꢂ
/
2 mg mlꢀ1. In clinical use, daily coumarin doses
200 mg have been administered (Burgos et
4. Experimental part
/
al., 1999; Shilling et al., 1969). On the assumption
that normally about 4% of the administered
coumarin will be metabolized via coumarin-3,4-
epoxide, N-acetyl-S-(3-coumarinyl)cysteine pre-
sent in 24 urine will be below the detection limit.
While the majority of humans metabolize cou-
marin to 7-hydroxycoumarin, it has also been
reported that a deficiency in this pathway was
observed in some individuals, apparently related to
a genetic polymorphism in CYP2A6. Such indivi-
duals might metabolize coumarin preferentially by
the 3,4-epoxidation pathway and possibly other
alternative pathways (Oscarson et al., 1998, 1999;
Hadidi et al., 1997; Ford et al., 2001; van Iersel et
4.1. Chemistry
4.1.1. General
Coumarin, 2-hydroxybenzaldehyde, rhodanine,
serine methyl ester, triethylamine, phosphorus
pentachloride, acetyl chloride, and solvents were
obtained from commercial sources. All reagents
were of analytical grade.
Melting points were measured using Buchi 510
¨
and are not corrected. Elemental analysis was
carried out by Perkin Elmer 2400 CHN-Elemen-
tal-Analyzer; 1H-NMR- and 13C-NMR-spectra by
Bruker AM 400; IR-spectra by Perkin Elmer FT-

G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ
/
258
253
Fig. 1. 1H-NMR-spectra of N-acetyl-S-(3-coumarinyl)cysteine methyl ester (1) (upper) and N-acetyl-S-(4-coumarinyl)cysteine methyl
ester (2) (below).
IR 90; UV-spectra by Kontron-Uvikon 810; and
mass spectra by Finnigan MAT 90 mass spectro-
meter.
acetonitrile (60 ml) under argon atmosphere. The
mixture was stirred for 4 h at 50 8C. Until
termination of the reaction (DC control; DC-
Alufoils, Kieselgel 60 F 254 Merck). The solvent
was removed in vacuo; 1 was washed with n-
hexane and water to yield a white, crystalline
4.2. N-Acetyl-S-(3-coumarinyl)-D,L-cysteine
methyl ester (1)
powder (400 mg, 47%), m.p. 175 8C.*
C15H15NO5S (321.3) calc. C, 56.0; H, 4.7; N, 4.4.
Found: C, 55.9; H, 4.8; N, 4.0%.*1
H-NMR (d6-
DMSO): d (ppm)ꢃ8.58 (d, 1H, NH), 7.96 (s, 1H,
H-4), 7.56ꢂ7.35 (m, 4H, aromat), 4.60 (m, 1H, NÃ
/
Freshly distilled triethylamine (0.9 ml) was
slowly added to a solution of 3-mercaptocoumarin
/
/
(3) (500 mg, 3 mmol) and N-acetyl-3-chloro-
D,L-
alanine methyl ester (4) (500 mg, 3 mmol) in
/
/

254
G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ258
/
Fig. 2. HPLC chromatogram of a standard mixture composed of 3 mg N-acetyl-S-(4-coumarinyl)-
N-acetyl-S-(3-coumarinyl)cysteine methyl ester (1) in 1 ml urine. UV-detector wavelength: 300 nm (right panel), 330 nm (left panel).
(b) HPLC chromatogram of N-acetyl-S-(3-coumarinyl)- -cysteine methyl ester (1), coumarin (11), 7-hydroxycoumarin (14), and 3-
hydroxycoumarin (15). UV-Detector wavelength: 330 nm.
L
-cysteine methyl ester (2) and 2 mg
D,L
CH Ã
SCH2), 3.26 (dd, 1H, SCH2), 1.85 (s, 3H,
NCOCH3).*13
C-NMR (d6-DMSO): d (ppm)ꢃ
170.7 (s, CO2), 169.6 (s, NCO), 158.4 (s, C2),
151.3 (s, C10), 136.0 (d, C4), 130.7 (d, C8), 127.1
(d, C7), 125.4 (s, C3), 124.9 (d, C6); 119.3 (s, C9),
116.0 (d, C5), 52.3 (q, CO2CH3), 50.8 (d, NHCH),
/
CO2), 3.66 (s, 3H, CO2CH3), 3.41 (dd, 1H,
4.3. 3-Bromocoumarin (12)
Coumarin (10 g, 68 mmol) was dissolved in 100
ml chloroform and bromine (10.9 g, 68 mmol) was
added into the solution in an ice bath. The reaction
mixture was stirred overnight until colorless,
triethylamine (20 ml) was then added and the
organic solution was washed two times with water.
Compound 12 was obtained as a yellowish solid
/
/
31.5 (t, SCH2), 22.2 (q, NCOCH3).*
yꢃ
3296 cmꢀ1 (amide), 1764 (ester), 1704 (lac-
tone), 1530 (aromat), 754 (1,2-substituted
aromat).*UV (CH3OH): lꢃ330 nm (oꢃ
10 307), lꢃ235 nm (oꢃ6154).*MS: m/zꢃ
321.4 [Mꢁ], 276.6 [Mꢁꢀ
COCH2], 246.6, 224.5.
/IR (KBr):
/
(11.5 g, 75%), m.p. 108 8C.*1
(ppm)ꢃ8.15 (s, 1H, H-4), 7.85ꢂ
aromat). The bromine substitution at position 3
/
H-NMR (CDCl3): d
7.15 (m, 4H,
/
/
/
/
/
/
/
/
/
1
/
is indicated by the signal at 8.15 ppm in the H-

G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ
/
258
255
Fig. 2 (Continued)
NMR spectrum, showing the presence of a proton
at position 4.
N, 4.6. Found: C, 53.7; H, 4.2; N, 4.5%.*1
NMR (d6-DMSO): (ppm)ꢃ
CO2H), 8.55 (d, 1H, NH), 7.79ꢂ
/
H-
d
/
13.22 (s, 1H,
7.39 (m, 4H,
/
aromat), 6.42 (s, 1H, H-3), 4.61 (m, 1H,
NCHCO2), 3.62 (dd, 1H, SCH2), 3.43 (dd, 1H,
SCH2); 1.89 (s, 3H, NCOCH3). The protons of the
methylene group present as two peaks at 3.62 and
3.43 ppm, indicating the occurrence of epimeriza-
4.4. N-Acetyl-S-(4-coumarinyl)-L-cysteine (10)
3-Bromocoumarin (12) (10 g, 44 mmol) and N-
acetyl- -cysteine (13) (7.3 g, 44 mmol) were
L
dissolved in dry methanol (100 ml). Triethylamine
(15 ml) was added into the reaction mixture and
stirred for 4 h at room temperature. After remov-
ing half of the solvent the reaction mixture was
acidified with hydrochloric acid. The precipitate
was collected on a filter, washed with n-hexane
and water, and dried over P2O5 to yield 10 as a
1
tion during the reaction. The H-NMR data of 10
agree with those described by Huwer et al. (Huwer
et al., 1991).*13
/
C-NMR (d6-DMSO): d (ppm)ꢃ
/
171.3 (s, CO2H), 169.6 (s, NCO); 157.9 (s, C-2);
154.7 (s, C-4); 151.5 (s, C-10); 132.7 (d, C-8);
124.5 (d, C-7); 123.7 (d, C-6); 117.4 (s, C-9); 116.9
(d, C-5); 107.1 (d, C-3); 50.6 (d, NHCH); 31.5 (t,
white, crystalline powder (6.1 g, 45%), m.p. 215ꢂ
/
220 8C. C14H13NO5S (307.3) calc. C, 54.7; H, 4.3;
SCH2); 22.3 (q, NCOCH3).*
/
UV (CH3OH): lꢃ
/

256
G. Eisenbrand et al. / Toxicology 190 (2003) 249ꢂ258
/
300 nm (oꢃ
/
11 960); lꢃ
/
278 nm (oꢃ
/
10 413).*
/
4.6.2. HPLC determinations
MS: m/zꢃ
/
307.4 [Mꢁ], 276.6 [Mꢁꢀ
/COCH2].
Fractions containing 10 were combined and
lyophilized. The dry residue was dissolved in 100
ml of a 25% BF3/methanol solution and heated up
to 70 8C for 10 min. Thereafter it was made up to 1
ml with water. Compound 2 was determined by
HPLC under similar conditions as described for
10, using methanol/buffer (pH 2.5) 1:1 (v/v) as
solvent.
Compounds 1 (2 mg mlꢀ1) and 2 (3 mg mlꢀ1) in
urine were isolated by solid extraction as described
and determined by HPLC under the same condi-
tions as described for 2 with an UV-detector (300
and 330 nm, respectively).
4.5. N-Acetyl-S-(4-coumarinyl)-L-cysteine methyl
ester (2)
50% boron trifluoride solution in methanol (1
ml) was added into a suspension of 10 (500 mg, 2
mmol) in dry methanol (10 ml). The mixture was
heated up to 70 8C until it became a clear solution.
Water (20 ml) was added after cooling. The
precipitate was collected by filtration and dried
in vacuo to give 2 as a white crystalline powder
(420 mg, 82%), m.p. 155 8C.*
(321.3) calc. C, 56.0; H, 4.7; N, 4.4. Found: C,
55.5; H, 4.5; N, 4.1%.*1
H-NMR (d6-DMSO): d
(ppm)ꢃ8.65 (d, 1H, NH), 7.76ꢂ7.36 (m, 4H,
/
C15H15NO5S
For determination of 11, 14, 15, and 1 (2 mg per
1 ml) in urine, extraction and fractionation was as
described for 10, HPLC: column: RP18, 250ꢄ4
/
/
/
/
aromat), 6.38 (s, 1H, H-3); 4.66 (m, 1H,
NCHCO2), 3.69 (s, 3H, CO2CH3), 3.62 (dd, 1H,
SCH2), 3.42 (dd, 1H, SCH2), 1.87 (s, 3H,
mm, particle size 5 mm (Merck LiChorCart);
elution solvent: methanol/buffer (pH 2.5)/tetrahy-
drofurane/acetonitrile 153:20:17:10 (v/v); flow
rate: 1 ml minꢀ1; UV-detector (300/330 nm).
NCOCH3).*13
/
C-NMR (d6-DMSO): d (ppm)ꢃ
/
170.4 (s, CO2), 169.7 (s, NCO), 157.9 (s, C-2),
154.5 (s, C-4), 151.5 (s, C-10), 132.7 (d, C-8),
124.5 (d, C-7), 123.7 (d, C-6), 117.4 (s, C-9), 116.9
(d, C-5), 107.2 (d, C-3), 52.4 (q, CO2CH3), 50.5 (d,
Acknowledgements
NHCH), 31.2 (t, SCH2), 22.2 (q, NHCOCH3).*
IR (KBr): yꢃ
3308 cmꢀ1 (amide), 1760 (ester),
1712 (lactone), 1654 (amide), 1530 (aromat), 760ꢂ
/
We thank Mrs I. Hemm for her help in the
preparation of the manuscript.
/
/
740 (1,2-substituted aromat).
4.6. Determination of N-acetyl-S-(3-
coumarinyl)cysteine methyl ester (1) in urine in a
model experiment
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