Metabolism of N,N-dialkylated amphetamines, including deprenyl, by CYP2D6 expressed in a human cell line

Article abstract of DOI:10.1080/004982500237686

1. Five N,N-dialkylated amphetamines, N-methyl-N-propargylamphetamine (deprenyl; DEP), N-benzyl-N-methylamphetamine (benzphetamine; BPA), N-allyl-N-methylamphetamine (AMA), N,N-diallylamphetamine (DAA) and N-methyl-N-propyl-amphetamine (MPA), were metabol

Full text of DOI:10.1080/004982500237686

xenobiotica
vol
no
. 30, . 3, 297±306
, 2000,
-
Metabolism of N,N dialkylated amphetamines,
including deprenyl, by CYP2D6 expressed in a
human cell line
‹
‹
Œ
M. V. BACH , R. T. COUTTS * and G. B. BAKER
Neurochemical Research Unit, ‹Faculty of Pharmacy and Pharmaceutical Sciences, and
ŒDepartment of Psychiatry, University of Alberta, Edmonton, Alberta T6G 2N8,
Canada
Received 21 June 1999
-
-
-
-
-
-
1. Five N,N dialkylated amphetamines, N methyl N propargylamphetamine (de
-
-
-
-
-
prenyl; DEP), N benzyl N methylamphetamine (benzphetamine ; BPA), N allyl N
-
-
-
-
-
methylamphetamine (AMA), N,N diallylamphetamine (DAA) and N methyl N propyl
amphetamine (MPA), were metabolized in vitro with a microsomal preparation from cells
-
expressing human CYP2D6 to determine what in uence the N,N dialkyl substituents had
}
-
on the extent of N dealkylation and or aromatic ring oxidation.
2. The results obtained from experiments with the Ž rst two substrates, DEP and BPA,
å
-
-
were surprisingly di erent. WhereasDEP was N demethylated and N depropargylatedby
the CYP2D6 enzyme system, no metabolites were formed from BPA. Subsequently, it was
-
-
-
determined that AMA, DAA and MPA also underwent CYP2D6 catalysed N dealky
-
-
-
lation. Both N methyl and N allylamphetamine were identiŽ ed as products of AMA
-
-
-
-
metabolism; similarly, metabolism of MPA produced both N methyl and N propargyl
-
amphetamine, and N allylamphetamine was the sole metabolite of DAA.
-
3. No N,N didealkylated product (i.e. amphetamine) was isolated from incubates of
-
any of the Ž ve substrates, and none of the N,N dialkylated substrates was metabolized to
-
a ring hydroxylated product.
-
4. Rates of these CYP2D6 catalysed reactions were dependent on the nature and
-
degree of unsaturation of the N substituents.
Introduction
Our interest in the involvement of CYP2D6 in the N dealkylation of amphet
et al
-
-
-
amine derivatives (Bach . 1999) has been extended to include N,N dialkylated
-
derivatives. In a preliminary study, benzphetamine [( 1 ) N benzyl N methyl
-
-
-
-
-
-
2
-
- -
amphetamine; BPA] and ( ) deprenyl[( ) N methyl N propargylamphetamine;
2
DEP] were metabolized with a fortiŽ ed expressed CYP2D6 enzyme preparation
using experimental conditions that had previously been employed successfully to
metabolize other amphetamine derivatives and some tricyclic antidepressants
et al
. 1994a, Bach
et al
. 1999). Surprisingly, whereas DEP was extensively
(Coutts
metabolized to two major products in this system, no metabolites were produced
et al et al
from BPA. Others (Grace
. 1994, Wacher . 1996) have since identiŽ ed
-
-
-
DEP’s two CYP2D6 catalyzed metabolites as N methylamphetamine(MA) and N
propargylamphetamine (PgA), but there was an appreciable diåerence in the
relative amounts of both metabolites produced by recombinant CYP2D6 and
-
-
CYP2D6 containing liver microsomes. PgA formation was favoured 13 fold over
et al
MA formation in the studies by Grace
. (1994), but both metabolites were
et al
et al
.
isolated by Wacher
. (1996) in approximately equal quantities. Grace
-
* Author for correspondence; e mail: ronald.coutts!ualberta.ca
}
ISSN 0049 8254 print ISSN 1366 5928 online ’ 2000 Taylor & Francis Ltd
Xenobiotica
±
}}
±
}
} }
http: www.tandf.co.uk journals tf 00498254.html

M V Bach
et al.
298
.
.
-
-
-
Figure 1. Structures of N,N dialkylamphetamine analogues and N dealkylated metabolites. Abbrevi
ations are identiŽ ed in the text.
(1994) described this metabolism of DEP as being an ‘atypical’ dealkylation,
-
-
presumably because very few examples of CYP2D6 catalyzed N dealkylation of
basic drugs were known at that time. It is now recognized that many drugs,
-
including ami amine, amitriptyline, desmethylcitalopram, haloperidol, imipra
- - -
mine, 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP), mianserin, tomoxe
-
-
-
-
-
tine, trazodone and venlafaxine, do undergo CYP2D6 catalyzed N dealkylation
et al
(Coutts
. 1994a, Coutts and Urichuk 1999), although in most instances
CYP2D6’s role in this important metabolic reaction is relatively minor.
-
-
Our observation that the N demethylation and N depropargylation of DEP
-
-
were catalysed to some extent by CYP2D6, whereas no N demethylation or N
debenzylation of BPA was catalysed by this enzyme, prompted the determination
}
-
-
-
-
whether other N methyl N alkylamphetamines could be N demethylated and or
-
-
N dealkylatedby the catalytic action of CYP2D6. Three additional N,N dialkylated
-
-
-
-
amphetamines, N allyl N methylamphetamine (AMA), N,N diallylamphetamine
-
- -
(DAA) and N methyl N propylamphetamine (MPA) were selected for this study.
They were readily synthesized and their structures (Ž gure 1) were conŽ rmed by
nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ESMS;
-
Ž gure 2). N allylamphetamine (AA), a potential metabolite of AMA and DAA,
and DEP’s metabolite, PgA, were also synthesized and similarly characterized.
-
-
-
(
) N,N dipropargylamphetamine (DPgA) was isolated in low yield as a by
2
-
product in the synthesis of PgA, but its CYP2D6 catalyzed metabolism was not
investigated at this time. Its mass spectrum was recorded for comparison with
those of the other amphetamine derivatives (Ž gure 2).
-
-
To determine whether the N allyl and N propyl analogues of DEP were suitable
-
-
in vitro
substrates for CYP2D6 catalyzed N dealkylation, the
metabolism of AMA,
DAA, MPA, and also DEP and BPA, under identical conditions, was studied using
CYP2D6 expressed in a human cell line. Since all of the compounds investigated,

-
Metabolism of N,N dialkylated amphetamines
299
-
-
-
Figure 2. Characteristic fragment ions in the electrospray mass spectra of N mono and N,N
dialkylated amphetamines.
-
-
except DAA, are N methylated and also N alkylated with another moiety, to avoid
confusion ‘demethylation’ has been used to indicate the loss of the methyl group,
and ‘dealkylation’ to indicate the loss of the other moiety.
Materials and methods
Instrumentation
-
3
3
GC analyses were conducted using a DB 5 fused capillary column, 13 m 0.25 mm i.d. 0.25 lm
Ž lm thickness (J&W ScientiŽ c, Folsom, CA, USA) on a HP 5890 GC (Hewlett Packard, PA, USA)
-
-
equipped with a nitrogen phosphorus detectorand a HP 3396Aintegrator.The carriergas was ultrapure
helium (Union Carbide, Edmonton, Canada) and its  ow rate was adjusted to maintain a column head
±
"
-
pressure of 10 psi. The make up gas at the detector was a mixture of hydrogen (3 ml min ) and air
(80 ml min± ). The injector and detector temperatures were 260 and 310 C respectively. Chroma
"
Ê
-
tographic peak areas were measured with a HP 3396A. GC conditions were: an initial column
Ê
temperature of 110 C held for 2 min and then raised by automatic temperature programming at
±
"
Ê
Ê
8 C min to 240 C.
Electrospray mass spectrometric analyses were performed on Fisons Trio 2000. Samples were
Ê
sprayed at 75 C at a probe voltage of 3.5 KV.
NMR spectra were recorded on a Bruker 300 MHz instrument using CDCl as a solvent and TMS
$
as an internal standard
Chemicals
-
-
-
\
Amphetamine sulphate and 4 methoxy N methylamphetamine (MMA) HCl were gifts from the
former Smith, Kline and French Laboratories (Philadelphia), and benzphetamine HCl was kindly
-
-
supplied by Professor A. H. Beckett and Dr D. A. Cowan, King’s College, London. N (n propyl)
\
-
\
amphetamine HCl and N methylamphetamine HCl were prepared by a reported method (Coutts et al.
1976, 1978). Other chemicals were purchased from various commercial sources: allyl bromide,
-
1 bromopropane, analytical grade tri uoroacetic acid (Aldrich Chemical Co., Milwaukee, WI, USA);

M V Bach
et al.
300
.
.
R ( ) deprenyl HCl (Research Biochemicals International, Natick, MA, USA); NADP+ sodium salt
-
-
\
2
-
-
-
from yeast, d glucose 6 phosphate monosodium salt (G6P) and glucose 6 phosphate dehydrogenase
(G6PD) type XII from Tortula yeast (Sigma Chemical Co., St Louis, MO, USA); potassium carbonate
-
-
-
(K CO ), methanol (CH OH), n hexane, HPLC grade acetonitrile (CH CN), isopropanol, dichloro
#
$
$
$
methane (CH Cl ), diethyl ether, toluene (BDH, Toronto, Canada). All solvents were distilled before
use. The K CO solution used was a 25% w v aqueous solution. The bu er solution used in all
experiments was 100 mm, pH 7.4.
#
#
}
å
#
$
Synthesis of potential substrates of CYP2D6 and their metabolites
-
-
-
-
-
-
(
) N allylamphetamine (AA) and ( ) N,N diallylamphetamine (DAA). ( ) Amphetamine sulph
6
6
6
}
ate (500 mg; 2.71 mmol) was suspended in CH CN (3 ml) and basiŽ ed with K CO solution (25% w v;
1.5 ml; 2.71 mmol). The CH CN solution was decanted into another  ask. The residue of K SO was
washed with CH CN (2.5 ml 2). To the combined organic solution, allyl bromide (117.45 ll;
1.36 mmol) was added, followed by K CO solution (1.5 ml). The reaction mixture was left to stir at room
$
#
$
$
#
%
3
$
#
$
temperature for 1 h during which time samples of the reaction mixture were examined by tlc using 10%
CH OH in CH Cl as the developing solvent. When the absence of allyl bromide was indicated, the
$
#
#
reaction mixture was evaporated (rotatory evaporator) and the residue obtained was dissolved in water
3
(40 ml). This solution was basiŽ ed with K CO solution (1.5 ml) and extracted with CH Cl (5 ml 3).
#
$
#
#
The combined extract was evaporated to dryness and the product was puriŽ ed by silica gel column
-
chromatography, using 2% CH OH in CH Cl as eluting solvent. Two products were obtained: (
)
6
$
#
#
-
- -
N,N diallylamphetamine (DAA; 24.80 mg) eluted Ž rst, followed by ( ) N allylamphetamine (AA;
112.20 mg). Both were liquids. Total yield was 67.4%. AA was converted to a salt by treating a solution
6
of it in diethyl ether with dry HCl gas, with cooling (solid CO in acetone). The melting point of AA.HCl
#
"
Ê
-
(recrystallized from ethyl acetate) was 168.5 169.5 C. H NMR (CDCl ) for AA, d: 7.22 7.17 (m, 5H,
±
±
$
Ph); 5.91 5.78 (m, 1H, allyl CH); 5.15 5.03 (m, 2H, terminal allyl CH ); 3.36 3.29 (m, 1H) and
3.24 3.16 (m, 1H) (allyl N CH ); 3.00 2.90 (m, 1H, N CH); 2.80 2.73 and 2.64 2.57 (two dd, 2H, J
±
±
±
#
-
-
±
±
±
±
gem
#
-
5 Hz; CH Ph); 1.07 1.05 (d, 3H, J 5 Hz, CH CH ).
±
5
# $
10 H , J
5
5
z
vic
The electrospray mass spectrum of AA was consistent with the proposed structure (Ž gure 2).
When this reaction was repeated using excess allyl bromide (four equivalents relative to the quantity
of amphetamine), a single product, DAA, was detected by tlc. The crude DAA was subjected to silica gel
}
-
column chromatography using a mixture of ethyl acetate and n hexane (1:4 v v ratio) as developing
solvent. The chromatographedproduct was convertedto its HCl salt as described above in the synthesis
-
Ê
±
of AA.HCl. ( ) DAA.HCl was obtained in 85% yield as a colourless solid, m.p. 162.0 163.0 C when
±
6
"
-
recrystallized from ethyl acetate. H NMR (CDCl ) for DAA, d: 7.25 7.14 (m, 5H, Ph); 5.88 5.75 (m,
2H, two allyl CH); 5.22 5.08 (m, 4H, two terminal allyl CH ); 3.22 3.06 (m, 5H, two allyl N CH
overlapping N CH); 2.96 2.90 (dd, 1H, J 8 Hz and J 6 Hz; CH Ph); 2.44 2.36 (dd, 1H, J 8 and
3 Hz, CH Ph); 0.95 0.93 (d, 3H, J 5 Hz, CH CH ).
±
$
-
±
±
±
#
#
-
±
5
5
5
#
-
±
5
#
$
The electrospray mass spectrum of DAA was consistent with the proposed structure (Ž gure 2).
- - - -
- -
6
(
) N allyl N methylamphetamine (AMA). ( ) N methylamphetamine .HCl (200.0 mg; 1.08 mmol)
6
was suspended in CH CN (10 ml) and convertedto its free base by the addition of K CO solution (1 ml).
To the base (isolated by the method used in the preparation of AA and DAA), an excess of allyl bromide
$
#
$
(2.69 mmol; 233 ll) was added, followed by K CO solution (1 ml). The resulting mixture was stirred at
#
$
"
room temperature for 45 min at which time tlc monitoring of the reaction mixture indicated that 90%
of MA had been consumed. The residue obtained after solvent evaporationwas dissolved in H O (10 ml)
#
and basiŽ ed with K CO solution, then extracted into CH Cl Chromatographic puriŽ cation of the
.
#
$
#
#
-
crude product on silica gel using 2.5% CH OH in CH Cl as eluting solvent yielded ( ) AMA base
(224.0 mg; liquid) in 92.1% yield. The base was converted to its HCl salt and recrystallized from ethyl
acetateas a colourless solid, m.p. 130.5 132 C. H NMR (CDCl ), d: 7.30 7.16 (m, 5H, Ph); 5.95 5.82
(m, 1H, allyl CH); 5.30 5.12(m, 2H, terminal allyl CH ); 3.17 3.15(d, 2H, allyl N CH ); 3.02 2.95(m,
6
$
#
#
"
Ê
-
±
±
±
$
-
±
±
±
#
#
-
-
2H, one H of CH Ph overlapping N CH); 2.45 2.37 (dd, one H of CH Ph); 2.30 (s, 3H, N CH );
±
#
#
$
-
0.96 0.94 (d, 3H, J 5 Hz, CH CH ).
±
5
$
The electrospray mass spectrum of AMA was consistent with the proposed structure (Ž gure 2).
- - - - -
) N methyl N propylamphetamine (MPA). This N dialkylamphetamine was synthesized by a
(
6
-
)
procedure similar to that used for the preparation of AMA. The amine and halide reagent used was (
6
-
-
N methylamphetamine.HCl (1.38 mmol; 257.0 mg) and 1 bromopropane (2.76 mmol; 251.66 ll)
respectively. After 1 day of stirring at room temperature,a tlc of the reaction mixture was developed with
-
7% CH OH in CH Cl . This showed that almost no reaction had occurred, so more 1 bromopropane
$
#
#
(121 ll) and K CO solution (1.02 ml) were added and the reaction continued for another day. The crude
product (isolated as described in the preparation of AA and DAA) was chromatographed on a silica gel
column, initially with 2%, and subsequently with 5% CH OH in CH Cl . Fractions were collected and
examined by tlc. Those containing the desired product were eluted by 5% CH OH in CH Cl .
Evaporation of the combined eluates gave MPA as a liquid in modest yield (40%). MPA.HCl salt,
#
$
$
#
#
$
#
#

-
Metabolism of N,N dialkylated amphetamines
301
"
Ê
-
prepared as described for AMA.HCl, was a colourless solid, m.p. 113.0 114.0 C. H NMR (CDCl ), d:
±
$
7.32 7.27 (m, 2H of Ph) and 7.22 7.17 (m, 3H of Ph); 3.10 2.86 (m, 2H, one H of CH Ph overlapping
N CH); 2.46 2.37(m, 3H, one H of CH Ph and 2H of N CH CH CH ); 2.32 (s, 3H, N CH ); 1.58 1.44
±
±
±
#
-
-
-
±
±
#
#
#
$
$
(m, 2H, CH CH ); 0.95 0.89 (overlapping d and t, 6H, CHCH and CH CH ).
±
#
$
$
#
$
The electrospray mass spectrum of MPA was consistent with the proposed structure (Ž gure 2).
- -
) N propargylamphetamine (PgA). PgA was synthesized according to the method of MacGregoret al.
(
2
-
(1988). The crude product obtained from a reaction of ( ) amphetamine sulphate (250 mg; 1.36 mmol)
2
3
with propargyl bromide (75 ll; 0.86 mmol) was puriŽ ed on a silica column (0.75 8 inches) using 2%
CH OH in CH Cl as eluting solvent. A small amount of DPgA as side product eluted Ž rst, followed by
$
#
#
PgA as a liquid in 75% yield. NMR data of the PgA coincided with reported literature values. PgA.HCl
and DPgA.HCl salts were prepared as described for AMA.HCl. Both products were colourless solids
Ê
162.5 163.5 C (m.p. reported by
±
when recrystallized from ethyl acetate. PgA.HCl had a m.p.
5
Ê
Ê
MacGregor et al. 158 160 C). The melting point of DPgA.HCl was 166 167 C. Their electrospray
±
±
mass spectra were consistent with the proposed structures (Ž gure 2).
Microsomal protein
Human CYP2D6 microsomalpreparationusedin this study was purchased from GentestCorporation
}
1
±
-
(Woburn, MA, USA). It was derived from a human AHH 1 TK
cell line transfected with
}
complementary DNA that encoded human CYP2D6. Total protein content was 10 mg ml in 100 mm
potassium phosphate (pH 7.4) and CYP2D6 content was 260 pmol mg protein. Control microsomes
}
obtained from the same human cell line that had not been transfected with speciŽ c cDNA, were
purchased from the same source.
-
NADPH generating system
+
-
An NADPH generating system was prepared by mixing freshly prepared stock solutions of NADP
(1.3 mm ; 1 mg ml), G6P (3.3 mm ; 1 mg ml), MgCl .6H O (3.3 mm ; 0.67 mg ml) and G6PD (50 U ml
bu er) in a 5:5:10:2 volume ratio.
}
}
}
}
#
#
å
In vitro microsomal incubation, extraction and derivatization
-
- -
Metabolism of N allyl N methylamphetamine (AMA). An incubation mixture containing AMA
å
(6.9 nmol), CYP2D6 (12.5 ll; 0.125 mg) and phosphate bu er to a volume of 97.5 ll was preheated at
Ê
-
37 C for 5 min before the enzymatic reaction was started by an addition of NADPH generating system
Ê
(27.5 ll). The resulting mixture (125 ll) was incubated at 37 C for 2 h. The reaction was terminated by
cooling in an ice bath, followed by the addition of K CO solution (100 ll). Internal standard (MMA;
#
$
}
0.7 nmol) was added and the mixture was extracted with a mixed organic solvent (2% v v isopropanol
-
3
in n hexane; 2 3.5 ml) by vortexing vigorously for 1 min, shaking mechanically for 5 min and then
-
centrifuging for 5 min at 2300 rpm in a Du Pont Sorvall GLC 2B General Laboratory Centrifuge
(Mississauga, ON, Canada). The organic layer was transferredto a clean tube and evaporatedto dryness
under a stream of nitrogen at room temperature. The residue was tri uoroacetylated under anhydrous
conditions as described below, and the derivatized sample was analyzed by GC as described above.
An identical incubation with controlmicrosomesinstead of CYP2D6 microsomeswas also performed
for comparison.
Derivatization procedure
The dried residue obtained at the end of each metabolic reaction was mixed with tri uoroacetic
anhydride (50 ll) and acetonitrile (25 ll) in a test tube which was capped tightly with a screw cap before
Ê
its insertion in a metal heating module at 60 C for 30 min. The reaction mixture was allowed to cool to
roomtemperaturebeforethe tube was opened and its contentsevaporatedto dryness at roomtemperature
under a stream of dry nitrogen. The residue was dissolved in toluene (30 ll) and aliquots (2 ll) were
analyzed by GC.
Quantitative analysis of metabolites
Known but varying amounts of authentic standards of all substrates (1.58 22.15 nmol) and
±
metabolites (0.09 1.05 nmol) and a Ž xed quantity of internal standard (0.7 nmol MMA) was added to
±
-
a series of tubes containing the same amount of NADPH generating system, control microsomes and
substrate as indicated in incubation samples. Extractionand derivatization procedures were then applied
to these tubes, and their tri uoroacetylatedproducts were analyzed by GC. Peak area ratios of metabolite
to internal standard were plotted against concentrations of metabolite to produce calibration curves. The
concentrationof each metabolite in incubation mixtures was determined from the equation of the straight
line derived from its calibration curve.

M V Bach
et al.
302
.
.
-
-
-
-
MetabolismofN methyl N propylamphetamine (MPA), N,N diallylamphetamine (DAA), deprenyl(DEP)
andbenzphetamine(BPA). The incubation proceduredescribed above was repeated,except that substrate
AMA was individually replaced with MPA, DAA, DEP or BPA (6.9 nmol).
The metabolism of DEP was also conducted in the presence of quinidine (0.638 nmol), an inhibitor
of CYP2D6.
Results
-
- -
The envisaged study required the synthesis of the substrates N allyl N
-
-
methylamphetamine, N,N diallylamphetamine (a by product of the synthesis of
-
-
-
-
-
N allylamphetamine) and N methyl N propylamphetamine, and metabolites N
-
allylamphetamine and N propargylamphetamine. The structures of these Ž ve
compounds were conŽ rmed by NMR and electrospray MS. Diagnostic ions (MH+,
}
m z
-
-
- -
119 and 91) were present in the mass spectra of all N mono and N,N di
-
substituted compounds (Ž gure 2). Most spectra also contained additional frag
ments of low abundance that were consistent with the structures from which they
were derived. These fragment ions are identiŽ ed in table 1.
-
When Ž ve N,N dialkylated amphetamines (AMA, MPA, DAA, DEP, BPA)
were individually incubated with the CYP2D6 isozyme fortiŽ ed with appropriate
-
-
Table 1. Identities of minor fragment ions in the electrospraymass spectra of the synthesized N mono
-
and N,N dialkylated amphetamines.
FragmentŒ
}
Compound‹
(m z; % rel. ab.)
Probable identity
CH CH N+HCH CH CH (MH+ C H )
AA
84 (5.8%)
74 (7.8%)
174 (4.7%)
98 (7.2%)
94 (17.9%)
E
( )
F
F
$
#
MPA
DAA
DAA
DPgA
CH CH CH N+H# CH
(MH+ C H CH CHCH )
E
F
’ & $
$
#
#
#
$
C H CH C(CH ) N+HCH CH CH (MH+ CH CH CH )
E
F
F
F
CH CHCH N+$H CH CH CH
(MH+ C H CH CHCH )
E
F
’ & $
’
&
#
#
#
$
#
F
F
#
#
#
#
#
CH3CHCH N+H CH C3CH
(MH+ C H CH CHCH )
E
F
’ & $
#
#
#
‹ Structures identiŽ ed in Fig. 1; Œrel. ab.,relative abundance ; C H
F
(
)
Figure 3. Diagnostic ions in the electrospray mass spectrum of the tri uoracetylated metabolite of
deprenyl (PgA). Figures in brackets are % relative abundances.
Table 2. Yields of metabolites formed when various amphetamines were incubated with CYP2D6 and
!
å
cofactors. Data are averages that di ered by 5% in duplicated experiments.
Substrate
(6.9 nmol incubation)
MA
Nor metabolite
(pmol incubation)
}
}
}
(pmol incubation)
AMA
MPA
DAA
DEP
BPA
98
44
(a)
624
0
279
73
594
1160
0
a
Metabolism of DAA to MA is not possible.

-
Metabolism of N,N dialkylated amphetamines
303
-
cofactors, no ring hydroxylation of any of these substrates was observed. N
dealkylated metabolites, however, were detected in the incubation mixtures that
-
contained AMA, MPA, DAA and DEP. AMA underwent two N dealkylations to
-
-
form the N demethylation and N deallylation products, AA and MA respectively.
-
Similarly, CYP2D6 catalyzed metabolism of MPA produced MA and PA, and
CYP2D6 promoted the production of MA and PgA from DEP.
et al
. 1994, Wacher
et al
. 1996), DEP
In agreement with literature data (Grace
-
-
was metabolized by N demethylation to nordeprenyl and by N depropargylation to
MA. As expected, these two metabolites were not observed when quinidine was
included in the metabolism reaction. DAA was metabolized to AA, but no
dideallylated product (amphetamine) was observed. The structures of these
metabolites were conŽ rmed by comparing their retention times with those of
authentic reference samples of MA, PA and AA, and by interpretation of the mass
-
spectrum of the N demethylated metabolite of DEP after its tri uoroacetylation.
}
m z
This spectrum had two prominent ions of
270 and 119, which are readily
-
identiŽ ed (Ž gure 3). The quantities of metabolitesformedare listed in table 2. No N
-
demethylated or N dealkylated metabolites were detected in incubations of these
substrates with control microsomes. This conŽ rmed that CYP2D6 catalyzed these
-
-
N demethylationsand N dealkylations.TypicalGCtraces of derivatized incubation
mixtures of DEP, AMA, MPA and DAA are shown in Ž gure 4a and b.
Discussion
The anticipated result of this study on amphetamine derivatives was that the
-
-
N,N dialkylated amphetamines bearing an N methyl substituent (AMA, MPA,
-
DEP, BPA) would at least be N demethylated since this pathway is known to be
catalyzed partially by CYP2D6 (Coutts and Urichuk 1999).Ring hydroxylationwas
-
also expected because CYP2D6 can mediate the ring oxidation of other amphet
-n-
-
amines such as N butylamphetamine, N ethylamphetamine and amphetamine
et al
(Bach
. 1999). Three of the compounds investigated (AMA, MPA, DEP) were
-
-
N demethylated to varying extents (table 2) but, surprisingly, no CYP2D6
-
-
catalyzed N demethylation or N dealkylation occurred when BPA was incubated
-
-
with CYP2D6 and none of the substrates was ring hydroxylated. Aromatic ring
hydroxylated metabolites of the Ž ve dialkylated amphetamines (AMA, BPA, DEP,
-
DAA, MPA) were not available to provide GC retention times of their O acetylated
derivatives and their mass spectra. However, it has been our experience that the GC
-
-
retention times of the O acetylated derivatives of many ring hydroxylated drugs,
et al et al et al
including imipramine (Coutts
. 1976, Su
. 1993), trimipramine (Bolaji
.
et al
1993),methoxyphenamine,an amphetaminederivative (Coutts . 1994b),and N
-
et al
. 1999) are always in close proximity to those of
alkylated amphetamines (Bach
-
the parent drug. If aromatic ring hydroxylated metabolites had been formed in the
present study, it probable that they would have been observed after acetylation in
the GC traces and would have been readily identiŽ ed by MS. No GC or MS
evidence in favour of their presence was found.
-
The major structural diåerence in the four N methylated substrates (AMA,
-
-
MPA, DEP, BPA) is the nature of the N alkyl groups (Ž gure 1). These N
substituents appeared to in uence the catalytic activity of CYP2D6 in the
-
N dealkylation pathway. Of the four alkyl groups, allyl (CH CH CH ), benzyl
F
#
#
@
n-
(PhCH ), propargyl (CH C CH: and propyl (CH CH CH ), the presence of
#
#
#
#
$

M V Bach
et al.
304
.
.
A
B
Figure 4. A. GC traces of tri uoracetylated dried extracts of DEP incubation with: (a) control
microsomal protein; and (b) CYP2D6 enzyme preparation. MMA is the internal standard. B. (a)
GC trace of tri uoroacetylated dried extract of AMA incubation with CYP2D6; (b) GC trace of
-
tri uoroacetylated dried extract of MPA incubation with CYP2D6; (c) GC trace of tri
 uoroacetylated dried extract of DAA incubation with CYP2D6. Incubations were carried
out under identical conditions. MMA is the internal standard.

-
Metabolism of N,N dialkylated amphetamines
305
the propargyl group had the greatest eåect on CYP2D6 activity. Indeed, DEP was
in vitro
-
substrate of CYP2D6 (table 2 and Ž gure 4a). The two N
a very good
dealkylated metabolites of DEP (MA, nordeprenyl) were formed at diåerent rates,
-
indicating that CYP2D6 selectively mediated the cleavage of N C bonds. When the
-
propargyl group of DEP was replaced with an allyl group in AMA, the extent of N
dealkylation was much reduced, and when the propargyl group was replaced with
n-
-
an propyl group in MPA, a further reduction in the extent of N dealkylation
was observed. Finally, replacing the propargyl group with a benzyl moiety in
-
-
benzphetamine resulted in a complete absence of CYP2D6 catalyzed metabolic N
-
demethylation or N dealkylation. It appears that when the N atom of amphetamine
-
contains both a 3 carbon substituent and a methyl group, the degree of unsaturation
-
-
of the 3 carbon unit has an important in uence on the extent of CYP2D6 catalyzed
-
-
N dealkylation. In addition, when the N methyl group of AMA was replaced by an
-
-
allyl substituent in DAA (which is N,N diallylated), the extent of N dealkylation
-
was again increased (table 2). These Ž ndings suggest that the aænities of N methyl,
-
-
-
N propyl, N allyl and N propargyl groups for CYP2D6 vary considerably.
In summary, this study has shown that human expressed CYP2D6 enzyme can
-
-
-
-
-
catalyze the N demethylation and N dealkylation of four N,N dialkylated amphet
amines, namely AMA, MPA, DAA and DEP, but that it does not mediate the N
-
dealkylation of benzphetamine. Rates of these N dealkylation reactions were clearly
-
dependent upon the nature and extent of unsaturation of the N substituents.
-
Another noteworthy observation from this study was that none of the N,N
-
dialkylated substrates was ring hydroxylated, in contrast to what was observed with
-
et al
N monoalkylated amphetamines (Bach
. 1999).
Results from the present study complemented those of previous investigators
et al et al
-
. 1993) who concluded that the N demethylation
(Guengerich
. 1986, Shet
in vitro
of benzphetamine was catalyzed
in humans by CYP3A4 and not by
-
CYP2D6. Also, four of the Ž ve N,N dialkylated amphetamines investigated were
racemates. Only DEP was a single enantiomer. It would be of interest to extend the
-
study to the enantiomers of all Ž ve N,N dialkylated amphetamines to determine
whether stereoselective metabolism is observed.
Acknowledgements
The authors are grateful to the Alberta Mental Health Fund and the Medical
Research Council of Canada for funding.
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