Synthesis, pharmacological evaluation and docking studies of pyrrole structure-based CB2 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.06.057

Abstract During the last years, there has been a continuous interest in the development of cannabinoid receptor ligands that may serve as therapeutic agents and/or as experimental tools. This prompted us to design and synthesize analogues of the CB₂ receptor antagonist N-fenchyl-5-(4-chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-1H-pyrazole-3-carboxamide (SR144528). The structural modifications involved the bioisosteric replacement of the pyrazole ring by a pyrrole ring and variations on the amine carbamoyl substituents. Two of these compounds, the fenchyl pyrrole analogue 6 and the myrtanyl derivative 10, showed high affinity (K_i in the low nM range) and selectivity for the CB₂ receptor and both resulted to be antagonists/inverse agonists in [³⁵S]-GTPγS binding analysis and in an in vitro CB₂ receptor bioassay. Cannabinoid receptor binding data of the series allowed identifying steric constraints within the CB₂ binding pocket using a study of Van der Waals' volume maps. Glide docking studies revealed that all docked compounds bind in the same region of the CB₂ receptor inactive state model.

Full text of DOI:10.1016/j.ejmech.2015.06.057

European Journal of Medicinal Chemistry 101 (2015) 651e667
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, pharmacological evaluation and docking studies of pyrrole
structure-based CB2 receptor antagonists
,
1
b
,
c
,
d
,
e
,
,
Giulio Ragusa a , María Gomez-Canas
1, Paula Morales f 1, Dow P. Hurst g,
ꢀ
~
d
Francesco Deligia a, Ruth Pazos b
, Gerard A. Pinna , Javier Fernandez-Ruiz
,
,
c
,
,
e
a
b, c, d, e
ꢀ
Pilar Goya f, Patricia H. Reggio g, Nadine Jagerovic f
,
,
***
b
,
c
,
d,
e,
**
,
, *
2, Gabriele Murineddu a
ꢀ
Moises García-Arencibia
Dipartimento di Chimica e Farmacia, Universita degli Studi di Sassari, via F. Muroni 23/A, 07100 Sassari, Italy
a
ꢁ
b
ꢀ
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Investigacion en Neuroquímica, Facultad de Medicina,
Universidad Complutense, 28040 Madrid, Spain
c Campus de Excelencia Internacional (CEI-Moncloa), Madrid, Spain
d
ꢀ
ꢀ
Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain
e
f
ꢀ
ꢀ
Instituto Ramon y Cajal de Investigacion Sanitaria (IRYCIS), Madrid, Spain
Instituto de Química Medica, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
ꢀ
g Center for Drug Discovery, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
During the last years, there has been a continuous interest in the development of cannabinoid receptor
ligands that may serve as therapeutic agents and/or as experimental tools. This prompted us to design
and synthesize analogues of the CB2 receptor antagonist N-fenchyl-5-(4-chloro-3-methyl-phenyl)-1-(4-
methyl-benzyl)-1H-pyrazole-3-carboxamide (SR144528). The structural modifications involved the
bioisosteric replacement of the pyrazole ring by a pyrrole ring and variations on the amine carbamoyl
substituents. Two of these compounds, the fenchyl pyrrole analogue 6 and the myrtanyl derivative 10,
showed high affinity (Ki in the low nM range) and selectivity for the CB2 receptor and both resulted to be
Received 18 December 2014
Received in revised form
23 June 2015
Accepted 23 June 2015
Available online 15 July 2015
Keywords:
Bioisosterism
Synthesis
Cannabinoid receptors
CB2 antagonism
Docking studies
antagonists/inverse agonists in [35S]-GTP
gS binding analysis and in an in vitro CB2 receptor bioassay.
Cannabinoid receptor binding data of the series allowed identifying steric constraints within the CB2
binding pocket using a study of Van der Waals' volume maps. Glide docking studies revealed that all
docked compounds bind in the same region of the CB2 receptor inactive state model.
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
signalling lipids, and the proteins responsible for their synthesis
and inactivation (reuptake and degradation) [1e3].
The endocannabinoid system is an intercellular communicating
system, active in the brain and in the periphery, that comprises the
G-protein coupled cannabinoid receptors CB1 and CB2, some
It appears well demonstrated that the CB2R (CB2 receptors) have
a different distribution than CB1R (CB1 receptors) in the body, the
former being preferentially found in the peripheral tissues (e.g.
immune tissues, bone) and the latter widely distributed in the
central nervous system (CNS) [4]. Recently, CB2R gene transcripts
and proteins have also been discovered in the CNS, preferentially
located in glial elements [5], particularly when they become acti-
vated by different types of insults. However, they can be found only
in small amounts in the CNS. In contrast, CB1R are abundant in most
neuronal cells, in concordance with their key role in regulation of
synaptic processes [6].
* Corresponding author.
** Corresponding author. Departamento de Bioquímica
y Biología Molecular,
ꢀ
Instituto Universitario de Investigacion en Neuroquímica, Facultad de Medicina,
Universidad Complutense, 28040 Madrid, Spain.
*** Corresponding author.
E-mail addresses: nadine@iqm.csic.es (N. Jagerovic), moisesgar@med.ucm.es
(M. García-Arencibia), muri@uniss.it (G. Murineddu).
The absence of psychoactive effects, given the poor presence of
CB2R in neuronal subpopulations, has increased the interest in
1
These three authors share the first position within the authorship.
Present address: Department of Biochemistry and Molecular Biology, Uni-
2
versidad de Las Palmas de Gran Canaria, ULPGC, Las Palmas, Spain.
http://dx.doi.org/10.1016/j.ejmech.2015.06.057
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
developing selective CB2R ligands. Selective agonists of this re-
ceptor may be useful for the treatment of neuropathic and in-
flammatory pain [7,8], and also for their anti-inflammatory/
neuroprotective properties in a number of neurodegenerative dis-
orders [9]. In addition, the selective activation of the CB2R may be
also useful for the treatment of certain types of cancer [10,11], as
well as immune disorders [12]. By contrast, CB2R antagonists may
be useful for bone disorders [13] as these receptors are expressed in
osteoblasts, osteoclasts, and osteocytes, in which they play a role in
the regulation of specific activities of these cells in the process of
bone formation/resorption. Particular attention has been paid to
the process of osteoclast formation and bone resorption, which has
been found to be inhibited by CB2R antagonists and enhanced by
CB2R activation. However, the role of CB2R in bone formation/
resorption is rather controversial, and other authors reported
opposite results using also pharmacological (agonists vs antago-
nists) and genetic (studies in CB2R-deficient mice) strategies [14].
This biopharmacological potential prompted us to design and
synthesize deaza-analogues of the potent CB2 ligand SR144528 [15]
(Fig. 1). To this end, structureeactivity relationships (SAR) studies
were conducted on two different regions of the template com-
pound, SR144528, by (a) replacing the pyrazole ring of the phe-
nylpyrazole moiety with a phenylpyrrole ring system (6), and (b)
substituting the amine carbamoyl group on the pyrazole ring with
other amines (7e22, 25 and 26). The objective of this work was also
to prepare a rigid analogue (39) incorporating the phenylpyrrole
backbone into a tricyclic ring system for conformational restriction
purpose.
reaction with hydroxylamine hydrochloride led to the corre-
sponding oxime 2. Further Michael addition of 2 with methyl
propiolate, followed by thermal cyclization under microwave irra-
diation of the resulting O-vinyl oxime (structure of intermediate
not reported), gave the pyrrole ester 3. And then, N-alkylation of 3
with 4-methylbenzyl chloride in presence of NaH (60% in mineral
oil) gave the N-methylbenzyl-1H-pyrrole 4 which was subse-
quently saponified to provide the carboxylic acid 5. Finally, acti-
vation of 5 with thionyl chloride followed by reaction with the
appropriate amines afforded the desired compounds 6, 8e22.
To evaluate the influence of the carboxy group of the fenchyl
pyrrole 6 on receptor binding, this latter was replaced by a meth-
ylene group. The corresponding compound 7 was readily prepared
by reduction of 6 with LiAlH4 (Scheme 1). Furthermore, two other
methylene compounds 25 and 26 were prepared using a different
strategy as outlined in Scheme 2. Reduction of 5 to alcohol 23 with
LiAlH4 followed by oxidation to aldehyde 24 using MnO2, and
finally reductive amination in the presence of NaCNBH4 led to
compounds 25 and 26.
Conformationally constrained analogue of the fenchyl pyrrole
analogue 6 in which the C20 of the di-substituted phenyl ring and
the C4 of the pyrazole were linked by a methylene bridge was
proposed. The target compound 39 was synthesized via the routes
illustrated in Scheme 3. Benzoic acid derivative 27 was reduced to
alcohol 28, and then it was oxidized to aldehyde 29. The Knoeve-
nagel condensation of 29 with malonic acid in pyridine gave the
derivative 30, whose reduction to 31 followed by Sandmeyer re-
action (32) and cyclization in presence of CH3SO3H yielded 4-
methyl-5-chloro-1-indanone 33. Subsequent bromination of 33 to
34 and its alkylation (35) with ethyl acetoacetate followed by
treatment with NH4OAc gave the tricyclic core 36. Following the
procedures used for the preparation of the series 6, 8e22 starting
from 5 as depicted in Scheme 1, N-alkylation of 36 with 4-
methylbenzyl chloride (37) followed by saponification (38) and
final reaction with fenchylamine, via acyl chloride, yielded the
desired conformationally constrained compound 39.
Previously, as part of a project with Seltzman's group, Reggio
[16] published studies on the fenchyl-pyrrole derivative of
SR144528 (compound 6). Meanwhile Seltzman prepared this pyr-
role derivative by cycloaddition of tosylbenzylisocyanide with ethyl
acrylate; in our group we had an ongoing project on the prepara-
tion of this pyrrole and derivatives by cycloaddition of acetophe-
none oxime with methyl propiolate. The study presented here
showed a large structure variation on the amine carbamoyl sub-
stituent. The in vitro binding affinities for CB2R and CB1R were
measured for all compounds (6e22, 25, 26 and 39) and selected
compounds 6 and 10 were also tested for their functional activities
2.2. CB1/CB2 receptor binding studies
in [35S]-GTP
gS binding analysis. To further study their interaction
Radioligand binding assays have been used to evaluate the af-
mode with CBRs, molecular docking studies were carried out. The
results of these studies are reported below.
finity
of
the
new
5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-pyrroles 6e22, 25, 26 and tricyclic congener 39 to
CBRs expressed in membrane fractions of human CB1 or CB2
transfected cells. They were first subjected to a preliminary
screening at a concentration of 40
18, 22 and amine 26, which was performed at 10
25 and carboxamide 39, performed at 5 M, due to solubility rea-
2. Results and discussion
mM, except for carboxamides 12,
mM, and for amine
2.1. Chemistry
m
sons. A complete doseeresponse curve was generated for the
compounds that displaced the radioligand by > 50% in the pre-
liminary screen in at least one of the two receptors analysed. The
CB2R and CB1R affinities of the new 5-(4-chloro-3-methylphenyl)-
1-(4-methylbenzyl)-pyrroles 6e22, 25, 26 and the tricyclic
congener 39 are reported in Table 1. For comparison, the Ki values of
the reference CB2 ligand SR144528 have been reported.
The desired compounds (see Table 1) were prepared according
to the reactions depicted in Schemes 1e3. A series of SR144528
analogues 6e22 was prepared (Scheme 1) from the 1H-pyrrole-3-
carboxylic acid 5. The key intermediate 5 was synthesized by a
four-step synthesis starting from the commercial ketone 1, whose
Bioisosteric replacement of the pyrazole ring of the lead com-
pound SR144528 by a pyrrole ring (6) retains an attractive CB2R
binding value (Ki ¼ 5.7 nM) and CB2 selectivity binding value (Ki
CB1/Ki CB2 ¼ 258), although not better than the parent compound
(Ki ¼ 0.6 nM, and Ki CB1/Ki CB2 ¼ 667) [15]. This is consistent with
the reduced, but not completely lost, affinity of the pyrrole 8
compared to its pyrazole counterpart (Ki ¼ 164 and 29 nM [16],
respectively). Thus, the replacement of the pyrazole N-1 nitrogen
by CH did not drastically modify affinity for CB2R.
As reported in the pyrazole series [16], we observed that the
amide group plays a role in the interaction with the CB2R.
Fig. 1. Annular equivalent: pyrazole/pyrrole.

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
653
Table 1
Structures and binding dataa for compounds 6e22, 25, 26, 39 and SR144528.
Compd
X
Y
R
R1
H
Ki CB2 (nM)
Ki CB1 (nM)
1470 179
6
C]O
H
5.7 0.7
7
8
CH2
H
H
H
H
343 24.1
164 11.2
NDb
C]O
368 45
9
C]O
C]O
H
H
H
H
>5000
ND
10
72.2 9.4
>5000
11
12
13
C]O
C]O
C]O
H
H
H
H
H
H
>5000
>5000
>5000
>5000
>5000
1100 91.5
14
15
C]O
C]O
H
H
H
H
>5000
>5000
492 54.4
ND
(continued on next page)

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G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
Table 1 (continued )
Compd
X
Y
R
R1
H
Ki CB2 (nM)
Ki CB1 (nM)
>5000
16
C]O
H
244 34.2
17
18
C]O
C]O
H
H
H
H
106 7.2
3070 15.5
1707 226
>5000
19
20
21
C]O
C]O
C]O
H
H
H
H
H
H
>5000
>5000
>5000
>5000
>5000
575 35
22
C]O
H
H
>5000
>5000
25
26
39
CH2
H
H
>5000
>5000
>5000
>5000
CH2
H
H
C]O
CH2
CH3
346 117
>5000
SR144528 [15]
0.6
400
a
Affinity of compounds for the CB1R and CB2R was assayed using RBHCB1M400UA and RBXCB2M400UA membranes respectively and [3H]-CP-55,940 as radioligand. Ki
values were obtained from three independent experiments carried out in triplicate and are expressed as mean standard error.
b
ND, not determined.
Substitution of the carboxy group in pyrrole 6 by a methylene group
(7) caused significant decrease in affinity. As well, pyrroles 25 and
26 that contain the methylene group resulted in loss of CB2R
binding. These data support the importance of hydrogen bond
between the amide group and a residue (D(275)) of the inactive
CB2R binding site proposed by Kotsikorou et al. [16] for SR144528.
Once confirmed the importance of the carbamoyl group, we
focused our interest on the carbamoyl substituent. Curiously, few
structural modifications have been reported in the literature on the
fenchyl part of the pyrazole SR144528. One of these modifications
was the substitution of the fenchyl by a bornyl group that causes a
change in Ki (CB2) value from 0.31 to 7.2 nM. In the pyrrole series,
the same structural modification, compound 8, lower the Ki (CB2)
value in the same extent (from 5.7 to 164 nM). Given the fact that
much less is known about the influence of the carbamoyl substit-
uent on CB2 binding, we first compared compounds bearing as
monoterpene moiety a bornyl (8), an isopinocampheyl (9) and a
myrtanyl (10) group. It is worthy to note the variation of affinity and
selectivity depending on the position of the carbamoylpyrrole on
the fenchyl moiety (Table 1). Derivative 8 showed 4-fold increase in
CB1R affinity and reduced affinity towards CB2R compared to the
fenchyl analogue 6, wheres the myrtanyl derivative 10 binds
selectively to the CB2R (Ki ¼ 72.2 nM). The presence of iso-
pinocamphene (9) as well as the bulky adamantanes (11 and 12) led

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
655
Scheme 1. Reagents and conditions: a) NH2OH$HCl, AcONa$3H2O, H2O, EtOH, reflux, 2.5 h; b) (i) HC]CCO2CH3, 1,4-diazabicyclo[2.2.2]octane (DABCO), toluene, MW, 80 ꢀC, 10 min,
(ii) MW, 170 ꢀC, 45 min; c) (i) DMFan, 60% NaH in mineral oil, r.t., 15 min, (ii) 4-MeeBnCl, THFanh, r.t., 4 h; d) 10% NaOHaq, reflux, 12 h; e) (i) SOCl2, toluene, reflux, 4 h, (ii) CH2Cl2, TEA,
ReNH2, r.t., 2 h; f) LiAlH4, THFanh, r.t., 12 h.
to a total loss of affinity for CB receptors. Indeed, bulky substituents
are not favourable for binding sites. To further explore the possible
steric effects of the amine cyclohexyl substituents on CB receptors
affinity, compounds 13e16 were evaluated. Among these, cyclo-
hexyl derivative 16 showed the highest affinity for the CB2R
(Ki ¼ 244 nM), whereas the introduction of the two enantiomers of
cyclohexylethylamine furnished the compound 14-(R), with no
affinity for CBRs, and its S-enantiomer 15 with a Ki value of 492 nM
for CB2R. The influence of the nature of heterocyclic ring containing
one or two nitrogen atoms on the carboxamide portion was also
explored. N1-Piperidine (17) retained CB2 affinity while N4-aryl-
piperazine (18) showed a clear decrease in CB2 affinity. None of the
piperazine derivatives (18, 25, 26) binds to CBRs except 18 with a Ki
value in the micromolar range for CB2R. Among the carbohydrazide
Scheme 2. Reagents and conditions: a) LiAlH4, THFanh, r.t., 4 h; b) MnO2, CH2Cl2, reflux, 12 h; c) arylpiperazine, MeOH, AcOH, NaCNBH4, 0e25 ꢀC, 5e12 h.

656
G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
Scheme 3. Reagents and conditions: a) NaBH4, THFanh, CH3SO3H, r.t., 12 h; b) MnO2, CH2Cl2, reflux, 12 h; c) CH2(COOH)2, pyridine, piperidine, reflux, 18 h; d) H2, EtOH, Pd/C 10%,
30 psi, r.t., 12 h; e) NaNO2, HCl, H2O, CuCl, r.t., 12 h; f) CF3SO3H, 5e25 ꢀC, 5 h; g) Br2, AcOH, r.t., 4 h; h) THFanh., CH3COCH2COOEt, 60% NaH in mineral oil, r.t., 24 h; i) NH4OAc, SiO2,
toluene, MW, 110 ꢀC, 2.5 h; j) (i) DMFanh, 60% NaH in mineral oil, r.t., 15 min, (ii) 4-MeeBnCl, THFanh, r.t., 12 h; k) 10% NaOHaq, reflux, 6 h; l) (i) SOCl2, toluene, reflux, 4 h, (ii) CH2Cl2,
TEA, fenchylamine, r.t., 4 h.
derivatives (19, 20 and 21), the most interesting regarding CB2R was
21 with a Ki value of 575 nM. Both, the introduction of the naph-
thalene system (22) and the substitution of the carboxamide
function with N1-methylene-N4-aryl-piperazine (25, 26) resulted
in a loss of affinity for CBRs.
is critical to its CB2 affinity. Glide docking studies suggested that the
SR144528 amide hydrogen interaction with EC-3 loop residue,
D(275), is the primary interaction for SR144528 at CB2R, with ar-
omatic stacking interactions in the TMH5/6 aromatic cluster of
CB2R also having importance [16]. Each compound in Table 1 for
which there was measurable CB2R affinity (6e8, 10, 13, 15e18, 21,
39) was docked using Glide here in our CB2R model of the inactive
The conformationally restricted analogue (39) of the fenchyl
compound 6 adopts a semi-planar geometry. Introduction of this
conformational constrain led to
a
decrease in CB2 affinity
state. This CB2R model was pre-equilibrated in a stearoyl-
(Ki ¼ 343 nM).
docosahexaenoylphosphatidylcholine (SDPC) bilayer for 300 ns to
allow it to adjust to a lipid environment [16]. Glide docking studies
revealed that all of these compounds bind in the same region of
CB2R as SR144528. Table A-1 in the supporting data presents the
docking information for all of the binding analogues. Below, we
present the Glide docking study of compound 10 at CB2R as an
example of these results.
As commented in this study, we observed significant CB2 affinity
differences related to the nature of the carbamoyl substituent. Since
this part of the corresponding binding site has been less explored,
we underwent studies on the Van der Waals (VdW) volume map of
the binding analogues and of the non-binding analogues.
2.3. Molecular modelling
2.3.1. Conformational analysis
Our previous studies of the binding site for the CB2 antagonist,
SR144528, have shown that the SR144528 amide functional group
Fig. 2a illustrates the global minimum-energy conformer of 10
compared to that of SR144528. Fig. 2b provides a numbering

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
657
10. All residues contributing energies of interaction of ꢁ2.0 kcal/
mol or less, are shown with brown carbons. K3.28, shown with
magenta carbons, contributes repulsive interactions.
Because the global minimum energy conformer of 10 has its
amide group oriented differently than SR144528, a higher energy
conformer of 10 (with the proper amide orientation) that is
0.4 kcal/mol above the global minimum was used for docking
studies. Fig. 4c illustrates the final energy minimized 10/CB2R
complex. In this complex, the amide hydrogen of 10 interacts with
the EC-3 loop residue, D(275). The hydrogen bond heteroatom
distance (NeO) and hydrogen bond (NeHeO) angle are 2.6 Å and
154ꢀ, respectively. The carboxamide and pyrrole are positioned
closer to TMH2/3 and the myrtanyl ring is packed against TMH7.
This orientation accommodates the steric bulk of the myrtanyl ring,
but does introduce a closer unfavorable proximity of the carbox-
amide to K3.28. Like SR144528, 10 forms aromatic stacking in-
teractions
with
W6.48(258)
and
W5.43(194).
The
chloromethylphenyl ring of compound 10 forms an offset parallel
aromatic stack with W6.48(258) with a ring centroid to ring
centroid distance of 4.3 Å with the 6-member ring and 5.5 Å with
the 5-member ring. The chloromethylphenyl group also forms an
aromatic T-stack with W5.43(194) (6-member ring) with a ring
centroid to centroid distance of 6.1 Å and a ring plane to plane angle
of 69ꢀ. Finally, the methylbenzyl ring forms an aromatic T-stack
interaction with W5.43(194). The ring centroid to centroid distance
is 6.3 Å with the 6-member ring and 6.9 Å with the 5-member ring
and the ring plane to ring plane angle is 53ꢀ. The final docked
conformational cost of 10 relative to its global minimum was
calculated to be 4.5 kcal/mol. The net Glide score for 10 docked in
CB2R was found to be ꢁ7.5 kcal/mol. This result is consistent with
the net Glide score for SR144528 (ꢁ9.2 kcal/mol; see Table A1) and
is also consistent with the fact that 10 has a larger Ki at CB2R
(Ki ¼ 72.2 nM) relative to SR144528 (Ki ¼ 0.6 nM).
Fig. 2. (a) Global minimum energy conformer of SR144528 (left), and 10 (right). (b)
Chemical drawing of 10 with atoms labeled to facilitate discussion of the conforma-
tional analysis.
system for 10 used in the discussion of dihedral angles below. The
myrtanyl derivative, 10 generates a higher number of conformers
compared to SR144528 due to the presence of an additional
rotatable bond. However, the pyrrole ring and the amide of the
global minimum energy conformer of 10 remain co-planar
(C2eC3eC10eN20 ¼ 177.8ꢀ). The methylbenzyl ring and the chlor-
omethylphenyl ring of 10 adopt a relative position with respect to
the pyrrole ring that is similar to SR144528. One major difference
between the global minima for SR144528 and 10 is that the amide
groups are oriented differently. Another difference between
SR144528 and 10 is that the bulky myrtanyl ring in 10 is positioned
towards the front face of the molecule and is almost perpendicular
2.4. Reasons for loss of measurable binding at CB2
The analogues that have no measurable binding in Table 1, fall
into two categories: (1) analogues larger in size (analogues 9, 11, 12,
14, 22, 25 and 26) than analogues with measurable CB2R binding
and (2) analogues in which the electrostatic distributions may
impact CB2R affinity (19 and 20). These sets are considered sepa-
rately below.
2.5. Size considerations: Active Analogue Approach
to the amide group (C10eN20eC30eC40
N20eC30eC40eH50 ¼ ꢁ54ꢀ).
¼
ꢁ105.3ꢀ and
Inspection of Table 1 suggests that enlargements of the amine
carbamoyl substituent beyond a certain size results in analogues
with no measurable CB2R binding (Ki > 5000 nM) (analogues 9, 11,
12, 14, 22, 25 and 26). We hypothesized that the loss of binding for
these analogues may be due to steric constraints within the CB2R
binding pocket. Therefore, to identify possible sterically occluded
regions, we used a modified version of the Active Analogue
Approach [17]. This approach identifies this region of space occu-
pied by conformers of analogues with no measurable CB2R affinity
that is not occupied by conformers of analogues with measurable
CB2R affinity. Fig. 4a shows the union of the Van der Waals (VdW)
volume maps for all accessible conformers of analogues with
measurable CB2R affinity (shown in green surface display). Fig. 4b
shows the union of the Van der Waals (VdW) volume maps for all
accessible conformers of analogues with no measurable CB2R af-
finity (shown in red surface display). Fig. 4c illustrates, in red col-
oured grid, this volume of space occupied by atoms of analogues
with no measurable CB2R affinity that is not occupied by atoms of
analogues with measurable CB2R affinities. The global minimum
conformation of the non-binding analogue 26 is shown in Fig. 4c as
2.3.2. Glide docking study
Glide docking studies were performed for all analogues with
measurable binding affinities (i.e., SR144528 and analogues 6, 7, 8,
10, 13, 15, 16, 17, 18, 21, 39). Because the docking positions and
interaction sites are quite similar among these compounds, we
show here the complete results for one of these, compound 10, as
an example. For information about Glide scores and Conforma-
tional Energy costs for the other analogues in this set, please see
Supplementary data.
Analogue 10 was docked into our previously published model of
the CB2R inactive state [16]. Our recent dock of SR144528 in this CB2
model revealed that SR144528 is a large ligand that modelling
studies predict to span the entire CB2 binding pocket with fenchyl
ring near TMH1/2/7, amide functionality near TMH3/7, and aro-
matic moieties near TMH3/5/6. Fig. 3 presents Glide docking results
for analogue 10 binding at CB2R from an extracellular view. Here,
EC-1 and -2 loops have been omitted for clarity. The EC-3 loop
residue D(275) (shown in yellow) is the primary interaction site for

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G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
Fig. 3. (Left) Extracellular view of the analogue 10/CB2R complex. The EC-1 and -2 loops have been removed for clarity. The EC-3 loop residue D(275) (shown in yellow) is the
primary interaction site for 10. All residues contributing energies of interaction of ꢁ2.0 kcal/mol or less, are shown with brown carbons. K3.28 is shown in magenta. (Right) TMH2-3
side view of the analogue 10/CB2R complex. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. (a) The union of the Van der Waals (VdW) volume maps for all accessible conformers of analogues with measurable CB2R affinity is shown in green surface display. (b) The
union of the Van der Waals (VdW) volume maps for all accessible conformers of analogues with no measurable CB2R affinity is shown in red surface display. (c) The red coloured
grid illustrates the calculated excluded volume. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
a structural reference. It is clear here that the analogues with no
measurable CB2R affinities do project into space not occupied by
analogues with measurable affinities. This is caused by the R groups
extending further away from the plane of the central pyrrole ring
than do the R groups of analogues with measurable affinities. In the
context of the full CB2R R bundle, this additional bulk prevents
these analogues from binding at CB2R due to steric overlaps with
TMH7 that cannot be relieved.
2.6. Electrostatic effects
Compounds 19, 20 and 21 form a unique set of analogues in
Fig. 5. The molecular electrostatic potential maps of compounds 10, 21, 20 and 19 are illustrated here. The positive dark blue regions correspond to the amide NH. The negative
yellow-red regions in 21, 20 and 19 correspond to the nitrogen adjacent to the amide NH. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
659
Table 1 because each has a nitrogen atom directly attached to the
amide NH. The result of such a substitution is that the electrostatic
potentials of 19, 20 and 21 will be different from high affinity
compounds such as 6 or 10. Because the primary interaction site for
6 or 10 is D(275) [16], (see above), we reasoned that the presence of
a negative electrostatic potential region immediately adjacent to a
negatively charged amino acid (D(275)) would be repulsive and
lead to reduced binding affinity of these compounds for CB2R. Fig. 5
shows a comparison of molecular electrostatic potential (MEP)
maps for 10, 21, 20 and 19. Global minimum energy conformers of
each compound have the ring nitrogen's lone pair of electrons
pointing in the same direction as the amide NH. Here the molecules
are arranged such that the amide NeH is pointing towards the
viewer. All analogue MEPs show a positive electrostatic potential
(dark blue regions) that correspond to the amide hydrogen. How-
ever, the MEPs of 21, 20 and 19 also show a negative potential re-
gion (red-yellow) immediately adjacent to the positive region. In
each case, this corresponds to the ring nitrogen connected to the
amide NH. The negative potential regions are stronger in 19 and 20
relative to 21. The reason for this appears to be due to the size of the
ring in which the nitrogen is incorporated. The rings (R substituent,
see Table 1) in 19, 20 and 21 progress in size from 5-, to 6-, to 7-
membered rings. As the size of the rings increase, the charge on
the ring nitrogen decreases, such that the negative potential region
in 21 is significantly diminished relative to 19. For analogues 19 and
20, electrostatic repulsion of D(275) may diminish the ability of
these analogues to interact at CB2R, while the electrostatics of 21
may still allow interaction with D(275).
inflammatory response. This response was quantitated by
measuring the concentration of prostaglandin E2 (PGE2) using an
ELISA immunoassay. Through this in vitro bioassay, it was possible
to determine if the new compounds behave as CB2 agonists,
reducing the inflammatory response (by attenuating LPS-induced
PGE2 release), or otherwise as antagonists/inverse agonists by
reversing the effects of an agonist and, even, by increasing the in-
flammatory response produced LPS. This can be found in a control
assay conducted with two well-known CB2R ligands, the non-
selective agonist WIN55,212-2, and the selective antagonist/in-
verse agonist SR144528. The data presented in Fig. 7 show how the
stimulation of BV-2 cells with LPS produced a 3-fold increase in
PGE2 release which was completely reversed by the co-incubation
with WIN55,212-2. The complete blockade of WIN55,212-2 effects
by SR144528 supports the fact that the PGE2 release is mediated
through CB2R activation.
Compounds 6 and 10 were examined in this bioassay and both
of them behaved again as antagonists/inverse agonist of CB2R. This
was concluded from the observation that they were not able to
reverse LPS-induced response, as did WIN55,212-2 in the control
assay, but both were able to reverse the effect of WIN55,212-2 at
the same extent as SR144528. In addition, both compounds
elevated PGE2 levels when combined with LPS and, in particular,
when combined with SR144528. These data are presented in Fig. 8.
4. Conclusions
In summary, a series of SR144528 derivatives were designed and
synthesized as cannabinoid ligands. Among this pyrrole series, the
closest structural SR144528 homologue (6) exhibited the best af-
finity for the CB2R although not better selectivity vs CB1R compared
to SR144528. Structural modifications on the amine group of 6
could modulate the binding and selectivity for CBRs. Examination
of the Van der Waals's volume maps of non-binding and binding
derivatives allowed identifying steric constraints within the CB2
binding site. Therefore, TMH7 represents the key region that may
sterically block non-binding compounds. Each compound with
measurable CB2R affinity (6e8, 10, 13, 15e18, 21, 39) was docked
using Glide in our CB2R model of the inactive state. These Glide
docking studies revealed that all of these compounds bind in the
same region of CB2R as SR144528. Besides binding to CBRs,
3. Determination of the functional activity at the CB2
receptor
According to their binding values, compounds
6
(KiCB2 ¼ 5.7 nM) and 10 (KiCB2 ¼ 72.2 nM) have been chosen in
order to determine their activity as agonist or antagonists on CB2R.
To this end, we conducted [35S]-GTP
g
S binding studies, which
demonstrated that they behave as antagonists/inverse agonists
with values of IC50 of 171.4 90.7 nM for compound 6 and of
1816.0 70.2 nM for compound 10 (see a representative curve for
compound 6 in Fig. 6). The differences of IC50 values for both
compounds correlate with their differences in binding affinity, in
both cases being in a range of 1e10.
To further confirm these properties, we also used an in vitro
bioassay in which the CB2R activity of titled compounds was
assayed against LPS-induced inflammatory responses in cultures of
mouse BV-2 microglial cell line. These cells express only CB2R and
selective agonists of this receptor reduce the intensity of this pro-
Fig. 7. Effects of WIN55,212-2 and SR144528 on the LPS-induced release of PGE2 in
cultured BV-2 cells. Data were assessed by one-way analysis of variance
(F(3,34)
¼
29.98, p < 0.0001; ***p < 0.005 versus controls (DMSO-exposed) or
Fig. 6. Representative curve of the [35S]-GTP
gS binding for compound 6.
WIN þ LPS; #p < 0.05 versus LPS).

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G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
Fig. 8. Effects of compounds 6 or 10, combined with WIN55,212-2 and/or SR144528, on the LPS-induced release of PGE2 in cultured BV-2 cells. Data were assessed by one-way
analysis of variance (F(5,52) ¼ 24.26, p < 0.0001 for compound 6; F(5,52) ¼ 39.27, p < 0.0001 for compound 10; *p < 0.05, **p < 0.01,***p < 0.005 versus controls (DMSO-
exposed) or WIN þ LPS; ##p < 0.01, ###p < 0.005 versus LPS or compounds 6 or 10 þ WIN þ LPS; @p < 0.05, @@p < 0.01 versus compounds 6 or 10 þ LPS).
functional studies on compounds 6 and 10 showed that they
behaved as antagonists/inverse agonists of the CB2R. Compounds 6
and 10 would deserve further investigation as potential therapeutic
agents in those conditions in which the selective blockade of the
CB2R may have beneficial effects. An interesting possibility may be
the treatment of certain bone disorders, as has been suggested in
the Introduction.
(DMSO-d6) as solvents. Chemical shifts (d
) for 1H and 13C NMR
spectra are reported in parts per million (ppm) using the residual
non-deuterated solvent resonance as the internal standard (for
chloroform-d: 7.26 ppm, 1H and 77.16 ppm, 13C; for DMSO-d6:
2.50 ppm, 1H, 39.52 ppm, 13C. Data are reported as follows:
chemical shift (sorted in descending order), multiplicity (s for
singlet, br s for broad singlet, d for doublet, t for triplet, q for
quadruplet, m for multiplet), integration and coupling constants (J)
in Hertz (Hz). LC/MS analyses were run on an Agilent 1100 LC/MSD
system consisting of a single quadrupole detector (SQD) mass
spectrometer (MS) equipped with an electrospray ionization (ESI)
interface and a photodiode array (PDA) detector. PDA range was
120e550 nm. ESI in positive mode was applied. Mobile phases: (A)
MeOH in H2O (8:2). Analyses were performed with: flow rate
0.9 mL/min; temperature 350 ꢀC. All final compounds displayed
ꢂ95% purity as determined by elemental analysis on a Per-
kineElmer 240-B analyser, for C, H, and N.
5. Experimental section
5.1. General procedures
All reactions involving air or moisture-sensitive compounds
were performed under argon atmosphere. Solvents and reagents
were obtained from commercial suppliers and were used without
further purification. Ketone 1 and amines for the synthesis of final
compounds were purchased by SigmaeAldrich®: fenchylamine [18]
and benzyl alcohol 28 [19] was synthesized according to the liter-
ature procedure. Microwave irradiation experiments were carried
out in a Biotage® Microwave Initiator Eight 2.5 in the standard
configuration as delivered, including proprietary software. All ex-
periments were carried out in sealed microwave process vials un-
der normal absorption. After completion of the reaction, the vial
was cooled down to 25 ꢀC via air jet cooling before opening. Re-
action temperatures were monitored by an IR sensor on the outside
wall of the reaction. Hydrogenations were carried out in the 4560
Parr Apparatus using a H2PEM-100 Parker Balston Hydrogen
Generator. Flash column chromatography was performed auto-
matically on Flash-master (Biotage®) with pre-packed Biotage®
SNAP silica gel cartridges or manually on silica gel (Kieselgel 60,
0.040e0.063 mm, Merck®). Thin layer chromatography (TLC) was
performed with Polygram SIL N-HR/HV254 pre-coated plastic sheets
(0.2 mm) on aluminium sheets (Kieselgel 60 F254, Merck®).
5.1.1. Synthesis of (E,Z)-1-(4-dichloro-3-methylphenyl)ethanone
oxime (2)
A mixture of ketone 1 (500 mg, 2.97 mmol, 1 eq), NH2OH$HCl
(1.7 eq) and AcONa$3H2O (3 eq) in 60% aqueous ethanol solution
(1.74 mL) was refluxed for 2.5 h. The suspension was cooled at room
temperature and the resulting precipitate was filtered, washed
(H2O), and then solved in Et2O. The organic solution was dried
(Na2SO4) and concentrated to give 2 as white solid (520 mg, 95.5%).
Rf ¼ 0.33 (petroleum ether/EtOAc 9:1); mp 104e106 ꢀC; 1H NMR
(CDCl3)
d 2.27 (s, 3H), 2.40 (s, 3H), 7.31e7.47 (m, 2H), 7.49 (s. 1H),
8.51 (br s, 1H, OH, exch. with D2O).
5.1.2. Synthesis of methyl-5-(4-chloro-3-methylphenyl)-1H-
pyrrole-3-carboxylate (3)
To a stirred solution of 1,4-diazabicyclo[2.2.2]octane (DABCO)
(0.1 eq) and oxime 2 (549 mg, 2.99 mmol, 1 eq) at ꢁ5 ꢀC in dry
toluene (5.25 mL) methyl propiolate (1 eq) was dropwise added.
The reaction mixture was allowed to warm to room temperature
and was subjected to a two-stage microwave irradiation sequence
(stage 1, 80 ꢀC, 10 min; stage 2, 170 ꢀC, 45 min). The mixture was
concentrated under reduced pressure, and the residue was purified
by gradient-flash chromatography (petroleum ether/EtOAc
95:5e7:3) to afford the pyrrole ester 3 as an orange solid (149 mg,
€
Melting points were obtained on a Kofler melting point apparatus
and are uncorrected. IR spectra were recorded as nujol mulls on
NaCl plates with a Jasco FT/IR 460 plus spectrophotometer and are
expressed in
n
(cmꢁ1). NMR experiments were run on a Varian
Unity 200 spectrometer (200.07 MHz for 1H, and 50.31 MHz for 13C)
and on a Bruker Avance III Nanoboy 400 system (400.13 MHz for 1H,
and 100.62 MHz for 13C). Spectra were acquired using deuterated
chloroform (chloroform-d) or deuterated dimethylsulfoxide

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
661
20%). Rf ¼ 0.28 (petroleum ether/EtOAc 8:2); mp 135e138 ꢀC; 1H
(ESI): C30H35ClN2O requires m/z 475, found 476 [M þ 1]þ; Anal.
calcd for C30H35ClN2O: C, 75.85; H, 7.43; Cl 7.46; N, 5.90. Found: C,
75.88; H, 7.45; Cl 7.43; N, 5.93.
NMR (CDCl3)
d 2.40 (s, 3H), 3.84 (s, 3H), 6.88 (s, 1H), 7.24e7.36 (m,
3H), 7.47 (s, 1H), 8.76 (br s, 1H, NH, exch. with D2O).
5.1.3. Synthesis of methyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxylate (4)
5.1.7. N-(R)-(þ)-Bornyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (8)
To a solution of pyrrole-ester 3 (299 mg, 1.20 mmol, 1 eq) in
anhydrous DMF (4 mL) under N2, was added portionwise 60% NaH
in mineral oil (1.2 eq). The solution was stirred at room temperature
for 15e20 min, then a solution of 4-methyl-benzyl chloride (1 eq)
in anhydrous THF (1.2 mL) was added dropwise: the resulting
mixture was stirred at room temperature for 3 h. The solution was
poured in H2O (8 mL) and extracted with CH2Cl2, which was
washed (H2O), dried (Na2SO4) and concentrated under reduced
pressure to furnish an oily brown residue, whose flash chroma-
tography purification (petroleum ether/EtOAc 95:5) gave derivative
4 as a white solid (254 mg, 60%). Rf ¼ 0.55 (petroleum ether/EtOAc
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(R)-(þ)-bornylamine into the title product. The
mixture was purified by flash chromatography (petroleum ether/
EtOAc 8:2) to afford 8 (73 mg, 53%) as a yellow solid. Rf ¼ 0.58
(petroleum ether/EtOAc 7:3); mp 96e100 ꢀC; IR 1635 (C]O), 3350
(NH); 1H NMR (CDCl3)
d 0.87 (s, 3H), 0.89 (s, 3H), 0.99 (s, 3H), 1.23
(d, 2H, J ¼ 8.8 Hz),1.38e1.60 (m, 3H),1.62e1.85 (m, 2H), 2.32 (s, 6H),
4.42 (t, 1H, J ¼ 8.4 Hz), 5.04 (s, 2H), 5.8 (d, 1H, J ¼ 8.4 Hz, NH, exch.
with D2O), 6.42 (s, 1H), 6.91 (d, 2H, J ¼ 7.4 Hz), 7.00e7.20 (m, 4H),
7.28e7.32 (m, 2H); 13C NMR (CDCl3)
d 13.72 (CH3),18.70 (CH3),18.86
(CH3), 20.04 (CH3), 21.05 (CH3), 28.09 (CH2), 28.43 (CH2), 37.88
(CH2), 44.93 (CH), 48.13 (C), 49.56 (C), 50.94 (CH2), 53.32 (CH),
106.89 (CH), 119.92 (CH), 125.35 (CH), 126.59 (CH ꢃ2), 127.57 (CH),
129.04 (C), 129.46 (CH ꢃ2), 130.65 (C), 131.57 (CH), 133.89 (C),
134.29 (C),134.47 (C),136.18 (C),137.48 (C),164.46 (C]O); MS (ESI):
8:2); mp 154e156 ꢀC; 1H NMR (CDCl3)
d 2.33 (s, 3H), 2.35 (s, 3H),
3.80 (s, 3H), 5.03 (s, 2H), 6.63 (d, 1H, J ¼ 1.6 Hz), 6.90 (d, 2H,
J ¼ 8.0 Hz), 7.00e7.16 (m, 4H), 7.30 (d, 1H, J ¼ 8.0 Hz), 7.35 (d, 1H,
J ¼ 1.8 Hz).
C
C
30H35ClN2O requires m/z 475, found 476 [M þ 1]þ; Anal. calcd for
5.1.4. Synthesis of 5-(4-chloro-3-methylphenyl)-1-(4-
30H35ClN2O: C, 75.88; H, 7.45; Cl 7.43; N, 5.93. Found: Anal. calcd
methylbenzyl)-1H-pyrrole-3-carboxylic acid (5)
for C30H35ClN2O: C, 75.85; H, 7.43; Cl 7.46; N, 5.90. Found: C, 75.87;
H, 7.45; Cl 7.44; N, 5.91.
A solution of pyrrole ester 4 (400 mg, 1.13 mmol) in a 10% hydro-
alcoholic NaOH solution (11.75 mL, 60% EtOH) was refluxed over-
night. The solution was cooled at room temperature and acidified
with 37% HCl. The precipitate was filtered, washed (H2O) and air-
dried to yield the analytically pure acid 5 (320 mg, 83.5%) as a
5.1.8. N-(1R,2R,3R,5S)-Isopinocampheyl-5-(4-chloro-3-
methylphenyl)-1-(4-methylbenzyl)-1H-pyrrole-3-carboxamide (9)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(1R,2R,3R,5S)-isopinocampheylamine into the
title product. The mixture was purified by flash chromatography
(petroleum ether/EtOAc 8:2) to afford 9 (89.5 mg, 65%) as a brown
solid. Rf ¼ 0.225 (petroleum ether/EtOAc 8:2); mp 85e89 ꢀC; IR
grey. Rf 0.18 (petroleum ether/EtOAc 8:2); mp 191e192 ꢀC; 1H
¼
NMR (CDCl3)
d
1.80 (br s, 1H, OH, exch. with D2O), 2.33 (s, 3H), 2.36
(s, 3H), 5.05 (s, 2H), 6.67 (s, 1H), 6.91 (d, 2H, J ¼ 7.8 Hz), 6.98e7.32
(m, 5H), 7.42 (s, 1H).
1634 (C]O), 3352 (NH); 1H NMR (CDCl3)
d 1.08 (s, 3H), 1.15 (d, 3H,
5.1.5. General procedure for the synthesis of carboxamides 6, 8e22
A mixture of the acid 5 (98 mg, 0.29 mmol, 1 eq) and thionyl
chloride (3 eq) in toluene (2.38 mL) was refluxed for 4 h. The sol-
vent and the excess of SOCl2 were removed under reduced pressure
and the resulting dark solid in CH2Cl2 (15 mL) was dropwise added
to a solution of requisite amine or hydrazine (1.5 eq) and Et3N (1.5
eq) in CH2Cl2 (15 mL) at 0 ꢀC. The mixture was refluxed for 4 h. The
mixture was then poured into a separatory funnel and brine was
added. The aqueous layer was separated and extracted with CH2Cl2.
The combined organic layer were washed (H2O), dried (Na2SO4)
and concentrated under reduced pressure. The analytically pure
product was isolated by flash chromatography purification.
J ¼ 6.8 Hz), 1.23 (s, 3H), 1.57e1.62 (m, 1H), 1.83e1.86 (m, 2H),
1.92e1.97 (m, 1H), 2.32 (s, 3H), 2.33 (s, 3H), 2.37e2.43 (m, 1H),
2.64e2.70 (m, 1H), 4.44 (t, 1H, J ¼ 7.2 Hz), 5.02 (s, 2H), 5.68 (d, 1H,
J ¼ 8.4 Hz, NH, exch. with con D2O), 6.42 (s, 1H), 6.90 (d, 2H,
J ¼ 7.6 Hz), 7.03e7.20 (m, 3H), 7.28e7.30 (m, 3H); 13C NMR (CDCl3)
d
20.07 (CH3), 20.80 (CH3), 21.08 (CH3), 23.40 (CH3), 28.06 (CH2),
35.34 (CH2), 37.54 (CH2), 38.46 (C), 41.71 (CH3), 46.61 (CH), 47.46
(CH), 47.89 (CH), 50.99 (CH2), 107.14 (CH), 119.98 (C), 125.33 (CH),
126.68 (CH), 126.74 (CH), 127.14 (CH), 127.60 (CH), 129.09 (CH),
129.52 (CH ꢃ2), 131.62 (CH), 133.91 (C), 134.36 (C), 134.52 (C),
135.48 (C), 136.21 (C), 137.54 (C), 163.88 (C]O); MS (ESI):
C
C
30H35ClN2O requires m/z 475, found 476 [M þ 1]þ; Anal. calcd for
30H35ClN2O: C, 75.88; H, 7.45; Cl 7.43; N, 5.93. Found: Anal. calcd
5.1.6. N-(1)-(S)-Fenchyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (6)
for C30H35ClN2O: C, 75.85; H, 7.43; Cl 7.46; N, 5.90. Found: C, 75.87;
H, 7.46; Cl 7.47; N, 5.92.
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(1)-(S)-fenchylamine into the title product. The
mixture was purified by flash chromatography (petroleum ether/
EtOAc 8:2) to afford 6 (96.5 g, 70%) as beige solid. Rf ¼ 0.44 (pe-
troleum ether/EtOAc 8:2); mp 155e156 ꢀC; IR 1633 (C]O), 3354
5.1.9. N-(1S,2R)-Myrtanyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (10)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(1S,2R)-myrtanylamine into the title product.
The mixture was purified by flash chromatography (petroleum
ether/EtOAc 8:2) to afford 10 (91 mg, 66%) as a beige solid. Rf ¼ 0.56
(petroleum ether/EtOAc 8:2); mp 72e76 ꢀC; IR 1635 (C]O), 3354
(NH); 1H NMR (CDCl3)
d 0.84 (s, 3H), 1.09 (s, 3H), 1.16 (s, 3H),
1.20e1.35 (m, 2H), 1.45e1.55 (m, 1H), 1.63e1.71 (m, 4H), 2.32 (s,
3H), 2.34 (s, 3H), 3.81 (d, 1H, J ¼ 8.0 Hz), 5.03 (s, 2H), 5.78 (d, 1H,
J ¼ 8.0 Hz, NH, exch. with D2O), 6.38 (s, 1H), 6.92 (d, 2H, J ¼ 8.0 Hz),
7.06 (d, 1H, J ¼ 8.0 Hz), 7.11 (d, 2H, J ¼ 8.0 Hz), 7.16e7.21 (m, 1H),
(NH); 1H NMR (CDCl3)
d
0.88 (d, 1H, J ¼ 8.0 Hz), 1.05 (s, 3H), 1.18 (s,
3H),1.52e1.56 (m,1H),1.81e1.96 (m, 7H), 2.31 (s, CH3), 2.31 (s, CH3),
3.36e3.42 (m, 2H), 5.02 (s, 2H), 5.78e5.85 (br s, 1H, NH, exch. with
D2O), 6.39 (s, 1H), 6.89 (d, 2H, J ¼ 8.0 Hz), 7.03 (d, 1H, J ¼ 8.0 Hz),
7.08 (d, 2H, J ¼ 8.0 Hz), 7.13 (s, 1H), 7.25e7.29 (m, 2H); 13C NMR
7.31 (d, 2H, J ¼ 7.0 Hz); 13C NMR (CDCl3)
d 19.67 (CH3), 20.06 (CH3),
21.04 (CH3), 21.25 (CH3), 26.01 (CH2), 27.34 (CH2), 30.78 (CH3),
39.36 (CH), 42.64 (CH2), 48.14 (CH), 48.56 (CH), 51.04 (CH2), 62.78
(CH), 106.73 (CH), 119.97 (C), 125.39 (CH), 126.74 (CH ꢃ2), 127.66
(CH), 129.09 (CH), 129.51 (CH ꢃ2), 130.72 (C), 131.66 (CH), 133.98
(C), 134.23 (C), 134.51 (C), 136.23 (C), 137.55 (C), 164.87 (C]O); MS
(CDCl3)
d 18.87 (CH2), 19.03 (CH3), 20.05 (CH3), 22.18 (CH3), 25.04
(CH2), 27.00 (CH3), 32.30 (CH2), 37.70 (C), 40.39 (CH), 40.63 (CH),
42.80 (CH), 43.93 (CH2), 49.96 (CH2), 106.10 (CH), 118.83 (C), 124.25

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G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
(CH), 125.66 (CH ꢃ2), 126.57 (CH), 128.05 (CH), 129.72 (CH ꢃ2),
130.57 (C), 132.87 (CH), 133.20 (C), 133.29 (C) 133.46 (C), 135.15 (C),
136.49 (C), 163.43 (C]O); MS (ESI): C30H35ClN2O requires m/z 475,
found 476 [M þ 1]þ; Anal. calcd for C30H35ClN2O: C, 75.88; H, 7.45;
Cl 7.43; N, 5.93. Found: Anal. calcd for C30H35ClN2O: C, 75.85; H,
7.43; Cl 7.46; N, 5.90. Found: C, 75.88; H, 7.45; Cl 7.45; N, 5.91.
calcd for C30H37ClN2O: C, 75.53; H, 7.82; Cl 7.43; N, 5.87. Found: C,
75.59; H, 7.90; Cl, 7.47; N, 5.90.
5.1.13. N-(R)-(ꢁ)-Cyclohexylethyl-5-(4-chloro-3-methylphenyl)-1-
(4-methylbenzyl)-1H-pyrrole-3-carboxamide (14)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(R)-(ꢁ)-cyclohexylethylamine into the title
product. The mixture was purified by flash chromatography (pe-
troleum ether/EtOAc 7:3) to afford 13 (41.7 mg, 32%) as a brown
solid. Rf ¼ 0.25 (petroleum ether/EtOAc 8:2); mp 211e214 ꢀC; IR
5.1.10. N-(Adamantan-1-yl)-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (11)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-1-adamantylamine into the title product. The
mixture was purified by flash chromatography (petroleum ether/
EtOAc 8:2) to afford 11 (64.5 mg, 47%) as a beige solid. Rf ¼ 0.41
(petroleum ether/EtOAc 8:2); mp 125e127 ꢀC; IR 1637 (C]O), 3354
1640 (C]O), 3350 (NH); 1H NMR (DMSO)
d
1.16 (d, 3H, J ¼ 6.8 Hz),
1.19e1.24 (m, 3H), 1.30e1.45 (m, 2H), 1.64e1.82 (m, 6H), 2.33 (s,
3H), 2.38 (s, 3H), 4.03e4.10 (m, 1H), 5.03 (s, 2H), 5.57 (d, 1H,
J ¼ 9.2 Hz, NH, exch. with D2O), 6.38 (d, 1H, J ¼ 1.6 Hz), 6.90 (d, 2H,
J ¼ 8.0 Hz), 7.04 (d,1H, J ¼ 8.4 Hz), 7.10 (d, 2H, J ¼ 8.0 Hz), 7.15 (d,1H,
(NH); 1H NMR (CDCl3)
d 1.67e1.73 (m, 7H), 2.08e2.12 (m, 8H), 2.32
J ¼ 1.6 Hz), 7.30 (d, 2H, J ¼ 8.0 Hz); 13C NMR (DMSO)
d 17.84 (CH3),
(s, 3H), 2.33 (s, 3H), 5.01 (s, 2H), 6.34 (s, 1H), 6.89 (d, 2H, J ¼ 8.0 Hz),
7.03 (d, 1H, J ¼ 8.0 Hz), 7.09 (d, 2H, J ¼ 8.0 Hz), 7.14 (s, 1H), 7.21 (br s,
1H, NH, exch. with D2O), 7.29 (d, 2H, J ¼ 8.0 Hz); 13C NMR (CDCl3)
19.51 (CH3), 20.59 (CH3), 25.75 (CH2 ꢃ2), 26.02 (CH2), 28.90 (CH2),
29.25 (CH2), 42.54 (CH), 48.20 (CH), 50.22 (CH2), 108.71 (CH), 119.92
(C), 125.98 (CH), 126.08 (CH ꢃ2), 127.16 (CH), 128.93 (CH), 129.13
(CH ꢃ2), 130.94 (CH), 131.19 (C), 132.15 (C), 132.75 (C), 135.11 (C),
135.63 (C), 136.56 (C), 162.52 (C]O); MS (ESI): C28H33ClN2O re-
quires m/z 449, found 450 [M þ 1]þ; Anal. calcd for C28H33ClN2O: C,
74.90; H, 7.41; Cl, 7.90; N, 6.24. Found: C, 74.72; H, 7.40; Cl, 7.88; N,
6.23.
d
20.07 (CH3), 21.08 (CH3), 29.56 (5 ꢃCH), 36.47 (CH2 ꢃ2), 41.95
(CH2 ꢃ2), 50.96 (CH2), 51.79 (CH2), 107.16 (CH), 121.05 (C), 125.08
(CH),126.66 (CH ꢃ2),127.60 (CH),129.49 (CH ꢃ2),130.80 (C),131.60
(CH), 133.87 (C), 134.39 (C), 135.90 (C), 136.17 (C), 137.50 (C), 163.76
(C]O); MS (ESI): C30H33ClN2O requires m/z 473, found 474
[M þ 1]þ; Anal. calcd for C30H35ClN2O: C, 75.88; H, 7.45; Cl 7.43; N,
5.93. Found: Anal. calcd for C30H33ClN2O: C,76.17; H, 7.03; Cl, 7.49;
N, 5.92. Found: C, 76.22; H, 7.09; Cl, 7.43; N, 5.90.
5.1.14. N-(S)-(þ)-Cyclohexylethyl-5-(4-chloro-3-methylphenyl)-1-
(4-methylbenzyl)-1H-pyrrole-3-carboxamide (15)
5.1.11. N-Adamantylmethane-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (12)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(S)-(þ)-cyclohexylethylamine into the title
product. The mixture was purified by flash chromatography (pe-
troleum ether/EtOAc 7:3) to afford 15 (100 mg, 77%) as a brown
solid. Rf ¼ 0.19 (petroleum ether/EtOAc 8:2); mp 205e207 ꢀC; IR
General procedure for the synthesis of carboxamides was used
to convert 5 and N-1-adamantylmethanamine into the title prod-
uct. The mixture was purified by flash chromatography (petroleum
ether/EtOAc 8:2) to afford 12 (90.4 mg, 64%) as a brown solid.
Rf ¼ 0.47 (petroleum ether/EtOAc 7:3); mp 78e81 ꢀC; IR 1633 (C]
1645 (C]O), 3340 (NH); 1H NMR (DMSO)
d 1.05e1.23 (m, 8H),
O), 3351 (NH); 1H NMR (CDCl3)
d 1.22e1.65 (m, 10H), 1.92e2.00 (m,
1.64e1.86 (m, 6H), 2.33 (s, 6H), 4.03e4.10 (m, 1H), 5.03 (s, 2H), 5.57
(d, 1H, J ¼ 9.2 Hz, NH, exch. with D2O), 6.38 (d, 1H, J ¼ 1.6 Hz), 6.91
(d, 2H, J ¼ 8.0 Hz), 6.98e7.20 (m, 4H), 7.21e7.33 (m, 2H); 13C NMR
4H) 2.32 (s, 3H), 2.34 (s, 3H), 3.10 (d, 1H, J ¼ 6.8 Hz), 5.03 (s, 2H),
5.81 (br s, 1H, NH, exch. with D2O), 6.40 (s, 1H), 6.91 (d, 2H,
J ¼ 7.6 Hz), 6.96e7.18 (m, 4H), 7.20e7.33 (m, 2H); 13C NMR (CDCl3)
(DMSO)
d
17.84 (CH3), 19.51 (CH3), 20.59 (CH3), 25.75 (CH2 ꢃ2),
26.02 (CH2), 28.90 (CH2), 29.25 (CH2), 42.54 (CH), 48.20 (CH), 50.22
(CH2), 108.71 (CH), 119.92 (C), 125.98 (CH), 126.08 (CH ꢃ2), 127.16
(CH),128.93 (CH),129.13 (CH ꢃ2),130.94 (CH), 131.19 (C), 132.30 (C),
132.75 (C), 135.10 (C), 135.63 (C), 136.56 (C), 162.52 (C]O); MS
(ESI): C28H33ClN2O requires m/z 449, found 450 [M þ 1]þ; Anal.
calcd for C28H33ClN2O: C, 74.90; H, 7.41; Cl, 7.90; N, 6.24. Found: C,
74.75; H, 7.40; Cl, 7.88; N, 6.23.
d
20.00 (CH3), 21.02 (CH3), 28.19 (CH2 ꢃ3), 33.97 (C), 36.89
(CH2 ꢃ3), 40.21 (CH ꢃ3), 50.62 (CH2), 50.91 (CH2), 106.96 (CH),
117.95 (C), 119.71 (CH), 125.37 (CH), 126.61 (CH ꢃ2), 127.51 (C),
129.00 (CH), 129.41 (CH ꢃ2), 130.77 (C), 131.52 (CH), 134.03 (C),
134.21 (C), 136.12 (C), 137.43 (C), 164.57 (C]O); MS (ESI):
C
31H35ClN2O requires m/z 487, found 488 [M þ 1]þ; Anal. calcd for
C31H35ClN2O: C, 76.44; H, 7.24; Cl, 7.28; N, 5.75. Found: C, 76.47; H,
7.26; Cl, 7.30; N, 5.79.
5.1.15. N-Cyclohexyl-5-(4-chloro-3-methylphenyl)-1-(4-
5.1.12. N-Menthyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (16)
methylbenzyl)-1H-pyrrole-3-carboxamide (13)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-cyclohexylamine into the title product. The
mixture was purified by flash chromatography (petroleum ether/
EtOAc 7:3) to afford 16 (79.3 mg, 65%) as a beige solid. Rf ¼ 0.35
(petroleum ether/EtOAc 7:3); mp 100e104 ꢀC; IR 1640 (C]O), 3360
General procedure for the synthesis of carboxamides was used
to convert 5 and N-menthylamine into the title product. The
mixture was purified by flash chromatography (petroleum ether/
EtOAc 8:2) to afford 13 (105 mg, 76%) as a brown solid. Rf ¼ 0.42
(petroleum ether/EtOAc 8:2); mp 98e102 ꢀC; IR 1635 (C]O), 3350
(NH); 1H NMR (CDCl3)
d 1.07e1.78 (m, 8H), 1.87e1.98 (m, 2H), 2.32
(NH); 1H NMR (CDCl3)
d
0.83 (d, 6H, J ¼ 7.6 Hz), 0.89 (d, 3H,
(s, 6H), 3.79e4.01 (m, 1H), 5.03 (s, 2H), 5.60 (d, 1H, NH, exch. with
D2O, J ¼ 8.0 Hz), 6.38 (d, 1H, J ¼ 2.0 Hz), 6.90 (d, 2H, J ¼ 7.8 Hz),
J ¼ 7.4 Hz), 0.98e1.25 (m, 2H), 1.45e1.79 (m, 5H), 1.83e2.18 (m, 2H),
2.33 (s, 6H), 3.90e4.10 (m, 1H), 5.03 (s, 2H), 5.41 (d, 1H, J ¼ 9.2 Hz,
NH, exch. with con D2O), 6.38 (s, 1H), 6.92 (d, 2H, J ¼ 7.4 Hz),
6.97e7.17 (m, 4H), 7.19e7.37 (m, 2H); 13C NMR (CDCl3)
d 19.79
(CH2), 20.05 (CH3), 21.07 (CH3), 25.00 (CH2), 25.65 (CH2), 30.92
(CH), 33.45 (CH2), 47.93 (CH2), 50.97 (CH2),107.22 (CH),120.03 (CH),
125.31 (CH), 126.66 (CH), 126.72 (C), 127.13 (CH), 129.08 (CH),
129.50 (CH), 130.77 (C), 130.91 (C), 131.60 (CH), 134.27 (C), 134.39
(CH), 135.45 (C), 136.19 (C), 137.52 (C), 163.63 (C]O); MS (ESI):
7.00e7.22 (m, 4H), 7.30 (d, 2H, J ¼ 8.0 Hz); 13C NMR (CDCl3)
d 16.25
(CH3), 20.03 (CH2), 21.18 (CH3 ꢃ2), 22.15 (CH2), 23.86 (CH2), 26.84
(CH), 31.84 (CH3), 34.55 (CH2), 43.35 (CH), 48.37 (CH2), 49.49 (CH),
50.92 (CH), 106.96 (CH), 118.02 (C), 119.84 (CH), 125.33 (CH), 126.67
(CH), 127.51 (CH ꢃ2), 129.01 (C), 129.43 (CH ꢃ2), 130.66 (C), 131.53
(CH), 134.27 (C), 134.96 (C), 136.12 (C), 137.47 (C), 163.63 (C]O); MS
(ESI): C30H37ClN2O requires m/z 477, found 478 [M þ 1]þ; Anal.
C
C
26H29ClN2O requires m/z 420, found 421 [M þ 1]þ; Anal. calcd for
26H29ClN2O: C, 74.18; H, 6.94; Cl, 8.42; N, 6.65. Found: C, 74.24; H,
7.01; Cl, 8.45; N, 6.69.

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
663
5.1.16. N-Piperidinyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (17)
(s, 6H), 2.67e2.90 (m, 4H), 5.03 (s, 2H), 6.11 (br s 1H, NH, exch. with
D2O), 6.37e6.42 (br s, 1H), 6.85e6.95 (m, 2H), 7.01e7.27 (m, 4H),
General procedure for the synthesis of carboxamides was used
to convert 5 and piperidine into the title product. The mixture was
purified by flash chromatography (petroleum ether/EtOAc 8:2) to
afford 17 (73.4 mg, 60%) as a brown solid. Rf ¼ 0.375 (petroleum
ether/EtOAc 6:4); mp 104e105 ꢀC; IR 1635 (C]O), 1703 (C]O),
7.50e7.67 (m, 2H); 13C NMR (CDCl3)
d 20.04 (CH3), 21.04 (CH3),
23.29 (CH2), 25.34 (CH2), 25.66 (CH2), 51.01 (CH2), 57.33 (CH2), 57.92
(CH2), 107.71 (CH), 118.17 (C), 126.68 (CH), 127.16 (CH), 127.59 (CH),
129.05 (CH), 129.47 (CH ꢃ2), 130.88 (CH), 130.97 (C), 131.59 (CH),
133.33 (C), 134.05 (C), 135.19 (C), 136.15 (C), 137.56 (C), 164.53 (C]
O); MS (ESI): C25H28ClN3O requires m/z 421, found 422 [M þ 1]þ;
Anal. calcd for C25H28ClN3O: C, 71.16; H, 6.69; Cl, 8.40; N, 9.96.
Found: C, 71.20; H, 6.73; Cl, 8.43; N, 9.94.
3350 (NH); 1H NMR (DMSO)
d 1.48e1.55 (m, 4H),1.58e1.64 (m, 2H),
2.24 (s, 3H), 2.30 (s, 3H), 3.55e3.60 (m, 4H), 5.18 (s, 2H), 6.36 (d, 1H,
J ¼ 2.0 Hz), 6.85 (d, 2H, J ¼ 7.6 Hz), 7.09 (d, 2H, J ¼ 7.6 Hz), 7.19 (d,
2H, J ¼ 8.0 Hz), 7.21e7.25 (m, 1H), 7.28 (s, 1H), 7.35e7.40 (m, 2H);
13C NMR (DMSO)
d
19.51 (CH3), 20.58 (CH3), 24.24 (CH2 ꢃ2), 25.83
5.1.20. N-Homopiperidinyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carbohydrazide (21)
(CH2), 50.11 (CH2 ꢃ3), 109.81 (CH), 118.17 (C), 126.21 (CH), 126.28
(CH ꢃ2), 126.46 (CH), 128.89 (CH), 129.10 (CH ꢃ2), 130.97 (C), 131.09
(CH), 133.33 (C), 134.36 (C), 135.14 (C), 135.61 (C), 136.52 (C), 164.53
(C]O); MS (ESI): C30H35ClN2O requires m/z 406, found 407
[M þ 1]þ; Anal. calcd for C25H27ClN2O: C, 73.79; H, 6.69; Cl, 8.71; N,
6.88. Found: C, 73.77; H, 6.68; Cl, 8.69; N, 6.86.
General procedure for the synthesis of carboxamides was used
to convert 5 and N-aminohomopiperidine into the title product.
The mixture was purified by flash chromatography (petroleum
ether/EtOAc 65:35) to afford 21 (50.6 mg, 40%) as a brown solid.
Rf ¼ 0.175 (petroleum ether/EtOAc 7:3); mp 220e224 ꢀC; IR 1669
(C]O), 3210 (NH); 1H NMR (CDCl3)
d 1.42e1.85 (m, 10H), 2.34 (s,
5.1.17. N-(4-(2-Chlorophenyl)piperazin-1-yl)-(5-(4-chloro-3-
6H), 3.02e3.22 (m, 2H), 5.03 (s, 2H), 6.38 (s 1H, NH, exch. with
D2O), 6.53 (s, 1H), 6.88e6.95 (m, 2H), 7.01e7.17 (m, 4H), 7.24e7.30
methylphenyl)-1-(4-methylbenzyl)-1H-pyrrole-3-carboxamide (18)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-(4-(2-chlorophenyl)piperazine) into the title
product. The mixture was purified by flash chromatography (pe-
troleum ether/EtOAc 7:3) to afford 18 (40 mg, 25%) as a brown solid.
Rf ¼ 0.16 (petroleum ether/EtOAc 7:3); mp 67e71 ꢀC; IR 1635 (C]
(m, 2H); 13C NMR (CDCl3)
d
19.64 (CH3), 20.66 (CH2 ꢃ2), 25.73
(CH3), 26.73 (CH2 ꢃ2), 50.47 (CH2), 57.80 (CH2 ꢃ2), 107.71 (CH),
118.17 (C), 126.27 (C), 126.35 (CH), 127.14 (CH), 128.65 (CH ꢃ2),
129.02 (CH ꢃ2), 131.05 (CH), 133.89 (CH), 134.07 (C), 135.62 (C ꢃ2),
136.13 (C), 137.54 (C), 166.36 (C]O); MS (ESI): C26H30ClN3O re-
quires m/z 435, found 436 [M þ 1]þ; Anal. calcd for C26H30ClN3O: C,
71.63; H, 6.94; Cl, 8.13; N, 9.64. Found: C, 71.60; H, 6.93; Cl, 8.13; N,
9.63.
O), 3350 (NH); 1H NMR (CDCl3)
d 2.32 (s, 3H), 2.33 (s, 3H), 3.06 (t,
4H, J ¼ 4.8 Hz), 3.95 (t, 4H, J ¼ 4.8 Hz), 5.03 (s, 2H), 6.39 (s, 1H), 6.91
(d, 2H, J ¼ 8.0 Hz), 7.01 (d, 2H, J ¼ 8.0 Hz), 7.05e7.11 (m, 4H),
7.17e7.22 (m, 2H), 7.25 (d,1H, J ¼ 8.0 Hz), 7.30 (d,1H, J ¼ 8.0 Hz); 13
C
NMR (CDCl3)
d
20.08 (CH3), 21.09 (CH3), 50.89 (CH2 ꢃ4), 51.58
5.1.21. N-(Naphthalen-1-yl)-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carboxamide (22)
(CH2), 109.53 (CH), 118.32 (C), 120.03 (CH), 124.16 (CH), 125.72 (CH),
126.72 (CH ꢃ2), 126.79 (CH), 128.94 (C), 129.10 (CH), 130.75
(CH ꢃ3), 131.64 (CH), 132.06 (CH), 133.88 (C ꢃ2), 133.90 (C), 136.23
(C), 137.55 (C), 148.87 (C), 166.32 (C]O); MS (ESI): C30H29Cl2N3O
requires m/z 517, found 518 [M þ 1]þ; Anal. calcd for C30H29Cl2N3O:
C, 69.50; H, 5.64; Cl, 13.68; N, 8.10. Found: C, 69.48; H, 5.62; Cl,
13.65; N, 8.08.
General procedure for the synthesis of carboxamides was used
to convert 5 and 1-naphtylamine into the title product. The mixture
was purified by flash chromatography (petroleum ether/EtOAc 7:3)
to afford 22 (93.8 mg, 70%) as a beige solid. Rf ¼ 0.54 (petroleum
ether/EtOAc 7:3); mp 170e172 ꢀC; IR 1640 (C]O), 3345 (NH); 1H
NMR (CDCl3)
d 2.32 (s, 3H), 2.35 (s, 3H), 5.03 (s, 2H), 6.62 (s, 1H),
6.91 (d, 1H, J ¼ 8.0 Hz), 7.10e7.12 (m, 3H), 7.19 (s, 1H), 7.32 (d, 1H,
J ¼ 8.0 Hz), 7.42e7.49 (m, 4H), 7.67 (d, 1H, J ¼ 8.0 Hz), 7.85 (d, 1H,
J ¼ 8.0 Hz), 7.91 (d, 1H, J ¼ 8.0 Hz), 7.95 (s, 1H), 7.99 (d, 1H,
5.1.18. N-Pyrrolidinyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carbohydrazide (19)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-aminopyrrolidine hydrochloride into the title
product. The mixture was purified by flash chromatography (pe-
troleum ether/EtOAc 3:7) to afford 19 (22 mg, 18%) as a brown solid.
Rf ¼ 0.375 (petroleum ether/EtOAc 3:7); mp 141e143 ꢀC; IR 1640
J ¼ 8.0 Hz); 13C NMR (CDCl3)
d 20.11 (CH3), 21.11 (CH3), 51.16 (CH2),
107.43 (CH), 119.72 (C), 120.86 (CH), 125.44 (CH), 125.87 (CH ꢃ2),
126.15 (CH), 126.20 (CH), 126.75 (CH ꢃ2), 126.82 (CH), 127.44 (CH),
127.74 (C), 128.74 (CH), 129.18 (CH), 129.58 (CH ꢃ2), 130.54 (C),
131.73 (CH), 132.74 (C), 134.13 (C), 134.15 (C), 134.17 (C), 134.97 (C),
136.33 (C), 137.68 (C), 163.14 (C]O); MS (ESI): C30H25ClN2O re-
quires m/z 464, found 465 [M þ 1]þ; Anal. calcd for C30H25ClN2O: C,
77.49; H, 5.42; Cl, 7.62; N, 6.02. Found: C, 74.24; H, 7.01; Cl, 8.45; N,
6.69.
(C]O), 3330 (NH); 1H NMR (CDCl3)
d 1.69e1.95 (m, 4H), 2.33 (s,
6H), 2.88e3.00 (m, 4H), 5.02 (s, 2H), 6.30e6.40 (br s 1H, NH, exch.
with D2O), 6.89e6.92 (m, 2H) 7.03e7.15 (m, 4H), 7.29e7.31 (m, 2H);
13C NMR (CDCl3)
d
20.20 (CH3), 21.32 (CH3), 23.12 (CH2 ꢃ2), 46.30
(CH2), 58.24 (CH2), 108.40 (CH), 112.29 (C), 123.45 (CH), 126.15 (CH),
127.32 (CH ꢃ2), 129.15 (CH ꢃ2), 129.67 (CH), 131.04 (C), 131.62 (C),
134.48 (C ꢃ2), 135.27 (C), 136.40 (C), 139.42 (C), 165.00 (C]O); MS
(ESI): C24H26ClN3O requires m/z 407, found 408 [M þ 1]þ; Anal.
calcd for C24H26ClN3O: C, 70.66; H, 6.42; Cl, 8.69; N, 10.30. Found: C,
70.71; H, 6.49; Cl, 8.72; N, 10.25.
5.1.22. Synthesis of 1-(5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-yl)methyl)-fenchylamine (7)
To a solution of carboxamide 6 (100 mg, 0.21 mmol, 1 eq) in
THFan (3 1 L) under N2 at 0 ꢀC, was portionwise added a solution of
2 M LiAlH4 in THFan (0.17 mL). The resulting solution was refluxed
for 12 h, then cooled to room temperature and added of 10% NaOH
(few drops). The resulting precipitate was removed under vacuum
and the filtrate extracted with EtOAc which was dried (Na2SO4) and
concentrated under reduced pressure to give derivative 7 as a
yellow solid (40 mg, 41%). Rf ¼ 0.18 (petroleum ether/EtOAc 7:3);
5.1.19. N-Piperidinyl-5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carbohydrazide (20)
General procedure for the synthesis of carboxamides was used
to convert 5 and N-aminopiperidine into the title product. The
mixture was purified by flash chromatography (petroleum ether/
EtOAc 6:4e4:6) to afford 20 (50.2 mg, 41%) as a beige solid. Rf ¼ 0.15
(petroleum ether/EtOAc 6:4); mp 82e84 ꢀC; IR 1649 (C]O), 3214
mp 108e111 ꢀC; IR 3390 (NH); 1H NMR (CDCl3)
d 0.96 (s, 3H),1.01 (s,
3H), 1.06 (s, 3H), 1.28e1.48 (m, 2H), 1.46e1.68 (m, 5H), 2.25e2.30
(br s, 1H, NH, exch. with D2O), 2.32 (s, 6H), 3.56 (d, 1H, J ¼ 13.2 Hz),
3.72 (d, 1H, J ¼ 12.8 Hz), 5.02 (d, 2H, J ¼ 8.8 Hz), 6.20 (s, 1H), 6.65 (s,
(NH); 1H NMR (CDCl3)
d 1.36e1.48 (m, 2H), 1.65e1.73 (m, 4H), 2.34

664
G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
1H), 6.91 (d, 2H, J ¼ 8.0 Hz), 7.02e7.12 (m, 3H), 7.15 (d, 2H, J ¼ 8.0);
13C NMR (CDCl3)
19.63 (CH3),19.71 (CH3), 21.02 (CH3), 25.98 (CH2),
5.1.27. 1-{[5-(4-Chloro-3-methylphenyl)-1-(4-methylbenzyl)-1H-
pyrrol-3-yl]methyl}-4-(3,4-dichlorophenyl)piperazine (26)
d
26.65 (CH2), 31.91 (CH3), 31.98 (CH3), 39.12 (CH2), 43.01 (CH2), 45.66
(CH2), 48.71 (C), 48.87 (CH), 50.57 (C), 70.95 (CH), 109.90 (CH),
123.13 (CH), 126.44 (CH ꢃ2), 127.03 (CH), 128.83 (CH), 128.98
(CH ꢃ2), 129.21 (CH), 133.33 (C), 134.41 (C), 135.35 (C ꢃ2), 135.68
(C), 137.16 (C ꢃ2); MS (ESI): C30H37ClN2 requires m/z 461, found 462
[M þ 1]þ; Anal. calcd for C30H37ClN2: C, 78.15; H, 8.09; Cl 7.69; N,
6.08. Found: C, 78.07; H, 8.08; Cl 7.68; N, 6.07.
General procedure for the synthesis of amines was used to
convert 24 and 1-(3,4-dichlorophenyl)piperazine into the title
product. The mixture was purified by flash chromatography (pe-
troleum ether/EtOAc 7:3) to afford 26 (80 mg, 47%) as a yellow
sticky solid. Rf ¼ 0.20 (petroleum ether/EtOAc 7:3); mp 170e172 ꢀC
(triturated with petroleum ether); IR 1640 (C]O), 3345 (NH); 1H
NMR (CDCl3)
d 2.32 (s, 6H), 2.60e2.65 (m, 4H), 3.19 (t, 4H,
J ¼ 9.6 Hz), 3.48 (s, 2H), 5.04 (s, 2H), 6.21 (s, 1H), 6.64 (s, 1H), 6.73
(dd, 1H, Jm ¼ 8.8 Hz, Jo ¼ 2.8 Hz), 6.90 (d, 1H, J ¼ 8.0 Hz), 6.94 (s, 1H),
5.1.23. Synthesis of [5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-yl]methanol (23)
7.06e7.15 (m, 3H), 7.18e7.28 (m, 3H); 13C NMR (CDCl3)
d 20.07
(CH3), 21.07 (CH3), 48.61 (CH2 ꢃ2), 50.46 (CH2), 52.48 (CH2 ꢃ2),
55.28 (CH2), 110.38 (CH), 115.19 (CH), 117.10 (CH), 119.49 (C), 122.74
(CH), 126.39 (CH), 126.41 (C), 126.55 (CH), 127.20 (CH ꢃ2), 128.97
(CH),129.39 (CH ꢃ2),130.38 (CH), 131.23 (CH),131.34 (C), 133.10 (C),
133.95 (C), 135.60 (C), 135.97 (C), 137.15 (C), 149.07 (C); MS (ESI):
To a solution of acid 5 (510 mg, 1.5 mmol, 1 eq) in anhydrous THF
(3 mL) under N2 at 0 ꢀC, was portionwise added a solution of 2 M
LiAlH4 in anhydrous THF (1.3 mL). The resulting solution was stirred
at room temperature for 4 h, then 10% NaOH was added. The pre-
cipitate was removed under vacuum and the filtrate extracted with
EtOAc, which dried (Na2SO4) and concentrated under reduced
pressure gave derivative 23 as yellow oil (400 mg, 82%). Rf ¼ 0.12
C
C
30H30Cl3N3 requires m/z 537, found 538 [M þ 1]þ; Anal. calcd for
30H30Cl3N3: C, 66.86; H, 5.61; Cl, 19.73; N, 7.80. Found: C, 66.85; H,
(petroleum ether/EtOAc 8:2); 1H NMR (CDCl3)
d 1.50e1.65 (br s, 1H,
5.60; Cl, 19.70; N, 7.79.
OH exch. with D2O), 2.32 (s, 6H), 4.56 (s, 2H), 5.01 (s, 2H), 6.25 (s,
1H), 6.71 (s, 1H), 6.91 (d, 2H, J ¼ 8.2 Hz), 7.0e7.16 (m, 4H), 7.28 (d,
1H, J ¼ 8.0 Hz).
5.1.28. Synthesis of 2-methyl-3-nitrobenzaldehyde (29) [20]
The compound was synthesized starting from a solution of
alcohol 28 [13] (1.10 g, 6.58 mmol, 1eq) in CH2Cl2 as reported in US
Patent 73850 [20]. The brown solid residue obtained after work up
was purified by flash chromatography (petroleum ether/EtOAc 8:2)
to obtain derivative 29 as a yellow solid (900 mg, 83.3%). Rf ¼ 0.28
5.1.24. Synthesis of 5-(4-chloro-3-methylphenyl)-1-(4-
methylbenzyl)-1H-pyrrole-3-carbaldehyde (24)
To a solution of alcohol 23 (400 mg, 1.22 mmol, 1eq) in CH2Cl2
(7 mL) was added MnO2 (10 eq) in small portions and the resulting
mixture was refluxed for 12 h. Then the solution was cooled to
room temperature and the catalyst removed by filtration on a bed
of celite®. The organic solution concentrate under reduced pressure
gave derivative 24 as yellow oil (310 mg, 82%). Rf ¼ 0.21 (petroleum
(petroleum ether/EtOAc 8:2); mp 54e57 ꢀC; 1H NMR (CDCl3)
d 2.78
(s, 3H), 7.53 (t, 1H, J ¼ 8.0 Hz), 7.98 (d, 1H, J ¼ 8.0 Hz), 8.06 (d, 1H,
J ¼ 8.0 Hz), 10.39 (s, 1H).
5.1.29. Synthesis of (E)-3-(2-methyl-3-nitrophenyl)propenoic acid
(30)
ether/EtOAc 8:2); 1H NMR (CDCl3)
d 2.34 (s, 6H), 5.06 (s, 2H), 6.66
To a mixture of aldehyde 29 (500 mg, 3 mmol, 1 eq) and malonic
acid (2.2 eq) in dry pyridine (11.5 mL) was added piperidine
(0.1 mL). The mixture was refluxed for 18 h, then was cooled to
room temperature and poured onto concd. HCl (8 mL) and ice. The
resulting precipitate was filtered, washed (5% aqueous HCl) and air
dried to give derivative 30 as a yellow solid (490 mg, 79%). Rf ¼ 0.04
(petroleum ether/EtOAc 7:3); mp 190e192 ꢀC; 1H NMR (CDCl3)
(d, 1H, J ¼ 1.8 Hz), 6.92 (d, 2H, J ¼ 7.8 Hz), 7.04e7.21 (m, 4H), 7.33 (d,
1H, J ¼ 8.4 Hz), 9.75 (s, 1H).
5.1.25. General procedure for the synthesis of amines 25e26
To a stirred solution of carbaldehyde 24 (1000 mg, 0.31 mmol,
1 eq) and the appropriate arylpiperazine (2 eq) in MeOH (5 mL),
AcOH was added until pH ¼ 5e6. The mixture was added of
NaCNBH4 (2 eq) at 0 ꢀC and the whole stirred at room temperature
for 5 (for 25) or 12 h (for 26). The solvent was removed under
reduce pressure and the resulting yellow oil dissolved in Et2O. The
organic solution was washed (H2O), dried (Na2SO4) and concen-
trated under reduced pressure. The analytically pure product was
isolated by flash chromatography purification.
d
2.54 (s, 3H), 6.40 (d, 1H, J ¼ 16.0 Hz), 7.38 (t, 2H, J ¼ 8.0 Hz), 7.77 (t,
1H, J ¼ 8.8 Hz), 8.08 (d, 1H, J ¼ 16.0 Hz).
5.1.30. Synthesis of 3-(3-amino-2-methylphenyl)propanoic acid
(31)
To a suspension of acid 30 (800 mg, 3.86 mmol, 1 eq) in EtOHabs
(7.2 mL) was added Pd/C-10% (0.1 eq) and the mixture was hy-
drogenated at 30 psi for 12 h at room temperature. Then the
catalyst was removed by filtration on bed of celite® and the filtrate
concentrated under reduce pressure to yield 31 as a brown solid
(635 mg, 93%). Rf ¼ 0.13 (petroleum ether/EtOAc 1:1); mp
5.1.26. 1-{[5-(4-Chloro-3-methylphenyl)-1-(4-methylbenzyl)-1H-
pyrrol-3-yl]methyl}-4-(4-chlorophenyl)piperazine (25)
General procedure for the synthesis of amines was used to
convert 24 and 1-(4-chlorophenyl)piperazine into the title product.
The mixture was purified by flash chromatography (CHCl3//MeOH
98:2) to afford 25 (60 mg, 38%) as a white solid. Rf ¼ 0.15 (petro-
158e161 ꢀC; 1H NMR (CDCl3)
2.70e2.85 (br s, 2H, NH2, exch. with D2O), 2.91 (t, 2H, J ¼ 8.8 Hz),
6.60 (d, 1H, J ¼ 8.0 Hz), 6.97 (s, 1H), 7.28 (s, 1H), 11.10 (s, 1H, OH,
exch. with D2O).
d
2.12 (s, 3H), 2.59 (t, 2H, J ¼ 8.8 Hz),
leum ether/EtOAc 1:1); mp 99e102 ꢀC; 1H NMR (CDCl3)
d 2.32 (s,
6H), 2.60e2.67 (m, 4H), 3.17 (t, 4H, J ¼ 9.6 Hz), 3.48 (s, 2H), 5.04 (s,
2H), 6.21 (s, 1H), 6.63 (s, 1H), 6.82 (d, 2H, 9.2 Hz), 7.02e7.13 (m, 3H),
5.1.31. Synthesis of 3-(3-chloro-2-methylphenyl)propanoic acid
(32)
7.15e7.23 (m, 3H), 7.24e7.25 (m, 1H); 13C NMR (CDCl3)
d 20.09
(CH3), 21.09 (CH3), 49.12 (CH2 ꢃ2), 50.46 (CH2), 52.70 (CH2 ꢃ2),
55.39 (CH2), 110.45 (CH), 117.15 (CH ꢃ3), 119.49 (C), 124.31 (CH),
126.41 (C), 126.56 (CH), 128.25 (CH), 129.14 (CH ꢃ3), 129.33
(CH ꢃ2), 129.40 (CH ꢃ2), 131.24 (C), 133.08 (C), 133.94 (C), 135.62
(C), 135.98 (C), 137.12 (C), 150.05 (C); MS (ESI): C30H31Cl2N3 requires
m/z 503, found 504 [M þ 1]þ; Anal. calcd for C30H31Cl2N3: C, 71.42;
H, 6.19; Cl, 14.05; N, 8.33. Found: C, 71.44; H, 6.20; Cl, 14.03; N, 8.32.
To a solution of acid 31 (500 mg, 2.79 mmol, 1eq) in conc. HCl
(5.3 mL) cooled at ꢁ5 ꢀC was dropwise added an aqueous solution
(5 mL) of NaNO2 (2.6 eq) and CuCl (1.25 eq). The mixture was stirred
at room temperature for 12 h, then slowly added of 10% NaOH until
pH 5e6. The precipitate was removed under vacuum and the
filtrate extracted with Et2O. The organic solution dried (Na2SO4)
and concentrated under reduced pressure to furnish a brown

G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
665
residue, whose flash chromatography purification (petroleum
5.1.36. Synthesis of ethyl 6-chloro-2,5-dimethyl-1-(4-
ether/EtOAc 4:6) gave derivative 32 as a yellow solid (349 mg, 63%).
methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxylate (37)
To a solution of tricyclic-ester 36 (405 mg, 1.40 mmol, 1 eq) in
anhydrous DMF (4.8 mL) under N2, was portionwise added 60%
NaH in mineral oil (1.2 eq). The solution was stirred at room tem-
perature for 15e20 min, then a solution of 4-methyl-benzyl chlo-
ride (1 eq) in anhydrous THF (1.5 mL) was added dropwise: the
resulting mixture was stirred at room temperature for 12 h. The
solution was poured in H2O (8.5 mL) and extracted with CH2Cl2,
which was washed (H2O), dried (Na2SO4) and concentrated under
reduced pressure to furnish an oily dark residue, whose flash
chromatography purification (petroleum ether/EtOAc 95:5) gave
derivative 37 as a purple solid (480 mg, 87%). Rf ¼ 0.21 (petroleum
Rf ¼ 0.20 (petroleum ether/EtOAc 4:6); mp 121e124 ꢀC; 1H NMR
(CDCl3)
d
2.29 (s, 3H), 2.65 (t, 2H, J ¼ 7.8 Hz), 3.02 (t, 2H, J ¼ 8.0 Hz),
6.81 (d, 1H, J ¼ 9.0 Hz), 7.06 (d, 1H, J ¼ 4.6 Hz), 7.91 (d, 1H,
J ¼ 8.8 Hz), 11.09 (s, 1H, OH, exch. with D2O).
5.1.32. Synthesis of 4-methy-5-chloro-indan-1-one (33)
A solution of acid 32 (2 g, 10 mmol) in CF3SO3H (10 mL) was
stirred at room temperature for 5 h. Then crushed ice was slowly
added and the solution extracted with Et2O. The organic layer
washed with 10% NaHCO3, H2O and brine, dried (Na2SO4) and
concentrated under reduced pressure to give a yellow solid, whose
flash chromatography purification (petroleum ether/EtOAc 8:2)
afforded derivative 33 as a pallid yellow solid (720 mg, 40%).
Rf ¼ 0.23 (petroleum ether/EtOAc 8/2); mp 100e102 ꢀC; 1H NMR
ether/EtOAc 8:2); 1H NMR (CDCl3)
d
1.40 (t, 3H, J ¼ 7.2 Hz), 2.32 (s,
3H), 2.47 (s, 3H), 2.61 (s, 3H), 3.64 (s, 2H), 4.36 (d, 2H, J ¼ 7.2 Hz),
5.18 (s, 2H), 6.97 (d, 2H, J ¼ 8.4 Hz), 7.02 (d, 1H, J ¼ 7.6 Hz), 7.15 (d,
2H, J ¼ 8.0 Hz), 7.25e7.28 (m, 1H).
(CDCl3)
d
2.38 (s, 3H), 2.72 (t, 2H, J ¼ 5.2 Hz), 3.06 (t, 2H, J ¼ 5.2 Hz),
7.38 (d, 1H, J ¼ 8.2 Hz), 7.53 (d, 1H, J ¼ 8.4 Hz).
5.1.37. Synthesis of 6-chloro-2,5-dimethyl-1-(4-methylbenzyl)-1,4-
dihydroindeno[1,2-b]pyrrole-3-carboxylic acid (38)
A solution of indenopyrrole-ester 37 (470 mg, 1.2 mmol) in a 10%
hydro-alcoholic NaOH solution (11.5 mL, 60% EtOH) was refluxed
6 h. The solution was cooled at room temperature and acidified
with 37% HCl. The resulting precipitate was filtered, washed (H2O)
and air-dried to give the acid 38 as an orange-red solid (270 mg,
65%). Rf ¼ 0.10 (petroleum ether/EtOAc 7:3); mp 193e196 ꢀC; 1H
5.1.33. Synthesis of 2-bromo-4-methy-5-chloro-indan-1-one (34)
To a solution of indanone 33 (1 g, 5.5 mmol, 1 eq) in AcOH
(4 mL), Br2 (1 eq) was dropwise added and the mixture stirred at
room temperature for 4 h. Then H2O (3 mL) was poured into re-
action flask, and the resulting precipitate filtered under vacuum,
washed (H2O) and air dried to furnish a yellow solid whose puri-
fication by flash chromatography (petroleum ether/EtOAc 95:5)
gave derivative 34 as a white solid (850 mg, 60%). Rf ¼ 0.12 (pe-
NMR (CDCl3) d 2.10 (br s, 1H, OH, exch. with D2O), 2.32 (s, 3H), 2.46
(s, 3H), 2.61 (s, 3H), 3.63 (s, 2H), 5.20 (s, 2H), 6.96 (d, 2H, J ¼ 8.2 Hz),
7.03 (d, 1H, J ¼ 7.6 Hz), 7.15 (d, 2H, J ¼ 8.0 Hz), 7.25e7.28 (m, 1H).
troleum ether/EtOAc 95:5); mp 131e134 ꢀC;
d 2.37 (s, 3H), 3.33 (d,
1H, J ¼ 15.6 Hz), 3.76 (dd, 1H, Jm ¼ 18 Hz, Jo ¼ 7.2 Hz), 6.66 (d, 1H,
5.1.38. Synthesis of N-(1)-(S)-fenchyl-6-chloro-2,5-dimethyl-1-(4-
methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide
(39)
J ¼ 7.6 Hz), 7.45 (d, 1H, J ¼ 8.4 Hz), 7.62 (d, 1H, J ¼ 8.0 Hz).
General procedure for the synthesis of carboxamides was used
to convert acid 38 and N-(1)-(S)-fenchylamine into the title prod-
uct. The mixture was refluxed for 4 h and purification by flash
chromatography (petroleum ether/EtOAc 85:15) afforded 39
(51 mg, 35%) as a red solid. Rf ¼ 0.10 (petroleum ether/EtOAc 85:15);
5.1.34. Synthesis of ethyl 2-(5-chloro-4-methyl-1-oxo-1H-indan-2-
yl)-3-oxobutanoate (35)
A solution of ethyl-acetoacetate (1.2 eq) in anhydrous THF
(1 mL) cooled to 0 ꢀC was slowly added 60% NaH in mineral oil
(3 eq) and the suspension was stirred at room temperature for
20 min, under N2. Then, a solution of bromo-indanone 34 (850 mg,
3.27 mmol, 1 eq) in anhydrous THF (4 mL) was dropwise added and
the mixture was stirred at room temperature for 24 h H2O was
added to the solution and extracted with Et2O. The organic phase
dried (Na2SO4) and concentrated at reduced pressure to give an oily
residue, which was purified by gradient flash chromatography
(petroleum ether/EtOAc 9:1e8:2) to give derivative 35 as a yellow
oil (650 mg, 65%); Rf ¼ 0.21 (petroleum ether/EtOAc 8:2); 1H NMR
(CDCl3) 1.34 (t, 3H, J ¼ 6.8 Hz), 2.30 (s, 3H), 2.42 (s, 3H), 2.88e3.00
(m, 1H), 3.15e3.40 (8m, 2H), 4.00e4.15 (m, 2H), 4.1 (q, 2H,
J ¼ 6.8 Hz), 7.38 (d, 1H, J ¼ 8.0 Hz), 7.54 (d, 1H, J ¼ 8.0 Hz).
mp 192e193 ꢀC; IR 1633 (C]O), 3354 (NH); 1H NMR (CDCl3)
d 0.89
(s, 3H), 1.09 (s, 3H), 1.16 (s, 3H), 1.20e1.28 (m, 3H), 1.65e2.00 (m,
6H), 2.33 (s, 3H). 2.57 (s, 3H), 2.61 (s, 3H), 3.88 (d, 1H, J ¼ 7.8 Hz),
5.15 (s, 2H), 6.55 (d, 1H, J ¼ 7.8 Hz), 6.99 (d, 2H, J ¼ 7.8 Hz), 7.07 (d,
1H, J ¼ 7.8 Hz), 7.16 (d, 2H, J ¼ 7.8 Hz), 8.17 (d, 1H, NH, exch. with
D2O); 13C NMR (CDCl3)
d 10.88 (CH3), 13.52 (CH3), 19.86 (CH3), 21.07
(CH3), 21.55 (CH3), 26.19 (CH2), 27.29 (CH2), 31.18 (CH3), 39.38 (C),
42.93 (CH2), 48.39 (CH2), 48.46 (CH), 48.60 (C ꢃ2), 63.79 (CH),
113.57 (C), 115.39 (CH), 119.99 (C), 125.85 (CH ꢃ2), 129.92 (CH ꢃ2),
131.93 (C), 132.00 (CH), 134.40 (C), 135.85 (C), 136.29 (C), 137.84 (C),
138.05 (C), 142.11 (C), 148.49 (C); MS (ESI): C32H37ClN2O requires m/
z 501, found 502 [M þ 1]þ; Anal. calcd for C32H37ClN2O: C, 76.70; H,
7.44; Cl 7.08; N, 5.59. Found: C, 76.68; H, 7.43; Cl 7.07; N, 5.57.
5.1.35. Synthesis of ethyl 6-chloro-2,5-dimethyl-1,4-dihydroindeno
[1,2-b]pyrrole-3-carboxylate (36)
5.2. Radioligand binding assays for CB1 and CB2 receptors
To a solution of keto-ester 35 (700 mg, 2.27 mmol, 1 eq) in
toluene (4.5 mL) were added NH4OAc (2.5 eq) and SiO2 (0.2 eq) and
the mixture of reaction was subjected to mW irradiation at 110 ꢀC
for 2.5 h. Then the suspension was filtered under vacuum, and the
residue washed with EtOAc. The filtrate was concentrated under
reduced pressure to furnish a dark solid whose purification by flash
chromatography (petroleum ether/EtOAc 8:2) gave derivative 36 as
a brown solid (400 mg, 61%); Rf ¼ 0.15 (petroleum ether/AcOEt
CB1R/CB2R binding studies were performed using membrane
fractions of human CB1R/CB2R transfected cells purchased from
PerkineElmer Life and Analytical Sciences (Boston, MA).
HEK293EBNA membranes were resuspended in Tris buffer (50 mM
TriseHCl, 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
8:2); mp ¼ 152e155 ꢀC; 1H NMR (CDCl3)
d
1.40 (t, 3H, J ¼ 7.2 Hz),
2.42 (s, 3H), 2.65 (s, 3H), 3.58 (s, 2H), 4.33 (d, 2H, J ¼ 7.2 Hz), 7.02 (d,
1H, J ¼ 7.6 Hz), 7.25e7.28 (m, 1H), 8.32 (br s, 1H, NH, exch. with
D2O).

666
G. Ragusa et al. / European Journal of Medicinal Chemistry 101 (2015) 651e667
for CB1 and 0.6 mL for CB2 of assay buffer (50 mM TriseHCl, 2.5 mM
EDTA, 5 mM MgCl2, 0.5 mg/mL BSA fatty acid free, pH 7.4).
used at a concentration 10-fold the Ki obtained in binding studies.
10 M WIN55,212-2 (Sigma) and 10 M SR144528 (Santa Cruz
m
m
Nonspecific binding was determined in the presence of 10
m
M
Biotechnology) were used as reference compounds because of their
capability to either activate or block the CB2R, respectively. Media
were then removed and used for the determination of PG-E2
release using the ELISA kit DetectX® Prostaglandin E2 (Arbor
Assays).
WIN55,212-2. Silanized tubes were used throughout the experi-
ment to minimize receptor binding loss due to tube adsorption. The
reaction was terminated by rapid vacuum filtration with a filter
mate Harvester apparatus (PerkineElmer) through Filtermat A GF/C
filters presoaked in 0.05% polyethylenimine (PEI). The filters were
washed nine times with ice-cold buffer for CB1 (50 mM TriseHCl,
2.5 mM EDTA, 5 mM MgCl2, 0.5 mg/mL BSA fatty acid free, pH 7.4)
for CB1 and CB2 (50 mM TriseHCl, 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
MicroBeta TriLux (PerkineElmer). The binding assay showed the
appropriate sensitivity to CB1 and CB2 ligands. Thus, WIN55,212-2
inhibited the binding with a Ki value of 36.2 nM (CB1R) and
WIN55,212-2 and HU-308 inhibited the binding with Ki values of
3.7 and 11.2 nM (CB2R), respectively. For all binding experiments,
competition binding curves were analysed by using an iterative
curve-fitting procedure GraphPad Prism version 5.02 (GraphPad
Software Inc., San Diego, CA, USA) and Ki values are expressed as
mean SEM of at least three experiments performed in triplicate
for each point.
5.5. Molecular modelling
5.5.1. Conformational analysis
Complete conformational analyses of compounds listed in
Table 1 were performed by first using the semi-empirical RM1
forcefield to conduct conformational search calculations and then
optimizing resulting unique conformers with ab initio Har-
treeeFock calculations at the 6-31G* level as encoded in Spartan '08
(Wavefunction, Inc., Irvine, CA). In each conformer search, local
energy minima were identified by rotation of a subject torsion
angle through 360ꢀ in 45ꢀ increments (8-fold search), followed by
semi-empirical RM1 energy minimization of each rotamer gener-
ated. Duplicate conformations were removed and HF 6-31G* en-
ergy minimizations of each unique conformer were performed. To
calculate the energy difference between the global minimal energy
conformer of each compound and its final docked conformation,
the single point energy of each was calculated in OPLS 2005 and
difference was calculated.
5.3. [35S]-GTP
g
S binding analysis
[
35S]-GTP
gS binding analyses were carried out for compounds 6
and 10 using CB2R-containing membranes (HTS020M2, Eurofins
Discovery Services). To this end, membranes (5 g/well) were
permeabilized by addition of saponin (SigmaeAldrich), then mixed
with 0.3 nM [35S]-GTP
S (PerkineElmer) and 10 M GDP (Sigma-
m
5.5.2. Unique volume map calculation
g
m
To probe the steric limits of the CB2 binding pocket, we used a
modification of the Active Analogue Approach [17]. Here, we
calculated that volume of space occupied by poor affinity
(Ki > 5000 nM) analogues in Table 1 that was not occupied by those
analogues in Table 1 with higher affinities (Ki < 5000 nM). All
conformers within 5.00 kcal/mol of the global minimum conformer
were considered to be accessible conformers. These conformers
were then superimposed at their central five membered ring. Using
eAldrich) in 20 mM HEPES (SigmaeAldrich) buffer containing
100 mM NaCl (Merck) and 10 mM MgCl2 (Merck), at pH 7.4. 30 nM
CP55,940 (SigmaeAldrich) and increasing concentrations of com-
pound 6 or 10 (from 10ꢁ11 to 10ꢁ5 M) were added in a final volume
of 100
was measured with 10
m
l and incubated for 30 min at 30 ꢀC. The non-specific signal
M GTP S (SigmaeAldrich). All 96-well
m
g
plates and the tubes necessary for the experiment were previ-
ously silanized with Sigmacote (SigmaeAldrich). The reaction was
terminated by rapid vacuum filtration with a filter mate Harvester
apparatus (PerkineElmer) through Filtermat A GF/C filters. The
filters were washed nine times with ice-cold filtration buffer
(10 mM sodium phosphate, pH 7.4), and bound radioactivity was
measured with a 1450 LSC & Luminiscence counter Wallac
€
the Surface facilities within Maestro 9.8 (Schrodinger Inc.), a den-
sity of 3.33 points per Å, and a probe radius of 1.4 Å, the UNION of
Van der Waals (VdW's) volume maps of each of the conformers
identified belonging to the binding group was calculated. The
UNION of the VdW's volume maps of the non-binding group was
separately calculated. Using a logical NOT operation, the region of
space that the conformers of the non-binding group did not share
with that of the binding group was then calculated.
MicroBeta TriLux (PerkineElmer). [35S]-GTP
gS binding data were
analysed to determine the IC50 values by using an iterative curve-
fitting procedure with the GraphPad Prism version 5.02 (GraphPad
Software Inc.). IC50 values are expressed as mean SEM of at least
three experiments performed in triplicate for each point.
5.5.3. Docking study in CB2R
Compound 10 was docked into the SR144528 CB2R binding site
previously identified [16] by Glide docking studies. This CB2 inac-
tive state receptor model was pre-equilibrated in a stearoyl-
docosahexaenoylphosphatidylcholine (SDPC) bilayer for 300 ns to
allow it to adjust to a lipid environment [16]. The selected
conformer was docked using Glide 6.6 and the dock with the best
Glide score modified by the strain energy (relative to the global
min) was chosen. Glide was used to generate a grid based on the
centroid of select residues in the binding pocket. The box size was
set to the default value of 14 ꢃ 14 ꢃ 14 Å, with the inner box size
was set to 10 ꢃ 10 ꢃ 10 Å. This default box size encompasses the
entire CB2R binding pocket both in width and depth. Standard
precision (SP) and flexible docking with ring sampling were
selected for the docking setup. Only trans amides were allowed.
After docking with ring sampling, a 500 step conjugate gradient
minimization was performed by Glide (dielectric ¼ 1).
5.4. Determination OF CB2 receptor-mediated functional activity in
a cultured cell-based bioassay
The functional activity of the new compounds for CB2R was also
evaluated in cultured BV-2 cells, a mouse microglial cell line. Cells
were plated at a density of 5 ꢃ 105 cells per well in 12-well culture
plates previously coated with 15 mg/ml Poly-L-ornithine (Sigma),
and incubated overnight in Dulbecco's Modified Eagle's Medium
(DMEM, Lonza) supplemented with 10% foetal bovine serum (FBS,
Lonza), 2 mM Ultraglutamine and antibiotics (Lonza) in a humidi-
fied atmosphere of 5% CO2 at 37 ꢀC. One hour before treatment,
medium was replaced with DMEM supplemented with 1% FBS,
2 mM Ultraglutamine and antibiotics. Cells were treated for 16 h
with 1
mg/ml Lipopolysaccharides (LPS from Escherichia coli 055:B5,
Sigma), alone or in combination with the investigated compound,

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Conflict of interest
None of the authors have a conflict of interest to declare.
Acknowledgements
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GM acknowledges Regione Autonoma della Sardegna for eco-
nomic support (grant n. CRP-26417, LR n. 7/2007 and INNOVA.RE-
POR FESR 2007-2013). PM is recipient of a CSIC fellowship JAE-
ꢀ
ꢀ
ꢀ
Pre-2010-01119 from Junta para la Ampliacion de Estudios co-
financed by FSE. MGA was recipient of a postdoctoral fellowship
from the PICATA Program, CEI-Moncloa. NJ thanks the Spanish
Ministry of Economy and Competitivity for the grant SAF2012-
40075, and with JFR the “Programa de Biomedicina, Comunidad de
Madrid” (S2011/BMD-2308).
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Appendix A. Supplementary data
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D. Oustric, M. Sarran, M. Bouaboula, B. Calandra, M. Portier, D. Shire, J. Breliere,
G. Le Fur, SR 144528, the first potent and selective antagonist of the CB2,
Cannabinoid Recept. 284 (1998) 644e650.
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dx.doi.org/10.1016/j.ejmech.2015.06.057.
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hydrogen bonding and aromatic stacking to the affinity and efficacy of
cannabinoid receptor CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-
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