Thiosemicarbazone Derivatives as Inhibitors of Amyloid-β Aggregation: Effect of Metal Coordination

Article abstract of DOI:10.1021/acs.inorgchem.0c00467

Three thiosemicarbazone derivatives, namely 4-(dimethylamino)benzaldehyde 4,4-dimethylthiosemicarbazone (HL¹), 4-(dimethylamino)benzaldehyde thiosemicarbazone (HL²), and 4-(dimethylamino)benzaldehyde 4-methylthiosemicarbazone (HL³), have been synthesized and characterized. The three palladium(II) complexes 1-3 were prepared respectively from HL¹, HL², and HL³. The crystal structures of two coordination compounds, namely Pd(L²)₂ (2) and Pd(L³)₂ (3), were obtained, which showed the expected square-planar environment for the metal centers. The ligand HL³ and the Pd(II) complexes 1-3, which are stable in buffered solutions containing up to 5% DMSO, exhibit remarkable inhibitory properties against the aggregation of amyloid-β, reducing the formation of fibrils. HL¹, HL³, 2, and 3 display IC₅₀ values (i.e., the concentrations required to reduce Aβ fibrillation by 50%) below 1 μM, lower that of the reference compound catechin (IC₅₀ = 2.8 μM). Finally, in cellulo studies with E. coli cells revealed that the palladium(II) compounds are significantly more efficient than the free ligands in inhibiting Aβ aggregation inside bacterial inclusion bodies, thus illustrating a beneficial effect of metal coordination.

Full text of DOI:10.1021/acs.inorgchem.0c00467

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Article
Thiosemicarbazone Derivatives as Inhibitors of Amyloid‑β
Aggregation: Effect of Metal Coordination
́
́
Ana I. Matesanz, Ana B. Caballero, Carmen Lorenzo, Alba Espargaro, Raimon Sabate,
Adoracion G. Quiroga,* and Patrick Gamez*
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ABSTRACT: Three thiosemicarbazone derivatives, namely 4-(dimethylamino)-
benzaldehyde 4,4-dimethylthiosemicarbazone (HL1), 4-(dimethylamino)-
benzaldehyde thiosemicarbazone (HL2), and 4-(dimethylamino)benzaldehyde 4-
methylthiosemicarbazone (HL3), have been synthesized and characterized. The
three palladium(II) complexes 1−3 were prepared respectively from HL1, HL2, and
HL3. The crystal structures of two coordination compounds, namely Pd(L2)2 (2)
and Pd(L3)2 (3), were obtained, which showed the expected square-planar
environment for the metal centers. The ligand HL3 and the Pd(II) complexes 1−3,
which are stable in buffered solutions containing up to 5% DMSO, exhibit
remarkable inhibitory properties against the aggregation of amyloid-β, reducing the
formation of fibrils. HL1, HL3, 2, and 3 display IC50 values (i.e., the concentrations
required to reduce Aβ fibrillation by 50%) below 1 μM, lower that of the reference
compound catechin (IC50 = 2.8 μM). Finally, in cellulo studies with E. coli cells
revealed that the palladium(II) compounds are significantly more efficient than the free ligands in inhibiting Aβ aggregation inside
bacterial inclusion bodies, thus illustrating a beneficial effect of metal coordination.
analogues.16,19−21 It can be stressed here that the potential of
TSCNs toward the inhibition of Aβ aggregation has not been
INTRODUCTION
■
Alzheimer’s disease (AD) is a neurodegenerative disorder
extensively explored; some recent studies have highlighted
characterized by the progressive loss of cholinergic neurons,
interesting neuroprotective properties of a variety of
resulting in cognitive decline.1 AD is the leading cause of
thiosemicarbazone compounds.22−25 Some metal TSCN
dementia, currently affecting about 50 million people world-
complexes have been reported as well for their antiaggregation
wide.2 To date, there are no efficient treatments against the
properties; for instance, some Ru(II) complexes bearing indole
disease or effective tools to diagnose it at its very early stage.3
thiosemicarbazones showed inhibitory potential against the
Therefore, the development of new diagnostic and therapeutic
aggregation of Aβ(1−42).26 Copper and zinc thiosemicarba-
strategies is of paramount importance, as the number of AD
zone complexes have also been described to reduce the levels
patients is rapidly increasing, which also causes a large
of Aβ, in vitro.27
economic burden on society.4
The antiaggregation properties of TSCN-based molecules
The extracellular aggregation of amyloid-β (Aβ) observed in
have been associated with their planarity, especially if they
AD brains represents one of the hallmarks of the disease.5,6
contain aromatic substituents.23,28 The presence of planar
This accumulation of amorphous Aβ deposits produces senile
aromatic moieties in the inhibitors of aggregation appears to be
plaques, which contain high amounts of Cu and Fe.7,8 It has
important for their hydrophobic interactions with Aβ: for
been demonstrated that these redox-active metal ions facilitate
instance, through π-stacking contacts.29 Recently, a series of
Aβ aggregation8,9 and mediate the generation of reactive
arene-containing TSCNs (namely with a naphthyl, anthracen-
oxygen species (ROS).10 Therefore, the development of
yl, or pyrenyl group) have been reported whose antiaggrega-
molecules that may control/avoid the abnormal accumulation
tion efficiencies, analyzed by docking studies, were ascribed to
of Aβ in the brain (i.e., amyloid cascade) may act as efficient
agents to treat AD.
Received: February 14, 2020
Thiosemicarbazones (TSCNs) and their metal complexes
have been used in many pharmaceutical applications:11,12 for
instance, as antiparasital,13 antibacterial,14 antiretroviral,15 and
anticancer agents.16−18 Triapine is a well-known thiosemicar-
bazone derivative, which has undergone clinical trials as a
potential chemotherapeutic drug, together with some of its
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their ability to interact hydrophobically with the aggregation-
prone stretch of the peptide; in particular, strong π interactions
were observed with particular amino acid residues, viz. Ser 26,
Asn 27, Phe 20, Leu 17, and Lys 16, together with hydrogen
bonds.22
Considering the highly promising results reported for
thiosemicarbazones in the area of anti-AD drug design (see
above),30,31 we have decided to design and prepare three new
4-(dimethylamino)benzaldehyde thiosemicarbazone deriva-
tives, viz. HL1, HL2, and HL3 (Scheme 1), comprising the
same aryl ring and distinct hydrogen-bonding capabilities
(provided by the terminal amine group; see R1 and R2 groups
in Scheme 1).
values (viz. the concentrations required to decrease Aβ
aggregation by 50%) in the nanomolar to low micromolar
range. Even more interestingly, in cellulo studies with
Escherichia coli cells expressing Aβ(1−42) in inclusion bodies35
showed drastically distinct behaviors between the free organic
molecules and the metal-containing compounds, the latter
being significantly more efficient.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of the Compounds.
The thiosemicarbazone ligands HL1, HL2, and HL3 were
prepared applying a known synthetic procedure,36 consisting of
a condensation reaction between 4-(dimethylamino)-
benzaldehyde and the corresponding thiosemicarbazide
derivative (see Experimental Section for details).
Scheme 1. Structures of the Thiosemicarbazone Ligands
and Synthetic Pathway to Their Corresponding
Palladium(II) Complexes
The palladium complexes Pd(L1)2 (1), Pd(L2)2 (2), and
Pd(L3)2 (3) were prepared through the reaction of a
methanolic solution of Li2PdCl4 (generated in situ) with 2
equiv of the corresponding ligand (HL1, HL2, or HL3)
suspended in methanol (see Scheme 1 and the Experimental
Section for details).
All compounds were fully characterized by analytical and
1
spectroscopic techniques. The H NMR spectra of complexes
1−3 showed similar profile, the AA′BB′ system for the phenyl
groups of the ligands being maintained upon coordination to
the metal center; the signals for the hydrogens H7 and H4/4′
are the most affected due to their proximity to the palladium
atom. The 13C NMR chemical shifts corresponding to the C
atoms adjacent to the N-donor atom move to lower values.
Analytical data including those of mass spectrometry agree
with the general formula proposed: i.e., Pd(L)2 (Scheme 1).
Moreover, single crystals of 2 and 3 were obtained and their X-
ray structures were determined, which are depicted in Figure 1.
Crystallographic and refinement parameters for 2 and 3 are
summarized in Table S1. Selected bond distances and angles
for 2 and 3 are given in Tables S2 and S3, respectively. The
solid-state structures of the two compounds are flat, as
anticipated; for both of them, the metal center is in a slightly
distorted square-planar environment formed by two sulfur
atoms (monoanionic form of the ligand) and two nitrogen
atoms belonging to two different ligands, which are trans to
each other. The metal−chelate rings and the dimethylamino
arenes are coplanar. It can be pointed out that 2 is isostructural
with its previously reported nickel analogue.37 While the
crystal lattice of 3 does not contain any solvent molecules, that
of 2 includes two molecules of DMSO (Figure 1), which are
hydrogen-bonded (through their oxygen atom) to the nitrogen
atom N3 of the two ligands (N3−H···O = 2.903(9) Å). The
crystal packing of Pd(L3)2 (3) reveals the occurrence of π−π
We subsequently prepared the corresponding palladium(II)
complexes, viz. Pd(L1)2 (1) Pd(L2)2 (2), and Pd(L3)2 (3)
(Scheme 1), with the objective to investigate and compare
their antiaggregation properties with those of the free ligands.
Palladium(II) was purposely chosen for the square-planar
geometry typically obtained upon coordination and was
preferred to platinum(II) due to the better solubility of
palladium complexes.32,33 The aim was to rigidify the systems
through metal binding and generate planar and extended
structures. Indeed, free TSCNs exhibit a certain degree of
rotational freedom, which is illustrated in Chart 1 with 4-
nitrobenzaldehyde thiosemicarbazone (TSCN-H), whose
crystal structure has been reported.34 While rotation is possible
around the N−N bond for the free ligand, the system becomes
rigid upon coordination with palladium (TSCN-Pd); fur-
thermore, the ligand changes its conformation from trans-trans
to cis-cis (see bonds in blue in Chart 1), as revealed by its
reported crystal structure.34
In vitro aggregation studies with Aβ(1−40) revealed
disparities between the free TSCN ligands HL1−HL3 and
the corresponding palladium(II) complexes 1−3, with IC50
a
Chart 1. Structure of Free 4-Nitrobenzaldehyde Thiosemicarbazone (TSCN-H) and of Its Palladium(II) Complex34
a
The red arrow illustrates the N−N rotation for the free ligand, and the bonds in blue show the change in conformation from the free to the
coordinated ligand.
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UV−vis measurements, stock solutions of 1−3 were prepared
in DMSO, which were then used to prepare buffered aqueous
solutions (Tris-HCl) of the complexes containing 5% DMSO.
The UV−vis spectra displayed in Figure S2 show three intense
bands in the UV region for the three complexes, ascribed to
intraligand (IL) and ligand to metal charge transfer (LMCT)
transitions. The absorption band around 260 nm is attributed
to π → π* (aromatic skeleton) transitions and that at about
350 nm to n → π* (thioamide skeleton) transitions. The
absorption band observed at ca. 400 nm is due to LMCT
transitions. The compounds do not seem to be altered in
solution; the same absorption bands are observed over time,
and no new ones develop. However, diminutions of the
absorbance intensities are noticed, which suggest that the
compounds are gradually precipitating, most likely due to their
poor solubility in Tris-HCl/5% DMSO. Spectroscopic studies
to verify that the integrity of 1−3 is retained in Tris-HCl/5%
DMSO were also carried out with egg-white lysozyme
(HEWL), named as lysozyme thereafter. It can be pointed
out here that, after 4 h, most of the compounds are in solution
(i.e., significant precipitation is only observed after 24 h),
hence allowing aggregation studies with Aβ(1−40) (see
below).
Figure 1. Representation of the X-ray structures of coordination
compounds 2 and 3. Symmetry operations: (a) 1 − x, 2 − y, −z (1);
(a) 1 − x, 1 − y, 1 − z (2); (a) −x, 1 − y, −z (3).
Interaction Studies with the Model Protein Lyso-
zyme. In a first instance, the potentially amyloidogenic protein
lysozyme (hereditary lysozyme amyloidosis38) was used to
investigate the interaction of the metal complexes with a
“model protein” that is broadly employed for interaction
studies with metallodrugs.39,40 The kinetic constants corre-
sponding to the interaction of the different compounds with
lysozyme were obtained considering a pseudo-first-order
reaction (see Experimental Section for details and Figure 3
and Figure S3). The values achieved (at λ = 433 nm) were
interactions between neighboring molecules, the shortest
contact distance being of 3.597 Å (Figure 2).
compared to that of the reference compound cisplatin; kobs
=
1.98 × 10−4 s−1 was determined for cisplatin under the same
experimental conditions (to those used for 1−3), which
matches reported values.41
Figure 2. Representation of the crystal packing of 3 showing the
stacking of molecules through π−π interactions.
For 1−3, the kinetic data were determined from the spectra
shown in Figure 3 and Figure S3. The kobs values for 1 and 2
are in the range (0.4−1) × 10−4 s−1 (Table 1). Complexes 1
and 3 show about 2 times more affinity for lysozyme in
comparison to 2. The three compounds exhibit kobs values 1
order of magnitude lower than that of cisplatin; 1−3 are
certainly binding to lysozyme but less efficiently than cisplatin.
Stability in Solution. The stability of the coordination
compounds in solution was assessed to determine the
appropriate conditions to perform assays in biological media.
Solution studies were thus carried out by using NMR and
UV−visible spectroscopy over 24 h. Complexes 1−3 were
dissolved in DMSO-d6 for the NMR experiments (Figure S1),
which did not reveal any spectral changes after 1 day. For the
Figure 3. (a) UV−visible spectra illustrating the interaction between complex 3 (10−5 M; λ = 433 nm) and lysozyme (ratio 3:1) as a function of
time at 37 °C and using 95% Tris-HCl (pH 7.2)/5% DMSO as a solvent. (b) Corresponding plot of the variation of the absorbance (λ = 433 nm)
as a function of time. UV−visible data for complex 3 are shown in Figure S2.
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phosphate-buffered saline (PBS). ThT is routinely used to
quantify the formation of amyloid fibrils (other probes have
been regularly described43−46); ThT rapidly interacts with
amyloid fibrils in a specific manner, resulting in an increase in
its fluorescence emission at 490 nm, on excitation at 440
nm.45−47
The kinetic curve for the aggregation of a 25 μM solution of
Aβ(1−40) monomers (CTR; black dots in Figure 5) shows
the typical sigmoidal shape, with a lag phase (nucleation) of
about 50 min, followed by a fast fibril elongation phase, which
reaches a plateau after 80 min.
Table 1. kobs Values Obtained for Coordination Compounds
1−3 Interacting with Lysozyme
complex
kobs (10−5 s−1
)
1
2
3
9.89
4.17
7.00
It has been shown that the electronic spectra of metal
complexes can be modulated by their interaction with proteins;
for instance, a hyperchromic effect can be observed.42 Hence,
potential changes of the UV−vis absorption properties of 1−3
upon interaction with lysozyme have been investigated. UV−
vis spectra of 1−3 have therefore been recorded with and
without the presence of lysozyme for 24 h. The corresponding
comparative data (absorption values at λ = 433 nm) are
depicted in Figure 4. In all cases, the absorptions of 1−3 in the
presence of lysozyme are higher than those without the
enzyme (Figure 4). The curves with or without lysozyme
follow the same pattern, but the decrease is clearly less
pronounced for the “enzyme” curves, especially for complex 3
(75 vs 37% decrease). The data thus suggest that the
complexes are not degraded in solution (95% Tris-HCl/5%
DMSO in the present case); hence, it appears that the
complexes are poorly soluble in aqueous solutions (however,
their solubility is apparently improved upon interaction with
lysozyme, most likely through the formation of lysozyme/
complex species). It can be pointed out that complex 3, whose
“solubilization” is sensibly improved in the presence of
lysozyme, is also the biologically most efficient compound
(see below).
Figure 5. Time-resolved measurements of the aggregation of Aβ(1−
40) through the emission of ThT at 490 nm (λexc = 440 nm) for free
Aβ(1−40) (CTR) and Aβ(1−40) incubated with the ligands HL1−
HL3 and their metal complexes 1−3. Conditions: [Aβ(1−40)] = 25
μM; [compound] = 1 μM. The respective antiaggregation efficiencies
(in percent) of the compounds are given in parentheses (comparison
with the control CTR).
In the presence of 1 μM of the different compounds (viz.
HL1−HL3, 1−3), the corresponding inhibitory activities
against Aβ(1−40) fibrillation were determined over 5 h. It
should be noted herein that compound 1 did not completely
dissolve in PBS containing 10% DMSO. Nevertheless, for
consistency, it was tested under the same conditions as for 2
and 3, namely using 5% DMSO; thus, the data achieved with 1
should be considered cautiously. The results obtained revealed
Interaction with Aβ(1−40). The effect of the thiosemi-
carbazone ligands HL1, HL2, and HL3 and their respective
palladium complexes (1−3) on the aggregation of Aβ(1−40)
was subsequently evaluated in vitro. The aggregation of
monomeric Aβ(1−40) was monitored through the changes
in the fluorescence intensity of thioflavin T (ThT; specific
amyloid dye) upon incubation with each compound in
Figure 4. Time-dependent UV−visible data for complexes 1−3 (10−5 M; λ = 433 nm) in the absence and presence of lysozyme (ratio 3:1). The
spectra were recorded at 37 °C using 95% Tris-HCl (pH 7.2)/5% DMSO as solvent.
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a
b
Table 2. Rate Constants (kn and ke) and Fitting Time Parameters (t0, t1/2, and t1) and Inhibition Percentages for Free
c
Ligands HL1−HL3 and Complexes 1−3 on the Aggregation of Aβ(1−40)
CTR
HL1
HL2
HL3
kn (min−1
)
1.24 × 10−7
8864
54.3
2.64 × 10−7
8676
1.09 × 10−10
11444
66.7
4.06 × 10−8
7684
70.7
ke (M−1 min−1
t0 (min)
)
51.6
t1/2 (min)
65.2
62.7
76.1
79.7
t1 (min)
76.1
73.8
85.4
88.8
inhibition (%)
0
80.5
41.3
86.6
1
2
3
kn (min−1
)
8.05 × 10−8
10192
50.1
58.6
67.1
5.06 × 10−11
12036
66.3
75.0
83.7
2.45 × 10−11
ke (M−1 min−1
t0 (min)
)
13764
60.1
67.9
75.8
91.1
t1/2 (min)
t1 (min)
inhibition (%)
11.6
68.5
a
b
kn = nucleation rate constant; ke = elongation rate constant (see Scheme S1). t0 = lag time; t1/2 = time corresponding to the aggregation of 50% of
c
the protein; t1 = end time (plateau). [Aβ(1−40)] = 25 μM. CTR corresponds to the fibrillation of free Aβ.
that the six new molecules could reduce Aβ(1−40) fibrillo-
genesis (in comparison with free Aβ; Figure 5), the final
inhibition percentages ranging from 11% for 1 to 91% for 3
(Table 2). Except for partially soluble 1, metal coordination of
the thiosemicarbazones improves the antiaggregation proper-
ties; indeed, 2 and 3 show higher antiaggregation activities (68
and 91%, respectively) than the corresponding free ligands
HL2 and HL3 (41 and 87%, respectively; Figure 5 and Table
2). In addition, HL1, HL3, 2, and 3 display inhibition
percentage values of higher than 50%, which correspond to
remarkable IC50 values below 1 μM. Actually, such inhibitory
data are indicative of potent antiamyloid properties since they
are in the range (or even below the range) found for known
polyphenolic inhibitors such as epicatechin (whose IC50
amounts to 2.8 μM).48 It can also be pointed out that
although ligands HL1−HL3 are structurally similar, they exhibit
different antiaggregation properties; in particular, HL2 is 2
times less efficient than HL1 and HL3. This disparity may arise
from the fact that HL2 is the sole ligand that contains an −NH2
group; thus, HL2 has three potential H-bond donors, HL1 only
one (from the hydrazine unit), and HL3 two (see Scheme 1).
Moreover, the −NMe2 group of HL1 can only act as an H-
bond acceptor. It thus appears that the hydrogen-bonding
properties as well as the hydrophilic character of these
molecules may represent important features regarding the
interaction with the protein.
The notable antiaggregation activities for all compounds
(except 1, probably as the result of its poor solubility) suggest
that the scaffold of the thiosemicarbazone moieties is an
important feature with regard to the observed behaviors; the
flat conjugated ligands most likely interact with Aβ(1−40)
through H-bonding and/or π−π interactions, as reported for
other TSCN compounds (see above),22 delaying the formation
of β sheets (i.e., the fibrils).48
The antiaggregating activity of the compounds was further
investigated by determining different kinetic parameters that
allowed an assessment of their effect on the various stages of
the Aβ fibrillation process (Scheme S1). The main phases of
the aggregation are nucleation, characterized by the constant
kn, and elongation, described by the constant ke (Scheme S1).
This evaluation considers the amyloid aggregation as a
nucleation−elongation process,49 which can be mathematically
described as an autocatalytic polymerization (see the
Experimental Section for details).50 In addition, the lag time
t0, the time for the formation of the first amyloid-like species
(viz. the first nuclei), t1/2, the time required to aggregate half of
the protein to its amyloid conformation, and the end time t1,
corresponding to the completion of the fibrillation process,
were determined for each compound (Scheme S1 and Table
2). All of the data obtained from the corresponding
aggregation curves depicted in Figure 5 are given in Table 2.
As evidenced in Figure 5 and in Table 2, in all cases, the
initiation of Aβ aggregation is not significantly delayed by the
presence of the different compounds; indeed, the lag time, i.e.
t0 (see Scheme S1), for free Aβ(1−40) (CTR) is around 54
min, while the maximum delay observed is ca. 71 min with
HL3 (Table 2). It can be pointed out that the t0 values are
comparable for the TSCN ligands and the corresponding
complexes (Table 2); only HL3 somewhat delays the
aggregation in comparison with 3 (71 vs 60 min). It can be
noticed that, except for HL1, the compounds retard the
nucleation (i.e., the phase during which oligomers are
generated), 2 and 3 being the most effective compounds
(see kn values in Table 2). The elongation phase, namely the
phase that results in fibrils, is also affected by the presence of
most of the compounds; HL1 and HL3 do not alter this phase,
as reflected by their elongation constants, namely ke, which are
similar to that of the control (Table 2). HL2 and 1−3 appear
to accelerate the elongation of fibrils, with ke values ranging
from 10192 (complex 1) to 13764 min−1 (compound 3); these
values should be compared with that of nontreated Aβ(1−40),
namely 8864 min−1 (Table 2). It thus appears that, if some
nuclei are generated, then these compounds facilitate their
fibrilliation. However, it should be stressed that all compounds
give rise to the generation of fewer amounts of fibrils at t1
(namely, at the final plateau; see Figure 5) with percentages of
inhibition ranging from 12 to 91% (Table 2); thus, the
interaction of the compounds with Aβ(1−40) seems to
generate stable monomeric/oligomeric species that can not
fibrillate. The effiency of the different compounds follows the
trend 3 > HL3 > HL1 > 2 ≫ HL2 ≫ 1. It can be noticed that
coordination of palladium to the ligands results in an
improvement of the inhibition properties, except for 1
(which is much less efficient than HL1); this disparity may
be explained by the poor solubility of 1 in the (biological)
solvent medium used for these experiments.
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In Cellulo assays with Aβ(1−42) in Bacterial Inclusion
Bodies. The inhibition efficacies of the TSCN derivatives were
then evaluated in cellulo with Escherichia coli cells expressing
Aβ(1−42). After overnight incubation of the compounds with
bacteria cultures, the aggregation of Aβ(1−42) was checked
using Thioflavin S (ThS) and compared with that of bacteria
cultured under the same conditions, but without added
compound, to determine the respective inhibition percentages
(Table 3). Under the conditions used (viz. final [complex] =
delivery vehicles for radioactive 64Cu ions was reported as well,
and it has been shown that Cu-TSCN complexes can cross the
blood−brain barrier.51
In the study reported herein, we have prepared a series of
TSCN ligands with the objective of comparing their inhibitory
abilities against Aβ aggregation with those of their palladium-
(II) complexes. The data obtained show that TSCN-based
molecules are indeed efficient antiaggregation agents that may
therefore be considered in research aimed at developing
potential anti-AD drugs. Moreover, in cellulo experiments with
E. coli expressing Aβ(1−42) revealed that the palladium(II)
complexes were notably more efficient inhibitors than the free
TSCN ligands, illustrating the valuable effect of metal
coordination and encouraging the common use of bacterial
inclusion bodies as an additional experiment to accompany in
vitro studies.
Table 3. Inhibition Percentages for Ligands HL1−HL3 and
Palladium Complexes 1−3 ([Compound] = 20 μM) in
Bacterial Cells Overexpressing Aβ(1−42) Monitored by
a
ThS Staining
compound
Aβ(1−42) inhibition (%)
b
control
0.0 (1.6)
0.8 (3.1)
3.2 (2.0)
4.7 (2.0)
9.3 (3.5)
22.5 (2.3)
HL1
HL2
HL3
1
EXPERIMENTAL SECTION
■
Materials and Methods. Solvents and reagents were commer-
cially available and were used as received. H NMR and 13C NMR
1
experiments were performed in DMSO-d6 using a Bruker AMX-300
(300 MHz) spectrometer at room temperature (25 °C). Elemental
analyses were performed on a PerkinElmer 2400 Series II micro-
analyzer. Fast atom bombardment (FAB) mass spectra (MS) were
recorded on a VG AutoSpec spectrometer. Infrared spectra (IR) were
obtained with a PerkinElmer Model 283 spectrophotometer equipped
with an ATR accessory (Miracle Single Reflection Horizontal). UV−
visible spectra were recorded with a Thermo Fisher Scientific
Evolution 260 Bio spectrophotometer.
2
3
37.2 (2.9)
a
b
The corresponding SEM values are given in parentheses. Aβ(1−42)
without compound added.
0.2 μM; see the Experimental Section), all of the compounds
tested were not toxic to the bacteria. The results achieved show
that the metal complexes 1−3 are much more efficient than the
free ligands HL1−HL3. For instance, the inhibition percentage
is 4.7% for HL3 and 37.2% for 3. In general, the coordination
compounds are 7−12 times more efficient than the free
TSCNs, following the trend 3 > 2 ≫ 1 > HL3 > HL2 ≫ HL1.
It is important to note that while the free ligands show
significant inhibition properties in vitro, they are almost
inactive in cellulo. For example, the inhibition percentage is
86.6% for HL3 in vitro (Table 2) while it is only 4.7% in cellulo
(Table 3); HL3 is 18 times less efficient with bacteria. Such a
drastic decrease in inhibition abilities may arise from
difficulties for the TSCNs to cross the bacterial membranes,
hence reducing the concentration of the effective compound
inside the cells; more (biological) studies are required,
however, to verify this hypothesis. In summary, the in cellulo
results are important for two main reasons. (i) Going from in
vitro conditions (with an isolated protein) to living cells
considerably modifies the behavior of an “active compound”;
thus, the use of bacteria is certainly a valuable and simple tool
that may be implemented on a more regular basis, in
association with in vitro studies. (ii) Coordination of palladium
to the TSCN ligands clearly improves the in cellulo inhibitory
activities, hence illustrating the (beneficial) role of the metal.
Synthesis of the Compounds. Thiosemicarbazones HL1−HL3.
The thiosemicarbazone ligands were prepared by following a general
procedure in which a methanolic solution (10 mL) of the
corresponding thiosemicarbazide (2 mmol) was added dropwise to
a methanolic solution (10 mL) of 4-(dimethylamino)benzaldehyde
(0.300 g, 2 mmol). The reaction mixture was refluxed with constant
stirring for 6 h. Next, the mixture was cooled to ambient temperature
and the solid that formed was filtered off, washed with cold methanol
and diethyl ether, and dried under reduced pressure.
4-(Dimethylamino)benzaldehyde 4,4-Dimethylthiosemicarba-
zone (HL1). Green solid. Yield: 92%. Anal. Calcd for C12H18N4S: C,
57.6; H 7.2; N 22.4; S, 12.8. Found: C, 57.5; H, 7.10; N, 22.4; S, 12.6.
MS (ESI) m/z: 251.13 [M + H]+. 1H NMR (300 MHz; DMSO-d6; δ
(ppm)): 2.95 (s, 6H, H1); 3.26 (s, 6H, H8/9); 6.75 (d, 2H, H3/3′);
7.46 (d, 2H, H4/4′); 8.04 (s, 1H, H6); 10.6 (s, 1H, NH). 13C NMR
(300 MHz; DMSO-d6; δ (ppm)): 40.4 (C1); 42.1 (C8/9); 111.8
(C3/3′); 121.8 (C5); 128.0 (C4/4′); 144.8 (C6); 151.2 (C2); 180.1
(C7). IR (ν; cm−1): 3286 (NH); 1612 (CN); 807 (CS). See
Scheme 1 for atom numbering.
4-(Dimethylamino)benzaldehyde Thiosemicarbazone (HL2). Pale
yellow solid. Yield: 89%. Anal. Calcd for C10H14N4S: C, 54.0; H 6.3;
N, 25.2; S, 14.4. Found: C, 53.6; H, 6.2; N, 25.2; S, 13.9. MS (ESI)
1
m/z: 223.10 [M + H]+. H NMR (300 MHz; DMSO-d6; δ (ppm)):
2.95 (s, 6H, H1); 6.71 (d, 2H, H3/3′); 7.55 (d, 2H, H4/4′); 7.75 (s, 1H,
H8/9); 7.92 (s, 1H, H6); 8.00 (s, 1H, H8/9); 11.10 (s, 1H, NH). 13C
NMR (300 MHz; DMSO-d6; δ (ppm)): 40.0 (C1); 111.6 (C3/3′);
121.8 (C5); 128.6 (C4/4′); 143.3 (C6); 151.3 (C2); 180.0 (C7). IR (ν;
cm−1): 3414, 3229 (NH); 1614 (CN); 808 (CS). See Scheme 1
for atom numbering.
4-(Dimethylamino)benzaldehyde 4-Methylthiosemicarbazone
(HL3). Pale yellow solid. Yield: 90%. Anal. Calcd for C11H16N4S: C,
55.9; H, 6.82; N, 23.7; S, 13.6. Found: C, 55.8; H 6.70; N, 22.9; S,
13.4. MS (ESI) m/z: 237.12 [M + H]+. 1H NMR (300 MHz; DMSO-
d6; δ (ppm)): 2.96 (s, 6H, H1); 3.00 (s, 3H, H9); 6.72 (d, 2H, H3/3′);
7.56 (d, 2H, H4/4′); 7.94 (s, 1H, H6); 8.26 (s, 1H, H8); 11.20 (s, 1H,
NH). 13C NMR (300 MHz; DMSO-d6; δ (ppm)): 30.4 (C9); 41.3
(C1); 111.4 (C3/3′); 121.3 (C5); 128.2 (C4/4′), 142.5(C6); 151.0 (C2);
179.8 (C7). IR (ν; cm−1): 3328, 3142 (NH); 1612 (CN); 812
(CS). See Scheme 1 for atom numbering.
CONCLUSIONS
■
Thiosemicarbazone derivatives have shown great promise for
medicinal applications, especially in the area of anticancer
agents. In the field of AD, a few studies have been reported,
which revealed great potential for thiosemicarbazone-based
compounds. Metal-free TSCNs have been used as efficient
metal chelators: for instance to capture copper(II) ions (and
potentially lessen the oxidative stress associated with AD)28 or
to inhibit Aβ aggregation.22 With regard to metal TSCN
complexes, a few compounds have been described that exhibit
interesting inhibitory properties.26,27 Their use as effective
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Coordination Compounds 1−3: Pd(L1)2 (1), Pd(L2)2 (2), and
Pd(L3)2 (3). The palladium(II) complexes were obtained by reaction
of a methanolic suspension of the corresponding ligand (0.4 mmol)
with a methanolic solution of lithium tetrachloridopalladate(II),
prepared in situ from palladium chloride(II) (0.2 mmol) and lithium
chloride (0.8 mmol). The reaction mixture was stirred for 24 h at
room temperature, and the solid obtained was subsequently filtered
off and washed with water, methanol, and diethyl ether. The isolated
solid was finally dried under reduced pressure.
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; 1 mL) with vigorous
shaking at room temperature for 1 h. The resulting solution was
sonicated for 30 min and subsequently shaken at room temperature
for an additional 1 h. The solution was then maintained at 4 °C for 30
min to avoid solvent evaporation during the collection of aliquots.
HFIP was evaporated under a gentle stream of nitrogen, and the
aliquots of soluble Aβ(1−40) were stored at −20 °C.
Amyloid-β Aggregation Studies. Aβ(1−40) aliquots were
resuspended in 30 μL of DMSO, and the monomers were solubilized
through sonication for 10 min. PBS buffer (pH 7.4), 500 μM
thioflavin T (ThT), and 0.2 mM stock solution (in DMSO) of the
compound to be tested were added to the Aβ(1−40) sample (final
concentration of compound: 1 μM). PBS buffer (pH 7.4) was then
added to obtain final solutions of 25 μM Aβ(1−40) and 34 μM ThT,
containing 4.4% (v/v) DMSO. Control experiments with nontreated
protein samples contained the same amount of DMSO as in the
samples containing the compounds investigated. For the kinetic
assays, the samples were placed in a 96-well plate at 37 °C and stirred
at 700 rpm with double-orbital mode. The course of the aggregation
was followed by measuring ThT fluorescence using a FLUOstar
OMEGA plate reader (BMG Labtech GmbH), with excitation and
emission filters of 440 and 490 nm, respectively.
Pd(L1)2 (1). Brown solid; Yield: 68%. Anal. Calcd for
PdC24H34N8S2: C, 47.6; H, 5.66; N, 18.5; S, 10.6. Found: C, 46.4;
1
H, 6.56; N, 18.3; S, 10.5. MS (ESI) m/z: 605.2 [M + H]+. H NMR
(300 MHz; DMSO-d6; δ (ppm)): 3.01 (s, 12H, H1); 3.19 (s, 12H,
H8/9); 6.73 (d, 4H, H3/3′); 7.22 (s, 2H, H6); 7.91 (d, 4H, H4/4′). IR
(ν; cm−1): 3276 (NH); 1584 (CN−NC); 711 (C−S). See
Scheme 1 for atom numbering.
Pd(L2)2 (2). Brown solid; Yield: 70%. Anal. Calcd for
PdC20H26N8S2·5H2O: C, 37.6; H, 5.68; N, 17.5; S, 10.0. Found: C,
37.3; H, 4.64; N, 17.3; S, 10.3. MS (ESI) m/z: 551.10 [M + H]+. 1H
NMR (300 MHz; DMSO-d6; δ (ppm)): 3.02 (s, 12H, H1); 6.67 (d,
4H, H3/3′); 6.95 (s, 4H, H8/9); 7.22 (s, 2H, H6); 8.00 (d, 4H, H4/4′).
13C NMR (300 MHz; DMSO-d6; δ (ppm)): 48.6 (C1); 110.9 (C3/3′);
118.5 (C5); 134.9 (C4/4′); 144.8 (C6); 151.2 (C2); 180.1 (C7). IR (ν;
cm−1): 3276 (NH); 1600 (CN−NC); 789 (C−S). Single
crystals suitable for X-ray diffraction analysis were obtained by
recrystallization from DMSO. See Scheme 1 for atom numbering.
Pd(L3)2 (3). Brown solid; Yield: 69%. Anal. Calcd for
PdC22H32N8S2·H2O: C, 44.4; H, 5.4; N, 18.8; S, 11.0. Found: C,
44.0; H, 5.1; N, 18.5; S, 10.4. MS (FAB) m/z: 576.11 [M + H]+. 1H
NMR (300 MHz; DMSO-d6; δ (ppm)): 2.80 (s, 6H, H9); 3.01 (s,
12H, H1); 6.70 (d, 4H, H3/3′); 7.16 (s, 2H, H6); 7.93 (d, 4H, H4/4′).
13C NMR (300 MHz; DMSO-d6; δ (ppm)): 32.3 (C9); 40.0 (C1);
Aggregation Assay Analysis. The amyloid aggregation can be
studied as an autocatalytic reaction using eq 1
ρ{exp[(1 + ρ)kt ] − 1}
f =
1 + ρ exp[(1 + ρ)kt ]
(1)
where f is the fraction of fibrillary Aβ and the rate constant k includes
the kinetic contributions arising from the formation of the nucleus
from monomeric Aβ and the elongation of the fibril, which are
described by the rate constants kn and ke, respectively. ρ is a
dimensionless parameter that describes the ratio of kn to k. Equation 1
is obtained under the boundary conditions of t = 0 and f = 0, where k
= kea (a is the protein concentration). By nonlinear regression of f
against t, values for ρ and k were obtained, and from them the rate
constants ke (elongation constant) and kn (nucleation constant) were
determined.50 The extrapolation of the linear portion of the sigmoid
curve to the abscissa (f = 0) and to the highest ordinate value of the
fitted plot afforded two values of time, namely t0 and t1, which
corresponded to the lag time and to the end time of the reaction,
respectively. The time at which half of the protein was aggregated
(i.e., when f = 0.5) was considered as the time of half an aggregation
(viz. t1/2).50
111.0 (C3/3′); 119.9 (C5); 128.5 (C4/4′); 134.1 (C6); 134.4(C2);
151.2 (C7). IR (ν; cm−1): 3409 (NH); 1600 (CN−NC); 754
(C−S). Single crystals suitable for X-ray diffraction analysis were
obtained by recrystallization from DMSO. See Scheme 1 for atom
numbering.
Crystallography. Data were collected on a Bruker Kappa Apex II
diffractometer. Crystallographic and refinement parameters are
summarized in Table S1. The software package SHELXTL was
used for space group determination, structure solution, and
refinement.52 The structures were solved by direct methods,
completed with difference Fourier syntheses, and refined with
anisotropic displacement parameters.
All details can be found in CCDC 1884751 (2) and 1884752 (3) ,
which contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge
Crystallographic Data Center via https://summary.ccdc.cam.ac.uk/
structure-summary-form.
Spectrophotometric Studies. The solution stabilities of
complexes 1−3 were evaluated by recording their UV−vis spectra
for 24 h at 37 °C and a concentration of 10−5 M. Time-dependent
UV−vis spectra of the complexes in the presence of a biomolecule,
namely lysozyme, have been registered as well.
Interaction with Lysozyme Followed by UV−vis Spectros-
copy. The interaction of the metal complexes with lysozyme (egg
white lysozyme; HEWL) was investigated using a 10−5 M solution of
the protein. Lysozyme is a widely used model protein for studying the
binding of potential metallodrugs; lysozyme is also a model amyloid-
forming protein. The integrity of the enzyme was checked in each
experiment, with a comparison of fresh and incubated samples. UV−
vis spectra were recorded before and after incubation with the
different complexes (dissolved in 95% Tris-HCl/5% DMSO) for 24 h
at 37 °C. A metal to protein ratio of 3:1 was used for these studies.
The experimental time-dependent profiles of the spectra were
analyzed as pseudo-first-order reactions by plotting the variation of
the absorbance as a function of time. The absorption profiles are
shown in Figure 3a (3) and Figure S3 (1 and 2).
The estimation of the IC50 values and of the kinetic data from the
autocatalytic eq 1 was carried out using GraphPad Prism (GraphPad
Software, La Jolla, CA, USA).
In Cellulo Assays of Aβ(1−42) Aggregation. Escherichia coli
competent cells BL21 (DE3) were transformed with the pET28a
vector from Novagen carrying the DNA sequence of Aβ(1−42). A 10
mL of M9 minimal medium containing 50 μg mL−1 of kanamycin was
inoculated with a single colony of BL21 (DE3) bearing the plasmid to
be expressed, and the bacteria were cultured overnight at 37 °C. For
Aβ(1−42) expression, 200 μL of the culture (incubated overnight)
was transferred into 1.5 mL microcentrifuge tubes with 790 μL of
fresh M9 minimal medium, giving final concentrations of 50 μg mL−1
of kanamycin and 25 μM of thioflavin S (ThS); 10 μL of 100 mM
isopropyl 1-thio-β-D-galactopyranoside (IPTG) was added or not, for
induced and noninduced cultures, respectively. Then, 10 μL of each
TSCN-based compound (different ligands and Pd complexes) with
the required concentration (20 μM) was added, giving a final complex
concentration of 0.2 μM. The samples were grown overnight at 37 °C
and at 300 rpm, using an incubator with orbital shaking. As a control
of maximal amyloid formation, samples for which the 10 μL of
compound solution was replaced by 10 μL of buffer were analyzed as
well. Aβ(1−42) aggregation was followed by fluorescence spectros-
copy, using thioflavin S (ThS). Variations in ThS fluorescence
intensities due to potential differences in bacterial growth between
samples (the growth of induced bacteria may slightly be lower than
In Vitro studies of Aβ(1−40) Aggregation. Preparation of
Aggregate-Free Amyloid-β. Aβ(1−40) (5 mg) was solubilized in
G
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that of noninduced bacteria), were corrected by tracking the
absorbance at 405 nm.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
ASSOCIATED CONTENT
■
Notes
sı
* Supporting Information
The authors declare no competing financial interest.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c00467.
ACKNOWLEDGMENTS
Financial support from the Spanish Ministerio de Ciencia
■
Crystallographic data for compounds 2 and 3, time-
dependent NMR spectra of 1−3 in DMSO-d6, time-
dependent UV−vis spectra for 1−3 in Tris-HCl/5%
DMSO, UV−vis spectra for 1−3 interacting with
lysozyme, NMR and mass spectra for compounds
HL1−HL3, and schematic representation of Aβ
fibrillation (PDF)
́
Innovacion, y Universidades (Projects CTQ2015-70371,
CTQ2015-68779-R and CTQ2017-88446-R AEI/FEDER,
UE and RED2018-102471-T) is kindly acknowledged. P.G.
́
acknowledges the Institucio Catalana de Recerca i Estudis
Avancats (ICREA).
̧
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AUTHOR INFORMATION
■
Corresponding Authors
́
Adoracion G. Quiroga − Institute for Advanced Research in
Chemical Sciences (IAdChem) and Department of Inorganic
́
Chemistry (M-07), School of Sciences, Universidad Autonoma
de Madrid, Madrid 28049, Spain; orcid.org/0000-0002-
9261-9542; Email: adoracion.gomez@uam.es
́
Patrick Gamez − nanoBIC, Departament de Quimica
́
̀
̀
Inorganica i Organica, Facultat de Quimica and Institute of
Nanoscience and Nanotechnology (IN2UB), Universitat de
Barcelona, 08028 Barcelona, Spain; Catalan Institution for
Research and Advanced Studies, 08010 Barcelona, Spain;
orcid.org/0000-0003-2602-9525; Phone: +34 934
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Authors
Ana I. Matesanz − Department of Inorganic Chemistry (M-07),
School of Sciences, Universidad Autonoma de Madrid, 28049
Madrid, Spain; orcid.org/0000-0002-6113-0656
Ana B. Caballero − nanoBIC, Departament de Quimica
Inorganica i Organica, Facultat de Quimica and Institute of
Nanoscience and Nanotechnology (IN2UB), Universitat de
Barcelona, 08028 Barcelona, Spain; orcid.org/0000-0001-
9294-9085
Carmen Lorenzo − Department of Inorganic Chemistry (M-
07), School of Sciences, Universidad Autonoma de Madrid,
́
́
́
̀
̀
́
28049 Madrid, Spain
́
́
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̀
̀
́
Farmacia i Ciencies de l’Alimentacio and Institute of
Nanoscience and Nanotechnology (IN2UB), Universitat de
Barcelona, 08028 Barcelona, Spain
́
́
Raimon Sabate − Departament de Fisicoquimica, Facultat de
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Farmacia i Ciencies de l’Alimentacio and Institute of
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Barcelona, 08028 Barcelona, Spain; orcid.org/0000-0003-
3894-2362
Complete contact information is available at:
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