Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates

Article abstract of DOI:10.1021/jm010045q

We report for the first time that small molecule-based radiodiodinated ligands, showing selective binding to Aβ aggregates, cross the intact blood-brain barrier by simple diffusion. Four novel ligands showing preferential labeling of amyloid aggregates of Aβ(1-40) and Aβ(1-42) peptides, commonly associated with plaques in the brain of people with Alzheimer's disease (AD), were developed. Two ¹²⁵I-labeled styrylbenzenes, (E,E)-1-iodo-2,5-bis(3-hydroxvcarbonyl-4-hydroxy)-styrylbenzene, 12 (ISB), and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene, 13 (IMSB), and two ¹²⁵I-labeled thioflavins, 2-[4′-(dimethylamino)phenyl]-6-iodobenzothiazole, 18a (TZDM), and 2-[4′-(4″-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, 18b (TZPI), were prepared at a high specific activity (2200 Ci/mmol). In vitro binding studies of these ligands showed excellent binding affinities with K_d values of 0.08, 0.13, 0.06, and 0.13 nM for aggregates of Aβ(1-40) and 0.15, 0.73, 0.14, and 0.15 nM for aggregates of Aβ(1-42), respectively. Interestingly, under a competitive-binding assaying condition, different binding sites on Aβ(1-40) and Aβ(1-42) aggregates, which are mutually exclusive, were observed for styrylbenzenes and thioflavins. Autoradiography studies of postmortem brain sections of a patient with Down's syndrome known to contain primarily Aβ(1-42) aggregates in the brain showed that both [¹²⁵I]18a and [¹²⁵I]18b labeled these brain sections, but [¹²⁵I] 13, selective for Aβ(1-40) aggregates, exhibited very low labeling of the comparable brain section. Biodistribution studies in normal mice after an iv injection showed that [¹²⁵I]18a and [¹²⁵I]18b exhibited excellent brain uptake and retention, the levels of which were much higher than those of [¹²⁵I]12 and [¹²⁵I]13. These findings strongly suggest that the new radioiodinated ligands, [¹²⁵I]12 (ISB), [¹²⁵I]13 (IMSB), [¹²⁵I]18a (TZDM), and [¹²⁵I]18b (TZPI), may be useful as biomarkers for studying Aβ(1-40) as well as Aβ(1-42) aggregates of amyloidogenesis in AD patients.

Full text of DOI:10.1021/jm010045q

J . Med. Chem. 2001, 44, 1905-1914
1905
Ra d ioiod in a ted Styr ylben zen es a n d Th iofla vin s a s P r obes for Am yloid
Aggr ega tes
Z.-P. Zhuang,† M.-P. Kung,† C. Hou,† D. M. Skovronsky,‡ T. L. Gur,‡ K. Plo¨ssl,† J . Q. Trojanowski,‡
V. M.-Y. Lee,‡,§ and H. F. Kung*,†,§
Departments of Radiology, Pathology and Laboratory Medicine, and Pharmacology, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received February 1, 2001
We report for the first time that small molecule-based radiodiodinated ligands, showing selective
binding to Aâ aggregates, cross the intact blood-brain barrier by simple diffusion. Four novel
ligands showing preferential labeling of amyloid aggregates of Aâ(1-40) and Aâ(1-42) peptides,
commonly associated with plaques in the brain of people with Alzheimer’s disease (AD), were
developed. Two 125I-labeled styrylbenzenes, (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)-
styrylbenzene, 12 (ISB), and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene,
13 (IMSB), and two 125I-labeled thioflavins, 2-[4′-(dimethylamino)phenyl]-6-iodobenzothiazole,
18a (TZDM), and 2-[4′-(4′′-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, 18b (TZPI), were
prepared at a high specific activity (2200 Ci/mmol). In vitro binding studies of these ligands
showed excellent binding affinities with Kd values of 0.08, 0.13, 0.06, and 0.13 nM for aggregates
of Aâ(1-40) and 0.15, 0.73, 0.14, and 0.15 nM for aggregates of Aâ(1-42), respectively. Inter-
estingly, under a competitive-binding assaying condition, different binding sites on Aâ(1-40)
and Aâ(1-42) aggregates, which are mutually exclusive, were observed for styrylbenzenes and
thioflavins. Autoradiography studies of postmortem brain sections of a patient with Down’s
syndrome known to contain primarily Aâ(1-42) aggregates in the brain showed that both
[
125I]18a and [125I]18b labeled these brain sections, but [125I]13, selective for Aâ(1-40)
aggregates, exhibited very low labeling of the comparable brain section. Biodistribution studies
in normal mice after an iv injection showed that [125I]18a and [125I]18b exhibited excellent
brain uptake and retention, the levels of which were much higher than those of [125I]12 and
[
[
125I]13. These findings strongly suggest that the new radioiodinated ligands, [125I]12 (ISB),
125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), may be useful as biomarkers for studying
Aâ(1-40) as well as Aâ(1-42) aggregates of amyloidogenesis in AD patients.
In tr od u ction
lular fibrillar Aâ aggregates in the brain may be a
pivotal event in AD pathogenesis.2,4-6 Various ap-
proaches in trying to inhibit the production and reduce
the extent of accumulation of fibrillar Aâ in the brain
are currently being evaluated as potential therapies for
AD.7-12 It is therefore of great interest to develop
ligands that specifically bind fibrillar Aâ aggregates.
Since extracellular SPs are accessible targets, these new
ligands could be used as in vivo diagnostic tools and as
probes to visualize the progressive deposition of Aâ in
studies of AD amyloidogenesis in living patients.
To this end, several interesting approaches for devel-
oping fibrillar Aâ aggregate specific ligands have been
reported.13-20 The most attractive approach is based on
highly conjugated chrysamine-G (CG) and Congo red
(CR) (see Figure 1), and the latter has been used for
fluorescent staining of SPs and NFTs in postmortem AD
brain sections.13,21 The inhibition constants (Ki) for
binding to fibrillar Aâ aggregates of CR, CG, and 3′-
bromo and 3′-iodo derivatives of CG are 2800, 370, 300,
and 250 nM, respectively.18 These compounds have been
shown to bind to Aâ(1-40) peptide aggregates in vitro
as well as to fibrillar Aâ deposits in AD brain sections.18
The unique interaction between fibrillar Aâ ag-
gregates and small negatively charged, highly conju-
gated ligands may represent a novel opportunity for
designing specific ligands for in vivo imaging of amy-
Alzheimer’s disease (AD) is a progressive neurode-
generative disorder characterized by cognitive decline,
irreversible memory loss, disorientation, and language
impairment. Postmortem examination of AD brain
sections reveals abundant senile plaques (SPs) com-
posed of amyloid-â (Aâ) peptides and numerous neu-
rofibrillary tangles (NFTs) formed by filaments of highly
phosphorylated tau proteins (for recent reviews and
additional citations, see ref 1-3). Familial AD (FAD) is
caused by multiple mutations in the Aâ precursor
protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2)
genes. While the exact mechanisms underlying AD are
not fully understood, all pathogenic FAD mutations
studied thus far increase production of the more amy-
loidogenic 42-43-amino acid form of the Aâ peptide.
Thus, at least in FAD, dysregulation of Aâ production
appears to be sufficient to induce a cascade of events
leading to neurodegeneration. Indeed, the amyloid
cascade hypothesis suggests that formation of extracel-
* To whom correspondence should be addressed: Department of
Radiology, University of Pennsylvania, Room 305, 3700 Market St.,
Philadelphia, PA 19104. Telephone: (215) 662-3096. Fax: (215) 349-
5035. E-mail: kunghf@sunmac.spect.upenn.edu.
† Department of Radiology.
‡ Department of Pathology and Laboratory Medicine.
§ Department of Pharmacology.
10.1021/jm010045q CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/04/2001

1906 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Zhuang et al.
F igu r e 1. Chemical structures of chrysamine G (CG), Congo Red (CR), X-34, thioflavin T, and 6 (BSB).
loid.15,16 Tracers labeled with 123I (T1/2 ) 13 h, 159 keV
γ-ray)18,22-25 or 99mTc (T1/2 ) 6 h, 140 keV γ-ray)20
may be useful as single-photon emission computed
tomography (SPECT) imaging agents for detection and
eventually quantification of fibrillar Aâ aggregates in
the brains of living patients. Initial attempts to de-
velop 99mTc-labeled CG and CR derivatives were re-
ported.14,20,26,27 They are too large (molecular weight >
700), making them unlikely candidates for penetrating
the intact blood-brain barrier, and therefore, they are
not suitable as imaging agents for detecting fibrillar Aâ
aggregates in patients with AD. Recently, radiolabeled
Aâ peptides have been reported as potential imaging
agents;28,29 however, the usefulness of these peptides as
brain imaging agents remains to be investigated.
Recently, a new compound, X-34, has been produced
by replacing the diazo group of CG with a simple vinyl
group and substituting one phenyl for the biphenyl
group (see Figure 1).30 X-34 showed excellent binding
to fibrillar Aâ aggregates, and in postmortem AD brain
tissue, it displays intense fluorescent staining associated
with SPs and NFTs. Significantly, this substitution has
several advantages: (i) decreased molecular weight,
which may increase the extent of penetration of tissue;
(ii) improved in vitro stability; and (iii) the fact that
derivatives could be prepared via a versatile reaction
scheme, which is amenable to additional substitution.
X-34 is a potentially useful ligand for studying fibrillar
Aâ aggregates, but the chemical synthesis of this
compound or the iodinated derivatives has not been
published. Thus, to develop better and more versatile
ligands for studies of fibrillar Aâ aggregates, we initially
prepared a bromo derivative, 6 (BSB), which displayed
exquisite properties as a novel fluorescent probe for
highly specific labeling of fibrillar Aâ aggregates.31 BSB
labels Aâ plaques in human AD brain sections with high
sensitivity and specificity, and it permeates living cells
in culture and binds specifically to intracellular Aâ
aggregates. Additionally, it crosses the blood-brain
barrier and labels numerous AD-like Aâ plaques (as
detected by fluorescence) throughout the brain of trans-
genic mice following iv injection. To characterize 6 (BSB)
further, we prepared the corresponding 125I-labeled
derivatives 12 (ISB) and 13 (IMSB).
in the AD brain.32 These compounds are known to bind
amyloid specifically. Similar series of thioflavin com-
pounds have been reported as potential antitumor
agents.33,34 They are based on benzothiazole, which is
relatively small in molecular size. However, the thiofla-
vins contain an ionic quaternary amine, which is
permanently charged and unfavorable for brain uptake.
Thus, two neutral derivatives of thiazoles, 2-[4′-(di-
methylamino)phenyl]-6-iodobenzothiazole, 18a (TZDM),
and 2-[4′-(4′′-methylpiperazin-1-yl)phenyl]-6-iodoben-
zothiazole, 18b (TZPI), were designed as potential
ligands for imaging Aâ aggregates in the AD brain.
Reported herein are syntheses and initial character-
ization of these novel radioiodinated ligands, [125I]12
(ISB), [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b
(TZPI), as selective probes that are useful for detecting
amyloid aggregates in the brain by in vivo and in vitro
techniques.
Resu lts a n d Discu ssion
The key step for synthesis of styrylbenzenes is the
Wittig reaction between aldehyde 135 (prepared from
5-formylsalicylic acid) and triphenyl phosphonium salt,
236 or bis(diethyl phosphonate), 3 (Scheme 1). Both
Wittig reagents, 2 and 3, were readily prepared from
2-bromo-p-xylene. When the triphenylphosphonium salt,
2, was used for the Wittig reaction, all four possible
isomers [(E,E)-4, (Z,Z)-4, (E,Z)-4, and (Z,E)-4] were
formed, of which (E,E)-4, the trans-trans isomer, pre-
cipitated from the reaction mixture in 11% yield.
Alternatively, when bis(diethyl phosphonate), 3, was
used instead of the triphenylphosphonium salt, 2, for
the Wittig-Horner reaction, (E,E)-4 was obtained as the
sole product in 45% yield, and no other isomers were
detected from the reaction mixture (Scheme 1). The
selectivity of producing only the trans (E) isomer of
stilbenes by using dialkyl phosphonates has been re-
ported previously.37 However, for this particular series
of styrylbenzenes, the chemical synthesis via a Wittig
reaction producing only the trans-trans (E,E) isomer by
using bis(diethyl phosphonate) has not been reported.
The later procedure using bis(diethyl phosphonate), 3,
not only increased the yield of the trans-trans isomer
but also eliminated the time-consuming chromato-
graphic purification. Therefore, it is the method of choice
to prepare the starting compound (E,E)-4. All of the
subsequent styrylbenzenes are in the trans-trans (E,E)
form; therefore, the (E,E) designation is discontinued
for all of the other compounds.
Potential ligands for detecting Aâ aggregates in the
living brain must cross the intact blood-brain barrier.
Thus, brain uptake can be improved by using ligands
with a smaller molecular size and increased lipophilic-
ity. To generate ligands with those properties, we looked
to highly conjugated thioflavins S and T, which are
commonly used as dyes for staining the Aâ aggregates
Compound 4 was readily converted to 6 (BSB) in two
steps: demethylation with BBr3 in CH2Cl2 and hydroly-

Probes for Binding Amyloid Aggregates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1907
Sch em e 1
Sch em e 2
sis of the methyl esters in KOH and EtOH. The
methoxy/acid derivative 9 was obtained by hydrolysis
of 4 by KOH and EtOH. When 4 was reacted with bis-
(tributyltin) using Pd(0) as the catalyst, the correspond-
ing tributyltin derivative 7 was obtained (21% yield).
The tributyltin derivative 7 was reacted with iodine in
chloroform at room temperature to give the iodo deriva-
tive 10 in 87% yield. Hydrolysis of 10 in KOH and EtOH
produced 13 (IMSB) in good yield (84%). The methyl
groups of the phenolic ether on 10 were readily removed
by reacting with BBr3 to give 11. Subsequently, 11 was
converted to 12 (ISB) using the same base-catalyzed
hydrolysis. The other tributyltin derivative 14 was
prepared from the corresponding bromo compound 5,
by use of a bromo to tributyltin exchange reaction
catalyzed by Pd(0) (25% yield). Hydrolysis of the ester
groups of 14 was again achieved in KOH and EtOH to
afford 15 in 95% yield (Scheme 1). It is important to
note that the tributyltin derivatives (7, 8, 14, and 15)
are generally sensitive to strong acid-catalyzed decom-
position; therefore, it is preferable to prepare the
tributyltin derivatives after demethylation in which the
acidic BBr3 was used (e.g., 14 and 15). The methyl ester
groups of 7 and 14 were removed by a base (KOH)-
catalyzed hydrolysis, which led to the desired com-
pounds, 8 and 15. The tributyltin derivatives 8 and 15
can be readily used as the starting materials for
radioiodination in preparation of [125I]12 (ISB) and [125I]-
13 (IMSB).
Preparation of benzothiazole derivatives was achieved
by reactions described in Scheme 2. Heating 5-bromo-
2-aminobenzenethiol38,39 and 4-(dimethylamino)benzal-
dehyde or 4-(4-methylpiperazin-1-yl)benzaldehyde40 in
DMSO produced benzothiazoles, 16a and 16b. Using the
same Pd(0)-catalyzed bromo to tributyltin exchange
reaction, these two bromo derivatives were successfully
converted to the corresponding tributyltin derivatives,
17a and 17b. They were successfully used in an iodo-
destannylation reaction to produce the corresponding
iodinated compounds, 18a and 18b (yields were between
25 and 35%; the reactions were not optimized). Thus,
the tributyltin derivatives served two useful purposes.
(i) They served as intermediates for converting bromo
to iodo derivatives, and (ii) they are also useful as
starting material for preparation of radioiodinated “hot”
ligands.
Ra d iola belin g. The novel radioiodinated ligands,
[
[
125I]12 (ISB), [125I]13 (IMSB), [125I]18a (TZDM), and
125I]18b (TZPI), were successfully prepared from the
corresponding tributyltin derivatives by an iododestan-
nylation reaction, which resulted in no-carrier-added
tracers (specific activity comparable to that of Na125I,
2200 Ci/mmol). The radiochemical identities of the
radioiodinated ligands were verified by co-injection with
the nonradioactive compounds by their HPLC profiles.
All of the radioiodinated ligands displayed excellent in
vitro stability for up to 2 months (>95% radiochemically
pure as determined by HPLC).

1908 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Zhuang et al.
F igu r e 2. Scatchard plots of four ligands, [125I]12 (ISB), [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), binding to
aggregates of Aâ(1-40) and Aâ(1-42) peptides showing very high affinity. Under the assaying condition, both of the styrylbenzenes,
[
125I]12 and [125I]13, detected about 2-3-fold more binding sites (Bmax values in femtomoles per nanomolar peptide as indicated
by the intercept on the x-axis) on Aâ(1-40) aggregates than on Aâ(1-42) aggregates, while the binding preference of thiazoles,
[
125I]18a and [125I]18b, displayed binding for both.
Ta ble 1. Inhibition Constants (Ki, nM) of Compounds on
Ligand Binding to Aggregates of Aâ(1-40) and Aâ(1-42) at
25 °Ca
Bin d in g to Aâ Aggr ega tes of Aâ(1-40) a n d Aâ-
(1-42). Using these four radioiodinated probes with
high specific activity, studies of the binding of the
ligands to aggregates of Aâ(1-40) and Aâ(1-42) pep-
tides in solution were carried out. All of the ligands
displayed a saturable binding. Transformation of the
saturation binding of all of the new ligands to Scatchard
plots gave linear plots suggesting one-site binding. The
Kd values estimated for [125I]12, [125I]13, [125I]18a , and
aggregates of Aâ(1-40) aggregates of Aâ(1-42)
vs [125I]13 vs[ 125I]18a vs [125I]13 vs [125I]18a
compound
(IMSB)
(IMSB)
chrysamine G 0.14 ( 0.04 >1000
0.4 ( 0.1 >2000
0.8 ( 0.2 >1000
13 (IMSB)
thioflavin T
16a
0.17 ( 0.03 >1000
>9000
>2000
>2000
>2000
>2000
116 ( 20 >4000
294 ( 40
1.9 ( 0.3 >3000
1.6 ( 0.5 >2400
0.9 ( 0.2 >5000
5.4 ( 0.7 >2000
0.8 ( 0.3
5.0 ( 0.8
2.2 ( 0.4
6.4 ( 0.7
[
125I]18b were 0.08, 0.13, 0.06, and 0.13 nM for ag-
16b
18a
18b
gregates of Aâ(1-40) and 0.15, 0.73, 0.14, and 0.15 nM
for aggregates of Aâ(1-42), respectively (Figure 2). More
importantly, under the assaying conditions, both of the
styrylbenzenes, [125I]12 and [125I]13, detected about
2-3-fold more binding sites (Bmax values in femtomoles
per nanomolar peptide as indicated by the intercept on
the x-axis) on Aâ(1-40) aggregates than on Aâ(1-42)
aggregates, while benzothiazoles, [125I]18a and [125I]18b,
displayed an opposite preference, showing 4-5-fold
more binding sites on Aâ(1-42) aggregates than on Aâ-
(1-40) aggregates (Figure 2).
a
Values are means ( standard error of the mean of three
independent experiments, each in duplicate.
(Table 1). In comparison, Ki values on competition of
CG and IMSB against [125I]13, as well as those for
benzothiazoles (i.e., 16a , 16b, 18a , and 18b) against
[
125I]18a , displayed the expected and consistent poten-
cies on both Aâ(1-40) and Aâ(1-42) aggregates. These
Ki values presented in Table 1 provide compelling
evidence that there are distinctive and mutually exclu-
sive binding sites on Aâ(1-40) and Aâ(1-42) aggregates
for these two series of compounds.
Interestingly, when styrylbenzene derivatives, i.e., CG
and IMSB, were evaluated for their competition against
[
125I]18a (a benzothiazole derivative) binding on Aâ(1-
40) and Aâ(1-42) aggregates, high Ki values were
observed (see Table 1), indicating poor binding competi-
tion. Similarly, high Ki values were obtained for 16a
and 16b, and 18a and 18b (benzothiazole derivatives),
against binding of [125I]13 (a styrylbenzene derivative)
The unique selectivity demonstrated by these two sets
of ligands based on structurally divergent compounds
(styrylbenzenes and benzothiazoles) is very striking. To
our knowledge, this is the first time such small molecule
probes demonstrated specific binding at a sub-nanomo-

Probes for Binding Amyloid Aggregates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1909
F igu r e 3. (A) Film autoradiography showing binding of [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI) to amyloid plaques
of the formalin-fixed brain sections of a Down’s syndrome patient containing mainly Aâ(1-42) aggregates. The plaque labeling
was clearly visualized in the brain sections labeled with [125I]18a (TZDM) and [125I]18b (TZPI), both of which primarily label
Aâ(1-42) aggregates, while an adjacent section labeled with [125I]13 (IMSB) showed very little labeling, because IMSB is more
selective toward Aâ(1-40) aggregates. (B) Comparable brain sections of the same subject were immunostained by antibodies
specific for Aâ(1-40) (Ba27) and Aâ(1-42) (BC05). The autoradiography images of [125I]18a (TZDM) and [125I]18b (TZPI) displayed
high concentrations of Aâ(1-42) aggregates in the brain section, consistent with the high concentration of Aâ(1-42) shown by
the immunostaining. (C) The level of plaque labeling of [125I]18a (TZDM) was significantly reduced in the presence of 20 µM
thioflavin T (TT), indicating TZDM competes with TT for the same binding sites.
lar concentration to Aâ(1-40) and Aâ(1-42) aggregates.
It is known that when Aâ(1-40) and Aâ(1-42) peptides
are aggregated, they assemble into a fibrillary coil
containing â-sheet structures. It is evident that under
the conditions used in this study Aâ(1-40) and Aâ(1-
42) peptides form aggregates containing two different
binding pockets for styrylbenzenes and benzothiazoles
(thioflavins). Clearly, there are significant structural
differences between these two aggregates, and at least
two different binding pockets, possibly more, exist on
these aggregates.41,42 No detailed structural information
is currently available on the potential binding sites, nor
do we know the full extent of functional linkages of these
styrylbenzene or benzothiazole binding sites on Aâ
aggregates. It has been suggested previously that bind-
ing of small molecules to the Aâ aggregates may prevent
further aggregation and reduce their toxicity.11,41-46
There are still many unanswered questions. Are there
any other specific sites with structural requirements
which differ from those of styrylbenzene and benzothia-
zole? Are these sites just “incidental inventions of
Mother Nature”? Further testing is warranted to in-
vestigate any functional or physiological roles of these
binding sites in amyloid plaque (aggregates) formation
and cellular toxicity. As such, the feasibility of using
these ligands with high binding affinity as potential
drugs for treatment of AD can be explored.
The radioiodinated probes, [125I]13 (IMSB) and [125I]-
18a (TZDM), may allow the use of high-throughput
screening for rapidly selecting agents with the requisite
binding affinity (at sub-nanomolar concentrations) and
selectivity for aggregated amyloid peptides in solution.
Indeed, this strategy may serve as the initial screen for
the selection of compounds with the potential to bind
to amyloid plaques in vivo.
La belin g of Aâ Aggr ega tes in Hu m a n Br a in
Section s w ith Ra d ioiod in a ted Liga n d s. Since the
in vitro binding assays demonstrated the selectivity of
the radioiodinated probes for aggregates composed of
synthetic Aâ(1-40) and Aâ(1-42) peptides, we asked
if these probes showed similar selectivity to Aâ ag-
gregates found in the human brain. To test this, we
performed autoradiography using tissue sections from
a postmortem Down’s syndrome brain. We chose to use
the Down’s syndrome brain since patients with Down’s
syndrome invariably develop neuropathological changes
characteristic of AD, and these changes begin with the
deposition of SPs containing predominantly Aâ(1-
42)47,48 (Figure 3). The sections were immunostained
with antibodies Ba27 and BC05 specific for Aâ(1-40)
and Aâ(1-42), respectively. It is apparent that [125I]13
(IMSB) displayed only weak binding to these plaques
(Figure 3) with only a few spots of plaque signals, but
no intense labeling. This is consistent with the fact that

1910 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Zhuang et al.
Ta ble 2. Biodistribution in Mice after iv Injection of 125I-Labeled Styrylbenzenes (12 or 13) or 125I-Labeled Thioflavins (18a or 18b)
and the Partition Coefficients (PC) of the Ligandsa
organ
5 min
30 min
125I]12 (ISB) (PC ) 35)
60 min
[
blood
heart
muscle
lung
kidney
spleen
liver
7.57 ( 1.23
0.33 ( 0.06
5.68 ( 0.79
0.81 ( 0.12
1.67 ( 0.42
0.25 ( 0.10
42.68 ( 2.50
4.02 ( 0.17
0.27 ( 0.04
2.98 ( 0.34
0.17 ( 0.02
3.90 ( 0.66
0.30 ( 0.03
0.77 ( 0.05
0.13 ( 0.01
30.16 ( 4.24
4.13 ( 0.44
0.06 ( 0.01
2.59 ( 0.18
0.13 ( 0.03
2.98 ( 0.25
0.25 ( 0.01
0.63 ( 0.17
0.07 ( 0.01
19.12 ( 3.11
3.66 ( 0.29
0.04 ( 0.01
skin
brain
[
125I]13 (IMSB) (PC ) 1.1)
5.19 ( 1.01
blood
heart
muscle
lung
kidney
spleen
liver
22.52 ( 6.03
0.43 ( 0.11
3.16 ( 1.37
0.89 ( 0.18
1.31 ( 0.25
0.17 ( 0.02
47.85 ( 4.61
1.56 ( 0.63
0.14 ( 0.03
2.17 ( 0.69
0.12 ( 0.04
1.30 ( 0.45
0.16 ( 0.05
0.23 ( 0.05
0.03 ( 0.01
6.14 ( 2.67
1.02 ( 0.34
0.02 ( 0.01
0.18 ( 0.04
1.82 ( 0.28
0.31 ( 0.08
0.46 ( 0.09
0.07 ( 0.03
24.47 ( 4.30
1.44 ( 0.36
0.03 ( 0.00
skin
brain
organ
2 min
30 min
60 min
6 h
24 h
[
125I]18a (TZDM) (PC ) 70)
blood
heart
muscle
lung
kidney
spleen
liver
15.74 ( 6.06
1.79 ( 0.39
17.81 ( 2.29
5.91 ( 1.39
5.42 ( 0.57
0.59 ( 0.04
31.62 ( 2.38
3.2 ( 0.45
3.26 ( 0.05
0.20 ( 0.01
12.89 ( 1.06
0.72 ( 0.16
2.24 ( 0.98
0.20 ( 0.05
10.93 ( 2.34
7.4 ( 0.60
3.79 ( 0.19
0.17 ( 0.02
12.81 ( 2.78
0.78 ( 0.07
2.23 ( 0.41
0.17 ( 0.06
9.21 ( 3.05
12.8 ( 1.13
1.57 ( 0.24
1.44 ( 0.05
0.05 ( 0.01
2.93 ( 0.02
0.21 ( 0.04
0.54 ( 0.12
0.04 ( 0.00
1.52 ( 0.30
3.8 ( 0.07
0.29 ( 0.09
0.01 ( 0.00
0.33 ( 0.08
0.03 ( 0.01
0.06 ( 0.00
0.01 ( 0.00
0.3 ( 0.07
0.2 ( 0.07
0.04 ( 0.01
skin
brain
0.6 ( 0.11
0.9 ( 0.29
0.65 ( 0.11
[
125I]18b (TZPI) (PC ) 312)
blood
heart
muscle
lung
kidney
spleen
liver
8.02 ( 0.82
2.19 ( 0.43
13.24 ( 3.08
12.80 ( 1.54
6.46 ( 1.80
0.89 ( 0.14
28.84 ( 3.77
4.06 ( 0.52
1.50 ( 0.10
5.15 ( 0.23
0.69 ( 0.02
18.82 ( 1.16
4.14 ( 0.41
8.06 ( 1.26
0.87 ( 0.22
21.22 ( 5.86
6.07 ( 0.43
1.59 ( 0.19
4.16 ( 0.28
0.66 ( 0.06
19.46 ( 0.30
3.38 ( 0.33
7.99 ( 1.45
0.60 ( 0.06
17.20 ( 2.49
5.63 ( 0.67
1.89 ( 0.43
1.49 ( 0.26
0.22 ( 0.06
6.65 ( 0.67
1.15 ( 0.31
3.62 ( 0.12
0.22 ( 0.08
5.79 ( 1.24
3.17 ( 0.27
1.08 ( 0.08
0.41 ( 0.09
0.08 ( 0.01
2.44 ( 0.37
0.44 ( 0.06
1.15 ( 0.22
0.08 ( 0.01
3.05 ( 0.87
1.64 ( 0.47
0.91 ( 0.08
skin
brain
a
The % dose/organ; average of three or four mice ( standard deviation.
this section contained only a very small number of Aâ-
(1-40) positive plaques [as confirmed by staining with
an Aâ(1-40) specific monoclonal antibody, Ba27], and
intramolecular hydrogen bonding between the OH group
of phenol and the carboxylic group of [125I]12 (ISB).
Intramolecular hydrogen bonding of similar structures
has been previously reported.49
[
125I]13 (IMSB) labels the Aâ(1-40) aggregates prefer-
entially. The level of nonspecific binding observed with
To test the permeability through the intact blood-
brain barrier, these new agents were injected into
normal mice. The brain uptake 5 min post iv injection
showed relatively low values (0.27 and 0.14% dose/organ
for [125I]12 and [125I]13, respectively). The blood level 5
min post iv injection was relatively high, which is very
unfavorable (Table 2). For a brain perfusion tracer, the
uptake in the rat brain at 2 min after a bolus iv injection
is expected to be 2-3% dose/organ (to be useful as
potential imaging agents of the brain, the minimum
brain uptake will be 0.5% dose/organ). The brain reten-
tion level of [125I]12 and [125I]13 at later time points was
low. The results suggest that these two styrylbenzenes
showed relatively low initial permeability on crossing
the blood-brain barrier. The potential usefulness of
these agents for in vivo imaging after a bolus iv injection
appears to be limited.
[
125I]12 (ISB) was relatively high; therefore, it was not
used for this part of the experiment. The distinctive
labeling of the plaques by [125I]18a (TZDM) and [125I]-
18b (TZPI) resulted in an excellent autoradiographic
visualization of the amyloid plaques [mainly Aâ(1-42)
aggregates, stained positively by BC05] in the sections
from this patient (Figure 3). The results are consistent
with the in vitro observation that these benzothiazoles
bind to Aâ(1-42) aggregates with a high affinity.
Biod istr ibu tion of New Liga n d s in Nor m a l Mice.
One of the key prerequisites for an in vivo imaging agent
of the brain is the ability to cross the intact blood-brain
barrier after a bolus iv injection. The lipophilicities of
[
125I]12 (ISB) and [125I]13 (IMSB) were 35 and 1.1,
respectively (measured by a partition between 1-octanol
and pH 7.4 phosphate buffer). It is interesting to note
that the [125I]ISB exhibited a higher partition coefficient
as compared to [125I]13 (IMSB), which has two more
methyl groups. This is likely due to the presence of
Two new compounds derived from benzothiazole were
prepared to improve the brain uptake after an iv
injection. These new compounds are derivatives of

Probes for Binding Amyloid Aggregates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1911
Hz, 2H), 3.95-4.14 (m, 8H), 7.20 (d, J ) 7.9 Hz, 1H), 7.40 (d,
d, J ) 2.0, 7.9 Hz, 1H), 7.50 (s, 1H).
thioflavins, but contain no quaternary ammonium ion;
therefore, they are relatively small in size, neutral, and
lipophilic (PC ) 70 and 312 for [125I]18a and [125I]18b,
respectively). Initial brain uptake of [125I]18a and [125I]-
18b in mice after an iv injection was 0.67 and 1.50%
dose/organ, respectively. The brain uptake peaked at 60
min for both compounds with a maximum brain uptake
of 1.57 and 1.89% dose/organ, respectively. The blood
levels are relatively low throughout the time course that
was evaluated. For this series of ligands, the level of
specific uptake in the brain is relatively high and the
retention in the brain is long. The new thioflavins may
provide better candidates for further development of the
in vivo imaging agents critically important for evalua-
tion of Alzheimer’s disease. A more detailed biodistri-
bution study using transgenic mice containing an excess
amount of Aâ aggregate deposition in the brain is
currently under way. The preliminary results of such a
study appear to further confirm the utility of these new
agents. The data will be published elsewhere soon.
Development of Aâ aggregate specific tracers may
lead to simple and effective tools for the early and
accurate diagnosis of AD as well as for monitoring the
progression of this disorder in patients.6,50 SPECT
imaging may also provide a simple technique for study-
ing the efficacy of drugs designed to retard or reverse
the formation of fibrillar Aâ aggregates in AD patients.
Currently, there is no specific imaging agent available
for direct mapping of fibrillar Aâ aggregates in vivo.
Given the highly pressing needs for such agents, the
timely development of imaging agents for monitoring
the burden of senile plaques in vivo is widely regarded
by basic and clinical investigators as one of the highest
priorities for AD research.50 We have demonstrated a
proof of concept that development of small molecule-
based tracers showing a high level of selective binding
to the Aâ aggregates in the brain may be feasible.
In conclusion, four novel ligands, [125I]12 (ISB), [125I]-
13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), were
developed. They may be useful as biomarkers for studies
of Aâ(1-40) as well as Aâ(1-42) aggregates for detect-
ing amyloid aggregates in the brain by in vivo and in
vitro techniques.
1-Br om o-2,5-bis(3-m eth oxyca r bon yl-4-m eth oxy)styr yl-
ben zen e (4). (1) Meth od A. To a mixture of 1 (1.16 g, 6.0
mmol) and 2 (2.6 g, 3.0 mmol) in MeOH (12 mL) was added a
solution of sodium methoxide (1.3 mL, 25 wt % in MeOH)
dropwise at 0 °C in an ice bath. The mixture was stirred at
room temperature overnight. The solid that formed was
filtered and washed with methanol to give (E,E)-4 (175 mg,
10.9%).
(2) Meth od B. To a mixture of 1 (2.7 g, 13.9 mmol) and 3
(3.2 g, 7.0 mmol) in MeOH (20 mL) was added a solution of
sodium methoxide (5 mL, 25 wt % in MeOH) dropwise at 0 °C
in an ice bath. The mixture was stirred under reflux overnight.
The precipitate was collected by filtration and washed with
1
methanol to give (E,E)-4 (1.67 g, 44.5%). H NMR (500 MHz,
CDCl3): δ 3.907 (s, 3H, CH3O), 3.909 (s, 3H), 3.915 (s, 3H),
3.919 (s, 3H), 6.91 (d, J ) 16.3 Hz, 1H), 6.97 (d, J ) 8.5 Hz,
1H), 6.98 (d, J ) 8.5 Hz, 1H), 6.99 (d, J ) 16.4 Hz, 1H), 7.03
(d, J ) 16.3 Hz, 1H), 7.34 (d, J ) 6.2 Hz, 1H), 7.40 (d, J ) 8.2
Hz, 1H), 7.59 (d, d, J ) 8.5, 2.1 Hz, 1H), 7.60 (d, J ) 8.3 Hz,
1H), 7.65 (d, d, J ) 8.7, 2.2 Hz, 1H), 7.69 (s, 1H), 7.94 (s, 1H),
7.95 (s, 1H). MS: m/z 559 (MNa+). HRMS: m/z calcd for
C
28H26BrO6 (MH+) 537.0913, found 537.0963.
1-Br om o-2,5-bis(3-m eth oxyca r bon yl-4-h yd r oxy)styr yl-
ben zen e (5). To a solution of (E,E)-4 (730 mg, 1.36 mmol) in
CH2Cl2 (250 mL) was added BBr3 (10.5 mL, 1 M in hexane)
dropwise at -78 °C in a dry ice/acetone bath. The mixture was
allowed to warm to room temperature. Water was added, while
the reaction mixture was cooled at 0 °C in an ice bath. The
mixture was extracted with CH2Cl2. The organic phase was
dried and filtered. The filtrate was concentrated to afford 636
mg of 5 (92%). The analytical sample was obtained by
preparative thin-layer chromatography (PTLC) (1:3 EtOAc/
1
hexane mixture). H NMR (200 MHz, CDCl3): δ 3.99 (s, 6H),
6.99 (d, J ) 16.3 Hz, 1H), 6.98 (d, J ) 16.5 Hz, 1H), 7.01 (d,
d, J ) 8.3, 2.6 Hz, 2H), 7.04 (d, J ) 16.3 Hz, 1H), 7.33 (d, J )
16.5 Hz, 1H), 7.40 (d, J ) 8.7 Hz, 1H), 7.60-7.73 (m, 4H), 7.97
(s, 2H), 10.81 (s, 1H), 10.83 (s, 1H). MS: m/z 509 (MH+).
1-Br om o-2,5-bis(3-h yd r oxyca r bon yl-4-h yd r oxy)styr yl-
ben zen e (6) (BSB). To a solution of 5 (534 mg, 1.0 mmol) in
EtOH (60 mL) was added KOH (1.2 g) in a solid form. The
mixture was stirred under reflux for 5 h. The cold mixture
was acidified with HCl (10% solution) and extracted with a
mixed solvent (9:1 CH2Cl2/MeOH mixture). The organic phase
was dried and filtered. The filtrate was concentrated to give
480 mg of 6 (BSB) (95%). An analytical sample was obtained
by PTLC (9:1:0.05 CH2Cl2/MeOH/HOAc mixture). 1H NMR
(200 MHz, MeOD): δ 6.96 (d, d, J ) 3.4, 8.7 Hz, 2H), 6.99 (d,
J ) 16.3 Hz, 1H), 7.12 (d, J ) 16.3 Hz, 1H), 7.17 (d, J ) 16.3
Hz, 1H), 7.34 (d, J ) 16.3 Hz, 1H), 7.56 (d, J ) 8.4 Hz, 1H),
7.73-7.80 (m, 4H), 8.02 (t, J ) 2.6 Hz, 2H). MS: m/z 480 (M+).
Anal. C24H17BrO6‚0.5H2O: C, H.
Exp er im en ta l Section
Reagents used in syntheses were purchased from Aldrich
Co. or Fluka Co., and were used without further purification
unless otherwise indicated. Anhydrous Na2SO4 was used as a
drying agent. 1H NMR spectra were obtained on Bruker
spectrometers (Bruker DPX 200 and AMX500). Chemical shifts
are reported as δ values (parts per million) relative to internal
TMS. Coupling constants are reported in hertz. The multiplic-
ity is defined by s (singlet), d (doublet), t (triplet), br (broad),
and m (multiplet). Mass spectrometry was performed by the
Mass Spectrometry Center, Department of Chemistry, Uni-
versity of Pennsylvania. Elementary analysis was performed
by the Analysis Laboratory, Department of Chemistry, Uni-
versity of Pennsylvania.
1-(Tr ibu tylstan n yl)-2,5-bis(3-m eth oxycar bon yl-4-m eth -
oxy)styr ylben zen e (7). A mixture of 4 (200 mg, 0.37 mmol),
bis(tributyltin) (0.4 mL), and Pd(Ph3P)4 (30 mg) in a mixed
solvent (20 mL, 3:1 dioxane/triethylamine mixture) was stirred
at 90 °C overnight. The solvent was removed, and the residue
was purified by PTLC (2:1 hexane/EtOAc mixture) to give 57
1
mg of product (21%). H NMR (200 MHz, CDCl3): δ 0.92 (t, J
) 7.1 Hz, 9H), 1.20-1.45 (m, 12H), 1.55-1.68 (m, 6H),3.94 (s,
12H), 6.90-7.08 (m, 6H), 7.40-7.70 (m, 5H), 7.95 (d, J ) 2.2
Hz, 1H), 7.98 (d, J ) 2.2 Hz, 1H).HRMS: m/z Calcd for C40H52
-
SnO6Na (MNa+): 771.2683; Found: 771.2686.
1-(Tr ibu tylsta n n yl)-2,5-bis(3-h yd r oxyca r bon yl-4-m eth -
oxy)styr ylben zen e (8). The same reaction as described above
for preparing 6 was employed, and 8 was obtained in 78% yield
from 7. 1H NMR (200 MHz, CDCl3): δ 0.93 (t, J ) 7.1 Hz,
9H), 1.06-1.40 (m, 12H), 1.52-1.68 (m, 6H), 3.92 (s, 6H),
6.93-7.03 (m, 6H), 7.45-7.68 (m, 6H), 7.97 (m, 1H), 8.03 (m,
1H). HRMS: m/z calcd for C38H48O6SnNa (MNa+) 743.2371,
found 743.2370.
2-Br om o-1,4-bis(d ieth ylp h osp h on a to)xylen e (3). A mix-
ture of 1-bromo-2,5-bis(bromomethyl)benzene36 (3.43 g, 10
mmol) and triethyl phosphite (3.32 g, 20 mmol) was stirred at
160 °C for 4 h. After the mixture was cooled to room
temperature, the mixture was treated under reduced pressure
to remove the low-boiling point material and the residue was
subjected to flash chromatography (95:5 CH2Cl2/MeOH mix-
1
ture) and gave 4.2 g of 3 (92%). H NMR (200 MHz, CDCl3):
1-Br om o-2,5-bis(3-h yd r oxyca r bon yl-4-m eth oxy)styr yl-
ben zen e (9). The same reaction as described above to prepare
δ 1.26 (m, 12H), 3.17 (d, J ) 20.6 Hz, 2H), 3.36 (d, J ) 20.9

1912 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Zhuang et al.
1
6 was employed, and 9 was obtained in 71% yield from 4. H
J ) 5.0 Hz, 4H), 6.96 (d, J ) 8.9 Hz, 2H), 7.54 (d, d, J ) 8.5,
1.9 Hz, 1H), 7.83 (d, J ) 8.5 Hz, 1H), 7.95 (d, J ) 8.9 Hz, 2H),
7.98 (s, 1H). HRMS: m/z calcd for C18H19BrN3S (MH+)
388.0483, found 388.0474.
NMR (200 MHz, DMSO-d6): δ 3.83 (s, 6H), 7.12 (d, d, J ) 7.7,
3.2 Hz, 1H), 7.13 (d, J ) 16.7 Hz, 2H), 7.27 (s, 1H), 7.34 (d, J
) 16.7 Hz, 2H), 7.61 (d, J ) 8.1 Hz, 1H), 7.74 (d, J ) 8.7 Hz,
2H), 7.86 (br, 4H). HRMS: m/z calcd for C26H22BrO6 (MH+)
509.0600, found 509.0635. Anal. C26H21BrO6: C, H.
2-[4′-(Dim eth yla m in o)p h en yl]-6-(tr ibu tylsta n n yl)ben -
zoth ia zole (17a ). To a solution of 2-[4′-(dimethylamino)-
phenyl]-6-bromobenzothiazole (16a ) (60 mg, 0.18 mmol) in 1,4-
dioxane (2 mL), toluene (2 mL), and triethylamine (2 mL) was
added (Bu3Sn)2 (0.2 mL) followed by Pd(Ph3P)4 (20 mg). The
mixture was stirred at 90 °C overnight. The solvent was
removed, and the residue was purified by PTLC (6:1 hexane/
EtOAc mixture) to give 33 mg of product (33.6% yield). 1H
NMR (200 MHz, CDCl3): δ 0.90 (t, J ) 7.1 Hz, 9H), 1.10 (t, J
) 8.0 Hz, 6H), 1.34 (hex, J ) 7.3 Hz, 6H), 1.57 (m, 6H), 3.05
(s, 6H), 6.74 (d, J ) 9.0 Hz, 2H), 7.50 (d, d, J ) 7.9, 0.9 Hz,
1H), 7.93 (s, 1H), 7.95 (d, J ) 8.5 Hz, 1H), 7.97 (d, J ) 9.0 Hz,
2H). HRMS: m/z calcd for C27H41N2SSn (MH+) 545.2012, found
545.2035.
1-Iod o-2,5-b is(3-m et h oxyca r b on yl-4-m et h oxy)st yr yl-
ben zen e (10). To a solution of 7 (50 mg, 0.07 mmol) in CHCl3
(20 mL) was added a solution of iodine in CHCl3 (0.5 mL, 1
M) at room temperature. The mixture was stirred at room
temperature for 1 h. The NaHSO3 solution (3 mL, 5% in water)
and KF (3 mL, 1 M in MeOH) were added successively. The
mixture was stirred for 5 min, and the organic phase was
separated. The aqueous phase was extracted with CH2Cl2, and
the combined organic phase was dried over Na2SO4, filtered,
and concentrated to give the crude product. PTLC (2:1 hexane/
1
EtOAc mixture) gave 34 mg of product (87% yield). H NMR
(200 MHz, CDCl3): δ 3.93 (s, 12H), 6.84-7.24 (m, 6H), 7.43-
7.70 (m, 4H), 7.95 (d, J ) 1.4 Hz, 2H), 7.98 (d, J ) 1.4 Hz,
1H). HRMS: m/z calcd for C28H25IO6Na (MNa+) 607.0594,
found 607.0579.
1-Iod o-2,5-b is(3-h yd r oxyca r b on yl-4-h yd r oxy)st yr yl-
ben zen e (11). The same reaction as described above to
prepare 5 was employed, and 11 was obtained in 75% yield
from 10. 1H NMR (200 MHz, CDCl3): δ 3.99 (s, 6H), 6.81-
7.22 (m, 6H), 7.46-7.70 (m, 4H), 7.95 (d, J ) 2.0 Hz, 2H), 7.98
(d, J ) 1.3 Hz, 1H), 10.80 (s, 1H), 10.83 (s, 1H).
1-Iod o-2,5-b is(3-h yd r oxyca r b on yl-4-h yd r oxy)st yr yl-
ben zen e (12). The same reaction as described above to
prepare 6 was employed, and 12 was obtained in 83% yield
from 11. 1H NMR (200 MHz, MeOD): δ 6.46-7.23 (m, 6H),
2-[4′-(4′′-Meth ylp ip er a zin -1-yl)p h en yl]-6-(tr ibu tylsta n -
n yl)ben zoth ia zole (17b). The procedure described above to
prepare 17a was employed, and 17b was obtained in 23% yield
1
from 16b. H NMR (200 MHz, CDCl3): δ 0.89 (t, J ) 7.2 Hz,
9H), 1.06 (t, J ) 8.2 Hz, 6H), 1.30 (hex, J ) 7.3 Hz, 6H), 1.57
(pen, J ) 7.2 Hz, 6H), 2.38 (s, 3H), 2.60 (m, 4H), 3.36 (t, J )
5.0 Hz, 4H), 6.96 (d, J ) 8.9 Hz, 2H), 7.52 (d, J ) 7.9 Hz, 1H),
7.93 (s, 1H), 7.95 (d, J ) 7.9 Hz, 1H), 7.98 (d, J ) 8.9 Hz, 2H).
HRMS: m/z calcd for C30H46N3SSn (MH+) 600.2434, found
600.2449.
2-[4′-(Dim eth ylam in o)ph en yl]-6-iodoben zoth iazole (18a,
TZDM). To a solution of 17a (45 mg, 0.08 mmol) in CHCl3 (10
mL) was added a solution of iodine (1 mL, 1 M in CHCl3)
dropwise at room temperature until it became dark red. The
resulting mixture was stirred at room temperature for 10 min.
The NaHSO3 solution (2 mL, 5% in water) and KF (1 mL, 1 M
in MeOH) were added successively. The mixture was stirred
for 5 min, and the organic phase was separated. The aqueous
phase was extracted with CH2Cl2, and the combined organic
phase was dried over Na2SO4, filtered, and concentrated to give
the crude product which was purified by PTLC (6:1 hexane/
EtOAc) to give 9 mg of the desired product (29% yield). 1H
NMR (200 MHz, CDCl3): δ 3.06 (s, 6H), 6.73 (d, J ) 9.0 Hz,
2H), 7.69 (s, 1H), 7.70 (s, 1H), 7.93 (d, J ) 9.0 Hz, 2H), 8.15
(s, 1H). HRMS: m/z calcd for C15H13N2IS (MH+) 380.9922,
found 380.9914. Anal. C15H14N3IS: C, H, N.
Under two different HPLC conditions (A and B), 18a
(TZDM) appeared as one single peak that was >95% pure.
System A included a Phenomenex Nucleosil Si, 10 µm, 250
mm × 4.6 mm column, a 3:1 hexane/ethyl acetate mixture as
the solvent, a flow rate of 0.8 mL/min, and a retention time of
4.9 min.
System B included a Phenomenex Luna C8, 5 µm, 250 mm
× 4.6 mm column, a 9:1 acetonitrile/5 mM 3,3-dimethylglut-
arate mixture as the solvent (pH 7.0), a flow rate of 1.0 mL/
min, and a retention time of 7.1 min.
7.49-7.74 (m, 4H), 7.98 (m, 3H). HRMS: m/z calcd for C24H17
-
IO6Na (MNa+) 550.9968, found 550.9941. Anal. C24H17IO6‚
0.5H2O: C, H.
1-Iod o-2,5-b is(3-h yd r oxyca r b on yl-4-m et h oxy)st yr yl-
ben zen e (13). The same reaction as described above to
prepare 6 was employed, and 13 was obtained in 84% yield
1
from 10. H NMR (200 MHz, CDCl3): δ 10 (s, 6H), 6.88-7.29
(m, 6H), 7.43 (d, J ) 8.2 Hz, 1H), 7.55 (d, J ) 8.2 Hz, 1H),
7.67 (d, d, J ) 9.0, 2.3 Hz, 1H), 7.77 (d, d, J ) 9.0, 2.3 Hz,
1H), 7.97 (d, J ) 1.4 Hz, 1H), 8.29 (d, J ) 1.4 Hz, 1H), 8.30 (d,
J ) 1.4 Hz, 1H). HRMS: m/z calcd for C26H21IO6Na (MNa+)
579.0281, found 579.0292. Anal. C26H21IO6: C, H.
1-(Tr ib u t ylst a n n yl)-2,5-b is(3-m et h oxyca r b on yl-4-h y-
d r oxy)styr ylben zen e (14). The same reaction as described
above to prepare 7 was employed, and 14 was obtained in 25%
yield from 5. 1H NMR (200 MHz, CDCl3): δ 0.80 (t, J ) 7.2
Hz, 9H), 1.00-1.36 (m, 12H), 1.43-1.62 (m, 6H), 3.92 (s, 6H),
6.85 (d, J ) 15.8 Hz, 2H), 6.94 (m, 2H), 6.95 (d, J ) 15.8 Hz,
2H), 7.39-7.64 (m, 5H), 7.89 (d, J ) 2.1 Hz, 2H), 10.71 (s,
2H).
1-(Tr ib u t ylst a n n yl)-2,5-b is(3-h yd r oxyca r b on yl-4-h y-
d r oxy)styr ylben zen e (15). The same reaction as described
above to prepare 6 was employed, and 15 was obtained in 26%
1
yield from 14. H NMR (200 MHz, MeOD): δ 0.86 (t, J ) 7.2
2-[4′-(4′′-Meth ylp ip er a zin -1-yl)p h en yl]-6-iod oben zoth i-
a zole 18b (TZP I). The same reaction as described above to
prepare 18a was employed, and 18b was obtained in 36% yield
Hz, 9H), 1.10-1.40 (m, 12H), 1.45-1.68 (m, 6H), 6.90-7.24
(m, 6H), 7.39-7.72 (m, 5H), 8.01 (s, 2H).
1
2-[4′-(Dim e t h yla m in o)p h e n yl]-6-b r om ob e n zot h ia z-
ole33,34 (16a ). A mixture of 5-bromo-2-aminobenzenethiol38,39
(306 mg, 1.5 mmol) and 4-(dimethylamino)benzaldehyde (224
mg, 1.5 mmol) in DMSO was heated at 180 °C for 15 min.
Water (10 mL) was added after the mixture was cooled. The
solid was collected by suction and recrystallized in ethyl
from 17b. H NMR (200 MHz, CDCl3): δ 2.42 (s, 3H), 263 (t,
J ) 4.8 Hz, 4H), 3.40 (t, J ) 4.9 Hz, 4H), 6.95 (d, J ) 9.0 Hz,
2H), 7.71 (s, 1H), 7.72 (s, 1H), 7.95 (d, J ) 8.9 Hz, 2H), 8.17 (t,
J ) 1.0 Hz, 1H). HRMS: m/z calcd for C18H19N3IS (MH+)
436.0344, found 436.0364. Anal. C18H18N3SI: C, H, N.
P r ep a r a tion of Ra d ioiod in a ted Liga n d s. The four de-
sired 125I-labeled compounds were prepared using iododestan-
nylation reactions with tributyltin precursors 8, 15, 17a , and
17b.51 Hydrogen peroxide (50 µL, 3% w/v) was added to a
mixture of 50 µL of the correspondent tributyltin precursor (1
mg/mL EtOH), 50 µL of 1 N HCl, and Na125I (1-5 µCi) in a
closed vial. The reaction was allowed to proceed for 10 min at
room temperature and terminated by addition of 100 µL of
saturated NaHSO3. The reaction mixture was either directly
extracted (styrylbenzenes) with ethyl acetate (3 × 1 mL) or
extracted after neutralization with a saturated sodium bicar-
bonate solution (thioflavins). The combined extracts were
1
acetate to give 340 mg of product (68%). H NMR (200 MHz,
CDCl3): δ 3.06 (s, 6H), 6.74 (d, J ) 9.0 Hz, 2H), 7.52 (d, d, J
) 8.7, 2.0 Hz, 1H), 7.82 (d, J ) 8.6 Hz, 1H), 7.93 (d, J ) 8.8
Hz, 2H), 7.95 (s, 1H). HRMS: m/z calcd for C15H14BrN2S (MH+)
333.0061, found 333.0072.
2-[4′-(4′′-Meth ylpiper azin -1-yl)ph en yl]-6-br om oben zoth i-
a zole (16b). The procedure described above to prepare 16a
was employed to give 57.2% of product 16b from 4-(4-
methylpiperazin-1-yl)benzaldehyde40 (204 mg, 1 mmol) and
5-bromo-2-aminobenzenethiol (204 mg, 1 mmol). 1H NMR (200
MHz, CDCl3): δ 2.38 (s, 3H), 2.60 (t, J ) 5.0 Hz, 4H), 3.38 (t,

Probes for Binding Amyloid Aggregates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1913
evaporated to dryness. For styrylbenzenes, the residues were
dissolved in 100 µL of EtOH and purified by HPLC using a
reversed phase column (Waters ubondpad, 3.9 mm × 300 mm)
with an isocratic solvent mixture of 65% acetonitrile and 35%
trifluoroacetic acid (0.1%) at a flow rate of 0.8 mL/min.
Thioflavins were purified on a C4 column (Phenomenex Inc.,
Torrance, CA) eluted with an isocratic solvent of 80% aceto-
nitrile and 20% 3,3-dimethylglutaric acid (5 mM, pH 7.0) and
a flow rate of 0.8 mL/min. The desired fractions containing
the product were collected, condensed, and re-extracted with
ethyl acetate. The no-carrier-added products were evaporated
to dryness and redissolved in 100% EtOH (1 µCi/µL). The final
125I probes, with a specific activity of 2200 Ci/mmol and a
radiochemical purity of >95%, were stored at -20 °C for up
to 6 weeks for in vitro binding and autoradiography studies.
40% EtOH (two 3 min washes) for [125I]18a . For [125I]18b, the
sections were washed with saturated Li2CO3 in 40% EtOH for
2 min. All sections were then washed in 40% EtOH for 2 min
followed by water for 30 s. After drying, the labeled sections
were exposed to Cronex MRF-34 film for 72 h. The films were
developed and digitized.
Im m u n osta in in g. Sections were deparaffinized in xylene
and rehydrated to 70% EtOH and incubated in a methanol/
30% H2O2 mixture for 30 min before being blocked in a 0.1 M
Tris/2% donor horse serum mixture for 5 min. The anti-Abeta
monoclonal antibodies Ba27 [selective antibody for Aâ(1-40),
Takeda Pharmaceuticals] and BC05 [selective antibody for Aâ-
(1-42), Takeda Pharmaceuticals] were applied at a 1:150000
dilution, and slides were incubated at 4 °C overnight. Sections
were washed and blocked again in a 0.1 M Tris/2% donor horse
serum mixture for 5 min, and biotinylated anti-mouse antibody
was applied for 1 h. Sections were washed, and staining was
visualized using the avidin-biotin peroxidase kit (Vector-stain
ABC kit, Vector Laboratories) and 3,3′-diaminobenzidine tet-
rahydrochloride.
In Vivo Biod istr ibu tion of New P r obes in Nor m a l
Mice. While under ether anesthesia, 0.15 mL of a saline
solution containing labeled agents (5-10 µCi) was injected
directly into the tail vein of ICR mice (2-3 months old, average
weight of 20-30 g). The mice were sacrificed by cardiac ex-
cision at various time points postinjection. The organs of in-
terest were removed and weighed, and the amount of radio-
activity was counted with an automatic γ-counter (Packard
5000). The percentage dose per organ was calculated by a com-
parison of the tissue counts to suitably diluted aliquots of the
injected material. Total activities of blood and muscle were
calculated under the assumption that they were 7 and 40% of
the total body weight, respectively.
P a r tition Coefficien t Deter m in a tion . Partition coef-
ficients were measured by mixing the 125I tracer with 3 g each
of 1-octanol and buffer (0.1 M phosphate, pH 7.4) in a test tube.
The test tube was vortexed for 3 min at room temperature,
followed by centrifugation for 5 min. Two weighed samples (0.5
g each) from the 1-octanol and buffer layers were counted in
a well counter. The partition coefficient was determined by
calculating the ratio of counts per minute per gram of 1-octanol
to that of buffer. Samples from the 1-octanol layer were
repartitioned until consistent partitions of coefficient values
were obtained. The measurement was carried out in triplicate
and repeated three times.
Bin d in g Assa ys Usin g th e Aggr ega ted Aâ(1-40) or Aâ-
(1-42) P ep tid e in Solu tion . The solid forms of peptides Aâ-
(1-40) and Aâ(1-42) were purchased from Bachem (King of
Prussia, PA). Aggregation of peptides was carried out by gently
dissolving the peptide [0.5 mg/mL for Aâ(1-40) and 0.25 mg/
mL for Aâ (1-42)] in a buffer solution (pH 7.4) containing 10
mM sodium phosphate and 1 mM EDTA. The solutions were
incubated at 37 °C for 36-42 h with gentle and constant
shaking. Binding studies were carried out in 12 mm × 75 mm
borosilicate glass tubes according to the procedure described
previously15 with some modifications. Aggregated fibrils (10-
50 nM in the final assay mixture) were added to the mixture
containing 50 µL of radioligands (0.01-0.5 nM in 40% EtOH)
and 10% EtOH in a final volume of 1 mL for saturation studies.
The final concentration of EtOH was 10%. Nonspecific binding
was defined in the presence of 800 nM CG for styrylbenzenes
or 2 µM thioflavin T for thioflavins. For inhibition studies, 1
mL of the reaction mixture contained 40 µL of inhibitors
(10-5-10-10 M in 10% EtOH) and 0.05 nM radiotracer in 40%
EtOH. The mixture was incubated at room temperature for 3
h, and the bound and free radioactivities were separated by
vacuum filtration through Whatman GF/B filters using a
Brandel M-24R cell harvester followed by 2 × 3 mL washes of
10% ethanol at room temperature. Filters containing the
bound 125I ligand were counted in a γ-counter (Packard 5000)
with 70% counting efficiency. Under the assay conditions, the
percent of the specifically bound fraction was less than 20%
of the total radioactivity. The results of saturation and
inhibition experiments were subjected to nonlinear regression
analysis using EBDA,52 by which Kd and Ki values were
calculated.
In Vitr o La belin g of Br a in Section s fr om a Dow n ’s
Syn d r om e P a t ien t b y F ilm Au t or a d iogr a p h y. Brain
samples from a patient with Down’s syndrome were obtained
at autopsy, and neuropathological diagnosis was confirmed by
current criteria (NIA-Reagan Institute Consensus Group,
1997). Serial sections (6 µm thick) of paraffin-embedded blocks
of cortex fixed by immersion in 10% neutral buffered formalin
were utilized for staining. Prior to radiolabeling, paraffin
sections were taken through two 5 min washes in xylene,
followed by 1 min washes sequentially with 100, 100, 95, 85,
and 70% EtOH. The sections were blow dried and labeled with
0.3 nM [125I]13 (IMSB) at room temperature for 3 h and
washed with saturated Li2CO3 in 40% EtOH for 30 s. Sections
labeled with [125I]18a or [125I]18b were incubated at a ligand
concentration between 0.03 and 0.06 nM at room temperature
for 1 h. The sections were then washed in 1% acetic acid in
Ack n ow led gm en t. We thank Ms. Laura Cesaro for
her editorial assistance. This work was supported by
grants from the National Institutes of Health (NS 18509
to H.F.K. and PO1 AG-11542 to V.M.-Y.L.), the Institute
for the Study of Aging (M.-P.K.), and a Medical Scientist
Training Program Predoctoral Fellowship from the
National Institutes of Health (D.M.S.). We also thank
Dr. N. Suzuki and Takeda Pharmaceutical, Inc., for
providing the monoclonal antibodies for Aâ(1-40) and
Aâ(1-42).
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