Forensic Science International 152 (2005) 157–173

www.elsevier.com/locate/forsciint

Some pyridine derivatives as ‘‘route-specific markers’’ in

4-methoxyamphetamine (PMA) prepared by the Leuckart method
Studies on the role of the aminating agent in their
distribution in the final product
Dariusz Błachuta,b, Krystyna Wojtasiewiczb, Zbigniew Czarnockib,*
a
Department of Criminalistics, Internal Security Agency, 1 Sierpnia 30A, 02-134 Warsaw, Poland
b
Faculty of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland

Received 23 January 2004; received in revised form 21 July 2004; accepted 25 July 2004
Available online 13 October 2004

Abstract

The two previously unknown isomeric aryl-methylpyridines were prepared and analysed. Both 2,6-dimethyl-3,5-(40 -
methoxyphenyl)pyridine and 2,4-dimethyl-3,5-di-(40 -methoxyphenyl)pyridine have been identified as a new by-product in
the crude 4-methoxyamphetamine (PMA) obtained via the Leuckart method. The synthesis of 2,6-dimethyl-3,5-diphenylpyr-
idine, which is connected to amphetamine chemistry, is also reported. It was also found that different reagents (formamide,
formamide/HCOOH, ammonium formate) used in the course of the Leuckart synthesis of PMA significantly affected the
impurity content. The presented results point out on the ‘‘high-boiling pyridines’’ as compounds especially useful in the
comparative analysis, since their profile seems to be independent on the purification procedure and may be conveyed from the
crude reaction mixture even into a carefully purified final product.
# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Heterocyclic impurities; Route-specific markers; Gas chromatography–mass spectrometry; Structure elucidation; Suzuki cross-
coupling

1. Introduction
The synthetic drugs, whose structures are based on the
common arylalkyl system Ar–C–C–N, are nowadays widely
abused by the young people in Poland. According to the poll
carried out by the Warsaw Neurology and Psychiatry Insti-
tute [1], a number of youths having occasional contact with
the most popular drugs, including phenylisopropylamines,
opiates, cocaine and LSD, has increased substantially,
* Corresponding author. Tel.: +48 228220211;
fax: +48 228225996.
E-mail address: czarnoz@chem.uw.edu.pl (Z. Czarnocki).
whereas the age of the persons trying drugs for the first
time has lowered.
Phenylisopropylamine derivatives due to their relatively
simple structures are very suitable for clandestine laboratory
manufacture, even in domestic and unprofessional condi-
tions. In order to overcome existing regulation system
against their production, illegal chemists make their struc-
tural modification leading to the so-called ‘‘designer drugs’’.
In this way, variously ring, side chain and nitrogen-substi-
tuted derivatives of different kind of pharmacological action,
potency and toxicity have been produced during the last two
decades [2]. Some of them, e.g. 3,4-methylenedioxy-
methamphetamine (MDMA) and 3,4-methylenedioxyethy-
lamphetamine (MDE) have gained considerable popularity,

0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2004.07.018
158 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
mainly because of their unique ability to facilitate inter-
personal communication and reduction of anxiety which
often accompanies discussion of emotionally painful issues
[3,4].
The modification of the parent arylisopropyl structure
can be accomplished by two general strategies. In the first
one, appropriately functionalised in b-position arylisopropyl
system with fixed substituents can be readily transformed
into the nitrogen-substituted homologues. The N-methyl, N-
ethyl, N,N-dimethyl and N–OH derivatives are most likely
produced, since further elongation of the N-alkyl chain
usually abolishes stimulant or hallucinogenic activity of
thus modified parent structure. In accordance with this
general scheme all members of Ecstasy family, including
MDMA, MDE, N,N-dimethyl MDA and N-hydroxy MDA,
have been prepared [5,6].
The ability of amphetamine analogues to elicit a wide
range of psychoactive effects steams mainly from the pre-
sence of the appropriate configuration of various substituents
within the aryl moiety [7]. Therefore, the second strategy
that allows the choice of the desired ring system seems to be
of special importance. The commercially available and still
not controlled ring-substituted benzaldehydes permit the
synthesis of a large number of ring-modified primary amines
through arylnitropropene intermediates. The additional pos-
sibility of transforming these intermediates into the corre-
sponding ketones, which are important precursors in the first
strategy, greatly extends the possible outcome of this
approach. The scope of the second strategy can be further
extended by the substitution of nitroethane with nitropro-
pane in a Knovenagel–Walter condensation. The resulting
arylnitrobutenes permit the preparation of various a-homo-
logues of arylisopropylamines and ketones [8–10].
One of the simplest ring modifications of amphetamine,
which can be easily prepared according to both strategies is
4-methoxyamphetamine, also known as p-methoxyamphe-
tamine (PMA). The first report of its appearance on illicit
market dates back to 1973 (Canada, Ontario) [11]. Since that
time, illicit preparations containing this agent were reported
in Spain [12], Australia [13], US [14] and Poland [15,16].
There were also reports on the cases of fatal intoxication
following the consumption of illegal tablets containing PMA
and its derivative, 4-methoxymethamphetamine (PMMA)
[17,18]. The individuals who overdosed 4-methoxyamphe-
tamine derivatives were observed to demonstrate numerous
symptoms characteristic for an acute poisoning, such as
convulsions, rhabdomyolysis, cardiac dyshrythmia, hyper-
pyrexia and disseminated intravascular coagulation. Death
was usually associated with a respiratory or cardiac failure
[19].
We have reported recently that 4-methoxyamphetamine
could be readily obtained in moderate yields from commer-
cially available 4-methoxyphenylacetone based on the well-
known Leuckart protocol [20]. The synthesis and identi-
fication of several Leuckart-connected impurities, including
the corresponding ring-substituted derivatives of N,N-di-(b-
Fig. 1. The chemical structures of 2,4-dimethyl-3,5-diphenylpyr-
idine 1, 2,4-dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 2, 2,6-
dimethyl-3,5-diphenylpyridine 3 and 2,6-dimethyl-3,5-di-(40 -meth-
oxyphenyl)pyridine 4.
phenylisopropyl)amine, N,N-di-(b-phenylisopropyl)methy-
lamine and N,N-di-(b-phenylisopropyl)formamide, have
been also described [15]. We have also mentioned the
presence of the methoxy derivatives of so-called ‘‘high-
boiling pyridines’’, which we tentatively identified in the
crude PMA on the basis of their mass spectra [15].
In this report we describe the synthesis and identification
of 2,4-dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 2 and
2,6-dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 4 as two
novel impurities from ‘‘pyridine family’’ in PMA produced
by the Leuckart method (Fig. 1).
Moreover, 2,4-dimethyl-3,5-diphenylpyridine 1 and 2,6-
dimethyl-3,5-diphenylpyridine 3 have been synthesised as
model compounds. Although both isomeric methyl–phenyl
pyridines have already been reported [21–23] in connection
with the study on the Leuckart amphetamine, their unequi-
vocal identification has not yet been supported by their
independent synthesis. This work describes also some of
our observations concerning the influence of the exchange of
the nitrogen-containing reagent during the Leuckart synth-
esis of PMA on the whole by-products content. Accordingly,
three different reagents have been selected to react with
4-methoxyphenylacetone, namely formamide alone, the
mixture of formamide and formic acid and ammonium
formate.
2. Experimental
2.1. Materials
All reagents and solvents employed were analytical or
HPLC grade. Phenylacetone, 4-methoxyphenylacetone,
phenylacetonitrile, (4-methoxyphenyl)acetonitrile were
obtained from Merck (Germany); 2,6-dimethyl-g-pyrone,
dibenzylketone were obtained from Fluka (Germany); 4-
methoxyphenylacetic acid was purchased from Aldrich
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
(Germany); tetrakis(triphenylphosphine)palladium(0) and
4-methoxybenzenboronic acid were obtained from Lancas-
ter (Germany). Commercial absolute ethanol and ethyl
acetate were dried by standard methods [24]. All the remain-
ing reagents and solvents were purchased from renowned
manufacturers and were used without additional purifica-
tion.
TLC was performed on the Merck Kieselgel 60 F-254
plate. The spots were visualised with UV lamp and/or iodine
vapour. Column chromatography was carried out at atmo-
spheric pressure using silica gel 70-230 mesh.
Melting points were determined on a capillary melting
points apparatus (Electrothermal, Model IA 9200) and are
uncorrected. NMR spectra were recorded on a Varian Unity
Plus spectrometer operating at 500 or 200 MHz for 1H NMR
and at 125 or 50 MHz for 13C NMR, respectively.
Resonances were measured in parts per million relative to
tetramethylsilane (TMS) (d = 0.0) for hydrogen, and chloro-
form (d = 77.0) for carbon). The abbreviation used are as
follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m,
multiplet.
Infrared spectra were recorded on a Bruker FTIR 113v
spectrometer equipped with IR microscope. Spectra were
taken from 400 to 4000 cm1 working in the reflection
mode. The spectral data were collected of 16 scans with
2 cm1 resolution.
The gas chromatographic analyses were carried out on
two GC systems.
The first one consisted of HP 6890 Series gas chromato-
graph coupled with HP 5973 mass-selective detector. The
capillary column HP-5MS, 30 m  0.25 mm ID with
0.25 mm film thickness was used with helium as a carrier
gas with constant flow F = 0.6 ml/min. The injector tem-
perature was 250 8C. The transfer line and ion source were
maintained at 280 and 230 8C, respectively. The mass
spectra were recorded from m/e 40 to 500. The ionisation
energy was 70 eV. Samples (0.2–1 ml) were introduced in a
split mode with a split ratio from 5:1 to 40:1, depending on
the concentration of analytes. The oven temperature was
programmed as follows: the initial temperature was set at
100 8C, held for 2 min and ramped at 12 8C/min to 300 8C
and held for 7 min.
Second GC system consisted of Agilent 6850 GC
System equipped with Agilent 7683 Series Injector and
flame-ionisation detector (FID). During analysis of impur-
ity extracts, reference samples, and intermediate products,
30 m long, HP-5 column with internal diameter 0.25 mm
and 0.25 mm film thickness was used. Gas flow-rate
(helium) was 1 ml/min, split ratio varied from 5:1 to
50:1, and injector and detector temperatures were main-
tained at 250 and 290 8C, respectively. The oven tempera-
ture program was as in the case of the GC–MS system. The
retention indices measurements were performed on the
25 m long, HP-1 capillary column with internal diameter
0.2 mm and 0.33 mm film thickness; as a reference stan-
dards straight chain alkanes, namely heptacosane and
159
octacosane (Fluka) were used; oven was operated in the
isothermal condition (275 8C).
2.2. Synthesis
2.2.1. PMA experiments: general procedure
The mixture of 4-methoxyphenylacetone (20 mmol) and
formamide (100 mmol) (Version 1) was heated under reflux
on the oil bath (185 8C) for 6 h. Alternatively, formamide/
98% formic acid mixture (100 mmol/50 mmol) (Version 2)
or ammonium formate (100 mmol) (Version 3) was used
instead of pure formamide. To the cooled reaction mixture,
14 ml of concentrated HCl was added and the resulting
solution was kept under reflux for 3 h. After cooling, the
reaction mixture was poured into cold water (50 ml) and
made alkaline with NaOH pellets with external cooling. The
mixture was extracted with chloroform (2  15 ml). The
combined extracts were dried over Na2SO4 and the solvent
was evaporated under reduced pressure. Before evaporation
step a 0.5 ml amount of the organic extract was collected and
analysed by GC–MS and GC–FID. The sulfate salts from
crude amines were obtained according to the procedure
described earlier [15]. The beige or brown material thus
obtained was dried under vacuum (25 8C) and next was kept
in desiccator. The average yield was 20%. Each version of
reaction was repeated four times to assure its reproducibility.
According to Version 1 the additional run was done in a
scale about five times larger then described above. After
workup, crude PMA (15.5 g) as a dark viscous oil was
obtained. The product was divided into two parts. The first
one (approx. 3 g) was immediately precipitated as the sulfate
salt. The second one was vacuum distilled to afford 3.7 g of
purified amine, as a colourless oil. The amine base was
precipitated to give PMA sulfate as a white crystalline
product. Further workup and storage proceeded as described
previously. Overall yield was 18%.
In order to investigate the influence of formaldehyde on
pyridines content, two runs according to Version 1 was
additionally repeated. For these experiments 4-methoxyphe-
nylacetone contained addition of 15 and 50 ml of formalde-
hyde solution (ca 37%, w/w aq. soln.) was used, respectively.
2.2.2. a-Phenylacetoacetonitrile 7
a-Phenylacetoacetonitrile 7 was obtained according to
[25], m.p. 87–88 8C (lit. [25], m.p. 88.5–89.5 8C).
2.2.3. 4,6-Dimethyl-3,5-diphenyl-2-pyridone 9
4,6-Dimethyl-3,5-diphenyl-2-pyridone 9 was obtained
according to [26], m.p. 294–296 8C (lit. [26], m.p. 296–
297 8C); 1H NMR (500 MHz, CDCl3) d: 1.73 (s, 3H, CH3 at
C-4), 2.01 (s, 3H, CH3 at C-6), 7.15–7.42 (m, 10H, Harom two
phenyls), 12.89 (s, 1H, NH); 13C NMR (125 MHz, CDCl3) d:
18.18, 19.91, 121.71, 127.19, 127.55, 128.08, 128.32,
128.88, 130.49, 130.52, 136.67, 137.89, 141.14, 149.22,
163.66; IR (n): 3080, 2877, 1641, 1575, 1493, 1443,
1388, 1330, 1220, 751, 697 cm1.
160 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
2.2.4. 2-Chloro-4,6-dimethyl-3,5-diphenylpyridine
2-Chloro-4,6-dimethyl-3,5-diphenylpyridine 11 was pre-
pared according to [26] in slightly modified manner. The
magnetically stirred mixture of 2 g (6.8 mmol) of pyridone 9
and 12 ml (130 mmol) of POCl3 was heated on the oil bath
(145 8C) in thick-walled Mini Flask (Supelco) closed with
solid screw cups with Teflon/silicone septum. After 8 h of
heating the excess of POCl3 was evaporated under vacuum
and the dark brown oily residue was treated with the mixture
of ice and water (approx. 40 ml). The resulting mixture was
allowed to stand for 1 h and was extracted with dichloro-
methane (2  10 ml). The combined extracts were washed
with sodium bicarbonate solution (2  5 ml) dried over
sodium sulfate and evaporated in vacuo. The crude product
was purified by column chromatography (chloroform) to
give chloropyridine 11 (1.35 g, 63%) as a white solid, m.p.
223–224 8C (benzene) (lit. [26], 219–220 8C); MS (EI,
70 eV): 293 (M+, 35Cl), 295 (M+, 37Cl), 242, 256, 215,
115, 258, 278; 1H NMR (500 MHz, CDCl3) d: 1.76 (s, 3H,
CH3 at C-4), 2.28 (s, 3H, CH3 at C-6), 7.16–7.18 (m, 2H,
Harom), 7.23–7.25 (m, 2H, Harom), 7.37–7.41 (m, 2H, Harom),
7.44–7.48 (m, 4H, Harom); 13C NMR (125 MHz, CDCl3) d:
19.40, 23.64, 127.82, 128.03, 128.79, 129.11, 129.12,
129.58, 134.17, 136.44, 137.62, 138.49, 147.33, 148.53,
155.79; IR (n): 3053, 1563, 1540, 1439, 1410, 1384,
1366, 1261, 1220, 885, 757, 701 cm1.
2.2.5. 2,4-Dimethyl-3,5-diphenylpyridine
2,4-Dimethyl-3,5-diphenylpyridine 1 was prepared from
11 as described in [27], analytical sample was obtained after
column chromatography, using chloroform–methanol
99:1 (v/v) as eluent, m.p. 102–103 8C (lit. [27,21], m.p.
99–100.5 and 104 8C, respectively); for MS data see Fig.
5; 1H NMR (200 MHz, CDCl3) d: 1.94 (s, 3H, CH3 at C-4),
2.31 (s, 3H, CH3 at C-2), 7.17–7.51 (m, 10H, Harom, two
phenyls), 8.35 (s, 1Hpyridyl, C-6); 13C NMR (50 MHz,
CDCl3) d: 18.15, 23.61, 127.38, 127.45, 128.39, 128.85,
128.93, 129.54, 138.43, 139.10, 143.20, 147.68, 154.81; IR
(n): 3055, 3024, 2957, 2919, 1600, 1573, 1439, 1392, 993,
914, 766, 700 cm1.
2.2.6. a-(4-Methoxyphenyl)acetoacetonitrile
a-(4-Methoxyphenyl)acetoacetonitrile 8 was prepared
accordingly to the procedure described for 7, m.p. 79–
81 8C (lit. [28], m.p. 80–81 8C; MS (EI, 70 eV): 147,
132, 43, 146, 76, 103, 189, 91.
2.2.7. 4,6-Dimethyl-3,5-di-(40 -methoxyphenyl)-2-pyridone
10
The procedure for the preparation of 10 was adopted
from lit. [26]. Thus, a mechanically stirred mixture of 11 g
(58 mmol) of 8 and 15 g (91 mmol) of 4-methoxyphenyla-
cetone 6, 13 ml of glacial acetic acid and 13 ml of concen-
trated sulfuric acid was gently refluxed for 3 h. Still hot
viscous dark mixture was poured into ice-cold water
(150 ml) and neutralised with solid sodium bicarbonate.
The dark brown precipitate thus formed was washed with
mixture of acetone/diethyl ether 1:1. The crude pyridone 10
was chromatographed to give 7.25 g (37%) of light yellow
solid, eluted with ethyl acetate/methanol 99:2 (v/v), m.p.
253–254 8C; 1H NMR (500 MHz, CDCl3) d: 1.74 (s, 3H,
CH3 at C-4), 2.02 (s, 3H, CH3 at C-6), 3.81 and 3.84 (two s,
each 3H, O–CH3), 6.92 and 7.06 (4Harom, AA0 XX0 , phenyl),
6.94 and 7.21 (4Harom, AA0 XX0 , phenyl), 12.83 (s, 1H, NH);
13
C NMR (125 MHz, CDCl3) d: 18.20, 19.99, 55.38, 55.41,
113.75, 114.21, 121.13, 127.56, 129.01, 130.16, 131.52,
131.65, 141.13, 149.52, 158.63, 158.90; IR (n): 2995,
2899, 2835, 1631, 1544, 1509, 1465, 1283, 1242, 1174,
828, 748 cm1.
2.2.8. 2-Chloro-4,6-dimethyl-3,5-di-(40 -
methoxyphenyl)pyridine 12
A mixture of 4.15 g (11.7 mmol) of pyridone 10 and
21 ml (227 mmol) of POCl3 was heated on the oil bath
(140 8C) in equipment as in the case of 11. After 14 h most of
the POCl3 was evaporated in vacuo and black residue was
treated with ice and water (approx. 100 ml). The resulting
mixture was allowed to stand for 1 h and was extracted with
dichloromethane (3  20 ml). The combined extracts were
washed with sodium bicarbonate solution (2  10 ml), dried
over sodium sulfate and evaporated in vacuo. The crude
product was purified by column chromatography to give
chloropyridine 12 (cyclohexane/ethyl acetate 1:3, v/v)
(3.21 g, 73%) as a light brown solid, m.p. 212–213 8C;
MS (EI, 70 eV): 353 (M+, 35Cl), 355 (M+, 37Cl), 338,
302, 301, 286, 189, 285; 1H NMR (500 MHz, CDCl3) d: 1.79
(s, 3H, CH3 at C-4), 2.28 (s, 3H, CH3 at C-6), 3.86 (s, 6H,
two O–CH3), 6.97–7.36 (m, 8H, Harom, two phenyls); 13C
NMR (125 MHz, CDCl3) d: 19.31, 23.45, 55.24, 55.28,
113.97, 114.30, 129.73, 130.06, 133.64, 135.95, 147.93,
148.63, 155.81, 158.96, 159.11; IR (n): 2938, 2838, 1608,
1564, 1509, 1368, 1286, 1246, 1176, 1029, 830, 756 cm1.
2.2.9. 2,4-Dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 2
A magnetically stirred mixture of 750 mg (2.12 mmol) of
chloropyridine 12 and 200 mg of KOH in 150 ml of anhy-
drous ethanol was hydrogenated over 510 mg of palladium
on charcoal (10% Pd–C). After 24 h the catalyst was filtered
off and the resulting solution was evaporated to dryness to
give pyridine 2 as a yellow solid. Purification by column
chromatography (cyclohexane/ethyl acetate 1:2, v/v)
afforded a pure pyridine 2 (450 mg, 66%) as a white solid,
m.p. 93–94 8C; for MS data see Fig. 3; 1H NMR (200 MHz,
CDCl3) d: 1.95 (s, 3H, CH3 at C-4), 2.30 (s, 3H, CH3 at C-2),
3.85 and 3.87 (two s, each 3H, O–CH3), 6.98 and 7.26
(4Harom, AA0 XX0 , phenyl), 7.00 and 7.21 (4Harom, AA0 XX0 ,
phenyl), 8.31 (s, 1Hpyridyl, C-6); 13C NMR (50 MHz, CDCl3)
d: 18.19, 23.77, 55.26, 55.77, 113.80, 114.19, 130.07,
130.64, 130.94, 131.49, 135.19, 136.63, 143.41, 147.94,
155.10, 158.75, 158.97; IR (n): 2956, 2834, 1610, 1511,
1450, 1287, 1247, 1176, 1107, 1036, 830 cm1; Kovat’s
retention index (275 8C) 2711.
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
2.2.10. 2,6-Dimethyl-3,5-diphenyl-4-pyrone
2,6-Dimethyl-3,5-diphenyl-4-pyrone 15 was prepared
from dibenzylketone 13 and acetic acid in polyphosphoric
acid according to [29]; m.p. 212–214 8C (lit. [29], m.p. 213–
214 8C); MS (EI, 70 eV): 275, 274, 115, 43, 89, 202, 90, 63;
1
H NMR (200 MHz, CDCl3) d: 2.24 (s, 6H, two CH3), 7.25–
7.42 (m, 10H, two phenyls); 13C NMR (50 MHz, CDCl3) d:
18.93, 126.58, 127.80, 128.29, 130.52, 132.98, 161.83; IR
(n): 1647, 1614, 1490, 1434, 1405, 1364, 1307, 1233, 982,
757, 700 cm1.
2.2.11. 2,6-Dimethyl-3,5-diphenyl-4-pyridone 16
A mixture of 1.9 g (6.9 mmol) of pyrone 15, 25 ml of
aqueous ammonia (25%), and 30 ml of ethanol was heated in
sealed tube at 105 8C for 190 h. After cooling the reaction
mixture was evaporated in vacuo to the half of its initial
volume. The light brown precipitate was filtered off, washed
with ethanol and recrystallized from a large volume of
methanol to give 1.68 g (88%) of pyridone 16 as a beige
crystals; m.p. >380 8C with decomp.; MS (EI, 70 eV): 274,
273, 275, 115, 215, 89, 257, 230; 1H NMR (200 MHz,
DMSO) d: 2.09 (s, 6H, two CH3), 7.12–7.35 (m, 10H, Harom,
two phenyls); 13C NMR (50 MHz, CDCl3) d: 17.66, 126.30,
127.50, 130.58, 136.18, 142.60; IR (n): 1647, 1614, 1490,
1434, 1405, 1364, 1307, 1233, 982, 757, 700 cm1.
2.2.12. 4-Chloro-2,6-dimethyl-3,5-diphenylpyridine
4-Chloro-2,6-dimethyl-3,5-diphenylpyridine 17 was
obtained from 1.45 g (5.5 mmol) of pyridone 16 and 9 ml
(96 mmol) of POCl3 according to procedure described for 9.
Crude product was purified by column chromatography to
afford 1.45 g (79%) of chloropyridine 17 as a white solid.
Analytical sample (GC purity better than 95%) was obtained
after recrystallization of a small sample from ethanol; m.p.
215–216 8C; MS (EI, 70 eV): 293 (M+, 35Cl), 292, 294, 295
(M+, 37Cl), 215, 258, 115, 202; 1H NMR (200 MHz,
CDCl3) d: 2.34 (s, 6H, two CH3), 7.22–7.30 (m, 4H, Harom),
7.40–7.51 (m, 6H, Harom); 13C NMR (50 MHz, CDCl3) d:
24.00, 127.79, 128.58, 129.36, 133.30, 137.47, 155.98; IR
(n): 3055, 3029, 1599, 1567, 1529, 1439, 1405, 1072, 1028,
843, 756, 698 cm1.
2.2.13. 2,6-Dimethyl-3,5-diphenylpyridine 3
A solution of 230 mg (0.78 mmol) of chloropyridine 17
in dry ethanol (15 ml) and ethyl acetate (20 ml) was treated
with palladium on charcoal (10% Pd–C, 180 mg) and stirred
under hydrogen atmosphere (1 atm.) during 24 h. After this
period, the solution was filtered and filtrate was evaporated
in vacuo to yield yellow oil which slowly solidified upon
standing in refrigerator. Purification by column chromato-
graphy (chloroform/methanol, 99:1, v/v) afforded pyridine 3
as a white solid (135 mg, 66%). Analytical sample (GC
purity better then 95%) was obtained after recrystallization
from ethanol; m.p. 103.5–105 8C (lit. [21], m.p. 103 8C); for
MS spectrum see Fig. 5; 1H NMR (200 MHz, CDCl3) d: 2.55
(s, 6H, two CH3), 7.33–7.50 (m, 11H, 10Hphenyl and 1Hpyridyl
161
at C-4); 13C NMR (50 MHz, CDCl3) d: 23.05, 127.31,
128.37, 129.14, 134.31, 138.58, 139.77, 153.75; IR (n):
3056, 2994, 2919, 1602, 1544, 1452, 1428, 1220, 1074,
1027, 765, 701 cm1.
2.2.14. Ethyl 4-methoxyphenyl acetate
Ethyl 4-methoxyphenyl acetate was prepared from 4-
methoxyphenylacetic acid by usual method [24], b.p. 98–
102 8C (0.4 mmHg) (lit. [30], b.p. 108–110 8C (1 mmHg).
2.2.15. 1,3-Di-(40 -methoxyphenyl)acetone
1,3-Di-(40 -methoxyphenyl)acetone 14 was prepared
from ethyl 4-methoxyphenyl acetate according to [30];
m.p. 85–86 8C (lit. [30], m.p. 86–86.2 8C).
2.2.16. 2,6-Dimethyl-4-pyridone
2,6-Dimethyl-4-pyridone 20 was prepared from 2,6-
dimethyl-4-pyrone 19 according to literature [31], m.p.
224–225 8C (lit. [31], m.p. 225 8C).
2.2.17. 3,5-Dibromo-2,6-dimethyl-4-hydroxypyridine
3,5-Dibromo-2,6-dimethyl-4-hydroxypyridine 21 was
synthesised based on the method presented in [32]. The
solution of 6.2 g (50 mmol) of pyridone 20 and 5.6 g of KOH
in 150 ml of water was cooled in the ice bath. To the
vigorously stirred solution a sample of 6 ml (112 mmol)
of bromine was added during 20 min. The precipitate of 21
was filtered off and washed with a copious amount of water.
The product was practically insoluble in water, methanol,
chloroform and acetone. After drying, the resulting dibro-
mohydroxypyridine 21 (13.3 g, 94%) was used for the next
step without further purification, m.p. >310 8C decomp. (lit.
[33], m.p. 288 8C with decomp.); MS (EI, 70 eV): 281 (M+,
Br Br), 279 (M+, 2  79Br), 283 (M+, 2  81Br), 51, 79,
79 81
145, 146, 200; IR (n): 3249, 3074, 3055, 3002, 2930, 1602,
1557, 1057, 1001, 833, 749 cm1.
2.2.18. 4-Chloro-3,5-dibromo-2,6-dimethylpyridine 22
A mixture of 3.7 g (13.1 mmol) of 21 and 15.5 ml
(167 mmol) of POCl3 was heated at 135 8C (Supelco equip-
ment) for 8 h. As in the case of compound 11 a dark brown
solid was obtained after the workup, which was purified by
column chromatography (cyclohexane/ethyl acetate 1:1) to
afford compound 22 (2.64 g, 67%). Analytical sample of 22
was obtained after recrystallization from ethanol, m.p. 105–
106 8C; MS (EI, 70 eV): 293, 292, 294, 295, 215, 258, 202,
115; 1H NMR (200 MHz, CDCl3) d: 2.66 (s, 6H, two CH3);
13
C NMR (50 MHz, CDCl3) d: 26.03, 119.09, 144.35,
156.60; IR (n): 3002, 2958, 1520, 1433, 1350, 1267,
1056, 1021, 982, 843, 720, 665 cm1.
2.2.19. 3,5-Dibromo-2,6-dimethylpyridine 23
A mixture of 2.42 g (8.1 mmol) of trihalogenopyridine
22, 20 ml of 57% aqueous HI, and 2.5 g (81 mmol) of red
phosphorus was refluxed for 16 h. After being cooled, the
mixture was poured carefully into the 250 ml of saturated
162 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
aqueous sodium bicarbonate. The solution was extracted
with dichloromethane (3  30 ml). The combined extracts
were dried with Na2SO4 and the solvent was evaporated to
give crude dibromopyridine 23 as a yellow solid. Purification
by column chromatography (hexane/ethyl acetate 6:1) gave
1.62 g (67%) of white solid; m.p. 65–67 8C (lit [34], m.p.
68–69 8C); MS (EI, 70 eV): 265 (M+, 79Br81Br), 263 (M+,
2  79Br), 267 (M+, 2  81Br), 63, 184, 104, 186, 103; 1H
NMR (200 MHz, CDCl3) d: 2.58 (s, 6H, two CH3), 7.91 (s,
1Hpyridil, C-4); 13C NMR (50 MHz, CDCl3) d: 24.50, 118.35,
142.48, 155.50.
2.2.20. 2,6-Dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 1
A magnetically stirred mixture of 210 mg (0.79 mmol)
of dibromopyridine 23, 277 mg (1.8 mmol) of 4-methox-
yphenylboronic acid, 35 mg (0.026 mmol) of Pd(PPh3)4,
and 550 mg (6.5 mmol) of NaHCO3 in solvent system
consisted of benzene (15 ml), EtOH (1.5 ml), and water
(3.5 ml) was heated at 80 8C (oil bath) under a nitrogen
atmosphere for 24 h (Supelco equipment). The cooled
mixture was partitioned between benzene (25 ml) and
10% aqueous NaHCO3 (15 ml). The aqueous solution
was additionally extracted with 10 ml of benzene. The
combined organic extracts were washed with brine
(10 ml), dried with Na2SO4, and evaporated in vacuo to
give 265 mg of a crude product as a brown solid. Purifica-
tion by column chromatography (cyclohexane/ethyl acetate
95:5 ! 75:25) gave GC (>95%) and TLC pure pyridine 4
(239 mg, 95%) as a white solid. Analytical sample was
obtained after recrystallization from ethanol; m.p. 107–
108 8C; for MS spectrum see Fig. 3; 1H NMR (200 MHz,
CDCl3) d: 2.53 (s, 6H, two CH3), 3.85 (s, 6H, two O–CH3),
6.91 and 7.28 (8Harom, AA0 XX0 ), 7.36 (s, 1Hpyridyl, C-4);
13
C NMR (50 MHz, CDCl3) d: 23.05, 55.34, 113.82,
130.24, 132.19, 133.96, 138.66, 153.53, 158.93; IR (n):
2999, 2836, 1610, 1513, 1439, 1290, 1248, 1177,
1036, 833, 802 cm1; Kovat’s retention index (275 8C)
2711.
2.3. Sample preparation for GC–MS analysis
Pyridines 1–4 were dissolved in chloroform to obtain
final concentration between 1 and 2 mg/ml. Samples taken
from reaction mixtures (Section 2.2.1) were analysed with-
out further treatment.
Extraction of impurities from PMA sulfate: a sample
of 50 mg of 4-methoxyamphetamine sulfate was dissolved
in 2 ml of 0.3 M phosphate buffer (pH 7.0). The solution
was extracted by vigorous shaking with 500 ml of toluene
for 10 min. After layers separation, 1 ml of the organic
extract was injected into the GC–MS and GC–FID
systems.
The extraction of impurities from amphetamine sulfate
was performed according to previously described procedure
[35]. For this experiment, a sample of the real amphetamine
sulfate seized in illegal laboratory was used.
3. Results and discussion
3.1. Synthesis
The group of compounds bearing methyl, phenyl and
benzyl substituents on the pyridine ring takes a place of
considerable significance as a result of its presence in
virtually all samples of amphetamine sulfate produced by
the Leuckart method. These compounds are often named
‘‘high-boiling pyridines’’, probably due to a high tempera-
ture that is needed for their elution during the gas chroma-
tography analysis. Because of their exclusive connection
with the Leuckart method, they have been considered as a
‘‘route-specific impurities’’ and play important role in the
comparative analysis of amphetamine sulfate samples. For
the first time, the presence of 2,4-dimethyl-3,5-diphenylpyr-
idine 1, 2,6-dimethyl-3,5-diphenylpyridine 3, 2-benzyl-4-
methyl-5-phenylpyridine, 2-benzyl-6-methyl-5-phenylpyri-
dine, 2-benzyl-4,6-dimethyl-5-phenylpyridine in the
‘‘Leuckart’’ amphetamine has been reported by van der
Ark et al. [21,22], after their isolation from the crude
material before a steam distillation. The compounds of
interest were isolated after several acidic and basic extrac-
tions with subsequent TLC runs. According to our knowl-
edge, the assignments of the structure were made exclusively
on the basis of the proton and carbon NMR spectroscopy.
However, it seems that these results were not confirmed by
comparison with the data obtained for independently synthe-
sised reference samples. Only in the case of compound 3 the
differentiation between two possible symmetrical isomers
was supported by the examination of the NMR spectra of
synthesised 3,5-dimethyl-2,6-diphenylpyridine.
At the early stage of our work we planned to prepare 4-
methoxyphenyl pyridines by a direct adaptation of the
synthetic procedures available for their des-methoxy deri-
vatives. Surprisingly, the literature search revealed that
among five pyridines reported previously by van der Ark,
only 1 was a goal of the synthetic efforts. In the two
independent publications compound 1 was obtained after
condensation of benzylmethylketone 5 with a-phenylace-
toacetonitrile 7 in the presence of polyphosphoric acid [27]
or the mixture of acetic and sulfuric acid [26] (Scheme 1).
The intermediate pyridone 9 was chlorinated with POCl3/
PCl5 mixture [27] or POCl3 alone at elevated pressure [26] to
give chloropyridine 11. Removal of the chlorine atom was
accomplished by the catalytic hydrogenolysis over palla-
dium. We found that AcOH/H2SO4 based cyclization pro-
cedure worked better giving relatively pure pyridone 9,
which could be used for the next step without additional
purification. In turn, the conversions of 9 into chloropyridine
11 by means of POCl3/PCl5 treatment brought about the
formation of the desired product contaminated with a num-
ber polychlorinated pyridines, as was shown by GC–MS
examination. These by-products were formed probably as
the result of a competitive oxidative chlorination of 4- and 6-
methyl groups. The chloropyridine 11 was isolated as a sole
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
Scheme 1. a: H2SO4/AcOH, 140 8C, 2 h; b: POCl3, 140 8C, 24 h; c:
H2/Pd, 20 8C, 24 h.
product when POCl3 alone was used as a chlorinating agent
at the temperature slightly above its boiling point. This
procedure was used successfully by us in the chlorination
of other 2- and 4-pyridones mentioned in this paper. Having
in hands established conditions for the preparation of pyr-
idine 1, we were able to repeat the synthetic cycle using a
building block necessary for the formation of its methoxy
derivative (Scheme 1).
Thus, b-ketonitrile 8 was cyclized with 4-methoxyphe-
nylacetone 6 to form 2-pyridone 10 in 37% yield. Treatment
of 10 with POCl3 at 140 8C (thick-walled Supelco flask)
gave chloropyridine 12, from which the desired final 2,4-
dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 2 was obtained
by the catalytic reduction over palladium on charcoal.
As a second goal of our synthetic efforts we chose 2,6-
dimethyl-3,5-di-(40 -methoxyphenyl)pyridine 4. As in the
case of pyridines 1 and 3, we decided to develop a synthetic
method leading to 2,6-dimethyl-3,5-diphenylpyridine first.
We assumed that both pyridines 3 and 4 might be obtained in
a three-step transformation from 3,5-diaryl-2,6-dimethyl-4-
pyrones synthesised from the corresponding 1,3-diarylke-
tones, which are either commercially available or conveni-
ently prepared by relatively simple procedures. As can be
seen from Scheme 2, the pyridone 16 derived from corre-
Scheme 2. a: PPA, AcOH, 1.5 h, 120–130 8C; b: NH3, EtOH, 105 8C, 190 h; c: POCl3, 140 8C, 16 h; d: H2/Pd, 20 8C, 24 h.
163
sponding pyrone 14 was easily converted to pyridine 3 in the
manner described for its 2,4-dimethyl-2-keto-isomers 11
and 12.
Our attempts to produce 2,6-dimethyl-3,5-di-(40 -meth-
oxyphenyl)-4-pyrone 18 from 1,3-di-(40 -methoxyphenyl)a-
cetone 14 led surprisingly only to a complex mixture of
unidentified products, among which no detectable amount of
the desired pyrone could be found by GC–MS. Further
experiments with a number of arylacetones confirmed that
the presence of electro-donating group in the phenyl ring
completely suppress the double acylation and cyclization
sequence, which is crucial for the 4-pyrone ring formation.
The difference in outcome between the condensation of
phenyl ketones and their congeners bearing electron-donat-
ing group was probably caused by a more facile enolization
of the latter in acidic environment.
Since it proved impossible to synthesise pyridine 4 by the
route outlined in the Scheme 2, we decided to seek an
alternative way for its preparation, e.g. the arylation of
the appropriately functionalised 2,6-dimethylpyridine.
Although 3,5-diarylpyridines systems might be created in
a variety of ways, the direct arylation of a pyridine nucleus
has several limitations due to the different nature of aryl
group and the lack of selectivity [36]. In order to develop an
attractive, highly effective, and regioselective arylation
sequence towards the desired arylmethylpyridines, we drew
our attention to palladium-catalysed cross-coupling reaction
between aryl pyridyl halides with various coupling partners
including stannanes, Grignard, zinc reagents and arylboronic
acids. Among these, the Suzuki reaction appeared to be
especially attractive, mainly due to commercial availability
of the several arylboronic acids, which are air and moisture
stable (contrary to zinc and magnesium organo-metallics)
and non-toxic (contrary to trialkylarylstannes). These advan-
tages as well as numerous examples of the successful double
cross-coupling reaction between dibromopyridines and phe-
nyl [37], 4-metxoxyphenyl [38] pyridyl [39], naphthyl [40]
and sterically hindered boronic acids [41] prompted us to
consider them in planned reaction sequence. We expected
that the presence of the additional two methyl groups at
164 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
2- and 6-positions would not interfere with the desire course
of this reaction.
According to Scheme 3, appropriately functionalised 2,6-
lutidine 23 was synthesised, starting from the commercially
available pyrone 19, which upon amination to pyridone 20
followed by bromination, formed dibromopyridine 21.
Chlorination of compound 21 led to the trihalogeno-
derivative 22, from which chlorine function was smoothly
removed by refluxing with 57% HI. Finally, the introduction
of both 4-methoxyphenyl groups was accomplished in good
yield by the Suzuki cross-coupling reaction as a key step. It
should be noted here that the method used does not require
initial synthesis of the open-chain precursors bearing appro-
priately substituted phenyl ring, as is in the case of com-
pounds 1 and 3. Since the variously substituted aryl groups
can be introduced in the last step and the overall process is
simple, efficient and runs in mild conditions therefore it
appears to be a general and attractive synthetic route for the
preparation of different 3,5-aryl substituted 2,6-dimethyl-
pyridines, including parent 3,5-diphenyl derivative 3 itself
and other forensically relevant pyridines.
The synthesised pyridines 1–4, as well as others newly
reported compounds have been fully characterised on the
basic of their 1H and 13C NMR, IR and MS spectra. The
lacking spectroscopic data for the previously reported com-
pounds have also been completed.
3.2. Identification of pyridines in the ‘Leuckart’
preparations
The crude 4-methoxyamphetamine was obtained by
treating 4-methoxyphenylacetone with formamide accord-
ing to the procedure described in Section 2 (Version 1). The
product was analysed by GC–MS yielding typical ‘‘reach’’
chromatogram with PMA and previously identified pyrimi-
dines [13], diasteromeric amines and formamides [15] as a
major components. Having in hands reference samples of
both regioisomeric 40 -methoxyphenyl methylpyridines 2 and
4, we were able to confirm their presence in thus obtained
Fig. 2. The selected fragment of TIC chromatogram from crude PMA showing newly identified pyridines 2 and 4.
Scheme 3. a: NH3, 20 8C, 48 h; b: Br2/H2O, 0 8C, 2 h; c: POCl3,
135 8C, 8 h; d: HVP(red), reflux, 12 h; e: (4-MeO-C2H4)B(OH)2,
Pd(PPh3)4, NaHCO3, benzene/H2O/EtOH, 80 8C, 24 h.
‘‘Leuckart’’ PMA. For clarity, only selected fragment of TIC
containing peaks of the newly identified pyridines was
presented in Fig. 2.
The evidence for the assignment of the peaks at 18.50 and
18.58 min as pyridines 2 and 4, respectively, came from their
mass spectra and chromatographic data comparison with
those recorded for the synthetic material. As can be seen in
Fig. 3, the mass spectra of both pyridine isomers are very
similar, nevertheless they can be readily distinguished on the
basis of different retention times.
In a similar way, the presence of previously mentioned
[15] des-methoxy pyridines 1 and 3 in the ‘‘Leuckart’’
amphetamine was also indicated (Figs. 4 and 5).
The characteristic feature in the mass spectra of all
pyridines 1–4 is the presence of the predominant peaks of
molecular ions. The appearance of M+-15 ions can be
readily explained by the loss of methyl radical. In the mass
spectra of 1 and 3 an intense ions corresponding to the
elimination of hydrogen radicals from the parent ions were
also observed. In the case of 2 and 4 the similar process gave
significantly lower peaks of ions m/z 318. This behaviour can
be attributed to the presence of an electron-donating meth-
oxy groups that are probably responsible for the extra
stabilization of the parent ions of 2 and 4.
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173 165

Fig. 3. The EI mass spectra of pyridines 2 and 4.

It is also interesting to note that both pyridine pairs are
eluted in a similar order; the symmetrical derivatives 3 and 4
precede their unsymmetrical counterparts 1 and 2. This
phenomenon was observed only during the chromatographic
analysis performed on the capillary column with the (5%-
phenyl)-dimethylpolysiloxane-based stationary phase film.
Our attempts to resolve isomeric pyridines on the 100%
dimethylsilicone column (HP-1) using various temperature
programs were unsuccessful. We therefore recommend the
HP-5 or equivalent column for the analysis of this kind of
compounds.
3.3. Nitrogen-donating agent—a comparison
3.3.1. General
The first comprehensive and interesting study on the
different factors that influence the results of the Leuckart
synthesis was carried out by Crossley and Moore [42].
Various experimental conditions, namely the ratio of
reagent/substrate, the temperature of reaction, time of heat-
ing during the condensation step, and time and reagents used
during the hydrolysis of intermediary formylamine were
investigated. The effect of various reagents as well as the

Fig. 4. The selected fragment of TIC chromatogram from impurity extract of amphetamine sulfate obtained by the Leuckart method.
166 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
Fig. 5. The mass spectra of pyridines 1 and 3.
type of carbonyl compound, were also checked. The effec-
tiveness of the reaction was estimated on the basis of the final
amine yield and the weight of tars. The best yield of 3-
phenyl-2-aminobutane was obtained when the reaction was
carried out with the mixture of formamide and formic acid or
ammonium formate at 160–170 8C. The prolongation of the
reaction time brought about lowering the yield, especially
when the condensation was carried out at elevated tempera-
ture (190–200 8C). In the case of benzylmethylketone, the
effect of reagents used was not investigated, whereas tem-
perature/time fluctuations seemed not to be a predominant
factor, since the yield of amphetamine did not change
significantly.
The appearance of illegally produced amphetamine as a
street drug renewed the interest of forensic chemists in
modifications that might affect the so-called chemical sig-
nature—e.g. impurities content in the final product. The
influence of the various factors during the amphetamine
synthesis by the Leuckart method was examined [43–47]. It
was found that the addition of formic acid to formamide at
the first step of the synthesis decreased considerably the
amount of pyridine and pyrimidine derivatives, whereas the
presence N,N-di-(b-phenylisopropyl)amine was observed in
a large amount. Different temperature, reagents/substrates
molar ratio, prolonged time of the condensation step as well
as the workup procedure was proved to affect, however in
different extent, the amount of impurities, which in the early
study [42] were described as tars. Obviously, modern chro-
matographic (GC, HPLC) and spectroscopic (MS) techni-
ques available today allow forensic community to collect
detailed information concerning the nature and quantity of
the important impurities, which are present sometimes in
trace amounts.
The second aim of our work was to investigate the
influence of the exchange of the ‘‘Leuckart’’ nitrogen-donat-
ing agent on the overall impurity pattern. We ran three series
of condensations of 4-methoxyphenylacetone with different
kind of nitrogen-donating reagent, namely formamide alone,
mixture of the formamide with formic acid and ammonium
formate. The ammonium formate was used in our experi-
ment because this reagent is widely used by illegal producers
of Polish amphetamine. For each variant of the synthesis, an
identical temperature and time during the condensation step
was applied. In order to discard other parameters that would
affect the by-product content and the level of impurities,
common purification procedure after the hydrolysis step was
omitted. Samples for GC–MS analysis were obtained by the
removal of aliquots from the reaction mixture after the
hydrolysis step with subsequent alkalisation and extraction
into chloroform.
For better clarity it seemed reasonable to split further
discussion in three parts. In the first one we show the changes
in whole chromatographic pattern derived from the
exchange of the reagent. In the second, we turn our attention
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173 167

to the interesting fluctuation within pyridine ‘‘family’’. In
the third, we try to give some explanation concerning our
observations.
3.3.2. Impurity profiles
The results of GC–MS analysis of the crude PMA
samples obtained after the treatment of 4-methoxyphenyla-
cetone with formamide, mixture of formamide/HCOOH,
and ammonium formate are shown in Fig. 6A–C, respec-
tively.
During the first synthesis in which formamide was used,
4-methyl-5-(40 -methoxyphenyl)pyrimidine and 4-(40 -meth-
oxybenzyl)pyrimidine were detected as main by-products.
The pyrimidine content was about 2–3 times greater then the

Fig. 6. Typical TIC chromatograms of crude PMA samples obtained by the Leuckart method using different reagents: A—formamide, B—
formamide/HCOOH, C—ammonium formate.
168 D. Błachut et al. / Forensic Science International 152 (2005) 157–173

methoxy derivatives of N,N-di-(b-phenylisopropyl)amine the nitrogen-donating reagent. The additional detailed

and N,N-di-(b-phenylisopropyl)formamide. Significant
amount of pyridine derivatives was also observed. The
addition of formic acid reduced the total intensity of the
profile. The general appearance was similar to those
obtained during the first series, with one exception for
methoxy derivative of N,N-di-(b-phenylisopropyl)amine,
which content rose reaching the level similar to that of
corresponding N,N-di-(b-phenylisopropyl)formamide. This
result is consistent in part with the reported by Huizer et al.
[44], who found that N,N-di-(b-phenylisopropyl)amine dia-
stereomers were the predominant by-products in ampheta-
mine obtained in similar conditions. The downward trend in
pyridines formation was noted accompanied by observed
reduction of the intensity by roughly two orders of magni-
tude.
A completely different result was obtained when analo-
gous experiment was carried out in the presence of ammo-
nium formate. Only a trace amounts of methoxypyrimidines
and pyridine derivatives could be detected, whereas methoxy
derivatives of N,N-di-(b-phenylisopropyl)amine and N,N-di-
(b-phenylisopropyl)formamide were predominant in the
reaction mixture.
3.3.3. Fluctuation in the pyridines profile
We have observed that quantity of pyridines as a whole
set of compounds seemed to be dependent on the nature of
inspection of TIC chromatogram indicated the presence
of interesting fluctuations within this group. We noticed
that three previously tentatively identified pyridines with
molecular weight Mw = 333 predominated over their analogs
(Mw = 319) in the case when ammonium formate was used in
the condensation step. For simplicity, the pyridines with
molecular weight at 319 will be further described as Pyr319,
whereas their analogs (Mw = 333) receive abbreviation
Pyr333 (for a full list of abbreviations, see footnote in
Table 1). In order to create data set that would be useful
in estimation of this phenomenon, the following procedure
was used. First, the two groups of pyridine derivatives were
created, following their identification by means of MS
spectra. The first set consisted of five compounds Pyr319,
including unambiguously identified compounds 2 and 4. The
second set grouped those compounds that were characterised
by the predominant peak m/z 333 (Pyr333). Next, we
extracted two ion chromatograms from the data file for
selected mass 319 and 333, respectively. This was necessary
for an exact peaks area calculation, since in certain cases the
peaks of interest slightly coeluted with those of unidentified
compound. After integration, the ratios of the summarised
peaks areas of Pyr333 and Pyr319 for each chromatographic
run were calculated and were summarised in Table 1.
The pyridines ratio SPyr333/SPyr319 obtained in the
case of ammonium formate modified reagent is significantly

Table 1
Comparison of pyridines area ratiosa
Experiment Mass spectrometric detectionb Flame-ionization detectionc, S4/S2 (S.D.)
SPyr333/SPyr319 (S.D.) S4/S2 (S.D.) Crude extract Sulfate
Crude extract Sulfate Crude extract Sulfate
Version 1/Run 1 0.20 (0.031) 0.25 (0.034) 0.70 (0.021) 0.80 (0.018) 0.64 (0.031)) 0.71 (0.046)
Version 1/Run 2 0.18 (0.025) 0.18 (0.032) 0.70 (0.019) 0.66 (0.016) 0.69 (0.038) 0.68 (0.043)
Version 1/Run 3 0.23 (0.029) 0.21 (0.034) 0.69 (0.019) 0.76 (0.021) 0.69 (0.035) 0.66 (0.053)
Version 1/Run 4 0.27 (0.036) 0.25 (0.031) 0.86 (0.020) 0.74 (0.017) 0.81 (0.036) 0.75 (0.051)
Version 1/Run 5d 0.13 (0.019) 0.120.15 e 0.81 (0.017) 0.82, 0.84f 0.71 (0.032) 0.72, 0.63f
Version 2/Run 1 0.09 (0.011) 0.09 (0.015) 2.27 (0.047) 2.32 (0.039) 2.76 (0.063) 2.05 (0.066)
Version 2/Run 2 0.09 (0.012) 0.09 (0.013) 2.12 (0.042) 2.16 (0.038) 2.62 (0.054) 2.23 (0.090)
Version 2/Run 3 0.06 (0.010) 0.04 (0.008) 2.03 (0.044) 2.24 (0.046) 2.62 (0.069) 1.62 (0.098)
Version 2/Run 4 0.05 (0.009) 0.04 (0.007) 1.99 (0.039) 2.12 (0.041) 1.64 (0.049) –g
Version 3/Run 1 0.83 (0.041) 1.07 (0.046) 0.47 (0.018) 0.52 (0.018) 0.52 (0.044) 0.45 (0.039)
Version 3/Run 2 2.55 (0.095) 5.06 (0.066) 0.55 (0.016) 0.42 (0.023) 0.48 (0.052) –g
Version 3/Run 3 2.67 (0.110) 2.98 (0.049) 0.55 (0.015) 0.67 (0.016) 0.36 (0.061) 0.41 (0.055)
Version 3/Run 4 2.63 (0.096) 2.24 (0.053) 0.60 (0.016) 0.66 (0.024) –g –g
a
The abbreviations are as follows: SPyr333—summarised peaks area of pyridines Pyr333; SPyr319—summarised peaks area of pyridines
Pyr333; S4—peak area of pyridine 4; S2—peak area of pyridine 2; S.D.—standard deviation; description ‘‘sulfate’’ is related to the crude,
unpurified PMA sulfate.
b
Mean value, manual injection (n = 3).
c
Mean value, automated injection (n = 4).
d
The crude PMA was purified by vacuum distillation (see Section 2.2.1).
e
The first number in line is related to the crude salt of PMA whereas the second is related to the purified product (see Section 2.2.1), in both
cases S.D. 0.023.
f
The first number in line is related to the crude salt of PMA whereas the second is related to the purified product (see Section 2.2.1). The
calculated standard deviations are as follows: MS detection—0.019 and 0.022; FID detection—0.042 and 0.045, respectively.
g
Because of the co-elution with unknown compound no useful data for computation were obtained.
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
Fig. 7. The ‘‘pyridine region’’ of extracted ion chromatograms of crude PMA samples obtained by the Leuckart method using different reagents.
greater than those measured for formamide and formamide/
formic acid based experiments. The later modifications give
the pyridines ratio within the ranges 0.13–0.27 and 0.05–
0.09, respectively. Another interesting phenomenon is con-
nected with the distribution of pyridines 2 and 4, whose
content was found to be also associated with reagents
constitution. The isomer ratios (S4/S2) measured for extracts
obtained according to Versions 1 and 3 are <1, whereas that
for HCOOH modified procedure, reached values greater than
2. For clear visualization, the relationships between selected
pyridines were presented additionally in a graphic form as
selected ion chromatograms (Fig. 7).
It should be noted that the observed pyridines ratios were
generally unchanged after the transformation of the crude
amine PMA into the sulfate salt. In the case of S4/S2 ratio
the GC–MS results were confirmed additionally by GC runs
with flame-ionisation detection. We then assumed that simi-
lar physical and chemical properties of the isomeric pyr-
idines would allow conveying them as a whole group even
over the accurate and extensive purification procedure. Thus,
the information of the nature of the aminating reagent used is
included in the specific profile of pyridine group and the
whole their profile is conveyed over the purification proce-
dure albeit with lower total intensity. In order to verify this
hypothesis an additional run (Version 1/Run 5) was per-
formed. Part of the crude PMA thus obtained was distilled
under vacuum to give pure amine base as clear oil which was
subsequently precipitated into the form of sulfate salt. As
can be seen from Table 1, the measured pyridines ratios
remained generally unchanged after the distillation process.
In contrast to this observation, the whole chromatographic
pattern (Fig. 8) has changed dramatically.
169
3.3.4. Attempted rationalization
The results of our experiments may be partially rationa-
lised on the basis of the following transformation scheme.
Apart from the desirable action of the nitrogen donating
reagents leading to N-formyl-4-methoxyamphetamine inter-
mediate, two competitive routes for the formation of by-
products exist (Scheme 4). In the first one (Route A) two
molecules of formamide and one of the ketone or/and two
molecules of the ketone and one of formamide undergo
condensation process giving methoxyphenyl/benzyl pyrimi-
dines and/or corresponding pyridines, respectively. In the
second one, the intermediate imine is reduced to secondary
dimeric amine with subsequent formylation and reduction
yielding N-formyl and N-methyl derivatives (Route B).
Thus, high content of formamide in the reaction mixture
favours heterocycles formation. Because of the ability of
HCOOH to reduce unstable dimeric imine, its addition to the
reaction mixture must shift the position of equilibrium into
the right side of the reaction, which in consequence results in
a higher amount of the secondary amine. Accordingly, the
absence of formamide during ammonium formate based
modification brings about the formation of crude PMA
practically devoid of pyridines and pyrimidines.
As was mentioned above, the pyridines were formed in
the reaction mixture in a condensation process in which two
molecules of a ketone and one of formamide take part. In
fact, this explanation is slightly simplified and clarifies
exclusively the way by which pyridine isomers bearing a-
hydrogen are formed. This kind of condensation is some-
times called 2-2-1 type, according to the arrangement of
two- and one-carbon ketonic components, which take part in
the heterocyclic ring construction [48] (Scheme 5).
170 D. Błachut et al. / Forensic Science International 152 (2005) 157–173

Fig. 8. The TIC chromatogram of PMA sulfate impurity extracts: A—crude salt, B—salt obtained after vacuum distillation.

The formation of compound 2 proceeds by the route
called 2–1–2 type and besides the arrangement of the two-
carbon donors, quite different a one-carbon donor is
required. In our opinion, the simplest and most logical
choice is formaldehyde alone. In order to verify the
idea that formaldehyde is involved as a C-4 donor in
the 2–1–2 type condensation, another test experiment
was designed. We therefore performed two additional
reactions according to Version 1, in which two samples
of 4-methoxyphenylacetone containing 15 and 50 ml of
formaldehyde solution (see Section 2.2.1) were employed,
respectively. As can be seen from Fig. 9, the experiment
brought about the formation of PMA with S4/S2 ratio
exceeded 2.

Scheme 4.
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173 171

Scheme 5.

Fig. 9. The ‘‘pyridine region’’ of extracted ion chromatograms (m/z 319) of crude PMA samples obtained from 4-methoxyphenylacetone mixed
with formalin. The amount of formalin: A—15 ml, B—50 ml.

Additionally, we observed the relation between the con-
centration of formaldehyde in 4-methoxyphenylacetone and
S4/S2 value. Having demonstrated that formaldehyde serves
as important C-4 atom donor, we still had a question to
answer about its origin in the reaction mixture. It is likely
that this aldehyde is formed from formic acid during the
course of the Leuckart reaction or/and is initially introduced
to the reaction mixture as formic acid impurity. It should be
stressed here that in the formamide and ammonium formate
based runs formic acid is formed during the decomposition
of the amide or disproportionation of the salt. It therefore
could be concluded from the above observations that the
2-1-2 type condensation process predominates when an
excess of formic acid is present in the reaction mixture
(Version 2) during the formation of pyridine 2 and 4. It
is additionally consistent with general rules described earlier
in [49].
The consistent explanation of differences in compounds
distribution within Pyr333 and Pyr319 families is still to be
done. Although we cannot give at the present moment the
complete rationalisation of this phenomenon, but it certainly
must be associated with the nature of reagent that is being
used during the condensation step. The exact factors that
contribute to the observed fluctuations will need additional
studies including the identification and synthesis of the other
members of the pyridine family.
4. Summary
The treatment of 4-methoxyphenylacetone with forma-
mide or ammonium formate under the Leuckart condition
leads to 4-methoxyamphetamine (PMA) accompanied with
a large number of impurities, including previously reported
172 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
derivatives of N,N-di-(b-phenylisopropyl)amine and pyri-
midines. In this study, two new products from the group of
‘‘high-boiling pyridines’’ were synthesised, characterised
and subsequently identified in crude as well as in the purified
PMA. The isomeric pyridines 2 and 4 were fully charac-
terised by means of MS, 1H NMR, 13C NMR, IR and GC
methods. The new preparation method leading to 2,6-
dimethyl-3,5-diphenylpyridine 3, which is known from
the amphetamine chemistry, is also reported.
We have also demonstrated that diverse nitrogen-donat-
ing reagents showed significant impact on the by-product
content in ‘‘Leuckart’’ 4-methoxyamphetamine. Unfortu-
nately, when determination of the reagent is made by
inspection of the overall chromatographic profile, it must
be limited only to the crude products e.g. crude amine base
or reaction mixtures submitted from clandestine laboratory.
Since the chromatographic profile as a whole can be strongly
affected by the workup procedure, the more promising
results have been obtained when the comparison was limited
to pyridine derivatives only. The relative amount of pyri-
dines Pyr333 and Pyr319 seems to be useful in discrimina-
tion between formamide and ammonium formate based
reaction while ratio of isomers 2 and 4 may be indicative
for formic acid modified procedure. The advantage of such
an approach may consist of fact that the information about
reagent included in the pyridines profile seems to be con-
veyed from the crude reaction mixture into the carefully
purified final product. In our opinion, the ability to find out
what reagent was used during the Leuckart synthesis of
PMA may serve as an additional important factor in the more
detailed forensic examination of the submitted samples.
Although we have not yet carried out similar comprehensive
study on another members of amphetamine family, a few
preliminary trial reactions have already been done. The first
results point out that it should be possible to apply this
approach during the analysis of another forensically relevant
phenylisopropylamines, including amphetamine itself.
Acknowledgments
A part of this paper was preliminary presented as a poster
at the Third European Academy of Forensic Science Meet-
ing on September 22–27, 2003, Istanbul, Turkey. This work
was supported by the Polish State Committee for Scientific
Research, grant 4 T09A 11222. The authors would like to
thank Mrs. Maria Lisiak and Mr. Sławomir Szczepańczyk,
for IR measurements. The authors also thank Dr. Aleksandra
Tucholska-Lenart for her kind support.
References
[1] K. Ostaszewski, Trends in psychoactive substance use among
adolescents. The Mokotów study. continuation, Alkoholizm i
Narkomania 14 (3) (2001) 387–406 (in Polish).
[2] L.A. King, A.J. Poortman van-der Meer, New synthetic drugs
in the European Union, Sci. Justice 41 (3) (2001) 200–202,
and references cited therein.
[3] A.T. Shulgin, D.E. Nichols, in: R.C. Stillman, R.E. Willette
(Eds.), The Psychopharmacology of Hallucinogens, Pergamon
Press, New York, 1987, p. 74.
[4] D.E. Nichols, A.J. Hoffman, R.A. Oberlender, P. Jacob III, A.T.
Shulgin, Derivatives of 1-(1,3-benzodioxol-5-yl)-2-butana-
mine: representatives of a novel therapeutic class, J. Med.
Chem. 29 (1986) 2009–2015.
[5] U. Braun, A.T. Shulgin, G. Braun, Centrally active N-sub-
stituted analogs of 3,4-methylenedioxyphenylisopropylamine
(3,4-methylenedioxyamphetamine), J. Pharm. Sci. 69 (1980)
192–195.
[6] T.A. Dal Cason, The characterization of some 3,4-methyle-
nedioxyphenylisopropylamine (MDA) analogs, J. Forensic
Sci. 34 (1989) 928–961.
[7] D.E. Nichols, R. Oberlender, Structure–activity relationships
of MDMA-like substances, in: K. Ashgar, E. De Souza (Eds.),
Pharmacology and Toxicology of Amphetamine Related
Drugs (NIDA Research Monograph 94), U.S Government
Printing Office, Washington, DC, 1989, pp. 1–29.
[8] F.T. Noggle Jr., C.R. Clark, S. Andurkar, J. DeRuiter, Methods
for the analysis of 1-(3,4-methylenedioxyphenyl)-2-butana-
mine and N-methyl-1-(3,4-methylenedioxyphenyl)-2-propa-
namine (MDMA), J. Chromatogr. Sci. 29 (1991) 103–
106.
[9] C.R. Clark, J. DeRuiter, A. Valaer, F.T. Noggle, Gas chroma-
tographic–mass spectrometric and liquid chromatographic
analysis of designer butanamines related to MDMA, J. Chro-
matogr. Sci. 33 (1995) 328–337.
[10] C.R. Clark, J. DeRuiter, F.T. Noggle, Chromatographic and
mass spectrometric methods for the differentiation of N-
methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine from
regioisomeric derivatives, J. Chromatogr. Sci. 34 (1996)
230–237.
[11] G. Cimbura, PMA deaths in Ontario, CMA J. 110 (1974)
1263–1267.
[12] C. Lora-Tamayo, T. Tena, A. Rodriguez, Amphetamine deri-
vative related deaths, Forensic Sci. Int. 85 (1997) 149–157.
[13] J.C. Coumbaros, K.P. Kirkbride, G. Klass, Application of
solid-phase microextraction to the profiling of an illicit drug:
manufacturing impurities in illicit 4-methoxyamphetamine, J.
Forensic Sci. 44 (1999) 1237–1242.
[14] T.A. Dal Cason, The identification of 4-methoxyamphetamine
(PMA) and 4-methoxymethamphetamine (PMMA), Micro-
gram 33 (8) (2000) 207–221.
[15] D. Błachut, K. Wojtasiewicz, Z. Czarnocki, Identification and
synthesis of some contaminants present in 4-methoxyamphe-
tamine (PMA) prepared by the Leuckart method, Forensic Sci.
Int. 127 (2002) 45–62.
[16] D. Błachut, The use of gas chromatography (GC), mass
spectrometry (MS), and Fourier transformation infrared spec-
trometry (FTIR) in analysis of p-methoxyamphetamine iso-
mers, Problemy Kryminalistyki 235 (2002) 7–20, issue
contains translation into English.
[17] R.W. Byard, J.D. Gilbert, R.A. James, R.J. Lokan, Ampheta-
mine derivative deaths in South Australia—is ecstasy the
culprit, Am. J. Forensic Med. Pathol. 3 (1998) 261–265.
[18] T.L. Martin, Three cases of fatal paramethoxyamphetamine
overdose, J. Anal. Toxicol. 25 (2001) 649–651.
 D. Błachut et al. / Forensic Science International 152 (2005) 157–173
[19] D.E. Nichols, M. Ilhan, J.P. Long, Comparison of cardiovas-
cular, hyperthermic, and toxic effects of para-methoxyamphe-
tamine (PMA) and 3,4-methylenedioxyamphetamine (MDA),
Arch. Int. Pharmacodyn. 214 (1975) 133–140.
[20] D. Błachut, J.K. Maurin, W. Starosta, K. Wojtasiewicz, Z.
Czarnocki, (2S)-1-(4-Methoxyphenyl)-N-[(1R)-2-(4-methox-
yphenyl)-1-methylethyl]-2-propanamine in crude p-methox-
yamphetamine (PMA) produced by the Leuckart method, Z.
Naturforsch. 57b (2002) 593–597.
[21] A.M. van der Ark, A. Sinnema, A.B.E. Theeuwen, J.M. van der
Toorn, A.M.A. Verweij, Impurities in illicit amphetamine, 3.
Isolation and identification of 2,4-dimethyl-3,5-diphenyl pyr-
idine, 2,6-dimethyl-3,5-diphenyl pyridine and 4-methyl-5-
phenyl-2-(phenylmethyl)pyridine, Pharmaceutisch Weekblad
113 (1978) 41–45.
[22] A.M. van der Ark, A. Sinnema, J.M. van der Toorn, A.M.A.
Verweij, Impurities in illicit amphetamine, 4. Isolation and
identification of 2-methyl-3-phenyl-6-(phenylmethyl)pyridine
2,4-dimethyl-3-phenyl-6-(phenylmethyl)pyridine, Pharma-
ceutisch Weekblad 113 (1978) 341–343.
[23] A.M. van der Ark, A.M.A. Verweij, A. Sinnema, Weakly basic
impurities in illicit amphetamine, J. Forensic Sci. 23 (1978)
693–700.
[24] A.J. Vogel, Textbook of Practical Organic Chemistry, Long-
man, New York, 1978.
[25] P.L. Julian, J.J. Oliver, R.H. Kimball, A.B. Pike, G.D. Jeffer-
son, a-Phenylacetoacetonitrile, Org. Synth. Coll. 2 (1943)
487–489.
[26] J.F.M. Wajon, J.F. Arens, Formation of a-pyridones from a-
acylbenzyl cyanides, Recl. Trav. Chim. Pays-Bas. 76 (1957)
65–74.
[27] C.R. Hauser, C.J. Eby, Cyclization of b-ketonitriles or b-
ketoamides with ketones by polyphosphoric acid to form
substituted 2-pyridones, J. Am. Chem. Soc. 79 (1957) 728–
731.
[28] C.E. Cook, R.C. Corley, M.E. Wall, I. Flavonoids, Synthesis of
2,2-dialkyl-D3-isoflavenes from coumarins, J. Org. Chem. 30
(1965) 4114–4120.
[29] T.L. Emmick, R.L. Letsinger, Org. Synth. 47 (1967) 54–56.
[30] S.B. Coan, D.E. Trucker, E.I. Becker, The absorption spectra
of tetracyclones, J. Am. Chem. Soc. 77 (1954) 60–66.
[31] K.N. Campbell, J.F. Ackerman, B.K. Campbell, Studies on g-
pyrones. II. Synthesis of 4-piperidinols from pyrones, J. Org.
Chem. 15 (1950) 337–342.
[32] O.S. Tee, M. Paventi, Kinetics and mechanism of the bromina-
tion of 4-pyridone and related derivatives in aqueous solution,
Can. J. Chem. 61 (1983) 2556–2562.
[33] Nippon Kagku Zasshi 79 (1958) 840–836; Chem. Abstr.
54:4553b.
173
[34] A.D. Dunn, S. Guillermic, The bromination of lutidines, Z.
Chem. 28 (1988) 59–60.
[35] D. Błachut, K. Wojtasiewicz, Z. Czarnocki, a-Phenylethyla-
mine in illegally produced amphetamine, Forensic Sci. Int. 123
(2001) 182–190.
[36] R.G. Micetich, in: R.A. Abramovitch (Ed.), The Chemistry of
Heterocyclic Compounds, vol. 14, Supplement Part 2, Wiley,
New York, 1974, p. 263.
[37] Y. Miura, S. Kurokawa, M. Nakatsuji, Pyridyl-substituted
thioaminyl stable free radicals: isolation, ESR spectra, and
magnetic characterization, J. Org. Chem. 63 (1998) 8295–
8303.
[38] F. Bracher, J. Daab, Total synthesis of the indolizidinium
alkaloid ficuseptine, Eur. J. Org. Chem. (2002) 2288–2291.
[39] M. Ishikura, T. Ohta, M. Terashima, A novel synthesis of 4-aryl
and 4-heteroarylpyridines via diethyl(4-pyridil)borane, Chem.
Pharm. Bull. 33 (1985) 4755–4763.
[40] C.M. Unrau, M.G. Campbell, V. Snieckus, Directed ortho
metalation–Suzuki cross-coupling connections, convenient
regiospecific routes to functionalized m- and p-teraryls and
m-quinquearyls, Tetrahedron Lett. 33 (1992) 2773–2776.
[41] F.C. Krebs, M. Jørgensen, A new versatile receptor platform, J.
Org. Chem. 66 (2001) 6169–6173.
[42] F.S. Crossley, M.L. Moore, Studies on the Leuckart reaction, J.
Org. Chem. 9 (1944) 529–536.
[43] D.G. Sanger, I.J. Humphreys, A.C. Patel, M. Japp, R.G.L.
Osborne, The significance of gas chromatographic impurity
patterns obtained from illicitly produced amphetamine, For-
ensic Sci. Int. 28 (1985) 7–17.
[44] H. Huizer, H. Brussee, A.J. Poortman-van der Meer, Di-(b-
phenylisopropyl)amine in illicit amphetamine, J. Forensic Sci.
30 (1985) 427–438.
[45] R. Kronstrad, The conditions of the first step of the Leuckart
synthesis of amphetamine and their influence on the impurity
profile, Report 22, The National Laboratory of Forensic
Science, Linköping, Sweden, 1990.
[46] Å. Johansson, Analysis of the impurity profile in Leuckart
amphetamine. Influence of variation in condensation and
purification steps, Report 23, The National Laboratory of
Forensic Science, Linköping, Sweden, 1991.
[47] A. Lindberg, Imitation of illicit amphetamine production using
an authentic recipe, Report 24, The National Laboratory of
Forensic Science, Linköping, Sweden, 1991.
[48] E. Klinsberg, Pyridine and Its Derivatives in The Chemistry of
Heterocyclic Compounds, Part One, Interscience Publishers,
New York, 1960, p. 474.
[49] E. Klinsberg, Pyridine and Its Derivatives in the Chemistry of
Heterocyclic Compounds, Part One, Interscience Publishers,
New York, 1960, p. 498.