Synthesis and evaluation of N-methyl and S-methyl 11C-labeled 6-methylthio-2-(4′-N,N-dimethylamino)phenylimidazo[1,2-a]pyridines as radioligands for imaging β-amyloid plaques in Alzheimer's disease

Article abstract of DOI:10.1021/jm700970s

6-Thiolato-substituted 2-(4′-N,N-dimethylamino)phenylimidazo[ 1,2-a]pyridines (RS-IMPYs; 1-4) were synthesized as candidates for labeling with carbon-11 (t_1/2 = 20.4 min) and imaging of A_β plaques in living human brain using positron

Full text of DOI:10.1021/jm700970s

148
J. Med. Chem. 2008, 51, 148–158
Synthesis and Evaluation of N-Methyl and S-Methyl 11C-Labeled 6-Methylthio-2-(4′-
N,N-dimethylamino)phenylimidazo[1,2-a]pyridines as Radioligands for Imaging ꢀ-Amyloid Plaques
in Alzheimer’s Disease
Lisheng Cai,*,† Jeih-San Liow,† Sami S. Zoghbi,† Jessica Cuevas,† Cesar Baetas,† Jinsoo Hong,† H. Umesha Shetty,†
Nicholas M. Seneca,† Amira K. Brown,† Robert Gladding,† Sebastian S. Temme,† Mary M. Herman,‡ Robert B. Innis,† and
Victor W. Pike†
Molecular Imaging Branch and Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892
ReceiVed August 4, 2007
6-Thiolato-substituted 2-(4′-N,N-dimethylamino)phenylimidazo[1,2-a]pyridines (RS-IMPYs; 1-4) were
synthesized as candidates for labeling with carbon-11 (t1/2 ) 20.4 min) and imaging of Aꢀ plaques in living
human brain using positron emission tomography (PET). Ki values for binding of these ligands to Alzheimer’s
disease brain homogenates were measured in vitro against tritium-labeled 6 (Pittsburgh compound B). MeS-
IMPY (3, Ki ) 7.93 nM) was labeled with carbon-11 at its S- or N-methyl position to give [11C]7 or [11C]8,
respectively. After injection into rats, [11C]7 or [11C]8 gave moderately high brain uptakes of radioactivity
followed by rapid washout to low levels. The ratio of radioactivity at maximal uptake to that at 60 min
reached 18.7 for [11C]7. [11C]7 behaved similarly in mouse and monkey. [11C]7 also bound selectively to
Aꢀ plaques in post mortem human Alzheimer’s disease brain. Although rapidly metabolized in rat by
N-demethylation, [11C]7 was stable in rat brain homogenates. The ex vivo brain radiometabolites observed
in rats have a peripheral origin. Overall, [11C]7 merits further evaluation in human subjects.
Introduction
A key event in the pathogenesis of Alzheimer’s disease
(ADa) is now believed to be the deposition of ꢀ-amyloid (Aꢀ)
plaques in brain.1,2 A “ꢀ-amyloid cascade” hypothesis has
emerged to account for various experimental facts, including
genetic variations related to the production and elimination of
Aꢀ.3,4 Four inherited genes5,6 (APP, presenilin-1, presenilin-2,
and SORL17) and one firmly established susceptibility gene
(Apo ꢀ4)8,9 have been identified. Also, more than a further dozen
potential AD susceptibility genes have been identified with
statistically significant allelic summary odds ratio.10,11 Neuritic
plaques and neurofibriliary tangles are accepted pathological
hallmarks of AD disease as confirmed at autopsy.12,13 Ante
Figure 1. Three radioligands for Aꢀ plaque imaging with PET and
mortem identification and quantification of Aꢀ plaques would
have value for both the early diagnosis of AD and the monitoring
of the clinical development of therapeutic drugs targeted to arrest
or eliminate Aꢀ plaques in living brain.14,15 At least three
radioligands for positron emission tomography (PET) and one
for single photon emission computed tomography (SPECT)16
have reached the clinical stage14,15,17 (Figure 1).
one for imaging with SPECT in clinical research.
Among these radioligands, [11C]PIB (6) has been the most
effective and the most thoroughly studied.18,19 There is a clear
distinction between AD subjects and normal controls in Aꢀ-
plaque load measured with this radioligand in PET imaging.18
The ratio of radioactivity of different regions of the gray matter
to that of cerebellum, the relatively plaque-free reference region,
reaches about 2.5 in AD patients. Although this is a significant
result in its own right, this ratio is not optimal for the two
targeted purposes for Aꢀ PET radioligands (early diagnosis and
drug monitoring). The average heavy load of Aꢀ plaques in the
AD patient (about 2 µM) should permit more sensitive radio-
ligands to be developed.20 We aimed to develop new PET
radioligands with potential to expand the dynamic range of the
radioactivity ratio between cortex and cerebellum, by increasing
either radioligand binding affinities or the rates of washout of
radioactivity from the brain.21 The former would increase the
binding potential (BP), which is proportional to the PET signal
produced, and the latter would decrease the nonspecific binding
(the background noise).
* To whom correspondence should be addressed. Address: Molecular
Imaging Branch, National Institute of Mental Health, National Institutes of
Health, 10 Center Drive, Building 10, Room B3 C346, Bethesda, MD 20892.
Phone: (301) 451-3905. Fax: (301) 480-5112. E-mail: cail@
intra.nimh.nih.gov.
†
Molecular Imaging Branch.
Clinical Brain Disorders Branch.
‡
aAbbreviations: Aꢀ, beta amyloid; AD, Alzheimer’s disease; APP,
amyloid precursor protein; BP, binding potential; CA, carrier-added;
cLogD7.4, calculated logarithm of distribution coefficient between octanol
and water at pH 7.4; EOS, end of synthesis; HRMS, high-resolution mass
spectra; ID, injected dose; NCA, no-carrier-added; PBS, phosphate buffered
saline; PET, positron emission tomography; PIB, Pittsburgh compound B;
RCY, decay-corrected radiochemical yield; SAR, structure–activity relation-
ship; SD, standard deviation; SORL1, sortolin related receptor; SPECT,
single photon emission computed tomography; TAC, time-activity curve;
nM, nanomolar; RT, room temperature; ppm, parts per million.
10.1021/jm700970s This article not subject to U.S. Copyright. Published 2008 by the American Chemical Society
Published on Web 12/14/2007

Pyridines as Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 149
Scheme 2
Scheme 1
For prospective high-affinity radioligands for imaging Aꢀ
plaques with PET, the ratios of radioactivity in normal animal
brains at maximal level and at a later specific time (e.g., 60
min after injection) are considered predictive of the signal-to-
noise ratio that might be achievable when Aꢀ plaques are
Pharmacological Screen. Ligand 3 was selective for ꢀ-amy-
loid plaques, since 10 µM compound caused <50% inhibition
of radioligand binding to all of the large number of tested central
nervous system receptors (NIMH Pharmacology Drug Screening
present.21 125I]IMPY (5) ([125I]4-(6-iodo-H-imidazo[1,2-a]py-
[
Program). These receptors included 5-HT1A,B,D,E,2A-C,3,5A,6,7
1a,b,2a, ꢀ2, D1–5, H1, and MOR.
Radiochemistry. Treatment of 4 with [11C]iodomethane
,
R
ridin-2-yl)-N,N-dimethylbenzeneamine) (Figure 1) is one of the
few radioligands showing a high ratio of radioactivity between
2 and 60 min17 and is superior to [11C]622 in this respect. IMPY
analogues also show scope for further study of structure-affinity
relationships (SAR) in order to select better ligands for Aꢀ
plaque imaging.23,24
under basic conditions (Scheme 3) gave chemically (>95%)
and radiochemically pure (>99%) [11C]7 in 10-13% decay-
corrected radiochemical yield (RCY) after formulation for
intravenous administration with a high specific radioactivity
(1.4-2.0 Ci/µmol) at the end of synthesis (EOS). The “loop”
technique reported by Wilson et al.27 for the preparation of
[11C]6 when applied to 2 gave [11C]8 in moderate RCY
(5.1-17.4%) and in high chemical purity (>90%) and radio-
chemical purity (>99%) and with a specific radioactivity of
2.3-11.9 Ci/µmol at EOS (Scheme 4).
WehavepreviouslyevaluatedtwoIMPYderivatives,[18F]FEM-
IMPY [N-(2-fluoroethyl)-4-(6-iodo-H-imidazo[1,2-a]pyridin-2-
yl)-N-methylbenzeneamine] and its 3-fluoropropyl analogue,
[18F]FPM-IMPY, as Aꢀ plaque PET radioligands.25 After
intravenous injection of either radioligand into rodent or monkey
there is a rapid and moderately high uptake of radioactivity into
the brain (∼160% SUV), followed by biphasic clearance with
a fast and very slow component. Metabolism is rapid and is
demonstrated in rat to involve dealkylation of the tertiary
aromatic amino group, culminating in defluorination and high
uptake of radioactivity ([18F]fluoride ion) into bone. Tetradeu-
teration of the fluoroethyl group in [18F]FEM-IMPY does not
lead to a significant reduction in the residual brain radioactivity
but reduces the bone uptake of radioactivity, presumably because
of an isotope effect on metabolism. With a view to avoiding
rapid defluorination and to eliminate residual radioactivity in
brain, we later decided to make use of an “isoelectronic effect”
in the design of further analogues of 5.24 We considered that
this strategy might retain high binding affinity while reducing
lipophilicity. Also, we have recently explored replacement of
the 6-iodo group in 5 with thiolato groups, thereby opening up
new possibilities for labeling with positron emitters [carbon-11
(t1/2 ) 20.4 min) and fluorine-18 (t1/2 ) 109.7 min)].24 Here,
we report the syntheses, 11C-labeling, and in vitro and in vivo
evaluation of new 6-thiolato-IMPY derivatives in our continuing
effort to develop useful radioligands for the detection of Aꢀ
plaques in AD patients with PET.
Stability of [11C]7 in the Formulated Dose. The radio-
chemical purity of [11C]7 ranged between 99.2% and 94.7%
immediately after radiosynthesis. This formulated radioligand (pH
∼ 7.5) was radiochemically stable for 3 h. However, when the pH
of the solution was reduced immediately after preparation to ∼4.5,
the radioligand decomposed to 51.2% radiochemical purity within
2 h. Immediate neutralization was therefore required after the HPLC
purification of [11C]7 under acidic conditions.
Autoradiography of Human Aꢀ Plaques with NCA
[11C]7 in Vitro. In vitro autoradiography of a confirmed AD brain
specimen exposed to no-carrier-added (NCA) [11C]7 showed
specific binding only to cortical areas containing neuritic plaques
(Figure 2A); the areas of specific binding of [11C]7 matched those
stained with thioflavin-S in the same tissue slides (data not shown).
Specific binding of [11C]7 to regions of cortical gray matter having
neuritic plaques was eliminated in an AD brain specimen pretreated
with nonradioactive 3 (Figure 2B). No specific binding of NCA
or carrier-added (CA) [11C]7 was observed in a control human
brain specimen (Figure 2C, D).
Stability of NCA [11C]7 in Rat Whole Blood, Rat
Brain, and Monkey Whole Blood in Vitro. NCA [11C]7 was
91% unchanged after 60 min of incubation in rat whole blood
and 93.6% unchanged after 60 min of incubation with rat brain
homogenate. The radioligand was 96.2% and 94.5% unchanged
after 30 and 90 min of incubation in monkey whole blood,
respectively.
Results
Chemistry. We used a combination of sodium methanethi-
olate and trimethyltin chloride to synthesize the IMPY deriva-
tives 1-3 from their corresponding iodo compounds (Scheme
1). To facilitate the radiolabeling of the S-methyl group in 3,
we designed compound 4 (Scheme 2), a masked arylthiol
compound, as the precursor for reaction with [11C]iodomethane.
This precursor was synthesized in high yield from 5 according
to a reported general method.26
Binding Affinities for Human AD Aꢀ Plaques and
Lipophilicities of Ligands. The Kd value of 6 in our in vitro
assay for binding to human Aꢀ plaques was 7.23 nM. Ligand 3
exhibited an affinity comparable to that of 6 or 5 (Table 1).
Ligands 1, 2, and 4 exhibited much lower affinity. Computed
(cLogD7.4) and measured lipohilicities (LogD7.4) were in fair
agreement for each of the ligands 1-4 (Table 1).
Evaluation of NCA [11C]7 in Normal Rodents in Vivo
with PET. After intravenous injection of a bolus of NCA [11C]7
into rat, there was a rapid and high uptake of radioactivity into
brain followed by a rapid and continuous washout (Figure 3A).
Similar time-activity curves (TACs) were observed for mice
(Figure 3B). Radioactivity levels were uniform across rat and
mouse brain. The ratios of brain radioactivity at maximal uptake
to that at 60 min after injection of [11C]7 were 18.7 for rat and
11.1 for mouse.
Evaluation of NCA [11C]8 in Normal Rat in Vivo with
PET. After intravenous injection of a bolus of NCA [11C]8 into
rat (Figure 4), there was rapid and high uptake of radioactivity

150 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Table 1. Ki and measured LogD7.4 Values of IMPY Derivatives
Cai et al.
a
b
ligand
R
R1
R2
LogD7.4
cLogD7.4
Ki (nM)c
1
2
3
4
Me
Me
Me
H
H
Me
Me
H
2.6 ( 0.1
3.6 ( 0.1
4.1 ( 0.1
3.6 ( 0.1
2.43 ( 1.3
3.24 ( 1.3
3.67 ( 1.3
3.33 ( 1.4
4.37 ( 0.88
3.33 ( 0.8
377 ( 225
80 ( 16
Me
Me
Me
7.93 ( 0.60
205 ( 56
MeO2CCH2CH2
5, IMPY
6, PIB
3.58 ( 0.4425
8.95 ( 0.72
1.322
7.23 ( 0.26d
a Values are mean ( SD (n ) 3). b Values are calculated with ACD/LogD, version 9.02 (Advanced Chemistry Development, Inc., Toronto, Canada).
c Values are mean ( SD (n ) 3). d Kd value is mean ( SD (n ) 4).
Scheme 3
raphy of extracts from perfused rat cerebellum, cerebrum, and
plasma showed 21.3%, 15.6%, and 0.1% of their total radio-
activities to be parent [11C]7 at 30 min. These values were thus
similar to those obtained in nonperfused rats. At 2.83 min the
total radioactivity in urine was very low and mainly composed
of radiometabolites A and B (Table 2).
Emergence of Radiometabolites of CA [11C]7 in Rat
Brain, Plasma, and Urine. The rat tissue concentrations of
[11C]7 and of its radiometabolites at 10 min after bolus injection
of CA [11C]7 (carrier dose, 22.94 µmol/kg) are shown in Table
3. The percentage of radioactivity represented by [11C]7 in
cerebellar, cerebral, and plasma extracts at 10 min after injection
was higher (80.9%, 83.1%, and 55.3%, respectively) in this CA
experiment than in the NCA experiment. Even the urine had a
higher percentage of [11C]7.
Scheme 4
Evaluation of NCA [11C]7 in Normal Monkey with
PET. TACs (time-activity curves) for brain regions of a 5 year
old rhesus monkey administered intravenously with a bolus of
NCA [11C]7 are shown in Figure 6A. The uptake of radioactivity
into all brain regions was rapid and high. Cerebellum showed
the highest uptake of radioactivity. The radioactivity washed
out continuously from all brain regions and at a similar rate all
over the duration of the scan. The washout was somewhat slower
than in rats and mice. The ratios of radioactivity of maximal
uptake to that at 60 min after injection were 9.9 for frontal
cortex, 8.3 for temporal cortex, 7.6 for parietal cortex, 7.8 for
occipital cortex, and 9.4 for cerebellum.
Evaluation of NCA [11C]8 in Normal Monkey with
PET. After intravenous injection of a bolus of NCA [11C]8 into
a 9.7 kg normal monkey there was rapid and high uptake of
radioactivity into the brain followed by washout to a low plateau
level (Figure 7B). The ratios of the maximal radioactivity uptake
to that at 60 min after injection for various brain regions were
3.5 for frontal cortex, 4.1 for temporal cortex, 3.1 for parietal
cortex, 3.4 for occipital cortex, and 4.2 for cerebellum.
Emergence of Radiometabolites of NCA [11C]7 in
Monkey Plasma. In two monkey experiments, anticoagulated
venous blood samples were drawn at 10 and 30 min after
injection of NCA [11C]7 into monkey, and radio-HPLC showed
at least three radiometabolites with retention times of 2.0, 3.7,
and 4.9 (vs 7.4 min for [11C]7) (Figure 7). Although [11C]7
was well-resolved from its radiometabolites, the radiometabolites
were not well-resolved from each other. Between 12.4% and
17.3% (8.1% and 15.2% SUV) of the plasma radioactivity was
[11C]7 at 10 min after its administration. At 30 min, 3.5–5.6%
(1.8–4.2% SUV) of the plasma was [11C]7.
into the brain. This uptake was followed by washout of the
majority of radioactivity over about 30 min, but thereafter
the decay-corrected radioactivity remained almost constant. The
ratio of radioactivity of maximal uptake to that at 60 min after
injection was 6.1 for rat.
Emergence of Radiometabolites of NCA [11C]7 in
Normal Rat Brain, Plasma, and Urine. The radiochemical
purities of the two preparations of NCA [11C]7 used in this
experiment were 98% and 99%. Recoveries of rat cerebellum,
forebrain, and plasma radioactivities into acetonitrile for HPLC
analysis were more than 86%. Recovery of all analyte radio-
activity from the HPLC was confirmed in an experiment with
samples from one rat. In all the tissue and plasma samples three
radiometabolites (A, B, and C) were detected, and these
appeared to be less lipophilic (tR ) 2.2, 3.7, and 5.0 min,
respectively) than parent radioligand (tR ) 7.6 min) in the
reverse phase HPLC analysis (Figure 5). The concentrations of
[11C]7 and its radiometabolites in rat tissue regions were
normalized for injected dose and rat weight by conversion to
% SUV (Table 2). At 2.83 min after the injection of [11C]7 into
three rats, the percentages of radioactivity represented by parent
radioligand in cerebellum, cerebrum, and plasma were 32.6,
30.1, and 6.5, respectively. Thirty minutes after the injection
of [11C]7, these values decreased to 18.6, 13.0, and 0.2,
respectively. At this time, residual parent radioligand concentra-
tions were very low (8.9, 5.1, and 0.2, SUV, respectively).
Therefore, the effect of residual blood on the accuracy of these
outcome measures was studied in one rat. Ex vivo chromatog-

Pyridines as Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 151
Figure 2. Autoradiographs of AD brain slices incubated with NCA [11C]7 (A) or CA [11C]7 (B) and of control human brain slices incubated with
NCA [11C]7 (C) or CA [11C]7 (D).
Figure 4. Time course of radioactivity (% SUV) in cerebrum and
cerebellum after administration of NCA [11C]8 to normal rats (n ) 2).
Figure 3. Time course of radioactivity (% SUV) in cerebrum and
cerebellum after administration of NCA [11C]7 to normal rats (n ) 4)
(A) and normal mice (n ) 4) (B). Unilateral error bars indicate the
Figure 5. Radiochromatogram of a rat brain extract at 2.83 min after
magnitude of SD.
injection of NCA [11C]7.
the HPLC column with tR ) 8.1 min (mean of three analyses).
MS-MS analysis of this metabolite generated product ions m/z
255, 237, and 223. In an MSn(3) experiment, trapping and
dissociation of the most abundant ion, m/z 255, resulted in
secondary product ions m/z 254, 240, 239, 228, 222, 211, 208,
151, and 131. These LC-MS data of metabolite B were in full
accord with those of the reference N-desmethyl compound 2.
LC-MS Analysis of Rat Brain Metabolites of 3. To
characterize the metabolites of 3, CA [11C]7 was administered
to a rat and HPLC fractions of brain homogenates corresponding
to the radiometabolites were collected and analyzed by LC-MS.
The total-ion chromatogram was searched for potential metabolite-
specific ions. Fraction 1 of the brain extract (metabolite A)
showed no protonated molecule, [M + H]+, for any metabolite
of 3 that might be generated by oxidative hydroxylation of an
N-methyl group to produce formaldehyde, formate, or carbonate.
Analysis of fraction 2 (metabolites B and C) showed a [M +
H]+ ion at m/z 270. This metabolite (metabolites B) eluted off
Discussion
We have previously developed a homogeneous catalytic
reaction for the substitution of an arylhalo substituent with a

152 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Cai et al.
Table 2. Distribution of [11C]7 and Its Radiometabolites (Mean ( SD) in Various Tissues in Rats That Were Sacrificed at Different Times after iv
Injection of NCA [11C]7
radioactivity distribution
time (min)
2.33
tissue
total radioactivity (%SUV)
% [11C]7
% Met A
1.50
23.7
5.85 ( 4.2
0.550 ( 0.55
36.6 ( 18
48.2
31.4 ( 5.7
28.9 ( 18
46.4 ( 11
% Met B
51.3
60.3
60.9 ( 0.75
68.6 ( 3.5
67.6 ( 7.5
24.0
28.3 ( 3.6
24.4 ( 1.6
34.4 ( 9.3
% Met C
cerebrum (n ) 1)
plasma (n ) 1)
cerebellum (n ) 2)
cerebrum (n ) 2)
plasma (n ) 2)
urine (n ) 1)
cerebellum (n ) 3)
cerebrum (n ) 3)
plasma (n ) 3)
668
115
41.9
9.57
5.08
6.43
2.83
261 ( 37
392 ( 12
81.3 ( 34
0.8
48.3 ( 0.9
40.0 ( 7.6
81.6 ( 14
31.9 ( 4.9
30.2 ( 4.5
9.15 ( 6.5
25.7
18.6 ( 9.2
13.0 ( 5.2
0.2 ( 0.1
1.5 ( 0.1
0.60 ( 0.5
1.6 ( 1.5
2.1
21.7 ( 7.1
33.7 ( 11
19.0 ( 2.1
30
Table 3. Distribution of CA [11C]7 and Its Radiometabolites (% SUV)
Since [11C]6 is the most thoroughly studied PET radioligand
for the detection of Aꢀ plaques in living human brain in vivo,
we selected [3H]6 as the reference radioligand for our in vitro
binding assay. Aꢀ plaques from synthetic or transgenic rodents
differ from that of AD brain in both topology and number of
binding sites.28 Therefore, we selected Aꢀ plaques from AD
brain homogenates as the closest mimic of human brain Aꢀ
plaques in vivo, for use in our assay. Binding affinity increased
with the number of N-methyl groups in this series of IMPY
derivatives (1-3) as observed for other IMPY derivatives.23
Thus, only ligand 3 was found to have a high binding affinity
similar to that of 6 and 5 (Table 1). Furthermore, the computed
and measured lipophilicities of this ligand were in the range
considered to be optimal for good penetration of the blood-brain
barrier without excessive nonspecific binding to brain tissue
(Table 1).29,30 Therefore 3 was selected for labeling with carbon-
11 and evaluation of its radioligand behavior in vitro and in
vivo.
in Various Rat Tissues and Fluids at 10 min after Bolus Injectiona
radioactivity distribution
total
radioactivity
(% SUV)
samplea
% [11C]7 % Met A % Met B % Met C
cerebellum
cerebrum
plasma
122
417
154
4.7
83.1
80.9
55.3
29.7
0.5
0.5
12.1
33.5
15.4
16.4
28.9
20.2
1.1
2.3
3.7
urine
16.7
a n ) 1.
A receptor screen revealed that ligand 3 is devoid of high-
affinity binding to a host of neuroreceptors and transporters in
the brain. Any significant PET imaging signal compared with
that of cerebellum, which is devoid of amyloid plaques in AD
patients, obtained with 11C-labeled 3 is therefore expected to
represent binding to Aꢀ plaques.
The successful 11C-labeling of an aryl methyl thiol ether
through treatment of a sulfanyl γ-propionic acid methyl ester
with [11C]iodomethane under basic conditions has been reported
previously.31 We sought to apply this method to the radiosyn-
thesis of [11C]7 and for this purpose synthesized the requisite
precursor 4 according to the general method that we have
described for the preparation of aryl thiolates (Scheme 2).26 The
choice of base for the 11C-methylation reaction was found to
be critical. The synthesis of aryl thiol ethers from masked thiol
esters has previously been realized with strong bases, such as
KOtBu at low temperature.32 Therefore, we initially evaluated
KOtBu and a variety of other inorganic bases, such as NaOH,
KOH, Cs2CO3, and K2CO3, for use in the labeling reaction.
Although some labeled product was observed, the radiochemical
Figure 6. Time course of radioactivity (% SUV) in brain regions of a
rhesus monkey after administration of NCA [11C]7 (A) or NCA [11C]8
(B).
thiolato group that avoids concomitant formation of reduced
products when using thiol or thiolate as reagent.26 Methanethiol
is a gas and inconvenient for easy handling. Therefore, to
synthesize S-methyl compounds 1-3, we modified the method
to use sodium methanethiolate and trimethyltin chloride as
reagents instead of thiol and trimethyltin dimethylamide (Scheme
1). Low to moderate yields were achieved. The observed
equivalence between thiol plus trimethyltin dimethylamide and
alkali thiolate plus trimethyltin chloride as reagents expands the
scope of our previously developed reaction.26
Figure 7. Radiochromatogram of a monkey plasma at 10 min after
injection of NCA [11C]7.

Pyridines as Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 153
yields varied dramatically. Precursor also disappeared com-
pletely during these reactions. The major side reaction was ester
hydrolysis to give the carboxylate salt, which was inert to any
further reaction. A large strong organic base (e.g., tert-
butylimine-tris(dimethylamino)phosphorane) was much more
successful in promoting a high and consistent radiochemical
yield of [11C]7. In the absence of water, such a base avoids
unwanted ester hydrolysis and favors removal of the ꢀ-proton
to generate the free thiolate ion with the desired high reactivity
toward [11C]iodomethane. A pre-equilibrium between precursor
and free thiolate ion is likely reached in the presence of such a
base (Scheme 3). Formulated [11C]7 at neutral pH was stable
but gradually decomposed under acidic conditions. Immediate
neutralization of the acid was therefore required after the HPLC
purification of [11C]7 under acid conditions.
rat were similar to those of control nonperfused animals, thus
confirming the reliability of the observations. Since the radio-
ligand was quite stable in brain homogenates, we deduced that
brain radiometabolites originated in the periphery. Therefore,
we can conclude that once [11C]7 enters the brain, it is protected
from further extensive metabolism. In the brain and in the
presence of target Aꢀ amyloid plaques, [11C]7 should bind to
the target with high affinity to deliver a significant signal that
should be detectable externally. Furthermore, when carrier 3
was added to the [11C]7 administered intravenously to rats, a
higher proportion of radioactivity in plasma at 10 min was parent
radioligand than in the NCA experiment (Table 3). This may
indicate that one or more saturable enzymes participated in the
metabolism of 3.
LC-MS analysis of rat brain extract, from the experiment
in which CA [11C]7 was injected into rat, showed a m/z 270
ion at the same retention time as the m/z 270 [M + H]+ ion of
the N-desmethyl compound 2. Furthermore, the product-ion
spectra from MS-MS and MSn(3) analyses of the brain sample
were virtually identical to those of 2. These data identify [11C]2
as the major radiometabolite of [11C]7 in rat brain. Metabolism
by N-dealkylation has been observed for other IMPY deriva-
tives.25 The enzyme CYP 3A4, which is highly abundant in
gut and liver, is a strong candidate for mediation of these
N-dealkylation reactions. The minor radiometabolites, observed
in chromatography, were not detected by LC-MS. The similari-
ties between the radiochromatograms of rat (data not shown)
and monkey plasma (Figure 7) after administration of NCA
[11C]7 imply the generation of a similar spectrum of radiome-
tabolites and that N-dealkylation is probably the route of
metabolism in monkey. This may be the expected route of
metabolism in human subjects. Entry of radiometabolites into
human brain may prove troublesome to the quantification of
specific binding of [11C]7 to Aꢀ plaques. Hence, the further
development of this and related 11C-labeled IMPY derivatives
may need to address the issue of rapid metabolism.
The technique27 of using [11C]methyl triflate to N-methylate
arylamines within a loop of narrow bore stainless steel tubing
is quite general. [11C]8 was prepared successfully in this manner
with a commercially available apparatus (Scheme 4).
NCA [11C]7 bound specifically to Aꢀ plaques in human AD
brain slices (Figure 2A). The specific binding was blocked in
the presence of excess 3 (Figure 2B), while no binding to normal
human brain tissue occurred (Figure 2C, D). These data were
encouraging for the further evaluation of carbon-11 labeled 3
as a PET radioligand in vivo.
Dynamic PET scanning in normal mouse, rat, and monkey
showed that both [11C]7 and [11C]8 gives high brain uptake
and quick washout of radioactivity (Figures 3, 4, and 6).
Cerebellum typically does not contain a significant amount of
Aꢀ plaque even in AD.18 The distribution of radioactivity across
normal brain was quite uniform for each radioligand in all three
species, as would be expected in the absence of Aꢀ plaques.
However, the different position of labeling in [11C]7 and [11C]8
led to a major difference in the brain radioactivity remaining at
60 min after injection. Correspondingly, the ratios of the
radioactivity of maximal uptake to that at 60 min after injection
were also quite different, with [11C]7 being superior in each
species. Ratios for [11C]7 were 18.7, 11.1, and 6.4 in rat, mouse,
and monkey, respectively. These ratios are higher than those
reported for [11C]6, namely, 12.7 and 10.3 for rat and mouse,
respectively.22 A dependence of radioligand pharmacodynamics
on position of radiolabel has been observed for some other PET
radioligands, a well-known example being [11C]WAY-100635.33,34
Such differences are invariably attributable to metabolism to
different radiometabolites. It was deduced that [11C]8 produces
radiometabolites that enter and persist in brain. Hence, [11C]8
was not studied further. Further experiments were focused on
the stability of [11C]7 in vitro and in vivo.
[11C]7 was stable during incubation in vitro with various
tissue homogenates, including whole blood and brain of the three
species evaluated. However, [11C]7 was rapidly metabolized in
various tissue compartments of living animals. At least three
radiometabolites (A-C) were detected in cerebellum, cerebrum,
plasma, and urine of rats (Table 2) and also in monkey plasma
(Figure 7). Each of these radiometabolites was less lipophilic
than 3 according to their faster elution from reverse-phase HPLC
(Figures 5 and 7). Radiometabolites A and B were generally
predominant in rat brain tissue, plasma, and urine between
measurements at 2.33 and 30 min after radioligand injection
(Table 2). Ratios of the cerebellar and cerebral parent concentra-
tions to those of the plasma are 55.6 ( 35.8 and 30.5 ( 12.6,
respectively. Parent radioligand is the only accumulated or
retained species in the brain tissues with its ratio to that in
plasma well above 1.0. Similar data obtained from a perfused
Conclusions
We identified [11C]7 as a prospective PET radioligand for
imaging Aꢀ plaques with PET. This radioligand is simply
prepared. It binds selectively and with high affinity to human
Aꢀ plaques in AD post mortem brain tissue slides. In healthy
rats in vivo the radioligand shows a high initial brain uptake of
radioactivity and a very fast washout, generating a ratio of the
maximal radioactivity to that at 60 min after injection of 18.
This ratio predicts a high maximal ratio of radioactivity in vivo
between the Aꢀ plaque-containing regions and a reference region
in AD patients. The analogous ratios in monkeys (7.5-10) well
exceed that of [11C]6 (about 5), an established radioligand for
Aꢀ plaque imaging. [11C]7 is slightly unstable in incubations
in vitro and is metabolized extensively in vivo, with at least
three less lipophilic radiometabolites emerging rapidly in rat
and monkey plasma. These radiometabolites are also present
in rat brain and urine. Although rapidly generated, these
radiometabolites are not retained in brain. Further evaluation
of [11C]7 in human subjects including AD patients is in progress
under an FDA-approved exploratory IND. These studies will
reveal whether a sizable specific quantifiable PET signal can
be obtained with this radioligand or whether further development
of related radioligands that avoid problems of metabolism will
be necessary.
Experimental Section
Materials. Common reagents were purchased from Aldrich
Chemical Co. (Milwaukee, WI), Fluka Chemical Co. (Milwaukee,

154 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Cai et al.
WI), Acros (Hampton, NH), or Strem Chemicals (Newburyport,
MA) and were used without further purification unless otherwise
indicated. DiPPF (1,1′-bis(diisopropylphosphino)ferrocene) was
from Strem Chemicals and Pd2dba3 [tris(dibenzylideneacetone)di-
palladium] from Aldrich. Parent IMPY derivatives were obtained
from BioAssay Systems (Hayward, CA). Water was purified
through a water purification system comprising a combination of
two filters, one Rio, one reservoir, and one Milli-Q synthesis system
(Millipore; Bedford, MA). Common solvents were obtained from
Fisher Scientific (Pittsburgh, PA). [N-methyl-3H]6 (80 Ci per mmol,
radiochemical purity >97%) was obtained from GE Healthcare
(Piscataway, NJ). Post mortem brain tissues from an autopsy-
confirmed AD case and a healthy control were obtained from the
Clinical Brain Disorders Branch, National Institute of Mental
Health. Experiments with this material were performed under the
regulations of the Ethics Committee of the National Institutes of
Health.
with a calibrated automatic well-type γ-counter (model 1480
Wizard, Perkin-Elmer; Waltham, MA) having an electronic window
set between 360 and 1800 keV (counting efficiency, 51.8%). Decay
corrections were performed with a half-life of 20.395 min.36 3
H
was measured with a liquid scintillation counter (Tri-Carb, Perkin-
Elmer).
All animal experiments were performed in accordance with the
Guide for the Care and Use of Laboratory Animals37 and were
approved by the National Institute of Mental Health Animal Care
and Use Committee.
Subsequent numerical data are expressed as the mean ( SD for
n > 2 or as the mean and range for n ) 2.
4-(6-(Methylthio)imidazo[1,2-a]pyridin-2-yl)aniline (1). 4-(6-
Iodo-H-imidazo[1,2-a]pyridin-2-yl)benzenamine (50 mg, 0.149
mmol), Pd2(dba)3 (42 mg, 0.0459 mmol), DiPPF (23.5 mg, 0.0562
mmol), NaSCH3 (12.6 mg, 0.180 mmol), and SnCl(CH3)3 (36 mg,
0.181 mmol) were added to a 10 mL microwave reaction tube. The
setup was transferred to a glovebox and anhydrous THF (1.5 mL)
added. Reaction was performed in the microwave at 110 °C, 250
psi, and 100 W for 10 min. After the reaction was confirmed to be
complete with analytical HPLC, it was quenched with aqueous
sodium bicarbonate solution (10% w/v). The mixture was extracted
with ethyl acetate (20 mL × 3). The collected organic phase was
dried to generate a brown solid, which was further purified through
reverse-phase HPLC on a preparative column (250 mm × 30 mm)
eluted with a gradient from 5% to 95% acetonitrile over 45 min in
a MeCN-H2O system, giving 1 (9.9 mg, 26%) (tR ) 26 min). Mp
124-127 °C; 1H NMR (400 MHz, CDCl3) δ 8.04 (dd, 4JHH ) 1.6
Hz, 5JHH ) 0.8 Hz, 1H, Ar-H), 7.71 (m, 2H, Ar-H), 7.65 (s, 1H,
Ar-H), 7.51 (d, 3JHH ) 9.4 Hz, 1H, Ar-H), 7.13 (dd, 3JHH ) 9.4
Hz, 4JHH ) 1.8 Hz, 1H, Ar-H), 6.72 (m, 2H, Ar-H), 2.45 (s, 3H,
S-CH3); 13C NMR (400 MHz, CDCl3) δ 146.7, 144.4, 127.6, 127.4
(s, 2C), 124.9, 124.1, 122.2, 117.1, 115.4 (s, 2C), 106.9, 18.7 (s,
1C, S-CH3); m/z (ES-MS) 391.3 (15%), 282.3 (13%), 257.1 (7%),
256.1 (100%, (M+ + H)), 149.0 (18%), 113.1 (10%). HRMS
(TOF+): calcd for C14H14N3S (M+ + H), 256.0908; found,
256.0912. Error (ppm): 1.6.
Instruments and General Methods. Analytical HPLC was
performed using a reverse-phase column (X-bridge C18, 5 µm, 10.0
mm × 250 mm, Waters Corp.; Milford, MA) eluted with concen-
trated ammonia (0.025%) in acetonitrile-water at 6.2 mL/min. The
chromatography system was fitted with a continuous wavelength
UV–vis detector (System Gold 168 detector, Beckman; Fullerton,
CA) and an autosampler (System Gold 508 autosampler, Beckman).
For semipreparative HPLC, a reverse-phase column (Atlantis C18,
5 µm, 30 mm × 150 mm, Waters Corp.) or silica gel column (10
µm, 30 mm × 250 mm, Phenomenex; Torrance, CA) was eluted
at 30 mL/min. The HPLC system was fitted with a manual injector
(5 mL loop) and a third delivery pump using acetonitrile or ethyl
acetate as eluent at 1 mL/min. The purities of compounds were
determined with HPLC monitored for absorbance at 254 nm and
expressed as area percentage of all peaks.
1
The H and 13C NMR spectra of all compounds were acquired
on a DRX 400 instrument (400 MHz for 1H and 100 MHz for 13C,
Bruker; Billerica, MA), using the chemical shifts of residual
deuterated solvent as the internal standard. Chemical shift (δ) data
for the proton and carbon resonances are reported in parts per
million (ppm) downfield relative to the internal standard. Mass
spectra were acquired using a LCQDECA LC-MS instrument
(Thermo Fisher Scientific; Waltham, MA) fitted with a Luna C18
column (5 µm, 2.0 mm × 150 mm, Phenomenex) eluted at 150
µL/min with a MeOH-H2O mixture.
N-Methyl-4-(6-(methylthio)imidazo[1,2-a]pyridin-2-yl)aniline
(2). The procedure was as in the synthesis of 1 except that the
iodo compound was 4-(6-iodo-H-imidazo[1,2-a]pyridin-2-yl)-N-
methylbenzenamine and gave 2 (20 mg, 50%). Mp 142-144 °C;
4
5
1H NMR (400 MHz, CD3OD) δ 8.24 (dd, JHH ) 1.8 Hz, JHH
)
High-resolution mass spectra (HRMS) were acquired from the
Mass Spectrometry Laboratory, University of Illinois at
UrbanasChampaign (Urbana, IL). Either electron spray ionization
or electron ionization was used for the ionization. Melting points
were measured with a Mel-Temp manual melting point apparatus
(Electrothermal, Fisher Scientific) and were uncorrected. A Discover
microwave system was used for microwave synthesis (CEM;
Matthews, NC).
0.9 Hz, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 7.57 (m, 2H, Ar-H),
7.33 (d, 3JHH ) 9.4 Hz, 1H, Ar-H), 7.16 (dd, 3JHH ) 9.3 Hz, 4JHH
) 1.8 Hz, 1H, Ar-H), 6.56 (m, 2H, Ar-H), 2.70 (s, 3H, N-CH3),
2.40 (s, 3H, S-CH3); 13C NMR (400 MHz, CDCl3) δ 149.6, 147.0,
144.4, 127.5, 127.3 (s, 2C), 124.9, 122.6, 122.1, 117.1, 112.6 (s,
2C), 106.7, 30.9 (s, 1C, N-CH3) 18.7 (s, 1C, S-CH3); m/z (ES-
MS) 271.1 (9%), 270.1 (100%, (M+ + H)). HRMS (TOF+) calcd
for C15H16N3S, 270.1065 (M+ + H); found, 270.1054. Error (ppm):
-4.1.
N,N-Dimethyl-4-(6-(methylthio)-H-imidazo[1,2-a]pyridin-2-
yl)benzenamine (3). A 10 mL microwave tube was charged with
4-(6-iodo-H-imidazo[1,2-a]pyridin-2-yl)-N,N-dimethylbenze-
namine (100 mg, 0.275 mmol), NaSMe (42.4 mg, 0.605 mmol),
Me3SnCl (120 mg, 0.605 mmol), Pd2(dba)3 (25.2 mg, 0.0275 mmol),
DiPPF (11.5 mg, 0.0275 mmol), and THF (4.0 mL) and placed in
the microwave at 120 °C, 300 psi, and 250 W for 10 min. The
reaction was monitored by analytical HPLC. At the end of the
reaction, the reaction mixture was partitioned between K2CO3
solution (2 M) and ethyl acetate. The combined organic phase was
dried with Na2SO4, filtered, and evaporated to leave brown oil. A
minimal amount of DMSO was used to dissolve the crude
compound, which was then loaded onto a preparative reverse-phase
HPLC column. The solvent of the collected peak was removed to
afford an oily product. To obtain a crystalline compound, diethyl
ether-hexane (1: 5 v/v) was added and the mixture stood at room
temperature for about 19 h. The supernatant liquid was removed
and the solid washed with excess diethyl ether and hexane and dried
overnight to give 3 (40 mg, 51%). Mp 155-164 °C; 1H NMR (400
MHz, CD3OD) δ 8.34 (s, 1H, Ar-H), 7.96 (s, 1H, Ar-H), 7.73
Methods for the analysis of radioligands and their metabolites
in biological samples were generally as previously published.35 The
following are specific details. The initial purity of [11C]7 to be
administered into animals and its stability over 2 h was assessed
by HPLC on a Novapak C18 column (100 mm × 8 mm, Waters
Corp.) housed within a Radial-Pak compression module (RCM-
100) with a sentry precolumn and eluted with MeOH/H2O/Et3N
(70:30:0.1 by volume) at 2.0 mL/min (general method A). Samples
were injected onto HPLC through nylon filters (13 mm × 0.45
µm, Iso-Disk, Supelco; Bellefonte, PA). This HPLC system was
equipped with an in-line photodiode array absorbance detector (λ
) 245 nm, Beckman) and a flow-through Na(Tl) scintillation
detector rate meter (Bioscan; Washington, DC). Data from analyses
were collected and stored with Bio-Chrome Lite software (Bioscan)
and analyzed after decay correction. Recovery of all radioactivity
from the HPLC column was checked by an injection of absolute
methanol (2 mL) at the end of the chromatography with continued
monitoring for radioactivity.
γ-Radioactivity from 11C (>1 µCi) was measured with a
calibrated dose calibrator (Atomlab 300, Biodex Medical Systems;
Shirley, NY). Low levels of radioactivity (<1 µCi) were measured

Pyridines as Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 155
(d, 3JHH ) 9.4 Hz, 2H, Ar-H), 7.44 (d, 3JHH ) 9.4 Hz, 1H, Ar-H),
7.26 (dd, 3JHH ) 9.4 Hz, 4JHH ) 2.2 Hz, 1H, Ar-H), 6.82 (d, 3JHH
) 8.9 Hz, 2H, Ar-H), 2.99 (s, 6H, NCH3), 2.51 (s, 3H, S-CH3);
13C NMR (400 MHz, CD3OD) δ 152.3, 147.6, 145.6, 129.0, 128.1,
125.9, 124.6, 122.8, 116.7, 113.9, 108.8, 40.9 (C-S), 17.9 (NCH3);
where [L] is the concentration (0.4 nM) and KD the equilibrium
constant of the reference radioligand ([3H]6). The latter was
determined with “Scatchard analysis of homologous displacement”
from multiple runs with self-displacement from AD brain tissue.
Production of Labeling Agents. NCA [11C]carbon dioxide was
produced by irradiation of nitrogen (∼225 psi) containing a low
concentration of oxygen (1%) for 40 min with a proton beam (16
MeV, 40 µA) produced from a PETtrace cyclotron (GE Medical
Systems; Milwaukee, WI). The irradiation produced about 1.4 Ci
of [11C]carbon dioxide. [11C]Carbon dioxide was converted into
[11C]iodomethane by reduction to [11C]methane and then high
temperature iodination within a Microlab module (GE MS PET
Systems AB, GE Healthcare; Piscataway, NJ). [11C]Methyl triflate
was produced by passing [11C]iodomethane in helium (30 mL/min)
over heated (190 °C) silver triflate.39
m/z (LC-MS): 286.2 (4%), 285.3 (13%), 284.2 (100%, (M+
+
H)). HRMS (TOF+) calcd for C16H18N3S (M+ + H), 284.1221;
found, 284.1215. Error (ppm): -2.4.
Methyl 3-(2-(4-(dimethylamino)phenyl)imidazo[1,2-a]pyridin-
6-ylthio)propanoate (4). 4-(6-Iodo-H-imidazo[1,2-a]pyridin-2-yl)-
N,N-dimethylbenzenamine (200 mg, 0.563 mmol), Pd2(dba)3 (75
mg, 0.0819 mmol), DiPPF (50 mg, 0.120 mmol), methyl 3-mer-
captopropanoate (80 µL, 0.738 mmol), and N-methyl-N-(trimeth-
ylstannyl)methanamine (110 µL, 0.674 mmol) were assembled in
a microwave tube within a glovebox. Anhydrous THF (1.0 mL)
was added and the mixture heated in the microwave oven at 100
°C, 250 psi, and 100 W for 30 min. The reaction was quenched
with water, and the mixture was extracted with CH2Cl2 (25 mL ×
3). The combined organic phases were dried, and the crude residue
was further purified by normal phase preparative HPLC eluted with
CHCl3 and ethyl acetate, with 0.025% TEA by volume in each
Preparation of [11C]7. Methyl 3-(2-(4-(dimethylamino)phe-
nyl)imidazo[1,2-a]pyridin-6-ylthio)propanoate (4, 0.5 mg, 1.4 µmol)
in acetonitrile (0.40 mL) and tert-butylimino-tris(dimethylamino-
)phosphorane (7 µL of 0.5 M stock solution in MeCN, 3.5 µmol)
were loaded into a septum-sealed reaction vial (1 mL neck vial,
Waters Corp.) of a Synthia apparatus (Uppsala University PET
Center, GE Healthcare; Uppsala, Sweden). [11C]Iodomethane was
then swept into the vial from the MicroLab module in a stream of
helium at 15 mL/min. After reaction was allowed to proceed at 80
°C for 5 min, [11C]7 was separated by HPLC on a Luna C18 column
(10 µm, 4.6 mm × 250 mm, Phenomenex) eluted at 6 mL/min
with MeCN/0.1% H3PO4 (20:80 v/v) changed linearly to 50:50 (v/
v) over 20 min. Eluate was monitored for absorbance at 350 nm
and radioactivity. The fraction containing [11C]7 (tR ) 9.3 min)
was collected and neutralized with aqueous NaHCO3 solution (8.4%
w/v) and then rotary-evaporated to dryness (80 °C water bath). The
residue of [11C]7 was formulated in sterile saline for injection USP
(0.9% w/v, 10 mL) plus ethanol USP (0.9 mL) containing
polysorbate 80 (20 mg) and then sterile-filtered into a sterile and
pyrogen-free dose vial.
This product was analyzed by HPLC on a Luna C18 column
(10 µm, 4.6 mm × 250 mm, Phenomenex) eluted with 0.1%
phosphoric acid (84% w/w) in water (pH ∼ 2.35) (A)/acetonitrile
(B) at 2 mL/min with mobile phase composition run at 70% A for
2 min and then decreased to 40% A over 15 min. Eluate was
monitored for absorbance at 350 nm and for radioactivity. Retention
times were as follows: 5.2 min for 4, 7.2 min for [11C]7, and 11
min for [11C]iodomethane. The response of the analytical system
was calibrated for mass of 3 to allow specific radioactivity to be
calculated.
1
solvent, to afford 4 (140 mg, 70%). Mp 116-118 °C; H NMR
(400 MHz, DMSO-d6) δ 8.60 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H),
3
3
7.76 (d, JHH ) 8.4 Hz, 2H, Ar-H), 7.50 (d, JHH ) 9.3 Hz, 1H,
Ar-H), 7.25 (dd, 3JHH ) 9.2 Hz, 1H, Ar-H), 6.78 (d, 3JHH ) 8.5
Hz, 2H, Ar-H), 3.57 (s, 3H, O-CH3), 3.10 (t, 3JHH ) 6.9 Hz, 2H,
SCH2), 2.94 (s, 6H, NCH3), 2.62 (t, 3JHH ) 6.9 Hz, 2H, CH2); 13
C
NMR (400 MHz, CD3OD) δ 171.6 (-CO2-), 150.1, 145.8, 143.6,
128.3, 126.5, 121.4, 117.4, 116.2, 112.2, 107.2, 51.5 (OCH3), 38.9
(NCH3), 33.7 (CH2), 29.9 (CH2); m/z (ES-MS): 358.1 (2%), 357.1
(9%), 356.1 (100%, (M+ + H)). HRMS (TOF+) calcd for
C19H21N3O2S (M+ + H), 356.1433; found, 356.1450. Error (ppm):
4.8.
LogD Measurement. Ligand (3-5 mg) was dissolved in PBS
(0.1 M, pH 7.4, 6.0 mL) and n-octanol (1.0 mL). The septum-
sealed mixture was vortexed for 1 min, shaken vigorously for
another 1 min, filtered, held at room temperature for 10 min, and
centrifuged at 5000g for 10 min. Aliquots of the organic and
aqueous layers were sampled through the septum by syringe. An
aliquot of octanol layer (5.0 µL) was added to acetonitrile (5.0 mL)
to prepare a stock solution. Both aqueous and organic phases (1.0
mL each) were analyzed for ligand by HPLC (X-bridge C18, 5
µm, 10.0 mm × 250 mm, Waters Corp.) eluted at 2.0 mL/min with
MeCN/water containing concentrated ammonia (0.025% by volume)
in each component, using a gradient from 5% to 95% MeCN over
15 min. Partition coefficients (P) were calculated as the ratio of
ligand concentrations between the organic and aqueous phases.
In Vitro Binding Assay. Human AD brain tissue was homog-
enized in phosphate-buffered saline (PBS) at 1:500 dilution in
volume, and aliquots of this suspension (100 µL) were added to
each of four tubes (total 48 in a rack). A solution of [3H]6 (2.7 ×
10-4 µCi/µL) in PBS (100 µL) was added to each tube. Nonra-
dioactive 6 or other displacer was dissolved in DMSO to give a 1
mM stock solution, which was further diluted with DMSO to give
solutions ranging from 1 × 10-5 to 10-10 M; 10 µL of solution
was added to each tube. After assembly of the components listed
above in each tube (made up to a total volume of 1.0 mL by PBS),
the tubes were vortexed and then incubated for 2 h at 37 °C. After
separation of tube contents with a cell harvester, the filter paper
(GF/B, Whatman, pretreated with 0.5% polyethyleneimine solution)
was washed with PBS (3 mL × 3). The filters were placed in 7
mL plastic vials, and scintillation fluid (4 mL) was added to each.
After overnight incubation, the scintillation vials were counted for
radioactivity. The collected data were analyzed using GraphPad
Prism 4, version 4.03 (GraphPad Software; San Diego, CA), with
“one site competition” curve-fitting. Ki values were calculated
according to the Cheng-Prusoff equation:38
Preparation of [11C]8. The labeling reaction between precursor
2 and [11C]methyl triflate was performed in a commercial loop
system (Bioscan).27 The helium flow through the apparatus was
adjusted to 30 mL/min. Precursor 2 (0.5 mg, 1.9 µmol) was
dissolved in methyl ethyl ketone (80 µL) and loaded into the loop
at about 1 min before the end of radionuclide production. [11C]M-
ethyl triflate was passed into the loop in helium at 30 mL/min, and
the reaction was allowed to proceed for 2 min at room temperature.
The reaction mixture was injected onto a C-18 Luna column (10
µm, 4.6 × 250 mm, Phenomenex) eluted with MeCN/50 mM
HCOONH4 (60:40 v/v, pH 7.2) at 6 mL/min. Eluate was monitored
for absorbance at 350 nm and radioactivity. The fraction containing
[11C]8 (tR ) 7.5 min) was well separated from [11C]iodomethane
(tR ≈ 3-4 min), 2 (tR ≈ 5.3 min) and other products and was
collected and then rotary-evaporated to dryness (80 °C water bath).
The residue was formulated in sterile saline (10 mL) containing
ethanol (5% v/v). This product was analyzed by radio-HPLC as
described for [11C]7.
Autoradiography of [11C]7 to Post Mortem Human Brain
Tissue. Coronal sections of cerebrum from a confirmed case of
AD (female, 59 year old, post mortem interval (PMI) of 47.5 h)
and a normal control (female, 64 year old, PMI of 28.5 h) were
frozen rapidly in an equal mixture of dry ice and isopentane, sealed
in a plastic bag, and stored at -76 °C before sectioning. Frozen
blocks of the medial temporal lobe (hippocampal region) were
sectioned at a thickness of 14 µm, mounted on gelatin-coated glass
IC50
Ki )
[L]
1 +
KD

156 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Cai et al.
slides, dried, and stored under desiccant at -76 °C. Before use,
slide-mounted tissue sections were removed from the freezer,
thawed at room temperature for 20 min, and air-dried.
center of the field of view. Tomographic images were analyzed
with PMOD 2.6 (pixelwise modeling computer software, PMOD
Group; Zurich, Switzerland).41 Two regions of interest, cerebrum
and cerebellum, were delineated, and time-activity curves were
calculated.
Ligand 3 (1.0 mg) was dissolved in DMSO (3.529 mL), resulting
in a stock solution of 1 mM, which was then further diluted with
DMSO to 0.1 mM and then diluted with PBS to 1 µM. The AD
tissue and normal tissue slides were pretreated with either a stock
solution of 3 or PBS for 20 min at room temperature. All slides
were incubated for 20 min at room temperature in [11C]7 formula-
tion solution (0.3 mCi). They were then dipped in ethanol for 5
min. The slides were dried on a hot plate with a stream of cold air
and placed in a cassette with the exposed sides facing up. The
phosphor imaging plate was held with the blue side down. The
entire cassette was placed in the dark at room temperature overnight
for 11C-labeling. Digital autoradiography was acquired using a FUJI
BAS 5000 phosphorimager (FUJI, Tokyo, Japan) with a resolution
of 25 µm.
Emergence of Radiometabolites of NCA [11C]7 in Normal
Rat. Various minutes (2.33, 2.83, and 30 min) after the penile
venous injection of NCA [11C]7 (0.82-3.6 mCi, SR 0.9-1.9 Ci/
µmol) into each of eight rats, a large blood sample (about 5-10
mL) was drawn from each and placed on ice until analysis. Urine
was aspirated from the urinary bladder and analyzed within a few
minutes with HPLC by general method A. The rat was then
immediately sacrificed by decapitation, and the cerebrum and
cerebellum were excised and placed on ice until analysis. One rat
was perfused with heparinized saline (0.9% w/v, 12 mL) through
the heart after severing the right aortic vessel. The perfusion was
continued until the perfusate ran clear of blood. The brain tissues
were placed in acetonitrile (1.5 mL) and measured for radioactivity
in the γ-counter. The tissues were then homogenized along with
nonradioactive 3 (50 µg). After the addition of water (500 µL), the
tissues were further homogenized and measured in the γ-counter.
The homogenates were centrifuged at 10000g for 1 min. The
supernatant liquids were then analyzed with HPLC (general method
A). After separation of the plasma from blood cells, plasma samples
(50 µL) were counted in the γ-counter and aliquots (450 µL) placed
in acetonitrile (700 µL) along with carrier 3 (5 µg). After the
samples were mixed well, water (100 µL) was added and the
samples were further mixed well and measured in a γ-counter.
The samples were then centrifuged and the clear supernatant liquids
analyzed by radio-HPLC (general method A). All precipitates were
measured in the γ-counter so that recoveries of radioactivity into
the supernatants could be calculated.
Stability of NCA [11C]7 in Rat and Monkey Whole Blood
in Vitro. Whole blood (1 mL) from monkey or rat was placed in
a polypropylene test tube (13 mm × 60 mm) with 25.0 µCi (30
µL) or 46.5 µCi (15 µL) of NCA [11C]7, respectively. The samples
were then incubated at 37 °C in a reciprocating shaker water bath
(model 25, Fisher Scientific; Pittsburgh, PA) at 60 oscillations per
minute. The radioactive whole blood was sampled (50-100 µL)
at 60 and 90 min and then placed in MeCN (300 µL) that had been
spiked with 3. The mixture was mixed well. Then water (1.0 mL)
was added and the solution mixed well again. All the MeCN
samples were counted in a γ-counter and then centrifuged at 10000g
for 1 min. The supernatant liquids were analyzed with HPLC
general method A. The precipitates were measured for radioactivity
in a γ-counter to allow calculation of the recovery of radioactivity
into acetonitrile.
Stability of NCA [11C]7 in Rat Brain in Vitro. One healthy
rat (350 g) was anesthetized with 1.5% isoflurane and 98.5% O2.
Blood (about 10 mL) was drawn by cardiac puncture and used in
the above-described whole blood stability experiment. The rat was
then perfused with saline (0.9% w/v, 12 mL) through the exposed
heart right atrium while the aorta was severed. The excised brain
(1.75 g) was homogenized with saline (3 mL) while cooled on ice
with a Tissue-Tearor (model 985-370, Biospec Products Inc.;
Bartlesville, OK). Formulated [11C]7 (186 µCi) was added to the
brain homogenate and then incubated in a reciprocating shaker at
37 °C. For HPLC analyses, aliquots (100 µL) from this mixture
were removed at 90 min and placed in acetonitrile (300 µL) that
had been spiked with 3. The mixture was mixed well, and then
water (100 µL) was added and the solution mixed well again. All
the acetonitrile samples were counted in a γ-counter and then
centrifuged at 10000g for 1 min. The supernatant liquids were
analyzed with HPLC general method A. The precipitates were then
counted in a γ-counter for calculating the recovery of radioactivity
into the acetonitrile.
Evaluation of Radioligands in Normal Rodents in Vivo
with PET. For radioligand injection, a 30 gauge needle was attached
to a polyethylene catheter (PE 10) and inserted into the tail vein of
a mouse or the penis vein of a male rat. The needle and catheter
were secured with tissue adhesive and tape. For dynamic scanning,
heads and bodies of mice and rats were fixed with tape and kept
under 1.5% isoflurane anesthesia via a nose cone. Body temperature
was monitored with a rectal probe and maintained between 36 and
37 °C. Then formulated NCA radioligand (400-450 µCi, volume
of 0.1-0.2 mL) was injected and flushed with heparinized saline
(0.070 mL).
The % SUV due to parent radioligand or radiometabolite only
was calculated by multiplying the total % SUV by the fraction
obtained by HPLC using the following equation:
% SUV )
tissue activity/tissue (g)
× 100 × body weight (g) × HPLC fraction
(
)
injected activity
Emergence of Rat Brain Metabolites of CA [11C]7 in Rat.
One healthy rat (311 g) was anesthetized with 1.5% isoflurane in
O2. The rat body temperature was maintained around 37 °C with a
heating pad. To ensure the expression of urine, the rat was infused
with saline (0.9% w/v, 5.0 mL) for 30 min. The rat was then injected
with CA [11C]7 (950 µCi, 7.14 µmol carrier, 22.9 µmol carrier per
kg, specific radioactivity 0.113 mCi/µmol) formulated in saline
(0.9% w/v, 2.0 mL) containing ethanol (10% v/v) and Tween-80
(5% v/v). Briefly, the dose was prepared as follows: 3 (2.02 mg,
7.135 nmol) was dissolved in ethanol (200 µL) and then Tween-
80 (100 µL) added. The mixture was vortexed. To the resulting
solution, saline (0.9% w/v) was added in increments of 200 µL
with vortexing on each addition until the total volume became 2.0
mL. Radioactivity was added to the vial that contained the
nonradioactive 3, mixed well, and measured in a dose calibrator.
The now low specific activity dose was then drawn, the vial
measured again, and the dose injected into the rat. The dose was
infused through the penile vein over 5.0 min. After 10 min, the
urinary bladder of the rat was exposed and urine (∼850 µL)
withdrawn into a syringe. The brain (1.6 g) and cerebellum (0.31
g) were excised, and each was placed in MeCN (1.5 mL) along
with carrier 3 (∼100 µg). The tissues were then homogenized with
the “Tissue-Tearor”. Water (500 µL) was added and the mixture
rehomogenized. The homogenates were centrifuged at 10000g for
1.0 min. The clear supernatant liquids were decanted and placed
in fresh polypropylene tubes. The precipitates were rehomogenized
with acetonitrile (1.5 mL) followed by water (500 µL) and
centrifuged as before. The second supernatants were combined with
the previous ones. The tissue radioactivities were further separated from
the parent by HPLC and the radiometabolite fractions I and II collected
for storage at -70 °C until LC-MS analysis. Aliquots (1 mL) from
rat brain extracts were concentrated with a SpeedVac evaporator
For PET imaging, we used the NIH Advanced Technology
Laboratory Animal Scanner (ATLAS) with an effective transaxial
field of view of 6.0 cm and an axial field of view of 2.0 cm.40
Dynamic scanning began at the time of injection and lasted for 90
min. Data were reconstructed into 17 coronal slices with a voxel
size of 0.56 mm × 0.56 mm × 1.12 mm. No attenuation or scatter
corrections were applied. The dual-layered phoswich detector design
of the scanner and 3D OSEM (ordered subset expectation maxi-
mization) reconstruction achieved a resolution of 1.6 mm at the

Pyridines as Radioligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 157
(Thermo Fisher Scientific) and the residues reconstituted in mobile
phase A (H2O/MeOH/AcOH, 90:10:0.5 by volume, 200 µL). The
samples were centrifuged (10000g, 1 min) and the supernatant liquids
transferred to an autosampler vial for injection into LC-MS. Because
only a small amount of radioactivity was excreted, the urine was diluted
2.5-fold with acetonitrile and injected onto the HPLC. A very small
volume was saved for LC-MS analysis.
A reverse-phase HPLC column (Synergi Fusion-RP, 4 µm,
150 mm × 2 mm, Phenomenex) was used for the separation of the
metabolites of CA [11C]7. A gradient analysis was performed with
mobile phase A (listed above) and mobile phase B composed of
methanol with 0.5% acetic acid. Initially, the column was equili-
brated with mobile phase 80% A and 20% B at 150 µL/min. The
sample (5 µL) was injected, and after 1 min the pump ran a linear
gradient reaching 20% A and 80% B over 10 min and then was
held isocratic at this composition for 3 min. At the end of the run,
the mobile phase was returned to the initial composition and
equilibrated with elution for 3 min at 250 µL/min.
The separated components from the brain extract were ionized
by electrospray with the following settings: sheath gas flow 64 units,
auxiliary gas flow 10 units, capillary temperature 260 °C, capillary
voltage 26 V, and spray voltage 5 kV. For MS analysis, ions ranging
between m/z 150 and 600 were acquired. MS-MS and MSn(3)
analyses of 3 and its metabolites were performed with an isolation
width of 1.5 amu and a collision energy level at 44%.
Reference solutions (2 ng/µL) of 3 and 2 were prepared in mobile
phase A and injected (1-2 µL) into LC-MS and analyzed in a
similar manner to brain samples.
Evaluation of Radioligands in Monkey in Vivo with PET. A
male rhesus monkey (15 kg) was initially immobilized with
ketamine (15 mg/kg) and subsequently anesthetized with isoflurane
(1.5%) for the duration of the experiment. The monkey was placed
prone in the PET camera (HRRT, Siemens/CPS; Knoxville, TN).
A fixation device was used to secure the monkey’s head during
scanning. A urinary catheter was inserted and clamped so that the
activity overlaying the bladder represented the total urinary excre-
tion during the scan. Electrocardiogram, body temperature, and heart
and respiration rates were measured throughout the experiment.
Body temperature was controlled and monitored by a forced-air
temperature management unit (Bair Hugger model 505, Arizant
Healthcare Inc.; MN). The scanner consisted of eight flat panel
detectors with a transaxial and axial coverage of 31.2 and 25.2 cm,
respectively. The scanner is also equipped with dual-layered
phoswich detector allowing depth of interaction. Dynamic PET
scans were acquired in 64-bit list mode format, following the
intravenous administration of [11C]7 or [11C]8 (2.9 mCi). The scan
lasted for 2 h containing 33 frames with duration ranging from
30 s to 5 min. Data were reconstructed into a 256 × 256 × 207
image matrix (voxel size 1.21 × 1.21 × 1.23 mm), using a 3D list
mode OSEM algorithm.42 The reconstructed image resolution was
2.5 mm. Transmission scan was acquired with a rotating 137Cs point
source for 6 min and used to correct for attenuation. A model-
based scatter correction was applied. Tomographic images were
analyzed with PMOD 2.6.41 Time-activity curves were calculated
in % SUV for volume of interest (VOIs), defined by coregistration
with MRI (see below) and compared for uptake and washout
between different brain regions.
basic pH with sodium bicarbonate. A second baseline (retest)
experiment was performed in the same monkey injected with [11C]7
(3.99 mCi, 1.93 Ci/µmol, 2.07 nmol carrier, 0.14 nmol carrier per
kg, pH 4.5-5.0). Heparinized venous blood samples (2.0 mL) from
both experiments were drawn at 10 and 30 min after injection.
Blood samples were centrifuged at 1800g for 1.5 min. Plasma
samples (450 µL) were then placed in MeCN (700 µL) and spiked
with 3. The MeCN-plasma mixture was measured for total
radioactivity in a γ-counter and then centrifuged at 10000g for 1
min and analyzed by radio-HPLC (general method A). The activities
remaining in the precipitates were used to calculate the percent
recovery of activity into the acetonitrile analytes. The % SUV values
for each component detected by radio-HPLC were calculated as
described above for the rat experiment.
Acknowledgment. This research was supported by the
Intramural Research Program of the National Institutes of
Health, specifically the National Institute of Mental Health. We
gratefully thank Dr. Shuiyu Lu (MIB, NIMH), Kun Park (MIB,
NIMH), and Edward Tuan (MIB, NIMH) for experimental
assistance and Dr. Joel E. Kleinman (CDBD, NIMH) for post
mortem human brain tissues and useful discussions. We also
thank the NIH PET Department for carbon-11 production and
successful completion of the scanning experiments, PMOD
Technologies for providing the image analysis software, and
the NIMH Psychoactive Drug Screening Program (PDSP) for
performing assays. The PDSP is directed by Bryan L. Roth,
Ph.D., with project officer Jamie Driscoll (NIMH) at the
University of North Carolina Chapel Hill (Contract No.
NO1MH32004).
Supporting Information Available: Purity data and HPLC
chromatograms of compounds 1-4. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy. Physiol.
ReV. 2001, 81, 741–766.
(2) Selkoe, D. J. Alzheimer disease: mechanistic understanding predicts
novel therapies. Ann. Intern. Med. 2004, 140, 627–638.
(3) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease:
progress and problems on the road to therapeutics. Science 2002, 297,
353–356.
(4) Cummings, J. L. Alzheimer’s disease. N. Engl. J. Med. 2004, 351,
56–67.
(5) Mattson, M. P. Pathways towards and away from Alzheimer’s disease.
Nature 2004, 430, 631–639.
(6) Wilquet, V.; De Strooper, B. Amyloid-beta precursor protein process-
ing in neurodegeneration. Curr. Opin. Neurobiol. 2004, 14, 582–588.
(7) Rogaeva, E.; Meng, Y.; Lee, J. H.; Gu, Y.; Kawarai, T.; Zou, F.;
Katayama, T.; Baldwin, C. T.; Cheng, R.; Hasegawa, H.; Chen, F.;
Shibata, N.; Lunetta, K. L.; Pardossi-Piquard, R.; Bohm, C.; Wakutani,
Y.; Cupples, L. A.; Cuenco, K. T.; Green, R. C.; Pinessi, L.; Rainero,
I.; Sorbi, S.; Bruni, A.; Duara, R.; Friedland, R. P.; Inzelberg, R.;
Hampe, W.; Bujo, H.; Song, Y. Q.; Andersen, O. M.; Willnow, T. E.;
Graff-Radford, N.; Petersen, R. C.; Dickson, D.; Der, S. D.; Fraser,
P. E.; Schmitt-Ulms, G.; Younkin, S.; Mayeux, R.; Farrer, L. A.;
George-Hyslop, P. The neuronal sortilin-related receptor SORL1 is
genetically associated with Alzheimer disease. Nat. Genet. 2007, 39,
168–177.
(8) Farrer, L. A.; Cupples, L. A.; Haines, J. L.; Hyman, B.; Kukull, W. A.;
Mayeux, R.; Myers, R. H.; Pericak-Vance, M. A.; Risch, N.; van Duijn,
C. M. Effects of age, sex, and ethnicity on the association between
apolipoprotein E genotype and Alzheimer disease. A meta-analysis.
APOE and Alzheimer Disease Meta Analysis Consortium. JAMA,
J. Am. Med. Assoc. 1997, 278, 1349–1356.
(9) Saunders, A. M.; Strittmatter, W. J.; Schmechel, D.; George-Hyslop,
P. H.; Pericak-Vance, M. A.; Joo, S. H.; Rosi, B. L.; Gusella, J. F.;
Crapper-MacLachlan, D. R.; Alberts, M. J. Association of apolipo-
protein E allele epsilon 4 with late-onset familial and sporadic
Alzheimer’s disease. Neurology 1993, 43, 1467–1472.
(10) Bertram, L.; Hsiao, M.; McQueen, M. B.; Parkinson, M.; Mullin, K.;
Blacker, D.; Tanzi, R. E. The LDLR locus in Alzheimer’s disease: a
family-based study and meta-analysis of case-control data. Neurobiol.
Aging 2007, 28, 18–21.
Coregistration of PET Data with MRI. All frames of the
original reconstructed PET data were summed and then coregistered
to a T1-weighted magnetic resonance (MR) image acquired
separately on a 1.5 T Signa MR scanner (GE Medical Systems)
with image analysis software MEDx (Sensor Systems Inc.; Sterling,
VA). The summed PET image was fused with the coregistered MR
image with an image fusion tool in PMOD. Several VOIs for the
source organs were then manually defined on this fused image with
anatomical structures identified on the MR image.
Emergence of Radiometabolites of NCA [11C]7 in Monkey
Plasma. A monkey (14.9 kg) was anesthetized with 1.6% isoflurane
in oxygen and as a baseline experiment (test) was administered
[11C]7 (2.31 mCi, 1.35 Ci/µmol, 1.71 nmol carrier, 0.11 nmol carrier
per kg, pH ∼ 8-9) formulated in 5% ethanolic saline adjusted to

158 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Cai et al.
(11) Bertram, L.; McQueen, M. B.; Mullin, K.; Blacker, D.; Tanzi, R. E.
Systematic meta-analyses of Alzheimer disease genetic association
studies: the AlzGene database. Nat. Genet. 2007, 39, 17–23.
(12) Braak, H.; Braak, E. Neuropathological stageing of Alzheimer-related
changes. Acta Neuropathol. 1991, 82, 239–259.
(13) Tanzi, R. E. Molecular genetics of Alzheimer’s disease and the amyloid
beta peptide precursor gene. Ann. Med. 1989, 21, 91–94.
(14) Engler, H.; Forsberg, A.; Almkvist, O.; Blomquist, G.; Larsson, E.;
Savitcheva, I.; Wall, A.; Ringheim, A.; Långström, B.; Nordberg, A.
Two-year follow-up of amyloid deposition in patients with Alzheimer’s
disease. Brain 2006, 129, 2856–2866.
(15) Small, G. W.; Kepe, V.; Ercoli, L. M.; Siddarth, P.; Bookheimer, S. Y.;
Miller, K. J.; Lavretsky, H.; Burggren, A. C.; Cole, G. M.; Vinters,
H. V.; Thompson, P. M.; Huang, S. C.; Satyamurthy, N.; Phelps, M. E.;
Barrio, J. R. PET of brain amyloid and tau in mild cognitive
impairment. N. Engl. J. Med. 2006, 355, 2652–2663.
(26) Cai, L.; Cuevas, J.; Peng, Y. Y.; Pike, V. W. Rapid palladium-catalyzed
cross-coupling in the synthesis of aryl thioethers under microwave
conditions. Tetrahedron Lett. 2006, 47, 4449–4452.
(27) Wilson, A. A.; Garcia, A.; Chestakova, A.; Kung, H.; Houle, S. A
rapid one-step radiosynthesis of the ꢀ-amyloid imaging radiotracer
N-methyl-[11C]-2-(4′-methylaminophenyl)-6-hydroxybenzothiazole([11C]-
6-OH-BTA-1). J. Labelled Compd. Radiopharm. 2004, 47, 679–682.
(28) Klunk, W. E.; Lopresti, B. J.; Debnath, M. L.; Holt, D. P.; Wang, Y.;
Huang, G.-F.; Shao, L.; Lefterov, I.; Koldamova, R.; Ikonomovic, M.;
DeKosky, S. T.; Mathis, C. A. Amyloid deposits in transgenic PS1/
APP mice do not bind the amyloid PET tracer, PIB, in the same
manner as human brain amyloid. Neurobiol. Aging 2004, 25, 232–
233.
(29) Pike, V. W. Positron-emitting radioligands for studies in vivo. Probes
for human psychopharmacology. J. Psychopharmacol. 1993, 7, 139–
158.
(16) Newberg, A. B.; Wintering, N. A.; Plossl, K.; Hochold, J.; Stabin,
M. G.; Watson, M.; Skovronsky, D.; Clark, C. M.; Kung, M. P.; Kung,
H. F. Safety, biodistribution, and dosimetry of 123I-IMPY: a novel
amyloid plaque-imaging agent for the diagnosis of Alzheimer’s disease.
J. Nucl. Med. 2006, 47, 748–754.
(17) Cai, L.; Innis, R. B.; Pike, V. W. Radioligand development for PET
imaging of ꢀ-amyloid-current status. Curr. Med. Chem. 2007, 14, 19–
52.
(18) Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.;
Holt, D. P.; Bergstrom, M.; Savitcheva, I.; Huang, G. F.; Estrada, S.;
Ausen, B.; Debnath, M. L.; Barletta, J.; Price, J. C.; Sandell, J.;
Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni, G.; Mathis, C. A.;
Långström, B. Imaging brain amyloid in Alzheimer’s disease with
Pittsburgh Compound-B. Ann. Neurol. 2004, 55, 306–319.
(19) Price, J. C.; Klunk, W. E.; Lopresti, B. J.; Lu, X.; Hoge, J. A.; Ziolko,
S. K.; Holt, D. P.; Meltzer, C. C.; DeKosky, S. T.; Mathis, C. A.
Kinetic modeling of amyloid binding in humans using PET imaging
and Pittsburgh Compound-B. J. Cereb. Blood Flow Metab. 2005, 25,
1528–1547.
(30) Waterhouse, R. N. Determination of lipophilicity and its use as a
predictor of blood-brain barrier penetration of molecular imaging
agents. Mol. Imaging Biol. 2003, 5, 376–389.
(31) Schou, M.; Pike, V. W.; Varrone, A.; Gulyas, B.; Farde, L.; Halldin,
C. Synthesis and PET evaluation of (R)-[S-methyl-11C]thionisoxetine,
a candidate radioligand for imaging brain norepinephrine transporters.
J. Labelled Compd. Radiopharm. 2006, 49, 1007–1019.
(32) Becht, J. M.; Wagner, A.; Mioskowski, C. Facile introduction of SH
group on aromatic substrates via electrophilic substitution reactions.
J. Org. Chem. 2003, 68, 5758–5761.
(33) Pike, V. W.; McCarron, J. A.; Lammerstma, A. A.; Hume, S. P.; Poole,
K.; Grasby, P. M.; Malizia, A.; Cliffe, I. A.; Fletcher, A.; Bench, C. J.
First delineation of 5-HT1A receptors in human brain with PET and
[
11C]WAY-100635. Eur. J. Pharmacol. 1995, 283, R1–R3.
(34) Hume, S. P.; Ashworth, S.; Opacka-Juffry, J.; Ahier, R. G.; Lam-
mertsma, A. A.; Pike, V. W.; Cliffe, I. A.; Fletcher, A.; White, A. C.
Evaluation of [O-methyl-3H]WAY-100635 as an in vivo radioligand
for 5-HT1A receptors in rat brain. Eur. J. Pharmacol. 1994, 271, 515–
523.
(20) Naslund, J.; Schierhorn, A.; Hellman, U.; Lannfelt, L.; Roses, A. D.;
Tjernberg, L. O.; Silberring, J.; Gandy, S. E.; Winblad, B.; Greengard,
P. Relative abundance of Alzheimer Aꢀ amyloid peptide variants in
Alzheimer disease and normal aging. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 8378–8382.
(21) Laruelle, M.; Slifstein, M.; Huang, Y. Relationships between ra-
diotracer properties and image quality in molecular imaging of the
brain with positron emission tomography. Mol. Imaging Biol. 2003,
5, 363–375.
(22) Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.; Debnath, M. L.;
Klunk, W. E. Synthesis and evaluation of 11C-labeled 6-substituted
2-arylbenzothiazoles as amyloid imaging agents. J. Med. Chem. 2003,
46, 2740–2754.
(23) Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.; Plossl, K.; Hou,
C.; Holtzman, D. M.; Kung, H. F. Structure-activity relationship of
imidazo[1,2-a]pyridines as ligands for detecting ꢀ-amyloid plaques
in the brain. J. Med. Chem. 2003, 46, 237–243.
(24) Cai, L.; Cuevas, J.; Temme, S.; Herman, M. M.; Dagostin, C.;
Widdowson, D. A.; Innis, R. B.; Pike, V. W. Synthesis and
structure-affinity relationships of new 4-(6-iodo-H-imidazo[1,2-
a]pyridin-2-yl)-N,N-dimethylbenzeneamine derivatives as ligands for
human betaamyloid plaques. J. Med. Chem. 2007, 50, 4746–4758.
(25) Cai, L.; Chin, F. T.; Pike, V. W.; Toyama, H.; Liow, J. S.; Zoghbi,
S. S.; Modell, K.; Briard, E.; Shetty, H. U.; Sinclair, K.; Donohue,
S.; Tipre, D.; Kung, M. P.; Dagostin, C.; Widdowson, D. A.; Green,
M.; Gao, W.; Herman, M. M.; Ichise, M.; Innis, R. B. Synthesis and
evaluation of two 18F-labeled 6-iodo-2-(4′-N,N-dimethylamino)phe-
nylimidazo[1,2-a]pyridine derivatives as prospective radioligands for
ꢀ-amyloid in Alzheimer’s disease. J. Med. Chem. 2004, 47, 2208–
2218.
(35) Zoghbi, S. S.; Shetty, H. U.; Ichise, M.; Fujita, M.; Imaizumi, M.;
Liow, J. S.; Shah, J.; Musachio, J. L.; Pike, V. W.; Innis, R. B. PET
imaging of the dopamine transporter with 18F-FECNT: a polar
radiometabolite confounds brain radioligand measurements. J. Nucl.
Med. 2006, 47, 520–527.
(36) Weber, D. A.; Eckerman, K. F.; Dillman, L. T.; Ryman, J. C. MIRD:
Radionuclide Data and Decay Schemes; Society of Nuclear Medicine:
New York, 1989.
(37) Clark, J. D.; Baldwin, R. L.; Bayne, K. A.; Brown, M. J.; Gebhart,
G. F.; Gonder, J. C.; Gwathmey, J. K.; Keeling, M. E.; Kohn, D. F.;
Robb, J. W.; Smith, O. A.; Steggerda, J.-A. D.; VandeBerg, J. L. Guide
for the Care and Use of Laboratory Animals; National Academy Press:
Washington, DC, 1996.
(38) Cheng, Y.; Prusoff, W. H. Relationship between inhibition constant
(K1) and concentration of inhibitor which causes 50% inhibition (IC50)
of an enzymatic-reaction. Biochem. Pharmacol. 1973, 22, 3099–3108.
(39) Jewett, D. M. A simple synthesis of [C-11] methyl triflate. Appl. Radiat.
Isot. 1992, 43, 1383–1385.
(40) Green, M. V.; Seidel, J.; Vaquero, J. J.; Jagoda, E.; Lee, I.; Eckelman,
W. C. High resolution PET, SPECT and projection imaging in small
animals. Comput. Med. Imaging Graphics 2001, 25, 79–86.
(41) Mikolajczyk, K.; Szabatin, M.; Rudnicki, P.; Grodzki, M.; Burger, C.
A JAVA environment for medical image data analysis: initial
application for brain PET quantitation. Med. Informatics (London)
1998, 23, 207–214.
(42) Carson, R. E.; Barker, W. C.; Liow, J.-S.; Yao, R.; Thada, S.; Zhao,
Y.; Iano-Fletcher, A.; Lenox, M. List-mode reconstruction for the
HRRT. J. Nucl. Med. 2004, (Suppl. 45), 105P.
JM700970S