Novel stilbenes as probes for amyloid plaques

Full text of DOI:10.1021/ja0167147

12740
J. Am. Chem. Soc. 2001, 123, 12740-12741
be 0.4 nM. Preliminary studies in humans appear to suggest that
[18F]FDDNP showed a higher retention in regions of the brain
suspected of having tangles and plaques, and the PET images
were consistent with autoradiography and staining of postmortem
brain samples.24 A neutral thioflavin (benzothiazole) derivative,
[3H]BTA-1, was recently reported. [3H]BTA-1 showed an excel-
lent affinity (Kd ) 3 nM) in in vitro binding assay using Aâ1-40
aggregates. When [11C]BTA-1 was injected intravenously into
mice, it showed an excellent brain penetration with an initial brain
uptake at 2 min of 3.0% dose/organ.25-27 Our laboratory has
reported two types of iodinated probes, styrylbenzenes (IMSB)
and thioflavins (benzothiazole, TZDM), for binding to Aâ
aggregates.28,29 In vitro binding studies of these ligands showed
excellent binding affinities with Kd values of 0.13 and 0.06 nM
for aggregates of Aâ1-40 and 0.73 and 0.14 nM for aggregates of
Aâ1-42, respectively. More importantly, under a competitive-
binding assaying condition, two different and distinctive binding
sites on Aâ1-40 and Aâ1-42 aggregates, which are mutually
exclusive, were observed for styrylbenzenes (SB) and thioflavins
(benzothiazole, TZ). Significantly, [125I]TZDM crossed intact
blood-brain barrier and localized in the brain of normal mice
after an intravenous injection.28 For in vivo imaging of Aâ
aggregates to succeed, it will be necessary to develop agents which
show good brain uptake in vivo. Brain penetration, a key factor
for consideration, is usually related to the molecular size,
neutrality, and lipophilicity. Further refinements of these probes
are necessary to improve the brain uptake and washout from the
normal brain regions and to achieve a high retention in the regions
rich in Aâ plaques.
Novel Stilbenes as Probes for Amyloid Plaques
Hank F. Kung,*,†,‡ Chi-Wan Lee,† Zhi-Ping Zhuang,†
Mei-Ping Kung,† Catherine Hou,† and Karl Plo¨ssl†
Departments of Radiology and Pharmacology
UniVersity of PennsylVania, Room 305
3700 Market Street, Philadelphia, PennsylVania 19104
ReceiVed July 26, 2001
Alzheimer’s disease (AD) is a neurodegenerative disease of
the brain characterized by dementia, cognitive impairment, and
memory loss. Formation and accumulation of aggregates of
â-amyloid (Aâ) peptides in the brain are critical factors in the
development and progression of AD. The fibrillar aggregates of
amyloid peptides, Aâ1-40 and Aâ1-42, are major metabolic
peptides derived from amyloid precursor protein found in senile
plaques and cerebrovascular amyloid deposits in AD patients.1
Prevention and reversal of Aâ plaque formation by immunization
with Aâ peptides,2,3 or by inhibitors of secretases,4-7 are being
targeted as a treatment for this disease.6,8,9 Currently, the only
definitive confirmation of AD is by postmortem histopathological
examination of amyloid deposits in the brain. Early appraisal of
clinical symptoms for diagnosis of AD is often difficult and
unreliable.10 Therefore, there is an urgent need for in vivo imaging
agents, which can specifically demonstrate the location and density
of amyloid plaques in the brain. The Aâ-plaque-specific imaging
agents will be useful for early detection or monitoring the
progression and effectiveness of treatment of AD.11 Imaging
agents may be labeled by using either one of the two types of
isotopes: 99mTc (T1/2, 6 h; 140 keV) and 123I (T1/2, 13 h; 159 keV)
are routinely used for single photon emission computed tomo-
To search for new Aâ probes, a large series of benzothiazole
and stilbenes (1-3) purchased from Aldrich Chemical Inc.
(Milwaukee, WI) was screened by in vitro binding assays.
Serendipitously, two of them (1 and 2) are analogues of TZDM
and showed strikingly high binding affinities for the TZ binding
sites (Ki ) 2.3 and 1.4 nM, respectively), while E-stilbene and 3
graphy (SPECT), while 11C (T1/2, 20 min; 511 keV) and 18F (T1/2
,
110 min; 511 keV) are commonly used for positron emission
tomography (PET). In addition, successful agents will have to
be relatively small in molecular size, neutral and lipophilic for
brain penetration, and show selective and high binding affinity
to Aâ aggregates in vivo. Many attempts on developing such
agents based on Chrysamine-G (CG) or Congo Red derivatives
have been reported,12-21 but the efforts have not yielded any useful
imaging agents. Recently, a small but highly lipophilic probe,
[18F]FDDNP, for binding tangles and plaques has been re-
ported.22,23 Using an in vitro fluorescent titration method, the
binding affinity of FDDNP to Aâ aggregates was determined to
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* To whom correspondence should be addressed. E-mail: kunghf@sunmac.
spect.upenn.edu.
(18) Lorenzo, A.; Yankner, B. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
12243-12247.
† Departments of Radiology.
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Lansbury, P. T., Jr. J. Med. Chem. 1999, 42, 2805-2815.
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Verbruggen, A. M. J. Labelled Compd. Radiopharm. 1999, 42, 309-324.
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Verbruggen, A. M. Eur. J. Nucl. Med. 1999, 26, 1392-1399.
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‡ Department of Pharmacology.
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10.1021/ja0167147 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/21/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 50, 2001 12741
Table 1. Inhibition Constants (Ki, nM) of Compounds on Ligand
Binding to Aâ1-40 Aggregates at 25 °C
affinities toward SB sites were very low (>1000 nM). The binding
affinity of p-Me2N-stilbenes is not sensitive to the position of
the iodo group; o-, m- or p-iodo substitution (4, 5, or 6) on one
of the benzene rings of stilbene displayed about equal potency
(2.0-7.7 nM). The p-F-substituted derivative, 7, showed slightly
lower affinity (Ki ) 22 nM). It is evident that these extremely
simple and small stilbenes containing an electron-donating group,
such as p-Me2N-, -OMe, or -OH, displayed superb binding
affinity to Aâ aggregates. Replacing the benzothiazole ring with
an iodo- or fluoro-substituted phenyl ring has no effect on binding
affinity at the TZ binding sites of Aâ aggregates. Binding affinity
appears to be determined by the pharmacophore on one side of
the stilbene molecule, suggesting that additional modifications
could be possible.
compounds
[
125I]IMSB (SB)a
[
125I]TZDM (TZ)a
CG
IMSB
thioflavin T
TZDM
1
2
3
0.14 ( 0.04
0.17 ( 0.03
>9000
>1000
>1000
116 ( 20
0.9 ( 0.2
2.3 ( 0.1
1.4 ( 0.2
>3000
535 ( 43
7.7 ( 0.8
4.5 ( 0.8
2.0 ( 0.4
22 ( 3
22 ( 6
32 ( 3
>2000
not tested
not tested
not tested
not tested
>3000
E-stilbene
4
5
6
7
8
9
>2000
>2000
>2000
To characterize these stilbenes further, [125I]5 was prepared by
converting the corresponding tributyltin derivative28 in the pres-
ence of Na[125I]I and hydrogen peroxide, by which the no-carrier-
added [125I]5 was obtained in excellent yield (radiochemical purity
>95%). The partition coefficient between 1-octanol and pH 7.4
buffer was 422. In vivo biodistribution study of [125I]5 in normal
mice after an iV injection suggested a good brain penetration. The
brain uptake was 0.72, 1.12, 1.08, and 0.19% dose/organ, and
the brain/blood gram ratio was 0.46, 1.46, 1.34, and 0.24, at 2,
30, 60, and 240 min after injection (the blood levels were
relatively low, 6.5-2.8% dose/organ, at all of the time points).
Binding of [125I]5 to the aggregates of Aâ1-40 is saturable, and
the dissociation constant (Kd) was 0.19 nM, which is similar to
that observed for [125I]TZDM.28 These results suggest that
molecular weight can be reduced (molecular weights of TZDM
and 5 were 380 and 349, respectively) while maintaining binding
affinity; as such it significantly enhances the flexibility on
designing new probes for imaging Aâ plaques in the brain. This
finding is of paramount importance, because it represents a
structural simplicity and suggests better alternatives for designing
probes for binding to Aâ aggregates: (1) These probes may
contain a simple stilbene-like structure. (2) One of the aromatic
rings contains an electron-donating group, p-Me2N-, -OMe, or
-OH, which appears to be essential for the binding affinity. (3)
There is a bulk tolerance for the second aromatic ring, on which
radiolabel, 99mTc, 123I, or 18F, can be readily attached without
detrimental effects on binding affinity to Aâ aggregates.
In conclusion, synthesis of novel stilbenes as probes for binding
to Aâ1-40 aggregates is reported. Derivatives of stilbenes contain-
ing a p-Me2N-, -OMe, or -OH group on one of the benzene
rings are simple, relatively small, neutral, and lipophilic. Based
on their exquisitely high binding affinity to Aâ1-40 aggregates,
these novel stilbenes are candidates as probes for in vivo
evaluation of amyloid plaques containing Aâ aggregates in the
brains of AD patients.
>1000
>2000
a Values (Ki, nM) are the mean ( SEM of three independent
experiments, each in duplicate.
Scheme 1. Scheme for the Synthesis of trans-Stilbenes
displayed low binding affinities (Ki ) 535 and >1000 nM,
respectively) (Table 1). Based on these unexpected results, we
have prepared and tested a series of stilbenes (4-9), all of which
contain an electron-donating group: p-Me2N, -OMe, or -OH.
These novel stilbenes (4-9) were successfully prepared by a
Wadsworth-Emmons reaction (Scheme 1). By using the diethyl
phosphonates as the starting material, the trans-stilbenes were
formed preferentially. Similar observation on synthesis of stilbenes
has been reported previously.30,31 The free phenol derivative, 9,
was obtained from the corresponding 8 by a demethylation
reaction using BBr3. Previously, our laboratory has demonstrated
that in vitro binding assay can be an effective way of screening
probes that bind to Aâ aggregates.28 The in vitro binding assay
uses preformed Aâ aggregates of synthetic Aâ1-40 peptides and
Acknowledgment. This work was supported by grants awarded from
the National Institutes of Health (NS 18509 and NS-24538, H.F.K.).
Authors thank Drs. John Q. Trojanowski and Virginia M. Y. Lee for
their helpful support and discussion.
[
125I]TZDM as the ligand. The novel stilbenes, 4-9, showed high
Supporting Information Available: Complete experimental proce-
dures for the synthesis, radiolabeling, and binding of compounds 4-9
(PDF). This material is available free of charge via the Internet at
http://pubs.acs.org.
binding affinities (Ki ) 2-32 nM) to the TZ sites, while the
(30) Bottin-Strzalko, T.; Seyden-Penne, J. Bull. Soc. Chim. Fr. 1984, 161-
163.
(31) March, J. AdVanced Organic Chemistry, 4th ed.; Wiley-Interscience:
New York, 1992.
JA0167147