Novel pyrazole derivatives as neutral CB1 antagonists with significant activity towards food intake

Article abstract of DOI:10.1016/j.ejmech.2012.12.056

In spite of rimonabant's withdrawal from the European market due to its adverse effects, interest in the development of drugs based on CB₁ antagonists is revamping on the basis of the peculiar properties of this class of compounds. In particular, new strategies have been proposed for the treatment of obesity and/or related risk factors through CB₁ antagonists, i.e. by the development of selectively peripherally acting agents or by the identification of neutral CB₁ antagonists. New compounds based on the lead CB₁ antagonist/inverse agonist rimonabant have been synthesized with focus on obtaining neutral CB₁ antagonists. Amongst the new derivatives described in this paper, the mixture of the two enantiomers (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1- hydroxyethyl)-4-methyl-1H-pyrazole ((±)-5), and compound 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl] -4-methyl-1H-pyrazole ((Z)-6), showed interesting pharmacological profiles. According to the preliminary pharmacological evaluation, these novel pyrazole derivatives showed in fact both neutral CB₁ antagonism behaviour and significant in vivo activity towards food intake.

Full text of DOI:10.1016/j.ejmech.2012.12.056

Accepted Manuscript
Novel pyrazole derivatives as neutral CB antagonists with significant activity towards
1
food intake
Ilaria Manca, Andrea Mastinu, Francesca Olimpieri, Matteo Falzoi, Monica Sani,
Stefania Ruiu, Giovanni Loriga, Alessandro Volonterio, Simone Tambaro, Mirko
Emilio Heiner Bottazzi, Matteo Zanda, Gérard Aimè Pinna, Paolo Lazzari
PII:
S0223-5234(13)00017-2
10.1016/j.ejmech.2012.12.056
EJMECH 5932
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 May 2012
Revised Date: 21 December 2012
Accepted Date: 25 December 2012
Please cite this article as: I. Manca, A. Mastinu, F. Olimpieri, M. Falzoi, M. Sani, S. Ruiu, G. Loriga,
A. Volonterio, S. Tambaro, M.E. Heiner Bottazzi, M. Zanda, G.A. Pinna, P. Lazzari, Novel pyrazole
derivatives as neutral CB antagonists with significant activity towards food intake, European Journal of
1
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2012.12.056.
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ACCEPTED MANUSCRIPT
Compd
Q
O
O
1
3
4
N
N
H
Q
N
N
Cl
OH
Cl
(
±)-5
Z)-6
E)-7
±)-8
F
(
Cl
(
F
CF
3
(
N
N
H

ACCEPTED MANUSCRIPT
Highlights
•
1
Renew the importance of drugs based on CB antagonists.
•
•
•
Fluorinated compounds based on the lead rimonabant have been synthesized.
Novel pyrazole derivatives showed neutral CB antagonism behaviour.
1
Coumpounds ±-5 and Z-6 have significant in vivo activity towards food intake.

ACCEPTED MANUSCRIPT
Novel pyrazole derivatives as neutral CB antagonists with significant
1
activity towards food intake.
a
b
c
d,e
f,g
Ilaria Manca , Andrea Mastinu , Francesca Olimpieri , Matteo Falzoi , Monica Sani ,
b
b
c,g
a
Stefania Ruiu , Giovanni Loriga , Alessandro Volonterio , Simone Tambaro , Mirko
b
g,h
i
a,f,i,*
Emilio Heiner Bottazzi , Matteo Zanda , Gérard Aimè Pinna , Paolo Lazzari
a
Neuroscienze PharmaNess S.c.a r.l., Edificio 5, Loc. Piscinamanna, 09010 Pula (CA), Italy
b
C.N.R. Istituto di Farmacologia Traslazionale, UOS Cagliari, Edificio 5, Loc. Piscinamanna, 09010 Pula
CA), Italy
(
c
d
e
f
Dipartimento di Chimica, Materiali, e Ingegneria Chimica ‘Giulio Natta’, Via Mancinelli 7, 20131 Milano, Italy
Consorzio ELPRO, Edificio 5, Loc. Piscinamanna, 09010 Pula (CA), Italy
Dipartimento di Scienze della Vita e dell’Ambiente, Lab. Genetica, Via T.Fiorelli 1, 09126 Cagliari, Italy
KemoTech S.r.l., Edificio 3, Loc. Piscinamanna, 09010 Pula (CA), Italy
C.N.R. Istituto di Chimica del Riconoscimento Molecolare, Via Mancinelli 7, 20131 Milano, Italy
Kosterlitz Centre for Therapeutics, Institute of Medical Sciences, College of Life Sciences and Medicine,
University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK
g
h
i
Dipartimento di Chimica e Farmacia, Università di Sassari, Via F. Muroni 23/A, 07100 Sassari, Italy
*
Corresponding author:
Paolo Lazzari, phone +39 07092433118, fax +39 07092433119, e_mail: paolo.lazzari@kemotech.it
1

ACCEPTED MANUSCRIPT
Abstract – In spite of rimonabant’s withdrawal from the European market due to its
adverse effects, interest in the development of drugs based on CB
1
antagonists is
revamping on the basis of the peculiar properties of this class of compounds. In particular,
new strategies have been proposed for the treatment of obesity and/or related risk factors
through CB
or by the identification of neutral CB
antagonist/inverse agonist rimonabant have been synthesized with focus on obtaining
neutral CB antagonists. Amongst the new derivatives described in this paper, the mixture
1
antagonists, i.e. by the development of selectively peripherally acting agents
1
antagonists. New compounds based on the lead CB
1
1
of the two enatiomers (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-
hydroxyethyl)-4-methyl-1H-pyrazole ((±)-5), and compound 5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl]-4-methyl-1H-pyrazole ((Z)-6), showed
interesting pharmacological profiles. According to the preliminary pharmacological
evaluation, these novel pyrazole derivatives showed in fact both neutral CB
behaviour and significant in vivo activity towards food intake.
1
antagonism
1
Keywords: cannabinoids, CB receptor, fluorinated ligands, food-intake.
1. Introduction
Different classes of cannabinoid CB antagonists have been developed in the last two
1
decades with principal focus on the discovery of new therapeutic strategies for obesity
treatment [1-11]. CB receptors (CB R), belonging to Class α G protein-coupled receptors
1
1
(GPCRs), are in fact involved in appetite regulation and energy homeostasis, as well as in
other physiological processes including intestinal motility, nociception, locomotive activity,
2

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1
learning and memory [12]. Considering the wide distribution of CB R at both peripheral
level (i.e. gut, eye, liver, pancreas, adipose tissue, testis) and Central Nervous System [4],
compounds able to interact with this cannabinoid receptor subtype have been also
proposed as potential candidates for the treatment or prevention of other disorders and
diseases such as pain [13], glaucoma [14], inflammation [15], drug and smoke addiction
[
16,17], vomiting [18,19], neurodegeneration (i.e. Parkinson’s and Huntigton’s diseases)
20,21], melanoma [22], and acute myocardial infarction [23]. In addition to obesity, CB1
[
antagonists have been in particular proposed as therapeutic agents for smoking cessation
16], drug abuse [24-26], liver fibrosis [27], obesity-associated hepatic steatosis [28],
sexual dysfunction [29], metabolic syndrome and dyslipidemia [30]. Cardiovascular and
[
metabolic risk factor improvements due to the administration of CB
been also highlighted in type 2 diabetes patients [31].
1
antagonists have
Despite initial enthusiasm in drug discovery for the identification and the selection of first
CB antagonist candidates able to reach the pharmaceutical market, with reference to the
well known history concerning the CB antagonist / CB inverse agonist lead product N-
piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
1
1
1
(
®
carboxamide (Acomplia , rimonabant, SR141716A, 1) (Figure 1) [32,33], industrial
®
attention has been recently reduced on this class. In fact, Acomplia , launched on the
European market by Sanofi Aventis for the treatment of obesity, was soon blocked by
European Medicines Agency (EMA) for side effects, i.e. anxiety and depression [34].
However, there is an increasing body of opinion that the use of cannabinoid derivatives as
therapeutic agents should be reevaluated on the basis of the interesting properties of
1
these compounds. In particular, for CB antagonists, new strategies have been proposed
for the treatment of obesity and/or related risk factors using cannabinoid agents with
reduced or no side effects, i.e. by the development of selectively peripherally acting agents
1
[35-37], or by the identification of neutral CB antagonists [38].
3

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It has been reported that CB
ways of interacting with the cannabinoid receptor. Particularly, hydrogen bond interaction
with 3.28(192) is often found with inverse agonists [39-42]. Rimonabant CB inverse
agonism is in particular related to the capability of the carboxamide oxygen to form
hydrogen bond with K3.28(192). CB neutral antagonism is instead assured by avoiding
1
neutral antagonists and inverse agonists show different
1
1
hydrogen bond with K3.28(192). This has been confirmed by the neutral antagonism
profile of VPSR (2) and VCHSR (3) compounds, the corresponding vinyl derivatives of 1
and
carboxamide, respectively (Figure 1) [39]. The different behavior of CB
antagonists and inverse agonists can be in part explained by the extended ternary
complex model. According to this model, CB agonists, neutral antagonists, and inverse
agonists interact with this receptor subtype in different ways involving a ground or
inactivate R state and an active R* state [39]. CB agonist derivatives show higher affinity
for R*, with a consequent receptor activation and shifting of the R / R* equilibrium towards
the R*. CB neutral antagonists show equal affinity for the two states, while CB inverse
N-(cyclohexyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
1
neutral
1
1
1
1
agonists have higher affinity for R state with an R / R* equilibrium unbalanced towards the
R state and a reduction of the signaling pathway activation. Equal affinity for R and R* has
been evidenced by modeling studies for the biarylpyrazole scaffold of rimonabant, being
this affinity due to the aromatic interaction with F3.36(200), Y5.39(275), and W5.43(279) of
the transmembrane helix (TMH)3-4-5-6 aromatic microdomain [39-41]. In contrast,
significant bias toward R state has been related to the carboxamide oxygen at the 3
position of pyrazole ring of rimonabant, as in the case of analogues bearing the same
substituent, even if with a cyclohexyl instead of the piperidinyl ring.
Within the class of pyrazole based cannabinoids, the compound 5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-3-(2-cyclohexyl-1-oxoethyl)-1H-pyrazole (4), namely ABD-395
4

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1
(Figure 1), has been recently characterized and its CB neutral antagonism behavior has
been confirmed by both in vitro e in ex-vivo assays [43].
FIGURE 1
The evidence for CB
1
R inverse agonism in in vitro assays is generally related to the
blockade of CB agonist action and to the opposite effects determined by CB inverse
1
1
agonists when administered alone compared to those detected in the case of CB
agonists. In contrast to CB agonists, CB inverse agonists determine for example the
increase of the Ca current in neurons expressing CB R [42], the enhancement of the
amplitude of electrically-evoked contractions of the mouse isolated vas deferens [43], the
1
1
1
2
+
1
3
5
inhibition of basal CB
release of acetylcholine, noradrenaline and γ-aminobutyric acid in hippocampal slices
44,45]. In the case of CB neutral antagonists, no effect is instead detected when they are
1
R activation by [ S]GTPγS binding assays, the rise of ongoing
[
1
administered alone. These compounds elicit their activity by counteracting the CB
effects.
1
agonist
We have now synthesized the alcohol (±)-5, obtained as an equimolar mixture of
enantiomers from the reduction of the ketone group of 4, the fluorine derivatives ((Z)-6 and
(E)-7) of 5-(4-chlorophenyl)-3-(2-cyclohexylethenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole (3) and the racemic trifluoroethylamine analogue (±)-8 of rimonabant (Figure 2),
with the aim of obtaining new neutral CB antagonists, that might be characterized by
1
reduced side effects compared to the lead compound 1. Neutral antagonism was
previously highlighted for reference compounds 2-4 [39,43], and for the 5-(4-bromophenyl)
analogues of both 4 and 5, respectively ABD-399 and ABD-402 [43].
FIGURE 2
5

ACCEPTED MANUSCRIPT
The adopted medicinal chemistry strategies for the obtainment of the new potential CB1
antagonist derivatives 5-8 with reduced or void side effects have been based on both the
the well-established bioisosteric replacement of carboxamide group with vinyl fluoride and
1
trifluoroethylamine moieties [46], and the neutral CB antagonism highlighted for reference
compounds 2-4 [39], as for the 5-(4-bromophenyl) analogue of 5 [43]. The new
compounds were compared to rimonabant (1), VCHSR (3), and ABD-399 (4), both in
terms of affinity to cannabinoid receptors and through intrinsic activity determination by ex-
vivo assays (isolated organs) and in vitro tests based on the use of a specific cell line
expressing CB
1
but not CB
preliminary [ S]GTPγS binding assays with CB
CHO) cells. Moreover the capability of the new compounds to induce food intake
2
receptors. Further investigations were carried out by
3
5
1
transfected Chinese hamster ovary
(
reduction was determined by using an acute animal model.
Interesting pharmacological profiles were determined, especially for compounds (±)-5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-hydroxyethyl)-4-methyl-1H-
pyrazole ((±)-5), and 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-
fluorovinyl]-4-methyl-1H-pyrazole ((Z)-6). According to preliminary pharmacological
assays, these new derivatives showed in fact both neutral CB
significant in vivo activity towards food intake.
1
antagonism behaviour and
2. Chemistry
The racemic carbinol (±)-5 was prepared by the method shown in Scheme 1. The method
was based on previously reported procedure for the obtainment of the corresponding
6

ACCEPTED MANUSCRIPT
ketone 4 [43], with revisions. Addition of the 4-chloropropiophenone (9) to a mixture of
diethyl oxalate (10) and sodium ethoxide in absolute ethanol at room temperature afforded
Claisen condensation product, the α,γ−diketoester 11 [47]. Subsequent reaction of 11 with
2
,4-dichlorophenylhydrazine hydrochloride (12) in refluxing absolute ethanol afforded the
ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylate (13)
39,48]. Weinreb amide 14 [43] was synthesized from the reaction of 13 with N,O-
[
dimethylhydroxylamine hydrochloride in the presence of trimethylaluminum. Grignard
reaction of 14 with (cyclohexylmethyl)magnesium bromide (15) gave ketone 4 in good
yield [43]. Finally, the carbonyl group of 4 was reduced by sodium borohydride at room
temperature to obtain the racemic alcohol (±)-5 (Scheme 1).
SCHEME 1
The preparation of derivatives (Z)-6 and (E)-7 is shown in Scheme 2. Weinreb amide 14
was reduced with LiAlH
diethylphosphite producing 17 in good yields. Fluorination of the hydroxy group of 17 using
diethylamino)sulfur trifluoride (DAST) afforded the fluoro derivative 18. The final step to
4
to afford the aldehyde 16, which was in turn condensed with
(
prepare the isomeric compounds (Z)-6 and (E)-7 consisted of the Horner-Wadsworth-
Emmons olefination between the stabilized ylide derived from 18 and the
cyclohexylcarboxaldehyde (19). The isomers (Z)-6 and (E)-7 were separated by flash
chromatography in the 1.0:5.4 molar ratio.
SCHEME 2
Finally, the racemic trifluoromethyl derivative 8 has been synthesized as depicted in
Scheme 3. The trifluoroethylalcohol 20, obtained after trifluoromethylation of the aldehyde
7

ACCEPTED MANUSCRIPT
16 with the Ruppert-Prakash reagent [49], was oxidized to the corresponding ketone 21 via
classical Swern oxidation [50-53]. Subsequently 21 was submitted to condensation with N-
aminopiperidine in refluxing toluene, with azeotropic removal of water using pyridinium p-
toluenesulfonate (PPTS) catalysis, affording the hydrazone 22 [54]. Such compound
proved to be pretty labile, especially toward water. Thus, a number of different reducing
4
agents, such as LiAlH [55], borane-THF complex [56], and triethylsilane in trifluoroacetic
acid (TFA) [57], were unsuccessfully tried. We achieved our best result using borane-
trimethylamine complex in toluene in the presence of gaseous HCl, which successfully led
to the synthesis of the racemic hydrazine hydrochloride (±)-8 [58].
SCHEME 3
According to previously reported synthetic process [39], we have also synthesized the vinyl
compound 3, starting from key intermediate 13.
3. Biology
3
.1. Receptor binding assays
The affinity of both the new and reference compounds for CB
was determined according to the previously reported procedures [59]. Assays for the
1
2 2
R and CB receptors (CB R)
evaluation of CB
1
R and CB
2
R affinity were performed using mouse brain (minus
3
cerebellum) and mouse spleen homogenates, respectively. [ H]-(1R,3R,4R)-3-[2-hydroxy-
3
4
-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol ([ H]CP-55,940) was
employed as radio-labeled ligand. The experimental data (IC50 values) were converted into
8

ACCEPTED MANUSCRIPT
K
i
values [60]. The affinity of the new compounds 5-8 was compared with that of
rimonabant 1 and the reference neutral CB antagonists 3,4.
1
3.2. Intrinsic activity by in vitro assays
Intrinsic activity of the new derivatives and reference compounds towards CB
1
R was
evaluated through in vitro tests based on the determination of phosphorylated ERK 1/2 (P-
ERK 1/2) expression in Mouse Neuroblastoma N1E-115 cell line, following the treatment of
these cells with the cannabinoid compounds. As previously reported, this cell line
expresses CB
1 2
but not CB receptors, and its exposure to the cannabinoid agonists ACEA
and WIN55-212,2 induces a rapid phosphorylation and activation of the ERK 1/2 [61]. This
effect is counteracted by a pre-treatment of N1E-115 cells with a CB
rimonabant.
1
antagonist such as
3
.3. Isolated organs (mouse vas deferens)
The mouse isolated vas deferens is a nerve-smooth muscle preparation widely adopted in
highly sensitive bioassays concerning CB receptor modulation studies [62]. CB agonists
produce concentration-related decreases in the amplitude of electrically evoked
contractions of the vas deferens. CB antagonist derivatives determine parallel dextral
shifts in CB agonist log concentration-response curves in electrically stimulated tissues.
Moreover it has been ascertained that CB neutral antagonists and inverse agonists can
1
1
1
1
1
be distinguished through this isolated organ technique. No significant increment in the
amplitude of the electrically evoked contractions is detected in the case of neutral
antagonists. In contrast, an increase of these contractions compared to the control is
instead observed following the administration of inverse agonists [43]. According to these
1
last reported behaviors, a preliminary indication of CB neutral antagonism was evaluated
9

ACCEPTED MANUSCRIPT
for the newly synthesized compounds in comparison with rimonabant and the reference
neutral CB antagonists 3 and 4.
1
3
5
3
.4. [ S]GTP
It has been reported that CB
G protein-coupled receptor activation [43-45]. A concentration-response curve with a
γ
S binding assays
3
5
1
agonist agents elicit [ S]GTPγS binding stimulation through
3
5
sigmoid shape is provided by following the action of CB
expressed as stimulation percentage, as their concentration increases. In contrast, no
substantial effect on GTP attachment to the receptors is caused by neutral CB antagonist
alone. In addition, CB antagonist derivatives determine shifting to the right of the
S]GTPγS binding concentration-response curves of CB agonists. CB inverse agonist
compounds show opposite effect to that detected in the case of CB agonist derivatives. In
particular, CB inverse agonists exert a detach action towards GTP binding to the
1
agonists on [ S]GTPγS binding,
1
1
3
5
[
1
1
1
1
3
5
receptors, with corresponding negative values of [ S]GTPγS binding stimulation
percentage.
3
5
Preliminary [ S]GTPγS binding assays were carried out to support intrinsic activity
behaviour determined by isolated organ experiments. Assays were carried out using CB
1
transfected Chinese hamster ovary (CHO) cells. Novel compounds 5 and (Z)-6, and
reference CB ligands 1 and 4, were assayed alone in order to evaluate their capability to
1
stimulate or to inhibit GTPγS binding.
3.5. Food intake
To preliminarily evaluate the potential application of the new CB
1
antagonist derivatives in
the control of food intake and, potentially, in obesity treatment, compounds (±)-5 and (Z)-6
were tested in an acute in vivo assay based on the animal model previously reported by Di
1
0

ACCEPTED MANUSCRIPT
Marzo [63]. C57Bl6/J mice were used. Activity of the new compounds after intraperitoneal
dosing was compared with that of rimonabant and of the reference neutral CB antagonists
1
3
and 4.
4. Results and Discussion
4
.1. Cannabinoid receptor affinities
As first step of the study, radioreceptor binding assays were carried out in order to assure
for the new derivatives both CB affinity and selectively vs CB R. The affinity values
expressed as K, of the reference compounds 1 and 3,4 were in accord to those previously
1
2
i
reported by other authors [39,43]. As showed in Table 1, the substitution of N-(piperidinyl)-
carboxamide group at position 3 of pyrazole ring of rimonabant (1) with the vinyl, fluorine-
vinyl, trifluoroethylamine, ketone, and its corresponding alcohol, produced a reduction in
CB
influenced by these replacements. However good CB
determined for both (Z)-6 and the corresponding hydrogen analogue 3, as well as for the
ketone derivative 4. The three compounds above showed K values only one order of
1
affinity. With the exclusion of compound 3, CB
1
vs CB
2
selectivity was also negatively
1
affinity and selectivity values were
i
magnitude higher than that of the lead 1. One further order of magnitude in CB affinity
1
was lost by the substitution of the carboxamide moiety of rimonabant with 1-hydroxy-2-
cyclohexylethyl group of (±)-5 or with 2,2,2-trifluoro-N-(piperidin-1-yl)ethylamine group of
(±)-8. The drop in affinity observed for (±)-8 is not surprising taking into account the well
known importance of the 3-carboxamide oxygen as a hydrogen bond acceptor (HBA) in
the binding mode of rimonabant-like ligands to the CB1R [10]. This confirms that a
trifluoroethylamine function is not a good carboxamide replacement when the oxygen atom
of the parent molecule plays an important HBA role, owing to the bad HBA properties of a
1
1

ACCEPTED MANUSCRIPT
1
trifluoromethyl group [64]. The determined reduction in CB affinity of (±)-5 compared to
that of the corresponding ketone 4, was of the same entity of that reported for the
analogues bearing a 4-bromophenyl instead of 4-chlorophenyl in position 5 of the pyrazole
ring [43]. Moreover it is important to note as the separation feasibility of the two isomers
(E) and (Z) of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-fluorovinyl)-4-
methyl-1H-pyrazole, allowed to ascertain the relevance of the spatial geometry of the vinyl
group to assure affinity towards CB R. Compound 6, the Z isomer, showed in fact CB
1
1
affinity 16 fold higher than that of the corresponding E isomer (7).
TABLE 1
4.2. Intrinsic activity by in vitro assays.
Except compound 7 showing K value for CB
i
1
R higher than 400 nM, all the new and
reference compounds were assayed to determine or confirm their CB antagonism. In
1
common with rimonabant and in contrast to the reference cannabinoid agonist WIN55,212-
2, no effect on the expression of P-ERK 1/2 was evidenced following N1E-115 cell line
treatments with compounds 3-6 and 8 (Figure 3).
FIGURE 3
In addition the up-regulation of P-ERK 1/2 expression induced on the same cells by the
administration of the reference cannabinoid agonist WIN55,212-2 (25 nM), was significantly
counteracted through a pre-treatment step with the assayed compounds. The detected CB
1
1
2

ACCEPTED MANUSCRIPT
antagonism effect of the tested derivatives was statistically significant starting from
concentrations of 125-500 nM for compounds 3, 4, and (Z)-6, while a higher concentration
(1 µM) was needed to have the same effect in the case of (±)-5 and (±)-8 which present
lower affinity for CB R (Figure 4).
1
FIGURE 4
4
.3. Isolated organs
The effect of CB agonists on mouse vas deferens is related to their action on naturally
expressed prejunctional neuronal CB to inhibit release of the contractile
1
1
R
neurotransmitters, noradrenaline and ATP, provoked by electrical stimulation [65]. Neutral
antagonism of the new compounds for CB R was preliminary evaluated by comparing the
1
amplitude of the electrically evoked contractions in mouse vas deferens assays in the
presence or in the absence of the derivatives (Figure 5). CB
1
inverse agonism of
rimonabant (1) was confirmed by this study at both 0.1 and 1.0 µM. According to
previously reported data by other authors [43], an increase in the amplitude of the evoked
contractions was in fact detected with the reference compound at a concentration of 0.1
µM, with enhancement percentage higher than 90% at 1 µM. In contrast to rimonabant, no
marked rise of these contractions compared to the control was instead detected following
the administration of the compounds 3-8 at concentrations 0.1 - 100 µM (Figure 5). The
same behavior was previously reported for both the reference derivative 4 and the
corresponding 5-(4-bromophenyl) analogue of racemic compound (±)-5, being the effect of
these compounds on isolated mouse vas deferens supported by the results obtained
1
3

ACCEPTED MANUSCRIPT
3
5
1
through [ S]GTPγS binding assays [43]. A CB R neutral antagonism profile of the new
compounds was therefore suggested by the detected profiles.
FIGURE 5
1
Mediated CB activity of the novel compounds on isolated mouse vas deferens was
ascertained by evaluating the capability of the synthesized derivatives to counteract the
contraction inhibition elicited by the reference cannabinoid agonist WIN 55,212-2.
According to previously reported behavior concerning CB
1
antagonists [66], compounds 3-
8
affected the sigmoidal curve obtained with WIN 55,212-2, by shifting it towards right. As
1
example of the detected results, the CB antagonism behavior of compound 6 towards
WIN 55,212-2 mouse vas deference activity has been reported in Figure 6.
FIGURE 6
3
5
4
.4. [ S]GTPγS binding assays
3
5
To further investigate intrinsic activity of the novel compounds, preliminary [ S]GTPγS
binding assays were carried out by using CB1 transfected Chinese hamster ovary (CHO)
cells. Experiments were performed following the procedures by Greig at al. [43].
Derivatives 5, (Z)-6, and reference compounds 1 and 4 were assayed.
3
5
The effect induced by different concentrations of each compound on [ S]GTPγS binding is
reported in Figure 7. The results are expressed as percentage of stimulation vs basal ±
SEM. According to previously reported results: rimonabant showed an inverse agonism
35
behavior by eliciting a significant reduction in [ S]GTPγS binding at concentrations higher
-
8
than 10 M (Figure 7a); in contrast to 1, no significant effect was instead detected for the
1
4

ACCEPTED MANUSCRIPT
reference neutral CB
1
antagonist compound 4 (Figure 7b). As in the case of compound 4,
3
5
both the novel derivatives 5 and (Z)-6 didn’t significantly affect [ S]GTPγS binding, at least
-
5
1
up to 10 M (Figure 7 c-d). CB R neutral antagonism profile of the new compounds
detected by isolated organ assays was therefore further supported by the results reported
in Figure 7.
FIGURE 7
4.5. Food intake
The activity of the new derivatives (±)-5 and (Z)-6, rimonabant (1), and the reference
compounds 3 and 4, in food intake inhibition was evaluated according to the procedure by
Di Marzo [63]. The results obtained during three hours from the presentation of food to the
animals after a fast period of 18 hours are reported in Figure 8. The acute treatment of
fasted C57Bl6/J mice with (Z)-6 at the doses of 10 and 20 mg/kg, compared to the vehicle,
induced a significant reduction of food intake starting from 3 and 2 hours from food
availability, respectively (Figure 8 a). Rimonabant significantly affected food intake in the
same animal model starting from the first hour of the evaluation. No significant variation
was detected for 1 between the doses of 3 and 10 mg/kg, although a trend related to an
increase of the effect with the dose was evidenced at third hour. It is important to note that
no higher dosage was adopted for rimonabant (1) due to suffering state, locomotion
blockage, and, in some case (one third of the tested mice), mortality, observed in the
animals treated with this compound at 20 mg/kg. The extent of food intake inhibition for
(Z)-6 at 20 mg/kg was comparable to that detected in the case of rimonabant
administration at 10 mg/kg. In contrast to the side effects related to 1 administration, no
observable adverse effect was evidenced in the case of treatments with (Z)-6 at 20 mg/kg.
1
5

ACCEPTED MANUSCRIPT
Analogous behaviors were highlighted on food intake after treatments with compounds 4
and the racemic (±)-5, even if these compounds evidenced their effect earlier than (Z)-6
1
(Figure 8 c-d). Considering the substantial lower affinity of (±)-5 for CB R if compared to
those of the other assayed compounds, these results suggest a significantly higher
availability of this derivative which might be ascribed to the presence of the hydroxyl
substituent at 3 position of the pyrazole ring. Further studies must be performed in the
future in order to separate the two enantiomers of 5, and successively to verify the
probable relevance of one of the two compounds in the reduction of food intake.
Starting from 2 hours of observation, the administration of compound 3 determined a
significant inhibition of food intake relative to the vehicle (Figure 8 b). However the activity
of 3 was significantly lower than that of 1 (10 mg/kg) and compounds 4-6, especially at 20
mg/kg. The improved activity of (Z)-6 compared to 3 suggests a possible enhancement of
bioavailability as a result of introducing the vinylic fluorine atom. An alternative explanation
of the detected in vivo behavior of (Z)-6 compared to 3 could be related to the possible
different metabolism of the two componds, which could be heavily influnced by the
presence of fluorine atom in the case of (Z)-6.
FIGURE 8
5. Conclusion
1
6

ACCEPTED MANUSCRIPT
We have described herein the synthesis of novel rimonabant derivatives with the goal of
obtaining neutral CB R antagonist compounds with reduced side effects relative to those
1
of the reference compound 1.
In particular, we have synthesized and tested the enantiomer mixture of alcohol derivative
(±)-5, the keto parent compound 4, the fluorine vinyl derivatives (Z)-6 and (E)-7, and the
racemic trifluoroethylamine rimonabant analogue (±)-8.
According to in vitro (radioreceptor binding assays and P-ERK 1/2 expression from N1E-
1
15 cell line) and in ex vivo assays (isolated organs), the newly synthesized derivatives
showed significant affinity for CB R (K values in the nanomolar range), with a biological
profile compatible with that of neutral CB R antagonist compounds.
In spite of their CB affinities, which were lower than those of the reference compounds 3-
, derivatives (±)-5 and (Z)-6 evidenced the most significant efficacy in the control of food
intake in the adopted acute animal model. The entity of food intake inhibition for (±)-5 and
Z)-6 was comparable to that detected in the case of rimonabant, although at higher
1
i
1
1
4
(
doses. However, it is important to note that no observable adverse effect was evidenced in
the case of treatments with (±)-5 and (Z)-6 at doses up to 20 mg/kg, in contrast to the side
effects highlighted in the case of administration of rimonabant at 20 mg/kg.
These results further support the relevance of the substituent at 3 position of the pyrazole
1 1 1
ring for determining both CB R affinity and neutral CB antagonism or inverse CB
agonism. Moreover, the unexpectedly high in vivo potency and efficacy of the compounds
may indicate improved bioavailability after i.p. administration.
In order to confirm and validate the improved properties of the new compounds compared
to that of the lead compound 1, their behavior will be further investigated, with particular
emphasis to the identification of reduced or, better, void adverse effects as induction of
anxiety and depression. Moreover, considering the interesting results obtained with (±)-5,
1
7

ACCEPTED MANUSCRIPT
the separation and the characterization of the two enantiomers will be carried out in the
future in order to identify the component with the best profile.
6. Experimental protocols
6.1. General methods
Melting points were obtained on a Stuart SMP10 Melting point apparatus and are
uncorrected. IR spectra were recorded on a Bruker Alpha-T FT-IR Spectrophotometer
-
1
equipped with an Alpha Eco ATR as sampling module and are expressed in ν (cm ). All
NMR spectra were taken on a Bruker Ultra Shield 400 Avance III NMR Spectrometer, with
1
13
19
1
H, C and F observed at 400, 100 and 377 MHz, respectively. Chemical shifts for H
1
3
19
and C NMR spectra are expressed in δ downfield from TMS (Me
spectra are expressed in δ from CFCl . Multiplicities are reported as s (singlet), bs (broad
singlet), d (doublet), t (triplet), q (quartet), qu (quintuplet), dd (doublet of doublets), m
multiplet). ESI mass spectra were recorded on a Bruker Esquire 3000 Plus Ion Trap mass
4
Si), for F NMR
3
(
spectrometer in the positive mode. All reactions involving air or moisture sensitive
compounds were performed under highly pure nitrogen atmosphere. All reagents and
solvents were purchased from Sigma-Aldrich Chemical Company and used as received.
Combustion analyses of compounds used in biological testing were performed by
Laboratorio di Microanalisi, Dipartimento di Chimica, Università di Sassari, Italy, and the
analyses indicated by the symbols of the elements were within ± 0.4 % of the theoretical
values.
6.2. Chemistry
1
8

ACCEPTED MANUSCRIPT
6.2.1. Ethyl 4-(4-chlorophenyl)-3-methyl-2,4-dioxobutanoate (11). Sodium metal (0.82 g,
35.58 mmol) was added in small portion to dry ethanol (30 mL) and stirred until all the
sodium had reacted. Diethyl oxalate (10) (3.62 mL, 26.69 mmol) was added, followed by
dropwise addition of a solution of 4-chloropropiophenone (9) (3.00 g, 17.79 mmol) in dry
ethanol (40 mL). The mixture was stirred at room temperature for 18 h and then it was
slowly poured into ice and 1N HCl was added. The resulting mixture was extracted with
Et O, dried (Na SO ), and concentrated to afford the analytically pure product in
2
2
4
quantitative yield as a yellowish oil: R
f
= 0.23 (petroleum ether/EtOAc, 9:1); bp: 68-70°C
1
[
55-65 °C /700 mmHg] [46], IR : ν = 1680, 1730, 3440; H NMR (CDCl
3
, 400 MHz) δ= 1.30
(t, 3H, J =7.1 Hz), 1.45 (d, 3H, J =7.1 Hz), 4.28 (q, 2H, J =7.2 Hz), 5.00 (q, 1H, J =7.1 Hz),
4
7.50 (d, 2H, J =8.8 Hz), 7.93 (d, 2H, J =8.8 Hz). Anal. C13H13ClO (C, H, N).
6.2.2. Ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylate
(13).
A
stirred mixture of diketoester 11 (3.00 g, 11.17 mmol) and 2,4-
dichlorophenylhydrazine hydrochloride (12) (2.62 g, 12.28 mmol) in EtOH (90 mL) was
heated under reflux for 16 h. The mixture was allowed to cool to room temperature, and
the solvent was removed under reduced pressure to give a red-orange solid, which was
purified by flash chromatography (petroleum ether/EtOAc, 9:1) to afford the analytically
f
pure product as an orange solid (2.20 g, 50%). R = 0.29 (petroleum ether/EtOAc, 9:1); IR :
1
ν = 1724; H NMR (CDCl
3
, 400 MHz)
δ
=1.43 (t, 3H, J =7.1 Hz), 2.33 (s, 3H), 4.45 (q, 2H, J
=
7.2 Hz), 7.08 (bd, 2H, J =8.5 Hz), 7.29 (dd, 1H, J =2.2, 8.4 Hz), 7.30 (bd, 2H, J =8.5 Hz),
1
3
7
6
1
3
.35 (d, 1H, J =8.4 Hz), 7.38 (d, 1H, J =2.2 Hz); C NMR (CDCl , 100 MHz) δ= 9.8, 14.6,
1.1, 119.2, 127.2, 127.9, 129.0, 130.2, 130.9, 131.0, 133.2, 135.1, 136.0, 136.1, 143.0,
43.1, 162.8; ESI-MS m/z: 408.1 [ESMS m/z: 409.1 (M + 1)] [38,47].
1
9

ACCEPTED MANUSCRIPT
6.2.3.
N-methoxy-N-methyl-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-
pyrazole-3-carboxamide (14). Trimethylaluminum (2.44 mL of 2M solution in hexane, 4.88
mmol) was added dropwise to a suspension of dimethylhydroxylamine (0.48 g, 4.88 mmol)
in dry CH
temperature for 40 min. To this solution, the pyrazole ester 13 (1.00 g, 2.44 mmol) in
CH Cl (6 mL) was added dropwise. Stirring was continued for 16 h at room temperature.
The reaction mixture was cooled to 0 °C , and 10% HCl was carefully added dropwise. The
mixture was extracted with CH Cl , washed with water, brine, dried over Na SO , and
filtered. After evaporation of the solvent, the residue was purified by flash chromatography
2 2
Cl (10 mL) at 0 °C . The solution was stirred at 0 °C fo r 45 min and then at room
2
2
2
2
2
4
f
(petroleum ether/AcOEt, 7:3) affording 0.72 g (80%) of compound 14 as a white solid. R =
1
0
.49 (petroleum ether/AcOEt, 7:3); IR : ν = 1681; H NMR (CDCl
3
, 400 MHz)
δ
= 2.23 (s,
3
H), 3.46 (s, 3H), 3.81 (s, 3H), 7.07 (bd, 2H, J =8.3 Hz), 7.16-7.34 (m, 4H), 7.43 (d, 1H, J
1.6 Hz). Anal. C19 (C, H, N).
=
3 3 2
H16Cl N O
6.2.4. 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-(2-cyclohexyl-1-oxoethyl)-1H-
pyrazole (4). To a solution of compound 14 (0.50 g, 1.18 mmol) in dry THF (10 mL) the
cyclohexylmethylmagnesium bromide (15) (7.06 ml of 0.5 M solution in THF, 3.53 mmol)
was added dropwise at 0 °C under nitrogen. The react ion mixture was gradually warmed to
room temperature and stirred at the same temperature for 24 h. Saturated aqueous NH
was added dropwise at 0 °C , the reaction mixture was diluted with Et O, and the layers
were separated. The aqueous layer was extracted with Et O, and the combined organic
layers were washed with water, dried (Na SO ), and filtered. After evaporation of the
4
Cl
2
2
2
4
solvent, the residue was purified by flash chromatography (petroleum ether/Et
2
O 9:1)
affording 0.37 g (69%) of compound 4 as a white solid. R
f
= 0.5 (petroleum ether/Et O,
2
1
9
:1); IR : ν = 1685; H NMR (CDCl
3
, 400 MHz) δ=0.98-1.11 (m, 2H), 1.14-1.36 (m, 4H),
2
0

ACCEPTED MANUSCRIPT
1.61-1.81 (m, 4H), 1.97-2.11 (m, 1H), 2.32 (s, 3H), 2.94 (d, 2H, J =7.0 Hz), 7.05 (bd, 2H, J
1
3
=
3
8.6 Hz), 7.24-7.32 (m, 4H), 7.43 (d, 1H, J =1.9 Hz); C NMR (CDCl , 100 MHz) δ= 9.8,
26.3, 26.4, 33.4, 34.3, 46.9, 118.0, 127.2, 127.9, 128.9, 130.4, 130.5, 130.9, 133.1, 134.9,
+
1
36.0, 136.1, 142.8, 149.6, 198.0; ESI (m/z) 485.1 [M +Na]. Anal. C H Cl N O (C, H, N).
2
4
23
3
2
6.2.5.
(±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-hydroxyethyl)-4-
methyl-1H-pyrazole ((±)-5). To a suspension of ketone 4 (0.15 g, 0.32 mmol) in MeOH (3
mL) was added sodium borohydride (25 mg, 0.67 mmol), and the mixture was stirred at
room temperature for 1 h. The reaction mixture was diluted with CHCl
water. The organic layer was dried over anhydrous Na SO , filtered, and concentrated to
= 0.27 (petroleum ether/AcOEt, 8:2);
3
and washed with
2
4
give 0.14 g (93%) of compound 5 as a white solid. R
f
1
IR : ν = 3315; H NMR (CDCl
3
, 400 MHz)
δ
= 0.92-1.08 (m, 2H), 1.14-1.33 (m, 4H), 1.53-
1
7
3
1
.94 (m, 7H),1.95-2.05 (m, 1H), 2.12 (s, 3H), 4.93-5.02 (m, 1H), 7.06 (bd, 2H, J =8.6 Hz),
1
3
.23-7.30 (m, 4H), 7.39 (bs, 1H); C NMR (CDCl , 100 MHz) δ= 8.6, 26.2, 26.4, 26.6,
3
2.8, 34.1, 34.2, 44.7, 66.3, 112.6, 127.7, 128.1, 128.7, 130.2, 130.7, 130.8, 133.2, 134.4,
+
+
35.3, 136.5, 142.0, 155.2; E SI (m/z) 463.2 [M ], 485.2 [M +Na]. Anal. C H Cl N O (C,
24 25 3 2
H, N).
6.2.6.
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbaldehyde
(16). The Weinreb amide 14 (0.70 g, 1.65 mmol) dissolved in 7 mL of dry THF was added
dropwise to a stirred suspension of LiAlH (0.08 g, 1.98 mmol) in dry THF (10 mL) under
4
nitrogen at 0 °C and the mixture was stirred for 20 minutes at the same temperature. The
suspension became dark brown. The reaction mixture was quenched by addition of 10%
HCl aq. and poured into brine. The mixture was extracted with Et
2
O. The organic layer was
dried over Na SO and the solvent was removed by rotary evaporation, affording the
2
4
2
1

ACCEPTED MANUSCRIPT
product 16 as a yellowish, viscous oil which formed crystals upon standing, in quantitative
1
yield. R
f
= 0.29 (petroleum ether/Et
2
O, 9:1); IR : ν = 1723; H NMR (CDCl
3
, 400 MHz)
δ
=
2.35 (s, 3H), 7.07 (d, 2H, J =8.3 Hz), 7.18-7.36 (m, 4H), 7.45 (bs, 1H), 10.13 (s, 1H).
6.2.7.
Diethyl
[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-
yl]hydroxymethylphosphonate (17). Triethylamine (0.23 mL, 1.68 mmol) was added to a
stirred mixture of diethyl phosphite (0.44 mL, 3.82 mmol) in toluene (5 mL) at room
temperature. After 5 minutes a solution of aldehyde 16 (0.57 g, 1.56 mmol) in toluene
(
5mL) was added. After 24 h at reflux, an aliquot of K
to this mixture which was then strirred for further 2 h at the same temperature. The mixture
was washed with water, extracted with Et O, dried with Na SO and the solvent
2 3
CO (0.43g, 3.12 mmol) was added
2
2
4
evaporated to give the crude product. Flash chromatography on silica gel (AcOEt/
petroleum ether, 7:3), gave 0.59 g (80%) of pure 17 as a viscous oil which became solid
1
upon standing. R = 0.22 (AcOEt/ petroleum ether, 7:3); IR : ν = 1245, 3590; H NMR
f
(
CDCl
17.6 Hz), 7.07 (d, 2H, J =13.6 Hz), 7.25-7.30 (m, 4H), 7.41 (bs, 1H). Anal.
P (C, H, N).
3
, 400 MHz) δ= 1.27-1.40 (m, 6H), 2.18 (s, 3H), 4.10-4.27 (m, 4H), 5.17 (d, 1H, J
=
21 3 2 4
C H22Cl N O
6
.2.8.
yl]fluoromethylphosphonate (18). To a stirred solution of alcohol 17 (0.42 g, 0.83 mmol) in
CH Cl (2 mL) at −78°C was added DAST (0.13 mL, 1.00 mmol). After 1h at −78 °C , the
reaction mixture was poured carefully into a saturated NaHCO solution. The aqueous
layer was extracted with CH Cl . The combined organic phases where dried over Na SO
Diethyl
[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-
2
2
3
2
2
2
4
,
filtered and concentrated to give the crude product that was purified by flash
chromatogarphy (petroleum ether/ AcOEt, 6:4) to give 0.33g (79%) of 18 as an orange oil.
2
2

ACCEPTED MANUSCRIPT
1
R
f
= 0.40 (petroleum ether/ AcOEt, 6:4); IR : ν = 1246; H NMR (CDCl
3
, 400 MHz)
δ
= 1.32
(
t, 3H, J =7.0 Hz), 1.37 (t, 3H, J =7.0 Hz), 2.23 (s, 3H), 4.07-4.24 (m, 2H), 4.24-4.34 (m,
H), 5.89 (dd, 1H, J =9.2 and 44.5 Hz), 7.07 (d, 2H, J =8.2 Hz), 7.22-7.32 (m, 4H), 7.39
bs, 1H). Anal. C21 FN P (C, H, N).
2
(
H
21Cl
3
2 3
O
6.2.9. 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl]-4-methyl-
1H-pyrazole ((Z)-6) and 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(E)-2-cyclohexyl-1-
fluorovinyl]-4-methyl-1H-pyrazole ((E)-7). To a stirred solution of the phosphite derivative
8 (0.37 g, 0.73 mmol) dissolved in 2.32 mL of THF the cyclohexyl carboxaldehyde (19)
0.11 mmol) was added under nitrogen. The resulting mixture was cooled to −78 °C , then
.48 mL (0.95 mmol) of a solution 2M of lithium diisopropylamide (LDA) in
THF/cyclohexane/ethylbenzene were added dropwise. The resulting solution was stirred at
78 °C for 30 minutes and then allowed to warm up to room temperature over 4 h and
1
(
0
−
stirred at that temperature overnight. The reaction mixture was poured into water, and the
water layer was extracted with ether. The combined organic materials were washed with
1
0% HCl aq. and then with water and brine, dried over Na SO and concentrated at
2 4
reduced pressure. The residue was purified by flash chromatogarphy (petroleum ether/
Et O, 96:4) to give 0.2g (59%) of (E)-7 as a viscous, colorless oil and 0.04 g (11%) of (Z)-6
2
f
as an oil that became solid upon standing. Compound (E)-7: R = 0.46 (petroleum ether/
1
Et
2
O, 96:4); H NMR (CDCl
3
, 400 MHz)
δ
= 1.06-1.36 (m, 4H), 1.53-1.73 (m, 4H), 1.74-1.85
(m, 2H), 2.13 (s, 3H), 2.49-2.64 (m, 1H), 5.42 (dd, 1H, J = 10.0 and 21.1 Hz), 7.07 (d, 2H,
J =8.3 Hz), 7.15 (d, 1H, J =8.3 Hz), 7.23 (dd, 1H, J= 1.6 and 8.3 Hz), 7.30 (d, 2H, J =8.3
1
3
Hz), 7.45 (d, 1H, J =1.6 Hz); C NMR (CDCl , 100 MHz) δ= 9.1, 9.2, 25.9, 26.0, 33.5,
3
33.6, 34.8, 34.9, 115.5, 117.6, 117.7, 127.6, 127.7, 128.9, 130.3, 130.4, 130.7, 133.1,
+
19
1
34.6, 135.3, 136.3, 141.3, 145.0, 145.4, 149.3, 151.7; ESI (m/z) 487.1 [M +Na]; F NMR
2
3

ACCEPTED MANUSCRIPT
(
CDCl
3
, 235 MHz,) δ= −112.83 (s, 1F). Anal. C24
H
22Cl
3
FN
2
(C, H, N). Compound (Z)-6: R
f
1
=
0.27 (petroleum ether/ Et
2
O, 96:4); H NMR (CDCl
3
, 400 MHz) δ
= 1.14-1.43 (m, 4H),
1.58-1.88 (m, 6H), 2.17 (s, 3H), 2.62-2.75 (m, 1H), 5.38 (dd, 1H, J = 9.4 and 38.5 Hz), 7.07
1
3
(
d, 2H, J =8.6 Hz), 7.25-7.34 (m, 4H), 7.39 (d, 1H, J= 1.9 Hz). C NMR (CDCl
δ= 9.7, 9.8, 25.9, 26.0, 33.1, 33.2, 33.6, 33.7, 113.8, 115.0, 115.1, 127.6, 127.8, 128.8,
30.2, 130.8, 130.9, 133.1, 134.7, 135.6, 136.2, 142.3, 145.6, 145.9, 149.5, 152.0; ESI
3
, 100 MHz)
1
+
19
(
(
3 3 2
m/z) 465.1 [M +H]; F NMR (CDCl , 235 MHz,) δ= −122.19 (s, 1F). Anal. C24H22Cl FN
C, H, N).
6.2.10.
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(1-hydroxy-2,2,2-trifluoroethyl)-4-
methyl-1H-pyrazole (20). To a solution of 16 (390 mg, 1.1 mmol) in dry THF (6 mL), a 1 M
tetrabutylammonium fluoride THF solution (55 ꢀL, 0.055 mmol) was added at 0 °C under
nitrogen atmosphere, followed by 2M trimethyl(trifluoromethyl)silane THF solution (1 mL, 2
mmol). The ice bath was then removed and the reaction was monitored by TLC analysis
until completion. Additional 1M tetrabutylammonium fluoride THF solution (55 µL, 0.055
mmol) was then added at room temperature. After 15 min stirring, saturated NH
aqueous solution was added and the mixture extracted with AcOEt. The combined organic
layers were dried over anhydrous Na SO , concentrated under vacuum and the crude
4
Cl
2
4
purified by flash chromatography (hexane/AcOEt, 8:2), yielding 20 as a pale yellow foam
1
(407 mg, 85%). R
f
= 0.30 (hexane/AcOEt, 8:2); H NMR (CDCl
3
, 400 MHz) δ= 2.11 (s, 3H),
3.47 (d, 1H, J =7.9 Hz), 5.16 (m, 1H), 7.70 (m, 2H), 7.28 (m, 4H), 7.42 (d, 1H, J =2.0 Hz);
1
3
C NMR (CDCl3,101 MHz) δ= 8.2, 67.4 (q, J = 33.5 Hz), 102.7, 114.2, 115.3, 122.8, 124.2
q, J = 282.8 Hz), 125.6, 127.2, 127.4, 127.7, 127.7, 128.8, 129.0, 130.2, 130.5, 130.6,
(
2
4

ACCEPTED MANUSCRIPT
1
9
1
30.7, 133.1, 134.9, 135.7, 135.8, 142.6, 146.0, 147.7; F NMR (CDCl
3
, 235 MHz) δ=
−
79.16 (d, 3F, J =6.1 Hz). Anal. C18 O (C, H, N).
3 3 2
H12Cl F N
6.2.11.
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-(1-oxo-2,2,2-trifluoroethyl)-
1H-pyrazole (21). To a solution of oxalyl chloride (550 ꢀL, 6.3 mmol) in dry DCM (20 mL)
dry DMSO (870 ꢀL, 12.1 mmol) was slowly added drop-wise at -78 °C u nder nitrogen
atmosphere. After 15 min stirring, a solution of 20 (706 mg, 1.62 mmol) in dry DCM (5 mL)
was slowly added and after further 15 min freshly distilled triethylamine (2.4 mL, 17.2
mmol) was added drop-wise. The cooling bath was then removed and the mixture was
allowed to warm up to room temperature. The mixture was then concentrated under
vacuum, diluted with water and finally extracted with AcOEt. The combined organic layers
were dried over anhydrous Na SO , concentrated under vacuum and the residue purified
2
4
f
by flash chromatography (DCM), affording 702 mg (99%) of product 21. R = 0.20
1
(
hexane/AcOEt, 95:5); H NMR (CDCl , 400 MHz) δ= 2.36 (s, 3H), 7.07 (d, 2H, J =8.4 Hz),
3
19
7
3
.34 (m, 4H), 7.44 (d, 1H, J =1.8 Hz); F NMR (CDCl , 235 MHz,) δ= −74.25 (s, 3F). Anal.
C H Cl F N O (C, H, N).
1
8
10
3
3
2
6.2.12.
(Z)-1-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]-2,2,2-
trifluoro-N-(piperidin-1-yl)ethanimine (22). To a solution of crude 21 (685 mg, 1.58 mmol)
in toluene (20 mL) pyridinium p-toluenesulfonate (79 mg, 0.31 mmol) was added followed
by N-aminopiperidine (258 µL, 2.39 mmol). The mixture was then refluxed 24h with a
Dean-Stark trap to remove water. After the mixture was cooled to room temperature and
concentrated under vacuum, the crude was purified by flash chromatography
f
(hexane/AcOEt, 95:5), affording 22 as a pale yellow foam (407 mg, 50%). R = 0.30
2
5

ACCEPTED MANUSCRIPT
1
(
Hex/AcOEt = 95:5); H NMR (CDCl
J =5.3 Hz), 7.08 (d, 2H, J =8.5 Hz), 7.31 (m, 4H), 7.41 (d, 1H, J =1.9 Hz,); C NMR
CDCl , 101 MHz) δ= 8.7, 23.7, 25.1, 54.3, 116.9, 117.9, 120.6, 122.0 (q, J =277.0 Hz),
23.3, 128.8, 129.0, 130.0, 130.3, 130.3, 130.4, 130.5, 130.7, 130.8, 133.0, 134.7, 135.7,
3
, 400 MHz) δ= 1.60 (m, 6H), 2.06 (s, 3H), 3.18 (t, 4H,
1
3
(
3
1
1
19
3 3 3 4
36.1, 141.6, 144.8; F NMR (CDCl , 235 MHz) δ= −66.74 (s, 3F). Anal. C23H20Cl F N (C,
H, N).
6.2.13. 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]-2,2,2-trifluoro-
N-(piperidin-1-yl)ethanamine hydrochloride ((±)-8). A solution of 22 (203 mg, 0.39 mmol)
and borane-trimethylamine complex (43 mg, 0.59 mmol) in toluene (6 mL) was saturated
with gaseous HCl for about 40 min. After stirring for 4h at room temperature, the reaction
was complete. Nitrogen was bubbled and the solvent removed under vacuum. The residue
was purified by flash chromatography (hexane/AcOEt, 95:5) affording (±)-8 as a white
1
foam (150 mg, 70%). R = 0.40 (hexane/AcOEt, 9:1); H NMR (CDCl , 400 MHz) δ= 1.36
f
3
(m, 2H), 1.62 (m, 4H), 2.12 (s, 3H), 2.66 (m, 4H), 3.34 (bs, 1H), 4.63 (q, 1H, J =7.7 Hz),
1
3
7
.08 (d, 2H, J = 7.3 Hz), 7.40 (s, 1H), 7.26 (dd, 4H, J =17.7 and 9.8 Hz,); C NMR (CDCl ,
3
101 MHz) δ= 8.6, 23.7, 26.1, 58.0, 59.5 (q, J =29.4 Hz), 114.6, 125.3 (q, J =281.9 Hz),
1
9
127.7, 127.9, 128.8, 130.3, 130.7, 130.8, 133.5, 134.8, 135.6, 136.5, 142.0, 147.3;
F
+
+
3
NMR (CDCl , 235 MHz) δ= −75.03 (bs, 3F); ESI (m/z) 517.2 [M -HCl], 539.2 [M -HCl+Na].
Anal. C H Cl F N · HCl (C, H, N).
2
3
22
3
3
4
6
.3. Pharmacology
.3.1. Chemicals and Drugs for in vitro and in vivo assays.
6
2
6

ACCEPTED MANUSCRIPT
3
[
H]-CP-55,940 (specific activity 180 Ci/mmol) was purchased from Perkin Elmer Italia.
WIN 55,212-2 was obtained from Sigma-Aldrich (Milan, Italy). N-piperidinyl-5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (rimonabant)
was purchased by KEMPROTECH Limited, Middlesbrough, UK.
6.3.2. Animals
All animal experiments were performed according to the UE guidelines for the care and
use of experimental animals (CEE N° 86/609). Animal s (Charles River, Calco, LC, Italy)
were housed in the animal care quarters; temperatures were maintained at 22 ± 2 °C
(humidity 55 ± 5%) on a 12 hrs light/dark cycle. Food and water were available ad libitum
before to start with the experimental procedures.
6.3.3. Receptor binding studies.
Male CD1 mice weighing 30-35 g were killed by cervical dislocation and the brain (minus
cerebellum) and spleen were rapidly removed and placed on an ice-cold plate. Brain
tissues were used for CB
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 MgCl , pH 7.4). The homogenates were centrifuged at
,086 x g for 10 min at 4 °C , and the resulting sup ernatants were centrifuged at 45,000 x g
for 30 min.
For radioreceptor binding experiments, drugs were dissolved in dimethyl-sulfoxide
DMSO). DMSO concentration in the different assays never exceeded 0.1% (v/v) and was
1 2
binding assays, while spleen tissues for CB affinity evaluation.
2
1
(
without effects.
3
[
H]-CP-55,940 binding was performed by the previously described method [58]. Briefly,
3
the membranes (30-80 µg of protein) were incubated with 0.5-1 nM of [ H]-CP-55,940 for
1
h at 30 °C in a final volume of 0.5 ml of TME buf fer containing 5 mg/ml of fatty acid-free
2
7