Novel Isatin Thiosemicarbazone Derivatives as Potent Inhibitors of β-Amyloid Peptide Aggregation and Toxicity

Article abstract of DOI:10.1021/acschemneuro.0c00208

Inhibition of β-amyloid peptide (Αβ) aggregation in Alzheimer's disease (AD) is among the therapeutic approaches against AD which still attracts scientific research interest. In the search for compounds that interact with Aβ and disrupt its typical aggreg

Full text of DOI:10.1021/acschemneuro.0c00208

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Article
Novel isatin thiosemicarbazone derivatives as potent
inhibitors of #-amyloid peptide (##) aggregation and toxicity
Marina Sagnou, Barbara Mavroidi, Archontia Kaminari, Nikos Boukos, and Maria Pelecanou
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00208 • Publication Date (Web): 29 Jun 2020
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Novel isatin thiosemicarbazone derivatives as potent inhibitors of β-amyloid peptide
(Αβ) aggregation and toxicity
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Marina Sagnou , Barbara Mavroidi , Archontia Kaminari , Nikos Boukos , Maria
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Pelecanou1
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1Institute of Biosciences & Applications, National Centre for Scientific Research
“Demokritos”, 15310 Athens, Greece
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2Institute of Nanoscience and Nanotechnology, National Centre for Scientific Research
“Demokritos”, 15310 Athens, Greece
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Corresponding Author
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Marina Sagnou, Institute of Biosciences &Applications, National Centre for Scientific
Research “Demokritos”, Patriarhou Grigoriou & Neapoleos 27, 15310, Athens, Greece;
E-mail: sagnou@bio.demokritos.gr
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ABSTRACT
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Inhibition of the amyloid β-peptide (Αβ) aggregation of Alzheimer's disease (AD) is
among the therapeutic approaches against AD which still attracts scientific research
interest. In the search for compounds that interact with Aβ and disrupt its typical
aggregation course towards oligomeric or polymeric toxic assemblies, small organic
molecules of natural origin, combining low molecular weight (necessary blood-brain
barrier penetration) and low toxicity (necessary for pharmacological application), are
greatly sought after. Isatin (1H-indoline-2,3-dione), a natural endogenous indole, and
many of its derivatives exhibit a wide spectrum of neuropharmacological and
chemotherapeutic properties. The synthesis and biological evaluation of four new isatins
as inhibitors of Aβ aggregation is presented herein. In these derivatives the N-phenyl
thiosemicarbazide moiety is joined at the 3-oxo position of isatin through Schiff base
formation, and substitutions are present at the indole nitrogen and position 5 of the isatin
core. Biophysical studies employing circular dichroism, thioflavin T fluorescence assay
and transmission electron microscopy reveal the potential of the ITSCs to alter the course
of Αβ aggregation, with two of the derivatives exhibiting outstanding inhibition of the
aggregation process, preventing completely the formation of amyloid fibrils.
Furthermore, in in vitro studies in primary neuronal cell cultures, the ITSCs were found
to inhibit the Aβ-induced neurotoxicity and reactive oxygen species (ROS) production, at
concentrations as low as 1 μM. Taken all together, the novel ITSCs can be considered as
privileged structures for further development as potential AD therapeutics.
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Keywords:
Alzheimer's disease; β-Amyloid peptide; Isatin thiosemicarbazones; Inhibitors of β-
amyloid aggregation, Circular dichroism spectropolarimetry; Primary neuronal cell
cultures; Antioxidant activity
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INTRODUCTION
Isatin (1H-indole-2,3-dione) (1, Figure 1) is a well-known natural product which can be
found in plants of Genus Isatis and Couroupita Guianensis. This indolic derivative is
known in the chemical industry for nearly 140 years, as it is a product of indigo dye
oxidation by means of nitric and chromic acid.1-5 It is an endogenous oxidized indole
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present in the mammalian brain, peripheral tissues and body fluids , originally identified
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in humans as one component of the monoamine oxidase inhibitory activity. Isatin has
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been extensively studied in behavioral and metabolic assays and >90 proteins have been
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identified through proteomic analysis as its potential biological targets . It acts as a
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potent inhibitor of human mitochondrial monoamine oxidase B (MAO-B) and
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administered exogenously was found to significantly increase the levels of acetylcholine,
choline and dopamine in rat brain tissues.10 It is a flexible and versatile synthetic
scaffold, consisting of an indole nucleus and two types of carbonyl groups, a keto and a
lactam group that render it a favorable building block for Schiff base reactions,
heterocyclic compound synthetic strategies and pharmacophore development.11
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Isatin derivatives possess a wide range of biological activities, including
antimicrobial, anti-HIV, antiviral, antimalarial, anticancer, anticonvulsant, anti-asthmatic,
antiinflammatory and analgesic activities.12,13 Isatin derivatives have also been studied as
potential therapeutic agents against Alzheimer’s disease (AD).14-20 More specifically, a
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library of N-substituted isatin 3-benzoylhydrazones has been explored as β-secretase
(BACE-1) inhibitors using a virtual high-throughput screening (HTS) approach.14
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Furthermore, out of eight compounds sorted out in a HTS based on the fluorescence of
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Aβ42-GFP fusions on a collection of 65,000 small molecules, compound D737 (2, Figure
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1), a 2-oxoindole derivative, proved to be the most effective in inhibiting Aβ42
aggregation, reducing Aβ42-induced toxicity in neuronal cells, as well as increasing the
life span and locomotive ability of flies in a Drosophila melanogaster model of AD.15
Campagna, Catto and their colleagues have extensively studied a series of isatin-3-
arylhydrazones16 (3, Figure 1) and other related molecules as inhibitors of Aβ40
aggregation revealing interesting structure activity relationships like 17-20 the role of
lipophilicity, the positive contribution of the 5-methoxy substitution on the isatin core and
the opportunity of alkylating the indole nitrogen for efficient aggregation inhibition.16
The findings are in agreement with the general notion that indole derivatives interact with
Aβ, blocking its self-assembly and fibril formation, by interfering with π–π interactions
or π-stacking, hydrophobic forces and electrostatic interactions between the Aβ side
chains and/or by effectively forming polar interactions and/or hydrogen bond with Aβ
moieties.21-23
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Based on the encouraging results of isatin-3-arylhydrazones and in view of our ongoing
investigation on isatin-3-thiosemicarbazones (ITSCs)24,25 and AD therapeutics26,27 we
report herein the synthesis and biological evaluation of four novel ITSCs as inhibitors of
Aβ aggregation and neuronal cell toxicity. The thiosemicarbazone (TSC) moiety has
been utilized in the synthesis of metal complexes able to interfere with Aβ peptide
aggregation or up-regulate Aβ peptide degrading enzymes. More specifically, copper-
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bis(thiosemicarbazonato) and Ru(II)‐p‐cymene thiosemicarbazone complexes have
shown interesting biological activities as potential Aβ aggregation inhibitors.28,29
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Furthermore, recent work by Kulkarni and his colleagues showed that a 3-acetylcoumarin
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compared to acetylcoumarin alone.30,31 Also, the TSC moiety provided additional benefit
to the acetylcoumarin molecule in rescuing Aβ42 induced toxicity in neuronal SH-SY5Y
cells and reduced its cell toxicity, whereas results from docking studies showed that the
thiosemicarbazone moiety plays an important role due to its capacity for formation of
hydrogen bonding contacts with peptide residues. Therefore, it was reasonable to
anticipate that the combination of the thiosemicarbazone moiety with the isatin core may
generate active agents with anti-amyloid activity. The four new compounds were
evaluated in detail with circular dichroism (CD), thioflavin T (ThT) fluorescence assay,
and transmission electron microscopy (TEM), for their potential to inhibit the assembly
of Aβ into oligomeric/polymeric aggregates and the eventual formation of amyloid
fibrils. Furthermore, the effect of the ITSCs on the cytotoxic and ROS producing activity
of Aβ was evaluated in primary neuronal cells.
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Figure 1. Chemical structures of isatin (1) and other active indole derivatives (2, 3) in
the literature.
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RESULTS AND DISCUSSION
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Design. A preliminary screening of 13 structurally related ITSCs (see Supporting
Information) for their potential to inhibit aggregation of Aβ40 was conducted by means
of CD. The structures were designed aiming to investigate the potential contribution of
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the isatin substitution at position 5- and the para- substitution of the phenyl
thiosemicarbazone moiety (Figure 2). Out of the 13 structurally related ITSCs only
compounds 4 and 5 (Figure 3), when co-incubated with Aβ peptide, resulted in a
considerable alterations of the characteristic CD spectrum of the Aβ peptide compared to
spectrum of the peptide alone. The rest of the ITSCs did not affect the CD spectrum of
the Aβ peptide at all, suggesting the lack of any interaction with the peptide.
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Figure 2. The design of the initial library of ITSCs
Consequently, ITSCs 4 and 5 were further converted to the piperidine N-Mannich base
derivatives 6 and 7, respectively (Figure 3). This was based on literature reports stating
that Mannich base moieties exhibit good antioxidant,32,33 anti-inflammatory and AChE
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inhibitory activities, rendering them useful in the anti-amyloid drug design.
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Figure 3. Chemical structures of the ITSC derivatives 4 - 7 of this work.
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Synthesis. Compounds 4 and 5 were synthesized in high yield reactions between 5-
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methoxy- or 5-fluoroisatin, respectively, with phenyl thiosemicarbazide, using acetic acid
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as a catalyst to initiate the reaction, based on reported methods (Scheme 1). In both
cases the desired product precipitated out of the reaction mixture and was collected in
pure form after a single recrystallization from ethanol. It is worth noting that this simple,
high yielding, and low-cost preparation of scaffolds with pharmacophoric potential is a
highly advantageous and desirable feature. The Mannich bases, 6 and 7, were
consequently prepared by condensing the acidic isatin NH-group of 4 and 5, respectively,
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with formaldehyde and piperidine. The Mannich reactions were of equally high yield as
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the preceding Schiff reactions (Scheme 1).
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All compounds were characterized by H and C NMR spectroscopy as well as MS-ESI
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(Supporting Information). The ITSC derivatives are soluble in CHCl , DMSO, DMF and
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they are stable in organic solvents for at least a month as witnessed by NMR.
Scheme 1. Synthetic route of the ITSC derivativesa
R
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O
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a.
R
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H
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, R = OMe
, R = F
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aReagents and conditions: (a) ethanol, cat. CH
COOH, Reflux; (b) formaldehyde, piperidine.
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CD studies of the inhibition of Aβ40 and Aβ42 aggregation. Circular dichroism (CD)
spectropolarimetry was employed to assess the effect of the ITSCs on the aggregation
process of Aβ40 and Aβ42 during a 30-day course. As can be seen in Figure 3 for the
Aβ40 (top left) and Aβ42 (top right) peptides, the typical aggregation process of Aβ
produces characteristic CD spectra over time reflecting the conformational changes that
take place in solution: the random coil Αβ monomers are gradually converted into β-sheet
assemblies, finally producing insoluble amyloid fibrils that precipitate from solution with
loss of CD signal. 38
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It is evident from the spectra in Figure 4 (left column) that the addition of compounds 4
and 7 in solutions of Aβ40 at a ratio of 1:1 strongly inhibited its aggregation. The CD
spectra essentially remained at random coil conformation (min at 198 nm) with a small
reduction in intensity as time proceeded and without any further transformation, even at
day 30. In the presence of derivatives 5 and 6, a decrease in the intensity of the random
coil trace was observed, suggesting the formation of amyloid fibrils, though with
considerable delay compared to solutions of plain Aβ40. More specifically, in the case of
derivative 5, a shift of the absorption minimum to higher wavelengths (200 nm) was
noted and the signal approached zero at day 30, while in derivative 6 the random coil
minimum at 198 nm remained stable and CD signal was still present at day 30. The
interaction of compounds 4 and 7 with Aβ42, which is more fibrillogenic and aggregates
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faster than Aβ40, was even more pronounced. Right from the beginning, composite
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remained unchanged in solution for the whole duration of the study with no sign of fibril
formation. Corresponding experiments in the presence of compounds 5 and 6 revealed
once again their effect to delay the typical Aβ aggregation process, though not preventing
fibril formation leading to almost complete signal loss at day 30.
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Figure 4. CD spectra of plain solutions of Aβ40 and Aβ42 (50 μM) as well as in the
presence of the ITSC derivatives (Αβ:ITSCs ratio 1:1). Spectra were recorded for a
period of 30 days at 37 °C. Representative spectra from n = 3 independent experiments
are presented.
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ThT test for the detection of typical amyloid fibrils. The aged for 30 days solutions of
the CD study were subsequently subjected to the thioflavin T (ThT) binding assay that
detects amyloids fibrils. Specifically, the ThT dye, which is negligibly fluorescent in
solution, displays significant fluorescence enhancement upon binding to typical amyloid
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fibrils. As it is apparent in Figure 5 (black line) the ThT assay of plain solutions of
Aβ40 and Aβ42 resulted in considerable fluorescence signal indicative of fibril
formation. This fluorescence signal was dramatically decreased when either Aβ40 or
Aβ42 were incubated with 4 or 7 for 30 days, a finding which indicates the lack of fibrils
in solution, and is in agreement with the CD data. The presence of 5 and 6 in solutions of
Aβ40 (Figure 5A) resulted in reduced fluorescence intensity, compared to plain Aβ40,
indicating reduction in fibril formation. Furthermore, the shift in fluorescence maximum
to higher wavelengths may suggest the presence of fibrils of differentiated structure
compared to typical Aβ40 fibrils. The presence of compounds 5 and 6 in solutions of
Aβ42 (Figure 5B) did not result in significant reduction of fluorescence intensity
indicating that fibril formation remains almost unaffected. Again, the ThT results for
compounds 5 and 6 are in complete agreement to the CD studies.
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Figure 5. Αβ aggregation assay using fluorescence emission of ThT upon binding to
aggregated Αβ (25 μΜ, 30 d). A lower intensity than that of plain Aβ solutions ThT
(black line) indicates lower concentration of Αβ fibrils. (A) Αβ40 solutions in the absence
and presence of ITSCs and (B) Αβ42 solutions in the absence and presence of ITSCs.
Fluorescence was monitored after excitation at λ = 440 nm. Representative spectra from
n = 3 independent experiments are presented.
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TEM microscopy images of amyloid fibrils. To evaluate the effect of ITSCs on the
occurrence and morphology of Aβ fibers, TEM microscopy was used employing the 30
day aged samples subjected to CD and ThT analysis. Images of plain Aβ42 solutions as
well as after incubation with compounds 4 and 7 are presented in Figure 6. In the absence
of any ITSCs the typical dense network of intertwined Aβ fibrils was observed.41
However, upon incubation of Aβ with either 4 or 7, the samples were devoid of fibrils
and only a very small amount of sporadic crystal-like formations could be observed
which may be attributed to a degree of self-organization of the ITSCs under the particular
experimental conditions. The complete lack of fibrils is an impressive finding. To the
best of our knowledge, it is the first time that a complete absence of Aβ typical fibrils or
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other type of lower amorphous aggregates has been reported in the presence of an
aggregation inhibitor. On the other hand, the corresponding aged Αβ42 samples
containing 5 and 6, displayed an extended fibrillar network, similar to the untreated
control containing Aβ peptide alone (Figure 6). The results form TEM microscopy are in
complete agreement with the CD and ThT findings providing further confirmation for the
potential of 4 and 7 to effectively intervene in the Aβ40 and Aβ42 aggregation process
and inhibit fibril formation.
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Figure 6. TEM images of the aged Aβ42 solutions (30 d, 50 µM) used for the CD
evaluation in the absence (A) and the presence of compounds 4 and 7 (50 µM, B and E),
where complete lack of fibrils was observed and 5 and 6 (50 µM, C and D) where fibril
formation has taken place. The scale bars correspond to 0.5 μm. Representative images
from n = 2 independent experiments are presented.
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In vitro studies of inhibition of Aβ induced neurotoxicity. Aβ is known to be cytotoxic
in neuronal cells and inhibitors/modulators of its aggregation are often effective in
reducing toxicity in vitro and in vivo.42,43 To investigate whether the ITSCs can protect
cells from Aβ-induced toxicity, the MTT cytotoxicity assay on primary mouse
hippocampal cells was performed. Hippocampal neuronal cells are a suitable model for
assessing Aβ-related toxicity since hippocampus plays a key role in learning and memory
and is particularly vulnerable to damages at early stages of Alzheimer’s disease.44
Treatment of the primary neurons with Aβ40 (1 μM, Figure 7A) and Aβ42 (1 μM, Figure
7B) alone, resulted in a drop of cell viability to 63.4% and 52.5% compared to control
(untreated cells). These viability measurements are in agreement with other literature
findings which also report the enhanced neural cytotoxicity of Aβ42 relative to that of
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Aβ40. The addition of ITSC derivatives in the culture medium resulted in inhibition of
neurotoxicity of both Aβ40 and Aβ42 in a dose-dependent manner. More specifically,
addition of 4 resulted in a considerable cell viability increase to 91.1% (Aβ40, 1:2 ratio)
and 93.9% (Aβ42, 1:2 ratio). In a similar fashion, in the presence of 7, cell survival was
recovered to nearly control levels (89.9% for Aβ40 and 95% for Aβ42, 1:2 ratio). In the
presence of 6 and 5 derivatives, the cell viability increase was lower, but still statistically
significant, reaching 82.3% and 80.5% for Aβ40, 85.1% and 81% for Aβ42, respectively.
The fact that all ITSCs were found to restrain Aβ induced neurotoxicity is in accordance
with their potential to interact early on with Aβ, as revealed by the CD study. The
stronger effect exhibited by 4 and 7 also correlates well with the stronger intervention of
the compounds in Aβ aggregation, as revealed by the CD, ThT and TEM studies, and is
in agreement with relevant studies.15 Our results are in good agreement with literature
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reports revealing the antiaggregation potential of the indole core, as exemplified by
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tabersonine and melatonine.
However, tabersonine exhibited increased cytotoxicity
and melatonine was used in much higher concentrations up to 50 μM to cause a
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comparable result.
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Figure 7. Effects of the ITSC derivatives (0.5, 1 and 2 μΜ) on the cytotoxicity of Aβ40
(A) or Αβ42 (B) (1 μM) in primary hippocampal neuronal cells after 24 h of incubation at
37 °C, as determined using the MTT assay (n = 3 independent experiments, each one
performed in six replicates). The red bars represent the effect on the cell viability of
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ITSCs alone. The data are presented as mean ± SEM, p≤0.05, p≤0.01, ***p≤0.001, ns
(not significant)>0.05 compared to Aβ (1 μM) treatment and p<0.01 and ##p<0.01,
###p<0.001 compared to control (untreated cells).
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In addition, qualitative assessment of the ITSC effect on cell viability and morphology of
hippocampal primary cells exposed to Aβ was assessed by phase-contrast microscopy
(Figure 8). Aβ-treated neurons (1 μΜ) demonstrated hallmarks of degeneration, such as
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fragmented neurites, non-developed axons, cell shrinkage and membrane blebbings, that
are in accordance with relevant literature data.49 Addition of the ITSCs in the Aβ
solutions in a 1:2 ratio alleviated the effects of Aβ toxicity and a substantial recovery of
the Aβ-induced alterations was observed. In agreement to the MTT cytotoxicity data, the
selected derivatives alone exerted no substantial alteration on the morphology of neuronal
cells (data not shown). Both MTT results and microscopic examinations indicate that the
ITSCs can effectively attenuate the cytotoxicity of Aβ in primary hippocampal cells.
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Figure 8. Phase-contrast microscopy images of primary hippocampal neuronal cells
exposed for 24 h at 37 °C to pre-incubated Aβ42 solutions (1 μΜ) in the absence or in the
presence of ITSC derivatives (2 μΜ). The scale bar corresponds to 50 μm.
Representative images from n = 2 independent experiments are presented.
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In vitro studies of inhibition of Aβ induced ROS production. Aβ induced oxidative
stress is linked to the pathogenesis and early development of AD. Elevated levels of Aβ
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are associated with increased levels of oxidation products from proteins, lipids and
nucleic acids in AD hippocampus and cortex50,51. Accumulated evidence suggests that
ROS may be important mediators of Aβ neuronal cell death in the development of AD.52
Hence, the effect of ITSCs on the levels of ROS generated by Aβ in hippocampal
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neuronal cells was evaluated by means of the DCFH-DA assay. The results of the ROS
assessment are summarized in Figure 9. Exposure of hippocampal cell cultures to Aβ
solutions (1 μM) resulted in the production of considerable amount of ROS, as evidenced
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by the significant increase in DCF fluorescence. Incubation of the cells with any of the
four compounds 4 - 7 alone did not cause any significant change in the DCF fluorescence
compared to untreated cells. On the contrary, as can be seen in Figure 9A for Aβ40, and
9B for Aβ42, co-incubation of the Aβ with 4 and 7 resulted in more than 50% reduction
in DCF fluorescence indicating an important decrease in ROS production. Co-incubation
of the Aβ solutions with derivatives 6 and 5 also resulted in lower intracellular ROS
generation, but to a lesser extent. The studied ITSCs show the same trend in reducing
ROS production and in protecting form Aβ cytotoxicity, with 4 and 7 showing the best
results and 5 and 6 following in efficacy. Even though the mechanism of Aβ cytotoxicity
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and ROS production are still not clear, our results provide a link between the potential
of inhibitors to intervene in Aβ aggregation and to decrease ROS production that may add
in the design of effective agents against AD insults.
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Figure 9. Effect of 2 μΜ of the ITSC derivatives on ROS generation induced by Aβ40
and Aβ42 (1 μΜ) in primary hippocampal neuronal cells after 24 h of incubation at 37
°C. ROS levels were measured by the DCF fluorescence assay (n = 3 independent
experiments, each performed in six replicates). The red bars represent the effect on ROS
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Overall, the combined analysis of CD and ThT data indicate interaction of both
Aβ40 and Aβ42 peptides with the new ITSCs, interaction which is particularly strong for
derivatives 4 and 7 and leads to almost complete inhibition of formation of Aβ fibrils. In
the case of Αβ40, where the random coil CD spectrum remains dominant for the duration
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of the study, it appears that the majority of the interaction takes place with the monomeric
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(or dimeric, small oligomeric ) peptide. In the case of Αβ42, which is more fibrillogenic
compared to Aβ40 and self-associates faster, the CD spectrum indicates the presence of a
degree of β-sheet arrangement, so interaction of 4 and 7 may be taking place with a
higher order Aβ42 assembly, which, nevertheless, does not further evolve into fibrils.
Interaction of the ITSCs 5 and 6 with the Aβ40 and Aβ42 peptides is weaker than that of
4 and 7 allowing the eventual formation of amyloid fibrils, as shown in the ThT test,
though in lower amounts and with considerable delay compared to solutions of plain
peptides. The stronger activity of 4 and 7 in all cytotoxicity assays indicates a link
between the inhibitory activity of Aβ aggregation and the reduction in Aβ toxicity which
has been observed in the literature and merits further investigation.
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This work falls into the worldwide effort to discover small-molecule based therapeutic
treatment for Alzheimer's disease. A great number of organic molecules, including small
peptides polyphenols, inositols, quinones and metal chelators have been reported in the
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literature as inhibitors of Aβ aggregation. Even though the recorded cell viability values
for Aβ solutions in the presence ITSCs are comparable to those observed in the presence
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of natural organic compounds, like olive biophenols , quercetin , rutin and
melatonin48 in cell rescuing studies against Αβ toxicity, the existing experimental
differences, including the cell line used, the incubation time, the concentration, protocol
applied, etc., do not allow for direct comparison of our results to those in the literature.
Despite the extensive in vitro, in vivo and in silico studies carried out to unravel the
mechanism of action of small molecule aggregation inhibitors and common structural
features underlying the process of inhibition, the effort has not produced a valid drug
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against AD. This is mainly due to the fact that the structural identity of Aβ aggregates is
not known, and Aβ aggregation process is very complex, to allow for rational design of
aggregation inhibitors. In addition, promising candidates that appear, after constructing
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and screening libraries of compounds, are usually limited by bioavailability. So the
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effort for novel inhibitors continues, with each study and its findings providing further
insight into the mechanism of Aβ aggregation and cytotoxicity, and serving as base for
design improvement. Within this framework, the present data safely establish ITSCs, and
more specifically compounds 4 and 7 as highly promising Αβ inhibitors that combine a)
full inhibition of Aβ aggregation as witnessed by the complete absence of Aβ typical
fibrils, b) strong cytoprotective effect against Αβ insult and oxidative stress in
hippocampal primary neurons and c) ease of synthesis in large amounts and in high
purity. Even though our results do not allow for a clear structure-activity–relationship to
be established, the thiosemicarbazone moiety emerges as an important linker for further
investigation. In particular, the ITSCs 4 and 7 presented herein can be considered as
privileged structures for further exploration as potential AD therapeutics and towards that
goal. It is very interesting to note that in the presence of the 5-OMe isatin substitution the
free indoline-2-one NH (compound 4) was found to be of the highest antiaggregation
activity, whereas the N- substituted piperidine Mannich base derivative (compound 5)
was considerably less active. Interestingly, in the case of the 5-F ITSCs (compound 6 and
7) the opposite structure-activity relationship was observed, making the N-substituted
derivative (compound 7) the most active one and of comparable activity to that of
compound 4. This may suggest that in the case of the phenyl ITSCs a reasonable balance
between the bulkiness and the electronegativity at position 5- of the isatin moiety with the
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polarity of the molecule, lipophilicity and hydrogen bonding of the indole NH- group are
critical for antiaggregation activity. The mechanistic investigation of the signaling
pathways that they affect in wild type and AD transgenic mice (5xFAD) is currently in
progress.
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MATERIALS AND METHODS
7
All reagents used in synthesis were purchased from Sigma Aldrich and Alfa Aesar
(USA), and were used without further purification. Amyloid peptides (Aβ40 and Aβ42,
>95% pure) were purchased from Eurogentec (Belgium). The media/agents for the
cultures of primary neuronal cells were purchased from Thermo Fisher Scientific (USA).
The MTT reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was
bought from Applichem (Germany) while 2′,7′-dichlorofluorescin diacetate (DCFH-DA),
thioflavin T (ThT) were bought from Sigma-Aldrich (Germany).
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NMR spectra were obtained in DMSO-d at 25 °C on a Bruker Avance DRX 500
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MHz ( H at 500.13 MHz and C at 125.77 MHz). Tetramethylsilane (TMS) was used as
the internal standard. Assignment of H and 13C chemical shifts was based on the
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combined analysis of a series of H - H and H - C correlation experiments recorded
using standard pulse sequences from the Bruker library. The IR spectra were recorded
using a Perkin-Elmer Spectrum 100 spectrometer with Universal ATR accessory (Perkin-
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Elmer, USA) over the range 4000 – 200 cm . The high resolution electrospray mass
spectra were recorded in the range of m/z 250 - 1400 on a TSQ 7000 Finnigan MAT
(Scientific Instrument Services, USA). For the mass spectrometric studies samples were
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dissolved in DMSO and the resulting solution was supplied to the electrospray capillary
through a syringe pump.
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The C57BL/6 mice that were used for the in vitro biological evaluation
experiments were obtained from the breeding facilities of the Institute of Bioscience &
Applications, NCSR "Demokritos". All animal procedures were carried out in
compliance with the Presidential Decree 56/2013 (published in the Official Government
Gazette of Greece 106 A/30-4-2013) that has transposed the EU Directive 2010/63 on the
protection of animals used for scientific purposes.
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Synthesis. ITSCs 4, 5 were synthesized according to previously reported procedures by
reacting equimolar amounts of the commercially available substituted isatins with 4-
phenyl-3-thiosemicarbazide in the presence of a catalytic amount of acetic acid. 24, 25, 36
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H and C NMR are given in Supporting Information and are in agreement with the
literature data.
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General synthesis of 6, 7 : A solution of the desired isatin thiosemicarbazone (2 mmol)
in ethanol (20 mL) was treated with formalin (37 %, 0.20 mL, 2.5 mmol) for 15 min and
then piperidine (2 mmol) was added dropwise. The reaction mixture was stirred overnight
and the resulting precipitate was filtered and crystallized from ethanol.
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2-(5-methoxy-2-oxo-1-(piperidin-1-ylmethyl)indolin-3-ylidene)-N
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phenylhydrazinecarbothioamide (6). This was synthesized according to the general
procedure by reacting 4 with piperidine under Mannich reaction conditions. Yield 68%.
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IR (cm ) 1515, 1470, 1017, 3199, 1626. H and 13C NMR are given in Supporting
–
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Information. H NMR (500 MHz, DMSO) δ 12.69 (s, 1H), 10.79 (s, 1H), 7.62 (d, J = 7.6
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Hz, 2H), 7.42 – 7.38 (m, 3H), 7.27 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 6.97 (d, J
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= 8.0 Hz, 1H), 4.44 (s, 2H), 3.75 (s, 3H), 1.43 (bs, 6H), 1.30 (bs, 2H), 1.10 (bs, 2H). C
NMR (126 MHz, DMSO) δ 177.16, 162.55, 156.54, 139.16, 138.49, 132.23, 129.13,
126.94, 126.57, 120.71, 118.19, 117.75, 112.81, 107.08, 62.71, 56.42, 52.08, 26.07,
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24.26; HRMS (ESI) [M+H] calcd for C H N O S, 424.1802, found, 424.1796.
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2-(5-fluoro-2-oxo-1-(piperidin-1-ylmethyl)indolin-3-ylidene)-N-
phenylhydrazinecarbothioamide (7). This was synthesized according to the general
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procedure and previously reported method by reacting 5 with piperidine under Mannich
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reaction conditions. H and C NMR are given in Supporting Information and are in
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agreement with the literature data. Yield 73%.
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Preparation of Aβ Stocks and Solutions. Aβ was gently dissolved without vortexing in
Type 1 (Milli-Q) water to prepare a 100 μM stock solution for CD, ThT and TEM
analysis and a 10 μM for cell viability and ROS measurements. Solutions of Aβ in
phosphate buffer (PB, 10 mM, pH 7.33) were prepared by adding equal volumes of PB to
aliquots of the aqueous solution stock to achieve the desired final concentration. For the
CD, ThT and TEM studies, solutions of Aβ in phosphate buffered saline (PBS, 10 mM,
pH 7.4) were prepared by adding equal volumes of PBS to aliquots of the stock to
achieve final concentration of 50 µM. Proper amount of the ITSCs (stock concentration
of 10 mM in DMSO) were added to the Aβ solutions to achieve a final concentration of
50 μM.
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Circular Dichroism Measurements. CD spectra were recorded on a JASCO J-715
spectropolarimeter (Jasco Co., Japan), at 37 °C in the range of 190 – 260 nm with a 1 mm
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path length quartz cuvette. Each spectrum was the average of three scans at a speed of
100 nm·min−1 and a resolution of 0.5 nm. CD spectra of the Aβ solutions were obtained
for 30 days by CD. During this period, the Aβ solutions remained at the incubator at 37
°C without stirring. Three independent experiments were performed for each condition
and in each case solutions of plain Aβ were run as control. The analysis of the CD data
was performed using the OriginPro 9 program.
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Thioflavin T Assay. For the ThT test 100 μL of the aged (30 days) 50 μM CD solutions
of Aβ (with or without ITSCs) were diluted to half with PB (10 mM, pΗ 7.33) to obtain
200 μL of final solutions with theoretical Aβ concentration of 25 μM. To these solutions,
a stock solution of ThT (Sigma, St. Louis, MO) in PB (10 mM, pH 7.33) was added to
achieve a final concentration of ThT of 5 μM. The mixture was well agitated with
pipetting and immediately thereafter, fluorescence was monitored after excitation at 440
nm (EM slit = 2.5 nm, PMT Voltage 700 V, response 0.4 s) with a HITACHI F-2500
spectrofluorometer. The analysis of the fluorescence data was performed using the
OriginPro 9 program.
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Transmission Electron Microscopy (TEM). For the TEM analysis, the aged (30 days)
50 μM CD solutions of Aβ42 (plain or with 100 μM ITSCs) was mixed well by pipetting.
A 2 μL aliquot of this solution was placed in a carbon coated film on 200-mesh copper
grids (Agar Scientific, UK) for 5 min.62 After adsorption, the grids were washed in
deionized water and negatively stained by applying a 2 μL drop of freshly prepared 1%
(w/v) uranyl acetate (Sigma–Aldrich) in Milli-Q water for 5 min. Excess fluid was
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blotted off and the grids were washed in deionized water and dried in air. Images were
recorded using a FEI CM20 electron microscope (FEI) with a Gatan GIF 200 imaging
filter (Gatan) equipped with a Peltier-cooled slow-scan charge-coupled device camera.
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Cell Cultures. Hippocampal neuronal cultures were obtained from postnatal day 1
female pups of C57BL/6 mice as previously described.63,64 Briefly, after being dissected,
the hippocampus was incubated with 0.25% trypsin for 15 min at 37 °C. The hippocampi
were then rinsed in 10 ml of Hibernate A containing 10% (v/v) heat-inactivated fetal
bovine serum (FBS). Cultures were maintained in Neurobasal-A medium containing 2%
B-27 supplement, 0.5 mM Gluta-MAX and 1% penicillin/streptomycin at 37 °C and 5%
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CO . Half of the medium was replaced twice a week. Neuronal hippocampal cells were
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measurements and 5 × 10 per well in 24-well for phase-contrast microscopy. After
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seven days of incubation in culture well plates, the primary hippocampal neurons were
harvested for the cell rescue from Aβ toxicity and ROS measurements.
Cell Rescue from Aβ Toxicity. Solutions of Aβ40 or Aβ42 (10 μM) in PBS in the
presence of the ITSCs (1:1 ratio of Aβ:compounds) preincubated (3 days for all Aβ40
solutions and 1 day for all Aβ42 solutions) at 37 °C were diluted with fresh medium and
added to individual wells at a final concentration of Aβ of 1 μM. Cell viability was
determined by the standard MTT assay. After 24 h exposure of cells to the Aβ solutions,
100 μL of a 0.5 mg/mL stock solution of MTT in Neurobasal-A was added to each well
of primary hippocampal neurons followed by a 3 h incubation at 37 °C. The medium was
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removed and the cells were diluted in DMSO. The relative formazan concentration was
measured by determination of the absorbance at 540 nm (Tecan well plate reader).
Results were expressed as the percentage of MTT reduction, assuming that the
absorbance of control (untreated) cells was 100%, and are the mean of three independent
experiments with six replicate wells for each condition. In each run the effect of
solutions of plain Αβ and ITSC derivatives was independently checked to serve as
internal standard. Induced cell death was also qualitatively examined by phase-contrast
microscopy (Axiovert 25 CFL; Carl Zeiss) using the above solutions. In each run, the
effect of solutions of plain ITSCs and plain Aβ42 was independently checked to serve as
internal control.
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Intracellular ROS Measurements. Primary hippocampal neurons were treated with Aβ
solutions following the procedure described in the Cell Rescue from Aβ Toxicity section.
After incubation for 24 h, cells were washed with PBS and incubated with 10 μM of
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DCFH-DA for 30 min at 37 °C in the incubator with 5% CO . The fluorescence intensity
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reader at the excitation wavelength of 485 nm and emission wavelength of 528 nm. ROS
levels are presented as arbitrary fluorescence units (AFU). Control groups consisted of
cells incubated with medium only and plain Aβ or plain ITSC derivatives.
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Statistical Analysis. Data in all assays are the mean of at least three independent
experiments. Graphs were analyzed using GraphPad Prism 5.0 software. In hippocampal
neuronal cultures the statistical significance of changes in different groups was evaluated
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by one-way analysis of variance (ANOVA) and Student’s t-tests, using GraphPad Prism
5.0 software. For each experiment, data are expressed as the mean ± standard error of the
mean (SEM), p≤0.05, p≤0.01, ***p≤0.001, ns (not significant)>0.05 compared to Aβ42
*
**
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(1 μM) treatment and p<0.01 and p<0.01, ###p<0.001 compared to control (untreated
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ASSOCIATED CONTENT
Supporting Information
13
Figure S1. Structures of the Initial isatin thiosemicarbazones Library screened by CD,
Synthesis, Figure S2. 1H NMR and 13C NMR spectra of 4, Figure S3. 1H NMR and
13C NMR spectra of 5, Figure S4. 1H NMR and 13C NMR spectra of 6, Figure S5. 1H
NMR and 13C NMR spectra of 7, Figure S6. FT-IR spectra, Figure S7. HRMS
ABBREVIATIONS
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MAO-B
Ach
Monoamine Oxidase B
Acetylcholine
DA
Dopamine
ITSCs
HTS
AD
Isatin-3-thiosemicarbazones
High throughput screening
Alzheimer disease
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