Bis-pyridylethenyl benzene as novel backbone for amyloid-β binding compounds

Article abstract of DOI:10.1016/j.bmc.2016.05.022

Detection of cerebral β-amyloid (Aβ) by targeted contrast agents is of great interest for in vivo diagnosis of Alzheimer's disease (AD). Partly because of their planar structure several bis-styrylbenzenes have been previously reported as potential Aβ imaging agents. However, these compounds are relatively hydrophobic, which likely limits their in vivo potential. Based on their structures, we hypothesized that less hydrophobic bis-pyridylethenylbenzenes may also label amyloid. We synthesized several bis-pyridylethenylbenzenes and tested whether these compounds indeed display improved solubility and lower Log?P values, and studied their fluorescent properties and Aβ binding characteristics. Bis-pyridylethenylbenzenes showed a clear affinity for Aβ plaques on both human and murine AD brain sections. Competitive binding experiments suggested a different binding site than Chrysamine G, a well-known stain for amyloid. With a Log?P value between 3 and 5, most bis-pyridylethenylbenzenes were able to enter the brain and label murine amyloid in vivo with the bis(4-pyridylethenyl)benzenes showing the most favorable characteristics. In conclusion, the presented results suggest that bis-pyridylethenylbenzene may serve as a novel backbone for amyloid imaging agents.

Full text of DOI:10.1016/j.bmc.2016.05.022

Accepted Manuscript
Bis-pyridylethenyl benzene as novel backbone for amyloid-β binding com-
pounds
Rob J.A. Nabuurs, Varsha V. Kapoerchan, Athanasios Metaxas, Sarah Hafith,
Maaike de Backer, Mick M. Welling, Wim Jiskoot, Adrianus M.C.H. van den
Nieuwendijk, Albert D. Windhorst, Herman S. Overkleeft, Mark A. van
Buchem, Mark Overhand, Louise van der Weerd
PII:
S0968-0896(16)30346-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.022
BMC 13006
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 August 2015
28 April 2016
14 May 2016
Please cite this article as: Nabuurs, R.J.A., Kapoerchan, V.V., Metaxas, A., Hafith, S., Backer, M.d., Welling, M.M.,
Jiskoot, W., van den Nieuwendijk, A.M.C., Windhorst, A.D., Overkleeft, H.S., van Buchem, M.A., Overhand, M.,
Weerd, L.v.d., Bis-pyridylethenyl benzene as novel backbone for amyloid-β binding compounds, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.022
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Bis-pyridylethenyl benzene as novel backbone for amyloid-β
binding compounds
Rob J.A. Nabuurs1
Varsha V. Kapoerchan2
Athanasios Metaxas3
Sarah Hafith1
Maaike de Backer1
Mick M. Welling1
Wim Jiskoot4
Adrianus M.C.H. van den Nieuwendijk2
Albert D. Windhorst3
Herman S. Overkleeft2
Mark A. van Buchem1
Mark Overhand2
Louise van der Weerd1,5
1Department of Radiology, Leiden University Medical Center, Leiden, Netherlands
2Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands
3Radionucleotide Laboratory, Free University, Amsterdam, Netherlands
4Division of Drug Delivery Technology, Leiden Academic Center for Drug Research, Leiden University, Leiden, Netherlands
5Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands
Address for Correspondence and institution of origin:
Louise van der Weerd, PhD
Leiden University Medical Center
Department of Radiology CS2
Albinusdreef 2, 2333 ZA Leiden
l.van_der_weerd@lumc.nl
Tel. +31-71-526 4376

Abstract
Detection of cerebral β-amyloid (Aβ) by targeted contrast agents is of great interest for in
vivo diagnosis of Alzheimer’s disease (AD). Partly because of their planar structure several
bis-styrylbenzenes have been previously reported as potential Aβ imaging agents. However,
these compounds are relatively hydrophobic, which likely limits their in vivo potential. Based
on their structures, we hypothesized that less hydrophobic bis-pyridylethenylbenzenes may
also label amyloid. We synthesized several bis-pyridylethenylbenzenes and tested whether
these compounds indeed display improved solubility and lower LogP values, and studied
their fluorescent properties and Aβ binding characteristics. Bis-pyridylethenylbenzenes
showed a clear affinity for Aβ plaques on both human and murine AD brain sections.
Competitive binding experiments suggested a different binding site than Chrysamine G, a
well-known stain for amyloid. With a LogP value between 3 and 5, most bis-
pyridylethenylbenzenes were able to enter the brain and label murine amyloid in vivo with
the bis(4-pyridylethenyl)benzenes showing the most favorable characteristics. In conclusion,
the presented results suggest that bis-pyridylethenylbenzene may serve as a novel backbone
for amyloid imaging agents.

Introduction
Cerebral accumulation of senile amyloid plaques plays an important role in the pathogenesis
of Alzheimer’s disease (AD), as amyloid deposits are thought to precede the onset of the first
clinical symptoms by up to two decades.[1-3] These typical deposits of fibrillar amyloid-β
peptides (Aβ) thereby constitute an important target for the development of imaging agents
capable of visualizing and quantifying them. Much progress has been made in the
development of such Aβ-targeting imaging ligands suitable for visualization by positron
emission tomography (PET), single positron emission tomography (SPECT), fluorescence
microscopy or magnetic resonance imaging (MRI). Due to the introduction of the first
radioligands in the clinic, the landscape of the landscape of research into amyloid-targeting
therapies is currently changing dramatically, with clinical trials involving subjects at risk for
Alzheimer Disease who have few or no symptoms, but who are positive for the presence of
amyloid plaques.
Pittsburgh compound B (PiB, 1, Figure 1), a [11C]-benzothiazole derivative, is the best
characterized in vivo PET radiotracer thus far. The short half-life of 11C however limits its use
to medical centers with an on-site cyclotron. In addition longer-lived 18F radiofluorinated
derivatives have been synthesized, like flutemetamol (2)[4], florbetapir (3) and
florbetaben(4)[5], of which the first two recently were approved by the U.S. Food and Drug
Administration for commercial use.[6,7] However improved Aβ-targeted imaging agents or
backbones that could potentially outperform previous agents, bind to specific subtypes of
cerebral amyloid or that are suitable for other clinical imaging modalities are still warranted.

Figure 1. Previously reported amyloid-β binding ligands
The precise mechanisms that cause these small molecules to bind specifically to amyloid
structures are still not completely understood.[8] An hypdrophobic planarized π system is
hypothesized as an important design feature for high binding specificity based on a well-
known histological amyloid dye, Congo Red (5).[9] In vivo application of Congo Red however
is not feasible due its limited brain entry. Its molecular structure served as backbone for the
development of bis-styrylbenzene imaging probes for PET, SPECT and 19F MRI, like 6
(Chrysamine G), 7 (X-34)[10], 8 (ISB)[11], 9 (FSB)[12]and 10 (Methoxy-X04)[13]. (Figure 1)
The acidic functional groups appeared to be unnecessary for high affinity Aβ binding, with
bis-styrylbenzenes lacking these groups showing Kd’s ~300 fold lower compared to 9.
Unfortunately, the use of amyloid targeting bis-styrylbenzenes is limited to preclinical
imaging studies that exploit their fluorescent properties.[15] The styrylbenzene backbone

has even been explored as a potential MRI contrast agent with addition of one or more
fluorine atoms, allowing in vivo detection of Aβ using 19F MRI. Despite the promising initial in
vivo 19F MRI performed with 9 the incorporation of multiple fluorine atoms thus far has not
sufficiently increased their sensitivity for the use as an MRI contrast agent.[3,14] In general,
many of these compounds are very hydrophobic. Their poor aqueous solubility and blood-
brain barrier (BBB) passage are likely to limit the in vivo imaging potential of these
compounds or lead to an unfavorable cerebral washout that causes non-specific signal of
unbound compound.
We envisioned that replacement of the phenyl outer rings of the (bis)styrylbenzene
backbone with pyridine rings may overcome some of these hurdles, as the presence of the
nitrogen atom is expected to improve solubility and lower LogP values. This study therefore
aims to investigate whether bis-pyridylethenyl benzene may serve as a new backbone for
the development of an amyloid-β targeting imaging agent. Synthesized compounds were
evaluated with respect to their in vitro Aβ binding and specificity, and their fluorescent
properties upon binding. Partition coefficients (LogP) were determined to assess
hydrophobicity. In vivo affinity for amyloid plaques and blood-brain-barrier passage was
evaluated following intravenous injection of the compound in a transgenic amyloid-
accumulating mouse model.
Design
We designed a series of six different bis-pyridylethenylbenzenes (11 – 16) with the amyloid-
targeting compound Methoxy-X04 (10)[13] set as starting point for the design and synthesis.
(Figure 2) With an affinity for Aβ plaques in the nanomolar range (Ki = 26.8 nM), this
fluorescent small molecule is frequently used for intravital microscopy studies in vivo
following intravenous or intraperitoneal injection.[13] Compounds 11 – 13 have no
substituents at all and can serve to evaluate whether the presence of the two nitrogen
atoms can compensate for the hydroxy- and methoxy functionalities in Methoxy-X04.
Compounds 14 – 16 contain a methoxy-substituent on the middle ring for a more direct
comparison with Methoxy-X04.

Figure 2. Envisioned bis-pyridylethenylbenzenes
All these compounds are accessable using the general synthesis route recently separately
published by ourselves[3] and the group of Boländer et al.[16], which is depicted in Scheme
1 with the key building blocks being diphosphonate C and an aldehyde D. p-xylene A is
subjected to radical bromination to yield dibromide B. This dibromide is then treated with
triethyl phosphite in an Arbuzov reaction to give diphosphonate C. A Horner-Wadsworth-
Emmons reaction between diphosphonate C and an aldehyde D yields (E,E)-bis-
pyridylethenylbenzene E.
Scheme 1. General synthesis route for the envisioned bis-pyridylethenyl benzenes
Reagents and conditions: a) N-bromosuccinimide, benzoyl peroxide, CCl4, reflux for 16 h. b) Triethyl phosphite,
at 150 °°C for 16 h. c) 1. KOtBu, THF at -10 °°C for 20 min. then compound D at room temperature for 16 h. For
detailed reaction conditions, see Materials & Methods.

The building blocks needed for the synthesis of compounds 11 – 16 are either easily
synthesized or commercially available. (Figure 3) With all building blocks in hand,
compounds 11 – 16 were synthesized. The synthesis proceeded uneventful, and all
compounds were obtained in reasonable yields (14 – 45%). The exact yield per compound
for each synthesis step can be found in the Synthesis procedures section.
Figure 3. The required building blocks
Results and Discussion
Fluorescent properties
All synthesized compounds are expected to have fluorescent properties based on their
conjugated ring structures. We therefore determined excitation and emission wavelength
maxima of 300 nM solutions. The emission intensities were compared to that of Methoxy-
X04. (Table 1) The intrinsic fluorescent intensity was typically higher for the bis(3-
pyridylethenyl)benzenes compared to the bis(2-pyridylethenyl)benzenes.
It has been reported that binding to amyloid may have a significant effect on fluorescence
properties[17], and therefore the fluorescence was also measured in the presence of
synthetic Aβ fibrils. The parent compound 10 showed the highest fluorescence yield with a
10-fold fluorescence increase in the presence of amyloid fibrils. All tested bis-
pyridylethenylbenzenes showed an increase of fluorescence after binding, with compound
12 being the brightest. (Table 1) Several previously reported Aβ-targeting fluorophores have
shown a clear red-shifted emission spectrum following binding[18,19], but such a spectral
shift was not observed for any of the tested bis-pyridylethenylbenzenes (Table 1).

Table 1 Fluorescent and binding characteristics
Fold increase on
binding to fibrillar
Aβ
Fluoresence Fluoresence intensity on
a
Ex vivo Aβ bindingc
Compound
MW
λex
λem
intensityb
binding to fibrillar Aβb
(all ± 1nm) (all ± 1nm)
(%)
100
70
(%)
982
227
505
312
130
359
189
Human
+
Murine
344.403
284.354
284.354
284.354
314.380
314.380
314.380
372
354
352
356
373
360
362
451
420
412
415
430
436
441
9.82
3.23
2.24
5.48
8.18
1.94
4.99
+
+++
++
+++
+
10
11
12
13
14
15
16
++
+
225
57
++
- -
16
185
38
- -
-
- -
+
aEx/Em wavelength maxima were determined of 300 nM solutions.
bFluoresence intensity was calculated relative to that of 10.
cStaining of amyloid plaques in human and APP-PS1 murine at 1 μM was scored whether the compound stained nothing (- -), less (-), similar
(+), slightly more (++) or much more (+++) for amyloid plaques in comparison to 10.
Qualitative assessment of human and murine amyloid plaques binding
Fluorescence microscopy was used for a qualitative ex vivo assessment of the amyloid-
binding properties. A concentration series of each compound (1 – 10 – 100 μM) was applied
to brain sections of AD patients and of aged APPswe-PS1 E9 (APP/PS1) mice, which have a
high amyloid plaque load. At 100 μM all compounds showed characteristic staining of
amyloid plaques on murine APP-PS1 brain sections. Although all compounds bound to
amyloid plaques at high concentration, differences in binding became visible at the lower
concentrations. (Table 1 and Figure 4) No non-specific background labeling or labeling of
neurofibrillary tangles (NFTs) was observed at these concentrations, suggesting their
specificity for amyloid.

Figure 4. Staining of human and APP/PS1 murine amyloid plaques
All bis-pyridylethenylbenzenes stained both human and murine amyloid plaques after applying 100 µM (data
not shown). At 1 µM concentration, differences in binding between the compounds become apparent
compared to each other or the negative controls (C). All images were taken with the same microscope settings.
Scale bar = 200 µm.
As stated in the previous paragraph, the fluorescence intensity with and without amyloid
fibrils differed significantly for the various compounds. Therefore, the fluorescence intensity
in the presence of fibrils was calculated and expressed relative to the intensity of Methoxy-
X04 (10). (Table 1) Assuming that the fluorescence yield in the presence of synthetic A is

representative for that in the presence of amyloid plaques, the staining data was interpreted
as follows. Without substitution on the inner ring, bis-pyridylethenylbenzenes 11 – 13
displayed improved amyloid labeling compared to Methoxy-X04 (10). This was already
shown for a similar bis-styrylbenzene lacking additional groups, suggesting that planarity
indeed plays an important role.[20] Although compounds 12 and 15 showed the highest
fluorescent intensity after synthetic amyloid binding of all tested bis-
pyridylethenylbenzenes, on brain sections these compounds were not performing as well as
e.g. compounds 11 and 13. This would suggest that incorporation of a nitrogen atom on the
3rd position results in less favorable binding characteristics. Incorporation of an additional
methoxy-group (14 and 16) resulted in a slightly reduced binding on the murine sections.
Compared to Methoxy-X04, however, their ability to depict murine amyloid seems better, as
the fluorescence yield of compounds 14 and 16 is lower. This suggests that the nitrogen
alone might be sufficient to replace the hydroxy groups present in Methoxy-X04 (10). Similar
to Methoxy-X04, the affinity of the ligands for human plaques appears to be less than for the
murine plaques. As amyloid plaques in humans and mice differ in composition and
compactness[21], this is likely the reason for the observed differences in staining between
human and murine AD sections. Nevertheless, staining of human tissue could still be clearly
observed for compounds 11 – 13, and was improved compared to Methoxy-X04 for
compound 11 and 13.
Affinity for synthetic Amyloid-β fibrils
A competition assay with tritiated amyloid dye Chrysamine G (6) was used to determine the
binding inhibition coefficient (Ki) for the compounds.[13] Unlike compound 10, none of the
bis-pyridylethenylbenzenes showed any competitive binding to [3H]Chrysamine G using
either synthetic amyloid fibrils (Figure 5) or murine brain homogenate. (Figure 6) This
implies that these compounds use a different binding site than Chrysamine G, and most
likely than Methoxy-X04 (10). Unfortunately, we were therefore unable to determine Ki
values for the synthesized compounds.

Figure 5. Competition binding with synthetic Aβ fibrils
Displacement of [3H]ChrysamineG binding by Methoxy-X04 (10) (closed circles) and compound 11 (open
circles), on fibrilized synthetic Aβ1-40. Data are presented as the mean ± SEM of one experiment, conducted in
triplicate, and replicated three to seven independent times. 10 inhibited the binding of 5 nM [3H]Chrysamine G
with a mean Ki value of 24.1 ± 8.0 nM.
Figure 6. Binding competition with APP/PS1 transgenic brain homogenates
Displacement of [3H]Chrysamine G binding by Methoxy-X04 (10) (A) and compound 11 (B), on brain cortex
homogenates of 11 months-old APP/PS1 transgenic and control mice. Data are presented as the mean ± SEM of
one experiment, conducted in duplicate and replicated three independent times. 10 inhibited the binding of 5
nM [3H]Chrysamine G in APP/PS1 mice (closed circles) with a Ki value of 27.5 ± 3.5 nM, which is in good
agreement with literature.[28]
LogP values
The blood-brain barrier (BBB) is formed by a tight layer of endothelial cells in the wall of
cerebral blood vessels that regulates the exit and entry of blood compounds into the brain. It
is traditionally stated that for optimal passive BBB passage, compounds should preferably
have moderately hydrophobicity (LogD or LogP 2.0 - 3.5), although several successful

radiopharmaceuticals do not meet this requirement.[22] Therefore LogD or LogP should not
be used as absolute selection criteria. Nevertheless, within one series of compounds it is
nonetheless a useful parameter to assess hydrophobicity. For each of the compounds 10 –
16, LogP values were determined with an HPLC-based method according to Benhaim et
al.[23-25] All compounds have a LogP below that of Methoxy-X04, although none of them
meets the before-mentioned criterion. (Table 2)
Table 2. LogP values and in vivo amyloid labeling
compound
LogP
LogP
In vivo amyloid labeling
(calc)a (determined)
5.55
3.94
3.94
4.12
3.86
4.03
4.16
4.84
3.93
3.73
3.75
3.73
4.32
4.25
+
+
+
+
+
+
+
10
11
12
13
14
15
16
aLogP values were calculated used web-based methods: www.molinspiration.com and http://intro.bio.umb.edu/111-
112/OLLM/111F98/jLogP/test.html.
The ability to label amyloid plaques in the brains of APP-PS1 mice following intravenous injection were scored absent (-) or present (+).
In vivo amyloid plaque labeling in transgenic AD mice
To assess the ability of the compounds to pass the BBB in vivo, solutions of each compound
were injected at 30 µmol/kg in aged transgenic APP/PS1 mice, which have extensive cerebral
amyloid plaques. One day after injection the mice were sacrificed, their brains were
removed and sections were studied using the same fluorescence microscopy set-up used for
the stained brain sections. All compounds were readily dissolved using a mixture of 0.05 M
compound dissolved in 1:1 DMSO:Cremophor (volume ratio) diluted up to 200 µl with PBS at
pH 7.2. Disolving bis(2-pyridylethenyl)benzenes 13 and 16 however resulted in a viscous
substance that was not well tolerated by the animals. In vivo targeting of amyloid plaque
however was observed for all compounds, showing their ability to pass the BBB. (Figure 7
and Table 2) No non-specific labeling was detected in the wild-type brains. Direct
comparison of the in vivo amyloid labeling between the different compounds remained
difficult, as the amyloid load may differ between the transgenic animals and the
fluorescence yield of the compounds varies considerably. Bis-pyridylethenylbenzene 11 and

13 showed a signal intensity in individual labeled plaques comparable to Methoxy-X04,
while having a 3 to 4-fold lower fluorescence yield. Also their methoxy variants, compound
14 and 16, clearly crossed the BBB in vivo to label amyloid plaques. The lower fluorescence
intensity is due to the almost 10-fold lower fluorescence yield of 14 and 16 after binding
compared to Methoxy-X04. Although compounds 12 and 15 do cross the blood-brain barrier
and label plaques, the fluorescence intensity is much less, despite their relatively high
fluorescence yield in vitro (Table 1).
Figure 7. In vivo amyloid labeling
Shown are thirty micrometer brain sections of APP/PS1 transgenic mice resected injected with compound 10 –
16. None of the wild type mice showed amyloid plaques or aspecific labeling. Scale bar = 200 µm
Summary and Conclusion

Several bis-pyridylethenylbenzenes have been synthesized and evaluated for their Aβ
binding properties and ability to pass the BBB. In general, all compounds showed affinity for
both human and murine fibrillar amyloid. Independent of the position of the nitrogen atom,
the bis-pyridylethenylbenzenes without any additional groups seemed to show a higher
affinity to amyloid compared to Methoxy-X04 (10). Besides the possible influence of the
nitrogen atom, this might be due to their planar structure, which was previously suggested
to be advantageous for a similar (bis)styrylbenzene with no further substituents.[20] The
methoxy group on the inner ring structure seemed to result in a decrease in amyloid binding,
particularly for human amyloid. However, in contrast to several known bis-styrylbenzenes,
like 10, the bis-pyridylethenylbenzenes did not share a common binding site with
Chrysamine G. In addition, all bis-pyridylethenylbenzenes seemed to cross the BBB and label
amyloid in vivo. Based on the above results, compound 11, (E,E)-1,4-bis(4-
pyridylethenyl)benzene, showed the most favorable ex and in vivo characteristics and as
such might serve as a starting point to further explore the potency of bis-
pyridylethenylbenzenes.
A direct application of bis-pyridylethenylbenzenes for amyloid targeting would be their use
in preclinical optical imaging. In addition we believe that the bis-pyridylethenylbenzene may
serve as a novel backbone for the development of amyloid targeting PET or SPECT probes. As
their binding sites may differ from existing amyloid targeting imaging probes, these
compounds may provide additional information regarding accumulation of cerebral amyloid.
Materials and Methods
Preparation of Aβ1-40 fibrils
Aβ fibrils were prepared by stirring a 0.5 mg/ml solution of Aβ1-40 peptide (RPeptide, Bogart,
GA) at 37 °C for 3 days, which resulted in a cloudy solution. The presence of fibrils was
confirmed by the presence of an emission peak at 482 nm (excitation 440 nm) upon addition
of a 5 μM solution in PBS of Thioflavin T (Sigma, Germany) to a small amount of fibrils.
Aliquots of 10 μl were transferred to Eppendorf vials and stored at -80 °C until the assay was
performed.

Fluorescence spectra
All compounds were dissolved in DMSO at 0.3 mM and diluted to 300 nM with 10 % ethanol
in PBS (pH 7.2). Fluorescence spectra were measured on a Varian Cary Eclipse fluorescence
spectrophotometer to obtain excitation and emission wavelength maxima, which were used
to select the correct fluorescence filter settings for further microscopic evaluation. All
measurements were carried out at 20 °C and in triplicate. 3D emission-excitation spectra
were obtained by adding 500 μl of the dissolved compounds to previously prepared Aβ
aliquots. After manual shaking for 30 sec, 300 μl samples were measured (Infinity M1000,
Tecan, Switzerland).
Staining of human and transgenic AD brain sections
Stock solutions of 3 mM compound in DMSO were diluted to 1 – 10 – 100 µM in 2:3 volume
ratio PBS:ethanol and sonicated for 15 minutes. Paraffin sections (8 µm) of human AD
cortex, 14-month-old transgenic murine APP/PS1 brain and age-matched controls were
deparaffinized prior to staining for 10 minutes in absolute darkness. After gently rinsing with
tap water, sections were placed in 0.1 % NaOH in 80% ethanol for 2 minutes, air dried and
coverslipped using Aqua / Polymount. Fluorescence of the stained sections was analyzed
using a microscopic slide scanner (Pannoramic MIDI, 3DHistech, Hungary) with a DAPI filter
cube (Ex 365 nm; Em 445/50 nm) using the same intensity settings throughout all
experiments.
In vivo Aβ plaque labeling in transgenic AD mice
Twelve-to-fourteen-month-old APP/PS1 mice or age-matched wildtype animals (n=2 per
compound) were injected intravenously with 0.05 M dissolved in 1:1 volume ratio
DMSO:Cremophor diluted with PBS (pH 7.2) to a total volume of 200 µl, resulting in a total
dose of 30 µmol/kg. One day after injection, animals were sacrificed using 200 µl Euthanasol
(AST Pharma) prior to transcardial perfusion with 4% paraformaldehyde in PBS (pH 7.2).
Brains were removed and cryoprotected in 4% PFA with 10% sucrose for 4 hours, followed
by immersion in 4% PFA with 30% sucrose overnight. Snap-frozen brains were cryosectioned
(30 µm) and fluorescence images were analyzed as described above.
Brain homogenates

Male, control and transgenic APP/PS1 mice (11-month-old) were killed by decapitation. The
cortices were removed and homogenized using a DUALL tissue homogenizer (20 strokes,
2000 rpm), in a 20-fold excess (v/w) of ice-cold 0.25 M sucrose. Homogenates were isolated
by centrifugation (40 min x 250,000 x g) in a refrigerated Beckman ultracentrifuge (rotor 60-
Ti). The resulting pellet was suspended in 20 fractions of 150 mM Tris + 20 % ethanol buffer
(pH 7.0) for competition binding experiments.
Competition binding assay
Competition binding experiments were conducted at room temperature, in a final volume of
1 ml assay buffer (150 mM Tris-HCl, 20% ethanol, pH 7.0). Compounds were dissolved as 3
mM stock solutions in DMSO, sonicated for 15 min, and used in a final concentration range
of 30 μM to 30 pM. 10 μl of unlabeled test compound was combined with 890 μl of assay
buffer and 50 μl of 100 nM [3H]Chrysamine G stock (specific activity 33.8 Ci/mmol). The
mixture was sonicated for 10 min, and the assay was subsequently started by the addition of
50 μl synthetic Aβ1-40 fibrils or by addition of 400 µl of the brain homogenate suspension.
Non-specific binding was determined in the presence of 1 μM of 10. Incubations were
terminated after 1 h via filtration through Whatman GF/B filters (pre-soaked in binding
buffer), using a 48-well Brandel harvester. The filters were washed two times with 3 ml of
ice-cold binding buffer (pH 7.0), and the radioactivity count was determined by liquid
scintillation spectrometry in 5 ml of Optiphase-HiSafe 3, at an efficiency of 40 %.
Ki values were determined by nonlinear regression analysis using the equation: logEC50 =
log[10^logKi*(1+RadioligandNM/HotKdNM)] where Kd = 200 nM, and radioligand = 5nM.
(GraphPad Software Inc., San Diego, CA)
Procedure for LogP determinations
LogP determinations were performed according to literature procedures.[23] The
measurements were performed on a Jasco HPLC-system (detection simultaneously at 214
and 254 nm) coupled to a Perkin Elmer Sciex API 165 mass instrument with a custom-made
Electrospray Interface (ESI). An analytical Gemini C18 column (Phenomenex, 50 x 4.60 mm, 3
micron) was used in combination with buffers A) phosphate buffer of pH = 7.0 (0.02 M
Na2HPO4 adjusted to pH = 7.0 with phosphoric acid) and B) 0.25% octanol in methanol.

Of all compounds to be evaluated, stock solutions of 0.5 mg ml-1 were prepared in methanol.
These stock solutions were then diluted with water, making sure that the volume percentage
of water was such that the compounds did not precipitate (max 40% water). A 0.25 mg ml-1
solution of NaNO3 in water was used as a non-retaining compound to determine the dead
time of the system. For calibration purposes known compounds were taken from the
literature. (Table 3) Of these compounds, 0.25 mg ml-1 solutions in either 75% H2O/MeOH or
50% H2O/MeOH were made.
For each sample, a series of four isocratic runs was performed, (for instance 55, 60, 65, 70%
B), and the retention times (from UV-detection at 214 nm) thus obtained were converted to
the retention factor k’ according to the formula k’ = (tR-t0)/t0, with tR being the retention
time of the compound and t0 being the retention time of NaNO3. The retention factors were
extrapolated to 0% B, yielding k’w. As there is a linear relationship between LogP and log k’w
plotting LogP values of known compounds against obtained log k’w values in this system
yields a calibration curve. From this curve, LogP values of unknown compounds were
calculated from their k’w values.
Table 3. Reference LogP values
Compound
LogP
Resorcinol
0.8
p-nitroaniline 1.39
Phenol
1.46
2
m-nitrophenol
2-naphtol
2.7
3.37
Naphthalene
Based on known literature values, the LogP of several reference compounds yield a required calibration curve to determine the LogP values
of our compounds.23-25
Synthesis Procedures
General reaction conditions and analysis procedures
Reagents and solvents were used as provided, unless stated otherwise. 2-
pyridinecarboxaldehyde (21) and 4-pyridinecarboxaldehyde (19) were purchased from
Acros, 3-pyridinecarboxaldehyde (20) was purchased from Sigma-Aldrich. THF was distilled
over LiAlH4 prior to use. Reactions were carried out under inert conditions and ambient

temperature, unless stated otherwise. Prior to performing a reaction, traces of water were
removed from the starting materials by repeated co-evaporation with anhydrous 1,4-
dioxane or anhydrous toluene. These solvents were stored over 4 Å molsieves. Reactions
were monitored by thin layer chromatography on aluminum coated silica sheets (Merck,
silica 60 F254), using visualization either with iodine, or spraying with a solution of 25 g
(NH4)2MoO4, 10 g (NH4)4Ce(SO4)4 in 100 ml H2SO4 and 900 ml H2O, or a solution of 20%
H2SO4 in ethanol, followed by charring at ~150 °C. Column chromatography was carried out
with silica gel (Screening Devices bv, 40-63 μm particle size, 60 Å), using technical grade
solvents. NMR spectra were recorded at 298K on a Bruker AV400 using deuterated solvents.
All carbon spectra are proton-decoupled. Chemical shifts (δ) are given in ppm, in 13C spectra
relative to the solvent peaks of CDCl3 (77.0 ppm) or CD3OD (49.0 ppm), in 1H spectra relative
to the solvent peak of tetramethylsilane (0.0 ppm) or CD3OD (3.31 ppm). Coupling constants
are given in Hz. LC-MS analysis was performed on a Finnigan Surveyor HPLC system with a
Gemini C18 50 × 4.6 mm column (3 micron, Phenomenex, Torrance, CA, USA) (detection at
200-600 nm), coupled to a Thermo Finnigan LCQ Advantage Max mass spectrometer (Breda,
The Netherlands) with electrospray ionization (ESI; system 1), with as eluents A: H2O; B:
MeCN and C: 1% aq. TFA. For RP-HPLC purification of the peptides, a Gilson GX-281
automated HPLC system (Gilson), supplied with a preparative Gemini C18 column
(Phenomenex, 150 x 21.2 mm, 5 micron) was used. The applied buffers were A: 0.2% aq. TFA
or 20 mM NH4OAc and B: MeOH.
IR spectra were recorded on a Perkin Elmer Paragon 1000 FT-IR Spectrometer. High
resolution mass spectra were recorded by direct injection (2 µL of a 2 µM solution in
H2O/MeCN; 50/50; v/v and 0.1% formic acid) on a mass spectrometer (Thermo Finnigan LTQ
Orbitrap) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV,
sheath gas flow 10, capillary temperature 250 C) with resolution R = 60000 at m/z 400 (mass
range m/z = 150-2000) and dioctylpthalate (m/z = 391.28428) as a “lock mass”. The high
resolution mass spectrometer was calibrated prior to measurements with a calibration
mixture (Thermo Finnigan). It should be noted that we did not obtain the suited HRMS data
for every compound. This finding is in agreement with related compounds as reported
earlier by us. Based on the other characterization data obtained for each compounds we
know that our compounds are sound. For clarity, we included the HMRS data that we did
find: compound 14: Exact mass: Calculated for [C21H19N2O]+: 315.14919. Found: 315.14901

[M + H]+. compound 15 Exact mass: Calculated for [C21H19N2O]+: 315.14919. Found:
315.14915 [M + H]+.
General procedure for the Horner-Wadsworth-Emmons reaction
The diphosphonate (0.95 mmol) was dissolved in THF (20 mL) and cooled to -10° C. KOtBu
(0.30 g, 2.45 mmol) was added and the black mixture stirred for 20 min. A solution of the
aldehyde (2.37 mmol) in THF (6 mL) was added and the reaction stirred at rt for 16 h. The
reaction was cooled to 0° C, quenched with water and extracted five times with EtOAc. The
combined organic layers were dried (Na2SO4), filtered and concentrated. The crude product
was subjected to column chromatography to yield the pure product.
(E,E)-1,4-bis(4-pyridylethenyl)benzene (11)
According to the general procedure, diphosphonate 17 (0.19 g, 0.5 mmol) was reacted with
4-pyridinecarboxaldehyde 19 (0.12 mL, 1.25 mmol). After work-up, the resulting yellow
solids were purified by column chromatography (50 → 70% EtOAc/light petroleum) and the
pure compound was obtained as a bright yellow solid (43 mg, 0.15 mmol, 30%).
1H NMR (CDCl3, 400 MHz): δ 8.59 (d, J = 5.6 Hz, 4H); 7.56 (s, 4H); 7.38 (d, J = 5.7 Hz, 4H); 7.30
(d, J = 16.3 Hz, 2H); 7.05 (d, J = 16.3 Hz, 2H). 13C NMR (CDCl3, 100 MHz): δ 150.1; 144.4;
136.5; 132.4; 127.5; 126.4; 120.8. IR (neat): 3442.0; 1591.6; 1414.1; 1217.5; 1118.9; 973.4;
868.4; 830.6; 595.9; 556.2. LC-MS retention time: 3.86 min (10 → 90% MeCN, 15 min run).
Mass (ESI): m/z 285.13 [M + H]+.
(E,E)-1,4-bis(3-pyridylethenyl)benzene (12)
According to the general procedure, diphosphonate 17 (0.19 g, 0.5 mmol) was reacted with
3-pyridinecarboxaldehyde 19 (0.12 mL, 1.25 mmol). After work-up, the resulting yellow
solids were purified by column chromatography (50 → 100% EtOAc/light petroleum) and the
pure compound was obtained as a bright yellow solid (64 mg, 0.23 mmol, 45%).
1H NMR (CDCl3, 400 MHz): δ 8.73 (s, 2H); 8.49 (d, J = 4.3 Hz, 2H); 7.83 (d, J = 8.0 Hz, 2H); 7.53
(s, 4H); 7.29 (dd, J = 8.2 Hz, J = 5.1 Hz, 2H); 7.16 (d, J = 16.4 Hz, 2H); 7.09 (d, J = 16.4 Hz, 2H).
13C NMR (CDCl3, 100 MHz): δ 148.5; 148.5; 136.5; 132.9; 132.6; 130.1; 127.0; 125.0; 123.5. IR
(neat): 3029.1; 1564.9; 1481.0; 1427.6; 1022.1; 965.9; 823.5; 704.8; 551.7. LC-MS retention
time: 3.87 min (10 → 90% MeCN, 15 min run). Mass (ESI): m/z 285.13 [M + H]+.

(E,E)-1,4-bis(2-pyridylethenyl)benzene (13)
According to the general procedure, diphosphonate 17 (0.19 g, 0.5 mmol) was reacted with
2-pyridinecarboxaldehyde 21 (0.12 mL, 1.25 mmol). After work-up, the resulting yellow
solids were purified by column chromatography (0 → 20% EtOAc/light petroleum) and the
pure compound was obtained as a bright yellow solid (20 mg, 0.07 mmol, 14%).
1H NMR (CDCl3, 400 MHz): δ 8.62 (d, J = 4.4 Hz, 2H); 7.70-7.64 (m, 2H); 7.65 (d, J = 16.2 Hz,
2H); 7.60 (s, 4H); 7.40 (d, J = 7.8 Hz, 2H); 7.20 (d, J = 16.0 Hz, 2H); 7.16 (dd, J = 7.0 Hz, J = 5.4
Hz, 2H). 13C NMR (CDCl3, 100 MHz): δ 155.5; 149.6; 136.7; 136.6; 132.2; 128.0; 127.5; 122.2;
122.1. IR (neat): 1634.1; 1581.5; 1559.8; 1467.9; 1431.7; 1333.4; 1205.7; 1145.3; 981.8;
873.8; 829.9; 763.9; 736.2; 609.6; 547.6. LC-MS retention time: 3.84 min (10 → 90% MeCN,
15 min run). Mass (ESI): m/z 285.13 [M + H]+.
(E,E)-1-methoxy-2,5-bis(4-pyridylethenyl)benzene (14)
According to the general procedure, diphosphonate 18 (0.20 g, 0.5 mmol) was reacted with
4-pyridinecarboxaldehyde 19 (0.12 mL, 1.25 mmol). After work-up, the resulting yellow
solids were purified by column chromatography (50% EtOAc/light petroleum → 2%
MeOH/EtOAc) and the pure compound was obtained as a bright yellow solid (20 mg, 0.07
mmol, 14%).
1H NMR (CDCl3, 400 MHz): δ 8.60-8.55 (m, 4H); 7.80 (s, 1H); 7.64 (d, J = 16.3 Hz, 1H); 7.61 (d,
J = 8.1 Hz, 1H); 7.42 (d, J = 6.1 Hz, 2H); 7.40-7.36 (m, 4H); 7.17 (s, 1H); 6.97 (d, J = 16.2 Hz,
1H); 3.98 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 156.5; 137.7; 131.7; 130.9; 128.7; 127.7;
127.3; 125.8; 120.9; 120.8; 119.9; 108.8; 55.9. IR (neat): 1633.9; 1590.5; 1463.7; 1417.6;
1389.6; 1323.3; 1297.7; 1116.6; 1029.2; 971.4; 819.7; 805.4; 616.0; 582.4; 548.1; 518.4. LC-
MS retention time: 4.14 min (10 → 90% MeCN, 15 min run). Mass (ESI): m/z 315.2 [M + H]+.
(E,E)-1-methoxy-2,5-bis(3-pyridylethenyl)benzene (15)
According to the general procedure, diphosphonate 18 (0.20 g, 0.5 mmol) was reacted with
3-pyridinecarboxaldehyde 20 (0.12 mL, 1.25 mmol). After work-up, the resulting yellow
solids were purified by column chromatography (50% → 100% EtOAc/light petroleum) and
the pure compound was obtained as a bright yellow solid (54 mg, 0.17 mmol, 34%).

1H NMR (CDCl3, 400 MHz): δ 8.79-8.65 (m, 2H); 8.48 (dd, J = 9.3 Hz, J = 4.7 Hz, 2H); 7.88-7.81
(m, 2H); 7.59 (d, J = 8.0 Hz, 1H); 7.52 (d, J = 16.6 Hz, 1H); 7.31-7.25 (m, 2H); 7.19-7.03 (m,
4H); 3.97 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 157.3; 148.6; 148.5; 148.3; 137.7; 133.6;
132.8; 132.6; 132.6; 130.5; 126.9; 125.9; 125.4; 125.2; 125.1; 123.5; 123.5; 119.5; 115.6;
108.9; 55.6. IR (neat): 1633.7; 1567.2; 1557.6; 1479.8; 1463.5; 1423.3; 1328.1; 1240.6;
1164.2; 1117.7; 1035.0; 1024.0; 964.2; 819.8; 799.3; 705.5; 643.8; 627.1; 549.8.
LC-MS retention time: 4.14 min (10 → 90% MeCN, 15 min run). Mass (ESI): m/z 315.2 [M +
H]+.
(E,E)-1-methoxy-2,5-bis(3-pyridylethenyl)benzene (16)
According to the general procedure, diphosphonate 18 (0.20 g, 0.5 mmol) was reacted with
2-pyridinecarboxaldehyde 21 (0.12 mL, 1.25 mmol). After work-up, the resulting yellow
solids were purified by column chromatography (0% → 30% EtOAc/light petroleum) and the
pure compound was obtained as a bright yellow solid (42 mg, 0.13 mmol, 27%).
1H NMR (CDCl3, 400 MHz): δ 8.61 (s, 2H); 7.93 (d, J = 16.3 Hz, 1H); 7.70-7.58 (m, 4H); 7.45 (d,
J = 8.2 Hz, 1H); 7.41 (d, J = 7.9 Hz, 1H); 7.31-7.08 (m, 6H); 3.95 (s, 3H). 13C NMR (CDCl3, 100
MHz): δ 157.6; 156.2; 155.5; 149.7; 149.5; 137.9; 136.6; 136.4; 132.4; 128.8; 128.2; 127.5;
127.4; 125.9; 122.1; 122.1; 121.8; 121.8; 119.9; 109.2; 55.5. IR (neat): 1584.0; 1562.5;
1467.9; 1430.5; 1248.3; 1036.0; 970.7; 769.3; 668.0. LC-MS retention time: 4.10 min (10 →
90% MeCN, 15 min run). Mass (ESI): m/z 315.07 [M + H]+.
Tetraethyl 1,4-xylylene diphosphonate (17)
As described previously by Kikuchi et al.[26], a solution of NaBrO3 (12.1 g, 80 mmol) in H2O
(40 mL) was added to a solution of p-xylene (2.47 mL, 20 mmol) in EtOAc (40 mL) and the
mixture was vigorously stirred. A solution of NaHSO3 (8.3 g, 80 mmol) in H2O (80 mL) was
added dropwise within 15 min and the reaction was stirred for 5 h. After the reaction
mixture was poured out in Et2O (300 mL), the layers were separated and the aqueous layer
extracted twice with Et2O. The combined organic layers were washed with 2 M Na2S2O3,
dried (Na2SO4), filtered and concentrated. The resulting white solids were subjected to
column chromatography (0 → 5% EtOAc/light petroleum) and the product was obtained as a
white solid (5.4 g), but not completely pure.

1H NMR (CDCl3, 400 MHz) δ 7.37 (s, 4H); 4.47 (s, 4H). 13C NMR (CDCl3, 100 MHz) δ 129.5;
32.8.
The impure 1,4-di(bromomethyl)benzene (5.4 g) was dissolved in triethyl phosphite (8.6 mL,
50 mmol) and the mixture was heated to 150 °C to reflux for 16 h. After cooling, the crude
product was applied to a silica column and purified (50% EtOAc/light petroleum → 2%
MeOH/EtOAc). The pure product was obtained as white fluffy powder (5.6 g, 14.8 mmol,
74% over two steps).
1H NMR (CDCl3, 400 MHz) δ 7.25 (s, 4H); 4.07-3.95 (m, 8H); 3.13 (d, J = 20.2 Hz, 4H); 1.24 (t, J
= 7.1 Hz, 12H). 13C NMR (CDCl3, 100 MHz) δ 130.2; 130.1; 130.1; 129.9; 129.8; 129.7; 62.0;
62.0; 61.9; 34.0; 33.0; 16.3; 16.3; 16.2. 31P NMR (CDCl3, 162 MHz): δ 26.7 (s).
Tetraethyl 2-methoxy-1,4-xylylene diphosphonate (18)
As described previously by Kumar et al.[27], 2,5-dimethylanisole (1.36 g, 10 mmol) was
dissolved in CCl4 (22 mL). N-bromosuccinimide (3.92 g, 22 mmol) and benzoyl peroxide (20
mg) were added and the mixture heated to reflux for 2 h. After cooling to rt, the reaction
mixture was filtered over Celite and concentrated. The resulting solids were purified by
column chromatography (0 → 5% EtOAc/light petroleum). The dibromide was obtained as
white solid (1.71 g, 5.8 mmol, 58%).
1H NMR (CDCl3, 400 MHz): δ 7.27 (d, J = 7.7 Hz, 1H); 6.92 (dd, J = 7.6 Hz, J = 1.2 Hz, 1H); 6.89
(s, 1H); 4.52 (s, 2H); 4.44 (s, 2H); 3.88 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 157.4; 139.7;
131.0; 126.3; 121.1; 111.5; 55.6; 33.2; 28.3.
Next, the 2,5-di(bromomethyl)-methoxybenzene (1.71 g, 5.8 mmol) was dissolved in triethyl
phosphite (2.5 mL, 14.5 mmol) and the mixture was heated to reflux for 16 h. The mixture
was cooled, and directly applied to a silica column (50% EtOAc/PE → 4% MeOH/EtOAc). The
pure product was obtained as a yellowish solid (1.55 g, 3.8 mmol, 66%).
1H NMR (CDCl3, 400 MHz): δ 7.25 (dd, J = 7.6 Hz, J = 2.9 Hz, 1H); 6.86 (s, 1H); 6.83 (d, J = 7.6
Hz, 1H); 4.07-3.95 (m, 8H); 3.84 (s, 1H); 3.21 (d, J = 21.4 Hz, 2H); 3.13 (d, J = 22.2 Hz, 2H);
1.27-1.20 (m, 12H). 13C NMR (CDCl3, 100 MHz): δ 157.2; 131.5; 131.1; 121.9; 121.900;
118.85; 112.1; 55.5; 34.4; 33.0; 27.0; 25.6; 16.3. 31P NMR (CDCl3, 162 MHz): δ 27.39 (d, J =
9.0 Hz), 26.77 (d, J = 9.1 Hz).

Reference List
[1] Frisoni GB, Fox NC, Jack CR, Jr., Scheltens P, Thompson PM (2010) The clinical use
of structural MRI in Alzheimer disease. Nat Rev Neurol 6, 67-77.
[2] Jack CR, Jr., Knopman DS, Jagust WJ, Shaw LM, Aisen PS, Weiner MW, Petersen
RC, Trojanowski JQ (2010) Hypothetical model of dynamic biomarkers of the
Alzheimer's pathological cascade. Lancet Neurol 9, 119-128.
[3] Nabuurs RJA, Kapourchan VV, Metaxas A, de Jongh S, de Backer M, Welling MM,
Jiskoot W, Windhorst AD, Overkleeft HS, van Buchem MA, Overhand M, van
der Weerd L (2014) Polyfluorinated bis-styrylbenzenes as amyloid-beta plaque
binding ligands. Bioorg Med Chem 22, 2469-2481.
[4] Vandenberghe R, Van LK, Ivanoiu A, Salmon E, Bastin C, Triau E, Hasselbalch S,
Law I, Andersen A, Korner A, Minthon L, Garraux G, Nelissen N, Bormans G,
Buckley C, Owenius R, Thurfjell L, Farrar G, Brooks DJ (2010) 18F-
flutemetamol amyloid imaging in Alzheimer disease and mild cognitive
impairment: a phase 2 trial. Ann Neurol 68, 319-329.
[5] Barthel H, Gertz HJ, Dresel S, Peters O, Bartenstein P, Buerger K, Hiemeyer F,
Wittemer-Rump SM, Seibyl J, Reininger C, Sabri O (2011) Cerebral amyloid-
beta PET with florbetaben (18F) in patients with Alzheimer's disease and
healthy controls: a multicentre phase 2 diagnostic study. Lancet Neurol 10,
424-435.
[6] No authors listed (2013) GE beta-amyloid agent approved. J Nucl Med 54, 10N.
[7] Yang L, Rieves D, Ganley C (2012) Brain amyloid imaging--FDA approval of
florbetapir F18 injection. N Engl J Med 367, 885-887.
[8] Buell AK, Esbjorner EK, Riss PJ, White DA, Aigbirhio FI, Toth G, Welland ME,
Dobson CM, Knowles TP (2011) Probing small molecule binding to amyloid
fibrils. Phys Chem Chem Phys 13, 20044-20052.
[9] Nesterov EE, Skoch J, Hyman BT, Klunk WE, Bacskai BJ, Swager TM (2005) In vivo
optical imaging of amyloid aggregates in brain: design of fluorescent markers.
Angew Chem Int Ed Engl 44, 5452-5456.
[10] Styren SD, Hamilton RL, Styren GC, Klunk WE (2000) X-34, a fluorescent derivative
of Congo red: a novel histochemical stain for Alzheimer's disease pathology. J
Histochem Cytochem 48, 1223-1232.
[11] Kung MP, Hou C, Zhuang ZP, Zhang B, Skovronsky D, Trojanowski JQ, Lee VM,
Kung HF (2002) IMPY: an improved thioflavin-T derivative for in vivo
labeling of beta-amyloid plaques. Brain Res 956, 202-210.
[12] Higuchi M, Iwata N, Matsuba Y, Sato K, Sasamoto K, Saido TC (2005) 19F and 1H
MRI detection of amyloid beta plaques in vivo. Nat Neurosci 8, 527-533.

[13] Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP, Debnath
ML, Holt DP, Wang Y, Hyman BT (2002) Imaging Abeta plaques in living
transgenic mice with multiphoton microscopy and methoxy-X04, a
systemically administered Congo red derivative. J Neuropathol Exp Neurol 61,
797-805.
[14] Flaherty DP, Walsh SM, Kiyota T, Dong Y, Ikezu T, Vennerstrom JL (2007)
Polyfluorinated bis-styrylbenzene beta-amyloid plaque binding ligands. J Med
Chem 50, 4986-4992.
[15] Bacskai BJ, Hickey GA, Skoch J, Kajdasz ST, Wang Y, Huang GF, Mathis CA,
Klunk WE, Hyman BT (2003) Four-dimensional multiphoton imaging of brain
entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic
mice. Proc Natl Acad Sci U S A 100, 12462-12467.
[16] Bolander A, Kieser D, Scholz C, Heyny-von HR, Mall G, Goetschy V, Czech C,
Schmidt B (2013) Synthesis of Methoxy-X04 Derivatives and Their
Evaluation in Alzheimer's Disease Pathology. Neurodegener Dis
[17] Saroja G, Soujanya T, Ramachandram B, Samanta A (1998) 4-Aminophthalimide
derivatives as environment-sensitive probes. Journal of Fluorescence 8, 405-
410.
[18] Raymond SB, Skoch J, Hills ID, Nesterov EE, Swager TM, Bacskai BJ (2008) Smart
optical probes for near-infrared fluorescence imaging of Alzheimer's disease
pathology. Eur J Nucl Med Mol Imaging 35 Suppl 1, S93-S98.
[19] Ran C, Xu X, Raymond SB, Ferrara BJ, Neal K, Bacskai BJ, Medarova Z, Moore A
(2009) Design, synthesis, and testing of difluoroboron-derivatized curcumins
as near-infrared probes for in vivo detection of amyloid-beta deposits. J Am
Chem Soc 131, 15257-15261.
[20] Flaherty DP, Kiyota T, Dong Y, Ikezu T, Vennerstrom JL (2010) Phenolic bis-
styrylbenzenes as beta-amyloid binding ligands and free radical scavengers. J
Med Chem 53, 7992-7999.
[21] Meadowcroft MD, Connor JR, Smith MB, Yang QX (2009) MRI and histological
analysis of beta-amyloid plaques in both human Alzheimer's disease and
APP/PS1 transgenic mice. J Magn Reson Imaging 29, 997-1007.
[22] Pike VW (2009) PET radiotracers: crossing the blood-brain barrier and surviving
metabolism. Trends Pharmacol Sci 30, 431-440.
[23] Benhaim D , Grushka E (2008) Effect of n-octanol in the mobile phase on lipophilicity
determination by reversed-phase high-performance liquid chromatography on
a modified silica column. J Chromatogr A 1209, 111-119.
[24] Lombardo F, Shalaeva MY, Tupper KA, Gao F, Abraham MH (2000) ElogPoct: a tool
for lipophilicity determination in drug discovery. J Med Chem 43, 2922-2928.

[25] Lombardo F, Shalaeva MY, Tupper KA, Gao F (2001) ElogD(oct): a tool for
lipophilicity determination in drug discovery. 2. Basic and neutral compounds.
J Med Chem 44, 2490-2497.
[26] Kikuchi D, Sakaguchi S, Ishii Y (1998) An Alternative Method for the Selective
Bromination of Alkylbenzenes Using NaBrO(3)/NaHSO(3) Reagent. J Org
Chem 63, 6023-6026.
[27] Kumar P, Zheng W, McQuarrie SA, Jhamadas JH, Wiebe LI (2005) 18F-FESB:
Synthesis and automated radiofluorination of a novel 18F-labelled PET tracer
for beta-amyloid plaques. J Labelled Comp Radiopharm, 983-996.
[28] Cohen AD, Ikonomovic MD, Abrahamson EE, Paljug WR, Dekosky ST, Lefterov IM,
Koldamova RP, Shao L, Debnath ML, Mason NS, Mathis CA, Klunk WE
(2009) Anti-Amyloid Effects of Small Molecule Abeta-Binding Agents in
PS1/APP Mice. Lett Drug Des Discov 6, 437-444.

Graphical abstract