Synthesis, molecular modeling and SAR study of novel pyrazolo[5,1-f][1,6]naphthyridines as CB2receptor antagonists/inverse agonists

Article abstract of DOI:10.1016/j.bmc.2016.08.055

Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affinity at CB₁and CB₂receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology, the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel compounds feature a p-tolyl (8a–i) or a 2,4-dichlorophenyl (8j) motif at the C₅-position of the tricyclic pyrazolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C₂-position includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, compound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the highest CB₂receptor affinity (K_i?=?33?nM) and a high degree of selectivity (K_iCB₁/K_iCB₂?=?173:1), whereas a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, K_i?=?53?nM) and the myrtanyl derivative 8j (K_i?=?67?nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were determined, showing antagonist/inverse agonist properties for such compounds. Docking studies conducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) disclosed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB₂inverse agonists.

Full text of DOI:10.1016/j.bmc.2016.08.055

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and SAR study of novel pyrazolo[5,1-f]
[1,6]naphthyridines as CB2 receptor antagonists/inverse agonists
a
b,
a
Antonio Dore a, Battistina Asproni a, , Alessia Scampuddu , Stefania Gessi , Gabriele Murineddu ,
⇑
⇑
Elena Cichero c, Paola Fossa c, Stefania Merighi b, Serena Bencivenni b, Gérard A. Pinna a
a Dipartimento di Chimica e Farmacia, Università degli Studi di Sassari, Via F. Muroni 23/a, 07100 Sassari, Italy
b Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università di Ferrara, Via Fossato di Mortara, 17-19, 44121 Ferrara, Italy
c Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV n. 3, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affin-
ity at CB1 and CB2 receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology,
the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophe-
nyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the
appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel com-
pounds feature a p-tolyl (8a–i) or a 2,4-dichlorophenyl (8j) motif at the C5-position of the tricyclic pyra-
zolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C2-position
includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, com-
pound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the
highest CB2 receptor affinity (Ki = 33 nM) and a high degree of selectivity (KiCB1/KiCB2 = 173:1), whereas
a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, Ki = 53 nM) and the
myrtanyl derivative 8j (Ki = 67 nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were
determined, showing antagonist/inverse agonist properties for such compounds. Docking studies con-
ducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) dis-
closed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine
scaffold as CB2 inverse agonists.
Received 27 May 2016
Revised 5 August 2016
Accepted 27 August 2016
Available online xxxx
Keywords:
Pyrazolo[5,1-f][1,6]naphthyridine
Cannabinoid receptors
CB2 antagonism/inverse agonism
Docking studies
Ó 2016 Published by Elsevier Ltd.
1. Introduction
cannabinol (
nabis sativa.6
D
9-THC) 3, the major psychoactive component of Can-
The endocannabinoid system indicates a whole signaling sys-
tem that comprises cannabinoid receptors, endogenous ligands
exemplified by N-arachidonoylethanolamine (AEA, anandamide)
1 and 2-arachidonoylglycerol (2-AG) 2 (Fig. 1) and enzymes for
ligand biosynthesis and inactivation.1,2 This signaling system plays
a crucial role in numerous physiological and pathological functions
and it is involved in maintaining the physiological steady state and
homeostasis.3
The CB1Rs are abundantly expressed in the central nervous sys-
tem (CNS) and are responsible for the psychotropic effects
observed with nonselective cannabinoid ligands.7–10 The CB2Rs
were initially identified as a peripherally restricted receptors pre-
dominantly expressed in cells of the human immune system,11–13
and therefore playing an important role in mediating the
immune-modulatory function.14 Subsequent data, however, have
also demonstrated the presence of CB2Rs in brain, albeit to a much
lesser extent,15 myocardium, cardiomyoblasts and endothelian
cells of various origins.16
Recent studies have demonstrated that CB2Rs are involved in
numerous diseases.17 Several selective CB2R agonists exhibited
analgesic activity in preclinical models of acute, inflammatory
and neuropathic pain.18–21 Furthermore, CB2R agonists have been
evaluated for efficacy in ameliorating disease progression in
in vivo models of multiple sclerosis,22 Alzheimer’s disease,23 as
well as amyotrophic lateral sclerosis.24 Recent findings highlight
There are two subtypes of cannabinoid receptors discovered so
far, the cannabinoid type 1 receptor (CB1R) and the cannabinoid
type 2 receptor (CB2R),4 which belong to the rhodopsin-like family
of G-protein-coupled receptors (GPCRs).5 Even before the
endogenous ligands were known, CBRs have been described to be
activated by terpenoid plant constituents, e.g., by
D
9-tetrahydro-
⇑
Corresponding authors. Tel.: +39 079 228749 (B.A.), +39 0532 455332 (S.G.).
E-mail addresses: asproni@uniss.it (B. Asproni), gss@unife.it (S. Gessi).
http://dx.doi.org/10.1016/j.bmc.2016.08.055
0968-0896/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

2
A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. The major endogenous ligands and
D
9-THC for cannabinoid receptors.
the emerging interest in the development of CB2R inverse agonists
effective for the control of immune cell mobility in arthritis and
autoimmune encephalomyelitis25 and for the treatment of osteo-
porosis via the inhibition of osteoclast differentiation.26 CB2R neu-
tral antagonist ligands have been recently described.27 Such
compounds could be useful to ascertain more in detail the role of
CB2 receptor in physiopathological conditions.
Because of the therapeutic potential of CB2 ligands, there has
been an increasing interest in identifying a huge diversity of chem-
ical scaffolds for CB2 interaction. Within this frame, hundreds of
bioactive CB2R ligands have been discovered17,18 and among them,
pyrazole derivatives 4 (SR144528)28 and 529 emerged as important
prototypical CB2 selective ligands (Fig. 2). Compound 4 from Sanofi
was identified as the first potent and selective antagonist/inverse
agonist of the CB2R. Molecular modeling studies carried out on
430 highlighted the importance of the C3 carboxamide moiety
together with N1-methylbenzyl and C5-chloromethylphenyl sub-
stituents for CB2R affinity. A fenchyl group as carboxamide sub-
stituent is important for productive CB2R interaction. Pyrazole
derivative 5 from our lab is a dihydroindenopyrazol-based com-
pound and belongs to one of the first tricycles to be described for
CBR affinity. Structure activity-relationship (SAR) studies per-
formed on compound 5 revealed that the flattening of the plane
of the tricyclic core was a determining factor in its very high
CB2-affinity, exceptional selectivity over CB1R, and CB2 agonist
activity in vitro. Tricyclic derivatives 631 and 732 feature, respec-
tively, a dihydrothienocyclopentapyrazole and benzofuropyrazole
scaffolds. Interestingly, both compounds kept the affinity for
CB2R in the nM range and were endowed with a good selectivity
over CB1R, even if it was lower than that elicited by compound 4
and 5.
Pursuing our interest in expanding SAR studies on CB2R, we
thought that the planar pyrazolo[5,1-f][1,6]naphthyridine
8
(Fig. 3) could be an attractive scaffold for designing novel CB2
ligands. Looking at the structure of compounds 4–7, we postulated
that the introduction of the carboxamide moiety and the pendant
N1-benzyl substituent into the tricyclic core 8 might provide novel
CB2 agonist, antagonist, inverse agonist ligands with potential ther-
apeutic value. Thus novel compounds that varied the carboxamide
moiety and the phenyl substitution were designed (Fig. 3 and
Table 1). In this paper we report the synthesis of compounds 8a–
k together with preliminary aspects of their receptor affinity, selec-
tivity, biological activities and molecular modeling studies. These
data are accompanied by a thorough in silico evaluation of the
pharmacokinetic and toxicity properties of the most potent deriva-
tives here disclosed, with the aim at gaining preliminary informa-
tion concerning their potentially drug-like profile.
2. Chemistry
The synthetic route to the title compounds 8a–k is outlined in
Scheme 1. Based on the AgOTf and proline-cocatalyzed multicom-
ponent methodology,33 the key intermediates ethyl 5-(p-tolyl)
pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl
5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-car-
boxylate (13) were synthesized from the appropriate o-alkyny-
laldehydes, p-toluenesulfonyl hydrazide (PTSH) and ethyl pyruvate.
Thus, 2-bromonicotinaldehyde 9 was allowed to react with
appropriate alkynes under the conventional Sonogashira condi-
tions to give the corresponding o-alkynylaldehydes 10,11 in good
yields. Compounds 10,11 were condensed in situ with PTSH in
the presence of AgOTf (10 mol%), DL-proline (10 mol%), to give
the corresponding hydrazones 100,110 (not isolated), which were
converted into the desired tricyclic pyrazoles 12,13 by sequential
treatment with ethyl pyruvate, heating the reaction mixture by
microwave irradiation to 50–60 °C, and with Na2CO3 at room
temperature. The starting o-alkynylaldehyde 10, bearing the elec-
tron-donating –CH3 group in para position to the triple bond of
the phenyl ring was found to be suitable to trigger the heterocy-
clization reaction,33 and provided the desired pyrazole ester 12
in 64% yield. Also the o-alkynylaldehyde 11, featuring the elec-
tron-withdrawing 2,4-dichlorophenyl group proved to be favorable
for the reaction, even if the corresponding pyrazole ester 13 was
obtained in 49% yield. The hydrolysis of 12,13 with NaOH in THF
afforded the acid precursors 14,15 in almost quantitative yield.
Finally, the acids 14 and 15 were treated with pivaloyl chloride
Figure 2. Chemical structures of CB2 selective ligands.
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Figure 3. Novel 5-aryl-pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamides 8a–k.
and then with the appropriate amine to provide the desired car-
3.2. Intrinsic activity by in vitro assay
boxamides 8a–k
(23–76% yield).
The compounds 8f, 8h, and 8j characterized by the best affinity
and selectivity toward CB2 receptors were investigated to evaluate
their activity as agonists or antagonists on CB2 receptors in func-
tional assays. In particular their ability to affect forskolin-stimu-
lated cAMP levels in human CB2 CHO cells has been evaluated
and compared to WIN 55,212-2. As shown in Figure 4, the refer-
3. Results and discussion
3.1. Cannabinoid receptor binding studies
ence agonist WIN 55,212-2 (0.1 lM) was able to inhibit forsko-
lin-stimulated cAMP levels of 52% and all the compounds under
examination were able to antagonize this effect in the range of
concentration 0.1–10 lM, with the 8h being the most potent com-
The CB1 and CB2 receptor binding affinities of the novel pyrazolo
[5,1-f][1,6]naphthyridine derivatives 8a–k were evaluated by radi-
oligand binding assays performed by using transfected human CB1
and CB2 Chinese hamster ovary (CHO) cells. [3H]CP-55,940 was
employed as radiolabeled ligand. The experimental data (IC50 val-
ues) were converted into Ki values.34 The receptor affinities are
shown in Table 1. For comparison, the Ki value of the mixed
CB1/CB2 ligand WIN 55,212-2 is reported. Our initial results showed
that the introduction of a fenchyl group at the C2 carboxamide por-
tion in combination with a p-tolyl group at the C5 position of the
pyrazolo[5,1-f][1,6]naphthyridine scaffold gave compound 8a
endowed with negligeable affinity for CB1R (KiCB1 = 2200 nM) and
low affinity for CB2R (KiCB2 = 800 nM). Further, replacement of
the N-fenchyl moiety of 8a with N-piperidinyl (8b) and N-cyclohe-
syl (8c) tail-pieces resulted in total loss of affinity for both CB1 and
CB2Rs. To further explore whether improvements in CBR-affinity
might be obtained by modifying the C2-carboxamide side group,
we next synthesized a small library of four monoterpene deriva-
tives (8d–g) and two adamantine compounds (8h,i). Interestingly,
all these compounds exhibited improved CB2R affinity, almost total
loss of affinity for CB1R, and improved CB1 to CB2 selectivity than
did 8a. Among the monoterpene series, compound 8f, featuring a
bornyl group, emerged for its high CB2R affinity (8f: Ki = 53 nM vs
8a: Ki = 800 nM) and CB1 to CB2 selectivity (8f: KiCB1/KiCB2 = 132:1
vs 8a: KiCB1/KiCB2 = 2.7:1). Concerning the adamantine derivatives,
compound 8h featuring a 1-adamantyl group, exhibited the higher
CB2R affinity and CB1 to CB2 selectivity among all the synthesized
compounds (8h: Ki = 33 nM, KiCB1/KiCB2 = 173:1). Finally, com-
pound 8j, derived from compound 8d by replacing the p-tolyl group
at the C5-position with a dichlorophenyl group, exhibited improved
CB2R affinity (8j: Ki = 67 nM vs 8d: Ki = 120 nM) and selectivity (8j:
KiCB1/KiCB2 = >149:1 vs 8d: KiCB1/KiCB2 = 83:1), highlighting the
versatility of the planar pyrazolo[5,1-f][1,6]naphthyridine scaffold
to determine CB2R affinity and selectivity.
pound, according to binding data.
To evaluate in more detail the potency of compounds to antag-
onize cAMP inhibition induced by WIN 55,212-2 a Schild analysis
was performed. Compound 8h shifted the concentration–response
curve of WIN 55,212-2 to the right, in a concentration-dependent
manner (Fig. 5A). A Schild plot of the data gave a line with a slope
near unity (1.05 + 0.06) and KB value of 316 + 47 nM (Fig. 5B). Anal-
ogously, compounds 8f and 8j present a KB value of 505 61 and
650 74 nM, respectively. Therefore, the compounds investigated
appear weaker in the functional assay respect to the binding test.
However, these shifts have been repeatedly reported in the litera-
ture for different GPCRs and also for CB2 receptors.35 Moreover, the
values of CB2 affinity in binding assay and functional tests have not
to be necessarily identical. What is interesting to note is that a
good correlation between receptor affinity (Ki) and efficacy (KB)
was observed because the order of potency between the affinity
and potency values is the same. Nevertheless, we could hypothe-
size that the discrepancy concerning the affinity values found
between binding and functional test derive from the different
experimental conditions between these assays, e.g., ionic strength
of the buffer, time and temperature of the incubations.
Furthermore 8f and 8j ligands were able to increase forskolin-
stimulated cAMP production at 1–10
lM, while 8h compound
started its significant effect at 0.1 M (Fig. 6). These results charac-
l
terize the compounds 8f, 8h, and 8j as antagonists/inverse agonists
at CB2R. Accordingly, it has been shown that also the commercially
available CB2 receptor ligand AM630 behaves as neutral antago-
nist, or agonist, or inverse agonist depending on the bioassay type
or cell line.36 This may be explained based on the two-state model
theory of GPCRs in which ligands depending on their affinity for
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

4
A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
Structures and binding dataa for compounds 8a–k and WIN 55,212-2
Compd
R
R0
Q
Receptor affinity (nM)
CB2 selectivity
KiCB1
KiCB2
KiCB1/KiCB2
8a
8b
8c
H
H
H
CH3
CH3
CH3
2200 190
>10,000
800 84
2.7
-
>1000
>10,000
2133 230
>4.7
8d
H
CH3
>10,000
120 15
240 28
>83
8e
8f
H
H
CH3
CH3
>10,000
>42
132
7000 680
53
7
8g
H
CH3
3300 270
280 30
12
8h
8i
H
H
CH3
CH3
5700 550
>10,000
33
2
173
>75
133 15
8j
Cl
Cl
Cl
Cl
>10,000
NDb
67
8
>149
8k
ND
WIN 55,212-2
13
2
2.5 0.3
5.2
a
Affinity of compounds to CB1R and CB2R was assayed using transfected human CB1 and CB2 CHO cells and [3H]CP-55,940 as radiolabeled ligand. Ki values are the
mean SEM of at least three independent experiments run in triplicate.
b
ND, not determined.
the receptor may shift it from an inactive to an active state,
when they are agonists, or vice versa, in the case of inverse
agonists. In addition, compounds not producing any shift in
active/inactive receptor ratio are defined neutral antagonists.37
Therefore, our compounds appear as inverse agonists
in the absence of WIN 55,212-2 while as antagonists in its
presence.
3.3. Molecular modeling studies
In this work, in order to rationalize the pharmacological results
concerning the most promising derivatives 8f, 8h and 8j and to
refine our computational model, which could guide further syn-
thetic efforts, docking studies on 8f, 8h and 8j were also performed
(see Supplementary data, Table 1). In this way, we disclosed the
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Scheme 1. Reagent and conditions: (i) p-tolylacetylene (for 10) or 2,4-dichlorophenylacetylene (for 11), Pd(PPh3)2Cl2, CuI, Et3N, dry DMF, MW 60 °C, 20 min; (ii) PTSH, AgOTf,
DL-proline, dry EtOH, rt 10 min. (iii) (a) Ethyl pyruvate, MW 60 °C, 3.5 h; (b) Na2CO3, rt 12 h; (iv) NaOH, THF, 40 °C, 3 h; (v) (a) pivaloyl chloride, Et3N, dry CH2Cl2, 0 °C to rt, 2 h;
(b) amine, CH2Cl2, Et3N, 0 °C to rt, 2.5 h.
of selectivity issue. Indeed, the CB2 V164 corresponds to an isoleu-
cine residue at the CB1 protein, determining specific steric require-
ments for the binding to CB1 and, therefore, for CB2 selectivity.
Notably, all these data are also supported by the computational
studies we performed within a series of tricyclic pyrazole carbox-
amides as CB2 inverse agonists.40 According to our calculations,
in this study, compounds 8f, 8h and 8j share quite a similar
behavior if compared with that of SR144528, displaying an overall
pattern of interactions including the required H-bonds and a
Figure 4. Inhibition of forskolin-stimulated cAMP levels (100%) by WIN 55,212-2
(0.1
(10
(10
lM) and antagonism by 8j, 8f, and 8h (0.1–10 lM) in hCB2CHO cells. Forskolin
lM) effect was set to 100%. The effect of compounds alone 8j, 8f, and 8h
l
M) is shown. Values are the mean SEM of four independent experiments
(N = 4). *P <0.05, compared with WIN 55,212-2; analysis was by ANOVA followed by
Dunnett’s test.
specific pattern of interactions probably related to the pyrazolo
[5,1-f][1,6]naphthyridine scaffold as CB2 inverse agonists.
This kind of approach relied on a ligand-based homology model
of the human CB2 receptor we previously built around the chemical
scaffold of the potent ligand 4 (SR144528), revealing a pool of key
residues involved in the antagonist binding.38 Notably, our results
were in agreement with those discussed by Montero and cowork-
ers about the putative antagonist binding site of the hCB2
receptor.39
Thus, according to our calculations and also in agreement with
recent data reported in literature, the interactions of the inverse
agonists with the receptor included H-bonds at least with S165
for weaker compounds, or both the T114 and S165 residues for
the most potent derivatives.35
In particular, the SR144528 4-chloro-3-methyl-phenyl and the
benzyl group were projected toward two hydrophobic pockets
including L195, Y190, W194 and I110, L169, respectively. On the
other hand, the fenchyl portion was surrounded by L160, V164,
F197 and F202.
Figure 5. (A) Concentration–response curves of WIN 55,212-2 in hCB2CHO cells in
the absence and presence of various concentrations of 8h. (B) Schild plot of 8h
against WIN 55,212-2. Abscissa, log of the molar concentration of the antagonist 8h.
Ordinate, log of the concentration ratio ꢀ1 (cr ꢀ1) of the agonist WIN 55,212-2.
Values are the mean SEM of four independent experiments (N = 4).
These findings allowed us to highlight the key role played by a
bulky group in proximity of the third hydrophobic pocket in terms
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

6
A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
*
*
*
*
*
*
*
Figure 6. Effect of 8j, 8f, and 8h (0.1–10
lM) expressed as% of increase of forskolin-
stimulated cAMP accumulation (100%) in hCB2CHO cells. Forskolin (10
l
M) effect
was set to 100%. Values are the mean SEM of four independent experiments
(N = 4). *P < 0.05, compared with Forskolin (10
lM); analysis was by ANOVA
followed by Dunnett’s test.
Figure 8. Docking poses of compound 8f (C atom: yellow) within the hCB2 inverse
agonist binding site. The most important residues are labeled.
number of p–p stacking and Van der Waals contacts with the three
aforementioned hydrophobic pockets (see Supplementary data,
Fig. 9). More in details, the most potent 8h derivative shows one
H-bond between the carbonyl oxygen atom and the S165 side-
chain, in tandem with a second H-bond with the T114 backbone
(Fig. 7).
In addition, the phenyl ring is engaged in
p–p stacking with
W194 while the tricyclic core proves to mimic the reference com-
pound fenchyl group. Accordingly, 8h is characterized by a very
favorable selectivity profile. Furthermore, the Q substituent is able
to play the same role of the SR144528 phenyl and benzyl sub-
stituents, partially occupying the region delimited by L107, I110,
L169, L182 and Y190.
As shown in Figures 8 and 9, the related analogues 8f and 8j dis-
play a highly similar binding mode, with the exception of the Q
group on 8j. Indeed, the presence of a methylene between the
nitrogen atom and the cycloaliphatic moiety increases the Q group
flexibility, thus it moves quite far from the receptor crevice sur-
rounded by L107, I110 and L182.
These results confirm that a promising affinity profile could be
obtained especially performing a proper set of H-bonds with T114
Figure 9. Docking poses of compound 8j (C atom: white) within the hCB2 inverse
agonist binding site. The most important residues are labeled.
and S165, while selectivity appears to be strictly related to the hin-
dered groups interacting with V164. On this basis, the computa-
tional studies here discussed are expected to pave the way for a
further design process and for the filtering of new more potent
and selective ligands.
3.4. In silico evaluation of pharmacokinetic properties
The computationally-driven prediction of descriptors related to
absorption, distribution, metabolism, excretion and toxicity prop-
erties (ADMET) allows to rely on a useful strategy accelerating
the lead compound discovery process.41
In this work, for the newly synthesized derivatives 8f, 8h, 8j and
also for 4, a series of ADMET properties were calculated. In details,
we took into account the logarithmic ratio of the octanol–water
partitioning coefficient (cLogP), extent of blood–brain barrier per-
meation (LogBB), rate of passive diffusion-permeability (LogPSA),
Figure 7. SR144528 (C atom: cyan) and compound 8h (C atom: deep magenta)
docking poses into the hCB2 inverse agonist binding site. The most important
residues are labeled.
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
human intestinal absorption (HIA), volume of distribution (Vd), the
role played by plasmatic protein binding (%PPB) and by the com-
pound affinity toward the human serum albumin (LogKHa SA), and
an overall perspective of the molecule oral bioavailability (%F).
In addition, we evaluated a number of descriptors predicting for
metabolism and toxicity profiles, including the compound poten-
tial behavior as P-glycoprotein substrate, the ability to act as cyto-
chrome P450 3A4 inhibitor or substrate, the median lethal dose
(LD50) related to oral administration.
NMR spectra were reported in d (ppm) downfield from tetram-
ethylsilane, and coupling constants (J) were expressed in Hertz.
Multiplicities are recorded as s (singlet), br s (broad singlet), d
(doublet), t (triplet), dd (doublet of doublets), ddd (doublet of dou-
blet douplets), m (multiplet). Atmospheric Pressure Ionization
Electrospray (API-ES) mass spectra were obtained on an Agilent
1100 series LC/MSD spectrometer. Elemental analyses were per-
formed with a PerkineElmer 2400 analyzer, and results were
within 0.40% of the calculated values. Microwave experiments
were carried out by a Biotage Iniziator-8-microwave system
(max pressure 20 bar, IR temperature sensor). TLC was performed
on Merck silica gel 60 TLC plates F254 and visualized using UV.
Flash chromatography (FC) was performed using Merck silica gel
60 (230–400 mesh ASTM).
As shown in Table 2, all the compounds are characterized by a
better favorable profile in terms of lipophilicity, being the calcu-
lated cLogP within 5 (Lipinski rules) for 8f and 8h and, in any case,
lower than that displayed by the reference compound 4, being
insoluble in water (solubility 6 0.05 mg/mL). Notably, all of them,
with the exception of 8j, show an overall adequate blood–brain
barrier permeation, showing LogBB and/or LogPSA values falling
in the recommended ranges (0 < LogBB < 1.5; ꢀ3 < LogPSA < ꢀ1).
Only 8j was characterized by a predicted weak ability to pass at
the central nervous system. All of them are able to be fully
adsorbed at the human intestinal membrane (HIA). The calculated
volume of distribution values and the potential binding to the plas-
matic proteins fall in the allowed ranges, being in any case consis-
tent with those shown for 4. As an overall consequence,
compounds 8f and 8h are endowed with a favorable or acceptable
bioavailability profile (%F), especially in comparison with 4.
Based on Table 3, none of the compounds here proposed should
be substrate of the P-glycoprotein, or be involved in cytochrome
P450 3A4 inhibition. On the contrary, they should be substrate
for the enzyme, showing comparable values with those displayed
by 4. Finally, 8f and 8h exhibit an acceptable toxicity profile, being
the estimated LD50 in the range of 320–430 mg/kg for mouse after
oral administration, a value highly comparable with that calculated
for the reference compound.
2-Bromonicotinaldehyde (9), amines and p-tolylacetylene were
purchased by Sigma–AldrichÒ. Fenchylamine42 and 2,4-
dichlorophenylacetylene43 were synthesized according to litera-
ture procedure. Title compounds 8a, 8h and 8i were tested as
hydrochloride salts.
5.1.2. General procedure I. Synthesis of o-alkynylaldehydes
(10,11)
A mixture of 2-bromonicotinaldehyde (9) (1 equiv, 2.68 mmol),
the appropriate alkyne (1.2 equiv, 3.22 mmol), Pd(PPh3)2Cl2
(0.04 equiv, 0.11 mmol), CuI (0.07 equiv, 0.20 mmol), Et3N
(1.5 equiv, 4.02 mmol), in dry DMF (16 mL), was stirred under
argon atmosphere using microwave irradiation at 60 °C for
20 min. The reaction mixture was cooled to room temperature,
quenched with Et2O (200 mL) and the solid filtered off. The organic
solution was evaporated to afford a crude residue which was puri-
fied by FC (petroleum ether/AcOEt 8:2).
5.1.2.1. 2-(p-Tolylethynyl)nicotinaldehyde (10).44
General
procedure I was used to convert 9 and p-tolylacetylene into the
title product. Yellow solid (0.485 g, 82%); Rf 0.37 (AcOEt/petroleum
ether 2:8); mp 97–99 °C (96–100 °C); 1H NMR (CDCl3) 2.39 (s, 3H),
7.20 (d, J = 7.6 Hz, 2H), 7.38 (dd, J = 4.8 Hz, J = 7.6 Hz, 1H), 7.63 (d,
J = 7.6 Hz, 2H), 8.20 (d, J = 8.0 Hz, 1H), 8.81 (d, J = 4.8 Hz, 1H),
10.66 (s, 1H); 13C NMR (CDCl3) 21.8, 84.4, 96.7, 118.3, 123.2,
129.5, 131.8, 132.3, 134.9, 140.5, 146.4, 154.6, 191.1; API-ES m/z:
[M+H]+ calcd for C15H12NO: 222.1, found: 222.1.
4. Conclusion
In conclusion, a small series of pyrazolo[5,1-f][1,6]naphthyridi-
nes 8 have been designed and synthesized for CB1/CB2R interac-
tion. Among the synthesized compounds, 8f, 8h and 8j exhibited
affinity levels for CB2R in the near nM range (33–67 nM) with a
high degree of selectivity for CB2 compared to CB1 (KiCB1/KiCB2:
132–173). According to in vitro assays based on the effects of for-
skolin-stimulated cAMP levels in human CB2 CHO cells, com-
pounds 8f, 8h and 8j act as antagonist/inverse agonists. Docking
studies carried out on such compounds and the reference 4
(SR144528) disclosed the specific pattern of interactions probably
related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB2
inverse agonists. Our preliminary results point out the versatility
of the planar pyrazolo[5,1-f][1,6]naphthyridine architecture to
provide novel chemical entities for CB2R interaction and pave the
way for a further design process and for the filtering of new more
potent and selective CB2 ligands. Further work on this interesting
class of CB2R ligands will be disclosed in due course.
5.1.2.2.
(11).
2-((2,4-Dichlorophenyl)ethynyl)nicotinaldehyde
General procedure I was used to convert 9 and 2,4-
dichlorophenylacetylene into the title product. Cream solid
(0.376 g, 51%); Rf 0.28 (AcOEt/petroleum ether 2:8); mp 160–
162 °C; 1H NMR (CDCl3) 7.29 (dd, J = 1.6 Hz, J = 8.4 Hz, 1H,), 7.44
(dd, J = 4.8 Hz, J = 8.0 Hz, 1H), 7.49 (d, J = 1.6 Hz, 1H), 7.62 (d,
J = 8.4 Hz, 1H), 8.23 (dd, J = 1.2 Hz, J = 8.0 Hz, 1H), 8.84 (dd,
J = 1.6 Hz, J = 4.8 Hz, 1H), 10.73 (s, 1H); 13C NMR (CDCl3) 90.3,
91.4, 120.2, 123.8, 127.5, 129.8, 132.4, 134.7, 134.9, 136.6, 137.6,
145.6, 154.7, 190.9; API-ES m/z: [M+H]+ calcd for C14H8Cl2NO:
275.9, found: 275.8. Anal. (C14H7Cl2NO) C, H, N.
5.1.3. General procedure II. Synthesis of pyrazole esters (12,13)
5. Experimental protocols
5.1. Chemistry
A
mixture of the appropriate o-alkynylaldehyde (1 equiv,
0.452 mmol), PTSH (1.05 equiv, 0.474 mmol), AgOTf (0.1 equiv,
0.0452 mmol), DL-proline (0.1 equiv, 0.0452 mmol), in dry EtOH
(4 mL) was stirred at room temperature for 10 min. Ethyl pyruvate
(10 equiv, 4.520 mmol) was added, and the whole heated by
microwave irradiation to 60 °C for 3.5 h. The reaction mixture
was cooled to room temperature; Na2CO3 (6 equiv, 2.712 mmol)
was added and the mixture stirred at room temperature for 12 h.
The solvent was evaporated to afford a crude residue which was
purified by FC (petroleum ether/AcOEt 6:4).
5.1.1. General methods
Melting points were obtained on a Koffler melting point appara-
tus and are uncorrected. IR spectra were recorded as KBr pellets
with a Jasco FT/IR 460 plus spectrophotometer and are expressed
in
Nanobody 400 MHz spectrometer with 1H and 13C being observed
at 400 and 100.6 MHz, respectively. Chemical shifts for 1H and 13
m
(cmꢀ1). 1H NMR spectra were taken on a Bruker AVANCE III
C
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

8
A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 2
Calculated ADMET descriptors related to absorption and distribution properties
Compd
cLogP
LogBBa
LogPSAb
HIA (%)c
Vd (l/kg)d
%PPB
LogKaHSA
%F (oral)
8f
8h
8j
5.00
4.89
6.12
6.19
0.44
0.27
0.00
0.37
ꢀ1.1
ꢀ1.1
ꢀ1.3
ꢀ1.3
100
100
100
100
2.7
2.7
3.6
4.0
97.92
98.26
99.00
98.96
4.68
4.94
5.47
5.39
97.3
78.6
34.6
89.0
SR144528 (4)
a
b
c
Extent of brain penetration based on ratio of total drug concentrations in tissue and plasma at steady-state conditions.
Rate of passive diffusion-permeability. PSA represents Permeability-Surface area product and is derived from the kinetic equation of capillary transport.
HIA represents the human intestinal absorption, expressed as percentage of the molecule able to pass through the intestinal membrane.
Prediction of volume of distribution (Vd) of the compound in the body.
d
Table 3
Calculated ADMET descriptors related to metabolism, excretion and toxicity properties
Compd
P-glycoprotein substrate (R.I. P 0.40)
CYP3A4
LD50 (mg/kg)a (R.I. P 0.40)
Inhibitor (IC50 < 10
lM) (R.I. P 0.40)
Substrate (R.I. P 0.40)
8f
8h
8j
Not
Not
Not
Not
0.50
0.32
0.21
0.39
0.99
0.98
0.98
0.99
430
320
270
440
SR144528 (4)
a
Acute toxicity (LD50) for mouse after oral administration (RI: reliability index). Borderline-allowed values for reliability parameter are P0.3, the most predictive fall in the
range 0.50–1.
5.1.3.1. Ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-
5.1.5. General procedure IV. Synthesis of carboxamides (8a–k)
A solution of appropriate acid (1 equiv. 0.495 mmol) and Et3N
(3 equiv. 1.50 mmol) in dry CH2Cl2 (10 mL) was cooled at 0 °C;
pivaloyl chloride (2.5 equiv. 1.237 mmol) was dropwise added
and the whole warmed to room temperature and stirred for 2 h.
This mixture was dropwise added at 0 °C to a solution of appropri-
ate amine or hydrazine (0.9 equiv. 0.990 mmol) and Et3N
(1.5 equiv. 1.500 mmol) in dry CH2Cl2 (5 mL) and the whole stirred
at room temperature for 2.5 h. The reaction mixture was quenched
with an aqueous saturated solution of NH4Cl and extracted with
CH2Cl2. The organic phase was dried (Na2SO4) and evaporated to
give a crude residue which was purified by FC (petroleum ether/
AcOEt 3:7).
carboxylate (12).
General procedure II was used to convert
10 into the title product. White solid (0.100 g, 64%); Rf 0.34
(AcOEt/petroleum ether 4:6); mp 176–179 °C; IR 1716 (CO); 1H
NMR (CDCl3) 1.44 (t, J = 7.2 Hz, 3H), 2.46 (s, 3H), 4.46 (q,
J = 7.2 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.46 (s, 1H), 7.50 (dd,
J = 4.8 Hz, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 8.40
(dd, J = 1.6 Hz, J = 8.0 Hz, 1H), 8.90 (dd, J = 1.6 Hz, J = 4.8 Hz, 1H);
13C NMR (CDCl3) 14.5, 21.6, 61.5, 102.0, 115.8, 119.8, 122.3,
129.4, 129.6, 129.7, 131.3, 139.7, 140.4, 142.6, 145.1, 146.4,
151.4, 162.7; API-ES m/z: [M+H]+ calcd for C20H18N3O2: 332.1,
found: 332.1. Anal. (C20H17N3O2) C, H, N.
5.1.3.2. Ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naph-
thyridine-2-carboxylate (13).
General procedure II was used
5.1.5.1. N-Fenchyl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-
to convert 11 into the title product. White solid (0.085 g, 49%); Rf
0.31 (AcOEt/petroleum ether 4:6); mp 231–234 °C; IR 1729 (CO);
1H NMR (DMSO-d6) 1.31 (t, J = 6.4 Hz, 3H), 4.34 (q, J = 6.4 Hz, 2H),
7.49 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.91
(s, 1H), 8.06 (s, 1H), 8.91–9.00 (m, 2H); 13C NMR (DMSO-d6) 14.2,
60.9, 103.1, 117.3, 119.9, 123.6, 127.8, 129.2, 130.7, 132.2, 133.5,
134.6, 135.5, 137.7, 138.7, 144.5, 144.8, 151.7, 161.6; API-ES m/z:
2-carboxamide (8a).
General procedure IV was used to con-
vert 14 and fenchylamine into the title product. White solid
(0.050 g, 23%). Rf 0.49 (AcOEt/petroleum ether 7:3); mp 220–
222 °C; IR 3404 (NH), 1673 (CO); 1H NMR (CDCl3) 0.86 (s, 3H),
1.11 (s, 3H), 1.14–1.22 (m, 4H), 1.23–1.30 (m, 1H), 1.29–1.39 (m,
1H), 1.43–1.55 (m, 1H), 1.66–1.77 (m, 2H), 1.80 (d, J = 2.8 Hz,
1H), 2.49 (s, 3H), 3.83 (d, J = 9.6 Hz, 1H), 7.23 (d, J = 9.6 Hz, 1H),
7.36 (d, J = 8.0 Hz, 2H), 7.46 (s, 1H), 7.50 (dd, J = 4.4 Hz, J = 8.0 Hz,
1H), 7.65 (s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 8.42 (d, J = 8.0 Hz, 1H),
8.89 (d, J = 4.4 Hz, 1H); 13C NMR (CDCl3) 19.8, 21.3, 21.7, 26.1,
27.3, 31.1, 39.7, 42.8, 48.3, 48.9, 63.3, 99.7, 115.2, 119.9, 122.4,
129.0, 129.6, 129.8, 131.5, 140.2, 140.4, 141.9, 146.4, 147.9,
151.3, 162.5; API-ES m/z: [M+H]+ calcd for C28H31N4O: 439.2,
found: 439.2. Anal. (C28H30N4O) C, H, N.
[M+H]+ calcd for C19H14Cl2N3O2: 386.0, found: 386.3. Anal. (C19
13Cl2N3O2) C, H, N.
-
H
5.1.4. General procedure III. Synthesis of pyrazole acids (14,15)
To a solution of appropriate pyrazole ester (1 equiv, 1.41 mmol)
in THF (10 mL), 10% aqueous NaOH solution (10 mL) was added
dropwise, and the whole stirred at 40 °C for 3 h. The solution
was cooled at room temperature and neutralized with 4N HCl.
The solid was filtered, washed with acetone to give a crude product
which was used in the following step with no further purification.
5.1.5.2. N-(piperidin-1-yl)-5-(p-tolyl)pyrazolo[5,1-f][1,6]naph-
thyridine-2-carboxamide (8b).
General procedure IV was
used to convert 14 and N-aminopiperidine into the title product.
Cream solid (0.078 g, 41%). Rf 0.11 (AcOEt/petroleum ether 7:3);
mp 207 °C; IR 3398 (NH), 1687 (CO); 1H NMR (CDCl3) 1.41–1.49
(m, 2H), 1.71–1.79 (m, 4H), 2.50 (s, 3H), 2.90 (t, J = 5.2 Hz, 4H),
7.39 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H), 7.51 (dd, J = 4.4 Hz, J = 8.0 Hz,
1H), 7.72 (s, 1H), 7.76 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 8.41 (dd,
J = 1.6 Hz, J = 8.0 Hz, 1H), 8.89 (dd, J = 1.6 Hz, J = 4.4 Hz, 1H); 13C
NMR (CDCl3) 21.7, 23.4, 25.5, 57.0, 100.6, 115.6, 119.8, 122.5,
129.3, 129.5, 129.9, 131.6, 140.0, 140.4, 142.0, 146.3, 147.3,
5.1.4.1. 5-(p-Tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxy-
lic acid (14).
General procedure III was used to convert 12
into the title product. White solid (0.443 g, 95%). IR 3422 (OH),
1636 (CO).
5.1.4.2.
dine-2-carboxylic acid (15).
5-(2,4-Dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyri-
General procedure III was used
to convert 13 into the title product. Yellow solid (0.352 g, 70%).
IR 3448 (OH), 1718 (CO).
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
151.4, 159.3; API-ES m/z: [M+H]+ calcd for C23H24N5O: 386.2,
162.0; API-ES m/z: [M+H]+ calcd for C28H31N4O: 439.2, found:
found: 386.0. Anal. (C23H23N5O) C, H, N.
439.2. Anal. (C28H30N4O) C, H, N.
5.1.5.7.
dine-2-carboxamide (8g).
N-Menthyl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyri-
General procedure IV was used
5.1.5.3.
ridine-2-carboxamide (8c).
N-Cyclohexyl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthy-
General procedure IV was used
to convert 14 and menthylamine into the title product. White solid
(0.165 g, 76%). Rf 0.58 (AcOEt/petroleum ether 7:3); mp 151–
152 °C; IR 3399 (NH), 1664 (CO); 1H NMR (CDCl3) 0.85–0.93 (m,
11H), 1.11–1.23 (m, 2H), 1.49–1.60 (m, 1H), 1.66–1.77 (m, 2H),
1.88–2.01 (m, 1H), 2.02–2.11 (m, 1H), 2.50 (s, 3H), 3.93–4.05 (m,
1H), 6.75 (d, J = 9.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H),
7.51 (dd, J = 4.4 Hz, J = 8.0 Hz, 1H), 7.68 (s, 1H), 7.86 (d, J = 8.0 Hz,
2H), 8.41 (d, J = 8.0 Hz, 1H), 8.89 (d, J = 4.4 Hz, 1H); 13C NMR
(CDCl3) 16.8, 21.1, 21.7, 22.3, 24.5, 27.3, 32.1, 34.7, 43.1, 48.2,
50.1, 100.1, 115.4, 119.9, 122.4, 129.3, 129.5, 130.0, 131.5, 140.1,
140.3, 142.1, 146.3, 148.1, 151.3, 161.2; API-ES m/z: [M+H]+ calcd
for C28H33N4O: 441.2, found: 441.5. Anal. (C28H32N4O) C, H, N.
to convert 14 and N-cyclohexylamine into the title product. White
solid (0.051 g, 27%). Rf 0.50 (AcOEt/petroleum ether 7:3); mp 229–
230 °C; IR 3406 (NH), 1670 (CO); 1H NMR (CDCl3) 1.16–1.32 (m,
3H), 1.35–1.48 (m, 2H), 1.60–1.69 (m, 1H), 1.71–1.80 (m, 2H),
1.97–2.06 (m, 2H), 2.49 (s, 3H), 3.92–4.05 (m, 1H), 6.89 (d,
J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.40 (s, 1H), 7.50 (dd,
J = 4.8 Hz, J = 8.0 Hz, 1H), 7.67 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 8.41
(d, J = 8.0 Hz, 1H), 8.88 (d, J = 3.20 Hz, 1H); 13C NMR (CDCl3) 21.6,
25.1, 25.7, 33.3, 48.3, 100.1, 115.4, 119.9, 122.4, 129.3, 129.5,
129.9, 131.5, 140.1, 140.3, 142.1, 146.3, 148.2, 151.3, 161.0; API-
ES m/z: [M+H]+ calcd for C24H25N4O: 385.2, found: 385.5. Anal.
(C24H24N4O) C, H, N.
5.1.5.8. N-Adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthy-
5.1.5.4. N-Myrtanyl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridi-
ridine-2-carboxamide (8h).
General procedure IV was used
ne-2-carboxamide (8d).
General procedure IV was used to
to convert 14 and 1-adamantylamine into the title product. White
solid (0.12 g, 55%). Rf 0.54 (AcOEt/petroleum ether 7:3); mp 238–
240 °C; IR 3390 (NH), 1676 (CO); 1H NMR (CDCl3) 1.68–1.77 (m,
6H), 2.10–2.18 (m, 9H), 2.48 (s, 3H), 6.80 (s, 1H), 7.37 (d,
J = 8.0 Hz, 2H), 7.39 (s, 1H), 7.49 (dd, J = 4.4 Hz, J = 8.0 Hz, 1H),
7.61 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 8.40 (d, J = 8.0 Hz, 1H), 8.88
(d, J = 4.4 Hz, 1H); 13C NMR (CDCl3) 21.6, 29.6, 36.5, 41.7, 52.1,
99.8, 115.2, 119.9, 122.3, 129.2, 129.6, 129.9, 131.5, 140.1, 140.3,
142.0, 146.3, 148.9, 151.2, 160.9; API-ES m/z: [M+H]+ calcd for
convert 14 and myrtanylamine into the title product. White solid
(0.128, 59%). Rf 0.53 (AcOEt/petroleum ether 7:3); mp 164–
166 °C; IR 3448 (NH), 1670 (CO); 1H NMR (CDCl3) 0.91 (d,
J = 9.6 Hz, 1H), 1.10 (s, 3H), 1.21 (s, 3H), 1.51–1.62 (m, 1H); 1.85–
2.01 (m, 5H), 2.28–2.40 (m, 2H), 2.49 (s, 3H), 3.41–3.53 (m, 2H),
7.08 (t, J = 5.8 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H), 7.51
(dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 7.68 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H),
8.42 (dd, J = 0.8 Hz, J = 8.0 Hz, 1H), 8.89 (d, J = 3.2 Hz, 1H); 13C
NMR (CDCl3) 19.9, 21.66, 23.4, 26.2, 28.1, 33.4, 38.9, 41.5, 41.7,
44.0, 44.9, 100.1, 115.4, 119.9, 122.4, 129.3, 129.6, 129.9, 131.6,
140.1, 140.4, 142.1, 146.3, 148.0, 151.3, 161.9; API-ES m/z: [M
+H]+ calcd for C28H31N4O: 439.2, found: 439.5. Anal. (C28H30N4O)
C, H, N.
C28H29N4O: 437.2, found: 437.5. Anal. (C28H28N4O) C, H, N.
5.1.5.9.
N-Adamant-2-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naph-
General procedure IV was
thyridine-2-carboxamide (8i).
used to convert 14 and 2-adamantylamine hydrochloride into the
title product. White solid (0.153 g, 71%). Rf 0.39 (AcOEt/petroleum
ether 7:3); mp 218–220 °C; IR 3419 (NH), 1672 (CO); 1H NMR
(CDCl3) 1.68 (d, J = 12.8 Hz, 2H), 1.78 (s, 2H), 1.83–1.92 (m, 8H),
2.05 (s, 2H), 2.48 (s, 3H), 4.26 (d, J = 8.0 Hz, 1H), 7.36 (d,
J = 8.0 Hz, 2H), 7.44 (s, 1H), 7.46–7.54 (m, 2H), 7.66 (s, 1H), 7.91
(d, J = 8.0 Hz, 2H), 8.41 (d, J = 8.0 Hz, 1H), 8.88 (d, J = 4.4 Hz, 1H);
13C NMR (CDCl3) 21.6, 27.3, 27.5, 32.1, 32.2, 37.2, 37.7, 53.2, 99.8,
115.2, 119.9, 122.3, 129.1, 129.6, 129.8, 131.5, 140.1, 140.4,
142.0, 146.4, 148.1, 151.3, 161.0; API-ES m/z: [M+H]+ calcd for
5.1.5.5. N-Isopinocampheyl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naph-
thyridine-2-carboxamide (8e).
General procedure IV was
used to convert 14 and isopinocampheylamine into the title prod-
uct. Yellow solid (0.074 g, 34%). Rf 0.40 (AcOEt/petroleum ether
7:3); mp 215 °C; IR 3402 (NH), 1662 (CO); 1H NMR (CDCl3) 0.95
(d, J = 10.0 Hz, 1H), 1.11 (s, 3H), 1.17 (d, J = 7.2 Hz, 3H), 1.25 (s,
3H), 1.68 (ddd, J = 2.0 Hz, J = 6.0 Hz, J = 14.0 Hz, 1H), 1.87 (t,
J = 5.6 Hz, 1H), 1.93 (t, J = 7.0 Hz, 1H), 1.96–2.02 (m, 1H), 2.40–
2.48 (m, 1H), 2.50 (s, 3H), 2.63–2.73 (m, 1H), 4.45–4.56 (m, 1H),
6.89 (d, J = 9.2 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.42 (s, 1H), 7.51
(dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 7.69 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H),
8.43 (d, J = 8.0 Hz, 1H), 8.90 (d, J = 4.0 Hz, 1H); 13C NMR (CDCl3)
21.0, 21.7, 23.6, 28.2, 35.3, 37.1, 38.6, 41.8, 46.2, 47.9, 48.0,
100.2, 115.5, 119.9, 122.4, 129.3, 129.5, 129.9, 131.5, 140.1,
140.4, 142.1, 146.3, 148.1, 151.3, 161.4; API-ES m/z: [M+H]+ calcd
for C28H31N4O: 439.2, found: 439.4. Anal. (C28H30N4O) C, H, N.
C28H29N4O: 437.2, found: 437.4. Anal. (C28H28N4O) C, H, N.
5.1.5.10. N-Myrtanyl-5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]
naphthyridine-2-carboxamide (8j). General procedure IV
was used to convert 15 and myrtanylamine into the title product.
White solid (0.090 g, 37%). Rf 0.38 (AcOEt/petroleum ether 6:4);
mp 216–218 °C; IR 3403 (NH), 1687 (CO); 1H NMR (CDCl3) 0.90
(d, J = 9.6 Hz, 1H), 1.09 (s, 3H), 1.19 (s, 3H), 1.49–1.61 (m, 1H),
1.80–2.0 (m, 5H), 2.26–2.39 (m, 2H), 3.40–3.51 (m, 2H), 6.93 (t,
J = 6.0 Hz, 1H), 7.36 (s, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.51–7.60 (m,
2H), 7.63 (s, 1H), 7.67 (s, 1H), 8.45 (d, J = 8.4 Hz, 1H), 8.92 (d,
J = 4.4 Hz, 1H); 13C NMR (CDCl3) 19.8, 23.4, 26.2, 28.1, 33.3, 38.8,
41.5, 41.6, 44.0, 45.0, 100.2, 117.2, 120.6, 123.0, 127.5, 130.1,
130.8, 131.7, 132.8, 135.6, 136.6, 138.4, 139.4, 145.7, 148.4,
151.4, 161.8; API-ES m/z: [M+H]+ calcd for C27H27Cl2N4O: 493.2,
found: 493.3. Anal. (C27H26Cl2N4O) C, H, N.
5.1.5.6. N-Bornyl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-
2-carboxamide (8f).
General procedure IV was used to con-
vert 14 and bornylamine into the title product. Yellow solid
(0.093 g, 43%). Rf 0.52 (AcOEt/petroleum ether 7:3); mp 224–
225 °C; IR 3390 (NH), 1676 (CO); 1H NMR (CDCl3) 0.90 (s, 3H),
0.91 (s, 3H), 0.95 (dd, J = 4.4 Hz, J = 13.6 Hz, 1H), 1.01 (s, 3H),
1.18–1.28 (m, 1H), 1.38–1.50 (m, 1H), 1.55–1.65 (m, 1H), 1.71 (t,
J = 4.4 Hz, 1H), 1.76–1.88 (m, 1H), 2.37–2.47 (m, 1H), 2.49 (s, 3H),
4.41–4.50 (m, 1H), 7.17 (d, J = 9.2 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H),
7.45 (s, 1H), 7.51 (dd, J = 4.4 Hz, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.92
(d, J = 8.0 Hz, 2H), 8.42 (d, J = 8.0 Hz, 1H), 8.90 (dd, J = 1.2 Hz,
J = 4.4 Hz, 1H);); 13C NMR (CDCl3): 13.9, 18.9, 20.0, 21.7, 28.2,
28.5, 37.7, 45.2, 48.3, 50.0, 53.7, 99.9, 115.2, 119.9, 122.4, 129.1,
129.6, 129.8, 131.6, 140.2, 140.5, 142.0, 146.4, 148.0, 151.3,
5.1.5.11. N-Fenchyl-5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]-
naphthyridine-2-carboxamide (8k).
General procedure IV
was used to convert 15 and fenchylamine into the title product.
Cream solid (0.011 g, 44%). Rf 0.42 (AcOEt/petroleum ether 6:4);
mp 249–251 °C; IR 3387 (NH), 1671 (CO); 1H NMR (CDCl3) 0.78
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

10
A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(s, 3H), 1.07 (s, 3H), 1.12–1.22 (m, 5H), 1.42–1.53 (m, 1H), 1.55–
1.62 (m, 1H), 1.63–1.73 (m, 2H), 1.75–1.82 (m, 1H), 3.78 (d,
J = 9.6 Hz, 1H), 7.07 (d, J = 10.0 Hz, 1H), 7.39 (s, 1H), 7.46 (d,
J = 8.4 Hz, 1H), 7.54–7.60 (m, 2H), 7.62 (s, 1H), 7.65 (s, 1H), 8.46
(d, J = 8.4 Hz, 1H), 8.93 (d, J = 4.4 Hz, 1H); 13C NMR (CDCl3): 19.8,
21.6, 26.1, 27.3, 31.1, 39.7, 42.7, 48.3, 48.9, 63.4, 99.9, 117.2,
120.6, 123.0, 127.4, 129.8, 130.8, 131.7, 132.8, 133.0, 136.7, 138.5,
139.4, 145.8, 148.4, 151.4, 162.3; API-ES m/z: [M+H]+ calcd for
5.3. Molecular modeling studies
Compounds 8f, 8h and 8j were built, parameterized (Gasteiger-
Huckel method) and energy minimized within MOE using MMFF94
forcefield.49 Docking studies were performed using the previously
built ligand-based human CB2 homology model.38 In particular, the
hCB2 inverse agonist binding site was defined taking into account
any amino acids placed at 5Å distance far from the key residue
S165, as described by mutagenesis data.50 Successively, flexible
docking studies were applied using the Surflex docking module
implemented in Sybyl-X1.0.51 Surflex-Dock uses an empirically
derived scoring function based on the binding affinities of X-ray
protein–ligand complexes.
The Surflex-Dock scoring function is a weighted sum of non-lin-
ear functions involving van der Waals surface distances between
the appropriate pairs of exposed protein and ligand atoms, includ-
ing hydrophobic, polar, repulsive, entropic and solvation and crash
terms represented in terms of a total score conferred to any calcu-
lated conformer.
Then, the best docking geometry (selected on the basis of the
SurFlex scoring functions) was refined by ligand/protein complex
energy minimization (CHARMM27) by means of the MOE software.
Finally, the protein–ligand complex stability was successfully
assessed using a short ꢁ1 ps run of molecular dynamics (MD) at
constant temperature, followed by an all-atom energy minimiza-
tion (LowModeMD implemented in MOE software). In this way,
an exhaustive conformational analysis of the ligand-receptor bind-
ing site complex was explored, as we already explained about
other case studies for a preliminary evaluation of the derived dock-
ing poses.52
C27H27Cl2N4O: 493.2, found: 493.4. Anal. (C27H26Cl2N4O) C, H, N.
5.2. Biological evaluation
5.2.1. Competition binding experiments for human CB1 and CB2
receptors
Competition binding experiments were carried out through [3H]
CP-55,940 (specific activity, 180 Ci/mmol) provided by Perkin-
Elmer Life and Analytical Sciences (U.S.). Human CB1 and CB2
receptors expressed in CHO cells were from Perkin-Elmer Life
and Analytical Sciences (U.S.). All other reagents were derived from
commercial sources.
CHO cells transfected with human CB1 and CB2 receptors were
cultured in Ham’s F12 containing 10% fetal bovine serum, penicillin
(100 U/mL), streptomycin (100 lg/mL), and Geneticin (G418,
0.4 mg/mL) at 37 °C in 5% CO2/95% air.45 Cellular membranes for
binding experiments were obtained as previously described.45
Briefly, the cells were scraped off in ice-cold hypotonic buffer
(5 mM Tris–HCl, 2 mM EDTA, pH 7.4), homogenized with a Poly-
tron, centrifuged for 10 min at 1000g, and the supernatant was fur-
ther centrifuged for 30 min at 100,000g. The pellet of membranes
was diluted in 50 mM Tris–HCl buffer, 0.5% BSA (pH 7.4) containing
5 mM MgCl2, 2.5 mM EDTA or 1 mM EDTA for hCB1 or hCB2 recep-
tor, respectively. Competition binding experiments were carried
out with [3H]CP-55,940 and the examined compounds at different
5.4. In silico evaluation of pharmacokinetic properties
The prediction of ADMET properties was performed using the
Advanced Chemistry Development (ACD) Percepta platform
(www.acdlabs.com).
Any ADMET descriptor was evaluated by Percepta, based on
training libraries implemented in the software, which include a
consistent pool of molecules whose pharmacokinetic and toxicity
profiles are experimentally known.
concentrations (1 nM to 10 lM) or WIN 55,212-2, the CB1/CB2
standard agonist for 90 or 60 min at 30 °C for CB1 or CB2 receptors,
respectively. Bound and free radioactivities were separated by fil-
tering the assay mixture through Whatman GF/C glass fiber filters
using a Brandel cell harvester (Brandel Instruments, Unterföhring,
Germany). The filter bound radioactivity was counted on a Perkin–
Elmer 2810 TR scintillation counter (Perkin–Elmer Life and Analyt-
ical Sciences, U.S.).
Acknowledgement
5.2.2. cAMP assay for human CB2 receptors
Authors acknowledge Regione Autonoma della Sardegna for
economic support (grant n. CRP-26417, LR n. 7/2007 and
INNOVA.RE- POR FESR 2007-2013).
CHO cells transfected with human CB2 receptors were preincu-
bated with 0.5 mM 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidi-
none (Ro 20-1724) as a phosphodiesterase inhibitor and the
effect of the novel CB compounds tested at increasing concentra-
Supplementary data
tions (0.1–10
lM) or WIN 55,212-2 (0.1
lM) was studied in the
presence of forskolin (10
(cAMP direct immunoassay kit, ab65355, abcam) according to the
manufacturer’s instructions.
l
M) by using the fluorimetric cAMP kit
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.08.055.
References and notes
5.2.3. Data analysis
The protein concentration was evaluated by a Bio-Rad method46
with bovine albumin as reference standard. Inhibitory binding con-
stants, Ki, were obtained from the IC50 values following the Cheng
and Prusoff equation: Ki = IC50/(1 + [C]/KD), where [C] is the con-
centration of the radioligand and KD its dissociation constant
(Cheng Prusoff).34 A weighted nonlinear least-squares curve fitting
program, LIGAND, was used for computer analysis of the inhibition
experiments.47 Schild analysis was used to calculate pKB values of
8f, 8h and 8j compounds.48 All the data are expressed as the
mean SEM of n = 4 independent experiments. Statistical analysis
of the data was performed using ANOVA followed by Dunnett’s
test.
1. Di Marzo, V.; Bifulco, M.; De Petrocellis, L. Nat. Rev. Drug Discov. 2004, 3, 771.
2. Boyd, S. T. 2006, 26, 218S.
3. DiPatrizio, N. V.; Piomelli, D. Trends Neurosci. 2012, 35, 403.
4. Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.; Casellas, P.; Devane, W. A.;
Felder, C. C.; Herkenham, M.; Mackie, K.; Martin, B. R.; Mechoulam, R.; Pertwee,
R. G. Pharmacol. Rev. 2002, 54, 161.
5. Reggio, P. H. Curr. Med. Chem. 2010, 17, 1468.
6. Gaoni, Y.; Mechoulam, R. J. Am. Chem. Soc. 1964, 86, 1646.
7. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I. Nature
1990, 346, 561.
8. Adams, I. B.; Martin, B. R. Addiction 1996, 91, 1585.
9. Pertwee, R. G.; Howlett, A. C.; Abood, M. E.; Alexander, S. P. H.; Di Marzo, V.;
Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos, G.; Mackie, K.; Mechoulam,
R.; Ross, R. A. Pharmacol. Rev. 2010, 62, 588.
10. Freund, T. F.; Katona, I.; Piomelli, D. Physiol. Rev. 2003, 83, 1017.
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055

A. Dore et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
11. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Nature 1993, 365, 61.
12. Galiegue, S.; Mary, S.; Marchand, J.; Dussossoy, D.; Carriere, D.; Carayon, P.;
Bouaboula, M.; Shire, D.; Le Fur, G.; Casellas, P. Eur. J. Biochem. 1995, 232, 54.
13. Schatz, A. R.; Lee, M.; Condie, R. B.; Pulaski, J. T.; Kaminski, N. E. Toxicol. Appl.
Pharmacol. 1997, 142, 278.
14. Lynn, A. B.; Herkenham, M. J. Pharmacol. Exp. Ther. 1994, 268, 1612.
15. (a) Van Sickle, M. D.; Duncan, M.; Kingsley, P. J.; Mouihate, A.; Urbani, P.;
Mackie, K.; Stella, N.; Makriyannis, A.; Piomelli, D.; Davison, J. S.; Marnett, L. J.;
Di Marzo, V.; Pittman, Q. J.; Patel, K. D.; Sharkey, K. A. Science 2005, 310, 329;
(b) Gong, J.-P.; Onaivi, E. S.; Ishiguro, H.; Liu, Q.-R.; Tagliaferro, P. A.; Brusco, A.;
Uhl, G. R. Brain Res. 2006, 1071, 10; (c) Onaivi, E. S.; Ishiguro, H.; Gong, J.-P.;
Patel, S.; Perchuk, A.; Meozzi, P. A.; Myers, L.; Mora, Z.; Tagliaferro, P.; Gardner,
E.; Brusco, A.; Akinshola, B. E.; Liu, Q.-R.; Hope, B.; Iwasaki, S.; Arinami, T.;
Teasenfitz, L.; Uhl, G. R. Ann. N. Y. Acad. Sci. 2006, 1074, 514; (d) Ellert-
Miklaszlewska, A.; Grajkowska, W.; Gabrusiewicz, K.; Kaminska, B.; Konarska,
L. Brain Res. 2007, 1137, 161.
Feng, Z.; Ma, S.; Zhang, Y.; Tong, Q.; Alqarni, M. H.; Gou, X.; Xie, X.-Q. J. Chem.
Inf. Model 2016, 56, 1152.
28. (a) Rinaldi-Carmona, M.; Barth, F.; Millan, J.; Derocq, J.-M.; Casellas, P.; Congy,
C.; Oustric, D.; Sarran, M.; Bouaboula, M.; Calandra, B.; Portier, M.; Shire, D.;
Brelière, J.-C.; Le Fur, G. J. Pharmacol. Exp. Ther. 1998, 284, 644; (b) Portier, M.;
Rinaldi-Carmona, M.; Pecceu, F.; Combes, T.; Poinot-Chazel, C.; Calandra, B.;
Barth, F.; Le Fur, G.; Casellas, P. J. Pharmacol. Exp. Ther. 1999, 288, 582.
29. Mussinu, J.-M.; Ruiu, S.; Mule, A. C.; Pau, A.; Carai, M. A. M.; Loriga, G.;
Murineddu, G.; Pinna, G. A. Bioorg. Med. Chem. 2003, 11, 251.
30. (a) Chen, J.-Z.; Wang, J.; Xie, X.-Q. J. Chem. Inf. Model 2007, 47, 1626; (b)
Kotsikorou, E.; Navas, F., III; Roche, M. J.; Gilliam, A. F.; Thomas, B. F.; Seltzman,
H. H.; Kumar, P.; Song, Z.-H.; Hurst, D. P.; Lynch, D. L.; Reggio, P. H. J. Med. Chem.
2013, 56, 6593.
31. Pinna, G.; Curzu, M. M.; Dore, A.; Lazzari, P.; Ruiu, S.; Pau, A.; Murineddu, G.;
Pinna, G. A. Eur. J. Med. Chem. 2014, 85, 747.
32. Pinna, G.; Loriga, G.; Lazzari, P.; Ruiu, S.; Falzoi, M.; Frau, S.; Pau, A.; Murineddu,
G.; Asproni, B.; Pinna, G. A. Eur. J. Med. Chem. 2014, 82, 281.
33. Dore, A.; Asproni, B.; Scampuddu, A.; Pinna, G. A.; Christoffersen, C. T.;
Langgård, M.; Kehler, J. Eur. J. Med. Chem. 2014, 84, 181.
16. Rajesh, M.; Mukhopadhyay, P.; Hasko, G.; Huffman, J. W.; Mackie, K.; Pacher, P.
Br. J. Pharmacol. 2008, 153, 347.
17. (a) Han, S.; Thatte, J.; Buzard, D. J.; Jones, R. M. J. Med. Chem. 2013, 56, 8224; (b)
Tabrizi, M. A.; Baraldi, P. G.; Borea, P. A.; Varani, K. Chem. Rev. 2016, 116, 519.
18. (a) Murineddu, G.; Asproni, B.; Pinna, G. A. Rec. Pat. CNS Drug Discovery 2012, 7,
4; (b) Murineddu, G.; Deligia, F.; Dore, A.; Pinna, G.; Asproni, B.; Pinna, G. A. Rec.
Pat. CNS Drug Discovery 2013, 8, 42.
34. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
35. Tabrizi, M. A.; Baraldi, P. G.; Ruggiero, E.; Saponaro, G.; Baraldi, S.; Poli, G.;
Tuccinardi, T.; Ravani, A.; Vincenzi, F.; Borea, P. A.; Varani, K. Eur. J. Med. Chem.
2016, 113, 11.
19. Malan, T. P., Jr; Ibrahim, M. M.; Lai, J.; Vanderah, T. W.; Makriyannis, A.;
Porreca, F. Curr. Opin. Pharmacol. 2003, 3, 62.
36. Bolognini, D.; Cascio, M. G.; Parolaro, D.; Pertwee, R. G. Br. J. Pharmacol. 2012,
165, 2561.
20. Quartilho, A.; Mata, H. P.; Ibrahim, M. M.; Vanderah, T. W.; Porreca, F.;
Makriyannis, A.; Malan, T. P., Jr Anesthesiology 2003, 99, 955.
21. Hollinshead, S. P.; Tidwell, M. W.; Palmer, J.; Guidetti, R.; Sanderson, A.;
Johnson, M. P.; Chambers, M. G.; Oskins, J.; Stratford, R.; Astles, P. C. J. Med.
Chem. 2013, 56, 5722.
22. (a) Arevalo-Martin, A.; Vela, J. M.; Molina-Holgado, E.; Borrell, J.; Guaza, C. J.
Neurosci. 2003, 23, 2511; (b) Zajicek, J. P.; Apostu, V. I. CNS Drugs 2011, 25, 187;
(c) Gijsen, H. J.-M.; De Cleyn, M. A.-J.; Surkyn, M.; Van Lommen, G. R.-E.;
Verbist, B. M.-P.; Nijsen, M. J. M.-A.; Meert, T.; Van Wauwe, J.; Aerssens, J.
Bioorg. Med. Chem. Lett. 2012, 22, 547; (d) Han, S.; Zhang, F.-F.; Qian, H.-Y.;
Chen, L.-L.; Pu, J.-B.; Xie, X.; Chen, J.-Z. J. Med. Chem. 2015, 58, 5751.
23. (a) Aso, E.; Juvés, S.; Maldonado, R.; Ferrer, I. J. Alzheimer Dis. 2013, 35, 847; (b)
Aso, E.; Ferrer, I. Front. Pharmacol. 2014, 5, 1; (c) Cao, C.; Li, Y.; Liu, H.; Bai, G.;
Mayl, J.; Lin, X.; Sutherland, K.; Nabar, N.; Cai, J. J. Alzheimer Dis. 2014, 42, 973.
24. Kim, K.; Moore, D. H.; Makriyannis, A.; Abood, M. E. Eur. J. of Pharmacol. 2006,
542, 100.
37. Kenakin, T. FASEB J. 2001, 15, 598.
38. Cichero, E.; Menozzi, G.; Guariento, S.; Fossa, P. Med. Chem. Commun. 2015, 6,
1978.
39. Montero, C.; Campillo, N. E.; Goya, P.; Páez, J. A. Eur. J. Med. Chem. 2005, 40, 75.
40. Deiana, V.; Gómez-Cañas, M.; Pazos, M. R.; Fernández-Ruiz, J.; Asproni, B.;
Cichero, E.; Fossa, P.; Muñoz, E.; Deligia, F.; Murineddu, G.; García-Arencibia,
M.; Pinna, G. A. Eur. J. Med. Chem. 2016, 112, 66.
41. van de Waterbeemd, H.; Gifford, E. Nat. Rev. Drug Discov. 2003, 2, 192.
42. Suchocki, J. A.; May, E. L.; Martin, T. J.; George, C.; Martin, B. R. J. Med. Chem.
1991, 34, 1003.
43. Zhao, M.; Kuang, C.; Yang, Q.; Cheng, X. Tetrahedron Letters 2011, 52, 992.
44. Verma, A. K.; Lotla, S. K. R.; Choudhary, D.; Patel, M.; Tiwari, R. K. J. Org. Chem.
2013, 78, 4386.
45. Merighi, S.; Simioni, C.; Gessi, S.; Varani, K.; Borea, P. A. Biochem. Pharmacol.
2010, 79, 471.
46. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
25. Lunn, C. A.; Reich, E.-P.; Fine, J. S.; Lavey, B.; Kozlowski, J. A.; Hipkin, R. W.;
Lundell, D. J.; Bober, L. Br. J. Pharmacol. 2008, 153, 226.
47. Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107, 220.
48. Gessi, S.; Varani, K.; Merighi, S.; Morelli, A.; Ferrari, D.; Leung, E.; Baraldi, P. G.;
Spalluto, G.; Borea, P. A. Br. J. Pharmacol. 2001, 134, 116.
49. MOE: Chemical Computing Group Inc. Montreal. H3A 2R7 Canada. http://www.
chemcomp.com].
50. Gouldson, P.; Calandra, B.; Legoux, P.; Kernéis, A.; Rinaldi-Carmona, M.; Barth,
F.; Le Fur, G.; Ferrara, P.; Shire, D. Eur. J. Pharmacol. 2000, 28, 17.
51. Sybyl-X 1.0 Tripos Inc 1699 South Hanley Road. St Louis. Missouri. 63144. USA
25.
52. (a) Fossa, P.; Cichero, E. Bioorg. Med. Chem. 2015, 23, 3215; (b) Franchini, S.;
Battisti, U. M.; Prandi, A.; Tait, A.; Borsari, C.; Cichero, E.; Fossa, P.; Cilia, A.;
Prezzavento, O.; Ronsisvalle, S.; Aricò, G.; Parenti, C.; Brasili, L. Eur. J. Med.
Chem. 2016, 112, 1.
26. (a) Yang, P.; Myint, K.-Z.; Tong, Q.; Feng, R.; Cao, H.; Almehizia, A. A.; Alqarni,
M. H.; Wang, L.; Bartlow, P.; Gao, Y.; Gertsch, J.; Teramachi, J.; Kurihara, N.;
Roodman, G. D.; Cheng, T.; Xie, X.-Q. J. Med. Chem. 2012, 55, 9973; (b) Yang, P.;
Wang, L.; Feng, R.; Almehizia, A. A.; Tong, Q.; Myint, K.-Z.; Ouyang, Q.; Alqarni,
M. H.; Wang, L.; Xie, X.-Q. J. Med. Chem. 2013, 56, 2045.
27. (a) Feng, Z.; Alqarni, M. H.; Yang, P.; Tong, Q.; Chowdhury, A.; Wang, L.; Xie, X.-
Q. J. Chem. Inf. Model 2014, 54, 2483; (b) Bertini, S.; Parkkari, T.; Savinainen, J.
R.; Arena, C.; Saccomanni, G.; Saguto, S.; Ligresti, A.; Allarà, M.; Bruno, A.;
Marinelli, L.; Di Marzo, V.; Novellino, E.; Manera, C.; Macchia, M. Eur. J. Med.
Chem. 2015, 90, 526; Bertini Chicca, A.; Arena, C.; Chicca, S.; Saccomanni, G.;
Gertsch, J.; Manera, C.; Macchia, M. Eur. J. Med. Chem. 2016, 116, 252; (d) Hu, J.;
Please cite this article in press as: Dore, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.055