Synthesis and Biological Evaluation of Dantrolene-Like Hydrazide and Hydrazone Analogues as Multitarget Agents for Neurodegenerative Diseases

Article abstract of DOI:10.1002/cmdc.202100209

Dantrolene, a drug used for the management of malignant hyperthermia, had been recently evaluated for prospective repurposing as multitarget agent for neurodegenerative syndromes, including Alzheimer's disease (AD). Herein, twenty-one dantrolene-like hydrazide and hydrazone analogues were synthesized with the aim of exploring structure-activity relationships (SARs) for the inhibition of human monoamine oxidases (MAOs) and acetylcholinesterase (AChE), two well-established target enzymes for anti-AD drugs. With few exceptions, the newly synthesized compounds exhibited selectivity toward MAO B over either MAO A or AChE, with the secondary aldimine 9 and phenylhydrazone 20 attaining IC₅₀ values of 0.68 and 0.81 μM, respectively. While no general SAR trend was observed with lipophilicity descriptors, a molecular simplification strategy allowed the main pharmacophore features to be identified, which are responsible for the inhibitory activity toward MAO B. Finally, further in vitro investigations revealed cell protection from oxidative insult and activation of carnitine/acylcarnitine carrier as concomitant biological activities responsible for neuroprotection by hits 9 and 20 and other promising compounds in the examined series.

Full text of DOI:10.1002/cmdc.202100209

Accepted Article
Title: Synthesis and biological evaluation of dantrolene-like
hydrazide and hydrazone analogues as multitarget agents for
neurodegenerative diseases
Authors: Isabella Bolognino, Nicola Giangregorio, Annamaria Tonazzi,
Antón L. Martínez, Cosimo D. Altomare, María I. Loza, Sara
Sablone, Saverio Cellamare, and Marco Catto
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202100209
Link to VoR: https://doi.org/10.1002/cmdc.202100209
01/2020

10.1002/cmdc.202100209
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Synthesis and biological evaluation of dantrolene-like hydrazide
and hydrazone analogues as multitarget agents for
neurodegenerative diseases
Dr. Isabella Bolognino,[a],§,# Dr. Nicola Giangregorio,[b],§ Dr. Annamaria Tonazzi,[b] Dr. Antón L.
Martínez,[c] Prof. Cosimo D. Altomare,[a] Prof. María I. Loza,[c] Dr. Sara Sablone,[d] Prof. Saverio
Cellamare[a] and Prof. Marco Catto[a],*
[a]
[b]
[c]
[d]
Dr. I. Bolognino, Prof. C. D. Altomare, Prof. S. Cellamare, Prof. M. Catto
Department of Pharmacy-Pharmaceutical Sciences, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy
E-mail: marco.catto@uniba.it
Dr. N. Giangregorio, Dr. A. Tonazzi
Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), National Research Council (CNR), via Amendola 122/O, 70126 Bari,
Italy
Dr. A. L. Martínez, Prof. M. I. Loza
BioFarma Research Group, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), University of Santiago de Compostela, Av.
Barcelona, Campus Vida, 15782 Santiago de Compostela, Spain
Dr. S. Sablone
Section of Legal Medicine, Interdisciplinary Department of Medicine, Bari Policlinico Hospital, University of Bari Aldo Moro, Piazza Giulio Cesare 11, 70124
Bari, Italy
§ These authors contributed equally.
# Current address: Department of Engineering and Applied Sciences, University of Bergamo, Viale G. Marconi 5, 24044 Dalmine (BG), Italy
Supporting information for this article is given via a link at the end of the document.
Abstract: Dantrolene, a drug used for the management of malignant
hyperthermia, had been recently evaluated for prospective
repurposing as multitarget agent for neurodegenerative syndromes,
including Alzheimer’s disease (AD). Herein, twenty-one dantrolene-
like hydrazide and hydrazone analogues were synthesized with the
aim of exploring structure-activity relationships (SARs) for the
inhibition of human monoamine oxidases (MAOs) and
acetylcholinesterase (AChE), two well-established target enzymes
for anti-AD drugs. With few exceptions, the newly synthesized
compounds exhibited selectivity toward MAO B over either MAO A
or AChE, with the secondary aldimine 9 and phenylhydrazone 20
attaining IC50 values of 0.68 and 0.81 M, respectively. While no
general SAR trend was observed with lipophilicity descriptors, a
molecular simplification strategy allowed the main pharmacophore
features to be identified, which are responsible for the inhibitory
activity toward MAO B. Finally, further in vitro investigations revealed
cell protection from oxidative insult and activation of
carnitine/acylcarnitine carrier as concomitant biological activities
responsible for neuroprotection by hits 9 and 20 and other promising
compounds in the examined series.
modulating the concentration of neurotransmitters, mostly in
some severe and chronic neurodegenerative pathologies. This
established reputation is strictly related to the substrate and
tissue specificity of both isoforms: MAO A selective inhibitors are
clinically administered as antidepressants,[2] while MAO
B
selective inhibition is commonly used for the treatment of the
early symptoms of Parkinson’s disease.[3] A new outcome of that
study was the discovery of the activation by DAN of the
carnitine/acylcarnitine carrier (CAC), with EC50 of 9.3 μM for the
purified recombinant wild type (WT) protein. This transporter
acts through reductive activation and is involved in trafficking of
acyl groups into the mitochondria, carried by L-carnitine. Treated
with DAN, this transport system facilitates, under oxidative
stress (OS) conditions, the restoring of ATP production and thus
cell vitality, but also the export of endogenous acetyl-L-carnitine
from mitochondria, with consequent neuroprotective effects.
Introduction
Dantrolene (DAN; Figure 1) is a drug specifically used in the
management of malignant hyperthermia,
a life-threatening
pathology with fatal course. In a recent work, we disclosed new
biological activities exerted by DAN, namely inhibition of
monoamine oxidase (MAO) B human enzyme with Ki value in
the low micromolar range, acetylcholinesterase (AChE), and
aggregation of beta amyloid-40 and hexapeptide tau protein
sequence PHF6, i.e. two probes of amyloid aggregation in
Alzheimer’s disease (AD) brain.[1] It is well known the crucial role
Figure 1. General strategy for the synthesis of hydrazide/hydrazone
derivatives.
of MAO isoforms
A and B as metabolizing enzymes in
1
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Scheme 1. Reagents and conditions: (i) HCl, 0 °C, NaNO2, rt, 30 min; (ii) 2-
furaldehyde, CuCl2, acetone, rt; (iii) amine or hydrazine or hydrazide,
DMF/water, HCl (cat.), rt, 24 h.
The above promising results prompted us to synthesize a
number of novel DAN analogues, with the aim of optimizing the
inhibitory activity against MAO B and AChE, ultimately improving
their pleiotropic pharmacological potential in the treatment of AD
and related neurodegenerative syndromes. In this work we
investigated in particular: i) the bioisosteric replacement of the
NO2 group, which may be toxicophore and precursor for the
production of reactive oxygen species (ROS), with CN group; ii)
the improvement of the aqueous solubility of DAN by replacing
the hydantoin moiety with other (hetero)cyclic moieties bearing
protonatable nitrogen(s); iii) the effect of molecular simplification
of the three-ring scaffold in DAN, for detecting the minimal
pharmacophoric features responsible for the dual activity on
MAO A/B and CAC (Figure 1). SARs were investigated as
thoroughly as possible, even considering the effect of
lipophilicity on the enzymes’ inhibition potency.
Scheme 2. Reagents and conditions: (i) HCl, 0 °C, NaNO2, rt, 30 min; (ii) 3-
methoxybenzaldehyde, CuCl2, acetone, rt; (iii) 2-thiophenecarboxylic acid
hydrazide, DMF/water, HCl (cat.), rt, 24 h.
Some of the prepared compounds retained the molecular motif
of hydantoin, present in DAN and, as thiohydantoin, in the
known hypoglycemic drugs rosiglitazone and pioglitazone
(Figure 2), which also behave as moderate MAO inhibitors. In
turn, the molecular pruning gave simple hydrazone and
hydrazide derivatives (2-rings series, compounds 14-21) whose
structural pattern could be related to that of isocarboxazide
(Figure 2), an early irreversible MAO inhibitor.
Scheme 3. Reagents and conditions: (i) hydrazine or hydrazide,
acetone/water, HCl (cat.), rt, 24 h.
Inhibition of MAOs and AChE
The results of in vitro inhibition tests on human MAOs and AChE
of the newly synthesized DAN analogues are summarized in
Tables 1 and 2, along with the inhibition data of pargyline and
galantamine used as positive controls for MAO (B-selective) and
AChE, respectively. Inhibition assays were also performed on
human butyrylcholinesterase (BChE), where all compounds
resulted inactive or poorly active (data not shown). IC50 values
were calculated for compounds displaying >60% inhibition in
one-point (10 M) concentration assay.
Figure 2. MAO inhibitors structurally related to dantrolene.
Compared with previously obtained results of DAN,[1] its CN
congener 7 showed a significant loss of inhibition potency
toward all three enzymes (approximately a four-fold decrease for
MAO B). On the contrary, the thiophene hydrazides 1 and 4
proved to be equipotent with DAN for MAO B inhibition, with a
noteworthy increase of MAO A activity and loss of MAO B/A
selectivity for the CN congener 4. The same modification in
compounds 2 and 5 resulted in almost overlapping activities,
even with a decrease of MAO B potency.
Compound 3, which along with 1 and 2 maintains the 4-
nitrophenyl substituent, was prepared with the aim of introducing
a strong basic moiety likely able to improve the affinity to
cholinesterases. Unfortunately, it resulted a poor AChE inhibitor,
while retaining fair MAO inhibition, although unselective, in the
low micromolar range. The same activity profile was featured by
the hydrazide 6 and 8, where the structural simplification of the
hydantoin ring of the close analogue 7 to acethydrazide (6) and
phenylacethydrazide (8) allowed the restoration of MAO
inhibitory activity.
Results and Discussion
Chemistry
The synthetic pathways chosen for the preparation of the
designed compounds are those explored by Snyder and
coworkers with slight modifications.[4] The chemical scaffolds
were selected to investigate a range of molecular diversity
around the structure of DAN, in terms of variation of stereo-
electronic and hydrophobic properties. The general strategy of
structural modifications is shown in Figure 1, whereas the
syntheses of compounds are shown in Schemes 1-3.
Condensations from suitable aldehydes and amino/hydrazide
derivatives were performed in DMF/water or acetone/water
mixtures, with acidic catalysis and agitation at room temperature.
Final compounds were obtained in moderate (20-50%) to good
(70%) yields, following a simple workup including filtration and
purification
through
either
crystallization
or
column
The introduction of terminal moieties with larger structural
diversity, as in case of compounds 9-13, produced contrasting
results. Concerning MAO B inhibition, we obtained an interesting
submicromolar value of IC50 for phenol derivative 9, which
turned out as the most potent MAO B inhibitor of this series, also
displaying 5-fold selectivity over MAO A.
chromatography.
2
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Figure 3. Michaelis-Menten plot (A) and Lineweaver-Burk linearization (B) of
inhibition kinetics of hMAO B with compound 9. Image is representative of a
single experiment.
Inhibition kinetics assessed for 9 a competitive mechanism, with
Ki equal to 0.50±0.06 µM (Figure 3). Taking into account the
possible hydrolytic degradation of imine (see stability studies
below), inhibition kinetics were determined without preincubation
with substrate, as usually done for MAO inhibition experiments.
Table 1. Inhibition of MAOs and AChE (compounds 1-13).
IC50 (µM) or % inhibition at 10 µM
log k’[b]
Entry
R
R’
Clog P[a]
hMAO A
hMAO B
hAChE
DAN
7
NO2
CN
14.0±1.0c
2.69±0.44c
4.19±0.73c
1.63
1.32
0.115
-
40% ±5
50% ±3
28%±4
1
4
2
5
NO2
CN
33%±2
1.46±0.21
7.89±1.1
6.62±0.71
3.67±0.92
2.63±0.42
3.56±0.36
12.6±0.9
40%±4
51%±3
30%±1
27%±2
4.18
3.12
2.63
2.32
0.947
-
NO2
CN
0.522
0.312
3
6
NO2
CN
5.61±1.11
4.25±0.45
3.15±0.03
2.65±0.11
44%±2
38%±4
2.58
1.88
0.752
0.236
8
CN
5.51±0.54
3.61±0.04
16%±1
3.43
0.803
9
CN
CN
3.46±0.25
0.68±0.05
30%±0.4
55±1
3.95
4.14
0.782
-
10
47%±4
29%±4
3
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11
12
CN
CN
11%±4
28%±3
39%±4
3.15
2.58
-
10.0±1.9
28%±4.0
1.67±0.19
0.466
13
29%±4
10.9±0.6
-
2.32±0.43
2.69±0.48
-
35%±5
-
3.19
0.549
Pargyline
-
-
-
-
Galantamine
0.72±0.15
[a] ChemDraw software 15.0. [b] Logarithm of the capacity factor k’ (defined as (tR-t0)/t0, where tR is the retention time and t0 the dead time) measured by RP-
HPLC using MeOH/phosphate buffer pH 7.4 gradient from 80:20 to 60:40 v/v. [c] Data taken from Ref. [1].
Table 2. Inhibition of MAOs and AChE (compounds 14-21).
IC50 (µM) or % inhibition at 10 µM
log k’[b]
Entry
R
Clog P[a]
-0.21
hMAO A
hMAO B
hAChE
14
11%±3
no inhibition
9.06±0.45
-1.018
15
16
16%±4
18%±3
13%±3
13%±4
7.18±0.07
1.59
1.27
-0.010
-
21%±5
17
18
22%±5
25%±5
22%±4
21%±3
52%±4
27%±5
1.89
1.03
-
-
19
20
19%±5
12.2±1.5
11.2±1.6
1.69
0.129
42%±2
8%±2
0.81±0.04
10.2±1.6
2.09
0.307
-
21
18%±5
28%±4
-0.08
[a], [b] see Footnotes of table 1.
The derivatization of furaldehyde bridge as 4-substituted
arylhydrazones (compounds 10-12) resulted in a decrease of
activity towards MAO isoforms. Nevertheless, sulfonamide
derivative 12 scored an unexpectedly remarkable inhibitory
activity towards AChE with an IC50 value of 1.67 µM.
While compound 4 was the most active inhibitor of MAO A (IC50
1.46 µM), the replacement of the central furan ring with a larger
and sterically hindered ring such as methoxybenzene in 13,
resulted in a strong decrease of this inhibitory activity, while
retaining a similar low value of IC50 for MAO B. This potency
reversal represents an interesting example of isoform selectivity,
but seems also contrasting with previous results,[5] assessing a
preferential MAO A affinity for molecules bearing bulky bridging
substituents.
A number of structurally simpler derivatives, based on two-ring
scaffolds, were also synthesized with the aim of gaining
4
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information on the minimal pharmacophore features[6] of the
compounds under examination. The effects of such a molecular
pruning on target interactions are shown in Table 2. Removal of
nitrophenyl group (compare 14 vs. DAN and 7, 15 vs. 1 and 4,
16 vs. 3, 17 vs. 8, 18 vs. 12) resulted in a marked loss of activity
towards MAOs, while only in few cases AChE inhibition was
sparingly retained, particularly in the case of isonicotinic acid
derivative 19, analogue of 2 and 5. It is worthy of note that
sulfonamide 18 lost the appreciable AChE inhibitory activity of its
analogue 12, likely because of the lack of favorable
hydrophobic/aromatic interactions by the nitrophenyl moiety in
the enzyme binding site. This hypothesis is strengthened by the
result of compound 20, which can be considered as a superior
homologue of 18. The increase of lipophilicity, along with the
introduction of an electron-donating substituent, through the
replacement of furan with the methoxybenzene ring determined
not only the restoring of the activity towards hAChE but also, in
line with expectations, the restoring of the affinity towards MAO
targets. Particularly in MAO B inhibition (IC50 = 0.81 µM),
sulfonamide 20 resulted as a potent and B/A selective candidate
within the entire molecular set herein presented.
Figure 4. Plots of pIC50 values determined toward MAO A (A) and B (B) versus
calculated Clog P values (ChemDraw); only compounds with finite IC50 values
are shown.
While the graphical analysis proved the absence of a general
correlation trend, the experimental log k’ values (Tables 1 and 2),
albeit limited to just over half of the compounds studied, suggest
a certain effect of lipophilicity on the inhibition potency. Indeed,
within the physicochemical property space explored, the
strongest MAO A/B inhibitors possess higher lipophilicity (i.e.,
with values comprised in the 0.0–1.0 range), whereas the most
active AChE inhibitors scored log k’ values close to or lower than
0.
Cell-Based Assay of Neuroprotection
In order to get further information about the potential
neuroprotective effects exerted by the most active compound of
the molecular series (phenol derivative 9) toward hMAO B, we
tested it in
a 2′,7′-dichlorofluorescein diacetate (DCF-DA)
fluorescence-based assay measuring the production of ROS
induced by dopamine in cultured SH-SY5Y cells.
Lastly the introduction of a sterically hindered ring such as 3,4,5-
The immortalized neuroblastoma cells are a widely used model
for neuroprotection assays, while dopamine at a concentration of
10 nM acts as an inducer of oxidative stress,[8],[9] being
metabolized by MAOs to form hydrogen peroxide. Once
hydrolyzed and oxidized, DCF acts as the fluorescing probe of
the oxidative stress (OS) state of cells. In the presence of an
antioxidant, or even of a MAO inhibitor, ROS burden is lowered
and DCF fluorescence decreased. Figure 5 shows that 9 was
effective in reducing ROS oxidation of DCF. Its activity is
superimposable to that of phenelzine, a nonselective MAO
inhibitor used as a positive control in this test.[10] The same
experiment performed on dantrolene in our previous work gave
similar results.[1]
trimetoxybenzene (21) caused
a loss of activity (8-28%
inhibition) towards both MAOs and AChE at the highest
concentration tested (10 M).
To investigate a possible correlation between MAO inhibitory
potency and lipophilicity,[7] we calculated log P with three
different programs (ChemDraw 15.0; ALOG PS 2.1;
ChemSketch 2017, see Table S1 in Supporting Information) and
compared the calculated values with experimental lipophilicity
indexes as assessed by reversed-phase (RP) HPLC in isocratic
conditions for the majority of compounds. The log of capacity
factors (log k’) measured by RP-HPLC using a mixture of
methanol/PBS (60:40, v/v) as the mobile phase are reported in
Tables 1 and 2, along with Clog P values calculated with
ChemDraw. These calculated lipophilicity descriptors correlated
with the experimental log k’ values (n = 13, r2 = 0.901) better
than the other log Ps calculated by ALOG PS and ChemSketch
computational tools (r2 equal 0.705 and 0.568, respectively). As
shown by the scatter plots in Figure 4 and Figure S1 in
Supporting Information, there is no evident correlation trend
between MAO A/B inhibition data and lipophilicity calculated by
the expert system implemented in ChemDraw for compounds
achieving finite IC50 values.
Figure 5. Neuroprotection of SH-SY5Y cells from oxidative insult; DCF-DA assay.
Green line, control cells; purple line, 10 nM dopamine; blue line, 10 nM dopamine +
10 nM phenelzine; black line, 10 nM dopamine + 10 nM 9. The data represent mean ±
SD of three independent experiments.
Hydrolytic Stability in Buffered Solution
The
hydrolytic
stability
of
some
representative
imino/hydrazone/hydrazide derivatives was determined in
buffered aqueous media. The stability studies were carried out
on compounds 3, 4 and 9, the most active MAO B inhibitors of
the 3-rings scaffold series, at a single concentration of 20 µM in
10 mM phosphate buffered saline (PBS) at physiological pH of
7.4, at 37 °C and for 6 h incubation time. The degradation
5
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profiles in Figure 6A confirmed a substantial stability for 3 and 4,
while the imine 9 resulted, at the end of the 6 h of incubation, in
a degradation percentage of 76%. This result is in line with the
nature of the functional group of this molecule: in fact, 9 is the
only Schiff base within the synthesized series and therefore by
itself less stable than the other hydrazone analogues. The
hydrolytic degradation of 9 in its starting reagents was confirmed
by the comparison of the chromatograms of 9 and 2-chloro-4-
hydroxyaniline, obtained in the same chromatographic
conditions (Figure 6B).
Figure 6. A, time-resolved stability of compounds 3, 4 and 9 at the concentration of 20 µM in PBS at 37 °C. Data are representative of three independent
experiments and values expressed as mean. B, overlapping chromatographic peaks of 9 at t 0 and t 4h and chromatogram of 2-Cl-4-OH aniline.
Activation of the Carnitine/Acylcarnitine Carrier (CAC)
Transport
activity of the oxidized protein.[1] Herein, we also investigated
whether some newly synthesized derivatives, namely the most
active MAO B inhibitors 9 and 20, the DAN homologue 14, and
the dual MAO B/AChE inhibitor 19, are effective in activating
CAC. To calculate the EC50 values from dose-response curves,
that is, the concentration which increases the transport activity of
the carrier by 50% compared to the control, a wide concentration
range (1–100 μM) was tested (Figure 7). The EC50 values
measured after 30 min of incubation were 8.2 ± 2.8 μM (comp.
9), 8.4 ± 1.6 μM (14), 8.2 ± 0.57 μM (19), 13 ± 1.8 μM (20),
whereas the whole activation of the WT protein was observed at
concentrations close to 100 μM for 8, 19 and 20, and 50 μM for
14.
CAC (SLC25A20) is essential for the transport into mitochondria
of acyl moieties as acylcarnitines, where they are processed by
β-oxidation pathway. The protein contains six cysteine residues,
but two of them, namely C136 and C155, based on the redox
state of the protein, are crucial for the regular function of the
carrier.[11],[12] In fact, the transporter is active when the two
cysteines are in reduced form, while it is inhibited when a C136-
C155 disulfide bridge is formed in conditions of OS. One or both
cysteines represent also specific targets for various chemical
and physiological thiol reducing agents,[13-17] allowing to
modulate the transport activity of the carrier. We demonstrated
that DAN led to a significant recovery of the CAC transport
Figure 7. Dose–response curves of activation of CAC (purified recombinant WT protein). The antiport rate was measured by adding 0.1 mM [3H]-carnitine to
proteoliposomes containing 15 mM internal carnitine and stopped after 30 min by the specific inhibitor N-ethylmaleimide (NEM). Compounds at increasing
concentrations were added 2 min before the transport assay. Values are mean ± SD from three independent experiments.
The tested molecules were able to improve the transport activity
of CAC compared to the control and at least three of them (9, 14
and 19) showed EC50 lower than that previously calculated for
DAN (9.3 μM) after the same exposure duration (30 min),
highlighting similar pharmacological effect. On the contrary, the
efficacy of these compounds, i.e., the power of the molecules to
achieve maximum effect, is about 5 times lower than DAN.[1]
6
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To demonstrate that the action of DAN analogues was exerted
on the cysteine residues of the CAC, 1 or 50 mM dithioerythritol
(DTE), a strong reducing agent, was added to the reconstitution
mixture (see Experimental section) in order to mimic the protein
at different states of oxidation. The bar plot in Figure 8 shows
that the tested molecules enabled the protein to recover a
significant transport activity, compared to the control, when the
protein is more oxidized, i.e., in the presence of 1 mM DTE.
transport activity after 30 min by NEM. The values are means ± SD from three
independent experiments, significantly different from the controls, as
calculated from Student's t-test analysis (*p < 0.01).
The new compounds confirmed a good stability in buffered
conditions, with the obvious exception of imine 9, and a safe
cellular activity in contrasting ROS cytotoxicity from oxidative
degradation of dopamine. Finally, some tested compounds
confirmed the activation of the mitochondrial carnitine trafficking
mediated by CAC, thus giving further evidence to the multitarget
profile of this class of molecules. The evaluation of possible
activity on ryanodine receptors will be a major concern to be
faced in the near future, in order to achieve a more complete
activity profile of these DAN analogues.
Conclusion
From our previous investigation of the multitarget activity exerted
by DAN,[1] we evidenced the potential of repurposing of this
orphan drug toward AD-related targets, but also its intrinsic
limitations, particularly its low aqueous solubility, that may
discourage further pharmacological evaluation. The DAN-like
molecules herein reported were designed and synthesized with
the aim of extending the knowledge of the SARs and improving
the pharmaceutical potential of this class of compounds. In
particular, our efforts were focused on modifying the structure of
DAN for increasing the aqueous solubility and replacing the
potentially toxicophore nitro group, as well as following an
approach of molecular simplification. The in vitro screening
proved that some of the newly investigated analogs improved
the MAO inhibitory potency, mostly for the 3-ring series
(compounds 1-13), although with low isoenzyme selectivity. The
phenol derivative 9 emerged as an outstanding, reversible MAO
B inhibitor (Ki 0.50 μM) with fair B/A selectivity. The drop of MAO
A activity of compound 13, compared to that of its close
congener 4, is a matter of evidence that would deserve further
investigations, considering the high B/A selectivity obtained with
this homologation. Among the 2-ring series, only the
sulfonamide hydrazone 20 resulted in a strong and selective
MAO B inhibitor (IC50 0.81 μM). As far as the AChE inhibition is
concerned, appreciable inhibition values were obtained only
sparsely, in line with results recently published for a related
class of DAN analogues.[18] While only sulfonamide 12 resulted
in good AChE inhibition, the best selectivity was achieved with
compounds 14 and 15 resulting from the molecular simplification
study. The limited molecular size of both 2-ring and 3-ring
scaffold hampered a significant inhibition of BChE.
Experimental Section
Chemistry
Chemicals, solvents and reagents used for the syntheses were
purchased from Sigma-Aldrich (Milan, Italy) or Alfa Aesar (Haverhill,
Massachusetts, USA) and used without any further purification. The
purity of all intermediates, checked by 1H NMR and HPLC, was always
higher than > 95%. Column chromatography was performed using Merck
silica gel 60 (0.063-0.200 mm, 70-230 mesh). All reactions were routinely
checked by TLC using Merck Kieselgel 60 F254 aluminum plates and
visualized by UV light. Nuclear magnetic resonance spectra were
recorded on a Varian Mercury 300 instrument (at 300 MHz) or on Agilent
Technologies 500 apparatus (at 500 MHz) at ambient temperature in the
specified deuterated solvent. Chemical shifts (δ) are quoted in parts per
million (ppm) and are referenced to the residual solvent peak. The
coupling constants J are given in Hertz (Hz). The following abbreviations
were used: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q
(quadruplet), qn (quintuplet), m (multiplet), br s (broad signal); signals
due to OH and NH protons were located by deuterium exchange with
D2O. High resolution mass spectrometry experiments were performed
with a dual electrospray interface (ESI) and a quadrupole time-of-flight
mass spectrometer (Q-TOF, Agilent 6530 Series Accurate-Mass
Quadrupole Time-of-Flight LC/MS, Agilent Technologies Italia S.p.A.,
Cernusco sul Naviglio, Italy). Full-scan mass spectra were recorded in
the mass/charge (m/z) range 50-3000 Da. Melting points (MP) for solid
final compounds were determined by the capillary method on a Stuart
Scientific SMP3 electrothermal apparatus and are uncorrected. RP-HPLC
analyses were performed on a system equipped with automatic injector
and a Waters Breeze 1525 pump coupled with a Waters 2489 UV
detector (Waters SpA, Sesto San Giovanni, Italy). The UV detection was
measured at λ 254 and 370 nm. Clog P values of the data set were
computed by using ChemDraw version 15.0 (PerkinElmer, Milan, Italy),
ALOGPS 2.1 (VCCLAB, Virtual Computational Chemistry Laboratory,
http://www.vcclab.org)
and
ChemSketch
2017
version
2.1
(ACD/ChemSketch, Advanced Chemistry Development, Inc., Toronto,
ON, Canada, www.acdlabs.com).
All compounds were synthesized following Snyder’s procedures[4] with
slight modifications. Compounds 2,[19] 14,[20] 15[21] and 19[22] have already
been described; their analytical data agreed with those reported in
quoted references.
Synthesis of compounds 1-12
Figure 8. Effects of DAN analogues on the recombinant WT CAC protein. The
proteoliposomes were prepared in two different reducing conditions, adding to
the reconstitution mixture 1 or 50 mM DTE. Thus, the antiport rate was
measured incubating the reconstituted protein with test molecules at 10 µM
final concentration together with 0.1 mM [3H]-carnitine and then stopping the
A suspension of 4-cyano or 4-nitroaniline (2 mmol) in 10 mL 6N HCl was
heated until the solid is solubilized, then the mixture cooled to 0 °C. A
solution of NaNO2 (0.14 g, 2 mmol) in 1 mL of water was added and the
mixture left under stirring for 30 min. In the order, solutions of 2-
furaldehyde (0.19 g, 2 mmol) in 2 mL of acetone and CuCl2 (0.04 g, 0.3
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100209
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FULL PAPER
mmol) in 1 mL of water were added and the mixture was kept under
stirring for 5 h. The precipitate formed was filtered and washed with
distilled water. This intermediate compound was then solubilized in DMF
(2 mL) and slowly added to an aqueous solution of the appropriate
amine/hydrazine/hydrazide derivative (1.8 mmol). A catalytic amount of
4N HCl was added and the solution was left under stirring at room
temperature for 24 h. The mixture was extracted with CHCl3 (3 x 15 mL),
the organic phase abundantly washed with H2O to remove the DMF and
dried with Na2SO4. The solvent was evaporated under reduced pressure
to give the crude product that was purified by column chromatography
with hexane/ethyl acetate: 6/4 or 7/3 v/v as the mobile phase.
DMSO-d6) δ 3.68 (s. 3H, OCH3), 6.76 (d, J = 4.1 Hz, 1H, furan), 6.84 (d,
J = 9.9 Hz, 2H, arom.), 7.00 (d, J = 9.9 Hz, 2H, arom.), 7.30 (d, J =3.5 Hz,
1H, furan), 7.72 (s, 1H, aldimine) 7.84-7.89 (m, 4H, arom.), 10.36 (brs,
1H, NH). ESI-MS (C19H14N3O2, [M–H]-) calcd. m/z = 316.1083, found
316.1099. MP 138-140 °C.
4-(5-((2-(4-(methylsulfonyl)phenyl)hydrazono)methyl)furan-2-
yl)benzonitrile (11). Dark-orange crystals; yield: 25%; 1H NMR (300
MHz, DMSO-d6) δ 3.10 (s. 3H, CH3), 6.95 (d, J = 4.1 Hz, 1H, furan), 7.19
(d, J = 8.7 Hz, 2H, arom.), 7.35 (d, J = 3.5 Hz, 1H, furan), 7.73 (d, J = 8.7
Hz, 2H, arom.), 7.72 (s, 1H, aldimine), 7.87-7.94 (m, 4H, arom.), 11.12
(brs, 1H, NH). ESI-MS (C19H16N3O3S, [M+H]+) calcd. m/z = 366.0909,
found 366.0906. MP 175 °C (dec).
N'-((5-(4-nitrophenyl)furan-2-yl)methylene)thiophene-2-
carbohydrazide (1). Yellow crystals; yield: 25%. 1H NMR (300 MHz,
DMSO-d6) δ 7.14 (d, J = 3.5 Hz, 1H, furan), 7.23 (t, J = 3.5 Hz, 1H,
thiophene), 7.47 (d, J = 3.5, 1H, furan), 7.89-8.40 (m, 7H arom.), 12.20
(brs, 1H, NH). ESI-MS (C16H10N3O4S, [M–H]-) calcd. m/z = 340.0390,
found: 340.0392. MP 241-243 °C.
((5‐ (4‐ cyanophenyl)furan‐ 2‐ yl))methylidene)hydrazin-1-
yl)benzene-1-sulfonamide (12). Dark-orange crystals; yield: 32%; 1H
NMR (300 MHz, DMSO-d6) δ 6.92 (d, J =3.5 Hz, 1H, furan), 7.08 (brs,
2H, NH2), 7.13 (d, J = 8.2 Hz, 2H, arom.), 7.35 (d, J = 3.5 Hz, 1H, furan),
7.67 (d, J = 8.7 Hz, 2H, arom.), 7.84-7.96 (m, 5H, arom., aldimine), 10.91
(brs, 1H, NH). ESI-MS (C18H13N4O3S, [M–H]-) calcd. m/z = 365.0706,
found 365.0699. MP 217-219 °C.
N-(4-methylpiperazin-1-yl)-1-(5-(4-nitrophenyl)furan-2-
yl)methanimine (3). Red crystals; yield: 50%. 1H NMR (300 MHz,
DMSO-d6) δ 2.21 (s, 3H, CH3), 2.41-2.50 (m, 4H, piperazine), 3.13 (t, J =
5.2 Hz, 4H, piperazine), 6.70 (d, J = 4.1 Hz, 1H, furan) 7.35 (d, J = 4.1 Hz,
1H, furan), 7.54 (s, 1H, aldimine), 7.91 (d, J = 8.5 Hz, 2H, ArNO2), 8.26
(d, J = 8.5 Hz, 2H, ArNO2). ESI-MS (C16H19N4O3, [M+H]+) calcd. m/z =
315.1453, found 315.1456. MP 122-124 °C.
Synthesis of compound 13
The procedure was the same as for compounds 1-12, using 3-
methoxybenzaldehyde (0.27 g, 2.0 mmol) instead of 2-furaldehyde, and
2-thiophenecarboxylic acid hydrazide (0.43 g, 3.0 mmol). After
evaporation of organic extract, the crude product obtained was purified
by crystallization from absolute ethanol.
N'-((5-(4-cyanophenyl)furan-2-yl)methylene)thiophene-2-
carbohydrazide (4). Yellow crystals; yield: 20%. 1H NMR (300 MHz,
DMSO-d6) δ 7.11 (d, J = 3.5 Hz, 1H, furan), 7.23 (t, J = 4.7 Hz, 1H,
thiophene), 7.40 (d, J = 3.5 Hz, 1H, furan), 7.80-8.05 (m, 6H, arom.), 8.37
(s, 1H, aldimine), 11.86 (brs, 1H, NH). ESI-MS (C17H12N3O2, [M+H]+)
calcd. m/z = 322.0648, found 322.0643. MP 245 °C (dec).
N'-[(4'-cyano-2-methoxy[1,1'-biphenyl]-4-yl)methylidene]thiophene-
2-carbohydrazide (13). White ivory crystals; yield: 24%. 1H NMR (300
MHz, DMSO-d6) δ 3.82 (s. 3H, OCH3), 7.01 (d, J =7.2 Hz, 1H, arom.),
7.19 (t, J = 3.5 Hz, 1H, thiophene), 7.30-8.20 (m, 8H, arom.), 8.39 (s, 1H,
aldimine), 11.86 (brs, 1H, NH). ESI-MS (C20H16N3O2S, [M+H]+) calcd. m/z
= 362.0690, found 362.0699. MP 255 °C (dec).
N'-((5-(4-cyanophenyl)furan-yl)methylene)isonicotinohydrazide (5).
1
Yellow crystals; yield: 30%. H NMR (300 MHz, DMSO-d6) δ 7.14 (d, J =
3.5 Hz, 1H, furan), 7.40 (d, J = 3.5 Hz, 1H, furan), 7.80 (d, J = 5.8 Hz, 2H,
pyridine), 7.91 (d, J = 8.5 Hz, 2H, ArCN), 7.96 (d, J = 8.5 Hz, 2H, ArCN),
8.03 (brs, 1H, NH), 8.40 (s, 1H, aldimine), 8.75 (d, J = 5.8 Hz, 2H,
pyridine). ESI-MS (C18H11N4O2, [M–H]-) calcd. m/z = 315.0880, found
315.0879. MP 240 °C (dec).
Synthesis of compounds 14-21
2.0 mmol of aldehyde (2-furaldehyde for 14-18; 3-methoxybenzaldehyde
for 19 and 20; 3,4,5-trimethoxybenzaldehyde for 21) were solubilized in 5
mL of acetone and slowly added to the aqueous solution of the
N'-((5-(4-cyanophenyl)furan-2-yl)methylene)acetohydrazide
(6).
appropriate amine/hydrazine/hydrazide derivative, with
a catalytic
1
Orange crystal; yield: 35%. H NMR (300 MHz, DMSO-d6) δ 2.18 (s, 3H,
CH3), 7.00 (d, J = 3.6 Hz, 1H, furan), 7.35 (d, J = 3.6 Hz, 1H, furan), 7.87-
7.94 (m, 4H, arom.), 8.07 (s, 1H, aldimine), 11.33 (brs, 1H, NH). ESI-MS
(C14H11N3O2, [M+Na]+) calcd. m/z = 276.0747, found 276.0743. MP
190 °C (dec).
amount of HCl 4N. The mixture was stirred at room temperature for 24 h,
then extracted with ethyl acetate (3 x 15 mL) and the organic layer dried
over anhydrous Na2SO4. Compound 21 was filtered after precipitation
from the acetone/water mixture. The solvent was evaporated under
reduced pressure to give the crude product that was purified by
crystallization from absolute ethanol.
N'-((5-(4-cyanophenyl)furan-2-yl)methylene)-2-phenyl-
acetohydrazide (8). Orange crystals; yield: 35%. 1H NMR (300 MHz,
DMSO-d6) δ 3.99 (s, 2H, CH2), 7.05 (d, J = 3.5 Hz, 1H, furan), 7.29-7.40
(m, 6H, arom.), 7.88-7.97 (m, 4H, arom.), 8.15 (s, 1H, aldimine), 11.45
(brs, 1H, NH). ESI-MS (C20H16N3O2, [M+H]+) calcd. m/z = 330.1239,
found 330.1236. MP 197 °C (dec).
1-(Furan-2-yl)-N-(4-methylpiperazin-1-yl)methanimine (16). Brown oil;
yield: 38%; 1H NMR (300 MHz, DMSO-d6) δ 2.39 (s, 3H, CH3), 2.66 (t, J
= 3.1 Hz, 4H, 2xCH2), 3.24 (t, J = 3.1 Hz, 4H, 2xCH2), 6.41 (t, J = 1.5 Hz,
1H, furan), 6.45 (dd, J = 1.5 Hz, 1H, furan), 7.26 (s, 1H, aldimine), 7.41 (d,
J = 2.0 Hz, 1H, furan). ESI-MS (C10H16N3O, [M+H]+) calcd. m/z =
194.1290, found 194.1286.
(4-(5-(((2-chloro-4-hydroxyphenyl)imino)methyl)furan-2-
yl)benzonitrile (9). Yellow crystals; yield: 25%. 1H NMR (500 MHz,
DMSO-d6) δ 6.76 (dd, J = 8.5 Hz, 1H, arom.), 6.90 (d, J = 2.9 Hz, 1H,
arom.), 7.22 (d. J = 8.8 Hz, 1H, arom.), 7.28 (d, J = 3.6 Hz, 1H, furan),
7.45 (d, J = 3.6 Hz, 1H, furan), 7.93 (d, J = 8.5 Hz, 2H, arom.), 7.99 (d, J
= 8.5 Hz, 2H, arom.), 8.41 (s, 1H, aldimine), 9.95 (brs, 1H, OH). ESI-MS
(C18H10ClN2O2, [M–H]-) calcd. m/z = 321.0429, found 321.0432. MP 196-
198 °C.
N'-((furan-2-yl)methylidene)-2-phenylacetohydrazide (17). White ivory
crystals; yield: 46%. 1H NMR (300 MHz, DMSO-d6) δ 3.50 (s, 2H, CH2),
6.50 (t, J = 2.1 Hz, 1H, furan), 6.86 (d, J = 2.0 Hz, 1H, furan), 7.18-7.30
(m, 5H, arom.), 7.80 (d, J = 2.1 Hz, 1H, furan), 7.88 (s, 1H, aldimine),
11.30 (brs, 1H, NH). ESI-MS (C13H13N2O2, [M+ H]+) calcd. m/z =
229.0974, found 229.0980. MP 156-160 °C.
4-(-2-((furan-2-yl)methylidene)hydrazinyl)benzene-1-sulfonamide
(18). Orange crystals; yield: 47%. 1H NMR (300 MHz, DMSO-d6) δ 6.57 (t,
J = 1.6 Hz, 1H, furan), 6.72 (d, J = 1.6 Hz, 1H, furan), 7.04 (brs, 2H, NH2),
4‐ ((5-(2‐ (4‐ methoxyphenyl)hydrazin‐ 1‐ ylidene)methyl)furan‐
2yl))benzonitrile (10). Orange crystals; yield: 40%; 1H NMR (300 MHz,
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100209
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FULL PAPER
7.05 (d, J = 5.3 Hz, 2H, arom.), 7.63 (d, J = 5.2 Hz, 2H, arom.), 7.71 (s,
1H, aldimine), 7.81 (d, J = 1.6 Hz, 1H, furan), 10.72 (brs, 1H, NH). ESI-
MS (C11H12N3O3S, [M+H]+) calcd. m/z = 266.0597, found 266.0593. MP
161-165 °C.
(from 10 to 250 M), and data analysed by means of the “Enzyme
kinetics” module of Prism.
Cell cultures
4-(2-((3-methoxyphenyl)methylidene)hydrazinyl)benzene-1-
sulfonamide (20). Orange crystals; yield: 25%; 1H NMR (300 MHz,
DMSO-d6) δ 3.79 (s, 3H), 6.89 (d, J = 4.1 Hz, 1H, arom.), 7.07 (brs, 2H,
NH2), 7.14 (d, J = 8.8 Hz, 2H, arom.), 7.21-7.34 (m, 3H, arom.), 7.65 (d, J
= 8.9 Hz, 2H, arom.), 7.90 (s, 1H, aldimine), 10.79 (brs, 1H, NH). ESI-MS
(C14H14N3O3S, [M–H]-) calcd. m/z = 304.0753, found 304.0756. MP 214-
218 °C.
Reagents for cell cultures were purchased from Life Technologies
(ThermoFisher Scientific, Waltham, MA, USA) unless otherwise stated.
The human tumour cell lines of neuroblastoma SH-SY5Y were obtained
from the National Cancer Institute, Biological Testing Branch (Frederick,
MD, USA) and were maintained in the logarithmic phase at 37˚C in a 5%
CO2 humidified air in RPMI 1640 + Glutamax medium supplemented with
10% fetal bovine serum, 1% penicillin and streptomycin and 50 µg/mL
gentamicin.
1-(((3,4,5-trimethoxyphenyl)methylidene)amino)imidazolidine-2,4-
dione (21). White solid; yield: 70%. 1H NMR (300 MHz, DMSO-d6) δ 3.68
(s, 3H, OCH3), 3.80 (s, 6H, 2xOCH3), 4.31 (s, 2H, CH2), 7.01 (s, 2H,
arom.), 7.73 (s, 1H, aldimine), 11.17 (brs, 1H, NH). ESI-MSv(C13H16N3O5,
[M+H]+) calcd. m/z = 294.1086, found 294.1083. MP 270 °C (dec.).
Measurement of reactive oxygen species levels: DCF-DA assay
ROS production in SH-SY5Y cell line was detected using Cellular
Reactive Oxygen Species Detection Assay Kit ab186027 (Abcam,
Cambridge, UK), as previously described.[1] Briefly, cells were seeded
into 384-well flat bottom transparent polystyrene microtiter plates
(Greiner) at a plating density of 12000 cells per well. After seeding,
microtiter plates was incubated overnight at 37 ˚C, then added with
phenelzine sulfate (positive control) or test compounds, and dopamine
HCl (10 nM) as ROS inductor. After incubation and washing, DCF-DA
was added, the plate was incubated, washed again with PBS, and the
fluorescence read every 15 min in a 60 min time interval at 535 nm
(excitation at 485 nm) using the Tecan Infinite M1000 Pro plate reader.
Chromatographic Measures
Stability studies in buffered solution were performed following an already
described procedure,[23] on a Phenomenex C18 column (150 x 4.6 mm
i.d., 3 µm particle size; Phenomenex, Castel Maggiore, Italy) using a
mobile phase consisting of a mixture of methanol-water (75:25 v/v, with
aqueous formic acid 0.1%). The used flow rate was 0.500 mL/min while
the injection volume was 20 µL. Wavelength of UV-Vis detector was
adjusted at 254 nm. The chemical stability was evaluated in a phosphate
-
buffer solution pH 7.4 (10 mM HPO42-/ H2PO4 ; 100 mM NaCl) at 37 °C.
Transport Measurement
Five different concentrations from 0.5 µM to 20 µM were studied in a 2 h
time range (data not shown). Each concentration (0.5-1-5-10 and 20 µM)
was tested in triplicate starting from 3 different stocks solution (1 mM)
prepared separately. The time range was extended to 6 h only for the
samples at 20 µM concentration (Figure 6a).
An already reported protocol,[1] based on the inhibitor-stop method,[25]
was used. The recombinant WT CAC protein was reconstituted into
liposomes as described previously.[1,26] Briefly, transport was started by
adding [3H]-carnitine to proteoliposomes and stopped by the addition of
N-ethylmaleimide. After removal of the external substrate, intraliposomal
radioactivity was measured by a liquid scintillation counter (Perkin Elmer,
Milan, Italy). EC50 values were calculated using AATBioquest EC50
calculator.[27] Statistical analysis was performed by Student’s t-test, as
Log k’ values (k’ = (tr − t0)/t0) and purity determinations were carried out
using a Phenomenex Gemini C18 4.6 × 150 mm, with 3 μm size particles,
built on a Waters double pump HPLC system in isocratic conditions.
Injection volumes were 10 μL, flow rate was 0.5 mL/min, and detection
was performed with UV (λ = 254 and 370 nm). Samples were prepared
by dissolving 0.1 mg/mL of the solute in 10% v/v DMSO and 90% v/v
methanol. Retention times (tr) were measured at least from three
separate injections, and dead time (t0) was the retention time of
deuterated methanol. The mobile phase was filtered through a Supelco
Nylon-66 membrane 0.45 μm (Merck Life Science Srl, Milan, Italy) before
use. For each reference compound, the average tr of three consecutive
injections of 10 μL of sample was used to calculate the log k’ values. The
eluent consisted of five different mixtures of methanol and PBS buffer 10
mM at pH 7.4, with methanol/buffer ratios ranging from 80:20 to 60:40 v/v.
indicated in figure legends. Values of
p < 0.05 were considered
statistically significant. Data points were derived from the mean of three
different experiments, as specified in the figure legends.
Keywords: dantrolene analogues • hydrazide and hydrazone
derivatives • multitarget activity • carnitine/acylcarnitine carrier •
Alzheimer’s disease
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Entry for the Table of Contents
Simpler is better: Structural modifications of the chemical structure of dantrolene, previously disclosed as a repositionable drug with
potential for Alzheimer’s disease, allowed preparing new small molecules with multitarget activity profile. Compared to dantrolene,
molecular simplification resulted in less toxic and more soluble compounds. Some of them had higher potency as selective MAO B
inhibitors, and were effective in cell protection from oxidative insult and in the activation of CAC carnitine carrier.
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