Novel analogs of PSNCBAM-1 as allosteric modulators of cannabinoid CB1 receptor

Article abstract of DOI:10.1016/j.bmc.2017.10.015

In this work, we explored the molecular framework of the known CB1R allosteric modulator PSNCBAM-1 with the aim to generate new bioactive analogs and to deepen the structure-activity relationships of this type of compounds. In particular, the introduction

Full text of DOI:10.1016/j.bmc.2017.10.015

Accepted Manuscript
Novel analogs of PSNCBAM-1 as allosteric modulators of cannabinoid CB1
receptor
Simone Bertini, Andrea Chicca, Francesca Gado, Chiara Arena, Daniela Nieri,
Maria Digiacomo, Giuseppe Saccomanni, Pingwei Zhao, Mary E. Abood,
Marco Macchia, Jürg Ghertsch, Clementina Manera
PII:
S0968-0896(17)31488-8
https://doi.org/10.1016/j.bmc.2017.10.015
BMC 14020
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 July 2017
13 October 2017
15 October 2017
Please cite this article as: Bertini, S., Chicca, A., Gado, F., Arena, C., Nieri, D., Digiacomo, M., Saccomanni, G.,
Zhao, P., Abood, M.E., Macchia, M., Ghertsch, J., Manera, C., Novel analogs of PSNCBAM-1 as allosteric
modulators of cannabinoid CB1 receptor, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/
j.bmc.2017.10.015
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Novel analogs of PSNCBAM-1 as allosteric
modulators of cannabinoid CB1 receptor
Leave this area blank for abstract info.
a
b
a
a
b
a
Simone Bertini , Andrea Chicca , Francesca Gado , Chiara Arena , Daniela Nieri , Maria Digiacomo , Giuseppe
a
c
c
a
b
a,
Saccomanni , Pingwei Zhao , Mary E. Abood , Marco Macchia , Jürg Ghertsch , Clementina Manera ∗
a
Department of Pharmacy, University of Pisa, 56126 Pisa
Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, Bühlstrasse 28, CH-
b
3
012 Bern, Switzerland
c
Center for Substance Abuse Research, Lewis Katz School of Medicine at Temple University, Philadelphia, PA
9140, USA
1
Cl
Cl
O
O
N
N
N
H
N
H
N
H
SC4a
CH
2
a+b
SC33
a
CH
a
b
Cl
Cl
O
Cl
O
O
N
N
c
a+c
N
H
N
H
N
H
N
H
N
H
N
N
H
N
H
N
H
N
N
c
PSNCBAM-1
FG45a
SN15b
NH

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Novel analogs of PSNCBAM-1 as allosteric modulators of cannabinoid CB1 receptor
a, 1
b, 1
a
a
b
a
Simone Bertini , Andrea Chicca , Francesca Gado , Chiara Arena , Daniela Nieri , Maria Digiacomo ,
a
c
c
a
b
Giuseppe Saccomanni , Pingwei Zhao , Mary E. Abood , Marco Macchia , Jürg Ghertsch , Clementina
a,
Manera ∗
a
Department of Pharmacy, University of Pisa, 56126 Pisa
b
Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland
c
Center for Substance Abuse Research, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In this work, we explored the molecular framework of the known CB1R allosteric modulator
PSNCBAM-1 with the aim to generate new bioactive analogs and to deepen the structure-
activity relationships of this type of compounds. In particular, the introduction of a NH group
between the pyridine ring and the phenyl nucleus generated the amino-phenyl-urea derivative
SN15b that behaved as a positive allosteric modulator (PAM), increasing the CB1R binding
affinity of the orthosteric ligand CP55,940. The functional activity was evaluated using serum
response element (SRE) assay, which assesses the CB1R-dependent activation of the
MAPK/ERK signaling pathway. SN15b and the biphenyl-urea analog SC4a significantly
inhibited the response produced by CP55,940 in the low µM range, thus behaving as negative
allosteric modulators (NAMs). The new derivatives presented here provide further insights about
the modulation of CB1R binding and functional activity by allosteric ligands.
Available online
Keywords:
Allosteric modulator
CB1 receptor
Endocannabinoid system
MAPK/ERK signaling pathway
Serum response element
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
The endocannabinoid system (ECS) is abundantly
ameliorating some pathological conditions such as pain, nausea,
9, 10
obesity, type 2 diabetes, glaucoma and substance abuse.
expressed throughout the body and it consists of cannabinoid
receptors, their endogenous ligands (endocannabinoids), and the
enzymes involved in the metabolism of endocannabinoids.
Several CB1R agonist and antagonists/inverse agonists
have been reported in literature with different potencies and
selectivities, showing effects in several preclinical disease
1
, 2
3
, 18-20
Since the discovery of the cannabinoid receptors (CB1R and
CB2R), several studies have been extensively devoted to their
synthetic ligands. In fact, the direct activation of these receptors
results in several beneficial effects, in the brain and in the
models.
However, the orthosteric action of CB1R agonists
21,
and antagonists/inverse agonists induces important side effects.
2
2
In particular, CB1R orthosteric agonists produce psychotropic
3
, 23
effects, addiction and cognitive impairment.
On the other
3-7
periphery. CB1R is the most expressed G-protein coupled
receptor (GPCR) in brain, where it transduces the
hand, CB1R orthosteric antagonists/inverse agonists are
associated with undesirable side effects, such as nausea, anxiety,
21, 24
endocannabinoid signaling and thereby acts as a modulator of
depression, and in some cases, suicidal tendencies.
8
neurotransmitter release. In the periphery, CB1R regulates
Recently, it has been suggested that CB1R contains (an)
metabolic processes including substrate storage and
allosteric binding site(s) topologically distinct from the
9
, 10
mobilization.
On the other hand, CB2R is mainly localised in
25-29
orthosteric site.
Compared to orthosteric ligands, allosteric
peripheral immune cells and tissues, though recent studies
modulators offer several advantages. In fact, they show greater
subtype selectivity due to the higher sequence divergence at
extracellular allosteric binding sites, respect to the conserved
orthosteric domains; furthermore, allosteric modulators show
tissue selectivity, because they exert effects only where
endocannabinoids are present; finally, the effect of allosteric
modulators is saturable because of their dependence on
showed its presence in CNS, and it has been investigated for
1
1-14
treating pain and inflammation.
CB1R is shown to be involved in regulation of several
physiological functions, such as food intake and energy balance,
cardiovascular and reproductive functions, immune modulation
15-17
and cell apoptosis.
Therefore, CB1R has been suggested as an
30, 31
important target of small-molecule pharmacotherapeutics, for
endogenous ligands.
For these reasons, allosteric modulators
—
——
∗
Corresponding author. Tel.: +39-0502219548; fax: +39-0502219608; e-mail: clementina.manera@unipi.it
1
These authors contributed equally to this work.

might offer an alternative strategy to pharmacologically modulate
CB1R without producing unwanted side effects associated with
CB1R orthosteric agonists and antagonists/inverse agonists.
favorable than secondary. Furthermore, the 4-position on the
phenyl group tolerates structural modifications, but electron-
withdrawing groups such as cyano or CF are preferred, and the
3
3
5
substitution of the urea group with other spacers such as
carbamate, methylated ureas or cyclic ureas, reduces the
activity.
Allosteric modulators can be classified as positive allosteric
modulators (PAMs) or negative allosteric modulators (NAMs),
depending on the type of modulation on the affinity and/or
36
32
efficacy of the orthosteric agonists. Various classes of
In an effort to deepen the knowledge on structural
requirements for CB1R allosteric modulation within the
PSNCBAM-1 template, we decided to introduce further
modifications on the structure of PSNCBAM-1 obtaining
compounds SC4a, SC33, SN15b and FG45a (Figure 3).
compounds have been reported as CB1R allosteric modulators,
25
such as the indole derivatives (e.g., the NAM Org27569), the
27
urea derivatives (e.g., the NAM PSNCBAM-1), and other
3
3
34
molecules (e.g the PAMs RTI-371 and ZCZ011 ) (Figure 1).
Cl
Cl
O
O
N
Cl
N
N
H
N
O
N
H
N
H
N
H
HN
O
N
N
N
H
N
H
Cl
SC4a
SC33
Org27569 (NAM)
Cl
PSNCBAM-1(NAM)
Cl
O
N
N
N
O
N
H
N
H
HN
NO
2
PSNCBAM-1
N
S
RT-371 (PAM)
ZCZ011 (PAM)
Cl
Cl
O
O
N
N
H
N
H
N
N
Figure 1. Chemical structures of some CB1R allosteric modulators.
N
H
N
N
H
N
H
H
FG45a
SN15b
Furthermore, recent works reported natural PAMs and NAMs
of CB1R, such as the lipoxin A4, the peptide pepcan-12 (RVD-
26, 28, 29
hemopessin), and pregnenolone (Figure 2).
Figure 3. Compounds SC4a, SC33, SN15b and FG45a structurally derived
from urea derivative PSNCBAM-1.
O
HO
OH
O
In particular, we investigated the role of the pyridine nitrogen
H
atom by replacing it with a carbon atom (SC4a, FG45a). The
OH
3
6
derivative SC4a was previously reported and its
HO
OH
pharmacological activity was determined by cAMP Hunter assay
and CNR1 PathHunter assay (β-arrestin), but competitive
radioligand experiments were not conducted. SC4a was prepared
in our laboratory following a different synthetic route respect to
that reported in Ref. 36, with an overall comparable yield.
Moreover, we verified the importance of the urea group by
replacing one NH group with a methylene group, thus obtaining
the carboxamide derivative SC33. Finally, we evaluated the
influence due to the introduction of a NH group between the
pyridine ring and phenyl nucleus (SN15b, FG45a). For all these
compounds, the 4-chlorophenyl group and the 2-pyrrolydyl
substituent have been selected based on previous results obtained
Pregnenolone
LipoxinA4
NH
2
O
O
HN
O
O
NH
2
O
O
H
O
N
H
N
H
H
N
HO
N
N
OH
N
H
N
H
N
H
N
H
O
O
O
O
O
HN
OH
O
NH
Pepcan-12
O
NH2
3
5
for PSNCBAM-1 analogs.
NH
NH
HN
In this work we reported the synthesis of compounds SC4a,
SC33, SN15b and FG45a and their biological evaluation on the
cannabinoid receptors, on the major metabolic enzymes of
endocannabinoids, fatty acid amide hydrolase (FAAH), and
monoacylglycerol lipase (MAGL) and on anandamide (AEA)
uptake. Furthermore, the activity of the target compounds at
CB1R was assessed in serum response element (SRE) assay.
2
Figure 2. Chemical structures of endogenous CB1R allosteric modulators.
PSNCBAM-1 was discovered by Prosidion Limited in 2007
through an high throughput screening of a small library. This
compound showed a pharmacological profile similar to
2
7
Org27569, increasing the binding of CB1R agonists but
decreasing their functional responses in several in vitro assays. In
an in vivo study of acute feeding model, PSNCBAM-1 showed to
2
. Methods
2
7
decrease food intake and body weight. Several analogs of
35-37
PSNCBAM-1
have been reported and structure-activity
2
.1. Chemical synthesis
relationship studies showed that alkyl substitution at the 2-
aminopyridine moiety is important for CB1R allosteric
modulation; in particular, tertiary amine substitution is more
The synthesis of compound SC4a and SC33 is described in
Scheme 1. Commercial 1,3-dibromobenzene 1 was subjected to a

monoamination with pyrrolidine in the presence of a catalytic
system constituted by tris(dibenzylideneacetone)dipalladium(0)
The synthesis of compound SN15b is described in Scheme 3.
Commercially available 1,3-diaminobenzene 6 and 2,6-
dibromopyridine were refluxed for 48 h in anhydrous toluene, in
the presence of an excess of t-BuOK, obtaining the intermediate
7, which was refluxed in an excess of pyrrolidine for 12 h,
affording compound 8. The subsequent reaction of 8 with 4-
(
Pd
2
3
dba ), 2,2’-bis(diphenylphosphino)-1,1’-binaphtalene
(
BINAP) and t-BuONa (mol ratio: 0.05:0.15:2) in toluene,
affording the intermediate 2. The subsequent Suzuki cross-
coupling reaction with 3-nitrophenylboronic acid, in the presence
of Pd(OAc)
2
, P(Ph)
3
and acqueous Na
2
CO
3
in 1,2-
chloro-phenylisocyanate in anhydrous CHCl
3
afforded the
dimethoxyethane (DME), afforded compound 3. Then the nitro
group was reduced with Raney nickel and hydrazine hydrate in
EtOH and the amino derivative 4 thus obtained was treated with
desired urea derivative SN15b.
H
H
H N
NH2
H
2
N
N
N
Br
H
2
N
N
N
N
2
4
-chloro-phenylisocyanate in anhydrous CHCl
desired urea derivative SC4a. The treatment of 4 with (4-
chlorophenyl)acetyl chloride in the presence of Et N and DMAP
3
, affording the
a
b
[16%]
[45%]
6
7
8
3
in anhydrous CH
2
Cl
2
afforded the final amide derivative SC33.
H
N
H
N
H
N
N
N
c
N
O
Br
Br
Br
N
O N
2
[28%] Cl
a
b
c
SN15b
[
99%]
[59%]
[>99%]
1
2
3
Scheme 3. Reagents and conditions: a) 2,6-dibromopyridine,
t-BuOK, anhydrous toluene, reflux, 48 h; b) pyrrolidine,
reflux, 12 h; c) 4-chloro-phenylisocyanate, anhydrous CHCl ,
3
Cl
O
N
N
d
H
2
N
N
H
N
H
[
39%]
RT, 12 h.
4
SC4a
e
[22%]
2
.2. Biological assays
Cl
O
Cannabinoid receptors binding was evaluated by incubating
the target compounds at different concentrations with membrane
N
N
H
preparations obtained from CHO-K1 cells overexpressing
hCB1R or hCB1R in presence of 0.5 nM of [ H]CP55,940.
3
38
SC33
The inhibitory activity of all four compounds and of
Scheme 1. Reagents and conditions: a) pyrrolidine, Pd
BINAP: t-BuONa (mol ratio: 0.05:0.15:2), toluene, 80 °C, 4.5
h; b) 3-nitrophenylboronic acid, Pd(OAc) , P(Ph) , aq.
NaHCO , DME, 95 °C, ovenight; c) Raney nickel,
NH NH ·H O, EtOH, 50 °C, 40 min - 1 h; d) 4-chloro-
phenylisocyanate, anhydrous CHCl , RT, 12 h; e) (4-
chlorophenyl)acetyl chloride, Et N, DMAP, anhydrous
CHCl , RT, 24 h.
2 3
(dba) :
compound PSNCBAM-1 on FAAH, MAGL and on AEA cell
uptake was evaluated using U937 cell homogenate for metabolic
enzymes and intact U937 cells for AEA uptake, in the presence
2
3
3
3
39, 40
of AEA and of [ethanolamine-1- H]AEA (0.5 nM) as tracer.
2
2
2
Finally the functional activity of the new compounds was
3
determined by SRE assay using HEK293 cells transfected with
3
41
CB1R.
3
The synthesis of compound FG45a is described in Scheme 2.
The intermediate 5 was obtained by treating 2 with commercial
3. Results and Discussion
3
5-37
Since the discovery of PSNCBAM-1, some SAR studies
1
,3-diaminobenzene 6, using the same reaction conditions
showed that CB1R allosteric modulators with improved affinity,
efficacy and potency, could be generated from PSNCBAM-1
template. With the aim to extend the knowledge about the
structural requirements of PSNCBAM-1 template as CB1R
allosteric modulator, in this work we studied some derivatives
designed by introducing various modifications on PSNCBAM-1
structure.
described for the first step of Scheme 1. The subsequent reaction
of 5 with 4-chloro-phenylisocyanate in anhydrous CHCl
afforded the desired urea derivative FG45a.
3
H
Br
N
H N
2
N
N
a
[43%]
2
5
Firstly, with the purpose of studying the role of the pyridine
nitrogen atom, we synthesized compound SC4a bearing a phenyl
ring in replacement of the pyridine nucleus. Pharmacological
characterization of SC4a showed its behavior as positive
H
N
H
N
H
N
N
b
3
O
allosteric modulator for the orthosteric ligand [ H]CP55,940 at
[
42%] Cl
FG45a
CB1R, inducing a dose-dependently binding increase by
approximately 15-20%. The estimated EC50 is 43 (5-520) nM
(Fig. 4).
Scheme 2. Reagents and conditions: a) 1,3-diaminobenzene,
Pd (dba) : BINAP: t-BuONa (mol ratio: 0.05:0.15:2), toluene,
0 °C, 4.5 h; b) 4-chloro-phenylisocyanate, anhydrous CHCl
RT, 12 h.
2
3
In the second step, with the aim to verify the importance of
the urea group, we synthesized compound SC33 characterized by
a carboxamide group. The results of pharmacological studies
8
3
,
3
showed that SC33 does not influence [ H]CP55,940 binding to
CB1R up to 10 µM, while only a minor binding (approx. 10%)
seems to occur at 100 µM (Fig. 4).

Furthermore, we evaluated the influence of NH group
placed between the pyridine ring and the phenyl nucleus,
synthesizing compound SN15b. This derivative behaves as a
transfected with both pGL4.33 [luc2P/SRE/Hygro] and hCB1R.
The next day cells were treated with ligands for 5 h, following
which transcriptional activation of the SRE was detected using
luminescence from the luciferase reporter (Ref 41 and
experimental procedures). The SRE assay assesses the
contribution of MAPK/ERK signalling pathway and thus
encompasses both G-protein dependent and G-protein
positive allosteric modulator at CB1R for the orthosteric ligand
3
[
H]CP55,940. The estimated EC50 is 1.3 (0.41-5.8) µM and the
binding is increased by 35% (Fig. 4).
Finally, we synthesized compound FG45a as an analog of
SC33 and characterized by the same spacer (NH) of SN15b
between the two phenyl rings. FG45a behaves as a positive
41
independent signalling. Recent findings showed the roles of
MAPK/ERK signaling pathways in human neurodegenerative
diseases including Alzheimer's disease, Parkinson's disease and
3
allosteric modulator by enhancing [ H]CP55,940 binding to
42
amyotrophic lateral sclerosis. Moreover the ERK signaling
CB1R with an EC50 value of 13.5 (5.1-35.3) µM and reaching
pathway plays a central role in several steps of cancer
1
82% of maximal binding (Fig. 4).
43
development. As shown in Figure 5, all the compounds
We also tested the progenitor compound PSNCBAM-1 which
inhibited the response produced by CP55,940 at 33 nM
(concentration corresponding to EC80). SC4a and SN15b were
the most potent, with IC50 values of 291 (101-848) nM and 182
(92-357) nM, respectively, which were comparable to that of
PSNCBAM-1 (IC50 value of 234 (161-337) nM). SC33 and
FG45 were less potent, with IC50 values of 1.9 (0.52-6.9) µM and
9.6 (0.36-25) µM, respectively.
3
increased [ H]CP55,940 binding at CB1R by 15-20% with an
EC50 value of 270 (140-520) nM, thus showing a similar behavior
as its analogs SC4a, SN15b and FG45a (Table 1 and Fig. 4)
1
1
1
20
10
00
140
1
20
00
80
1
9
0
0
8
-
10 -8 -6 -4
-10 -8 -6 -4
Log [SC4a] (M)
Log [SN15b] (M)
1
20
00
200
160
120
80
1
8
0
0
6
-
10 -8 -6 -4
-10 -8
-6
-4
Log [SC33] (M)
Log [FG45a] (M)
Figure 5. Compounds SC4a, SN15b, SC33 and FG45a were found to
decrease the SRE response induced by CB1 agonist CP55,940 (33 nM).
PSNCBAM-1 was used as a reference compound. Data show mean values ±
SD of at least 3 independent experiments each performed in triplicates.
1
1
1
20
10
00
Compounds were also tested for binding to CB2R and for
interaction with the other major targets of the ECS. As shown in
Figure 6, compound SC4a and SC33 weakly bound to CB2R
9
8
0
0
i
with K values of approximately 50 µM, while SN15b and
FG45a did not significantly interact with the receptor up to 100
µM. The binding curves suggest that SC4a and SC33 behave as
weak orthosteric CB2R ligands and do not interact with a distinct
allosteric pocket. PSNCBAM-1 did not show any significant
binding to CB2R up to 10 µM.
-
9
-8 -7 -6 -5
Log [PSNCBAM-1] )
Log (PSNCBAM-1)( M[ M]
Figure 4. CB1 receptor binding. Compounds SC4a, SN15b, SC33, FG45a
and PSNCBAM-1 were incubated at different concentrations with membrane
preparations obtained from CHO-K1 cells overexpressing hCB1R in the
In further experiments, all four compounds did not exhibit
any significant inhibition of FAAH, MAGL and AEA cell uptake
activity up to 10 µM. The progenitor compound PSNCBAM-1
was negative for MAGL and AEA cell uptake, but inhibited
FAAH activity with an IC50 value of 2.1 (1.9-2.4) µM (Table1).
Altogether, these data show that, analogously to PSNCBAM-1,
SN15b and SC4a are potent PAMs at CB1R for the orthosteric
3
presence of 0.5 nM of [ H]CP55,940. Data show mean values ± SD of at least
3
independent experiments each performed in triplicates.
Functional activity was evaluated using a serum response
element (SRE) reporter assay. HEK293 cells were transiently

3
ligand [ H]CP55,940 with high selectivity over CB2R and the
line with the complex receptor pharmacology previously
27, 44, 45
other targets of the ECS (Table 1). Furthermore all new
compounds decreased the functional response in SRE assay
showing to be NAMs.
described for PSNCBAM-1.
The obtained results showed
that the pyridine nitrogen atom in PSNCBAM-1 is not an
important feature for CB1R modulating activity, indeed the
compound SC4a, concentration-dependently increased
3
[
H]CP,55940 binding to CB1R. Furthermore, a spacer (NH
group) between the pyridine ring and phenyl nucleus (SN15b and
FG45a) is allowed for the modulation of CB1R activity. These
compounds showed also high selectivity over CB2R and the
other targets of the ECS. Moreover, all compounds inhibited the
response produced by CP55,940 in SRE assay, indicating a
behaviour as NAMs. In particular, SC4a and SN15b were the
most potent, reducing the MAPK/ERK signalling pathway with
an activity similar to that of PSNCBAM-1. The new urea
derivatives presented here provide further insights regarding the
modulation of CB1R binding and offer new opportunities for
modulating specific signalling pathway associated with CB1R,
avoiding unwanted side effects usually caused by CB1R
orthosteric modulation.
1
25
125
100
75
50
25
0
100
7
5
5
0
2
5
0
-
10-9 -8 -7 -6 -5 -4
-10-9 -8 -7 -6 -5 -4
Log [SC4a] (M)
Log [SN15b] (M)
125
125
100
75
50
25
0
100
7
5
5
. Experimental
5
0
2
5
5
.1. Chemistry
0
Commercially available reagents were purchased from Sigma
-
10-9 -8 -7 -6 -5 -4
-10-9 -8 -7 -6 -5 -4
1
Aldrich or Alfa Aesar and used without purification. H NMR
Log [SC33] (M)
Log [FG45a] (M)
1
3
and C NMR spectra were recorded on a Bruker AVANCE III™
4
00 spectrometer (operating at 400 MHz). Chemical shift (δ) are
Figure 6. CB2 receptor binding. Compound SC4a, SN15b, SC33 and FG45a
reported in parts per million related to the residual solvent signal,
while coupling constants (J) are expressed in Hertz (Hz). All
synthesized 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 278 nm was
employed. Analyses were performed on a reverse phase C18
column (Phenomenex 250 x 4.6 mm, 5 mm particle size,
Gemini). The mobile phase was constituted by a mixture of
were incubated at different concentrations with membrane preparations
obtained from CHO-K1 cells overexpressing hCB2 receptors in presence of
3
0
.5 nM of [ H]CP55,940. Data show mean values ± SD of at least 3
independent experiments each performed in triplicates.
a
Table 1. Summary data for SC33, SN15b, SC4a and FG45a.
2
H O/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 SC4a, SC33b and FG45a. For compound
SN15b was chosen an isocratic elution with 100% of B. The flow
rate was 1.0 ml/min. Evaporation was carried out in vacuo using
a rotating 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.
K
i
value
EC50 (µM)
IC50 (µM)
IC50
µM)
(
µM)
(
CB1R
CB1R
CB2R
Compound
FAAH
MAGL
>100
AEA
Binding
SRE assay
Binding
uptake
0
.27
0.23
2.1
PSNCBAM-1
>10
>10
>10
(
0.14-0.52)
(0.16-0.34)
(1.9-2.4)
1
.9
SC33
>10
>100
>10
>10
>10
>10
>10
>10
>10
>10
(0.52-6.9)
1
.3
0.18
52.6
SN15b
SC4a
>10
(
0.41-5.8)
(0.09-0.36)
(21.5-88.4)
0
.043
0.29
49.8
>10
(
0.005-0.52)
(0.10-0.84)
(12.9-91.5)
1
3.5
9.6
5.1.1. 1-(3-Bromophenyl)pyrrolidine (2)
FG45a
>100
>10
(
5.1-35.3)
(0.36 – 25)
Commercial 1,3-dibromobenzene 1 (200.0 mg, 0.85 mmol),
pyrrolidine (60.5 mg, 0.85 mmol) and 157.0 mg of a reagent
constituted by tris(dibenzylideneacetone)dipalladium(0), BINAP
and t-BuONa (mol ratio: 0.05:0.15:2) were suspended in toluene
a
Data show mean and 95% CI; n = 3.
(
2.5 mL) and allowed to react in a sealed tube, at 80 °C for 4.5 h,
4
. Conclusion
under magnetic stirring. After cooling to room temperature, the
mixture was filtered under reduced pressure and the resulting
solution evaporated in vacuo. The residue was dissolved in
With the aim to deepen the knowledge on structural
requirements for CB1R allosteric modulation, we have designed
and synthesized some analogs of urea derivative PSNCBAM-1, a
known allosteric modulator of CB1R. The new compounds
showed to be PAMs on binding affinity of orthosteric ligand
CHCl
3
and washed with a 10% aqueous solution of NaOH. The
SO , filtered,
organic phase was dried over anhydrous Na
evaporated in vacuo, and purified by flash column
2
4
(
CP55,940) but NAMs in functional assays. This behavior is in
chromatography on silica gel (petroleum ether), obtaining the

intermediate 2 as a transparent oil (190.5 mg, 0.84 mmol). Yield:
solvent was removed in vacuo, the residue dissolved in AcOEt
and washed with water. The organic phase was dried over
anhydrous Na SO , filtered, evaporated in vacuo, and purified by
2 4
flash column chromatography on silica gel (petroleum
1
9
9%. H NMR (CDCl
Hz), 6.76-6.74 (m, 1H), 6.68 (m, 1H), 6.46 (m, 1H), 3.27-3.24
m, 4H), 2.05-1.99 (m, 4H).
3
, 400 MHz) δ (ppm): 7.05 (t, 1H, J = 8.0
(
ether/AcOEt 6:4), obtaining the intermediate 7 as a brown oil
1
(
147.0 mg, 0.56 mmol). Yield: 16%. H NMR (CDCl
3
, 400 MHz)
5
.1.2. 1-(3’-Nitrobiphenyl-3-yl)pyrrolidine (3)
δ (ppm): 7.29 (td, 1H, J = 7.9 Hz, J = 0.3 Hz), 7.10 (td, 1H, J =
7
.7 Hz, J = 0.8 Hz), 6.86 (dd, 1H, J = 7.5 Hz, J = 0.6 Hz), 6.81
In a sealed tube, under a flux of nitrogen, were introduced
(
(
dd, 1H, J = 8.2 Hz, J = 0.6 Hz), 6.63-6.58 (m, 2H), 6.44-6.41
m, 1H), 4.00 (bs, 2H).
2 3
DME (40.0 mL), Pd(OAc) (158.1 mg, 0.24 mmol) and P(Ph)
(
307.9 mg, 1.17 mmol). The mixture was left under magnetic
stirring at room temperature for 15 min, allowing the in situ
formation of the catalyst. Then, compound 2 (701.0 mg, 3.10
mmol), NaHCO
3
(775.0 mg, 9.23 mmol), H
2
O (17.6 mL) and 3-
5.1.6. N-[6-(pyrrolidin-1-yl)pyridin-2-yl]benzene-
,3-diamine (8)
1
nitrophenylboronic acid (571.5 mg, 3.42 mmol) were added. The
mixture was allowed to react at 95 °C, overnight. After cooling to
room temperature, the solvent was removed in vacuo and the
residue partitioned between water and ethyl acetate. The organic
A mixture of 7 (362.0 mg, 1.37 mmol) and pyrrolidine
(2.17 mL) was refluxed for 12 h. After cooling to room
temperature, pyrrolidine was removed in vacuo, obtaining a
phase was dried over anhydrous Na
vacuo, and purified by flash column chromatography on silica gel
petroleum ether/CH Cl 7:3), obtaining the intermediate 3 as an
orange solid (491.0 mg, 1.83 mmol). Yield: 59%. H NMR
CDCl , 400 MHz) δ (ppm): 8.46-8.45 (m, 1H), 8.19-8.16 (m,
H), 7.94-7.91 (m, 1H), 7.58 (t, 1H, J = 7.9 Hz), 7.33 (t, 1H, J =
.8 Hz), 6.89 (d, 1H, J = 8.0 Hz), 6.75-6-74 (m, 1H), 6.65-6.63
2
SO
4
, filtered, evaporated in
brown oil that was dissolved in CHCl
The organic phase was dried over anhydrous Na
evaporated in vacuo, and purified by flash column
3
and washed with water.
2
SO , filtered,
4
(
2
2
1
chromatography on silica gel (petroleum ether/AcOEt 6:4),
(
1
7
3
obtaining the intermediate 8 as a white solid (153.0 mg, 0.60
1
mmol). Yield: 45%. H NMR (CDCl
3
, 400 MHz) δ (ppm): 7.29
(t, 1H, J = 7.9 Hz), 7.06 (t, 1H, J = 7.9 Hz), 6.87-6.84 (m, 1H),
6.72 (dd, 1H, J = 8.0 Hz, J = 1.9 Hz), 6.43 (bs, 1H), 6.32 (dd, 1H,
J = 7.8 Hz, J = 1.9 Hz), 6.12 (d, 1H, J = 7.8 Hz), 5.82 (d, 1H, J =
(
m, 1H), 3.38-3.35 (m, 4H), 2.07-2.03 (m, 4H).
8
.1 Hz), 3.47-3.42 (m, 4H), 3.20 (bs, 2H), 2.01-1.96 (m, 4H).
5
.1.3. 3’-(Pyrrolidin-1-yl)biphenyl-3-amine (4)
Hydrazine hydrate (0.2 mL, 66 mmol) was added to a
5
.1.7. General procedure for the synthesis of urea
derivatives SC4a, FG45a and SN15b.
solution of 3 (63.8 mg, 0.29 mmol) in ethanol (4.0 mL). The
mixture was stirred at 50 °C for 15 min. Then, an excess of
Raney nickel (∼ 60 mg) was added. After the bubbling ceased
the solution changed from orange to colorless), the mixture was
cooled to room temperature and filtered through a celite pad. The
4-Chloro-phenylisocyanate (1 eq) was added to a solution
(
of the suitable amine intermediate (1 eq) in the minimum amount
of freshly distilled CHCl . The resulting mixture was left at room
3
filtrate was evaporated in vacuo, affording intermediate 4 (58.5
temperature, under magnetic stirring, for 12 h. The solvent was
removed by evaporation in vacuo or by filtration and the residue
purified.
mg) as a white solid, which was used in the next step without
1
further purification. H NMR (CDCl
3
, 400 MHz) δ (ppm): 7.28
(
t, 1H, J = 7.8 Hz), 7.22 (t, 1H, J = 7.8 Hz), 7.03-7.01 (m, 1H),
6
6
.95-6.92 (m, 1H), , 6.88-6.85 (m, 1H), 6.75-6.73 (m, 1H), 6.69-
.65 (m, 1H), 6.59-6.55 (m, 1H), 3.36-3.33 (m, 4H), 2.05-1.99
5.1.8. 1-(4-Chlorophenyl)-3-[3’-(pyrrolidin-1-
yl)biphenyl-3-yl]urea (SC4a)
(
m, 4H).
Prepared from 4-chloro-phenylisocyanate (39.0 mg, 0.25
mmol) and intermediate 4 (58.5 mg, 0.25 mmol) dissolved in
5
.1.4. N-[3-(pyrrolidin-1-yl)phenyl]-1,3-diamino-
benzene (5)
Obtained by treating 2 (714.0 mg, 3.16 mol) with
commercial 1,3-diaminobenzene 6 (410 mg, 3.79 mmol) and
28.0 mg of a reagent constituted by
tris(dibenzylideneacetone)dipalladium(0), BINAP and t-BuONa
mol ratio: 0.05:0.15:2) in toluene (7.9 mL), following the same
10.2 mL of freshly distilled CHCl . Purification by flash column
3
chromatography on silica gel (petroleum ether/AcOEt 7:3). SC4a
1
(37.1 mg, 0.10 mmol). Yield: 39%. M. p.: 168-172 °C. H NMR
(DMSO-d
7.69 (m, 1H), 7.49 (AA’XX’, 2H, JAX = 8.9 Hz, JAA’XX’ = 1.6 Hz),
7.43-7.41 (m, 1H), 7.35-7.30 (m, 1H), 7.33 (AA’XX’, 2H, JAX
6
, 400 MHz) δ (ppm): 8.84 (s, 1H), 8.79 (s, 1H), 7.72-
6
=
(
8.9 Hz, JAA’XX’ = 1.6 Hz), 7.26-7.22 (m, 2H), 6.81 (d, 1H, J = 7.3
Hz), 6.72-6.68 (m, 1H), 6.55 (dd, 1H, J = 8.0 Hz, J = 1.7 Hz),
reaction and work-up conditions used for the synthesis of 2. The
crude was purified by flash column chromatography on silica gel
13
3.30-3.27 (m, 4H), 1.99-1.96 (m, 4H). C NMR (DMSO-d , 400
6
(
petroleum ether/AcOEt 9:1), obtaining 5 as an yellow oil (342.0
MHz) δ (ppm): 152.49, 148.07, 141.98, 141.15, 139.89, 138.71,
129.44, 129.16, 128.61, 125.32, 120.56, 119.75, 117.20, 116.71,
113.76, 110.96, 109.78, 41.33, 24.96. HPLC analysis: retention
time = 20.62 min; peak area, 99% (280 nm).
1
mg, 1.35 mmol). Yield: 43%. H NMR (CDCl
(
6
1
3
, 400 MHz) δ
ppm): 7.11 (t, 1H, J = 8.0 Hz), 7.02 (t, 1H, J = 8.0 Hz), 6.50-
.42 (m, 3H), 6.32-6.31 (m, 1H), 6.25-6.20 (m, 2H), 5.58 (bs,
H), 3.66 (bs, 2H), 3.28-3.25 (m, 4H), 2.00-1.97 (m, 4H).
5.1.9. 2-(4-Chlorophenyl)-N-[3’-(pyrrolidin-1-
yl)biphenyl-3-yl]acetamide (SC33)
5
(
.1.5. N-(6-bromopyridin-2-yl)benzene-1,3-diamine
7).
The reaction was conducted under nitrogen atmosphere. Et N
3
A mixture of 1,3-diaminobenzene 6 (2.60 g, 24.0 mmol),
,6-dibromopyridine (7.11 g, 30.0 mmol), t-BuOK (6.16 g, 55.2
mmol) and anhydrous toluene (130.0 mL) was refluxed for 48 h,
(29.3 mg, 0.29 mmol) and DMAP (35.4 mg, 0.29 mmol) were
added to a solution of 4 (69.4 mg, 0.29 mmol) in anhydrous
2
2 2
CH Cl (2.0 mL). Then, the mixture was cooled to -5 °C (ice-
under magnetic stirring. After cooling to room temperature, the
NaCl bath) and (4-chlorophenyl)acetyl chloride (54.8 mg, 0.29

mmol) was added. The reaction mixture was left, under magnetic
stirring, at room temperature for 24 h. The crude was washed
tubes. Membranes were incubated with the compounds at
different concentrations (1 nM-100 µM) in presence of 0.5 nM of
3
-1
twice with an aqueous saturated solution of NaHCO
3
and purified
[ H]CP55,940 (168 Ci·mmol ) for 1.5 h at 30 °C. Non-specific
binding of the radioligand was determined in the presence of 10
µM of (R)-WIN55,512-2. Non-specific binding was around 10%.
After the incubation time, membrane suspensions were rapidly
filtered through a 0.05% polyethyleneimine presoaked 96-well
microplate bonded with GF/B glass fiber filters (UniFilter-
96GF/B, Perkin Elmer Life Sciences) under vacuum and washed
10 times with 167 µL of ice-cold washing buffer (50 mM Tris-
by flash column chromatography on silica gel (petroleum
ether/AcOEt 8:2), obtaining the desired product SC33 as a solid
1
(
(
7
24.8 mg, 0.06 mmol). Yield: 22%. M. p.: 138-140 °C. H NMR
CDCl
.47 (m, 1H), 7.40-7.30 (m, 4H), 7.30-7.23 (m, 2H), 6.86 (d, 1H,
J = 7.6 Hz), 6.75-6.71 (m, 1H), 6.59 (dd, 1H, J = 8.1 Hz, J = 1.9
3
, 400 MHz) δ (ppm): 7.68 (bs, 1H), 7.64 (bs, 1H), 7.52-
1
3
Hz), 3.67 (s, 2H), 3.35-3.32 (m, 4H), 2.04-2.01 (m, 4H).
NMR (CDCl , 400 MHz) δ (ppm): 168.66, 141.84, 137.90,
33.75, 133.02, 130.98, 129.67, 129.43, 129.36, 123.77, 118.85,
18.78, 44.21, 29.85, 25.56. HPLC analysis: retention time =
0.75 min; peak area, 98% (280 nm).
C
3
2
HCl, 2.5 mM EDTA, 5 mM MgCl , 0.5% fatty acid free BSA,
pH 7.4). Filters were added to 3 mL of MicroScint 20
1
1
2
scintillation liquid and radioactivity was measured with the 1450
Micro Beta Trilux top counter. The non-specific binding was
3
subtracted and results were expressed as [ H]CP55,940 bound as
%
of binding in vehicle-treated samples.
5.1.10. 1-(4-Chlorophenyl)-3-(3-{[3-(pyrrolidin-1-
yl)phenyl]amino}phenyl)urea (FG45a)
Prepared from 4-chloro-phenylisocyanate (31.8 mg, 0.21
5.2.2. FAAH, MAGL and AEA uptake assays
mmol) and intermediate 5 (52.5 mg, 0.21 mmol) dissolved in 8.5
mL of freshly distilled CHCl
chromatography on silica gel (CHCl
crystallization from EtOH. FG45a (34.7 mg, 0.09 mmol). Yield:
3
. Purification by flash column
FAAH and MAGL activity assay and AEA uptake assays
3
) and subsequent
3
9,40
were performed as previously described.
Briefly, FAAH and
1
MAGL activity assays were performed using U937 cell
homogenate (100 µg) which were diluted in 200 µL of Tris–HCl
4
(
2
(
8
(
4
2%. M. p.: 168-171 °C. H NMR (DMSO-d
ppm): 8.73 (s, 1H), 8.60 (s, 1H), 7.97 (s, 1H); 7.46 (AA’XX’,
H, JAX = 9.0 Hz, JAA’XX’ = 2.6 Hz), 7.36-7.35 (m, 1H), 7.31
AA’XX’, 2H, JAX = 8.9 Hz, JAA’XX’ = 2.6 Hz), 7.07 (t, 1H, J =
.0 Hz), 7.00 (t, 1H, J = 8.0 Hz), 6.79-6.77 (m, 1H), 6.66-6.64
m, 1H), 6.36-6.30 (m, 2H), 6.08-6.07 (m, 1 H), 3.22-3.19 (m,
6
, 400 MHz) δ
1
0 mM, EDTA 1 mM, pH 8 containing 0.1% fatty acid-free
BSA. Compounds were added at the screening concentration of
0 µM and incubated for 30 min at 37 °C. Then, 100 nM of AEA
1
3
containing 1 nM of [ethanolamine-1- H]AEA as a tracer 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
1
3
H), 1.94-1.92 (m, 4H). C NMR (DMSO-d , 400 MHz) δ
6
(
ppm): 152.35, 148.57, 144.44, 143.78, 140.17, 138.80, 129.43,
3
CHCl :MeOH (1:1), samples were vortexed and rapidly
1
1
1
29.25, 128.60, 125.20, 119.59, 110.64, 109.17, 106.04, 105.18,
04.29, 100.78, 47.24, 24.95. HPLC analysis: retention time =
6.53 min; peak area, 97% (280 nm).
centrifuged at 16’000 x g for 10 min. at 4 °C. The aqueous
phases were collected and the radioactivity was measured for
tritium content by liquid scintillation spectroscopy. For AEA
6
uptake assay 0.5 x 10 of intact U937 cells were suspended in
5
00 µL of serum-free medium in silanized glass tubes and
5
.1.11. 1-(4-Chlorophenyl)-3-{[6-(pyrrolidin-1-
preincubated with a screening concentration (10 µM) of the
compounds for 20 min at 37 °C. Then, the cells were incubated
for 5 min at 37 °C with 100 nM of AEA and [ethanolamine-1-
yl)pyridin-2-yl]amino}phenyl)urea (SN15b)
Prepared from 4-chloro-phenylisocyanate (20.3 mg, 0.13
mmol) and intermediate 8 (33.8 mg, 0.13 mmol) dissolved in 5.4
mL of freshly distilled CHCl
from EtOH. SN15b (14.8 mg, 0.04 mmol). Yield: 28%. M. p.:
3
H]AEA (0.5 nM) was added. The uptake process was stopped by
3
. Purification by crystallization
transferring the tubes on ice and by rapid filtration over
UniFilter®-96 GF/C filters pre-soaked with PBS supplemented
with 0.2% BSA. Cells were washed three times with 100 µL ice-
cold PBS supplemented with 1% fatty acid free BSA. After
drying, 50 µL MicroScint 20 scintillation cocktail was added to
the wells. The radioactivity was measured using a Trilux
MicroBeta 1450.
1
dec. at 220 °C. H NMR (DMSO-d
6
, 400 MHz) δ (ppm): 8.81 (s,
1
2
7
H), 8.67 (s, 1H), 8.54 (s, 1H), 7.88-7.82 (m, 1H), 7.50-7.47 (m,
H), 7.44-7.42 (m, 1H), 7.33-7.31 (m, 2H), 7.28-7.24 (m, 1H),
.11-7.07 (m, 1H), 6.81 (d, 1H, J = 8.1 Hz), 6.03 (d, 1H, J = 7.9
Hz), 5.77 (d, 1H, J = 7.6 Hz), 3.44-3.37 (m, 4H), 1.96-1.90 (m,
13
4
1
1
H). C NMR (DMSO-d , 400 MHz) δ (ppm): 155.99, 154.65,
6
52.31, 142.80, 139.52, 138.88, 138.03, 128.58, 125.11, 119.52,
11.76, 110.06, 107.86, 97.03, 95.56, 46.42, 25.01. HPLC
5
.2.3. Serum response element (SRE) assay
analysis: retention time = 4.65 min; peak area, 95% (280 nm).
HEK293 cells were transiently transfected with hCB1R (5
5
5
.2. Biological assays
ng/well) and pGL4.33 [luc2P/SRE/Hygro] vector (100 ng/well)
reporter (Promega) plasmids using Lipofectamine 2000 as
46
described by the manufacturer (Invitrogen). Transfected
HEK293 cells were seeded (60,000ꢀcells per well) in 96-well
plates. Five hours later, medium was changed to 1%
.2.1. Radioligand binding assays on CB1R and
CB2R
FBS/DMEM. Cells were incubated overnight. The next day cells
were treated with ligands for 5ꢀh in serum-free DMEM medium
at 37 °C. After treatment, cells were lysed by 1X lysis buffer for
10 min. at room temperature. Plates were read to record
bioluminescent after the injection of 40µl Luciferin (≥ 250 µM)
per well. Luminescence was measured in an Envision 2104
Multilabel Reader (Perkin Elmer). Luminescence values are
Cannabinoid receptors affinity was determined using
membrane preparations obtained from CHO-K1 cells stably
3
8
transfected with hCB1 or hCB2 as previously described.
Briefly, 20 mg of membrane preparation were resuspended in
00 µL of binding buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5
mM MgCl , 0.5% fatty acid free BSA, pH 7.4) in silanized glass
5
2

given as relative light units. Concentration-effect curves for
agonist-mediated receptor activation were analyzed by nonlinear
regression techniques using GraphPad Prism 5.0 software
18. Palmer SL, Thakur GA, Makriyannis A. Cannabinergic ligands.
Chem Phys Lipids 2002; 121; 3−19.
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9. Thakur GA, Bajaj S, Paronis C, Peng Y, Bowman AL, Barak LS,
Caron MG, Parrish D, Deschamps JR, Makriyannis A. Novel
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2013; 56: 3904−3921.
(
GraphPad) and data were fitted to sigmoidal dose-response
curves to obtain EC50 values.
2
0. Dixon DD, Tius MA, Thakur GA, Zhou H, Bowman AL, Shukla
VG, Peng Y, Makriyannis A. C3-heteroaroyl cannabinoids as
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