New Coumarin derivatives as cholinergic and cannabinoid system modulators

Article abstract of DOI:10.3390/molecules26113254

In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer’s di

Full text of DOI:10.3390/molecules26113254

molecules
Article
New Coumarin Derivatives as Cholinergic and Cannabinoid
System Modulators
Serena Montanari 1,†, Marco Allarà 2, Laura Scalvini 3 , Magdalena Kostrzewa 2 , Federica Belluti 1
,
Silvia Gobbi 1 , Marina Naldi 1 , Silvia Rivara 3 , Manuela Bartolini 1 , Alessia Ligresti 2
,
Alessandra Bisi 1,
*
and Angela Rampa 1,
*
1
Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, Via Belmeloro 6,
40126 Bologna, Italy; serena.montanari5@unibo.it (S.M.); federica.belluti@unibo.it (F.B.);
silvia.gobbi@unibo.it (S.G.); marina.naldi@unibo.it (M.N.); manuela.bartolini3@unibo.it (M.B.)
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council,
Via Campi Flegrei 34, 80078 Pozzuoli, Italy; mallara@icb.cnr.it (M.A.); m.kostrzewa@icb.cnr.it (M.K.);
aligresti@icb.cnr.it (A.L.)
2
3
Department of Pharmacy, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy;
laura.scalvini@unipr.it (L.S.); silvia.rivara@unipr.it (S.R.)
*
†
Correspondence: alessandra.bisi@unibo.it (A.B.); angela.rampa@unibo.it (A.R.)
Present Address: Department for Life Quality Studies, Alma Mater Studiorum University of Bologna,
Corso d’Augusto 237, 47921 Rimini, Italy.
Abstract: In the last years, the connection between the endocannabinoid system (eCS) and neuropro-
tection has been discovered, and evidence indicates that eCS signaling is involved in the regulation
of cognitive processes and in the pathophysiology of Alzheimer’s disease (AD). Accordingly, phar-
macotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease
such as AD, opening a new perspective for the development of active agents with multitarget po-
tential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed
and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets.
Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases
and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Montanari, S.; Allarà, M.;
Scalvini, L.; Kostrzewa, M.; Belluti, F.;
Gobbi, S.; Naldi, M.; Rivara, S.;
Bartolini, M.; Ligresti, A.; et al. New
Coumarin Derivatives as Cholinergic
and Cannabinoid System Modulators.
Molecules 2021, 26, 3254. https://
doi.org/10.3390/molecules26113254
CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Aβ42
was assessed. Compounds and , bearing a carbamate function, emerged as promising inhibitors of
hAChE, hBuChE, FAAH and Aβ42 self-aggregation, albeit with moderate potencies, while the amide
also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an
)
2
3
6
Academic Editor: Robert J. Doerksen
encouraging multitarget profile, deserving further evaluation.
Received: 26 April 2021
Accepted: 26 May 2021
Published: 28 May 2021
Keywords: Alzheimer’s disease; AChE; BuChE; FAAH; CB receptors; Aβ42 self-aggregation; carba-
mate; amide
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Alzheimer’s disease (AD), currently considered the most common form of dementia,
is a fatal illness inducing a progressive decline in memory and in other aspects of cogni-
tion. The complexity of its onset and progression poses one of the most difficult medical
challenges of our time [
environmental factors; however, the main risk factor is by far age. As a consequence,
the current aging population trend makes the disease prevalence more likely to rise [ ].
Three classical neuropathological changes in the brains of patients typically define AD:
the accumulation of extracellular aggregated -amyloid protein (to form senile plaques)
and intracellular aggregation of hyperphosphorylated tau protein (to form neurofibrillary
tangles), together with a severe loss of cholinergic innervation in the cerebral cortex [ ].
1]. The risk of developing AD is influenced by both genetic and
Copyright:
© 2021 by the authors.
2
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
β
3
Indeed, a marked reduction of acetylcholine (ACh), causing a loss of cholinergic tone,
is responsible for the severe cognitive impairment characteristic of AD: the “cholinergic
Molecules 2021, 26, 3254. https://doi.org/10.3390/molecules26113254
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3254
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hypothesis”, formulated in 1982, was then the first and most studied approach that de-
scribed AD pathophysiology [4]. In particular, the inhibition of cholinesterases (ChEs), the
enzymes catalyzing the degradation of ACh, remains to date the most successful approach
for the symptomatic treatment of the disease. In human brain, two main forms of ChEs are
involved in ACh hydrolysis, namely acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE). In healthy brain, AChE is the principal responsible for ACh breakdown, while
in AD-affected brain AChE levels decline, and BuChE activity progressively becomes
prevailing. Consequently, in AD patients, the regulation of central ACh levels relies on the
activity of both enzymes.
Currently, three drugs approved by FDA act at a synaptic level by inhibiting ChEs [5],
and despite the limited clinical outcomes, research on the central cholinergic system is still
thriving. Indeed, recent studies suggest that long-term use of these drugs may also have
disease-modifying benefits [6]. Although the precise mechanisms of cholinergic deficit in
AD remain to be deeply elucidated, a growing body of evidence suggests that complex
reciprocal interactions exist between central cholinergic changes and other pathophysio-
logical features of AD, including abnormal Aβ protein, tau cascade and inflammation [7].
A better elucidation of these interconnections could contribute to a reconsideration of the
cholinergic hypothesis, leading to a proper targeting of the cholinergic system, which may
also be involved in modifying AD disease progression [
found to be responsible for up-regulating the expression of the amyloid precursor protein
(APP) in cell membranes which undergoes cleavage by - and -secretase enzymes leading
to A production in a process called the amyloidogenic pathway. Thereby, inhibition of
8]. In particular, BuChE has been
β
γ
β
BuChE could be regarded as an additional approach for the treatment of moderate forms of
AD, as it would result in the increase of ACh synaptic levels and the decrease of neurotoxic
Aβ fibrils [9].
The term “endocannabinoid system” (ECS) refers to the two cannabinoid receptors
CB1 and CB2, the endocannabinoids (eCBs), and a group of enzymes involved in their
synthesis and breakdown. eCBs constitute a relatively small group of fatty acid-derived
endogenous ligands of CB1 and CB2 receptors [10]. The first two eCBs discovered, anan-
damide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG), are still
the most studied. In neurons, eCBs are synthesized and released “on demand”, in several
physiological and pathophysiological conditions, via different multi-step processes and are
catabolized by enzymatic hydrolysis [11]. Fatty acid amide hydrolase (FAAH, now also
known as FAAH-1), distributed throughout the brain as an integral membrane protein, is
the major catabolic enzyme of AEA. Hence, it has been considered a promising therapeutic
target in different inflammatory diseases, pain and other disorders [12]. 2-AG is mainly
hydrolyzed via the enzyme monoacylglycerol lipase (MAGL), but also by FAAH [13].
In recent years, the involvement of eCBs in neuroprotection has been assessed, and a
number of studies have proven that activation of eCB signaling can suppress microglial
activation and hamper the neurodegenerative processes involved in several neurological
diseases [14,15]. Accordingly, targeted pharmacotherapeutic manipulations of eCB signal-
ing have been undertaken in order to attain neuroprotective outcomes; in particular, it
may be indirectly enhanced to therapeutic levels through FAAH inhibition, making FAAH
an attractive target and selective FAAH inhibitors promising drug candidates for various
neurological and neurodegenerative/neuroinflammatory disorders, including AD [16].
Indeed, FAAH inhibitors seem more appropriate to exploit the neuroprotective nature of
eCB signaling compared to chronic application of direct CB1 agonists, thanks to a lower
risk of psychotropic (or other) adverse effects.
The lack of an effective treatment for AD is mainly related to the involvement of
multiple and complex pathways in its onset and evolution, making any drug endowed
with a single target-mechanism of action essentially ineffective. The polypharmacology
approach, i.e., the delivery of a “cocktail” of two or more drugs, could thus be beneficial for
treating such complex pathologies, even if poor patient compliance and pharmacokinetic
interferences may negatively affect this therapeutic strategy. Alternatively, a single chem-

Molecules 2021, 26, 3254
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ical compound could be purposely designed to simultaneously engage different targets
and/or pathways involved in the multifactorial nature of the disease. This multitarget
approach would greatly enhance the probability of disease modification and would be
more appreciated by patients [17,18].
In this respect, we recently reported a series of compounds as first dual ChE/FAAH
inhibitors, with activity in the nanomolar range [19
,20]. A structure-activity relationship
(SAR) study was performed and compound (Figure 1) emerged as one of the most potent
1
BuChE inhibitors reported to date (IC50 = 1.36 nM) and as a well-balanced AChE and
FAAH inhibitor (IC50 = 37.4 nM e 28.5 nM, respectively). From this study, some significant
structural data resulted, in particular:
•
the coumarin moiety gave the best results as core structure. Notably, this natural-
derived scaffold is recognized as “privileged structure” in medicinal chemistry, and it
is widely used in drug design [21,22];
•
•
•
the carbamate function is the key fragment for the interaction with the catalytic sites
of ChEs and FAAH where it is supposed to carbamoylate the catalytic serine residue;
the N,N’-benzylmethylamino group gave potent enzyme inhibition and was proposed
as the recognition element for the anionic sites of both AChE and BuChE;
a 3-methylene units linker between the coumarin and the amino function provided
high and balanced inhibition activity.
Figure 1. Structure of lead compound 1 and design strategies for the new compounds.
In this paper, appropriately designed modifications were performed on our lead com-
pound
and outline a more complete picture of their SARs (Figure 1), focusing on the carbamate
group. In detail, this portion was modified by enclosing the heptyl chain of in a 5- or
6-membered ring (compounds and , respectively), or by introducing a N-naphthyl
substituent (compound ). These modifications, aimed at maintaining or reducing the con-
1, in order to further explore the chemical space of these multi-target inhibitors
1
2
3
4
siderable lipophilicity of the parent compound, could improve the steric interactions with
the binding pocket of the target enzymes; notably, the cyclohexyl carbamate is also present
in the selective FAAH inhibitor URB597 [23]. Moreover, a small series of derivatives was
designed and synthesized replacing the carbamate with an amide group (compounds 5–7),
a favourable moiety widely reported in drugs, intermediates, pharmaceuticals, and natural
products [24], due to its ability to form important hydrogen bonding interactions. This
substitution aimed at elucidating the role of the carbamate function in enzyme blockade
and evaluating the feasibility of attaining reversible inhibitors of ChEs and FAAH.

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2. Results
2.1. Chemistry
The synthesis of compounds
2–
7
was accomplished as shown in Scheme 1. The
starting synthon 8, synthesized according to our previously described procedure [20],
was treated with the selected isocyanate, in the presence of NaH, to give the desired
compounds . Alternatively, underwent an alkylation reaction with the selected 2-
iodo-N-alkylacetamide 12 14, in the presence of K2CO3, to obtain compounds . The
2-iodo-N-alkylacetamide intermediates 12 14 were prepared by reacting 2-chloroacetyl
chloride with the selected amine in the presence of K2CO3 to obtain amides 11, which
14, more reactive species
2–
4
8
–
5–7
–
9–
were then reacted with NaI to obtain the iodinated analogues 12
–
for the following Williamson reaction.
Scheme 1. Synthesis of compounds 2–7. Reagents and conditions: (a) RNCO, NaH, toluene, rt, 24 h; (b) K2CO3, acetone,
reflux, 10 h; (c) NaI, MeCOEt, reflux; 3 h; (d) 12–14, K2CO3, acetone, reflux, 24 h.
2.2. Biological Evaluation
2.2.1. Predicted Pharmacokinetic Parameters
Lipophilicity (QPlogPo/w) and solubility (QPlogS) were calculated for compounds
1–7 and are reported in Table 1.
Table 1. Lipophilicity, solubility and blood-brain barrier permeation calculated for target compounds
and rivastigmine.
QPlogBB a,b
Comp
QPlogPo/w a
QPlogS a
1
2
3
4
5.46
4.16
4.47
5.27
2.46
4.89
3.85
4.15
−7.55
−5.74
−6.17
−6.94
−2.25
−6.07
−5.14
−5.52
−1.65
−1.01
−1.02
−1.11
0.41
−1.72
−1.22
−1.22
Riva
5
6
7
Calculated with QikProp (Schrodinger LLC); b Brain/blood partition coefficient.
a
Compared to the reference heptyl derivative 1, the new compounds 2–7 show lower
QPLogPo/w, which leads to higher predicted solubility and potential improved in vivo
distribution.

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2.2.2. Cholinesterases and FAAH Inhibition by Compounds 2–7
The newly synthesized compounds were evaluated against human recombinant AChE
(hAChE) and BuChE from human serum (hBuChE), using the method of Ellman [25] and
the inhibitory potencies, expressed as IC50 values, are reported in Table 2 (compounds
2–4) and 3 (compounds 5–7). Inhibition values (IC50s) for compound 1 and rivastigmine,
the only marketed carbamate drug approved for AD treatment, are also listed in Table 2
for comparison.
Regarding carbamates 2–4, some considerations can be drawn. As already mentioned,
previously reported SAR studies [20] identified the simple coumarin core as the best
nucleus for activity. Therefore, it was retained in the new derivatives. In order to evaluate
the effects of lipophilicity and size of the carbamic substituent, the linear heptyl chain of the
lead
1
was enclosed in a 5- or 6- membered ring, to obtain
2
and
3, showing a lower logP
value and an increased steric hindrance. In compound
group was introduced.
4, the bulkier and planar naphthyl
The inhibition of ChEs by carbamates involves the formation of a reversible complex
with subsequent formation of a covalent adduct [26]. It is also well known that carbamoy-
lation rate is influenced by the nature of the substituent at the N-carbamoyl group, as
previously shown for
1 [20]. Hence, by bearing different R groups, derivatives 2–4 may
require different (incubation) time to form the covalent adduct and inhibit AChE and
BuChE activities. Given these premises, we initially investigated the time-dependent ChEs
inhibition by
As shown for
2
1
–
[
4
by evaluating the time required to achieve a plateau in the inhibition plot.
20] and other carbamates [19 27], due to the larger enzyme gorge (790 Å vs.
,
1133 Å [28]) that allows accommodating bulkier substituents, the carbamoylation kinetics
was faster on hBuChE than hAChE, independently from the nature of the R substituent
(see Figure 2).
Table 2. Inhibitory activities against hAChE, hBuChE, rFAAH (IC50), and Aβ42 self-aggregation (% inhibition) values of
compounds 2–4 and of the marketed anti-AD drug Rivastigmine (Riva).
IC50 FAAH
with 20 min
Pre-incubation
µM
% Inhibition
IC50 hAChE
(±SEM)
nM a
IC50 FAAH
(m ± sd)
µM
IC50 hBuChE
Aβ self
42
Comp.
R
b
(±SEM) nM a
Aggreg.
(±SEM)
1
2
37.4 ± 1.9 c
521 ± 14 d
1.36 ± 0.07 c
8.42 ± 0.13 e
0.161 ± 0.027 c
6.09 ± 1.55
0.029 ± 0.001 c
nd
2.53 ± 0.55
22.9 ± 1.3
18.9 ± 2.1
104 ± 21 f
34.9 ± 4.2 d
3
4
2.55 ± 0.33
1.33 ± 0.28
264 ± 0.05 f
1535 ± 64 g
2.00 ± 0.20 d
301 ± 14 g
1.58 ± 0.26
0.63 ± 0.08
13.9 ± 3.8
Riva
nd
nd
nd
a
Human recombinant AChE and BuChE from human serum were used. IC50 values represent the concentration of inhibitor required to
decrease enzyme activity by 50% and are the mean of two/three independent measurements, each performed in duplicate. SEM = standard
error of the mean; b [A
β
42] = [inhibitor] = 50
µ
M; c Data from reference [20]; d Incubation time 60 min; e Incubation time 45 min; f Incubation
time 90 min; g Data from reference [27]; nd stands for not determined.

Molecules 2021, 26, 3254
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(a)
(b)
Figure 2. Time-dependent inhibition of (
a) hAChE and (
b) hBuChE by carbamates 2–4 (black lines) and lead compound 1
(grey line) at their IC50 concentrations.
The fastest carbamoylation rate was observed in both cholinesterase enzymes with
the cyclopentyl-derivative , with the following trends: cyclopentyl- ( ) > cyclohexyl ( ) >
naphtyl-derivative ( ), and cyclopentyl- ( ) > naphtyl ( ) > cyclohexyl-derivative ( ) for the
2
2
3
4
2
4
3
carbamoylation of hAChE and hBuChE, respectively. When the carbamoylation kinetics of
new derivatives were compared to that previously determined for 1, it was observed that
replacement of the heptyl chain of
in detail, for AChE inhibition, replacement of the heptyl chain of
or a cyclohexyl ( ) group eased the carbamoylation reaction, while no significant difference
was observed between and . Conversely, for hBuChE inhibition, all new carbamates
showed a significantly slower carbamoylation kinetics compared to that of . Hence, as
1
led to opposite effects on hAChE and hBuChE. More
1
with a cyclopentyl (2)
3
1
4
1
a consequence of this opposite trend, although a difference still remains, the inactivation
rates of the two cholinesterases by 2–4 were, in general, less divergent than those observed
for
the last aspect, hAChE carbamoylation by the drug rivastigmine occurs in a time frame
close to that observed for , being the pseudo-first order kobs values for rivastigmine and
determined at their IC50 value, 0.059 min−1 [27] and 0.038 min−1, respectively.
1, making an in vivo inhibition of both cholinesterases more likely to happen. As for
2
2,
Considering that time clearly affects the inhibition rate, in order to achieve a proper
comparison of the inhibitory potencies, in the determination of the IC50 values the incu-
bation time was adjusted according to the time required to each carbamate to form the
covalent adduct, following the same approach previously used for the determination of
the inhibitory potency of 1 [20]. This strategy also allowed a better definition of the SARs
of this new class of carbamates.
All the tested compounds turned out to inhibit hBuChE more effectively than hAChE,
and the potency trend observed was naphthyl > cyclopentyl > cyclohexyl. In particular,
going from the cyclopentyl derivative
in activity can be observed, whereas the introduction of the planar naphthyl group in
derivative allowed obtaining the most potent BuChE inhibitor in the series, with activity
in the low nanomolar range and comparable to that of compound . Conversely, these
modifications negatively affected the inhibitory activity on hAChE, with potency decreasing
from cyclohexyl to naphthyl and cyclopentyl, with the most potent derivative showing a
three-fold reduction in potency with respect to . Remarkably, all carbamic compounds
proved to be more active than the marketed drug rivastigmine, with compound showing a
2 to the homologue cyclohexyl 3, a four-fold decrease
4
1
3
1
3
fifteen-fold increase in potency. The peculiar selectivity profile of this subset of compounds
makes them of particular interest in treating moderate forms of AD, where the progressive
reduction of AChE levels in the cholinergic synapses upgrades the role of BuChE, which
becomes the main responsible for central cholinergic tone regulation.

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Looking at the inhibitory activity on FAAH, expressed as IC50 values against AEA
hydrolysis by FAAH in rat brain membranes and reported in Table 2, a ten-fold reduc-
tion in activity can be noticed with respect to
1. As reported for BuChE, the cycloalkyl
carbamates and were the least active, with the cyclohexyl derivative 3
2
3
slightly more
potent. Surprisingly, these coumarin-based derivatives seem not to benefit from the intro-
duction of cycloalkyl groups in the carbamic function, unlike biphenyl derivatives such as
URB597 [23]. Notably, compound 4, bearing the aromatic naphthyl group, again proved to
be the most promising, with potency in the low micromolar range.
A docking study has been performed (see below) to rationalize SARs and to get
insights into the molecular interactions with the targets. In this respect, the discrepancy
between the IC50 values observed with or without the pre-incubation lapse seems to
strongly support a covalent inhibition mechanism.
The selectivity towards the other regulator of eCBs levels, MAGL, was also evaluated
and all the compounds turned out to be inactive (data not shown). This finding is actually
convenient, as dual FAAH/MAGL inhibition was reported to induce cannabimimetic side
effects [29].
Taking into account the increasing relevance of amide-containing compounds in
medicinal chemistry [24], a small set of easily affordable amide derivatives 5–7 were
designed and synthesized in order to evaluate the significance of the carbamic group in
enzymatic inhibition. As reported in Table 3, the results of biological evaluation clearly
indicated a pivotal role for the carbamic function in enzyme inhibition, showing amide
derivatives reduced activities on both ChEs and FAAH.
Table 3. Inhibitory activity against hAChE, hBuChE, rFAAH and Aβ42 self-aggregation.
hAChE
hBuChE IC50
(±SEM)
µM a
IC50 FAAH
(m ± sd)
µM
% Inhibition Aβ
42
% Inhibition
Comp.
R
Self-Aggregation b
(±SEM) a
(±SEM)
[I] = 40 µM
5
6
13.6 ± 5.1
182 ± 28
>1000
>1000
n.a.
41.7 ± 0.5 c
11.6 ± 0.8
20.3 ± 1.9
7
34.0 ± 3.2
12.8 ± 2.1
>1000
13.5 ± 5.0
a
Human recombinant AChE and BuChE from human serum were used. IC50 values represent the concentration of inhibitor required to
decrease enzyme activity by 50% and are the mean of two/three independent measurements, each performed in duplicate. SEM = standard
error of the mean; b [A
tested concentration.
β42] = [inhibitor] = 50
µ
M; c IC50 = (59.4
±
1.0) µM; na stands for not active, i.e., % inhibition lower than 10% at the
In particular compound 5, bearing the same linear heptyl chain of our lead 1, demon-
strated an impressive decrease in inhibitory potency towards hBuChE, while the cy-
clopentyl and cyclohexyl analogues retained IC50 values in the low micromolar range.
On hAChE, only compound 6, bearing the cyclopentyl group, exhibited an interesting
inhibitory activity profile. The structural requirements for FAAH inhibition seemed even
more limiting, being all amide compounds inactive both on this target and on MAGL (data
not shown).

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2.2.3. BuChE and FAAH Docking Studies
Docking studies were performed to explore the binding mode of
2–
4
within the
binding site of hBuChE (PDB: 4TPK [30]), for which they showed nM inhibitory potencies.
The three compounds gave binding poses consistent with carbamoylation of the catalytic
residue S198, with the carbamate group close to the catalytic triad (S198, H438 and E325)
and forming hydrogen bonds with the oxyanion-hole residues (G116 and G117). The
compounds adopt a folded conformation, with the propylaminomethyl linker and the
coumarin group occupying the entrance of the gorge. The carbamate O-phenyl ring
is accommodated at the bottom of the binding site, delimited by the indole nucleus of
W82. The amino group, modelled in its protonated state, takes polar contacts with the
hydroxyl group of T120. The binding of the three compounds is stabilized by different
polar interactions between hydrophilic residues situated at the entrance of the gorge and
the coumarin portion. The acyl chain binding pocket of hBuChE, delimited by L286 and
V288, contains the carbamate N-substituent. In particular, the naphthyl nucleus of
4 is
well accommodated in this pocket, occupying the same region as the naphthamide moiety
of a selective, non-covalent BuChE inhibitor co-crystallized within the X-ray structure
4TPK (Figure 3A). Notably, this hydrophobic cleft is characterized by a reduced volume
in hAChE, with L286 and V288 of hBuChE replaced by the bulkier residues F295 and
F297, which hamper the accommodation of large N-substituents. The reduced volume
can account for the selectivity observed for the new inhibitors, with a 100-fold difference
in IC50 values observed for the naphthyl derivative 4. When docked in the substrate
binding site of hAChE (PDB: 4EY6 [31]), this compound is not able to properly insert the
carbamate group in the catalytic site due to steric clashes of the naphthyl ring with the two
phenylalanines and the non-optimal accommodation of the inhibitor is likely reflected in
the lower inhibitory potency toward hAChE (Figure 3B).
Figure 3. (A) Docking of compound 4 (green carbons) in the substrate binding site of hBuChE (grey ribbons). The coumarin
head points towards the entrance of the gorge undertaking polar contacts with the backbone of N68. The N-naphthyl group
fits the acyl chain binding pocket, overlapping the position of the naphthamide group of the inhibitor crystallized in the
X-ray structure 4TPK (purple carbons). (B) Docking of compound 4 (green carbons) in the substrate binding site of hAChE
(cyan ribbons). Given the presence of the bulkier F295 and F297, the N-naphthyl group cannot occupy the acyl chain binding
pocket. Hydrogen bonds are indicated with orange dotted lines.
Docking studies were also performed to investigate the binding mode of compounds
2–4 within the active site of rat FAAH (PDB: 1MT5 [32]). We started from the hypothesis,
supported by the lack of activity of amide analogues 5–7 and the increase in potency
observed with inhibitor pre-incubation (Table 2), that these compounds carbamoylate the
active serine, similarly to what had been proposed for compounds of the same series [20].
We thus analyzed the induced-fit-docking poses that accommodate the carbamate carbonyl

Molecules 2021, 26, 3254
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within the oxyanion site of the enzyme. The coumarin nucleus and the propylaminomethyl
linker occupy a wide region situated at the interface between the two subunits of the FAAH
homodimer. This region, also referred to as the cytosolic port, is lined by hydrophilic
residues and represents the exit route of the ethanolamine released upon cleavage of the
substrate anandamide. More specifically, 2–4 occupy the upper region of the cytosolic
port cleft, similar to the arrangement seen for other carbamate FAAH inhibitors having
the same O-substituent. The cyclic N-substituents are positioned in the hydrophobic
acyl chain binding (ACB) channel, which is involved in the recruitment of the substrate
from the membrane, and is occupied, in a crystallized structure (PDB: 3LJ7 [33]), by the
cyclohexyl fragment of the reference FAAH inhibitor URB597 covalently bound to S241
(Figure 4A). The cycloalkyl carbamates 2 and 3 showed lower FAAH inhibitory potencies
than previously described analogues with primary alkyl chains at the nitrogen atom. This
can be ascribed to the greater steric bulk, which influence the overall accommodation of
the inhibitor. In fact, the docking poses of 2 and 3 do not allow the formation of a hydrogen
bond between the methylamino group of the ligands and T236, contrary to what had been
observed for primary N-alkyl derivatives. On the other hand, the naphthyl nucleus is
better tolerated at FAAH binding site, with a partial recovery of inhibitory potency. This
apparently contrasts with the lack of activity observed for the p-hexylphenyl-carbamate in a
previous work [20]. Actually, the docking pose (Figure 4A) shows that the naphthyl nucleus
is accommodated within the first portion of the ACB pocket, similarly to the N-cyclohexyl
group of other covalent inhibitors (Figure 4B). As the ACB channel makes a turn just after
this first portion of space, bulky substituents at the para position of a phenyl ring would
cause steric clashes.
Figure 4. (A) Binding mode of 4 (green carbons) within the active site of rFAAH. The coumarin ring occupies the cytosolic
port at the interface between the two subunits of the FAAH homodimer (chain A, light blue ribbons; chain B, beige
ribbons). The N-naphthyl substituent fits the ACB channel and overlaps the cyclohexyl fragment of URB597 (purple
carbons) covalently bound to the catalytic serine in the X-ray structure 3LJ7, superposed to the docking complex. Polar
interactions are marked with orange dotted lines. (B) Surface of the ACB channel of FAAH with docked 4.
2.2.4. CB Receptors Binding Assay for Compounds 5–7
Taking advantage of the widespread placement of the amide function in potent CB
ligands, the ability of the newly synthesized compounds 5–7 to bind these receptors was
also evaluated. The binding affinities of compounds 5–7 at human recombinant CB1 and
CB2 receptors were determined by a competition assay using a high affinity radioligand,
i.e., [3H]-CP-55,940 and results are reported in Table 4.

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Table 4. Effect of compounds 5–7 on [3H]-CP55940 binding to the human recombinant CB1 and CB2 receptors.
Max Tested on CB1
(% Displacement)
(m ± sd)
Max Tested on CB2
Ki on CB1
(m ± sd)
µM
Ki on CB2
(m ± sd)
µM
(% Displacement)
(m ± sd)
Comp.
µM
µM
5
6
7
>10
>10
>10
10 (35.57 ± 0.43)
10 (35.75 ± 0.65)
10 (39.62 ± 10.48)
>10
>10
>10
10 (41.41 ± 0.87)
10 (48.33 ± 1.73)
10 (14.3 ±3.83)
Derivatives
5
–
7
appear weak ligands, with IC50/Ki in the
µM range, showing com-
pounds 5 and 6 the highest potency on CB2 and compound 7 a slight selectivity for CB1.
2.2.5. Inhibition of Aβ42 Self-Aggregation for Compounds 2–7
Finally, in a multitarget perspective, the compounds were tested to evaluate their A
anti-aggregation properties. Indeed, FAAH proved to co-localize with A rich plaques and
activated astrocytes, suggesting a correlation between FAAH expression and AD-related
neuropathologic changes [34]. The results indicate a weak inhibitory activity on self-A
aggregation, allowing some interesting considerations: the nature of the terminal function
(carbamic or amidic) appears irrelevant for activity ( vs. and vs. ), whereas the
β
β
β
2
6
3
7
N-substituent seems to play a more prominent role, being the cycloaliphatic groups more
effective than the naphthyl one in the carbamate series and the cyclopentyl more than the
cyclohexyl one in the amide series.
3. Discussion
Starting from a previously reported lead compound 1, suitably designed modifications
were applied in a multitarget perspective, with a particular focus on the carbamate moiety.
Besides, in a small number of compounds the carbamate was replaced with an amide
group, widely reported in medicinal chemistry. The new derivatives were evaluated for
their activities on a panel of the selected target embroiled in AD. The results proved that
the carbamic derivatives
AChE, BuChE, FAAH and Aβ42 self-aggregation, combined with calculated brain-to-blood
ratios lower than rivastigmine (QPLogBB in Table 1), but better than the n-heptyl lead
2 and 3 were endowed with promising inhibitory activities on
1.
Undoubtedly, the potencies related to the single activities of these twocompounds appear
moderate, but, remarkably, all the selected targets are involved in the pathogenesis and
progression of the same disease and, from a multitarget point of view, this behaviour
acquires a pivotal relevance. Indeed, the derivatives may counteract AD by simultaneously
modulating different networked pathways involved in the disease development. The
cholinergic system and ACh play a crucial role in learning and in the formation of memory.
A vicious circle is recognized between A
and facilitates its precipitation around plaques, A
tion and a severe loss of AChE. Additionally, an excess of A
β
and AChE: AChE directly interacts with soluble
induce cholinergic system dysfunc-
can simultaneously affect
Aβ
β
β
AEA production and degradation, by both reducing the availability of its biosynthetic
precursor NArPE and by overexpressing FAAH, respectively [35].
It should be noted that levels of BuChE, the enzyme that compensates for AChE in
hydrolyzing central ACh, increase as the disease progresses. Moreover, recent studies
demonstrate that BuChE can hydrolyze 2-AG to arachidonic acid, which may give evidence
for a more specific role of this enzyme in endocannabinoid regulation by CB receptors
activation [36]. This action will be added to the inhibition of endocannabinoid inactivation
by FAAH, obtaining beneficial effects in terms of both anti-inflammatory and neuropro-
tective effects and, subsequently, anti-amnesic actions. On the other hand, activation of
CB1 was suggested to contribute to the amnesic actions of A
tions, depending on the timing of administration, and to inhibit A
levels increase in AD and its stimulation counteracts microglia activation induced by A
β
or to counteract these ac-
β
toxicity in vitro. CB2
β,

Molecules 2021, 26, 3254
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a beneficial effect found in both in vitro and in vivo models and stimulates A
β
removal
by macrophages.
In the amide series, compound 6 emerged as an interesting multipotent inhibitor of
AChE, BuChE and Aβ42 self-aggregation, as well as a potential CB1/CB2 receptors ligand.
In summary, some of these compounds show an encouraging multitarget profile and
deserve further pharmacological characterization to deeper exploit their therapeutic potential.
4. Materials and Methods
4.1. Chemistry
4.1.1. General Methods
Starting materials, unless otherwise specified, were used as high grade commercial
products. Solvents were of analytical grade. Reaction progress was followed by thin
layer chromatography (TLC) on precoated silica gel plates (Silica Gel 60 F254, Merck,
Darmstadt, Germany and then visualized with a UV254 lamplight. Melting points were
measured in glass capillary tubes on a SMP-20 apparatus (Büchi Italia, Assago, MI, Italy )
1
and are uncorrected. H-NMR and 13C-NMR experiments were recorded in CDCl3, unless
differently indicated, on a VXR 400 MHz instrument (Varian, Palo Alto, CA). Chemical
shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS), and spin
multiplicities are given as s (singlet), d (doublet), t (triplet), m (multiplet) or br (broad).
Direct infusion ESI-MS spectra were recorded on a Micromass ZQ 4000 apparatus (Waters,
San Diego, CA, USA). Chromatographic separations were performed on silica gel columns
(Kieselgel 40, 0.040 0.063 mm; Merck) by flash chromatography. Compounds were named
−
relying on the naming algorithm developed by CambridgeSoft Corporation (Waltham, MA,
USA) and used in ChemDraw Professional 15.0 (PerkinElmer Inc., Waltham, MA, USA).
4.1.2. General Method for the Preparation of Carbamates 2–4
A mixture of 7-(3-((3-hydroxybenzyl)(methyl)amino)propoxy)-2H-chromen-2-one
(8) (1.0 mmol), the selected isocyanate (1.0 mmol) and NaH (10 mg) in dry toluene
(20 mL) was stirred at rt for 24 h, quenched with water and then extracted with
dichloromethane (3 × 20 mL). The organic layer was washed with water, dried and
evaporated under reduced pressure. The residue was purified by flash column chro-
matography (toluene/acetone 3:2).
3-((Methyl(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)amino)methyl)phenyl
cyclopentylcarbamate (2)
Using the previous procedure and starting from
8
and cyclopentane isocyanate com-
1
pound
2
was obtained. Yield 30 %, oil. H-NMR
δ
7.60 (d, J = 9.6 Hz, 1H), 7.31 (d, J = 9.2 Hz,
1H), 7.24–7.19 (m, 1H), 7.07 (t, J = 7.6 Hz, 2H), 6.94 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 7.8 Hz,
2H), 6.21 (d, J = 9.6 Hz, 1H), 5.13 (br, 1H, NH), 4.02 (t, J = 6.3 Hz, 2H, OCH2), 3.47 (s, 2H,
NCH2-Ph), 2.50 (t, J = 6.4 Hz, 2H, NCH2), 2.22 (s, 3H, NCH3), 2.01–1.93 (m, 5H), 1.69–1.62
(m, 2H), 1.60–1.51 (m, 2H), 1.49–1.46 (m, 2H). 13C-NMR
δ 162.5, 161.6, 155.7, 153.8, 151.3,
143.2, 140.4, 128.8, 126.0, 122.3, 120.4, 116.7, 113.5, 113.0, 112.6, 101.2, 66.6, 62.4, 55.2, 52.2,
42.8, 33.9, 33.6, 27.1, 25.6, 25.4. ESI-MS m/z: 451 (M + H+).
3-((Methyl(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)amino)methyl)phenyl
cyclohexylcarbamate (3)
Using the previous procedure and starting from
8 and cyclohexyl isocyanate, com-
1
pound
3
was obtained. Yield 80 %, oil. H-NMR
δ
7.64 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 8.8 Hz,
1H), 7.27–7.24 (m, 1H), 7.12 (d, J = 9.2 Hz, 2H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 6.8 Hz,
2H), 6.23 (d, J = 9.6, 1H), 5.04 (br, 1H, NH), 4.07 (t, J = 6.4 Hz, 2H), 3.55 (s, 2H), 2.53 (t,
J = 6.4 Hz, 2H), 2.29 (s, 3H), 2.18–1.93 (m, 4H), 1.76–1.72 (m, 3H), 1.64–1.61 (m, 2H), 1.43–
1.32 (m, 4H). 13C-NMR
δ 162.4, 161.4, 155.8, 153.8, 151.1, 143.6, 140.4, 128.6, 125.6, 122.1,
120.3, 116.2, 113.1, 112.9, 112.8, 101.3, 66.7, 62.0, 53.2, 50.2, 42.3, 33.9, 33.2, 26.8, 25.6, 25.4,
24.8. ESI-MS m/z: 465 (M + H+)

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3-((Methyl(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)amino)methyl)phenyl
naphthalen-2-ylcarbamate (4)
Using the previous procedure and starting from
8
and 2-isocyanatonaphthalene,
7.83–7.79 (m, 4H), 7.55
compound
4
was obtained. Yield 50 %, mp 190–192 ◦C. 1H-NMR
δ
(d, J = 9.6 Hz, 1H), 7.49–7.45 (m, 2H), 7.31–7.25 (m, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.11 (d,
J = 7.6 Hz, 1H), 7.04 (d, J = 7.6, 1H), 6.81–6.79 (m, 2H), 6.19 (d, J = 9.6 Hz, 1H), 5.81 (br, 1H,
NH), 4.04 (t, J = 6.8 Hz, 2H), 3.51 (s, 2H), 2.52 (t, J = 6.8 Hz, 2H), 2.28 (s, 3H), 2.01–1.94 (m,
2H). 13C-NMR
δ 161.4, 155.8, 154.4, 151.7, 143.4, 135.3, 133.5, 133.0, 130.6, 129.1, 128.8, 128.3,
127.9, 127.8, 126.5, 126.4, 126.2, 126.1, 125.9, 124.7, 123.7, 113.6, 113.2, 112.1, 102.2, 65.4, 59.3,
52.6, 40.0, 24.2. ESI-MS m/z: 509 (M + H+).
4.1.3. General Method for the Preparation of Acetamides 5–7
To a solution of 8 (1.0 mmol) in acetone (30 mL), the selected iodoacetamide (12–14,
1.0 mmol) and K2CO3 were added and the mixture was stirred and refluxed for 24 h. Upon
reaction completion, the mixture was hot filtered and the solvent was evaporated under re-
duced pressure. The resulting crude product was purified by flash column chromatography
(toluene/acetone 4:1), and then triturated with petroleum ether.
N-Heptyl-2-(3-((methyl(3-((2-oxo-2H-chromen-7-
yl)oxy)propyl)amino)methyl)phenoxy)acetamide (5)
Using the previous procedure and starting from
8 and N-heptyl-2-iodoacetamide (12),
◦
the final acetamide
5
was obtained. Yield 30 %, mp 40–42 C. 1H-NMR
δ 7.63 (d, J = 9.6 Hz,
1H), 7.36 (d, J = 8.4 Hz, 1H), 7.27–7.21 (m, 1H), 6.96 (d, J = 7.2 Hz, 1H), 6.91 (s, 1H), 6.82–6.77
(m, 3H), 6.81 (br, 1H), 6.25 (d, J = 9.6 Hz, 1H), 4.44 (s, 2H), 4.09 (t, J = 6.4 Hz, 2H), 3.50 (s,
2H), 3.35–3.30 (m, 2H), 2.56 (t, J = 6.8 Hz, 2H), 2.25 (s, 3H), 2.03–1.98 (m, 2H), 1.55–1.50 (m,
2H), 1.34–1.26 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H). 13C-NMR
δ 162.6, 161.7, 156.4, 156.0, 143.8,
140.4, 129.5, 128.8, 121.2, 116.2, 114.6, 113.3, 112.9, 112.5, 101.5, 67.5, 66.7, 53.6, 42.4, 39.2,
31.8, 29.7, 29.1, 26.9, 22.7, 14.2, 11.3. ESI-MS m/z: 495 (M + H+).
N-Cyclopentyl-2-(3-((methyl(3-((2-oxo-2H-chromen-
7yl)oxy)propyl)amino)methyl)phenoxy)-acetamide (6)
Using the previous procedure and starting from
13), the final acetamide
8
and N-cyclopentyl-2-iodoacetamide
1
(
6
was obtained. Yield 45 %, oil. H-NMR
δ
7.65 (d, J = 9.6 Hz,
1H), 7.36 (d, J = 8.4 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.91 (s, 1H), 6.88 (s, 1H), 6.84–6.80
(m, 2H), 6.75–6.73 (m, 1H), 6.26 (d, J = 9.2 Hz, 1H), 4.11 (t, J = 6.2 Hz, 2H), 3.48 (s, 2H),
2.57 (t, J = 6.4 Hz, 2H), 2.25 (s, 3H), 2.03–1.98 (m, 3H), 1.65–1.58 (m, 4H), 1.44–1.36 (m, 4H).
13C-NMR
δ 162.6, 161.7, 156.4, 156.0, 143.8, 140.4, 129.5, 128.8, 121.2, 116.2, 114.6, 113.3,
112.9, 112.5, 101.5, 66.9, 62.5, 54.8, 53.6, 42.2, 42.3, 33.2, 27.2, 26.9, 23.8, 23.7. ESI-MS m/z:
465 (M + H+).
N-Cyclohexyl-2-(3-((methyl(3-((2-oxo-2H-chromen-7-
yl)oxy)propyl)amino)methyl)phenoxy)-acetamide (7)
Using the previous procedure and starting from
8
and N-cyclohexyl-2-iodoacetamide 14
,
the final acetamide
7
was obtained. Yield 25 %, oil. 1H-NMR
δ
7.63 (d, J = 9.6 Hz, 1H), 7.35
(d, J = 7.6 Hz, 1H), 7.22 (t, J = 8 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 6.91 (s, 1H), 6.80–6.78 (m,
3H), 6.42 (d, J = 8 Hz, 1H), 6.24 (d, J = 9.5 Hz, 1H), 4.42 (s, 2H), 4.08 (t, J = 6 Hz, 2H), 3.86–3.84
(m, 1H), 3.50 (s, 2H), 2.58 (t, J = 6.9 Hz, 2H), 2.25 (s, 3H), 2.03–2.00 (m, 2H), 1.92–1.90 (m, 2H),
1.68–1.61 (m, 4H), 1.41–1.32 (m, 2H), 1.25–1.16 (m, 2H). 13C-NMR
δ 167.3, 162.3, 161.4, 157.4,
155.9, 143.6, 141.1, 129.6, 128.8, 122.7, 115.3, 113.4, 113.1, 112.9, 112.5, 101.6, 67.4, 66.7, 62.3, 53.6,
47.9, 42.3, 33.2, 33.1, 26.9, 25.5, 25.0, 24.9. ESI-MS m/z: 479 (M + H+).

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4.1.4. General Method for the Preparation of Amide Derivatives 9–11
To a solution of selected amine (4.34 mmol) in acetone (20 mL), anhydrous K2CO3
(1.0 g) and 2-chloroacetyl chloride (4.34 mmol) were added and the mixture was stirred
and refluxed for 24 h. Upon reaction completion, the mixture was hot filtered and the
solvent was evaporated under reduced pressure. The residue was purified by flash column
chromatography (petroleum ether/ethyl acetate 4:1). The compounds are already reported
in literature with different synthetic procedures. The full characterization can be found in
the corresponding reference.
2-Chloro-N-heptylacetamide (9)
Using the previous procedure and starting from heptylamine, compound
9 was
obtained [37]. Yield 89 %, oil. 1H-NMR
δ
6.65 (br, 1H), 4.02 (s, 2H), 3.30–3.25 (m, 2H),
1.56–1.49 (m, 2H), 1.32–1.28 (m, 8H), 0.88 (t, J = 7.2 Hz, 3H)
2-Chloro-N-cyclopentylacetamide (10)
Using the previous procedure and starting from cyclopentylamine, compound 10 was
obtained [38]. Yield 95 %, oil. 1H-NMR
6.47 (br, 1H), 4.25–4.20 (m, 1H), 4.02 (s, 2H),
δ
2.06–1.98 (m, 2H), 1.75–1.62 (m, 4H), 1.48–1.40 (m, 2H).
2-Chloro-N-cyclohexylacetamide (11)
Using the previous procedure and starting from cyclohexylamine, compound 11 was
obtained [39]. Yield 85 %, oil. 1H-NMR
δ 6.51 (br, 1H), 4.03 (s, 2H), 3.54–3.41 (m, 1H),
1.94–1.88 (m, 2H), 1.46–1.41 (m, 4H), 1.21–1.11 (m, 4H).
4.1.5. General Method for the Preparation of Iodo Derivatives 12–14
A mixture of the selected chloro derivative (1.0 mmol) and NaI (1.0 mmol) in
methylethylketone (15 mL) was refluxed for 3 h, then it was concentrated under
reduced pressure. The crude was dissolved in dichloromethane and washed with water.
The organic layer was evaporated under reduced pressure, affording compounds 12–14
that were used for the next reaction without any further purification.
4.1.6. General Method for the Preparation of Isocyanates
A mixture of selected benzoic acid (1.0 eq) and SOCl2 (10 mL) was refluxed for 5 h,
then SOCl2 was removed. To an ice-cold mixture of NaN3 (1.4eq) previously dissolved
in water (2 mL), a solution of the selected benzoyl chloride in acetone (2 mL) was added,
◦
keeping the temperature below 10 C. The mixture was stirred for 1 h, then the layers were
separated and the lower aqueous layer was disc◦arded; the upper layer was slowly and
dropwise added to 30 mL of benzene heated to 60 C and the mixture was kept at 60–70 ◦C
until gas production ceased; then it was filtered and the solvent was removed. The obtained
oil was used as a crude, without any further purification, for next step of reaction.
4.2. Biological Evaluation Methods
4.2.1. Determination of the Inhibitory Potency towards Human AChE and BuChE
The capacity of 2–7 to inhibit human ChE activity was assessed using the Ellman’s
◦
method [25]. Initial rate assays were performed at 37 C with a V-530 double beam
spectrophotometer (Jasco Corporation, Cremella (LC), Italy city, state/prov abbrev if
USA/Canada, country). The rate of increase in the absorbance at 412 nm was followed
for 180 s. AChE stock solution was prepared by dissolving human recombinant AChE
lyophilized powder (Sigma-Aldrich, Merck group, Milan, Italy) in 0.1 M phosphate buffer
at pH 8.0 containing 0.1% Triton X-100. Stock solution of BuChE was prepared by diluting
commercial stock solution of BuChE from human serum (Sigma) in an aqueous solution
of gelatin (0.2%) in order to have a final activity comprised between 0.11–0.15 ∆Abs/min.
Stock solutions of inhibitors (1–2 mM) were prepared in methanol and diluted in methanol.
For IC50 assessment, five/six increasing concentrations of the inhibitor were evaluated, able

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to give an inhibition of the enzymatic activity in the range of 20–80%. The assay solution
consisted of a 0.1 M phosphate buffer pH 8.0, with the addition of 340
µM (Ellman’s reagent,
0.02 unit/mL of human recombinant AChE or BuChE from human serum and 550
µM of
substrate (acetylthiocholine iodide-ATCh or butyrylthiocholine iodide–BTCh for AChE
and BuChE, respectively). Aliquots of the tested inhibitor at increasing concentration
(or methanol) were added to the assay solution and incubated with the enzyme at 37 ◦C
for 20 min for non-covalent derivatives 5–7, and for the time required to reach a stable
inhibition value (carbamoylation plateau) for carbamates 2–4, before the addition of the
substrate. Incubation times were: 60 min for 2 and 90 min for 3 and 4 on hAChE assay,
and 45 min for 2 and 60 min for 3 and 4 on hBuChE assay, as assessed on the preliminary
determination of the carbamoylation rate. Blank solutions containing all components
except the enzyme were prepared in order to account for the non-enzymatic hydrolysis
of the substrate. The reaction rates obtained in the absence and presence of inhibitor
were compared and the percent inhibition due to the presence of inhibitor was calculated.
Each concentration was analyzed in duplicate/triplicate, and IC50 values were determined
graphically from log concentration–inhibition curves (GraphPad Prism 4.03 software,
GraphPad Software Inc., San Diego, CA). Two independent experiments were performed
for the determination of each IC50 value or for the calculation of the % of inhibition where
the IC50 value could not be determined because of low inhibitory activity.
4.2.2. Determination of Carbamoylation Events for Carbamic Derivatives 2–4
A stopped time assay was performed in order to define the time required for enzyme
carbamoylation. In detail, residual enzyme activity upon selected incubation times was
assessed by following the same protocol described in section “Determination of the in-
hibitory potency towards human AChE and BuChE”. Briefly, the specific cholinesterase
enzyme (either hAChE or hBuChE) was mixed in the assay buffer at pH 8.0 in the presence
of DTNB (340 µM final concentration) with a concentration of inhibitor equal to its IC50
value. The mixture was incubated at 37 ◦C for given times (from 5 to 90 min) before the
addition of the substrate (AChT or BChT, respectively) and the determination of residual
enzyme activity by Ellman’s method. A parallel control, with no inhibitor, allowed to
adjust activities measured at various time.
4.2.3. Inhibition of Aβ42 Self-Aggregation
As reported in a previously published protocol [40], 1,1,1,3,3,3-hexafluoro, 2-propanol
pretreated Aβ42 samples (Bachem AG, Bubendorf, Switzerland) were solubilized with
a CH3CN/0.3 mM Na2CO3/250 mM NaOH (48.4:48.4:3.2) mixture. Experiments were
performed by incubating the peptide in 10 mM phosphate buffer (pH = 8.0) containing
◦
10 mM NaCl, at 30 C for 24 h (final A
β
concentration = 50
µM) with and without inhibitor
(50 µM, Aβ/inhibitor = 1/1). Blanks containing the tested inhibitors were also prepared
and tested. To quantify amyloid fibrils formation, the thioflavin T (ThT) fluorescence
method was used [41]. After incubation, samples were diluted to a final volume of 2.0 mL
with 50 mM glycine-NaOH buffer (pH 8.5) containing 1.5
fluorescence intensity was carried out ( exc = 446 nm; em = 490 nm, FP-6200 fluorometer,
Jasco Europe), and values at plateau were averaged after subtracting the background
fluorescence of 1.5 M ThT solution. The fluorescence intensities were compared and the
µM ThT. A 300 s-time scan of
λ
λ
µ
percent inhibition due to the presence of the inhibitor.
4.2.4. FAAH Assay
The effect of compounds on FAAH activity was detected by coincubating them with
the 10,000
×
g membrane fraction of rat brain (70 µg/sample) and synthetic N-arachidonoyl-
[14C]-ethanolamine (110 mCi/mmol, ARC, St. Louis, MO, USA) properly diluted with
AEA (TocrisBioscience, Avonmouth, Bristol, UK) in Tris
at 37 C for 30 min. For the preincubation protocol, compounds were preincubated at
−
HCl 50 mM, at pH 9.00 10.00
−
◦
various concentrations in the presence of the enzyme (10,000
×
g membrane fraction of rat

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brain, 70
µ
g/sample) for 20 min at 37 ◦C. The reaction was then initiated by adding the
substrate. After incubation, the amount of [14C]-ethanolamine produced was measured by
scintillation counting of the aqueous phase after extraction of the incubation mixture with 2
volumes of CHCl3/MeOH 1:1 (by vol.). Data are expressed as means
separate experiments of the concentration exerting 50% inhibition of [14C]-AEA hydrolysis
(IC50) calculated by fitting sigmoidal concentration response curves by GraphPad.
±
SD of at least three
4.2.5. Competition Binding Assay
Competition binding assay for CB1 and CB2 receptors was performed as previously
reported [42]. Briefly, membranes from HEK-293 cells over-expressing the respective hu-
man recombinant CB1R (Bmax = 2.5 pmol/mg protein) and human recombinant CB2R
(Bmax = 4.7 pmol/mg protein) were incubated with [3H]-CP-55,940 (0.14 nM/Kd = 0.18 nM
and 0.084 nM/Kd = 0.31 nM, respectively, for CB1R and CB2R) as the high affinity ligand.
Competition curves were performed by displacing [3H]-CP-55,940 with increasing con-
centration of compounds (0.1 nM–10 µM). Nonspecific binding was defined by 10 µM of
WIN55,212-2 as the heterologous competitor (Ki values 9.2 nM and 2.1 nM, respectively,
for CB1R and CB2R). All compounds were tested following the procedure described by
the manufacturer (Perkin Elmer, Milan, Italy). Displacement curves were generated by
incubating drugs with [3H]-CP-55,940 for 90 min at 30 ◦C.
4.2.6. Computational Studies
Lipophilicity (QPlogPo/w), solubility (QPlogS) and brain/blood partition coefficient
(QPlogBB) were calculated with QikProp using the default settings. Molecular modelling
studies were performed using the Schrodinger 2018-2 software suite [43–46]. Ligands were
built using Maestro 11.6 [47] and prepared with LigPrep 4.2 [48]. Protein structures were
prepared using the Protein Preparation Wizard tool. Induced Fit Docking (IFD) studies
were conducted using Glide 7.9 applying the SP scoring function [49].
4.2.7. Protein Preparation
Chains A and B of the X-ray structure of rat FAAH covalently bound to methylarachi-
donyl phosphonate (MAP; PDB 1MT5) were used to perform IFD calculations. The covalent
inhibitor was removed, and missing hydrogen atoms were added. The overall hydrogen
bonding network was optimized by adjusting histidine tautomers and by sampling the
orientation of hydroxyl and thiol groups and the side chain amides of asparagine and
glutamine residues. Acid and basic amino acids were modelled in their charged state,
while K142, which is part of the catalytic triad of FAAH, was maintained neutral. The
structure was submitted to a minimization performed with the OPLS2005 force field [50
]
during which only hydrogen atoms were allowed to move. A second minimization run
was performed restraining the position of heavy atoms to an RMSD value of 0.3 Å from
the X-ray structure. All the co-crystallized water molecules were removed before running
docking studies.
Chain A of the crystallographic structure of hBuChE in complex with a non-covalent
inhibitor (4TPK [30]) and chain B of hAChE in complex with galantamine (4EY6 [31]) were
used to perform IFD studies. The proteins were prepared following the same procedure
described above, removing the inhibitor and water molecules before docking studies.
4.2.8. rFAAH Induced Fit Docking Studies
Compounds were docked in their neutral state, following a protocol previously applied
to similar inhibitors [20]..In fact, coumarin analogues with the same propylaminomethyl
linker had shown a significant loss of potency at pH 7.4, compared to pH 9.0, which supports
the hypothesis that these derivatives bind FAAH in their neutral state [20]. The docking grid
was built using Glide 7.9. In the first step of the IFD protocol, a softened-potential docking
run was performed setting van der Waals scaling factors to 0.7 and 0.5 for the protein and
ligand non-polar atoms, respectively, and the side chain of L278 was temporarily mutated

Molecules 2021, 26, 3254
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to alanine. Hydrogen bond constraints between the carbamate group of the ligands and
the backbone NH groups of I238 and G239, and the backbone oxygen atom of M191 were
imposed to obtain complexes consistent with the carbamoylation-based mechanism of these
compounds. The resulting complexes were thus submitted to a refinement process, in which
the side chain of L278 was restored and the side chains of amino acids within 5 Å from the
ligand were refined through a conformational search using Prime 5.2 [46]. The resulting
complexes were minimized according to the Prime routine. In the last docking step, each
ligand was optimized in the field of the receptor and scored with default Glide settings.
The complexes were ranked according to their IFD score and the best-ranked complexes
were energy-minimized applying the OPLS2005 force field implemented in MacroModel
12.0 [51] using the Polak-Ribiere conjugate gradient method to a convergence threshold of
0.05 kJ mol−1
Å
−1. During minimization, only the ligands and the surrounding residues
within 10 Å were free to move, while the backbone of other residues was kept fixed.
4.2.9. hBuChE and hAChE Induced Fit Docking Studies
Docking studies were performed following the protocol described in [20]. Compounds
were docked in their protonated state, given the ability of cholinesterases to accommodate
positively charged groups. The docking grid was built using Glide 7.9. A preliminary
softened-potential docking run was performed setting van der Waals radii scaling of protein
and ligand non-polar atoms to 0.7 and 0.6, respectively. A positional constraint was applied
on the carbamate group, to obtain complexes consistent with the carbamoylation-based
mechanism of these compounds. The ligand-protein complexes were then submitted to a
refinement procedure, in which the side chains of amino acids within 5 Å from the ligand,
with the exception of S198, H438, E325, W82 for hBuChE and S203, H447, E334, W86 for
hAChE, were refined through a conformational search. After minimization, in the final
docking step each inhibitor was optimized in the field of the receptor and scored according
to the default Glide settings. Finally, the complexes with best IFD score were energy-
minimized applying the OPLS2005 force field implemented in MacroModel 12.0 using the
Polak-Ribiere conjugate gradient method to a convergence threshold of 0.05 kJ mol−1 Å−1
.
During minimization, only the ligands and the surrounding residues within 10 Å were free
to move, while the backbone of other residues was kept fixed.
Author Contributions: Conceptualization, A.R. and A.B.; methodology, S.M., S.G. and F.B.; software,
L.S.; validation, M.B., S.R. and A.L.; formal analysis, M.N.; investigation, M.A. and M.K.; data
curation, S.R.; writing—original draft preparation, A.R. and A.B.; writing—review and editing, F.B.,
S.G., S.R., M.B., A.L.; supervision, A.R. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are unavailable from the authors.
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