Polyfluorinated bis-styrylbenzenes as amyloid-β plaque binding ligands

Article abstract of DOI:10.1016/j.bmc.2014.02.054

Detection of cerebral β-amyloid (Aβ) by targeted contrast agents remains of great interest to aid the in vivo diagnosis of Alzheimer's disease (AD). Bis-styrylbenzenes have been previously reported as potential Aβ imaging agents. To further explore their potency as ¹⁹F MRI contrast agents we synthetized several novel fluorinated bis-styrylbenzenes and studied their fluorescent properties and amyloid-β binding characteristics. The compounds showed a high affinity for Aβ plaques on murine and human brain sections. Interestingly, competitive binding experiments demonstrated that they bound to a different binding site than chrysamine G. Despite their high logP values, many bis-styrylbenzenes were able to enter the brain and label murine amyloid in vivo. Unfortunately initial post-mortem ¹⁹F NMR studies showed that these compounds as yet do not warrant further MRI studies due to the reduction of the ¹⁹F signal in the environment of the brain.

Full text of DOI:10.1016/j.bmc.2014.02.054

Bioorganic & Medicinal Chemistry 22 (2014) 2469–2481
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Polyfluorinated bis-styrylbenzenes as amyloid-b plaque binding
ligands
Rob J. A. Nabuurs a, , , Varsha V. Kapoerchan b, , Athanasios Metaxas c, Sanne de Jongh a,
⇑
Maaike de Backer a, Mick M. Welling a, Wim Jiskoot d, Albert D. Windhorst c, Hermen S. Overkleeft b,
Mark A. van Buchem a, Mark Overhand b, Louise van der Weerd a,e
a Department of Radiology CS2, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, Netherlands
b Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands
c Radionucleotide Laboratory, Free University, Amsterdam, Netherlands
d Division of Drug Delivery Technology, Leiden Academic Center for Drug Research, Leiden University, Netherlands
e Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Detection of cerebral b-amyloid (Ab) by targeted contrast agents remains of great interest to aid the
in vivo diagnosis of Alzheimer’s disease (AD). Bis-styrylbenzenes have been previously reported as poten-
tial Ab imaging agents. To further explore their potency as 19F MRI contrast agents we synthetized several
novel fluorinated bis-styrylbenzenes and studied their fluorescent properties and amyloid-b binding
characteristics. The compounds showed a high affinity for Ab plaques on murine and human brain sec-
tions. Interestingly, competitive binding experiments demonstrated that they bound to a different bind-
ing site than chrysamine G. Despite their high logP values, many bis-styrylbenzenes were able to enter
the brain and label murine amyloid in vivo. Unfortunately initial post-mortem 19F NMR studies showed
that these compounds as yet do not warrant further MRI studies due to the reduction of the 19F signal in
the environment of the brain.
Received 17 October 2013
Revised 25 February 2014
Accepted 28 February 2014
Available online 7 March 2014
Keywords:
Amyloid b
Fluorine
Imaging
Alzheimer’s disease
Contrast agent
Ó 2014 Published by Elsevier Ltd.
1. Introduction
For clinical use, the [11C]-benzothiazole derivative Pittsburgh
compound B (PiB, 1, Fig. 1) is the best characterized in vivo PET radi-
Alzheimer’s disease (AD) is the predominant form of dementia
in the aging population. The disease is marked by neuronal degen-
eration associated with deposits of tau proteins in intraneuronal
neurofibrillary tangles (NFTs) and of amyloid-b (Ab) peptides in
extracellular amyloid plaques.1 Although the precise role of amy-
loid in AD pathology is still not completely understood, accumula-
tion of amyloid plaques is thought to precede the onset of the first
clinical symptoms by up to two decades.2,3 A clinical imaging tech-
nique capable of visualizing and quantifying these early changes
thus may enable early diagnosis and better understanding of the
pathophysiology.
Over the past years progress has been made in the development
of Ab-targeting imaging ligands suitable for visualization by posi-
tron emission tomography (PET), single positron emission tomog-
raphy (SPECT), fluorescence microscopy or magnetic resonance
imaging (MRI).
otracer. However, as the short half-life of 11C limits its use to med-
ical centers with an on-site cyclotron, alternatives are desired.
Therefore, several longer-lived 18F radiofluorinated derivatives
have been designed, like flutemetamol (2),4 florbetapir (3) and
florbetaben(4),5 of which the first two recently have been the first
to be admitted for commercial use with the last one expected to fol-
low soon thereafter.6,7 Despite the inherent high sensitivity of PET,
the development of Ab-targeted imaging probes suitable for clinical
MRI remains attractive as this would lower the threshold for per-
forming amyloid scans given the wider availability of MRI systems
as compared to PET systems, the lack of ionizing irradiation and the
lower costs involved in performing clinical MRI as compared to PET.
Furthermore, such agents could be used in one scan session com-
prising a comprehensive structural and functional scan protocol
as well as a scan to detect the molecular imaging tracers, whereas
one PET examination only provides one biomarker.
Based on congo red (5), several bis-styrylbenzenes have been
reported to show strong Ab binding affinities and serve as potential
backbones for in vivo PET or SPECT imaging probes, like 6 (chrys-
amine G), 7 (X-34),8 8 (ISB)9 and 10 (Methoxy-X04)10 (Fig. 1 and
Scheme 1). The styrylbenzene backbone has also been explored
⇑
Corresponding author. Tel.: +31 71 526 4376.
E-mail address: r.j.a.nabuurs@lumc.nl (R.J.A. Nabuurs).
Authors have contributed to this work equally.
http://dx.doi.org/10.1016/j.bmc.2014.02.054
0968-0896/Ó 2014 Published by Elsevier Ltd.

2470
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18F
HO
S
N
HO
S
N
NH
NH
1
PIB
2
Flutemetamol
HN
HN
O
O
18F
O
O
O
18F
O
N
3
4
Florbetapir
Florbetaben
NH2
H2N
N
N
N
N
N
N
HO
N
N
OH
O
O
O
S
S
HO
O
HOOC
COOH
5
6
OH
Congo Red
Chrysamine G
I
O11CH3
COOH
HO
HO
HOOC
OH
COOH
HOOC
7
X-34
8
ISB
F
OCH3
HO
OH
COOH
HO
OH
HOOC
9
FSB
10
Methoxy-X04
Figure 1. Previously reported amyloid-b binding ligands.
for the development as an MRI contrast agent. The initial break-
through came with the design of compound 9 (FSB), which was
specifically designed for in vivo detection of Ab using 19F MRI.11
Since normal biological tissue completely lacks fluorine, 19F MRI
would allow direct imaging of the amyloid binding compound,
without any endogenous background signal (‘hot spot imaging’).
Initial in vivo animal studies with compound 9 showed promising
results. Unfortunately, 19F MRI in vivo experiments suffer from the
inherently low sensitivity of MRI with a detection limit in the
micromolar to millimolar range depending merely on the voxel
size and magnetic field strength. As initial 19F compound only car-
ried a single fluorine atom this makes 19F MRI a technical challenge
requiring long acquisition times. The incorporation of multiple
magnetically equivalent 19F atoms could aid with respect to this
aspect, as the spin of each individual 19F nucleus directly adds to
the MR signal. The study therefore aimed to further exploit the
bis-styrylbenzene backbone as a specific amyloid-b targeting MR
contrast agent by increasing the number of fluorine atoms posi-
tioned to maintain favorable NMR characteristics as well as solu-
bility. Synthetized compounds were evaluated with respect to
their Ab binding and specificity, and their fluorescent properties.
Partition coefficients (logP) were determined to assess hydropho-
bicity. Compounds were injected systemically in transgenic AD
mice to determine BBB passage and affinity for amyloid plaques
in vivo. For the most promising compound, in vitro and post-mor-
tem 19F NMR studies were performed.
2. Design
Previously Flaherty et al. have designed a similar series of poly-
fluorinated compounds, based on a bis-styrylbenzene structure
with either a non-substituted core with fluorine substitutions on
the outer rings, or four 19F atoms on the inner ring with a polar
substituent on the outer ring.12 These compounds were found to
target Ab in vivo. As was already proven for Methoxy-X04 (10,
Scheme 1), acidic functional groups are not required for high affin-
ity Ab binding, with the apparent Kd’s of these fluorinated com-
pounds being ꢀ300 fold lower compared to 9. Despite their
ability to label amyloid in vivo, however, the Flaherty compounds
are very hydrophobic, and therefore their solubility and blood-
brain barrier (BBB) passage are likely to limit their full potential
as a 19F MRI agent. Furthermore, positioning of the 19F group di-
rectly on the planar backbone may have a detrimental effect on
its relaxometry following binding, while it has been suggested that
in addition to the inherent sensitivity of MRI also 19F relaxivity
plays an important role in the detection, due to a reduction of
the transverse relaxation time (T2) caused by binding to amyloid,
or by a hydrophobic environment like brain tissue.13 Therefore,

R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
2471
R1
R1
R1
O
Br
P(OEt)2
a
b
Br
(EtO)2P
R2
R2
R2
C
A
B
O
R1
R4
c
R3
R3
O
R2
H
R4
R3 = OTBDMS
R3 = OH
E
F
d
R3
R4
D
R1
R2
R3
R4
10 (Methoxy-
OMe
H
OH
H
X04)
11
OMe
OMe
OMe
OMe
OMe
OH
OH
OH
CF3
OMe
H
H
H
H
H
H
H
H
H
H
H
H
OH
CH2OH
CF3
OCF3
CF2CF2H
CF3
OCF3
CF2CF2H
OH
OH
OH
OH
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
12
13
14
15
16
17
18
19
20
21
22
23
24
OCH2CF3
O(CH2)3CF3
OC(CF3)3
OH
OMe
H
Scheme 1. Reagents and conditions: (a) N-bromosuccinimide, benzoyl peroxide, CCl4, reflux, 16 h; (b) triethyl phosphite, 150 °C, 16 h; (c) (1) KOtBu or NaH, THF, ꢁ10 or 0 °C,
20 min then D, rt, 16 h (d) tetra-n-butylammonium fluoride, THF, 0 °C, rt, 16 h.
we set out to extend the existing bis-styrylbenzene library by add-
ing one or more polar moieties to improve solubility and increase
the number of (magnetically equivalent) fluorine atoms positioned
such to maintain favorable NMR characteristics.
The building blocks needed for the synthesis of compounds 10–
24 are depicted in Figure 2, and were synthetized or commercially
available. (For synthesis of all building blocks, see Supporting
information.)
As a starting point, Methoxy-X04 (10) was chosen.10 With an
affinity for Ab in the nanomolar range (Ki = 26.8 nM), this fluores-
cent small molecule is frequently used for intravital microscopy
studies as it has high affinity for Ab plaques in vivo following
intravenous or intraperitoneal injection.14 We designed a series
of bis-styrylbenzenes with incorporating multiple preferably mag-
netically equivalent fluorine atoms (11–24) or additional minor
modifications (Scheme 1).
3. Fluorescent properties
All synthesized compounds are expected to have fluorescent
properties based on their conjugated ring structures. We therefore
determined excitation and emission wavelengths of 300 nM solu-
tions and the corrected emission intensities were compared to that
of Methoxy-X04 (Table 1).
Compounds 11–24 are accessible from the general synthesis
route given in Scheme 1, with the key building blocks being a
diphosphonate (C) and an aromatic aldehyde (D). Substituted p-xy-
lene (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
(HWE) reaction between diphosphonate (C) and an aldehyde (D)
yields the (E,E)-bis-styrylbenzene (E). If necessary, an additional
deprotection step with TBAF is carried out to remove the silyl
protecting groups (only in the cases where the final compounds
have hydroxyl substituents). Independently, a similar synthesis
route for the design of NFT and amyloid probes based on
styrylbenzenes was recently published by Boländer et al.15
It has been reported that binding to amyloid may have a signif-
icant effect on fluorescence properties,16 and therefore the fluores-
cence was also measured in the presence of synthetic Ab fibrils.
The parent compound 10 showed the highest intrinsic fluorescence
with a 10-fold increase in the presence of amyloid fibrils. A similar
increase was typically only observed for those compounds that
only had minor modifications on the outer rings. Several previously
reported Ab-targeting fluorophores have shown a clear red-shifted
emission spectrum following binding.17,18 Some of our compounds
showed a similar red-shift; though resulting in multiple emission
peaks in the spectrum (Fig. 3). This observation suggests the pres-
ence of multiple binding sites, similar to what has previously been
reported, for example for Thioflavin.19

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R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
O
O
O
O
OMe
OMe
TBDPSO
CF3
P(OEt)2
P(OEt)2
P(OEt)2
P(OEt)2
(EtO)2P
O
(EtO)2P
(EtO)2P
(EtO)2P
MeO
O
O
O
25
26
27
28
CF3
O
CF3
O
F3C CF3
F3C
CF3
F3C
O
O
O
O
O
O
P(OEt)2
P(OEt)2
P(OEt)2
P(OEt)2
(EtO)2P
O
(EtO)2P
(EtO)2P
(EtO)2P
O
O
O
29
33
30
31
32
O
O
O
O
H
H
H
H
H
TBDMSO
F3C
TBDMSO
F3CO
34
37
35
O
O
H
F2C
HF2C
O
MeO
CF3
38
36
Figure 2. Structures of the building blocks 25–38.
for that in the presence of amyloid plaques, the data in Figure 4 can
be interpreted as follows. Despite having a lower fluorescent inten-
sity for the bound compound, compounds 16–18 and 22 labeled
more human amyloid, thus suggesting an improved binding com-
pared to Methoxy-X04. Similar analysis of the stained APP/PS1 sec-
tions revealed the affinity of all compounds, except 11 and 21, to
be significantly higher than that of our lead compound Methoxy-
X04 (Kd 26.8 nM). Compounds 13 and 15 were found to have the
most efficient binding properties for murine amyloid plaques. With
the lowest fluorescent intensity after binding to amyloid, being
100ꢂ less than Methoxy-X04, the murine amyloid plaques none-
theless appeared very bright.
4. Qualitative assessment of human and murine amyloid
plaques binding
Fluorescence microscopy was used for a qualitative assessment
of the amyloid-binding properties. A concentration series of each
compound (1–10–100
lM) was applied to brain slices of APP/PS1
mice and human AD patients. At 100
lM all compounds, except
11 and 21, showed characteristic staining of amyloid plaques on
both human and murine sections. At the lower concentrations,
however, clear differences in affinity were found (Table 1 &
Fig. 4). Amyloid plaques in humans and mice differ in composition
and compactness.20 This is likely the reason that all compounds
showed higher affinity for murine plaques as they were visible
5. Affinity for synthetic amyloid-b fibrils
even after staining with 1 lM concentration, whereas compounds
19–21 and 23–24 showed no detectable amyloid plaques in the hu-
man sections at this concentration. To illustrate these differences,
A competition assay with [3H]chrysamine G (6) was used to
determine the binding inhibition coefficient (Ki) for the com-
pounds. Based on this assay reproducible results could only be ob-
tained for those compounds with a hydroxyl substituent on the
outer rings (Fig. 5). The ex vivo stainings clearly showed that many
of the other compounds show affinity for amyloid, implying that
these compounds probably use different binding sites than chrys-
amine G. Even the provided Ki values for compounds 19–23 only
reflect competition against the [3H]chrysamine G binding sites,
and therefore most likely underestimate the overall affinity of
these compounds for synthetic Ab fibrils.
Table 1 and Figure 4 highlight the results of the 10
lM staining
on human sections and the 1
murine sections.
lM concentration used to stain the
In general, planarity is considered one of the criteria for binding
to Ab plaques.21 The introduction of an additional substitution on
the middle ring, which disturbs the planar conformation, has a det-
rimental effect on the binding affinity, as virtually no binding was
seen using 11.
Although previous studies have reported possible interaction of
styrylbenzenes with NFTs,15 for none of our compounds NFTs
staining was observed in the human AD cortex at these concentra-
tions. Furthermore, with no background staining in either human
or murine control brain sections this suggested their specificity
to amyloid. 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 Ab is representative
6. LogP values
The blood–brain barrier (BBB) is a tight layer of endothelial cells
in the wall of cerebral blood vessels that limits the passage of blood
compounds into the brain. It is traditionally stated that for optimal
passive BBB passage, compounds should preferably have moder-
ately hydrophobicity (logD or logP 2.0–3.5) however a number of
successful radiopharmaceuticals do not meet this requirement.22

R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
2473
Table 1
Fluorescent and binding characteristics
b
Compound MW
Ex vivo Ab
bindinga
kex
kem
Fluorescence
intensityc
Fluorescence intensity on binding to Increase on binding to
Ki
fibrillar Abd
fibrillar Ab
(da)
Human Murine (all 1 nm) (all 1 nm) (%)
(nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
344.403
+
ꢁ
+
+
+
+
+
ꢁ
372
n.d.
360
356
354
354
351
331
325
354
360
360
355
355
355
451
n.d.
436
471
456
456
464
458
487
466
450
465
472
472
494
100
n.p.
63
9
31
10
6
17
7
18
16
5
100
n.p.
26
1
3
1
9.8
n.p.
4.1
ꢀ1
24,2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
244
39.3
56.4
84.48
n.d.
n.d.
374.429
372.456
448.400
480.399
544.433
434.374 ++
466.372 ++
530.407 ++
382.375
412.401
440.454
548.397 ++
494.426
508.452
++
++
++
++
++
++
++
++
+
ꢀ1
ꢀ1
2
3.6
7.4
n.d.
10.7
4.1
4.3
17.8
ꢀ1
13
n.d.
19
7
2
38
2
ꢁ
ꢁ
ꢁ
ꢁ
++
++
++
21
17
37
ꢁ
ꢁ
4
ꢀ1
n.d. = not detected; n.p. = not performed.
a
Staining of amyloid plaques in human (10 lM) and APP-PS1 murine (1 lM) was scored whether the compound stained less (ꢁ), similar (+) or more (++) for amyloid
plaques in comparison to 10.
b
Ex/Em wavelength maxima were determined of 300 nM solutions.
Fluoresence intensity was calculated relative to 10.
Fluoresence intensity was calculated relative to intensity of 10 after binding to fibrillar Ab.
c
d
Therefore logP or logD should be considered carefully as selection
criterion, but it is a valid parameter for selection nonetheless when
applied within one series of compounds. For each of our com-
pounds 10–24, log P values were determined with an HPLC-based
method according to Benhaim and Grushka.23–25 The found logP
values are shown in Table 2. The limitation of this method is that
logP values > 7 cannot be measured reliably. Not surprisingly,
many of the compounds actually do show logP values > 7, which
is a logical consequence of the fact that Methoxy-X04 (10) itself al-
ready has a logP value of 5.05 and that the introduction of fluorine
makes the molecules more hydrophobic.
extensive cerebral amyloid plaques. The mice were sacrificed, their
brains were removed and post-mortem sections were studied using
the same fluorescence microscopy set-up as for the stained brain
sections. In vivo labeling of amyloid plaque was observed for almost
all compounds, except 11, 23 and 24, showing their ability to pass
the BBB. (Fig. 4 and Table 2) Apparently, a high logP value does not
necessarily prohibit BBB passage. Some compounds, particularly
13, showed a comparable signal intensity after intravenous injection
compared to Methoxy-X04, despite having a 100-fold lower fluores-
cence yield. This suggests that these compounds have a high affinity
and/or cross the BBB more efficiently than Methoxy-X04.
7. In vivo amyloid plaque labeling in transgenic AD mice
8. In vitro and post-mortem fluorine NMR
To assess the ability of the compounds to pass the BBB in vivo and
subsequently bind to amyloid, solutions of each compound were
injected intravenously in living transgenic APP/PS1 mice that had
Based on the above experiments, compounds 13 and 22 were
identified as the most promising leads, based on favorable binding,
and positive staining after intravenous injection in vivo indicating
300 nM of 22
300 nM of 22 with fibrillar Aβ
200000
200000
180000
160000
140000
120000
100000
80000
180000
160000
140000
120000
100000
80000
60000
60000
40000
20000
0
40000
20000
0
360
360
Excitation (nm)
Excitation (nm)
500
500
Emission (nm)
Emission (nm)
Figure 3. Excation and emission spectra of compound 22. Shown are the excitation/emission spectra of compound 22 to illustrate possible effects on the fluorescent
properties following binding of amyloid.

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R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
Figure 4. Staining for murine and human amyloid plaques. Shown paraffin embedded 8
lm thick human AD sections are stained using 10 lM, whereas APP/PS1 murine
sections are stained using 1 M to best depict the differences in amyloid staining between the different compounds. Thirty micrometer thick murine APP/PS1 sections
l
following in vivo administration are shown with two times less magnification to allow visualization of a larger cortical region. All images were digitized using the same
settings.

R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
2475
Figure 5. Non-linear fitting to assess affinity for synthetic amyloid-b fibrils by a competition assay with [3H]chrysamine G.
mouse brain. This is contrary to the initial publication of a 19F amy-
loid ligand by Higuchi et al.,11 but in agreement with the findings
of Amatsubo et al.13 Our design balanced between the planarity
needed for amyloid binding and free rotation for the fluorine
groups to maintain favourable NMR characteristics, however, this
data still suggests that the hydrophobic interaction between the
brain and the 19F groups are responsible for line broadening. As re-
cently pointed out in vivo by Yanagisawa et al. this problem might
be overcome by the use of a polyethylene glycol (PEG) linker to at-
tach the fluorine groups further away from the amyloid binding
core.26
Table 2
LogP values and scoring of amyloid labeling following intravenous injection
Compound
LogP
LogP
(determined)
In vivo amyloid labeling
(calcd)a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5.55
5.46
5.53
9.06
8.44
10.04
8.74
8.12
9.72
6.74
6.83
7.37
10.22
8.57
8.89
4.84
5.00
4.99
>7
>7
>7
6.18
>7
>7
5.64
5.25
5.79
>7
7.17
>7
+
ꢁ
+
+
+
+
+
+
+
+
+
+
+
ꢁ
ꢁ
9. Summary and conclusion
In this work, a series of 15 analogs of Methoxy-X04 (10) with
various number of fluorine atoms has been synthesized and evalu-
ated for their Ab binding properties and ability to pass the BBB. The
incorporation of suitably placed fluoro substitutions could improve
the current (MRI) contrast agents for the diagnosis of Alzheimer’s
disease. It was concluded that the introduction of a second substi-
tution on the inner ring was not well tolerated, whereas single
bulky modifications on both the outer and inner rings were well
tolerated. Despite the observed high logP values brain entry did
not seem to be inhibited for most compounds. Based on all find-
ings, compounds 13 and 22 were considered most promising for
the development of Ab imaging agents. However, the post-mortem
NMR results leave us to conclude that there seems to be no role for
these compounds as MR imaging agents for the diagnosis of AD. To
our opinion it remains doubtful whether the incorporation of other
fluorine moieties or higher field magnetic field strength will help
to overcome these hurdles next to those set by the inherent
relatively low sensitivity of 19F MRI in combination with known
cerebral Ab concentrations.
Nevertheless, this study expands the existing knowledge on bis-
styrylbenzenes as amyloid targeting agents in general and creates
opportunities for their application as fluorescent amyloid ligands
for preclinical optical imaging. Recent advances in fluorine chemis-
try create further opportunities to radiolabel compounds of our
series with 18F.27,28 These compounds might provide additional
information regarding accumulation of cerebral amyloid, espe-
cially since we have found that our series of compounds bind to
a distinct binding site.
The ability to label amyloid plaques in the brains of APP–PS1 mice following
intravenous injection were scored absent (ꢁ) or present (+).
a
LogP values were calculated used web-based methods: www.molinspira-
tion.com and http://intro.bio.umb.edu/111-112/OLLM/111F98/jlogp/test.html.
good BBB passage. Initial 19F NMR studies were only conducted
with compound 22, which had the highest number of magnetically
equivalent 19F atoms. NMR spectra were acquired for 1.92 mM of
22 with or without an excessive amount of fibrillar Ab. Binding
to fibrillar amyloid did not result in significant line broadening of
the NMR spectrum. (Fig. 6A) The NMR spectrum corresponding
to compound 22 mixed with homogenized APP/PS1 brain, how-
ever, revealed a small chemical shift of 0.03 ppm and a severe
reduction in T2 as observed by the line broadening of the peak.
As suggested previously, most likely the lipophilic environment
of the brain tissue itself results in a reduced relaxation time and
thereby signal loss.13
Finally, 30 lmol/kg of 22 was injected intravenously in APP/PS1
mice. The brains were removed after 24 h and a 19F NMR spectrum
was obtained of the brain homogenate. No 19F signal was observed,
although antemortem intravital microscopy showed clear labeling
of cerebral amyloid (Fig. 6B–C).
The severe attenuation of the 19F NMR signal due the hydropho-
bic nature of both the fluorinated compounds as well as the brain
itself seemed to hampered the detection of the 19F signal in the

2476
R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
Figure 6. In vitro and post-mortem 19F NMR analyses of compound 22. (A) Shows the NMR spectra corresponding to 1.92 mM of compound 22 in solution without (-) or with
the presence of 22 M fibrillar Ab (- -) or mixed with a homogenized aged APP/PS1 mouse brain. All spectra are relative to trifluoroacetic acid set at 0 ppm. (B) shows a NMR
spectra of a homogenized APP/PS1 mouse brain made 24 h post-injection of 30 mol/kg of dissolved 22. The corresponding intravital microscopic image presented in (C)
clearly shows the antemortem labeling of cerebral amyloid (blue) in both vasculature as well as parenchymal amyloid plaques. Image dimensions are 615 ꢂ 615 m.
l
l
l
ethanol for 2 min, air dried and coverslipped using Aqua/Poly-
mount. Fluorescence of the stained sections was analyzed using a
whole microscopic slide scanner (Pannoramic MIDI, 3DHistech,
Hungary) with a DAPI filter cube (Ex 365 nm; Em 445/50 nm) using
the same intensity setting throughout all experiments.
10. Experimental section
10.1. Preparation of Ab1–40 fibrils
Ab fibrils were prepared by stirring a 0.5 mg/ml solution of Ab
peptide (1–40) (RPeptide, Bogart, GA) at 37 °C for 3 days, which re-
sulted in a cloudy solution. The presence of fibrils was confirmed
by the appearance of an emission peak at 482 nm (excitation
10.4. In vivo Ab 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 intrave-
nously with 0.05 M dissolved in 1:1 DMSO/Cremophor diluted
440 nm) upon addition of a 5
T (Sigma, Germany) to a small amount of fibrils. Aliquots of 10
lM solution in PBS of Thioflavin
l
l
were transferred to Eppendorf vials and stored at ꢁ80 °C until
with PBS to a total volume of 200
30 mol/kg. One day after injection, animals were sacrificed using
200 l Euthanasol (AST Pharma) prior to transcardial perfusion
ll, resulting in a total dose of
the assay was to be performed.
l
l
10.2. Fluorescence spectra
with 4% paraformaldehyde in PBS. Brains were removed and cryo-
protected in 4% PFA with 10% sucrose for 4 hours followed by
immersion in 4% PFA with 30% sucrose overnight. Snap-frozen
All compounds were dissolved in DMSO at 0.3 mM and diluted
to 300 nM with 9:1 PBS/ethanol. Fluorescence spectra were mea-
sured on a Varian Cary Eclipse fluorescence spectrophotometer to
obtain peak excitation and emission wavelengths, 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 fluorescence spectra were obtained by
brains were cryosectioned (30
analyzed as described above.
lm) and fluorescence images were
10.5. 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
adding 500 ll of the dissolved compounds to previously prepared
Ab aliquots. After manual shaking for 30 s, 300 ll samples were
measured (Infinity M1000, Tecan, Switzerland).
a final concentration range of 30
test compounds was combined with 890
50 stock (specific activity
of 100 nM [3H]-chrysamine
33.8 Ci/mmole). The mixture was sonicated for 10 min, and the
l
M to 30 pM. 10
ll of unlabeled
10.3. Staining of human and transgenic AD brain sections
ll of assay buffer and
l
l
G
Stock solutions of 3 mM in DMSO were diluted to 1–10–100 lM
in 2:3 PBS/ethanol and sonicated for 15 min. Paraffin sections
assay was subsequently started by the addition of 50 ll synthetic
(8 m) of the medial temporal lobe cortex of human AD subject
l
Ab1-40 fibrils, to achieve final concentrations of 5 nM [3H]chrys-
that was assigned as Braak IV, 14 months old transgenic murine
APP/PS1 brain and age-matched control cortices were deparaffi-
nized prior to staining for 10 min in absolute darkness. After gently
rinsing with tap water, sections were placed in 0.1% NaOH in 80%
amine G and 50 nM fibrils. Nonspecific binding was determined
in the presence of
1 lM Methoxy-X04. Incubations were
terminated after 1 h via filtration through Whatman GF/B filters

R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
2477
(pre-soaked in binding buffer), using a 48-well Brandel harvester.
Roche Diagnostics) added to every 10 ml. Aliquots (500
the solvent, the solvent containing 22
brain homogenate were added to 20
DMSO/Cremophor to achieve a final concentration of 1.92 mM of
compound 22. The mixtures were transferred to a standard 5 mm
NMR tube. NMR spectra were obtained using a single pulse se-
quence with a 22,573 Hz spectral width (SW) and 100 scans. The
chemical shifts of the 19F NMR signals were identified by setting
the reference trifluoroacetic acid (TFA) at 0 ppm.
l
l) of only
The filters were washed two times with 3 ml of ice-cold binding
buffer (pH 7.0), and radioactivity was determined by liquid scintil-
lation 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 = 5 nM. (GraphPad
Software Inc., San Diego, CA).
l
l
M of aggregated Ab or the
l of 0.05 M of 22 in 1:1
10.6. Procedure for logP determinations
10.8. Post-mortem 19F NMR in APP/PS1 mice
LogP determinations were performed using literature proce-
dures.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 (Phe-
nomenex, 50 ꢂ 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 mlꢁ1 were prepared in methanol. These stock solutions
were then diluted with water, making sure that the volume per-
centage of water was such that the compounds did not precipitate
(max 40% water). A 0.25 mg mlꢁ1 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 ta-
ken from the literature. Of these compounds, 0.25 mg mlꢁ1 solu-
tions 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 k0 according to the formula k0 = (tR ꢁ t0)/t0, with
tR being the retention time of the compound and t0 being the reten-
tion time of NaNO3. The retention factors were extrapolated to 0%
B, yielding k0w. As there is a linear relationship between logP and
logk0w plotting logP values of known compounds (Table 3) against
obtained logk0w values in this system yields a calibration curve.
From this curve, logP values of unknown compounds can be calcu-
lated from their k0w values.
Twelve-to-fourteen-month-old APP/PS1 mice (n = 2) received
an intravenous injection with 0.05 M of compound 22 dissolved
in 1:1 DMSO/Cremophor diluted with PBS to a total volume of
200 ll to achieve a total dose of 30 lmol/kg. Prior injection the ani-
mals underwent cranial window surgery.14 Twenty four hours post
injection the animals underwent in vivo multiphoton microcopy
(LSM 710 MP, Carl Zeiss, Germany) operating at 750 nm to validate
the presence and labeling of cerebral amyloid. After perfusion the
resected brain was snap frozen and prepared for NMR similar to
the above brain homogenates. A similar fluorine NMR spectrum
was obtained using a single pulse sequence with a 94,340 Hz SW
and 1200 scans.
10.9. Synthetic procedures
10.9.1. General
Reagents and solvents were used as provided, unless stated
otherwise. 4-Trifluoromethylbenzaldehyde (35), 4-trifluorometh-
oxybenzaldehyde (36), 4-(1,1,2,2-tetrafluoroethyl)benzaldehyde
(37), 3-trifluoromethyl-4-methoxybenzaldehyde (38) (Fig. 2) were
purchased at standard suppliers. The other building blocks as
shown in Figure 2 were prepared following literature procedures
(see supporting info). 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 reac-
tion, traces of water were removed from the starting materials by
repeated coevaporation 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)4-
Ce(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 chroma-
tography was carried out with silica gel (Screening Devices bv, 40–
10.7. In vitro 19F NMR analyses
To investigate the possible effect on its NMR properties caused
by either binding to amyloid or the lipophilic environment of the
brain several samples were prepared similar to the protocol de-
scribed previously.13 The in vitro 19F NMR analyses were per-
63 lm particle size, 60 Å), using technical grade solvents. NMR
spectra were recorded at 298 K on a Bruker AV400 using deuterated
solvents. All carbon spectra are proton-decoupled. Chemical shifts
(d) are given in ppm, in 13C spectra relative to the solvent peaks
of CDCl3 (77.0 ppm), CD3OD (49.0 ppm), DMF-d7 (29.76 ppm), ace-
tone-d6 (29.9 ppm) or DMSO-d6 (39.51 ppm), in 1H spectra relative
to the solvent peak of tetramethylsilane (0.0 ppm), CD3OD
(3.31 ppm), DMF-d7 (2.75 ppm), acetone-d6 (2.05 ppm) or DMSO-
d6 (2.50 ppm), in 19F spectra relative to the solvent peak of TFA
(0 ppm). Coupling constants are given in Hz. IR spectra were re-
corded on a Perkin Elmer Paragon 1000 FT-IR Spectrometer. High
formed using
a Bruker DMX400 NMR spectrometer (Bruker,
Germany). All samples were diluted with 10% D2O in 0.1 M PBS
with one EDTA-free protease inhibitor tablet (Complete Mini,
Table 3
Reference logP values
Compound
LogP
resolution mass spectra were recorded by direct injection (2
ll of
Resorcinol
p-Nitroaniline
phenol
m-Nitrophenol
2-Naphtol
0.8
1.39
1.46
2
2.7
3.37
a 2 M solution in H2O/MeCN; 50:50; v/v and 0.1% formic acid)
l
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 res-
olution R = 60,000 at m/z 400 (mass range m/z = 150–2000) and
dioctylpthalate (m/z = 391.28428) as a ‘‘lock mass’’. The high reso-
lution mass spectrometer was calibrated prior to measurements
with a calibration mixture (Thermo Finnigan). It should be noted
Naphtalene
Based on known literature values, the logP of
several reference compounds yield a required
calibration curve to determine the logP values
of our compounds.21–23

2478
R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
that, with the exception of compound 9 (data not shown) and only
certain intermediates ionized correctly, none of the target com-
pounds provided useful (HR)MS data.
LC–MS analysis was performed on a Finnigan Surveyor HPLC
system with a Gemini C18 50 ꢂ 4.6 mm column (3 micron, Phe-
nomenex, Torrance, CA, USA) (detection at 200–600 nm), coupled
to a Thermo Finnigan LCQ Advantage Max mass spectrometer (Bre-
da, The Netherlands) with electrospray ionization (ESI; system 1),
with as eluents (A): H2O; (B): MeCN and (C): 1% aq TFA.
1.25 mmol). The crude product was subjected to column chroma-
tography (0 ? 2% EtOAc/light petroleum) to yield the intermediary
bis-TBDMS protected styrylbenzene (0.28 g, 0.48 mmol, 96%) as a
bright yellow solid. 1H NMR (CDCl3, 400 MHz):
d 7.58 (d,
J = 8.1 Hz, 1H); 7.51 (d, J = 6.1 Hz, 2H); 7.49 (d, J = 6.3 Hz, 2H);
7.44 (s, 1H); 7.34-7.28 (m, 4H); 7.16–7.13 (m, 2H); 7.13–7.09 (m,
2H); 7.03 (d, J = 1.4 Hz, 1H); 4.75 (d, J = 2.4 Hz, 4H); 3.95 (s, 3H);
0.95 (s, 9H); 0.95 (s, 9H); 0.11 (s, 6H); 0.11 (s, 6H). 13C NMR (CDCl3,
100 MHz): d 157.0; 141.0; 140.7; 137.9; 136.7; 136.0; 128.7;
128.4; 128.1; 126.4; 126.4; 126.4; 126.0; 122.7; 119.3; 108.6;
64.9; 64.8; 55.6; 26.0; 18.4; -5.2. IR (neat): 2953.7; 2927.5;
2855.3; 1593.8; 1553.7; 1515.3; 1458.3; 1421.9; 1377.6; 1248.7;
1206.0; 1084.2; 1037.1; 1005.7; 967.3; 837.4; 773.9; 668.1;
504.2. The bis-TBDMS protected (E,E)-styrylbenzene (0.27 g,
0.46 mmol) was treated with TBAF according to general procedure
C. Compound 12 was obtained by column chromatography (50%
EtOAc/light petroleum ? 10% MeOH/EtOAc) as a bright yellow so-
lid (0.14 g, 0.37 mmol, 79%).
10.9.1.1. General procedure A: Horner–Wadsworth–Emmons
reaction with NaH. The diphosphonate (0.83 mmol) was dis-
solved in THF (2.6 mL) and cooled to 0 °C. NaH (60% wt. dispersion
in mineral oil, 0.17 g, 4.17 mmol) was added and the mixture stir-
red for 30 min at 0 °C. The aldehyde (2.08 mmol) was dissolved in
THF and added to the reaction mixture, which was subsequently
stirred for 16 h at rt. After cooling and quenching with water, the
mixture was extracted three times with EtOAc and the combined
organic layers were washed with satd aq NaHCO3, dried (Na2SO4),
filtered and concentrated. The crude product was subjected to col-
umn chromatography to yield the pure product.
1H NMR (DMF-d7, 400 MHz): d 7.68 (d, J = 8.0 Hz, 1H); 7.59 (d,
J = 8.1 Hz, 2H); 7.55 (d, J = 8.1 Hz, 2H); 7.49 (d, J = 16.6 Hz, 1H);
7.39–7.34 (m, 4H); 7.31 (d, J = 5.0 Hz, 2H); 7.28 (d, J = 4.9 Hz,
1H); 7.25–7.21 (m, 2H). 13C NMR (DMF-d7, 100 MHz): d 157.7;
142.6; 142.3; 138.8; 137.2; 136.6; 129.0; 128.9; 128.4; 127.4;
126.8; 126.8; 126.6; 126.0; 122.9; 119.7; 109.3; 63.8; 55.6. IR
(neat): 1592.0; 1557.6; 1515.8; 1455.9; 1418.0; 1340.0; 1269.9;
1243.7; 1211.9; 1158.1; 1112.4; 1034.2; 1011.7; 956.3; 825.7;
624.2.
10.9.1.2. General procedure B: Horner–Wadsworth–Emmons
reaction with KOtBu. The diphosphonate (0.95 mmol) was dis-
solved 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 con-
centrated. The crude product was subjected to column chromatog-
raphy to yield the pure product.
10.9.1.7. (E,E)-1-Methoxy-2,5-bis(4-trifluoromethyl)styrylben-
zene (13). Following general procedure A, diphosphonate 26
(0.20 g, 0.5 mmol) was reacted with aldehyde 35 (0.22 g,
1.25 mmol). After work-up, the crude product was purified by col-
umn chromatography (0 ? 1% EtOAc/light petroleum) and the
compound 13 was obtained as a bright yellow solid (80 mg,
0.18 mmol, 36%).
10.9.1.3. General procedure C: silyl deprotection.
A solution of
protected bis-styrylbenzene (0.63 mmol) in THF (2 mL) was cooled to
0 °C and TBAF (1 M in THF, 3.14 ml, 3.14 mmol) was added. The blood-
red solution was stirred for 16 h after which water was added and the
reaction mixture was extracted with EtOAc. To the aqueous layer 1 N
HCl was added, followed by two times extracting with EtOAc. The
combined organic layers were dried (Na2SO4), filtered and concen-
trated. The pure product was obtained by column chromatography
(0 ? 25% EtOAc/light petroleum).
1H NMR (acetone-d6, 400 MHz): d 7.82 (d, J = 8.2 Hz, 2H); 7.79
(d, J = 8.5 Hz, 2H); 7.76–7.69 (m, 5H); 7.66 (d, J = 16.7 Hz, 2H);
7.43 (s, 2H); 7.38 (d, J = 16.4 Hz, 2H); 7.29 (dd, J = 8.1 Hz,
J = 1.3 Hz, 1H); 4.00 (s, 3H). 13C NMR (acetone-d6, 100 MHz): d
158.5; 143.0; 142.4; 139.3; 132.1; 128.3; 128.3; 127.9; 127.9;
127.8; 126.7; 126.5; 126.5; 126.5; 120.7; 110.3; 56.1. 19F NMR
(acetone-d6, 375 MHz): d 12.70 (s, 3F); 12.65 (s, 3F). IR (neat):
1608.4; 1464.0; 1420.1; 1323.6; 1246.5; 1158.5; 1116.6; 1105.9;
1065.5; 1036.4; 1014.1; 967.0; 954.2; 866.5; 848.8; 829.9; 759.2;
744.8; 622.2; 593.3; 508.4.
10.9.1.4. (E,E)-1-Methoxy-2,5-bis(4-hydroxy)styrylbenzene; Meth-
oxy-X04 (10). Methoxy-X04 was prepared according to general pro-
cedures A and C, using diphosphonate 25 and aldehyde 33. Physical
data corresponded to those reported in Ref. 10
.
10.9.1.8. (E,E)-1-Methoxy-2,5-bis(4-trifluoromethoxy)styrylben-
zene (14). Following general procedure A, diphosphonate 25
(0.20 g, 0.5 mmol) was reacted with aldehyde 36 (0.24 g,
1.25 mmol). After work-up, the product 14 was obtained by col-
umn chromatography (0 ? 1.5% EtOAc/light petroleum) as a bright
yellow solid (83 mg, 0.17 mmol, 35%).
10.9.1.5. (E,E)-1,4-Dimethoxy-2,5-bis(4-hydroxy)styrylbenzene
(11). Following general procedure A, diphosphonate 26 (0.34 g,
0.78 mmol) was reacted with aldehyde 33(0.46 g, 1.9 mmol). A
bright yellow solid was isolated (221 mg) by column chromatogra-
phy (10 ? 30% EtOAc/light petroleum) of which 100 mg was puri-
fied by preparative HPLC (40:60 ? 20:80 of 20 mM NH4OAc/
MeOH), to yield 22 mg (0.059 mmol, 8%) of compound 11.
1H NMR (CDCl3, 400 MHz): d 7.56 (d, J = 8.0 Hz, 1H); 7.54–7.50
(m, 4H); 7.44 (d, J = 16.5 Hz, 1H); 7.19 (t, J = 7.8 Hz, 4H); 7.14–
7.10 (m, 2H); 7.09–7.05 (m, 2H); 7.01 (d, J = 1.3 Hz, 1H); 3.94 (s,
3H). 13C NMR (CDCl3, 100 MHz): d 157.2; 137.7; 136.7; 135.9;
127.7; 127.5; 127.2; 126.6; 125.9; 124.1; 121.2; 121.1; 119.4;
108.5; 55.5. 19F NMR (CDCl3, 375 MHz): d 19.80 (s, 3F); 19.79 (s,
3F). IR (neat): 1593.1; 1557.6; 1510.2; 1421.9; 1254.6; 1199.8;
1159.4; 1104.9; 1034.0; 1015.7; 962.2; 921.6; 838.6; 673.8;
620.1; 530.0; 505.9.
1H NMR (CD3OD, 400 MHz): d 7.43 (d, J = 8.6 Hz, 4H); 7.33 (d,
J = 16.5 Hz, 2H); 7.27 (s, 2H); 7.19 (d, J = 16.5 Hz, 2H); 6.82 (d,
J = 8.6 Hz, 4H); 3.93 (s, 6H). 13C NMR (CD3OD, 100 MHz): d 158.5;
152.6;130.9; 129.7;128.9; 127.6; 121.1; 116.6;109.9; 56.8. IR(neat):
3359.6; 1605.3; 1515.7; 1495.6; 1463.6; 1435.4; 1408.7; 1260.0;
1196.0; 1171.8; 1022.4; 958.3; 849.6; 819.7; 790.1; 685.9; 551.0;
521.6.LC–MSretentiontime:8.99 min(10 ? 90%MeCN,15 minrun).
10.9.1.9. (E,E)-1-Methoxy-2,5-bis(4-{1,1,2,2-tetrafluoroethyl})
styrylbenzene (15). Following general procedure A, diphospho-
nate 25 (0.20 g, 0.5 mmol) was reacted with aldehyde 37 (0.28 g,
10.9.1.6. (E,E)-1-Methoxy-2,5-bis(4-hydroxymethyl)styrylben-
zene (12).
Following general procedure A, diphosphonate
25 (0.2 g, 0.5 mmol) was reacted with aldehyde 34 (0.31 g,

R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
2479
1.25 mmol). After work-up, compound 15 was isolated by column
chromatography (0 ? 7.5% EtOAc/light petroleum) as a yellow so-
lid (0.14 g, 0.25 mmol, 50%).
column chromatography (0 ? 4% EtOAc/light petroleum) and the
intermediary bis-TBDMS protected styrylbenzene was obtained
as a bright yellow solid (0.36 g, 0.59 mmol, 71%). 1H NMR (CDCl3,
400 MHz): d 7.77–7.72 (m, 2H); 7.61 (d, J = 8.1 Hz, 1H); 7.44–7.37
(m, 4H); 7.31 (dd, J = 16.1 Hz, J = 1.8 Hz, 1H); 7.11 (d, J = 16.3 Hz,
1H); 7.05 (d, J = 16.0 Hz, 1H); 7.00-6.92 (m, 1H); 6.88-6.81 (m,
4H). 13C NMR (CDCl3, 100 MHz): d 156.0, 155.9, 136.5, 135.0;
131.7, 130.3, 130.2, 129.6, 129.0, 128.1, 127.9, 126.9, 125.0,
123.8, 122.0, 120.4, 29.7, 25.7, ꢁ4.4. 19F NMR (CDCl3, 375 MHz):
d 18.17 (s, 3F). IR (neat): 1598.9; 1508.0; 1471.8; 1327.9; 1314.0;
1251.5; 1170.0; 1154.7; 1131.4; 1115.5; 1102.1; 1051.1; 962.1;
938.5; 906.8; 834.5; 778.2; 700.2; 667.7; 660.3; 638.4; 554.5;
534.7.
1H NMR (CDCl3, 400 MHz): d 7.56 (d, J = 7.3 Hz, 1H); 7.55–7.49
(m, 4H); 7.33 (d, J = 16.5 Hz, 1H); 7.24–7.16 (m, 4H); 7.13–7.04 (m,
4H); 7.01 (d, J = 2.3 Hz, 1H); 6.07–5.76 (m, 2H); 3.94 (s, 3H). 13C
NMR (CDCl3, 100 MHz): d 157.1; 148.5; 148.2; 137.7; 136.4;
135.6; 129.2; 128.7; 127.9; 127.8; 127.6; 127.5; 127.2; 126.6;
125.8; 122.4; 121.9; 121.8; 121.8; 121.7; 119.4; 119.1; 116.7;
116.4; 116.2; 115.5; 110.2; 108.7; 108.1; 107.7; 107.3; 55.5. 19F
NMR (CDCl3, 375 MHz): d -10.5 (s, 4F); -59.06 (t, J = 5.5 Hz, 2F); -
59.20 (t, J = 5.5 Hz, 2F). IR (neat): 1510.5; 1463.9; 1421.9; 1392.9;
1302.4; 1274.0; 1195.2; 1115.4; 1035.7; 1015.7; 961.8; 837.0;
784.1; 766.2; 709.4; 623.3; 600.0; 544.1.
The bis-TBDMS protected styrylbenzene (0.34 g, 0.54 mmol)
was treated with TBAF according to general procedure C, and fol-
lowing column chromatography (0 ? 30% EtOAc/light petroleum),
the impure product was purified by HPLC (CN column,
35:65 ? 10:90 of 0.2% aq TFA/MeOH) and product 19 was obtained
as a yellow solid (83 mg, 0.22 mmol, 40%).
10.9.1.10. (E,E)-1-Hydroxy-2,5-bis(4-trifluoromethyl)styrylben-
zene (16). Following general procedure B, diphosphonate 27
(0.85 g, 1.34 mmol) was reacted with aldehyde 35 (0.59 g,
3.36 mmol). After work-up, the crude product was purified by col-
umn chromatography (0 ? 6% EtOAc/light petroleum) and product
16 was obtained as a bright yellow solid (0.2 g, 0.48 mmol, 36%).
1H NMR (CD3OD, 400 MHz): d 7.67–7.52 (m, 10H); 7.21 (d,
J = 16.5 Hz, 1H); 7.14 (d, J = 2.2 Hz, 2H); 7.07 (dd, J = 8.1 Hz,
J = 1.5 Hz, 1H); 7.02 (d, J = 1.6 Hz, 1H). 13C NMR (CD3OD,
100 MHz): d 156.2; 142.7; 141.9; 138.6; 131.7; 129.2; 127.8;
127.8; 127.5; 127.4; 127.2; 126.9; 126.2; 126.2; 126.1; 126.1;
125.0; 119.5; 114.3. 19F NMR (CD3OD, 375 MHz): d 14.98 (s, 3F);
14.91 (s, 3F). IR (neat): 1611.5; 1428.3; 1319.9; 1163.2; 1107.4;
1066.6; 1014.2; 967.4; 956.5; 872.2; 831.8; 757.9; 750.6; 624.8;
592.8; 507.7.
1H NMR (CD3OD, 400 MHz): d 7.81 (d, J = 8.2 Hz, 1H); 7.73 (s,
1H); 7.70 (d, J = 8.5 Hz, 1H); 7.42 (d, J = 8.0 Hz, 2H); 7.38 (d,
J = 8.0 Hz, 2H); 7.23 (d, J = 16.0 Hz, 1H); 7.14 (t, J = 16.9, 2H); 6.99
(d, J = 16.3 Hz, 1H); 6.80 (dd, J = 7.8 Hz, J = 3.5 Hz, 4H).
13C NMR (CD3OD, 100 MHz): d 159.1; 158.9; 138.3; 136.2;
133.3; 131.1; 130.2; 130.0; 129.3; 129.2; 128.4; 128.0; 127.5;
125.0; 124.9; 124.7; 124.7; 124.6; 124.6; 121.7; 116.7; 116.6. 19F
NMR (CD3OD, 375 MHz): d 17.64 (s, 3F). IR (neat): 1605.0;
1512.1; 1441.0; 1313.9; 1257.1; 1239.9; 1199.4; 1171.9; 1153.6;
1108.8; 1079.5; 1049.9; 959.8; 869.9; 837.2; 812.7; 671.7; 552.7;
522.1.
10.9.1.11. (E,E)-1-Hydroxy-2,5-bis(4-trifluoromethoxy)styrylben-
zene (17). Following general procedure B, diphosphonate 27
(0.99 g, 1.56 mmol) was reacted with aldehyde 36 (0.74 g,
3.91 mmol). The crude product was purified by column chromatogra-
phy(0 ? 8%EtOAc/lightpetroleum)andproduct17 wasobtainedasa
bright yellow solid (0.47 g, 1.01 mmol, 65%).
10.9.1.14. (E,E)-1-(2,2,2-Trifluoroethoxy)-2,5-bis(4-hydroxy)sty-
rylbenzene (20). Following general procedure B, dipshospho-
nate 29 (0.45 g, 0.95 mmol) was reacted with aldehyde 33
(0.56 g, 2.37 mmol). After work-up, the crude product was sub-
jected to column chromatography (0 ? 5% EtOAc/light petroleum)
and the bis-TBDMS protected styrylbenzene was obtained as a
bright yellow solid (0.4 g, 0.63 mmol, 66%). 1H NMR (CDCl3,
400 MHz): d 7.59–7.51 (m, 4H); 7.43 (d, J = 16.5 Hz, 1H); 7.31 (d,
J = 7.7 Hz, 1H); 7.69 (d, J = 8.0 Hz, 1H); 7.24 (d, J = 16.4 Hz, 1H);
7.18 (d, J = 16.3 Hz, 1H); 7.10-7.06 (m, 2H); 7.00 (dd, J = 8.6 Hz,
J = 2.0 Hz, 4H); 4.56 (q, J = 8.2 Hz, J = 8.1 Hz, 2H); 1.16 (s, 18H);
0.38 (s, 6H); 0.38 (s, 6H). 13C NMR (CDCl3, 100 MHz): d 155.6;
155.5; 154.7; 137.9; 131.8; 131.0; 130.3; 129.3; 128.6; 127.7;
126.7; 126.7; 125.8; 121.0; 120.4; 120.3; 120.1; 110.7; 67.2;
66.8; 66.5; 66.1; 29.7; 25.6; -4.4. 19F NMR (CDCl3, 375 MHz): d
3.80 (t, J = 8.1 Hz, 3F).
1H NMR (CD3OD, 400 MHz): d 7.63 (d, J = 5.8 Hz, 2H); 7.61 (d,
J = 5.6 Hz, 2H); 7.48 (d, J = 16.5 Hz, 1H); 7.40–7.34 (m, 1H); 7.27–
7.18 (m, 5H); 7.12 (d, J = 3.2 Hz, 2H); 7.07 (dd, J = 8.1 Hz,
J = 1.5 Hz, 1H); 7.02 (d, J = 1.6 Hz, 1H). 13C NMR (CD3OD,
100 MHz): d 156.7; 149.7; 149.4; 149.4; 139.2; 139.0; 138.1;
133.0; 130.8; 129.0; 128.8; 128.1; 127.9; 127.7; 126.0; 125.4;
122.3; 122.2; 121.6; 119.7; 114.4. 19F NMR (CD3OD, 375 MHz): d
18.85 (s, 6F). IR (neat): 1604.3; 1557.6; 1515.8; 1505.7; 1269.9;
1158.4; 1099.9; 1015.6; 967.6; 928.0; 839.2; 676.6; 624.0; 525.1.
10.9.1.12. (E,E)-1-Hydroxy-2,5-bis(4-{1,1,2,2-tetrafluoroethyl})
styrylbenzene (18). Following general procedure B, diphospho-
nate 27 (0.74 g, 1.17 mmol) was reacted with aldehyde 37
(0.65 g, 2.92 mmol). After work-up, the crude product was sub-
jected to column chromatography (0 ? 10% EtOAc/light petro-
leum) to yield product 18 as a bright yellow solid (0.27 g,
0.5 mmol, 43%).
The bis-TBDMS protected styrylbenzene (0.4 g, 0.63 mmol) was
treated with TBAF according to general procedure C. After work-up,
the crude product was purified by column chromatography
(0 ? 25% EtOac/light petroleum) and product 20 was obtained as
a bright yellow solid (0.127 g, 0.31 mmol, 49%).
1H NMR (CD3OD, 400 MHz): d 7.51 (d, J = 8.1 Hz, 1H); 7.40–7.29
(m, 4H); 7.20 (d, J = 16.5 Hz, 1H); 7.13 (dd, J = 8.1 Hz, J = 1.0 Hz,
1H); 7.05 (d, J = 16.5 Hz, 1H); 7.03 (d, J = 16.3 Hz, 1H); 7.00 (d,
J = 1.1 Hz, 1H); 6.87 (d, J = 16.3 Hz, 1H); 6.81-6.75 (m, 4H); 4.48
(q, J = 8.3 Hz, J = 8.3 Hz, 2H). 13C NMR (CD3OD, 100 MHz): d
157.8; 157.6; 155.5; 138.9; 130.4; 130.0; 129.7; 129.5; 128.6;
128.5; 127.2; 125.7; 121.5; 120.0; 116.2; 116.2; 111.4; 67.4;
67.0; 66.7. 19F NMR (CD3OD, 375 MHz): d 6.89 (t, J = 7.5 Hz, 3F).
IR (neat): 3331.9; 1606.2; 1515.7; 1505.9; 1455.8; 1427.9;
1238.9; 1169.7; 1118.3; 964.1; 828.9; 667.9; 617.9; 518.9.
1H NMR (CD3OD, 400 MHz): d 7.61 (d, J = 5.3 Hz, 2H); 7.59 (d,
J = 5.2 Hz, 2H); 7.56 (d, J = 8.1 Hz, 1H); 7.48 (d, J = 16.6 Hz, 1H);
7.25–7.19 (m, 5H); 7.12 (d, J = 5.5 Hz, 2H); 7.08 (dd, J = 7.9 Hz,
J = 1.7 Hz, 1H); 7.02 (d, J = 1.5 Hz, 1H); 6.46-6.14 (m, 2H). 13C
NMR (CD3OD, 100 MHz): d 156.6; 149.2; 139.2; 138.4; 137.6;
130.4; 128.8; 128.6; 128.1; 128.0; 127.9; 125.6; 125.4; 123.0;
123.0; 119.7; 114.3.
10.9.1.13. (E,E)-1-Trifluoromethyl-2,5-bis(4-hydroxy)styrylben-
zene (19). Following general procedure A, diphosphonate 28
(0.37 g, 0.83 mmol) was reacted with aldehyde 33 (0.49 g,
2.1 mmol). After work-up, the crude product was purified by
10.9.1.15. (E,E)-1-(4,4,4-Trifluorobutoxy)-2,5-bis(4-hydroxy)sty-
rylbenzene (21). Following general procedure B, diphosphonate
30 (0.50 g, 1 mmol) was reacted with aldehyde 33 (0.59 g,

2480
R. J. A. Nabuurs et al. / Bioorg. Med. Chem. 22 (2014) 2469–2481
2.5 mmol). After work-up, the crude product was purified by col-
umn chromatography (0 ? 2% EtOAc/light petroleum) and the
bis-TBDMS protected styrylbenzene was obtained as a bright yel-
low solid (0.3 g, 0.46 mmol, 46%). 1H NMR (CDCl3, 400 MHz): d
7.53 (d, J = 8.1 Hz, 1H); 7.38 (d, J = 8.4 Hz, 4H); 7.28 (d,
J = 16.5 Hz, 1H); 7.12–7.05 (m, 2H); 7.03 (d, J = 16.2 Hz, 1H); 6.95
(d, J = 1.1 Hz, 1H); 6.92 (d, J = 16.3 Hz, 1H); 6.86-6.80 (m, 4H);
4.13 (t, J = 6.0 Hz, 2H); 2.46-2.31 (m, 2H); 2.20-2.10 (m, 2H); 0.99
(s, 18H); 0.21 (s, 12H). 13C NMR (CDCl3, 100 MHz): d 155.9;
155.6; 155.4; 137.9; 131.3; 130.6; 128.7; 128.3; 127.7; 127.6;
126.5; 126.5; 126.1; 120.9; 120.4; 120.3; 119.6; 109.6; 66.6;
31.1; 30.8; 29.4; 25.7; 22.4; 22.3; 18.2; -4.4. 19F NMR (CDCl3,
375 MHz): d 11.39 (t, J = 10.9 Hz, 3F). IR (neat): 1599.9; 1508.7;
1471.7; 1250.8; 1166.6; 1153.9; 1027.7; 908.7; 833.2; 798.6;
779.1; 700.5; 661.8; 623.5; 531.4; 504.4.
1H NMR (acetone-d6, 400 MHz): d 7.80-7.71 (m, 4H); 7.54 (d,
J = 8.0 Hz, 1H); 7.39 (d, J = 16.6 Hz, 1H); 7.26-7.16 (m, 3H); 7.13
(d, J = 16.4 Hz, 1H); 7.10–6.98 (m, 3H); 3.90 (s, 6H). 13C NMR (ace-
tone-d6, 100 MHz): d 156.2; 139.0; 132.5; 132.2; 131.9; 131.1;
129.0; 127.8; 127.6; 127.4; 125.8; 125.7; 125.6; 125.5; 125.0;
124.1; 119.3; 114.3; 113.9; 56.6; 56.6. 19F NMR (acetone-d6,
375 MHz): d 7.62 (s, 3F); 7.60 (s, 3F). IR (neat): 3523.0; 1615.5;
1512.0; 1501.5; 1428.0; 1327.6; 1274.3; 1261.6; 1119.8; 1058.0;
1017.1; 963.1; 823.7; 667.6; 645.9; 569.8; 544.1.
10.9.1.18. (E,E)-1-Methoxy-2,5-bis(3-trifluoromethyl-4-methoxy)
styrylbenzene (24). Following general procedure B, diphospho-
nate 25 (0.41 g, 1 mmol) was reacted with aldehyde 38 (0.51 g,
2.5 mmol). After work-up, the crude product was purified by col-
umn chromatography (0 ? 10% EtOAc/light petroleum) and product
24 was obtained as a bright yellow solid (0.14 g, 0.27 mmol, 27%).
1H NMR (DMSO-d6, 400 MHz): d 7.97-7.83 (m, 3H); 7.81 (dd,
J = 7.8 Hz, J = 1.7 Hz, 1H); 7.76 (d, J = 1.7 Hz, 1H); 7.65 (d,
J = 8.1 Hz, 1H); 7.46 (d, J = 16.8 Hz, 1H); 7.40–7.20 (m, 6H); 3.93
(s, 3H); 3.92 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): d 156.1;
137.9; 131.8; 131.5; 130.0; 129.7; 129.2; 128.8; 127.8; 127.3;
127.0; 126.6; 125.6; 125.1; 122.4; 122.2; 120.7; 119.3; 117.5;
117.2; 114.6; 113.4; 109.1; 56.3; 56.2.
The silylated bis-styrylbenzene (0.3 g, 0.46 mmol) was depro-
tected according to general procedure C. After work-up, the crude
product was purified using column chromatography (10 ? 22.5%
EtOAc/light petroleum) and product 21 was obtained as a bright
yellow solid (0.12 g, 0.27 mmol, 58%).
1H NMR (CD3OD, 400 MHz): d 7.50 (d, J = 8.1 Hz, 1H); 7.38 (d,
J = 8.6 Hz, 2H); 7.34 (d, J = 8.6 Hz, 2H); 7.24 (d, J = 16.5 Hz, 1H);
7.11–7.02 (m, 4H); 6.92 (d, J = 16.3 Hz, 1H); 6.80–6.75 (m, 4H);
4.13 (t, J = 6.0 Hz, 2H); 2.50–2.35 (m, 2H); 2.16–2.06 (m, 2H). 13C
NMR (CD3OD, 100 MHz): d 158.4; 158.2; 157.3; 139.4; 131.1;
130.4; 129.7; 129.5; 128.9; 128.6; 127.3; 127.1; 126.6; 121.1;
120.4; 116.5; 110.8; 67.8; 32.1; 31.8; 31.5; 31.2; 23.5; 23.4; 23.4.
19F NMR (CD3OD, 375 MHz): d 3.48 (t, J = 11.3 Hz, 3F). IR (neat):
3325.9; 1605.69; 1593.7; 1515.5; 1505.9; 1447.9; 1385.8;
1338.1; 1241.0; 1171.3; 1026.0; 961.8; 826.7; 621.1; 519.8.
19F NMR (DMSO-d6, 375 MHz): d 17.26 (s, 3F); 17.2 (s, 3F). IR
(neat): 1615.6; 1510.4; 1505.8; 1463.9; 1328.5; 1260.0; 1118.3;
1054.9; 1021.0; 959.3; 818.0; 667.8; 645.5; 541.1.
Acknowledgments
This research was performed within the framework of CTMM,
the Center for Translational Molecular Medicine (www.ctmm.nl),
project LeARN (grant 02N-101).
10.9.1.16. (E,E)-1-(Nonafluoro-tert-butoxy)-2,5-bis(4-hydroxy)
styrylbenzene (22). Following general procedure B, diphospho-
nate 32 (0.55 g, 0.89 mmol) was reacted with aldehyde 33
(0.53 g, 2.2 mmol). After work-up, the crude product was purified
by column chromatography (0 ? 2% EtOAc/light petroleum) and
the bis-TBDMS protected styrylbenzene was obtained as a bright
yellow solid (0.22 g, 0.28 mmol, 32%). 1H NMR (CDCl3, 400 MHz):
d 7.27–7.20 (m, 4H); 7.20–7.12 (m, 2H); 6.90–6.81 (m, 2H); 6.79–
6.73 (m, 2H); 6.72–6.64 (m, 4H); 7.48 (d, J = 8.2 Hz, 1H); 0.84 (s,
1H); 0.06 (s, 6H); 0.06 (s, 6H). 13C NMR (CDCl3, 100 MHz): d
155.8; 155.8 150.5; 137.9; 130.5; 130.5; 130.2; 130.0; 129.3;
127.9; 127.8; 126.1; 125.3; 124.2; 120.5; 119.0; 31.9; 29.4; 25.7;
ꢁ4.4. 19F NMR (CDCl3, 375 MHz): d 8.81 (s, 9F). IR (neat): 1600.0;
1508.4; 1472.0; 1249.8; 1169.5; 1155.1; 1124.8; 998.7; 965.7;
904.5; 833.8; 779.5; 726.0; 700.6; 537.4; 504.5. The bis-TBDMS
protected styrylbenzene was treated with TBAF according to gen-
eral procedure C. The crude product was subjected to column chro-
matography (10 ? 25% EtOAc/light petroleum) and product 22
was obtained as a bright yellow solid (72 mg, 0.13 mmol, 47%).
1H NMR (CD3OD, 400 MHz): d 7.72 (d, J = 8.3 Hz, 1H); 7.46–7.34
(m, 5H); 7.31 (s, 1H); 7.22 (d, J = 16.5 Hz, 1H); 7.05 (d, J = 16.4 Hz,
1H); 7.10 (d, J = 12.4 Hz, 1H); 6.96–6.90 (m, 1H); 6.82–6.77 (m,
4H). 13C NMR (CD3OD, 100 MHz): d 159.0; 158.9; 151.6; 139.6;
131.8; 131.7; 130.9; 130.1; 129.9; 129.3; 129.1; 127.4; 125.5;
125.1; 123.0; 120.1; 119.8; 118.7; 116.7; 116.6. 19F NMR (CD3OD,
375 MHz): d 10.93 (s, 9F). IR (neat): 3332.1; 1606.3; 1515.6;
1250.3; 1171.8; 1123.0; 1000.3; 965.1; 829.3; 727.4; 668.0; 529.1.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.054.
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