Tricyclic pyrazoles part 7. Discovery of potent and selective dihydrothienocyclopentapyrazole derived CB2 ligands

Article abstract of DOI:10.1016/j.ejmech.2014.08.042

A series of dihydrothienocyclopentapyrazole-based derivatives was synthesized and evaluated for the affinity at CB1 and CB2 receptors. The major term, the 6-methyl-1-(1,4-dichlorophenyl)-N-piperidinyl)-1,4- dihydrothieno[2′,3′-4,5]cyclopenta[1,2-c]pyrazole-3-carboxamide (6a), displayed a high affinity and good selectivity for CB2 receptors (Ki values of 2.30 nM for CB2 receptor and 440 nM for CB1 receptors respectively). Subsequent analogue preparation resulted in the identification of compounds such as 6b, 6d, 6e, 6k, 6l, 6m, 6s and 6t that showed 1.3-485 fold selectivity for CB2 receptors with potencies in the 1.1-7.2 nM range. These compounds profiled as full agonists at CB2 receptor in an inhibition assay of P-ERK 1/2 up regulation in HL-60 cells.

Full text of DOI:10.1016/j.ejmech.2014.08.042

European Journal of Medicinal Chemistry 85 (2014) 747e757
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tricyclic pyrazoles part 7. Discovery of potent and selective
dihydrothienocyclopentapyrazole derived CB2 ligands
,
, c
Giansalvo Pinna a *, Maria Michela Curzu a, Antonio Dore a, Paolo Lazzari b
,
d
a
a
a
ꢀ
Stefania Ruiu , Amedeo Pau , Gabriele Murineddu , Gerard A. Pinna
Dipartimento di Chimica e Farmacia, Universita degli Studi di Sassari, Via F. Muroni 23/A, 07100 Sassari, Italy
a
ꢁ
b
ꢁ
PharmaNess S.c.a r.l., Edificio 5, Localita Piscinamanna, 09010 Pula, CA, Italy
c KemoTech Srl, Edificio 3, Loc. Piscinamanna, 09010 Pula, CA, Italy
d
ꢁ
CNR, Istituto di Farmacologia Traslazionale, UOS di Cagliari, Edificio 5, Localita Piscinamanna, 09010 Pula, CA, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihydrothienocyclopentapyrazole-based derivatives was synthesized and evaluated for the
affinity at CB1 and CB2 receptors. The major term, the 6-methyl-1-(1,4-dichlorophenyl)-N-piperidinyl)-
1,4-dihydrothieno[20,30-4,5]cyclopenta[1,2-c]pyrazole-3-carboxamide (6a), displayed a high affinity and
good selectivity for CB2 receptors (Ki values of 2.30 nM for CB2 receptor and 440 nM for CB1 receptors
respectively). Subsequent analogue preparation resulted in the identification of compounds such as 6b,
6d, 6e, 6k, 6l, 6m, 6s and 6t that showed 1.3e485 fold selectivity for CB2 receptors with potencies in the
1.1e7.2 nM range. These compounds profiled as full agonists at CB2 receptor in an inhibition assay of P-
ERK 1/2 up regulation in HL-60 cells.
Received 16 April 2014
Received in revised form
29 July 2014
Accepted 12 August 2014
Available online 13 August 2014
Keywords:
Cannabinoid receptors
Dihydrothienocyclopentapyrazole
CB2 agonists
© 2014 Elsevier Masson SAS. All rights reserved.
Amides
Thiophene tricycles
1. Introduction
In recent years drug discovery organizations have taken an
increased interest in CB2 ligands to develop potential drugs for the
The cannabinoid subtype 2 receptor (CB2), member of the su-
perfamily of G protein-coupled receptors, is expressed at high
density on immune cells [1]. CB2 receptors are also expressed in
peripheral tissues and organs, i.e. spleen, tonsils, and pancreas [2].
Moreover, the presence of this cannabinoid receptor subtype has
also been evidenced in rat microglia cells, human perivascular
microglial cells, and neuritic plaques-associated microglial cells in
brain humans of patients affected by Alzheimer's disease [3].
The CB2 receptor together with CB1 receptor [4e6], endogenous
ligands (anandamide-AEA [7] and 2-arachidonoylglycerol-2AG
[8,9]), anabolic and catabolic enzymes [10e12] and
endocannabinoid-binding protein and transporters [13,14] are
components of the physiological endocannabinoid system.
This signaling system plays a crucial role in numerous physio-
logical and pathological functions and it is involved in maintaining
the physiological steady state and homeostasis [15].
treatment of multiple diseases such as cancer [16,17], multiple
sclerosis [18e20], Alzheimer's disease [3], neuropathic and in-
flammatory pain [3,18e22], diabetes [23] and autoimmune uveor-
etinitis [24].
As already described in preceding publications [25e27], our
search for CB2 receptor ligands started by screening condensed
tricyclic pyrazole compounds as conformational restricted ana-
logues of SR144528 (1) [28], the CB2 selective congener of the CB1
antagonist/inverse agonist compound SR141716A (Rimonabant, 2)
[29] (See Fig. 1).
These efforts provided three main lead derivatives: 6-methyl-1-
(20,40-dichlorophenyl)-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]
pyrazole-3-carboxamide (3) [25], the corresponding 6-chlorine
derivative (4) [25], and 6-chloro-7-methyl-1-(20,40-dichlor-
ophenyl)-N-fenchyl-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxamide (5) [27]. Particularly, compounds 3 and 4 exhibited
excellent affinity and selectivity to CB2 receptors (affinity
expressed as Ki in the order of 0.037e0.34 nM; selectivity towards
CB1 receptors up to 9810) (See Fig. 2).
According to widest structure activity relationship (SAR) studies
carried out in our laboratories based on three main series
* Corresponding author.
E-mail addresses: gsvpinna@gmail.com, pinlab@uniss.it (G. Pinna).
http://dx.doi.org/10.1016/j.ejmech.2014.08.042
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

748
G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
O
N
O
N
N
NH
NH
N
N
Cl
Cl
Cl
Cl
2
SR141716A (Rimonabant)
Ki CB2 = 514 nM
1
SR144528
Ki CB2 = 0.6 nM
Ki CB1 = 400 nM
Ki CB1 = 1.8 nM
Ki CB1 / Ki CB2 (selectivity ratio) = 0.0035:1
Ki CB1 / Ki CB2 (selectivity ratio) = 666:1
Fig. 1. Structures and cannabinoid receptor affinities of reference compounds 1 and 2.
characterized respectively by five, six, and seven carbon atoms in
the central carbocyclic ring of the condensed tricyclic pyrazole
compounds, the planar dihydroindenopyrazole ring system
appeared as important feature to assure CB2 affinity. In contrast,
the enlargement of the size of the central ring in the condensed
tricyclic scaffold determined a shifting to CB1 affinity and selec-
tivity [30e32]. This behavior was further supported by the results
of other groups, although with marked differences in absolute
values of Ki CB1 for some of the lead compounds [33,34].
To further investigate the relevance of these condensed tricyclic
compounds in the development of novel CB2 ligands with
improved pharmacological profiles, we have recently oriented our
focus on the replacing the benzene ring of the tricyclic inden-
opyrazole scaffold in compounds 3e5 with a ring-fused thiophene
moiety to obtain a novel dihydrothienocyclopentapyrazole scaffold
as illustrated in Fig. 3.
values to cannabinoid receptors, and preliminary data concerning
their intrinsic activity.
2. Chemistry
According to the retrosynthetic analysis in Scheme 1, the target
dihydrothienocyclopentapyrazoles 6aet could be prepared using a
multistep route starting from substituted thienoindanones 7 [35]
and 8. Thus, methylthienoindanones were transformed to ethyl
thienoindanonoxoacetate intermediates 9, 10 using a Claisen re-
action and subsequent condensation with an aromatic/methylar-
ylhydrazine afforded the tricyclic dihydrocyclopentapyrazoles
11e14. Final elaboration to the designed compounds 6 was
accomplished by functional interconversion of the esters 11e14 via
acids 15e18 to the amides/hydrazides.
Preparation of the 3-methyl-thienoindan-4-one 8 started with
3-methyl-2-thiophen-carbaldehyde 19 and malonic acid that gave
the propenoic acid 20 [36], that was converted to the correspond-
ing propanoic acid 21 [36] and cyclized to the desired ketone under
poliphosphoric acid conditions in 40% yield (Scheme 2).
It was postulated that bioisosteric approach used in this case
could be a rational and efficient strategy to develop novel thieno-
cyclopentapyrazole CB2 selective ligands and probably a more
detailed insight into CB2 subtype-specific receptor pharmacology
might by expected.
Claisen reaction of ketones 7,8 with diethyl oxalate and sodium
ethoxide provided the diketoesters 9,10 in good yields. Subsequent
cyclization with 2,4-dichlorophenylhydrazine hydrochloride or p-
In this paper we report the synthesis of thiophene based ana-
logues of 3e5 with the general structure 6 (Fig. 4), the affinity
O
N
O
N
N
NH
O
NH
NH
Cl
Cl
N
N
N
N
N
Cl
Cl
Cl
Cl
5
3
4
Ki CB2 = 23 nM
Ki CB1 = 4500 nM
Ki CB2 = 0.037 nM
Ki CB1 = 363 nM
Ki CB2 = 0.34 nM
Ki CB1 = 2050 nM
Ki CB1 / Ki CB2 = 195.6:1
Ki CB1 / Ki CB2 = 9810:1
Ki CB1 / Ki CB2 = 6029:1
Fig. 2. Structures and cannabinoid receptor affinities of reference compounds 3, 4, and 5.

G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
749
phosphorylated ERK 1/2 (P-ERK 1/2) expression in human pro-
myelocytic leukemia HL-60 (HL-60) cell line, following the treat-
ment of the cells with the cannabinoid compounds. As previously
reported, this cell line expresses CB2 but not CB1 receptors, and it
represents a reliable platform for the evaluation of intrinsic activity
of CB2 ligands [38]. In fact, the exposure of HL-60 cells to the
cannabinoid agonists CP-55,940, as well as to other synthetic se-
lective CB2 agonists, or to the endogenous cannabinoid ligand 2-
AG, elicited rapid phosphorylation and activation of the ERK 1/2
[26,39]. This up-regulation of P-ERK 1/2 was counteracted by a pre-
treatment of HL-60 cells with the reference CB2 selective antago-
nist SR144528, with the subsequent evidence of CB2 mediated ef-
fect of the cannabinoid agonist on the adopted cells.
S
N
N
N
N
H
H
Dihydroindenopyrazole
scaffold
Dihydrothienocyclopentapyrazole
scaffold
Fig. 3. Comparison of dihydroindenopyrazole scaffold with the bioisosteric dihy-
drothienocyclopentapyrazole one.
O
Q
NH
S
G and Q
as indicated in
Table 1
N
R1
N
G
4. Results and discussion
R2
4.1. Cannabinoid receptor affinities
6
Fig. 4. General structures of novel thienocyclopentapyrazole compounds.
The goal of this work was to prepare
a series of 1,4-
dihydrocyclopentapyrazole carboxamide analogues of CB2 ligands
3, 4 and 5 and to determine their affinities at CB1 and CB2 re-
ceptors. According to the obtained binding data of the novel com-
pounds 6aet, the replacement of the indenopyrazole tricyclic
system of 3e5 induced a significant impact on cannabinoid re-
ceptor affinity (Table 1).
In general, all the investigated thienocyclopentapyrazole de-
rivatives evidenced poor binding affinity for CB1 receptors, with the
exception of compound 6e which showed Ki CB1 lower than 10 nM.
Comparison of 6a respect to 3 suggested that CH]CH/S bio-
isosteric replacement elicited a marked effect on potency at CB2
receptor subtype leading to 62-fold decrease in CB2 binding
affinity.
methylbenzylhydrazine hydrochloride provided the dihy-
drothienocyclopentapyrazole esters 11e14 in 53e64% overall yield.
Hydrolisis of tricyclic esters 11e14 provided the acids 15e18.
The acids 15e18 were directly converted to the corresponding
acid chlorides which were treated with appropriate amines/hy-
drazines to furnish target compounds 6aet (Scheme 3).
3. Biology
3.1. Radioreceptor binding assays
The affinity of the novel compounds for CB1 and CB2 receptors
were determined according to the previously reported procedures
[26]. Assays were performed by using mouse brain (minus cere-
bellum) and mouse spleen homogenates for CB1 and CB2 receptors
respectively. [3H]-(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)
phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol ([3H]CP-55,940) was
employed as radio-labeled ligand. The experimental data (IC50
values) were converted into Ki values [37].
Compound 6a, however, was chosen as a convenient benchmark
for all of the others, in terms of presenting structure binding affinity
relationship.
Initially, the effect of changing the size of the carboxamide
moiety was examined. The affinity for the CB2 receptor of the
compound 6b having a seven-membered ring was only slightly
increased (compare 6a vs 6b) but with good improvement in CB2
selectivity (Ki CB1/Ki CB2 ¼ 401). On the contrary the compound
having a five membered ring (6c) had markedly decreased CB2
affinity (16.6 fold reduction: compare 6c vs 6a). Replacement of the
piperidine nitrogen with a methylene group also led to a three-fold
decrease in binding at CB2 receptor subtype (compare 6a vs 6d).
3.2. CB2 intrinsic activity by in vitro assays
Intrinsic activity of the novel derivatives towards CB2 receptors
was evaluated through in vitro tests based on the determination of
O
O
NH
Q
O
S
S
Q
H2N
N
N
R1
R1
N
N
R2
G
R2
G
6a-t
11-14
O
S
O
S
R1
HCl
NHNH2
R1
G
O
R2
O
R2
O
9, 10
7, 8
Scheme 1. Retrosynthetic pathways to the target dihydrothienocyclopentapyrazoles 6aet.

750
G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
O
S
S
S
S
i
ii
iii
HOOC
HOOC
O
21
8
19
20
Scheme 2. Synthesis of compound 8. Reagents and conditions: (i) CH(COOH)2, pyridine, piperidine, 115 ꢀC, overnight, 65%; (ii) H2, Pd(OH)2, EtOH, 15 psi, 25 ꢀC, 18 h, 89%; (iii) PPA,
75 ꢀC, 1 h, 32%.
Interestingly, introduction of a bulky alkyl group at the car-
boxamide nitrogen afforded compound 6e which showed a 2.2-fold
decrease in Ki CB2 value and as well as increased CB1 affinity
(Ki ¼ 6.5 nM), thereby producing reduced selectivity. In contrast to
the bioisosteric N-piperidine derivative 6a, compound 6e repre-
sents an unselective cannabinoid ligand.
In order to investigate the effect of methyl shifting from C6 to C7
of the tricyclic platform on CB receptor affinities, compounds 6fel
with simple or branched rings on C3-carboxamide function were
examined.
receptor affinity (compounds 6met). Compound 6m, which
contain a 6-methyl group in the tricyclic system and a menthyl
motif in the C3 carbamoyl function showed CB2 affinity similar to
that of the homologue derivative 6e. In the mean time, the
replacement of substituent at 1 position of pyrazole ring showed a
27-fold reduction of CB1 affinity (6m Ki CB1 ¼ 180 nM vs 6e Ki
CB1 ¼ 6.5 nM) with a consequent improvement of CB2 selectivity.
According to the 1-(2,4-dichlorophenyl) analogue derivatives
(compounds 6fel), novel derivatives 6net bearing a 7-methyl
group evidenced low affinity to CB1 receptor as well as reduced
CB2 receptor affinity. However, as for the reference homologue sub-
class, menthyl and fenchyl groups on C3-carboxamide function
determined significant affinity for CB2 receptor subtype.
When the simple carboxamide substitution pattern of 6ner was
replaced with a menthyl moiety, the resulting compound, 6s, had
2.75 fold increase in CB1 receptor affinity with Ki CB1 from 440 nM
for 6a to 160 nM for 6s.
On the other hand, this structural modification resulted in a
similar potency toward CB2 affinity with a Ki CB2 value of 2.3 nM
for 6a and 2.7 nM for 6s. This result may indicate a sensitivity of
both receptors to steric bulk of the carboxamide moiety for con-
geners possessing a methylbenzyl motif at N1 position of the
thienocyclopentapyrazole template.
Thus compound 6e represents an unselective CB ligand in
contrast to the bioisosteric N-piperidin derivative 6a. Very high Ki
values indicating low CB1 affinity for CB1 receptors were deter-
mined (Ki > 2000) and much lower affinity for CB2 receptor were
observed compared to 6a with the exception of derivatives 6k, l.
Compounds 6k, l contain in the carboxamide function an alkyl
bulky residue such as menthyl or fenchyl group in place of a simple
ring as in 6fej. This led to a moderate decrease in binding affinities
respect to 6a, with Ki values of 6.7 and 5.3 nM at CB2, respectively.
As a result, the menthyl analogue 6k had the highest CB1eCB2
selectivity (CB1:CB2 ratio ¼ 485).
We also explored the effect related to the replacement of the N1-
dichlorophenyl group with p-methylbenzyl moiety on cannabinoid
O
S
O
S
R1
O
i
ii
R1
O
S
R2
N
O
R1
O
R2
N
G
O
R2
11-14
9
R1 = CH3; R2 = H
7 R1 = CH3; R2 = H
8 R1 = H; R2 = CH3
10 R1 = H; R2 = CH3
iii
O
O
NH
Q
OH
S
iv
S
N
N
R1
R1
N
G
N
R2
G
R2
R1, R2, G and Q
as indicated in
table 1
15-18
6a-t
11, 15 R1 = CH3; R2 = H; G = 2,4-Cl2C6H3
12, 16 R1 = H; R2 = CH3; G = 2,4-Cl2C6H3
13, 17 R1 = CH3; R2 = H; G = CH2C6H4-p-CH3
14, 18 R1 = H; R2 = CH3; G = CH2C6H4-p-CH3
Scheme 3. Synthesis of compounds 6aet. Reagents and conditions: (i) Na, EtOH, (COOEt)2, r.t., 5 h; (ii) 2,4-dichlorophenylhydrazine hydrochloride or p-methylbenzylhydrazine
hydrochloride, AcOH, 110 ꢀC, 8 h; (iii); EtOH/H2O 1:1, NaOH, 80 ꢀC 5 h; (iv) SOCl2, CH2Cl2, 40 ꢀC, 5 h, then NH2-Q, CH2Cl2, 40 ꢀC, 2 h.

G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
751
Table 1
Structures and binding data of 1,4-dihydrothieno[20,30-4,5]cyclopenta[1,2-c]pyrazole derivatives 6.
,
b
Compd
R1
R2
G
Q
Receptor affinitya (nM)
CB2 ratio Ki CB1/Ki CB2
,b
,c
Ki CB1a
Ki CB2a
6a
6b
6c
6d
6e
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
440 25
442 38
1386 330
447 63
6.5 13
2.3 0.3
1.1 0.1
38.3 3.2
7.2 1.3
5.1 0.6
191.3:1
401.8:1
36.2:1
62.1:1
1.3:1
6f
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
3918 107
3798 709
4731 161
3076 594
4705 294
3255 274
578 83
321 75
817 136
501 76
747 200
6.7 0.9
6.8:1
11.8:1
5.8:1
6g
6h
6i
6.1:1
6j
6.3:1
6k
485.8:1
6l
H
CH3
H
2245 71
180 32
5.3 1.0
4.8 0.8
423.6:1
37.5:1
6m
CH3
6n
6o
H
H
CH3
CH3
377 37
125 31
69 14
3.0:1
1276 55
18.5:1
6p
6q
6r
H
H
H
CH3
CH3
CH3
4500 500
1977 474
4500 250
515 96
191 44
8.7:1
10.4:1
1758 474
2.6:1
(continued on next page)

752
G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
Table 1 (continued )
,
b
Compd
R1
R2
G
Q
Receptor affinitya (nM)
CB2 ratio Ki CB1/Ki CB2
,
b
,c
Ki CB1a
Ki CB2a
6s
H
H
CH3
CH3
160 37
420 64
2.7 0.2
2.4 0.1
59.3:1
6t
175.0:1
a
Ki values were obtained from five independent experiments run in triplicate and are expressed as the mean of the five values standard error.
Affinity of compounds was assayed using mouse brain (minus cerebellum) homogenate and [3H]-CP-55,940.
Affinity of compounds was evaluated using mouse spleen homogenate and [3H]-CP-55,940.
b
c
However the replacement of the C3 carboxamide menthyl sub-
thionyl chloride (3.0 equiv) in CH2Cl2 (10 mL) was refluxed for 5 h.
stituent with a fenchyl group to give 6t elicited a similar binding
profile for CB receptor if compared to the major term 6a.
The solvent and the excess of SOCl2 were removed under reduced
pressure. The resulting dark solid in CH2Cl2 (10 mL) was dropwise
added to a solution of requisite amine or hydrazine (1.5 equiv) in
CH2Cl2 (15 mL) at 0 ꢀC. The mixture was warmed to room tem-
perature and refluxed for 2 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 with water, dried over Na2SO4 and concentrated
under reduced pressure.
The analytically pure product was isolated by an appropriate
method of purification as below indicated.
6.1.1.1.1. 6-Methyl-1-(2,4-dichlorophenyl)-N-(piperidinyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6a). General procedure I was used to convert 15 and N-ami-
nopiperidine into the title product. Purification by flash chro-
4.2. Intrinsic activity by in vitro assays
To evaluate intrinsic activity of the novel CB2 ligands, all the
novel compounds showing Ki values for CB2 receptors lower than
10 nM were assayed by in vitro test based on HL-60 cells. According
to the profile determined with the reference cannabinoid agonist
WIN55,212-2, significant increasing of P-ERK 1/2 expression in HL-
60 cells has been evidenced for all the tested compounds (Table 1;
P-ERK 1/2 values in absence of AM630). CB2 agonist of the novel
derivatives has been confirmed through the inhibition of the
induced P-ERK 1/2 up-regulation by the pre-treatment of HL-60
cells with the reference selective CB2 antagonist AM630 (Table 1;
P-ERK 1/2 values with AM630 at the concentration of 75 nM).
matography afforded 6a (0.217 g, 82%) as
a yellow solid.
Rf ¼ 0.32 (CHCl3/MeOH 99:1); mp 177e178 ꢀC; IR (
n
/cmꢁ1):
3089, 1662; 1H NMR (CDCl3, ppm/
d): 1.38e1.60 (m, 2H),
5. Conclusions
1.64e1.90 (m, 4H), 2.49 (s, 3H), 2.80e2.98 (m, 4H), 3.85 (s, 2H),
6.46 (s, 1H), 7.35e7.40 (m, 1H), 7.42 (dd, J ¼ 8.4 Hz, 2.2 Hz, 1H),
7.52 (d, J ¼ 8.4 Hz, 1H), 7.63 (d, J ¼ 2.2 Hz, 1H); 13C NMR (CDCl3,
In this report we have identified
dihydrothienocyclopentapyrazole-based cannabinoid receptor li-
gands that are selective for CB2 receptor subtype.
a
novel series of
ppm/d): 15.9, 23.2, 25.3, 28.9, 57.0, 115.4, 128.0, 129.2, 130.1,
130.3, 130.7, 134.6, 135.3, 135.8, 141.1, 143.6, 148.6, 149.2, 159.0;
Anal. Calcd for C21H20Cl2N4OS: C, 56.38; H, 4.51; N, 12.52. Found:
C, 56.41; H, 4.55; N, 12.45.
The more potent and selective analogues in this series have
binding affinity of 1.1e7.2 nM at CB2 receptor and selectivity over
CB1 of up to 485-fold. According to in vitro assays based on the
evaluation of P-ERK 1/2 expression in HL-60 cells, the novel com-
pounds act as CB2 agonists.
6.1.1.1.2. 6-Methyl-1-(2,4-dichlorophenyl)-N-(homopiperidinyl)-
1,4-dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6b). General procedure I was used to convert 15 and N-amino-
homopiperidine into the title product. Purification by flash chro-
matography afforded 6b (0.218 g, 79%) as a yellow solid. Rf ¼ 0.36
6. Experimental protocols
(CHCl3/MeOH 99:1); mp 148e151 ꢀC; IR ( /cmꢁ1): 3401, 1672; 1H
n
6.1. Chemistry
NMR (CDCl3, ppm/d): 1.55e1.90 (m, 8H), 2.49 (s, 3H), 3.18 (t,
J ¼ 5 Hz, 4H), 3.84 (s, 2H), 6.46 (s, 1H), 7.43 (d, J ¼ 8.4 Hz, 1H), 7.52
6.1.1. General procedures
(d, J ¼ 8.4 Hz, 1H), 7.63 (s, 1H), 8.00e8.10 (bs, 1H); 13C NMR (CDCl3,
All reagents and solvents were purchased from Aldrich and used
as received. Low boiling petroleum ether was the fraction collected
between 40 and 60 ꢀC. Melting points were determined on a Büchi
510 capillary apparatus and are uncorrected. The NMR spectra were
obtained with a Varian VXR-200 spectrometer at 200 for 1H and
50.0 MHz for 13C. Chemical shifts are reported in ppm downfield
from internal Me4Si in CDCl3. IR spectra were recorded on a JASCO
FT/IR-460 plus equipment. The following abbreviations were used
to describe peak patterns where appropriate: singlet (s), doublet
(d), triplet (t), multiplet (m) and broad (br). Elemental analyses
were performed on a PerkineElmer 240 B analyser. TLC was per-
formed on Merck silica gel 60 TLC plates F254 and visualized using
UV or phosphomolibdic acid. Flash chromatography was carried
out on silica gel (230e400 mesh).
ppm/d): 15.9, 23.2, 25.3, 28.9, 58.2, 115.4, 128.0, 129.2, 130.1, 130.4,
130.8,134.6,135.4,135.9,141.2,143.7,148.6,149.2,159.4; Anal. Calcd
for C22H22Cl2N4OS: C, 57.27; H, 4.81; N, 12.14. Found: C, 57.32; H,
4.80; N, 12.17.
6.1.1.1.3. 6-Methyl-1-(2,4-dichlorophenyl)-N-(pyrrolidinyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6c). General procedure I was used to convert 15 and N-amino-
pyrrolidine hydrochloride into the title product adding 1.5 equiv of
triethylamine to the mixture. Purification by flash chromatography
afforded 6c (0.192 g, 74%) as a yellow solid. Rf ¼ 0.25 (CHCl3/MeOH
99:1); mp 100e103 ꢀC; IR (
ppm/
n
/cmꢁ1): 3434, 1670; 1H NMR (CDCl3,
): 1.80e2.00 (m, 4H), 2.49 (s, 3H), 2.90e3.15 (m, 4H), 3.86 (s,
d
2H), 6.46 (s, 1H), 7.41 (dd, J ¼ 9.6 Hz, 1.2 Hz, 1H), 7.53 (d, J ¼ 9.6 Hz,
1H), 7.63 (d, J ¼ 1.2 Hz, 1H); 13C NMR (CDCl3, ppm/
d): 16.0, 22.3,
29.0, 55.4, 115.4, 128.1, 129.2, 130.1, 130.4, 130.8, 134.6, 135.4, 135.9,
141.2, 143.7, 148.7, 149.2, 159.2; Anal. Calcd for C20H18Cl2N4OS: C,
55.43; H, 4.19; N, 12.93; Found: C, 55.40; H, 4.24; N, 13.00.
6.1.1.1. General procedure I: synthesis of carboxamides and hydra-
zides. A mixture of the appropriate 1,4-dihydrothieno[20,30-4,5]
cyclopenta[1,2c]pyrazole-3-carboxylic acid 15e18 (0.6 mmol) and

G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
753
6.1.1.1.4. 6-Methyl-1-(2,4-dichlorophenyl)-N-cyclohexyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6d). General procedure I was used to convert 15 and cyclohexyl-
amine into the title product. Purification by flash chromatography
afforded 6d (0.244 g, 91%) as a yellow solid. Rf ¼ 0.64 (CHCl3/MeOH
ppm/d): 14.4, 22.2, 28.7, 29.6, 55.4, 124.3, 127.9, 128.1, 130.0, 130.4,
130.5, 133.7, 135.6, 136.5, 136.9, 140.8, 149.9, 151.7, 159.9. Anal. Calcd
for C20H18Cl2N4OS: C, 55.43; H, 4.19; N, 12.93; Found: C, 55.43; H,
4.27; N, 13.01.
6.1.1.1.9. 7-Methyl-1-(2,4-dichlorophenyl)-N-cyclohexyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide (6i).
General procedure I was used to convert 16 and cyclohexylamine
into the title product. Purification by flash chromatography affor-
ded 6i (0.220 g, 82%) as a yellow solid. Rf ¼ 0.60 (CHCl3/MeOH
99:1); mp 103e105 ꢀC; IR (
NMR (CDCl3, ppm/
n
/cmꢁ1): 3405, 3301, 3100, 1662; 1H
d
): 1.20e1.43 (m, 4H), 1.66e1.73 (m, 4H),
1.95e2.10 (m, 2H), 2.49 (s, 3H), 3.84 (s, 2H), 3.84e4.00 (m, 1H), 6.46
(s, 1H), 6.80 (d, J ¼ 7.5 Hz, 1H, NH exch. D2O), 7.42 (dd, J ¼ 8.7 Hz,
2.1 Hz, 1H), 7.54 (d, J ¼ 8.7 Hz, 1H), 7.63 (d, J ¼ 2.1 Hz, 1H); 13C NMR
99:1); mp 102e103 ꢀC; IR (
NMR (CDCl3, ppm/d): 1.10e1.65 (m, 6H), 1.73 (s, 3H), 1.90e2.10 (m,
n
/cmꢁ1): 3403, 3315, 3102, 1660; 1H
(CDCl3, ppm/
d
): 16.0, 24.9, 25.5, 28.9, 33.1, 48.0, 115.4, 128.8, 129.3,
129.7, 130.4, 130.8, 134.8, 135.4, 136.0, 142.0, 143.6, 148.5, 149.4,
160.9; Anal. Calcd for C22H21Cl2N3OS: C, 59.20; H, 4.74; N, 9.41.
Found: C, 59.27; H, 4.73; N, 9.48.
4H), 3.85 (s, 2H), 3.90e4.00 (m, 1H), 6.63 (bs, 1H), 6.80 (s, 1H), 7.43
(dd, J ¼ 8.4 Hz, 2.2 Hz, 1H), 7.55 (d, J ¼ 8.4 Hz, 1H), 7.60 (s, 1H); 13
C
NMR (CDCl3, ppm/d): 14.5, 24.9, 25.6, 28.6, 33.1, 48.1, 124.3, 127.9,
6.1.1.1.5. 6-Methyl-1-(2,4-dichlorophenyl)-N-menthyl-1,4-
128.2, 130.0, 130.2, 130.5, 133.7, 135.8, 136.2, 137.0, 141.8, 150.1, 151.7,
160.9. Anal. Calcd for C22H21Cl2N3OS: C, 59.20; H, 4.74; N, 9.41.
Found: C, 59.22; H, 4.76; N, 9.49.
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6e). General procedure I was used to convert 15 and L-menthyl-
amine into the title product. Purification by flash chromatography
afforded 6e (0.175 g, 58%) as a yellow solid. Rf ¼ 0.75 (CHCl3/MeOH
6.1.1.1.10. 7-Methyl-1-(2,4-dichlorophenyl)-N-morpholinyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide (6j).
General procedure I was used to convert 16 and N-amino-
morpholine into the title product. Purification by flash chroma-
tography afforded 6j (0.110 g, 41%) as a yellow solid. Rf ¼ 0.17
99:1); mp 170e172 ꢀC; IR ( /cmꢁ1): 3399, 1658; 1H NMR (CDCl3,
n
ppm/d): 0.75e1.00 (m, 9H), 1.10e1.30 (m, 2H), 1.43e1.80 (m, 5H),
1.90e2.10 (m, 2H), 2.49 (s, 3H), 3.85 (s, 2H), 3.90e4.00 (m, 1H), 6.46
(s, 1H), 6.65 (d, J ¼ 7.3 Hz, 1H, NH exch. D2O), 7.42 (dd, J ¼ 8.4 Hz,
2.1 Hz, 1H), 7.52 (d, J ¼ 8.4 Hz, 1H), 7.64 (d, J ¼ 2.1 Hz, 1H); 13C NMR
(CHCl3/MeOH 99:1); mp 165e168 ꢀC; IR ( /cmꢁ1): 3401, 3246, 3091,
n
1670; 1H NMR (CDCl3, ppm/
d): 1.72 (s, 3H), 2.88e3.10 (m, 4H),
(CDCl3, ppm/
d
): 16.0, 16.2, 21.1, 22.1, 23.8, 26.7, 28.9, 31.9, 34.5, 43.1,
3.78e4.02 (m, 6H), 6.81 (s, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H), 7.55 (d,
47.8, 49.7, 115.4, 128.0, 129.2, 129.7, 130.4, 130.7, 134.7, 135.3, 135.9,
J ¼ 8.4 Hz, 1H), 7.62 (d, J ¼ 2.0 Hz, 1H), 7.66 (s, 1H); 13C NMR (CDCl3,
142.0, 143.6, 148.5, 149.4, 161.0; [
Calcd for C26H29Cl2N4OS: C, 62.15; H, 5.82; N, 8.36. Found: C, 62.19;
H, 5.77; N, 8.32.
a
]
20: ꢁ2.18ꢀ (CHCl3, c ¼ 0.14); Anal.
ppm/d): 14.4, 28.5, 29.6, 56.0, 66.4, 124.4, 128.0, 128.1, 130.0, 130.2,
D
130.4, 130.6, 133.7, 135.7, 136.7, 136.8, 150.0, 151.8, 159.2. Anal. Calcd
for C20H18Cl2N4O2S: C, 53.46; H, 4.04; N, 12.47. Found: C, 53.50; H,
4.08; N, 12.40.
6.1.1.1.11. 7-Methyl-1-(2,4-dichlorophenyl)-N-menthyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
6.1.1.1.6. 7-Methyl-1-(2,4-dichlorophenyl)-N-(piperidinyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide (6f).
General procedure I was used to convert 16 and N-aminopiperidine
into the title product. Purification by flash chromatography affor-
ded 6f (0.220 g, 82%) as a yellow solid. Rf ¼ 0.27 (CHCl3/MeOH
(6k). General procedure I was used to convert 16 and L-menthyl-
amine into the title product. Purification by flash chromatography
afforded 6k (0.187 g, 62%) as a yellow solid. Rf ¼ 0.74 (CHCl3/MeOH
99:1); mp 165e167 ꢀC; IR (
n
/cmꢁ1): 3390, 3315, 3247, 3091, 1675;
1H NMR (CDCl3, ppm/
d): 1.38e1.68 (m, 2H), 1.60e1.90 (m, 7H),
99:1); mp 104e105 ꢀC; IR ( /cmꢁ1): 3390, 3278, 3066, 1662; 1H
n
2.88e3.00 (m, 4H), 3.86 (s, 2H), 6.81 (s, 1H), 7.32e7.40 (m, 1H), 7.42
(dd, J ¼ 8.4 Hz, 2.2 Hz, 1H), 7.52 (d, J ¼ 8.4 Hz,1H), 7.63 (d, J ¼ 2.2 Hz,
NMR (CDCl3, ppm/d): 0.83 (s, 3H), 0.87 (s, 3H), 0.91 (s, 3H),
0.70e1.02 (m, 2H), 1.05e1.25 (m, 2H), 1.60e1.80 (m, 3H), 1.72 (s,
3H), 1.98e2.19 (m, 2H), 3.86 (s, 2H), 3.85e4.00 (m, 1H), 6.59 (d,
J ¼ 8.2 Hz, 1H), 6.80 (s, 1H), 7.41e7.61 (m, 3H); 13C NMR (CDCl3,
1H); 13C NMR (CDCl3, ppm/
d): 14.4, 23.3, 25.3, 28.6, 57.0, 124.3,
127.9, 128.1, 130.0, 130.4, 130.6, 133.7, 135.7, 136.5, 136.9, 140.9,
149.9, 151.7, 159.0. Anal. Calcd for C21H20Cl2N4OS: C, 56.38; H, 4.51;
N, 12.52. Found: C, 56.40; H, 4.57; N, 12.57.
ppm/d): 14.5, 16.1, 21.1, 22.1, 23.8, 26.7, 28.6, 31.9, 34.5, 43.0, 47.8,
49.7, 124.2, 127.9, 128.1, 130.0, 130.1, 130.5, 133.7, 135.7, 136.5, 137.0,
6.1.1.1.7. 7-Methyl-1-(2,4-dichlorophenyl)-N-(homopiperidinyl)-
1,4-dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6g). General procedure I was used to convert 16 and N-amino-
homopiperidine into the title product. Purification by flash chro-
matography afforded 6g (0.213 g, 77%) as a yellow solid. Rf ¼ 0.34
141.7, 150.0,151.6, 161.0; [
for C26H29Cl2N4OS: C, 62.15; H, 5.82; N, 8.36. Found: C, 62.21; H,
5.79; N, 8.32.
a
]
20: ꢁ26.35ꢀ (CHCl3, c ¼ 0.16). Anal. Calcd
D
6.1.1.1.12. 7-methyl-1-(2,4-dichlorophenyl)-N-fenchyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide (6l).
General procedure I was used to convert 16 and 1R,2S,4R-fenchyl-
amine into the title product. Purification by flash chromatography
afforded 6l (0.180 g, 60%) as a yellow solid. Rf ¼ 0.72 (CHCl3/MeOH
(CHCl3/MeOH 99:1); mp 150e152 ꢀC; IR (
n
/cmꢁ1): 3401, 3300,
d): 1.51e1.91 (m, 11H),
3247, 3077, 1673; 1H NMR (CDCl3, ppm/
3.15e3.21 (m, 4H), 3.79e4.00 (m, 2H), 6.81 (s, 1H), 7.40e7.47 (m,
1H), 7.50e7.58 (m, 1H), 7.61 (s, 1H), 8.02 (bs, 1H); 13C NMR (CDCl3,
99:1); mp 85e87 ꢀC; IR (
ppm/d): 0.87 (s, 3H), 1.11 (s, 3H), 1.18 (s, 3H), 1.20e1.26 (m, 2H),
n
/cmꢁ1): 3415, 3091, 1668; 1H NMR (CDCl3,
ppm/
d
): 14.4, 26.3, 26.9, 28.5 29.6, 58.2, 124.3, 127.9, 128.1, 130.0,
130.4, 130.5, 133.6,135.6, 136.5, 136.9, 140.9, 149.8, 151.6, 159.3. Anal
Calcd for C22H22Cl2N4OS: C, 57.27; H, 4.81; N, 12.14. Found: C, 57.30;
H, 4.86; N, 12.14.
1.31e1.48 (m, 2H), 1.60e1.80 (m, 3H), 1.73 (s, 3H), 3.76e3.90 (m,
1H), 3.85 (s, 2H), 6.80 (s, 1H), 6.95 (d, J ¼ 10.0 Hz, 1H), 7.44 (dd,
J ¼ 9.0 Hz, 1.8 Hz, 1H), 7.57 (d, J ¼ 9.0 Hz, 1H), 7.60 (s, 1H); 13C NMR
6.1.1.1.8. 7-Methyl-1-(2,4-dichlorophenyl)-N-(pyrrolidinyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6h). General procedure I was used to convert 16 and N-amino-
pyrrolidine hydrochloride into the title product adding 1.5 equiv of
triethylamine to the mixture. Purification by flash chromatography
afforded 6h (0.171 g, 66%) as a yellow solid. Rf ¼ 0.30 (CHCl3/MeOH
(CDCl3, ppm/d): 14.6, 19.8, 21.3, 26.0, 27.3, 28.6, 30.9, 39.5, 42.7, 48.1,
48.6, 63.1, 124.2, 127.8, 128.1, 129.9, 130.6, 133.7, 135.9, 136.3, 137.1,
141.6, 149.9, 151.5, 162.5; [
for C26H27Cl2N3OS: C, 62.40; H, 5.44; N, 8.40. Found: C, 62.48; H,
5.46; N, 8.38.
a
]
20: ꢁ6.08ꢀ (CHCl3, c ¼ 0.23). Anal. Calcd
D
6.1.1.1.13. 6-Methyl-1-(4-methylbenzyl)-N-menthyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6m). General procedure I was used to convert 17 and L-menthyl-
99:1); mp 170e180 ꢀC; IR (
NMR (CDCl3, ppm/
n
/cmꢁ1): 3397, 3309, 3234, 1666; 1H
): 1.71 (s, 3H), 1.85e1.98 (m, 4H), 2.95e3.15 (m,
d
4H), 3.87 (s, 2H), 6.81 (s,1H), 7.44 (dd, J ¼ 8.6 Hz, 2.2 Hz,1H), 7.55 (d,
amine into the title product. Purification by flash chromatography
afforded 6m (0.138 g, 50%) as a yellow solid. Rf ¼ 0.63 (CHCl3/MeOH
J ¼ 8.6 Hz,1H), 7.61 (d, J ¼ 2.2 Hz,1H), 8.08 (bs,1H); 13C NMR (CDCl3,

754
G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
99:1); mp 67e70 ꢀC; IR (
ppm/d): 0.83e0.93 (m, 9H), 1.10e1.25 (m, 2H), 1.64e1.78 (m, 5H),
n
/cmꢁ1): 3399, 3303,1658; 1H NMR (CDCl3,
aminomorpholine into the title product. Purification by flash
chromatography afforded 6r (0.181 g, 74%) as a yellow solid.
2.03e2.15 (m, 2H), 2.34 (s, 3H), 2.45 (s, 3H), 3.74 (s, 2H), 3.85e4.00
(m, 1H), 5.38 (s, 2H), 6.33 (s, 1H), 6.60 (d, J ¼ 9.9 Hz, 1H), 7.10e7.21
Rf ¼ 0.14 (CHCl3/MeOH 99:1); mp 218e222 ꢀC; IR (
n
/cmꢁ1): 3222,
1646; 1H NMR (CDCl3, ppm/
d): 2.29 (s, 3H), 2.32 (s, 3H), 2.95e3.05
(m, 4H); 13C NMR (CDCl3, ppm/
d
): 16.0, 16.2, 21.1, 22.1, 23.8, 26.7,
(m, 4H), 3.79 (s, 2H), 3.80e3.98 (m, 4H), 5.56 (s, 2H), 6.85 (s, 1H),
28.8, 31.9, 34.5, 43.0, 47.8, 49.6, 55.0, 115.0, 127.3, 129.5, 129.6, 132.8,
134.5, 137.9, 139.8, 143.4, 147.8, 161.3; [
6.98 (d, J ¼ 8.0 Hz, 1H), 7.13 (d, J ¼ 8.0 Hz, 2H), 7.64 8 (s, 1H); 13C
a]
20: ꢁ26.47ꢀ (CHCl3,
NMR (CDCl3, ppm/d): 15.7, 21.0, 28.3, 55.3, 55.9, 66.4, 124.3, 125.8,
D
c ¼ 0.13). Anal. Calcd for C28H35N3OS: C, 72.85; H, 7.64; N, 9.10.
127.6, 129.4, 130.9, 133.6, 135.5, 137.4, 139.0, 148.1, 151.2, 159.6. Anal.
Calcd for C22H24N4O2S: C, 64.68; H, 5.92; N, 13.71. Found: C, 64.74;
H, 5.99; N, 13.70.
6.1.1.1.19. 7-Methyl-1-(4-methylbenzyl)-N-menthyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
Found: C, 72.87; H, 7.60; N, 9.14.
6.1.1.1.14. 7-Methyl-1-(4-methylbenzyl)-N-(piperidinyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6n). General procedure I was used to convert 18 and N-amino-
piperidine into the title product. Purification by flash chromatog-
raphy afforded 6n (0.193 g, 88%) as a yellow solid. Rf ¼ 0.26 (CHCl3/
(6s). General procedure I was used to convert 18 and L-menthyl-
amine into the title product. Purification by flash chromatography
afforded 6s (0.197 g, 71%) as a yellow solid. Rf ¼ 0.73 (CHCl3/MeOH
MeOH 99:1); mp 213e218 ꢀC; IR ( /cmꢁ1): 3234, 1648; 1H NMR
n
(CDCl3, ppm/
d): 1.38e1.50 (m, 2H), 1.60e1.81 (m, 4H), 2.28 (s, 3H),
99:1); mp 158e165 ꢀC; IR ( /cmꢁ1): 3397, 3297, 3100, 1658; 1H
n
2.31 (s, 3H), 2.87 (t, J ¼ 5.2 Hz, 4H), 3.79 (s, 2H), 5.55 (s, 2H), 6.83 (s,
NMR (CDCl3, ppm/d d): 0.83e0.93 (m, 9H),
): 1H NMR (CDCl3, ppm/
1H), 6.97 (d, J ¼ 8.0 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 7.59 (s, 1H); 13
C
1.10e1.26 (m, 2H), 1.64e1.78 (m, 5H), 1.95e2.15 (m, 2H), 2.27 (s,
3H), 2.32 (s, 3H), 3.79 (s, 2H), 3.80e4.00 (m, 1H), 5.56 (s, 2H), 6.60
(d, J ¼ 10.0 Hz, 1H), 6.83 (s, 1H), 6.97 (d, J ¼ 7.8 Hz, 2H), 7.13 (d,
NMR (CDCl3, ppm/d): 15.7, 21.0, 23.3, 25.3, 28.6, 55.3, 57.0, 124.2,
125.8, 127.6, 129.4, 130.9, 133.7, 135.6, 137.4, 139.3, 148.0, 151.2,
159.4. Anal. Calcd for C23H26N4OS: C, 67.95; H, 6.45; N, 13.78.
Found: C, 67.99; H, 6.43; N, 13.81.
J ¼ 7.8 Hz, 2H); 13C NMR (CDCl3, ppm/
d): 15.7, 16.2, 21.0, 21.1, 22.1,
23.9, 26.7, 28.4, 31.9, 34.5, 43.1, 47.8, 49.5, 55.3, 124.1, 125.7, 127.7,
129.4, 130.4, 133.9, 135.7, 137.3, 140.2, 148.1, 151.2, 161.4;
6.1.1.1.15. 7-Methyl-1-(4-methylbenzyl)-N-(homopiperidin-yl)-
1,4-dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6o). General procedure I was used to convert 18 and N-amino-
homopiperidine into the title product. Purification by flash chro-
matography afforded 6o (0.227 g, 90%) as a yellow solid. Rf ¼ 0.29
[a
]
20: ꢁ38.89ꢀ (CHCl3, c ¼ 0.30). Anal. Calcd for C28H35N3OS: C,
D
72.85; H, 7.64; N, 9.10. Found: C, 72.90; H, 7.68; N, 9.18.
6.1.1.1.20. 7-Methyl-1-(4-methylbenzyl)-N-fenchyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide (6t).
General procedure I was used to convert 18 and 1R,2S,4R-fenchyl-
amine into the title product. Purification by flash chromatography
afforded 6t (0.098 g, 59%) as a yellow solid. Rf ¼ 0.73 (CHCl3/MeOH
(CHCl3/MeOH 99:1); mp 188e191 ꢀC; IR ( /cmꢁ1): 3365, 3270,
n
1654; 1H NMR (CDCl3, ppm/
d): 1.55e1.82 (m, 8H), 2.28 (s, 3H), 2.31
(s, 3H), 3.12e3.19 (m, 4H), 3.79 (s, 2H), 5.54 (s, 2H), 6.83 (s, 1H), 6.97
(d, J ¼ 8.0 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 8.03 (s, 1H); 13C NMR
99:1); mp 58e62 ꢀC; IR ( /cmꢁ1): 3417, 1668; 1H NMR (CDCl3, ppm/
n
(CDCl3, ppm/
d
): 15.8, 21.0, 26.7, 27.0, 28.4, 55.4, 58.2, 124.2, 125.9,
d): 0.86 (s, 3H), 1.11 (s, 3H), 1.17 (s, 3H), 1.20e1.25 (m, 2H), 1.35e1.45
127.7, 129.5, 130.8, 133.8, 135.7, 137.4, 139.4, 148.0, 151.3, 159.9. Anal
Calcd for C24H28N4OS: C, 68.54; H, 6.71; N,13.32. Found: C, 68.60; H,
6.75; N, 13.33.
(m, 2H), 1.60e1.80 (m, 3H), 2.26 (s, 3H), 2.31 (s, 3H), 3.75e3.86 (m,
1H), 3.79 (s, 2H), 5.58 (s, 2H), 6.82 (s, 1H), 6.96e6.70 (m, 1H), 6.98
(d, J ¼ 7.8 Hz, 2H), 7.12 (d, J ¼ 7.8 Hz, 2H); 13C NMR (CDCl3, ppm/
d):
6.1.1.1.16. 7-Methyl-1-(4-methylbenzyl)-N-(pyrrolidinyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6p). General procedure I was used to convert 18 and N-amino-
pyrrolidine hydrochloride into the title product adding 1.5 equiv of
triethylamine to the mixture. Purification by flash chromatography
afforded 6p (0.174 g, 74%) as a yellow solid. Rf ¼ 0.21 (CHCl3/MeOH
15.7, 19.8, 21.0, 21.3, 26.0, 27.3, 28.4, 30.9, 39.4, 42.7, 48.1, 48.6, 55.4,
63.0, 124.1, 125.8, 127.7, 129.4, 130.3, 134.0, 135.8, 137.3, 139.9, 148.1,
151.2, 162.8; [
a
]
20: ꢁ7.76ꢀ (CHCl3, c ¼ 0.32). Anal. Calcd for
D
C28H33N3OS: C, 73.17; H, 7.24; N, 9.14. Found: C, 73.22; H, 7.28; N,
9.10.
99:1); mp 177e185 ꢀC; IR (
(CDCl3, ppm/
n
/cmꢁ1): 3234, 3052, 1662; 1H NMR
6.1.1.2. General procedure II: synthesis of acids. To a mixture of an
ester 11e14 (7.00 mmol) in ethanol (36 mL) was added a solution of
sodium hydroxide (10 equiv) in H2O (36 mL). The resulting mixture
was heated under refluxed 5 h. The mixture was allowed to cool to
room temperature and then poured into water and acidified with
1 N hydrochloric acid. The precipitate was filtered, washed with
water and air-dried to yield the analytically pure acid.
6.1.1.2.1. 6-Methyl-1-(2,4-dichlorophenyl)-1,4-dihydrothieno
[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxylic acid. General pro-
cedure II was used to convert 11 into the title product 15 (2.311 g,
d
): 1.85e1.99 (m, 4H), 2.28 (s, 3H), 2.31 (s, 3H),
2.90e3.15 (m, 4H), 3.80 (s, 2H), 5.55 (s, 2H), 6.83 (s, 1H), 6.96 (d,
J ¼ 8.0 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 7.56 (br. s, 1H); 13C NMR
(CDCl3, ppm/d): 15.8, 21.0, 22.2, 28.4, 55.4, 124.2, 125.8, 127.7, 129.5,
130.8, 133.7, 135.6, 137.4, 139.2, 148.1, 151.3, 160.3. Anal. Calcd for
22H24N4OS: C, 67.32; H, 6.16; N, 14.27; Found: C, 67.42; H, 6.19; N,
14.20.
6.1.1.1.17. 7-Methyl-1-(4-methylbenzyl)-N-cyclohexyl-1,4-
C
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
(6q). General procedure I was used to convert 18 and cyclohexyl-
amine into the title product. Purification by flash chromatography
afforded 6q (0.124 g, 51%) as a yellow solid. Rf ¼ 0.61 (CHCl3/MeOH
98%) as
255e258 ꢀC; IR (
(s, 3H), 3.79 (s, 2H), 6.55 (s, 1H), 7.50e7.95 (m, 3H), 12.72 (brs, 1H);
13C NMR (DMSO, ppm/
): 15.6, 28.8, 115.4, 128.7, 130.1, 130.2, 130.7,
134.3, 134.8, 135.6, 139.3, 143.8, 147.6, 148.3, 162.9. Anal. Calcd for
16H10Cl2N2O2S: C, 52.62; H, 2.76; N, 7.67. Found: C, 52.68; H, 2.80;
N, 7.69.
a
white solid. Rf
¼
0.38 (CHCl3/MeOH 8:2); mp:
n
/cmꢁ1): 3400,1705; 1H NMR (DMSO, ppm/
d
): 2.43
99:1); mp 188e194 ꢀC; IR (
n
/cmꢁ1): 3332, 3064, 1639; 1H NMR
d
(CDCl3, ppm/d): 1.20e1.45 (m, 4H),1.60e1.73 (m, 4H),1.95e2.05 (m,
2H), 2.28 (s, 3H), 2.31 (s, 3H), 3.78 (s, 2H), 3.83e3.98 (m,1H), 5.55 (s,
2H), 6.75 (d, J ¼ 8.2 Hz, 1H), 6.82 (s, 1H), 6.96 (d, J ¼ 7.8 Hz, 2H), 7.12
C
(d, J ¼ 7.8 Hz, 2H); 13C NMR (CDCl3, ppm/
d
): 15.8, 21.0, 25.0, 25.6,
6.1.1.2.2. 7-Methyl-1-(2,4-dichlorophenyl)-1,4-dihydrothieno
[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxylic acid. General pro-
cedure II was used to convert 12 into the title product 16 (2.147 g,
28.4, 29.7, 33.2, 48.0, 55.4, 124.2, 125.8, 127.7, 129.5, 130.5, 133.9,
135.8,137.4, 140.2, 148.1, 151.2, 161.3. Anal. Calcd for C24H27Cl2N3OS:
C, 71.08; H, 6.71; N, 10.36. Found: C, 71.16; H, 6.79; N, 10.31.
6.1.1.1.18. 7-Methyl-1-(4-methylbenzyl)-N-morpholinyl-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxamide
91%) as
a
white solid. Rf
¼
0.41 (CHCl3/MeOH 8:2); mp:
200e202 ꢀC; IR (
n
/cmꢁ1): 3400, 3089, 1714; 1H NMR (DMSO, ppm/
d
): 1.62 (s, 3H), 3.79 (s, 2H), 7.10 (s, 1H), 7.71 (d, J ¼ 7.8 Hz, 1H), 7.86
(6r). General procedure
I
was used to convert 18 and N-
(d, J ¼ 7.8 Hz, 1H), 7.99 (s, 1H), 13.1 (brs, 1H); 13C NMR (DMSO, ppm/

G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
755
d
): 13.9, 28.4, 125.4, 127.4, 128.6, 129.6, 131.2, 132.6, 135.4, 135.7,
95%) as a gray solid. Rf ¼ 0.45 (CHCl3); mp 61e63 ꢀC; IR (
n
/cmꢁ1):
136.7, 139.0, 148.9, 150.7, 162.9. Anal. Calcd for C16H10Cl2N2O2S: C,
52.62; H, 2.76; N, 7.67. Found: C, 52.70; H, 2.81; N, 7.70.
6.1.1.2.3. 6-methyl-1-(4-methylbenzyl)-1,4-dihydrothieno[20,30-
4,5]cyclopenta[1,2c]pyrazole-3-carboxylic acid. General procedure II
was used to convert 13 into the title product 17 (1.599 g, 90%) as a
3100, 1716; 1H NMR (CHCl3, ppm/
d
): 1.42 (t, J ¼ 7.2 Hz, 3H); 2.33 (s,
3H), 2.44 (s, 3H), 3.68 (s, 2H), 4.43 (q, J ¼ 7.2 Hz, 2H), 5.47 (s, 2H),
6.26 (s, 1H), 7.17 (dd, J ¼ 7.8 Hz, 4H); 13C NMR (CHCl3, ppm/
d): 14.4,
15.7, 21.0, 28.4, 55.7, 60.8, 124.3, 125.9, 127.6, 129.0, 132.2, 133.6,
135.1, 137.2,137.3, 147.7, 150.6,162.5. Anal. Calcd for C20H20N2O2S: C,
68.16; H, 5.72; N, 7.95. Found: C, 68.21; H, 5.72; N, 7.89.
white solid. Rf ¼ 0.45 (CHCl3/MeOH 8:2); mp: 240e243 ꢀC; IR (
n/
cmꢁ1): 3600, 3440, 1685; 1H NMR (DMSO, ppm/
d): 2.26 (s, 3H), 2.47
(s, 3H), 3.65 (s, 2H), 5.49 (s, 2H), 6.93 (s,1H), 7.00-7.40 (m, 4H),12.72
6.1.1.4. General procedure IV: synthesis of diketoesters. Sodium (2.0
equiv) was added in small portion to dry ethanol (5 mL) and stirred
until all the sodium had reacted. Ethyl oxalate (1.0 equiv) was
added, followed by dropwise addition of a solution of appropriate
thienoindanone (7,8) starting material (6 mmol) in dry ethanol
(30 mL). The solution was stirred at room temperature for 5 h. The
mixture was slowly poured over 2 N hydrochloride acid and the
resulting precipitate was collected by filtration and washed with a
small volume of ice-cold ethanol and water. The air-dried residue
afforded the analytically pure product.
(brs, 1H); 13C NMR (DMSO, ppm/
d): 15.8, 20.7, 28.7, 52.2, 115.8,
127.6, 129.3, 130.7, 133.7, 134.4, 137.2, 143.2, 143.3, 146.4, 163.1. Anal.
Calcd for C18H16N2O2S: C, 66.65; H, 4.97; N, 8.64. Found: C, 66.70; H,
5.02; N, 8.61.
6.1.1.2.4. 7-methyl-1-(4-methylbenzyl)-1,4-dihydrothieno[20,30-
4,5]cyclopenta[1,2c]pyrazole-3-carboxylic acid. General procedure II
was used to convert 13 into the title product 17 (1.724 g, 97%) as a
white solid. Rf ¼ 0.46 (CHCl3/MeOH 8:2); mp: 200e202 ꢀC; IR (
n/
cmꢁ1): 3400, 3100, 1714; 1H NMR (DMSO, ppm/
d): 2.23 (s, 6H), 3.67
(s, 2H), 5.60 (s, 2H), 6.93 (s,1H), 7.00e7.40 (m, 4H); 13C NMR (DMSO,
ppm/ ): 15.2, 20.6, 28.3, 55.9, 117.6, 124.9, 125.8, 127.6, 129.3, 131.7,
6.1.1.4.1. Ethyl 2-(2-methyl-4-oxo-5,6-dihydro-4H-cyclopenta[b]
thiophen-5-yl)-2-oxoacetate. General procedure IV was used to
convert 7 into the title product 9 (1.027 g, 62%) as a yellow solid.
d
134.5,135.6,136.7,146.8,150.0,163.4. Anal. Calcd for C18H16N2O2S: C,
66.65; H, 4.97; N, 8.64. Found: C, 66.69; H, 5.05; N, 8.63.
Rf ¼ 0.68 (CHCl3/MeOH 9:1); mp: 136e138 ꢀC; IR (
n
/cmꢁ1): 3100,
): 1.41 (t, J ¼ 7.2 Hz, 3H); 2.53 (s,
1716, 1666; 1H NMR (CHCl3, ppm/
d
6.1.1.3. General procedure III: synthesis of esters. To
a
stirred
3H), 3.97 (s, 2H), 4.39 (q, J ¼ 7.2 Hz, 2H), 6.90 (s, 1H), 12.80 (brs, 1H);
mixture of diketoester 9,10 (3.17 mmol) and the requisite phenyl
hydrazine hydrochloride (1.2 equiv) in AcOH (6 mL) was heated
under reflux for 8 h. The reaction was allowed to cool to room
temperature and poured onto ice-water. The precipitate was
filtered, washed with water and air-dried to yield the analytically
pure esters.
13C NMR (CHCl3, ppm/
d): 14.2, 16.1, 30.2, 62.1, 117.0, 119.8, 145.4,
146.4, 150.5, 162.7, 165.1, 191.9. Anal. Calcd for C12H12O4S: C, 57.13;
H, 4.79. Found: C, 57.19; H, 4.83.
6.1.1.4.2. Ethyl 2-(3-methyl-4-oxo-5,6-dihydro-4H-cyclopenta[b]
thiophen-5-yl)-2-oxoacetate. General procedure IV was used to
convert 8 into the title product 10 (1.093 g, 66%) as a white solid.
6.1.1.3.1. Ethyl 6-methyl-1-(2,4-dichlorophenyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxylate.
General procedure III was used to convert 9 into the title product 11
(1.123 g, 97%) as a bright yellow solid. Rf ¼ 0.42 (CHCl3); mp:
Rf ¼ 0.64 (CHCl3/MeOH 9:1); mp: 84e87 ꢀC; IR (
n
/cmꢁ1): 3417,
): 1.42 (t, J ¼ 7.2 Hz, 3H);
3097, 1718, 1660; 1H NMR (CHCl3, ppm/
d
2.28 (s, 3H), 3.77 (s, 2H), 4.41 (q, J ¼ 7.2 Hz, 2H), 7.55 (s, 1H), 12.90
(brs, 1H); 13C NMR (CHCl3, ppm/
d): 13.5, 14.1, 19.7, 62.1, 115.6, 120.3,
162e166 ꢀC; IR (
n
/cmꢁ1): 1716; 1H NMR (CHCl3, ppm/
d): 1.43 (t,
132.5, 144.2, 150.5, 162.6, 166.5, 192.9. Anal. Calcd for C12H12O4S: C,
57.13; H, 4.79. Found: C, 57.20; H, 4.85.
J ¼ 7.4 Hz, 3H); 2.49 (s, 3H), 3.81 (s, 2H), 4.45 (q, J ¼ 7.0 Hz, 2H), 6.46
(s, 1H), 7.40 (dd, J ¼ 8.4 Hz, 2.2 Hz, 1H), 7.55 (d, J ¼ 8.4 Hz, 1H), 7.61
6.1.1.4.3. 3-Methyl-5,6-dihydro-4H-cyclopenta[b]thiophen-4-one
(d, J ¼ 2.2 Hz, 1H); 13C NMR (CHCl3, ppm/
d
): 14.4, 16.0, 29.0, 61.2,
(8). 3-(4-methylthiophen-2-yl)propanoic
acid
(21)
(1
g,
115.6,128.0,129.7, 130.1, 130.9,131.2,135.0, 135.6, 136.0,139.1,143.9,
148.0, 149.2, 162.3. Anal. Calcd for C18H14Cl2N2O2S: C, 54.97; H,
3.59; N, 7.12. Found: C, 55.02; H, 3.55; N, 7.10.
5.88 mmol) was added in small portions to warm (75 ꢀC) poly-
phosphoric acid (8.5 g). After the addition was complete, heating
was continued at 75 ꢀC for 1 h. Cooling to room temperature was
followed by dilution with water and extraction with CH2Cl2. The
combined organics were dried over Na2SO4 and concentrated un-
der reduced pressure. Flash chromatography (PEt/EtOAc 1:1) gave 8
as a bright yellow solid (0.28 g, 32%). Rf ¼ 0.89 (CHCl3/MeOH 9:1);
6.1.1.3.2. Ethyl 7-Methyl-1-(2,4-dichlorophenyl)-1,4-
dihydrothieno[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxylate.
General procedure III was used to convert 9 into the title product 12
(1.123 g, 92%) as a white solid. Rf ¼ 0.32 (CHCl3); mp: 170e173 ꢀC; IR
(
n
/cmꢁ1): 3095, 1731; 1H NMR (CHCl3, ppm/
d
): 1.43 (t, J ¼ 7.2 Hz,
mp: ꢀC; 55e58 ꢀC; IR ( /cmꢁ1): 1672; 1H NMR (CHCl3, ppm/
n d): 2.49
3H); 1.73 (s, 3H), 3.82 (s, 2H), 4.45 (q, J ¼ 7.2 Hz, 2H), 6.82 (s, 1H),
(s, 3H), 2.89 (t, J ¼ 5.1 Hz, 2H), 3.11 (t, J ¼ 5.1 Hz, 2H), 6.79 (s,1H); 13
C
7.41 (dd, J ¼ 8.4 Hz, 2.2 Hz, 1H), 7.50e7.70 (m, 2H); 13C NMR (CHCl3,
NMR (CHCl3, ppm/d): 13.5, 24.0, 41.8, 125.3, 132.4, 144.28, 171.0,
ppm/
d
): 14.4, 16.0, 28.6, 61.2, 124.4, 127.8, 128.3, 129.8, 130.6, 131.6,
198.8. Anal. Calcd for C8H8OS: C, C, 63.13; H, 5.30. Found: C, 63.15; H,
5.32.
133.7, 135.9, 136.6, 136.9, 138.9, 149.8, 151.0, 162.1. Anal. Calcd for
18H14Cl2N2O2S: C, 54.97; H, 3.59; N, 7.12. Found: C, 55.03; H, 3.58;
N, 7.17.
6.1.1.3.3. Ethyl 6-methyl-1-(4-methylbenzyl)-1,4-dihydrothieno
[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxylate. General
proce-
C
6.2. Pharmacology
6.2.1. Animals
dure III was used to convert 10 into the title product 13 (0.884 g,
All animal experiments were performed according to the UE
guidelines for the care and use of experimental animals (CEE Nꢀ 86/
609). Animals (Charles River, Calco, LC, Italy) were housed in the
86%) as a white solid. Rf ¼ 0.29 (CHCl3); mp: 128e132 ꢀC; IR (
n/
cmꢁ1): 1719; 1H NMR (CHCl3, ppm/
d
): 1.42 (t, J ¼ 7.2 Hz, 3H); 2.33 (s,
3H), 2.44 (s, 3H), 3.68 (s, 2H), 4.43 (q, J ¼ 7.2 Hz, 2H), 5.47 (s, 2H),
animal care quarters; temperatures were maintained at 22
2
ꢀC
6.26 (s, 1H), 7.17 (dd, J ¼ 7.8 Hz, 4H); 13C NMR (CHCl3, ppm/
d
): 14.4,
(humidity 55 5%) on a 12 h light/dark cycle. Food and water were
available ad libitum before to start with the experimental
procedures.
16.0, 21.1, 28.8, 55.6, 60.8, 115.4, 127.6, 129.5, 131.6, 132.6, 134.8,
136.8, 138.0, 143.5, 147.0, 147.4, 162.5. Anal. Calcd for C20H20N2O2S:
C, 68.16; H, 5.72; N, 7.95. Found: C, 68.17; H, 5.77; N, 7.90.
6.1.1.3.4. Ethyl 7-methyl-1-(4-methylbenzyl)-1,4-dihydrothieno
[20,30-4,5]cyclopenta[1,2c]pyrazole-3-carboxylate. General
dure III was used to convert 10 into the title product 14 (1.021 g,
6.2.2. Receptor binding studies
Male CD1 mice weighing 30e35 g were killed by cervical
dislocation and the brain (minus cerebellum) and spleen were
proce-

756
G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
rapidly removed and placed on an ice-cold plate. Brain and spleen
tissues were used for CB1 and CB2 binding assays, respectively.
After thawing, tissues were homogenated in 20 vol. (wt/v) of ice-
cold TME buffer (50 mM TriseHCl, 1 mM EDTA and 3.0 mM
MgCl2, pH 7.4). The homogenates were centrifuged at 1086 ꢂ g for
10 min at 4 ꢀC, and the resulting supernatants were centrifuged at
45,000 ꢂ g for 30 min.
40 m
g. Separation was performed by 10% BiseTris Gel NuPAGE®
Novex (Invitrogen) and nitrocellulose membranes (Bio-Rad). Blots
were blocked through ChemiBLOCKER™ (Millipore) in TBST (0.1%
Tween 20 in Tris borate saline, Bio-Rad) and probed with Anti-
phospho-ERK 1/2 recombinant clone AW39R (Millipore) with a
1:1000 dilution in 1% BSA (Bovine Serum Albumin) in TBST
(10 min). After three cycles of washing in TBST, membranes were
probed with the secondary antibody Goat anti-Rabbit IgC e HRP
(Invitrogen) with a 1:2000 dilution in ChemiBLOCKER™-TBST 1:4.
After three cycles of washing in TBST, blots were treated for 5 min
with Immobilon Western Chemiluminescent HRP substrate (Milli-
pore) and immunoreactive bands were visualized by a Fujifilm Las-
For radioreceptor binding experiments, the novel compounds
were dissolved in dimethyl-sulfoxide (DMSO). DMSO concentration
in the different assays never exceeded 0.1% (v/v) and was without
effects.
[3H]-CP-55,940 binding was performed by the previously
described method [30]. [3H]-CP-55,940 (specific activity 180 Ci/
mmol) was purchased from Perkin Elmer Italia. Briefly, the mem-
€
1000 analyzer (Raytest Isotopenmessgerate GmbH). Immunoreac-
tive band optical density was measured using AIDA 2.11 software
€
branes (30e80
m
g of protein) were incubated with 0.5e1 nM of
(Raytest Isotopenmessgerate GmbH).
[3H]-CP-55,940 for 1 h at 30 ꢀC in a final volume of 0.5 mL of TME
Data were expressed as mean percentage of vehicle SEM (re-
sults of five separate experiments). Statistical analysis was per-
formed by One-way ANOVA followed by Dunnet's multiple
comparison using Graph Pad Prism 5 program (San Diego).
In a first step P-ERK 1/2 was determined after 15 min treatment
of the HL-60 cells with the compounds to be assayed. Successively,
to confirm the capability of the novel derivatives to act as CB2 ag-
onists, a 5 min pre-treatment with the selective CB2 receptor
antagonist AM630 (75 nM) was carried out before the exposure to
the cells to the compounds to be tested or to the reference
cannabinoid agonist WIN55,212-2 (75 nM).
buffer containing 5 mg/mL of fatty acid-free bovine serum albumin.
Non-specific binding was estimated in the presence of 1 mM of CP-
55,940. All binding studies were performed in glass tubes pre-
treated with Sigma-Cote (Sigma Chemical Co. Ltd., Poole, UK), in
order to reduce non-specific binding. The reaction was blocked by
rapid filtration through Whatman (GF/C filters presoaked in 0.5%
polyethyleneimine (PEI) using a Brandell 36-sample harvester
(Gaithersburg, MD, USA). Filters were washed five times with 4 mL
aliquots of ice cold Tris HCl buffer (pH 7.4) containing 1 mg/mL BSA.
The filter bound radioactivity was measured in a liquid scintillation
counter (Trisarb 2900, Packard, Meridien, USA) with 4 mL of scin-
tillation fluid (Ultima Gold MV, Packard).
Acknowledgments
Protein determination was performed by means of Bradford
protein assay using BSA as a standard according to the protocol of
the supplier (Bio-Rad, Milan, Italy).
The authors pay their thanks to Regione Autonoma della Sar-
degna (RAS) for the financial support (RAS: project: CRP-26417).
All experiments were performed in triplicate and results were
confirmed in at least five independent experiments. Data from
radioligand inhibition experiments were analyzed by nonlinear
regression analysis of a Sigmoid Curve using Graph Pad Prism
program. IC50 values were derived from the calculated curves and
converted to Ki values according to previously described pro-
cedures [39].
References
[1] S. Munro, K.L. Thomas, M. Abu-Shaar, Molecular characterization of a pe-
ripheral receptor for cannabinoids, Nature 365 (1993) 61e65.
ꢁ
ꢁ
[2] S. Galiegue, S. Mary, J. Marchand, D. Dussossoy, D. Carriere, P. Carayon,
M. Bouaboula, D. Shire, G. Le Fur, P. Casellas, Expression of central and pe-
ripheral cannabinoid receptors in human immune tissues and leukocyte
subpopulations, Eur. J. Biochem. 232 (1995) 54e61.
~
ꢀ
[3] C. Benito, E. Núnez, R.M. Tolon, E.J. Carrier, A. Rabano, C.J. Hillard, J. Romero,
Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively
overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains,
J. Neurosci. 23 (2003) 11136e11141.
6.2.3. Intrinsic activity by in vitro assays
HL-60 cells were grown at 37 ꢀC in humidified 5% CO2 in RPMI
1640 Medium (Gibco-BRL, Gaithensburg, MA) supplemented with
[4] L.A. Matsuda, S.J. Lolait, M.J. Brownstein, A.C. Young, T.I. Bonner, Structure of a
cannabinoid receptor and functional expression of the cloned cDNA, Nature
346 (1990) 561e564.
[5] J.A. Wagner, Z. Jarai, S. Batkai, G. Kunos, Hemodynamic effects of cannabi-
noids: coronary and cerebral vasodilation mediated by cannabinoid CB1 re-
ceptors, Eur. J. Pharmacol. 423 (2001) 203e210.
[6] N.E. Kaminski, M.E. Abood, F.K. Kessler, B.R. Martin, A.R. Schatz, Identification
of a functionally relevant cannabinoid receptor on mouse spleen cells that is
involved in cannabinoid-mediated immune modulation, Mol. Pharmacol. 42
(1992) 736e742.
[7] W.A. Devane, L. Hanus, A. Breuer, R.G. Pertwee, L.A. Stevenson, G. Griffin,
D. Gibson, A. Mandelbaum, A. Etinger, R. Mechoulam, Isolation and structure
of a brain constituent that binds to the cannabinoid receptor, Science 258
(1992) 1946e1949.
[8] R. Mechoulam, S. Ben-Shabat, L. Hanus, M. Ligumsky, N.E. Kaminski,
A.R. Schatz, A. Gopher, S. Almog, B.R. Martin, D.R. Compton, Identification of an
endogenous 2-monoglyceride, present in canine gut, that binds to cannabi-
noid receptors, Biochem. Pharmacol. 50 (1995) 83e90.
[9] T. Sugiura, S. Kondo, A. Sukagawa, S. Nakane, A. Shinoda, K. Itoh, A. Yamashita,
K. Waku, 2-Arachidonoylglycerol: a possible endogenous cannabinoid recep-
tor ligand in brain, Biochem. Biophys. Res. Commun. 215 (1995) 89e97.
[10] T. Sugiura, Y. Kobayashi, S. Oka, K. Waku, Biosynthesis and degradation of
anandamide and 2-arachidonoylglycerol and their possible physiological
significance, Prostaglandins Leukot. Essent. Fat. Acids 66 (2002) 173e192.
[11] B.F. Cravatt, D.K. Giang, S.P. Mayfield, D.L. Boger, R.A. Lerner, N.B. Gilula,
Molecular characterization of an enzyme that degrades neuromodulatory
fatty-acid amides, Nature 384 (1996) 83e87.
10% Fetal Bovine Serum (FBS), 1
2 mM -Glutamine, 2.5 g/mL Amphotericin B, and 50
Gentamicin. Cells lines from the European Collection of Cell Cul-
tures (ECACC), FBS, Penicillin-Streptomycin, -Glutamine, Ampho-
tericin B, Gentamicin, D-PBS and DMSO were purchased from
SigmaeAldrich (Milan, Italy).
HL-60 cells were treated with the compounds to be assayed or
the reference compounds WIN55,212-2 or AM630 (SigmaeAldrich,
Milan, Italy). Tested and reference compounds were dissolved in
culture medium with 1% DMSO and treatments were performed in
m
g/mL Penicillin-Streptomycin,
L
m
mg/mL
ꢀ
ꢀ
L
a volume of 10 mL/mL of cell suspension.
P-ERK 1/2 expression was determined after HL-60 cell treat-
ment. Western blot analysis was carried out according to the pre-
viously reported general procedure [30]. After appropriate time of
exposure, cells were collected by centrifugation at 1000 ꢂ g and the
resulting pellets were washed in ice-cold PBS buffer by centrifu-
gation at 1000 ꢂ g. The pellet was lysed at 4 ꢀC in HEPES based
buffer containing other components [30]. The extracts were
centrifuged at 10,000 ꢂ g for 15 min at 4 ꢀC. The resulting super-
natant was collected as total cell extracts. Extracted total proteins
were quantified using a Quant-iT™ Protein Assay Kit (Invitrogen™)
by a Qubit Quantitation platform system (Invitrogen™). Each
western blot assay was carried out at fixed total protein amount of
[12] N. Ueda, K. Yamanaka, Y. Terasawa, S. Yamamoto, An acid amidase hydro-
lyzing anandamide as an endogenous ligand for cannabinoid receptors, FEBS
Lett. 454 (1999) 267e270.

G. Pinna et al. / European Journal of Medicinal Chemistry 85 (2014) 747e757
757
[13] A. Hermann, M. Kaczocha, D.G. Deutsch, 2-Arachidonoylglycerol (2-AG)
membrane transport: history and outlook, AAPS J. 8 (2006) 409e412.
[14] C.J. Fowler, Anandamide uptake explained? Trends Pharmacol. Sci. 33 (2012)
181e185.
Dihydroindeno[1,2-c]pyrazole CB2 ligands using molecular hybridization
based on scaffold hopping, Open Med. Chem. J. 6 (2012) 1e14.
[28] M. Portier, M. Rinaldi-Carmona, F. Pecceu, T. Combes, C. Poinot-Chazel,
B. Calandra, F. Barth, G. Le Fur, P.J. Casellas, SR 144528, an antagonist for the
peripheral cannabinoid receptor that behaves as an inverse agonist,
J. Pharmacol. Exp. Ther. 288 (1999) 582e589.
[29] M. Rinaldi-Carmona, F. Barth, M. Heaulme, D. Shire, B. Calandra, C. Congy,
S. Martinez, J. Maruani, G. Neliat, D. Caput, SR141716A, a potent and selective
antagonist of the brain cannabinoid receptor, FEBS Lett. 350 (1994) 240e244.
[30] S. Ruiu, G.A. Pinna, G. Marchese, J.M. Mussinu, P. Saba, S. Tambaro, P. Casti,
R. Vargiu, L. Pani, Synthesis and characterization of NESS 0327: a novel pu-
tative antagonist of the CB1 cannabinoid receptor, J. Pharmacol. Exp. Ther. 306
(2003) 363e370.
[31] G. Murineddu, S. Ruiu, G. Loriga, I. Manca, P. Lazzari, R. Reali, L. Pani, L. Toma,
G.A. Pinna, Tricyclic pyrazoles. Part 3. Synthesis, biological evaluation and
molecular modelling of analogues of the cannabinoid antagonist NESS 0327,
J. Med. Chem. 48 (2005) 7351e7362.
[32] G. Murineddu, S. Ruiu, J.M. Mussinu, G. Loriga, G.E. Grella, M.A.M. Carai,
P. Lazzari, L. Pani, G.A. Pinna, Tricyclic pyrazoles. Part 2. Synthesis and bio-
logical evaluation of novel 4,5-dihydro-1H-benzo[g]indazole-based ligands
for cannabinoid receptors, Bioorg. Med. Chem. 13 (2005) 3309e3320.
[33] A.R. Stoit, J.H.M. Lange, A.P. den Hartog, E. Ronken, K. Tipker, H.H. van Stui-
venberg, J.A.R. Dijksman, H.C. Wals, C.G. Kruse, Design, synthesis and bio-
logical activity of rigid cannabinoid CB1 receptor antagonists, Chem. Pharm.
Bull. 50 (2002) 1109e1113.
[15] N.V. DiPatrizio, D. Piomelli, The thrifty lipids: endocannabinoids and the
neural control of energy conservation, Trends Neurosci. 35 (2012) 403e411.
[16] J. Flygare, K. Gustafsson, E. Kimby, B. Christensson, B. Sander, Cannabinoid
receptor ligands mediate growth inhibition and cell death in mantle cell
lymphoma, FEBS Lett. 579 (2005) 6885e6889.
ꢀ
[17] B. Herrera, A. Carracedo, M. Diez-Zaera, M. Guzman, G. Velasco, p38 MAPK is
involved in CB2 receptor-induced apoptosis of human leukemia cells, FEBS
Lett. 579 (2005) 5084e5088.
[18] G. Pryce, D. Baker, Emerging properties of cannabinoid medicines in man-
agement of multiple sclerosis, Trends Neurosci. 28 (2005) 272e276.
[19] C. Benito, J.P. Romero, R.M. Tolon, D. Clemente, F. Docagne, C.J. Hillard,
C. Guaza, J. Romero, Cannabinoid CB1 and CB2 receptors and fatty acid amide
hydrolase are specific markers of plaque cell subtypes in human multiple
sclerosis, J. Neurosci. 27 (2007) 2396e2402.
[20] D. Baker, G. Pryce, J.L. Croxford, P. Brown, R.G. Pertwee, J.W. Huffman,
L. Layward, Cannabinoids control spasticity and tremor in a multiple sclerosis
model, Nature 404 (2000) 84e87.
[21] G. Murineddu, B. Asproni, G.A. Pinna, Different classes of CB2 ligands poten-
tially useful in the treatment of pain, Recent Pat. CNS Drug Discov. 7 (2012)
4e24.
[22] L. Luogo, E. Palazzo, S. Tambaro, C. Giordano, L. Gatta, M.A. Scafuro, F. Rossi,
P. Lazzari, L. Pani, V. de Novellis, M. Malcangio, S. Maione, 1-(20,40-dichlor-
ophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxamide, a novel CB2 agonist, alleviates neuropathic pain through func-
tional microglial changes in mice, Neurobiol. Dis. 37 (2010) 177e185.
[34] Y. Zhang, J.P. Burgess, M. Brackeen, A. Gilliam, S.W. Mascarella, K. Page,
H.H. Seltzman, B.F. Thomas, Conformationally constrained analogues of N-
(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyr-
azole-3-carboxamide (SR141716): design, synthesis, computational analysis,
and biological evaluations, J. Med. Chem. 51 (2008) 3526e3539.
ꢀ
[23] F.J. Bermudez-Silva, I. Sanchez-Vera, J. Suarez, A. Serrano, E. Fuentes, P. Juan-
ꢀ
ꢀ
Pico, A. Nadal, F. Rodríguez de Fonseca, Role of cannabinoid CB2 receptors in
glucose homeostasis in rats, Eur. J. Pharmacol. 565 (2007) 207e211.
[24] H. Xu, C.L. Cheng, M. Chen, A. Manivannan, L. Cabay, R.G. Pertwee, A. Coutts,
J.V. Forrester, Anti-inflammatory property of the cannabinoid receptor-2-
selective agonist JWH-133 in a rodent model of autoimmune uveoretinitis,
J. Leukoc. Biol. 82 (2007) 532e541.
[35] N. Goudreau, D.R. Cameron, R. Deziel, B. Hache, A. Jakalian, E. Malenfant,
J. Naud, W.W. Ogilvie, J. O'Meara, P.W. White, C. Yoakima, Optimization and
determination of the absolute configuration of a series of potent inhibitors of
human papillomavirus type-11 E1eE2 proteineprotein interaction: a com-
bined medicinal chemistry, NMR and computational chemistry approach,
Bioorg. Med. Chem. 15 (2007) 2690e2700.
ꢀ
[25] J.M. Mussinu, S. Ruiu, A.C. Mule, A. Pau, M.A.M. Carai, G. Loriga, G. Murineddu,
[36] B.G. Raju, R. Giabatti, S.I. Maffioli, U. Singh, G. Romano, E. Michelucci, P.S.
Tiseni, G. Candiani, B.K. O'Dowd U.S. Patent 0211603, Sep 21, 2006.
[37] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and
the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
[38] O. Jbilo, J.M. Derocq, M. Segui, G. Le Fur, P. Casellas, Stimulation of peripheral
cannabinoid receptor CB2 induces MCP-1 and IL-8 gene expression in human
promyelocytic cell line HL60, FEBS Lett. 448 (1999) 273e277.
[39] M. Bouaboula, D. Dussossoy, P.J. Casellas, Regulation of peripheral cannabi-
noid receptor CB2 phosphorylation by the inverse agonist SR 144528. Impli-
cations for receptor biological responses, Biol. Chem. 274 (1999)
20397e20405.
G.A. Pinna, Tricyclic pyrazoles. I. Synthesis and biological evaluation of novel
1,4-dihydroindeno[1,2-c]pyrazol-based ligands for CB1 and CB2 cannabinoid
receptors, Bioorg. Med. Chem. 11 (2003) 251e263.
[26] G. Murineddu, P. Lazzari, S. Ruiu, A. Sanna, G. Loriga, I. Manca, M. Falzoi,
C. Dessì, M.M. Curzu, G. Chelucci, L. Pani, G.A. Pinna, Tricyclic pyrazoles. 4.
Synthesis and biological evaluation of analogues of the robust and selective
CB2 cannabinoid ligand 1-(20,40-dichlorophenyl)-6-methyl-N-piperidin-1-yl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide, J. Med. Chem. 49 (2006)
7502e7512.
[27] G. Murineddu, B. Asproni, S. Ruiu, F. Deligia, M. Falzoi, A. Pau, B.F. Thomas,
Y. Zhang, G.A. Pinna, L. Pani, P. Lazzari, Tricyclic pyrazoles. Part 5. Novel 1,4-