Azobioisosteres of Curcumin with Pronounced Activity against Amyloid Aggregation, Intracellular Oxidative Stress, and Neuroinflammation

Article abstract of DOI:10.1002/chem.202005263

Many (poly-)phenolic natural products, for example, curcumin and taxifolin, have been studied for their activity against specific hallmarks of neurodegeneration, such as amyloid-β 42 (Aβ42) aggregation and neuroinflammation. Due to their drawbacks, arising from poor pharmacokinetics, rapid metabolism, and even instability in aqueous medium, the biological activity of azobenzene compounds carrying a pharmacophoric catechol group, which have been designed as bioisoteres of curcumin has been examined. Molecular simulations reveal the ability of these compounds to form a hydrophobic cluster with Aβ42, which adopts different folds, affecting the propensity to populate fibril-like conformations. Furthermore, the curcumin bioisosteres exceeded the parent compound in activity against Aβ42 aggregation inhibition, glutamate-induced intracellular oxidative stress in HT22 cells, and neuroinflammation in microglial BV-2 cells. The most active compound prevented apoptosis of HT22 cells at a concentration of 2.5 μm (83 % cell survival), whereas curcumin only showed very low protection at 10 μm (21 % cell survival).

Full text of DOI:10.1002/chem.202005263

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doi.org/10.1002/chem.202005263
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&
Natural Products
Azobioisosteres of Curcumin with Pronounced Activity against
Amyloid Aggregation, Intracellular Oxidative Stress, and
Neuroinflammation
Julian Hofmann,[a] Tiziana Ginex,[b] Alba Espargaró,[c] Matthias Scheiner,[a] Sandra Gunesch,[a]
Marc Aragó,[b] Christian Stigloher,[d] Raimon SabatØ,[c] F. Javier Luque,*[b] and
Michael Decker*[a]
Abstract: Many (poly-)phenolic natural products, for exam-
ple, curcumin and taxifolin, have been studied for their activ-
ity against specific hallmarks of neurodegeneration, such as
amyloid-b 42 (Ab42) aggregation and neuroinflammation.
Due to their drawbacks, arising from poor pharmacokinetics,
rapid metabolism, and even instability in aqueous medium,
the biological activity of azobenzene compounds carrying a
pharmacophoric catechol group, which have been designed
as bioisoteres of curcumin has been examined. Molecular
simulations reveal the ability of these compounds to form a
hydrophobic cluster with Ab42, which adopts different folds,
affecting the propensity to populate fibril-like conforma-
tions. Furthermore, the curcumin bioisosteres exceeded the
parent compound in activity against Ab42 aggregation in-
hibition, glutamate-induced intracellular oxidative stress in
HT22 cells, and neuroinflammation in microglial BV-2 cells.
The most active compound prevented apoptosis of HT22
cells at a concentration of 2.5 mm (83% cell survival), where-
as curcumin only showed very low protection at 10 mm
(21% cell survival).
Introduction
sition of senile plaques, which consist of aggregates of amy-
loid-b (Ab) peptides, generally containing 40 (Ab40) or 42
(Ab42) residues.[2] These plaques are linked to neurotoxicity, ox-
idative stress, and neurodegeneration.[3,4] Neuroinflammation
also contributes to neurodegeneration and accelerates the pro-
gression of AD.[5] Because the aggregation of Ab peptides is
believed to be the initial event of AD, the identification of po-
tential inhibitors of amyloid aggregation has attracted much
interest.[6] Among these compounds, curcumin (Figure 1) is a
diarylheptanoid natural product that has shown positive ef-
fects on counteracting oxidative stress and inflammation, as
well as preventing Ab aggregation.[7] Structure–activity rela-
tionship (SAR) studies have shown that methylation of the free
hydroxy groups of curcumin leads to a loss of activity.[8] Never-
theless, the therapeutic potential of curcumin is limited by
poor pharmacokinetics, high rate of metabolism, and low sta-
bility in an aqueous environment.[9] Additionally, curcumin is
considered as a pan-assay interference compound (PAIN),[9]
which can possibly interfere with the assay readout or bind
nonspecifically to proteins, leading to false positive results.[10]
Other (poly-)phenolic compounds, such as apigenin, querce-
tin, and taxifolin (Figure 1), have also shown positive effects in
counteracting the causative events of neurodegeneration.[7c,11]
In particular, flavonoids, a class of polyphenolic natural prod-
ucts, are promising compounds against amyloid aggregation,
neuroinflammation, and oxidative stress.[12] Recent studies
have shown that chemical hybrids of taxifolin exhibit pro-
nounced neuroprotectivity in vitro and in vivo.[13] Furthermore,
through the development of chemical probes for proteomic
Alzheimer’s disease (AD) is the most common form of demen-
tia and causes progressive deterioration in cognitive behav-
ior.[1] One of the main pathogenic hallmarks of AD is the depo-
[a] J. Hofmann, M. Scheiner, S. Gunesch, Prof. Dr. M. Decker
Pharmaceutical and Medicinal Chemistry, Institute of
Pharmacy and Food Chemistry, University of Würzburg, Am Hubland
97074 Würzburg (Germany)
E-mail: michael.decker@uni-wuerzburg.de
[b] Dr. T. Ginex, M. Aragó, Prof. Dr. F. J. Luque
Department of Nutrition Food Science and Gastronomy
Faculty of Pharmacy, Institute of Theoretical and Computational
Chemistry and Institute of Biomedicine, Campus Torribera
University of Barcelona, Santa Coloma de Gramenet 08921 (Spain)
E-mail: fjluque@ub.edu
[c] Dr. A. Espargaró, Prof. Dr. R. SabatØ
Pharmacy and Pharmaceutical Technology and Physical-Chemistry
School of Pharmacy Institute of Nanoscience and Nanotechnology
(IN2UB), University of Barcelona, 08028, Barcelona (Spain)
[d] Prof. Dr. C. Stigloher
Imaging Core Facility, Biocenter/Theodor-Boveri-Institute
University of Würzburg, Am Hubland, 97074 Würzburg (Germany)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.202005263.
ꢀ 2021 The Authors. Chemistry - A European Journal published by Wiley-
VCH GmbH. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which permits
use and distribution in any medium, provided the original work is properly
cited, the use is non-commercial and no modifications or adaptations are
made.
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A common strategy to improve the pharmacological profile
of bioactive molecules is bioisosterism. This applies changes in
the molecular structure of a lead compound to improve their
physicochemical properties, while preserving the relevant
pharmacophoric features of the lead structure.[23] If combined
with photopharmacology, which represents an emerging strat-
egy that enables the photochemical control of biologically
active molecules and biosensors,[24] bioisoteric compounds
might offer a fine-tuning of the antiaggregation activity. In par-
ticular, our strategy in this study has been to characterize the
pharmacological profile of azobenzene bioisosteres of curcu-
min suitably modified to incorporate the pharmacophoric cate-
chol moiety of flavonoids. These compounds were conceived
by hybridizing relevant structural elements present in curcu-
min and taxifolin by following the rationale summarized in
Figure 2. This led to a new class of compounds that successful-
ly incorporated the previously cited Ab-related pharmacologi-
cal properties (bioactive catechol ring, aromaticity, and planari-
ty).
Figure 1. Chemical structures of curcumin, taxifolin, quercetin, apigenin, and
epigallocatechin gallate (EGCG).
studies, it was shown that these compounds seemed to specif-
ically address mitochondrial targets.[14] Several studies revealed
the importance of the catechol unit of flavonoids for their ac-
tivity against age-related disease processes, especially in the
context of AD.[15] However, flavonoids suffer from similar draw-
backs to those of curcumin, such as poor pharmacokinetics
and low metabolic stability.[16]
A clear understanding of the precise mechanism of action of
Ab42 aggregation inhibitors is challenging due to the com-
plexity of the conformational space of Ab42 monomers, the
occurrence of distinct oligomeric species in early aggregates,
and the timescale of different events implicated in the forma-
tion of Ab42 fibrils. Furthermore, whether a given compound
can exert inhibitory activity acting at different stages of Ab42
aggregation is also unclear.[17] The antiaggregating activity of
small organic compounds has been related to specific chemical
features, such as the hydroxylation profile, the presence of car-
boxyl moieties that may form salts bridges with Ab42, and the
molecular planarity conferred by aromatic rings.[18] Thus, it has
been proposed that curcumin could intercalate in Ab42 assem-
blies and destabilize preformed fibrils;[19] this effect is improved
upon through 1) expansion of the aromatic rings; 2) integra-
tion of large conjugated structures; 3) the presence of aromat-
ic rings connected by nitrogen-containing bridge; and 4) hy-
droxyl groups on aromatic, conjugated rings.[19] Apart from
that, (poly-)phenolic compounds bearing a catechol unit, such
as taxifolin, were observed to undergo autoxidation, and thus,
Figure 2. Rationalization for the azobioisostere prototype from the Ab42 in-
hibitors curcumin and taxifolin. HBD=hydrogen-bond donor; HBA=hydro-
gen-bond acceptor.
Herein, we report the design and synthesis of azobenzene-
containing bioisosteric analogues of curcumin and investigate
their antiaggregation ability against Ab42 in vitro and in a bac-
terial model.[25] Furthermore, we evaluate their neuroprotective
properties against intracellular oxidative stress in murine hip-
pocampal HT22 cells and their anti-neuroinflammatory poten-
tial in microglial BV-2 cells.
produce a site-specific covalent inhibition of Ab42 aggregation Results and Discussion
by acting on K16 and/or K28 residues of preformed amyloid fi-
brils, according to an aza-Michael addition mechanism.[20]
Synthesis of the target compounds
EGCG (Figure 1), another catechol type (poly-)phenolic com-
pound, naturally occurring in green tea, was observed to pro-
duce covalent adducts through a Schiff base mechanism.[21] In-
terestingly, the reduced form of EGCG was proposed to act at
early aggregation stages by redirecting toxic Ab oligomers to-
wards off-pathway nontoxic oligomers.[22]
The synthesis of the target compounds started with the Frie-
del–Crafts acylation of 4-nitrobenzoyl chloride (1) with dime-
thoxybenzene (2) to yield the corresponding acetophenone 3.
Ether cleavage of the methoxy groups was achieved in a mix-
ture of concentrated hydrobromic acid and acetic acid, fol-
lowed by hydrogenation of the keto group with H2 on Pd/C, to
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Scheme 1. Synthesis of target compounds 8a–f and comparison compound 8g. Reagents and conditions: i) FeCl3, 608C, 16 h; ii) 48% HBr, AcOH, reflux, 3.5 h;
iii) H2, Pd/C, MeOH, 10 bar, RT, 16 h; iv) oxone, CH2Cl2/H2O, RT, 3.5 h; v) AcOH, RT, 16 h, vi) nitrosobenzene, AcOH, RT, 16 h.
obtain compound 5a as the first building block. Partial oxida-
Table 1. In vitro antiamyloid activity of taxifolin, curcumin, and 8a–g.
tion of anilines with oxone yielded the respective nitroso deriv-
atives, which were combined with 5a in a Baeyer–Mills reac-
tion to form the desired target compounds 8a–f (Scheme 1).
Compound 8g, with both catechol hydroxyl groups methylat-
ed, was also synthesized to explore the role of the catechol
moiety by comparison (see below) with the activity of target
compounds 8a–f. It was synthesized in analogy to compounds
8a–f, but without cleavage of the methoxy groups of com-
pound 3.
E. coli overexpresses the respective protein, which forms IBs and can be
quantified by Th-S staining. Compounds were tested at 10 mm.
Compound
Ab42
inhibition [%]
tau
SEM[a]
inhibition [%]
SEM[a]
control
taxifolin
curcumin
8a
8b
8c
8d
8e
8 f
8g
0.0
4.9
2.0
4.0
2.7
2.1
3.4
1.6
4.2
2.9
4.3
3.9
0.0
1.1
2.1
4.4
3.2
2.1
2.4
3.9
2.9
3.6
4.1
3.8
37.8
80.4
78.2
81.3
63.1
67.5
73.3
9.6
35.2
71.0
65.1
58.0
66.6
73.6
78.3
5.7
In vitro inhibition of Ab42 and tau aggregation
Because the aggregation of amyloidogenic proteins, such as
Ab42, and deposits of hyperphosphorylated tau protein in neu-
rofibrillary tangles are associated with neurodegenerative dis-
eases, such as AD,[26,27] compounds with antiaggregation prop-
erties may be a viable option for modifying the disease. To
evaluate the antiaggregation activity of the target compounds,
a rapid in vitro screening method in bacterial cells was ap-
plied.[7c,25] This method is based on Escherichia coli overexpress-
ing the respective protein (Ab42, human tau), which forms in-
clusion bodies (IBs). IBs are consequently stained by thioflavin-
S (Th-S) to assess the amount of aggregated protein.
[a] SEM=standard error of the mean.
ed the antiamyloid effect of these natural products against
Ab42 and tau. As shown in Table 1, a similar antiaggregation
activity was observed for Ab42 and tau, whereas taxifolin dis-
played practically no activity (<5%) and curcumin was found
to have a moderate inhibitory effect (38%). Remarkably, the ac-
tivity of the target compounds greatly exceeds the potency of
curcumin and taxifolin, revealing the suitability of the bioiso-
steric design.
The evaluation of the antiamyloid aggregation activity of
the novel compounds displayed good potencies, with an ag-
gregation inhibition between 65 and 80% tested at a concen-
tration of 10 mm (Table 1). In general, similar antiaggregation
activity was found against Ab42 and tau. Compounds 8a and
8 f display an average (Ab42 and tau) inhibition of 75.8 and
75.7%, respectively, against these proteins. Compound 8c, and
to a lesser extent 8b, showed, however, a higher inhibitory po-
tency against aggregation of Ab42. Interestingly, compound
8g, which has a protected catechol moiety, displays practically
no activity (<10%) in the bacterial system.
In vitro inhibition of Ab42 detected by TEM
The detection of amyloid fibrils by fluorescent dyes can be
biased by compounds with absorptive and fluorescent proper-
ties such as the molecules investigated in this study.[28] Hence,
the inhibitory effect of these compounds was further examined
by resorting to TEM, which provided a dye-independent ap-
proach to assess the antiaggregating effect of the compounds.
The results clearly confirmed the inhibitory effect on fibril for-
mation of Ab42 at 10 mm for curcumin and compounds 8a–f
(cf. Figure 3 and Figure S5 in the Supporting Information).
Because the new compounds were conceived as bioisosteric
mimics of curcumin and taxifolin (Figure 1), we also investigat-
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Figure 3. TEM analysis of the inhibitory effect on Ab42. The Ab monomer (100 mm) was incubated at 378C in phosphate-buffered saline (PBS) for 24 h with or
without 10 mm of the respective compound. A) Control; B) curcumin; C) 8g; D) 8 f. Scale bar: 300 nm.
Interaction of the target compounds with Ab42
lected and the first five replicas, corresponding to the
Ab42mon–8 f system at 315, 316.7, 318.4, 320.1, and 321.8 K,
were analyzed (see the Experimental Section for a more com-
plete discussion of the computational protocol).
In light of these results, molecular simulations that combine
docking, classical molecular dynamics (MD) and replica-ex-
change molecular dynamics (REMD) were carried out to investi-
gate the potential mechanism of action responsible for Ab42
aggregation inhibition. In particular, our aim was to examine
the ability of the catechol-containing target compounds to in-
terfere with both early (oligomerization) and late (fibrillation)
stages of Ab42 aggregation.
Secondary-structure analysis for the first five T-replicas of
Ab42mon–8 f is reported in Figure 4. The results highlight the
large conformational flexibility of Ab42mon, which can adopt a
variety of conformations that mediate the interaction with
compound 8 f. In general, turn/coil are the most populated
states, followed by b-sheet and a-helix arrangements. A high
a-helix content, especially for residues 15–18 and 24–36 of
Ab42mon, is observed at the beginning of all simulated replicas,
although the a-helical content is lost during the first 50–
100 ns of REMD simulation. A transient a-helix to b-sheet con-
version of the central (18–24) and C-terminal residues is ob-
served for the first three replicas. Interestingly, a different pro-
file is observed for the fourth replica, in which a stable confor-
mer characterized by an a-helical motif for residues 14–24 and
b-sheet fold for residues at the N and C termini is found. This
conformation seems to be the most populated one, as noted
by the 2D root-mean-square (RMS) analysis (see Figure S2D in
the Supporting Information). Finally, a higher degree of confor-
mational flexibility is observed for the last replica, in which the
lack of well-defined secondary structures is generally observed.
Along the trajectories sampled in REMD simulations, com-
pound 8 f exhibits a tendency to interact with the middle and
C-terminal regions of the Ab42mon sequence (cf. Figure 3), thus
affecting its conformational assembly. Interestingly, our data
are in agreement with those observed by Zhang et al.,[22a] who
performed REMD simulations to study the conformational be-
havior of the Ab42–EGCG complex. The present results, howev-
er, emphasize the role of hydrophobic interactions formed be-
tween 8 f and the apolar residues of Ab42mon. Generally, the
main interactions are formed with residues 12–20 and 32–38
of Ab42mon (Ab42mon Ca–8 f distances <1.0 nm), although there
are differences between the different replicas (see Figure S3 in
the Supporting Information).
Formation of covalent adducts with Ab42 fibrils
To examine the ability of the target compounds to covalently
interfere with Ab42 aggregation, compounds 8a–f were
docked in their oxidized (o-quinone) form into the 10 confor-
mational states for the solid-state NMR spectroscopy model of
Ab42 (PDB ID: 5KK3)[29] by using Glide.[30] The three top-scoring
docking solutions for compound 8c were further investigated
by means of 100 ns MD simulations with Amber18.[31] The
choice of 8c was motivated by the presence of the nitro
group, which would help to stabilize the binding mode
through electrostatic interactions with the protonated amino
group of K16 residues along the binding groove. This would
enhance the residence time around the reactive site and facili-
tate the proper arrangement for covalent adduct formation, as
described for the aza-Michael addition observed for taxifolin.[32]
However, none of the three simulated poses for compound 8c
were able to maintain a proper orientation around the reactive
site delimited by K16 and D22 in the binding groove (cf. dis-
tances d1 and d2 in Figure S1 in the Supporting Information).
These results led us to exclude the possibility of a covalent
Ab42 inhibition mechanism for this class of compounds.
Interaction of the target compounds with Ab42 monomer
As an alternative mechanism, we explored the ability of com-
pound 8 f, one of the most potent Ab42 inhibitors found in
this study, on the early stage of Ab42 aggregation by means of
REMD simulations. Following previous studies,[22] the Ab42
monomer (Ab42mon), which crystallized in a nonpolar environ-
ment (PDB ID: 1IYT),[33] was selected to model the interaction
with compound 8 f. A total of 30 ms of MD trajectory was col-
Collectively, these data are in line with a putative Ab42 anti-
aggregation mechanism, in which the presence of compound
8 f redirects the conformational landscape of Ab42 oligomers
toward less structured/off-pathway oligomers. No evidence of
stable and well-formed b-sheet configuration emerged from
our simulations. In fact, the N terminus of Ab42mon is mainly
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Figure 4. Left: Secondary-structure analysis for the first five T-replicas of Ab42mon–8 f, according to the DSSP algorithm. Right: Representative geometries for
Ab42mon–8 f at 0, 250, and 500 ns of simulation. The N- and C-terminal edges of Ab42mon are reported as blue and red spheres, respectively.
unstructured, a transient a-helix propensity is often observed
for residues 15–24, and a partial b-sheet-turn-b-sheet propensi-
ty is observed for the central and C-terminal region. Com-
pound 8 f is suggested to have a significant impact on this as-
sembly because this compound would stably form contacts
with the C-terminal region of the Ab42mon, intercalating the b-
sheet-turn-b-sheet region.
ed the flavonol quercetin, which served as a positive control
and prevented cell death at 25 mm. Curcumin and 8g did not
show distinct neuroprotection. At only 10 mm, weak protection
with 21% cell survival was observed. These data show the im-
portance of the free catechol and are in good agreement with
results reported by Maher et al., who showed that chemical al-
ternation of the catechol structure of flavonoids of the plant
Eriodictyon californicum (also known as yerba santa) led to a
drastic reduction in activity in different phenotypic screening
assays, including the oxytosis assay.[15a]
Neurotoxicity and neuroprotection in HT22 cells
The effect of the target compounds on neuroprotection and
neurotoxicity was examined by using the sensitivity of the
murine hippocampal neuronal cell line HT22 to glutamate.
High extracellular concentrations of glutamate lead to oxida-
tive glutamate toxicity, so-called oxytosis, a form of pro-
grammed cell death.[34] The inhibition of the cystine/glutamate
antiporter causes glutathione (GSH) depletion, followed by ac-
cumulation of reactive oxygen species (ROS), calcium influx,
and finally cell death by oxidative stress.[35] Similar features are
observed in the brain during aging and are accelerated in
AD.[36] Compounds 8a–f showed very strong protection
against intracellular oxidative stress at concentrations between
2.5 and 7.5 mm (Figure 5). The target compounds even exceed-
DPPH radical scavenging assay
To evaluate (and exclude) unspecific protection against oxida-
tive stress by radical scavenging, the direct antioxidant capaci-
ty was tested in a cell-free system. The widely applied DPPH
radical scavenging assay uses the stable radical 2,2-diphenyl-1-
picrylhydrazyl, which is decolorized upon reduction.[37] The
known antioxidant ascorbic acid (vitamin C) served as a posi-
tive control with an IC50 value of 8.4 mm. The parent compound
curcumin had an IC50 value of 10.5 mm. The target compounds
were active over a similar range, from 5 to 10 mm (Table 2).
Compound 8g did not show any activity because there was
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Figure 5. Neuroprotection and neurotoxicity were determined in HT22 cells. 5 mm glutamate (red) induced cell death, 25 mm quercetin (yellow) served as a
positive control for cell survival: A) neurotoxicity of curcumin and 8a–c; B) neuroprotection of curcumin and 8a–c; C) neurotoxicity of 8d–g; D) neuroprotec-
tion of 8d–g. Data are presented as meansÆSEM of three independent experiments and results refer to untreated control cells (black). Statistical analysis
was performed by using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post-test by using GraphPad Prism 5, with refer-
ence to cells treated with 5 mm glutamate. Level of significance: ***p<0.001, **p<0.01, *p<0.05.
no functionality to react with the free DPPH radical. Com-
pound 8a shows strong cellular protection against intracellular
oxidative stress, but a weak radical scavenging activity com-
pared with the other target compounds. This suggests that the
neuroprotection in HT22 cells is based on a target-specific
mode of action. The pronounced differences in the activity of
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Table 2. Free radical scavenging capacity determined by the DPPH assay.
Compound
EC50 [mm]
SEM
ascorbic acid
curcumin
8a
8b
8c
8d
8e
8 f
8.4
10.5
9.1
7.7
5.4
5.6
9.6
5.4
0.5
0.2
0.3
0.4
0.5
0.1
0.4
0.1
8g
not active
curcumin in the oxytosis assay (10 mm, 21% cell survival) in
comparison with the EC50 value of 10.5 mm in the DPPH assay
strongly indicate a specific intracellular protective mechanism,
rather than an unspecific protection due to radical scavenging.
The lack of activity of dimethoxy compound 8g in the DPPH
radical scavenging assay shows the necessity of a catechol (or
monohydroxyl) unit for reaction with free radicals.
Figure 6. Effect of compounds 8c, 8 f, 8g, and curcumin on the production
of NO as an inflammation marker. BV-2 cells were treated with 50 ngmLÀ1
LPS alone or with the respective compound. NO was determined by the
Griess assay in the supernatant. Data are presented as meansÆSEM of three
independent experiments and results refer to LPS-treated cells. Statistical
analysis was performed by using one-way ANOVA followed by Dunnett’s
multiple comparison post-test by using GraphPad Prism 5. Level of signifi-
cance: ***p<0.001, **p<0.01, *p<0.05.
Conclusion
Anti-inflammatory effect on BV-2 cells
Apart from amyloid plaques, neuroinflammation represents a
key hallmark of AD.[38] Microglia cells act as a major immune
defense in the central nervous system.[39] Activation by, for ex-
ample, bacterial endotoxins to their proinflammatory pheno-
type results in the production of NO and several other inflam-
mation-promoting factors, such as cytokines, free radicals, and
excitatory neurotransmitters.[38] Although active microglia cells
are important for brain repair processes and response to
immune challenge, chronic activation, such as that in AD, leads
to neurodegeneration caused by inflammation and oxidative
stress.[39b,40] Therefore, inhibition of the proinflammatory micro-
glia state is important in the context of AD.[41]
In this study, a series of azobioisosteres of curcumin, suitably
modified to incorporate the pharmacophoric catechol moiety
of flavonoids, have been synthesized and their pharmacologi-
cal profile against amyloid aggregation, intracellular oxidative
stress, and neuroinflammation has been characterized. The
synthetic bioisosteric compounds have shown higher aggrega-
tion inhibition of Ab42 relative to the parent compound, cur-
cumin. This could be observed in a bacterial in vitro assay with
Th-S staining, as well as dye-independently in TEM experi-
ments. Additionally, the compounds showed strong activity in
AD-related cell assays. In particular, there was higher protec-
tion against glutamate-induced intracellular oxidative stress in
murine hippocampal HT22 cells than that of curcumin. More-
over, the compounds revealed pronounced anti-inflammatory
properties in microglial BV-2 cells. The observed effects
seemed to underlie a specific mechanism, as the activity of the
compounds in the DPPH radical scavenging assay did not
show substantial differences.
Mouse microglial BV-2 cells were used to evaluate a possible
anti-inflammatory effect. Cells were treated with bacterial lipo-
polysaccharide (LPS) to induce inflammation and the produc-
tion of NO was quantified in the Griess assay. All compounds
reduced the NO production dose, with the strongest anti-in-
flammatory effect at 10 mm (Figure 6). Similar to the results for
neuroprotectivity on HT22 cells, the target compounds exceed-
ed the activity of curcumin. Compound 8c was the most
active compound, with a decrease of inflammation down to
17% relative to the LPS control. The other compounds tested
reduced NO production in a similar manner to 31–42% (com-
pound 8 f is shown as a representative example; for more de-
tailed information, see the Supporting Information). It is not
surprising that compound 8 f was not the most active com-
pound, as in the oxytosis assay or Ab fibrilization inhibition,
since the respective modes of action may well differ from each
other. Nevertheless, protection of the catechol with methoxy
groups (compound 8g) led to a dramatic loss of activity, simi-
lar to in the other assays applied in this study. No effect on the
production of NO was observed at 2.5 and 5 mm. At 10 mm,
compound 8g reduced the amount of NO to 65% in compari-
son with the control.
Until now, drugs targeting Ab have failed in clinical trials.
The reasons for this failure include fluorescence interference
during the commonly used thioflavin T assay, poor reproduci-
bility of Ab experiments in general, and the overall complexity
of amyloid aggregation processes in AD with still unknown as-
pects of toxicity of amyloid species.[42] Therefore, the experi-
mental design of this work includes a dye-based readout for
Ab aggregation that applies to a highly replicable assay, and a
dye-independent setup, that is, electron microscopy. In addi-
tion to their antiaggregation properties, the compounds pre-
sented herein also act against oxidative stress and neuroin-
flammation.
It must be taken into account that azobenzenes are com-
monly suspected to cause long-term toxicity due to instability
towards bacterial azoreductases, which cleave the azobenzene
in anilines, but there are several examples of food colorants
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À2
2 kcalmolÀ1
Š
was also applied to the backbone atoms of the
and drugs, which support the safe use of molecules containing
azobenzene moieties.[43]
first and last Ab42 monomers to preserve the structural integrity of
the fibrils. These restraints were gradually eliminated during the
first 50 ns of the MD simulation. The SHAKE algorithm[51] was ap-
plied to constrain bonds involving hydrogen atoms. Periodic boun-
dary conditions were used during the MD simulations and a cutoff
of 10 Š for the nonbonded interactions and the particle mesh
Ewald (PME) method[52] was used for the treatment of electrostatic
interactions beyond the cutoff. Langevin dynamics with a collision
frequency of 1.0 psÀ1 were applied for temperature regulation
during heating. Finally, 100 ns of the MD production in the NVT en-
semble (300 K) were run by using the weak-coupling algorithm[53]
(with a time constant of 10.0 ps) for each of the three complexes.
The analysis was performed for the set of snapshots saved every
2 fs along the trajectories.
Finally, because azobenzene compounds can undergo cis–
trans photoisomerization upon irradiation with light of an ap-
propriate wavelength, this paves the way to the photoinduced
control of the antiaggregating activity of azobenzene bioisos-
teres. Currently, the use of the target compounds in a photo-
pharmacological approach is under investigation.
Experimental Section
Computational methods
Docking and MD simulations of complexes with Ab42 fibrils:
The solid-state NMR structure of the Ab42 fibril (PDB ID 5KK3),[29]
corresponding to a double S-shaped Ab42 fibrillar assembly, was
used to model the interaction of compounds 8a–f with Ab42 fi-
brils, following the same protocol as that adopted in previous
studies.[32] Briefly, 20 docking runs, one for each of the two mono-
mers of the 10 NMR models deposited for 5KK3, were performed
with Glide.[30] Protonation states for the protein were set at pH 7.4.
According to previous pKa studies,[32] one K16 located in the
middle of the fibril assembly was simulated in its neutral form. For
each of the six tested compounds, the oxidized o-quinone form
was generated and used during docking. A total of 2000 poses
were generated, and thus, analyzed to identify suitable candidates
for further MD studies.
Among the best-ranked poses obtained for compound 8c, three
docked arrangements were selected and investigated by means of
MD simulations. The choice of this compound as a suitable candi-
date for MD simulation was dictated by the presence of a nitro
group, which would be able to stabilize the protein–ligand com-
plex by interacting with K16 residues along the Ab fibrils. MD sim-
ulations were run with Amber18.[31] The Amber ff14SB-ILDN force
field[44] was used for the protein and modified parameters (see
above) from the general Amber force field (GAFF)[45] were used to
parameterize the ligand. Partial charges were derived at the B3LYP/
6-31G(d) level, after preliminary geometry optimization, by using
the restrained electrostatic potential[46] fitting procedure. Torsional
parameters for the C-C-N-N dihedral angle, which defined the con-
formation of the benzene ring relative to the diazo group, were re-
fined by using 4-({4-[(1E)-2-(3-hydroxyphenyl)diazen-1-yl]phenyl}-
methyl)cyclohexa-3,5-diene-1,2-dione (see Table S1 in the Support-
ing Information) as a reference model in quantum mechanical
(QM) calculations. To this end, the MM torsional potential energy
of C-C-N-N torsion were fitted to the QM-derived potential energy
profile obtained from a relaxed scan performed at the M062X/6-
31G(d) level in the gas phase with Gaussian 09.[47] The python
package pyevolve[48] was used to fit the two profiles (see the Sup-
porting Information)
REMD simulation of the interaction between 8 f and the Ab42
monomer: The solution NMR spectroscopy structure of the Ab42
peptide (PDB ID: 1IYT)[33] was used to model the interaction be-
tween Ab42 monomer and compound 8 f, which was simulated in
its reduced (catechol) form. The ligand parameters were adopted
from the GAFF force field, although both atomic charges (RESP
charges derived from B3LYP/6-31G(d) calculations) and torsional
(C-C-N-N) parameters were adjusted, and the ff14SB-ILDN force
field was used for the protein. Standard protonation states at
pH 7.4 were adopted for ionizable residues. The system was em-
bedded in a truncated octahedron box of TIP3P water molecules
and counterions (Na+, ClÀ) were added at a salt concentration of
0.15m. The final system (18,386 atoms) contained an Ab42 mono-
mer, one molecule of 8 f randomly placed around the monomer,
5891 water molecules, 20 Na+ ions, and 17 ClÀ ions.
REMD simulations were carried out on 60 T-replicas ranging from
315 to 430 K with Gromacs2018.[54] The systems were energy mini-
mized by applying 50000 steps of the steepest descent algorithm
followed by 5000 steps of conjugate gradient algorithm. 1 ns of
MD simulation in the NVT ensemble by using the velocity-rescaling
thermostat (0.1 ps time coupling constant)[55] was run to heat the
system to the final temperatures for production. Positional re-
straints with a force constant of 1000 kJmolÀ1 mnÀ2 were applied
to the Ab42 backbone atoms to avoid unnatural distortions during
heating. Finally, 5 ns of MD simulation in the NPT ensemble by
using the Parrinello–Rahman barostat[56] with a 0.5 ps time con-
stant for coupling were run to properly equilibrate density.
500 ns of REMD simulation in the NPT ensemble by using the Parri-
nello–Rahman barostat[56] under periodic boundary conditions
were run for each T-replica, leading to a total of 30 ms of sampled
MD trajectory. The LINCS method[57] was applied to constraint
bonds involving hydrogen atoms. A cutoff of 1.2 nm was used to
treat short-range nonbonded interactions, whereas the PME
method was applied to manage long-range electrostatic interac-
tions.[52] A time step of 2 fs was applied to collect trajectories
during the simulation. Exchanges between T-replicas were at-
tempted every 100 MD steps, leading to an acceptance ratio of
about 45%.
The three 8c–Ab42 fibrils were solvated with TIP3P[49] water mole-
cules by using a truncated octahedron box with a layer of 20 Š
and neutralized by adding Na+ ions.[50] The systems were energy
minimized in a three-stage protocol, which involved hydrogen
atoms, then water molecules, and finally the whole system, with a
maximum number of 20000 minimization cycles for the last stage.
Then, the systems were gradually heated from 0 to 300 K in six
steps; the first was performed at a constant volume and the rest at
constant pressure. To avoid artifactual alterations in the ligand
pose due to thermal equilibraÀti2on, harmonic restraints with
Demuxed trajectories for the first five replicas were considered for
final analysis. Here, time-dependent evolution of the secondary
structure of Ab42 was calculated by using the DSSP algorithm.[58]
Finally, mdmap and rms commands implemented in Gromacs2018
were applied to generate the contact map and 2D-RMS plots, re-
spectively.
a force constant of 10 kcalmolÀ1
Š
were applied during equilibra-
tion to selected ligand–protein contacts. A Cartesian restraint of
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123.4 (+, 2”Ph-C), 116.6 (+, Ph-C), 115.2 ppm (+, Ph-C); ESI-MS:
m/z: 259.90 [M+H]+.
General
All reagents were used without further purification and bought
from common commercial suppliers. TLC was performed on silica
gel 60 (alumina foils with fluorescent indicator 254 nm). UV light
(254 and 366 nm) was used for detection. For column chromatog-
raphy, silica gel 60 (particle size 0.040–0.063 mm) was used. NMR
spectra were recorded with a Bruker AV-400 spectrometer (Bruker,
Karlsruhe, Germany) in CDCl3 or [D6]DMSO, and chemical shifts are
expressed in ppm relative to CDCl3 (d=7.26 ppm for 1H and d=
77.16 ppm for 13C) or [D6]DMSO (d=2.50 ppm for 1H and d=
39.52 ppm for 13C). Spectral data reported refer to the thermody-
namically more stable trans isomer. The purity of the synthesized
products was determined by means of HPLC (Shimadzu Products),
containing a DGU-20A3R degassing unit, a LC20AB liquid chroma-
tograph, and an SPD-20A UV/Vis detector. UV detection was mea-
sured at 254 nm. Mass spectra were obtained by using a LCMS
2020 instrument (Shimadzu Products). As a stationary phase, a Syn-
ergi 4U fusion-RP (150 mm”4.6 mm) column was used and, as a
mobile phase, a gradient of methanol/water with 0.1% formic acid
was used . Parameters: A=water, B=methanol, V(B)/[V(A)+V(B)]=
from 5 to 90% over 10 min, V(B)/[V(A)+V(B)]=90% for 5 min,
V(B)/[V(A)+V(B)]=from 90 to 5% over 3 min. The method was per-
formed at a flow rate of 1.0 mLminÀ1. Compounds were only used
for biological evaluation if the purity was ꢀ95%. Melting points
were determined by using an OptiMelt automated melting point
system (Scientific Instruments GmbH, Gilching, Germany).
4-(4-Aminobenzyl)benzene-1,2-diol (5a)
Hydrogenation of 4 (200 mg, 0.772 mmol) was performed at room
temperature for 16 h under a hydrogen atmosphere (10 bar) in
methanol (10 mL) by using 20 wt% Pd/C. The reaction mixture was
filtered through Celite and the solvent was removed under re-
duced pressure. The crude product was purified by column chro-
matography on silica gel by using a mixture of dichloromethane/
methanol/triethylamine (40:1:0.1) as the eluent. The product was
1
obtained as a light brown solid (76 mg, 45%). M.p. 2058C; H NMR
(400 MHz, [D6]DMSO): d=8.67 (s, 1H; OH), 8.55 (s, 1H; OH), 6.81 (d,
3J=8.02 Hz, 2H; Ph), 6.60 (d, 3J=7.9 Hz, 1H; Ph), 6.51 (d, 4J=
2.1 Hz, 1H), 6.47 (d, 3J=8.3 Hz, 2H; Ph), 6.41 (dd, 3J=8.02, 4J=
2.10 Hz, 1H; Ph), 4.82 (s, 2H; NH2), 3.55 ppm (s, 2H; CH2); 13C NMR
(100 MHz, [D6]DMSO) d=146.4 (Cq, Ph-C), 144.9 (Cq, Ph-C), 143.1
(Cq, Ph-C), 133.2 (Cq, Ph-C), 129.0 (+, 2”Ph-C), 128.9 (Cq, Ph-C),
119.1 (+, Ph-C), 115.9 (+, Ph-C), 115.3 (+, Ph-C), 113.9 (+, 2”Ph-
C), 39.8 ppm (À, CH2); ESI-MS: m/z: 216.00 [M+H]+.
4-(3,4-Dimethoxybenzyl)aniline (5b)
Hydrogenation of 3 (400 mg, 1.57 mmol) was performed at room
temperature for 16 h under a hydrogen atmosphere (10 bar) in
methanol (30 mL) by using 20 wt% Pd/C. The reaction mixture was
filtered through Celite and the solvent was removed under re-
duced pressure. The crude product was purified by column chro-
matography on silica gel by using a mixture of ethyl acetate/cyclo-
hexane (1:1) as the eluent. The product was obtained as a colorless
(3,4-Dimethoxyphenyl)(4-nitrophenyl)methanone (3)
4-Nitrobenzoyl chloride (500 mg, 2.69 mmol) was added to a sus-
pension of FeCl3 (436 mg, 2.69 mmol) in veratrole (3 mL) and the
reaction mixture was heated to 608C for 16 h. Water and methanol
were added until the precipitant was dissolved, and the suspen-
sion was extracted with dichloromethane. The combined organic
layers were washed with water and dried over Na2SO4. The solvent
was removed under reduced pressure and the crude product was
filtered through silica gel by using dichloromethane as the eluent.
The solvent was removed under reduced pressure and the residue
was kept in the fridge to crystallize the product. The precipitant
was washed with ethanol and the product was obtained as a
1
3
oil (225 mg, 56%). H NMR (400 MHz, CDCl3): d=7.12 (d, J=8.46,
4
3
4
2H), 6.93 (d, J=1.9 Hz, 1H), 6.86 (dd, J=8.2, J=2.0 Hz, 1H), 6.80
3
3
(d, J=8.2 Hz, 1H), 6.62 (d, J=8.46, 2H), 3.86 (s, 3H; -OCH3), 3.82
(s, 3H, -OCH3), 3.49–2.92 ppm (s, 2H); 13C NMR (100 MHz, CDCl3):
d=148.9 (Cq, Ph-C), 148.2 (Cq, Ph-C), 145.8 (Cq, Ph-C), 137.07 (Cq,
Ph-C), 134.32 (Cq, Ph-C), 127.8 (+, 2”Ph-C), 118.7 (+, Ph-C), 115.1
(+, 2”Ph-C), 110.9 (+, Ph-C), 109.7 (+, Ph-C), 75.6 (À, CH2), 55.9
(+, -OCH3), 55.8 ppm (+, -OCH3); ESI-MS: m/z: 242.95 [M+H]+.
1
yellow solid (316 mg, 41%). M.p. 1488C; H NMR (400 MHz, CDCl3):
3
3
d=8.32 (d, J=8.7 Hz, 2H; Ph), 7.88 (d, J=8.7 Hz, 2H; Ph), 7.50 (d,
4J=2.0 Hz, 1H; Ph), 7.30 (dd, 3J=8.4 Hz, 4J=2.0 Hz, 1H), 6.90 (d,
3J=8.4 Hz, 1H; Ph), 3.97 (s, 3H; OCH3), 3.95 ppm (s, 3H; OCH3);
13C NMR (100 MHz, CDCl3): d=193.6 (Cq), 154.0 (+, Ph-C), 149.6 (+,
Ph-C), 149.6 (+, Ph-C), 143.9 (+, Ph-C), 130.4 (+, 2”Ph-C), 129.1
(+, Ph-C), 125.9 (+, Ph-C), 123.5 (+, 2”Ph-C), 111.8 (+, Ph-C),
110.0 (+, Ph-C), 56.3 (+, -OCH3), 56.2 ppm (+, -OCH3); ESI-MS: m/z:
288.28 [M+H]+.
(E)-4-[4-(Phenyldiazenyl)benzyl]benzene-1,2-diol (8a)
Compound 5a (48 mg, 0.22 mmol) and nitrosobenzene (28.7 mg,
0.27 mmol) were stirred in acetic acid (3 mL) at room temperature
for 16 h. Ethyl acetate (50 mL) was added, and the organic layer
was washed with a 1m aqueous solution of NaOH (50 mL) and
water (50 mL). The solvent was removed under reduced pressure
and the crude product was purified by column chromatography
on silica gel by using a mixture of ethyl acetate and cyclohexane
(1:2!1:1!2:1) as the eluent. The product was obtained as an
(3,4-Dihydroxyphenyl)(4-nitrophenyl)methanone (4)
1
orange solid (17 mg, 25%). M.p. 1218C; H NMR (400 MHz, CDCl3):
Compound 3 (1.00 g, 3.48 mmol) was suspended in acetic acid
(10 mL), 48% HBr (20.0 mL) was added, and the reaction mixture
was heated to reflux for 3.5 h. After cooling, the precipitant was fil-
tered, washed with water, and dried under vacuum. The product
was obtained as a yellowish green solid (733 mg, 85%). M.p.
d=7.92–7.87 (m, 2H; Ph), 7.86–7.80 (m, 2H; Ph), 7.55–7.43 (m, 3H;
Ph), 7.34–7.28 (m, 2H; Ph), 6.80 (d, 3J=8.00 Hz, 1H; Ph), 6.70 (d,
3
4
4J=2.1 Hz, 1H; Ph), 6.65 (dd, J=8.1, J=2.1 Hz, 1H), 5.20 (s, 2H;
OH), 3.93 ppm (s, 2H; CH2); 13C NMR (100 MHz, CDCl3): d=152.8
(Cq, Ph-C), 151.2 (Cq, Ph-C), 145.0 (Cq, Ph-C), 144.0 (Cq, Ph-C), 142.4
(Cq, Ph-C), 140.7 (Cq, Ph-C), 133.3 (+, Ph-C), 130.9 (+, Ph-C), 129.6
(+, Ph-C), 129.1 (+, Ph-C), 128.7 (+, Ph-C), 127.8 (+, Ph-C), 127.1
(+, Ph-C), 123.1 (+, Ph-C), 122.8 (+, Ph-C), 121.2 (+, Ph-C), 116.1
(+, Ph-C), 115.5 (+, Ph-C), 41.23 ppm (À, CH2); ESI-MS: m/z: 304.95
[M+H]+; HPLC purity: 98% (retention time: cis=9.18 min, trans=
10.47 min).
1
1658C; H NMR (400 MHz, [D6]DMSO): d=10.14 (s, 1H; OH), 9.56 (s,
3
3
1H; OH), 8.34 (d, J=8.7 Hz, 2H), 7.86 (d, J=8.8 Hz, 2H; Ph), 7.26
(d, 4J=2.2 Hz, 1H; Ph), 7.11 (dd, 3J=8.3, 4J=2.1 Hz, 1H; Ph),
6.87 ppm (d, 3J=8.3 Hz, 1H; Ph); 13C NMR (100 MHz, [D6]DMSO):
192.9 (Cq), 151.5 (Cq, Ph-C), 148.8 (Cq, Ph-C), 145.5 (Cq, Ph-C), 144.1
(Cq, Ph-C), 130.0 (+, 2”Ph-C), 127.3 (Cq, Ph-C), 124.0 (+, Ph-C),
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General procedure for the partial oxidation of aromatic
amines
(E)-4-{4-[(3-Chlorophenyl)diazenyl]benzyl}benzene-1,2-diol
(8d)
Amine 6b–f (1 equiv) was dissolved in dichloromethane (0.3m)
and an aqueous solution of oxone (1 equiv, 0.17m; commercially
available mixture of 2KHSO5·KHSO4·K2SO4) was added. The mixture
was stirred for 3.5 h at room temperature. The aqueous layer was
extracted with dichloromethane (50 mL); the combined organic
layers were washed with a 5% solution of HCl (50 mL), water
(25 mL), and brine (25 mL); and dried over Na2SO4. The solvent was
removed under reduced pressure and the crude product was di-
rectly used for the next reaction.
The crude product was purified by column chromatography on
silica gel by using a mixture of ethyl acetate and cyclohexane (1:3)
as the eluent. The product was obtained as an orange powder
(150 mg, 64%). M.p. 1498C; 1H NMR (400 MHz, CDCl3): d=7.90–
3
3
7.79 (m, 4H; Ph), 7.44 (d, J=6.9 Hz, 2H; Ph), 7.32 (d, J=8.1 Hz,
3
4
2H; Ph), 6.81 (d, J=8.0 Hz, 1H; Ph), 6.71 (d, J=2.0 Hz, 1H), 6.67
3
4
(dd, J=8.1, J=2.1 Hz, 1H; Ph), 5.07 (s, 1H; OH), 4.99 (s, 1H; OH),
3.95 ppm (s, 2H; CH2); 13C NMR (100 MHz, CDCl3): 148.8 (Cq, Ph-C),
145.2 (Cq, Ph-C), 141.8 (Cq, Ph-C), 130.2 (+, Ph-C), 129.7 (+, 2”Ph-
C), 123.3 (+, Ph-C), 121.6 (+, 2”Ph-C), 117.1 (+, Ph-C), 115.6,
41.2 ppm (À, CH2); ESI-MS: m/z: 335.05 [M+H]+; HPLC purity: 97%
(retention time: cis=9.65 min, trans=10.95 min).
General procedure for the Mills reaction
The crude product of partial oxidation and 5a were dissolved in
acetic acid and stirred at room temperature for 16 h. After that
time, the solvent was removed under reduced pressure; ethyl ace-
tate was added; and the organic layer was washed with a saturat-
ed aqueous solution of NaHCO3, water, and brine. The solvent was
removed under reduced pressure and the crude product was puri-
fied by column chromatography on silica gel by using a mixture of
ethyl acetate and cyclohexane or cyclohexane and dichlorometh-
ane as the eluent.
(E)-4-{4-[(3-Methoxyphenyl)diazenyl]benzyl}benzene-1,2-diol
(8e)
The product was purified by column chromatography on silica gel
by using a mixture of ethyl acetate and cyclohexane (1:4) as the
eluent. The product was obtained as an orange solid (17 mg, 25%).
1
3
M.p. 1228C; H NMR (400 MHz, CDCl3): d=7.84 (d, J=8.4 Hz, 2H;
3
Ph), 7.47–7.39 (m, 3H; Ph), 7.31 (d, J=7.95 Hz, 2H; Ph), 7.05–7.01
(m, 1H; Ph), 6.80 (d, J=8.1, 1H; Ph), 6.71 (d, J=2.1 Hz, 1H; Ph),
3
4
3
4
6.66 (dd, J=8.10 Hz, J=2.0 Hz, 1H), 5.38 (s, 2H; OH), 3.94 (s, 2H;
CH2), 3.90 (s, 3H; OCH3); 13C NMR (100 MHz, CDCl3): 159.7 (Cq, Ph-C),
154.2 (Cq, Ph-C), 151.01 (Cq, Ph-C), 144.89 (Cq, Ph-C), 143.55 (Cq, Ph-
C), 142.1 (Cq, Ph-C), 133.6 (Cq, Ph-C), 129.9 (+, Ph-C), 129.7 (+, 2”
Ph-C), 123.2 (+, 2”Ph-C), 121.6 (+, Ph-C), 117.7 (+, Ph-C), 117.1
(+, Ph-C), 116.2 (+, Ph-C), 115.6 (+, Ph-C), 105.8 (+, Ph-C), 55.6 (À,
OCH3), 41.2 ppm (À, CH2); ESI-MS: m/z: 335.00 [M+H]+; HPLC
purity: 96% (retention time: cis=9.31 min, trans=10.57 min).
(E)-4-{4-[(3-Ethoxyphenyl)diazenyl]benzyl}benzene-1,2-diol
(8b)
The product was purified by column chromatography on silica gel
by using a mixture of ethyl acetate and cyclohexane (1:4) as the
eluent. The product was obtained as an orange solid (24 mg, 21%).
1
3
M.p. 1268C; H NMR (400 MHz, CDCl3): d=7.83 (d, J=8.4 Hz, 2H;
3
3
Ph), 7.51 (d, J=7.8, 1H; Ph), 7.42 (d, J=7.95 Hz, 2H; Ph), 7.30 (d,
3J=8.4 Hz, 2H; Ph), 7.02 (ddd, J=8.2, J=2.6, J=1.0 Hz, 1H; Ph),
3
4
4
3
4
6.80 (d, J=8.1 Hz, 1H; Ph), 6.70 (d, J=2.0 Hz, 1H; Ph), 6.66 (dd,
3J=8.0, 4J=2.1 Hz, 1H; Ph), 5.15 (s, 2H; OH), 4.13 (q, 3J=7.0 Hz,
2H; OCH2CH3), 3.94 (s, 2H; CH2), 1.45 ppm (t, 3J=7.0 Hz, 3H;
OCH2CH3); 13C NMR (100 MHz, CDCl3): 159.8 (Cq, Ph-C), 154.0 (Cq,
Ph-C), 151.2 (Cq, Ph-C), 144.8 (Cq, Ph-C), 143.7 (Cq, Ph-C), 142.1 (Cq,
Ph-C), 133.7 (Cq, Ph-C), 129.8 (+, Ph-C), 129.7 (+, 2”Ph-C), 123.1
(+, 2”Ph-C), 121.5 (+, Ph-C), 118.2 (+, Ph-C), 117.0 (+, Ph-C),
116.2 (+, Ph-C), 115.5 (+, Ph-C), 106.5 (+, Ph-C), 63.8 (À,
OCH2CH3), 41.2 (À, CH2), 14.9 (+, OCH2CH3); ESI-MS: m/z: 349.00
[M+H]+; HPLC purity: 96% (retention time: cis=9.63 min, trans=
10.83 min).
(E)-4-{4-[(3-Isopropoxyphenyl)diazenyl]benzyl}benzene-1,2-
diol (8 f)
The product was purified by column chromatography on silica gel
by using a mixture of ethyl acetate and cyclohexane (1:4) as the
eluent. The product was obtained as an orange solid (37 mg, 16%).
1
3
M.p. 1278C; H NMR (400 MHz, CDCl3): d=7.82 (d, J=8.1 Hz, 2H;
3
3
Ph), 7.49 (d, J=7.8, 1H; Ph), 7.44–7.36 (m, 2H; Ph), 7.30 (d, J=
8.1 Hz, 2H), 7.00 (m, 1H; Ph), 6.80 (d, J=8.0 Hz, 1H; Ph), 6.69 (d,
4J=1.9 Hz, 1H; Ph), 6.65 (dd, J=8.1, J=2.0 Hz, 1H; Ph), 5.23 (s,
3
4
3
2H; OH), 4.66 (p, J=6.1 Hz, 1H; OiPr-H), 3.93 (s, 2H; CH2), 1.39 (s,
3H; OiPr-CH3), 1.37 ppm (s, 3H; OiPr-CH3); 13C NMR (100 MHz,
CDCl3): 158.7 (Cq, Ph-C), 154.1 (Cq, Ph-C), 151.2 (Cq, Ph-C), 144.8 (Cq,
Ph-C), 143.7 (Cq, Ph-C), 142.1 (Cq, Ph-C), 133.7 (Cq, Ph-C), 129.9 (+,
Ph-C), 129.6 (+, 2”Ph-C), 123.1 (+, 2”Ph-C), 121.6 (+, Ph-C), 119.3
(+, Ph-C), 116.6 (+, Ph-C), 115.6 (+, Ph-C), 108.2 (+, Ph-C), 70.3 (+,
OiPr-CH), 41.2 (À, CH2), 22.1 (+, 2”OiPr-CH3); ESI-MS: m/z: 363.15
[M+H]+; HPLC purity: 96% (retention time: cis=9.74 min, trans=
10.84 min).
(E)-4-{4-[(3-Nitrophenyl)diazenyl]benzyl}benzene-1,2-diol
(8c)
The product was purified by column chromatography on silica gel
by using a mixture of ethyl acetate and cyclohexane (1:4!1:3) as
the eluent. The product was obtained as an orange solid (59 mg,
1
4
52%). M.p. 1258C; H NMR (400 MHz, CDCl3): d=8.72 (t, J=2.1 Hz,
1H; Ph), 8.31 (ddd, 3J=8.2, 4J=2.3, 4J=1.0 Hz, 1H; Ph), 8.23 (dt,
3J=7.9, J=1.0 Hz, 1H; Ph), 7.89 (d, J=8.32, 2H; Ph), 7.69 (t, J=
8.0 Hz, 1H; Ph), 7.35 (d, J=8.1 Hz, 2H; Ph), 6.81 (d, J=8.0 Hz, 1H;
4
3
3
(E)-1-[4-(3,4-Dimethoxybenzyl)phenyl]-2-phenyldiazene (8g)
3
3
Ph), 6.72 (3d, J=2.0 Hz, 1H; Ph), 6.67 (dd, J=8.1, J=2.0 Hz, 1H),
5.22 (s, 2H; OH), 3.96 ppm (s, 2H; CH2); 13C NMR (100 MHz, CDCl3):
d=153.2 (Cq, Ph-C), 150.8 (Cq, Ph-C), 146.2 (Cq, Ph-C), 143.8 (Cq, Ph-
C), 142.1 (Cq, Ph-C), 133.5 (Cq, Ph-C), 130.0 (+, Ph-C), 129.8 (+, 2”
Ph-C), 129.3 (+, Ph-C), 124.8 (+, Ph-C), 123.6 (+, 2”Ph-C), 121.6
(+, Ph-C), 117.1 (+, Ph-C), 116.2 (+, Ph-C), 115.6 (+, Ph-C),
41.3 ppm (À, CH2); ESI-MS: m/z: 350.10 [M+H]+; HPLC purity: 97%
(retention time: cis=11.60 min, trans=13.52 min).
The product was purified by column chromatography on silica gel
by using a mixture of ethyl acetate and cyclohexane (1:2!1:1!
2:1) as the eluent. The product was obtained as an orange oil
3
4
1
(17 mg, 25%). H NMR (400 MHz, CDCl3): d=7.93–7.88 (m, 4H; Ph),
7.60–7.43 (m, 5H; Ph), 6.95–6.81 (m, 3H; Ph), 3.88–3.83, 3.87 (m,
2H; CH2), (s, 3H; -OMe), 3.85 ppm (s, 3H; -OMe); 13C NMR
(100 MHz, CDCl3): 152.8 (Cq, Ph-C), 152.2 (Cq, Ph-C), 149.2 (Cq, Ph-C),
149.1 (Cq, Ph-C), 143.2 (Cq, Ph-C), 132.4 (Cq, Ph-C), 131.2 (+, Ph-C),
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129.2 (+, 2”Ph-C), 127.6 (+, 2”Ph-C), 123.1 (+, 2”Ph-C), 123.01
(+, 2”Ph-C), 120.2 (+, Ph-C), 111.1 (+, Ph-C), 110.8 (+, Ph-C), 76.5
(À, CH2), 56.0 (+, OMe); ESI-MS: m/z: 331.95 [M+H]+; HPLC purity:
97% (retention time: cis=7.66 min, trans=11.28 min).
378C and 250 rpm. Once the cell density reached OD600=0.6, an
aliquot of culture (980 mL) was transferred into 1.5 mL Eppendorf
tubes with solutions (10 mL) of each compound to be tested in
DMSO and arabinose (10 mL, 25%). The final concentration of com-
pound was fixed at 10 mm. The samples were grown overnight at
378C and 1400 rpm by using a Thermomixer (Eppendorf, Hamburg,
Germany). As a negative control (maximal presence of tau), the
same amount of DMSO without drug was added to the sample. In
parallel, noninduced samples (in the absence of arabinose) were
also prepared and used as positive controls (absence of tau). In ad-
dition, these samples were used to assess the potential intrinsic
toxicity of the compounds and to confirm the correct bacterial
growth.
Ab42 aggregation inhibition— TEM analysis
The procedure was adapted from a method reported by Murakami
et al.[59] Ab42, purchased from Amatek, was dissolved in a 0.1% so-
lution of NH4OH at a concentration of 1 mgmLÀ1. Each compound
was dissolved in ethanol at a concentration of 1 mgmLÀ1. Samples
were diluted to a final concentration of 100 mm Ab42, and the de-
sired concentration of compound (10 mm/50 mm) in PBS (50 mm
sodium phosphate and 100 mm NaCl, pH 7.4). After 24 h incuba-
tion at 378C, solutions were applied on 200 mesh, fixed with
1.25% glutaraldehyde solution, and negatively stained with 1%
uranyl acetate. The aggregates were observed with a JEOL JEM-
2100 transmission electron microscope. Images on the transmis-
sion electron microscope were acquired with a TemCamF416
camera (Tietz Video and Imaging Processing Systems, Gauting, Ger-
many).
Th-S steady-state fluorescence: Th-S (T1892) and other chemical
reagents were purchased from Sigma (St. Louis, MO). Th-S stock so-
lution (2500 mm) was prepared in double-distilled water purified
through a Milli-Q system (Millipore, USA). Th-S fluorescence and
absorbance were tracked by using a DTX 800 plate reader multi-
mode detector equipped with multimode analysis software (Beck-
man-Coulter, USA). Filters of 430/35 and 485/20 nm were used for
the excitation and emission wavelengths, respectively. 535/25 nm
filters were also used for absorbance determination. To normalize
the Th-S fluorescence as a function of the bacterial concentration,
OD600 was obtained by using a Shimadzu UV-2401 PC UV/Vis
spectrophotometer (Shimadzu, Japan). Notably, fluorescence nor-
malization was carried out by considering the Th-S fluorescence of
the bacterial cells expressing the peptide or protein in the absence
of drug as 100% and the Th-S fluorescence of the bacterial cells
nonexpressing the peptide or protein as 0%.
Inhibition assay in E. coli cells overexpressing Ab42 and tau
Cloning and overexpression of Ab42 peptide: E. coli competent
cells BL21 (DE3) were transformed with the pET28a vector (Nova-
gen, Inc., Madison, WI, USA) carrying the DNA sequence of Ab42.
Because of the addition of the initiation codon ATG in front of
both genes, the overexpressed peptide contained an additional
methionine residue at its N terminus. For overnight culture prepa-
ration, M9 minimal medium (10 mL) containing 50 mgmLÀ1 kana-
mycin was inoculated with a colony of BL21 (DE3) bearing the
plasmid to be expressed at 378C. For expression of the Ab42 pep-
tide, the required volume of overnight culture to obtain 1:500 dilu-
tion was added to fresh M9 minimal medium containing
50 mgmLÀ1 kanamycin and 250 mm Th-S. The bacterial culture was
grown at 378C and 250 rpm. Once the cell density reached
OD600=0.6, an aliquot of culture (980 mL) was transferred into
1.5 mL Eppendorf tubes with solutions (10 mL) of each compound
to be tested in DMSO and isopropyl 1-thio-b-d-galactopyranoside
(IPTG; 10 mL) at 100 mm. The final concentration of drug was fixed
at 10 mm. The samples were grown overnight at 378C and
1400 rpm by using a Thermomixer (Eppendorf, Hamburg, Germa-
ny). As a negative control (maximal amyloid presence), the same
amount of DMSO without drug was added to the sample. In paral-
lel, noninduced samples (in the absence of IPTG) were also pre-
pared and used as positive controls (nonamyloid presence). In ad-
dition, these samples were used to assess the potential intrinsic
toxicity of the compounds and to confirm the correct bacterial
growth.
Cell culture general procedures
HT22 cells were grown in Dulbecco’s modified Eagle medium
(DMEM; Sigma Aldrich, Munich Germany) supplemented with 10%
(v/v) fetal calf serum (FCS) and 1% (v/v) penicillin–streptomycin.
BV-2 cells were grown in low-glucose DMEM (Invitrogen, Carlsbad,
CA, USA) supplemented with 10% FCS and 1% (v/v) penicillin–
streptomycin. Cells were subcultured every 2 days and incubated
at 378C with 5% CO2 in a humidified incubator. Compounds were
dissolved in DMSO (Sigma Aldrich, Munich, Germany) as stock solu-
tions and diluted further into culture medium. To determine cell vi-
ability, a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide (MTT; Sigma Aldrich, Munich, Germany) assay was
used. MTT solution (4 mgmLÀ1 in PBS) was diluted 1:10 with
medium and added to the wells after removal of the old medium.
Cells were incubated for 3 h and then lysis buffer (10% sodium do-
decyl sulfide) was applied. The next day, the absorbance at 560 nm
was determined with a multiwell plate photometer (Tecan, Spectra-
Max 250).
Cloning and overexpression of tau protein: E. coli BL21 (DE3)
competent cells were transformed with pTARA containing the RNA
polymerase gen of T7 phage (T7RP) under the control of the pro-
moter PBAD. E. coli BL21 (DE3) with pTARA competent cells were
transformed with pRKT42 vector encoding four repeats of tau pro-
tein in two inserts. For overnight culture preparation, M9 medium
(10 mL) containing 0.5% glucose, 50 mgmLÀ1 ampicillin, and
12.5 mgmLÀ1 chloramphenicol were inoculated with a colony of
BL21 (DE3) bearing the plasmids to be expressed at 378C. For the
expression of tau protein, the required volume of overnight culture
to obtain 1:500 dilution was added to fresh M9 minimal medium
containing 0.5% glucose, 50 mgmLÀ1 ampicillin, 12.5 mgmLÀ1 chlor-
amphenicol, and 250 mm Th-S. The bacterial culture was grown at
Neurotoxicity and neuroprotection (oxytosis)
For the toxicity and oxytosis assay, 5”103 cells per well were
seeded into sterile 96-well plates and incubated overnight. For the
neurotoxicity assay, medium was removed and 1, 5, 10, 25, or
50 mm of the compound diluted with medium from a 0.1m stock
solution was added to the wells. DMSO (0.05%) in DMEM served as
a control. Cells were incubated for 24 h if neurotoxicity was deter-
mined by using a colorimetric MTT assay.
For the oxytosis assay, 5 mm glutamate (monosodium l-glutamate,
Sigma Aldrich, Munich, Germany) together with 1, 2.5, 5, 7.5, or
10 mm of the respective compounds were added to the cells and
incubated for 24 h. As a positive control, a mixture of 25 mm quer-
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Chemistry—A European Journal
cetin (Sigma Aldrich, Munich, Germany) and 5 mm glutamate was
used. After 24 h incubation, cell viability was determined by using
a colorimetric MTT assay, as described above. Results are presented
as percentage of untreated control cells. Data are expressed as
meansÆSEM of three independent experiments. Analysis was ac-
complished by using GraphPad Prism 5 software by applying one-
way ANOVA followed by Dunnett’s multiple comparison post-test.
Levels of significance: *p<0.05; **p<0.01; ***p<0.001.
Laboratory, The Salk Institute for Biological Studies, La Jolla,
USA) for providing HT22 and BV-2 cells. We thank Claudia
Gehrig and Daniela Bunsen (Biocenter/Theodor-Boveri-Insti-
tute) for technical assistance. Open access funding enabled
and organized by Projekt DEAL.
Conflict of interest
DPPH radical scavenging assay
The authors declare no conflict of interest.
Stock solutions of standard antioxidant, ascorbic acid, and com-
pounds were prepared in DMSO (3 mm). DPPH solution was freshly
prepared in methanol daily and stored in the dark. A dilution row
of compound in methanol ranging over nine dilutions (1–500 mm)
was prepared in a 96-well plate by using a multichannel pipette.
The blank was measured at 517 nm. To 100 mL compound dilution,
a solution of DPPH (33.3 mL, 200 mm) in ethanol was added by
using a multichannel pipette. The 96-well plate was incubated at
room temperature in the dark for 30 min. After incubation, the ab-
sorbance was measured at 517 nm. Methanol (100 mL) and DPPH
(33.3 mL, 200 mm) served as the negative control. The percentage
of DPPH radical scavenging activity (SCV) was calculated by using
Equation ():
Keywords: amyloid beta · bioisosterism · natural products ·
neuroprotectivity · replica-exchange molecular dynamics
[1] E. Babusikova, A. Evinova, J. Jurecekova, M. Jesenak, D. Dobrota in Ad-
vanced Understanding of Neurodegenerative Diseases, IntechOpen, 2011.
[2] a) G. G. Glenner, C. W. Wong, Biochem. Biophys. Res. Commun. 1984, 120,
885–890; b) C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L.
McDonald, K. Beyreuther, Proc. Natl. Acad. Sci. USA 1985, 82, 4245–
4249.
[3] C. Haass, D. J. Selkoe, Nat. Rev. Mol. Cell Biol. 2007, 8, 101–112.
[4] L. M. Sayre, G. Perry, M. A. Smith, Chem. Res. Toxicol. 2008, 21, 172–188.
[5] M. Prior, C. Chiruta, A. Currais, J. Goldberg, J. Ramsey, R. Dargusch, P. A.
Maher, D. Schubert, ACS Chem. Neurosci. 2014, 5, 503–513.
[6] a) S. Giorgetti, C. Greco, P. Tortora, F. A. Aprile, Int. J. Mol. Sci. 2018, 19,
2677; b) F. Panza, M. Lozupone, G. Logroscino, B. P. Imbimbo, Nat. Rev.
Neurosci. 2019, 15, 73–88.
Aneg:control À Ablank1 À Asampe1 À Ablank2
% SCV
 100
1
Aneg:control À Ablank1
[7] a) L. Fang, S. Gou, X. Liu, F. Cao, L. Cheng, Bioorg. Med. Chem. Lett.
2014, 24, 40–43; b) M. Venigalla, S. Sonego, E. Gyengesi, M. J. Sharman,
G. Münch, Neurochem. Int. 2016, 95, 63–74; c) A. Espargaró, T. Ginex,
M. d. M. Vadell, M. A. Busquets, J. Estelrich, D. MuÇoz-Torrero, F. J.
Luque, R. Sabate, J. Nat. Prod. 2017, 80, 278–289.
Concentration-dependent SCV curves were calculated by using a
nonlinear fit and EC50 values were then determined graphically by
using GraphPad Prism 5 software.
[8] A. A. Reinke, J. E. Gestwicki, Chem. Biol. Drug Des. 2007, 70, 206–215.
[9] K. M. Nelson, J. L. Dahlin, J. Bisson, J. Graham, G. F. Pauli, M. A. Walters,
J. Med. Chem. 2017, 60, 1620–1637.
Microglial activity
For the anti-neuroinflammation assay, 1”106 cells per well were
seeded in a sterile six-well plate. After overnight incubation, the
medium was exchanged for fresh medium. The cells were pretreat-
ed with the respective compounds at the indicated concentrations
for 30 min after 50 ngmLÀ1 bacterial LPS was added. After 24 h in-
cubation, the medium was collected, spun briefly to remove float-
ing cells, and the supernatant (100 mL) was assayed for nitrite by
using the Griess Reagent (100 mL) in a 96-well plate. After incuba-
tion for 10 min at room temperature, the absorbance at 550 nm
was read on a microplate reader. Results were normalized to cell
number as assessed by the MTT assay, as described above. Analysis
was accomplished by using GraphPad Prism 5 software by apply-
ing one-way ANOVA followed by Dunnett’s multiple comparison
post-test. Levels of significance: *p<0.05; **p<0.01; ***p<0.001.
[10] J. B. Baell, G. A. Holloway, J. Med. Chem. 2010, 53, 2719–2740.
[11] a) F. Li, Q. Gong, H. Dong, J. Shi, Curr. Pharm. Des. 2012, 18, 27–33; b) S.
Gunesch, S. Schramm, M. Decker, Future Med. Chem. 2017, 9, 711 –713;
c) J. Vrba, R. Gazµk, M. Kuzma, B. Papouskovµ, J. Vacek, M. Weiszenstein,
V. Kren, J. Ulrichovµ, J. Med. Chem. 2013, 56, 856–866; d) F. Yang, G. P.
Lim, A. N. Begum, O. J. Ubeda, M. R. Simmons, S. S. Ambegaokar, P. P.
Chen, R. Kayed, C. G. Glabe, S. A. Frautschy, J. Biol. Chem. 2005, 280,
5892–5901; e) G. Henríquez, A. Gomez, E. Guerrero, M. Narayan, ACS
Chem. Neurosci. 2020, 11, 2915–2934.
[12] F. Pohl, P. Kong Thoo Lin, Molecules 2018, 23, 3283.
[13] S. Gunesch, C. Kiermeier, M. Hoffmann, W. Fischer, A. F. M. Pinto, T.
Maurice, P. Maher, M. Decker, Redox Biol. 2019, 101378.
[14] S. Gunesch, D. Soriano-Castell, S. Lamer, A. Schlosser, P. Maher, M.
Decker, ACS Chem. Neurosci. 2020, 11, 3823–3837.
[15] a) W. Fischer, A. Currais, Z. Liang, A. Pinto, P. Maher, Redox Biol. 2019, 21,
101089; b) N. Taguchi, M. Yuriguchi, T. Ando, R. Kitai, H. Aoki, T. Kunisa-
da, Biol. Pharm. Bull. 2019, 42, 1446–1449; c) M. Sato, K. Murakami, M.
Uno, H. Ikubo, Y. Nakagawa, S. Katayama, K.-i. Akagi, K. Irie, Biosci. Bio-
tech. Biochem. 2013, 77, 1100–1103.
Acknowledgements
[16] M. Singh, M. Arseneault, T. Sanderson, V. Murthy, C. Ramassamy, J. Agric.
Food Chem. 2008, 56, 4855–4873.
Financial support by the Graduate School of Life Science
(GSLS) Würzburg and the COST action CA15135 (Multitarget
Paradigm for Innovative Ligand Identification in the Drug Dis-
covery Process MuTaLig) to J.H. is gratefully acknowledged. We
are also grateful for financial support by the Spanish Ministerio
de Economía y Competitividad (MDM2017-0767; AEI/FEDER
UE) and the Generalitat de Catalunya (2017SGR1746;
2019LLAV00016). The Barcelona Supercomputer Center is ac-
knowledged for providing computational resources (BCV-2021-
1-0022). We thank Dr. Pamela Maher (Cellular Neurobiology
´
[17] T. C. T. Michaels, A. Saric, G. Meisl, G. T. Heller, S. Curk, P. Arosio, S. Linse,
C. M. Dobson, M. Vendruscolo, T. P. J. Knowles, Proc. Natl. Acad. Sci. USA
2020, 117, 24251–24257.
[18] K. Murakami, K. Irie, Molecules 2019, 24, 2125.
[19] E. Chainoglou, D. Hadjipavlou-Litina, Int. J. Mol. Sci. 2020, 21, 1975.
[20] M. Sato, K. Murakami, M. Uno, Y. Nakagawa, S. Katayama, K.-i. Akagi, Y.
Masuda, K. Takegoshi, K. Irie, J. Biol. Chem. 2013, 288, 23212–23224.
[21] F. L. Palhano, J. Lee, N. P. Grimster, J. W. Kelly, J. Am. Chem. Soc. 2013,
135, 7503–7510.
[22] a) T. Zhang, J. Zhang, P. Derreumaux, Y. Mu, J. Phys. Chem. B 2013, 117,
3993–4002; b) H. Minh Hung, M. T. Nguyen, P.-T. Tran, V. K. Truong, J.
Chem. Eur. J. 2021, 27, 6015 –6027
www.chemeurj.org
6026 ꢀ 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH

Full Paper
doi.org/10.1002/chem.202005263
Chemistry—A European Journal
Chapman, L. H. Quynh Anh, P. Derreumaux, V. V. Vu, S. T. Ngo, J. Chem.
Inf. Model. 2020, 60, 1399–1408.
[43] J. Broichhagen, J. A. Frank, D. Trauner, Acc. Chem. Res. 2015, 48, 1947–
1960.
[23] L. M. Lima, E. J. Barreiro, Curr. Med. Chem. 2005, 12, 23–49.
[24] a) M. J. Fuchter, J. Med. Chem. 2020, 63, 11436–11447; b) W. A. Velema,
W. Szymanski, B. L. Feringa, J. Am. Chem. Soc. 2014, 136, 2178–2191;
c) D. A. Rodríguez-Soacha, M. Decker, Adv. Ther. 2018, 1, 1800037; d) L.
Agnetta, M. Decker in Design of Hybrid Molecules for Drug Development
(Ed.: M Decker), Elsevier, Amsterdam, 2017, pp. 279–311.
[25] A. Espargaró, A. Medina, O. Di Pietro, D. MuÇoz-Torrero, R. Sabate, Sci.
Rep. 2016, 6, 23349.
[26] F. Chiti, C. M. Dobson, Annu. Rev. Biochem. 2017, 86, 27–68.
[27] G. Invernizzi, E. Papaleo, R. Sabate, S. Ventura, Int. J. Biochem. Cell Biol.
2012, 44, 1541–1554.
[28] S. A. Hudson, H. Ecroyd, T. W. Kee, J. A. Carver, FEBS J. 2009, 276, 5960–
5972.
[29] M. T. Colvin, R. Silvers, Q. Z. Ni, T. V. Can, I. Sergeyev, M. Rosay, K. J. Do-
novan, B. Michael, J. Wall, S. Linse, R. G. Griffin, J. Am. Chem. Soc. 2016,
138, 9663–9674.
[30] S. Release, Schrçdinger, LLC, New York, NY 2018.
[31] Amber18, T. Darden, R. Duke, D. Ghoreishi, M. Gilson, H. Gohlke, A.
Goetz, D. Greene, R. Harris, N. Homeyer, S. Izadi, University of California,
San Francisco 2018.
[44] K. Lindorff-Larsen, S. Piana, K. Palmo, P. Maragakis, J. L. Klepeis, R. O.
Dror, D. E. Shaw, Proteins 2010, 78, 1950–1958.
[45] J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case, J. Comput.
Chem. 2004, 25, 1157–1174.
[46] J. Wang, P. Cieplak, P. A. Kollman, J. Comput. Chem. 2000, 21, 1049–
1074.
[47] Gaussian 09, Revision D1, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.
Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Men-
nucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian,
A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro,
M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Ko-
bayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyen-
gar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B.
Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,
S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cio-
slowski, D. J. Fox, Gaussian Inc., Wallingford CT, 2016.
[32] T. Ginex, M. Trius, F. J. Luque, Chem. Eur. J. 2018, 24, 5813–5824.
[33] O. Crescenzi, S. Tomaselli, R. Guerrini, S. Salvadori, A. M. D’Ursi, P. A. Te-
mussi, D. Picone, Eur. J. Biochem. 2002, 269, 5642–5648.
[34] S. Tan, D. Schubert, P. Maher, Curr. Top. Med. Chem. 2001, 1, 497–506.
[35] A. Currais, P. Maher, Redox. Signal. 2013, 19, 813–822.
[36] J. A. Sonnen, J. C. Breitner, M. A. Lovell, W. R. Markesbery, J. F. Quinn, T. J.
Montine, Free Radical Biol. Med. 2008, 45, 219–230.
[37] M. S. Blois, Nature 1958, 181, 1199–1200.
[38] T. Wyss-Coray, J. Rogers, Cold Spring Harbor Perspect. Med. 2012, 2,
a006346.
[48] C. S. Perone, SIGEVOlution 2009, 4, 12–20.
[49] W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein,
J. Chem. Phys. 1983, 79, 926–935.
[50] I. S. Joung, T. E. Cheatham, J. Phys. Chem. B 2008, 112, 9020–9041.
[51] J.-P. Ryckaert, G. Ciccotti, H. J. Berendsen, J. Comput. Phys. 1977, 23,
327–341.
[52] T. Darden, D. York, L. Pedersen, J. Chem. Phys. 1993, 98, 10089–10092.
[53] H. J. C. Berendsen, J. P. M. Postma, W. F. v. Gunsteren, A. DiNola, J. R.
Haak, J. Chem. Phys. 1984, 81, 3684–3690.
[54] M. Abraham, D. Van Der Spoel, E. Lindahl, B. Hess, GROMACS user
manual version 2014, 5, 1–298.
[55] G. Bussi, D. Donadio, M. Parrinello, J. Chem. Phys. 2007, 126, 014101.
[39] a) G. W. Kreutzberg, Trends Neurosci. 1996, 19, 312–318; b) L. Minghetti,
G. Levi, Prog. Neurobiol. 1998, 54, 99–125.
[40] a) F. Gonzµlez-Scarano, G. Baltuch, Annu. Rev. Neurosci. 1999, 22, 219– [56] M. Parrinello, A. Rahman, J. Appl. Phys. 1981, 52, 7182–7190.
240; b) K. M. Lenz, L. H. Nelson, Front. Immunol. 2018, 9, .
[41] M. Scheiner, D. Dolles, S. Gunesch, M. Hoffmann, M. Nabissi, O. Marinelli,
M. Naldi, M. Bartolini, S. Petralla, E. Poeta, B. Monti, C. Falkeis, M. Vieth,
H. Hübner, P. Gmeiner, R. Maitra, T. Maurice, M. Decker, J. Med. Chem.
2019, 62, 9078–9102.
[57] B. Hess, H. Bekker, H. J. C. Berendsen, J. G. E. M. Fraaije, J. Comput.
Chem. 1997, 18, 1463–1472.
[58] W. Kabsch, C. Sander, Biopolymers 1983, 22, 2577–2637.
[59] K. Murakami, K. Irie, A. Morimoto, H. Ohigashi, M. Shindo, M. Nagao, T.
Shimizu, T. Shirasawa, J. Biol. Chem. 2003, 278, 46179–46187.
[42] a) A. J. Doig, P. Derreumaux, Curr. Opin. Struct. Biol. 2015, 30, 50–56;
b) A. Abbott, E. Dolgin, Nature 2016, 540, 15–16; c) A. J. Doig, M. P. del
Castillo-Frias, O. Berthoumieu, B. Tarus, J. Nasica-Labouze, F. Sterpone,
P. H. Nguyen, N. M. Hooper, P. Faller, P. Derreumaux, ACS Chem. Neurosci.
2017, 8, 1435–1437.
Manuscript received: December 9, 2020
Revised manuscript received: January 14, 2021
Version of record online: March 5, 2021
Chem. Eur. J. 2021, 27, 6015 –6027
www.chemeurj.org
6027 ꢀ 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH