Synthesis of isothiocyanate-derived mercapturic acids

Article abstract of DOI:10.1016/S0223-5234(03)00141-7

Twelve mercapturic acids derived from saturated and unsaturated aliphatic and aromatic isothiocyanates were synthesised, by adding isothiocyanate to a solution of N-acetyl-L-cysteine and sodium bicarbonate, in a typical yield of 77%. Isothiocyanates were synthesised first by adding the corresponding alkyl bromide to phthalimide potassium salt. The obtained N-alkyl-phthalimide was hydrazinolysed yielding the alkyl amine, which subsequently was reacted with thiophosgene yielding the isothiocyanate with an overall yield of 16%. Mercapturic acids in urine can serve as a biomarker of intake to determine the health promoting potential of isothiocyanates present in cruciferous vegetables.

Full text of DOI:10.1016/S0223-5234(03)00141-7

European Journal of Medicinal Chemistry 38 (2003) 729ꢀ737
/
www.elsevier.com/locate/ejmech
Laboratory note
Synthesis of isothiocyanate-derived mercapturic acids
a,
Martijn Vermeulen *, Binne Zwanenburg , Gordon J.F. Chittenden ,
b
b
a,c
Hans Verhagen
a
Department of Food and Food Supplement Analysis, TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, The Netherlands
b
NSR-Center for Molecular Structure, Design and Synthesis, Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, 6525 ED
Nijmegen, The Netherlands
c
Unilever Health Institute, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands
Received 14 February 2003; received in revised form 16 June 2003; accepted 16 June 2003
Abstract
Twelve mercapturic acids derived from saturated and unsaturated aliphatic and aromatic isothiocyanates were synthesised, by
adding isothiocyanate to a solution of N-acetyl- -cysteine and sodium bicarbonate, in a typical yield of 77%. Isothiocyanates were
L
synthesised first by adding the corresponding alkyl bromide to phthalimide potassium salt. The obtained N-alkyl-phthalimide was
hydrazinolysed yielding the alkyl amine, which subsequently was reacted with thiophosgene yielding the isothiocyanate with an
overall yield of 16%. Mercapturic acids in urine can serve as a biomarker of intake to determine the health promoting potential of
isothiocyanates present in cruciferous vegetables.
#
´
2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
Keywords: Nucleophilic addition; Glucosinolates; Isothiocyanates; Mercapturic acids; Biomarker
1
. Introduction
to be strong cancer chemopreventors [11ꢀ13]. Many
/
different isothiocyanates (more than 25) block the
carcinogenic effects of more than 12 chemically different
types of carcinogens in at least 10 different target sites in
three species of rodents [14]. Phenethyl isothiocyanate is
a particularly effective inhibitor of lung tumor induction
by the tobacco-specific nitrosamine-4-(methylnitrosa-
mino)-1-(3-pyridyl)-1-butanone and, therefore, is cur-
rently being developed as a chemopreventive agent
against lung cancer [14]. Human intervention trials
with large quantities of cruciferous vegetables gave
similar effects on phase I and phase II enzymes [15,16].
Isothiocyanates are conjugated to glutathione in the
body and excreted into the urine as their corresponding
mercapturic acids (Fig. 1) as was demonstrated in rats
Consumption of fruits and vegetables is associated
with a reduced risk on degenerative diseases such as
cancer and cardiovascular diseases, as indicated by
epidemiological studies [1,2]. A reasonable estimate of
the overall extent to which dietary modification may be
expected to reduce cancer risk is 30ꢀ40% [3]. In
/
particular, cruciferous vegetables appear to have bene-
ficial health potential [4,5]. Since cruciferous vegetables
differ from other vegetables by the presence of glucosi-
nolates, these health promoting effects seem to be
attributable to these phytochemicals or breakdown
products thereof [6]. Glucosinolates are broken down
into indoles, nitriles and isothiocyanates (Fig. 1) by a
thioglucosidase (myrosinase, EC 3.2.3.1) present in
cruciferous vegetables [7,8] and to a lesser degree by
microbes present in the human gut [9,10]. Isothiocya-
nates are strong inhibitors of phase I enzymes and
inducers of phase II enzymes, and therefore are thought
[
17], guinea pigs and rabbits [18] and in humans [19,20].
Mercapturic acids reflect the intake of glucosinolates
present in cruciferous vegetables [21ꢀ23] and could be
/
used as a selective biomarker for cruciferous vegetable
intake. Different cruciferous vegetables can botanically
be differentiated by the variety and amount of glucosi-
nolates [24]. Broccoli, for instance, is rich in 4-methyl-
sulfinylbutyl glucosinolate, whereas Brussels sprouts are
rich in 2-propenyl and 2-hydroxy-3-butenyl glucosino-
* Corresponding author.
E-mail address: m.vermeulen@voeding.tno.nl (M. Vermeulen).
´
0
223-5234/03/$ - see front matter # 2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00141-7

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M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ737
/
cyanate, introduced as a drug for the treatment of
infections of the respiratory and urinary tract under the
†
trademark Tromacaps , resulted in 43ꢀ
/
60% excretion
of the compound in the urine as its mercapturic acid
20]. Sulforaphane is the bioactive compound found in
broccoli [27]. This synthesis is described in Scheme 1.
[
3. Results and discussion
Twelve isothiocyanate mercapturic acids have been
Fig. 1. Isothiocyanates are enzymatically hydrolysed from glucosino-
lates, in the body conjugated to glutathione and excreted as mercap-
turic acids in the urine.
synthesised (Fig. 2, Table 1). The synthesis of isothio-
cyanates from phthalimide is a multistep reaction with
an overall yield of 16%. The synthesis from N-acetyl-L-
cysteine (NAC) and isothiocyanate was a convenient
one-step reaction which proceeds in a typical yield of
late (progoitrin), and cress is rich in aromatic glucosi-
nolates [7].
77%. Before adding isothiocyanate to NAC, we con-
Several reports describing the synthesis of isothiocya-
nates and the corresponding mercapturic acids are
available (Table 1, comments in Section 3). We here
describe the synthesis of 12 mercapturic acids derived
from saturated and unsaturated aliphatic and aromatic
isothiocyanates, namely methyl, butyl, isopropyl, 1-
methylpropyl, 2-propenyl, 3-butenyl, 4-pentenyl, phen-
yl, benzyl, phenylethyl, 4-methylthiobutyl and 4-methyl-
verted NAC to its sodium salt using sodium bicarbo-
nate, thus enhancing the reaction rate. After the reaction
was completed, the mercapturic salt was converted into
the poorly water-soluble mercapturic acid using hydro-
chloric acid. Several solvents were tested for recrystalli-
sation. The products dissolved best in boiling ethyl
acetate which allowed crystallisation on cooling. The
mercapturic acids derived from isopropyl and of 1-
methylpropyl isothiocyanate were obtained in a yield of
sulfinylbutyl isothiocyanate (Fig. 2). The HPLCꢀMS/
/
MS analysis of isothiocyanate-derived mercapturic acids
in urine is described in Ref. [41].
50%. The solubility of these products in ethyl acetate is
perhaps higher than, for instance, the mercapturic acid
derived from benzyl isothiocyanate. A better solvent for
recrystallisation could not be found.
2
. Chemistry
The synthesis of 3-butenyl isothiocyanate has pre-
viously been described by Kjær et al. [28] starting from
allyl cyanide. The boiling point observed by us and that
reported [28] are the same (60 8C at 12 mmHg). Ettlinger
and Hodgkins [29] started from allylcarbinol and
reported the boiling point to be 77.5 8C at 28 mmHg.
Leoni et al. [30] obtained 3-butenyl isothiocyanate by
enzymatic hydrolysis of gluconapin. Our NMR data are
identical with that reported. 4-Pentenyl isothiocyanate
has been synthesised previously by Kjær and Jensen [31]
and in the same way by Gilbert and Nursten [32]. Only
the boiling point, density and elemental analysis were
mentioned. 4-Methylthiobutyl isothiocyanate (trivial
name erucin) has first been synthesised by Schmid and
Karrer [33] and later by Kjær and Gmelin [34]. Our
boiling point and NMR data are identical with those
reported. 4-Methylsulfinylbutyl isothiocyanate (trivial
name sulforaphane) has first been synthesised by
Schmid and Karrer [33] who also described the synthesis
In cruciferous plants, glucosinolates are formed from
amino acids [25]. Tissue disruption by, e.g. chewing
starts the breakdown of glucosinolates into isothiocya-
nates and this provides protection from plants towards
insects as isothiocyanates are pungent metabolites. In
humans, chewing of Brussels sprouts releases 39% of the
glucosinolates as isothiocyantes, measured in the urine,
whereas no chewing results in the excretion of 26% of
isothiocyanates [26]. Ingestion of pure benzyl isothio-
of optically pure L- and D-sulforaphane. Sulforaphane
was later synthesised by Zhang et al. [27] and Kuhnert et
al. [35] (conversion of amine into the isothiocyanate unit
precedes the oxidation of sulfur). The physical and
spectral data are identical. All authors who published
their results between 1948 and 1972 used pyridine and
benzene. These toxic compounds can be replaced by
Fig. 2. Structures of 12 synthesised mercapturic acids derived from
isothiocyanate.

M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ
/737
731
Table 1
Trivial names of isothiocyanate yielding glucosinolates, structures of the corresponding isothiocyanates, (main) dietary source and literature
references to isothiocyanate and mercapturic acid synthesis
a
Glucosinolate
Isothiocyanate
Main dietary
source
Previous isothiocyanate synth- Previous isothiocyanate mercapturic acid
b
b
esis by others
synthesis by others
Glucocapparin
Unknown
H
H
3
CNCS
C(CH
cauliflower, capers
cabbage, horserad-
ish
1
2
commercially available
commercially available
(36)
(36)
3
2
)
3
NCS
)NCS
Glucoputranjivin
H
3
CCH(CH
3
Brussels sprouts,
turnip
3
commercially available
not yet reported
c
Glucocochlearin
Sinigrin
H
H
3
CCH CH(CH
2
3
)NCS black mustard
cabbage, brown
mustard
4
5
commercially available
commercially available
not yet reported
(36, 37)
2
CꢀCHCH
/
2
NCS
Gluconapin
Glucobrassicanapin
Unknown
H
H
2
Cꢀ
/
CH(CH
2
2
)
)
2
NCS
NCS
Chinese cabbage
Chinese cabbage
6
7
8
9
(28, 29)
(31, 32)
not yet reported
not yet reported
not yet reported
(17)
2
Cꢀ
/CH(CH
3
d
C
C
C
6
H
5
5
5
NCS
CH NCS
(CH NCS
NCS
ambiguous
commercially available
commercially available
Glucotropaeolin
Gluconasturtiin
Glucoerucin
6
H
H
2
garden cress
water cress
broccoli, salad
rocket
6
2
)
2
10 commercially available
11 (27, 33, 34)
(38, 39)
(40)
H
3
CS(CH
2
)
4
Glucoraphanin
H
3
CS(ꢀ
/
O)(CH
2
)
4
NCS cabbage
12 (27, 33, 35)
(40)
a
Numbers refer to structures in Fig. 2.
For comments, see Section 3.
Not optically pure.
b
c
d
Presence in nature is unlikely.
aqueous ethanol and heptane, respectively. The presence
in nature and isolation from plants of isothiocyanates
was summarised by Kjær [7].
conjugates. They reported an m.p. of 58ꢀ62 8C for 9,
/
which is very different from ours (136.12 8C). Since
optical rotation, elemental analysis and NMR data are
in agreement with ours, the product obtained by
Br u¨ sewitz is probably contaminated by some solvent
which lowers the m.p. Toxic solvents can be replaced by
non-toxic. Eklind et al. [38] synthesised 10, by adding
Mennicke et al. [36] described the synthesis of 1, 2 and
. Physical data were only given for the dicyclohexyla-
5
mine salt. Ioannou et al. [37] (only brief description of
the synthesis) described the isolation of 5 from rat urine
1
4
and the synthesis of C radio-labeled 5 using column
chromatography to obtain the pure compound. All
NMR data are comparable with ours. Br u¨ sewitz et al.
the isothiocyanate to a solution of N-acetyl-L-cysteine in
methanol in poor yield (39%). The NMR data are the
same as ours. Jiao et al. [39] used the method of
synthesis described by Br u¨ sewitz, however, their NMR
data for the obtained product (10) differ from those
reported by Eklind and found by us. Kassahun et al. [40]
prepared 11 and 12 by adding the isothiocyanate to an
[
17] described the synthesis of benzyl isothiocyanate
aqueous ethanol solution of N-acetyl-L-cysteine at basic
pH. These products were purified by column chromato-
graphy, the NMR data (in D O/CDCl ) are in agree-
ment with ours.
2
3
In our synthesis of 11 and 12, phthalimide potassium
salt was added to three equivalents of 1,4-dibromobu-
tane to minimise the formation of 1,4-diphthalimidyl-
butane. A top piece containing potassium hydroxide was
used to avoid moisture and to prevent the reaction of
sodium methylmercaptide with carbon dioxide to give
methylmercaptan. We compared triethylamine, sodium
hydroxide and sodium bicarbonate as the basic reagent
in the formation of isothiocyanate from the amine and
thiophosgene. Three equivalents of the base were used,
after which chloroform and excess of thiophosgene were
evaporated and the residue was pored in water and
diethyl ether. This resulted in black solids when
Scheme 1. Synthesis of N-acetyl-S-(N-4-methylsulfinylbutylthiocar-
bamoyl)- -cysteine (sulforaphane mercapturic acid).
L

732
M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ737
/
triethylamine was used. Because of gas forming after
adding sodium bicarbonate, we preferred the use of
sodium hydroxide as a base. Distillation of the iso-
thiocyanate formed was performed immediately after
evaporating chloroform and thiophosgene in order to
prevent residues of thiophosgene to react with the
isothiocyanate. 4-Methylsulfinylbutyl isothiocyanate
was obtained by oxidation of 4-methylthiobutyl iso-
thiocyanate. 4-Methylsulfinylbutylamine could be ob-
tained by oxidising N-(4-methylthiobutyl)-phthalimide
with MCPBA, followed by hydrazinolysis. Subsequent
reaction of the amine with thiophosgene led to complete
reduction of the sulfinyl group yielding 4-methylthiobu-
tylamine rendering this route of synthesis impossible.
This deoxygenation in the absence of a reducing agent
has been observed also by Kuhnert et al. [35] and cannot
be explained. Thiophosgene used to convert amines to
isothiocyanates can be replaced by di-2-pyridyl thiono-
carbonate which is not toxic and easier to handle. We
were unable to derive optimal conditions for this
conversion using 1,1?-thiocarbonyl diimidazole.
Product 7 has an brownish-orange color, while all
other mercapturic acids are colored yellow to white. The
reaction mixture containing 4-pentenyl isothiocyanate
and NAC also contained some black residue which may
be due to side products resulting from thiophosgene.
Isothiocyanate-derived mercapturic acids and its pre-
cursors can be separated using TLC. Isothiocyanates
and their mercapturic acids are UV 254 nm active,
whereas NAC is not UV active. Potassium dichromate
followed by heat colors all products and precursors and
has the advantage that it colors NAC orange before heat
used as internal standard for the analysis of mercapturic
acids obtained from isothiocyanates. Future research
will include the development of a liquid chromato-
graphic method to analyse mercapturic acids in urine
and validation of mercapturic acids as biomarkers to
measure the effective dose of isothiocyanates absorbed.
5. Experimental protocols
5.1. Chemistry
Methyl, 2-propenyl, phenyl, benzyl and phenylethyl
isothiocyanate as well as N-acetyl-L-cysteine (NAC),
1,4-dibromobutane, 4-bromo-1-butene and 5-bromo-1-
pentene were purchased from Acros Organics. Butyl
isothiocyanate, iso-propyl isothiocyanate and sodium
methylmercaptide were purchased from Sigma-Aldrich
Co., 1-methylpropyl isothiocyanate was obtained from
Maybridge Chemical Company Ltd. and phthalimide
potassium salt was purchased from Merck. Solvents
were dried using the following methods. Dichloro-
methane was distilled from P O . Diethyl ether was
2
5
distilled from NaH. Hexane and heptane were distilled
from CaH . All other chemicals were of analytical
2
grade. All chemical and physical data are mentioned
in Table 2.
1
H-NMR (100 MHz) spectra were recorded on a
1
Bruker AC 100 spectrometer, 300 MHz H-NMR and
1
3
C-NMR spectra were recorded on a Bruker AC 300
1
spectrometer, 400 MHz H-NMR spectra were recorded
1
on a Varian Unity-400 spectrometer, and 600 MHz H-
treatment. In ethyl acetateꢀ
60:35:18, v/v/v), all components showed only minor
retention, NAC R ꢁ0.78, phenyl mercapturic acid
0.90 and phenyl isothiocyanate R ꢁ0.99. Adding
just enough acetic acid (8 mL) to mix 80 mL of ethyl
acetate with 10 mL of water gave a satisfactory retention
and separation of the components.
The melting of the mercapturic acid derived from
phenyl isothiocyanate resulted in the explosion of a
DSC sample cell. This is probably due to an exothermal
reaction with formation of gas following the melt.
Melting of NAC also resulted in the formation of gas
but the reaction products could not be analysed.
/
waterꢀ
/
formic acid
NMR spectra were recorded on a Bruker Avance
spectrometer with DMSO-d as internal standard unless
(
6
/
stated otherwise (Aldrich, dꢁ
respectively, J in Hz). Optical rotation (OR) was
measured on a PerkinꢀElmer 241 Polarimeter at 589.3
/2.49 and 39.7 ppm,
F
R ꢁ
/
/
F
F
/
ꢂ1
nm, products were dissolved in methanol (10 mg mL ),
measured three times and the average was taken.
Melting points (m.p.) were determined with a Reichert
Thermopan microscope and figures between brackets on
a differential scanning calorimeter (DSC-2920, TA
Instruments), both are uncorrected. Elemental analyses
(EA) were performed on a Carlo Erba instruments
CHNS-O 1108 elemental analyser at the Department of
Microanalysis of the University of Nijmegen. The
formula weight (FW) is the average molecular weight.
The exact molecular mass was calculated using the
atomic masses of the most abundant isotopes. Mass
spectra (MS) were collected on a Finigan MAT LCQ
mass spectrometer coupled to a Waters 2690 liquid
chromatograph (HPLC). All isothiocyanate-derived
mercapturic acids were separated on a C18 column
using a gradient of acetonitrile in water (both with 0.1%
formic acid) and detected using an LCQ ion-trap MS
with electrospray ionisation (positive mode). Mercaptu-
4
. Conclusions
In conclusion, we have synthesised 12 mercapturic
acids derived from isothiocyanates in good yield. These
2 compounds were prepared because they probably
1
represent the most important urinary excretion products
after eating a cruciferous vegetable meal. Phenyl gluco-
sinolate is not a natural compound so that the mercap-
turic acid derived from phenyl isothiocyanate can be

Table 2
Chemical and physical data of the synthesised isothiocyanate-derived mercapturic acids
1
No. Melting
a
Optical ro-
tation (8)
H-NMR (d in ppm, J in Hz)
Elemental analysis calculated/found
Mass
UV (absorbance
maximum in nm)
b
point (8C)
1
2
3
4
144ꢀ
/
146
ꢂ
/
35.0
19.2
17.6
14.2
(300 MHz) 1.82 (s, 3H, C(O)CH
1
3
), 3.00 (d, 3H, Jꢁ
), 3.75 (dd, 1H, JAX
8.1 Hz, OCNH), 9.96 (t, 1H, SCNH), 11.87 27.29
/
4.4 Hz, NCH
3
), 3.30 (dd,
7 12 2 3 2
C H N O S ; C 35.58, H 5.12, N 11.85, FW 236.316; ex- 267 (1st max); 249
H, JAB
ꢁ/13.6 Hz, JBX
ꢁ/
9.2 Hz, CH
B
ꢁ
/
5.0 Hz, CH
A
), S 27.14; C 35.82, H 5.01, N 11.70, S
act 236.0; MS
237.0
(2nd max)
4
X
.38 (m, 1H, CH ), 8.30 (d, 1H, Jꢁ
/
(
s, 1H, OH)
(100 MHz) 0.86 (t, 3H, NCCCCH
NCCH ), 1.82 (s, 3H, C(O)CH ), 3.27 (dd, 1H, JAB
), 3.54 (t, 2H, NCH ), 3.76 (dd, 1H, JAX 5.1 Hz, CH
), 8.32 (d, 1H, Jꢁ8.0 Hz, OCNH), 10.00 (t, 1H, SCNH)
6.5 Hz, C(CH )CH ), 1.81 (s, 3H, C(O)CH
), 3.75 (dd, 1H, JAB 13.6 Hz, JAX 5.0 Hz, CH ), 4.41 (m, 2H, S 24.26; C 40.85, H 6.12, N 10.40, S
and CH(C)C), 8.31 (d, 1H, Jꢁ7.9 Hz, OCNH), 9.88 (d, 1H, Jꢁ7.3 Hz, 24.01
rt
ꢂ/
3
), 1.23 (m, 2H, NCCCH
13.6 Hz, JBX
), 4.37 (m, 1H,
2
), 1.45 (m, 2H,
9.2 Hz, 10.06, S 23.04; C 43.61, H 6.81, N 9.67, act 278.1; MS
A
S 21.64 279.0
C
10
H
18
N
2
O
3
S
2
; C 43.14, H 6.52, N
FW 278.397; ex- 251 (1st max); 270
2
3
ꢁ/
ꢁ/
(2nd max)
CH
CH
B
2
ꢁ/
X
/
165ꢀ
/
169
ꢂ/
(100 MHz) 1.13 (d, 6H, Jꢁ
/
3
3
3 9 16 2 3 2
), 3.26 C H N O S ; C 40.89, H 6.10, N 10.60, FW 264.370; ex- 253 (1st max); 270
(
m, 1H, CH
B
ꢁ
/
ꢁ/
A
act 264.1; MS
265.0
(2nd max)
CH
X
/
/
SCNH), 12.83 (s, 1H, OH)
140ꢀ
/
144
ꢂ/
(100 MHz) 0.86 (t, 3H, C(C)CCH
3
), 1.15 (d, 3H, Jꢁ
), 3.34 (dd, 1H, JAB
5.1 Hz, CH ), 4.41 (m, 2H, CH
8.0 Hz, OCNH), 9.89 (d, 1H, Jꢁ
/
6.6 Hz, C(CH
3
)CC), 1.52
C
10
H
18
N
2
O
3
S
2
; C 43.14, H 6.52, N
10.06, S 23.04; C 43.13, H 6.63, N 10.09, act 278.1; MS
and S 22.66 279.0
FW 278.397; ex- 253 (1st max); 270
(
dd, 2H, C(C)CH
9.1 Hz, CH
CH(C)CC), 8.36 (d, 1H, Jꢁ
2.87 (s, 1H, OH)
(100 MHz) 1.82 (s, 3H, C(O)CH
2
C), 1.87 (s, 3H, C(O)CH
3
ꢁ
/
13.3 Hz,
(2nd max)
J
BX
ꢁ/
B
), 3.80 (dd, 1H, JAX
ꢁ/
A
X
/
/7.7 Hz, SCNH),
1
5
6
7
rt
rt
rt
ꢂ
/
21.7
3
), 3.29 (dd, 1H, JAB
5.0 Hz, CH ), 4.20 (dd, 2H, NCH
C), 5.84 (m, 1H, CꢀCH), 8.32 (d, 1H, Jꢁ
ꢁ/
13.6 Hz, JBX
), 4.39 (m, 1H, S 24.44; C 40.53, H 5.47, N 10.26, S
8.1 Hz, 23.40
ꢁ
/
9 14 2 3 2
9.2 Hz, C H N O S ; C 41.20, H 5.38, N 10.68, FW 262.354; ex- 251 (1st max); 269
CH
CH
B
), 3.77 (dd, 1H, JAX
), 5.14 (dd, 2H, CH
ꢁ
/
A
2
act 262.1; MS
263.0
(2nd max)
X
2
ꢀ
/
/
/
OCNH), 10.17 (t, 1H, SCNH), 12.85 (s, 1H, OH)
(100 MHz) 1.82 (s, 3H, C(O)CH ), 2.31 (m, 2H, NCCH
3.6 Hz, JBX 9.2 Hz, CH ), 3.61 (m, 2H, NCH ), 3.75 (dd, 1H, JAX
CH ), 4.38 (m, 1H, CH ), 5.05 (dd, 2H, CH CH), 8.31 S 22.19
C), 5.78 (m, 1H, Cꢀ
d, 1H, Jꢁ8.0 Hz, OCNH), 10.03 (t, 1H, SCNH), 12.84 (s, 1H, OH)
(100 MHz) 1.66 (m, 2H, NCCH ), 1.82 (s, 3H, C(O)CH ), 2.03 (m, 2H,
NCCCH ), 3.28 (dd, 1H, JAB 13.6 Hz, JBX 9.2 Hz, CH ), 3.54 (m, 2H,
NCH ), 3.76 (dd, 1H, JAX 5.0 Hz, CH ), 4.37 (m, 1H, CH ), 5.01 (dd, 2H,
CH CH), 8.32 (d, 1H, Jꢁ8.1 Hz, OCNH), 10.02 (t, 1H,
C), 5.77 (m, 1H, Cꢀ
SCNH), 12.85 (s, 1H, OH)
(400 MHz) 1.83 (s, 3H, CH
CH ), 3.81 (dd, 1H, JAX
ꢂ
/
21.8
3
2
), 3.28 (dd, 1H, JAB
5.1 Hz, 10.14, S 23.20; C 42.95, H 5.89, N 9.94, act 276.1; MS
277.0
ꢁ
/
C
10
H
16
N
2
O
3
S
2
; C 43.46, H 5.84, N
FW 276.381; ex- 251 (1st max); 269
1
ꢁ/
B
2
ꢁ/
(2nd max)
A
X
2
ꢀ
/
/
(
/
missing
2
3
C
11
H
18
N
2
O
3
S
2
; missing
FW 290.41; exact 251 (1st max); 269
290.1; MS 291.0 (2nd max)
2
ꢁ/
ꢁ/
B
2
ꢁ/
A
X
2
ꢀ
/
/
/
8
9
1
176ꢀ
/
178
ꢂ
/
16.7
15.8
14.9
3
), 3.28 (dd, 1H, JAB
ꢁ
/
13.7 Hz, JBX
), 4.46 (m, 1H, CH ), 7.22 (m, 1H,
8.0
ꢁ
/
10.0 Hz,
C
12
H
14
N
2
O
3
S
2
; C 48.30, H 4.73, N 9.39, FW 298.387; ex- 277
(176.50)
B
ꢁ/
4.8 Hz, CH
A
X
S 21.49; C 48.36, H 4.72, N 9.29, S 20.77 act 298.1; MS
299.0
aromatic), 7.40 (m, 2H, aromatic), 7.70 (m, 2H, aromatic), 8.39 (d, 1H, Jꢁ
Hz, OCNH), 11.70 (s, 1H, SCNH), 12.90 (s, 1H, OH)
(100 MHz) 1.82 (s, 3H, CH ), 3.32 (dd, 1H, JAB 13.7 Hz, JBX
CH ), 3.79 (dd, 1H, JAX 4.8 Hz, CH ), 4.41 (m, 1H, CH ), 4.82 (d, 2H, Jꢁ
.3 Hz, NCH ), 7.29 (m, 5H, aromatic), 8.33 (d, 1H, Jꢁ8.0 Hz, OCNH), 10.50
t, 1H, SCNH), 12.85 (s, 1H, OH)
(300 MHz) 1.83 (s, 3H, CH ), 2.88 (t, 2H, Jꢁ
9.0 Hz, CH ), 3.75 (m, 1H, JAX
), 4.39 (m, 1H, CH ), 7.25 (m, 5H, aromatic), 8.28 (d, 1H, Jꢁ
/
133ꢀ
/
135
ꢂ/
3
ꢁ/
ꢁ/10.0 Hz,
13 16 2 3 2
C H N O S ; C 49.98, H 5.16, N 8.97, FW 312.414; ex- 251 (1st max); 270
(136.12)
B
ꢁ/
A
X
/
S 20.53; C 50.10, H 5.37, N 9.06, S 20.50 act 312.1; MS
313.0
(2nd max)
5
2
/
(
0
50ꢀ/58
ꢂ/
3
/
7.6 Hz, ØCH
2
), 3.30 (dd, 1H,
), 3.75 (m, S 19.64; C 50.92, H 5.67, N 8.46, S 18.82 act 326.1; MS
8.0 327.0
C
14
H
18
N
2
O
3
S
2
; C 51.51, H 5.56, N 8.58, FW 326.441; ex- 253 (1st max); 270
J
AB
ꢁ/13.6 Hz, JBX
ꢁ/
B
ꢁ
/
5.0 Hz, CH
A
(2nd max)
2
H, NCH
2
X
/
Hz, OCNH), 10.17 (t, 1H, SCNH), 12.86 (s, 1H, OH)

734
M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ737
/
ric acids were detected as their [Mꢃ
MS data mentioned in the syntheses [41]. Ultraviolet
UV) spectra were collected on a Waters 996 Diode-
/
H]ꢃ ion which is the
(
array detector (DAD) after chromatographic separation
as described for MS. Thin-layer chromatography (TLC)
was carried out on Merck precoated silica gel 60 F254
plates (0.25 mm) using the eluents indicated. Spots were
visualised with UV and using a potassium dichromate
spray. Potassium dichromate colors NAC orange, but
not its isothiocyanate mercapturic acid, the latter is
made visible by heating the TLC plate with hot air.
5.1.1. General procedure for commercially available
isothiocyanates
Compounds 1, 2, 3, 4, 5, 8, 9, and 10 were synthesised
by gradually adding the diluted isothiocianate to a
solution of N-acetyl-
The reaction was monitored by TLC with ethyl acetateꢀ
waterꢀacetic acid (80:10:8, v/v/v) as eluent.
L-cysteine and sodium bicarbonate.
/
/
5.1.1.1. N-Acetyl-S-(N-methylthiocarbamoyl)-L-
cysteine (1), mercapturic acid derived from methyl
isothiocyanate. Methyl isothiocyanate (16.5 mmol) was
dissolved in 20 mL of ethanol and gradually added to a
solution of N-acetyl-
sodium bicarbonate (15.8 mmol) in 20 mL of water.
TLC: NAC R ꢁ0.48, 1 R ꢁ0.43. All NAC had
L-cysteine (NAC, 15.0 mmol) and
/
/
F
F
reacted immediately after methyl isothiocyanate was
added. Using a Dowex 50X8-200 cation exchange
column, the sodium salt of the mercapturic acid was
converted to the free carboxylic acid. The product was
crystallised from ethyl acetate yielding 11.0 mmol
(73.3%) of 1 as bright white crystals.
5
.1.1.2. N-Acetyl-S-(N-butylthiocarbamoyl)-L-cysteine
2), mercapturic acid derived from butyl isothiocyanate.
(
Butyl isothiocyanate (14.0 mmol) was dissolved in 15
mL of ethanol and gradually added to a solution of
NAC (12.8 mmol) and sodium bicarbonate (13.4 mmol)
in 15 mL of water. TLC: NAC R ꢁ
/
0.47, 2 R ꢁ0.57.
/
F
F
All NAC has reacted within a few hours after butyl
isothiocyanate was added. The aqueous ethanol was
evaporated and the residue was dissolved in 15 mL of
brine. The excess of butyl isothiocyanate was extracted
with heptaneꢀ
was acidified. The mercapturic acid was extracted with
5 mL of ethyl acetate, concentrated until dryness and
/diethyl ether (10:5, v/v) and the mixture
1
traces of ethyl acetate were removed by subsequent
washing and evaporating with dichloromethane yielding
9.3 mmol (72.7%) of 2 as a yellow sticky product.
5.1.1.3. N-Acetyl-S-(N-isopropylthiocarbamoyl)-L-
cysteine (3), mercapturic acid derived from isopropyl
isothiocyanate. Isopropyl isothiocyanate (27.2 mmol)
was dissolved in 20 mL of ethanol and gradually added
to a solution of NAC (24.8 mmol) and sodium

M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ
/737
735
bicarbonate (26.0 mmol) in 20 mL of water. TLC: NAC
R ꢁ0.45, 3 R ꢁ0.53. All NAC has reacted immedi-
ately after isopropyl isothiocyanate was added. Ethanol
was evaporated, the mixture was acidified with HCl and
the product was crystallised from boiling ethyl acetate
yielding 12.3 mmol (49.6%) of 3 as bright white crystals.
All NAC had reacted within a few hours after benzyl
isothiocyanate was added. Ethanol was evaporated and
the mixture was acidified with HCl. After crystallisation,
the product was washed on filter with cold water and
dried on filter with gentle suction, yielding 11.5 mmol
(84.6%) of 9 as yellowish-white crystals.
/
/
F
F
5
.1.1.4. N-Acetyl-S-(N-1-methylpropylthiocarbamoyl)-
-cysteine (4), mercapturic acid derived from 1-
5.1.1.8. N-Acetyl-S-(N-phenylethylthiocarbamoyl)-L-
L
cysteine (10), mercapturic acid derived from phenylethyl
isothiocyanate. Phenylethyl isothiocyanate (13.8 mmol)
was gradually added to a solution of NAC (12.5 mmol)
and sodium bicarbonate (13.1 mmol) in 25 mL of
methylpropyl isothiocyanate. 1-Methylpropyl isothiocya-
nate (105.1 mmol) was dissolved in 50 mL of ethanol
and gradually added to a solution of NAC (100.0 mmol)
and sodium bicarbonate (110.0 mmol) in 50 mL of
aqueous 70% (v/v) ethanol. TLC: NAC R ꢁ
/
0.41, 10
0.53. All NAC had reacted within a few hours after
F
water. TLC: NAC R ꢁ
/
0.44, 4 R ꢁ
/
0.54. All NAC has
R ꢁ
/
F
F
F
reacted within a few hours after 1-methylpropyl iso-
thiocyanate was added. Ethanol was evaporated and the
excess of 1-methylpropyl isothiocyanate was extracted
with heptane. The mixture was acidified with HCl and
the product was crystallised from boiling ethyl acetate,
yielding 49.7 mmol (49.7%) of 4 as bright white crystals.
phenylethyl isothiocyanate was added. Ethanol was
evaporated, the excess of phenylethyl isothiocyanate
was extracted with hexane, the remaining mixture was
acidified with HCl and the mercapturic acid was
extracted with ethyl acetate. Traces of organic solutes
were finally evaporated using high vacuum, yielding 9.1
1
3
mmol (72.8%) of 10 as white crystals. C-NMR: 195.6
O), 139.0 (C-1 Ø),
5
.1.1.5. N-Acetyl-S-(N-2-propenylthiocarbamoyl)-
L
-
(Cꢀ
/
S), 172.2 (COOH), 169.5 (Cꢀ
/
cysteine (5), mercapturic acid derived from 2-propenyl
isothiocyanate. 2-Propenyl isothiocyanate (57.3 mmol)
was dissolved in 50 mL of ethanol and gradually added
to a solution of NAC (51.2 mmol) and sodium
bicarbonate (56.3 mmol) in 50 mL of water. TLC:
128.8 (C-3 Ø), 128.6 (C-2 Ø), 126.5 (C-4 Ø), 51.9
(CCOOH), 48.3 (ØCCH ), 35.9 (CH S), 33.5 (ØCH ),
22.6 (CH3).
2
2
2
5.1.2. General procedure for the complete synthesis
NAC R ꢁ
/
0.46, 5 R ꢁ
/
0.57. All NAC has reacted
Compounds 6, 7, 11, and 12 were synthesised by first
adding the corresponding alkyl bromide to phthalimide
potassium salt. The obtained N-alkyl-phthalimide was
hydrazinolysed, yielding the alkylamine, which was
subsequently reacted with thiophosgene to produce the
isothiocyanate. The diluted isothiocyanate was gradu-
F
F
within a few hours after 2-propenyl isothiocyanate was
added. Ethanol was evaporated and the mixture was
acidified with HCl which gave a yellow oily precipitate.
The mercapturic acid was extracted with ethyl acetate
and traces of organic solutes were evaporated using high
vacuum, yielding 42.5 mmol (83.0%) of 5 as a yellow
sticky product.
ally added to a solution of N-acetyl-
sodium bicarbonate which was monitored by TLC with
ethyl acetateꢀwaterꢀacetic acid (80:10:8, v/v/v) as
eluent.
L-cysteine and
/
/
5
.1.1.6. N-Acetyl-S-(N-phenylthiocarbamoyl)-L-
cysteine (8), mercapturic acid derived from phenyl
isothiocyanate. Phenyl isothiocyanate (13.3 mmol) was
5.1.2.1. N-Acetyl-S-(N-3-butenylthiocarbamoyl)-L-
dissolved in 15 mL of ethanolꢀ
gradually added to a solution of NAC (12.3 mmol) in 70
mL of tetrahydrofuran (THF). TLC: NAC R ꢁ0.39, 8
0.46. Sodium bicarbonate (14.0 mmol) and 30 mL
of water were added to the mixture which started the
reaction. All NAC had reacted within 24 h. THF was
evaporated, the mixture was acidified with HCl and the
/
water (8:2, v/v) and
cysteine (6), mercapturic acid derived from 3-butenyl
isothiocyanate. Phthalimide potassium salt (224.0 mmol)
was slowly added to a solution of 25 mL of 4-bromo-1-
butene (246.3 mmol) in 75 mL of dimethylformamide
(DMF). All phthalimide potassium salt had reacted
after overnight stirring. DMF and the excess of 4-
bromo-1-butene were evaporated. With heat, the pro-
duct and by-product (potassium bromide) were dis-
/
F
R ꢁ
/
F
product was crystallised from boiling ethyl acetateꢀ
/
methanol, yielding 8.9 mmol (72.4%) of 8 as white
crystals.
solved in 400 mL of ethyl acetateꢀwater (1:1, v/v). The
/
ethyl acetate layer was evaporated until dryness and the
product was crystallised from boiling diisopropyl ether,
yielding 183.0 mmol of N-(3-butenyl)-phthalimide as
5
.1.1.7. N-Acetyl-S-(N-benzylthiocarbamoyl)-
L-
1
white, needle-shaped crystals. H-NMR (in CDCl , 100
cysteine (9), mercapturic acid derived from benzyl
isothiocyanate. Benzyl isothiocyanate (14.9 mmol) was
gradually added to a solution of NAC (13.6 mmol) and
sodium bicarbonate (13.6 mmol) in 33 mL of aqueous
3
MHz, d in ppm relative to TMS): 7.78 (m, 4H), 5.82 (m,
1H), 5.07 (dd, 2H), 3.78 (t, 2H), 2.46 (m, 2H). Hydrazine
monohydrate (233 mmol) was added to a solution of
179.0 mmol of N-(3-butenyl)-phthalimide in 200 mL of
8
2% (v/v) ethanol. TLC: NAC R ꢁ
/
0.42, 9 R ꢁ0.55.
/
F
F

736
M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ737
/
ethanol under nitrogen. After 2 h of reflux at 75 8C, HCl
was added and the mixture was further heated for 1 h at
Crystallisation from diisopropyl ether yielded 9.9 mmol
of N-(4-methylthiobutyl)-phthalimide as white, needle-
1
shaped crystals. H-NMR (in CDCl , 300 MHz, d in
1
00 8C. The mixture was left to cool overnight, filtered
3
and the residue was washed with water. The filtrate
containing 3-butenylamine was extracted twice with an
equal volume of diethyl ether, distillated, and dissolved
in 100 mL of chloroform. Slowly 1.1 molar equivalent of
thiophosgene was added to the mixture with stirring,
followed by 3.0 molar equivalents of sodium hydroxide.
The reaction was monitored by TLC with chloroform as
ppm relative to TMS): 7.83 (m, 2H), 7.70 (m, 2H), 3.71
(t, 2H), 2.54 (t, 2H), 2.09 (s, 3H), 1.80 (m, 2H), 1.66 (m,
2H). Hydrazinolysis yielded 4-methylthiobutylamine
which was distilled at reduced pressure, b.p. 72.9 8C
1
(12.0 mmHg), yielding 21.1 mmol. H-NMR (in CDCl ,
3
100 MHz, d in ppm): 2.72 (t, 2H), 2.52 (t, 2H), 2.11 (s,
3H), 1.59 (m, 4H), 1.16 (s, 2H). After reaction with
thiophosgene, 4-methylthiobutyl isothiocyanate was
obtained by distillation at 14.5 mmHg, b.p. 135 8C
eluent, a spot was formed at R ꢁ0.60. The R of butyl
/
F
F
isothiocyanate was 0.61. The mixture was left overnight
at room temperature. The chloroform and thiophosgene
were evaporated and 3-butenyl isothiocyanate was
obtained by distillation, b.p. 60 8C 12 mmHg (60 8C 12
(136 8C at 12 mmHg [34]; 130ꢀ140 8C at 9 mmHg [33]),
/
1
yielding 12.5 mmol of a yellow oil. H-NMR (in CDCl ,
3
100 MHz, d in ppm): 3.56 (t, 2H), 2.54 (t, 2H), 2.11 (s,
3H), 1.78 (m, 4H). A singlet peak at 2.58 ppm belonging
to a methyl adjacent to a sulfinyl group and a singlet at
2.91 belonging to a methyl adjacent to a sulfonyl group
were also observed. 4-Methylthiobutyl isothiocyanate
(10.5 mmol) was dissolved in 10 mL of ethanol and
gradually added to a solution of NAC (9.5 mmol) and
sodium bicarbonate (10.0 mmol) in 20 mL of water.
mmHg [28]; 77.5 8C 28 mmHg [29]), yielding 28.9 mmol.
1
H-NMR (in CDCl , 100 MHz): 5.80 (m, 1H), 5.18 (dd,
3
2
(
H), 3.57 (t, 2H), 2.45 (m, 2H). 3-Butenyl isothiocyanate
28.2 mmol) was dissolved in 20 mL of ethanol and
gradually added to a solution of NAC (27.8 mmol) and
sodium bicarbonate (29.2 mmol) in 20 mL of water.
TLC: NAC R ꢁ
/
0.49, 6 R ꢁ0.59. All NAC had
/
F
F
reacted within 1 h after 3-butenyl isothiocyanate was
added. Ethanol was evaporated and the excess of 3-
butenyl isothiocyanate was extracted with heptane. The
mixture was acidified with HCl, the mercapturic acid
was extracted with ethyl acetate and ethyl acetate was
evaporated yielding 22.9 mmol (10.9%) of 6 as a white
sticky product.
TLC: NAC R ꢁ
and extraction, 7.1 mmol (13.0%) of 11 was yielded as a
yellow sticky product.
/
0.45, 11 R ꢁ0.59. After acidification
/
F
F
5.1.2.4. N-Acetyl-S-(N-4-
methylsulfinylbutylthiocarbamoyl)-
L
-cysteine
(12),
mercapturic acid derived from 4-methylsulfinylbutyl iso-
thiocyanate. To a solution of 4-methylthiobutyl isothio-
cyanate (10.3 mmol, prepared as described above) in 15
mL of DCM, a solution of m-chloroperbenzoic acid
5
.1.2.2. N-Acetyl-S-(N-4-pentenylthiocarbamoyl)-L-
cysteine (7), mercapturic acid derived from 4-pentenyl
isothiocyanate. 5-Bromo-1-pentene was used. N-4-pen-
1
tenyl-phthalimide was dissolved in boiling ethanol. H-
(MCPBA, 12.6 mmol) in 15 mL of DCM was gradually
NMR (in CDCl , 100 MHz, d in ppm relative to TMS):
3
added (Scheme 1). The reaction was monitored by TLC
with ethyl acetate as eluent (4-methylthiobutyl isothio-
7
2
2
5
1
.78 (m, 4H), 5.80 (m, 1H), 4.98 (dd, 2H), 3.70 (t, 2H),
.00 (m, 4H). The yield of 4-pentenyl isothiocyanate was
cyanate R ꢁ
/
0.64, 4-methylsulfinylbutyl isothiocyanate
0.06). All isothiocyanate was oxidised after 30 min.
The DCM layer was separated and evaporated, yielding
0.4 mmol (100%) of 4-methylsulfinylbutyl isothiocya-
1
4.4 mmol. H-NMR (in CDCl , 100 MHz, d in ppm):
.78 (m, 1H), 5.08 (dd, 2H), 3.53 (t, 2H), 2.21 (m, 2H),
F
3
R ꢁ
/
F
.80 (m, 2H). TLC: NAC R ꢁ
/
0.48, 7 R ꢁ0.64. The
/
F
F
1
yield of 7 was 14.6 mmol (12.0%) as an brownish-orange
sticky product.
1
nate as a yellow oil. H-NMR (in CDCl , 100 MHz, d in
3
ppm relative to TMS): 1.92 (m, 4H), 2.61 (s, 3H), 2.74
(
m, 2H), 3.61 (t, 2H). 4-Methylsulfinylbutyl isothiocya-
nate reacted completely after overnight stirring with
NAC. TLC: NAC R ꢁ0.36, 12 R ꢁ0.09. The yield of
5.1.2.3. N-Acetyl-S-(N-4-
methylthiobutylthiocarbamoyl)-
L-cysteine
(11),
/
/
F
F
mercapturic acid derived from 4-methylthiobutyl isothio-
cyanate. 1,4-Dibromobutane was used (Scheme 1). N-(4-
bromobutyl)-phthalimide (103.0 mmol) was yielded as
mercapturic acid 12 was 6.3 mmol (69.5%) as white
crystals.
1
white, needle-shaped crystals. H-NMR (in CDCl , 100
3
MHz, d in ppm): 7.79 (m, 4H), 3.73 (m, 2H), 3.45 (m,
2
H), 1.88 (m, 4H). N-(4-bromobutyl)-phthalimide (14.1
mmol) was slowly added to a solution of sodium
methylmercaptide (14.8 mmol) in 10 mL of ice-cooled
DMF. The reaction was complete within a few hours.
The mixture was pored into 100 mL of ice cold water
while stirring, resulting in crystallisation of the product.
Acknowledgements
We thank Mr. Henk Regeling and Dr. G e´ rard H.L.
Nefkens (University of Nijmegen) for coaching the
laboratory work.

M. Vermeulen et al. / European Journal of Medicinal Chemistry 38 (2003) 729ꢀ
/737
737
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