New cannabinoid receptor antagonists as pharmacological tool

Article abstract of DOI:10.1016/j.bmc.2020.115672

Synthesis and pharmacological evaluation of a new series of cannabinoid receptor antagonists of indazole ether derivatives have been performed. Pharmacological evaluation includes radioligand binding assays with [³H]-CP55940 for CB1 and CB2 receptors and functional activity for cannabinoid receptors on isolated tissue. In addition, functional activity of the two synthetic cannabinoids antagonists 18 (PGN36) and 17 (PGN38) were carried out in the osteoblastic cell line MC3T3-E1 that is able to express CB2R upon osteogenic conditions. Both antagonists abolished the increase in collagen type I gene expression by the well-known inducer of bone activity, the HU308 agonist. The results of pharmacological tests have revealed that four of these derivatives behave as CB2R cannabinoid antagonists. In particular, the compounds 17 (PGN38) and 18 (PGN36) highlight as promising candidates as pharmacological tools.

Full text of DOI:10.1016/j.bmc.2020.115672

Bioorganic&MedicinalChemistry28(2020)115672
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New cannabinoid receptor antagonists as pharmacological tool
Pedro González-Naranjoa, Concepción Péreza, Rocío Girónc, Eva M. Sánchez-Roblesc,
María I. Martín-Fontellesc, Natalia Carrillo-Lópezd, Julia Martín-Vírgalad, Manuel Navesd,
⁎
⁎
Nuria E. Campillob, , Juan A. Páeza,
a Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
b Centro de Investigaciones Biológicas Margarita Salas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain
c Área de Farmacología, Nutrición y Bromatología, Unidad Asociada al IQM y al CIAL (CSIC), Departamento de C.C. Básicas de la Salud, Facultad de Ciencias de la Salud,
Universidad Rey Juan Carlos, Avda. Atenas s/n, 28922 Alcorcón, Spain
d U.G.C de Metabolismo Óseo, RedinREN del ISC III, Hospital Universitario Central de Asturias, Instituto de Investigaciones Sanitarias del Principado de Asturias, Edificio
FINBA, Planta primera F1.1 (Aula 14), Avenida de Roma s/n, 33011 Oviedo, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
Synthesis and pharmacological evaluation of a new series of cannabinoid receptor antagonists of indazole ether
derivatives have been performed. Pharmacological evaluation includes radioligand binding assays with [3H]-
CP55940 for CB1 and CB2 receptors and functional activity for cannabinoid receptors on isolated tissue. In
addition, functional activity of the two synthetic cannabinoids antagonists 18 (PGN36) and 17 (PGN38) were
carried out in the osteoblastic cell line MC3T3-E1 that is able to express CB2R upon osteogenic conditions. Both
antagonists abolished the increase in collagen type I gene expression by the well-known inducer of bone activity,
the HU308 agonist. The results of pharmacological tests have revealed that four of these derivatives behave as
CB2R cannabinoid antagonists. In particular, the compounds 17 (PGN38) and 18 (PGN36) highlight as pro-
mising candidates as pharmacological tools.
Cannabinoid receptors
Antagonist
Indazole ether
Bone disease
Pharmacological tool
1. Introduction
present in highest concentration in olfactory and cortical brain regions,
hippocampus, amygdale, basal ganglia, thalamic and hypothalamic
Identification and target validation are key steps of drug develop-
ment and pharmacological research. To possess high selective and well-
defined molecule mode of action, chemical probes are essential to study
and characterize completely the action mechanism of any drugs. In this
context, the cannabinoid system is a very interesting and intriguing
biological system.
nuclei, cerebellar cortex, and brainstem nuclei and in peripheral tissues
such as testis, eye, urinary bladder, ileum, adipose tissue, liver, skeletal
muscles and pancreas.10–13
Meanwhile, the CB2 receptor (CB2R) is mainly expressed in cells of
the immune system in peripheral tissues, the thymus, tonsils, bone
marrow, spleen, pancreas, peripheral nerve terminals, microglial cells,
glioma and skin tumour cells.10–12 It has been also described the ex-
istence of CB2R in the central nervous system (CNS) under both pa-
thological14 and physiological conditions.15
The endogenous cannabinoid system includes transmembrane can-
nabinoid CB1 and CB2 receptors, endogenous ligands (the en-
docannabinoids), and the processes responsible for their biosynthesis,
cellular uptake, and metabolism.1–3 This system has been implicated in
a variety of biological processes, both in the central and peripheral
nervous systems and in peripheral organs. As a consequence, an array of
potential therapeutic targets is currently being studied including spe-
cific cannabinoid agonists, and antagonists/inverse agonists4–8 as well
as compounds that interrupt the synthesis, uptake or metabolism of the
endocannabinoids.9
In relation to CB1R antagonists, the main principal applications that
have been proposed for clinic are the treatment of obesity and meta-
bolic syndrome, as well as the treatment of addictions.16,17
One of the main representative example is the SR1417A (rimona-
bant) a 1,2-diarylpyrazole derivative reported in 1994 by the Sanofi
group as a selective CB1 antagonist.18 However, rimonabant is now
described as an inverse agonist at the CB1R, displaying negative in-
trinsic activity.19
The CB1 receptor (CB1R) is one of the most abundant G pro-
tein–coupled receptors located within the mammalian brain and are
In 2006, rimonabant was approved by the European Commission as
⁎ Corresponding authors.
E-mail addresses: nuria.campillo@csic.es (N.E. Campillo), jpaez@iqm.csic.es (J.A. Páez).
https://doi.org/10.1016/j.bmc.2020.115672
Received 18 April 2020; Received in revised form 15 July 2020; Accepted 18 July 2020
Availableonline29July2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

P. González-Naranjo, et al.
Bioorganic&MedicinalChemistry28(2020)115672
an anti-obesity agent. However, rimonabant was withdrawn by
European Medicine Agency in 2008 because of its substantial CNS risk
factors including depression and anxiety, including suicidal tendency.
Therefore, the development of anti-obesity drugs targeting CB1R in the
brain has been suspended and/or terminated globally, although there is
some controversy over the origin of these side effects. According to a
recently published meta-analysis, obesity increased the risk of depres-
sion and this increased the chances for developing obesity.17 Other
studies suggest that 50% of patients who are looking for anti-obesity
treatment report depression symptoms.20,21 If the adverse effects of
rimonabant are attributed to its inverse agonist character, it was sug-
gested that a neutral antagonist would be an alternative approach for
treating obesity, related metabolic risk factors22 or against drug ad-
diction. Thus, Tudge et al.23 have performed a clinical study with tet-
rahydrocannabivarin, a neutral antagonist, showing that treatment
with this CB1 neutral antagonist increases neural responding to re-
warding and aversive stimuli. This effect profile suggests therapeutic
activity in obesity, perhaps with a lowered risk of depressive side ef-
fects. However, other authors suggest that neutral antagonism is a
concept that probably does not exist in real world; therefore, apparent
neutral antagonists are probably inverse agonists that have not yet been
unmasked.24
cannabinoids,29,30 we proposed to develop cannabinoid antagonists.
Recently, we have published a new family of indazole ethers as a
novel class of cannabinoid multitarget drugs with potential application
for the treatment of Alzheimer's disease.29 The agonist activity of these
indazole ether derivatives is function of the substituents at positions 1,
3 and 5 of heterocyclic system. Continuing with the study of the
properties of this family of indazole derivatives other substituents has
been considered to obtain new indazole ethers behaving as cannabinoid
antagonists.
In this work, we have addressed the synthesis and biological char-
acterization of a family of indazole ethers that behave as pure CB2R
antagonist. We have performed isolated tissue assays in order to char-
acterize the biological profile of the cannabinoid. Later, we have stu-
died the ability of these antagonists to revert the agonist effect of
HU308 on primary osteoblasts assays.
2. Results and discussion
2.1. Chemistry
Based on previous results we performed the synthesis of a re-
presentative set of compounds (1–22) in order to found cannabinoid
antagonists (Table 1).
As for the CB2R, the first selective antagonists, the pyrazole
SR14452825 and AM63019 are two of the most commonly used CB2
selective antagonists and have been generally used as pharmacological
tools in order to demonstrate specificity of CB2 selective agonists.
However, SR144528 has been described both as an antagonist25 and as
an inverse agonist,26,27 probably due to the different experimental
protocols used. Regarding the therapeutics applications of antagonist
CB2R, laboratory data suggest that antagonist/inverse agonists selec-
tive for the CB2R may have therapeutic potential. The expression of the
receptor by immune cells, both in the periphery and in the CNS suggests
that modulation of inflammations or allergies could be achieved with
appropriate CB2R antagonists.28
The synthetic strategy used depends of the desired substituents at
positions N-1, C-5 and at the hydroxyl group of the indazole system.
Thus, there are three possible strategies or synthetic routes that are
presented in Scheme 1.
The route A, by direct reaction of the indazolol derivatives with the
corresponding halides in acetone or butanone and caesium or po-
tassium carbonate as base, is only useful to obtain disubstituted deri-
vatives with identical group at both positions (N-1, O-3). For deriva-
tives with different groups at these positions, the synthetic route B is a
two-step procedure involving, first, the preparation of the indazolol
derivatives with the benzyl or methyl substituents at position 1, and
then introduction of the substituent at O-3 position using different ar-
ylalkyl or cycloalkylmethyl halides.
For all these reasons, the description of alternative antagonists is
very interesting, in order to characterize the action mechanism of new
cannabinoids agonists. Encouraged by this challenge and continuing
with our efforts in the design and development of new
A more versatile and general synthetic route for the formation of
substituted indazoles, the route C, comprises three steps involving
Table 1
Binding affinities (Ki, µM) and selectivity for CB1 and CB2 receptors of indazole derivatives 1–22.
Compd
R1
R2
R3
Ki CB1 (μM)
Ki CB2 (μM)
Selectivity Ki CB1/Ki CB2
1
H
H
4-MeOPh
1-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
cyclohexyl
Ph
21
6
4.5
0.8
0.08
4.7
2
H
H
1.6
21.7
4
0.1
20.8
0.44
7
3.6
3
H
H
2
3.1
4
H
CH3
2
10.1
> 40
1.3
2.4
0.4
5
H
(CH2)2-cyclohexyl
CH2-cyclohexyl
CH2-Ph
H
0.15
0.2
2.6
> 40
0.09
0.2
0.9
< 0.004
0.2
6
H
0.6
7
H
3.5
0.1
0.2
0.2
0.7
8
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NH2
NH2
4-MeOPh
1-naphthyl
2-naphthyl
(CH2)Ph
1.8
> 22.2
> 44.4
2.7
9
H
> 40 (38%)
0.9
10
H
13.6
5.4
5
1
11
CH3
> 40 (25%)
> 40
1.2
0.5
2.5
> 33.3
> 11.4
≥160.0
1.9
12
CH3
2-naphthyl
1-naphthyl
(CH2)3CH3
4-MeOPh
2-naphthyl
2-naphthyl
cyclohexyl
Ph
3.5
13
CH3
≥40
0.25
0.68
0.49
1.37
0.47
0.09
0.4
0.06
14
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)2CH3
CH2-Ph
CH2-Ph
CH2-Ph
(CH2)2CH3
CH2-Ph
1.3
0.6
0.2
0.1
0.04
0.06
0.06
0.04
0.03
15
0.8
1.6
16
1.6
1.2
17 (PGN38)
0.356
0.006
0.8
18 (PGN36)
> 40 (39%)
> 444.4
3.0
19
20
21
22
1.2
> 10
1.6
4
0.3
0.2
0.1
2-naphthyl
2-naphthyl
Ph
0.4
0.2
0.7
0.3
> 25.0
0.6
2.9
1
1.6
2.5
2

P. González-Naranjo, et al.
Bioorganic&MedicinalChemistry28(2020)115672
Scheme 1. Synthetic routes to prepare indazole derivatives 1–25.
protection of position N-1, formation of the corresponding ether deri-
vative, subsequent deprotection and introduction of the second desired
arylmethyl or alkyl substituents at the N-1 position.
respectively.
The second step is the formation of the ether function and sub-
sequent deprotection. Thus, the reaction of 28 with the corresponding
halides afforded the 5-nitro ether derivatives of 2-naphthylmethoxy
10,29 1-naphthylmethoxy 9 and 4-methoxybenzyloxy 8. Similarly, the
preparation of 1-naphthyl, 2-naphthyl and 4-methoxyphenyl deriva-
tives 2, 3, and 129 were carried out in the same conditions from in-
dazolol 27. All these products were carried out using the procedure
described for us.29
Following the route A, different N-1 substituted indazole ethers
were prepared by reaction of indazolol 23 or 5-nitroindazolol 2431 with
the corresponding halides in acetone or butanone and caesium or po-
tassium carbonate as base (Table 1, Scheme 2). Thus, the synthesis of
indazole ethers 629 and 7 were carried out starting from 23 and cy-
clohexylmethyl or benzyl bromides, respectively. Similarly, the 5-ni-
troindazole ethers 1429 and 1932 were prepared by reaction of 5-nitro-
3-indazolol 24 with the corresponding pentyl or benzyl halides, re-
spectively.
The last step of route C is to introduce the appropriate group in
position N1 in order to obtain alkyl N1-substituted indazole ethers.
Thus, the preparation of the 3-(2-naphthylmethoxy)indazoles 4 and 5
were carried out from 329 with the corresponding alkyl halide to afford
the 1-methyl derivative 4 and 1-cyclohexylethyl derivative 5. Similarly,
the reaction of the 5-nitro-2-naphthylmethoxyindazole 1029 with the
corresponding alkyl iodides afforded the methyl, propyl and pentyl
derivatives, 12, 17 (PGN38) and 16.29 The 4-methoxybenzylderivative
1529 was carried from 8 with pentyl iodide.
To synthesize 1,3-disubstituted indazoles with different groups at N-
1 position and ether function the synthetic route B were followed
starting from 5-nitro-3-hydroxyindazole 24. Thus, the reaction of 5-
nitroindazole 24 with methyl iodide or benzyl bromide afforded 2533
and 26,34 respectively. Then, subsequent O-alkylation of the indazoles
25 and 26 with the corresponding halides were carried out to obtain the
5-nitroindazoles 11, 13, 18,29 and 20.29
Finally, the 5-amino derivative 21, was obtained by catalytic re-
duction with ferric oxyhydroxide (FeO(OH))of 5-nitroderivative 17
Thus, the preparation of indazole ether 20 29 was carried out with 2-
naphthylmethyl bromide from 5-nitro-1-benzylindazole 26. Following
the same route B, the 1-methyl derivatives 11 and 13 were obtained
from 5-nitro-3-indazolol 25 with phenethyl bromide or with 1-naph-
thylmethyl chloride in acetone, using K2CO3 as base, respectively. Si-
milarly, the reaction of 1-benzyl-3-indazole 26 with cyclohexylmethyl
bromide afforded the corresponding indazole ether 18 (PGN36).
As mentioned before the route C has been especially useful to the
preparation of indazole ethers bearing aminoethyl groups at N-1 posi-
tion.29 Therefore, route C is the more versatile and suitable procedure
to introduce any group and has been used for the preparation of the
indazole ethers 4, 5, 12, 15–17. Thus, the first step is the reaction of
ethyl chloroformate with the indazole derivatives 23 and 24 afforded
the corresponding 1-ethoxycarbonyl derivatives 2734 and 28,32
(PGN38) with hydrazine, following the method described for 4-amino-
29
3- benzyloxy-1-benzylindazole 18
(Scheme 2).
2.2. Biological assays
2.2.1. In vitro binding studies in cannabinoid receptors.
Radioligand displacement assays were used to evaluate the affinity
of the new compounds 1–22 using membranes from cells
(HEK293EBNA) transfected with the CB1R or the CB2R and [3H]-
CP55940 as radioligand. Several indazole ethers showed low solubility
at concentrations greater than 10 μM under test conditions (see mate-
rial and methods section), therefore precluding the determination of
their quantitative value of Ki for these compounds.
3

P. González-Naranjo, et al.
Bioorganic&MedicinalChemistry28(2020)115672
are shown in Table S1. Compounds 1–3, 5–8, 10, 11, 14, 16, and
19–21 were devoid of antagonist properties when added to the organ
bath 10 min before the addition of WIN 55212-2 at increasing con-
centrations. However, cannabinoids 4, 15, 17 (PGN38), 22 and, spe-
cially, selective CB2R indazoles 9, 12, 13, and 18 (PGN36), antag-
onized the effect of WIN 55212-2 as shown in Figs. 1 and 2. These
results indicate that in the MVD compounds 9, 12, 13, and 18 (PGN36)
behave as cannabinoid receptor antagonists with similar or higher ef-
fect to the reference cannabinoid receptor antagonist AM251.
Considering that the indazole derivatives 4, 9, 12, 13, 15, 17
(PGN38), 18 (PGN36), and 22 showed an interesting profile as poten-
tial cannabinoid receptor antagonists, they were tested at the same
concentrations used to WIN 55212-2 (in the range from 10-7 to
1.82 × 10-5 M). The results of these experiments with AM251 and with
the cited indazoles indicate that they did not induce significant mod-
ification of contractile responses at the tested concentrations and
therefore, it could be established that these indazoles did not show any
intrinsic activity (Fig. 3, Table S2).
Scheme 2. Synthetic route for 5-amino indazole ethers.
All synthesized compounds together with their binding affinity for
the CB1R and CB2R are gathered in Table 1. Examination of these data
indicates that all of the evaluated derivatives bind to the CB2R, except
the 1-cyclohexylmethyl derivative 5. The obtained data shown that the
3-cyclohexylmethoxy derivative 18 (PGN36) is the most potent and the
derivative with greatest affinity in CB2R. In relation to CB1R, all 5H
derivatives assays showed some affinity. Regarding, the 5-ni-
troindazoles 8, 12, and 13 not exhibit affinity to 40 µM. However, the
1-alkyl-5-nitro derivatives 15 and 17 showed the lowest values of Ki.
Regarding CB2 selectivity, it is interesting to mark several com-
pounds as the 5-nitro compounds 8, 11, 20, the 5H-indazole 4, the 5-
amine derivative 18 and especially the derivatives 9 and 18 (PGN36)
and the 1-methyl-indazoles 12, 13, due to interesting pharmacological
profiles that they will be discussed later.
The obtained data suggest that these indazoles behave as antago-
nists with a better profile than AM630 and that they do not display the
inverse agonist activity described for other cannabinoid receptor an-
tagonists and then, considering these results and those obtained in the
binding studies, 9, 12, 13, and 18 (PGN36) could be considered as
neutral cannabinoid CB2R antagonists.
2.2.3. Tool as antagonist ligands
Endocannabinoids are present in the skeleton suggesting that ske-
letal endocannabinoid system plays an important role in the regulation
of bone mass.42,43 Bone cells express CBRs and the machinery for en-
docannabinoid metabolism, thereby indicating that endocannabinoids
influence bone remodelling acting on CB1R and CB2R expressed on
bone cells. It has been recently reported that bone remodelling is sub-
jected to CNS,44–47 which is also associated with the regulation of en-
docannabinoid brain levels.48 However, the role of the CBRs in bone
disorder is not clear due to the contradictory results of published
works.43 Different reasons could be responsible for these experimental
inconsistencies, such as the species differences, off-target effects of CBR
ligands at different concentrations, the activity of endogenous canna-
binoids and the interactions of ligands with other receptors such as
GPR55.49 GPR55 is a G protein-coupled receptor that is activated by
certain cannabinoids and by lysophosphatidylinositol (LPI). This re-
ceptor that has been expressed in human and mouse osteoclasts and
osteoblasts is implicated in bone physiology by regulating osteoclast
number and function.50
Receptors binding studies were performed using membrane frac-
tions of human CB1R or CB2R transfected cells (HEK293EBNA). Ki
value of WIN55212-2 is 36.2 nM in CB1 receptor. Ki values of
WIN55212-2 and HU308 are 3.7 and 11.2 nM in CB2, respectively.
2.2.2. Cannabinoid activity: isolated tissue assays
According to the objectives, all derivatives that showed activity as
cannabinoid ligands have been studied in detail in isolated tissues.
Thus, the functional activity of the new compounds 1–22 has been
tested on mouse vas deferens (MVD), a tissue commonly used to study
and characterize cannabinoid effects.29,30,35,36 In MVD cannabinoid
agonists, acting at prejunctional cannabinoid receptors, reduce ATP and
noradrenaline release and inhibit the electrically evoked smooth muscle
contractions. In this tissue, cannabinoid receptor antagonists oppose the
inhibitory effect of cannabinoid receptor agonists in a competitive and
surmountable manner. CB1 and CB2-like cannabinoid receptors seem to
be involved in this effect.37–41
The ability of indazoles 1–22 to inhibit the effect of WIN 55212-2 in
this tissue was investigated. Variations produced by 1–22, on the in-
hibition of electrically induced contractions evoked by WIN 55212-2,
In this sense, having the biochemical tools necessary to get unravel
the role of the cannabinoid ligands is of the utmost importance.
To confirm the antagonist activity of these compounds and therefore
Fig. 1. Antagonist effect of the new derivatives
in mouse isolated vas deferens (MVD). Lines
show the
mean
%
of inhibition (expressed as
S.E.M., n = 6–8) of the electrically
induced contractions of the MVD by addition of
increasing concentrations of WIN 55,212–2 in
control tissues (WIN) or in tissues incubated with
the new compounds 4, 9, 12, 13, 15, 17
(PGN38), 18 (PGN36), 22 or the cannabinoid
receptor antagonist AM251 (10−6 M) that were
added to the organ bath 10 min before each
concentration of WIN. A two-way ANOVA fol-
lowed by Bonferroni post-hoc test was used for
statistical analysis. vs. WIN (μ,*,ω,ϕ,$p < 0.05; @
,&,
*
,$p < 0.01; #,@,&,$p < 0.001): ω22 + WIN,
μ15 + WIN, ϕ17 (PGN38) + WIN, *13 + WIN,
#18 (PGN36) + WIN, @12 + WIN, $9 + WIN, &
AM251 + WIN.
4

P. González-Naranjo, et al.
Bioorganic&MedicinalChemistry28(2020)115672
Fig. 2. Antagonist effect of indazoles 9, 12, 13,
and 18 (PGN36) in mouse vas deferens (MVD).
The graph shows the modification induced by
the new indazoles on the inhibitory effect of WIN
55,212–2 (expressed as mean % of inhibition of
the contractions
S.E.M., n = 6–8) in tissues
incubated with the new compounds or the can-
nabinoid receptor antagonists AM251 or AM630
(10−6 M) and in comparison with the effect of
WIN in control tissues. A two-way ANOVA fol-
lowed by Bonferroni post-hoc test was used for
statistical analysis. vs. WIN (*,$ p < 0.05; @,&,
*
,
$p < 0.01; #,@,&,$p < 0.001): *13 + WIN, #18
(PGN36) + WIN, @12 + WIN, $9 + WIN,
&
AM251 + WIN.
Fig. 3. Effect of WIN 55,212–2 (WIN), AM251, 4, 9, 12, 13, 15, 17 (PGN38), 18 (PGN36) and 22 in isolated mouse vas deferens. Lines show the mean %
(n = 6–8) of inhibition of the electrically induced contraction of this tissue by addition of increasing concentrations to the organ bath.
S.E.M.
the ability to be used as biochemical tools, we used them in the mouse
osteoblastic MC3T3-E1 cell line to study the osteoblast proliferation in
assay on primary osteoblasts (obtained from the calvaria of new born
mice). The osteoblastic cell line MC3T3-E1, a cellular system able to
express CBR2 upon osteogenic conditions,51 was used to study the effect
of 17 (PGN38) and 18 (PGN36) on bone cell activity. The concentration
of 10-6 M used of both CBR antagonists did not affect cell viability
(Fig. 4).
bone activity. By contrast, HU308 10-8 M significantly increased the
collagen type I gene expression compared with the control group but
also with the other two concentrations used (10-6 M and 10-7 M, Fig. 6).
This effect of HU308 on bone activity has been reported by some au-
thors using similar concentrations to the used in our in vitro system.52,53
While cells treated only with HU308 10-8 M significantly increased
the collagen type I gene expression, preincubations during the first 24 h
with 17 (PGN38) 10-6 M or 18 (PGN36) 10-6 M totally abolished this
effect showing similar values to the observed in the control group
(Fig. 7).
Collagen type I gene expression was used as marker of bone activity.
Neither 17 (PGN38) nor 18 (PGN36) were able to modify collagen type
I gene expression (Fig. 5), indicating no effect of both compounds on
Fig. 4. Cell viability in MC3T3-E1 cells treated with 17 (PGN38) 10-6M, 18
(PGN36) 10-6M, and vehicle. Experiments were carried out three times in
sextuplicate.
Fig. 5. Gene expression of Collagen type I in MC3T3-E1 cells treated with 17
(PGN38) 10-6M, 18 (PGN36) 10-6M, and vehicle. UR
= relative units.
Experiments were carried out three times in triplicates.
5

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Bioorganic&MedicinalChemistry28(2020)115672
4. Materials and methods
4.1. Synthesis
4.1.1. General
All starting materials were purchased from common commercial
suppliers, mostly Sigma-Aldrich and Alfa Aesar, and were used without
further purification. All the reactions were monitorized by TLC.
Analytical thin-layer chromatography (TLC) was carried out using
Merck silica gel 60 F254 plates and the compounds were visualized
under UV light (λ = 254 or 365 nm). 1H NMR spectra (300 MHz) and
13C NMR spectra (75 MHz) were recorded on a Bruker Avance 300
spectrometer and are reported in ppm. The signal of the solvent was
used as reference. Flash column chromatography was carried out using
Merck silica gel 60 (230–400 mesh). HPLC-MS was performed using a
Waters 2695 apparatus with a diodo array UV/Vis detector Waters
2996 and coupled to a Waters micromass ZQ using a Sunfire C18
(4.6 × 50 mm, 3.5 mM) column at 30 °C, with a flow rate of 1 mL /
min. The mobile phases used were with different gradients of CH3CN
with 0.1% of formic acid in H2O. The initial conditions, time of gradient
(gt) and time of retention (rt) are specified in each case. Electrospray in
positive mode was used for ionization. The sample injection volume
was set 5 μL of a solution of 1 mg / mL CH3CN. Melting points were
determined with a Reichert-Jung Kofler apparatus. Elemental analysis
were performed on a Heraeus CHN-O Rapid Analysis apparatus. The
purity of all compounds was > 95% prior to biological testing. 1H-3-
indazol-ol (23) was purchased on Alfa Aesar and used without further
purification. 5-nitro-3-indazol-ol (24) was prepared from the procedure
reported by Pfannstiel.31 1-benzyl-3-benzyloxy-5-nitroindazole (19)
and 1-ethoxycarbonyl-5-nitro-3-indazolone (28) were prepared from
the procedure reported by Arán et al.32 1-benzyl-5-nitro-3-indazolone
(26) and 1-ethoxycarbonyl-3-indazolone (27) were prepared from the
procedure reported by Palazzo.34 The compounds 11 and 25 were
Fig. 6. Gene expression of Collagen type I in MC3T3-E1 cells treated with
HU308 10-6M, 10-7M and 10-8M. ap
< 0.05 compared with control group;
bp
<
0.05 compared with HU308 10-7M and 10-8M. UR = relative units.
Experiments were carried out three times in triplicates.
Fig. 7. Gene expression of Collagen type I in MC3T3-E1 cells pre-treated 24 h
with 18 (PGN36) 10-6M, 17 (PGN38) 10-6M and HU308 10-8M and 48 addi-
tional hours with HU308 10-8M. ap
< 0.05 compared with control group;
bp < 0.05 compared with 18 (PGN36) 10-6M; cp < 0.05 compared with 17
(PGN38) 10-6M. Experiments were carried out three times in triplicates.
synthesized by methods described in reference 33. The compounds 1–3,
29
6–10, 14, 15, 16, 18, 20, and 22 were described in reference
.
3. Conclusions
4.1.2. General procedure for the synthesis of the products 4, 5, 12, 17
To a suspension of 1H-3-indazolylether derivative in butanone,
K2CO3 were added. The suspension was stirred and heated to reflux and
the corresponding halide in excess was added and the reaction was
maintained at reflux until the complete elimination of starting material.
Then, it was cooled and filtered to remove inorganic salts. The re-
maining solvent was evaporated under reduced pressure and the pro-
duct was purified by crystallization or by silica gel column chromato-
graphy using the appropriate solvents. Reaction times, conditions and
specific treatments are described individually for each compound.
Indazole derivatives have been synthesized and identified as a new
class of cannabinoid antagonists. Structural requirements for activity
are the presence of one aromatic group, one H or alkyl group at position
1 and the nitro group at position 5. According to binding studies and
tissues assays there are four mixed CB1/CB2 cannabinoid antagonists
and four are selective agonist CB2R. In particular compound 18
(PGN36) showed greater affinity and selectivity by CB2R.
An interesting idea suggested about pharmacological application of
cannabinoid antagonists is related to the treatment of certain bone
disorders. Nevertheless, the performed studies have shown that can-
nabinoids antagonists either by CB1 or by CB2 action have no effect in
MC3T3-E1 celular assays.
4.1.3. 1-Methyl-3-(2-naphthylmethoxy)indazole (4)
From 3 (0.30 g, 1.10 mmol), methyl iodide (0.18 mL, 3.40 mmol)
and K2CO3 (0.33 g, 2.34 mmol) in 2-butanone (20 mL). The final pro-
duct was purified by chromatography column using as eluent methy-
lene chloride: hexane (1:2, 2:1). Reaction time: 24 h. Yield: (0.06 g,
52%). Oil. 1H NMR (300 MHz, DMSO‑d6) δ: 8.04 (bs, 1H, Ar); 7.96–7.91
(m, 3H, Ar); 7.66–7.62 (m, 2H, 4-H, Ar); 7.55–7.47 (m, 3H, 7-H, Ar);
7.38 (t, J = 7.8 Hz, 1H, 6-H); 7.03 (t, J = 7.8 Hz, 1H, 5-H); 5.55 (s, 2H,
CH2); 3.87 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ: 155.6 (C-3);
142.0 (C-7a); 127.3 (C-6); 120.1 (C-5); 118.9 (C-4); 112.5 (C-3a); 108.5
(C-7); 70.7 (CH2); 35.1 (CH3); 134.5 (Ar); 133.3 (Ar); 133.1 (Ar); 128.2
(Ar); 128.1 (Ar); 127.7 (Ar); 126.8 (Ar); 126.1 (Ar); 126.0 (Ar); 125.8
(Ar). HPLC-MS (ES+): CH3CN/H2O 20:80, gt: 18 min; rt: 16.50,
[M + H]+ = 289.3.
However, the selective CB2R cannabinoid agonist as HU308 sti-
mulates osteoblast proliferation in assay on primary osteoblasts (ob-
tained from the calvaria of new born mice) being completely abolish by
the antagonist PGN36 and PGN38.
In conclusion, we present the CB2 antagonist 18 (PGN36) and the
non-selective antagonist 17 (PGN38) that are able to reverse the effect
of HU308 in assay on primary osteoblasts. Moreover, some antagonist
described in this work as the compounds 13 (PGN8) and 9 (PGN70)
already were used as pharmacological tool to demostrate that the
agonist effect of WIN 55212-2 on multiple myeloma (MM) cells is
mediated through CB2R.54 Thus, the pro-apoptotic effect of WIN
55212-2 is inhibited through the pre-incubation with 13 (PGN8) in
both U266 and RPMI cell lines.
Therefore, this new family of cannabinoid receptor antagonists are
promising candidates as pharmacological tool.
4.1.4. 1-(2-Cyclohexyl)ethyl-3-(2-naphthylmethoxy)indazole (5)
From 3 (0.48 g, 1.85 mmol), 2-(cyclohexyl)ethyl bromide (0.30 mL,
1.92 mmol) and K2CO3 (0.62 g, 4.5 mmol) in 2-butanone (50 mL). The
final product was purified by chromatography column using as eluent
6

P. González-Naranjo, et al.
Bioorganic&MedicinalChemistry28(2020)115672
methylene chloride: hexane (1:2, 5:1). Reaction time: 96 h. Yield:
(0.42 g, 60%). Oil. 1H NMR (300 MHz, CDCl3) δ: 8.05 (bs, 1H, Ar);
7.95–7.91 (m, 3H, Ar); 7.70–7.62 (m, 2H, Ar, 4-H); 7.54–7.48 (m, 3H,
7-H, Ar); 7.37 (t, 1H, 6-H); 7.02 (t, 1H, 5-H); 5.59 (s, 2H, O-CH2); 4.23
(t, J = 7.1 Hz, 2H, N1-CH2); 1.78–1.57 (m, 7H, CH2Cy); 1.30–1.15 (m,
4H, Cy); 0.98–0.90 (m, 2H, Cy). 13C NMR (75 MHz, CDCl3) δ: 155.4 (C-
3); 141.2 (C-7a); 126.9 (C-6); 120.1 (C-5); 118.8 (C-4); 112.5 (C-3a);
108.5 (C-7); 70.7 (O-CH2); 46.3 (N1-CH2); 35.9 (C, Cy); 34.1 (CH2Cy);
32.1 (2C, Cy); 25.5 (C, Cy); 25.2 (2C, Cy); 134.6 (Ar); 133.3 (Ar); 133.1
(Ar); 128.1 (Ar); 128.0 (Ar); 127.7 (Ar); 127.0 (Ar); 126.1 (Ar); 126.0
(Ar); 125.9 (Ar). HPLC-MS (ES+): CH3CN/H2O 10:90, gt: 8 min; rt:
7.45, [M + H]+ = 385.4.
added K2CO3 (0.14 g, 0.98 mmol). The suspension was stirred and
heated to reflux and cyclohexylmethyl bromide (0.10 mL, 0.70 mmol)
was added. The reaction was maintained at reflux until the complete
elimination of starting material. Then, it was cooled and filtered to
remove inorganic salts. The remaining solvent was evaporated under
reduced pressure and the product was purified by silica gel column
chromatography using as eluent a mixture methylene chloride: hexane
(1:1, 5:1). Reaction time: 24 h. Yield: (0.08 g, 60%). mp. 72–75 °C (2-
propanol). 1H NMR (300 MHz, CDCl3) δ: 8.67 (d, 1H, 4-H); 8.17 (dd,
1H, 6-H); 7.30 (d, 1H, 7-H); 7.29–7.15 (m, 5H, Ar); 5.41 (s, 2H, N1-
CH2); 4.20 (d, 2H, O-CH2); 1.93–1.09 (m, 11H, CyHex). 13C NMR
(75 MHz, CDCl3) δ: 158.7 (C-3); 143.1 (C-5); 141.3 (C-7a); 137.6 (C,
Ar); 129.3 (2C, Ar); 128.4 (C, Ar); 127.5 (2C, Ar); 123.0 (C-6); 119.2 (C-
4); 113.2 (C-3a); 109.2 (C-7); 71.5 (O-CH2); 53.3 (N1-CH2); 38.0 (CH2);
30.1 (2C, CH2); 26.9 (CH3); 26.2 (2C, CH2). HPLC-MS (ES+): CH3CN/
H2O 10:90, gt: 8 min; rt: 7.07, [M + H]+ = 366.4.
4.1.5. 1-Methyl-3-(2-naphthylmethoxy)-5-nitroindazole (12)
From 10 (0.15 g, 3.93 mmol), methyl iodide (0.90 g, 4.01 mmol)
and K2CO3 (2.57 g, 18.57 mmol), in 2-butanone (60 mL). The final
product was purified by chromatography column using as eluent me-
thylene chloride: hexane (1:1, 5:1). Reaction time: 24 h. Yield: (0.10 g,
58%). mp. 131–132 °C (2-propanol). 1H NMR (300 MHz, CDCl3) δ: 8.72
(d, J = 2.2 Hz, 1H, 4-H); 8.24 (dd, J = 9.3 Hz, J = 2.2 Hz, 1H, 6-H);
7.99 (bs, 1H, Ar); 7.92–7.85 (m, 3H, Ar); 7.64–7.61 (m, 1H, Ar);
7.53–7.48 (m, 2H, Ar); 7.24 (d, J = 9.2 Hz, 1H, 7-H); 5.60 (s, 2H, CH2);
3.95 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ: 157.5 (C-3); 143.0 (C-
7a); 140.9 (C-5); 122.6 (C-6); 118.7 (C-4); 112.0 (C-3a); 108.5 (C-7);
71.19 (CH2); 35.6 (CH3) 133.6 (Ar); 133.2 (Ar); 128.4 (Ar); 128.0 (Ar);
127.8 (Ar); 127.4 (Ar); 126.3 (Ar); 126.2 (Ar); 125.7 (Ar). HPLC-MS
(ES+): CH3CN/H2O 10:90, gt: 8.00 min, rt: 6.16, [M + H]+ = 334.1.
4.1.9. 5-Amino-3-(2-naphthylmethoxy)-1-propylindazole (21)
To a suspension of 17 (44 mg, 0.12 mmol), and FeO(OH) (3 mg,
0.03 mmol) in methanol (15 mL) is added monohydrated hydrazine
(3.0 mL, 7.9 mmol) under Argon atmosphere. The suspension was
stirred and heated at 70 °C until the complete elimination of starting
material. Then, the suspension is filtered over zelite to eliminate the
catalyst. After evaporate the solvent under reduced pressure, the crude
was suspended on water and extracted with diethylic ether. The organic
solvent was removed at reduced pressure to obtain 21 as oil. Reaction
time: 3 h. Yield: (0.028 g, 70%). Oil. 1H NMR (300 MHz, CDCl3) δ: 7.97
(bs, 1H, Ar); 7.88–7.84 (m, 3H, Ar); 7.63 (dd, J = 8.2 Hz, J = 1.5 Hz,
1H, Ar); 7.50–7.47 (m, 2H, Ar); 7.09 (d, J = 8.8 Hz, 1H, 7-H); 6.93 (d,
J = 2.1 Hz, 1H, 4-H); 6.84 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H, 6-H); 5.56
(s, 2H, O-CH2); 4.10 (t, J = 7.0 Hz, 2H, N1-CH2); 3.18 (bs, 2H, NH2);
1.86 (m, 2H, CH2); 0.89 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (75 MHz,
CDCl3) δ: 153.5 (C-3); 137.8 (C-5); 136.4 (C-7a); 118.2 (C-6); 111.9 (C-
3a); 108.5 (C-7); 102.0 (C-4); 69.7 (O-CH2); 49.2 (N1-CH2); 22.2 (CH2);
10.4 (CH3); 133.8 (Ar); 132.3 (Ar); 132.1 (Ar); 127.1 (Ar); 127.0 (Ar);
126.7 (Ar); 125.9 (Ar); 125.1 (Ar); 125.0 (Ar); 124.9 (Ar). HPLC-MS
(ES+): CH3CN/H2O 10:90, gt: 5.00 min, rt: 3.90, [M + H]+ = 333.1.
4.1.6. 3-(2-Naphthylmethoxy)-5-nitro-1-propylindazole (17)
From 10 (0.17 g, 0.52 mmol), 1-propyl iodide (0.10 mL, 1.02 mmol)
and K2CO3 (0.35 g, 2.53 mmol) in 2-butanone (30 mL). The product
was isolated by recrystallization from 2-propanol. Reaction time: 48 h.
Yield: (0.10 g, 55%). mp. 95–97 °C (2-propanol). 1H NMR (300 MHz,
CDCl3) 8.71 (d, J = 2.2 Hz, 1H, 4-H); 8.22 (dd, J = 9.2 Hz, J = 2.2 Hz,
1H, 6-H); 7.99 (bs, 1H, Ar); 7.91–7.85 (m, 3H, Ar); 7.65–7.62 (m, 1H,
Ar); 7.52–7.49 (m, 2H, Ar); 7.24 (d, J = 9.1 Hz, 1H, 7-H); 5.61 (s, 2H,
O-CH2); 4.19 (t, J = 6.9 Hz, 2H, N1-CH2); 1.94 (q, 2H, CH2); 0.92 (t,
J = 7.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ: 157.8 (C-3); 142.1
(C-7a); 141.1 (C-5); 122.8 (C-6); 119.2 (C-4); 112.3 (C-3a); 108.9 (C-7);
71.5 (O-CH2); 50.9 (N1-CH2); 23.4 (CH2); 11.7 (CH3); 134.1 (Ar); 133.6
(Ar); 128.7 (Ar); 128.4 (Ar); 128.1 (Ar); 127.6 (Ar); 126.7 (Ar); 126.6
(Ar); 126.2 (Ar). HPLC-MS (ES+): CH3CN/H2O 15:95, gt: 8.00 min, rt:
6.87, [M + H]+ = 362.4.
5. Biological methods
5.1. Radioligand binding assays for CB1 and CB2 receptors
CB1/CB2 receptor binding studies of indazole ether derivatives
1–22 (Table 1) were performed using membrane fractions of human
CB1/CB2 receptor transfected cells purchased from Perkin-Elmer Life
and Analytical Sciences (Boston, MA). HEK293EBNA membranes were
resuspended in Tris buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM
MgCl2, 0.5 mg/mL BSA fatty acid free, pH 7.4). Fractions of the final
membrane suspension (about 0.415 mg/mL of protein for CB1 and
about 0.18 mg/mL of protein for CB2) were incubated at 30 °C for
90 min with 0.54 nM [3H]-CP55940 (139.6 Ci/mmol) for CB1 and
0.33 nM [3H]-CP55940 (139.6 Ci/mmol) for CB2, in the presence or
absence of several concentrations of the competing drug, in a final
volume of 0.2 mL for CB1 and 0.6 mL for CB2 of assay buffer (50 mM
Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.5 mg/mL BSA fatty acid free,
pH 7.4). Nonspecific binding was determined in the presence of 10 μM
WIN 55,212–2. Silanized tubes were used throughout the experiment to
minimize receptor binding loss due to tube adsorption. The reaction
was terminated by rapid vacuum filtration with a filter mate Harvester
apparatus (Perkin-Elmer) through Filtermat A GF/C filters presoaked in
0.05% polyethylenimine (PEI).
4.1.7. 1-Methyl-3-(1-naphthylmethoxy)-5-nitroindazole (13)
To a solution of 25 (0.06 g, 0.19 mmol) in acetone (60 mL), was
added Cs2CO3 (0.06 g, 0.40 mmol). The suspension was stirred and
heated to reflux and 1-naphthylmethyl chloride (0.06 g, 0.32 mmol)
was added. The reaction was maintained at reflux until the complete
elimination of starting material. Then, it was cooled and filtered to
remove inorganic salts. The remaining solvent was evaporated under
reduced pressure and the product was purified by chromatography
column using as eluent methylene chloride: hexane (1:1, 5:1). Reaction
time: 24 h. Yield: (0.04 g, 67%). mp 152–155 °C. 1H NMR (300 MHz,
CDCl3) δ: 8.64 (d, J = 2.2 Hz, 1H, 4-H); 8.24 (dd, J = 9.1 Hz,
J = 2.1 Hz, 1H, 6-H); 8.15 (d, 1H, Ar); 7.98–7.87 (m, 2H, Ar); 7.70 (d,
1H, Ar); 7.60–7.48 (m, 3H, Ar); 7.24 (d, J = 9.2 Hz, 1H, 7-H); 5.89 (s,
2H, CH2); 3.99 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ: 157.5 (C-3);
143.0 (C-7a); 140.8 (C-5); 122.6 (C-6); 118.7 (C-4); 112.0 (C-3a); 108.5
(C-7); 69.5 (CH2); 35.6 (CH3); 133.8 (Ar); 131.7 (Ar); 131.6 (Ar); 129.4
(Ar); 128.7 (Ar); 127.8 (Ar); 126.6 (Ar); 126.0 (Ar); 125.3 (Ar). HPLC-
MS (ES+): CH3CN/H2O 10:90, gt: 8.00 min, rt: 6.12, [M + H]+
=
The filters were washed nine times with ice-cold buffer for CB1
(50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.5 mg/mL BSA fatty
acid free, pH 7.4) for CB2 (50 mM Tris-HCl, 2.5 mM EGTA, 5 mM
MgCl2, 1 mg/mL BSA fatty acid free, pH 7.5), and bound radioactivity
was measured with a 1450 LSC & Luminiscence counter Wallac
334.1.
4.1.8. 1-Benzyl-3-cyclohexylmethoxy-5-nitroindazole (18)
To a solution of 26 (0.10 g, 0.38 mmol) in 2-butanone (60 mL), was
7

P. González-Naranjo, et al.
Bioorganic&MedicinalChemistry28(2020)115672
MicroBeta TriLux (Perkin-Elmer). The binding assay showed the ap-
propriate sensitivity to CB1 and CB2 ligands. Thus, WIN 55,212–2 in-
hibited the binding with a Ki value of 36.2 nM (CB1R) and WIN
55,212–2 and HU308 inhibited the binding with Ki values of 3.7 and
11.2 nM (CB2R), respectively. For all binding experiments, competition
binding curves were analyzed by using an iterative curve-fitting pro-
cedure GraphPad,55 which provided IC50 values for test compounds. Ki
values were determined by the method of Cheng and Prusoff.56,57
5.1.4. Collagen type I gene expression
In 6-well plates, cells were placed in DMEM culture medium with
10% fetal bovine serum. After 24 h, unattached cells were removed, and
the attached cells were cultured in osteogenic medium containing
DMEM, 10% fetal bovine serum, 50 µg/mL L-ascorbic acid, 10-9
M
dexamethasone, and 10 mM b-glycerophosphate (Saint Louis, MO,
USA). The cells were treated with 18 (PGN36) 10-6 M, 17 (PGN38) 10-6
M, HU308 (10-6 to 10-8 M) and vehicle and maintained at 37 °C in a
fully humidified atmosphere at 5% CO2 in air during 72 h.
5.1.1. Functional activity for cannabinoid receptors on isolated tissue
The functional activity of the compounds 1–22 for CBRs was eval-
uated on the mouse vas deferens preparation. This is a nerve-smooth
muscle preparation that serves as a highly sensitive and quantitative
functional in vitro bioassay for cannabinoid receptor agonists. These
ligands induce concentration-related decreases in the amplitude of
electrically evoked contractions of the vas deferens by acting on natu-
rally expressed prejunctional neuronal cannabinoid receptors to inhibit
release of the contractile neurotransmitters, noradrenaline and ATP,
that is provoked by the electrical stimulation.38
In order to evaluate if 18 (PGN36) and 17 (PGN38) antagonize the
effect of the agonist HU308, cells were placed in 6-wells plates with
DMEM culture medium supplemented with 10% fetal bovine serum.
After 24 h, the attached cells were pre-treated with 18 (PGN36) 10-6 M,
17 (PGN38) 10-6 M and HU308 10-8 M and cultured in the same os-
teogenic medium. After 24 h, the medium of cells was replaced by the
osteogenic medium containing only the agonist HU308 (10-8 M) except
the control group that received vehicle. The cells were maintained at
37 °C in a fully humidified atmosphere at 5% CO2 in air during 48
additional hours.
For this study, male ICR mice weighing 25–30 g were used. Mouse
vas deferens were isolated as described by Hughes.58 Tissues were
suspended in a 10 mL organ bath containing 5 mL of Krebs solution
(NaCl 118; KCl 4.75; CaCl2 2.54; KH2PO4 1.19; MgSO4 1.2; NaHCO3 25;
glucose 11 mM) that was continuously gassed with carbogen (95% O2
and 5% CO2). Tissues were kept under 0.5 g of resting tension at 37 °C
and were electrically stimulated through two platinum ring electrodes.
They were subjected to alternate periods of stimulation (trains of five
rectangular pulses of 70 V, 15 Hz and 2 ms duration each were applied
every minute) and rest (10 min). The isometric force was monitored by
computer using a MacLab data recording and analysis system.
Total RNA from MC3T3-E1 cells was extracted using TRI reagent
(Sigma-Aldrich). cDNA was synthesized from 2 mg of total RNA using
the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems,
Foster City, CA). Quantitative real-time polymerase chain reaction
(qPCR) reactions were performed in triplicate using the Stratagene
Mx3005P QPCR System (Agilent Technologies, Santa Clara, CA),
FastStart Universal Probe Master (Roche Applied Science, Indianapolis,
IN), and predeveloped assays (Applied Biosystems) for collagen type I
and GAPDH. Relative quantification of target genes was performed by
comparing threshold cycles using the ΔΔCTmethod, as described pre-
viously.59
The effect of the synthetic cannabinoid agonist WIN 55212-2 and
that of the new compounds (10-7–1.82 × 10-5 M) was tested by con-
structing concentration-response curves in a step-by-step manner.
Curves were carried out by the following protocol: WIN 55212-2 or the
new compounds were added at a concentration to the organ bath
50 min after the beginning of electrical stimulation and their effect on
the electrically induced contractions was evaluated 10 min after their
addition. Then, the electrical stimulation was stopped, Krebs solution
was replaced and the following concentration of the compound was
added. This protocol was repeated for every concentration of the curve.
In order to check the antagonist profile of the new compounds, they
were added to the organ bath at a concentration of 10−6 M 10 min
before each addition of the increasing concentrations of WIN 55212-2
and their effect were compared with that of the cannabinoid antago-
nists AM251 and AM630. For some compounds, the antagonist effect
was evaluated in experiments where the concentration-response curve
of WIN 55212-2 ranged between 3 × 10-8 and 8.1 × 10-6 M.
Author contributions
The manuscript was realizad through contributions of all authors.
All authors have approved this version of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported from Ministerio de Ciencia e Innovación
under Grant RTI2018-096100B-100, Plan Nacional de I + D + I 2013-
2016, ISCIII–FEDER (FIS16/00637), Plan de Ciencia, Tecnología e
Innovación 2013-2017 and 2018-2022 del Principado de Asturias
(GRUPIN14-028, IDI-2018-000152), RedInRen from ISCIII (RD16/
0009/0017). Natalia Carrillo López was supported by GRUPIN14-028
and IDI-2018-000152. The authors would like to thank to Guadalupe
Pablo for their excellent technical assistant.
5.1.2. Functional activity of cannabinoids on bone
The mouse osteoblastic MC3T3-E1 cell line (ATCC® CRL2593™) was
cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza,
Vervier, Belgium) supplemented with 10% fetal bovine serum (Lonza),
100 µg/mL streptomycin (Lonza) and 100 U/mL penicillin (Lonza) and
at 37 °C in a humidified 5% CO2 atmosphere. Cells were grown to
confluence and then were placed in 96-wells and 6-wells plates with the
same culture medium.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115672.
5.1.3. Cell viability assay
To confirm whether the 18 (PGN36) and 17 (PGN38) compounds at
10-6 M concentrations were able to modify cell viability a MTT assay
was carried out. MTT reduction only occurs in metabolically active
cells. In 96-wells plates, cells were placed in a culture medium con-
taining DMEM with 10% fetal bovine serum during 24 h. After that, the
medium was replaced by DMEM culture medium supplemented with 18
(PGN36) 10-6 M, 17 (PGN38) 10-6 M or vehicle (control group) during
48 h.
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