Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling

Article abstract of DOI:10.1016/j.bmc.2010.11.002

In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the pipe

Full text of DOI:10.1016/j.bmc.2010.11.002

Bioorganic & Medicinal Chemistry 19 (2011) 504–512
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin,
Concerta): Rational design, synthesis, pharmacological evaluation,
and dopamine transporter photoaffinity labeling
David J. Lapinsky a, , Ranganadh Velagaleti a, Nageswari Yarravarapu a, Yi Liu a, Yurong Huang a,
⇑
Christopher K. Surratt a, John R. Lever b,c, James D. Foster d, Rejwi Acharya d, Roxanne A. Vaughan d,
Howard M. Deutsch e
a Division of Pharmaceutical Sciences, Duquesne University Mylan School of Pharmacy, 600 Forbes Avenue, Pittsburgh, PA 15282, USA
b Departments of Radiology, and Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65212, USA
c Harry S. Truman Veterans Administration Medical Center, 800 Hospital Drive, Columbia, MO 65201, USA
d Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA
e School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photo-
affinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this
knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphen-
idate was substituted with a benzyl group bearing a photoreactive azide. Analog ( )-3a demonstrated
modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and
Received 24 August 2010
Revised 25 October 2010
Accepted 1 November 2010
Available online 4 November 2010
hDAT expressed in cultured cells. Co-incubation of ( )-3a with nonradioactive D-(+)-methylphenidate
Keywords:
Methylphenidate
Ritalin
or (ꢀ)-2-b-carbomethoxy-3-b-(4-fluorophenyl)tropane (b-CFT, WIN-35,428, a cocaine analog) blocked
DAT labeling. Compound ( )-3a represents the first successful example of a DAT photoaffinity ligand
based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane
ligand-binding pockets within plasma membrane monoamine transporters.
Concerta
Photoaffinity labeling
Dopamine transporter
Cocaine
Ó 2010 Elsevier Ltd. All rights reserved.
Attention-deficit hyperactivity disorder
1. Introduction
norepinephrine into the presynaptic neuron.3 However, in sharp
contrast to the beneficial therapeutic effects associated with meth-
Threo-methylphenidate (( )-1a, Scheme 1) is a well-known
stimulant acting through dopaminergic and adrenergic pathways.
Both immediate-release (Ritalin) and long-acting (Concerta) prep-
arations of methylphenidate remain the mainstay of treatment for
adult attention-deficit hyperactivity disorder (ADHD), showing
more than 75% efficacy in controlling the symptoms of the dis-
ease.1 In addition to being prescribed for narcolepsy and a number
of off-label uses including lethargy, depression, and obesity, meth-
ylphenidate analogs continue to attract significant attention
towards the development of cocaine abuse therapeutics.2
In contrast to amphetamines, which cause a direct release of
norepinephrine and dopamine into the synapse, methylphenidate
acts as a mild central nervous system stimulant by inhibiting the
dopamine and norepinephrine transporter proteins (DAT and
NET, respectively), thus blocking the reuptake of dopamine and
ylphenidate, behavioral and pharmacological studies have also
implicated the DAT as the primary target associated with the
reward/reinforcing properties of cocaine and amphetamines as
abused psychostimulants.4,5 As a result, the long-term goal of our
research is to understand how the DAT discriminates abused ver-
sus therapeutic compounds at the molecular level.6
The lack of clinically available medications to battle psycho-
stimulant abuse can be linked, in part, to limited information on
the 3-D structure and function of the DAT. Indeed, the search for
therapeutics has resulted in a host of structurally disparate ligands
capable of selectively binding to the DAT and inhibiting dopamine
uptake;7 however, details regarding discrete ligand-binding pock-
ets and the transport inhibition mechanism remain poorly under-
stood. Furthermore, inhibitor structure–activity relationships
(SAR) and site-directed mutagenesis studies imply that structurally
divergent DAT inhibitors bind to different domains/binding sites
within the DAT, or with differential conformational preferences,
both of which could affect their behavioral profile in cocaine abuse
animal models.8–10 Radiolabeled (3H, 125I) molecular probes serv-
⇑
Corresponding author. Tel.: +1 412 396 6069; fax: +1 412 396 5593.
E-mail address: lapinskyd@duq.edu (D.J. Lapinsky).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.002

D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
505
R
N3
X
X
I
molecular
HN
O
N
O
N
O
p
p
+
4
4
4
hybridization
3
o
o
3
3
m
m
OMe
OMe
OMe
(+)-1a, X = -H
(+)-2a, X = R = -H
(+)-3a, p-N3-4-I
(+)-3b, m-N3-4-I
(+)-3c, o-N3-4-I
(+)-3d, p-N3-3-I
(+)-3e, m-N3-3-I
(+)-3f, o-N3-3-I
(+)-1b, X = 4-I
(+)-1c, X = 3-F
(+)-1d, X = 3-Cl
(+)-1e, X = 3-Br
(+)-1f, X = 3-I
(+)-2b, X = 3-Cl, R = -H
(+)-2c, X = 3,4-diCl, R = -H
(+)-2d, X = -H, R = p-Cl
(+)-2e, X = -H, R = p-OMe
(+)-2f, X = -H, R = m-Cl
(+)-2g, X = -H, R = p-NO2
Scheme 1. Rational design of ( )-threo-N-(azido-benzyl)-iodomethylphenidates (3) via molecular hybridization of halogenated methylphenidates (1b, 1f) with N-
benzylmethylphenidates (2).
ing as photoaffinity (–N3) and affinity (–NCS) ligands represent
important tools towards determining DAT conformational states
and mapping of inhibitor/substrate-binding sites.
radiotracer tag (i.e., 3-iodomethylphenidate (( )-1f)). Given these
established structure–activity relationships for methylphenidate
analogs, a series of photoaffinity ligands (( )-3) was designed via
molecular hybridization of iodomethylphenidates (( )-1b, ( )-1f)
with N-benzylmethylphenidates (( )-2). These target probes feature
systematic placement of a photoreactive azide on the aromatic ring
of the N-benzyl group (Scheme 1).
The chemical development of DAT affinity and photoaffinity li-
gands has focused on cocaine and benztropine analogs as tropane-
based ligands11–19 or their conformationally flexible piperidine20
and piperazine21–26 analogs. In contrast and due to having received
significantly less attention,27 the work herein focused on develop-
ing DAT photoaffinity ligands based on therapeutically relevant
non-tropane compounds.28 These ligands expand the battery of
complementary chemical probes that may be useful for mapping
DAT inhibitor-binding pockets at the molecular level. With the
crystal structure of the bacterial homolog LeuT serving as a tem-
plate for homology modeling of DAT 3-D structure, such high res-
olution ligand-binding mapping is finally in sight.29 Herein we
report our initial studies with respect to rational design, synthesis,
pharmacological evaluation, and photoaffinity labeling of methyl-
phenidate-based DAT irreversible ligands.
Thepreparationof 4-iodomethylphenidate(( )-1b)hasbeenpre-
viously described;33 however, this synthesis affords a poor yield (six
steps, 9% overall yield) and originates from methylphenidate itself, a
relatively expensive starting material. Towards synthesizing a vari-
ety of probes based on either ( )-1b or ( )-1f, we first demanded
short syntheses of these iodomethylphenidates involving cheap,
readily available starting materials. The application of methodology
originally developed by Axten et al.,34 and later modified by Gutman
et al.,35 met our needs (Scheme 2). Ketoamide 4a was prepared from
its corresponding ethyl ester derivative36 by treatment with piperi-
dine in ethanol,34 whereas ketoamide 4b was accessed via regiose-
lective meta-iodination of 1-(phenylglyoxylyl)piperidine35 using
N-iodosuccinimide. Tosylhydrazones 5a and 5b were subsequently
generated by allowing the ketoamides to react with p-toluenesulfo-
nyl hydrazide in acidic ethanol under reflux. Next, thermal cycliza-
tion of the tosylhydrazones in refluxing toluene was performed
under basic aqueous conditions using Aliquat 336 as a phase transfer
catalyst.35 Subsequent recrystallization from ether provided diaste-
reomerically pure racemic threo-b-lactams ( )-6 as the major prod-
uct in moderate yield (55–60%). In turn, these threo-b-lactams were
converted to iodomethylphenidates ( )-1b and ( )-1f by ring open-
ing with methanol under acidic conditions. These threo-iodomethyl-
phenidates were determined to be P95% diastereomerically pure by
1H NMR upon comparison to the known data for enantiomerically
pure threo- and erythro-methylphenidate and its para-substituted
derivatives.37 Finally, target photoaffinity probes ( )-3 were ac-
cessed from the iodomethylphenidates by either N-alkylation with
anazidobenzylbromide38 (e.g., ( )-3a), orasequenceofN-alkylation
with a nitrobenzyl bromide, nitro reduction, and diazotization fol-
lowed by azide displacement (e.g., ( )-3e).
2. Results and discussion
2.1. Probe design and synthesis
With respect to photoaffinity probe design based on DAT
inhibitors, the overwhelming number of known compounds in this
group contains an aromatic 4-azido-3-iodo-substituted ring
motif.12–17,19–21,24,25,28 However, this motif may inherently lead
to low efficiency (e.g., <1%) of incorporation of the aryl azide into
the protein due to its juxtaposition with the sterically bulky
iodine.30 Towards overcoming this potential problem, we decided
to focus on the design of compounds in which the photoreactive
azide group and 125I radiotracer tag are on different parts of the
methylphenidate scaffold.
We chose 4-iodomethylphenidate (( )-1b, Scheme 1) as a lead
compound given that this analog displays ꢁ6-fold higher DAT affin-
ity versus methylphenidate (DAT IC50 for inhibition of [3H]-WIN-
35,428 (a cocaine analog) binding, ( )-1a = 83.0 7.9 nM, ( )-
1b = 14.0 0.1 nM).31 We envisioned the 4-position of methylphe-
nidate’s aromatic ring as a logical place to anchor a bulky iodine as
a future radiotracer tag within rationally designed photoaffinity
probes. Additionally, we considered N-benzylmethylphenidate ana-
logs (( )-2) as lead compounds since they display either improved
(( )-2a = 52.9 2.3 nM, ( )-2b = 41.2 3.4 nM, ( )-2c = 76.3 2.7
nM, ( )-2d = 31.2 5.7 nM, ( )-2e = 79.1 1.4 nM) or retained (( )-
2f = 106 24 nM, ( )-2g = 113 3.0 nM) DAT affinity versus meth-
ylphenidate.32 Furthermore, improved DAT affinity was observed
for methylphenidate analogs bearing halogens at the 3-position of
the aromatic ring (( )-1c = 40.5 4.5 nM, ( )-1d = 5.10 1.6 nM,
( )-1e = 4.18 0.17 nM).31 Thispromptedustoinvestigatethisposi-
tion as another logical place to anchor a bulky iodine as a future
2.2. Pharmacology and structure–activity relationships
With the target methylphenidate compounds in hand, ligand
affinities (Ki values) were determined for inhibition of [3H]-WIN-
35,428 binding to a human dopamine transporter (hDAT) stably
expressed in N2A neuroblastoma cells. [3H]-Dopamine uptake
inhibition potencies in the same cells under the same conditions
were also determined (Table 1). Racemic threo-methylphenidate
(( )-1a) was synthesized34,35 and pharmacologically evaluated for
comparison to the novel compounds. A ꢁ5.5-fold improvement
in DAT affinity was observed upon substituting the 3-position of
methylphenidate’s aromatic ring with iodine (i.e., ( )-1f), further

506
D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
X
N
N
N
a
HN
HCl•
b
c
O
O
4
X
O
NNHTs
O
O
3
OMe
3
3
4
3
X
X
4
4
(+)-1b, X = 4-I, 100%
(+)-6a, X = 4-I, 60%
(+)-6b, X = 3-I, 55%
(+)-1f, X = 3-I, 100%
5a, X = 4-I, 74%
5b, X = 3-I, 72%
4a, X = 4-I
4b, X = 3-I
X
N
O
HN
HCl•
N
O
d
e
4
N3
I
O
3
OMe
OMe
N3
OMe
I
(+)-3a, 67%
(+)-1b, X = 4-I
(+)-3e, 54% (three steps)
(+)-1f, X = 3-I
Scheme 2. Synthesis of iodomethylphenidates ( )-1b and ( )-1f and representative approaches to target probes 3. Reagents and conditions: (a) TsNHNH2, H2SO4, EtOH,
reflux; (b) Aliquat 336, 50% aq NaOH, toluene, reflux then Et2O recrystallization; (c) HCl, MeOH, reflux; (d) (using (+)-1b) p-N3-BnBr, K2CO3, DMF, rt; (e) (using (+)-1f) (1)
m-NO2-BnBr, K2CO3, DMF, rt, (2) SnCl2, MeOH, rt, (3) NaNO2, HCl, H2O, 0 °C then NaN3, 0 °C.
Table 1
Inhibition of [3H]-WIN-35,428 binding and [3H]-dopamine uptake by compounds at hDAT N2A neuroblastoma cells
[3H]-WIN Binding Inhibition, Kia (nM)
[3H]-DA Uptake Inhibition, IC50 (nM)
a
Compound
( )-1a, threo-methylphenidate
( )-1b, 4-I-methylphenidate
( )-1f, 3-I-methylphenidate
25
1
156 58
14 3*
4.5 1*
11 2**
14 5**
( )-3a, p-N3-N-Bn-4-I-methylphenidate
( )-3b, m-N3-N-Bn-4-I-methylphenidate
( )-3c, o-N3-N-Bn-4-I-methylphenidate
( )-3d, p-N3-N-Bn-3-I-methylphenidate
( )-3e, m-N3-N-Bn-3-I-methylphenidate
( )-3f, o-N3-N-Bn-3-I-methylphenidate
363 28*
2754 169*
517 65*
658 70*
2056 73*
1112 163*
2764 196**,***
7966 348**,***
1232 70**,***
1828 261**,***
4627 238**,***
2696 178**,***
a
Each Ki or IC50 value represents data from at least three independent experiments with each data point on the curve performed in duplicate.
P <0.05 versus Ki of ( )-1a, threo-methylphenidate.
P <0.05 versus IC50 of ( )-1a, threo-methylphenidate.
*
**
***
P <0.05 versus its corresponding Ki.
supporting the observation that halogens are tolerated at this posi-
tion31 and suggesting an anchoring position for 125I as a radiotracer
tag. The affinity of 4-iodomethylphenidate (( )-1b) was approxi-
mately twofold higher than methylphenidate in our hands,
whereas previous pharmacological results indicated an approxi-
mate sixfold increase in DAT affinity.31 However, substituting the
piperidine nitrogen of these iodomethylphenidates with a N-ben-
zyl group bearing an aromatic azide resulted in a significant de-
crease in DAT binding affinity (ꢁ15-fold to 110-fold lower
binding affinity than methylphenidate). Out of the series of hybrid
compounds, N-(p-azido-benzyl)-4-iodomethylphenidate (( )-3a)
displayed the highest DAT affinity (Ki = 363 28 nM). With respect
to DAT binding affinity and the position of the azide group on the
N-benzyl aromatic ring, the para position appears to be about the
same (15- and 26-fold lower binding affinity than methylpheni-
date) as compared to the ortho position (21- and 44-fold lower
binding affinity than methylphenidate). Substitution of the azide
at the meta position resulted in a much greater decrease in DAT
binding affinity (110- and 82-fold lower binding affinity than
methylphenidate). There was no obvious advantage in terms of
DAT binding affinity associated with the position of the iodine in
these hybrid analogs (average Ki: 3-iodo compounds = 1280 nM;
4-iodo compounds = 1210 nM). Thus, these designed azido-N-ben-
zyl-iodomethylphenidates behaved unlike previously reported
methylphenidates in which N-benzyl substitution either increased
or retained DAT affinity.32
for each compound even though the two assays were conducted
under identical conditions. Using the Cheng–Prusoff equation, con-
version of uptake inhibition constants from IC50 to Ki did not signif-
icantly change the value, allowing for direct comparison of binding
and uptake results. A 3–4-fold difference between inhibitor bind-
ing and dopamine uptake inhibition potencies has been previously
observed with rDAT/CHO cells in this laboratory for WIN-35,428,
cocaine, mazindol, and methylphenidate.10 Interestingly, 4-iodom-
ethylphenidate (( )-1b) displayed essentially the same value for
binding and uptake inhibition (Table 1), a pattern previously seen
for benztropine and the related compounds GBR-12,909 and rim-
cazole.10 Cocaine and benztropine have been suggested to occupy
non-identical DAT sites or conformations;8–10 the present result
may imply that 4-iodomethylphenidate also interacts with the
DAT in a fashion different from the other compounds in Table 1.
With respect to identifying candidate probes for photoaffinity
labeling experiments, ideal compounds are those that are bioactive
in the same range as the parent compound. However, compounds
with as much as 1000-fold lower activity can still be useful.39 With
this in mind and the observation that compound ( )-3a retained
modest DAT affinity, we decided to further pursue development
of this compound into a potential DAT photoaffinity probe.
Wash-resistant binding experiments on nonradioactive azido
compounds frequently give false positives in the assessment of
covalent attachment.17 Therefore, the synthesis of radioiodo com-
pound [125I]-( )-3a was attempted to determine if photoactivation
produced covalent ligation to the DAT. Synthesis of [125I]-( )-3a
was achieved by converting nonradioactive iodo compound
With respect to inhibition of dopamine uptake (Table 1), IC50
values were typically 2–8-fold higher than the binding Ki values

D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
507
N
N3
X
O
OMe
Pd(PPh3)2Br2
((n-Bu)3Sn)2
toluene, 105°C
[
125I]-NaI
[
125I]-(+)-3a,
(+)-3a, X = -I
(+)-7, X = -Sn(n-Bu)3,
Chloramine-T
MeOH, AcOH
X = -I125
61%
,
41%
Scheme 3. Radiosynthesis of [125I]-( )-3a.
Figure 1. Panel A: Reversed-phase HPLC chromatogram for isolation of [125I]-( )-3a. The radioligand exhibited an appropriate retention time based on the non-radioactive
standard and was well resolved from radioactive and non-radioactive side-products. Panel B: Co-elution of purified [125I]-( )-3a and a standard sample of ( )-3a under the
same HPLC conditions used for preparative work.
( )-3a into a tributylstannyl derivative (( )-7) followed by radioio-
dination with [125I]-NaI (1.53 mCi) in the presence of Chloramine-T
(Scheme 3). This sequence ended with reversed-phase HPLC isola-
tion to provide [125I]-( )-3a in 61% isolated yield, high purity (98%),
and high specific activity (2099 mCi/lmol). The radioligand exhib-
ited a chromatographic profile identical to that of non-radioactive
( )-3a (Fig. 1). [125I]-( )-3a (tR = 18.4 min) was well resolved from
radioactive and non-radioactive side-products by preparative HPLC
(Figure 1A). The major non-radioactive material was assigned as
the chloro analog of ( )-3a (tR = 13.3 min) based upon model stud-
ies conducted using an excess of Chloramine-T but no radioiodine.
Purified [125I]-( )-3a co-eluted with a fully characterized sample of
non-radioactive ( )-3a under the same reversed-phase HPLC con-
ditions (Fig. 1B).
2.3. DAT photoaffinity labeling
Figure 2. Photoaffinity labeling of DAT with [125I]-( )-3a. Rat striatal membranes
were photoaffinity labeled with 30 nM [125I]-( )-3a in the absence or presence of
10 lM or 100 lM b-CFT or D-MPH. Membranes were solubilized and DATs were
immunoprecipitated followed by analysis by SDS-PAGE and autoradiography. The
relevant portion of a representative autoradiograph is pictured followed by a
histogram that quantitates relative band intensities. Mean SE of three indepen-
To determine if the DAT was able to be irreversibly labeled with
[
125I]-( )-3a, we used procedures previously developed for the anal-
ysis of other DAT photoaffinity labels.13,14,17,20,28,30,40–43 Briefly, rat
striatal membranes and HEK 293 cells expressing 6Xhis-hDAT were
incubated with 30 nM [125I]-( )-3a in the absence or presence of
***
P <0.001 versus control.
dent experiments is shown;

508
D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
10
l
M or 100
l
M b-CFT ((ꢀ)-2-b-carbomethoxy-3-b-(4-fluoro-
-(+)-methylphe-
-MPH). The membranes and cells were then detergent-
(Hz). HRMS samples were analyzed at Old Dominion University
phenyl)tropane, WIN-35,428, a cocaine analog) or
nidate (
D
(Norfolk, VA) by positive ion electrospray on a Bruker 12 Tesla
APEX-Qe FTICR-MS with an Apollo II ion source. Combustion anal-
yses of selected solid compounds were performed by Atlantic
Microlab, Inc. (Norcross, GA) and agree within 0.4% of calculated
values. Melting point determinations were conducted using a Tho-
mas-Hoover melting point apparatus and are uncorrected. Infrared
spectra were recorded using a Perkin–Elmer Spectrum RZ I FT-IR
spectrometer. On the basis of NMR and combustion data, all com-
pounds were P95% pure. A radioisotope dose calibrator (Capintec
CRC-15W) was used for radioactivity measurements and similar
counting geometries were employed for each determination.
D
solubilizedandthe lysateswere immunoprecipitated with DATanti-
body and analyzed by SDS–PAGE/autoradiography. Labeled proteins
of ꢁ80 kDa were obtained from both rat striatal tissue (Fig. 2) and
HEK hDAT cells (not shown), demonstrating the incorporation of
[
125I]-( )-3a into the DAT. Incorporation of the ligand was blocked
by 75–90%by bothb-CFTand D-MPH, demonstratingtheappropriate
pharmacological specificity of [125I]-( )-3a attachment to the DAT.
Similar to results previously reported for tropane, GBR, and benztro-
pine DAT photoaffinity ligands,42,43 analysis of total cell lysates
showed that several proteins undergo adduction with [125I]-( )-3a
(not shown). However, these do not represent the DAT because they
do not immunoprecipitate with DAT antibody as shown for the pro-
tein in Figure 2.
4.1. 1-(4-Iodophenylglyoxylyl)piperidine (4a)
A solution of ethyl 2-(4-iodophenyl)-2-oxoacetate36 (0.23 g,
0.75 mmol) in EtOH (3 mL) was added to a stirred mixture of piper-
idine (0.19 g, 2.25 mmol) in EtOH (3 mL). The reaction mixture was
stirred at 70 °C for 3 h, cooled to room temperature, diluted with
H2O then 1 M aq HCl, and extracted with EtOAc. The organic layer
was dried (MgSO4), filtered, concentrated, and chromatographed
(EtOAc/hexanes, 1:9) to provide 0.22 g of 4a as a colorless oil
(63%). Rf = 0.23 (EtOAc/hexanes, 1:9). 1H NMR (CDCl3, 400 MHz): d
7.82 (d, 2H, J = 8.2 Hz), 7.59 (d, 2H, J = 8.2 Hz), 3.63 (t, 2H,
J = 5.6 Hz), 3.21 (t, 2H, J = 5.5 Hz), 1.63–1.59 (m, 4H), 1.50–1.45 (m,
2H). 13C NMR (CDCl3, 100 MHz): d 191.0, 164.8, 138.3, 132.5,
130.7, 103.2, 47.0, 42.2, 26.2, 25.4, 24.3. HRMS calcd for C13H14INO2-
Na+ 365.9961, found 365.9961.
3. Conclusions
We have designed, synthesized, and pharmacologically evalu-
ated novel photoaffinity ligands based on the well-known ADHD
drug threo-methylphenidate. In particular, ( )-3a represents the
first successful example of a DAT photoaffinity ligand based on
the methylphenidate scaffold, thus representing an important con-
tribution to the growing arsenal of probes useful for characterizing
DAT function and 3-D structure. There is evidence that structurally
diverse DAT inhibitors bind to non-identical DAT sites or confor-
mations,8–10 suggesting that novel irreversible ligands based on
methylphenidate may yield new monoamine transporter struc-
ture-function information. The 3-iodo analog (( )-1f) was synthe-
sized as a new DAT inhibitor and pharmacologically found to
possess high affinity. Our results potentially warrant the explora-
tion of an 123I-labeled derivative of this compound as a prospective
SPECT radiopharmaceutical for brain dopamine transporters.33
Additionally, we found that ligand ( )-3a bound with modest affin-
ity to the DAT and its 125I analog was shown to bind covalently to
rDAT and hDAT expressed in cultured cells. Since the (R,R)-(+)-
enantiomer of threo-methylphenidate has been found to be the
more biologically active compound,44 future directions include
resolving ( )-3a into its enantiomerically pure components to-
wards obtaining a more specific and improved DAT photoaffinity
probe. Additional DAT irreversible ligands based on methylpheni-
date (particularly those with improved DAT affinity), their pharma-
cological characterization, binding site prediction via docking
within 3-D DAT homology models,29 and detailed elucidation of
DAT binding domains for comparison to established tropane-based
probes will be investigated in due course.
4.2. 1-(3-Iodophenylglyoxylyl)piperidine (4b)
N-Iodosuccinimide (0.23 g, 1 mmol) was added to 96% H2SO4
(3 mL) and cooled to 0 °C. The resulting suspension was stirred for
20–30 min until it became a homogeneous black solution. 1-(Phen-
ylglyoxylyl)piperidine34,35 (0.11 g, 0.5 mmol) was added to the solu-
tion and the mixture was stirred at 0 °C for 20 min. The reaction
mixture was poured into 10 mL of an ice-H2O mixture and treated
with Na2SO3 to adjust the pH to 7. The mixture was then extracted
with CH2Cl2, washed with brine, dried (MgSO4), and concentrated
to obtain 0.13 g of 4b as a yellow oil (77%). Rf = 0.45 (EtOAc/hexanes,
3:7). 1H NMR (CDCl3, 400 MHz): d 8.27 (s, 1H), 7.95 (d, 1H, J = 7.8 Hz),
7.89 (d, 1H, J = 7.8 Hz), 7.26 (t, 1H, J = 7.8 Hz), 3.69 (t, 2H, J = 4.8 Hz),
3.27(t, 2H, J = 5.5 Hz), 1.80–1.60(m, 4H), 1.60–1.50(m, 2H). 13CNMR
(CDCl3, 100 MHz): d 190.2, 164.5, 143.2, 138.0, 134.9, 130.6, 128.7,
94.5, 47.0, 42.2, 26.1, 25.4, 24.2. HRMS calcd for C13H14INO2Na+
365.9961, found 365.9960.
4.3. 1-(4-Iodophenylglyoxylyl)piperidine p-toluenesulfonylhyd
razone (5a)
4. Experimental
A solution of ketone 4a (0.93 g, 2.71 mmol) in EtOH (10 mL) was
added to a stirred mixture of p-toluenesulfonyl hydrazide (0.55 g,
2.93 mmol) in ethanol (10 mL) containing H2SO4 (4 mg, 0.04 mmol).
The reaction was refluxed at 85 °C overnight then cooled to room
temperature. The white precipitated solid was filtered, washed with
cold MeOH and cold hexanes, then dried under reduced pressure to
give 1.01 g of 5a (74%). Rf = 0.45 (EtOAc/hexanes, 4:6). 1H NMR
(CDCl3, 400 MHz): d 8.40 (s, 1H), 7.83 (d, 2H, J = 8.3 Hz), 7.71 (d,
2H, J = 8.7 Hz), 7.31 (d, 2H, J = 8.6 Hz), 7.26 (d, 2H, J = 8.1 Hz), 3.70–
3.66 (m, 2H), 3.17 (t, 2H, J = 5.6 Hz), 2.38 (s, 3H), 1.66–1.64 (m, 4H),
1.50–1.40 (m, 2H). 13C NMR (CDCl3, 100 MHz): d 161.5, 149.5,
144.2, 137.9, 135.0, 131.7, 129.6, 127.9, 127.7, 97.2, 47.3, 42.3,
26.3, 25.5, 24.1, 21.6. HRMS calcd for C20H22IN3O3SNa+ 534.0319,
found 534.0318. Anal. Calcd for: C20H22IN3O3S: C, 46.97; H 4.34; N,
8.22; I, 24.82; S, 6.27. Found: C, 46.92; H 4.31; N, 8.23; I, 24.63; S,
6.42. Mp: 137–138 °C.
Reaction conditions and yields were not optimized. All reactions
were performed in flame-dried glassware under argon unless
otherwise noted. All solvents and chemicals were purchased from
Aldrich Chemical Co. or Fisher Scientific and used without further
purification. Flash column chromatography was performed accord-
ing to the method of Still et al.45 using Fisher S826-25 silica gel sor-
bent (70–230 mesh) and eluting solvent mixtures as specified.
Thin-layer chromatography (TLC) was performed using TLC Silica
Gel 60 F254 plates obtained from EMD Chemicals, Inc. and com-
pounds were visualized under UV light and/or I2 stain. Proportions
of solvents used for TLC are by volume. 1H and 13C NMR spectra
were recorded on either a Bruker 400 or 500 MHz spectrometer.
Chemical shifts for 1H and 13C NMR spectra are reported as parts
per million (d ppm) relative to tetramethylsilane (0.00 ppm) as
an internal standard. Coupling constants are measured in hertz

D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
509
4.4. 1-(3-Iodophenylglyoxylyl)piperidine p-toluenesulfonylhyd
razone (5b)
1H, J = 9.6 Hz), 3.83 (t, 1H, J = 9.9 Hz), 3.73 (s, 3H), 3.45 (d, 1H,
J = 12.4 Hz), 3.10 (t, 1H, J = 11.8 Hz), 1.89–1.71 (m, 3H), 1.53–1.40
(m, 3H). 13C NMR (CD3OD, 100 MHz): d 172.8, 139.6, 134.9, 131.7,
95.0, 58.9, 54.7, 53.6, 46.7, 27.6, 23.3, 22.8. HRMS calcd for C14H18I-
NO2Na+ 382.0274, found 382.0275. Mp: 188–189 °C.
Ketone 4b (7.3 g, 21.2 mmol) was added to a stirred mixture
of p-toluenesulfonyl hydrazide (4.26 g, 22.89 mmol) in EtOH
(194 mL) containing H2SO4 (0.03 g, 0.3 mmol). The mixture was re-
fluxed at 85 °C overnight then cooled to room temperature. The
precipitated solid was filtered, washed with cold MeOH and cold
hexanes, then dried under reduced pressure to give 7.8 g of 5b
(72%). Rf = 0.1 (EtOAc/hexanes, 3:7). 1H NMR (CDCl3, 400 MHz): d
8.93 (s, 1H), 7.94 (s, 1H), 7.78 (d, 2H, J = 8.3 Hz), 7.69 (d, 1H,
J = 7.9 Hz), 7.51 (d, 1H, J = 7.9 Hz), 7.19 (d, 2H, J = 8.1 Hz), 7.09 (t,
1H, J = 7.9 Hz), 3.7–3.6 (m, 2H), 3.2–3.1 (m, 2H), 2.34 (s, 3H),
1.76–1.52 (m, 4H), 1.48–1.38 (m, 2H). 13C NMR (CDCl3,
100 MHz): d 161.3, 148.4, 144.1, 139.2, 135.0, 134.7, 134.2, 130.3,
129.4, 127.9, 127.9, 125.5, 94.5, 47.2, 42.3, 26.2, 25.4, 24.0, 21.5.
HRMS calcd for C20H22IN3O3SNa+ 534.0319, found 534.0314. Mp:
158–159 °C.
4.8. ( )-threo-3-Iodomethylphenidate Hydrochloride (( )-1f)
( )-threo-b-Lactam 6b (0.19 g, 0.58 mmol) was refluxed in
1.25 M HCl in MeOH (41 mL) for 7 h then concentrated under re-
duced pressure to afford 229 mg of ( )-1f (100%). This threo-iodom-
ethylphenidatewasdeterminedto beP95%diastereomericallypure
by 1H NMR upon comparison to the known data for enantiomerically
pure threo- and erythro-methylphenidate and its para-substituted
derivatives.37 1H NMR (CD3OD, 400 MHz): d 7.74 (d, 1H, J = 9.6 Hz),
7.70 (t, 1H, J = 1.7 Hz), 7.31 (d, 1H, J = 8.4 Hz), 7.18 (t, 1H,
J = 7.8 Hz), 3.89–3.81 (m, 2H), 3.74 (s, 3H), 3.43 (d, 1H, J = 12.8 Hz),
3.10 (t, 1H, J = 12.9 Hz), 1.91–1.79 (m, 2H), 1.74–1.63 (m, 1H),
1.57–1.46 (m, 2H), 1.41–1.32 (m, 1H). 13C NMR (CD3OD, 100 MHz):
d 172.8, 139.0, 138.6, 137.4, 132.2, 128.9, 95.6, 58.9, 54.7, 53.6,
46.7, 27.8, 23.3, 22.7. HRMS calcd for C14H18INO2Na+ 382.0274,
found 382.0271. Mp: 191–192 °C.
4.5. ( )-7-(4-Iodophenyl)-1-azabicyclo[4.2.0]octan-8-one (( )-6a)
Hydrazone 5a (1 g, 2 mmol) was added to a stirred mixture of
50% aq NaOH (0.17 mL, 2.08 mmol) and Aliquat 336 (8 mg,
0.02 mmol) in toluene (20 mL). The reaction was refluxed at
130 °C overnight then cooled to room temperature, diluted with
H2O, and extracted with toluene. The organic layer was dried
(MgSO4), filtered, concentrated, and chromatographed (EtOAc/hex-
anes, 2:8) to afford ( )-6a as a yellow solid. Recrystallization from
Et2O provided 0.39 g of ( )-6a (60%). 1H NMR (CDCl3, 500 MHz): d
7.64 (d, 2H, J = 8.3 Hz), 7.03 (d, 2H, J = 8.4 Hz), 3.92 (d, 1H,
J = 4.3 Hz), 3.91–3.88 (m, 1H), 3.34–3.30 (m, 1H), 2.82–2.76 (m,
1H), 2.18–2.14 (m, 1H), 1.94–1.91 (m, 1H), 1.71–1.67 (m, 1H),
1.45–1.38 (m, 3H). 13C NMR (CDCl3, 100 MHz): d 165.5, 137.7,
135.3, 129.3, 92.6, 62.8, 56.5, 39.0, 30.4, 24.4, 22.1. HRMS calcd
for C13H14INONa+ 350.0012, found 350.0009. Anal. Calcd for:
4.9. ( )-threo-N-(p-Azido-benzyl)-4-iodomethylphenidate (( )-3a)
( )-threo-4-Iodomethylphenidatehydrochloride (( )-1b) (0.11 g,
0.27 mmol) was added to a suspension of K2CO3 (0.15 g, 1.08 mmol)
inDMF(6 mL). Theresulting suspensionwas stirred at roomtemper-
ature for 10 min, then p-N3-N-BnBr38 (60 mg, 0.29 mmol) was added
and the mixture was allowed to stir at room temperature in the dark
for 30 h. Et2O (20 mL) was added and the mixture was decanted fol-
lowed by rinsing with Et2O (2 ꢂ 20 mL). The combined organic lay-
ers were washed with H2O, dried (MgSO4), filtered, concentrated,
and chromatographed (EtOAc/hexanes, 5:95) to give 90 mg of ( )-
3a as a yellow solid (67%). Rf = 0.26 (EtOAc/hexanes, 5:95). 1H NMR
(CDCl3, 400 MHz): d 7.64 (d, 2H, J = 8.3 Hz), 7.27 (d, 2H, J = 8.3 Hz),
7.14 (d, 2H, J = 8.4 Hz), 6.97 (d, 2H, J = 8.4 Hz), 4.11 (d, 1H,
J = 11.5 Hz), 3.89 (d, 1H, J = 13.6 Hz), 3.74 (d, 1H, J = 13.6 Hz), 3.65
(s, 3H), 3.45–3.41 (m, 1H), 2.97–2.91 (m, 1H), 2.55–2.50 (m, 1H),
1.58–1.47 (m, 4H), 1.33–1.25 (m, 1H), 1.06–1.02 (m, 1H). 13C NMR
(CDCl3, 100 MHz): d 173.5, 138.4, 137.7, 137.1, 136.6, 130.7, 129.9,
118.7, 93.1, 62.3, 55.9, 52.6, 51.9, 44.8, 21.1, 20.7, 19.5. HRMS calcd
for C21H23IN4O2H+ 491.0938, found 491.0930. Anal. Calcd for:
C13H14INO: C, 47.73; H 4.31; N, 4.28; I, 38.79. Found: C, 47.84; H
4.29; N, 4.23; I, 38.57. Mp: 127–128 °C.
4.6. ( )-7-(3-Iodophenyl)-1-azabicyclo[4.2.0]octan-8-one (( )-6b)
50% aq NaOH (1.3 mL, 15.96 mmol) was added to a stirred mix-
ture of hydrazone 5b (7.8 g, 15.2 mmol) and Aliquat 336 (61 mg,
0.16 mmol) in toluene (140 mL). The mixture was refluxed at
130 °C overnight then cooled to room temperature, diluted with
H2O, and extracted with toluene. The organic layer was dried
(MgSO4), filtered, concentrated, and chromatographed (EtOAc/hex-
anes, 2:8) to afford ( )-6b as a yellow solid. Further recrystallization
from Et2O provided 2.7 g of ( )-6b (55%). Rf = 0.13 (EtOAc/hexanes,
2:8). 1H NMR (CDCl3, 400 MHz): d 7.63–7.57 (m, 2H), 7.25 (d, 1H,
J = 7.7 Hz), 7.05 (t, 1H, J = 7.8 Hz), 4.05–3.80 (m, 2H), 3.40–3.30 (m,
1H), 2.85–2.70 (m, 1H), 2.20–2.10 (m, 1H), 2.05–1.90 (m, 1H),
1.72–1.65 (m, 1H), 1.45–1.35 (m, 3H). 13C NMR (CDCl3, 100 MHz):
d 165.4, 137.9, 136.3, 136.1, 130.4, 126.6, 94.6, 62.6, 56.5, 39.0,
30.4, 24.3, 22.1. HRMS calcd for C13H14INONa+ 350.0012, found
350.0013. Mp: 126–127 °C.
C
21H23IN4O2: C, 51.44; H 4.73; N, 11.43; I, 25.88. Found: C, 51.72;
H 4.67; N, 11.48; I, 25.62. Mp: 108–110 °C. IR: azide, 2109 cmꢀ1
.
4.10. ( )-threo-N-(m-Azido-benzyl)-4-iodomethylphenidate
(( )-3b)
( )-threo-4-Iodomethylphenidatehydrochloride (( )-1b) (0.15 g,
0.38 mmol)wasaddedto asuspensionofK2CO3 (0.13 g, 0.9 mmol)in
DMF (5 mL). The mixture was stirred at room temperature for
10 min, then m-NO2-N-BnBr (82 mg, 0.38 mmol) was added. The
reaction was allowed to stir at room temperature in the dark for
30 h. Et2O (20 mL) was added then the mixture was decanted fol-
lowed by rinsing with Et2O (2 ꢂ 20 mL). The combined organic lay-
ers were washed with H2O, dried (MgSO4), filtered, concentrated,
and chromatographed (CHCl3/hexanes, 1:1) to give 0.13 g of ( )-
threo-N-(m-nitro-benzyl)-4-iodomethylphenidate as a yellow oil
(69%). Rf = 0.21 (CHCl3/hexanes, 1:1). 1H NMR (CDCl3, 400 MHz): d
8.18 (s, 1H), 8.10 (d, 1H, J = 8.1 Hz), 7.65–7.60 (m, 3H), 7.47 (t, 1H,
J = 7.9 Hz), 7.16 (d, 2H, J = 8.4 Hz), 4.13 (d, 1H, J = 11.6 Hz), 4.05 (d,
1H, J = 14.3 Hz), 3.86 (d, 1H, J = 14.3 Hz), 3.72 (s, 3H), 3.48–3.44 (m,
1H), 3.03–2.96 (m, 1H), 2.54–2.51(m, 1H), 1.60–1.50 (m, 4H), 1.37–
1.34 (m, 1H), 1.10–1.06 (m, 1H). 13C NMR (CDCl3, 100 MHz): d
173.6, 148.4, 142.7, 137.8, 136.4, 134.5, 130.7, 128.9, 123.3, 122.0,
4.7. ( )-threo-4-Iodomethylphenidate Hydrochloride (( )-1b)33
( )-threo-b-Lactam 6a (90 mg, 0.28 mmol) was refluxed in
1.25 M HCl in MeOH (20 mL) at 85 °C for 7 h then concentrated un-
der reduced pressure to afford 107 mg of ( )-1b as a yellow solid
(100%). This threo-iodomethylphenidate was determined to be
P95% diastereomerically pure by 1H NMR upon comparison to the
known data for enantiomerically pure threo- and erythro-methyl-
phenidate and its para-substituted derivatives.37 1H NMR (CD3OD,
400 MHz): d 7.76 (d, 2H, J = 7.9 Hz), 7.11 (d, 2H, J = 8.0 Hz), 3.95 (d,

510
D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
93.2, 62.7, 56.0, 52.4, 52.1, 44.9, 21.2, 20.5, 19.5. HRMS calcd for
21H23IN2O4Na+ 517.0594, found 517.0594. ( )-threo-N-(m-nitro-
the dark for 30 h. Et2O (20 mL) was added and the mixture was
decanted followed by rinsing with Et2O (2 ꢂ 20 mL). The combined
organic layers were washed with H2O, dried (MgSO4), filtered, con-
centrated, and chromatographed (EtOAc/hexanes, 5:95) to give
0.11 g of ( )-3d as a yellow gum (63%). Rf = 0.23 (EtOAc/hexanes,
5:95). 1H NMR (CDCl3, 400 MHz): d 7.74 (s, 1H), 7.60 (d, 1H,
J = 7.9 Hz), 7.36 (d, 1H, J = 7.8 Hz), 7.26 (d, 2H, J = 8.6 Hz), 7.05 (t,
1H, J = 7.8 Hz), 6.97 (d, 2H, J = 8.4 Hz), 4.08 (d, 1H, J = 11.5 Hz),
3.89 (d, 1H, J = 13.6 Hz), 3.74 (d, 1H, J = 13.6 Hz), 3.66 (s, 3H),
3.46–3.42 (m, 1H), 2.97–2.90 (m, 1H), 2.53–2.49 (m, 1H), 1.58–
1.48 (m, 5H), 1.07–1.03 (m, 1H). 13C NMR (CDCl3, 100 MHz): d
173.4, 139.2, 138.4, 137.0, 136.6, 130.3, 129.9, 128.0, 118.7, 94.5,
62.5, 55.9, 52.5, 51.9, 44.6, 21.1, 20.6, 19.4. HRMS calcd for C21H23I-
C
benzyl)-4-iodomethylphenidate (0.1 g, 0.2 mmol) was added to
MeOH (5 mL) at 0 °C followed by concentrated HCl (1 mL) and SnCl2
(0.15 g, 0.78 mmol). The mixture was allowed to stir at room tem-
peratureovernightthenquenchedwithH2O(5 mL). ThepHwasthen
brought to 11 with 1 M aq NaOH and extracted with EtOAc. The or-
ganic layer was washed with brine, dried (MgSO4), filtered, concen-
trated, and chromatographed (CHCl3/MeOH, 9:1) to give 90 mg of
( )-threo-N-(m-amino-benzyl)-4-iodomethylphenidate as a color-
less oil (81%). Rf = 0.29 (CHCl3/MeOH, 9:1). 1H NMR (CDCl3,
400 MHz): d 7.63 (d, 2H, J = 8.4 Hz), 7.15 (d, 2H, J = 8.4 Hz), 7.09 (t,
1H, J = 7.7 Hz), 6.68–6.65 (m, 2H), 6.56 (d, 1H, J = 7.8 Hz), 4.10 (d,
1H, J = 11.4 Hz), 3.82 (d, 1H, J = 13.5 Hz), 3.69 (d, 1H, J = 13.4 Hz),
3.66 (s, 3H), 3.48–3.40 (m, 1H), 2.97–2.91 (m, 1H), 2.57–2.52 (m,
1H), 1.54–1.42 (m, 4H), 1.32–1.29 (m, 1H), 1.05–1.02 (m, 1H). 13C
NMR (CDCl3, 100 MHz): d 173.6, 146.3, 141.4, 137.7, 136.8, 130.8,
128.9, 118.9, 115.4, 113.7, 93.0, 62.5, 56.4, 52.7, 51.9, 45.1, 21.3,
N4O2H+ 491.0938, found 491.0945. IR: azide, 2111 cmꢀ1
.
4.13. ( )-threo-N-(m-Azido-benzyl)-3-iodomethylphenidate (( )-3e)
( )-threo-3-Iodomethylphenidate hydrochloride (( )-1f) (0.3 g,
0.76 mmol) was added to a suspension of K2CO3 (0.25 g, 1.8 mmol)
in DMF (5 mL). The mixture was stirred at room temperature for
10 min then m-NO2-N-BnBr (0.16 g, 0.76 mmol) was added. The
reaction was allowed to stir at room temperature in the dark for
30 h. Et2O (20 mL) was added then the mixture was decanted fol-
lowed by rinsing with Et2O (2 ꢂ 20 mL). The combined organic lay-
ers were washed with H2O, dried (MgSO4), filtered, concentrated,
and chromatographed (CHCl3/hexanes, 1:1) to give 0.26 g of ( )-
threo-N-(m-nitro-benzyl)-3-iodomethylphenidate as a yellow oil
(70%). Rf = 0.31 (CHCl3/hexanes, 1:1). 1H NMR (CDCl3, 400 MHz): d
8.18 (s, 1H), 8.10 (d, 1H, J = 8.1 Hz), 7.76 (s, 1H), 7.60 (d, 2H,
J = 7.9 Hz), 7.47 (t, 1H, J = 7.9 Hz), 7.37 (d, 1H, J = 7.8 Hz), 7.06 (t,
1H, J = 7.8 Hz), 4.10 (d, 1H, J = 11.6 Hz), 4.05 (d, 1H, J = 14.3 Hz),
3.86 (d, 1H, J = 14.3 Hz), 3.72 (s, 3H), 3.48–3.45 (m, 1H), 3.01–2.96
(m, 1H), 2.54–2.50 (m, 1H), 1.61–1.52 (m, 4H), 1.38–1.34 (m, 1H),
1.10–1.07 (m, 1H). 13C NMR (CDCl3, 100 MHz): d 173.4, 148.3,
142.6, 138.9, 137.4, 136.7, 134.4, 130.4, 128.9, 127.9, 123.2, 122.0,
94.6, 62.8, 56.0, 52.4, 52.1, 44.7, 21.2, 20.5, 19.4. HRMS calcd for
20.9, 19.6. HRMS calcd for
C
21H25IN2O2Na+ 487.0852, found
487.0859. A solution of ( )-threo-N-(m-amino-benzyl)-4-iodometh-
ylphenidate (62 mg, 0.13 mmol) in concentrated HCl (0.5 mL) and
H2O (5 mL) at 0 °C was treated with NaNO2 (10 mg, 0.15 mmol).
The mixture was stirred in the dark for 10 min at 0 °C then carefully
treated with NaN3 (18 mg, 0.27 mmol). The reaction was allowed to
stir in the dark for 2 h at 0 °C then diluted with H2O and CHCl3. The
organic layer was separated, washed with brine, dried (MgSO4), fil-
tered, concentrated, and chromatographed (CHCl3) to give 65 mg
of ( )-3b as a yellow oil (85%). Rf = 0.30 (CHCl3). 1H NMR (CDCl3,
400 MHz): d 7.63 (d, 2H, J = 8.4 Hz), 7.29–7.25 (m, 1H), 7.14 (d, 2H,
J = 8.4 Hz), 7.05–7.01 (m, 2H), 6.89 (d, 1H, J = 7.9 Hz), 4.11 (d, 1H,
J = 11.5 Hz), 3.91 (d, 1H, J = 13.8 Hz), 3.76 (d, 1H, J = 13.8 Hz), 3.68
(s, 3H), 3.45–3.41 (m, 1H), 2.99–2.93 (m, 1H), 2.55–2.50 (m, 1H),
1.64–1.48 (m, 4H), 1.33–1.25 (m, 1H), 1.06–1.03 (m, 1H). 13C NMR
(CDCl3, 100 MHz): d 173.3, 142.4, 139.9, 137.7, 136.6, 130.7, 129.3,
125.1, 119.0, 117.6, 93.1, 62.5, 56.3, 52.5, 52.0, 44.9, 21.1, 20.6,
19.5. HRMS calcd for C21H23IN4O2H+ 491.0938, found 491.0940. IR:
azide, 2109 cmꢀ1
.
C
21H23IN2O4H+ 495.0775, found 495.0780. ( )-threo-N-(m-Nitro-
benzyl)-3-iodomethylphenidate (0.26 g, 0.53 mmol) was added to
MeOH (5 mL) at 0 °C then treated with concentrated HCl (2 mL)
and SnCl2 (0.39 g, 2.05 mmol). The mixture was allowed to stir at
room temperature overnight then quenched with H2O (5 mL). The
pH was brought to 11 with 1 M aq NaOH and extracted with EtOAc.
The organic layers were washed with brine, dried (MgSO4), filtered,
concentrated, and chromatographed (CHCl3/MeOH, 95:5) to give
0.2 g of ( )-threo-N-(m-amino-benzyl)-3-iodomethylphenidate as
a colorless oil (81%). Rf = 0.27 (CHCl3/MeOH, 95:5). 1H NMR (CDCl3,
400 MHz): d 7.75 (s, 1H), 7.59 (d, 1H, J = 8.5 Hz), 7.37 (d, 1H,
J = 7.8 Hz), 7.09–7.02 (m, 2H), 6.68–6.65 (m, 2H), 6.56 (d, 1H,
J = 7.9 Hz), 4.08 (d, 1H, J = 11.4 Hz), 3.82 (d, 1H, J = 13.6 Hz), 3.69
(d, 1H, J = 13.7 Hz), 3.66 (s, 3H), 3.48–3.42 (m, 1H), 2.97–2.90 (m,
1H), 2.57–2.52 (m, 1H), 1.56–1.46 (m, 4H), 1.33–1.28 (m, 1H),
1.06–1.02 (m, 1H). 13C NMR (CDCl3, 100 MHz): d 173.4, 146.3,
141.3, 139.2, 137.5, 136.4, 130.2, 128.8, 127.9, 118.7, 115.2, 113.5,
94.5, 62.5, 56.3, 52.6, 51.0, 44.8, 21.2, 20.8, 19.5. HRMS calcd for
4.11. ( )-threo-N-(o-Azido-benzyl)-4-iodomethylphenidate (( )-3c)
( )-threo-4-Iodomethylphenidate hydrochloride (( )-1b) (80 mg,
0.2 mmol)wasaddedto asuspensionofK2CO3 (0.11 g, 0.81 mmol)in
DMF (5 mL). The mixture was stirred at room temperature for
10 min then o-N3-N-BnBr38 (42 mg, 0.2 mmol) was added. The
reaction was allowed to stir at room temperature in the dark for
30 h. Et2O (20 mL) was added and the mixture was decanted
followed by rinsing with Et2O (2 ꢂ 20 mL). The combined organic
layers were washed with H2O, dried (MgSO4), filtered, concentrated,
and chromatographed (hexanes) to give 48 mg of ( )-3c as a color-
less oil (49%). Rf = 0.28 (hexanes). 1H NMR (CDCl3, 400 MHz): d
7.63 (d, 2H, 8.4 Hz), 7.38 (d, 1H, J = 7.6 Hz), 7.29–7.25 (m, 1H),
7.14–7.09 (m, 4H), 4.09 (d, 1H, J = 11.5 Hz), 3.92 (d, 1H,
J = 14.4 Hz), 3.68 (d, 1H, J = 14.4 Hz), 3.59 (s, 3H), 3.43–3.38 (m,
1H), 2.99–2.93 (m, 1H), 2.58–2.52 (m, 1H), 1.62–1.46 (m, 4H),
1.38–1.33 (m, 1H), 1.06–1.03 (m, 1H). 13C NMR (CDCl3, 100 MHz):
d 173.4, 138.0, 137.7, 136.6, 131.5, 130.7, 130.3, 127.9, 124.6,
117.8, 93.0, 62.4, 52.6, 51.9, 50.7, 45.5, 21.3, 20.7, 19.9. HRMS calcd
C
21H25IN2O2H+ 465.1033, found 465.1026. A solution of ( )-threo-
N-(m-amino-benzyl)-3-iodomethylphenidate (0.18 g, 0.39 mmol)
in 2 N HCl (6 mL) at 0 °C was treated with NaNO2 (30 mg,
0.43 mmol). The mixture was stirred in the dark for 10 min at 0 °C,
carefully treated with NaN3 (50 mg, 0.78 mmol), stirred in the dark
at 0 °C for 2 h, then diluted with H2O and CHCl3. The organic layer
was separated, washed with brine, dried (MgSO4), filtered, concen-
trated, and chromatographed(CHCl3) to give 0.16 g of ( )-3e as a yel-
low oil (95%). Rf = 0.35 (CHCl3). 1H NMR (CDCl3, 400 MHz): d 7.75 (s,
1H),7.59(d, 1H, J = 8.4 Hz), 7.37(d, 1H, J = 7.9 Hz), 7.29–7.25(m, 1H),
7.06–7.01 (m, 3H), 6.89 (d, 1H, J = 7.9 Hz), 4.08 (d, 1H, J = 11.5 Hz),
3.93 (d, 1H, J = 13.8 Hz), 3.76 (d, 1H, J = 13.8 Hz), 3.68 (s, 3H),
for C21H23IN4O2H+ 491.0938, found 491.0930. IR: azide, 2116 cmꢀ1
.
4.12. ( )-threo-N-(p-Azido-benzyl)-3-iodomethylphenidate (( )-3d)
( )-threo-3-Iodomethylphenidate hydrochloride (( )-1f) (0.14 g,
0.35 mmol) was added to
a suspension of K2CO3 (0.19 g,
1.43 mmol) in DMF (7 mL). The mixture was stirred at room tem-
perature for 10 min then p-N3-N-BnBr38 (80 mg, 0.39 mmol) was
added. The reaction was allowed to stir at room temperature in

D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
511
3.46–3.41 (m, 1H), 2.98–2.92 (m, 1H), 2.55–2.50 (m, 1H), 1.64–1.48
(m, 4H), 1.35–1.31 (m, 1H), 1.07–1.03 (m, 1H). 13C NMR (CDCl3,
100 MHz): d 173.3, 142.4, 139.9, 139.1, 137.5, 136.6, 130.3, 129.3,
127.9, 125.0, 118.9, 117.5, 94.5, 62.5, 56.2, 52.5, 52.0, 44.7, 21.1,
rate of 3 mL/min. The HPLC system was equipped with a flow-
through radioactivity detector and a UV absorbance detector
(254 nm). Radioactive material (tR = 18.4 min) corresponding to
[
125I]-( )-3a was well resolved from non-radioactive and radioac-
20.6, 19.4. HRMS calcd for
C
21H23IN4O2Na+ 513.0757, found
tive side products. [125I]-( )-3a was collected in 5 mL of HPLC mo-
bile phase, diluted to 40 mL with distilled water, and then passed
through an activated (MeOH/water) solid phase extraction car-
tridge (Waters Sep-Pak Light t-C-18) that was flushed with water
(2.0 mL) and then with air. The cartridge retained essentially all
radioactivity. Elution with MeOH (1.5 mL) gave 0.93 mCi (61%) of
513.0763. IR: azide, 2113 cmꢀ1
.
4.14. ( )-threo-N-(o-Azido-benzyl)-3-iodomethylphenidate (( )-3f)
( )-threo-3-Iodomethylphenidate hydrochloride (( )-1f) (0.1 g,
0.27 mmol) was added to
a
suspension of K2CO3 (0.15 g,
[
125I]-( )-3a. This material co-eluted with a standard sample of
1.08 mmol) in DMF (5 mL). The mixture was stirred at room tem-
perature for 10 min then o-N3-N-BnBr38 (58 mg, 0.27 mmol) was
added. The reaction was allowed to stir at room temperature in
the dark for 30 h. Et2O (20 mL) was added then the mixture was
decanted followed by rinsing with Et2O (2 ꢂ 20 mL). The combined
organic layers were washed with H2O, dried (MgSO4), filtered, con-
centrated, and chromatographed (CH2Cl2/hexanes, 1:1) to give
67 mg of ( )-3f as a yellow oil (51%). Rf = 0.23 (CH2Cl2/hexanes,
7:3). 1H NMR (CDCl3, 400 MHz): d 7.74 (t, 1H, J = 1.7 Hz), 7.59 (d,
1H, J = 7.9 Hz), 7.39 (t, 2H, J = 8.3 Hz), 7.32–7.27 (m, 1H), 7.14–
7.09 (m, 2H), 7.03 (t, 1H, J = 7.8 Hz), 4.07 (d, 1H, J = 11.5 Hz), 3.93
(d, 1H, J = 14.5 Hz), 3.67 (d, 1H, J = 14.5 Hz), 3.60 (s, 3H), 3.44–
3.40 (m, 1H), 2.99–2.92 (m, 1H), 2.57–2.51 (m, 1H), 1.58–1.52
(m, 4H), 1.39–1.33 (m, 1H), 1.07–1.03 (m, 1H). 13C NMR (CDCl3,
100 MHz): d 173.3, 139.3, 137.9, 137.6, 136.5, 131.5, 130.3, 128.0,
127.9, 124.6, 117.8, 94.5, 62.5, 52.6, 51.9, 50.7, 45.4, 21.3, 20.7,
19.8. HRMS calcd for C21H23IN4O2H+ 491.0938, found 491.0932.
( )-3a under the HPLC conditions described above and displayed
98% radiochemical purity. After 20 weeks, >2 half-lives, of storage
at ꢀ20 °C in the dark, about 80% radiochemical purity was ob-
served by HPLC. A specific radioactivity of 2099 mCi/lmol was cal-
culated for
[
125I]-( )-3a using HPLC to determine the mass
associated with the absorbance peak for carrier in a purified sam-
ple of known radioactivity (0.21 mCi). The UV response for non-
radioactive ( )-3a was linear (r2 = 1.0) over a six-point standard
curve (30–750 pmol). The major non-radioactive product observed
during HPLC purification was tentatively assigned as the chloro
analog of ( )-3a (tR = 13.3 min) based upon HPLC analyses of model
reactions using an excess of Chloramine-T but no radioiodine.
4.17. Pharmacology
[3H]-WIN-35,428 binding inhibition assays were performed
with N2A neuroblastoma cells stably transfected with human wild-
type DAT cDNA. Cell monolayers were grown to confluence at
37 °C, 5% CO2 in 24 well plates with Opti-MEM media (Invitrogen)
supplemented with 10% fetal bovine serum, 100 U/mL penicillin,
IR: azide, 2115 cmꢀ1
.
4.15. ( )-threo-N-(p-Azido-benzyl)-4-(tri-n-butylstannyl)methyl
phenidate (( )-7)
100 U/mL streptomycin (from Fisher Scientific), and 450 lg/mL
G-418 (Clontech). Confluent monolayers were first washed
2 ꢂ 1 mL with 22 °C ‘KRH buffer’ (25 mM HEPES, pH 7.3, 125 mM
NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4,
and 5.6 mM glucose) supplemented with 50 mM ascorbic acid
(KRH/AA). The washed and aspirated monolayers were incubated
A mixture of ( )-threo-N-(p-azido-benzyl)-4-iodomethylpheni-
date (( )-3a) (105 mg, 0.21 mmol), Pd(PPh3)2Br2 (10 mg,
0.02 mmol), and bis(tri-n-butyltin) (0.15 mL, 0.39 mmol) in tolu-
ene (10 mL) was heated at 105 °C for 6 h. The mixture was then
cooled to room temperature, diluted with satd aq K2CO3 solution,
and extracted with EtOAc. The organic phase was washed with
brine, dried (MgSO4), filtered, concentrated, and chromatographed
(EtOAc/hexanes, 1:9) to give 49 mg of ( )-7 as a colorless oil (41%).
Rf = 0.35 (EtOAc/hexanes, 1:9). 1H NMR (CDCl3, 400 MHz): d 7.39
(d, 2H, J = 7.9 Hz), 7.32 (d, 2H, J = 7.9 Hz), 7.28 (d, 2H, J = 8.4 Hz),
6.97 (d, 2H, J = 8.4 Hz), 4.11 (d, 1H, J = 11.5 Hz), 3.90 (d, 1H,
J = 13.6 Hz), 3.76 (d, 1H, J = 13.6 Hz), 3.65 (s, 3H), 3.49–3.45 (m,
1H), 2.99–2.94 (m, 1H), 2.54–2.51 (m, 1H), 1.56–1.49 (m, 9H),
1.34–1.29 (m, 9H), 1.03 (t, 6H, J = 8.0 Hz), 0.88 (t, 9H, J = 7.3 Hz).
13C NMR (CDCl3, 100 MHz): d 174.1, 141.0, 138.3, 137.3, 136.7,
136.4, 129.9, 128.2, 118.7, 62.5, 55.8, 53.1, 51.8, 44.8, 29.0, 27.4,
with 500
itor (0.1 nM–10
aspiration and 2 ꢂ 1 mL KRH/AA washes. Non-specific binding
was determined by using 10 M mazindol as the competitor. Mon-
l
L [3H]-WIN-35,428 (1 nM) and nonradioactive compet-
M) for 15 min at 22 °C, followed by removal by
l
l
olayers were solubilized by incubation with 1 mL 1% SDS at 22 °C
for 1 h with gentle shaking. Cell lysates were transferred into vials
containing 5 mL ScintiSafe fluid (Fisher), mixed thoroughly by
inversion and vortexing, and analyzed via scintillation counting
for determination of remaining tritium radioactivity.
[3H]-Dopamine uptake inhibition assays were conducted identi-
cal to the [3H]-WIN-35,428 binding inhibition assays described
above with the exceptions that 10 nM [3H]-dopamine replaced
the WIN radioligand and the nonradioactive inhibitor drug was
added 10 min before the 5 min [3H]-dopamine uptake interval
commenced.
21.1, 20.8, 19.5, 13.7, 9.5. HRMS calcd for
C
33H50N4O2SnH+
655.3029, found 655.3033. IR: azide, 2110 cmꢀ1
.
4.16. [125I]-( )-threo-N-(p-Azido-benzyl)-4-iodomethylpheni
Ki values for nonlinear regression of [3H]-WIN-35,428 displace-
ment curves were determined with GraphPad Prism 5.0 (GraphPad,
La Jolla, CA). The algorithm converts Ki values from IC50 values
using the Cheng–Prusoff equation: Ki = IC50/1 + [ligand]/Kd.46 The
regression best fit the data points using one site competitive bind-
ing curves. Irreversible binding was not otherwise addressed in
these assays (it is unknown whether the time course employed is
adequate for covalent linkage, nor was UV irradiation employed).
date ([125I]-( )-3a)
[
125I]-NaI (15
butylstannyl precursor ( )-7 (25
aqueous N-chloro-4-toluenesulfonamide (Chloramine-T) trihy-
drate (15 L, 5.0 mM) and a solution of MeOH (85 L) containing
3% glacial HOAc. After 2 min at ambient temperature, the mixture
was quenched with Na2S2O5 (10 L, 50 mM) then taken up in a syr-
inge along with a rinse (200 L) of the vessel with the ternary HPLC
l
L, 1.53 mCi) was treated with a solution of tri-n-
l
L, 6.0 mM) in MeOH followed by
l
l
l
l
4.18. DAT photoaffinity labeling
mobile phase consisting of MeOH (35%), CH3CN (35%), and an
aqueous solution (30%) containing Et3N (2.1% v/v) and HOAc
(2.8% v/v). A Waters C-18 Nova-Pak column (radial compression
Assessment of irreversible labeling of the DAT by [125I]-( )-3a was
performed using previously published procedures.13,14,17,20,28,30,40–43
Rat striatal membranes were prepared as previously described
module, 8 ꢂ 100 mm, 6
lm) was employed for separation at a flow

512
D. J. Lapinsky et al. / Bioorg. Med. Chem. 19 (2011) 504–512
14. For
(ꢀ)-N-[4-(30-[125I]iodo-40-azidophenyl)butyl]-2b-carbomethoxy-3b-(4-
and suspended at 20 mg/mL original wet weight in sucrose
phosphate buffer (SP, 0.32 M sucrose, 10 mM Na2HPO4, pH 7.4).
HEK-293 cells expressing 6Xhis hDAT were grown to 90% conflu-
ency in 6-well plates and washed with KRH buffer. For both prep-
arations, [125I]-( )-3a was added to a final concentration of 30 nM
followed by incubation for 1 h at 4 °C. For pharmacological dis-
chlorophenyl)tropane (MFZ-2-24) and (ꢀ)-N-[4-(30-[125I]iodo-40-isothiocyano-
phenyl)butyl]-2b-carbomethoxy-3b-(4-chlorophenyl)tropane (MFZ-3-37) see:
Lever, J. R.; Zou, M. F.; Parnas, M. L.; Duval, R. A.; Wirtz, S. E.; Justice, J. B.;
Vaughan, R. A.; Newman, A. H. Bioconjugate Chem. 2005, 16, 644.
15. For azido and isothiocyanate analogs of (S)-2b-substituted-3a-(bis[4-fluoro-
phenyl]methoxy)tropanes and (R)-2b-substituted-3b-(3,4-dichlorophenyl)-
tropanes see: Zou, M. F.; Kopajtic, T.; Katz, J. L.; Newman, A. H. J. Med. Chem.
2003, 46, 2908.
placement studies, 10 lM or 100 lM b-CFT or D-(+)-methylpheni-
16. For azido and isothiocyanate analogs of
3a
-[bis(40-fluorophenyl)-
date was included in the binding mixture. Membranes or cells
were irradiated with shortwave ultraviolet light (254 nm,
Fotodyne UV Lamp model 3-6000) for 45 s at a distance of
15–20 mm to photoactivate the radioligand. Membranes or cells
were washed twice with 1 mL of ice-cold SP or KRH buffer and
lysed with RIPA buffer (50 mM NaF, 2 mM EDTA, 125 mM Na3PO4,
1.25% Triton X-100, and 1.25% sodium deoxycholate) for 15 min at
0 °C. Lysates were centrifuged at 20,000 g for 15 min at 4 °C to re-
move insoluble material and the supernatants were transferred to
clean tubes for immunoprecipitation. Lysates were subjected to
immunoprecipitation as described previously13,14,17,20,28,30,40–43
using antiserum 16 generated against amino acids 42–59 of rDAT
or anti-his monoclonal antibody (Sigma–Aldrich) for his-tagged
hDAT. Immunoprecipitated samples were separated on 4–20%
SDS–polyacrylamide gels followed by autoradiography using
Hyperfilm MP film (GE Healthcare) for 1–4 days at ꢀ80 °C. Band
densities were quantified using LumiAnalyst software (Roche)
and expressed as a fraction of control samples that received no
inhibitor.
methoxy]tropane see: Zou, M. F.; Kopajtic, T.; Katz, J. L.; Wirtz, S.; Justice, J.
B.; Newman, A. H. J. Med. Chem. 2001, 44, 4453.
17. For an azido N-substituted
3a
-[bis(40-fluorophenyl)methoxy]tropane see:
Agoston, G. E.; Vaughan, R.; Lever, J. R.; Izenwasser, S.; Terry, P. D.; Newman,
A. H. Bioorg. Med. Chem. Lett. 1997, 7, 3027.
18. For azido and isothiocyanate substituted 3-carbamoylecgonine methyl ester
analogs see: Kline, R. H.; Eshleman, A. J.; Wright, J.; Eldefrawi, M. E. J. Med.
Chem. 1994, 37, 2249.
19. For irreversible ligands derived from (ꢀ)-cocaine or 3b-phenyltropan-2b-
carboxylic acid methyl ester (WIN-35, 065-2) see: Carroll, F. I.; Gao, Y.;
Abraham, P.; Lewin, A. H.; Lew, R.; Patel, A.; Boja, J. W.; Kuhar, M. J. J. Med.
Chem. 1992, 35, 1813.
20. For a 4-[2-(diphenylmethoxy)ethyl]-1-benzyl piperidine based on GBR-12909
see: Dutta, A. K.; Fei, X. S.; Vaughan, R. A.; Gaffaney, J. D.; Wang, N. N.; Lever, J.
R.; Reith, M. E. A. Life Sci. 2001, 68, 1839.
21. For azido and isothiocyanato analogs of [3-(4-phenylalkylpiperazin-1-
yl)propyl]bis(4-fluorophenyl)amines see: Cao, J.; Lever, J. R.; Kopajtic, T.;
Katz, J. L.; Pham, A. T.; Holmes, M. L.; Justice, J. B.; Newman, A. H. J. Med. Chem.
2004, 47, 6128.
22. For isothiocyanate analogs of 9-[3-(cis-3,5-dimethyl-1-piperazinyl)propyl]-
carbazole (Rimcazole) see: Husbands, S. M.; Izenwasser, S.; Loeloff, R. J.;
Katz, J. L.; Bowen, W. D.; Vilner, B. J.; Newman, A. H. J. Med. Chem. 1997, 40,
4340.
23. For an azido-diphenylpiperazine analog, 3-azido[3H]GBR-12935, see: Berger, S.
P.; Martenson, R. E.; Laing, P.; Thurkauf, A.; Decosta, B.; Rice, K. C.; Paul, S. M.
Mol. Pharmacol. 1991, 39, 429.
Acknowledgements
24. For
1-(2-[bis-(4-fluorophenyl)-methoxyethyl)-4-(2-[4-azido-3-[125I]iodo-
phenyl]ethyl)piperazine (FAPP) see: Sallee, F. R.; Fogel, E. L.; Schwartz, E.;
Choi, S. M.; Curran, D. P.; Niznik, H. B. FEBS Lett. 1989, 256, 219.
25. For
This work was funded by a Hunkele Dreaded Disease Award
(D.J.L.), the Mylan School of Pharmacy at Duquesne University
(D.J.L.), and NIH Grants DA27081 (D.J.L.), DA16604 (C.K.S.), and
DA15175 (R.A.V.). We thank NIDA Drug Supply for contribution
of certain nonradioactive DAT ligand compounds.
125I–1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethyl]-
piperazine (DEEP) see: Grigoriadis, D. E.; Wilson, A. A.; Lew, R.; Sharkey, J. S.;
Kuhar, M. J. J. Neurosci. 1989, 9, 2664.
26. For irreversible affinity labels based on aryl 1,4-dialkylpiperazines related to
GBR-12783 see: Deutsch, H. M.; Schweri, M. M.; Culbertson, C. T.; Zalkow, L. H.
Eur. J. Pharmacol. 1992, 220, 173.
27. For Fourphit, an isothiocyanate phencyclidine derivative that inhibits the
binding of [3H]-methylphenidate to rDAT see: Schweri, M. M.; Thurkauf, M. V.;
Mattson, M. V.; Rice, K. C. J. Pharmacol. Exp. Ther. 1992, 261, 936.
References and notes
28. For
a photoaffinity ligand based on pyrovalerone, a structural variant of
1. Nair, R.; Moss, S. B. Neuropsychiatr. Dis. Treat. 2009, 5, 421.
2. For recent examples see: (a) Kim, D. I.; Deutsch, H. M.; Ye, X.; Schweri, M. M. J.
Med. Chem. 2007, 50, 2718; (b) Froimowitz, M.; Gu, Y.; Dakin, L. A.; Nagafuji, P.
M.; Kelley, C. J.; Parrish, D.; Deschamps, J. R.; Janowsky, A. J. Med. Chem. 2007,
50, 219.
3. Solanto, M. V. Behav. Brain Res. 1998, 94, 127.
4. Fleckenstein, A. E.; Volz, T. J.; Riddle, E. L.; Gibb, J. W.; Hanson, G. R. Annu. Rev.
Pharmacol. Toxicol. 2007, 47, 681.
5. Woolverton, W. L.; Johnson, K. M. Trends Pharmacol. Sci. 1992, 13, 193.
6. (a) Beuming, T.; Kniazeff, J.; Bergmann, M. L.; Shi, L.; Gracia, L.; Raniszewska, K.;
Newman, A. H.; Javitch, J. A.; Weinstein, H.; Gether, U.; Loland, C. J. Nat.
Neurosci. 2008, 11, 780; (b) Bisgaard, H.; Larsen, M. A. B.; Mazier, S.; Beuming,
T.; Newman, A. H.; Weinstein, H.; Shi, L.; Loland, C. J.; Gether, U.
Neuropharmacology 2011, 60, 182; (c) Loland, C. J.; Desai, R. I.; Zou, M. F.;
Cao, J.; Grundt, P.; Gerstbrein, K.; Sitte, H. H.; Newman, A. H.; Katz, J. L.; Gether,
U. Mol. Pharmacol. 2008, 73, 813.
7. (a) Runyon, S. P.; Carroll, F. I. Curr. Top. Med. Chem. 2006, 6, 1825; (b)Dopamine
Transporters: Chemistry, Biology, and Pharmacology; Trudell, M. L., Izenwasser, S.,
Eds.; Wiley: Hoboken, NJ, 2008; pp 123–304.
8. Reith, M. E. A.; Berfield, J. L.; Wang, L.; Ferrer, J. V.; Javitch, J. A. J. Biol. Chem.
2001, 276, 29012.
bupropion (Wellbutrin, Zyban), see: Lapinsky, D. J.; Aggarwal, S.; Huang, Y.;
Surratt, C. K.; Lever, J. R.; Foster, J. D.; Vaughan, R. A. Bioorg. Med. Chem. 2009,
17, 3770.
29. Indarte, M.; Madura, J. D.; Surratt, C. K. Proteins 2008, 70, 1033.
30. Vaughan, R. A.; Parnas, M. L.; Gaffaney, J. D.; Lowe, M. J.; Wirtz, S.; Pham, A.;
Reed, B.; Dutta, S. M.; Murray, K. K.; Justice, J. B. J. Neurosci. Methods 2005, 143,
33.
31. Deutsch, H. M.; Shi, Q.; Gruszecka-Kowalik, E.; Schweri, M. M. J. Med. Chem.
1996, 39, 1201.
32. Misra, M.; Shi, Q.; Ye, X.; Gruszecka-Kowalik, E.; Bo, W.; Liu, Z.; Schweri, M. M.;
Deutsch, H. M.; Venanzi, C. A. Bioorg. Med. Chem. 2010, 18, 7221.
33. Pan, D.; Gatley, S. J.; Chen, R.; Ding, Y.-S. J. Labelled Compd. Radiopharm. 1996,
38, 523.
34. Axten, J. M.; Krim, L.; Kung, H. F.; Winkler, J. D. J. Org. Chem. 1998, 63, 9628.
35. Gutman, A.; Zaltsman, I.; Shalimov, A.; Sotrihin, M.; Nisnevich, G.; Yudovich, L.;
Fedotev, I. Patent US2004180928(A1).
36. Kiesewetter, D. O. Tetrahedron: Asymmetry 1993, 4, 2183.
37. Thai, D. L.; Sapko, M. T.; Reiter, C. T.; Bierer, D. E.; Perel, J. M. J. Med. Chem. 1998,
41, 591.
38. Mornet, R.; Leonard, N. J.; Theiler, J. B.; Doree, M. J. Chem. Soc., Perkin Trans. 1
1984, 5, 879.
39. Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64.
40. Parnas, M. L.; Gaffaney, J. D.; Zou, M. F.; Lever, J. R.; Newman, A. H.; Vaughan, R.
A. Mol. Pharmacol. 2008, 73, 1141.
41. Vaughan, R. A.; Sakrikar, D. S.; Parnas, M. L.; Adkins, S.; Foster, J. D.; Duval, R. A.;
Lever, J. R.; Kulkarni, S. S.; Newman, A. H. J. Biol. Chem. 2007, 282, 8915.
42. Vaughan, R. A.; Agoston, G. E.; Lever, J. R.; Newman, A. H. J. Neurosci. 1999, 19,
630.
9. Newman, A. H.; Kulkarni, S. S. Med. Res. Rev. 2002, 22, 1.
10. Ukairo, O. T.; Bondi, C. D.; Newman, A. H.; Kulkarni, S. S.; Kozikowski, A. P.; Pan,
S.; Surratt, C. K. J. Pharmacol. Exp. Ther. 2005, 314, 575.
11. For naphthyl-containing phenylisothiocyanate tropane analogs see: (a)
Murthy, V.; Davies, H. M. L.; Hedley, S. J.; Childers, S. R. Biochem. Pharmacol.
2007, 74, 336; (b) Murthy, V.; Martin, T. J.; Kim, S.; Davies, H. M. L.; Childers, S.
R. J. Pharmacol. Exp. Ther. 2008, 326, 587.
12. For 3-iodo-4-azidococaine and characterization of its binding site within the
sigma-1 receptor see: Chen, Y.; Hajipour, A. R.; Sievert, M. K.; Arbabian, M.;
Ruoho, A. E. Biochemistry 2007, 46, 3532.
43. Vaughan, R. A.; Kuhar, M. J. J. Biol. Chem. 1996, 271, 21672.
44. Shafi’ee, A.; Hite, G. J. Med. Chem. 1969, 12, 266.
45. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
46. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
13. For
[
125I]-3-b-(40-azido-30-iodo-biphenyl-4-yl)-8-methyl-8-aza-bicyclo-
[3.2.1]octane-2b-carboxylic acid methyl ester (JHC-2-48) see: Newman, A.
H.; Cha, J. H.; Cao, J.; Kopajtic, T.; Katz, J. L.; Parnas, M. L.; Vaughan, R.; Lever, J.
R. J. Med. Chem. 2006, 49, 6621.