2-Styrylindolium based fluorescent probes visualize neurofibrillary tangles in Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2012.09.109

We evaluated 2-styrylindolium derivatives (6-11) as novel and selective probes for neurofibrillary tangles (NFTs) on brain sections of AD patients. The staining experiments indicated that these compounds may bind selectively to NFTs in the presence of ss-amyloid (Ass) plaques. Cell free binding assays confirmed that 2-[2-[4-(1-pyrrolidinyl)phenyl]ethenyl]-1,3,3-trimethyl-3H- indolium iodide (9) and 2-[2-[4-(diethylamino)phenyl]ethenyl]-1-butyl-3,3- dimethyl-3H-indolium iodide (11) display excellent affinities to Tau-aggregates (IC₅₀ values of 5.1 and 1.4 nM, respectively) in the displacement of Thiazin Red R. These probes have good solubility in distilled water and low or no cytotoxicity in zebrafish embryo and liver hepatocellular carcinoma cell assays.

Full text of DOI:10.1016/j.bmcl.2012.09.109

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7667–7671
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Styrylindolium based fluorescent probes visualize neurofibrillary tangles
in Alzheimer’s disease
Jiamin Gu a, Upendra Rao Anumala a, Fabio Lo Monte a, Thomas Kramer a, Roland Heyny von Haußen b,
Jana Hölzer c, Valérie Goetschy-Meyer d, Gerhard Mall b, Ingrid Hilger c, Christian Czech d, Boris Schmidt a,
⇑
a Clemens Schöpf Institute of Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstrasse 22, Darmstadt 64287, Germany
b Institute of Pathology, Klinikum Darmstadt, Grafenstrasse 9, Darmstadt 64283, Germany
c Institute of Diagnostic and Interventional Radiology I, Jena University Hospital—Friedrich Schiller Universität Jena, Erlanger Allee 10, Jena 07747, Germany
d F. Hoffmann-La Roche AG, Grenzacherstrasse 124, Gebäude 93/3.44, Basel 4070, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2012
Revised 27 September 2012
Accepted 29 September 2012
Available online 6 October 2012
We evaluated 2-styrylindolium derivatives (6–11) as novel and selective probes for neurofibrillary tan-
gles (NFTs) on brain sections of AD patients. The staining experiments indicated that these compounds
may bind selectively to NFTs in the presence of ß-amyloid (Aß) plaques. Cell free binding assays con-
firmed that 2-[2-[4-(1-pyrrolidinyl)phenyl]ethenyl]-1,3,3-trimethyl-3H-indolium iodide (9) and 2-[2-
[4-(diethylamino)phenyl]ethenyl]-1-butyl-3,3-dimethyl-3H-indolium iodide (11) display excellent affin-
ities to Tau-aggregates (IC50 values of 5.1 and 1.4 nM, respectively) in the displacement of Thiazin Red R.
These probes have good solubility in distilled water and low or no cytotoxicity in zebrafish embryo and
liver hepatocellular carcinoma cell assays.
Keywords:
Alzheimer’s disease
Neurofibrillary tangles
Zebrafish
Ó 2012 Elsevier Ltd. All rights reserved.
Cytotoxicity
Imaging
Alzheimer’s disease (AD) is the most common form of dementia,
which is a progressive neurodegenerative disorder associated with
memory loss, spatial disorientation, and gradual deterioration of
intellectual capacity.1–3 Age is the major risk factor for disease pro-
gression, and most individuals with the disease are age 65 or older.
After age 65, the possibility of developing dementia roughly dou-
bles every five years. It was estimated that 36 million people live
with Alzheimer’s disease and other dementias worldwide in
2011. This number is expected to rise to 66 million by 2030 and
115 million by 2050.4
The presence of two abnormal proteins in patients’ brains: se-
nile plaques (SPs) and neurofibrillary tangles (NFTs) are the charac-
teristic hallmarks of AD.5,6 SPs are composed of ß-amyloid peptide
(Aß), a fragment of the amyloid peptide precursor protein (APP),
whereas the intracellular NFTs are comprised of hyperphosphoryl-
ated aggregates of the twisted strands of Tau protein.7–9 Diagnostic
probes which specifically bind to Aß plaques and NFTs are consid-
ered to be a significant advance not only to diagnosis but as an en-
abling tool for drug development. Several probes have been
developed in order to detect Aß plaques pathology in the brains
of patients with AD by either single-photon emission computed
tomography (SPECT) or positron emission tomography (PET),10–15
whereas few probes for NFTs have been reported yet.16–19,41 Non-
invasive in vivo imaging of Aß plaques in the brain is well estab-
lished, yet limited by the late onset of detectable amyloid
deposition and a plateau phase in plaque load in late stage AD.
The presence of ß-amyloid in the human retina was demonstrated
by several groups and monitored in the mouse retina by fluores-
cence microscopy after high dosage of curcumin.20,21 Systemic
application of curcumin to humans at the necessary concentration
is likely to cause adverse events as curcumin is photo labile and
toxic to most human cell lines >50 lmol/L. Recent reports suggest
Tau pathology to be a more reliable biomarker for AD.8,22 These re-
sults showed that the number of NFTs in the brain correlates better
with the progress of dementia in AD patients and does not reach a
plateau as reported for amyloid pathology. Thus, in vivo imaging of
NFTs or Aß plaques in the human retina may provide an efficient
diagnosis of AD in the early, presymptomatic stages. Current data
suggest [18F]FDDNP,16 Methoxy-X04,17 11C]PIB,18 benzimidazole
[
and quinoline derivatives19 as candidates for in vivo imaging of
Tau pathology. However, these probes display equipotent affinity
for both Aß plaques and NFTs. Moreover, these probes do not dis-
play the appropriate fluorescence to be applied to human tissue or
they display undesirable pharmacokinetic properties, for example
the very low clearance of Methoxy-XO4 in rodents. This lack of
suitable NFTs-specific fluorescent probes imposes a limit on the
in vivo quantification of neurofibrillary pathology. Therefore, the
⇑
Corresponding author.
E-mail address: schmidt_boris@t-online.de (B. Schmidt).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.109

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J. Gu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7667–7671
challenge remains to develop fluorescent probes with improved
affinity and selectivity to NFTs. In this study, we present a series
of highly water-soluble 2-styrylindolium probes with excellent
affinities to NFTs and moderate toxicity. The probes may be applied
to the imaging of retinal NFTs in AD animal models.(see Fig. 1)
In order to eliminate potentially unsafe compounds in the early
development process and to prioritize compounds for further stud-
ies, we profiled the fluorescent probes in the in vitro liver hepato-
cellular carcinoma cell assay (HepG2) and in the zebrafish embryo
development assay, which has become a major model in neurobi-
ology and toxicology.23,24 The incubation with liver cells provides
information on the detoxification of the fluorophores in vivo in hu-
man tissue. The transparent body of the zebrafish embryo consti-
tutes an important advantage of this model as it enables to
observe and analyze several parameters by microscopy. The mor-
phological and molecular basis tissues are identical or similar to
other vertebrates including human beings. However, this model
does not include pathological Tau-aggregates.25–27
The lead structure was selected from a set of established highly
fluorescent dyes and evaluated for the silk dying properties versus
wool reserve, which indicates ß-sheet binding of the dye. The cya-
nines were advanced due to their structural analogy to known Tau
ligands and evaluated against 5 other lead structures, such as
bis-stilbenes41 and squaric acid derivatives. The bis-stilbenes
displayed limited solubility and the squaric acid derivatives did
not display sufficient binding affinity.28 The synthesis of the
pal sections of human AD brain tissue and analyzed by fluores-
cence microscopy. This low throughput screen secured binding of
the probes to Tau-aggregates in human AD pathology, as different
Tau isoforms may result in different binding site on in vitro aggre-
gates or Tau transgenic mouse models.34 The 2-styryl-indolium
probes stain NFTs in human tissue (Fig. 3) and provide a good con-
trast versus Aß plaques and background, especially probes 9 and
11. These probes displayed excellent staining of NFTs (Fig. 3, D
and F) in comparison to Aß plaques (Fig. 3, G and H). The results
suggested a good affinity to the NFTs, which required quantifica-
tion in a ligand binding assay to both aggregated amyloid and
Tau. Remarkably, the probes 6–11 stained NFTs in human tissue
strongly as observed by fluorescent microscopy, whereas plaques
in the same brain preparations were stained just weakly. This
observation stands in contrast to the activity in the Thiazine Red
R displacement assay, where compound 11 displayed almost equi-
potent affinity to Aß40-aggregates and Tau-aggregates (4R). The
lack of potent plaque staining in the human tissue preparations
may be due to interference with the fluorescence properties upon
plaque binding, e.g. a fluorescence reduction or changes in the
Stokes shift, which limit the detection by the DAPI- or FITC-filter
employed in fluorescence microscopy. However, these results indi-
cated that 2-styrylindolium probes 6–11 may be useful for the post
mortem detection of NFTs in the brain of AD patients.
Probes with appropriate lipophilicity and low molecular weight
may display sufficient permeability to cross the blood–brain bar-
rier (BBB) and rapid clearance from the body when used as an
in vivo probes.12,35 The calculated logP values of all these com-
pounds were found to be in the range of 0.81 and 3.58. Besides,
the molecular weight of that 2-styrylindolium iodide salts are be-
low 505 Daltons (Table 1).36 These properties are in the range of
accepted lipophilicity criteria for BBB permeation after intravenous
injection. The solubilities of the N-methylated probes 6–10 are
higher than 0.5 g/L in distilled water. The N-butylated probe 11
displays a solubility of >0.1 g/L at 25 °C. The solubility of estab-
lished imaging agents: FDDNP, Methoxy-X04 and BF-158, is in
the same range or inferior to the solubility of these 2-styrylindoli-
um derivatives.
2-styrylindolium derivatives employed known methods or slight
29–33
modifications
thereof, which are depicted in Scheme 1. The
phenylhydrazine hydrochloride and 3-methyl-2-butanone were
condensed to form the intermediate hydrazone and converted by
Fischer indole synthesis to obtain the 2,3,3-trimethylindolenine
3. Methylation of the indolenine in acetonitrile under reflux
formed the indolium 4 in quantitative yield. The indolenine and
1-iodobutane were refluxed in ethylmethylketone to obtain the
indolium 5. The key transformation to the target 2-styrylindolium
derivatives (6–11) compounds was achieved by the Knoevenagel
condensation of the N-alkylated indolium backbone with the
corresponding aldehyde in ethanol under reflux. The products
were isolated in good yields ranging from 77 to 96% (Table 1) by
standard chromatographic purification on silica gel. The 1H NMR
spectrum revealed that the two doublets at 8.40–8.26 and 7.54–
7.19 ppm correspond to trans configured olefinic protons with a
large coupling constant (15.5–16.5 Hz).
The affinity of the probes to aggregated tau protein was deter-
mined by displaced of the reference dye Thiazine Red R, hence
IC50 data obtained in the competition assay are reported rather
than Ki values. Thiazine Red R was selected as reference ligand
for the displacement assay, because it was previously shown to
To ensure that the tissues contain both tau and Aß aggregates,
we conducted a immunohistochemical staining of the tissues. Fig-
ure 2 shows that the tissues contain a rich amount of both tau and
Aß aggregates. The qualitative selective binding of the fluorescent
probes to NFTs was evaluated in the primary screen on hippocam-
be superior to thioflavin S or immune histochemical detection
37,41
of aggregated Tau and Aß40
.
It was shown to be an accurate
maker for ß-pleated sheet structures and to reliably stain Aß pla-
ques and diffuse NFTs (Kd for aggregated Aß40 = 49 nM and Kd for
aggregated tau = 18 nM). It was reported that the in vitro binding
Figure 1. Chemical structures of [18F]FDDNP, Methoxy-X04, [11C]PIB and BF-170.

J. Gu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7667–7671
7669
Scheme 1. Synthesis of 2-styrylindolium derivatives (6–11). Reagents and conditions: (a) 1.0 equiv 1, 1.25 equiv 2, CH3COOH, 120 °C, 16 h; (b) 1.0 equiv 3, 2.0 equiv methyl
iodide, CH3CN, reflux, 12 h; (c) 1.0 equiv 3, 1.5 equiv 1-iodobutane, 2-butanone, reflux, 12 h; (d) 1.0 equiv 4 or 5, 1.1 equiv corresponding aldehyde, EtOH, reflux, 12 h.
Table 1
man microtubule associated tau protein purified from
Escherichia coli and aggregated synthetic Aß40 as reported previ-
Yields, properties and toxicity (EC50) of probes 6–11
ously.41 Thiazine Red R was added at the concentration corre-
sponding to the Kd of the respective aggregated protein binding
site to induce a fluorescent signal that can be inhibited by the
addition of a displacer compound. (Kd for aggregated tau = 18 nM,
and Kd for aggregated Ab = 49 nM.) To determine the affinity of a
displacer compound to the thiazine red binding sites of the aggre-
gated proteins, the compound was added at different concentra-
tions to the assay mixture ranging from 0.1 to 10 000 nM.
Compounds were also measured together with the aggregated
proteins, but in the absence of Thiazine Red R. Net fluorescence
was calculated by subtracting the fluorescence of the wells with-
out Thiazine Red R from the fluorescence of wells containing Thi-
azine Red R.
Compound
Yielda
MWb
cLogPc
EC50 l
Md
6
7
8
9
10
11
92%
83%
77%
96%
91%
96%
460.39
405.27
419.30
458.38
487.42
502.47
2.14
0.81
1.34
1.79
1.28
3.58
8.57
ND
10.93
8.31
ND
0.93
ND, not determined.
a
Isolated yields.
Determined by CS ChemOffice 10.0.
LogP values were calculated using the Molinspiration Cheminformatics soft-
b
c
ware (V2011. 04).
d
EC50 declares the dye concentration in which 50% of the cells died after incu-
bation with the probe.
Remarkably, the pyrrolidyl substituent results in higher affinity
than the N,N-diethyl derivative. This may be due to the locked
conformation of the N-alkyl chain. The displacement ability of
these 2-styrylindolium derivatives was compared against the
known probes: Methoxy-X04, FDDNP, PIB and BF-158 under the
same assay conditions (Table. 2). Significantly, the 2-styrylindolium
derivatives 9 and 11 displayed ꢀ50–7000 fold higher affinity to
Tau-aggregates. It indicates sufficient affinity of the probes for
imaging of Tau-aggregates in vivo. Furthermore, the series
may be radiolabeled with 11CH3, 18F and 123I at either the O or N
position and evaluated as positron emission tomography (PET)
and single photon emission tomography (SPECT) radioligands for
NFT-imaging in vivo.
affinity (Ki) of Methoxy-X04 is 26.8 nM.17 The same compound
was measured in Thiazine Red R assay and IC50 values are shown
in Table. 2. Unfortunately, the affinity of most probes to Tau-
aggregates could not be quantified due to spectral overlap with
the reference ligand Thiazine Red R. Moreover it interfered with
the acquisition of dose dependent fluorescence increase of the
bound probe, which was employed to exclude potent protein
disaggregating dyes. The affinity of the probes 9 and 11 to Tau-
aggregates was determined indirectly (IC50 values of 5.1 and
1.4 nM) in the displacement assay for Tau affinity versus the ref-
erence ligand Thiazine Red R using aggregated recombinant hu-
Figure 2. Immunohistochemical staining of hippocampal sections:
s
antibody AT8 (B) Ab-plaques with antibody amyloid A4 (A). See Fig. 3.

7670
J. Gu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7667–7671
Figure 3. Neuropathological staining of brain sections from the hippocampus of an AD patient (A–H). Probes 6 (A), 7 (B), 8 (C), 9 (D), 10 (E) and 11 (F) are clearly stained
neurofibrillary tangles. In contrast, the probes 9 (G) and 11 (H) faintly stained with Aß plaques. (tissues: hippocampus; patient: male, 71 years old, CERAD-score: 3; NFTs-
level: V).
Table 2
9 and 11 showed EC50 values of 8.57, 10.93, 8.31 and 0.93 lM,
Competitive Thiazine Red R displacement from tau-/Ab40-aggregates for probes 9, 11,
Methoxy-X04, FDDNP, PIB and BF158
respectively, after 24 h incubation (Table. 1). Compound 11 dis-
played higher cytotoxicity in both assays than other compounds
in this series. Both cytotoxicity studies suggested that 2-styrylin-
dolium probes have no or negligible cytotoxicity against zebrafish
embryos and HepG2 at the concentration employed for the histol-
Compound
Aggr.Tau-IC50/nM
Aggr. Aß40-IC50/nM
9
11
5.10
1.41
246
1635
3255
>10000
ND
1.37
140
1467
5190
>10000
Methoxy-X04
FDDNP
PIB
ogy experiments on human AD brain tissue (1 lM).
Taken together these results suggest that 2-styrylindolium
compounds should be advanced to in vivo mice experiments in
transgenic mice. However, the 4R Tau transgenic mice available
to the project are all based on the 4R Tau repeat and deposit very
little amounts of Tau. Moreover, these 4R Tau mouse models seem
to be void of the Tau-aggregate binding site present in humans.38,39
The presence of at least two different binding sites on Tau-
aggregates was demonstrated for the Tau probe THK523 recently.40
In conclusion, we have developed a series of water soluble 2-
styrylindolium probes for fluorescent imaging of NFTs in human
Alzheimer’s disease brain. The results of the in vitro fluorescent
staining protocol using brain sections of AD patient indicated po-
tent NFT imaging and low nonspecific binding. The cytotoxicity
experiments with zebrafish embryos and HepG2 cells suggest that
these probes may be suitable for further in vivo applications. This
indicates sufficient safety for in vivo evaluation in mouse models.
BF-158
ND, not determined due to spectral overlap with the reference compound in the
displacement assay.
Four of the 2-styrylindolium compounds (6, 8, 9 and 11) have
been further evaluated at different concentrations in the zebrafish
embryo development assay. The embryos developed well and no
lesions were found at the different concentrations up to 10 lM at
24 hours post-fertilization (hpf). After 72 hpf, only the juvenile fish
incubated with compound 11 showed small lesions in the yolk sac
(Fig. 4, J). The HepG2 cell proliferation assay of these four com-
pounds was used to evaluate the cytotoxicity in human cells. The
effective doses (EC50) were calculated via the best-fitted trendline
of cell vitality as a function of dye concentration. Compounds 6, 8,
Fig. 4. In vivo cytotoxicity studies with embryos of zebrafish after 24 (A–E) and 72 (F–G) hpf at 10
11 (E and J).
lM resp.. Controls (A and F), Probe 6 (B and G), 8 (C and H), 9 (D and I) and

J. Gu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7667–7671
7671
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.109. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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