Synthesis and in vivo evaluation of halogenated N,N-dimethyl-2-(2′- amino-4′-hydroxymethylphenylthio)benzylamine derivatives as PET serotonin transporter ligands

Article abstract of DOI:10.1021/jm0707929

N,N-Dimethyl-2-(2′-amino-4′-hydroxymethylphenylthio)benzylamine (38), substituted on ring A, was reported to display high binding affinity and selectivity to the human brain serotonin transporter (SERT). In an attempt to explore the potential of compounds substituted on ring B of the phenylthiophenyl core structure, three derivatives of 38 were synthesized: N,N-dimethyl-2- (2′-amino-4′-hydroxymethyl-phenylthio)-5-fluorobenzylamine (35), N,N-dimethyl-2-(2′-amino-4′-hydroxymethyl-phenylthio) -5-bromobenzylamine (36), and N,N-dimethyl-2-(2′-amino-4′- hydroxymethyl-phenylthio)-5-iodobenzylamine (37). The in vitro binding studies in cells transfected with human SERT, norepinephrine transporter (NET), and dopamine transporter (DAT) showed that 35, 36, and 37 exhibited high SERT affinity with K_is, (SERT) = 1.26, 0.29, and 0.31 nM (vs [ ³H]citalopram), respectively. [¹¹C]-(35), [ ¹¹C]-(36), and [¹¹C]-(37) were prepared by methylation of their monomethyl precursors 16, 17, and 18, with [¹¹C]iodomethane in 28, 11, and 14% radiochemical yields, respectively. The microPET images of [¹¹C]-(35), [¹¹C]-(36), and [¹¹C]-(37) showed high uptake in the monkey brain regions rich in SERT with peak midbrain to cerebellum ratios of 3.41, 3.24, and 3.00 at 85 min post-injection, respectively. In vivo bindings of [¹¹C]-(35), [¹¹C]-(36), and [¹¹C]-(37) were shown to be specific to the SERT as displacement with citalopram (a potent SERT ligand) reduced radioactivity in SERT-rich regions to the cerebellum level. These results suggest that [ ¹¹C]-OS), [¹¹C]-(36), and [¹¹C]-(37) could be potential agents for mapping human SERT by PET and radiolabeling 37 with iodine-123, which could afford the first SPECT SERT imaging agent exhibiting fast kinetics.

Full text of DOI:10.1021/jm0707929

J. Med. Chem. 2008, 51, 271–281
271
Synthesis and In Vivo Evaluation of Halogenated
N,N-Dimethyl-2-(2′-amino-4′-hydroxymethylphenylthio)benzylamine Derivatives as PET
Serotonin Transporter Ligands
Nachwa Jarkas,† Ronald J. Voll,† Larry Williams,† John R. Votaw,† Mike Owens,‡ and Mark M. Goodman*,†,‡
Department of Radiology and Department of Psychiatry and BehaVior Sciences, Emory UniVersity, Atlanta, Georgia 30322
ReceiVed July 3, 2007
N,N-Dimethyl-2-(2′-amino-4′-hydroxymethylphenylthio)benzylamine (38), substituted on ring A, was reported
to display high binding affinity and selectivity to the human brain serotonin transporter (SERT). In an attempt
to explore the potential of compounds substituted on ring B of the phenylthiophenyl core structure, three
derivatives of 38 were synthesized: N,N-dimethyl-2-(2′-amino-4′-hydroxymethyl-phenylthio)-5-fluoroben-
zylamine (35), N,N-dimethyl-2-(2′-amino-4′-hydroxymethyl-phenylthio)-5-bromobenzylamine (36), and N,N-
dimethyl-2-(2′-amino-4′-hydroxymethyl-phenylthio)-5-iodobenzylamine (37). The in vitro binding studies
in cells transfected with human SERT, norepinephrine transporter (NET), and dopamine transporter (DAT)
showed that 35, 36, and 37 exhibited high SERT affinity with Kis (SERT) ) 1.26, 0.29, and 0.31 nM (vs
[3H]citalopram), respectively. [11C]-(35), [11C]-(36), and [11C]-(37) were prepared by methylation of their
monomethyl precursors 16, 17, and 18, with [11C]iodomethane in 28, 11, and 14% radiochemical yields,
respectively. The microPET images of [11C]-(35), [11C]-(36), and [11C]-(37) showed high uptake in the
monkey brain regions rich in SERT with peak midbrain to cerebellum ratios of 3.41, 3.24, and 3.00 at 85
min post-injection, respectively. In vivo bindings of [11C]-(35), [11C]-(36), and [11C]-(37) were shown to
be specific to the SERT as displacement with citalopram (a potent SERT ligand) reduced radioactivity in
SERT-rich regions to the cerebellum level. These results suggest that [11C]-(35), [11C]-(36), and [11C]-(37)
could be potential agents for mapping human SERT by PET and radiolabeling 37 with iodine-123, which
could afford the first SPECT SERT imaging agent exhibiting fast kinetics.
Introduction
developed and evaluated various SPECT and PET radiotracers
for measurements of various components of the serotonin
system.5–11 Many of these compounds from the tropane motif,
except 2ꢀ-carbomethoxy-3ꢀ-(4′-((Z-2-iodo-ethenyl)phenyl)nortro-
pane,12,13 2ꢀ-carbomethoxy-3ꢀ-(4′-((Z-2-iodo-ethenyl) phenyl)
tropane,14 and 2ꢀ-carbomethoxy-3ꢀ-(3′-((Z-2-iodo-ethenyl)phe-
nyl)nortropane15 have failed to show promising imaging prop-
erties in the nonhuman primate and human brain. The most
common limitation has been a combination of deficient selectiv-
ity, inappropriate kinetics, and inadequate in vivo binding
specificity.
Depression is a chronic debilitating mental disease affecting
more than 10% of the general population. The need for more
effective treatment of depression continues to command attention
in neuropsychopharmacology and within the psychotherapeutic
community.1 Theories on the pathophysiology of depression
have emphasized deficiencies of various aspects of serotonergic
transmission controlled in part by the serotonin transporter
(SERT).2 The principal function of the SERT is to terminate
the action of the serotonin (indoleamine, 5-hydroxytryptamine
[5-HT]) by removing it from the synaptic cleft and returning it
to the presynaptic neuron where the neurotransmitter can be
degraded or retained for rerelease at a later time.3 To date, there
is evidence that abnormalities in the brain central serotonin
function are involved in several neuropsychiatric conditions,
including major depression, anxiety, and obsessive compulsive
disorder.3 Because of the perceived importance of the brain
serotonin system in these diseases, as well as a variety of normal
brain functions like appetite and sleep, considerable interest has
been focused on the development of functional neuroimaging
techniques (e.g., positron emission tomography [PET] and
single-photon emission computed tomography [SPECT]) that
permit assessment of serotonin neurons in the living human
brain.4 Neuroimaging research has emerged as a valuable tool
in shaping our understanding of the pathophysiology of psy-
chiatric disorders. To this end, several laboratories have
In the past few years, there has been considerable interest in the
phenylthiophenyl core structure of the serotonin reuptake inhibitor
403U76 (Figure 1)16 that led to a new class of compounds: the
substituted diaryl sulfides. From this same class, a large collection
of carbon-11, fluorine-18,17–20 and iodine-12321,22 labeled radio-
ligands have been synthesized and evaluated as potential PET and
SPECT SERT imaging agents. The carbon-11 radiotracers included
[11C]-(38),23,24 [11C]-(39),23,25 [11C]-(40),26–28 ([11C]-(40) is 11C-
DASB, a well established PET SERT imaging agent widely used)
[11C]-(41),26 [11C]-(42),23,29,30 [11C]-(43),27,31 [11C]-(44),27
[11C]-(45),27,32 [11C]-(46),33 and [11C]-(47)34 (Figure 1). Ra-
diolabeled with iodine-123, 44 was reported to bind selectively
to the human brain SERT.35–37 However, this radioligand
exhibited slow brain kinetics, reaching a near steady state 3 h
after tracer injection.37 In our laboratory, preclinical studies
indicated that [11C]-(38) exhibited high affinity and selectivity
for the SERT in vitro and that, in vivo, it showed selective
binding to the SERT in the regions of interest of a rhesus
monkey brain.24 Furthermore, [11C]-(38) displayed better tissue-
to-cerebellum ratios relative to [11C]-(40) and an earlier time
to reach maximal uptake relative to [11C]-(45).24 Also, low
* To whom correspondence should be addressed. Mark M. Goodman,
Ph.D., Emory University, School of Medicine, Department of Radiology,
1364 Clifton Road, NE, Atlanta, GA 30322. Phone: (404) 727-9366. Fax:
(404) 727-3488. E-mail: mgoodma@emory.edu.
†
Department of Radiology.
Department of Psychiatry and Behavior Sciences.
‡
10.1021/jm0707929 CCC: $40.75
2008 American Chemical Society
Published on Web 12/18/2007

272 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Jarkas et al.
Figure 1. Substituted diaryl sulfide compounds.
Scheme 1. Synthesis of 5-Iodo-2-mercaptobenzoic Acid (6)a
is presented in Schemes 1-3. Compounds 2, 4, 5, and 19 were
commercially available. Compound 124 was prepared from
4-bromo-3-nitro-toluene (19) using previously described pro-
cedures.24 Diazotization40 of 2-amino-5-iodobenzoic acid (2)
followed by a reaction with sodium disulfide, gave 2,2′-dithiobis-
(5-iodobenzoic) acid (3; Scheme 1). Disulfide (3) was reduced
with sodium borohydride in anhydrous tetrahydrofuran to give
the 5-iodo-2-mercaptobenzoic acid (6) in very good yield
(Scheme 1).
a Conditions: (i) H2O, HCl, NaNO2, Na2S2; (ii) NaBH4, THF, 0 °C to rt,
15 min.
The synthesis of fluoro-, bromo-, and iodo-monomethy-
lamines 16, 17, and 18 was initiated by the coupling41 of 124
with thiosalicylic acids 4, 5, and 6 to afford 7, 8, and 9,
respectively, in good yields (Scheme 2). Treatment of 7, 8, and
9 with thionyl chloride in refluxing dichloromethane (DCM)
and a catalytic amount of dimethylformamide (DMF) gave the
crude acid chlorides, which were reacted, without further
purification, with methylamine hydrochloride in anhydrous
dichloromethane to yield the desired monomethylbenzamides
10, 11, and 12, respectively, in low yields (Scheme 2). Reduction
of the amide and acetoxy groups of 10, 11, and 12 with the
diborane-tetrahydrofuran (1 M) complex provided 13, 14, and
15, which, after reduction of the nitro group with tin chloride,
were transformed to 16, 17, and 18, respectively.
Under the same reaction conditions used to prepare 7, 8, and
9, condensation of 19 with thiosalicylic acids 4, 5, and 6
proceeded smoothly in dimethylformamide, yielding 20, 21, and
22, respectively, which, under the appropriate conditions, were
converted to dimethylbenzamides 23, 24, and 25 (Scheme 3).
Radical bromination with N-bromosuccinimide of 23, 24, and
25 proceeded smoothly in carbon tetrachloride,42 yielding 26,
27, and 28, which were subsequently transformed to 29, 30,
and 31 by nucleophilic substitution with potassium acetate. The
target compounds 35, 36, and 37 were obtained from 29, 30,
and 31, respectively, following the same conditions used to
prepare 16, 17, and 18 (Scheme 3).
concentrations of radioactive metabolites in arterial plasma
samples of rhesus monkey blood, relative to the parent
compound, suggested that this radioligand holds significant
potential for use in evaluating the status of the SERT in clinical
settings. Indeed, it has since advanced to imaging applications
in humans.38 Most of the diaryl sulfides reported are derivatives
having substitutions on phenyl ring A. Recently, few derivatives
of the diaryl sulfide class with substitutions on phenyl ring B
have been reported.39 The 4′-iodo-biphenylthiol derivative
peaked at 6–12 h after tracer injection, which might be too slow
for SPECT imaging.
The desire of further exploring the potential of compounds
substituted on ring B and the interesting results shown by 38
prompted us to think about incorporating a halogen atom (F,
Br, and I) onto the 5-position of ring B of the phenylthiophenyl
structure of 38 (Figure 1). In this paper, we report the synthesis,
radiolabeling, pharmacological characterization, and microPET
imaging studies, in cynomolgus monkeys, of 35, 36, and 37,
three halogeno-derivative compounds of 38.
Results and Discussion
Chemistry. The synthesis of the new target ligands N,N-
dimethyl-2-(2′-amino-4′-hydroxymethyl-phenylthio)-5-fluoroben-
zylamine (35), N,N-dimethyl-2-(2′-amino-4′-hydroxymethyl-
phenylthio)-5-bromobenzylamine (36), and N,N-dimethyl-2-(2′-
amino-4′-hydroxymethyl-phenylthio)-5-iodobenzylamine (37;
Scheme 3), as well as the monomethyl derivatives N-methyl-
2-(2′-amino-4′-hydroxymethylphenylthio)-5-fluoro-benzyl-
amine (16), N-methyl-2-(2′-amino-4′-hydroxymethylphenylthio)-
5-bromo-benzylamine (17), and N-methyl-2-(2′-amino-4′-
hydroxymethylphenylthio)-5-iodo-benzylamine (18; Scheme 2),
required as precursors for radiolabeling with [11C]iodomethane,
Radiochemistry. The labeling of [11C]-(35), [11C]-(36), and
[11C]-(37) was accomplished by alkylation of precursors 16,
17, and 18 with [11C]iodomethane, as depicted in Scheme 4.
The monomethylbenzylamines 16, 17, and 18 reacted with
cyclotron produced [11C]iodomethane, at room temperature for
5 min, on a standard High Performance Liquid Chromatography
(HPLC) loop injector in a manner similar to the previously

PET Serotonin Transporter Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 273
Scheme 2. Synthesis of the Monomethyl Precursors 16, 17, and 18a
a Conditions: (i) K2CO3, DMF, 130 °C; (ii) 1-SOCl2, DCM, reflux; 2-DCM/Et3N, CH3NH2, HCl, -70 °C; (iii) BH3-THF, reflux, 5 h, rt, 18 h; (iv) SnCl2,
cc.HCl/CH3OH, 0 °C to rt.
Scheme 3. Synthesis of the Target N,N-Dimethyl Benzylamines 35, 36, and 37a
a Conditions: (i) K2CO3, DMF, 130 °C; (ii) 1-SOCl2, DCM, reflux; 2-(CH3)2NH/THF, -70 °C; (iii) NBS, AIBN, CCl4, reflux; (iv) CH3CO2K, DMF; (v)
BH3-THF, reflux, 5 h, rt, 18 h; (vi) SnCl2, cc.HCl/CH3OH, 0 °C to rt.
Scheme 4. Synthesis of [11C]-(35), [11C]-(36), and [11C]-(37)
decay-corrected radiochemical yield (based on [11C]iodomethane
production). The yields are for final formulated products,
corrected for decay and normalized for a 60 min (40 µA)
bombardment that produces approximately 500 mCi of
[11C]iodomethane from the GE MicroLab methyl iodide system.
The amount of radioactivity formed (500 mCi) was measured
by trapping the [11C]iodomethane released in 200 µL of DMF
in a sealed vial immersed in an ice/water bath. Analytical HPLC
showed that the labeled products [11C]-(35), [11C]-(36), and
[11C]-(37) had chemical and radiochemical purities of >99%.
The lipophilicities (n-octanol/0.02 M phosphate buffer partition)
(log P7.4) of these radiotracers were measured24,25 and are
presented in Table 1. Log P7.4 is an important parameter for
correlating structure with brain penetrance and ligand-transporter
kinetic behavior. [11C]-(35), [11C]-(36), and [11C]-(37) had log
P7.4 values of 2.06, 2.63, and 1.82, respectively (Table 1). [11C]-
(37) (1.82) exhibited lower log P7.4 than [11C]-(35) (2.06), and
[11C]-(37) (2.63). Comparison between the parent 5-unsubsti-
tuted compound 38 and the 5-halogeno-substituted compounds
35, 36, and 37 demonstrated that a halogen (F, Br, and I)
reported “loop method”.43 [11C]-(35), [11C]-(36), and [11C]-(37)
were purified by reversed-phase HPLC. For in vivo studies, the
HPLC fractions containing [11C]-(35) were combined and the
desired product was formulated in 10% ethanol in saline using
a C18 solid-phase-extraction cartridge.24,44 [11C]-(36) and [11C]-
(37) were formulated in a similar manner. The entire labeling,
purification, and formulation procedure required approximately
45 min from end of bombardment. [11C]-(35), [11C]-(36), and
[11C]-(37) were prepared with a specific activity of 1550 ( 610
mCi/µ mol (n ) 3), 750 ( 320 mCi/µ mol (n ) 3), and 663 (
350 mCi/µ mol (n ) 3; at time of injection), in 28 ( 13% (n
) 3), 11 ( 4% (n ) 3), and 14 ( 5% (n ) 3), respectively,

274 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Jarkas et al.
Table 1. Binding Affinities (Ki (nM)) of Candidate SERT Ligands in Cells Expressing Human Transporters and Lipophilicities (log P7.4
)
cmpd
SERTa (n)b
DATc (n)
NETd (n)
log P7.4
DAT/SERT
NET/SERT
35
36
1.26 ( 0.06 (2)
0.29 ( 0.01 (3)
0.31 ( 0.02 (2)
0.57 ( 0.05 (3)
>2000 (2)
221 ( 18 (2)
>2000 (2)
>1000 (3)
618 ( 120 (2)
725 ( 30 (2)
467 ( 29 (2)
303 ( 226 (3)
2.06 ( 0.03
2.63 ( 0.2
1.82 ( 0.1
1.60 ( 0.01
>1000
762
>1000
>1000
490
>2000
>1000
532
37
3824
a Competitive binding versus [3H]citalopram in human kidney cells transfected with human serotonin transporters. b Number of assays. Values were
obtained from the mean ( SD of at least two independent assays each in duplicates. c Competitive binding versus [125I]RTI-55 in canine kidney cells
transfected with human dopamine transporters. d Competitive binding versus [3H]nisoxetine in human kidney cells transfected with human norepinephrine
transporters.
Figure 2. MicroPET time-activity curves of a male cynomolgus
Figure 3. MicroPET time-activity curves of a male cynomolgus
monkey brain regions, thalamus, midbrain, pons, medulla, frontal cortex,
monkey brain regions, caudate, putamen, thalamus, midbrain, pons,
and cerebellum after i.v. injection of 15.26 mCi of [11C]-(35).
medulla, cerebellum, occipital cortex, and frontal cortex after i.v.
injection of 11.36 mCi of [11C]-(36).
increased log P7.4 by 0.22–1.03. Furthermore, the 1.82 value of
[11C]-(37) was most similar to the measured value of 1.60 of
38 (Table 1), which achieved brain penetrance and good specific
(midbrain) to nonspecific binding (cerebellum) ratios greater
than 4.24
In Vitro Competition Assays. The affinities of N,N-dimethyl
derivatives 35, 36, and 37 for the human SERT (hSERT), human
DAT (hDAT), and human NET (hNET) were determined
through in vitro competition assays according to a known
procedure previously reported.23 These data are shown in Table
1 along with the previously determined Ki values of 38.24 The
data in Table 1 showed that 35, 36, and 37 displayed high
affinity and selectivity for the SERT over the DAT and NET.
The rank of order of hSERT affinity (Ki in nM) was 36 (0.29)
> 37 (0.31) > 38 (0.57)24 > 35 (1.26). Furthermore, 35, 36,
and 37 exhibited low affinities for the hDAT and hNET (Ki >
200 nM; Table 1). These findings demonstrated that the
incorporation of a halogen (F, Br, and I) onto the 5-position of
ring B of the diphenyl sulfide structure of 38 resulted in minor
changes in the binding potency to the SERT.
MicroPET Nonhuman Primate Imaging. The regional brain
distribution of [11C]-(35), [11C]-(36), and [11C]-(37) was
Figure 4. MicroPET time-activity curves of a male cynomolgus
monkey brain regions, caudate, putamen, thalamus, midbrain, pons,
medulla, frontal cortex, occipital cortex, and cerebellum after i.v.
injection of 18.20 mCi of [11C]-(37).
determined in an anesthetized male cynomolgus monkey using
a Concorde MicroPET P4. MicroPET imaging studies were
performed to determine the uptake of [11C]-(35), [11C]-(36), and
[11C]-(37) in the brain regions rich in SERT. The regional
distribution of radioactivity in the brain following administration
of [11C]-(35), [11C]-(36), and [11C]-(37) showed the highest
uptake in the midbrain such as hypothalamus, a region where
the SERT density is high, whereas the lowest uptake was
detected in the SERT poor cerebellum (Figures 2, 3, 4, and 5).
MicroPET time-activity curves for the thalamus, midbrain,
pons, medulla, frontal cortex, and cerebellum in a cynomolgus
monkey after [11C]-(35) injection are presented in Figure 2.
Following administration of [11C]-(35), the rank of order of
uptake of radioactivity in the cynomolgus monkey brain was
midbrain > pons > thalamus ∼ medulla, regions rich with

PET Serotonin Transporter Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 275
Figure 5. Summed (0–120 min) transaxial (A), and sagittal (B) images
from microPET acquisitions in a male cynomolgus monkey after i.v.
injection of 18.20 mCi of [11C]-(37).
Table 2. Region-of-Interest to Cerebellum Activity Ratios at 85 min
after Injection of [11C]-(35), [11C]-(36), and [11C]-(37)
Figure 6. MicroPET time-activity curves of a male cynomolgus
monkey brain regions, caudate, putamen, thalamus, midbrain, pons,
medulla, frontal cortex, occipital cortex, and cerebellum after i.v.
injection of 18.10 mCi of [11C]-(35). Citalopram (1.5 mg/kg body
weight) was administered at 45 min post-injection of [11C]-(35).
region
caudate
putamen
thalamus
midbrain
pons
medulla
occipital cortex
frontal cortex
[
11C]-(35)
[
11C]-(36)
[
11C]-(37)
1.70
2.40
2.30
3.24
2.80
2.60
1.34
1.06
1.80
2.30
2.06
3.00
1.65
2.40
1.20
1.09
2.30
3.40
2.80
2.10
These small differences in the uptake of radioactivity in the
cynomolgus monkey brain between [11C]-(35), [11C]-(36), and
[11C]-(37) in comparison to [11C]-(38) suggest that the hydroxy-
lmethyl substituent on the 4-position of ring A of the biphe-
nylthiol motif has the dominant influence on SERT binding.
The faster binding kinetics exhibited by [11C]-(35) in comparison
to [11C]-(36) and [11C]-(37) may reflect a lower binding affinity
to the SERT (Table 1). Furthermore, the lower midbrain-to-
cerebellum ratios, at 85 min, of [11C]-(35) (3.41), [11C]-(36)
(3.24), and [11C]-(37) (3.00; Table 2) and the slower kinetics
relative to [11C]-(38) (3.70∼4.00)24 at the same time may be
explained in part by the fact that [11C]-(35), [11C]-(36), and
[11C]-(37) have higher log P7.4 values, 2.06, 2.63, and 1.82,
respectively, than [11C]-(38) (1.60; Table 1). Furthermore, for
[11C]-(37), the uptake kinetics, peaking between 12.5 and 22.5
min, may serve for translation of 37 to SPECT imaging when
labeled with iodine-123. [123I]-(37) has the potential to be the
first example of an iodinated radioligand with fast kinetics, based
on the biphenylthiol core structure, to image the SERT by
SPECT.
1.04
SERT. Lower uptake was measured in the frontal cortex,
whereas the lowest uptake was detected in the cerebellum
(Figure 2). Surprisingly, the caudate and putamen regions were
not readily visible and could not be included in Figure 2. The
regional distribution of radioactivity in the brain following
administration of [11C]-(35) was consistent with binding to
SERT-rich sites (Figure 2). The peak uptake of radioactivity in
the midbrain occurred between 12.5 and 27.5 min after injection
and then declined with a T1/2 ) 90 min. The brain regions of
interest (ROIs) to cerebellum ratios at 85 min after [11C]-(35)
injection are presented in Table 2.
Figure 3 shows the time-activity curves obtained after
injection of [11C]-(36) into a cynomolgus monkey. The uptake
of radioactivity occurred in the following order: midbrain >
pons > medulla > putamen > thalamus > caudate. The caudate
and putamen regions are rich with both DAT and SERT. Lower
uptake was measured in the occipital cortex and frontal cortex,
whereas the lowest uptake was observed in the cerebellum
(Figure 3). The peak uptake of radioactivity in the midbrain
was achieved between 15.5 and 32.5 min and then declined
slowly. The brain regions of interest to cerebellum ratios at 85
min after [11C]-(36) injection are presented in Table 2.
The regional distribution of radioactivity in the brain of a
cynomolgus monkey following administration of [11C]-(37) is
depicted in the time-activity curves for brain ROIs (Figure 4).
The rank order of uptake of radioactivity in the cynomolgus
brain was midbrain > medulla ∼ putamen > thalamus > pons
∼ caudate. Lower uptake was observed in the occipital cortex
and frontal cortex. The lowest uptake occurred in the cerebellum
(Figure 4). The radioactivity uptake in the midbrain peaked
between 12.5 and 22.5 min and then declined with a T1/2 ) 90
min. The brain regions of interest to cerebellum ratios at 85
min after [11C]-(37) injection are presented in Table 2. The
summed microPET images acquired from 0 to 120 min in a
male cynomolgus monkey following [11C]-(37) accumulation
in the midbrain are shown in Figure 5.
To demonstrate that the brain regional [11C]-(35), [11C]-(36),
and [11C]-(37) binding was specific to the SERT, in vivo
microPET displacement studies by the SERT ligand R,S-
citalopram·HBr were performed on the same male cynomolgus
monkey. A dose (1.5 mg/kg body weight) of R,S-cit-
alopram·HBr was administered intravenously at 45 min post
[11C]-(35) and at 60 min post [11C]-(36) and [11C]-(37) and
resulted in a significant reduction of radioactivity in the SERT-
rich brain regions, caudate, putamen, thalamus, midbrain, pons,
medulla, frontal cortex, and occipital cortex, while the uptake
in the cerebellum was not affected (Figures 6, 7, and 8).
The reduction in the tissue-to-cerebellum ratios of [11C]-(35),
[11C]-(36), and [11C]-(37) in the regions of interest of the chase
scan before (at 45 min for [11C]-(35) and at 55 min for [11C]-
(36) and [11C]-(37)) and after (at 85 min for [11C]-(35) and (at
115 min for [11C]-(36) and [11C]-(37)) citalopram administration
(Table 3) indicated that regional [11C]-(35), [11C]-(36), and
[11C]-(37) uptake reflected specific SERT binding.
Conclusion
A halogen (F, Br, and I) was incorporated onto the 5-position
of phenyl ring B of the diphenyl sulfide core structure of 38 to

276 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Jarkas et al.
uptake in the midbrain, thalamus, pons, medulla, caudate,
putamen (except for [11C]-(35), where the caudate and putamen
were not visible in the PET images), and cortical regions of
the brain, with a peak uptake established around 12–27 min.
Citalopram chase studies with [11C]-(35), [11C]-(36), and [11C]-
(37) confirmed the binding of these radiotracers to the monkey
brain SERT. Furthermore, the current data also suggested that
[11C]-(37) has attractive kinetic characteristics as a PET SERT
ligand when radiolabled with carbon-11, and when radiolabeled
with iodine-123, this radiotracer may serve as the first candidate
of the diphenyl sulfide series, with fast kinetics, to image the
SERT in vivo by SPECT.
Experimental Section
Proton nuclear magnetic resonance (NMR) spectra were run on
a Varian Unity 400 at 400 MHz (1H) in CDCl3 with tetramethyl-
silane (TMS) as internal standard. Elemental analyses were
performed by Atlantic Microlabs, Inc. (Atlanta, GA) and were
within (0.4% of theoretical values. Compound 124 was prepared
and purified by methods previously reported.24 All other chemicals
were purchased from Aldrich Chemical Co. and were used without
further purification. Purification and analyses of radioactive com-
pounds by HPLC were performed with in-line UV (254 nm)
detectors in series with a radioactivity detector. The HPLC columns
used were either a Waters XTerra C18 reverse phase (19 × 100
mm; column A) for purification or a Waters C18 reverse phase (3.9
× 150 mm; column B) for quality control.
Figure 7. MicroPET time-activity curves of a male cynomolgus
monkey brain regions, caudate, putamen, thalamus, midbrain, pons,
medulla, cerebellum, occipital cortex, and frontal cortex after i.v.
injection of 6.90 mCi of [11C]-(36). Citalopram (1.5 mg/kg body weight)
was administered at 60 min post-injection of [11C]-(36).
2,2′-Dithiobis(5-iodobenzoic) Acid (3). A solution of 2-amino-
5-iodobenzoic acid (2; 10.00 g, 38.00 mmol) in a mixture of water
(76 mL) and concentrated hydrochloric acid (cc. HCl; 13 mL) was
diazotized at 0 °C with a solution of sodium nitrite (NaNO2; 4.50 g,
65.22 mmol) in water (25 mL) added dropwise. The solution was
stirred 1 h at 0 °C and then added to a solution of Na2S2 [prepared
by heating sodium sulfide ·nonahydrate (Na2S·9H2O; 16.70 g, 69.53
mmol) and S (2.20 g, 68.62 mmol) in water (200 mL), treating the
yellow clear solution with NaOH (2.50 g, 62.50 mmol) in water
(20 mL) and cooling] containing ice. The mixture was allowed to
stand for 3 h at room temperature and acidified with cc. HCl, and
the precipitated crude product was filtered. It was dissolved in a
warm solution of Na2CO3 and filtered with charcoal through celite,
and the filtrate was acidified again with cc. HCl. The precipitated
product was filtered, washed with water, and dried: orange solid
(10.58 g, 50%). It was used in the next step without further
purification.
5-Iodo-2-mercaptobenzoic Acid (6). To a 50 mL, round-bottom
flask capped with a rubber septum was added anhydrous tetrahy-
drofuran (THF; 7 mL) under argon (g). To this was added 3 (1.00
g, 1.79 mmol) to give a yellow solution of the disulfide in the
solvent. The flask was immersed in an ice/water bath. To the
reaction mixture was slowly and portionwise added sodium
borohydride (NaBH4; 0.27 g, 7.17 mmol). The color of the mixture
turned to red-brown immediately after addition of the hydride
reagent and the mixture became homogeneous. After 15 min, the
reaction was quenched with cold water and acidified with cc. HCl.
The precipitated product was filtered, washed with water, and dried
to give the desired thiol (6) as a light yellow solid. (0.48 g, 96%).
1H NMR (CDCl3, δ): 4.66 (s, 1H, SH), 7.08 (d, 1H, J ) 8.0 Hz,
ArH), 7.64 (dd, 1H, J ) 2.0, 8.0 Hz, ArH), 8.42 (d, 1H, J ) 1.6
Hz, ArH). MS m/z 278.90 (M – H)+.
Figure 8. MicroPET time-activity curves of a male cynomolgus
monkey brain regions, caudate, putamen, thalamus, midbrain, pons,
medulla, frontal cortex, occipital cortex, and cerebellum after i.v.
injection of 8.97 mCi of [11C]-(37). Citalopram (1.5 mg/kg body weight)
was administered at 60 min post-injection of [11C]-(37).
Table 3. Region-of-Interest to Cerebellum Activity Ratios Post-Injection
of [11C]-(35), [11C]-(36), and [11C]-(37) Before and After Citalopram
Administration
[
11C]-(35)
[
11C]-(36)
[
11C]-(37)
at 45 min
(85 min)
at 55 min
(115 min)
at 55 min
(115 min)
region
caudate
putamen
thalamus
midbrain
pons
medulla
occipital cortex
frontal cortex
1.60 (1.06)
1.80 (1.20)
1.90 (1.30)
2.30 (1.20)
2.00 (1.40)
2.00 (1.10)
1.30 (1.04)
1.06 (1.00)
1.40 (1.00)
1.60 (1.20)
1.80 (1.40)
2.40 (1.20)
2.00 (1.23)
2.30 (1.10)
1.35 (1.10)
1.00 (1.00)
1.80 (1.30)
1.90 (1.40)
2.00 (1.63)
2.40 (1.70)
1.90 (1.50)
2.50 (1.60)
1.20 (1.10)
1.00 (1.00)
2-(4′-Acetoxymethyl-2′-nitrophenylthio)-5-fluorobenzoic Acid
(7). To a solution of 1 (0.30 g, 1.09 mmol) in DMF (7 mL) was
added fluorothiosalicylic acid (4; 0.19 g, 1.09 mmol) followed by
K2CO3 (0.45 g, 3.28 mmol). The reaction mixture was stirred at
130 °C for 18 h. After cooling down to room temperature, the
reaction mixture was poured into cold water and extracted with
ethylacetate (EtOAc). Aqueous layers were acidified with concd
HCl and extracted with EtOAc. Organic layers were washed with
water, dried over Na2SO4, and concentrated under reduced pressure
to give the crude product as a yellow solid. Purification by silica
give the fluoro-, bromo-, and iodo-diphenyl sulfides 35, 36, and
37, respectively. In vitro and in vivo studies demonstrated that
the three carbon-11-labeled compounds were selective and
potent radioligands suitable to image the SERT by PET. In vivo
microPET imaging studies in a male cynomolgus monkey
showed that [11C]-(35), [11C]-(36), and [11C]-(37) displayed high

PET Serotonin Transporter Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 277
gel flash column chromatography (DCM/MeOH: 9/1) afforded 7
as an orange oil (0.33 g, 82%). 1H NMR (CDCl3, δ): 2.12 (s, 3H,
COCH3), 5.11 (s, 2H, CH2O), 6.92 (d, 1H, J ) 8.0 Hz, ArH), 7.29
(m, 1H, ArH), 7.37 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.53 (m, 1H,
ArH), 7.77 (dd, 1H, J ) 2.8, 8.8 Hz, ArH), 8.17 (d, 1H, J ) 1.6
Hz, ArH).
NCH3), 5.09 (s, 2H, CH2O), 6.20 (s, 1H, NH), 6.90 (d, 1H, J )
8.4 Hz, ArH), 7.29 (d, 1H, J ) 8.0 Hz, ArH), 7.37 (dd, 1H, J )
2.0, 8.4 Hz, ArH), 7.82 (dd, 1H, J ) 2.0, 8.0 Hz, ArH), 8.07 (d,
1H, J ) 1.6 Hz, ArH), 8.20 (d, 1H, J ) 2.0 Hz, ArH). Anal.
(C17H15IN2O5S) C, H, N. MS m/z 486.98 (M + H)+.
N-Methyl-2-(5′-hydroxymethyl-2′-nitrophenylthio)-5-fluoro-
benzylamine (13). To a solution of 10 (0.10 g, 0.26 mmol) in dry
THF (5 mL), BH3-THF complex (1 M soln in THF; 2.64 mL,
2.64 mmol) was added dropwise at 5 °C. The mixture was refluxed
5 h and left at room temperature 18 h. The reaction was quenched
by addition of concd HCl at 0 °C and the solvent was removed under
reduced pressure. To the residue, water (5 mL) was added and the
mixture was refluxed for 30 min with vigorous stirring. After
cooling down to room temperature, a saturated solution of Na2CO3
was added to neutralize the solution to pH 6∼7. The resulting
aqueous solution was extracted with EtOAc. Combined organic
layers were dried over Na2SO4, and the solvent was removed under
reduced pressure. Silica gel flash column chromatography purifica-
tion (DCM/MeOH: 9/1) gave 13 as a yellow oil (66.30 mg, 78%).
1H NMR (CDCl3, δ): 2.39 (s, 3H, NCH3), 3.78 (s, 2H, CH2N),
4.72 (s, 2H, CH2OH), 6.63 (d, 1H, J ) 8.4 Hz, ArH), 7.08 (m, 1H,
ArH), 7.37 (m, 2H, ArH), 7.57 (m, 1H, ArH), 8.27 (d, 1H, J ) 1.6
Hz, ArH). Anal. (C15H15FN2O3S) C, H, N. MS m/z 323.08 (M +
H)+.
2-(4′-Acetoxymethyl-2′-nitrophenylthio)-5-bromobenzoic Acid
(8). Compound 8 was prepared by the method described for 7, by
condensation of 1 (3.18 g, 11.61 mmol) with 5 (2.00 g, 11.61 mmol)
in DMF (20 mL) in the presence of K2CO3 (4.82 g, 34.85 mmol)
as a base. Silica gel flash column chromatography purification
1
(DCM/MeOH: 9/1) afforded 8 as an orange oil (2.37 g, 48%). H
NMR (CDCl3, δ): 2.11 (s, 3H, COCH3), 5.13 (s, 2H, CH2O), 7.06
(d, 1H, J ) 8.4 Hz, ArH), 7.16 (d, 1H, J ) 8.4 Hz, ArH), 7.31
(dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.55 (dd, 1H, J ) 2.4, 8.8 Hz,
ArH), 7.88 (d, 1H, J ) 1.2 Hz, ArH), 8.21 (d, 1H, J ) 2.4 Hz,
ArH).
2-(4′-Acetoxymethyl-2′-nitrophenylthio)-5-iodobenzoic Acid
(9). Compound 9 was prepared by the method described for 7, by
condensation of 1 (0.20 g, 0.73 mmol) with 6 (0.20 g, 0.73 mmol)
in DMF (5 mL) in the presence of K2CO3 (0.30 g, 2.19 mmol) as
a base. Silica gel flash column chromatography purification (DCM/
MeOH: 9/1) afforded 9 as an orange solid (0.21 g, 61%). 1H NMR
(CDCl3, δ): 2.13 (s, 3H, COCH3), 5.13 (s, 2H, CH2O), 7.03 (d,
1H, J ) 8.8 Hz, ArH), 7.19 (d, 1H, J ) 8.0 Hz, ArH), 7.45 (m,
2H, ArH), 7.90 (d, 1H, J ) 1.2 Hz, ArH), 8.19 (d, 1H, J ) 2.4 Hz,
ArH).
N-Methyl-2-(5′-hydroxymethyl-2′-nitrophenylthio)-5-bromo-
benzylamine (14). Amide (11; 0.15 g, 0.34 mmol) was treated with
BH3-THF (1 M soln in THF; 1.70 mL, 1.70 mmol) as described
for 13. Silica gel flash column chromatography (DCM/MeOH: 9/1)
N-Methyl-2-(5′-acetoxymethyl-2′-nitrophenylthio)-5-fluoro-
benzamide (10). To a suspension of 7 (0.31 g, 0.85 mmol) in
anhydrous DCM (8 mL) were added thionyl chloride (0.25 g, 2.12
mmol) and a few drops of DMF. The reaction mixture was refluxed
for 90 min under argon. The solvent was evaporated and the solid
was used without further purification.
1
gave 14 as a yellow oil (74.27 mg, 57%). H NMR (CDCl3, δ):
2.38 (s, 3H, NCH3), 3.77 (s, 2H, CH2N), 4.70 (s, 2H, CH2OH),
6.66 (d, 1H, J ) 8.4 Hz, ArH), 7.34 (dd, 1H, J ) 1.6, 8.0 Hz,
ArH), 7.42 (d, 1H, J ) 8.0 Hz, ArH), 7.50 (dd, 1H, J ) 2.4, 8.4
Hz, ArH), 7.78 (d, 1H, J ) 2.0 Hz, ArH), 8.25 (d, 1H, J ) 2.0 Hz,
ArH). Anal. (C15H15BrN2O3S) C, H, N.
A solution of triethylamine (0.67 g, 6.67 mmol) and methylamine
hydrochloride (0.22 g, 3.33 mmol) in anhydrous DCM (10 mL)
was cooled down to -70 °C. The acid chloride (0.32 g, 0.85 mmol;
freshly prepared) in DCM (5 mL) was added dropwise under argon.
The reaction mixture was allowed to warm up to room temperature,
stirred for 30 min, and mixed with cold water. The organic phase
was washed with saturated Na2CO3 solution followed by water,
and the resulting organic layers were combined, dried over Na2SO4,
and concentrated under reduced pressure. The product was purified
by silica gel flash column chromatography (DCM/EtOAc: 8/2) to
yield 10 as a yellow oil (0.11 g, 35%). 1H NMR (CDCl3, δ): 2.11
(s, 3H, COCH3), 2.87 (d, 3H, NCH3), 5.08 (s, 2H, CH2O), 6.37 (s,
1H, NH), 6.83 (d, 1H, J ) 8.4 Hz, ArH), 7.23 (m, 1H, ArH), 7.37
(dd, 1H, J ) 1.6, 8.0 Hz, ArH), 7.52 (dd, 1H, J ) 2.8, 8.4 Hz,
ArH), 7.61 (m, 1H, ArH), 8.22 (d, 1H, J ) 2.0 Hz, ArH). Anal.
(C17H15FN2O5S) C, H, N.
N-Methyl-2-(5′-hydroxymethyl-2′-nitrophenylthio)-5-iodo-
benzylamine (15). Amide 12 (53.0 mg, 0.11 mmol) was treated
with BH3-THF (1 M soln in THF; 1.09 mL, 1.09 mmol) as
described for 13. Silica gel flash column chromatography (DCM/
1
MeOH: 9/1) gave 15 as a yellow oil (20.0 mg, 42%). H NMR
(CDCl3, δ): 2.38 (s, 3H, NCH3), 3.74 (s, 2H, CH2N), 4.71 (s, 2H,
CH2OH), 6.67 (d, 1H, J ) 8.4 Hz, ArH), 7.25 (d, 1H, J ) 8.0 Hz,
ArH), 7.34 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.70 (dd, 1H, J ) 2.0,
8.0 Hz, ArH), 7.97 (d, 1H, J ) 2.0 Hz, ArH), 8.25 (m, 1H, ArH).
Anal. (C15H15IN2O3S) C, H, N.
N-Methyl-2-(2′-amino-4′-hydroxymethylphenylthio)-5-fluoro-
benzylamine (16). To a solution of 13 (59.20 mg, 0.18 mmol)
dissolved in concd HCl (1.42 mL) and methanol (13 mL), SnCl2
(207.0 mg) was added at 10 °C. The resulting reaction mixture
was stirred at room temperature for 15 h. The solvent was
evaporated to dryness and the yellow residue was taken into water.
A saturated solution of Na2CO3 was added to adjust the pH to 6∼7.
The clouded solution was extracted three times with EtOAc. The
combined organic layers were dried over Na2SO4, and the solvent
was removed under reduced pressure. Purification by silica gel flash
column chromatography (DCM/MeOH: 9/1) afforded 16 as a
N-Methyl-2-(5′-acetoxymethyl-2′-nitrophenylthio)-5-bromo-
benzamide (11). Acid 8 (0.20 g, 0.47 mmol) was treated with
thionyl chloride (0.14 g, 1.17 mmol), as described for 10. The crude
acid chloride (0.21 g, 0.47 mmol; freshly prepared) in DCM (10
mL) was treated with methylamine hydrochloride (0.13 g, 1.89
mmol) in the presence of triethylamine (0.38 g, 3.77 mmol). The
methylamide derivative (11) was purified by silica gel flash column
chromatography (DCM/EtOAc: 8/2) to give a yellow oil (44.0 mg,
1
colorless oil (18.20 mg, 34%). H NMR (CDCl3, δ): 2.50 (s, 3H,
1
NCH3), 3.88 (s, 2H, CH2N), 4.39 (s, 2H, NH2), 4.64 (s, 2H,
CH2OH), 6.72 (d, 1H, J ) 8.0 Hz, ArH), 6.79 (m, 3H, ArH), 7.07
(dd, 1H, J ) 2.4, 9.6 Hz, ArH), 7.35 (d, 1H, J ) 8.0 Hz, ArH).
Anal. (C15H17FN2OS) C, H, N. MS m/z 293.11 (M + H)+.
21%). H NMR (CDCl3, δ): 2.09 (s, 3H, COCH3), 2.83 (d, 3H,
NCH3), 5.07 (s, 2H, CH2O), 6.39 (s, 1H, NH), 6.90 (d, 1H, J )
8.4 Hz, ArH), 7.35 (m, 1H, ArH), 7.43 (d, 1H, J ) 4.0 Hz, ArH),
7.60 (m, 1H, ArH), 7.84 (d, 1H, J ) 2.4 Hz, ArH), 8.17 (d, 1H, J
) 2.0 Hz, ArH). Anal. (C17H15BrN2O5S) C, H, N.
N-Methyl-2-(2′-amino-4′-hydroxymethylphenylthio)-5-bromo-
benzylamine (17). Amide 14 (23.80 mg, 0.062 mmol) was treated
with SnCl2 (60.0 mg) as described for 16. The desired compound
17 was obtained following purification by silica gel flash column
chromatography (DCM/MeOH: 9/1) as a yellow oil (19.70 mg,
90%). 1H NMR (CDCl3, δ): 2.50 (s, 3H, NCH3), 3.87 (s, 2H,
CH2N), 4.63 (s, 2H, CH2OH), 6.71 (m, 1H, ArH), 6.80 (d, 1H, J
) 1.6 Hz, ArH), 7.16 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.36 (dd,
1H, J ) 2.4, 8.0 Hz, ArH), 7.45 (d, 1H, J ) 2.4 Hz, ArH). Anal.
(C15H17BrN2OS) C, H, N. MS m/z 353.03 (M + H)+.
N-Methyl-2-(5′-acetoxymethyl-2′-nitrophenylthio)-5-iodo-ben-
zamide (12). Acid 9 (0.19 g, 0.40 mmol) was treated with thionyl
chloride (0.12 g, 1.00 mmol), as described for 10. The crude acid
chloride (0.20 g, 0.41 mmol; freshly prepared) in DCM (8 mL)
was treated with methylamine hydrochloride (0.11 g, 1.63 mmol)
in the presence of triethylamine (0.33 g, 3.25 mmol). The
methylamide derivative (12) was purified by silica gel flash column
chromatography (DCM/EtOAc: 8/2) to give a yellow oil (57.0 mg,
1
28%). H NMR (CDCl3, δ): 2.11 (s, 3H, COCH3), 2.85 (d, 3H,

278 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Jarkas et al.
N-Methyl-2-(2′-amino-4′-hydroxymethylphenylthio)-5-iodo-
benzylamine (18). Amide 15 (25.0 mg, 0.058 mmol) was treated
with SnCl2 (78.66 mg), as described for 16. The desired compound
18 was obtained following purification by silica gel flash column
chromatography (DCM/MeOH: 9/1) as a light orange oil (11.70
mg, 51%). 1H NMR (CDCl3, δ): 2.50 (s, 3H, NCH3), 3.85 (s, 2H,
CH2N), 4.64 (s, 2H, CH2OH), 6.50 (d, 1H, J ) 8.4 Hz, ArH), 6.72
(dd, 1H, J ) 1.2, 7.6 Hz, ArH), 6.80 (d, 1H, J ) 1.6 Hz, ArH),
7.34 (d, 1H, J ) 2.0 Hz, ArH), 7.37 (m, 1H, ArH), 7.63 (d, 1H, J
) 2.4 Hz, ArH). Anal. (C15H17IN2OS) C, H, N. MS m/z 401.02
(M + H)+.
5-Fluoro-2-(5′-methyl-2′-nitrophenylthio)benzoic Acid (20).
Compound 20 was prepared by the method described for 7, by
condensation of 19 (0.63 g, 2.90 mmol) with 4 (0.50 g, 2.90 mmol)
in DMF (4 mL) in the presence of K2CO3 (1.20 g, 8.71 mmol) as
a base. Organic layers were washed with water, dried over Na2SO4,
and concentrated under reduced pressure. Purification by silica gel
flash column chromatography (DCM/MeOH: 9/1) gave 20 as a light
brown solid (0.55 g, 62%). 1H NMR (CDCl3, δ): 2.41 (s, 3H, CH3),
6.97 (d, 1H, J ) 8.4 Hz, ArH), 7.23 (m, 2H, ArH), 7.36 (m, 1H,
ArH), 7.78 (dd, 1H, J ) 3.2, 9.2 Hz, ArH), 7.93 (d, 1H, J ) 1.2
Hz, ArH).
5-Bromo-2-(5′-methyl-2′-nitrophenylthio)benzoic Acid (21).
Compound 21 was prepared by the method described for 7, by
condensation of 19 (1.25 g, 5.78 mmol) with 5 (0.99 g, 5.78 mmol)
in DMF (10 mL) in the presence of K2CO3 (2.40 g, 17.36 mmol)
as a base. Organic layers were washed with water, dried over
Na2SO4, and concentrated under reduced pressure. Purification by
silica gel flash column chromatography (DCM/MeOH: 9/1) gave
21 as an orange solid (1.10 g, 62%). 1H NMR (CDCl3, δ): 2.44 (s,
3H, CH3), 7.06 (d, 1H, J ) 8.4 Hz, ArH), 7.16 (d, 1H, J ) 8.4 Hz,
ArH), 7.31 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.55 (dd, 1H, J ) 2.4,
8.8 Hz, ArH), 7.88 (d, 1H, J ) 1.2 Hz, ArH), 8.21 (d, 1H, J ) 2.4
Hz, ArH).
5-Iodo-2-(5′-methyl-2′-nitrophenylthio)benzoic Acid (22). Com-
pound 22 was prepared by the method described for 7, by
condensation of 19 (0.38 g, 1.78 mmol) with 6 (0.50 g, 1.78 mmol)
in DMF (5 mL) in the presence of K2CO3 (0.74 g, 5.35 mmol) as
a base. Organic layers were washed with water, dried over Na2SO4,
and concentrated under reduced pressure. Purification by silica gel
flash column chromatography (DCM/MeOH: 9/1) gave 22 as an
orange oil (0.46 g, 62%). 1H NMR (CDCl3, δ): 2.35 (s, 3H, CH3),
6.91 (d, 1H, J ) 8.4 Hz, ArH), 7.23 (d, 1H, J ) 11.60 Hz, ArH),
7.36 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.75 (d, 1H, J ) 2.0 Hz,
ArH), 7.78 (dd, 1H, J ) 2.0, 8.0 Hz, ArH), 8.16 (d, 1H, J ) 1.6
Hz, ArH).
M soln in THF; 7.24 mL, 14.48 mmol) as described for 23. The
dimethylamide derivative 24 was purified by silica gel flash column
chromatography (DCM/EtOAc: 8/2) to give an orange oil (0.56 g,
49%). 1H NMR (CDCl3, δ): 2.35 (s, 3H, CH3), 2.87 (s, 3H, NCH3),
3.02 (s, 3H, NCH3), 6.85 (d, 1H, J ) 8.4 Hz, ArH), 7.20 (dd, 1H,
J ) 1.6, 8.0 Hz, ArH), 7.42 (d, 1H, J ) 8.0 Hz, ArH), 7.56 (m,
1H, ArH), 7.57 (m, 2H, ArH), 7.98 (d, 1H, J ) 1.6 Hz, ArH).
Anal. (C16H15BrN2O3S) C, H, N.
N,N-Dimethyl-2-(5′-methyl-2′-nitrophenylthio)-5-iodo-benza-
mide (25). The crude acid chloride (0.12 g, 0.28 mmol; freshly
prepared) in THF (2 mL) was treated with (CH3)2NH-THF (2.0
M soln in THF; 0.69 mL, 1.38 mmol) as described for 23. The
dimethylamide derivative 25 was purified by silica gel flash column
chromatography (DCM/EtOAc: 8/2) to give a yellow oil (60.0 mg,
50%). 1H NMR (CDCl3, δ): 2.34 (s, 3H, CH3), 2.85 (s, 3H, NCH3),
3.00 (s, 3H, NCH3), 6.86 (d, 1H, J ) 8.4 Hz, ArH), 7.19 (dd, 1H,
J ) 1.6, 8.0 Hz, ArH), 7.25 (d, 1H, J ) 8.0 Hz, ArH), 7.48 (m,
2H, ArH), 7.95 (d, 1H, J ) 0.8 Hz, ArH). Anal. (C16H15IN2O3S)
C, H, N.
N,N-Dimethyl-2-(5′-bromomethyl-2′-nitrophenylthio)-5-fluoro-
benzamide (26). A mixture of 23 (0.27 g, 0.81 mmol), N-
bromosuccinimide (0.21 g, 1.21 mmol), and AIBN (27.0 mg) was
refluxed for 24 h in CCl4 (12 mL). The solvent was removed under
reduced pressure and the residue was purified by silica gel flash
column chromatography using DCM/EtOAc (8/2) as eluent. Com-
1
pound 26 was obtained as a yellow oil (0.15 g, 45%). H NMR
(CDCl3, δ): 2.87 (s, 3H, NCH3), 3.04 (s, 3H, NCH3), 4.43 (s, 2H,
CH2Br), 6.88 (d, 1H, J ) 8.4 Hz, ArH), 7.18 (m, 2H, ArH), 7.39
(dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.60 (m, 1H, ArH), 8.23 (d, 1H,
J ) 2.0 Hz, ArH).
N,N-Dimethyl-2-(5′-bromomethyl-2′-nitrophenylthio)-5-bromo-
benzamide (27). Compound 24 (0.55 g, 1.39 mmol) was treated
with N-bromosuccinimide (0.37 g, 2.09 mmol) and AIBN (16.0
mg) as described for 26. The reaction mixture was refluxed for
18 h in CCl4 (13 mL) and the dimethylamide derivative 27 was
purified by silica gel flash column chromatography (DCM/EtOAc:
1
8/2) to give an orange residue (0.38 g, 57%). H NMR (CDCl3,
δ): 2.87 (s, 3H, NCH3), 3.02 (s, 3H, NCH3), 4.43 (s, 2H, CH2Br),
6.87 (d, 1H, J ) 8.4 Hz, ArH), 7.41 (dd, 1H, J ) 2.0, 8.8 Hz,
ArH), 7.57 (m, 2H, ArH), 7.62 (dd, 1H, J ) 2.4, 8.4 Hz, ArH),
8.22 (d, 1H, J ) 2.0 Hz, ArH).
N,N-Dimethyl-2-(5′-bromomethyl-2′-nitrophenylthio)-5-iodo-
benzamide (28). Compound 25 (59.0 mg, 0.13 mmol) was treated
with N-bromosuccinimide (35.62 mg, 0.20 mmol) and AIBN (6.0
mg) as described for 26. The reaction mixture was refluxed for
24 h in CCl4 (3 mL) and the dimethylamide derivative 28 was
purified by silica gel flash column chromatography (DCM/EtOAc:
N,N-Dimethyl-2-(5′-methyl-2′-nitrophenylthio)-5-fluoro-ben-
zamide (23). To a suspension of 20 (0.54 g, 1.76 mmol) in
anhydrous DCM (12 mL) were added thionyl chloride (0.52 g, 4.39
mmol) and a few drops of DMF. The reaction mixture was refluxed
for 90 min under argon. The solvent was evaporated and the solid
was used without further purification.
1
8/2) to give an orange oil (45.0 mg, 65%). H NMR (CDCl3, δ):
2.87 (s, 3H, NCH3), 3.01 (s, 3H, NCH3), 4.43 (s, 2H, CH2Br), 6.86
(d, 1H, J ) 8.4 Hz, ArH), 7.20 (d, 1H, J ) 8.4 Hz, ArH), 7.39
(dd, 1H, J ) 1.6, 8.0 Hz, ArH), 7.78 (m, 2H, ArH), 8.21 (d, 1H,
J ) 2.0 Hz, ArH).
A solution of dimethylamine in tetrahydrofuran ((CH3)2
NH-THF; 2.0 M soln in THF; 2.22 mL, 4.45 mmol) was added
dropwise to a stirred solution of the freshly prepared acid chloride
(0.29 g, 0.89 mmol) in dry THF (7 mL) at –70 °C. The reaction
mixture was allowed to stand overnight at room temperature, then
the solvent was removed under reduced pressure. The orange residue
was extracted with EtOAc. The organic phase was washed with
saturated Na2CO3 solution, followed by water, and the resulting
organic layers were combined, dried over Na2SO4, and concentrated
under reduced pressure. The product was purified by silica gel flash
column chromatography (DCM/EtOAc: 8/2) to yield the desired
N,N-Dimethyl-2-(5′-acetoxymethyl-2′-nitrophenylthio)-5-fluoro-
benzamide (29). A mixture of compound 26 (0.15 g, 0.36 mmol)
and CH3CO2K (35.0 mg, 0.36 mmol) was stirred at 120 °C for
18 h in DMF (6 mL). The mixture was cooled to room temperature,
poured into cold water, and extracted several times with EtOAc.
The combined organic layers were washed thoroughly with water,
dried over Na2SO4, and concentrated under reduced pressure. Silica
gel flash column chromatography purification (DCM/EtOAc: 8/2)
1
afforded 29 as a yellow oil (69.80 mg, 50%). H NMR (CDCl3,
δ): 2.09 (s, 3H, COCH3), 2.87 (s, 3H, NCH3), 3.03 (s, 3H, NCH3),
5.06 (s, 2H, CH2O), 6.90 (d, 1H, J ) 8.4 Hz, ArH), 7.16 (m, 2H,
ArH), 7.36 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.58 (m, 1H, ArH),
8.20 (d, 1H, J ) 2.0 Hz, ArH). Anal. (C18H17FN2O5S) C, H, N.
MS m/z 393.09 (M + H)+.
N,N-Dimethyl-2-(5′-acetoxymethyl-2′-nitrophenylthio)-5-bromo-
benzamide (30). Compound 27 (0.37 g, 0.78 mmol) was treated
with CH3CO2K (76.58 mg, 0.78 mmol) as described for 29. The
reaction mixture was heated at 145 °C for 20 h in DMF (5 mL)
and the dimethylamide derivative 30 was purified by silica gel flash
1
compound (23) as an orange oil (0.27 g, 90%). H NMR (CDCl3,
δ): 2.34 (s, 3H, CH3), 2.85 (s, 3H, NCH3), 3.02 (s, 3H, NCH3),
6.80 (d, 1H, J ) 8.8 Hz, ArH), 7.18 (m, 3H, ArH), 7.57 (m, 1H,
ArH), 7.98 (d, 1H, J ) 1.2 Hz, ArH). Anal. (C16H15FN2O3S) C, H,
N.
N,N-Dimethyl-2-(5′-methyl-2′-nitrophenylthio)-5-bromo-ben-
zamide (24). The crude acid chloride (1.12 g, 2.89 mmol; freshly
prepared) in THF (10 mL) was treated with (CH3)2NH-THF (2.0

PET Serotonin Transporter Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 279
column chromatography (DCM/EtOAc: 8/2) to give an orange oil
(0.18 g, 51%). 1H NMR (CDCl3, δ): 2.08 (s, 3H, COCH3), 2.86 (s,
3H, NCH3), 3.01 (s, 3H, NCH3), 5.06 (s, 2H, CH2O), 6.91 (d, 1H,
J ) 8.4 Hz, ArH), 7.35 (dd, 1H, J ) 2.0, 8.4 Hz, ArH), 7.43 (d,
1H, J ) 8.4 Hz, ArH), 7.58 (m, 2H, ArH), 8.17 (d, 1H, J ) 1.6
Hz, ArH). Anal. (C18H17BrN2O5S) C, H, N.
N,N-Dimethyl-2-(5′-acetoxymethyl-2′-nitrophenylthio)-5-iodo-
benzamide (31). Compound 28 (45.0 mg, 0.086 mmol) was treated
with CH3CO2K (8.50 mg, 0.086 mmol) as described for 29. The
reaction mixture was heated at 110 °C for 20 h in DMF (2 mL)
and the dimethylamide derivative 31 was purified by silica gel flash
column chromatography (DCM/EtOAc: 8/2) to give a yellow oil
(22.0 mg, 51%). 1H NMR (CDCl3, δ): 2.09 (s, 3H, COCH3), 2.87
(s, 3H, NCH3), 3.01 (s, 3H, NCH3), 5.07 (s, 2H, CH2O), 6.93 (d,
1H, J ) 8.4 Hz, ArH), 7.28 (d, 1H, J ) 8.0 Hz, ArH), 7.36 (dd,
1H, J ) 2.0, 8.4 Hz, ArH), 7.76 (d, 1H, J ) 1.6 Hz, ArH), 7.80
(dd, 1H, J ) 2.0, 8.0 Hz, ArH), 8.18 (d, 1H, J ) 2.0 Hz, ArH).
Anal. (C18H17IN2O5S) C, H, N.
N,N-Dimethyl-2-(5′-hydroxymethyl-2′-nitrophenylthio)-5-fluoro-
benzylamine (32). Amide 29 (67.40 mg, 0.17 mmol) was treated
with BH3-THF complex (1 M soln in THF; 3.43 mL, 3.43 mmol)
as described for 13. Silica gel flash column chromatography
purification (DCM/MeOH: 9/1) gave 32 as a yellow oil (13.20 mg,
23%). 1H NMR (CDCl3, δ): 2.19 (s, 6H, NCH3), 3.49 (s, 2H,
CH2N), 4.72 (s, 2H, CH2OH), 6.61 (d, 1H, J ) 8.4 Hz, ArH), 7.05
(m, 1H, ArH), 7.33 (m, 1H, ArH), 7.46 (dd, 1H, J ) 2.0, 8.4 Hz,
ArH), 7.54 (m, 1H, ArH), 8.26 (d, 1H, J ) 1.6 Hz, ArH). Anal.
(C16H17FN2O3S) C, H, N.
N,N-Dimethyl-2-(5′-hydroxymethyl-2′-nitrophenylthio)-5-bro-
mo-benzylamine (33). Amide 30 (0.18 g, 0.39 mmol) was treated
with BH3-THF (1 M soln in THF; 1.98 mL, 1.98 mmol) as
described for 13. Silica gel flash column chromatography (DCM/
MeOH: 9/1) gave 33 as an orange oil (0.16 g, 99%). 1H NMR
(CDCl3, δ): 2.27 (s, 6H, NCH3), 3.59 (s, 2H, CH2N), 4.73 (s, 2H,
CH2OH), 6.65 (d, 1H, J ) 8.4 Hz, ArH), 7.35 (dd, 1H, J ) 2.0,
8.4 Hz, ArH), 7.41 (d, 1H, J ) 8.4 Hz, ArH), 7.49 (dd, 1H, J )
1.6, 8.0 Hz, ArH), 7.94 (m, 1H, ArH), 8.26 (d, 1H, J ) 1.6 Hz,
ArH). Anal. (C16H17BrN2O3S) C, H, N.
desired compound 37 was obtained following purification by silica
gel flash column chromatography (DCM/MeOH: 9/1) as an orange
oil (7.20 mg, 36%). 1H NMR (CDCl3, δ): 2.46 (s, 6H, NCH3),
3.65 (s, 2H, CH2N), 4.65 (s, 2H, CH2OH), 6.56 (d, 1H, J ) 8.4
Hz, ArH), 6.72 (d, 1H, J ) 8.0 Hz, ArH), 6.79 (m, 1H, ArH), 7.39
(m, 1H, ArH), 7.67 (m, 1H, ArH), 7.77 (d, 1H, J ) 8.4 Hz, ArH).
Anal. (C16H19IN2OS) C, H, N. MS m/z 415.03 (M + H)+.
Radiochemistry. (1) Radiosynthesis of [11C]-(35). [11C]-(35)
was prepared in a manner similar to the previously reported “loop
method”.43 A commercial front load HPLC injector is equipped
with a commercial stainless steel loop (2 mL). A trap is connected
to the load waste port of the HPLC injector to catch the untrapped
[11C]iodomethane. With the valve in the LOAD position, a solution
of precursor 16 (0.8–1.0 mg) in DMF (70 µL) is injected slowly
(5–10 s) into the clean, dry injection loop using a 100 µL syringe.
The loading of the loop with precursor solution can be made at
least up to 10 min before end of bombardment (EOB). The
[11C]iodomethane product line of the GE MicroLab methyl iodide
system was inserted into the load port of the injector.
[11C]iodomethane, produced from 11CO2,24 was swept into the
HPLC loop coated with precursor solution by a stream of helium
gas (25 mL/min) at room temperature. Radioactivity trapped on
the loop is detected by a proximal radiation detector. When
radioactivity had peaked in the loop (2–3 min), the flow of helium
is stopped and the reaction allowed to proceed (5 min). The contents
of the loop were then quantitatively injected onto the HPLC
purification column (A) by simply changing the position of the
injection valve to INJECT. The semipreparative HPLC solvent was
50% absolute ethanol (abs. EtOH)/50% H2O/0.1% triethylamine
(Et3N), and the flow rate ) 5 mL/min (rt of [11C]-(35) ) 12.57
min, rt of 16 ) 8.15 min). The appropriate fractions were collected
and condensed via solid phase extraction (SPE) method.44 An
aliquot (0.1 mL) of the formulated solution was used to establish
the chemical and radiochemical purities and specific activity of the
final solution by analytical HPLC: column B, 70% MeOH/30%
H2O/0.1% Et3N, 1 mL/min (rt of [11C]-(35) ) 3.72 min). The
quality control tests showed that the final product was sterile,
pyrogen-free, and contained no precursor. Further evidence for the
identity of the radiolabeled product was achieved by coinjection
with authentic “cold” material on column B. [11C]-(36) and [11C]-
(37) were prepared in a similar manner: for ligand [11C]-(36),
column A, 8 mL/min (rt of [11C]-(36) ) 14.72 min, rt of 17 )
7.85 min); column B, 1 mL/min (rt of [11C]-(36) ) 5.42 min). For
ligand [11C]-(37), column A, 8 mL/min (rt of [11C]-(37) ) 12.87
min, rt of 18 ) 7.60 min); column B, 1 mL/min (rt of [11C]-(37)
) 5.82 min).
N,N-Dimethyl-2-(5′-hydroxymethyl-2′-nitrophenylthio)-5-iodo-
benzylamine (34). Amide 31 (22.0 mg, 0.044 mmol) was treated
with BH3-THF (1 M soln in THF; 0.22 mL, 0.22 mmol) as
described for 13. Silica gel flash column chromatography (DCM/
1
MeOH: 9/1) gave 34 as a yellow oil (11.00 mg, 56%). H NMR
(CDCl3, δ): 2.27 (s, 6H, NCH3), 3.47 (s, 2H, CH2N), 4.72 (s, 2H,
CH2OH), 6.67 (d, 1H, J ) 8.4 Hz, ArH), 7.66 (dd, 1H, J ) 2.0,
8.0 Hz, ArH), 7.75 (d, 1H, J ) 2.0 Hz, ArH), 7.79 (dd, 1H, J )
2.0, 8.0 Hz, ArH), 8.04 (d, 1H, J ) 2.0 Hz, ArH), 8.24 (d, 1H, J
) 1.6 Hz, ArH). Anal. (C16H17IN2O3S) C, H, N.
(2) Solid Phase Extraction. The 2.5 mL fractions of [11C]-(35)
collected from the semipreparative HPLC column were diluted with
sterile water (5 mL/fraction) and combined in one flask. After
stirring, the homogeneous solution was transferred with vacuum,
through a Cook line, to a C18 Sep Pak (previously activated with
abs. EtOH (10 mL) and water (10 mL)), the eluate being directed
to the waste. After two successive washings, one with saline (0.9%
NaCl, 50 mL) and one with abs. EtOH (0.5 mL), all directed to
the waste, the [11C]-(35) was eluted with abs. EtOH (1.5 mL) and
driven to a sterile 30 mL vial containing 0.9% NaCl solution (13.5
mL). The residue was pushed with argon through a Millipore filter
(pore size 1.0 µm), followed by a smaller one (pore size 0.2 µm),
to a 30 mL sterile empty vial for quality control tests and microPET
nonhuman primate imaging studies. [11C]-(36) and [11C]-(37) were
formulated in a similar manner.
(3) Log P Measurements. The determination of partition
coefficients of radioactive compounds [11C]-(35), [11C]-(36), and
[11C]-(37) between 1-octanol and 0.02 M phosphate buffer at pH
7.4 was performed as reported previously.23
In Vitro Competition Assays. Competition assays were per-
formed based on methods reported previously.23–25 Cell membranes
from HEK-293 cells stably expressing hSERT or hNET (gift from
Dr. Randy Blakely, Ph.D., Vanderbilt University) and MDCK
(Madin Darby Canine Kidney) cells stably expressing hDAT (gift
from Dr. Gary Rudnick, Yale University) were used in these assays.
N,N-Dimethyl-2-(2′-amino-4′-hydroxymethylphenylthio)-5-
fluoro-benzylamine (35). Amide 32 (13.20 mg, 0.040 mmol) was
treated with SnCl2 (44.27 mg) as described for 16. The desired
compound 35 was obtained following purification by silica gel flash
column chromatography (DCM/MeOH: 9/1) as a colorless oil (8.40
mg, 70%). 1H NMR (CDCl3, δ): 2.32 (s, 6H, NCH3), 3.56 (s, 2H,
CH2N), 4.63 (s, 2H, CH2OH), 6.71 (dd, 1H, J ) 2.0, 8.0 Hz, ArH),
6.76 (m, 1H, ArH), 6.81 (dd, 1H, J ) 3.2, 8.4 Hz, ArH), 6.87 (m,
1H, ArH), 7.03 (d, 1H, J ) 7.2 Hz, ArH), 7.42 (d, 1H, J ) 7.6 Hz,
ArH). Anal. (C16H19FN2OS) C, H, N. MS m/z 307.12 (M + H)+.
N,N-Dimethyl-2-(2′-amino-4′-hydroxymethylphenylthio)-5-
bromo-benzylamine (36). Amide 33 (75.0 mg, 0.19 mmol) was
treated with SnCl2 (0.18 g) as described for 16. The desired
compound 36 was obtained following purification by silica gel flash
column chromatography (DCM/MeOH: 9/1) as a light orange oil
1
(62.0 mg, 89%). H NMR (CDCl3, δ): 2.31 (s, 6H, NCH3), 3.53
(s, 2H, CH2N), 4.61 (s, 2H, CH2OH), 6.70 (d, 1H, J ) 8.0 Hz,
ArH), 6.82 (m, 2H, ArH), 7.15 (dd, 1H, J ) 2.0, 8.4 Hz, ArH),
7.39 (m, 2H, ArH). Anal. (C16H19BrN2OS) C, H, N. MS m/z 367.04
(M + H)+.
N,N-Dimethyl-2-(2′-amino-4′-hydroxymethylphenylthio)-5-
iodo-benzylamine (37). Amide 34 (21.40 mg, 0.048 mmol) was
treated with SnCl2 (45.0 mg, 0.20 mmol) as described for 16. The

280 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Jarkas et al.
Cells were grown to confluency in DMEM (Dulbecco’s modifica-
tion of Eagles’s medium) containing 10% fetal bovine serum and
Geneticin sulfate and then harvested using pH 7.4 phosphate-
buffered saline (PBS) containing 0.53 mM ethylenediaminetetracetic
acid (EDTA) at 37 °C. Cell pellets were prepared through
centrifugation at 2000 rpm for 10 min, the supernatant was decanted,
and the pellets were homogenized with a Polytron PT3000
(Brinkman, Littau, Switzerland) at 11000 rpm for 12 s in 30
volumes of PBS. The resulting cell membrane suspensions were
centrifuged at 43000 g for 10 min, the supernatants were decanted
and the resulting pellets were stored at -70 °C until used in assays.
Competition assays were performed in 13 × 100 mm polystyrene
tubes in a 2.0 mL final volume consisting of 1.7 mL of assay buffer,
100 µL of competing ligand in assay buffer, 100 µL of radioligand
in assay buffer, and 100 µL of cell membrane suspension (corre-
sponding to 30–70 µg protein) in assay buffer. Cell membrane
pellets were characterized prior to competition assays to determine
membrane concentrations that gave optimal signal while not
significantly affecting the concentration of free radioligand. The
cell membrane pellets were resuspended in the appropriate volume
of assay buffer through brief homogenization using a Polytron
PT3000. Competing ligands were assayed in triplicate at 12
concentrations ranging from 10-13 to 10-5 M. To ensure solubility,
the competing ligands were dissolved in 1:1 ethanol/5 mM HCl
and then serially diluted in 5 mM HCl.
Pharmacological Displacement in Nonhuman Primates. The
validity of in vivo [11C]-(35), [11C]-(36), and [11C]-(37) binding
to brain regions rich in SERT of a male cynomolgus monkey was
assessed by their displacements by the potent selective SERT ligand
R,S-citalopram.24 R,S-Citalopram (1.5 mg/kg) was administered as
a bolus over 30 s at 45 min following injection of [11C]-(35) and
at 60 min following injection of [11C]-(36) or [11C]-(37). Emission
data were collected continuously for either 90 min after injection
of [11C]-(35) or 120 min after injection of [11C]-(36) or [11C]-
(37). Prior to the [11C]-(35), [11C]-(36), or [11C]-(37) imaging
protocol, a transmission scan was obtained with a germanium-68
source for attenuation correction of the emission data. Regions of
interest were drawn as described above.
Acknowledgment. This research was sponsored by the
National Institute of Mental Health (R21 MH66622-01).
Supporting Information Available: Theoretical and experi-
mental elemental analyses for compounds 16-18 and 35-37 and
the actual uptake data plotted in Figures 2, 3, 4, 6, 7, and 8.
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For SERT assays, the assay buffer consisted of 53 mM Tris
buffer, 126 mM NaCl, and 5.3 mM KCl (pH 7.9 at room
temperature) and the equilibrium incubation time was 2 h at room
temperature. The radioligand used for SERT assays was [3H]-
citalopram, obtained from Dupont NEN (Boston, MA; 3100 GBq/
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[
125I]RTI-55, obtained from Dupont NEN (Boston, MA, 2200 Ci/
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the addition of the cell membrane suspension.
At the end of the incubation, the assays were terminated by the
addition of ∼5 mL of assay buffer at 4 °C followed by rapid vacuum
filtration with 3 × 5 mL washes with assay buffer at 4 °C through
GF/B filters (Whatman, Inc., Clifton, NJ) presoaked in assay buffer
containing 0.3% polyethyleneimine. The data from the competition
curves were analyzed and Ki values calculated using GraphPad
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duplicates, for each compound.
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system (Knoxville, TN).24 Briefly, the microPET imaging studies
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or [11C]-(37) with or without injection of pharmacological doses
of monoamine transporter ligands. Emission data were collected
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(35) or [11C]-(37) and then binned into either 18 (90 min) or 20
(120 min) time frames ranging in duration from 30 s to 10 min for
analysis. Acquired emission data were subject to iterative recon-
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postfiltering to provide images with an isotropic resolution of 3
mm fwhm. For generation of time-activity curves, regions of
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landmarks visible in reconstructed PET images using ASIPro
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standard uptake values (SUV). SUV were defined as the pixel value
in µCi/mL multiplied by [weight of the animal (g)]/[dose (µ Ci)].
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PET Serotonin Transporter Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 281
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