Modification on the 1,2-dihydro-2-oxo-pyridine-3-carboxamide core to obtain multi-target modulators of endocannabinoid system

Article abstract of DOI:10.1016/j.bioorg.2019.103353

Several preclinical evidence indicate that the modulation of the endocannabinoid system (ECS) represents a promising therapeutic approach for different diseases. However, only few modulators of this system have reached so far an advanced stage of clinical development, mainly due to limited efficacy and CB1 receptor-dependent side effects. Those limitations might be overcome by multi-target compounds that exert pro-cannabinoid activities through the modulation of two or more targets in the ECS. This approach can offer a safer and more effective pharmacological strategy as compared to the modulation of a single target. In this work, we report the synthesis and biological characterization of new 6-aryl-1,2-dihydro-2-oxo-pyridine-3-carboxamide derivatives. Our results identified several compounds exhibiting interesting multi-target profiles within the ECS. In particular, compound B1 showed moderate-to-high affinity for cannabinoid receptors (Ki CB1R = 304 nM, partial agonist, Ki CB2R = 3.1 nM, inverse agonist) and a potent inhibition of AEA uptake (IC₅₀ = 62 nM) with moderate inhibition of FAAH (IC₅₀ = 2.9 μM). The corresponding 2-alkoxypyridine analogue B14 exhibited significant inhibitor activity on both FAAH (IC₅₀ = 69 nM) and AEA uptake (IC₅₀ = 76 nM) without significantly binding to both cannabinoid receptor subtypes. Molecular docking analysis was carried out on the three-dimensional structures of CB1R and CB2R and of FAAH to rationalize the structure-activity relationships of this series of compounds.

Full text of DOI:10.1016/j.bioorg.2019.103353

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Modification on the 1,2-dihydro-2-oxo-pyridine-3-carboxamide core to
obtain multi-target modulators of endocannabinoid system
Francesca Gadoa,1, Chiara Arenaa,b,1, Cristiana La Faucib, Ines Reynoso-Morenob, Simone Bertinia,
Maria Digiacomoa, Serena Meinia, Giulio Polia, Marco Macchiaa, Tiziano Tuccinardia,
⁎
⁎
Jürg Gertschb, Andrea Chiccab, , Clementina Maneraa,
a Department of Pharmacy, University of Pisa, 56126 Pisa, Italy
b NCCR TransCure, Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Several preclinical evidence indicate that the modulation of the endocannabinoid system (ECS) represents a
promising therapeutic approach for different diseases. However, only few modulators of this system have
reached so far an advanced stage of clinical development, mainly due to limited efficacy and CB1 receptor-
dependent side effects. Those limitations might be overcome by multi-target compounds that exert pro-canna-
binoid activities through the modulation of two or more targets in the ECS. This approach can offer a safer and
more effective pharmacological strategy as compared to the modulation of a single target. In this work, we report
the synthesis and biological characterization of new 6-aryl-1,2-dihydro-2-oxo-pyridine-3-carboxamide deriva-
tives. Our results identified several compounds exhibiting interesting multi-target profiles within the ECS. In
particular, compound B1 showed moderate-to-high affinity for cannabinoid receptors (Ki CB1R = 304 nM,
partial agonist, Ki CB2R = 3.1 nM, inverse agonist) and a potent inhibition of AEA uptake (IC50 = 62 nM) with
moderate inhibition of FAAH (IC50 = 2.9 μM). The corresponding 2-alkoxypyridine analogue B14 exhibited
significant inhibitor activity on both FAAH (IC50 = 69 nM) and AEA uptake (IC50 = 76 nM) without significantly
binding to both cannabinoid receptor subtypes. Molecular docking analysis was carried out on the three-di-
mensional structures of CB1R and CB2R and of FAAH to rationalize the structure-activity relationships of this
series of compounds.
Endocannabinoid system
Cannabinoid receptors
Fatty acid amide hydrolase
α/β-Hydrolase domain
Anandamide cellular uptake
Multitarget
1. Introduction
nuclear receptors (e.g. peroxisome proliferator-activated receptor;
PPAR) [5,6]. AEA is the first EC found in the CNS, biosynthetized on-
The endocannabinoid system (ECS) is a neuromodulatory retro-
grade lipid signaling system [1] and it consists of two G protein-coupled
receptors (CBRs), CB1R and CB2R, a class of lipid mediators called
endocannabinoids (ECs), the most studied of which are anandamide
(AEA) and 2-arachidonoylglycerol (2-AG), the EC membrane trans-
porter (EMT) and several enzymes involved in the biosynthesis and
degradation of ECs. CB1R is localized mainly in the central nervous
system (CNS) and it is one of the most abundant GPCR in mammalian
brain [2], while CB2R is expressed primarily in cells of the immune and
hematopoietic systems [3], but it was also found to be present in the
brain [4]. Interestingly ECs do not only interact with CBRs, but also
with other G-protein-coupled receptors like GPR55 or with other intra-
and extracellular hydrophobic receptor sites including ion channels
(e.g. transient receptor potential vanilloid receptor type 1; TRPV1) and
demand from phospholipid precursors and released in the synaptic
cleft. AEA exerts the highest activity in the hippocampus, followed by
thalamus, cortex and striatum, while the lowest in the cerebellum, pons
and medulla [7]. 2-AG is the second EC which was initially found in
intestinal tissue and later in hippocampus, in concentration 170-folds
higher than AEA [8]. It is already known that AEA and 2-AG are de-
graded by fatty acid amide hydrolase (FAAH) and monoacylglycerol
lipase (MAGL) enzymes, respectively [9]. A minor proportion of 2-AG
in the CNS is hydrolyzed by two degrading enzymes belonging to the α/
β-hydrolase domain (ABHD) family, ABHD6 and ABHD12 [10]. AEA
and 2-AG are transported across plasma membranes mainly via a fa-
cilitated diffusion mechanism mediated by a not yet identified mem-
brane transporter [11,12].
Over the past few years, the ECS has emerged as a crucial player for
⁎ Corresponding authors.
E-mail addresses: andrea.chicca@ibmm.unibe.ch (A. Chicca), clementina.manera@unipi.it (C. Manera).
1 These author contributed equally in this work.
https://doi.org/10.1016/j.bioorg.2019.103353
Received 27 August 2019; Received in revised form 3 October 2019; Accepted 9 October 2019
0045-2068/©2019PublishedbyElsevierInc.
Pleasecitethisarticleas:FrancescaGado,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103353

F. Gado, et al.
BioorganicChemistryxxx(xxxx)xxxx
the regulation of different diseases such as cancer, cachexia, seizures,
neuropathic and inflammatory pain, obesity, neuropsychiatric and
neurodegenerative diseases [13]. For this reason, many CB1R and CB2R
ligands have been synthesized and tested in vitro and in vivo as possible
therapeutic strategy [14]. Unfortunately, the direct activation or
blockage of CB1R was found to be responsible of numerous side effects
in the central nervous system (CNS) including abuse potential, cogni-
tive dysfunction, impairment of learning and memory, anxiety, suicidal
ideation and psychotropic effects, that have hampered the clinical de-
velopment of this class of compounds [15,16] In order to overcome
those problems, inhibitors of the 2-AG and AEA degrading enzymes
(covalent FAAH and MAGL inhibitors) were developed and showed
positive results in several animal disease models including promising
results in initial human trials. However, genetic and pharmacological
(covalent inhibition) ablation of FAAH or MAGL activity may be asso-
ciated to CB1R desensitization in the brain (repeated administrations of
MAGL inhibitors) [17] and potential side effects on cardiovascular and
metabolic system [18]. With the aim to investigate alternative strate-
gies to modulate the ECS, a new approach is based on multi-target
compounds that mildly modulates more than one target in the ECS.
Multi-target compounds exert pro-cannabinoid activities through more
than one mechanism of action [19]. Multi-target approach can re-
present a promising strategy to modulate the ECS in a safer and more
effective manner as compared to strong modulators of a single target
[20]. Finally, multi-target compounds could be useful to explore the
polypharmacology of the ECS and, eventually, to discover its potential
advantages in the treatment of different pathologies [19,21].
and CB2R and of FAAH for the most active compounds.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds B1–B18 was accomplished as depicted
in Scheme 1. Acetophenone or 4-methoxyacetophenone reacted with
dimethylformamide dimethyl acetal in refluxing xylene to afford the
corresponding enaminones 1 or 2. The subsequent treatment with ethyl
cyanoacetate in refluxing ethanol gave the amide derivatives 3 or 4,
which reacted with glacial acetic acid in refluxing toluene (Dean-Stark
apparatus), affording the cyclized ester derivatives 5 or 6 respectively.
Exposure of these compounds to cycloheptylamine at 150 °C in a mi-
crowave reactor for 30 min, led to carboxamide derivatives 7 and 8
respectively, that were subjected to a N-alkylation by treatment with
cesium fluoride in anhydrous DMF, at room temperature for 1 h and
then with 4-fluorobenzyl chloride affording the desired N-alkylated
derivatives B1 and B10 together the corresponding O-alkylated com-
pounds B14 and B18. The two structural isomers were purified by flash
chromatography. The synthesis of compounds B2 and B11 was then
accomplished starting from derivatives 7 and 8 that by treatment with
Br2 in CHCl3, afforded derivatives 9 and 10 which were subsequently
alkylated using the same procedure above described to obtain the de-
sired compounds B2 and B11. The compounds B3-B6, B12 and B13
were obtained from compounds B2 and B11 by a Suzuki-Miyaura cross-
coupling reaction with the suitable boronic acid or ethyl acrylate in the
presence of tetrakis(trisphenylphosphine)-palladium(0) as the catalyst
and the appropriate base, refluxing for 48 h. Finally, the synthesis of
compounds B7–B9 was accomplished starting from derivative 5 which
by treatment with Br2 in CHCl3 gave the 5-bromo derivative 11 which
was treated with the suitable amine in a microwave reactor in the same
conditions above described to give compounds 12–14. These deriva-
tives were then N-alkylated with 4-fluorobenzyl chloride affording the
desired compounds B7–B9. Their purification through flash column
chromatography allowed the isolation of the corresponding O-alkylated
isomers B15–B17.
Previously our group reported the synthesis and the biological
evaluation on the different targets of the ECS, of a series of 1,2-dihydro-
2-oxo-pyridine-3-carboxamides with general structure A (Fig. 1) char-
acterized by a phenyl or p-methoxy phenyl in position 4 [22]. As a
follow-up of our previous study, and aiming to optimizing/improving
the biological activities and further exploring the structure-activity-re-
lationship (SAR) of this class of compounds, we developed a new series
of derivatives, B, in which the bulky substituent in 4 was shifted in
position 6 (Fig. 1).
The new compounds are characterized by the presence of the p-
fluorobenzyl moiety at the N-1 position of the 1,2-dihydro-2-oxo-pyr-
idine ring that was chosen on the basis of the best results obtained with
the previous series, [22,23] and considering that fluorine can be of
crucial importance in affecting potency and pharmacokinetic properties
of bioactive compounds [24]. Furthermore, the substituent in position 5
of compounds A and the cycloheptyl carboxamide in position 3 were
either retained or replaced by groups with different steric and lipophilic
properties. Finally, the substituent in the N-1 position was shifted to the
oxygen in position 2 of the heterocyclic nucleus.
2.2. Biology
2.2.1. CB1R and CB2R affinity and activity
Binding affinities (Ki values) to CB1R and CB2R were evaluated
with competitive radioligand displacement assays against [3H]CP-
55,940 using membrane preparations obtained in-house from stable
transfected CHO-hCB1 and CHO-hCB2 cells. (R)-WIN55,212 was used
as positive control for both CB1R and CB2R binding experiments and
In summary, a small library of eighteen new derivatives (B1–B18)
whose structures are collected in Table 1 was synthesized. All com-
pounds were assayed for their binding and functional activity on both
CBRs and for all the other main targets of the ECS. Moreover, in order to
rationalize the experimentally observed SAR, a molecular docking
studies was carried out into the three-dimensional structure of CB1R
showed Ki value of 2.5
CB2R.
0.9 nM for CB1R and 1.6
0.5 nM for
The results summarized in Table 1 indicate that the new compounds
B1–B18 bind to CB1R and/or CB2R with nanomolar or sub-micromolar
affinities, with exception of the N-substituted derivative B7 and the O-
alkylated compounds B14, B15 and B18 that did not show any sig-
nificant binding to both CBRs at the concentration of 10 µM. Generally,
all ligands exhibited a higher affinity for CB2R, with a selectivity factor
toward CB1R in the range of 4- to 417-times. This behavior is in
agreement with our previous results obtained for derivatives of scaffold
A (Fig. 1, [22]). The only exceptions were the O-alkylated derivatives
B16 and B17 which showed a low-to-moderate binding affinity only for
CB1R with Ki values of 4704
2608 nM and 158
94 nM, respec-
tively. Interestingly, N-alkylated derivatives tend to have a higher af-
finity and selectivity for CB2R unlike O-alkylated derivatives. These
data highlight that the shift of the p-fluorobenzyl group from position
1–2 reduces the activity on both CBRs especially towards CB2R. Among
the 6-phenyl derivatives bearing a N-cycloheptyl carboxamide group at
the 3-position (B1–B6), the most potent ligand for CB2R was B1 (un-
substituted in position 5) with a Ki of 3.1 nM followed by B2 (bromide
Fig. 1. General structure of compounds A and B.
2

F. Gado, et al.
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Table 1
Data of 1,2-dihydro-2-oxopyridine- and pyridine-3-carboxamide derivatives B1–B18.a
Ki value (mean
CB1R
SD, nM)
CB2R
IC50 (mean
FAAH
SD, μM)
MAGL
Cmpd
R1
R2
ABHD6
ABHD12
AEA uptake
B1
H
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
morpholine
304
160
191
214
3.1
1.8
2.9
2.7
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
0.53
> 10
0.095
0.39
0.062
> 10
> 10
> 10
> 10
> 10
> 10
> 10
1.9
0.03
B2
Br
32.3
24.2
2420
476
317
14.8
> 10
> 10
> 10
2.4
0.1
0.30
0.80
0.64
1.9
B3
p-OCH3 phenyl
> 10000
> 10000
> 10000
12.0
542
138
99
B4
pyran-2-yl
0.29
B5
thiophen-2yl
2.1
0.59
B6
acrylate
1980
718
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
0.07
1.062
> 10
B7
Br
> 10000
> 10000
B8
Br
benzyilamine
3321
1017
1172
998
876
273
33
65.1
39.7
88
325
5.0
2.8
0.03
B9
Br
isobutilamine
347
967
101
0.1
1.6
B10
B11
B12
B13
B14
B15
B16
B17
B18
H
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
Morpholine
32.1
52.9
587
> 10
0.52
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
0.076
> 10
> 10
> 10
0.096
Br
> 10000
> 10000
> 10000
> 10000
> 10000
0.35
phenyl
p-OCH3 phenyl
1308
H
> 10000
> 10000
> 10000
> 10000
> 10000
0.03
0.05
Br
Br
Br
H
> 10
> 10
> 10
0.064
Benzylamine
4704
158
2608
94
isobutilamine
cycloheptylamine
> 10000
0.02
0.25
0.09
a
Results are expressed as mean
SD calculated from at least 3 experiments each performed in triplicate.
Scheme 1. Synthetic pathway followed for the synthesis of 1,2-dihydro-2-oxo-pyridine derivatives B1–B13 and 2-substituted pyridine derivatives B14–B18.
Reagents and conditions: (i) DMF-DMA, xylene, 180 °C, 48 h; (ii) ethyl cyanoacetate, EtOH, reflux, 48 h; (iii) glacial AcOH, dry toluene, reflux, 24 h; (iv) Br2, CHCl3,
rt, 12 h; (v) Suitable amine 150 °C, 48 h or mw, 30 min, 130 °C (140 W, 100 psi); (vi) 1) dry DMF, CsF, rt, 1 h; 2) 4-fluorobenzyl chloride, 50 °C, 12 h; (vii) suitable
arylboronic acid, Pd(OAc)2, PPh3, dry toluene, anh. K2CO3, 110 °C, 12 h; (viii) suitable arylboronic acid/ester, Pd(OAc)2, PPh3, MeOH, Na2CO3, dioxane, 110 °C, 12 h;
(ix) Ethylacrylate, Pd(OAc)2, PPh3, Et3N, ACN, 100 °C, 12 h.
3

F. Gado, et al.
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Table 2
Summary of functional activity ([35S]GTPγS assay) of compounds B1–B18 at CB1R and CB2R. Data are express as mean
SD obtained from at least three ex-
periments performed in triplicate.
EC50 value (mean
CB1R
SD, µM)
Cmpd
R1
R2
CB2R
B1
H
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
morpholine
0.39
0.28 (partial agonist)
0.05
0.08
0.23
0.28
0.20
0.22
n.d.
0.03 (inverse agonist)
0.1 (inverse agonist)
0.27 (inverse agonist)
0.14 (inverse agonist)
0.51 (inverse agonist)
0.34 (inverse agonist)
B2
Br
n.a. (antagonist)
B3
p-OCH3 phenyl
> 10 (inverse agonist)
B4
pyran-2-yl
n.d.
B5
thiophen-2yl
n.d.
B6
acrylate
> 10 (inverse agonist)
n.d.
B7
Br
B8
Br
Benzyilamine
> 10 (inverse agonist)
0.21
0.01
0.09
0.18
0.13 (inverse agonist)
0.02 (inverse agonist)
0.04 (inverse agonist)
0.12 (partial agonist)
B9
Br
isobutilamine
2.1
0.5 (inverse agonist)
B10
B11
B12
B13
B14
B15
B16
B17
B18
H
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
cycloheptylamine
morpholine
> 10 (inverse agonist)
Br
n.d.
phenyl
n.d.
> 10 (inverse agonist)
p-OCH3 phenyl
n.d.
0.43
0.21 (inverse agonist)
H
n.d.
> 10 (inverse agonist)
Br
Br
Br
H
n.d.
n.d.
Benzylamine
> 10 (inverse agonist)
n.d.
isobutilamine
0.033
n.d.
0.41 (inverse agonist)
> 10 (inverse agonist)
n.d.
cycloheptylamine
aIn brackets the behavior on CB1R and CB2R is reported from the [35S]GTPγS assay: Part Ago: partial agonist; Full Ago: full agonist; Inv Ago: inverse agonist; Antago:
antagonist. N.a.: not applicable; n.d.: not determined because no significant binding at 10 µM (Ki > 10 µM).
in position 5) and B3 (p-OCH3phenyl in position 5). Interestingly, B3 is
the only molecule that combined a low nanomolar affinity for CB2R (Ki
of 24.2 nM) with high selectivity over CB1R (417-times). Furthermore,
the replacement of the cycloheptyl carboxamide in position 3 with
other amide chains (B7–B9), led to a significant decrease in the affinity
towards both CBR subtypes. Finally, the replacement of the phenyl
group in position 6 (B1–B9) with a p-OCH3phenyl group (B10–B13) led
to different effects on binding affinities and selectivity between CB1R
and CB2R. The C5 unsubstituted derivative B10 showed 10-times lower
affinity for CB2R and 4-times lower affinity for CB1R as compared to
the corresponding 6-phenyl analogue B1. This effect was inverted for
the C5 bromine substituted derivative B11 which showed a 62-times
lower affinity for CB1R with no significant loss of affinity for CB2R as
compared to the 6-phenyl analogue B2. Finally, the C5 p-OCH3 phenyl
derivative B13 did not show any change on CB1R affinity, while it
significant lost affinity to CB2R (54-times) as compared to the corre-
sponding 6-phenyl derivative B3.
new compounds (B series, Tables 1 and 2) is independent from the
nature of the substituent in position 5 of the pyridine ring when this is
combined with of a phenyl group in position 6. If the phenyl is replaced
by p-OCH3 phenyl group, the type of substituent in position 5 seems to
play a role for the functional activity at CB2R. In particular, the absence
of substituent (B10) as well as the presence of a bulky substituent (B12
and B13) generate inverse agonists, while the presence of a bromine
switches the activity towards partial agonism (B11).
2.2.2. Polypharmacology in the ECS
In order to further characterize the pharmacology of these new
compounds, we tested their effects on the main components of the ECS.
Our results identified interesting polypharmacological properties
among the tested compounds (Table 1).
Our results identified B14 and B18, both O-alkylated and without
substituent in position 5, as relatively potent FAAH inhibitors with IC50
values of 0.069 µM and 0.064 µM, respectively (Table 1). Concerning
the N-alkylated derivatives, only compounds B1 and B5 showed a weak
inhibition of FAAH with IC50 values in the µM range (IC50 = 2.9 µM and
2.4 µM, respectively, Table 1). Our data suggest that O-alkylated deri-
vatives that are unsubstituted in position 5 offer an optimal combina-
tion for a relatively potent inhibition of FAAH. This seems to be in-
dependent from the presence of a phenyl or p-OCH3 phenyl in position
6.
Altogether the results suggest that shift of the phenyl group from
position 4 to 6 on the 2-oxo-pyridine-3-carboxamide leads to a relevant
increase of affinity for CB2R as compared to the corresponding deri-
vatives of the previous series A [22].
Compounds that showed the best results in terms of binding prop-
erties to CBRs were tested for their functional activity, using the
[
35S]GTPγS assay. The results are summarized in Table 2 and show that
the most bioactive compounds behaved as inverse agonists at CB1R
with the exception of B1 and B2 that behaved as partial agonist and
antagonist, respectively. Similarly, all the new compounds acted as
inverse agonists at CB2R, with the exception of a compound B11 which
bears a bromine in position 5 combined with cycloheptyl carboxamide
in position 3 and p-OCH3 phenyl in position 6.
None of the new compounds (B series) significantly inhibited MAGL
activity at the concentration of 10 µM (Table 1) similarly to our pre-
viously reported results for compounds of scaffold A ([22]).
In contrast with MAGL, some of our new compounds inhibited the
other 2-AG degrading enzymes ABHD6 and ABHD12. For ABHD6, the
O-alkylated, C5-unsubstituted C3-cycloheptyl carboxamide B18 was the
only derivative to show a moderate inhibition of the enzymatic activity
with an IC50 value of 0.53 µM (Table 1). Its N-alkylated analogue B10
Altogether, our data indicate that in contrast with the corresponding
compounds of scaffold A (Fig. 1 and [22]), the functional activity of the
4

F. Gado, et al.
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was inactive at the concentration of 10 µM, suggesting that the position
of the p-fluorobenzyl group may play a role for the interaction with
ABHD6.
Interestingly, in contrast with ABHD6, N-alkylated derivatives with
a phenyl group in position 6 and a substituent in position 5 inhibited
ABHD12 with nanomolar IC50 values (Table 1). In particular, the 5-
bromine derivatives B2 and B9 resulted the most potent inhibitors with
IC50 values of 0.095 µM and 0.097 µM, respectively. In general, the
presence of a p-OCH3 phenyl group in position 6 led to a loss of activity
on ABHD12 (IC50 > 10 µM, Table 1), with the exception of the 5-
bromine derivative B11 that retains a moderate inhibition of ABHD12
(IC50 = 0.52 µM, Table 1).
Finally, the effect of all the compounds on AEA cellular uptake was
also investigated. Compounds B1, B2 and B18 potently inhibited AEA
uptake with EC50 values of 0.063 µM, 0.076 µM and 0.096 µM, respec-
tively (Table 1). However, B2 and B18 showed also a potent inhibition
of FAAH which could contributed to AEA uptake inhibition. Indeed, in
our assay format, it is impossible to undoubtedly differentiate between
FAAH-dependent and FAAH-independent AEA uptake inhibition
[11,12,25]. Nonetheless, compound B1 showed a 46-times lower EC50
for AEA uptake (0.063 µM) as compared to FAAH (IC50 = 2.9 µM), thus
indicating a possible independent inhibition of AEA uptake (at the
plasma membrane) and FAAH inhibition (intracellular). Similarly,
compound B9 showed a low micromolar IC50 value for AEA uptake
(EC50 = 1.89 µM) without a significant inhibition of FAAH at 10 µM
(Table 1).
2.3. Molecular modeling studies
With the aim of rationalizing the experimental results obtained from
the biological evaluation of the synthesized compounds, molecular
docking studies into the structures of CB1R and CB2R were performed.
Compound B1, which showed the highest affinity for CB2R, was used as
a reference and subjected to docking analyses. The binding disposition
of the ligand into this receptor subtype was evaluated using the recently
deposited X-ray structure of the human CB2R in complex with the an-
tagonist AM10257 (PDB code: 5ZTY) [26], while the docking studies
into the CB1R subtype were performed using the X-ray structure of the
human CB1R in complex with the antagonist AM6538 (PDB code:
5TGZ) [27]. By employing a robust AUTODOCK 4.2 protocol, 200 dif-
ferent docking poses were generated for each docking evaluation (see
the Experimental section for details). The obtained binding conforma-
tions were clustered using a 2.0 Å root-mean squared deviation (RMSD)
cut-off and the top-scored solutions were then considered as the pre-
dicted binding modes. As shown in Fig. 2A, compound B1 was pre-
dicted to bind the CB2R by interacting with five out of the seven
transmembrane domains (TM) of the receptor (namely TM 2–3 and
5–7) and occupying multiple sub-pockets within the CB2R binding site
where extensive hydrophobic and aromatic interactions can be ob-
served. In particular, the central 2-oxo-pyridine ring of the ligand and
its substituents in position 3 and 6 lay among TM 2–3 and 6–7, while
the p-fluorobenzyl group, oriented orthogonally to the rest of the mo-
lecule, extends toward TM 5. The cycloheptyl group of the ligand is
placed into a wide lipophilic pocket mainly delimited by F87, F91, F94,
H95 and F183, forming hydrophobic interactions with all these re-
sidues. The amide nitrogen of the ligand forms an intramolecular H-
bond with the 2-oxo-pyridine core, creating a sort of bicyclic system
that shows T-shaped stacking with F87 and F183, as well as further
lipophilic interactions with V261 and F281. The remaining portions of
the compound are placed within two adjacent hydrophobic sub-
pockets. The ligand p-fluorobenzyl group well fits a cavity formed by
I110, T114, F117, F183, W194 and M265, showing a strong π-π in-
teraction with F183, while the adjacent cavity comprised among F117,
W194, W258, V261, L262 and M265, is occupied by the phenyl ring,
which forms T-shaped stacking with F117, W194 and W258. The
docking result obtained for compound B1 into CB1R (Fig. 2B)
Fig. 2. Predicted binding modes of B1 into CB2R (A) and CB1R (B).
highlights that the ligand cannot assume the same binding mode pre-
dicted within CB2R. This is due to the fact that two out of the three
lipophilic pockets occupied by the ligand in CB2R result to be occluded
in the binding cavity of CB1R. Precisely, in the CB1R the side chain of
F102 is oriented in the middle of the wide cavity that hosted the cy-
cloheptyl group of B1 in CB2R, while the pocket occupied by the phenyl
ring of the ligand in CB2R is partially occluded in CB1R, since F200 and
W356 (homologues of F117 and W258 in CB2R) undergo a coupled
conformational switch in which W356 rotate inside the cavity. For this
reason, the compound was predicted to assume a different binding
conformation in CB1R, in which the phenyl ring is oriented toward the
entrance of the binding site, in a wider and more solvent-exposed cavity
where it shows reduced lipophilic interactions and no evident π-π
stacking. The cycloheptyl moiety of the compound is placed among
W279, F286, W356 and M363, forming hydrophobic interactions with
these residues. The p-fluorophenyl ring of the ligand is placed in a
different pocket, essentially formed by G166, F170, V196, C386 and
L387, thus taking different lipophilic contacts and showing a T-shaped
stacking with F170. Finally, the 2-oxo-pyridine core is placed among
F102, F286 and F379, but its orientation with respect to these residues
prevents the formation of good stacking interactions. Moreover, the
intramolecular H-bond between the carbonyl group of the 2-oxo-pyr-
idine ring and the amide nitrogen appears to be completely lost. The
lack of many of the aromatic interactions formed in CB2R and the ab-
sence of the intramolecular H-bond may explain the reduced binding
affinity of B1 for CB1R with respect to CB2R.
Compound B14, the 2-alkoxypyridine analogue of B1 that showed
5

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agonist was reported in a mouse model of mild traumatic brain injury,
suggesting a possible anti-inflammatory activity, due to its ability of
reducing the pro-inflammatory M1 phenotype and increasing the anti-
inflammatory M2 phenotype of human and murine microglia in vitro
[29]. Another study has recently reported that CB2R inverse agonists
are able to strongly reduce iNOS expression (M1 marker) induced by
LPS treatment, without changing TREM2 expression (M2 marker) [30].
This new series of compounds (B) has also shown to inhibit other
targets in the ECS as compared to the corresponding 4-substituted de-
rivatives A which were totally inactive [22]. In particular, C5-un-
subsituted O-alkylated derivatives inhibited FAAH with nanomolar
potency, without affecting the major 2-AG degrading enzyme MAGL.
Similarly, the inhibition of ABHD6 (one of the minor 2-AG degrading
enzymes) seems to be correlated to O-alkylated derivatives that bear a
p-OCH3 phenyl in position 6. On the contrary, the inhibition of the
other minor 2-AG degrading enzyme ABHD12 seems to be affected by
the presence of N-alkylation in combination with a bromine in position
5 which is independent from a phenyl or p-OCH3 phenyl in position 6.
Finally, the derivative B1, characterized by a hydrogen in position 5
and a cycloheptyl carboxamide in position 3 was the most potent
(IC50 = 0.062 µM) and selective (46-times higher affinity versus FAAH)
AEA uptake inhibitor.
Fig. 3. Predicted binding mode of B14 into FAAH.
nanomolar inhibitory activity toward FAAH, was subjected to docking
studies on this target using the same protocol applied for B1. In its
predicted binding conformation, the ligand is able to form two different
H-bonds through the amide carbonyl group, which is placed in the
proximity of the oxyanion hole of the enzyme binding site (Fig. 3). In
particular, an H-bond is established with the catalytic residue S241 and
the other one is formed with the backbone nitrogen of I238. The cy-
cloheptyl group is located into the inner part of the binding site and
shows lipophilic interactions with F192, Y194, V491 and M495, while
the phenyl ring in position 6 of the pyridine core is oriented toward the
entrance of the catalytic pocket and predominantly interacts with M191
and L278. Finally, the p-fluorobenzyl group of B14 is positioned into a
lipophilic pocket constituted by F192, I238, V270, L380 and F388, thus
forming hydrophobic interactions with all these residues and, in par-
ticular, a π-π stacking with F192. Notably, the predicted binding mode
of B14 is consistent with the experimental values of FAAH inhibitory
activities determined for the other synthesized 2-alkoxypyridine deri-
vatives. In fact, there is no available space in the binding site region of
the protein around position 5 of the ligand pyridine core; therefore,
compounds bearing in this position any possible substituent other than
a hydrogen would be unable to adopt the same binding mode. In
agreement with this consideration, only the 5-unsubstituted analogue
B18 showed a binding affinity for FAAH comparable to B14.
Altogether, despite the limited number of compounds tested, this
new series of 1,2-dihydro-2-oxo-pyridine-3-carboxamides 6-aryl sub-
stituted exhibited multi-target profiles in the ECS.
Intriguingly, the 6-phenyl N-alkylated derivatives produced
a
variety of multi-target compounds depending on the type of substituent
in position 5 and with different carboxamides in position 3. For ex-
ample, we identified a nanomolar CB1R partial agonist/CB2R inverse
agonist that potently inhibited AEA uptake and moderately blocked
FAAH (B1); a nanomolar CB1R antagonist/CB2R inverse agonist that
potently inhibited ABHD12 (B2); a potent and selective CB2R inverse
agonist that inhibited ABHD12 (B3) and a CB1R/CB2R inverse agonist
that potently inhibited ABHD12 and moderately inhibited AEA uptake
(B9). Among the O-alkylated derivatives, compound B11 represents the
only potent and selective CB2R partial agonist that also inhibited
ABHD12. Finally, the 6-p-OCH3 phenyl O-alkylated derivative B18 did
not show any binding propertied to CB1R and CB2R at 10 µM, while
being the most potent FAAH inhibitor and a moderate ABHD6 inhibitor.
Altogether, our results provide important insights towards the
synthesis of new analogues that differentially target the complex net-
work of lipid pathways characterizing ECS and they may be useful to
better understand endocannabinoid function.
3. Conclusion
4. Experimental section
In this paper the biological profile of 1,2-dihydro-2-oxopyridine-
(B1–B13) and pyridine-3-carboxamide derivatives (B14–B18), 6-sub-
stituted and 5,6-disubstituted was assessed on the major components of
the ECS. Overall, our results show that the shift of the phenyl group
from position 4 to 6 led to an increased affinity for both CBRs, in
particular for CB2R as compared to our previous series (compounds of
scaffold A, [22]). The presence of a bulky substituent in 6 exerted a
significant impact on the functional activity of those compounds at
CB2R. In particular, 6-phenyl and 6-p-OCH3 phenyl derivatives shifted
their behavior from partial/full agonists (4-phenyl derivatives (A) [22])
to inverse agonists (B series, Table 2). An interesting exception is re-
presented by 5-bromo-N-cycloheptyl-1-(4-fluorobenzyl)-6-(4-methox-
yphenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (B11), which was
the only compound of the B series to retain a partial agonist behavior at
CB2R. Interestingly N-alkylated derivatives presented a higher se-
lectivity for CB2R while O-alkylated compounds were more selective
for CB1R. Thanks to the pro-homeostatic effects exerted by the ECS,
CB2R inverse agonists may represent an interesting approach for cer-
tain pathological conditions beside classic CB2R agonists [28]. For ex-
ample, the beneficial effect of a potent and selective CB2R inverse
4.1. Chemistry
Commercially available reagents were purchased from Sigma
Aldrich, Alfa Aesar or Tokyo Chemical Industry and used without pur-
ification. 1H NMR and 13C NMR were recorded at 400 and 100 MHz
respectively, on a Bruker AVANCE III™ 400 spectrometer. Chemical shift
(δ) are reported in parts per million related to the residual solvent signal,
while coupling constants (J) are expressed in Hertz (Hz). Microwave-
assisted reactions were run in a Biotage® Initiator + microwave synthe-
sizer. All final compounds were analyzed by HPLC, showing
a
purity ≥ 95%. A Bechman HPLC instrument equipped with a System
Gold Solvent Delivery module (Pumps) 125, System Gold UV/VIS
Detector 166, Detector set to 280 nm, was employed. Analyses were
performed on a reverse phase C18 column (Phenomenex 250 × 4.6 mm,
5 mm particle size, Gemini). The mobile phase was constituted by a
mixture of H2O/AcOH (0.1% v/v) (eluent A) and ACN (eluent B). A
gradient starting from 50% of B, changing to 100% of B over 20 min, and
returning to the initial conditions over 10 min, was used for compounds.
The flow rate was 1.0 mL/min. Organic solutions were dried over an-
hydrous Na2SO4. Evaporation was carried out in vacuo using a rotating
6

F. Gado, et al.
BioorganicChemistryxxx(xxxx)xxxx
evaporator. Silica gel flash chromatography was performed using silica
gel 60 Å (0.040–0.063 mm; MERK). Reactions was monitored by TLC on
Merck aluminium silica gel (60 F254) plates that were visualized under a
UV lamp (λ = 254 nm). Melting points were determined on a Kofler hot-
stage apparatus and are uncorrected.
After cooling, the reaction was poured in an ice bath and treated with a
10% HCl solution until pH 4. The solid precipitate was collected by
filtration, washed with water and dried under vacuum affording the
pure amide.
4.1.4.1. N-cycloheptyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-
carboxamide (7). Brown solid (1360 mg, 4.38 mmol, yield: 99%). 1H
NMR (CDCl3) δ (ppm): 10.99 (bs, 1H), 9.35 (bs, 1H), 8.68 (d, 1H,
J = 7.6 Hz), 7.23 (m, 2H), 7.56 (m, 3H), 6.78 (d, 1H, J = 7.6 Hz), 4.10
(m, 1H), 1.79 (m, 12H).
4.1.1. General procedure for the synthesis of compounds 1 and 2
A solution of the suitable starting material (16.6 mmol) and N,N-
dimethylformamide dimethyl acetal (6.65 mL, 49.9 mmol) in xylene
(21.2 mL) was left at reflux for 48 h. After cooling to room temperature,
the solvent was evaporated and the solid obtained was triturated with
petroleum ether and filtrated under vacuum.
4.1.4.2. N-cycloheptyl-2-oxo-6-(4-methoxyphenyl)-1,2-dihydropyridine-
3-carboxamide (8). Beige solid (1358 mg, 3.99 mmol, yield: 90%). 1H
NMR (CDCl3) δ (ppm): 11.09 (bs, 1H), 9.15 (bs, 1H), 8.60 (d, 1H,
J = 7.6 Hz), 7.67 (dd, 2H, JAX = 8.9 Hz, JAA’XX’ = 2.6 Hz), 7.05 (dd,
2H, JAX = 8.9 Hz, JAA’XX’ = 2.6 Hz), 6.68 (d, 1H, J = 7.6 Hz), 4.10
(m, 1H), 3.88 (s, 3H), 1.73 (m, 12H).
4.1.1.1. (E)-3-(dimethylamino)-1-phenylprop-2-en-1-one
(1). Brown
solid (3160 mg, 17.9 mmol, yield: > 99%). 1H NMR (CDCl3) δ (ppm):
7.89 (m, 2H), 7.83 (d, 1H, J = 12.0 Hz), 7.42 (m, 3H), 5.72 (d, 1H,
J = 12.0 Hz), 3.15 (s, 3H), 2.93 (s, 3H).
4.1.1.2. (E)-3-(dimethylamino)-1-(4-methoxyphenyl)prop-2-en-1-one
(2). Brown solid (2453 mg, 11.95 mmol, yield: 72%). 1H NMR (CDCl3)
δ (ppm): 7.90 (dd, 2H, JAX = 9.2 Hz, JAA’XX’ = 2.6 Hz), 7.79 (d, 1H,
J = 12.4 Hz), 6.91 (dd, 2H, JAX = 9.2 Hz, JAA’XX’ = 2.6 Hz), 5.71 (d,
1H, J = 12.4 Hz) 3.88 (s, 3H), 3.15 (bs, 3H), 2.92 (bs, 3H).
4.1.4.3. 5-bromo-3-(morpholino-4-carbonyl)-6-phenylpyridin-2(1H)-one
(12). Colorless oil (521 mg, 1.33 mmol, yield: 30%). 1H NMR (CDCl3) δ
(ppm): 13.15 (bs, 1H), 7.83 (s, 1H), 7.52 (m, 5H), 3.61 (m, 4H), 3.31
(m, 2H), 3.13 (m, 2H).
4.1.4.4. N-benzyl-5-bromo-2-oxo-6-phenyl-1,2-dihydropyridine-3-
carboxamide (13). Brown solid (797 mg, 2.08 mmol, yield: 47%). 1H
NMR (CDCl3) δ (ppm): 12.39 (bs, 1H), 9.45 (m, 1H), 8.77 (s, 1H), 7.54
(m, 2H), 7.36 (m, 8H), 4.56 (d, 2H, J = 6.0 Hz).
4.1.2. General procedure for the synthesis of compounds 3 and 4
A solution of compound 1 or 2 (17.9 mmol) and ethyl cyanoacetate
(2.10 mL, 19.7 mmol) in EtOH (18.4 mL) was left refluxing for 48 h.
After cooling to room temperature, the solid obtained was filtrated
under vacuum and washed with diethyl ether and ethyl acetate.
4.1.4.5. 5-bromo-N-isobutyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-
carboxamide (14). Yellow solid (866 mg, 2.48 mmol, yield: 56%). 1H
NMR (CDCl3) δ (ppm): 13.0 (bs, 1H), 9.05 (bs, 1H), 8.74 (s, 1H), 7.57
(m, 5H), 3.10 (m, 2H), 1.65 (m, 1H), 0.83 (d, 6H, J = 6.4 Hz).
4.1.2.1. Ethyl
(2E,4E)-2-carbamoyl-5-(dimethylamino)-5-phenylpenta-
2,4-dienoate (3). Yellow solid (2217 mg, 7.69 mmol, yield: 43%). 1H
NMR (CDCl3) δ (ppm): 8.47 (bs, 1H), 7.47 (m, 3H), 7.34 (s, 1H), 7.22
(m, 3H), 5.26 (bs, 1H), 3.93 (q, 2H, J = 7.2 Hz), 3.03 (m, 6H), 1.00 (t,
2H, J = 7.2 Hz).
4.1.5. General procedure for the synthesis of 5-bromo-3-carboxamide
compounds 9–11
The suitable starting compound 5, 7 or 8 (1.20 mmol) was dissolved
in CHCl3 (2.5 mL) and a solution of bromine (0.093 mL, 1.8 mmol) in
CHCl3 (1.2 mL) was added dropwise. The mixture was stirred at room
temperature for 12 h and then diluted with CHCl3. The solution was
treated with a saturated solution of sodium thiosulfate and the was
washed with water. The organic phase was dried over anhydrous
Na2SO4, filtered and evaporated under reduce pressure affording the
desired compound.
4.1.2.2. Ethyl (2E,4E)-2-carbamoyl-5-(dimethylamino)-5-(4-methoxyphenyl)
penta-2,4-dienoate (4). Yellow solid (3077 mg, 9.67 mmol, yield: 54%). 1H
NMR (CDCl3) δ (ppm): 8.40 (bs, 1H), 7.50 (m, 3H), 7.21 (m, 3H), 5.31 (bs,
1H) 4.23 (q, 2H, J = 7.2 Hz),), 3.88 (s, 3H), 3.05 (m, 6H), 1.42 (t, 3H,
J = 7.2 Hz).
4.1.3. General procedure for the synthesis of compounds 5 and 6
Acetic acid (0.4 mL, 7.76 mmol) was added to a solution of com-
pound 3 or 4 (7.76 mmol) in dry toluene (34.4 mL). The reaction con-
tents were left refluxing for 24 h. After cooling, the reaction flask was
placed in an ice bath to allow the precipitation of the desired compound
as solid which was subsequently isolated by filtration under vacuum.
4.1.5.1. 5-bromo-N-cycloheptyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-
carboxamide (9). Yellow solid (175 mg, 0.45 mmol, yield: 38%). 1H
NMR (CDCl3) δ (ppm): 11.90 (bs, 1H), 9.03 (bs, 1H), 8.73 (s, 1H), 7.58
(m, 5H), 4.03 (m, 1H), 1.69 (m, 12H).
4.1.3.1. Ethyl 2-oxo-6-phenyl-1,2-dihydropyridine-3-carboxylate (5). Brown
solid (1075 mg, 4.42 mmol, yield: 57%). 1H NMR (CDCl3) δ (ppm): 8.27
(d, 1H, J = 8.0 Hz), 8.01 (m, 2H), 7.49 (m, 3H), 7.22 (d, 1H, J = 8.0 Hz),
4.44 (q, 2H, J = 7.2 Hz), 1.43 (t, 3H, J = 7.2 Hz).
4.1.5.2. 5-bromo-N-cycloheptyl-2-oxo-6-(4-methoxyphenyl)-1,2-
dihydropyridine-3-carboxamide (10). Beige solid (327.1 mg, 0.78 mmol,
yield: 65%). 1H NMR (CDCl3) δ (ppm): 9.04 (bs, 1H), 8.71 (bs, 1H),
7.55 (m, 2H), 7.02 (m, 2H), 4.07 (m, 1H), 3.88 (s, 3H), 1.73 (m, 12H).
4.1.3.2. Ethyl 6-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carboxylate
(6). Brown solid (1738 mg, 6.36 mmol, yield: 82%). 1H NMR (CDCl3) δ
(ppm): 8.23 (d, 1H, J = 8.0 Hz), 7.97 (dd, 2H, JAX = 8.8 Hz, JAA’XX’ =
2.6 Hz), 7.13 (d, 1H, J = 8.0 Hz), 7.00 (dd, 2H, JAX = 8.8 Hz, JAA’XX’ =
2.6 Hz), 4.43 (q, 2H, J = 7.2 Hz), 3.88 (s, 3H), 1.42 (t, 3H).
4.1.5.3. Ethyl 5-bromo-2-oxo-6-phenyl-1,2-dihydropyridine-3-carboxylate
(11). Beige solid (367 mg, 1.14 mmol, yield: 95%). 1H NMR (CDCl3)
δ (ppm): 8.44 (s, 1H), 7.73 (m, 2H), 7.47 (m, 3H), 4.48 (q, 2H,
J = 7.2 Hz), 1.45 (t, 3H, J = 7.2 Hz).
4.1.6. General procedure for the synthesis of N-substituted derivatives
B1–B13 and O-substituted derivatives B14–B18
4.1.4. General procedure for the synthesis of 3-carboxamide compounds 7,
8, 12–14
Cesium Fluoride (135 mg, 0.880 mmol) was added to a solution of
the proper amide 7–10, 12–14 (0.440 mmol) in anhydrous DMF
(2.00 mL). After 1 h, p-fluoro benzyl chloride (0.88 mmol, 0.120 mL)
was added, and the resulting mixture was left under stirring at 50 °C for
12 h. After cooling to room temperature the reaction mixture was
A mixture of the suitable 2-oxo-1,2-dihydropyridine-3-carboxilate
5, 6 or 11 (4.43 mmol) and the suitable amine (9.1 mmol) was heated at
150 °C in an oil bath for 48 h (compounds 12–14) or at 130 °C by mi-
crowave radiation for 30 min (140 W, 100 psi) (compounds 7 and 8).
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concentrated under reduce pressure treated with ice-water, and then
extracted with chloroform. The organic layers were dried over anhy-
drous Na2SO4, filtered and evaporated under pressure to give a residue
which was purified by flash chromatography.
4.1.6.4. 5-bromo-N-cycloheptyl-1-(4-fluorobenzyl)-6-(4-methoxyphenyl)-
2-oxo-1,2-dihydropyridine-3-carboxamide (B11). Prepared from amide
10. Purification by flash chromatography on silica gel (petroleum
ether/ethyl acetate 8:2). Yellow solid (83.54 mg, 0.158 mmol, yield:
36%). Mp: 94–97 °C (hexane). 1H NMR (CDCl3) δ (ppm): 9.69 (bs, 1H),
8.74 (s, 1H), 6.91 (m, 6H), 6.80 (m, 2H), 5.16 (s, 2H), 4.17 (m, 1H),
3.85 (s, 3H), 2.01 (m, 2H), 1.62 (m, 10H). 13C NMR (100 MHz, CDCl3):
162.05 (d, J = 245 Hz), 161.70, 161.30, 160.63, 150.99, 146.47,
131.82 (d, J = 3.0 Hz), 129.95, 128.53 (d, J = 8.0 Hz), 126.14,
121.58, 115.38 (d, J = 22 Hz), 114.16, 102.57, 55.34, 50.59, 49.98,
34.83, 28.06, 24.15. HPLC analysis: retention time = 16.68 min; peak
area 99.10% (280 nm).
4.1.6.1. N-cycloheptyl-1-(4-fluorobenzyl)-2-oxo-6-phenyl-1,2-
dihydropyridine-3-carboxamide
(B1)
and
N-cycloheptyl-2-((4-
fluorobenzyl)oxy)-6-phenyl-1,2-dihydropyridine-3-carboxamide
(B14). Prepared from amide 7. Purification by flash chromatography
on silica gel (petroleum ether/ethyl acetate 8:2). B1: white solid
(73.66 mg, 0.176 mmol, yield: 40%). Mp: 139–142 °C (hexane). 1H
NMR (CDCl3) δ (ppm): 9.77 (bs, 1H), 8.57 (d, 1H, J = 7.5 Hz), 7.44 (m,
1H), 7.36 (m, 2H), 7.11 (m, 2H), 6.90 (m, 2H), 6.82 (m, 2H), 6.34 (d,
J = 7.5 Hz), 5.20 (s, 2H), 4.15 (m, 1H), 2.01 (m, 2H), 1.61 (m, 10H).
13C NMR (100 MHz, CDCl3): 163.07, 162.88, 162.37 (d, J = 245 Hz),
153.59, 143.11, 134.91, 132.56 (d, J = 3 Hz), 130.16, 128.92, 128.82
(d, J = 8 Hz), 128.68, 120.84, 115.79 (d, J = 21 Hz), 109.42, 50.85,
48.92, 35.31, 28.44, 24.61. HPLC analysis: retention time = 18.98 min;
peak area 99.63% (280 nm). B14: white solid (55.24 mg, 0.132 mmol,
yield: 30%). Mp: 110–113 °C (petroleum ether). 1H NMR (CDCl3) δ
(ppm): 8.58 (d,1H, J = 7.8 Hz), 8.08 (m, 2H), 7.94 (bs, 1H), 7.54 (d,
2H, J = 7.8 Hz), 7.48 (m, 5H), 7.12 (m, 2H), 5.60 (s, 2H), 4.17 (m, 1H),
1.86 (m, 2H), 1.54 (m, 2H), 1.40 (m, 8H). 13C NMR (100 MHz, CDCl3):
162.88 (d, J = 246 Hz), 162.35, 159.52, 156.68, 142.52, 137.82,
132.22 (d, J = 4.0 Hz), 130.68 (d, J = 9.0 Hz), 129.76, 128.78,
127.04, 115.70 (d, J = 21 Hz), 114.68, 114.02, 68.21, 49.98, 34.68,
28.27, 23.68. HPLC analysis: retention time = 19.03 min; peak area
99.78% (280 nm).
4.1.6.5. 5-bromo-1-(4-fluorobenzyl)-3-(morpholine-4-carbonyl)-6-
phenylpyridin-2(1H)-one (B7) and 5-bromo-2-(4-fluorobenzyloxy)-3-
(morfolin-4-carbonyl)-6-phenyl-pyridin-2(1H)-one (B15). Prepared from
amide 12. Purification by flash chromatography on silica gel
(petroleum ether/ethyl acetate 3:7). B7: oil (58.06 mg, 0.123 mmol,
yield: 28%). 1H NMR (CDCl3) δ (ppm): 7.79 (s, 1H), 7.48 (m, 1H), 7.42
(m, 2H), 7.01 (m, 2H), 6.85 (m, 2H), 6.77 (m, 2H), 5.06 (s, 2H), 3.77
(m, 6H), 3.42 (m, 2H). 13C NMR (100 MHz, CDCl3): 164.72, 162.30 (d,
J = 245 Hz), 158.82, 149.64, 143.33, 134.13, 132.03 (d, J = 4.0 Hz),
130.19, 129.27 (d, J = 8.0 Hz), 129.02, 128.89, 128.38, 115.42 (d,
J = 22 Hz), 100.61, 67.08, 66.82, 49.90, 47.68, 42.70, 29.83. HPLC
analysis: retention time = 6.78 min; peak area 98.23% (280 nm). B15:
white oil (120.28 mg, 0.255 mmol, yield: 58%). 1H NMR (CDCl3) δ
(ppm): 7.89 (s, 1H), 7.70 (m, 2H), 7.47 (m, 3H), 7.38 (m, 2H), 7.04 (m,
2H), 5.39 (s, 2H), 3.74 (m, 4H), 3.52 (m, 2H), 3.29 (s, 2H). 13C NMR
(100 MHz, CDCl3): 164.65, 162.68 (d, J = 246 Hz), 157.00, 155.99,
143.12, 138.73, 132.40, 130.25 (d, J = 8.0 Hz), 129.47, 128.05,
119.49, 115.54 (d J = 21 Hz), 110.77, 67.84, 66.80, 47.48, 42.45.
HPLC analysis: retention time = 10.82 min; peak area 98.86%
(280 nm).
4.1.6.2. N-cycloheptyl-1-(4-fluorobenzyl)-6-(4-methoxyphenyl)-2-oxo-
1,2-dihydropyridine-3-carboxamide (B10) and N-cycloheptyl-2-((4-
fluorobenzyl)oxy)-6-(4-methoxyphenyl)-1,2-dihydropyridine-3-
carboxamide (B18). Prepared from amide 8. Purification by flash
chromatography on silica gel (petroleum ether/ethyl acetate 8:2).
B10: yellow solid (19.74 mg, 0.04 mmol, yield: 10%). Mp: 121–124 °C
(hexane). 1H NMR (CDCl3) δ (ppm): 9.78 (bs, 1H), 8.56 (d, 1H,
J = 7.4 Hz), 7.05 (m, 2H), 6.90 (m, 6H), 6.34 (d, 1H, J = 7.4 Hz),
5.23 (s, 2H), 4.16 (m, 1H), 3.84 (s, 3H), 2.02 (m, 2H), 1.60 (m, 5H),
1.31 (m, 5H). 13C NMR (100 MHz, CDCl3): 162.98, 162.81, 162.14 (d,
J = 244 Hz), 160.79, 153.51, 142.89, 132.49 (d, J = 3 Hz), 129.92,
128.51 (d, J = 8 Hz), 127.03, 115.99, 115.62 (d, J = 22 Hz), 114.09,
109.46, 55.55, 50.66, 48.84, 35.11, 28.24, 24.41. HPLC analysis:
retention time = 14.45 min; peak area 97.19% (280 nm). B18: yellow
solid (55.26 mg, 0.123 mmol, yield: 28%). Mp: 135–138 °C (hexane). 1H
NMR (CDCl3) δ (ppm): 8.54 (d, 1H, J = 8.0 Hz), 8.04 (m, 2H), 7.92 (bd,
1H), 7.50 (m, 2H), 7.46 (d, 1H, J = 8.0 Hz), 7.11 (m, 2H), 7.00 (m, 2H),
5.58 (s, 2H), 4.16 (m, 1H), 3.88 (s, 3H), 1.85 (m, 2H), 1.54 (m, 2H),
1.39 (m, 8H). 13C NMR (100 MHz, CDCl3): 162.77 (d, J = 245 Hz),
162.40, 161.02, 159.37, 156.34, 142.27, 132.25 (d, J = 3 Hz), 130.57
(d, J = 8 Hz), 130.31, 128.37, 115.58 (d, J = 22 Hz), 114.04, 113.69,
112.98, 68.01, 55.26, 49.87, 34.61, 28.20, 23.61. HPLC analysis:
retention time = 18.53 min; peak area 99.41% (280 nm).
4.1.6.6. N-benzyl-5-bromo-1-(4-fluorobenzyl)-2-oxo-6-phenyl-1,2-
dihydropyridine-3-carboxamide (B8) and N-benzyl-5-bromo-2-((4-
fluorobenzyl)oxy)-6-phenyl-1,2-dihydropyridine-3-carboxamide
(B16). Prepared from amide 13. Purification by flash chromatography
on silica gel (petroleum ether/ethyl acetate 3:7) B8: white solid
(101.61 mg, 0.206 mmol, yield: 47%). Mp: 140–143 °C (hexane). 1H
NMR (CDCl3) δ (ppm): 10.05 (bs, 1H), 8.80 (s, 1H), 7.39 (m, 8H), 7.01
(m, 2H), 6.89 (m, 2H), 6.76 (m, 2H), 5.12 (s, 2H), 4.67 (d, 2H,
J = 5.6 Hz). 13C NMR (100 MHz, CDCl3): 162.76, 162.26 (d,
J = 245 Hz), 161.73, 151.40, 147.09, 138.50, 133.97, 131.71 (d,
J = 4 Hz), 130.30, 129.03, 128.75, 128.73, 128.63 (d, J = 9 Hz),
127.92, 127.38, 121.52, 115.58 (d, J = 21 Hz), 102.08, 50.24, 43.83.
HPLC analysis: retention time = 13.02 min; peak area 99.32%
(280 nm). B16: white solid (93.0 mg, 0.189 mmol, yield: 43%). Mp:
148–150 °C (petroleum ether). 1H NMR (CDCl3) δ (ppm): 8.81 (s, 1H),
8.12 (m, 1H), 7.76 (m, 2H), 7.48 (m, 3H), 7.25 (m, 7H), 6.94 (m, 2H),
5.43 (s, 2H), 4.59 (d, 2H, J = 5.2 Hz). 13C NMR (100 MHz, CDCl3):
162.82 (d, J = 246 Hz), 162.35, 158.15, 157.22, 146.96, 138.49,
137.79, 131.58 (d, J = 4 Hz), 130.38 (d, J = 9 Hz), 129.59, 129.48,
128.88, 128.07, 127.91, 127.69, 115.82 (d, J = 22 Hz), 115.79, 111.68,
68.80, 44.32. HPLC analysis: retention time = 16.80 min; peak area
99.34% (280 nm).
4.1.6.3. 5-bromo-N-cycloheptyl-1-(4-fluorobenzyl)-2-oxo-6-phenyl-1,2-
dihydropyridine-3-carboxamide (B2). Prepared from amide 9.
Purification by flash chromatography on silica gel (petroleum ether/
ethyl acetate 9:1). Brown solid (87.54 mg, 0.176 mmol, yield: 40%).
Mp: 85–88 °C (hexane). 1H NMR (CDCl3) δ (ppm): 9.68 (bs, 1H), 8.74 (s,
1H), 7.46 (m, 1H), 7.39 (m, 2H); 6.98 (m, 2H), 6.88 (m, 2H), 6.76 (m,
2H), 5.12 (s, 2H), 4.17 (m, 1H), 2.01 (m, 2H), 1.59 (m, 10H). 13C NMR
(100 MHz, CDCl3): 162.27 (d, J = 245 Hz), 161.78, 161.36, 150.99,
146.76, 134.06, 131.82 (d, J = 3.4 Hz), 130.23, 128.99, 128.71 (d,
J = 8.2 Hz), 128.64, 121.97, 115.56 (d, J = 21.8 Hz), 102.12, 50.77,
50.11, 35.01, 28.21, 24.32. HPLC analysis: retention time = 16.77 min;
peak area 98.08% (280 nm).
4.1.6.7. 5-bromo-1-(4-fluorobenzyl)-N-isobutyl-2-oxo-6-phenyl-1,2-
dihydropyridine-3-carboxamide (B9) and 5-bromo-2-((4-fluorobenzyl)
oxy)-N-isobutyl-6-phenyl-1,2-dihydropyridine-3-carboxamide
(B17). Prepared from amide 14. Purification by flash chromatography
on silica gel (petroleum ether/ethyl acetate 3:7). B9: brown solid
(76.47 mg, 0.167 mmol, yield: 38%). Mp: 94–97 °C(hexane). 1H NMR
(CDCl3) δ (ppm): 9.75 (m, 1H), 8.77 (s, 1H), 7.47 (m, 1H), 7.41 (m,
2H), 7.00 (m, 2H), 6.90 (m, 2H), 6.77 (m, 2H), 5.14 (s, 2H), 3.30 (t, 2H
J = 6.8 Hz), 1.91 (m, 1H, J = 6,8 Hz), 0.98 (d, 6H, J = 6,8 Hz). 13C
8

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NMR (100 MHz, CDCl3): 162.67, 162.23 (d, J = 246 Hz), 161.79,
151.07, 146.81, 134.00, 131.78 (d, J = 3 Hz), 130.23, 128.97, 128.70
(d, J = 8 Hz), 128.60, 121.75, 115.54 (d, J = 21 Hz), 102.07, 50.14,
47.22, 28.61, 20.42. HPLC analysis: retention time = 13.18 min; peak
area 99.05% (280 nm). B17: white solid (116.7 mg, 0.255 mmol, yield:
58%). Mp: 101–102 °C. 1H NMR (CDCl3) δ (ppm): 8.77 (s, 1H), 7.85 (m,
1H), 7.77 (m, 2H), 7.48 (m, 2H), 7.43 (m, 2H), 7.09 (m, 2H), 5.48 (s,
2H), 3.31 (m, 2H), 1.93 (m, 1H), 0.99 (d, 6H, J = 6,8 Hz). 13C NMR
(100 MHz, CDCl3): 163.02 (d, J = 246 Hz), 162.38, 158.08, 156.89,
146.85, 138.50, 131.78 (d, J = 3 Hz), 130.88 (d, J = 9 Hz), 129.56,
129.39, 128.02, 116.09, 115.84 (d, J = 22 Hz), 111.61, 68.88, 47.33,
28.25, 20.06. HPLC analysis: retention time = 17.13 min; peak area
99.88% (280 nm).
4.1.8. N-cycloheptyl-5-(3,6-dihydro-2H-piran-4)-1-(4-fluorobenzyl)-2-
oxo-6-phenyl-1,2-dihydropyridin-3-carboxamide (B4)
A mixture of Pd(OAc)2 (3.00 mg, 0.020 mmol) and PPh3 (22.0 mg,
0.085 mmol) in dioxane (3.20 mL) was stirred at room temperature for
10–15 min, to allow the formation of the catalyst tetrakis(triphenyl-
phosphine)palladium-(0). Then 3,6-dihydro-2H-pyran-4-boronic acid
pinacol ester (81.0 mg, 0.388 mmol), Na2CO3 2 M (0.700 mL) and B2
(150.0 mg, 0.289 mmol) were added. The reaction was heated at 110 °C
under stirring for 12 h. after cooling the mixture was filtered under
vacuum using celite, evaporated under reduced pressure treated with
water and extracted with CHCl3. Combined organic layers were dried
over Na2SO4, evaporated and purified by flash column chromatography
to give the desired compound B4. Beige solid (87.0 mg, 0.170 mmol,
yield: 59%). Mp: 132–134 °C (hexane). 1H NMR (CDCl3) δ (ppm): 9.83
(bs, 1H), 8.51 (s, 1H), 7.40 (m, 1H), 7.32 (m, 2H), 6.96 (m, 2H), 6.89
(m, 2H), 6.78 (m, 2H), 5.49 (m, 1H), 5.16 (s, 2H), 4.19 (m, 1H), 3.99
(m, 2H), 3.50 (m, 2H,), 2.02 (m, 2H), 1.75(m, 2H), 1.63 (m, 10H). 13C
NMR (CDCl3) δ (ppm): 162.45, 162.03, 162.00 (d, J = 245 Hz), 149.55,
144.45, 133.15, 132.43, 132.27 (d, J = 3 Hz), 129.64, 129.24, 128.52
(d, J = 8 Hz), 128.37, 127.79, 122.55, 120.35, 115.34 (d, J = 21 Hz),
65.24, 63.93, 50.47, 48.88, 34.93, 29.39, 28.13, 24.22. HPLC analysis:
retention time = 14.77 min; peak area 99.50% (280 nm).
4.1.7. General procedure for the synthesis of derivatives B3, B12 and B13
A mixture of Pd(OAc)2 (7.34 mg, 0.030 mmol) and PPh3 (36.7 mg,
0.14 mmol) in anhydrous toluene (2.00 mL) was stirred for 10–15 min
at room temperature under nitrogen flux, to allow the formation of the
catalyst tetrakis(triphenylphosphine) palladium(0). Then the suitable
boronic acid (3.80 mmol), K2CO3 (196.3 mg, 1.42 mmol) and the bro-
mine derivative (B2 or B11) (1.90 mmol) were added The reaction was
heated at 110 °C under stirring for 12 h. The mixture was filtered under
vacuum using celite, evaporated under reduced pressure, treated with
water and extracted with CHCl3. Combined organic layers were dried
over anhydrous Na2SO4 and evaporated under reduced pressure to
obtain a residue which was purified by flash chromatography.
4.1.9. N-cycloheptyl-1-(4-fluorobenzyl)-2-oxo-6-phenyl-5-(tiophen-2-il)-
1,2-dihydro pyridin-3-carboxamide (B5)
A mixture of Pd(OAc)2 (10.00 mg, 0.040 mmol) and PPh3 (53.0 mg,
0.200 mmol) in dioxane (2.00 mL) was stirred under nitrogen flux at
room temperature for 10–15 min, to allow the formation of the catalyst
tetrakis(triphenylphosphine)-palladium(0). Then phenyl boronic acid
(102.0 mg, 0.800 mmol), Na2CO3 2 M (0.850 mL), methanol (1.00 mL)
and B2 (0.400 mmol) were added. Reaction contents were allowed to
stir at 110 °C for 12 h. The mixture was filtered under vacuum using
celite, evaporated under reduced pressure and extracted with CHCl3/
water. Combined organic phase was dried over Na2SO4 and evaporated
obtaining a brown oil purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate 8:2) affording the desired compound
B5. Brown solid (36 mg, 0.06 mmol, yield: 15%). Mp: 148–149 °C. 1H
NMR (CDCl3) δ (ppm): 9.79 (bs, 1H), 8.84 (s, 1H), 7.40 (m, 1H), 7.39
(m, 2H), 7.06 (m, 1H), 6.98 (m, 2H), 6.90 (m, 2H), 6.80 (m, 3H), 6.70
(m, 1H), 5.17 (s, 2H), 4.20 (m, 1H), 2.03 (m, 2H), 1.65 (m, 10H). 13C
NMR (100 MHz, CDCl3): 162.15, 162.04 (d, J = 244 Hz), 161.86,
149.54, 145.05, 138.84, 132.88, 132.06, 129.95, 129.61, 128.70,
128.44 (d, J = 8 Hz), 126.91, 126.62, 126.08, 120.61, 115.40, 115.38
(d, J = 21 Hz), 50.58, 49.19, 34.93, 28.11, 24.24. HPLC analysis: re-
tention time = 17.33 min; peak area 98.45% (280 nm).
4.1.7.1. N-cycloheptyl-1-(4-fluorobenzyl)-6-phenyl-2-oxo-5-(4-
methoxyphenyl)-1,2-dihydropyridine-3-carboxamide
(B3). Purification
by flash chromatography on silica gel (petroleum ether/ethyl acetate
8:2). Yellow solid (837.30 mg, 1.60 mmol, yield: 84%). Mp: 187–190 °C
(hexane). 1H NMR (CDCl3) δ (ppm): 9.87 (bs, 1H), 8.66 (s, 1H), 7.29
(m, 1H), 7.18 (m, 2H), 6.88 (m, 8H), 6.62 (m, 2H), 5.21 (s, 2H), 4.20
(m, 1H), 3.70 (s, 3H), 2.04 (m, 2H), 1.61 (m, 10H). 13C NMR (100 MHz,
CDCl3): 162.11 (d, J = 244 Hz), 162.63, 162.18, 158.45, 150.04,
145.79, 133.40, 132.45 (d, J = 3 Hz), 130.75, 129.82, 129.77,
129.35, 128.67 (d, J = 8 Hz), 128.41, 121.93, 120.46, 115.48 (d,
J = 21 Hz), 113.50, 55.21, 50.63, 49.21, 35.09, 28.25, 24.36. HPLC
analysis: retention time = 16.93 min; peak area 96.63% (280 nm).
4.1.7.2. N-cycloheptyl-1-(4-fluorobenzyl)-6-(4-methoxyphenyl)-2-oxo-5-
phenyl-1,2-dihydropyridine-3-carboxamide (B12). Purification by flash
column chromatography on silica gel (petroleum ether/ethyl acetate
8:2). Yellow solid (618 mg, 1.18 mmol, yield: 62%). Mp: 185–187 °C. 1H
NMR (CDCl3) δ (ppm): 9.86 (bs, 1H), 8.66 (s, 1H), 7.11 (m, 3H), 6.90
(m, 6H), 6.79 (m, 2H), 6.68 (m, 2H), 5.22 (s, 2H), 4.20 (m, 1H), 3.74 (s,
3H), 2.02 (m, 2H), 1.61 (m, 10H). 13C NMR (100 MHz, CDCl3) δ (ppm):
162.28, 161.35 (d, J = 249 Hz), 150.28, 145.49, 137.68, 132.49 (d,
J = 3 Hz), 131.14, 129.58, 128.50 (d, J = 8 Hz), 128.05, 126.81,
125.38, 122.52, 120.29, 115.43 (d, J = 22 Hz), 113.71, 55.28, 50.57,
49.18, 35.03, 28.18, 24.29. HPLC analysis: retention time = 17.58 min;
peak area 95.37% (280 nm).
4.1.10. Ethyl (E)-3-(5-(cycloheptylcarbamoyl)-1-(4-fluorobenzyl)-6-oxo-
2-phenyl-1,6-dihydropyridin-3-yl)acrylate (B6)
A mixture of Pd(OAc)2 (3.00 mg, 0.012 mmol) and PPh3 (22.0 mg,
0.085 mmol) in ACN (0.660 mL) was stirred at room temperature for
10–15 min, to allow the formation of the catalyst tetrakis(triphenyl-
phosphine)-palladium(0). Then ethyl acrylate (180.0 mg, 1.70 mmol),
Et3N (0.660 mL) and B2 (150.0 mg, 0.289 mmol) were added. The re-
action was heated at 100 °C under stirring for 12 h. After cooling the
reaction mixture was filtered under vacuum using celite, evaporated
under reduced pressure to obtain residue which was treated with water
and extracted with CHCl3. Combined organic layers were dried over
Na2SO4 and evaporated to give an oil residue which was purified by
flash chromatography on silica gel (petroleum ether/AcOEt 2:8) to
obtain the desired compound B6. Brown solid (83.0 mg, 0.162 mmol,
yield: 56%). Mp: 199–202 °C. 1H NMR: (CDCl3) δ (ppm): 9.64 (bs, 1H),
8.93 (s, 1H), 7.50 (m, 1H), 7.41 (m, 2H), 6.93 (m, 5H), 6.77 (m, 2H),
6.33 (d, 1H, J = 15.6 Hz), 5.12 (s, 2H), 4.20 (m, 1H), 4.11 (q, 2H,
J = 7.2 Hz), 2.02 (m, 2H), 1.61 (m, 10H), 1.22 (t, 3H, J = 7.2 Hz). 13C
NMR: (CDCl3) δ (ppm): 166.66, 162.33, 162.23 (d, J = 245 Hz),
161.91, 153.22, 140.44, 138.95, 131.88 (d, J = 3 Hz), 131.52, 130.46,
4.1.7.3. N-cycloheptyl-1-(4-fluorobenzyl)-5,6-di(4-methoxyphenyl)-2-
oxo-1,2-dihydropyridine-3-carboxamide (B13). Purification by flash
column chromatography on silica gel (petroleum ether/ethyl acetate
8:2) to give the desired compound. Yellow solid (611.23 mg,
1.10 mmol, yield: 58%). Mp: 200–203 °C. 1H NMR (CDCl3) δ (ppm):
9.87 (bs, 1H), 8.65 (s, 1H), 6.86 (m, 8H), 6.67 (m, 4H), 5.21 (s, 2H),
4.19 (m, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 2.03 (m, 2H), 1.63 (m, 10H).
13C NMR (100 MHz, CDCl3) δ (ppm): 162.66, 162.24, 162.04 (d,
J = 245 Hz), 160.04, 158.32, 150.08, 145.64, 132.55 (d, J = 3 Hz),
131.13, 130.68, 130.02, 128.49 (d, J = 8 Hz), 125.60, 122.18, 120.22,
115.43 (d, J = 21 Hz), 113.76, 113.50, 55.31, 55.17, 50.57, 49.18,
35.05, 28.20, 24.32. HPLC analysis: retention time = 17.12 min; peak
area 97.83% (280 nm).
9

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129.07 (d, J = 8 Hz), 128.64, 128.56, 121.40, 118.13, 115.57 (d,
J = 22 Hz), 114.98, 60.53, 50.73, 49.35, 35.01, 28.23, 24.32, 14.27.
HPLC analysis: retention time = 16.95 min; peak area 96.87%
(280 nm).
4.2.4. AEA uptake
AEA uptake inhibition was measured in U937 cells as previously
described in detail [12,33]. Briefly, 0.5 × 106 U937 cells per sample
were incubated with the indicated compounds in RPMI, 37 °C for
15 min. A mixture of [ethanolamine-1-3H]AEA (1 nM) and unlabeled
AEA (final 100 nM) was added for 15 min. The uptake process was
stopped by rapid filtration and washing with ice-cold PBS supple-
mented with 1% fatty acid-free BSA. All experiments were performed at
least three times in triplicate and data are reported as mean values
SD.
4.2. Biological assays
4.2.1. CB1R and CB2R binding assays
Receptor binding experiments were performed with membrane
preparations as previously reported [31]. Briefly, in-house generated
membrane preparations overexpressing hCB1 or hCB2 were re-sus-
pended in binding buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2,
0.5% fatty acid-free bovine serum albumin (BSA), pH 7.4) and in-
cubated with vehicle or compounds and 0.5 nM of [3H]CP55,940 for
90 min at 30 °C. Non-specific binding was determined in the presence of
10 μM of WIN55,512. After incubation, membranes were filtered
through a pre-soaked 96-well microplate bonded with GF/B filters
under vacuum and washed twelve times with 150 μL of ice-cold binding
buffer. The radioactivity was measured and the results expressed as
[3H]CP55,940 binding.
4.3. Docking studies
Compounds B1 and B14 were constructed with Maestro and sub-
jected to energy minimization in a water environment (employing the
Generalized-Born/surface-area model) by using Macromodel. The
minimization was performed until a convergence value of 0.05 kcal/
(Å·mol), using the conjugated gradient method, the MMFFs as force
field and a distance-dependent dielectric constant of 1.0. The ligand
was docked into the CB1R and CB2R binding pockets by means of
AUTODOCK4.2 [34], employing the structures of human CB1R in
complex with the antagonist AM6538 (PDB code 5TGZ) [27] and
human CB2R in complex with the antagonist AM10257 (PDB code:
5ZTY) [26], respectively. The docking sites were defined setting the
bound ligands as the center of a grid of 56, 56 and 50 points, respec-
tively, in the x, y and z directions. A grid spacing of 0.375 Å and a
distance-dependent function of the dielectric constant were used for the
energy map calculations. The compound was subjected to 200 runs of
the AUTODOCK search using the Lamarckian genetic algorithm with
10′000′000 steps of energy evaluation; as previously described [35] The
cluster analysis of the docking results was performed using an RMS
tolerance of 2.0 Å. The best docking pose belonging to the top-ranked
cluster was considered. The same docking procedure was applied to
dock compound B14 into the catalytic site of FAAH enzyme (PDB code
3QJ8) [36].
4.2.2. Functional activity at CB1R and CB2R
Assays were performed as previously described [32]. Briefly, hCB1-
and hCB2-overexpressing membranes (5 μg) were diluted in binding
buffer (50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EDTA, and 100 mM NaCl
at pH 7.4 plus 0.5% fatty acid-free BSA) in the presence of 10 μM of
GDP and 0.1 nM of [35S]GTPγS. The mixture was kept on ice until the
binding reaction was started by adding the vehicle or tested com-
pounds. Non-specific binding was measured in the presence of 10 μM of
GTPγS. The tubes were incubated at 30 °C for 90 min. The reaction was
stopped by rapid filtration through a 96-well microplate bonded with
GF/B filters previously pre-soaked with washing buffer (50 mM of Tris-
HCl pH 7.4 plus 0.1% fatty acid-free BSA). The filters were washed six
times with 180 μL of washing buffer under vacuum. The radioactivity
was measured, and the results were expressed as [35S]GTPγS binding.
Declaration of Competing Interest
A. Chicca and J. Gerstch are co-founders of Synendos Therapeutics.
The authors declare that they have no known competing financial in-
terests or personal relationships that could have appeared to influence
the work reported in this paper.
4.2.3. Enzymatic assays
FAAH, MAGL and ABHDs activity assays were performed as pre-
viously described [12]. Briefly, FAAH and MAGL activity assays were
performed using U937 cell homogenate (100 μg) which were diluted in
200 μL of Tris-HCl 10 mM, EDTA 1 mM, pH 8 containing 0.1% fatty
acid-free BSA. Compounds were added at the screening concentration
of 10 μM and incubated for 30 min at 37 °C. Then, 100 nM of AEA
containing 1 nM of [ethanolamine-1-3H]AEA as a tracer for FAAH or
10 μM of 2-oleoyl glycerol (2-OG) containing 1 nM of [glycerol-
1,2,3-3H]2-OG was added to the homogenates and incubated for 15 min
at 37 °C. The reaction was stopped by the addition of 400 μL of ice-cold
CHCl3: MeOH (1:1), samples were vortexed and rapidly centrifuged at
16000g for 10 min at 4 °C. The aqueous phases were collected and the
radioactivity was measured for tritium content by liquid scintillation
spectroscopy. hABHD6 and hABHD12 activity was determined using
cell homogenates from HEK-293 cells stably transfected with hABHD6
and hABHD12. Compounds were pre-incubated with 40 μg of cell
homogenate for 30 min at 37 °C in assay buffer (Tris 1 mM, EDTA
10 mM plus 0.1% fatty-acid free BSA, pH 7.6). DMSO was used as ve-
hicle control and WWL70 10 μM or THL 20 μM as positive controls for
ABHD6 and ABJHD12, respectively. Then, 10 μM of 2-OG was added
and incubated for 5 min at 37 °C. The reaction was stopped by the ad-
dition of 400 μL of ice-cold CHCl3:MeOH (1:1). The samples were vor-
texed and centrifuged (16000g, 10 min, 4 °C). Aliquots (200 μL) of the
aqueous phase were assayed for tritium content by liquid scintillation
spectroscopy. Blank values were recovered from tubes containing no
enzyme. Basal 2-OG hydrolysis occurring in non-transfected HEK293
cells was subtracted.
Acknowledgements
This research was supported by the Fondazione Italiana Sclerosi
Multipla (FISM) under 2017/R/16.
Competitive grant to Clementina Manera.
Andrra Chicca and Jürg Gertsch were supported by NCCR-
TransCure and University of Bern. Tiziano Tuccinardi was supported by
the Italian Ministry of Healt – Ricerca Finalizzata 2016 - NET-2016-
02363765.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103353.
References
[1] I. Katona, T.F. Freund, Multiple functions of endocannabinoid signaling in the
brain, Annu. Rev. Neurosci. 35 (2012) 529–558, https://doi.org/10.1146/annurev-
neuro-062111-150420.
[2] A. Busquets-Garcia, J. Bains, G. Marsicano, CB(1) Receptor signaling in the brain:
extracting specificity from ubiquity, Neuropsychopharmacology 43 (2018) 4–20,
https://doi.org/10.1038/npp.2017.206.
[3] Y. Lu, H.D. Anderson, Cannabinoid signaling in health and disease, Can. J. Physiol.
10

F. Gado, et al.
BioorganicChemistryxxx(xxxx)xxxx
Pharmacol. 95 (2017) 311–327, https://doi.org/10.1139/cjpp-2016-0346.
therapies, Methods Enzymol. 593 (2017) 175–198, https://doi.org/10.1016/bs.
[4] P. Pacher, R. Mechoulam, Is lipid signaling through cannabinoid 2 receptors part of
a protective system? Prog. Lipid Res. 50 (2011) 193–211, https://doi.org/10.1016/
j.plipres.2011.01.001.
mie.2017.06.021.
[22] A. Chicca, C. Arena, S. Bertini, F. Gado, E. Ciaglia, M. Abate, M. Digiacomo,
M. Lapillo, G. Poli, M. Bifulco, M. Macchia, T. Tuccinardi, J. Gertsch, C. Manera,
Polypharmacological profile of 1,2-dihydro-2-oxo-pyridine-3- carboxamides in the
endocannabinoid system, Eur. J. Med. Chem. 154 (2018) 155–171, https://doi.org/
10.1016/j.ejmech.2018.05.019.
[5] R.G. Pertwee, A.C. Howlett, M.E. Abood, S.P. Alexander, V. Di Marzo, M.R. Elphick,
P.J. Greasley, H.S. Hansen, G. Kunos, K. Mackie, R. Mechoulam, R.A. Ross,
International Union of basic and clinical pharmacology. LXXIX. Cannabinoid re-
ceptors and their ligands: beyond CB(1) and CB(2), Pharmacol. Rev. 62 (2010)
588–631, https://doi.org/10.1124/pr.110.003004.
[23] V. Lucchesi, T. Parkkari, J.R. Savinainen, A.M. Malfitano, M. Allarà, S. Bertini,
F. Castelli, S. Del Carlo, C. Laezza, A. Ligresti, G. Saccomanni, M. Bifulco, V. Di
Marzo, M. Macchia, C. Manera, 1,2-Dihydro-2-oxopyridine-3-carboxamides: The C-
5 substituent is responsible for functionality switch at CB2 cannabinoid receptor,
Eur. J. Med. Chem. 74 (2014) 524–532, https://doi.org/10.1016/j.ejmech.2013.10.
070.
[6] N. Bernabo‘, B. Barboni, M. Maccarrone, Systems biology analysis of the endo-
cannabinoid system reveals a scale-free network with distinct roles for anandamide
and 2-arachidonoylglycerol, OMICS 17 (2013) (2013) 646–654, https://doi.org/10.
1089/omi.2013.0071.
[7] W.A. Devane, J. Axelrod, Enzymatic synthesis of anandamide, an endogenous li-
gand for the cannabinoid receptor, by brain membranes, Proc. Natl. Acad. Sci. USA
91 (1994) 6698–6701, https://doi.org/10.1073/pnas.91.14.6698.
[8] N. Stella, P. Schweitzer, D. Piomelli, A second endogenous cannabinoid that mod-
ulates long-term potentiation, Nature 388 (1997) 773–778, https://doi.org/10.
1038/42015.
[24] W.K. Hagmann, The many roles for fluorine in medicinal chemistry, J. Med. Chem.
51 (2008) 4359–4369, https://doi.org/10.1021/jm800219f.
[25] A. Chicca, J. Marazzi, S. Nicolussi, J. Gertsch, Evidence for bidirectional en-
docannabinoid transport across cell membranes, J. Biol. Chem. 287 (2012)
34660–34682, https://doi.org/10.1074/jbc.M112.373241.
[26] X. Li, T. Hua, K. Vemuri, J.H. Ho, Y. Wu, L. Wu, P. Popov, O. Benchama, N. Zvonok,
K. Locke, L. Qu, G.W. Han, M.R. Iyer, R. Cinar, N.J. Coffey, J. Wang, M. Wu,
V. Katritch, S. Zhao, G. Kunos, L.M. Bohn, A. Makriyannis, R.C. Stevens, Z.J. Liu,
Crystal structure of the human cannabinoid receptor CB2, Cell 176 (2019) 459–467,
https://doi.org/10.1016/j.cell.2018.12.011.
[9] M. Feledziak, D.M. Lambert, J. Marchand-Brynaert, G.G. Muccioli, Inhibitors of the
endocannabinoid-degrading enzymes, or how to increase endocannabinoid’s ac-
tivity by preventing their hydrolysis, Recent Pat. CNS Drug Discov. 7 (2012) 49–70,
https://doi.org/10.2174/157488912798842223.
[10] C.C. Lord, G. Thomas, J.M. Brown, Mammalian alpha beta hydro- lase domain
(ABHD) proteins: lipid metabolizing enzymes at the interface of cell signaling and
energy metabolism, Biochim. Biophys. Acta. 2013 (1831) 792–802, https://doi.
org/10.1016/j.bbalip.2013.01.002.
[27] T. Hua, K. Vemuri, M. Pu, L. Qu, G.W. Han, Y. Wu, S. Zhao, W. Shui, S. Li, A. Korde,
R.B. Laprairie, E.L. Stahl, J.H. Ho, N. Zvonok, H. Zhou, I. Kufareva, B. Wu, Q. Zhao,
M.A. Hanson, L.M. Bohn, A. Makriyannis, R.C. Stevens, Z.J. Liu, Crystal structure of
the human cannabinoid receptor CB1, Cell 167 (2016) 750–762, https://doi.org/
10.1016/j.cell.2016.10.004.
[11] S. Nicolussi, J. Gertsch, Endocannabinoid transport revisited, Vitam Horm. 98
(2015) 441–485, https://doi.org/10.1016/bs.vh.2014.12.011.
[28] C.A. Lunn, E.-P. Reich, J.S. Fine, B. Lavey, J.A. Kozlowski, R.W. Hipkin,
D.J. Lundell, L. Bober, Biology and therapeutic potential of cannabinoid
CB2receptor inverse agonists, Br. J. Pharmacol. 153 (2008) 226–239, https://doi.
org/10.1038/sj.bjp.0707480.
[12] A. Chicca, S. Nicolussi, R. Bartholomäus, M. Blunder, A. Aparisi Rey, V. Petrucci,
I. Reynoso-Moreno, J.M. Viveros-Paredes, M. Dalghi Gens, B. Lutz, H.B. Schiöth,
M. Soeberdt, C. Abels, R.P. Charles, K.H. Altmann, J. Gertsch, Chemical probes to
potently and selectively inhibit endocannabinoid cellular reuptake, Proc. Natl.
Acad. Sci. USA, 114 2017, pp. E5006–E5015, , https://doi.org/10.1073/pnas.
1704065114.
[29] W. Bu, H. Ren, Y. Deng, N. Del Mar, N.M. Guley, B.M. Moore, M.G. Honig,
A. Reiner, Mild traumatic brain injury produces neuron loss that can be rescued by
modulating microglial activation using a CB2 receptor inverse agonist, Front.
Neurosci. 10 (2016) 449, https://doi.org/10.3389/fnins.2016.00449.
[30] S. Montanari, A.M. Mahmoud, L. Pruccoli, A. Rabbito, M. Naldi, S. Petralla,
I. Moraleda, M. Bartolini, B. Monti, I. Iriepa, F. Belluti, S. Gobbi, V. Di Marzo,
A. Bisi, A. Tarozzi, A. Ligresti, A. Rampa, Discovery of novel benzofuran-based
compounds with neuroprotective and immunomodulatory properties for
Alzheimer's disease treatment, Eur. J. Med. Chem. 178 (2019) 243–258, https://
doi.org/10.1016/j.ejmech.2019.05.080.
[13] P. Pacher, G. Kunos, Modulating the endocannabinoid system in human health and
disease successes and failures, FEBS J. 280 (2013) 1918–1943, https://doi.org/10.
1111/febs.12260.
[14] T. Jourdan, G. Szanda, A.Z. Rosenberg, J. Tam, B.J. Earley, G. Godlewski, R. Cinar,
Z. Liu, J. Liu, C. Ju, P. Pacher, G. Kunos, Overactive cannabinoid 1 receptor in
podocytes drives type 2 diabetic nephropathy, Proc. Natl. Acad. Sci. USA 111
(2014) E5420–E5428, https://doi.org/10.1073/pnas.1419901111.
[15] B. Gatta-Cherifi, D. Cota, New insights on the role of the endocannabinoid system in
the regulation of energy balance, Int. J. Obes. (Lond) 40 (2016) 210–219, https://
doi.org/10.1038/ijo.2015.179.
[31] A. Chicca, D. Caprioglio, A. Minassi, V. Petrucci, G. Appendino, O. Taglialatela-
Scafati, J. Gertsch, Functionalization of b-caryophyllene generates novel poly-
pharmacology in the endocannabinoid system, ACS Chem. Biol. 9 (2014)
1499–1507, https://doi.org/10.1021/cb500177c.
[16] V. Di Marzo, New approaches and challenges to targeting the endocannabinoid
system, Nat Rev Drug Discov. 17 (2018) 623–639, https://doi.org/10.1038/nrd.
2018.115 Erratum. In: Nat Rev Drug Discov. 17 (2018) 688.
[32] V. Petrucci, A. Chicca, S. Glasmacher, J. Paloczi, Z. Cao, P. Pacher, J. Gertsch,
Pepcan-12 (RVD-hemopressin) is a CB2 receptor positive allosteric modulator
constitutively secreted by adrenals and in liver upon tissue damage, Sci. Rep. 7
(2017) 9560, https://doi.org/10.1038/s41598-017-09808-8.
[17] J.E. Schlosburg, J.L. Blankman, J.Z. Long, D.K. Nomura, B. Pan, S.G. Kinsey,
P.T. Nguyen, D. Ramesh, L. Booker, J.J. Burston, E.A. Thomas, D.E. Selley, L.J. Sim-
Selley, Q.S. Liu, A.H. Lichtman, B.F. Cravatt, Chronic monoacylglycerol lipase
blockade causes functional antagonism of the endocannabinoid system, Nat.
Neurosci. 9 (2010) 1113–1119, https://doi.org/10.1038/nn.2616.
[18] F.F. Hoyer, M. Khoury, H. Slomka, M. Kebschull, R. Lerner, B. Lutz, H. Schott,
D. Lütjohann, A. Wojtalla, A. Becker, A. Zimmer, G. Nickeni, Inhibition of en-
docannabinoid-degrading enzyme fatty acid amide hydrolase increases athero-
sclerotic plaque vulnerability in mice, J. Mol. Cell. Cardiol. 66 (2014) 126–132,
https://doi.org/10.1016/j.yjmcc.2013.11.013.
[33] S. Nicolussi, A. Chicca, M. Rau, S. Rihs, M. Soeberdt, C. Abels, J. Gertsch,
Correlating FAAH and anandamide cellular uptake inhibition using N-alkyl- car-
bamate inhibitors: from ultrapotent to hyperpotent, Biochem. Pharmacol. 92 (2014)
669–689, https://doi.org/10.1016/j.bcp.2014.09.020.
[34] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791, https://doi.org/10.
1002/jcc.21256.
[19] S. Maione, B. Costa, V. Di Marzo, Endocannabinoids: a unique opportunity to de-
velop multitarget analgesics, Pain 154 (2013) S87–S93, https://doi.org/10.1016/j.
pain.2013.03.023.
[35] M. De Leo, C.G. Huallpa, B. Alvarado, C. Granchi, G. Poli, N. De Tommasi, A. Braca,
New diterpenes from Salvia pseudorosmarinus and their activity as inhibitors of
monoacylglycerol lipase (MAGL), Fitoterapia 130 (2018) 251–258.
[36] G. Bononi, C. Granchi, M. Lapillo, M. Giannotti, D. Nieri, S. Fortunato,
M.E. Boustani, I. Caligiuri, G. Poli, K.E. Carlson, S.H. Kim, M. Macchia,
A. Martinelli, F. Rizzolio, A. Chicca, J.A. Katzenellenbogen, F. Minutolo,
T. Tuccinardi, Discovery of long-chain salicylketoxime derivatives as mono-
acylglycerol lipase (MAGL) inhibitors, Eur J Med Chem. 157 (2018) 817–836.
[20] A. Chicca, C. Arena, C. Manera, Beyond the direct activation of cannabinoid re-
ceptors: new strategies to modulate the endocannabinoid system in CNS-related
diseases, Recent Pat. CNS Drug Discov. 10 (2015) 1–20, https://doi.org/10.2174/
1574889810999160603185126.
[21] J. Fernàndez-Ruiz, M. Gòmez-Ruiz, C. García, M. Hernàndez, J.A. Ramos, Modeling
neurodegenerative disorders for developing cannabinoid-based neuroprotective
11