Synthesis and evaluation of three 18F-labeled aminophenylbenzothiazoles as amyloid imaging agents

Article abstract of DOI:10.1021/jm900871v

We have developed three fluorine-18 labeled 6-(methyl)amino-2-(4′- fluorophenyl)-1,3-benzothiazoles, which display high in vitro binding affinity for human amyloid β plaques (K_i ≤ 10 nM). The radiolabeled probes were synthesized by aromatic nucleophilic substitution of the corresponding nitro precursor with ¹⁸F-fluoride, followed by deprotection of the BOC group if required. Determination of the octanol/water partition coefficient, biodistribution studies in mice, and in vivo μPET studies in rats and a rhesus monkey showed that initial brain uptake was high and brain washout was fast in normal animals. Radiometabolites were quantified in plasma and brain of mice and in monkey plasma using HPLC. Of the tested compounds, [¹⁸F]2 (6-amino-2-(4′-[¹⁸F]fluorophenyl)- 1,3-benzothiazole) shows the most favorable brain kinetics in mice, rats, and a monkey. Its polar plasma radiometabolites do not cross the blood-brain barrier. The preliminary results strongly suggest that this new fluorinated compound is a promising candidate as a PET brain amyloid imaging agent. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900871v

7090 J. Med. Chem. 2009, 52, 7090–7102
DOI: 10.1021/jm900871v
Synthesis and Evaluation of Three 18F-Labeled Aminophenylbenzothiazoles as Amyloid Imaging Agents
Kim Serdons,*,† Koen Van Laere,‡ Peter Janssen,§ Hank F. Kung, Guy Bormans,† and Alfons Verbruggen†
†Laboratory for Radiopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium, ‡Department of Nuclear Medicine, U.Z. Gasthuisberg,
Leuven, Belgium, §Department of Neurophysiology, Katholieke Universiteit Leuven, Leuven, Belgium, and Department of Radiology,
University of Pennsylvania, Pennsylvania
Received June 15, 2009
We have developed three fluorine-18 labeled 6-(methyl)amino-2-(40-fluorophenyl)-1,3-benzothiazoles,
which display high in vitro binding affinity for human amyloid β plaques (Ki e 10 nM). The radiolabeled
probes were synthesized by aromatic nucleophilic substitution of the corresponding nitro precursor with
18F-fluoride, followed by deprotection of the BOC group if required. Determination of the octanol/
water partition coefficient, biodistribution studies in mice, and in vivo μPET studies in rats and a rhesus
monkey showed that initial brain uptake was high and brain washout was fast in normal animals.
Radiometabolites were quantified in plasma and brain of mice and in monkey plasma using HPLC. Of
the tested compounds, [18F]2 (6-amino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole) shows the most
favorable brain kinetics in mice, rats, and a monkey. Its polar plasma radiometabolites do not cross
the blood-brain barrier. The preliminary results strongly suggest that this new fluorinated compound is
a promising candidate as a PET brain amyloid imaging agent.
Introduction
Amyloid plaques are one of the pathological hallmarks of
Alzheimer’s disease (ADa), which is a progressive and fatal
neurodegenerative brain disorder associated with progressive
memory loss and decrease of cognitive functions.1 The plaques
consist of extracellular aggregates of amyloid β (Aβ) peptide
and are surrounded by dystrophic axons and dendrites, reactive
astrocytes, and activated microglia.2 Several hypotheses have
been postulated to explain the molecular mechanisms leading
to AD, but the amyloid cascade hypothesis, stating an imbal-
ance between the production and removal of Aβ, seems to be
the most prominent theory at this time.3 A strong argument
supporting a causal role of amyloid in Alzheimer’s disease is the
fact that new treatment strategies, which are currently in clinical
trials, are aimed to delay the disease onset or slow the disease
progression by preventing Aβ deposition or increasing
Aβ solubilization.4 Early detection and quantification of the
amyloid plaques in brain with noninvasive techniques such as
positron emission tomography (PET) would allow not only
presymptomatic identification of patients but also monitoring
of the effectiveness of these novel treatments.
Figure 1. Chemical structures of [11C]PIB and [18F]KS28.
Pittsburgh compound B or [11C]PIB is up to now the most
widely used PET radioligand for amyloid visualization in
brain.5,6 However, the short half-life of carbon-11 (20.4 min)
restricts the use of [11C]PIB to PET centers equipped with an
on-site cyclotron. An 18F-labeled amyloid imaging agent is
needed to permit a more widespread application of in vivo
amyloid imaging using PET. The longer half-life of fluorine-
18(109.8 min) shouldprovide multipleinjections froma single
production batch.
Several 18F-labeled tracers for in vivo visualization of
amyloid have been proposed,7-13 and some have been tested
in humans. The PIB analogue [18F]KS28 (6-methyl-2-(40-
[18F]fluorophenyl)-1,3-benzothiazole, Figure 1)14 and the
naphthalene derivative [18F]FDDNP,15 which also labels
τ tangles, provide a signal of low specificity. The stilbene
derivative [18F]BAY94-9172 showsa similar uptake pattern as
[11C]PIB.16 Mathis et al. also reported a fluorine-18 labeled
PIB analogue, which is still under clinical investigation.17,18
We have now synthesized and evaluated three new
18F-labeled 2-phenylbenzothiazoles, which all contain a fluo-
rine-18 label directly attached to the 2-phenyl ring and a
(methyl substituted) amino group on the benzothiazole part.
These electron-donating amino substituents, which are pre-
sent inall reported promising amyloid tracers, are supposed to
contribute significantly to specific in vivo binding to amyloid,
as they may increase the binding affinity by introducing a
positive electrostatic interaction around the sulfur atom of the
phenylbenzothiazole.19 Their position on the amyloid tracers
*To whom correspondence should be addressed. Phone: þ32 16
330441. Fax: þ32 16 330449. E-mail: kim.serdons@pharm.kuleuven.be.
a Abbreviations: AD, Alzheimer’s disease; Aβ, amyloid β; BBB, blood-
-brain barrier; BOC, butyloxycarbonyl; cpm, counts per minute; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; HPLC, high performance
liquid chromatography; ID, injected dose; ID/g, injected dose per gram of
tissue; IMPY, 6-iodo-2-(40-dimethylamino)phenylimidazo[1,2]pyridine;
LSO, lutetium oxyorthosilicate; MCI, mild cognitive impairment; Mp,
melting point; NMR, nuclear magnetic resonance; P, partition coefficient;
PBS, phosphate buffered saline; p.i., postinjection; PET, positron emission
tomography; RP-HPLC, reversed phase high performance liquid chroma-
tography; ROI, region of interest; tR, retention time; rt, room temperature;
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.
r
pubs.acs.org/jmc
Published on Web 10/30/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7091
seems to have no influence on the binding characteristics. We
herereport thesynthesis, radiolabeling, invitroaffinity, andin
vivo biological evaluation in normal mice, rat, and monkey of
three new 18F-labeled 2-phenylbenzothiazoles.
1-bromo-4-fluorobenzene or 1-bromo-4-nitrobenzene, res-
pectively (Schemes 1-3).20 The yields of this one-step reac-
tion varied between 3% and 41%.
The nitro group of 6-nitro-2-(40-fluorophenyl)-1,3-benzo-
thiazole (1) was reduced with Sn2þ ions in ethanol, and the
resulting 6-amino-2-(40-fluorophenyl)-1,3-benzothiazole (2)
was monomethylated using the deprotonating agent sodium
methoxide, paraformaldehyde, and the reducing agent
sodium borohydride in methanol (Scheme 1, method A).21
6-Methylamino-2-(40-fluorophenyl)-1,3-benzothiazole (3) was
also synthesized by direct coupling of 6-methylaminobenzo-
thiazole (10) with 1-bromo-4-fluorobenzene with a yield of
17% (method B).
Results and Discussion
Chemistry. All described 2-fluorophenyl- or 2-nitrophe-
nyl-1,3-benzothiazoles were synthesized by direct coupling
of the corresponding benzothiazoles with the aryl bromide
Scheme 1. Synthesis of 6-Amino-2-(40-fluorophenyl)-1,3-ben-
zothiazole (2) and 6-Methylamino-2-(40-fluorophenyl)-1,3-ben-
zothiazole (3)a
6-Dimethylamino-2-(40-fluorophenyl)-1,3-benzothiazole (6)
and precursor 6-dimethylamino-2-(40-nitrophenyl)-1,3-ben-
zothiazole (7) were synthesized starting from 6-dimethylami-
nobenzothiazole (5), which was obtained by reduction of
6-nitrobenzothiazole (Scheme 2)22 followed by dimethylation
using formaldehyde in the presence of sulfuric acid and pow-
dered iron.23
The precursors 6-tert-butoxycarbonylamino-2-(40-nitro-
phenyl)-1,3-benzothiazole (9) and 6-tert-butoxycarbonyl-
methylamino-2-(40-nitrophenyl)-1,3-benzothiazole (12) were
synthesized by direct coupling of 1-bromo-4-nitrobenzene
with 6-tert-butoxycarbonylaminobenzothiazole (8) or 6-tert-
butoxycarbonylmethylaminobenzothiazole (11), respectively
a (i) Cs2CO3, CuBr, Pd(OAc)2, P(t-Bu)3, DMF, 150 °C, 3 h; (ii) SnCl2
2H2O, ethanol, reflux, 4 h; (iii) method A, sodium methoxide, parafor-
maldehyde, sodium borohydride, methanol, reflux, 3 h.
3
Scheme 2. Synthesis of 6-Dimethylamino-2-(40-fluorophenyl)-1,3-benzothiazole (6) and 6-Dimethylamino-2-(40-nitrophenyl)-1,
3-benzothiazole (7)a
a (i) SnCl2 2H2O, concentrated HCl, methanol, reflux, 30 min; (ii) H2SO4, Fe, formaldehyde, THF, reflux, 30 min; (iii) method B, Cs2CO3, CuBr,
3
Pd(OAc)2, P(t-Bu)3, DMF, 150 °C, 30 min.
Scheme 3. Synthesis of 6-tert-Butoxycarbonylamino-2-(40-nitrophenyl)-1,3-benzothiazole (9) and 6-tert-Butoxycarbonylmethyl-
amino-2-(40-nitrophenyl)-1,3-benzothiazole (12)a
a (i) Method C, Zn(ClO4)2 6H2O, (BOC)2O, tert-butanol, 80 °C, 3 h; (ii) method B, Cs2CO3, CuBr, Pd(OAc)2, P(t-Bu)3, DMF, 150 °C, 30 min;
(iii) method A, sodium methoxide, paraformaldehyde, sodium borohydride, reflux, 3 h.
3

7092 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Serdons et al.
Scheme 4. Synthesis of 6-Amino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole [18F]2, 6-Methylamino-2-(40-[18F]fluorophenyl)-1,3-
benzothiazole [18F]3, and 6-Dimethylamino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole [18F]6a
a (i) 18F-, K2CO3, Kryptofix, DMSO, 150 °C, 20 min; (ii) 1 M HCl, 100 °C, 5 min.
Table 1. Ki Values for Human Amyloid, log P Values, and Radiochemical Characteristics of 2, 3, 6, KS28, and PIB
Kia (nM)
compd
log P b
tR of nitro precursor (min)
tR of 18F/F product (min)
radiochemical yieldg (%)
2
10.0 ( 1.0
4.1 ( 0.3
3.8 ( 0.4
5.7 ( 1.8
2.8 ( 0.5
3.08 ( 0.04
2.40 ( 0.30
3.34 ( 0.13
2.52 ( 0.27
2.48 ( 0.06
9c
7.5c
12d
7.5e
41f
10.5 ( 5.2
45.0 ( 12.5
19.3 ( 9.0
29.4 ( 3.2
ndh
3
8d
6
11e
48f
ndh
KS28
PIB
ndh
a Ki expressed as mean ( SEM (standard error of the mean) in an [125I]IMPY competition study (Kd([125I]IMPY) = 5.3 ( 1.0 nM). b log P expressed as
mean ( SD (standard deviation, n = 6). c tR (retention time) determined on HPLC. Stationary phase: XTerra Prep RP18 (3.5 μm, 3.0 mm ꢀ 100 mm).
Mobile phase:0.05 M ammonium acetate and ethanol/THF (75:25) (60:40 v/v and after 5 min 40:60 v/v). Flow rate: 0.35 mL/min. Detection: UV 254 nm.
d tR (retention time) determined on HPLC. Stationary phase: XTerra Prep RP18 (3.5 μm, 3.0 mm ꢀ 100 mm). Mobile phase: 0.05 M ammonium acetate
and ethanol/THF (75:25) (55:45 v/v). Flow rate: 0.35 mL/min. Detection: UV 254 nm. e tR (retention time) determined on HPLC. Stationary phase:
XTerra Prep RP18 (3.5 μm, 3.0 mm ꢀ 100 mm). Mobile phase: 0.05 M ammonium acetate and ethanol/THF (75:25) (45:55 v/v). Flow rate: 0.35 mL/min.
Detection: UV 254 nm. f 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 234 nm. g Decay corrected radiochemical yield expressed
as mean ( SD (standard deviation, n > 7). h nd = not determined.
(Scheme 3). The butyloxycarbonyl (BOC) protecting group of
these benzothiazoles was introduced on the amino or methyl-
amino group of 6-aminobenzothiazole (4) or 6-methylamino-
benzothiazole (10), respectively, using themildLewis acid zinc
perchlorate hexahydrate as catalyst.24 Several attempts to
introduce the BOC-protecting group directly on the 6-amino-
phenylbenzothiazole failed. 10wassynthesizedstarting from4
using the selective monomethylation method (method A).
The overall yields of the synthesis pathways depicted in
Schemes 1-3 were rather low (<10%), but several other
attempts to synthesize the described 2-phenyl-1,3-benzo-
thiazoles 2, 3, 6, 7, 9, and 12 failed. Synthesis of the phenyl-
benzothiazoles by direct coupling of the corresponding
thiophenol with 4-nitrobenzoic acid or 4-fluorobenzoic
acid in polyphosphoric acid25 appeared not possible, as the
synthesis of the required thiophenols was not successful.
Synthesis of the phenylbenzothiazoles 2, 6, 7, and 9 via the
three-step pathway as described previously26 failed because
the final ring closure using K3Fe(CN)6 in 10% NaOH was
not successful.
radiolabeled 6-amino-2-(40-[18F]fluorophenyl)-1,3-benzo-
thiazole ([18F]2) and 6-methylamino-2-(40-[18F]fluoro-
phenyl)-1,3-benzothiazole ([18F]3) after removal of the
BOC-protecting group with
1 M hydrochloric acid
(Scheme 4). The crude mixtures were purified using pre-
parative reversed phase HPLC (RP-HPLC). The tetra-
hydrofuran (THF) present in the HPLC eluent was
removed by solid phase extraction using a Sep-Pak Plus
C18 cartridge.
Radiolabeling of the BOC-protected precursors using a
microwave cavity was not successful, as the BOC group was
found to be removed prior to the nucleophilic substitution.
Radiolabeling of dimethylaminonitrophenylbenzothiazole 7
using a microwave cavity surprisingly resulted in lower yields
compared to conventional heating.
The purified tracer products were analyzed on an analy-
tical C18 column, and their radiochemical purity was found
to be more than 99%. The identity of the tracers was
confirmed by coelution with the authentic nonradioactive
compounds after co-injection on the same analytical HPLC
system.
Affinity. The in vitro affinity of the nonradioactive refer-
ence compounds for amyloid β fibrils was determined using
an [125I]IMPY binding competition experiment with human
AD brain homogenates.27 All compounds (2, 3, and 6) were
found to have a low Ki value (e10 nM) and thus show good
affinity for Aβ plaques (Table 1).
Partition Coefficient. As an estimate of the potential of the
compounds to cross the blood-brain barrier (BBB) by
passive diffusion, the lipophilicity of the RP-HPLC purified
radiolabeled products was determined by partitioning bet-
ween 1-octanol and 0.025 M phosphate buffer, pH 7.4 (n =
6). The log of the partition coefficient (P) values of [18F]2,
[18F]3, and [18F]6 were 3.08 ( 0.04, 2.40 ( 0.30 and 3.34 (
0.13, respectively (Table 1). The log P values of [18F]2 and
[18F]6 are slightly higher than the optimal value (log P
between 1 and 3) for passive diffusion of a compound
through the BBB.28
Radiochemistry. To introduce the 18F-label on the
2-phenyl ring of the phenylbenzothiazoles, an aromatic
nucleophilic substitution of a [18F]fluorine atom for the nitro
group was achieved by heating the solution of the precursor
with K[18F]F-Kryptofix complex in dimethyl sulfoxide
(DMSO) at 150 °C for 20 min.25 Radiolabeling of 6-amino-
2-(40-nitrophenyl)-1,3-benzothiazole and 6-methylamino-2-(40-
nitrophenyl)-1,3-benzothiazole failed probably because in the
alkaline labeling conditions a partial negative charge is induced
on the N-atom at position 6. As a result, the benzothiazole
substituent no longer acts as electron-withdrawing substituent
and replacement of the 40-nitro group by [18F]fluoride using a
nucleophilic substitution reaction is not possible. To overcome
this problem, we introduced a BOC protecting group on the
amine at position 6. Labeling of these precursors, 6-tert-
butoxycarbonylamino-2-(40-nitrophenyl)-1,3-benzothiazole
(9) and 6-tert-butoxycarbonylmethylamino-2-(40-nitrophenyl)-
1,3-benzothiazole (12), resulted in the corresponding fluorine-18

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7093
Table 2. Tissue Distribution of 6-Amino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole ([18F2) after iv Injection in Normal Mice at 2 and 60 min p.i.a
% ID ( SD % ID/g tissue ( SD SUV ( SD
60 min p.i.
organ
2 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
urine
kidneys
liver
0.2 ( 0.1
8.3 ( 0.5
39.8 ( 2.3
1.4 ( 0.2
2.0 ( 0.4
0.7 ( 0.1
11.1 ( 1.2
1.6 ( 0.2
4.56 ( 0.16
1.74 ( 0.41
5.3 ( 2.2
4.9 ( 1.1
1.0 ( 0.3
10.7 ( 1.1
0.3 ( 0.0
0.3 ( 0.1
0.1 ( 0.0
68.6 ( 3.2
0.6 ( 0.5
0.32 ( 0.04
0.17 ( 0.07
1.3 ( 0.7
13.3 ( 1.2
20.0 ( 3.6
6.8 ( 0.6
8.9 ( 1.4
5.9 ( 0.4
1.8 ( 0.5
5.6 ( 0.6
1.1 ( 0.1
1.2 ( 0.1
0.7 ( 0.1
4.2 0.3
0.6 0.2
6.4 ( 0.9
2.2 ( 0.2
2.8 ( 0.4
1.9 ( 0.1
1.8 ( 0.2
0.3 ( 0.0
0.4 ( 0.0
0.2 ( 0.0
spleen þ pancreas
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
13.97 ( 0.63
15.48 ( 0.59
2.4 ( 0.9
0.97 ( 0.17
1.34 ( 0.31
0.6 ( 0.3
4.47 ( 0.10
4.96 ( 0.16
0.8 ( 0.3
0.31 ( 0.05
0.42 ( 0.10
0.2 ( 0.1
a n = 4 at each time point.
Table 3. Tissue Distribution of 6-Methylamino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole ([18F]3) after iv Injection in Normal Mice at 2 and 60 min p.i.a
% ID ( SD % ID/g tissue ( SD SUV ( SD
60 min p.i.
organ
2 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
urine
kidneys
liver
0.2 ( 0.1
8.2 ( 0.5
42.4 ( 3.0
1.4 ( 0.2
1.8 ( 0.4
0.8 ( 0.2
11.0 ( 0.5
2.3 ( 0.6
3.42 ( 0.25
1.19 ( 0.44
1.5 ( 1.3
13.0 ( 3.3
3.2 ( 1.3
17.5 ( 6.1
0.3 ( 0.1
0.3 ( 0.1
0.2 ( 0.1
37.2 ( 10.8
1.6 ( 1.8
0.44 ( 0.13
0.12 ( 0.05
2.2 ( 1.6
12.6 ( 0.7
20.8 ( 1.5
5.9 ( 0.3
7.3 ( 0.9
5.8 ( 1.0
4.6 ( 1.6
8.3 ( 3.9
0.9 ( 0.4
1.4 ( 0.4
1.0 ( 0.3
4.3 ( 0.6
7.0 ( 0.7
2.0 ( 0.2
2.5 ( 0.4
2.0 ( 0.3
1.6 ( 0.5
2.8 ( 1.2
0.3 ( 0.1
0.5 ( 0.1
0.4 ( 0.1
spleen þ pancreas
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
12.13 ( 0.48
11.76 ( 0.66
0.7 ( 0.6
1.39 ( 0.23
1.26 ( 0.25
0.9 ( 0.7
4.10 ( 0.53
3.97 ( 0.42
0.2 ( 0.2
0.48 ( 0.08
0.44 ( 0.13
0.3 ( 0.2
a n = 4 at each time point.
Table 4. Tissue Distribution of 6-Dimethylamino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole ([18F]6) after iv Injection in Normal Mice at 2 and 60 min p.i.a
% ID ( SD % ID/g tissue ( SD SUV ( SD
60 min p.i.
organ
2 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
urine
kidneys
liver
0.1 ( 0.0
8.5 ( 0.6
41.9 ( 4.1
1.5 ( 0.1
2.2 ( 0.3
1.2 ( 0.1
10.0 ( 0.6
1.4 ( 0.2
2.95 ( 0.33
0.77 ( 0.39
2.8 ( 1.3
7.6 ( 2.6
4.8 ( 1.9
18.9 ( 2.6
0.3 ( 0.1
0.6 ( 0.2
0.2 ( 0.0
28.0 ( 3.6
1.2 ( 1.3
0.65 ( 0.06
0.22 ( 0.10
2.4 ( 0.2
11.7 ( 0.8
18.9 ( 1.0
5.5 ( 0.7
8.0 ( 0.9
7.4 ( 0.7
6.5 ( 2.5
8.0 ( 1.2
1.0 ( 0.1
1.9 ( 0.3
1.3 ( 0.2
4.3 ( 0.4
6.8 ( 0.1
2.0 ( 0.2
2.9 ( 0.5
2.7 ( 0.1
2.4 ( 0.9
3.0 ( 0.4
0.4 ( 0.0
0.7 ( 0.1
0.5 ( 0.1
spleen þ pancreas
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
8.84 ( 0.91
9.23 ( 0.29
1.1 ( 0.6
1.94 ( 0.20
2.24 ( 0.56
0.9 ( 0.1
3.20 ( 0.23
3.35 ( 0.22
0.4 ( 0.2
0.72 ( 0.08
0.83 ( 0.21
0.3 ( 0.0
a n = 4 at each time point.
Biodistribution in Normal Mice. A biodistribution study of
[18F]2, [18F]3, and [18F]6 was performed in normal male
NMRI mice at 2 and 60 min postinjection (p.i.) to assess
the brain pharmacokinetics. Tables 2, 3, and 4 list the tracer
uptake in the most important organs. All compounds
showed a very high initial brain uptake. The initial uptake
in cerebrum of [18F]2 is the highest of the three compounds
(13.97 ( 0.63% ID/g). [18F]3 and [18F]6 show an initial
uptake in the cerebrum of 12.13 ( 0.48% ID/g and 8.84 (
0.91% ID/g, respectively. Brain uptake of all three com-
pounds at 2 min p.i. is significantly higher (p < 0.05, two-
sided) than that of [11C]PIB (Table 5, 3.60 ( 1.40% ID/g)
and [18F]KS28 (Table 6, 5.33 ( 0.74% ID/g). Brain uptake of
[18F]2 is significantly higher (p < 0.05, two-sided) than that
of [18F]3 and [18F]6, and brain uptake of [18F]3 is significantly
higher (p < 0.05, two-sided) than that of [18F]6. With respect
to brain washout (expressed as the ratio of % ID in cerebrum
at 2 min over % ID in cerebrum at 60 min), [18F]2 shows a
higher value(14.2) than[18F]3 (7.8) and[18F]6 (4.5). In healthy
mice, a compound with favorable characteristics for plaque
imaging not only should have a high affinity for amyloid but
also should show a high initial brain uptake followed by a
rapid washout, indicating absence of nonspecific binding to
any brain structure devoid of amyloid plaques. On the basis
of these results, compound [18F]2 seems to be the compound
with the most promising characteristics to image AD in vivo
in view of the highest initial brain uptake, which is 4 times
higher than that of [11C]PIB and almost 3 times higher than
that of [18F]KS28, and the fastest brain washout value. We
also studied the biodistribution of [18F]2 at 60 min p.i. in
mice pretreated with reference compound 2. There was no
significant difference (p < 0.05, two-sided) between the

7094 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Serdons et al.
Table 5. Tissue Distribution of 6-Hydroxy-2-(40-N-[11C]methylaminophenyl)-1,3-benzothiazole ([11C]PIB) after iv Injection in Normal Mice at 2 and
60 min p.i.a
% ID ( SD
60 min p.i.
% ID/g tissue ( SD
SUV ( SD
organ
2 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
urine
kidneys
liver
0.2 ( 0.1
10.3 ( 3.8
15.6 ( 4.5
0.6 ( 0.1
0.9 ( 0.2
0.6 ( 0.1
12.0 ( 2.4
1.1 ( 0.2
1.08 ( 0.44
0.17 ( 0.02
7.9 ( 0.5
24.4 ( 10.1
4.8 ( 3.9
9.8 ( 2.6
0.1 ( 0.1
0.3 ( 0.2
0.3 ( 0.2
42.5 ( 7.5
0.3 ( 0.2
0.18 ( 0.06
0.03 ( 0.02
2.7 ( 0.8
19.9 ( 9.1
7.9 ( 2.6
4.7 ( 2.4
3.6 ( 0.6
4.2 ( 0.3
9.1 ( 7.1
5.0 ( 1.7
0.7 ( 0.4
1.3 ( 0.7
2.1 ( 2.1
7.4 ( 2.6
2.9 ( 0.6
1.8 ( 0.2
1.3 ( 0.2
1.5 ( 0.7
3.3 ( 2.6
1.8 ( 0.6
0.2 ( 0.2
0.4 ( 0.2
0.7 ( 0.7
spleen þ pancreas
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
3.60 ( 1.40
1.60 ( 0.09
3.1 ( 0.2
0.60 ( 0.21
0.31 ( 0.13
1.1 ( 0.3
1.33 ( 0.08
0.59 ( 0.05
1.13 ( 0.11
0.22 ( 0.08
0.11 ( 0.05
0.4 ( 0.1
a n = 6 at each time point.
Table 6. Tissue Distribution of 6-Methyl-2-(40-[18F]fluorophenyl)-1,3-benzothiazole ([18F]KS28) after iv Injection in Normal Mice at 2 and 60 min p.i.a
% ID ( SD % ID/g tissue ( SD SUV ( SD
60 min p.i.
organ
2 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
urine
kidneys
liver
0.2 ( 0.1
5.8 ( 1.4
24.8 ( 3.3
1.3 ( 0.2
3.6 ( 1.0
0.9 ( 0.1
8.9 ( 1.0
1.3 ( 0.2
1.62 ( 0.29
0.42 ( 0.16
5.7 ( 2.4
3.3 ( 1.8
2.3 ( 0.2
51.8 ( 1.8
0.1 ( 0.0
0.3 ( 0.1
0.1 ( 0.0
19.3 ( 1.3
1.1 ( 0.6
0.07 ( 0.02
0.04 ( 0.02
2.8 ( 2.2
9.3 ( 2.0
12.0 ( 0.7
4.0 ( 0.7
14.4 ( 2.0
5.4 ( 0.5
3.6 ( 0.4
24.6 ( 2.1
0.4 ( 0.1
1.3 ( 0.2
0.5 ( 0.1
3.4 ( 0.8
4.5 ( 0.5
1.5 ( 0.2
5.3 ( 0.7
2.0 ( 0.1
1.4 ( 0.1
9.2 ( 0.7
0.2 ( 0.0
0.5 ( 0.1
0.2 ( 0.0
spleen þ pancreas
lungs
heart
intestines
stomach
cerebrum
cerebellum
blood
5.33 ( 0.74
5.99 ( 0.70
2.2 ( 0.9
0.27 ( 0.06
0.39 ( 0.06
1.1 ( 0.9
1.97 ( 0.29
2.21 ( 0.23
0.8 ( 0.4
0.10 ( 0.02
0.15 ( 0.03
0.4 ( 0.3
a n = 6 at each time point.
Figure 2. Standard uptake values (SUVs) of [11C]PIB, [18F]KS28, [18F]2, [18F]3, and [18F]6 in the frontotemporal cortex of a normal rat.
biodistribution values of the untreated and pretreated mice,
indicating that the residual brain activity of [18F]2 at 60 min
p.i. is due to nonspecific binding.
amino derivative at 60 min p.i.). Only a small fraction of the
radioactivity was excreted with the urine (ranging from
5.9%ID for the amino derivative to 16%ID for the methyl-
amino derivative at 60 min p.i.). In accordance with previous
reports,29 there is no real correlation between the lipophilicity
of the compounds (log P) and the degree of renal or hepato-
biliary clearance. Except for the liver, intestines, and kidneys,
the amount of radioactivity in other major organs was
negligible 60 min after injection of the tracers.
Blood levels of the three compounds were relatively low at
all time points measured. The clearance of [18F]2 from blood
(expressed as the ratio % ID in blood at 2 min over % ID in
blood at 60 min, 4.1) was faster than that of [11C]PIB (2.9) and
[18F]KS28 (2.0). As can be expected from the relatively high
log P value, the compounds were cleared from plasma mainly
bythe hepatobiliary system (rangingfrom 47%IDinliver and
intestines for the dimethylamino derivative to 79% ID for the
μPET Study in a Normal Rat. To compare the brain
pharmacokinetics of [18F]2, [18F]3, and [18F]6 with those of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7095
Figure 3. Dynamic μPET study of [11C]PIB, [18F]2, [18F]3, and [18F]6 in a normal rhesus monkey. Transverse, sagittal, and coronal MRI and
summed PET images between 0 and 20 min p.i. (left) and between 45 and 90 min p.i. for [11C]PIB, between 168 and 198 min for [18F]2, and
between 120 and 180 min p.i. for [18F]3 and [18F]6 (right).
Figure 4. Standard uptake values (SUVs) of [11C]PIB, [18F]2, [18F]3, and [18F]6 in the cerebellum, frontal cortex, parietal cortex, pons,
thalamus, and white matter of a normal rhesus monkey.

7096 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Serdons et al.
Figure 5. Ratio cerebellum/white matter of [11C]PIB, [18F]2, [18F]3, and [18F]6 in a normal rhesus monkey.
Figure 6. Plasma radiometabolite analysis of [18F]2, [18F]3, and [18F]6: RP-HPLC radiochromatogram of a plasma sample from a normal
mouse collected at 10 min p.i. The 1 min fractions were collected and counted.
Table 7. Percentage of Intact [18F]KS28, [18F]2, [18F]3, and [18F]6 ( SD
[11C]PIB and [18F]KS28, the compounds were injected in
a normal rat and brain μPET images were acquired for
120 min. Figure 2 shows the time-activity curves (TACs)
for [11C]PIB, [18F]KS28, [18F]2, [18F]3, and [18F]6 in the
frontotemporal cortex of a normal rat. Values are expressed
as standard uptake values (SUVs).
The three new fluorine-18 labeled PIB derivatives all
showed an initial brain uptake more than 2 times higher
than that of [11C]PIB and [18F]KS28. When we compare the
SUV values of mice (cerebrum, Tables 1-6) and rats
(frontotemporal cortex), we see that the relative proportion
of initial tracer uptake was identical for [18F]2, [18F]3, and
[18F]6. However, when we compare the brain activity present
at 60 min p.i., we see that the values of [18F]2, [18F]3, and
[18F]6 in the cerebrum of mice are lower than the values in the
frontotemporal cortex of the rat. This slower clearance of the
fluorine-18 labeled aminophenylbenzothiazoles in rats 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.
In a pretreatment study a rat was intraperitoneally injected
with reference compound 2 (0.35 mg) 30 min prior to
iv injection of [18F]2. Similar to the biodistribution study in
mice, there was no significant difference (p < 0.05, two-sided)
between the SUV values of this pretreated rat and the untreated
rat, indicating that brain retention of [18F]2 is nonspecific.
in Plasma of Normal Mice at 2, 10, 30, and 60 min p.i.a
% intact tracer in plasma mice
2 min p.i.
10 min p.i.
30 min p.i.
60 min p.i.
[
[
[
[
18F]KS28b
18F]2c
52.2 ( 13.2
70.0 ( 5.4
54.4 ( 13.3
77.0 ( 3.8
14.0 ( 6.2
41.7 ( 4.9
17.4 ( 3.4
37.0 ( 1.1
7.9 ( 2.8
33.4 ( 7.1
7.9 ( 2.4
7.7 ( 2.7
3.2 ( 1.7
16.5 ( 2.0
4.1 ( 1.4
6.0 ( 6.4
18F]3c
18F]6c
a n = 3 at each time point. b Plasma samples were analyzed on an
Oasis HLB column (25 μm, 4.6 mm ꢀ 20 mm, Waters). c Plasma samples
were analyzed on a Chromolith Performance RP-18 column (3 mm ꢀ
100 mm, Merck).
μPET Study in a Normal Rhesus Monkey. Because of the
observed interspecies differences between mice and rats with
regard to the brain washout of the studied compounds and
to demonstrate a high initial brain uptake in nonhuman
primates, we decided to further evaluate the three new
18F-labeled phenylbenzothiazoles in a normal rhesus mon-
key prior to selection of a compound for further clinical
evaluation. Brain μPET images were acquired for 180 min
for the 18F compounds [18F]2, [18F]3, and [18F]6 and for
90 min for [11C]PIB. Figure 3 shows the MRI and summed
PET images between 0 and 20 min p.i. for [11C]PIB, [18F]2,
[18F]3, and [18F]6 (left) and between 45 and 90 min p.i. for
[11C]PIB and 120 and 180 min p.i. for [18F]3 and [18F]6
(right). Note that because of technical reasons for [18F]2 the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7097
Figure 7. Brain radiometabolite analysis of [18F]2, [18F]3, and [18F]6: RP-HPLC radiochromatogram of a brain sample from a normal mouse
collected at 10 min p.i. The 1 min fractions were collected and counted.
Table 9. Percentage of Intact [11C]PIB, [18F]2, [18F]3, and [18F]6 in
Table 8. Percentage of Intact [18F]KS28, [18F]2, [18F]3, and [18F]6 ( SD
Plasma of Normal Rhesus Monkey at 2, 10, 30, and 60 min p.i.a
in Brain of Normal Mice at 2, 10, 30, and 60 min p.i.a
% intact tracer plasma monkey
% intact tracer brain mice
2 min 10 min 30 min 60 min 120 min 180 min
p.i.
2 min p.i.
10 min p.i.
30 min p.i.
60 min p.i.
p.i.
p.i.
p.i.
p.i.
p.i.
[
[
[
[
18F]KS28
18F]2
18F]3
18F]6
a
89.2 ( 0.4
98.8 ( 0.5
92.7 ( 1.0
90.4 ( 1.8
nd
nd
86.2 ( 2.5
86.3 ( 0.0
60.3 ( 3.9
59.2 ( 17.9
[
[
[
[
11C]PIB 94.9
61.5
62.2
nd
nd
nd
97.7 ( 0.5
84.0 ( 3.3
79.9 ( 5.4
94.8 ( 1.8
69.1 ( 4.0
68.7 ( 17.6
18F]2
18F]3
18F]6
91.8
89.7
93.5
50.6
42.7
54.1
57.5
25.1
24.4
62.9
16.9
21.1
66.3
17.7
15.8
66.9
20.8
nd
[
18F]KS28: n = 2 at each time point. [18F]2, [18F]3, and [18F]6: n = 3
at each time point. nd = not determined.
a n = 1 at each time point. nd = not determined.
late images encompass 168-198 min p.i. Also, in the monkey
no tracer uptake was observed in the skull for any of the new
tracers.
The data of [18F]KS28 were obtained in a previous study.26
The fraction of radiometabolites in plasma was the lowest for
compound [18F]2. Comparison of the percentages of intact
[18F]2 with those of [18F]KS28 demonstrates that [18F]2 is
metabolized to a lesser extent than [18F]KS28. An in vivo
metabolic breakdown of [18F]KS28 was suggested as one of
the possible reasons for its poor sensitivity as amyloid tracer
in AD patients.14 Figure 6 shows that [18F]2 is converted to at
least two polar metabolites. [18F]3, on the other hand, is
converted to at least three polar metabolites, one of which
was identified as the demethylated derivative of [18F]3,
namely, [18F]2. [18F]6 is converted to at least four polar
metabolites of which two were identified as the mono- and
bis-demethylated derivatives [18F]2 and [18F]3. Identification
of the radiometabolites was done by co-injection of the
nonradioactive reference compounds.
As can be expected, the radiometabolites resulting from
demethylation of [18F]3 and [18F]6 were also found in the
brain (Figure 7). The percentage of brain afctivity in the form
of intact [18F]3 and [18F]6 was in both cases about 69% at
30 min p.i. and 60% at 60 min p.i. (Table 8). As these
metabolites also have high, but different, affinity for human
brain amyloid, their presence may complicate the quantifica-
tion of the PET brain images. For [18F]2 only relatively low
amounts (<15%) of radiometabolites were found in the
brain at 60 min p.i. (86.3% intact [18F]2), indicating that no
substantial metabolism of [18F]2 occurs in brain and that its
radiometabolites do not cross the BBB to a high extent in
mice.
Regions of interest (ROIs; cerebellum, frontal cortex,
parietal cortex, pons, thalamus, and white matter) were
drawn on the initial (influx-weighted) PET images of the
first 20 min p.i., which were coregistered to the monkey’s
individual volumetric T1 weighted MRI, and the TACs were
calculated using PMOD software (Figure 4). These results
confirm the high initial brain uptake of [18F]2 (almost 2 times
higher than that of [18F]6 and [11C]PIB). However, [18F]3
showed the highest initial brain uptake with a remarkable
difference between uptake in the frontal and parietal cortex.
[11C]PIB seems to have the most favorable uptake pattern.
Its clearance from all indicated brain regions is the fastest
indicating that [11C]PIB has the lowest nonspecific binding.
A favorable characteristic of [18F]2 is its fast clearance out of
the cerebellum, a region that is often used as reference region.
The overall brain kinetics of [18F]2 seems to be the fastest
compared to the other two 18F-labeled phenylbenzothia-
zoles. The activity ratio between the cerebellum and the
white matter (Figure 5) is the highest for [18F]2, indicating
that this compound may have a lower nonspecific binding in
the white matter in comparison with the cerebellum, a
favorable characteristic for tracers agents used for in vivo
amyloid imaging in AD patients.
Biostability of [18F]2, [18F]3, and [18F]6 in Normal Mice.
The in vivo metabolic stability of [18F]2, [18F]3, and [18F]6
was studied by HPLC analysis of plasma and brain of normal
mice obtained at different time points after iv injection of the
18F-labeled phenylbenzothiazoles. The samples were ana-
lyzed on a monolithic C18 column, and the recovery of
radioactivity was high (87.0 ( 6.0%, n = 17).
Plasma Radiometabolite Analysis after Injection of [18F]2,
[18F]3, and [18F]6 in a Normal Rhesus Monkey. The in vivo
metabolic stability of [11C]PIB, [18F]2, [18F]3, and [18F]6 was
also studied in a rhesus monkey by analyzing plasma samples
collected at different time points during the μPET scan (2, 10,
30, 60, 120, or 180 min p.i.). Table 9 shows the percentage of
intact [11C]PIB, [18F]2, [18F]3, and [18F]6 in plasma of a
rhesus monkey as a function of time. Also in this animal
An example of plasma analysis of [18F]2, [18F]3, and [18F]6
10 min after injection is shown in Figure 6. Table 7 shows the
percentage of intact [18F]KS28, [18F]2, [18F]3, and [18F]6 in
plasma of normal mice as a function of time postinjection.

7098 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Serdons et al.
Figure 8. Percentage of intact [18F]KS28, [18F]2, [18F]3, and [18F]6 in plasma of normal mice (left) and percentage of intact [11C]PIB, [18F]2,
18F]3, and [18F]6 in plasma of normal rhesus monkey (right) as a function of time.
[
species, [18F]2 seems to be the most stable agent and its rate of
metabolism is comparable with that of [11C]PIB. When the
decline in percentage of intact tracer as a function of time is
compared between normal mice and the monkey, a slower
metabolism is observed in the rhesus monkey (Figure 8).
traceragents, compound[18F]2 hasthe highestbrain uptakein
healthy mice, which is 4 times higher than that of [11C]PIB,
and the fastest brain washout. μPET studies in normal rats
and a normal rhesus monkey confirmed that [18F]2 has the
most favorable brain kinetics. In addition, this agent seems
also to be the most stable in vivo and in a monkey its rate of
metabolism in plasma was found to be comparable with that
of [11C]PIB. As [18F]2 cannot be converted to demethylated
metabolites, which would also cross the BBB and might
complicate quantification of PET brain images, the results
of the present study clearly suggest that especially [18F]2 is a
promising candidate as Aβ plaque imaging agent for studying
AD patients with PET.
Conclusion
To allow noninvasive in vivo imaging of amyloid plaques
for early stage diagnosis of Alzheimer’s disease and monito-
ring of the disease progression and treatment efficacy with
PET, the search for a clinically useful radiolabeled PET tracer
is the focus of worldwide research. Up to now, the most
promising clinical results have been obtained with [11C]PIB.
The limitations associated with the shorthalf-life of carbon-11
may be overcome by introducing a fluorine-18 label, and on
the condition of maintenance of favorable biological charac-
teristics, this may provide a tracer agent useful for a wide-
spread clinical application. In a previous study, we evaluated
the PIB analogue [18F]KS28 (6-methyl-2-(40-[18F]fluoro-
phenyl)-1,3-benzothiazole) in two AD patients. Despite the
promising preclinical data, this 18F-labeled phenylbenzothia-
zole showed poor results in a clinical pilot study probably
because of its higher nonspecific binding and the slower
Aβ binding kinetics in comparison with [11C]PIB.
Experimental Section
Chemicals and Reagents. 6-Nitrobenzothiazole was obtained
from Alfa Aesar (Karlsruhe, Germany). 1-Bromo-4-fluoroben-
zene and 1-bromo-4-nitrobenzene were obtained from Acros
Organics (Geel, Belgium). All other reagents and solvents were
obtained commercially from Acros Organics, Aldrich (Sigma-
Aldrich, Bornem, Belgium), Alfa Aesar, Fischer Bioblock
Scientific (Tournai, Belgium), Fluka (Bornem, Belgium), Merck
€
(Darmstadt, Germany), or Riedel-de Haen (Seelze, Germany).
They were all used without further purification.
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. Evaporation of
organic solvents under reduced pressure was done with a Buchi
rotovapor (Buchi, 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, Macherey-Nagel, Duren,
Germany) as the stationary phase. Thin layer chromatography
(TLC) was done on precoated silica TLC plates (DC-Alufolien-
Kieselgel, Fluka, Buchs, Switzerland). The structure of the
synthesized products was confirmed with 1H nuclear magnetic
resonance (NMR) spectroscopy on a Gemini 200 MHz spectro-
meter (Varian, Palo Alto, CA) or a Bruker AVANCE 300 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
were determined with an IA9100 digital melting point apparatus
(Electrothermal, Essex, U.K.) in open capillaries and are re-
ported uncorrected. Exact mass measurements were performed
on a time-of-flight mass spectrometer (LCT, Micromass,
We have now synthesized and evaluated three new
18F-labeled 2-phenylbenzothiazoles, which all contain a fluo-
rine-18 label directly attached to the 2-phenyl ring and a
(methyl substituted) amino group on the benzothiazole part.
Such amine substituent is also present in the Pittsburgh
compound and in other promising fluorine-18 labeled PET
agents, which are under clinical evaluation for diagnosis of
AD. The position of the substituent on the amyloid tracers
seems to have no influence on the binding characteristics. In
2-phenylbenzothiazoles, an electron-donating amine substi-
tuent is supposed to contribute significantly to a specific in
vivo binding to amyloid, as it may increase the binding affinity
by introducing a positive electrostatic interaction around
the sulfur atom of the benzothiazole moiety. All three
new 6-amino-2-(40-fluorophenyl)-1,3-benzothiazoles, with or
without methyl substituent(s) on the amine, show high in vitro
affinity for amyloid paques present in post mortem human
AD brain homogenates (Ki e 10 nM). The fluorine-18 labeled
agents could be obtained in reasonable yields from suitable
nitro precursors and were found to pass efficiently the blood-
brain barrier in normal mice, rats, and a monkey. Of the new

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7099
Manchester, U.K.) equipped with an orthogonal electrospray
ionization interface, operating in positive mode (ESþ). 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, Milford, MA). Purity
of the synthesized compounds was determined using analytical
high performance liquid chromatography (HPLC) and was
found to be more than 95%.
Reversed phase high performance liquid chromatography
(RP-HPLC) purification and analysis was performed on a
Merck Hitachi L6200 or L7100 or VWR Hitachi L2400 pump
(Hitachi, Tokyo, Japan) 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.) or GINA Star data acquisi-
tion system (Raytest, Straubenhardt, Germany). 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-Lab Select, Mechelen, Belgium) after passage
through a UV detector.
successively with water, 2 M NaOH, and water to remove the
tin. The organic phase was dried over MgSO4, filtered, and
evaporated to dryness. The obtained yellow oil was purified with
silica column chromatography using gradient mixtures of hex-
ane and ethyl acetate (up to 20%) as eluent to yield 78 mg of 2 as
1
a gray powder (0.32 mmol, 89%). H NMR (CDCl3-d6, 200
MHz): δ 3.78 (s, 2H, NH2), 6.78 (dd, 1H, 3J = 8.8 Hz, 4J = 2.6
Hz, 5-H), 7.06 (dd, 2H, 3J = 5.5 Hz, 4J = 3.3 Hz, 30-H 50-H),
7.19 (s, 1H, 7-H), 7.75 (d, 1H, 3J = 8.8 Hz, 4-H), 7.93 (dd, 2H,
4
3J = 8.8 Hz, J = 5.4 Hz, 20-H 60-H). Accurate MS ESþ m/z
[M þ H]þ 245.0553 (calculated for C13H9N2FS 245.0543). Mp:
145-146 °C.
6-Methylamino-2-(40-fluorophenyl)-1,3-benzothiazole (3). Method
A. To a solution of 2 (37 mg, 0.15 mmol) in methanol (10 mL) was
added sodium methoxide (8 mg, 0.15 mmol) and paraformaldehyde
(4 mg, 0.15 mmol), and the mixture was refluxed for 2 h. The reaction
mixture was cooled to 0 °C, sodium borohydride (5 mg, 0.15 mmol)
was added in portions, and the mixture was refluxed further for 1 h.
The mixture was then poured into crushed ice and extracted with
ethyl acetate. The combined ethyl acetate layers were dried
over MgSO4, concentrated, and purified with silica column chroma-
tography using gradient mixtures of hexane and ethyl acetate (up to
30%) as eluent to yield 5 mg of 3 as a yellow solid (0.02 mmol, 13%).
1H NMR (CDCl3, 300 MHz): δ 2.90 (s, 3H, NCH3), 3.95 (s, 1H,
NH), 6.77 (dd, 1H, 3J=8.8Hz, 4J=2.2Hz, 5-H),6.98(d,1H,4J=
2.1 Hz, 7-H), 7.14 (t, 2H, 3J = 8.6 Hz, 30-H 50-H), 7.81 (d, 1H, 3J =
8.8 Hz, 4-H), 7.99 (dd, 2H, 3J = 8.6 Hz, 4J = 5.4 Hz, 20-H 60-H).
Accurate MS ESþ m/z [M þ H]þ 259.0679 (calculated for
C14H11N2FS 259.0700). Mp: 115.4-116.7 °C.
Gas chromatography was performed on a DI 200 gas
chromatograph (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.30
6-Aminobenzothiazole (4). A mixture of 6-nitrobenzothia-
zole (58.558 g, 0.325 mol) and stannous chloride dihydrate
(586.638 g, 2.600 mol) in a mixture of concentrated hydrochloric
acid (880 mL) and methanol (880 mL) was refluxed for 30 min.
After removal of the methanol by evaporation under reduced
pressure, the mixture was poured into crushed ice. The pH of the
aqueous solution was adjusted to 10 using 10 M NaOH, and the
mixture was extracted with ethyl acetate. The combined ethyl
acetate layers were dried over MgSO4 and concentrated to yield
39.055 g of 4 as a yellow solid (0.260 mol, 80%), which was used
without further purification. 1H NMR (CDCl3-d6, 300 MHz): δ
3.86 (s, 2H, NH2), 6.86 (dd, 1H, 3J = 8.7 Hz, 4J = 2.3 Hz, 3-H),
7.15 (d, 1H, 4J = 2.3 Hz, 7-H), 7.88 (d, 1H, 3J = 8.7 Hz, 4-H),
8.68 (s, 1H, 2-H). Accurate MS ESþ m/z [M þ H]þ 151.0334
(calculated for C7H6N2S 151.0324). Mp: 86.9-87.4 °C.
Quantitative determination of radioactivity in samples was
done using an automatic γ-counter coupled to a multichannel
analyzer (Wallac 1480 Wizard, 3 in., Wallac, Turku, Finland).
The results were corrected for background radiation and
physical decay during counting.
Dynamic μPET imaging was performed using a lutetium
oxyorthosilicate (LSO) detector-based tomograph (microPET
Focus 220; Siemens Medical Solutions, Knoxville, TN), which
has a nominal 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. The MRI scan
was performed on a Siemens Trio 3T scanner (Siemens Medical
Solutions).
Synthesis. 6-Nitro-2-(40-fluorophenyl)-1,3-benzothiazole (1).
A suspension of 6-nitrobenzothiazole (0.218 g, 1.21 mmol),
1-bromo-4-fluorobenzene (0.16 mL, 1.45 mmol), dry cesium
carbonate (Cs2CO3, 0.394 g, 1.21 mmol), copper(I) bromide
(CuBr, 34 mg, 0.24 mmol), palladium(II) acetate (Pd(OAc)2,
13 mg, 0.06 mmol), and tri-tert-butylphosphine (P(t-Bu)3,
24 mg, 0.12 mmol) in 10 mL of dimethylformamide (DMF)
was heated at 150 °C under nitrogen while stirring in a sealed
tube for 3 h. After cooling to room temperature (rt), the mixture
was diluted with ethyl acetate, washed several times with water,
and dried on MgSO4 and in a vacuum oven. The residue was
purified with silica column chromatography using gradient
mixtures of hexane and ethyl acetate (up to 20%) as eluent to
yield 0.136 g of 1 as a white solid (0.50 mmol, 41%). 1H NMR
6-Dimethylaminobenzothiazole (5). A solution of 4 (30.660 g,
0.203 mol) in tetrahydrofuran (THF, 390 mL) was slowly added
to a stirred mixture of 37% formaldehyde (161 mL, 2.030 mol)
and 4 M H2SO4 (161 mL, 0.608 mol). To the resultant mixture
was added powdered iron (90.589 g, 1.622 mol), and the mixture
was stirred mechanically for 30 min. The precipitate of iron was
removed by filtration and washed with ethyl acetate. The filtrate
was made strongly alkaline (pH > 11) with 10% NaOH and was
extracted with ethyl acetate. The combined ethyl acetate extracts
were dried over MgSO4 and concentrated and the oily residue
was purified with silica column chromatography using gradient
mixtures of hexane and ethyl acetate (up to 15%) as eluent
to yield 1.087 g of 5 as a slightly brown solid (6.10 mmol, 3%).
1H NMR (CDCl3-d6, 300 MHz): δ 3.02 (s, 6H, 2CH3), 6.99 (dd,
1H, 3J = 9.1 Hz, 4J = 2.5 Hz, 3-H), 7.14 (d, 1H, 4J = 2.5 Hz,
7-H), 7.94 (d, 1H, 3J = 9.0 Hz, 4-H), 8.66 (s, 1H, 2-H). Accurate
MS ESþ m/z [M þ H]þ 179.0623 (calculated for C9H10N2S
179.0638). Mp: 65.8-66.7 °C.
3
(CDCl3-d6, 200 MHz): δ 7.26 (t, 2H, J = 8.0 Hz, 30-H 50-H),
8.14 (d, 2H, 3J = 8.8 Hz, 20-H 60-H), 8.17 (d, 1H, 3J = 7.7 Hz, 4-
H), 8.40 (dd, 1H, 3J = 9.2 Hz, 4J = 2.2 Hz, 5-H), 8.86 (d, 1H,
4J = 2.2 Hz, 7-H). Accurate MS ESþ m/z [M þ H]þ 275.0280
(calculated for C13H7N2O2FS 275.0285). Mp: 176.4-177 °C.
6-Amino-2-(40-fluorophenyl)-1,3-benzothiazole (2). A solution
of 1 (0.100 g, 0.36 mmol) and stannous chloride dihydrate
6-Dimethylamino-2-(40-fluorophenyl)-1,3-benzothiazole
(6). Method B. A mixture of 5 (0.339 g, 1.90 mmol), 1-bromo-
4-fluorobenzene (0.25 mL, 2.28 mmol), dry cesium carbonate
(0.619 g, 1.90 mmol), copper(I) bromide (55 mg, 0.38 mmol),
and palladium(II) acetate (20 mg, 0.09 mmol) in 12 mL of
DMF was placed under nitrogen in a sealed tube at 150 °C.
Tri-tert-butylphosphine (38 mg, 0.19 mmol) was added, and the
suspension was stirred for 30 min. The DMF was removed
(SnCl2 2H2O, 0.411 g, 1.82 mmol) in dry ethanol (25 mL) was
3
refluxed under nitrogen for 4 h. After the mixture was cooled
to room temperature, the ethanol was removed by evapora-
tion under reduced pressure and the residual yellow solid
was dissolved in ethyl acetate. The solution was extracted

7100 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Serdons et al.
under reduced pressure and the obtained residue was purified
with silica column chromatography using gradient mixtures of
hexane and ethyl acetate (up to 15%) as eluent to yield 31 mg of 6
MS ESþ m/z [M þ H]þ 386.1167 (calculated for C19H19N3O4S
386.1169). Mp: 138.6-139.2 °C.
Production of [18F]Fluoride and Radiosynthesis of 6-Amino-
1
as a slighty yellow solid (0.11 mmol, 6%). H NMR (CDCl3,
2-(40-[18F]fluorophenyl)-1,3-benzothiazole 18F]2, 6-Methyl-
[
300 MHz): δ 3.04 (s, 6H, 2CH3), 6.96 (dd, 1H, 3J = 9.0 Hz, 4J =
2.6 Hz, 5-H), 7.10 (d, 1H, 4J = 2.5 Hz, 7-H), 7.14 (t, 2H, 3J =
8.7 Hz, 30-H 50-H), 7.88 (d, 1H, 3J = 9.1 Hz, 4-H), 8.00 (dd, 2H,
amino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole [18F]3, and
6-Dimethylamino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole [18F]6.
[18F]Fluoride was produced via a [18O(p,n)18F] reaction by irradia-
tion of 0.5-2.0 mL of 97% enriched H218O (Rotem HYOX18,
Rotem Industries, Beer 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-Krypto-
fix, 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-amino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole ([18F]2) and
6-methylamino-2-(40-[18F]fluorophenyl)-1,3-benzothiazole ([18F]3),
0.3 mL of 1 M HCl was added after the radiolabeling reac-
tion and the mixture was heated at 100 °C for 5 min. To
neutralize the reaction mixture, 0.4 mL of 0.5 M Na2HPO4
was added. The crude mixtures were applied onto an XTerra
Prep RP18 10 mm ꢀ 250 mm column (Waters) that was eluted
with an isocratic mixture of 45% 0.05 M NH4OAc and 55%
ethanol/tetrahydrofuran (75:25 v/v) at a flow rate of 3 mL/min.
Radiolabeled 6-amino-2-(40-[18F]fluorophenyl)-1,3-benzothia-
zole ([18F]2), 6-methylamino-2-(40-[18F]fluorophenyl)-1,3-benzo-
thiazole ([18F]3), and 6-dimethylamino-2-(40-[18F]fluorophenyl)-
1,3-benzothiazole ([18F]6) were collected at 20, 16, and 22 min,
respectively. Their corresponding precursors 9, 12, and 7 eluted at
26, 20, and 28 min, respectively. 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 [18F]2 was analyzed using an
XTerra RP18 3.5 μm, 3.0 mm ꢀ 100 mm column (Waters) eluted
with an isocratic mixture of 60% 0.05 M NH4OAc and 40%
ethanol/tetrahydrofuran (75:25 v/v) and after 5 min with an
isocratic mixture of 40% 0.05 M NH4OAc and 60% ethanol/
tetrahydrofuran (75:25 v/v) at a flow rate of 0.35 mL/min
(tR([18F]2) = 7.5 min). For purity analysis of [18F]3 and [18F]6,
the same column was eluted with an isocratic mixture of 55%
0.05 M NH4OAc and 45% ethanol/tetrahydrofuran (75:25 v/v)
or 45% 0.05 M NH4OAc and 55% ethanol/tetrahydrofuran
(75:25 v/v), respectively, at a flow rate of 0.35 mL/min (tR([18F]3)
= 12 min and tR([18F]6) = 7.5 min). Decay corrected radio-
chemical yields, corrected for the fraction of [18F]fluoride retained
on the Sep-Pak Light Accell plus QMA anion exchange cartridge,
were 10.5 ( 5.2% for [18F]2, 45.0 ( 12.5% for [18F]3, and 19.3 (
9.0% for [18F]6. The overall synthesis time to obtain the pure
products was between 75 and 100 min. The average specific activity
was found to be 72 GBq/μmol for [18F]2, 82 GBq/μmol for [18F]3,
and 24 GBq/μmol for [18F]6 at the end of synthesis.
4
3J = 8.8 Hz, J = 5.3 Hz, 20-H 60-H). Accurate MS ESþ m/z
[M þ H]þ 273.0857 (calculated for C15H13N2FS 273.0856). Mp:
146.4-147.1 °C.
6-Dimethylamino-2-(40-nitrophenyl)-1,3-benzothiazole (7). 7
was synthesized following method B starting from 5 (0.713 g,
4.00 mmol), 1-bromo-4-nitrobenzene (0.968 g, 4.79 mmol), dry
cesium carbonate (1.304 g, 4.00 mmol), copper(I) bromide
(0.114 g, 0.79 mmol), palladium(II) acetate (45 mg, 0.20 mmol),
and tri-tert-butylphosphine (81 mg, 0.40 mmol) in 24 mL of
DMF. Purification with silica column chromatography using
gradient mixtures of hexane and ethyl acetate (up to 10%)
as eluent and crystallization from ethanol yielded 36 mg of
7 as an orange solid (0.12 mmol, 3%). 1H NMR (CDCl3,
3
300 MHz): δ 3.08 (s, 6H, 2CH3), 7.00 (dd, 1H, J = 9.1 Hz,
4J = 2.6 Hz, 5-H), 7.10 (d, 1H, 4J = 2.5 Hz, 7-H), 7.94 (d, 1H,
3J = 9.1 Hz, 4-H), 8.17 (d, 2H, 3J = 9.0 Hz, 20-H 60-H), 8.31 (d,
2H, 3J = 9.0 Hz, 30-H 50-H). Accurate MS ESþ m/z [M þ H]þ
300.0793 (calculated for C15H13N3O2S 300.0801). Mp: 230-
230.4 °C.
6-tert-Butoxycarbonylaminobenzothiazole (8). Method C. To
a solution of 4 (0.376 g, 2.50 mmol) in 10 mL of tert-butanol at
80 °C were added zinc perchlorate hexahydrate (Zn(ClO4)2
3
6H2O, 45 mg, 0.12 mmol) and di-tert-butyl dicarbonate
((BOC)2O, 0.709 g, 3.25 mmol), and the mixture was stirred
for 3 h. After removal of the tert-butanol by evaporation under
reduced pressure, the obtained oily residue was purified with
silica column chromatography using gradient mixtures of hexa-
ne and ethyl acetate (up to 15%) as eluent to yield 0.580 g of 8 as
a white oil (2.32 mmol, 93%). 1H NMR (CDCl3-d6, 300 MHz):
δ 1.49 (s, 9H, C(CH3)3), 7.42 (dd, 1H, 3J = 8.8 Hz, 4J = 2.1 Hz,
3-H), 7.85 (d, 1H, 4J = 2.0 Hz, 7-H), 8.08 (d, 1H, 3J = 8.8 Hz,
4-H), 8.97 (s, 1H, 2-H). Accurate MS ESþ m/z [M þ H]þ
251.0836 (calculated for C12H14N2O2S 251.0849).
6-tert-Butoxycarbonylamino-2-(40-nitrophenyl)-1,3-benzo-
thiazole (9). 9 was synthesized following method B star-
ting from 8 (0.576 g, 2.30 mmol), 1-bromo-4-nitrobenzene
(0.558 g, 2.76 mmol), dry cesium carbonate (0.749 g, 2.30
mmol), copper(I) bromide (66 mg, 0.46 mmol), palladium(II)
acetate (25 mg, 0.11 mmol), and tri-tert-butylphosphine
(46 mg, 22.80 mmol) in 15 mL of DMF. Purification with
silica column chromatography using gradient mixtures of
hexane and ethyl acetate (up to 15%) as eluent and crystal-
lization from ethanol yielded 22 mg of 9 as a yellow solid
(0.06 mmol, 3%). 1H NMR (CDCl3, 300 MHz): δ 1.56 (s, 9H,
C(CH3)3), 6.69 (s, 1H, 7-H), 7.21 (dd, 1H, 3J = 6.6 Hz, 4J =
1.4 Hz, 5-H), 7.99 (d, 1H, 3J = 6.6 Hz, 4-H), 8.22 (d, 2H, 3J =
3
6.5 Hz, 20-H 60-H), 8.34 (d, 2H, J = 6.6 Hz, 30-H 50-H).
Accurate MS ESþ m/z [M þ H]þ 372.4250 (calculated for
C
18H17N3O4S 372.4247). Mp: 200.4-201.3 °C.
6-tert-Butoxycarbonylmethylamino-2-(40-nitrophenyl)-1,3-ben-
zothiazole (12). 12 was synthesized following method B starting
from 11 (5.290 g, 20.00 mmol), 1-bromo-4-nitrobenzene (4.840 g,
24.00 mmol), dry cesium carbonate (6.520 g, 20.00 mmol),
copper(I) bromide (0.598 g, 3.96 mmol), palladium(II) acetate
(0.223 g, 0.99 mmol), and tri-tert-butylphosphine (0.401 g,
1.98 mmol) in 120 mL of DMF. Purification with silica column
chromatography using gradient mixtures of hexane and ethyl
acetate (up to 15%) as eluent and crystallization from ethanol
yielded 1.670 g of 12 as a yellow solid (4.33 mmol, 22%).
1H NMR (CDCl3, 300 MHz): δ 1.49 (s, 9H, C(CH3)3), 3.36 (s,
3H, NCH3), 7.45 (dd, 1H, 3J = 8.8 Hz, 4J = 1.8 Hz, 5-H), 7.85 (s,
Binding Studies. Binding studies were carried out according
to a method described previously.27 125I]IMPY (6-[125I]iodo-
[
2-(40-dimethylamino)phenyl-imidazo[1,2]pyridine) with a speci-
fic activity of 81.4 TBq/mmol and greater than 95% radio-
chemical purity was prepared using a standard iododestannyla-
tion reaction and purified using a simplified C-4 minicolumn.
Binding assays were carried out in 12 mm ꢀ 75 mm borosili-
cate glass tubes. The reaction mixture contained 50 μL of post
mortem AD brain homogenate (20-50 μg), 50 μL of [125I]IMPY
3
3
1H, 7-H), 8.05 (d, 1H, J = 8.8 Hz, 4-H), 8.24 (d, 2H, J =
8.8 Hz, 20-H 60-H), 8.35 (d, 2H, 3J = 8.7 Hz, 30-H 50-H). Accurate

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7101
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 were 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
were subjected to nonlinear regression analysis using EBDA,31
from which Ki values were calculated.
Partition Coefficient Determination. The lipophilicity of the
RP-HPLC isolated 18F-complexes was determined using a
modification of the method described by Yamauchi and
co-workers.32 A 25 μL aliquot of the RP-HPLC isolated solu-
tion of the 18F-labeled compound was added to a tube contain-
ing 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 centrifugation at 3000 rpm (1837 g) for
10 min (Eppendorf centrifuge 5810, Eppendorf, Westbury, NY).
Aliquots of 61 and 500 μL were drawn from the 1-octanol and
buffer phases, respectively, taking care to avoid cross-conta-
mination between the two phases and weighed. The radioactivity
in the aliquots was counted using an automatic γ counter. After
correction for density and mass difference between the two phases,
the partition coefficient (P) was calculated using the following
equation:
and time-activity curves (TACs) were drawn. In a previous
study26 another rat was injected with 35.3 MBq [11C]PIB and
6.2 MBq [18F]KS28 and dynamic μPET images were acquired
for 90 min (4 ꢀ 15 s, 4 ꢀ 60 s, 5 ꢀ 180 s, 14 ꢀ 300 s) and 120 min
(4 ꢀ 15 s, 4 ꢀ 60 s, 5 ꢀ 180 s, 8 ꢀ 300 s, 6 ꢀ 600 s), respectively.
μPET Study in a Normal Rhesus Monkey. One juvenile male
rhesus monkey (maccaca mulatta, 5.0 kg) was sedated with
ketamine (Ketalar) and xylazine (Rompun) (intramuscular
injection) and anesthetized during the μPET experiment with
isoflurane (1.0-1.5%). The blood concentration of O2 and CO2,
the breathing frequency, and heartbeat were monitored during
the entire experiment. The monkey was injected with 109.4 MBq
[18F]2, 81.2 MBq [18F]3, 96.3 MBq [18F]6, or 152.9 MBq
[11C]PIB via the vena saphena with a time interval of 1 day
between each tracer injection. Dynamic μPET images were
acquired for 180 min (5 ꢀ 60 s, 5 ꢀ 120 s, 9 ꢀ 300 s, 12 ꢀ 600 s)
for [18F]2, [18F]3, and [18F]6 and for 90 min (5 ꢀ 60 s, 5 ꢀ 120 s, 9 ꢀ
300 s, 3 ꢀ 600 s) for [11C]PIB. Reconstruction of the images and
data analysis were performed in the same way as described for
the μPET study in a normal rat.
Plasma Radiometabolite Analysis after Injection of [18F]2,
[18F]3, and [18F]6 in Normal Mice. After intravenous injection
of about 9 MBq of [18F]2, [18F]3, or [18F]6 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, NJ). 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 2, 3, or 6
(1 mg/mL acetonitrile), and 200 μL of the mixture was injected
onto a Chromolith Performance RP-18 column (3 mm ꢀ
100 mm, Merck) eluted with a gradient mixture of 0.05 M
ammonium acetate/acetonitrile (0 min, 2:98 v/v; 2 min,
2:98 v/v; 12 min, 40:60 v/v; 17 min, 40:60 v/v; 22 min, 98:2 v/v;
25 min, 98:2 v/v) at a flow rate of 1 mL/min. After passage
through an in-line UV detector (254 nm), the HPLC eluate was
collected in 1 mL fractions and their radioactivity was measured
using a γ counter.
cpm=ðmL of octanolÞ
P ¼
cpm=ðmL of bufferÞ
with cpm = counts per minute.
Biodistribution in Normal Mice. A solution of [18F]2, [18F]3,
or [18F]6 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 in the final prepara-
tion did not exceed 10% and the concentration of THF did not
exceed 0.02%, as determined by gas chromatography. Biodis-
tribution was studied in male NMRI mice (body mass 30-40 g).
The mice were anesthetized with isoflurane (1.5-2.5%) in
oxygen at a flow rate of 1-2 L/min, and an aliquot of 0.1 mL
of the diluted tracer solution was injected via a tail vein. 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 back-
ground radioactivity and are expressed as percentage of the
injected dose (% ID), percentage of the injected dose per gram
tissue (% ID/g), or standard uptake value (SUV). SUVs were
calculated as (radioactivity in cpm in organ/weight of the
organ)/(total counts recovered/body weight). For calculation
of total radioactivity in blood, blood mass was assumed to be
7% of the total body mass.
Brain Radiometabolite Analysis after Injection of [18F]2,
[18F]3, and [18F]6 in Normal Mice. After intravenous injection
of about 9 MBq of [18F]2, [18F]3, or [18F]6 via a tail vein, normal
male NMRI mice (n = 3) were sacrificed by decapitation at 2,
10, 30, or 60 min p.i. The brain was removed, 100 μL of solution
containing the authentic nonradioactive 2, 3, or 6 (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 ammonium acetate/acetonitrile ([18F]2, 60:40 v/v at
a flow rate of 1.2 mL/min; [18F]3, 50:50 v/v at a flow rate of
1.5 mL/min; [18F]6, 45:55 v/v at a flow rate of 1.5 mL/min). After
passage through an in-line UV detector (254 nm), the HPLC
eluate was collected in 1 min fractions and their radioactivity
was measured using a γ counter.
Plasma Radiometabolite Analysis after Injection of [18F]2,
[18F]3, and [18F]6 in a Normal Rhesus Monkey. Blood was
collected during the μPET scan via vena saphena at different
time points after injection (2, 10, 30, 60, 120, or 180 min p.i.), and
samples were analyzed in the same way as described for plasma
radiometabolite analysis in normal mice.
μPET Study in a Normal Rat. A male Wistar rat (500 g) was
anesthetized with isoflurane (1.5-2.5%) in oxygen at a flow rate
of 1-2 L/min and was injected with 28.5 MBq [18F]2, 17.3 MBq
[18F]3, or 20.4 MBq [18F]6 via a tail vein with a time interval of
1 or 3 days between each tracer injection. The rat was also
injected with 29.0 MBq [18F]2 30 min after intraperitoneal
injection of 0.35 mg of reference compound 2 and 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, 10 ꢀ 360 s), and reconstruction was
done using filtered back projection with a RAMP filter. Data
were analyzed using PMOD2.7 software (Zurich, Switzerland),
volumes of interest (VOIs) were defined on the summed images,
Acknowledgment. This study was funded in part by the
EC-FP6-Project DiMI, LSHB-CT-2005-512146.
Supporting Information Available: Experimental details for
10 and 11 and additional data from 1H NMR, MS, and HPLC

7102 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Serdons et al.
analyses for the synthesized final products. This material is
available free of charge via the Internet at http://pubs.acs.org.
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biodistribution and radiation dosimetry of 18F-GE067, a radioli-
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(19) Kim, M. K.; Choo, I. H.; Lee, H. S.; Woo, L. I.; Chong, Y.
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ꢀ
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