Synthesis and evaluation of18F-labeled 2-phenylbenzothiazoles as positron emission tomography imaging agents for amyloid plaques in alzheimer's disease

Article abstract of DOI:10.1021/jm8013376

Imaging agents targeting amyloid β(Aβ) may be useful for early diagnosis and follow-up of treatment of patients with Alzheimer's disease (AD). Three of five tested 2-(4′-fluorophenyl)-1,3-benzothiazoles displayed high binding affinities for Aβ plaques in

Full text of DOI:10.1021/jm8013376

1428
J. Med. Chem. 2009, 52, 1428–1437
18
Synthesis and Evaluation of F-Labeled 2-Phenylbenzothiazoles as Positron Emission
Tomography Imaging Agents for Amyloid Plaques in Alzheimer’s Disease
Kim Serdons,*,† Christelle Terwinghe,‡ Peter Vermaelen,‡ Koen Van Laere,‡ Hank Kung,§ Luc Mortelmans,‡ Guy Bormans,†
and Alfons Verbruggen†
Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences, Katholieke UniVersiteit LeuVen, LeuVen, Belgium, Department of Nuclear
Medicine, U. Z. Gasthuisberg, LeuVen, Belgium, and Department of Radiology, UniVersity of PennsylVania, Philadelphia
ReceiVed October 23, 2008
Imaging agents targeting amyloid ꢀ (Aꢀ) may be useful for early diagnosis and follow-up of treatment of
patients with Alzheimer’s disease (AD). Three of five tested 2-(4′-fluorophenyl)-1,3-benzothiazoles displayed
high binding affinities for Aꢀ plaques in AD human brain homogenates (Ki between 2.2 and 22.5 nM).
They all contained the 18F-label directly attached to the aromatic ring and were synthesized starting from
the nitro precursor. Determination of the partition coefficient, biodistribution studies in normal mice, and in
vivo µPET studies in normal rats showed that their initial brain uptake was high and brain washout was
fast. The most promising compound [18F]5, or 6-methyl-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole, seemed
to be metabolically stable in the brain, and its plasma radiometabolites, which do not cross the blood-brain
barrier, were determined. The preliminary results strongly suggest that this new fluorinated compound is a
promising candidate as an Aꢀ plaque imaging agent for the study of patients with AD.
Introduction
chrysamine G, thioflavin S, or thioflavin T), amyloid ꢀ antibod-
ies, or a modified Bielschowsky technique (silver stain) can give
direct information but only post mortem.4 Although these highly
conjugated fluorescent molecules display desirable properties,
such as high binding affinity and moderate lipophilicity, their
charge and large structure prevent them from crossing the intact
blood-brain barrier (BBB). To allow noninvasive in vivo
imaging and diagnosis of AD in an early-to-moderate stage and
monitoring of the progression and effectiveness of treatment,
the search for a clinically useful radiolabeled derivative of one
of these dyes has become subject of worldwide research.
Alzheimer’s disease (ADa) is a progressive and fatal neuro-
degenerative brain disorder associated with progressive memory
loss and decrease of cognitive functions, which may be
accompanied by behavioral symptoms including agitation,
anxiety, depression, or psychosis. It is the most common form
of dementia affecting millions of elderly people. Neurohisto-
pathologically, AD is characterized by the presence of extra-
cellular depositions of amyloid ꢀ (Aꢀ), intracellular neurofibril-
lary tangles (NFTs), activated microglia, and reactive astrocytes.1
This triade of amyloid plaques, neurofibrillary tangles, and
dementia was first described by Alois Alzheimer in 1907.2
The presence of amyloid plaques seems to play a major role
in the AD pathogenesis.3 Early detection and quantification of
these hallmarks in brain with noninvasive techniques such as
positron emission tomography (PET) would allow presymp-
tomatic identification of patients and monitoring the effective-
ness of novel treatments.
In the past years, radiolabeled derivatives of a number of the
higher mentioned dyes have been tested and reported (Figure
1). X-34 derivatives consisting of only half the X-34 molecule
without carboxylic groups, the so-called stilbenes (SBs), showed
promising results.5 The recently reported carbon-11 labeled SB-
13 has already been tested in subjects with mild to moderate
AD and healthy controls, showing a high accumulation in the
frontal and temporoparietal cortex of patients with AD but not
in age-matched control subjects.6 Barrio and co-workers, on the
other hand, developed [18F]FDDNP, a naphthalene derivative
that binds to amyloid ꢀ at a different binding site and also to
the NFTs.7 This compound has been tested in subjects with AD,
mild cognitive impairment (MCI, which may progress to AD),
and no cognitive dysfunction. Small et al. reported a significantly
higher binding in subjects with AD than in those with MCI or
control subjects and higher binding in MCI patients than in
control subjects, indicating the potential usefulness of this
compound for early diagnosis of AD.8 The most promising of
the reported compounds are uncharged derivatives of thiofla-
vin-T such as 125I-TZDM9 and the carbon-11 labeled 2-phenyl-
benzothiazoles BTA and 6-OH-BTA, also known as Pittsburgh
compound-B (PIB).10 Of these two, PIB has the most favorable
pharmacokinetics and has already been tested intensively in
several clinical studies showing clear differences between AD,
MCI, and control subjects.11,12 Other reported compounds with
high affinity for amyloid ꢀ are iodine-123 and fluorine-18
labeled IBOX13 and IMPY,14 fluorine-18 labeled acridine orange
analogue BF-108,15 and the recently reported fluorine-18 labeled
Clinical diagnosis of AD is now done by neuropsychological
tests (Mini-Mental State Examination), but this only yields
indirect information. Immunohistochemical fluorescent staining
of brain tissue with highly conjugated dyes (Congo red,
* To whom correspondence should be addressed. Address: Herestraat
49 bus 821 BE, 3000 Leuven, Belgium. Phone: +32 16 330441. Fax: +32
16 330449. E-mail: kim.serdons@pharm.kuleuven.be.
†
Laboratory for Radiopharmacy.
Department of Nuclear Medicine.
Department of Radiology.
‡
§
a Abbreviations: AD, Alzheimer’s disease; Aꢀ, amyloid ꢀ; BBB,
blood-brain barrier; cpm, counts per minute; DMSO, dimethyl sulfoxide;
ID, injected dose; ID/g, injected dose per gram tissue; IMPY, 6-iodo-2-
(4′-dimethylamino)phenylimidazo[1,2]pyridine; LSO, lutetium oxyortho-
silicate; MCI, mild cognitive impairment; Mp, melting point; NFT,
neurofibrillary tangle; NMP, N-methylpyrrolidone; NMR, nuclear magnetic
resonance; P, partition coefficient; PBS, phosphate buffered saline; p.i., post
injection; PET, positron emission tomography; PPA, polyphosphoric acid;
RP-HPLC, reversed phase high performance liquid chromatography; tR,
retention time; rt, room temperature; SB, stilbene; SD, standard deviation;
SEM, standard error of the mean; SUV, standard uptake value; TAC,
time-activity curve; THF, tetrahydrofuran; TLC, thin layer chromatography;
VOI, volume of interest; DMF, dimethylformide.
10.1021/jm8013376 CCC: $40.75
2009 American Chemical Society
Published on Web 02/13/2009

18F-Labeled 2-Phenylbenzothiazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1429
Figure 1. Chemical structures of amyloid imaging agents previously reported.
Scheme 1. Synthesis of Phenylbenzothiazoles 3a-ha
a Method B: Lawesson’s reagent, 1,4-dioxane or chlorobenzene, reflux, 3 h. Method C: 10% NaOH, K3Fe(CN)6, 90 °C, 2 h. Method D: BBr3, N2, CH2Cl2,
-70 °C, 2 h. Method E: NaH, CH3OCH2Cl, DMF, rt, 5 h.
stilbenes,16 benzothiophenes,17 biphenyltrienes,18 styrylben-
zenes,19 styrylpyridines,20 and curcumin derivatives.21
Up to now, by far the most promising and clinically useful
results have been obtained with [11C]PIB. However, this tracer
agent remains suboptimal in view of its labeling with the short-
lived carbon-11 (T1/2 ) 20.39 min), which limits its availability
to centers equipped with a cyclotron. The limitations associated
with the short half-life of carbon-11 may be overcome by
introducing a fluorine-18 label which has a longer half-life (T1/2
) 109.8 min) and thus may provide a tracer agent that is useful
for a widespread clinical application. This paper describes the
synthesis, labeling, and biological evaluation of three 2-(4′-
[18F]fluorophenyl)-1,3-benzothiazoles with the 18F-label directly
attached to the 2-phenyl ring.
We adapted the described procedures to synthesize the 2-(4′-
fluorophenyl)-1,3-benzothiazoles 3a-d and the nitro precursors
3e-h. A schematic overview of the synthetic pathways is
depicted in Scheme 1. The benzamides 1a-d were prepared
by reaction of the corresponding substituted anilines with
4-fluorobenzoyl chloride or 4-nitrobenzoyl chloride in boiling
pyridine (method A). The benzamides were converted to the
corresponding thiobenzamides 2a-d using Lawesson’s reagent
in 1,4-dioxane (method B). Lawesson’s reagent or 2,4-bis(4-
methoxyphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide is
useful as a thiation reagent to replace the carbonyl oxygen of
ketones, amides, and esters with sulfur.25 For the synthesis of
2b chlorobenzene was used as solvent, as the conversion in 1,4-
dioxane failed. The thiobenzamides were cyclized to their
corresponding phenylbenzothiazoles by a Jacobsen synthesis
using the oxidizing agent potassium ferricyanide in aqueous
sodium hydroxide (method C). Demethylation of the methoxy-
Results and Discussion
Chemistry. The synthesis of a number of 2-(4′-aminophenyl)-
1,3-benzothiazoles has already been described previously.22-24

1430 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Serdons et al.
Scheme 2. Synthesis of 6-Methyl-2-(4′-fluorophenyl)-1,3-benzothiazole 5a
a (i) 50% KOH, reflux, 3 days; (ii) PPA, 220 °C, 4 h.
Scheme 3. Synthesis of
Scheme 4. Synthesis of
6-Methoxy-2-(4′-[18F]Fluorophenyl)-1,3-benzothiazole [18F]3a
and 6-Methyl-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole [18F]5a
6-Carboxy-2-(4′-fluorophenyl)-1,3-benzothiazole 8a
a (i) 18F-, K2CO3, Kryptofix, DMSO, 150 °C, 20 min.
way, as relatively high yields of the corresponding [18F]fluo-
rophenylbenzothiazoles were obtained (Scheme 4). The labeling
of 6-hydroxy-2-(4′-nitrophenyl)-1,3-benzothiazole (3f) failed
because the alkaline labeling conditions induce a partial negative
charge at position 6. As a result, the benzothiazole substituent
no longer acts as electron withdrawing substituent but induces
a partial negative charge at the N-atom, and therefore, the 4′-
nitro group can no longer be substituted by [18F]fluoride. To
overcome this problem, we introduced a protecting group on
the phenol at position 6. The use of an acetate protective group
on the phenol was not successful, as labeling of this precursor
failed because of instability of the ester in the alkaline labeling
conditions. Use of the more stable methoxymethyl protecting
group overcame this problem, and labeling of this precursor,
6-methoxymethoxy-2-(4′-nitrophenyl)-1,3-benzothiazole (3g),
resulted in the corresponding fluorine-18 radiolabeled derivative
that was hydrolyzed with a mixture of methanol/concentrated
HCl (2:1 v/v) to yield 6-hydroxy-2-(4′-[18F]fluorophenyl)-1,3-
benzothiazole ([18F]3b) with a yield of 38% (Scheme 5, Table
1).
Because of the low solubility of the nitro precursors, labeling
reactions were performed in dimethyl sulfoxide (DMSO). A
rather common challenge for fluorine-18 labeled tracers prepared
starting from a nitro precursor is the difficult separation between
the nitro precursor and the desired 18F-labeled compound using
high performance liquid chromatography (HPLC). After trying
various mobile phase compositions, we found that the use of
an XTerra C18 column eluted with an isocratic mixture of 50%
0.05 M NH4OAc and 50% ethanol/tetrahydrofuran (THF) (75:
25 v/v) allowed an efficient separation for all of the tested
compounds. Before purification on preparative HPLC, unreacted
[18F]F- was removed by passing the crude reaction mixture over
a Sep-Pak Plus C18 column. The THF present in the HPLC
eluent was removed by solid phase extraction using a Sep-Pak
Plus C18 cartridge that was first rinsed with water to remove
all THF and then eluted with ethanol to provide the pure
radiolabeled compound. In a different approach, we synthesized
the trimethylammonium precursor of [18F]5 instead of the nitro
precursor, but labeling failed because of decomposition of this
precursor in the alkaline reaction conditions.
a (i) CH3OH, Br2, -5 °C, 2 h; (ii) KOH, N2, reflux, 4 h; (iii) NMP, conc
HCl, 100 °C, 8 h.
phenylbenzothiazoles 3a, 3c, and 3e was accomplished with
excess boron tribromide in dichloromethane at -70 °C to yield
the required hydroxylated derivatives 3b, 3d, and 3f (method
D).
To protect precursor 3f with a methoxymethyl protecting
group, the compound was first deprotonated using NaH and was
then reacted with chloromethyl methyl ether and potassium
carbonate at room temperature (rt) (method E, 88% yield).26 In
the absence of sodium hydride only low yields (10%) were
obtained even after heating and extended reaction times.
6-Methyl-2-(4′-fluorophenyl)-1,3-benzothiazole (5) was syn-
thesized via a direct route22 by condensation of 2-amino-5-
methylthiophenol (4), which was synthesized by hydrolytic
cleavage of 2-amino-6-methylbenzothiazole with aqueous potas-
sium hydroxide at 100 °C,27 with commercially available
4-fluorobenzoic acid in polyphosphoric acid (PPA) (Scheme 2).
The role of PPA is dual: it serves not only as solvent for the
reagents but also as a dehydrating agent, thus shifting the
reaction toward the formation of 5.
6-Carboxy-2-(4′-fluorophenyl)-1,3-benzothiazole (8) was syn-
thesized by condensation of 4-amino-3-mercaptobenzoic acid
(7) with p-fluorothiobenzamide in N-methylpyrrolidone (NMP)
in the presence of 2 equiv of concentrated hydrochloric acid28
(Scheme 3). 4-Amino-3-mercaptobenzoic acid (7) was synthe-
sized following an improved method described by Lang et al.29
This method does not require stannous chloride to prevent
disulfide formation, as the hydrolysis of 2-aminobenzothiazole-
6-carboxylic acid (6) proceeds in the absence of light and air.
6 was synthesized by adding bromine to a stirred suspension
of sodium thiocyanate and 4-aminobenzoic acid.
Radiochemistry. To introduce a 18F-label on the 2-phenyl
ring of the phenylbenzothiazoles, an aromatic nucleophilic
substitution reaction of the nitro group with [18F]fluoride was
achieved by heating the solution of the precursor with K[18F]F-
Kryptofix complex in DMSO at 150 °C for 20 min. Aromatic
substitution of a nitro group with fluoride generally requires
other electron withdrawing groups on the ortho- or para-position.
The presence of an electron withdrawing benzothiazole sub-
stituent apparently activates the 4′-nitro group in a sufficient
The purified tracer products were analyzed by reversed phase
HPLC (RP-HPLC) on an analytical C18 column employing an
isocratic mixture of 50% 0.05 M NH4OAc and 50% ethanol/
tetrahydrofuran (75:25 v/v) as mobile phase at a flow rate of 1
mL/min. The radiochemical purity was found to be more than

18F-Labeled 2-Phenylbenzothiazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1431
Scheme 5. Synthesis of 6-Hydroxy-2-(4′-[18F]Fluorophenyl)-1,3-benzothiazole [18F]3ba
a (i) 18F-, K2CO3, Kryptofix, DMSO, 150 °C, 20 min; (ii) CH3OH/conc HCl 2:1 v/v, 125 °C, 5 min.
Table 1. Ki Values, log P Values, and Radiochemical Characteristics of
3a, 3b, 3d, 5, 8, and PIBa
Table 2. Tissue Distribution of
6-Methoxy-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole ([18F]3a) after iv
Injection in Normal Mice at 2 and 60 min p.i. (n ) 4 at Each Time
Point)
tR
nitro
tR 18F/
precursord F productd radiochemical
% ID ( SD
% ID/g tissue ( SD
compd Kib (nM)
log Pc
(min)
(min)
yielde (%)
organ
2 min p.i. 60 min p.i. 2 min p.i. 60 min p.i.
3a
3b
3d
5
2.2 ( 0.5 2.29 ( 0.24
22.5 ( 4.5 2.73 ( 0.23
776 ( 173 nd
24
24
nd
48
nd
nd
19
19
nd
41
nd
nd
24.1 ( 2.3
38.0 ( 0.2
nd
29.4 ( 3.2
nd
urine
kidneys
liver
0.2 ( 0.2 16.9 ( 8.6
8.6 ( 0.8
9.4 ( 6.5 13.9 ( 0.9 14.2 ( 9.6
19.9 ( 2.4 13.6 ( 3.6
9.9 ( 1.1
4.1 ( 0.3
9.9 ( 1.2
3.9 ( 1.4
6.7 ( 2.2
0.4 ( 0.1
2.1 ( 0.8
0.6 ( 0.2
5.7 ( 1.8 2.52 ( 0.27
spleen + pancreas 1.3 ( 0.2
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
0.2 ( 0.0
0.6 ( 0.3
0.1 ( 0.0
8
>4000
nd
2.3 ( 0.2
0.8 ( 0.2
PIB 2.8 ( 0.5 2.48 ( 0.06
nd
a nd ) not determined. b Ki expressed as mean ( SEM (standard error
of the mean) in an [125I]IMPY competition study (Kd [125I]IMPY ) 5.3 (
1.0 nM). c log P expressed as mean ( SD (standard deviation, n ) 6). d tR
(retention time) determined on HPLC: stationary phase, XTerra Prep RP18
(10 µm, 10 mm × 250 mm); mobile phase, 0.05 M ammonium acetate and
ethanol/THF (75:25) (50:50 v/v); flow rate, 3 mL/min; detection, UV 254
nm. e Decay corrected radiochemical yield expressed as mean ( SD
(standard deviation, n > 3).
8.4 ( 0.6 34.3 ( 6.1
1.1 ( 0.3 1.2 ( 0.8
1.57 ( 0.18 0.13 ( 0.04 5.10 ( 0.40 0.43 ( 0.12
0.56 ( 0.11 0.07 ( 0.04 5.12 ( 0.51 0.68 ( 0.29
4.9 ( 1.8
4.4 ( 1.8
2.0 ( 0.8
1.7 ( 0.6
Table 3. Tissue Distribution of
6-Hydroxy-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole ([18F]3b) after iv
Injection in Normal Mice at 2 and 60 min p.i. (n ) 3 at Each Time
Point)
95% for each of the compounds. The identity of the tracers was
confirmed by co-elution with the authentic nonradioactive
compounds after co-injection on the same analytical HPLC
system.
% ID ( SD
% ID/g tissue ( SD
organ
2 min p.i. 60 min p.i. 2 min p.i. 60 min p.i.
Affinity. The in vitro affinity of the nonradioactive reference
compounds for amyloid ꢀ fibrils was determined using a
[
urine
kidneys
liver
0.1 ( 0.1 11.3 ( 2.4
7.0 ( 1.0 5.9 ( 0.6
12.6 ( 1.4 11.8 ( 2.6
28.1 ( 1.6 24.6 ( 9.3 13.3 ( 0.7 11.9 ( 4.5
4.0 ( 0.1 1.8 ( 0.7
28.1 ( 3.6 5.5 ( 2.0
5.8 ( 1.1 1.3 ( 1.4
125I]IMPY binding competition experiment with human AD
spleen + pancreas 1.2 ( 0.1 0.5 ( 0.3
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
brain homogenates. The observed Ki values (Table 1) depend
on the nature and position of the substitution groups present on
the phenyl ring of the benzothiazole moiety. Compounds 3a,
3b, and 5 have a low Ki value and thus show good affinity for
Aꢀ plaques. Substitution of the methoxy group at position 6 in
3a with a hydroxyl group (3b) resulted in a 10-fold lower
affinity. Compounds 3d and 8 only have a low affinity for Aꢀ
plaques. Compounds 3a and 5 maintained the best affinity values
in the nanomolar range of Ki values. Their affinity is comparable
with that of 11C-labeled PIB, which has a Ki value of 2.8 ( 0.5
nM, determined using the same competition assay.
7.6 ( 2.1 1.5 ( 0.4
0.9 ( 0.2 0.1 ( 0.0
7.1 ( 0.7 36.7 ( 10.6
1.1 ( 0.2 0.5 ( 0.1
1.35 ( 0.25 0.16 ( 0.10 4.70 ( 0.48 0.57 ( 0.36
0.55 ( 0.07 0.08 ( 0.07 5.13 ( 0.54 0.76 ( 0.49
10.0 ( 3.7 3.3 ( 2.8
4.0 ( 1.6
1.3 ( 1.1
Table 4. Tissue Distribution of
6-Methyl-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole ([18F]5) after iv
Injection in Normal Mice at 2 and 60 min p.i. (n ) 6 at Each Time
Point)
% ID ( SD
% ID/g tissue ( SD
Partition Coefficient. As an estimate of the potential of the
compounds to cross the BBB by passive diffusion, the lipophi-
licity of the RP-HPLC purified radiolabeled products was
determined by partitioning between 1-octanol and 0.025 M
phosphate buffer, pH 7.4 (n ) 6). The log of the partition
coefficient (P) values of [18F]3a, [18F]3b, and [18F]5 were 2.29
( 0.24, 2.73 ( 0.23, and 2.52 ( 0.27, respectively (Table 1),
and are within the optimal range (log P between 1 and 3) for
passive diffusion of a compound through the BBB.30
Biodistribution in Normal Mice. A biodistribution study of
[18F]3a, [18F]3b, and [18F]5 was performed in normal male
NMRI mice at 2 and 60 min post injection (p.i.) to assess the
brain pharmacokinetics. Tables 2-4 list the tracer uptake in
the most important organs. All compounds showed a relatively
high initial brain uptake. The initial uptake in cerebrum of
[18F]3a (5.1 ( 0.4% ID/g) and [18F]5 (5.3 ( 0.7% ID/g) was
slightly higher than that of [18F]3b (4.7 ( 0.5% ID/g). The brain
uptake of [18F]5 at 2 min p.i. is significantly higher (p < 0.05,
two-sided) than that of PIB (Table 5, 3.6 ( 1.4% ID/g). With
respect to brain washout (expressed as % ID in cerebrum at 2
min/% ID in cerebrum at 60 min), [18F]5 shows a higher value
organ
2 min p.i. 60 min p.i. 2 min p.i. 60 min p.i.
urine
0.2 ( 0.1
5.8 ( 1.4
3.3 ( 1.8
2.3 ( 0.2
kidneys
liver
9.3 ( 2.0
3.6 ( 0.4
24.8 ( 3.3 51.8 ( 1.8 12.0 ( 0.7 24.6 ( 2.1
spleen + pancreas 1.3 ( 0.2
0.1 ( 0.0
0.3 ( 0.1 14.4 ( 2.0
0.1 ( 0.0 5.4 ( 0.5
4.0 ( 0.7
0.4 ( 0.1
1.3 ( 0.2
0.5 ( 0.1
lungs
heart
3.6 ( 1.0
0.9 ( 0.1
intestines
stomach
cerebrum
cerebellum
blood
8.9 ( 1.0 19.3 ( 1.3
1.3 ( 0.2 1.1 ( 0.6
1.62 ( 0.29 0.07 ( 0.02 5.33 ( 0.74 0.27 ( 0.06
0.42 ( 0.16 0.04 ( 0.02 5.99 ( 0.70 0.39 ( 0.06
5.7 ( 2.4
2.8 ( 2.2
2.2 ( 0.9
1.1 ( 0.9
(23.1) than [18F]3a (12.1) and [18F]3b (8.4). In healthy mice, a
compound with favorable characteristics for plaque imaging
should not only have a high affinity for amyloid but also show
a high initial brain uptake followed by a rapid washout,
indicating absence of nonspecific binding to any brain structure
devoid of amyloid plaques. Compound [18F]5 with the highest
initial brain uptake and the fastest brain washout value, which
is almost 4 times higher than that of [11C]PIB, seems to be the

1432 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Serdons et al.
Table 5. Tissue Distribution of 6-Hydroxy-2-(4′-N-[11C]methyl-
aminophenyl)-1,3-benzothiazole ([11C]PIB) after iv Injection in Normal
Mice at 2 and 60 min p.i. (n ) 6 at Each Time Point)
the Oasis column, the proteins and other hydrophilic matrix
components were washed from the column using water. The
analytes were then eluted from the Oasis column and led over
a reversed phase analytical column, the eluate of which was
collected in 1.5 mL fractions followed by counting of all
fractions for radioactivity.31 The recoveries of HPLC/Oasis
column injected radioactivity during these analyses were found
to be high with mean recovery of 82.0 ( 4.1% (n ) 6). An
example of plasma analysis is shown in Figure 3. Table 6 shows
the percentage of intact [18F]5 in plasma of normal mice as a
function of time. At 2 min p.i., already 40% of the tracer agent
was converted into at least three unidentified radiolabeled polar
metabolites. At 60 min p.i., only 1.6% of the intact compound
was found. This was not the case with [11C]PIB, which is much
more stable, with more than 80% intact tracer in plasma at 60
min p.i. (data not shown). Only relatively low amounts (<15%)
of radiometabolites were found in the brain at 2 or 60 min p.i.
(% intact tracer at 2 min p.i., 89.2 ( 0.4; % intact tracer at 60
min p.i., 86.2 ( 2.5; n ) 2), indicating that no substantial
metabolism of [18F]5 occurs in brain and that its radiometabolites
do not cross the BBB to a high extent in mice.
% ID ( SD
% ID/g tissue ( SD
organ
2 min p.i. 60 min p.i. 2 min p.i. 60 min p.i.
urine
0.2 ( 0.1 24.4 ( 10.1
kidneys
liver
10.3 ( 3.8
15.6 ( 4.5
4.8 ( 3.9 19.9 ( 9.1
9.1 ( 7.1
5.0 ( 1.7
0.7 ( 0.4
1.3 ( 0.7
2.1 ( 2.1
9.8 ( 2.6
0.1 ( 0.1
0.3 ( 0.2
0.3 ( 0.2
7.9 ( 2.6
4.7 ( 2.4
3.6 ( 0.6
4.2 ( 0.3
spleen + pancreas 0.6 ( 0.1
lungs
heart
0.9 ( 0.2
0.6 ( 0.1
intestines
stomach
cerebrum
cerebellum
blood
12.0 ( 2.4 42.5 ( 7.5
1.1 ( 0.2 0.3 ( 0.2
1.08 ( 0.44 0.18 ( 0.06 3.60 ( 1.40 0.60 ( 0.21
0.17 ( 0.02 0.03 ( 0.02 1.60 ( 0.09 0.31 ( 0.13
7.9 ( 0.5
2.7 ( 0.8
3.1 ( 0.2
1.1 ( 0.3
compound with the most promising characteristics to image AD
in vivo. The new 18F-labeled 2-phenylbenzothiazoles do not
show a significant difference between the uptake in the cerebrum
and cerebellum, whereas [11C]PIB does. As the cerebellum
contains more white matter, it is possible that the 18F-labeled
2-phenylbenzothiazoles bind more to the white matter of the
brain. The consequence of this with respect to imaging may
be a lower signal/noise ratio because there may be more white
matter retention. Blood levels of the three compounds were
relatively low at all time points measured. Clearance of [18F]5
from the blood (expressed as % ID in blood at 2 min/% ID in
blood at 60 min, 2.0) was between the clearance values of
[18F]3a (1.1) and [18F]3b (3.0) and in the same range as that of
PIB (2.9). The distribution of these tracer agents in other organs
is of a lesser concern. As could be expected from the relatively
high log P value, the compounds were cleared mainly by the
hepatobiliary system (ranging from 48% ID for the methoxy
derivative to 71% ID for the methyl derivative at 60 min p.i.)
and to a lesser extent to the urine (ranging from 5.6% ID for
the methyl derivative to 26% ID for the methoxy derivative at
60 min p.i.). Except for the liver, intestines, and kidneys, the
amount of radioactivity in other major organs was negligible
60 min after injection of the tracers. This indicates a good
clearance of the tracer agents from the major organs.
µPET Study in a Normal Rat. To compare the brain
pharmacokinetics of [18F]3a, [18F]3b, and [18F]5 with those of
[11C]PIB, the compounds were also injected in a normal rat and
µPET images were acquired for 120 min. Figure 2 shows the
time-activity curves (TACs) for [18F]3a, [18F]3b, and [18F]5
and [11C]PIB in the frontotemporal cortex of a normal rat. Values
are expressed as standard uptake values (SUVs).
The tested fluorine-18 labeled PIB derivatives all showed a
similar initial brain uptake followed by a relatively rapid
washout comparable with that of [11C]PIB. The lower variability
of the brain pharmacokinetics of the tested compounds observed
in rats compared to the values observed in mice may be
explained by interspecies differences. As no tracer uptake was
observed in the skull, we can conclude that the new 18F-labeled
2-phenylbenzothiazoles do not show substantial defluorination
in vivo.
Biostability in Brain of a Normal Rat. After intravenous
injection of [18F]5 in a normal rat, a polar radiolabeled
metabolite that reached 22% of the total radioactivity was
observed in brain at 2 min p.i. (78.4% intact tracer, n ) 1).
These findings are in agreement with the reported formation of
a radiolabeled metabolite of [11C]PIB, namely, 6-sulfato-PIB,
in rat brain, but also for this compound no radiometabolites
were formed in mouse or human brain homogenates.32 This
similar biological behavior of [11C]PIB and [18F]5 suggests that
replacement of the N-methyl substituent on the 2-phenyl ring
of PIB by a fluorine atom does not affect significantly the
pharmacokinetics. The only difference is the slightly higher
relative uptake in the cerebellum.
Conclusion
To allow noninvasive in vivo imaging of amyloid plaques
for early stage diagnosis of Alzheimer’s disease and monitoring
of the disease progression and treatment efficacy with PET, the
search for a clinically useful radiolabeled derivative of thiofla-
vin-T has become a subject of worldwide research. Up to now,
by far the most promising and clinically useful results have been
obtained with [11C]PIB. The limitations associated with the short
half-life of carbon-11 may be overcome by introducing a
fluorine-18 label and thus may provide a tracer agent that is
useful for a widespread clinical application.
We synthesized five 2-(4′-fluorophenyl)-1,3-benzothiazoles
of which three show high in vitro affinity for amyloid paques
present in human AD post mortem brain homogenates. The
corresponding nitro precursors were synthesized, and successful
18F-labeling was performed by heating for 20 min at 150 °C in
DMSO. The studied 18F-labeled phenylbenzothiazoles show a
log P value between 2.2 and 2.8. Their ability to pass the
blood-brain barrier was confirmed by a biodistribution study
in normal mice. All compounds show a high initial uptake in
cerebrum of normal mice. Compound [18F]5 has the highest
brain uptake, significantly higher than that of the 11C-labeled
PIB. The compound also showed a faster brain washout than
[11C]PIB. In vivo biostability of [18F]5 was studied in plasma
and brain of normal mice. The compound was extensively
metabolized, as large amounts of three unidentified radiolabeled
polar metabolites were found in plasma but not in brain. These
preliminary results strongly suggest that this new fluorinated
Biostability of [18F]5 in Normal Mice. Because [18F]5
showed the most promising results (highest brain uptake and
fastest washout in normal mice), the in vivo metabolic stability
of [18F]5 was studied by analyzing plasma and brain of normal
mice after injection of [18F]5 using RP-HPLC analysis. The
nonradioactive reference compound 5 was co-injected on HPLC
to assess the retention time of intact parent tracer.
For the analysis, online solid phase extraction using an Oasis
HLB column was employed. After injection of the samples on

18F-Labeled 2-Phenylbenzothiazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1433
Figure 2. Standard uptake values (SUVs) of [18F]3a, [18F]3b, and [18F]5 and [11C]PIB in the frontotemporal cortex of a normal rat.
Figure 3. Plasma radiometabolite analysis of [18F]5: RP-HPLC radiochromatogram of a plasma sample from a normal mouse collected 2 min p.i.
The 1 min fractions were collected and counted.
Table 6. Percentage of Intact [18F]5 in Plasma of Normal Mice at 2, 10,
30, and 60 min p.i. (n ) 3 at Each Time Point)
were obtained commercially from Acros Organics, Fischer Bioblock
Scientific (Tournai, Belgium), Fluka, Merck (Darmstadt, Germany),
or Riedel-de Hae¨n (Seelze, Germany). They were all used without
further purification.
time (min)
% intact
SD
2
52.15
13.95
7.88
13.15
6.23
2.80
1.66
Apparatus, Instruments, and General Conditions. MgSO4 was
used as drying agent. pH values of nonradioactive solutions were
measured with a P600 pH meter (Consort, Turnhout, Belgium)
provided with a glass electrode. pH determination of radioactive
solutions was done with pH strips (pH strips 0-14, Merck,
Darmstadt, Germany). Evaporation of organic solvents under
reduced pressure was done with a Bu¨chi rotovapor (Bu¨chi, Flawil,
Switzerland). Purification of reaction mixtures was done by column
chromatography using silica gel with a particle size varying between
0.04 and 0.063 mm (230-400 mesh, MN Kieselgel 60M, Mach-
erey-Nagel, Du¨ren, Germany) as the stationary phase. Thin layer
chromatography (TLC) was done on precoated silica TLC plates
(DC-Alufolien-Kieselgel, Fluka, Buchs, Switzerland). The structures
of the synthesized products were confirmed with 1H nuclear
magnetic resonance (NMR) spectroscopy on a Gemini 200 MHz
spectrometer (Varian, Palo Alto, CA) or a Bruker AVANCE 300
10
30
60
3.19
compound is a promising candidate as a Aꢀ plaque imaging
agent for studying patients with AD.
Experimental Section
Chemicals and Reagents. p-Anisidine, 4-fluorobenzoyl chloride,
4-nitrobenzoyl chloride, and p-toluidine were obtained from Acros
Organics (Geel, Belgium). 2-Amino-6-methylbenzothiazole and
ethyl 4-aminobenzoate were obtained from Aldrich (Sigma-Aldrich,
Bornem, Belgium). 4-Fluorobenzoic acid was purchased from
Avocado (Karlsruhe, Germany). 3,5-Dimethoxyaniline was obtained
from Fluka (Bornem, Belgium). All other reagents and solvents

1434 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Serdons et al.
MHz spectrometer (Bruker AG, Faellanden, Switzerland). Chemical
shifts are reported as δ-values (parts per million) relative to
tetramethylsilane (δ ) 0). Coupling constants are reported in hertz.
Splitting patterns are defined by s (singlet), d (doublet), dd (double
doublet), t (triplet), and m (multiplet). Melting points (Mp’s) were
determined with an IA9100 digital melting point apparatus (Elec-
trothermal, Essex, U.K.) in open capillaries and are reported
uncorrected. Exact mass measurement was performed on a time-
of-flight mass spectrometer (LCT, Micromass, Manchester, U.K.)
equipped with an orthogonal electrospray ionization interface and
operating in positive mode (ES+). Accurate mass determination was
done by co-injection with a compound with known mass as an
internal calibration standard (lock mass). Acquisition and processing
of data were done using Masslynx software (version 3.5, Waters).
Reversed phase high performance liquid chromatography (RP-
HPLC) purification and analysis were performed either on a Merck
Hitachi L6200 pump (Hitachi, Tokyo, Japan) or on a Waters 600
pump (Waters, Milford) connected to a UV spectrometer (Waters
2487 dual λ absorbance detector). The output signal was recorded
and analyzed using a RaChel data acquisition system (Lablogic,
Sheffield, U.K.). For analysis of radiolabeled compounds, the HPLC
eluate was led over a 3 in. NaI(Tl) scintillation detector connected
to a single channel analyzer (Medi-Laboratory Select, Mechelen,
Belgium) after passing through an UV detector.
6-Methoxy-2-(4′-fluorophenyl)-1,3-benzothiazole (3a). Me-
thod C. A solution of 2a (13.06 g, 0.050 mol) in a mixture of
ethanol (20 mL) and 10% (mol/v) sodium hydroxide (100 mL) was
slowly added over a period of 2 h to a solution of potassium
ferricyanide (65.85 g, 0.200 mol) in water (125 mL) at 90 °C. The
obtained suspension was stirred for 2 h at 90 °C and then cooled
to 4 °C. The precipitate was filtered off, washed with water, and
dried in a vacuum oven. The dry residue was dispersed in a mixture
of dichloromethane/ethanol (75:25 v/v, 1 L). The dispersion was
filtered off, and the filtrate was concentrated by vacuum evaporation.
The residue was purified with silica column chromatography using
gradient mixtures of hexane and ethyl acetate (up to 50%) as eluent
to yield 1.29 g of 3a as a yellow solid (0.005 mol, 10%). 1H NMR
3
(CDCl3, 200 MHz): δ 3.89 (s, 3H, CH3O), δ 7.09 (dd, 1H, J )
8.7 Hz, 4J ) 2.4 Hz, 5-H), δ 7.16 (t, 2H, 3J ) 8.8 Hz, 3′-H 5′-H),
δ 7.34 (d, 1H, 4J ) 2.6 Hz, 7-H), δ 7.93 (d, 1H, 3J ) 8.8 Hz, 4-H),
3
4
δ 8.02 (dd, 2H, J ) 7.8 Hz, J ) 3.6 Hz, 2′-H 6′-H). Accurate
MS ES+ m/z [M + H]+ 260.0536 (calculated for C14H10NOFS
260.0540). Mp: 180.5-181.2 °C.
6-Hydroxy-2-(4′-fluorophenyl)-1,3-benzothiazole (3b). Me-
thod D. A dispersion of 3a (1.56 g, 0.0060 mol) in dichloromethane
(60 mL) was cooled to -70 °C under nitrogen. Over a period of
1 h, 0.027 mol of BBr3 (27 mL of a 1 M solution in dichlo-
romethane) was added slowly, after which the mixture was stirred
for another hour at -70 °C. The suspension was allowed to warm
to room temperature and was stirred for 45 h. The suspension was
cooled to -70 °C, and methanol (60 mL) was added slowly until
no more gas evolved. After warming to room temperature, the
suspension was extracted three times with 2 M sodium hydroxide
(100 mL). The aqueous layer was separated and neutralized with 6
M hydrochloric acid and again extracted three times with a mixture
of dichloromethane/methanol (4:1 v/v, 200 mL). The combined
organic layers were dried over MgSO4, filtered, and evaporated to
dryness. Crystallization from methanol yielded 1.22 g of 3b (0.0050
Gas chromatography was performed on a DI 200 gas chromato-
graph (Delsi Instruments, Suresnes, France) with a Porapak QS 80/
100 column (Alltech, Deerfield, IL) of 180 cm × 0.25 in.
Animal studies were performed according to the Belgian code
of practice for the care and use of animals, after approval from the
university ethics committee for animals.
[11C]PIB was synthesized using [11C]methyl triflate following a
described procedure.33
Quantitative determination of radioactivity in samples was done
using an automatic γ-counter coupled to a multichannel analyzer
(Wallac 1480 Wizard 3”, Wallac, Turku, Finland). The results were
corrected for background radiation and physical decay during
counting.
Dynamic µPET imaging was performed using a lutetium oxy-
orthosilicate (LSO) detector-based tomograph (microPET Focus
220; Siemens Medical Solutions, Knoxville, TN), which has a
transaxial resolution of 1.35 mm in full width at half-maximum.
Data were acquired in a 128 × 128 × 95 matrix with a pixel width
of 0.475 mm and a slice thickness of 0.796 mm. The coincidence
window width was set at 6 ns.
1
mol, 83%) as white crystals. H NMR (DMSO-d6, 200 MHz): δ
3
4
3
7.00 (dd, 1H, J ) 8.8 Hz, J ) 2.2 Hz, 5-H), δ 7.37 (t, 2H, J )
4
8.8 Hz, 3′-H 5′-H), δ 7.41 (d, 1H, J ) 2.2 Hz, 7-H), δ 7.85 (d,
1H, 3J ) 8.8 Hz, 4-H), δ 8.05 (dd, 2H, 3J ) 7.7 Hz, 4J ) 3.2 Hz,
2′-H 6′-H), δ 9.91 (s, 1H, OH). Accurate MS ES+ m/z [M + H]+
246.0361 (calculated for C13H8NOFS 246.0383). Mp: 224.0-224.8
°C.
6-Methoxymethoxy-2-(4′-nitrophenyl)-1,3-benzothiazole (3g).
Method E. To a solution of 3f (0.44 g, 0.0016 mol) in dry
dimethylformide (DMF) (15 mL) was added slowly NaH (0.043
g, 0.0018 mol), and the resulting mixture was stirred at room
temperature for 30 min. After cooling the reaction mixture to 0
°C, chloromethyl methyl ether (0.5 mL, 0.0066 mol) was added
and the pH of the reaction mixture was adjusted to 9 using
potassium carbonate (2 g, 0.014 mol). After being stirred at room
temperature for 5 h, the reaction mixture was poured into water
and extracted with ethyl acetate. The organic layer was dried over
MgSO4, filtered, and evaporated to dryness. The residue was purified
with silica column chromatography using a mixture of ethylacetate
and hexane (1:3 v/v) as eluent to yield 0.46 g of 3g as a yellow
Synthesis. N-4′-Methoxyphenyl-4-fluorobenzamide (1a).
Method A. A solution of p-anisidine (16.50 g, 0.134 mol) and
4-fluorobenzoyl chloride (21.25 g, 0.134 mol) in 70 mL of pyridine
was refluxed for 1 h. After cooling to room temperature (rt), the
mixture was poured into water and the precipitate was filtered off,
washed with water, and dried in a vacuum oven. Crystallization
from methanol yielded 26.73 g of 1a as white crystals (0.109 mol,
1
81%). H NMR (DMSO-d6, 200 MHz): δ 3.73 (s, 3H, CH3O), δ
6.92 (dd, 2H, 3J ) 7.0 Hz, 4J ) 2.2 Hz, 3′-H 5′-H), δ 7.33 (t, 2H,
3J ) 9.0 Hz, 3-H 5-H), δ 7.64 (dd, 2H, 3J ) 9.2 Hz, 4J ) 2.2 Hz,
2′-H 6′-H), δ 8.01 (dd, 2H, 3J ) 8.8 Hz, 4J ) 3.2 Hz, 2-H 6-H), δ
10.16 (s, 1H, NH-CO). Accurate MS ES+ m/z [M + H]+ 246.0912
(calculated for C14H12NO2F 246.0925). Mp: 185.6-186.2 °C.
1
solid (0.0014 mol, 88%). H NMR (CDCl3, 200 MHz): δ 3.46 (s,
3H, CH3O), δ 5.19 (s, 2H, CH2O), δ 7.16 (dd, 1H, 3J ) 8.8 Hz, 4J
4
) 2.2 Hz, 5-H), δ 7.53 (d, 1H, J ) 2.2 Hz, 7-H), δ 7.93 (d, 1H,
3J ) 8.8 Hz, 4-H), δ 8.12 (dd, 2H, 3J ) 6.8 Hz, 4J ) 2.0 Hz, 2′-H
6′-H), δ 8.25 (d, 2H, 3J ) 6.8 Hz, 4J ) 2.4 Hz, 3′-H 5′-H). Accurate
MS ES+ m/z [M + H]+ 317.0576 (calculated for C15H12N2O4S
317.0591). Mp: 133.3-133.5 °C.
N-4′-Methoxyphenyl-4-fluorothiobenzamide (2a). Method
B. A solution of 1a (21.83 g, 0.089 mol) and Lawesson’s reagent
(16.18 g, 0.040 mol) in 1,4-dioxane (130 mL) was heated under
reflux for 3 h, after which it was cooled to room temperature and
poured into water. The precipitate was filtered off, washed with
water, and dried in a vacuum oven. Crystallization from methanol
yielded 17.84 g of 2a as yellow needles (0.068 mol, 76%). 1H NMR
2-Amino-5-methylthiophenol (4). A mixture of 2-amino-6-
methylbenzothiazole (13.14 g, 0.080 mol) and 50% (mol/v) KOH
(800 mL) was refluxed during 3 days. The reaction mixture was
poured into water, and acetic acid was added to attain pH 6.5. The
yellow precipitate was extracted with ethyl acetate, dried over
MgSO4, filtered, and evaporated to dryness. The resulting residue
was purified with silica column chromatography using dichlo-
romethane as eluent to yield 5.98 g of 4 as a yellow solid (0.043
3
(DMSO-d6, 200 MHz): δ 3.77 (s, 3H, CH3O), δ 6.97 (dd, 2H, J
) 7.0 Hz, 4J ) 2.2 Hz, 3′-H 5′-H), δ 7.28 (t, 2H, 3J ) 8.3 Hz, 3-H
5-H), δ 7.67 (dd, 2H, 3J ) 6.8 Hz, 4J ) 2.4 Hz, 2′-H 6′-H), δ 7.90
(dd, 2H, 3J ) 7.7 Hz, 4J ) 3.2 Hz, 2-H 6-H), δ 10.16 (s, 1H, NH-
CS). Accurate MS ES+ m/z [M + H]+ 262.0681 (calculated for
C14H12NOFS 262.0696). Mp: 159.2-159.6 °C.
1
mol, 54%). H NMR (CDCl3, 200 MHz): δ 2.13 (s, 3H, CH3), δ
4.17 (s, 2H, NH2), δ 6.63 (d, 1H, 3J ) 8.0 Hz, 3-H), δ 6.95 (s, 1H,

18F-Labeled 2-Phenylbenzothiazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1435
4-H), δ 6.99 (d, 1H, 4J ) 2.2 Hz, 6-H). Accurate MS ES+ m/z [M
+ H]+ 140.0528 (calculated for C7H9NS 140.0522). Mp: 85.8-87.3
°C.
Sheva, Israel) in a niobium target using 18 MeV protons from a
Cyclone 18/9 cyclotron (Ion Beam Applications, Louvain-la-Neuve,
Belgium). The aqueous solution of [18F]F- was transferred to a
synthesis module where [18F]F- was separated from H218O using a
Sep-Pak Light Waters Accell Plus CM cartridge (Waters). [18F]F-
was then eluted from the cartridge into a reaction vial with a solution
containing 2.5 mg of potassium carbonate and 27.9 mg of Kryptofix
2.2.2 dissolved in 0.75 mL of water/acetonitrile (5:95 v/v). After
evaporation of the solvent from the reaction vial under a stream of
helium at 115 °C for 7 min, [18F]F- was further dried by azeotropic
distillation of traces of water using 1 mL of anhydrous acetonitrile
(115 °C, 5 min). A solution of 1.5 mg of precursor in 0.5 mL of
anhydrous DMSO was added to the radioactive residue of K[18F]F-
Kryptofix, and the mixture was heated at 150 °C for 20 min in a
closed vial to provide the crude radiolabeled compound. For the
synthesis of 6-hydroxy-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole
([18F]3b), 0.5 mL of a mixture of methanol/concentrated HCl (2:1
v/v) was added after the radiolabeling reaction, and the mixture
was heated at 125 °C for 5 min. To neutralize the reaction mixture,
1 mL of 2 M sodium acetate was added. The crude mixtures were
allowed to cool to room temperature and were diluted with 0.5 mL
of Tween 1% in water and applied onto a Sep-Pak Plus C18
cartridge (Waters) that was first rinsed two times with 2 mL of
water/ethanol (75:25 v/v) and then eluted with 600 µL of ethanol.
After dilution with an equal volume of water, the resulting water/
ethanol mixture was applied onto an XTerra Prep RP18 10 mm ×
250 mm column (Waters) that was eluted with an isocratic mixture
of 50% 0.05 M NH4OAc and 50% ethanol/tetrahydrofuran (75:25
v/v) at a flow rate of 3 mL/min. Radiolabeled 6-methoxy-2-(4′-
[18F]fluorophenyl)-1,3-benzothiazole ([18F]3a) and 6-hydroxy-2-(4′-
[18F]fluorophenyl)-1,3-benzothiazole ([18F]3b) were collected at 19
min, whereas their precursors 3e and 3f eluted at 24 min.
Radiolabeled 6-methyl-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole
([18F]5) was collected at 41 min, whereas the precursor 3h eluted
at 48 min. The fractions containing the isolated radioactive
compound were diluted with an equal volume of water and were
then applied on an activated Sep-Pak Plus C18 cartridge (Waters)
that was first rinsed with water and then eluted with 1 mL of
ethanol. The purity of the labeled tracers was analyzed using an
XTerra RP18 5 µm, 4.6 mm × 250 mm column (Waters) eluted
with an isocratic mixture of 50% 0.05 M NH4OAc and 50% ethanol/
tetrahydrofuran (75:25 v/v) at a flow rate of 1 mL/min (tR [18F]3a
) 20.65 min, tR [18F]3b ) 12.81 min, and tR [18F]5 ) 31.37 min).
Decay corrected radiochemical yields, corrected for the fraction of
[18F]fluoride retained on the Sep-Pak Light Accell plus QMA anion
exchange cartridge, were 24.1 ( 2.3% for [18F]3a, 38.0 ( 0.2%
for [18F]3b, and 29.4 ( 3.2% for [18F]5. The overall synthesis time
to obtain the pure product was between 60 and 80 min. The average
specific activity was found to be 116 GBq/µmol at end of synthesis.
Binding Studies. Binding studies were carried out according to
6-Methyl-2-(4′-fluorophenyl)-1,3-benzothiazole (5). A mixture
of 4 (2.78 g, 0.02 mol) and 4-fluorobenzoic acid (2.80 g, 0.02 mol)
in polyphosphoric acid (PPA, 75 g) was stirred at 220 °C for 4 h.
After cooling to room temperature, the reaction mixture was poured
into a 10% (mol/v) Na2CO3 solution. The precipitate was filtered
off, washed with water, and dried in a vacuum oven. The residue
was purified with silica column chromatography using gradient
mixtures of hexane and ethyl acetate (up to 10%) as eluent to yield
0.64 g of 5 as a yellow solid (0.0026 mol, 13%). 1H NMR (CDCl3,
3
300 MHz): δ 2.47 (s, 3H, CH3), δ 7.14 (t, 2H, J ) 8.6 Hz, 3′-H
3
4
5′-H), δ 7.28 (dd, 1H, J ) 8.4 Hz, J ) 1.4 Hz, 5-H), δ 7.65 (s,
3
3
1H, 7-H), δ 7.91 (d, 1H, J ) 8.6 Hz, 4-H), δ 8.03 (dd, 2H, J )
7.8 Hz, 4J ) 3.6 Hz, 2′-H 6′-H). Accurate MS ES+ m/z [M + H]+
244.0498 (calculated for C14H10FNS 244.0591). Mp: 150.0-150.9
°C.
2-Aminobenzothiazole-6-carboxylic Acid Hydrochloride
(6). To a stirred suspension of sodium thiocyanate (13.00 g, 0.160
mol) and 4-aminobenzoic acid (20.00 g, 0.146 mol) in methanol
(75 mL) was slowly added bromine (7.5 mL, 0.146 mol) while the
temperature was maintained below -5 °C. After addition, the
mixture was stirred for 2 h at the same temperature. The precipitated
solid was collected and washed with water. The isolated solid was
suspended in hydrochloric acid (1 M, 70 mL), and the suspension
was refluxed for 30 min and then filtered while hot. Concentrated
hydrochloric acid (30 mL) was added to the filtrate, producing a
white solid after cooling in a refrigerator. The white solid was
collected and dried under vacuum to yield 21.59 g of 6 (0.094 mol,
64%). The crude product was used without further purification. 1H
NMR (DMSO-d6, 200 MHz): δ 7.60 (d, 1H, 3J ) 8.8 Hz, 4-H), δ
7.80 (dd, 1H, 3J ) 8.5 Hz, 4J ) 1.9 Hz, 5-H), δ 8.51 (d, 1H, 4J )
1.6 Hz, 7-H). Accurate MS ES+ m/z [M + H]+ 195.0217 (calculated
for C8H6N2O2S 195.0223). Mp: 294.2-295.7 °C.
4-Amino-3-mercaptobenzoic Acid Hydrochloride (7). A sus-
pension of 6 (6.00 g, 0.026 mol) and potassium hydroxide (26.32
g, 0.496 mol) in degassed demineralized water (50 mL) was
shielded from light (aluminum foil) and then refluxed under nitrogen
for 4 h. After the mixture was cooled to room temperature,
concentrated hydrochloric acid (45 mL) was added dropwise under
nitrogen. The resulting mixture was cooled to 5 °C and stirred for
30 min. The precipitate was collected under a nitrogen blanket and
dried under vacuum to give 4.14 g of the desired product 7 as a
white solid (0.024 mol, 92%). The product was stored under light-
1
and oxygen-free conditions (-5 °C). H NMR (DMSO-d6, 200
3
4
MHz): δ 6.76 (d, 1H, J ) 8.4 Hz, 5-H), δ 7.46 (d, 1H, J ) 2.2
Hz, 2-H), δ 7.62 (dd, 1H, 3J ) 8.6 Hz, 4J ) 2.0 Hz, 6-H). Accurate
MS ES+ m/z [M + H]+ 170.0284 (calculated for C7H7NO2S
170.0270). Mp: 288.5-290.0 °C.
a method described previously.34
[
125I]IMPY (6-iodo-2-(4′-
6-Carboxy-2-(4′-fluorophenyl)-1,3-benzothiazole (8). To a
solution of 7 (2.20 g, 0.013 mol) and p-fluorothiobenzamide (1.55
g, 0.010 mol) in dry N-methylpyrrolidone (NMP, 20 mL) was added
concentrated hydrochloric acid (2 mL), and the reaction mixture
was protected from light and heated (100 °C) for 8 h. After cooling
to room temperature, the mixture was poured over crushed ice. The
precipitate was filtered off, washed with water, and dried in a
vacuum oven. The residue was purified with silica column
chromatography using dichloromethane as eluent to yield 0.36 g
dimethylamino)phenylimidazo[1,2]pyridine) with a specific activity
of 81.4 TBq/mmol and greater than 95% radiochemical purity was
prepared using a standard iododestannylation reaction and purified
using a simplified C-4 minicolumn as described previously.14
Binding assays were carried out in 12 mm × 75 mm borosilicate
glass tubes. The reaction mixture contained 50 µL of post mortem
AD brain homogenate (20-50 µg), 50 µL of [125I]IMPY solution
(0.04-0.06 nM diluted in phosphate buffered saline (PBS)), and
50 µL of solutions of the test compound (10-5-10-10 M diluted
serially in PBS containing 0.1% bovine serum albumin) in a final
volume of 1 mL. Nonspecific binding was defined in the presence
of 600 nM nonradioactive IMPY in the same assay tubes. The
mixtures were incubated at 37 °C for 2 h, and the bound and free
radioactivity was separated by vacuum filtration through Whatman
GF/B filters using a Brandel M-24R cell harvester (Brandel,
Gaithersburg, MD) followed by two 3-mL washes with PBS at room
temperature. Filters containing the bound iodine-125 labeled ligand
were counted in a γ counter with 70% counting efficacy. Under
the assay conditions, the specifically bound fraction was less than
15% of total radioactivity. The results of inhibition experiments
1
of 8 as a yellow solid (0.0013 mol, 13%). H NMR (DMSO-d6,
3
200 MHz): δ 7.44 (t, 2H, J ) 8.8 Hz, 3′-H 5′-H), δ 8.11 (s, 1H,
4-H), δ 8.20 (dd, 2H, 3J ) 8.5 Hz, 4J ) 2.6 Hz, 2′-H 6′-H), δ 8.32
(d, 1H, 4J ) 1.0 Hz, 5-H), δ 8.78 (s, 1H, 7-H). Accurate MS ES+
m/z [M + H]+ 274.0339 (calculated for C14H8FNO2S 274.0333).
Mp: 133.7-134.2 °C.
Production of [18F]Fluoride and Radiosynthesis of 6-Meth-
oxy-2-(4′-[18F]fluorophenyl)-1,3-benzothiazole [18F]3a, 6-Hydroxy-
2-(4′-[18F]fluorophenyl)-1,3-benzothiazole [18F]3b, and 6-Methyl-
2-(4′-[18F]fluorophenyl)-1,3-benzothiazole [18F]5. [18F]Fluoride
was produced via a [18O(p,n)18F] reaction by irradiation of 0.5 mL
of 97% enriched H218O (Rotem HYOX,18 Rotem Industries, Beer

1436 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Serdons et al.
were subjected to nonlinear regression analysis using EBDA,35 from
which Ki values were calculated.
an in-line UV detector (234 nm), the HPLC eluate was collected
in 1.5 mL fractions and their radioactivity and the activity in fraction
1 were measured using a γ counter.
Partition Coefficient Determination. The lipophilicity of the
RP-HPLC isolated 18F complexes was determined using a modifica-
Brain Radiometabolite Analysis after Injection of [18F]5 in
Normal Mice. After intravenous injection of 5.55 MBq of [18F]5
via a tail vein, normal male NMRI mice (n ) 3) were sacrificed
by decapitation at 2 or 60 min p.i. The brain was removed, and
100 µL of solution containing the authentic nonradioactive 5 (1
mg/mL acetonitrile), 2 mL of acetonitrile, and 2 mL of water were
added, and the mixture was homogenized. After centrifugation at
3000 rpm (1837g) for 5 min at 4 °C (Eppendorf centrifuge 5810),
1 mL of supernatant was filtered (0.22 µm pore size/10 mm
diameter), and the filtrate was analyzed on an XTerra RP C18
column (5 µm, 4.6 mm × 250 mm) eluted with a mixture of 0.05
M sodium acetate (pH 5.5)/acetonitrile (40:60 v/v) at a flow rate
of 1.5 mL/min. After passing through an in-line UV detector (234
nm), the HPLC eluate was collected in 1.5 mL fractions and their
radioactivity was measured using a γ counter.
tion of the method described by Yamauchi and co-workers.36
A
25 µL aliquot of the RP-HPLC isolated solution of the 18F-labeled
compound was added to a tube containing 2 mL of 1-octanol and
2 mL of 0.025 M phosphate buffer, pH 7.4 (n ) 6). The test tube
was vortexed at room temperature for 3 min followed by centrifu-
gation at 3000 rpm (1837g) for 10 min (Eppendorf centrifuge 5810,
Eppendorf, Westbury). Aliquots of 61 and 500 µL were drawn from
the 1-octanol and buffer phases, respectively, taking care to avoid
cross-contamination between the two phases and weighed. The
radioactivity in the aliquots was counted using an automatic γ
counter. After correction for the density and the mass difference
between the two phases, the partition coefficient (P) was calculated
using the following equation:
(cpm) ⁄ (mL of octanol)
P )
Brain Radiometabolite Analysis after Injection of [18F]5 in
Normal Rat. After intravenous injection of 7.4 MBq [18F]5 via a
tail vein, a normal male Wistar rat was sacrificed with an overdose
Nembutal (pentobarbital) at 2 min p.i. After the heart and blood
vessels were rinsed with saline, the brain was removed. The 1 mL
of a solution containing the authentic nonradioactive compound 5
(1 mg/mL acetonitrile), 2 mL of acetonitrile, and 2 mL of water
were added and the mixture was homogenized. After centrifugation
at 3000 rpm (1837g) for 5 min at 4 °C (Eppendorf centrifuge 5810),
1 mL of supernatant was filtered (0.22 µm/10 mm) and the filtrate
was analyzed on an XTerra RP C18 column (5 µm, 4.6 mm × 250
mm) eluted with a mixture of 0.05 M sodium acetate (pH 5.5)/
acetonitrile (40:60 v/v) at a flow rate of 1.5 mL/min. After passing
through an in-line UV detector (234 nm), the HPLC eluate was
collected in 1.5 mL fractions and their radioactivity was measured
using a γ counter.
(cpm) ⁄ (mL of buffer)
with cpm ) counts per minute.
Biodistribution in Normal Mice. A solution of [18F]3a, [18F]3b,
and [18F]5 obtained after RP-HPLC purification was diluted using
0.9% NaCl in water for injection to a concentration of 3.7 MBq/
mL. The concentration of ethanol did not exceed 10%, and the
concentration of THF did not exceed 0.02%, as determined by gas
chromatography. The biodistribution was studied in male NMRI
mice (body mass of 30-40 g). An aliquot of 0.1 mL of the diluted
tracer solution was injected into the mice via a tail vein, after
intraperitoneal injection of 0.1 mL Hypnorm (fentanyl citrate 63
µg/mL and fluanisone 2 mg/mL). The mice were sacrificed by
decapitation at 2 or 60 min p.i. Blood was collected in a tared tube
and weighed. All organs and other body parts were dissected and
weighed, and their radioactivity was counted in a γ counter. Results
were corrected for background radioactivity and are expressed as
percentage of the injected dose (% ID) or as percentage of the
injected dose per gram of tissue (% ID/g). For calculation of total
radioactivity in blood, blood mass was assumed to be 7% of the
total body mass.
Supporting Information Available: Additional 1H NMR, MS,
and HPLC analysis data for the synthesized final products. This
material is available free of charge via the Internet at http://
pubs.acs.org.
µPET Study in a Normal Rat. A rat (300 g, male Wistar rat)
was anesthesized with isoflurane (1.5-2.5%) in oxygen at a flow
rate of 1-2 L/min and was injected with 1.41 MBq [18F]3a, 9.69
MBq [18F]3b, or 6.18 MBq [18F]5 via a tail vein with a time interval
of 5 days between each tracer injection. The rat was breathing
spontaneously throughout the entire experiment. Dynamic µPET
images were acquired for 120 min (4 × 15 s, 4 × 60 s, 5 × 180 s,
8 × 300 s, 6 × 600 s), and reconstruction was done using filtered
back-projection with a RAMP 0.5 filter. Data were analyzed using
PMOD2.7. Volumes of interest (VOIs) were defined on the summed
images, and time-activity curves (TACs) were drawn. The same
rat was injected 2 weeks earlier with 35.3 MBq [11C]PIB in a
separate study where dynamic µPET images were acquired for 90
min (4 × 15 s, 4 × 60 s, 5 × 180 s, 14 × 300 s).
References
(1) Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy. Phys.
ReV. 2001, 81, 741–766.
(2) Alzheimer, A. Uber eine eigenartige erkrankung der hirnrinde. Allg.
Z. Psychiatr. 1907, 64, 146–148.
(3) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease:
progress and problems on road to therapeutics. Science 2002, 297,
353–356.
(4) Vallet, P. G.; Guntern, R.; Hof, P. R.; Golaz, J.; Delacourte, A.;
Robakis, N. K.; Bouras, C. A comparative study of histological and
immunohistochemical methods for neurofibrillary tangles and senile
plaques in Alzheimer’s disease. Acta Neuropathol. 1992, 83, 170–
178.
(5) Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.; Verhoeff, P.; Zhuang,
Z. P.; Kung, M. P.; Kung, H. F. 11C-labeled stilbene derivatives as
Aꢀ-aggregate-specific PET imaging agents for Alzheimer’s disease.
Nucl. Med. Biol. 2003, 30, 565–571.
(6) Verhoeff, N.; Wilson, A.; Takeshita, S.; Trop, L.; Hussey, D.; Singh,
K.; Kung, H. F.; Kung, M. P.; Houle, S. In-vivo imaging of
Alzheimer’s disease ꢀ-amyloid with [11C]SB-13 PET. Am. J. Geriatr.
Psychiatr. 2004, 12, 584–595.
(7) Agdeppa, E.; Kepe, V.; Liu, J.; Flores-Torres, M.; Satyamurthy, N.;
Petric, A.; Cole, G.; Small, G.; Huang, S. C.; Barrio, J. Binding
characteristics of radiofluorinated 6-dialkylamino-2-naphthylethylidene
derivatives as positron emission tomography imaging probes for
ꢀ-amyloid plaques in Alzheimer’s disease. J. Neurosci. 2001, 21, 1–
5.
(8) Small, G.; Kepe, V.; Ercoli, L.; Siddarth, P.; Bookheimer, S.; Miller,
K.; Lavretsky, H.; Burggren, A.; Cole, G.; Vinters, H.; Thompson,
P.; Huang, S. C.; Satyamurthy, N.; Phelps, M.; Barrio, J. PET of brain
amyloid and tau in mild cognitive impairment. N. Engl. J. Med. 2006,
355, 2652–2663.
Plasma Radiometabolite Analysis after Injection of [18F]5
in Normal Mice. After intravenous injection of 5.55-7.40 MBq
[18F]5 via a tail vein, normal male NMRI mice were sacrificed by
decapitation at 2, 10, 30, or 60 min p.i. (n ) 3 at each time point).
Blood was collected into a BD vacutainer (containing 7.2 mg of
K2EDTA; BD, Franklin Lakes). The samples were centrifuged at
3000 rpm (1837g) for 5 min at 4 °C (Eppendorf centrifuge 5810)
to separate plasma. The supernatant plasma sample was mixed with
25 µL of solution containing the authentic nonradioactive compound
5 (1 mg/mL acetonitrile), and the mixture was injected onto an
Oasis HLB column (25 µm, 4.6 mm × 20 mm, Waters) that was
preconditioned by successive washings with acetonitrile and water.
The proteins of the biological matrix were washed from the Oasis
column with 10 mL of water (fraction 1). The outlet of the Oasis
column was then connected to an XTerra RP C18 column (5 µm,
4.6 mm × 250 mm), and both columns in series were then eluted
using 0.05 M sodium acetate (pH 5.5)/acetonitrile (40:60 v/v) as
the mobile phase at a flow rate of 1.5 mL/min. After passing through
(9) Zhuang, Z. P.; Klunk, W. E.; Wang, J.; Huang, G. F.; Debnath, M. L.;
Holt, D. P.; Mathis, C. A. Radioiodinated styrylbenzenes and thiofla-
vins as probes for amyloid aggregates. J. Med. Chem. 2001, 44, 1905–
1914.

18F-Labeled 2-Phenylbenzothiazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1437
(10) Klunk, W. E.; Wang, Y.; Huang, G.; Debnath, M. L.; Holt, D. P.;
Mathis, C. A. Uncharged thioflavin-T derivatives bind to amyloid-
beta protein with high affinity and readily enter the brain. Life Sci.
2001, 69, 1471–1484.
(11) Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.;
Holt, D.; Bergstro¨m, M.; Savitcheva, I.; Huang, G.; Estrada, S.; Ause´n,
B.; Debnath, M.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B.;
Wall, A.; Koivisto, P.; Antoni, G.; Mathis, C.; Langstro¨m, B. Imaging
brain amyloid in Alzheimer’s disease with Pittsbugh compound-B.
Ann. Neurol. 2004, 55, 306–319.
(12) Archer, H.; Edison, P.; Brooks, D.; Barnes, J.; Frost, C.; Yeatman,
T.; Fox, N.; Rossor, M. Amyloid load and cerebral atrophy in
Alzheimer’s disease: an 11C-PIB positron emission tomography study.
Ann. Neurol. 2006, 60, 145–147.
(13) Zhuang, Z. P.; Kung, M. P.; Hou, C.; Plossel, K.; Skovronsky, D.;
Gur, T. L.; Trojanowski, J. Q.; Lee, V.; Kung, H. F. IBOX(2-(4′-
dimethylaminophenyl)-6-iodobenzonxazole): a ligand for imaging
amyloid plaques in the brain. Nucl. Med. Biol. 2001, 28, 887–894.
(14) Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.; Plo¨ssl, K.; Hou,
C.; Holtzman, D.; 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.
Antitumor benzothiazoles. 8. Synthesis, metabolic formation, and
biological properties of the C- and N-oxidation products of antitumor
2-(4-aminophenyl)-benzothiazoles. J. Med. Chem. 1999, 42, 4172–
4184.
(24) Hutchinson, I.; Chua, M-S; Browne, H.; Trapani, V.; Bradshaw, T.;
Westwell, A; Stevens, M. Antitumor benzothiazoles. 14. Synthesis
and in vitro biological properties of fluorinated 2-(4-aminophenyl)
benzothiazoles. J. Med. Chem. 2001, 44, 1446–1455.
(25) Cava, M.; Levinson, M. Thionation reactions of Lawesson’s reagents.
Tetrahedron 1985, 41, 5061–5087.
(26) von Manfred, S.; Siegfried, J; Manfred, H. Synthese und reaktions-
verhalten 2′-substituierter isoflavone. HelV. Chim. Acta 1992, 75, 457–
470.
(27) Lin, A.; Kasina, S. Synthesis of 3-substituted 7-(3,3-dimethyl-1-
triazeno)-10-methylphenothazines as potential antitumor agents. J. Het-
erocycl. Chem. 1981, 18, 759–761.
(28) Mashraqui, S.; Kumar, S.; Traˆn Huu Daˆu, E. Synthesis, molecular
structure and optical spectral studies of syn-dithia benzothiazolophane
and anti-bis-lactone benzothiazolophane. J. Mol. Struct. 2004, 697,
221–230.
(29) Lang, R.; Williams, C.; Garson, M. An improved preparation of
4-amino-3-mercaptobenzoic acid. Org. Prep. Proced. Int. 2003, 35,
520–524.
(30) Dishino, D.; Welch, M.; Kilbourn, M.; Raichle, M. Relationship
between lipophilicity and brain extraction of C-11-labeled radiophar-
maceuticals. J. Nucl. Med. 1983, 24, 1030–1038.
(15) Suemoto, T.; Okamura, N.; Shiomitsu, T.; Suzuki, M.; Shimadzu, H.;
Akatsu, H.; Yamamoto, T.; Kudo, Y.; Sawada, T. In vivo labeling of
amyloid with BF-108. Neurosci. Res. 2004, 48, 65–74.
(16) Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D.; Kung, H. F.
F-18 polyethyleneglycol stilbenes as PET imaging agents targeting
Aꢀ aggregates in the brain. Nucl. Med. Biol. 2005, 32, 799–809.
(17) Chang, Y.; Jeong, J.; Lee, Y. S.; Kim, H.; Rai, G.; Kim, Y.; Lee, D.;
Chung, J. K.; Lee, M. Synthesis and evaluation of benzothiophene
derivatives as ligands for imaging ꢀ-amyloid plaques in Alzheimer’s
disease. Nucl. Med. Biol. 2006, 33, 811–820.
(18) Zhuang, Z. P.; Kung, M. P.; Kung, H. F. Synthesis of biphenyltrienes
as probes for ꢀ-amyloid plaques. J. Med. Chem. 2006, 49, 2841–2844.
(19) Sato, K.; Higuchi, M.; Iwata, N.; Saido, T.; Sasamoto, K. Fluoro-
substituted and 13C-labeled styrylbenzene derivatives for detecting
brain amyloid plaques. Eur. J. Med. Chem. 2004, 39, 573–578.
(20) Zhuang, W.; Kung, M. P.; Oya, S.; Hou, C.; Kung, H. F. 18F-labeled
styrylpyridines as PET agents for amyloid plaque imaging. Nucl. Med.
Biol. 2007, 34, 89–97.
(21) Ryu, E.; Choe, Y.; Lee, K. H.; Choi, Y.; Kim, B. T. Curcumin and
dehydrozingerone derivatives: synthesis, radiolabelling, and evaluation
for ꢀ-amyloid plaque imaging. J. Med. Chem. 2006, 49, 6111–6119.
(22) Shi, D.; Bradshaw, T.; Wrigley, S; McCall, C.; Lelieveld, P.; Fichtner,
I; Stevens, M. Antitumor benzothiazoles. 3. Synthesis of 2-(4-
aminophenyl)benzothiazoles and evaluation of their activities against
breast cancer cell lines in vitro and in vivo. J. Med. Chem. 1996, 39,
3375–3384.
(31) Chitneni, S. K.; Serdons, K.; Evens, N.; Fonge, H.; Celen, S.; Deroose,
C. M.; Debyser, Z.; Mortelmans, L.; Verbruggen, A. M.; Bormans,
G. M. Efficient purification and metabolite analysis of radiotracers
using high-performance liquid chromatography and on-line solid-phase
extraction. J. Chromatogr., A 2008, 1189, 323–331.
(32) Mathis, C. A.; Holt, D.; Wang, Y.; Huang, G.-F.; Debnath, M.; Shao,
L.; Klunk, W. E. Species-dependent formation and identification of
the brain metabolites of the amyloid imaging agent [11C]PIB.
Neurobiol. Aging 2004, 25 (S2), 277.
(33) Solbach, C.; Uebele, M.; Reischl, G.; Machulla, H.-J. Efficient
radiosynthesis of carbon-11 labelled uncharged thioflavin T derivatives
using [11C]methyl triflate for ꢀ-amyloid imaging in Alzheimer’s
disease with PET. Appl. Radiat. Isot. 2005, 62, 591–595.
(34) Kung, M. P.; Hou, C.; Zhuang, Z. P.; Skovronsky, D.; Kung, H. F.
Binding of two potential imaging agents targeting amyloid plaques in
postmortem brain tissues of patients with Alzheimer’s disease. Brain
Res. 2004, 1025, 89–105.
(35) Munson, P. J.; Rodbard, D. Ligand: a versatile computerized approach
for characterisation of ligand-binding systems. Anal. Biochem. 1980,
107, 220–239.
(36) Yamauchi, H.; Takahashi, J.; Seri, S.; Kawashima, H.; Koike, H.; Kato-
Azuma, M. In Technetium and Rhenium in Chemistry and Nuclear
Medicine; Nicolini, M., Bandoli, G., Mazzi, U., Eds.; Cortina
International: Verona, Italy, 1989; pp 475-502; Vol. 3.
(23) Kashiyama, E.; Hutchinson, I.; Chua, M.-S.; Stinson, S.; Phillips, L.;
Kaur, G.; Sausville, E.; Bradshaw, T.; Westwell, A.; Stevens, M.
JM8013376