Development of oxygen-bridged pyrazole-based structures as cannabinoid receptor 1 ligands

Article abstract of DOI:10.3390/molecules24091656

In this work, the synthesis of the cannabinoid receptor 1 neutral antagonists 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c] pyrazole-3-carboxamide 1a and its deaza N-cyclohexyl analogue 1b has led to a deepening of the structure-activity studies of this class of compounds. A series of novel 4,5-dihydrobenzo-oxa-cycloheptapyrazoles analogues of 1a,b, derivatives 1c-j, was synthesized, and their affinity towards cannabinoid receptors was determined. Representative terms were evaluated using in vitro tests and isolated organ assays. Among the derivatives, 1d and 1e resulted in the most potent CB₁ receptor ligands (K_iCB₁ = 35 nM and 21.70 nM, respectively). Interestingly, both in vitro tests and isolated organ assays evidenced CB₁ antagonist activity for the majority of the new compounds, excluding compound 1e, which showed a CB₁ partial agonist behaviour. CB₁ antagonist activity of 1b was further confirmed by a mouse gastrointestinal transit assay. Significant activity of the new CB₁ antagonists towards food intake was showed by preliminary acute assays, evidencing the potentiality of these new derivatives in the treatment of obesity.

Full text of DOI:10.3390/molecules24091656

molecules
Article
Development of Oxygen-Bridged Pyrazole-Based
Structures as Cannabinoid Receptor 1 Ligands
Gabriele Murineddu 1,
*
, Battistina Asproni 1 , Paola Corona 1, Sandra Piras 1, Paolo Lazzari 2,
Stefania Ruiu 3 , Laura Legnani 4, Lucio Toma 5 and Gérard A. Pinna 1
1
Department of Chemistry and Pharmacy, University of Sassari, Via F. Muroni 23/A, 07100 Sassari, Italy;
asproni@uniss.it (B.A.); pcorona@uniss.it (P.C.); piras@uniss.it (S.P.); pinger@uniss.it (G.A.P.)
KemoTech SrL, Build 3, Loc. Piscinamanna, 09010 Pula, Italy; paolo.lazzari@kemotech.it
National Research Council (C.N.R.)-Institute of Translational Pharmacology, U.O.S. of Cagliari, Science and
2
3
Technology Park of Sardinia Polaris, 09010 Pula, Italy; stefania.ruiu@ift.cnr.it
Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy;
4
laura.legnani@unict.it
5
Department of Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy; lucio.toma@unipv.it
*
Correspondence: muri@uniss.it; Tel.: +39-079-228-740
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Spyros P. Nikas
Received: 22 March 2019; Accepted: 24 April 2019; Published: 27 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In this work, the synthesis of the cannabinoid receptor 1 neutral antagonists
8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]
pyrazole-3-carboxamide 1a and its deaza N-cyclohexyl analogue 1b has led to
deepening of the structure-activity studies of this class of compounds. A series of novel
4,5-dihydrobenzo-oxa-cycloheptapyrazoles analogues of 1a , derivatives 1c , was synthesized, and
a
,b
–j
their affinity towards cannabinoid receptors was determined. Representative terms were evaluated
using in vitro tests and isolated organ assays. Among the derivatives, 1d and 1e resulted in the
most potent CB1 receptor ligands (KiCB1 = 35 nM and 21.70 nM, respectively). Interestingly, both
in vitro tests and isolated organ assays evidenced CB1 antagonist activity for the majority of the new
compounds, excluding compound 1e, which showed a CB1 partial agonist behaviour. CB1 antagonist
activity of 1b was further confirmed by a mouse gastrointestinal transit assay. Significant activity of
the new CB1 antagonists towards food intake was showed by preliminary acute assays, evidencing
the potentiality of these new derivatives in the treatment of obesity.
Keywords: cannabinoid receptor 1 neutral antagonists; synthesis; structure-activity relationship
studies; food intake; gastrointestinal transit; modelling studies
1. Introduction
Cannabinoid receptors [1], CB1R [2] and CB2R [3], are part of the endocannabinoid system (ECS).
This system consists of cannabinoid receptors, endogenous ligands, and several proteins responsible for
their synthesis and degradation. Cannabinergic neurotransmission is closely associated with cognition,
reward, appetite, pain perception and neuroexcitability, to name just a few putative physiological
functions. Thus, it appears that dysfunction of the ECS contributes to several pathophysiological
conditions that have been associated with the aforementioned biological processes [4].
Pharmacologic interest in CB1 antagonist derivatives is particularly related to their established
effects on food intake and weight control, improvement of cardiovascular risk factors, smoking cessation
and treatment of alcohol and drug abuse [5], liver fibrosis [6], and erectile dysfunction [7]. Among
synthetic cannabinoid compounds, the CB1 inverse agonist pyrazole derivative rimonabant (SR14176A)
produced by Sanofi-Aventis reached the market in Europe (as Acompliat®) for the treatment of obesity
Molecules 2019, 24, 1656; doi:10.3390/molecules24091656
www.mdpi.com/journal/molecules

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and related metabolic risk factors. Unfortunately, the European Medicines Agency blocked its sales due
to evidence of related adverse effects, in particular increased incidence of depression and suicides [ ].
8
This event encouraged pharmaceutical companies to block or reduce investments in cannabinoid-based
drug discovery. However, an imposing body of studies is in progress which considers the significant
potentiality of this class of substances. Particular attention is focused on the individuation of new CB1
antagonist compounds which acts like rimonabant on the reduction of appetite and weight control but
has reduced or neglected side effects, i.e., by acting at the peripheral level without having an effect on
the central nervous system [8].
Recently we have reported two neutral CB1R antagonists, compound 1a
[9] and its deaza analogue
1b
[10], as seen in Figure 1, demonstrating that the chronic treatment of obese mice with 1a, endowed
with poor blood-brain barrier (BBB) permeability, induces the reduction of food intake and body weight
without inducing the side effects responsible for withdrawal of rimonabant, whereas 1b reduces the
food intake in rats through a dopamine-dependent mechanism, suggesting an action on the hedonic
component of food intake.
Figure 1. Rimonabant and CB1 neutral antagonist 1a and its analogue 1b.
In general, it has been highlighted that chronic treatment with compound 1a results in both
anti-obesity effects and cardiovascular risk factor improvement in C57BL/6N diet-induced obesity
(DIO) mice and fat diet (FD) mice [9]. Moreover, in contrast to SR141716A treatment, the chronic
administration of 1a does not change mRNA expression of both monoaminergic transporters and
neurotrophins, which are highly related to anxiety and mood disorders. These previously reported
results [9] suggest that compound 1a reduces body weight and can restore the disrupted expression
profile of genes linked to the hunger-satiety circuit without altering monoaminergic transmission,
probably avoiding SR141716A side effects.
Food intake, food self-administration, and electrophysiological studies were carried on the
deaza-analogue of 1a, compound 1b. Behavioural studies evidenced that at the i.p. dose of 0.125 mg/kg,
1b reduced both food intake and body weight. Furthermore, the i.v. administration of high doses
of 1b reverted the effect of the agonist WIN-55,212-2 on the activity of ventral tegmental area (VTA)
dopamine neurons projecting to the NAc shell, confirming the CB1 antagonism of these novel
4,5-dihydrobenzo-oxa-cycloheptapyrazoles [10].
Hence, the novel CB1 neutral antagonists 1a and 1b may represent useful candidate agents for the
treatment of obesity and its metabolic complications.
Encouraged by these results, our efforts have been aimed towards the design and synthesis of
new CB1R neutral antagonists unable to cross the BBB, to avoid adverse effects determined by the use
of SR141716A. Thus, we assumed compound 1a as the lead compound for the further characterization
of the structure-activity relationship studies within the 1a class of compounds, with an aim to disclose
new potential substitutes of SR141716A with reduced side effects. Moreover, we wanted to further

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confirm the CB1 antagonism effect of these templates by employing the gastrointestinal test. We report
the preparation, cannabinoid affinity evaluation and in vitro, in ex-vivo and in vivo assessment of
novel 4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazoles (1c–j) bearing different substituents in
position 3 of the pyrazole ring (Table 1).
Table 1. Structure and binding data for compounds 1a–j.
Receptor Affinity
Selectivity
Compound 1
R
KiCB1 (nM) a
KiCB2/KiCB1
KiCB2(nM) b
a [9]
10.25 ± 0.41
>5000
-
N
b [10]
40.0 ± 7.0
55.10 ± 5.0
35.0 ± 7.6
3725 ± 25
4500 ± 500
2000 ± 50
93:1
82:1
57:1
c
N
d
N
e
21.70 ± 3.0
325 ± 38
15:1
H3C CH3
f
g
h
i
233.0 ± 33.0
318.0 ± 36.0
429.36 ± 49.0
330.0 ± 45.0
4125 ± 620
>5000
18:1
Cl
CH3
OCH3
CF3
-
-
-
>5000
>5000
j
459.0 ± 57.0
1.80 ± 0.075
>5000
-
SR141716A
514 ± 30
285:1
a
Affinity of compounds for the CB1 receptor was evaluated using mouse brain (minus cerebellum) homogenate
and [3H]-CP 55,940. Affinity of compounds for the CB2 receptor was assayed using mouse spleen homogenate
b
and [3H]-CP-55,940. Ki values were obtained from five independent experiments carried out in triplicate, and these
are expressed as mean ± standard error.
2. Results and Discussion
2.1. Chemistry
The six-step synthetic pathway for the preparation of the 8-chloro-1-(2,4-dichlorophenyl)-4,5-
dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazole-3-carboxamides 1a–j is outlined in Scheme 1.

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Scheme 1.
Synthetic route to compounds 1a
–
j
.
Reagents and conditions: (i) NaOH,
gamma-butyrrolactone, 170 ◦C, 5 h; (ii) Polyphosphoric acid, 90 ◦C, 1.5 h; (iii) Na, dry EtOH, (COOEt)2,
room temperature, 1.5 h; (iv) 2,4-Cl2-C6H3NHNH2 HCl, EtOH, reflux., 1.5 h; (v) KOH, MeOH/H2O,
·
r.f.x., 12 h; (vi) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole,
CH2Cl2, r.t., 0.5 h, then R-NH2, triethylamine, dichloromethane, r.t., 1–2 h.
The reaction between m-chloro-phenol (
upon cyclization with polyphosphoric acid (PPA) afforded the ketone
ketone was converted to the hydroxyketoester using a Claisen reaction with diethyl oxalate in the
presence of sodium ethylate. The ketoester was then treated with 2,4-dichlorophenylhydrazine
hydrochloride and refluxing in ethanol to furnish the pyrazole . Intermediate was hydrolysed to
give the corresponding acid . The acid reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
2
) and gamma-butyrrolactone gave the acid
3, which
4
[
11]. The readily available
5
5
6
6
7
7
hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt) and the appropriate amine or hydrazine to
afford the desired derivatives 1a–j.
2.2. Biology
Affinities at CB1R and CB2R for compounds 1c–j
were assessed by competition of [3H]-CP-55,940
in mouse brain (minus cerebellum) and spleen homogenates, respectively. Radioligand binding
experimental procedures previously reported by us [12] were adopted. The results were compared
with the Ki values of the leads and reference compound, 1a, 1b and SR141716A, respectively.
Agonist or antagonist activity of the new derivatives towards CB1Rs were assayed through
in vitro testing based on the determination of phosphorylated ERK 1/2 (P-ERK 1/2) expression in a
mouse neuroblastoma N1E-115 cell line. As previously reported [13], this cell line expresses CB1Rs
but not CB2Rs; moreover, it has been shown that the exposure of N1E-115 cells to the cannabinoid
agonists arachidonyl-2-chloroethylamide (ACEA) and WIN 55-212,2 induces a rapid phosphorylation

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and activation of the ERK 1/2. The addition of CB1 antagonists as SR141716A abolishes the response
induced by the cannabinoid agonists.
For all of the new derivatives which had good CB1 affinity (Ki lower than 60 nM), the in vitro test
results were successively confirmed and examined carefully by both isolated organ assays (mouse vas
deferens) and in vivo capability to affect gastrointestinal motility. The mouse isolated vas deferens is a
nerve-smooth muscle preparation that serves as a highly sensitive and quantitative functional in vitro
bioassay for cannabinoid CB1R agonists [14]. The effect of CB1 agonists is related to the action on
naturally expressed prejunctional neuronal CB1Rs to inhibit release of the contractile neurotransmitters,
noradrenaline and ATP, which is provoked by electrical stimulation [15]. It has been shown that CB1
antagonist derivatives are able to block or reduce the inhibition of the electrically evoked contraction
amplitude of the vas deferens induced by CB1 agonists, with parallel dextral shifts in CB1R agonist
log concentration-response curves in electrically stimulated tissues [16]. According to previous
studies concerning cannabinoid agents, a gastrointestinal transit test was adopted to confirm CB1
antagonism [17]. In fact, CB1Rs are significantly expressed in the enteral system of various mammalians
and the gastrointestinal motility is modulated by their action [18]. Moreover, the capability of CB1
antagonist compounds both to block the constipation effect induced by cannabinoid agonists and to
themselves increase themselves gastrointestinal motility has been reported [19].
To further evaluate the potential application of these CB1 antagonists in the control of food intake
and in obesity treatment, compound 1b was preliminarily tested in comparison with SR141716A in an
acute in vivo assay using C57Bl6/J mice. The evaluation was carried out according to the animal model
previously reported by Di Marzo [19].
2.2.1. Radioligand Binding Assays
Investigation of the cannabinoid receptors affinities of the 8-chloro-1-(2,4-dichlorophenyl)-4,5-
dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazole core was focused on the carboxamide moiety by
introduction of different types of substituents. Two main subclasses of the derivatives were investigated
and are reported in Table 1: compounds 1c
moieties or aryl substituents in position 3 of the pyrazole ring, respectively.
The N-piperidinyl carboxamide 1a ], with a CB1-affinity Ki value of 10.25 nM, was assumed
as the lead compound for the pharmacomodulation of this new tricyclic system. First, we wanted to
assess the effect of the homologation of the piperidine ring of 1a, synthesizing the pyrrolidine (1c) [20
–e and 1f–j bear saturated heterocyclic amines and myrtanyl
[
9
]
and homopiperidine (1d) analogues. This simple variation of a methylene unit in the aza-cycloalkyl
substituent in position 3 of the pyrazole ring resulted in a 5.4-fold and 3.5-fold CB1-affinity decrease,
respectively. To further evaluate the effect of an alicyclic moiety in the carbamoyl group on the tricyclic
core, we designed the replacement of the cyclohexyl ring of 1b
[10] with the introduction of a myrtanyl
moiety (1e). Among the reported derivatives, all of the heterocycloalkyl substituted compounds (1c,d
)
showed both good affinity (KiCB1 < 60 nM) and significant selectivity (KiCB2/KiCB1 > 57) toward
CB1Rs, whereas the myrtanyl derivative (1e) allowed the maintaining of CB1 affinity at the same level
of the derivatives 1a–d, even if with a loss of CB1R versus CB2R selectivity.
To examine the influence of the variously substituted benzene ring in position 3 of the pyrazole
ring on the CB1R affinity of the new tricyclic template, compounds 1f–j were synthesized. The simple
replacement of the piperidine group with the phenyl ring (1f) in the carboxamide moiety determined a
23-fold CB1-affinity decrease (KiCB1 = 233 nM). This trend was observed for all other aryl carboxamides.
In particular, the introduction of a –Cl atom (1g) in the para position of the phenyl ring or a –OCH3
group (1i) produced a CB1 affinity decrease compared to 1f. In addition, the para-substitution with a
–CH3 (1h) or a –CF3 group (1j) resulted in a marked unfavourable effect on the CB1 affinity. All tested
compounds showed a good or high CB1 versus CB2 selectivity.

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2.2.2. In Vitro Assays
Contrary to WIN 55,212-2, no effect on the P-ERK 1/2 expression was detected when mouse
neuroblastoma N1E-115 cells were treated with compounds 1b at concentrations up to 1 M,
–
d
µ
evidencing no agonist activity of these derivatives towards the CB1 receptor. On the contrary, according
to the rimonabant profile, CB1 antagonist activity of these ligands was highlighted through their
counteraction to the increase of P-ERK 1/2 expression due to the effect of the reference cannabinoid
agonist WIN 55,212-2. The results we have obtained with compounds 1b
(panels A and B) for derivative 1b.
–d are exemplified in Figure 2
Figure 2.
of exposure to reference cannabinoid agonist WIN 55,212-2 (25 nM) and different concentrations of
1b. ( ) Competition study of P-ERK 1/2 expression in N1E-115 cells mediated by a 5 min period of
pre-treatment with the CB1R antagonist rimonabant (1 M; SR) or 1b (200 nM), followed by a 10 min
period of exposure to reference cannabinoid agonist WIN 55,212-2 (25 nM). The results are compared
to those reported in (a) for WIN 55,212-2 (25 nM), SR (1 M), and 1b (200 nM). As for rimonabant,
inhibition of WIN 55,212-2 action suggests CB1 antagonist activity of 1b. Data are expressed as a mean
percentage of vehicle
SEM (standard error measurement). * p < 0.05 versus vehicle; # p < 0.05 versus
(A) Dose response study of P-ERK 1/2 expression in N1E-115 cells following a 10 min period
B
µ
µ
±
WIN 55,212-2. Representative western blots of P-ERK expression are shown at the top of the histograms.
Different results have been instead detected, with derivative 1e bearing a myrtanyl group in
position 3 of the pyrazole ring. In this case, as for WIN 55,212-2, a dose response effect on P-ERK
1/2 expression was highlighted up to a concentration of 1e of 75 nM (Figure 3). CB1 agonism of this
new compound was confirmed through the detected profile from the competition study of P-ERK 1/2
expression in N1E-115 cells carried out with a pre-treatment step with the CB1 receptor antagonist

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rimonabant, followed by a 10 min exposure to derivative 1e or the reference cannabinoid agonist WIN
55,212-2 (Figure 3).
Figure 3.
of exposure to reference cannabinoid agonist WIN 55,212-2 (25 nM) and different concentrations of
1e. ( ) Competition study of P-ERK 1/2 expression in N1E-115 cells mediated by a 5 min period
of pre-treatment with the CB1R antagonist rimonabant (1 M; SR), followed by a 10 min period of
exposure to reference cannabinoid agonist WIN 55,212-2 (25 nM) or 1e (5nM). The results are compared
to those reported in (a) for WIN 55,212-2 (25 nM), SR (1 M), and 1e (5 nM). As for WIN 55,212-2, the
SEM.
(A) Dose response study of P-ERK 1/2 expression in N1E-115 cells following a 10 min period
B
µ
µ
results suggest CB1 agonist activity of 1e. Data are expressed as a mean percentage of vehicle
±
* p < 0.05 versus vehicle; ** p < 0.01 versus vehicle; # p < 0.05 versus WIN55,212-2; § p < 0.05 versus 1e
.
Representative western blots of P-ERK expression are shown on the top of the histograms.
2.2.3. Isolated Organs (Mouse Vas Deferens)
The neutral antagonism of the new synthesized compounds 1b–d was evaluated through an
analysis of the profile of the electrically-evoked contractions on mouse vas deferens after a 30 min
period of incubation with the different CB1 ligands. The analysis was carried out according to the
previously reported procedure concerning other cannabinoid agents, rimonabant and corresponding
analogues [21–23]. Compounds 1b–d didn’t evoke significant changes compared to the basal contractile
responses up to a concentration of 10−6 M, indicating a neutral antagonist profile. By contrast, the
inverse agonist rimonabant elicited an increase of about 30% in the electrically evoked responses at a
concentration of 10−7 M (data not shown).

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Compound 1b was further studied as a representative derivative of the subclass 1b
–
d. No agonist
activity was detected for 1b following the amplitude of electrically evoked contractions of the vas
deferens up to a concentration of 10−6 M. The CB1 antagonist behaviour of 1b through the capability
to antagonize the effect of the reference cannabinoid agonist WIN 55,212-2 has been highlighted in
Figure 4. The inhibition of the contractions due to the activity of the reference cannabinoid agonist has
been counteracted by 1b, with a consequent shifting towards the right of the curves obtained with
WIN 55,212-2 in the presence of the assayed compound if compared to that determined with WIN
55,212-2 alone. The antagonist potency of 1b, expressed in terms of pA2, was 9.10. The obtained pA2
value is comparable to that of rimonabant (pA2 = 9.02; curve % of inhibition not shown) and of the
lead compound 1a (pA2 = 8.10; curve % of inhibition not shown), evidencing the potentiality of these
4,5-dihydrobenzo-oxa-cycloheptapyrazole derivatives for the treatments of diseases and disturbances
related to CB1Rs.
Figure 4. Cumulative concentration-response curves for WIN 55,212-2 on the amplitude of twitch
contractions elicited by electrical field stimulation of the mouse vas deferens obtained in the presence
of its vehicle (control) or in the presence of different concentrations of 1b. Each symbol represents the
mean value SEM of the inhibition of electrically evoked contractions of the vas deferens expressed as a
percentage of the amplitude of the twitch response measured before the first addition of WIN 55,212-2
to the organ bath (n = 6–8 different preparations). 1b was added 20 min before the first addition of
WIN 55,212-2.
According to the in vitro test results, an activation of CB1 receptors due to compound 1e was
highlighted through isolated organ assays. In particular, partial agonism, rather than a total agonism
related profile, was detected for compound 1e. In fact, this CB1 ligand produced concentration-related
decreases in the amplitude of electrically-evoked contractions of the vas deferens, with a maximum
inhibition around 60% rather than approximately 100% as for a total agonist as WIN 55,212-2 (Figure 5).

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Figure 5. Cumulative concentration-response curves for WIN 55,212-2 or 1e for the amplitude of twitch
contractions elicited by electrical field stimulation of the mouse vas deferens. Each symbol represents
the mean value SEM of the inhibition of electrically evoked contractions of the vas deferens expressed
as a percentage of the amplitude of the twitch response measured before the first addition of WIN
55,212-2 or 1e to the organ bath (n = 6–8 different preparations).
2.2.4. In Vivo Assays: Gastrointestinal Transit (GIT)
Administration of the compound 1b (0.5–1.0 mg/kg i.p.) produced an increase in gastrointestinal
transit of up to approximately 65–70% in comparison to vehicle-treated mice (one-way ANOVA F(5,104)
= 11.18, * p < 0.01). Results obtained with compound 1b are reported in Figure 6a. Analogue results
were obtained with derivatives 1c and 1d (data not shown). As shown in Figure 6b, the in vivo effect
of the assayed compound was significantly blocked by the administration of cannabinoid reference
agonist derivative WIN 55,212-2 (0.5 mg/kg i.p.). Considering the selectivity of the new compound
for CB1R, the detected effect on GIT was attributed to this cannabinoid receptor subtype rather than
to CB2.
Figure 6. Cont.

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Figure 6.
motility. Assays were performed as described in the experimental methods section. Each bar is the
mean SEM of 10–15 mice: one-way ANOVA F(3, 19) = 7.76; (*) p < 0.01 with respect to vehicle-treated
mice (Newman Keuls test). ( ) Effect of 1b (0.01–1 mg/kg i.p.) in combination with WIN 55,212-2
(0.5 mg/kg i.p.). Assays were performed as described in the experimental methods section. Each bar
represents the mean SEM of 15–20 mice: one-way ANOVA F(6, 62) = 26.03; (*) p < 0.01 with respect
(A) Bar graphs showing the effect of compound 1b (0.5–5.0 mg/kg i.p.) alone on gastrointestinal
±
−
B
±
to vehicle-treated mice (Newman−Keuls test).
2.2.5. In Vivo Assays: Food Intake
It is well known that CB1Rs mediate the hyperphagic effect of cannabinoids. The selective CB1R
inverse agonist rimonabant counteracts these effects, and, when administered alone, decreases standard
chow intake and caloric consumption, presumably by antagonizing the actions of endogenously
released endocannabinoids such as anandamide and 2-arachidonoylglycerol [24]. To test the potential
anti-obesity effect of the new CB1 antagonist 1b we performed food intake experiments according to
the acute animal model described by Di Marzo [19]. For a comparison we also reported the results
concerning the assumed lead compound of this new series, derivative 1a.
The assayed compound significantly reduced food intake compared to the vehicle, starting from a
dose of 3 mg/kg (Figure 7a). In spite of the difference of about one order of magnitude of the KiCB1 of
both 1a and 1b compared to that of rimonabant, the induced decrease in food intake was analogous to
that of the reference compound. Significant increments of the activity of 1a and 1b compared to that of
the reference CB1 inverse agonist were highlighted at a dose of 10 mg/kg (Figure 7b), and this effect
was sustained over time: indeed, both compounds still markedly decreased food intake at 2 h and 3 h,
whereas the effect of rimonabant fades away. Moreover, no observable side effects were detected in the
case of this new CB1 antagonist, even at 20 mg/kg. On the contrary, according to previously reported
results by other authors [25,26], marked behavioural effects have been observed with rimonabant at
the same dosage of 20 mg/kg, i.e., decreased frequency and duration of locomotion, rearing, grooming,
sniffing and scratching.
Previously obtained results [9,10] and these latest results evidence the potentiality of these new
derivatives in the control of appetite and in obesity treatment, with further potential application in
the improvement of cardiovascular and metabolic risk factors. In fact, it has been demonstrated that
the blocking of CB1Rs involves adiponectin up-regulation with improvement of cardiovascular risk
factors and decrease of hyper-insulinemia and dyslipidemia, with reduction of obesity-associated
hepatic steatosis [27,28]. Moreover, CB1 antagonists/inverse agonists significantly improve multiple
cardiometabolic risk markers, inducing reductions in both intra-abdominal and liver fat in abdominally
human obese patients [29].

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Figure 7. The mean hourly food intake of fasted C57BL/6J mice i.p. treated with vehicle, rimonabant,
1a and 1b at 3 mg/kg ( ) or 10 mg/kg ( ). Values are expressed as mean SEM. Two-way ANOVA was
A
B
±
used for statistical analysis followed by a Bonferroni post hoc test. *** p < 0.0001, ** p < 0.001, * p < 0.05
versus vehicle; ### p < 0.0001, # p < 0.05 versus rimonabant at the corresponding time point. N = 7 for
each group.
2.3. Modelling
In order to try to relate the affinity and the activity data of the new compounds with their
structural and geometrical features, a modelling study was performed in comparison with the reference
compounds rimonabant and 1a on their analogues 1b–e and on a representative term of derivatives
bearing an aryl substituent in position 3 of the pyrazole ring, compound 1f. All the minimum energy
conformations of the compounds were located through optimizations using DFT (density functional
theory) methods at the B3LYP level [30,31] with the 6-311+G(d,p) basis set used for all atoms but
chlorine, for which the 6-311+G(2df,p) basis set was used.
First, the results obtained for compound 1a are described in detail. Its oxygenated heptacyclic ring
can assume three geometries characterized by different folds of the ethyleneoxy chain and by particular
values of the torsional angle that defines the deviation of the chlorophenyl and the pyrazole rings from
planarity. In Figure 8 these three geometries (1a-A, 1a-B, 1a-C) are reported together with their relative
energy values. The geometry of the substituent in position 3 of the pyrazole ring is characterized by a
neat preference of the carboxamide function for the s-trans orientation with respect to pyrazole and by
a piperidine ring in a chair conformation that can assume two different orientations, both of which are
characterized by a mean plane of the heterocyclic ring almost perpendicular to the plane of the amide

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but differing for the orientation around the N-N bond that allows the ring nitrogen lone pair to point
either in the opposite direction as the amide oxygen or in the same direction, the former geometry
being preferred over the latter by 0.6–0.7 kcal/mol (see, for example, 1a-A and 1a-D in Figure 8).
Figure 8. Three-dimensional plot of significant conformers of compounds 1a
kcal/mol is given in parentheses).
–f (the relative energy in
The conformational behaviour of the substituent in position 3 of rimonabant is superimposable
onto that of 1a as they are characterized by the same piperazine ring. Their structural differences
derive from the absence of the ethyleneoxy chain in rimonabant where the chlorophenyl group is
connected by a single bond to pyrazole. Its minimum energy conformation is characterized by an
angle of
The crystal structure of the human CB1 cannabinoid receptor has been recently published [32
preferred conformation of compound 1a 1a-A, Figure 8) is quite superimposable onto the geometry of
rimonabant, as found in its favourite pose determined through docking calculations [32].
−
53◦ between the two rings, even if it can deviate from this value at a very low energy cost.
33]. The
,
(
As expected, the tricyclic moiety of compounds 1b–f shows exactly the same conformational
behaviour as in 1a. The structural differences in the substituent in position 3 of the pyrazole ring of
compounds 1a–f account for their geometrical and conformational differences. In 1b the two located
conformers resemble those of 1a, except for the presence of a methine CH which replaces the ring
nitrogen atom and for an opposite energy ranking (Figures 8 and 9). In this case the geometry with the
methine hydrogen atom pointing in the same direction as the carboxamide oxygen, 1b-D, is preferred
over the opposite one, 1b-A, by about 2 kcal/mol.
The pyrrolidine of 1c and the homopiperidine of 1d behave quite similarly to the piperidine of 1a
.
Two perpendicular orientations of the mean plane of the heterocyclic ring and that of the amide are
found also in these cases (1c-A and 1c-D, 1d-A and 1d-D, Figure 8) and the heterocyclic rings occupy
about the same region of space (Figure 9).

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1a-A vs 1b-A
1a-A vs 1c-A
1a-A vs 1e-B
1a-A vs 1d-A
1a-A vs 1f-A
1a-A vs 1e-A
Figure 9. Superimposition, through the best fit of the atoms of the pyrazole ring, of 1a-A (minimum
energy conformer of compound 1a) (red) with significant conformers of compounds 1b–f (blue). The
hydrogen atoms are omitted for clarity.
Compound 1e, bearing a cis-myrtanyl, shows a number of conformers, all of which present a
large bicycloalkyl group, apart from the carboxamide plane. In some conformers this group is on one
side but in other ones is on the other side of this plane (Figure 8), determining an unfavourable steric
interaction with the residues surrounding the piperidine ring of rimonabant in the binding site of
the CB1 receptor [32]. Hence, the cis-myrtanyl group in 1e occupies a region of space different from
that occupied by the (hetero)cycloalkyl moiety in 1a–d (Figure 9), which accounts for the different
agonist/antagonist behaviour of 1e with respect to the other analogues.
Finally, the aryl compound 1f shows its phenyl group lying on the same plane as the carboxamide
function (Figures 8 and 9), contrarily to the orthogonal arrangement of the (hetero)cycle found in 1a
thus suggesting a possible explanation for the much lower affinity of the aryl analogues.
–d,
In Table S1 of Supplementary Materials are reported the relative energy and selected geometrical
data of conformers of compounds 1a–f.
3. Experimental Section
3.1. General Procedures
Melting points were obtained on a Köfler melting point apparatus and are uncorrected. IR
spectra were recorded as thin films (for oils) or nujol mulls (for solids) on NaCl plates with a Perkin
Elmer 781 IR spectrophotometer and are expressed in
ν
(cm−1). All NMR spectra were taken on a
Varian XL-200 NMR spectrometer with 1H and 13C being observed at 200 and 50 MHz respectively.
1
Chemical shifts for H and 13C NMR spectra were reported in
δ or ppm downfield from TMS
[(CH3)4Si]. Multiplicities were recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q
(quartet), qu (quintuplet), dd (doublet of doudlets), and m (multiplet). Atmospheric pressure ionization
electrospray (API-ES) mass spectra, when reported, were obtained using an Agilent 1100 series LC/MSD
spectrometer. Combustion analyses on target compounds were performed and all compounds showed
≥
95% purity. All reactions involving air or moisture-sensitive compounds were performed under an
argon atmosphere. Flash chromatography was performed using Merck silica gel 60 (230–400 mesh
ASTM). Thin layer chromatography (TLC) was performed with Polygram SIL N-HR/HV254 precoated
plastic sheets (0.2 mm). The 3-chlorophenol 2 was purchased from Sigma-Aldrich.
3.1.1. Synthesis of 4-(3-Chlorophenoxy)butyrric Acid (3)
To 3-chlorophenol (23.31 mmol, 1 equiv), NaOH pellets (1 equiv) were added and the whole was
heated at 170 ◦C until all base was dissolved. To the mixture gamma-butyrrolactone (1.4 equiv) was
added dropwise and the solution heated at the same temperature for 5 h, then poured into ice, acidified
with 6N HCl and extracted with CHCl3, dried (using Na2SO4) and concentrated. The residue was

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purified by flash chromatography (petroleum ether/EtOAc 8:2) to give the acid
3
as a yellow solid.
Yield: 2.34 g (46.87%). Rf = 0.15 (petroleum ether/EtOAc 8:2); m.p.: 47 ◦C (51–53 ◦C) [11]. IR ( /cm−1):
ν
3223 (OH), 1709 (CO). 1H-NMR (200 MHz, CDCl3):
δ 2.11 (qu, 2H, J = 6.8 Hz), 2.58 (t, 2H, J = 7.4 Hz),
4.0 (t, 2H, J = 5.6 Hz), 6.76 (dd, 1H, Jm = 1.0 Hz, Jo = 8.2 Hz), 6.88 (s, 1H), 6.92 (d, 1H, J = 8.8 Hz), 7.18 (t,
1H, J = 7.8 Hz), 10.20 (br s, OH exch. with D2O).
3.1.2. Synthesis of 8-Chloro-1-oxo-2,3,4,5-tetrahydrobenzocycloeptan-5-one (4)
A mixture of 4-(3-chlorophenoxy)butanoic acid 3 (27.96 mmol) and polyphosphoric acid (48 g)
was heated at 90 ◦C for 1.5 h. The whole was poured into ice, extracted with CH2Cl2, washed (Na2CO3
aq. 10%), dried (using Na2SO4) and concentrated. The residue was purified by flash chromatography
(petroleum ether/EtOAc 9:1) to give ketone
4
as an orange oil. Yield: 2.46 g (45.53%); b.p.: 46–47 ◦C/27
2.22 (qu, 2H, J = 6.4 Hz), 2.89 (t, 2H,
1
mm Hg (107 ◦C/0.4 Torr) [11]. H-NMR (200 MHz, CDCl3):
δ
J = 6.8 Hz), 4.25 (t, 2H, J = 6.6 Hz), 7.05–7.16 (m, 2H), 7.71 (d, 1H, J = 7.0 Hz).
3.1.3. Synthesis of Ethyl 2-(8-Chloro-5-hydroxy-2,3-dihydrobenzo[b]oxepin-4-yl)-2-oxoacetate (5)
Sodium metal (2.0 equiv) was added in one small portion to dry EtOH (5 mL) and the mixture
stirred until all the sodium had reacted. Ethyl oxalate (1 equiv) was added, followed by dropwise
addition of a solution of ketone
4 (1 equiv, 6.0 mmol) in dry EtOH (30 mL). The solution was stirred at
room temperature for 1.5 h and then was slowly poured into ice, after which 2N HCl was added. The
solution was extracted with EtOAc, washed (H2O), dried (Na2SO4) and concentrated. The residue
was purified by flash chromatography (petroleum ether/EtOAc 8:2) to give
1.58 g (88.74%). Rf = 0.46 (petroleum ether/ EtOAc 8:2); m.p.: 135 ◦C. IR ( /cm−1): 1823 (CO), 1713 (CO),
1683 (CO). 1H-NMR (200 MHz, CDCl3/DMSO):
1.40 (t, 3H, J = 7.2 Hz), 2.78–2.98 (m, 2H), 4.22–4.50
5 as an orange solid. Yield:
υ
δ
(m, 4H), 7.08 (s, 1H), 7.15 (d, 1H, J = 8.2 Hz), 7.81 (d, 1H, J = 8.4 Hz), 15.76 (br s, OH exch. with D2O).
3.1.4. Synthesis of Ethyl 8-chloro-1-(2,4-dichlorophenyl)-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]
pyrazole-3-carboxylate (6)
A stirred mixture of hydroxyketoester
hydrochloride (1.1 equiv) in EtOH (50 mL) was heated under reflux for 1.5 h. After concentration of
the solvent, the analytically pure ester was isolated by flash chromatography (petroleum ether/EtOAc
9:1) as an orange solid. Yield: 2.84 g (47.48%). Rf = 0.32 (petroleum ether/ EtOAc 9:1); m.p.: 135–136 ◦C.
IR ( 1.42 (t, 3H, J = 7.0 Hz), 3.44 (t, 2H, J = 5.4 Hz),
/cm−1): 1712 (CO). 1H-NMR (200 MHz, CDCl3):
5 (13.68 mmol, 1 equiv) and 2,4-dichlorophenylhydrazine
6
υ
δ
4.35–4.51 (m, 4H), 6.65 (d, 1H, J = 8.6 Hz), 6.81 (dd, 1H, Jm = 2.2 Hz, Jo = 8.6 Hz), 7.14 (s, 1H) 7.39–7.44
(m, 2H), 7.49 (s, 1H).
3.1.5. Synthesis of 8-Chloro-1-(2,4-dichlorophenyl)-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]
pyrazole-3-carboxylic acid (7)
To a mixture of ester 6 (2 mmol, 1 equiv) in CH3OH (10 mL) was added a solution of potassium
hydroxide in pellets (2 equiv) in CH3OH (7 mL) and a few drops of H2O. The resulting solution
was heated under reflux for 12 h. The mixture was poured into ice and acidified with 1N HCl. The
precipitate was filtered, washed with water and air-dried to yield the analytically pure acid
yellow solid. Yield: 0.75 g (91.23%). Rf = 0.38 (CHCl3/MeOH 9:1); m.p.: 230–231 ◦C; IR ( /cm−1): 1689
(CO). 1H-NMR (200 MHz, CDCl3/DMSO):
3.10–3.45 (br s, 3H, 1 OH exch. with D2O), 4.30–4.48 (m,
7 as a
υ
δ
2H), 6.67 (d, 1H, J = 8.4 Hz), 6.83 (dd, 1H, Jm = 3.0 Hz, Jo = 8.2 Hz), 7.13 (s, 1H), 7.44–7.50 (m, 3H).
3.1.6. General Procedure for the Synthesis of Carbohydrazides (1c,d) and Carboxamides (1e–j)
A mixture of the 8-chloro-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-6-oxa-cyclohepta[1,2-c]pyrazole-
3-carboxylic acid
7 (6.4 mmol, 1 equiv), EDC (1.2 equiv) and HOBt (1.2 equiv) in dichloromethane
(20 mL) was stirred at room temperature for 30 min. A solution of the requested amine or hydrazine
(2 equiv) in dichloromethane (20 mL) was dropwise added and the whole was stirred at room

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temperature for 1–2 h. The solution was concentrated under reduced pressure and the analytically pure
product was isolated after purification by flash chromatography and trituration with an appropriate
solvent as indicated below.
3.1.7. 8-Chloro-1-(2,4-dichlorophenyl)-N-pyrrolidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta
[1,2-c]pyrazole-3-carbohydrazide (1c) [20]
A general procedure was used to convert 7 and N-aminopyrrolidine hydrochloride into the title
product. Because of an excess of the hydrochloride salt, an amount of triethylamine (2 equiv) was
used in this reaction. The mixture was stirred for 2 h at room temperature, then purified by flash
chromatography (petroleum ether/EtOAc 7:3) and triturated with petroleum ether to afford 1c as a
cream-coloured solid. Yield: 1.11 g (36.20%). Rf = 0.24 (petroleum ether/EtOAc 7:3); m.p.: 175 ◦C;
IR (
4H), 3.45–3.55 (m, 2H), 4.30–4.40 (m, 2H), 6.60 (d, 1H, J = 9.2 Hz), 6.80 (d, 1H, J = 9.0 Hz), 7.15 (s, 1H),
7.24–7.40 (m, 3H), 7.53 (br s, 1H, NH exch. with D2O). 13C-NMR (50 MHz, CDCl3):
22.26 (CH2 2),
26.94 (CH2), 55.43 (CH2 2), 73.53 (CH2), 118.06 (C), 119.88 (C), 120.46 (C), 122.79 (CH), 123.45 (CH),
υ δ 1.80–1.95 (m, 4H), 2.95–3.05 (m,
/cm−1): 3215 (NH), 1668 (CO). 1H-NMR (200 MHz, CDCl3):
δ
×
×
127.98 (CH), 128.35 (CH), 130.30 (CH), 130.69 (CH), 132.92 (C), 134.72 (C), 136.13 (C), 137.08 (C), 141.54
(C), 159.63 (C), 160.48 (CO). API-ES: calcd. for 477.77; found 476.00; anal. calcd, for C22H19Cl3N4O2: C,
55.31; H, 4.01; N, 11.73; found: C, 55.43; H, 4.02; N, 11.82.
3.1.8. 8-Chloro-1-(2,4-dichlorophenyl)-N-homopiperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-
cyclohepta[1,2-c]pyrazole-3-carbohydrazide (1d)
A general procedure was used to convert 7 and N-aminohomopiperidine into the title product. The
mixture was stirred for 1.5 h at room temperature, then purified by flash chromatography (petroleum
ether/EtOAc 7:3) to afford 1d as a yellow solid. Yield: 1.14 g (35.29%). Rf = 0.31 (petroleum ether/EtOAc
7:3); m.p.: 137–138 ◦C. IR ( /cm−1): 3289 (NH), 1678 (CO). 1H-NMR (200 MHz, CDCl3):
υ δ 1.60–1.80 (m,
8H), 3.14 (t, 4H, J = 5.4 Hz), 3.49 (t, 2H, J = 5.2 Hz), 4.39 (t, 2H, J = 5.4 Hz), 6.60 (d, 1H, J = 8.8 Hz), 6.80
(dd, 1H, Jm = 2.2 Hz, Jo = 8.6 Hz), 7.15 (s, 1H), 7.30–7.38 (m, 3H), 7.54 (br s, 1H, NH, exch. with D2O).
13C-NMR (50 MHz, CDCl3):
δ
26.22 (CH2
×
2), 26.92 (CH2
×
3), 58.37 (CH2
×
2), 73.56 (CH2), 116.05
(C), 119.93 (C), 120.46 (C), 122.78 (CH), 123.45 (CH), 127.99 (CH), 128.33 (CH), 130.30 (CH), 130.67 (CH),
132.88 (C), 134.69 (C), 136.07 (C), 137.07 (C), 138.99 (C), 143.74 (C), 159.95 (CO); API-ES: calcd. for
505.82; found 505.05; anal. calcd. for C24H23Cl3N4O2: C, 56.99; H, 4.58; N, 11.08; found: C, 57.13; H,
4.60; N, 11.13.
3.1.9. 8-Chloro-1-(2,4-dichlorophenyl)-N-(6,6-dimethyl-bicyclo[3.1.1]hept-2-yl-methyl)-4,5-
dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazole-3-carboxamide (1e)
A general procedure was used to convert
7 and cis-myrtanylamine into the title product. The
mixture was stirred for 2 h at room temperature, then purified by flash chromatography (petroleum
ether/EtOAc 9:1) to afford 1e as a white solid. Yield: 0.89 g (25.47%). Rf = 0.31 (petroleum ether/EtOAc
9:1); m.p.: 142–143 ◦C; IR ( /cm−1): 3403 (NH), 1676 (CO). 1H-NMR (200 MHz, CDCl3):
υ δ 1.07 (s, 3H),
1.19 (s, 3H), 1.45–1.62 (m, 1H), 1.82–2.04 (m, 6H), 2.21–2.43 (m, 2H), 3.26–3.57 (m, 4H), 4.35–4.42 (m,
2H), 6.60 (d, 1H, J = 8.6 Hz), 6.81 (d, 1H, J = 8.6 Hz), 6.93 (t, 1H, NH, exch. with D2O), 7.14 (s, 1H),
7.31–7.39 (m, 2H), 7.54 (s, 1H). 13C-NMR (50 MHz, CDCl3)
δ
19.84 (CH3), 23.22 (CH3), 26.00 (CH2),
27.08 (CH2), 27.96 (CH3), 33.26 (CH2), 38.70 (C), 41.32 (CH), 41.50 (CH), 43.80 (CH), 44.57 (CH2), 73.56
(CH2), 120.03 (C 2), 122.75 (CH), 123.44 (CH), 128.01 (CH), 128.33 (CH), 130.32 (CH), 130.67 (CH),
×
132.93 (C), 134.61 (C), 136.05 (C), 137.16 (C), 139.05 (C), 144.51 (C), 159.62 (C), 162.25 (CO); API-ES:
calcd. for 544.90; found 544.20; anal. calcd. for C28H28Cl3N3O2: C, 61.72; H, 5.18; N, 7.71; found: C,
61.86; H, 5.21; N, 7.73.

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3.1.10. 8-Chloro-1-(2,4-dichlorophenyl)-N-phenyl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]
pyrazole-3-carboxamide (1f)
A general procedure was used to convert 7 and alanine into the title product. The mixture was
stirred for 1 h at room temperature, then purified by flash chromatography (petroleum ether/EtOAc
9:1) to afford 1f as a yellowish solid. Yield: 1.35 g (43.48%). Rf = 0.28 (petroleum ether/EtOAc 9:1);
m.p.: 130 ◦C. IR (
υ
/cm−1): 3386 (NH), 1688 (CO). 1H-NMR (200 MHz, CDCl3):
δ
3.57 (t, 2H, J = 6.0 Hz),
4.42 (t, 2H, J = 6.0 Hz), 6.69 (d, 2H, J = 8.4 Hz), 6.82 (d, 1H, J = 8.4 Hz), 7.12–7.20 (m, 3H), 7.32–7.42
(m, 3H), 7.67 (d, 2H, J = 7.6 Hz), 8.76 (br s, 1H, NH, exch. with D2O). 13C-NMR (50 MHz, CDCl3):
27.22 (CH2), 73.41 (CH2), 119.72 (CH 2), 119.79 (C), 120.48 (C), 122.80 (CH), 123.54 (CH), 124.16 (CH),
δ
×
128.05 (CH), 128.40 (CH), 129.00 (CH × 2), 130.29 (CH), 130.75 (CH), 132.97 (C), 134.85 (C), 136.29 (C),
137.06 (C), 137.66 (C), 139.49 (C), 144.31 (C), 159.78 (C), 160.13 (CO); API-ES: calcd. for 484.76; found
484.10. Anal. calcd. for C24H16Cl3N3O2: C, 59.46; H, 3.33; N, 8.67; found: C, 59.65; H, 3.34; N, 8.70.
3.1.11. 8-Chloro-1-(2,4-dichlorophenyl)-N-(p-chlorophenyl)-4,5-dihydrobenzo-1H-6-oxa-cyclohepta
[1,2-c]pyrazole-3-carboxamide (1g)
A general procedure was used to convert
7 and p-chloroaniline into the title product. The mixture
was stirred for 1 h at room temperature, then purified by flash chromatography (petroleum ether/EtOAc
8:2) to afford 1g as a green solid. Yield: 0.58 g (17.39%). Rf = 0.37 (petroleum ether/EtOAc 8:2); m.p.:
υ δ 3.55 (t, 2H, J = 6.4 Hz), 4.42
239 ◦C. IR ( /cm−1): 3388 (NH), 1688 (CO). 1H-NMR (200 MHz, CDCl3):
(t, 2H, J = 6.0 Hz), 6.64 (d, 1H, J = 8.4 Hz), 6.82 (dd, 1H, Jm = 2.2 Hz, Jo = 8.4 Hz), 7.16 (s, 1H), 7.31
(d, 2H, J = 8.8 Hz), 7.37–7.41 (m, 3H), 7.63 (d, 2H, J = 9.0 Hz), 8.76 (br s, 1H, NH, exch. with D2O).
13C-NMR (50 MHz, CDCl3):
(CH), 123.58 (CH), 128.05 (CH), 128.43 (CH), 129.02 (CH 2), 130.26 (CH), 130.77 (CH), 132.97 (C),
134.95 (C), 136.28 (C), 136.38 (C), 137.00 (C), 139.61 (C), 141.54 (C), 144.08 (C), 159.81 (C), 160.09 (CO).
API-ES: calcd. for 519.21; found 518.80. Anal. calcd. for C24H15Cl4N3O2: C, 55.52; H, 2.91; N, 8.09;
found: C, 55.60; H, 2.92; N, 8.11.
δ
27.20 (CH2), 73.34 (CH2), 119.70 (C), 120.51 (C), 120.88 (CH
×
2), 122.83
×
3.1.12. 8-Chloro-1-(2,4-dichlorophenyl)-N-tolyl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]
pyrazole-3-carboxamide (1h)
A general procedure was used to convert
7 and p-toluidine into the title product. The mixture
was stirred for 1 h at room temperature, then purified by flash chromatography (petroleum ether/
EtOAc 8:2) to afford 1h as a yellow solid. Yield: 0.89 g (27.90%). Rf = 0.33 (petroleum ether/EtOAc 8:2);
υ δ 2.33 (s, 3H), 3.56 (t,
m.p.: 181 ◦C. IR ( /cm−1): 3383 (NH), 1688 (CO). 1H-NMR (200 MHz, CDCl3):
2H, J = 6.4 Hz), 4.41 (t, 2H, J = 6.4 Hz), 6.64 (d, 1H, J = 8.8 Hz), 6.82 (d, 1H, J = 8.6 Hz), 7.13–7.18 (m,
3H), 7.34–7.44 (m, 2H), 7.53–7.57 (m, 3H), 8.69 (br s, 1H, NH, exch. with D2O). 13C-NMR (50 MHz,
CDCl3)
δ
20.86 (CH3), 27.22 (CH2), 73.41 (CH2), 119.70 (CH), 119.72 (C), 119.79 (C), 120.48 (C), 122.80
2), 130.29 (CH), 130.75 (CH),
(CH), 123.54 (CH), 124.16 (CH), 128.05 (CH), 128.40 (CH), 129.00 (CH
×
132.97 (C), 134.85 (C), 136.29 (C), 137.06 (C), 137.66 (C), 139.49 (C), 144.31 (C), 159.78 (C), 160.13 (CO).
API-ES: calcd. for 498.79; found 498.20. Anal. calcd. for C25H18Cl3N3O2: C, 60.20; H, 3.64; N, 8.42;
found: C, 60.29; H, 3.65; N, 8.45.
3.1.13. 8-Chloro-1-(2,4-dichlorophenyl)-N-(p-methoxyphenyl)-4,5-dihydrobenzo-1H-6-oxa-
cyclohepta[1,2-c]pyrazole-3-carboxamide (1i)
A general procedure was used to convert
7 and p-methoxyaniline into the title product. The
mixture was stirred for 1 h at room temperature, then purified by flash chromatography (petroleum
ether/ EtOAc 7:3) to afford 1i as an orange solid. Yield: 0.57 g (17.40%). Rf = 0.27 (petroleum
ether/EtOAc 8:2); m.p.: 214 ◦C. IR ( /cm−1): 3378 (NH), 1682 (CO). 1H-NMR (200 MHz, CDCl3):
υ δ
3.53–3.59 (m, 2H), 3.80 (s, 3H), 4.40–4.44 (m, 2H), 6.64 (d, 1H, J = 8.6 Hz), 6.79–6.91 (m, 3H), 7.16 (s, 1H),
7.38–7.41 (m, 2H), 7.56–7.60 (m, 3H), 8.65 (br s, 1H, NH, exch. with D2O). 13C-NMR (50 MHz, CDCl3)
27.19 (CH2), 55.47 (CH3), 73.43 (CH2), 114.14 (CH 2), 119.83 (C), 120.39 (C), 121.46 (CH 2), 122.79
δ
×
×

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(CH), 123.27 (C), 123.51 (CH), 128.05 (CH), 128.40 (CH), 130.29 (CH), 130.73 (CH), 130.78 (C), 132.97
(C), 134.81 (C), 136.25 (C), 137.08 (C), 144.41 (C), 156.29 (C), 159.76 (C), 159.97 (CO). API-ES: calcd. for
514.79; found 514.10. Anal. calcd. for C25H18Cl3N3O3: C, 58.33; H, 3.52; N, 8.16; found: C, 58.42; H,
3.53; N, 8.19.
3.1.14. 8-Chloro-1-(2,4-dichlorophenyl)-N-(p-trifluoromethylphenyl)-4,5-dihydrobenzo-1H-6-oxa-
cyclohepta[1,2-c]pyrazole-3-carbohydrazide (1j)
A general procedure was used to convert
7 and p-trifluoroaniline into the title product. The
mixture was stirred for 1 h at room temperature, then purified by flash chromatography (petroleum
ether/EtOAc 9:1) to afford 1j as a yellow solid. Yield: 1.39 g (39.36%). Rf = 0.20 (petroleum ether/EtOAc
9:1); m.p.: 249–250 ◦C. IR ( /cm−1): 3379 (NH), 1698 (CO). 1H-NMR (200 MHz, CDCl3):
υ δ 3.53–3.57 (m,
2H), 4.40–4.44 (m, 2H), 6.65 (d, 1H, J = 8.4 Hz), 6.84 (d, 1H, J = 8.6 Hz), 7.17 (s, 1H), 7.34–7.46 (m, 2H),
7.57–7.63 (m, 3H), 7.80 (d, 2H, J = 8.4 Hz), 8.92 (br s, 1H, NH, exch. with D2O). 13C-NMR (50 MHz,
CDCl3)
δ
27.22 (CH2), 73.29 (CH2), 119.23 (CH
×
2), 119.63 (C), 120.62 (C), 122.85 (CH), 123.61 (CH),
124.84 (C), 126.25 (CH), 126.32 (CH), 128.04 (CH), 128.46 (CH), 130.24 (CH), 130.78 (CH), 132.97 (C),
135.03 (C), 136.46 (C), 136.96 (C), 139.73 (C), 140.76 (C), 141.55 (C), 143.89 (C), 159.84 (C), 160.28 (CO).
API-ES: calcd. for 552.76; found 552.20. Anal. calcd. for C25H15Cl3F3N3O2: C, 54.32; H, 2.75; N, 7.60;
found: C, 54.41; H, 2.74; N, 7.63.
3.2. Evaluation of Biological Activity
3.2.1. Chemicals and Drugs for in Vitro and in Vivo Assays
[3H]-CP-55,940 (specific activity 180 Ci/mmol) was purchased from Perkin Elmer
Italia.
CP-55,940 and WIN 55,212-2 were obtained from Tocris Cookson Ltd.
(Bristol,
UK). N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(rimonabant) was purchased from KEMPROTECH Limited, Middlesbrough, UK.
For both radioreceptor binding and isolated organ experiments, drugs were dissolved in
dimethyl-sulfoxide (DMSO). DMSO concentration in the different assays never exceeded 0.1% (v/v)
and was without effects.
In vivo assays were carried out by solubilizing compounds in saline solution containing Tween 80
and Ethanol (both at 2.5 wt%). Microemulsion systems of the compounds have been obtained. All
animal experiments were performed according to the EU guidelines for the care and use of experimental
animals (CEE N◦ 86/609). Animals (Charles River, Calco, LC, Italy) were housed in animal care quarters;
temperatures were maintained at 22
water were available ad libitum before starting with the experimental procedures.
±
2 ◦C (humidity 55
±
5%) on a 12 h light/dark cycle. Food and
3.2.2. Radioligand Binding Methods
Male CD1 mice weighing 30–35 g were killed by cervical dislocation and the brain (minus
cerebellum) and spleen were rapidly removed and frozen. After thawing, tissues were homogenated
in 20 vol. (wt/v) of ice-cold TME buffer (50 mM Tris-HCl, 1 mM EDTA and 3.0 mM MgCl2, pH 7.4). The
homogenates were centrifuged at 1,086
g for 10 min at 4 ◦C, and the resulting supernatants were
×
centrifuged at 45,000 ×g for 30 min.
[3H]-CP-55,940 binding was performed using the method previously described by Ruiu et al. [16].
Briefly, the membranes (30–80
30 ◦C in a final volume of 0.5 mL of TME buffer containing 5 mg/mL of fatty acid-free bovine serum
albumin. Non-specific binding was estimated in the presence of 1 M of CP-55,940. All binding studies
µ
g of protein) were incubated with 0.5–1 nM of [3H]-CP-55,940 for 1 h at
µ
were performed in disposable glass tubes pre-treated with Sigma-Cote (Sigma Chemical Co., Ltd.,
Poole, UK), in order to reduce non-specific binding. The reaction was terminated by rapid filtration
through Whatman GF/C filters presoaked in 0.5% polyethyleneimine (PEI) using a Brandell 36-sample
harvester (Gaithersburg, MD, USA). Filters were washed five times with 4 mL aliquots of ice-cold Tris

Molecules 2019, 24, 1656
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HCl buffer (pH 7.4) containing 1 mg/mL BSA. The filter bound radioactivity was measured in a liquid
scintillation counter (Tricarb 2900, Packard, Meridien, ID, USA) with 4 mL of scintillation fluid (Ultima
Gold MV, Packard).
Protein determination was performed by means of a Bradford protein assay using BSA as a
standard according to the protocol of the supplier (Bio-Rad, Milan, Italy) [34].
All experiments were performed in triplicate and results were confirmed in at least five independent
experiments. Data from radioligand inhibition experiments were analysed by nonlinear regression
analysis of a Sigmoid Curve using Graph Pad Prism program. IC50 values were derived from the
calculated curves and converted to Ki values as previously described [35].
3.2.3. In Vitro Assays
N1E-115 cells were grown at 37 ◦C in humidified 5% CO2 in Dulbecco’s Modified Eagle’s Medium
(high glucose supplemented with 10% fetal bovine serum (FBS), 1
µg/mL penicillin-streptomycin,
2 mM l-glutamine, 2.5 g/mL amphotericin B and 50 g/mL gentamicin). N1E-115 cells were treated
µ
µ
with the compounds to be assayed and reference compounds WIN55,212-2 (cannabinoid agonist) and
rimonabant (CB1 selective antagonist). Tested and reference compounds were dissolved in Dulbecco’s
phosphate buffered saline (D-PBS) with 1% DMSO and treatments were performed in a volume of
10 µL/mL of cell suspension.
Cells lines from the European Collection of Cell Cultures (ECACC), growth medium, FBS,
penicillin-streptomycin, l-glutamine, amphotericin B, gentamicin, D-PBS and DMSO were purchased
from Sigma-Aldrich (Milano, Italy).
P-ERK 1/2 expression was determined through Western blot analysis according to the previously
reported procedure [36]. One-way ANOVA was performed as a statistical analysis using the Graph
Pad Prism program (San Diego, CA, USA).
To verify whether the observed effects were CB1 specific, a 5 min pre-treatment with the CB1
receptor antagonist rimonabant (1 µM) was carried out before exposure to the compounds that
displayed a significant induction of P-ERK (as shown by Western blot analysis). The concentration of
rimonabant was chosen among the concentrations lacking an intrinsic activity, providing an appropriate
block of the CB1 receptor. The same procedure was adopted to determine CB1 specific antagonism for
compounds which had no effect on P-ERK 1/2 expression themselves. In these last cases, WIN55,212-2
at 25 nM as a reference cannabinoid agonist agent and the new CB1 antagonists at 150–200 nM
were employed.
3.2.4. Isolated Organs
Vas deferens was obtained from male albino CD1 mice weighing 30 to 40 g. Tissue was mounted
in a 10 mL organ bath at an initial tension of 0.5 g using a previously described method [16]. The
bath contained Krebs-Henseleit solution (118.2 mM NaCl, 4.75 mM KCl, 1.19 mM KH2PO4, 25.0 mM
NaHCO3, 11.0 mM glucose, and 2.54 mM CaCl2), which was kept at 37 ◦C and bubbled with 95% O2
and 5% CO2. Isometric contractions were evoked by stimulation with 0.5 sec trains of three pulses of
110% maximal voltage (train frequency, 0.1 Hz; pulse duration, 0.5 msec) through platinum electrodes
attached to the upper end of each bath and a stainless-steel electrode attached to the lower end. Stimuli
were generated using a Grass S88K stimulator and then amplified (multiplexing pulse booster 316S;
Ugo Basile, Comerio, Italy) and divided to yield separate outputs to four organ baths. Contractions
were monitored using a computer equipped with a data recording and analysis system (PowerLab 400)
linked via preamplifiers (QuadBridge) to an F10 transducer (Biological Instruments, Besozzo, Italy).
Each tissue was subject to several periods of stimulation. The first of these stimulation periods began
after the tissue had equilibrated in the buffering medium but before drug administration, and continued
for 10 min. The stimulator was then switched off for 15 min, after which the tissues were subjected
to further periods of stimulation each lasting 5 min and separated by a stimulation-free period. The
compounds to be assayed were added once the contractile responses to electrical stimulation were

Molecules 2019, 24, 1656
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reproducible. Preparations were exposed to cumulative increasing concentrations of the compounds
to obtain concentration-response curves in the case of CB1 agonist (and partial agonist) compounds.
When no inhibition action on contractile response was evidenced, CB1 antagonism was evaluated by
carrying out experiments with the reference cannabinoid agonist WIN 55,212-2. The compounds to be
assayed were added at a fixed concentration 20 min before the first concentration of WIN 55,212-2.
Inhibition of the electrically evoked twitch response was expressed in percentage terms and was
calculated by comparing the amplitude of the twitch response after each addition of the agonist
compound (alone or in the presence of a potential CB1 antagonist) with the amplitude detected in
absence of the compounds to be assayed (basal value). The pA2 values for competitive antagonism
were calculated by Schild regression analysis [37]. Data were plotted as log antagonist concentration
versus log [(antagonist concentration/agonist concentration)-1].
3.2.5. Gastrointestinal Transit
The same procedure previously reported by Murineddu et al. [17] was adopted to evaluate the
effect of the compounds on GIT. CD1 mice weighing 30–35 g were used. Carmine (Sigma Chemical Co,
St. Louis, MO, USA) was suspended (6% wt/v) in distilled water containing 0.5% methylcellulose and
administered by gavage at the dose of 0.3 mL/mouse. Briefly, after 20 min from carmine administration,
mice were sacrificed by cervical dislocation and the stomach and small intestine were removed and the
omentum was separated, avoiding stretching. The length of the intestine was measured starting from
the pyloric sphincter to the ileocecal junction. The distance travelled by the head of the marker was
measured and expressed as a percent of the total length of the small intestine. Using this procedure,
the marker was consistently found at approximately 50% of the small intestine, 20 min after its
administration to naive mice.
WIN 55,212-2 and compounds to be assayed were administered i.p. 20 min before the i.g.
administration of the marker. 20 min later, mice were killed by dislocation cervical and intestines
were removed. In the antagonism test, the assayed compounds were administered i.p. 30 min
prior to the i.g. administration of the marker and 10 min prior to the i.p. injection of WIN 55,212-2
(0.5 mg/kg). The length of the intestine from the pyloric sphincter to the ileocecal junction and the
distance travelled by the carmine front were both measured and recorded. Groups of n = 10 mice were
used in each experiment.
Statistical analyses were performed by one-way ANOVA followed by Newman-Keuls
post-hoc comparison.
3.2.6. Food Intake
A widely described method was used for the screening of the new CB1 antagonists in the food
intake animal model [19]. C57Bl6/J mice weighing 20–25 g were housed individually on a reverse
light-dark cycle in a room with temperature and humidity control. The mice were fasted for 18 h before
testing. Vehicle or CB1 antagonists were administered by an intraperitoneal injection and 15 min later
a known amount of food was presented. Food pellets were weighed after each hour for three hours.
Rimonabant was used as internal positive control in order to evaluate the efficacy of each compound.
All animal experiments were performed according to the UE (CEE N◦ 86/609) guidelines for the care
and use of experimental animals.
Two-way ANOVA with Bonferroni post-testing was performed using GraphPad Prism version
5.00 for Windows (GraphPad Software, San Diego CA, USA).
3.3. Computational Methods
All calculations were carried out using the Gaussian 09 [38] program package. The conformational
space of the compounds was explored through optimizations at the B3LYP level [30,31] with the
6-311+G(d,p) basis set (6-311+G(2df,p) for the chlorine atoms). First, a starting geometry was optimized.
Then, the energy profiles for rotation around the single bonds of the substituent in position 3 of pyrazole,

Molecules 2019, 24, 1656
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as well as of the aryl groups, were determined with a step size of 30◦, and ring flexibility of the tricyclic
moiety was considered. All the combinations of the observed minima were used to generate starting
geometries optimized as above. Vibrational frequencies were computed at the same level of theory to
verify that the optimized structures were minima.
4. Conclusions
Ten novel 4,5-dihydrobenzo-oxa-cycloheptapyrazole carboxamides were synthesized. They were
all found to bind to cannabinoid receptors and those bearing a (hetero)cyclic amine and a myrtanyl
group in the C3-carboxamide moiety bind with high affinities to CB1 receptors as compared to aryl
amides analogues. In particular, compound 1a revealed the highest CB1 affinity, whereas derivative 1b
showed the highest CB1 selectivity, demonstrating that aliphatic carboxamides seem to be the optimum
for CB1 receptors.
Interestingly, whereas carbamoyl derivatives with an aliphatic monocyclic ring 1b–d act as
antagonists at the CB1 receptor according to parent compound 1a, as evidenced by both in vitro and in
ex-vivo assays and further confirmed by in vivo tests, the introduction in the C3-carboxamide position
of a bicyclic bulky group as myrtanyl was found to be responsible for the partial agonist activity of
1e, as confirmed by both in vitro studies and isolated organ assays. Modelling studies evidenced
that the large bicycloalkyl group of 1e occupies a region of space different from that occupied by the
(hetero)cycloalkyl moiety in 1a–d and this could account for the different agonist/antagonist behaviour
of 1e with respect to the other analogues.
CB1 affinity of the new derivatives was also lower than that of the reference CB1 inverse agonist
rimonabant. However, despite the differences in KiCB1 values, it is important to note that the effect
on food intake of the compound with the highest CB1 affinity among the studied class, compound
1b, was comparable or significantly more marked than that obtained with the reference compounds
rimonabant and 1a, respectively. The potentiality of the compound 1b has been further supported by
the absence of observable side effects, even at the highest assayed dosage.
Supplementary Materials: Table S1 with the relative energy and selected geometrical data of conformers of
compounds 1a–f has been made available.
Author Contributions: Conceptualization, G.A.P. and G.M.; methodology, B.A. and P.C.; software, L.L.; validation,
P.C. and S.P.; formal Analysis, B.A. and S.P.; biological Investigation, P.L. and S.R.; resources, B.A.; data curation,
G.M, P.L., S.R. and L.T.; writing—original draft preparation, G.M., P.L. and L.T.; writing—review and editing, G.M.;
visualization, G.M.; supervision, G.M. and G.A.P.; project administration, G.M. and G.A.P.; funding acquisition,
P.L. and G.A.P.
Funding: This research was funded by Regione Autonoma della Sardegna: grant number CRP-26417, LR n. 7/2007
and INNOVA.RE-POR FESR 2007–2013.
Acknowledgments: The authors would like to thank C. Dessì, A. Pilleri and G. Manca for technical support in
radioreceptor binding assays and in vivo tests.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).