Synthesis and in vitro evaluation of fluorinated styryl benzazoles as amyloid-probes

Article abstract of DOI:10.1016/j.bmc.2011.09.065

The formation of proteinaceous aggregates is a pathognomonic hallmark of several neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. To date, the final diagnostic for these diseases can only be achieved by immunostaining of post-mortem brain tissues with the commonly used congo red and Thioflavin T/S amyloid-dyes. The interest in developing amyloid-avid radioprobes to be used for protein aggregates imaging by positron emission tomography has grown substantialy, due to the promise in assisting diagnosis of these disorders. To this purpose, the present work describes the synthesis and characterization of four novel fluorinated styryl benzazole derivatives 1-4 by means of the Wittig reaction, as well as their in vitro evaluation as amyloid-probing agents. All compounds were obtained as mixtures of geometric E and Z isomers, with the preferable formation of the E isomer. Photoisomerization reactions allowed for the maximization of the minor Z isomers. The authentic 1-4E/Z isomers were isolated after purification by column chromatography under dark conditions. Profiting from the fluorescence properties of the different geometric isomers of 1-4, their binding affinities towards amyloid fibrils of insulin, α-synuclein and β-amyloid peptide were also measured. These compounds share similarities with Thioflavin T, interacting specifically with fibrillary species with a red-shift in the excitation wavelengths along with an increase in the fluorescence emission intensity. Apparent binding constants were determined and ranged between 1.22 and 23.96 μM^-1. The present data suggest that the novel fluorinated styryl benzazole derivatives may prove useful for the design of ¹⁸F-labeled amyloid radioprobes.

Full text of DOI:10.1016/j.bmc.2011.09.065

Bioorganic & Medicinal Chemistry 19 (2011) 7698–7710
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of fluorinated styryl benzazoles
as amyloid-probes
Goreti Ribeiro Morais a, , Hugo Vicente Miranda b, , Isabel C. Santos a, Isabel Santos a, Tiago F. Outeiro b,c,d,
,
⇑
Antonio Paulo a,
⇑
a Unidade de Ciências Químicas Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Estrada Nacional 10, 2686 953 Sacavém, Portugal
b Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Av. Prof. Egas Moniz, 1649 028 Lisboa, Portugal
c Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
d Department of Neurodegeneration and Restaurative Research, University Medizin Göttingen, Waldweg 33, 37073 Göttingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2011
Revised 23 September 2011
Accepted 28 September 2011
Available online 18 October 2011
The formation of proteinaceous aggregates is a pathognomonic hallmark of several neurodegenerative
disorders such as Alzheimer’s and Parkinson’s diseases. To date, the final diagnostic for these diseases
can only be achieved by immunostaining of post-mortem brain tissues with the commonly used congo
red and Thioflavin T/S amyloid-dyes. The interest in developing amyloid-avid radioprobes to be used
for protein aggregates imaging by positron emission tomography has grown substantialy, due to the
promise in assisting diagnosis of these disorders. To this purpose, the present work describes the synthe-
sis and characterization of four novel fluorinated styryl benzazole derivatives 1–4 by means of the Wittig
reaction, as well as their in vitro evaluation as amyloid-probing agents. All compounds were obtained as
mixtures of geometric E and Z isomers, with the preferable formation of the E isomer. Photoisomerization
reactions allowed for the maximization of the minor Z isomers. The authentic 1–4E/Z isomers were iso-
lated after purification by column chromatography under dark conditions. Profiting from the fluorescence
properties of the different geometric isomers of 1–4, their binding affinities towards amyloid fibrils of
Keywords:
Benzazoles
Styryl derivatives
Wittig reaction
Fluorescence
Amyloid fibrils
Binding affinity
insulin,
a-synuclein and b-amyloid peptide were also measured. These compounds share similarities
with Thioflavin T, interacting specifically with fibrillary species with a red-shift in the excitation wave-
lengths along with an increase in the fluorescence emission intensity. Apparent binding constants were
determined and ranged between 1.22 and 23.96 l
Mꢀ1. The present data suggest that the novel fluori-
nated styryl benzazole derivatives may prove useful for the design of 18F-labeled amyloid radioprobes.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
late prior to the overt manifestation of the typical clinical symp-
toms of these diseases, would enable early diagnosis and the
Neurodegenerative diseases, such as Alzheimer’s (AD) or Par-
kinson’s diseases (PD), are highly debilitating conditions affecting
millions of people worldwide. These disorders are commonly
known as ‘protein misfolding disorders’ as they are characterized
by the accumulation of insoluble protein aggregates. Although
the molecular processes underlying these pathologies are still un-
known, b-amyloid (Ab) plaques and tau neurofibrillary tangles in
evaluation of the effects of anti-amyloidogenic based therapies.
Positron emission tomography (PET) is among the best suited
molecular imaging modalities to achieve such a goal, if available
amyloid-avid radioprobes can cross the blood brain barrier. Several
planar and nonionic molecules (Fig. 1) were designed, labeled with
the PET radioisotopes 11C (t1/2 = 20 min) and 18F (t1/2 = 110 min),
and clinically evaluated in humans, in order to generate radiop-
robes for the in vivo targeting of Ab plaques in the brain. Their
molecular target is not the Ab peptide sequence per se, but the gen-
eric b-sheet structure forming amyloid folds, which can also be
AD, and Lewy bodies mainly composed of alpha-synuclein (
a-
syn) in PD, are thought to play a central role in these disorders.
To date, definitive confirmation of AD or PD is only possible
upon post-mortem histopathologic studies, using antibodies and
dyes such as Thioflavin T (ThT) (Fig. 1) or Congo red that stain such
deposits.1 Thus, the in vivo visualization by noninvasive molecular
imaging techniques of the deposits, which are thought to accumu-
formed by a range of other proteins (e.g.,
a
-syn, and insulin).2
Hence, the amyloid binding profile of the compounds is a crucial
issue in the development of PET radiotracers for in vivo imaging
of amyloid aggregates.
The benzothiazole derivative [11C]PIB (Fig. 1), designated com-
monly as Pittsburgh compound B (PIB), is the best characterized
PET radiotracer for in vivo b-amyloid imaging.3 However, the short
half-life of 11C limits its use to centers with an on-site cyclotron.
⇑
Corresponding authors. Tel.: +351 21 9946196; fax: +351 21 994 6185 (A.P).
E-mail addresses: touteiro@gmail.com (T.F. Outeiro), apaulo@itn.pt (A. Paulo).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.065

G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
7699
Figure 1. Chemical structures of relevant Ab binding agents.
Such limitation prompted several authors to search for alternative
compounds radiolabeled with the long-lived 18F, namely radioflu-
orinated stilbenes and styrylpyridines (see compounds IV and V
in Fig. 1).4 Like benzothiazoles, these styryl derivatives are planar
Reaction of
8
and
9
with triphenylphosphine in refluxing
toluene afforded, in excellent yields, benzothiazolylmethyltri-
phenylphosphonium chloride 11 and benzoxazolylmethyltri-
phenylphosphonium chloride 12, respectively. The more polar
benzimidazolylmethyltriphenylphosphonium chloride 13 was ob-
tained in a similar way but the reaction was carried out in acetoni-
trile. The benzoxazolylmethyltriphenylphosphonium chloride 12
is a new compound that has been fully characterized by NMR tech-
niques (1H, 13C and 31P), and by X-ray diffraction analysis (Fig. 3).
For the benzothiazole- and benzoxazole-containing com-
pounds, the Wittig reaction leading to the styryl benzazoles (see
below) could be performed using the O-tosylate derivative 1525 ob-
tained from the commercially available 14 (Scheme 2). By contrast,
for the benzimidazoles, the Wittig reaction was performed using
the O-tert-butylcarbonate derivative 16, prepared as described in
Scheme 2. After the condensation reaction, removal of the base-
resistant O-protective group was followed by the preparation of
tosylated derivatives, as described below. These strategies allowed
for a convergent synthesis of the desired fluorinated styryl benzaz-
ole derivatives 1–4 via nucleophilic substitution (Schemes 2–4).
Treatment of 11 and 12 with potassium tert-butoxide in ben-
zene gave the respective phosphoranes 17 and 18. Like the previ-
ously reported 17,21 the new phosphorane 18 is not very stable
and was prepared just prior to use. The Wittig reactions of 17
and 18 with 15 afforded the styrylbenzothiazole 19 and styryl-
benzoxazole 20 in 89% and 65% yields, respectively (Scheme 3).
Compounds 19 and 20 were isolated as a mixture of E (19-E and
20-E) and Z (19-Z and 20-Z) isomers in a ratio of around 95:5. Z
and E geometry was confirmed by the proton–proton coupling con-
stants (JH,H) of 12.3 and 16.5 Hz that were found for the respective
olefinic protons. Reaction of 19-E/19-Z and 20-E/20-Z with tetrabu-
tylammonium fluoride (TBAF) in refluxing THF gave the respective
mixtures of 1-E/1-Z and of 2-E/2-Z in good yield.
The synthesis of the structurally related styryl benzimidazole 3
involved the reaction of the corresponding phosphorane, prepared
in situ by treating 13 with sodium methoxide in dry methanol with
the Boc-protected benzaldehyde 16 (Scheme 4). This reaction affor-
ded the styryl benzimidazole derivative 21 in good yield, which was
obtained as a mixture of the geometric isomers 21-E and 21-Z in a E/Z
ratio of 70:30. Hydrolysis of the Boc-protecting group of compounds
21-E/21-Z (mixture) under acid catalysis, followed by neutralization
of the reaction mixture with triethylamine and by a 30 min reaction
with p-toluenesulfonyl chloride, afforded authentic 22-E in a yield of
32%. The low yield of 22 was due to the short reaction time used to
prevent the N-tosylation of the benzimidazole ring. Under these
conditions only the less steric hindered E isomer was transformed
into the tosylate derivative. The reaction of 22-E with TBAF in reflux-
ing THF gave the final fluorinated styryl benzimidazole 3, which was
obtained as a mixture of E/Z isomers with a poor yield of 16%. This
low yield could be explained by competitive nucleophilic alkylation
reactions involving the deprotonated amine of the benzimidazole
ring, which led to the formation of unidentified by-products.
molecules with extended
p-delocalized systems, which enable
their insertion between the b sheets of amyloid aggregates. Some
of these styryl derivatives were able to target in vivo Ab plaques
but exhibited to some extent nonspecific retention in the brain.
Therefore, it is important to identify better performing PET probes
for selective in vivo imaging of amyloid deposits.
We focused on fluorinated styryl benzazole derivatives as small,
planar and p
-delocalized compounds to design 18F-labeled probes
for PET imagingof amyloid aggregates. Herein, we report the synthe-
sis and characterization of four novel fluorinated styryl-containing
compounds (1–4) of the benzothiazole, benzoxazole and benzimid-
azole type (Fig. 2), which were isolated as the respective E and Z iso-
mers. We also report on the fluorescence properties of the different
geometric isomers of 1–4, as well as on their binding affinities to-
wards amyloid aggregates of insulin, a-syn and Ab(1–42) peptide,
which were measured by in vitro binding assays using fluorescence
spectroscopy. We assessed the influence of the different heteroat-
oms (N vs O vs S) on the affinity of styryl-benzazole derivatives to-
wards different types of amyloid structures, aiming to have a first
insight into the relevance of these compounds for the design of
18F-labeled probes for in vivo targeting of amyloid aggregates.
2. Results and discussion
2.1. Synthesis
Several approaches can be undertaken to prepare styryl benzaz-
ole derivatives, namely those based on the Knoevenagel, Horner–
Wadsworth–Emmons and Wittig reaction.5–20 In this work, the ole-
fin bond of the styryl benzazole derivatives 1–4 was fashioned via
the Wittig reaction of benzazolylmethylenetriphenylphosphoranes
with substituted benzaldehydes. This involved the synthesis of ben-
zazolylmethyltriphenylphosphonium salts, 11–13, as key interme-
diates (Scheme 1) to generate the corresponding phosphoranes.
Compounds 11 and 13 have been previously used for the synthesis
of styryl derivatives.6,21,22 The synthesis of 11–13 started with the
preparation of the respective 2-chloromethylbenzazoles 8–10 by
condensation of the commercially available o-substituted phenyl
amines 5–7 with chloroacetic acid, using suitable catalysts.6,23,24
Figure 2. Structure of the new fluorinated styryl benzazole derivatives.

7700
G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
Scheme 1. Synthesis of the phosphonium derivatives.
tosylation of 23-E/23-Z afforded 24-E/24-Z compounds in a similar
manner as described for 22-E. Due to the absence of competitive N-
alkylation reactions, compounds 24-E/24-Z were obtained with a
yield of 69%. Finally, the desired fluorinated styryl N-methyl benz-
imidazole 4 was obtained in excellent yield (80%) when 24 was re-
acted with TBAF in refluxing THF.
To improve the final yield of 3 we have explored other strategies.
First, we have studied the synthesis of the O-tosylated intermediate
22 by the base-free direct condensation of 2-methylbenzimidazole
with 15 at 200 °C in a sealed tube, to overcome the competitive N-
tosylation.8 However, this reaction did not proceed with the forma-
tion of 22 as shown by the 1H NMR analysis of the reaction crude. An-
other alternative consisted on the reaction of o-phenylidenediamine
7 with the fluorinated cinnamic acid derivative 25, which was ob-
tained from 15 in three steps (Scheme 5).26 This condensation reac-
tion, catalyzed by PPA, also formed 3 in low yield (10–20%) and,
therefore, was not a better alternative for the synthesis of this com-
pound. This bad outcome was not improved by using a larger molar
excess of 25, longer reaction times or higher temperatures (200 °C).
Figure 3. ORTEP view of the cation of 2-benzoxazolylmethyltriphenylphosphoni-
um chloride 12.
2.2. Photochemical interconversion of E/Z isomers
As described, the final fluorinated styryl-benzazoles 1–4 were
obtained as a mixture of geometric E/Z isomers, being dominant
the E isomer. The E/Z ratio of the different styryl-benzazoles de-
scribed in this work changed when the compounds were kept in
solution and exposed to day light, in agreement with the ability of
styryl benzazoles to undergo photoisomerization.15,27 For instance,
1H NMR analysis of a solution of 4 in chloroform showed that the ini-
tial 4-E/4-Z ratio of 2.6:1 changed to a 1:19 ratio, after 2 days of light
exposure. After this time the E/Z ratio reached equilibrium. A similar
behavior was observed for the other fluorinated styryl-benzazoles
1–3 in chloroform solution. For all compounds, there is a preference
towards the formation of the Z isomer by photoisomerization. Thus,
the formation of the minor Z isomer of 1–4 was maximizedby expos-
ing the mixtures E-1–4/Z-1–4 to light for 48 h. With the exception of
3-Z, each isomer was purified by column chromatography under
dark conditions, as described in the experimental section.
Scheme 2. Synthesis of O-substituted benzaldehyde derivatives.
Treatment of the mixture 21-E/21-Z with 1.5 equiv of methyl io-
dide under phase transfer conditions9 gave the respective N-meth-
ylated analogs 23-E/23-Z in quantitative yield (Scheme 4). The O-
Scheme 3. Synthesis of fluorinated styryl benzazoles 1 and 2.

G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
7701
Scheme 4. Synthesis of fluorinated styryl benzimidazoles 3 and 4.
Scheme 5. Alternative synthesis of the fluorinated styryl benzimidazole 3.
2.3. Intrinsic fluorescence characteristics and interaction with
amyloid fibrils
shift. On the other hand, we observed a blue-shift upon binding
to insulin fibrils of compounds 4-Z and 4-E, which did not suffer
any change on their emission wavelength following the interaction
Profiting from the intrinsic fluorescence properties of the fluori-
with a-syn. Unlike 3-E, all the other fluorinated styryl-benzazoles
nated styryl-benzazole derivatives 1–4, we tested their binding
undergo a blue-shift in emission upon binding to Ab(1–42) fibrils.
To directly measure the fibril-binding properties of the com-
pounds, fluorescence intensity binding assays were performed by
ability towards amyloid fibrils formed by insulin,
a-syn and
Ab(1–42), all of which are known to form typical amyloid aggre-
gates with common ‘cross-b’ architecture. Fibrillization was in-
duced by standard procedures and the interaction of authentic E
and Z isomers of 1–4 with the resulting aggregated species was
studied in order to assess the influence of isomerism on the bind-
ing abilities of the compounds. This influence has been previously
assessed for styrylbenzene derivatives,28 but, so far, no similar
studies were reported for styryl-benzazole derivatives. The isomer
3-Z was not evaluated in this study, as its counterpart 3-E was ob-
tained in a quite poor yield and its photoisomerization did not pro-
vide the required amount of 3-Z.
The authentic E and Z isomers share similar fluorescence prop-
erties, displaying similar maximum excitation and emission wave-
lengths for the interaction with monomers and fibrils (Table 1).
Upon interaction with fibrils, we observed an increase in fluores-
cence intensity that was proportional to the concentration of amy-
loid fibrils (Fig. 4, Table 2), in agreement to what is observed upon
ThT binding to amyloid fibrils.29,30 For all the compounds, the
interaction with the different fibrillary species led to a red-shift
in the excitation wavelength. By contrast, the emission properties
varied slightly depending on the type of amyloid fibrils. The inter-
action of the E and Z isomers of 1 and 2 and isomer 3-E with insulin
fibrils caused a red-shift in their emission maximum, while for the
using a fixed concentration of fibrils (3.5 to 11.35
lM) and varying
concentrations of ligands (2.25 to 13.5 M) (Fig. 5). The binding
l
constants were obtained by single site saturated curve fitting to
the fluorescence data as a function of the ligand concentration.
The calculated Kd values are in the
l
M range spanning between
Mꢀ1 (Table 3) while for ThT the range is between
Mꢀ1. These apparent Kd values are in the same or-
1.22 and 23.96
2.43 and 11.93
l
l
der of magnitude as those previously reported for ThT, the most
widely used dye for cross b-sheet structure detection.31,32 More-
over, the Kd values of compounds 1–4 are in the same order of
magnitude as the values reported for other compounds with po-
tential as amyloid-avid PET radioprobes, when obtained directly
by intrinsic fluorescence intensity binding assays.33,34 There is an
influence of the nature of the heteroatom and type of geometric
isomer on the binding abilities of the compounds but a clear trend
could not be identified due to a few exceptions. In general, the ben-
zothiazole derivatives tend to exhibit the lowest affinity towards
the different types of amyloid fibrils. However, among all the
tested compounds the styryl-benzothiazole 1-E has shown the
highest affinity for
a-syn aggregates. For the different amyloid fi-
brils the E isomers show a higher affinity than the Z counterparts,
being the affinity of 4-Z and 4-E towards aggregates of Ab(1–42)
the unique exception to this trend. However, the Kd values found
same compounds the interaction with
a-syn fibrils led to a blue

7702
G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
Table 1
Fluorescence maximum excitation and emission wavelengths of the E and Z geometric isomers of compounds 1–4 in the presence of monomeric and fibrillary insulin,
Ab(1–42) and unbound dye (free)
a-syn, and
Insulin
a
-syn
Ab(1–42)
Free
Monomer
kexcitation kemission
Fibrils
Monomer
Fibrils
kexcitation kemission
Monomer
kexcitation kemission
Fibrils
kexcitation kemission
kexcitation kemission
kexcitation kemission
kexcitation kemission
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
(nm)
1-
2-
3-
Z
538
400
Z
508
384
416
1-E
542
384
2-E
508
372
4-Z
538
416
430
538
576
430
416
572
538
416
430
544
518
430
400
518
542
400
418
542
506
418
400
510
508
384
416
508
522
416
384
526
508
384
400
508
498
400
394
500
510
394
400
510
492
400
384
492
E
516
490
430
435
534
516
454
372
522
484
384
384
488
482
392
372
488
484
380
386
496
488
384
394
490
for each isomer are of the same order of magnitude and, most
probably, the occurrence of geometric isomerism should not affect
the amyloid-targeting ability of the reported compounds.
cally with fibrillary species with a red-shift in the excitation wave-
lenghts along with an increase in the fluorescence emission
intensity. Despite some influence of the azole heteroatom and isom-
erism, the binding of all compounds to different amyloid fibrils is
Although the interaction of the different compounds with
monomers does not result in an increase in the fluorescence inten-
sity signal, we also performed similar titration experiments with a
nonamiloydogenic protein (BSA). We have found that no hyper-
bolic or linear fitting to the experimental data is possible
(Fig. S1), given that increasing amounts of dyes concentration do
not lead to an increase in the fluorescence intensity. This suggests
that 1–4 undergo a specific interaction with amyloid fibrils.
In summary, the styryl benzazole derivatives 1–4 offer the pos-
sibility to bridge the gap between in vitro and in vivo studies
involving the targeting of amyloid aggregates. This is due to their
intrinsic fluorescence properties and results also from the possibil-
ity to obtain the radiofluorinated counterparts, readily available
from the tosylated precursors reported herein. Importantly, the
lipophilic character of compounds 1–4, displaying logP values
ranging from 3.95 and 4.83, warrants that the compounds should
cross the blood brain barrier, a crucial issue on their ability to reach
intracerebral deposits of amyloid. By themselves, they do not
emerge as alternative amyloid-fluorescence sensors for the
in vitro characterization of amyloid aggregates, as their fluores-
cence properties are less favorable than those presented by re-
ported fluorescent probes.35 Nevertheless, the compounds
reported herein offer the advantage of a modular synthesis that en-
ables for a versatile introduction of substituents, namely electron-
donating groups that can be appended at different positions of the
benzazole ring to improve the fluorescence and amyloid-binding
properties of the compounds.36
characterized by Kd values in the
that these compounds display rather similar binding properties to
the different insulin, -syn and Ab(1–42) fibrillary species, in agree-
ment with the ThT broad cross b-sheet structure recognition.
In conclusion, the present study suggests that the newly syn-
thesized styryl benzazole derivatives hold promise as amyloid fi-
bril detection agents that, once labeled with the positron-emitter
18F, may be explored to design amyloid-avid radioprobes for
in vivo detection of amyloid structures typical of neurodegenera-
tive diseases such as AD or PD.
lM range. These findings indicate
a
4. Materials and methods
All chemicals and solvents were of reagent grade and were used
without purifications unless stated otherwise. NMR spectra were
recorded on a Varian Unity 300 NMR spectrometer at the frequen-
cies of 300 MHz (1H), 75 MHz (13C), 121 MHz (31P) and 282 MHz
(
19F). Chemical shifts are reported in parts per million. 1H and
13C chemical shifts were referenced with the residual solvent res-
onances relative to tetramethylsilane. 31P chemicals shifts were
referenced with external 85% H3PO4 solution. 19F chemical shifts
were referenced externally to
a, ,
a0 a00-trifluorotoluene (0.05% in
C6D6; d = ꢀ63.3). Thin-layer chromatography (TLC) was performed
on plates of precoated silica plates 60 F254 (Merck). Visualization of
the plates was carried out using UV light (254 and 365 nm) and/or
iodine chamber. Gravity column chromatography was carried out
on silica gel (Merck, 70–230 mesh).
3. Conclusion
All geometric isomers of the styryl benzazole derivatives were
purified by column chromatography under dark. Only authentic E
and Z isomers of 1–4 were used to evaluate their fluorescence
properties and their binding to the different amyloid fibrils.
The lipophilicity of the final compounds 1–4 was estimated by
calculating the respective octanol/water partition coefficient (log-
Po/w). The log of the octanol/water partition coefficient (logPo/w)
of compounds 1–4 was calculated using Molecular Operating Envi-
ronment (MOE) software (Chemical Computing Group, Canada)
from the structures optimized with a modified MMFF94.37 The log-
P(o/w) was calculated by MOE from a linear atom type model (with
r2 = 0.931, RMSE = 0.393 on 1,827 molecules).
A series of novel fluorinated styryl benzazole derivatives have
been synthesized as potential amyloid-binding agents. Formation
of the styryl group has been successfully fashioned by the Wittig
reaction, which represents an unprecedented approach in the case
of styrylbenzoxazole derivatives. The final fluorinated styryl-ben-
zazole derivatives 1–4 were obtained as a mixture of geometric
isomers, being the E isomer formed preferably. Compounds 1–4
are prone to photoisomerize in solution but the extension of isom-
erization depends on the nature of the azole ring. E-benzimidazole
styryl derivatives fully isomerize into the Z isomer while the coun-
terpart benzothiazole and benzoxazole derivatives undergo only
partial isomerization. Taking advantage of the Z/E photoisomeriza-
tion ability of these compounds, the different E and Z isomers of
compounds 1–4 could be isolated and their binding capabilities to-
wards different types of amyloid fibrils were evaluated in vitro.
Like the benzothiazole dye ThT, the newly synthesized fluori-
nated derivatives interacted with different amyloid fibrils. These
compounds share similarities with thioflavin T, interacting specifi-
4.1. 2-Benzoxazolylmethyltriphenylphosphonium chloride (12)
A solution of 9 (173 mg, 1.0 mmol) and triphenylphosphine
(263 mg, 1.0 mmol) in toluene (1.6 mL) was refluxed overnight.
Thereafter, the formed precipitate was filtered off and was washed
several times with ethyl acetate to give 12 (340 mg, 79%)—1H NMR
(CD3OD, 300 MHz): d = 5.76 (d, J = 15.4 Hz, 2H), 7.48–8.10 (m,

G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
7703
Figure 4. Fluorescence spectra of the E and Z geometric isomers of the fluorinated styrylbenzazole derivatives 1–4 (9
lM) in the absence/presence of monomeric and
fibrillary
a-syn, insulin and Ab(1–42) species (2.25 lM). The excitation and emission spectra were taken with fixed maximum excitation and emission wavelengths for the
different species according to Table 1.
19H); 13C NMR (CD3OD, 75 MHz): d = 111.65, 118.46, 119.63,
121.05, 126.30, 127.34, 131.39, 131.56, 135.14, 135.28, 136.73,
136.77, 141.88; 31P NMR (CD3OD, 121 MHz): d = 22.77; ES+ MS
(C26H21ONP)Cl (429) m/z 394 (100) [M]+; HRMS (ES+) [M]+ calcd
for C26H21ONP: 394.1355, found: 394.1358
ture was extracted with sat. sol. NaHCO3 (50 mL). The organic
phase was dried over MgSO4, filtered and the filtrate was concen-
trated by vacuum evaporation. Column chromatography on silica
gel (n-hexane/EtOAc 2:1) gave 15 (1.34 g, 73%) as a light yellow so-
lid—Rf = 0.58 (n-hexane/EtOAc 1:1); 1H NMR (CDCl3, 300 MHz):
d = 2.36 (s, CH3, 3H), 2.97 (s, NCH3, 3H), 3.69 (t, J = 5.7 Hz, OCH2,
2H) 4.17 (t, J = 5.7 Hz, NCH2, 2H), 6.55 (d, 3J = 8.7 Hz, 1H), 7.20 (d,
3J = 8.7 Hz, 1H), 7.64 (d, 3J = 8.7 Hz, 2H), 9.71 (s, CHO, 1H); 13C
NMR (CDCl3, 75 MHz): d = 21.59, 39.23, 50.84, 66.44, 111.06 (2C),
125.87, 127.73 (2C), 129.81 (2C), 131.91 (2C), 132.39, 145.09,
152.61, 190.20; ES+ MS C17H19O4NS (333.04) m/z 334.2 (5)
[M+H]+; HRMS (ES+) [M+H]+ calcd for C18H20O4NS: 334.1108,
found: 334.1104.
4.2. N-Methyl-N-(2-tosyloxyethyl)-4-aminobenzaldehyde (15)
A solution of N-methyl-N-(2-hydroxyethyl)-4-aminobenzalde-
hyde (14) (1.00 g, 5.6 mmol) and Et3N (1.15 mL, 7.3 mmol) in
dichloromethane (50 mL) was stirred for 10 min at rt. Thereafter,
p-toluenesulfonyl chloride (1.6 g, 8.4 mmol) was added and the
reaction mixture was stirred for more 16 h. Then, the reaction mix-

7704
G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
Fig. 4 (continued)
4.3. N-Methyl-N-((2‘-O-tert-butylcarbonate)ethyl)-4-
aminobenzaldehyde (16)
A solution of N-Methyl-N-(2-hydroxyethyl)-4-aminobenzalde-
hyde (14) (1.0 g, 5–6 mmol), di-tert-butyl-dicarbonate (2.0 g,
9.2 mmol) and DMAP (33 mg, 0.3 mmol) in THF (30 mL) was stir-
red at room temperature for 2 h. Then, MeOH (50 mL) was added
and the reaction mixture was stirred for 30 min more. Thereafter,
the solvent was removed and the residue was submitted to column
chromatography on silica gel (n-hexane/EtOAc 1:1) to give 16
(1.5 g, 95%)—Rf = 0.85 (n-hexane/EtOAc 1:1); 1H NMR (CDCl3,
300 MHz): d = 1.39 (s, 9H), 3.05 (s, 3H), 3.67 (t, J = 6.0 Hz, 2H),
4.22 (t, J = 6.0 Hz, 2H), 6.71 (d, 3J = 8.8 Hz, 2H), 7.68 (d, 3J = 8.8 Hz,
2H), 9.70 (s, 1H); 13C NMR (CDCl3, 75 MHz): d = 27.39 (3C), 38.55,
50.47, 63.06, 82.16, 110.67, 110.86, 125.42, 131.78 (2C), 153.03,
153.11, 189.90; ES+ MS C15H21NO4 (279) m/z 302.1 (100) [M+Na]+.
Table 2
Fluorescence intensity fold increase of E/Z isomers (2.25 lM) in the presence of
3.5 lM of fibrillary insulin, a-syn and Ab(1–42) in comparison to the corresponding
equimolar monomeric specie
Insulin
a-syn
Ab(1–42)
1-Z
1-E
2-Z
2-E
3-E
4-Z
4-E
9.48 0.53
4.48 0.49
1.63 0.17
1.98 0.25
4.50 0.36
2.84 0.12
2.23 0.15
2.98 0.17
3.30 0.23
1.60 0.22
1.98 0.13
2.49 0.16
2.84 0.11
3.63 0.21
11.30 1.40
7.41 0.50
1.28 0.15
2.69 0.29
2.71 0.18
5.9 0.57
5.17 0.27

G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
7705
Figure 5. Fluorescence intensity binding assays in the presence of
(B) 1-E (2.25–9 M), (C) 2-Z (4.5–11.25 M), (D) 2-E (4.5–11.25 M), (E) 3-E (2.25 and 9
7.4. All spectra were recorded at fixed maximum emission and excitation wavelengths for the different species according to Table 1.
a
-syn, insulin and Ab(1–42) fibrillar species (7
l
M) with varying concentrations of (A) 1-Z (4.5–11.25
lM),
l
l
l
l
M), (F) 4-Z (4.5–11.25 M) and (G) 4-E (2.25 and 9 l
l
M) in 50 mM Tris–HCl buffer pH

7706
G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
4.18 (t, J = 5.7 Hz, 2H), 6.54 (d, 3J = 8.7 Hz, 2H), 7.18 (d, EJ = 16.2 Hz,
1H), 7.30–7.45 (m, 7H), 7.67 (d, 3J = 8.7 Hz, 2H), 7.81 (d, 3J = 7.6 Hz,
1H), 7.93 (d, 3J = 7.6 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d = 21.69,
39.12, 50.97, 66.96, 111.89, 117.47, 121.40, 122.35, 123.88,
124.92, 126.24, 127.80, 129.01, 129.81, 132.49, 138.13, 145.00,
149.07; ES+ MS C25H24N2O3S2 (464.60) m/z 487.2 (100) [M+Na]+;
HRMS (ES+) [M+H]+ calcd for C25H25N2O3S2: 465.1301, found:
465.1295.
4.5. 2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benzoxa
zole (20)
Compound 20 was prepared as described above for compound
19, starting from 12 (2.0 g, 4.6 mmol). Its purification was carried
out by column chromatography on silica gel (n-hexane/CHCl3/
EtOAc 3:1:1) to give the mixture of 20-Z and 20-E (550 mg, 65%).
4.5.1. Z-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benz
oxazole (20-Z)
Rf = 0.52 (n-hexane/CHCl3/EtOAc 3:1:1); 1H NMR (CDCl3,
300 MHz): d = 2.35 (s, 3H), 2.95 (s, 3H), 3.66 (t, J = 6.0 Hz, 2H),
4.18 (t, J = 6.0 Hz, 2H), 6.29 (d, 3JZ = 13.0 Hz, 1H), 6.55 (d,
3J = 9.0 Hz, 2H), 6.85 (d, 3JZ = 13.0 Hz, 1H), 7.23 (d, 3J = 7.2 Hz, 2H),
7.28–7.31 (m, 2H), 7.43–7.46 (m, 1H), 7.67–7.72 (m, 3H), 7.82 (d,
3J = 9.0 Hz, 2H); 13C NMR (CDCl3, 75 MHz): d = 21.59, 39.06,
50.97, 66.79, 109.60, 110.28, 111.11 (2C), 111.89, 119.78, 124.29,
124.94, 127.81 (2C), 129.83 (2C), 132.18 (2C), 132.61, 139.81,
142.11, 144.96, 148.82, 149.92, 162.42; ES+ MS C25H24N2O4S
(448.53) m/z 471 (100) [M+Na]+; HRMS (ES+) [M+Na]+ calcd for
Fig. 5 (continued)
4.4. 2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benzothia
zole (19)
To a stirred solution of 11 (1.57 g, 3.5 mmol) in benzene (25 mL)
was added KtBuO (400 mg, 3.6 mmol) at rt. After 3 h, the reaction
mixture was diluted with EtOAc (100 mL) and was extracted with
water (100 mL). The organic phase was dried over MgSO4, filtered
and the filtrate was dried under vacuum. Then, the resulting phos-
phorane (17) and 6 (450 mg, 1.4 mmol) were dissolved in anhy-
drous THF (24 mL) and refluxed overnight. Thereafter, the solvent
was concentrated and the reaction crude was re-dissolved in
CH2Cl2 (100 mL). The organic phase was extracted with sat. sol.
of NaHCO3 (100 mL). The organic extract was dried over MgSO4, fil-
tered and the filtrate was concentrated. The crude product was
submitted to column chromatography on silica gel (n-hexane/
EtOAc 2:1) to give a mixture of 19-Z and 19-E (580 mg, 89%)
C25H24N2O4NaS: 471.1347, found: 471.1336.
4.5.2. E-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benz
oxazole (20-E)
Rf = 0.30 (n-hexane/CHCl3/EtOAc 3:1:1); 1H NMR (CDCl3,
300 MHz): d = 2.38 (s, 3H), 2.94 (s, 3H), 3.66 (t, J = 6.0 Hz, 2H),
4.18 (t, J = 6.0 Hz, 2H), 6.55 (d, 3J = 8.7 Hz, 2H), 6.83 (d, 3JE = 16.5 Hz,
1H), 7.21–7.29 (m, 4H), 7.40 (d, 3J = 8.7 Hz, 2H), 7.46–7.49 (m, 1H),
8.64–7.71 (m, 4H); 13C NMR (CDCl3, 75 MHz): d = 21.76, 39.14,
50.98, 66.65, 108.89, 110.12, 111.87 (2C), 119.32, 123.72, 124.38,
124.68, 127.80 (2C), 129.24 (2C), 129.81 (2C), 132.53, 139.86,
142.06, 139.86, 142.06, 145.00, 149.34, 150.29, 163.78; ES+ MS
4.4.1. Z-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benzo
thiazole (19-Z)
C
25H24N2O4S (448.53) m/z 471 (100) [M+Na]+; HRMS (ES+)
Rf = 0.66 (n-hexane/EtOAc 1:1); 1H NMR (CDCl3, 300 MHz):
d = 2.37 (s, CH3, 3H), 2.92 (s, NCH3, 3H), 3.65 (t, J = 5.7 Hz, 2H),
4.18 (t, J = 5.7 Hz, 2H), 6.53 (d, 3J = 8.5 Hz, 2H), 6.72 (d, ZJ = 12.3 Hz,
1H), 6.93 (d, ZJ = 12.3 Hz, 1H), 7.25–7.44 (m, 6H), 7.70 (d,
3J = 8.5 Hz, 2H), 7.73 (d, 3J = 8.1 Hz, 1H), 7.97 (d, 3J = 8.1 Hz, 1H);
13C NMR (CDCl3, 75 MHz): d = 21.65, 38.96, 51.00, 66.79, 111.52,
120.85, 121.30, 122.72, 123.77, 125.17, 125.99, 127.83, 129.83,
130.85, 132.60, 137.95, 144.95, 148.55; ES+ MS C25H24N2O3S2
(464.60) m/z 487.2 (100) [M+Na]+; HRMS (ES+) [M+H]+ calcd for
[M+Na]+ calcd for C25H24N2O4: NaS 471.1347, found: 471.1336.
4.6. 2-[N-methyl-N-(2‘-O-tert-butylcarbonatethyl)
-4‘-aminostyryl]benzimidazole (21)
To a solution of phosphonium salt 13 (3.4 g, 7.8 mmol) and 16
(860 mg, 3.1 mmol) in anhydrous methanol (32 mL) was added
NaOMe (300 mg, 2.7 mmol) at rt under a nitrogen atmosphere.
Then, the reaction mixture was stirred at 50 °C overnight. At the
end of the reaction, the solvent was removed under vacuum and
the crude was taken upon CH2Cl2 (100 mL). The organic phase
was washed with a sat. sol. of NaHCO3 (100 mL), dried over Na2SO4,
filtered and the filtrate was concentrated. The residue was submit-
ted to column chromatography on silica gel (n-hexane/EtOAc 2:1)
to give the mixture of 21-Z and 21-E (810 mg, 67%)
C
25H25O3N2S2: 465.1301, found: 465.1295.
4.4.2. E-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benzo
thiazole (19-E)
Rf = 0.72 (n-hexane/EtOAc 1:1); 1H NMR (CDCl3, 300 MHz):
d = 2.37 (s, CH3, 3H), 2.93 (s, NCH3, 3H), 3.65 (t, J = 5.7 Hz, 2H),
Table 3
Amyloid binding constants (
l
Mꢀ1) towards aggregates of insulin,
1-E 2-Z
1.79 0.46
a-syn and Ab(1–42) for E and Z geometric isomers of compounds 1–4 and ThT
1-Z
2-E
3-E
4-Z
4-E
ThT
2.54 0.39
11.93 0.94
2.56 0.29
Insulin
-syn
Ab (1–42)
8.59 0.58
7.35 0.42
23.96 3.57
3.24 0.16
7.43 0.29
5.99 0.56
2.28 0.20
3.24 0.16
4.48 0.38
1.54 0.15
3.31 0.34
3.63 0.18
3.86 0.36
1.85 0.24
3.52 0.17
1.22 0.24
1.81 0.31
5.19 0.57
a
1.38 0.33
10.67 1.55

G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
7707
4.6.1. Z-2-[N-methyl-N-(2‘-O-tert-butylcarbonatethyl)-4‘-amino
styryl]benzimidazole (21-Z)
3J = 9.0 Hz, 2H), 7.13–7.26 (m, 6H), 7.47 (d, 3J = 9.0 Hz, 2H), 7.69–
7.72 (m, 1H), 7.74–7.77 (m, 1H), 7.86 (d, 3JE = 15.6 Hz, 1H); 13C
NMR (CDCl3, 75 MHz): d = 27.62 (3C), 38.62, 50.74, 63.40, 82.31,
107.97, 108.82, 109.33, 111.47, 111.79 (2C), 112.40, 118.78,
119.48, 121.86, 122.23, 124.34, 128.75 (2C), 130.31, 135.93,
137.43, 137.99, 143.16, 149.41, 152.11, 153.28; ES+ MS
Rf = 0.54 (n-hexane/EtOAc 2:1); 1H NMR (CDCl3, 300 MHz):
d = 1.45 (s, 9H), 3.02 (s, 3H), 3.65 (t, J = 6.0 Hz, 2H), 4.25 (t,
J = 6.0 Hz, 2H), 6.55 (d, 3JZ = 12.3 Hz, 1H), 6.70 (d, 3J = 8.4 Hz, 2H),
6.89 (d, 3JZ = 12.3 Hz, 1H), 7.18–7.22 (m, 4H), 7.35 (d, 3J = 8.4 Hz,
2H); 13C NMR (CDCl3, 75 MHz): d = 27.68 (3C), 38.68, 50.87,
63.46, 82.37, 111.95 (2C), 116.88, 123.91, 129.93 (2C), 135.61,
148.95, 150.05, 153.33; ES+ MS C23H27N3O3 (393) m/z 394.2 (100)
[M+H]+; HRMS (ES+) [M+H]+ calcd for C23H28N3O3: 394.2125
found: 394.2126.
C
24H29N3O3 (407) m/z 430.2 (60) [M+Na]+; HRMS (ES+) [M+H]+
calcd for C24H30N3O3: 408.2282, found; 408.2282.
4.9. 1-Methyl-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]
benzimidazole (24)
4.6.2. E-2-[N-methyl-N-(2‘-O-tert-butylcarbonatethyl)-4‘-amino
styryl]benzimidazole (21-E)
Compound 24 was prepared as described above for compound
22, starting from 21-Z/21-E (680 mg, 1.7 mmol). Its purification
was carried out by column chromatography on silica gel (n-hex-
ane/EtOAc 1:1) to give the mixture of 24-Z and 24-E (540 mg, 69%).
Rf = 0.42 (n-hexane/EtOAc 2:1); 1H NMR (CDCl3, 300 MHz):
d = 1.45 (s, 9H), 2.96 (s, 3H), 3.59 (t, J = 6.0 Hz, 2H), 4.19 (t,
J = 6.0 Hz, 2H), 6.58 (d, 3J = 8.4 Hz, 2H), 6.99 (dd, 4J = Hz,
3JE = 16.5 Hz, 1H), 7.21–7.28 (m, 4H), 7.59–7.64 (m, 3H); 13C NMR
(CDCl3, 75 MHz): d = 27.65 (3C), 38.60, 50.75, 63.51, 82.35,
111.83 (2C), 111.87 (2C), 122.47, 124.02, 128.57 (2C), 135.90,
149.32, 152.43, 153.33; ES+ MS C23H27N3O3 (393) m/z 394.2 (100)
[M+H]+; HRMS (ES+) [M+H]+ calcd for C23H28N3O3: 394.2125
found: 394.2126.
4.9.1. Z-1-Methyl-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminost
yryl]benzimidazole (24-Z)
Rf = 0.38 (n-hexane/EtOAc 2:1); 1H NMR (CDCl3, 300 MHz):
d = 2.35 (s, 3H), 2.83 (s, 3H), 3.43 (s, 3H), 3.54 (t, J = 6.0 Hz, 2H),
4.17 (t, J = 6.0 Hz, 2H), 6.34 (m, 3H), 6.87 (d, 3JZ = 12.0 Hz, 1H),
7.07 (d, 3J = 8.7 Hz, 2H), 7.18–7.30 (m, 4H), 7.64 (d, 3J = 9.0 Hz,
2H), 7.70–7.75 (m, 2H); 13C NMR (CDCl3, 75 MHz): d = 21.56,
30.26, 38.79, 50.81, 66.77, 109.39, 111.48 (2C), 112.67, 119.51,
122.01, 122.34, 124.36, 128.85 (2C), 130.35 (2C), 132.56 (2C),
135.33, 137.88, 143.26, 144.94, 148.22, 151.45; ES+ MS
4.7. E-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-aminostyryl]benz
imidazole (22-E)
A solution of 21-E/21-Z (810 mg, 2.1 mmol) in CH2Cl2 (12 mL)
was stirred with TFA (6 mL) at rt for 90 min. Then, the solvent
was removed under vaccum. The resulting crude was re-dissolved
in CH2Cl2 (50 mL) and was reacted with Et3N (0.4 mL) and p-TsCl
(500 mg, 2.6 mmol) for 30 min. Thereafter, the reaction mixture
was diluted with CH2Cl2 (100 mL) and was washed with a sat.
sol. of NaHCO3 (100 mL). The organic phase was dried over Na2SO4,
was filtered and the filtrate was evaporated. The residue was sub-
mitted to column chromatography on silica gel (n-hexane/EtOAc
1:2) to give 22-E (295 mg, 32%)—Rf = 0.19 (n-hexane/EtOAc 1:1);
1H NMR (CDCl3, 300 MHz): d = 2.31 (s, 3H), 3.06 (s, 3H), 3.56 (t,
J = 5.4 Hz, 2H), 3.85 (t, J = 5.4 Hz, 2H), 6.78 (d, 3J = 8.7 Hz, 2H),
7.18 (d, 3J = 7.9 Hz, 2H), 7.28–7.30 (m, 2H), 7.53 (d, 3J = 8.7 Hz,
2H), 7.60–7.63 (m, 1H), 7.67 (d, 3JE = 15.6 Hz, 1H), 7.75 (d,
3J = 7.9 Hz, 2H), 7.83 (d, 3JE = 15.6 Hz, 1H), 8.02–8.05 (m, 1H); 13C
NMR (CDCl3, 75 MHz): d = 21.60, 38.97, 54.70, 60.27, 109.29,
112.25 (2C), 113.86, 119.37, 124.34, 124.41, 125.09, 126.81 (2C),
129.43 (2C), 130.07 (2C), 133.08, 135.41, 140.15, 142.90, 145.68,
150.69, 152.31; ES+ MS C25H25O3N3 (447) m/z 470.22 (95)
[M+Na]+; HRMS (ES+) [M+H]+ calcd for C25H26N3O3S: 448.1689,
found: 448.1691.
C
26H27N3O3S (461) m/z 462.2 (100) [M+H]+; HRMS (ES+) [M+H]+
calcd for C26H27N3O3S: 462.1846; found: 462.1834.
4.9.2. E-1-Methyl-2-[N-methyl-N-(2‘-tosyloxyethyl)-4‘-amino
styryl]benzimidazole (24-E)
Rf = 0.18(n-hexane/EtOAc 2:1); 1H NMR (CDCl3, 300 MHz):
d = 2.37 (s, 3H), 2.92 (s, 3H), 3.64 (t, J = 6.0 Hz, 2H), 3.82 (s, 3H),
4.17 (t, J = 6.0 Hz, 2H), 6.54 (d, 3J = 8.4 Hz, 2H), 6.86 (d, 3JE = 15.9 Hz,
1H), 7.18–7.30 (m, 4H), 7.43 (d, 3J = 8.4 Hz, 2H), 7.67 (d, 3J = 8.7 Hz,
2H), 7.70–7.75 (m, 2H), 7.86 (d, 3JE = 15.9 Hz, 1H); 13C NMR (CDCl3,
75 MHz): d = 21.62, 29.63, 38.97, 50.96, 66.77, 108.30, 108.91,
111.84 (2C), 118.81, 122.01, 122.34, 124.67, 127.75 (2C), 128.71
(2C), 129.78 (2C), 135.95, 137.36, 140.09, 144.94, 148.78, 152.01;
ES+ MS C26H27N3O3S (461) m/z 462.2 (100) [M+H]+; HRMS (ES+)
[M+H]+ calcd for C26H27N3O3S: 462.1846; found: 462.1834.
4.10. 2-[N-methyl-N-(2‘-fluoroethyl)-4‘-aminostyryl]benzothia
zole (1)
To a solution of 19-Z/19-E (210 mg, 0.45 mmol) in anhydrous
THF (23 mL) was added anhydrous TBAF (1.8 mL, 1.8 mmol, 1.0 M
in THF). The reaction mixture was refluxed for 30 min. Thereafter
the solvent was removed under vacuum; chloroform (50 mL) was
added to the residue and was washed with sat sol NaHCO3
(50 mL). The organic phase was dried over MgSO4, filtered and
the filtrate was concentrated. Column chromatography on silica
gel (n-hexane/EtOAc 5:1) gave the mixture of 1-Z and 1-E
(104 mg, 74%).
4.8. E/Z-1-Methyl-2-[N-methyl-N-(2‘-O-tert-butylcarbon
atethyl)-4‘-aminostyryl]benzimidazole (23)
To a solution of NaOH (535 mg, 13.4 mmol) in water (0.75 mL)
were added 21-Z/21-E (715 mg, 1.8 mmol), acetone (7.5 mL) and
methyl iodide (145 lL, 2.3 mmol), dropwise with the flask cooling
in ice water. The reaction mixture was stirred for 1 h at rt. Then,
acetone was evaporated, the reaction mixture was diluted with
water (100 mL) and was extracted with CH2Cl2 (100 mL). The or-
ganic phase was dried over Na2SO4, filtered and the filtrate was
concentrated under vacuum to give the mixture of 23-Z and 23-E
(710 mg, 96%)—Rf = 0.60 (n-hexane/EtOAc 2:1); 1H NMR (CDCl3,
300 MHz): d = 1.41 (s, 9H), 1.42 (s, 9H), 2.91 (s, 3H), 3.00 (s, 3H),
3.41 (s, 3H), 3.54 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 6.0 Hz, 2H), 3.77 (s,
3H), 4.13 (t, J = 6.0 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 6.32 (d,
3JZ = 12.3 Hz, 1H), 6.49 (d, 3J = 9.0 Hz, 2H), 6.59 (d, 3J = 9.0 Hz, 2H),
6.83 (d, 3JE = 15.6 Hz, 1H), 6.87 (d, 3JZ = 12.3 Hz, 1H), 7.10 (d,
4.10.1. Z-2-[N-methyl-N-(2‘-fluoroethyl)-4‘-aminostyryl]
benzothiazole (1-Z)
Rf = 0.36 (n-hexane/EtOAc 4:1); logP(o/w) = 4.83; 1H NMR
(CDCl3, 300 MHz): d = 3.05 (s, 3H, CH3), 3.68 (dt, JH,H = 5.1 Hz,
JH,F = 24.3 Hz, 2H), 4.61 (dt, JH,H = 5.1 Hz, JH,F = 47.4 Hz, 2H), 6.67
(d, 3J = 8.7 Hz, 2H), 6.72 (d, ZJ = 12 Hz, 1H), 6.95 (d, ZJ = 12 Hz,
1H), 7.28 (ddd, 4J = 1.2 Hz, 3J = 7.2 Hz, 3J = 7.8 Hz, 1H), 7.41 (ddd,
4J = 1.2 Hz, 3J = 7.2 Hz, 3J = 8.1 Hz, 1H), 7.47 (d, 3J = 8.7 Hz, 2H),
7.72 (ddd, 5J = 0.4 Hz, 4J = 1.2 Hz, 3J = 7.8 Hz, 1H), 7.98 (ddd,
5J = 0.4 Hz 4J = 1.2 Hz, 3J = 8.1 Hz, 1H); 13C NMR (CDCl3, 75 MHz):

7708
G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
d = 39.07, 52.45 (d, JC,F = 20.8 Hz), 81.72 (d, JC,F = 168.6 Hz), 111.68
(2C), 120.87, 121.25, 122.78, 123.65, 125.07, 125.89, 130.89 (2C),
135.10, 137.95, 149.18, 152.84, 165.27, 19F (CDCl3, 282 MHz):
d = ꢀ223.09 (m); ES+ MS C18H17N2FS (312.11) m/z 313.2 (100)
[M+H]+; HRMS (ES+) [M+H]+ calcd for C18H18N2FS: 313.1169,
found: 313.1167.
4.12.1. Z-2-[N-methyl-N-(2‘-fluorethyl)-4‘-aminostyryl]benzi
midazole (3-Z)
Rf = 0.35 (n-hexane/EtOAc 1:1); logP(o/w) = 3.95; 1H NMR
(CDCl3, 300 MHz): d = 3.07 (s, 3H), 3.68 (dt, JH,H = 4.8 Hz,
JH,F = 29.7 Hz, 2H), 4.62 (dt, JH,H = 4.8 Hz, JH,F = 47.1 Hz, 2H) 6.55
(d, 3JZ = 12.3 Hz, 1H), 6.70 (d, 3J = 8.7 Hz, 2H), 6.89 (d, 3JZ = 12.3 Hz,
1H), 7.15–7.20 (m, 2H), 7.33 (d, 3J = 8.7 Hz, 2H), 7.42–7.50 (m, 2H)
13C NMR (CDCl3, 75 MHz): d = 38.90, 52.39 (d, JC,F = 20.9 Hz), 81.64
(d, JC,F = 169.2 Hz), 112.09 (2C), 116.99, 122.65 (2C), 123.99 (2C),
129.92 (2C), 135.56, 149.00, 149.97; 19F (CDCl3, 282 MHz):
d = d = ꢀ223.00 (m); ES+ MS C18H18FN3 (295) m/z 296.2 (100)
[M+H]+; HRMS (ES+) [M+H]+ calcd for C18H19N3F: 296.1558, found:
296.1562.
4.10.2. E-2-[N-methyl-N-(2‘-fluoroethyl)-4‘-amino styryl]benzo
thiazole (1-E)
Rf = 0.24 (n-hexane/EtOAc 4:1); logP(o/w) = 4.83; 1H NMR
(CDCl3, 300 MHz): d = 3.06 (s, 3H, CH3), 3.69 (dt, JH,H = 5.1 Hz,
2
3JH,F = 24.6 Hz, 2H), 4.60 (dt, JH,H = 5.1 Hz, JH,F = 47.1 Hz, 2H), 6.70
(d, 3J = 9.0 Hz, 2H), 7.19 (d, EJ = 16.2 Hz, 1H), 7.31 (ddd,
4J = 1.2 Hz, 3J = 7.6 Hz, 3J = 7.8 Hz, 1H), 7.39–7.47 (m, 3H), 7.80 (d,
3J = 7.8 Hz, 1H), 7.92 (d, 3J = 8.1 Hz, 1H); 13C NMR (CDCl3,
75 MHz): d = 39.12, 52.37 (d, JC,F = 21.45 Hz), 81.66 (d,
JC,F = 169.2 Hz), 112.03 (2C), 117.57, 121.36, 122.41, 123.80,
124.81, 126.12, 129.05 (2C), 134.06, 138.09, 149.71, 153.85,
168.10; 19F (CDCl3, 282 MHz): d = ꢀ223.15 (m); ES+ MS C18H17N2FS
(312.11) m/z 313.2 (100) [M+H]+; HRMS (ES+) [M+H]+ calcd for
4.12.2. E-2-[N-methyl-N-(2‘-fluorethyl)-4‘-aminostyryl]
benzimidazole (3-E)
Rf = 0.18 (n-hexane/EtOAc 1:1); logP(o/w) = 3.95; 1H NMR
(CDCl3, 300 MHz): d = 3.03 (s, 3H), 3.66 (dt, JH,H = 5.1 Hz,
JH,F = 24.6 Hz, 2H), 4.58 (dt, JH,H = 5.1 Hz, JH,F = 47.1 Hz, 2H), 6.64
(d, 3J = 8.7 Hz, 2H), 6.91 (d, 3JE = 16.5 Hz, 1H), 7.18–7.21 (m, 2),
7.37 (d, 3J = 8.7 Hz, 2H), 7.48–7.60 (m, 3H); 13C NMR (CDCl3,
75 MHz): d = 39.10, 52.40 (d, JC,F = 21.4 Hz), 81.67 (d,
JC,F = 169.2 Hz), 111.94 (2C), 112.05 (2C), 122.61, 124.12 (2C),
128.55 (2C), 125.55, 149.43, 151.81; 19F (CDCl3, 282 MHz):
d = ꢀ223.04 (m); ES+ MS C18H18FN3 (295) m/z 296 (100) [M+H]+;
HRMS (ES+) [M+H]+ calcd for C18H19N3F: 296.1558, found:
296.1562.
C18H18N2FS: 313.1169, found: 313.1167.
4.11. 2-[N-methyl-N-(2‘-fluoroethyl)-4‘-amino styryl]benzo
xazole (2)
Compound 2 was prepared as described above for compound 1,
starting from 20-Z/20-E (250 mg, 0.55 mmol). Its purification was
carried out by column chromatography on silica gel (n-hexane/
EtOAc 4:1) to give the mixture of 2-Z and 2-E (124 mg, 76%).
4.13. 1-Methyl-2-[N-methyl-N-(2‘-fluorethyl)-4‘-aminostyryl]
benzimidazole (4)
4.11.1. Z-2-[N-methyl-N-(2‘-fluoroethyl)-4‘-amino styryl]benzo
xazole (2-Z)
Compound 4 was prepared as described above for compound 1,
starting from 24-Z/24-E (300 mg, 0.6 mmol). The organic extract
was dried over Na2SO4. Its purification was carried out by column
chromatography on silica gel (n-hexane/EtOAc 2:1) to give the
mixture of 4-Z and 4-E (160 mg, 80%).
Rf = 0.52 (n-hexane/EtOAc 4:1); logP(o/w) = 4.01; 1H NMR
(CDCl3, 300 MHz): d = 3.07 (s, 3H), 3.70 (dt, JH,H = 5.1 Hz,
2
3JH,F = 24.3 Hz, 2H), 4.61 (dt, JH,H = 5.1 Hz, JH,F = 47.2 Hz, 2H), 6.28
(d, 3JZ = 12.9 Hz, 1H), 6.70 (d, 3J = 8.4 Hz, 2H), 6.87 (d, 3JZ = 12.9 Hz,
1H), 7.27–7.32 (m, 2H), 7.43–7.46 (m, 1H), 7.69–7.72 (m, 1H), 7.88
(d, 3J = 8.4 Hz, 2H); 13C NMR (CDCl3, 75 MHz): d = 39.09, 52.31 (d,
JC,F = 20.8 Hz), 81.69 (d, JC,F = 169.7 Hz), 109.43, 110.23, 111.23
(2C), 119.78, 123.81, 124.23, 124.85, 132.25 (2C), 139.91, 142.17,
149.49, 149.94, 162.52; 19F (CDCl3, 282 MHz): d = ꢀ223.18 (m),
ES+ MS C18H17ON2F (296.13) m/z 297.2 (100) [M+H]+; HRMS (ES+)
[M+H]+ calcd for C18H18ON2F: 297.1398, found: 297.1404.
4.13.1. Z-1-Methyl-2-[N-methyl-N-(2‘-fluorethyl)-4‘-amino
styryl]benzimidazole (4-Z)
Rf = 0.29 (n-hexane/EtOAc 2:1); logP(o/w) = 4.34; 1H NMR
(CDCl3, 300 MHz): d = 2.95 (s, 3H), 3.45 (s, 3H), 3.57 (dt,
JH,H = 5.1 Hz, JH,F = 24.3 Hz, 2H), 4.51 (dt, JH,H = 5.1 Hz, JH,F = 47.4 Hz,
2H), 6.33 (d, 3JZ = 12.3 Hz, 1H), 6.49 (d, 3J = 8.7 Hz, 2H), 6.88 (d,
3JZ = 12.3 Hz, 1H), 7.13 (d, 3J = 8.7 Hz, 2H), 7.24–7.26 (m, 3H),
7.75–7.78 (m, 1H); 13C NMR (CDCl3, 75 MHz): d = 30.24, 38.88,
52.25 (d, JC,F = 20.8 Hz), 81.60 (d, JC,F = 169.12 Hz), 109.31, 111.62
(2C), 112.47, 119.49, 121.94, 122.26, 124.17, 130.37 (2C), 135.31,
137.97, 143.26, 148.84, 151.53; 19F (CDCl3, 282 MHz):
d = ꢀ223.03 (m); ES+ MS C19H20FN3 (309) m/z 310 (10) [M+H]+;
HRMS (ES+) [M+H]+ calcd for C19H21N3F: 310.1714, found:
310.1717.
4.11.2. E-2-[N-methyl-N-(2‘-fluoroethyl)-4‘-aminostyryl]benzo
xazole (2-E)
Rf = 0.30 (n-hexane/EtOAc 4:1); logP(o/w) = 4.01; 1H NMR
(CDCl3, 300 MHz): d = 3.07 (s, 3H), 3.69 (dt, JH,H = 5.1 Hz,
2
3JH,F = 24.6 Hz, 2H), 4.61 (dt, JH,H = 5.1 Hz, JH,F = 47.1 Hz, 2H), 6.70
(d, 3J = 8.7 Hz, 2H), 6.83 (d, 3JE = 16.2 Hz, 1H), 7.26–7.30 (m, 2H),
7.46–7.49 (m, 3H), 7.64–7.67 (m, 1H), 7.70 (d, 3JE = 16.2 Hz, 1H);
13C NMR (CDCl3, 75 MHz): d = 39.11, 52.28 (d, JC,F = 20.8 Hz),
81.62 (d, JC,F = 169.7 Hz), 108.97, 110.06, 112.03 (2C), 119.37,
123.67, 124.27, 124.55, 129.27 (2C), 139.76, 142.36, 149.99,
150.35, 163.87; 19F (CDCl3, 282 MHz): d = ꢀ223.17 (m); ES+ MS
4.13.2. E-1-Methyl-2-[N-methyl-N-(2‘-fluorethyl)-4‘-amino
styryl]benzimidazole (4-E)
Rf = 0.21 (n-hexane/EtOAc 2:1); logP(o/w) = 4.34; 1H NMR
(CDCl3, 300 MHz): d = 3.05 (s, 3H), 3.68 (dt, JH,H = 5.1 Hz,
JH,F = 24.3 Hz, 2H), 3.81 (s, 3H), 4.60 (dt, JH,H = 5.1 Hz, JH,F = 47.1 Hz,
2H), 6.69 (d, 3J = 9.0 Hz, 2H), 6.86 (d, 3JE = 15.6 Hz, 1H), 7.20–7.29
(m, 3H), 7.49 (d, 3J = 9.0 Hz, 2H), 7.70–7.73 (m, 1H), 7.87 (d,
3JE = 15.6 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d = 29.67, 39.08,
52.43 (d, JC,F = 21.37 Hz), 81.68 (d, JC,F = 169.20 Hz), 108.28,
108.86, 112.04(2C), 118.93, 121.98, 121.32, 124.64, 128.81 (2C),
136.02, 137.48, 143.27, 149.47, 152.14; 19F (CDCl3, 282 MHz):
d = ꢀ223.02 (m); ES+ MS C19H20FN3 (309) m/z 310 (10) [M+H]+;
HRMS (ES+) [M+H]+ calcd for C19H21N3F: 310.1714, found:
310.1717.
C
18H17ON2F (296.13) m/z 8) 297.2 (100) [M+H]+; HRMS (ES+)
[M+H]+ calcd for C18H18ON2F: 297.1398, found: 297.1404
4.12. 2-[N-methyl-N-(2‘-fluorethyl)-4‘-aminostyryl]benzimi
dazole (3)
Compound 3 was prepared as described above for compound 1,
starting from 22-E (280 mg, 0.6 mmol). The organic extract was
dried over Na2SO4. Its purification was carried out by column chro-
matography on silica gel (n-hexane/EtOAc 1:1) to give the mixture
of 3-Z and 3-E (30 mg, 16%).

G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
7709
4.14. X- ray diffraction analysis
centrifugation at 40,000g for 1 h at 4 °C and supernatant collected
for protein quantification. To determine fibril concentrations, super-
natant protein concentration was assayed and the difference be-
tween the starting and remaining supernatant concentration was
assumed as that of the fibrils, an approximation that is widely used
in the field. The formation of fibrils was confirmed by Thioflavin T
binding.45 Fibrils were used upon inducing production or aliquoted
and stored at ꢀ80 °C. For all species, frozen and fresh material did
not show any difference in ligand binding behavior.
White crystals of compound 12 suitable for X-ray diffraction
studies were obtained by diffusion of diethyl ether into a concen-
trated methanolic solution of 12. The X-ray diffraction analysis of
12 was performed on a Bruker AXS APEX CCD area detector diffrac-
tometer, using graphite monochromated Mo
K
a
radiation
(0.71073 Å). Empirical absorption correction was carried out using
SADABS.38 Data collection and data reduction were done with the
SMART and SAINT programs.39 The structures were solved by direct
methods with SIR9740 and refined by full-matrix least-squares
analysis with SHELXL-9741 using the WINGX.42 All nonhydrogen
atoms were refined anisotropically. The remaining hydrogen atoms
were placed in calculated positions. Molecular graphics were pre-
pared using ORTEP3.43
4.18. Compound preparation
Stock solutions (34.9 mM) in DMSO of the E and Z geometric
isomers of the fluorinated styryl benzazole 1–4 isomers were pre-
pared in the dark, before dilution into assay buffer (Tris–HCl,
50 mM, pH 7.4).
4.15. Crystallographic data
4.19. Intrinsic Fluorescence Characterization
(C26H21NOP)+Clꢀ, Mr 429.86, triclinic, space group p1, Z = 2,
ꢀ
Dcalc = 1.299 g/cm3, a = 9.7244 (3) Å, b = 10.8474(3) Å, c = 12.6058(4) Å,
The excitation and emission wavelength scan of the E and Z iso-
mers of compounds 1–4 were recorded in a plate reader (Tecan Infi-
nite 200, Mannendorf, Switzerland) in the presence of monomeric or
fibrillary species of insulin, a-syn, Ab(1–42) peptide and BSA (5 lM).
The maximum excitation and emission wavelength was determined
a
l
= 68.2390(10)°, b = 71.740(2)°,
(MoKa
c = 64.9080(10)°, m
= 1098.98(6) Å3,
) 0.264 cmꢀ3, F(000) = 448, k = 0.71073 Å. Data were col-
lected using a crystal of 0.40 ꢁ 0.30 ꢁ 0.20 mm3. A total number of
9047 of reflections were collected for 2.58° < h < 25.68° and
ꢀ11< = h< = 11, ꢀ13< = k< = 11, ꢀ15< = l< = 15. There were 4146
according to the maximum fluorescence intensity level.
independent reflections and 3681 reflections with I > 2
r
(I) were
used in the refinement. The final
R
indices were [I > 2 (I)]
r
4.20. Intrinsic fluorescence intensity binding assays
R1 = 0.0330, wR2 = 0.0858 and (all data) R1 = 0.0380, wR2 = 0.0886.
The goodness-of-fit on F2 was 1.072 and the largest difference peak
Intrinsic fluorescence intensity changes associated with ligand
binding to insulin, a-syn, Ab(1–42) peptide fibrils and BSA were re-
corded in a plate reader (Tecan Infinite 200, Mannendorf, Switzer-
and hole was 0.350 and ꢀ0.304 eÅꢀ3
.
The crystallographic data of 12 have been deposited with the
Cambridge Crystallographic Data Center (CCDC 826044).
land). The assays were performed using different fixed
concentrations of fibrils (3.5–11.35
lM) and varying concentrations
4.16. Expression and purification of human
a-syn
of ligands 1–4 E/Z isomers (0–20 M) at the maximum determined
l
excitation and emission wavelengths (table 1, and supplementary
data Table 1). To correct for the minimal fluorescence background,
the intrinsic fluorescence of the dye blanks were determined at each
explored concentration and subtracted to all data presented.32,46,47
Resulting corrected intensities were plotted and the Kd values deter-
mined by fitting a single site saturated curve to the data using Sig-
maPlotTM 11.0 software. For purposes of comparison the binding
assays were also performed for ThT.
The expression and purification procedure of human a-syn was
adapted from the literature.44 Briefly, E. coli BL21 codon plus RIL
strain (Stratagene, Santa Clara, USA) was transformed with human
a
-syn PT7-7 construct (a kind gift from Hilal Lashuel, EPFL, Lau-
sanne) and expression was induced for 3 h with D-thiogalactopyra-
noside (IPTG, Calbiochem, Darmstadt, Germany) at final
a
concentration of 0.3 mM. Cells were harvested and resuspended
in hyperosmotic buffer (40% Sucrose, 30 mM Tris-HCl, 2 mM EDTA,
pH 7.3). The pellet was placed in a hypotonic solution, vortexed
and the 35% ammonium sulfate salt resultant precipitate dis-
carded. The supernatant was further precipitated in 60% ammo-
nium sulfate, and the resulting pellet was separated by ion-
exchange chromatography (HiTrap Q sepharose, GE Healthcare,
NJ, USA) in Tris–HCl 30 mM pH 7.3 buffer with a NaCl gradient.
Acknowledgements
The authors thank Dr Joaquim Marçalo for Mass Spectroscopy
measurements, and Prof Giuseppe Ermondi and Dr Mauro Ravera
for calculations of the octanol/water coefficient partition (LogPo/
w). This work was funded by Fundação para a Ciência e Tecnologia
(FCT), Portugal (PTDC/QUI/102049/2008). TFO is also supported by
a Marie Curie International Reintegration Grant from the European
Commission (Neurofold) and by an EMBO Installation Grant. GRM
thanks the FCT for the ‘Ciência 2008’ program and DAAD program
(0811983). HVM is supported by the FCT, Portugal (SFRH/BPD/
64702/2009)
Resulting 15 KDa
a-syn positive samples, as judged by 15% SDS–
PAGE (Bio-Rad, Hercules, California, USA, according to the sup-
plier’s instructions) were collected and further purified by gel fil-
tration chromatography (Superdex 200, Ge Healthcare, NJ, USA).
SDS–PAGE, followed by western-blot analysis (using standard pro-
cedures), confirmed the monomeric purification of
BD Transduction Laboratories, CA, USA).
a-syn (Syn-1
Supplementary data
4.17. Polymerization of insulin, a-syn and Ab 1–42 fibrils
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.065.
Human insulin was prepared in Tris–HCl 50 mM at 3 mg mLꢀ1
,
pH 3. Purified
-syn was resuspended at 3 mg mLꢀ1 in Tris–HCl
a
50 mM pH 7.4. Human Ab-(1–42)-peptide (rPeptide, Leipzig, Ger-
many) was prepared at 0.5 mg/ml in 1% ammonium hydroxide (Sig-
ma) in Mili-Q type II water (Millipore, Billerica, MA, USA). Samples
were incubated at 37 °C for 6 days in a thermomixer (Eppendorph,
Hamburg, Germany) at 1400 rpm. Fibrils were collected after ultra-
References and notes
1. Bacskai, B. J.; Hickey, G. A.; Skoch, J.; Kajdasz, S. T.; Wang, Y.; Huang, G. F.;
Mathis, C. A.; Klunk, W. E.; Hyman, B. T. Proc Natl Acad Sci U.S.A. 2003, 100,
12462.

7710
G. Ribeiro Morais et al. / Bioorg. Med. Chem. 19 (2011) 7698–7710
2. Ye, L.; Velasco, A.; Fraser, G.; Beach, T. G.; Sue, L.; Osredkar, T.; Libri, V.;
Spillantini, M. G.; Goedert, M.; Lockhart, A. J. Neurochem. 2008, 105, 1428.
3. Rowe, C. C.; Ng, S.; Ackermann, U.; Gong, S. J.; Pike, K.; Savage, G.; Cowie, T. F.;
Dickinson, K. L.; Maruff, P.; Darby, D.; Smith, C.; Woodward, M.; Merory, J.;
Tochon-Danguy, H.; O’Keefe, G.; Klunk, W. E.; Mathis, C. A.; Price, J. C.; Masters,
C. L.; Villemagne, V. L. Neurology 2007, 68, 1718.
23. Sharghi, H.; Asemani, O. Synth. Commun. 2009, 39, 860.
24. Burkholder, C. R.; Dolbier, W. R.; Medebielle, M. J. Fluorine Chem. 2000, 102,
369.
25. Qu, W. C.; Kung, M. P.; Hou, C.; Benedum, T. E.; Kung, H. F. J. Med. Chem. 2007,
50, 2157.
26. Watanabe, M.; Morais, G. R.; Mataka, S.; Ideta, K.; Thiemann, T. Z. Naturfor. B
2005, 60, 909.
4. Kung, H. F.; Choi, S. R.; Qu, W.; Zhang, W.; Skovronsky, D. J. Med. Chem. 2010, 53,
933.
27. Suemoto, T.; Okamura, N.; Shiomitsu, T.; Suzuki, M.; Shimadzu, H.; Akatsu, H.;
Yamamoto, T.; Kudo, Y.; Sawada, T. Neurosci. Res. 2004, 48, 65.
28. Lee, C. W.; Zhuang, Z. P.; Kung, M. P.; Plossl, K.; Skovronsky, D.; Gur, T.; Hou, C.;
Trojanowski, J. Q.; Lee, W. M. Y.; Kung, H. F. J. Med. Chem. 2001, 44, 2270.
29. Vassar, P. S.; Culling, C. F. Arch. Pathol. 1959, 68, 487.
30. Biancalana, M.; Koide, S. Biochim. Biophys. Acta 2010, 1804, 1405.
31. Groenning, M. J. Chem. Biol. 2010, 3, 1.
5. van den Berg, D.; Zoellner, K. R.; Ogunrombi, M. O.; Malan, S. F.; Terre’Blanche,
G.; Castagnoli, N.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. 2007, 15, 3692.
6. Prousek, J. Collect Czech. Chem. Commun. 1991, 56, 1358.
7. Zhang, G. H.; Wu, F. I.; Jiang, X. Q.; Sun, P. P.; Cheng, C. H. Synth. Met. 2010, 160,
1906.
8. Hranjec, M.; Kralj, M.; Piantanida, N.; Sedic, M.; Suman, L.; Pavelic, K.;
Karminski-Zamola, G. J. Med. Chem. 2007, 50, 5696.
32. LeVine, H., 3rd Protein Sci. 1993, 2, 404.
9. Popov, I. I. Chem. Heterocycl. Compd. 1997, 33, 949.
33. Lockhart, A.; Ye, L. A.; Morgenstern, J. L.; Lamb, J. R. Biochem. Biophys. Res.
Commun. 2006, 347, 669.
34. Ye, L.; Morgenstern, J. L.; Gee, A. D.; Hong, G.; Brown, J.; Lockhart, A. J. Biol.
Chem. 2005, 280, 23599.
35. Bertoncini, C. W.; Celej, M. S. Curr. Protein Pept. Sci. 2011, 12, 205.
36. Kung, H. F.; Lee, C. W.; Zhuang, Z. P.; Kung, M. P.; Hou, C.; Plossl, K. J. Am. Chem.
Soc. 2001, 123, 12740.
10. Petzer, J. P.; Steyn, S.; Castagnoli, K. P.; Chen, J. F.; Schwarzschild, M. A.; Van der
Schyf, C. J.; Castagnoli, N. Bioorg. Med. Chem. 2003, 11, 1299.
11. Wang, L. Y.; Zhang, X. G.; Li, F. M.; Zhang, Z. X. Synth. Commun. 2004, 34, 2245.
12. Kabatc, J. J. B.; Orlinski, P.; Paczkowski, J. Spectrochim. Acta Part A 2005, 62, 115.
13. Caleta, I.; Mrvos-Sermek, M. G. D.; Cetina, M.; Tralic-Kulenovic, V.; Pavelic, K.;
Karminski-Zamola, G. Il Farmaco 2004, 59, 297.
14. Pavlovic, G. S. Z.; Popovic, Z.; Tralic-Kulenovic, V. Polyhedron 2007, 26, 5162.
15. Vasilev, A.; Deligeorgiev, T.; Gadjev, N.; Kaloyanova, S.; Vaquero, J. J.; Alvarez-
Builla, J.; Baeza, A. G. Dyes Pigments 2008, 77, 550.
37. Halgren, T. J. Comp. Chem. 1996, 17, 490.
38. SADABS, W. Area-Detector Absorption Correction. Brukee AXS Inc., Madison, WI
2004.
16. Shimadzu, H.; Suemoto, T.; Suzuki, M.; Shiomitsu, T.; Okamura, N.; Kudo, Y.;
Sawada, T. J. Labelled Comp. Radiopharm. 2004, 47, 181.
39. SAINT, M. Area-Detector Integration Software. (version 7.23) Brukee AXS Inc.,
Madison, WI 2004.
17. Amatsubo, T.; Morikawa, S.; Inubushi, T.; Urushitani, M.; Taguchi, H.; Shirai, N.;
Hirao, K.; Kato, M.; Morino, K.; Kimura, H.; Nakano, I.; Yoshida, C.; Okada, T.;
Sano, M.; Tooyama, I. Neurosci. Res. 2009, 63, 76.
40. Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giacovazzo, C.;
Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr.
1999, 32, 115.
18. Fayed, T. A. J. Photochem. Photobiol. A 1999, 121, 17.
19. Cho, C. S.; Kim, D. T.; Zhang, J. Q.; Ho, S. L.; Kim, T. J.; Shim, S. C. J. Heterocycl.
Chem. 2002, 39, 421.
20. Yang, X.-L.; Xu, C.-M.; Lin, S.-M.; Chen, J.-X.; Ding, J.-C.; Wu, H.-Y.; Su, W.-K. J.
Braz. Chem. Soc. 2010, 21, 37.
21. Dondoni, A.; Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P. Tetrahedron 1988,
44, 2021.
22. Lau, C. K.; Dufresne, C.; Gareau, Y.; Zamboni, R.; Labelle, M.; Young, R. N.;
Metters, K. M.; Rochette, C.; Sawyer, N.; Slipetz, D. M.; Charette, L.; Jones, T.;
Mcauliffe, M.; Mcfarlane, C.; Fordhutchinson, A. W. Bioorg. Med. Chem. Lett.
1995, 5, 1615.
41. Sheldrick, G. M. SHELXL-97: Program for the refinement of Crystal Structure;
University of Gottingen: Germany, 1997.
42. Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837.
43. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
44. Huang, C.; Ren, G.; Zhou, H.; Wang, C. C. Protein Expr. Purif. 2005, 42, 173.
45. Nilsson, M. R. Methods 2004, 34, 151.
46. Ahmad, A.; Uversky, V. N.; Hong, D.; Fink, A. L. J. Biol. Chem. 2005, 280, 42669.
47. Kumar, S.; Singh, A. K.; Krishnamoorthy, G.; Swaminathan, R. J. Fluoresc. 2008,
18, 1199.