1,2-Dihydro-2-oxopyridine-3-carboxamides: The C-5 substituent is responsible for functionality switch at CB2 cannabinoid receptor

Article abstract of DOI:10.1016/j.ejmech.2013.10.070

The relevance of CB2R-mediated therapeutic effects is well-known for the treatment of inflammatory and neuropathic pain and neurodegenerative disorders. In our search for new cannabinoid receptor modulators, we report the optimization of a series of 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives as CB2R ligands. In particular, N-cycloheptyl-5-(4-methoxyphenyl)-1-(4- fluorobenzyl)-pyridin-2(1H)-on-3-carboxamide (17) showed high CB2R affinity (K_i = 1.0 nM), accompanied by interesting K_i(CB1R)/K _i(CB2R) selectivity ratio (SI = 43.4). Compound 17 was also identified as a potent CB2R neutral antagonist/weak partial inverse agonist. Finally we found that the functionality activity of the series of 1,2-dihydro-2-oxopyridine is controlled by the presence of a substituent in position 5 of the heterocyclic nucleus. In fact when the hydrogen atom in position 5 of the unsubstituted compound 1 was replaced with a phenyl group (compound 18) the CB2R activity was shifted from agonism to inverse agonism whereas the introduction in the same position of p-methoxyphenyl group lead to compound 17 which showed a behavior as CB2R neutral antagonist/weak partial inverse agonist.

Full text of DOI:10.1016/j.ejmech.2013.10.070

European Journal of Medicinal Chemistry 74 (2014) 524e532
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,2-Dihydro-2-oxopyridine-3-carboxamides: The C-5 substituent is
responsible for functionality switch at CB2 cannabinoid receptor
,
c
,
c
d
Valentina Lucchesi a, Teija Parkkari b
, Juha R. Savinainen , Anna Maria Malfitano ,
**
Marco Allarà f, Simone Bertini a, Francesca Castelli a, Sara Del Carlo a, Chiara Laezza e,
Alessia Ligresti f, Giuseppe Saccomanni a, Maurizio Bifulco d, Vincenzo Di Marzo f,
Marco Macchia a, Clementina Manera a
,
*
a Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b University of Eastern Finland, Faculty of Health Sciences, School of Pharmacy, PO Box 1627, 70211 Kuopio, Finland
c University of Eastern Finland, Faculty of Health Sciences, Institute of Biomedicine, School of Medicine, PO Box 1627, 70211 Kuopio, Finland
d Dipartimento di Scienze Farmaceutiche, Università di Salerno, 84084 Fisciano, SA, Italy
e Institute of Endocrinology and Experimental Oncology, IEOS, CNR, Naples, Italy
f Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The relevance of CB2R-mediated therapeutic effects is well-known for the treatment of inflammatory
and neuropathic pain and neurodegenerative disorders. In our search for new cannabinoid receptor
modulators, we report the optimization of a series of 1,2-dihydro-2-oxopyridine-3-carboxamide de-
rivatives as CB2R ligands. In particular, N-cycloheptyl-5-(4-methoxyphenyl)-1-(4-fluorobenzyl)-pyridin-
2(1H)-on-3-carboxamide (17) showed high CB2R affinity (Ki ¼ 1.0 nM), accompanied by interesting
Ki(CB1R)/Ki(CB2R) selectivity ratio (SI ¼ 43.4). Compound 17 was also identified as a potent CB2R neutral
antagonist/weak partial inverse agonist. Finally we found that the functionality activity of the series of
1,2-dihydro-2-oxopyridine is controlled by the presence of a substituent in position 5 of the heterocyclic
nucleus. In fact when the hydrogen atom in position 5 of the unsubstituted compound 1 was replaced
with a phenyl group (compound 18) the CB2R activity was shifted from agonism to inverse agonism
whereas the introduction in the same position of p-methoxyphenyl group lead to compound 17 which
showed a behavior as CB2R neutral antagonist/weak partial inverse agonist.
Received 11 July 2013
Received in revised form
30 September 2013
Accepted 28 October 2013
Available online 21 January 2014
Keywords:
Cannabinoid
CB1 receptor
CB2 receptor
CB2 receptor neutral antagonist
CB2 receptor inverse agonist
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
responsible for most of the overt pharmacological effects of can-
nabinoids [10,11]. Although CB2R was originally identified from
The endocannabinoid system (ECS) consists of two cannabinoid
receptors, a putative transporter, endogenous ligands, and diverse
enzymes involved in the synthesis and degradation of endocanna-
binoids [1]. An increasing body of evidence implicates a role of the
ECS in a variety of physiological and pathophysiological conditions,
including immunomodulation, metabolic regulation, bone growth,
pain, cancer, and psychiatric disorders [2e8]. Two cannabinoid re-
ceptors (CBRs), CB1R and CB2R, have been identified to date as G-
protein-coupled receptors (GPCRs) [9]. CB1R is found predomi-
nantly in the central nervous system and is thought to be
macrophages present in the spleen and was initially considered to
be expressed primarily by the immune system, it is now well
accepted that CB2R is expressed by activated microglia and other
macrophages in the brain [12e14]. As CB1R probably mediates most,
if not all, of the psychoactive effects of cannabinoids [15], CB2R se-
lective ligands are attractive as therapeutics because they would
presumably lack this psychoactivity [16,17]. Several studies have
found that selective CB2R agonists may exhibit anti-inflammatory
and analgesic properties in animal models [6,18,19], and they
could be useful for the treatment of neurodegenerative diseases,
including Alzheimer’s and Parkinson’s disease [1,20], amyotrophic
lateral sclerosis [21], and Huntington’s disease [22]. Furthermore,
CB2R agonists could have cardio-protective effects and be effective
in the treatment of cancer, e.g. gliomas [23e26]. Fewer compounds
have been described as selective antagonists/inverse agonists. An-
tagonists/inverse agonists may possess anti-inflammatory activity
[27], and may inhibit osteoclast formation and activity in vitro
* Corresponding author. Tel.: þ39 (0)502219548; fax: þ39 (0)502219605.
** Corresponding author. University of Eastern Finland, Faculty of Health Sciences,
School of Pharmacy, PO Box 1627, 70211 Kuopio, Finland. Tel.: þ358 (0)403553885;
fax: þ358 (0)17162424.
E-mail addresses: Teija.Parkkari@uef.fi (T. Parkkari), manera@farm.unipi.it
(C. Manera).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.070

V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
525
[28,29]. However, a recent study showed that the CB2R activation
enhances osteoclast activity [30]. Moreover, it was also reported that
CB2R antagonists may represent a new therapeutic approach for the
treatment of obesity-associated inflammation, insulin resistance
and non-alcoholic fatty liver disease [31].
and 18 which showed high CB2R affinity and interesting CB2R
versus CB1R selectivity, were also investigated by the GTP
gS
binding assay. The data obtained indicate that the substitution at
the position C-5 of the 2-oxopyridine nucleus has a very interesting
effect on the activity on the CB2R and permit the identification of
the “key” position to be substituted on the 1,2-dihydro-2-oxo-
pyridine scaffold responsible for functionality switch within this
series of compounds. Similar results were reported by other au-
thors for pyridine-based compounds [41,42].
In a research program aimed at obtaining CB2R selective li-
gands [32e36], we have recently described the synthesis and
pharmacological characterization of 1,2-dihydro-2-oxopyridine-3-
carboxamide derivatives that exhibited good CB2R affinity and
antiproliferative effects in several cancer cell lines [36]. On the basis
of our previous results and in an effort to develop structure activity
relationship (SAR) for both subtypes CBRs we chose derivative 1 for
the development of a new set of 1,2-dihydro-2-oxopyridine-3-
carboxamide derivatives 2e22 (Table 1). For these compounds
the p-fluorobenzyl group in N1 position and cycloheptyl carbox-
amide in position 3 of 1 were either retained or replaced by groups
with different steric and lipophilic properties, and various sub-
stituents at C5 position were introduced. Furthermore, the sub-
stituent in N-1 position was shifted to the oxygen in position 2 of
the heterocyclic nucleus (23e26, Table 1). Similar analogs were
recently reported as CB2R ligands [37e40].
2. Chemistry
The synthesis of 2-oxopyridine derivatives 2e22 and 2-
substituted pyridines 23e26 was accomplished as depicted in
Schemes 1,2. The methyl ester 27 [36], was heated with the 4-
methylcyclohexylamine or cycloheptylamine at 150 ꢀC to give the
carboxamide derivative 28 and 29 respectively [36] (Scheme 1). N-
Alkylation of 28 and 29 in anhydrous DMF with the suitable benzyl
chloride or alkyl halide or 4-(2-chloroethyl)-morpholine in the
presence of NaH afforded the desired 2-oxopyridine derivatives 2e
6 and 8e13. In the case of the reaction with benzyl chloride or p-
fluoro-benzyl chloride the O-substituted derivatives 23e25 were
also isolated in according to previously reported [36]. Alkaline
hydrolysis of 6 followed by acidification gave the carboxylic acid 7
(Scheme 1).
The new compounds were tested on membranes prepared from
HEK-293 cells expressing the human CB1R and CB2R, to determine
their affinities towards both subtypes of CBRs. Finally the CBRs
activity of the reference compound 1 and of the new derivatives 17
Table 1
Structures and radioligand binding data of 1,2-dihydro-2-oxopyridine- and pyridine-3-carboxamide derivatives.a
R1
R2
R3
Ki (nM)
b
c
CB1
CB2
7.8
SId
5.5
1
2
Cycloheptyl
Cycloheptyl
p-Fluorobenzyl
Benzyl
H
H
43
56 ꢁ 2.8
55 ꢁ 2.6
1270 ꢁ 11
1830 ꢁ 9.5
>10,000
>10,000
70 ꢁ 2.8
386 ꢁ 8.1
>10,000
1623 ꢁ 9.6
>10,000
>10,000
8.8 ꢁ 0.4
243 ꢁ 1.6
23 ꢁ 1.1
63 ꢁ 2.9
35 ꢁ 1.7
24 ꢁ 0.1
2734 ꢁ 13.3
24 ꢁ 0.1
24.8 ꢁ 0.1
1920 ꢁ 9.5
884 ꢁ 4.4
3093 ꢁ 15.4
>10,000
437
8 ꢁ 0.4
7
13.1
6.8
4.6
e
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
p-Chlorobenzyl
p-Iodobenzyl
m-Iodobenzyl
(CH2)3COOEt
(CH2)3COOH
p-Fluorobenzyl
Benzyl
n-Pr
n-Bu
(CH2)2OH
(CH2)3OH
p-Fluorobenzyl
Ethylmorpholino
n-Bu
p-Fluorobenzyl
p-Fluorobenzyl
p-Fluorobenzyl
Ethylmorpholino
p-Fluorobenzyl
p-Fluorobenzyl
Benzyl
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
4.2 ꢁ 0.2
187 ꢁ 2.4
397 ꢁ 2.3
1832 ꢁ 5.6
>10,000
4.8 ꢁ 1.8
53 ꢁ 2.6
583 ꢁ 2.5
104 ꢁ 2.1
>10,000
>10,000
2.2 ꢁ 0.08
65 ꢁ 2.2
5.1 ꢁ 0.2
1.0 ꢁ 0.03
1.2 ꢁ 0.04
2.8 ꢁ 0.1
63 ꢁ 0.3
7.9 ꢁ 0.04
13.7 ꢁ 0.07
481 ꢁ 2.4
80 ꢁ 0.4
594 ꢁ 2.8
396 ꢁ 1.9
0.6
Cycloheptyl
e
4-CH3-cyclohexyl
4-CH3-Cyclohexyl
4-CH3-Cyclohexyl
4-CH3-Cyclohexyl
4-CH3-Cyclohexyl
4-CH3-Cyclohexyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
14.6
7.3
e
15.6
e
e
4
3.7
4.5
63
29
8.6
43.4
3.0
1.8
4
p-Methoxyphenyl
Phenyl
p-Fluorophenyl
p-Fluorophenyl
Trans-phenylvinyl
Ethylacrylate
H
H
H
Br
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
4-CH3-cyclohexyl
4-CH3-cyclohexyl
Cycloheptyl
p-Fluorobenzyl
Benzyl
n-Bu
11
5.2
e
SR144528
JWH 133
677
3
a
Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM), for CB1R and CB2R binding assays.
Affinity of compounds for CB1R was evaluated using membranes from HEK-293 cells transfected and [3H]CP55,940.
Affinity of compounds for CB2R was evaluated using membranes from HEK-293 cells transfected and [3H]CP-55,940.
Selectivity index for CB2R calculated as Ki(CB1R)/Ki(CB2R) ratio.
b
c
d

526
V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
using human recombinant CB1R and CB2R overexpressed in HEK-
293 cells, as previously described [35]. The results are reported in
Table 1 with the Ki values of the previously studied N-cyclohepthyl-
1-(p-fluorobenzyl)-1,2-dihydro-2-oxo-pyridine-3-carboxamide (1)
[36] and the reference compounds SR144528 [43] and JWH 133
[44].
The results indicate that 1,2-dihydro-2-oxopyridine-3-
carboxamide derivatives 2e22 exhibit higher affinity for the CB2R
than for the CB1R. In fact, in no case was the Ki(CB1)/Ki(CB2) ratio
lower than 1. These data are in agreement with those previously
reported [36].
Regarding to the structural modifications in the position 1 of the
2-oxo-pyridine nucleus, substitution of the p-fluorobenzyl group of
the reference compound 1 with benzyl (2) or p-chlorobenzyl group
(3) was important in determining the maintenance in the affinity
towards both receptor subtypes. On the contrary, the presence of p-
iodobenzyl (4) or m-iodobenzyl (5) decreases affinity towards both
CBRs being more drastic in the case of m-iodobenzyl derivative 5.
Furthermore, the substitution of the benzyl group with ethyl
butyric ester or butyric acid led to compounds 6 and 7, respectively,
which exhibited very low affinity.
Replacement of the cycloheptylamide with 4-methylcyclohexyl
amide in position 3 of the 2-oxo-pyridine nucleus of compound 1
led to compound 8, which preserved the receptor affinities found
for the reference compound. Moreover, if 4-methylcyclo
hexylamide in the position 3 was kept and in position 1 the p-flu-
orobenzyl was replaced with benzyl, alkyl or hydroxyalkyl (9e13),
the affinity towards both CBRs decreased. In particular, this
decrement was drastic for the hydroxyalkyl derivatives 12 and 13.
Regarding the introduction of substituent on C-5 of the 2-oxo-
pyridine nucleus, the CBRs affinities were different depending on
the nature of substituent. In particular, the presence of bromine
atom (14) caused an increase in affinity against both CBRs, in
respect to that of observed for the reference compound 1. The
substitution of the p-fluorobenzyl group of 14 with ethylmorpho-
line reduced receptor affinities, especially against CB1R, as is clear
from a comparison of Ki values of compounds 15 with that of 14. On
the contrary, the derivative 16, characterized by n-butyl group in
position 1 of the heterocyclic nucleus, possessed receptor affinities
comparable to that of compound 14. The introduction of trans-
phenylvinyl or ethylacrylate on C5 led to compounds 21 and 22,
respectively, which showed a maintenance (21) or a very low
Scheme 1. Reagents and conditions: (i) R1NH2, 150 ꢀC, 48 h; (ii) DMF, NaH, R2Cl, r.t. or
70 ꢀC, 24 h; (iii) NaOH 10%, reflux, 2 h, HCl.
As described in Scheme 2, the cycloheptyl carboxamide 29 [36]
was refluxed with Br2 and sodium acetate in glacial acetic acid for
8 h to afford 5-bromo-N-cycloheptyl-piridin-2(1H)-on-3-carboxa
mide (30) which was purified by crystallization from toluene. The
treatment of 30 in anhydrous DMF with NaH for 2 h at room
temperature or at 70 ꢀC and then with p-fluoro-benzyl chloride or
4-(2-chloroethyl)morpholine or n-butyl bromide at 70 ꢀC for 24 h
afforded crude mixture which was purified by crystallization or by
flash chromatography to obtain the desired compounds 14e16. In
the case of the reaction of 30 with n-butyl bromide, the O-
substituted derivative 26 was also isolated. The cross-coupling re-
action under Suzuki conditions between 5-bromo derivative 30 and
the appropriate boronic acid carried out under microwave irradi-
ation afforded 5-substituted carboxamide derivatives 31e34 which
by treatment with NaH and then with p-fluoro-benzyl chloride or
4-(2-chloroethyl)-morpholine, gave the desired compounds 17e21.
The 5-ethyl-acrylate derivative 22 was prepared from the N-
cycloheptyl-5-bromo-1-(4-fluorobenzyl)-pyridin-2(1H)-on-3-
carboxamide (14) with ethylacrylate by Heck vinylation under
microwave irradiation according to Scheme 2.
3. Results and discussion
3.1. CB1 and CB2 receptor affinity
The binding affinities (Ki values) of the compounds 2e26 were
evaluated by competitive binding assays against [3H]CP-55,940
Scheme 2. Reagents and conditions: (i) glacial AcOH, AcONa, Br2, reflux, 8 h; (ii) DMF, NaH, 1e2 h at r.t. or 70 ꢀC, RCl/RBr, 70 ꢀC, 24 h; (iii) Dioxane, Ph3P, Pd(OAc)2, MeOH, Na2CO3
2 M, R1B(OH)2, microwave, 200 W, 100 psi, 150 ꢀC, 15 min; (iv) DMF, NaH, R2Cl/R2Br, 70 ꢀC, 24 h; (v) acetonitrile, Ph3P, Pd(OAc)2, Et3N, ethyl-acrylate, microwave, 200 W, 100 psi,
110 ꢀC, 20 min.

V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
527
decrease (22) affinity on CBRs compared to the reference com-
pound 1. On the contrary, the presence of phenyl (18) or p-fluo-
rophenyl (19) group in the same position led to an increase in
affinity against both CBRs which, however, was greater towards
CB2R. Also, in the case of compound 19 the substitution of the p-
fluorobenzyl in position 1 of the 2-oxo-pyridine nucleus with
ethylmorpholine reduced the CBRs affinities as can be seen by a
comparison of Ki values of compounds 19 with that of 20.
Furthermore, the introduction on C-5 of p-methoxyphenyl group
led to compound 17 which showed higher CB2R affinity than that of
the reference compound 1 (Ki ¼ 1.0 nM vs Ki ¼ 7.8 nM). In particular,
derivative 17 was proved to have the highest CB2R affinity and
selectivity in this series (Ki(CB1)/Ki(CB2) ¼ 63).
Finally, the O-substituted derivatives (23e26) showed lower
affinity on both CBRs than that of the corresponding N1-substituted
derivatives. This result is in agreement with the results previously
reported [36].
3.2. CB1 and CB2 receptor functional activity
Fig. 2. Compound 18 is a more potent compound in inhibiting constitutive CB2R ac-
tivity in hCB2-CHO-membranes than an established inverse agonist SR144528. The
results are from three separate experiments performed in duplicates.
CBR-mediated G protein activities of the reference compound 1
and of the new derivatives 17 and 18, which showed the highest
CB2R affinity together with an interesting selectivity, were inves-
tigated by the [35S]GTP
gS binding assay, essentially as previously
(Fig. 2). However, its ability to reverse the response of the inverse
agonist (SR144528, IC50-concentration 3 nM) indicates that would
rather be a neutral CB2R antagonist (Fig. 3A). Interestingly, further
analysis revealed that 17 is a competitive antagonist in inhibiting
HU-210-stimulated activity (pA2 value 7.0) (Fig. 3B), suggesting
altogether that it would be a potent competitive CB2R antagonist
with properties typical of neutral antagonist and/or weak partial
inverse agonist [46]. Furthermore, in rat cerebellar membranes,
compound 17 did not stimulate or antagonize CB1R-mediated G
described [45,46]. The results revealed that compound 1 acts as
a potent CB2R agonist (ꢂlog EC50 ¼ 8.2 ꢁ 0.3, n ¼ 3, Fig. 1A)
when tested in membranes prepared from hCB2-transfected
CHO-cells. However, it also showed CB1R activity at rat
cerebellar membranes, albeit with w200-fold lower potency
(ꢂlog EC50 ¼ 5.8 ꢁ 0.1, n ¼ 3, Fig. 1B). Strikingly, substituent at the
position C-5 has a very dramatic effect on the activity of the human
CB2R. Indeed, when the C-5 hydrogen of compound 1 was replaced
with a phenyl group (compound 18) the CB2R activity was shifted
from agonism to inverse agonism (Fig. 2). Moreover, compound 18
appeared to be as potent inverse agonist (ꢂlog IC50 ¼ 8.8 ꢁ 0.1,
n ¼ 3) as a well-established CB2R-inverse agonist SR144528
(ꢂlog IC50 ¼ 8.5 ꢁ 0.2). Substitution also increased selectivity over
the CB1R as it did not stimulate or antagonize CB1R-mediated G
protein-activity at concentrations below 10
mM (evoked non-
specific effects at higher concentrations) suggesting that it is
CB2R-selective compound. All the tested compounds were silent
when tested in mock-transfected-cell membranes.
protein-activity at concentrations below 10
mM (evoked non-
4. Conclusion
specific effects at higher concentrations). However, maybe the
most interesting compound in the series appeared to be the C-5 p-
methoxyphenyl derivative 17. Unlike the reference compound 1,
derivative 17 was not able to evoke any agonist response, but was
capable of antagonizing CB2R-agonist HU-210 (1 nM) -stimulated
activity in CB2ReCHO-membranes (Fig. 3A). Alone, it showed a
weak (partial) inverse agonist property (88 ꢁ 3% basal, n ¼ 3 (SEM))
compared to that of full inverse agonist SR144528 (75 ꢁ 1% basal)
In this study we have described a new series of 1,2-dihydro-2-
oxopyridine-3-carboxamide derivatives with high CB2R affinity
and interesting selectivity. The obtained results showed that the
nature of substituents at the position 1 of the 2-oxo-pyridine nu-
cleus is crucial for CBRs affinity. Moreover, the introduction of
substituents at the position 5 of the heterocyclic nucleus generally
increased CB2R affinity. In particular, the p-methoxyphenyl
Fig. 1. Doseeresponse curves for the compound 1 at hCB2-CHO cell membranes (A) and at CB1R using rat cerebellar membranes (B). The results are from three separate exper-
iments performed in duplicates.

528
V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
Fig. 3. Compound 17 is a silent compound in evoking CB2R-agonist activity in hCB2-CHO-membranes. However, it dose-dependently reversed agonist (HU210, 1 nM (wEC50-
value)) and also inverse agonist (SR144528, 3 nM (wIC50-value)) responses to the same final level (A). Derivative 17 competitively antagonizes dose-responses of HU210 at the
human CB2R. pA2-value (7.0) for the compound was calculated from the Schild-plot (the slope 1.10 ꢁ 0.1 does not significantly differ from unity (P < 0.05)) (B). The results are from
least three separate experiments performed in duplicates.
derivative 17 showed high affinity (Ki ¼ 1.0 nM) accompanied by
interesting good Ki(CB1R)/Ki(CB2R) selectivity. Furthermore, we
found that the functional activity of this series is controlled by the
presence of a substituent at the position 5 of the 2-oxopyridine
nucleus. Briefly, a hydrogen atom at this position makes the com-
pound a CB2R agonist (1) whereas a corresponding phenyl deriv-
acetonitrile:water (70:30 v/v) with 0.6 ml/min flow rate. Wave-
lengths were set at 220 and 320 nm. The purity of each compound
was >96% in either analysis.
5.1.1. N-(4-Methylcyclohexyl)-2-hydroxypyridine-3-carboxamide
(28)
ative (18) leads to
a highly potent inverse CB2R agonist.
A mixture of methyl 2-hydroxypyridine-3-carboxylate (27)
(0.590 g, 3.86 mmol) and 4-methylcyclohexylamine (1.32 g,
11.6 mmol) was heated at 150 ꢀC for 48 h. After cooling the reaction
mixture was poured into ice and treated with HCl 5% until pH ¼ 4.
The precipitated solid was collected by filtration and purified by
crystallization from ethyl acetate to give the pure amide 28 (0.89 g,
Importantly, a p-methoxyphenyl group at this position makes the
compound a competitive CB2R neutral antagonist/weak partial
CB2R inverse agonist. All the three compounds described have
great potential to serve selective and unique tools for both in vitro
and in vivo studies examining biological functionality of CB2R.
Accordingly to recently reported literature [41,42], these results
confirm that small structural changes can control functional ac-
tivity. It will be interesting to study the influence on the CB2R ac-
tivity of other substituents in the same position (5) and/or in other
positions (eg 4 and/or 6) of the 2-oxo-pyridine nucleus and if this is
due to steric or electronic effects.
yield 74%); m.p. 248e250 ꢀC; 1H NMR (DMSO)
d 12.47 (br, 1H, OH),
10.01 and 9.87 (2d, J ¼ 7.5 Hz, 1H, NH), 8.30 (m, 1H, Ar), 7.69 (m, 1H,
Ar), 6.46 (m, 1H, Ar), 3.75 and 3.54 (2m, 1H, NCH), 1.92e0.83 (m,
12H, cyclohexyl þ CH3). Anal. C13H18N2O2 (C, H, N).
5.1.2. N-Cycloheptyl-5-bromo-pyridin-2(1H)-on-3-carboxamide
(30)
To
a
suspension of N-cyclohepthyl-pyridin-2(1H)-on-3-
5. Experimental
carboxamide (29) (1.50 g; 6.40 mmol), sodium acetate (2.0 g;
25.62 mmol) in 15 ml of glacial acid acetic was added dropwise a
solution of bromine (0.8 ml; 16.0 mmol) in 10 ml of glacial acetic
acid. The mixture was refluxed for 8 h. The crude mixture was
treated with an aqueous solution of sodium bisulfate and extracted
with dichloromethane. The organic layer was washed with water,
dried over anhydrous Na2SO4 and evaporated to dryness under
reduced pressure to obtain a solid residue which was collected by
filtration and washed with small portions of isopropyl ether to give
pure 30 (1.0 g, yield 50%); mp 185e187 ꢀC (crystallized from
5.1. Chemistry
Reagents were purchased from commercial suppliers and used
without further purification. Merck silica gel 60 was used for flash
chromatography (230e400 mesh). Melting points were deter-
mined on a Kofler hot stage apparatus and are uncorrected. IR
spectra in Nujol mulls were recorded on an ATI Mattson Genesis
Series FTIR spectrometer. 1H NMR and 13C NMR spectra were
recorded with a Varian Gemini 200 spectrometer in
d units from
toluene); MS m/z 312 (Mþ); 1H NMR (DMSO)
d 12.81 (br, 1H, OH),
TMS as an internal standard. Microwave-assisted reactions were
run in a CEM microwave synthesizer. Mass spectra were performed
with a Thermo Quest Finningan GCQ plus. Elemental analyses (C, H,
N) were within ꢁ0.4% of theoretical values, and were performed on
a Carlo Erba elemental analyzer model 1106 apparatus. The
chemical purity of the target compounds was determined under
the following conditions: the HPLC system was an LC Workstation
Prostar (Varian, Inc., Walnut Creek, CA, USA) consisting of high
pressure mixer pump (ProStar, model 230), DAD detector (ProStar,
9.75 (d, J ¼ 6.1 Hz, 1H, NH), 8.27 (d, J ¼ 2.7 Hz, 1H, Ar), 8.00 (d,
J ¼ 2.7 Hz, 1H, Ar), 4.01 (m, 1H, CH), 1.81e1.40 (m, 12H, cycloheptyl).
Anal. C13H17BrN2O2 (C, H, N).
5.1.3. General procedures for the synthesis of compounds 31e34
A mixture of triphenylphosphine (83.5 mg, 0.32 mmol) and
palladium acetate (14.3 mg; 0.06 mmol) in 3 ml of dioxane was
stirred under nitrogen at room temperature. After 10 min a solution
of N-cycloheptyl-5-bromo-pyridin-2(1H)-on-3-carboxamide (30)
(0.200 g; 0.64 mmol) and suitable boronic acid (1.27 mmol) in
MeOH (1.5 ml) and Na2CO3 2 M (1.3 ml) were added. The reaction
mixture was heated by the microwave radiation for 15 min at
150 ꢀC (200 W, 100 psi). The reaction mixture was thereafter cooled
model 330) and a loop of 20 ml. Data were processed by a Star LC
Workstation (Varian, Inc.). Chromatographic separation was per-
formed on a Luna C18 ODS2 analytical column (150 ꢃ 4.6 mm inner
diameter, 3
mm particle size, Phenomenex, Torrance, CA, USA)
maintained at 25 ꢀC. The mobile phase consisted of

V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
529
down to room temperature treated with water and extracted with
dichloromethane. The combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, and evaporated. The
residue was purified by flash chromatography on silica gel (31, 34)
or by crystallization (32, 33).
5.1.4.2. N-Cycloheptyl-1-(4-chlorobenzyl)-pyridin-2(1H)-on-3-
carboxamide (3). Yield: 70%; mp 108e110 ꢀC (crystallized from
chloroform); MS m/z 358 (Mþ); 1H NMR (DMSO)
d 9.73 (d,
J ¼ 6.2 Hz, 1H, NH), 8.34 (dd, J ¼ 7.3 and 2.2 Hz, 1H, Ar), 8.26 (dd,
J ¼ 6.6 and 2.2 Hz, 1H, Ar), 7.42 (d, J ¼ 8.3 Hz, 2H, Ar), 7.31 (d,
J ¼ 8.3 Hz, 2H, Ar), 6.59 (m, 1H, Ar), 5.22 (s, 2H, CH2), 3.99 (m, 1H,
CH), 1.96e1.20 (m, 12H, cycloheptyl). 13C NMR (CDCl3)
d 162.44,
5.1.3.1. N-Cycloheptyl-5-phenyl-pyridin-2(1H)-on-3-carboxamide
(31). Yield: 25%; mp 185e190 ꢀC (crystallized from toluene); MS m/
162.20, 143.73, 140.20, 134.52, 134.23, 129.51, 129.44, 129.17, 122.60,
107.17, 52.27, 50.71, 35.19, 28.38, 24.51. Anal. C20H23ClN2O2 (C, H, N).
z 310 (Mþ); 1H NMR (DMSO)
1H, NH), 8.61 (d, J ¼ 2.7 Hz, 1H, Ar), 8.02 (d, J ¼ 2.7 Hz, 1H, Ar), 7.62
(m, 2H, Ar) 7.41 (m, 3H, Ar), 4.10 (m, 1H, CH), 2.00e1.00 (m, 12H,
cycloheptyl). Anal. C19H22N2O2 (C, H, N).
d
12.80 (br, 1H, OH), 9.92 (d, J ¼ 6.6 Hz,
5.1.4.3. N-Cycloheptyl-1-(4-iodobenzyl)-pyridin-2(1H)-on-3-
carboxamide (4). Yield: 81%; mp 88e90 ꢀC; (crystallized from
chloroform); MS m/z 450 (Mþ); 1H NMR (DMSO)
d 9.73 (d,
J ¼ 6.8 Hz, 1H, NH), 8.33 (dd, J ¼ 7.1 and 1.9 Hz, 1H, Ar), 8.16 (dd,
J ¼ 6.7 and 2.2 Hz, 1H, Ar), 7.72 (d, J ¼ 8.3 Hz, 2H, Ar), 7.31 (d,
J ¼ 8.3 Hz, 2H, Ar), 6.58 (m, 1H, Ar), 5.19 (s, 2H, CH2), 4.00 (m, 1H,
5.1.3.2. N-Cycloheptyl-5-(4-methoxyphenyl)-pyridin-2(1H)-on-3-
carboxamide (32). Purified by flash chromatography (hexane/ethyl
acetate 3:2). Yield: 28%; mp 207e209 ꢀC (crystallized from
CH), 1.99e1.15 (m, 12H, cycloheptyl). 13C NMR (DMSO)
d 162.44;
toluene); MS m/z 340 (Mþ); 1H NMR (DMSO)
d 12.70 (br, 1H, OH),
162.20; 143.81; 140.26; 138.36; 135.38; 130.00; 122.54; 107.25;
94.30; 52.48; 50.73; 35.20; 28.41; 24.51. Anal. C20H23IN2O2 (C, H, N).
9.95 (d, J ¼ 6.4 Hz, 1H, NH), 8.56 (d, J ¼ 2.9 Hz, 1H, Ar), 7.93 (d,
J ¼ 2.9 Hz, 1H, Ar), 7.52 (d, J ¼ 8.7 Hz, 2H, Ar), 7.00 (d, J ¼ 8.8 Hz, 2H,
Ar), 4.06 (m, 1H, CH), 3.78 (s, 3H, OCH3), 2.00e1.00 (m, 12H,
cycloheptyl). Anal. C20H24N2O3 (C, H, N).
5.1.4.4. N-Cycloheptyl-1-(3-iodobenzyl)-pyridin-2(1H)-on-3-
carboxamide (5). Purified by flash chromatography (hexane/ethyl
acetate 1:4). Yield: 75%; mp 128e130 ꢀC; (crystallized from chlo-
5.1.3.3. N-Cycloheptyl-5-(4-fluorophenyl)-pyridin-2(1H)-on-3-
carboxamide (33). Yield: 51%; mp 205e207 ꢀC (crystallized from
roform); MS m/z 450 (Mþ); 1H NMR (DMSO)
NH), 8.35 (dd, J ¼ 7.3 and 2.2 Hz,1H, Ar), 8.20 (dd, J ¼ 6.6 and 2.2 Hz,
1H, Ar), 7.67 (m, 2H, Ar), 7.26 (m, 2H, Ar), 6.56 (m, 1H, Ar), 5.20 (s,
2H, CH2), 3.98 (m,1H, CH),1.90e1.25 (m,12H, cycloheptyl). 13C NMR
d
9.71 (d, J ¼ 7.8 Hz,1H,
toluene); MS m/z 328 (Mþ); 1H NMR (DMSO)
d 12.70 (br, 1H, OH),
9.88 (d, J ¼ 7.9 Hz, 1H, NH), 8.57 (d, J ¼ 2.9 Hz, 1H, Ar), 8.01 (d,
J ¼ 2.9 Hz, 1H, Ar), 7.65 (m, 2H, Ar), 7.27 (m, 2H, Ar), 4.00 (m, 1H,
CH), 2.00e1.30 (m, 12H, cycloheptyl). Anal. C19H21FN2O2 (C, H, N).
(DMSO)
d 162.42; 162.22; 143.80; 140.36; 137.36; 135.38; 134.34;
131.20; 130.45; 124.54; 107.55; 94.20; 52.44; 50.70; 35.21; 28.44;
24.51. Anal. C20H23IN2O2 (C, H, N).
5.1.3.4. N-Cycloheptyl-5-[(E)-2-phenylethenyl]-pyridin-2(1H)-on-3-
carboxamide (34). Purified by flash chromatography (hexane/ethyl
acetate 1:1). Yield: 22%; mp 128e130 ꢀC (crystallized from
5.1.4.5. Ethyl 4-(3-(cycloheptylcarbamoyl)-2-oxopyridin-1(2H)-yl)
butanoate (6). Purified by flash chromatography (hexane/ethyl
acetate 1:1, v/v) as oil. Yield: 66%; MS m/z 348 (Mþ); 1H NMR
toluene); MS m/z 336 (Mþ); 1H NMR (DMSO)
d 12.35 (br, 1H, OH),
9.86 (d, J ¼ 6.7 Hz, 1H, NH), 8.68 (d, J ¼ 2.5 Hz, 1H, Ar), 7.89 (d,
J ¼ 2.5 Hz,1H, Ar), 7.56 (d, J ¼ 8.0 Hz, 1H, vinyl), 7.36 (m, 2H, Ar) 7.28
(m, 3H, Ar), 7.18 (d, J ¼ 8.0 Hz,1H, vinyl), 4.07 (m,1H, CH), 2.00e1.30
(m, 12H, cycloheptyl). Anal. C21H24N2O2 (C, H, N).
(DMSO)
d
9.86 (d, J ¼ 7.8 Hz,1H, NH), 8.30 (dd, J ¼ 7.0 and 2.2 Hz,1H,
Ar), 7.99 (dd, J ¼ 6.5 and 2.2 Hz, 1H, Ar), 6.51 (m, 1H, Ar), 3.96 (m,
5H, CH þ CH2), 2.30 (t, J ¼ 7.2 Hz, 2H, CH2), 1.98e1.47 (m, 14H,
cycloheptyl þ CH2), 1.13 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR (DMSO)
d
173.71, 162.42; 161.23; 143.82; 134.34; 131.20; 107.55; 61.43;
5.1.4. General procedure for the synthesis of N1- (2e6, 8e21) and
O-substituted pyridin-3-carboxamide derivatives (23e26)
An amount of 2.88 mmol of NaH was added to a solution of
0.96 mmol of appropriate pyridin-3-carboxamide 28e34 in 12 ml
of dry DMF. After 1 h at 70 ꢀC or at room temperature (for com-
pounds 4e6, 15, 17, 18 and 21) 1.92 mmol of the suitable chloride or
bromide was added and the mixture was stirred for 24 h at 50 ꢀC or
at room temperature (4e6). After cooling the reaction mixture was
concentrated under reduced pressure, treated with water to give a
residue which was purified by flash chromatography or by
crystallization.
50.70; 45.69; 35.21; 31.43; 28.44; 24.51; 23.35; 15.63. Anal.
19H28N2O4 (C,H, N).
C
5.1.4.6. N-(4-Methylcyclohexyl)-1-(4-fluorobenzyl)-pyridin-2(1H)-
on-3-carboxamide (8) and N-(4-Methylcyclohexyl)-2-(4-
fluorobenzyloxy)-pyridin-2(1H)-on-3-carboxamide (24). Purified by
flash chromatography (hexane/ethyl acetate 2:1). 8 as oil. Yield:
25%; MS m/z 342 (Mþ); 1H NMR (CDCl3)
d 9.98 and 9.59 (2d,
J ¼ 6.9 Hz,1H, NH), 8.37 (m,1H, Ar), 8.20 (m,1H, Ar), 7.36 (m, 2H, Ar),
7.23 (m, 2H, Ar), 6.41 (m, 1H, Ar), 5.24 and 5.21 (2s, 2H, CH2), 4.13
and 3.85 (2m, 1H, CH), 2.03e0.98 (m, 12H, cyclohexyl þ CH3). 13C
NMR (CDCl3)
d 162.89, 162.26, 160.92, 145.58, 134.79, 137.83, 131.33,
5.1.4.1. N-Cycloheptyl-1-benzyl-pyridin-2(1H)-on-3-carboxamide (2)
and N-cycloheptyl-2-benzyloxy-pyridin-2(1H)-on-3-carboxamide
(23). Purified by flash chromatography (hexane/ethyl acetate
128.64, 115.34, 111.71, 50.12, 47.36, 44.89, 34.92, 33.66, 32.54, 31.97,
30.54, 29.78, 22.56, 21.95. Anal. C20H23FN2O2 (C, H, N). 24 as oil.
Yield: 10%; (crystallized from hexane); MS m/z 342 (Mþ); 1H NMR
2:1). 2 as oil. Yield: 38%; MS m/z 324 (Mþ); 1H NMR (CDCl3)
d
9.80
(DMSO)
d
8.37 (m, 2H, NH þ Ar), 8.06 (m, 1H, Ar), 7.61 (m, 2H, Ar),
(d, J ¼ 6.4 Hz, 1H, NH), 8.54 (dd, J ¼ 7.1 and 2.2 Hz, 1H, Ar), 7.46 (dd,
J ¼ 6.7 and 2.2 Hz, 1H, Ar), 7.36 (m, 5H, Ar), 6.41 (m, 1H, Ar), 5.24 (s,
2H, CH2), 4.16 (m,1H, CH), 2.03e1.27 (m,12H, cycloheptyl). 13C NMR
7.18 (m, 3H, Ar), 5.44 and 5.41 (2s, 2H, CH2), 4.03 and 3.85 (2m,
1H, CH), 1.92e0.69 (m, 12H, cyclohexyl þ CH3). Anal. C20H23FN2O2
(C, H, N).
(CDCl3)
129.51, 129.44, 122.60, 107.67, 52.77, 51.21, 35.22, 28.18, 24.32. Anal.
20H24N2O2 (C, H, N). 23 as oil. Yield: 15%; MS m/z 324 (Mþ); 1H
NMR (DMSO)
and 2.2 Hz, 1H, Ar), 8.03 (d, J ¼ 7.9 Hz, 1H, NH), 7.45 (m, 5H, Ar), 7.13
(m, 1H, Ar), 5.45 (s, 2H, CH2), 3.85 (m, 1H, CH), 1.92e1.07 (m, 12H,
cycloheptyl). Anal. C20H24N2O2 (C, H, N).
d 162.44, 162.20, 143.73, 140.20, 134.52, 134.23, 130.15,
5.1.4.7. N-(4-Methylcyclohexyl)-1-benzyl-pyridin-2(1H)-on-3-
carboxamide (9) and N-(4-Methylcyclohexyl)-2-benzyloxy-pyridin-
2(1H)-on-3-carboxamide (25). Purified by flash chromatography
(hexane/ethyl acetate 2:1). 9 as oil. Yield: 28%; MS m/z 324 (Mþ); 1H
C
d
8.32 (dd, J ¼ 7.3 and 2.2 Hz, 1H, Ar), 8.13 (dd, J ¼ 6.7
NMR (CDCl3)
d
10.02 and 9.69 (2d, J ¼ 6.8 Hz, 1H, NH), 8.36 (m, 1H,
Ar), 8.18 (m, 1H, Ar), 7.31 (m, 5H, Ar), 6.52 (m, 1H, Ar), 5,28 and 5.24

530
V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
(2s, 2H, CH2), 4,15 and 3.75 (2m, 1H, CH), 2.01e0.97 (m, 12H,
from toluene); MS m/z 425 (Mþ); 1H NMR (CDCl3)
d 9.72 (d,
cyclohexyl þ CH3). 13C NMR (CDCl3)
d
162.34, 160.68, 145.49, 141.76,
J ¼ 6.2 Hz, 1H, NH), 8.57 (d, J ¼ 2.8 Hz, 1H, Ar), 7.67 (d, J ¼ 2.8 Hz, 1H,
Ar), 4.06 (m, 3H, CH and morpholine), 3.70 (m, 4H, morpholine),
2.70 (t, J ¼ 5.9 Hz, 2H, CH2), 2.50 (m, 4H, morpholine), 1.89e1.22 (m,
135.01, 131.83, 128.93, 127.87, 126.43, 111.86, 50.03, 47.28, 44.56,
34.89, 33.38, 32.78, 31.76, 30.34, 29.68, 22.25, 21.82. Anal.
C
20H24N2O2 (C, H, N). 25 as oil. Yield: 15%; MS m/z 324 (Mþ); 1H
12H, cycloheptyl). 13C NMR (CDCl3)
d 162.10; 161.20; 139.82; 131.41;
NMR (DMSO)
d
8.33 (m, 2H, NH þ Ar), 8.10 (m, 1H, Ar), 7.43 (m, 2H,
123.44; 99.67; 67.3; 55.30; 52.11; 51.27; 50.76; 35.10; 28.37; 24.43.
Anal. C19H28BrN3O3 (C, H, N).
Ar), 7.22 (m, 3H, Ar), 7.12 (m, 1H, Ar), 5,47 and 5.44 (2s, 2H, CH2),
4.00 and 3.75 (2m, 1H, CH), 1.98e0.63 (m, 12H, cyclohexyl þ CH3).
Anal. C20H24N2O2 (C, H, N).
5.1.4.14. N-Cycloheptyl-5-bromo-1-butyl-pyridin-2(1H)-on-3-
carboxamide (16) and N-cycloheptyl-5-bromo-2-butoxy-pyridin-3-
carboxamide (26). Purified by flash chromatography (hexane/
ethyl acetate 4:1). 16 as oil. Yield: 10%; MS m/z 368 (Mþ); 1H NMR
5.1.4.8. N-(4-Methylcyclohexyl)-1-propyl-pyridin-2(1H)-on-3-
carboxamide (10). Purified by flash chromatography (hexane/ethyl
acetate 1:1.5). Yield: 71%; MS m/z 276 (Mþ); 1H NMR (CDCl3)
d 9.65
(CDCl3)
d
9.77 (d, J ¼ 6.3 Hz, 1H, NH), 8.57 (d, J ¼ 2.9 Hz, 1H, Ar), 7.60
and 10.03 (2d, J ¼ 7.5 Hz, 1H, NH), 8.55 (dd, J ¼ 7.1 and 2.2 Hz, 1H,
(d, J ¼ 2.9 Hz, 1H, Ar), 4.21 (m, 1H, CH), 4.03 (t, J ¼ 7.5 Hz, 2H, CH2),
1.98e1.10 (m, 19H, cycloheptyl þ CH2 þ CH3). 13C NMR (CDCl3)
Ar), 7.46 (dd, J ¼ 6.6 and 2.2 Hz, 1H, Ar), 6.40 (m, 1H, Ar), 3.94e4.03
(m, 3H, CH þ CH2), 0.92e1.89 (m, 17H, cyclohexyl þ CH2 þ CH3). 13
C
d
162.10; 161.20; 139.82; 131.41; 123.44; 99.67; 50.76; 47.34; 35.10;
NMR (CDCl3)
d 162.66, 160.78, 145.28, 134.72, 131.64, 111.33, 50.12,
31.77; 28.36; 24.42; 22.78; 17.23. Anal. C17H25BrN2O2 (C, H, N). 26 as
oil. Yield: 12%, MS m/z 368 (Mþ); 1H NMR (CDCl3)
d
8.62 (d,
47.36, 38.95, 34.79, 33.82, 32.87, 31.67, 30.66, 30.34, 29.91, 24.62,
22.81, 21.75, 14.65. Anal. C16H24N2O2 (C, H, N).
J ¼ 2.6 Hz, 1H, Ar), 8.27 (d, J ¼ 2.6 Hz, 1H, Ar), 8.00 (d, J ¼ 6.3 Hz, 1H,
NH), 4.47 (t, J ¼ 6.6 Hz, 2H, CH2), 4.20 (m, 1H, CH), 2.00e1.00 (m,
19H, cycloheptyl þ CH2 þ CH3). Anal. C17H25BrN2O2 (C, H, N).
5.1.4.9. N-(4-Methylcyclohexyl)-1-butyl-pyridin-2(1H)-on-3-
carboxamide (11). Purified by flash chromatography (hexane/ethyl
acetate 2:1) as oil. Yield: 57%; MS m/z 290 (Mþ); 1H NMR (CDCl3)
5.1.4.15. N-Cycloheptyl-5-(4-methoxyphenyl)-1-(4-fluorobenzyl)-
pyridin-2(1H)-on-3-carboxamide (17). Purified by flash chroma-
tography (hexane/ethyl acetate 5:1) Yield: 34%; mp: 159e161 ꢀC
(crystallized from toluene); MS m/z 448 (Mþ); 1H NMR (DMSO)
d
9.65 and 10.03 (2d, J ¼ 7.5 Hz, 1H, NH), 8.52 (dd, J ¼ 7.1 and 2.2 Hz,
1H, Ar), 7.48 (dd, J ¼ 6.6 and 2.2 Hz, 1H, Ar), 6.41 (m, 1H, Ar), 3.97e
4.15 (m, 3H, CH
þ
CH2), 0.92e2.05 (m, 19H, 4-
methycyclohexyl þ 2CH2 þ CH3). 13C NMR (CDCl3)
d 162.48,
d
9.80 (d, J ¼ 6.3 Hz, 1H, NH), 8.59 (s, 2H, Ar), 7.55 (d, J ¼ 8.8 Hz, 2H,
160.76, 145.44, 134.73, 131.84, 111.63, 50.01, 47.39, 38.87, 34.79,
33.87, 32.59, 31.73, 30.69, 30.48, 29.85, 29.73, 24.52, 22.68, 21.87,
14.76. Anal. C17H26N2O2 (C, H, N).
Ar), 7.50e7.10 (m, 4H, Ar), 7.04 (d, J ¼ 8.8 Hz, 2H, Ar), 5.29 (s, 2H,
CH2), 4.00 (m, 1H, CH), 3.79 (s, 3H, OCH3), 2.00e1.20 (m, 12H,
cycloheptyl). 13C NMR (CDCl3)
d 162.49, 161.39, 160.36, 159.65,
143.08, 136.45, 131.66, 131.61, 130.11, 129.95, 128.16, 127.35; 122.08,
121.21, 116.49, 116.06, 114.80, 55.64, 52.45, 50.76; 35.20, 28.42,
24.52. Anal. C27H29FN2O3 (C, H, N).
5.1.4.10. N-(4-Methylcyclohexyl)-1-(2-hydroxyethyl)-pyridin-2(1H)-
on-3-carboxamide (12). Purified by flash chromatography (hexane/
ethyl acetate 1:2) as oil. Yield: 66%; MS m/z 278 (Mþ); 1H NMR
(CDCl3)
d
9.62 and 10.01 (2d, J ¼ 7.5 Hz,1H, NH), 8.39 (dd, J ¼ 7.3 and
5.1.4.16. N-Cycloheptyl-5-phenyl-1-(4-fluorobenzyl)-pyridin-2(1H)-
on-3-carboxamide (18). Purified by flash chromatography (hexane/
ethyl acetate 4:1) as oil. Yield: 37%; MS m/z 418 (Mþ); 1H NMR
2.1 Hz, 1H, Ar), 7.58 (dd, J ¼ 6.3 and 2.1 Hz, 1H, Ar), 6.35 (m, 1H, Ar),
3.51e3.99 (m, 3H, CH2 þ OH), 4.15e4.23 (m, 3H, CH þ CH2), 0.89e
2.05 (m, 12H, cyclohexyl þ CH3). 13C NMR (CDCl3)
d 162.66, 160.78,
(DMSO)
d
9.78 (d, J ¼ 7.5 Hz,1H, NH), 8.66 (d, J ¼ 2.9 Hz,1H, Ar), 8.63
145.28, 134.72,131.64,111.33, 61.78, 50.12, 47.36, 41.95, 34.86, 33.79,
32.92, 31.88, 30.49, 29.86, 22.65, 21.88. Anal. C15H22N2O3 (C, H, N).
(d, J ¼ 2.9 Hz, 1H, Ar), 7.63 (m, 2H, Ar), 7.50 (m, 4H, Ar), 7.20 (m, 3H,
Ar), 5.31 (s, 2H, CH2), 4.09 (m, 1H, CH), 2.00e1.20 (m, 12H, cyclo-
heptyl). 13C NMR (CDCl3)
d 162.47, 161.19, 160.40, 143.18, 137.15,
5.1.4.11. N-(4-Methylcyclohexyl)-1-(3-hydroxypropyl)-pyridin-
2(1H)-on-3-carboxamide (13). Purified by flash chromatography
(toluene/ethyl acetate 1:4) as oil. Yield: 47%; MS m/z 292 (Mþ); 1H
131.88, 131.41, 130.01, 129.75, 128.36, 127.65; 122.08, 116.49; 116.36;
52.35, 50.66; 35.20, 28.45, 24.50. Anal. C26H27FN2O2 (C, H, N).
NMR (CDCl3)
d
9.61 and 10.02 (2d, J ¼ 7.5 Hz, 1H, NH), 8.52 (dd,
5.1.4.17. N-Cycloheptyl-5-(4-fluorophenyl)-1-(4-fluorobenzyl)-pyr-
idin-2(1H)-on-3-carboxamide (19). Purified by flash chromatog-
raphy (hexane/ethyl acetate 5:1). as oil. Yield: 60%; MS m/z 436
J ¼ 7.3 and 2.2 Hz, 1H, Ar), 7.52 (dd, J ¼ 6.4 and 2.2 Hz, 1H, Ar), 6.38
(m, 1H, Ar), 3.43e3.89 (m, 3H, OH þ CH2), 4.01e4.24 (m, 3H,
CH þ CH2), 0.89e2.08 (m, 14H, cyclohexyl þ CH2 þ CH3). 13C NMR
(Mþ); 1H NMR (DMSO)
d
9.76 (d, J ¼ 7.5 Hz, 1H, NH), 8.65 (d,
(CDCl3) d 162.34, 160.63, 145.54, 134.72, 131.44, 111.63, 62.07, 49.78,
J ¼ 2.9 Hz, 1H, Ar), 8.63 (d, J ¼ 2.9 Hz, 1H, Ar), 7.67 (m, 2H, Ar), 7.49
47.41, 40.46, 34.85, 33.98, 32.72, 31.80, 30.63, 30.54, 29.44, 22.74,
21.86. Anal. C16H24N2O3 (C, H, N).
(m, 2H, Ar), 7.28 (m, 4H, Ar), 5.29 (s, 2H, CH2), 4.03 (m, 1H, CH),
2.00e1.20 (m, 12H, cycloheptyl). 13C NMR (CDCl3)
d 165.33; 162.36,
161.45, 160.40, 143.06, 136.96, 131.66, 131.86, 131.47, 130.17, 129.66,
128.04, 127.87; 122.28, 120.55, 116.57, 116.15, 115.60, 52.55, 50.82;
35.18, 28.40, 24.49. Anal. C26H26F2N2O2 (C, H, N).
5.1.4.12. N-Cycloheptyl-5-bromo-1-(4-fluorobenzyl)-pyridin-2(1H)-
on-3-carboxamide (14). Yield: 90%; mp 166e168 ꢀC; (crystallized
from chloroform); MS m/z 420 (Mþ); 1H NMR (DMSO)
d 9.68 (d,
J ¼ 6.2 Hz,1H, NH), 8.60 (d, J ¼ 2.9 Hz,1H, Ar), 8.29 (d, J ¼ 2.9 Hz,1H,
Ar), 7.36 (m, 2H, Ar), 7.18 (m, 2H, Ar), 5.18 (s, 2H, CH2), 3.91 (m, 1H,
5.1.4.18. N-Cycloheptyl-5-(4-fluorophenyl)-1-(2-morpholin-4-
ylethyl)-pyridin-2(1H)-on-3-carboxamide (20). Purified by flash
chromatography (hexane/ethyl acetate 2:3). as oil. Yield: 70%; MS
CH), 1.89e1.20 (m, 12H, cycloheptyl). 13C NMR (CDCl3)
d 162.34;
162.10; 161.21; 146.37; 139.82; 135.95; 130.41; 130.24; 123.45;
116.66; 116.22; 99.47; 52.41; 50.87; 35.10; 28.37; 24.43. Anal.
m/z 441 (Mþ); 1H NMR (DMSO)
d
9.79 (d, J ¼ 7.6 Hz,1H, NH), 8.58 (d,
C
20H22BrFN2O2 (C, H, N).
J ¼ 2.9 Hz, 1H, Ar), 8.38 (d, J ¼ 2.9 Hz, 1H, Ar), 7.63 (m, 2H, Ar), 7.33
(m, 2H, Ar), 4.25 (t, J ¼ 6.0 Hz, 2H, CH2), 4.03 (m, 1H, CH), 3.52 (m,
4H, morpholine), 2.58 (m, 6H, morpholine þ CH2), 1.85e1.35 (m,
5.1.4.13. N-Cycloheptyl-5-bromo-1-(2-morpholin-4-ylethyl)-pyridin-
2(1H)-on-3-carboxamide (15). Purified by flash chromatography
(hexane/ethyl acetate 1:3). Yield: 25%; mp 166e168 ꢀC (crystallized
12H, cycloheptyl). 13C NMR (CDCl3)
d 162.54, 161.34, 160.22, 142.79,
138.60, 132.20, 128.86, 127.89, 121.50, 119.53, 116.58, 116.15, 67.22,

V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
531
57.13, 53.95, 50.67, 47.65; 35.18, 28.42, 24.47. Anal. C25H32FN3O3
(C, H, N).
2.1 nM, respectively, for CB1R and CB2R). All compounds were
tested following the procedure described by the cell membrane
manufacturer. Displacement curves were generated by incubating
drugs with [3H]CP55,940 for 90 min at 30 ꢀC. Ki values were
calculated by applying the ChengePrusoff equation [49] to the IC50
values (obtained by GraphPad) for the displacement of the bound
radioligand by increasing concentrations of the test compound.
Data are the mean (SEM of at least n ¼ 3 experiments).
5.1.4.19. N-Cycloheptyl-5-[(E)-2-phenylethenyl]-1-(4-fluorobenzyl)-
pyridin-2(1H)-on-3-carboxamide (21). Purified by flash chroma-
tography (hexane/ethyl acetate 3:1). Yield: 26%; mp 135e137 ꢀC
(crystallized from hexane); MS m/z 444 (Mþ); 1H NMR (DMSO)
d
9.78 (d, J ¼ 6.7 Hz, 1H, NH), 8.70 (d, J ¼ 2.6 Hz, 1H, Ar), 8.41 (d,
J ¼ 2.6 Hz,1H, Ar), 7.58 (d, 1H, J ¼ 8.0 Hz, vinyl), 7.40 (m, 5H, Ar) 7.20
(m, 5H, Ar þ vinyl) 5.26 (s, 2H, CH2), 4.01 (m, 1H, CH), 2.00e1.30 (m,
5.3. Functional activity at CB1 and CB2 receptor in vitro
12H, cycloheptyl). 13C NMR (CDCl3)
d 161.83, 161.71, 160.40, 159.87,
141.18, 140.31, 138.58, 131.2, 130.95, 130.25, 128.72, 128.52, 125.45,
122.69, 116.87, 115.80, 52.45, 50.80; 35.12, 28.36, 24.41. Anal.
The [35S]GTP
to the reported procedures [39,40]. The results were calculated
using GraphPad Prism 5.0 (California, USA). The GTP S binding
gS binding experiments were performed according
C
28H29FN2O2 (C, H, N).
g
studies were conducted in three independent experiments per-
formed in duplicates. IC50 and EC50 values are presented as
mean ꢁ s.e.m.
5.1.5. Acid 4-(N-cycloheptyl)-pyridin-2(1H)-on-3-(carboxamide)
butanoic (7)
A solution of 150 mg (0.43 mmol) of ester 6 in 5 ml of NaOH 10%
was heated at 100 ꢀC. After 2 h the reaction mixture was treated
with water, acidified with HCl conc. until acid pH and it was
extracted with dichloromethane. The organic phase was dried over
anhydrous sodium sulfate and evaporated to dryness under
reduced pressure to obtain a solid residue which was treated with
Et2O and filtered to give 135 mg the desired compound 7. Yield:
Acknowledgments
Authors want to thank Mrs. Tiina Koivunen, Mrs. Minna Glad
and Mrs. Helly Rissanen for technical assistance. This work was
supported by National Interest Research Projects (PRIN 2010-2011,
Grant 20105YY2HL_008) and by the Academy of Finland (grants
127653, 139140, 139620).
98%; mp 84e86 ꢀC; MS m/z 320 (Mþ); 1H NMR (DMSO)
d 12.09 (br,
1H, OH), 9.82 (d, J ¼ 7.9 Hz, 1H, NH), 8.30 (dd, J ¼ 7.1 and 2.2 Hz, 1H,
Ar), 8.15 (d, J ¼ 6.8 and 2.2 Hz, 1H, Ar), 6.52 (m, 1H, Ar), 4.04 (m, 3H,
CH þ CH2), 2.24 (t, 2H, CH2), 1.93e1.54 (m, 14H, cycloheptyl þ CH2).
References
13C NMR (DMSO)
d 177.71, 162.32, 161.29, 143.72, 134.31, 131.20,
[1] P. Pacher, S. Batkai, G. Kunos, Pharmacol. Rev. 58 (2006) 389e462.
[2] D.P. Finn, Immunobiology 215 (2010) 629e646.
[3] I. Bab, A. Zimmer, Br. J. Pharmacol. 153 (2008) 182e188.
[4] S. Oesch, J. Gertsch, J. Pharm. Pharmacol. 61 (2009) 839e853.
[5] M. Karsak, E. Gaffal, R. Date, L. Wang-Eckhardt, J. Rehnelt, S. Petrosino, Science
316 (2007) 1494e1497.
[6] J. Guindon, A.G. Hohmann, Br. J. Pharmacol. 153 (2008) 319e334.
[7] A.M. Malfitano, E. Ciaglia, G. Gangemi, P. Gazzerro, C. Laezza, M. Bifulco, Expert
Opin. Ther. Targets 15 (2011) 297e308.
[8] F. Cianchi, L. Papucci, N. Schiavone, M. Lulli, L. Magnelli, M.C. Vinci,
L. Messerini, C. Manera, E. Ronconi, P. Romagnani, M. Donnini, G. Perigli,
G. Trallori, E. Tanganelli, S. Capaccioli, E. Masini, Clin. Cancer Res. 14 (2008)
7691e7700.
[9] S. Munro, K.L. Thomas, M. Abu-Shaar, Nature 365 (1993) 61e65.
[10] R.G. Pertwee, Curr. Med. Chem. 6 (1999) 635e664.
[11] H.H. Seltzman, Curr. Med. Chem. 6 (1999) 685e704.
[12] A.B. Lynn, M. Herkenham, J. Pharmacol. Exp. Ther. 268 (1994) 1612e1623.
[13] E.S. Onaivi, H. Ishiguro, J.P. Gong, S. Patel, A. Perchuk, P.A. Meozzi, L. Myers,
Z. Mora, P. Tagliaferro, E. Gardner, A. Brusco, B.E. Akinshola, Q.R. Liu, B. Hope,
S. Iwasaki, T. Arinami, L. Teasenfitz, G.R. Uhl, Ann. N.Y. Acad. Sci. 1074 (2006)
514e536.
107.50, 50.70, 45.69; 35.22; 31.40; 28.46, 24.51, 23.35. Anal.
17H24N2O4 (C,H, N).
C
5.1.6. (E)-Ethyl-3-[3-(N-cycloheptyl carboxamide)-1-(4-fluorobenzyl)-
pyridin-2(1H)-on-5-yl]acrylate (22)
A mixture of triphenylphosphine (31.0 mg; 0.20 mmol) and
palladium acetate (5.0 mg; 0.024 mmol) in 1.50 ml acetonitrile was
stirred for 10 min under nitrogen. Then N-cycloheptyl-5-bromo-1-
(4-fluorobenzyl)-pyridin-2(1H)-on-3-carboxamide (14) (100.0 mg;
0.64 mmol), 1.50 ml of triethylamine, 1.27 ml of ethylacrylate
(3.84 mmol) were added. The reaction mixture was heated by the
microwave radiation at 110 ꢀC for 20 min (power 200 W, pressure
100 psi, stirring on). The organic layer was evaporated under
reduced pressure and the residue was purified by flash chroma-
tography (hexane/ethyl acetate 7:3) tþo obtain derivative 22. Yield:
32%; mp 136e139 ꢀC; MS m/z 440 (M ); 1H NMR (DMSO)
d 9.55 (d,
[14] Y. Yiangou, P. Facer, P. Durrenberger, I.P. Chessell, A. Naylor, C. Bountra,
R. Banati, P. Anand, BMC Neurol. 6 (2006) 12.
[15] D.R. Compton, K.C. Rice, B.R. De Costa, R.K. Razdan, L.S. Melvin, M.R. Johnson,
B.R. Martin, J. Pharmacol. Exp. Ther. 265 (1993) 218e226.
[16] T.P. Malan Jr., M.M. Ibrahim, J. Lai, T.W. Vanderah, A. Makriyannis, F. Porreca,
Curr. Opin. Pharmacol. 3 (2003) 62e67.
[17] C. Manera, T. Tuccinardi, A. Martinelli, Mini Rev. Med. Chem. 8 (2008) 370e
387.
[18] G.T. Whiteside, G.P. Lee, K.J. Valenzano, Curr. Med. Chem. 14 (2007) 917e936.
[19] P. Anand, G. Whiteside, C.J. Fowler, A.G. Hohmann, Brain Res. Rev. 60 (2009)
255e266.
J ¼ 7.4 Hz,1H, NH), 8.75 (d, J ¼ 2.5 Hz, 1H, Ar), 8.62 (d, J ¼ 2.5 Hz, 1H,
Ar), 7.58 (d, J ¼ 16 Hz,1H, CH), 7.45 (m, 2H, Ar), 7.20 (m, 2H, Ar), 6.45
(d, J ¼ 16 Hz, 1H, CH), 5.20 (s, 2H, CH2), 4.18 (q, J ¼ 7.0 Hz, 2H, CH2),
3.98 (m, 1H, CH), 2.00e1.28 (m, 12H, cycloheptyl), 1.25 (t, J ¼ 7.0 Hz,
3H, CH3). 13C NMR (CDCl3)
d 166.68; 165.44, 161.83, 161.71, 160.40,
143.06, 141.08, 140.31, 138.58, 130.95, 130.25, 122.69, 116.87, 115.80,
60.90, 52.45, 50.80; 35.12, 28.38, 24.43, 14.55. Anal. C25H29FN2O4 (C,
H, N).
[20] R.G. Pertwee, AAPS J. 7 (2005) E625eE654.
[21] J.L. Shoemaker, K.A. Seely, R.L. Reed, J.P. Crow, P.L. Prather, J. Neurochem. 101
(2007) 87e98.
5.2. CB1 and CB2 receptor binding assays
[22] O. Sagredo, M. Garc-Arencibia, E. de Lago, S. Finetti, A. Decio, J. Fernández-
Ruiz, Mol. Neurobiol. 36 (2007) 82e91.
[23] C. Benito, R.M. Tolon, M.R. Pazos, E. Nunez, A.I. Castillo, J. Romero, Br. J.
Pharmacol. 153 (2008) 277e285.
[24] B.G. Ramirez, C. Blazquez, T. Gomez del Pulgar, M. Guzman, M.L. de Ceballos,
J. Neurosci. 25 (2005) 1904e1913.
[25] M. Body-Malapel, J. El Bakali, E. Mérour, E. Stern, R. Millet, P. Desreumaux,
Gastroenterology 134 (2008) Ae107.
[26] C. Benito, J.P. Romero, R.M. Tolón, D. Clemente, F. Docagne, C.J. Hillard,
C. Guaza, J. Romero, J. Neurosci. 27 (2007) 2396e2402.
[27] K. Mackie, R.A. Ross, Br. J. Pharmacol. 153 (2008) 177e178 and references
cited therein.
The new compounds were evaluated in CB1R and CB2R binding
assays using membranes from HEK-293 cells transfected with
cDNAs encoding the human recombinant CB1R (Bmax) 2.5 pmol/
mg protein and human recombinant CB2R (Bmax) 4.7 pmol/mg
protein (PerkineElmer, Italy). These membranes were incubated
with [3H]CP55,940 [47] (0.14 nM/kd ¼ 0.18 nM and 0.084 nM/kd ¼
0.31 nM for CB1 and CB2 receptors, respectively) as the high affinity
ligand and displaced with 100 nM of WIN55212-2 [48] as the
heterologous competitor for nonspecific binding (Ki values 9.2 and

532
V. Lucchesi et al. / European Journal of Medicinal Chemistry 74 (2014) 524e532
[28] A.I. Idris, A. Sophocleous, E. Landao-Bassonga, R.J. van’t Hof, S.H. Ralston,
Endocrinology 149 (2008) 5619e5626.
[38] K. Kusakabe, Y. Iso, Y. Tada, M. Sakagami, Y. Morioka, N. Chomei,
S. Shinonome, K. Kawamoto, H. Takenaka, K. Yasui, H. Hamana, K. Hanasaki,
Bioorg. Med. Chem. 21 (2013) 3154e3163.
[39] K. Kusakabe, Y. Tada, Y. Iso, M. Sakagami, Y. Morioka, N. Chomei,
S. Shinonome, K. Kawamoto, H. Takenaka, K. Yasui, H. Hamana, K. Hanasaki,
Bioorg. Med. Chem. 21 (2013) 2045e2055.
[40] Y. Tada, Y. Iso, K. Hanasaki, PCT/JP2001/011427 US6977266 B2, 2005.
[41] E. Stern, G.G. Muccioli, B. Bosier, L. Hamtiaux, R. Millet, J.H. Poupaert,
J.P. Henichart, P. Depreux, J.F. Goossens, D.M. Lambert, J. Med. Chem. 50
(2007) 5471e5484.
[42] S. Pasquini, A. Ligresti, C. Mugnaini, T. Semeraro, L. Cicione, M. De Rosa,
F. Guida, L. Luongo, M. De Chiaro, M.G. Cascio, D. Bolognini, P. Marini,
R. Pertwee, S. Maione, V. Di Marzo, F. Corelli, J. Med. Chem. 53 (2010) 5915e
5928.
[29] W. Schuehly, J.M. Viveros Paredes, J. Kleyer, A. Huefner, S. Anavi-Goffer,
S. Raduner, K.-H. Altmann, J. Gertsch, Chem. Biol. 18 (2011) 1053e1064.
[30] F. Rossi, D. Siniscalco, L. Luongo, L. De Petrocellis, G. Bellini, S. Petrosino,
M. Torella, C. Santoro, B. Nobili, V. Di Marzo, S. Maione, Bone 44 (2009)
476e484.
[31] V. Deveaux, T. Cadoudal, Y. Ichigotani, F. Teixeira-Clerc, A. Louvet, S. Manin,
J. Tran-Van Nhieu, M.P. Belot, A. Zimmer, P. Even, P.D. Cani, C. Knauf,
R. Burcelin, A. Bertola, Y. Le Marchand-Bruste, P. Gual, A. Mallat, S. Lotersztajn,
PLoS ONE 6 (2009) e5844.
[32] P.L. Ferrarini, V. Calderone, T. Cavallini, C. Manera, G. Saccomanni, L. Pani,
S. Ruiu, G.L. Gessa, Bioorg. Med. Chem. 12 (2004) 1921e1933.
[33] C. Manera, V. Benetti, M.P. Castelli, T. Cavallini, S. Lazzarotti, F. Pibiri,
G. Saccomanni, T. Tuccinardi, A. Vannacci, A. Martinelli, P.L. Ferrarini, J. Med.
Chem. 49 (2006) 5947e5957.
[34] C. Manera, M.G. Cascio, V. Benetti, M. Allara`, T. Tuccinardi, A. Martinelli,
G. Saccomanni, E. Vivoli, C. Ghelardini, V. Di Marzo, P.L. Ferrarini, Bioorg. Med.
Chem. Lett. 17 (2007) 6505e6510.
[43] F. Rinaldi-Carmona, J. Barth, J.M. Millan, P. Derocq, C. Casellas, D. Congy,
M. Oustric, M. Sarran, B. Bouaboula, M. Calandra, D. Portier, J.C. Shire,
G.L. Breliere, J. Le Fur, Pharmacol. Exp. Ther. 284 (1998) 644e650.
[44] J.W. Huffman, J. Liddle, S. Yu, M.M. Aung, M.E. Abood, J.L. Wiley, B.R. Martin,
Bioorg. Med. Chem. 7 (1999) 2905e2914.
[35] C. Manera, G. Saccomanni, B. Adinolfi, V. Benetti, A. Ligresti, M.G. Cascio,
T. Tuccinardi, V. Lucchesi, A. Martinelli, P. Nieri, E. Masini, V. Di Marzo,
P.L. Ferrarini, J. Med. Chem. 52 (2009) 3644e3651.
[36] C. Manera, G. Saccomanni, A.M. Malfitano, S. Bertini, F. Castelli, C. Laezza,
A. Ligresti, V. Lucchesi, T. Tuccinardi, F. Rizzolio, M. Bifulco, V. Di Marzo,
A. Giordano, M. Macchia, A. Martinelli, Eur. J. Med. Chem. 52 (2012)
284e294.
[37] M. Odan, N. Ishizuka, Y. Hiramatsu, M. Inagaki, H. Hashizume, Y. Fujii,
S. Mitsumori, Y. Morioka, M. Soga, M. Deguchi, K. Yasui, A. Arimura, Bioorg.
Med. Chem. Lett. 22 (2012) 2803e2806.
[45] J.R. Savinainen, S.M. Saario, R. Niemi, T. Järvinen, J.T. Laitinen, Br. J. Pharmacol.
140 (2003) 1451e1459.
[46] J.R. Savinainen, T. Kokkola, O.M. Salo, A. Poso, T. Järvinen, J.T. Laitinen, Br. J.
Pharmacol. 145 (2005) 636e645.
[47] S.J. Gatley, R. Lan, B. Pyatt, A.N. Gifford, N.D. Volkow, A. Makriyannis, Life Sci.
61 (1997) 191e197.
[48] J.W. Huffman, G. Zengin, M.J. Wu, J. Lu, G. Hynd, K. Bushell, A.L. Thompson,
S. Bushell, C. Tartal, D.P. Hurst, P.H. Reggio, D.E. Selley, M.P. Cassidy, J.L. Wiley,
B.R. Martin, Bioorg. Med. Chem. 13 (2005) 89e112.
[49] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099e3108.