Identification of the First Synthetic Allosteric Modulator of the CB2 Receptors and Evidence of Its Efficacy for Neuropathic Pain Relief

Article abstract of DOI:10.1021/acs.jmedchem.8b00368

The direct activation of cannabinoid receptors (CBRs) results in several beneficial effects; therefore several CBRs ligands have been synthesized and tested in vitro and in vivo. However, none of them reached an advanced phase of clinical development due

Full text of DOI:10.1021/acs.jmedchem.8b00368

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Article
2
Identification of the first synthetic allosteric modulator of the CB
receptors and evidence of its efficacy for neuropathic pain relief
Francesca Gado, Lorenzo Di Cesare Mannelli, Elena Lucarini, Simone Bertini,
Elena Cappelli, Maria Digiacomo, Lesley A. Stevenson, marco macchia, Tiziano
Tuccinardi, Carla Ghelardini, Roger G. Pertwee, and Clementina Manera
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00368 • Publication Date (Web): 10 Jul 2018
Downloaded from http://pubs.acs.org on July 10, 2018
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Identification of the first synthetic allosteric
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modulator of the CB2 receptors and evidence of its
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efficacy for neuropathic pain relief
Francesca Gado,⊥,# Lorenzo Di Cesare Mannelli,§, # Elena Lucarini,§ Simone Bertini,⊥ Elena
Cappelli,⊥ Maria Digiacomo,⊥ Lesley A Stevenson,ψ Marco Macchia,⊥ Tiziano Tuccinardi,⊥
,†
,
Carla Ghelardini,§ Roger G. Pertwee,ψ Clementina Manera⊥ *
⊥Department of Pharmacy, University of Pisa, 56126 Pisa. §Department of Neuroscience,
Psychology, Drug Research and Child Health, Section of Pharmacology and Toxicology, University
of Florence, 50139 Florence, Italy. ψSchool of Medicine, Medical Sciences and Nutrition, Institute
of Medical Sciences, University of Aberdeen, AB25 2ZD Aberdeen, Scotland, UK. †Sbarro Institute
for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and
Technology, Temple University, Philadelphia, Pennsylvania 19122, United States.
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Abstract
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The direct activation of cannabinoid receptors (CBRs) results in several beneficial effects, therefore
several CBRs ligands have been synthesized and tested in vitro and in vivo. However none of them
reached an advanced phase of clinical development due mainly to side effects on the CNS.
Medicinal chemistry approaches are now engaged to develop allosteric modulators that might offer
a novel therapeutic approach to achieve potential therapeutic benefits avoiding inherent side effects
of orthosteric ligands. Here we identify the first ever synthetized positive allosteric modulator
(PAM) that targets CB2Rs. The evidence for this was obtained using [3H]CP55940 and [35S]GTPγS
binding assays. This finding will be useful for the characterization of allosteric binding site(s) on
CB2Rs which will be essential for the further development of CB2R allosteric modulators.
Moreover, the new CB2R PAM displayed antinociceptive activity in vivo in an experimental mouse
model of neuropathic pain, raising the possibility that it might be a good candidate for clinical
development.
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Introduction
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The endocannabinoid system (ECS) is a ubiquitous neuromodulatory system with wideꢀranging
actions.1 Cannabinoid receptors (CB1Rs, CB2Rs) are members of Gꢀproteinꢀcoupled receptors
family. Several findings indicate that CB2R ligands, which do not share the ability of CB1R agonists
to produce psychotropic effects or of selective CB1R antagonists to produce other serious adverse
effects,2ꢀ4 appear to be promising drugs for treating several diseases.5ꢀ7 Medicinal chemistry
approaches are now being adopted to develop more efficacious agonists, and in particular, to
investigate the possibility of developing allosteric modulators (Figure 1) that might offer a novel
therapeutic approach with minimal sideꢀeffects. It has been reported that CBRs have spatially
distinct allosteric sites that can be targeted by exogenous/endogenous substances modulating the
receptors’ functional state.8ꢀ15 Allosteric modulators offer numerous advantages respect to
orthosteric ligands, such as a greater subtype selectivity for the higher sequence divergence at
allosteric sites, than the orthosteric domains; furthermore, allosteric modulators possess tissue
selectivity because they exert effects only where endocannabinoids are present and eventually they
might provide synergizing effects with other coꢀadministered ECS modulators.16ꢀ19 Moreover, they
could have the advantage of activating or inhibiting the biased signaling properties of these GPCRs.
For these reasons, allosteric modulators might represent a valid alternative method to modulate the
ECS and they could be useful for the development of future drugs. They are classified in: positive
allosteric modulators (PAMs), negative allosteric modulators (NAMs) which activate or inhibit the
receptor response, respectively, neutral or silent allosteric modulators, which do bind to the
allosteric site without affecting the response of the endogenous agonist and bitopic ligands that bind
to both the orthosteric and allosteric sites.
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POSITIVE ALLOSTERIC
MODULATOR (PAM)
NEGATIVE ALLOSTERIC
MODULATOR (NAM)
SILENT ALLOSTERIC
MODULATOR (SAM)
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β
α
γ
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γ
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Unaltered
signalling
Signalling
Signalling
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Orthosteric ligand
Allosteric ligand
Allosteric modulator interaction
G protein
β
γ
α
Figure 1. General classification of allosteric modulators: PAMs, NAMs and SAMs.
To date, few allosteric modulators for CB1Rs have been developed. Among them, indole
derivatives,9,20 urea derivatives21 and certain other molecules have a PAM profile (Figure 2).22 Up to
now, the only CB2R allosteric modulator reported in the literature is the endogenous Pepcanꢀ12
(Figure 2) which was reported also to be a CB1R NAM.23
Cl
N
O
H
HN
O
N
N
N
N
N
HN
H
H
NO2
Cl
ORG27569
PSNCBAM-1
S
NH2
ZCZ011
Cl
N
O
HN
O
O
O
N
O
NH2
H
O
O
O
HO
H
H
H
N
N
N
N
OH
N
N
H
N
N
N
H
O
H
H
O
O
O
O
HN
OH
O
RT371
NH
Pepcan-12
O
NH2
NH
HN
NH2
Figure 2. Representative CBRs allosteric modulators.
In this work, we present evidence that the novel synthetic compound C2 (Figure 3), is a positive
allosteric modulator (PAM) of the CB2Rs. This evidence was obtained by carrying out
[3H]CP55940 and [35S]GTPγS binding assays. C2 is a member of a group of compounds that were
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obtained by modifying the structure of 2ꢀoxopyridineꢀ3ꢀcarboxamides derivatives of general
structure A and B (Figure 3) that had been previously shown to be orthosteric CBR ligands.24 With
the aim of deeply exploring the structureꢀactivity relationships of derivatives A and B, we
synthesized derivatives C1ꢀC5, D1ꢀD6 and E1 in which the amide group in position Cꢀ3 of the 2ꢀ
oxopyridineꢀ3ꢀcarboxamides A and B was substituted with the corresponding retroꢀamide (Figure
3). The substituents of derivatives C and D have been selected on the basis of the best results
achieved with the series A and B. Moreover, compound E1, characterized by an OH group in
position 4, has been synthesized, to understand the role of the methyl group and, at the same time,
to define the effect on the affinity of binding to CBRs of a polar group in the same position (Figure
3). Finally, derivative C2 displayed antinociceptive activity in vivo in an experimental mouse model
of neuropathic pain.
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Figure 3. Design of retroꢀamides C1ꢀC5, D1ꢀD6 and E1.
Results and Discussion
Chemistry. The synthesis of 2ꢀoxopyridines C, D and E1 was obtained as reported in Schemes
1ꢀ3.
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The cycloheptanecarboxamide derivatives C1ꢀC5 were synthesized (Scheme 1) starting from the
2ꢀhydroxyꢀ4ꢀmethylꢀ3ꢀnitropyridine which underwent a catalytic hydrogenation to afford derivative
1. The reaction between the amine 1 and the cycloheptanecarbonyl chloride in toluene, DMF and
triethylamine, carried out initially at 0 °C and then at 30 °C for 48 hours, afforded the amide 2. The
acyl chloride was prepared from reaction between cycloheptanecarboxylic acid with SOCl2 at 76 °C
for 3 hours. The derivative 2 was reacted with CsF and then with pꢀfluorobenzyl chloride, yielding
the desired Nꢀalkylated compound C1. The derivatives C2ꢀC5 were synthesized starting from the
amide 2 which was treated with a solution of bromine in CHCl3, affording the corresponding 5ꢀ
bromo derivative 3. The treatment of 3 with pꢀfluorobenzyl chloride via a similar method to that
used for the preparation of C1, afforded the desired Nꢀalkylated compound C2 (Scheme 1). The 5ꢀ
aryl derivatives C3 and C4 were yielded starting from the 5ꢀbromo derivative C2 via a Suzukiꢀ
Miyaura crossꢀcoupling reaction with the suitable boronic acids in dry toluene and Pd(PPh3)4 as
catalyst (Scheme 1). The 5ꢀchloro derivative C5 was obtained from amide 2 which was treated with
Nꢀchlorosuccinimide to give compound 4. The Nꢀalkylation of 4, carried out following the same
method used to yield C1ꢀC4, afforded the desired compound C5 (Scheme 1).
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As reported in Scheme 2, the compounds D1ꢀD6 were obtained starting from 2ꢀhydroxyꢀ6ꢀ
methylꢀ3ꢀnitropyridine which underwent a catalytic hydrogenation, using Pd/C as catalyst, to afford
compound 5. Subsequently, the condensation of 5 with cycloheptanecarboxylic acid using TBTU as
condensing reagent in the presence of triethlylamine, yielded the carboxamide 6 which, for Nꢀ
alkylation with NaH, LiBr and pꢀfluoroꢀbenzylchloride, gave D1. The reaction of 6 with a solution
of bromine in CHCl3 produced the corresponding 5ꢀbromo derivative 7 which was Nꢀalkylated by
the same procedure previously described, to yield the desired compound D2. The derivatives D3
and D4 were obtained from D2 by Suzuki crossꢀcoupling reaction with the suitable arylboronic acid
(Scheme 2). The reaction of compound 6 with Nꢀchlorosuccinimide in acetonitrile at reflux gave the
5ꢀchloro derivative 8 which was Nꢀalkylated using the procedure already described, to afford
compound D5 (Scheme 2). The carboxamide 6 was also treated with 1ꢀchloromethylꢀ4ꢀfluoroꢀ1,4ꢀ
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diazoniabicyclo[2.2.2] octanebis(tetrafluoroborate) in acetonitrile at reflux for 12 hours, yielding
4
5
the 5ꢀfluorinated derivative 9 which was Nꢀalkylated to afford the desired compound D6 (Scheme
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2).
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Scheme 1. Synthesis of compounds C1ꢀC5
H
H
NO2
O
N
NH2
O
N
i
ii
iii
O
O
N
N
O
N
N
O
2
C1
H
H
H
1
iv
vi
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H
H
N
Br
N
Cl
O
O
N
O
N
O
3
4
H
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iii
iii
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N
H
Br
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Cl
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O
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C5
F
F
v
v
O
H
N
H
N
O
O
N
O
N
O
C4
C3
F
F
Reagents and conditions: (i) H2, Pd/C, MeOH, rt, 12 h; (ii) 1) cycloheptancarboxylic acid,
SOCl2, 76 °C, 3 h; 2) acyl chloride, toluene, DMF, NEt3, 0ꢀ32 °C, 48 h; (iii) 1) DMF, CsF, rt, 1 h;
2) pꢀfluorobenzyl chloride, 50 °C, 12 h; (iv) Br2, CHCl3, 30 °C, 12 h; (v) triphenylphosphine,
Pd(OAc)2, K2CO3, suitable arylboronic acid, 110 °C, 12 h; (vi) Nꢀchlorosuccinimide, ACN, reflux,
12 h.
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Scheme 2. Synthetic pathway for compounds D1ꢀD6
H
N
H
N
NH2
O
NO2
O
i
ii
iii
O
O
N
O
N
N
N
H
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D1
H
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5
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iv
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Br
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H
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H
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H
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iii
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H
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O
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O
D2
D6
D5
F
F
F
v
v
O
H
N
H
N
O
O
N
O
N
O
D3
D4
F
F
Reagents and conditions: (i) H2, Pd/C, MeOH, rt, 12 h; (ii) cycloheptancarboxylic acid, TBTU,
NEt3, DMF, 0 °C rt, overnight; (iii) NaH 60%, LiBr, pꢀfluorobenzyl chloride, DMF, DME, 65 °C,
12 h; (iv) Br2, CHCl3, rt, 12 h; (v) triphenylphosphine, Pd(OAc)2, K2CO3, suitable arylboronic acid,
110 °C, overnight; (vi) Nꢀchlorosuccinimide, ACN, reflux, 12 h; (vii) 1ꢀchloromethylꢀ4ꢀfluoroꢀ1,4ꢀ
diazoniabicyclo[2.2.2] octanebis(tetrafluoroborate), ACN, reflux, 12 h.
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Finally, compound E1 was synthesized as reported in Scheme 3, starting from 4ꢀhydroxyꢀ3ꢀ
nitroꢀ2ꢀoxoꢀ1,2ꢀdihydropyridine which underwent a catalytic hydrogenation to yield derivative 10.
Reaction of 10 with cycloheptanecarbonyl chloride prepared as reported above, gave compound 11
which, by treatment with a solution of bromine in CHCl3, yielded compound 12. The Nꢀalkylation
of 12 carried out with CsF and pꢀfluorobenzyl chloride, afforded the derivative E1.
Scheme 3. Synthetic pathway for compound E1
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OH
OH
OH
OH
OH
H
N
H
N
H
N
Br
Br
NH2
NO2
O
iii
ii
i
iv
O
O
O
N
O
N
H
O
N
H
O
N
H
O
N
H
E1
10
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12
F
Reagents and conditions: (i) H2, Pd/C, MeOH, rt, 12 h; (ii) 1) cycloheptancarboxilic acid, SOCl2,
76 °C, 3 h; 2) Toluene, DMF, NEt3, 0ꢀ32 °C, 48 h; (iii) Br2, CHCl3, 30 °C, 12 h; (iv) 1) DMF, CsF,
rt, 1 h; 2) pꢀfluorobenzylchloride, 50 °C, 12 h.
[3H]CP55940 Binding Assays. Compounds C1ꢀC5, D1ꢀD6 and E1 were evaluated for CBRs
binding by incubating these compounds with membrane preparations obtained from Chinese
hamster ovary (CHO) cells overexpressing hCB1Rs or hCB2Rs in the presence of 0.7 nM
[3H]CP55940, a highꢀaffinity orthosteric CB1R and CB2R radioligand. Also, the N1ꢀunsubsituted
analogue of C2, derivative 3, was assayed with the aim of exploring the influence of the substituent
in position 1.
Regarding the CB1Rs, initially, all compounds were screened at 100 nM. The results (Figure S1
in Supporting Information) showed that only compounds C1, D1, D5 and derivative 3 displaced
[3H]CP55940. Rather unexpectedly, all the other compounds enhanced the binding of the
radioligand to CB1Rs. In particular, compounds C2, C3 and D3 were found to be effective as
enhancers at 100 nM (Figure S1 in Supporting Information) and their concentrationꢀdependent (1
nM to 1 µM) CB1R binding curves (C3 and D3 Figure S2 in Supporting Information) indicated
compound C2 (Figure 4) to be the best enhancer, inducing approximately a 40% increase in the
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binding of [3H]CP55940 up to a concentration of 1 µM. However, no increase in such binding was
induced by C2 at 10 µM. These data suggest that C2 may be a CB1R PAM (Figure 4).
In the case of the CB2Rs, concentrationꢀdependent binding curves (Figure S3 in Supporting
Information) indicate that D1ꢀD6, C1, C3ꢀC5 and 3 were either weak orthosteric ligands or
completely inactive. Derivative E1 was found to enhance weakly the binding of [3H]CP55940
(Figure S3 in Supporting information), whereas compound C2 strongly increased the binding of
[3H]CP55940 at very low concentrations (Figure 4) raising the possibility that it may be a CB2R
PAM.
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-10 -9 -8 -7 -6 -5 -4
C2 (logM)
-10 -9 -8 -7 -6 -5 -4
C2 (log M)
Figure 4. Effects of compounds C2 (at 1 nM to 10 µM) on [3H]CP55940 binding to CB1R (left)
and CB2R (right). 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 means ± SEM of six independent
experiments, each performed in duplicate.
[35S]GTPγS assays. To evaluate the functional activity of C2 on CBRs, [35S]GTPγS assays were
carried out in the presence of [35S]GTPγS (0.1 nM), GDP (30 µM), GTPγS (30 µM), with CHO cell
membranes (1 mg/ml) overexpressing hCB1Rs or hCB2Rs and in the presence of CP55940, a full
agonist of both CBRs. The choice of a C2 concentration of 100 nM for these experiments was
influenced by CB1R and CB2R binding data. The effects of C2 on the modulation of CBRꢀmediated
signaling was studied by measuring [35S]GTPγS receptor binding (Figure 5).
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DMSO
DMSO
100 nM C2
100 nM C2
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Figure 5. CB1R [35S]GTPγS binding assays (left) and CB2R [35S]GTPγS binding assays (right)
performed with CP55940 and compound C2. . Asterisks indicate mean values significantly different
from zero (*P < 0.05; **P < 0.01; ***P < 0.001) (Student’s unpaired t test). Data are expressed as
means ± SEM of six and twelve independent experiments for CB1R and CB2R respectively, each
performed in duplicate.
The CP55940ꢀinduced stimulation of [35S]GTPγS CB1R binding was not significantly altered by
100 nM C2 (Figure 5). This result suggests that although this concentration of compound C2
increases the binding of the orthosteric ligand [3H]CP55940 to CB1Rs, this enhancement does not
significantly affect the ability of CP55940 to activate CB1Rs. However, the current data is not
sufficient to indicate C2 as SAM for CB1R.
Conversely C2 appeared to target CB2Rs in a very promising and interesting manner (Figure 5).
Thus, the ability of CP55940 to stimulate [35S]GTPγS binding to CB2Rs was significantly enhanced
by 100 nM C2. Furthermore as shown in Figure S4 (Supporting Information), in the absence of the
orthosteric ligand, compound C2 did not produce any stimulation or inhibition of [35S]GTPγS
binding to CB2Rs. Collectively, these data suggest that C2 acts as a PAM of CB2Rs.
Further studies should be carried out with the aim to investigate the allosteric effects of C2 more
fully (e.g. more concentrations and additional functional in vitro assays) and to highlight different
behaviors at other CB1Rꢀ and CB2Rꢀmediated signaling pathways (e.g. cAMP production, βꢀ
arrestin, ion channels).
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Consequently, the study of this compound was extended by performing [35S]GTPγS receptor
binding assays using one or the other of the two major endocannaboinoids, anandamide (AEA) and
2ꢀarachidonoylglycerol (2ꢀAG), as CB2R agonists, rather than CP55940. As shown in Figure 6, 100
nM of C2 significantly enhanced the stimulation of [35S]GTPγS binding to CB2R induced by 2ꢀAG,
but not the enhancement induced by AEA. Thus, it is likely that C2 would be able to facilitate the
activation of CB2Rs by low concentrations of endogenouslyꢀreleased 2ꢀAG, although not by
endogenouslyꢀreleased AEA.
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Figure 6. CB2R [35S]GTPγS binding assays performed in the presence of C2 with 2ꢀ
arachidonoylglycerol (2ꢀAG) (left) or anandamide (AEA) (right). Asterisks indicate mean values
significantly different from zero (*P < 0.05; **P < 0.01) (Student’s unpaired t test). Data are
expressed as means ± SEM of six independent experiments, each performed in duplicate.
Dissociation Kinetic Assays. In order to better characterize the postulated allosteric action of
derivative C2, some dissociation kinetic assays in the absence or in the presence of C2 (Figure 7)
were performed. These assays were carried out with the CB1/CB2R ligand [3H]CP55940 (1 µM), C2
(100 nM) and CHO cell membranes (1 mg/ml) overexpressing hCB2Rs. Dissociation was monitored
from +0.5 to +120 min at 25 °C. As shown in Figure 7, [3H]CP55940 dissociation was completely
prevented by 100 nM of C2 for at least 2 hours. The absence of [3H]CP55940 dissociation from
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CB2Rs was in line with the increased binding of [3H]CP55940 induced by C2 in binding
experiments. These findings support our hypothesis that C2 targets an allosteric binding site of the
CB2Rs. Each point on the graph represents a mean of 4 replicates.
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CP55940 + DMSO
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Figure 7. Dissociation of [3H]CP55940 from CB2Rs in the presence or absence of C2. Data are
expressed as means ± SEM of four independent experiments, each performed in duplicate.
Inhibitory activity on MAGL. Finally, in line with the hypothesis that C2 enhanced 2ꢀAGꢀ
induced stimulation of [35S]GTPγS binding to the CB2Rs by acting as a CB2R PAM, evidence was
obtained that C2 did not induce this enhancement by inhibiting monoacylglycerol lipase (MAGL),
the main 2ꢀAG degrading enzyme. This evidence was obtained from experiments in which the
enzymatic activity of human isoforms of MAGL was measured by using a colorimetric assay that
exploits 4ꢀnitrophenylacetate (4ꢀNPA) as a nonꢀspecific chromogenic substrate for MAGL and by
measuring the absorbance values at 405 nm due to 4ꢀnitrophenol (PNP) released from 4ꢀNPA, upon
enzymatic hydrolysis.25 The obtained results showed that C2 produced no significant inhibition of
MAGL at < 10 µM.
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Antinociceptive Effects of C2 in Animal Models of Neuropathic Pain. Neuropathic pain may
be induced by traumatic injury, metabolic challenges and chemotherapeutic agents.
Pharmacotherapies used to treat neuropathic pain produce inadequate pain relief and/or unwanted
side effects. Thus, the identification of novel therapeutic approaches with limited side effect profiles
remains an urgent medical need. Cannabinoids suppress behavioural responses to noxious
stimulation and suppress nociceptive transmission through activation of CB1Rs and CB2Rs.
Moreover, the CB2Rs are upregulated in the CNS and dorsal root ganglia by pathological pain states
and they were identified as a therapeutic target for treating pathological pain states.26 These
observations prompted us to examine the ability of C2 to alleviate signs of neuropathic pain in a
mouse model of nociceptive behavior caused by the chemotherapeutic agent, oxaliplatin (OXP).
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A daily treatment with the neurotoxic compound oxaliplatin (2.4 mg kgꢀ1 intraperitoneally ꢀ i.p.)
progressively decreased the pain threshold of mice evaluated as hypersensitivity to a cold nonꢀ
noxious stimulus (allodyniaꢀlike measurements; cold plate test).27 The licking latency decreased to
9.7 ± 0.9 s in comparison to control mice (18.2 ±0.6 s) treated with vehicle (Figure 8). The effect of
single oral (p.o.) administration of four different doses of derivative C2 (1, 5, 10, 20 mg kgꢀ1) on
oxaliplatinꢀtreated mice is shown in Figure 8. C2 significantly increased licking latency in the
animals starting from 5 mg kgꢀ1. The doses of 5 and 10 mg kgꢀ1 of C2 were equally effective
between 30 and 60 min after administration (Figure 8). The effect of the higher 20 mg kgꢀ1 dose was
detectable within 15 min and lasted 60 min, reducing animal pain threshold back to the value of
controls between +30 and +45 min.
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Figure 8. Neuropathic pain modulation by C2. Hypersensitivity was induced by repeated
treatment with oxaliplatin, the response to a thermal stimulus was evaluated by the cold plate test
measuring the latency (s) to painꢀrelated behaviors (lifting or licking of the paw). Tests were
performed on day 15. C2 or vehicle were administered p.o., measurements were performed 15, 30,
45, 60 and 75 min. after treatment. Each point on the graph represents a mean of 16 individual
values obtained from 16 mice analyzed in 2 different experimental sets. ^^P < 0.01 vs vehicle +
vehicle treated mice. *P < 0.05 and **P < 0.01 vs oxaliplatin + vehicle treated mice. ANOVA
followed by Bonferroni test was performed.
Additional experiments were performed to establish if an increase in endocannabinoid levels
induced by a monoacylglycerol lipase (MAGL) inhibitor would potentiate the effect of C2. The
used MAGL inhibitor F (compound 4a in the reference28) was previously reported in literature and
it showed to be efficacious in the same model of neuropathic pain.28 It was administered p.o. at a
minimum active dose of 1 mg kgꢀ1 15 min before compound C2 (used at the completely inactive
dose 1 mg kgꢀ1, and the partially effective dose 10 mg kgꢀ1). The effect observed after the coꢀ
administration of C2 with the MAGL inhibitor, was significantly increased in comparison to that
produced by each compound administered alone (Figure 9). The lowest dose of C2 (1 mg kgꢀ1)
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became able to fully revert oxaliplatinꢀinduced hypersensitivity when administered after the MAGL
inhibitor F (1 mg kgꢀ1). The analysis of synergism performed by the ChouꢀTalalay method29
revealed a combination index (CI; CompuSyn software) 0.20123 considered as “synergism”.
MAGL inhibitor, administered alone at dose 1 mg kgꢀ1 had a partial pain relieving efficacy (Figure
9).
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Figure 9. Neuropathic pain. Effect of C2 administered 15 min after the MAGL inhibitor F.
Measurements were performed 15, 30, 45, 60 and 75 min after the administration (cold plate test).
Control mice were treated with vehicle. Each point on the graph represents a mean of 16 individual
values obtained from 16 mice analyzed in 2 different experimental sets. ^^P < 0.01 vs vehicle +
vehicle treated mice. *P < 0.05 and **P < 0.01 vs oxaliplatin + vehicle treated mice. °°P < 0.01 vs
oxaliplatin + C2 treated mice. ANOVA followed by Bonferroni test was performed.
Finally, the possible predominance of a CBR subtype in the antiꢀneuropathic effect of C2 was
studied. Selective CB1R (SR141716A) and CB2R (SR144528) antagonists were administered i.p. 15
min before C2 (20 mg kgꢀ1 p.o.; Figure 10). The efficacy of C2 was strongly decreased by preꢀ
treating with SR144528 (10 mg kgꢀ1 i.p.), whereas SR141716A only reduced the duration of C2
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effect without affecting its antiꢀhyperalgesic efficacy. A main involvement of CB2Rs is indicated as
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pharmacodynamic mechanism of C2 efficacy against chemotherapyꢀinduced neuropathic pain.
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Figure 10. Neuropathic pain. Effects of CB1R and CB2R antagonism on C2 pain relieving
efficacy. Measurements were performed 15, 30, 45, 60 and 75 min after the administration of C2.
Control mice were treated with vehicle. Each point on the graph represents a mean of 16 individual
values obtained from 16 mice analyzed in 2 different experimental sets. ^^P < 0.01 vs vehicle +
vehicle treated mice. *P < 0.05 and **P < 0.01 vs oxaliplatin + vehicle treated mice. ANOVA
followed by Bonferroni test was performed.
Conclusion
In conclusion, we have presented evidence that compound C2 is the first ever synthesized CB2R
PAM. This compound was obtained from the CBR orthosteric ligands, 2ꢀoxopyridineꢀ3ꢀ
carboxamide, simply by changing its amide side chain into a retroꢀamide side chain.
Derivative C2 behaved as a potent CB2R PAM as indicated by its ability, at 1 nM to 1 µM, to
produce marked increases in the binding of [3H]CP55940 to CB2Rs. At 100 nM, C2 also
significantly enhanced the ability of CP55940 and 2ꢀAG, but not of AEA, to stimulate [35S]GTPγS
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binding to CB2Rs. As expected for an allosteric modulator, C2 did not affect signs of CB2R
receptor signalling in the [35S]GTPγS assay, in the absence of a CB2R agonist. Interestingly, C2
significantly improved the ability of 2ꢀAG, but not of AEA, to stimulate [35S]GTPγS binding to
CB2Rs. Thus, derivative C2 would be able to activate CB2Rs by low concentrations of
endogenouslyꢀreleased 2ꢀAG and also it could potentiate the pharmacological activity of MAGL
inhibitors.
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Although we studied only a small compound library, the obtained results suggest that the C and
D series of compounds display interesting SARs. Undoubtedly, it was the introduction of a retroꢀ
amide sidechain into compounds A and B that converted them into CB2R allosteric modulators (C
and D). Also the bromo substituent in position 5 seems to be important, because its replacement
with an H or with other groups abolished all signs of allosteric activity. Furthermore, the
substitution of the methyl group in position 4 with an OH group (compound E1) reduced signs of
allosteric activity. The display of such activity also seems to require the presence of a substituent at
position 1 of the pyridine ring, as suggested by the finding that compound 3 lacks any detectable
allosteric activity.
The new CB2R PAM, C2, was tested in cold allodynia assays to investigate its effect on
neuropathic pain. Results showed that derivative C2, after oral administration, doseꢀdependently
reversed the lowering of the threshold to cold stimuli (cold plate test) induced by oxaliplatin. As
demonstrated by pharmacological antagonism, this effect is predominantly mediated by the CB2Rs.
Finally, C2 potentiated the antiꢀallodynic effect of the MAGL inhibitor F, which previously
showed to be efficacious in the same model of neuropathic pain.28
Our findings support our hypothesis that compound C2 acts as a CB2R PAM both in vitro and in
vivo. Moreover, C2 could beneficially influence the CNS damages induced by neuropathies.
Several cell types (including microglia, astrocytes and oligodendrocytes) are involved in the
maladaptive plasticity of the nervous tissue that is thought to be the basis of chronic pain states.30
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The widespread expression (for anatomic areas and cell types)31 of the CB2Rs makes its regulation a
powerful therapeutic approach. The antinociceptive effects of C2 need to be further explored as this
compound might be a good candidate for clinical development. Recently, based on a homology
model, an allosteric binding pocket was postulated to exist adjacent to the CB2R orthosteric binding
site.8,14 The CB2R PAM C2 could be useful for the characterization of such putative binding site(s),
a crucial step for the future development of CB2R allosteric modulators. In addition, a better
understanding of the physiological consequences of CB2R allosteric modulation might lead to novel
pharmacotherapies that rely on this pharmacological action.
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EXPERIMENTAL SECTION
Chemistry. Commercially available reagents were purchased from Sigma Aldrich or Alfa Aesar
and used without purification. 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE
III™ 400 spectrometer (operating at 400 MHz). Chemical shift (δ) are reported in parts per million
related to the residual solvent signal, while coupling constants (J) are expressed in Hertz (Hz)
Catalitic hydrogenation was performed with CLAIND H2 generator, type HG2200 (flow meter: 200
ml/min; outlet pressure: 1ꢀ6 bar; operating temperature: 5ꢀ40°C). 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 x 4.6 mm, 5 mm particle size, Gemini). The mobile phase was
constituted by a H2O/AcOH (0.1% v/v) (eluent A) and ACN (eluent B). A linear 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 all the compounds. For the HPLC analysis, all target compounds (i.e., assessed
in biological assays) were ≥95% pure. Evaporation was carried out in vacuo using a rotating
evaporator. Silica gel flash chromatography was performed using silica gel 60 Å (0.040ꢀ0.063 mm;
MERK). Reactions was monitored by TLC on Merck aluminum silica gel (60 F254) plates that
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were visualized under a UV lamp (
λ = 254 nm). Melting points were determined on a Kofler hotꢀ
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stage apparatus and are uncorrected.
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3ꢀAminoꢀ2ꢀhydroxyꢀ4ꢀmethylpyridine (1). Pd/C (100.0 mg) was added to a solution of the
commercially available 2ꢀhydroxyꢀ4ꢀmethylꢀ3ꢀnitropyridine (1.00 g, 6.49 mmol) in methanol. The
reaction mixture was stirred under hydrogen at room temperature for 12 h and then was filtered
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under vacuum using celite. The filtrate was evaporated under reduced pressure to afford the desired
1
compound 1 as solid (0.80 g, 99%). HꢀNMR (CDCl3 δ): 12.36 (bs,1H), 6.57 (d, 1H,
J
=7.2 Hz),
5.88 (d, 1H, J=7.2 Hz), 3.97 (bs, 2H), 2.25 (s, 3H).
Nꢀ(1,2ꢀdihydroꢀ4ꢀmethylꢀ2ꢀoxopyridinꢀ3ꢀyl)cycloheptanecarboxamide
(2).
Cycloheptanecarboxylic acid (0.700 ml, 4.99 mmol) was dissolved in SOCl2 (2.60 ml, 36.5 mmol)
in a vial under nitrogen flux. The solution was stirred at 76 °C for 3 hours and then the excess of
SOCl2 was removed by evaporation under nitrogen flux. The obtained acyl chloride was added
dropwise to a solution of the amino derivative 1 (310 mg, 2.50 mmol) and triethylamine (0.700 ml,
4.99 mmol) in anhydrous toluene (46.5 ml) and anhydrous DMF (6.20 ml) in a round bottom flask
at 0 °C. The reaction mixture was stirred at room temperature for 48 hours. Then the solvent was
removed under reduced pressure to give a residue which was dissolved in CHCl3 and washed three
times with water. Subsequently, the organic phase was dried over Na2SO4, filtered and evaporated
under reduced pressure. The brown solid obtained was purified by flash chromatography on silica
gel (ethyl acetate, 2% acetic acid) to give a solid which was triturated in hexane to afford 2 as white
1
solid (372 mg, 60%). Mp: dec. >150 °C (crystallized from hexane). HꢀNMR (CDCl3 δ): 7.62 (bs,
1H), 7.10 (d, 1H,
2H), 1.81ꢀ1.71 (m, 4H), 1.59ꢀ1.53 (m, 6H).
ꢀ(5ꢀbromoꢀ1,2ꢀdihydroꢀ4ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl)cycloheptanecarboxamide (3).
J=6.4 Hz), 6.26 (d, 1H, J=6.8 Hz), 2.53ꢀ2.48 (m, 1H), 2.16 (s, 3H), 2.02ꢀ1.98 (m,
N
A
solution of Br2 (0.11 ml, 2.08 mmol) in CHCl3 (2.1 ml) was added dropwise to a solution of the
compound 2 (518 mg, 2.08 mmol) in CHCl3 (3.5 ml) at room temperature. Reaction mixture was
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stirred at room temperature overnight and then was washed four times with a saturated aqueous
solution of Na2S2O3. The organic phase was dried over Na2SO4, filtered and evaporated under
reduced pressure. The solid residue was crystallized from hexane to give 3 as a white solid (578 mg,
85%). Mp: 217ꢀ219 °C. 1HꢀNMR (CDCl3 δ): 7.40 (bs, 1H), 7.39 (s, 1H), 2.56ꢀ2.49 (m, 1H), 2.21 (s,
3H), 2.06ꢀ2.00 (m, 2H), 1.85ꢀ1.73 (m, 4H), 1.61ꢀ1.55 (m, 6H). 13CꢀNMR (CDCl3 δ): 176.32,
158.04, 134.75, 128.17, 127.41, 100.81, 48.21, 31.57, 28.40, 26.47, 19.68. HPLC analysis: retention
time = 5.1 min; peak area, 95.5% (280 nm).
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Nꢀ(5ꢀchloroꢀ1,2ꢀdihydroꢀ4ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl)cycloheptanecarboxamide (4).
A
solution of ꢀchlorosuccinimide (270 mg, 2.02 mmol) in acetonitrile (2.0 mL) was added to a
N
suspension of the derivative 2 (500 mg, 2.02 mmol) in acetonitrile (3.0 mL). The reaction mixture
was refluxed overnight. Then, the solvent was removed under reduced pressure and the residue was
treated with water and extracted with ethyl acetate. The organic phase was dried over Na2SO4,
filtered and concentrated under reduced pressure to give a yellow solid purified by flash
chromatography on silica gel using ethyl acetate/petroleum ether 7:3 and 1% acetic acid as eluent,
to give a yellow solid which was triturated in hexane to afford compound 4 as white solid (274 mg,
1
48%). Mp: 200 °C (dec), (crystallized from hexane). HꢀNMR (CDCl3 δ): 8.73 (bs, 1H), 7.52 (s,
1H), 7.29 (bs, 1H), 2.54 (m, 1H), 2.18 (s, 3H), 2.04ꢀ2.01 (m, 2H), 1.83ꢀ1.73 (m, 4H), 1.66ꢀ1.49 (m,
6H).
3ꢀaminoꢀ2ꢀhydroxyꢀ6ꢀmethylpyridine (5). Compound 5 was prepared as described for
1
compound 1, starting from 2ꢀhydroxyꢀ6ꢀmethylꢀ3ꢀnitropyridine. Yield: 99%. HꢀNMR (CDCl3 δ):
12.20 (bs, 1H), 6.78 (d, 1H, J=6.8 Hz), 6.09 (d, 1H, J=6.8 Hz), 4.03 (bs, 2H), 2.10 (s, 3H).
Nꢀ(1,2ꢀdihydroꢀ6ꢀmethylꢀ2ꢀoxopyridinꢀ3ꢀyl)cycloheptanecarboxamide (6). A solution of
cycloheptanecarboxylic acid (0.870 ml, 6.32 mmol) in anhydrous DMF (15.70 ml) was stirred in an
iceꢀbath. TBTU (2440 mg, 7.59 mmol) and triethylamine (2.40 ml, 17.70 mmol) were added at 0
°C. After 30 minutes, compound 5 (785 mg, 6.32 mmol) was added and the reaction mixture was
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stirred at 0 °C for 30 minutes and then at room temperature overnight. The solvent was removed
under reduced pressure. The solid residue was dissolved in CHCl3 and washed with water. The
organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure. The brown
solid thus obtained was purified by flash chromatography on silica gel using ethyl acetate/petroleum
ether 8:2 as eluent to give a yellow solid which was crystallized from hexane to afford compound 6
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1
as white solid (1.290 g). Yield: 82 %. Mp: 182ꢀ185 °C. HꢀNMR (CDCl3 δ): 11.77 (bs, 1H), 8.39
(d, 1H,
(m, 2H), 1.83ꢀ1.71 (m, 4H), 1.62ꢀ1.49 (m, 6H).
ꢀ(5ꢀbromoꢀ1,2ꢀdihydroꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl)cycloheptanecarboxamide
J=7.6 Hz), 8.16 (bs, 1H), 6.09 (d, 1H, J=7.6 Hz), 2.47ꢀ2.44 (m, 1H), 2.32 (s, 3H), 2.01ꢀ1.95
N
(7).
Compound 7 was prepared starting from derivative 6, as described for compound 3 and it was
purified by flash chromatography on silica gel using ethyl acetate/petroleum ether 7:3. Yield: 89%.
1HꢀNMR (CDCl3 δ): 12.25 (bs, 1H), 8.64 (s, 1H), 8.10 (s, 1H), 2.50ꢀ2.41 (m, 1H), 2.01 (s, 3H),
1.75ꢀ1.61 (m, 6H), 1.84ꢀ1.76 (m, 4H), 1.99ꢀ1.94 (m, 2H).
N
ꢀ(5ꢀchloroꢀ1,2ꢀdihydroꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl)cycloheptanecarboxamide
(8).
Compound 8 was prepared starting from derivative 6, as described for compound 4 and it was
purified by flash chromatography on silica gel using ethyl acetate/petroleum ether 1:1, Yield: 19%.
Mp: 240ꢀ246 °C (crystallized from hexane). 1HꢀNMR (CDCl3 δ): 11.99 (bs, 1H), 8.55 (s, 1H), 8.11
(bs, 1H), 2.50ꢀ2.48 (m, 1H), 2.39 (s, 3H), 2.01ꢀ1.96 (m, 2H), 1.85ꢀ1.81 (m, 4H), 1.79ꢀ1.54 (m, 6H).
N
ꢀ(1,2ꢀdihydroꢀ5ꢀfluoroꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl)cycloheptanecarboxamide (9). 1ꢀ
chloromethylꢀ4ꢀfluoroꢀ1,4ꢀdiazoniabicyclo[2.2.2] octanebis(tetrafluoroborate) (Selectfluor®)(1.200
g, 3.39 mmol) was added at room temperature to a suspension of compound 6 (700 mg, 2.82 mmol)
in acetonitrile. Reaction mixture was refluxed for 12 hours. Then, the solvent was removed under
reduced pressure and the residue was treated with ethyl acetate and washed three times with water.
The organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure. The
black solid thus obtained was purified by flash chromatography on silica gel using petroleum
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ether/ethyl acetate 1:1 as eluent, to give compound 9 (52.0 mg, 0.196 mmol) as a black solid. Yield:
4
5
1
7% Mp: 95ꢀ98 °C (crystallized from hexane). HꢀNMR (CDCl3 δ): 8.48 (d, 1H,
J
=10.4 Hz), 8.16
6
7
8
9
(bs, 1H), 2.51ꢀ2.46 (m, 1H), 2.30 (d, 3H,
1.53 (m, 6H).
J=2.8 Hz), 2.01ꢀ1.95 (m, 2H), 1.81ꢀ1.75 (m, 4H), 1.62ꢀ
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3ꢀaminoꢀ2,4ꢀdihydroxypyridine (10). Compound 10 was prepared starting from 2,4ꢀdihydroxyꢀ
3ꢀnitropyridine, as described for compound 1. Yield: 33%. 1HꢀNMR (MeOD δ): 12.26 (bs,1H), 6.84
(d, 1H,
J= 7.2 Hz); 6.13 (d, 1H, J= 7.2 Hz), 4.03 (bs, 2H).
Nꢀ(1,2ꢀdihydroꢀ4ꢀhydroxyꢀ2ꢀoxopyridinꢀ3ꢀyl)cycloheptanecarboxamide (11). Compound 11
was prepared starting from derivative 10, as described for compound 2 and it was purified by
trituration with MeOH and then with Et2O. Yield: 25%. Mp: dec. >130 °C (crystallized from
hexane). 1HꢀNMR (CDCl3 δ): 12.71 (bs, 1H); 11.28 (bs, 1H); 8.39 (s, 1H), 7.03 (d, 1H,
J
= 7.2 Hz),
6.08 (d, 1H,
6H).
J= 7.2 Hz) 2.58ꢀ2.57 (m, 1H), 2.02ꢀ1.98 (m, 2H), 1.83ꢀ1.72 (m, 4H), 1.65ꢀ1.50 (m,
Nꢀ(5ꢀbromoꢀ1,2ꢀdihydroꢀ4ꢀhydroxyꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl)cycloheptanecarboxamide
(12).
Compound 12 was prepared starting from derivative 11, as described for compound 3 and it was
purified by flash chromatography on silica gel using ethyl acetate/petroleum ether 7:3. Yield: 68 %.
1HꢀNMR (CDCl3 δ): 8.34 (bs, 1H), 7.35 (s, 1H), 2.60ꢀ2.55 (m, 1H), 2.02ꢀ1.98 (m, 2H), 1.83ꢀ1,71
(m, 4H), 1.61ꢀ1.51 (m, 6H).
Nꢀ[1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ4ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3yl]cycloheptanecarboxamide
(C1). An amount of 214.0 mg of CsF (1.41 mmol) was added to a solution of of 1,2ꢀdihydroꢀ
pyridinꢀ3ꢀcycloheptanecarboxamide 2 (117.0 mg, 0.470 mmol) in anhydrous DMF (1.40 ml). After
1 hour at room temperature, 4ꢀfluorobenzyl chloride (0.17 ml, 1.41 mmol) was added dropwise and
the mixture was stirred at 50 °C overnight. After cooling, the reaction mixture was concentrated
under reduced pressure, treated with iceꢀwater and then extracted with CH2Cl2. The organic phase
was dried over Na2SO4, filtered and evaporated under reduced pressure. The yellow oil obtained
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was purified by flash chromatography on silica gel using petroleum ether/ethyl acetate 1:1), to give
4
5
1
C1 as white solid (67.0 mg, 40%). Mp: 160ꢀ165 °C (crystallized from hexane). HꢀNMR: (CDCl3
6
7
8
9
δ): 7.43 (bs, 1H), 7.28ꢀ7.25 (m, 2H), 7.04ꢀ7.00 (m, 3H), 6.10 (d, 1H,
J=7.2 Hz), 5.07 (s, 2H), 2.53ꢀ
2.46 (m, 1H), 2.12 (s, 3H), 2.07ꢀ2.00 (m, 2H), 1.83ꢀ1.72 (m, 4H), 1.59ꢀ1.52 (m, 6H). 13CꢀNMR:
10
11
12
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(CDCl3 δ): 176.06, 162.65 (d,
(d, =9.1 Hz), 125.85, 115.95 (d,
HPLC analysis: retention time = 8.6 min; peak area 99.7% (280 nm).
ꢀ[5ꢀbromoꢀ1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ4ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ
J
=246.0 Hz), 159.56, 142.40, 132.04 (d,
J=3.2 Hz), 131.67, 130.02
J
J
=22.0 Hz), 110.21, 51.99, 48.04, 31.92, 28.27, 26.77, 19.83.
N
3yl]cycloheptanecarboxamide (C2). Compound C2 was prepared starting from derivative 3, as
described for compound C1 and it was purified by flash chromatography on silica gel using
petroleum ether/ethyl acetate 7:3 as eluent. Yield: 58% Mp: 167ꢀ170 °C (crystallized from hexane).
1HꢀNMR (CDCl3 δ): 7.48 (bs, 1H), 7.33 (s, 1H), 7.30ꢀ7.28 (m, 2H), 7.06ꢀ7.02 (m, 2H), 5.05 (s, 2H),
13
2.53ꢀ2.47 (m, 1H), 2.15 (s, 3H), 2.06ꢀ1.99 (m, 2H), 1.81ꢀ1.71 (m, 4H), 1.59ꢀ1.52 (m, 6H). Cꢀ
NMR (CDCl3 δ): 176.08, 162.77 (d,
130.21 (d, =9.1 Hz), 126.60, 116.12 (d,
20.67. HPLC analysis: retention time = 9.5 min; peak area 95.5 % (280 nm).
ꢀ[1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ5ꢀ(4’ꢀmethoxyphenyl)ꢀ4ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
J
=246.0 Hz), 158.42, 142.07, 132.10, 131.34 (d,
J=4.1 Hz),
J
J
=22.0 Hz), 104.08, 52.06, 47.94, 31.81, 28.22, 26.70,
N
yl]cycloheptanecarboxamide (C3). A mixture of Pd(OAc)2 (2.20 mg, 0.009 mmol) and PPh3
(11.80 mg, 0.045 mmol) in 2.50 mL of toluene was stirred under nitrogen at room temperature.
After 15 minutes, the suitable boronic acid (37.9 mg, 0.600 mmol), K2CO3 (62,2 mg, 0.450 mmol)
and compound C2 (130.6 mg, 0.300 mmol) were added. Reaction mixture was stirred at 110 °C
overnight. After cooling, the reaction was filtered under vacuum using celite, and the filtrate was
evaporated under reduced pressure. The residue was treated with water and extracted with CHCl3.
The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and
evaporated to give a residue which was purified by flash chromatography on silica gel using
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petroleum ether/ethyl acetate 8:2 as eluent affording C3 as oil (100 mg, 0.216 mmol). Yield: 72%.
4
5
1HꢀNMR (CDCl3 δ): 7.57 (bs, 1H), 7.32ꢀ7.29 (m, 2H), 7.17ꢀ7.15 (m, 2H), 7.05ꢀ7.00 (m, 3H), 6.92ꢀ
6
7
8
9
6.90 (m, 2H), 5.12 (s, 2H), 3.83 (s, 3H), 2.56ꢀ2.51 (m, 1H), 2.07ꢀ2.02 (m, 2H), 1.98 (s, 3H), 1.81ꢀ
13
1.76 (m, 4H), 1.60ꢀ1.58 (m, 6H). CꢀNMR (CDCl3 δ): 176.15, 162.18 (d,
J
=245.0 Hz), 159.26,
=8.6 Hz), 129.05, 125.85,
=22.0 Hz), 113.95, 55.42, 52.00, 47.89, 31.88, 28.26, 26.70, 18.50. HPLC
analysis: retention time = 13.2 min; peak area 98.2 % (280 nm).
ꢀ[1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ4ꢀmethylꢀ2ꢀoxoꢀ5ꢀphenylꢀpyridinꢀ3ꢀ
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158.70, 142.25, 135.89, 131.97 (d,
123.46, 115.92 (d,
J=32 Hz), 130.92, 130.82, 130.07 (d, J
J
N
yl]cycloheptanecarboxamide (C4). Compound C4 was prepared from compound C2, as described
for compound C3 and it was purified by crystallization from hexane. Yield: 45%. Mp: 143ꢀ146 °C
(crystallized from hexane). 1HꢀNMR: (CDCl3 δ): 7.54 (bs, 1H), 7.41ꢀ7.29 (m, 5H), 7.25ꢀ7.24 (m,
2H), 7.05ꢀ7.01 (m, 3H), 5.12 (s, 2H), 2.56ꢀ2.51 (m, 1H), 2.07ꢀ2.02 (m, 2H), 1.99 (s, 3H), 1.83ꢀ1.77
(m, 4H), 1.60ꢀ1.53 (m, 6H). 13CꢀNMR (CDCl3 δ): 176.05, 162.58 (d,
J
=243.8 Hz), 158.72, 141.85,
=8.6 Hz), 129.67, 128.52, 127.72, 126.02,
=21.8 Hz), 51.98, 47.81, 31.85, 28.25, 26.67, 18.41. HPLC analysis: retention
136.78, 131.93 (d,
123.77, 115.89 (d,
J=3.2 Hz), 131.00, 130.04 (d, J
J
time = 13.5 min; peak area 99.6 % (280 nm).
ꢀ[5ꢀchloroꢀ1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ4ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
N
yl]cycloheptanecarboxamide (C5). Compound C5 was prepared from compound 4, as described
for compound C1 and it was purified by flash chromatography on silica gel using petroleum
ether/ethyl acetate 7:3 as eluent to give a light yellow solid which was triturated in hexane to afford
1
C5 as white solid. Yield: 39%. Mp: 197ꢀ199 °C (crystallized from hexane). HꢀNMR (CDCl3 δ):
7.49 (bs, 1H), 7.30ꢀ7.27 (m, 2H), 7.21 (s, 1H), 7.06ꢀ7.02 (m, 2H), 5.06 (s, 2H), 2.53ꢀ2.48 (m, 1H),
2.14 (s, 3H), 2.06ꢀ2.00 (m, 2H), 1.81ꢀ1.74 (m, 4H), 1.58ꢀ1.51 (m, 6H). 13CꢀNMR (CDCl3 δ):
176.06, 162.72 (d,
J
=246.0 Hz), 158.24, 141.17, 131.33 (d, J=3.2 Hz), 130.17 (d, J=8.6 Hz),
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129.62, 126.64, 116.05 (d,
J
=22.0 Hz), 115.75, 52.01, 47.83, 31.77, 28.19, 26.66, 17.86. HPLC
4
5
6
7
analysis: retention time = 11.6 min; peak area 96.8 % (280 nM).
ꢀ[1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3yl]cycloheptanecarboxamide
N
8
9
(D1). An amount of 35.2 mg (0.879 mmol) of NaH (60% mineral oil dispersion) was added
portionwise to a solution of 1,2ꢀdihydropyridinꢀ3ꢀcycloheptanecarboxamide 6 (212 mg, 0.854
mmol) in DME (1.700 ml) and anhydrous DMF (0.400 ml). After 10 minutes at 0 °C, LiBr (147.6
mg, 1.700 mmol) was added and the reaction mixture was stirred at room temperature for 15
minutes. After that, 4ꢀfluorobenzylchloride (0.200 ml, 1.650 mmol) was added dropwise and the
mixture was stirred at 65 °C overnight. After cooling, the solvent was removed under reduced
pressure and the obtained residue was dissolved in CHCl3 and washed with brine. The organic
phase was dried over Na2SO4, filtered and evaporated under reduced pressure to obtain a residue
which was purified by flash chromatography on silica gel using petroleum ether/ethyl acetate 8:2 as
eluent giving D1 as oil (79.1 mg, 0.220 mmol). Yield: 26% 1HꢀNMR (CDCl3 δ): 8.31 (bs, 1H), 8.30
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(d, 1H,
J=7.6 Hz), 7.11ꢀ7.08 (m, 2H), 6.99ꢀ6.96 (m, 2H), 6.09 (d, 1H, J=7.6 Hz), 5.31 (s, 2H), 2.44ꢀ
2.43 (m, 1H), 2.26 (s, 3H), 1.99ꢀ1.95 (m, 2H), 1.79ꢀ1.70 (m, 4H), 1.58ꢀ1.48 (m, 6H). 13CꢀNMR
(CDCl3 δ): 176.44, 162.27 (d, J=245.0 Hz), 158.75, 137.97, 131.86, 128.25 (d, J=8.6 Hz), 127.36,
122.34, 115.93 (d, J=22.1 Hz), 107.32, 48.58, 47.62, 31.69, 28.31, 26.64, 20.14. HPLC analysis:
retention time = 13.2 min; peak area 97.1 % (280 nm).
Nꢀ[5ꢀbromoꢀ1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
y]cycloheptanecarboxamide (D2). Compound D2 was prepared from derivative 7, as described for
compound D1 and it was purified by flash column chromatography on silica gel using petroleum
ether/ethyl acetate 8:2 as eluent. Yield: 40%. Mp: 124ꢀ127 °C (crystallized from hexane). 1HꢀNMR
(CDCl3 δ): 8.61 (s, 1H), 8.31 (bs, 1H), 7.12ꢀ7.09 (m, 2H), 7.04ꢀ7.00 (m, 2H), 5.39 (s, 2H), 2.48ꢀ
13
2.43 (m, 1H), 2.42 (s, 3H), 2.02ꢀ1.94 (m, 2H), 1.82ꢀ1.71 (m, 4H), 1.63ꢀ1.54 (m, 6H). CꢀNMR
(CDCl3 δ): 176.33, 162.19 (d,
J
=246.0 Hz), 157.76, 135.64, 131.20 (d, J=3.2 Hz), 128.05 (d, J=8.1
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Hz), 127.46, 125.78, 115.91 (d,
J
=22.0 Hz), 101.53, 48.77, 48.23, 31.42, 28.18, 26.39, 19.49.
4
5
6
7
HPLC analysis: retention time = 17.6 min; peak area 98.1 % (280 nM).
ꢀ[1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ5ꢀphenylꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀyl]cycloheptane
N
8
9
carboxamide (D3). Compound D3 was prepared starting from derivative D2, as described for
compound C3 and it was purified by flash column chromatography on silica gel using petroleum
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ether/ethyl acetate 8:2 as eluent. Yield: 13%. HꢀNMR (CDCl3 δ): 8.45 (s, 1H), 8.38 (bs, 1H),
7.38−7.28 (m, 4H), 7.25ꢀ7.23 (m, 3H), 7.17ꢀ7.01 (m, 2H), 5.44 (s, 2H), 2.46−2.43 (m, 1H), 2.24 (s,
3H), 2.01−1.95 (m, 2H), 1.79−1.71 (m, 4H), 1.59−1.49 (m, 6H). 13CꢀNMR (CDCl3 δ): 176.47,
162.29 (d,
127.43, 126.75, 125.23, 121.35, 115.99 (d,
HPLC analysis: retention time = 18.0 min; peak area 95.8 % (280 nm).
ꢀ[1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ5ꢀ(4’ꢀmethoxyphenyl)ꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
J
=244.0 Hz), 158.22, 139.27, 135.05, 132.00, 129.84, 128.53, 128.25 (d,
J=8.6 Hz),
J
=22.0 Hz), 48.46, 48.16, 31.68, 28.42, 26.60, 17.56.
N
yl]cycloheptanecarboxamide (D4). Compound D4 was prepared starting from compound D2, as
described for compound C3 and it was purified by flash column chromatography on silica gel using
petroleum ether/ethyl acetate 8:2 as eluent. Yield: 19%. 1HꢀNMR (CDCl3 δ): 8.43 (s, 1H), 8.38 (bs,
1H), 7.17ꢀ7.13 (m, 4H), 7.05ꢀ7.01 (m, 2H), 6.90ꢀ6.88 (m, 2H), 5.43 (s, 2H), 3.83 (s, 3H), 2.48ꢀ2.43
13
(m, 1H), 2.23 (s, 3H), 2.00ꢀ1.97 (m, 2H), 1.80ꢀ1.74 (m, 4H), 1.52ꢀ1.50 (m, 6H). CꢀNMR (CDCl3
δ): 176.43, 162.24 (d,
(d, =8.6 Hz), 126.61, 125.47, 120.95, 115.94 (d,
28.38, 26.58, 17.53. HPLC analysis: retention time = 17.3 min; peak area, 99.0 % (280 nm).
ꢀ[5ꢀchloroꢀ1,2ꢀdihydroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
J
=244.0 Hz), 158.93, 158.14, 135.03, 131.99 (d,
J=3.2 Hz), 130.89, 128.22
J
J
=21.8 Hz), 113.90, 55.42, 48.44, 48.16, 31.65,
N
yl]cycloheptanecarboxamide (D5). Compound D5 was prepared starting from compound 8, as
described for compound D1 and it was purified by crystallization from hexane. Yield: 36%. Mp:
1
116ꢀ119 °C (crystallized from hexane). HꢀNMR (CDCl3 δ): 8.49 (s, 1H), 8.33 (bs, 1H), 7.12ꢀ7.09
(m, 2H), 7.04ꢀ7.00 (m, 2H), 5.37 (s, 2H), 2.46ꢀ2.43 (m, 1H), 2.38 (s, 3H), 2.04ꢀ1.94 (m, 2H), 1.81ꢀ
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1.71 (m, 4H), 1.61ꢀ1.48 (m, 6H). CꢀNMR (CDCl3 δ): 176.39, 162.24 (d,
J
=245.0 Hz), 157.56,
=22.0 Hz), 113.35,
134.35, 131.26 (d, =3.2 Hz), 128.11 (d, =8.1 Hz), 127.37, 123.48, 115.95 (d, J
J
J
6
7
8
9
48.50, 48.30, 31.47, 28.21, 26.44, 16.68. HPLC analysis: retention time = 16.9 min; peak area 98.2
% (280 nm).
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N
ꢀ[1,2ꢀdihydroꢀ5ꢀfluoroꢀ1ꢀ(4’ꢀfluorobenzyl)ꢀ6ꢀmethylꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
yl]cycloheptanecarboxamide (D6). Compound D6 was prepared starting from compound 9, as
described for compound D1, and it was purified by flash column chromatography on silica gel
1
using petroleum ether/ethyl acetate 8:2. Yield: 24%. HꢀNMR (CDCl3 δ): 8.43 (d, 1H,
J
=10.4 Hz),
8.40 (bs, 1H), 7.14ꢀ7.11 (m, 2H), 7.04ꢀ7.00 (m, 2H), 5.32 (s, 2H), 2.48ꢀ2.43 (m, 1H), 2.25 (d, 3H,
13
J
=3.2 Hz), 2.05ꢀ1.95 (m, 2H), 1.80ꢀ1.74 (m, 4H), 1.55ꢀ1.52 (m, 6H). CꢀNMR (CDCl3 δ): 176.54,
162.33 (d,
J
=245.0 Hz), 156.74, 145.52 (d,
J
=224.2 Hz), 131.48 (d,
J
=3.2 Hz), 128.30 (d,
J=8.6
Hz), 127.23 (d,
J
=13.0 Hz), 123.12 (d, =29.1 Hz), 116.02 (d,
J
J
=21.0 Hz), 114.34 (d, J
=32.4 Hz),
48.45, 47.78, 31.56, 28.28, 26.56, 11.78. HPLC analysis: retention time = 15.1 min; peak area 96.3
% (280 nm).
Nꢀ(5ꢀbromoꢀ1,2ꢀdihydroꢀ1ꢀ(4ꢀfluorobenzyl)ꢀ4ꢀhydroxyꢀ2ꢀoxoꢀpyridinꢀ3ꢀ
yl)cycloheptanecarboxamide (E1). Compound E1 was prepared from compound 12, as described
for compound C1 and it was purified by flash column chromatography on silica gel using petroleum
1
ether/ethyl acetate 8:2 as eluent. Yield: 8%. Mp: 73ꢀ76 °C (crystallized from hexane). HꢀNMR
(CDCl3 δ): 13.46 (bs, 1H), 8.51 (bs, 1H), 7.30 (s, 1H), 7.28ꢀ7.24 (m, 2H), 7.08ꢀ7.03 (m, 2H), 5.06
(s, 2H), 2.61ꢀ2.52 (m, 1H), 2.05ꢀ1.97 (m, 2H), 1.85ꢀ1.68 (m, 4H), 1.62ꢀ1.44 (m, 6H). 13CꢀNMR
(CDCl3 δ): 178.70, 161.56, 132.50, 131.40, 129.94 (d, J=8.6 Hz), 116.19 (d, J=21.8 Hz), 112.40,
97.30, 51.88, 47.72, 31.85, 28.09, 26.50. HPLC analysis: retention time = 15.6 min; peak area
95.8% (280 nM).
Biological Evaluations
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Page 29 of 39
Journal of Medicinal Chemistry
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CHO cells. CHO cells stably transfected with cDNA encoding human cannabinoid CB1Rs or
CB2Rs were maintained at 37°C and 5% CO2 in Gibco™ Ham’s Fꢀ12 Nutrient Mix supplied by
Fisher Scientific UK Ltd that was supplemented both with 2 mM Lꢀglutamine, 10% FBS and 0.6%
penicillinꢀstreptomycin, all also supplied by Fisher Scientific UK Ltd, and with the disulphate salt
of G418 [(2R,3S,4R,5R,6S)ꢀ5ꢀaminoꢀ6ꢀ{[(1R,2S,3S,4R,6S)ꢀ4,6ꢀdiaminoꢀ3ꢀ{[(2R,3R,4R,5R)ꢀ3,5ꢀ
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dihydroxyꢀ
5ꢀmethylꢀ4ꢀ(methylamino)oxanꢀ2ꢀyl]oxy}ꢀ2ꢀhydroxy
cyclohexyl]oxy}ꢀ2ꢀ[(1R)ꢀ1ꢀ
hydroxyethyl]oxaneꢀ3,4ꢀdiol; 600 mg·mLꢀ1] supplied by SigmaꢀAldrich UK. All cells were exposed
to 5% CO2 in their respective media, and were passaged twice a week using nonꢀenzymatic cell
dissociation solution. For membrane preparation, cells were removed from flasks by scraping,
centrifuged, and then frozen as a pellet at ꢀ20 °C until required. Before use in a radioligand binding
assay, cells were defrosted, diluted in Tris buffer (50 mM Tris–HCl and 50 mM Tris–base).
Radioligand displacement assay. The assays were carried out with [3H]CP55940 and Tris
binding buffer (50 mM Tris–HCl, 50 mM Tris–base, 0.1% BSA, pH 7.4), total assay volume 500
ꢁl. Binding was initiated by the addition of transfected human CB1 or CB2 CHO cell membranes
(50 ꢁg protein per well). All assays were performed at 37 °C for 60 min before termination by the
addition of iceꢀcold Tris binding buffer, followed by vacuum filtration using a 24ꢀwell sampling
manifold (Brandel Cell Harvester; Brandel Inc, Gaithersburg, MD, USA) and Brandel GF/B filters
that had been soaked in wash buffer at 4°C for at least 24 h. Each reaction well was washed six
times with a 1.2 mL aliquot of Trisꢀbinding buffer. The filters were ovenꢀdried for 60 min and then
placed in 3 mL of scintillation fluid (Ultima Gold XR, PerkinElmer, Seer Green, Buckinghamshire,
UK). Radioactivity was quantified by liquid scintillation spectrometry. Specific binding was
defined as the difference between the binding that occurred in the presence and absence of 1 ꢁM
unlabelled CP55940. The concentration of [3H]CP55940 used in our displacement assays was 0.7
nM. The compounds used in this investigation were stored as stock solutions of 10 mM in DMSO,
the vehicle concentration in all assay wells being 0.1% DMSO. Each point on each graph represents
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