Synthesis and evaluation of dopamine and serotonin transporter inhibition by oxacyclic and carbacyclic analogues of methylphenidate

Article abstract of DOI:10.1021/jm0205292

Methylphenidate (Ritalin) binds stereoselectively and enantioselectively to the dopamine transporter (DAT) and inhibits dopamine reuptake with in vitro and in vivo potency similar to that of cocaine. Unlike cocaine, it manifests little, if any, tolerance or addiction liability. Since this compound has a substantial clinical history, it provides an excellent template from which to design potential medications for cocaine abuse. It has long been assumed that a nitrogen, such as exists in cocaine and methylphenidate, is essential for interaction with monoamine transporters. We previously demonstrated that an amine nitrogen in phenyltropane analogues of cocaine is not necessary for conferring high DAT binding affinity. We now report the synthesis of oxacyclic and carbacyclic analogues of methylphenidate, including the four enantiomerically pure isomers of 2-(3,4-dichlorophenyl)-2-(tetrahydropyran-2-yl)acetic acid methyl ester. The threo isomers are potent and selective inhibitors of the DAT. This is the first generalization of the principle that the presence of nitrogen is not a necessity for DAT inhibition.

Full text of DOI:10.1021/jm0205292

1538
J . Med. Chem. 2003, 46, 1538-1545
Syn th esis a n d Eva lu a tion of Dop a m in e a n d Ser oton in Tr a n sp or ter In h ibition
by Oxa cyclic a n d Ca r ba cyclic An a logu es of Meth ylp h en id a te
Peter C. Meltzer,*,† Pinglang Wang,† Paul Blundell,† and Bertha K. Madras‡
Organix Inc., 240 Salem Street, Woburn, Massachusetts 01801, and Department of Psychiatry, Harvard Medical School and
New England Regional Primate Research Center, Southborough, Massachusetts 01772
Received November 21, 2002
Methylphenidate (Ritalin) binds stereoselectively and enantioselectively to the dopamine
transporter (DAT) and inhibits dopamine reuptake with in vitro and in vivo potency similar to
that of cocaine. Unlike cocaine, it manifests little, if any, tolerance or addiction liability. Since
this compound has a substantial clinical history, it provides an excellent template from which
to design potential medications for cocaine abuse. It has long been assumed that a nitrogen,
such as exists in cocaine and methylphenidate, is essential for interaction with monoamine
transporters. We previously demonstrated that an amine nitrogen in phenyltropane analogues
of cocaine is not necessary for conferring high DAT binding affinity. We now report the synthesis
of oxacyclic and carbacyclic analogues of methylphenidate, including the four enantiomerically
pure isomers of 2-(3,4-dichlorophenyl)-2-(tetrahydropyran-2-yl)acetic acid methyl ester. The
threo isomers are potent and selective inhibitors of the DAT. This is the first generalization of
the principle that the presence of nitrogen is not a necessity for DAT inhibition.
In tr od u ction
Methylphenidate1 (Figure 1), marketed as Ritalin (DL-
threo-methylphenidate) for the treatment of attention-
deficit hyperactivity disorder (ADHD), binds stereose-
lectively and enantioselectively to the dopamine trans-
porter (DAT) (IC50 ) 34-83 nM).2-4 It inhibits dopamine
reuptake with in vitro and in vivo potency similar to
that of cocaine5 yet, unlike cocaine, manifests little, if
any, tolerance or addiction liability.5,6 Patrick et al.7
have shown the D-threo-methylphenidate to be the
pharmacologically active isomer. Ding et al.8 reported
a positron emission tomography (PET) study in which
they confirmed that the biological activity of meth-
ylphenidate resides in the (2R,2′R)-D-threo isomer,
which is distributed to sites in the basal ganglia. In
contrast, they showed that the (2S,2′S)-L-threo isomer
is distributed nonspecifically in both baboon and human
brains. In view of this biological enantioselectivity, there
has been a considerable effort devoted to the develop-
F igu r e 1.
ment of enantioselective synthetic pathways to (2R,2′R)-
threo-methylphenidate.9-12
date. Inhibition of binding to the dopamine transporter
is most often measured by displacement studies in
which either [3H]RTI55 or [3H]WIN 35,428 are used as
radioligands. A desirable attribute of a potential an-
tagonist-based cocaine medication is an ability to inhibit
DAT binding of cocaine while allowing dopamine to
transport intracellularly. Deutsch et al. have reported2
a methylphenidate analogue that manifests a difference
in inhibition of [3H]WIN 35,428 versus dopamine re-
uptake at the DAT, and they postulated that this
compound may provide a lead for a dopamine-sparing
cocaine antagonist.
Methylphenidate has a mode of action (inhibition of
the DAT) similar to that of cocaine and the 3-aryl-8-
azabicyclo[3.2.1]octanes (3-aryltropanes).3,13 Methylpheni-
date’s substantial clinical history, combined with mini-
mal, if any, addiction liability, suggested that this might
provide an excellent template from which to design
potential medications for cocaine abuse. This has been
recognized by researchers, and Gatley et al.4,14 have
reported aromatic ring brominated analogues that
manifest greater potency at the DAT than the parent
methylphenidate, while Deutsch et al.2 have evaluated
a series of ring-substituted analogues of methylpheni-
Prior to our work on 8-oxabicyclo[3.2.1]octanes15,16
and 8-carbabicyclo[3.2.1]octanes (Figure 1),17 it had been
assumed that a nitrogen, such as exists in cocaine, the
3-aryltropanes, and methylphenidate, was essential for
interaction with monoamine transporters. Since we
have shown unequivocally that the nitrogen “anchor”
* To whom correspondences should be addressed. Phone: 781-932-
4142. Fax: 781-933-6695. E-mail: Meltzer@organixinc.com.
† Organix Inc.
‡ Harvard Medical School and New England Regional Primate
Research Center.
10.1021/jm0205292 CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/11/2003

Methylphenidate Analogue Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1539
Sch em e 1. Synthesis of Aryltetrahydropyranyl Methyl Estersa
a
Reagents: (i) LDA, Et2O; (ii) column chromatography.
Ta ble 1. Inhibition of [3H]WIN 35,428 Binding to the
Dopamine Transporter and [3H]Citalopram Binding to the
Serotonin Transporter in Rhesus (Macaca mulatta) or
Cynomolgus Monkey (Macaca fascicularis) Caudate-Putamena
hydrolyzed to the (()-threo-6. Hydrolysis under basic
conditions (LiOH) leads to racemization because the
benzylic proton is labile. Therefore, nonbasic conditions
were needed for the cleavage of the methyl ester.
Hydrolysis was readily effected with trimethylsilyl
iodide (TMSI).19 The enantiomeric pair 6 was then
transformed into diastereomeric menthyl esters 8 and
9 by treatment of their acid chlorides with optically pure
L-menthol. Column chromatography then allowed sepa-
ration of the two diastereomeric menthyl esters 8 and
9. Separation was confirmed by 1H NMR. The 2′-H
doublet for 8 appeared at δ 7.465 and δ 7.459 and that
for 9 at δ 7.508 and δ 7.502. On an expanded spectrum
of a mixture of 8 and 9, these sharp resonances can be
Organix DAT IC50
compd
no.
(nM)
SERT
DL-threo methylphenidateb
17 ( 2.0
>100000
>100000
1000
4a
4b
4c
5a
5b
5c
12
13
14
15
16
17
18
19
O-2602 3000
O-2618 430
O-1730 29.1 ( 5.05 2180 ( 226
O-2601 13000
O-2617 4000
>100000
10000
O-1731 286 ( 10.5 7795 ( 1840
O-1794 34 ( 8.6
O-1783 17 ( 1.3
O-1793 736 ( 59
O-1792 193 ( 3.5
O-2170 127
1655
>10000
7800
>10000
8000
12000
10000
7000
1
clearly distinguished. The H NMR spectrum for pure
8 shows no resonances due to 9. Correspondingly, the
spectrum for pure 9 shows no resonances attributable
to 8. Therefore, the enantiomeric purity of 8 and 9 is
estimated at >98% ee. These two menthyl esters were
each separately hydrolyzed, again with TMSI, to give
the two optically pure acids (2S,2′S)-6′ and (2R,2′R)-6′
in good yield (74% and 77%, respectively). Acid (2S,2′S)-
6′ was crystallized, and its absolute configuration was
confirmed by X-ray crystallography. The configuration
of (2S,2′S)-6′ was thus proved. Therefore (2R,2′R)-6′ was
confirmed as the 2R,2′R enantiomer. These acids were
then methylated with trimethylsilyl diazomethane to
furnish optically pure target molecules (2S,2′S)-12′ and
(2R,2′R)-13′. The compounds had equal and opposite
optical rotations.
O-2169 146
O-2171 128
O-2172 47
a
Each value is the mean of three or more independent experi-
ments each conducted in different brains and in triplicate. Errors
generally do not exceed 15% between replicate experiments.
b
Highest doses tested were generally 10-100 µM. Schweri et al.3
reported D-threo-(2R,2′R)- and L-threo-(2S,2′S)-methylphenidate
IC50 values of 88 nM and 1.2 µM, respectively.
point is not needed for potent biological activity in the
bicyclo[3.2.1]octane series,18 we wished to probe the
generalizability of exchange of oxygen, or carbon, for
nitrogen in monoamine uptake inhibitors. We therefore
explored the substitution of the nitrogen in the piperi-
dine ring of methylphenidate for an oxygen and a
carbon. Herein, we describe the synthesis of 10 oxygen
and 4 carbon analogues of methylphenidate. We report
that the 3,4-dichlorophenyloxamethylphenidate ana-
logues prove to be quite potent inhibitors of the dopa-
mine transporter. The synthesis and binding of the four
enantiomerically pure isomers of 2-(3,4-dichlorophenyl)-
2-(tetrahydropyran-2-yl)acetic acid methyl ester are also
described.
Conversion of (()-erythro-5c, as described above,
provided the (()-erythro acids 7. The menthyl esters of
the pair of acids 7 proved to be difficult to separate by
column chromatography. Instead, the racemic acids 7
were converted to the diastereomeric indanyl esters 10
and 11 by reaction with (S)-(+)-1-indanol, which were
then resolved by medium-pressure flash column chro-
matography. Separation was again confirmed by
1H
NMR. The doublet for 10 (δ 3.544 and 3.515) and that
for 11 (δ 3.557 and 3.528) were distinguishable on an
expanded 1H NMR spectrum. Each purified diastere-
omer showed less than 2% contribution from the other
diastereomer. Therefore, the enantiomeric purity of each
of 10 and 11 was estimated at >96% ee. The esters were
hydrolyzed (TMSI) and methylated (as above) to provide
the two enantiomerically pure methyl esters (2S,2′R)-
14′ and (2R,2′S)-15′, which again, had equal and op-
posite optical rotations.
Ch em istr y
The synthesis of the racemic diastereomers 4 and 5
is presented in Scheme 1. 2-Chlorotetrahydropyran was
reacted with the enolate of the appropriate methylaryl
acetate. The mixture of diastereomers 4 and 5 thus
obtained was separated by column chromatography to
provide the racemic threo isomers 4a -c and the racemic
erythro isomers 5a -c. The 3,4-dichloroaryl compounds
4c and 5c manifested nanomolar inhibition of the DAT
(Table 1) and were therefore selected for enantiomeric
resolution. The pairs of separated diastereomers 4c and
5c were then resolved into their (+)- and (-)-enanti-
omers as shown in Scheme 2. The (()-threo-4c was
A sample of the optically pure acid that provided 15
was also allowed to react with 4-nitrophenol to obtain
the 4-nitrophenyl ester (Figure 2). This compound was
recrystallized, and X-ray crystallographic analysis con-

1540 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Meltzer et al.
Sch em e 2. Resolution of threo- and erythro-3,4-Dichlorophenyltetrahydropyranyl Acetic Acid Methyl Esters 12-15a
a
Reagents: (i) TMSI, CHCl3; (ii) (COCl)2, DMF, CH2Cl2; (iii) pyridine, L-menthol, THF; (iv) pyridine, (S)-(+)1-indanol, THF; (v) TMSI,
CCl4; (vi) TMSCHN2.
Sch em e 3. Synthesis of 3,4-Dichlorophenylcycloalkyl-
acetic Acid Methyl Esters 17 and 19a
F igu r e 2. Absolute configuration of the 4-nitrophenyl ester
of (2R,2′S)-15.
firmed its configuration as 2R,2′S. Therefore, the methyl
ester derived from that acid was compound (2R,2′S)-
15′. Therefore, the remaining enantiomer was confirmed
as (2S,2′R)-14′.
The 3,4-dichlorophenyl carbacyclic analogues of meth-
ylphenidate were prepared (Scheme 3) by reaction of
either cyclohexyl bromide or cyclopentyl bromide with
a
Reagents: (i) t-BuOK, cycloalkyl bromide; (ii) HCl, MeOH.
3,4-dichlorophenylacetonitrile to provide 16 and 18.
Hydrolysis and esterification then gave the desired
binding was measured with fluoxetine (10 µM).16 Stud-
carbacyclic analogues 17 and 19, respectively.
used to label the serotonin transporter, and nonspecific
ies were conducted in monkey striatum because this
tissue is used in an ongoing investigation of structure-
Biology
The affinities (IC50) for inhibition of the dopamine and
serotonin transporters were determined in competition
studies and are presented in Table 1. The dopamine
transporter was labeled with [3H]3â-(4-fluorophenyl)-
tropane-2â-carboxylic acid methyl ester ([3H]WIN 35,428
or [3H]CFT (1 nM)), and nonspecific binding was mea-
sured with (-)-cocaine (30 µM).20 [3H]Citalopram was
activity relationships at the DAT and meaningful
comparisons with an extensive database can be made.18
Competition studies were conducted with a fixed con-
centration of radioligand and a range of concentrations
of the test drug. All drugs inhibited [3H]WIN 35,428 and
[3H]citalopram binding in a concentration-dependent
manner.

Methylphenidate Analogue Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1541
The (()-threo isomers 4a -c inhibited [3H]WIN 35,428
binding to the DAT with a potency of IC50 ) 29 nM to
3 µM, while the (()-erythro isomers manifested an
inhibitory potency range of 0.28-13 µM. The 3,4-
dichlorophenyl compounds were most potent, and 4c
(3,4-dichlorophenyltetrahydrofuran methyl ester) had
DAT (IC50 ) 29.1 nM) with poor inhibition of the SERT
(IC50 ) 2.2 µM). The carbacyclic analogues 16-19
manifested DAT potency in the nanomolar range
(IC50 ) 47-146 nM) with considerable selectivity (SERT
IC50 ) 7-12 µM).
4c is a 3,4-dichlorophenyl analogue, it cannot be com-
pared directly with DL-threo-methylphenidate itself
(IC50 ) 17 nM). However, the 3,4-dichlorophenyl ana-
logue of methylphenidate has been reported2 to manifest
an IC50 of 5.3 nM at the DAT. These IC50 values are
obtained in different laboratories and therefore are not
directly comparable; however, it is apparent that the
oxa analogue 4c is at least about 6-fold less potent than
its aza counterpart. Notwithstanding this apparent loss
in inhibitory potency, the dichlorophenyl oxameth-
ylphenidate remains an extremely potent inhibitor of
the DAT and is certainly as potent as methylphenidate
itself.
Discu ssion
It is notable that in all cases the threo isomers (4)
are, as with methylphenidate itself, more potent dia-
stereomers than the erythro compounds (5). As evi-
denced in the 3,4-dichlorophenyl series, the biological
diastereoselectivity for these oxa analogues is similar
to that of the aza counterparts. Thus, the (()-threo-3,4-
dichlorophenyl analogues 4c are about an order of
magnitude more potent at the DAT than their (()-
erythro counterparts 5c (IC50((()-threo) ) 29.1 ( 5.1
nM vs IC50((()-erythro) ) 286 ( 10.5 nM). Also, the
(2R,2′R)-(-)-threo-13 (IC50 ) 17 ( 1.3 nM) is about twice
as potent as the (2S,2′S)-(+)-threo-12 (IC50 ) 34 ( 8.6
nM). Thus, it is interesting to note that the biological
enantioselectivity seen in the (2R,2′R)-threo- versus
(2S,2′S)-threo-methylphenidate is about 13.6-fold,3
whereas for the oxygen analogues presented here, that
ratio is reduced to about 2-fold. Finally, both (-)-13 and
(+)-12 threo analogues exhibit substantial selectivity for
the DAT over the SERT.
The carbacyclic analogues show surprising potency.
The cyclohexyl analogues, as either a methyl ester 17
or a nitrile 16, exhibit similar binding potency at the
DAT (IC50 ) 127-146 nM) and SERT (IC50 ) 8-12 µM).
While the cyclopentylnitrile analogue 18 is similarly
potent (IC50 ) 128 nM), the methyl ester 19 manifests
substantial binding potency at the DAT (IC50 ) 47 nM)
with substantial selectivity (150-fold) over the SERT.
It remains to be determined whether biological enan-
tioselectivity is also manifested by this compound.
Methylphenidate inhibits [3H]WIN 35,428 binding to
the DAT with substantial potency (IC50 ) 17 nM) and
selectivity (SERT IC50 g 100 mM). In particular, the
(2R,2′R)-threo isomer is about 14-fold more potent than
its (2S,2′S)-threo counterpart.3 We wished to investigate
whether similar potency and biological enantioselectiv-
ity would be evidenced by analogues in which the amine
had been exchanged for an ether. To further investigate
whether a heteroatom was required for binding of these
compounds to the DAT, we planned to synthesize and
evaluate carbacyclic analogues of our most potent oxa-
methylphenidates, namely, the 3,4-dichlorophenyl com-
pounds. The investigation was conducted in two phases.
In the first phase, we prepared the oxa analogues with
three different aromatic substitutions [phenyl (4a and
5a ), 2-naphthyl (4b and 5b), and 3,4-dichlorophenyl (4c
and 5c)] and evaluated them as pairs of enantiomers.
On the basis of the binding potencies (Table 1) of each
of the (()-threo-(4) and (()-erythro-(5) racemates, we
selected the most potent of these (4c and 5c) to
determine the biological enantio- and stereoselectivity
in this new class of non-nitrogen DAT inhibitors. The
asymmetric synthesis of an oxacyclic furanyl analogue
of methylphenidate has been reported;21 however, our
general synthetic route (Scheme 1) provided access to
all isomers simultaneously, and we judged that the most
expeditious route to each isomer would be by subsequent
separations, as already described. Thus, the enantiopure
3,4-dichlorophenyl analogues 12-15 were obtained.
Synthesis of the enantiomeric pairs of the carbacyclic
analogues 17 and 19 was achieved through the inter-
mediate nitriles 16 and 18. This afforded us the op-
portunity to evaluate a 2-cyano carbacyclic analogue of
methylphenidate as well as the C2 methyl esters.
Indeed, we prepared both the six-membered (16 and 17)
and the five-membered (18 and 19) analogues with some
surprising biological results.
The exchange of the nitrogen for an oxygen or a
carbon in these analogues of methylphenidate has
proved to be extremely interesting. These compounds
retain biological activity. The unsubstituted phenyl
compounds 4a and 5a manifest micromolar potency at
the DAT. For comparison, DL-threo-methylphenidate has
an IC50 of 17 nM at the DAT. Therefore the DL-threo-
oxa analogue 4a is about 175-fold less potent. The
naphthyl analogues 4b and 5b are slightly more potent
than the unsubstituted compounds 4a and 5a , respec-
tively. The 3,4-dichlorophenyl compounds 4c and 5c
prove to be the most potent in this oxacyclic series.
Indeed, the racemic threo-dichlorophenyl analogue 4c
manifests an IC50 of 29 nM. It should be noted that since
Con clu sion
Oxacyclic and carbacyclic analogues of methylpheni-
date have been synthesized. Evaluation of their binding
potency at both DAT and SERT has shown that these
compounds manifest substantial inhibitory potency. We
had reported that 3,4-dichlorophenyl substitution in
8-aza-, 8-oxa-, and 8-carbatropanes had provided espe-
cially potent inhibitors,18 and this is evident in this new
series as well. The four isomers of (3,4-dichlorophenyl)-
(tetrahydropyran-2-yl)acetic acid methyl ester were
prepared, and their absolute stereochemistry was es-
tablished. It was shown that the threo isomers are about
1 order of magnitude more potent as DAT inhibitors
than the erythro compounds. Furthermore, as with
methylphenidate, the (2R,2′R)-threo isomer is more
potent than its (2S,2′S)-threo counterpart.
Exp er im en ta l Section
NMR spectra were recorded in CDCl3, unless otherwise
mentioned, on a J EOL 300 NMR spectrometer operating at
300.53 MHz for 1H and at 75.58 MHz for 13C. TMS was used
as the internal standard. Melting points are uncorrected and

1542 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Meltzer et al.
were measured on a Gallenkamp melting point apparatus.
Thin-layer chromatography (TLC) was carried out on Baker
Si250F plates. Visualization was accomplished with either UV
exposure or treatment with phosphomolybdic acid (PMA).
Flash chromatography was carried out on Baker silica gel, 40
µM. Elemental analyses were performed by Atlantic Microlab,
Atlanta, GA. Optical rotations were measured on a Perkin-
Elmer 241 polarimeter. All reactions were conducted under
an inert (N2) atmosphere. [3H]WIN 35,428 (2â-carbomethoxy-
3â-(4-fluorophenyl)-N-[3H]methyltropane, 79.4-87.0 Ci/mmol)
and [3H]citalopram (86.8 Ci/mmol) were purchased from
DuPont-New England Nuclear (Boston, MA). A Beckman 1801
scintillation counter was used for scintillation spectrometry.
Bovine serum albumin (0.1%) was purchased from Sigma
Chemicals. (R)-(-)-Cocaine hydrochloride for the pharmaco-
logical studies was donated by the National Institute on Drug
Abuse [NIDA]. Room temperature is ca. 22 °C. TMSI is
trimethylsilyl iodide. Yields have not been optimized.
evaporation, and H2O (150 mL) was added. It was extracted
with EtOAc (300 mL × 3). The combined organic phase was
dried (Na2SO4), filtered, and evaporated to dryness. Distillation
(130 °C, 0.5 mmHg) furnished a white solid (22.0 g, 84%). H
NMR δ 7.88-7.78 (m, 3H), 7.72 (s, 1H), 7.53-7.39 (m, 3H),
3.80 (s, 2H), 3.71 (s, 3H).
1
LDA (0.054 mol, 1.2 equiv, 2.0 M in heptane) was introduced
into a 250 mL round-bottom flask and was cooled to 0 °C in
an ice/water bath. The 2-naphthylacetic acid methyl ester
prepared above (9.0 g, 0.045 mol) in anhydrous THF (20 mL)
was added dropwise to the LDA solution, and the mixture was
stirred at 0 °C for 2 h. It was then cooled to -78 °C, and
2-chlorotetrahydropyran (6.5 g, 0.054 mol) in THF (20 mL) was
added dropwise. Stirring was continued for 1 h at -78 °C. The
mixture was then allowed to warm to room temperature, and
stirring was continued overnight. THF was removed in vacuo,
and water (400 mL) was added. The mixture was extracted
with EtOAc (3 × 300 mL), and the combined organic phases
were dried (Na2SO4), filtered, and evaporated. The residue was
then dried in vacuo to provide an oil (10.0 g). The crude product
was purified by column chromatography (1% EtOAc in hex-
anes) to provide 5b (1.2 g) and 4b (2.8 g) in a total yield of
32%. 4b: 1H NMR δ 7.70-7.90 (m, 4H), 7.40-7.60 (m, 3H),
3.95-4.20 (m, 2H), 3.69 (s, 3H), 3.45-3.80 (m, 2H), 1.10-1.80
(m, 6H). Anal. (C18H20O3) C, H. 5b: 1H NMR δ 7.75-7.90 (m,
4H), 7.50-7.60 (m, 1H), 7.35-7.50 (m, 2H), 3.25-4.15 (m, 4H),
3.66 (s, 3H), 1.30-2.00 (m, 6H). Anal. (C18H20O3‚0.1H2O)
C, H.
2-Ch lor otetr a h yd r op yr a n . This compound was prepared
by a modification of the method of Ficini.22 Dry HCl gas was
bubbled for a period of 2 h through a solution of 3,4-dihydro-
2H-pyran (34.1 g, 0.41 mol) in anhydrous ether (150 mL), and
the mixture was cooled in a dry ice/acetone bath. The solution
was sparged with N2 to remove free HCl. Ether was removed
by evaporation. Fractional distillation of the residue under
reduced pressure (bp 36-39 °C, 18 mmHg) furnished a
colorless oil (36.7 g; 75%): 1H NMR δ 6.27 (t, J ) 0.54 Hz,
1H), 3.9-4.1 (m, 1H), 3.7-3.8 (m, 1H), 1.9-2.2 (m, 3H), 1.4-
1.8 (m, 3H). This compound is not stable and is best used
immediately.
(()-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)a ce-
tic Acid (6). The racemic methyl ester 4c (8.5 g, 28 mmol)
obtained from column chromatography was dissolved in an-
hydrous CHCl3 (10 mL) at 22 °C. TMSI (14 g, 70 mmol, 2.5
equiv) was added dropwise with stirring. The solution was
heated to 80 °C and stirred at that temperature overnight. It
was cooled to 22 °C, and volatiles were removed in vacuo.
Aqueous Na2SO3 (10 mL, 1%) and Et2O (25 mL) were added
to the residue. The Et2O phase was concentrated and purified
by gradient column chromatography (30-50% EtOAc in hex-
ane). The racemates 6 were obtained (5.2 g. 65% yield). Mp
139.1-140.1 °C. 1H NMR δ 7.45 (d, 1H), 7.40 (d, 1H), 7.18 (dd,
1H), 4.05 (m, 1H), 3.83 (m, 1H), 3.51 (d, 1H), 3.49 (m, 1H),
1.9-1.1 (m, 6H). 13C NMR δ 177.8, 134.8, 132.9, 132.2, 130.6,
128.2, 78.9, 68.9, 57.2, 29.1, 25.5, 22.9.
(()-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)a ce-
tic Acid Meth yl Ester s (4c a n d 5c). n-Butyllithium (20.8
mL, 52 mmol, 2.5 M in hexane) was added dropwise to a
solution of diisopropylamine (4.8 g, 48 mmol) in anhydrous
THF (100 mL) at 0 °C. The yellow solution was stirred at 0 °C
for 1.5 h. Methyl 3,4-dichlorophenylacetate (9.6 g, 44 mmol)
in THF (20 mL) was added dropwise over 30 min. After the
addition, the black solution was stirred for 2 h at 0 °C and
then cooled to -78 °C and stirred for a further 20 min. A
solution of 2-chlorotetrahydropyran (5.3 g, 44 mmol) in THF
(40 mL) was then added dropwise over 1 h. The mixture was
warmed slowly to 22 °C and stirred overnight. Cold (0 °C),
aqueous HCl (104 mL, 0.5 N) was added, followed by EtOAc
(400 mL). The organic layer was washed with brine and dried
over anhydrous Na2SO4. TLC showed two major spots (20%
EtOAc in hexane; Rf ) 0.59, 0.50) in a ratio of 1:1.5 (1H NMR).
The crude product was purified by gradient column chroma-
tography (5-15% EtOAc in hexane). Compounds 5c eluted first
(1.6 g, oil), followed by 4c (3.4 g, solid, mp 65 °C) (total yield,
75%). 5c: 1H NMR δ 7.47 (d, 1H), 7.38 (d, 1H), 7.21 (dd, 1H),
3.93-3.80 (m, 2H), 3.67 (s, 3H), 3.55 (d, J ) 11.5 Hz, 1H),
3.40-3.26 (m, 1H), 1.90-1.20 (m, 6H). 13C NMR δ 171.7, 136.6,
132.4, 131.6, 130.9, 130.3, 128.5, 78.2, 68.95, 56.9, 52.3, 29.9,
25.7, 23.2. Anal. (C14H16O3Cl2) C, H, Cl. For 4c: 1H NMR δ
7.47 (d, 1H), 7.38 (d, 1H), 7.19 (dd, 1H), 3.99 (m, 1H), 3.91 (m,
1H), 3.71 (s, 3H), 3.50 (d, 1H), 3.47 (m, 1H) 1.9-1.0 (m, 6H).
13C NMR δ 172.7, 135. 5, 132.8, 132.1, 130.6, 128.2, 79.2, 68.9,
57.4, 52.4, 29.2, 25.7, 23.1. Anal. (C14H16O3Cl2) C, H, Cl.
(()-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)a ce-
tic Acid (7). The combined racemates of 7 were obtained from
5c as described above for 6 (36%). Mp 124.1-125.1 °C. 1H NMR
δ 7.48 (d, 1H), 7.38 (d, 1H), 7.20 (dd, 1H), 4.0-3.8 (m, 2H),
3.60 (d, J ) 7.41 Hz, 1H), 3.39 (m, 1H), 1.9-1.2 (m, 6H). 13C
NMR δ 176.7, 135.5, 132.4, 131.8, 131.2, 130.2, 128.7, 77.8,
68.9, 56.6, 29.6, 25.5, 23.0.
(3,4-Dich lor oph en yl)(tetr ah ydr opyr an -2-yl)acetic Acid
Men th yl Ester s (8 a n d 9). The racemic mixture of (3,4-
dichlorophenyl)(tetrahydropyran-2-yl)acetic acid 6 (0.86 g, 3.0
mmol) was dissolved in anhydrous CH2Cl2 (80 mL). Three
drops of DMF were added, followed by the dropwise addition
of oxalyl chloride (0.58 g, 4.8 mmol, 1.6 equiv) at 22 °C. After
3 h, volatiles were removed and anhydrous THF (75 mL) was
introduced, followed by pyridine (0.5 g). l-Menthol (0.47 g, 3.0
mmol) in THF (5 mL) was added dropwise. The reaction
mixture was stirred overnight and then poured into water (100
mL). Et2O (150 mL) was added. The layers were separated,
and the aqueous phase was extracted with Et2O. The combined
organic phases were washed with brine and dried over Na2-
SO4. TLC showed two major components (Rf ) 0.54 and 0.50,
10% EtOAc in hexane). After column chromatography (hexane
800 mL, 1% EtOAc in hexane 800 mL, and finally 3% EtOAc
in hexane 800 mL), 400 mg of the first product 9 and 425 mg
of the second product 8 were obtained (65% yield). 8: 1H NMR
δ 7.46 (d, 1H), 7.38 (d, 1H), 7.20 (dd, 1H), 4.69 (td, J ) 4.4,
10.9 Hz, 1H), 3.94 (dt, J ) 2.2, 11.2 Hz, 1H), 3.83 (td, J ) 2.2,
10.2 Hz, 1H), 3.44 (td, J ) 3.0, 12.1 Hz, 1H), 3.45 (d, J ) 9.9
Hz, 1H), 2.0-0.9 (m, 15H), 0.88 (d, J ) 6.7 Hz, 3H), 0.78 (d,
J ) 6.9 Hz, 3H), 0.61 (d, J ) 6.9 Hz, 3H). 13C NMR δ 171.2,
(()-(P h en yl)(tetr a h yd r op yr a n -2-yl)a cetic Acid Meth yl
Ester (4a a n d 5a ). The procedure described above was
followed to obtain 4a and 5a . 4a : 1H NMR δ 7.20-7.40 (m,
5H), 3.97-4.06 (m, 1H), 3.92 (td, J ) 10.4, 3.45 Hz, 1H), 3.68
(s, 3H), 3.54 (d, J ) 10.1 Hz, 1H), 3.52 (td, J ) 11.5, 3.0 Hz,
1H), 1.0-1.8 (m, 6H). Anal. (C14H18O3) C, H. 5a : 1H NMR δ
7.20-7.50 (m, 5H), 3.84-3.95 (m, 2H), 3.63 (s, 3H), 3.65 (t,
J ) 8.3 Hz, 1H), 3.25-3.40 (m, 1H), 1.30-2.00 (m, 6H). Anal.
(C14H18O3) C, H.
(()-(2-Na p h t h yl)(t et r a h yd r op yr a n -2-yl)a cet ic Acid
Meth yl Ester s (4b a n d 5b). 2-Naphthylacetic acid (24.5 g,
0.132 mol) was dissolved in methanol (180 mL), and concen-
trated H2SO4 (2 mL) was added. The mixture was warmed to
45 °C and stirred for 18 h. It was then cooled to 22 °C and
neutralized with NaHCO3 to pH 7. Methanol was removed by

Methylphenidate Analogue Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1543
135.8, 132.6, 131.7, 130.5, 130.4, 128.1, 78.9, 75.0, 68.5, 57.3,
47.2, 40.7, 34.2, 31.4, 29.0, 25.8, 25.6, 23.1, 22.0, 20.7, 15.9. 9:
1H NMR δ 7.50 (d, 1H), 7.38 (d, 1H), 7.22 (dd, 1H), 4.70 (td,
J ) 4.4, 11.0 Hz, 1H), 3.94 (dt, J ) 2.2, 11.3 Hz, 1H), 3.79 (td,
J ) 2.2, 10.4 Hz, 1H), 3.45 (td, J ) 2.7, 9.1 Hz, 1H), 3.46 (d,
J ) 9.8 Hz, 1H), 2.0-0.9 (m, 15H), 0.88 (d, J ) 7.7 Hz, 3H),
0.86 (d, J ) 6.6 Hz, 3H), 0.72 (d, J ) 6.9 Hz, 3H). 13C NMR δ
171.6, 135.7, 132.6, 131.7, 130.5, 130.4, 128.2, 79.4, 74.9, 68.5,
57.8, 47.0, 40.5, 34.2, 31.4, 28.9, 25.7, 25.6, 23.0, 22.0, 20.9,
15.9.
(2S,2′R)-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)-
a cetic Acid ((2S,2′R)-7′). 1H NMR δ 7.47 (d, 1H), 7.40 (d, 1H),
7.21 (dd, 1H), 3.97 (dd, J ) 10.8, 2.2 Hz, 1H), 3.92-3.83 (m,
1H), 3.60 (d, J ) 7.1 Hz, 1H), 3.45-3.36 (m, 1H), 1.9-1.2 (m,
6H). 13C NMR δ 176.7, 135.5, 132.4, 131.8, 131.2, 130.2, 128.7,
77.8, 68.9, 56.6, 29.6, 25.5, 23.0. Mp 124.2-125.2 °C. [R]20
D
14.0° (c 1.0, CHCl3).
(2R,2′S)-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)-
a cetic Acid ((2R,2′S)-7′). NMR data are identical to acid
(2S,2′R)-7′. Mp 124.1-125.1 °C. [R]20 -13.9° (c 1.0, CHCl3).
D
2-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)a cet ic
Acid In d a n yl Ester s (10 a n d 11). The racemic mixture of
2-(3,4-dichlorophenyl)(tetrahydropyran-2-yl)acetic acid 7 (4.0
g, 13.8 mmol) was dissolved in anhydrous CH2Cl2 (80 mL), and
4 drops of DMF were added. Oxalyl chloride (3.5 g, 27.7 mmol,
2 equiv) was added dropwise while the solution was vigorously
stirred. Evolution of bubbles was observed. After completion
of addition, the light-yellow solution was stirred at room
temperature for a further 2.5 h.
Gen er a l P r oced u r e for th e Meth yla tion of Acid s To
P r ovid e Meth yl Ester s (12-15). The following procedure is
representative. (2S,2′R)-(3,4-Dichlorophenyl)(tetrahydropyran-
2-yl)acetic acid (2S,2′R)-7′ (90 mg, 0.31 mmol) was dissolved
in anhydrous toluene (4 mL) and anhydrous CH3OH (1 mL).
Trimethylsilyldiazomethane (0.63 mL, 2.0 M in hexane, 4
equiv) was slowly added at 22 °C, and the mixture was stirred
for 5 h. Volatiles were removed in vacuo. The residue (100 mg)
was purified by column chromatography (2% EtOAc in hexane)
to provide 14 as an oil (61 mg, 64%).
Solvent was removed by evaporation, and the residue was
dried in vacuo. (S)-(+)-1-Indanol (1.87 g, 13.9 mmol) was
dissolved in anhydrous THF (25 mL), and dry pyridine (25 mL)
was added. The solution was cooled to 0 °C. The acid chloride,
prepared as above, in THF (50 mL) was added dropwise. The
mixture was stirred at 0 °C for 2 h, and then it was warmed
(2S,2′S)-(+)-(3,4-Dich lor op h en yl)(tetr a h yd r op yr a n -2-
1
yl)a cetic Acid Meth yl Ester (12). H NMR δ 7.48 (d, 1H),
7.39 (d, 1H), 7.25 (dd, 1H), 4.03-3.95 (m, 1H), 3.84 (td, J )
10.7, 2.2 Hz, 1H), 3.70 (s, 3H), 3.50 (d, J ) 9.9 Hz, 1H), 3.47
(td, J ) 11.3, 3.3 Hz, 1H), 1.9-1.0 (m, 6H). [R]20 +29.8° (c
1
D
to room temperature and stirred overnight. H NMR showed
1.0, CHCl3). Anal. (C14H16Cl2O3) C, H, Cl.
that the ratio of 10 (Rf ) 0.71 in 10% EtOAc, 90% hexane,
developed 3 times) to 11 (Rf ) 0.67) was 4:5 based on
resonances at δ 3.53 and δ 3.54. The mixture was evaporated
to remove most of the solvent. The residue was taken up in a
mixture of hexane/ethyl acetate (10:5) (80 mL) to provide a
light-yellow suspension. The mixture was loaded on a short
silica gel column and chromatographed with hexane/ethyl
acetate (10:1). The product fractions were combined, evapo-
rated, and dried. A yellow oil was obtained (4.0 g, 71%). It was
further purified by column chromatography (300 g of silica gel,
0.4% of ethyl acetate, 99.6% of hexane, 4 L, then 0.8% ethyl
acetate in hexane, 5 L). A total of 0.6 g of 10, 1.0 g of a mixture
of 10 and 11, and 0.5 g of 11 were obtained. 10: 1H NMR δ
7.49 (d, J ) 2.2 Hz, 1H), 7.39-7.10 (m, 6H), 6.20 (m, 1H),
3.92-3.80 (m, 2H), 3.544 and 3.515 (d, J ) 8.8 Hz, 1H), 3.34-
3.25 (m, 1H), 3.1-3.0 (m, 1H), 2.9-2.8 (m, 1H), 2.5-2.4 (m,
1H), 2.0-1.1 (m, 6H). 13C NMR δ 171.07, 144.31, 140.56,
136.56, 132.19, 131.39, 130.90, 130.08, 129.04, 128.50, 126.73,
125.34, 124.85, 79.09, 78.16, 68.80, 57.08, 31.99, 30.13, 29.81.
11: 1H NMR δ 7.47 (d, J ) 2.2 Hz, 1H), 7.37 (d, J ) 8.3 Hz,
1H), 7.31-7.18 (m, 5H), 6.17 (m, 1H), 3.92-3.80 (m, 2H), 3.557
and 3.528 (d, J ) 8.5 Hz, 1H), 3.40-3.27 (m, 1H), 3.14-3.03
(m, 1H), 2.95-2.82 (m, 1H), 2.59-2.40 (m, 1H), 2.2-1.2 (m,
6H). 13C NMR δ 171.1, 144.3, 140.4, 136.5, 132.2, 131.4, 130.9,
130.1, 129.0, 128.5, 126.7, 125.3, 124.8, 79.2, 78.2, 68.8, 57.0,
32.2, 30.1, 29.8, 25.6, 23.1.
(2R,2′R)-(-)-(3,4-Dich lor op h en yl)(tetr a h yd r op yr a n -2-
yl)a cetic Acid Meth yl Ester (13). 1H NMR data are identical
to 12. [R]20 -29.1° (c 1.0, CHCl3). Anal. (C14H16Cl2O3) C, H,
D
Cl.
(2S,2′R)-(-)-(3,4-Dich lor op h en yl)(tetr a h yd r op yr a n -2-
1
yl)a cetic Acid Meth yl Ester (14). H NMR δ 7.47 (d, 1H),
7.39 (d, 1H), 7.22 (dd, 1H), 3.92-3.81 (m, 1H), 3.68 (s, 3H),
3.56 (d, J ) 8.5 Hz, 1H), 3.39-3.20 (m, 1H), 1.9-1.1 (m, 6H).
[R]20 -1.8° (c 1.0, CHCl3). Anal. (C14H16Cl2O3) C, H, Cl.
D
(2R,2′S)-(+)-(3,4-Dich lor op h en yl)(tetr a h yd r op yr a n -2-
1
yl)a cetic Acid Meth yl Ester (15). H NMR identical to 14.
[R]20 +2.0° (c 1.0, CHCl3). Anal. (C14H16Cl2O3) C, H, Cl.
D
(()-2,2-(3,4-Dich lor oph en yl)cycloalkylacetic Acid Meth -
yl Ester s (17 a n d 19). The following procedure is representa-
tive. To a stirred solution of t-BuOK (11.0 mL, 1.0 M in THF),
a solution of 3,4-dichlorophenylacetonitrile (1.86 g, 10.0 mmol)
in THF (20 mL) was slowly added. The mixture was stirred
for 0.5 h, and cyclopentyl bromide (1.57 g, 10.5 mmol) in THF
(10 mL) was added. The dark-brown solution was stirred at
22 °C for 1 h and then heated to reflux overnight. After cooling,
the solution was transferred to a separatory funnel and EtOAc
(200 mL) and water (150 mL) were added. The organic phase
was separated and washed consecutively with H2O and brine,
dried (Na2SO4), concentrated, and purified by column chro-
matography (1-2% EtOAc in hexane) to yield 2,2-(3,4-dichlo-
rophenyl)cyclopentylacetonitrile 18 as an oil (1.78 g, 70%). 1H
NMR δ 7.45 (d, 1H), 7.43 (d, 1H), 7.18 (dd, 1H), 3.69 (d, J )
7.7 Hz, 1H), 2.4-1.2 (m, 9H). Anal. (C13H13Cl2N) C, H, N, Cl.
The nitrile 18 (1.2 g, 4.7 mmol) was dissolved in HCl/
methanol solution (65 mL, 10.3 M), sealed with a stopper, and
stirred at 22 °C for 2 days. A 6 N hydrochloric acid (30 mL,
exothermic!) solution was slowly added to the mixture, which
was stirred for 10 min and evaporated to dryness. A further
50 mL of HCl/methanol solution (10.3 M) was added, and
stirring continued for 4 days. An additional 30 mL of 6 N
hydrochloric acid was added, and the mixture was brought to
reflux for 2 days. After the mixture was cooled, EtOAc (200
mL) and water (150 mL) were added. The organic phase was
washed with water followed by brine, dried (Na2SO4), and
concentrated. The oil obtained (Rf ) 0.45, 10% EtOAc in
hexane,) was purified by column chromatography (0.5-1%
EtOAc in hexane) to provide 19 as a colorless oil (1.1 g, 82%).
1H NMR δ 7.45 (d, 1H), 7.35 (d, 1H), 7.19 (dd, 1H), 3.66 (s,
3H), 3.23 (d, J ) 11.3 Hz, 1H), 2.6-2.4, m, 1H), 2.0-1.8 (m,
1H), 1.8-0.8 (m, 7H). Anal. (C14H16O2Cl2) C, H, Cl.
Gen er a l P r oced u r e for th e Hyd r olysis of Men th yl a n d
In d a n yl Ester s (8-11) to th e Cor r esp on d in g Acid s. The
following experiment is representative (yields 38-55%). In-
danyl ester 10 (1.65 g, 4.07 mmol) was dissolved in anhydrous
CCl4 (25 mL). Trimethylsilyl iodide (2.4 g, 12 mmol, 3 equiv)
was added. The mixture was heated to 90 °C and stirred for
18 h, then cooled to 0 °C. Cold water (20 mL) and CH2Cl2 (50
mL) were added, and the layers were separated. The aqueous
phase was washed with CH2Cl2 (50 mL × 2). The combined
organic phases were dried (Na2SO4) and filtered. The filtrate
was evaporated to dryness. The residue was purified by column
chromatography (10% CH3OH in CH2Cl2) to obtain (2S,2′R)-
7′ (600 mg, 55%).
(2S,2′S)-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)-
a cetic Acid ((2S,2′S)-6′). 1H NMR δ 7.45 (d, 1H), 7.40 (d, 1H),
7.18 (dd, 1H), 4.06 (dt, J ) 11.2, 1.9 Hz, 1H), 3.9-3.7 (m, 1H),
3.52 (d, J ) 9.3 Hz, 1H), 3.50 (td, J ) 11.2, 3.3 Hz, 1H), 1.9-
1.1 (m, 6H). Mp 138.9-139.9 °C. [R]20 18.8° (c 1.0, CHCl3).
D
(2R,2′R)-(3,4-Dich lor op h en yl)(tetr a h yd r op yr a n -2-yl)-
a cetic Acid ((2R,2′R)-6′). NMR data are identical to those of
(2S,2′S)-6′. Mp 139.1-140.1 °C. [R]20 -19.1° (c 1, CHCl3)
D

1544 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
2,2-(3,4-Dich lor oph en yl)cyclh exylaceton itr ile (16). Yield,
Meltzer et al.
mL for [3H]WIN 35,428 or [3H]citalopram assay in buffer just
before assay and was dispersed with a Brinkmann Polytron
homogenizer (setting no. 5) for 15 s. All experiments were
conducted in triplicate, and each experiment was repeated in
each of two to three preparations from individual brains.
1
46%. H NMR δ 7.45 (d, 1H), 7.39 (d, 1H), 7.42 (dd, 1H), 3.60
(d, J ) 6.3 Hz, 1H), 1.9-1.1 (m, 11H). Anal. (C14H15Cl2N) C,
H, N, Cl.
2,2-(3,4-Dich lor op h en yl)cycloh exyla cetic Acid Meth yl
1
Ester (17). Yield, 40%. H NMR δ 7.43 (d, 1H), 7.38 (d, 1H),
Dop a m in e Tr a n sp or ter Assa y. The dopamine transporter
was labeled with [3H]WIN 35,428 ([3H]CFT, (1R)-2â-car-
bomethoxy-3â-(4-fluorophenyl)-N-[3H]methyltropane, 81-84
Ci/mmol, DuPont-NEN). The affinity of [3H]WIN 35,428 for
the dopamine transporter was determined in experiments by
incubating tissue with a fixed concentration of [3H]WIN 35,428
and a range of concentrations of unlabeled WIN 35,428. The
assay tubes received, in Tris-HCl buffer (50 mM, pH 7.4 at
0-4 °C, NaCl 100 mM), the following constituents at a final
assay concentration: WIN 35,428, 0.2 mL (1 pM to 100 or 300
nM), [3H]WIN 35,428 (0.3 nM); membrane preparation 0.2 mL
(4 mg of original wet weight of tissue/mL). The 2 h incubation
(0-4 °C) was initiated by addition of membranes and termi-
nated by rapid filtration over Whatman GF/B glass fiber filters
presoaked in 0.1% bovine serum albumin (Sigma Chemical
Co.). The filters were washed twice with 5 mL of Tris-HCl
buffer (50 mM) and were incubated overnight at 0-4 °C in
scintillation fluor (Beckman Ready-Value, 5 mL). Radioactivity
was measured by liquid scintillation spectrometry (Beckman
1801). The cpm data were converted to dpm following deter-
mination of the counting efficiency (>45%) of each vial by
external standardization.
Total binding was defined as [3H]WIN 35,428 bound in the
presence of ineffective concentrations of unlabeled WIN 35,428
(1 or 10 pM). Nonspecific binding was defined as [3H]WIN
35,428 bound in the presence of an excess (30 µM) of (-)-
cocaine. Specific binding was the difference between the two
values. Competition experiments to determine the affinities
of other drugs at [3H]WIN 35,428 binding sites were conducted
using procedures similar to those outlined above. Stock solu-
tions of water-soluble drugs were dissolved in water or buffer.
Stock solutions of other drugs were made as follows. Oils were
drawn up in preweighed capillary tubes and dissolved in
ethanol. Stock solutions of relatively insoluble drugs were
made in >95% ethanol or an ethanol/HCl combination. Several
of the drugs were sonicated to promote solubility. The stock
solutions were diluted serially in the assay buffer and added
(0.2 mL) to the assay medium as described above. IC50 values
were computed by the EBDA computer program and are the
mean values of experiments conducted in triplicate.
Ser oton in Tr a n sp or ter Assa y. The serotonin transporter
was assayed in caudate-putamen membranes using conditions
similar to those for the dopamine transporter. The affinity of
[3H]citalopram (specific activity of 82 Ci/mmol, DuPont-NEN)
for the serotonin transporter was determined in experiments
by incubating tissue with a fixed concentration of [3H]citalo-
pram and a range of concentrations of unlabeled citalopram.
The assay tubes received, in Tris-HCl buffer (50 mM, pH 7.4,
at 0-4 °C, NaCl 100 mM), the following constituents at a final
assay concentration: citalopram, 0.2 mL (1 pM to 100 or 300
nM), [3H]citalopram (1 nM); membrane preparation 0.2 mL
(4 mg of original wet weight of tissue/mL). The 2 h incubation
(0-4 °C) was initiated by addition of membranes and termi-
nated by rapid filtration over Whatman GF/B glass fiber filters
presoaked in 0.1% polyethyleneimine. The filters were washed
twice with 5 mL of Tris-HCl buffer (50 mM) and incubated
overnight at 0-4 °C in scintillation fluor (Beckman Ready-
Value, 5 mL). Radioactivity was measured by liquid scintil-
lation spectrometry (Beckman 1801). The cpm data were
converted to dpm following determination of the counting
efficiency (>45%) of each vial by external standardization.
Total binding was defined as [3H]citalopram bound in the
presence of ineffective concentrations of unlabeled citalopram
(1 or 10 pM). Nonspecific binding was defined as [3H]citalo-
pram bound in the presence of an excess (10 µM) of fluoxetine.
Specific binding was the difference between the two values.
Competition experiments to determine the affinities of other
drugs at [3H]citalopram binding sites were conducted using
procedures similar to those outlined above. IC50 values were
7.28 (dd, 1H), 3.66 (s, 3H), 3.19 (d, J ) 10.7 Hz, 1H), 2.1-0.7
(m, 11H). Anal. (C14H18Cl2O2) C, H, Cl.
(2R,2′S)-(3,4-Dich lor op h en yl)(t et r a h yd r op yr a n -2-yl)-
a cetic Acid 4-Nitr op h en yl Ester [(4-Nitr op h en yl Ester
of (2R,2′S)-7]. Compound (2R,2′S)-7 obtained from 15 (100
mg, 0.33 mmol) was dissolved in anhydrous CH2Cl2 (5 mL).
One drop of DMF was added. Oxalyl chloride (0.14 g) was
slowly added, and the reaction mixture was stirred at room
temperature for 3 h. Solvent was then removed by evaporation.
4-Nitrophenol (56 mg, 0.40 mmol) was dissolved in anhy-
drous THF (3 mL), and pyridine (85 mg) was added. The
mixture was cooled to 0 °C in an ice/water bath. To this cooled,
stirred solution was added the acid chloride prepared above
in 2 mL of THF over 10 min. The mixture was warmed to room
temperature and stirred overnight. The crude product was
purified by column chromatography (0.6% EtOAc in hexane).
The product (0.105 g) was obtained as a gummy material
(75%). Rf ) 0.46 (20% ethyl acetate in hexane). 1H NMR δ 8.3-
8.2 (m, 2H), 7.55 (d, J ) 2.2 Hz, 1H), 7.45 (d, J ) 8.3 Hz, 1H),
7.32-7.18 (m, 3H), 4.07-3.91 (m, 2H), 3.83 (d, J ) 7.4 Hz,
1H), 3.46-3.37 (m, 1H), 2.0-1.2 (m, 6H). Crystals were
obtained from pentane. X-ray structural analysis showed the
enantiomerically pure p-nitrophenyl ester to be (2R,2′S)-7.
Sin gle-Cr ysta l X-r a y An a lysis of (2S,2′S)-(3,4-Dich lo-
r op h en yl)(tetr a h yd r op yr a n -2-yl)a cetic Acid (6). Mono-
clinic crystals of (2S,2′S)-6, obtained from (+)-12, were ob-
tained from pentane. A representative crystal was selected,
and a data set was collected at room temperature. Pertinent
crystal, data collection, and refinement parameters are the
following: crystal size, 0.56 mm × 0.40 mm × 0.22 mm; cell
dimensions, a ) 11.988(1) Å, b ) 8.222(1) Å, c ) 14.539(1) Å,
R ) 90°, â ) 104.35(1)°, γ ) 90°; formula, C13H14Cl2O3; formula
weight ) 289.14; volume ) 1388.4(2) Å3; calculated density )
1383 Mg/m-3; space group ) P2(1); number of reflections )
2231, of which 2048 were considered independent (Rint
)
0.0185). Refinement method was full-matrix least-squares on
F2. The final R indices were [I > 2σ(I)] R1 ) 0.0465 and
wR2 ) 0.1263.
Sin gle-Cr ysta l X-r a y An a lysis of (2R,2′S)-(3,4-d ich lo-
r op h en yl)(tetr a h yd r op yr a n -2-yl)a cetic Acid (7) 4-Nitr o-
p h en yl Ester . Orthorhombic crystals of the purified p-nitro-
phenyl ester of 7 were obtained from 90% hexane/10% EtOAc.
A representative crystal was selected, and a data set was
collected at room temperature. Pertinent crystal, data collec-
tion, and refinement parameters are the following: crystal
size, 0.49 mm × 0.08 mm × 0.06 mm; cell dimensions, a )
6.844 (1) Å, b ) 11.516(2) Å, c ) 23.922(6) Å, R ) 90°, â ) 90°,
γ ) 90°; formula, C19H17Cl2NO5; formula weight ) 410.24;
volume ) 1885.3(7) Å3; calculated density ) 1.445 Mg/m-3
;
space group ) P212121; number of reflections ) 1580, of which
1529 were considered independent (Rint ) 0.0215). Refinement
method was full-matrix least-squares on F2. The final R indices
were [I > 2σ(I)] R1 ) 0.0504 and wR2 ) 0.1190.
Tissu e Sou r ces a n d P r ep a r a tion . Brain tissue from adult
male and female cynomolgus monkeys (Macaca fascicularis)
and rhesus monkeys (Macaca mulatta) was stored at -85 °C
in the primate brain bank at the New England Regional
Primate Research Center. We recently cloned the DAT and
SERT from both species and found them to have virtually
identical protein sequences. The caudate-putamen was dis-
sected from coronal slices and yielded 1.4 ( 0.4 g of tissue.
Membranes were prepared as described previously. Briefly,
the caudate-putamen was homogenized in 10 volumes (w/v)
of ice-cold Tris-HCl buffer (50 mM, pH 7.4, at 4 °C) and
centrifuged at 38000g for 20 min in the cold. The resulting
pellet was suspended in 40 volumes of buffer, and the entire
procedure was repeated twice. The membrane suspension (25
mg of original wet weight of tissue/mL) was diluted to 12 mL/

Methylphenidate Analogue Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1545
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computed by the EBDA computer program and are the mean
values of experiments conducted in triplicate.
Ack n ow led gm en t. This work was supported by the
National Institute on Drug Abuse ((P.M.) Grant DA7-
8081; Grant DA11542; (B.K.M.) Grants DA06303,
DA11558, DA00304, RR00168). We thank Clifford George
and J udith Flippen-Anderson of the Office of Naval
Research for the X-ray structural analyses. The authors
thank the reviewers of a prior version of this manuscript
for their suggestion that evaluation of the unsubstituted
phenyl oxacyclic analogues may provide insights into
the SAR of this novel series.
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Su p p or tin g In for m a tion Ava ila ble: ORTEP drawings
of (2S,2′S)-(3,4-dichlorophenyl)(tetrahydropyran-2-yl)acetic acid
(6) and (2R,2′S)-(3,4-dichlorophenyl)(tetrahydropyran-2-yl)-
acetic acid (7) 4-nitrophenyl ester, crystal data and refinement
parameters, coordinates, anisotropic temperature factors,
distances, and angles This material is available free of charge
via the Internet at http://pubs.acs.org.
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