Synthesis of [13C6]-labelled phenethylamine derivatives for drug quantification in biological samples

Article abstract of DOI:10.1002/jlcr.3193

The availability of high-quality ¹³C-labelled internal standards will improve accurate quantification of narcotics and drugs in biological samples. Thus, the synthesis of 10 [¹³C₆]-labelled phenethylamine derivatives, namely amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 4-methoxyamphetamine, 4-methoxymethamphetamine, 3,5-dimethoxyphenethylamine 4-bromo-2,5- dimethoxyphenethylamine and 2,5-dimethoxy-4-iodophenethylamine, have been undertaken. [¹³C₆]-Phenol proved to be an excellent starting material for making ¹³C-labelled narcotic substances in the phenethylamine class, and a developed Stille-type coupling enabled an efficient synthesis of the 3,4-methylenedioxy and 4-methoxy derivatives. The pros and cons of alternative routes and transformations are also discussed. The [ ¹³C₆]-labelled compounds are intended for use as internal standards in forensic analysis, health sciences and metabolomics studies by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Copyright

Full text of DOI:10.1002/jlcr.3193

Research Article
Received 21 November 2013,
Revised 23 January 2014,
Accepted 29 January 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3193
13
Synthesis of [ C6]-labelled phenethylamine
derivatives for drug quantification in
biological samples
Morten Karlsen,a,b HuiLing Liu,b Thomas Berg,c Jon Eigill Johansen,b
a
*
and Bård Helge Hoff
The availability of high-quality 13C-labelled internal standards will improve accurate quantification of narcotics and drugs in
biological samples. Thus, the synthesis of 10 [13C6]-labelled phenethylamine derivatives, namely amphetamine,
methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-
ethylamphetamine, 4-methoxyamphetamine, 4-methoxymethamphetamine, 3,5-dimethoxyphenethylamine 4-bromo-2,5-
dimethoxyphenethylamine and 2,5-dimethoxy-4-iodophenethylamine, have been undertaken. [13C6]-Phenol proved to be
an excellent starting material for making 13C-labelled narcotic substances in the phenethylamine class, and a developed
Stille-type coupling enabled an efficient synthesis of the 3,4-methylenedioxy and 4-methoxy derivatives. The pros and cons
of alternative routes and transformations are also discussed. The [13C6]-labelled compounds are intended for use as internal
standards in forensic analysis, health sciences and metabolomics studies by gas chromatography-mass spectrometry and
liquid chromatography-tandem mass spectrometry.
Keywords: [13C6]-labelled standards; amphetamine; methamphetamine; 4-methoxymethamphetamine; 3,4-methylenedioxymethamphetamine;
Stille coupling
methamphetamine (1b) is often synthesized by reduction of
Introduction
ephedrine or by reductive amination of phenyl-2-propanone.5
Amphetamine (1a) and methamphetamine (1b) are some of the
(E)-1-Methoxy-4-(1-propenyl)benzene (anethole) has been
most abused drugs in Scandinavia and occur frequently in blood
reported as the main precursor of the para methoxy derivatives
samples from car drivers suspected for driving under influence.
PMA (2a) and PMMA (2b), which is widely available as the main
component (95%) in anise oil.6 Illegal production of MDMA (3b)
The prevalence of amphetamines in a roadside survey made in
Norway (2013) of 12000 volunteer candidates shoved 0.3% positive
relies among others on 3,4-methylenedioxyphenyl-2-propanone
samples.1 Also, other designer drugs from the class of substituted
(12) and 5-(2-propenyl)-1,3-benzodioxole (safrole) as starting
materials.4 For many of the synthetic phenethylamines such as
phenethylamines like 3,4-methylenedioxymethamphetamine
(MDMA, 3b), 3,4-methylenedioxyamphetamine (MDA, 3a) and 3,4-
2C-B (5a), 2C-I (6a) and PMA (2a), synthetic procedures have been
methylenedioxy-N-ethylamphetamine (MDEA, 3c), popularized
listed with biological activity in the book PIHKAL: A Chemical Love
Story,7 and these methods are also used in clandestine laboratories.
through the rave culture as ecstasy, pose a serious problem
worldwide.2 Probably because of restriction on supply of piperonal
The method of choice for analysis of drugs in biological
and safrole used in the production of the methylenedioxy
samples is liquid chromatography-tandem mass spectrometry
derivatives 3a–c, an increased illegal production and use of the
highly toxic analogues 4-methoxyamphetamine (2a) and
4-methoxymethamphetamine (2b), currently being sold as fake
(LC-MS/MS) due to speed of analysis, sensitivity, selectivity and
ease of sample preparation.8,9 LC-MS/MS instruments frequently
show ion suppression or alteration due to matrix compounds,
other drugs, preservation agents or other unknown substances
ecstasy, is seen. As a result, a series of fatal intoxications has
been observed in the Scandinavian countries.3 4-Bromo-2,5-
dimethoxyphenethylamine (5a) and 2,5-dimethoxy-4-iodo-
aDepartment of Chemistry, Norwegian University of Science and Technology,
Høgskoleringen 5, NO-7491 Trondheim, Norway
phenethylamine (6a) first emerged as legal substitutes of MDMA
(3b) because of their somewhat similar narcotic effects but are
now both listed as illegal drugs in most parts of the world (Figure 1).
Besides the classical methods of synthesis, few ‘modern’
reports on the preparation of these molecules exist. On the other
bChiron AS, Stiklestadveien 1, 7041 Trondheim, Norway
cDivision of Forensic Toxicology and Drug Abuse, Norwegian Institute of Public
hand, impurity profiles from seized materials have been used to Health, PO Box 4404 Nydalen, N-0403 Oslo, Norway
determine the preferred routes of synthesis in clandestine
*Correspondence to: Bård Helge Hoff, Department of Chemistry, Norwegian
University of Science and Technology, Høgskoleringen 5, NO-7491 Trondheim,
Norway.
laboratories.4–6 The Leuckart reaction seems to be the most
common transformation in amphetamine laboratories giving
access to the amine function via ketone precursors, while E-mail: bard.helge.hoff@chem.ntnu.no
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M. Karlsen et al.
Phenol was purchased from Campro Scientific (Berlin, Germany) with
isotopic purity >99%. All solvents and chemicals were used as is without
further purification if otherwise is not stated. Anhydrous solvents were
used as is and stored over activated molecular sieves. The silica gel used
for flash chromatography was Merck silica gel 60 (230–400 mesh). For
chromatography, thin-layer chromatography (TLC) silica gel 60 F254
Merck plates/sheets were employed with visualization under ultraviolet
light at 254 nm. 1H and 13C NMR spectra were recorded from Bruker
Advance DPX instruments (400/100 MHz) (Coventry, United Kingdom).
Chemical shifts (δ) are reported in parts per million relative to
tetramethylsilane. Because of the high intensity of the 13C-labelled
carbons as compared with those unlabelled, some NMR resonances were
not detected. In this study, the non-labelled benzylic carbons
experiencing extensive coupling, and often also appearing in the solvent
region, were difficult to detect. Isotopic purity and accurate mass
determination/high resolution mass spectrometry (HRMS) in positive and
negative modes on the final product was performed on a ‘Synapt G2-S’
quadrupole time-of-flight mass spectrometer (Q-TOF) instrument from
Waters (En Yvelines Cedex, France), with a resolution of 5 ppm. Samples
were ionized by the use of an atmospheric pressure solids analysis probe
(ASAP), and no chromatography separation was used previous to the mass
analysis. HPLC analysis was performed on an Agilent 1200 with atmospheric
pressure chemical ionization (APCI)/electrospray ionization (ESI) multimode
ionization and acetonitrile as the mobile phase in combination with water
buffered at pH 3 with formic acid (Santa Clara, United States). The column
used was XBridge™ C18, 5μm, 4.6× 150mm from Waters or Hilic Plus,
3.5 μm, 4.6× 100 mm from Agilent. GC-MS analysis was performed on
Agilent 6890 with electron impact (EI) ionization, Agilent column HP5MS,
30m, 0.25mm internal diameter, 0.25μm film. GC analyses of the final
products (both free amines and hydrochloric acid salts) were performed
as their trifluoroacetyl (TFA) derivatives.
Figure 1. Structure of the synthesized [13C6]-labelled compounds.
contaminating the biological sample.10 This type of ion
suppression/alteration effect generally limits the ruggedness
and accuracy of LC-MS/MS methods.11 Stable isotope internal
standards (SIL IS) are added to correct for error in the sample
preparation and matrix effects.12 IS are used to quantify the level
of each analyte in the sample. These analyses can be performed
using deuterium-labelled IS. However, disadvantages associated
with deuterium labelling include the risk of deuterium hydrogen
exchange, and that the deuterium-labelled compounds often
elute differently from the native, non-labelled compounds in
liquid chromatography making them at risk to overlap with
matrix compounds.13 In addition, the deuterium-labelled
compounds may have a response factor, which is slightly
different from the response factor of the native, non-labelled
compounds. Changes in the physicochemical properties of
deuterium-labelled compounds as compared with hydrogen
analogues can lead to differences in the MS ionization causing
ion suppression or alteration of the IS giving false results.14 This
is caused by the isotope effect, lowering the zero point energy of
the deuterated IS.15 13C-labelled IS have much smaller
differences in physicochemical properties16 and behave almost
identical to the unlabelled substance giving no difference in
retention times or response factor. Moreover, there is also no risk
of exchange. The 13C-labelled IS are particularly suitable for
minimizing ion suppression effects in LC-MS/MS analysis;
therefore, the quantitative analysis in various biological samples
is particularly accurate and reproducible. However, natural
matter contains approximately 1.1% 13C; thus, there is a risk of
‘overlap’ with the natural 13C in the native compound in MS
detection. Therefore, the number of labelled atoms must
preferably be at least three. Recently, Berg et al.17 demonstrated
the success of substituting the deuterated IS for 13C IS in their
routine analysis of amphetamine (1a) and methamphetamine
(1b) by SIL IS synthesized in our laboratory. Moreover, the use
of 13C as label in analytes for use as IS is widely known and
appreciated also in other fields of analytical chemistry.11,13,18–20
We herein wish to report our effort to make 10 [13C6]-labelled
compounds intended for use as IS in forensic analysis, health
sciences and metabolomics studies. To the best of our
knowledge, these compounds have not been reported
synthesized before with more than three labelled 13C atoms.
Results and discussion
[13C6]-Phenol is readily available from several distributors and
relatively cheap compared with other 13C-labelled compounds,
and was therefore selected as the general starting material for
the synthesis of the phenylethylamine derivatives.
Our first approach to the substituted phenethylamines started by
converting phenol to bromobenzene using hydrobromic acid,
followed by
a
modified Bouveault synthesis with N-
methylformanilide21 giving [13C6]-benzaldehyde (Scheme 1).
However, also [13C6]-benzaldehyde can be bought at a relatively
low price so [13C6]-benzaldehyde was used in our later tests to save
time. [13C6]Benzaldehyde was then condensed in a classic Henry
reaction with nitroethane to give the nitrostyrene 7. [13C6]-
Amphetamine (1a) was obtained by a clean Red-AlTM reduction of
7 in toluene. More by-products and lower yields were experienced
when using lithium aluminium hydride in this transformation.
Further, 1a was formylated quantitatively using ethyl formate
in a closed pressure reactor, and reduction with lithium
aluminium hydride without purification of the formate gave
[13C6]-methamphetamine (1b) in good yield. Purification of 1b
was performed by crystallization of its hydrochloride salt.
In an alternative strategy, electrolytic reduction of the
nitrostyrene 7 with iron in acetic acid gave 1-phenylpropan-2-
one.22 This ketone could successfully be converted to
amphetamine (1a) by reaction with hydroxylamine to the
corresponding ketoxime followed by reduction with sodium
metal in ethanol23 or reductive amination.24 This route, however,
was more time-consuming and also gave lower overall yield.
The 3,4-methylenedioxy derivatives 3a–c were initially targeted
by the same type of chemistry as shown in Scheme 1. However,
difficulties in preparing the target aldehyde in a straightforward
Experimental
Chemicals and analysis
Bulk solvents were purchased either from LabScan (Gliwice, Poland) or
Merck (Darmstadt, Germany). Deuterated solvents were purchased from
CDN Isotopes Inc. All chemicals or reagents used were of highest purity
available and purchased from Sigma-Aldrich (Oslo, Norway) or Acros
(Geel, Belgium). [13C6]-Benzaldehyde was purchased from Cambridge
Isotope Laboratories with isotopic purity >99% (Andover, MA). [13C6]- fashion made us look for alternative routes. Different methods
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M. Karlsen et al.
Scheme 1. Synthesis of [13C6]-amphetamine (1a) and [13C6]-methamphetamine (1b). *All aromatic carbons as 13C.
were tested, and in our hands, a new strategy, relying on a Stille were tested with different dihalomethanes. We found that the use
coupling,25 was found to be most useful (Scheme 2).
of dichloromethane was preferable over diiodomethane or
Our first attempt at synthesizing [13C6]-catechol (9) was by the dibromomethane because of easier product purification. The most
procedure of Ji et al.,26 but this procedure was time-consuming efficient transformation was achieved with cesium carbonate as
and gave low yield. We then tried the procedure of Thakur et al.,27 the base leading to 95% yield of good purity product.31 The use of
but this method gave minimal conversion even after 2 weeks and sodium hydride in hexamethylphosphoramide as reported by
was also abandoned. [13C6]-Phenol was instead converted to Castillo et al.32 also gave good conversion but resulted in a more
[13C6]-2-hydroxybenzaldehyde (8) in 95% yield by an ortho-specific difficult purification.
formylation with magnesium methoxide and formaldehyde with
Compound 10 was then converted to the bromide 11 with N-
continuous removal of methanol by a toluene azeotrope.This bromosuccinimide33 and taken further to the key intermediate
reaction was superior to several other formylation methods 12 by a Stille-type coupling. This reaction where the tributyltin
tested.28,29 Compound 8 could then easily be converted to [13C6]- enolate is generated in situ gave the ketone 12 in one step in
catechol (9) by a Dakin oxidation.30 The crude product 9 was dark over 90% yield. The precursor 12 was then transformed to the
in colour, but could be purified by sublimation, and NMR analysis respective final products [13C6]-MDMA (3b) and [13C6]-MDEA
indicated high purity. [13C6]-1,2-Methylenedioxybenzene (10) was (3c) by imine formation in the presence of molecular sieves,
then synthesized by a methenylation reaction, and several bases and reduction with sodium borohydride in methanol or ethanol,
Scheme 2. Synthesis of the [13C6]-3,4-methylenedioxyamphetamine derivatives 3a–c. *All aromatic carbons as 13C.
Scheme 3. Synthetic route to derivatives [13C6]-PMA (2a) and [13C6]-PMMA (2b). *All aromatic carbons as 13C.
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M. Karlsen et al.
Scheme 4. Synthetic route to the [13C6]-labelled derivatives 4a, 5a and 6a. *All aromatic carbons as 13C.
Figure 2. Ultra performance liquid chromatography (UPLC) chromatogram showing overlap of the [13C6]-labelled (yellow) and unlabelled compounds (green). This figure
is available in colour online at wileyonlinelibrary.com/journal/jlcr
methylation with dimethylsulfate. Although both steps were high
yielding, this pathway was judged more labour-intensive than the
Rieche formylation.
which facilitates the reaction in one pot. MDA (3a) was
synthesized by reductive amination using sodium
cyanoborohydride and ammonium acetate as amine source,
giving lower yield than the method discussed earlier. [13C6]-
MDMA (3b) was also made from 3a by formylation and
reduction in 84% yield.
The amphetamine derivatives 2a–b, 4a, 5a and 6a were all
made via 4-bromo-[13C6]-anisole (14) obtained by methylation
of [13C6]-phenol with methylsulfate and N-bromosuccinimide
bromination (Scheme 3). To prepare the 4-methoxy derivatives
2a–b, 14 was converted to the key intermediate 15 by a Stille
reaction in quantitative yield. Compound 15 was then
transformed to the respective amines [13C6]-PMA (2a) and
[13C6]-PMMA (2b) as described in the case of 3a–b.
a
The aldehyde 17 was further condensed with nitromethane
in a Henry reaction, and the nitrostyrene 18 was reduced in
two steps to the target 4a. Direct hydrogenation of 18 to the
primary amine 4a with palladium on barium sulfate, palladium
on carbon and platinum (IV) oxide was also tested. In the best
cases using platinum (IV) oxide, the yield was around 45%, and
several non-identified by-products were seen. Compound 4a
was then selectively brominated and iodinated36 in position
4 yielding [13C6]-2C-B (5a) and [13C6]-2C-I (6a), respectively.
For all the final products, yields were sacrificed because of the
need of achieving a final purity >99%. Crystallization from
various solvents including diethyl ether, acetonitrile and alcohols
and a mixture of these was efficient. The identity of all products
and intermediates were confirmed by co-elution with unlabelled
Synthesis of 2,5-dimethoxy-[13C6]-phenethylamine (4a), 4-bromo-
2,5-dimethoxy-[13C6]-phenethylamine (5a) and 2,5-dimethoxy-4-
iodo-[13C6]-phenethylamine (6a) is shown in Scheme 4. First, 4-
bromo-[13C6]-anisole (14) was transformed to 1,4-dimethoxy-[13C6]-
benzene (16) in a copper iodide/ethyl acetate-catalysed nucleophilic
aromatic substitution.34 Then, a Rieche formylation with titanium
tetrachloride and dichloromethyl methyl ether by the procedure of
materials, high-resolution MS and NMR spectroscopy.
representative UPLC chromatogram is seen in Figure 2.
A
Conclusion
Mancini et al.35 gave the aldehyde 17. Compound 17 was also Synthetic pathways have been designed for [13C6]-
synthesized by an ortho formylation of 4-hydroxyanisole and amphetamine, [13C6]-methamphetamine, [13C6]-MDA, [13C6]-
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M. Karlsen et al.
MDMA, [13C6]-PMA, [13C6]-PMMA, [13C6]-2C-B, [13C6]-2C-I and (m), 49.1, 126.5–130.5 (m, 5C), 134.7–137.3 (m); HRMS (ES+): calcd
[13C6]-2C-H. To the best of our knowledge, these 13C6-labelled for 13C6C3H14N [M]+: 142.1322; found 142.1328.
compounds have been prepared for the first time. Several
Synthesis of DL-[13C6]-methamphetamine hydrochloride (1b)
methods were tested at each step to find synthetic routes that
were efficient, fast, high yielding and allowing for
a
Amphetamine (1a) free base (100 mg, 0.71 mmol) was dissolved
in ethyl formate (10 mL). The solution was heated in an ACE
pressure reactor (Vineland, New Jersey, United States) for 2 h at
100 °C. The reaction mixture was cooled to ambient temperature,
and the solvent was evaporated under reduced pressure. The
resulting carbamate derivative was dissolved in dried diethyl
ether (5 mL). The solution was added drop wise to a suspension
of lithium aluminium hydride (40.4 mg, 1.07 mmol) in dry diethyl
ether (10 mL). The reaction mixture was refluxed for 5 h. After
cooling to 0 °C, water was carefully added. The lithium salt was
filtered over Celite™ in a Büchner funnel. The granules obtained
were washed thoroughly with excess diethyl ether. The
combined ether phases were washed with 5% sodium hydroxide
and brine before being dried with magnesium sulfate. The
solvent was evaporated to yield 96 mg of 1b as free base. The
free base was re-dissolved in diethyl ether (10 mL), and the
solution was cooled down to 10 °C before hydrochloric acid gas
dissolved in 2-propanol (1.85 mL) was added in small portions
until the pH reached 4. The resulting hydrochloride salt was
filtered off, washed with diethyl ether and dried to yield to
110 mg (0.57 mmol, 81%) of the product; mp. 133.5–134.1 °C;
purity > 98.8% (TFA derivative, GC-MS); MS-EI (m/z): 154.0 (100),
124.1 (26), 110.0 (28), 97.1 (16), 69.0 (11); 1H NMR (400MHz,
D2O) δ: 1.19 (d, J= 6.6 Hz, 3H), 2.62 (s, 3H), 2.74–3.08 (m, 2H), 3.41–
3.51 (m, 1H), 6.81–7.74 (m, 5H); 13C NMR (100 MHz, D2O) δ: 14.8,
30.0, 38.5–39.0 (m), 56.4, 126.4–30.2 (m, 5C), 134.5–136.7 (m); HRMS
(ES+): calcd for 13C6C4H16N [M]+: 156.1479; found 156.1491.
straightforward purification. The identified processes rely on
[13C6]-phenol as starting material and use some common
intermediates and similar process steps, thus allowing for this
series of compounds to be prepared in a fast and economic way.
Synthesized [13C6]-amphetamine and [13C6]-methamphetamine
have been found superior over deuterated analogues for use as
SIL IS in the field of forensic analysis and toxicology.
Synthesis of DL-[13C6]-amphetamine (1a) and DL-[13C6]-
methamphetamine (1b)
Synthesis of 2-nitro-1-propene-1-yl-[13C6]-benzene (7)
[13C6]-Benzaldehyde (1.00 g, 8.92 mmol) was added to
nitroethane (5 mL) in a 20-mL round-bottom flask with a magnetic
stirrer. Anhydrous ammonium acetate (0.16 g, 2.1 mmol) was
added, and the solution was warmed to 80 °C before methylamine
(33% in ethanol, 0.1 mL) was added in one portion. After 2 h, full
conversion was obtained as observed by TLC (silica gel eluting with
toluene) and by GC-MS. The solvent from the reaction mixture was
evaporated on a rotary evaporator, and the resulting crude product
was recrystallized from hot 2-propanol (3 mL). After slowly cooling
to 4 °C overnight, the crystals were isolated on a Büchner funnel
and washed with a small amount of cold 2-propanol. The light
yellow crystals obtained were dried thoroughly to remove volatiles,
yielding 1.10 g (6.51 mmol, 73%) based on [13C6]-benzaldehyde.
MS-EI (m/z): 169.1 (18), 152.1 (12), 137.0 (6), 121.1 (100), 111.0 (30),
1
95.1 (16); H NMR (400 MHz, CDCl3) δ: 2.47 (s, 3H), 7.15–7.34 (m,
3H), 7.55–7.75 (m, 2H), 8.10 (br.t, J= 3.7 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ: 14.0, 128.0–130.0 (m, 4C), 131.7–133.2 (m), 147.8.
Synthesis of DL-[13C6]-4-methoxyamphetamine derivatives
2a–b
Synthesis of [13C6]-anisole (13)
Synthesis of DL-[13C6]-amphetamine sulfate (1a)
[13C6]-Phenol (3.00 g, 30.0 mmol) was dissolved in water (30 mL).
Sodium hydroxide (1.21 g, 30.3 mmol) was dissolved in water
(20 mL) and added to the phenol solution under an argon
atmosphere. The reaction was stirred for 30 min and was then
cooled to 0 °C. Dimethyl sulfate (3.82 g, 30.3 mmol) was added
slowly through a cannula and septum. The reaction mixture
was gradually warmed to 50 °C and stirred at this temperature
for 1 h. Then, the reaction mixture was extracted with
dichloromethane (3 × 20 mL), and the combined organic phases
were washed with 5% sodium hydroxide solution (20 mL) and
brine (20 mL), and dried over magnesium sulfate. The solvent
was evaporated to yield 3.38 g (29.7 mmol, 99%) of [13C6]-anisole
(13) as a clear liquid. MS-EI (m/z): 114.1 (100), 99.1 (14), 84.1 (57),
70.1 (45); 1H NMR (400 MHz, CDCl3) δ: 3.77 (d, J = 4.2 Hz, 3H),
6.61–6.79 (m, 1H), 7.00–7.19 (m, 1H), 6.7 (dm, 156 Hz, 1H), 7.47
(quin, 8.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ: 55.1 (dt, J = 6.6,
2.2 Hz), 113.1–114.9 (m, 2C), 120.6 (tdt, J = 56.1, 9.1, 2.8 Hz),
129.5 (tdd, J = 56.4, 9.4, 2.2 Hz, 2C), 159.6 (td, J = 66.7, 9.1 Hz).
2-Nitro-1-propene-1-yl-[13C6]-benzene (7) (1.10 g, 6.50 mmol)
was dissolved in dry toluene (20 mL). The resulting solution
was added drop wise to a mixture of Vitride (Red-Al™, sodium
bis(methoxyethoxy)aluminium hydride) solution (70% in toluene,
5 mL) and dry toluene (22.5 mL) at 70 °C. After all the nitrostyrene
solution had been added, the resulting reaction mixture was
stirred for overnight at room temperature for the completion
of the reaction. Sodium hydroxide (5% aqueous solution) was
then added carefully, and the toluene layer was separated. The
water phase was washed with toluene (2 × 25 mL), and the
combined organic phases were washed with saturated sodium
bicarbonate and brine, and dried over anhydrous magnesium
sulfate. The solvent was evaporated to yield 930 mg of the free
base as light yellow oil. This oil was distilled on a Kugelröhr
apparatus. The distilled free base was dissolved in anhydrous
diethyl ether (20 mL), followed by
a slow addition of
concentrated sulfuric acid in diethyl ether (10%) until the pH
was 4.5. The precipitated salt was isolated by filtration and
washed with cold diethyl ether (2 × 10 mL). After drying under
reduced pressure, 751 mg (3.14 mmol, 48%) of the product was
Synthesis of 4-bromo-[13C6]-anisole (14)
obtained as white powdery sulfate salt; mp. 310–311 °C; purity [13C6]-Anisole (3) (3.03 g, 26.6 mmol) was dissolved in chloroform
99% (TFA derivative, GC-MS); MS-EI (m/z): 140.0 (100), 124.1 (15 mL) and N,N-dimethylformamide (2 mL). Then, N-
(90), 97.1 (55), 69.0 (28); 1H NMR (400 MHz, D2O) δ: 1.22 (d, bromosuccinimide (4.73 g, 26.6 mmol) was added in small
J = 6.6 Hz, 3H), 2.82–2.90 (m, 2H), 3.55 (qd, J = 6.9, 2.3 Hz, 1H), portions at 70 °C with intensive magnetic stirring. Upon reaction
6.87–7.75 (m, 5H); 13C NMR (100 MHz, D2O) δ: 17.5, 39.0–40.1 completion, more chloroform (50 mL) was added, and the
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M. Karlsen et al.
organic phase was washed with water (50 mL). The solvent was compound; HRMS (ES+): calcd for 13C6C4H16NO [M]+: 172.1428;
evaporated, and the crude product was purified by bulb-to-bulb found 172.1441.
distillation (70 °C, 1.7 mbarÀ1) yielding 4.21 g (21.8mmol, 82%) of
Synthesis of DL-[13C6]-4-methoxymethamphetamine hydrochloride
clear oil. MS-EI (m/z): 194.0 (100), 192.0 (100), 179.0 (45), 177.0
(DL-[13C6]-PMMA-HCl, 2b)
4-Methoxy-[13C6]-phenyl-2-propanone (15) (300 mg, 1.76 mmol)
1
(45), 150.0 (36), 148.0 (36); H NMR (400 MHz, CDCl3) δ: 3.77 (d,
J = 4.2 Hz, 3H), 6.56 (dm, J = 169.0Hz, 2H), 7.15 (dm, J = 169.0Hz,
2H); 13C NMR (100 MHz, CDCl3) δ: 55.4 (td, J = 6.6, 2.2Hz), 112.8
was dissolved in methanol (10 mL), and molecular sieves 4 Å
(200 mg) were added together with methylamine (322 mg,
3.53 mmol) in ethanol (33 wt.%). The reaction was stirred for
2 h, followed by addition of sodium borohydride (84 mg,
2.22 mmol) in small portions. Then, the reaction mixture was
stirred overnight, and the quenched by adding 3 M hydrochloric
acid (50 mL) followed by additional stirring for 1 h. The water
phase was washed with diethyl ether (30 mL) and basified with
50% sodium hydroxide solution (pH 11). The product was
extracted from the water phase with diethyl ether (3 × 30 mL).
The organic phase was washed with brine (30 mL) and dried over
magnesium sulfate. The solvent was evaporated yielding DL-
[13C6]-4-methoxymethamphetamine free base, which was
dissolved in anhydrous diethyl ether (10 mL), and hydrochloric
acid gas was slowly bubbled through the solution until pH was
4. The solvent was evaporated, and the solid obtained was
recrystallized twice from 2-propanol/diethyl ether yielding
292 mg (1.32 mmol, 75%) of [13C6]-PMMA hydrochloride as white
crystals; mp. 176.6–176.7 °C; purity > 99% (GC-MS). MS-EI of TFA
(tdt, J = 64.7, 9.5, 1.8Hz), 115.7 (tm, J = 64.7, 6.6 Hz, 2C), 132.2 (tm,
J = 62.7, 2.2 Hz, 2C), 158.7 (td, J = 68.1, 9.5Hz).
Synthesis of [13C6]-4-methoxyphenyl-2-propanone (15)
4-Bromo-[13C6]-anisole (14) (1.00 g, 5.18 mmol) was dissolved in
toluene (20 mL) in a 100-mL reaction flask with a stirring bar
and septum. Tri-(o-tolyl)phosphine (94 mg, 0.31 mmol),
tributyltinmethoxide (2.48 g, 7.72 mmol) and iso-propenyl
acetate (0.79 g, 27.2 mmol) were added, and the flask was
flushed with argon by the aid of ultrasound degassing and
vacuum. Palladium(II) chloride (27 mg, 0.15 mmol) was added
rapidly under a blanket of argon, and the mixture was heated
at 90 °C for 4 h. The solvent was then evaporated under vacuum,
and the crude product was purified by dry flash chromatography
on a plug of silica gel (60 mL) using heptane/ethyl acetate (85/
15). This gave 873 mg (5.13 mmol, 99%) of [13C6]-4-
methoxyphenyl-2-propanone as an oil; purity > 99% (GC-MS).
1
MS-EI (m/z): 170.1 (16), 127.1 (100), 96.1 (5), 83.1 (10); H NMR
1
derivative (m/z): 281.1 (3), 154.1 (100), 127.1 (65), 110.1 (15); H
(400 MHz, CDCl3) δ: 2.13 (s, 3H), 3.62 (q, J = 5.4 Hz, 2H), 3.78 (d,
J = 4.2 Hz, 3H), 6.62–6.72 (m, 1H), 6.90 (q, J = 8.7 Hz, 1H), 7.01–
7.11 (m, 1H), 7.25–7.36 (m, 1H); 13C NMR (100 MHz, CDCl3) δ:
29.1, 50.1 (ddt, J = 44.7, 7.5, 3.4 Hz), 55.2 (td, J = 4.0, 2.2 Hz),
113.4–115.0 (m, 2C), 126.2 (tdt, J = 58.1, 8.7, 2.2 Hz), 129.7–131.1
(m, 2C), 158.7 (td, J = 67.3, 8.7 Hz), 206.9.
NMR (400 MHz, DMSO-d6) δ: 1.08 (d, J = 6.6 Hz, 3H), 2.57 (s, 3H),
2.54–2.62 (m, 2H), 3.01–3.11 (m, 1H), 3.74 (d, J = 4.0 Hz, 3H),
6.66–6.76 (m, 1H), 6.91–7.04 (m, 1H), 7.06–7.15 (m, 1H), 7.30–
7.42 (m, 1H), 8.69 (br.s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
17.7, 40.8, 58.3, 59.5, 114.0 (ddd, J = 65.9, 55.5, 8.3 Hz, 2C),
127.4–131.2 (m, 3C), 158.1 (td, J = 66.6, 8.1 Hz); the CH3-N signal
could not be found; HRMS (ES+): calcd for 13C6C5H18NO [M]+:
186.1584; found 186.1598.
Synthesis of DL-[13C6]-4-methoxyamphetamine hydrochloride (DL-
[13C6]-PMA-HCl, 2a)
Synthesis of DL-[13C6]-3,4-methylenedioxyamphetamine
derivatives 3a–c
4-Methoxy-[13C6]-phenyl-2-propanone (15) (300 mg, 1.76 mmol)
was dissolved in methanol (10 mL), and ammonium acetate
was added (1.36 g, 17.6 mmol) under an argon atmosphere. To
this solution was added sodium cyanoborohydride (113 mg,
Synthesis of [13C6]-o-salicylaldehyde (8)
1.8 mmol) in small portions under stirring, and the reaction was Magnesium turnings (627 mg, 25.8 mmol) were added to a three-
further stirred overnight at 22 °C. Then, the solvent was necked round-bottom flask together with methanol (7.8 mL) and
evaporated, and hydrochloric acid (3 M, 30 mL) was added. The a small iodine crystal. When all the metal had dissolved, the
water phase was then washed with diethyl ether (30 mL) and solution was cooled to 40 °C, and [13C6]-phenol (5.00 g,
basified by addition of a concentrated sodium hydroxide 49.9 mmol) was added slowly as a solution in toluene (40 mL)
solution until pH was 10. The product free base was extracted under argon atmosphere. The reaction mixture was warmed to
from the aqueous phase with diethyl ether (3 × 30 mL). The 75 °C, and most of the methanol was distilled out. When the
combined organic phases were dried over magnesium sulfate, temperature reached 95 °C, paraformaldehyde (4.60 g,
and the solvent was evaporated yielding DL-[13C6]-4- 155 mmol) was added in small portions, and the temperature
methoxyamphetamine free base. The compound was dissolved was controlled at 95 °C. Vacuum was applied for short intervals
in anhydrous diethyl ether (10 mL), and hydrochloric acid gas to assure that the methanol was distilled out continuously, and
was slowly bubbled through the solution until pH was 4. The toluene was added to maintain solvent volume. After 30 min at
solvent was evaporated and the product recrystallized twice 100 °C, full conversion was confirmed by TLC analysis (heptane/
from acetonitrile yielding 233 mg (1.12 mmol, 64%) of [13C6]- acetone, 6/4). The mixture was cooled to 20 °C, 10% sulfuric acid
PMA hydrochloride as white crystals; mp. 200.6–201.2 °C; purity solution (90 mL) was added and the product was extracted with
99% (GC-MS); MS-EI of TFA derivative (m/z): 267.1 (8), 154.1 toluene (3 × 30 mL). The combined organic phases was washed
(35), 127.1 (100); 1H NMR (400 MHz, DMSO-d6) δ: 1.09 (d, with water and dried over magnesium sulfate. After solvent
J = 6.6 Hz, 3H), 2.54–2.65 (m, 2H), 2.85–2.96 (m, 1H), 3.74 (d, evaporation, the residue was dry flashed with silica gel
J = 4.3 Hz, 3H), 6.66–6.74 (m, 1H), 6.91–7.02 (m, 1H), 7.06–7.14 (heptane/acetone, 4/1) yielding 5.25 g (40.97 mmol 95%) of
(m, 1H), 7.29–7.39 (m, 1H), 7.93 (br.s, 2H); 13C NMR (100 MHz, [13C6]-o-salicylaldehyde; purity: 98% (GC-MS); MS-EI (m/z): 128.1
DMSO-d6) δ: 17.7, 48.1, 55.9, 113.9 (ddd, J = 65.9, 55.5, 8.3 Hz, (100), 127.1 (99), 110.1 (17), 99.1 (20), 82.1 (15), 70.1 (24); 1H
2C), 127.6–131.1 (m, 3C), 158.1 (td, J = 66.6, 8.1 Hz); the benzylic NMR (400 MHz, CDCl3) δ: 6.76–6.88 (m, 1H), 7.14–7.41 (m, 2H),
carbon was not detected in 13C NMR of the [13C6]-labelled 7.68–7.81 (m, 1H), 9.86–9.95 (dm, J = 19.9 Hz, 1H), 11.01–11.07
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J. Label Compd. Radiopharm 2014

M. Karlsen et al.
(m, 1H); 13C NMR (100 MHz, CDCl3) δ: 117.5 (dddt, J = 66.4, 58.3, Synthesis of 3,4-methylenedioxy-[13C6]-phenyl-2-propanone (12)
6.6, 2.9 Hz), 119.8 (tm, J = 57.1 Hz), 120.6 (tm, J = 58.9 Hz), 133.7
3,4-Methylenedioxy-[13C6]-1-bromobenzene
(11)
(3.66 g,
(tdd, J = 58.8, 6.5, 1.5 Hz), 136.9 (dddd, J = 58.3, 54.5, 6.5, 1.5 Hz),
161.6 (ddd, J = 66.4, 60.8, 8.2 Hz), 196.5 (dtd, J = 54.5, 5.4, 2.2 Hz).
17.7 mmol) was dissolved in toluene (30 mL) in a 100-mL reaction
flask with a stirring bar and septum. Tri-(o-tolyl)phosphine
(322 mg, 1.06 mmol), tributyltinmethoxide (8.51 g, 26.5 mmol)
and iso-propenyl acetate (2.72 g, 27.17 mmol) were added, and
argon was flushed in by the aid of ultrasound degassing and
vacuum. Palladium(II) chloride (94 mg, 0.53 mmol) was added
rapidly under a blanket of argon. The reaction mixture was then
heated at 90 °C for 4 h, followed by evaporation of solvent. The
crude product was purified by dry flash chromatography on a
plug of silica gel in a Büchner funnel using heptane/ethyl acetate
(85/15) as the mobile phase. The yield of 3,4-methylenedioxy-
[13C6]-phenyl-2-propanone (12) was 2.95 g (16.0 mmol, 91%) as
a colourless oil. MS-EI (m/z): 184.1 (35), 141.1 (100), 111.1 (5),
82.1 (11); 1H NMR (400 MHz, CDCl3) δ: 2.14 (s, 3H), 3.59 (q,
J = 5.9 Hz, 2H), 5.93 (t, J = 2.0 Hz, 2H), 6.39–7.03 (m, HC); 13C
NMR (100 MHz, CDCl3) δ: 29.1, 50.2–50.9 (m), 101.3 (t, J = 3.7 Hz),
107.6–110.5 (m, 2C), 122.5 (dddd, J = 61.8, 57.8, 6.6, 1.5 Hz),
127.8 (dddt, J = 61.8, 59.1, 7.3, 2.2 Hz), 146.6 (ddtd, J = 66.0, 65.5,
7.3, 1.5 Hz), 147.9 (ddtd, J = 69.7, 65.5, 8.1, 2.2 Hz), 206.4.
Synthesis of 1,2-dihydroxy-[13C6]-benzene (9)
To a stirred solution of [13C6]-o-salicylaldehyde (8) (4.87 g,
38.0 mmol) in water (50 mL) under an argon atmosphere was
rapidly added sodium hydroxide (1.52 g, 38.0 mmol) in water
(50 mL). A solution of hydrogen peroxide (4.63 g, 47.6 mmol,
35%) in water was then added slowly from a dropping funnel,
and the temperature was controlled with a water bath so that
the temperature did not exceed 40 °C. The reaction was then
stirred at 30 °C for 30 min before quenching with 15% sulfuric
acid solution to pH 3–4. The water phase was then saturated
with magnesium sulfate, and the product was extracted out with
diethyl ether (5 × 50 mL). The organic phase was dried with
magnesium sulfate and stirred overnight with active carbon
(0.5 g). The black suspension was filtered, the solvent evaporated
and the residue was crystallized from toluene (30 mL). The
crystals were then sublimated (105 °C, 1.4 mbar) to a final yield
of 2.77 g (23.9 mmol, 63%) as a white crystalline solid; mp.
104.7–105.9 °C; 1H NMR (400 MHz, DMSO-d6) δ: 6.26–6.57 (m,
2H), 6.68–6.98 (m, 2H), 8.76 (br.s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ: 114.4–116.8 (m, 2C), 118.3–120.1 (m, 2C), 145.2
(dtt, J = 27.3, 23.0, 3.6 Hz, 2C).
Synthesis of DL-[13C6]-3,4-methylenedioxyamphetamine hydro-
chloride (DL-[13C6]-MDA-HCl, 3a)
3,4-Methylenedioxy-[13C6]-phenyl-2-propanone (12) (313.4 mg,
1.70 mmol) was dissolved in methanol (10 mL), and ammonium
acetate was added (1.31 g, 17.0 mmol). An argon atmosphere
was introduced at this stage. To this solution was added sodium
cyanoborohydride in small portions under stirring (113 mg,
1.80 mmol). The reaction was then stirred overnight at room
temperature. The solvent was evaporated, and hydrochloric acid
(3 M, 30 mL) was added. The water phase was then washed with
diethyl ether (30 mL) and basified by addition of a concentrated
sodium hydroxide solution until pH was 10. The product free
base was extracted from the aqueous phase with diethyl ether
(3 × 30 mL). The combined organic phases were dried over
magnesium sulfate, and the solvent was evaporated yielding
DL-[13C6]-MDA free base (394 mg). This was dissolved in
anhydrous diethyl ether (10 mL), and hydrochloric acid gas was
slowly bubbled trough the solution until pH was 4. The solvent
was evaporated, and the obtained solid was recrystallized twice
from acetonitrile yielding 122.0 mg (0.55 mmol, 32%) [13C6]-
MDA hydrochloride as glistening white crystals; mp. 181.1–
181.6 °C; purity > 99% (MS-EI, TFA derivative); MS-EI (m/z): 281.1
(25), 168.1 (46), 141.1 (100), 111.0 (5), 82.1 (10); 1H NMR
(400 MHz, DMSO-d6) δ: 1.11 (d, J = 6.3 Hz, 3H), 2.55–2.66 (m, 1H),
2.81–2.93 (m, 1H), 3.59 (tq, J = 7.2, 6.3 Hz, 1H), 5.99 (t, J = 2.0 Hz,
2H), 6.40–7.21 (m, 3H), 7.93 (br.s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ: 17.7, 50.2–50.9 (m), 100.9 (t, J = 3.6 Hz), 107.6–110.5
(m, 2C), 122.8 (ddd, J = 60.0, 57.1, 7.3 Hz), 130.7 (dddt, J = 60.0,
58.3, 7.3, 2.2 Hz), 145.7–148.7 (m, 2C); the benzylic carbon was
not detected in 13C NMR of the [13C6]-labelled compound/within
solvent region; HRMS (ES+): calcd for 13C6C4H14NO2 [M]+:
186.1220; found 186.1234.
Synthesis of 3,4-methylenedioxy-[13C6]-benzene (10)
To a stirred solution of 1,2-dihydroxy-[13C6]-benzene (9) (2.76 g,
23.8 mmol) in dimethylsulfoxide (30 mL), cesium carbonate
(15.50 g, 47.6 mmol) was added under an argon atmosphere.
The reaction was then stirred at 80 °C for 2 h. The reaction was
quenched by ice water (120 mL), and then, the product was
steam distilled from the water suspension by two 100-mL water
portions. The water/product distillate was extracted with diethyl
ether (3 × 50 mL). The combined ether extracts were dried over
magnesium sulfate, and the diethyl ether was evaporated. This
yielded 3.04 g (23.8 mmol, 99%) based on 1,2-dihydroxy-[13C6]-
benzene. MS-EI (m/z): 128 (62), 127 (100), 68.1 (20); 1H NMR
(400 MHz, CDCl3) δ: 5.91 (t, J = 2.0 Hz, 2H), 6.49–6.81 (m, 2H),
6.82–7.11 (m, 2H); 13C NMR (100 MHz, CDCl3) δ: 100.6 (t,
J = 3.5 Hz), 107.5–109.8 (m, 2C), 120.8–122.5 (m, 2C), 146.5–
148.3 (m, 2C).
Synthesis of 3,4-methylenedioxy-[13C6]-1-bromobenzene (11)
3,4-Methylenedioxy-[13C6]-benzene (10) (3.04 g, 23.8 mmol) was
dissolved in chloroform (15 mL). N-Bromosuccinimide (4.23 g,
23.8 mmol) was added in small portions with intensive magnetic
stirring. The reaction mixture was refluxed for 2 h. The obtained
solids were removed by filtration and washed with two small
portions of cold chloroform. The combined organic fractions
were evaporated, and diethyl ether (150 mL) was added before
washing with water (100 mL). The organic phase was purified
by dry flash chromatography on a plug of silica gel in a Büchner
funnel using diethyl ether as the mobile phase. The yield of 3,4-
methylenedioxy-[13C6]-1-bromobenzene was 4.38 g (21.2 mmol,
89%) based on methylenedioxy-[13C6]-benzene. MS-EI (m/z):
Synthesis of DL-[13C6]-3,4-methylenedioxy-N-methylamphetamine
hydrochloride (DL-[13C6]-MDMA-HCl, 3b) from 3a
206.0 (100), 147.1 (5), 68.1 (35); 1H NMR (400 MHz, CDCl3) δ: DL-[13C6]-MDA (3a) as a free base (100 mg, 0.54 mmol) was
5.96 (t, J = 2.0 Hz, 2H), 6.42–7.20 (m, 3H); 13C NMR (100 MHz, dissolved in ethyl formate (10 mL) and heated in an ACE pressure
CDCl3) δ: 101.6 (t, J = 3.1 Hz), 108.7–110.3 (m), 111.3–113.9 (m, reactor for 2 h at 100 °C. The reaction mixture was cooled to 22 °
2C), 123.45–125.1 (m), 146.1–149.5 (m, 2C).
C, and the solvent was evaporated under reduced pressure. The
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M. Karlsen et al.
carbamate derivative obtained was dissolved in dry diethyl ether stirred for 2 h. Sodium borohydride (87 mg, 2.30 mmol) was
(5 mL) and added drop wise to a suspension of lithium added in small portions, and the reaction mixture was stirred
aluminium hydride (31 mg, 0.81 mmol) in diethyl ether (10 mL). overnight. Hydrochloric acid (3 M, 50 mL) was added, and the
The reaction mixture was refluxed for 5 h and cooled to 0 °C, reaction was stirred for 1 h. The water phase was washed with
and then, water was added carefully. The lithium salt formed diethyl ether (30 mL) and basified with 50% sodium hydroxide
was filtered through Celite™ on a Büchner funnel, and the solution (pH 11). The product was extracted from the water
granules were washed thoroughly using an excess of diethyl phase with diethyl ether (3 × 30 mL), and organic phase was
ether. The ether phases were combined and washed with 5% washed with brine (30 mL) and dried over magnesium sulfate.
sodium hydroxide and brine before drying over magnesium The solvent was evaporated yielding DL-[13C6]-MDEA free base.
sulfate. The solvent was evaporated to yield 3b as a free base This was dissolved in anhydrous diethyl ether (10 mL), and
(96 mg). The product was re-dissolved in diethyl ether and hydrochloric acid gas was slowly bubbled through the solution
cooled down to 10 °C before hydrochloric acid dissolved in 2- until pH was 4. The solvent was evaporated, and the solid
propanol (1.85 M) was added in small portions until the pH was obtained was recrystallized twice from 2-propanol/diethyl ether
4. The salt formed was filtered off, washed with diethyl ether (15 mL/6 mL) yielding 260 mg (1.04 mmol, 58%) [13C6]-MDEA
and dried to yield DL-[13C6]-MDMA hydrochloride (110 mg, hydrochloride as white crystals; mp. 200.2 °C (dec); purity
0.47 mmol, 86%) based on DL-[13C6]-MDA; mp. 249.5–250.1 °C;
99% (GC-MS); MS-EI of TFA derivative (m/z): 309.1 (9), 168.1
purity > 99% (GC-MS, TFA derivative). MS-EI of TFA derivative (100), 140.0 (24), 82.1 (6); 1H NMR (400 MHz, DMSO-d6) δ: 1.09
(m/z): 295.1 (16), 168.1 (86), 154.0 (100), 141.1 (59), 110.0 (30), (d, J = 6.6 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H), 2.09 (s, 2H), 2.92–3.13
1
82.1 (10); H NMR (400 MHz, DMSO-d6) δ: 1.09 (d, J = 6.6 Hz, 3H), (m, 3H), 6.01 (t, J = 2.0 Hz, 2H), 6.41–7.18 (m, 3H), 8.61 (br.s, 1H);
2.55 (br.s, 4H), 2.97–3.13 (m, 1H), 3.26–3.30 (m, 1H), 6.01 (s, 2H), 13C NMR (100 MHz, DMSO-d6) δ: 11.7, 17.7, 32.7, 50.7, 100.9 (t,
6.40–7.20 (m, 3H), 8.84 (br.s, 1H); 13C NMR (100 MHz, DMSO-d6) J = 3.6 Hz), 107.9–110.7 (m, 2C), 122.8 (dddd, J = 61.2, 57.1, 7.3,
δ: 17.8, 29.6, 50.2–50.9 (m), 100.9 (t, J = 3.6 Hz), 107.7–110.6 (m, 1.5 Hz), 130.7 (dddt, J = 61.2, 59.1, 7.3, 2.2 Hz), 145.6–148.7 (m,
2C), 122.4 (dddd, J = 61.8, 57.8, 6.6, 1.5 Hz), 130.2 (dddd, J = 61.8, 2C); the benzylic carbon was not detected in 13C NMR of the
59.1, 7.3, 2.2 Hz), 146.0 (tt, J = 65.4, 7.1 Hz), 146.7 (tt, J = 65.1, [13C6]-labelled compound/within solvent region; HRMS (ES+):
7.1); the benzylic carbon was not detected in 13C NMR of the calcd for 13C6C6H18NO2 [M]+: 214.1533; found 214.1548.
[13C6]-labelled compound/within solvent region; HRMS (ES+):
calcd for 13C6C5H14NO2 [M]+: 200.1377; found 200.1391.
Synthesis of 2,5-dimethoxy-[13C6]-phenethylamine
derivatives 4a, 5a and 6a
Synthesis of DL-[13C6]-3,4-methylenedioxy-N-methylamphetamine
hydrochloride (DL-[13C6]-MDMA-HCl, 3b) from 12
Synthesis of 1,4-dimethoxy-[13C6]-benzene (16)
4-Bromo-[13C6]-anisole (1.30 g, 6.73 mmol) (14) was dissolved in
3,4-Methylenedioxy-[13C6]-phenyl-2-propanone (12) (500 mg,
2.72 mmol) was dissolved in methanol (10 mL), and molecular
methanol (10 mL) and N,N-dimethylformamide (10 mL). To this
sieves 4 Å (1.00 g) were added together with methylamine
solution, freshly made sodium methoxide (10 mL, 5 M) was
(511 mg, 5.43 mmol) in ethanol (33 wt.%). The reaction was
added together with ethyl acetate (2 mL). Copper iodide (1.4 g,
stirred for 2 h. Sodium borohydride (130 mg, 3.44 mmol) was
7.4 mmol) was added and the reaction stirred at 80 °C in a sealed
added in small portions, and the reaction mixture was stirred
tube under an argon atmosphere for 48 h. Sulfuric acid (aq 10%,
overnight. Water was added (60 mL) followed by careful addition
100 mL) was added, and the product was extracted with diethyl
of hydrochloric acid (3 M, 50 mL), and the reaction was stirred for
ether (3 × 30 mL). The organic phase was washed with water
(2 × 20 mL) and dried over magnesium sulfate. The solvent was
1 h. The water phase was washed with diethyl ether (30 mL) and
basified with 50% sodium hydroxide solution (pH 11). The
evaporated yielding 837 mg (5.81 mol, 86%) of 2,4-dimethoxy-
product was extracted from the water phase with diethyl ether
[13C6]-benzene as white platelets after drying; purity: 99% (GC-
(3 × 30 mL), and organic phase was washed with brine (30 mL)
MS); MS-EI (m/z): 144.1 (80), 129.1 (100), 114.1 (3), 100.1 (30),
and dried over magnesium sulfate. The solvent was evaporated
1
DL-[13C6]-3,4-methylenedioxy-N-methylamphetamine
85.1 (5), 69.1 (8); H NMR (400 MHz, CDCl3) δ: 3.77 (d, J = 4.3 Hz,
yielding
6H), 6.62 (dm, J = 160.0 Hz, 4H); 13C NMR (100 MHz, CDCl3) δ:
55.8–55.9 (m, 2C), 113.7–115.5 (m, 4C), 152.5–155.0 (m, 2C).
free base, which was purified by short path vacuum distillation
to (533 mg, 2.67 mmol). The product was re-dissolved in diethyl
ether and cooled down to 10 °C before hydrochloric acid
dissolved in 2-propanol (1.85 M) was added in small portions
Synthesis of 2,4-dimethoxy-[13C6]-benzaldehyde (17)
until the pH was 4. The salt formed was filtered off, washed with 2,4-Dimethoxy-[13C6]-benzene (16) (837 mg, 5.81 mmol) was
acetone (5 mL) and diethyl ether (10 mL), recrystallized from dissolved in dichloromethane (50 mL) in a septum-sealed flask,
isopropanol/ether and dried to yield DL-[13C6]-MDMA and dichloromethyl methyl ether (667 mg, 5.8 mmol) was added.
hydrochloride as white needles (540 mg, 2.29 mmol); purity This solution was cooled to À5 °C, and argon was flushed in.
99% (GC-MS, TFA derivative); the product had the same Titanium tetrachloride (1.82 g, 9.60 mmol) was added slowly with
chromatographic and spectroscopic data as that described good stirring through a cannula. After 1 h at À5 °C, the reaction
earlier.
was allowed to stir for 16 h at 4 °C. After this time, the reaction
mixture was quenched rapidly over crushed ice and allowed to
stir for 1 h. The product was extracted with dichloromethane
(3 × 30 mL), and the combined organic phases were washed with
Synthesis of DL-[13C6]-3,4-methylenedioxy-N-ethylamphetamine
hydrochloride (DL-[13C6]-MDEA-HCl, 3c)
3,4-Methylenedioxy-[13C6]-phenyl-2-propanone (12) (338 mg, brine (20 mL) and dried over magnesium sulfate. The solvent was
1.84 mmol) was dissolved in methanol (10 mL), and molecular evaporated and the crude product purified by silica-gel flash
sieves 4 Å (1.00 g) were added together with ethylamine chromatography (toluene/ethyl acetate, 92/8), followed by
(752 mg, 3.67 mmol) in ethanol (22 wt.%). The reaction was crystallization from pentane. This gave 912 mg (5.30 mmol,
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J. Label Compd. Radiopharm 2014

M. Karlsen et al.
91%) of 2,4-dimethoxy-[13C6]-benzaldehyde as white needles; Synthesis of 2,5-dimethoxy-[13C6]-phenethylamine hydrochloride
purity: 99% (GC-MS); MS-EI (m/z): 172.1 (100), 157.0 (32), 143.0 (4a)
1
(10), 126.1 (18), 112.1 (15), 100.1 (20); H NMR (400 MHz, CDCl3)
2-Nitro-1-propane-1-yl-2,4-dimethoxy-[13C6]-benzene
(19)
δ: 3.82 (d, J = 4.2 Hz, 3H) 3.91 (d, J = 4.0 Hz, 3H), 6.94 (dm,
J = 152.8 Hz, 1H), 7.15 (dm, J = 159.3 Hz, 1 H), 7.34 (dm,
J = 154.1 Hz, 1H), 10.46 (ddd, J = 22.5, 3.0, 1.5 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ: 55.8 (td, J = 4.0, 2.2 Hz), 56.2 (td, J = 4.8,
2.7 Hz), 110.4 (ddtd, J = 69.5, 62.3, 4.5, 1.5 Hz), 113.3 (ddt,
J = 66.5, 60.40, 4.4 Hz), 123.5 (ddt, J = 68.0, 60.5, 4.7 Hz), 124.9
(ddtd, J = 69.7, 62.3, 4.5, 1.5 Hz), 153.6 (dddd, J = 69.5, 68.0, 7.0,
1.5 Hz), 156.7 (dddd, J = 69.7, 66.3, 7.0, 1.5 Hz), 189.1–189.9 (m).
(240 mg, 1.11 mmol) was dissolved in methanol (20 mL), and
palladium on carbon (10%, 72 mg) was added. Hydrogen gas
was added to the mixture for 4 h. The reaction mixture was
filtered using a plug of Celite™, and hydrochloric acid gas
dissolved in methanol was added until the pH was 3, measured
on a moist pH paper. The reaction mixture was evaporated,
and the residue was recrystallized with 2-propanol (1 mL) and
diethyl ether (3 mL). The crystals formed were then isolated by
filtration and recrystallized from 2-propanol (2 mL) and ethanol
(5 mL) yielding 218 mg (1.16 mmol, 88%) of 2,5-dimethoxy-
[13C6]-phenethylamine hydrochloride salt as white needles; mp.
136.5–137.3 °C; purity > 99% (GC-MS); MS-EI of TFA derivative
Synthesis of 2-nitro-1-propene-1-yl-2,4-dimethoxy-[13C6]-benzene
(18)
2,4-Dimethoxy-[13C6]-benzaldehyde (17) (130 mg, 0.76 mmol) (m/z): 283.1 (100), 170.0 (92), 157.1 (45), 127.1 (49), 97.1 (11),
was dissolved in nitromethane (10 mL), and ammonium acetate 83.1 (7); 1H NMR (400 MHz, DMSO-d6) δ: 2.77–2.88 (m, 2H),
(33.5 mg, 0.44 mmol) was added. The reaction was warmed at 2.88–3.02 (m, 2H), 3.70 (d, J = 4.3 Hz, 3H), 3.75 (d, J = 4.3 Hz, 3H),
70 °C for 2 h. After this, the reaction was poured over a plug of 6.78 (dm, J = 157.0 Hz, 2H), 6.90 (dm, J = 157.0 Hz, 1H), 7.97 (br.s,
silica gel (50 mL) wetted with heptanes in a Büchner funnel, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 39.0, 55.3, 55.8, 111.3–
and the product was eluted with (20 mL) fractions of toluene. 113.5 (m), 116.8 (tm, J = 64.2 Hz), 119.1 (tm, J = 64.9 Hz), 126.5
The fractions containing the product were concentrated yielding (tm, J = 65.5 Hz), 150.8–154.4 (m, 2C); the benzylic carbon could
166 mg (0.77 mmol, ≥100%) as yellow crystals. Some trace not be identified; HRMS (ES+): calcd for 13C6C4H16NO2 [M]+:
1
amount of toluene was detected by H NMR. MS-EI (m/z): 215.1 188.1377; found 188.1390.
(100), 168.0 (45), 153 (36), 139.0 (45), 124.0 (15), 110.0 (15), 95.1
Synthesis of 4-bromo-2,5-dimethoxy-[13C6]-phenethylamine hydro-
chloride (5a)
(10), 81.0 (17); 1H NMR (400 MHz, CDCl3) δ: 3.80 (d, J = 4.3 Hz,
3H), 3.90 (d, J = 4.2 Hz, 3H), 6.69–7.28 (m, 3H), 7.85 (dd,
J = 13.6 Hz, 1H), 8.12 (dt, J = 13.6, 5.5 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ: 55.8 (td, J = 4.0, 2.2 Hz), 56.0 (td, J = 4.8, 2.7 Hz), 112.4
(ddt, J = 68.3, 60.0, 4.7 Hz), 116.2 (ddtd, J = 68.8, 62.9, 4.7,
2.1 Hz), 119.1 (ddt, J = 67.5, 60.0, 4.6 Hz), 119.5 (ddtd, J = 69.8,
62.9, 5.1, 1.6 Hz), 153.5 (dddd, J = 68.8, 67.5, 7.1, 1.6), 154.0 (dddd,
J = 69.8, 68.3, 7.1, 2.1 Hz). The sp2 carbons residing at 135 and
138 ppm in the unlabelled compound could not be observed
in this analysis.
2,5-Dimethoxy-[13C6]-phenethylamine hydrochloride salt (4a)
(103 mg, 0.46 mmol) was dissolved in acetic acid (10 mL). Bromine
(73.5 mg, 0.46 mmol) was added as a solution in acetic acid (1mL)
over 30 min, and the reaction was stirred for 16h at 22 °C. The
reaction mixture was then poured onto a 15% sodium hydroxide
solution (40 mL), and the product was extracted with
dichloromethane (2× 30 mL). The combined organic phases were
washed with brine (20 mL) and dried over magnesium sulfate.
The solvent was evaporated and the crude product dissolved in
dry diethyl ether. The pH was adjusted with hydrochloric acid
gas in 2-propanol to 4 where the product precipitated as a white
solid. The solid was isolated by filtration and then recrystallized
from acetonitrile containing some methanol. Filtration and drying
gave 80.9 mg (0.27 mmol, 57%) of 4-bromo-2,5-dimethoxy-[13C6]-
phenethylamine as the hydrochloride salt; mp. 224–225 °C (dec);
purity> 99% (HPLC); 1H NMR (400MHz, DMSO-d6) δ: 2.79–2.87
(m, 2H), 2.93–3.02 (m, 2H), 3.77 (d, J = 4.0Hz, 3H), 3.80 (d,
J = 4,0Hz, 3H), 6.06–7.94 (m, 2H), 7.96 (br.s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 55.2, 56.0, 108.8 (tm, J = 68.4 Hz), 115.4
(qm, J = 67.3 Hz, 2C), 125.4 (tm, J = 62.2 Hz), 149.3 (tm, J = 73.2 Hz),
151.5 (tm, J = 66.6 Hz); the two methylene carbons were not
identified; NHRMS (ES+): calcd for 13C6C4H15NO2Br [M]+: 266.0482;
found 266.0493.
Synthesis of 2-nitro-1-propane-1-yl-2,4-dimethoxy-[13C6]-benzene
(19)
Sodium borohydride (92 mg, 2.44 mmol) was dissolved in a
mixture of ethyl acetate (10 mL) and ethanol (2 mL) and cooled
to 0 °C. Then, 2-nitro-1-propene-1-yl-2,4-dimethoxy-[13C6]-
benzene (18) (175 mg, 0.81 mmol) was added slowly as a
solution in ethyl acetate (5 mL). The reaction was stirred at 22 °
C for 1 h, and a water solution of sulfuric acid (30 mL, 10%) was
added. The product was isolated by extraction with diethyl ether
(2 × 20 mL), and the combined organic phases were washed with
brine (20 mL) and dried over magnesium sulfate. The solvent was
evaporated to give 160 mg (0.74 mmol, 91%) of yellow oil, which
was pure enough to be used in the next step; purity: 97% (GC-
MS); MS-EI (m/z): 217.1 (83), 170.1 (100), 156.1 (72), 141.1 (36),
1
Synthesis of 4-iodo-2,5-dimethoxy-[13C6]-phenethylamine hydro-
chloride (6a)
127.1 (21), 111.1 (11); H NMR (400 MHz, CDCl3) δ: 3.23–3.34 (m,
2H), 3.76 (d, J = 4.3 Hz, 3H), 3.81 (d, J = 4.3 Hz, 3H), 4.61 (td,
J = 7.3, 3.5 Hz, 2H), 6.49–6.69 (m, 2H), 6.88–7.07 (m, 1H); 13C
NMR (100 MHz, CDCl3) δ: 39.5, 55.7 (dd, J = 5.1, 2.2 Hz), 56.0 (td,
J = 4.8, 2.7 Hz), 74.6, 111.1 (ddtd, J = 66.2, 59.5, 4.7, 4.7, 1.9 Hz)
112.9 (ddtd, J = 67.2, 59.5, 4.5, 4.5, 2.5 Hz) 116.9 (ddtd, J = 68.2,
62.4, 4.5, 4.5, 1.9 Hz) 124.8 (ddt, J = 68.8, 62.4, 4.7, 4.7 Hz) 151.6
(dddd, J = 68.8, 67.2, 7.5, 1.9 Hz) 153.5 (dddd, J = 68.2, 66.2, 7.5,
2.5 Hz). This reaction was repeated to provide sufficient material
for further synthesis.
2,5-Dimethoxy-[13C6]-phenethylamine
hydrochloride
(4a)
(99.3 mg, 0.45 mmol) was dissolved in ethanol (10 mL) under
argon atmosphere. To this solution was added under constant
stirring silver sulfate (140.3 mg, 0.45 mmol) and iodine (114 mg,
0.45 mmol). The conversion halted after stirring for 24 h; thus,
additional silver sulfate (140 mg, 0.45 mmol) and iodine
(114 mg, 0.45 mmol) were added, followed by 24-h extended
reaction time. The reaction mixture was then poured on a 5%
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

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sodium hydroxide solution (40 mL), and the product was
extracted with dichloromethane (2 × 30 mL). The combined
organic phases were washed with brine (20 mL) and dried over
magnesium sulfate. The solvent was evaporated, and the crude
product dissolved in dry diethyl ether (10mL). The pH was adjusted
with hydrochloric acid gas in 2-propanol to 4 where the product
precipitated as a white solid. This solid was re-dissolved in hot 2-
propanol (10 mL) and acetonitrile (4 mL) and crystallized at 20 °C
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dimethoxy-[13C6]-phenethylamine hydrochloride was 75 mg
(0.21 mmol, 48%) as fluffy white needles; mp. 244.0 °C (dec); purity
99% (HPLC); 1H NMR (400 MHz, DMSO-d6) δ: 2.79–2.87 (m, 2H),
2.93–3.02 (m, 2H), 3.77 (d, J= 4,0 Hz, 3H), 3.80 (d, J= 4,0 Hz, 3H),
6.63–7.58 (m, 2H), 7.86 (br.s, 2H); 13C NMR (100 MHz, DMSO-d6) δ:
39.1, 55.3, 55.6, 113.9 (tm, J= 64.2 Hz), 119.1 (tm, J= 62.2 Hz), 121.3
(tm, J= 68.1Hz), 126.3 (tm, J= 66.6 Hz), 151. 9 (tm, J= 62.2 Hz, 2C);
the benzylic carbon could not be identified; HRMS (ES+): calcd for
13C6C4H15NO2I [M]+: 314.0343; found 314.0357.
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Acknowledgements
We would like to thank the Norwegian Research Council (NFR)
and Innovation Norway for financial support. We would also like
to thank Dag Helge Strand at the Norwegian Institute of Public
Health (NIPH), Oslo, Norway, for good cooperation and testing
of the final products in their routine analysis of urine samples.
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Conflict of Interest
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The authors did not report any conflict of interest.
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