11C-labelled PIB analogues as potential tracer agents for in vivo imaging of amyloid β in Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2008.09.038

Pittsburgh Compound-B (PIB) is currently being evaluated clinically for in vivo visualization of amyloid plaques in patients with Alzheimer's disease (AD). We have synthesized three structural isomers of 6-hydroxy-2-(4′-aminophenyl)-1,3-benzothiazole, per

Full text of DOI:10.1016/j.ejmech.2008.09.038

European Journal of Medicinal Chemistry 44 (2009) 1415–1426
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
11C-labelled PIB analogues as potential tracer agents for in vivo imaging of
amyloid
b
in Alzheimer’s disease
,
a
a
b
a
K. Serdons a , T. Verduyckt , D. Vanderghinste , P. Borghgraef , J. Cleynhens ,
*
F. Van Leuven b, H. Kung c, G. Bormans a, A. Verbruggen a
a Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences, K.U. Leuven, O&N2, Herestraat 49 – Bus 821, 3000 Leuven, Belgium
b Laboratory of Experimental Genetics and Transgenesis, K.U. Leuven, O&N1, Herestraat 49 – Bus 602, 3000 Leuven, Belgium
c Department of Radiology, University of Pennsylvania, Market Street 3700 – Room 305, Philadelphia, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2008
Received in revised form 18 September 2008
Accepted 18 September 2008
Available online 7 October 2008
Pittsburgh Compound-B (PIB) is currently being evaluated clinically for in vivo visualization of amyloid
plaques in patients with Alzheimer’s disease (AD). We have synthesized three structural isomers of 6-
hydroxy-2-(40-aminophenyl)-1,3-benzothiazole, performed radiolabelling with carbon-11 and investi-
gated their in vivo and in vitro properties. Specific binding to amyloid plaques was demonstrated in vitro
using post-mortem brain homogenates of AD patients, transgenic AD mice brain sections and post-
mortem human AD brain sections. In normal mice, initial brain uptake (at 2 min p.i.) was high and was
followed by a fast wash-out. The three structural analogues have a high potential as tracer agents for in
vivo visualization of amyloid plaques in AD patients.
Keywords:
Alzheimer’s disease
Amyloid
Ó 2008 Elsevier Masson SAS. All rights reserved.
Diagnosis
PET
Carbon-11
PIB
Phenylbenzothiazole
1. Introduction
neurofibrillary tangles (NFTs), activated microglia and reactive
astrocytes [2]. Although scientists are uncovering more and more of
The increasing mean life span of the global population empha-
sizes the need for accurate diagnostic tools and therapies for age-
related diseases such as Alzheimer’s disease (AD) which has an
exponentially increasing incidence with age [1]. Alzheimer’s
disease is the most frequent form of dementia and is characterized
by a progressive neurodegeneration. The hallmarks of AD are
the pathogenesis of AD, it is still not entirely clear what the exact
correlation is between the neuritic plaques and the NFTs [3–5].
Nevertheless, the amyloid cascade hypothesis presented by Hardy
and Higgins in 1991 [6] is still up-to-date and highlights the
importance of Ab deposits and NFTs as potential targets for thera-
pies against AD and as diagnostic tracer agents.
an extracellular deposition of amyloid
uritic plaques, an intraneuronal cytoplasmatic deposition of
b
(A
b
) in the form of ne-
Until now, post-mortem histological staining of amyloid plaques
with specific dyes (silver staining, Congo red, thioflavin-S, thio-
flavin-T (ThT),.) or using amyloid
b antibodies is the only way to
obtain a definitive diagnosis of AD. To allow non-invasive in vivo
diagnosis of AD, the search for a radiolabelled derivative of one of
these dyes has become subject of worldwide research. There are,
however, a number of challenges to reach this goal. First of all, the
tracer agent has to be able to cross the blood-brain barrier (BBB).
This requires a neutral molecule with a molecular mass not
exceeding 600 [7]. Furthermore, the log octanol-buffer partition
coefficient (log P), which is a measure of the lipophilicity of the
compound, should be preferably between 1 and 2.5 [8]. The ability
to pass the BBB has to be combined with a high affinity for amyloid
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b; APP, amyloid precursor
protein; BBB, blood-brain barrier; ESI, electrospray ionization; FCS, fetal calf serum;
ID, injected dose; IMPY, 6-iodo-2-(40-dimethylamino)phenyl-imidazo[1,2]pyridine;
LC–MS, liquid chromatography hyphenated to mass spectrometry; Mp, melting
point; MPLC, medium pressure liquid chromatography; NFT, neurofibrillary tangle;
NMP, N-methyl-2-pyrrolidone; NMR, nuclear magnetic resonance; P, partition
coefficient; PBS, phosphate buffered saline; PBST, PBS containing 0.05% TweenÒ 20;
PET, positron emission tomography; PIB, Pittsburgh Compound-B, 6-hydroxy-2-(40-
N-[11C]methylaminophenyl)-1,3-benzothiazole; PPA, polyphosphoric acid; RP-
HPLC, reversed phase high performance liquid chromatography; RT, room
temperature; ThT, thioflavin-T; TLC, thin layer chromatography.
b
plaques and the compound must be radiolabelled with a suitable
* Corresponding author. Tel.: þ3216330441; fax: þ3216330449.
radionuclide to allow detection of the radiation emitted by the
E-mail address: kim.serdons@pharm.kuleuven.be (K. Serdons).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.038

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K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
tracer agent outside the patient’s body using a gamma or positron
emission tomography (PET) camera ultimately providing quanti-
2.1.2. Synthesis of 4-hydroxy-2-(40-methylaminophenyl)-1,3-
benzothiazole (6a) and 7-hydroxy-2-(40-methylaminophenyl)-1,3-
benzothiazole (6c)
tative in vivo images of amyloid
b plaque deposition in the brain.
In the past years, radiolabelled derivatives of several of the
higher mentioned dyes have been tested and reported [9–18]. Up to
now, by far the most promising and clinically useful results have
A small amount of 6a and 6c was synthesized by methylation of
5a and 5c, respectively, using iodomethane (Scheme 2). Mass
spectrometric analysis of the reaction mixture showed the pres-
ence of both the N-dimethylated and N-monomethylated 6.
Reversed phase high performance liquid chromatography (RP-
HPLC) analysis further showed that the obtained monomethylated
compound had a longer retention time than the methoxy-isomer
(4), indicating that methylation had occurred at the amino group.
O-methylation normally requires deprotonation of the phenol and
thus requires more alkaline reaction conditions. Purification was
done using medium pressure liquid chromatography (MPLC).
been obtained with
[
11C]PIB (6-hydroxy-2-(40-N-[11C]methyl-
aminophenyl)-1,3-benzothiazole, Fig. 1), but early clinical evalua-
tion of fluorine-18 labelled derivatives is also ongoing [12,19]. In
a search for derivatives with improved characteristics we synthe-
sized and labelled in the present study three structure analogues of
this compound, namely the 4-hydroxy, 5-hydroxy and 7-hydroxy
structural isomers (Fig. 1). We compared their in vivo and in vitro
biological characteristics with those of the parent compound
[
11C]PIB to obtain structure activity information related to the
position of the OH-group which would also be relevant for the
2.1.3. Synthesis of 5-hydroxy-2-(40-methylaminophenyl)-1,3-
benzothiazole (6b)
development of new 18F-labelled PIB derivatives.
To avoid a difficult MPLC separation of the mono- and dime-
thylamino derivative resulting from the methylation reaction, 6b
was prepared using the method described by Lin (Scheme 3) [23].
The benzothiazole ring of commercially available 5-methoxy-2-
methyl-benzothiazole was opened using 10 M sodium hydroxide in
order to obtain 2-amino-4-methoxy-thiophenol (7), which then
was reacted with 4-(methylamino)benzoic acid in polyphosphoric
acid (PPA) to obtain 8 after further purification using column
chromatography. The methoxy group was then converted to
a hydroxy group using BBr3 to obtain 6b in a 69% yield.
2. Results and discussion
2.1. Synthesis
2.1.1. Synthesis of 4-hydroxy-, 5-hydroxy- and 7-hydroxy-2-(40-
aminophenyl)-1,3-benzothiazole (5a–c)
The three compounds were synthesized using a similar pathway
(Scheme 1) [20]. For the synthesis of 4-hydroxy-2-(40-amino-
phenyl)-1,3-benzothiazole (5a), ring closure of the benzothiazole
does not yield two isomers and further separation was not required.
Briefly, o-anisidine was first reacted with p-nitrobenzoyl chloride
to form N-20-methoxyphenyl-4-nitrobenzamide (1a). The amide
was then converted to the thiobenzamide (2a) using Lawesson’s
reagent (2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-
2,4-disulphide) which is a useful thiation reagent to replace the
carbonyl oxygen atoms of ketones, amides and esters by sulphur. In
the presence of potassium ferricyanide, 2a was cyclized to the 2-(40-
nitrophenyl)-benzothiazole 3a. After reduction of the nitro to an
amine group using SnCl2, the methyl ether was demethylated using
boron tribromide (BBr3) in dichloromethane at 70 ꢀC, yielding 5a.
For the synthesis of 5-hydroxy-2-(40-aminophenyl)-1,3-benzo-
thiazole (5b) and 7-hydroxy-2-(40-aminophenyl)-1,3-benzothia-
zole (5c), ring closure can lead to the formation of two isomers and
therefore the procedure described by Hutchinson [21] was used.
When a halogen atom (typically chlorine or bromine) is present in
the position where the ring closure should occur and sodium
hydride (NaH) or sodium methoxide (NaOMe) is used in combi-
nation with N-methyl-2-pyrrolidone (NMP) as a solvent, the ring
closure is specific for the intended position. Using this procedure,
we were able to obtain 5b and 5c in sufficient yield. For the
synthesis of 7-hydroxy-2-(40-aminophenyl)-1,3-benzothiazole (5c)
the starting compound 2-bromo-3-aminoanisole was obtained
following a reported procedure [22]. However, for the synthesis of
5b, the commercially available hydrochloride salt of 6-chloro-m-
anisidine was used as starting product resulting in a much higher
overall yield.
2.2. Radiolabelling
2.2.1. Synthesis of 4-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-
benzothiazole ([11C]6a), 5-hydroxy-2-(40-[11C]methylaminophenyl)-
1,3-benzothiazole ([11C]6b) and 7-hydroxy-2-(40-[11C]
methylaminophenyl)-1,3-benzothiazole ([11C]6c)
11C-methylation of precursor 5a was performed with a 25% yield
by bubbling [11C]CH3OSO2CF3 (methyltriflate) with a stream of
helium through a solution of the precursor (Scheme 4). Pre-puri-
fication of the reaction mixture was done with the aid of an acti-
vated C-18 Sep-PakÒ cartridge, which retained [11C]6a whereas
more hydrophilic compounds (such as [11C]methyltriflate) were
eluted from the cartridge. After rinsing the cartridge with water, the
labelled compound [11C]6a was eluted with methanol. The eluate
containing [11C]6a was further purified by RP-HPLC on a semi-
preparative C18 column. The use of a semi-preparative column was
necessary to allow injection of the entire prepurified reaction
mixture. During isolation of the desired 11C-labelled benzothiazole
care was taken to start the collection of the peak only after the UV-
signal had returned to baseline, in order to prevent contamination
of the carbon-11 labelled benzothiazole with the non-radioactive
precursor. Identity confirmation was done using radio-HPLC
combined with mass spectrometry (radio-LC–MS) (experimental
mass: 257 Da, theoretical mass: 257 Da in ESþ, data not shown) and
by comparison of the retention times of the authentic analogue 6a
and the 11C-labelled [11C]6a on RP-HPLC. Starting from 5b and 5c,
H
N
N
11CH3
S
HO
PIB
OH
HO
N
S
H
11CH3
N
S
H
N
S
H
N
N
N
11CH3
11CH3
11C]6c
[
11C]6a
[
11C]6b
[
OH
Fig. 1. Structure of PIB, 4-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole [11C]6a, 5-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole [11C]6b and 7-hydroxy-
2-(40-[11C]methylaminophenyl)-1,3-benzothiazole [11C]6c.

K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
1417
NO2
NO2
1,4-dioxane
Lawesson's reagent
reflux, 3 h
H
N
H
N
R
R
S
O
R1
R1
R1
R
R1
R
2a
2b
2c
2'-OCH3
5'-OCH3
3'-OCH3
H
2'-Cl
2'-Br
1a
1b
1c
2'-OCH3
5'-OCH3
3'-OCH3
H
2'-Cl
2'-Br
NaOH, EtOH
K3Fe(CN)6
90°C, 2 h
NMP,NaH
110°C, 12 h
EtOH
SnCl2.2H2O
reflux, N2, 4 h
N
N
S
NO2
NH2
R
R
S
R
R
BBr3, CH2Cl2
-70°C, 12 h
4a
4b
4c
4-OCH3
5-OCH3
7-OCH3
3a
3b
3c
4-OCH3
5-OCH3
7-OCH3
N
NH2
R
S
R
5a
5b
5c
4-OH
5-OH
7-OH
Scheme 1. Synthesis of 4-hydroxy-2-(40-aminophenyl)-1,3-benzothiazole (5a), 5-hydroxy-2-(40-aminophenyl)-1,3-benzothiazole (5b) and 7-hydroxy-2-(40-aminophenyl)-1,3-
benzothiazole (5c).
the same procedure as for [11C]6a was followed to prepare [11C]6b
and [11C]6c, respectively. Despite the low labelling yield of 5% and
7%, respectively (not decay corrected), sufficient quantities of the
11C-labelled tracer were obtained to perform the biological studies
and no attempts were made to increase the labelling yield.
intramolecular hydrogen bond formation is possible, the lip-
ophilicity of the 4-hydroxy derivative on the one hand and the
5-hydroxy and 7-hydroxy derivatives on the other hand is
approximately the same, as demonstrated by an identical retention
time of 4-methoxy-2-(40-aminophenyl)-1,3-benzothiazole and
6-methoxy-2-(40-aminophenyl)-1,3-benzothiazole using RP-HPLC
(data not shown).
2.3. Partition coefficient
The log P values of [11C]6a, [11C]6b and [11C]6c were found to be
3.18 ꢁ 0.18, 2.48 ꢁ 0.063 and 2.45 ꢁ 0.096, respectively. The differ-
ence in log P values of [11C]6a on the one hand and [11C]6b and
2.4. Affinity of the authentic non-radiolabelled derivatives 6a, 6b
and 6c for post-mortem human AD brain homogenates
[
11C]6c on the other hand can be explained by an assumed forma-
In order to obtain information on the in vitro affinity of these
tion of an intramolecular hydrogen bond in compound [11C]6a
tracers for amyloid
b fibrils, determination of their binding to
(Fig. 2), making the compound more lipophilic. Indeed, if no
human AD brain homogenates is a useful test. The Ki values
CH3CN, CH3I
80°C, 36 h
N
S
N
S
H
NH2
R
N
R
CH3
R
R
5a
5c
4-OH
7-OH
6a
6c
4-OH
7-OH
Scheme 2. Synthesis of 4-hydroxy-2-(40-methylaminophenyl)-1,3-benzothiazole (6a) and 7-hydroxy-2-(40-methylaminophenyl)-1,3-benzothiazole (6c).

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K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
Ethylene glycol
10 M NaOH
reflux, N2, 2 h
H3CO
H3CO
N
S
NH2
SH
CH3
7
H
PPA
180°C, 3 h
HOOC
N
CH3
BBr3, CH2Cl2, N2
RT, 12 h
H3CO
HO
H
N
S
H
N
S
N
N
CH3
CH3
8
6b
Scheme 3. Synthesis of 5-hydroxy-2-(40-methylaminophenyl)-1,3-benzothiazole (6b).
obtained for the authentic non-radioactive compounds are given in
simultaneously, also enables to obtain useful co-localization data.
The results of the incubation of 8- m microtome sections of
transgenic AD mouse brain and post-mortem human AD brain with
either 6a, 6b or 6c at a 1- M concentration and of the immuno-
histochemical staining of neighbouring sections is shown in Figs. 3
and 4. The region of the mouse brain shown is the subiculum,
Table 1. Compounds with a low Ki value (ꢂ20 nM) show good
m
affinity for Ab plaques. Pittsburgh Compound-B showed a Ki value
of 2.8 ꢁ 0.5 nM in this test, indicating that the new compounds
m
have a similar affinity for amyloid as PIB.
b
a structure rich in Ab plaques in AD mice that is located in the
2.5. Binding of 6a, 6b and 6c to amyloid plaques in mouse and
hippocampal region. The region shown in the human brain is part
of the cortex. From both figures it can be seen that 6a as well as 6b
and 6c stain plaques in transgenic AD mouse brain as well as in
human AD brain. Comparable images were obtained using the PIB-
compound (data not shown). This is in accordance with the findings
from the in vitro affinity test. Comparison of the images of the
immunohistochemical staining with the images obtained with
human AD brain sections
The potential of binding to amyloid
6b and 6c was also evaluated in post-mortem brain sections of
transgenic AD mice and in post-mortem brain sections of an AD
patient. Both 40-mm vibratome sections and 8-mm paraffin sections
of brain can be used for this purpose. Vibratome sections normally
are incubated with the non-radioactive compound in free floating
conditions, maximizing the contact surface between the solution
and the brain section. Paraffin sections benefit from the fact that
they are thinner than vibratome sections. Here they were used for
performing ‘co-localization’ experiments. In such experiments,
a section is incubated with one of the non-radioactive reference
compounds, which can be visualized with fluorescence microscopy
as the bound benzothiazole is fluorescent, while an adjacent serial
b plaques of compounds 6a,
fluorescence microscopy shows that the A
body stain more A plaques than the benzothiazoles. This is
a consequence of the much higher affinity of the A N25 and pan A
antibody for amyloid plaques in comparison to the affinity of 6a,
6b and 6c. From Fig. 4 it also appears that the benzothiazoles
primarily stain the core of the amyloid plaque. To further evaluate
this, a vibratome section was incubated first with the A N25 anti-
bN25 and pan Ab anti-
b
b
b
b
b
b
body and then with 6b. This co-localization is shown in Fig. 5. In
these images, the outer rim of the plaque is only stained with the
section can be stained immunohistochemically with the A
pan A antibody that binds to fibrillar amyloid . If the non-
radioactive compound binds to fibrillar amyloid on a particular
section, the localization of the fluorescence on this section has to be
almost identical to the localization of the antibody on the adjacent
section. Such a co-localization experiment is almost impossible to
bN25 or
Ab
N25 antibody, which colors both the dense and diffuse parts of
the plaque, while the core of the amyloid plaque shows intensive
fluorescence. This is probably a consequence of the higher amount
of -pleated sheets in the core of the plaque and proves the
assumption that these benzothiazoles primarily label the core of
the amyloid plaque. Other vibratome sections from transgenic AD
b
b
b
b
b
b
perform with vibratome sections since such sections are 40
thick whereas the typical diameter of an amyloid plaque is 100
mm
mouse brain also show that 6b and 6c can be used to visualize
vascular amyloid as can be seen in Fig. 6. In these images the
background signal is higher in the right section stained with 6c
mm.
However, incubation of a vibratome section with the fluorescent
compounds (6a, 6b or 6c) and the AbN25 (or pan Ab) antibody
[
11C]CH3OSO2CF3
H
11CH3
N
RT, 30sec
N
NH2
R
N
R
S
S
R
R
11C]6a 4-OH
11C]6b 5-OH
11C]6c 7-OH
5a 4-OH
5b 5-OH
5c 7-OH
[
[
[
Scheme 4. Radiosynthesis of 4-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole ([11C]6a), 5-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole ([11C]6b) and 7-
hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole ([11C]6c).

K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
1419
Table 1
O
H
Ki values of 6a, 6b and 6c competing with 125I-IMPY binding to amyloid plaques in
human AD brain homogenates.
11CH3
N
S
Compound
Kia (nM, mean ꢁ SEM)
N
6a
6b
6c
18.8 ꢁ 3.8
11.5 ꢁ 3
11.2 ꢁ 5
2.8 ꢁ 0.5
H
Fig. 2. Formation of an intramolecular hydrogen bond in compound [11C]6a.
PIB
a
Ki ¼ IC50=ð1 þ L=KdÞ with IC50 ¼ inhibition constant 50%, L ¼ concentration
radioactive ligand and Kd ¼ dissociation constant of the radioactive ligand.
than in the left section stained with 6b. Both compounds highlight
the presence of vascular amyloid, but the fluorescent signal with 6b
is more intense than that of 6c. Therefore, the test was repeated
technique uses micromolar amounts of the non-radioactive coun-
terparts instead of the nanomolar tracer amounts used in vivo. At
micromolar concentration also lower affinity binding sites may be
labelled and furthermore fluorescence intensity may depend on the
target that the studied compound is binding to so that fluorescence
images provide qualitative information rather than quantitative
information.
using a 0.1 mM concentration of the compounds (data not shown).
As could be expected from Fig. 6, this lower concentration still
yielded useful fluorescence images with 6b with a lower back-
ground signal, while the fluorescence signal was close to the
detection limit for 6c. The three compounds stain amyloid plaques
in human AD brain as well as in transgenic AD mouse brain.
Compound 6a not only binds to the plaques, but also to neurofi-
brillary tangles (Fig. 7). This behaviour has also been observed with
other compounds, such as the 18F-labelled FDDNP and BF-108
[12,18,24]. The reason, according to Suemoto et al. [18], is that
2.6. Biodistribution in healthy mice
plaques and neurofibrillary tangles share the
structure.
b-sheet secondary
Biodistribution of the three new tracer agents and PIB was
studied in normal mice. Tables 2–5 list the tracer uptake in the most
important organs. The initial brain uptake (2 min p.i.) of the three
new compounds is in the same range as that of PIB. The initial
uptake is slightly lower for the 7-hydroxy derivative [11C]6c and
slightly higher for the 5-hydroxy derivative [11C]6b. The 5-hydroxy
and 7-hydroxy derivatives [11C]6b and [11C]6c, respectively, show
equal initial uptake in cerebrum and cerebellum (expressed as
% ID/g). The 4-hydroxy derivative [11C]6a shows a higher activity
concentration in the cerebrum compared with the cerebellum,
which is also the case for PIB. With respect to brain wash-out (% ID
in cerebrum at 2 min/% ID in cerebrum at 60 min), the 4-hydroxy
and 7-hydroxy derivatives [11C]6a and [11C]6c, respectively, show
a wash-out which is 2 times faster than that of PIB. The 5-hydroxy
derivative [11C]6b even has a brain wash-out that is 8 times faster
Both in vitro tests show promising results for the 11C-labelled
PIB analogues. After intravenous injection of tracer amounts of the
11C-labelled molecules it can be expected that specific binding will
only occur at high affinity binding sites in the amyloid
b plaques.
However, for most of the reported amyloid PET tracers, a substan-
tial fraction of the tracer also shows non-specific binding to white
matter, decreasing both the image contrast and amyloid detection
sensitivity. Determination of the in vitro affinity using amyloid
containing human brain homogenate yields quantifiable affinity
constant values whereas the clearance of non-specifically bound
tracer is difficult to predict in vitro. High resolution fluorescence
microscopy can be used to determine whether binding of the
studied compounds is limited to amyloid binding sites, but this
Fig. 3. Immunohistochemical and fluorescent staining of 8-
mm microtome sections of transgenic mouse brain (left) and post-mortem human AD brain (right). Top images are of
sections immunohistochemically stained with pan A
b
antibody, the bottom images are of sections incubated with 6a and investigated with fluorescence microscopy. Bar ¼ 100
mm.

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K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
Fig. 4. Fluorescence microscopic images after incubation of transgenic AD mouse brain (left) and human AD brain (right) with 6b (top) or 6c (bottom). Middle row is immuno-
histochemically stained with A m. From top to bottom are three serial sections. Amyloid plaques in AD mouse brain highlighted with white arrowheads (top/
N25. Bar ¼ 100
bottom) or red arrowheads (middle). Amyloid
b
m
b
b
plaques in human AD brain marked with # for 6b (corresponding plaques immunohistochemically stained marked with red #) or
with * for 6c (corresponding plaques immunohistochemically stained marked with yellow *). (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
than that of PIB over the studied time interval. In healthy mice,
a compound with favourable characteristics for plaque imaging
should show a high initial uptake followed by a rapid wash-out,
thus without non-specific binding to any brain structure devoid of
amyloid plaques. In view of these requirements, the 5-hydroxy
derivative [11C]6b, with its high initial brain uptake and very fast
wash-out seems to be the most suitable tracer agent for in vivo
diagnosis of AD, based on the mouse data. The distribution of these
Fig. 5. Section of a vibratome section of a transgenic AD mouse brain stained with A
bN25 (brown) in combination with 6b (blue). Depicted are two enlarged plaques from an entire
section. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
1421
Fig. 6. Fluorescence microscopic images of vibratome sections of transgenic AD mouse brain cortex stained with a 1 mM solution of 6b (left) or 6c (right). Arrows indicate vascular
amyloid.
tracer agents in other organs is of a lesser concern, except for the
clearance of the activity from blood, which should be rapid in order
to increase the target-to-non-target activity ratio. For the three new
11C-labelled phenylbenzothiazoles, the clearance of the activity
from blood (% ID/g blood at 2 min/% ID/g blood at 60 min) is rela-
tively fast, ranging from 6 for the 4-hydroxy derivative [11C]6a to 12
for the 7-hydroxy derivative [11C]6c as compared to 3 for PIB, and is
in the same range as the brain wash-out.
which also showed the fastest blood clearance. This illustrates the
effect of small structural differences on the biological behaviour of
this type of compounds. Furthermore, the log P values of the 5- and
7-hydroxy derivatives [11C]6b and [11C]6c, respectively, are almost
identical, while their biological behaviour is diverging. Although
the log P value of the 4-hydroxy analogue [11C]6a is higher, its in
vivo values for initial brain uptake, hepatobiliary clearance and
urinary clearance always are in between the values of the other two
new compounds.
The studied 11C-labelled phenylbenzothiazoles also show
a moderate uptake in the kidneys and excretion in the urine. The
total activity in the renal system (kidneys þ urine) at 60 min p.i.
varies from 15% ID for the 5-hydroxy derivative [11C]6b to almost
30% ID for the 7-hydroxy derivative [11C]6c. No correlation could be
found between the lipophilicity of these compounds (log P) and
their degree of renal handling. The hepatobiliar excretion to the
intestines is also rather fast. The total activity in the hepatobiliary
system at 60 min p.i. varies from about 59% of ID for the 4-hydroxy
derivative [11C]6a to 78% for the 5-hydroxy derivative [11C]6b,
3. Conclusion
Three structural isomers of 6-hydroxy-2-(40-aminophenyl)-1,3-
benzothiazole have been synthesized and radiolabelled with a 11C-
labelled methyl group. We studied the biological characteristics of
the new tracer agents with a focus on their potential use as PET
probes for in vivo visualization of amyloid
b. The log P values of 5-
hydroxy-2-(40-[11C]methylamino)-1,3-phenylbenzothiazole [11C]6b
Fig. 7. Fluorescence microscopic images after incubation of 8-mm sections of post-mortem human AD brain with 6a. The top left image shows an overview of the stained section.
The top right image is a magnification of a section of the top left picture, showing a NFT. The bottom images are magnifications of plaques.

1422
K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
Table 2
Table 4
Tissue distribution of 4-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole
Tissue distribution of 7-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole
[
11C]6a after i.v. injection in normal mice at 2 and 60 min p.i. (n ¼ 4 at each time
[
11C]6c after i.v. injection in normal mice at 2 and 60 min p.i. (n ¼ 4 at each time
point).
point).
% ID ꢁ s.d.
% ID/g tissue ꢁ s.d.
% ID ꢁ s.d.
% ID/g tissue ꢁ s.d.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
Urine
Kidneys
Liver
Intestines
Cerebrum
Cerebellum
Blood
0.2 ꢁ 0.2
10.6 ꢁ 3.6
21.2 ꢁ 4.1
12.0 ꢁ 1.0
1.2 ꢁ 0.3
22.7 ꢁ 6.3
0.6 ꢁ 0.2
6.4 ꢁ 2.4
52.3 ꢁ 4.6
0.1 ꢁ 0.1
0.0 ꢁ 0.0
2.3 ꢁ 0.9
–
–
Urine
Kidneys
Liver
Intestines
Cerebrum
Cerebellum
Blood
0.58 ꢁ 1.1
9.0 ꢁ 2.6
24.8 ꢁ 2.9
2.5 ꢁ 0.98
7.1 ꢁ 0.59
54.7 ꢁ 2.8
0.05 ꢁ 0.01
0.01 ꢁ 0.01
1.2 ꢁ 0.17
–
–
19.4 ꢁ 6.3
11.1 ꢁ 2.4
–
3.8 ꢁ 0.9
2.7 ꢁ 2.1
6.5 ꢁ 0.5
1.2 ꢁ 0.4
3.7 ꢁ 1.5
–
0.3 ꢁ 0.3
0.6 ꢁ 0.4
1.0 ꢁ 0.4
16.9 ꢁ 4.0
14.5 ꢁ 2.0
–
2.6 ꢁ 0.76
2.8 ꢁ 0.49
4.6 ꢁ 0.40
4.8 ꢁ 1.8
27.8 ꢁ 2.4
9.4 ꢁ 2.2
4.0 ꢁ 0.38
–
0.83 ꢁ 0.23
0.22 ꢁ 0.05
9.8 ꢁ 0.78
0.16 ꢁ 0.03
0.10 ꢁ 0.14
0.57 ꢁ 0.08
0.2 ꢁ 0.1
13.8 ꢁ 1.0
and 7-hydroxy-2-(40-[11C]methylamino)-1,3-phenylbenzothiazole
11C]6c are similar to the value obtained for PIB, whereas that of the
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 60 M, Macherey-Nagel, Du¨ren, Germany) as
the stationary phase or by medium pressure liquid chromatog-
raphy (MPLC). The MPLC-system consisted of a Bu¨chi, model B-680
pump and a column (internal diameter 2.5 cm, length 45 cm) filled
[
4-hydroxy analogue [11C]6a is significantly higher. The possibility of
forming an intramolecular hydrogen bond is assumed to be the
reason for this difference in log P value. The log P values of the 5-
and 7-hydroxy analogues [11C]6b and [11C]6c, respectively, are in
good range for passive diffusion over the BBB, while the log P value
of the 4-hydroxy compound [11C]6a is rather high, which might
lead to residual background activity in the brain because of non-
specific binding to white matter. The affinity of the three structure
with octadecylsilyl silica gel (LiChroprepÒ, 15–25
mm, mean pore
diameter 100 Å, Merck, Darmstadt, Germany). The column was
eluted using mixtures of acetonitrile and 0.05 M ammonium
acetate and the eluate was monitored for UV absorbance at 254 nm
(Econo UV monitor model EM-1, Biorad, Hercules, CA). For thin
layer chromatography (TLC) precoated silica TLC plates were used
(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
spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are
isomers for amyloid
b on the other hand is roughly the same. They
all show suitable binding affinity for post-mortem human AD brain
homogenates as well as affinity to sections of post-mortem trans-
genic AD mouse brain and human AD brain. The 5-hydroxy
analogue [11C]6b seems to have the most favourable in vivo phar-
macokinetics in brain. It shows the highest brain uptake of the
three isomers at 2 min p.i. and its brain wash-out within the 2-min
to 60-min p.i. interval is 8 times faster than that of PIB. Accordingly,
the 5-hydroxy analogue [11C]6b shows a rapid blood clearance, 4
times faster than that of PIB. Significant differences have been
observed in general biodistribution characteristics of the respective
agents, not necessarily in line with their log P value. These differ-
ences indicate that very subtle structural modifications in this class
of ThT-derivatives can have a significant influence on the in vivo
behaviour. Overall, the results indicate that these analogues
deserve further evaluation as potential useful tracer agents for in
vivo visualization of amyloid plaques.
reported as
lane (
d-values (parts per million) relative to tetramethylsi-
¼ 0). Coupling constants are reported in hertz. Splitting
d
patterns are defined by s (singlet), d (doublet), dd (double doublet),
t (triplet) and m (multiplet). Melting points (Mps) were deter-
mined with an IA9100 digital melting point apparatus (Electro-
thermal, Essex, UK) in open capillaries and are reported
uncorrected.
Carbon-11 was produced by irradiation of N2-gas (containing 1%
O2) with 10-MeV protons generated by a Cyclone 10/5 cyclotron
(Ion Beam Applications, Louvain-La-Neuve, Belgium).
Quantitative determination of radioactivity in samples was done
using an automatic gamma counter coupled to a multi-channel
analyzer (Wallac 1480 WizardÒ 300, Wallac, Turku, Finland).
The reversed phase high performance liquid chromatography
(RP-HPLC) system consisted of a Merck–Hitachi ternary gradient
pump (model L-6200A intelligent pump, Merck) and an XTerraÔ RP
4. Experimental protocols
4.1. Materials and general procedures
C18 column (5
m
m, 250 mm ꢃ 4.6 mm) (Waters, Milford, MA, USA).
All reagents and solvents used in synthesis were obtained from
Acros Organics (Geel, Belgium), Aldrich, Fluka or Sigma (Sigma–
Aldrich, Bornem, Belgium) and were used without further purifi-
cation. MgSO4 was used as drying agent. Evaporation of organic
solvents under reduced pressure was done with a Bu¨chi Rotovapor
(Bu¨chi, Flawil, Switzerland). Purification of the reaction mixtures
The eluate was monitored for UV absorbance at 254 nm with a dual
wavelength absorbance detector (Waters 2487) and for radioac-
tivity with a 3-inch NaI(Tl) scintillation detector coupled to a single
channel analyzer (ACEMateÔ amplifier and bias supply, EG&G
ORTEC). The mobile phase consisted of a mixture of acetonitrile and
Table 3
Table 5
Tissue distribution of 5-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-benzothiazole
Tissue distribution of 6-hydroxy-2-(40- [11C]methylaminophenyl)-1,3-benzothiazole
or [11C]PIB after i.v. injection in normal mice at 2 and 60 min p.i. (n ¼ 4 at each time
point).
[
11C]6b after i.v. injection in normal mice at 2 and 60 min p.i. (n ¼ 4 at each time
point).
% ID ꢁ s.d.
% ID/g tissue ꢁ s.d.
% ID ꢁ s.d.
% ID/g tissue ꢁ s.d.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
Urine
Kidneys
Liver
Intestines
Cerebrum
Cerebellum
Blood
0.35 ꢁ 0.23
8.1 ꢁ 1.5
14.6 ꢁ 2.2
0.70 ꢁ 0.70
4.2 ꢁ 1.5
–
–
Urine
Kidneys
Liver
Intestines
Cerebrum
Cerebellum
Blood
0.2 ꢁ 0.1
10.3 ꢁ 3.8
15.6 ꢁ 4.5
12.0 ꢁ 2.4
1.1 ꢁ 0.4
24.4 ꢁ 10.1
4.8 ꢁ 3.9
9.8 ꢁ 2.6
42.5 ꢁ 7.5
0.2 ꢁ 0.1
0.0 ꢁ 0.0
2.7 ꢁ 0.8
–
–
14.2 ꢁ 1.8
12.1 ꢁ 1.7
–
4.3 ꢁ 0.45
4.4 ꢁ 0.54
2.5 ꢁ 0.30
1.2 ꢁ 1.2
19.9 ꢁ 9.1
7.9 ꢁ 2.6
–
3.6 ꢁ 1.4
1.6 ꢁ 0.1
3.1 ꢁ 0.2
9.1 ꢁ 7.1
5.0 ꢁ 1.7
–
0.6 ꢁ 0.2
0.3 ꢁ 0.1
1.1 ꢁ 0.3
25.8 ꢁ 2.4
12.9 ꢁ 2.7
1.3 ꢁ 0.25
0.57 ꢁ 0.08
6.0 ꢁ 0.70
2.2 ꢁ 0.91
73.7 ꢁ 3.3
0.03 ꢁ 0.00
0.01 ꢁ 0.00
0.50 ꢁ 0.06
–
0.09 ꢁ 0.02
0.08 ꢁ 0.07
0.21 ꢁ 0.03
0.2 ꢁ 0.0
7.9 ꢁ 0.5

K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
1423
0.05 M ammonium acetate (unless otherwise stated) and gradient
as well as isocratic elution were used. Data were collected with
a RaChel data acquisition system (Lablogic, Sheffield, UK).
Radio-HPLC combined with mass spectrometry (radio-LC-MS)
was performed on a system consisting of a Waters Alliance 2690
separation module coupled to a reverse phase XTerraÔ MS C18
Accurate MS ESþ m/z [M þ H]þ 287.0493 (calculated for
C14H10N2O3S 287.0485). Mp: 208.5–210.5 ꢀC.
4.2.1.4. 4-Methoxy-2-(40-aminophenyl)-1,3-benzothiazole (4a), method
C. A dispersion of 3a (10.02 g, 35 mmol) and stannous chloride
dihydrate (23.7 g, 105 mmol) in ethanol (400 ml) was refluxed
under nitrogen for 4 h. After cooling to RT, ethanol was removed by
vacuum evaporation. The residue was dissolved in ethyl acetate
(1500 ml) and the organic layer was washed with 2 M sodium
hydroxide (500 ml), water (2 ꢃ 250 ml) and brine (250 ml). After
drying with MgSO4, the solvent was removed by vacuum evapo-
ration. Purification was done using column chromatography with
dichloromethane as eluent. Finally, 5.5 g of 4a was obtained as
3.5
mm column (2.1 mm ꢃ 50 mm) (Waters). The column eluate was
monitored for UV absorbance (Waters 2487 Dual wavelength
absorbance detector) and for radioactivity (3-inch NaI(Tl)-crystal
coupled to a single channel analyzer (The Nucleus, Oak Ridge, TE,
USA)) and then analyzed in a time-of-flight mass spectrometer
(LCT, Micromass, Manchester, England) equipped with an electro-
spray ionization (ESI) source. Acquisition and processing of data
were performed with Masslynx software (version 3.5).
a yellow powder (21 mmol, 60%). 1H NMR (CDCl3, 200 MHz):
d
4.05
3
Gas chromatography was performed with a DI 200 gas chro-
matograph (Delsi Instruments, Suresnes, France) with a Porapak QS
80/100 column (Alltech, Lokeren, Belgium) of 180 cm ꢃ 0.25 inch.
(3H, s, OCH3);
d
6.69 (2H, d, J ¼ 8.8 Hz, 30-H 50-H);
d 6.87 (1H ,d,
3J ¼ 8.2 Hz, 5-H);
d
7.25 (1H, dd, 3J ¼ 8.1 Hz, 4J ¼ 2.2 Hz, 6-H);
d 7.42
(1H, d, 3J ¼ 8.0 Hz, 7-H);
d
7.92 (2H, d, J ¼ 8.8 Hz, 20-H 60-H).
3
Accurate MS ESþ m/z [M þ H]þ 257.0730 (calculated for C14H13N2OS
4.2. Synthesis
257.0743). Mp: 142.5–143 ꢀC.
4.2.1. Synthesis of 4-hydroxy-2-(40-aminophenyl)-1,3-
benzothiazole (5a)
4.2.1.5. 4-Hydroxy-2-(40-aminophenyl)-1,3-benzothiazole (5a), method
D. A dispersion of 4a (3.85 g, 15 mmol) in dichloromethane
(180 ml) was cooled to ꢄ70 ꢀC under nitrogen. Over a period of 1 h,
36 mmol BBr3 (36 ml of a 1 M solution in dichloromethane) was
added slowly, after which the mixture was stirred for another hour
at ꢄ70 ꢀC. The suspension was allowed to warm up to RT and was
stirred for 12 h. The suspension was cooled to ꢄ70 ꢀC and methanol
was added slowly until no more gas evolved. After warming up to
RT, 2 M sodium hydroxide (400 ml) was added. The aqueous layer
was separated and neutralized with 3 M hydrochloric acid. The
precipitate was filtered off and dried in a vacuum oven to yield 3.4 g
4.2.1.1. N-20-Methoxyphenyl-4-nitrobenzamide (1a), method A. A
solution of o-anisidine (24.63 g, 200 mmol) and p-nitrobenzoyl
chloride (37.11 g, 200 mmol) in pyridine (250 ml) was heated under
reflux for 2 h. After cooling to room temperature (RT), the resulting
solution was poured into water. The precipitate was filtered off,
washed with water and dried in a vacuum oven. Crystallization
from methanol yielded 43 g of 1a (158 mmol, 79%) under the form
of light yellow flakes. 1H NMR (DMSO-d6, 200 MHz):
d 3.83 (3H, s,
OCH3);
H);
d
6.98 (1H, dd, 3J ¼ 7.5 Hz, 40-H);
d
7.01 (1H, d, 3J ¼ 8.4 Hz, 30-
d
7.22 (1H, dd, 3J ¼ 7.7 Hz, 50-H);
d
7.71 (1H, d, 3J ¼ 7.6 Hz, 60-H);
of 5a (14 mmol, 93%) as a brown powder. 1H NMR (CDCl3,
3
d
8.17 (2H, d, 3J ¼ 8.8 Hz, 2-H 6-H);
d
8.34 (2H, d, 3J ¼ 7.6 Hz, 3-H 5-
200 MHz):
d
6.67 (2H, d, J ¼ 8.4 Hz, 30-H 50-H);
d
6.84 (1H, d,
H). Accurate MS ESꢄ m/z [M ꢄ H]ꢄ 271.0705 (calculated for
3J ¼ 8.0 Hz, 5-H);
d
7.15 (1H, dd, 3J ¼ 7.9 Hz, 6-H);
d 7.39 (1H, d,
C14H11N2O4 271.0724). Mp: 129–130 ꢀC.
3J ¼ 7.6 Hz, 7-H);
d
7.74 (2H, d, 3J ¼ 8.4 Hz, 20-H 60-H);
d 9.93 (1H, s,
OH). Accurate MS ESþ m/z [M þ H]þ 243.0599 (calculated for
4.2.1.2. N-20-Methoxyphenyl-4-nitrothiobenzamide (2a), method
B. A solution of 1a (40.84 g, 150 mmol) and Lawesson’s reagent
(40.04 g, 90 mmol) in 1,4-dioxane (350 ml) was heated under reflux
for 3 h, after which it was cooled to RT and poured into water. The
precipitate was filtered off, washed with water and dried in
a vacuum oven. Purification was done using column chromatog-
raphy with dichloromethane/hexane (50:50 v/v) as eluent.
Compound 2a was obtained as 34.6 g of a red powder (120 mmol,
C13H11N2OS 243.0587). Mp: 210–212 ꢀC.
4.2.2. Synthesis of 5-hydroxy-2-(40-aminophenyl)-1,3-
benzothiazole (5b)
4.2.2.1. N-(20-Chloro-50-methoxyphenyl)-4-nitrobenzamide
(1b). Compound 1b was synthesized following method A starting
from 6-chloro-m-anisidine hydrochloride (10 g, 52 mmol) and p-
nitrobenzoyl chloride (9.75 g, 52 mmol) in pyridine (50 ml). This
yielded 12.28 g of 1b (40 mmol, 77% yield). 1H NMR (DMSO-d6,
80%). 1H NMR (DMSO-d6, 200 MHz):
d 3.82 (3H, s, OCH3); d 7.03 (1H,
dd, 3J ¼ 7.4 Hz, 40-H);
d
7.16 (1H, d, 3J ¼ 7.8 Hz, 30-H);
d
7.36 (1H, dd,
8.04 (2H, d,
200 MHz):
d
3.85 (3H, s, OCH3);
d
6.70 (1H, dd, 3J ¼ 7.5 Hz, 40-H);
d
7.31
8.21
8.45
3
3
3J ¼ 7.4 Hz, 50-H);
d
7.52 (1H, d, J ¼ 7.4 Hz, 60-H);
d
(1H, d, J ¼ 8.4 Hz, 30-H);
d
8.07 (2H, d, 3J ¼ 8.4 Hz, 2-H 6-H);
8.38 (2H, d, 3J ¼ 7.6 Hz, 3-H 5-H);
d
3J ¼ 8.4 Hz, 2-H 6-H);
d
8.30 (2H, d, 3J ¼ 8.4 Hz, 3-H 5-H). Accurate
(1H, d, J ¼ 8.8 Hz, 60-H);
d
d
3
MS ESꢄ m/z [M ꢄ H]ꢄ 287.0478 (calculated for C14H11N2O3S
(1H, s, NH-CO). Accurate MS ESþ m/z [M þ H]þ 307.0480 (calculated
287.0496). Mp: 132.5–134 ꢀC.
for C14H11ClN2O4 307.0469).
4.2.1.3. 4-Methoxy-2-(40-nitrophenyl)-1,3-benzothiazole(3a). A solu-
tion of 2a (28.83 g, 100 mmol) in a mixture of ethanol (50 ml) and
10% (m/v) sodium hydroxide (300 ml) was slowly added over
a period of 2 h to a solution of potassium ferricyanide (131.73 g,
400 mmol) in water (400 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). The dispersion was filtered off and the filtrate was
concentrated by vacuum evaporation. Purification was done by
recrystallization from methanol/chloroform (75:25 v/v) to yield
13.7 g of 3a as yellow needles (48 mmol, 48%). 1H NMR (CDCl3,
4.2.2.2. N-(20-Chloro-50-methoxyphenyl)-4-nitrothiobenzamide
(2b). Compound 2b was synthesized from 1b (12.28 g, 40 mmol)
following method B. A bright, orange solid was obtained (12 g,
37 mmol, 93%). 1H NMR (DMSO-d6, 200 MHz):
d
3.82 (3H, s, OCH3);
3
3
d
6.83 (1H, dd, J ¼ 7.4 Hz, 40-H);
d
7.38 (1H, d, J ¼ 7.8 Hz, 30-H);
d
7.97 (2H, d, 3J ¼ 7.4 Hz, 2-H 6-H);
d
8.28 (2H, d, 3J ¼ 8.4 Hz, 3-H
5-H);
d
8.51 (1H, s, 6’-H);
d
9.4 (1H, s, NH-CS). Accurate MS ESþ m/z
[M þ H]þ 323.0252 (calculated for C14H11ClN2O3S 323.0240). Mp:
136.2–138.2 ꢀC.
4.2.2.3. 5-Methoxy-2-(40-nitrophenyl)-1,3-benzothiazole (3b), method
E. Sodium (368 mg, 16 mmol) was dissolved in methanol (20 ml),
the solvent was evaporated under reduced pressure and the residue
was added to a solution of 2b (3.23 g, 10 mmol) in N-methyl-2-
pyrrolidone (NMP, 50 ml). The mixture was stirred overnight at
200 MHz):
d
4.11 (3H, s, OCH3);
d
6.97 (1H, d, 3J ¼ 7.9 Hz, 5-H);
d 7.41
(1H, dd, 3J ¼ 8.1 Hz, 6-H);
d
7.52 (1H, d, 3J ¼ 8.0 Hz, 7-H);
d 8.29 (2H,
3
3
d, J ¼ 5.6 Hz, 20-H 60-H);
d
8.31 (2H, d, J ¼ 9.6 Hz, 30-H 50-H).

1424
K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
110 ꢀC and then poured into water (200 ml). The precipitate was
filtered off, dried under reduced pressure and purified using
column chromatography with hexane/ethyl acetate (90:10 v/v) as
eluent. This yielded 150 mg of 3b (0.52 mmol, 5%). 1H NMR (CDCl3,
Accurate MS ESþ m/z [M þ H]þ 257.0723 (calculated for C14H12N2OS
257.0743). Mp: 151.1–154 ꢀC.
4.2.3.5. 7-Hydroxy-2-(40-aminophenyl)-1,3-benzothiazole (5c). Com-
pound 5c was synthesized from 4c (513 mg, 2 mmol) following
method D. The product was purified using MPLC to yield 182 mg of
200 MHz):
d
3.91 (3H, s, OCH3);
d
7.11 (1H, dd, 3J ¼ 9.0 Hz,
4J ¼ 2.4 Hz, 6-H);
d
7.58 (1H, d, 4J ¼ 2.2 Hz, 4-H);
d
7.78 (1H, d,
3J ¼ 8.8 Hz, 7-H);
d
8.21 (2H, d, 3J ¼ 8.8 Hz, 20-H 60-H);
d
8.33 (2H, d,
5c (0.75 mmol, 38%). H NMR (DMSO-d6, 200 MHz):
d
6.67 (2H, d,
7.26 (1H, t,
7.75 (2H, d,
1
3J ¼ 8.8 Hz, 30-H 50-H). Accurate MS ESþ m/z [M þ H]þ 287.0493
3J ¼ 8.4 Hz, 30-H 50-H);
d
6.77 (1H, d, J ¼ 7.8 Hz, 6-H);
d
3
(calculated for C14H10N2O3S 287.0485). Mp: 195–196 ꢀC.
3J ¼ 7.7 Hz, 5-H);
d
7.38 (1H, d, 3J ¼ 7.8 Hz, 4-H);
d
3J ¼ 8.0 Hz, 20-H 60-H). Accurate MS ESþ m/z [M þ H]þ 243.0581
4.2.2.4. 5-Methoxy-2-(40-aminophenyl)-1,3-benzothiazole (4b). Com-
pound 4b was synthesized from 3b (115 mg, 0.4 mmol) following
method C, yielding 77 mg of 4b (0.3 mmol, 75%). 1H NMR (CDCl3,
(calculated for C13H10N2OS 243.0587). Mp: 253.2–255 ꢀC.
4.2.4. Synthesis of 4-hydroxy-2-(40-methylaminophenyl)-1,3-
benzothiazole (6a), method F
200 MHz):
d
3.88 (3H, s, OCH3);
d
6.72 (2H, d, 3J ¼ 8.6 Hz, 30-H 50-H);
d
6.97 (1H, dd, 3J ¼ 8.9 Hz, 4J ¼ 2.6 Hz, 4-H);
d
7.50 (1H, d, 4J ¼ 2.6 Hz,
A solution of 5a (303 mg, 1.25 mmol) and methyl iodide (800 ml,
3-H);
d
7.68 (1H, d, 3J ¼ 8.8 Hz, 5-H);
d
7.87 (2H, d, 3J ¼ 8.4 Hz, 20-H 60-
12 mmol) in 50 ml of acetonitrile was heated at 80 ꢀC for 36 h. The
organic solvent wasremoved under reduced pressure and the residue
was purified using MPLC. This yielded 16 mg of 6a (0.062 mmol, 5%).
H). Accurate MS ESþ m/z [M þ H]þ 257.0725 (calculated for
C14H12N2OS 257.0743). Mp: 108–109 ꢀC.
1H NMR (CDCl3, 200 MHz):
d
3.01 (3H, s, CH3);
d
6.64 (2H, d,
4.2.2.5. 5-Hydroxy-2-(40-aminophenyl)-1,3-benzothiazole (5b). Com-
pound 5b was synthesized from 4b (77 mg, 0.3 mmol) following
method D, yielding 55 mg of 5b (0.23 mmol, 77%). 1H NMR (DMSO-
3J ¼ 8.8 Hz, 30-H 50-H);
d
6.93 (1H, d, 3J ¼ 7.8 Hz, 7-H);
d 7.21 (1H, dd,
3J ¼ 7.9 Hz, 4J ¼ 3.0 Hz, 6-H);
d
7.34 (1H, d, 3J ¼ 8.0 Hz, 5-H);
d 7.89 (2H,
3
d, J ¼ 8.8 Hz, 20-H 60-H). Accurate MS ESþ m/z [M þ H]þ 257.0753
d6, 200 MHz):
d
5.85 (2H, s, NH2);
d
6.66 (2H, d, 3J ¼ 8.4 Hz, 30-H 50-
(calculated for C14H11N2O2S 257.0743). Mp: 193–195 ꢀC.
3
H);
d
6.85 (1H, dd, 3J ¼ 7.9 Hz, 6-H);
d
7.25 (1H, d, J ¼ 8.0 Hz, 4-H);
d
7.72 (1H, d, 3J ¼ 7.6 Hz, 7-H);
d
7.75 (2H, d, 3J ¼ 8.4 Hz, 20-H 60-H);
4.2.5. Synthesis of 5-hydroxy-2-(40-methylaminophenyl)-1,3-
benzothiazole (6b)
d
9.67 (1H, s, OH). Accurate MS ESþ m/z [M þ H]þ 243.0569 (calcu-
lated for C13H10N2OS 243.0587). Mp: 250.5–252.4 ꢀC.
4.2.5.1. 2-Amino-4-methoxy-thiophenol (7). To
a
mixture of
ethylene glycol (23 ml) and 10 M sodium hydroxide (23 ml), 5-
methoxy-2-methyl-benzothiazole (5 g, 28 mmol) was added and
the mixture was refluxed for 2 h under nitrogen. After cooling to RT,
the mixture was neutralized with concentrated hydrochloric acid
and extracted with ethyl acetate (3 ꢃ 50 ml). The organic layer was
dried on MgSO4, filtered and evaporated to dryness under reduced
pressure. The obtained product 7 was used without further
purification.
4.2.3. Synthesis of 7-hydroxy-2-(40-aminophenyl)-1,3-
benzothiazole (5c)
4.2.3.1. N-(20-Bromo-30-methoxyphenyl)-4-nitrobenzamide
(1c). Compound 1c was synthesized following method A starting
from 2-bromo-3-aminoanisole (5.05 g, 25 mmol) and p-nitrobenzoyl
chloride (4.64 g, 25 mmol) in pyridine (100 ml). This yielded 5.02 g of
1c (14.3 mmol, 57%). 1H NMR (DMSO-d6, 200 MHz):
d 3.89 (3H, s,
OCH3);
d
7.07 (1H, d, 3J ¼ 8.2 Hz, 60-H);
d
7.17 (1H, d, 3J ¼ 8.2 Hz, 40-H);
3
d
7.41 (1H, t, J ¼ 8.1 Hz, 50-H);
d
8.21 (2H, d, 3J ¼ 8.4 Hz, 2-H 6-H);
4.2.5.2. 5-Methoxy-2-(40-methylaminophenyl)-1,3-benzothiazole
d
8.39 (2H, d, 3J ¼ 8.8 Hz, 3-H 5-H);
d
10.38 (1H, s, NH–CO). Accurate
(8). A dispersion of
7
(2.4 g, 28 mmol) and 4-(methyl-
MS ESþ m/z [M þ H]þ 352.9951 (calculated for C14H11BrN2O4
amino)benzoic acid (2 g, 28 mmol) in polyphosphoric acid (PPA)
(6 g) was stirred at 180 ꢀC for 3 h. After cooling to RT, 10% (m/v)
sodium carbonate solution (100 ml) was added and the water layer
was extracted with ethyl acetate (2 ꢃ 100 ml). The combined
organic fractions were washed with brine, dried on MgSO4, filtered
and evaporated to dryness under reduced pressure. Purification
was done using column chromatography with hexane/ethyl acetate
(80:20 v/v) as eluent. This yielded 81 mg of 8 (0.3 mmol, 1%). 1H
352.9956). Mp: 242-243.6 ꢀC.
4.2.3.2. N-(20-Bromo-30-methoxyphenyl)-4-nitrothiobenzamide (2c).
Compound 2c was synthesized from 1c (4.56 g, 13 mmol)
following method B, yielding 4.55 g of 2c (12.4 mmol, 95%). 1H
NMR (DMSO-d6, 200 MHz):
d
3.89 (3H, s, OCH3);
d
7.07 (1H, d,
d 7.40 (1H, t,
3
3J ¼ 8.0 Hz, 60-H);
d
7.13 (1H, d, J ¼ 8.0 Hz, 40-H);
3J ¼ 5.4 Hz, 50-H);
d
8.21 (2H, d, 3J ¼ 8.8 Hz, 2-H 6-H);
d
8.38 (2H, d,
NMR (DMSO-d6):
d
2.78 (3H, s, NCH3);
d
3.73 (3H, s, OCH3);
d
4.00
7.06 (1H, d,
d 7.74 (1H, s,
3
3J ¼ 8.8 Hz, 3-H 5-H);
d
10.37 (1H, s, NH-CS). Accurate MS ESþ m/z
(1H, s, NH);
d
6.49 (2H, d, J ¼ 8.0 Hz, 30-H 50-H);
d
[M þ H]þ 368.9739 (calculated for C14H11BrN2O3S 368.9727). Mp:
3J ¼ 7.8 Hz, 6-H);
d
7.26 (2H, d, 3J ¼ 8.8 Hz, 20-H 60-H);
174.5–176 ꢀC.
4-H);
d
8.01 (1H, d, 3J ¼ 7.8 Hz, 7-H). Accurate MS ESþ m/z [M þ H]þ
271.0900 (calculated for C15H14N2OS 271.0879).
4.2.3.3. 7-Methoxy-2-(40-nitrophenyl)-1,3-benzothiazole (3c). Com-
pound 3c was synthesized from 2c (4.04 g, 11 mmol) following
method E, yielding 2.00 g of 3c (7 mmol, 64%). 1H NMR (DMSO-d6,
4.2.5.3. 5-Hydroxy-2-(40-methylaminophenyl)-1,3-benzothiazole
(6b). Compound 6b was synthesized from 8 (70 mg, 0.26 mmol)
following method D, yielding 45 mg of 6b (0.18 mmol, 69%). 1H
200 MHz):
d
3.89 (3H, s, OCH3);
d
7.15 (1H, d, 3J ¼ 8.4 Hz, 6-H);
d 7.58
(1H, t, 3J ¼ 8.1 Hz, 5-H);
d
7.77 (1H, d, 3J ¼ 7.8 Hz, 4-H);
d
8.21 (2H, d,
NMR (DMSO-d6): d 2.75 (3H, s, CH3); d 6.40 (1H, s, NH); d 6.63
3
3J ¼ 8.8 Hz, 20-H 60-H);
d
8.39 (2H, d, 3J ¼ 5.1 Hz, 30-H 50-H). Accurate
(2H, d, J ¼ 8.8 Hz, 20-H 60-H);
d
6.84 (1H, d, 3J ¼ 7.8 Hz, 6-H);
MS ESþ m/z [M þ H]þ 287.0506 (calculated for C14H10N2O3S
d
7.25 (1H, s, 4-H);
d
7.73 (1H, d, 3J ¼ 7.8 Hz, 7-H);
d 7.77 (2H, d,
287.0485). Mp: 199.9–201.3 ꢀC.
3J ¼ 8.0 Hz, 30-H 50-H);
d
9.64 (1H, s, OH). Accurate MS ESþ m/z
[M þ H]þ 257.0743 (calculated for C14H12N2OS 257.0743). Mp:
4.2.3.4. 7-Methoxy-2-(40-aminophenyl)-1,3-benzothiazole (4c). Com-
pound 4c was synthesized from 3c (1.80 g, 6.3 mmol) following
method C, yielding 1.12 g of a yellow solid (3.9 mmol, 62%). 1H NMR
260–261 ꢀC.
4.2.6. Synthesis of 7-hydroxy-2-(40-methylaminophenyl)-1,3-
benzothiazole (6c)
Compound 6c was synthesized from 5c (30 mg, 0.12 mmol)
following method F, yielding 5.7 mg of 6c (0.022 mmol, 18% yield).
(CDCl3, 200 MHz):
d
3.90 (3H, s, OCH3);
d
6.67 (2H, d, 3J ¼ 8.6 Hz, 30-H
50-H);
d
6.77 (1H, d, 3J ¼ 8.0 Hz, 6-H);
d
7.38 (1H, t, 3J ¼ 8.3 Hz, 5-H);
3
3
d
7.63 (1H, d, J ¼ 8.0 Hz, 4-H);
d
7.90 (2H, d, J ¼ 8.4 Hz, 20-H 60-H).

K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
1425
1H NMR (DMSO-d6):
d
2.51 (3H, s, CH3);
d
5.80 (1H, s, NH);
d
d
6.64 (2H,
7.26 (1H,
81.4 TBq/mmol and more than 95% radiochemical purity [16].
Binding assays were carried out in 12 mm ꢃ 75 mm borosilicate
glass tubes. For saturation studies, the reaction mixture contained
d, 3J ¼ 10.9 Hz, 30-H 50-H);
d
6.76 (1H, d, 3J ¼ 9.3 Hz, 6-H);
t, 3J ¼ 7.0 Hz, 5-H);
3J ¼ 10.9 Hz, 4-H);
d
7.38 (2H, d, 3J ¼ 6.2 Hz, 20-H 60-H);
d 7.80 (1H, d,
d
9.30 (1H, s, OH). Accurate MS ESþ m/z [M þ H]þ
50
m
l of post-mortem AD brain homogenate (20–50
mg), 50 ml of
257.0722 (calculated for C14H12N2OS 257.0743). Mp: 245–245.8 ꢀC.
a [125I]IMPY solution in PBS (0.02–0.04 nM) and 40
ml of a solution
of the test compound (6a, 6b and 6c; 10ꢄ7–10ꢄ10 M diluted serially
in PBS containing 0.1% bovine serum albumin) in a final volume of
1 ml. Non-specific binding was defined in the presence of 600 nM
non-radioactive IMPY in the same assay tubes. For the competition
binding studies, concentrations of 10ꢄ5–10ꢄ10 M of the compounds
and 0.06 nM [125I]IMPY were used. The mixtures were incubated at
37 ꢀC for 2 h, and the bound and the free radioactivity were sepa-
rated by vacuum filtration through Whatman GF/B filters using
a Brandel M-24R cell harvester (London, England) followed by two
3-ml washes with PBS at room temperature. Filters containing the
bound iodine-125 ligand were counted in a gamma counter
(Packard 5000, Ramsey, Minnesota, USA) with 70% counting effi-
ciency. Under the assay conditions, the specifically bound fraction
was less than 15% of the total radioactivity. Protein determinations
were performed with Lowry’s method [28] using bovine serum
albumin as a standard. The results of saturation and inhibition
experiments were subjected to non-linear regression analysis using
EBDA by which Kd and Ki values were calculated.
4.3. Radiolabelling
4.3.1. Synthesis of 4-hydroxy-2-(40-[11C]methylaminophenyl)-1,3-
benzothiazole ([11C]6a), 5-hydroxy-2-(40-[11C]methylaminophenyl)-
1,3-benzothiazole ([11C]6b) and 7-hydroxy-2-(40-[11C]
methylaminophenyl)-1,3-benzothiazole ([11C]6c)
With a stream of He, [11C]CH3OSO2CF3 [25] was bubbled
through 0.3 ml of a solution of 5a (3 mg/ml in anhydrous acetoni-
trile) at RT for 30 s. The reaction mixture was diluted with 5 ml of
water and passed over an activated Sep-PakÒ light C18 cartridge
(Waters). The cartridge was rinsed with 5 ml of water and [11C]6a
was eluted from the cartridge with 0.75 ml of methanol, after
which the eluate was applied on an Econosphere C18 column
(250 mm ꢃ 10 mm; 10
mm, Alltech) which was eluted with
an isocratic mixture of 0.05 M ammonium acetate/acetonitrile
(50:50 v/v) at a flow rate of 3 ml/min. The peak containing [11C]6a
was identified using radiometric and UV detection (254 nm), and
isolated. For the synthesis of [11C]6b and [11C]6c, 0.5 ml of a solu-
tion (1 mg/ml in acetone) of 5b or 5c, respectively, was used.
4.6. Binding to amyloid plaques in mouse and human AD
brain sections
4.4. Partition coefficient
4.6.1. Paraffin sections
Transgenic mice overexpressing human amyloid precursor
protein (APP) (London mutation [29]) were anaesthetized with
The lipophilicity of the RP-HPLC isolated 11C-complexes was
determined using a modification of the method described by
Yamauchi and coworkers [26]. To a test tube containing 2 ml of 1-
NembutalÒ (pentobarbital, intraperitoneal injection, 2
ml/g) and the
brain was flushed transcardially with ice-cold saline. The brain was
removed and post-fixed overnight with 4% paraformaldehyde in
PBS. One part of the brain was stored in 0.1% sodium azide in PBS at
4 ꢀC. The other part was processed for paraffin embedding. Sagittal
octanol and 2 ml of 0.025 M phosphate buffer pH 7.4, 25 ml of the
RP-HPLC isolated 11C-complex was added. The test tube was vor-
texed at room temperature for 3 min followed by centrifugation at
2700 g for 10 min. Aliquots of 60 ml and 500 ml were drawn from the
sections (8 mm) were cut using a Microm HM 340 E microtome
1-octanol and buffer phases, respectively, and weighed. The
radioactivity in each aliquot was counted using a gamma counter
and the partition coefficient P was calculated using the following
equation:
(Microm, Walldorf, Germany). Prior to incubation, paraffin sections
were taken through two 5-min washes with xylol, followed by 1-
min sequential washes with 100%, 100%, 90%, 70%, 50% ethanol and
PBS. After rehydration, the sections were incubated for 1 h with one
of the cold compounds (6a, 6b or 6c) at a concentration of 1 mM.
cpm=ml octanol
cpm=ml buffer
P ¼
with cpm ¼ counts per minute
After 1 h, the brain sections were rinsed with tap water and cover-
slipped with Mowiol-DABCO. For the preparation of the Mowiol-
DABCO mixture, 10 g of Mowiol (Calbiochem, Darmstadt, Germany)
and 90 ml of PBS pH 7.4 was stirred for 4–6 h, then 3.25 g DABCO (1,4
diazabicyclo[2.2.2]octane; Sigma–Aldrich) was added followed by
40 ml of glycerin and the mixture was stirred overnight. The
mixture was centrifuged at 15 000 rpm for 30 min, aliquoted and
stored at ꢄ20 ꢀC. The aliquots were thawed before use and can be
refrozen after use. For immunohistochemical staining, the same
deparaffinization protocol was used. The sections were boiled for
10 min in a microwave (Miele M 326 G) with 0.01 M citrate buffer
pH 6 as antigen retrieval. After cooling down for 20 min the sections
were rinsed with PBS followed by treating them with 1.5% H2O2 in
PBS/methanol (50:50 v/v) to inhibit endogenous peroxidase. Non-
specific binding was blocked by treatment with PBS containing
0.05% TweenÒ 20 (PBST) and 10% fetal calf serum (FCS). The sections
Experiments were performed in triplicate.
4.5. Determination of the affinity of the authentic non-radiolabelled
derivatives for post-mortem human AD brain homogenates
4.5.1. Preparation of brain tissue homogenates
Post-mortem brain tissues were obtained from four AD patients
and four age-matched control persons at autopsy, and neuropath-
ological diagnosis was confirmed by current criteria (NIA-Reagan
Institute Consensus Group, 1997). The cerebellum and the affected
temporal or parietal cortex of AD patients together with the cor-
responding regions from control persons were isolated. Gray
matter was carefully separated from white matter from the cortical
tissues. Homogenates were then prepared in phosphate buffered
saline (PBS, pH 7.4) at a concentration of approximately 100 mg
wet tissue/ml (motor-driven glass homogenizer with setting of 6
for 30 s). The homogenates were aliquoted into 1-ml portions and
stored at ꢄ70 ꢀC for 3–6 months without loss of binding signal.
were incubated overnight with pan b-amyloid (AB-1) polyclonal
antibodies (Oncogene Research Products, San Diego, CA, U.S.A.)
diluted 1/1000 in PBST 10% FCS at 4 ꢀC for compound 6a. For
compounds 6b and 6c, the sections were incubated overnight with
A
b
N25 monoclonal antibody, diluted 1/500 in PBST with 10% FCS at
4 ꢀC. After rinsing in PBST, the sections were incubated for 1 h with
1/500 diluted goat anti-mouse biotin (goat anti-rabbit for pan Ab)
4.5.2. Binding studies
Binding assays were carried out according to
a
method
described previously [27] using [125I]IMPY (6-iodo-2-(40-dimethy-
lamino)phenyl-imidazo[1,2]pyridine) with a specific activity of
(Vector Laboratories, Burlingame, CA, U.S.A.) in PBST with 10% FCS.
Then sections were incubated with Avidin Biotin Complex

1426
K. Serdons et al. / European Journal of Medicinal Chemistry 44 (2009) 1415–1426
(Vectastain Elite ABC-peroxidase kit, Vector Laboratories) dissolved
in PBST with 10% FCS for 30 min at room temperature. Peroxidase
activity was developed with 3,30-diaminobenzidine, after which
the sections were counterstained with haematoxylin. Paraffin
embedded human AD brain was stained following the same
procedure as described for mouse brain. AD brain was treated
according to the regulations of the Ethical Committee of the
hospital.
Acknowledgments
The authors wish to acknowledge the help of Christelle Ter-
winghe (Radiopharmacy, UZLeuven, Leuven, Belgium) for per-
forming the biodistribution studies.
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(fentanyl citrate 63
m
g/ml þ fluanisone 2 mg/ml) and injected with
the compound via a tail vein. The mice were sacrificed by decapi-
tation at 2 or 60 min p.i. (n ¼ 4 at each time point). Blood was
collected in a tared tube and weighed. All organs and other body
parts were dissected and weighed and their activity was counted
using a gamma counter. Results were corrected for background
activity and are expressed as percentage of the injected dose per
organ (% ID) or as percentage of the injected dose per gram tissue
(% ID/g). To calculate the activity in the blood, blood mass was
assumed to be 7% of body mass.