0022-3565/02/3012-527535$7.

00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 301:527535, 2002

Vol. 301, No. 2

4691/977159
Printed in U.S.A.

Biochemical and Behavioral Characterization of Novel

Methylphenidate Analogs
M. M. SCHWERI, H. M. DEUTSCH, A.T. MASSEY,1 AND S. G. HOLTZMAN
Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia (M.M.S., A.T.M.); School of Chemistry and
Biochemistry, Georgia Institute of Technology, Atlanta, Georgia (H.M.D.); and Department of Pharmacology, Emory University School of
Medicine, Atlanta, Georgia (S.G.H.)
Received October 29, 2001; accepted January 10, 2002

This article is available online at http://jpet.aspetjournals.org

Illicit use of cocaine is a major public health problem worldwide. There is an urgent need for treatment agents that could
be used to block the pleasurable effects of cocaine and/or
reduce craving for the drug, without causing deleterious side
effects. To this end, our research efforts have been directed
toward the synthesis of compounds that will interact with the
DAT, the site in the brain thought to subserve the reinforcing
effects of cocaine (Ritz et al., 1987; Howell and Wilcox, 2001).
Although cocaine is known to block norepinephrine and serotonin transport, as well as conductance through sodium
channels (Reith, 1988), its abuse potential is generally attribSupported by a grant from the National Institute on Drug Abuse (DA06305)
to H.M.D., M.M.S., and S.G.H., and by a Senior Scientist Award (DA00008) to
S.G.H.
1
Current address: Instructor, Division of Natural Sciences and Mathematics, Macon State College, Macon, GA.

uptake did not attenuate inhibition by cocaine of synaptosomal

[3H]DA transport. The compounds were significantly less potent
in displacing [3H]citalopram binding from the serotonin transporter. In cocaine discrimination studies in rats, all but two of
the analogs (both N-substituted) completely generalized with
the cocaine stimulus. Robust positive correlations were observed between potency in the drug discrimination assay and
activity at the dopamine transporter, but not the serotonin
transporter. When tested for their ability to alter cocaine discrimination, four of the analogs (three of which had Nsubstitutions and shallow dose-response curves as cocaine
substitutes) actually enhanced cocaine discrimination, often at
combined doses of cocaine and test compound that were
inactive when given separately. Taken together, the results
suggest that TMP analogs may have potential as substitution
therapies for the treatment of cocaine abuse.

uted to its inhibition of the reuptake of the neurotransmitter

DA at nerve terminals of the mesolimbic system. To increase
the probability that a proposed compound will target the
DAT, our approach has been to use psychomotor stimulant
agents (in this case, TMP) as the starting point for structural
modification. TMP was selected for several reasons: 1) it has
somewhat more inhibitory potency at the DAT than at the
norepinephrine transporter or the SERT (Ritz et al., 1987); 2)
it has several important chemical and structural properties
in common with cocaine; and 3) based both on widespread
clinical experience with the drug as an oral treatment agent
for attention deficit disorder, and on several recent studies on
its use as replacement therapy for cocaine addicts
(Grabowski et al., 1997; Roache et al., 2000), TMP appears to
have low abuse potential and few serious side effects. The

ABBREVIATIONS: DAT, dopamine transporter; DR, discrimination ratio; [3H]CIT, [3H]citalopram; [3H]DA, [3H]dopamine; [3H]WIN, [3H]WIN 35,428,
3
H-()-2--carbomethoxy-3--(4-fluorophenyl)tropane 1,5-naphthanedisulfonate; SERT, serotonin transporter; 3CTMP, ()-threo-3-chloromethylphenidate; 3CTMPNMe, ()-threo-N-methyl-3-chloromethylphenidate; 4ATMP, ()-threo-4-aminomethylphenidate; 4FTMP, ()-threo-4fluoromethylphenidate; 4MeTMP, ()-threo-4-methylphenidate; 4MeTMPNMe, ()-threo-N-methyl-4-methyl-methylphenidate; 3,4CTMP,
()-threo-3,4-dichloromethylphenidate; TMP, ()-threo-methylphenidate; TMPNMe, ()-threo-N-methyl-methylphenidate; TMPNBn, ()-threoN-benzyl-methylphenidate; TROHNBn, ()-threo-N-benzylritalinol; TROMeNBn, ()-threo-N-benzylritalinol methyl ether; ANOVA, analysis of
variance.
527

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ABSTRACT
As part of a project to develop treatment agents for cocaine
abuse, ()-threo-methylphenidate (TMP) and 11 analogs were
characterized biochemically and behaviorally to assess their
potential as anti-cocaine medications. The compounds contained aryl and/or nitrogen substitutions, and/or replacement of
the ester function by an alcohol or ether. All of the analogs,
except for the N-methyl-substituted compounds, showed increased inhibitory potency against 3H-()-2--carbomethoxy3- -(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate
([3H]WIN 35,428) ([3H]WIN) binding to the dopamine transporter, compared with TMP. In general, parallel results were
obtained for inhibition of [3H]dopamine ([3H]DA) uptake. Although compounds with N-substitutions were proportionally
less potent at blocking DA uptake than WIN binding (compared
with the unsubstituted compounds), one such compound that
was 6-fold more potent against [3H]WIN binding than [3H]DA

528

Schweri et al.

Materials and Methods

Chemicals
Synthetic Compounds. The syntheses of ()-threo-methylphenidate
(TMP), ()-threo-3,4-dichloromethylphenidate (3,4CTMP), ()-threo-4fluoromethylphenidate (4FTMP), ()-threo-3-chloromethylphenidate
(3CTMP), ()-threo-4-aminomethylphenidate (4ATMP), ()-threo-4methylphenidate (4MeTMP), ()-threo-N-methyl-3-chloromethylphenidate (3CTMPNMe), ()-threo-N-methyl-4-methyl-methylphenidate
(4MeTMPNMe), ()-threo-N-methyl-methylphenidate (TMPNMe), ()threo-N-benzyl-methylphenidate (TMPNBn), ()-threo-N-benzylritalinol
methyl ether (TROMeNBn), and ()-threo-N-benzylritalinol (TROHNBn)
have been described previously (Deutsch et al., 1996; Froimowitz et al.,
1997; Deutsch, 1998). 3CTMPNMe and TMPNMe were prepared as free
bases; all other synthesized compounds were HCl salts. NMR analysis
suggested that 3CTMPNMe contained approximately 10% of the ()erythro enantiomers; this was likely due to epimerization that occurred at
the N-methylation step.
Other Chemicals. The TMP used in the in vitro assays was a gift
of Ciba-Geigy (Basel, Switzerland). ()-Cocaine HCl used in the drug
discrimination studies was obtained from the National Institute on
Drug Abuse (Bethesda, MD); that used in the in vitro assays was
purchased from Sigma Chemical Co. (St. Louis, MO). Citalopram
was a gift of H. Lundbeck A/S (Copenhagen, Denmark). All radioisotopes were purchased from PerkinElmer Life Sciences (Boston, MA);
they are: [ring-2,5,6-3H]dopamine; [N-methyl-3H]citalopram, and
[N-methyl-3H]WIN 35,428. All other drugs were purchased from
standard commercial sources.

In Vitro Studies
Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN),
weighing 150 to 300 g, were anesthetized using CO2 gas and sacrificed by decapitation. Their brains were quickly removed and placed
in ice-cold 0.32 M sucrose. Tissue preparations were prepared from
those areas of the brain as required for the individual assays described below. This protocol was approved by the Institutional Animal Care and Use Committee of Mercer University and is in accord
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
[3H]WIN Binding to the DAT. These assays were conducted
using rat striatal tissue as previously described (Deutsch et al.,
1999). IC50 values (that concentration of drug which inhibits 50% of
specific binding of the radioligand) and Hill coefficients were determined by least-squares nonlinear regression analysis of sigmoidal
dose-response curves using the GraphPad Prism (v.2) program
(GraphPad Software, Inc., San Diego, CA).
[3H]CIT Binding to the SERT. The method used here was
modified from binding assays described elsewhere (DAmato et al.,
1987; Dutta et al., 1996). Cortical tissue from all brain areas except
the olfactory tubercles and medial cortex extending to approximately
1 mm on either side of the interhemispheric fissure was combined
and homogenized in 20 volumes (based on wet weight) of 0.32 M
sucrose, using 10 up/down strokes of a motorized Potter-Elvehjem
homogenizer. The homogenized tissue was centrifuged at 1,000g for
10 min at 0C, and the resulting supernatant was further centrifuged at 20,000g for 20 min. The resulting pellet (P2 fraction) was
suspended in 16 volumes of ice-cold assay buffer (25 mM sodium
phosphate buffer, pH 7.7 at 0C) using an Ultra-Turrax tissue homogenizer. Binding was initiated by addition of 150 l of the P2
suspension to samples containing 750 l of assay buffer, 50 l of the
test compound, 25 l of water or clomipramine (to define nonspecific
binding; final concentration, 1 M) and 25 l of [3H]CIT (final
concentration, 2 nM). Test compounds were dissolved in water, diluted dimethyl sulfoxide, or diluted methanol, with the concentration
of organic solvent in any assay tube limited to 0.3% by volume.
Usually, a range of seven drug concentrations was used to generate
the dose-response curve; triplicate samples were run at each concen-

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goal of this research is to create novel TMP analogs that will

retain their selectivity for the DAT, and either serve as
replacement therapy for cocaine (full or partial cocaine agonists) or prevent its effects by directly blocking its binding to
the DAT (cocaine antagonists). Ideally, potential antagonists
would inhibit the binding of cocaine to its recognition site on
the DAT but not interfere with the binding and subsequent
uptake of DA. Although this was once thought to be an
unattainable goal, a large body of evidence now supports its
feasibility (see references in Deutsch and Schweri, 1994).
Although the reinforcing effects of cocaine are not attributable to its action on serotonin transport, serotonin can
modulate its subjective effects (Walsh and Cunningham,
1997). Serotonergic systems affect stimulant-induced behaviors in a somewhat complex manner. For instance,
specific 5HT uptake blockers enhance cocaine discrimination (Kleven and Koek, 1998, and references therein), but
serotonergic agonists attenuate some cocaine-reinforced
behaviors and self-administration of amphetamine (see
references in Ritz et al., 1987). Therefore, because alteration of the SERT activity of the TMP analogs could influence their utility as cocaine treatment agents, their inhibitory potency against [3H]CIT binding to the SERT was
also monitored.
We now report on the testing of TMP and 11 of its
derivatives, which contain amino, methyl, or halide substituents on the aryl ring, methyl or benzyl substitutions
on the piperidine nitrogen, and/or modifications of the
methyl ester function. This is the first time, to our knowledge, that the effect of more radical structural changes
(i.e., a disubstituted aryl ring, N-substitutions, and/or replacement of the ester function by an ether or alcohol) have
been examined. Previous reports correlating the biochemical and behavioral effects of MP analogs have been limited
to derivatives containing only monosubstituted aryl rings
or modification of the ester function (e.g., Patrick et al.,
1981; Schweri et al., 1985; Gatley et al., 1996a; Thai et al.,
1998). Preliminary characterization of these novel compounds was conducted, using both in vitro and in vivo
techniques. The inhibitory potencies of the compounds
against [3H]WIN binding to the cocaine recognition site on
the DAT, [3H]CIT binding to the SERT, and [3H]DA uptake
were determined, if not assessed previously. Based on the
results, ()-threo-N-methyl-4-methyl-methylphenidate
(4MeTMPNMe) was selected as a representative compound
for further in vitro tests designed to examine the nature of
its interaction with the DAT, as well as its ability to
attenuate the inhibition of DA uptake by cocaine. All of the
analogs were tested for cocaine-like discriminative stimulus effects in rats trained to discriminate between saline
and 10 mg/kg cocaine; selected compounds were further
tested for their ability to block the discriminative effects of
cocaine. The drug discrimination assay is a proven animal
model of the subjective effects of drugs in humans, with
predictive value for abuse potential (Holtzman, 1990). Possible candidates for further study were identified, and the
combined data were evaluated with the aim of determining
useful characteristics and possible correlations between
the in vitro and in vivo results that might guide the design
of future pharmacotherapies for cocaine abuse.

Methylphenidate Analogs as Treatments for Cocaine Abuse

Drug Discrimination Studies

Subjects. Male rats of Sprague-Dawley descent (Charles River
Laboratories, Raleigh, NC) were used in the study. They weighed
250 to 300 g when discrimination training began and were housed in
pairs in a vivarium that had a 12-h light/dark cycle. Food and water
always were available in the home cage. The vivarium was part of a
facility that was accredited by the American Association for Accreditation of Laboratory Animal Care. The care and testing of the
animals conformed to a protocol that was approved by the Institutional Animal Care and Use Committee of Emory University and
were in accord with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals.
Drug Discrimination. Rats were trained to discriminate between i.p. injections of 10 mg/kg cocaine and saline in a two-choice
discrete-trial avoidance/escape procedure (Shannon and Holtzman,
1976). Injections of cocaine or saline were given 15 min before a
session, which was conducted in a standard testing chamber. The
beginning of a trial was signaled by the onset of white noise and
illumination of the house light in the chamber. Five seconds after a
trial began, a constant electrical current (1.0 1.5 mA) was applied to
the grid floor of the chamber for 1.0 sec every 3.0 sec. To end a trial,
the rat had to complete a two-response chain: press an observing
lever in one wall of the chamber and then press one of two choice
levers mounted in the opposite wall. The choice levers were 15 cm
apart and were separated by a 5.0-cm-wide Plexiglas partition that
extended from the floor to the ceiling. A response on the observing
lever turned off the white noise and enabled a response on the correct
choice lever to extinguish the house light and end the trial. A trial
was recorded as correct if the rat pressed the choice lever appropriate
for what was injected beforthtee the session (i.e., cocaine or saline)
right after it pressed the observing lever. A trial was recorded as
incorrect (and did not end) if the rat pressed the choice lever that was
not appropriate for what was injected before the session between
pressing the observing lever and pressing the appropriate choice
lever. A trial also ended if the rat did not complete either the correct
or incorrect sequence of responses within 30 sec and was recorded as
an incomplete trial. Trials were separated by a 50-sec period, during
which the chamber was illuminated by a red stimulus lamp. A

session consisted of 20 trials and, with fully trained rats, usually

lasted 19 21 min.
Approximately half the animals were trained to press the left
choice lever in training sessions that followed an injection of 10
mg/kg cocaine and to press the right choice lever in training sessions
that followed an injection of saline. The designation of choice levers
was reversed for the rest of the animals. Rats were considered to be
under the stimulus control of cocaine and saline when they completed the response sequence of observing lever-appropriate choice
lever in at least 18 trials of 20 in four consecutive training sessions
and two consecutive test sessions, half preceded by cocaine and half
by saline. Test sessions differed from training sessions in one respect: after a response on the observing lever, a response on either
choice lever ended a trial.
After rats met the criterion for acquisition of the discrimination,
they all were tested first with graded doses of cocaine. Doses were
injected i.p. 15 min before a session in a random sequence that also
included saline. Other drugs were then tested in a nonsystematic
order in groups of four to five rats. They were injected i.p. 30 min
before a session based upon the time course of action of methylphenidate; each dose was administered once to each rat in the group. Drug
test sessions in which both choice levers were activated usually were
conducted twice weekly, 3 to 4 days apart. Training sessions in which
only the pretreatment-appropriate choice lever was activated were
conducted three times each week to maintain stable discrimination
performance. Saline and cocaine were injected on alternate days
before a training session. If an animal failed to complete at least 18
trials correctly in a training session, testing was suspended until it
had done so again in four consecutive training sessions.
Drugs. In all cases, doses refer to the free base. Cocaine was
dissolved in normal saline solution; 3CTMPNMe, 4MeTMPNMe, and
TMPNMe were dissolved in one part dimethyl sulfoxide followed by
three parts distilled water; all other compounds were dissolved in
distilled water. Drugs usually were injected in a volume of 1.0 ml/kg
of body weight; however, twice this volume was used for some of the
higher doses.
Data Analysis. Data from stimulus-generalization tests are presented as the average number of trials completed on the cocaineappropriate choice lever in a 20-trial session; the remaining trials of
the session were completed on the choice lever appropriate for the
drug vehicle. The dose of a drug that resulted in the selection of the
cocaine-appropriate choice lever in 10 trials of a session (i.e., ED50)
was calculated for individual rats. This was done by linear regression
of the ascending limb of the stimulus-generalization curve, using
log10 dose and at least three points. In those instances when only two
points defined the ascending portion of the curve, ED50 values were
derived by simple interpolation. The individual ED50 values were
averaged to obtain a group mean and 95% confidence limits. Slopes
of stimulus-generalization curves also were determined by linear
regression of the log-dose data. Comparisons among ED50 values and
among slopes were made by analysis of variance, followed, where
appropriate, by the Student-Newman-Keuls test for multiple comparisons among all means. The alpha level was set at 0.05.

Results
In Vitro Studies. The potencies of the compounds as
inhibitors of [3H]WIN binding, [3H]CIT binding, and [3H]DA
uptake in vitro are shown in Table 1, expressed as IC50
values. The compounds show a hundred-fold variation in
potency against [3H]WIN binding, ranging from IC50 values
of about 5 nM for both 3CTMP and 3,4CTMP to about 500 nM
for TMPNMe. A 200-fold range of potency was observed
against [3H]DA uptake, from an IC50 of 7.0 0.6 nM for
3,4CTMP to 1435 5 nM for TMPNMe. The most potent
compounds at the SERT are N-benzyl-substituted compounds in which the ester function has been converted to an

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tration. The samples were incubated for 3 h at 0C. The reaction was
terminated by addition of 5 ml of ice-cold assay buffer to the assay
tubes, followed by rapid filtration through glass microfiber filter
strips (presoaked in 0.05% polyethyleneimine) under reduced pressure using a Brandel 30-port cell harvester (Brandel Inc., Gaithersburg, MD). Assay tubes were washed with an additional 5-ml aliquot
of ice-cold assay buffer. The filters were transferred to scintillation
vials, shaken vigorously for 30 min in the presence of 8 ml of ReadySafe cocktail (Beckman Coulter, Fullerton, CA), and counted on a
Beckman LS6000IC scintillation counter. Data were analyzed using
GraphPad Prism, as described above.
[3H]DA Uptake. The effect of test compounds on the accumulation of [3H]DA was determined using a crude synaptosomal preparation of rat striatal tissue that was preincubated for 10 min in the
presence of the test compound, then exposed to 30 nM [3H]DA over a
2-min period at 37C, as described previously (Deutsch et al., 1999).
In those experiments in which the effect of 4MeTMPNMe on the
ability of cocaine to inhibit [3H]DA uptake was determined, both
4MeTMPNMe and cocaine were present for the entire preincubation
period. Studies to characterize the effect of 4MeTMPNMe on the
Michaelis-Menten kinetics of [3H]DA uptake were performed under
the same conditions, except that the [3H]DA concentrations ranged
from 25 to 800 nM. Km and Vmax were determined by nonlinear
least-squares regression fit of specific [3H]DA uptake versus free
[3H]DA to a rectangular hyperbola function using the GraphPad
Prism program (see above). The resulting values were then used to
generate the equations for the straight lines to allow plotting of the
data in the Lineweaver-Burk format.

529

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Schweri et al.

TABLE 1
Effect of compounds on [3H]WIN and [3H]CIT binding and [3H]DA uptake

[3H]WIN Binding

Substitution
Compound
R1

R2

R3

R4

IC50a

nH

nM

5.1 1.6
5.3 0.7
17.8 1.1
23.7 3.3
33.0 1.2
34.5 4.0
35.0 3.0
52.9 2.3
83.0 7.9
140 9
160 18
499 25
160 15

Discrimination
Ratioc

nM

0.95 0.12 23.0 3.0

2.08 0.05
7.0 0.6
1.12 0.13
88 14
1.08 0.06 181 32
1.05 0.02 126 1
0.96 0.09 114 10
0.97 0.02 142 2
1.08 0.02 273 28
0.90 0.09 224 19
1.02 0.03 920 145
0.96 0.04 441 89
1.00 0.01 1435 5
1.03 0.01 404 26

4.5
1.3
5.0
7.7
3.8
3.3
4.1
5.2
2.7
6.6
2.7
2.9
2.5

Mean S.E.M. from two or more independent experiments in which triplicate samples were run
Mean S.E.M. from two or more independent experiments in which duplicate samples were run
Discrimination ratio is IC50 for [3H]DA uptake/IC50 for [3H]WIN binding.
d
Mean S.E.M. from two or more independent experiments in which triplicate samples were run
e
Mean S.E.M. from two or more independent experiments in which the inhibition due to 10 M
a
b

[3H]CIT Binding
IC50d

nH

Inhibition by
10 M
Compounde

nM

10,000
1,064 62
166 8
204 9
10,000
10,000
10,000
1,201 6
10,000
1,109 40
10,000
10,000
401 27
9.2 2.3

45.5 3.7
93.3 2.2

0.99 0.01
1.03 0.09
0.85 0.01

1.02 0.04
0.78 0.08
1.27 0.01
1.00 0.07

94.1 3.2
45.0 0.5
7.9 2.6
36.9 0.8
85.2 1.9
19.6 2.5
74.2 3.2
33.3 6.1
25.2 1.2

IC50 [3H]CIT/
IC50 [3H]WIN

2000
213
9
9
300
300
300
23
120
8
60
20
2.5

at each of six different concentrations of test compound.

at each of five different concentrations of test compound.

ether (TROMeNBn; IC50, 166 8 nM) or an alcohol

(TROHNBn; IC50, 204 9 nM); the least potent is 4ATMP,
with an IC50 well over 10,000 nM.
All of the analogs are selective for the [3H]WIN over the
[3H]CIT binding site (Table 1). The parent compound, TMP,
exhibits well over 120-fold selectivity for the DAT. For the
most part, N-substituted derivatives show the least separation in binding affinities at the two transporters (e.g., 4MeTMPNMe, TROMeNBn, and TROHNBn are only 8- to 9-fold
more potent against [3H]WIN binding than [3H]CIT binding),
whereas derivatives with single substitutions on the aromatic ring exhibit the greatest degree of selectivity (e.g.,
3CTMP bound with more than 2000-fold affinity to the DAT
over the SERT).
Although the potency against [3H]DA uptake parallels that
found for [3H]WIN binding, the correlation is not perfect
(Pearson r 0.9380, p 0.0001). This modest deviation from
perfect linearity results in discrimination ratios (DRs; the
ratio of the IC50 of a given compound against [3H]DA uptake
to its IC50 against [3H]WIN binding) ranging from 1.3 for
3,4CTMP to 7.7 for TROHNBn. The DR was calculated as a
possible tool for use in the identification of compounds with
potential as partial or full cocaine antagonists. In theory,
structural modifications that increase the DR of a compound
should result in greater antagonist properties, because they
reflect the improved ability of the compound to block the
binding of cocaine to its recognition site, while leaving DA
uptake largely unaffected. Interestingly, when the analogs
containing a substituent on the nitrogen were compared with
unsubstituted analogs, it was found that the N-substituted

at each of seven different concentrations of test compound.

compound was determined in triplicate.

compounds had significantly higher DRs (5.0 0.8 versus

3.3 0.5, one-tailed independent t test, p 0.05).
As a compound with one of the higher DRs in this series
(6.6), 4MeTMPNMe was selected for further study to determine the nature of its interaction with the DAT, as well as its
ability to affect the inhibition by cocaine of DA uptake. In the
first series of studies, the effects of 300 and 600 nM 4MeTMPNMe on the Km and Vmax of [3H]DA uptake were examined. The saturation curves obtained in the presence and
absence of 4MeTMPNMe were analyzed according to
Michaelis-Menten kinetics. Data from a representative experiment are shown as a Lineweaver-Burk plot in Fig. 1;
results from three independent experiments are summarized
in Table 2. Results characteristic of a competitive inhibitor
were obtained; i.e., the apparent Km increased with increasing concentrations of 4MeTMPNMe, whereas the apparent
Vmax values did not differ significantly from the corresponding control values.
The ability of 4MeTMPNMe to block the inhibition of
[3H]DA uptake by cocaine was examined using the method
described by Simoni et al. (1993). In these experiments, the
effect of 200, 500, and 1000 nM 4MeTMPNMe on the IC50 of
cocaine was determined (Table 3). If cocaine and 4MeTMPNMe competed for the same domain on the DAT, the IC50 for
cocaine would be expected to rise by a predicted theoretical
amount. On the other hand, if cocaine and 4MeTMPNMe
acted at two distinct and unrelated sites, the IC50 for cocaine
in the presence of added 4MeTMPNMe would be expected to
change very little from its value in the absence of 4MeTMPNMe, whereas if the compounds acted at two overlapping but

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3CTMP
H
CO2CH3 Cl
H
3,4CTMP
H
CO2CH3 Cl
Cl
TROMeNBn
Bn CH2OCH3 H
H
TROHNBn
Bn
CH2OH H
H
4MeTMP
H
CO2CH3 H CH3
4ATMP
H
CO2CH3 H NH2
4FTMP
H
CO2CH3 H
F
TMPNBn
Bn
CO2CH3 H
H
TMP
H
CO2CH3 H
H
4MeTMPNMe CH3
CO2CH3 H CH3
3CTMPNMe
CH3
CO2CH3 Cl
H
TMPNMe
CH3
CO2CH3 H
H
()-Cocaine
Citalopram

[3H]DA
Uptake
IC50b

Methylphenidate Analogs as Treatments for Cocaine Abuse

531

Fig. 1. Lineweaver-Burk analysis of the effect of 4MeTMPNMe on [3H]DA uptake by rat striatal synaptosomes. Accumulation of [3H]DA (25 800 nM) was measured over a
2-min period in an S1 fraction of rat striatal tissue that had
been mixed with 25 mM phosphate buffer, then preincubated for 10 min at 37C with water (controls), 300 nM
4MeTMPNMe, or 600 nM 4MeTMPNMe before the addition of the [3H]DA. In this representative experiment, the
Vmax values were 134.8 (12.5314.42), 134.2 (11.5215.31),
and 152.5 (14.6715.84) pmol/(mg protein 2 min) and the
apparent Km values were 180 (146 214), 239 (157322),
and 333 (304 361) nM for the water, 300 nM 4MeTMPNMe, and 600 nM 4MeTMPNMe treatment groups, respectively (95% confidence intervals are shown in parentheses).
The results from three such experiments are presented in
Table 2.

Km and Vmax values (mean S.E.M.) are from three separate experiments, calculated by nonlinear regression analysis of synaptosomal [3H]DA uptake as a function
of free [3H]DA concentration (see Materials and Methods for details).
[3H]DA Uptake Parameters
[4MeTMPNMe]
Apparent Kma
nM

0
300
600

Vmaxb
pmol/mg protein/2 min

157 11
235 23*
326 6**

153 21
159 29
175 27

a
Significant effect of [4MeTMPNMe] on apparent Km by one-way ANOVA with
repeated measures (F2,4 41.04, p 0.002).
b
Significant effect of [4MeTMPNMe] on Vmax by one-way ANOVA with repeated
measures (F2,4 7.43, p 0.045), but no significant difference between samples with
and without 4MeTMPNMe by t test with Bonferroni correction.
* Significantly different from control by t test with Bonferroni correction (p
0.05).
** Significantly different from control by t test with Bonferroni correction (p
0.01).

nonidentical regions of the transporter, the IC50 for cocaine

in the presence of a fixed concentration of 4MeTMPNMe
would be expected to exceed the theoretical value that was
calculated, assuming a straightforward competition for the
same binding site. The results show that, although the IC50
for cocaine increased significantly as the concentration of
4MeTMPNMe in the samples rose from 200 to 1000 nM, it
never differed significantly from the theoretical value that
would be predicted if cocaine and 4MeTMPNMe were directly
competing for the same site.
Drug Discrimination Studies. A total of 23 rats were
trained to discriminate between 10 mg/kg cocaine and saline
in a median of 32 sessions (range: 1390). Doses of cocaine
from 1.0 to 10 mg/kg occasioned orderly increases in the
number of trials completed on the cocaine-appropriate choice
lever (Fig. 2). The group averaged more than 19 trials on the
cocaine-appropriate choice lever at the training dose of cocaine and at the highest dose tested, 17.5 mg/kg. TMP also
occasioned dose-dependent responding on the cocaine-appropriate choice lever and was three times more potent than
cocaine in this regard (Fig. 2; Table 4).
With the exceptions of 3CTMPNMe and TMPNBn, all of

TABLE 3
Effect of 4MeTMPNMe on the inhibition of [3H]DA uptake by cocaine
Cocaine IC50 (nM)
[4MeTMPNMe]
Observed

Theoretical (IC50)b,c

nM

0
200
500
1000

454 38
632 73
861 7
1208 58

593 53
801 75
1149 113

a
The observed IC50 values for cocaine differed in the presence of varying concentrations of 4MeTMPNMe (one-way ANOVA with repeated measures; F3,6 67.1, p
0.00001). The observed IC50 for cocaine increased in the presence of increasing
concentrations of 4MeTMPNMe, with the following rank order as a function of
[4MeTMPNMe]: 1000 nM 500 nM 200 nM 0 nM, by Scheffe multiple
comparisons.
b
IC50 was calculated as described in Simoni et al. (1993) and expressed as
mean S.E.M. from three separate experiments. For two inhibitors acting at the
same site, IC50 KCOC[1 [DA]/KDA [4MeTMPNMe/K4MeTMPNMe], where KCOC
and K4MeTMPNMe are the equilibrium dissociation constants for cocaine and 4MeTMPNMe, respectively, calculated from the Cheng-Prusoff equation for competitive
inhibitors (Cheng and Prusoff, 1973), using the IC50 values for cocaine and 4MeTMPNMe obtained in each individual experiment. (Ki S.E.M. from the three experiments were 382 32 nM and 553 21 nM for cocaine and 4MeTMPNMe, respectively.) [DA] was 29.5 0.1 nM. KDA is the Michaelis constant for DA uptake
determined under the same conditions (157 nM; see Table 2).
c
No significant difference was found between the observed and theoretical IC50
values for cocaine at any concentration of 4MeTMPNMe.

the analogs of TMP occasioned dose-dependent increases in

the number of trials completed on the cocaine-appropriate
choice lever; at the highest dose they generalized completely
(i.e., 18 trials to the cocaine-appropriate choice lever) or
almost completely with the training dose of cocaine (Fig. 3).
3CTMPNMe, tested over a dose range of 0.1 to 3.0 mg/kg,
occasioned completion of a maximum average of 10.6 trials on
the cocaine-appropriate lever at the highest dose, but with
considerable variability among the five rats: 0, 3, 12, 18, and
20 trials. The amount of 3CTMPNMe synthesized was not
sufficient to test higher doses. TMPNBn (1.0 30 mg/kg) occasioned the completion of few trials to the cocaine-appropriate lever, the group average reaching only 6.0 at 30 mg/kg
(Fig. 3), the highest dose available for testing. The results of
ANOVA indicated a difference among the slopes of the stimulus-generalization curves for the 12 compounds listed in
Table 4 that occasioned a group average of at least 10 trials

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TABLE 2
Effect of 4MeTMPNMe on kinetic parameters of [3H]DA uptake

532

Schweri et al.

TABLE 4
Potency of methylphenidate and analogs for producing cocaine-like
discriminative effects
ED50 (95% Confidence Limits)a
Compound
mg/kg

mol/kg

3,4CTMP
0.38 (0.20.73)
0.43 (0.131.36)c
4FTMP
0.26 (0.180.36)
1.03 (0.721.43)c,d
3CTMP
0.38 (0.200.73)
1.42 (0.752.73)c,d,e
4ATMP
0.39 (0.091.79)
1.57 (0.367.22)c,d,e
TMP
0.80 (0.611.05)
3.43 (2.624.51)d,e,f
4MeTMP
1.15 (0.255.33)
4.65 (1.0121.6)e,f
3CTMPNMe
2.00*
7.10*
Cocaine
2.58 (2.033.28)
8.45 (6.6610.8)
TROMeNBn
8.28 (2.4627.9) 26.7 (7.9490.0)g
TROHNBn
9.40*
31.6*
4MeTMPNMe
9.06 (3.2425.4) 34.7 (12.497.3)g
TMPNMe
11.4 (3.4737.2)
46.1 (14.0151)g
TMPNBn
30
92

Relative
Potencyb

7.98
3.33
2.42
2.18
1.00
0.74
0.48
0.41
0.13
0.11
0.10
0.07
0.04

a
Dose resulting in selection of the cocaine-appropriate choice lever in an average
of 10 trials per session [n 5 for all drugs except TROHNBn (n 4) and cocaine (n
23)].
b
Potency relative to methylphenidate, based upon ED50 values (mol/kg).
c g
ED50 values that do not have a superscript in common are significantly
different from each other, p 0.05.
* Estimated from group means and not included in the ANOVA of ED50 values.

to the cocaine-appropriate choice lever (F[11,259] 1.92, p

0.037). However, post hoc tests failed to identify differences
between specific compounds.
Comparison of the slopes of the stimulus-generalization
curves obtained for the N-substituted compounds (8.5 1.4
trials/log dose) with the slopes obtained for the compounds
containing no N-substitution (13.2 1.5 trials/log dose) revealed that the average slope for the unsubstituted compounds was about 55% higher than the average slope for the
N-substituted compounds, although the difference did not
quite reach statistical significance (t9 2.196; p 0.056).
The slope value for TMPNBn was not included in these
calculations because a reliable slope could not be calculated
from the available data.

Fig. 3. Stimulus generalization curves for methylphenidate and 11 analogs

in rats trained to discriminate between 10 mg/kg cocaine and saline. Drugs
were administered i.p. 30 min before a session. Points are means based upon
one observation in each of five or four (TROHNBn) rats. Solid lines indicate
compounds that have a substitution on the ring nitrogen, and dashed lines
indicate compounds that do not. The curve for methylphenidate (TMP) is
reproduced from Fig. 2. Other details are the same as in Fig. 2.

3,4CTMP was the most potent of the analogs, 8 times more

potent than TMP; TMPNMe was the least potent of the
compounds for which an ED50 could be calculated, 1/14 as
potent as TMP (Table 4). Thus, relative to the parent compound, the analogs spanned a potency range of 107-fold. The
inclusion of TMPNBn increases the potency range to more
than 200-fold. The relative potencies of TMP and the 10
analogs for which ED50 values could be determined as cocaine-like discriminative stimuli correlated significantly
with their order of potency for displacing the binding of
[3H]WIN and for inhibiting [3H]DA uptake in striatal preparations (Fig. 4): Correlation coefficients of 0.61 (p 0.048)
and 0.75 (p 0.008) were obtained between the log ED50 for
cocaine discrimination and the log of the [3H]WIN binding
IC50 or [3H]DA uptake IC50, respectively. The correlation
between the log ED50 for cocaine discrimination and the log
of the DR approached significance (Pearson r 0.60; p
0.0534). No correlation was observed between the log of the
ED50 values for cocaine discrimination and the log of inhibitory potencies against [3H]CIT binding to the SERT, based
on the four analogs for which [3H]CIT binding IC50 values
could be calculated (Pearson r 0.534, p 0.46).
On the basis of stimulus-generalization curves, DR values,
and chemical structures, four of the TMP analogs were selected for testing in combination with cocaine: 4ATMP,
4MeTMPNMe, TROHNBn, and TMPNBn. 4ATMP is a more
potent compound than cocaine that required a 100-fold range
of doses to define the ascending limb of the stimulus-generalization curve. 4MeTMPNMe is a less potent compound
than cocaine that occasioned a low level of drug-appropriate
responding over a 30-fold dose range. The last three compounds had the highest DR values of all of the analogs
studied in this series, suggesting that a dose might be found
at which they could inhibit cocaine binding to the transporter
but not block transport of the substrate. The last two com-

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Fig. 2. Stimulus-generalization curves for methylphenidate and cocaine

in rats trained to discriminate between 10 mg/kg cocaine and saline in a
two-choice discrete-trial avoidance/escape procedure. Drugs were administered i.p. either 30 (methylphenidate) or 15 min (cocaine) before a
session. Points are means based upon one observation in each of 5
(methylphenidate) or 23 (cocaine) rats. They indicate the number of trials
completed on the cocaine-appropriate choice lever in a 20-trial session;
the remaining trials of the session were completed on the choice lever
appropriate for saline (Veh). The upper and lower dashed horizontal lines
indicate the minimum level of performance maintained in training sessions that followed an injection of 10 mg/kg cocaine or saline, respectively.

Methylphenidate Analogs as Treatments for Cocaine Abuse

533

Fig. 5. Interactions of four analogs

of methylphenidate with cocaine in
rats discriminating between 10
mg/kg cocaine and saline. Cocaine
(n 79) was administered i.p. 15
min before a session, and each analog (n 5) was administered i.p.
30 min before a session. Points
above Veh represent the effect of
an injection of the indicated dose of
the methylphenidate analog followed 15 min later by an injection
of saline, the vehicle for cocaine.
Other details are the same as in
Fig. 2.

pounds have N-benzyl substitutions, and one, TMPNBn, occasioned little cocaine-appropriate responding up to a dose of 30
mg/kg. Administered 15 min before cocaine, each of the TMP
analogs tended to shift the cocaine stimulus-generalization
curve upward and to the left (Fig. 5). This effect is particularly
notable with 3.0 mg/kg TROHNBn (Fig. 5, upper right) and 10
mg/kg TMPNBn (Fig. 5, lower right), doses that occasioned
little or no cocaine-appropriate responding when tested alone.
The ED50 of cocaine was lowered from 3.27 (2.10 5.08)
mg/kg to 1.38 (0.375.14) and 1.43 (0.36 5.70) mg/kg after
pretreatment with 0.03 or 0.1 mg/kg 4ATMP, respectively. It
was lowered from 2.60 (1.823.72) mg/kg to 2.25 (0.99 5.10)
and 1.58 (0.435.79) mg/kg, after pretreatment with 1.0 or
3.0 mg/kg 4MeTMPNMe, respectively. However, none of

these changes was statistically reliable, according to

ANOVA. Pretreatment with 3.0 mg/kg TMPNBn lowered the
cocaine ED50 nonsignificantly from 2.72 (1.69 4.36) mg/kg to
a group mean of 1.87 mg/kg.
ED50 values could not be calculated for cocaine after pretreatment with 10 mg/kg 4MeTMPNMe, 3.0 mg/kg TROHNBn, or 10
mg/kg TMPNBn because the threshold for detection of cocaine
was reduced so markedly. However, a reduction in ED50 can be
inferred from the fact that pretreatment with each of the four
compounds resulted in a flattening of the stimulus-generalization curve for cocaine. ANOVA confirmed significant differences
among the three curves in the 4ATMP (F[2,15] 5.69, p 0.015)
and TMPNBn (F[2,15] 14.32; p 0.001) series as well as
among the four curves in the 4MeTMPNMe series (F[3,20]

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Fig. 4. The order of potency of methylphenidate and 10 analogs to produce cocaine-like discriminative effects in rats discriminating between 10 mg/kg
cocaine and saline correlates significantly with their order of potency to inhibit [3H]WIN binding to the DAT (left) and [3H]DA uptake (right) in rat
striatal preparations. E, the N-substituted analogs; F, the analogs with no N-substitution. TMPNBn (open square with arrow) was not included in the
regression analysis, because it did not generalize sufficiently to the cocaine stimulus to allow a valid calculation of its ED50 (see Table 4). It is shown
here to underscore its unique activity and allow comparison with the other N-substituted compounds.

534

Schweri et al.

5.84, p 0.005); Students t test did the same for the slopes of
the curves for cocaine alone and with TROHNBn (t10 5.32;
p 0.001).

Discussion

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The methylphenidate analogs described here exhibit many

cocaine-like properties, both in vitro and in vivo, suggesting
that they may ultimately prove useful as substitution therapies for the treatment of cocaine addiction. Like cocaine,
they potently inhibit both the binding of [3H]WIN to the
DAT, as well as the uptake of [3H]DA by striatal synaptosomes. With the exception of 3CTMPNMe and TMPNBn, all
of the compounds fully substituted for 10 mg/kg cocaine in
cocaine discrimination studies. The analogs with and without
N-substitutions exhibit some unique differences that may
influence their usefulness as drug therapy for cocaine abuse.
All of the derivatives retained activity at the DAT in vitro,
despite the various structural modifications made to the
methylphenidate molecule. Substitutions on the aryl ring, or
introduction of an N-benzyl group (with or without simultaneous modification of the ester function) increased the affinities of the resulting compounds for the WIN binding site,
compared with the parent compound. On the other hand,
introduction of an N-methyl substitution (with or without
simultaneous substitution on the aryl ring) caused up to a
6-fold loss in affinity. Hill coefficients of approximately 1
were obtained for all of the compounds in the WIN binding
assay, with the notable exception of 3,4CTMP, which had a
nH of 2, as reported previously (Deutsch et al., 1996). For the
most part, parallel results were obtained for the inhibition of
[3H]DA uptake by the various derivatives.
In contrast to their activity against [3H]WIN binding to the
DAT, the compounds were much less potent as inhibitors of
[3H]CIT binding to the SERT. Comparison of the IC50 values
of the analogs in the two assays showed that the derivatives
ranged from 8- to more than 2000-fold less potent against
[3H]CIT binding than against [3H]WIN binding (Table 1).
Cocaine exhibited much less selectivity than the methylphenidate derivatives for the DAT: its IC50 against [3H]WIN
binding was only 2.5-fold less than its IC50 against [3H]CIT.
In agreement with previous reports (Ritz et al., 1987; Gatley
et al., 1996b), TMP had little inhibitory activity at the SERT.
N-Benzyl modification increased affinity for the [3H]CIT
binding site, and simultaneous conversion of the ester function to an alcohol or methyl ether increased it even more
(10-fold). In contrast to the effect of ring halogenation on
[3H]WIN binding, where 3-Cl and 3,4-Cl modifications produced equivalent increases in potency, 3,4CTMP is much
more potent than 3CTMP against [3H]CIT binding. Although
dichloro substitution increased potency at the SERT compared with unmodified TMP, it did not raise the nH to 2, as it
did against [3H]WIN binding. Because the [3H]WIN and
[3H]CIT binding assay conditions are so similar, this finding
suggests that the nH obtained for this compound in the
[3H]WIN assay is valid and not an artifact caused by its high
lipophilicity.
In general, the in vitro interactions of these agents with
the DAT are predictive of their activity in the in vivo cocaine
discrimination studies (Fig. 4), consistent with the generally
accepted notion that inhibition of DA uptake is the chief
internal cue for identification of cocaine-like drugs (Kleven

and Koek, 1998, and references therein; Howell and Wilcox,

2001). In accord with this are the positive correlations observed between the logs of the [3H]WIN binding IC50, [3H]DA
uptake IC50, or DR, and the log ED50 for cocaine discrimination. However, results from in vitro assays were not perfect
predictors of potency in vivo. Although the low in vivo potency of the N-Me-substituted compounds reflects their low
potency in vitro, this was not the case with the N-Bn-substituted compounds. TROMeBn, for instance, was approximately 5 times more potent than TMP against [3H]WIN
binding and two and a half times more potent against [3H]DA
uptake than TMP; yet it was almost 8-fold less potent than
TMP as a substitute for cocaine. TMPNBn, the low potency of
which in the cocaine discrimination studies precluded a calculation of its ED50, was comparable with TMP in the
[3H]WIN binding and [3H]DA uptake assays. Five of the six
N-substituted compounds lie above the regression line,
whereas all of the compounds without an N-substitution fall
on or below the line (Fig. 4). As a group, the N-substituted
compounds had shallower stimulus-generalization curves,
leading to higher ED50 values than would have resulted had
their slopes resembled those of the unsubstituted compounds. N-substituted analogs are more hydrophobic than
their secondary amine counterparts. It is possible, therefore,
that the shallower dose-response curves of this group are
attributable to differences in onset of action caused by pharmacokinetic factors, such as absorption, binding to plasma
proteins, and subsequent transfer across the blood-brain barrier. There was no correlation between the potencies of compounds to substitute for cocaine and their inhibitory potencies against [3H]CIT binding to the SERT.
As a group, the N-substituted analogs also have DR values
that are approximately 50% higher than those obtained for
compounds without substitutions at the piperidinyl nitrogen.
The DR is an empirical measure utilized in our laboratory as
a preliminary screen to identify potential cocaine antagonists. Because it represents the ratio of the IC50 of a compound against [3H]DA uptake to its corresponding IC50
against [3H]WIN binding, high values would be expected to
be associated with compounds that are more effective at
blocking cocaine binding than DA uptake. For example, a
compound that inhibited 80% of [3H]WIN binding sites while
blocking only 10% of [3H]DA uptake would theoretically have
a DR value of 36 (Deutsch et al., 1999). The DR is utilized
only as one means of comparison of compounds assayed under rigorously maintained experimental conditions: even
small variations in the assay conditions can drastically alter
the DR value (Rothman et al., 1993; Xu et al., 1995). Although the DR values of the tertiary amines were significantly higher than those for the unsubstituted analogs, their
mean absolute value (5.0 0.8) nevertheless was quite low
relative to the value theoretically required for a compound to
exhibit predominantly antagonist-like characteristics
against cocaine; this suggests that these compounds will
have greatest utility as substitution therapy for cocaine.
In an effort to learn more about the interaction of these
compounds with cocaine, both in vitro and in vivo studies
were conducted. In vitro, the effect of 4MeTMPNMe on cocaine inhibition of [3H]DA uptake into synaptosomes was
examined. 4MeTMPNMe was selected because of its relatively high DR (6.6) and its shallow dose-response curve in
the cocaine discrimination assay. In the absence of cocaine,

Methylphenidate Analogs as Treatments for Cocaine Abuse

Acknowledgments

We thank Monica Stafford and Adam Eason for assistance in the

in vitro assays, and Christine Engels for assistance in the drug
discrimination assays.
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Address correspondence to: Dr. Margaret M. Schweri, Division of Basic

Medical Sciences, Mercer University School of Medicine, 1550 College Street,
Macon, GA 31207. E-mail: schweri_mm@mercer.edu

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4MeTMPNMe acted as a classic competitive antagonist of

[3H]DA uptake. When tested in combination with cocaine,
4MeTMPNMe appeared to act additively with cocaine at the
same or similar site(s) to block the transport of [3H]DA; it did
not prevent access of cocaine to its binding site on the transporter in such a fashion as to raise the IC50 of cocaine over
and above that predicted for the combination of two inhibitors acting at the same site.
When examined in vivo in combination with cocaine, most
of the analogs tested appeared to potentiate cocaine discrimination, even at doses that by themselves had little or no
cocaine-like discriminative stimulus activity. It is unlikely
that these compounds potentiate effects of cocaine in vivo by
interfering with its metabolism, because even the nonesterified analogs (which would not be expected to compete for
metabolism) caused this effect. It is conceivable that the
potency of these compounds against serotonin transport underlies their potentiation of the discriminative stimulus effects of cocaine. Although it is generally acknowledged that
inhibition of DA transport is the internal cue responsible for
the discrimination of cocaine by rats (Cunningham and Callahan, 1991), serotonin transport inhibitors increase the sensitivity for detection of this cue, even though they cannot by
themselves substitute for cocaine (Kleven and Koek, 1998,
and references therein). Indeed, TROHNBn, which potentiated cocaine discrimination by 8-fold when rats were pretreated with approximately 0.3 ED50 of it, was one of the
most potent analogs against [3H]CIT binding, whereas approximately the same relative dose of 4ATMP, which has
virtually no activity at the SERT, caused only a 3-fold
increase in cocaine discrimination. On the other hand,
synergism with cocaine has also been reported for 1-{2[bis(4-fluorophenyl-)methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR-12909) and 4-chlorobenztropine, structurally diverse dopamine uptake inhibitors with little activity at
the SERT (Tolliver et al., 1999; Holtzman, 2001).
In summary, structural modification of TMP yields compounds with high to moderate potency as inhibitors of the
DAT, and varying degrees of activity at the SERT, that may
prove useful as substitution therapy for cocaine abuse. Further systematic studies, several of which are under way in
our laboratories, are needed to investigate and exploit the
unique (although sometimes conflicting) properties identified
in this study for the N-substituted analogs. Their shallow
dose-response curves may augment their utility as substitution therapy by providing a broader therapeutic dosing range
before the threshold for full cocaine-like character is reached.
If further chemical modification can generate compounds
with even higher DR values, clinically useful partial agonists/antagonists may result. On the other hand, if the leftshift of the cocaine discrimination curve seen with these
agents reflects their ability to potentiate the rewarding effects of cocaine, their appeal would diminish.

535