PSNCBAM-1 analogs: Structural evolutions and allosteric properties at cannabinoid CB1 receptor

Article abstract of DOI:10.1016/j.ejmech.2020.112606

Allosteric modulation of the CB1Rs could represent an alternative strategy for the treatment of diseases in which these receptors are involved, without the undesirable effects associated with their orthosteric stimulation. PSNCBAM-1 is a reference diaryl urea derivative that positively affects the binding affinity of orthosteric ligands (PAM) and negatively affects the functional activity of orthosteric ligands (NAM) at CB1Rs. In this work we reported the design, synthesis and biological evaluation of three different series of compounds, derived from structural modifications of PSNCBAM-1 and its analogs reported in the recent literature. Almost all the new compounds increased the percentage of binding affinity of CP55940 at CB1Rs, showing a PAM profile. When tested alone in the [³⁵S]GTPγS functional assay, only a few derivatives lacked detectable activity, so were tested in the same functional assay in the presence of CP55940. Among these, compounds 11 and 18 proved to be functional NAMs at CB1Rs, dampening the orthosteric agonist-induced receptor functionality by approximately 30percent. The structural features presented in this work provide new CB1R-allosteric modulators (with a profile similar to the reference compound PSNCBAM-1) and an extension of the structure-activity relationships for this type of molecule at CB1Rs.

Full text of DOI:10.1016/j.ejmech.2020.112606

European Journal of Medicinal Chemistry 203 (2020) 112606
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
PSNCBAM-1 analogs: Structural evolutions and allosteric properties at
cannabinoid CB1 receptor
,
,
Serena Meini a 1, Francesca Gado a 1, Lesley A. Stevenson b, Maria Digiacomo a,
Alessandro Saba c, Simone Codini c, Marco Macchia a, Roger G. Pertwee b,
Simone Bertini a *, Clementina Manera a
,
a Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy
b School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD Aberdeen, Scotland, UK
c Department of Surgical Pathology, Molecular Medicine and of the Critical Area, University of Pisa, Via Savi 10, 56126, Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Allosteric modulation of the CB1Rs could represent an alternative strategy for the treatment of diseases
in which these receptors are involved, without the undesirable effects associated with their orthosteric
stimulation. PSNCBAM-1 is a reference diaryl urea derivative that positively affects the binding affinity of
orthosteric ligands (PAM) and negatively affects the functional activity of orthosteric ligands (NAM) at
CB1Rs. In this work we reported the design, synthesis and biological evaluation of three different series
of compounds, derived from structural modifications of PSNCBAM-1 and its analogs reported in the
recent literature. Almost all the new compounds increased the percentage of binding affinity of CP55940
Received 15 April 2020
Received in revised form
17 June 2020
Accepted 18 June 2020
Available online 12 July 2020
Keywords:
at CB1Rs, showing a PAM profile. When tested alone in the [35S]GTP
gS functional assay, only a few
Allosteric modulator
CB1 receptors
Endocannabinoid system
PSNCBAM-1
Diaryl urea
GTP
derivatives lacked detectable activity, so were tested in the same functional assay in the presence of
CP55940. Among these, compounds 11 and 18 proved to be functional NAMs at CB1Rs, dampening the
orthosteric agonist-induced receptor functionality by approximately 30%. The structural features pre-
sented in this work provide new CB1R-allosteric modulators (with a profile similar to the reference
compound PSNCBAM-1) and an extension of the structure-activity relationships for this type of molecule
at CB1Rs.
S
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
liver, pancreas, bone and skeletal muscle [4]. CB1Rs have a role in
inhibiting the release of neurotransmitters in the CNS, affecting
The endocannabinoid system (ECS) is ubiquitously distributed
in the human organism and involved in important physiological
and pathological mechanisms [1]. It consists of specific G protein-
coupled receptors (cannabinoid receptor type 1 and 2, called
CB1Rs and CB2Rs respectively) endogenous ligands, called endo-
cannabinoids (the two main ones are anandamide and 2-
arachidonoylglycerol) and it also comprises all the enzymes and
transporters responsible for the biosynthesis, degradation and
transport of endocannabinoids [1e3].
processes like memory, learning, motor functions and pain trans-
mission. On the other hand, peripheral CB1Rs are involved in
metabolism, energy balance, regulation of food intake and body
weight gain [5e7]. CB2Rs are mostly present in immune cells
where they regulate the release of pro-inflammatory cytokines
[8,9], and they are also expressed in some brain regions [7,10,11].
In spite of their theorical usefulness, the use of CB1R orthosteric
ligands in therapy is limited because of the onset of severe side
effects. In particular, CB1R orthosteric agonists, although being
effective in the treatment of pain, nausea, inflammation, cardio-
vascular disorders, multiple sclerosis and cancer, can also produce
psychotropic effects, strong mood alteration, acute psychosis, and
cognitive and motor impairments [12,13]. On the other hand, CB1R
orthosteric antagonists/inverse agonists, besides their useful
application in counteracting obesity, insulin resistance, osteopo-
rosis, nicotine and alcohol addiction, can also give rise to
CB1Rs are mainly expressed in the central nervous system
(CNS), although they can also be found in peripheral districts such
as adipose tissue, cardiovascular system, gastro-intestinal tract,
* Corresponding author.
E-mail address: simone.bertini@unipi.it (S. Bertini).
S. M. and F. G. contributed equally.
1
https://doi.org/10.1016/j.ejmech.2020.112606
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
depression, mood alterations and suicidal tendency [14e16].
For this reason, the need of an alternative therapeutic strategy in
order to avoid these side effects and the discovery on CB1Rs of an
allosteric binding site in 2005, led to further investigations in this
field [17e19]. Allosteric modulators (AMs), binding to a topo-
graphically distinct site, stabilize a unique conformation of the re-
ceptor, influencing the binding affinity and/or the efficacy of
orthosteric ligands [20e22]. They are characterized by a saturable
effect, giving less risk of overdose, since the extent of their efficacy
is dependent on the local concentration of endogenous ligands
(endocannabinoids in the case of CBRs). This feature is also related
to an improved selectivity, because AMs can provide an effect only
in the tissues and at the time endocannabinoids are released,
without interfering with the physiological signaling pathways [23].
Moreover, given the lower conservation of allosteric binding sites,
the development of more selective compounds acting at these sites
should be more easily achievable [20,24]. AMs can be classified as
positive (PAMs) if they enhance the binding affinity and/or the ef-
ficacy of the orthosteric ligand, or negative (NAMs) if the binding
affinity and/or the efficacy of the orthosteric ligand is decreased
[25,26]. The well-known CB1R allosteric modulator PSNCBAM-1
(compound 1, Fig. 1), characterized by a diaryl urea structure,
showed divergent properties, affecting the binding affinity and the
efficacy of orthosteric ligands in opposite directions at CB1R (PAM
in binding and NAM in functionality) [27]. Given its usefulness in
counteracting obesity, influencing food intake without affecting
behavior, PSNCBAM-1 is a reference compound which can be taken
as a starting point for structural modifications in order to achieve
improved pharmacological properties [28].
To deepen the knowledge of the structure-activity relationships
at CB1R for this class of compound, in this work we reported the
design, synthesis and biological evaluation of three different series
of compounds, derived from PSNCBAM-1 and its analogs reported
in the recent literature [29,30]. The structure activity relationships
studies performed up to date showed that the pyridinyl ring can be
successfully replaced by a phenyl ring, giving the active compound
2 (Fig. 1) with a biological profile resembling the activity of
PSNCBAM-1 at CB1R [29]. This consideration led us to the design of
“A” series, comprising compounds 10, 11 and 12 (Fig. 1). 10 and 11
are characterized by different substituents at the extremities of the
molecule, to better understand the stereo-electronic requirements
of these portions at CB1R. On the other hand, 12, homologue of
compound 3 (Fig. 1), recently reported in literature [29], was
designed to establish if increasing the degrees of freedom of the
molecule, could improve its activity. Another structural feature
taken in account, is the insertion of an amine spatial linker in the
biaryl system, similar to compounds 4 and 5 (Fig. 1), which dis-
played good properties as allosteric modulators with a behavior
similar to that of PSNCBAM-1 [29]. On these bases, compounds 13
and 14 (“B” series, Fig. 1) were designed. Both compounds possess
two pyridine rings to fully elucidate the importance of the nature of
the biaryl system components. Another recent study proved that
the pyrrolidinyl ring is not required for CB1R allosteric modulation
and the pyridinyl ring could be replaced by other aromatic rings
[30]. Some of the most promising derivatives are reported in Fig. 1
(compounds 6e9). All these new derivatives displayed a PAM
profile in binding assays and behaved as NAMs in functional assays,
like the parental compound. Combining these structural features
with the presence of the amine spatial linker, we designed com-
pounds 15e19 (“C” series, Fig. 1). In the following paragraphs we
described the synthetic routes of the new derivatives and their
biological evaluation obtained by carrying out [3H]CP55940 and
[
35S]GTP
gS binding assays.
2. Results and discussion
2.1. Chemistry
The synthetic procedure to obtain compounds of “A” series is
reported in Scheme 1.1,3-dibromobenzene (20) was heated at 80 ꢀC
in toluene with Pd2(dba)3, BINAP, t-BuONa and the suitable sec-
ondary cyclic amine, to afford intermediates 21 or 22. These de-
rivatives were subjected to a cross-coupling reaction with 3-
nitrophenylboronic acid in presence of Pd(OAc)2, PPh3 and an
aqueous solution of NaHCO3 in DME at 95 ꢀC, to obtain compounds
23 and 24. The nitro group was then reduced with Ni-Raney and
hydrazine hydrate in a mixture of ethanol and water at 50 ꢀC
affording derivatives 25 and 26 that were treated with the suitable
phenylisocyanate to give the desired products 10 and 11, respec-
tively. For the preparation of 12, the intermediate 25 was reacted
with the acyl chloride 27 (synthesized from commercial 3-(4-
chlorophenyl)propionic acid (28) and thionyl chloride), in the
presence of Et3N and DMAP in anhydrous DCM.
The synthesis of compounds of “B” series is reported in Scheme
2. Commercial 2,6-diaminopyridine (29) was reacted with com-
mercial 2,6-dibromopyridine in presence of t-BuOK in refluxing
Fig. 1. Design of novel structural modifications of PSNCBAM-1 based on the derivatives recently reported in the literature [29,30].

S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
3
reflux, affording derivative 40. This was then treated with p-
chlorophenylisocyanate as described above to give the desired
compound 17.
2.2. [3H]CP55940 binding assay (CB1R)
To evaluate CB1R binding, different concentrations of com-
pounds 10e19 (from 1 nM up to 10 mM) were incubated with hCB1-
CHO cell membrane preparations in presence of [3H]CP55940
(0.700 nM), a high-affinity orthosteric CB1R and CB2R radioligand.
Almost all the compounds belonging to “A” series (characterized
by a biphenyl moiety) and “B” series (characterized by a bipyridinyl
system separated by an amine linker) (Figs. 2 and 3) resulted PAMs
in binding assays, enhancing the binding of [3H]CP55940 by
approximately 70% (10), 60% (13) and 50% (14) up to 10
dose-dependent trend. Compound 11 displayed an enhancement of
binding (about 40%) up to 1 M, being completely ineffective at
10 M. This behavior might be possibly due to a strong decrease of
solubility which occurs when the concentration of 11 is raised to
10 M. Compound 12, the only amide derivative, did not influence
mM in a
m
m
Scheme 1. Synthetic route for the preparation of compounds of “A” series. Reagents
and conditions: a) pyrrolidine or piperidine, Pd2(dba)3, BINAP, t-BuONa (mol ratio:
0.05:0.15:2), toluene, 80 ꢀC, 4.5 h; b) 3-nitrophenylboronic acid, Pd(OAc)2, PPh3, aq.
NaHCO3, DME, 95 ꢀC, overnight; c) Ni-Raney, NH2NH2$H2O, abs. EtOH, 50 ꢀC, 40 mine1
h; d) p-chlorophenylisocyanate or p-fluorophenylisocianate, anhydrous CHCl3, rt,
overnight; e) SOCl2, rt, overnight; f) Et3N, DMAP, anhydrous DCM, rt, overnight.
m
the binding and behaved as a weak orthosteric ligand at 10
(Fig. S1 in Supplementary Data).
mM
As to “C” series (Fig. 4), compounds 15, 18 and 19 showed a PAM
profile, dose-dependently increasing the binding percentage of [3H]
CP55940 by approximately 170% (15) and 90% (18 and 19). 15 dis-
played this trend up to 10
mM, whereas compounds 18 and 19
revealed a slow decrease of the activity above 1 mM. Finally, com-
pounds 16 and 17 affected the binding of [3H]CP55940 only at a
neglectable extent (Fig. S2 in Supplementary Data).
2.3. [35S]GTP
gS assay
Different concentrations of compounds 10, 11, 13e15, 18 and 19
(from 1 nM up to 10 S assay, using
M) were tested in the [35S]GTP
m
g
hCB1-CHO membrane preparations, in order to determine their
ability to affect CB1R functionality on their own. This evaluation
was performed in order to establish if the compounds do produce a
functional effect by themselves, since this behavior would preclude
their pure allosteric nature.
Scheme 2. Synthetic route for the preparation of compounds of “B” series. Reagents
and conditions: a) 2,6-dibromopyridine, t-BuOK, anhydrous toluene, reflux, 48 h; b)
pyrrolidine or piperidine, reflux, 12 h; c) p-chlorophenylisocianate, anhydrous CHCl3,
rt, overnight.
Compounds 13 and 14 did not significatively alter the cellular
response at CB1R up to 10 mM (Fig. 5).
On the other hand, compound 10, 15 and 19 proved to decrease
the cellular response by 45% (10) and approximately 30% (15 and
19) showing an inverse agonist profile (Fig. S3 in Supplementary
Data).
Compound 11 and 18 showed no significant alteration of re-
ceptor functionality up to 1 mM and a slight decrease only above
anhydrous toluene. The obtained intermediate 30 was refluxed
with the suitable cyclic amine to afford compounds 31 and 32,
which were then reacted with p-chlorophenylisocyanate at room
temperature in anhydrous chloroform to give the desired products
13 and 14, respectively.
The synthetic route for the preparation of compounds of “C”
series is described in Scheme 3. For the synthesis of compounds 15,
18 and 19, commercial 1,3-diaminobenzene (33) was refluxed with
the suitable aryl halide in presence of t-BuOK in anhydrous toluene,
to obtain intermediates 34e36. These were then reacted with p-
chlorophenylisocyanate at room temperature in anhydrous chlo-
roform to give the desired compounds 15, 18 and 19.
For the synthesis of compound 16, commercial 2,6-
diaminopyridine (29) was reacted with iodobenzene in presence
of t-BuOK and copper iodide, in anhydrous 1,4-dioxane at 100 ꢀC, to
afford the intermediate 37 that was treated with p-chlor-
ophenylisocyanate as described above to give the desired com-
pound 16.
For the synthesis of compound 17, commercial 1-bromo-3-
nitrobenzene (38) was treated with 4-aminopyridine, xantphos
and Pd2(dba)3 in anhydrous 1,4-dioxane in presence of Cs2CO3, at
95 ꢀC, affording the derivative 39, which was reduced with iron
powder and ammonium chloride in a mixture of ethanol/water, at
this concentration, by approximately 30% and 35%, respectively
(Fig. 6).
Because of the above results, compounds 11, 13, 14 and 18 (at a
chosen concentration of 1
assays, against different concentrations of CP55940 (from 0.1 nM
up to 1 M), to evaluate their ability to modulate the CB1R-
m gS functional
M) were tested in [35S]GTP
m
mediated functional activity induced by the orthosteric agonist.
While 13 and 14 did not influence the activity of the orthosteric
ligand (Fig. S4 in Supplementary Data), when CP55940 was co-
administered with 11 and 18, the receptor functionality was
dampened by approximately 30%, suggesting that 11 and 18 behave
as functional NAMs at CB1R. Compound 18, albeit only to a minor
extent, retained its activity in this assay also at 100 nM (Fig. 7).
2.4. [3H]CP55940 binding assay (CB2R)
Compounds 11 and 18 were also tested in CB2R binding exper-
iments (from 1 nM up to 10 m
M) in the presence of [3H]CP55940

4
S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
Scheme 3. Synthetic route for the preparation of compounds of “C” series. Reagents and conditions: a) 2-brompyridine or 4-bromofluorobenzene or iodobenzene, t-BuOK,
anhydrous toluene, reflux, 48 h; b) iodobenzene, t-BuOK, CuI, anhydrous 1,4-dioxane, 100 ꢀC, 24 h; c) 4-aminopyridine, xantphos, Pd2(dba)3, Cs2CO3, anhydrous 1,4-dioxane, 95 ꢀC,
24 h; d) Fe0 powder, NH4Cl, EtOH/H2O, reflux, 4 h; e) p-chlorophenylisocianate, anhydrous CHCl3, rt, overnight.
to clarify its receptor subtype selectivity and its pharmacological
effects. On the other hand, compound 18 did not affect the binding
affinity of CP55940 at any concentration, displaying a high selec-
tivity for CB1R over CB2R.
3. Conclusions
In this work we have reported the design, synthesis and bio-
logical evaluation of new derivatives of the known CB1R allosteric
modulator PSNCBAM-1. The structural modifications are based on
recent structural analysis described in literature, with the purpose
of extending information about the structure activity relationships
at CB1R of diaryl urea derivatives. As to the “A” series (characterized
by a biphenyl system) compounds 10 and 11 behaved as PAMs in
radioligand binding assays against CP55940, whereas 12, the only
Fig. 2. Effects of compounds 10 and 11 (“A” series) (at 1 nM to 10 m
M) on [3H]CP55940
binding to CB1R. Asterisks indicate mean values significantly different from zero
(*P < 0.05; **P < 0.01; ****P < 0.0001) (one sample t-test). Data are expressed as the
mean SEM of six independent experiments, each performed in duplicate.
amide derivative, did not display any detectable activity in this
binding assay. These results suggest that the replacement of the
chlorine atom on phenyl “a” with a fluorine atom (Fig. 1), combined
with a biphenyl system, is tolerated and the substitution of pyrro-
lidine with piperidine does not remarkably change the activity. On
the other hand, the urea group seems fundamental for the activity,
as was previously claimed by Bertini et al. [29], and the increase of
the degrees of freedom of this portion of the molecule does not
provide any improvement. Compound 10 behaved as an inverse
agonist in [35S]GTP
receptor functionality only above 10
g
S functional assays, whereas 11 inhibited the
mM, indicating that the strong
electro-withdrawing (EWG) effect of fluorine might stabilize a re-
ceptor conformation endowed with higher constitutional activity,
at least of this signaling pathway [31,32]. Moreover, 11 (1
mM)
displayed a strong inhibition of the cellular response in the pres-
ence of CP55940, indicating a NAM behavior in functionality, with a
profile similar to PSNCBAM-1. Regarding “B” series, where a
bipyridinyl system is separated by an amine spatial linker, both
compounds 13 and 14 behaved as PAMs in binding assay, not
influencing the cellular response in [35S]GTP
gS functional assays
either in the absence and in the presence of the orthosteric com-
pound CP55940. This outcome confirms that the difference be-
tween pyrrolidine and piperidine on aryl ring “c” (Fig. 1) is
negligible and might suggest that the presence of the bipyridinyl
system together with the amine linker does not affect the influence
on binding, but does stabilize a receptor conformation which
cannot couple to G protein. Further investigations on this class of
compounds should involve the functional assessment of other
Fig. 3. Effects of compounds 13 and 14 (“B” series) (at 1 nM to 10 m
M) on [3H]CP55940
binding to CB1R. Asterisks indicate mean values significantly different from zero
(*P < 0.05; **P < 0.01) (one sample t-test). Data are expressed as the mean SEM of
six independent experiments, each performed in duplicate.
(0.700 nM), using hCB2-CHO cell membrane preparations, in order
to assess their receptor subtype selectivity (Fig. 8). Derivative 11
produced a displacement of the orthosteric agonist above 1
mM, so
signaling pathways, such as
b-arrestins recruitment and ERK
further experiments in hCB2-CHO cells would be necessary in order
phosphorylation, which could be differently influenced [33,34]. As

S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
5
Fig. 4. Effects of compounds 15, 18 and 19 (“C” series) (at 1 nM to 10 m
M) on [3H]CP55940 binding to CB1R. Asterisks indicate mean values significantly different from zero
(*P < 0.05; **P < 0.01; ***P < 0.001 ****P < 0.0001) (one sample t-test). Data are expressed as the mean SEM of six independent experiments, each performed in duplicate.
replaces phenyl ring “b” (Fig. 1) and the results may indicate that a
nitrogen atom is not tolerated at this position, in this kind of biaryl
systems. A similar assumption can be made for 17, which bears a
nitrogen atom at position 4 of aryl ring “c” (Fig. 1). All the other
compounds of “C” series behaved as PAMs in binding assays, but
only 18 was unable to influence the receptor functionality on its
own, decreasing the cellular response only in presence of CP55940.
The fluorine atom, already related to a good activity both in terms
of binding and of functionality and also to an increased metabolic
stability by Nguyen et al. [30], might positively contribute thanks to
its EWG effect combined with its small size, which makes it steri-
cally comparable to a hydrogen with the ability to significantly
change the lipophilicity and conformation of the molecule [31,32].
The remaining compounds of the series, 15 and 19, behaved as
inverse agonists in functional assays indicating that a biaryl system
consisting of a phenyl and a pyridine separated by an amine linker
Fig. 5. [35S]GTP
gS assays performed with compounds 13 and 14 (“B” series). Data are
(15) changes the kind of activity displayed by the compounds 4 and
PSNCBAM-1 [29]. A similar behavior is displayed by 19, which is
directly derived from the combination of the structural features of 2
and 4 [29].
expressed as the mean
duplicate.
SEM of six independent experiments, each performed in
The new structural features analyzed in this work can be taken
as starting points for further modifications of this kind of de-
rivatives, providing an extension of the structural requirements at
CB1R and potential novel strategies for developing new and
improved allosteric modulators of CB1R.
4. Experimental section
4.1. Chemistry
Commercially available reagents were purchased from Sigma
Aldrich or TCI chemicals and used without purification, if not
differently indicated in the procedures. 1H NMR and 13C NMR
spectra were recorded on a Bruker AVANCE III™ 400 spectrometer
(operating at 400 MHz or 100 MHz, respectively). Chemical shifts
(d) are reported in parts per million related to the residual solvent
Fig. 6. [35S]GTP
Asterisks indicate mean values significantly different from zero (**P
****P < 0.0001) (one sample t-test). Data are expressed as the mean
g
S assays performed with compound 11 (“A” series) and 18 (“C” series).
0.01;
SEM of six
signal, while coupling constants (J) are expressed in Hertz (Hz). All
final compounds were analyzed by HPLC, showing a purity in the
range 96e99%. A Bechman HPLC instrument equipped with a Sys-
tem Gold Solvent Delivery module (Pumps) 125, System Gold UV/
VIS Detector 166 (set to 278 nm) was employed. Analyses were
<
independent experiments, each performed in duplicate.
to compounds of “C” series, characterized by the presence of the
amine spatial linker and the removal of the pyrrolidine moiety, only
compounds 16 and 17 were ineffective in influencing the binding of
CP55940 and the functionality at CB1R. 16 is the first derivative
reported in the literature up to date, in which a pyridine ring
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,

6
S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
Fig. 7. CB1R [35S]GTP
g
S experiments with compounds 11 (“A” series) and 18 (“C” series) against increasing concentrations of CP55940. Asterisks indicate mean values significantly
different from zero (*P < 0.05; **P < 0.01; ***P < 0.001 ****P < 0.0001) (one sample t-test). Data are expressed as the mean SEM of six independent experiments, each performed
in duplicate.
per 0.239 mmol of aryl amine) in a round bottom flask, under ni-
trogen atmosphere. The mixture was left under magnetic stirring
overnight at room temperature.
4.1.2. General procedure B
The suitable phenylisocyanate (1.2 equiv) was added to a solu-
tion of aryl amine (1.0 equiv) and THF (2.7 mL per 0.440 mmol of
aryl amine) in a round bottom flask. The mixture was refluxed for
1 h, then cooled to room temperature and the solvent was removed
under reduced pressure. The residue was triturated with ethanol.
4.1.3. General procedure C
A mixture of the suitable aryl amine (1.0 equiv), t-BuOK (2.3
equiv) and the proper amount of aryl halide (1.0 equiv) were
refluxed in anhydrous toluene (100.0 mL per 18.5 mmol of aryl
amine) for 48 h, under inert atmosphere. After cooling to room
temperature, the solvent was removed in vacuo, the residue dis-
Fig. 8. Effects of compounds 11 (“A series) and 18 (“C” series) (at 1 nM to 10 mM) on
solved in AcOEt and washed with water. The organic phase was
dried over Na2SO4, filtered and evaporated under reduced pressure.
[3H]CP55940 binding to CB2R. Asterisks indicate mean values significantly different
from zero (**P < 0.01) (one sample t-test). Data are expressed as the mean SEM of six
independent experiments, each performed in duplicate.
4.1.4. General procedure D
In a round bottom flask, the suitable nitroarene (1.0 equiv) and
hydrazine hydrate (0.14 equiv) were suspended in absolute ethanol
(4.0 mL per 0.24 mmol of nitroarene). The mixture was stirred at
50 ꢀC for 15 min to promote the complete solubilization of the
reagents. Then, an excess of an aqueous suspension of Raney nickel
was added (about 200 mg per 0.24 mmol of nitroarene) and the
reaction, which changed from orange to colorless, was left under
magnetic stirring at 50 ꢀC until the bubbling ended. The mixture
was filtered through a celite pad and washed with ethanol. The
solution collected was evaporated under reduced pressure to
obtain the desired product.
was used for compounds 10, 12, 13, 15e19. For compounds 11 and
14 was chosen an isocratic elution with 80% of B and 20% of A. The
flow rate was 1.0 mL/min. Retention times (HPLC, tR) are given in
minutes. Mass experiments were performed with an AB Sciex API
3000 triple quadrupole mass spectrometer, equipped with a Tur-
boIonSpray source. Compounds were analyzed both in positive and
negative mode; in Q1 scan mode, for the detection of pseudo-
molecular ions [MþH]þ and [M-H]-; to obtain structural informa-
tion, MS-MS experiments were performed in product ion scan
mode. Samples were dissolved in DMSO and diluted 1:1000 with an
ACN/H2O 50:50 (v/v) solution. The instrumental parameters used
were: Curtain Gas: 12.0; Ion Spray Voltage: 5500 V; Temperature:
450 ꢀC; Declustering Potential: 20.0; Entrance Potential: 10.0.
Evaporation was carried out in vacuo using a rotating evaporator.
Silica gel flash chromatography was performed using silica gel 60 Å
(0.040e0.063 mm; MERK). Reactions was monitored by TLC on
Merck aluminum silica gel (60 F254) plates that were visualized
under a UV lamp (ε ¼ 254 nm). Melting points were determined on
a Kofler hot-stage apparatus and are uncorrected.
4.1.5. General procedure E
In a sealed tube, under nitrogen flux, commercial triphenyl-
phosphine (0.4 equiv) and Pd(OAc)2 (0.08 equiv) were dissolved in
DME (5.0 mL per 0.3 mmol of triphenylphosphine) and the mixture
was stirred for 15 min at room temperature. A solution of the
suitable aryl halide (1.0 equiv) in DME (6.0 mL per 0.79 mmol of aryl
halide) was added to the reaction tube, followed by the addition of
NaHCO3 (3.0 equiv), distilled water (4.5 mL per 0.79 mmol of aryl
halide) and 3-nitrophenylboronic acid (1.0 equiv). The reaction
mixture was stirred at 95 ꢀC, overnight. After the removal of DME
under reduced pressure, the residue was partitioned between
AcOEt and distilled water. The organic phase was dried over
4.1.1. General procedure A
The suitable phenylisocyanate (1.0 equiv) was added to a solu-
tion of aryl amine (1.0 equiv) and anhydrous chloroform (10.0 mL

S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
7
Na2SO4, filtered and evaporated under reduced pressure to obtain a
dark oil.
0.24 mmol) according to general procedure D. 25 (58.5 mg,
0.233 mmol). Yield: 97%. 1H NMR: (CDCl3)
(ppm): 7.30 (t, 1H,
d
J ¼ 7.8 Hz), 7.23 (t, 1H, J ¼ 7.8 Hz), 7.05e7.02 (m, 1H), 6.95e6.93 (m,
1H), 6.89e6.86 (m, 1H), 6.76e6.75 (m, 1H), 6.70e6.67 (m, 1H),
6.59e6.56 (m,1H), 3.36e3.33 (m, 4H), 2.06e2.01 (m, 4H).
4.1.6. General procedure F
In a sealed tube, commercial 1,3-dibromobenzene (1.0 equiv)
was suspended in toluene (2.1 mL per 0.85 mmol of 1,3-
dibromobenzene), followed by the addition of the suitable cyclic
secondary amine (1.0 equiv) and the suitable amount of a reagent
(157.2 mg of reagent per 0.85 mmol of starting material) consti-
tuted by tris(dibenzylideneacetone)dipalladium(0), BINAP and t-
BuONa (mol ratio: 0.05:0.15:2). The reaction mixture was stirred at
80 ꢀC for 4.5 h. After cooling to room temperature, the mixture was
filtered under reduced pressure and the solution collected was
evaporated under reduced pressure. The resulting residue was
diluted with CHCl3 and washed with a 10% aqueous solution of
NaOH. After anhydrification over Na2SO4, the organic phase was
filtered and evaporated under reduced pressure.
4.1.13. 3’-(piperidin-1-yl)[1,10-biphenyl]-3-amine (26)
Compound 26 was prepared from 24 (73.8 mg, 0.263 mmol)
according to general procedure D. 26 (64.6 mg, 0.229 mmol) Yield:
87%. 1H NMR: (CDCl3)
d (ppm): 7.31e7.25 (m, 1H), 7.22e7.19 (m,1H),
7.14e7.10 (m,1H), 7.03e6.96 (m, 2H), 6.95e6.88 (m, 2H), 6.68e6.64
(m,1H), 3.21e3.16 (m, 4H), 1.74e1.66 (m, 6H).
4.1.14. N-(4-fluorophenyl)-N’-[3’-(pyrrolidin-1-yl)[1,10-biphenyl]-3-
yl]urea (10)
Compound 10 was prepared from 25 (68.0 mg, 0.285 mmol),
following general procedure A. Purification by flash column chro-
matography on silica gel (petroleum ether/AcOEt 7:3). 10 (28.7 mg,
4.1.7. General procedure G
0.076 mmol). Yield: 27%. M. p.: 185 ꢀC. 1H NMR: (DMSO‑d6)
d (ppm):
The suitable aryl halide (1.0 equiv) was refluxed with the suit-
able cyclic secondary amine (19.3 equiv) for 12 h. After cooling to
room temperature, the excess of unreacted amine was removed
under reduced pressure. The brown oil obtained was dissolved in
CHCl3 and washed with distilled water. The organic phase was then
dried over Na2SO4, filtered and evaporated under reduced pressure.
8.74 (m, 2H), 7.70e7.69 (m, 1H), 7.49e7.45 (m, 2H), 7.43e7.41 (m,
1H), 7.33 (t,1H, J ¼ 7.7 Hz), 7.23 (AA’XX’, 2H, JAX ¼ 8.0 Hz,
JAA’XX’ ¼ 1.5 Hz), 7.12 (AA’XX’, 2H, JAX ¼ 8.0 Hz, JAA’XX’ ¼ 1.5 Hz),
6.82e6.80 (m,1H), 6.71e6.69 (m,1H), 6.56e6.53 (m,1H), 3.30e3.27
(m, 4H), 1.99e1.96 (m, 4H). 13C NMR: (DMSO‑d6)
d (ppm): 157.34 (d,
J ¼ 228.0 Hz), 152.65, 148.02, 141.93, 141.14, 140.01, 135.98 (d,
J ¼ 2.0 Hz), 129.40, 129.10, 120.38, 119.97 (d, J ¼ 7.0 Hz), 117.09,
116.61, 115.22 (d, J ¼ 22.0 Hz), 113.73, 110.91, 109.75, 47.30, 24.93.
MS: the experiment in full scan mode showed: m/z 376 ([MþH]þ,
100); MSeMS of the ion at m/z 376 showed: m/z 265 ([MþH]þ ‒ p-
Cl-C6H4-NH, 100), 239 ([MþH]þ ‒ p-Cl-C6H4-NHCO, 18). HPLC, tR
15.23.
4.1.8. 1-(3-bromophenyl)pyrrolidine (21)
Compound 21 was prepared from 1,3-dibromobenzene (0.10 mL,
0.85 mmol) and pyrrolidine according to general procedure F. Pu-
rification by flash column chromatography on silica gel, eluting
with petroleum ether. 21 (190.5 mg, 0.843 mmol). Yield: 99%. 1H
NMR (CDCl3)
d
(ppm): 7.06 (t, 1H, J ¼ 8.0 Hz), 6.78e6.76 (m, 1H),
6.69e6.71 (m, 1H), 6.50e6.49 (m, 1H), 3.28e3.25 (m, 4H),
2.03e1.99 (m, 4H).
4.1.15. N-(4-chlorophenyl)-N’-[3’-(piperidin-1-yl)[1,10-biphenyl]-3-
yl]urea (11)
Compound 11 was prepared from 26 (59.1 mg, 0.234 mmol),
following general procedure A. Purification by two subsequent
flash column chromatographies on silica gel (CHCl3/MeOH 99:1). 11
(53.4 mg, 0.131 mmol). Yield: 56%. M. p.: 99 ꢀC. 1H NMR: (DMSO‑d6)
4.1.9. 1-(3-bromophenyl)piperidine (22)
Compound 22 was prepared from 1,3-diaminobenzene
(0.50 mL, 4.24 mmol) and piperidine according to general proced-
ure F. Purification by flash column chromatography on silica gel,
eluting with petroleum ether and petroleum ether/AcOEt 9:1. 22
d
(ppm): 8.94 (bs, 1H), 8.88 (bs, 1H), 7.70e7.68 (m, 1H), 7.50 (AA’XX’,
2H, JAX ¼ 9.0 Hz, JAA’XX’ ¼ 2.6 Hz), 7.44e7.42 (m, 1H), 7.36e7.34 (m,
1H), 7.32 (AA’XX’, 2H, JAX ¼ 9.0 Hz, JAA’XX’ ¼ 2.6 Hz), 7.30e7.26 (m,
1H), 7.25e7.22 (m, 1H), 7.09e7.08 (m, 1H), 6.99e6.92 (m, 2H),
3.21e3.18 (m, 4H), 1.67e1.61 (m, 4H), 1.57e1.55 (m, 2H). 13C NMR:
(676.3 mg, 2.81 mmol). Yield: 66%. 1H NMR: (CDCl3)
d (ppm): 7.08
(t, 1H, J ¼ 8.0 Hz), 7.04e7.03 (m, 1H), 6.92e6.89 (m, 1H), 6.84e6.81
(m, 1H), 3.17e3.14 (m, 4H), 1.71e1.66 (m, 4H), 1.61 (m, 2H).
(DMSO‑d6)
d (ppm): 152.46, 152.06, 141.60, 141.05, 139.88, 138.65,
4.1.10. 1-(30-nitro[1,10-biphenyl]-3-yl)pyrrolidine (23)
129.37, 129.13, 128.53, 125.29, 120.51, 119.73, 117.24, 116.92, 116.70,
115.07, 114.03, 48.52, 25.19, 23.86. MS: the experiment in full scan
mode showed: m/z 406 ([MþH]þ, 100); MSeMS of the ion at m/z
406 showed: m/z 279 ([MþH]þ ‒ p-Cl-C6H4-NH, 100). HPLC, tR 4.70.
Compound 23 was prepared from 21 (179.1 mg, 0.793 mmol)
according to general procedure E. Purification by flash column
chromatography on silica gel (petroleum ether/CH2Cl2 7:3). 23
(115.0 mg, 0.43 mmol). Yield: 54%. 1H NMR (CDCl3)
d (ppm):
8.46e8.45 (m, 1H), 8.19e8.16 (m, 1H), 7.94e7.91 (m, 1H), 7.58 (t, 1H,
J ¼ 8.0 Hz), 7.33 (t, 1H, J ¼ 8.0 Hz), 6.89 (d, 1H, J ¼ 8.0 Hz), 6.75e6-74
(m, 1H), 6.65e6.63 (m, 1H), 3.38e3.35 (m, 4H), 2.07e2.03 (m, 4H).
4.1.16. 3-(4-Chlorophenyl)-N-[3’-(pyrrolidin-1-yl)biphenyl-3-yl]
propanamide (12)
In a vial, commercial 3-(4-chlorophenyl)propionic acid 28
(119.0 mg, 0.645 mmol) was dissolved in thionyl chloride (1.56 mL,
0.645 mmol) under nitrogen flux. The vial was sealed and left under
magnetic stirring at room temperature, overnight, to allow the
formation of the acyl chloride (27), then the excess of unreacted
thionyl chloride was removed under nitrogen flux. In a round
bottom flask under nitrogen atmosphere, triethylamine (0.1 mL,
75.2 mg, 0.745 mmol) and DMAP (90.5 mg, 0.741 mmol) were
added to a solution of 25 (70.1 mg, 0.745 mmol) in the minimum
amount of anhydrous CH2Cl2. The flask was cooled to ꢂ5 ꢀC (ice-
NaCl bath) and the crude acyl chloride was added dropwise. After
24 h at room temperature, under magnetic stirring, the reaction
was heated at 40 ꢀC for 6 h. The reaction mixture was partitioned
between CH2Cl2 and an aqueous saturated solution of NaHCO3.
4.1.11. 1-(30-nitro[1,10-biphenyl]-3-yl)piperidine (24)
Compound 24 was prepared from 22 (89.8 mg, 0.37 mmol) ac-
cording to general procedure E. Purification by two subsequent
flash column chromatographies on silica gel (petroleum ether/
AcOEt 9:1 and petroleum ether/CH2Cl2 8:2). 24 (73.8 mg,
0.263 mmol). Yield: 70%. 1H NMR: (CDCl3)
d (ppm): 8.44e8.43 (m,
1H), 8.19e8.17 (m,1H), 7.91e7.89 (m,1H), 7.58 (t, 1H, J ¼ 7.9 Hz), 7.36
(t, 1H, J ¼ 7.9 Hz), 7.15e6.98 (m, 1H), 7.07e6.98 (m, 2H), 3.26e3.23
(m, 4H), 1.78e1.71 (m, 6H).
4.1.12. 3’-(pyrrolidin-1-yl)[1,10-biphenyl]-3-amine (25)
Compound 25 was prepared starting from 23 (63.8 mg,

8
S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
After anhydrification over MgSO4, the organic phase was filtered
and evaporated. Purification by two subsequent flash column
chromatographies on silica gel (petroleum ether/AcOEt 7:3 and
CHCl3/acetone 8:2). 12 (22.0 mg, 0.054 mmol). Yield: 8%. M. p.:
8%. M. P.: dec. 244 ꢀC. 1H NMR (DMSO‑d6)
d (ppm): 7.56 (t, 1H,
J ¼ 8.0 Hz), 7.53 (AA’XX’, 2H, JAX ¼ 8.8 Hz, JAA’XX’ ¼ 2.0 Hz), 7.39 (t,
1H, J ¼ 8.0 Hz), 7.33 (AA’XX’, 2H, JAX ¼ 8.8 Hz, JAA’XX’ ¼ 2.0 Hz), 7.27
(d, 1H, J ¼ 7.6 Hz), 6.84 (d, 1H, J ¼ 8.0 Hz), 6.71 (d, 1H, J ¼ 7.6 Hz),
133 ꢀC. 1H NMR: (CDCl3)
d
(ppm): 7.62 (bs, 1H), 7.51e7.50 (m, 2H),
6.30 (d, 1H, J ¼ 8.4 Hz), 3.62e3.58 (m, 5H), 1.78e1.74 (m, 5H). 13
C
7.34e7.33 (m, 2H), 7.28e7.22 (m, 3H), 7.15e7.12 (m, 2H), 6.84e6.82
(m, 1H), 6.71 (m, 1H), 6.57e6.54 (m, 1H), 3.33e3.30 (m, 4H),
3.02e2.99 (m, 2H), 2.64e2.60 (m, 2H), 2.01e1.98 (m, 4H). 13C NMR:
NMR (DMSO‑d6) d (ppm): 157.95, 152.40, 152.37, 152.15, 150.92,
139.55, 139.00, 138.06, 128.42, 126.00, 120.99, 104.53, 102.51, 99.59,
98.63, 45.65, 25.10, 24.31. MS: the experiment in full scan mode
showed: m/z 421 ([M-H]-, 35), 385 ([M-H]- ‒ Cl, 100); MSeMS of the
ion at m/z 421 showed: m/z 294 ([M-H]- ‒ p-Cl-C6H4-NH, 20), 268
([M-H]- ‒ p-Cl-C6H4-NHCO, 100), 126 [p-Cl-C6H4-NH]-, 10). HPLC, tR
4.97.
(CDCl3)
d (ppm): 170.23, 148.31, 143.27, 141.76, 139.29, 138.08,
132.12, 129.87, 129.55, 129.26, 128.74, 123.45, 118.86, 118.79; 114.64,
111.02, 110.49, 47.78, 30.87, 29.79, 25.55. MS: the experiment in full
scan mode showed: m/z 405 ([MþH]þ, 100); MSeMS of the ion at
m/z 405 showed: m/z 279 ([MþH]þ ‒ p-Cl-C6H4-CH2, 25), 197
([MþH]þ ‒ p-Cl-C6H4-CH2CH2 ‒ pyrrolidine, 67). HPLC, tR 15.23.
4.1.22. N1-(pyridin-2-yl)benzene-1,3-diamine (34)
Compound 34 was obtained from 1,3-diaminobenzene (1.00 g,
9.25 mmol, 1 equiv) and 2-bromopyridine (0.5 equiv) according to
general procedure C. Purification by flash column chromatography
on silica gel (n-hexane/AcOEt 1:9). 34 (50.0 mg of a mixture con-
taining approximately 85% of the desired product. Percentage ratio
calculated comparing corresponding integration of 1H NMR peaks).
4.1.17. N2-(6-bromopyridin-2-yl)pyridine-2,6-diamine (30)
Compound 30 was obtained from 2,6-diaminopyridine (2.60 g,
24.0 mmol, 1 equiv) and 2,6-dibromopyridine (1.25 equiv) accord-
ing to general procedure C. The crude product was purified by flash
column chromatography on silica gel (petroleum ether/AcOEt 6:4).
30 (147.0 mg, 0.560 mmol). Yield: 30%. 1H NMR: (CDCl3)
d
(ppm)
Yield: 2%. 1H NMR: (CD3OD)
d (ppm): 8.05e8.03 (m,1H), 7.54e7.50
8.40 (bs, 1H), 7.52 (dd, 1H, J ¼ 8.0 Hz, J ¼ 0.4 Hz), 7.37 (m, 2H), 6.94
(dd, 1H, J ¼ 7.6 Hz, J ¼ 0.4 Hz), 6.59 (dd, 1H, J ¼ 7.6 Hz, J ¼ 0.4 Hz),
6.09 (dd, 1H, J ¼ 8.0 Hz, J ¼ 0.4 Hz), 4.87 (bs, 2H).
(m, 1H), 7.02 (t,1H, J ¼ 8.0 Hz), 6.90 (t,1H, J ¼ 2.0 Hz), 6.86e6.84 (m,
1H), 6.73e6.69 (m, 2H), 6.41e6.38 (m, 1H).
4.1.23. N1-(4-fluorophenyl)benzene-1,3-diamine (35)
4.1.18. N2-[6-(pyrrolidin-1-yl)pyridin-2-yl]pyridine-2,6-diamine
(31)
Compound 31 was prepared from 30 (0.768 g, 2.90 mmol) and
pyrrolidine according to general procedure G. Purification by flash
liquid column chromatography on silica gel, eluting with petroleum
ether/AcOEt 4:6. 31 (0.550 g, 2.14 mmol). Yield: 74%. 1H NMR:
Compound 35 was obtained from 1,3-diaminobenzene (2.00 g,
18.5 mmol) and 4-bromofluorobenzene (3.24 g, 18.5 mmol) ac-
cording to general procedure C. Purification by flash column
chromatography on silica gel (n-hexane/AcOEt 9:1 and AcOEt). 35
(52.0 mg, 0.257 mmol). Yield: 1%. 1H NMR: (CDCl3)
d (ppm):
7.06e7.02 (m, 3H), 7.00e6.95 (m, 2H), 6.37 (dd, 1H, J ¼ 8.0 Hz,
J ¼ 1.2 Hz), 6.31 (t, 1H, J ¼ 2.0 Hz), 6.25e6.23 (m, 1H), 5.50e5.47 (bs,
1H), 3.69e3.59 (bs, 2H).
(CDCl3)
d (ppm): 7.37e7.25 (m, 3H), 7.07 (bs, 1H), 6.36 (d, 1H,
J ¼ 7.6 Hz), 6.01 (dd, 1H, J ¼ 8.0 Hz, J ¼ 0.8 Hz), 5.87 (d, 1H,
J ¼ 8.0 Hz), 4.30 (bs, 2H), 3.47e3.44 (m, 4H), 1.99e1.96 (m, 4H).
4.1.24. N1-phenylbenzene-1,3-diamine (36)
4.1.19. N2-[6-(piperidin-1-yl)pyridin-2-yl]pyridine-2,6-diamine
(32)
Compound 32 was prepared from 30 (0.837 g, 3.17 mmol) and
piperidine according to general procedure G. Purification by flash
liquid column chromatography on silica gel, eluting with petroleum
ether/AcOEt 6:4. 32 (0.231 g, 0.900 mmol). Yield: 30%. 1H NMR
Compound 36 was obtained from 1,3-diaminobenzene (1.00 g,
9.25 mmol) and iodobenzene (1.89 g, 9.25 mmol) according to
general procedure C. Purification by flash column chromatography
on silica gel (petroleum ether/AcOEt 7:3). 36 (33.0 mg, 0.179 mmol).
Yield: 2%. 1H NMR: (CDCl3)
d (ppm): 7.29e7.24 (m, 2H), 7.09e7.03
(m, 3H), 6.93 (t, 1H, J ¼ 7.2 Hz), 6.48e6.46 (m, 1H), 6.43e6.42 (m,
(CDCl3)
d
(ppm): 7.38e7.33 (m, 2H), 6.99 (dd,1H, J ¼ 8.0 Hz,
1H), 6.29e6.26 (m, 1H), 5.64 (bs, 1H), 3.55 (bs, 2H).
J ¼ 0.8 Hz), 6.97 (bs, 1H), 6.57 (d,1H, J ¼ 7.6 Hz), 6.17 (d, 1H,
J ¼ 8.0 Hz), 6.03 (dd, 1H, J ¼ 7.6 Hz, J ¼ 0.4 Hz), 4.28 (bs, 2H),
3.51e3.49 (m, 4H), 1.64e1.63 (m, 6H).
4.1.25. N2-phenyl-2,6-diaminopyridine (37)
Commercial t-BuOK (1.37 g, 12.2 mmol), iodobenzene (1.0 mL,
10.7 mmol) and CuI (227.0 mg, 1.19 mmol) were added to a solution
of 2,6-diaminopyridine (1.00 g, 9.16 mmol) crystalized from
toluene, in anhydrous 1,4-dioxane (18.0 mL), under nitrogen at-
mosphere. The reaction mixture was stirred at 100 ꢀC for 24 h.
Then, it was diluted with AcOEt and brine (30 mL) to allow the
partition of the crude product. The organic phase was anhydrified
over Na2SO4, filtered and evaporated under reduced pressure. The
purification was performed by flash column chromatography on
silica gel (Et2O). 37 (84.0 mg, 0.453 mmol). Yield: 5%. 1H NMR:
4.1.20. N-(4-chlorophenyl)-N’-(6-{[6-(pyrrolidin-1-yl)pyridin-2-yl]
amino}pyridin-2-yl)urea (13)
Compound 13 was prepared from 31 (0.114 g, 0.440 mmol) ac-
cording to general procedure B. 13 (0.0392 g, 0.0955 mmol). Yield:
22%. M. P.: dec. 242 ꢀC. 1H NMR: (DMSO‑d6)
d (ppm): 10.7 (bs, 1H),
9.31 (bs, 1H), 9.23 (bs, 1H), 7.62e7.55 (m, 4H), 7.38e7.31 (m, 3H),
6.77 (d, 1H, J ¼ 7.6 Hz), 6.52 (d, 1H, J ¼ 7.6 Hz), 5.93 (d, 1H,
J ¼ 8.0 Hz), 3.37e3.35 (m, 4H), 1.93e1.90 (m, 4H). 13C NMR
(DMSO‑d6)
d
(ppm): 152.31, 152.23, 150.90, 139.67, 138.50, 138.45,
(CDCl3) d (ppm): 7.33e7.28 (m, 4H), 7.27e7.26 (m, 1H), 7.03e7.00
137.96, 128.62, 128.39, 126.11, 121.27, 119.85, 104.64, 98.27, 97.92,
46.37, 24.94. MS: the experiment in full scan mode showed: m/z
409 ([MþH]þ, 100); MSeMS of the ion at m/z 354 showed: m/z 282
([MþH]þ ‒ p-Cl-C6H4-NH, 100), 256 ([MþH]þ ‒ p-Cl-C6H4-NHCO,
79). HPLC, tR 4.76.
(m, 1H), 6.32 (bs, 1H), 6.26 (d, 1H, J ¼ 8.0 Hz), 5.98 (dd, 1H, J ¼ 8.0,
J ¼ 0.4 Hz), 4.28 (bs, 2H).
4.1.26. N-(3-nitrophenyl)pyridin-4-amine (39)
200 mg (2.12 mmol) of 4-aminopyridine were dissolved in
7.1 mL of freshly distilled 1,4-dioxane. Then, 358.0 mg (1.77 mmol)
of 1-bromo-3-nitrobenzene, 1.44 g (4.43 mmol) of Cs2CO3, 162.0 mg
(0.177 mmol) of xantphos and 81.0 mg (0.089 mmol) of Pd2(dba)3
were added to the solution and the reaction mixture was heated
under magnetic stirring at 95 ꢀC for 24 h. After cooling to room
4.1.21. N-(4-chlorophenyl)-N’-(6-{[6-(piperidin-1-yl)pyridin-2-yl]
amino}pyridin-2-yl)urea (14)
Compound 14 was prepared from 32 (0.0912 g, 0.355 mmol)
according to general procedure B. 14 (0.0116 g, 0.0274 mmol). Yield:

S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
9
temperature, the mixture was partitioned between distilled water
and AcOEt. The organic phase was dried over Na2SO4, filtered and
evaporated under reduced pressure, to obtain an orange-brown
solid. Purification by flash liquid column chromatography on sil-
ica gel, eluting with AcOEt/MeOH 9:1. 39 (311.0 mg, 1.45 mmol).
NH, 100), 184 ([M-H]- ‒ p-Cl-C6H4-NHCO, 68), 126 [p-Cl-C6H4-NH]-,
24). HPLC, tR 2.13.
4.1.31. N-(4-chlorophenyl) eN’-{3-[(4-fluorophenyl) amino]
phenyl} urea (18)
Yield: 82%. 1H NMR: (CDCl3)
d
(ppm): 8.41e8.39 (m, 2H), 8.04e8.03
Compound 18 was prepared from 35 (49.0 mg, 0.242 mmol)
according to general procedure A. 18 (55.0 mg, 0.154 mmol). Yield:
(m, 1H), 7.94e7.92 (m, 1H), 7.54e7.48 (m, 2H), 6.91e6.89 (m, 2H),
6.28 (bs, 1H).
64%. M. p.: 160 ꢀC. 1H NMR: (DMSO‑d6)
d (ppm): 8.74 (s, 1H), 8.62 (s,
1H), 8.13 (s, 1H), 7.46 (AA’XX’, 2H, JAX ¼ 9.0 Hz, JAA’XX’ ¼ 2.8 Hz), 7.31
(AA’XX’, 2H, JAX ¼ 9.0 Hz, JAA’XX’ ¼ 2.8 Hz), 7.25e7.24 (m, 1H),
7.11e7.08 (m, 5H), 6.83e6.81 (m, 1H), 6.62e6.60 (m, 1H). 13C NMR:
4.1.27. N1-(pyridin-4-yl)benzene-1,3-diamine (40)
Compound 39 (150.0 mg, 0.697 mmol) was dissolved in a
mixture of EtOH/H2O (8.6 mL: 4.3 mL). After the addition of
430.0 mg (0.697 mmol) of iron powder and 215.0 mg (4.02 mmol)
of NH4Cl, the reaction mixture was stirred at reflux for 4 h. After
cooling to room temperature, the reaction mixture was filtered over
a celite pad. The filtrate was evaporated in vacuo and the residue
was dissolved in CHCl3 and washed with 10% aqueous NaOH. The
organic phase was dried over Na2SO4, filtered and evaporated. 40
(DMSO‑d6)
d
(ppm): 156.40 (d, J ¼ 235.0 Hz), 152.35, 144.45, 140.41,
139.65 (d, J ¼ 2.0 Hz), 138.77, 129.47, 128.64, 125.25, 119.64, 119.16
(d, J ¼ 7.0 Hz), 115.67 (d, J ¼ 22.0 Hz), 109.93, 109.55, 105.62. MS: the
experiment in full scan mode showed: m/z 354 ([M-H]-, 21);
MSeMS of the ion at m/z 354 showed: m/z 227 ([M-H]- ‒ p-Cl-C6H4-
NH, 100), 201 ([M-H]- ‒ p-Cl-C6H4-NHCO, 96), 126 [p-Cl-C6H4-NH]-,
15). HPLC, tR 10.15.
(86.0 mg, 0.464 mmol). Yield: 67%. 1H NMR: (CDCl3)
d (ppm):
8.27e8.25 (m, 2H), 7.12 (t, 1H, J ¼ 7.9 Hz), 6.81e6.80 (m, 2H),
6.58e6.55 (m, 1H), 6.51 (t, 1H, J ¼ 2.1 Hz), 6.45 (ddd, 1H, J ¼ 8.0 Hz,
J ¼ 2.2 Hz, J ¼ 0.8 Hz), 5.98 (bs, 1H), 3.71 (bs, 2H).
4.1.32. N-(3-anilinophenyl)-N’-(4-chlorophenyl)urea (19)
Compound 19 was prepared starting from 36 (27.0 mg,
0.147 mmol) according to general procedure A. 19 (8.0 mg,
0.025 mmol). Yield: 17%. M. p.: 170 ꢀC. 1H NMR: (DMSO‑d6)
d (ppm):
4.1.28. N-(4-chlorophenyl) eN’-{3-[(pyridin-2-yl) amino]phenyl}
urea (15)
8.74 (s, 1H), 8.62 (s, 1H), 8.16 (s, 1H), 7.46 (AA’XX’, 2H, JAX ¼ 8.8 Hz,
JAA’XX’ ¼ 2.8 Hz), 7.31 (AA’XX’, 2H, JAX ¼ 9.2 Hz, JAA’XX’ ¼ 2.6 Hz),
7.34e7.30 (m, 1H), 7.25e7.21 (m, 2H), 7.13e7.07 (m, 3H), 6.86e6.80
Compound 15 was prepared from 34 (43.0 mg, 0.232 mmol)
according to general procedure A. 15 (55 mg, 0.169 mmol). Yield:
(m, 2H), 6.69e6.66 (m, 1H). 13C NMR: (DMSO‑d6)
d (ppm): 152.32,
73%. M. p.: 190 ꢀC. 1H NMR: (DMSO‑d6)
d
(ppm): 9.02 (s, 1H), 8.77 (s,
143.91, 143.29, 140.30, 138.73, 129.36, 129.09, 128.82, 128.59, 125.21,
119.81, 116.99, 110.60, 109.77, 106.27. MS: the experiment in full
scan mode showed: m/z 336 ([M-H]-, 23); MSeMS of the ion at m/z
336 showed: m/z 209 ([M-H]- ‒ p-Cl-C6H4-NH, 100), 183 ([M-H]- ‒
p-Cl-C6H4-NHCO, 25), 126 [p-Cl-C6H4-NH]-, 5). HPLC, tR 11.99.
1H), 8.65 (s, 1H), 8.15e8.14 (m, 1H), 7.80 (s, 1H), 7.58e7.54 (m, 1H),
7.49e7.47 (m, 2H), 7.34e7.29 (m, 3H), 7.15 (t, 1H, J ¼ 8.0 Hz),
7.06e7.04 (m, 1H), 6.83 (d, 1H, J ¼ 8.4 Hz), 6.75e6.72 (m, 1H). 13
C
NMR: (DMSO‑d6)
d (ppm): 155.92, 152.36, 147.19, 142.14, 139.73,
138.81, 137.19, 128.65, 125.21, 119.58, 119.49, 114.25, 112.06, 110.60,
110.48, 107.83. MS: the experiment in full scan mode showed: m/z
337 ([M-H]-, 21); MSeMS of the ion at m/z 337 showed: m/z 210
([M-H]- ‒ p-Cl-C6H4-NH, 100), 126 ([p-Cl-C6H4-NH]-, 14). HPLC, tR
4.60.
4.2. Biological assays
4.2.1. Cell membrane preparations
CHO cells stably transfected with cDNA encoding human
cannabinoid CB1Rs or CB2Rs were maintained at 37 ꢀC and 5% CO2
in Gibco Ham’s F-12 nutrient mix supplied by Fisher Scientific UK
4.1.29. N-(6-anilinopyridin-2-yl)-N’-(4-chlorophenyl)urea (16)
Compound 16 was prepared from 37 (80.0 mg, 0.432 mmol)
according to general procedure A. Purification by flash column
chromatography on silica gel (CHCl3/CH3OH 95:5). 16 (17.0 mg,
Ltd. that was supplemented with 2 mM L-glutamine, 10% FBS, and
0.6% penicillinꢂstreptomycin, all also supplied by Fisher Scientific
UK Ltd., and with the disulfate salt of G418 [(2R,3S,4R,5R,6S)-5-
amino-6-{[(1R,2S,3S,4R,6S)-4,6-diamino-3-{[(2R,3R,4R,5R)-3,5-
0.050 mmol). Yield: 11%. M. p.: 187 ꢀC. 1H NMR: (CD3OD)
d (ppm):
7.91 (s, 1H), 7.50 (t, 1H, J ¼ 8.0 Hz), 7.37e7.36 (m, 2H), 7.27e7.23 (m,
dihydroxy-5-methyl-4
(methylamino)oxan-2-yl]oxy}-2-
2H), 7.12e7.10 (m, 2H), 7.03e7.01 (m, 1H), 6.96e6.94 (m, 2H),
hydroxycyclohexyl]oxy}-2-[(1R)-1-hydroxyethyl]oxane-3,4-diol;
600 mg mLꢂ1] supplied by Sigma-Aldrich UK. All cells were
exposed to 5% CO2 in their respective media and were passaged
twice a week using nonenzymatic cell dissociation solution. To
prepare the membranes, cells were scraped from flask, centrifuged
at 1800 rpm for 5 min and the pellet obtained was frozen at ꢂ20 ꢀC.
To perform a radioligand binding assays, cells were defrosted,
6.43e6.37 (m, 2H). 13C NMR: (CD3OD)
d (ppm): 155.78, 152.59,
142.19, 141.11, 138.51, 130.45, 129.38, 129.00, 123.83, 122.62, 122.19,
106.48, 104.27, 102.13. MS: the experiment in full scan mode
showed: m/z 337 ([M-H]-, 21); MSeMS of the ion at m/z 337
showed: m/z 210 ([M-H]- ‒ p-Cl-C6H4-NH, 11), 184 ([M-H]- ‒ p-Cl-
C6H4-NHCO, 100), 126 [p-Cl-C6H4-NH]-, 24). HPLC, tR 9.97.
diluted with 500 mL of Tris buffer (composed by 50.0 mM of Tris-
4.1.30. N-(4-chlorophenyl)-N’-{3-[(pyridin-4-yl)amino]phenyl}
urea (17)
HCl and 50 mM of Tris-base).
Compound 17 was prepared from 40 (43.0 mg, 0.232 mmol)
according to general procedure A. 17 (67.0 mg, 0.198 mmol). Yield:
4.2.2. Radioligand displacement assay
The total assay volume was 500 m
L and for the procedure [3H]
85%. M. p.: 220 ꢀC. 1H NMR: (DMSO‑d6)
d
(ppm): 8.80 (s, 1H), 8.79 (s,
CP55940 (0.700 nM) and Tris binding buffer (0.1% BSA in Tris buffer,
with a pH of 7.4) were employed. Different concentrations of the
1H), 8.74 (s, 1H), 8.19e8.18 (m, 2H), 7.48 (AA’XX’, 2H, JAX ¼ 9.0 Hz,
JAA’XX’ ¼ 2.6 Hz), 7.48e7.46 (m, 1H), 7.33e7.31 (AA’XX’, 2H,
JAX ¼ 9.0 Hz, JAA’XX’ ¼ 2.6 Hz), 7.23 (t, 1H, J ¼ 8.0 Hz), 7.02e7.00 (m,
1H), 6.93e6.91 (m, 2H), 6.81e6.79 (m, 1H). 13C NMR: (CD3OD)
compounds (from 1 nM to 10
solutions in DMSO) were incubated at 37 ꢀC with hCB1- or hCB2-
CHO cell membranes (50.0 g of protein per tube) and DMSO
mM, obtained from 10.00 mM stock
m
d
(ppm): 155.00, 153.80, 149.84, 142.07, 141.75, 139.53, 131.00,
vehicle (0.1% per tube) for 60 min. The assay was terminated by
adding ice-cold Tris binding buffer and filtrating under vacuum
(24-well sampling manifold, Brandel Cell Harvester; Brandel Inc,
Gaithersburg, MD, USA). The filters used (Brandel GF/B filters) had
129.98, 128.91, 121.91, 116.95, 115.80, 113.39, 110.74. MS: the
experiment in full scan mode showed: m/z 337 ([M-H]-, 23);
MSeMS of the ion at m/z 337 showed: m/z 210 ([M-H]- ‒ p-Cl-C6H4-

10
S. Meini et al. / European Journal of Medicinal Chemistry 203 (2020) 112606
been soaked in Tris binding buffer at 4 ꢀC for at least 24 h. The
filtration was carried out by washing each reaction well with
1.200 mL of Tris binding buffer for six times. After oven-drying for
60 min, the filters were placed in 3.00 mL of scintillation fluid
(Ultima Gold XR, PerkinElmer, Seer Green, Buckinghamshire, UK)
and subjected to liquid scintillation spectrometry in order to
quantify the radioactivity. Specific binding was defined as the dif-
ference between the binding that occurred in the presence and
significant. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgments
absence of 1
m
M unlabeled CP55940.
This research was supported by grant from University of Pisa,
Italy (Progetti di Ricerca di Ateneo, PRA_2018_20).
4.2.3. [35S]GTP
g
S assays
The total assay volume was 500
GTP S (0.1 nM), GDP (30 M), GTP
m
L and for the procedure [35S]
S (30 M and binding buffer
Appendix A. Supplementary data
g
m
g
m
(50 mM Tris, 10 mM MgCl2, 100 mM NaCl, 0.2 mM EDTA and 1 mM
DTT (dithiothreitol), with a pH of 7.4) were employed. Different
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112606.
concentrations of the compounds (from 1 nM to 10
from 10.00 mM stock solutions in DMSO) were incubated at 30 ꢀC
with hCB1-CHO cell membranes (50.0 g of protein per tube) and
mM, obtained
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