Sequential metabolism of secondary alkyl amines to metabolic-intermediate complexes: Opposing roles for the secondary hydroxylamine and primary amine metabolites of desipramine, (S)-fluoxetine, and N-desmethyldiltiazem

Article abstract of DOI:10.1124/dmd.110.032391

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine ? primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d₃-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation. Copyright

Full text of DOI:10.1124/dmd.110.032391

0090-9556/10/3806-963–972$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:963–972, 2010
Vol. 38, No. 6
32391/3586530
Printed in U.S.A.
Sequential Metabolism of Secondary Alkyl Amines to Metabolic-
Intermediate Complexes: Opposing Roles for the Secondary
Hydroxylamine and Primary Amine Metabolites of Desipramine,
(S)-Fluoxetine, and N-Desmethyldiltiazem
Kelsey L. Hanson, Brooke M. VandenBrink, Kantipudi N. Babu, Kyle E. Allen,
Wendel L. Nelson, and Kent L. Kunze
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington
Received January 22, 2010; accepted March 3, 2010
ABSTRACT:
Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], amine metabolites, which accumulate in incubations of the second-
and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the cor- ary amines, inhibit MI complex formation. Mass balance studies pro-
responding secondary hydroxylamines [N-hydroxydesipramine, (S)- vided estimates of the product ratios of N-dealkylation to N-hydroxy-
N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cyto- lation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19],
chromes P450 2C11, 2C19, and 3A4, respectively. The expected and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are
primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, significant metabolites of the P450-mediated metabolism of second-
and N,N-didesmethyldiltiazem, are also observed. The formation of ary alkyl amines. Parallel studies with N-methyl-d3-desipramine and
metabolic-intermediate (MI) complexes from these substrates and CYP2C11 demonstrated significant isotopically sensitive switching
metabolites was examined. In each example, the initial rates of MI from N-demethylation to N-hydroxylation. These findings demon-
complex accumulation followed the order secondary hydroxyl- strate that the major pathway to MI complex formation from these
amine > secondary amine ϾϾ primary amine, suggesting that the secondary amines arises from N-hydroxylation rather than N-dealky-
primary amine metabolites do not contribute to formation of MI com- lation and that the primary amines are significant competitive inhibi-
plexes from these secondary amines. Furthermore, the primary tors of MI complex formation.
The metabolism of alkyl amine-containing drugs leads to the for- al., 1979; Franklin, 1995). Further oxidation of these metabolites leads
to time-dependent inhibition (TDI) of cytochrome P450 (P450) en-
zymes because of the formation of metabolic-intermediate (MI) com-
plexes (Fig. 1). MI complexes exhibit a signature Soret absorbance at
approximately 455 nm (Franklin, 1974). Predictions of the magnitude
of drug-drug interactions (DDIs) for diltiazem, fluoxetine, and clar-
ithromycin have been reported using results from in vitro kinetic
studies and from measured alkyl amine concentrations in vivo (May-
mation of primary and secondary metabolites, which include N-
dealkylated, N-hydroxylated, and N-oxygenated species (Lindeke et
This work was supported by the National Institutes of Health National Institute
of General Medical Sciences [Project Program Grant P01-GM32165, Pharmaco-
logical Sciences Training Grant T32-GM007750] (to B.M.V. and K.E.A.).
K.L.H. and B.M.V. contributed equally to this work.
Parts of this work were previously presented at the following conference: hew et al., 2000; Galetin et al., 2006; Zhang et al., 2009b).
Hanson KL, Zhao P, Babu KN, Nelson WL, Kalhorn TF, and Kunze KL (2008)
Mapping the oxidative pathways that drive MI complex formation
and defining the roles of the metabolites will be beneficial in devising
better methods for the prediction of DDIs. The complexity of the alkyl
amine metabolic pathways is a barrier to the rational prediction of
Time-dependent inhibition of CYP3A by diltiazem: the role of the sequentially-
formed metabolite, N-hydroxydesmethyl diltiazem, in MI complex formation and
VandenBrink BM, Babu KN, Isoherranen N, Nelson WL, Kalhorn TF, and Kunze KL
(2008) Stereoselective metabolism of fluoxetine results in irreversible inhibition of
DDIs (Fig. 1). The study of the pathways of metabolism in single
CYP2C19 through the formation of an MI complex. 15th Annual International
Society for the Study of Xenobiotics Meeting; 2008 Oct 12–16; San Diego, CA.
enzyme systems is a logical first step to improve current predictive
efforts. Proof that a single P450 catalyzes all of the steps required for
International Society for the Study of Xenobiotics, Washington, DC.
MI complex formation from a tertiary amine was obtained more than
three decades ago (Kawalek et al., 1976). More recent results from
several laboratories show that a number of alkyl amine metabolites
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.032391.
ABBREVIATIONS: TDI, time-dependent inhibition; P450, cytochrome P450; MI complex, metabolic-intermediate complex; DDI, drug-drug
interaction; DES, desipramine; (S)-FLX, (S)-fluoxetine; MA, N-desmethyldiltiazem; UPLC, ultraperformance liquid chromatography; LC, liquid
chromatography; MS, mass spectroscopy; DBOMF, dibenzyloxymethylfluorescein; BOMR, benzylresorufin; EMOCC, 7-methoxy-4-trifluorometh-
ylcoumarin; CHCl3, chloroform; MeOH, methanol; DMSO, dimethyl sulfoxide; m-CPBA, m-chloroperbenzoic acid; KPi, potassium phosphate
buffer; ACN, acetonitrile; (S)-FLXOH, (S)-N-hydroxyfluoxetine; MAOH, N-hydroxy-N-desmethyldiltiazem; DDES, N-desmethyldesipramine; (S)-
NFLX, (S)-norfluoxetine; MD, N,N-didesmethyldiltiazem; DESOH, N-hydroxydesipramine; HLM, human liver microsome.
963

964
HANSON ET AL.
FIG. 1. Metabolism of alkyl amines by cytochrome P450 enzymes
to MI complexes. Metabolism of these secondary amines leads to
two primary metabolites: a primary amine (pathway a) and a sec-
ondary hydroxylamine (pathway b), both of which may contribute
to MI complex formation. Secondary branching of the nitrone has
been suggested (Jeffery and Mannering, 1983; Cerny and Hanzlik,
2005).
cause MI complex formation in microsomes or single enzyme systems mulate to concentrations that will significantly inhibit MI complex
(Jeffery and Mannering, 1983; Polasek and Miners, 2008; Zhang et formation by secondary amines.
al., 2009a).
Materials and Methods
The reaction sequence to MI complex is characterized by a major
metabolic branch point, namely oxidation of the secondary amine to
the primary amine via N-dealkylation (Fig. 1, pathway a) or to the
secondary hydroxylamine by N-hydroxylation (Fig. 1, pathway b).
P450-catalyzed N-dealkylation has been studied extensively; how-
ever, N-hydroxylation has received less attention. In addition, it is
clear that both products are formed from a single substrate by multiple
enzymes, which suggests that N-hydroxylation may be common (Baba
et al., 1988).
Chemicals. All chemicals from commercial sources were of analytical
grade. Solvents for Waters ACQUITY UPLC were LC-MS Optima grade.
Human CYP3A4, rat CYP2C11, and human CYP2C19 Supersomes, coex-
pressed with cytochrome b5 and cytochrome P450 reductase, were purchased
from BD Biosciences (San Jose, CA). Desipramine, (S)-fluoxetine, and dilti-
azem were purchased from Sigma-Aldrich (St. Louis, MO). The internal
standard, ring-labeled desipramine-d4, was purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA). The internal standards, fluoxetine-d6 and
norfluoxetine-d6, were purchased from Cerilliant Corporation (Round Rock,
TX). The fluorescent probe substrates and metabolites DBOMF (Vivid Green),
BOMR (Vivid Red), EMOCC (Vivid Blue), fluorescein (DBOMF metabolite),
A survey of the results of microsomal studies indicates that the
relative rates of MI complex formation from intermediates tend to
rank in the order primary hydroxylamine Ͼ nitrone Ͼ secondary and 7-hydroxy-4-trimethylcoumarin (EMOCC metabolite) were purchased from
Invitrogen (Carlsbad, CA). Resorufin was purchased from Sigma-Aldrich. All
other chemicals were purchased from Sigma-Aldrich unless specified.
hydroxylamine Ͼ secondary amine ϾϾ primary amine (Gumbrecht
and Franklin, 1979; Jo¨nsson and Lindeke, 1992; Bensoussan et al.,
1995). The primary amine pathway (Fig. 1, pathway a) is often cited
as the major pathway to MI complex from alkyl amines (Correia and
Ortiz de Montellano, 2005). The conversion of primary amines to MI
complexes has received considerable attention in which the slow step
is thought to be N-hydroxylation of the primary amine (Buening and
Franklin, 1976). However, the rate of MI complex formation from
primary amines is significantly slower than the rate observed from the
corresponding secondary amines. For example, Lindeke et al. (1979)
studied the intermediates to MI complex formation during the metab-
olism of methamphetamine. They concluded that the secondary hy-
droxylamine pathway (Fig. 1, pathway b) is largely responsible for the
formation of an MI complex in rat liver microsomes. It is reasonable
to expect that the relative contributions of these two pathways to MI
complex formation will depend on a number of factors including the
enzyme-substrate pair, the amount and stability of the metabolites
formed, and the effects of other enzymes on the generation of pathway
intermediates in the systems of interest.
The relative importance of the two pathways from the secondary
amines on the rate of MI complex formation in single enzyme systems
has not been reported previously. This report addresses this gap by
studying the metabolism of three secondary alkyl amines, each paired
with a different P450 enzyme (Fig. 2). The enzyme-substrate pairs are
DES with rat CYP2C11, (S)-FLX with human CYP2C19, and MA
with human CYP3A4. In agreement with the proposal of Lindeke et
al. (1979), we show that the secondary hydroxylamine pathway con-
trols the rate of MI complex formation from secondary amines. In
Chemical Syntheses of Primary Amine and Secondary Hydroxylamine
Metabolites and Deuterated Internal Standards of DES, (S)-FLX, and
MA. Preparation of deuterated 5-[3-aminopropyl]iminodibenzyl (desmethyl-
desipramine-d4). A solution of N-desmethyldesipramine (0.504 g, 2.00 mmol)
in 10% DCl-D2O (15 ml) was heated at 80°C for 8 h in a nitrogen-filled sealed
heavy glass tube, according to the method reported previously (Baba et al.,
1988). The reaction mixture was then cooled and adjusted to pH 10 with
aqueous 2 N NaOH at 0°C and extracted with ether (three 100-ml washes). The
combined extracts were washed with saturated NaCl solution and dried over
Na2SO4. After evaporation of solvent under reduced pressure, the crude
product was chromatographed on silica gel eluting with CHCl3 and a CHCl3-
MeOH gradient to afford 0.416 g (82%) of desmethyldesipramine-d4, as a
thick gum. 1H NMR (free base, 500 MHz) CDCl3 ␦: 1.75 (quintet, 2H, J ϭ 7
Hz, –N–CH2–CH2–CH2–N–), 2.76 (t, 2H, J ϭ 7 Hz, –N–CH2–CH2–CH2–N–),
3.17 (s, 4H, Ar–CH2–CH2–Ar), 3.80 (t, 2H, J ϭ 6.5 Hz, Ar–N–CH2–),
7.11–7.14 (m, 4H, ArH); MS [MHϩ] 257.1 (C17H16D4N2 requires 256.3).
Preparation of deuterated 5-[3-(N-hydroxyl-N-methylamino)propyl]imi-
nodibenzyl (N-hydroxydesipramine-d4). An aqueous formaldehyde solution
(37%, 90.6 mg, 3.02 mmol) was added to a solution of ring-deuterated
5-[3-(N-hydroxylaminopropyl]iminodibenzyl (330 mg, 1.2 mmol) in benzene
(35 ml), and the reaction mixture was refluxed for 9 h with a Dean-Stark
apparatus until no more water was collected. After evaporation of solvent, the
crude product was purified by flash chromatography on silica gel, eluting with
a hexane-ethyl acetate gradient to give 0.245 g (71%) of the deuterated
N-methylene nitrone as a pale yellow oil. A stirred solution of deuterated
N-methylene nitrone (0.150 g, 0.52 mmol) in MeOH (10 ml) was cooled to
Ϫ10°C, and the reaction mixture was adjusted to pH 4 with 1 N HCl. Sodium
cyanoborohydride (82.6 mg, 1.3 mmol) was added to the reaction at Ϫ10°C.
The mixture was warmed to room temperature and stirred for 9 h. The reaction
addition, we demonstrate that primary amine metabolites can accu- mixture was quenched by addition of 1 N HCl (1 ml), and the MeOH was
FIG. 2. Chemical structures of the three alkyl amine drugs used in
this study.

SEQUENTIAL METABOLISM OF SECONDARY AMINES TO MI COMPLEXES
965
removed under reduced pressure. The residue was dissolved in water (10 ml), –CH2–NH–CH3), 3.59–3.68 (m, 1H, –CH2–NH–CH3), 3.83 (s, 3H, –OCH3),
made alkaline (pH 10) with 2 N NaOH solution, and then extracted with ether
(three 40-ml washes). The combined organic extracts were washed with
4.17–4.27 (m, 1H, –CO–N–CH2–), 4.45–4.52 (m, 1H, –CO–N–CH2–), 5.09
(d, 2H, J ϭ 7.8 Hz, –CH–O–COCD3 and –S–CH–), 6.88 (d, 2H, J ϭ 8.7 Hz,
saturated NaCl solution and dried (Na2SO4). Evaporation of the solvent gave ArH), 7.41–7.47 (m, 3H, ArH), 7.62–7.67 (m, 2H, ArH), 7.80 (d, 1H, J ϭ 8.1
the crude product, which was purified by silica gel flash column chromatog-
raphy eluting with CHCl3 followed by a CHCl3-MeOH gradient, which af-
forded 0.125 g (83%) of a thick liquid product, which was converted to its
Hz, ArH); MS [MHϩ] 418.3 (C22H23D3N2O4S requires 417.2).
Preparation of N-desmethyldiltiazem-d3. Diltiazem was N-demethylated
according to a method described previously (Alexander, 1993). In brief,
diltiazem-d3 (0.600 g, 1.15 mmol free base) was refluxed with ␣-chloroethyl
oxalic acid salt, obtained as a white solid, m.p. 121°C. The nondeuterated
1
compound has been reported (Beckett et al., 1983). H NMR (free base) (500 chloroformate (0.365 g, 2.75 mmol) in 20 ml of 1,2-dichloroethane for 12 h.
MHz) CDCl3 ␦: 1.86 (quintet, 2H, J ϭ 7 Hz, –N–CH2–CH2–CH2–N–), 2.59 (s, Solvent and ␣-chloroethyl chloroformate were evaporated under reduced pres-
3H, –N–CH3), 2.72 (t, 2H, J ϭ 7 Hz, –N–CH2–CH2–CH2–N–), 3.17 (s, 4H, sure to obtain the carbamate ester. This ester was dissolved in 20 ml of MeOH
Ar–CH2–CH2–Ar), 3.82 (t, 2H, J ϭ 6.5 Hz, Ar–N–CH2–), 6.92–6.96 (m, 1H, and refluxed for 2 h. After evaporation of the MeOH, the gummy mass
ArH), 7.06–7.16 (m, 2H, ArH); MS [MHϩ] 287 (d4) (70), 288.4 (100), 289 obtained was dissolved in a mixture of ethyl acetate-ether (1:1, 30 ml) and
(94), 290 (55) (C18H18D4N2O requires 286.2).
allowed to stand for 30 min. The resultant white solid was filtered, washed
Preparation of 5-[3-(N-trideuteromethylamino)propyl]iminodibenzyl (desi- with ether, dried, and crystallized from EtOAc to afford 0.514 g (89%) of
pramine-methyl-d3). A solution of 5-(3-chloropropyl)iminodibenzyl (0.160 g, N-desmethyldiltiazem-d3 HCl as a white solid, m.p. 133°C. 1H NMR (500
0.59 mmol) in MeOH (10 ml) in a 60-ml sealed heavy glass tube was cooled
MHz) CDCl3 ␦: 2.81 (t, 3H, J ϭ 5.0 Hz, –CH2–N–CH3), 3.37–3.48 (m, 1H,
to 0°C. Methylamine-d3 hydrochloride (Cambridge Isotope Laboratories) –CH2–N–CH3), 3.51–3.57 (m, 1H, –CH2–NH–CH3), 3.86 (s, 3H, –OCH3),
(0.125 g, 1.77 mmol) was added at 0°C followed by an aqueous solution of
sodium carbonate (0.250 g, 2.3 mmol in 2 ml of water). The reaction mixture
was saturated with N2 gas, sealed, and heated at 110°C for 14 h. After cooling
4.34–4.46 (m, 2H, –CO–N–CH2–), 5.04 (d, 1H, J ϭ 7.5 Hz, –CH–O–
COCD3), 5.11 (d, 1H, J ϭ 7.5 Hz, –S–CH–), 6.93 (d, 2H, J ϭ 9.0 Hz, ArH),
7.31–7.36 (m, 1H, ArH), 7.40 (d, 2H, J ϭ 8.5 Hz, ArH), 7.59 (d, 2H, J ϭ 4.0
the pressure vessel tube at 0°C over 4 h, it was opened, and the MeOH was Hz, ArH), 7.74 (d, 1H, J ϭ 8.0 Hz, ArH), 9.53 (bs, 1H, –NH, exchangeable
evaporated under reduced pressure. Water (10 ml) was added, and the mixture
was extracted with CHCl3 (three 15-ml washes). The combined organic ex-
with D2O); MS [MHϩ] 404.2 (C21H21D3N2O4S requires 403.2).
Preparation of N-hydroxydesipramine oxalate. To a stirred solution of
tracts were washed with water and saturated NaCl solution and then dried desipramine (free base, 0.500 g, 1.87 mmol) in MeOH (15 ml), acrylonitrile
(Na2SO4). Evaporation of solvent gave the crude product, which was purified (0.5 g, 9.4 mmol) was added. The resulting solution was stirred for 24 to 32 h
by flash column chromatography on silica gel eluting with CHCl3 followed by at room temperature. After the MeOH was evaporated, the resultant gummy
a CHCl3-MeOH gradient, afforded 0.137 g (86%) of the desipramine-d3, mass was purified by flash chromatography on silica gel eluting with CHCl3 to
obtained as a thick gummy liquid. 1H NMR (300 MHz) CDCl3 ␦: 1.91 (m, 2H, yield 0.540 g (90%) of the N-cyanoethyl intermediate as a thick gummy liquid.
–N–CH2–CH2–CH2–N–), 2.75 (t, 2H, J ϭ 7.2 Hz, –N–CH2–CH2–CH2–N–), 1H NMR (300 MHz) CDCl3 ␦: 1.95 (m, 2H, –N–CH2–CH2–CH2–N–), 2.40 (s,
3.17 (s, 4H, Ar–CH2–CH2–Ar), 3.84 (t, 2H, J ϭ 6.6 Hz, Ar–N–CH2–), 6.93 3H, –N–CH3), 2.69–2.77 (m, 4H, –N–CH2–CH2–CN), 2.88 (t, 2H, J ϭ 6.9 Hz,
(dt, 2H, ArH), 7.07–7.16 (m, 6H, ArH); MS [MHϩ] 270.1 (C18H19D3N2 –N–CH2–CH2–CH2–N–), 3.17 (s, 4H, Ar–CH2–CH2–Ar), 3.85 (t, 2H, J ϭ 6.6
requires 269.2).
Hz, Ar–N–CH2–), 6.96 (broad dt, 2H, ArH), 7.07–7.19 (m, 6H, ArH); MS
Preparation of (S)-fluoxetine and (S)-norfluoxetine. The (S)-enantiomers of [MHϩ] 320.2 (C21H25N3 requires 319.2).
fluoxetine and norfluoxetine were prepared from commercially available (S)-3-
A stirred solution of N-cyanoethyldesipramine (0.502 g, 1.57 mmol) in dry
chloro-1-phenylpropanol by conversion to the corresponding 3-iodo compound CH2Cl2 (20 ml) was cooled to Ϫ78°C and m-CPBA (77%, 0.185 g, 0.828
and subsequent displacement with ammonia or methylamine, respectively (Corey
mmol) was added. The reaction mixture was stirred for 24 h at Ϫ78°C under
and Reichard, 1989). Subsequent O-arylation of 4-chlorotrifluoromethylbenzene argon and then washed sequentially with ice-cold aqueous 10% K2CO3 solu-
was accomplished using sodium hydride in DMSO (norfluoxetine) (Mitchell and tion (three 10-ml washes), water (three 10-ml washes), and saturated NaCl
Koening, 1995) or in dimethylacetamide (fluoxetine) (Kamal et al., 2002). The
tartrate salts of norfluoxetine enantiomers were prepared by the method described
previously (Hilborn et al., 2001).
(three 10-ml washes) and dried (Na2SO4). Evaporation of solvent gave crude
product, which was purified by silica gel flash column chromatography eluting
with CHCl3 followed by a CHCl3-MeOH gradient to afford 0.327 g (74%) of
N-hydroxydesipramine as a thick liquid. The product was converted to its
oxalate salt, m.p. 121°C. 1H NMR (oxalate salt) (300 MHz) CD3OD ␦: 2.02
(m, 2H, –N–CH2–CH2–CH2–N–), 2.99 (s, 3H, –N–CH3), 3.16 (s, 4H, Ar–
CH2–CH2–Ar), 3.26–3.34 (m, 2H, –N–CH2–CH2–N–), 3.88 (t, 2H, J ϭ 6.6
Preparation of diltiazem-d3. To a stirred solution of diltiazem (0.650 g, 1.44
mmol) in ethanol (195 ml) was added 13 ml of aqueous 5% NaOH. The
resulting clear solution was stirred for 26 h at room temperature. The reaction
mixture was then neutralized with cold aqueous 10% HCl, the solvents were
removed under reduced pressure, and the residue was treated with aqueous 5% Hz, Ar–N–CH2–), 6.91–6.96 (m, 2H, ArH), 7.10–7.19 (m, 6H, ArH); 1H NMR
NaOH and extracted with CHCl3 (three 30-ml washes). The combined organic
(base) (500 MHz) CDCl3 ␦: 1.88 (q, 2H, J ϭ 7.0 Hz, –N–CH2–CH2–CH2–N–),
extracts were washed with water and brine and then dried (Na2SO4). Removal 2.60 (s, 3H, N–CH3) 3.18 (s, 4H, Ar–CH2–CH2–Ar), 2.75 [t, 2H, J ϭ 7.0 Hz,
of solvent gave crude product that was purified by flash column chromatog- –CH2–N(OH)–], 3.82 (t, J ϭ 7.0 Hz, Ar-N-CH2–), 6.93–6.96 (m, 2H, ArH),
raphy on silica gel eluting with CHCl3, affording 0.530 g (98%) of O- 7.09–7.15 (m, 6H, ArH); MS [MHϩ] 283.2 (C18H22N2O requires 282.2).
desacetyldiltiazem as thick liquid. 1H NMR (300 MHz) CDCl3 ␦: 2.69 [s, 6H,
Preparation of (S)-N-hydroxyfluoxetine. To a stirred solution of (S)-fluox-
–N(CH3)2], 2.92–3.10 (m, 1H, –CH2–NH–CH3), 3.18–3.30 (m, 1H, –CH2– etine (0.500 g, 1.61 mmol) in MeOH (15 ml) acrylonitrile (0.429 g, 8.05 mmol)
NH–CH3), 3.84 (s, 3H, –OCH3), 4.18–4.25 (m, 1H, –CO–N–CH2–), 4.30– was added, and the mixture was treated as described for the first step in the
4.39 (m, 1H, –CO–N–CH2–), 4.61–4.72 (m, 1H, –CH–OH), 4.92 (d, 1H, J ϭ preparation of N-hydroxydesipramine. The intermediate (S)-N-cyanoethyl-
7.5 Hz, –S–CH–), 6.89–6.94 (m, 2H, ArH), 7.30–7.39 (m, 3H, ArH), 7.45 (dd,
fluoxetine (0.514 g, 88%) was obtained as a thick liquid. 1H NMR (500 MHz)
1H, J ϭ 1.5 and 7.1 Hz, ArH), 7.56 (ddd, 1H, J ϭ 1.5 and 7.2 Hz, ArH), 7.72
CDCl3 ␦: 1.99–2.19 (m, 1H, –CH–CH2–CH2–), 2.18–2.28 (m, 1H, –CH–
(dd, 1H, J ϭ 1.5 and 7.5 Hz, ArH); MS [MHϩ] 373.3 (C20H24N2O3S requires CH2–CH2–), 2.35 (s, 3H, –N–CH3), 2.46–2.60 (m, 3H, –N–CH2–CH2–CN),
372.3).
2.65–2.81 (m, 3H, –CH2–N(CH3)–CH2–CH2–CN), 5.41 (dd, 1H, J ϭ 4.5 and
The intermediate O-desacetyldiltiazem (0.600 g, 1.61 mmol) was dissolved 8.0 Hz, Ph–CH–CH2–), 6.94 (d, 2H, J ϭ 8.5 Hz, ArH), 7.30–7.40 (m, 5H,
in CH2Cl2 (50 ml). Acetic anhydride-d6 (0.885 g, 8.19 mmol) and 4-N,N- ArH), 7.45 (d, 2H, J ϭ 8.5 Hz, ArH); MS [MHϩ] 363.2 (C20H21F3N2O
dimethylaminopyridine (0.426 g, 3.49 mmol) were added. The reaction mix-
ture was stirred for 24 h at room temperature and then heated at reflux for 2 h.
The solvent was then evaporated under reduced pressure, and the thick liquid
obtained was purified by flash column chromatography on silica gel eluting
requires 362.2).
The (S)-N-cyanoethylfluoxetine intermediate (0.300 g, 0.828 mmol) was treated
with m-CPBA (77%, 0.185 g, 0.828 mmol) in dry CH2Cl2 at Ϫ78°C as described
for the second step in the preparation of N-hydroxydesipramine. Workup and
with CHCl3 to give 0.662 g (98%) of diltiazem-d3 as a thick liquid, which was chromatography as described therein afforded 0.247 g (92%) of (S)-N-
converted into its oxalic acid salt, obtained as a white solid, m.p. 150–151°C. hydroxyfluoxetine as a thick gummy liquid. 1H NMR (500 MHz) CDCl3 ␦:
1H NMR (300 MHz) CD3OD ␦: 2.98 [s, 6H, –N–(CH3)2], 3.32–3.41 (m, 1H, 2.14–2.21 (m, 1H, –CH–CH2–CH2–), 2.28–2.36 (m, 1H, –CH–CH2–CH2–), 2.67

966
HANSON ET AL.
(s, 3H, –N–CH3), 2.78–2.85 (m, 2H, –CH2–N(OH)–CH3), 5.34 (dd, 1H, J ϭ 5 and
8.5 Hz, Ph–CH–CH2–), 6.93 (d, 2H, J ϭ 8.5 Hz, ArH), 7.29–7.37 (m, 5H, ArH),
MI Complex Formation. The formation of MI complexes was monitored
on an Olis-modernized Aminco DW-2 spectrophotometer (Olis, Bogart, GA).
7.45 (d, 2H, J ϭ 8.5 Hz, ArH); MS [MHϩ] 326.2 (C17H18F3NO2 requires 325.1). Sample and reference cuvettes contained 0.11 ␮M enzyme, 0.1 M KPi buffer
Preparation of N-hydroxyfluoxetine-d5. To a stirred solution of fluox- (pH ϭ 7.4), and 10 ␮M secondary hydroxylamine, primary amine, or second-
etine-d5 (phenyl-d5) (0.100 g, 0.32 mmol) (C/D/N Isotopes Inc., Pointe-Claire,
QC, Canada) in MeOH (15 ml) was added acrylonitrile (0.168 g, 3.2 mmol). NADPH (10 mM, 0.05 ml) and KPi buffer (0.1 M, 0.05 ml) were added to the
The mixture was treated as described for the first step in the preparation of sample and reference cuvettes, respectively. The spectrophotometer was set to
ary amine to a total volume of 0.45 ml. After 3 min of preincubation at 37°C,
N-hydroxydesipramine. The intermediate N-cyanoethylfluoxetine-d5 (0.110 g, scan, repetitively, from 495 to 430 nm (5-nm intervals) until MI complex
94%) was obtained as a thick gum. 1H NMR (500 MHz) CDCl3 ␦: 1.98–2.12 formation reached completion. The concentrations of MI complexes were
(m, 1H, –CH–CH2–CH2–), 2.18–2.28 (m, 1H, –CH–CH2–CH2–), 2.35 (s, 3H, estimated using 65 cmϪ1 mMϪ1, the extinction coefficient for the absorbance
–N–CH3), 2.47–2.60 (m, 2H, –CH2–CN), 2.65–2.83 [m, 4H, –CH2–N(CH3)– difference between 490 and 455 nm (Liu and Franklin, 1985). Initial rates of
CH2–CH2–CN], 5.41 (dd, 1H, J ϭ 4.5 and 8.0 Hz, Ph–CH–CH2–), 6.94 (d, 2H, enzyme inactivation and maximum absorbance were estimated by fitting the
J ϭ 8.5 Hz, ArH), 7.45(d, 2H, J ϭ 8.5 Hz, ArH); MS [MHϩ] 368.3 data to the standard monoexponential function Abst ϭ Absmax (1 Ϫ eϪkt),
(C20H26F3N2O, requires 367.2).
where k is equal to the initial rate of MI complex formation. After each MI
The N-cyanoethylfluoxetine-d5 intermediate (0.100 g, 0.272 mmol) was
complex formed to completion, quasi-reversibility was tested by adding po-
treated with m-CPBA (77%, 61 mg, 0.272 mmol) in dry CH2Cl2 at Ϫ78°C and tassium ferricyanide (100 mM, 5 ␮l) to the sample and reference cuvettes.
further treated as described for the second step in the preparation of N-
Stoichiometry of P450 Oxidation of the Secondary Amines. MI complex
hydroxydesipramine. Workup and chromatography as described therein af-
formation resulting from metabolism of the secondary amines by their respec-
forded N-hydroxyfluoxetine-d5 (83 mg, 92%) as a thick gum. 1H NMR (500 tive P450 enzymes was monitored as described previously until the reactions
MHz) CDCl3 ␦: 2.14–2.22 (m, 1H, –CH–CH2–CH2–), 2.27–2.37 (m, 1H, were complete. For analysis of metabolite concentrations by LC-MS/MS
–CH–CH2–CH2–), 2.68 (s, 3H, –NH–CH3), 2.78–2.81 [m, 2H, –CH2–N(OH)– [(S)-FLX and MA] or LC-MS (DES), aliquots were removed from the sample
CH3], 5.31 (m, 1H, Ph–CH–CH2–), 6.92 (d, 2H, J ϭ 8.0 Hz, ArH), 7.45 (d, 2H,
J ϭ 8.5 Hz, ArH); MS [MHϩ] 331.3 (C17H18D5F3NO2 requires 330.2).
and reference cuvettes and added to an equal volume of ACN containing
internal standards. The concentrations of alkyl amines and enzymes were DES
Preparation of N-hydroxy-N-desmethyldiltiazem oxalate. To a stirred solu- or DES-d3 (100 ␮M) and CYP2C11 (0.10 ␮M), (S)-FLX (10 ␮M) and
tion of N-desmethyldiltiazem (0.458 g, 1.14 mmol) in MeOH (15 ml) was CYP2C19 (0.10 ␮M), and MA (10 ␮M) and CYP3A4 (0.25 ␮M). The
added acrylonitrile (0.303 g, 5.72 mmol). The mixture was treated as described
for the first step in the preparation of N-hydroxydesipramine. The intermediate concentrations (50 ␮M) of ring-deuterated DES (DES-d4) and DES-d3.
N-cyanoethyl-N-desmethyldiltiazem (0.488 g, 94%) was obtained as a thick
competitive isotope effect experiment incubations contained a mixture of equal
Cytochrome P450 Activity Assays. Dealkylation of fluorescent probe
liquid. 1H NMR (300 MHz) CDCl3 ␦: 1.92 (s, 3H, –COCH3), 2.37 (s, 3H, substrates BOMR, EMOCC, and DBOMF by CYP2C11, CYP2C19, and
–N–CH3), 2.45–2.88 (m, 6H, H3C–N–CH2–CH2–CN and –CH2–N–CH3), 3.84 CYP3A4, respectively, was assayed fluorometrically (plate reader) using ex-
(s, 3H, –OCH3), 4.28–4.37 (m, 1H, –CO–N–CH2–), 4.77–4.80 (m, 1H, citation and emission wavelengths as suggested by Invitrogen. The Km value
–CO–N–CH2–), 5.01 (d, 1H, J ϭ 7.8 Hz, –S–CH–), 5.14 (d, 1H, J ϭ 7.8 Hz, for each probe substrate-enzyme pair was determined (BOMR-CYP2C11 ϭ
–CH–O–COCH3), 6.89–6.93 (m, 2H, ArH), 7.28–7.32 (m, 1H, ArH), 7.41–
7.45 (m, 2H, ArH), 7.50–7.56 (m, 2H, ArH), 7.70–7.73 (m, 1H, ArH); MS
[MHϩ] 454.3 (C24H27N3O4S requires 453.2).
0.13 ␮M, EMOCC-CYP2C19 ϭ 0.90 ␮M, and DBOMF-CYP3A4 ϭ 1.5 ␮M).
Substrate concentrations equal to the Km values determined were used in
assays for changes in enzyme activity in the TDI studies. Probe substrates were
The N-cyanoethyl-N-desmethyldiltiazem intermediate (0.250 g, 0.55 mmol) incubated for a time period that fell within the linear range of product
was mixed with m-CPBA (77%, 0.123 g, 0.55 mmol) in dry CH2Cl2 at Ϫ78°C formation: BOMR-2C11 15 min, EMOCC-2C19 8 min, and DBOMF-3A4 4
and further treated as described for the second step in the preparation of
N-hydroxydesipramine. Workup and chromatography as described therein
min.
Inhibition of MI Complex Formation Caused by Coincubation with the
afforded N-hydroxy-N-desmethyldiltiazem (0.193 g, 84%) as a thick liquid. Primary Amines. Time-dependent inhibition of CYP2C11, CYP2C19, and
The oxalic acid salt was obtained as a white solid, m.p. 145°C. 1H NMR (300 CYP3A4 activity was assayed using a 96-well format. Each inactivation assay
MHz) CD3OD ␦: 1.88 (s, 3H, –COCH3), 2.87 (s, 3H, –N–CH3), 3.05–3.15 (m, well contained 0.05 ␮M enzyme, 0.1 M KPi buffer, and secondary alkyl amine
1H, –CH2–N–CH3), 3.36–3.43 (m, 1H, –CH2–NH–CH3), 3.82 (s, 3H,
–OCH3), 4.00–4.09 (m, 1H, –CO–N–CH2), 4.46–4.55 (m, 1H, –CO–N–
substrate [50 ␮M DES, 10 ␮M (S)-FLX, or 2 ␮M MA] or secondary hydrox-
ylamine substrate [1 ␮M DOH, 5 ␮M (S)-FLXOH, or 0.5 ␮M MAOH] alone
CH2–), 5.09 (d, 2H, J ϭ 7.8 Hz, –CH–O–COCH3 and –S–CH–), 6.91–6.95 (m, or with the respective primary amine [1 ␮M DDES, 0.1 ␮M (S)-NFLX, or 0.2
2H, ArH), 7.38–7.46 (m, 3H, ArH), 7.60–7.68 (m, 2H, ArH), 7.76–7.79 (m,
1H, ArH); MS [MHϩ] 417.3 (C21H24N2O5S, requires 416.1).
Preparation of N-hydroxy-N-desmethyldiltiazem-d3. To a stirred solution of
␮M MD]. Control wells, without inhibitor, were treated in the same manner.
Inactivation was initiated by addition of NADPH (10 mM, 0.02 ml) after a
3-min equilibration period at 37°C. Aliquots (0.02 ml, 1:10 dilution of the
N-desmethyldiltiazem-d3 (0.330 g, 0.81 mmol) in MeOH (15 ml) was added inactivation assay) were removed from the inactivation assay wells at timed
acrylonitrile (43.4 mg, 5.72 mmol). The mixture was treated as described for intervals (0.5, 1, 2, 4, and 7 min) and transferred to activity assay wells to
the first step in the preparation of N-hydroxydesipramine. The intermediate determine enzyme activity. The activity assay wells contained KPi buffer (0.1
N-cyanoethyl-N-desmethyldiltiazem-d3 (0.340 g, 91%) was obtained as a thick
M), NADPH (1 mM), and the appropriate concentration of probe substrate.
liquid. 1H NMR (500 MHz) CDCl3 ␦: 2.42–2.84 (m, 10H, –N–CH2–CH2–CN, The activity assay wells were incubated over the appropriate time interval and
–CH2–N–CH3, –CH2–N–CH3, and –CO–N–CH2), 3.86 (s, 3H, –OCH3), 4.30– were quenched with 0.1 ml of isopropanol. The contents of the activity assay
4.45 (m, 1H, –CO –N–CH2–), 5.04 (d, 1H, J ϭ 7.5 Hz, –CH–O–COCD3), 5.17 wells were then transferred to a second 96-well plate and fluorescent metab-
(d, 1H, J ϭ 8.0 Hz, –S–CH–), 6.92 (d, 2H, J ϭ 8.50 Hz, ArH), 7.31–7.35 (m,
olite concentrations were determined using a Microplate Spectrophotometer
2H, ArH), 7.43 (d, 2H, J ϭ 8.5 Hz, ArH), 7.54 (d, 1H, J ϭ 3.0 Hz, ArH), 7.73 Gemini XPS (Molecular Devices, Sunnyvale, CA). Rates of enzyme inactiva-
(d, 1H, J ϭ 7.5 Hz, ArH); MS [MHϩ] 457.3 (C24H30N3O4S, requires 456.2). tion were estimated from the slopes of the semilog plots of percent remaining
The N-cyanoethyl-N-desmethyldiltiazem-d3 intermediate (0.220 g, 0.48
mmol) was treated with m-CPBA (77%, 0.084 g, 0.48 mmol) in dry CH2Cl2 at
enzyme activity in the presence and absence of inactivator with time.
LC-MS and LC-MS/MS Methods. Synthetic and stable-labeled internal
Ϫ78°C as described for the second step in the preparation of N- standards for substrates and metabolites were used to develop LC methods, to
hydroxydesipramine. Workup and chromatography as described therein af- optimize MS conditions, and to identify new metabolites [(S)-FLXOH and
forded N-hydroxy-N-desmethyldiltiazem-d3 (0.162 g, 81%) as a thick liquid. MAOH].
The oxalic acid salt was a white solid, m.p. 150–152°C. 1H NMR (500 MHz)
DES-related assays. LC-MS analysis of the metabolites of DES, DESOH,
CD3OD ␦: 2.73 (s, 3H, –N–CH3), 2.94 (m, 2H, –CH2–N–CH3), 3.71 (s, 3H, and DDES was performed on a Micromass Platform LCZ (Waters, Milford,
–OCH3), 3.92 (m, 1H, –CO–N–CH2–), 4.38 (m, 1H, –CO–N–CH2–), 6.80 (m, MA). The Micromass Platform LCZ, equipped with Shimadzu LC-10AD
2H, ArH), 7.28–7.66 (m, 6H, ArH); MS [MHϩ] 420.2 (C21H21D3N2O5S pumps and autoinjector (Shimadzu Scientific Instruments, Inc., Columbia,
requires 419.2).
MD), was operated in the electrospray positive ion mode. Analyte separation

SEQUENTIAL METABOLISM OF SECONDARY AMINES TO MI COMPLEXES
967
was achieved using a Zorbax SB-C3 column (150 ϫ 2.1 mm, 5 ␮m particle
size) at a flow rate of 0.4 ml/min. Solvent A was Nanopure water with 0.025%
acetic acid and 0.01% n-propylamine. Solvent B was HPLC-grade ACN with
0.025% acetic acid and 0.01% n-propylamine. The solvent program was 70%
A:30% B (0–0.5 min), linear gradient to 40% B (0.5–3.5 min), and linear
gradient to 80% B (3.5–6 min), which was held constant from 6 to 7 min. The
total run time was 12 min. The retention times were 5.2 and 7.2 min for DDES
(MHϩ: m/z ϭ 253.3 d0 and m/z ϭ 256.3 d3 substrates) and DESOH (MHϩ:
m/z ϭ 283.3 d0 and m/z ϭ 286.3 d3 substrate), respectively. For quantification,
standard curves were constructed using ring-labeled (DES-d4, DDES-d4, and
by their respective major metabolites in the presence of CYP2C11,
CYP2C19, and CYP3A4. Representative spectra obtained for the
secondary amines, secondary hydroxylamines, and primary amines
are shown in Fig. 3. The absorbance maxima (␭max) were DES,
DDES, DOH-CYP2C11 (␭
ϭ 455 nm), (S)-FLX, (S)-NFLX, and
max
(S)-FLXOH-CYP2C19 (␭max ϭ 458 nm). In the MA-CYP3A4 series,
the tentative MI complex spectrum of MD was red-shifted (␭
ϭ
max
460 nm) and asymmetric compared with the spectra for MA and
MAOH (␭ ϭ 452 nm). The theoretical concentrations of the MI
max
DESOH-d4) internal standards. Data were acquired in the SIM mode, and peak complexes were calculated based on the literature extinction coeffi-
cient of 65 mMϪ1 cmϪ1 for the 455 to 490 nm absorbance difference
areas were corrected for ion overlap as required. The cone voltage was 25 V
for all DES-related compounds. In the competitive isotope effect experiments,
(Liu and Franklin, 1985). The P450 concentrations provided by BD
peak area ratios of DESOH and DDES metabolites for coincubated ring-
Gentest were confirmed by CO difference spectra and then were used
labeled DES-d4 and DES-d3 were corrected for measured substrate concentra-
to calculate the theoretical yield of MI complex. The concentrations of
tions and ion overlap based on the ion envelopes of the substrates as necessary.
the MI complexes from incubations with the secondary hydroxy-
All other incubation samples were analyzed for metabolites using a Micromass
lamines were 100, 98, and 115% of theoretical for DESOH, (S)-
FLXOH, and MAOH, respectively. The corresponding values for the
Premier XE (Waters) equipped with an ACQUITY UPLC system and an autoin-
jector integral to the ACQUITY UPLC system. Chromatographic separations were
secondary amines were 80, 90, and 118% of theoretical values,
whereas the values for the primary amines were much lower (11, 8,
achieved on an ACQUITY UPLC C18 BEH column (2.1 ϫ 50 mm, 1.7 ␮m;
Waters). The mass spectrometer was operated in the positive ion mode.
(S)-FLX-related assays. These substrates and metabolites were separated and 23%, respectively). After each MI complex formed to completion
using a linear gradient at 0.3 ml/min and mobile phases A (0.1% formic acid
in water) and B (0.1% formic acid in ACN). The gradient started at 90% A
(0–0.5 min). Solvent B was linearly increased from 10% at 0.5 min to 100%
at 4 min. The total run time was 5 min. The parent and product ions monitored
for (S)-FLX were 310 Ͼ 44 m/z, for (S)-NFLX were 296 Ͼ 134 m/z, and for
(S)-FLXOH were 326 Ͼ 60 m/z. The parent and product ions for internal
standards were FLX-d6 316 Ͼ 44 m/z, NFLX-d6 302 Ͼ 140 m/z, and
FLXOH-d5 331 Ͼ 60 m/z. The cone and collision energies for (S)-FLX and
FLX-d6 were 20 V and 15 kV, for (S)-NFLX and NFLX-d6 were 10 V and 5
kV, and for (S)-FLXOH and FLXOH-d5 were 12 V and 10 kV.
for each inhibitor-enzyme pair, potassium ferricyanide was added to
MA-related assays. These compounds were separated using a linear gradient at
0.3 ml/min and mobile phases A (0.1% formic acid in water) and B (0.1% formic
acid in ACN). The gradient started with 80% A (0–0.5 min). Solvent B was
linearly increased to 100% over 3.5 min and held constant at 100% for 0.5 min.
The total run time was 5.5 min. The parent and product ions monitored for MA
were 401 Ͼ 178 m/z, for MD were 387 Ͼ 178 m/z, and for MAOH were 416 Ͼ
178 m/z. Internal standards for MA-d3 were 404 Ͼ 178 m/z, for MAOH-d3
were 419 Ͼ 178 m/z, and for alprazolam were 309 Ͼ 205 m/z. The cone and
collision energies for MA and MA-d3 were 25 V and 25 kV, for MAOH and
MAOH-d3 were 30 V and 20 kV, for MD were 25 V and 25 kV, and for
alprazolam were 30 V and 35 kV.
Results
Synthesis of Metabolites and Internal Standards. The structures
of the secondary amines used in this study are given in Fig. 2.
Synthesis of the secondary hydroxylamines from the corresponding
secondary amines was accomplished by the method of O’Neil et al.
(2001), in which N-alkylation of the secondary amine with acryloni-
trile is followed by N-oxidation with m-CPBA and Cope elimination
of the protecting group. (S)-FLXOH and MAOH have not been
reported previously. DESOH and MAOH were prepared as the oxalate
salts. Stable-labeled standards of the substrates and metabolites were
prepared or purchased, with the exception of MD. DDES-d4 was
obtained by acid-catalyzed exchange of four or more deuterium atoms
in the aromatic rings by the method of Baba et al. (1988). DESOH-d4
was synthesized from deuterated imidodibenzyl-d4, exchanged by the
same method. DES-d3, labeled on the N-methyl carbon, was synthe-
sized by reaction of trideuteromethylamine with 3-chloroimidodiben-
zylpropane. FLX-d6 and NFLX-d6 were purchased, and FLXOH-d5
was prepared from commercially available FLX-d5. Internal standards
MA-d3 and MAOH-d3,with three deuterium atoms in the acetoxy
methyl group, were synthesized.
FIG. 3. Final MI complex spectra obtained by incubation of each compound at a
concentration 10 ␮M. a, DES series incubated with CYP2C11. b, FLX series
incubated with CYP2C19. c, MA series incubated with CYP3A4. MI complex
formation by each secondary amine (——), primary amine(– – –), and secondary
hydroxylamine (⅐ ⅐ ⅐ ⅐ ⅐). The absorption (ABS) maximum for each complex ranged
from 452 to 460 nm. Scans were recorded for 15 min total (20-s intervals). After MI
complex formation with each inhibitor-enzyme pair was complete, the potassium
ferricyanide reversal test was used. In each case, loss of peak at approximately 455
MI Complex Formation. MI complexes were observed upon in-
cubation of the three secondary amines, DES, (S)-FLX, and MA, and nm was observed after addition of potassium ferricyanide.

968
HANSON ET AL.
the sample and reference cuvettes to test for quasi-reversibility of the primary amines but noticeably slower than the rates observed from the
secondary hydroxylamines. MI complex formation from the primary
amines, DDES and (S)-NFLX, was barely discernable and incomplete
(11 and 8% of the theoretical yield, respectively). For incubations of MD
with CYP3A4, a rapid burst in absorbance at 460 nm was observed in the
first two scans, which accounted for 18% of theoretical yield, followed by
a gradual increase to 23% of theoretical yield by 15 min. This unusual
biphasic accumulation profile could result from a contaminant present in
the primary amine MD; however, identical results were obtained with a
sample of MD purified by semipreparative HPLC. Overall, the relative
rates of MI complex formation follow the consistent order secondary
hydroxylamine Ͼ secondary amine ϾϾ primary amine.
MI complex. In each case, loss of the peak at approximately 455 nm
was observed.
The accumulation of MI complexes from these alkyl amines and
metabolites are shown in Fig. 4. In each series, MI complex formation
rates for the secondary amines were markedly faster than from the
Stoichiometry of P450 Oxidation of the Secondary Amines. The
product stoichiometry of the P450-catalyzed oxidation of the secondary
amines was determined from the concentrations of the corresponding
primary amine and secondary hydroxylamine metabolites and MI com-
plexes (Table 1). The concentrations of the alkyl amines and P450s used
in these experiments are in the legend to Table 1. Metabolites were
quantitated by LC-MS or LC-MS/MS using authentic standards, and
standard curves were constructed using stable-labeled internal standards
or, in the case of MD, alprazolam. The primary and secondary amines
were stable in solution during the sample workup and subsequent anal-
ysis. Stable-labeled internal standards were used for quantitation of the
secondary hydroxylamines because of concerns of stability through sam-
ple workup and analysis. Structure assignments for the metabolites were
made by comparison to the mass spectra and retention times of authentic
standards obtained from commercial sources (DDES, NFLX, and MD) or
by synthesis [DESOH, (S)-NFLXOH, and MAOH].
Metabolite formation was NADPH-dependent and negligible
amounts of the metabolites were observed in control incubations.
From each substrate, the primary amine and the secondary hydroxyl-
amine metabolites were observed, and the primary amine was the
major metabolite. The MI complexes accounted for 7, 17, and 37% of
total products from DES-CYP2C11, (S)-FLX-CYP2C19, and MA-
CYP3A4, respectively. The ratio of metabolites (primary amine/
secondary hydroxylamine) (Table 1) observed ranged from 2.4:1
(MA-CYP3A4) to 17:1 [(S)-FLX-CYP2C19].
A second branching ratio [primary amine/(secondary hydroxyl-
amine ϩ MI complex)] (Table 1) was also calculated. The branching
ratio is particularly meaningful if further metabolism of the secondary
hydroxylamines, rather than the primary amines, leads to MI complex
formation because it provides an improved estimate of the true ratio of
the rates of N-dealkylation to N-hydroxylation. The calculated ratios
ranged from 0.80:1 (MA-CYP3A4) to 3.6:1 [(S)-FLX-CYP2C19].
Although the branching ratios are probably different from the “true”
ratios of product formation rates, it seems likely from these data that
secondary hydroxylamine formation from secondary amines may oc-
casionally exceed rates of N-dealkylation.
FIG. 4. The accumulation of MI complexes by secondary amines (X), primary
amines (E), and secondary hydroxylamines (F). a, DES series. b, FLX series. c, MA
series. The theoretical concentration of MI complex was calculated based on total
P450 enzyme using the molar extinct coefficient of 65 mMϪ1 cmϪ1 (Liu and
Franklin, 1985).
TABLE 1
Final concentrations of primary metabolites and MI complexes formed from desipramine, (S)-fluoxetine, and N-desmethyldiltiazem
The concentrations of the three products, primary amine, secondary hydroxylamine, and MI complex, from incubations with 100 ␮M DES, 10 ␮M FLX, 10 ␮M MA, and 100 ␮M DES-d3.
The concentration of enzyme in each experiment was 0.10 ␮M, except for CYP3A4 for which the concentration was 0.25 ␮M. Numbers in parentheses are the S.D. of triplicate experiments or
S.E. of the ratios.
Substrate/Enzyme
Primary Amine
Secondary Hydroxylamine
MI Complex
MRa
BRb
␮M
␮M
␮M
Desipramine/CYP2C11
(S)-Fluoxetine/CYP2C19
N-Desmethyldiltiazem/CYP3A4
Desipramine-methyl-d3/CYP2C11
0.89 (0.10)
0.50 (0.01)
0.23 (0.02)
0.40 (0.04)
0.23 (0.01)
0.030 (0.001)
0.10 (0.01)
0.27 (0.02)
0.08 (0.01)
0.11 (0.01)
0.19 (0.01)
0.07 (0.02)
3.9 (0.5)
17 (1)
2.4 (0.3)
1.5 (0.2)
2.9 (0.4)
3.6 (0.4)
0.80 (0.09)
1.2 (0.4)
a Metabolite ratio (MR): primary amine/secondary hydroxylamine.
b Branching ratio (BR): primary amine/(secondary hydroxylamine ϩ MI complex).

SEQUENTIAL METABOLISM OF SECONDARY AMINES TO MI COMPLEXES
969
The DES-CYP2C11 system was probed for isotope effects in primary amine (Fig. 3). The primary amines are the major metabolites
N-dealkylation, N-hydroxylation, and MI complex formation by com- observed and accumulate to significant concentrations in the incuba-
parison of the product profiles from separate incubations with DES tions with the secondary amines [DDES ϭ 0.89 ␮M with 0.10 ␮M
and DES-d3. A significant normal isotope effect of 2.2 (0.3) was 2C11; (S)-NFLX ϭ 0.50 ␮M with 0.10 ␮M 2C19; and MD ϭ 0.23
observed for final DDES concentrations, whereas an inverse isotope ␮M with 0.25 ␮M 3A4] (Table 1).
effect was observed for the concentrations of DOH [0.85 (0.07)]. The
The primary amine metabolites, although poor inactivators of the
apparent isotope effect ratio for the concentrations of MI complex in enzymes, may serve as competitive inhibitors of MI complex forma-
the incubations was 1.1 (0.4). The calculated product ratios, although tion by competing with the substrates and products of the secondary
reflecting the effects of the underlying isotope effects, are not true hydroxylamine pathway. The reversible Ki for inhibition of CYP3A4
kinetic isotope effects because they were not obtained at steady state. activity by MD in HLMs is 0.2 ␮M (Zhao et al., 2007). The calculated
The initial rates of MI complex formation for DES and DES-d3 I/Ki value based on the final concentration of MD in our incubations
were estimated by fitting the progress curves to the function Abst ϭ (0.23/0.2 ␮M) is approximately 1, suggesting that inhibition of inac-
Absmax(1 Ϫ eϪkt). The estimates of the initial rates of MI complex tivation may have occurred in our studies. Reversible Ki estimates
formation were not significantly different [kd0 ϭ 0.40 (0.01) minϪ1 were also determined for DDES-CYP2C11 (0.8 ␮M) and (S)-NFLX-
versus kd3 0.44 (0.07) minϪ1; p Ͼ 0.05, two-tailed t test], and the CYP2C19 (0.3 ␮M) from which I/Ki values for the final concentrations
calculated isotope effect was slightly inverse [Hk/Dk ϭ 0.91 (0.10)], of the primary amines of 1.1 and 1.6 can be calculated, respectively.
but not significantly different from 1.
Because these ratios are all near 1, it is likely that the primary amines will
In addition, we performed a competitive experiment with equal compete with substrates and other pathway metabolites for active enzyme
molar amounts of DES and DES-d3. The DES used in this experiment and progressively slow the rates of enzyme inactivation.
was d4 ring-labeled to distinguish the products of N-dealkylation from
To test for this possibility, we performed a series of studies to
DES-d3 and DES-d0. The respective competitive isotope effects on examine the effects of the primary amines on enzyme inactivation by
product ratios were 1.8 (0.1) for N-demethylation (DDES) and 0.52 both secondary amines and their secondary hydroxylamine metabo-
(0.01) for N-hydroxylation (DESOH).
lites. The concentrations of the secondary amines in the inactivation
These results compare favorably with those of a previous report, in assays and estimates of reversible affinities for their respective en-
which deuterium isotope effects and metabolic switching for the zymes were as follows: DES ϭ 50 ␮M with CYP2C11 (IC50 ϭ 5 ␮M,
metabolism of cyclohexylamine to cyclohexanone and cyclohexylhy- data not shown), (S)-FLX ϭ 10 ␮M with CYP2C19 (IC50 ϭ 4.7 ␮M
droxylamine in rabbit liver microsomes of DV ϭ 1.75 C–H bond in HLMs) (Di Marco et al., 2007), and MA ϭ 2 ␮M with CYP3A4
oxidation and DV ϭ 0.85 for N-hydroxylation in steady-state experi- (Ki ϭ 2 ␮M in HLMs) (Zhao et al., 2007). The secondary hydroxyl-
ments (Kurebayashi, 1989). Together, these metabolic switching re- amine substrate concentrations used were lower to facilitate data
sults indicate that N-dealkylation and N-oxygenation probably arise acquisition and curve-fitting: DESOH ϭ 1 ␮M, (S)-FLXOH ϭ 5 ␮M,
from a common P450 cycle intermediate (Chunsen et al., 2009).
and MAOH ϭ 0.5 ␮M. The initial rates of the loss of enzyme activity
These studies provide insights into the metabolism of these sec- caused by the secondary amines and secondary hydroxylamines were
ondary amine drugs with respect to MI complex formation. First, determined in triplicate in the presence and absence of fixed concen-
primary amines and secondary hydroxylamines are formed in signif- trations of the primary amines at their respective Ki or IC50 values.
icant quantities from each of these secondary alkyl amines. Second,
The results of these experiments are given in Fig. 5, which shows
the MI complex comprises a significant fraction of the total products the initial rates of inactivation as a function of the inhibitor(s) in the
measured, suggesting that the pathways to MI complex are efficient inactivation assays. Inactivation caused by the secondary amines and
(low partition ratio). In the cases of (S)-FLX and MA metabolism, the secondary hydroxylamines were, in all cases, significantly (p Ͻ 0.05)
final concentrations of the secondary hydroxylamines were less than inhibited by coincubation with the respective primary amines. Note
the corresponding MI complexes (27 and 51%, respectively). These that the concentrations of primary amines (S)-NFLX and MD required
results imply that these secondary hydroxylamines have a high affin- to inhibit inactivation by roughly 50% were less than their concen-
ity for the enzymes and that the “slow step” in the secondary hydrox- trations measured during formation of MI complexes caused by the
ylamine pathway could be N-hydroxylation of the secondary amines. secondary amines. These results show that the primary amine metab-
The isotope effect experiments with DES provide additional in- olites generated from the secondary amines will cause meaningful
sights. First, deuterium substitution at the N-methyl group of DES inhibition of the formation of MI complexes from the metabolism of
provided significant normal isotope effects in both the competitive the secondary amines (Fig. 6).
and noncompetitive experiments. The inverse isotope effect in the
competitive experiment demonstrates that metabolic switching occurs
Discussion
between N-dealkylation and N-hydroxylation. A more complete
The pathways of P450-catalyzed metabolism of the alkyl amine
mechanistic interpretation of these results would require experiments moiety include N-dealkylation and N-hydroxylation reactions that
in the steady state, as well as a consideration of the effects of result in the formation of a catalytically inactive MI complex (Fig. 1).
subsequent isotope effects for further metabolism of the secondary A significant number of DDIs are believed to arise from the formation
hydroxylamine. Second, in the noncompetitive design, the isotope of MI complexes by alkyl amine-containing drugs (Backman et al.,
effects on DDES concentration were normal (2.2), whereas the iso- 1994; Mayhew et al., 2000; Hall et al., 2003; Zhang et al., 2010).
tope effects for initial MI complex formation rate and the final Considerable effort has been directed toward development of a model
concentrations of DESOH and MI complex were not significantly to predict the magnitude of DDIs using in vitro data. Mayhew et al.
different from 1. The lack of a significant isotope effect on the initial (2000) developed a model to account for interactions caused by
rate of complex formation indicates that the primary amine pathway fluoxetine, N-desmethyldiltiazem, and clarithromycin with CYP3A.
must play a minor role in MI complex formation from DES.
This model assumed a hyperbolic relationship between alkyl amine
Inhibition of MI Complex Formation by the Primary Amines. concentration and MI complex formation rates in vitro and in vivo.
The rates of accumulation of MI complex in each series follow the The original model was subsequently modified to better predict the
general trend of secondary hydroxylamine Ͼ secondary amine ϾϾ magnitude of interactions in the liver and gut and to account for the

970
HANSON ET AL.
FIG. 5. The initial rates of P450 inactivation caused by the second-
ary amine (dark gray) or secondary hydroxylamine (light gray) in
control experiments. The striped bars immediately to the right of
each show the effect of coincubation with the primary amines of
each series. The concentrations were as follows: DES-CYP2C11
series (50 ␮M DES, 1 ␮M DESOH, and 1 ␮M DDES); (S)-FLX-
CYP2C19 series [10 ␮M (S)-FLX, 5 ␮M (S)-FLXOH, and 0.1 ␮M
(S)-NFLX]; MA-CYP3A4 series (2 ␮M MA, 0.5 ␮M MAOH, and
0.2 ␮M MD). Primary amines caused significant (p Ͻ 0.05) inhi-
bition of inactivation by the secondary amines and secondary hy-
droxylamines in all cases.
combined effects of tertiary amines and their secondary amine metabo- the formation of MI complexes in rat liver microsomes by N-meth-
lites (Galetin et al., 2006; Zhang et al., 2009a,b). A key assumption
implicit to these models is that the fates of the relevant downstream
metabolites (hydroxylamines, nitrones, and nitroso compounds) formed
in vivo are reproduced in the in vitro experiments, which are used to
generate the apparent kinetic parameters (KI and kinact). A second
assumption of these models is that the contribution of these metabo-
lites in the promotion or inhibition of enzyme inactivation is known.
The metabolic scheme for the formation of MI complexes from
tertiary alkyl amines includes multiple pathways and branching reac-
tions that complicate analysis even in single enzyme systems (Fig. 1).
Here, the metabolism of secondary amines to primary amines and
secondary hydroxylamines was studied. There is ample literature
precedent to show that metabolism of these two products of secondary
amine metabolism can lead to the formation of MI complexes. The
rates of MI complex formation by a secondary amine and its primary
metabolites, where each step in the pathway is catalyzed by the same
enzyme, have not been directly compared in single enzyme systems. We
examined three secondary amine-enzyme systems: DES-CYP2C11, (S)-
FLX-CYP2C19, and MA-CYP3A4. In each case, incubation of the
secondary amines with their respective P450 enzyme caused rapid
formation of MI complexes. The relative rates of MI complex forma-
tion followed the order secondary hydroxylamine Ͼ secondary amine ϾϾ
primary amine (Fig. 3). Of particular note, MI complex formation by
the primary amines was slow and incomplete. We also found that the
primary amines inhibit rather than promote enzyme inactivation
caused by the secondary amines. Lindeke et al. (1979) suggested that
ylamphetamine arises via the secondary hydroxylamine pathway
rather than via the formation of the primary amine. Likewise, Jeffery
and Mannering (1983) concluded that MI complex formation from the
metabolism of N-benzylamphetamine arises via the secondary hy-
droxylamine pathway rather than through the formation of the primary
amine. By these criteria and the literature precedent, we propose that
the secondary hydroxylamine metabolites, rather than the primary
amines from DES, (S)-FLX, and MA, are on the major pathway to MI
complex formation.
Two additional lines of evidence support this conclusion. First, the
secondary hydroxylamines (DESOH, (S)-FLXOH, and MAOH) are
observed as metabolites of the secondary amines. The formation of
DESOH from imipramine and desipramine has been demonstrated in
rabbit liver microsomes (Beckett et al., 1983), whereas (S)-FLXOH
and MAOH are newly identified metabolites. Second, deuterium
substitution on the methyl group of DES results in a 2-fold reduction
of formation of DDES from DES by CYP2C11 but has no significant
effect on the final concentration of DESOH or the MI complex.
Furthermore, there is no significant isotope effect on the initial rate of
MI complex formation, which rules out a significant contribution of
the primary amine pathway to MI complex formation.
The factors that govern the rates of formation of the primary amine
and secondary hydroxylamine metabolites from a secondary amine
will be important determinants of the formation rates for MI com-
plexes and the magnitude of DDIs caused by alkyl amines. The
clearance of primary amines can be slow, and primary amine metab-
olites can accumulate in vivo. The N-hydroxylation reaction, on the
other hand, has received less mechanistic attention and the fate of
hydroxylamines in vivo and in vitro is not well understood. In addi-
tion, although N-dealkylation is generally catalyzed by P450 enzymes,
N-hydroxylation reactions may be catalyzed by other enzymes present
in more complex systems such as microsomes, hepatocytes, and the
intact liver. For example, it has been shown that the formation of an
MI complex in microsomes by a secondary cyclopropylamine re-
quired the flavin-containing monooxygenase-catalyzed conversion to
a secondary hydroxylamine (Cerny and Hanzlik, 2005).
Little information about the product ratios and reaction rates for the
generation of primary amines and secondary hydroxylamines from
secondary amines by P450 enzymes has been reported. However, data
are available that suggest that N-hydroxylation and N-dealkylation are
mechanistically linked. For instance, metabolism of the N-ethylam-
phetamine isomers results in a ratio of N-dealkylation to N-hydroxy-
FIG. 6. The major roles of the primary amine and secondary hydroxylamine me-
tabolites in the metabolism of the three secondary amines to MI complexes. The
formation of MI complexes occurs via the secondary hydroxylamine pathway.
Formation of MI complexes by the primary amine pathway is negligible. The
primary amine metabolites accumulate in incubations to significant levels that cause
significant inhibition of MI complex formation by the secondary amines.

SEQUENTIAL METABOLISM OF SECONDARY AMINES TO MI COMPLEXES
971
lation (sum of secondary hydroxylamine and ethylene nitrone) of 5:1 formation of MI complexes by N-desmethylclarithromycin and N,N-
for the (S)-enantiomer and 3:1 for the (R)-enantiomer in rabbit liver didesmethylclarithromycin in dexamethazone-induced rat liver micro-
microsomes (Beckett and Haya, 1978). In addition, the metabolism of somes. In this instance, the rates of MI complex formation seem to
methamphetamine by five purified rat enzymes results in product be similar for the primary and secondary amines incubated at the same
ratios (primary amine/secondary hydroxylamine) that range from 2 concentration (Ohmori et al., 1993). The cycloalkylamine substructure
of clarithromycin and erythromycin may selectively promote the
primary hydroxylamine pathway. This possibility is supported by
studies with analogs of the amphetamine series, which clearly dem-
onstrate that the rate of MI complex formation by primary amines is
increased by the presence of a branched lipophilic group on the
␣-carbon of the alkyl amine (Lindeke et al., 1982). Although struc-
ture-activity relationship studies have not been reported for the cor-
responding effects of ␣-carbon substitution on the conversion of
secondary amines to MI complexes and/or secondary hydroxylamines,
studies in microsomes also show that substitution on the ␣-carbon
substantially increase catalytic efficiency (Vmax/Km) for N-demethyl-
ation of secondary amines (Duncan et al., 1983). Thus, it is feasible
that the presence of branched lipophilic groups on the ␣-carbon,
exemplified by the cycloalkylamine structures of erythromycin and
clarithromycin, will serve to increase the likelihood that the primary
amine pathway will contribute to MI complex formation.
Fluoxetine causes TDI of CYP2C19 activity in Supersomes, HLMs,
hepatocytes, and profound inhibition in vivo (McGinnity et al., 2006).
Thus, we conclude that the inactivation of CYP2C19 both in vitro and
most likely in vivo is due to formation of the secondary hydroxyl-
amine metabolite of fluoxetine, which subsequently forms an MI
complex to inactivate the enzyme. The CYP3A4-diltiazem interaction
has received considerable clinical, experimental, and theoretical at-
tention. We conclude that formation of an MI complex with CYP3A4
in vitro and possibly in vivo is through the secondary hydroxylamine
metabolite of diltiazem. In addition, we show that the primary amine
metabolite protects CYP3A4 from MI complex formation by accu-
mulating in solution and competing for active enzyme (Ki ϭ 0.2 ␮M)
(Zhao et al., 2007). However, similar roles in vivo cannot be assigned
to these metabolites because the fate of the primary amine and
secondary hydroxylamine of diltiazem is unknown.
These studies show that secondary hydroxylamine metabolites of
DES, (S)-FLX, and MA promote P450 MI complex formation. In
these cases, the primary amine metabolites inhibit MI complex for-
mation. Further characterization of alkyl amine metabolism by P450
enzymes in single enzyme and multiple enzyme preparations is nec-
essary before establishment of guidelines for prediction. It seems
prudent to include studies in hepatocytes, in which the metabolites are
exposed to the full complement of enzymes, when seeking to eliminate or
confirm the possibility of DDIs with alkyl amines (Zhao et al., 2005;
McGinnity et al., 2006). Levels of primary amines and hydroxylamines in
vitro and in vivo may be significantly different, and the prediction of
primary and secondary metabolite concentrations in vivo from in vitro
data is problematic at best and warrants further investigation.
(CYP2B2) to 14 (CYP1A1) (Baba et al., 1988). The branching ratio
estimates calculated from our studies [primary amine/(secondary hy-
droxylamine ϩ MI complex)] range from less than 1 (MA-
CYP3A4 ϭ 0.8) to 3.6 [(S)-FLX-CYP2C19]. Multiple P450 enzymes,
including CYP3A4, CYP2C9, and CYP2D6, catalyze the N-demeth-
ylation of the fluoxetine isomers. In addition, we have found that
N-demethylation is always accompanied by N-hydroxylation in single
enzyme systems and in microsomes (B. M. VandenBrink and K. L.
Kunze, unpublished data). Theoretical calculations for the P450-
catalyzed oxidation of trimethylamine and dimethylaniline to the cor-
responding secondary amines and N-oxides indicate that N-demethylation
and N-oxygenation are both catalyzed by the perferryl oxygen. N-Deal-
kylation is predicted to be favored over N-oxygenation by 2.6 kcal/mol
(Chunsen et al., 2009). The above experimental results for the analogous
P450-catalyzed reactions with secondary amines indicate that the differ-
ence in activation energies for the two competing reactions is low and
variable with enzyme and substrate.
MI complex formation by DES, (S)-FLX, and MA is inhibited by
the corresponding primary amine metabolites. Significant inhibition
of MI complex formation by the primary amine metabolites would be
expected to influence the progress curves for MI complex formation.
Silverman (1988) considered the possibility that a metabolite of a
conventional one-step mechanism-based inhibitor will have a signif-
icant affinity for the enzyme and concluded that the observed rate of
inactivation will be time-dependent. The resulting conventional semi-
log plots will, in some cases, be curved as reproduced in Fig. 7.
Likewise, Zhang et al. (2009a) have considered the combined effects
of tertiary amines and their secondary amine metabolites in vitro and
in vivo and concluded that both species (e.g., diltiazem and MA) must
be explicitly considered as substrates and inhibitors of inactivation to
achieve accurate predictions. Our data show that the models may need
to be modified to incorporate terms that account for the effects of the
primary amine metabolites and that reflect the roles of the other
branched metabolites.
Although our results favor MI complex formation through the
secondary hydroxylamine metabolite, the fact that primary amines can
cause formation of MI complexes is well established. One example for
which the primary amine pathway may be more important is in the
Acknowledgments. We acknowledge Tom Kalhorn for help in
development of the LC-MS and LC-MS/MS assays. We thank Ping
Zhao for helpful discussions regarding this work.
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