Design and synthesis of H2S-donor hybrids: A new treatment for Alzheimer's disease?

Article abstract of DOI:10.1016/j.ejmech.2019.111745

Hydrogen sulphide (H₂S) is an endogenous gasotransmitter, largely known as a pleiotropic mediator endowed with antioxidant, anti-inflammatory, pro-autophagic, and neuroprotective properties. Moreover, a strong relationship between H₂S and aging has been recently identified and consistently, a significant decline of H₂S levels has been observed in patients affected by Alzheimer's disease (AD). On this basis, the use of H₂S-donors could represent an exciting and intriguing strategy to be pursued for the treatment of neurodegenerative diseases (NDDs). In this work, we designed a small series of multitarget molecules combining the rivastigmine-scaffold, a well-established drug already approved for AD, with sulforaphane (SFN) and erucin (ERN), two natural products deriving from the enzymatic hydrolysis of glucosinolates contained in broccoli and rocket, respectively, endowed both with antioxidant and neuroprotective effects. Notably, all new synthetized hybrids exhibit a H₂S-donor profile in vitro and elicit protective effects in a model of LPS-induced microglia inflammation. Moreover, a decrease in NO production has been observed in LPS-stimulated cells pre-treated with the compounds. Finally, the compounds showed neuroprotective and antioxidant activities in human neuronal cells. The most interesting compounds have been further investigated to elucidate the possible mechanism of action.

Full text of DOI:10.1016/j.ejmech.2019.111745

European Journal of Medicinal Chemistry 184 (2019) 111745
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of H2S-donor hybrids: A new treatment for
Alzheimer's disease?
Simona Sestito a, Letizia Pruccoli b, Massimiliano Runfola a, Valentina Citi a,
,
c
,
,
c
,
, **
Alma Martelli a d, Giuseppe Saccomanni a, Vincenzo Calderone a d, Andrea Tarozzi b
,
, d, *
Simona Rapposelli a
a Department of Pharmacy, University of Pisa, Italy
b Department for Life Quality Studies, University of Bologna, Italy
c Interdepartmental Research Centre “Nutraceuticals and Food for Health (NUTRAFOOD)”, University of Pisa, Italy
d Interdepartmental Research Centre of Ageing Biology and Pathology, University of Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2019
Received in revised form
12 September 2019
Accepted 26 September 2019
Available online 27 September 2019
Hydrogen sulphide (H2S) is an endogenous gasotransmitter, largely known as a pleiotropic mediator
endowed with antioxidant, anti-inflammatory, pro-autophagic, and neuroprotective properties. More-
over, a strong relationship between H2S and aging has been recently identified and consistently, a sig-
nificant decline of H2S levels has been observed in patients affected by Alzheimer's disease (AD). On this
basis, the use of H2S-donors could represent an exciting and intriguing strategy to be pursued for the
treatment of neurodegenerative diseases (NDDs).
In this work, we designed a small series of multitarget molecules combining the rivastigmine-scaffold,
a well-established drug already approved for AD, with sulforaphane (SFN) and erucin (ERN), two natural
products deriving from the enzymatic hydrolysis of glucosinolates contained in broccoli and rocket,
respectively, endowed both with antioxidant and neuroprotective effects.
Notably, all new synthetized hybrids exhibit a H2S-donor profile in vitro and elicit protective effects in
a model of LPS-induced microglia inflammation. Moreover, a decrease in NO production has been
observed in LPS-stimulated cells pre-treated with the compounds. Finally, the compounds showed
neuroprotective and antioxidant activities in human neuronal cells. The most interesting compounds
have been further investigated to elucidate the possible mechanism of action.
Keywords:
Hydrogen sulphide
Multitarget approach
Alzheimer's disease
Sulforaphane
Erucin
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
emerged as a pleiotropic mediator endowed with antioxidant [2],
anti-inflammatory [3], pro-autophagic [4] and neuroprotective
Hydrogen sulphide (H2S) is an endogenous gasotransmitter,
which acts as a signaling molecule in the central nervous system
(CNS) as well as in many other districts. In the CNS it is mainly
properties [5]. The existence of a strong relationship between H2S
and aging [6] has been proved for the first time in C. elegans in
which the exposure to low concentrations of exogenous H2S
induced beneficial effects on lifespan [7]. Up to date, the design of
new H2S-donors as novel pharmacological agents for the treatment
of neurodegenerative diseases (NDDs) represents an exciting and
intriguing task to be pursued.
As for many others NDDs characterized by a complex etiology,
the treatment of AD still represents one of the major therapeutic
areas where the discovery of new effective drugs remains a major
challenge. To date, scientists are currently taking advantage of the
‘multi-target approach’ as a promising option to develop new drugs
for the treatment of AD [8e10]. Consistently, in the last decade our
group made big efforts in the development of therapeutically useful
produced by cystathionine
b-synthase (CBS) by the use of the
amino acids -cysteine and homocysteine as substrates. Altered
L
levels of CBS have been found in subjects affected by neurode-
generative diseases. For instance, in patients affected by Alz-
heimer's disease (AD) the expression of CBS is drastically decreased,
resulting in a significant decline of H2S levels [1]. Basically, H2S has
* Corresponding author. Department of Pharmacy, University of Pisa, Italy.
** Corresponding author.
E-mail addresses: andrea.tarozzi@unibo.it (A. Tarozzi), simona.rapposelli@unipi.
it (S. Rapposelli).
https://doi.org/10.1016/j.ejmech.2019.111745
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
molecules for NDD following this approach [11e13]. In particular,
we have recently synthetized the first neuroprotective H2S-hybrid
agent by combining the molecular scaffold of memantine (NMDA
antagonist) with an isothiocyanate group able to release H2S
through a cysteine-mediated mechanism, thus generating mem-
antine [14]. To further expand the class of new H2S-releasing agents
we developed new chemical entities based on the combination of
rivastigmine an acetylcholinesterase (AChE) inhibitor with brain-
region selectivity [15] and FDA approved for AD, with antioxidant
natural products. In particular, we identified sulforaphane (SFN)
and erucin (ERN), mainly deriving from broccoli and rocket gluco-
sinolates, as two interesting compounds to be included in the
design of new H2S-hybrids [16] (Fig. 1). Several studies revealed the
therapeutic and prophylactic properties of both SFN and ERN
[17,18], including strong antioxidant [19,20], anti-inflammatory
activity and neuroprotective effects [21]. The pleiotropic role of
both isothiocyanates has been linked to their ability to address
several targets (i.e. NO, GSH, Nrf2/ARE pathway) and to modulate
different pathways in neuronal and glial cells [22].
Within the complexity and multifactorial etiology of AD,
hyperactivation of microglia has emerged as a pivotal player in the
pathogenesis of late AD. The significant reduction of cholinergic
neurotransmission promotes microglial activation and subse-
quently, the release of pro-inflammatory factors [23]. Moreover, the
close correlation between the inflammatory state of microglia and
the severity of neurodegeneration [24e26] suggests microglia's
inflammation as a consistent biological target.
effects induced on NO-production. In addition, the neuroprotective
and antioxidant activities in human neuronal cells have been
determined. The most interesting compounds have been further
investigated to elucidate the potential mechanism of action.
2. Materials and Methods
2.1. Chemistry
The synthesis of the final products 1e6 was carried out
following the synthetic procedure showed in Scheme 1. The reac-
tion between phthalimide potassium 7 and 1,4-dibromobutane in
acetone gave the bromo-derivative 8, which was then reacted with
potassium thioacetate in THF to give the thioester 9. The following
hydrolysis furnished the thiol 10 which was alkylated with the
appropriate chloroacetamide 11a-c [27] in the presence of KOH to
afford the corresponding thioethers 12e14. The subsequent
hydrazinolysis provided respectively the intermediates 15e17,
which were treated with thiophosgene and NaOH to give the iso-
thiocyanates 1e3. Finally, oxidation of compounds 1e3 by Oxone®
led to sulphoxides 4e6.
3. Results and discussion
3.1. H2S-release and anti-inflammatory effects in microglia BV-
2 cells
With the aim to design new protective agents against neuronal
inflammation and able to induce a substantial drop in ROS pro-
duction, we synthesized original H2S-releasing hybrids combining
the rivastigmine-scaffold with SFN/ERN pharmacophores through a
multitarget approach. The present work deals with the design and
synthesis of new multitarget chemical entities, and with the eval-
uation of their biological profile. In particular, we evaluated their
capability to release H2S and to elicit protective effects in a model of
LPS-induced inflammation of microglia. We also assessed the
Intracellular H2S-release of compounds 1e6 was tested in a
murine microglia cell line (BV-2) using WSP-1, a fluorescent dye
indicating the generation of intracellular H2S [28]. Administration
of 100
increase in WSP-1 fluorescence levels reaching the plateau in about
45 min. Concentration of 100 M was selected according to dye
mM of the tested compounds to BV-2 caused a significant
m
manufacturer, due to its relatively short time of recording. The area
under the curve (AUC) of the H2S-release (expressed as fluores-
cence index, FI) was calculated for each compound, and the amount
Fig. 1. Structures of ERN and SFN-rivastigmine hybrids.

S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
3
Scheme 1. Reagents and conditions: i) 1,4-dibromobutane, acetone, reflux, 12 h; ii) MeCOSeKþ, THF, reflux, 12 h; iii) HCl 37%, MeOH, reflux, 12 h; iv) 11a-c, KOH, acetone, reflux,
12 h; v) NH2NH2 ꢀ H2O, EtOH, reflux, 1 h; vi) thiophosgene, NaOH, DCM, r.t., vii) Oxone/H2O, MeOH/THF (1:1), r.t., 1 h.
of H2S released from the hybrid compounds and native molecule
rivastigmine, as negative control, were compared with H2S levels
produced by 100 mM of diallyl disulfide (DADS), a well-known slow
H2S-donor. All tested compounds exhibited a slow H2S-donor
profile. Moreover, the amount of H2S generated by almost all the
hybrid compounds, was comparable to diallyl disulfide (DADS),
except for compound 2 which exhibited the highest levels of
intracellular H2S-release (Fig. 2).
Isothiocyanates like ERN and SFN were described as H2S-donors
in several previous works [29e32]. Remarkably, H2S exhibits two
opposite concentration-dependent behaviors. This feature, named
“hormesis”, consists in a protective and pro-proliferative effect at
low concentrations that switch to anti-proliferative and anti-cancer
ones at high concentrations [33]. Hence, the new hybrids and
rivastigmine were tested at the concentration of 1
mM to evaluate
their cytoprotective effect on microglial cells against LPS- induced
Fig. 2. Intracellular H2S-release in BV-2 cells. The histograms show the total amount of H2S released by vehicle, DADS and the tested compounds (1e6) 100 mM during 45 min,
expressed as AUC. The vertical bars represent the standard error (n ¼ 9). The asterisks indicate a statistically significant difference from vehicle (**p < 0.01 and ***p < 0.001 vs
vehicle). Cell viability in microglia BV-2 cells.

4
S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
neuroinflammatory stimuli. The administration of LPS (5
m
g/ml for
intracellular ROS, SH-SY5Y cells were incubated with the tested
compounds for 24 h and then with hydrogen peroxide (H2O2) or
tert-butyl hydroperoxide (t-BuOOH) for 30 min. The ROS formation
in SH-SY5Y cells was evaluated using the fluorescent probe 20e70
dichlorodihydrofluorescein diacetate (DCFH-DA). Compounds 1
and 2 showed the highest antioxidant activity against the ROS
formation elicited by both H2O2 and t-BuOOH (Fig. 6). To under-
stand the molecular mechanisms involved in the antioxidant ac-
tivity, we evaluated the ability of hybrid compounds to induce the
formation of the antioxidant mediator glutathione (GSH).
24 h) to BV2 cells, led to approximately a 20% decrease in cell
viability (80.6 1.2%). The pre-incubation of almost all the tested
compounds, except compound 6, induced a significant reduction of
LPS-induced damage preserving cell viability. In particular, the
native compound rivastigmine induced a slight although significant
increase in cell viability (84.0 1.0%) but, notably, the 1 h pre-
incubation with all hybrid compounds led to a further increase in
BV-2 viability, compared both to LPS treated cells and rivastigmine
pre-incubation. In particular, this increase reached a statistical
significance for compound 1 (93.0 3.2%) (Fig. 3).
The GSH levels in SH-SY5Y cells were evaluated after 24 h of
A typical effect of a sub-clinic inflammation is represented by
the increase in ROS production in cells subjected to pro-
inflammatory stimuli [34]. Accordingly, a 24 h LPS administration
elicited a significant increase of ROS vs vehicle (154 3.8%) in BV-
2 cell lines. All the tested compounds, except rivastigmine, exhibi-
ted a clear antioxidant effect with a significant reduction of ROS of
about 30e35% after 1 h of pre-incubation and 24 h of co-incubation
with LPS. Rivastigmine did not show any significant effect on LPS-
induced ROS increase (Fig. 4).
The LPS-induced neuroinflammation or other pro-inflammatory
stimuli leads to an increase in nitric oxide levels inside cells as
marker of inflammation. In particular, the incubation of BV-2 with
LPS for 24 h in our experimental protocol induced a significant in-
crease of NO levels of about 46% (145.7 4.4%) compared to vehicle.
This increase was counteracted by all the tested compounds even if
only hybrid compounds 1 (117.4 11.9%), 2 (125.9 6.9%), and 3
(98.8 26.5%) led to a significant decrease of NO levels (Fig. 5).
Although further investigations are required, we can speculate that
the different decrease in NO levels observed for ERN- and SFN-
derivatives could be affected by the H2S-releasing properties.
The neurotoxicity of compounds 1, 2 and 4e6 was evaluated in
SH-SY5Y cells using the MTT assay. The 24 h incubation with
incubation with the hybrid compounds 1, 2 and 4e6 (5 mM) using
the fluorescent probe monochlorobimane (MCB). As depicted in
Fig. 7, compounds 1 and 2 recorded the best capacity to increase the
basic levels of GSH with an increase percentage of 35.60% and
25.73%, respectively. We speculated that this effect could be related
to activation of Nrf2 pathway, as previously reported for SFN and
ERN [35]. Thus, we decided to assess the capacity of the hybrid
compounds 1 and 2 to activate the Nrf2 pathway which is a tran-
scription factor that once activated binds to the antioxidant
response element (ARE), thus initiating the transcription of cyto-
protective genes.
In particular, the Nrf2 activation at nuclear level in SH-SY5Y
cells, was evaluated using the TransAM Nrf2 kit after different
times of incubation with compounds 1 and 2. Only compound 1
was able to activate Nrf2 suggesting that its antioxidant activity is
due to an increase of genetic transcription of GSH (Fig. 8).
The neuroprotective activity against the neurotoxicity induced
by Ab1e42 oligomers (OAb1e42) in SH-SY5Y cells was also evaluated
after 24 h of incubation with the hybrid compounds 1, 2 and 4e6
(5 mM) and 4 h of incubation with OAb1e42 using the MTT formazan
exocytosis assay. All the tested compounds did not exert neuro-
protective effects against the neurotoxicity induced by OAb1e42 (see
SI).
This result confirms that the neuroprotective effects observed
for the new hybrid compounds are finely due to the H2S-releasing
different concentrations of the tested compounds (1.25e40
recorded that concentrations up to 5 M did not affect the neuronal
viability (data not shown). Therefore, all the hybrids were used at
the maximum concentration of 5 M in the following experiments.
To investigate the antioxidant activity in terms of inhibition of
mM)
m
m
properties and not to a direct inhibition of the Ab-aggregation.
Fig. 3. The histograms show the viability of BV-2 cells treated with vehicle, LPS 5
percentage of viability recorded for BV-2 cells treated with vehicle. The vertical bars represent the standard error (n ¼ 9). The x indicate a statistically significant difference from
vehicle (xxxp < 0.001) The asterisks indicate a statistically significant difference from LPS 5 g/ml (*p < 0.05, **p < 0.01, ***p < 0.001). ROS production in microglia BV-2 cells.
mg/ml, tested compounds þ LPS 5
mg/ml (1e6) or rivastigmine 1 mM. Data are expressed as a
m

S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
5
Fig. 4. The histograms show the ROS amount of BV-2 cells treated with vehicle, LPS 5
m
g/ml, tested compounds þ LPS 5
m
g/ml (1e6) or rivastigmine 1
m
M. Data are expressed as a
percentage of ROS production recorded for BV-2 cells treated with vehicle. The vertical bars represent the standard error (n ¼ 9). The x indicate a statistically significant difference
from vehicle (xxxp < 0.001). The asterisks indicate a statistically significant difference from LPS 5 g/ml (**p < 0.01 and ***p < 0.001). NO formation in microglia BV-2 cells.
m
Fig. 5. The histograms show the NO amount of BV-2 cells treated with vehicle, LPS 5
m
g/ml, tested compounds þ LPS 5
mg/ml (1e6) or rivastigmine 1 mM. Data are expressed as a
percentage of NO production recorded for BV-2 cells treated with vehicle. The vertical bars represent the standard error (n ¼ 9). The x indicate a statistically significant difference
from vehicle (xxxp < 0.001) The asterisks indicate a statistically significant difference from LPS 5
mg/ml (*p < 0.05). Antioxidant and neuroprotective effects in neuronal SH-SY5Y
cells.
3.2. Determination of lipophilicity
if these theoretical data are only preliminary, we hypothesise that
the new compounds could be able to cross the BBB and exert a
neuroprotective and H2S-mediated effect on both neuronal and
microglia cells.
Brain selectivity is strongly related to the specific capability of a
drug to cross BBB. Therefore, since lipophilicity represents one of
the main requirements requested to address this issue, especially in
AD, we predicted the theoretical logP of compounds 1e6 (Table 1)
compared with their native drug. Calculations revealed that com-
pounds 1e3 possess a logP higher than rivastigmine (logP ¼ 2,41),
already described as drug with brain oriented selectivity [15]. Even
4. Conclusions
The goal of our study was to develop and preliminarily evaluate
a new class of multitarget H2S-donor hybrids. These new chemical

6
S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
Fig. 6. Antioxidant activity of compounds 1, 2 and 4e6 against the ROS formation induced by H2O2 or t-BuOOH in SH-SY5Y cells. Cells were incubated with the tested compounds
(5 M) for 24 h and then with H2O2 or t-BuOOH (50 M) for 30 min. At the end of incubation, intracellular ROS formation was detected using the fluorescent probe DCFH-DA, as
m
m
described in the Materials and Methods section. Data are expressed as antioxidant activity in terms of inhibition percentage of ROS formation induced by H2O2 or t-BuOOH and
reported as mean SEM of three independent experiments; n.a. ¼ not active. Effects induced on GSH levels in neuronal SH-SY5Y cells.
profile of our newly synthetized hybrids in microglia and neuronal
cell lines. Results showed that both SFN- and ERN-derivatives show
significant anti-inflammatory and antioxidant activities in micro-
glia cell line, and induce the expression of proteins (i.e. GSH)
involved in the antioxidant defense in neuronal cell line. In
particular, all hybrids produced a significant decrease in ROS pro-
duction elicited by pro-inflammatory stimulus compared to the
native compound rivastigmine, which completely lacks of antioxi-
dant activity. The new compounds were also able to reduce NO-
release in microglia BV-2 cells whereas rivastigmine showed no
effect. This result is mainly due to the multiple mechanisms of
actions of SFN and ERN moieties and their capability to release H2S.
Moreover, the most active compounds 1 and 2 increased GSH level
in neuronal SH-SY5Y cells. Notably, these two compounds present
favorable logP values, suggesting their potential ability to reach the
brain. In order to elucidate the mechanism of indirect antioxidant
action at molecular level, we further explored the influence of
compounds 1 and 2 on the activation of Nrf2 pathway in SH-SY5Y
Fig. 7. Effects of compounds 1, 2 and 4e6 on GSH levels in SH-SY5Y cells. Cells were
incubated with the tested compounds (5 mM) for 24 h. At the end of incubation, GSH
cells. While compound 2 showed a negligible activity, compound 1
exerted a time-dependent Nrf2 activation. We speculate that the
lack of Nrf2 activation for compound 2 could be partially due to the
high amount of gasotransmitter released inside the cells. Exoge-
nous H2S has been shown to have hormetic effects with antioxidant
(involving Nrf2) and pro-oxidant activities at low and high con-
centrations, respectively [36,37].
In conclusion, we developed a set of original H2S releasing hy-
brids starting from rivastigmine, a brain selective AchEI provided of
a slight microglial neuroprotective capability against LPS-induced
neuroinflammation, and SFN/ERN as natural-occurring H2S
releasing moieties. Globally, our biological evaluation indicates
compound 1 as a new well-balanced anti-inflammatory and anti-
oxidant agent. This promising pharmacological profile could be
partially due to the slow H2S release and to the favorable logP,
which promotes a cellular-specific activity.
levels were detected using the fluorescent probe MCB, as described in the Materials
and Methods section. Data are expressed as increase percentage of GSH level and
reported as mean SEM of three independent experiments; n.a. ¼ not active.
entities were obtained combining natural occurring H2S-releasing
moieties with the pharmacophore portion of rivastigmine, one of
the few drugs currently used in AD therapy reported to be brain-
selective.
As we recently reviewed, the main drawback in designing new
clinically suitable H2S-donor hybrids is to ensure a tissue specific
and controlled cellular H2S-release, avoiding systemic toxic effects.
Therefore, in this work we initially aimed to validate the use of SFN
and ERN as valuable H2S releasing portions through the combina-
tion with the acetylcholinesterase inhibitor rivastigmine, to obtain
new chemical entities able to target AD altered neuronal setting
and microglia.
Then, we explored the pharmacological profile of this limited set
of molecules in terms of anti-inflammatory and antioxidant activ-
ity, effects mainly related to the presence of H2S releasing moiety, in
order to validate the proof of concept of using ERN and SFN as
natural occurring H2S donor moieties for the design and synthesis
of new H2S-releasing drugs.
Future in vitro investigation (i.e. permeability assay, AchE inhi-
bition) will be of help in defining more in depth the pharmaco-
logical profile of compound 1 and will guide the in vivo evaluation
in specific models of AD.
5. Experimental section
Since the induction of a pro-inflammatory state may correlate
with grade of severity in neurodegeneration, we firstly investigated
the H2S-donor, anti-inflammatory, antioxidant and neuroprotective
Chemistry. General Material and Methods. Melting points were
determined on a Kofler hot-stage apparatus and are uncorrected.
Chemical shifts (d) are reported in parts per million downfield from

S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
7
Fig. 8. Compound 1, but not compound 2, activates the Nrf2 protein in SH-SY5Y cells. Cells were incubated with compounds 1 and 2 (5
mM) for 1, 3 and 6 h. At the end of incubation,
nuclear extraction and detection of active Nrf2 protein level were performed using the Nuclear Extract and TransAM Nrf2 Kit, respectively, as described in the Materials and
Methods section. Data are expressed as fold increase and reported as mean SEM of three independent experiments (**p < 0.01 and ***p < 0.001 vs untreated cells at two-way
ANOVA with Bonferroni post hoc test).
Table 1
(0.03 ml, 0.32 mmol) and NaOH (10.9 mg, 0.27 mmol). The resulting
suspension was stirred for 4 h at r.t. Then the solution was evapo-
rated to dryness, rinsed with new DCM and filtered on Celite. The
solution was dried and the crude product purified through flash
Calculated logP per compounds 1e6 and their native drug
rivastigmine. logP values were calculated by using the logP
predictor plugin (Consensus Method) included in Marvin-
Sketch 19.18 provided by ChemAxon.
chromatography.
Compound
Calculated logP
1
2
3
4
5
6
3,27
2,98
3,27
1,67
1,37
1,66
2,41
5.1.1. 3-(2-((4-isothiocyanatobutyl)thio)acetamido)phenyl
ethyl(methyl)carbamate (1)
Amine 15 (30.6 mg, 0.09 mmol) was reacted with thiophosgene
(0.03 mL, 0.28 mmol) in the presence of NaOH (9.6 mg, 0.24 mmol).
The crude product was purified through flash chromatography
using PE/AcOEt 1:1 as eluting mixture to give derivative 1 (30.9 mg,
rivastigmine
0.081 mmol, yield 90%). 1H NMR (CDCl3):
d
1.19 (t, J ¼ 7.0 Hz, 1.5H,
CH3 rotamer); 1.22 (t, J ¼ 7.0 Hz, 1.5H, CH3 rotamer); 1.73e1.83 (m,
4H, CH2); 2.63 (t, J ¼ 6.8 Hz, 2H, CH2S); 2.99 (s, 1.5H, CH2 rotamer);
3.06 (s, 1.5H, CH2 rotamer); 3.35 (s, 2H, CH2CO); 3.40 (q, J ¼ 7.0 Hz,
1H, CH2 rotamer); 3.47 (q, J ¼ 7.0 Hz, 1H, CH2 rotamer); 3.54 (t,
J ¼ 6.2 Hz, 2H, CH2N); 6.87e6.94 (m, 1H, Ar); 7.29e7.3 (m, 2H, Ar);
7.41e7.48 (m, 1H, Ar); 8.60 (br s, 1H, NH) ppm. 13C NMR (CDCl3):
tetramethylsilane and referenced from solvent references; coupling
constants J are reported in hertz. 1H NMR and 13C NMR spectra of
all compounds were obtained with a Bruker TopSpin 3.2400 MHz
spectrometer.13C NMR spectra were fully decoupled. The following
abbreviations are used: singlet (s), doublet (d), triplet (t),
doubleꢁdoublet (dd), and multiplet (m). The elemental composi-
tions of the compounds agreed to within 0.4% of the calculated
values. Chromatographic separation was performed on silica gel
columns by flash (Kieselgel 40, 0.040e0.063 mm; Merck) or gravity
column (Kieselgel 60, 0.063e0.200 mm; Merck) chromatography.
HPLC purity determination was performed on a Varian Pro Star
330PDA detector, a binary HPLC pump Varian 9012 and a Rehodyne
d
167.08, 154.66, 151.98, 138.52, 129.64, 118.02, 117.95, 116.51, 113.67,
44.71, 44.24, 36.91, 34.39 (CH2 rotamer), 33.97 (CH2 rotamer), 32.16,
29.03, 26.12, 13.34 (CH3 rotamer), 12.59 (CH3 rotamer) ppm. HPLC
purity: 97%.tR ¼ 17.32 min.
5.1.2. 3-((2-((4-isothiocyanatobutyl)thio)acetamido)methyl)phenyl
ethyl(methyl)carbamate (2)
injector with 20
ml loop. A Thermos Scientific ™ Hypersil™ C18 ODS
Amine 16 (35.4 mg, 0.10 mmol) was reacted with thiophosgene
(0.03 mL, 0.32 mmol) in the presence of NaOH (10.9 mg,
0.27 mmol). The crude product was purified through flash chro-
matography using PE/AcOEt 1:1 as eluting mixture to give deriva-
(5
m
m, 250 ꢀ 4.6 mmID) HPLC column was used (detection at
220 nm). Chromatographic separation was carried out with a
gradient of 20e80% acetonitrile in water (containing 0.1% TFA) over
20 min and a gradient of 80e20% over 10 min. Reactions were fol-
lowed by thin-layer chromatography (TLC) on Merck aluminum
silica gel (60 F254) sheets that were visualized under a UV lamp.
Evaporation was performed in vacuo (rotating evaporator). Sodium
sulfate was always used as the drying agent. Commercially available
chemicals were purchased from Sigma-Aldrich or Alfa Aesar.
tive 2 (11.9 mg, 0.03 mmol, 30% yield). 1H NMR (CDCl3):
d 1.19 (t,
J ¼ 7.0 Hz, 1.5H, CH3 rotamer); 1.23 (t, J ¼ 7.0 Hz, 1.5H, CH3 rotamer);
1.65e1.77 (m, 4H, CH2); 2.55 (t, J ¼ 7.8 Hz, 2H, CH2S); 2.98 (s, 1.5H,
CH3 rotamer); 3.06 (s, 1.5H, CH3 rotamer); 3.26 (s, 2H, CH2S); 3.41
(q, J ¼ 7.0 Hz, 1H, CH2 rotamer); 3.44e3.50 (m, 3H, CH2
rotamer þ CH2NCS); 4.46 (d, J ¼ 6.0 Hz, 2H, CH2NH); 7.03e7.13 (m,
4H, Ar þ NH); 7.31e7.35 (m,1H, Ar) ppm. 13C NMR (CDCl3):
d 168.73,
5.1. General procedure for the synthesis of isothiocyanates (1e3)
154.58, 154.42, 151.97, 139.52, 129.74, 124.70, 121.47, 121.24, 44.68,
44.25, 43.62, 36.21, 34.39 (CH2 rotamer), 33.97 (CH2 rotamer),
32.26, 29.82, 29.00, 26.18, 13.37 (CH3 rotamer), 12.59 (CH3 rotamer)
ppm. HPLC purity: 95%.tR ¼ 22.06 min.
To a solution of amine 15e17 (0.10 mmol) in DCM (1 ml), under
N2 atmosphere and cooled to 0 ꢂC, were added thiophosgene

8
S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
5.1.3. 3-(2-(2-((4-isothiocyanatobutyl)thio)acetamido)ethyl)phenyl
ethyl(methyl)carbamate (3)
rotamer),
96%.tR ¼ 16.51 min.
12.58
(CH3
rotamer)
ppm.
HPLC
purity:
Amine 17 (36.8 mg, 0.10 mmol) was reacted with thiophosgene
(0.03 mL, 0.32 mmol) in the presence of NaOH (10.9 mg,
0.27 mmol). The crude product was purified through flash chro-
matography using PE/AcOEt 1:1 as eluting mixture to give deriva-
5.2.3. 3-(2-(2-((4-isothiocyanatobutyl)sulfinyl)acetamido)ethyl)
phenyl ethyl(methyl)carbamate (6)
Compound 6 (17.0 mg, 0.04 mmol, 33% yield) was obtained from
isothiocyanate 3 (49.1 mg, 0.12 mmol) through oxidation with
Oxone® (43.0 mg, 0.07 mmol). The crude product was purified
through flash chromatography using AcOEt as the eluent. 1H NMR
tive 3 (10.2 mg, 0.025 mmol, 25% yield). 1H NMR (CDCl3):
d 1.19 (t,
J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.25 (t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer);
1.59e1.75 (m, 4H, CH2CH2); 2.43 (t, J ¼ 7.0 Hz, 2H, CH2S); 2.85 (t,
J ¼ 6.8 Hz, 2H, CH2Ph); 2.98 (s, 1.5H, CH3 rotamer); 3.06 (s, 1.5H, CH3
rotamer); 3.16 (s, 2H, CH2S); 3.41 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer);
3.46e3.51 (m, 3H, CH2 rotamer þ CH2NCS); 3.57 (q, J ¼ 6.4 Hz, 2H,
CH2NH); 6.80 (br s, 1H, NH); 6.98e7.04 (m, 3H, Ar); 7.27e7.31 (m,
(CDCl3):
d
1.20 (t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.25 (t, J ¼ 7.2 Hz,
1.5H, CH3 rotamer); 1.79e1.89 (m, 4H, CH2CH2); 2.71e2.82 (m, 2H,
CH2S); 2.86 (t, J ¼ 7.4 Hz, 2H, CH2Ph); 2.97 (s, 1.5H, CH3 rotamer);
3.06 (s, 1.5H, CH3 rotamer); 3.30 (d, J ¼ 14.0 Hz, 1H, CH2SO); 3.41 (q,
J ¼ 7.2 Hz, 1H, CH2 rotamer); 3.50 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer);
3.56e3.60 (m, 4H, CH2NH þ CH2NCS); 3.61 (d, J ¼ 14.0 Hz, 1H,
CH2SO); 6.96e7.00 (m, 3H, Ar þ br NH); 7.04 (d, J ¼ 7.6 Hz, 1H, Ar);
1H, Ar) ppm. 13C NMR (CDCl3):
d 168.75, 154.65, 154.49, 151.92,
140.16, 129.53, 125.60, 122.33, 120.15, 44.71 (CH3 rotamer), 44.23
(CH3 rotamer), 40.56, 36.10, 35.28, 34.38 (CH2 rotamer), 33.96 (CH2
rotamer), 32.05, 29.81, 28.96, 26.10, 13.37 (CH3 rotamer), 12.60 (CH3
rotamer) ppm. HPLC purity: 95%.tR ¼ 18.07 min.
7.28e7.30 (m, 1H, Ar) ppm. 13C NMR (CDCl3):
d 163.87, 154.75,
151.76, 140.07, 131.22, 129.50, 125.72, 122.44, 120.09, 54.47, 50.67,
44.66, 44.22, 40.69, 35.27, 34.37 (CH2 rotamer), 33.94 (CH2
rotamer), 29.01, 20.37, 13.34 (CH3 rotamer), 12.58 (CH3 rotamer)
ppm. HPLC purity: 97%.tR ¼ 17.05 min.
5.2. General procedure for the synthesis of isothiocyanates (4e6)
A solution of Oxone® (43.0 mg, 0.07 mmol) in H2O (0.33 ml) was
added to isothiocyanates 1e3 (0.12 mmol) dissolved in CH3OH/THF
(1:1, 0.70 ml) and cooled to 0 ꢂC. The reaction mixture was stirred
for 1 h at r.t. Then the organic solvent was evaporated and the
residue aqueous phase extracted several times with AcOEt. Then
the organic layer was washed with brine, dried over anhydrous
Na2SO4 and evaporated to dryness.
5.3. General procedure for the synthesis of derivatives (12e15)
To a solution of thiol 10 (232.9 mg, 0.99 mmol) and KOH (111 mg,
1.99 mmol) in acetone (5 mL) was added the appropriate chlor-
oderivative 11a-c (0.99 mmol) [13]. The resulting mixture was
refluxed for 12 h, then the solid was filtered off and the solution
evaporated to dryness.
5.2.1. 3-(2-((4-isothiocyanatobutyl)sulfinyl)acetamido)phenyl
ethyl(methyl)carbamate (4)
Compound 4 (9.1 mg, 0.023 mmol, 46% yield) was obtained from
isothiocyanate 1 (19.1 mg, 0.05 mmol) through oxidation with
Oxone® (19.4 mg, 0.03 mmol). The crude product was purified
through flash chromatography using a gradient of PE/AcOEt (from
5.3.1. 3-(2-((4-(1,3-dioxoisoindolin-2-yl)butyl)thio)acetamido)
phenyl ethyl(methyl)carbamate (12)
Carbamate 12 (122 mg, 0.26 mmol, 26% yield) was synthetized
starting from thiol 10 (232.9 mg, 0.99 mmol) and chloroderivative
11a (268.0 mg 0.99 mmol). The crude product was purified by flash
chromatography using PE/AcOEt (1:1) as eluting mixture. 1H NMR
7:3 to 0:10) as the eluent. 1H NMR (CDCl3):
d
1.22 (t, J ¼ 7.2, 1.5H,
CH3 rotamer); 1.27 (t, J ¼ 7.0 Hz, 1.5H, CH3 rotamer); 1.85e1.99 (m,
4H, CH2); 2.89e2.98 (m, 2H, CH2SO); 3.01 (s, 1.5H, CH2 rotamer);
3.09 (s, 1.5H, CH2 rotamer); 3.43 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer);
3.48 (q, J ¼ 7.0 Hz, 1H, CH2 rotamer); 3.51 (d, J ¼ 14 Hz, 1H, CH2CO);
3.61 (t, J ¼ 6.0 Hz, 2H, CH2N); 3.84 (d, J ¼ 14 Hz, 1H, CH2CO);
6.83e6.92 (m, 1H, Ar); 7.21e7.30 (m, 2H, Ar); 7.42e7.49 (m, 1H, Ar);
(CDCl3):
d
1.18 (t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.25 (t, J ¼ 7.2 Hz,
1.5H, CH3 rotamer); 1.65e1.71 (m, 2H, CH2); 1.76e1.81 (m, 2H, CH2);
2.64 (t, J ¼ 7.4 Hz, 2H, CH2S); 2.97 (s, 1.5H, CH2 rotamer); 3.06 (s,
1.5H, CH2 rotamer); 3.33 (s, 2H, CH2CO); 3.36 (q, J ¼ 6.8 Hz, 1H, CH2
rotamer); 3.44 (q, J ¼ 6.8 Hz, 1H, CH2 rotamer); 3.70 (t, J ¼ 7.0 Hz,
2H. CH2N); 6.86e6.94 (m, 1H, Ar); 7.27e7.34 (m, 2H, Ar); 7.44e7.49
(m, 1H, Ar); 7.70e7.72 (m, 2H, Ar); 7.81e7.87 (m, 2H, Ar); 8.74 (br s,
1H, NH) ppm.
9.19 (br s, 1H, NH) ppm. 13C NMR (CDCl3):
d: 162.05, 151.97, 152,
138.35, 131.45, 129.65, 118.35, 117.06, 114.18, 54.10, 50.66, 44.70
(CH3 rotamer), 44.25 (CH3 rotamer), 39.99 (CH2 rotamer), 39.10,
34.42 (CH2 rotamer), 29.82, 29.09, 13.35 (CH3 rotamer), 12.60 (CH3
rotamer) ppm. HPLC purity: 95%.tR ¼ 20.02 min.
5.3.2. 3-((2-((4-(1,3-dioxoisoindolin-2-yl)butyl)thio)acetamido)
methyl)phenylethyl(methyl)carbamate (13)
Carbamate 13 (193.4 mg, 0.4 mmol, 40% yield) was synthetized
starting from thiol 10 (232.9 mg, 0.99 mmol) and chloroderivative
11b (281.9 mg, 0.99 mmol). The crude product was purified by flash
chromatography using PE/AcOEt (7:3) as eluting mixture. 1H NMR
5.2.2. 3-((2-((4-isothiocyanatobutyl)sulfinyl)acetamido)methyl)
phenyl ethyl(methyl)carbamate (5)
Compound 5 (11.9 mg, 0.029 mmol, 24% yield) was obtained
from isothiocyanate 2 (47.5 mg, 0.12 mmol) through oxidation with
Oxone® (43.0 mg, 0.07 mmol). The crude product was purified
through flash chromatography using a gradient of PE/AcOEt (from
(CDCl3):
d
1.16 (t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.20 (t, J ¼ 7.2 Hz,
1.5H, CH3 rotamer); 1.59e1.63 (m, 2H, CH2); 1.70e1.76 (m, 2H, CH2);
2.57 (t, J ¼ 7.2 Hz, 2H, CH2S); 2.96 (s, 1.5H, CH3 rotamer); 3.04 (s,
1.5H, CH3 rotamer); 3.25 (s, 2H, CH2S); 3.39 (q, J ¼ 7.2 Hz, 1H, CH2
rotamer); 3.44 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer); 3.64 (t, J ¼ 7.0 Hz,
2H, CH2N); 4.47 (d, J ¼ 6.0 Hz, 2H, CH2NH); 7.00e7.04 (m, 2H, Ar);
7.11 (d, J ¼ 7.6 Hz, 1H, Ar); 7.28e7.32 (m, 1H, Ar); 7.68e7.73 (m, 2H,
Ar); 7.80e7.84 (m, 2H, Ar) ppm.
9:1 to 0:10) as the eluent. 1H NMR (CDCl3):
d
1.20 (t, J ¼ 7.2 Hz, 1.5H,
CH3 rotamer); 1.24 (t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.79e1.88 (m,
4H, CH2); 2.70e2.77 (m, 1H, CH2SO); 2.81e2.87 (m, 1H, CH2SO);
2.98 (s, 1.5H, CH3 rotamer); 3.06 (s, 1.5H, CH3 rotamer); 3.30 (d,
J ¼ 14.2 Hz, 1H, CH2SO); 3.40 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer); 3.45
(q, J ¼ 7.2 Hz, 1H, CH2 rotamer); 3.54 (t, J ¼ 5.8 Hz, 2H, CH2NCS);
3.71 (d, J ¼ 14.2 Hz, 1H, CH2SO); 4.39e4.44 (m, 1H, CH2Ph);
4.52e4.57 (m, 1H, CH2Ph); 7.02e7.16 (m, 3H, Ar); 7.30e7.34 (m, 1H,
5.3.3. 3-(2-(2-((4-(1,3-dioxoisoindolin-2-yl)butyl)thio)acetamido)
ethyl)phenylethyl(methyl)carbamate (14)
Carbamate 14 was synthetized starting from thiol 10 (232.9 mg,
0.99 mmol) and chloroderivative 11c (295.8 mg, 0.99 mmol). The
crude product was purified by flash chromatography using AcOEt as
Ar) ppm. 13C NMR (CDCl3):
d
163.84, 154.63, 154.47, 151.89, 139.30,
129.65, 124.87, 121.60, 121.22, 54.39, 50.56, 44.63, 44.24, 43.55,
34.37 (CH2 rotamer), 33.96 (CH2 rotamer), 29.00, 20.37, 13.35 (CH3

S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
9
the eluent (238.6 mg, 0.48 mmol, 48% yield). 1H NMR (CDCl3):
d
1.16
2H, Ar) ppm.
(t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.22 (t, J ¼ 7.2 Hz, 1.5H, CH3
rotamer); 1.50e1.58 (m, 2H, CH2); 1.66e1.74 (m, 2H, CH2); 2.45 (t,
J ¼ 7.4 Hz, 2H, CH2S); 2.81 (t, J ¼ 7.0 Hz, 2H, CH2Ph); 2.94 (s, 1.5H,
CH3 rotamer); 3.02 (s,1.5H, CH3 rotamer); 3.13 (s, 2H, CH2S); 3.37 (q,
J ¼ 7.2 Hz, 1H, CH2 rotamer); 3.41 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer);
3.52 (q, J ¼ 7.0 Hz, 2H, CH2NH); 3.62 (t, J ¼ 7.0 Hz, 2H, CH2N);
6.95e7.00 (m, 3H, Ar); 7.22e7.26 (m, 1H, Ar); 7.67e7.69 (m, 2H, Ar);
7.79e7.81 (m, 2H, Ar) ppm.
5.4.5. S-(4-(1,3-dioxoisoindolin-2-yl)butyl) ethanethioate (9)
Bromoderivative 8 (601.0 mg, 2.13 mmol) dissolved in THF
(26 mL) was reacted with potassium thioacetate (729 mg,
6.38 mmol). The mixture was refluxed for 12 h, then cooled and
evaporated under vacuum. The crude residue was diluted with H2O
and extracted with AcOEt; the organic layer was then washed
several times with brine, dried over anhydrous Na2SO4 and evap-
orated to dryness to provide the desired product 9 (479 mg,
5.4. General procedure for the synthesis of derivatives (15e17)
1.73 mmol 81% yield). 1H NMR (CDCl3):
d 1.61e1.66 (m, 2H, CH2);
1.71e1.79 (m, 2H, CH2); 2.31 (s, 3H, CH3); 2.90 (t, J ¼ 7.2 Hz, 2H,
CH2S); 3.69 (t, J ¼ 7.0 Hz, 2H, CH2N); 7.70e7.72 (m, 2H, Ar);
7.83e7.85 (m, 2H, Ar) ppm.
To a solution of the opportune derivative 12e14 (0.22 mmol) in
MeOH (1.3 ml) was added hydrazine monohydrate (0.54 mmol,
0.03 ml); the resulting mixture was refluxed for 4 h. Once verified
the disappearance of starting material by TLC, the solution was
cooled, diluted with AcOEt and washed with NaOH 1 N. The organic
layer was then dried over Na2SO4 and evaporated to dryness.
5.4.6. 2-(4-mercaptobutyl)isoindoline-1,3-dione (10)
To a solution of compound 9 (496 mg, 1,79 mmol) in dry MeOH
(5.37 mL), under N2 atmosphere, was added HCl 37% (0.72 mL). The
resulting solution was refluxed for 5 h, then diluted with H2O and
extracted with AcOEt. Finally, the organic layer dried over anhy-
drous Na2SO4 and evaporated to dryness to provide the desired
5.4.1. 3-(2-((4-aminobutyl)thio)acetamido)phenyl ethyl(methyl)
carbamate (15)
Amine 15 (44.1 mg, 0.13 mmol, 75% yield) was obtained starting
from derivative 12 (79.8 mg, 0.17 mmol). 1H NMR (CDCl3):
d
1.18 (t,
product 10 (371 mg, 1.58 mmol 88% yield). 1H NMR (CDCl3):
d 1.35
J ¼ 7.0 Hz, 1.5H, CH3 rotamer); 1.25 (t, J ¼ 7.0 Hz, 1.5H, CH3 rotamer);
1.65e1.71 (m, 4H, CH2); 2.58 (t, J ¼ 6.4 Hz, 2H, CH2S); 2.70e2.78 (m,
2H, NH2); 2.97 (s, 1.5H, CH2 rotamer); 3.06 (s, 1.5H, CH2 rotamer);
3.39 (s, 2H, CH2CO); 3.48 (q, J ¼ 6.8 Hz, 1H, CH2 rotamer); 3.48 (q,
J ¼ 6.4 Hz, 1H, CH2 rotamer); 6.80e6.91 (m, 1H, Ar); 7.28e7.31 (m,
1H, Ar); 7.40e7.48 (m, 2H, Ar); 9.30 (br s, 1H, NH) ppm.
(t, J ¼ 7.5 Hz, 1H, SH); 1.64e1.70 (m, 2H, CH2); 1.76e1.84 (m, 2H,
CH2); 2.57 (q, J ¼ 7.5 Hz, 2H, CH2S); 3.70 (t, J ¼ 7.0 Hz, 2H, CH2N);
7.70e7.72 (m, 2H, Ar); 7.83e7.85 (m, 2H, Ar) ppm.
5.5. Cell culture
The immortalized murine microglial cell line BV-2 (IRCCS
Ospedale Policlinico San Martino-IST Istituto Nazionale per la
Ricerca sul Cancro, Genova, Italy) was cultured in RPMI 1640
(Sigma-Aldrich) supplemented with 2 mM Glutamine (Sigma-
Aldrich), 5% Fetal Bovine Serum (FBS) (Sigma-Aldrich) and 1% of
100 units/ml penicillin and 100 mg/ml streptomycin (Sigma
Aldrich) in tissue culture flasks at 37 ꢂC in a humidified atmosphere
and 5% CO2.
5.4.2. 3-((2-((4-aminobutyl)thio)acetamido)methyl)phenyl
ethyl(methyl)carbamate (16)
Amine 16 (60.0 mg, 0.17 mmol, 75% yield) was obtained starting
from derivative 13 (106.4 mg, 0.22 mmol). 1H NMR (CDCl3):
d 1.18 (t,
J ¼ 7.0 Hz, 1.5H, CH3 rotamer); 1.23 (t, J ¼ 7.0 Hz, 1.5H, CH3 rotamer);
1.46e1.56 (m, 2H, CH2); 1.58e1.62 (m, 2H, CH2); 2.52 (t, J ¼ 7.4 Hz,
2H, CH2S); 2.64 (t, J ¼ 6.8 Hz, 2H, CH2NH2); 2.97 (s, 1.5H, CH3
rotamer); 3.06 (s, 1.5H, CH3 rotamer); 3.26 (s, 2H, CH2S); 3.39 (q,
J ¼ 7.0 Hz, 1H, CH2 rotamer); 3.46 (q, J ¼ 7.0 Hz, 1H, CH2 rotamer);
4.46 (d, J ¼ 6.0 Hz, 2H, CH2NH); 7.02e7.05 (m, 2H, Ar); 7.12 (d,
J ¼ 7.6 Hz, 1H, Ar); 7.30e7.34 (m, 2H, Ar þ NH) ppm.
Human neuronal (SH-SY5Y) cells were routinely grown in Dul-
becco's modified Eagle's Medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, 50 U/mL penicillin and 50 mg/mL
streptomycin at 37 ꢂC in a humidified incubator with 5% CO2.
5.4.3. 3-(2-(2-((4-aminobutyl)thio)acetamido)ethyl)phenyl
ethyl(methyl)carbamate (17)
5.6. Evaluation of H2S release on BV-2
Amine 17 (77.2 mg, 0.21 mmol, 95% yield) was obtained starting
from derivative 14 (109.4 mg, 0.22 mmol). 1H NMR (CDCl3):
d
1.20
The evaluation of H2S release into the cytosol of BV-2 was
assessed by spectrofluorometric method as already described
[33,38]. Briefly, BV-2 were cultured up to about 90% confluence. 24
h before the experiment, cells were seeded onto a 96-well black
plate, at density of 72 ꢀ 103 per well in serum free medium. After
(t, J ¼ 7.2 Hz, 1.5H, CH3 rotamer); 1.25 (t, J ¼ 7.2 Hz, 1.5H, CH3
rotamer); 1.43e1.47 (m, 2H, CH2); 1.48e1.60 (m, 2H, CH2); 2.43 (t,
J ¼ 7.2 Hz, 2H, CH2S); 2.67 (t, J ¼ 6.8 Hz, 2H, CH2NH2); 2.85 (t,
J ¼ 7.0 Hz, 2H, CH2Ph); 2.98 (s, 1.5H, CH3 rotamer); 3.06 (s, 1.5H, CH3
rotamer); 3.18 (s, 2H, CH2S); 3.42 (q, J ¼ 7.2 Hz, 1H, CH2 rotamer);
3.46 (q, J ¼ 7.2 Hz,1H, CH2 rotamer); 3.56 (q, J ¼ 6.8 Hz, 2H, CH2NH);
6.93 (br s, 1H, NH); 6.98e7.04 (m, 3H, Ar); 7.27e7.31 (m, 1H, Ar)
ppm.
24 h the cells were pre-loaded with a 100 mM solution of the fluo-
rescent dye WSP-1 (Washington State Probe-1, Cayman Chemical).
In particular, WSP-1 was first incubated with BV-2 for 30 min
(allowing cells to up-load the dye), then the supernatant was
removed and replaced with a solution of the tested compounds
(1e6), reference drug or vehicle (dimethyl sulfoxide, DMSO 1%).
5.4.4. 2-(4-bromobutyl)isoindoline-1,3-dione (8)
Potassium phthalimide salt 7 (572 mg, 3.09 mmol) was added to
a solution of 1,4-dibromobutane (2.0 g, 1.11 ml, 9.26 mmol) in
acetone (5 mL). The reaction mixture was refluxed for 12 h; later
the solid was filtered off and the resulting solution was evaporated
under reduced pressure. The crude product was purified through
flash chromatography using EP/AcOEt 9:1 as eluting mixture to
afford derivative 8 (696.9 mg, 2.47 mmol, 80% yield). 1H NMR
The tested compounds (100
mM) and the reference drug diallyl di-
sulfide (DADS) 100 M [39], were first dissolved in DMSO and then
m
diluted in buffer standard (HEPES 20 mM, NaCl 120 mM, KCl 2 mM,
CaCl2$2H2O 2 mM, MgCl2$6H2O 1 mM, Glucose 5 mM, pH 7.4, at
room temperature). When WSP-1 reacts with H2S, it releases a
fluorophore detectable by a spectrofluorometer at
l
¼ 465e515 nm.
The increase of the fluorescence index (FI) was monitored for
45 min, through a spectrofluorometer (EnSpire, PerkinElmer) and
expressed as the area under the curve (AUC).
(CDCl3):
d
1.83e1.93 (m, 4H, CH2); 3.44 (t, J ¼ 6.4 Hz, 2H, CH2Br);
3.72 (t, J ¼ 6.8 Hz, 2H, CH2N); 7.71e7.73 (m, 2H, Ar); 7.83e7.86 (m,

10
S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
5.7. Analysis of cell viability, ROS and nitric oxide (NOx)
evaluated by the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) to its insoluble for-
mazan, as previously described [40]. Briefly, the treatment medium
was replaced with MTT in Hank's Balanced Salt Solution (HBSS)
(0.5 mg/mL) for 2 h at 37 ꢂC in 5% CO2. After washing with HBSS,
formazan crystals were dissolved in isopropanol. The amount of
formazan was measured (570 nm, reference filter 690 nm) using
the multilabel plate reader VICTOR™ X3 (PerkinElmer, Waltham,
MA, USA). The quantity of formazan was directly proportional to
the number of viable cells. Data are expressed as percentage of
neuronal viability versus untreated cells.
5.7.1. General conditions
BV-2 were seeded onto 6 well plates in serum free medium at a
density per well of 5 ꢀ 105 and after 24 h, the cells were treated
with the tested compounds (1e6) at the concentration of 1 mM,
with rivastigmine 1
mM or with vehicle (DMSO 0.1%). After 1 h, cells
were stimulated with lipopolysaccharide 5
mg/mL (LPS, Sigma-
Aldrich, O111:B4 E. coli) for 24 h. Cell viability, ROS and NOx were
assessed as follow with the Muse Cell Analyzer: cells were de-
tached, centrifuged for 5 min 1100 rpm and resuspended at the
density of 1 ꢀ 106 to 1 ꢀ 107 cells/ml in the provided 1X Assay
Buffer.
5.7.7. Determination of antioxidant activity
The intracellular antioxidant activity of the studied compounds
was evaluated in SH-SY5Y cells as previously described [41]. Briefly,
cells were seeded in a 96-well plate at 2 ꢀ 104 cells/well, incubated
for 24 h and subsequently incubated with the hybrid compounds 1,
5.7.2. Cell viability
20
mL of the cell suspension (prepared as previously described:
see general conditions) were added to 380
m
L of Muse™ Count &
2 and 4e6 (5
bation, the treatment medium was removed and 100
rescent probe, 20e70 dichlorodihydrofluorescein diacetate (DCFH-
DA) (10 g/mL), was added to each well. After 30 min of incubation
at room temperature, DCFH-DA solution was replaced with a so-
lution of hydrogen peroxide (H2O2) (50 M) or tert-butyl hydro-
peroxide (t-BuOOH) (50 M) for 30 min. The reactive oxygen
m
M) for 24 h at 37 ꢂC in 5% CO2. At the end of incu-
L of a fluo-
Viability Reagent and incubated protected from light at room
temperature for 5 min. Each sample was analyzed by the Muse™
Cell Analyzer using “Count and Cell Viability” protocol. Data are
expressed as percentage of cell viability recorded for BV-2 cells
treated with vehicle.
m
m
m
5.7.3. ROS
m
The Muse® Oxidative Stress Reagent working solution for cell
staining has been prepared immediately before use by diluting
Muse® Oxidative Stress Reagent stock solution 1:8000 in 1X Assay
Buffer following the manufacture's instruction. 10
suspension (see general conditions) were added to 190
species (ROS) formation was measured (excitation at 485 nm and
emission at 535 nm) using the multilabel plate reader VICTOR™ X3
(PerkinElmer). The antioxidant activity is expressed as inhibition
percentage of ROS formation induced by H2O2 or t-BuOOH.
mL of the cell
mL of Muse®
Oxidative Stress Reagent working solution and incubated protected
from light at 37 ꢂC for 30 min. Each sample was analyzed by the
Muse™ Cell Analyzer using “Oxidative stress” protocol. Data are
expressed as percentage of ROS recorded for BV-2 cells treated with
vehicle.
5.7.8. Determination of glutathione levels
The glutathione (GSH) levels were evaluated in SH-SY5Y cells as
previously described [42]. Briefly, cells were seeded in a black 96-
well plate at 2 ꢀ 104 cells/well, incubated for 24 h and subse-
quently incubated with the hybrid compounds 1, 2 and 4e6 (5 mM)
for 24 h at 37 ꢂC in 5% CO2. At the end of incubation, the treatment
medium was removed and 100 L of a fluorescent probe, mono-
5.7.4. Nitric oxide
m
The Muse® Nitric Oxide working solution was prepared
immediately before use by diluting the Muse® Nitric Oxide Reagent
stock solution 1:1000 in 1X Assay Buffer, while the Muse® 7-AAD
working solution was obtained by diluting the Muse® 7-AAD
stock solution 1:45 in 1X Assay Buffer (see manufacturer's in-
chlorobimane (MCB), was added to each well. After 30 min of in-
cubation at 37 ꢂC in 5% CO2, the GSH levels were measured
(excitation at 355 nm and emission at 460 nm) using the multilabel
plate reader VICTOR™ X3 (PerkinElmer). Data are expressed as
increase percentage of GSH levels.
structions). 10
added to 100
and each sample was incubated for 30 min in the 37 ꢂC incubator
with 5% CO2. Then, 90 L of Muse® 7-AAD working solution were
mL of cell suspension (see general conditions) were
mL of Muse® Nitric Oxide Reagent working solution
5.7.9. Nuclear extraction and determination of active Nrf2 protein
level
m
SH-SY5Y cells were seeded in 100 mm dishes at 2 ꢀ 106 cells/
added to each sample and incubate at room temperature for 5 min,
protected from light. Then, samples were analyzed by the Muse™
Cell Analyzer using “Nitric Oxide” protocol. Data are expressed as
percentage of NOx recorded for BV-2 cells treated with vehicle.
dish and incubated with the hybrid compounds 1 and 2 (5 mM) for 1,
3 and 6 h at 37 ꢂC in 5% CO2. At the end of incubation, nuclear
extraction and determination of active Nrf2 protein level were
performed using the Nuclear Extract and TransAM Nrf2 Kit (Active
Motif, Carlsbad, CA, USA), respectively, according to the manufac-
turer's guidelines. The TransAM Nrf2 Kit is a DNA-binding ELISA
able to determine the active Nrf2 protein level in nuclear extract.
The primary antibody of the kit is able to recognize an epitope on
Nrf2 protein upon ARE binding. The active Nrf2 protein levels in the
treated cells are expressed as fold increase with respect to corre-
sponding untreated cells.
5.7.5. Statistical analysis
Experiments were performed in triplicate each in three repli-
cates. All data are expressed as mean standard error (SEM); Stu-
dent t-test (followed by Bonferroni post hoc test, when required)
was selected as statistical analysis, p < 0.05 was considered repre-
sentative of significant statistical differences (software: GraphPad
Prism 6.0).
5.7.6. Determination of neuronal viability
5.7.10. Ab1e42 oligomers preparation
To establish the range of concentrations not associated with
neurotoxicity, SH-SY5Y cells were seeded in a 96-well plate at
2 ꢀ 104 cells/well, incubated for 24 h and subsequently incubated
with different concentrations of the hybrid compounds 1, 2 and
A
b1e42
peptide
was
first
dissolved
in
1,1,1,3,3,3-
hexafluoroisopropanol to 1 mg/mL, sonicated, incubated at room
temperature for 24 h and lyophilized. The resulting unaggregated
A
b1e42 peptide film was dissolved with dimethyl sulfoxide and
4e6 (1.25e40
m
M) for 24 h at 37 ꢂC in 5% CO2. The neuronal
stored at ꢁ20 ꢂC until use. The Ab1e42 peptide aggregation to
viability, in terms of mitochondrial metabolic function, was
oligomeric form was prepared as previously described [43].

S. Sestito et al. / European Journal of Medicinal Chemistry 184 (2019) 111745
11
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Alzheimer's Disease, Sci. Rep. 9 (2019) 4612.
5.7.11. MTT formazan exocytosis assay
The neuroprotection of the studied compounds against Ab1-42
was evaluated in SH-SY5Y cells as previously described [44]. Cells
were seeded in a 96-well plate at 3 ꢀ 104 cells/well, incubated for
24 h and subsequently incubated with the hybrid compounds 1, 2
and 4e6 (5 mM) for 24 h and then with Ab1-42 oligomers (10 mM) for
4 h at 37 ꢂC in 5% CO2. The neuroprotective activity, in terms of
increase in intracellular MTT granules, was measured by MTT for-
mazan exocytosis assay. Briefly, the treatment medium was
replaced with MTT in HBSS (0.5 mg/mL) for 1 h at 37 ꢂC in 5% CO2.
After the incubation, intracellular MTT granules were completely
solubilized in Tween-20 (10% v/v). The absorbance of Tween-20
soluble MTT was measured at 570 nm (reference filter 690 nm)
using the multilabel plate reader VICTOR™ X3 (PerkinElmer). Data
are expressed as percentage of neurotoxicity.
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Funding sources
[20] A. Tarozzi, F. Morroni, C. Bolondi, G. Sita, P. Hrelia, A. Djemil, G. Cantelli-Forti,
Neuroprotective effects of erucin against 6-hydroxydopamine-induced
oxidative damage in a dopaminergic-like neuroblastoma cell line, Int. J. Mol.
Sci. 13 (2012) 10899e10910.
[21] Q. Zhou, B. Chen, X. Wang, L. Wu, Y. Yang, X. Cheng, Z. Hu, X. Cai, J. Yang,
X. Sun, Sulforaphane protects against rotenone-induced neurotoxicity in vivo:
involvement of the mTOR, Nrf2, and autophagy pathways, Sci. Rep. 6 (2016)
32206.
This work was supported by grant from International Society of
Drug Discovery (ISDD), Milan and from the University of Pisa Pro-
getto di Ricerca di Ateneo 2018 (PRA_2018_20 to SR).
Acknowledgements
[22] A. Tarozzi, C. Angeloni, M. Malaguti, F. Morroni, S. Hrelia, P. Hrelia, Sulfo-
raphane as a Potential Protective Phytochemical against Neurodegenerative
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The authors thank the COST action CA15135 (Multitarget Para-
digm for Innovative Ligand Identification in the Drug Discovery
Process MuTaLig) for support. They also thank POR FSE 2014e2020
e Regione Toscana and the University of Pisa for financing the
fellowship to SS.
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