Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains

Article abstract of DOI:10.1016/j.bmc.2013.06.039

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC₅₀ = 19 nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC₅₀ values of lower than 100 nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72 h post fertilization.

Full text of DOI:10.1016/j.bmc.2013.06.039

Accepted Manuscript
Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alz‐
heimer’s disease brains
Upendra Rao Anumala, Jiamin Gu, Fabio Lo Monte, Thomas Kramer, Roland
Heyny-von Haußen, Jana Hölzer, Valerie Goetschy-Meyer, Christian Schön,
Gerhard Mall, Ingrid Hilger, Christian Czech, Jochen Herms, Boris Schmidt
PII:
S0968-0896(13)00570-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.06.039
BMC 10932
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 April 2013
11 June 2013
16 June 2013
Please cite this article as: Anumala, U.R., Gu, J., Monte, F.L., Kramer, T., Haußen, R.H-v., Hölzer, J., Goetschy-
Meyer, V., Schön, C., Mall, G., Hilger, I., Czech, C., Herms, J., Schmidt, B., Fluorescent rhodanine-3-acetic acids
visualize neurofibrillary tangles in Alzheimer’s disease brains, Bioorganic & Medicinal Chemistry (2013), doi:
http://dx.doi.org/10.1016/j.bmc.2013.06.039
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in
Alzheimer’s disease brains
Upendra Rao Anumalaa, Jiamin Gua,† Fabio Lo Montea,‡ Thomas Kramera, Roland Heyny-von Haußenb,
Jana Hölzerc, Valerie Goetschy-Meyerd, Christian Schöne, Gerhard Mallb, Ingrid Hilgerc, Christian Czechd,
Jochen Hermse, Boris Schmidta,*
aClemens Schöpf-Institute of Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstrasse 22, 64287, Darmstadt, Germany
bInstitute of Pathology, Klinikum Darmstadt, Grafenstrasse 9, Darmstadt 64283, Germany
cInstitute of Diagnostic and Interventional Radioloygy I, Jena University Hospital – Friedrich Schiller University Jena, Erlanger Allee 101, 07747 Jena,
Germany
dF. Hoffmann-La Roche AG, Grenzacherstrasse 124, Gebäude 93/3.44, Basel 4070, Switzerland
eDepartment of Translational Brain Research, DZNE, German Center for Neurodegenerative Diseases, Munich, Germany
*Corresponding author. Tel.: +49 6151 164531; fax: +49 6151 163278.
E-mail address: schmidt_boris@t-online.de (B. Schmidt).
†Present address: Alberta Glycomics Centre, Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary Alberta T2N 1N4, Canada
‡ Present address: Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
There is a high demand for the development of an imaging agent for neurofibrillary tangles
(NFTs) detection in Alzheimer’s diagnosis. In the present study, a series of rhodanine-3-acetic
acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD
patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid
plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay
is not confirmed by the histopathological evaluation, which indicates significant differences in
the binding sites in the assays. Probe 6 showed binding affinity (IC50 = 19 nM) to tau aggregates
which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100
nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five
probes with human liver carcinoma cells revealed that these compounds excert negligible
cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24
and 72 hours post fertilization.
Available online
Keywords:
Alzheimer’s disease
Neurofibrillary tangles
Zebrafish
Cytotoxicity
Fluorescence imaging
2009 Elsevier Ltd. All rights reserved.
deep in tissue.4 Nevertheless it is limited by a time-consuming
1. Introduction
Alzheimer’s disease (AD) is one of the most common forms
of dementia. This disease is a progressive neurodegenerative
disorder associated with cognitive decline, disorientation and
language impairment. Incidence of new AD cases worldwide is
2
growing with the age of the baby boomer generation.1, The
major cause is still unknown for the disease but it is usually
accepted that the formation of two abnormal proteins; extra
cellular senile plaques (SPs) and neurofibrillary tangles (NFTs)
are the two key pathological findings in postmortem histology.
They are observed in hippocampus and cerebral cortex but also in
other areas of the brain. SPs are composed of aggregated β-
amyloid (Aβ) peptides and NFTs are formed by the aggregated
microtubule associated tau protein.3
Non-invasive imaging is vital for early diagnosis of AD.
Currently there are few positron emission tomography (PET)
imaging agents available for the early diagnosis. PET imaging is
a very well-known technique which provides good sensitivity
Figure 1. Structure of PET imaging probes for the detection of
NFTs and Aβ plaques in AD brain tissues

Table 1
General properties, optical properties, competitive Thiazine Red R displacement from tau-/Aβ40-aggregates for probes 1-7 and
cytotoxicity.
abs
em
Compound
Yielda
M.Wtb
cLogPc
λmax
λmax
Aggr.Tau-
IC50/nM
Aggr. Aβ40-
IC50/nM
Cytotox. EC50
(nm)d
445
497
444
391
468
466
455
(nm)d
511
645
525
533
555
651
645
(M)e
1
2
3
4
5
6
79
61
53
83
85
71
345.39
348.44
367.49
391.48
348.44
512.71
414.52
2.70
2.19
3.03
3.11
1.70
7.12
3.41
662
32
5002
55
>100*
>100*
>100*
>100*
>100*
18
43
292
91
28
63
218
91
19
7
69
238
1156
>100*
a
b,c
e
Isolated yields Determined by CS Chemoffice 10.0. d Measured in ethanol. EC50 declares the dye concentration in which 50% of the cells died after
incubation with the probe after 24 h of exposure. * a decrease of cell viability ≥ 50% was not observed at the tested concentrations. The IC50 data were obtained
from the average of technical replicates and are thus presented without standard deviation
data acquisition process and exposure to radioactivity,
insufficient spatial resolution along with expensive equipment
and need of highly skilled personnel. Pittsburgh compound B
(PiB) and Florbetapir F18 are well studied PET ligands targeting
SPs.5, 6 Florbetapir F18 is a PET probe for the imaging of Aβ
plaque density in AD patients and other causes of cognitive
decline.7 Florbetapir is the most widely used amyloid marker for
Aβ PET-imaging. PET ligands for imaging of NFTs were also
reported. These include [11C]-BF-158, [18F]-THK 523 and [18F]-
FPPDB (Figure 1).8-10 Moreover, Aβ imaging cannot differentiate
between different forms of frontotemporal lobe degeneration and
Aβ plaque load plateaus as disease progresses. Additionally, PIB
imaging is negative in up to 25% of the patients diagnosed with
AD.11 Furthermore, the formation of neurofibrillary tangles brain
correlates better with disease progression in AD.12 Fluorescence
imaging is a relatively new modality that offers real time, non-
radioactive, inexpensive in vivo imaging.13 It is frequently
rejected to be a non-viable modality in humans, however the
recent reports on pathological changes in the retina and the
human olfactory system suggest to investigate a non-invasive
access to amyloid and tau deposits by either scanning of the
retina or endoscopic nasal examination.14, 15
presence of hyperphosphorylated non-fibrillar tau was reported
for both the retina of P301S transgenic mice and the human
retina.17 Thus, there is an immediate need for fluorescent NFT-
imaging agents, which may complement the established amyloid
PET imaging tools.
Rhodanine based compounds are known for their ability to
inhibit tau aggregation in vitro and in vivo.18-20 Rhodanine and
thiohydantoin derivatives were reported as single photon
emission computed tomography (SPECT) imaging agents for
NFTs.21 Rhodanine-3-acetic acids are known as amyloid imaging
agents, dye sensitized solar cells and anthrax lethal factor
protease inhibitors.22-25 In the present study we synthesized and
studied the fluorescence imaging properties of rhodanine-3-acetic
acid (RA) derivatives as fluorescent probes for NFTs and
evaluated their affinities in the Thiazine red R assay. We
evaluated the effects of these probes on cell metabolism on
human hepato-cellular carcinoma cells (HepG2) and zebrafish
embryo development. These compounds were evaluated in
human and transgenic P301 mice retinae for their ability as
imaging agents. However, the absence of fibrillar tau deposits in
the retina of both species excluded a proof of concept.17
Fluorescence imaging of a boron dipyrromethane derivative
(BAP-1) and curcumin has been reported for Aβ deposits.16 The
Figure 2. Cell viabilities of HepG2 cells after 24 hours of exposure with
the probes 2 to 6 at 0.1, 1 and 10 M concentrations after 24 hours of
exposure. +/- SD
Scheme 1. Synthesis of rhodanine-3-acetic acids and 10-alkyl-10H-
phenothiazine-3-carbaldehyde.

Figure 3. Neuropathological staining of brain sections from the hippocampus of an AD patient (A-F). Probe 1 (A), 2 (B), 3 (C), 4 (D), 5 (E) clearly
stained flame shaped neurofibrillary tangles and Probe 6 (F) showed photo bleaching and high background tissue staining to NFTs. (NFTs are shown
with arrow). Tissues: hippocampus; patient: female, 80 years old, CERAD Score: 3; NFTs-level: V.
Initial immune histochemical staining confirmed that these
tissues contain both NFTs and SPs (supporting info). The RA
derivatives were used to stain these protein aggregates on brain
sections of the same patient. All of these compounds showed
selective binding to NFTs of post mortem AD brain tissues. Our
observations with RA derivatives showed selective staining to
NFTs rather than that of Aβ plaques, which stands in contrast
with the selectivity observed in the Thiazine red R displacement
assay. This striking difference indicates a significant difference
between the binding sites in the protein aggregation assay and the
human brain. A related discrepancy was reported for amyloid
binding agents in filtration assays and upon binding to human
tissue.27 Thus the affinity data obtained from the displacement
assays must be analyzed with care. The introduction of a bis-
thiophene moiety on RA leads to probe 3, which displayed good
fluorescence. Under the fluorescence microscope, this compound
displayed excellent staining of NFTs in brain tissues. Most of
these compounds showed reduced background staining in
comparison to the staining of NFTs. Probe 1 stained NFTs in
good contrast to the background, whereas photo bleaching was
observed with probe 6. (Figure 3).
2. Results and discussion
RA derivatives were synthesized by known methods using a
Knoevenagel condensation between RA and corresponding
aldehydes using sodium acetate in acetic acid.26 Most of the
aldehydes used in the present study were commercial but a few
aldehydes had to be synthesized by known methods. Probes 6
and 7 were synthesized in a three step procedure. In the first step,
alkylation of phenathiazine in the presence of sodium hydride
and dimethyl formamide was performed. The N-alkyl
phenathiazine was formylated in the second step employing the
Vilsmeier-Haack reaction of phosphorous oxychloride and
dimethylformamide. In the final step
a
Knoevenagel
condensation was carried out between the RA derivatives and N-
alkyl phenathiazine aldehyde to obtain the probes 6 and 7. The
yields of the Knoevenagel condensation for probes 1-7 varied
from 61-85% depending on the aldehydes. The presence of
electron donating groups increased the reactivity whereas
electron withdrawing groups decreased the reactivity and yield of
derivative formation.
All of these compounds were further evaluated for their ability
to visualize NFTs in post mortem human AD brain sections.
Figure 4. In vivo cytotoxicity studies with embryos of zebrafish after 24 (A-F) and 72 (G-L) hpf. Controls (A and G), Probe 2 (B and H), 3 (C and
I), 4 (D and J), 5 (E and K) at 10 M concentration and Probe 6 (F and L) at 5 M concentration.

The in vitro affinity studies of these compounds were
tested concentrations (100 µM was the highest concentration
tested). The EC50 of probe 6 after 24 h of incubation is about 18
µM. Figure 2 shows the cell viability of these compounds in
HepG2 cells at 24 h after incubation.
28, 29
performed by the Thiazine red R displacement assay.15,
Thiazine red R is known for its binding to tau aggregates in vitro
and for its superiority over Thioflavin T.30 Aggregates of
recombinant human-microtubule associated tau protein, which
was purified from Escherichia coli, and synthetic Aβ peptides
were used in this study. The in vitro aggregates of A40 and A42
display differences in the binding sites for amyloid ligands,
which depend on the aggregation conditions and differ from the
binding situation observed in human tissue. Thus we selected
A40 for the aggregation assay as it is the predominant
component in amyloid plaques and thus likely to be engaged in
binding of the probes.
It was recently reported that fibrillar tau inclusions can be
observed in retinal ganglion cells of P301S mice in and ex vivo.
17, 33 However, we could not visualize fibrillar tau aggregates with
these compounds (Probe 1, 2, 3 and 4) in the retina of P301S
mice. These results suggest that the tau aggregates in the retina
of P301S mice may be distinctly different from human tau
aggregates that can be stained with compounds 1-4. This may be
due to the fact that the human brain produces a mixture of 3R and
4R isoforms of tau, whereas the P301S mice do produce one
isoform only.
IC50 values of Thiazine red R are depicted in Table 1. All of
these probes have higher affinity to aggregated tau than
aggregated Aβ40 in the Thiazine red R assay. However, the
apparent selectivity in the histology was even higher than
indicated by the affinity assays. This suggests fundamental
differences between the protein aggregates. Caveat: chemically
induced synthetic tau fibrils are not a reliable surrogate for native
NFTs, they differ in size, morphology and binding properties.31
Five compounds out of seven showed IC50 values of less than 100
nM. Probe 6 showed almost five fold higher affinity to tau
aggregates (19 nM) than to amyloid-β aggregates (91 nM), the
highest selectivity to tau aggregates in this series. Probe 3
showed around 7-fold higher affinity to aggregated tau but
weaker binding in comparison to probe 6.
3. Conclusion
In summary, RA derivatives provide selective visualization
of NFTs over Aβ plaques in brain sections of post mortem AD
patient. Excellent affinity to fibrillar tau aggregates was observed
in the Thiazine red R displacement assay and no or negligible
cytotoxicity of these compounds in HepG2 cells or in zebrafish
embryo development. Hyperphosporylated, non-fibrillar Tau
aggregates as present in aggresomes or mouse retina are not
stained by these dyes, this observation indicates selective binding
to fibrillar tau. Further evaluation of these compounds is needed
in mammals that produce a similar isoform mixture of tau
aggregates as humans.
Probes 2, 3, 4 and 5 displayed IC50 values of 32 nM, 43 nM,
28 nM and 63 nM, respectively. Probes 1 and 7 display poor
affinity compared to the other probes in this series. Apparently,
the binding affinities of RA derivatives are higher to tau
aggregates than to Aβ40 aggregates. It is generally known that
compounds with a cLogP lesser than 3 and a molecular weight
less than 500 Daltons are most favourable for blood brain barrier
(BBB) penetration.32 RA derivatives were designed according to
these cLogP values. According to the cLogP value, Probe 5
(cLogP value of 1.7) has the lowest cLogP value in this series
and other probes except probe 6 are in the range of 2.0 to 3.5.
Probe 6 is characterized by a cLogP of 7.12. Despite the high
affinity of probe 6 to tau aggregates the lipophilicity, poor
solubility and photo bleaching make it unsuitable for in vivo
studies.
4. Experimental
4.1. General
All commercial chemicals, reagents, solvents were purchased
from Sigma-Aldrich. All reactions were performed under argon
atmosphere using dry solvents unless otherwise specified. H-
1
NMR spectra were recorded on a Bruker AC300, ARX300 and
DRX 500 spectrometer at 300 MHz and 500 MHz respectively.
The 13C-NMR spectra were recorded on a Bruker AC300,
ARX300 and DRX 500 spectrometer at 75 MHz and 125 MHz
respectively. Chemical shifts values were reported as % values
(ppm) downfield from Me4Si. UV-Vis spectra were carried out
by Shimadzu UV-2401PC. Fluorescence emission experiments
were carried out by TECAN Infinite® M1000 PRO. Mass
spectrometry was performed on a MAT 95 double focusing
sector field MS. Flash column chromatography was carried out
with Merck silica gel 60 (15-40 mm). Acid protons are not
visible in 1H-NMR for some compounds measured in DMSO-d6.
As safety is one of the most important factors during the
development of an imaging agent, the effects of these compounds
on HepG2 cell proliferation and zebrafish embryo development
were evaluated. Except for probes 1 and 7, all other probes were
tested for their cytotoxicity in zebrafish embryos. The
cytotoxicity evaluation of probes 2-5 clearly showed that most of
these probes have no or negligible cytotoxicity at concentrations
up to 10 M. Probe 6 showed cytotoxicity at 10 M
concentration. Yet, no lesions in embryos of zebrafish were
observed at 5 M concentration at 24 and 72 hours post
fertilization (hpf), which indicates that all of these compounds
display negligible cytotoxicity up to a concentration of 5 M. It
was observed that zebrafish embryos developed normally in
comparison with control for all RA derivatives at 5 M. Further
experiments with liver HepG2 cells were performed and the
effective doses (EC50) were calculated via the best-fitted trend
line of cell viability as a function of dye concentration. Probes
within the range of 1-100 nM affinity to tau aggregates were
considered for the cytotoxicity studies. Probes 2, 3, 4, 5 and 6
were studied in vitro for their cytotoxicity in HepG2 cells. Apart
from compound 6, the EC50 values of all compounds are > 100
µM and loss of cell viability ≥ 50 % was not observed at the
4.2. General procedure for the synthesis of RA derivatives
4.2.1. (Z)-2-(4-Oxo-5-((5-phenylfuran-2-yl)methylene)-2-
thioxothiazolidin-3-yl)acetic acid (1)
To a solution of 5-phenylfuran-2-carbaldehyde (1.0 mmol,
1
equiv.) and rhodanine-3-acetic acid (1.0 mmol, 1 equiv) in glacial
acetic acid (5 ml) was added of sodium acetate (3.0 mmol, 3
equiv) and the reaction mixture was stirred for 7 hours at
100oC, forming a precipitate after cooled to room temperature.
The resulted crude product was recovered by filtration and
recrystallized from acetone/water mixture to give 272 mg of the
titled compound as an orange solid; 1H-NMR (500 MHz,
DMSO-d6) δ = 13.42 (s, 1H), 7.91 – 7.85 (m, 2H), 7.77 (s, 1H),
7.61 – 7.55 (m, 2H), 7.49 – 7.44 (m, 1H), 7.42 (d, J = 3.8 Hz,
1H), 7.36 (d, J = 3.8 Hz, 1H), 4.75 (s, 2H); 13C-NMR (125 MHz,
DMSO-d6) δ = 194.24, 167.78, 166.48, 158.78, 149.61, 129.91,
129.85, 128.97, 124.96, 124.18, 119.62, 118.28, 110.84, 45.46;

MS (EI, 70 eV) m/z = 345 [M+]; UV/Vis (Ethanol) λmax = 445
nm.
δ = 192.70, 167.25, 166.28, 147.68, 143.72, 133.13, 130.98,
129.11, 128.08, 126.99, 126.92, 123.45, 122.86, 120.99, 118.69,
115.32, 115.20, 45.02, 35.50; MS (EI, 70 eV) m/z = 414 [M+];
UV/Vis (Ethanol) λmax = 455 nm.
4.2.2. 2-((Z)-5-((E)-3-(4-(dimethylamino)phenyl)allylidene)-4-
oxo-2-thioxothiazolidin-3-yl)acetic acid (2)
4.3. Methods
Further purification of this compound was carried out using
column chromatography. A red solid; 1H-NMR (500 MHz,
DMSO-d6): δ = 7.58 (dd, J = 7.2, 4.8 Hz, 3H), 7.35 (d, J = 14.8
Hz, 1H), 6.87 – 6.78 (m, 1H), 6.74 (d, J = 9.0 Hz, 2H), 4.69 (s,
2H), 3.02 (s, 6H); 13C-NMR (125 MHz, DMSO-d6): δ = 192.84,
167.89, 166.03, 152.34, 148.57, 136.69, 130.91, 123.49, 118.62,
118.53, 112.35, 45.37. 39.65 (NMe2 peak un resolved from
4.3.1. Immunohistochemical staining
Immunohistochemical staining was carried out on four
micrometers thick sections of the AD patient tissues by a
Ventana Benchmark automated stainer (Ventana, Tuscon, AZ).
The antibodies are anti-PHF-Tau 75 clone AT8 mAb (Thermo
Scientific Pierce Protein Research Products, Rockford, IL), TAU
Ab-3 (Neomarkers, Freemont, CA), and amyloid A4 (BAM10,
Sigma, St. Louis, MO) and the Ultraview Universal DAB
Detection Kit (Ventana, Tuscon, AZ) were used in staining
experiments.
+
DMSO); HRMS m/z [M+] calcd for C16H16N2O3S2 : 348.0602,
found 348.0603; UV/Vis (Ethanol) λmax = 497 nm.
4.2.3. (Z)-2-(5-(2,2'-Bithiophen-5-ylmethylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (3)
1
4.3.2. Neuropathological staining of AD brain sections
A brick red solid; H-NMR (500 MHz, DMSO-d6) δ = 8.14
(s, 1H), 7.78 (dd, J = 4.0 , 0.5 Hz, 1H), 7.70 (dd, J = 5.0 , 1.0 Hz,
1H), 7.59 (dd, J = 3.7,1.0 Hz, 1H), 7.54 (d, J = 4.0 Hz, 1H), 7.18
(dd, J = 3.6, 1.4 Hz, 1H), 4.73 (s, 2H); 13C-NMR (125 MHz,
DMSO-d6) δ = 192.18, 167.72, 166.41, 145.64, 138.39, 136.11,
135.66, 129.40, 128.52, 127.12, 126.91, 126.32, 118.98, 45.63;
MS (EI, 70 eV) m/z = 367 [M+]; UV/Vis (Ethanol) λmax = 444 nm.
Post mortem brain tissues from an AD patient (80-year old
female) were obtained at autopsy. Four micrometer thick paraffin
embedded serial sections of the hippocampus area were
deparaffinized with xylene and ethanol. These sections were then
hydrated in distilled water. Ethanol solution (500 L) of probes 1
to 7 with concentration of 1 mM was poured on tissue slide and
waited for 10 min. These sections were washed with methanol
and differentiated in 1% acetic acid solution for 20 min and
washed with water. These tissue sections were finally treated
with Roti – Mount Fluor Care (from Sigma-Aldrich) and covered
with coverslip. Fluorescence microscopic examination was
performed using a Axioskop microscope with a HBO100
fluorescence illuminator (Carl Zeiss, Oberkochen, Germany)
with band pass filter set 09 BP450-490, FT510, LP515, the filter
set 02 G365, FT395, LP420 and the filter set 15 BP546, FT580,
LP590.
4.2.4. (Z)-2-(5-((5-(4-Methoxyphenyl)thiophen-2-
yl)methylene)-4-oxo-2-thioxothiazolidin -3-yl)acetic acid (4)
1
A maroon solid; H-NMR (500 MHz, DMSO-d6) δ = 8.08
(s, 1H), 7.77 (d, J = 4.5 Hz, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.64
(d, J = 4.0 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 4.56 (s, 2H), 3.82
(s, 3H); 13C-NMR (125 MHz, DMSO-d6) δ = 192.33, 167.35,
166.70, 160.67, 152.66, 138.38, 135.88, 127.88, 126.74, 125.62,
125.11, 118.96, 115.26, 55.86, 46.98; MS (EI, 70 eV): m/z = 391
[M+]; UV/Vis (Ethanol): λmax = 451 nm.
4.2.5. (Z)-2-(4-Oxo-5-(4-(pyrrolidin-1-yl)benzylidene)-2-
thioxothiazolidin-3-yl)acetic acid (5)
4.3.3. In vitro cell proliferation assay
The cytotoxicity data were determined via CellTiter
96®AQueous non-radioactive cell proliferation assay (Promega,
Madison, USA), which is based on the reduction of a tetrazolium
salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-
2-(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] into
formazan product by intrinsic dehydrogenases of living cells.
1
A maroon solid; H-NMR (500 MHz, DMSO-d6): δ = 7.69
(s, 1H), 7.49 (t, J = 5.8 Hz, 2H), 6.71 (t, J = 5.9 Hz, 2H), 4.54
(s, 2H), 3.36 (t, J = 6.6 Hz, 4H), 1.99 (t, 4H); 13C-NMR (125
MHz, DMSO-d6): δ = 192.94, 167.39, 167.13, 149.93, 135.24,
133.84, 120.04, 113.81, 112.97, 47.86, 46.82, 25.37; MS
(EI, 70 eV): m/z = 348 [M+]; UV/Vis (Ethanol): λmax = 468 nm.
a
In short, human liver hepatocellular carcinoma cells HepG2,
maintained in DMEM/F12 1+1 and supplemented with 10 %
fetal bovine serum, were seeded in 96-well plates and incubated
with 0.1 µM, 1 µM, 10 µM and 100 µM of probe 2, 3, 4, 5 and 6
for 24 hours. After washing, CellTiter 96®AQueous non-
radioactive cell proliferation assay reagent was added to the cells
and the number of metabolically active cells was calculated by
measuring the amount of formazan product via photometric
analysis at λ = 492 nm. The absorbance of treated samples was
normalized to untreated controls (percent of non-treated
controls), which resulted in cell viability. Finally effective doses
(EC50) that induced a loss in cell viability of 50 % were
calculated via the best-fitted trend line of cell viability as a
function of dye concentration.
4.2.6. (Z)-2-(5-((10-Octyl-10H-phenothiazin-3-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (6)
1
A maroon solid; H-NMR (500 MHz, DMSO-d6) δ = 7.77
(s, 1H), 7.48 (dd, J = 8.8, 2.2 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H),
7.25 – 7.20 (m, 1H), 7.18 – 7.14 (m, 2H), 7.09 – 7.05 (m, 1H),
7.00 (td, J = 7.5, 1.0 Hz, 1H), 4.73 (s, 2H), 3.93 (t, J = 7.0 Hz,
2H), 1.73 – 1.64 (m, 2H), 1.43 – 1.34 (m, 2H), 1.30 – 1.17
(m, 8H), 0.82 (t, J = 6.9 Hz, 3H); 13C-NMR (125 MHz,
DMSO-d6) δ = 193.17, 167.75, 166.78, 147.71, 143.43, 133.58,
131.30, 129.95, 128.42, 127.71, 127.37, 124.38, 123.94, 122.68,
119.04, 116.86, 116.62, 47.30, 45.49, 31.53, 29.03, 28.89, 26.51,
26.39, 22.47, 14.39; MS (EI, 70 eV): m/z = 512.0 [M+]; UV/Vis
(Ethanol) λmax = 466 nm.
4.3.4. Thiazine Red R displacement assay
4.2.7. (Z)-2-(5-((10-Methyl-10H-phenothiazin-3-
Recombinant human-microtubule associated 4R tau protein
purified from Escherichia coli and Synthetic Aß40 was used in
this assay. Tau protein was aggregated at 5 uM concentration
with arachidonic acid (100 uM) in Tris 10 mM pH = 8, 24h at
37°C, whereas Aß40 was aggregated at a concentration of 50 µM
with arachidonic acid (100 µM) in Tris 10 mM pH = 8, for three
yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (7)
1
A dark red solid; H-NMR (500 MHz, DMSO-d6) δ = 7.79
(s, 1H), 7.51 (dd, J = 2.20, 8.59 Hz, 1H), 7.43 (d, J = 2.17 Hz,
1H), 7.26 (td, J = 1.60, 7.70 Hz, 1H), 7.19 (dd, J = 1.56, 7.82 Hz,
1H), 7.11 (d, J = 8.62 Hz, 1H), 7.03 (dd, J = 5.68, 8.03 Hz, 2H),
4.73 (s, 2H), 3.39 (s, 3H); 13C-NMR (125 MHz, DMSO-d6):

days at 37°C. For the displacement assay, Thiazine red R was
added at same concentration of the Kd to the respective
aggregated binding sites (Kd for aggregated Tau = 18 nM, Kd for
aggregated Aß = 49 nM). To determine the affinity of the
required probe, the probe was added at different concentrations in
the assay ranging from 0.1 nM to 10000 nM. Auto fluorescence
of the probe was measured together with aggregated proteins.
Negative controls were obtained from Thiazine red R and
aggregated proteins. Assay was performed in Perkin Elmer
OptiPlate 384, black, 45 ul assay volume, assay buffer was DPBS
no CaCl2 no MgCl2 (GIBCO N. 14020). Tested compounds were
diluted in DMSO and 2.25 µl was added to the assay (5% DMSO
final). Assay experiment was initiated by the addition of the
aggregated protein (competitive condition). Plates were shortly
shacked (1 min with Sterico variomag teleshake) and incubated
for 30 min at room temperature. Measurements were performed
with En:Vision (Perkin Elmer), at excitation 531 nm/emission
595 nm. Corresponding IC50 were calculated by excel fit.
Schneider, J.; Arora, A.; Carpenter, A.; Flitter, M.; Joshi, A.;
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La Joie, R.; Tafani, M.; Hommet, C.; Chételat, G.; Mondon, K.; de La
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Vercouillie, J.; Vellas, B.; Eustache, F.; Guilloteau, D. Eur. J. Nucl. Med.
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4.3.5. In vivo zebrafish embryo development assay
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Zebra fish were bred in 2L spawning tank and maintained
according to the methods described by Christiane Nuesslein-
Volhard and Ralf Dahm. Briefly, zebrafish were raised on 14 h
light 10 h dark cycle at 26.0 ± 0.5°C. The embryos were obtained
via natural mating and cultured in the water. The embryos were
collected and placed into 24-well plates, every ten embryos per
well. When the embryos older than 6 hpf, more than 50%
epiboly, they were treated with 1, 5, 10 μM of RA derivatives in
E2 solution. The phenotypes were observed and images were
captured using the Axio Scope A1 microscope system from Carl
Zeiss at 24 and 72 hpf. All experiments in this study were carried
out according to the ethical and welfare principles in legislation
on animal research in Germany.
11. Takeuchi, J.; Shimada, H.; Ataka, S.; Kawabe, J.; Mori, H.; Mizuno, K.;
Wada, Y.; Shiomi, S.; Watanabe, Y.; Miki, T. Dement. Geriatr. Cogn.
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4.3.6. In vivo scanning of the mouse retina
Mixed genders of homozygous mice expressing human
mutant P301S tau, those were backcrossed for at least 7
generations to obtain animals on a pure C57Bl/6 background
were used in this study. The ophthalmological examinations of
the mouse retinas were performed using a modified Spectralis
HRA + OCT system (Heidelberg Engineering, Dossenheim,
Germany) like described elsewhere.17 In short, mice received 24
h before the imaging sessions i.p. injections of the fluorescent
probes (28-31 mM in DMSO). Imaging was performed using two
different lasers wavelengths for the excitation of the fluorophores
(450 and 488 nm). Emission filters used in this study were LP
458 nm, BP 512/25, BP 550/49 nm and BP 617/73 nm. To
exclude false positive signals from retinal autofluorescence, pre-
examinations of the retinas before the application of the
fluorophores were performed.
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Motta, C.; Taliani, S.; Visse, R.; Nagase, H.; da Settimo, F.; Novellino,
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5. Acknowledgement
This work was supported by grants from the German Federal
Ministry of Education and Research (Bundesministerium für
Bildung und Forschung, Germany, 13N10636) and the Hans and
Ilse Breuer-foundation.
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Fluorescent rhodanine-3-acetic acids
visualize neurofibrillary tangles in
Alzheimer’s disease brains
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Upendra Rao Anumalaa, Jiamin Gua, Fabio Lo Montea, Thomas Kramera, Roland Heyny-Von Haußenb, Jana
Hölzerc, Valerie Goetschy-Meyerd, Christian Schöne, Gerhard Mallb, Ingrid Hilgerc, Christian Czechd, Jochen
Hermse, Boris Schmidta,*