2-Propargylamino-naphthoquinone derivatives as multipotent agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.113112

Alzheimer's disease is a progressive brain disorder with characteristic symptoms and several pathological hallmarks. The concept of “one drug, one target” has not generated any new drugs since 2004. The new era of drug development in the field of AD build

Full text of DOI:10.1016/j.ejmech.2020.113112

Journal Pre-proof
2-Propargylamino-Naphthoquinone Derivatives as Multipotent Agents for the
Treatment of Alzheimer's Disease
Eva Mezeiova, Jana Janockova, Rudolf Andrys, Ondrej Soukup, Tereza Kobrlova,
Lubica Muckova, Jaroslav Pejchal, Miriama Simunkova, Jiri Handl, Petra Micankova,
Jan Capek, Tomas Rousar, Martina Hrabinova, Eugenie Nepovimova, Jose Luis
Marco-Contelles, Marian Valko, Jan Korabecny
PII:
S0223-5234(20)31084-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.113112
EJMECH 113112
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 November 2020
Revised Date: 14 December 2020
Accepted Date: 14 December 2020
Please cite this article as: E. Mezeiova, J. Janockova, R. Andrys, O. Soukup, T. Kobrlova, L. Muckova,
J. Pejchal, M. Simunkova, J. Handl, P. Micankova, J. Capek, T. Rousar, M. Hrabinova, E. Nepovimova,
J.L. Marco-Contelles, M. Valko, J. Korabecny, 2-Propargylamino-Naphthoquinone Derivatives as
Multipotent Agents for the Treatment of Alzheimer's Disease, European Journal of Medicinal Chemistry,
https://doi.org/10.1016/j.ejmech.2020.113112.
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inhibition of
amyloid-beta
aggregation
monoamine
oxidase B
inhibition
N-propargyl-naphthoquinone/
N-(methyl)propargyl-naphthoquinone
metal-chelating
properties
anti-infammatory
action
IL-6
IL-6
Cu(II)
Cu(II)
TNFα
TNFα
TNFα
Cu(II)
Cu(II)
IL-6

2-Propargylamino-Naphthoquinone Derivatives as Multipotent
Agents for the Treatment of Alzheimer's Disease
Eva Mezeiova,1 Jana Janockova,1 Rudolf Andrys,2 Ondrej Soukup,1 Tereza Kobrlova,1 Lubica
Muckova,1,3 Jaroslav Pejchal,3 Miriama Simunkova,4 Jiri Handl,5 Petra Micankova,5 Jan Capek,5
Tomas Rousar,5 Martina Hrabinova,1,3 Eugenie Nepovimova,2 Jose Luis Marco-Contelles,6,*
Marian Valko,4,* Jan Korabecny1,3,*
1 University Hospital Hradec Kralove, Biomedical Research Centre, Sokolska 581, 500 05 Hradec Kralove, Czech Republic
2 University Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 500 03 Hradec Kralove, Czech
Republic
3
University of Defence in Brno, Faculty of Military Health Sciences, Department of Toxicology and Military Pharmacy,
Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
4 Slovak University of Technology, Faculty of Chemical and Food Technology, Radlinskeho 9, 812 37 Bratislava, Slovakia
5
University of Pardubice, Faculty of Chemical Technology, Department of Biological and Biochemical Sciences, Studentska
573, 532 10 Pardubice, Czech Republic
6 Institute of General Organic Chemistry, Laboratory of Medicinal Chemistry, Juan de la Cierva 3, 28006 Madrid, Spain
Corresponding authors: J.K. - phone: +420-973-255-167; E-mail: jan.korabecny@fnhk.cz; ORCID ID:
0000-0001-6977-7596; M.V. - phone: +421-2593-25-750; E-mail: marian.valko@stuba.sk; ORCID ID:
0000-0001-7483-0267; J.L.M.-C. – phone: +34-91-2587554; E-mail: jlmarco@iqog.csic.es; ORCID ID:
0000-0003-0690-0328
Abstract: Alzheimer's disease is a progressive brain disorder with characteristic symptoms
and several pathological hallmarks. The concept of "one drug, one target" has not generated
any new drugs since 2004. The new era of drug development in the field of AD builds upon
rationally designed multi-target directed ligands that can better address the complexity of
AD. Herewith, we designed ten novel derivatives of 2-propargylamino-naphthoquinone. The
biological evaluation of these compounds includes inhibition of monoamine oxidase A/B,
inhibition of amyloid-beta aggregation, radical-scavenging, and metal-chelating properties.
Some of the compounds possess low cytotoxicity profile with an anti-inflammatory ability in
the lipopolysaccharide-stimulated cellular model. All these features warrant their further
testing in the field of AD.
Keywords: Alzheimer's disease; amyloid-beta; antioxidant; inflammation; monoamine
oxidase; multi-target directed ligand; naphthoquinone

1. Introduction
Alzheimer's disease (AD) is a progressive brain disorder with characteristic symptoms and
pathological hallmarks. One of the most critical findings of AD is that the individual
pathological features are tightly intertwined, portraying the disorder as a highly complex
condition. Aberrant processing of amyloid-beta (Aβ) and tau proteins, oxidative stress,1
inflammation,2 disrupted neurotransmission,3 and biometal dyshomeostasis4 are among
frequently pronounced mediators in the cascade of AD pathology. The overall imbalance of
the system functioning in the brain ultimately leads to a dramatic loss of neurons and
synapses and the death of AD patients.
The amyloid hypothesis first postulated that the neurodegeneration in AD is caused by the
toxicity of Aβ oligomers and excessive accumulation of Aβ into plaques in various brain
areas.5,6 The triggering mechanism of amyloidogenic processing generating toxic species of
Aβ remains elusive. However, the factors that can contribute to this process are well
described, including mutations in PSEN1, APO-ε4 and APP genes.7 Mechanistically,
aggregation and accumulation of Aβ are associated with metal dyshomeostasis and oxidative
stress.8,9 The N-terminus of Aβ peptides contains metal-binding sites capable of coordinating
transition metal ions, such as Cu(II), Fe(III), and Zn(III). Accordingly, metal ions significantly
affect the overall aggregation behavior of Aβ.8,9 Oxidative stress reflects the imbalance
between the generation of reactive oxygen species (ROS) and antioxidant defense that
damages the mitochondrial function.10 The generation of ROS is also strongly associated with
redox-active metal ions, especially Cu(II) and Fe(III). Cycling between the redox couples (like
Cu(I)/Cu(II), and Fe(II)/Fe(III)) can facilitate the activation of molecular oxygen, which is
mediated mainly by Aβ, in other words Cu(II)- and Fe(III)-Aβ complexes are able to produce
H2O2 in the presence of oxygen.9 Moreover, the reduced form of the metalloproteins
catalyzes the generation of highly toxic hydroxyl radicals (OH·).11 The connection between
metal-induced Aβ aggregation and oxidative stress with metal dyshomeostasis reinitiated
the emergence of metal-targeted therapeutics in the management of AD.
Herein, in the design of novel potential drugs for AD, we turned our attention to agents
endowed with monoamine oxidase-B (MAO-B) inhibition capacity and simultaneous
mitigation of Aβ aggregation. MAO (E.C. 1.4.3.4) is a flavoprotein present in the outer
mitochondrial membranes of neuronal, glial and other cells. Monoaminergic insult along

with downstream MAO-A/B dysfunction, severely contributes to the pathogenesis of AD.12
Furthermore, MAO-B isoform is also implicated in oxidative stress producing H2O2.13 From a
drug design perspective, it has been well-documented that either N-propargylamine or
N-(methyl)propargylamine moieties can deliver MAO inhibition ability.14,15 Thus, we took the
structural characteristics of several well-known compounds (Fig. 1) to confront AD on a
quadruple front by a) inhibiting Aβ aggregation directly, b) reducing oxidative stress via
inhibition of MAO-B, c) introducing metal-chelating properties, and d) mitigating
inflammatory response. Namely, we were inspired by rasagiline, ladostigil, and selegiline
(Fig. 1), all being neuroprotective agents that display cognitive improvement in
AD/Parkinson's disease by modulating the proteolytic cleavage of amyloid precursor protein
(APP) and decreasing brain levels of Aβ.16 The design is also corroborated by the structural
features of memoquin17 (Fig. 1), one of the first rationally designed multipotent drug
candidate for the treatment of AD, and several other related analogues like tacrine-
naphthoquinone hybrids18 or naphthoquinone-tryptophan derivatives.19 The top-ranked
representatives of the latter series, compounds 118 and 219, are displayed in Figure 1. We
envisaged that the attachment of N-propargylamine or N-(methyl)propargylamine moieties
to the naphthoquinone (NQ) scaffold would provide promising multipotent features.
The developed N-propargyl-naphthoquinones (N-PNQs) and N-(methyl)propargyl-
naphthoquinones (N-(M)PNQs) were subjected to biological evaluation to determine their
inhibitory activities against MAOs and Aβ-aggregation, cytotoxicity on SH-SY5Y cell line, anti-
inflammatory action in the lipopolysaccharide (LPS)-stimulated SIM-A9 cells, and to predict
the ability to cross the blood-brain barrier (BBB). We were also interested in their
antioxidant capacity and metal-chelating ability since these two properties have a strong
potential to modulate the progression of AD. We investigated their structure-activity
relationship (SAR) regarding structure modification in four different regions, specifically a)
substitution by chlorine atom at position 2- of NQ, b) substitution at position 5- by bulky
functional group or the presence of two substituents at positions 5- and 8- at NQ core, c) the
presence of hydrogen or methyl group at the nitrogen of propargylamine moiety, and d) the
position of these moieties with respect to basic NQ core (substitutions at positions 2- or 3-).

Figure 1. The design strategy of N-PNQs or N-(M)PNQs exploiting rasagiline, ladostigil and selegiline as
representatives with MAO inhibition activity, memoquin as AChE/Aβ-dual inhibition blocker, and 1 and 2 as
agents from tacrine-naphthoquinone hybrids and naphthoquinone-tryptophan families, respectively.
2. Results and Discussion
2.1. Synthesis of N-PNQs and N-(M)PNQs
N-PNQs and N-(M)PNQs were synthesized in one or two steps (Scheme 1). The 1,4-NQ (3),
2,3-dichloro-1,4-NQ (4), 5-hydroxy-1,4-NQ (5), and 5,8-dihydroxy-1,4-NQ (6) were
commercially available. Derivatives 7-9 were synthesized from 5 in one step according to
previously published methods.20,21 Final products 12, 13 and 16-21 were obtained by the
reaction of 3, or 5-9 either with N-propargylamine (10) or N-(methyl)propargylamine (11) in
CH3CN at room temperature. The compounds containing chlorine at C3 (14, 15) were
produced by the reaction between 4 and 10 or 11 using EtOH as solvent at reflux. Derivatives
12-21 were prepared in moderate to good yields (30 – 79%). The lower yields of the
reactions can be attributed to the low stability of some of the representatives as observed
during the work-up, thin-layer chromatography (TLC) and repeated purification by column
chromatography.

For compounds 16-20, we expected the formation of two isomers with N-propargyl or N-
(methyl)propargyl group in positions 2- or 3-. However, we isolated only one isomer for each
derivative. 2D NMR experiments determined the position of N-propargyl group of
derivatives 16 and 19 (see Supporting Information). Interestingly, attachment of 10 in 16
bearing free phenolic group was placed at position 2-, while in 19 containing methoxy
substituent, 10 was attached at position 3-. Likewise, the attachment of 11 was directed to
the C3 in 20. Compounds 13 and 15 were previously prepared.22–26
All the derivatives were submitted to biological evaluation except for low-stable derivatives
16 and 20.
Scheme 1. Synthesis of N-PNQs and N-(M)PNQs 12-21. Reagents and conditions: a) for compounds
12, 13, 16-21: corresponding propargylamine, CH3CN, r.t., 12, 42%, 13, 49%, 16, 31%, 17, 79%, 18,
87%, 19, 43%, 20, 30%, 21, 52%; b) for compounds 14 and 15, corresponding propargylamine,
ethanol, 78 °C, 14, 53%, 15, 74%.
2.2. Inhibition activity of Aβ aggregation
Disruption of the cellular amyloid metabolism followed by Aβ accumulation in the brain is
suspected to be one of the most considerable hallmarks in the pathogenesis and progression
of AD. Assembled Aβ peptide forms extracellular senile plaques originating from the
proteolytic cleavage of APP.27,28 APP is cleaved sequentially by β- and γ-secretases, which
generates Aβ peptide of different length. Specifically, 40- and 42-long Aβ sequences,
denoted as Aβ1-40 and Aβ1-42, respectively, are the most abundant ones. Aβ1-42 isoform is
more hydrophobic, tending to self-aggregate more rapidly, and is also considered to be more
toxic.29–31 Aβ monomers then aggregate into different types of assemblies, such as soluble

oligomers and protofibrils, which can further assemble into insoluble, misfolded fibrils and
ultimately into amyloid plaques. Some studies proposed that the neurotoxic effects of Aβ
species impose cell injury, oxidative stress, ion channels and membranes disruption,
inflammatory responses, and apoptosis.32,33 Accordingly, the potential to block Aβ
aggregation, especially its oligomerization, represents a promising way for effective therapy.
Inhibition experiments were performed by incubating Aβ1-42 at 50 μM in the presence of
studied compounds 12-15, 17-19 and 21 at 1:1 ratio. The formation of Aβ1-42 aggregates was
monitored by using thioflavin T (ThT) fluorimetric assay. ThT is a fluorescent dye that binds
specifically to a β-sheet rich region of a protein structure, and thus increase of fluorescence
emission is indicative of binding to amyloid aggregates.34 Fluorescence intensity was
monitored every 10 min for 72 h aggregation kinetic under given conditions. The obtained
data are presented in Table 1. Determination of the inhibitory potency (%) was carried out
by comparing fluorescence values in the presence and in the absence of the inhibitor. All
studied compounds inhibited Aβ1-42 aggregation in the range of 11.2% - 67.4%. The results
were compared with positive controls myricetin and doxycycline (˃ 90%), well-known Aβ1-42
aggregation inhibitors.34,35 The most active inhibitor was derivative 12 (67.4%), followed by
14 (40.5%). All of these compounds were 2-times more active than their counterparts
without the methyl group at nitrogen of the N-propargylamine moiety (derivatives 13
(24.0%), 18 (17.5%), and 15 (18.4%), respectively). The presence of chlorine atom and
substitutions at position 5- and/or 8- decreased Aβ inhibition capability.
Table 1. Inhibition of Aβ1-42 aggregation produced by the tested compounds at 50 μM concentration.
Inhibition of Aβ1-42 aggregates
Compound
± SEM (%)a
12
13
14
15
17
18
19
67.4 ± 5.7
24.0 ± 6.4
40.5 ± 6.7
18.4 ± 3.7
11.2 ± 3.9
17.5 ± 3.8
15.5 ± 4.9
21.7 ± 5.8
92.7 ± 3.4
93.1 ± 9.8
21
Myricetin
Doxycycline
a The results are the mean of at least three independent measurements each performed in duplicates.
2.3. Inhibition of MAOs

Derivatives 12-15, 17-19 and 21 were initially screened at two concentrations (1 and 10 μM)
against both isoforms of human MAOs (Table S1; Supplementary Information). The IC50
values of the most active compounds 12-15 were then determined (Table 2). Clorgyline (an
irreversible and selective inhibitor of MAO-A) and pargyline (an irreversible and selective
inhibitor of MAO-B) were used as references. All tested derivatives inhibited MAO-A at
micromolar range with IC50 values between 6.6 – 10.0 μM, being less active than clorgyline
(IC50 = 0.05 μM), except for 12 (IC50 = 8.6 μM, SI = 0.77). Importantly, all compounds were
more effective on MAO-B than MAO-A. The most potent inhibitor of MAO-B was 15 (IC50
=
0.05 μM) being comparable to pargyline (IC50 = 0.08 μM). Compound 15 was also the most
selective inhibitor of MAO-B with SI = 155. From SAR analysis, it can be deduced that any
substituent at position 5- and/or 8 is detrimental for the anti-MAOs activity.
Table 2. Inhibition of 12-15 and reference compounds against both isoforms of human MAO.
IC50 (μM) ± SD
SI for MAO-Bb
Compound
MAO-Aa
MAO-Ba
12
13
14
6.64 ± 0.41
9.81 ± 0.71
10.0 ± 0.74
7.76 ± 0.61
0.05 ± 0.00
8.62 ± 0.46
0.27 ± 0.02
0.14 ± 0.01
0.05 ± 0.00
0.77
36
71
15
155
c
c
Clorgyline
Pargyline
–
–
–
c
c
–
0.08 ± 0.01
a IC50 values are the mean ± SEM of at least three different experiments, b SI = selectivity index, determined as
IC50(MAO-A) / IC50(MAO-B), c not determined.
Additional experiments were carried out to determine the enzyme kinetic of MAO-B
inhibition for derivatives 13 and 15. Accordingly, the time-dependent binding rates of 13 and
15 were established, providing insight into the compounds' inhibition mechanism.
Time-dependent inhibition of 13 showed a rapid decrease of enzymatic activity in 1 min, and
the enzyme activity remained almost unchanged for 60 minutes (Fig. 2). This could be
explained by the non-covalent interaction between tested compounds and enzyme. Such
interactions are in general very rapid as there is no chemical reaction involved. On the other
hand, compound 15 showed mixed behavior. The decrease in enzyme activities showed
patterns of both covalent and non-covalent inhibitions. This was manifested by a relatively
rapid initial inhibition (non-covalent interaction) followed by a gradual decrease in enzyme
activity (covalent interaction).

Figure 2. Time-dependent enzyme inhibition progress for compounds 13 and 15 on MAO-B.
The inhibition mechanism of MAO-B for 13 and 15 were further investigated. The results
(Fig. 3) showing the reversibility of inhibition are consistent with the results of time-
dependent inhibition progress. The MAO-B enzyme activity could be recovered to the initial
state after the treatment with 13. This result suggests that 13 does bind to the enzyme
through a non-covalent interaction. In contrast, the MAO-B enzyme activity inhibited by 15
could not be completely reactivated. This result suggests that a portion of the 15 molecule
bound to the enzyme covalently, causing partial irreversible inhibition. These results were
further corroborated by the determination of the enzyme inhibition kinetics (Fig. 4). It is
apparent that the inhibition of the MAO-B enzyme by 13 is determined by a change in both
Km and Vmax. As the inhibitor concentration increases, Km value increases and Vmax value
decreases. This behavior corresponds to a mixed type of inhibition. On the other hand, the
inhibition of the enzyme by 15 is manifested by a change in Km (increasing values), but a
constant value of Vmax. This phenomenon would correspond to competitive inhibition.

Figure 3. MAO-B inhibition activity of 13 and 15 prior- and after-dialysis.
Figure 4. Determination of the enzyme inhibition kinetics (KM – Michaelis' constant, Vmax – maximum rate
achieved by the system) for compounds 13 and 15 on MAO-B.
2.4. Metal-chelating and scavenging properties of selected compounds
Interaction between Cu(II), a model of free uncomplexed metal ion, and studied compounds
was studied using cw-EPR spectroscopy. The EPR spectra of studied compounds in the
presence of Cu(II) ions (Fig. 5) were significantly different from the spectrum of Cu(II)
chloride, model of free uncomplexed metal, (spectrum is not shown), confirming thus their
interaction with Cu(II) ions. The EPR spectra of Cu(II) complexes bearing 15 and 18 as
coordinating ligands revealed resolved parallel hyperfine splitting pattern. The EPR spectrum
of Cu(II)-14 complex showed monomeric nature of the interaction between Cu(II) and drug.
Remaining compounds upon coordination with Cu(II) ions exhibited broad bands centered at
about
3600 Gauss, confirming the formation of dimeric or even polymeric species originating most

probably from the aggregation of Cu(II)-drug complexes upon cooling to liquid nitrogen
temperatures. The Cu(II) coordination sites in cases of 15 and 18 may involve nitrogen
functional groups and in the case of clioquinol may involve proximal –OH group and
heterocyclic nitrogen. Taken together, all studied drugs interacted with Cu(II) ions and form
either monomeric species (14) or aggregated species (15, 18, and clioquinol) following their
interaction with Cu(II) ions. This finding obtained by EPR spectroscopy is in agreement with
the data obtained by the ABTS assay (see below).
K1411 + Cu
K1410 + Cu
15 + Cu
14 + Cu
2600 2800 3000 3200 3400 3600 3800
Magnetic field (Gauss)
2600 2800 3000 3200 3400 3600 3800
Magnetic field (Gauss)
Clioquinol + Cu
K1413 + Cu
18 + Cu
2600 2800 3000 3200 3400 3600 3800
Magnetic field (Gauss)
2600 2800 3000 3200 3400 3600 3800
Magnetic field (Gauss)
Figure 5. The EPR spectra (drug-to-Cu ratio 1:1) of studied drugs with Cu(II) ions. Concentration of Cu(II) was 1
mM. EPR data: Cu-14, g1 = 2.075, g2 = 2.082, g3 = 2.228; Cu-15, g = 2.075, gII = 2.228, AII = 179 Gauss; Cu-18, g1
= 2.075, g2 = 2.083, g3 = 2.3244, AII = 120 Gauss; Cu-clioquinol, g = 2.101.
The time decay of ABTS radical cation in the presence of studied compounds revealed
relatively efficient radical scavenging activity (Fig. 6). As expected, the most profound radical
scavenging activity was observed for clioquinol in the absence of Cu(II) ions since this drug
contains a hydroxyl group in the structure. Interaction of clioquinol with Cu(II) ions
(stoichiometric ratio 1:1) resulted in the blockage of the hydroxyl group by Cu(II) which in

turn has been seen in the ABTS assay as a significant decrease of its radical scavenging
activity (by 40%). Drugs 14 and 15 exhibited similar and significantly lower radical scavenging
activity than it was observed for clioquinol. With respect to 14 and 15, compound 18 showed
reduced radical scavenging activity by 15%. Interaction of both 14 and 18 with Cu(II) ions
resulted in reduced radical scavenging activity (pro-oxidant behavior) by 5% and 10%,
respectively. Reduced radical scavenging activity of 14 and 18 in the presence of Cu(II), with
respect to free compounds, is probably due to the saturation of nitrogen functional groups
by Cu(II) ions containing hydrogen donor atoms. The structure of derivative 18 contains two
proximal oxygen atoms capable of chelating Cu(II) ion. Thus upon coordination of Cu(II) to
18, nitrogen atom from N-propargylamine is left free and available for interaction with
radical species. In line with this, compound 18 showed more profound radical scavenging
activity in the presence of Cu(II) ions than it was observed for clioquinol.
1.0
0.9
0.8
.
ABTS +
14
14 + Cu(II)
15
0.7
15 + Cu(II)
18
18 + Cu(II)
0.6
Clioq
Clioq + Cu(II)
0.5
0
100
200
300
400
500
600
700
800
900
time / s
•
Figure 6. Time decay of ABTS .+ in the presence of studied drugs and their Cu(II) complexes.
2.5. Determination of in vitro BBB permeation

AD drug development builds on the drug capable to cross the BBB and acting in the CNS. The
BBB regulates the transport of molecules into and out of CNS, maintaining controlled
chemical composition of the neuronal milieu required for proper neuronal functioning.36
Parallel artificial membrane permeability assay (PAMPA) was used to determine the ability of
N-PNQs and N-(M)PNQs to cross the BBB and the results are summarized in Table 3. The
data indicate that all newly prepared compounds have a high potential to cross the BBB via
passive diffusion readily. The validation of the assay has been performed using standard
compounds whose central availability or unavailability was experimentally predicted in vitro
and confirmed in vivo.37–39
Table 3. Prediction of BBB penetration of studied compounds expressed as Pe ± SEM.
Compound
Pe ± SEM
(×10-6 cm s-1)
18.5 ± 1.95
20.8 ± 1.82
14.0 ± 0.28
29.4 ± 7.84
18.5 ± 7.30
25.5 ± 3.77
18.8 ± 2.39
43.3 ± 0.06
5.90 ± 0.67
21.9 ± 2.06
18.7 ± 2.03
18.0 ± 4.32
1.14 ± 0.54
0.19 ± 0.07
0.04 ± 0.02
0.09 ± 0.05
na
CNSb (+/-)
12
13
14
15
17
18
19
21
2
2
2
3
2
2
2
2
8
6
4
3
3
3
2
2
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (-)
CNS (-)
CNS (-)
CNS (-)
Tacrine
Donepezil
Rivastigmine
Ibuprofen
Chlorothiazide
Furosemide
Ranitidine
Sulfasalazine
a n = number of measurements; b CNS + (high BBB permeation predicted): Pe (×10-6 cm s-1) > 4.0, CNS - (low BBB
permeation predicted): Pe (×10-6 cm s-1) < 2.0, CNS +/- (BBB permeation uncertain): Pe (×10-6 cm s-1) from 4.0 to
2.0.
2.6. Determination of cytotoxic profile
The cytotoxic profiles of 12-15, 17-19 and 21 were evaluated by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human neuroblastoma (SH-SY5Y) and
murine microglial (SIM-A9) cell lines. Rasagiline was used as a standard yielding very low
cytotoxicity profile against both cell lines. All the data are summarized in Table 4. The lowest
cytotoxicity from the newly developed compounds revealed 13, 17, and 18 with the
N-propargylamine group and protected oxygen at C5. The IC50 values are higher than 64 µM,

which was also the highest tested concentration for some of the compounds given their
solubility profile. The most cytotoxic species were 14 (IC50 = 3.26 µM) and 15 (IC50 = 4.59 µM)
bearing chlorine atom at position 2-.
Compounds 12-15, 17-19 and 21 were further subjected to cytotoxicity measurement
against SIM-A9, a microglial cell line representing a major component of the active immune
system in the brain.40 The most toxic derivative was 21 (IC50 = 5.3 µM) with two deprotected
hydroxyl groups, followed by 14 (IC50 = 7.3 µM) and 15 (IC50 = 10.4 µM), the latter having
chlorine atom at position 2-. Maximally tolerated concentrations (MTCs) for this cell line
were also determined. The safety of MTCs was additionally confirmed by the flow-
cytometric method. The values served as an input into the ongoing measurement of anti-
inflammatory action.
Table 4. Cytotoxic profiles of tested compounds on SH-SY5Y and SIM-A9 and MTC in SIM-A9 after 24 h.
SH-SY5Y
IC50 ± SD (µM)a
SIM-A9
Compound
MTCb (µM)
12
13
14
15
17
22.0 ± 0.7
> 64
3.3 ± 0.30
4.6 ± 0.10
> 64
36.1 ± 2.3
8
4
2
4
32
8
34.6 ± 2.3
7.3 ± 0.6
10.4 ± 0.6
> 64
18
> 64
> 32
19
21
> 32
15.0 ± 1.5
115.6 ± 7.3
5.3 ± 0.4
8
0.4
rasagiline
2700 ± 110
2900 ± 110
–
a
b
Results are expressed as the mean of at least three experiments; MTC represents the highest non-toxic
concentration that did not produce any significant difference in cell viability when compared with control. MTC
was determined using MTT assay and further confirmed by microcapillary flow cytometry.
2.7. The anti-inflammatory action of tested compounds
SIM-A9 cells can be stimulated by LPS to produce pro-inflammatory cytokines like
interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα). The anti-inflammatory action of
tested compounds was measured at three concentrations corresponding to 1.0, 0.5, and
0.25 MTC. Derivative 18 was the only one demonstrating anti-inflammatory effect at
concentrations corresponding to 1.0 MTC (8 µM) or 0.5 MTC (4 µM). Data representing the
decrease in LPS-stimulated IL-6 and TNFα production as a response to compound 18
administration in comparison to a positive control (i.e. cell treated solely with LPS) are
summarized in Table 5. Dexamethasone was used as a negative control mitigating the LPS-

induced insult. Compounds 12, 19 and 21 did not affect inflammatory response stimulated
by LPS. On the other hand, treatment of SIM-A9 with 13-15, and 17, even aggravated IL-6
and/or TNFα production. The mechanisms underlying this observation remains unknown.
However, naphthoquinone itself or its close analogues may sometimes produce oxidative
stress, stimulate pro-inflammatory pathways and aggravate inflammation.41,42 For more
details about the effect of all tested compounds on LPS-stimulated IL-6 and TNFα production
in the SIM-A9 cell line, see Supplementary Information (Table S2).
Table 5. The anti-inflammatory action of compound 18 in SIM-A9 after 24 h incubation expressed as % of
positive control (cell treated solely with LPS) ± SD
Cytokine production
decrease in % ± SD
drug (concentration)
IL-6
TNFα
18 (1.0 MTC = 8 µM)
18 (0.5 MTC = 4 µM)
18 (0.25 MTC = 2 µM)
49 ± 15a
74 ± 7a
95 ± 8b
40 ± 15a
46 ± 8a
74 ± 17
76 ± 17
60 ± 21a
dexamethasone (1 µg.mL-1)
statistically significant compared with the positive control (cells treated solely with LPS): p ≤ 0.05;
a
b
statistically significant compared with the negative control (cells treated with dexamethasone and LPS): p ≤
0.05.
2.8. Effect of 18 on neuronal cells
Compound 18 showed anti-inflammatory action and was therefore selected for further tests
regarding its effect on intracellular dehydrogenase activity and glutathione levels. The SH-
SY5Y cells were treated with 18 (0.1–100 µM) for 24 h. To evaluate an effect of 18 treatment
on cells, we measured total intracellular dehydrogenase activity as a marker of cellular
viability, glutathione levels for evaluation of oxidative stress, and mitochondrial membrane
potential and mitochondrial respiration to characterize mitochondrial function. After 24 h of
treatment, we detected a slight decrease in dehydrogenase activity in 10 and 100 µM 18
treated cells to 89 ± 8% (p<0.001) and 88 ± 7% (p<0.001), respectively, compared to control
cells (Fig. 7A). Similar results showing negligible cellular effects of 18 treatment on SH-SY5Y
cells were found after analysis of glutathione levels. Slight but significant glutathione
depletion was detected only in cells treated with 10 µM of 18 (89 ± 8%; p<0.001) after
comparison with control cells (Fig. 7B).

Figure 7. Measurements of dehydrogenase activity of 18 treatment (0.1–100 µM; 24 h) in SH-SY5Y cells. A)
Intracellular dehydrogenase activity; B) Glutathione levels. The results are expressed as mean ± SD (control =
100%; n = 20; ***, p<0.001 vs. control).
In addition, the effect of 18 treatment on mitochondrial function was tested. We used a
probe JC-1 detecting the mitochondrial potential in SH-SY5Y cells. We did not find any
significant changes of R/G ratio induced by 18 treatment compared to control cells (Fig. 8A).
Based on the results on cellular viability, an effect of 10 µM 18 on mitochondria was
estimated using high-resolution respirometry experiments (Fig. 8B), which confirmed that no
change of any of the tested respiratory states rates (i.e. ROUTINE, LEAK, ETS, ROX) occurred.
In conclusion, the treatment of SH-SY5Y with 18 did not cause any significant damage to
these cells within the tested range of concentrations.

Figure 8. Estimation of changes in mitochondrial activity after 18 treatment (0.1–100 µM; 24 h) in SH-SY5Y
cells. A) Mitochondrial membrane potential. The results are expressed as R/G ratio, mean ± SD (n = 20).
B) Mitochondrial respiration. The results are expressed as the respiration of untreated cells (white) and 10 µM
18 treated cells (grey). The data are expressed as mean ± SD (n = 8). (ROUTINE – basal respiration of intact cells,
LEAK – state after enzymatic inhibition of the phosphorylation system; ETS – maximal respiratory capacity; ROX
– residual oxygen consumption)
3. Conclusions
Current therapy of AD builds on cholinergic and glutamatergic systems leading to temporary
symptomatic alleviation. Nevertheless, the development of new AD pharmacotherapy
should be more complexed, ideally addressing targets plausibly responsible for the disease
onset. Such targets can be classified and found within the amyloid cascade hypothesis
(generation of Aβ species), oxidative stress, inflammation, and biometals dyshomeostasis,
representing the pivotal mediators.2 To pursue the aim of developing new potent AD
therapeutics able to modulate all of the contributors mentioned above, we have constructed
several N-propargylamine-NQ derivatives. These N-PNQs and N-(M)PNQs were tested on
inhibition activity against Aβ aggregation and MAOs. We were also interested in the ability of
prepared compounds to cross the BBB, to affect the inflammatory response, and to chelate
and scavenge biometals. From the tested compounds, we are highlighting 14, 15, and 18.
Especially derivatives 14 and 15 with chlorine atom at position 2- expressed high MAO-B
inhibition and metal-chelating properties for Cu(II). Besides, 14 also showed satisfactory
inhibition activity against Aβ aggregation. Compound 18 with benzyloxy group was the only
one demonstrating anti-inflammatory effects at concentrations corresponding to 1.0 or 0.5
MTC in SIM-A9 cell line, and showed more profound radical scavenging activity in the
presence of Cu(II) ions than it was observed for standard clioquinol. Compound 18 was also
selected for further tests regarding its cytotoxicity and it did not cause any significant
damage in SH-SY5Y cells in therapeutically relevant concentrations. From the perspective of
MAO inhibition and metal-chelating properties, we are highlighting derivative 15. These
findings warrant further investigation of N-PNQs and N-(M)PNQs as disease-modifying
agents in AD treatment.

4. Materials and Methods
4.1. General Chemistry Methods
All reagents and solvents were purchased from Sigma-Aldrich (Prague, Czech Republic) and
were used in the highest available purity without further purification. The reactions were
monitored by thin layer chromatography (TLC) performed on aluminium sheets precoated
with silica gel 60 F254 (Merch, Prague, Czech Republic). The spots were visualized by
ultraviolet light (at wavelength 254 nm). Purifications of crude products were performed by
column chromatography on silica gel 100 (particle size 0.063–0.200 mm, 70–230 mesh
ASTM, Fluka, Prague, Czech Republic). NMR spectra were recorded in deuterated chloroform
(CDCl3) or dimethyl sulfoxide (DMSO-d6) on a Varian S500 spectrometer operating at 500
MHz for 1H and 126 MHz for 13C. Chemical shifts (δ) are reported in parts per millions (ppm)
and spin multiplicities are given as singlet (s), bold singlet (bs), doublet (d), triplet (t), quartet
(q), doublet of doublet (dd), doublet of triplet (dt), triplet of doublet (td), or multiplet (m).
Coupling constants (J) are reported in Hz. The assignment of chemical shifts of compounds
1
16 and 19 are based on standard 2D NMR experiments (1H-1H-COSY, H-13C HSQC, HMBC).
The synthesized compounds were analyzed by LC-MS system consisting of UHLPC Diones
Ultimate 3000 coupled with Q Exactive Plus Orbital mass spectrometer to obtain high-
resolution mass spectra (Thermo Fisher Scientific, Bremen, Germany). The purity of all the
final compounds established by LC-UV (254 nm) analysis was higher than 95%. Melting
points were measured using a M-565 automated melting point recorder (Büchi, Flawil,
Switzerland).
Derivatives 5-7 were prepared according to the previously published procedures from
5-hydroxy-1,4-naphthoquinone (5).20,21 The 1H and 13C NMR spectra were in good agreement
with published data. Final compounds 12-15, 17-19 and 21 were synthesized according to
general synthetic procedure A or B.
General synthetic procedure A. Either N-propargylamine (2.0 eq) or N-
methylpropargylamine (2.0 eq) were added to a solution of starting compound (1.0 eq) in
CH3CN (0.3 M). The reaction mixture was stirred at room temperature for 24 h. The solvent
was evaporated under reduced pressure and the crude product was purified by column
chromatography in the mixture of eluents (petroleum ether(PE):ethyl acetate(EA) = 5:1 or
4:1).

General synthetic procedure B. Either N-propargylamine (1.0 eq) or N-
methylpropargylamine (1.0 eq) were added to a solution of starting compound (1.0 eq) in
ethanol (0.3 M). The reaction mixture was stirred at 78 °C for 6 h. The solvent was
evaporated under reduced pressure and the crude product was purified by column
chromatography in the mixture of eluents (PE:EA = 3:1).
2-[Methyl(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione
(12):
red
solid,
decomposition at 90 °C. Prepared from 3 with 11 by general synthetic procedure A in 49%
yield. 1H NMR (500 MHz, DMSO-d6): δ 7.94 (dd, J = 7.6, 1.4 Hz, 1H), 7.90 (dd, J = 7.5, 1.5 Hz,
1H), 7.81 (td, J = 7.5, 1.4 Hz, 1H), 7.76 (td, J = 7.5, 1.5 Hz, 1H), 5.94 (s, 1H), 4.40 (d, J = 2.5 Hz,
2H), 3.34 (t, J = 2.4 Hz, 1H), 3.08 (bs, 3H). 13C NMR (126 MHz, DMSO-d6): δ 183.1, 182.4,
152.7, 134.6, 133.1, 132.8, 132.3, 126.6, 125.3, 109.2, 75.9, 76.1, 43.2, 40.1. HRMS: m/z
calcd. for C14H11NO2: 225.0790, [M+H]+ found: 226.0864.
2-[(Prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (13): light brown solid,
decomposition at 182 °C. Prepared from 3 with 10 by general synthetic procedure A in 42%
yield. The NMR spectra were in good agreement with previously published data.22–24 1H NMR
(500 MHz, DMSO-d6): δ 7.99 (dd, J = 7.7, 1.3 Hz, 1H), 7.95 (dd, J = 7.7, 1.3 Hz, 1H), 7.84 (td, J
= 7.5, 1.4 Hz, 1H), 7.75 (td, J = 7.5, 1.4 Hz, 1H), 5.79 (bs, 1H), 4.05 (dd, J = 6.0, 2.5 Hz, 2H),
3.27 (t, J = 2.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δ 182.1, 181.9, 148.3, 135.3, 133.3,
132.9, 130.8, 126.4, 125.9, 101.8, 79.4, 75.1, 31.6. HRMS: m/z calcd. for C13H9NO2: 211.0633,
[M+H]+ found: 212.0712.
2-Chloro-3-[methyl(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (14): red solid,
decomposition at 62 °C. Prepared from 4 with 11 by general synthetic procedure B in 53%
1
yield. H NMR (500 MHz, DMSO-d6): δ 7.99–7.96 (m, 2H), 7.84–7.79 (m, 2H), 4.27 (d, J = 2.5
Hz, 2H), 3.35 (t, J = 2.5 Hz, 1H), 3.18 (bs, 3H). 13C NMR (126 MHz, DMSO-d6): δ 181.5, 177.9,
151.1, 134.6, 134.0, 131.8, 131.3, 127.2, 126.2, 122.5, 80.4, 76.0, 44.6, 41.5. HRMS: m/z
calcd. for C14H10ClNO2: 259.0400, [M+H]+ found: 260.0461.
2-Chloro-3-[(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (15): red solid,
decomposition at 104 °C. Prepared from 4 with 10 by general synthetic procedure B in 74%
yield. The NMR spectra were in good agreement with previously published data.25 1H NMR
(500 MHz, DMSO-d6): δ 7.99–7.97 (m, 2H), 7.84 (td, J = 7.6, 1.5 Hz, 2H), 7.77 (td, J = 7.6, 1.4

Hz, 1H), 7.69 (t, J = 6.7 Hz, 1H), 4.47 (dd, J = 6.8, 2.5 Hz, 2H), 3.27 (t, J = 2.4 Hz, 1H). 13C NMR
(126 MHz, DMSO-d6): δ 180.4, 176.2, 145.3, 135.4, 133.4, 132.1, 130.4, 127.0, 126.3, 81.8,
75.0, 34.2. HRMS: m/z calcd. for C13H8ClNO2: 245.0244, [M+H]+ found: 246.0311.
5-Hydroxy-2[(prop-2-yn-1-yl)amino]-1,4-dihydronaphtalene-1,4-dione (16). Prepared from 5
with 10 by general synthetic procedure A in 15% yield. 1H NMR (500 MHz, DMSO-d6): δ 13.43
(s, OH), 8.20 (t, J = 5.94 Hz, 1H), 7.60 (dd, J = 8.3, 7.5 Hz, 1H), 7.52 (dd, J = 7.5, 1.2 Hz, 1H),
7.29 (dd, J = 8.3, 1.2 Hz, 1H), 5.74 (s, 1H), 4.07 (dd, J = 6.0, 2.5 Hz, 1H), 3.29 (t, J = 2.5 Hz). 13C
NMR (126 MHz, DMSO-d6): δ 188.3, 180.6, 160.2, 149.1, 134.5, 130.5, 125.2, 118.7, 114.2,
100.1, 78.6, 74.9, 31.3.
8-Ethoxy-2-[(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (17): dark brown
solid, decomposition at 114 °C. Prepared from 7 with 10 by general synthetic procedure A in
1
79% yield. H NMR (500 MHz, DMSO-d6): δ 7.74–7.70 (m, 2H), 7.56 (dt, J = 7.6, 1.1 Hz, 1H),
7.38 (dt, J = 8.5, 1.0 Hz, 1H), 5.67 (d, J = 0.8 Hz, 1H), 4.18 (q, J = 6.9 Hz, 2H), 4.00 (dd, J = 6.1,
2.5 Hz, 2H), 3.24 (td, J = 2.4, 0.9 Hz, 1H), 1.39 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-
d6): δ 181.4, 179.4, 159.1, 149.1, 136.1, 135.3, 118.0, 117.9, 99.8, 79.2, 74.6, 64.8, 31.3, 14.7.
HRMS: m/z calcd. for C15H13NO2: 255.0895, [M+H]+ found: 256.0976.
8-(Benzyloxy)-2-[(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (18): dark brown
solid, decomposition at 99 °C. Prepared from 8 with 10 by general synthetic procedure A in
1
87% yield. H NMR (500 MHz, DMSO-d6): δ 7.81 (t, J = 6.0 Hz, 1H), 7.78–7.75 (m, 1H), 7.62–
7.59 (m, 3H), 7.52–7.50 (m, 1H), 7.43–7.41 (m, 2H), 7.35–7.31 (m, 1H), 5.70 (d, J = 0.9 Hz,
1H), 5.31 (s, 2H), 4.01 (dd, J = 6.1, 2.4 Hz, 1H), 3.24 (td, J = 2.4, 0.8 Hz, 1H). 13C NMR (126
MHz, DMSO-d6): δ 181.6, 179.9, 159.0, 149.3, 137.1, 136.5, 135.6, 128.8, 128.1, 127.3, 118.6,
100.2, 79.4, 74.9, 70.5, 31.6. HRMS: m/z calcd. for C20H15NO3: 317.1052, [M+H]+ found:
318.1127.
8-Methoxy-2-[(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (19): red solid,
decomposition at 138 °C. Prepared from 9 with 10 by general synthetic procedure A in 43%
1
yield. H NMR (500 MHz, DMSO-d6): δ 7.75 (ddd, J = 8.5, 7.5, 0.9 Hz, 1H), 7.63 (t, J = 6.0 Hz,
1H), 7.57 (dd, J = 7.5, 1.1 Hz, 1H), 7.41 (dd, J = 8.5, 1.2 Hz, 1H), 5.68 (s, 1H), 4.01 (dd, J = 6.1,
2.5 Hz, 2H), 3.91 (s, 3H), 3.24 (m, 1H). 13C NMR (126 MHz, DMSO-d6): δ 181.3, 179.4, 159.8,

149.0, 136.2, 135.3, 118.0, 117.9, 117.2, 99.8, 79.2, 74.7, 56.5, 31.3. HRMS: m/z calcd. for
C14H11NO3: 241.0739, [M+H]+ found: 242.0813.
5,8-Dihydroxy-2-[methyl(prop-2-yn-1-yl)amino]-1,4-dihydronaphthalene-1,4-dione (21): dark
red solid, decomposition at 106 °C. Prepared from 6 with 10 by general synthetic procedure
1
A in 52% yield. H NMR (500 MHz, DMSO-d6): δ 13.24 (s, 1H), 11.73 (s, 1H), 7.29 (d, J = 9.3
Hz, 1H), 7.20 (d, J = 9.3 Hz, 1H), 5.91 (s, 1H), 4.45 (d, J = 2.5 Hz, 2H), 3.38–3.37 (m, 1H), 3.13
(s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 186.5, 185.2, 156.4, 154.9, 152.5, 129.7, 126.9,
113.1, 111.1, 108.4, 79.0, 76.0, 43.5, 40.5. HRMS: m/z calcd. for C13H9NO4: 257.0688, [M+H]+
found: 258.0763.
4.2. Inhibition of Aβ aggregation
Preparation of samples: 1 mg of Aβ1-42 (HFIP-treated, BACHEM) was dissolved in DMSO to
obtain a stable stock solution, aliquoted and stored at -20 °C. For the assay, Aβ1-42 stock
solution was then diluted to a final 50 µM concentration with 10 mM phosphate buffer (PB)
pH = 7.4 containing 150 mM NaCl by brief sonication and vortexing. 1 mg of ThT was
dissolved in methanol to obtain a stock solution which was subsequently diluted in 50 mM
glycine-NaOH (pH = 8.6) to 0.4 mM. The assay mixture contains 20 µM ThT. Stock solution of
inhibitors was prepared in DMSO.
Aβ1-42 inhibition assay: Aβ1-42 self-aggregation was performed by incubating of 50 µM Aβ1-42
solution at 30 °C in the assay conditions without any stirring in black, clear bottom 96-well
plate (Greiner) by a multi plate reader (Synergy HT, Biotek, Winooski, Vermont, United
States) using ThT fluorimetric assay. The final volume of assay mixture was 100 µL. The
excitation and emission wavelengths were set at 440/30 and 485/20 nm, respectively.
Inhibition experiments were monitored by incubating Aβ1-42 at the given conditions in the
presence of 50 µM 12-15, 17-19 and 21. As a positive control 50 µM myricetin and 50 µM
doxycycline were used. Fluorescence data were recorded every 10 min during 72 h
incubation time without any stirring. Each inhibitor was assayed in duplicates in at least
three independent experiments and the presented values were averaged and are expressed
as the mean ± SEM . Firstly, the ratio was calculated after subtraction of fluorescence of
unbound ThT according to equation 1:
ꢀ
R= ꢀ
(equation 1),
ꢁꢂꢃꢄꢅꢃꢆꢇ ꢂꢃꢈ
ꢀ
ꢂꢃꢈ

where Flag and Fplateau are fluorescence intensities of Aβ1-42 in lag and plateau phase of
aggregation kinetic at five time points every 30 min, respectively. After that, the percent
inhibition of Aβ1-42 aggregation was calculated as follow (equation 2):
ꢏ
ꢐ
% ꢉꢊℎꢉꢋꢉꢌꢉꢍꢊ = 100 − ꢎꢏ ꢒ × 100 (equation 2),
ꢑ
where RI and R0 are ratios according to equation 1 with or without studied compounds,
respectively.
4.3. Determination of MAOs inhibition activity
The reaction mixture contains 2.5 μg/mL MAO-A or 6.25 μg/mL MAO-B enzyme (Merck,
Germany) and inhibitor in final concentrations of 1, 5, 8, 10, 15, 30, 50, and 80 μM in 50 mM
potassium phosphate buffer with 20% (v/v) glycerol (pH 7.5). The mixture was pre-incubated
at 37 °C for 5 min and subsequently, substrate kynuramine was added to the final
concentration of 60 μM in the case of MAO-A and 30 μM in the case of MAO-B. The final
volume of the reaction mixture was 0.1 mL. The whole reaction mixture was incubated at 37
°C for 30 min. The reaction was stopped by the addition of 200 μL acetonitrile:methanol
mixture (1:1) and cooling down to 0 °C. The sample was then centrifuged (16.500 g) for 10
min.
The deamination product of kynuramine formed during the enzymatic reaction 4-
hydroxyquinoline (4-HQ) was determined by UHPLC–MS on a Zorbax RRHD Eclipse plus C18
column (2.1 mm x 50 mm, 1.8 µm) (Agilent Technologies, USA), by using a 6470 Series Triple
Quadrupole mass spectrometer (Agilent Technologies, USA) as detector with electrospray
ionisation - positive ion mode. Three m/z MRM transitions were followed for kynuramine
(165.1=>30.2, 165.1=>118.0, 165.1=>136.0) and 4-HQ (146.1=>51.1, 146.1=>77.0,
146.1=>91.0). Eluents: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile. IC50
of individual compounds were determined by nonlinear regression using GraphPad Prism 8.0
(GraphPad Software, USA).
Determination of time-dependent enzyme inhibition: The time-dependent binding rates of
compounds 13 and 15 were analyzed on MAO-B enzyme. The enzyme-compound mixtures
were preincubated for different time periods (0, 1, 5, 15, 30, 45, and 60 min) to achieve

enzyme inhibitions ≥ 50 %. The levels of enzyme inhibitions were determined as described
previously.
Inhibition mechanism: The reversibility of MAO-B inhibition was determined by 30 minutes
incubation of the enzyme with a high concentration of tested compounds (at least four times
higher than IC50 value where possible). It was followed by overnight dialysis at 4°C of the
enzyme-inhibitor complex against 25 mM sodium phosphate buffer (pH 7). Subsequently,
the enzyme catalytic activity recovery was determined. The remaining enzymatic activity was
calculated as a percent of non-inhibited control reactions after dialysis and the data of the
residual enzyme activity are shown as means and standard errors from two independent
experiments. Compounds 13 and 15 were further analyzed regarding its inhibition kinetics
parameters (inhibition mechanism). Thus activity assay (previously described) was carried
out at various concentrations of substrate kynuramine (ranging from 5 μM to 500 μM) and
various concentrations of the tested compounds. The inhibition mechanism was determined
by non-linear regression using GraphPad Prism 8.
4.4. Determination of chelation properties
The DMSO was of the highest purity, dry and was obtained from Merck (Germany). ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and copper chloride were obtained
from Sigma-Aldrich (Germany). ABTS was measured using a UV-vis-NIR spectrometer
Shimadzu (Shimadzu, Kyoto, Japan). EPR spectra of studied compounds in the presence of
Cu(II) ions in DMSO were studied on a spectrometer Bruker EMX at X-band (cca 9.4 GHz) at
77 K using quartz cylindrical tubes.
ABTS assay is based on the monitoring of the decay of absorption band at 734nm originating
from radical cation of ABTS (ABTS•+) in the presence of studied compounds alone or their
copper complexes (1:1). The solution of ABTS•+ was prepared by dissolving 17.2 mg of ABTS
and 3.3 mg of K2S2O8 in 5 mL of deionized water. The prepared solution of ABTS radical
cation was kept at ambient temperature for 24 hours in order to fully oxidize the ABTS
compound. A stock solution of radical cation of ABTS was prepared by diluting 1 mL of ABTS
radical cation solution with 60 mL of deionized water. Studied compounds and their Cu(II)
complexes were dissolved in DMSO (final concentration 1 mM). The decay of ABTS•+ in the
presence of studied compounds/their copper complexes was recorded at a wavelength of
734 nm for 15 min.

4.5. Determination of in vitro BBB permeation
PAMPA assay is a high-throughput screening tool applied to predict the passive transport of
potential drugs across the BBB.37,43 It has been used as the non-cell-based in vitro assay
carried out in a coated 96-well membrane filter. The filter membrane of the donor plate was
coated with polar brain lipids (PBL, Avanti, USA) in dodecane (4 µL of 20 mg/mL PBL) and the
acceptor well was filled with 300 µL of PBS buffer (phosphate-buffered saline, pH 7.4; VA).
Tested compounds were dissolved first in DMSO and then diluted with PBS pH 7.4 to reach
the final concentration 30 - 50 µM in the donor well. The concentration of DMSO did not
exceed 0.5% (V/V) in the donor solution. 300 µL of the donor solution (VD) was added to the
donor wells and the donor filter plate was carefully put on the acceptor plate so that coated
membrane was "in touch" with both donor solution and acceptor buffer. Test compound
diffused from the donor well through the polar brain lipid membrane (Area = 0.28 cm2) to
the acceptor well. The concentration of the tested compound in both donor and the
acceptor wells was assessed after 3, 4, 5, and 6 hours of incubation respectively in
quadruplicate using the UV plate reader Synergy HT (Biotek, USA) at the maximum
absorption wavelength of each compound. There were also prepared solutions at the
theoretical equilibrium of given compound (i.e. theoretical concentration if the donor and
acceptor compartment were simply combined). The concentration of the compounds in the
donor and acceptor well and equilibrium concentration were calculated from the standard
curve and expressed as the permeability (Pe) according to the equation:
ꢘ
ꢝ
ꢃꢞꢞꢅꢁꢄꢟꢠ
ꢙꢚꢛꢜ
ꢓꢔ = ꢕ × −ꢖꢊ ꢗ1 − ꢘ
ꢥ,
ꢝ
ꢙꢚꢛꢜ
ꢅꢡꢆꢢꢂꢢꢣꢠꢢꢆꢤ
where
ꢦ
ꢫ
×ꢦ
ꢨ
ꢧ
ꢕ = ꢎꢩ
×ꢬꢚꢭꢮ×ꢯꢰꢱꢭꢒ.
ꢦ
ꢪꢦ
ꢧ
ꢨ
4.6. Evaluation of cytotoxicity by MTT assay and determination of MTC
Cytotoxicity of tested compounds was evaluated using human neuroblastoma derived cells
(SH-SY5Y, ATCC, Mannassas, VA, USA) and murine microglial cells (SIM-A9, ATCC, Manassas,
VA, USA). The SH-SY5Y cells were plated at a density of 30×103 per well and SIM-A9 cells
were plated at a density of 7.5×103 per well in a 96-well plate in Dulbecco's Modified Eagle's
Medium with Nutrient Mixture F-12 (DMEM/F-12; Biosera, Nuaille, France) with 10% FBS
(Biosera) and were left to attach overnight. The incubation was performed under condition

37 °C, 5% CO2 and 80 – 95% air humidity. Stock solutions of tested compounds were
prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and then serially diluted in two-fold
dilution scale in DMEM medium. The final concentration of DMSO was less than 0.25%.
The cell viability was detected using (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT; Sigma – Aldrich) assay44 after 24 h of incubation with compounds.
Subsequently, the medium was aspirated and 100 µL MTT solution (0.5 mg/mL) in serum-
and fenol red-free DMEM medium was added to each well. Cells were then incubated for 1
hour. The medium was then discarded and purple crystals of MTT formazan were dissolved
in 100 μl DMSO under shaking. The absorbance was measured with Multi-Mode Reader
Microplate Synergy 2 reader BioTek Instruments at a test wavelength of 570 nm.
The safety of MTC for SIM-A9 cells was also evaluated under inflammatory conditions. For
that, the cells were plated into 96-well plates at the density of 7.5 × 103 cells per well and
allowed to adhere in the CO2 incubator at 37 °C overnight. The cells were treated either with
DMSO (untreated cells and positive control treated solely with LPS; Sigma-Aldrich),
dexamethasone (1 µg/mL; negative control; Sigma-Aldrich) or tested compounds at 1, 0.5,
and 0.25 MTC. To simulate inflammatory conditions, LPS was added into the media at the
final concentration of 1 µg/mL 1 h after the treatment. MTT assay was evaluated 24 h after
the application of tested compounds.
The safety of MTC under inflammatory conditions was further confirmed using
microcapillary flow cytometry (Muse™ Cell Analyzer, Merck Millipore, Hayward, CA) which
determined the cell death. This technique allows quantitative analysis of non-apoptotic,
early apoptotic, late apoptotic and necrotic cells. The Muse™ Annexin V & Dead Cell Assay
(Merck Millipore) is based on the detection of phosphatidylserine on the surface of
apoptotic cells. The reagent contains also the membrane non-permeable dye 7-
aminoactinomycin D, which is utilized to distinguish necrotic cells from early apoptotic or
live cells. The measurement of cell death was performed after 24-h of incubation with tested
compounds. The results were obtained from three independent experiments.
The IC50 values were calculated using four parametric nonlinear regressions with statistic
software GraphPad Prism (GraphPad Software, Inc., San Diego, USA). Data were obtained
from three independent experiments performed in triplicates. IC50 values were expressed as
mean ± SD.

4.7. Evaluation of anti-inflammatory action
SIM-A9 cells were plated at the density of 7.5×103 per well in 96-well plates and were left to
attach overnight. Then, the cells were treated with either DMSO (untreated cells or positive
control treated solely with LPS), dexamethasone (negative control; Sigma-Aldrich) or tested
compounds at 1, 0.5, and 0.25 MTC. After 1 h incubation, LPS was added into the media to
the final concentration of 1 µg/mL. Subsequently, 23 h later, the anti-inflammatory action of
tested compounds was evaluated using anti-mouse TNF-α (cat. No 88-7324-8888; Thermo
Fischer Scientific, Waltham, MA, USA) and anti-mouse IL-6 (cat. No ab47596; Abcam,
Cambridge, United Kingdom) ELISA kits according to the manufactures' instructions. The
absorbance was measured with Multi-Mode Reader Microplate Synergy 2 reader at a test
wavelength of 450 nm. Data were statistically evaluated using one-way ANOVA (GraphPad
Software, Inc.). The results were obtained from four independent experiments performed in
hexaplicates. Values were expressed as % of positive control ± SD.
4.8. Toxicity testing of 18 in SH-SY5Y cells
The SH-SY5Y human neuroblastoma cells were cultivated in Dulbecco's Modified Eagle's
Medium/Nutrient Mixture F-12 (with/without phenol red). Fetal bovine serum, nonessential
amino acids, glutamine, penicillin, streptomycin and all other chemicals, if not otherwise
specified, were purchased from Invitrogen-Gibco (USA). The SH-SY5Y cell line was purchased
from the American Type Culture Collection (ATCC, Manassas, VA, USA) and all experiments
were conducted using SH-SY5Y cells (passages 14.–18.). The cells were Mycoplasma-free and
the STR authentication confirmed the origin of the SH-SY5Y cells. The cells were seeded into
96-well plates at the density of 2.5×104 cells/well for 24 h. After exchange of the culture
medium, the cells were treated with 18 diluted in the cell culture medium for 24 h to obtain
0.1-100 μM 18. Then, WST-1 test, glutathione assay, JC-1 assay and high resolution
respirometry were performed.
Dehydrogenase activity was evaluated by WST-1 test (Sigma-Aldrich, USA). The WST-1 test
detects the activity of intra- and extramitochondrial dehydrogenases. After the treatment,
the cells were incubated with the WST-1 reagent according to the manufacturer's
instructions for 1 h. The cells were incubated at 37 °C for 60 min and the absorbance change
(0-1 h) was measured spectrophotometrically at wavelength of 440 nm using a Tecan Spark

plate reader (Tecan, Austria). The dehydrogenase activity was expressed as the percentage
of total cellular dehydrogenases activity relative to that in control cells (control = 100%).
The glutathione (GSH) levels were measured using an optimized bimane assay.45 The
working solutions of monochlorobimane (MCB, Sigma-Aldrich, USA) were prepared fresh at
the time of analysis by dilution in Dulbecco's phosphate buffer and tempered at 37 °C. After
18 treatment, 20 μL of the MCB solution was added to the cells in 96-well plates and the
measurement started immediately (final concentration of MCB in a well was 40 µM). The
fluorescence intensity (394/490 nm) was measured kinetically for 20 min using a Tecan Spark
plate reader (Tecan, Austria). The fluorescence was expressed as the slope of a fluorescence
change over time. The GSH levels were expressed as the percentage relative to the GSH
levels in control cells (= 100%).
Mitochondrial membrane potential was measured using a JC-1 intracellular probe (Enzo,
USA). The working solution of JC-1 was prepared fresh at the time of analysis by dilution in
Dulbecco's phosphate buffer. After 18 treatment, 20 µL of the JC-1 solution was added to
cells. The final concentration of JC-1 in a well was 10 µg/mL. The SH-SY5Y cells were loaded
for 20 min and then washed with PBS. The fluorescence (red: 485/595 nm; green: 485/535
nm) was measured using a Tecan Spark fluorescence reader. The rate of mitochondrial
membrane potential was expressed as the red/green ratio (= R/G).
Oxygen consumption was measured by a high-resolution respirometer Oxygraph-2k
(Oroboros Instruments, Innsbruck, Austria) in a glass chamber at 37 °C according to the
coupling control protocol for intact cells. Each chamber contained 2 mL of MiRO5 media and
5×105 SH-SY5Y cells. Four respiratory conditions were measured: ROUTINE = basal
respiration of intact cells, LEAK = state after enzymatic inhibition of the phosphorylation
system, ETS = maximal respiratory capacity, ROX = residual oxygen consumption. LEAK state
was gained after treatment with oligomycin (2.5 µM). ETS state was induced by an uncoupler
carbonylcyanide-m-chlorophenyl hydrazone (1 µM). Finally, mitochondrial respiration was
inhibited with antimycin A (2.5 µM). For evaluation of oxygen consumption, the Oroboros
software DatLab 7.4 was used.
All cellular experiments were repeated at least three times independently. The results are
expressed as mean ± SD. The differences among groups of samples were after normality
testing analyzed using one-way analysis of variance (ANOVA; p = 0.05) followed by
Bonferroni test (OriginPro 9.0.0, USA; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Supplementary Information: Supplementary data consists of NMR spectra of intermediates
and final compounds, as well as LC-MS and LC-UV spectra, the summary of anti-
inflammatory effects, anti-MAOs activity screening and antioxidant efficiencies of tested
compounds.
Funding: This work was supported by a grant of the Ministry of Defence "Long Term
Development Plan" Medical Aspects of Weapons of Mass Destruction of the Faculty of
Military Health Sciences, University of Defence; by the Ministry of Education, Youth and
Sports of Czech Republic (project ERDF IT4N no. CZ.02.1.01/0.0/0.0/18_069/0010054), by EU
COST Action (CA15135: "Multi-target paradigm for innovative ligand identification in the
drug discovery process (MuTaLig)"), and by MH CZ - DRO (University Hospital Hradec
Kralove, No. 00179906). We also thank the Scientific Grant Agency (VEGA Project 1/0482/20)
and Research and Development Support Agency (APVV-15-0079 and APVV-19-0087). Marvin
was used for drawing, displaying, and characterizing chemical structures, substructures and
reactions, Marvin 20.15.0, ChemAxon (https://www.chemaxon.com).
Author Contributions: Organic synthesis; E.M.; NMR measurement; NMR spectra
interpretation, E.M.; measurement of anti-Aβ aggregation activity, J.J.; BBB permeation
prediction, O.S., T.K.; in vitro measurement of anti-MAOs activity, R.A.; cytotoxicity
screening, L.M., J.H., J.C., T.R.; high resolution respirometry, J.M., T.R.; measurement of anti-
inflammation potency, L.M., J.P.; determination of metal-chelating and scavenging
properties, M.S., M.V.; writing-original draft preparation, E.M., J.J., M.V.; editing, J.K., E.M.;
supervision, J.K., J.L.M.-C. All authors have read and agreed to the published version of the
manuscript.
Conflict of interest: The authors declare no conflict of interest. The funder had no role in the
design of the study, in the collection, analyses, or interpretation of data, in the writing of the
manuscript, or in the decision to publish the results.
Abbreviations: 4-HQ – 4-hydroxyquinoline; ABTS – 2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid); AD - Alzheimer's disease; APP – amyloid precursor protein; Aβ – amyloid-beta;
BBB – blood-brain barrier; CNS – central nervous system; DMSO – dimethyl sulfoxide; EA –
ethyl acetate; EPR – electron paramagnetic resonance; ETS – maximal respiratory capacity;
GSH – glutathione; IL-6 – interleukin-6; LC-MS – liquid chromatography-mass spectrometry;
LC-UV – liquid chromatography with ultraviolet detection; LEAK – state after enzymatic

inhibition of the phosphorylation system; LPS – lipopolysaccharide; MAO - monoamine
oxidase; MCB – monochlorobimame; MS – mass spectra; MTC – maximally tolerated
concentration; MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MRM –
multiple reaction monitoring; N-(M)PNQ – N-(methyl)propargyl-naphthoquinone; NMR –
nuclear magnetic resonance; N-PNQ – N-propargyl-naphthoquinone; PAMPA – parallel
artificial membrane permeability assay; PBS – phosphate-buffered saline; PE – petroleum
ether; ROS – reactive oxygen species; ROUTINE – basal respiration of intact cells; ROX –
residual oxygen consumption; SAR – structure-activity relationship; SH-SY5Y – human
neuroblastoma cell line; SIM-A9 – murine microglial cell line; ThT – thioflavin T; TLC – thin
layer chromatography; TNFα – tumor necrosis factor-alpha; UHPLC – ultra-high performance
liquid chromatography.
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