Polyfluorinated bis-styrylbenzene β-amyloid plaque binding ligands

Article abstract of DOI:10.1021/jm070085f

β-Amyloid (Aβ) binding affinities and specificities for six bis-styrylbenzenes with multiple magnetically equivalent fluorine atoms in the form of a tetrafluorophenyl core or symmetrical trifluoromethyl and trifluoromethoxy groups were determined by means of fluorescence titrations with amyloid peptide Aβ_1-40 and a novel in vitro fluorescence-based assay using APP/PS1 transgenic mouse brain sections. Bisstyrylbenzenes with a tetrafluorophenyl core had increased Aβ binding affinities compared to their monofluorophenyl or phenyl counterparts. Bis-styrylbenzenes with carboxylic acid functional groups had lower Aβ binding affinities than their neutral counterparts. Selected bis-styrylbenzenes were demonstrated to have good blood - brain barrier penetration capabilities. These data extend the SAR of bis-styrylbenzene Aβ binding and provide direction for the development of a noninvasive probe for early detection of Alzheimer's disease using ¹⁹F MRI.

Full text of DOI:10.1021/jm070085f

4986
J. Med. Chem. 2007, 50, 4986-4992
Polyfluorinated Bis-styrylbenzene â-Amyloid Plaque Binding Ligands
Daniel P. Flaherty,† Shannon M. Walsh,‡ Tomomi Kiyota,‡ Yuxiang Dong,† Tsuneya Ikezu,‡ and Jonathan L. Vennerstrom*,†
UniVersity of Nebraska Medical Center, College of Pharmacy, 986025 Nebraska Medical Center, Omaha, Nebraska 68198, and
UniVersity of Nebraska Medical Center, College of Medicine, 985880 Nebraska Medical Center, Omaha, Nebraska 68198
ReceiVed January 22, 2007
â-Amyloid (Aâ) binding affinities and specificities for six bis-styrylbenzenes with multiple magnetically
equivalent fluorine atoms in the form of a tetrafluorophenyl core or symmetrical trifluoromethyl and
trifluoromethoxy groups were determined by means of fluorescence titrations with amyloid peptide Aâ1-40
and a novel in vitro fluorescence-based assay using APP/PS1 transgenic mouse brain sections. Bis-
styrylbenzenes with a tetrafluorophenyl core had increased Aâ binding affinities compared to their
monofluorophenyl or phenyl counterparts. Bis-styrylbenzenes with carboxylic acid functional groups had
lower Aâ binding affinities than their neutral counterparts. Selected bis-styrylbenzenes were demonstrated
to have good blood-brain barrier penetration capabilities. These data extend the SAR of bis-styrylbenzene
Aâ binding and provide direction for the development of a noninvasive probe for early detection of
Alzheimer’s disease using 19F MRI.
Introduction
a number of bis-styrylbenzenes with strong Aâ plaque binding.
These include 1 (X-34),15 2 (FSB),16 3 (BSB),17,18 4 (IMSB),19,20
and 5 (K114).21 Importantly, intravenously administered 2 or 3
can cross the blood-brain barrier (BBB) and bind to Aâ plaques
deposited in APP or APP/PS1 mice.16,17,22 Early data suggested
that interactions between cationic amino acid residues of the
Aâ peptide with the sulfonic acid functional groups of Congo
red and the carboxylic acid functional groups of Chrysamine G
and 1-3 were necessary for Aâ-ligand binding.14 However, as
illustrated by 5, acidic functional groups are not necessarily
required for good Aâ binding.21
Alzheimer’s disease (AD),1 a relatively common neurode-
generative disorder in the elderly,2,3 is characterized by an
accumulation of senile plaques containing aggregated protein
deposits of amyloid (Aâ) fibrils, numerous neurofibrillary
tangles (NFTs), reactive astrocytes, and activated microglia in
the neocortex and hippocampus.4,5 Senile plaques can contain
either a diffuse amyloid deposition (presumably early senile
plaques) or a dense core of insoluble Aâ with neuritic structures
(mature senile plaques). Although Aâ deposition and NFTs are
commonly observed in nondemented elderly, 80 years and older,
increased levels of senile plaques are seen in AD brains.6,7 The
degree of both Aâ deposition and NFT formation are correlated
with cognitive decline.6,8-10 The molecular pathogenesis of AD
is centered on Aâ production and its clearance. Aâ is generated
by the processing of amyloid precursor protein (APP) by
processing enzymes, and is cleared from the brain by its
diffusion, export to vascular system, phagocytosis, or degrada-
tion. The amyloid cascade hypothesis11 for AD pathogenesis
proposes that accumulation and aggregation of the Aâ triggers
a cascade that leads to the characteristic pathologies of AD.
The strongest support for this hypothesis comes from genetic
studies of familial AD. These studies reveal that Aâ accumula-
tion or aggregation is elevated by mutations of the APP, PS1,
and PS2 genes directly related to Aâ production and other risk
factors (apoE).12 Therefore, it would be beneficial to identify
Aâ-binding ligands with high specificity and sensitivity that
could be used for early in vivo detection of AD and for
monitoring the progression of the disease.
Design. Monofluoro bis-styrylbenzene 216 has been proposed
as a potential in vivo probe for Aâ plaques using 19F MRI.22
To address the low 19F MRI sensitivity inherent in the structure
of 2, we designed bis-styrylbenzene ligands with multiple
magnetically equivalent fluorine atoms in the form of a
tetrafluorophenyl core (6-8) or symmetrical trifluoromethyl (9,
10) and trifluoromethoxy (11, 12) groups (Schemes 1 and 2).
FSB (2) and the nonfluorinated bis-styrylbenzene 13 served as
controls. For each of these, Aâ binding affinity and specificity
were determined. We also assessed the potential of selected bis-
styrylbenzenes (8, 11, 14) to penetrate the BBB. These data
that we present in this paper extend the SAR of bis-styrylben-
zene Aâ binding and provide direction for the development of
a noninvasive probe for AD using 19F MRI.
Chemistry. Bis-styrylbenzene 2 was obtained using the
procedure of Sato et al.16 with two modifications. First, the
dimethylation of 5-formylsalicylic acid to afford 16 was
achieved in 78% yield using dimethyl sulfate rather than methyl
iodide (65% yield). Second, the key 1-fluoro-2,5-bis(bromo-
methyl)benzene intermediate was obtained in a one-step benzylic
bromination of 3-fluoro-4-methylbenzyl bromide (49% yield)
using N-bromosuccinimide (NBS)/2,2′-azobis(isobutyronitrile)
The discovery of the fluorescent Aâ-binding diazo dye Congo
red13 provided the starting point for the synthesis of numerous
compounds with potential as in vivo imaging agents using
positron emission tomography (PET), single photon emission
computed tomography (SPECT), and magnetic resonance imag-
ing (MRI). The diazo disalicylate Chrysamine G14 was one of
the first Congo red derivatives to be investigated as a candidate
for PET and SPECT imaging. This soon led to the synthesis of
* To whom correspondence should be addressed. Tel.: 402-559-5362.
Fax: 402-559-9543. E-mail: jvenners@unmc.edu.
† College of Pharmacy.
‡ College of Medicine.
10.1021/jm070085f CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/11/2007

Amyloid Plaque Binding Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4987
Scheme 1. Synthesis of Bis-styrylbenzenes 6-8
Scheme 2. Synthesis of Bis-styrylbenzenes 9-14
(AIBN) rather than the low yielding (6% overall) two-step
procedure starting with 2-fluoro-p-xylene using diazonium
chemistry.16
capturing setting,28 we were able to calculate a signal/noise (S/
N) ratio, a measure of Aâ binding specificity. This Aâ binding
specificity represents the relative affinity of a compound for
Aâ plaques compared to normal brain tissue.
As illustrated in Schemes 1 and 2, bis-styrylbenzenes 7, 9-13,
and 20 were obtained in yields of 58-91% using classic Wittig
(Horner-Wadsworth-Emmons) couplings between benzalde-
hydes and the ylides derived from bisdiethylphosphonates. Bis-
styrylbenzene 1323 has also been obtained using Heck chem-
istry.24 Phenolic bis-styrylbenzenes 8 (97%), 14 (68%), and 21
(62%) were obtained by subsequent boron tribromide demethyla-
tion. Finally, bis-styrylbenzene 6 was obtained in 83% yield
by ester hydrolysis of 21. As expected, clean singlets were
obtained in the 19F NMR spectra of target polyfluorinated bis-
styrylbenzenes 6-12.
â-Amyloid (Aâ) Binding Affinity and Specificity. â-Amy-
loid (Aâ) binding affinity and specificity for bis-styrylbenzenes
6, 8-12, and 14, with bis-styrylbenzene 2 and thioflavin T as
controls, was determined by means of fluorescence titrations25
with amyloid peptide Aâ1-40 and a novel in vitro fluorescence-
based assay using APP/PS1 transgenic mouse brain sections.
We could not obtain data for 6 and 13 due to their low solubility
in phosphate buffered saline (PBS)/ethanol cosolvent mixtures.
The APP/PS1 transgenic mice were developed by crossing the
Tg2576 strain,26 which express the K670N/M671L mutant of
APP695 found as Swedish familial AD gene, and the M146L
6.1 strain,27 which express the M146L mutant of PS1 found as
the early onset familial AD gene. By measuring the fluorescent
intensities of Aâ plaques (specific signal) and background
regions (noise; Figure 1) using the same fluorescence image
In considering the Aâ binding affinity (Kd) and specificity
(S/N ratio) data (Table 1), we note that the Kd of 560 nM that
we obtained for thioflavin T was quite similar to the value (Kd
) 750 nM) reported by Lockhart et al.25 Although varying
amounts of ethanol were required to solubilize the bis-
styrylbenzenes (9:1 PBS/ethanol for 2, 6, and 14 and 2:3 PBS/
ethanol for 8-12), the proportion of ethanol had little effect on
the measured binding affinities. For example, the Kd (nM) values
in 9:1 versus 2:3 PBS/ethanol for 2 were 3400 ( 200 versus
3300 ( 100 and for 6 were 9.4 ( 0.8 versus 6.4 ( 0.4.
The FSB control (2) had the weakest binding affinity of all
of the bis-styrylbenzenes, although it did have high specificity,
exceeded only by that of 14. The binding affinity for tetrafluo-
rophenyl bis-styrylbenzene 6 increased 360-fold compared to
its monofluorophenyl counterpart 2, albeit with a 4-fold loss in
specificity. Similarly, the binding affinity of 8, with its tet-
rafluorophenyl core, was 370-fold greater than 14, its phenyl
counterpart; this was accompanied by a 2-fold lower specificity.
As revealed by the 13C NMR signals for the central aromatic
carbon atoms of 8 (144.0 ppm) and 14 (126.5 ppm),29 the more
electropositive tetrafluorophenyl core in 6 and 8 apparently
provides a stronger interaction with Aâ compared to the
corresponding monofluorophenyl (2) or phenyl (14) cores. In
addition, the multiple fluorine atoms of 6 and 8 can form
hydrogen bonds and interact with cations,30 both of which could

4988 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Flaherty et al.
Figure 1. Fluorescence imaging of Aâ plaque in aged APP/PS1 mouse brain. Frozen brain sections (10 µm thickness) were stained with 50 µM
of each compound for 8 min in 1:1 PBS/ethanol, washed successively with 75% aq ethanol, 95% aq ethanol, and xylene. Fluorescence images were
taken using a Nikon TE-2000, 40× Pan Fluor objective, and Roper HQ CCD camera (400× original magnification). Ex/Em wavelengths are
488/520 (FITC filter set) for thioflavin T (ThT), and 360/460 (DAPI filter set) for 2, 6, 8-12, and 14. Original magnification: 400×.
Table 1. Amyloid Peptide Aâ1-40 Binding Affinity (Kd) and Aâ Plaque
Binding Specificity (S/N Ratio) to APP/PS1 Transgenic Mouse Brain
Slices for Thioflavin T and Bis-styrylbenzenes 2, 6, 8-12, and 14
In Vivo BBB Delivery of Bis-styrylbenzenes into Aâ
Plaque-Bearing APP/PS1 Mouse Brain. We next determined
whether intravenously administered 8, 11, and 14 (with 2 as a
control) could cross the BBB and bind to Aâ plaque. Bis-
styrylbenzenes 2, 8, 14 (10 mg/kg), or 11 (5 mg/kg) were
injected (100 µL total volume) into 11-month-old APP/PS1 mice
through the tail vein. The 10 mg/kg dose was based on previous
investigations.17,22,31,32 For 11, the highest dose possible was 5
mg/kg, as its solubility was 10-fold lower than the other bis-
styrylbenzenes. The animals were sacrificed at 48 h after
injection, and the frozen brain sections were stained with
hematoxylin for histology. As shown in Figure 3, all of the bis-
styrylbenzenes crossed the BBB and bound to Aâ plaques in
vivo. APP/PS1 mice brain sections treated with 14 (A, B, E,
F), 2 (C, D, G, H), 8 (I, J, M, N), and 11 (K, L, O, P) each
showed intense Aâ plaque signals. Each fluorescent signal is
Aâ plaque-specific as determined by hematoxylin counterstain-
ing (see arrows in the matched fluorescent and histology
images: A and E, B and F, C and G, D and H, etc.). Control
nontransgenic animals showed no specific signals in the brain
for any of the tested compounds after intravenous injection (data
not shown). These data confirm that these bis-styrylbenzene
derivatives retain the BBB penetration capabilities of 2 and that
8 and 11 have the potential for Aâ detection using 19F MRI.
MRI experiments with 8 and 11 are ongoing and will be reported
in due course.
cmpd
Kda (nM)
S/N ratiob
thioflavin T
2
6
8
560 ( 20
6.1 ( 0.5
14 ( 0.8
3.6 ( 0.2
10 ( 1.3
8.5 ( 0.5
10 ( 1.3
8.6 ( 0.2
7.8 ( 1.3
17 ( 1.3
3400 ( 200
9.4 ( 0.8
0.030 ( 0.001
9.5 ( 0.3
26 ( 1
9
10
11
12
14
10 ( 1
16 ( 1
11 ( 2
a Values represent the average ( SD of three determinations. b Values
represent the average ( SD of five determinations.
contribute to the higher binding affinities of 6 compared to 2
and of 8 compared to 14. The superior binding affinity and
specificity for the neutral 8 versus the acidic 6 is consistent
with previous data,21 suggesting that acidic functional groups
are not required for good Aâ binding. The data for 11 versus
14 indicate that phenol functional groups are also not required
for good Aâ binding. The data for 9 versus 10 and 11 versus
12 suggest that para- is marginally superior to meta-substitution
on the outer phenyl rings.
While the binding specificity data (Table 1) obtained from
the APP/PS1 transgenic mouse brain sections (Figure 1) were
useful, this model did not permit us to differentiate compound
binding to sheet structures other than Aâ such as NFTs. To
this end, we stained human AD brain sections containing both
Aâ senile plaques and NFTs (Figure 2) with 2, 8, 11, and 14.
These data show that 8 (D, E) and 11 (G, H) stain mostly Aâ
plaques, 14 (J, K) stains both Aâ plaques and NFTs, and 2 (A,
B) rather preferentially stains NFTs. There was no specific
staining by any of the compounds in age-matched control brain
sections (C, F, I, L), demonstrating the binding specificity of
these bis-styrylbenzenes for AD sheet structures. The preferential
staining of 2 to NFTs is consistent with its low binding affinity
for Aâ (Table 1).
Summary. These data extend the SAR of bis-styrylbenzene
Aâ binding and provide direction for the development of a
noninvasive probe for early detection of AD using 19F MRI.
Although 14, one of the nonfluorinated bis-styrylbenzene control
compounds, is obviously not a candidate for a 19F MRI AD
imaging probe, its structure provides a core skeleton for the
discovery of additional polyfluorinated derivatives.
Experimental Section
General. Starting materials were purchased from Aldrich, TCI,
Acros, Avocado, or Apollo Scientific. All reactions were run under
a positive pressure of Ar. Melting points were determined on a

Amyloid Plaque Binding Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4989
Figure 2. Fluorescence staining of AD and control brain sections by 2, 8, 11, or 14. Paraffin sections (7 µm thickness) of AD cortex (left and
middle columns) and age-matched control cortex (right column) were deparaffinized, chemically bleached for autofluorescence, and stained with
10 µM of 2 (A-C), 8 (D-F), 11 (G-I), or 14 (J-L). Arrows indicate representative Aâ plaques, and * denotes representative neurofibrillary tangles
(B, E, H, K). Original magnifications: 200× (left and right columns) and 400× (middle column).
Mel-Temp apparatus and are uncorrected. 1H (500 MHz), 13C (125.7
MHz), and 19F (470 MHz) NMR spectra were measured on a Varian
spectrometer using CDCl3 and DMSO-d6 as solvents. All chemical
shifts are reported in parts per million (ppm) and are relative to
Reactions. (E,E)-1,2,4,5-Tetrafluoro-3,6-bis(4-methoxy)styryl-
benzene (7). A 30 wt % solution of sodium methoxide in methanol
(0.260, 0.87 mL, 4.62 mmol) was added to a stirred mixture of
1,2,4,5-tetrafluoro-3,6-bis(diethylphosphonylmethyl)benzene (19;
1.040 g, 2.31 mmol) and p-anisaldehyde (15; 0.705 g, 4.62 mmol)
in DMF (10 mL) at rt. This mixture was then heated to 80 °C for
2 h. The reaction was then quenched with 2:1 DMF/H2O (15 mL),
and the precipitate was filtered and rinsed with DMF and ether
1
internal (CH3)4Si for H, CDCl3 (77.0 ppm) and DMSO-d6 (39.7
ppm) for 13C NMR, and C6H5CF3 (-63.72 ppm) for 19F NMR.
Microanalyses were performed by M-H-W Laboratories, Phoenix,
AZ.
1
(E,E)-1,2,4,5-Tetrafluoro-3,6-bis(3-hydroxycarbonyl-4-hy-
droxy)styrylbenzene (6). To (E,E)-1,2,4,5-tetrafluoro-3,6-bis(3-
methoxycarbonyl-4-hydroxy)styrylbenzene (21; 0.105 g, 0.209
mmol) was added 1.0 M NaOH (0.126 g, 3.14 mL, 3.14 mmol)
and the solution was stirred at 100 °C for 5.5 h. After cooling to
rt, the solution was quenched with 5 mL of water and 1 mL of 6.0
M HCl to afford, after filtering and drying, 6 (0.082 g, 83%): mp
affording 7 (0.556 g, 58%): mp 197-200 °C. H NMR (CDCl3)
3.85 (s, 6H), 6.93 (d, J ) 8.8 Hz, 5H), 6.97 (s, 1H), 7.46 (d, J )
16.6 Hz, 2H), 7.50 (d, J ) 8.3 Hz, 4H). 13C NMR (CDCl3) 55.4,
111.9, 114.3, 114.8 (brs), 128.3, 129.7, 136.3, 144.6 (brd, J ) 259.6
Hz), 160.2. 19F NMR (C6H5CF3) -146.14 (s, 4F). Anal. (C24H18F4O2)
C, H.
(E,E)-1,2,4,5-Tetrafluoro-3,6-bis(4-hydroxy)styrylbenzene (8).
A 1.0 M solution of boron tribromide in dichloromethane (0.924
g, 3.69 mL, 3.69 mmol) was added dropwise over 10 min to a
stirred suspension of 7 (0.255 g, 0.62 mmol) in chloroform (10
mL) at rt. The reaction mixture was stirred for an additional 2 h at
rt before quenching with ∼25 mL of water to afford 8 (0.206 g,
97%) as a precipitate, which was filtered, washed with water and
ethyl acetate, and dried: mp 275-277 °C dec. 1H NMR (DMSO-
1
329-332 °C dec. H NMR (DMSO-d6) 6.71 (d, J ) 8.3 Hz, 2H),
6.85 (d, J ) 16.6 Hz, 2H), 7.38 (d, J ) 16.6 Hz, 2H), 7.56 (d, J )
8.3 Hz, 2H), 7.92 (d, J ) 2.4 Hz, 2H). 13C NMR (DMSO-d6) 109.1,
114.5 (brs), 117.3, 119.2, 124.6, 129.2, 131.2, 137.4, 144.0 (d, J
) 240 Hz), 165.0, 171.1. 19F NMR (C6H5CF3) -147.87 (s, 4F).
Anal. (C24H14F4O6‚7/6H2O) C, H; H: calcd, 3.32; found, 2.89.
General Procedure for the Horner-Wadsworth-Emmons

4990 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Flaherty et al.
Figure 3. In vivo labeling of Aâ plaques via intravenous injection of 2, 8, 11, or 14 in aged APP/PS1 mice. Eleven-month-old APP/PS1 mice were
injected with 10 mg/kg of 2, 8, 11, or 14 in 100 µL volumes via the tail vein and sacrificed at 48 h after injection. Fixed frozen sections (10 µm
thickness) were hematoxylin stained, and both fluorescence and histological images were captured using 4× (A, C, E, G), 20× (I, K, M, O), or 40×
(B, D, F, H, J, L, N, P) objectives on a Nikon TE-300 with CCD camera. Arrows indicate colocalization of fluorescence-labeled Aâ deposition and
hematoxylin staining in cortex (Ctx), corpus callosum (CC), or hippocampus (Hp; A, C, E, G). High-power magnification images (20× and 40×)
show representative fluorescent staining of Aâ plaques by 2, 8, 11, or 14.
d6) 6.81 (d, J ) 8.3 Hz, 4H), 6.91 (d, J ) 16.6 Hz, 2H), 7.38 (d,
J ) 16.6 Hz, 2H), 7.52 (d, J ) 7.8 Hz, 4H). 13C NMR (DMSO-d6)
110.1, 114.6 (brs), 116.0, 127.4, 128.9, 137.2, 144.0 (ddd, J )
249.6, 22.1, 5.7 Hz), 158.8. 19F NMR (C6H5CF3) -145.7 (s, 4F).
Anal. (C22H14F4O2) C, H.
(CDCl3) 7.07 (d, J ) 13.0 Hz, 2H), 7.11 (d, J ) 19.6 Hz, 2H),
7.21 (d, J ) 8.3 Hz, 4H), 7.52 (s, 4H), 7.53 (d, J ) 8.8 Hz, 4H).
13C NMR (CDCl3) 120.5 (q, J ) 257.2 Hz), 121.2, 127.0, 127.2,
127.7, 129.2, 136.0, 136.6, 148.5. 19F NMR (C6H5CF3) -58.81 (s,
6F). Anal. (C24H16F6O2) C, H.
(E,E)-1,4-Bis(4-trifluoromethyl)styrylbenzene (9). Compound
9 was prepared as described above from 1,4-bis(diethylphospho-
nylmethyl)benzene (26; 0.706 g, 1.87 mmol) and 4-trifluoromethyl-
benzaldehyde (22; 0.65 g, 3.74 mmol) to afford 9 (0.593 g, 77%):
(E,E)-1,4-Bis(3-trifluoromethoxy)styrylbenzene (12). Com-
pound 12 was prepared as described above from 26 (0.50 g, 1.32
mmol) and 3-trifluoromethoxybenzaldehyde (25; 0.506 g, 2.64
mmol) to yield 12 (0.55 g, 99%): mp 168-170 °C. 1H NMR
(CDCl3) 7.11 (d, J ) 13 Hz, 2H), 7.13 (d, J ) 11.2 Hz, 2H), 7.15
(d, J ) 15.0 Hz, 2H), 7.38 (s, 2H), 7.40 (t, J ) 8.3 Hz, 2H), 7.46
(d, J ) 11.2 Hz, 2H), 7.55 (s, 4H). 13C NMR (CDCl3) 118.7, 119.9,
120.5 (q, J ) 257.2 Hz), 124.9, 127.1, 127.3, 129.8, 130.0, 136.5,
139.4, 149.7. 19F NMR (C6H5CF3) -58.65 (s, 6F). Anal. (C24H16F6O2)
C, H.
1
mp 272-275 °C. H NMR (CDCl3) 7.15 (d, J ) 16.6 Hz, 2H),
7.20 (d, J ) 16.6, 2H), 7.55 (s, 4H), 7.62 (s, 8H). 13C NMR (CDCl3)
124.2 (q, J ) 282.2 Hz), 126.7 (q, J ) 3.8 Hz), 126.6, 127.2, 127.4,
129.4 (q, J ) 31.67 Hz), 130.6, 136.6, 140.7. 19F NMR (C6H5CF3)
-63.46 (s, 6F). Anal. (C24H16F6) C, H.
(E,E)-1,4-Bis(3-trifluoromethyl)styrylbenzene (10). Compound
10 was prepared as described above from 26 (0.700 g, 1.85 mmol)
and 3-trifluoromethylbenzaldehyde (23; 0.645 g, 3.7 mmol) to afford
(E,E)-1,4-Bis(4-methoxy)styrylbenzene (13).23,24 Compound 13
was prepared (as described for 7) from 26 (0.701 g, 1.85 mmol)
and p-anisaldehyde (15; 0.505 g, 3.70 mmol) to afford 13 (0.58 g,
91%): mp 303-305 °C (lit.23 mp 310-311 °C). 1H NMR (CDCl3)
3.84 (s, 6H), 6.91 (d, J ) 8.8 Hz, 4H), 6.97 (d, J ) 16.1 Hz, 2H),
7.08 (d, J ) 16.1 Hz, 2H), 7.46 (d, J ) 8.8 Hz, 4H), 7.47 (s, 4H).
(E,E)-1,4-Bis(4-hydroxy)styrylbenzene (14).24 A 1.0 M solution
of boron tribromide in dichloromethane (2.19 g, 8.73 mL, 8.73
mmol) was added dropwise to 13 (0.498 g, 1.45 mmol) in
chloroform (20 mL) at 0 °C. The reaction proceeded for 1 h and
was quenched with ∼30 mL of water to form a precipitate that
was filtered and identified as 14 (0.31 g, 68%): mp 310-312 °C
1
10 (0.56 g, 72%): mp 138-140 °C. H NMR (CDCl3) 7.15 (d, J
) 17.0 Hz, 2H), 7.19 (d, J ) 17.0 Hz, 2H), 7.48 (t, J ) 7.3 Hz,
2H), 7.52 (d, J ) 11.5 Hz, 2H), 7.55 (s, 4H), 7.69 (d, J ) 7.8,
2H), 7.77 (s, 2H). 13C NMR (CDCl3) 122.0 (q, J ) 285.2 Hz),
123.1 (q, J ) 3.8 Hz), 124.1 (q, J ) 3.4 Hz), 125.2, 127.1, 127.3,
129.2, 129.6, 129.9, 131.2 (q, J ) 32.1 Hz), 136.6, 138.0. 19F NMR
(C6H5CF3) -63.77 (s, 6F). Anal. (C24H16F6) C, H.
(E,E)-1,4-Bis(4-trifluoromethoxy)styrylbenzene (11). Com-
pound 11 was prepared as described above from 26 (0.50 g, 1.32
mmol) and 4-trifluoromethoxybenzaldehyde (24; 0.506 g, 2.64
mmol) to yield 11 (0.47 g, 85%): mp 235-238 °C. 1H NMR
1
dec (lit.23 mp 360 °C). H NMR (DMSO-d6) 6.77 (d, J ) 8.3 Hz,

Amyloid Plaque Binding Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4991
4H), 7.00 (d, J ) 16.6 Hz, 2H), 7.15 (d, J ) 16.6 Hz, 2H), 7.43
(d, J ) 8.8 Hz, 4H), 7.52 (s, 4H), 9.57 (s, 2H). 13C NMR (DMSO-
d6) 115.8, 125.0, 126.5, 128.0, 128.2, 128.3, 136.5, 157.6.
with ligand binding to aggregated Aâ1-40 were recorded on a Cary
Eclipse fluorescence spectrophotometer (Varian) using excitation
wavelengths determined for each compound. Fixed concentrations
of Aâ1-40 (500 nM for thioflavin T and 2; 40 nM for 14; 25 nM
for 9-12; 10 nM for 6; 1 nM for 8) were diluted to 500 µL in 9:1
(thioflavin T and 14) or 2:3 (2, 6, 8-12) PBS/ethanol in a 10 mm
quartz fluorescence cuvette. With the exception of 8, concentrations
of Aâ1-40 were selected to be no more than 10-fold higher than
compound binding affinities to obtain accurate Kd values. This
indicates that the measured Kd for 8 is a minimum value, and the
true binding affinity may be higher. To the Aâ1-40 PBS/ethanol
solution in the cuvette, aliquots of test compounds in PBS/ethanol
were titrated using a 2.0 µL Hamilton syringe with a reproducibility
adapter along a concentration gradient of compound (0.8 nMs
saturation). Fluorescence spectra were recorded until the fluores-
cence no longer increased with increasing compound concentration
(saturation). The FLINT at these wavelengths were plotted versus
compound concentrations to yield binding isotherms. Compound
Aâ1-40 Kd values were determined (Prism 4.0c software, GraphPad,
Inc.) by means of the standard thermodynamic relationship for
single-site ligand/receptor binding: B ) K(L)/1 + K(L).
In Vitro Aâ Plaque Binding Specificity Using APP/PS1
Transgenic Mouse Brain Slices. Thioflavin T, 2, 6, 8-12, and
14 were dissolved in chloroform or DMSO at 25 mM, diluted to
0.50 mM with ethanol and then diluted with 1:1 PBS/ethanol to
prepare a solution of 50 µM. The fluorescence spectra of 6, 8-12,
and 14 were determined to obtain peak excitation and emission
wavelengths (Ex/Em nm) to select the fluorescence filter/dichroic
mirror settings of the microscope (Nikon TE-2000U). Eleven-
month-old transgenic APP/PS1 mice derived from crossing Tg2576
expressing APP Swedish mutant and M146L 6.1 line expressing
presenilin-1 mutant26,27 were anesthetized and transcardically per-
fused with 4% paraformaldehyde in PBS under the guidance of
Institutional Animal Care and Use Committee. The fixed brain
samples were cryoprotected in 20% sucrose in PBS and subjected
to cryostat sectioning (Leica). Frozen brain sections (10 µm
thickness) of aged APP/PS1 transgenic mice (three sections per
dilution point) were stained with the compound solutions for 30
min, and then washed successively with 75% aq ethanol, 95% aq
ethanol, and xylene. Fluorescence imaging of the stained and
washed brain sections were carried out using a DAPI filter (Chroma)
and a Roper HQ CCD camera (original magnification: 400×)
following a standard BSB/FSB staining protocol.18,22 Fluorescence
images of one plaque per section were systematically captured using
the same image acquisition setting (laser power, capturing time,
photomultiplier setting) to obtain comparable fluorescent intensities
of five plaque regions (specific signal) and five background regions
(noise signal) to obtain signal-to-noise (S/N) ratios.
3-Methoxycarbonyl-4-methoxybenzaldehyde (16).16 5-Formyl-
salicylic acid (1.47 g, 8.82 mmol) was dissolved in acetone (125
mL) with stirring. Dimethyl sulfate (2.780 g, 22.05 mmol) and
potassium carbonate (2.67 g, 19.5 mmol) were added, and the
reaction was stirred at reflux for 5.5 h. The solid potassium sulfate
was filtered and the solvent was removed in vacuo to give a solid
crude product that was crystallized in two crops from ethanol to
1
afford 16 (1.343 g, 78%): mp 83-85 °C. H NMR (DMSO-d6)
3.83 (s, 3H), 3.94 (s, 3H), 7.38 (d, J ) 8.8 Hz, 1H), 8.09 (dd, J )
8.8 Hz, 2.4, 1H), 8.20 (d, J ) 2.4 Hz, 1H), 9.92 (s, 1H). 13C NMR
(CDCl3) 52.3, 56.5, 112.3, 120.6, 129.1, 134.4, 134.5, 163.6, 165.5,
190.0.
1,2,4,5-Tetrafluoro-3,6-bis(bromomethyl)benzene (18).33 To
a stirred solution of 2,3,5,6-tetrafluoro-p-xylene (17; 4.90 g, 0.0275
mol) in CHCl3 (300 mL) at rt was added NBS (16.08 g, 0.0903
mol) and 2′2′-azobis(isobutyronitrile) (0.23 g, 1.4 mmol). The
reaction mixture was heated to reflux for 15 h. The cooled reaction
mixture was then washed with H2O (2 × 300 mL) and dried with
MgSO4. After removal of the CHCl3 solvent in vacuo, the residue
was crystallized from ethanol to afford 18 (5.34 g, 58%): mp 88-
1
90 °C (lit.33 mp 125-126 °C). H NMR (CDCl3) 4.51 (s, 4H).
1,2,4,5-Tetrafluoro-3,6-bis(diethylphosphonylmethyl)ben-
zene (19).33 A mixture of triethyl phosphite (5.212 g, 3.14 mmol)
and 18 (5.266 g, 15.7 mmol) were heated to 160 °C for 4 h. The
solid residue was crystallized from ethyl ether to afford 19 (4.079
1
g, 58%): mp 73-75 °C (lit.33 mp 41-42 °C). H NMR (CDCl3)
1.30 (t, J ) 6.8 Hz, 12H), 3.26 (d, J ) 20.5 Hz, 4H), 4.06-4.18
(m, 8H).
(E,E)-1,2,4,5-Tetrafluoro-3,6-bis(3-methoxycarbonyl-4-meth-
oxy)styrylbenzene (20). Compound 20 was prepared as described
above from 16 (0.748 g, 3.85 mmol) and 19 (0.867 g, 1.93 mmol)
1
to yield 20 (0.91 g, 89%): mp 251-253 °C. H NMR (CDCl3)
3.94 (s, 6H), 3.95 (s, 6H), 7.00 (d, J ) 17.1 Hz, 2H), 7.01 (d, J )
8.8 Hz, 2H), 7.46 (d, J ) 16.6 Hz, 2H), 7.66 (dd, J ) 8.8 Hz, 2.4
Hz, 2H), 7.99 (d, J ) 2.4 Hz, 2H). 13C NMR (CDCl3) 52.2, 56.2,
112.4, 113.1, 114.9 (brs), 120.5, 129.1, 130.3, 131.8, 135.4, 144.6
(d, J ) 237.0 Hz), 159.5, 166.4. 19F NMR (C6H5CF3) -145.63 (s,
4F). Anal. (C28H22F4O6) C, H.
(E,E)-1,2,4,5-Tetrafluoro-3,6-bis(3-methoxycarbonyl-4-hy-
droxy)styrylbenzene (21). To a stirred suspension of 20 (0.510 g,
0.961 mmol) in chloroform (34 mL) was added a 1.0 M solution
of boron tribomide in methylene chloride (1.45 g, 5.77 mmol) at 0
°C. The reaction mixture was stirred for 10 min, warmed to rt, and
stirred for an additional 1 h before quenching with water (30 mL)
to form a yellow precipitate. Filtration and washing with water and
1
ethyl acetate afforded 21 (0.299 g, 62%): mp 265-267 °C. H
Fluorescence Staining of AD and Control Brain Sections by
2, 8, 11, or 14. Paraffin sections (7 µm thickness) of AD cortex
and age-matched control cortex were deparaffinized before incuba-
tion with 3% KMnO4 for 4 min according to the autofluorescence
chemical bleaching method of Sun et al.34 The slides were then
rinsed with water, treated with a solution of 1% K2S2O5 and 1%
oxalic acid until the brown color faded, washed again with water,
treated with freshly prepared 1% NaBH4 in water for 5 min, and
washed three times with water and PBS. The slides were then
stained with 10 µM of 2, 8, 11, and 14 as described in the previous
section.
In Vivo BBB Delivery of 2, 8, 11, or 14 into Aâ Plaque-
Bearing APP/PS1 Mouse Brain. Eleven-month-old APP/PS1 mice
were injected with 10 mg/kg of 2, 8, 11, or 14 in 100 µL volumes
of PBS with 10% DMSO via the tail vein and sacrificed at 48 h
after injection by transcardial perfusion of 4% paraformaldehyde
in PBS. After cryoprotection and cryosectioning, fixed frozen brain
sections (10 µm thickness) were hematoxylin stained, and both
fluorescence and histological images were captured with a Nikon
TE-300 Magnafire CCD camera using a DAPI filter for fluores-
cence. The fluorescent images depict the in vivo labeling of Aâ
plaques by the intravenously injected 2, 8, 11, or 14.
NMR (CDCl3) 4.08 (s, 6H), 6.99 (d, J ) 33.7 Hz, 2H), 7.02 (d, J
) 25.4 Hz, 2H), 7.44 (d, J ) 16.6 Hz, 2H), 7.71 (dd, J ) 8.3 Hz,
2.4 Hz, 2H), 8.00 (d, J ) 2.0 Hz, 2H). 13C NMRstoo insoluble to
generate data. 19F NMR (C6H5CF3) -145.72 (s, 4F). Anal.
(C26H18F4O6) C, H.
1-Fluoro-2,5-bis(bromomethyl)benzene.16 3-Fluoro-4-methyl-
benzyl bromide (3.00 g, 14.8 mmol) and NBS (2.89 g, 16.2 mmol)
in chlorform (300 mL) were heated to reflux at which time 2′2-
azobis(isobutyronitrile) (0.036 g, 0.22 mmol) was added. The
reaction mixture was refluxed for 10 h, cooled, and washed with
water. The chloroform layer was dried with MgSO4 and filtered,
and the solvent was removed in vacuo to afford an off-white solid
that was crystallized from ethanol (15 mL) to afford 1-fluoro-2,5-
bis(bromomethyl)benzene (1.79 g, 42%): mp 322-325 °C dec
1
(lit.16 mp 294 °C dec). H NMR (CDCl3) 4.43 (s, 2H), 4.49 (s,
2H), 7.12 (dd, J ) 10.3, 1.5 Hz), 7.15 (dd, J ) 7.8, 1.5 Hz), 7.37
(t, J ) 7.3 Hz).
In Vitro Aâ1-40 Binding Affinity Determination by Fluores-
cence Titration. Amyloid (Aâ) binding affinities (Kd) for 6, 8-12,
and 14, with 2 and thioflavin T as controls, were determined by
means of fluorescence titrations,25 with amyloid peptide Aâ1-40 at
23 °C. Intrinsic fluorescence intensity (FLINT) changes associated

4992 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Flaherty et al.
(18) Schmidt, M. L.; Schuck, T.; Sheridan, S.; Kung, M. P.; Kung, H.;
Zhuang, Z. P.; Bergeron, C.; Lamarche, J. S.; Skovronsky, D.;
Giasson, B. I.; Lee, V. M.; Trojanowski, J. Q. The Fluorescent Congo
Red Derivative, (trans,trans)-1-Bromo-2,5-bis-(3-hydroxycarbonyl-
4-hydroxy)styrylbenzene (BSB), Labels Diverse Beta-Pleated Sheet
Structures in Postmortem Human Neurodegenerative Disease Brains.
Am. J. Pathol. 2001, 159, 937-943.
(19) Kung, M. P.; Hou, C.; Zhuang, Z. P.; Skovronsky, D. M.; Zhang,
B.; Gur, T. L.; Trojanowski, J. Q.; Lee, V. M.; Kung, H. F.
Radioiodinated Styrylbenzene Derivatives as Potential SPECT Imag-
ing Agents for Amyloid Plaque Detection in Alzheimer’s Disease.
J. Mol. Neurosci. 2002, 19, 7-10.
Acknowledgment. This investigation received financial
support from the Vada Oldfield Alzheimer Research Foundation
(T.I.), the National Institute of Health (P01NS043985; T.I.), and
the Department of Pharmaceutical Sciences, College of Phar-
macy, University of Nebraska Medical Center (J.L.V.). We
thank K. Ashe and K. Duff for providing Tg2576 and
PS1M146L mice, J. Buescher for transgenic mouse colony
maintenance and genotyping, Paul A. Keifer and Edward L.
Ezell for assistance with NMR experiments, and Tessa Smolik
and Amanda Mausbach for synthesis of intermediates.
(20) Zhuang, Z. P.; Kung, M. P.; Skovronsky, D. M.; Gur, T. L.; Plo¨ssl,
K.; Trojanowski, J. Q.; Lee, V. M.-Y.; Kung, H. F. Radioiodinated
Styrylbenzenes and Thioflavins as Probes for Amyloid Aggregates.
J. Med. Chem. 2001, 44, 1905-1914.
Supporting Information Available: Elemental analysis data
for 6-12, 20, and 21, the proton decoupled 13C NMR spectra of 8
and 14, HMBC and HSQC spectra of 14, the fluorescence properties
of 2, 6, 8-12, and 14, and representative Aâ1-40 binding isotherms
for thioflavin T and 8. This material is available free of charge via
the Internet at http://pubs.acs.org.
(21) Crystal, A. S.; Giasson, B. I.; Crowe, A.; Kung, M. P.; Zhuang, Z.
P.; Trojanowski, J. Q.; Lee, V. M. Y. A Comparison of Amyloid
Fibrollogenesis using the Novel Fluorescent Compound K114. J.
Neurochem. 2003, 86, 1359-1368.
(22) Higuchi, M.; Iwata, N.; Matsuba, Y.; Sato, K.; Sasamoto, K.; Saido,
References
1
T. C. 19F and H MRI Detection of Amyloid Beta Plaques In Vivo.
Nat. Neurosci. 2005, 8, 527-533.
(1) Goedert, M.; Spillantini, G. A Century of Alzheimer’s Disease.
Science 2006, 314, 777-781.
(2) Hebert, L. E.; Scherr, P. A.; Bienias, J. L.; Bennett, D. A.; Evans,
D. A. Alzheimer Disease in the U.S. Population: Prevalence
Estimates Using the 2000 Census. Arch. Neurol. 2003, 60, 1119-
1122.
(3) Brookmeyer, R.; Gray, S.; Kawas, C. Projections of Alzheimer’s
Disease in the United States and the Public Health Impact of Delaying
Disease Onset. Am. J. Public Health 1998, 88, 1337-1342.
(4) Alzheimer, A. Ueber eine eigenartige Erkrankung der Hirnrinde. Allg.
Z. Psychiatr. 1907, 64, 146-148.
(5) Selkoe, D. J. Alzheimer’s Disease: Genotypes, Phenotypes, and
Treatments. Science 1997, 275, 630-631.
(6) Berg, L.; McKeel, D. W., Jr.; Miller, J. P.; Baty, J.; Morris, J. C.
Neuropathological Indexes of Alzheimer’s Disease in Demented and
Nondemented Persons Aged 80 Years and Older. Arch. Neurol. 1993,
50, 349-358.
(7) Schmitt, F. A.; Davis, D. G.; Wekstein, D. R.; Smith, C. D.; Ashford,
J. W.; Markesbery, W. R. “Preclinical” AD Revisited: Neuropa-
thology of Cognitively Normal Older Adults. Neurology. 2000, 55,
370-376.
(8) Wilcock, G. K.; Esiri, M. M. Plaques, Tangles and Dementia. A
Quantitative Study. J. Neurol. Sci. 1982, 56, 343-356.
(9) Braak, H.; Braak, E. Staging of Alzheimer’s Disease-Related
Neurofibrillary Changes. Neurobiol. Aging 1995, 16, 271-284.
(10) 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. JAMA,
J. Am. Med. Assoc. 2000, 283, 1571-1577.
(11) Hardy, J.; Selkoe, D. J. The Amyloid Hypothesis of Alzheimer’s
Disease: Progress and Problems on the Road to Therapeutics. Science
2002, 297, 353-356.
(12) Strittmatter, W. J; Saunders, A. M.; Schmechel, D; Pericak-Vance,
M.; Enghild, J.; Salvesen, G. S.; Roses, A. D. Apolipoprotein E:
High-Avidity Binding to Amyloid and Increased Frequency of Type
4 Allele in Late-Onset Familial Alzheimer Disease. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 1977-1981.
(13) Puchtler, H.; Sweat, F.; Levine, M. On the Binding of Congo Red
by Amyloid. J. Histochem. Cytochem. 1962, 10, 355-364.
(14) 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.
(15) 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.
(16) Sato, K.; Higuchi, M.; Iwata, N.; Saido, T. C.; Sasamoto, K. Fluoro-
Substituted and 13C-Labeled Styrylbenzene Derivatives for Detecting
Brain Amyloid Plaques. Eur. J. Med. Chem. 2004, 39, 573-578.
(17) Skovronsky, D. M.; Zhang, B.; Kung, M. P.; Kung, H. F.; Trojan-
owski, J. Q.; Lee, V. M. Y. In Vivo Detection of Amyloid Plaques
in a Mouse Model of Alzheimer’s Disease. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 7609-7614.
(23) Tewari, R.; Kumari, N.; Kendurkar, P. Studies on Ylides: Stereospe-
cific Synthesis of trans,trans-Distyrylbenzenes and trans,trans-
Divinylbenzenes. Indian J. Chem., Sect B: Org. Chem. Incl. Med.
Chem. 1977, 15, 753-755.
(24) Bayly, S. R.; Humphrey, E. R.; de Chair, H.; Paredes, C. G.; Bell,
Z. R.; Jeffery, J. C.; McCleverty, J. A.; Ward, M. D.; Totti, F.;
Gatteschi, D.; Courric, S.; Steele, B. R.; Screttas, C. G. Electronic
and Magnetic Metal-Metal Interactions in Dinuclear Oxomolybde-
num(V) Complexes Across bis-Phenolate Bridging Ligands with
Different Spacers Between the Phenolate Termini: Ligand-Centered
vs Metal-Centered Redox Activity. J. Chem. Soc., Dalton Trans.
2001, 9, 1401-1414.
(25) Lockhart, A.; Ye, L.; Judd, D. B.; Merritt, A. T.; Lowe, P. N.;
Morgenstern, J. L.; Hong, G.; Gee, A. D.; Brown, J. Evidence for
the Presence of Three Distinct Binding Sites for the Thioflavin T
Class of Alzheimer’s Disease PET Imaging Agents on Amyloid
Peptide Fibrils. J. Biol. Chem. 2005, 280, 7677-7684.
(26) Hsiao, K.; Chapman, P.; Nilsen, S.; Eckman, C.; Harigaya, Y.;
Younkin, S.; Yang, F.; Cole, G. Correlative Memory Deficits, Abeta
Elevation, and Amyloid Plaques in Transgenic Mice. Science 1996,
274, 99-102.
(27) Duff, K.; Eckman, C.; Zehr, C.; Yu, X.; Prada, C. M.; Perez-tur, J.;
Hutton, M.; Buee, L.; Harigaya, Y.; Yager, D.; Morgan, D.; Gordon,
M. N.; Holcomb, L.; Refolo, L.; Zenk, B.; Hardy, J.; Younkin, S.
Increased Amyloid-beta42(43) in Brains of Mice Expressing Mutant
Presenilin 1. Nature 1996, 383, 710-713.
(28) The S/N ratio is independent of a target compound’s fluorescence
Ex/Em and extinction coefficient (ꢀex; see Supporting Information).
This is illustrated by the complete lack of correlation (r ) -0.045,
P ) 0.90) between ꢀex and S/N ratio for the compounds in Table 1.
(29) Peak assigment from HSQC and HMBC experiments (see Supporting
Information).
(30) Razgulin, A. V.; Mecozzi, S. Binding Properties of Aromatic Carbon-
Bound Fluorine. J. Med. Chem. 2006, 49, 7902-7906.
(31) Hoefert, V. B; Aiken, J. M.; McKenzie, D.; Johnson, C. J. Labeling
of the Scrapie-Associated Prion Protein In Vitro and In Vivo.
Neurosci. Lett. 2004, 371, 176-180.
(32) Ishikawa, K.; Doh-ura, K.; Kudo, Y.; Nishida, N.; Murakami-Kubo,
I.; Ando, Y.; Sawada, T.; Iwaki, T. Amyloid Imaging Probes are
Useful for Detection of Prion Plaques and Treatment of Transmissible
Spongiform Encephalopathies. J. Gen. Virol. 2004, 85, 1785-1790.
(33) Krebs, F. C.; Jensen, T. Fluorinated Molecules Relevant to Conduct-
ing Polymer Research. J. Fluorine Chem. 2003, 120, 77-84.
(34) Sun, A.; Nguyen, X. V.; Bing, G. Comparative Analysis of an
Improved Thioflavin-S Stain, Gallyas Silver Stain, and Immunohis-
tochemistry for Neurofibrillary Tangle Demonstration on the Same
Sections. J. Histochem. Cytochem. 2002, 50, 463-472.
JM070085F