Synthesis and evaluation of 11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents.

Article abstract of DOI:10.1021/jm030026b

The synthesis and evaluation of a series of neutral analogues of thioflavin-T (termed BTA's) with high affinities for aggregated amyloid and a wide range of lipophilicities are reported. Radiolabeling with high specific activity [(11)C]methyl iodide provided derivatives for in vivo evaluation. Brain entry in control mice and baboons was high for nearly all of the analogues at early times after injection, but the clearance rate of radioactivity from brain tissue varied by more than 1 order of magnitude. Upon the basis of its rapid clearance from normal mouse and baboon brain tissues, [N-methyl-(11)C]2-(4'-methylaminophenyl)-6-hydroxybenzothiazole (or [(11)C]6-OH-BTA-1) was selected as the lead compound for further evaluation. The radiolabeled metabolites of [(11)C]6-OH-BTA-1 were polar and did not enter brain. The binding affinities of [N-methyl-(3)H]6-OH-BTA-1 for homogenates of postmortem AD frontal cortex and synthetic Abeta(1-40) fibrils were similar (K(d) = 1.4 nM and 4.7 nM, respectively), but the ligand-to-Abeta peptide binding stoichiometry was approximately 400-fold higher for AD brain than Abeta(1-40) fibrils. Staining of AD frontal cortex tissue sections with 6-OH-BTA-1 indicated the selective binding of the compound to amyloid plaques and cerebrovascular amyloid. The encouraging in vitro and in vivo properties of [(11)C]6-OH-BTA-1 support the choice of this derivative for further evaluation in human subject studies of brain Abeta deposition.

Full text of DOI:10.1021/jm030026b

2740
J . Med. Chem. 2003, 46, 2740-2754
Syn th esis a n d Eva lu a tion of 11C-La beled 6-Su bstitu ted 2-Ar ylben zoth ia zoles a s
Am yloid Im a gin g Agen ts
Chester A. Mathis,*,† Yanming Wang,† Daniel P. Holt,† Guo-Feng Huang,† Manik L. Debnath,‡ and
William E. Klunk‡
Departments of Radiology and Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
Received J anuary 16, 2003
The synthesis and evaluation of a series of neutral analogues of thioflavin-T (termed BTA’s)
with high affinities for aggregated amyloid and a wide range of lipophilicities are reported.
Radiolabeling with high specific activity [11C]methyl iodide provided derivatives for in vivo
evaluation. Brain entry in control mice and baboons was high for nearly all of the analogues
at early times after injection, but the clearance rate of radioactivity from brain tissue varied
by more than 1 order of magnitude. Upon the basis of its rapid clearance from normal mouse
and baboon brain tissues, [N-methyl-11C]2-(4′-methylaminophenyl)-6-hydroxybenzothiazole (or
[11C]6-OH-BTA-1) was selected as the lead compound for further evaluation. The radiolabeled
metabolites of [11C]6-OH-BTA-1 were polar and did not enter brain. The binding affinities of
[N-methyl-3H]6-OH-BTA-1 for homogenates of postmortem AD frontal cortex and synthetic
Aâ(1-40) fibrils were similar (Kd ) 1.4 nM and 4.7 nM, respectively), but the ligand-to-Aâ
peptide binding stoichiometry was ∼400-fold higher for AD brain than Aâ(1-40) fibrils. Staining
of AD frontal cortex tissue sections with 6-OH-BTA-1 indicated the selective binding of the
compound to amyloid plaques and cerebrovascular amyloid. The encouraging in vitro and in
vivo properties of [11C]6-OH-BTA-1 support the choice of this derivative for further evaluation
in human subject studies of brain Aâ deposition.
In tr od u ction
entry, but not to the degree typically necessary to
provide a good noninvasive positron emission tomogra-
phy (PET) imaging agent.13 To overcome the low brain
penetration of the styrylbenzenes, we developed neutral
carbon-11-labeled derivatives of thioflavin-T, termed
BTA’s, that displayed both high affinity for aggregated
Aâ and provided the high brain entry necessary for PET
imaging.15,16 Kung and co-workers developed similar
radioiodinated thioflavin-T derivatives as potential Aâ
imaging agents for single photon emission computed
tomography (SPECT), and several of these compounds
(e.g., IBOX and IMPY) show promise for this purpose
with high binding affinities for aggregated Aâ and good
penetration in mouse brain.17,18 Another neutral and
highly lipophilic tracer, fluorine-18-labeled FDDNP, for
binding both amyloid plaques and NFTs has recently
been reported.19,20 The absolute retention of [18F]-
FDDNP demonstrated by time-radioactivity curves in
an AD patient showed cortical areas to exceed the
reference region, the pons, by only 10-30% at late
imaging times, indicating the need to develop improved
Aâ agents with higher signal-to-background ratios for
PET imaging.
The presence and regional abundance of amyloid
plaques and neurofibrillary tangles (NFTs) in human
postmortem brain are the defining characteristics of
Alzheimer’s disease (AD).1-3 Amyloid plaques are com-
prised principally of amyloid-beta (Aâ) peptide fibrils,
while NFTs consist primarily of filaments of hyperphos-
phorylated tau protein. A validated biomarker of amy-
loid deposition in brain would likely prove useful to
identify and follow individuals at risk for AD and to
assist in the evaluation of new antiamyloid therapies
currently under development.4-8 A number of groups
have worked to develop radiolabeled amyloid-specific
imaging agents (Chart 1), but efforts have been limited
by poor brain entry, high levels of nonspecific binding,
or low levels of specific binding in brain regions known
to contain high concentrations of Aâ. We reported that
while Chrysamine G (CG) possessed high affinity for Aâ
fibrils and plaques,9 the penetration of an 125I-labeled
CG derivative into normal rat brain was low.10 Similar
results were reported using a 99mTc-labeled CG deriva-
tive.11 Other more chemically distant analogues of CG
composed of styrylbenzene salicylic acids, such as X-34
(Chart 1), provided similar results of high in vitro
binding affinity to aggregated Aâ and low brain pen-
etration in normal rodent brain.12-14 Further modifica-
tion of the structure by removing the carboxylic acid
groups to provide MeO-X-04 resulted in improved brain
We report here the systematic in vitro and in vivo
evaluation of a series of neutral thioflavin-T derivatives,
whose structures are shown in Figure 1. These com-
pounds are potential PET amyloid imaging agents when
radiolabeled with the 20.4 min half-life, positron-emit-
ting radionuclide carbon-11. Our short-hand nomencla-
ture of these 2-arylbenzothiazole (BTA) compounds has
previously been described.15 Briefly, the 6-position sub-
stituent (R6) is written before “BTA” and the number
of methyl groups on the 4′-amino group is written after
* To whom correspondence should be addressed: PET Facility,
B-938 UPMC Presbyterian, 200 Lothrop Street, Pittsburgh, PA 15213-
2582. Telephone: (412) 647-0736. Fax: (412) 647-0700. E-mail:
mathisca@msx.upmc.edu.
† Department of Radiology.
‡ Department of Psychiatry.
10.1021/jm030026b CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/24/2003

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2741
Ch a r t 1
O-demethylated at the 6-position to afford 19. Similarly,
condensation of the 5-substituted o-aminothiophenols
with p-(dimethylamino)benzaldehyde gave the tertiary
amine compounds 20 and 21. Approach A was more
versatile than approach B, as a wide variety of substi-
tuted anilines were readily available. When a specific
2-amino-6-substituted benzothiazole was readily acces-
sible, approach B was more straightforward, particu-
larly for the synthesis of secondary amine derivatives
as a result of the relatively low monomethylation yields
of primary amines with methyl iodide.
F igu r e 1. Structure of 6-substituted benzothiazole aniline
(BTA) derivatives. Compounds reported here include the
following: R6 ) H, CH3, OCH3, OH, CN, Br; R4′ ) NH2,
NHCH3, N(CH3)2 (total of 18 BTA analogues).
“BTA”. Thus 2-(4′-dimethylaminophenyl)-6-methylben-
zothiazole, a compound with a 6-methyl group and a 4′-
N(CH3)2 group, is referred to as “6-Me-BTA-2” (Chart
1).
Representative examples of 11C-methylation condi-
tions to produce a primary amine ([O-methyl-11C]6-
MeO-BTA-0; [11C]7), a secondary amine ([N-methyl-
11C]6-OH-BTA-1; [11C]19), and a tertiary amine ([N-
methyl-11C]6-MeO-BTA-2; [11C]18) are described in the
Experimental Section and shown in Scheme 2. Radi-
omethylation yields were higher for O-methylation
(about 30% at end of synthesis (EOS)) compared to
N-methylation (about 15% at EOS). This may be at-
tributed to the fact that alkoxide group at the 6-position
generated under basic conditions is a stronger nucleo-
phile than the amino group at the 4′-position. It is
worthwhile noting that no detectable [11C]N-methyla-
tion was observed when 6-OH-BTA-0 was reacted with
[11C]methyl iodide (for about 10 min) in the presence of
potassium carbonate, and only [O-methyl-11C]6-MeO-
BTA-0 was formed in relatively high yield. Thus, forma-
tion of [N-methyl-11C]6-OH-BTA-1 required protection
of the hydroxy group to block [11C]O-methylation at the
6-position.The [11C]N-methylated derivative was achieved
in an overall modest yield following removal of the
MOM-protecting group. For the [11C]N-methylation of
Ch em istr y
Two different approaches were taken to synthesize
the BTA precursors and standard compounds (Scheme
1). In approach A,21 substituted anilines were trans-
formed into p-nitrobenzoyl amides (1 and 2) and sub-
sequently to the corresponding thioamides (3 and 4),
which underwent cyclization to form 2-(4′-nitrophenyl)-
benzothiazoles (5 and 6). Subsequent reduction of nitro
group afforded 2-(4′-aminophenyl)benzothiazoles (7 and
8), from which compounds 9-14 were derived by me-
thylation, demethylation, or aromatic substitution. In
approach B, a series of 5-substituted o-aminothiophenols
(15-17), which were prepared by base hydrolysis of the
corresponding 2-amino-6-substituted-benzothiazoles,22
were reacted with commercially available p-(methy-
lamino)benzoic acid or (dimethylamino)benzaldehyde to
form the secondary and tertiary amine derivatives,
respectively. Thus, condensation of the 5-methoxy-
substituted o-aminothiophenol with p-(methylamino)-
benzoicacidin thepresenceofpolyphosphoricacid23,24provided
the secondary amine 18, which was then selectively

2742 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
Sch em e 1. Synthesis of 6-Substituted 2-(4′-Aminophenyl)benzothiazoles
Sch em e 2. Examples of Radiolabeling Reactions with [11C]Methyl Iodide To Provide
[O-Methyl-11C]2-(4′-aminophenyl)-6-methoxybenzothiazole ([11C]7), [N-Methyl-11C]6-OH-BTA-1 ([11C]19), and
[N-Methyl-11C]2-(4′-dimethylaminophenyl)-6-methoxybenzothiazole ([11C]9). Also Shown Is the Synthesis of the
MOM-Protected Precursor 24 for Subsequent Radiolabeling and Deprotection To Provide [11C]19
6-MOMO-BTA-0, the use of potassium hydroxide re-
sulted in a considerably higher radiochemical yield than
was achieved using potassium carbonate. However,
relatively high N-methylation yields were achieved
using potassium carbonate and nonradiolabeled (cold)
methyl iodide for extended (16 h) reaction times at
elevated temperatures.
Resu lts
Table 1 shows the apparent lipophilicities (log PC18
values) of the eighteen BTA derivatives included in this
study. For the four compounds in which the global
lipophilicities (log Poct) were determined by more stan-
dard shake-flask methods (shown in parentheses in

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2743
Ta ble 1. Lipophilicity (log PC18) and Aâ(1-40) Binding
Affinities (Ki) of 6-Substituted BTA Analogues
different species. Neuroreceptor radioligands that have
been used successfully as PET tracers typically have
(%ID-kg)/g values in the range of 0.1-0.5 within min-
utes after iv injection (e.g., refs 28-32). For mice whose
body weight is about 30 g, this equals a brain concen-
tration range of between 3.3 and 16.6%ID/g. All but two
of the BTA derivatives were within this range at 2 min
after injection. [11C]BTA-1 showed the highest brain
entry with a value of 0.43 (%ID-kg)/g. The highly
lipophilic derivatives 6-Br-BTA-2 and 6-Me-BTA-2
showed the poorest brain entry, with (%ID-kg)/g values
of only 0.054 and 0.078, respectively, and this may be a
result of the phenomenon of blood element binding by
highly lipophilic compounds (log P > 3).25 The 2 min
brain level of the BTA derivatives studied showed the
classic parabolic relationship with log PC18 (Figure 2)
described by Dishino and co-workers.25 The only com-
pound that appeared to not fit a parabolic-type relation-
ship was [11C]BTA-1, which demonstrated a consider-
ably higher brain entry than any of the 12 other 11C-
labeled BTA derivatives.
a
log PC18 R4
Ki (nM) R4′
a
R6
NH2
NHCH3
N(CH3)2 NH2 NHCH3 N(CH3)2
H
CH3
OCH3 1.9
OH
CN
Br
2.0
2.4
2.7 (2.7)b,c
3.1 (3.4)b,c
2.6 (2.7)b
3.4
3.8
3.3
2.0
3.2
4.4
37
9.5
7.0
46
64
7.2
11c
4.0
64
1.9
4.4
11
2.9
10c
4.9
0.66 1.2 (1.3)b
1.8
2.9
4.3
8.6
1.7
2.5
3.6
a
R6 and R4′ refer to substituent positions shown in Figure 1.
b
The numbers in parentheses are log Poct values determined by
conventional octanol-buffer partitioning. c Previously published
values shown here for comparison.16
Table 1), log PC18 correlated closely with log Poct (r )
0.99). The log PC18 values of the BTA derivatives
spanned and exceeded the log Poct range of about 1-3
for compounds expected to enter the brain readily.25,26
It should be noted that the log PC18 values for this group
of BTA derivatives, relative to BTA-0, could be predicted
fairly accurately using the substituent constants devel-
oped by Hansch and Leo.27 For example, addition of an
N-methyl group consistently increased the log PC18 by
0.7 units as predicted by Hansch substituent constants.
The one notable exception was the effect of adding a
hydroxyl group at the 6-position (R6 in Figure 1). Instead
of the expected decrease in log PC18 of 0.7 units, a
decrease of ∼1.4 was consistently observed. This may
be a result of the greater ionization of the phenol in
these BTA compounds compared to the simpler phenols
used by Hansch and Leo in determining their substitu-
ent constants. Using methods described by Klunk and
co-workers,15 the pKa of the 6-OH-BTA-1 phenol was
determined to be 9.3, while the pKa of the (protonated)
+NH2CH3 group of 6-OH-BTA-1 was 3.0.
Experiments to assess brain penetration of the ra-
diolabeled derivatives were performed in young, wild
type Swiss-Webster mice that had no amyloid deposits
in their brain. Thus, this study reflects brain entry and
clearance from normal brain tissue. A necessary crite-
rion for a good PET imaging agent is rapid clearance
from brain areas that do not contain the targeted
binding site, particularly for relatively short-lived 11C-
labeled radioligands. The (%ID-kg)/g values of the BTA
derivatives are shown in Table 2 and indicate that all
but the two most lipophilic compounds, 6-Br-BTA-2 and
6-CH3-BTA-2, provided lower 30 min brain values than
2 min values. The compound with the lowest 30 min
brain (%ID-kg)/g value was [11C]6-OH-BTA-1, with a
more than 3-fold lower concentration than any other
derivative. Another measure of clearance for the BTA
derivatives is shown by the ratio of the 2 min-to-30 min
(%ID-kg)/g values in Table 2. J udged by this measure,
both [11C]BTA-1 and [11C]6-OH-BTA-1 stand out as
particularly promising compounds, with [11C]6-OH-
BTA-1 demonstrating the highest 2 min-to-30 min brain
radioactivity ratio. Interestingly, there was a significant
correlation between log PC18 and the clearance as
expressed by the 2 min-to-30 min ratio (r ) 0.79). The
least lipophilic compounds tended to clear from brain
the fastest (Figure 2), while the most lipophilic com-
pounds accumulated in brain over 30 min.
Table 1 also shows the binding affinities (Ki values)
of the BTA derivatives for aggregated Aâ(1-40) fibrils
in competition with [N-methyl-3H]BTA-1. Affinities
ranged from 1.7 to 64 nM, and all the BTA derivatives
demonstrated much higher affinity for Aâ(1-40) fibrils
than was previously observed for thioflavin-T (Ki ) 580
nM).16 Within a series with the same R6 substituent,
the more lipophilic secondary and tertiary amines were
typically more potent than the primary amines. An
exception to this trend was 6-CH3-BTA-2 [R6 ) CH3; R4′
) N(CH3)2], which appeared much less potent than
predicted. This may be related to the fact that this very
lipophilic compound (log PC18 ) 3.8) was poorly soluble
in the PBS buffer used for the binding assays.
PET imaging studies in baboons were performed with
five different 11C-labeled BTA-1 derivatives including
[11C]BTA-1, [11C]6-OH-BTA-1, [11C]6-MeO-BTA-1, [11C]6-
CN-BTA-1, and [11C]6-Me-BTA-1. All of these radiotrac-
ers were injected in two or more baboon PET imaging
studies to demonstrate consistent findings. Figure 3
Table 2 shows the brain entry of the BTA derivatives
in Swiss-Webster mice in terms of percent injected dose
per gram of brain (%ID/g) normalized to body weight
(in kg) or (%ID-kg)/g. This latter unit can be directly
compared across animals of very different size or across
Ta ble 2. Mouse Brain Entry and Clearance of 6-Substituted BTA Analogues
(%ID-kg)/g in brain at 2 min R4′ (%ID-kg)/g in brain at 30 min R4′
ratio of 2 min/30 min R4′
R6
NH2
NHCH3
N(CH3)2
NH2
NHCH3
N(CH3)2
NH2
NHCH3
N(CH3)2
H
-
-
0.43
0.22
0.33
0.21
0.32
0.12
0.19
0.078
0.16
0.32
0.24
0.054
-
0.057
0.083
0.10
0.018
0.063
0.12
0.078
0.15
0.14
0.10
0.097
0.11
-
-
7.6
2.7
3.2
12
5.0
1.0
2.5
0.52
1.1
3.0
2.5
CH3
OCH3
OH
CN
Br
-
0.32
0.084
3.8
-
-
-
-
-
-
-
-
-
0.49

2744 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
F igu r e 2. (A) Relationship between the brain entry of the 11C-labeled BTA derivatives at 2 min after injection in control mice
(Table 2) and the corresponding log PC18 value, a measure of the lipophilicity of the derivative (Table 1). (B) Relationship between
the ratio of brain radioactivity concentrations ((%ID-kg)/g) at 2 and 30 min after injection (Table 2) and the lipophilicity of the
11C-labeled BTA derivatives (Table 1).
F igu r e 4. PET time-activity curves of the regional distribu-
tion of radioactivity in baboon brain following the injection of
[11C]BTA-1.
F igu r e 3. (top row) Summed PET images (0-9 min after
injection) of the distribution of [11C]BTA-1 in baboon brain at
three different transverse levels. (second row) Coregistered
SPGR MRI anatomical brain images of the same baboon. (third
row) Summed images (0-9 min after injection) of the distribu-
tion of [11C]6-OH-BTA-1 in baboon brain at three different
levels. (bottom row) Coregistered SPGR MRI anatomical brain
images of the same baboon.
shows typical early summed PET images (0-9 min after
injection) in baboons for the two most promising deriva-
tives from the mouse brain entry studies, [11C]BTA-1
and [11C]6-OH-BTA-1, with their corresponding coreg-
istered MR images. At early time points, radioactivity
appeared to be relatively uniformly distributed through-
out the baboon brains, and these results were consistent
with the expected absence of amyloid plaques in the
brains of these control animals. However, time-activity
plots of the regional concentration of radioactivity from
0 to 60 min indicated a heterogeneous distribution of
brain radioactivity that was most apparent at later
times (Figures 4 and 5). Regions of brain containing
F igu r e 5. PET time-activity curves of the regional distribu-
tion of radioactivity in baboon brain following the injection of
[11C]6-OH-BTA-1.
higher levels of white matter (such as pons) contained
20-30% higher concentrations of radioactivity at 60 min
than regions that were dominated by gray matter such
as temporal, mesial-temporal, and occipital cortex. The
regional heterogeneity of radioactivity concentration
was greater for [11C]BTA-1 than for [11C]6-OH-BTA-1,

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2745
F igu r e 7. Comparison of the entry and clearance of radioac-
tivity in baboon cerebellum of four reference PET radioligands
([11C]raclopride, [carbonyl-11C]WAY100635, [11C](+)-McN5652,
and [18F]altanserin) relative to that for [11C]BTA-1 and [11C]6-
OH-BTA-1. The shaded area is the desired target zone bounded
by the cerebellar concentrations of the successful PET neu-
roreceptor radioligands [18F]altanserin and [carbonyl-11C]-
WAY100635. [11C]6-OH-BTA-1 demonstrated fairly rapid clear-
ance of free and nonspecifically bound radioactivity from
baboon cerebellum and was near the upper bound of the target
zone, while [11C]BTA-1 cleared relatively slowly from baboon
cerebellum and was clearly out of the zone.
F igu r e 6. Comparison of the PET time-activity curves of
radioactivity concentration in baboon cortex (summed over
frontal, temporal, parietal, and occipital cortices) following the
injection of five different 11C-labeled BTA derivatives.
and the pons was particularly elevated for [11C]BTA-1
as shown in Figure 4. The concentration of radioactivity
in baboon cortex was nearly identical to that in the
cerebellar cortex at all time points for all of the deriva-
tives studied.
A comparison of typical time-activity curves for the
11C-labeled BTA derivatives in the cortex of baboons is
shown in Figure 6. The maximum brain radioactivity
concentration (%ID-kg/g) in baboons correlated signifi-
cantly with 2 min (%ID-kg)/g values in mice (r ) 0.85),
with the maximum brain concentration of radioactivity
resulting from [11C]BTA-1 > [11C]6-MeO-BTA-1 > [11C]6-
CN-BTA-1 > [11C]6-Me-BTA-1 > [11C]6-OH-BTA-1. The
brain radioactivity concentrations (%ID-kg/g) of [11C]-
BTA-1 and [11C]6-OH-BTA-1 were remarkably similar
in mice and baboons (0.43 vs 0.45 for [11C]BTA-1 and
0.21 vs 0.27 for [11C]6-OH-BTA-1). The rate of clearance
of radioactivity was considerably slower from baboon
brain than from mouse brain, although the rank order
of clearance rate was similar in mice and baboons, with
[11C]6-OH-BTA-1 the fastest clearing and [11C]6-Me-
BTA-1 the slowest clearing derivative. Monoexponential
fits of radioactivity loss from baboon cortices resulted
in the following clearance half-times (t1/2 values): [11C]6-
Me-BTA-1 ) 111 min, [11C]6-MeO-BTA-1 ) 55 min,
[11C]6-CN-BTA-1 ) 51 min, [11C]BTA-1 ) 20 min,
[11C]6-OH-BTA-1 ) 13 min. As in the mice studies,
[11C]6-OH-BTA-1 and [11C]BTA-1 resulted in the highest
ratios of early-to-late brain radioactivity concentrations
in baboons (4.6 and 2.8, respectively). Comparison of the
in vivo behavior of the two lead compounds, [11C]6-OH-
BTA-1 and [11C]BTA-1, in baboon brain to that of the
entry and clearance of other successful PET radioli-
gands in a reference brain region devoid of specific
binding sites (i.e., cerebellum) was helpful in selecting
the lead 11C-labeled BTA derivative to take into human
studies. The cerebellar time-activity curves in baboons
of [11C]raclopride, [carbonyl-11C]WAY100635, [11C](+)-
McN5652, and [18F]altanserin are compared to those of
[11C]6-OH-BTA-1 and [11C]BTA-1 in Figure 7. The
relatively rapid nonspecific binding clearance rates of
[carbonyl-11C]WAY100635, [11C]raclopride, and [18F]-
altanserin are important in the success of these PET
radioligands for imaging the serotonin 5-HT1A, dopam-
ine D2, and serotonin 5-HT2A receptor systems.33-38 In
contrast, the relatively slow in vivo clearance of [11C]-
(+)-McN5652 has limited the usefulness of this radio-
ligand for imaging the serotonin transporter system.39,40
We reasoned that a 11C-labeled BTA compound with
similarly rapid nonspecific binding clearance properties
within the range bracketed by [carbonyl-11C]WAY100635
and [18F]altanserin might prove useful as an in vivo
imaging agent for amyloid plaques. The brain clearance
properties of [11C]6-OH-BTA-1 indicated that the rela-
tively rapid rate of nonspecific clearance of this ra-
diotracer (t1/2 ) 13 min) was similar to that of other
useful PET neuroreceptor imaging agents and those of
[11C]BTA-1 (t1/2 ) 20 min) were too slow (Figure 7).
Clearly, this comparison ignores the specific binding
properties of the 11C-labeled BTA derivatives, which
could be as or more important than the rate of clearance
of free and nonspecifically bound radioactivity from
brain tissues,41 and these in vivo specific binding studies
are currently in progress. For this study, it was only
possible to critically assess the rates of in vivo clearance
of the free and nonspecifically bound radioactivity of the
11C-labeled BTA derivatives in the brains of rodent and
nonhuman primate animal models.
Because of the promising properties of [11C]6-OH-
BTA-1 in the brains of control mice and baboons, we
further evaluated its in vivo and in vitro properties and
compared them to those previously reported for [11C]-
BTA-1.16 Scatchard analysis of [N-methyl-3H]6-OH-
BTA-1 binding to aggregated Aâ(1-40) fibrils showed
a single binding component (Figure 8A) having a Kd of
4.7 nM and a Bmax of 2.7 pmol of 6-OH-BTA-1 per nmol
of Aâ(1-40) peptide. The binding affinity of 6-OH-BTA-1
to homogenates of frontal cortex from postmortem
advanced AD brain also was determined. Again, only a

2746 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
F igu r e 8. (A) Scatchard plot of the binding of [N-methyl-3H]6-OH-BTA-1 to synthetic Aâ(1-40) fibrils. The plot identifies a
single binding component with a Kd of 4.7 nM and a Bmax of 2.7 pmol of 6-OH-BTA-1 bound per nmol of Aâ(1-40). (B) Scatchard
plot of the binding of [N-methyl-3H]6-OH-BTA-1 to homogenates of frontal cortex from postmortem AD brain. The plot identifies
a single binding component with a Kd of 1.4 nM and a Bmax of 1900 pmol/g tissue (1.9 µM), a value consistent with other assay
techniques for Aâ in the frontal cortex of advanced AD subjects.
F igu r e 9. Fluorescence micrographs of sections of AD brain (8 µm thick) stained with 1 µM 6-OH-BTA-1. The section on the left
shows numerous compact plaques stained by 6-OH-BTA-1. The section on the right shows several plaques and cross-sections of
blood vessels laden with cerebrovascular amyloid, also stained by 6-OH-BTA-1 (marked by arrows).
single binding component was evident, which had a
binding affinity (Kd ) 1.4 nM) very similar to that
observed in Aâ(1-40) fibrils. The Bmax of 1900 pmol/g
tissue (or 1.9 µM) was within the range of 1000-3000
pmol/g total Aâ (1-3 µM) found in AD frontal cortex
brain by Naslund and co-workers using biochemical
analysis methods.42 This suggests that the binding
stoichiometry of 6-OH-BTA-1 to Aâ in AD brain is close
to 1:1 under saturating conditions. It is not clear why
the binding stoichiometry of 6-OH-BTA-1 appears to be
considerably lower (∼1:370) for synthetic Aâ(1-40)
fibrils aggregated in vitro than for homogenates of AD
cortical tissue. There was no appreciable specific binding
of [N-methyl-3H]6-OH-BTA-1 to homogenates of frontal
cortex from age-matched control brain (data not shown).
In addition, there was no appreciable specific binding
of [N-methyl-3H]6-OH-BTA-1 to homogenates of cer-
ebellum from age-matched control brain or to homoge-
nates of cerebellum from AD subjects. These findings
are in very good agreement with those previously
reported for [3H]BTA-1 binding to post-mortem AD and
control brain.43 The absence of [N-methyl-3H]6-OH-
BTA-1 binding to cerebellar tissues is consistent with
histopathological findings in postmortem AD brain that
demonstrate the relatively low density of compact Aâ
plaques in the cerebellum.44 This low level of specific
binding in the cerebellum makes this region a good
candidate for a reference tissue for human PET studies.
Additional experiments were performed using the
resources of the NIMH Psychoactive Drug Screening
Program to assess whether 6-OH-BTA-1 interacted with
any of a representative array of neurotransmitter recep-
tors and transporters.45,46 The array has been previously
reported45 and includes adrenergic, serotonergic, mus-
carininc, dopaminergic, opiate, vasopressin, oxytocin,
GABA, glutamate, and imidazoline receptor sites. Using
10 µM 6-OH-BTA-1, no inhibition was observed at any
receptor site.
As a qualitative measure of specificity for amyloid
deposits, nonradiolabeled (cold) 6-OH-BTA-1 was used
to stain paraffin sections of postmortem AD brain
(Figure 9). Like thioflavin-T, nearly all of the BTA
derivatives are fluorescent compounds, and the staining
of 6-OH-BTA-1 was localized to both Aâ plaques and
cerebrovascular amyloid (CVA) in frontal cortex. The
identity of the plaques and CVA stained by 6-OH-BTA-1
was confirmed by staining serial sections with an
antibody to Aâ (data not shown). Aâ plaques and CVA
are both composed predominantly of Aâ peptides.47-52
Note the very low background staining of normal brain
structures.
The peripheral metabolism of the lead compound
[11C]6-OH-BTA-1 in control mice was rapid, with un-
metabolized [11C]6-OH-BTA-1 comprising 73 ( 12% of
total plasma radioactivity at 2 min after injection and
6.4 ( 2.0% at 30 min. All of the radiolabeled plasma

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2747
metabolites of [11C]6-OH-BTA-1 were polar species and
were not expected to enter brain. To confirm this, mice
were injected with [11C]6-OH-BTA-1, and their brains
were removed at 2 and 10 min after injection and
homogenized. Radiolabeled species were extracted from
the homogenate with g94% efficiency using a modified
Folch extraction procedure16 and analyzed using radio-
HPLC. Radioactivity in the homogenates was deter-
mined to be >95% unmetabolized [11C]6-OH-BTA-1,
indicating very low brain entry of radiolabeled metabo-
lites of [11C]6-OH-BTA-1 and little metabolism of the
parent compound in brain parenchyma. Attempts to
assay the brain concentration of radiolabeled [11C]6-OH-
BTA-1 metabolites at 30 min proved unsuccessful as a
result of the very low concentration of radioactivity in
the mouse brain at this time point (Table 2). The
peripheral metabolism of [11C]6-OH-BTA-1 in baboons
was similar to that of mice, with unmetabolized [11C]6-
OH-BTA-1 comprising 85 ( 3% of total plasma radio-
activity at 2 min after injection and 4.3 ( 0.3% after 30
min. The radiolabeled plasma metabolites of [11C]6-OH-
BTA-1 in baboon plasma were polar species and dis-
played similar retention times as the mouse metabolites
on reverse phase HPLC analysis.
in AD. However, it is possible that the concentration of
potential Aâ binding sites in very early or presymptom-
atic AD brain is 1 order of magnitude less than 2 µM
and that some of the Aâ binding sites may not be readily
accessible to radioligand binding when found in compact
cored or neuritic plaques. Thus, compounds with Kd
values <20 nM remain potentially attractive as Aâ
radioligands until in vivo experiments prove otherwise.
This requirement would eliminate only three of the 18
BTA derivatives listed in Table 1.
While the binding affinities of the BTA derivatives
were previously reported to be very similar for both Aâ-
(1-40) fibrils and frontal cortex from AD brain,43 the
Bmax values obtained by binding to Aâ fibrils and AD
brain homogenates suggested that the stoichiometry of
binding was several orders of magnitude different in
these two systems. The very low Bmax value obtained
with [N-methyl-11C]6-OH-BTA-1 binding to synthetic Aâ
fibrils was consistent with the low Bmax observed with
other closely related BTA derivatives such as [N-methyl-
11C]BTA-1 (unpublished data), and the basis for the low
B
max value obtained for BTA derivatives with synthetic
Aâ fibrils is unknown. One possibility is that in vitro
aggregation results in a low percentage of fibrils that
accurately mimic those found in AD brain. This could
explain the observation that the Bmax value of BTA
derivatives determined in homogenates taken from
postmortem AD brain were strikingly higher (∼400-fold)
than for synthetic Aâ(1-40) fibrils and resulted in a
ligand-to-Aâ peptide stoichiometry of about 1-to-1.
The tissue staining findings utilizing nonradiolabeled,
fluorescent 6-OH-BTA-1 are similar to those reported
for BTA-1.43 Both amyloid plaques and cerebrovascular
amyloid were stained by 6-OH-BTA-1 in a manner
similar to serial sections stained with an antibody to
Aâ, and relatively little tissue background staining was
observed. In a qualitative way, these findings support
the specificity of 6-OH-BTA-1 for Aâ deposits, even in
the complex milieu of human brain tissue.
The highest control mouse brain entry at early time
points was achieved with derivatives possessing log PC18
values in the range of 2-3, but the clearance rate of
free and nonspecifically bound radioactivity varied
inversely with the log PC18 values, complicating the
identification of the “optimal” radioligand for use as a
PET tracer for imaging brain amyloid. Two derivatives,
[11C]BTA-1 and [11C]6-OH-BTA-1, clearly demonstrated
superior in vivo clearance rates from mouse brain.
Baboon brain studies provided qualitatively similar
results as the mouse studies, but the clearance rate of
the 11C-labeled derivatives was slower from baboon
brain. The two lead compounds from the mouse brain
studies were also the best compounds in the baboon
brain studies, but [11C]6-OH-BTA-1 exhibited free and
nonspecifically bound radioactivity clearance properties
most similar to other successful PET radioligands.
Equally important, [11C]6-OH-BTA-1 exhibited less
regional heterogeneity in normal baboon brain when
compared to [11C]BTA-1.
Discu ssion
This study was limited to the examination of the
properties of a series of 6-substituted 2-(4′-aminophe-
nyl)benzothiazoles. The parent compound of BTA de-
rivatives (thioflavin-T) possesses a 6-methyl substituent,
and we have reported the properties of several other
6-substituted BTA's including 6-Me-BTA-0, 6-Me-BTA-
1, 6-Me-BTA-2, and BTA-1.15,16 The studies reported
here were intended to more thoroughly explore the
effects of 6-position substitution with polar and lipo-
philic groups on the in vitro and in vivo properties of
the BTA derivatives. The syntheses of the precursors
and standard 6-substituted BTA derivatives were rela-
tively straightforward, and two different synthetic
strategies were employed. Radiolabeling the derivatives
with high specific activity [11C]methyl iodide was readily
accomplished, although the primary amines provided
relatively low radiomethylation yields.
The 18 6-substituted BTA derivatives provided a wide
range of log PC18 values, varying over nearly 4 orders
of magnitude (0.7-4.4) and spanning the log P range
of 1-3 predicted for good brain entry. The use of brain
concentration (%ID/g) normalized to whole body weight
((%ID-kg)/g) proved useful in comparing the brain
penetration of the BTA derivatives across species with
body weights varying about 1000-fold. The binding
affinities of the BTA derivatives for synthetic Aâ(1-40)
ranged from 2 to 64 nM. Conventional wisdom has been
that successful PET radioligands should possess Kd
(binding dissociation constant) values e1 nM. However,
it has been pointed out that this requirement could be
subject to modification for binding targets that are
either in very low or very high concentrations.53 Another
rule of thumb for successful imaging agents is attaining
a Bmax/Kd ratio g10, although a lower ratio may be
sufficient for PET applications.54 Given the high con-
centration of potential Aâ binding sites in AD brain, this
would mean a radioligand with a Kd of about 200 nM (2
µM/200 nM ) 10) could be sufficient for imaging amyloid
Con clu sion
The selection of the most promising agent to take
forward into human PET imaging studies was made
based largely upon the available experimental data

2748 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
eluted with an acetonitrile/aqueous buffer (50 mM triethy-
lammonium (TEA) phosphate buffer, pH 7.2) mobile phase
mixture or a Phenomenex Silica 5 µm 4.6 × 250 mm normal
phase column eluted with an ethyl acetate/hexane/diethy-
lamine (DEA), and both columns were eluted at flow rates of
2.0 mL/min. Both chromatography systems were fitted with a
UV detectors (Waters Model 996 set at 350 nm). For detection
of radiolabeled compounds, a γ-ray detector (Bioscan Flow-
Count fitted with a NaI(Tl) detector) was used in series with
the UV detector. Semipreparative HPLC was performed using
a Phenomenex Prodigy 10 µm ODS-prep 10 × 250 mm reverse
phase column eluted with an acetonitrile/aqueous buffer (50
mM TEA phosphate buffer, pH 7.2) mobile phase mixture, flow
rate varied from 5 to 15 mL/min, and UV (Waters Model 481
set at 350 nm) and γ-ray (Bioscan Flow-Count fitted with a
PIN detector) detectors. Data acquisition for both the analyti-
cal and preparative systems was accomplished using a Waters
Millenium Chromatography System.
obtained from in vitro binding studies and in vivo
studies performed in wild-type mice and normal ba-
boons. The overall encouraging in vitro and in vivo
properties of [11C]6-OH-BTA-1 led to the choice of this
agent (also referred to as Pittsburgh Compound B or
PIB) for further evaluation in human subjects.55 In vivo
studies of both BTA-116 and 6-OH-BTA-156 have been
performed in transgenic mouse models of AD using
multiphoton fluorescence microscopy. The one micron
resolution afforded by multiphoton microscopic imag-
ing57 allows the identification of individual amyloid
plaques and amyloid-laden cerebral vessels. However,
multiphoton fluorescence microscopy requires the ad-
ministration of compounds at doses >1000-fold higher
than those used in PET studies. Nevertheless, the
multiphoton approach constitutes an important comple-
ment to help interpret PET studies that, by their nature,
are performed with spatial resolutions insufficient to
distinguish individual plaques. In vivo microPET stud-
ies of transgenic mouse models of AD with [11C]6-OH-
BTA-1, along with the in vitro and in vivo data described
above, may become important points of reference in the
development of other amyloid-imaging agents labeled
with longer-lived positron-emitting radionuclides, such
as 110 min half-life 18F. Such studies are currently being
initiated as part of our ongoing program to develop
amyloid-imaging compounds.
Syn th esis of P r ecu r sor s a n d Sta n d a r d Sa m p les. 2-(4′-
(Dim eth yla m in o)p h en yl)ben zoth ia zole (BTA-2). A mix-
ture of 2-aminothiophenol (0.5 g, 4.0 mmol), 4-(dimethylamino)-
benzoic acid (0.66 g, 4.0 mmol), and polyphosphoric acid (PPA)
(10 g) was heated to 220 °C for 4 h. The reaction mixture was
cooled to room temperature and poured into 10% potassium
carbonate solution (∼400 mL). The precipitate was collected
by filtration under reduced pressure to give 0.96 g (95%) of
the product, which was ca. 90% pure based upon 1H NMR
analysis. Recrystallization of 100 mg of the product in metha-
nol gave 80 mg of the pure product. Analytical HPLC: reverse
phase (60/40 acetonitrile/triethylammonium (TEA) phosphate
buffer pH 7.2) k′ ) 7.0, purity 95.3%; normal phase (10/90/0.2
ethyl acetate/hexane/diethylamine (DEA)) k′ ) 2.1, purity
95.7%. 1H NMR (300 MHz, acetone-d6) δ: 7.12 (d, J ) 7.7 Hz,
1 H), 7.01 (d, J ) 9.0 Hz, 1 H), 6.98 (d, J ) 9.1 Hz, 2 H), 6.56
(t, J ) 7.8 Hz, J ) 7.3 Hz, 1 H), 5.92 (d, J ) 8.9 Hz, 1 H), 2.50
(s, 6 H). HRMS (EI) m/z calcd for C15H14N2S 254.0878, found
254.0879.
Exp er im en ta l Section
Gen er a l Meth od s. The syntheses of BTA-0 and BTA-1 (R6
) H in Figure 1) and 6-Me-BTA-1 (R6 ) Me), and the
radiosyntheses of [N-methyl-11C]6-Me-BTA-1 and [N-methyl-
11C]BTA-1 were described previously.15,16 Compounds 6-Me-
BTA-0 and 6-Me-BTA-2 were obtained from Aldrich (Milwau-
kee, WI). The purities of BTA-0, BTA-1, 6-Me-BTA-0, 6-Me-
BTA-1, and 6-Me-BTA-2 were determined using reverse phase
and normal phase HPLC (see below), and all five compounds
were determined to be greater than 95% pure. Here we report
the chemical syntheses of the remaining compounds shown
in Figure 1 and the radiolabeling of representative examples
of a primary amine ([O-methyl-11C]6-MeO-BTA-0), a secondary
amine ([N-methyl-11C]6-OH-BTA-1), and a tertiary amine ([N-
methyl-11C]6-CH3O-BTA-2) shown in Scheme 2. The reported
chemical yields were not optimized.
All commercial reagents and solvents were used without
further purification unless otherwise specified. [11C]Methyl
iodide was synthesized according to literature methods using
the reaction of [11C]carbon dioxide with lithium aluminum
hydride followed by hydroiodic acid.58 High-resolution mass
spectra were acquired under electron ionization (EI) conditions
using a double-focusing high-resolution mass spectrometer
(AUTOSPEC, Micromass Inc.), and samples were introduced
by a direct insertion probe. The 1H NMR spectra of all the
compounds were recorded on Bruker 300 or 500 MHz spec-
trometers using tetramethylsilane (TMS) as an internal
standard, and chemical shift (δ) data for the proton resonances
were reported in parts per million (ppm) relative to internal
standard TMS. Elemental analyses of selected compounds
were carried out by Midwest Microlab (Indianapolis, IN) or
Galbraith Laboratories, Inc. (Knoxville, TN). Thin-layer chro-
matography (TLC) was performed using Silica Gel 60 F254
plates from EM Science and visualized by UV light. Flash
chromatography was carried out using Mallinckrodt silica gel
60 (230-400 mesh; column sizes: 54 cm × 2.5 cm, 36.5 cm ×
2.5 cm, or 39.2 cm × 2.2 cm) with hexanes and ethyl acetate
(EtOAc) as eluents with chromatographic solvent proportions
expressed on a volume:volume basis.
Syn th esis of 2-(4′-Am in op h en yl)-6-m eth oxyben zoth ia -
zole (7; 6-MeO-BTA-0). N-(4′-Meth oxyp h en yl)-4-n itr oben -
za m id e (1). To a solution of anisidine (12.3 g, 0.1 mmol) in
pyridine (150 mL) was added 4-nitrobenzoyl chloride (18.5 g,
0.1 mmol). The reaction mixture was heated to reflux for 2 h.
Upon cooling to room temperature, the reaction was quenched
with water. The precipitate was filtered under reduced pres-
sure. The filtrate was washed with 5% sodium bicarbonate and
water and dried at room temperature to give 20.0 g (74%) of
1
N-(4′-methoxyphenyl)-4-nitrobenzamide. H NMR (300 MHz,
DMSO-d6) δ: 10.47 (s, 1 H, NH), 8.37 (d, J ) 8.5 Hz, 2 H),
8.18 (d, J ) 8.4 Hz, 2 H), 7.69 (d, J ) 8.6 Hz, 2 H), 6.96 (d, J
) 8.4 Hz, 2 H), 3.77 (s, 3 H).
N-(4′-Meth oxyp h en yl)-4-n itr oth ioben za m id e (3). To a
solution of N-(4′-methoxyphenyl)-4-nitrobenzamide (1) (20.0 g,
73.5 mmol) in chlorobenzene (50 mL) was added Lawesson’s
reagent (17.8 g, 44 mmol, 0.6 equiv). The reaction mixture was
heated to reflux for 3 h. Upon cooling, the products slowly
precipitated out from the reaction mixture and filtered under
reduced pressure. The filtrate was recrystallized from metha-
nol to give 13.6 g (64%) of N-(4′-methoxyphenyl)-4-nitrothioben-
zamide. 1H NMR (300 MHz, DMSO-d6) δ: 8.29 (d, J ) 8.5 Hz,
2 H), 8.00 (d, J ) 8.4 Hz, 2 H), 7.76 (d, J ) 8.6 Hz, 2 H), 7.03
(d, J ) 8.4 Hz, 2 H), 3.80 (s, 3 H, OCH3).
2-(4′-Nitr op h en yl)-6-m eth oxyben zoth ia zole (5). N-(4′-
Methoxyphenyl)-4-nitrothiobenzamide (3) (13.6 g, 47.2 mmol)
was first wetted with ethanol (∼2 mL), and 30% aqueous
sodium hydroxide solution (15 g, 8 equiv) was added. The
mixture was diluted with water to provide a final suspension
of 10% aqueous sodium hydroxide. Aliquots of this mixture
were added at 1 min intervals to a stirred solution of potassium
ferricyanide (62 g, 4 equiv) in water (200 mL) at 80-90 °C.
The reaction mixture was heated for a further 30 min and then
allowed to cool to room temperature. The participate was
filtered under reduced pressure and washed with water to give
12.5 g of crude product, which was used in next step without
Analytical HPLC was performed using either a Phenomenex
Prodigy 5 µm ODS(3) 4.6 × 250 mm reverse phase column

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2749
1
further purification. H NMR (300 MHz, acetone-d6) δ: 8.36
2-(4′-Am in op h en yl)-6-h yd r oxyben zoth ia zole (11; 6-OH-
BTA-0). To a suspension of 2-(4′-aminophenyl)-6-methoxy-
benzothiazole (7) (500 mg, 1.95 mmol) in CH2Cl2 (20 mL) was
injected BBr3 (1 M in CH2Cl2, 5 mL, 5 mmol). The reaction
mixture was stirred at room temperature for 16 h and then
quenched with 5% HCl. The mixture was neutralized with
saturated NaCO3 and extracted with ethyl acetate (6 × 50 mL).
The organic layers were combined and washed with water and
dried over MgSO4. Evaporation of the solvent gave ca. 500 mg
of the crude product, which was purified by flash chromatog-
raphy (silica gel, hexanes:ethyl acetate ) 2:1) to give 360 mg
(76%) of 2-(4′-aminophenyl)-6-hydroxybenzothiazole as a solid.
Analytical HPLC: reverse phase (35/65 acetonitrile/TEA
phosphate buffer pH 7.2) k′ ) 2.3, purity 99.8%; normal phase
(90/10/0.2 ethyl acetate/hexane/DEA) k′ ) 11.1, purity 99.0%.
1H NMR (300 MHz, Acetone-d6) δ: 7.86 (s, 1H, OH), 6.95 (d,
J ) 9.1 Hz, 2H), 6.91(d, J ) 8.8 Hz, 1H), 6.55 (d, J ) 2.5 Hz,
1 H), 6.17 (dd, J 1 ) 8.8 Hz, J 2 ) 2.5 Hz, 1 H), 5.95 (d, J ) 9.1
Hz, 2 H), 4.45 (s, 2 H, NH2). HRMS (EI) m/z calcd for C13H10N2-
OS 242.0514, found 242.0523.
(m, 4 H), 7.08 (d, J ) 8.5 Hz, 1 H), 7.61 (s, 1 H), 7.13 (d, J )
8.5 Hz, 1H), 3.93 (s, 3 H).
2-(4′-Am in oph en yl)-6-m eth oxyben zoth iazole (7; 6-MeO-
BTA-0). To a solution of 2-(4′-nitrophenyl)-6-methoxyben-
zothiazole (5) (22 mg, 0.077 mmol) in ethanol (10 mL) was
added tin(II) chloride dehydrate (132 mg, 0.45 mmol). The
reaction mixture was refluxed under nitrogen for 4 h. Ethanol
was then evaporated, and the residue was dissolved in ethyl
acetate (10 mL). The resulting solution was washed with 1 N
sodium hydroxide (2 mL) followed by water (5 mL) and dried
over MgSO4. Evaporation of the solvent gave 19 mg (97%) of
the product as yellow solid. Analytical HPLC: reverse phase
(35/65 acetonitrile/TEA phosphate buffer pH 7.2) k′ ) 6.2,
purity 98.8%; normal phase (50/50/0.2 ethyl acetate/hexane/
DEA) k′ ) 2.1, purity 98.7%. 1H NMR (300 MHz, CDCl3) δ:
7.85 (d, J ) 8.7 Hz, 2 H, H-2′ 6′), 7.75 (dd, J 1 ) 8.8 Hz, J 2
)
1.3 Hz, 1 H, H-4), 7.31 (d, J ) 2.4 Hz, 1 H, H-7), 7.03 (dd, J 1
) 8.8 Hz, J 2 ) 2.4 Hz, H-5), 6.56 (d, J ) 7.6 Hz, 2 H, H-3′ 5′),
3.84 (s, 3 H, OCH3). HRMS (EI) m/z calcd for C14H12N2OS
256.0670, found 256.0679.
2-(4′-(Met h yla m in o)p h en yl)-6-h yd r oxyb en zot h ia zole
(19; 6-OH-BTA-1). To a suspension of 2-(4′-methylaminophe-
nyl)-6-methoxybenzothiazole (18) (3.0 g, 10.4 mmol) in CH2-
Cl2 (100 mL) was injected BBr3 (1 M in CH2Cl2, 35 mL, 35
mmol). The reaction mixture was stirred at room temperature
for 16 h. After quenching with water, the reaction mixture was
extracted with ethyl acetate (6 × 100 mL). The organic layers
were combined, washed with water, and dried over MgSO4.
Evaporation of the solvent gave 2.5 g of the crude product,
which was purified by flash chromatography (silica gel, hex-
anes:ethyl acetate ) 1:8, followed by THF and MeOH) to give
2.1 g (75%) of 2-(4′-methylaminophenyl)-6-hydroxybenzothia-
zole. Analytical HPLC: reverse phase (35/65 acetonitrile/TEA
phosphate buffer pH 7.2) k′ ) 7.5, purity 99.6%; normal phase
(90/10/0.2 ethyl acetate/hexane/DEA) k′ ) 10.0, purity 97.9%.
1H NMR (300 MHz, acetone-d6) δ: 7.88 (d, J ) 9.1 Hz, 2 H),
7.73 (d, J ) 8.8 Hz, 1 H), 7.38 (d, J ) 2.5 Hz, 1 H), 6.99 (dd,
J 1 ) 8.8 Hz, J 2 ) 2.5 Hz, 1 H), 6.83 (d, J ) 9.1 Hz, 2 H), 2.83
(d, J ) 9.5 Hz, 3 H). HRMS (EI) m/z calcd for C14H12N2OS
Syn th esis of 2-(4′-(Meth yla m in o)p h en yl)-6-m eth oxy-
ben zoth ia zole (18; 6-MeO-BTA-1). 2-Am in o-5-m eth oxy-
th iop h en ol (15). 2-Amino-6-methoxy-benzothiazole (30.0 g,
172 mmol) was suspended in 50% KOH (180 g KOH dissolved
in 180 mL water) and ethylene glycol (40 mL). The suspension
was heated to reflux for 48 h. Upon cooling to room temper-
ature, toluene (300 mL) was added and the reaction mixture
was neutralized with acetic acid (180 mL). The organic layer
was separated, and the aqueous layer was extracted with
another 200 mL of toluene. The toluene layers were combined
and washed with water and dried over MgSO4. Evaporation
of the solvent gave 15.3 g of 2-amino-5-methoxythiophenol as
1
yellow solid. H NMR (300 MHz, acetone-d6) δ: 6.66 (d, J )
2.6 Hz, 1 H), 6.57 (d, J ) 8.5 Hz, 1 H), 6.43 (dd, J 1 ) 2.6 Hz,
J 2 ) 8.5 Hz, 1 H), 3.77 (s, 3 H).
2-(4′-(Met h yla m in o)p h en yl)-6-m et h oxyb en zot h ia zole
(18; 6-MeO-BTA-1). 4-Methylaminobenzoic acid (11.5 g, 76.2
mmol) and 2-amino-5-methoxythiophenol (15) (12.5, g, 80
mmol) were mixed together with PPA (∼30 g) and heated to
170 °C under N2 atmosphere for 1.5 h. The reaction mixture
was cooled to room temperature and poured into 10% K2CO3
solution. The precipitate was filtered under reduced pressure.
The filtrate was first purified by recrystallization from acetone/
water followed by treatment with actived charcoal in THF/
water gave 4.6 g (21%) of 2-(4′-methylaminophenyl)-6-meth-
oxybenzothiazole as a yellow solid. Analytical HPLC: reverse
phase (35/65 acetonitrile/TEA phosphate buffer pH 7.2) k′ )
11.9, purity 98.8%; normal phase (10/90/0.2 ethyl acetate/
hexane/DEA) k′ ) 14.7, purity 98.5%. 1H NMR (300 MHz,
acetone-d6) δ: 7.84 (d, J ) 9.0 Hz, 2 H), 7.80 (d, J ) 9.0 Hz,
1 H), 7.51 (d, J ) 3.0 Hz, 1 H), 7.05 (dd, J 1 ) 9.0 Hz, J 2 ) 3.0
Hz, 1 H), 6.70 (d, J ) 9.0 Hz, 2 H), 3.88 (s, 3 H), 2.85 (d, J )
6.0, 3 H). HRMS (EI) m/z calcd for C15H14N2OS 270.0827, found
270.0828.
256.0670, found 256.0661. Anal. Calcd for C14H12N2OS:
C
65.60; H 4.72; N 10.93; S 12.51. Found: C 65.26; H 5.15; N
10.11; S 11.52.
2-(4′-(Dim et h yla m in o)p h en yl)-6-h yd r oxyb en zot h ia z-
ole (10; 6-OH-BTA-2). To a solution of 2-(4′-dimethyami-
nophenyl)-6-methoxybenzothiazole (9) (3 mg) in CH2Cl2 (an-
hydrous, 3 mL) was added BBr3 (200 µL, 1.0 M CH2Cl2
solution) was injected at -78°C under N2 atmosphere. The
reaction mixture was allowed to warm slowly and stirred at
room temperature for another 16 h. After water was added,
the reaction mixture was stirred for another 0.5 h. The organic
layer was separated, and the aqueous layer was extracted with
CH2Cl2 (3 × 5 mL). The organic layers were combined and
dried with MgSO4. The solvent was evaporated and the residue
was purified with preparative TLC to give 1 mg (34%) of 2-(4′-
dimethylaminophenyl)-6-hydroxybenzothiazole. Analytical
HPLC: reverse phase (35/65 acetonitrile/TEA phosphate buffer
pH 7.2) k′ ) 14.3, purity 99.5%; normal phase (65/35/0.2 ethyl
acetate/hexane/DEA) k′ ) 12.6, purity 98.8%. 1H NMR (300
MHz, DMSO) δ: 9.58 (s, 1 H, OH), 7.85 (d, J ) 8.5 Hz, 2 H),
7.71 (d, J ) 8.7 Hz, 1 H), 7.33 (s, 1 H), 6.95 (d, J ) 8.7 Hz, 1
H), 6.82 (d, J ) 8.5 Hz, 2 H), 3.04 (s, 6 H). HRMS (EI) m/z
calcd for C15H14N2OS 270.0827, found 270.0828.
2-(4′-(Dim et h yla m in o)p h en yl)-6-m et h oxyb en zot h ia z-
ole (9; 6-MeO-BTA-2). To a solution of 2-(4′-aminophenyl)-
6-methoxybenzothiazole (7) (150 mg, 0.59 mmol) in DMSO
(anhydrous, 2 mL) were added methyl iodide (297 mg, 2.09
mmol) and K2CO3 (500 mg, 3.75 mmol). The reaction mixture
was heated at 140 °C for 16 h. Upon cooling to room temper-
ature, the reaction mixture was poured into water and
extracted with ethyl acetate (3 × 10 mL). The organic layers
were combined, and the solvent was evaporated. The residue
was purified by flash column (silica gel, hexanes:ethyl acetate
) 9:1) to give 63 mg (38%) of 2-(4′-dimethylaminophenyl)-6-
methoxybenzothiazole. Analytical HPLC: reverse phase (60/
40 acetonitrile/TEA phosphate buffer pH 7.2) k′ ) 6.3, purity
99.8%; normal phase (10/90/0.2 ethyl acetate/hexane/DEA) k′
Syn th esis of 2-(4′-Am in op h en yl)-6-br om oben zoth ia z-
ole (8; 6-Br -BTA-0). N-(4′-Br om op h en yl)-4-n itr oben za -
m id e (2). To a solution of 4-bromoaniline (1.0 g, 5.8 mmol) in
benzene (20 mL) was added dropwise a solution of 4-nitroben-
zoyl chloride (1.08 g, 5.8 mmol) in benzene (20 mL). The
reaction mixture was stirred at room temperature for 23 h.
The precipitate was filtered under reduced pressure. The
filtrate was washed with water thoroughly and recrystallized
from ethanol to give 1.6 g (86%) of N-(4′-bromophenyl)-4-
nitrobenzamide as light yellow solid. 1H NMR (300 MHz,
1
) 3.4, purity 95.3%. H NMR (300 MHz, acetone-d6) δ: 7.85
(d, J ) 8.9 Hz, 2 H), 7.74 (d, J ) 8.8 Hz, 1 H), 7.49 (d, J ) 2.4
Hz, 1 H), 7.15 (dd, J 1 ) 8.8 Hz, J 2 ) 2.4 Hz, 1 H), 6.78 (d, J )
8.9 Hz, 2 H), 3.83 (s, 3 H), 3.01 (s, 6 H). HRMS (EI) m/z calcd
for C16H16N2OS 284.0983, found 284.0985.

2750 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
DMSO-d6) δ: 10.69 (s, NH), 8.37 (d, J ) 8.8 Hz, 2 H), 8.18 (d,
J ) 8.8 Hz, 2 H), 7.77 (d, J ) 8.6 Hz, 2 H), 7.57 (d, J ) 8.6 Hz,
2 H).
of the solvent gave a residue that was purified by flash column
(hexanes:ethyl acetate ) 4:1) to give 179 mg (54%) of the
product. Analytical HPLC: reverse phase (60/40 acetonitrile/
TEA phosphate buffer pH 7.2) k′ ) 17.8, purity 99.8%; normal
phase (3/97/0.2 ethyl acetate/hexane/DEA) k′ ) 3.4, purity
99.4%. 1H NMR (300 MHz, acetone-d6) δ: 8.20 (d, J 1 ) 2.0
Hz, 1 H), 7.95 (d, J ) 8.8 Hz, 2 H), 7.83 (d, J ) 8.8 Hz, 1 H),
7.60 (dd, J 1 ) 8.8 Hz, J 2 ) 2.0 Hz, 1 H), 6.84 (d, J ) 8.8 Hz,
2 H), 3.08 (s, 6H). HRMS (EI) m/z calcd for C15H13N2SBr
331.9983, found 331.9970.
N-(4′-Br om op h en yl)-4-n itr oth ioben za m id e (4). To a
solution of N-(4′-bromophenyl)-4-nitrobenzamide (2) (2.8 g, 8.7
mmol) in chlorobenzene (5 mL) was added Lawesson’s reagent
(2.4 g, 5.9 mmol). The reaction mixture was heated at 120 °C
under N2 atmosphere for 6 h. Upon cooling to room temper-
ature, an orange solid precipitated out from the reaction
mixture. The precipitate was filtered and washed with chlo-
robenzene (1 mL). The filtrate was recrystallized from metha-
nol to give 2.1 g (71%) of N-(4′-bromophenyl)-4-nitrothioben-
2-(4′-Am in op h en yl)-6-cya n oben zoth ia zole (12; 6-CN-
BTA-0). To a solution of 6-bromo-2-(4′-aminophenyl)ben-
zothiazole (8) (27 mg, 0.088 mmol) in 1-methylpyrilidinone (1
mL) was added CuCN (65 mg, 0.71 mmol). The reaction
mixture was heat to 200 °C for 18 h. After the mixture was
cooled to room temperature, water (5 mL) was added followed
by ethylenediamine (0.3 mL). The reaction mixture turned to
dark blue and was extracted with ethyl acetate (6 × 10 mL).
The organic layers were combined, washed with water, and
dried over MgSO4. Evaporation of the solvent afforded a
residue, which was purified by preparative TLC (hexanes:ethyl
acetate ) 2:1) to give 15 mg (68%) of 2-(4′-aminophenyl)-6-
cyanobenzothiazole. Analytical HPLC: reverse phase (35/65
acetonitrile/TEA phosphate buffer pH 7.2) k′ ) 12.0, purity
99.4%; normal phase (50/50/0.2 ethyl acetate/hexane/DEA) k′
1
zamide. H NMR (300 MHz, DMSO-d6) δ: 12.18 (s, NH), 8.31
(d, J ) 8.5 Hz, 2 H), 8.03 (d, J ) 8.5 Hz, 2 H), 7.86 (d, J ) 8.6
Hz, 2 H), 7.66 (d, J ) 8.6 Hz, 2 H).
2-(4′-Nitr op h en yl)-6-br om oben zoth ia zole (6). N-(4′-Bro-
mophenyl)-4-nitro-thiobenzamide (4) (1.0 g, 2.97 mmol) was
first wetted with ethanol (∼0.5 mL), and 30% aqueous sodium
hydroxide solution (950 mg 23.8 mmol, 8 equiv) was added.
The mixture was diluted with water to provide a final
suspension of 10% aqueous sodium hydroxide. Aliquots of this
mixture were added at 1 min intervals to a stirred solution of
potassium ferricyanide (3.9 g, 11.9 mmol, 4 mol equiv) in water
(10 mL) at 80-90 °C. The reaction mixture was heated for
another 0.5 h. After cooled to room temperature, the precipi-
tate was filtered under reduced pressure and washed with
water. The crude product (0.88 g) was recrystallized from
methanol/acetone to give 352 mg (35%) of the 2-(4-nitrophe-
1
) 3.0, purity 95.9%. H NMR (300 MHz, acetone-d6) δ: 8.58
(d, J ) 1.7 Hz, 1H), 7.98 (d, J ) 8.5 Hz, 1 H), 7.82 (dd, J 1
)
1.7 Hz, J 2 ) 8.5 Hz, 1 H), 7.79 (d, J ) 10.1 Hz, 2 H), 6.65 (d,
J ) 10.1 Hz, 2 H). HRMS (EI) m/z calcd for C14H9N3S 251.0517,
found 251.0519.
1
nyl)-6-bromobenzothiazole. H NMR (300 MHz, DMSO-d6) δ:
8.56 (d, J ) 1.9 Hz, 1 H), 8.41 (d, J ) 8.5 Hz, 2 H), 8.37 (d, J
) 8.5 Hz, 2 H), 8.09 (d, J ) 8.7 Hz, 1 H), 7.76 (dd, J 1 ) 2.0 Hz,
J 2 ) 8.7 Hz, 1 H).
2-(4′-(Meth yla m in o)p h en yl)-6-cya n oben zoth ia zole (14;
6-CN-BTA-1). To a solution of 2-(4′-methylaminophenyl)-6-
bromobenzothiazole (13) (54 mg, 0.17 mmol) in 1-methylpy-
rilidinone (1 mL) was added CuCN (110 mg, 1.4 mmol). The
reaction mixture was heat to 170 °C for 17 h. Upon cooling to
room temperature, the mixture was diluted with water and
the product precipitated out. The precipitate was filtered under
reduced pressure and purified by preparative TLC (hexanes:
ethyl acetate ) 3:2) to give 25 mg (67%) of 6-cyano-2-(4′-
methylaminophenyl)benzothiazole. Analytical HPLC: reverse
phase (60/40 acetonitrile/TEA phosphate buffer pH 7.2) k′ )
3.3, purity 99.2%; normal phase (25/75/0.2 ethyl acetate/
hexane/DEA) k′ ) 4.7, purity 97.5%. 1Η ΝΜR (300ΜΗ,
actetone-d6) δ: 8.17(d, J ) 1.6 Hz, 1 H), 8.04 (d, J ) 7.9 Hz,
1 H), 7.96 (d, J ) 8.8 Hz, 2 H), 7.70 (dd, J 1 ) 1.6 Hz, J 2 ) 7.9
Hz, 1 H), 6.68 (d, J ) 8.8, 2 H), 2.97 (s, 3 H). HRMS (EI) m/z
calcd for C15H11N3S 265.0674, found 265.0668.
2-(4′-Am in op h en yl)-6-b r om ob en zot h ia zole (8; 6-Br -
BTA-0). To a solution of 2-(4-nitrophenyl)-6-bromobenzothia-
zole (32 mg, 0.1 mmol) in ethanol (15 mL) was added tin(II)
chloride dehydrate (165 mg, 0.8 mmol). The reaction mixture
was refluxed under nitrogen for 1.5 h. After ethanol was
evaporated, the residue was dissolved in ethyl acetate (10 mL),
washed with 1 N sodium hydroxide (2 mL) and water (5 mL),
and dried over MgSO4. Evaporation of the solvent gave a
residue, which was purified with preparative TLC (hexanes:
ethyl acetate ) 70:30) to give 11 mg (38%) of 2-(4′-aminophe-
nyl)-6-bromobenzothiazole as yellow solid. Analytical HPLC:
reverse phase (60/40 acetonitrile/TEA phosphate buffer pH 7.2)
k′ ) 4.5, purity 99.2%; normal phase (25/75/0.2 ethyl acetate/
hexane/DEA) k′ ) 9.0, purity 95.3%. 1H NMR (300 MHz,
DMSO-d6) δ: 8.28 (d, J 1 ) 1.7 Hz, 1 H), 7.80 (d, J ) 8.6 Hz,
1 H), 7.73 (d, J ) 8.5 Hz, 2 H), 7.57 (dd, J 1 ) 8.6 Hz, J 2 ) 1.7
Hz, 1 H), 6.65 (d, J ) 8.5 Hz, 2 H), 5.96 (s, 2 H). HRMS (EI)
m/z calcd for C13H9N2SBr 303.9670, found 303.9665.
2-(4′-(Meth yla m in o)p h en yl)-6-br om oben zoth ia zole (13;
6-Br -BTA-1). To a solution of 2-(4′-aminophenyl)-6-bromoben-
zothiazole (8) (4 mg, 0.013 mmol) in DMSO (anhydrous, 0.5
mL) was added methyl iodide (3.9 mg, 0.027 mmol) and
anhydrous K2CO3 (100 mg, 0.75 mmol). The reaction mixture
was heated at 100 °C for 16 h. Upon cooling to room temper-
ature, the reaction mixture was directly purified by normal
phase preparative TLC (hexanes ethyl acetate ) 7:3) to give
2.0 mg (50%) of 6-bromo-2-(4′-methylaminophenyl)benzothia-
zole. Analytical HPLC: reverse phase (60/40 acetonitrile/TEA
phosphate buffer pH 7.2) k′ ) 8.7, purity 99.1%; normal phase
(10/90/0.2 ethyl acetate/hexane/DEA) k′ ) 9.2, purity 96.2%.
1H NMR (300 MHz, acetone-d6) δ: 8.19 (d, J 1 ) 2.0 Hz, 1 H),
7.09 (d, J ) 8.8 Hz, 2 H), 7.81 (d, J ) 8.8 Hz, 1 H), 7.59 (dd,
J 1 ) 8.8 Hz, J 2 ) 2.0 Hz, 1 H), 6.70 (d, J ) 8.8 Hz, 2 H), 5.79
(s, 1 H), 3.17 (s, 3 H).
2-(4′-((Dim et h yla m in o))p h en yl)-6-cya n ob en zot h ia z-
ole (21; 6-CN-BTA-2). To a solution of 2-amino-5-cyan-
othiophenol (17) (30 mg, 0.5 mmol) in DMSO (1 mL) was added
4-(dimethylamino)benzaldehyde (30 mg, 0.5 mmol). The reac-
tion mixture was heated to 170 °C for 20 min and cooled to
room temperature. After it was poured into water, the mixture
was extracted with ethyl acetate (3 × 10 mL). The organic
layers were combined, washed with water, and dried over
MgSO4. Evaporation of the solvent afforded a residue, which
was purified by preparative TLC (hexanes:ethyl acetate ) 4:1)
to give 9 mg (15%) of 2-(4′-dimethylaminophenyl)-6-cyanoben-
zothiazole. Analytical HPLC: reverse phase (60/40 acetonitrile/
TEA phosphate buffer pH 7.2) k′ ) 7.3, purity 98.7%; normal
phase (25/75/0.2 ethyl acetate/hexane/DEA) k′ ) 1.7, purity
1
99.3%. H NMR (300 MHz, DMSO) δ: 8.85 (d, J ) 1.6 Hz, 1
H), 8.41 (s, 4 H), 8.30 (d, J ) 8.5 Hz, 1 H), 8.00 (dd, J 1 ) 1.6
Hz, J 2 ) 8.5 Hz, 1 H), 3.35 (s, 6 H). HRMS (EI) m/z calcd for
C
16H13N3S 279.0830, found 279.0822.
Ra d iosyn th esis of Rep r esen ta tive 6-Su bstitu ted BTA
2-(4′-(Dim eth yla m in o)p h en yl)-6-br om oben zoth ia zole
(20; 6-Br -BTA-2). A mixture of 2-amino-5-bromothiophenol
(16) (204 mg, 1.0 mmol) and 4-(dimethylamino)benzaldehyde
(149 mg, 1.0 mmol) in DMSO (1 mL) was heated to 170 °C for
20 min. The reaction mixture was cooled to room temperature
and poured into water. The organic component was extracted
with ethyl acetate (3 × 10 mL). The combined organic layers
were washed with water and dried over MgSO4. Evaporation
An a logu es. [O-Meth yl-11C]2-(4′-a m in op h en yl)-6-m eth oxy-
ben zoth ia zole ([11C]7; [11C]6-MeO-BTA-0). The radiolabel-
ing precursor, 6-OH-BTA-0 (11) (1.0 mg, 4.1 µmoles), was
dissolved in 400 µL of DMSO. Dry K2CO3 (∼5 mg) was added,
and the 3 mL V-vial was vortexed for 5 min. No-carrier-added
[11C]methyl iodide was bubbled through the solution at 30 mL/
min at room temperature. The reaction was heated for 5 min
at 95°C using an oil bath. The reaction product was purified

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2751
by semipreparative HPLC using an ODS-Prep column eluted
with 35/65 acetonitrile/triethylammonium phosphate buffer pH
7.2 (flow at 5 mL/min for 0-13 min, increased to 10 mL/min
for 13-18 min, then increased to 15 mL/min for 18-30 min).
[O-Methyl-11C]6-MeO-BTA-0 eluted at about 24 min. No
[N-methyl-11C]6-OH-BTA-1 side-product was detected under
these reaction conditions. The fraction containing [O-methyl-
11C]6-MeO-BTA-0 was diluted with 50 mL of water and eluted
through a Waters C18 SepPak Plus cartridge. The C18 SepPak
was washed with 10 mL of water, and the product was eluted
with 1 mL of ethanol (absolute) into a sterile vial followed by
14 mL of saline. Radiochemical and chemical purities were
>95% as determined by analytical HPLC (k′ ) 6.2 using the
ODS(3) analytical column eluted with 35/65 acetonitrile/
triethylammonium phosphate buffer pH 7.2). The radiochemi-
cal yield averaged 28.2% at end of synthesis (EOS) based on
[11C]methyl iodide, and the specific activity averaged 210 GBq/
µmol (5.7 Ci/µmol) at EOS.
determine percent incorporation for [N-methyl-11C]6-MOMO-
BTA-1 (k′ ) 6.6 for 6-MOMO-BTA-1 on the Prodigy ODS(3) 5
µm analytical column eluted with 50/50 acetonitrile/triethy-
lammonium phosphate buffer pH 7.2). A solution of MeOH/
HCl (500 µL, 2/1 MeOH/concentrated HCl) was added to the
V-vial, and the reaction was heated an additional 5 min at
125°C. The reaction was neutralized by adding 1 mL of 2.0 M
sodium acetate and purified by semipreparative HPLC using
a Prodigy ODS-Prep column eluted with 35% acetonitrile/65%
triethylammonium phosphate buffer pH 7.2 (flow at 5 mL/min
for 0-2 min then increased to 15 mL/min for 2-30 min). [11C]6-
HO-BTA-1 eluted at about 11.5 min. The fraction containing
[11C]6-OH-BTA-1 was diluted with 50 mL of water and eluted
through a Waters C18 SepPak Plus cartridge. The C18 SepPak
was washed with 10 mL of water, and the product was eluted
with 1 mL of ethanol (absolute) into a sterile vial followed by
14 mL of saline. Radiochemical and chemical purities were
>95% as determined by analytical HPLC (k′ ) 7.5 using the
Prodigy ODS(3) analytical column eluted with 35/65 acetoni-
trile/triethylammonium phosphate buffer pH 7.2). The radio-
chemical yield averaged 12.1% at EOS based on [11C]methyl
iodide, and the specific activity averaged 85 GBq/µmol (2.3 Ci/
µmol) at EOS.
[N-Meth yl-11C]2-(4′-(m eth yla m in o)p h en yl)-6-h yd r oxy-
ben zoth ia zole ([11C]19;[11C]6-OH-BTA-1). 2-(4′-Am in op h e-
n yl)-6-m eth oxym eth oxyben zoth iazole (24; 6-MOMO-BTA-
0). 2-(4′-Nitr op h en yl)-6-h yd r oxyben zoth ia zole (22). To a
suspension of 2-(4′-nitrophenyl)-6-methoxybenzothiazole (5)
(5.0 g, 18.7 mmol) in CH2Cl2 (150 mL) was injected BBr3 (1 M
in CH2Cl2, 50 mL, 50 mmol). The reaction mixture was stirred
at room temperature for 24 h. The reaction mixture was
quenched with water and extracted with ethyl acetate (3 ×
100 mL). The extracts were combined and washed with water,
dried over MgSO4, and evaporated. The residue was purified
by flash column (silica gel, hexanes:ethyl acetate ) 4:1) to give
[N-Meth yl-3H]2-(4′-(Meth yla m in o)p h en yl)-6-h yd r oxy-
ben zoth ia zole ([3H]19; [3H]6-OH-BTA-1). The radiosynthe-
sis of [3H]6-OH-BTA-1 was accomplished in a manner similar
to that of [11C]6-OH-BTA-1 given above, except that 6.0 GBq
of [3H]CH3I were utilized to radiomethylate 6-MOMO-BTA-0
(2 mg) rather than [11C]CH3I. The final product was obtained
in a 15% radiochemical yield with a radiochemical purity >96%
and a specific activity of 2.1 GBq/µmol.
1
960 mg (20%) of the product as a yellow solid. H NMR (300
Ra d iosyn th esis of [N-Meth yl-11C]2-(4′-(Dim eth yla m i-
n o)p h en yl)-6-m eth oxyben zoth ia zole ([11C]9; [11C]6-MeO-
BTA-2). Compound 18 (6-CH3O-BTA-1) (1.0 mg, 3.7 µmoles)
was dissolved in 400 µL of DMSO. Dry KOH (10 mg) was
added, and the 3 mL V-vial was vortexed for 5 min. No-carrier-
added [11C]methyl iodide was bubbled through the solution at
30 mL/min at room temperature. The reaction was heated for
5 min at 95° C using an oil bath. The reaction product was
purified by semipreparative HPLC using a Prodigy ODS-Prep
column eluted with 60% acetonitrile/40% triethylammonium
phosphate buffer pH 7.2 (flow at 5 mL/min for 0-7 min then
increased to 15 mL/min for 7-30 min). The fraction containing
[N-methyl-11C]6-CH3O-BTA-2 (at about 15 min) was collected
and diluted with 50 mL of water and eluted through a Waters
C18 SepPak Plus cartridge. The C18 SepPak was washed with
10 mL of water, and the product was eluted with 1 mL of
ethanol (absolute) into a sterile vial followed by 14 mL of
saline. Radiochemical and chemical purities were >95% as
determined by analytical HPLC (k′ ) 4.4 using the Prodigy
ODS(3) analytical column eluted with 65/35 acetonitrile/
triethylammonium phosphate buffer pH 7.2). The radiochemi-
cal yield averaged 17.3% at EOS based on [11C]methyl iodide,
and the specific activity averaged about 160 GBq/µmol (4.3 Ci/
µmol) at EOS.
Deter m in a tion of Octa n ol-Bu ffer P a r tition Coeffi-
cien ts (P oct) a n d Rever se P h a se HP LC P a r tition Coef-
ficien ts (P C18). Measurement of the octanol-buffer partition
coefficients (Poct) of selected 11C-labeled compounds (Table 1)
were performed as previously described.15 HPLC retention
times of all unlabeled (cold) BTA's were determined using
reverse phase C18 chromatography [Phenomenex Prodigy
ODS(3) 3 µm 4.6 × 250 mm column eluted with 1.4 mL/min
acetonitrile/aqueous buffer (60/40 v/v; 50 mM triethylammo-
nium phosphate buffer, pH 7.2)] and UV detection at 365 nm.
For example, the retention time (tR) of BTA-1 under these
conditions was 9.98 min and the solvent front appeared at 1.75
min. The retention factor (k′C18) was determined using the
equation:
MHz, acetone-d6) δ: 9.02 (s, OH), 8.41 (d, J ) 9.1 Hz, 1H),
8.33 (d, J ) 9.1 Hz, 1H), 7.96 (d, J ) 8.6 Hz, 1H), 7.53 (d, J )
2.4 Hz, 1H), 7.15 (dd, J 1 ) 8.6 Hz, J 2 ) 2.4 Hz, 1H).
2-(4′-Nit r op h en yl)-6-m et h oxym et h oxyb en zot h ia zole
(23). To a solution of 2-(4′-nitrophenyl)-6-hydroxybenzothiazole
(22) (960 mg, 3.5 mmol) dissolved in acetone (120 mL, dried
over K2CO3) was injected methoxymethyl chloride (5 mL) at
room temperature. The reaction mixture was monitored with
TLC every 30 min, and additional methoxymethyl chloride (ca.
0.5 mL) was added until the reaction was completed. The
reaction mixture was then filtered, the mother liquid was
concentrated and dried over the high vacuum pump to give
1.0 g of the crude product, which was purified by flash column
(hexanes:ethyl acetate ) 80:20) to give 833 mg (75%) of the
pure product. 1H NMR (300 MHz, acetone-d6): 8.42 (d, J )
10.9 Hz, 2 H),8.35 (d, J ) 10.9 Hz, 2 H), 8.04 (d, J ) 9.0 Hz,
1 H), 7.97(d, J ) 2.4 Hz, 1 H), 7.30(dd, J 1 ) 9.0 Hz, J 2 ) 2.4
Hz, 1 H), 5.33 (s, 2 H), 3.49 (s, 3 H).
2-(4′-Am in op h e n yl)-6-m e t h oxym e t h oxyb e n zot h ia z-
ole (24; 6-MOMO-BTA-0). To a mixture of 2-(4′-nitrophenyl)-
6-methoxymethoxybenzothiazole (23) (360 mg, 1.14 mmol) and
Cu(OAc)2 (207 mg, 1.14 mmol) in anhydrous ethanol (50 mL)
was added NaBH4 (866 mg, 22.8 mmol, 20 equiv) while stirring
at room temperature. The reaction mixture was stirred for
another 1 h. The solvent was then removed under reduced
pressure. The residue was dissolved in water and extracted
with ethyl acetate (3 × 50 mL). The extracts were combined,
dried over MgSO4, and evaporated, and the residue was
purified by flash column (hexanes:ethyl acetate ) 80:20 to 50:
1
50) to give 306 mg (93%) of the product as a yellow solid. H
NMR (300 MHz, Acetone-d6):δ 7.81 (d, J ) 8.8 Hz, 3H), 7.64
(d, J ) 2.5 Hz, 1H), 7.15 (dd, J 1 ) 8.8 Hz, J 2 ) 2.5 Hz, 1H),
6.77(d, J ) 8.8 Hz, 2H), 5.33 (s, 2H), 3.47 (s, 3H). Anal. Calcd
for C 62.92; H 4.93; N 9.78; S 11.20. Found: C 63.03; H 5.26;
N 9.43; S 10.85.
[N-Meth yl-11C]2-(4′-(Meth yla m in o)p h en yl)-6-h yd r oxy-
ben zoth ia zole ([11C]19;[11C]6-OH-BTA-1). 6-MOMO-BTA-0
(24) (1.5 mg, 5.2 µmol) was dissolved in 400 µL of DMSO. Dry
KOH (∼10 mg) was added, and the 3 mL V-vial was vortexed
for 5 min. No-carrier-added [11C]methyl iodide was bubbled
through the solution at 30 mL/min at room temperature. The
reaction was heated for 5 min at 125° C using an oil bath. A
sample of the crude was analyzed by analytical HPLC to
k′C18 )(tR - 1.75)/(1.75)
From a series of approximately 10 structurally related BTA
derivatives (including those of 6-H, 6-CH3, 6-OCH3, and 6-OH-

2752 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
BTA-1 (Table 1)), a correlation of experimentally determined
log Poct and log k′C18 values was performed resulting in a slope
of 2.35, a y-intercept of 1.11, and a correlation coefficient of r
) 0.95. Similar to the method of Salminen and co-workers59
these values were used in the following equation to determine
throughout the course of the imaging study. Blood pressure,
heart and respiratory rates, and expired CO2 and oxygen
saturation levels were monitored continuously during the PET
studies. The baseline rectal body temperature (∼37 °C) was
maintained using a heating blanket (Gaymar, Orchard Park,
NY) and temperature regulator (Yellow Springs Instruments,
Yellow Springs, OH). Prior to scanning, the baboon’s head was
fixed so that the image planes were acquired approximately
parallel to the orbital-meatal line.
log PC18, which represents an approximation of the log Poct
:
log PC18 ) 2.35(logk′C18) + 1.11
PET data were acquired using an ECAT HR+ PET scanner
(CTI PET Systems, Knoxville, TN) in 3D imaging mode (63
parallel slices; 15.2 cm axial field-of-view; 4.1 mm full-width
half-maximum in-plane resolution). A Neuro-Insert (CTI PET
Systems) was used to reduce the contribution of scattered
photon events. After the baboons were positioned in the PET
scanner, a windowed transmission scan (10-15 min) was
obtained for attenuation correction of the PET emission data
using rotating 68Ge/68Ga rods. Each of the PET tracers was
administered iv over 20 s, and a dynamic series of PET scans
were acquired over 60 min using 23 frames of increasing length
(6 × 20 s; 4 × 30 s; 6 × 60 s; 4 × 5 min; 3 × 10 min).
Approximately 185 MBq (∼5 mCi) of a high specific activity
(>14.8 GBq/µmol) 11C-labeled BTA radiotracer was injected
in the baboons, including either [N-methyl-11C]BTA-1, [N-meth-
yl-11C]6-Me-BTA-1, [N-methyl-11C]6-OMe-BTA-1 ([11C]18),
[N-methyl-11C]6-CN-BTA-1 ([11C]14), or [N-methyl-11C]6-OH-
BTA-1 ([11C]19). In addition in other studies, 148-296 MBq
(4-8 mCi) of a high specific activity (>18.5 GBq/µmol) refer-
ence PET radiotracer was injected, including either [11C]-
raclopride,60 [11C](+)-McN5652,39 [carbonyl-11C]WAY100635,33,34
or [18F]altanserin.61 The PET data were reconstructed using
a Hanning filter (Nyquist cutoff) and corrected for decay,
photon attenuation, and scatter.
An MRI scan was obtained for each baboon using a 1.5T
GE Signa scanner (GE Medical Systems, Milwaukeee, WI)
equipped with a standard head coil. A volumetric spoiled
gradient recalled (SPGR) MR sequence with parameters for
high contrast among gray matter, white matter, and CSF was
acquired in the coronal plane (TE ) 5, TR ) 24, flip angle )
40°, slice thickness ) 1.5 mm, NEX ) 2, field of view 12 cm,
voxel size ) 0.94 × 1.25 × 1.5 mm). Each individual baboon’s
MR image was coregistered to the PET data using the
automated image registration (AIR) algorithm for cross-
modality image alignment and reslicing.62 The initial 16
frames (0-9 min pi) of the dynamic PET images were summed
together into images consisting of a single frame. Prior to
coregistration, both the MR and summed PET images were
edited using the ANALYZE software package (Mayo Clinic,
Rochester, MN) to remove extracerebral tissues that could
possibly confound the coregistration process. The edited MR
images were then coregistered to the summed PET image and
resliced to yield MR images in the same spatial orientation
and resolution as the summed PET images. The coregistration
of MR and PET datasets in the baboon has been demonstrated
to be a reliable and robust application of the AIR method.63,64
When limited to compounds with similar structures, this
method can yield an accurate representation of relative
lipophilicities.59 In addition, this method avoids the problems
encountered in determining log Poct values for compounds
having very high or very low lipophilicities and avoids the need
to radiolabel each compound.
In Vitr o Bin d in g Assa ys w ith Aâ(1-40) F ibr ils a n d
Br a in Hom ogen a tes (Ki a n d Kd d eter m in a tion s). The Ki
values of the unlabeled (cold) BTA derivatives for inhibiting
[3H]BTA-1 binding to aggregated fibrils of synthetic Aâ(1-
40) (BACHEM Bioscience, Inc., King of Prussia, PA) were
determined utilizing methods previously reported.15,16 The
aggregation of Aâ(1-40) and the radiosynthesis of [3H]BTA-1
also were previously described.15,16
Scatchard analyses were performed to determine the Kd and
Bmax values of [11C]6-OH-BTA-1 ([11C]19) for both aggregated
fibrils of synthetic Aâ(1-40) and brain tissue homogenates
from postmortem medial-frontal cortex gray matter obtained
from an autopsy-confirmed AD case through the University
of Pittsburgh Alzheimer’s Disease Research Center Brain
Bank. These Scatchard analyses were performed employing
standard methods that have been previously described in
detail.43
Neu r or ecep tor Scr een in g. Binding assays for receptors
and transporters were performed as previously described using
the resources of the National Institute of Mental Health
Psychoactive Drug Screening Program.45,46
In Vivo Mou se Br a in En tr y Stu d ies. Studies were
performed in female Swiss-Webster mice (23-35 g) in ac-
cordance with the Guide for the Care and Use of Laboratory
Animals adopted by NIH and with the approval of the local
Institutional Animal Care and Use Committee. The mice were
injected in a lateral tail vein with 0.37-3.7 MBq (10-100 µCi)
of a high specific activity (>7.4 GBq/µmol) 11C-labeled BTA
derivative (Table 2) contained in e0.10 mL of a solution of
93% isotonic saline and 7% ethanol. The mice were anesthe-
tized and killed by cardiac excision following cardiac puncture
to obtain arterial blood samples at 2 or 30 min postinjection.
The mouse brains were rapidly excised and divided into
cerebellum and remaining whole brain (including brain stem)
fractions. The brain samples were counted in a gamma well-
counter, and the counts were decay-corrected to the time of
injection relative to 11C standards prepared from the injection
solution to determine the percent injected dose (%ID) in the
samples. The brain samples were weighed to determine the
percent injected dose per gram tissue (%ID/g), and this
quantity was multiplied by the whole body weight (in kg) to
determine the body-weight normalized radioactivity concen-
tration [(%ID-kg)/g] of each tissue sample.
In Vivo Ba boon Br a in En tr y Stu d ies. PET studies were
performed in six adult (four female) baboons (Papio anubis)
(weight 15-35 kg, ages 6-12 years) in accordance with the
Guide for the Care and Use of Laboratory Animals adopted
by NIH and with the approval of the local Institutional Animal
Care and Use Committee. Prior to PET imaging, the animals
were initially sedated with ketamine (10-15 mg/kg, im), given
atropine (0.5 mg, i.m.) to control salivation and heart rate, and
intubated. The baboons were subsequently maintained on a
ventilator with isofluorane (0.5-1.25%) anesthesia and medi-
cal air. Pancuronium bromide was administered as necessary
(iv, up to 0.06 mg/kg/h, titrated to effect) to keep the animals
immobilized during the study. A femoral artery catheter was
inserted to monitor blood pressure and sample arterial blood,
and an intravenous catheter was placed in an antecubital vein
for radiotracer injection and to administer fluids as necessary
Regions of interest (ROIs) were defined on the coregistered
MR image and applied to the dynamic PET datasets to
determine regional time-activity data for pons, white matter
(cerebral white matter posterior to prefrontal cortex and
anterior to lateral ventricles), striatum (caudate and putamen),
mesial temporal cortex (amygdala and hippocampus), cerebel-
lum (cerebellar cortex), occipital cortex, and lateral temporal
cortex. The ROIs and time-activity curves were created using
an internally developed tool based on the interactive data
language platform (IDL, Research Systems, Inc., Boulder, CO)
and correspond to left and right hemisphere averages (exclud-
ing pons). The PET time-activity data were converted to units
of microcuries per milliliter using a phantom-based calibration
factor and were subsequently normalized to the injected dose
and body mass of the animal ((%ID-kg)/g).
Meta bolite Deter m in a tion s of [11C]6-OH-BTA-1 ([11C]-
19). Following the injection of [11C]6-OH-BTA-1 ([11C]19),
arterial whole blood samples were obtained in mice (via cardiac
puncture) and baboons (via femoral artery catheter). The

2-Arylbenzothiazoles as Amyloid Imaging Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2753
(12) Styren S. D.; Hamilton R. L.; Styren G. C.; Klunk W. E. X-34, a
fluorescent derivative of Congo red: a novel histochemical stain
for Alzheimer’s disease pathology. J . Histochem. Cytochem. 2000,
48, 1223-1232.
(13) Klunk W. E.; Bacskai B. J .; Mathis C. A.; Kajdasz S. T.; McLellan
M. E.; Frosch M. P.; Debnath M. L.; Holt D. P.; Wang Y.; Hyman
B. T. Imaging Abeta plaques in living transgenic mice with
multiphoton microscopy and methoxy-X04, a systemically ad-
ministered Congo red derivative. J . Neuropathol. Exp. Neurol.
2002, 61, 797-805.
(14) Wang, Y.; Mathis, C. A.; Huang, G.-F.; Holt, D. P.; Debnath, M.
L.; Klunk, W. E. Synthesis and C-11-Labeling of (E, E)-1-(3′, 4′-
Dihydroxystyryl)-4-(3′-Methoxy-4′-Hydroxystyryl) Benzene for
PET Imaging of Amyloid Deposits. J . Labelled Compd. Radiop-
harm. 2002, 45, 647-664.
(15) Klunk, W. E.; Wang, Y.; Huang, G.-F.; Debnath, M. L.; Holt, D.
P.; Mathis, C. A. Uncharged thioflavin-T derivatives bind to
amyloid-beta protein with high affinity and readily enter the
brain. Life Sci. 2001, 69, 1471-1484.
(16) Mathis, C. A.; Bacskai, B. J .; Kajdasz, S. T.; McLellan, M. E.;
Frosch, M. P.; Hyman, B. T.; Holt, D. P.; Wang, Y.; Huang, G.-
F.; Debnath, M. L.; Klunk, W. E. A lipophilic thioflavin-T
derivative for positron emission tomography (PET) imaging of
amyloid in brain. Bioorg. Med. Chem. Lett. 2002, 12, 295-298.
(17) Zhuang Z. P.; Kung M. P.; Hou C.; Plossl K.; Skovronsky D.;
Gur T. L.; Trojanowski J . Q.; Lee V. M.; Kung H. F. IBOX (2-
(4′-dimethylaminophenyl)-6-iodobenzoxazole): a ligand for imag-
ing amyloid plaques in the brain. Nucl. Med. Biol. 2001, 28, 887-
894.
(18) Zhuang Z. P.; Kung M. P.; Wilson A.; Lee C. W.; Plossl K.; Hou
C.; Holtzman D. M.; Kung H. F. Structure-activity relationship
of imidazo[1,2-a]pyridines as ligands for detecting beta-amyloid
plaques in the brain. J . Med. Chem. 2003, 46, 237-243.
(19) Agdeppa E. D.; Kepe V.; Liu J .; Flores-Torres S.; Satyamurthy
N.; Petric A.; Cole G. M.; Small G. W.; Huang S. C.; Barrio J . R.
Binding characteristics of radiofluorinated 6-dialkylamino-2-
naphthylethylidene derivatives as positron emission tomography
imaging probes for beta-amyloid plaques in Alzheimer’s disease.
J . Neurosci. 2001, 21, RC189-.
(20) Shoghi-J adid, K.; Small, G. W.; Agdeppa, E. D.; Kepe, V.; Ercoli,
L. M.; Siddarth, P.; Read, S.; Satyamurthy, N.; Petric, A.; Huang,
S. C.; Barrio, J . R. Localization of neurofibrillary tangles and
beta-amyloid plaques in the brains of living patients with
Alzheimer disease. Am. J . Geriatr. Psych. 2002, 10, 24-35.
(21) Shi, D. F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J .; Lelieveld,
P.; Fichtner, I.; Stevens, M. F. G. Antitumor Benzothiazoles. 3.
Synthesis of 2-(4-aminophenyl)benzothiazoles and evaluation of
their activities against breast cancer cell lines in vitro and in
vivo. J . Med. Chem. 1996, 39, 3375-3384.
(22) Kawaoka, Y.; Takashita, K. Preparation of 2-aminothiophenols.
J apanese Patent 07278100, 1995.
(23) Tsuruoka, A.; Kaku Y.; Kakinuma H.; Tsukada I.; Yanagisawa
M., Nara K., Naito T. Synthesis and antifungal activity of novel
thiazole-containing triazole antifungals. II. optically active ER-
30346 and its derivatives. Chem. Pharm. Bull. 1998, 46, 623-
630.
(24) Hasebe, N. Use of polyphosphoric acid in the synthesis of
2-arylbenzothiazoles. Yamagata Daigaku Kiyo, Shizen Kagaku
1972, 8, 63-67.
(25) Dishino, D. D.; Welch, M. J .; Kilbourn, M. R.; Raichle, M. E.
Relationship between lipophilicity and brain extraction of C-11-
labeled radiopharmaceuticals. J . Nucl. Med. 1983, 24, 1030-
1038.
(26) Vera, D. R.; Eckelman, W. C. Receptor 1980 and Receptor 2000:
twenty years of progress in receptor-binding radiotracers. Nucl.
Med. Biol. 2001, 28, 475-476.
(27) Hansch, C.; Leo, A. Substituent Constants for Correlation
Analysis in Chemistry and Biology; J ohn Wiley and Sons: New
York, 1979.
(28) Lemaire, C.; Cantineau, R.; Guillaume, M.; Plenevaux, A.;
Christiaens, L. Fluorine-18-altanserin: a radioligand for the
study of serotonin receptors with PET: radiolabeling and in vivo
biologic behavior in rats. J . Nucl. Med. 1991, 32, 2266-2272.
(29) Scheffel, U.; Pogun, S.; Stathis, M.; Boja, J . W.; Kuhar, M. J . In
vivo labeling of cocaine binding sites on dopamine transporters
with [3H]WIN 35,428. J . Pharmacol. Exp. Ther. 1991, 257, 954-
958.
(30) Hume, S. P.; Myers, R.; Bloomfield, P. M.; Opacka-J uffry, J .;
Cremer, J . E.; Ahier, R. G.; Luthra, S. K.; Brooks, D. J .;
Lammertsma, A. A. Quantitation of carbon-11-labeled raclopride
in rat striatum using positron emission tomography. Synapse
1992, 12, 47-54.
plasma was separated from cellular elements, and the radio-
labeled species in the plasma were analyzed as previously
described.16 In addition, the procedures employed to assess
radioactivity in the brains of mice 2 and 30 min after injection
of about 18.5 MBq (500 µCi) of [11C]6-OH-BTA-1 also have been
described.16
Tissu e Sta in in g. Postmortem brain tissues from autopsy-
confirmed AD cases were obtained through the University of
Pittsburgh Alzheimer’s Disease Research Center Brain Bank.
Hippocampus and entorhinal cortex were dissected from 1 cm
thick coronal slices. Tissue was processed for staining and
quenching of autofluorescence was accomplished according to
previously described methods.12 Quenched tissue sections were
taken from PBS into a solution of 1 µM (cold) 6-OH-BTA-1 in
PBS pH 7.0 for 45 min. The sections were then dipped for
approximately 5 s into water before coverslipping with Fluo-
romount-G (Electron Microscopy Sciences, Fort Washington,
PA). Fluorescent sections were viewed using an Olympus
Vanox AH-RFL-LB fluorescence microscope using a UV filter
set: U-filter (excites 360-370 nm, dichroic mirror DM400, 420
nm long-pass filter).
Ack n ow led gm en t. This work was supported by
grants from the National Institute on Aging (AG18402
to C.M. and AG01039 and AG20226 to W.K.), the
Alzheimer’s Association (IIRG-95-076 and TLL-01-3381
to W.K. and NIRG-00-2355 to Y.W.), and ISOA/AFAR
(210304 to Y.W.). Brain tissue for binding and staining
assays was provided through the University of Pitts-
burgh Alzheimer’s Disease Research Center Brain Bank
(AG05133) by Drs. Steven T. DeKosky and Ronald L.
Hamilton. We thank Dr. Bryan Roth of Case Western
Reserve University and Dr. Linda Brady of NIMH for
allowing us access to the NIMH Psychoactive Drug
Screening Program.
Refer en ces
(1) Braak, H.; Braak, E. Neuropathological stageing of Alzheimer-
related changes. Acta Neuropathol. (Berl.) 1991, 82, 239-259.
(2) Mirra, S. S.; Heyman, A.; McKeel, D.; Sumi, S. M.; Crain, B. J .;
Brownlee, L. M.; Vogel, F. S.; Hughes, J . P.; van Bell, G.; Berg,
L. The consortium to establish a registry for Alzheimer’s disease
(CERAD). Part II. Standardization of the neuropathologic as-
sessment of Alzheimer’s disease. Neurology 1991, 41, 479-486.
(3) Klunk, W. E.; Abraham, D. J . Filamentous proteins in Alzhe-
imer’s disease: new insights through molecular biology. Psych.
Dev. 1988, 6, 121-152.
(4) Schenk, D. B.; Rydel, R. E.; May, P.; Little, S.; Panetta, J .;
Lieberburg, I.; Sinha, S. Therapeutic approaches related to
amyloid-beta peptide and Alzheimer’s disease. J . Med. Chem.
1995, 38, 4141-4154.
(5) Schenk, D. B.; Seubert, P.; Lieberburg, I.; Wallace, J . Beta-
peptide immunization: a possible new treatment for Alzheimer
disease. Arch. Neurol. 2000, 57, 934-936.
(6) Holtzman, D. M.; Bales, K. R.; Paul, S. M.; DeMattos, R. B. Abeta
immunization and anti-Abeta antibodies: potential therapies for
the prevention and treatment of Alzheimer’s disease. Adv. Drug
Deliv. Rev. 2002, 54, 1603-1613.
(7) Olson, R. E.; Copeland, R. A.; Seiffert, D. Progress towards
testing the amyloid hypothesis: inhibitors of APP processing.
Curr. Opin. Drug Discov. Dev. 2001, 4, 390-401.
(8) Matsuoka, Y.; Saito, M.; LaFrancois, J .; Saito, M.; Gaynor, K.;
Olm, V.; Wang, L.; Casey, E.; Lu, Y.; Shiratori, C.; Lemere, C.;
Duff, K. Novel therapeutic approach for the treatment of
Alzheimer’s disease by peripheral administration of agents with
an affinity for â-amyloid. J . Neurosci. 2003, 23, 29-33.
(9) Klunk W. E.; Debnath M. L.; Pettegrew J . W. Chrysamine-G
binding to Alzheimer and control brain: autopsy study of a new
amyloid probe. Neurobiol. Aging 1995, 16, 541-548.
(10) Mathis C. A.; Mahmood K.; Debnath M. L.; Klunk W. E.
Synthesis of a lipophilic radioiodinated ligand with high affinity
to amyloid protein in Alzheimer’s disease brain tissue. J .
Labelled Compd. Radiopharm. 1997, 40, 94-95.
(31) Suehiro, M.; Scheffel, U.; Dannals, R. F.; Ravert, H. T.; Ricaurte,
G. A.; Wagner, H. N. J r. A PET radiotracer for studying
serotonin uptake sites: carbon-11-McN-5652Z. J . Nucl. Med.
1993, 34, 120-127.
(11) Dezutter, N. A.; Dom, R. J .; de Groot, T. J .; Bormans, G. M.;
Verbruggen, A. M. 99mTc-MAMA-Chrysamine G, a Probe for beta-
Amyloid Protein of Alzheimer’s Disease. Eur. J . Nucl. Med. 1999,
26, 1392-1399.

2754 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Mathis et al.
(32) Mathis, C. A.; Simpson, N. R.; Mahmood, K.; Kinahan, P. E.;
Mintun, M. A. [11C]WAY 100635: a radioligand for imaging
5-HT1A receptors with positron emission tomography. Life Sci.
1994, 55, PL403-407.
(33) Pike, V. W.; McCarron, J . A.; Lammertsma, A. A.; Osman, S.;
Hume, S. P.; Sargent, P. A.; Bench, C. J .; Cliffe, I. A.; Fletcher,
A.; Grasby, P. M. Exquisite delineation of 5-HT1A receptors in
human brain with PET and [carbonyl-11C]WAY-100635. Eur. J .
Pharmacol. 1996, 301, R5-7.
(47) Allsop, D.; Landon, M.; Kidd, M.; Lowe, J . S.; Reynolds, G. P.;
Gardner, A. Monoclonal antibodies raised against a sub-sequence
of senile plaque core protein react with plaque cores, plaque
periphery and cerebrovascular amyloid in Alzheimer’s disease.
Neurosci. Lett. 1986, 68, 252-256.
(48) Glenner, G. G.; Wong, C. W. Alzheimer’s disease: initial report
of the purification and characterization of a novel cerebrovas-
cular amyloid protein. Biochem. Biophys. Res. Commun. 1984,
120, 885-890.
(34) Drevets, W. C.; Frank, E.; Price, J . C.; Kupfer, D. J .; Holt, D.;
Greer, P. J .; Huang, Y.; Gautier, C.; Mathis, C. PET imaging of
serotonin 1A receptor binding in depression. Biol. Psych. 1999,
46, 1375-1387.
(35) Farde, L.; Halldin, C.; Stone-Elander, S.; Sedvall, G. PET
Analysis of human dopamine receptor subtypes using 11C-SCH
23390 and 11C-raclopride. Psychopharmacology (Berl.) 1987, 92,
278-284.
(36) Farde, L.; Pauli, S.; Hall, H.; Eriksson, L.; Halldin, C.; Hogberg,
T.; Nilsson, L.; Sjogren, I.; Stone-Elander, S. Stereoselective
binding of 11C-raclopride in living human brain-a search for
extrastriatal central D2-dopamine receptors by PET. Psychop-
harmacology (Berlin) 1988, 94, 471-478.
(37) Smith, G. S.; Price, J . C.; Lopresti, B. J .; Huang, Y.; Holt, D.;
Mason, N. S.; Simpson, N. R.; Sweet, R.; Meltzer, C. C.; Sashin,
D.; Mathis, C. A. Test-retest variability of serotonin 5-HT2A
receptor binding measured with positron emission tomography
(PET) and [F-18]altanserin in the human brain. Synapse 1998,
30, 380-392.
(49) Ikeda, S.; Wong, C. W.; Allsop, D.; Landon, M.; Kidd, M.;
Glenner, G. G. Immunogold labeling of cerebrovascular and
neuritic plaque amyloid fibrils in Alzheimer’s disease with an
anti-beta protein monoclonal antibody. Lab. Investig. 1987, 57,
446-449.
(50) Okamoto, K.; Yamaguchi, H.; Hirai, S.; Shoji, M.; Inoue, K.;
Takatama, M. Immunogold electron microscopic study of cere-
brovascular and senile plaque amyloid using anti-beta protein
antibody. Prog. Clin. Biol. Res. 1989, 317, 953-963.
(51) Wong, C. W.; Quaranta, V.; Glenner, G. G. Neuritic plaques and
cerebrovascular amyloid in Alzheimer disease are antigenically
related. Proc. Natl. Acad. Sci. U. S .A. 1985, 82, 8729-8732.
(52) Yamaguchi, H.; Hirai, S.; Morimatsu, M.; Shoji, M.; Nakazato,
Y. Diffuse type of senile plaques in the cerebellum of Alzheimer-
type dementia demonstrated by beta protein immunostain. Acta
Neuropathol. 1989, 77, 314-319.
(53) Eckelman, W. C. The use of in vitro models to predict the
distribution of receptor binding radiotracers in vivo. Nucl. Med.
Biol. 1989, 16, 233-245.
(38) Price, J . C.; Lopresti, B. J .; Mason, N. S.; Holt, D. P.; Meltzer,
C. C.; Smith, G. S.; Gunn, R. N.; Huang, Y.; Mathis, C. A.
Analyses of [18F]altanserin bolus injection PET data II: consid-
eration of radiolabeled metabolites in humans. Synapse 2001,
41, 11-21.
(39) Lopresti, B. J .; Mathis, C. A.; Price, J . C.; Villemagne, V. L.;
Meltzer, C. C.; Holt, D. P.; Smith, G. S.; Moore, R. Y. Serotonin
transporter binding in vivo: further examination of [11C]-
McN5652, In Molecular and Pharmacological Brain Imaging
with Positron Emission Tomography; Gjedde A., Ed.; Academic
Press: San Diego, 2001; pp 265-271.
(54) Eckelman, W. C.; Gibson, R. E. The design of site directed
radiopharmaceuticals for use in drug discovery. In Nuclear
Imaging in Drug Discovery, Development and Approval; Burns,
H. D., Gibson, R. E., Dannals, R. F., Siegl P. K. S., Eds.;
Birkhauser: Boston, Mass., 1992; pp 113-134.
(55) Engler, H.; Blomqvist, G.; Bergstrom, M.; Langstrom, B.; Klunk,
W.; Debnath, M.; Holt, D.; Wang, Y.; Huang, G.-F.; Mathis, C.
First human study with a benzothiazole amyloid-imaging agent
in Alzheimer’s disease and control subjects. Neurobiol. Aging
2002, 23 (Suppl.), S1568.
(56) Hyman, B. T.; Bacskai, B. J .; Kajdasz, S. T.; Mclellan, M. E.;
Frosch, M. P.; Debnath, M. L.; Holt, D. P.; Wang, Y.; Mathis, C.
A.; Klunk, W. E. Visualizing amyloid plaques. Neurobiol. Aging
2002, 23 (1S), S149.
(57) Bacskai, B. J .; Klunk, W. E.; Mathis, C. A.; Hyman, B. T.
Imaging amyloid-beta deposits in vivo. J . Cereb. Blood Flow
Metab. 2002, 22, 1035-1041.
(58) Dannals, R. F.; Ravert, H. T.; Wilson, A. A.; Wagner, H. N. J r.
An improved synthesis of (3-N-[11C]methyl)spiperone. Int. J .
Radiat. Appl. Isot. 1986, 37, 433-434.
(59) Salminen, T.; Pulli, A.; Taskinen, J . Relationship between
immobilised artificial membrane chromatographic retention and
the brain penetration of structurally diverse drugs. J . Pharm.
Biomed. Anal. 1997, 15, 469-477.
(40) Huang, Y.; Hwang, D. R.; Narendran, R.; Sudo, Y.; Chatterjee,
R.; Bae, S. A.; Mawlawi, O.; Kegeles, L. S.; Wilson, A. A.; Kung,
H. F.; Laruelle, M. Comparative evaluation in nonhuman
primates of five PET radiotracers for imaging the serotonin
transporters:
[
11C]McN 5652, [11C]ADAM, [11C]DASB, [11C]-
DAPA, and [11C]AFM. J . Cereb. Blood Flow Metab. 2002, 22,
1377-1398.
(41) Eckelman, W. C. Sensitivity of New Radiopharmaceuticals. Nucl.
Med. Biol. 1998, 25, 169-173.
(42) Naslund, J .; Haroutunian, V.; Mohs, R.; Davis, K. L.; Davies,
P.; Greengard, P.; Buxbaum, J . D. Correlation between elevated
levels of amyloid beta-peptide in the brain and cognitive decline.
J . Am. Med. Assoc. 2000, 283, 1571-1577.
(43) Klunk, W. E.; Wang, Y.; Huang, G.-F.; Debnath, M. L.; Holt, D.
P.; Shao, L.; Hamilton, R. L.; Ikonomovic, M.; DeKosky, S. T.;
Mathis, C. A. The binding of 2-(4′-methylaminophenyl)-ben-
zothiazole to postmortem brain homogenates is dominated by
the amyloid component. J . Neurosci. 2003, 23, 2086-2092.
(44) Wolf, D. S.; Gearing, M.; Snowdon, D. A.; Mori, H.; Markesbery,
W. R.; Mirra, S. S. Progression of regional neuropathology in
Alzheimer disease and normal elderly: findings from the nun
study. Alzh. Disease Assoc. Disorders 1999, 13, 226-231.
(45) Roth, B. L.; Ernsberger, P.; Steinberg, S. A.; Rao, S.; Rauser,
L.; Savage, J .; Hufeisen, S.; Berridge, M. S.; Muzic, R. F. J r.
The in vitro pharmacology of the beta-adrenergic receptor PET
ligand (S)-fluorocarazolol reveals high affinity for cloned beta-
adrenergic receptors and moderate affinity for the human
5-HT1A receptor. Psychopharmacology (Berlin) 2001, 157, 111-
114.
(60) Drevets, W. C.; Price, J . C.; Kupfer, D. J .; Kinahan, P. E.;
Lopresti, B.; Holt, D.; Mathis, C. PET measures of amphetamine-
induced dopamine release in ventral versus dorsal striatum.
Neuropsychopharmacology 1999, 21, 694-709.
(61) Price, J . C.; Lopresti, B. J .; Mason, N. S.; Holt, D. P.; Huang,
Y.; Mathis, C. A. Analyses of [18F]altanserin bolus injection PET
data. I: consideration of radiolabeled metabolites in baboons.
Synapse 2001, 41, 1-10.
(62) Woods, R. P.; Mazziotta, J . C.; Cherry, S. R. MRI-PET registra-
tion with an automated algorithm. J . Comput. Assist. Tomogr.
1993, 17, 536-546.
(63) Greer, P. J .; Villemagne, V. L.; Ruszkiewicz, J .; Graves, A. K.;
Meltzer, C. C.; Mathis, C. A.; Price, J . C. MR atlas of the baboon
brain for functional neuroimaging. Brain Res. Bull. 2002, 58,
429-438.
(64) Price, J . C.; Drevets, W. C.; Ruszkiewicz, J .; Greer, P. J .;
Villemagne, V. L.; Xu, L.; Mazumdar, S.; Cantwell, M.; Mathis,
C. A. Sequential H215O PET studies in baboons: before and after
amphetamine. J . Nucl. Med. 2002, 43, 1090-1100.
(46) Rothman, R. B.; Baumann, M. H.; Savage, J . E.; Rauser, L.;
McBride, A.; Hufeisen, S. J .; Roth, B. L. Evidence for possible
involvement of 5-HT2B receptors in the cardiac valvulopathy
associated with fenfluramine and other serotonergic medications.
Circulation 2000, 102, 2836-2841.
J M030026B