Design and Synthesis of CNb-targeted Flavones and Analogues with Neuroprotective Potential Against H2O2- and Aβ1-42-Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells

Article abstract of DOI:10.3390/ph12020098

With the lack of available drugs able to prevent the progression of Alzheimer’s disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the widely reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and their C-glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical properties. To complement our analvsis logD_7.4 was determined. From all screened compounds, the p-morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma cells against both H₂O₂ A β_1-42-induced cell death. Cholinesterase inhibition was also evaluated, and modest inhibitory activities were found. This work highlights the potential of C-glucosylflavones as neuroprotective agents, and presents the p-morpholinyl C-glucosylflavone 37, which did not show any cytotoxicity towards HepG2 and Caco-2 cells at 100 ΜM, as a new lead structure for further development against AD.

Full text of DOI:10.3390/ph12020098

pharmaceuticals
Article
Design and Synthesis of CNS-targeted Flavones and
Analogues with Neuroprotective Potential Against
H2O2- and Aβ -Induced Toxicity in SH-SY5Y
Human Neuro1b-4la2 stoma Cells
Ana M. de Matos 1,2,3, Alice Martins 1, Teresa Man 4, David Evans 4, Magnus Walter 5,
Maria Conceição Oliveira 6 , Óscar López 7, José G. Fernandez-Bolaños 7, Philipp Dätwyler 8,
Beat Ernst 8, M. Paula Macedo 3,9,10 , Marialessandra Contino 11, Nicola A. Colabufo 11 and
Amélia P. Rauter 1,2,
*
1
Center of Chemistry and Biochemistry, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, Campo
Grande, 1749-016 Lisboa, Portugal; amjgmatos@gmail.com (A.M.d.M); aimartins@fc.ul.pt (A.M.)
Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, Campo Grande,
1749-016 Lisboa, Portugal
MEDIR: Metabolic Disorders, CEDOC Chronic Diseases, Nova Medical School, Campus Sant’Ana,
Rua Câmara Pestana, 6, Lab 3.8, 1150-082 Lisboa, Portugal; paula.macedo@nms.unl.pt
Department of Chemistry, Erl Wood Manor, Eli Lilly, Windlesham, Surrey GU20 6PH, UK;
man_teresa@lilly.com (T.M.); evans_david_de@lilly.com (D.E.)
Abbvie Germany, Knollstr. 51, 67061 Ludwigshafen, Germany; magnus.walter@abbvie.com
Centro de Química Estrutural, Instiuto Superior Técnico, Ulisboa, Av. Rovisco Pais, 1049-001 Lisboa,
Portugal; conceicao.oliveira@tecnico.ulisboa.pt
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203,
E-41071 Sevilla, Spain; osc-lopez@us.es (Ó.L.); bolanos@us.es (J.G.F.-B.)
Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel,
Switzerland; philipp.daetwyler@roche.com (P.D.); beat.ernst@unibas.ch (B.E.)
Department of Medical Sciences, IBIMED, Universidade de Aveiro, 3810-193 Aveiro, Portugal
APDP-ERC, APDP-Diabetes Portugal, Rua do Salitre, Nº 118-120, 1250-203 Lisboa, Portugal
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari/Biofordrug, Via Edoardo
Orabona, 4-70125 Bari, Italy; marialessandra.contino@uniba.it (M.C.);
2
3
4
5
6
7
8
9
10
11
nicolaantonio.colabufo@uniba.it (N.A.C.)
*
Correspondence: aprauter@fc.ul.pt; Tel.: +35-196-8810971
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 April 2019; Accepted: 6 June 2019; Published: 21 June 2019
Abstract: With the lack of available drugs able to prevent the progression of Alzheimer’s disease
(AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is
presently a matter of extreme importance and urgency. Here, we were inspired by the widely
reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and
their C-glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable
pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane
permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely
the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would
be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical
properties. To complement our analysis, logD7.4 was determined. From all screened compounds,
the p-morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma
cells against both H2O2- and Aβ1-42-induced cell death. Cholinesterase inhibition was also evaluated,
and modest inhibitory activities were found. This work highlights the potential of C-glucosylflavones
as neuroprotective agents, and presents the p-morpholinyl C-glucosylflavone 37, which did not show
any cytotoxicity towards HepG2 and Caco-2 cells at 100
development against AD.
µM, as a new lead structure for further
Pharmaceuticals 2019, 12, 98; doi:10.3390/ph12020098
www.mdpi.com/journal/pharmaceuticals

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Keywords: Alzheimer’s disease; Aβ1-42; cholinesterase inhibitors; flavones; chromen-4-ones;
C-glucosyl flavonoids; PAMPA
1. Introduction
Alzheimer’s disease (AD) is a chronic neurodegenerative condition currently affecting more
than 40 million people worldwide [1]. Age, genetic background, and type 2 diabetes (T2D)
are well-established risk factors for the development of this pathology, which leads to severe
memory impairment, language problems, extreme apathy, unpremeditated aggression, and delusional
symptoms [
factor not only for AD patients, but also for relatives and caregivers. But more importantly, with no
drugs being able to stop disease progression [ ], the hope and quality of life for people living with
AD is inevitably compromised unless new effective therapies are rapidly discovered.
At the molecular level, AD is a multifactorial disease [ ]. Though the amyloid
commonly placed in the center of AD aetiology, the scientific community has been conducting research to
link A with many other molecular players and processes known to contribute to disease development
and progression, including the cellular prion protein (PrPC) [
], tau hyperphosphorylation [ ], oxidative
stress, neuroinflammation [ ], and insulin resistance [ ], among others. Natural products, including
flavonoids and their C-glucosyl derivatives, have been widely studied and found to interfere with one
or more of these features [10]. Examples include chrysin ( ) and 8- -d-glucosylgenistein ( ) (Figure 1).
2]. The loss of independence in the performance of the simplest tasks is a major stress
3,4
5
β (Aβ) protein is
β
6
7
8
9
1
β
2
Both compounds have been studied by our group and were found to inhibit the formation of small
Aβ1-42 oligomers [11] or to interact with Aβ1-42 peptides [12], respectively.
OH
HO
OH
O
HO
HO
H
HO
O
O
O
O
OH
OH
OH
1
2
Figure 1. Chemical structure of chrysin (1) and 8-β-d-glucosylgenistein (2), two natural flavonoids with
potential against Alzheimer’s disease (AD). Chrysin was used as the prototype structure for chemical
modification in the present work.
In this work, we were inspired by the therapeutic potential of flavonoids against AD, having chrysin
1) as the lead structure [11] We focused on projecting and generating a small library of flavonoids
(
with neuroprotective potential, while displaying a suitable physicochemical profile. For that purpose,
we used the 5,7-dihydroxychromen-4-one unit as the basic building block of all flavonoid structures
(aglycones and C-glucosyl derivatives) for two reasons: (a) it is present in many flavonoids exhibiting
neuroprotective activities, including not only chrysin [13,14], but also apigenin [15], luteolin [16],
and vitexin [17 18], among others, and (b) its synthetic precursor, 2,4,6-trihydroxyacetophenone, has
,
the ideal electron-donating capacity to act as the sugar acceptor in C-glycosylation reactions, in contrast
with other polyphenols such as hydroquinone or catechol, which favor the accomplishment of highly
effective synthetic routes.
In the light of our previously published data [11], we planned on executing a thorough replacement
of the substituent at C-2 of the 5,7-dihydroxychromen-4-one core, with the ultimate goal of generating
new compounds with potential for the establishment of interactions with Aβ1-42. Furthermore,

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by synthesizing a number of flavonoids and their respective C-glucosyl derivatives, we were aiming
at creating a substantial pool of data that would help us to understand the influence of the sugar
moiety in their activity and/or bioavailability. Indeed, having been reported as good stabilizers of
non-amyloidogenic Aβ aggregates when combined with polyphenol aglycones [19], sugars may also
potentiate antioxidant and antidiabetic activities of these flavonoids [20], which is also an important
feature in light of the well-established relationship between AD and type 2 diabetes (T2D) [21].
By screening our compounds against H2O2- and Aβ1-42-induced cell death in SH-SY5Y human
neuroblastoma cells, we have herein established a concise structure–activity relationship study on the
neuroprotective effects of the molecular scaffold under investigation, focusing on the substituent at C-2
and the importance of the sugar moiety.
2. Results
Database assembly and compound selection. Following the same synthetic route developed for
chrysin (1) in a previous study [11], we were now interested in the base-catalysed Claisen–Schmidt
aldol condensation reaction for introducing structural diversity into the new flavone analogues.
On the basis of the commercially available aldehydes, we then generated a database collection of
98 compounds (the aglycones), which were submitted to a selection process using the Central Nervous
System (CNS)-MultiParameter Optimization (MPO) algorithm [22]. This mathematical tool enables
the alignment of six key drug-like attributes: partition coefficient (ClogP), distribution coefficient
(ClogD), acidity constant (pKa), molecular weight (MW), topological polar surface area (TPSA), and
the number of hydrogen bond donors (HBD). Once estimated, these parameters are processed by the
algorithm, which generates a desirability score in a scale from 1 to 6. This process allows the selection
of compounds with chemical features that make them the most suitable to enter the central nervous
system, while displaying favorable permeability, P-gp efflux, metabolic stability, and safety [22].
Using the Molecular Operating Environment (MOE) software, the required physicochemical
parameters were calculated for each database compound and were subsequently processed through
the CNS-MPO algorithm to generate a set of output scores. According to the creators of this tool,
only molecules having a CNS-MPO desirability score above 4 are adequate for further development.
Following this restriction, a diverse group of flavone analogues with different alkyl, aryl, and heteroaryl
substituents at C-2 was selected (see Supplementary Materials, Table S1) and synthesized.
Synthesis. Chromones and flavones were prepared starting from MOM-diprotected acetophenone
14 (see Supplementary Materials, Scheme S1), which base-catalysed Claisen–Schmidt aldol
condensation reaction with commercially available aldehydesgenerated chalcone and chalcone
analogue intermediates in very good reaction yields (63–95%). Interestingly, the isomerization
acyclic/cyclic product, resulting from chalcone/flavanone equilibrium, could be detected by liquid
chromatography-mass spectrometry (LCMS) and the flavanone was the single product isolated,
by reaction of the starting material with cyclobutylcarboxaldehyde (see Supplementary Materials).
Subsequently, chalcones and flavanones were submitted to iodine-promoted oxidation in pyridine,
followed by p-TsOH catalyzed deprotection to give the final products 4–12 in yields ranging from 38%
to 95% (Figure 2 and Supplementary Materials).
Chrysin (1) and 5,7-dihydroxychromen-4-one (13) were also synthesized, as described in a previous
study published by our group [11], with the purpose of bioactivity comparison.
For the generation of C-glucosylflavones and analogues, acetophenone C-glucosylation and
selective benzylation were carried out prior to the aldol condensation step, as previously described12
(Scheme S2, Supplementary Materials). Then, aldol condensation gave the intermediate chalcones
that reacted with iodine and were debenzylated with BCl3 at a low temperature to afford the
target glucosylchromones and glucosylflavones 15–21 (see Figure 2 and Supplementary Materials for
compound synthesis, and structure elucidation of intermediate compounds). Notably, some of the target
glucosylflavones could not be obtained, either due to the high reactivity of the intermediates, or due to
the extreme hydrophilic character of the final product, as in the case of the 2-(pyridin-4-yl)chromone,

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which made purification virtually unfeasible even when using reverse phase column purification
techniques such as HPLC.
R1O
R2
OH
R1O
R2
O
R3
R1O
O
OR1
O
R
3
14
R1=R2= H
1 = MOM, R2=H
4
12
=
R1
R
-
2 = H
15 - 21 R1= H, R2 = -D-glucosyl
O
R3
=
H
N
N
X
N
N
O
5
6
7/16
8/19
4/15
(X=H)
17 (X=OH)
18
9/20
10
11/21
13/22
12
(X=F)
Figure 2. Structure of the new chromones, flavones and their C-glucosyl derivatives studied in this
work (for the synthetic approach followed, see Supplementary Materials).
The formation of 6-glucosyl-5,7-dihydroxychromen-4-one (22) required a different methodology
as that described for its analogues. For this task, we applied the same protocol used for generating its
aglycone. Dibenzylated acetophloroglucinol derivatized with a perbenzylglucosyl group (Scheme S2B,
Supplementary Materials) reacted with sodium hydride in ethyl formate at 0 ◦C to give an intermediate,
that was subsequently dehydrated in acid medium, under reflux, affording perbenzylglucosylchromone
in 84% yield. Further deprotection with BCl3 in dichloromethane at a low temperature gave compound
22 in good yield (Scheme S2B in Supplementary Materials, that also include intermediates’ synthesis
and structure elucidation).
Parallel Artificial Membrane Permeability Assay (PAMPA) and log D7.4 determination. Finding new
molecules exhibiting adequate physicochemical properties for permeating the blood-brain barrier is
often a challenging drawback in CNS drug discovery. Indeed, in order to fully evaluate the therapeutic
potential of CNS-targeted compounds, such as those generated in the present study, the evaluation
of their physicochemical and pharmacokinetic profiles is key. Hence, all synthesized compounds
were tested in the parallel artificial membrane permeability assay (PAMPA) in order to measure and
rationalize their potential to cross membrane barriers. Testosterone was used as the positive control in
this assay. To complete our analysis, the partition coefficient at physiological pH (logD7.4) was also
determined. Ideally, log D values should be located between 1 and 4 for a good compromise between
solubility and membrane permeability allowing oral availability, good cell permeation, and low
metabolic susceptibility [23]. Results are presented in Table 1.
Neuroprotective activity assays against H2O2-induced toxicity. On the basis of previously described
protocols [24–26], the flavones and analogues herein synthesized were screened for their neuroprotective
effects against H2O2-induced oxidative stress and neuronal damage in SH-SY5Y human neuroblastoma
cells by means of a thiazolyl blue tetrazolium bromide (MTT) reduction-based cell viability assay.
This screening protocol aimed not only at comparing the activity of aglycones versus C-glucosyl
derivatives when tested at the same concentration (50
best candidates amongst amines, aliphatic or heteroaryl derivatives, or aromatic derivatives with
electron-withdrawing substituents. In addition, all compounds were directly compared to compound
µM), but also at distinguishing between the
2
,
a natural molecule conjectured to display neuroprotective effects [12], but with no cell-based evidence
reported up to this point. Results are presented in Figure 3. All compounds were tested in the same
experimental conditions without the neurotoxic agent beforehand, and none were found to significantly
decrease cell viability when compared to non-treated controls (see Supplementary Materials, Table S2).

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Table 1. Effective permeability (log Pe) and partition coefficient at pH 7.4 (log D7.4) of the synthesized
flavones and analogues.
a
b
Compound Nr.
Log Pe
Log D7.4
1
4
5
6
7
8
9
10
11
12
13
15
16
17
18
19
−4.65 ± 0.09
−4.66 ± 0.09
−4.51 ± 0.06
−4.48 ± 0.04
−4.37 ± 0.12
−4.56 ± 0.04
−5.31 ± 0.12
−4.70 ± 0.14
−4.93 ± 0.20
−4.64 ± 0.02
−4.76 ± 0.02
Below detection limit
−8.94 ± 1.83
−8.70 ± 1.50
Below detection limit
−7.08 ± 0.91
−6.52 ± 0.41
−6.94 ± 0.50
−6.76 ± 0.11
−4.42 ± 0.09
3.6 ± 0.4
2.9 ± 0.1
>2.5
>2.5
>2.5
>2.5
n.d.c
3.4 ± 0.2
>2.5
c
n.d.
2.4 ± 0.1
−0.6 ± 0.2
0.8 ± 0.3
0.1 ± 0.1
−0.2 ± 0.1
1.2 ± 0.1
1.8 ± 0.2
−0.2 ± 0.1
−2.0 ± 0.2
-
20
21
22
Testosterone
a: effective permeability; b: partition coefficient at pH 7.4; c: not determined.
Figure 3. Neuroprotective effects of compound
2, flavone derivatives and corresponding aglycones
against H2O2-induced toxicity in human SH-SY5Y neuroblastoma cells via a MTT cell viability assay.
(
A
) Effects caused by amine moieties in para-position of ring B; (
ring B with heteroaromatic groups; ( ) effects caused by electron withdrawing groups in para-position
of ring B; ( ) effects caused by the replacement of ring B with aliphatic moieties. Cells were incubated
with 100 M) or absence of each compound. The tests
M H2O2 for 24 h at 37 ◦C, in the presence (50
were performed in triplicate with a final concentration of 0.5% DMSO. Results are presented as means
B) effects caused by the replacement of
C
D
µ
µ
±
standard error. Statistical differences between groups were assessed by one-way ANOVA followed by
a Tukey’s post-test. * p < 0.05, and ** p < 0.01 versus cell control; & p < 0.05, && p < 0.01
,
p < 0.001
&&&
and &&&& p < 0.0001 versus H2O2 control; §§ p < 0.01 and §§§ p < 0.001 versus another compound.
At 100
µM, H2O2 caused over 50% of cell viability loss, which is consistent with earlier published
data (Figure 3) [25]. While compound
2
was not found to display neuroprotective effects against

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the observed H2O2-induced cell death in this cell line, at the tested concentration, amine derivatives
were generally well-succeeded in restoring cell viability (Figure 3A). Compounds 9, and 19 were
8,
in fact able to produce statistically significant differences when compared to H2O2 controls (P < 0.5,
P < 0.001 and P < 0.0001, respectively). C-glucosides 17 and 21 were also able to rescue cells from
oxidative damage caused by H2O2 (P < 0.001 and P < 0.01 respectively) (Figure 3B,C), while neither
the remaining glycosides nor the remaining aglycones were able to lead to similar results.
Neuroprotective activity assays against Aβ1-42-induced toxicity. From all compounds tested in the
above presented assay, the pair
8 and 19 presented the strongest neuroprotective potential, with
convincing proof on the importance of the aglycone for the desired neuroprotective effects against
H2O2-induced cell injury. Both compounds were henceforth selected to be further explored, together
with compound
for comparison purposes. Here, the MTT assay was used once again to assess cell viability of SH-SY5Y
cells treated with 20 M of Aβ1-42, in the presence of 50 M of each compound. Similar protocols have
been used to screen candidates for AD therapy [27 28]. Moreover, based on the existing evidence that
2, the C-analogue of chrysin (16), vitexin (17), and the pair of 4’-fluoroflavones (7 and 18)
µ
µ
,
in situ spontaneous fibrillization of Aβ1-42 in the incubation mixture is important in the triggering
of neurotoxic effects [29], we added the Aβ1-42 peptide fragment dissolved in DMSO to the culture
medium prior to incubation, together with each compound in study. Results are displayed in Figure 4.
Even though there is published evidence that undifferentiated SH-SY5Y cells are not as sensitive to
A
β1-42-induced neurite degeneration and apoptosis as differentiated ones [29], with 20 µM of Aβ1-42 we
were able to observe a significant decrease in cellular MTT reduction capacity, corresponding to roughly
half of the cell viability rates observed in the non-treated control. The 4-morpholinyl derivative 19
exhibited, once more, the result with the highest significance when compared to controls. Compound
8 also displayed relevant neuroprotective effects against Aβ1-42, contrarily to the 4’-fluoroflavone 7.
Figure 4. Neuroprotective effects of compound
human SH-SY5Y neuroblastoma cells via a MTT cell viability assay. Cells were incubated with 20
β1-42 for 24 h at 37 ◦C, in the presence (50
M) or absence of each compound. The tests were performed
in triplicate with 1% DMSO (A ) or 1.5% DMSO (A + compound maximum DMSO percentage
presented in the graph). Results are presented as means standard error. Statistical differences
2 and analogues against Aβ1-42-induced toxicity in
µM
A
µ
β
β
−
±
between groups were assessed by one-way ANOVA followed by a Tukey’s post-test. *** p < 0.001 and
**** p < 0.0001 versus cell control; & p < 0.05, && p < 0.01 and &&&& p < 0.0001 Aβ control.

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3. Discussion
As predicted by our computational calculations, all aglycones (
1,
4
–
13) presented an excellent
membrane permeation capacity, as shown by the measured effective permeability for these molecules
(Log Pe > 5.7). Yet, compounds and 10 displayed log D7.4 values slightly above the desired upper
limit, and thus they may be associated with the risk of being retained in cell membranes and/or fat
tissues. The data for C-glucosyl derivatives 15 22 exhibited much more variability. Compounds 15 and
18 displayed no detectable permeability in PAMPA. Even though compounds 16 and 17 have a slightly
improved effective permeability (Log Pe ~ 9), it is still very limited to allow passive diffusion over
−
2
–
−
membranes. Notably, the 4-pyrrolidinyl derivative (38) presented the best effective permeability from
all C-glucosylflavones (log Pe = −6.520 ± 0.408) and a determined log D7.4 (1.8 ± 0.2) within the ideal
value range. Compounds 19
derivative 19 had a desirable log D7.4 value (1.8
,
21, and 22 fell in the middle (log Pe ~ 7); however, only the 4-morpholinyl
−
±
0.2). Indeed, it seems that only certain aglycones are
able to compensate for the hydrophilicity of the sugar moiety, either owing to their intrinsic lipophilicity,
or to the resulting molecular conformation of the C-glycoside as a whole. Nevertheless, it is important
to mention that, as previously described for other C-glucosyl flavonoids [30], the glucosyl moiety in
these compounds might prompt their affinity towards GLUT-1 transporters in the blood-brain barrier
(BBB), which may ultimately contribute to enhance their concentration near the therapeutic targets in
the CNS.
The neuroprotective activity of many lead candidates, especially from natural origin, has been
assessed against cellular damage induced by H2O2 in SH-SY5Y and other relevant neuronal cell
lines over the past decades [24–26,31]. Indeed, this neurotoxic agent has been reported to cause
mitochondrial and cell membrane damage, with the depletion of antioxidant enzymatic machinery
leading to increased levels of reactive oxygen species (ROS) and associated neuroinflammatory
processes [25]. Ultimately, H2O2-induced oxidative stress and inflammation trigger neuronal apoptosis
through increased expression of pro-apoptotic factors such as caspase 3, and depletion of anti-apoptotic
factors such as Bcl-2, which have been linked to the development and progression of AD [25,32].
As described for melittin, orientin, and bikaverin, among others, protection mechanisms offered by
small molecules against H2O2-promoted cell damage and apoptosis may include the downregulation
of pro-inflammatory transcription factors such as the nuclear factor-kappaB (NF
mRNA and protein expression, inhibition of caspase expression and activity, and replenishment of
neuronal pro-oxidative/antioxidant enzyme balance [24 26]. In addition, provided that A oligomers
κB), increased Bcl-2
–
β
have been described to enhance ROS levels and protein oxidation in neurons [33], it is important
to bear in mind that other possible neuroprotective mechanisms with impacts on oxidative stress
can be associated with the inhibition of Aβ oligomerization or regulation of Aβ production and/or
clearance itself.
Compounds
H2O2 controls. What’s more, the amine moiety was found to be critical for the neuroprotective activity,
as shown by the absence of significant effects displayed by chrysin ( ) and its C-glucosyl analogue 16
8, 9, and 19 were able to produce statistically significant differences when compared to
1
.
Yet, it is herein important to distinguish between aglycones and C-glucosyl derivatives, as a consistent
correlation between the presence of the sugar moiety and stimulation of cell survival could not be
observed. If, on the one hand, amine aglycones
cell viability when compared to controls, on the other hand only compound 19 produced the desired
effect, with dramatic differences from that of its 4-pyrrolidinyl analogue 20. Since both aglycones
and are active, it is likely that the C-glucosyl moiety is detrimental in the second case. Conversely,
the fact that the 4-morpholinyl group in compounds and 19 induced protective effects regardless of
8 and 9 were able to cause significant improvements in
8
9
8
the presence of the sugar moiety could mean that this substituent, perhaps through the presence of
an endocyclic oxygen atom, is preferred for this type of activity. Interestingly, aglycone 10 was not
significantly active, reinforcing that the presence of a nitrogen-containing ring in para-position of ring
B should, indeed, be beneficial. All in all, this group of derivatives presented a major improvement

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when compared to compound
2, and both unsubstituted flavones
1
and 16, with compounds
8
and 19
as the most promising pair of flavone derivatives.
From the heteroaryl derivatives, only the C-glucosyl flavone analogue containing the 2-furanyl
moiety 21 was able to produce a significant improvement in cell viability, contrarily to its aglycone 11
or the 4-pyridinyl derivative 12 (Figure 3B). Contrasting with the pair of 4-pyrrolidinyl derivatives
(9 and 20, Figure 3A), in the case of compounds 11 and 21 it appears to be the combination of the
sugar moiety with the modified aglycone that triggers the neuroprotective effect, and not the aglycone
per se. Still, this combination was able to improve the activity of the C-glucosyl analogue of chrysin
16, indicating that the replacement of ring B with a heteroaromatic ring in the presence of the sugar
moiety may be advantageous. Ultimately, compound 21 succeeded at improving the activity of the
lead compound 2.
Regarding the compounds with electron-withdrawing groups in para-position of ring B (Figure 3C),
it is interesting to note that vitexin (17) was able to fully recover cell viability, while compound
2 and
C-glucosylchrysin (16) were not. These results indicate that the C-glucosylflavone scaffold may be more
effective in terms of neuroprotective effects than the corresponding isoflavone, and that the hydroxy
group in para-position of ring B is in fact beneficial for activity. What’s more, from both electron
withdrawing groups in para-position of ring B (OH- in vitexin and F- in derivatives 18 and
hydroxy group was able to produce a relevant neuroprotective effect, which may be attributable to: (a)
the formation of a phenoxyl radical stabilized by resonance due to the presence of the -unsaturated
7) only the
α,β
ketone if the neuroprotective effect results from direct chemical ROS inactivation, or (b) the formation
of hydrogen bonds with a macromolecular target, e.g., an antioxidant enzyme.
Accordingly, no significant differences between cell viability in the presence of either of the two
5,7-dihydroxychromen-4-ones (22 and 13) and the cell viability of H2O2 controls were observed in
our study (Figure 3D). Even though all compounds with aliphatic substituents (4, 5, and 6 led to an
increase in MTT reduction when compared to both chromen-4-ones, the increase was not significant.
From the compounds selected to be tested against Aβ1-42-induced neurotoxicity, the 4-morpholinyl
derivative 19 again presented the result with the highest significance when compared to the
controls. Compound
to the 4’-fluoroflavone
compound is active per se, but its effects are maintained or potentiated by the presence of the
sugar moiety. Provided that the pair of compounds and 19 was active in H2O2-induced toxicity
assay as well, whether the observed effects against Aβ1-42 are due to a direct interaction with A
8
also displayed relevant neuroprotective effects against Aβ1-42 (contrarily
7
) which once more supports the hypothesis that in this particular case,
8
8
β
oligomers or to the inhibition of Aβ1-42-promoted oxidative stress (or both) remains to be clarified.
Nonetheless, compound 19, the best in the series of flavones and analogues presented in this work,
also showed some inhibitory activity of acetylcholinesterase (18% at 100 µM, see Supplementary
Materials, Table S3). Moreover, all compounds tested inhibited either acetylcholinesterase (AChE)
and/or butyrylcholinesterase (BuChE). Chrysin was the most active butyrylcholinesterase inhibitor,
while amongst the selective BuChE inhibitors, the chromen-4-one derivative 6 was indeed the most
promising one. Nonetheless, compound 19 stood out as the lead molecule with potential for AD. It was
tested in human epithelial colorectal adenocarcinoma (Caco-2) and liver hepatocarcinoma (HepG2)
cells from concentrations ranging from 0.1 µM to 100 µM, and no significant cytotoxic effects could be
observed (IC50 ≥ 100 µM).
4. Materials and Methods
General Methods. HPLC grade solvents and reagents were obtained from commercial suppliers and
were used without further purification. Chrysin ( ), compound 2, and 5,7-dihydroxychromen-4-one (13
were synthesized according to the methodologies previously described by us.11,12 LCMS experiments
were performed in a column XBridge C18 3.5u 2.1
50 mm at 1.2 mL/min and 50 ◦C; 10 mM ammonium
bicarbonate pH 9/ACN, gradient 10 > 95% ACN in 1.5 min + 0.5 min hold. Reactions affording
compounds 37 and 17 were followed by TLC, carried out on aluminum sheets (20 20 cm) coated
1
)
×
×

Pharmaceuticals 2019, 12, 98
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with silica gel 60 F-254, 0.2 mm thick (Merck, Darmstadt, Germany) with detection by charring with
10% H2SO4 in ethanol. Flash column chromatography was performed using CombiFlash® Rf200
(Teledyne Isco, Lincoln, CA, USA). Preparative HPLC was performed in a Gilson apparatus using
either Phenomenex Gemini NX, C18, 5
µ
m 30
×
100 mm or Phenomenex Gemini NX, C18, 10 µm
50 × 150 mm columns. NMR spectra for compound characterization were recorded on a Bruker AV III
HD Nanobay spectrometer running at 400.13 MHz equipped with a room temperature 5 mm BBO
Smartprobe. Chemical shifts are expressed in δ (ppm) and the proton coupling constants J in Hertz (Hz).
NMR data were assigned using appropriate COSY, DEPT, HMQC, and HMBC spectra (representative
examples are provided in the Supporting information appendix). Optical rotations were measured with
a Perkin–Elmer 343 polarimeter. Melting points were measured using a Stuart SMP30 melting point
apparatus. High-resolution mass spectra of final compounds were acquired on a Bruker Daltonics
HR QqTOF Impact II mass spectrometer (Billerica, MA, USA). The nebulizer gas (N2) pressure was
set to 1.4 bar, and the drying gas (N2) flow rate was set to 4.0 L/minute at a temperature of 200 ◦C.
The capillary voltage was set to 4500 V and the charging voltage was set to 2000 V. Tested compounds
have
reported in Supplementary Materials, together with their LCMS data, physical and NMR data.
General procedure for the synthesis of non-glycosylated chalcones/flavones. Each compound 23 30 was
≥
95% purity as determined by LCMS. Synthesis of intermediate compounds 23–31 and 35–42 is
–
dissolved in dry pyridine (0.248 mmol in 7.33 mL). Then, catalytic amounts of I2 (0.087 mmol, 0.35
eq.) were added and the mixture was stirred under reflux for 24 h–72 h. All reactions were followed
by LCMS. Once the starting material was fully consumed, the mixture was allowed to reach room
temperature and the pyridine was co-evaporated with toluene under reduced pressure. The residue
was resuspended in dichloromethane, washed first with a saturated solution of sodium thiosulfate,
and then with brine. The flavone was extracted with dichloromethane (3
×
30 mL), dried over MgSO4,
and the solution filtered and concentrated under vacuum. The residue was then resuspended in
ethanol (15 mL) and p-TsOH (12% in AcOH, 0.1 mL) was added. The reaction was stirred under reflux
for 2–24 h. After having reached completion by LCMS, the solvent was evaporated under vacuum and
the residue purified using the most adequate purification method(s) to afford compounds 4–12.
2-Cyclopropyl-5,7-dihydroxy-4H-chromen-4-one (
two steps: 53%; LCMS: RT = 0.52 min, m/z = 219.0 [M + H]+ (high pH method); white solid;
m.p. = 201.2–202.9 ◦C. 1H NMR (MeOD)
(ppm) 6.26 (d, 1H, Jmeta = 2.1 Hz, H-8), 6.17 (d, 1H, Jmeta = 2.1
Hz, H-6), 6.10 (s, 1H, H-3), 2.02–1.96 (m, 1H, H-10), 1.18–1.09 (m, 4H, H-20, H-30). 13C NMR [MeOD]
4). Purified by preparative HPLC. Reaction yield over
δ
δ
(ppm) 181.5 (C-4), 173.5 (C-2), 166.0 (C-7), 163.4 (C-5), 159.6 (C-8a), 106.5 (C-3), 100.2 (C-6), 95.0 (C-8),
15.4 (C-10), 9.5 (C-20, C-30). HRMS-ESI (m/z): [M + H]+ calcd for C12H11O4 219.0652, found 219.0642.
2-Cyclobutyl-5,7-dihydroxy-4H-chromen-4-one (
two steps: 85%; LCMS: RT = 0.72 min, m/z = 233.0 [M + H]+ (high pH method); white solid;
m.p. = 200.3–201.5 ◦C. 1H NMR (MeOD)
(ppm) 6.34 (d, 1H, Jmeta = 2.1 Hz, H-8), 6.18 (d, 1H, Jmeta = 2.1
Hz, H-6), 6.03 (s, 1H, H-3), 3.53 (td, 1H, J = 8.0 Hz, H-10), 2.37–2.31 (m, 4H, H-20, H-40), 2.17–2.06 (m,
5). Purified by preparative HPLC. Reaction yield over
δ
1H, H-30a), 1.99–1.91 (m, 1H, H-30). 13C NMR [(CD3)OD]
δ (ppm) 183.4 (C-4), 174.1 (C-2), 166.0 (C-7),
163.3 (C-5), 159.9 (C-8a), 106.5 (C-3), 100.0 (C-6), 94.9 (C-8), 39.5 (C-10), 27.5 (C-20, C-40), 19.0 (C-30).
HRMS-ESI (m/z): [M + H]+ calcd for C13H13NO4 233.0808, found 233.0804.
5,7-Dihydroxy-2-(1-methylpropyl)-4H-chromen-4-one (
over three steps: 67%; LCMS: RT = 1.10 min, m/z = 235.0 [M + H]+ (low pH method); brown solid;
m.p. = 192.5–193.8 ◦C. 1H NMR (MeOD)
(ppm) 6.33 (d, 1H, Jmeta = 2.2 Hz, H-8), 6.20 (d, 1H, Jmeta = 2.2
Hz, H-6), 6.05 (s, 1H, H-3), 2.65 (sextet, 1H, J1 -1”~1 -2 = 7.4 Hz, H-10), 1.80–1.59 (m, 2H, H-20), 1.30 (d,
6). Purified by preparative HPLC. Reaction yield
δ
0
0
0
3H, J1”-1 = 7.4 Hz, H-1”), 0.95 (t, 3H, J3 -2 = 7.4 Hz, H-30). 13C NMR [MeOD]
δ (ppm) 184.1 (C-4),
0
0
0
175.8 (C-2), 166.0 (C-7), 163.3 (C-5), 159.9 (C-8a), 107.6 (C-3), 105.4 (C-4a), 100.0 (C-6), 94.8 (C-8), 41.7
(C-10), 28.6 (C-20), 18.2 (C-1”), 11.9 (C-30). HRMS-ESI (m/z): [M + H]+ calcd for C13H15O4 235.0965,
found 235.0962.

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40-Fluoro-5,7-dihydroxyflavone (
7
). Purified by preparative HPLC. Reaction yield over two steps: 38%;
LCMS: RT = 1.10 min, m/z = 273.0 [M + H]+ (low pH method); white solid; m.p. = 264.8–265.5 ◦C.
1H NMR [MeOD]
δ
(ppm) 8.05 (dd, 2H, Jortho = 8.9 Hz, J2 -F=6 -F = 5.2 Hz, H-20, H-60), 7.31 (t, 2H,
0
0
ortho~J3 -F=5 -F = 8.7 Hz, H-30, H-50), 6.73 (s, 1H, H-3), 6.50 (d, 1H, Jmeta = 2.2 Hz, 1H, H-8), 6.24 (d, 1H,
0
0
J
J
meta = 2.2 Hz, H-6). 13C NMR [MeOD]
δ (ppm) 183.8 (C-4), 166.3 (C-7), 164.7 (C-5), 163.3 (d, JC-F = 259.3
Hz, C-40), 159.5 (C-8a), 130.1 (d, JC-F = 9.0 Hz, C-20, C-60), 127.6 (C-10), 117.3 (d, JC-F = 22.5 Hz, C-30,
C-50), 106.0 (C-3, C-4a), 100.3 (C-6), 95.2 (C-8). HRMS-ESI (m/z): [M + H]+ calcd for C15H10FO4 273.0558,
found 273.0554.
5,7-Dihydroxy-40-(morpholin-4-yl)flavone (
68%; LCMS: RT = 1.07 min, m/z = 340.0 [M + H]+ (low pH method); orange solid; m.p. = 234.0–235.6 ◦C.
1H NMR (MeOD) (ppm) 7.87 (d, 2H, Jortho = 9.0 Hz, H-20 and H-60), 7.06 (d, 2H, H-30 and H-50),
6.58 (s, 1H, H-3), 6.45 (d, 1H, Jmeta = 2.2 Hz, H-8), 6.21 (d, 1H, Jmeta = 2.1 Hz, H-6), 3.86–3.83 (m, 4H,
8). Purified by preparative HPLC. Reaction yield over two steps:
δ
NCH2CH2O), 3.34 (NCH2CH2O, overlapped with metanol-d4 peak). 13C NMR (MeOD)
δ (ppm) 183.8
(C-4), 165.9 (C-2), 163.7 (C-7 and C-5), 159.4.0 (C-8a), 155.3 (C-40), 128.9 (C-20, C-60), 121.8 (C-10), 115.4
(C-30, C-50), 105.3 (C-4a), 103.3 (C-3), 100.1 (C-6), 95.0 (C-8), 67.7 (NCH2CH2O), 48.1 (NCH2CH2O).
HRMS-ESI (m/z): [M + H]+ calcd for C19H18NO5 340.1179, found 340.1175.
5,7-Dihydroxy-40-(pyrrolidin-1-yl)flavone (
95%; LCMS: RT = 1.08 min, m/z = 324.0 [M + H]+ (high pH method); orange solid; m.p. = 282.4–283.6 ◦C.
1H NMR [(CD3)2CO] (ppm) 7.89 (d, 2H, Jortho = 9.0 Hz, H-20 and H-60), 6.70 (s, 1H, H-3), 6.65 (d,
2H, H-30 and H-50), 6.46 (d, 1H, Jmeta = 2.2 Hz, H-8), 6.16 (d, 1H, Jmeta = 2.2 Hz, H-6), 3.35–3.33 (m,
9). Purified by preparative HPLC. Reaction yield over two steps:
δ
NCH2CH2), 2.00–1.97 (m, 4H, NCH2CH2). 13C NMR [(CD3)2CO]
δ (ppm) 181.5 (C-4), 164.6 (C-2), 163.9
(C-7), 161.5 (C-5), 157.2 (C-8a), 150.1 (C-40), 128.0 (C-20, C-60), 116.0 (C-10), 111.7 (C-30, C-50), 103.6
(C-4a), 100.9 (C-3), 98.7 (C-6), 93.9 (C-8), 47.4 (NCH2CH2), 25.0 (NCH2CH2). HRMS-ESI (m/z): [M + H]+
calcd for C19H18NO4 324.1230, found 324.1225.
40-Dimethylamino-5,7-dihydroxyflavone (10). Purified by preparative HPLC. Reaction yield over two steps:
54%; LCMS: RT = 1.12 min, m/z = 298.0 [M + H]+ (low pH method); orange solid; m.p. = 291.3–292.7 ◦C.
1H NMR [(CD3)2SO]
δ
(ppm) 13.11 (s, 1H, OH-5), 7.88 (d, 2H, Jortho = 9.0 Hz, H-20 and H-60), 6.80
(d, 2H, H-30 and H-50), 6.71 (s, 1H, H-3), 6.46 (d, 1H, Jmeta = 2.1 Hz, H-8), 6.17 (d, 1H, Jmeta = 2.1 Hz,
H-6), 3.03 [s, 6H, N(CH3)2]. 13C NMR [(CD3)2SO]
δ (ppm) 181.5 (C-4), 164.3 (C-2), 163.8 (C-7), 161.4
(C-5), 157.2 (C-8a), 152.6 (C-40), 127.8 (C-20, C-60), 116.5 (C-10), 111.6 (C-30, C-50), 103.6 (C-4a), 101.2
(C-3), 98.6 (C-6), 93.8 (C-8), 39.6 [N(CH3)2]. HRMS-ESI (m/z): [M + H]+ calcd for C17H16NO4 298.1074,
found 298.101.
2-(Furan-2-yl)-5,7-dihydroxy-4H-chromen-4-one (11). Purified by preparative HPLC. Reaction yield
over two steps: 59%; LCMS: RT. = 0.99 min, m/z = 298.0 [M + H]+ (low pH method); white solid;
m.p. = 239.3–240.8 ◦C. 1H NMR [(CD3)2CO]
δ (ppm) 12.87 (s, 1H, OH-5), 7.92 (d, 1H, J5 -4 = 1.6 Hz,
0 0
H-50), 7.34 (d, 1H, J4 -3 = 3.5 Hz, H-30), 6.77 (dd, 1H, J5 -4 = 3.5 Hz, J3 -4 = 1.8 Hz, H-40), 6.49–6.48 (m,
0
0
0
0
0
0
2H, H-3, H-8), 6.27 (d, 1H, Jmeta = 1.8 Hz, H-6). 13C NMR [(CD3)2CO]
δ (ppm) 182.7 (C-4), 164.9 (C-2),
156.3 (C-7)*, 163.1 (C-5), 158.0 (C-8a)*, 147.8 (C-50), 146.9 (C-20), 114.8 (C-30), 113.9 (C-40), 104.0 (C-4a),
103.9 (C-3), 100.1 (C-6), 95.0 (C-8). HRMS-ESI (m/z): [M + H]+ calcd for C13H19O5 245.0444, found
245.0438. *Permutable signals.
5,7-Dihydroxy-2-(pyridin-4-yl)-4H-chromen-4-one (12). Purified by preparative HPLC. Reaction yield
1
over three steps: 87%; LCMS: RT = 0.43 min, m/z = 256.0 [M + H]+ (low pH method);: orange oil. H
NMR (MeOD)
H-3), 6.30 (d, 1H, Jmeta = 1.8 Hz, H-8), 6.10 (d, 1H, Jmeta = 1.8 Hz, H-6). 13C NMR (MeOD)
180.7 (C-4), 171.1 (C-2), 169.8 (C-7), 161.0 (C-5), 155.6 (C-8a), 151.6 (C-10), 143.3 (C-30, C-50), 128.1 (C-20,
δ
(ppm) 8.79 (d, 2H, H-30, H-50), 8.37 (d, 2H, Jortho = 6.6 Hz, H-20, H-60), 6.74 (s, 1H,
δ
(ppm)

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C-60), 103.4 (C-3), 103.2 (C-4a), 99.6 (C-6), 92.7 (C-8). HRMS-ESI (m/z): [M + H]+ calcd for C14H10NO4
256.0604, found 256.0600.
General procedure for the synthesis of C-glucosylflavones. Each C-glucosylchalcone 35–41 was dissolved
in dry pyridine (0.172 mmol in 5.11 mL). Then, catalytic amounts of I2 (0.060 mmol, 0.35 eq.) were
added and the mixture was stirred under reflux for 48–72 h. All reactions were followed by LCMS.
Once the starting material was fully consumed, the mixture was allowed to reach room temperature and
the pyridine was co-evaporated with toluene under reduced pressure. The residue was resuspended
in dichloromethane, washed first with a saturated solution of sodium thiosulfate, and then with
brine. The flavone was extracted with dichloromethane (3
solution filtered and concentrated under vacuum. The residue was then resuspended in extra dry
dichloromethane (7.10 mL) and stirred at
78 ◦C under N2 saturated atmosphere. A 1 M solution
×
30 mL), dried over MgSO4, and the
−
of BBr3 in dichloromethane (1.72 mL, 1.72 mmol, 10 eq.) was added in a dropwise manner over
5 min, and the reaction stirred for 2–4 h. After having reached completion by LCMS, the reaction was
quenched with a 1:1 mixture of dichloromethane/methanol (ca. 15 mL) and the reaction was stirred
for approximately 20 min at room temperature. The solvent was evaporated under vacuum and the
residue purified using the most adequate purification method(s) to afford compounds 15–21.
2-Cyclopropyl-8-(β-d-glucopyranosyl)-5,7-dihydroxy-4-chromen-4-one (15). Purified by preparative HPLC.
Reaction yield over two steps: 37%; LCMS: RT = 0.51 min, m/z = 381.0 [M + H]+ (low pH method);
1
yellowish oil. H NMR (MeOD)
δ (ppm) 6.23 (s, 1H, H-6), 6.20 (s, 1H, H-3), 4.86 (H-1”, overlapped
with the methanol-d6 water peak), 3.92–3.87 (m, 2H, H-2”, H-6”a), 3.75–3.64 (m, 1H, H-6”b), 3.47–3.42
(m, 3H, H-3”, H-4”, H-5”), 2.06–2.00 (m, 1H, H-10), 1.46–1.43 (m, 1H, H-20a)*, 1.28–1.22 (m, 1H, H-30a)*,
1.18–1.09 (m, 2H, H-20b, H-30b). 13C NMR (MeOD)
δ (ppm) 183.6 (C-4), 173.3 (C-2), 164.4 (C-7), 162.7
(C-5), 160.4 (C-8a), 106.8 (C-8), 105.5 (C-3), 104.9 (C-4a), 99.3 (C-6), 82.8 (C-5”), 80.1 (C-3”), 74.9 (C-1”),
73.3 (C-2”), 72.6 (C-4”), 63.4 (C-6”), 15.7 (C-10), 10.0, 9.6 (C-20, C-30). *Permutable signals. HRMS-ESI
(m/z): [M + H]+ calcd for C18H21O9 381.1180, found 381.1176.
8-(
β-d-Glucopyranosyl)-5,7-dihydroxyflavone (16). Purified by preparative HPLC, followed by Isolute
SCX-2 column chromatography (Biotage). Reaction yield over two steps: 88%; LCMS: RT = 0.49 min,
m/z = 414.80 [M
−
H]− (high pH method); yellow solid; m.p. = 188.1–189.2 ◦C. 1H NMR (MeOD)
δ
(ppm) 8.13, 8.03 (d, 2H, Jortho = 7.1 Hz, H-20 and H-60)*, 7.58–7.54 (m, 3H, H-30, H-40 and H-50), 6.75 (s,
1H, H-3), 6.30 (s, 1H, H-6), 5.00 (d, 1H, J1”-2” = 9.9 Hz, H-1”)*, 4.11 (t, 1H, J2”-1”~2”-3” = 9.3 Hz, H-2”),
3.97 (br d, 1H, J6”a-6”b = 12.1 Hz, H-6”a)*, 3.81 (dd, 1H, J6”b-6”a = 12.1 Hz, J6”b-5” = 5.3 Hz, H-6”a), 3.68
(t, 1H, J4”-3”~4”-5” = 9.2 Hz, H-4”), 3.55–3.48 (m, 2H, H-3” and H-5”). 13C NMR (MeOD)
δ (ppm) 184.2
(C-4), 166.0 (C-2), 164.8 (C-7), 162.8 (C-5), 158.2 (C-8a), 133.1 (C-30 and C-50), 132.8 (C-10), 130.2 (C-40),
128.1, 127.8 (C-20 and C-60)*, 105.8 (C-3), 105.1 (C-8), 104.6 (C-4a), 99.6 (C-6), 82.9 (C-5”), 80.2 (C-3”),
75.3 (C-1”), 72.8 (C-2”), 72.3, 71.5 (C-4”)*, 63.1, 62.7 (C-6”)*. *Two peaks were observed due to the
presence of rotamers. HRMS-ESI (m/z): [M + H]+ calcd for C21H21O9 417.1180, found 417.1174.
8-(
β
-d-Glucopyranosyl)-5,7,40-trihydroxyflavone (17). Purified by column chromatography (DCM/MeOH
1
1:0 to 5:1). Isolated yield over two steps: 7%; Rf = 0.43 (EtOAc/EtOH 6:1). H NMR (MeOD)
7.93 (d, 2H, Jortho = 8.6 Hz, H-20, H-60), 6.91 (d, 2H, Jortho = 8.8 Hz, H-30, H-50), 6.49 (s, 1H, H-3), 6.14
δ
(ppm)
(s, 1H, H-6), 5.05 (d, 1H, J1”-2” = 9.7 Hz, H-1”), 4.17–4.10 (m, 1H, H-2”), 3.94 (d, 1H, J6”a-6”b = 12.0 Hz,
H-6”a), 3.79 (dd, 1H, J6”b-6”a = 12.1 Hz, J6”b-5” = 5.5 Hz, H-6”b), 3.65 (t, 1H, J4”-3” = J
= 9.2 Hz,
4”-5”
H-4”), 3.56–3.48 (m, 2H, H-3”, H-5”). 13C NMR (MeOD)
δ
(ppm) 182.1 (C-4), 168.2 (C-40), 164.4 (C-7),
163.3 (C-2), 161.2 (C-5), 160.8 (C-8a), 129.8 (C-20, C-60), 124.6 (C-10), 117.3 (C-30, C-50), 106.2 (C-4a), 104.5
(C-8), 102.8 (C-3), 99.3 (C-6), 82.8 (C-2”), 80.7 (C-5”), 78.5 (C-3”), 74.4 (C-1”), 72.5 (C-4”), 61.4 (C-6”).
HRMS-ESI (m/z): [M + H]+ calcd for C21H21O10 433.1129, found 433.1120.

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40-Fluoro-8-(
β-d-glucopyranosyl)-5,7-dihydroxyflavone (18). Purified by preparative HPLC. Reaction
yield over two steps: 45%; LCMS: RT= 0.71 min, m/z = 433.00 [M - H]- (low pH method); colorless
1
oil. H NMR (MeOD)
δ
(ppm) 8.12 (dd, 2H, Jortho = 8.20 Hz, JH-F = 5.9 Hz, H-20, H-60), 7.27 (t, 2H,
Jortho~H-F = 8.7 Hz, H-30, H-50), 6.52 (s, 1H, H-3), 6.02 (s, 1H, H-6), 5.04 (d, 1H, J1”-2” = 9.3 Hz, H-1”),
4.11 (t, 1H, J2”-1” = J 2”-3” = 9.2 Hz, H-2”), 3.89 (dd, 1H, J6”a-6”b = 12.3 Hz, J6”a-5” = 1.8 Hz, H-6”a), 3.80
(dd, 1H, J6”b-6”a = 12.1 Hz, J6”b-5” = 4.8 Hz, H-6”b), 3.65 (t, 1H, J4”-3” = J 4”-5” = 9.3 Hz, H-4”), 3.51 (t, 1H,
J
3”-2” = J 3”-4” = 9.0 Hz, H-3”), 3.48–3.44 (m, 1H, H-5”). 13C NMR (MeOD)
δ (ppm) 182.5 (C-4), 167.5
(C-2), 166.0 (d, JC-F = 251.2 Hz, C-40), 163.3 (C-7), 161.9 (C-5), 159.1 (C-8a), 130.3 (d, JC-F = 8.8 Hz, C-20,
C-60), 129.9 (d, JC-F = 3.2 Hz, C-10), 117.0 (d, JC-F = 22.5 Hz, C-30, C-50), 105.6 (C-4a), 104.8 (C-3), 104.4
(C-6), 104.1 (C-8), 82.6 (C-5”), 80.9 (C-3”), 76.0 (C-1”), 73.8 (C-2”), 72.1 (C-4”), 62.8 (C-6”). HRMS-ESI
(m/z): [M + H]+ calcd for C21H20FO9 435.1086, found 435.1083.
8-(
Reaction yield over two steps: 74%; LCMS: RT = 0.57 min, m/z = 500.0 [M
orange solid; m.p. = 210.5–211.4 ◦C; [α]D20 = + 10 (c 0.5 MeOH); 1H NMR (MeOD)
β
-d-Glucopyranosyl)-5,7-dihydroxy-40-(morpholin-4-yl)flavone (19). Purified by preparative HPLC.
H]− (high pH method);
(ppm) 7.94, 7.84
−
δ
(d, 2H, Jortho = 8.3 Hz, H-20 and H-60)*, 7.01 (d, 2H, Jortho = 8.6 Hz, H-30 and H-50), 6.52 (s, 1H, H-3),
6.26 (s, 1H, H-6), 5.05, 4.99 (d, 1H, J1”-2” = 9.9 Hz, H-1”)*, 4.14 (t, 1H, J2”-1” = J 2”-3” = 9.5 Hz, H-2”),
3.98–3.79 (m, 6H, H-6”a, H-6”b and NCH2CH2O), 3.70 (t, 1H, J4”-3” = J 4”-5” = 9.6 Hz, H-4”), 3.57–3.53
(m, 1H, H-3”), 3.49–3.46 (m, 1H, H-5”), 3.32–3.20 (NCH2CH2O, superimposed with the MeOD peak).
13C NMR (MeOD) (ppm) 184.0 (C-4), 166.5 (C-2), 164.4 (C-7), 162.6 (C-5), 158.0 (C-8a), 155.1 (C-40),
δ
129.5, 129.1 (C-20 and C-60)*, 122.0 (C-10), 115.3 (C-30 and C-50), 105.8 (C-4a), 105.2 (C-8), 103.0 (C-3),
99.4 (C-6), 82.8 (C-5”), 80.3 (C-3”), 75.3 (C-1”), 72.9 (C-2”), 72.3 (C-4”), 67.7 (NCH2CH2O), 63.1 (C-6”),
40.4 (NCH2CH2O). *Peaks were observed due to the presence of rotamers. HRMS-ESI (m/z): [M + H]+
calcd for C25H28NO10 502.1708, found 502.1695.
8-(β
-d-Glucopyranosyl)-5,7-dihydroxy-40-(pyrrolidin-1-yl)flavone (20). Purified by preparative HPLC.
Reaction yield over two steps: 80%; LCMS: RT = 0.86 min, m/z = 486.00 [M + H]+ (low pH method);
1
orange oil. H NMR (MeOD)
δ
(ppm) 7.93 (d, 2H, Jortho = 8.2 Hz, H-20 and H-60)*, 6.67 (d, 2H, Jortho = 8.3
Hz, H-30 and H-50), 6.50 (s, 1H, H-3), 6.25 (s, 1H, H-6), 4.99 (d, 1H, J1”-2” = 9.9 Hz, H-1”)*, 4.16 (t, 1H,
J2”-1” =J2”-3” = 9.4 Hz, H-2”), 3.98 (d, 1H, J6-a”- 6-b” = 11.8 Hz, H-6”a)*, 3.80 (dd, 1H, J6-b”-6-a” = 11.9
Hz, J6-b”,5” = 5.8 Hz, H-6”b), 3.70 (t, 1H, J4”-3” = J 4”-5” = 9.2 Hz, H-4”), 3.56–3.46 (m, 2H, H-3”, H-5”),
3.38–3.35 (m, 4H, NCH2CH2), 2.07 (s, 4H, NCH2CH2). 13C NMR (MeOD)
δ (ppm) 181.6 (C-4), 167.6
(C-2), 165.5 (C-7), 162.6 (C-5), 160.4 (C-8a), 156.4 (C-40), 129.7 (C-20 and C-60), 121.7 (C-10), 112.8 (C-30
and C-50), 105.0 (C-4a), 104.5 (C-8), 101.5 (C-3), 97.9 (C-6), 82.9 (C-5”), 80.3 (C-3”), 75.3 (C-1”), 72.9
(C-2”), 72.4 (C-4”), 63.2 (C-6”), 49.1 (NCH2CH2, overlapped with the MeOD peak), 26.4 (NCH2CH2).
*Peaks were observed due to the presence of rotamers. HRMS-ESI (m/z): [M + H]+ calcd for C25H28NO9
486.1759, found 486.1743.
2-(Furan-2-yl)-8-(β-d-glucopyranosyl)-5,7-dihydroxy-4H-chromen-4-one (21). Purified by preparative HPLC.
Reaction yield over two steps: 56%; LCMS: RT = 0.62 min, m/z = 404.80 [M
−
H]− (low pH method);
1
yellowish oil. H NMR (MeOD)
δ
(ppm) 7.82 (d, 1H, J2 -3 = 3.5 Hz, H-20), 7.33 (br s, 1H, H-40), 6.72 (dd,
0
0
1H, J3 -4 = 1.8 Hz, H-30), 6.53 (s, 1H, H-3), 6.28 (s, 1H, H-6), 4.96 (d, 1H, J1”-2” = 9.4 Hz, H-1”), 4.16 (t,
1H, J2”-1” = J2”-3” = 9.4 Hz, H-2”), 3.89 (d, 1H, J6-a”,6-b” = 11.6 Hz, H-6”a), 3.73 (dd, 1H, J6-b” = J 5” = 5.1
Hz, H-6”b), 3.64 (t, 1H, J4”-3” = J 4”-5” = 9.4 Hz, H-4”), 3.53 (t, 1H, J4”-3”~4”-5” = 9.2 Hz, H-3”), 3.46–3.41
0
0
(m, 1H, H-5”). 13C NMR (MeOD)
δ 183.7 (C-4), 167.2 (C-2), 164.9 (C-7), 162.9 (C-5), 157.3 (C-8a), 148.0
(C-20), 147.3 (C-10), 115.6 (C-40), 113.9 (C-30), 107.2 (C-4a), 106.0 (C-8), 103.5 (C-3), 99.6 (C-6), 82.6 (C-5”),
80.1 (C-3”), 75.0 (C-1”), 72.9 (C-2”), 72.3 (C-4”), 63.0 (C-6”). HRMS-ESI (m/z): [M + H]+ calcd for
C19H19NO10 407.0973, found 407.0965.
8-(
β
-d-Glucopyranosyl)-5,7-dihydroxy-4H-chromen-4-one (22). Compound 42 (0.195 g, 0.221 mmol, 1 eq.)
78 ◦C, and BCl3 (1M solution
was dissolved in anhydrous DCM (5 mL). The mixture was stirred at
−

Pharmaceuticals 2019, 12, 98
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in DCM, 2.21 mL, 2.21 mmol, 10 eq.) was added in a dropwise manner over 5 min. The reaction
was complete after 1 h, as detected by LCMS, and was quenched with a 1:1 mixture of DCM/MeOH
(40 mL). The mixture was stirred for approximately 20 min at room temperature. The solvent was
evaporated under vacuum and the residue purified by column chromatography (DCM-MeOH 1:0 to
4:1). Compound 22 was obtained as a white solid; m.p. = 192.5–193.0 ◦C; [α]D20 = + 16
º
(c 0.5 MeOH).
1
Reaction yield: 45%; LCMS: RT = 0.41 min, m/z = 338.80 [M - H]- (low pH method). H NMR (MeOD)
δ
(ppm) 8.04 (d, 1H, Jcis = 5.8 Hz, H-2), 6.28 (s, 1H, H-6), 6.24 (d, 1H, Jcis = 5.9 Hz, H-3), 4.91 (d,
1H, J1”-2” = 9.9 Hz, H-1”), 4.07 (t, 1H, J2”-1”~2”-3” = 9.3 Hz, H-2”), 3.88 (dd, 1H, J6-a”~6-b” = 12.1 Hz,
6-a”~5” = 2.1 Hz, H-6”a), 3.71 (dd, 1H, J6-b”~6-a” = 12.0 Hz, J6-b”~5” = 5.4 Hz, H-6”b), 3.50–3.41 (m, 3H,
H-3”, H-4”, H-5”). 13C NMR (MeOD)
183.7 (C-4), 165.0 (C-7), 163.0 (C-5), 158.0 (C-8a and C-2), 111.5
J
δ
(C-3), 106.8 (C-4a), 104.9 (C-8), 100.5 (C-6), 82.6 (C-5”), 80.1 (C-3”), 75.4 (C-1”), 72.8 (C-2”), 71.8 (C-4”),
62.9 (C-6”). HRMS-ESI (m/z): [M + H]+ calcd for C15H17NO9 341.0867, found 341.0865.
Log D7.4 determination. The in-silico prediction tool ALOGPS [4] was used to estimate the
octanol-water partition coefficients (log P) of the compounds. Depending on these values, the
compounds were classified either as hydrophilic (log P below zero), moderately lipophilic (log P
between zero and one), or lipophilic (log P above one) compounds. For each category, two different
ratios (volume of octan-1-ol to volume of buffer) were defined as experimental parameters (Table 2).
Table 2. Compound classification based on estimated log P values.
Compound Category
log P
Ratios (Octan-1-ol:Buffer)
hydrophilic
moderately lipophilic
lipophilic
<0
0–1
>1
30:140, 40:130
70:110, 110:70
3:180, 4:180
Equal amounts of phosphate buffer (0.1 M, pH 7.4) and octan-1-ol were mixed and shaken
vigorously for 5 min to saturate the phases. The mixture was left until separation of the two phases,
and the buffer was retrieved. Stock solutions of the test compounds were diluted with buffer to a
concentration of 1
performed in different wells of a 96-well plate. The respective volumes of buffer-containing analyte
(1 M) were pipetted to the wells and covered by saturated octan-1-ol according to the chosen
µM. For each compound, three determinations per octan-1-ol:buffer ratio were
µ
volume ratio. The plate was sealed with aluminum foil, shaken (1350 rpm, 25 ◦C, 2 h) on a Heidolph
Titramax 1,000 plate-shaker (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) and
centrifuged (2,000 rpm, 25 ◦C, 5 min, 5804 R Eppendorf centrifuge, Hamburg, Germany). The aqueous
phase was transferred to a 96-well plate for analysis by liquid chromatography-mass spectrometry
(LCMS, see below). Log P coefficients were calculated from the octan-1-ol:buffer ratio (o:b), the initial
concentration of the analyte in buffer (1 µM), and the concentration of the analyte in buffer (cB)
according to the following equation:
!
1 µM − cB
1
o : b
log P = log
Results are presented as the mean
×
(1)
cB
±
SD of three independent experiments. If the mean of two
independent experiments obtained for a given compound did not differ by more than 0.1 units, the
results were accepted.
Parallel artificial membrane permeability assay (PAMPA). Effective permeability (log Pe) was
determined in a 96-well format with PAMPA [34]. For each compound, measurements were performed
at pH 7.4 in quadruplicates. Four wells of a deep well-plate were filled with 650
µL of PRISMA HT
universal buffer, adjusted to pH 7.4 by adding the requested amount of NaOH (0.5 M). Samples (150
µL)
were withdrawn from each well to determine the blank spectra by UV/Vis-spectroscopy (190 to 500 nm,
SpectraMax 190, Molecular Devices, Silicon Valley, CA, USA). Then the analyte, dissolved in DMSO

Pharmaceuticals 2019, 12, 98
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(10 mM), was added to the remaining buffer to yield 50
µM solutions. To exclude precipitation,
the optical density (OD) was measured at 650 nm, and solutions exceeding OD 0.01 were filtrated.
Afterwards, samples (150 L) were withdrawn to determine the reference spectra. Further 200
were transferred to each well of the donor plate of the PAMPA sandwich (pIon, P/N 110 163). The filter
membranes at the bottom of the acceptor plate were infused with 5 L of GIT-0 Lipid Solution and
200 L of Acceptor Sink Buffer were filled into each acceptor well. The sandwich was assembled,
placed in the GutBoxTM, and left undisturbed for 16 h. Then, it was disassembled and samples (150
L)
µ
µL
µ
µ
µ
were transferred from each donor and acceptor well to UV-plates for determination of the UV/Vis
spectra. Effective permeability (log Pe) was calculated from the compound flux deduced from the
spectra, the filter area, and the initial sample concentration in the donor well with the aid of the PAMPA
Explorer Software (pIon, version 3.5).
LC-MS measurements. Analyses were performed using a 1100/1200 Series HPLC System coupled to
a 6410 Triple Quadrupole mass detector (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped
with electrospray ionization. The system was controlled with the Agilent MassHunter Workstation
Data Acquisition software (version B.01.04). The column used was an AtlantisR T3 C18 column
(2.1 × 50 mm) with a 3
µm-particle size (Waters Corp., Milford, MA, USA). The mobile phase consisted
of eluent A: 10 mM ammonium acetate, pH 5.0 in 95:5, H2O:MeCN; and eluent B: MeCN containing
0.1% formic acid. The flow rate was maintained at 0.6 mL/min. The gradient was ramped from 95%
A/5% B to 5% A/95% B over 1 min, and then held at 5% A/95% B for 0.1 min. The system was then
brought back to 95% A/5% B, resulting in a total duration of 4 min. MS parameters such as fragmentor
voltage, collision energy, and polarity were optimized individually for each drug, and the molecular
ion was followed for each compound in the multiple reaction monitoring mode. The concentrations
of the analytes were quantified by the Agilent Mass Hunter Quantitative Analysis software (version
B.01.04).
Neuroprotective assays in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells were grown in
Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Life Technologies) supplemented with 10% fetal
bovine serum (FBS, Biochrom◦GmbH) and 1% Penicillin-Streptomycin (Gibco, Life Technologies) in
a humidified incubator at 37 C, 5% CO2. For the neuroprotective activity assay, undifferentiated
SGSY-5Y cells were plated onto 96-well flat-bottomed microtiter plates at a density of 1
in DMEM supplemented with 2% FBS and preincubated for 24 h at 37 C, 5% CO2. Compounds
×
104 cells/well
◦
(stored as 10 mM solutions in DMSO at
50 M and, after 30 min, cells were incubated in the presence or absence of 20
1-42 (Sigma-Aldrich, dissolved in DMSO and stored in 2 mM aliquots at
20 ◦C) or 100
−
20 ◦C) were then added to achieve a final concentration of
µ
µM A
β
protein fragment
M of H2O2
−
µ
(Sigma-Aldrich, dissolved to 10 mM in 0.9% NaCl aqueous solution immediately prior to the assay)
overnight at 37 ◦C, 5% CO2. The final DMSO percentage was 0.5% for compounds in the presence
of H2O2, 1% for cells incubated only with A
β
, or 1.5% for cells incubated with compounds and A
β,
thus, controls for each DMSO percentage were also run. The following morning, 20
µL of a 5 mg/mL
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) in PBS
(Gibco, Life Technologies) was added to each well and the plates were further incubated for 4 h at 37 ◦C,
followed by the addition of DMSO (200 µL) to each well in order to dissolve the resulting formazan
crystals. After 2 h incubating at 37 ◦C, the optical density (OD) at 540 nm (with a 620 nm reference filter)
was measured in an Amersham Biosciences Biotrak II Plate Reader. The percentage of MTT reduction
was determined according to Equation (1). All experiments were performed in triplicate and results
are presented as means
±
standard error. Differences between experimental conditions were compared
for statistical significance by one-way ANOVA followed by a Tukey’s post-test, an analysis carried out
using GraphPad Prism Software (LA Jolla, CA, USA). Differences were considered significant when
P < 0.05. In order to exclude direct MTT reduction, compounds were also tested in (a) the absence of
cells and (b) in the absence of both cells and culture medium, using the same experimental conditions
described above.

Pharmaceuticals 2019, 12, 98
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"
#
ODsample − ODmedium
MTT Reduction (% of Control) =
× 100
(2)
ODcell control − ODmedium
Cell viability assay in human epithelial colorectal adenocarcinoma (Caco-2) and human liver hepatocellular
(HepG2) cells. Caco-2 and HepG2 Cells were cultured in DMEM-Dulbecco’s modified Eagle’s medium
(Sigma-Aldrich) supplemented with 10% (v/v) inactivated fetal bovine serum (PAA Laboratories GmbH),
2 mM l-glutamine (Sigma-Aldrich), 100 U/mL penicillin and 100
in a humidified incubator at 37 ◦C with a 5% CO2 atmosphere. Cells (3
into 96-well plates and incubated at 37 ◦C in 5% CO2 atmosphere. After 24 h, compounds were added
at different concentrations (0.1–100 M) and incubated in the same conditions. DMSO controls were
µ
g/mL streptomycin (Sigma-Aldrich),
×
104 cells/well) were seeded
µ
performed to evaluate a possible solvent cytotoxicity, while pure DMSO was used as a cytotoxic drug.
After the established incubation time with compounds, MTT (5 mg/mL) in PBS (Sigma-Aldrich) was
added (10
The formazan crystals were solubilized using ethanol/DMSO (1:1) (100
µ
L) to each well. After 3 h incubation at 37 ◦C, in 5% CO2, the supernatant was removed.
µL), and the absorbance values
were determined at 570 nm on the microplate reader Victor3 from PerkinElmer Life Sciences.
5. Conclusions
In the present work, we have generated a library of chemically diverse flavone analogues and
their C-glucosyl derivatives, and explored the structural requirements for neuroprotective activity and
therapeutic potential against AD.
With imposed physicochemical properties upon their design as CNS-targeted agents,
all synthesized aglycones were found to have good effective permeability and adequate logD7.4
values, indicating that, as expected, they have adequate pharmacokinetic and safety profiles for
administration, with the ability to cross cell membrane barriers, including gastrointestinal epithelia and
the BBB. Even though not all C-glucosyl derivatives were able to achieve the same outcomes due to the
presence of the polar sugar moiety, some of them presented a promising compromise between effective
permeability and lipophilicity. From these compounds, the p-morpholinyl derivative 37 stood out for
having fully rescued SH-SY5Y human neuroblastoma cells from H2O2- and Aβ1-42-induced toxicity in a
screening MTT assay. Importantly, it was the only compound in which the aglycone (
pointing towards aglycone specificity for the desired activity. The ability of the sugar moiety to maintain
or even potentiate the activity in this compound ( 19) definitely comes across as a benefit, since
according to previously published indications, the sugar may enhance the antioxidant properties of the
compound as a whole, while helping to maintain Aβ1-42 peptides in their disaggregated state [19 20].
8) was also active,
8
/
,
Furthermore, our results support the use of C-glucosylflavones for neuroprotective applications in
detriment of the corresponding isoflavones by providing a direct comparison between two scaffolds
from natural origin—compound 2 and vitexin 17—in more than one cell-based assay. Compound 19
also exhibits a modest acetylcholinesterase inhibitory activity, thus coming across as a new multitarget
lead compound for AD. This promising and non-toxic morpholinyl flavone derivative ultimately offers
a relevant improvement when compared to the natural products chrysin (1) and 2, which served as the
inspiration for our work owing to their reported neuroprotective potential.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/12/2/98/s1,
Table S1: Structure, physicochemical properties and CNS-MPO score of the flavone analogues selected for
synthesis. All physicochemical properties were calculated using MOE software; Table S2: Cytotoxic activity of
each compound assessed in SH-SY5Y neuroblastoma cells via an MTT cell viability assay; Experimental procedures
for cholinesterase inhibition assays; Table S3: Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
inhibitory efficacy of chrysin (1), 8-glucosylgenistein (2) and some of the synthesized flavone analogues at 100 µM;
Synthetic approaches for chromones, flavones and the corresponding C-glucosyl derivatives; Scheme S1: Synthesis
of selected flavones and analogues (A) via chalcone formation and (B) via flavanone formation; Scheme S2:
Synthesis of (A) C-glucosyl flavones and analogues and (B) the 6-glucosyl-5,7-dihydroxychromen-4-one (41);
Preparation procedures, physical and LCMS data, and NMR spectra of intermediate compounds.
Author Contributions: Compound design, synthesis and characterization were carried out by A.M.d.M., under
the supervision of M.W., T.M., D.E. (Eli Lilly) and A.P.R. PAMPA and logD determinations were conducted by

Pharmaceuticals 2019, 12, 98
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A.M.d.M. and P.D. under the supervision of B.E. Neuroprotective activity assays were performed by A.M.d.M.
under the supervision of M.P.d.M. Cholinesterase inhibition assays were conducted by .L. and J.G.F.-B. HRMS of
Ó
final compounds was accomplished by M.C.O. Cytotoxicity assays were conducted by A.M. under the supervision
of M.C. and N.A.C. This paper was written by A.M.d.M., revised by D.E., M.P.M., and B.E., revised and modified
by A.P.
Funding: This research was funded by the FP7—Seventh framework programme of the European Community,
FP7-PEOPLE-2013-IAPP, grant number 612347, and by Fundação para a Ciência e a Tecnologia, project
UID/Multi/0612/2019 and grant number SFRH/BD/93170/2013.
Acknowledgments: The authors thank Fundação para a Ciência e a Tecnologia for financial support through the
project UID/Multi/0612/2019 and for the PhD grant attributed to Ana Marta de Matos (SFRH/BD/93170/2013).
The European Union is also gratefully acknowledged for the support of the project entitled “Diagnostic and Drug
Discovery Initiative for Alzheimer’s Disease” (D3i4AD), FP7-PEOPLE-2013-IAPP, GA 612347.
Conflicts of Interest: There are no conflicts of interest to declare.
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