Derivatives of tenuazonic acid as potential new multi‐target anti‐alzheimer’s disease agents

Article abstract of DOI:10.3390/biom11010111

Alzheimer’s disease (AD) is generally recognized as a multifactorial neurodegenerative pathology with an increasing impact on society. Tenuazonic acid (TA) is a natural compound that was recently identified as a potential multitarget ligand with anti‐cholinesterase, anti-amyloidogenic and antioxidant activities. Using its structure as a chemical scaffold, we synthesized and evaluated new derivatives (1–5), including tenuazonic‐donepezil (TA‐DNP) hybrids (4 and 5) due to the clinical importance of the anti‐AD drug donepezil. These novel compounds all achieved activity in the micromolar range towards all selected targets and demonstrated to be potentially orally absorbed. Moreover, a selected compound (1) was further investigated as a chelating agent towards copper (II), zinc (II) and iron (III) and showed good chelating ability (pFe = 16.6, pCu = 11.6, pZn = 6.0 at pH 7.4). Therefore, the TA motif can be considered an interesting building block in the search for innovative multi‐functional anti‐neurodegenerative drugs, as exemplified by hybrid 5, a promising non‐cytotoxic lead compound adequate for the early stages of AD, and capable of ameliorating the oxidative status of SH‐SY5Y human neuroblastoma cells.

Full text of DOI:10.3390/biom11010111

biomolecules
Article
Derivatives of Tenuazonic Acid as Potential New Multi-Target
Anti-Alzheimer’s Disease Agents
Viviana Poliseno 1,2 , Sílvia Chaves 2, Leonardo Brunetti 1 , Fulvio Loiodice 1, Antonio Carrieri 1
,
Antonio Laghezza 1 , Paolo Tortorella 1 , João D. Magalhães 3 , Sandra M. Cardoso 3,4, M. Amélia Santos 2,
*
and Luca Piemontese 1,
*
1
Department of Pharmacy and Pharmaceutical Sciences, University of Bari “A. Moro”, via E. Orabona 4,
70125 Bari, Italy; v.poliseno1@studenti.uniba.it (V.P.); leonardo.brunetti@uniba.it (L.B.);
fulvio.loiodice@uniba.it (F.L.); antonio.carrieri@uniba.it (A.C.); antonio.laghezza@uniba.it (A.L.);
paolo.tortorella@uniba.it (P.T.)
2
3
4
Centro de Química Estrutural and Departamento de Engenharia Química, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; silvia.chaves@tecnico.ulisboa.pt
CNC—Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal;
joaoduartemagalhaes7@gmail.com (J.D.M.); cardoso.sandra.m@gmail.com (S.M.C.)
Institute of Molecular and Cell Biology, Faculty of Medicine, University of Coimbra,
3000-548 Coimbra, Portugal
*
Correspondence: masantos@tecnico.ulisboa.pt (M.A.S.); luca.piemontese@uniba.it (L.P.)
Abstract: Alzheimer’s disease (AD) is generally recognized as a multifactorial neurodegenerative
pathology with an increasing impact on society. Tenuazonic acid (TA) is a natural compound that
was recently identified as a potential multitarget ligand with anti-cholinesterase, anti-amyloidogenic
and antioxidant activities. Using its structure as a chemical scaffold, we synthesized and evaluated
new derivatives (1–5), including tenuazonic-donepezil (TA-DNP) hybrids (4 and 5) due to the clinical
importance of the anti-AD drug donepezil. These novel compounds all achieved activity in the
micromolar range towards all selected targets and demonstrated to be potentially orally absorbed.
Moreover, a selected compound (1) was further investigated as a chelating agent towards copper
(II), zinc (II) and iron (III) and showed good chelating ability (pFe = 16.6, pCu = 11.6, pZn = 6.0 at
pH 7.4). Therefore, the TA motif can be considered an interesting building block in the search for
innovative multi-functional anti-neurodegenerative drugs, as exemplified by hybrid 5, a promising
non-cytotoxic lead compound adequate for the early stages of AD, and capable of ameliorating the
oxidative status of SH-SY5Y human neuroblastoma cells.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Poliseno, V.; Chaves, S.;
Brunetti, L.; Loiodice, F.; Carrieri, A.;
Laghezza, A.; Tortorella, P.;
Magalhães, J.D.; Cardoso, S.M.;
Santos, M.A.; et al. Derivatives of
Tenuazonic Acid as Potential New
Multi-Target Anti-Alzheimer’s
Disease Agents. Biomolecules 2021, 11,
111. https://doi.org/10.3390/
biom11010111
Keywords: Alzheimer’s disease; tenuazonic acid; donepezil; neurodegenerative; metal chelation;
multifunctional drugs; acetylcholinesterase; antioxidant; amyloid
Received: 28 October 2020
Accepted: 13 January 2021
Published: 15 January 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases,
characterized by a drastic and progressive decline in memory and cognitive abilities [1].
It represents the main cause of dementia worldwide, and it is also recognized as a socio-
economic problem especially due to the constant care needed by patients and their fami-
lies [
and is poised to rise sharply in the coming decades, as a result of increasing life expectancies
worldwide [ ]. No effective treatments are currently available for this form of dementia,
despite billions of dollars having been invested towards this goal, the United States being
the single world region responsible for most clinical trials [ ]. Only five symptomatic drugs
were approved in the EU and US, four acetylcholine esterase (AChE) inhibitors (tacrine,
donepezil, Figure 1a, rivastigmine and galantamine), and memantine, an antagonist of the
NMDA receptor [2].
2]. The number of people affected by AD is substantial, reaching 46 million globally,
Copyright:
© 2021 by the authors.
3
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
4
Biomolecules 2021, 11, 111. https://doi.org/10.3390/biom11010111
https://www.mdpi.com/journal/biomolecules

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Figure 1. (a) Donepezil (DNP); (b) Tenuazonic acid (TA).
The etiology of neurodegeneration in AD is complex, and it is now clear that it involves
a plethora of factors which must be tackled at the same time in order to achieve a valid ther-
apeutic effect. These factors include a reduced level of acetylcholine (ACh), the formation
of extracellular aggregates of amyloid
reactive oxygen species (ROS) resulting in oxidative stress. Dyshomeostasis of metal ions,
particularly Cu2+, Zn2+ and Fe3+, is thought to be responsible for the precipitation of A
and for the onset of neuroinflammatory processes which contribute to oxidative stress [5–7].
β (Aβ) and an overproduction of free radicals and
β
In accordance with this multifaceted nature, numerous researchers have focused on the
development of drugs aiming at potential one-molecules-several-target therapies, including
AChE inhibition, metal chelation and antioxidant effects [2,8–11]. In the last few years, our
research group has made several discoveries regarding potential scaffolds for the devel-
opment of multi-target agents, resulting in compounds with interesting activity profiles,
especially in regards to AChE inhibition and metal chelation [12–14]. Following the multi-
target approach, one innovative hypothesis that could lead to a future successful therapy
entails the correlation between metabolic function (or rather dysfunction) of neurons and
glial cells and neurodegeneration during the progression of AD [15–20].
In this context, natural compounds play an important role in the development of novel
multitarget agents for the treatment of many pathologies, including AD, and have been
garnering increasing attention in the last few years [17
polyphenols and other natural molecules such as fungal metabolites [19] may also prevent
neuronal damage due to their metal chelating action, reducing the aggregation of A into
,18]. Furthermore, plant-derived
β
amyloid plaques and also the formation of ROS [20]. Semisynthetic and fully synthetic
derivatives of such natural compounds have been taken into consideration as potential
drug candidates [21,22].
In a recent study, we selected six natural compounds on the basis of their chemical
structures (tenuazonic acid, epi-radicinol, mycophenolic acid, 6-methoxymellein, radicinin,
visoltricin/fungerin). These compounds were tested against a series of targets involved in
AD onset. Thus, some of them showed a multi-target activity profile, which allowed us
to consider them as starting hit compounds for the development of future potential anti
AD-drugs [19].
Tenuazonic acid (TA, Figure 1b) was the most interesting scaffold for the development
of new potential drugs for the treatment of AD due to its properties as inhibitor of AChE
and as an antioxidant, as well as for its metal chelation capacity [19].
The herein developed compounds (Figure 2) were designed by structural modification
of the TA scaffold (compounds
1–3) or by hybridization with a donepezil-mimetic-moiety
(compounds and , as tenuazonic-donepezil (TA-DNP) hybrids) trying, in the latter
4
5
case, to improve inhibitory activity towards AChE as well: the inclusion of the DNP-like
benzylpiperazine moiety should optimize the interactions formed by the inhibitors with
the enzyme pocket, allowing the occupation of the catalytic active site (CAS). Moreover,
in order to characterize their biological activity, our novel compounds were assayed for
acetylcholine esterase inhibition, antioxidant activity, inhibition of Aβ self-aggregation, cell
viability and neuroprotection. It was also interesting to investigate the chelating capacity of
these compounds towards Fe(III), Cu(II), Zn(II): thus, compound
to determine this potential activity.
1 was chosen as a model

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Figure 2. Structural formula of the developed compounds.
2. Results and Discussion
2.1. Chemistry
Tenuazonic (TA) congeners and Tenuazonic-Donepezil (TA-DNP) hybrids were syn-
thesized as racemic mixtures according to Scheme 1. In particular, starting from commercial
aminoacids and ethyl malonyl chloride, we have synthesized the linear intermediates 1a
3a, which were subsequently cyclized in presence of sodium ethoxide under reflux to afford
23 28]. Compounds and were obtained through reaction of and , respectively,
with the crucial intermediate 6 [13 23 29]. The intermediate 6 was synthesized starting
–
1–3
[
–
4
5
1
2
,
,
from the commercial N-aminoethylpiperazine as previously reported [12,29].
Scheme 1. Reagents and conditions: (i) ethyl malonyl chloride, triethylamine, anhydrous CH2Cl2,
◦
◦
N2, 0 C, 24 h; (ii) Na, abs. ethanol, reflux 4 h; (iii) phthalic anhydride, 160 C, 4 h; (iv) benzyl
◦
bromide, KOH, 96 ethanol, 24 h RT; (v) aq solution of MeNH2 (40% w/w), 60 h, RT; (vi)
1 or 2, THF,
5 h, reflux.
2.2. AChE Inhibition, Radical Scavenging Activity and Inhibition of Aβ1-42 Aggregation
Important biological assays, as well as measurements of radical scavenging activity,
were performed on the newly synthesized compounds, whose results are depicted in

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Table 1. In particular, the classical panel of tests (AChE inhibition, radical scavenging
activity and inhibition of Aβ1-42 aggregation, with or without bivalent copper) was useful
to explore the possible structure-activity relationship of tenuazonic acid (TA) derivatives
and of tenuazonic acid—donepezil(TA-DNP) hybrids.
Table 1. Results from AChE inhibition, radical scavenging and inhibition of Aβ aggregation assays.
eeAChEi a
IC50 (µM)
Antioxidant Activity EC50
Inhib. Aβ Aggreg.(%) c,d
Self-Induced
Cu2+-Induced
Compound
(µmol/µmol of DPPH ± SEM) b
1
2
3
4
5
42 ± 3
57 ± 4
44 ± 4
16 ± 2
24 ± 3
10.8 ± 0.2
6.3 ± 0.1
>30
32.9 36.6
12.4
57.4
72.0
78.4
28.2
63.8
-
-
>30
>30
65 ± 4
8.13 ± 0.08 [19]
0.026 [13]
0.11 ± 0.01
-
25.2
Tenuazonic acid
-
2.6 ± 0.2 [19]
50 ± 8 e [19]
Donepezil
Tacrine
>30
-
-
-
-
31.2
a
The values are the mean of three independent experiments
±
SEM (standard error of the mean); AChE inhibition from electric eel; b EC50
M)
M). The thioflavin-T fluorescence method was used and the measurements were carried out in the presence of
M); d The values are the means of three independent measurements in duplicate (SEM < 10%); e Cinhibitor = 100
M [19].
values for the DPPH assay (Mean
with or without copper (40
an inhibitor (40
±
SEM of 3 independent experiments); c Inhibition of self-mediated Aβ1-42 aggregation (% at 40
µ
µ
µ
µ
All new racemic compounds were tested as acetylcholinesterase inhibitors via an
in vitro assay, following a modification of Ellman’s method [30] in three independent
experiments, using tacrine as a reference compound. These biological activities were
also referred to tenuazonic acid and donepezil, included in the results table (Table 1) for
comparison purposes. TA was previously assayed for the inhibition of electric eel AChE,
but in slightly different conditions, obtaining an IC50 of 8.13
of AChE inhibitory activity obtained for the TA analogues (
±
0.08
µ
M [19]. The IC50 values
1–3) are slightly better, but
comparable with those obtained for the parent compound (TA) in the same experimental
conditions (65 ± 4 µM).
Interestingly, but not surprisingly, the TA-DNP hybrids (4 and 5) showed enhanced
AChE inhibitory profiles. This can be attributed with certainty to the benzylpiperazine
moiety (DNP pharmacophore derivative), due to the important and well-known binding
interaction within the active site (cationic active site, CAS) of the enzyme [12
–14]. As
expected, compounds and are less effective than donepezil, probably due to the absence
4
5
of the fused dimethoxybenzene ring of the DNP indanone moiety, and concomitantly lower
interactions with the peripheral anionic site (PAS) of the enzyme. Furthermore, multi-target
ligands are very often less active than compounds with single activities, since the presence
of multiple pharmacophores makes ligand optimization much more difficult.
Since some of the compounds (1–3) bear an enolic hydroxyl group, their antioxidant
activity was anticipated, namely because the weak OH bond can favor free radical inactiva-
tion. Therefore, they were assayed for radical scavenging activity using the DPPH method.
The results are consistent with this assumption, considering the moderate antioxidant
activity showed in the tested conditions, at least for compounds 1 and 2 (Table 1).
The anti-amyloidogenic activity of all the compounds was also evaluated in vitro, in
the absence and in presence of Cu(II), by measuring the fluorescence emission of Thioflavin
T (ThT) [31]. Inhibition of Cu2+-induced A
since these compounds do not contain a metal chelating moiety (see below). The A
self-aggregation inhibitory activity shown by all compounds is higher than that of parent
compound TA, with the exception of compound (probably due to solubility issues at
the experimental conditions of the assays). The fact that the reference value previously
reported by us for TA (50 8%) [19] was obtained for quite different conditions, including a
much higher inhibitor concentration (100 M instead of 40 M) is noteworthy. TA was also
preliminarily tested for inhibition of Cu2+-induced A
aggregates but did not demonstrate
β aggregates by 4 and 5 was not performed
β
2
±
µ
µ
β

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a significant difference of effect (data not shown). This may be due to different electronic
contributions between the ketone and the ester functions. Therefore, the ester derivatives
seem to be better chelators compared to TA.
It is important to note that the increase in inhibitory potential in the case of the TA-
DNP hybrids, apparently confirms that the extra benzylpiperazine moieties of compounds
4
and
with the peptide. Moreover, the chelating compounds
inhibition of A aggregation in the presence of copper, in accordance with the fact that
5
, can interfere with the formation of A
β
sheets by enabling further interactions
1–3
also present a relatively higher
β
they are copper chelators with only average activity (see Section 2.3).
The results contained in Table 1 and those in Section 2.3. suggest that the inhibition of
A
A
β
β
aggregation must be mainly explained by ligand intercalation between the
fibrils, as previously found for other compounds [12 14], and not by competition for
peptide. In fact, the conditional dissociation con-
calculated at pH 7.4 is 83 M, much higher than the
β-sheets of
–
copper between the compounds and A
stant (K’d) for the copper complex of
β
1
µ
proposed range (K’d = 1–10 picomolar) corresponding to chelators able to retrieve copper
from the Aβ peptide (K’d = 10 picomolar–100 nanomolar for Cu(Aβ) complexes) [32].
Aiming to gain proper indications on how the TA derivatives’ absolute configuration
could affect their binding properties, and to challenge at the same time any molecular
similarity with known anti-Alzheimer agents, dockings of the most interesting derivative
were enrolled for both the (R) and (S) configuration of compound
5 using the eeAChE
complex with the (R) enantiomer of donepezil (PDB entry 1EVE) [33]. As a first but crucial
step, a blind docking carried out on the whole receptor surface was performed to determine
whether our derivatives were able to primary recognize the same binding site of donepezil.
Once evidence of this was gathered, dockings were carried out exploring a smaller grid
box focused on the AChE binding sites.
In both instances, as shown in Figure 3, the benzylpiperazine moiety is well oriented
and consequently accommodated at the choline binding site, facing Trp84 for an optimized
face-to-face π-π interaction, largely resembling the pose of the native ligand, while the
cationic piperazine ring is oriented towards the catalytic triad of CAS (Ser200, His440, Glu
327), similarly to the piperidine moiety of DNP (Figure 3, bottom). Simultaneously, the
whole molecular architecture is further stabilized by a network of water mediated polar
contacts involving the phenoxy group of Tyr121.
Nonetheless, the most relevant indication is perceived at the entrance of the enzyme
active-site gorge, where the amide function of the five members heterocyclic ring stabilizes
the binding, engaging through more than one water molecule the backbone of Ser286,
Phe288 and Arg289, and the isopropyl group of the stereocenter retrieves additional
hydrophobic interactions with the side chains of Trp279 and Tyr121, paralleling those
found for the indanone moiety of DNP. This also guarantees good interactions with the
peripheral anionic site (PAS).
It might be postulated, quite undoubtedly, that comparable interactions are gained by
both the enantiomers, and as a further justification the estimated free energy of binding
present very tight values gained for both configurations (
and (S), respectively).
−
8.58 and
−
9.07 kcal/mol for (R)

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Figure 3. Top: docking poses of (R), (top left, green carbon atoms), and (S), (top right, orange carbon atoms) enantiomers of
compound :
5
into the eeAChE binding site (polar interactions are drawn in yellow, π − π stacking in magenta). Bottom
molecular superposition of the (R) and (S) with the donepezil X-ray pose (yellow carbon atoms).
2.3. Metal Complexation
Metal ions are deeply involved in the pathogenesis of neurodegenerative diseases,
namely Alzheimer’s disease (AD) [34]. Cu(II) binds to A peptides, thereby contributing
β
to enable their aggregation, while deregulated redox active metal ions, Cu(II) and Fe(III),
stimulate the overproduction of reactive oxygen species (ROS) with concomitant degrada-

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tion of several cellular biomolecules (e.g., proteins, deoxyribonucleic acid (DNA), lipids).
Therefore, one of the goals of candidate anti-AD drugs is their ability to remove deleterious
ions from the affected region, restoring ionic balance without interfering with physiological
ion homeostasis [35].
Thus, a model compound (1) containing a chelating moiety similar to that of TA was
chosen to evaluate the Fe(III), Cu(II) and Zn(II) chelating ability of this series of compounds.
2.3.1. Acid-Base Properties
In order to determine the metal complexation capacity of model compound 1, its
acid-base behavior was first studied, since it must be taken into account in the metal
complexation models. A 15% w/w DMSO/H2O medium was adopted to perform the
acid-base and chelating studies because the compound was insoluble in water. Although
this medium does not seem to be compatible with biological applications, in fact, in terms
of cellular studies the ligand concentration is considerably lower (<7 mM) and, therefore,
the final concentration of DMSO employed in culture media would be even lower (<1%),
leading to negligible alterations in biological tissues. The protonation constant of the
compound was determined by pH-potentiometry and UV-Vis spectrophotometry (see
Figure 4 and Table 2) by using Hyperquad 2008 [36] and Psequad [37], respectively.
Figure 4.
CL = 6.7 × 10
(
a
)−P4otentiometric titration curves of compound
1
(I = 0.1 M KCl, T = 25.0
(M(II) = Cu(II), Zn(II)) systems in
) Spectrophotometric
10−5 M, 15% DMSO/water) [28].
±
0.1 ◦C,
M
) and corresponding 1:3 Fe(III)/1 and 1:2 M((II)/1
15% w/w DMSO/water (a represents moles of added base per mole of ligand); (
absorption spectra of compound (2.49 < pH < 6.95) (CL = 4.0
The arrow shows spectra evolution when pH increases.
b
1
×

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Table 2. Stepwise protonation constant of compound
1
and global formation constants a of its Fe(III), Cu(II) and Zn(II)
◦
complexes (T = 25.0 ± 0.1 C, I = 0.1 M KCl, 15% w/w DMSO/water) and pM b values.
MmHhLl
(m,h,l)
Compound
log K
log β(FemHhLl)
log β(CumHhLl)
log β(ZnmHhLl)
(011)
2.334(5) c
2.85(4) d
(101)
(1-21)
(102)
(1-12)
(1-22)
(103)
pM
3.16(8) c
9.46(7) d
-
4.08(8) d
−11.75(5) c
−4.15(7) d
14.30(7) d
-
-
-
-
-
-
6.0
6.56(6) d
5.83(8) d
19.4(1) d
16.6
-
-
1
11.6
a
βMmHhL1 = [MmHhLl]/[M]m[H]h[L]l; b pM =
−
log[M] at pH 7.4 (CL/CM = 10, CM = 10−6 M) [38]; c potentiometric values; d spectropho-
tometric values.
The compound was isolated in its mono-protonated neutral form (HL in Figure 4
)
and it has only one dissociable proton. The corresponding protonation constant was
determined both by potentiometric (log K = 2.334) and spectrophotometric (log K = 2.85)
titrations (see Table 2), and the obtained values are slightly lower than the value reported
in the literature for TA (log K = 3.523). This difference can probably be attributed to
differences in the working media with different abundances of the ketonic and enolic form:
compound
that group is
1
(Figure 2) has an enolic hydroxyl group directly attached to the ring, while
-attached to the ring in the most abundant tautomer and rotamer of TA [39
β
]
(Figure 5). Both compounds can establish intramolecular H-bonds with 6-member-ring
intermediates, but the considerable increase in the acidity of the hydroxylic proton of
compound
TA forms H-bonds under predominantly two tautomeric forms, they seem weaker than
in the case of compound because they involve ketone and amide groups (with known
1 suggests a stronger interaction in this case. Although it has been admitted that
1
electron delocalization pushed by the amide nitrogen atom), instead of the more electron
rich ester group contained in compound 1.
Figure 5. Different rotamers of TA as enolic tautomer [39].
Therefore, the acidity of TA and TA derivatives
effect resulting from the existence of different intramolecular H-bonds. Figure 6 shows
the species distribution curves for compound at the experimental conditions used in the
1–3 can be explained by the stabilizing
1
spectrophotometric titration of the compound. At physiological pH 7.4, the compound is
in its deprotonated negative form (L−).
The fact that only a low percentage of DMSO (15%) was needed in the mixed DMSO/
water medium chosen to perform the solution equilibrium studies can be explained by
the predominance of the mono-charged L− species at physiological pH, although the
lipo-hydrophilic character of a compound is determined by both the molecular charge and
the ability to establish solute-solvent interactions.

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Figure 6. Species distribution curves for compound
DMSO/water [28].
1
(CL = 4
×
10−5 M) in 15% w/w
2.3.2. Metal Chelation
The chelating ability of compound
1 towards Fe(III), Cu(II) and Zn(II) was evaluated
through the determination of the global formation constants of the complexes via pH-
potentiometric and spectrophotometric titrations of the respective 1:1, 1:2 and 1:3 M/L
systems in 15% w/w DMSO/water. The fitting analysis of the respective pH-potentiometric
curves was done via Hyperquad 2008 [36] while that of the spectrophotometric absorption
spectra was performed using Psequad [37], and the found equilibrium models are presented
in Table 2. Compound 1 was chosen because of its better profile after preliminary assays
(i.e., good AChE inhibition and antioxidant activity, as well as good ability to inhibit
Cu2+-induced Aβ aggregation).
A significant change in the deprotonation profile of the ligand titration curve due to the
presence of the metal ion was observed (see Figure 4). In fact, the curves for the M/L systems,
especially for the Fe(III)/L system, lie below that of t−he ligand for 0 < a < 2, showing the
formation of metal complexes with the deprotonated (L ) form of the ligand as well as mixed
ligand-hydroxo metal complexes with relative stability order Fe > Cu > Zn.
TA complexes with several metal ions, such as Fe(III), Cu(II), Ni(II) and Mg(II) were
studied in detail by different techniques (microanalysis, mass spectrometry, IV spectroscopy,
voltammetry) and M/L stoichiometric ratios were established: 1:2 (M = Cu, Ni, Mg) and
1:3 (M = Fe) [40].
The regioselectivity of metal complexation by TA has also been a matter of discussion,
since two coordination sites have been pointed out: (i) between the external carbonyl (exo-
enol) and the amidic carbonyl, as observed in Cu(TA)2; (ii) between the external carbonyl
and the internal carbonyl (endo-enol), as observed to some degree in Fe(TA)3 [40,41].
Therefore, owing to their low log K value, TA and its derivatives like compound
are expected to be deprotonated in vivo, so compound can behave as a bidentate (O,O)
chelator for different metal ions, leading to six-membered coordination rings.
1
1
To the best of our knowledge, the determination of stability formation constants of
the complexes with TA has not yet been done and so the herein calculated values for
compound
equilibrium models of compound
1
cannot be compared with those from the literature. The metal complexation
were determined by the following process: for the Fe/L
1
and Cu/L systems, only spectrophotometric data was used due to the better quality of the
experimental results obtained [the 1:1 titration (e.g., Figure 7)] allowed the determination
of the stability constant corresponding to the ML species and then this value, as well as
the respective spectra, was introduced in the fitting of Fe/L 1:3 or Cu/L 1:2 data]; for
the Zn/L system, potentiometric data was used. For the found metal complex models
contained in Table 2, MH-2L, MH-1L2 and MH-2L2 correspond to mixed ligand-hydroxo
metal complexes. Figure 8 shows the species distribution curves for the 1:3 Fe(III)/L, 1:2
Cu(II)/L and 1:2 Zn(II)/L systems at the used experimental conditions.

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Figure 7. Spe−ct5rophotometric absorption spectra of the Fe3+/
/
1
(1:1) system (2.77 < pH < 7.87)
(
CL = 4.0 × 10
M, 15% DMSO/water) [28]. The arrow shows spectra evolution when pH increases.
−5
−5
10 M)
Figure 8. Species distribution curves for the systems: (
a
) Fe3+
/
1
(1:3) (CL = 4
×
10 M), (
b
) Cu2+
/
1
(1:2) (CL = 4
×
−4
and (c) Zn2+/1 (1:2) (CL = 6.7 × 10 M) in 15% w/w DMSO/water.
Figure 8 provides evidence that, at the concentrations used in the experimental condi-
tions, complexation of Fe(III), Cu(II) and Zn(II) begins at pH below 2.5, with the formation
of ML species. For the Zn(II)/L system, some competition also exists with metal hydroxide
species above pH 7. Moreover, both 1:1 (CuL, CuH-2L, ZnL), 1:2 (FeL2, FeH-2L2, CuH-1L2)
and 1:3 (FeL3) species are formed, although a 1:2 complex was not found by fitting the
acquired potentiometric data for the Zn(II)/L system. The mixed hydroxo-ligand com-
plexes (MH-2L, MH-1L2 and MH-2L2) are also present and denote the fact that a preferred
5-coordination core is preferential for the complex of copper with compound
1 than a
square-planar geometry, while for both CuH-2L and ZnH-2L maybe a tetragonal geometry
is accomplished by coordination of two water molecules.
Figure 9 reports possible structures for the Cu(II)/1 system at the physiological pH,
evidencing the formation of 6-member chelating rings. The behavior of compounds
2 and
3, which have an identical pharmacophore, should be the same.

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Figure 9. Structures of the Cu(II) complexes with compound 1 at the physiological pH [34].
Finally, it can be concluded that the TA derivatives herein studied are good iron
and copper chelators (pFe( ) = 16.6, pCu(1) = 11.6), although only moderate for zinc
pZn (1) = 6.0). This stability order in metal complexation found for compound is in
1
(
1
accordance with the fact that hard bidentate (O,O) compounds prefer to coordinate to
hard metal ions, like Fe(III), instead of softer ones like Cu(II) and Zn(II), following the
Pearson´s principle of “hard and soft acids bases” [6]. Therefore, these TA derivatives
can be considered selective iron chelators, although also good copper chelators without
interfering with the biological Zn2+ ion.
2.4. Cell Viability and Neuroprotection
Since an effective drug targeting Aβ oligomerization is yet to be found, the emergence
of new disease-modifying agents is crucial for the development of new therapies. With this
in mind, we performed a dose-response screening to determine the highest non-cytotoxic
concentration of selected therapeutic candidates, namely
1 and 2, and their respective
derivatives and (Figure 10). This assay was also useful to demonstrate that, at the active
5
4
concentrations, these molecules do not show (except for compound
tested cell line.
4) cytotoxicity on the
Figure 10. Dose-response screening of compounds
Overnight cultures were treated with a range of concentrations from 10
cellular viability was determined by employing the MTT reduction assay. Results are expressed
relatively to SH-SY5Y untreated cells, with the mean SEM derived from at least three different
1
,
2,
4
and
5
on SH-SY5Y neuronal cell line.
µ
M to 100 M for 24 h and
µ
±
experiments. * p < 0.05; ** p < 0.01, significantly different when compared with control, untreated
SH-SY5Y cells.

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At the molecular level, this multifactorial disease is characterized by the intracellular
formation of neurofibrillary tangles and by a marked extracellular deposition of senile
plaques. These plaques are fibrillar depositions mainly composed of Aβ1-42 peptides,
which in turn oligomerize into fibrils and are then externalized by neurons. Hence, the
administration of Aβ1-42 peptides to SH-SY5Y cells is a useful cellular model to investigate
the pathophysiology of AD. Indeed, administration of Aβ1-42 was sufficient to induce cell
toxicity in cells (Figure 11). Our results demonstrate that the tested compounds did not
directly protect from Aβ1-42-induced toxicity (Figure 11).
Figure 11. Selected compounds do not confer protection against Aβ1-42 -induced toxicity on SH-SY5Y
cells. Overnight cultures were treated with Aβ1-42 peptide (2.5 µM) for 24 h in the absence or in the
presence of the compounds. When compounds were present, a pre-incubation of 1 h preceded the
Aβ1-42 treatment. Cellular viability was determined by employing the MTT reduction assay. Results
are expressed relatively to SH-SY5Y untreated cells, with the mean SEM derived from at least three
±
different experiments. **** p < 0.0001, significantly different when compared with SH-SY5Y untreated
cells. (compounds: 1, 2–100 µM; 4–10 µM; 5–20 µM).
However, it must be emphasized that oxidative stress derived from intense production
of reactive oxygen species is a another important hallmark of AD [42]. Interestingly, A
β
plaques deposition is preceded by an increase in ROS production [43]. In fact, mitochon-
drial disruption leads to an increase in ROS production, leading to an oxidative-stress
mediated deposition of amyloid plaques [44]. Additionally, oxidative stress has a mean-
ingful impact in AD, since the levels of oxidized proteins and DNA are increased in the
brain of AD patients [45]. As such, we sought to utilize the combination of Fe/Asc to
induce oxidative stress in our cellular model, in order to study a possible positive effect of
selected compounds in the early stages of the pathology. As observed in Figure 11, Fe/Asc
was able to arrest SH-SY5Y proliferation. In our assay, compound
5 presented the best
capacity to scavenge oxygen radicals and conferred neuroprotection against the Fe/Asc
challenge (Figure 12). This is preferable because drugs that target enzymes involved in the
redox state regulation are a better option than radical scavengers in antioxidant therapy.
Since compound
5 was not able to directly protect SH-SY5Y cells from Aβ1-42-induced cell
toxicity, we believe that it can be beneficial in the earlier stages of AD by ameliorating the
oxidative status of cells.

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Figure 12. Neuroprotective effect of compounds after Ferrous Sulfate (Fe)/L-Ascorbic Acid (AscH(-))
insult. SH-SY5Y cells were treated with Fe/Asc (2.5 M/5 mM, respectively) for 24 h in the absence or
in the presence of compounds. When compounds were present, a pre-incubation of 1 h preceded the
Fe/Asc treatment. Cellular viability was determined by employing the MTT reduction assay. Results
are expressed relatively to SH-SY5Y untreated cells, with the mean
±
SEM derived from at least
three different experiments. **** p < 0.0001, significantly different when compared with SH-SY5Y
untreated cells; # p < 0.05, significantly different when compared with Fe/Asc treated SH-SY5Y cells.
(compounds: 1, 2–100 µM; 4–10 µM; 5–20 µM).
2.5. Pharmacokinetic Properties
Pharmacokinetic studies were performed using QikProp v.2.5. [46], in an effort to
assess the drug-likeness of the novel compounds and their potential to penetrate important
membranes such as the blood–brain barrier (BBB). Moreover, analysis of parameters such
as calculated log P (clog P) can be useful to address the design of new compounds. From
the analysis of the results reported in Table 3, it is evident that all the compounds do not
show violations of “Lipinski’s rule of five” [47] and this indicates a potential good oral
bioavailability. The results calculated for the BBB permeability (log BB), and the octanol-
water partition coefficient (clog P, are within the range of values calculated for known
drugs, respectively, (
present good CACO-2 permeability (absorption from the intestinal tract to the blood)
but low CNS activity. These are still encouraging results, in view of expected further
structural derivatizations. On the other hand, compounds and , presenting a high CNS
activity, can be considered potentially effective as final anti-AD drug candidates.
−
3–1.2) and (
−
2–6.5) [46]. Furthermore, the small sized compounds
1–3
4
5
Table 3. Pharmacokinetic properties of the compounds as predicted in silico by software QikProp v.2.5 [46].
Caco2 Permeability
(nm/s)
Violations of Lipinski’s
Comp.
MW
clog P
log BB
CNS
Rule of 5
1
2
3
4
5
213.233
261.277
185.179
390.527
342.483
1.662
2.614
0.862
2.623
1.327
−0.979
−0.931
−0.938
−0.128
0.070
417
525
335
19
–
–
–
+
+
0
0
0
0
0
19
Moreover, this assumption was corroborated further thanks to drug-likeness pre-
dictions made with the Brain Or IntestinaL EstimateD permeation method [48], which
suggested that all studied compounds are well absorbed in the gastrointestinal tract due
to their lipophilicity and polarity (measured by WLOGP and TPSA, respectively), shown
in the white ellipse of the BOILED-Egg plot (see Figure 13). This graph confirms that
compound
4 (and probably 5 as well) might be able to pass the blood-brain barrier and
could therefore be active in the Central Nervous System. Consequently, these molecules can
be definitely considered as promising hit compounds in view of a future structure–activity
relationship study.

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Figure 13. Pharmacokinetic profile of compounds 1–5 according to BoiledEgg. BBB = blood-brain barrier permeation;
HIA = human intestinal absorption; PGP+ = potential substrate of P-glycoprotein, PGP
P-glycoprotein.
−
= non potential substrate of
3. Materials and Methods
3.1. Chemicals
Chemicals were purchased from Merck Life Science (Milan, Italy) and were utilized
without any further purification. The reactions were monitored via thin-layer chromatogra-
phy (TLC; silica gel, F254) with UV light (short-wave ultraviolet 254 and 365 nm). Inert
atmosphere of nitrogen was used to carry out all reactions that required an anhydrous
environment. Column chromatography was conducted using 60 Å silica gel (63–200 µm,
Fluka, Milan, Italy). Mass spectrometry was conducted on a HP MS 6890-5973 MDS spec-
trometer, electron impact 70 eV, equipped with a HP ChemStation or with an Agilent
6530 Series Accurate-Mass Quadrupole Time-of-FLIGHT (Q-TOF) LC/MS (Agilent, Santa
Clara CA, USA). A Bruker (Billerica, MA, USA) micro-TOF QII mass spectrometer equipped
with an electrospray (ESI) ion source was used to carry out HRMS (high-resolution mass
1
spectroscopy) analyses. H NMR spectra were obtained deuterated solvents on a Varian
Mercury 300 (Varian Inc., Palo Alto, CA, USA) and an Agilent VNMRS500 spectrometer
(Agilent, Santa Clara CA, USA). Chemical shifts (δ) are reported as parts per million (ppm)
and coupling constants (J) in Hertz (Hz). Melting points are uncorrected and were mea-
sured on a Gallenkamp electrothermal apparatus (Fisons Erba Science Ltd., Guildford, UK)
in open capillaries.
3.2. Synthesis of Chemical Compounds
3.2.1. Synthesis of 2-(2-4-Benzylpiperazinil-1-yl)ethyl)isoindoline-1,3-dione
Phthalic anhydride (10 mmol) and 1-(2-aminoethyl)-piperazine (10 mmol) were heated
◦
◦
at 160 C for 4 h [13]. The resulting dark brown solid was dissolved in 96 ethanol (25 mL)
and added with KOH (11 mmol) and benzyl bromide (10 mmol). The mixture was stirred
at room temperature (RT) for 24 h. Then, the ethanol was removed in vacuo and the residue
was partitioned between diethyl ether and H2O. The aqueous layer was further extracted

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with diethyl ether (3 times), and the collected organic phases were washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure obtaining
1
a pale yellow solid. Yield: 53%. H-NMR (400 MHz, CDCl3),
δ (ppm): 2.39–2.52 (m, 8H,
piperazine); 2.60 (t, 2H, J = 6.7 Hz, phthalimide CH2CH2N); 3.44 (s, 2H, NCH2Ph); 3.78 (t,
2H, J = 6.7 Hz, phthalimide CH2CH2N); 7.19–7.27 (m, 5H, aromatics, PhCH2); 7.67–7.69
and 7.80–7.82 (m, 4H, phthalimide). GC-MS m/z (%): 349 (7) [M]+, 189 (100), 91 (47). HRMS
[M + H]+: calculated 350.1863; found 350.1858.
3.2.2. Synthesis of 2-(4-Benzyl-1-piperazinyl)ethanamine (6)
2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (1 mmol) was dissolved in
an aqueous solution of MeNH2 40% (w/w, 5 mL). Then, the mixture was stirred at room
temperature for 60 h. Subsequently, an aq. solution of 20% (w/w) NaOH (5 mL) was
added and the resulting mixture was stirred for 2 h. Then, NaCl (1 mmol) was added
and the solution was extracted with dichloromethane and the organic layer washed with
water, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure
1
affording a pale-yellow oil. Yield: 85%. H-NMR (400 MHz, CDCl3),
δ (ppm): 2.35–2.43 (m,
10H, piperazine, NCH2CH2N); 2.73 (t, 2H, J = 6.1 Hz, NCH2CH2N); 3.46 (s, 2H, NCH2Ph);
7.18–7.27 (m, 5H, aromatics, PhC). GC-MS m/z (%): 219 (1) [M]+, 189 (100), 91 (94). HRMS
[M + H]+: calculated 220.1808; found 220.1809.
3.2.3. General Procedure for the Synthesis of Compounds 1a–3a
Ethyl malonyl chloride (5 mmol), was added at 0 ◦C to a solution of a commercial
aminoacid (alanine, or phenylalanine, or valine) methyl ester (6 mmol) and triethylamine
(TEA, 11.5 mmol) in anhydrous CH2Cl2 and the reaction mixture was stirred for 24 h,
under nitrogen atmosphere at room temperature. Then, the solution was washed with
NaHCO3 and brine solutions, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure, affording a yellow oil.
Methyl 2-((3-ethoxy-3-oxopropanamido)methyl)-3-methyl-butanoate (1a). Obtained starting
1
from valine methyl ester hydrochloride. Yield: 95%. H-NMR (300 MHz, CDCl3),
δ (ppm):
0.91 (t, 6H, CH3CHCH3); 1.28 (t, 3H, OCCH3); 1.88-1.95 (m, 1H, CH); 3.33 (s, 3H, OCH3);
3.72 (s, 2H, COCH2CO); 4.21 (q, 2H, OCH2C); 4.58 (dd, 1H, NHCH); 7.59 (d, 1H, NH).
GC-MS m/z (%): 245 (1) [M]+; 186 (100); 88 (25); 72 (42).
Ethyl 3-((2-benzyl-3-methoxy-3-oxopropyl)amino)-3-oxopro-panoate (2a). Obtained starting from
phenylalanine methyl ester hydrochloride. Yield: 90%. 1H-NMR (300 MHz, CDCl3),
δ
(ppm): 1.26 (t, 3H, OCCH3); 3.09 (dd, 1H, PhCCH) and 3.17 (dd, 1H, PhCH2C); 3.72 (s, 3H,
OCH3); 4.18 (q, 2H, OCH2); 4.87 (m, 1H), 7.12–7.29 (m, 5H, aromatics) and 7.44 (1H, d, NH).
GC-MS m/z (%): 293 (1) [M]+; 162 (100); 88 (48).
Ethyl 3-((3-methoxy-2-methyl-3-oxopropyl)amino)-3-oxopro-panoate (3a). Obtained starting
1
from alanine methyl ester hydrochloride. Yield: 71%. H-NMR (300 MHz, CDCl3),
δ
(ppm):
1.28 (t, 3H, CCH2); 1.42 (d, 3H, CH3C); 3.32 (s, 2H, COCH2CO); 3.74 (s, 3H, OCH3); 4.20
(q, 2H, OCH2C); 4.59 (m, 1H); 7.61 (s, 1H, NH). GC-MS m/z (%): 217 (1) [M]+; 158 (100);
44 (79).
3.2.4. General Procedure for the Synthesis of Compounds 1–3
Sodium ethoxide was prepared dissolving Na (4 mmol) in absolute ethanol (9 mL).
Then the suitable substrate (1a–3a, 1 mmol) was added and the resulting mixture was
stirred and refluxed for 4 h. The absolute ethanol was removed under reduced pressure
and the crude was diluted with distilled water and acidified with 2N HCl to pH 2 and
extracted with CH2Cl2. The collected organic phases were then washed with brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure to obtain the title
compounds as orange solids.
Ethyl 4-hydroxy-5-isopropyl-2-oxo-2,5-dihydro-1H-pyrrole-3-carboxylate (
1). Yield: 51%.
m.p. = 100–103 ◦C 1H-NMR (300 MHz, CDCl3),
.
δ
(ppm): 0.89 and 1.05 (2 d, 3H + 3H,

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CH3CCH3); 1.39 (t, 3H, CH3C); 2.17–2.24 (m, 1H, CCHC); 4.08 (d, 1H, NHCH); 4.36–
4.44 (q, 2H, CCH2). ESI-MS m/z: 214 [M + 1]+. HRMS (C10H15NO4+Na+): calculated:
236.0893 found: 236.0893.
Ethyl 5-benzyl-4-hydroxy-2-oxo-2,5-dihydro-1H-pyrrole-3-carboxylate (
2
).
(ppm): 1.39 (t, 3H, CH3C); 2.70
(dd, 1H, PhCCH); 3.28 (dd, 1H, PhCCH); 4.33–4.43 (m, 2H, CCH2 + 1H CH); 5.63 (s, 1H,
Yield: 79%.
◦
1
m.p. = 105–107 C. H-NMR (300 MHz, CD3OD),
δ
OH); 7.19–7.32 (m, 5H, aromatics). ESI-MS m/z: 260 [M
calculated: 284.0893 found: 284.0894.
−
1]−. HRMS (C14H15NO4+Na+):
Ethyl 4-hydroxy-5-methyl-2-oxo-2,5-dihydro-1H-pyrrole-3-carboxylate (
3
). Yield: 41%.
m.p. = 103–105 ◦C 1H-NMR (300 MHz, CD3OD),
. δ (ppm): 1.37–1.46 (m, 6H, 2 CH3);
4.21 (q, 1H, CHC); 4.42 (q, 2H, CCH2); 5.88 (s, 1H, OH). ESI-MS m/z: 186 [M + 1]+ 208
[M + Na]+. HRMS (C8H11NO4+Na+): calculated: 208.0580 found: 208.0571.
3.2.5. General Procedure for the Synthesis of Compounds 4 and 5
Intermediate 6 was added to a solution of compound
2 or 1 in THF [23]. After 5 h
reflux, the solution was dried to evaporate the THF affording a brown oil. Then, the
crude was purified affording the title compounds 4 and 5 as white solids. The com-
pound
4
(R = CH2Ph) was purified through crystallization with n-hexane and chloro-
form. The compound
5
(
R = isopropyl) was purified through chromatography column
(CH2Cl2:MeOH:TEA 98:2:0.1) and then crystallization with ethyl acetate and chloroform to
obtain a white solid.
5-Benzyl-4-((2-(4-benzylpiperazin-1-yl)ethyl)amino)-1,5-di-hydro-2H-pyrrol-2-one (
lized from n-hexane and chloroform. Yield: 26%. m.p. = 173–174 ◦C. 1H-NMR (300 MHz,
CD3OD), (ppm): 2.47–2.59 (m, 8H piperazine + 2H CH2piperazine); 2.76 (dd, 2H, CHCPh);
4). Crystal-
δ
3.00 (dd, 2H, CH2 piperazine); 3.08 (m, 2H, CH2); 3.53 (s, NCH2Ph); 4.21 (q, 1H, CH); 4.60
(s, 1H); 4.97 (s, 1H, NH); 5.17 (s, 1H, NH); 7.22-7.31 (m, 10H, aromatics). ESI-MS m/z (%):
413 [M + Na]+, HRMS (C24H30NO4+Na+): calculated: 413.2312 found: 413.2317.
4-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-5-isopropyl-1,5-dihydro-2H-pyrrol-2-one (5). Purified
through chromatography column (CH2Cl2:MeOH:TEA◦98:2:0.1) and then crystallization
with acetate and chloroform. Yield: 25%. m.p. = 168–170 C. 1H-NMR (300 MHz, CD3OD),
δ
(ppm): 0.77 and 1.05 (2 d, 3H + 3H, CH3CCH3); 1.90 (m, 1H, CCHC); 2.49–2.65 (m, 11H,
8H piperazine + 2H CCH2Ph + 1H CH); 3.10 (m, 2H CH2); 3.52 (s, 2H, NCH2Ph); 4.65 (s,
1H, CH); 4.96 (s, 1H, NH); 5.17 (s, 1H, NH); 7.26–7.32 (m, 5H, aromatics). GC-MS m/z
(%): 342 (1) [M]+; 189 (100) [C12H17N2]+; 91 (33) [C7H7]+. ESI-MS m/z (%): 365 [M + Na]+,
− −
1] . HRMS (C20H30NO4-H ): calculated: 341.2341 found: 341.2354.
343 [M + 1]+, 341 [M
−
3.3. Bioanalytical Procedures
3.3.1. Acetylcholinesterase (AChE) Inhibition
Enzyme inhibition assays were performed using an adaptation of Ellman’s method [30].
The readings were made in a spectrophotometer Perkin-Elmer Lambda 35 UV-Vis. Enzyme
activity is measured by following the increase in yellow color produced from thiocholine
when it reacts with dithiobisnitrobenzoate ion (DTNB). DTNB solution was obtained dis-
solving the reagent in HEPES buffer with salts, namely NaCl (50 mM) and MgCl2 (20 mM),
obtaining a 3 mM solution. The enzyme AChE of electroforus electricus (electric eel) stock
(500 U) was dissolved in TRIS 50 mM, pH 8.0, being posteriorly diluted to assay concentra-
tions (5 U), obtaining an aliquot of 1 mL. The enzyme substrate used was acetylthiocholine
iodide (AThCh, 16 mM), which was prepared with deionized water. The compounds tested
−3
were diluted in MeOH or DMSO (1 mg/mL, 2–7
×
10 M), with further dilution, and five
different concentrations were used for each compound, obtaining different percentages of
AChE inhibition. The assay consists in the addition of HEPES (50 mM, pH = 8), inhibitor,
AChE and MeOH in a 1 mL cuvette. The mixture was left to incubate for 15 min and, after
that, AThCh and DTNB were added. The enzymatic reaction was followed through the
measurements of absorbencies at 405 nm (Perkin Elmer Lambda 35 spectrophotometer

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equipped with a temperature programmer PTP 1þ1 Peltier System, using thermostated
◦
(T = 25.0
±
0.1 C) 1 cm path length cells) during 5 min. A blank solution containing
all the components except the enzyme, that was substituted for equal volume of HEPES,
was used. A control reaction was used using MeOH instead of the new compounds and
was considered the reaction corresponding to 100% enzymatic activity. Each assay was
performed in quintupled [12–14].
3.3.2. Radical Scavenging Capacity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was used to determine radical
scavenging activity consisting in the addition of different volumes of stock solution of
compound (4
×
10−4
−
6
×
10−3 M in MeOH) to a methanolic solution of 2.5 mL of
DPPH [13]. The final volume of the assay was 3.5 mL, corrected with MeOH to obtain
different concentrations of our compound in each assay. The control solution was composed
of MeOH and DPPH [14]. After 30 min, at RT, protected from light, the absorbance
was measured at 517 nm (Perkin Elmer Lambda 35 spectrophotometer equipped with a
temperature programmer PTP 1þ1 Peltier System, using thermostated (T14 25.0
±
0.1 ◦C)
1 cm path length cells), against a blank solution (MeOH). The assays were performed in
triplicate, in which the EC50 was obtained through the graphical representation of the %AA
vs. the inhibitor concentration.
3.3.3. Inhibition of Aβ Aggregation
The spectrofluorimetric method, based on the fluorescence emission of thioflavin T (ThT),
was used to study the Aβ1-42 aggregation inhibition. Firstly, Aβ1-42 was dissolved in HFIP
(1,1,1,3,3,3-Hexafluoro-2-propanol) and kept overnight at room temperature [12
to obtain a dry film. Then, the film was re-dissolved in CH3CN-Na2CO3 (300
(250 M) (48.3/48.3/3.4 mL v/v/v) and then phosphate buffer (pH = 8) was added. The
ligand solutions were prepared with MeOH (1mg/mL) and further with phosphate buffer.
For the inhibition studies of copper-induced A aggregation, a solution of CuCl2 240
was used. As a control, a solution of 30 L of phosphate buffer and 30 L of Aβ1-42 was
used to observe the aggregation without any inhibition, while the ligand assay solutions were
composed of 20 L of phosphate buffer, 30 L of Aβ1-42 and 10 L of ligand. The Cu-induced
aggregation studies were similarly conducted, adding 10 L of CuCl2 to induce the fibril
–14] in order
µM)/NaOH
µ
β
µM
µ
µ
µ
µ
µ
µ
aggregation. Each experiment was composed of an assay and a blank sample, where the blank
sample lacks Aβ1-42, to monitor the effect of the compounds on the fluorescence emission.
The samples were left to incubate in a CERTOMAT WR water bath for 24 h at 37 ◦C with
shaking. After 24 h, a Glycine-NaOH buffer (pH = 8.5) containing ThT were added to each
sample in a microplate. The fluorescence signal was monitored at 446 nm (
λexc) and 486 nm
(λem) with a Varian Cary Eclipse fluorimeter. Amyloid β peptide (Aβ1-42) was purchased
from GeneCust as a lyophilised powder and stored at −20 ◦C [12–14].
3.3.4. Metal Complexation
Materials and Equipment
General analytical grade reagents were purchased from current suppliers and they
were used without further purification. The aqueous iron(III) (0.0177 M), copper(II)
(0.015 M) and zinc(II) (0.0156 M) stock solutions were prepared from 1000 ppm stan-
dards (Titrisol) and its metal content was evaluated by atomic absorption. The 0.1 M HCl
solution used in calibration of the glass electrode was prepared from a Titrisol ampoule.
The iron stock solution was prepared in an HCl excess, to prevent hydrolysis, and its exact
concentration in HCl was accurately determined by the usual standard-addition method
using 0.1 M HCl (Titrisol). The titrant used in pH-potentiometric and spectrophotometric
titrations was prepared from carbonate free commercial concentrate (Titrisol, KOH 0.1 M
ampoules). The KOH solution was standardized by titration with a solution of potassium
hydrogen phthalate and was discarded whenever the percentage of carbonate, determined
by Gran’s method [49] was greater than 0.5% of the total amount of base. The potentio-

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metric studies were performed with an automated potentiometric apparatus containing
a CrisonmicropH 2002 milivoltimeter, a Crison microBu 2031 burette and a Haake ther-
mostatic bath (T = 25.0
titrations were done using a Perkin-Elmer Lambda 35 spectrophotometer.
±
0.1 ◦C), controlled by PASAT program. The spectrophotometric
Potentiometric and Spectrophotometric Studies
Titrations of the selected compound
accomplished in a 15% w/w DMSO/H2O medium, at T = 25.0
1
, alone or in the presence of the metal ions, were
±
0.1 ◦C and ionic strength
(I) 0.1 M KCl, by using 0.1 M KOH as titrant. Both glass and Ag/AgCl reference electrodes
were previously conditioned in different DMSO/H2O mixtures of increasing DMSO %
composition and the response of the glass electrode was evaluated by strong acid–strong
base (HCl/KOH) calibrations with the determination of the Nernst parameters by Gran’s
method [49]. The measurements were performed in a final volume of 30.00 mL and the
ligand concentrations (CL) were 6.7
×
10−4 M for the potentiometric studies and 4 × 10−5 M
for the spectrophotometric titrations, under different CM/CL (M = Fe, Cu, Zn) ratios: 0:1 (L),
1:1, 1:2 and 1:3. The spectrophotometric measurements were carried out in a 250–320 nm
wavelength range at pH ca 2.5-8.0. Under the stated experimental conditions, the pKw
value (13.6) was determined and subsequently used in the computations. The stepwise
protonation constants of the ligands, Ki = [HiL]/[Hi-1L][H], and the overall metal–complex
stability constants,
β
(MmHhL1) = [MmHhLl]/[M]m[H]h[L]l, were calculated by fitting the
pH-potentiometric and spectrophotometric data with, respectively, Hyperquad 2008 [36
]
and Psequad [37] programs. The metal hydrolysis model was determined under the defined
experimental conditions (I = 0.1 M KCl, 15% w/w DMSO/H2O, T = 25.0 ± 0.1 ◦C) and the
following values of stability constants were included in the fitting of the experimental data
towards the equilibrium models related to the Fe(III)/L, Cu(II)/L and Zn(II)/L systems:
log
log
β
(FeH-1) =
(ZnH-1) =
−
1.79, log
8.6, log
β
(FeH-2) =
−
4.9, log
β
(FeH-4) =
−
20.0; log
β
(Cu2H-2) =
−
9.5;
β
−
β
(ZnH-3) =
−
27.2, log
β
(Zn2H-1) =
−
8.6. The species distribution
curves were obtained with the Hyss program [36].
3.3.5. Cell Viability and Neuroprotection
SH-SY5Y human neuroblastoma cells (ATCC-CRL-2266) were seeded in DMEM/F12
(Dulbecco’s Modified Eagle Medium/Ham’s F-12 Nutrient Mixture; Thermo Fisher Sci-
entific) with 10% heat inactivated fetal bovine serum, 50 U/mL penicillin and 50◦U/mL
streptomycin under a humidified atmosphere with 95% relative humidity at 37 C at a
0.16
×
106 cells/mL density. Stock solutions from tested compounds (1, 2, 4 and 5) were
prepared in DMSO at a 25 mM concentration and stored at −20 ◦C until needed. In order
to select the highest non-toxic concentration, a dose-response screening was performed,
where SH-SY5Y cells were exposed to the compounds for 25 h. The selected concentrations
for each compound were: 75
µM final concentration for 1 and 2; 10 µM final concentration
for 4 and 20 M final concentration for 5. The final concentration of DMSO did not exceed
µ
0.05% (v/v) and no alterations on cells were observed. For the neuroprotection assays,
the selected compounds were added to the cell media 1 hour prior to the incubation with
A
β1-42 or L-ascorbic acid/ferrous sulphate. Aβ1-42 or L-ascorbic acid/ferrous sulphate
treatment was maintained for another 24 h. Aβ1-42 was prepared as 276.9 M stock in
sterile water and administrated in the medium at a 2.5 M final concentration. Ferrous
µ
µ
sulphate was freshly prepared as 0.36 M stock in water and added to the medium at 2.5 mM
final concentration. L-ascorbic acid was freshly prepared as 80 mM stock in water and
added to the medium at 5 mM final concentration. Aβ1-42 was purchased from Bachem
(Torrance, CA, USA) and ferrous sulphate and L-ascorbic acid from Sigma Chemical Co (St.
Louis, MO, USA).
Cell viability was measured by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) reduction assay. This test is based on the presence of succinate dehy-
drogenase enzyme in viable cells, that allows them to metabolize MTT into a formazan.
After treatment, the medium was removed, and cells were washed with 1x PBS one time

Biomolecules 2021, 11, 111
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to remove dead cells. 200
µL of MTT solution (0.5 mg/mL) was added to each well and
incubated for 2 h protected from light. Formazan precipitates were then dissolved in
DMSO and absorbance measured at 570 nm. The ability to reduce MTT was expressed as a
percentage of untreated control cells.
Pharmacokinetics Properties
Pharmacokinetics properties were calculated using the software QikProp v.2.5 [46
]
for parameters clog P (lipophilic character), log BB (the capacity to cross the blood brain
barrier), Caco2 permeability (the velocity of intestinal absorption), the activity in the CNS
(central nervous system) and the verification of the Lipinski’s rule. To calculate these
parameters, the optimized structures of the compounds obtained in Maestro [50] were
submitted to the software QikProp v.2.5. Drug-likeness predictions were made with the
SwissADME web service (http://www.swissadme.ch) [51].
3.4. Molecular Docking
Compounds
5, with standard values of bond lengths and valence angles, were built
within Maestro software package [50] as protonated skeleton, and passed to Open Babel [52
]
for a 10,000 steps of Steepest Descent minimization using the Universal Force Field. Chain
A of eeAChE (PDB 1EVE) enzyme structure was passed to the Protein Preparation Wizard
interface of Maestro, where water molecules were removed, and hydrogen atoms added,
optimizing their position, and determining the protonation states of residues according to
PROPKA prediction at pH 7.0. AMBER UNITED force field electrostatic charges [53] were
loaded on the protein structure, while the molcharge complement of QUACPAC [54] was
used in order to achieve Marsili-Gasteiger charges for the inhibitors. Affinity maps were
calculated firstly on a 0.500 Å spaced 100
and afterwards on 0.375 Å spaced 85
crystallized structures, and the accessibility of the binding site was exploited throughout
1000 runs of Lamarckian Genetic Algorithm (LGA) implemented in AUTODOCK 4.2.6 [55
×
100
×
100 Å3 cubic box in the blind test dockings,
85 Å3 cubic box, centered on thedonepezil
×
85
×
]
using the GPU-OpenCL algorithm version [56]. Explicit water contribution was taken into
account according to the hydration force field of AUTODOCK [57], and the population size
and the number of energy evaluations figures were set to 300 and 10000000, respectively.
Among all the plausible binding poses, the most similar pose using donepezil as query
was selected by means of the shape matching algorithm ROCS [58].
4. Conclusions
Alzheimer’s disease is generally considered a multifactorial pathology. For this reason,
a good amount of recent research effort has focused on the development of multi-target
drugs. The main goal of this work was to design and prepare Tenuazonic acid (TA) deriva-
tives (
for AChE inhibition for compounds
to TA, these compounds present improved inhibition of A
1
–
3
) starting from the structure of the natural scaffold. Though the results obtained
are in the micromolar range and relatively similar
aggregation, metal chelation
1–3
β
capacity, potential oral absorbance and are non-cytotoxic towards SH-SY5Y human neurob-
lastoma cell line which makes them good building blocks for the development of future
anti-neurodegenerative drugs. In fact, the selected compound 1 shows a good chelating
ability towards copper, with desired selectivity as compared to zinc, and unsurprisingly but
not uninterestingly, it is also a strong iron chelator, confirming to be a promising scaffold for
the development of new multi-target molecules. Moreover, two hybrids with a donepezil-
mimetic moiety (
of AChE (IC50 16–24
besides keeping the potential oral bioavailabilty. Molecular docking of compound
4
and
5
) were also synthesized and showed significantly higher inhibition
aggregation (72.0–78.4% at 40 M),
also
µM) and excellent inhibition of A
β
µ
5
showed how its length is comparable to that of Donepezil and highlighted the positive
interactions that occur between the compound and the enzyme. Moreover, although there
is no direct correlation between AChE inhibition and cell protection from A
β toxicity,
compound seems to prevent A aggregation itself. The fact that all the tested compounds
5
β

Biomolecules 2021, 11, 111
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are not per se cytotoxic on the human neuroblastoma cell line of choice is also a positive
result that will contribute to further develop this class of potential therapeutic agents.
All tested compounds, while not showing activity on all desired targets, still show
activity on at least two of them, demonstrating they are all promising hit compounds for
further drug design and development. Therefore, the newly developed compounds can be
considered as an interesting starting point for the development of future multi-functional
drugs potentially useful for the treatment of AD.
Author Contributions: Conceptualization, L.P.; methodology, L.P., M.A.S., S.C., J.D.M.; software,
S.C., A.C.; investigation, V.P., L.B., J.D.M.; data curation, V.P., S.C., A.L., L.B., A.C.; writing—original
draft preparation, V.P., L.B., S.C., J.D.M., A.C.; writing—review and editing, L.P., F.L., P.T., S.C.,
M.A.S., S.M.C.; supervision, L.P., A.L., S.C., S.M.C.; funding acquisition, L.P., F.L., P.T., M.A.S. All
authors have read and agreed to the published version of the manuscript.
Funding: Luca Piemontese acknowledges the Università degli Studi di Bari “Aldo Moro” and
Regione Puglia (Fondo di Sviluppo e Coesione 2007–2013, APQ Ricerca Regione Puglia “Programma
regionale a sostegno della specializzazione intelligente e della sostenibilità sociale ed ambientale—
FutureInResearch”—Project ID: I2PCTF6). The authors acknowledge the Portuguese Fundação para
a Ciência e Tecnologia (FCT) for the projects UID/QUI/00100/2019 and also the Portuguese NMR
(IST-UL Center) and Mass Spectrometry Networks (Node IST-CTN) for providing access to their
facilities. Cell based experiments were funded by FCT-Fundação para a Ciência e a Tecnologia project
POCI-01-0145-FEDER-030712 (Sandra M Cardoso). The author V.P. is thankful to the Global Thesis
Prize for providing fellowship.
Acknowledgments: We thank Sara Occhineri from University of Bari “Aldo Moro” for technical
support and Michele Solfrizzo from CNR-ISPA, Bari (Italy) for providing the reference compound
Tenuazonic acid.
Conflicts of Interest: The authors declare no conflict of interest.
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