Phenolic bis-styrylbenzenes as β-amyloid binding ligands and free radical scavengers

Article abstract of DOI:10.1021/jm1006929

Starting from bisphenolic bis-styrylbenzene DF-9 (4), β-amyloid (Aβ) binding affinity and specificity for phenolic bis-styrylbenzenes, monostyrylbenzenes, and alkyne controls were determined by fluorescence titration with β-amyloid peptide Aβ_1-40 and a fluorescence assay using APP/PS1 transgenic mouse brain sections. Bis-styrylbenzene SAR is derived largely from work on symmetrical compounds. This study is the first to describe Aβ binding data for bis-styrylbenzenes unsymmetrical in the outer rings. With one exception, binding affinity and specificity were decreased by adding and/or changing the substitution pattern of phenol functional groups, changing the orientation about the central phenyl ring, replacing the alkene with alkyne bonds, or eliminating the central phenyl ring. The only compound with an Aβ binding affinity and specificity comparable to 4 was its 3-hydroxy regioisomer 8. Like 4, 8 crossed the blood-brain barrier and bound to Aβ plaques in vivo. By use of a DPPH assay, phenol functional groups with para orientations seem to be a necessary, but insufficient, criterion for good free radical scavenging properties in these compounds.

Full text of DOI:10.1021/jm1006929

7992 J. Med. Chem. 2010, 53, 7992–7999
DOI: 10.1021/jm1006929
Phenolic Bis-styrylbenzenes as β-Amyloid Binding Ligands and Free Radical Scavengers
Daniel P. Flaherty,† 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, United States, and
‡University of Nebraska Medical Center, College of Medicine, 985880 Nebraska Medical Center, Omaha, Nebraska 68198, United States
Received June 8, 2010
Starting from bisphenolic bis-styrylbenzene DF-9 (4), β-amyloid (Aβ) binding affinity and specificity
for phenolic bis-styrylbenzenes, monostyrylbenzenes, and alkyne controls were determined by fluores-
cence titration with β-amyloid peptide Aβ1-40 and a fluorescence assay using APP/PS1 transgenic
mouse brain sections. Bis-styrylbenzene SAR is derived largely from work on symmetrical compounds.
This study is the first to describe Aβ binding data for bis-styrylbenzenes unsymmetrical in the outer
rings. With one exception, binding affinity and specificity were decreased by adding and/or changing the
substitution pattern of phenol functional groups, changing the orientation about the central phenyl ring,
replacing the alkene with alkyne bonds, or eliminating the central phenyl ring. The only compound with
an Aβ binding affinity and specificity comparable to 4 was its 3-hydroxy regioisomer 8. Like 4, 8 crossed
the blood-brain barrier and bound to Aβ plaques in vivo. By use of a DPPH assay, phenol functional
groups with para orientations seem to be a necessary, but insufficient, criterion for good free radical
scavenging properties in these compounds.
Alzheimer’s disease (ADa)1 is a common neurodegenera-
tive disorder in the elderly2 characterized by an accumulation
of β-amyloid (Aβ) plaques in the brain parenchyma and neuro-
fibrillary tangles (NFTs) in the neuron.3,4 Although Aβ and
NFTs are commonly observed in nondemented elderly, 80
years and older, the molecular pathogenesis of AD is believed
to be affected by Aβ production and its clearance.5,6 The
amyloid cascade hypothesis7 for AD pathogenesis proposes
that accumulation and aggregation of Aβ trigger a cascade
that leads to the characteristic pathologies of AD. Evidence in
support of this hypothesis is obtained from studies of familial
AD, which reveal that Aβ accumulation and aggregation are
elevated by mutations of the APP, PS1, and PS2 genes.8
The discovery of the diazo dye Congo red as an Aβ binding
ligand9 provided a starting point in the search for potential
in vivo imaging agents using positron emission tomography
(PET), single photon emission computed tomography (SPECT),
and magnetic resonance imaging (MRI). InvestigationofCongo
red led to the discovery of bis-styrylbenzenes as a class of
compounds with strong Aβ plaque binding properties. These
compounds are exemplified by the salicylate bis-styrylben-
zenes X-34 (1)10 and FSB (2),11 which have been shown to
penetrate the blood-brain barrier (BBB) and bind to Aβplaques
deposited in APP or APP/PS1 mice.11 As shown by K114 (3)
and DF-9 (4), acidic functional groups are not necessary for
good Aβ binding.12,13 Several stilbene-based compounds, such
as SB-13 and BAY94-9172, have been studied for early detec-
tion in AD.14,15 While the structure-activity relationship
(SAR) of bis-styrylbenzene compounds has been extensively
investigated, the common thread in these studies is that Aβ-
binding SAR has been elucidated based largely on symmet-
rical bis-styrylbenzenes.
Design
*To whom correspondence should be addressed. Phone: 402-559-
5362. Fax: 402-559-9543. E-mail: jvenners@unmc.edu.
Previously, we showed that the bis-styrylbenzene, (E,E)-
1,4-bis(4-hydroxystyryl)benzene (4), binds to Aβ with high
affinity and specificity.13 In recent years, many16,17 have
recognized the antioxidant and potential anti-AD properties
of the polyphenolic styrylbenzene (stilbene) resveratrol (5), a
well-known component of red wine. Therefore, we also included
structural elements of 5 in the design of target compounds
a Abbreviations: APP/PS1, amyloid precursor protein/presenlin 1;
PS2, presenilin 2; PDAPP, plate-derived growth factor-β chain promo-
ter driving amyloid presursor protein transgenic mice; AD, Alzheimer’s
disease; Aβ, amyloid β; NFT, neurofibrillary tangles; PET, positron
emission tomography; SPECT, single photon emission computed tomo-
graphy; BBB, blood-brain barrier; SAR, structure-activity relation-
ship; MRI, magnetic resonance imaging; MOM, methoxymethyl; FLINT,
fluorescence intensity.
r
pubs.acs.org/jmc
Published on Web 11/01/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7993
Figure 1. Target compounds 6-17.
Scheme 1. Synthesis of Bis-styrylbenzenes 4 and 6-10a
a Reagents and conditions: (a) triethyl phosphite, 140 °C, 4 h; (b) NaOCH3, DMF, 80 °C, 1 h; (c) HCl, CHCl3/MeOH (3:1), room temp, 24 h.
(Figure 1), especially since oxidative stress is considered to
play an important role in AD pathology.18
those (49% and 28%) previously reported20,21 for these com-
pounds. 1,3- and 1,2-Bis(diethylphosphonylmethyl)benzenes
19b (96%) and 19c (93%) were synthesized using the Arbuzov
reaction with o- and m-xylylene dibromide. Bis-diethylphospho-
nates 19a-c were coupled with methoxymethoxy (MOM)
substituted benzaldehydes 20a-d to afford 4a and 6a-10a in
48-84% yields, which were then deprotected to afford 4 and
6-10 in 64-99% yields.
Compounds 11-15 (Scheme 2) were synthesized via suc-
cessive Heck and Horner-Wadsworth-Emmons reactions
using a new one-pot procedure.22 Heck couplings between 4-iodo-
benzyl phosphonate (21) and styrenes 22a-c were accom-
plished with palladium(II) acetate, phenylurea, and potassium
carbonate in DMF at 110 °C for 22 h.22 The reaction mixtures
were cooled to 80 °C followed by addition of benzaldehyde
20a, 20b, 20d, or 20e and 30% w/v sodium methoxide in MeOH
and then stirred for an additional 1 h to afford 11a-15a in
52-79% yield. HCl deprotection of 11a-15a afforded 11-15
in 70-94% yield after filtration and washing. Although 11
was previously synthesized in 31% overall yield in a five-step
procedure,24 we were able to synthesize this same compound
in 73% overall yield using this new one-pot method. Com-
pounds 16 and 17 were synthesized by modified literature25,26
procedures (see Supporting Information).
Little has been studied regarding substitution patterns in
the central benzene ring of Aβ-binding bis-styrylbenzenes.
For this purpose we designed target compounds6 and 7 with a
1,3 and 1,2 orientation about the central benzene ring as
opposed to the more extended 1,4 orientation in 4 and other
reported bis-styrylbenzenes.11,12 Target compounds 8 and 9
are the meta and ortho bis-phenolic regioisomers of 4. Tetra-
phenol 10 is a symmetrical bis-styrylbenzene that combines
the resorcinol substructures of 5. Unsymmetrical target com-
pounds 11-15 contain from one to three phenol functional
groups, but each maintains at least one of the para phenols of
prototype 4. The final target compounds are the monostyryl
(16) and bis-alkyne (17) derivatives of 4. A secondary objec-
tive in designing these unsymmetrical bis-styrylbenzenes was
an attempt to increase the solubility of this class of compounds,
as we have observed that symmetrical bis-styrylbenzenes have
poor aqueous solubilities. For each target compound, we
determined Aβ-binding affinity and specificity and antioxi-
dant properties in addition to BBB penetration for selected
compounds. We suggest that these data will be useful in the
design and discovery of new early diagnosis probes for AD
using PET, SPECT, or MRI and potentially for AD thera-
peutics.
β-Amyloid (Aβ) Binding Affinity and Specificity
Chemistry
β-Amyloid (Aβ) binding affinity and specificity for bis-
styrylbenzenes 6-17, with 4 and 5 as controls, were deter-
mined by means of fluorescent titrations27 with aggregated
amyloid peptide Aβ1-40 and an in vitro fluorescence-based
assay using APP/PS1 transgenic mouse brain sections.13 The
APP/PS1 transgenic mice were developed by crossing the
Compounds 413 and 6-10 (Scheme 1) were obtained using
a previously reported procedure.13 The preparation of 6 was
an improvement on the previously published procedure19 in
which a low-yielding (20%) double-Heck coupling was used.
The overall yields of 8 (69%) and 10 (42%) were better than

7994 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Scheme 2. Synthesis of Bis-styrylbenzenes 11-15a
Flaherty et al.
a Reagents and conditions: (a) Pd(OAc)2, phenylurea, K2CO3, DMF, 110 °C, 22 h; (b) NaOCH3, DMF, 80 °C, 1 h; (c) HCl, CHCl3/MeOH (3:1), room
temp, 24 h.
Table 1. Amyloid Peptide Aβ1-40 Binding Affinity (Kd) and Aβ Plaque
Binding Specificity (S/N Ratio) to APP/PS1 Transgenic Mouse Brain
Slices for 4-17
compd
Kd a (μM)
S/N ratiob
4
0.0095 ( 0.0003c
0.50 ( 0.04
2.5 ( 0.1
14 ( 1.1
3.5 ( 0.7
3.1 ( 0.7
5.1 ( 1.4
9.6 ( 3.0
11 ( 2.8
9.6 ( 1.8
8.1 ( 2.3
9.0 ( 1.5
9.1 ( 0.6
3.8 ( 0.9
5.6 ( 1.3
5.2 ( 1.2
5.9 ( 0.4
5
6
7
6.3 ( 0.1
8
0.0053 ( 0.0003
0.80 ( 0.02
0.16 ( 0.01
0.16 ( 0.01
3.5 ( 0.2
0.26 ( 0.02
0.19 ( 0.01
0.20 ( 0.02
9.2 ( 0.2
9
10
11
12
13
14
15
16
17
Figure 2. Fluorescence imaging of in vitro mouse and human brain
sections: (A) Aβ plaque in (Aβ) aged APP/PS1 mouse and (B)
human AD brain sections. Frozen brain sections (10 μm thickness)
were stained with 50 μM of 4, 8, and 10 for 30 min in 9:1 PBS/EtOH,
washed successively with 75% aqueous EtOH, 95% aqueous EtOH,
and xylene. Fluorescence images were taken using a Nikon TE-
2000, 60Â Pan Fluor objective, and Roper HQ CCD camera (600Â
original magnification). A 360/460 (DAPI filter set) was used for
imaging.
0.054 ( 0.005
a Values represent the average ( SD of 3 determinations. b Values
represent the average (SD of 10 determinations. c Reported Kd =11(2nM
and S/N = 17 ( 1.3.13
Tg2576 strain,28 which expresses the K670N/M671L mutant
of APP695 found as the Swedish familial AD gene, and the
M146L 6.1 strain,29 which expresses the M146L mutant of
PS1 found as the early onset familial AD gene. The specificity
is determined by measuring the fluorescent intensity of Aβ
plaques (specific signal) compared to that of background
normal brain tissue (noise) (Figure 2).
The only compound with an Aβ binding affinity and
specificity comparable to that of 4 was its 3-hydroxy regio-
isomer 8, which had a measured Kd of 5.3 ( 0.3 nM and a
specificity of 9.6 ( 3.0 (Table 1). In contrast, 9, the 2-hydroxy
regioisomer of 4, had an 84-fold weaker Aβ binding affinity,
although it had good binding specificity. Apparently, the 1,4
orientation about the central benzene ring in compounds 4, 8,
and 9 endows them with the extended geometry required to
maintain strong binding interactions in the hydrophobic
binding site(s) of the Aβ fibril, but only 4 and 8 have the
required phenol functional group orientations to potentially
form strong H-bonding interactions. However, 12, the un-
symmetrical regioisomeric hybrid of 4 and 8, had an unex-
pectedly much weaker Aβ binding affinity (370- and 660-fold)
than 4 and 8.
The 2 orders of magnitude decrease in Aβ binding affinity
for 6 and 7 show that the optimal geometry about the central
benzene ring is a 1,4 orientation as opposed to 1,3 (6) or 1,2
(7). Even though the antiaggregation properties of 1,3-bis-
styrylpyridines and 1,3-bis-styrylbenzenes30 and Aβ -binding
properties of 1,3-bis-styrylquinaldines31 have been investigated,
this is the first time that Aβ-binding affinity and specificity
hasve been assessed as a function of the central ring substitution
pattern of bis-styrylbenzenes.
As evidenced by the 17- to 27-fold loss in Aβ binding
affinity for the more polar tetraphenol 10 and triphenols 13
and 14, additional phenolfunctional groupsdecreased affinity
and tended to decrease specificity. Compared to bis-phenol 4,
monophenol 11 showed a 16-fold reduction in binding affi-
nity, due most likely to the loss of a H-bond formed by the
second phenol functional group in 4. Compared to 4, the more
rigid linear alkyne analogue 17 had a 6-fold loss in Aβ binding
affinity and a 2.4-fold loss in specificity, possibly due to an
inability to attain a conformer orientation complementary to
the Aβ fibril surface. Of all of the target compounds, the
symmetrical monostyrylbenzene 16 had the weakest Aβ binding

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7995
Table 2. Free Radical Scavenging Activity Measured by DPPH Assay
for Quercetin and 4-17
compd
DPPH IC50 (μM)a
quercetin
4
1.3 ( 1.1b
3.1 ( 1.2
62 ( 1.4c
3.4 ( 1.1
4.0 ( 1.1
>100
5
6
7
8
9
4.0 ( 1.1
5.1 ( 1.0
5.6 ( 1.1
3.5 ( 1.2
>100
10
11
12
13
14
15
16
17
4.5 ( 1.1
9.1 ( 1.1
1.8 ( 1.2
45 ( 1.0
a Values represent the average ( SD of three determinations. b Reported
Figure 3. In vivo labeling of Aβ plaques via intravenous injec-
tion of 4, 8, or 10 in PDAPP mice. Ten-month-old PDAPP mice
were injected intraperitoneally with vehicle only (Aβ) (A), or single
50 mg/kg doses of 4 (B), 8 (C), or 10 (D) and sacrificed at 48 h after
injection. Fluorescence images of the CA1 and dentate gyrus (DG)
fields of the hippocampi were captured using 10Â or 40Â (insets)
objectives on a Nikon TE-300 with a digital camera. Insets show
representative fluorescent staining of Aβ plaques by 4, 8, or 10.
Bar=200 or 50 μm (insets).
IC50 = 9.1 μM.35 cReported IC50 = 74 ( 5.3 μM.43
measured using the stable free radical 1,1-diphenyl-2-picryl-
hydrazyl (DPPH) following the procedure of Yang et al.32
with some modifications (Table 2). Compounds 4, 6, 12, and
16 were no less effective than quercetin (IC50=1.3 ( 1.1 μM)
inDPPHfree radical scavenging activity, whereas compounds
7, 9, 10, 14, and 15 were 3- to 7-fold less potent (p < 0.05) than
quercetin. Aside from 8 and 13, which had no antioxidant
activity (IC50 > 100 μM) in this assay, monostyrylbenzene 5
and bis-alkyne 17 were the least effective radical scavengers
with IC50 values of 62 and 45 μM, respectively. No particular
SAR is apparent from these data aside from the general
observation that a phenol functional group with a para
orientation seems to be a necessary, but insufficient, criterion
for good free radical scavenging properties of bis-styrylben-
zenes. With polyphenolic antioxidants such as 517 of increas-
ing interest in AD research, this set of compounds may prove
beneficial in future research.
affinity, 3 orders of magnitude weaker than that of 4. This could
be attributed either to the loss of the central benzene ring elimi-
nating a critical binding interaction or to the shorter distance
between phenol functional groups, reducing the effectiveness of
16 to form strong H-bonds with the Aβfibril surface or a combi-
nation of the two. Surprisingly, monostyrylbenzene 5 displayed
only a 52-fold loss in binding affinity compared to 4. Conceiv-
ably, the 3,5-dihydroxy substituted 5, but not the 4-hydroxy
substituted 16, provides conformers with the geometry required
to form strong H-bonds to the aggregated Aβ fibril.
Human AD Brain Section Staining
Summary
Bis-styrylbenzenes 8 and 10, along with the control bis-
styrylbenzene 4, were used to stain human AD brain sections
(Figure 2). Each compound was shown to bind specifically to
Aβ plaques in human brain sections. Age matched controls
were also stained and showed no staining of normal brain
tissue (data not shown).
These data extend the SAR of bis-styrylbenzene Aβ binding
and free-radical scavenging activity and provide further direc-
tion for the development of noninvasive Aβ binding ligands
for early detection of AD. One new bis-styrylbenzene proto-
type (8) was identified that could serve as a building block for
the design and discovery of new early diagnosis probes for AD
using PET, SPECT, or MRI and potentially for AD thera-
peutics. Finally, we observed that a phenol functional group
with a para orientation seems to be a necessary, but insuffi-
cient, criterion for good free radical scavenging properties in
these bis-styrylbenzenes.
In Vivo BBB Delivery in Plaque-Bearing Mice
We next determined whether interperitoneally administered
8 and 10 (with 4 as a control) could cross the BBB and bind to
Aβ plaques. Bis-styrylbenzenes 4, 8, and 10 (50 mg/kg) were
injected into 10-month-old plate-derived growth factor-β chain
promoter driving APP (PDAPP) transgenic mice. As shown in
Figure 3, all compounds crossed the BBB and bound to Aβ
plaques in vivo. Each section showed intense Aβ plaque signals
with high specificities. Control nontransgenic animals showed
no specific signals in the brain for any of the tested compounds
after intraperitoneal injection (data not shown). These data
confirm that these bis-styrylbenzene derivatives retain the BBB
penetration capabilities of 4 and that 8 and 10 have the
potential as lead compounds for early Aβ detection research.
Experimental Section
General. Starting materials were purchased from Aldrich,
TCI, or Acros. All reactions were run under a positive pressure
of Ar. Melting points were determined on a Stanford Research
Systems E-Z Melt apparatus and are uncorrected. 1H (500 MHz)
and 13C (125.7 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 internal (CH3)4Si for 1H and CDCl3 (77.0 ppm) and
DMSO-d6 (39.7 ppm) for 13C NMR. Elemental analyses were
performed by M-H-W Laboratories, Phoenix, AZ. All target
compounds had purities of g95%.
Free Radical Scavenging Activity
(E,E)-1,4-Bis(4-hydroxystyryl)benzene (4).13 Step 1. A 30 wt %
solution of sodium methoxide in methanol (1.39 mL, 7.40 mmol)
The relative free radical scavenging properties of the target
bis-styrylbenzenes, along with quercetin as a control, were

7996 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Flaherty et al.
was added to a stirred mixture of 20a (0.983 g, 5.92 mmol) and 1,4-
bis(diethylphosphonylmethyl)benzene (19a) (1.12 g, 2.96 mmol) in
DMF (15 mL) at room temperature. This mixture was heated to
80 °C for 2 h. The reaction was then quenched with H2O (10 mL)
and the solid precipitate was filtered and rinsed with ether to afford
(E,E)-1,4-bis(4-methoxymethoxy)styrylbenzene 4a (0.858 g, 72%).
1H NMR (CDCl3) δ 3.50 (s, 6H), 5.20 (s, 4H), 6.98 (d, J =
16.1 Hz, 2H), 7.04 (d, J = 8.8 Hz, 4H), 7.08 (d, J = 16.1, 2H), 7.45
(d, J = 8.3 Hz, 4H), 7.48 (s, 4H). 13C NMR (CDCl3) δ 56.0, 94.4,
116.4, 126.6, 126.7, 127.7, 127.8, 131.3, 136.6, 156.8.
2H), 7.13 (d, J = 7.8 Hz, 2H), 7.22 (td, J = 8.3 Hz, 1.5 Hz, 2H),
7.52 (d, J = 15.1 Hz, 2H), 7.53 (s, 4H), 7.63 (dd, J = 7.8 Hz, 1.5
Hz, 2 H). 13C NMR (CDCl3) δ 56.2, 94.8, 114.9, 122.1, 123.1,
126.2, 126.8, 127.3, 128.6, 128.9, 137.1, 154.6.
Step 2. As described above for 4, 9a (0.634 g, 1.58 mmol) was
deprotected using 12.0 M HCl (0.230 mL, 2.70 mmol) to yield 9
(0.316 g, 64%): mp 262-265 °C (lit.33 mp 230-231 °C). 1H NMR
(DMSO-d6) δ 6.82 (t, J = 7.8 Hz, 2H), 6.88 (d, J = 7.8 Hz, 2H),
7.10 (t, J = 7.8 Hz, 2H), 7.21 (d, J = 16.1 Hz, 2H), 7.43 (d, J =
16.6 Hz, 2H), 7.55 (s, 4H), 7.59 (d, J = 7.8 Hz, 2H), 9.75 (s, 2H).
13C NMR (DMSO-d6) δ 116.0, 119.5, 123.5, 124.0, 126.6, 126.8,
127.6, 128.8, 136.9, 155.2. Anal. (C22H18O2) C, H.
Step 2. 4a (0.490 g, 1.22 mmol) and 4:1 CHCl3/MeOH (25 mL)
were stirred at room temperature until 4a was completely
dissolved before dropwise addition of 12.0 M HCl (0.812 mL,
9.74 mmol) at room temperature. The reaction was quenched
with H2O (20 mL) after 24 h. The CHCl3/MeOH layer was
removed in vacuo to afford 4 as a white solid (0.379 g, 99%): mp
(E,E)-1,4-Bis(3,5-dihydroxystyryl)benzene (10).21 Step 1. As
described above for 4a, 20d (0.173 g, 0.765 mmol) and 19a (0.145 g,
1
0.382 mmol) yielded 10a (0.096 g, 48%): mp 101-106 °C. H
NMR (CDCl3) δ 3.51 (s, 12H), 5.20 (s, 8H), 6.66 (t, J = 2.4 Hz,
2H), 6.88 (d, J = 2.4 Hz, 4H), 7.04 (d, J = 16.1 Hz, 2H), 7.09 (d,
J = 16.1 Hz, 2H), 7.50 (s, 4H). 13C NMR (CDCl3) δ 56.1, 94.5,
104.3, 107.8, 126.9, 128.3, 128.9, 136.5, 139.5, 158.5.
1
dec 312 °C. H NMR (DMSO-d6) δ 6.77 (d, J = 8.3 Hz, 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) δ
115.8, 125.0, 126.5, 128.0, 128.2, 128.3, 136.5, 157.6.
Step 2. As described above for 4, 10a (0.084 g, 0.16 mmol) was
deprotected using 12.0 M HCl (0.214 mL, 2.57 mmol) to yield 10
(0.049 g, 88%) mp 290-305 °C (lit.21 mp >300 °C). 1H NMR
(DMSO-d6) δ 6.16 (s, 2H), 6.45 (s, 4H), 7.04 (d, J = 16.1 Hz, 2H),
7.09 (d, J = 16.1 Hz, 2H), 7.57 (s, 4H). 13C NMR (DMSO-d6) δ
104.9, 127.0, 127.7, 129.0, 136.5, 139.0, 158.7.
(E,E)-1,3-Bis(4-hydroxystyryl)benzene (6).18 Step 1. As de-
scribed above for 4a, 20a (0.677 g, 4.07 mmol) and 19b (0.773 g,
2.04 mmol) yielded 6a (0.660 g, 80%): mp 123-126 °C. 1H NMR
(DMSO-d6) δ 3.39 (s, 6H), 5.22 (s, 4H), 7.05 (d, J = 8.8 Hz, 4H),
7.14 (d, J = 16.1 Hz, 2H), 7.28 (d, J = 16.6, 2H), 7.36 (t, J = 7.3
Hz, 1H), 7.45 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.8 Hz, 4H), 7.80
(s, 1H). 13C NMR (DMSO-d6) δ 55.8, 94.0, 116.6, 124.0, 125.6,
126.7, 127.9, 128.4, 129.2, 130.9, 137.8, 156.7.
(E,E)-1-Styryl-4-(4-hydroxystyryl)benzene (11).22 As described
above for 4, 11a (0.654 g, 1.91 mmol) was deprotected using 12.0 M
HCl (1.05 mL, 12.6 mmol) to afford 11 (0.53 g, 93%): mp 281-
1
Step 2. As described above for 4, 6a (0.406 g, 1.01 mmol) was
deprotected using 12.0 M HCl (0.672 mL, 8.07 mmol) to yield 6
(0.309 g, 97%): mp dec 218-220 °C (lit.18 mp 232-234 °C). 1H
NMR (DMSO-d6) δ 6.78 (d, J = 8.8 Hz, 4H), 7.03 (d, J = 16.1
Hz, 2H), 7.21 (d, J = 16.1 Hz, 2H), 7.32 (t, J = 6.8 Hz, 1H), 7.40
(d, J = 7.3 Hz, 2H), 7.44 (d, J = 8.3 Hz, 4H), 7.72 (s, 1H), 9.59
(s, 2H). 13C NMR (DMSO-d6) δ 116.0, 124.0, 125.2, 125.4,
285 °C. H NMR (DMSO-d6) δ 6.78 (d, J = 8.3 Hz, 2H), 7.03
(d, J = 16.1 Hz, 2H), 7.18 (d, J = 16.6 Hz, 2H), 7.26 (s, 2H), 7.27
(t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.44 (d, J = 8.3 Hz,
2H), 7.55(d, J = 8.3 Hz, 2H), 7.59 (d, J= 8.3 Hz, 2H), 7.61 (d, J=
7.9 Hz, 2H), 9.59 (s, 1H). 13C NMR (DMSO-d6) δ 115.8, 124.9,
126.5, 126.6, 127.0, 128.1, 128.3, 128.7, 128.9, 131.8, 131.9, 135.9,
137.2, 137.3, 157.6, 167.2.
128.23, 128.5, 129.0, 129.3, 138.3, 157.8. Anal. (C22H18O2 1/3H2O)
(E,E)-1-Styryl-4-[4-(methoxymethoxy)styryl]benzene (11a).22
Diethyl 4-iodobenzylphosphonate (21) (0.414 g, 1.17 mmol) and
22a (0.260 g, 2.34 mmol) were mixed with palladium(II) acetate
(0.008 g, 0.035 mmol), phenylurea (0.010 g, 0.070 mmol), and
potassium carbonate (0.323 g, 2.34 mmol) in DMF (5 mL) and
stirred at 110 °C for 22 h. The mixture was then cooled to 80 °C,
and to this mixture were added 20a (0.195 g, 1.17 mmol) and
30% w/v sodium methoxide in methanol (0.440 mL, 2.34 mmol).
The mixture was stirred for 1 h at 80 °C and then cooled to room
temperature and quenched with H2O (15 mL). The solid was
then filtered and rinsed with H2O and ether to afford 11a (0.317
g, 79%): mp 240-243 °C. 1H NMR (CDCl3) δ 3.50 (s, 3H), 5.20
(s, 2H), 6.99 (d, J = 16.6 Hz, 1H), 7.04 (d, J = 8.3 Hz, 2H), 7.09
(d, J = 16.6 Hz, 1H), 7.12 (s, 2H), 7.26 (t, J = 7.3 Hz, 1H), 7.36
(t, J = 7.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 7.8 Hz,
2H), 7.49 (s, 4H), 7.52 (d, J = 7.8 Hz, 2H). 13C NMR (CDCl3) δ
56.0, 94.4, 116.4, 126.5, 126.6, 126.7, 126.8, 127.6, 127.7, 128.0,
128.3, 128.4, 128.7, 131.3, 136.3, 136.9, 137.3, 156.9.
(E,E)-1-(4-Hydroxystyryl)-4-(3-hydroxystyryl)benzene (12).
As described above for 4, 12a (0.280 g, 0.696 mmol) was
deprotected using 12.0 M HCl (0.696 mL, 8.35 mmol) to afford
12 (0.161 g, 74%): mp 297-301 °C. 1H NMR (DMSO-d6) δ 6.68
(d, J = 8.3 Hz, 1H), 6.78 (d, J = 8.8 Hz, 2H), 6.98 (s, 1H), 7.02
(d, J = 16.6 Hz, 1H), 7.04 (d, J = 7.3 Hz, 1H), 7.12-7.22
(m, 4H), 7.44 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.58
(d, J = 8.8 Hz, 2H). 13C NMR (DMSO-d6) δ 113.2, 115.0, 115.8,
117.7, 125.0, 126.6, 127.1, 128.1, 128.3, 128.6, 129.8, 135.9,
137.1, 138.7, 157.6, 157.8.
3
C, H.
(E,E)-1,2-Bis(4-hydroxystyryl)benzene (7). Step 1. As de-
scribed above for 4a, 20a (0.310 g, 1.87 mmol) and 19c (0.353 g,
0.993 mmol) yielded 7a (0.169 g, 45%): mp 67-70 °C. 1H NMR
(CDCl3) δ 3.50 (s, 6H), 5.20 (s, 4H), 6.95 (d, J = 16.1 Hz, 2H),
7.04 (d, J = 8.8 Hz, 4H), 7.26 (dd, J1 = 5.8 Hz, J2 = 3.4 Hz, 2H),
7.33 (d, J = 16.1 Hz, 2H), 7.46 (d, J = 8.8 Hz, 4H), 7.55 (dd,
J1 = 5.9 Hz, J2 = 3.4 Hz, 2H). 13C NMR (CDCl3) δ 55.0, 94.3,
116.4, 125.0, 126.5, 127.4, 127.8, 130.6, 131.5, 136.0, 156.9.
Step 2. As described above for 4, 7a (0.151 g, 0.375 mmol) was
deprotected using 12.0 M HCl (0.250 mL, 3.00 mmol) to yield 7
1
(0.108 g, 92%): mp 262-265 °C. H NMR (DMSO-d6) δ 6.78
(d, J = 7.8 Hz, 4H), 7.00 (d, J = 16.1 Hz, 2H), 7.25 (brs, 2H),
7.42 (d, J = 16.1 Hz, 2H), 7.50 (d, J = 7.8 Hz, 4H), 7.62 (brs,
2H), 9.58 (s, 2H). 13C NMR (DMSO-d6) δ 115.6, 122.7, 126.0,
127.4, 128.4, 128.5, 130.8, 135.7, 157.6. Anal. (C22H18O2) C, H.
(E,E)-1,4-Bis(3-hydroxystyryl)benzene (8).19 Step 1. As de-
scribed above for 4a, 20b (0.388 g, 2.33 mmol) and 19a (0.442 g,
1.17 mmol) yielded 8a (0.359 g, 76%): mp 136-140 °C. 1H NMR
(DMSO-d6) δ 3.41 (s, 6H), 5.24 (s, 4H), 6.92-6.95 (m, 2H),
7.24-7.28 (m, 8H), 7.31 (t, J = 7.8 Hz, 2H), 7.62 (s, 4H). 13C
NMR (DMSO-d6) δ 56.0, 94.4, 114.0, 115.6, 120.3, 126.9, 128.3,
128.6, 129.7, 136.6, 138.8, 157.6.
Step 2. As described above for 4, 8a (0.136 g, 0.338 mmol) was
deprotected using 12.0 M HCl (0.230 mL, 2.70 mmol) to yield 8
1
(0.097 g, 91%): mp 281-283 °C (lit.19 mp 240 °C). H NMR
(DMSO-d6) δ 6.69 (dd, J = 7.8 Hz, 2.0 Hz, 2H), 6.98 (brs, 2H),
7.04 (d, J = 7.8 Hz, 2H), 7.12-7.22 (m, 6H), 7.60 (s, 4H).
13C NMR (DMSO-d6) δ 113.2, 115.1, 117.8, 127.1, 128.0, 128.7,
129.8, 136.5, 138.6, 157.8. Anal. (C22H18O2) C, H.
(E,E)-1-[4-(Methoxymethoxy)styryl]-4-[3-(methoxymethoxy)-
styryl}benzene (12a). As described above for 11a, 21 (0.312 g,
0.880 mmol) and 22b (0.289 g, 1.76 mmol) were mixed with
palladium(II) acetate (0.006 g, 0.03 mmol), phenylurea (0.007 g,
0.05 mmol), and potassium carbonate (0.243 g, 1.76 mmol) and
stirred for 22 h at 110 °C in DMF. After the mixture was cooled
to 80 °C, compound 20b (0.146 g, 0.880 mmol) and sodium
(E,E)-1,4-Bis(2-hydroxystyryl)benzene (9).33 Step 1. As de-
scribed above for 4a, 20c (1.05 g, 6.31 mmol) and 19a (1.19 g,
3.17 mmol) yielded 9a (1.07 g, 84%): 1H NMR (CDCl3) δ 3.53
(s, 6H), 5.26 (s, 4H), 7.03 (t, J = 7.3 Hz, 2H), 7.11 (d, J = 16.6 Hz,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7997
methoxide solution (0.330 mL, 1.76 mmol) were added. The
mixture was stirred for 1 h at 80 °C and worked up as described
J = 7.8 Hz, 1H), 7.11 (d, J = 16.6 Hz, 1H), 7.16 (s, 2H), 7.17
(t, J = 8.3 Hz, 1H), 7.54 (d, J = 7.8 Hz, 2H), 7.57 (d, J = 8.3 Hz,
2H). 13C NMR (DMSO-d6) 113.2, 113.5, 115.0, 115.9, 117.7,
118.9, 124.8, 126.6, 127.0, 128.1, 128.3, 128.9, 129.0, 129.8,
135.9, 137.1, 138.7, 145.6, 145.9, 157.8.
1
above to yield 12a (0.23 g, 65%): mp 142-152 °C. H NMR
(CDCl3) δ 3.50 (s, 3H), 3.51 (s, 3H), 5.20 (s, 2H), 5.22 (s, 2H),
6.95 (dd, J1 = 7.8 Hz, J2 = 2.0 Hz, 1H), 6.99 (d, J = 16.6 Hz,
1H), 7.04 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 16.1 Hz, 1H), 7.09
(s, 2H), 7.18 (d, J = 7.3 Hz, 1H), 7.21 (s, 1H), 7.28 (t, J = 8.3 Hz,
1H), 7.45 (d, J = 8.8 Hz, 2H), 7.50 (s, 4H). 13C NMR (CDCl3) δ
55.9, 94.2, 94.3, 113.9, 115.4, 116.3, 120.3, 126.6, 126.8, 127.6,
127.9, 128.0, 128.6, 129.6, 131.2, 136.1, 136.9, 138.8, 156.8, 157.5.
(E,E)-1-(4-Hydroxystyryl)-4-(3,4-dihydroxystyryl)benzene (13).
As described above for 4, 13a (0.100 g, 0.216 mmol) was de-
protected using 12.0 M HCl (0.252 mL, 3.03 mmol) to afford 13
(0.050 g, 70%): mp 341-350 °C. 1H NMR (DMSO-d6) δ 6.73 (d,
J = 7.8 Hz, 1H), 6.77 (d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.3 Hz,
1H), 6.91 (d, J = 16.6 Hz, 1H), 7.01 (d, J = 16.6 Hz, 1H), 7.08 (d,
J = 16.6 Hz, 1H), 7.15 (d, J = 16.6 Hz, 1H), 7.43 (d, J = 8.3 Hz,
2H), 7.51 (s, 4H). 13C NMR (DMSO-d6) δ 113.5, 115.8, 115.9,
118.8, 124.9, 125.1, 126.6, 126.6, 128.1, 128.2, 128.4, 128.6, 129.0,
136.4, 136.5, 145.6, 145.8, 157.5.
(E,E)-1-[3-(Methoxymethoxy)styryl]-4-[3,4-di(methoxymethoxy)-
styryl]benzene (15a). As described above for 11a, 21 (0.359 g,
1.01 mmol) and 22c (0.333 g, 2.03 mmol) were mixed with
palladium(II) acetate (0.007 g, 0.03 mmol), phenylurea (0.008
g, 0.06 mmol), and potassium carbonate (0.281 g, 2.03 mmol)
and stirred for 22 h at 110 °C in DMF. After the mixture was
cooled to 80 °C, compound 20e (0.229 g, 1.01 mmol) and sodium
methoxide solution (0.381 mL, 2.03 mmol) were added. The
mixture was stirred for 1 h at 80 °C and worked up as described
1
above to yield 15a (0.244 g, 52%): mp 94-101 °C. H NMR
(CDCl3) δ 3.51 (s, 3H), 3.53 (s, 3H), 3.56 (s, 3H), 5.22 (s, 2H),
5.26 (s, 2H), 5.30 (s, 2H), 6.95 (dd, J1 = 7.8 Hz, J2 = 2.4 Hz,
1H), 6.98 (d, J = 16.6 Hz, 1H), 7.06 (d, J = 16.1 Hz, 1H), 7.09
(s, 2H), 7.12 (dd, J1 = 8.8 Hz, J2 = 2.0 Hz, 1H), 7.16 (d, J =
8.3 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 7.21 (s, 1H), 7.28 (t, J =
7.8 Hz, 1H), 7.36 (d, J = 1.5 Hz, 1H), 7.49 (s, 4H). 13C NMR
(CDCl3) δ 56.0, 56.2, 94.4, 95.3, 95.4, 114.0, 114.2, 115.5, 116.6,
120.3, 121.1, 126.7, 126.8, 127.1, 128.0, 128.1, 128.7, 129.7,
132.1, 136.3, 136.8, 138.9, 146.9, 147.4, 157.6.
(E,E)-1-[4-(Methoxymethoxy)styryl]-4-[3,4-di(methoxymethoxy)-
styryl]benzene (13a). As described above for 11a, 21 (0.346 g,
0.977 mmol) and 22b (0.321 g, 1.95 mmol) were mixed with
palladium(II) acetate (0.007 g, 0.03 mmol), phenylurea (0.008 g,
0.06 mmol), and potassium carbonate (0.270 g, 1.95 mmol) and
stirred for 22 h at 110 °C in DMF. After the mixture was cooled
to 80 °C, compound 20e (0.221 g, 0.977 mmol) and sodium
methoxide solution (0.366 mL, 1.95 mmol) were added. The
mixture was stirred for 1 h at 80 °C and worked up as described
In Vitro Aβ1-40 Binding Affinity Determination by Fluorescence
Titration. Amyloid (Aβ) binding affinities (Kd) for 5-17 and 4 as
a control were determined by means of fluorescence titrations,13,27
with amyloid peptide Aβ1-40 at 23 °C. Intrinsic fluorescence
intensity (FLINT) changes associated with ligand binding to
aggregated Aβ1-40 were recorded on a Cary Eclipse fluorescence
spectrophotometer (Varian) using excitation wavelengths deter-
mined for each compound. Fixed concentrations of Aβ1-40
(500 nM for 5-7, 9, 10-16; 40 nM for 4 and 17; 20 nM for 8)
were diluted to 500 μL in 9:1 PBS/EtOH in a 10 mm quartz
fluorescence cuvette. Concentrations of Aβ1-40 were selected to be
no more than 10-fold higher than compound binding affinities to
obtain accurate Kd values. To the Aβ1-40 PBS/EtOH solution in
the cuvette, aliquots of test compounds in PBS/EtOH were titrated
using a 2.0 μL of Hamilton syringe with a reproducibility adapter
along a concentration gradient of test compound (0.8 nM satura-
tion). Fluorescence spectra were recorded until the fluorescence no
longer increased with increasing compound concentration (satura-
tion). The FLINT at these wavelengths was plotted versus com-
pound concentrations to yield binding isotherms. Aβ1-40 Kd values
were determined (Prism 4.0c software, GraphPad, Inc.).
In Vitro A Plaque Binding Specificity Using APP/PS1 Trans-
genic Mouse Brain Slices. DMSO stock solutions of 4-17 were
diluted to 0.50 mM with EtOH and then diluted with 9:1 PBS/
EtOH to prepare 50 μM solutions. Eleven-month-old transgenic
APP/PS1 mice derived from crossing the Tg2576 line expressing
APP Swedish mutant and the M146L 6.1 line expressing pre-
senilin-1 mutant28,29 were anesthetized and transcardially per-
fused with 4% paraformaldehyde in PBS under the guidance of
Institutional Animal Care and Use Committee at the University
of Nebraska Medical Center. Fixed brain samples were cryo-
protected 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 compound solutions for 30 min and
then washed successively with 75% aqueous EtOH, 95% aqu-
eous EtOH, 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 magnifica-
tion: 400Â) following a standard FSB staining protocol.34
Fluorescence images of three plaques per section were system-
atically captured using the same image acquisition setting (laser
power, capturing time, photomultiplier setting) to obtain com-
parable fluorescent intensities of five plaque regions (specific
signal) and five background regions (noise signal) to calculate
signal-to-noise (S/N) ratios.
1
above to yield 13a (0.35 g, 77%): mp 128-138 °C. H NMR
(CDCl3) δ 3.50 (s, 3H), 3.53 (s, 3H), 3.56 (s, 3H), 5.20 (s, 2H),
5.26 (s, 2H), 5.30 (s, 2H), 6.98 (d, J = 16.1, 1H), 6.98 (d, J = 16.1
Hz, 1H), 7.04 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 16.1 Hz, 1H), 7.08
(d, J = 16.1 Hz, 1H), 7.12 (dd, J1 = 8.3 Hz, J2 = 2.0 Hz, 1H),
7.15 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 1.5 Hz, 1H), 7.46 (d, J =
8.8 Hz, 2H), 7.48 (s, 4H). 13C NMR (CDCl3) δ 56.0, 56.2, 94.4,
95.3, 95.4, 114.2, 116.4, 116.6, 121.0, 126.6, 126.7, 127.2, 127.6,
127.8, 127.8, 131.3, 132.2, 136.4, 136.7, 146.9, 147.4, 156.8.
(E,E)-1-(4-Hydroxystyryl)-4-(3,5-dihydroxystyryl)benzene (14).
As described above for 4, 14a (0.113 g, 0.244 mmol) was
deprotected using 12.0 M HCl (0.285 mL, 3.42 mmol) to afford
14 (0.076 g, 94%): mp 295-305 °C. 1H NMR (DMSO-d6) δ 6.16
(s, 1H), 6.45 (d, J = 2.0 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 7.02
(d, J = 16.6 Hz, 1H), 7.04 (s, 1H), 7.08 (d, J = 16.6 Hz, 1H), 7.17
(d, J = 16.1 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 8.3
Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 9.27 (s, 2H), 9.61 (s, 1H). 13C
NMR (DMSO-d6) δ 102.5, 104.9, 115.8, 125.0, 126.6, 127.0,
127.8, 128.1, 128.4, 128.6, 128.7, 135.9, 137.1, 139.1, 157.6, 158.8.
(E,E)-1-[4-(Methoxymethoxy)styryl]-4-[3,5-di(methoxymethoxy)-
styryl]benzene (14a). As described above for 11a, 21 (0.342 g,
0.965 mmol) and 22b (0.317 g, 1.93 mmol) were mixed with
palladium(II) acetate (0.007 g, 0.03 mmol), phenylurea (0.008 g,
0.06 mmol), and potassium carbonate (0.267 g, 1.93 mmol) and
stirred for 22 h at 110 °C in DMF. After the mixture was cooled to
80 °C, compound 20d (0.218 g, 0.965 mmol) and sodium meth-
oxide solution (0.362 mL, 1.93 mmol) were added. The mixture
was stirred for 1 h at 80 °C and worked up as described above to
yield 14a (0.285 g, 64%): mp 114-119 °C. 1H NMR (CDCl3) δ
3.49 (s, 3H), 3.51 (s, 6H), 5.20(s, 6H), 6.66 (s, 1H), 6.88(d, J = 2.0
Hz, 2H), 6.98 (d, J =16.1 Hz, 1H), 7.03 (d, J = 16.6 Hz, 1H), 7.04
(d, J = 8.8 Hz, 2H), 7.08 (d, J = 16.6 Hz, 2H), 7.45 (d, J = 8.8
Hz, 2H), 7.48 (s, 4H). 13C NMR (CDCl3) δ 56.1, 94.3, 94.4, 104.3,
107.8, 116.4, 126.6, 126.9, 127.7, 128.0, 128.1, 129.0, 131.2, 136.1,
137.0, 139.5, 156.9, 158.5.
(E,E)-1-(3-Hydroxystyryl)-4-(3,4-dihydroxystyryl)benzene (15).
As described above for 4, 15a (0.114 g, 0.246 mmol) was
deprotected using 12.0 M HCl (0.246 mL, 2.96 mmol) to afford
15 (0.072 g, 89%): mp 285-289 °C. 1H NMR (DMSO-d6) δ 6.69
(d, J = 7.8 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 8.3 Hz,
1H), 6.93 (d, J = 16.1 Hz, 1H), 6.98 (s, 1H), 7.01 (s, 1H), 7.04 (d,

7998 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Flaherty et al.
Fluorescence Staining of AD and Control Brain Sections by 4,
8, and 10. Human adult AD brain sections and age-matched
controls were stained with compounds 4, 8, and 10. The frozen
brain sections (10 μm thickness) were stained with compound
solutions (same concentrations and PBS/EtOH content as
mouse brain section staining solutions) for 30 min and then
washed successively with 75% aqueous EtOH, 95% ethanol,
and xylene. Fluorescence imaging was performed as described in
the previous section.
In Vivo BBB Delivery of 4, 8, and 10 to Aβ Plaque-Bearing
PDAPP Mouse Brain. Ten-month-old plate-derived growth
factor-β chain promoter driving (PDAPP) mice were injected
intraperitoneally (ip) with vehicle only (10% DMSO/PBS) or
with single 50 mg/kg doses ip of 4, 8, or 10 and sacrificed at 48 h
after injection by transcardial perfusion with 4% paraformal-
dehyde (PFA) in PBS. After cryoprotection, fixed frozen brains
were sectioned with 30 mm thickness, postfixed with 4% PFA
for 5 min, washed in PBS, and mounted on microscope slides
with coverslips using VECTASHIELD mounting medium
(Vector Laboratories, Burlingame, CA). Fluorescence images
were captured with a digital Olympus DP71 camera (Olympus,
Center Valley, PA) connected to a TE-300 microscope (Nikon,
Garden City, NJ).
DPPH Free Radical Scavenging Assay. Relative free radical
quenching for 4-17, along with quercetin as a control, was
assayed spectrophotometrically using the stable free radical 1,1-
diphenyl-2-picrylhydrazyl (DPPH) following the procedure of
Yang et al.32,34 with some modifications. Abstraction of a
proton from a free radical scavenger (target compound) de-
creases the DPPH absorption at 517 nm. Reactions were run in a
96-well plate, and the absorbance at 517 nm was recorded on a
plate reader. All compounds were first solublized in DMSO to
form concentrated stock solutions (∼15 mM) that were then
diluted into working solutions in MeOH. Mixtures in the wells
contained increasing concentrations of target compounds (0 nM
to 5 mM), 100 μM DPPH, and MeOH (300 μL total volume).
Each concentration was performed in triplicate, and IC50 values
were determined using the sigmoidal dose-response fit in Prism
4.0c software (GraphPad, Inc.)
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Compound 4 was prepared previously in our lab using a Horner-
Wadsworth-Emmons coupling with 1,4-bis(diethyl phosphonylmethyl)-
benzene and p-anisaldehyde to form (E,E)-1,4-bis(4-methoxy)styryl-
benzene which was deprotected with 1.0 M boron tribromide in DCM to
yield 4.
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Acknowledgment. This investigation received financial
support from the Vada Oldfield Alzheimer Research Founda-
tion (T.I, T.K.), National Institutes of Health (Grant P01
NS043985 (T.I.), Grant R01 MH083523 (T.I.), Grant R21
AG032600 (T.I.)), UNMC Brain Bank Core Fund (T.I.), an
American Foundation of Pharmaceutical Education (AFPE)
predoctoral fellowship (D.P.F.), a Josiah Kirby Lilly, Sr.
Memorial AFPE predoctoral fellowship (D.P.F), a Nancy
and Ronald Reagan Alzheimer’s Scholarship Award (D.P.F.),
and the Department of Pharmaceutical Sciences, College of
Pharmacy, University of Nebraska Medical Center (D.P.F.).
We thank Dr. L. Mucke for providing PDAPP mice (line J20).
We also thank Sarah A. Flaherty for preparing figures of
brain staining and in vivo data.
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Supporting Information Available: Elemental analysis data
for 4a, 6a-10a, 12a-15a, 6-16, and 29; synthetic procedures
for 16,25 17,26 19b,37 19c,36 20a-e,22,32,37-39 22b,22 22c,40 24,41
25,41 and 28.42 This material is available free of charge via the
Internet at http://pubs.acs.org.
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