Synthesis and pharmacology of ethylphenidate enantiomers: The human transesterification metabolite of methylphenidate and ethanol

Article abstract of DOI:10.1021/jm0490989

Ethanol elevates methylphenidate (1) plasma concentrations and yields the metabolite ethylphenidate (2). The therapeutic implications are tinder investigation. The IC₅₀ for dopamine reuptake inhibition by (+)-2 was 27 nM compared to 367 nM for cocaine and 1730 nM for (-)-2. Binding selectivity for dopamine versus norepinephrine transporters was greater for (+)-2 than for cocaine. Intraperitoneal (+)-2 was approximately half as active as (+)-1 in stimulating mouse motor activity at 5 mg/kg, but (+)-2 was as active as (+)-1 at 10 mg/kg.

Full text of DOI:10.1021/jm0490989

2876
J. Med. Chem. 2005, 48, 2876-2881
Synthesis and Pharmacology of Ethylphenidate Enantiomers: The Human
Transesterification Metabolite of Methylphenidate and Ethanol
Kennerly S. Patrick,*,† Robin L. Williard,†,‡ Adam L. VanWert,† Justin J. Dowd,† John E. Oatis, Jr.,§ and
Lawrence D. Middaugh#
Departments of Pharmaceutical Sciences, Physiology and Neuroscience, Pharmacology and Experimental Therapeutics, and
Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina 29425
Received November 9, 2004
Ethanol elevates methylphenidate (1) plasma concentrations and yields the metabolite
ethylphenidate (2). The therapeutic implications are under investigation. The IC50 for dopamine
reuptake inhibition by (+)-2 was 27 nM compared to 367 nM for cocaine and 1730 nM for
(-)-2. Binding selectivity for dopamine versus norepinephrine transporters was greater for
(+)-2 than for cocaine. Intraperitoneal (+)-2 was approximately half as active as (+)-1 in
stimulating mouse motor activity at 5 mg/kg, but (+)-2 was as active as (+)-1 at 10 mg/kg.
Introduction
Attention-deficit hyperactivity/disorder (ADHD) is the
most frequently diagnosed childhood neurobehavioral
health problem, and (()-threo-methylphenidate [(()-1,
Figure 1] is the drug most widely prescribed for its
treatment.1 Since the 1980s, the persistence of ADHD
Figure 1. Left: (+)-(2R,2′R)-threo-methylphenidate [(+)-1, R
into adolescence and adulthood has been increasingly
) CH3)], (+)-(2R,2′R)-threo-ethylphenidate [(+)-2, R ) CH2-
recognized.2 Appropriate drug therapy for this older
CH3], and (2R,2′R)-threo-ritalinic acid [(2R,2′R)-3, R ) H].
ADHD population requires a special consideration of
lifestyle comorbidity. For example, substance/alcohol
Right: (-)-(2S,2′S)-threo-methylphenidate [(-)-1, R ) CH3)],
(-)-(2S,2′S)-threo-ethylphenidate [(-)-2, R ) CH2CH3], and
abuse and dependence appear to be over-represented
in adult ADHD.3,4 Accordingly, prescribing psycho-
stimulants to this population has generated concern.5
In this context, nothing is known regarding the phar-
macology of concomitant 1 and ethanol use or abuse.
This concern is justified by numerous recent reports of
1-ethanol coabuse6,7 and by emergency department
statistics on 1-ethanol drug combination episodes. In
1997 and 1999, respectively, there were 553 and 422
1-ethanol emergency episodes documented.8
The metabolic hydrolysis of methyl ester 1 yields
inactive9 ritalinic acid (3, Figure 1). This facile process
limits the absolute bioavailability of the enantiomers
of 1 to <50% for the (+)-isomer and usually <5% for
the (-)-isomer, and results in the relatively short 2-3
h elimination half-life of 1.1 However, consumption of
ethanol while receiving 1 results in the methyl ester also
being transesterified to yield the ethyl ester ethylpheni-
date (2, Figure 1).1,10-13 The specific carboxylesterase
isoform primarily responsible for the hydrolysis of 1 may
similarly catalyze this ethanolysis pathway.14-16 The
formation of 2 in humans dosed with 1 and ethanol
occurs enantioselectively.1,12,13 Further, concomitant
ethanol results in significantly elevated plasma concen-
trations of the parent drug 1.1,13
(2S,2′S)-threo-ritalinic acid [(2S,2′S)-3, R ) H].
the activity of racemic 2. Portoghese and Malspeis17
reported that (()-2 induces locomotor activity in mice
with approximately 80% of the potency of (()-1, and
Schweri et al.18 found that (()-2 exhibits approximately
50% of the potency of (()-1 in inhibiting [3H]-(()-1
binding to rat striatal synaptosomal membranes. This
inhibition assay models the putative stimulant mode of
action of 1, i.e., uptake inhibition of impulse released
dopamine by the presynaptic dopamine transporter
(DAT).19-21
Any potential pharmacodynamic significance regard-
ing the enantioselective formation of metabolite 2 after
concomitant 1 and ethanol cannot be assessed without
first characterizing the activity of each enantiomer of
2. Accordingly, enantiomeric 2 and racemic 2 were
synthesized and evaluated in vivo for dose-response
effects on locomotor activity in C57BL/6J mice, as well
as in vitro for monoamine uptake inhibition activity and
receptor binding affinity. The behavioral effects of
metabolites (+)-2 and (-)-2 were gauged against the
enantiomers of parent drug 1.
The receptor pharmacology of (+)-2 and (-)-2 was
evaluated in context to the prototypic DAT inhibitor
cocaine22 and of racemic 1.
The separate enantiomers of 2 have not been phar-
macologically characterized, and little is known about
Results
Chemistry. Enantiomeric and racemic 2 were syn-
thesized by standard methods. Gas chromatography-
mass spectrometry (GC-MS) of (S)-prolyl diastereo-
meric derivatives of 2 established the enantiopurity of
the synthesized compounds.19
* To whom correspondence should be addressed. Phone: (843) 792-
8429. Fax: (843) 792-1617. E-mail: patrickk@musc.edu.
† Department of Pharmaceutical Sciences.
‡ Department of Physiology and Neuroscience.
§ Department of Pharmacology and Experimental Therapeutics.
# Department of Psychiatry and Behavioral Sciences.
10.1021/jm0490989 CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/25/2005

Ethylphenidate Enantiomers
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2877
Table 1. In Vitro Monoamine Activities: Ethylphenidate (2) Isomers Compared to (()-Methylphenidate (1) and Cocainea
IC50/Ki (nM)
(()-1b
(+)-2
27 ( 6
(-)-2
(()-2
cocaine
DAT uptake inhibition
DAT binding
NET uptake inhibition
NET binding
SERT uptake inhibition
SERT binding
19.9 ( 7.7
121.7 ( 5.7
51 ( 21
788 ( 98
ND
1730( 180
2700 ( 540
>10 µM
>10 µM
>10000
95 ( 18
289 ( 38
367 ( 45
462 ( 89
2,990 ( 300
289 ( 28
433 ( 16
230 ( 100
290 ( 120
3700( 580
ND
319 ( 98
480 ( 200
>7200
>10000
7540 ( 400
>10000
>10000
>9500
a DAT, dopamine transporter; NET, norepinephrine transporter; SERT, serotonin transporter; ND, not determined. b Dr. Richard Kline,
NIDA, personal communication.
Figure 2. Motor activity effects of enantiomeric [(+) and (-)] and racemic (() methylphenidate‚HCl (1; MPH) and ethylphenidate‚
HCl (2; EPH). Values represent cumulative 90 min responses to 2.5 (left), 5 (center), and 10 mg/kg (right) intraperitonal injections
in C57B/L mice (n ) 10/group). Vehicle was normal saline.
In Vitro Experiments: Monoamine Transport-
ers. The Cocaine Treatment Discovery Program of the
National Institute on Drug Abuse (NIDA) performed the
binding affinity and uptake inhibition evaluations of
(+)-, (-)-, and (()-2. Their monoamine transporter
studies used human embryonic kidney (HEK) cell
cultures expressing the human transporters for dopa-
mine, norepinephrine (hNET), or serotonin (hSERT).
These activities were compared against those of the
prototypic DAT inhibitor cocaine.22
conducted because Ki was greater than 10 µM (289 (
28 nM for cocaine).
In Vitro Experiments: Monoamine Receptors.
No significant postsynaptic receptor function activity
was found for isomers of 2 when evaluated by the
standardized NIDA protocols for D1-3 and 5-HT1A,2A,2C
interactions.
In Vivo Experiments. The effects of a range of (+)-,
(-)-, and (()-1 and (+)-, (-)-, and (()-2 doses on motor
activity are summarized in Figure 2. Because motor
activity had reached constant low levels of activity by
90 min after injection for all compounds at all doses,
only cumulative activity prior to this time point was
subjected to analyses. As noted in Figure 2, both the
(+)- and (()-forms of each compound produced dose-
dependent increases in motor activity in comparison to
vehicle values. No statistically significant effects were
produced by the (-)-isomers of 1 and 2. At the 5.0 mg/
kg intraperitoneal dose, motor activity over the 90-min
test period was elevated less by (+)- and (()-2 than by
(+)- and (()-1. At the 10 mg/kg dose, the increases in
motor activity were similar for (+)-1 versus (+)-2 and
for (()-1 versus (()-2.
To facilitate behavioral comparisons of the metabolite
2 with the parent drug 1, motor activity data generated
by the isomeric compounds were subjected to indepen-
dent 2 × 3 (2 vs 1) × (isomer) analyses of variance
(ANOVAs) for each drug dose. The 2.5 mg/kg doses of
the compounds did not elevate motor activity in mice
relative to saline controls [F(1,54) < 1]. Activity did differ
on the basis of isomeric state, with greater activity for
the (+)-enantiomer and for racemic modifications in
comparison to the (-)-enantiomer [F(2,54) ) 5.863, p <
0.01; Duncan’s post hoc tests, p < 0.05]. The (+)- and
(()-derivatives elevated motor activity at the 5 mg/kg
dose [F(1,53) ) 7.720, p < 0.01], unlike the (-)-isomers
As with the enantiomers of parent drug 1,19 the (R,R)-
configuration found in metabolite (+)-2 provided for the
greatest in vitro activities in the DAT and NET cellular
systems (Table 1). On the basis of NIDA archival values
for racemic 1, the racemate of 2 was less active in these
catecholaminergic transporter models. (+)-2 exhibited
approximately an order of magnitude greater potency
than cocaine in inhibiting [3H]dopamine uptake: IC50
) 27 nM for (+)-2 versus 235 nM for cocaine. (+)-2 was
marginally more active than cocaine in displacing the
potent radiolabeled DAT ligand 3â-(4-iodophenyl)tro-
pan-2â-carboxylic acid methyl ester ([125I]RTl-55) from
DAT binding: 230 nM for (+)-2 versus 367 nM for
cocaine. (-)-2 showed very little activity in these in vitro
evaluations, while the racemate of 2 exhibited the
anticipated intermediate activity.
The binding selectivity for the hDAT relative to the
hNET was approximately 10-fold greater for (+)-2 than
for cocaine (Table 1). [3H]Norepinephrine uptake inhibi-
tion assays demonstrated similar potencies for (+)-2 and
cocaine: IC50 ) 290 nM for (+)-2 compared to IC50
462 nM for cocaine.
)
In HEK-hSERT cells, the binding affinity of (+)-2 for
the SERT was much less than for cocaine; Ki for the
displacement of [125I]RTl-55 was >10 µM for (+)-2
versus 433 nM for cocaine. Uptake assays of 2 were not

2878 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Patrick et al.
[F(2,53) ) 19.01, p < 0.01; Duncan’s post hoc tests, p <
0.05]. The effects of 1 and 2 at the 10.0 mg/kg dose did
not differ [F(1,54) < 1]; however, there were differences
associated with the isomeric state [F(2,54) ) 31.045, p <
0.001]. At this dose, the enantiomeric forms differed
from each other, with activity being greater for the (+)-
enantiomer than for the racemate and the racemate
activity being greater than for the (-)-form [Duncan’s
post hoc tests, p < 0.05].
trolled clinical studies of 1-ethanol metabolic11-13 and
pharmacodynamic (unpublished) interactions. The me-
tabolite 2 was found in human plasma primarily as the
(-)-isomer, where it reached low ng/mL values while
(+)-2 concentrations were in the pg/mL range.1,13 These
studies also demonstrated that ethanol significantly
elevates maximum plasma concentrations of (+)-1 and
the area under the plasma concentration-time profile
of (+)-1.13 The clinical implications of these findings are
under active investigation.
Ethyl ester 2 has served as an internal standard in
pharmacokinetic studies of 1.35 However, in view of the
slower rate of chemical hydrolysis of 2 relative to 1,17 it
cannot control for possible hydrolytic loss of 1 during
sample processing. This concern with differential rates
of 1 vs 2 hydrolysis in biological samples, in addition to
former problems of baseline chromatographic resolution
between 1 and 2,36 has been overcome through the
utilization of piperidine deuterated 1 as an internal
standard and analytical separation by mass spectrom-
etry.13,36 Owing to 2 now being recognized as a metabo-
lite of 1, its role as an internal standard becomes even
more problematic.
In the present study, the human metabolite (+)-2
produced potent effects on DAT model systems (Table
1). Racemic 2 was marginally less active than racemic
1 in these tests, though (+)-2 was approximately an
order of magnitude more potent than cocaine in inhibit-
ing [3H]dopamine uptake. (+)-2 also exhibited greater
selectivity than cocaine in inhibiting transmitter uptake
at the DAT relative to the NET.
Both (+)-1 and (+)-2 elevated motor activity with
respect to saline controls (Figure 2). The effect of these
eutomers differed at the middle dose, 5 mg/kg, with 2
inducing approximately half the activity recorded for 1.
Neither (-)-1 nor (-)-2 was associated with significant
behavioral activity. The dose-response evaluation yielded
floor and ceiling effects; i.e., differences between the
drugs were not observed at the low (2.5 mg/kg) and high
(10 mg/kg) doses.
The results of the motor activity experiments are in
concordance with the results of the in vitro studies. (-)-2
was inactive, (+)-2 was highly active, and (()-2 was of
intermediate activity. The effects of (+)-1 and (+)-2 on
motor activity may be driven through activation of
dopaminergic systems in the nucleus accumbens, par-
ticularly its core.37 Our observations of stereotypic
behavior at the high 10 mg/kg dose are consistent with
a dose-dependent breakthrough of (+)-2 effects into
striatal dopaminergic systems.38
The present study provides evidence for a molecular
level basis/mechanism underlying the behavioral effects
induced by the new metabolite 2. Further characteriza-
tion of behavioral profiles for 1 and 2 is in progress.
These analyses, when complemented with measure-
ments of plasma and brain concentrations, should yield
meaningful pharmacokinetic/pharmacodynamic correla-
tions. Exploration of the rewarding and discriminative
properties of metabolite 2 could contribute to a funda-
mental understanding of the abuse liability associated
with 1 combined with ethanol. These studies are ulti-
mately directed toward the rational optimization of drug
therapy for adult ADHD patients with or without
comorbid alcohol use disorder. Ironically, metabolite 2
Discussion
Upon development of a preparative-scale method for
resolving (()-1,19 it was established that (+)-1 was the
component enantiomer of the racemic drug responsible
for increasing motor activity,19 elevating blood pres-
sure,19 and reducing appetite23 in rats. In mechanistic
studies, inhibition of [3H]dopamine uptake into striatal
synaptosomes was also limited to the (+)-isomer.19
Imaging investigations in humans have since reveled
that specific binding to ADHD-relevant dopaminergic
brain regions is associated with only (+)-1.24 Additional
clinical investigations have demonstrated that (+)-1
produces the therapeutic effects of 1 in ADHD25 and
narcoleptic26 patients. These, among additional find-
ings,1 prompted the development of the new “chiral or
racemic switch” product (+)-1 (Focalin, Novartis), re-
leased in 2002 as an alternative to the several existing
(()-1 formulations used to treat ADHD. It now appears
that in the absence of (-)-1, the pharmacodynamic
actions of (+)-1 are prolonged.27
Baldessarini and Cambell28 recently patented (-)-1
as an antidote to stimulant overdose based on the
finding that (-)-1 antagonizes behavioral effects of (+)-
1, cocaine, or apomorphine in rats. In other preclinical
studies, Davids et al.29 reported that (-)-1 may not
merely be a passive isomeric component of racemic 1.
The (-)-enantiomer opposed the actions of (+)-1 in
6-hydroxydopamine (a catecholaminergic neurotoxin)
lesioned rats. Further, examining the behavioral effects
of (-)-1 and (+)-1 in nonlesioned rats revealed that
females were more sensitive than males to some effects
of the (-)-isomer (and that females were more sensitive
to both isomers in other elements of an observational
battery).30
Metabolic transesterification of methyl ester 1 to ethyl
ester 2 appears to be a biotransformation analogous to
the well-established cocaine-ethanol interaction where
human esterase(s) transesterify the methyl ester of
cocaine to form cocaethylene (the ethyl ester of co-
caine).31 Ethanol has been reported to elevate plasma
cocaine concentrations,32 much as ethanol elevates
plasma 1 concentrations.1,13 X-ray crystallography de-
picts both (+)-1 and cocaine displaying a near-super-
imposable pharmacophore: the amine, phenyl ring, and
methyl ester moiety.33 Such structural similarities
between 1 and cocaine may pertain to a common
enzymatic transesterification pathway for esters 1 and
cocaine, both yielding ethyl esters.
Bourland and co-workers34 incubated (()-1 with etha-
nol in a rat liver preparation and reported the detection
of the product 2. Subsequently, Markowitz et al.10
quantified 2 in postmortem blood and liver from two
suicide cases involving concomitant 1 and ethanol. The
lethal drug doses were unknown. This prompted con-

Ethylphenidate Enantiomers
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2879
3.0%), 115 (M - 132, 3.0%); [R]25 +65.10 (c 1.08, methanol).
has recently been patented as an ADHD medication
with diminished abuse potential.39
D
Anal. (C15H22Cl NO2) C, H, N.
(-)-(2S,2S′)-threo-Ethylphenidate Hydrochloride [(-)-
2‚HCl, Figure 1]. (-)-2‚HCl was synthesized as above,
yielding a white solid (86%): mp 220-222; GC (TFP deriva-
tive), tR ) 11.6 min (98+% ee); [R]25D -66.87 (c 1.24, methanol).
Anal. (C15H22Cl NO2) C, H, N.
Experimental Section
Chemistry. GC-MS was performed using an Agilent 6890
GC-5973N MS (Wilmington, DE) instrument. The GC used a
5% phenylpolysiloxane 30 m × 0.25 mm, 0.25 µm film
thickness fused-silica column (J & W Scientific, Palo Alto, CA),
with the helium linear velocity at 42 cm/s. For nonderivatized
samples, the GC oven was held at 150 °C for 6 min, followed
by a 15 °C/min ramp, then held at 240 °C. MS ionization was
by electron impact at -70 eV, acquiring m/z 50-700. Chiral
derivatized samples were treated with (S)-N-trifluoroacetyl-
prolyl (TFP) chloride (0.1 M in dichloromethane, 97% ee,
Aldrich, Milwaukee, WI) at 40 °C for 45 min. The GC oven
was held at 125 °C for 1.5 min, followed by a 20 °C/min ramp
to 280 °C while acquiring m/z 277 (N-trifluoroprolylpiperidyl
fragment).12,19 The minor contribution of the trace impurity
(R)-TFP chloride to the chromatographically separated dia-
steriomeric products from (+)-2 or (-)-2 was corrected for by
analysis of enantiopure methamphetamine derivatized with
TFP chloride, followed by subtraction of the resulting ∼1%
peak area ratio of (R)-TFP-/(S)-TFP-methamphetamine as
described previously.19
Both proton and carbon NMR spectra were obtained on a
Varian INOVA spectrometer operating at 400 and 100 MHz,
respectively. Proton assignments were made by employing the
double quantum filtered COSY (DQ-COSY) experiment ac-
quired in the phase-sensitive mode, and 2 × 256 free induction
decays (FIDs) were acquired. Digital resolution in F1 was
increased by linear prediction to 1024 points, processed using
the Gaussian weighting function and then Fourier trans-
formed. The chemical shifts of unresolved multiplets were
based on the chemical shifts of the cross-peaks. Carbon
resonances were assigned using gradient versions of the
heteronuclear single quantum coherence (gHSQC) and het-
eronuclear multibond correlation (gHMBC) experiments. In
the gHSQC, 128 FIDs were acquired, then use of linear
prediction increased the points in F1 to 512. After Gaussian
weighting, the FIDs were then Fourier transformed to produce
the 2D contour plot. In the gHMBC, 400 FIDs were acquired,
then linear prediction increased the points in F1 to 1200. Upon
sine-bell weighting, the FIDs were then Fourier transformed
to produce the 2D contour plot.
(()-threo-Ethylphenidate Hydrochloride [(()-2‚HCl,
Figure 1]. (()-threo-Ritalinic acid [(()-3,9 Figure 1] was
esterified by the above method used to synthesize the enan-
tiomers of 2, yielding a white solid: mp 197-199 °C; GC tR
)
10.1 min underivatized (99%). Anal. (C15H22ClNO2) C, H, N.
In Vitro Pharmacology. All in vitro testing was conducted
by NIDA through their standard Cocaine Treatment Testing
Program screening.
Biogenic Amine Transporter Binding. (+)-, (-)-, and
(()-2‚HCl in DMSO (10 mM) was diluted to 50 µM in assay
buffer for binding (or to 1 mM for uptake). Further dilutions
gave DMSO concentrations of 0.1-0.25%.
Inhibition of [125I]RTI-55 Binding. HEK293 cells ex-
pressing hDAT, hSERT, or hNET inserts were grown to 80%
confluence. Cell membranes were washed with phosphate-
buffered saline (10 mL). Lysis buffer (10 mL, 2mM HEPES
with 1 mM EDTA) was added, then cells were centrifuged
(30000g). The pellet was resuspended in 0.32 M sucrose (12-
32 mL) to reflect binding of 10% or less of the total radioactiv-
ity. Each assay tube contained 50 µL of membrane preparation
(10-15 µg of protein), 25 µL of test compound or compound
used to define nonspecific binding or buffer (Krebs-HEPES,
pH 7.4, 122 mM NaCl, 2.5 mM CaCI2, 1.2 mM MgSO4, 10 µM
pargyline, 100 µM tropolone, 0.2% glucose, and 0.02% ascorbic
acid, buffered with 25 mM HEPES), 25 µL of [125I]RTI-55 (40-
80 pM), and additional buffer sufficient to bring up the final
volume to 250 µL was added. Membranes are preincubated
with test compounds for 10 min prior to the addition of [125I]-
RTI-55, then incubated at 25 °C for 90 min. Binding was
terminated by filtration over GF/C filters using a Tomtec 96-
well cell harvester, and when the sample was washed, scintil-
lation fluid was added to each square and filter radioactivity
determined using a Wallac µ- or â-plate reader. Specific
binding was defined as the difference in binding observed in
the presence and absence of 5 µM mazindol (HEK-hDAT and
HEK-hNET) or 5 µM imipramine (HEK-hSERT). Two or three
independent competition experiments were conducted with
duplicate determinations. GraphPAD Prism was used to
analyze the data, with IC50 values converted to Ki values using
the Cheng-Prusoff equation [Ki ) IC50/(1 + ([RTI-55]/Kd(RTI-
55)))].
Elemental analyses, optical rotations, and melting points
were performed by Quantitative Technologies Inc. (White-
house, NJ). (+)- and (-)-1‚HCl were generous gifts from
Celgene Corporation (Warren, NJ). (()-1‚HCl was provided by
NIDA. Ethanol for synthesis was from AAPER (Shelbyville,
KY) and that for GC was from Aldrich (Milwaukee, WI).
Diethyl ether and HCl gas were also from Aldrich.
Filtration Assay for Inhibition of [3H]Neurotransmit-
ter Uptake in HEK293 Cells Expressing Recombinant
Biogenic Amine Transporters. Cells were grown to conflu-
ence as described above and then washed. Krebs-HEPES
buffer was added, and then the mixture was triturated.
Specific uptake was defined as the difference in uptake
observed in the presence and absence of 5 µM mazindol (HEK-
hDAT and HEK-hNET) or 5 µM imipramine (HEK-hSERT).
Cells (50 µL) were added and preincubated with the unknowns
for 10 min. The assay was initiated by the addition of [3H]-
dopamine, [3H]serotonin, or [3H]norepinephrine (50 µL, 20 nM
final concentration). Filtration through Whatman GF/C filters
presoaked in 0.05% polyethylenimine was used to terminate
uptake after 10 min. IC50 values were calculated by applying
the GraphPAD Prism program to triplicate curves made up
of six test compound concentrations each. Two or three
independent determinations of each curve were made.
Numbers represent the mean ( SEM from at least three
independent experiments, each conducted with duplicate (for
binding assays) or triplicate (for uptake assays) determina-
tions. When the Ki or the IC50 for the test compound was
greater than 10 µM, only two experiments were conducted and
no standard error was reported.
(+)-(2R,2R′)-threo-Ethylphenidate Hydrochloride [(+)-
2‚HCl, Figure 1]. (+)-1‚HCl (627 mg, 2.32 mmol) was dis-
solved in 10% aqueous HCl (60 mL) and refluxed with stirring
for 24 h. The solution was evaporated to dryness under reduced
pressure, then the flask was purged with nitrogen and
desiccated in vacuo overnight. The product (+)-3‚HCl was used
without further purification. Ethanolic HCl (100 mL) was
added to the flask containing (2R,2′R)-3‚HCl. After refluxing
for 24 h under nitrogen, the solution was evaporated to dryness
under reduced pressure. Crystallization from ethanol-diethyl
ether yielded white solid (+)-2‚HCl (66%): mp 224-226 °C;
1
as the TFP derivative, tR ) 11.0 min (98% ee); H NMR (400
MHz, CD3OD) δ 7.40 (m, 2H, H-3′′,5′′), 7.38 (m, 1H, H-4′′), 7.30
(m, 2H, H-2′′,6′′), 4.20 (m, 2H, CH2), 3.83 (m, 1H, H-2), 3.82
(m, 1H, H-2′), 3.43, 3.10 (m, 2H, H-6′), 1.87, 1.67 (m, 2H, H-5′),
1.51, 1.35 (m, 2H, H-3′), 1.79, 1.28 (m, 2H, H-4′), 1.16 (t, 3H,
J ) Hz, CH3); 13C NMR (100 MHz, CD3OD) δ 172.9 (C-1), 135.4
(C-1′′), 130.6 (C-3′′,5′′), 129.9 (C-4′′), 129.7 (C-2′′,6′′), 63.2 (CH2),
59.3 (C-2′), 55.6 (C-2), 46.7 (C-6′), 27.8 (C-3′), 23.5 (C-5′), 22.8
(C-4′), 14.2 (CH3); MS-EI m/z 246 (M - 1, <1%), 84 (M - 163,
100%), 91 (M - 156, 30%), 55 (M - 192, 6.0%), 164 (M - 83,
6.0%), 56 (M - 191, 5.0%), 85 (M - 162, 5.0%), 65 (M - 182,
In Vivo Pharmacology. Animals. Male C57BL/6 mice
were obtained from the Jackson Laboratories (Bar Harbor,
ME) at 49 days of age. They were individually housed in a

2880 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Patrick et al.
colony room on a 12-h light/dark cycle (light: 0700-1900) for
at least 4 days prior to the start of behavioral tests. All
experiments were approved by and conducted within the
guidelines of the Institutional Animal Care and Use Commit-
tee (IACUC) at the Medical University of South Carolina and
followed the guidelines of the NIH Guide for the Care and Use
of Laboratory Animals (NIH Publication No. 80-23, revised
1996).
Drugs and Treatment Groups. Animals were assigned
to 1 of 18 treatment groups (n ) 10/group). Treatment groups
consisted of low (2.5 mg/kg), medium (5.0 mg/kg), and high
(10.0 mg/kg) doses of each of the six compounds [(+)-, (-)-,
and (()-1‚HCl and (+)-, (-)-, and (()-2‚HCl]. All drugs were
dissolved in 0.9% saline. An additional control group (n ) 20)
received injections of saline and provided basal levels of motor
activity during 3 h of motor activity tests. All drugs were
administered at 0.01 mL/g body weight by the intraperitoneal
route immediately before testing.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
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Acknowledgment. The authors appreciate the gen-
erous donation of (+)- and (-)-1‚HCl by the Celgene
Corporation, with special thanks to Dr. Steve Teo and
Ms. Elizabeth Pensabene. We thank NIDA for supplying
(()-1 and especially for the involvement of their Cocaine
Treatment Discovery Program investigators Drs. Jane
Acri and Richard Kline and the associated laboratories
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Carolina NMR Resource Facility, for Institutional Re-
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