A novel 18F-labeled imidazo[2,1- b ]benzothiazole (IBT) for high-contrast PET imaging of β-amyloid plaques

Article abstract of DOI:10.1021/ml200123w

¹⁸F-labeled imidazo[2,1-b]benzothiazole ([¹⁸F]8) was synthesized and evaluated as a tracer for cerebral β-amyloid deposits (Aβ) by means of positron emission tomography (PET). [¹⁸F]8 exhibits a high affinity to Aβ and suit

Full text of DOI:10.1021/ml200123w

LETTER
pubs.acs.org/acsmedchemlett
A Novel 18F-Labeled Imidazo[2,1-b]benzothiazole (IBT) for
High-Contrast PET Imaging of β-Amyloid Plaques
Behrooz H. Yousefi,*,† Alexander Drzezga,† Boris von Reutern,† Andrꢀe Manook,† Markus Schwaiger,†
Hans-J€urgen Wester,‡ and Gjermund Henriksen*,†
†Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universit€at M€unchen, Ismaninger Strasse 22, 81675 M€unchen,
Germany
‡Lehrstuhl f€ur Pharmazeutische Radiochemie, Walther-Meissner-Strasse 3, 85748 Garching, Germany
S Supporting Information
b
ABSTRACT: 18F-labeled imidazo[2,1-b]benzothiazole ([18F]8)
was synthesized and evaluated as a tracer for cerebral β-amyloid
deposits (Aβ) by means of positron emission tomography
(PET). [18F]8 exhibits a high affinity to Aβ and suitable brain
uptake kinetics combined with a high metabolic stability in
the brain. In a double transgenic APP/PS1 mouse model of
Alzheimer's disease, we demonstrated a specific uptake of [18F]8 in Aβ-containing telencephalic brain regions. The specific binding
of [18F]8 to Aβ was confirmed by regional brain biodistribution and autoradiography and correlated to immunohistochemistry
staining. Analysis of brain sections of APP/PS1 mouse injected with a cocktail of [18F]8 and reference compound [3H]PiB revealed
that the two tracers bind to Aβ plaques in the brain of mouse in a comparable binding pattern. [18F]8 represents the first high-
contrast PET imaging agent for detection of Aβ plaques in transgenic mouse model of Alzheimer's disease and holds promise for
transfer to a clinical evaluation.
KEYWORDS: Alzheimer's disease, 18F-labeled tracer for β-amyloid, IBT, β-amyloid plaques, positron emission tomography,
autoradiography, APP/PS1transgenic mice, neuroimaging
urrent tracer development for the noninvasive imaging of
Alzheimer's disease (AD) is focused on markers of senile
Recently, we reported the synthesis and evaluation of 11C-
labeled imidazo[2,1-b]benzothiazoles (IBTs) as a new pharma-
cophore for imaging Aβ.17 Here, we report the synthesis and
evaluation of a novel 18F-labeled 2-(p-methylaminophenyl)-7-(2-
fluoroethoxy)imidazo[2,1-b]benzothiazole ([18F]IBT, [18F]8).
Compound 8 was compared with 1a for binding affinity to Aβ
fibrils in vitro, and [18F]8 was used for the evaluation of brain
uptake kinetics in Balb-C mice. Furthermore, the properties of
[18F]8 were investigated in an APP/PS1 transgenic mouse
model of AD by means of in vivo μPET/CT, dual-tracer
autoradiography, regional brain biodistribution, and immuno-
histochemistry.
The IBTs 3ꢀ8, obtained in moderate to excellent isolated
yield (35ꢀ95%) in a high purity (>95% by HPLC), were
characterized by means of LC-MS and NMR. The IBT derivative
3 was synthesized by direct coupling of 6-methoxybenzo[d]-
thiazol-2-amine and N-(4-(2-bromoacetyl)phenyl)acetamide 2
in ethanol at reflux temperature (Scheme 2). Compound 3 was
deacetylated by treatment with 2 M NaOH at 100 °C for 30 min
under microwave heating. Intermediate 4 was methylated using
MeI in DMF to yield 5. Compound 5 was demethylated by BBr3
C
plaques (SP) that consist of β-amyloid peptides (Aβ) with posi-
tron emission tomography (PET).1ꢀ4 Several 11C- and 18F-
labeled PET tracers have been provided, including (N-[11C-
methyl])-6-OH-BTA-1, Pittsburgh compound B ([11C]PiB, 1a),5
its 18F-labeled analogue derivative 30-[18F]FPiB (Flutemetamol, GE-
067, 1b),6,7 [11C]SB-13 (1c),8 [18F]Florbetaben (1d).9,10 [18F]-
Florbetapir (1e),11ꢀ13 [18F]BF228 (1f),14 and [18F]FDDNP15
(1g) (Scheme 1).
Currently available 18F-labeled compounds (t1/2 = 109.7 min)
are hampered by a higher unspecific binding, i.e. to white matter,
as compared to their 11C-labeled analogues (t1/2 = 20.3 min).
Thus, despite the high concentrations of Aβ in advanced AD
cases, the tracer uptake ratios AD patients/healthy controls of
[18F]FDDNP, 18[F]Florbetaben, and [11C]PiB in brain regions
known to contain Aβ have been found to be 1.3,15 1.5,9 and 2,5,16
respectively.
Further development in the field of Aβ imaging aims at
providing tracers for Aβ with improved opportunity for detection
of less extensive amyloid pathology, that is, in patients that are
not yet in an advanced clinical stage. Therefore, new pharmaco-
phores suitable for 18F-labeling with improved brain uptake and
clearance kinetics, combined with high metabolic stability in vivo
and high binding affinity to Aβ-plaques, are required for advances
in the field of Aβ-targeted radiopharmaceuticals.
Received: March 30, 2011
Accepted: July 19, 2011
Published: July 19, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ml200123w ACS Med. Chem. Lett. 2011, 2, 673–677
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Scheme 1. PET Tracers Evaluated in Clinical Trials
Scheme 2. Synthesis and Radiosynthesis of IBT 8a
a Reagents and conditions: (i) EtOH, reflux, 2 h. (ii) 2 M NaOH, 100 °C, MW, 30 min. (iii) DMF, MeI, 90 °C. (iv) BBr3 in CH2Cl2, MW, 120 °C, 30 min.
(v) NaH, DMF, [18F]FEtTs, 90 °C, 5 min. (vi) NaH in DMF, F(CH2)2Br, 80 °C, 15 min. (vii) K2CO3 in DMF, ethylene glycol ditosylate, 100 °C,
15 min. (viii) DMF:CH3CN (1:5), [K+/2.2.2]18Fꢀ, 120 °C, 20 min.
in CH2Cl2 using microwave irradiation at 120 °C for 30 min to
yield 6.
58 ( 4%; total synthesis time, 55 min). Both procedures provided
[18F]8 in a high radiochemical (>99%) and chemical purity.
For the determination of binding affinity of 8 to Aβ, fibrils of
Aβ1ꢀ40 and Aβ1ꢀ42 were prepared from the respective mono-
mers according to a published procedure.18 The presence of
fibrils was confirmed by transmission electron microscopy at 10
and 100 μM concentration. The inhibition constants, Ki, of 8 and
of 1a versus [3H]1a are presented in Table 1.
The IBT scaffold is an electron-rich heteroaromatic system
with a lipophilicity value17 suitable for in vivo imaging of targets
in the brain. A further advantage is the opportunity of introducing
the 18F-label by means of an 18F-fluoroalkyl group connected to the
phenolic oxygen. In this study, compound 6 was directly reacted
with F(CH2)2Br to yield 8 and also with ethylene glycol ditosylate
to yield the precursor for one-step radiofluorination (7) in DMF.
For the radiosynthesis, 7 was reacted with [K+/2.2.2]18Fꢀ to yield
[18F]8 after 20 min at 120 °C in DMF:CH3CN (1:5). The one-
step procedure provided [18F]8 in a moderate radiochemical yield
(24 ( 5%). Alternatively, [18F]8 was prepared in a high radio-
chemical yield via a two-step 18F-fluorination (radiochemical yield,
The brain uptake kinetics of [18F]8 was compared to that of
[11C]1a at 5 and 30 min postinjection (Figure 1) in Balb-C mice
(n g 4). The measure of lipophilicity of [18F]8 was determined at
pH 7.4: log Poct/PBS = 1.92 (n = 6).
For comparison, the key preclinical data of a currently advanc-
ing 18F-labeled tracer for Aβ, [18F]Florbetapir, were reportedly13
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an in vitro binding affinity to Aβ aggregates in postmortem AD
brain homogenates of 2.87 ( 0.17 nM, an initial uptake of 6.2%
ID/g at 2 min pi and 1.84% ID/g at 60 min in male mice. The
brain uptake kinetics measurements in Balb-C mice at 5 and
30 min pi of [18F]8 and [11C]1a show the desirable characteristics
for an in vivo amyloid imaging agent with an excellent initial brain
uptake and rapid clearance properties.
The metabolic stabilities of [18F]8 and [11C]1a from in vivo
experiments with Balb-C mice at 10 and 30 min pi in samples
of blood and brain tissue were determined as reported else-
where,17,19 and the results are given in Table 2.
In vivo Aβ imaging with [18F]8 in Tg mice and cross-
confirmation of its imaging properties with ex vivo experiments
were performed by employing homozygous animals of the APP/
PS1 AD mouse model.20 A multimodal approach was followed,
including μPET/CT, ex vivo regional brain biodistribution, and
dual-tracer digital autoradiography. For the PET studies, Tg
animals (24.0 ( 0.4 months old; body weight, 34.5 ( 3.6 g;
n = 7) were injected with a single bolus of [18F]8 (11.7 ( 4.1
MBq). Age-matched C57B6/J (25 ( 2 months old; body weight,
34.8 ( 3.0 g; n = 3) were used as controls and received a single
bolus injection of [18F]8 (11.5 ( 3.4 MBq). The PET images
(Figure 2) were generated as summed frames from 36 to 45 min
pi. To obtain the anatomical reference, the CT image obtained in the
PET/CT sequence was overlaid with a cerebral MRI template of an
age-, gender-, and weight-matched Tg/control mouse (Figure 2).
The brain uptake kinetics in Tg animals and controls as
measured by means of μPET are presented in Figure 3A. The
timeꢀradioactivity curves (TACs) from whole cortex and cere-
bellum and the cortex/cerebellum ratio curves in Tg and
C57BL/6J control mice were calculated (Figure 3B).
Table 1. Ki Values (nM; x ( SD, n = 2) Determined for
Inhibition of [3H]1a Binding to Aβ Fibrils
compound
8
1a
Ki Aβ1ꢀ40
Ki Aβ1ꢀ42
2.1 ( 0.8
3.2 ( 0.6
12.0 ( 3.2
7.7 ( 2.0
For the multimodal analysis, a 25 months old Tg mouse was
coinjected with a mixture of 13.5 MBq [18F]8 and 4.0 MBq
[3H]1a into a tail vein and first scanned with a Siemens Inveon
μPET/CT for 45 min in list mode. The control (28 months old)
mouse received a mixture of 10.0 MBq [18F]8 and 3.7 MBq
[3H]1a. Animals were killed at 45 min postinjection. Analysis of
the data from dual-label autoradiography (tritium and fluorine-
18) and brain biodistribution verified the cortical uptake of
[18F]8 seen in the PET studies and also that the uptake of
[18F]8 represents a true binding of [18F]8 to cortical Aβ plaques.
Furthermore, the binding profile of fluorine-18 autoradiography
channels is consistent with that of 1a (Figure 4) as well as with
that of the results from Aβ immunohistochemistry (IHC).
On the basis of the results from μPET/CT and ex vivo
experiments in this Tg mouse model, we conclude that [18F]8
has a specific binding to Aβ plaques in hippocampal and cortical
Figure 1. Brain uptake of [18F]8 as compared to that of the reference
[11C]1a in male Balb-C mice at 5 and 30 min postinjection (mean ( SD,
n g 4).
Table 2. Speciation of Radioactivity in Brain and Blood of
Mice Injected with [18F]8 and [11C]1a (% Intact Tracera)
blood
brain
tissue
10 min
30 min
10 min
30 min
[
[
18F]8
11C]1a
12 ( 3
20 ( 5
5 ( 4
92 ( 4
96 ( 1
88 ( 6
92 ( 3
11 ( 2
a The extraction efficiency from the blood and brain homogenate
samples was 64ꢀ95%.
Figure 2. Orthogonal μPET images superimposed onto a MRI template. The PET signal represents the summed frames 36ꢀ45 min postinjection of
[18F]8 in (A) Tg and (B) control (coregistered μPET images of all animals in each experimental group were summed to generate these images).
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Figure 3. (A) μPET mean TACs from cortical and cerebellar VOIs in Tg (n = 7) and control mice (n = 3) of [18F]8. (B) Mean of ratios in Tg (n = 7) and
control mice (n = 3) of [18F]8.
justify the further evaluation of this compound for the detection
of amyloid plaques in the living brain at an early stage of the
disease.
’ ASSOCIATED CONTENT
S
Supporting Information. Full experimental details for
b
compounds synthesized, procedures for radiosynthesis, log Poct/PBS
measurements, description of assays, metabolite analyses, HPLC
purity tests, animal studies, PET imaging, and ex vivo evaluation of
control mouse. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Authors
*Tel: +49 89 4140-6340. Fax: +49 89 4140-6493. E-mail:
b.yousefi@tum.de (B.H.Y.). Tel: +49 89 4140-4586. Fax: +49
89 4140-4841. E-mail: G.Henriksen@lrz.tum.de (G.H.).
Funding Sources
Figure 4. Ex vivo dual-tracer autoradiography of a 12 μm thick axial
section of Tg mouse brain killed 45 min after coinjection of [18F]8 and
[3H]1a superimposed to the optical image. (A) Separated image of
[18F]8 autoradiography. (B) Separated image of [3H]1a autoradiogra-
phy. (C) Fused IHC images of anti-Aβ40 and anti-Aβ42 (gray scale).
This work was supported by grants from Deutsche Forschungs-
gemeinschaft (DFG) (HE4560/1-2, DR 445/3-1, DR 445/4-1,
and IRTG 1373).
’ ACKNOWLEDGMENT
regions. The timeꢀactivity curves (TACs) evidenced an excel-
lent brain uptake and clearance profile. From 5 min pi on to the
end of the PET examination (45 min), an excellent differentia-
tion of cortex and cerebellum was observed in Tg animals. In
contrast, the ratio of tracer uptake in cortex relative to cerebellum
in controls animals approaches unity. The potential of [18F]8 as a
tracer for Aβ is strengthened by the results from the ex vivo
regional brain biodistribution and comparison to [3H]1a in dual-
tracer autoradiography experiments. It was confirmed that [18F]8
and [3H]1a bind in a similar manner to brain of APP/PS1 Tg mice.
A PET study of the performance of [18F]8 in a younger cohort of
Tg mice is currently in progress.
This proof of concept study demonstrates that the imidazo-
[2,1-b]benzothiazole [18F]8 allows high-contrast imaging of Aβ
in an APP/PS1 mouse model of AD by means of μPET. The
favorable properties of an efficient and rapid 18F-labeling com-
bined with an excellent brain entry/clearance kinetics as well as a
high affinity for the amyloid-β plaques and high in vivo stability
We thank Katrina McGuire, Andrea Alke, Monika Beschorner,
Sybille Reder, Axel Weber, and Markus Lehmann for their
excellent technical support; Antje Willuweit, Michael Schoor,
Heinz von der Kammer for provision of APP/PS1 (Arte10)
animals and their scientific support for IHF; and Prof. Axel Walch
and his group for excellent technical support for fluorescence
microscopy.
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