Novel pyrrolocycloalkylpyrazole analogues as CB1 ligands

Article abstract of DOI:10.1111/cbdd.13069

Novel 1,4-dihydropyrazolo[3,4-a]pyrrolizine-, 4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-3-carboxamide-based compounds were designed and synthesized for cannabinoid CB₁ and CB₂ receptor interactions. Any of the new synthesized compounds showed high affinity for CB₂ receptor with K_i values superior to 314?nm, whereas some of them showed moderate affinity for CB₁ receptor with K_i values inferior to 400?nm. 7-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-carboxamide (2j) exhibited good affinity for CB₁ receptor (K_iCB₁?=?81?nm) and the highest CB₂/CB₁ selectively ratio (>12). Docking studies carried out on such compounds were performed using the hCB₁ X-ray in complex with the close pyrazole analogue AM6538 and disclosed specific pattern of interactions related to the tricyclic pyrrolopyrazole scaffolds as CB₁ ligands.

Full text of DOI:10.1111/cbdd.13069

Received: 17 March 2017
DOI: 10.1111/cbdd.13069
Revised: 16 May 2017
Accepted: 24 June 2017
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R E S E A R C H A R T I C L E
Novel pyrrolocycloalkylpyrazole analogues as CB1 ligands
Battistina Asproni1
Ilaria Manca2
Giansalvo Pinna1
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Elena Cichero3
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Paola Fossa3
Gabriele Murineddu1
Paolo Lazzari2
Giovanni Loriga4
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Gérard A. Pinna1
1Dipartimento di Chimica e
Farmacia, Università degli Studi di Sassari,
Sassari, Italy
Novel 1,4-dihydropyrazolo[3,4-a]pyrrolizine-, 4,5-dihydro-1H-pyrazolo[4,3-g]in-
dolizine-and1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-3-carboxamide-
based compounds were designed and synthesized for cannabinoid CB1 and CB2
receptor interactions. Any of the new synthesized compounds showed high affinity
for CB2 receptor with Ki values superior to 314 nm, whereas some of them showed
moderate affinity for CB1 receptor with Ki values inferior to 400 nm. 7-Chloro-1-(2,
4-dichlorophenyl)-N-(homopiperidin-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]
indolizine-3-carboxamide (2j) exhibited good affinity for CB1 receptor
(KiCB1 = 81 nm) and the highest CB2/CB1 selectively ratio (>12). Docking studies
carried out on such compounds were performed using the hCB1 X-ray in complex
with the close pyrazole analogue AM6538 and disclosed specific pattern of interac-
tions related to the tricyclic pyrrolopyrazole scaffolds as CB1 ligands.
2KemoTech Srl, Pula, CA, Italy
3Dipartimento di Farmacia, Università di
Genova, Genova, Italy
4Consiglio Nazionale delle Ricerche, Istituto
di Farmacologia Traslazionale, UOS
Cagliari, Pula, CA, Italy
Correspondence
Battistina Asproni, Dipartimento di
Chimica e Farmacia, Università degli Studi
di Sassari, Sassari, Italy.
Email: asproni@uniss.it
K E Y W O R D S
binding affinity, cannabinoid receptors, docking studies, pyrrolocycloalkylpyrazole
responsible of psychotropic effects.[10] CB1R antagonists are
potential drugs for the therapy of drug and alcohol addiction
1
INTRODUCTION
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The cannabinoid CB1 and CB2 receptors (CB1R and CB2R)
belong to the rhodopsin-like family of G protein-coupled re-
ceptors (GPCRs) and are key components of the endocanna-
binoid system.[1–3] The CB1R is abundantly expressed in the
central nervous system (CNS) but also is present in periph-
eral tissues, including lungs, liver, kidneys and adipocytes.[4]
CB2R is found most abundantly in the periphery, predom-
inantly expressed in cells of the human immune system as
spleen, tonsils and thymus,[4] and to a much lesser extent in
CNS.[5] CBRs are activated by terpenoid plant constituents, for
example, by Δ9-tetrahydrocannabinol, the major psychoactive
component of Cannabis sativa.[6] Recent studies have demon-
strated that CB2Rs are involved in numerous diseases.[4,7]
Several selective CB2R agonists exhibited analgesic activity
in preclinical models of acute, inflammatory and neuropathic
pain,[4,8,9] whereas CB1R activation mediates analgesia, stim-
ulation of appetite and euphoria, among other effects,[4] and is
as well as for the treatment of obesity. In this regard, rimon-
abant (Figure 1) was the first potent CB1R antagonist/inverse
agonist[11] approved by European Commission as an antiobe-
sity agent; however, it was soon withdrawn by EMEA for its
serious psychiatric disorders including anxiety, depression
and suicidal tendency.[12] Within the search for new and safe
antiobesity agents, recent medicinal chemistry approaches are
oriented towards the obtainment of new peripherally selective
CB1 antagonists, by designing ligands that do not cross the
blood–brain barrier and have low brain penetration.[13–15] The
relevance of CBRs as emerging target of pharmacotherapy
is documented also by the discovery of peripherally mixed
CB1R/CB2R agonists as antiglaucoma agents.[16]
During the past decade, numerous ligands endowed with
high affinity and subtype selectivity for both receptors were
synthesized, and within each chemo-type the structure–activ-
ity relationship (SAR) studies were explored.
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Chem Biol Drug Des. 2017;1–13.
wileyonlinelibrary.com/journal/cbdd
© 2017 John Wiley & Sons A/S
1

2
ASPRONI et Al.
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O
O
N
Q
NH
NH
( )
n
N
N
R
N
N
Cl
Cl
R1
1
Cl
Rimonabant
n = 1(A); n = 2(B); n = 3(C)
KiCB1 = 1.8 n
M
KiCB2 = 514 n
M
CB1selectivity = KiCB2/KiCB1 = 285.5
O
O
O
N
N
N
NH
NH
NH
R
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1C
KiCB1 = 4.2 n
KiCB2 = 55.7 n
1A
1B
KCB = 14.8 n
KiiCB12 = 227 n
CB1 selectivity KiCB2/KiCB1 = 15.3
M
M
1Aa: R = Cl KiCB1 = 2050 n
KiCB2 = 0.34 n
CB1 selectivity = KiCB2/KiCB1 = 0.000166
M
M
M
M
CB1 selectivity KiCB2/KiCB1 = 13.3
1Ab: R = CH3 KiCB1 = 363 n
M
KiCB2 = 0.037 n
M
CB1 selectivity KiCB2/KiCB1 = 0.000102
O
O
N
NH
NH
O
O
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
NESS06SM
SM-11
KiCB1 = 40.0 n
KiCB2 = > 3725 n
CB1 selectivity KiCB2/KiCB1 = > 93.1
M
KiCB1 = 10.2 n
KiCB2 = > 5000 n
CB1 selectivity KiCB2/KiCB1 = > 490.2
M
M
FIGURE 1 Chemical structures of
M
CBRs ligands
The 4-alkyl-5-arylpyrazole skeleton of rimon-
abant has been modified by us (Figure 1), giving rise to
1,4-dihydroindeno[1,2-c]pyrazole 1A,[17] 4,5-dihydro-1H-
benzo[g]indazole 1B[18] and 1,4,5,6-tetrahydrobenzo[6,7]cy-
clohepta[1,2-c]pyrazole-based ligands 1C.[19] Such tricyclic
systems feature a carbamoyl group at position 3 and simple
substituents (halogen, methyl, methoxy) on the aryl moieties.
These compounds displayed interesting cannabinoid binding
affinity and subtype selectivity. In particular, changes in the
size and shape of the tricyclic unit in ligands 1 revealed in-
triguing effects on the biological activity. Thus, increasing
the length of the carbon bridge between C4 of the pyrazole
and the 4-chlorophenyl group from one to three methylene
units led to a marked increase in the CB1 binding affinity and
selectivity. Moreover, the presence of a substituent, such as
F, Cl, Br or CH3, on the phenyl ring of the tricyclic system
generally gave an increase in the affinity and selectivity for
CB2R. Compounds 1Aa, 1Ab, 1B and 1C are representatives
of this class of CB ligands. The pharmacological relevance of
these compounds emerged from the ability of the CB2 agonist
1Ab in alleviating neuropathic pain through functional mi-
croglia changes in mice.[20] Furthermore, the analogues CB1
antagonist compounds NESS06SM and SM-11, featuring the
4,5-dihydrobenzo-oxa-cycloheptapyrazole skeleton could
represent useful candidate agents for the treatment of obesity
and its metabolic complications.[21]
Continuing with our interest in expanding SAR studies
on CBRs,[22] we have undertaken a study to prepare new
CB ligands related to both rimonabant and its derivatives
1. Thus, driven by our understanding of the biological and
pharmacological behaviour of the tricyclic pyrazoles, we
explored a series of new tricyclic scaffolds incorporating the
biologically interesting pyrrole and pyrazole moieties with a
central ring that can be modulate in size (Table 1; Figure 2),
namely 1,4-dihydropyrazolo[3,4-a]pyrrolizines (2a–h),
4,5-dihydro-1H-pyrazolo[4,3-g]indolizines (2i–l), 1,4,5,6-te
trahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepines (2m–p) that
varied the carbamoyl unit with the Cl or CH3 substituents
on the pyrrole moiety. Such bioisosteric benzene/pyrrole re-
placement might give access to pharmaceutically interesting

ASPRONI et Al.
3
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TABLE 1 Structures and binding data for compounds 2a–p
Receptor affinity
CB1 selectivity
Compound
R
n
Q
KiCB1 (nm)a
KiCB2 (nm)b
KiCB2/KiCB1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Cl
Cl
Cl
Cl
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1
1
1
1
1
1
1
1
2
2
2
2
3
3
3
3
a
b
c
d
a
b
c
d
a
b
c
d
a
b
c
902 75
230 36
715 80
355 39
>1 μm
>1 μm
>1.10
2.67
1.05
0.88
—
615 48
750 66
314 33
>1 μm
>1 μm
>1 μm
731 75
>1 μm
>1 μm
>1 μm
>1 μm
895 70
>1 μm
>1.11
142 30
209 25
81 12
154 22
>1 μm
190 30
244 35
544 48
257 30
5.15
>4.78
>12.35
>6.49
—
>5.26
>4.10
1.76
>1 μm
>1 μm
958 68
>1 μm
d
>3.89
aAffinity of compounds for the CB1 receptor was evaluated using mouse whole-brain membranes and [3H]CP 55940. Ki values were obtained from five independent
experiments carried out in triplicate.
bAffinity of compounds for the CB2 receptor was evaluated using CHO cell membranes transfected with hCB2 receptors and [3H]CP 55940. Ki values were obtained from
five independent experiments carried out in triplicate.
O
O
Q
NH
Q
NH
In this article, we report the synthesis of compounds
2a–p together with preliminary aspects of their affinity and
selectivity on CBRs and molecular modelling studies. These
data are accompanied by a thorough in silico evaluation of
the pharmacokinetic properties, with the aim at gaining pre-
liminary information concerning their potentially drug-like
profile.
R
Bioisosteric
replacement
( )
( )
n
n
N
N
N
R
N
N
Cl
R
1
1
Cl
2a-p
(see Tab. 1)
2
MATERIALS AND METHODS
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FIGURE 2 Design of CBR ligands by bioisosteric replacement
benzene/pyrrole
2.1
Chemistry
General
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2.1.1
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compounds with modified physical–chemical properties,
because the introduction of the pyrrole ring makes the tri-
cyclic system more electron-rich. Moreover, it increases the
polar surface area and decreases the lipophilicity.
Melting points were obtained on a Koffler melting point
apparatus and are uncorrected. IR spectra were recorded
as Nujol mulls on NaCl plates with a Jasco FT/IR 460 plus

4
ASPRONI et Al.
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spectrophotometer and are expressed in ν (cm−1). NMR ex-
periments were run on a Bruker AVANCE III Nanobody
acid (1 mmol) in CH2Cl2 (15 ml). After 1 hr, the suspension
became a solution and then the appropriate amine (2 mmol)
was added and stirring was continued at room temperature
overnight. The organic phase was washed with water, dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was purified by FC using the appropri-
ate eluents.
400 MHz spectrometer with H and 13C being observed at
1
400 and 100.6 MHz, respectively. Spectra were acquired
using CDCl3 as solvent. Chemical shifts for H and 13C
1
NMR spectra were reported in δ (ppm) downfield from te-
tramethylsilane, and coupling constants (J) were expressed
in Hertz. Multiplicities are recorded as s (singlet), d (dou-
blet), t (triplet), dd (doublet of doublets), m (multiplet).
Specific rotation was recorded with a PerkinElmer 241
apparatus, using the sodium D line (589 nm), and CHCl3
as solvent. Atmospheric pressure ionization electrospray
(API-ES) mass spectra were obtained on an Agilent 1100
series LC/MSD spectrometer. Elemental analyses were
performed with a PerkinElmer 2400 analyser, and results
were within 0.40% of the calculated values. TLC was per-
formed on Merck silica gel 60 TLC plates F254 and visual-
ized using UV. Flash chromatography (FC) was performed
using Merck silica gel 60 (230–400 mesh ASTM).
6-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-
1-yl)-1,4-dihydropyrazolo[3,4-a]pyrrolizine-3-
carboxamide (2b)
Compound 2b was obtained from 16 and 1-aminohomopi-
peridine. The crude residue was purified by FC (AcOEt/pe-
troleum ether 3/7) to afford 2b (0.29 g, 63%) as yellow solid;
Rf 0.25 (AcOEt/petroleum ether 2/8); m.p. 167–168°C. IR
1
1,645 (C=O). H NMR 7.62 (s, 1H), 7.53 (d, J = 8.6 Hz,
1H), 7.42 (d, J = 8.6 Hz, 1H), 7.40–7.30 (m, 1H), 6.09 (d,
J = 3.6 Hz, 1H), 5.92 (d, J = 3.6 Hz, 1H), 4.89 (s, 2H), 3.30–
3.00 (m, 4H), 1.90–1.50 (m, 8H). 13C NMR 160.1, 141.9,
135.7, 130.8, 129.3, 128.0, 125.3, 124.7, 116.2, 109.3, 100.7,
58.6, 44.0, 27.0, 26.1. API-ES m/z: [M + H]+ calcd for
C21H21Cl3N5O: 464.1, found: 464.1. Anal. (C21H20Cl3N5O)
C, H, N.
Chemical intermediates 3–18 and final compounds 2a, 2e,
2i and 2m (Table 1; Scheme 1) were synthesized according
to literature procedure.[23] 1-Aminohomopiperidine, cyclo-
hexylamine and (−)-cis-myrtanylamine were purchased from
Sigma-Aldrich®.
6-Chloro-1-(2,4-dichlorophenyl)-N-(cyclohexyl-1-yl)-
1,4-dihydropyrazolo[3,4-a]pyrrolizine-3-carboxamide
(2c)
2.1.2
General procedure for the
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amidation of 15–18
Compound 2c was obtained from 16 and cyclohexylamine.
The crude residue was purified by FC (AcOEt/petroleum
ether 15/85) to afford 2c (0.29 g, 65%) as light brown solid;
Rf 0.61 (AcOEt/petroleum ether 2/8); m.p. 194–196°C. IR
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (1.2
mmol) and 1-hydroxybenzotriazole (HOBt) (1.2 mmol) were
added in sequence to a suspension of the appropriate tricyclic
O
R
O
( )
n
O
O
N
R
( )
N
n
R
( )
ii
n
N
N
Cl
iii
i
N
O
Cl
O
O
7 R = CH ; n = 1
11 R = CH ; n = 1
3 R = CH ; n = 1
3
3
3
8 R = Cl; n = 1
9 R = Cl; n = 2
10 R = Cl; n = 3
12 R = Cl; n = 1
13 R = Cl; n = 2
14 R = Cl; n = 3
4 R = Cl; n = 1
5 R = Cl; n = 2
6 R = Cl; n = 3
O
N
O
Q
NH
R
OH
R
( )
n
( )
n
N
N
iv
N
N
N
R, Q, and n as
indicated in Tab. 1
Cl
SCHEME 1 Reagents and conditions:
(i) Na, EtOH, (COOEt)2, r.t., 1 hr; (ii)
2,4-Cl2C6H3NHNH2·HCl, EtOH, 80°C,
8 hr; (iii) THF, LiOH 0.8 m, 60°C, 3 hr; (iv)
EDC, HOBt, CH2Cl2, r.t., 1 hr, then NH2-Q,
overnight
Cl
15 R = CH ; n = 1
3
Cl
Cl
2a-p
16 R = Cl; n = 1
17 R = Cl; n = 2
18 R = Cl; n = 3

ASPRONI et Al.
5
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1,645 (C=O). 1H NMR 7.62 (s, 1H), 7.53 (d, J = 8.0 Hz, 1H),
7.42 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 7.6 Hz, 1H), 6.08 (d,
J = 2.8 Hz, 1H), 5.92 (d, J = 2.8 Hz, 1H), 4.91 (s, 2H), 4.08–
3.78 (m, 1H), 2.15–1.88 (m, 2H), 1.88–1.71 (m, 2H), 1.71–
1.50 (m, 1H), 1.50–1.34 (m, 2H), 1.36–1.10 (m, 3H). 13C
NMR 160.2, 141.9, 135.9, 130.8, 129.1, 128.3, 125.3, 124.7,
116.1, 109.4, 100.7, 48.3, 45.6, 33.3, 25.7, 25.1. API-ES
m/z: [M + H]+ calcd for C21H20Cl3N4O: 449.1, found: 449.2.
Anal. (C21H19Cl3N4O) C, H, N.
J = 8.6 Hz, 1H), 7.41 (dd, J = 8.6, 1.6 Hz, 1H), 6.80 (d,
J = 8.0 Hz, 1H), 5.94 (d, J = 3.0 Hz, 1H), 5.88 (d, J = 3.0 Hz,
1H), 4.83 (s, 2H), 4.04–3.84 (m, 1H), 2.27 (s, 3H), 2.07–1.97
(m, 2H), 1.80–1.72 (m, 2H), 1.68–1.58 (m, 1H), 1.50–1.35
(m, 2H), 1.35–1.03 (m, 3H). 13C NMR 160.5, 145.5, 142.0,
135.9, 135.7, 130.8, 130.7, 129.4, 129.0, 128.2, 125.1, 125.0,
109.6, 99.9, 48.3, 44.6, 33.3, 25.7, 25.1, 12.0. API-ES m/z:
[M + H]+ calcd for C22H23Cl2N4O: 429.1, found: 429.0.
Anal. (C22H22Cl2N4O) C, H, N.
6-Chloro-1-(2,4-dichlorophenyl)-N-(cis-myrtanyl-
1-yl)-1,4-dihydropyrazolo[3,4-a]pyrrolizine-3-
carboxamide (2d)
6-Methyl-1-(2,4-dichlorophenyl)-N-(cis-myrtanyl-
1-yl)-1,4-dihydropyrazolo[3,4-a]pyrrolizine-3-
carboxamide (2h)
Compound 2d was obtained from 16 and (−)-cis-
myrtanylamine. The crude residue was purified by FC
(AcOEt/petroleum ether 15/85) to afford 2d (0.28 g, 55%) as
light brown solid; m.p. 250–253°C; Rf 0.55 (AcOEt/petro-
leum ether 2/8). IR 1,645 (C=O). 1H NMR 7.72 (s, 1H), 7.53
(d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.00–6.85 (m,
1H), 6.08 (d, J = 4.0 Hz, 1H), 5.92 (d, J = 4.0 Hz, 1H), 4.91
(s, 2H), 3.46–3.29 (m, 2H), 2.42–2.11 (m, 2H), 2.00–1.40
(m, 6H), 1.20 (s, 3H), 1.08 (s, 3H), 0.90 (d, J = 9.6 Hz, 1H).
13C NMR 160.3, 142.5, 141.6, 135.7, 130.8, 130.2, 129.1,
128.7, 128.3, 125.0, 124.7, 116.1, 109.3, 100.9, 44.9, 44.8,
44.1, 41.7, 41.5, 38.9, 33.6, 28.0, 26.1, 23.4, 20.3. [α]D
−2.41 (c 0.1678, CHCl3). API-ES m/z: [M + H]+ calcd for
C25H26Cl3N4O: 503.1, found: 503.2. Anal. (C25H25Cl3N4O)
C, H, N.
Compound 2h was obtained from 15 and (−)-cis-
myrtanylamine. The crude residue was purified by FC
(AcOEt/petroleum ether 15/85) to afford 2h (0.17 g, 35%)
as white solid; Rf 0.62 (AcOEt/petroleum ether 2/8); m.p.
1
178–179°C; IR 1,651 (CO). H NMR 7.62 (d, J = 2.0 Hz,
1H), 7.53 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 8.6, 2.0 Hz, 1H),
6.98 (t, J = 6.0 Hz, 1H), 5.94 (d, J = 3.6 Hz, 1H), 5.88 (d,
J = 3.6 Hz, 1H), 4.84 (s, 2H), 3.56–3.33 (m, 2H), 2.40–2.29
(m, 2H), 2.27 (s, 3H), 2.03–1.96 (m, 2H), 1.96–1.81 (m,
3H), 1.64–1.51 (m, 1H), 1.21 (s, 3H), 1.09 (s, 3H), 0.91 (d,
J = 9.6 Hz, 1H). 13C NMR 161.5, 145.6, 141.8, 135.9, 135.7,
130.9, 130.8, 129.4, 128.9, 128.2, 125.2, 124.9, 109.6, 99.9,
44.9, 44.7, 43.9, 41.7, 41.5, 38.9, 33.4, 28.1, 26.2, 23.4,
20.0, 12.0. [α]D −3.08 (c 0.2048, CHCl3). API-ES m/z:
[M + H]+ calcd for C26H29Cl2N4O: 483.2, found: 483.3.
Anal. (C26H28Cl2N4O) C, H, N.
6-Methyl-1-(2,4-dichlorophenyl)-N-(homopiperidin-
1-yl)-1,4-dihydropyrazolo[3,4-a]pyrrolizine-3-
carboxamide (2f)
7-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-
1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-
carboxamide (2j)
Compound 2f was obtained from 15 and 1-aminohomopi-
peridine. The crude residue was purified by FC (AcOEt/pe-
troleum ether 3/7) to afford 2f (0.20 g, 45%) as white solid;
Rf 0.25 (AcOEt/petroleum ether 2/8); m.p. 154–155°C.
Compound 2j was obtained from 17 and 1-aminohomopi-
peridine. The crude residue was purified by FC (AcOEt/pe-
troleum ether 3/7) to afford 2j (0.17 g, 36%) as white solid;
Rf 0.28 (AcOEt/petroleum ether 2/8); m.p. 150–152°C. IR
1,645 (C=O). 1H NMR 8.01 (s, 1H), 7.63 (s, 1H), 7.46–7.38
(m, 2H), 5.93 (d, J = 3.8 Hz, 1H), 5.41 (d, J = 3.8 Hz, 1H),
4.10 (t, J = 6.8 Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H), 3.14 (t,
J = 5.6 Hz, 4H), 1.80–1.45 (m, 8H). 13C NMR 161.5, 143.6,
137.1, 136.8, 136.2, 133.9, 130.7, 130.6, 128.3, 120.7, 119.1,
112.7, 107.2, 104.4, 58.5, 42.3, 26.9, 26.0, 20.7. API-ES
m/z: [M + H]+ calcd for C22H23Cl3N5O: 478.1, found: 478.0.
Anal. (C22H22Cl3N5O) C, H, N.
1
IR 1,648 (C=O). H NMR 8.26 (s, 1H), 7.61 (s, 1H), 7.52
(d, J = 8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 5.98–5.91
(m, 1H), 5.92–5.81 (m, 1H), 4.82 (s, 2H), 3.35–3.03 (m,
4H), 2.27 (s, 3H), 1.87–1.77 (m, 4H), 1.73–1.61 (m, 4H).
13C NMR 158.9, 145.4, 135.7, 135.6, 130.6, 129.3, 128.8,
128.1, 125.1, 124.9, 124.8, 109.6, 99.9, 58.5, 44.4, 26.9,
26.0, 11.9. API-ES m/z: [M + H]+ calcd for C22H24Cl2N5O:
444.1, found: 444.2. Anal. (C22H23Cl2N5O) C, H, N.
6-Methyl-1-(2,4-dichlorophenyl)-N-(cyclohexyl-1-yl)-
1,4-dihydropyrazolo[3,4-a]pyrrolizine-3-carboxamide
(2g)
7-Chloro-1-(2,4-dichlorophenyl)-N-(cyclohexyl-1-
yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-
carboxamide (2k)
Compound 2g was obtained from 15 and cyclohexylamine.
The crude residue was purified by FC (AcOEt/petroleum
ether 15/85) to afford 2g (0.21 g, 49%) as white solid; Rf
0.60 (AcOEt/petroleum ether 2/8); m.p. 130–133°C. IR
Compound 2k was obtained from 17 and cyclohexylamine.
The crude residue was purified by FC (AcOEt/petroleum
ether 15/85) to afford 2k (0.18 g, 40%) as light brown solid Rf
0.55 (AcOEt/petroleum ether 2/8); m.p. 190–192°C. IR 1,650
1
1,653 (C=O). H NMR 7.61 (d, J = 1.6 Hz, 1H), 7.53 (d,

6
ASPRONI et Al.
|
1
(C=O). H NMR 7.62 (s, 1H), 7.44 (s, 2H), 6.85–6.58 (m,
1H), 5.94 (d, J = 4.0 Hz, 1H), 5.40 (d, J = 4.0 Hz, 1H), 4.10
(d, J = 6.8 Hz, 2H), 4.01–3.84 (m, 1H), 3.40 (d, J = 6.8 Hz,
2H), 2.01 (d, J = 12.0 Hz, 2H), 1.82–1.66 (m, 2H), 1.68–1.60
(m, 1H), 1.46–1.12 (m, 5H). 13C NMR 161.5, 143.6, 137.0,
136.9, 136.0, 133.8, 130.7, 130.5, 128.3, 120.7, 119.0, 112.7,
107.2, 104.4, 48.2, 42.3, 33.3, 25.7, 25.1, 20.9. API-ES m/z:
[M + H]+ calcd for C22H22Cl3N4O: 463.1, found: 463.2. Anal
(C22H21Cl3N4O) C, H, N.
(C=O). 1HNMR7.53(s, 1H), 7.41(s, 2H), 6.83(d, J = 7.6 Hz,
1H), 5.89 (d, J = 2.4 Hz, 1H), 5.33 (d, J = 2.4 Hz, 1H),
4.20–4.05 (m, 2H), 4.00–3.83 (m, 1H), 3.42–3.24 (m, 2H),
2.24–2.10 (m, 2H), 2.10–1.88 (m, 2H), 1.87–1.50 (m, 4H),
1.44–1.36 (m, 4H). 13C NMR: 163.2, 146.5, 136.2, 135.9,
133.7, 133.6, 130.7, 130.4, 128.2, 121.8, 117.9, 116.4, 108.4,
106.9, 48.3, 44.7, 44.6, 33.3, 26.9, 25.7, 24.0, 25.1. API-ES
m/z: [M + H]+ calcd for C23H24Cl3N4O: 477.1, found: 477.0.
Anal. (C23H23Cl3N4O): C, H, N.
7-Chloro-1-(2,4-dichlorophenyl)-N-(cis-myrtanyl-
1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-
carboxamide (2l)
8-Chloro-1-(2,4-dichlorophenyl)-N-(cis-myrtanyl-1-
yl)-1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]
azepine-3-carboxamide (2p)
Compound 2l was obtained from 17 and (−)-cis-myrta-
nylamine. The crude residue was purified by FC (AcOEt/
petroleum ether 15/85) to afford 2l (0.31 g, 61%) as light
brown solid; Rf 0.60 (AcOEt/petroleum ether 2/8); m.p.
192–193°C. IR 1,645 (C=O). 1H NMR 7.63 (s, 1H), 7.45 (s,
2H), 7.00–6.80 (m, 1H), 5.95 (d, J = 3.0 Hz, 1H), 5.41 (d,
J = 3.0 Hz, 1H), 4.10 (t, J = 7.0 Hz, 2H), 3.40 (t, J = 7.0,
2H), 3.50–3.31 (m, 2H), 2.41–2.23 (m, 2H), 2.01–1.84 (m,
5H), 1.73–1.52 (m, 1H), 1.20 (m, 3H), 1.07 (s, 3H), 1.00–
0.80 (m, 1H). 13C NMR 161.5, 143.6, 137.0, 136.9, 136.0,
133.8, 130.7, 130.5, 128.3, 120.7, 119.0, 112.7, 107.2, 104.4,
44.9, 44.5, 43.9, 41.5, 41.1, 38.6, 33.1, 28.7, 26.2, 23.4, 20.1.
[α]D −1.86 (c 0.20, CHCl3). API-ES m/z: [M + H]+ calcd for
C26H28Cl3N4O: 517.1, found: 517.2. Anal. (C26H27Cl3N4O):
C, H, N.
Compound 2p was obtained from 18 and (−)-cis-myrta-
nylamine. The crude residue was purified by FC (AcOEt/
petroleum ether 15/85) to afford 2p (0.16 g, 30%) as light
brown solid; Rf 0.68 (AcOEt/petroleum ether 2/8); m.p. 154–
155°C. IR 1,645 (C=O). 1H NMR 7.53 (s, 1H), 7.42–7.30 (m,
2H), 7.10–6.90 (m, 1H), 5.89 (s, 1H), 5.34 (s, 1H), 4.23–4.05
(m, 2H), 3.44–3.23 (m, 2H), 3.10–3.02 (m, 2H), 2.41–2.10
(m, 4H), 2.00–1.88 (m, 2H), 1.88–1.76 (m, 3H), 1.60–1.48
(m, 1H), 1.19 (s, 3H), 1.07 (s, 3H), 0.80–1.00 (m, 1H). 13C
NMR 163.1, 146.5, 136.4, 136.1, 133.8, 133.6, 130.7, 130.5,
128.5, 121.8, 118.0, 116.4, 108.3, 107.1, 44.7, 44.2, 44.1,
42.0, 41.4, 38.8, 33.4, 28.2, 26.1, 26.0, 24.0, 23.5, 20.3. [α]D
−2.10 (c 0.2017, CHCl3). API-ES m/z: [M + H]+ calcd for
C27H30Cl3N4O: 531.1, found: 531.2. Anal. (C27H29Cl3N4O)
C, H, N.
8-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-
1-yl)-1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]
azepine-3-carboxamide (2n)
2.2
Biological assays
|
2.2.1
Radioligand-binding assays
|
Compound 2n was obtained from 18 and homopiperidine.
The crude residue was purified by FC (AcOEt/petroleum
ether 3/7) to afford 2n (0.20 g, 40%) as pink solid; Rf 0.21
(AcOEt/petroleum ether 2/8); m.p. 160–162°C. IR 1,645
General procedures binding experiments
Affinities at CB1R and CB2R for 2a–p were assessed by
competition for [3H]CP-55940 binding in mouse whole-brain
membranes and Chinese hamster ovary (CHO) cell mem-
branes transfected with hCB2, respectively.[24]
1
(C=O). H NMR 8.04 (s, 1H), 7.54 (s, 1H), 7.46–7.35
(m, 2H), 5.89 (d, J = 3.8 Hz, 1H,), 5.33 (d, J = 3.8 Hz,
1H,), 4.20–4.05 (m, 2H), 3.40–3.20 (m, 3H), 3.19–3.05
(m, 3H), 2.31–2.22 (m, 2H), 1.82–1.54 (m, 8H). 13C NMR
(101 MHz, CDCl3) 163.2, 146.5, 136.2, 135.9, 133.9,
133.6, 130.7, 130.4, 128.2, 121.8, 117.9, 116.4, 108.4,
106.9, 58.3, 44.7, 26.9, 26.2, 23.9. API-ES m/z: [M + H]+
calcd for C23H25Cl3N5O: 492.1, found: 492.2. Anal.
(C23H24Cl3N5O) C, H, N.
Mouse brain membranes[25]
Whole-mouse brains from four adult male MF1 mice were
suspended in centrifugation buffer (320 mm sucrose, 2 mm
Tris–HCl, 2 mm Tris base, 2 mm EDTA, 5 mm MgCl2 at pH
7.4) and the tissue homogenized with an Ultra-Turrex ho-
mogenizer. Tissue homogenates were centrifuged at 1,600 g
for 10 min and the resulting supernatant collected. The pellet
was resuspended in centrifugation buffer, centrifuged as be-
fore and the supernatant collected. Supernatants were com-
bined before undergoing further centrifugation at 28,000 g
for 20 min. The supernatant was discarded and the pellet re-
suspended in 20 ml of buffer A (50 mm Tris–HCl, 50 mm Tris
base, 2 mm EDTA, 5 mm MgCl2 at pH 7.0) and incubated at
37°C for 10 min. Following the incubation, the suspension
8-Chloro-1-(2,4-dichlorophenyl)-N-(cyclohexyl-1-
yl)-1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]
azepine-3-carboxamide (2o)
Compound 2o was obtained from 18 and cyclohexylamine.
The crude residue was purified by FC (AcOEt/petroleum
ether 15/85) to afford 2o (0.20 g, 42%) as pink solid; Rf 0.21
(AcOEt/petroleum ether 1/1); m.p. 150–152°C. IR 1,645

ASPRONI et Al.
7
|
was centrifuged for 20 min at 23,000 g. After resuspending
the pellet in another 20 ml of buffer A, the suspension was
incubated for 40 min at room temperature before a final cen-
trifugation for 15 min at 11,000 g. The final pellet was re-
suspended in 2 ml of buffer B (50 mm Tris–HCl, 50 mm Tris
base, 1 mm EDTA, 3 mm MgCl2 at pH 7.4) to give a protein
concentration of 1 mg/ml and stored at 80°C. All centrifuga-
tion procedures were carried out at 4°C.
(Bmax) and 2.31 nm (Kd) in mouse brain membranes and
72,570 fmol/mg protein (Bmax) and 4.3 nm (Kd) in hCB2-
transfected cells.
2.3
Molecular modelling
|
2.3.1
Ligand preparation and
|
docking studies
CHO cell membranes[24]
All the compounds were built, parameterized (Gasteiger–
Huckel method) and energy minimized within moe using
MMFF94 forcefield.1
CHO cells stably transfected with cDNA encoding human
cannabinoid CB2R (Bmax = 72.6 pmol/mg protein) were
maintained at 37°C and 5% CO2 in Dulbecco’s modified
Eagle’s medium (DMEM) nutrient mixture F-12 HAM
supplemented with 10% foetal bovine serum (FBS), 3 ml
penicillin–streptomycin and 4 ml G-418. These CHO hCB2
cells were passed twice a week using a nonenzymatic cell
dissociation solution (5 ml EDTA). The hCB2-transfected
cells were removed from flasks by scraping and then fro-
zen as a pellet at −20°C until required. Before use in a
radioligand-binding assay, cells were defrosted, diluted in
50 mm Tris-binding buffer (see radioligand displacement
assay) and homogenized with a 1 ml hand-held homog-
enizer. Protein assays were performed using a Bio-Rad
Dc kit.
Docking calculations within the X-ray structure of the
CB1R (pdb code = 5TGZ; resolution = 2.8 Å)[26] were
performed using the leadit 2.1.8 software suite.2 This tool
includes the FlexX scoring algorithm, which is based on
calculation of the binding free energy by means of Gibbs–
Helmholtz equation.[27] The software detects the binding
site defining a radius of 10 Å far from the co-crystallized
ligand, to set up a spherical search space for the docking
approach.
The standard setting as docking strategy was followed,
choosing the so-called Hybrid Approach (enthalpy and en-
tropy criteria), the related scoring function evaluation is de-
scribed in the literature.[28] The derived docking poses were
prioritized taking into account the score values of the lowest
energy pose of the compounds docked to the protein struc-
ture. All ligands were further refined and rescored by assess-
ment with the algorithm HYDE, included in the leadit 2.1.8
software. The HYDE module considers dehydration enthalpy
and hydrogen bonding.[29]
Radioligand competition assay[24]
Binding experiments were carried out with [3H]CP-55940,
Tris-binding buffer (50 mm Tris–HCl, 50 mm Tris base;
0.1% BSA, pH 7.4), total assay volume 500 ml, using the
filtration procedure described previously by Ross et al.[19]
Binding was initiated by the addition of mouse brain
(CB1 assay) or CHO cells (CB2 assay) membranes (50 μg
protein per tube). All assays were performed at 37°C for
60 min before termination by addition of ice-cold Tris-
binding buffer and vacuum filtration using a 12-well sam-
pling manifold (Cell Harvester; Brandel) and Whatman
GF/B glass-fibre filters that had been soaked in wash
buffer at 4°C for 24 hr. Each reaction tube was washed five
times with the wash buffer. The filters were oven-dried
for 60 min and then placed in 5 ml of scintillation fluid.
Radioactivity was quantified by liquid scintillation spec-
trometry. Specific binding was defined as the difference
between the binding that occurred in the presence and ab-
sence of 1 μm unlabelled CP-55940. The concentration of
[3H]CP-55940 used in our displacement assays was 0.7 nm.
Each unlabelled cannabinoid tested was stored as a stock
solution of 10 mm in DMSO, the vehicle concentration in
all assay tubes being 0.1% DMSO. Protein assays were
performed using a Bio-Rad Dc kit. The binding param-
eters for [3H]CP-55940, determined by fitting data from
saturation-binding experiments to a one-site saturation
plot using GraphPad Prism, were 2,336 fmol/mg protein
Then, the stability of the selected protein–ligand com-
plexes was assessed using a short ~1 ps run of molecular
dynamics (MD) at constant temperature, followed by an all-
atom energy minimization (LowModeMD implemented in
moe software). This kind of module allowed to perform an
exhaustive conformational analysis of the ligand–receptor
binding site subset, as we previously reported about other
case studies.[22e,30]
2.3.2
In silico evaluation of
|
pharmacokinetic properties
The prediction of ADMET properties was performed using
the Advanced Chemistry Development (ACD) Percepta plat-
form (www.acdlabs.com).
Any ADMET descriptor was evaluated by Percepta based
on training libraries implemented in the software, which in-
cludes a consistent pool of molecules whose pharmacokinetic
and toxicity profiles are experimentally known.
TPSA descriptor, representing the polar surface area (Å2)
of the molecules, was evaluated by means of moe software.
TPSA is calculated using group contributions to approximate

8
ASPRONI et Al.
|
the polar surface area from connection table information
only. The parameterization is that of Ertl et al.[31]
with respect to compound 2b. Changing the chlorine atom
on the pyrrole ring of the 1,4-dihydropyrazolopyrrolizine
scaffold with a methyl group, compounds 2e–h, showed a
quite negative impact on CBRs affinity, with the excep-
tion of compound 2h. The myrtanyl derivative 2h had the
highest CBRs affinity among the four methyl substituted
analogues with a CB1R affinity that is 1,6-fold higher to
that of 2b. CB2R affinity of 2h mirrored the ground term
2b. Therefore, within the subseries 2e–h, the introduction
of bulky hydrophobic myrtanyl unit proved to be suitable
for CB2R interaction, resulting in a slight improvement
of CB2/CB1 selectivity ratio with respect to 2b. However,
compound 2h and all pyrrolizine analogues presented in
this study did not reach the interesting binding profile ex-
hibited by reference compounds 1Aa and 1Ab (Figure 1),
indicating that the bioisosteric replacement benzene/pyr-
role has a negative impact on CBR affinity.
3
RESULTS AND DISCUSSION
Chemistry
|
3.1
|
The strategy followed for pyrrolocycloalkylpyrazole-based
compounds 2 is outlined in Scheme 1 and started with sub-
stituted pyrrolocycloalkanones 3–6.[23] Ketones were first
transformed to 7–10 using a Claisen reaction and next con-
densed with 2,4-dichlorophenylhydrazine to give the tricy-
clic pyrrolocycloalkylpyrazoles 11–14. Final elaboration to
designed compounds 2a–p was accomplished by functional
interconversion of esters 11–14 via acid 15–18 to the amides/
hydrazides.
The 4,5-dihydro-1H-pyrazolo[4,3-g]indolizines 2i–l had
higher CB1R affinity with the only exception of 2l as com-
pared to 2b. By contrast, ligands 2i–l showed no binding
preference for CB2R. Among these derivatives, the homo-
piperidine substituent analogue, 2j, had a good affinity for
CB1R (KiCB1 = 81 nm) together with the highest CB2/CB1
selectively ratio (>12).
The 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]aze-
pines 2m–p displayed a similar pattern of binding to CBR
affinities and no significant CB2R affinity. Among these de-
rivatives, the piperidine substituted analogue 2m exhibited
moderate affinity for CB1R with a Ki value similar to that of
2b.
3.2
Cannabinoid receptor binding studies
|
Affinities at CB1R and CB2R for compounds 2 were assessed
by competition for [3H]CP-55940 binding in mouse whole-
brain membranes and Chinese hamster ovary (CHO) cell
membranes transfected with hCB2, respectively. The experi-
mental data (IC50 values) were converted into Ki values[32]
and are shown in Table 1.
To investigate how bioisosteric replacement benzene/
pyrrole can modify binding profile of the tricyclic scaffold,
three series of tricyclic pyrrolopyrazoles 2a–p structurally
correlated to compounds 1Aa, 1Ab, 1B and 1C, were synthe-
sized. Any of the new synthesized compounds showed high
affinity for CB2R with Ki values superior to 314 nm, whereas
some of them showed moderate affinity for CB1R with Ki
values inferior to 400 nm.
The 1,4-dihydropyrazolo[3,4-a]pyrrolizine compounds
2a–d with a chlorine atom at C5 showed moderate bind-
ing affinity to CB1R and values that were from 314 nm to
>1 μm range for CB2R subtype. The highest affinity for
both CBRs was shown by the ligand bearing the homo-
piperazine carbamoyl unit. Compound 2b, the best of this
group with a CB1R affinity of 230 nm and CB2R affinity
of 615 nm, was used as reference term for all of the others
to present SAR studies. Compound 2a, having a pipera-
zine ring replacement of the homopiperazine ring of the
carbamoyl unit, showed much lower affinity for CB1R
as compared to 2b and no significant affinity for CB2R
(Ki > 1 μm). Compound 2c with a cyclohexyl moiety in the
carbamoyl unit exhibited little change in binding affinity
between CBRs, however, resulting in a threefold to 1.2-fold
reduction. Compound 2d, in which the size and shape of
amine unit were markedly changed by introduction of the
myrtanyl moiety, showed a slight increase of CB2R affinity
(Ki 314 nm) and decrease in CB1R affinity (Ki 355 nm) with
K1CB2/KiCB1 values lying in the same order of magnitude
According to these SAR studies based on three main se-
ries of compounds characterized by five, six and seven atoms
in the central ring of the condensed tricyclic pyrrolopyrazole
system, the planar dihydropyrazolo[3,4-a]pyrrolizine archi-
tecture appeared an important feature to assure moderate
CB1/CB2R affinity. By contrast, the enlargement of the size
of the central ring in the condensed tricyclic scaffold engen-
ders conformational changes that promote the affinity for
CB1R and improve the CB2/CB1 selectivity ratio (vide infra,
molecular modelling section).
3.3
Molecular modelling
|
Nowadays, the rational design and discovery of CB1 li-
gands were efficiently driven by deepening computational
studies, including homology modelling of the biological
target,[33] as well as ligand-based analyses.[34] The recent
X-ray crystallographic structures of the cannabinoid CB1R
became available (pdb code = 5U09; resolution = 2.6 Å),[35]
(pdb code = 5TGZ; resolution = 2.8 Å),[26] providing use-
ful guidelines for the rational design of novel derivatives.
These experimental data disclosed the binding mode of the
co-crystallized propanamide-based (pdb code = 5U09) and

ASPRONI et Al.
9
|
pyrazole-containing (pdb code = 5TGZ) derivatives, shed-
ding light for the design of further series of analogues and/
or isosteres.
In this work, we performed docking studies of the newly
synthesized compounds, choosing as reference compound
the pyrazole ligand (AM6538) co-crystallized in hCB1 (pdb
code = 5TGZ), because of the structural similarity to the de-
rivatives here investigated (Figure 3; Table S1).
bearing the 1,4-dihydropyrazolo[3,4-a]pyrrolizines (2a–h),
4,5-dihydro-1H-pyrazolo[4,3-g]indolizines (2i–l) and the 1,
4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepines (2m–
p) containing derivatives within the human CB1R. In par-
ticular, the SAR for the three series of compounds has been
clarified, when the tricyclic core is coupled with hydrazide or
carboxamide moieties as Q group.
Thus, all of them displayed a common positioning, mov-
ing the tricyclic core and the phenyl ring towards the two phe-
nyl rings linked to the position 5 and to the position 1 of the
pyrazole scaffold in AM6538, in order to gain the same hy-
drophobic contacts. On the other hand, the hydrazide or the
carboxamide moieties exhibited a comparable docking mode,
sometimes detecting H-bonds with S383. In particular, the pres-
ence of one acid hydrogen atom onto the nitrogen one of the
carboxamide (or hydrazide) moiety resulted to be a key-feature
for H-bonding S383 side chain, especially in presence of a
bulky hydrophobic group in Q. Among the three series of com-
pounds, the 4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and
the 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-
containing derivatives were well suited and preferred to the
1,4-dihydropyrazolo[3,4-a]pyrrolizines. Indeed, compounds
2i–2p moved the tricyclic core much more in proximity of the
aforementioned phenyl ring of compound AM6538 (Figure 5,
compound 2j is shown, and Figure 6, compound 2n is shown).
Conversely, the 1,4-dihydropyrazolo[3,4-a]pyrrolizine
experienced a much more rigid conformation, leading the
compound to quite differently accommodate within the
human CB1 binding site (Figure 7, compound 2b is shown).
Notably, the improved CB1-targeting ability occur-
ring when the pyrrolizine scaffold (2a–h; hCB1 = 142 nm
to >1,000 μm) was modified to the indolizine core (2i–l;
hCB1 = 81 nm to >1,000 μm) and to the pyrroloazepine one
(2m–p;hCB1 = 190–544 nm)resemblestheCB1 affinitytrend
drawn by the bioisosteres 1,4-dihydroindeno[1,2-c]pyrazole
As shown in the following Figures 4–7, AM6538 exhib-
ited one H-bond between the hydrogen atom of the carbox-
amide group and the side chain of S383, moving the piperidyl
substituent towards I119, F174, A380, M384. The two aro-
matic centres occupied a deep hydrophobic pocket including
I169, L193, V196, F268, W279, L359, W356, M363, L387.
In this way, a number of Van der Waals contacts and π–π
stacking were detected. Similarly, the well-known CB1 an-
tagonist rimonabant was able to display a highly comparable
docking positioning if compared to the co-crystallized com-
pound AM6538 (Figure 4).
Docking calculations on the newly synthesized
molecules (2a–2p) allowed us to explore the puta-
tive binding mode exhibited by the cannabinoid ligands
O
N
NH
N
N
Cl
O
N
Cl
AM6538
O
O
FIGURE 3 Chemical structure of pyrazole ligand AM6538
FIGURE 4 Docking mode of
rimonabant (C atom; pink) within the X-
ray crystallographic co-ordinates of hCB1
receptor in complex with AM6538 (C
atom; green). The most important residues
are labelled. H-bonds detected by the two
CB1 antagonists are shown by dot lines (in
yellow)

10
ASPRONI et Al.
|
FIGURE 5 Docking mode of 2j
(C atom; magenta) within the X-ray
crystallographic co-ordinates of hCB1
receptor in complex with AM6538 (C
atom; green). The most important residues
are labelled. H-bonds detected by the
4,5-dihydro-1H-pyrazolo[4,3-g]indolizine
compound are shown by dot lines (in
yellow)
FIGURE 6 Docking mode of
2n (C atom; yellow) within the X-ray
crystallographic co-ordinates of hCB1
receptor in complex with AM6538
(C atom; green). The most important
residues are labelled. H-bonds detected
by the 1,4,5,6-tetrahydropyrazolo[3,4-c]
pyrrolo[1,2-a]azepine compound are shown
by dot lines (in yellow)
FIGURE 7 Docking mode of 2b
(C atom; magenta) within the X-ray
crystallographic co-ordinates of hCB1
receptor in complex with AM6538 (C
atom; green). The most important residues
are labelled. H-bonds detected by the
1,4-dihydropyrazolo[3,4-a]pyrrolizine
compound are shown by dot lines (in
yellow)

ASPRONI et Al.
11
|
1Aa (hCB1 = 2050 nm), 4,5-dihydro-1H-benzo[g]indazole
1B (hCB1 = 14.8 nm) and 1,4,5,6-tetrahydrobenzo[6,7]cy-
clohepta[1,2-c]pyrazole ligands 1C (hCB1 = 4.2 nm).
Nevertheless, based on a perspective of the docking results
for all the three series of derivatives, adequate CB1 antago-
nism could be achieved with the introduction of proper bulky
hydrophobic groups in Q. Indeed, in any case, this substituent
fall within a protein narrow cavity, delimited by I119, F174,
A380, M384. Then, lipophilic cores, such as the cyclohexyl
or myrtanyl moieties, proved to be the most suitable in sim-
ulate the binding mode of the co-crystallized ligand, making
the compound promising in CB1-targeting.
the newly proposed derivatives, with respect to the prototypes
1Aa, 1Ab, 1B and 1C, by prediction of the most important
descriptors related to absorption, blood–brain barrier (BBB)
permeation and distribution. In details, we took into account
the topological polar surface area (Å2), the logarithmic ratio
of the octanol–water partitioning coefficient (cLogP), extent
of blood–brain barrier permeation (LogBB), rate of passive
diffusion-permeability (LogPS), human intestinal absorption
(HIA), volume of distribution (Vd), the role played by plas-
matic protein binding (%PPB) and by the compound affin-
ity towards the human serum albumin (LogKaHSA), and an
overall perspective of the molecule oral bioavailability (%F).
As shown in Table 2, the bioisosteric replacement ben-
zene/pyrrole experienced by 2a–p led to compounds endowed
with higher polar surface area and decreased lipophilicity. In
particular, compounds 2a, 2e, 2i and 2m (TPSA = 55.09 Å2;
cLogP = 4.67–5.46) displayed higher TPSA and lower cLogP
values if compared with the strictly related analogues 1Aa,
3.4
In silico evaluation of
|
pharmacokinetic properties
In the search of more druggable compounds, we deemed in-
teresting to evaluate in silico the pharmacokinetic profile of
TABLE 2 Calculated pharmacokinetic descriptors related to absorption and distribution properties
Compound
TPSAa (Å2)
cLogP
LogBBb
LogPSc
HIA (%)d
Vd (L/kg)e
%PPB
pKaHSA
%F (oral)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
1Aa
1Ab
1B
1C
NESS06SM
SM-11
AM6538
Rimonabant
55.09
55.09
51.85
51.85
55.09
55.09
51.85
51.85
55.09
55.09
51.85
51.85
55.09
55.09
51.85
51.85
50.16
50.16
50.16
50.16
59.39
56.15
105.21
50.16
5.02
5.47
6.20
7.51
4.67
5.15
5.79
7.25
5.26
5.69
6.45
7.77
5.46
5.93
6.13
7.79
5.31
5.35
5.73
5.94
5.25
6.09
5.94
5.45
0.47
0.72
0.60
0.23
0.57
0.83
0.63
0.46
0.01
0.01
0.10
−0.38
0.54
0.74
0.38
0.24
−0.02
0.00
−0.01
0.46
0.50
0.27
−0.29
0.07
−1.1
−1.2
−1.8
−2.6
−1.1
−1.1
−1.5
−2.4
−1.2
−1.2
−1.8
−2.7
−1.2
−1.3
−1.6
−2.6
−1.1
−1.1
−1.2
−1.2
−1.2
−1.3
−1.5
−1.2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
2.8
2.9
2.9
4.1
2.7
2.9
2.8
4.0
2.9
3.7
3.6
4.5
3.0
3.8
3.8
4.7
2.9
2.9
3.1
3.9
3.0
3.7
3.8
2.9
97.83
96.92
97.92
99.16
96.37
95.44
97.67
98.55
99.33
99.13
99.36
99.75
97.96
97.07
98.76
99.08
99.62
99.40
99.57
98.76
98.39
99.00
99.73
99.28
6.43
6.21
5.64
5.90
6.15
6.06
5.36
5.70
6.66
6.43
5.95
6.05
5.61
5.44
5.04
4.87
6.68
6.33
6.82
5.76
5.68
5.11
5.98
6.45
52.2
21.2
34.6
7.6
54.6
21.2
43.9
13.4
49.2
12.4
25.2
4.4
90.0
46.0
17.1
12.6
14.2
49.2
10.7
36.4
46.1
37.6
23.4
48.7
aTPSA represents the polar surface area (Å2) of the molecule.
bExtent of brain penetration based on ratio of total drug concentrations in tissue and plasma at steady-state conditions.
cRate of passive diffusion-permeability PS represents permeability-surface area product and is derived from the kinetic equation of capillary transport.
dHIA represents the human intestinal absorption, expressed as percentage of the molecule able to pass through the intestinal membrane.
ePrediction of volume of distribution (Vd) of the compound in the body.

12
ASPRONI et Al.
|
1Ab, 1B and 1C (TPSA = 50.16 Å2; cLogP = 5.31–5.94)
and with rimonabant (TPSA = 50.16 Å2; cLogP = 5.45). On
the other hand, 2a, 2e, 2i and 2m were endowed with a more
drug-like lipophilicity over AM6538 (TPSA = 105.21 Å2;
cLogP = 5.94) but featuring lower TPSA values. In addition,
these piperidine derivatives were characterized by the better
pharmacokinetic profile with respect to the other congeners,
exhibiting also increased oral bioavailability (%F = 49.2–
90.0) with respect to the previous prototypes (%F = 10.7–
49.2). Moreover, the piperidine-containing molecules
experienced an improved bioavailability also in comparison
with the references AM6538 and rimonabant (%F = 23.4–
48.7). On the other hand, 2a, 2e, 2i and 2m, as well as most
of the other analogues and the related prototypes, proved to
pass the blood–brain barrier displaying adequate logBB and
logPS values (recommended ranges as follows: 0 < LogBB
< 1.5; −3 < LogPS < −1). According to our calculations,
only AM6538 was predictive inactive in terms of BBB per-
meation ability. Notably, only some compounds exhibited
unfavoured BBB permeation probably because of the too
high cLogP, deeply over 5 (Lipinski rules). In particular, a
number of derivatives bearing cyclohexyl or myrtanyl moi-
eties (2d, 2h, 2k, 2l, 2p) were predicted to be accompanied
by elevated lipophilicity (cLogP = 6.45–7.79) and therefore
are predicted to be anchored at the cellular membranes, so
as to highly bind to plasmatic proteins. As consequence, 2d,
2h, 2k, 2l, 2p displayed low bioavailability (%F = 4.4–13.4).
All the newly synthesized derivatives are able to be fully ad-
sorbed at the human intestinal membrane (HIA).
Based on all these data together with those coming from
molecular docking studies, the bioisosteric replacement ben-
zene/pyrrole arranged by 2a–p with respect to 1A–C appears
to be a proper choice when accompanied by an adequate Q
substituent, such as the piperidine one, in order to improve
the pharmacokinetic profile. On the contrary, more hydro-
phobic moieties, such as cycloalkyl groups, could lead to
unfavoured lipophilicity values. Thus, pharmacodynamics
properties of any further new analogues need to be carefully
optimized accordingly, by a proper selection of a branched
and polar moiety to be inserted in Q, in order to achieve a
good hydrophilicity–lipophilicity balance.
for CB2 receptor with Ki values superior to 314 nm, whereas
some of them showed moderate affinity for CB1 receptor with
Ki values inferior to 400 nm. Compound 2j exhibited good af-
finity for CB1 receptor (KiCB1 = 81 nm) and the highest CB2/
CB1 selectively ratio (>12).
Docking studies carried out on such compounds were
performed using the hCB1 X-ray in complex with the close
pyrazole analogue AM6538 and disclosed specific pattern
of interactions related to the tricyclic pyrrolopyrazole scaf-
folds as CB1 ligands. Our preliminary results point out the
versatility of the 1,4-dihydropyrazolo[3,4-a]pyrrolizine-,
4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and 1,4,5,6-tetra
hydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine architectures to
provide novel compounds for CB1 interaction. The results re-
ported in the present work pave the way for a further design
process towards more potent and selective CB1 ligands.
CONFLICT OF INTEREST
None of the authors have conflict of interest to declare.
ENDNOTES
1 MOE: Chemical Computing Group Inc. Montreal. H3A 2R7 Canada.
http://www.chemcomp.com.
2 www.biosolveit.com.
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SUPPORTING INFORMATION
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How to cite this article: Asproni B, Manca I, Pinna
G, et al. Novel pyrrolocycloalkylpyrazole analogues
as CB1 ligands. Chem Biol Drug Des. 2017;00:1–13.
https://doi.org/10.1111/cbdd.13069