Rational design, synthesis, and pharmacological properties of new 1,8-naphthyridin-2(1H)-on-3-carboxamide derivatives as highly selective cannabinoid-2 receptor agonists

Article abstract of DOI:10.1021/jm801563d

The CB₂ receptor activation can be exploited for the treatment of diseases such as chronic pain and tumors of immune origin, devoid of psychotropic activity. On the basis of our already reported 1,8-naphthyridin- 4(1H)-on-3-carboxamide derivati

Full text of DOI:10.1021/jm801563d

3644
J. Med. Chem. 2009, 52, 3644–3651
Rational Design, Synthesis, and Pharmacological Properties of New
1,8-Naphthyridin-2(1H)-on-3-Carboxamide Derivatives as Highly Selective Cannabinoid-2
Receptor Agonists
Clementina Manera,† Giuseppe Saccomanni,*,† Barbara Adinolfi,‡ Veronica Benetti,† Alessia Ligresti,§ Maria Grazia Cascio,§
Tiziano Tuccinardi,*,†,| Valentina Lucchesi,† Adriano Martinelli,† Paola Nieri,‡ Emanuela Masini, Vincenzo Di Marzo,§ and
Pier Luigi Ferrarini†
Dipartimento di Scienze Farmaceutiche and Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniVersita` di Pisa, Via
Bonanno 6, 56126 Pisa, Italy, Dipartimento di Farmacologia Preclinica e Clinica, UniVersita` di Firenze, Viale G. Pieraccini 6, 50139 Firenze,
Italy, Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078
Pozzuoli, Napoli, Italy, and Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and
Technology, Temple UniVersity, Philadelphia, PennsylVania 19122
ReceiVed December 10, 2008
The CB2 receptor activation can be exploited for the treatment of diseases such as chronic pain and tumors
of immune origin, devoid of psychotropic activity. On the basis of our already reported 1,8-naphthyridin-
4(1H)-on-3-carboxamide derivatives, new 1,8-naphthyridin-2(1H)-on-3-carboxamide derivatives were de-
signed, synthesized, and tested for their affinities toward the human CB1 and CB2 cannabinoid receptors.
Some of the reported compounds showed a subnanomolar CB2 affinity with a CB1/CB2 selectivity ratio
greater than 200 (compounds 6, 12, cis-12, 13, and cis-13). Further studies revealed that compound 12,
which presented benzyl and carboxy-4-methylcyclohexylamide substituents bound in the 1 and 3 positions,
exerted a CB2-mediated inhibitory action on immunological human basophil activation. On the human T
cell leukemia line Jurkat the same derivative induced a concentration-dependent decrease of cell viability.
The obtained results suggest that 1,8-naphthyridin-2(1H)-on-3-carboxamides represent a new scaffold very
suitable for the development of new promising CB2 agonists.
Introduction
receptor was expressed exclusively in peripheral tissues, par-
ticularly under pathological conditions. Specifically, CB2R has
been demonstrated in cells and tissues of the immune system,
such as the marginal zone of the spleen.6 In addition, it has
been recently recognized that CB2R may play a functionally
relevant role in the central nervous system. This role is mediated
primarily through microglia, a resident population of cells related
to macrophages.7 Intracellular CB2-dependent signaling path-
ways include Gi/o-dependent inhibition of adenylyl cyclase,
stimulation of mitogen-activated protein kinase and phosphoi-
nositide 3-kinase pathways, and activation of de novo ceramide
production or cyclooxygenase-2 induction.8
Following the discovery of the endocannabinoid system
(ECSa), it has become apparent that research in the cannabinoid
field has promise of becoming therapeutically important. The
ECS includes cannabinoid receptors, several endocannabinoids
(e.g., 2-arachidonoylglycerol, N-arachidonoylethanolamine), and
the enzymes responsible for their production and inactivation
(fatty acid amide hydrolases I and II, monoacylglycerol lipase,
and N-acylethanolamine acid amidase).1,2 To date, two G-
protein-coupled seven-transmembrane receptors, namely, CB1
(CB1R) and CB2 (CB2R), have been identified. On the basis of
pharmacological evidence of cannabinoid action in CB1R- and
CB2R-deficient mice, recent studies support the presence of yet
uncloned cannabinoid receptors.3
Over the past few years, the CB2R has emerged as a critical
target for regulation of inflammation,9 pain,10 bone loss,11
dermatitis,12 and more recently, liver pathophysiology.13 Fur-
thermore, the activation of CB2R has been proposed in the
treatment of cancers of different origin.14,15 Finally, CB2
selective agonists could be exploited for prevention of Alzhe-
imer’s disease by blocking ꢀ-amyloid peptide-induced activation
of microglial cells16 and possibly by providing neuroprotection
through blockade of microglial activation.17 For all these
reasons, the development of potent, selective CB2 molecules
that are devoid of CB1R mediated psychotropic side effects has
gained interest as a new target in drug discovery.
The CB1R appears to be responsible for most of the centrally
mediated effects of cannabinoids.4 This receptor is concentrated
in areas of the brain that control movement, coordination,
sensory perception, learning and memory, reward and emotions,
hormonal function, and body temperature.
The human CB2R is a 360-amino-acid protein that shares 44%
homology with the CB1R.5 Initial studies revealed that this
* To whom correspondence should be addressed. For G.S.: phone,
+39(0)502219580; fax, +39(0)502219605; e-mail: gsacco@farm.unipi.it.
For T.T.: phone, +39(0)502219595; fax, +39(0)502219605; e-mail:
tuccinardi@farm.unipi.it.
We have previously reported the synthesis and binding
activity of a series of 1,8-naphthyridin-4(1H)-on-3-carboxamide
derivatives of general structure A (Figure 1).18-20 These
compounds generally exhibited a remarkable CB2 affinity, with
a Ki value in the nanomolar range. This affinity was ac-
companied by a high selectivity with respect to CB1R. Moreover,
the [35S]GTPγ binding assay and functional studies on human
basophils indicated that these derivatives behaved as CB1 and
†
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
‡
nologie, Universita` di Pisa.
§
Istituto di Chimica Biomolecolare.
Temple University.
|
Universita` di Firenze.
a Abbreviations: ECS, endocannabinoid system; CB1, cannabinoid
subtype 1 receptor; CB2, cannabinoid subtype 2 receptor; RT-PCR, reverse
transcription polymerase chain reaction.
10.1021/jm801563d CCC: $40.75
2009 American Chemical Society
Published on Web 05/12/2009

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3645
On the basis of these results, a series of 1,8-naphthyridin-
2(1H)-on-3-carboxamide derivatives were synthesized.
Chemistry
The synthetic routes to obtain the target 1,8-naphthyridin-
2(1H)-on-3-carboxamide derivatives 2-13 are depicted in
Scheme 1. All substituents are summarized in Table 1.
The heating of 2-aminopyridin-3-carboxaldehyde with ethyl
malonate and a few drops of piperidine at 90 °C for 20 h
afforded the ethyl 1,8-naphthyridin-2(1H)-on-3-carboxylate
derivative 14.24 The reaction of ethyl ester 14 with the
appropriate amine in microwave for 1 h (power 200 W, pressure
100 psi, stirring on) provided the desired carboxamide deriva-
tives 15-19. N-Alkylation of 15-10 in anhydrous DMF with
the suitable benzylchloride or arylalkyl chloride or 4-(2-
chloroethyl)morpholine in the presence of NaH afforded the
desired 1,8-naphthyridin-2-one derivatives 2-13.
Figure 1. General structures of the 1,8-naphthyridin-4(1H)-on-3-
carboxamide (A) and 1,8-naphthyridin-2(1H)-on-3-carboxamide (B)
derivatives and structure of compound 1.
CB2 receptor agonists. The docking study of this class of
cannabinoid ligands highlighted the ligand-receptor interactions
that determined an increase in affinity and selectivity.21 In
particular, this analysis suggested that the preservation of a good
CB2/CB1 selectivity and the improvement of the CB2 affinity
seemed to require the presence of a lipophilic central core
connected with a nonaromatic carboxamide group in position
3 capable of interacting in the CB2R with the nonconserved
residue F5.46(197) and a lipophilic substituent in position 1 with
an H-bond acceptor atom, capable of interacting in the CB2R
with the nonconserved S3.31(112). The important role of
S3.31(112) and F5.46(197) has also been confirmed by site-
directed mutagenesis data.5,21
The separation of cis and trans isomers of 12 and 13 was
obtained by flash chromatography on a silica gel using hexane/
AcOEt/MeOH, 10:1:0.1, to obtain cis-12, trans-12, cis-13, and
trans-13. The faster eluting peak was identified as the cis isomer
and the slower peak as the corresponding trans-isomer based
1
on H NMR spectroscopy data.
The purity of each isomer was determined by analytical
HPLC. We analyzed the mixture to evaluate the best analysis
condition and the retention times of the isomers. For both
isomers, 1.0 mg was dissolved in 1.0 mL of MeOH and an
amount of 20 µL was injected in analytical HPLC. The retention
times for the cis-12 and trans-12 isomers were about 28 and
32 min, and for the cis-13 and trans-13 isomers they were about
46 and 52 min, respectively. Comparing the chromatograms,
we were able to estimate the purity of each isomer that appeared
>96% (see Figures S1 and S2 in the Supporting Information).
Following this binding hypothesis, we investigated the
possible modifications of the 1,8-naphthyridin-4(1H)-one central
scaffold, and the present paper describes the synthesis and the
pharmacological properties of a set of 1,8-naphthyridin-2(1H)-
on-3-carboxamide derivatives of general structure B (Figure 1).
The aliphatic carboxamide groups in position 3 and the
substituents in position 1 have been selected on the basis of the
binding results obtained for the derivatives of general structure
A.18-20
Results and Discussion
CB1 and CB2 Receptor Affinity. The binding affinities (Ki
values) of compounds 2-13 for human recombinant CB1 and
CB2 receptors are reported in Table 1. The Ki values of 5-(4-
chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-
[(1S,4R,6S)-1,5,5-trimethyl-6-bicyclo[2.2.1]heptanyl]pyrazole-
3-carboxamide (20, SR144528)25 and (6aR,10aR)-3-(1,1-
dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-
dibenzo[b,d]pyran (21, JWH133)26 as reference compounds for
both CB1 and CB2 cannabinoid receptors have also been
included in Table 1.
Among the newly synthesized 1,8-naphthyridin-2(1H)-on-3-
carboxamide derivatives, compound 7 possessed a solubility that
hindered its biological testing in DMSO/water solution during
the cannabinoid receptor binding assay. With regard to the CB1
affinity, the other 1,8-naphthyridin-2(1H)-on-3-carboxamide
derivatives 2-6 and 8-13 have high variability of affinity, with
Ki values in the range of 6947 nM (8) to 9.6 nM (10).
Concerning the CB2R, the new compounds 2-6 and 8-13
proved to be high affinity CB2R ligands with Ki values ranging
from 0.7 nM (10 and cis-13) to 69.8 nM (8). The presence of
a fluorine atom para to the benzyl group in position 1 of the
1,8-naphthyridine nucleus increased affinity. This finding was
in agreement with a previous report concerning 1,8-naphthyri-
din-4(1H)-on-3-carboxamide derivatives.18-20 The increased
affinity was evident when comparing compounds 4, 10, 13,
trans-13, and cis-13 to their corresponding benzyl derivatives
3, 9, 12, trans-12, and cis-12. In particular, the (p-fluorobenzyl)-
1,8-naphthyridine derivatives 10 and cis-13 proved to be the
ones with the highest CB2R affinity in this series, with Ki values
The new compounds were tested in competitive binding
assays toward both human recombinant CB1 (hCB1) and CB2
(hCB2) cannabinoid receptors expressed in CHO cells and were
found to be selective for CB2R. Furthermore, a functional study
on human basophils22,23 was performed to determine the
functionality of one of the most interesting newly synthesized
CB2R ligands. Finally, the same compound was evaluated for
its in vitro effect as anticancer agent on human cell lines
expressing CB2Rs, i.e., Jurkat and U87MG cells.
Theoretical Studies
In order to investigate possible modifications of the 1,8-
naphthyridin-4(1H)-one central scaffold, the published com-
pound N-cyclohexyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-
4(1H)-on-3-carboxamide (1, see Figure 1) (CB1R Ki > 1000 nM;
CB2R Ki ) 48.6 nM)19 was chosen as a lead for the development
of new derivatives. The superimposition between this reference
compound and the N-cyclohexyl-1-(2-morpholin-4-ylethyl)-1,8-
naphthyridin-2(1H)-on-3-carboxamide (2) analogue encouraged
further experimental studies. As shown in Figure 2, the
superimposition between the two compounds highlighted a
satisfactory overlap of the structural features deemed important
for the CB2 affinity, i.e., the lipophilic central core, the
cyclohexyl, and the morpholine substituents.

3646 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Manera et al.
Figure 2. 3D structure and solvent-excluded molecular surface of compound 1 (A), 2 (B), and their superimposition (C).
Table 1. Radioligand Binding Data of Compounds 2-13a
Scheme 1. Synthesis of the 1,8-Naphthyridin-2(1H)-
on-3-carboxamide Derivatives 2-13a
Ki (nM)
Ki(CB1)/
Ki(CB2)
b
c
compd
R1
R2
CB1 CB2
2
3
4
5
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
ethylmorpholino 560 7.9
71
18
25
80
526
benzyl
560 31
56 2.2
5600 70
p-fluorobenzyl
n-butyl
6
4-methylcyclohexyl ethylmorpholino 1000 1.9
7
8
9
10
11
12
phenethyl
benzyl
benzyl
benzyl
p-fluorobenzyl
NT
6947 69.8
960 22
NT
4-fluorophenethyl
cycloheptyl
cycloheptyl
cycloheptyl
100
44
14
9.6
0.7
3
ethylmorpholino 18
6
4-methylcyclohexyl benzyl
1600 7.8
5255 39.8
1519 5.8
200 0.9
300 9.0
200 0.7
437 0.6
205
132
262
222
33
286
728
226
trans-12 4-methylcyclohexyl benzyl
cis-12
13
4-methylcyclohexyl benzyl
4-methylcyclohexyl p-fluorobenzyl
trans-13 4-methylcyclohexyl p-fluorobenzyl
cis-13
4-methylcyclohexyl p-fluorobenzyl
2025
2126
677
3
a Reagents and conditions: (i) EMME, 120 °C, 20 h; (ii) R1NH2,
microwave, 140 °C, 1 h 200 W; (iii) NaH, R2Cl.
a Data represent mean values for at least three separate experiments
performed in duplicate and are expressed as Ki (nM) for CB1 and CB2
binding assays. Values of standard error of the mean (SEM) are not shown
for the sake of clarity and were never higher than 5% of the means. NT )
not tested, as it is insoluble in the solvent normally used in binding assays.
b Affinity of compounds for CB1 receptor was evaluated using membranes
from HEK-293 cells transfected with CB1 receptor and [3H]CP55,940.
c Affinity of compounds for CB2 receptor was evaluated using membranes
from HEK-293 cells transfected with CB2 receptor and [3H]CP55,940.
of 0.7 nM. Finally, the compounds with an ethylmorpholino
group in position 1 exhibited CB2R affinity higher (Ki values
lower) than that of the corresponding benzyl derivatives and
lower (Ki values higher) than that of the corresponding p-
fluorobenzyl derivatives. The presence of the n-butyl substituent
(5) in the same position reduces the affinity for CB2R.
With regard to the structural modification in position 3 of
the 1,8-naphthyridine nucleus, the presence of the cyclohepty-
lamido or the 4-methylcyclohexylamido substituents led to
compounds that exhibited the highest affinity. Furthermore, the
1,8-naphthyridin-2(1H)-on-3-carboxamide derivatives 12 and 13
showed a stereoselectivity for the binding to the CB2R. In fact,
cis-12 and cis-13 showed 7-fold and 13-fold increases in their
affinity for the CB2R when compared with the corresponding
trans-12 and trans-13, respectively. Finally, the substitution of
the cycloheptylamido or the 4-methylcyclohexylamido with
cyclohexylamido or 4-fluorophenethylamido substituents re-
duced the affinity.
bound in the 1 and 3 positions of the 1,8-naphthyridin-2-one
nucleus, showed a CB1/CB2 selectivity ratio of 526. In no case
was the Ki(CB1)/Ki(CB2) ratio lower than 1. Therefore, no
compound exhibited a reversed selectivity.
CB2 Functional Activity. The functional activity of com-
pound 12 was assessed by using functional studies on human
basophils. In fact, activation of CB2 receptors is known to down-
regulate the immunological activation of guinea pig mast cells
and human basophils.22,23 Human basophils pretreated with
N-(4-methylcyclohexyl)-1-benzyl-1,8-naphthyridin-2(1H)-on-3-
carboxamide (12) (0.1-100 nM, 30 min of preincubation, 37
°C) showed a reduced expression of CD203c in response to
immunological stimulation (anti-IgE, 1 µg/mL, 30 min, 37 °C).
CD203c is a glycosilated type II transmembrane molecule that
belongs to the family of ectanucleotide pyrophosphatase/
phosphodiesterase enzymes. It has recently been demonstrated
as a specific activation marker of basophils that is rapidly up-
regulated after allergen challenge.27 The inhibition was reversed
With regard to the CB2R selectivity, the 1,8-naphthyridin-
2(1H)-on-3-carboxamide derivatives 2-6 and 8-13 generally
showed good selectivity. In particular, 6, 12, cis-12, and cis-13
exhibited very significant selectivity, with Ki(CB1)/Ki(CB2) ratios
greater than 200. In particular, compound 6, with ethylmor-
pholino and carboxy-4-methylcyclohexylamide substituents

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3647
Table 2. Cytotoxic Activity of 12 and 23 on Jurkat and U87MG Cell
Linesa
IC50 (µM)
compd
Jurkat
U87MG
12
23
5.98 ( 2.78
9.60 ( 4.12
NR
NR
a Data are the mean ( SE from three experiments carried out in triplicate.
NR: not reached.
Figure 3. Expression of CD203c by human basophils activated with
anti-IgE (1 µg/mL) is reduced by compound 12 in a dose-related
fashion. The inhibitory effect of compound 12 on basophil CD203c
expression is reverted by 20 (100 nM), a CB2 antagonist, but not by
22 (100 nM), a CB1 antagonist. The values are the mean ( SEM of
six independent experiments performed in triplicate: (**) P < 0.001 vs
compound 12 plus 22.
Figure 5. Effect of 12 (10 µM) and combination of 12/24 (1 µM) on
Jurkat cell viability. Data are the mean ( SE of two experiments carried
out in triplicate.
(Figure 4), 12 and 23 only induced a poor effect (about 20% of
cell viability inhibition) and only at the highest dose used (10
µM).
Conclusions
In our study, we investigated possible modifications of the
1,8-naphthyridin-4(1H)-one central scaffold in order to improve
CB2R affinity and selectivity. On the basis of theoretical studies,
a series of 1,8-naphthyridin-2(1H)-on-3-carboxamides was
designed, synthesized, and tested on CB1R and CB2R. The new
compounds showed CB2R affinity and selectivity that were
higher than corresponding 1,8-naphthyridin-4(1H)-on-3-car-
boxamide derivatives previously studied19 and, in some cases
(i.e., compound 6), among the highest ever obtained for CB2R
ligands. In particular, the (p-fluorobenzyl)-1,8-naphthyridine
derivatives 10 and cis-13 exhibited the highest affinity, with Ki
values of 0.7 nM. The derivatives 6, 12, cis-12, 13, and cis-13
showed very remarkable selectivity with a Ki(CB1)/Ki(CB2) ratio
>200.
Figure 4. CB2 and GAPDH mRNA levels were determined by RT-
PCR on Jurkat and U87MG cell lines. On U87MG cells, CB2 fragment
was obtained after a further amplification of PCR product: (I) single
amplification; (II) reamplification.
by the selective CB2 antagonist 20 (100 nM, 30 min of
preincubation, 37 °C) but not by the selective CB1 antagonist
1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-piperidin-
1-ylpyrazole-3-carboxamide (22, AM251)28 (100 nM, 30 min
of preincubation, 37 °C) (see Figure 3). These results suggest
that the derivative 12 exerts a CB2-mediated inhibitory action
on immunological human basophil activation.
Furthermore, the concentration-dependent activity shown by
derivative 12 in human basophils and in Jurkat cells strongly
suggests that this compound and the other 1,8-naphthyridin-
2(1H)-on-3-carboxamides possess agonist properties at CB2R.
These results indicate that 1,8-naphthyridin-2(1H)-on-3-
carboxamides represent new scaffolds that may be suitable for
the development of new potent and selective CB2R agonists to
be used in several therapeutic areas.
Cytotoxicity Assay. The derivative 12 was also evaluated
for its anticancer activity on the human T cell leukemia line
Jurkat and the human glioblastoma cell line U87MG. Both lines
were obtained from the ICLC (Interlab Cell Line Collection)
cell bank (Genoa, Italy). Compound 12 was evaluated in parallel
with the known selective CB2R agonist (2-methyl-1-propyl-1H-
indol-3-yl)-1-naphthalenylmethanone (23, JWH015).29 Jurkat
cells are a cell line with a good level of CB2R expression (Figure
4). Exposure to 12 in the range 0.1-10 µM for 48 h induced a
concentration-dependent decrease in cell viability. A similar
behavior was observed with the known CB2 agonist 23, whose
cytotoxicity matched that previously reported.29 The 1,8-
naphthyridin-2(1H)-on-3-carboxamide derivatives 12 and 23
showed IC50 values that were not statistically different (Table
2). In addition, 1 µM [6-iodo-2-methyl-1-(2-morpholin-4-
ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (24, AM630)30
significantly antagonized the cytotoxic effect of 12 on Jurkat
cells (Figure 5), confirming its CB2-mediated mechanism.
Experimental Section
Chemistry. Melting points were determined 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 spectra
were recorded with a Bruker AC-200 spectrometer in δ units with
TMS as an internal standard. Microwave-assisted reactions were run
in a CEM microwave synthesizer. Mass spectra were performed with
a Hewlett-Packard MS system 5988. 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 analytical HPLC system consisted of a Thermo Finnigan
SpectraSystem SN4000 system controller, coupled to a P2000 pump,
a SCM1000 degasser, and a UV2000 UV detector at an operation
wavelength of 280 nm (Thermo Finnigan, Waltham, MA). Separation
On U87MG cells, which express CB2R at lower levels (a PCR
electrophoretic band was evident only after reamplification)

3648 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Manera et al.
was performed on a 150 mm × 4.6 mm ChromoSep column packed
with 5 µm C18 particles, preceded by a ChromSep guard column
Inertsil 5 ODS-3 (Varian). The mobile phase, delivered at a flow rate
of 1.0 mL/min, consisted of methanol and water (75:25, v/v). HPLC
grade methanol was acquired from Sigma-Aldrich (Sydney, Australia),
and the water used was of Milli-Q grade purified by a Milli-Q UV
purification system (Millipore Corporation, MA).
N-Cyclohexyl-1-benzyl-1,8-naphthyridin-2(1H)-on-3-carboxa-
mide (3). Yield 61%; mp 154-156 °C (crystallized from hexane);
MS m/z 361 (M+); 1H NMR (DMSO) δ 9.58 (d, 1H, NH), 8.96 (s,
1H, H4), 8.76 (dd, 1H, H7), 8.54 (dd, 1H, H5), 7.45 (m, 1H, H6),
7.24 (m, 5H, Ar), 5.72 (s, 2H, CH2), 3.85 (m, 1H, CH), 1.89-1.30
(m, 10H, cyclohexyl). Anal. (C22H23N3O2) C, H, N.
N-Cyclohexyl-1-(p-fluorobenzyl)-1,8-naphthyridin-2(1H)-on-
3-carboxamide (4). Yield 8.44%; mp 203-205 °C (crystallized
from hexane); MS m/z 379 (M+); 1H NMR (DMSO) δ 9.56 (d,1H,
NH) 8.95 (s, 1H, H4), 8.77 (dd, 1H, H7), 8.53 (dd, 1H, H5), 7.47
(m, 1H, H6), 7.31 (m, 2H, Ar), 7.10 (m, 2H, Ar), 5.68 (s, 2H, CH2),
3.80 (m, 1H, CH), 1.88-1.22 (m, 10H, cyclohexyl). Anal.
(C22H22FN3O2) C, H, N.
1,8-Naphthyridin-2(1H)-on-3-carboxylic Acid Ethyl Ester
(14).24 A solution of 1.0 g (8.19 mmol) of 2-aminopyridin-3-
carboxaldehyde, 1.85 mL of diethyl malonate (12.18 mmol), and
few drops of piperidine was heated at 120 °C for 20 h. After the
mixture was cooled, the precipitate formed was treated with ethyl
ether and crystallized from ethyl acetate to obtain 14 (1.26 g, 76%)
1
mp 182-185 °C; H NMR (DMSO) δ 8.60 (dd, 1H, H7), 8.50 (s,
N-Cyclohexyl-1-butyl-1,8-naphthyridin-2(1H)-on-3-carboxa-
mide (5). Yield 55%; mp 152-154 °C (crystallized from hexane);
MS m/z 327 (M+); 1H NMR (DMSO) δ 9.69 (d, 1H, NH), 8.88 (s,
1H, H4), 8.79 (dd, 1H, H7), 8.49 (dd, 1H, H5), 7.45 (m, 1H, H6),
4.47 (t, 2H, CH2), 3.82 (m, 1H, CH), 1.89-0.88 (m, 17H,
cyclohexyl + 2CH2 + CH3). Anal. (C19H25N3O2) C, H, N.
N-(4-Methylcyclohexyl)-1-(2-morpholin-4-ylethyl)-1,8-naph-
thyridin-2(1H)-on-3-carboxamide (6). Yield 33%; crystallized
1H, H4), 8.26 (dd, 1H, H5), 7.29 (m, 1H, H6), 4.37 (q, 2H, CH2),
1.29 (t, 3H, CH3).
General Procedure for the Preparation of N1-Substituted
1,8-Naphthyridin-2(1H)-on-3-carboxamides 15-19. A mixture
of 1 mmol of ethyl ester 14 and 10 mmol of the appropriate amine
was heated in microwave at 140 °C for 1 h (power 200 W, pressure
100 psi, stirring on). After cooling, the reaction mixture was treated
with ethyl ether to give a solid residue which was collected by
filtration and purified by crystallization.
1
from ethyl acetate; MS m/z 398 (M+); H NMR (DMSO) δ 9.90
and 9.52 (2d, 1H, NH), 8.90 (s, 1H, H4), 8.79 (dd, 1H, H7), 8.50
(dd, 1H, H5), 7.45 (m, 1H, H6), 4.65 (t, 2H, CH2), 4.00 and 3.75
(2m, 1H, CH), 3.51 (m, 4H, morpholine), 2.56 (m, 6H, morpholine
+ CH2), 1.90-0.97 (m, 9H, cyclohexyl), 0.92 and 0.88 (2d, 3H,
CH3). Anal. (C22H30N4O3) C, H, N.
N-Cyclohexyl-1,8-naphthyridin-2(1H)-on-3-carboxamide (15).
1
Yield 88%; mp 278-280 °C (crystallized from ethyl acetate); H
NMR (DMSO) δ 12.03 (br,1H, NH), 9.70 (d, 1H, NH), 8.85 (s,
1H, H4), 8.66 (dd, 1H, H7), 8.42 (dd, 1H, H5), 7.36 (m, 1H, H6),
3.85 (m, 1H, CH), 1.89-1.27 (m, 10H, cyclohexyl). Anal.
(C15H17N3O2) C, H, N.
N-(ꢀ-Phenylethyl)-1-benzyl-1,8-naphthyridin-2(1H)-on-3-car-
boxamide (7). Yield 98%; mp 220-222 °C (crystallized from ethyl
acetate); MS m/z 383 (M+); 1H NMR (DMSO) δ 9.64 (t, 1H, NH),
8.96 (s, 1H, H4), 8.77 (dd, 1H, H7), 8.53 (dd, 1H, H5), 7.47 (m,
1H, H6), 7.23 (m, 10H, Ar), 5.72 (s, 2H, CH2), 3.59 (m, 2H, CH2),
2.86 (t, 2H, CH2). Anal. (C24H21N3O2) C, H, N.
N-(4-Methylcyclohexyl)-1,8-naphthyridin-2(1H)-on-3-carbox-
1
amide (16). Yield 87%; crystallized from ethyl acetate; H NMR
(DMSO) δ 11.85 (br,1H, NH), 10.0 and 9.60 (2d, 1H, NH), 8.85
and 8.87 (2s, 1H, H4), 8.66 (dd, 1H, H7), 8.41 (dd, 1H, H5), 7.36
(m, 1H, H6), 4.11 and 3.85 (2m, 1H, CH), 2.00-1.05 (m, 9H,
cyclohexyl), 0.92 and 0.89 (2d, 3H, CH3). Anal. (C16H19N3O2) C,
H, N.
N-(4-Fluorophenylethyl)-1-benzyl-1,8-naphthyridin-2(1H)-on-
3-carboxamide (8). Yield 87%; mp 183-185 °C (crystallized from
1
ethyl acetate); MS m/z 401 (M+); H NMR (DMSO) δ 9.72 (brs,
N-Cycloheptyl-1,8-naphthyridin-2(1H)-on-3-carboxamide (17).
Yield 71%; mp 203-204 °C (crystallized from dichloromethane);
1H NMR (DMSO) δ 11.95 (br,1H, NH), 9.75 (d, 1H, NH), 8.84 (s,
1H, H4), 8.65 (dd, 1H, H7), 8.40 (dd, 1H, H5), 7.35 (m, 1H, H6),
4.10 (m, 1H, CH), 1.90-1.45 (m, 12H, cycloheptyl). Anal.
(C16H19N3O2) C, H, N.
1H, NH), 8.93 (s, 1H, H4), 8.78 (dd, 1H, H7), 8.43 (dd, 1H, H5),
7.36 (m, 8H, Ar + H6), 7.07 (m, 2H, Ar), 5.82 (s, 2H, CH2) 3.69
(m, 2H, CH2), 2.95 (t, 2H, CH2). Anal. (C24H20FN3O2) C, H, N.
N-Cycloheptyl-1-benzyl-1,8-naphthyridin-2(1H)-on-3-carbox-
amide (9). Yield 88%; mp 171-173 °C (crystallized from ethanol);
MS m/z 375 (M+); 1H NMR (DMSO) δ 9.62 (d, 1H, NH), 8.95 (s,
1H, H4), 8.76 (dd, 1H, H7), 8.53 (dd, 1H, H5), 7.46 (m, 1H, H6),
7.25 (m, 5H, Ar), 5.72 (s, 2H, CH2), 4.09 (m, 1H, CH), 1.90-0.85
(m, 12H, cycloheptyl). Anal. (C23H25N3O2) C, H, N.
N-Phenethyl-1,8-naphthyridin-2(1H)-on-3-carboxamide (18).
1
Yield 87%; mp 270-272 °C (crystallized from ethyl acetate); H
NMR (DMSO) δ 12.15 (br,1H, NH), 9.71 (t, 1H, NH), 8.86 (s,
1H, H4), 8.65 (dd, 1H, H7), 8.42 (dd, 1H, H5), 7.32 (m, 6H, H6 +
Ar), 3.59 (m, 2H, CH2), 2.85 (t, 2H, CH2). Anal. (C17H15N3O2) C,
H, N.
N-Cycloheptyl-1-(p-fluorobenzyl)-1,8-naphthyridin-2(1H)-on-
3-carboxamide (10). Yield 97.%; mp 161-163 °C (crystallized
1
from ethanol); MS m/z 393 (M+); H NMR (DMSO) δ 9.78 (d,
N-(4-Fluorophenethyl)-1,8-naphthyridin-2(1H)-on-3-carbox-
amide (19). Yield 84%; mp >300 °C (crystallized from ethanol);
1H NMR (DMSO) δ 11.15 (br,1H, NH), 9.68 (t, 1H, NH), 8.85 (s,
1H, H4), 8.65 (dd, 1H, H7), 8.40 (dd, 1H, H5), 7.33 (m, 3H, H6 +
Ar), 7.12 (m, 2H, Ar), 3.58 (m, 2H, CH2), 2.85 (t, 2H, CH2). Anal.
(C17H14FN3O2) C, H, N.
General Procedure for the Synthesis of N1-Substituted 1,8-
Naphthyridin-2(1H)-on-3-carboxamides 2-13. NaH (0.10 g, 2.00
mmol, 50% in mineral oil) was added to a solution of suitable 1,8-
naphthyridin-2(1H)-on-3-carboxamide derivatives 15-19 (0.81
mmol) in 6.5 mL of dry DMF. After 1 h, a suitable chloride (0.81
mmol) was added and the mixture was stirred for 24 h at room
temperature for compounds 2-5, 7-9, 12, and 13 or at 50 °C for
compounds 6 and 10 or at 70 °C for compound 11. After the mixture
was cooled, water was added and the solid obtained was collected
by filtration and was purified by crystallization.
N-Cyclohexyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-
2(1H)-on-3-carboxamide (2). Yield 56%; mp 148-151 °C (crys-
tallized from hexane); MS m/z 384 (M+); 1H NMR (DMSO) δ 9.75
(d, 1H, NH), 8.88 (s, 1H, H4), 8.78 (dd, 1H, H7), 8.50 (dd, 1H,
H5), 7.45 (m, 1H, H6), 4.64 (t, 2H, CH2), 3.90 (m, 1H, CH), 3.51
(m, 4H, morpholine), 2.57 (m, 6H, morpholine + CH2), 1.89-1.22
(m, 10H, cyclohexyl). Anal. (C21H28N4O3) C, H, N.
1H, NH), 8.88 (s, 1H, H4), 8.75 (dd, 1H, H7), 8.49 (dd, 1H, H5),
7.47 (m, 1H, H6), 7.32 (m, 2H, Ar), 7.05 (m, 2H, Ar), 5.78 (s, 2H,
CH2), 4.10 (m, 1H, CH), 1.88-1.01 (m, 12H, cycloheptyl). Anal.
(C23H24FN3O2) C, H, N.
N-Cycloheptyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-
2(1H)-on-3-carboxamide (11). Yield 64%; mp 154-158 °C
1
(crystallized from hexane); MS m/z 398 (M+); H NMR (CDCl3)
δ 9.77 (d, 1H, NH), 8.88 (s, 1H, H4), 8.68 (dd, 1H, H7), 8.08 (dd,
1H, H5), 7.28 (dd, 1H, H6), 4.78 (t, 2H, CH2), 4.10 (m, 1H, CH),
3.70 (m, 4H, morpholine), 2.72 (m, 6H, morpholine + CH2),
2.05-0.85 (m, 12H, cycloheptyl). Anal. (C22H30N4O3) C, H, N.
N-(4-Methylcyclohexyl)-1-benzyl-1,8-naphthyridin-2(1H)-on-
3-carboxamide (12). Yield 75%; crystallized from hexane; MS m/z
1
375 (M+); H NMR (DMSO) δ 9.84 and 9.47 (2d, 1H, NH), 8.97
(s, 1H, H4), 8.77 (dd, 1H, H7), 8.53 (dd, 1H, H5), 7.46 (m, 1H, H6),
7.30 (m, 5H, Ar), 5.73 and 5.78 (2s, 2H, CH2), 4.10 and 3.75 (2m,
1H, CH), 1.94-0.99 (m, 9H, cyclohexyl), 0.94 and 0.89 (2d, 3H,
CH3). Anal. (C23H25N3O2) C, H, N.
trans-N-(4-Methylcyclohexyl)-1-benzyl-1,8-naphthyridin-2(1H)-
on-3-carboxamide (trans-12) and cis-N-(4-Methylcyclohexyl)-
1-benzyl-1,8-naphthyridin-2(1H)-on-3-carboxamide (cis-12). Com-
pounds trans-12 and cis-12 were obtained from derivative 12 by
flash chromatography on a silica gel using hexane/AcOEt/MeOH,

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3649
10:1:0.1. trans-12: yield 25%; mp 152-154 °C (crystallized from
hexane); MS m/z 375 (M+); 1H NMR (DMSO) δ 9.47 (d, 1H, NH),
8.97 (s, 1H, H4), 8.77 (dd, 1H, H7), 8.53 (dd, 1H, H5), 7.46 (m,
1H, H6), 7.30 (m, 5H, Ar), 5.73, (s, 2H, CH2), 3.75 (m, 1H, CH),
1.94-0.99 (m, 9H, cyclohexyl), 0.89 (d, 3H, CH3). Anal.
(C23H25N3O2) C, H, N. cis-12: yield 20%; mp 157-159 °C
residual leukocyte-rich preparation was diluted 1:4 with a buffer
with the following composition: 20 mM HEPES, 130 mM NaCl, 5
mM KCl, 5 IU/mL sodium heparin, and 1.5 mg/mL bovine serum
albumin (BSA), at a pH of 7.4 (washing buffer). Aliquots of 10
mL were then carefully layered over 10 mL of Ficoll-Paque in 30
mL conical tubes (25 mm diameter) and centrifuged at room
temperature at 420g. After removal of the supernatant plasma, the
basophil-rich Ficoll-Paque layer was separated and the neutrophil-
rich buffy coat was discarded. The suspension was washed twice
with the washing buffer and centrifuged at 200g at 20 °C for 10
min. The pellets were then resuspended in a calcium-free mainte-
nance buffer composed of 20 mM HEPES, 130 mM NaCl, 5 mM
KCl, 5 mM Na3EDTA, and 1.5 mg/mL bovine serum albumin
(BSA) at a pH of 7.4. The pellets were then further processed as
described below. Upon isolation, cell viability (determined by trypan
blue exclusion) was always greater than 95%. The procedure used
resulted in the low handling of basophils. Low handling prevented
their aspecific activation, which could have occurred if high-
purifying procedures such as specific antibody-coated magnetic
beads had been used. Before the experiments were started, samples
from each basophil-rich leukocyte preparation were challenged for
their ability to respond to anti-IgE by flow cytometric assay
(described below). Poorly responsive preparations were discarded.
Flow Cytometric Analysis. Basophil-rich leukocyte pellets were
labeled with a saturating concentration of anti-IgE fluoresceine
isothiocyanate (FITC) conjugated antibodies and anti-CD203c
phycoerythrin (PE) conjugated antibodies. The fluorescent antibod-
ies were incubated with the pellets for 20 min at 4 °C. The cells
were then washed with buffer, centrifuged at 200g for 10 min at
room temperature, and resuspended in buffer. After the lysis of
residual erythrocytes, the leukocyte suspensions were analyzed by
a flow cytometer (Coulter XL, Coulter Cytometry, Hialeah, FL).
Because the separation technique provides a leukocyte preparation
with no more than 70% basophils, it was necessary to sort the
basophil-related events using appropriate electronic gates. Basophils
were recognized by their high expression of membrane-bound IgE
resulting in a high signal related to FITC fluorescence (emission
peak at 530 nm). IgE-negative cells were then gated out by
electronic subtraction. The fluorescent signal of PE (emission peak
at 575 nm) was used to characterize activated and nonactivated
cells. Before activation human basophils showed a low expression
of CD203c, which was strongly up-regulated after the activation
of the cells.
1
(crystallized from hexane); MS m/z 375 (M+); H NMR (DMSO)
δ 9.84 (d, 1H, NH), 8.97 (s, 1H, H4), 8.77 (dd, 1H, H7), 8.53 (dd,
1H, H5), 7.46 (m, 1H, H6), 7.30 (m, 5H, Ar), 5.78 (s, 2H, CH2),
4.10 (m, 1H, CH), 1.94-0.99 (m, 9H, cyclohexyl), 0.94 (d, 3H,
CH3). Anal. (C23H25N3O2) C, H, N.
N-(4-Methylcyclohexyl)-1-(p-fluorobenzyl)-1,8-naphthyridin-
2(1H)-on-3-carboxamide (13). Yield 80%; crystallized from
1
hexane; MS m/z 393 (M+); H NMR (DMSO) δ 9.85 and 9.43
(2d, 1H, NH), 8.95 (s, 1H, H4), 8.77 (dd, 1H, H7), 8.52 (dd, 1H,
H5), 7.47 (dd, 1H, H6), 7.31 (m, 2H, Ar), 7.10 (m, 2H, Ar), 5.72
and 5.69 (2s, 2H, CH2), 4.10 and 3.70 (2m, 1H, CH), 1.94-0.99
(m, 9H, cyclohexyl), 0.92 and 0.88 (2d, 3H, CH3). Anal.
(C23H24FN3O2) C, H, N.
trans-N-(4-Methylcyclohexyl)-1-(p-fluorobenzyl)-1,8-naphthy-
ridin-2(1H)-on-3-carboxamide (trans-13) and cis-N-(4-Methyl-
cyclohexyl)-1-(p-fluorobenzyl)-1,8-naphthyridin-2(1H)-on-3-
carboxamide (cis-13). Compounds trans-13 and cis-13 were
obtained from derivative 13 by flash chromatography on a silica
gel using hexane/AcOEt/MeOH, 10:1:0.1. trans-13: yield 22%; mp
1
161-163 °C (crystallized from hexane); MS m/z 393 (M+); H
NMR (DMSO) δ 9.43 (d, 1H, NH), 8.95 (s, 1H, H4), 8.77 (dd, 1H,
H7), 8.52 (dd, 1H, H5), 7.47 (m, 1H, H6), 7.31 (m, 2H, Ar), 7.10
(m, 2H, Ar) 5.69 (s, 2H, CH2), 3.70 (2m, 1H, CH), 1.94-0.99 (m,
9H, cyclohexyl), 0.88 (d, 3H, CH3). Anal. (C23H24FN3O2) C, H, N.
cis-13: yield 18%; mp 170-172 °C (crystallized from hexane); MS
m/z 393 (M+); 1H NMR (DMSO) δ 9.85 (d, 1H, NH), 8.95 (s, 1H,
H4), 8.77 (dd, 1H, H7), 8.52 (dd, 1H, H5), 7.47 (m, 1H, H6), 7.31
(m, 2H, Ar), 7.10 (m, 2H, Ar) 5.72 (s, 2H, CH2), 4.10 (2m, 1H,
CH), 1.94-0.99 (m, 9H, cyclohexyl), 0.92 (d, 3H, CH3). Anal.
(C23H24FN3O2) C, H, N.
CB1 and CB2 Receptor Binding Assays. 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) (Perkin-Elmer,
Italy). These membranes were incubated with [3H]-(-)-cis-3-[2-
hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropy-
l)cyclohexanol (25, [3H]CP55,940)31 (0.14 nM/Kd ) 0.18 nM and
0.084 nM/Kd ) 0.31 nM for CB1 and CB2 receptors, respectively)
as the high affinity ligand and displaced with 100 nM [(3R)-5-
methyl-3-(morpholin-4-ylmethyl)-2,3-dihydro[1,4]oxazino[2,3,4-hi]in-
dol-6-yl](2-naphthyl)methanone (26, WIN55212-2)32 as the heterol-
ogous competitor for nonspecific binding (Ki values 9.2 and 2.1
nM, respectively, for CB1R and CB2R). All compounds were tested
following the procedure described by the cell membrane manufac-
turer.33 Displacement curves were generated by incubating drugs
with 25 for 90 min at 30 °C. Ki values were calculated by applying
the Cheng-Prusoff equation34 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 ) 3 experiments.
Cytotoxicity Assay. Jurkat cells were maintained in RPMI 1640
medium supplemented with L-glutamine (2 mM), 15% fetal bovine
serum, and 1% of a 1:1 mixture of penicillin (50 IU/mL) and
streptomycin (50 µg/mL) (Roche Molecular Biochemicals, Milan,
Italy). U87MG cells were maintained in DMEM medium supple-
mented with 10% fetal bovine serum, 1% of a 1:1 mixture of
penicillin (50 IU/mL) and streptomycin (50 µg/mL) (Roche
Molecular Biochemicals, Milan, Italy). Exponentially growing
Jurkat (2 × 104) and U87MG cells (4 × 103) were seeded into
96-well plates in serum-free medium to avoid interactions of serum
proteins and cannabinoid molecules (Z. M. Zheng et al., 1993).
After 2 h of incubation (for in suspension Jurkat cells) and 24 h of
incubation (for adherent U87MG cells), they were exposed to
compound 12 or to the known selective CB2R agonist 23.
Preparation of Basophil-Rich Leukocyte Samples. Twenty
healthy donors were recruited in the transfusion unit of Careggi
General Hospital (Florence, Italy). The subjects did not suffer from
allergic diseases and had not taken any drug during the previous 4
weeks. They gave explicit informed consent to their enrollment in
this study. About 400 mL of venous blood was collected from each
subject, and 64 mL of a citrate solution (CPD) was added as an
anticoagulant. The blood was centrifuged at 3500 rpm (11 min, 20
°C) in a slow-stop centrifuge (Sorvall RC 12 BP, Kendro Laboratory
Products). Plasma was removed by an automatic press (NPBI
Compomat 64). After 24 h of gentle stirring in a platelet incubator
(Helmer) at 22 °C to reduce cell stress, the buffy coat was
centrifuged at 900 rpm (9 min, 20 °C). Platelet-rich plasma was
removed by the same automatic press. A volume of 30 mL of the
Compounds 12 (10 mM), 23 (10 mM), and 24 (1 mM) were
dissolved in DMSO. Mother solutions were diluted to working
concentrations with the appropriate medium (without serum). Cell
viability was measured using a method based on the cleavage of
the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-
benzene disulfonate (WST-1) to formazan by mitochondrial dehy-
drogenase activity (cell proliferation reagent WST-1; Roche, Milan,
Italy). Following drug exposure, WST-1 was added to each well.
After 60 min of incubation at 37 °C, the absorbance at 450 nm
was measured by a microplate reader (Wallac Victor II, Perkin-
Elmer, MA). Inhibition of cell viability was calculated after 48 h
of drug exposure, by comparing the number of viable cells after
treatment to the number of viable cells exposed to solvent alone
(controls). The IC50 value represents the drug concentration at which

3650 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Manera et al.
the absorbance subtracted from the relative blank (drug/solvent in
medium w/o cells) is 50% of that in controls. All experiments were
performed in triplicate, and results are expressed as the mean (
SE. Statistical difference among IC50 values was evaluated by means
of ANOVA analysis of variance and Bonferroni post-test. A p-value
less than 0.05 was taken to be significant.
Supporting Information Available: Chromatogram of 12, trans-
12, cis-12 (Figure S1); chromatogram of 13, trans-13, cis-13 (Figure
S2); elemental analysis data of the compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
Reverse Transcription Polymerase Chain Reaction (RT-
PCR) Analysis of CB2R Expression. To confirm the expression
of CB2R in the specific clones of both Jurkat and U87MG cell lines
used in this study, we performed an RT-PCR analysis. RNA was
isolated from (5 × 106) Jurkat and (2 × 106) U87MG cell lines
using the “SV Total RNA Isolation System” kit (Promega S.r.l.;
Milan, Italy). cDNAs were subsequently obtained from 1 µg of
total RNA using the “Quantitect Reverse Transcription” kit (Qiagen;
Milan, Italy). A DNase digestion step was included.
CB2 forward (5′-CTGGCTCCTGTTCATCGCCT-3′) and CB2
reverse (5′-GCTTCTTCTTTTGCCTCTGAC-3′) primers yielding
a 417 bp product (705-1121 position in Genbank accession
NM_001841.1) were used. The glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) gene was used as an expression standard and
was amplified with forward (5′-GTGAAGGTCGGTGTCAACG-
3′) and reverse (5′-GGTGAAGACGCCAGTAGACTC-3′) primers.
This process yielded a 300 bp product (85-384 position in Genbank
accession NM_002046.2).
PCR reactions were performed using the following parameters:
for CB2, 95 °C 15′ (one cycle), 95 °C 1′, 58.4 °C 1′, 72 °C 1′ (35
cycles), and 72 °C 10′ (one cycle); for GAPDH, 95 °C 15′ (one
cycle), 95 °C 1′, 55 °C 2′, 74 °C 1′ (36 cycles), and 74 °C 9′
(one cycle). The “HotStarTaq Master Mix” kit (Qiagen; Milan, Italy)
was used for all PCR reactions. All protocols were carried out in
a MyCycler thermal cycler (Bio-Rad, Milan, Italy), and PCR
products were run on ethidium bromide-stained 1% agarose gel.
Gel images were acquired under UV (Euroclone SpA, Siziano PV,
Italy) transillumination. The identity of PCR-products was con-
firmed by sequencing.
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shape score with the color score for the appropriate overlap of
groups with similar properties (donor, acceptor, hydrophobe, cation,
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default values. For compound 2, the number of conformations was
about 800, generated using Omega 2.1.0.39 Default parameters were
used with the following exceptions: the rms parameter was set to
0.2 and the intramolecular H bond21 was maintained activating the
fixfile parameter. The result of the docking study for compound 1
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Carboxamide DeriVatiVes as Agonists
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