Synthesis, central and peripheral benzodiazepine receptor affinity of pyrazole and pyrazole-containing polycyclic derivatives

Article abstract of DOI:10.1016/j.farmac.2004.05.008

A series of new pyrazole-condensed 6,5,5 tricyclic compounds were synthesized and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Some 1-aryl-5- phenylpyrazole derivatives were also prepared and tested for comparison with their corresponding rigid tricyclic analogs. Among the newly synthesized 1-aryl-1,4-dihydro-indeno[1,2-c]pyrazoles bearing both an ethoxycarbonyl group at position 3 and a carbonyl function at the position 4, compound 4b emerged as a new potent (IC₅₀ = 26.4 nM) and selective CBR ligand. The 4-oxo-1-aryl-1,4-dihydro-indeno[1,2-c]pyrazole diethylamide derivative 14a was instead identified as a relatively potent (IC₅₀ = 124 nM) but highly selective PBR ligand.

Full text of DOI:10.1016/j.farmac.2004.05.008

IL FARMACO 59 (2004) 849–856
http://france.elsevier.com/direct/FARMAC/
Original article
Synthesis, central and peripheral benzodiazepine receptor affinity
of pyrazole and pyrazole-containing polycyclic derivatives
Francesco Campagna a,*, Fausta Palluotto a, Angelo Carotti a, Elisabetta Maciocco b
a Dipartimento Farmacochimico, Università di Bari, via Orabona 4, 70126 Bari, Italy
b Dipartimento di Biologia Sperimentale, Sezione di Neuropsicofarmacologia, Istituto Nazionale di Neuroscienze, Università degli Studi di Cagliari, Italy
Received 15 March 2004; accepted 14 May 2004
Available online 15 September 2004
Abstract
A series of new pyrazole-condensed 6,5,5 tricyclic compounds were synthesized and tested to evaluate their binding affinities at both
central (CBR) and peripheral (PBR) benzodiazepine receptors. Some 1-aryl-5-phenylpyrazole derivatives were also prepared and tested for
comparison with their corresponding rigid tricyclic analogs.Among the newly synthesized 1-aryl-1,4-dihydro-indeno[1,2-c]pyrazoles bearing
both an ethoxycarbonyl group at position 3 and a carbonyl function at the position 4, compound 4b emerged as a new potent (IC50 = 26.4 nM)
and selective CBR ligand. The 4-oxo-1-aryl-1,4-dihydro-indeno[1,2-c]pyrazole diethylamide derivative 14a was instead identified as a
relatively potent (IC50 = 124 nM) but highly selective PBR ligand.
© 2004 Elsevier SAS. All rights reserved.
Keywords: 1-Aryl-1,4-dihydro-indeno[1,2-c]pyrazoles; Central benzodiazepine receptor; Peripheral benzodiazepine receptor; Binding assay; Structure–
activity relationships
1. Introduction
activities. Antagonists do not exhibit, per se, any relevant
biological effect but antagonize the action of agonists and
inverse agonists [6].
Benzodiazepines bind two main classes of receptors, the
central-type benzodiazepine receptor (CBR) and the
peripheral-type benzodiazepine receptor (PBR).
Benzodiazepines, b-carbolines, imidazopyridines, pyra-
zoloquinolines, and imidazoquinoxalines represent major fa-
milies of compounds with high affinity for CBR [7,8].
Among them, flunitrazepam I (Chart 1) and pyrazoloquino-
lines, such as CGS 9896 II, exhibit high affinity for CBR but
not for PBR, while benzodiazepine III (diazepam) and imi-
dazopyridine IV (alpidem) bind with high affinity to both
CBR and PBR.
Several pharmacophore models have been proposed for
CBR, and among them, the one developed by Cook et al. [7]
for agonists, antagonists and inverse agonists received much
consensus. It consists of four essential features: a lipophilic
region (L1), two hydrogen-bond donor sites (HB1 and HB2)
and a hydrogen-bond acceptor site (A2). Lipophilic regions
named L2, L3 and LDi have been identified as three additio-
nal, but not essential, binding regions. A schematic represen-
tation of this pharmacophore model is reported in Fig. 1.
PBR is ubiquitously expressed throughout the body espe-
cially in steroid producing tissues, such as adrenal, testis,
placenta and brain. It is localised mainly in the mitochondria
CBR, which is present exclusively in the central nervous
system (CNS), has been identified as part of GABAA-
receptor/Cl– ionophore supramolecular complex [1,2], a pen-
tameric protein, formed by different combinations of 21 dis-
tinct subunits (6a, 4b, 4c, 1e, 1d, 3q 1ϖ, and 1p), 16 of which
have been found in the mammalian CNS [3–5]. Allosteric
modulation of GABAA by CBR ligands involves three dis-
tinct events: ligand binding to recognition site, transduction
of the signal to the GABA effector site, modification in
GABA-gated conductance.
Agonists (GABA-positive ligands), increasing the fre-
quency of Cl– channel opening, induce sedative/hypnotic,
muscle relaxant, anticonvulsant and anxiolytic activities. In-
verse agonists (GABA-negative ligands) decrease channel
open frequency and display (pro)convulsant and anxiogenic
* Corresponding author. Fax: +39-080-544-2724.
E-mail address: Campagna@farmchim.uniba.it (F. Campagna).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.05.008

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F. Campagna et al. / IL FARMACO 59 (2004) 849–856
Chart 1
.
and its role in several biological functions, such as the regu-
lation of cell proliferation, mitochondrial oxidative phospho-
rylation, steroidogenesis, immune and inflammatory respon-
ses, has been recognized [9–16].
Many compounds have been discovered which bind to
PBR with varying affinities. Among them, Ro 5-4864 V
(Chart 1) and PK 11195 VI are noteworthy, due to their high
PBR affinity and PBR/CBR selectivity. The pharmacophore
models recently developed for PBR ligands converge in iden-
tifying four relevant features: three hydrophobic moieties
(L1, L3 and L4) and a hydrogen-bond donor group (HB2) as
shown in Fig. 2 [17–19].
In recent years, we have extensively investigated new
central and peripheral benzodiazepine receptors ligands built
on the pyridazino[4,3-b]indole or indeno[1,2-c]pyridazine
nuclei (Fig. 3). The highest CBR affinity and selectivity was
Fig. 3. Pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines.
found for 2-aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)-
ones, which possess structural features fitting the pharmaco-
phore requirements. Interestingly, these compounds showed
in vivo a full spectrum of intrinsic activities spanning from
full agonism to inverse agonism [20–22].
2-Aryl-pyridazino[4,3-b]indole and 2-aryl-indeno[1,2-
c]pyridazine derivatives bearing an ethoxycarbonyl or a car-
boxamido group at C-4 did not show any affinity for CBR,
whereas in contrast they showed a selective, but weak, bin-
ding to PBR. Within that research program, relatively good
PBR binding affinities had been found for compounds belon-
ging to the 3-arylmethyloxy-pyridazinoindole series [23].
As a continuation of our ongoing research in this field, we
synthesized new pyrazole-condensed 6,5,5 tricyclic com-
pounds (3a–e, 4a–e, 8b, 9d, 10d, 13a, 14a,b, and 18a–c)
and measured their binding affinity at both CBR and PBR
(Table 2). 1-Aryl-5-phenylpyrazole derivatives 21a–e, were
also prepared and tested for comparison with their correspon-
ding rigid tricyclic analogs.
Fig. 1. Schematic representation of CBR pharmacophore model proposed
by Cook [7]. The molecular structure of flunitrazepam is sketched to help
interpretation.
2. Chemistry
Scheme 1 outlines the synthetic pathway leading to ethyl
1-aryl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxylates 3a–e
and 4a–e. In short, ethyl (3-hydroxy-1H-inden-2-yl)(oxo)-
acetate 2 was prepared by reacting the 1-indanone 1 with
Fig. 2. Schematic representation of PBR pharmacophore model as proposed
by different groups [16–18]. The molecular structure of PK11195 is sket-
ched to help interpretation.

F. Campagna et al. / IL FARMACO 59 (2004) 849–856
851
Table 1
Physical and spectroscopic data of newly synthesized compounds (3b–e, 4a–d, 7b, 8b, 9d, 10d, 12a–b, 13a, 14a–b, 16, 17a–c, 18a–c, 21c)
Compound
m.p. (°C)
IR mmax (cm–1
)
1H-NMR, d (ppm), J (Hz)
(crystallization solvent)
215–216
(ethanol)
3b
1730, 1500
1.71 (t, 3H, CH3, J = 7.1); 4.10 (s, 2H, CH2); 4.73 (q, 2H, CH2, J = 7.1);
7.50–7.60 (m, 2H, Arom); 7.65–7.70 (m, 1H, Arom); 7.75–7.85 (m, 3H,
Arom); 7.90–8.00 (m, 2H, Arom)
3c
141–142
(ethanol)
1725, 1595
1.45 (t, 3H, CH3, J = 7.1); 3.84 (s, 2H, CH2); 4.48 (q, 2H, CH2, J = 7.1);
7.25–7.35 (m, 2H, Arom): 7.40–7.60 (m, 4H, Arom); 7.65–7.70 (m, 1H,
Arom); 7.80–7.85 (m, 1H, Arom)
3d
3e
131–133
(ethanol)
201–202
(ethanol)
1735, 1535, 1520
1725, 1500
1.45 (t, 3H, CH3, J = 7.1); 3.85 (s, 2H, CH2), 4.45 (q, 2H, CH2, J = 7.1);
6.95–7.00 (m, 1H,Arom), 7.15–7.35 (m, 3H,Arom); 7.40–7.65 (m, 4H,Arom)
1.45 (t, 3H, CH3, J = 7.1); 3.83 (s, 2H, CH2); 4.47 (q, 2H, CH2, J = 7.1);
7.25–7.40 (m, 2H, Arom); 7.40–7.50 (m, 1H, Arom); 7.50–7.60 (m, 1H,
Arom); 7.60–7.80 (m, 4H, Arom)
4a
4b
4c
207–209
1740, 1720, 1610, 1520, 1480 1.46 (t, 3H, CH3, J = 7.1); 4.48 (q, 2H, CH2, J = 7.1); 7.10-7.40 (m, 4H, Arom);
7.50-7.75 (m, 5H, Arom)
(chloroform/hexane)
197–200 °C
(ethanol)
175–180 °C
(ethanol)
1740, 1720, 1600
1.44 (t, 3H, CH3, J = 7.2); 4.47 (q, 2H, CH2, J = 7.2); 7.10–7.15 (m, 1H,Arom);
7.30–7.40 (m, 2H,Arom); 7.50–7.60 (m, 2H,Arom); 7.60–7.70 (m, 3H,Arom)
1.47 (t, 3H, CH3, J = 7.1); 4.50 (q, 2H, CH2, J = 7.1); 7.15–7.20 (m, 1H,Arom);
7.35–7.40 (m, 2H, Arom); 7.50–7.55 (m, 2H, Arom); 7.60–7.70 (m, 2H,
Arom); 7.75–7.80 (m, 1H, Arom)
1735, 1715, 1600, 1590
4d
7b
162–164 °C
(ethanol)
193–197 °C
(ethanol)
1730, 1715, 1525
3250, 1705, 1650
1.45 (t, 3H, CH3, J = 7.1); 4.50 (q, 2H, CH2, J = 7.1); 6.65–6.75 (m, 1H,Arom);
7.25–7.35 (m, 3H, Arom); 7.45–7.70 (m, 4H, Arom)
6.34 (s, 1H, NH); 6.70–6.80 (m, 2H, Arom); 7.20–7.30 (m, 2H, Arom);
7.60–7.70 (m, 2H, Arom); 7.70–7.90 (m, 2H, Arom); 7.91(s, 1H, CH); 9.90–
10.20 (br, 1H, NH)
8b
9d
220–221 °C
(ethanol)
129–130 °C
(diethyl ether)
1715, 1610, 1520
1775, 1725, 1610
7.10–7.20 (m, 1H, Arom); 7.30–7.35 (m, 2H, Arom); 7.50–7.65(m, 5H,
Arom); 7.65 (s, 1H, CH)
1.05 (t, 3H, CH3, J = 7.1); 1.20–1.30 (m, 2H, CH2); 1.41 (d, 3H, CH3, J = 6.3);
5.10–5.30 (m, 1H, CH); 6.60–6.80 (m, 1H, Arom); 7.20–7.35 (m, 2H, Arom);
7.45–7.60 (m, 2H, Arom); 7.60–7.70 (m, 3H, Arom)
10d
12a
12b
13a
124–125 °C
(diethyl ether)
236–238 °C
(dioxane)
299–300 °C dec
(chloroform)
124–126 °C
(ethanol)
1735, 1720, 1615
3420, 1725, 1600
3500–2500 br, 1725, 1600
1730, 1630
1.24 (d, 6H, 2CH3, J = 6.8); 1.55–1.60 (m, 1H, CH); 4.21 (d, 2H, CH2, J = 6.8);
7.20–7.35 (m, 3H,Arom); 7.45–7.60 (m, 2H,Arom); 7.60–7.70 (m, 3H,Arom)
7.09–7.18 (m, 1H, Arom); 7.35–7.50 (m, 2H, Arom); 7.50–7.70 (m, 4H,
Arom); 7.70–7.80 (m, 2H, Arom)
7.17 (d, 1H, Arom, J = 7.0); 7.40–7.50 (m, 2H, Arom); 7.57 (d, 2H, Arom,
J = 6.70); 7.73 (d, 1H, Arom, J = 8.6); 7.83 (d, 2H, Arom, J = 8.6)
1.20–1.40 (m, 6H, 2CH3); 3.60 (q, 2H, CH2, J = 7.1); 3.85 (s, 2H, CH2); 3.95
(q, 2H, CH2, J = 7.1); 7.25–7.35 (m, 3H, Arom); 7.40–7.60 (m, 4H, Arom);
7.70–7.80 (m, 2H, Arom).
1.22 (t, 3H, CH3, J = 7.1); 1.25 (t, 3H, CH3, J = 7.1); 3.55 (q, 2H, CH2, J = 7.1),
3.61 (q, 2H, CH2, J = 7.1); 7.20-7.40 (m, 4H, Arom); 7.50-7.80 (m, 5H, Arom)
1.20 (t, 3H, CH3, J = 7.1); 1.27 (t, 3H, 3H, CH3, J = 7.1); 3.52 (q, 2H, CH2,
J = 7.1); 3.59 (q, 2H, CH2, J = 7.1); 7.15–7.20 (m, 1H, Arom); 7.30–7.35 (m,
2H, Arom); 7.52–7.60 (m, 2H, Arom); 7.60–7.70 (m, 3H, Arom)
1.50 (t, 3H, CH3, J = 7.0); 4.56 (q, 2H, CH2, J = 7.0); 7.20–7.35 (m, 1H,Arom);
7.48 (d, 1H, Arom, H, J = 8.4); 7.55–7.655 (m, 1H, Arom); 7.84 (d, 1H, Arom,
J = 8.6); 11.83 (s, 1H, OH)
0.76 (t, 3H, CH3, J = 7.1); 4.00 (q, 2H, CH2, J = 7.1); 5.31 (s, 1H, NH);
7.00–7.15 (m, 2H, Arom); 7.20–7.40 (m, 4H, Arom); 7.60–7.80 (m, 2H,
Arom), 12.53 (s, 1H, OH)
0.78 (t, 3H, CH3, J = 7.1); 4.00 (q, 2H, CH2, J = 7.1); 5.28 (s, 1H, NH);
7.10–7.50 (m, 6H, Arom); 7.60–7.80 (m, 2H, Arom), 12.53 (s, 1H, OH)
0.75 (t, 3H, CH3, J = 7.1); 4.01 (q, 2H, CH2, J = 7.1); 5.27 (s, 1H, NH);
7.10–7.70 (m, 8H, Arom); 12.53 (s, 1H, OH)
14a
14b
113–115 °C
(chloroform/hexane)
145–146 °C
1715, 1645
1715, 1630
(ethyl acetate)
16
132–133 °C
(ethanol)
1690, 1640, 1620, 1600
3210, 1725, 1700, 1615
3450, 1730, 1685
17a
129–131 °C
(ethanol)
17b
17c
18a
18b
18c
21c
135–137 °C
(ethanol)
164–166 °C
(ethanol)
3600–3200, 1716, 1700,
1615
1730, 1595
193–194 °C
(ethanol)
1.50 (t, 3H, CH3, J = 7.1); 4.54 (q, 2H, CH2, J = 7.1); 7.30–8.0 (m, 9H, Arom)
203–206 °C
(ethanol)
1730, 1500
1730, 1595
1710, 1590
1.49 (t, 3H, CH3, J = 7.1); 4.53 (q, 2H, CH2, J = 7.1); 7.3–7.9 (m, 8H, Arom)
153–154 °C
(ethanol)
1.50 (t, 3H, CH3, J = 7.1); 4.56 (q, 2H, CH2, J = 7.1); 7.25–7.55 (m, 4H,Arom);
7.65–7.75 (m, 1H,Arom); 7.80–7.90 (m, 2H,Arom); 7.95–8.00 (m, 1H,Arom)
1.43 (t, 3H, CH3, J = 7.1); 4.46 (q, 2H, CH2, J = 7.1); 7.00–7.50 (m, 10H,
Arom)
71–73 °C
(ethanol)
1H-NMR spectra were recorded in CDCl3 (3b–e, 4a–d, 7b, 8b, 9d, 10d, 13a, 14a–b, 16, 17a–c, 18a–c, 21c) or in DMSO-d6 (12a,b).

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F. Campagna et al. / IL FARMACO 59 (2004) 849–856
Table 2
Binding data of pyrazoles and pyrazole-containing polycyclic derivatives
Compound
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
8b
9d
10d
13a
14a
14b
18a
18b
18c
21a
21b
21c
X
Y
R
H
CBR a
PBR b
CH2 COOEt
CH2 COOEt
CH2 COOEt
CH2 COOEt
CH2 COOEt
11 0.5%
45 4%
40 3%
31 3%
26 1.3%
6% 0.4
750 60
10 1%
671 45
12 1.1%
25 0.8%
7 0.5%
435 35%
23 2%
552 37
124 10
460 25
5 0.3%
17 1.4%
4 0.3%
2 0.1%
3 0.2%
8 0.5%
2 0.2%
4–Cl 0%
3–Cl 4 0.3%
2–Cl 33 2% c
4–Br 0%
Scheme 2
.
CO
CO
CO
CO
CO
CO
CO
CO
COOEt
COOEt
COOEt
COOEt
COOEt
H
H
137 10
4–Cl 26.4 1.5
3–Cl 51.5 2.5
2–Cl 670 50
4–Br 141 12
4–Cl 9 0.5%
2–Cl 30 1.5% c
2–Cl 29 2 c
tic acid to give the corresponding 4-oxo derivatives 4a–e. The
already known compounds 3a [26] and 4e [27] were obtained
through the reported procedure [26] and through the new
method described herein, respectively.
The synthesis of 1-(4-chlorophenyl)-indeno[1,2-c]-
pyrazol-4(1H)-one 8b was accomplished according to the
method reported in Ref. [28] for the preparation of the
4-phenyl derivative (Scheme 2).
Sec and isobutyl esters 9d and 10d were prepared by
transesterification of the ethyl ester 4d with sodium sec- and
isobutoxide, respectively, in anhydrous toluene (Scheme 3).
Diethylcarboxamides 13a and 14a,b were prepared
through a preliminary hydrolysis of esters 3a and 4a,b to
carboxylic acids 11a [26] and 12a,b, respectively, followed
by a transformation to the corresponding acid chlorides and,
finally, a reaction with diethylamine (Scheme 3).
COO–sBut
COO–iBut
CH2 CONEt2
H
H
0%
0%
CO
CO
O
O
O
–
CONEt2
CONEt2
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
4–Cl 0%
H
38 2.5%
4–Cl 0%
3–Cl 22 1.2%
H
581 50
–
4–Cl 0%
3–Cl 5 0.3%
4–Br 0%
11.5 1.1
–
21e
Diazepam
PK 11195
–
3.3 0.5
Ethyl
1-aryl-1H-[1]benzofuro[3,2-c]pyrazol-3-carbo-
Each value is the mean SEM of three determinations.
xylates 18a–c were prepared as outlined in Scheme 4. Ethyl
(3-hydroxy-1-benzofuran-2-yl) (oxo) acetate 16, obtained
from 1-benzofuran-3-(2H)-one 15 and diethyl oxalate, was
condensed with the appropriate arylhydrazine hydrochloride
a Inhibition of [3H] flunitrazepam binding expressed as IC50 (nM) or as
percentage of inhibition at 1 µM concentration.
b Inhibition of [3H] PK 11195 binding expressed as IC50 (nM) or as
percentage of inhibition at 1 µM concentration.
c Percentage of inhibition at 10 µM concentration.
diethyl oxalate in the presence of sodium ethoxide according
to reported methods [24,25].
Compound 2 was subsequently condensed with the appro-
priate arylhydrazine hydrochloride to afford the ethyl 1-aryl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxylates
3a–e,
which were oxidized by chromic anhydride in refluxing ace-
Scheme 1
.
Scheme 3.

F. Campagna et al. / IL FARMACO 59 (2004) 849–856
853
carbonyl group at position 3 and the carbonyl function at the
position 4, bind with a good affinity to the CBR. In fact,
indanones 4a–e are more active than the corresponding in-
deno 3a–e and benzofuropyrazoles analogs 18a–c, both lac-
king the carbonyl group at position 4.
Interestingly, also compound 8b, in which the ethoxycar-
bonyl group at position 3 is missing, was also inactive. The
role played by the above chemical groups in the receptor
binding is noteworthy. As a matter of fact, when a methylene
group (compounds 3a–e) or an oxygen atom (compounds
18a–c) are present at position 4 or when position 3 is unsubs-
tituted (i.e., compound 8b), the receptor affinity dramatically
decreases, and this may indicate that two hydrogen bonds,
likely involving the carbonyl oxygens at the 3 and 4 positions
and the HB1 and HB2 donor sites on the receptor, may be
formed.
Scheme 4
.
to afford the arylhydrazones 17a–c, which furnished the
ethyl 1-aryl-1H-[1]benzofuro[3,2-c]pyrazole-3-carboxy-
Another important observation can be made by analyzing
the different effect arising form the introduction of a chloro
substituent in the N-1 phenyl ring. For ligands 4, the intro-
duction of a Cl substituent at the para and meta positions (but
not at the ortho position) determined a significant improve-
ment of the binding affinity whereas for the other two classes
of ligands the affinity remained unchanged or even dimi-
nished (compare 3b vs. 3a, 14b vs. 14a and 18b vs. 18a).
Taking these results into account, we could hypothesize that
the N-1 aryl ring probably interacts with L1 lipophilic region
of CBR.
lates 18a–c, by refluxing in toluene in the presence of
p-toluenesulfonic acid.
Ethyl 1,5-diaryl-1H-pyrazole-3-carboxylates 21a–c,e
were synthesized (Scheme 5) by condensation of the corres-
ponding arylhydrazine hydrochloride with the ethyl (3Z)-4-
hydroxy-2-oxo-4-phenylbut-3-enoate 20, prepared from ace-
tophenone and diethyloxalate according to a reported method
[29]. Compounds 21a,b,e have been already prepared by
others through a different route [30,31].
Physical and spectroscopic data of the newly synthesized
compounds are reported in Table 1.
Bulky substituents in position 3 do not favour CBR bin-
ding since the replacement of the ethoxycarbonyl substituent
with 3-sec(iso)-butyloxycarbonyl-(compounds 9d, 10d) or
with 3-diethylcarboxamido-(compounds 14a,b) groups re-
sulted in a marked drop of the CBR affinity. Conversely, an
increase of affinity was observed at the PBR. In particular, a
carboxamide function similar to that present in PK11195,
showed a preferential binding to PBR. A comparison of the
binding data of amides 14a,b with those of esters 4a,b sug-
gests that N,N-disubstituted amidic groups might engage a
better interaction with the lipophilic region L4 of PBR.
The presence of a carbonyl function at position 4 results
less critical for binding to PBR than CBR. However, it must
say that a significantly higher affinity was shown by the
indanone derivatives 4a,c and 14a (IC50, 124 nM) in compa-
rison with the corresponding indeno derivatives 3a,c and 13a
(IC50, 552 nM).
3. Binding studies
All the synthesized compounds were evaluated for their in
vitro binding affinity at CBR and PBR by means of a binding
assay using [3H]flunitrazepam and [3H]PK11195, respecti-
vely, as radioligands and cerebral cortices from male Spra-
gue–Dawley rats as receptor source [32,33]. Binding data,
reported in Table 2, are expressed as IC50 (nM) or as percen-
tage of inhibition of specific radioligand binding at 1 µM
compound concentration.
4. Results and discussion
From the data shown in Table 2 it appears that only the
indeno[1,2-c]pyrazole derivatives, bearing both the ethoxy-
Unlike the 6,5,5 rigid tricyclic analogs, pyrazole-1,5-
diphenyl derivatives 21a–e did not display any CBR and/or
PBR affinity.
5. Conclusions
Among the newly synthesized 1-aryl-1,4-dihydro-
indeno[1,2-c]pyrazoles, compound 4b emerged as a new
potent (IC50 = 26.4 nM) and selective CBR ligand whereas
the 4-oxo-1-aryl-1,4-dihydro-indeno[1,2-c]pyrazole diethy-
lamide derivative 14a was identified as a less potent
(IC50 = 124 nM) but highly selective PBR ligand.
Scheme 5
.

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F. Campagna et al. / IL FARMACO 59 (2004) 849–856
The key importance of the ethoxycarbonyl substituent in
indeno[1,2-c]pyrazole-3-carboxylate 3a–e (0.5 mmol) in
acetic acid (12.5 ml) and the mixture refluxed for 48 h. After
cooling, the reaction mixture was poured on ice and extracted
with chloroform. The extracts were dried over anhydrous
sodium sulfate and evaporated to dryness giving a oil residue,
which was purified by chromatography (4a: eluent mixture,
ethyl acetate/petroleum ether 3:7, Rf = 0.32, 0.034 g, 21%
yield; 4b: eluent mixture, ethyl acetate/petroleum ether 3:7,
Rf = 0.3, 0.035 g, 20% yield; 4c: eluent mixture, ethyl
acetate/petroleum ether 3:7, Rf = 0.37, 0.035 g, 20% yield;
4d: eluent mixture, ethyl acetate/petroleum ether 4:6,
Rf = 0.42; 0.035 g, 20% yield; 4e: eluent mixture ethyl
acetate/petroleum ether 3:7, Rf = 0.43, 0.040 g, 22% yield).
position 3 and of a carbonyl group in position 4 for a high
CBR affinity and of a N,N-diethylamidic group in position
3 and, to a lesser extent, of a 4-carbonyl group for an accep-
tably high PBR affinity was clearly pointed out.
As far as the pharmacophore for CBR ligands is concer-
ned, our findings support the key role of a lipophilic interac-
tion at the L1 region and of hydrogen bond formation at the
HB1, HB2 donor sites. The good CBR activity of compound
4b would deserve in vivo pharmacological studies, whereas
the switching of the selectivity toward PBR triggered by the
replacement of the COOEt with the CON(Et)2 group (com-
pare 4a vs. 14a and 4b vs. 14b) call for further structural
modifications of the N,N-alkylamidic groups aimed at an
optimization of the lipophilic interaction at the L4 region of
PBR. We are presently working along these two research
lines.
6.1.3. 2-{[2-(4-Chlorophenyl)hydrazono]methylene}-1H-
indene-1,3(2H)-dione 7b
4-Chlorophenylhydrazine (0.596 g, 4.2 mmol) in
1-butanol (4 ml) was slowly added with stirring to a solution
of 2-[(dimethylamino)methylene]-1H-indene-1,3(2H)-dione
6 [28] (0.804 g, 4 mmol) in 1-butanol (16 ml) and acetic acid
(0.6 ml). The resulting solution was stirred at 0 °C for 1 h.
The precipitate was filtered and recrystallized from ethanol
to furnish compound 7b (0.597 g, 50% yield).
6. Experimental
6.1. Chemistry
Melting points were taken on a Gallenkamp MFB
595010 M, apparatus and are uncorrected. Elemental analy-
ses were performed on a Carlo Erba 1106 analyser for C, H,
N; experimental results agreed to within 0.40% of the
theoretical values. IR spectra were recorded using potassium
bromide disks on a Perkin Elmer 283 spectrophotometer,
only the most significant and diagnostic absorption bands
being reported. 1H NMR spectra were recorded on a Bruker
AM 300 WB 300 MHz or on a Varian 300 Mercury spectro-
meters. Chemical shifts are expressed in d (ppm) and the
coupling constants J in Hz. The following abbreviations were
used: s, singlet; d, doublet; t, triplet; m, multiplet; dd, double
doublet. Exchange with deuterium oxide was used to identify
OH and NH protons, which in some cases gave broad signals
widely spread on the base line and thus very difficult to
detect. Reagents and solvents were purchased from Sigma
(Aldrich).
6.1.4. 1-(4-Chlorophenyl)-indeno[1,2-c]pyrazol-4(1H)-one
8b
A
suspension of 2-{[2-(4-chlorophenyl)hydrazono]-
methylene}-1H-indene-1,3(2H)-dione 7b (0.343 g,
1.15 mmol) and p-toluensulfonic acid (0.006 g) in anhydrous
toluene (17 ml) was refluxed in a Dean–Stark apparatus for
3 h, cooled, washed with 1 N sodium hydroxide and water,
dried over sodium sulfate and evaporated under reduced
pressure. The residue was crystallized from ethanol (0.273 g,
85% yield).
6.1.5. Sec-butyl 1-(2-chlorophenyl)-4-oxo-1,4-dihydro-
indeno[1,2-c]pyrazole-3-carboxylate 9d and isobutyl 1-(2-
chlorophenyl)-4-oxo-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxylate 10d
The solution of sodium butoxide in anhydrous
toluene—prepared from anhydrous toluene (17 ml), sodium
(0.008 g) and isobutyl alcohol (0.77 ml) or sec-butyl alcohol
(0.77 ml)—was added drop wise to a suspension of ethyl
1-(2-chlorophenyl)-4-oxo-1,4-dihydroindeno[1,2-c]pyrazole-
3-carboxylate 4d (0.088 g, 0.25 mmol) in anhydrous toluene
(0.84 ml). The mixture was stirred at room temperature for
2–3 h, then was diluted with chloroform and washed with
cold water. The organic phase was evaporated to dryness,
dried over anhydrous sodium sulfate and the residue purified
by chromatography, using ethyl acetate/petroleum ether 4:6
as the eluent mixture (9d: 0.029 g, 30% yield, Rf = 0.53; 10d:
0.073 g, 77% yield, Rf = 0.53).
6.1.1. Ethyl 1-aryl-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxylates 3b–e
Arylhydrazine hydrochloride (6 mmol) was added to a
solution of ethyl (3-hydroxy-1H-inden-2-yl)(oxo)acetate 2
[23–25] (1.16 g, 5 mmol) in anhydrous ethanol (33 ml). The
reaction mixture was refluxed for 1 h 30 min. After cooling,
the precipitate was filtered, washed with ethanol and recrys-
tallized from ethanol affording: 3b 1.34 g, 77% yield; 3c
1.28 g, 75% yield; 3d 1.45 g, 85% yield; 3e 1.10 g, 58%
yield).
6.1.2. Ethyl 1-aryl-4-oxo-1,4-dihydroindeno[1,2-
c]pyrazole-3-carboxylates 4a–e
6.1.6. 1-Aryl-4-oxo-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxylic acids 12a,b
A solution of CrO3 in acetic acid (10%, 4 ml) was added
drop wise to a solution of ethyl 1-aryl-1,4-dihydro-
A solution of NaOH (0.618 g, 15.5 mmol) in ethanol
(77 ml) was added to a suspension of ethyl 1-aryl-4-oxo-

F. Campagna et al. / IL FARMACO 59 (2004) 849–856
855
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxylate
4a,b
(1.55 mmol). The reaction mixture was refluxed for 2 h. The
solvent was evaporated under reduced pressure and the resi-
due suspended in water.After acidification with concentrated
HCl, the precipitate was filtered, washed with water and
recrystallized from ethanol (12a: 0.313 g, 70% yield; 12b:
0.452 g, 90% yield).
6.1.11. Ethyl 1-aryl-1H-[1]benzofuro[3,2-c]pyrazole-3-
carboxylate 18a–c
To a solution of ethyl (2Z)-(3-hydroxy-1-benzofuran-2-
yl)(arylhydrazono) acetate 17a–c (0.80 mmol) in toluene
(5 ml), p-toluenesulfonic acid (catalytic amount) was added.
The solution was refluxed for 3 h. After cooling to 0 °C, the
resulting precipitate was filtered, washed with toluene and
crystallized from ethanol (18a, 0.134 g, 55% yield; 18b,
0.136 g, 50% yield; 18c, 0.040 g, 32% yield).
6.1.7. N,N-diethyl-1-phenyl-1,4-dihydroindeno[1,2-c]pyra-
zole-3-carboxamide 13a
The mixture of 1-phenyl-1,4-dihydroindeno[1,2-c]pyra-
zole-3-carboxylic acid 11a (0.150 g, 0.543 mmol) and thio-
nyl chloride (7.1 ml, 97 mmol) was refluxed for 7 h. The
unreacted thionyl chloride was evaporated and to the residue,
suspended in anhydrous THF (10 ml), the diethylamine
(0.021 ml, 0.4 mmol) was added. The mixture was stirred at
room temperature for 2–3 h. After washing with diluted HCl,
the organic phase was evaporated to dryness and the residue
purified by crystallization from ethanol to give 13a (0.130 g,
72% yield).
6.1.12. Ethyl 1,5-diaryl-1H-pyrazole-3-carboxylate
21a–c,e
Arylhydrazine hydrochloride (0.9 mmol) was added to a
solution of ethyl (3Z)-4-hydroxy-2-oxo-4-phenylbut-3-
enoate 20 [29]. in anhydrous ethanol (5 ml). The mixture was
refluxed for 1 h. After cooling at 0 °C, the resulting precipi-
tate was filtered and crystallized from ethanol (21a: 0.081 g,
40% yield; 21b: 0.091 g, 40% yield, 21c: 0.096 g, 42% yield,
21e: 0.114 g, 50% yield).
6.1.8. N,N-diethyl-1-aryl-4-oxo-1,4-dihydroindeno[1,2-
c]pyrazole-3-carboxamide 14a,b
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