The synthesis of [phenyl-2H5]gluconasturtiin and its metabolites for metabolic studies

Article abstract of DOI:10.1002/jlcr.1004

The synthesis of a deuterium labelled glucosinolate, [²H ₅]gluconasturtiin, is described starting from [²H ₅]bromobenzene. The potential metabolites of the glucosinolate, namely the [phenyl-²H₅

Full text of DOI:10.1002/jlcr.1004

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 897–907.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1004
Research Article
The synthesis of [phenyl-2H5]gluconasturtiin and its
metabolites for metabolic studies
John J. Morrison and Nigel P. Botting*
School of Chemistry, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
Summary
The synthesis of a deuterium labelled glucosinolate, [2H5]gluconasturtiin, is described
starting from [2H5]bromobenzene. The potential metabolites of the glucosinolate,
namely the [phenyl-2H5]phenethyl isothiocyanate, nitrile, thiocyanate, amine and the
mercapturic acid conjugate of phenethyl isothiocyanate are also described. This series
of compounds has been prepared for use in feeding studies to examine the mammalian
metabolism of gluconasturtiin and search for new biomarkers of exposure. Copyright
# 2005 John Wiley & Sons, Ltd.
Key Words: glucosinolates; isothiocyanates; gluconasturtiin; cancer prevention
Introduction
There is convincing epidemiological evidence that consumption of broccoli
and other cruciferous vegetables is associated with a decreased risk of cancer
and this association is strongest for cancers of the gastrointestinal and
respiratory tracts.1 The anti-cancer effects have been attributed to the presence
of glucosinolates in these vegetables. Glucosinolates (1) are a class of naturally
occurring thioglucosides which are metabolized by the plant enzyme
myrosinase during food preparation, cooking and chewing (Scheme 1).2,3
The major product of this metabolism is the corresponding isothiocyanate (2),
formed via a Lossen-type rearrangement of the unstable thiohydroximate-O-
sulphonate aglycone initially produced. In plants glucosinolates and myr-
osinase are compartmentalized and only interact following tissue damage. In
mammals there also appears to be myrosinase activity in intestinal bacteria
which may contribute to glucosinolate degradation in vivo.4
*Correspondence to: Nigel P. Botting, School of Chemistry, University of St. Andrews, St. Andrews, Fife
KY16 9ST, UK. E-mail: npb@st-andrews.ac.uk
Contact/grant sponsor: Food Standards Agency (FSA); contract/grant number: T01027
Copyright # 2005 John Wiley & Sons, Ltd.
Received 8 July 2005
Revised 19 July 2005
Accepted 21 July 2005

898
J. J. MORRISON AND N. P. BOTTING
-
-
HO
HO
HO
HO
OSO3
N
OSO3
Lossen
O
O
-S
Rearrangment
S
Myrosinase HO
HO
N
OH
R
R
N C
4
S
N
OH
1
OH
R
R
2
3
?
ESP
Fe2+
S
C
R C
N
6
5
Scheme 1. General metabolism for glucosinolates
The anti-cancer activity appears to reside with the isothiocyanate5 although
the mechanism is still controversial and could be a result of inhibition of the
Phase 1 detoxification enzymes,6 upregulation of the Phase 2 detoxification
enzymes7 or even induction of apoptosis of tumour cells.8 There are also many
unanswered questions regarding the metabolism and bioavailability of
glucosinolates and isothiocyanates. It is known that the aglycone can also
break down to give nitriles (5) and thiocyanates (6), depending on the
conditions and presence of other factors such as ESP (epithiospecifier protein)
and ferrous ions.9 The biological importance and metabolic fate of some of
these metabolites is as yet unknown.
A project is being developed to employ a stable isotopically labelled
derivative of gluconasturtiin, which has a phenethyl side chain and is one of
the most common glucosinolates, in feeding studies with rats. These studies are
aimed at further elucidating glucosinolate metabolism and identifying possible
new biomarkers of glucosinolate exposure.
Results and discussion
In previous studies in our laboratory a deuterated desulfogluconasturtiin was
prepared as an internal standard for glucosinolate analysis.10 This was
synthesized from [2H5]bromobenzene, commonly used as a solvent for NMR
spectroscopy and fairly inexpensive. It was thus decided to repeat this
synthesis but carry it through to the required [phenyl-2H5]gluconasturtiin (7).
Similarly labelled versions of potential gluconasturtiin metabolites were also
needed as internal standards, producing a family of synthetic targets. These
were the isothiocyanate (8), amine (9), thiocyanate (10), nitrile (11) and the
mercapturic acid conjugate of the isothiocyanate (12), a known human
metabolite (Figure 1).
[2H5]Bromobenzene (13) was thus converted to the Grignard reagent which
was reacted with acrolein diethyl acetal (Scheme 2). The conjugate addition
took place to give a mixture of E and Z isomers of the enol ether (14), in
quantitative yield over the two steps.10 There was no need to separate the two
isomers and the mixture was directly hydrolysed in aqueous acid to give the
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 897–907

SYNTHESIS OF [PHENYL-2H5]GLUCONASTURTIIN
899
-
HO
HO
HO
OSO3
N
D
O
N C S
NH2
8; R =
9; R =
S
D
D
R
OH
S C N
C N
10; R =
11; R =
D
7
D
D
D
D
D
D
D
D
D
S
D
D
H
S
N
H
CO2H
NHAc
12
Figure 1. Synthetic targets
OH
N
D
D
D
D
D
D
Br
D
D
D
D
a
b, c, d
OEt
Cl
e
D
D
D
D
D
13
AcO
D
S
7
15
14
OH
-
HO
HO
HO
OSO3
O
O
AcO
AcO
S
D
f, g
N
D
N
D
OAc
D
OH
D
16
D
D
D
D
Scheme 2. Synthesis of [phenyl-2H5]Gluconasturtiin. Reagents and conditions:
(a) Mg, THF, then 5% CuBr, THF then acrolein diethyl acetal (100%); (b)
Acetone/H2O (1:4), HCl (93%); (c) NH2OH.HCl, NaOAc, EtOH, reflux
(88%); (d) N-Chlorosuccinimide, pyridine, CHCl3 (82%); (e) Et3N, THF,
tetraacetyl thioglucose (74%); (f) ClSO3H, pyridine, DCM (20%); (g) KOMe,
MeOH (99%)
aldehyde, converted to the oxime using hydroxylamine hydrochloride and then
chlorinated using N-chlorosuccinimide. The oximyl chloride (15) was used
without purification in the coupling reaction employing modified literature
procedures,11 whereby under basic conditions elimination gives the nitrile
oxide which is trapped out by the thiolate from tetraacetyl thioglucose to give
the protected desulfoglucosinolate (16) in 74% yield. The Z-isomer is obtained
exclusively due to stereoelectronic effects.12 For the synthesis of the complete
glucosinolate sulfonation was carried out using conditions established in our
laboratory employing chlorosulfonic acid in pyridine.13 The yield at this stage
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 897–907

900
J. J. MORRISON AND N. P. BOTTING
D
D
D
D
D
D
Br
D
D
D
OH
D
D
OMs
D
D
NH2
a
b
f
c, d
D
D
D
D
D
17
D
18
D
9
13
D
D
D
D
D
D
D
N C S
e
S
H
D
S
N
H
D
D
CO2H
NHAc
12
8
Scheme 3. Synthesis of gluconasturtiin metabolites. Reagents and conditions: (a)
Mg, THF, then ethylene oxide (99%); (b) MsCl, Et3N, DCM (99%); (c) NaN3,
DMF (95%); (d) LiAlH4, Et2O (81%); (e) CSCl2, Na2CO3, DCM, H2O (82%);
(f) N-Acetylcysteine, aqueous buffer (pH 6.6) (82%)
(20%) is the lowest on the sequence, although it reflects difficulties in
purification rather than incomplete reaction. Final deprotection was carried
out using potassium methoxide in methanol to give the [phenyl-2H5]gluconas-
turtiin as its potassium salt. The 1H and 13C NMR data were identical to those
of the unlabelled compounds except for the lack of any signal for the aromatic
protons and electrospray mass spectrometry confirmed the molecular mass.
In order to obtain the same labelling pattern for each metabolite
[2H5]bromobenzene (13) was retained as the starting material. It was thought
that the nitrile (11) would be accessible via a similar conjugate addition
reaction to that used to make the aldehyde intermediate but using acrylonitrile
instead of the acrolein diethyl acetal. However this turned out to be a very
poor reaction and was abandoned. An alternative, more flexible approach
involved reaction of the Grignard reagent with ethylene oxide to give the
alcohol (17) (Scheme 3). This reaction could be carried out on a large scale to
provide material for further reactions as 17 can then be modified to prepare all
the required metabolites. Mesylation with mesyl chloride and triethylamine
gave 18 in 99% yield. Nucleophilic substitution using azide ion as the
nucleophile followed by reduction with LiAlH4 then gave the required amine
(8), in 81% yield. Conversion of the amine to the [phenyl-2H5]phenethyl
isothiocyanate (9) was achieved in high yield using thiophosgene in a two-
phase system of dichloromethane (DCM) and aqueous sodium carbonate
solution. Synthesis of the deuterium labelled mercapturic acid conjugate (12)
was carried out by reaction of the isothiocyanate and N-acetylcysteine in an
aqueous buffer at pH 6.6 and the product purified by recrystallization.14
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 897–907

SYNTHESIS OF [PHENYL-2H5]GLUCONASTURTIIN
901
D
D
D
D
OH
D
D
CN
a, b
D
D
D
D
11
17
Scheme 4. Synthesis of nitrile. Reagents and conditions: (a) Ph3P, CBr4, DCM
(36%); (b) KCN, 16-crown-6, MeCN (73%)
D
D
KSCN, MeOH,
reflux
D
D
OMs
D
D
SCN
D
D
70%
D
D
18
10
Scheme 5. Synthesis of phenethyl thiocyanate
The nitrile (11) was synthesized from the same alcohol (17) in two steps via
conversion to the bromide and nucleophilic substitution using potassium
cyanide in acetonitrile (MeCN), with 18-crown–6 to assist solubilization
(Scheme 4).
The final metabolite required was the thiocyanate (10). This molecule can be
prepared using thiocyanate anion as a nucleophile, which is an ambident
nucleophile and can react either through sulphur to give the thiocyanate or
through nitrogen to give the isothiocyanate. The product distribution has been
shown to depend on a number of factors including the solvent employed.
It was found that reaction of potassium thiocyanate with the mesylate
(18) in refluxing acetonitrile gave a 70% yield of the desired thiocyanate (10)
after purification (Scheme 5). Under these conditions only a trace of the
alternative isothiocyanate was produced and this was easily removed on
purification.
All the unlabelled compounds have been previously reported in the
literature and the spectral data obtained herein were identical apart from
the absence of a signal for the aromatic protons in the 1H NMR spectrum and
an increase of 5 mass units in the observed molecular ion. The compounds are
now being employed in feeding studies using rats to examine the metabolism
and search for novel biomarkers. The experiments involve comparing the
products derived from rats fed with unlabelled compounds and rats fed with
the equivalent deuterium labelled compounds. LC–MS provides a rapid search
Copyright # 2005 John Wiley & Sons, Ltd.
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J. J. MORRISON AND N. P. BOTTING
method to look for compounds with a 5 unit mass difference between the two
sets of experiments which will then be further investigated. Feeding studies are
being carried out using the glucosinolate, the glucosinolate plus added
myrosinase, the isothiocyanate and the nitrile. The other compounds are being
used as reference standards.
Experimental
General
Melting points were determined in open capillary tubes and are uncorrected.
1H and 13C NMR spectra were recorded at 300 and 75.46 MHz, respectively,
using Bruker Avance 500 and Varian Gemini 2000 spectrometers. The
chloroform peaks (7.27 ppm for 1H, 77.00 ppm for 13C) were used as
references. The EI and CI mass spectra were obtained on a VG Autospec
mass spectrometer, and the ESI and APCI mass spectra on a Micromass LCT
mass spectrometer. Diethyl ether and tetrahydrofuran were distilled over
sodium and dichloromethane over calcium hydride. DMSO was freshly
distilled from calcium hydride.
2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl-[phenyl-2H5]gluconasturtiin (16)
Chlorosulfonic acid (4.2 ml, 62.6 mmol) was diluted with dry DCM (50 ml) and
this mixture was slowly added under argon to a solution of pyridine (62.0 ml,
626 mmol) in DCM (50 ml) and allowed to stir for 40 min. 2,3,4,
6-tetra-O-acetyl-b-d-glucopyranosyl-1-[phenyl-2H5]phenethyl thiohydroximate
(15) (2.80 g, 5.4 mmol) was dissolved in DCM (100 ml) and added dropwise
and the mixture was stirred for 24 h. Potassium hydrogen carbonate (6.26 g,
62.6 mmol in 100 ml water) was added and the mixture stirred for 30 min prior
to evaporation of all the liquids below 408C and addition of small aliquots of
toluene to maintain a pyridine:toluene azeotrope. This provided a yellow gum
which was purified by column chromatography on silica using 1:5
methanol:ethyl acetate as the eluant to give a white amorphous solid
(0.680 g, 20%); Rf ¼ 0:61 (1:4 methanol-ethyl acetate); dH (300 MHz; CD3OD)
1.87 (3H, s, OC(O)CH3), 2.00–2.10 (9H, 3 ꢀ s, OC(O)CH3), 2.92–3.02 (2H, m,
H-8), 3.02–3.18 (2H, m, H-9), 3.90–4.00 (1H, m, H-5), 4.10–4.20 (2H, m, H-6),
4.95–5.15 (2H, m, H-2, 4), 5.25 (2H, t, J ¼ 10, H-1, 3); dC (75.4 MHz;
CD3OD) 20.7 (4 ꢀ OC(O)CH3), 34.4 (CH2), 35.6 (CH2), 63.6 (C-6), 69.7 (C-4),
71.5 (C-2), 75.1 (C-3), 77.0 (C-5), 80.9 (C-1), 140.1 (C-10), 158.9 (C¼¼N),
172.0,171.3, 171.6 and 172.3 (4 ꢀ OC(O)CH3); Mpt 168–1708C decomp. (Lit15
198–2008C decomp [from recryst in EtOH]); M.S. (ꢁve ion electrospray)
C23H23D5O13NS2 required 595.1334 found 595.1326.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 897–907

SYNTHESIS OF [PHENYL-2H5]GLUCONASTURTIIN
903
[phenyl-2H5]Gluconasturtiin (7)
2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl-[phenyl-2H5]gluconasturtiin
(16)
(0.73 g, 1.2 mmol) was dissolved in dry methanol (10 ml) and potassium metal
(about 100 mg) was carefully added under a flow of nitrogen. The mixture was
stirred at room temperature and after 4 h TLC showed all the starting material
had been consumed. Amberlite IR 120 resin (1 g) was added and the mixture
stirred for 1 h then the mixture was concentrated by evaporation and purified
by column chromatography on silica using 1:4 methanol:ethyl acetate as the
eluant to give a pale yellow amorphous solid (0.53 g, 99%); Rf ¼ 0:23; dH
(300 MHz; D2O) 2.95–3.18 (4H, m, CH2CH2), 3.41–3.58 (4H, m, H-2,3,4,5),
3.65–3.75 (1H, m, H-6a), 3.75–3.90 (1H, m, H-6b), 4.85 (1H, d, J ¼ 12:0, H-1);
M.S. (ꢁve ion electrospray) C15H15D5O9NS2 required 427.0889 found
427.0903.
[phenyl-2H5]Phenethyl alcohol (17)
[2H5]Bromobenzene (13) (20 g, 123 mmol in 50 ml dry THF) was added
dropwise under N2 onto magnesium turnings (3.36 g, 140 mmol in 75 ml dry
THF). The reaction was activated with a few iodine crystals. The addition was
regulated to maintain reflux and when addition was complete the mixture was
heated under reflux for a further 4 h then cooled over ice. Ethylene oxide (9.0 g,
200 mmol) was decanted into ice cold dry THF (100 ml) and slowly added to
the cooled Grignard reagent. A slight exotherm was observed on this addition.
This procedure was done with due care as ethylene oxide boils at 118C. The
mixture was allowed to warm to room temperature then stirred overnight
prior to cooling over ice then hydrolysed by careful addition of HCl (100 ml
10%). The mixture was extracted with EtOAc (2 ꢀ 100 ml), then washed with
brine, dried over MgSO4 and evaporated to give a mixture found to contain
the desired product and 2-bromoethanol which was removed on the
evaporator at 858C to give [phenyl-2H5]phenethyl alcohol (15.6 g, 99%) as a
colourless oil. dH (300 MHz; CDCl3) 1.62(1H, s, OH) 2.90 (2H, t, J ¼ 6:8,
PhCH2CH2), 3.88 (2H, t, J ¼ 6:8, CH2CH2OH); dC (75.4 MHz; CDCl3) 38.7
(PhCH2CH2OH), 63.3 (CH2CH2OH), 138.65 (Ar-C1); M.S. (C.I.) C8H5D5O
required 127.1045 found 127.1048.
[phenyl-2H5]Phenethyl methanesulfonate (18)
[phenyl-2H5]Phenethyl alcohol (17) (9.53 g, 75 mmol) and methanesulfonyl
chloride (7.74 ml, 100 mmol) and dry triethylamine (14 ml, 100 mmol) were
stirred in DCM (100 ml) under nitrogen at 08C for 4 h. The products were
washed swiftly with HCl (150 ml, 0.5 M), then saturated NaHCO3 followed by
brine. The organic layer was dried over anhydrous Na2SO4 and evaporated to
give the title compound as a colourless oil (15.4 g, 100%); dH (300 MHz;
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CDCl3) 2.85 (3H, s, CH3), 3.08 (2H, t, J ¼ 7:6, PhCH2CH2), 4.44 (2H, t,
J ¼ 7:6, CH2CH2O); dC (75.4 MHz; CDCl3) 35.5 (PhCH2CH2CN), 37.2
(CH3), 70.3 (CH2CH2O), 136.1 (Ar-C1); M.S. (+ve ion electrospray)
C9H7D5O3NaS required 228.0719 found 228.0729.
[phenyl-2H5]Phenethyl azide
[phenyl-2H5]Phenethyl methanesulfonate (18) (15.46 g, 75 mmol) and sodium
azide (4.47 g, 106 mmol) were heated to 708C overnight in DMF (300 ml) then
cooled by adding ice. The mixture was diluted with water (600 ml) and
extracted with diethyl ether (3 ꢀ 200 ml). The extracts were washed with HCl
(250 ml, 0.1 M) and brine and then dried over anhydrous Na2SO4 and
evaporated to give a pale yellow oil (10.9 g, 95%); dH (300 MHz; CDCl3) 2.91
(2H, t, J ¼ 7:2, PhCH2CH2), 3.52 (2H, t, J ¼ 7:2, CH2CH2N3); dC (75.4 MHz;
CDCl3) 35.3 (PhCH2CH2 N3), 52.5 (CH2CH2 N3), 137.8 (Ar-C1). The azide
was used in the next step without further purification.
[phenyl-2H5]Phenethylamine (9)
Lithium aluminium hydride (5.0 g, 132 mmol) was added portionwise to dry
diethyl ether (200 ml) and stirred under nitrogen until all effervescence had
ceased. [phenyl-2H5]Phenethyl azide (5.0 g, 33 mmol) was dissolved in dry
diethyl ether (80 ml) and added dropwise with care to ensure the rate of gas
evolution did not increase to the point where excessive frothing would result.
Once all the azide had been added the mixture was heated under reflux for 3 h
then stirred overnight. The solvent was evaporated off and replaced with
DCM (50 ml) and the organic layer filtered through celite washing with
copious DCM, then washed with water, then brine, prior to evaporation of the
DCM. The residue was purified by column chromatography on silica using 2:1
DCM:MeOH as the eluant. This gave the product as a colourless oil (3.78 g,
91%); dH (300 MHz; CDCl3) 1.32 (2H, s, NH2), 2.77 (2H, t, J ¼ 6:8,
PhCH2CH2), 2.99 (2H, t, J ¼ 6:8, CH2CH2NH2); dC (75.4 MHz; CDCl3) 39.9
(PhCH2CH2NH2), 43.5 (CH2CH2NH2), 139.5 (Ar-C1); M.S. (C.I.)[M++H]
C8H7D5 required 127.1284 found 127.1286.
[phenyl-2H5]Phenethyl isothiocyanate (8)
Thiophosgene (34.5 g, 300 mmol) and sodium carbonate (6.89 g, 65 mmol)
were stirred in water (100 ml) at room temperature until dissolved.
[phenyl-2H5]Phenethylamine (9) was dissolved in 90 ml DCM and the mixture
stirred vigorously. CO2 evolution is quite rapid at first and slowly decreases
over 5 h so the mixture was allowed to stir overnight. More DCM was added
before the mixture was washed with HCl (100 ml 1 M) and brine prior to the
organic layer being dried over anhydrous Na2SO4 and evaporated. The residue
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF [PHENYL-2H5]GLUCONASTURTIIN
905
was purified by column chromatography on silica using 1:1 petroleum
ether:diethyl ether as eluant to give the product as a light yellow oil (4.12 g,
82%); dH (300 MHz; CDCl3) 3.00 (2H, t, J ¼ 7:2, PhCH2CH2), 3.76 (2H, t,
J ¼ 7:2, CH2CH2NCS); dC (75.4 MHz; CDCl3) 36.8 (PhCH2CH2NCS), 46.8
(CH2CH2NCS), 128.2 (NCS), 137.2 (Ar-C1); (C.I.)[M++H] C9H5D5NS
required 169.0844 found 169.0847.
Mercapturic acid conjugate of [phenyl-2H5]phenethyl isothiocyanate (12)14
[phenyl-2H5]Phenethyl isothiocyanate (8) (0.672 g, 4 mmol) and N-acetyl
cystine (1.31 g, 8 mmol) were stirred in aqueous phosphate buffer at pH 6.6.
The oily isothiocyanate eventually dissolved and the product was precipitated
out by adding 2 M HCl dropwise. The product was extracted into DCM and
washed with HCl (100 ml, 0.01 M) then dried over anhydrous Na2SO4 and
NaHCO3. The DCM was evaporated at reduced pressure and the residue
freeze dried from water to give light brown flakes (1.09 g, 82%); dH (300 MHz;
CDCl3) 1.92 (3H, s, COCH3), 2.90 (2H, t, J ¼ 7:2, PhCH2CH2), 3.58–3.72
(2H, m, SCH2CH) 3.78–3.92 (2H, m, PhCH2CH2NCS), 4.58–4.70 (1H, m,
SCH2CH), 7.42 and 8.25 (2 ꢀ 1 H, br,s 2 ꢀ NH) 9.95 (1H, br,s, COOH); dC
(75.4 MHz; CDCl3) 22.8 (COCH3), 33.9 (PhCH2CH2NCS), 34.6 (SCH2CH),
48.8 (CH2CH2NCS), 53.9 (SCH2CH), 137.8 (Ar-C1), 172.0 and 172.7 (2C,
COCH3 and COOH), 197.4 (NCSS); M.S. (ꢁve ion electrospray)
C14H13D5O3N2S2 required 331.1073 found 331.1076.
[phenyl-2H5]Phenethyl bromide
[phenyl-2H5]Phenethyl alcohol (17) (2.5 g, 19.7 mmol) triphenylphosphine
(10.5 g, 40 mmol) and carbon tetrabromide (13.26 g, 40 mmol) were dissolved
in dry DCM (100 ml) over ice then allowed to warm to room temperature and
stirred overnight. The original yellow solution gave a thick milky white
precipitate that did not dissolve on addition of saturated NaHCO3. The
mixture was filtered through celite and the celite washed with copious EtOAc.
The solvents were evaporated and the residue purified by column chromato-
graphy on silica using 1:1 petroleum ether:diethyl ether as eluant to remove the
phosphines. The products were further purified by column chromatography on
silica using 9:1 petroleum ether:DCM as eluant to give the product as a
colourless oil (1.36 g, 36%); dH (300 MHz; CDCl3) 3.16 (2H, t, J ¼ 7:5,
PhCH2CH2), 3.56 (2H, t, J ¼ 7:5, CH2CH2Br). The bromide was used in the
next step without further purification.
3-[phenyl-2H5]Phenylpropionitrile (11)
[phenyl-2H5]Phenethyl bromide (0.89 g, 4.7 mmol), 18-crown-6 (1.88 g, 5.2 mmol)
and potassium cyanide (0.34 g, 5.2 mmol) were combined in acetonitrile (20 ml)
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J. J. MORRISON AND N. P. BOTTING
and heated under reflux for 4 h. The solvent was evaporated off and replaced
with DCM (50 ml) and the organic layer washed with water then brine prior to
evaporation of the DCM. The residues were purified by bulb to bulb
distillation at 2408C at 6 mmHg to give the product as a colourless oil (0.46 g,
73%); dH (300 MHz; CDCl3) 2.53 (2H, t, J ¼ 7:5, CH2CH2CN), 2.88 (2H, t,
J ¼ 7:5, PhCH2CH2); dC (75.4 MHz; CDCl3) 19.3 (CH2CH2CN), 31.4
(PhCH2CH2CN), 119.1 (CH2CH2CN), 137.8 (Ar-C1); M.S. (C.I.)[M++H]
C9H4D5N required 136.1049 found 136.1053.
[phenyl-2H5]Phenethyl thiocyanate
[phenyl-2H5]Phenethyl methanesulfonate (18) (2.0 g, 10 mmol) and potassium
thiocyanate (1.1 g, 11 mmol) were heated to reflux in acetonitrile (50 ml)
overnight. The acetonitrile was evaporated and the residue dissolved in warm
diethyl ether and filtered. The diethyl ether was evaporated off and the residues
purified by column chromatography on silica using 1:1 petroleum ether:diethyl
ether as eluant. This gave the product as a pale yellow oil (1.18 g, 70%); dH
(300 MHz; CDCl3) 3.10–3.21 (4H, m, PhCH2CH2); dC (75.4 MHz; CDCl3)
35.1 and 35.9 (PhCH2CH2SCN and PhCH2CH2SCN), 112.0 (SCN), 138.0
(Ar-C1); M.S. (C.I.)[M++H] C9H5D5NS required 169.0844 found 169.0848.
Acknowledgements
This work was funded by the Food Standards Agency (FSA) under contract
T01027.
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