2-Arylpyrazolo[1,5-a]pyrimidin-3-yl acetamides. New potent and selective peripheral benzodiazepine receptor ligands

Article abstract of DOI:10.1016/S0968-0896(01)00192-4

A new class of N,N-diethyl-(2-arylpyrazolo[1,5-a]pyrimidin-3-yl)acetamides (3f-y), as azaisosters of Alpidem, was prepared following a novel synthetic method and their affinities for both the peripheral (PBR) and the central (CBR) benzodiazepine receptors were evaluated. Binding assays were carried out using both [³H]PK 11195 and [³H]Ro 5-4864 as radioligands for PBR, whereas [³H]Ro 15-1788 was used for CBR, in rat kidney and rat cortex, respectively. The tested compounds exhibited a broad range of binding affinities from as low as 0.76 nM to inactivity and most of them proved to be high selective ligands for PBR. The preliminary SAR studies suggested some of the structural features required for high affinity and selectivity; particularly the substituents on the pyrimidine moiety seemed to play an important role in PBR versus CBR selectivity. A subset of the highest affinity compounds was also tested for their ability to stimulate steroid biosynthesis in C6 glioma rat cells and some of these were found to increase pregnenolone formation with potency similar to Ro 5-4864 and PK 11195.

Full text of DOI:10.1016/S0968-0896(01)00192-4

Bioorganic & Medicinal Chemistry 9 (2001) 2661–2671
2-Arylpyrazolo[1,5-a]pyrimidin-3-yl Acetamides. New Potent
and Selective Peripheral Benzodiazepine Receptor Ligands
Silvia Selleri,a Fabrizio Bruni,a,* Camilla Costagli,a Annarella Costanzo,a
Gabriella Guerrini,a Giovanna Ciciani,a Barbara Costab and Claudia Martinib
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Firenze, Via G. Capponi 9, 50121 Firenze, Italy
`
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b
Received 6 February 2001; accepted 23 May 2001
Abstract—A new class of N,N-diethyl-(2-arylpyrazolo[1,5-a]pyrimidin-3-yl)acetamides (3f–y), as azaisosters of Alpidem, was pre-
pared following a novel synthetic method and their affinities for both the peripheral (PBR) and the central (CBR) benzodiazepine
receptors were evaluated. Binding assays were carried out using both [3H]PK 11195 and [3H]Ro 5-4864 as radioligands for PBR,
whereas [3H]Ro 15-1788 was used for CBR, in rat kidney and rat cortex, respectively. The tested compounds exhibited a broad
range of binding affinities from as low as 0.76 nM to inactivity and most of them proved to be high selective ligands for PBR. The
preliminary SAR studies suggested some of the structural features required for high affinity and selectivity; particularly the sub-
stituents on the pyrimidine moiety seemed to play an important role in PBR versus CBR selectivity. A subset of the highest affinity
compounds was also tested for their ability to stimulate steroid biosynthesis in C6 glioma rat cells and some of these were found to
increase pregnenolone formation with potency similar to Ro 5-4864 and PK 11195. # 2001Elsevier Science Ltd. All rights
reserved.
Introduction
although non-mitochondrial distribution of the PBR
has also been described in various tissues.15 The PBR is
designated as a receptor because of the binding of spe-
cific endogenous ligands implicated in several physi-
ological functions,16,17 including steroidogenesis,18
calcium homeostasis,19 cell growth and differentiation,20
regulation of immune function (chemotaxis, apopto-
sis),21,22 mitochondrial respiratory control,23 and
alteration of protooncogene expression.24 Although the
pharmacological role of this receptor remains to be fully
clarified, some evidence indicates its involvement in
important cellular functions such as the production of
neurosteroids (pregnenolone, progesterone, dehy-
droepiandrosterone, their metabolites and sulfate
esters).25,26 Indeed the PBR, when occupied by the
proper ligand, has been shown to facilitate the rate-lim-
iting translocation of cholesterol from the outer to the
inner mitochondrial membrane. In the mitochondria,
cholesterol is converted by cytochrome P450scc into
pregnenolone, which is the parent molecule of
endogenous steroids.
It is well known that, besides binding to the GABAA
receptors in the central nervous system (CNS), benzo-
diazepines (BZs) also bind to various sites located in
peripheral tissues. This second class of recognition sites
is often called the peripheral benzodiazepine receptor
(PBR), since it was originally discovered outside the
CNS.1 Other names have been used to describe this
same class of drug binding sites: mitochondrial benzo-
diazepine receptor (MBR),2,3 mitochondrial DBI recep-
tor complex (mDRC),4,5 o3 receptor,6 PBS,7 and p-
site.8 The PBR is pharmacologically distinct from the
central benzodiazepine receptor (CBR) which is asso-
ciated with GABAA-regulated ion-channel in the CNS.9
Although the Bz GABA-independent receptor was
identified in the CNS, mainly in the astrocytes,10,11 it is
particularly plentiful in endrocrine tissues:12 testis,
ovary, and adrenal cortex, but also in kidney, heart,13
and erytrocytes.14 Its subcellular location was reported
to be mainly in the mitochondrial outer membrane,
It is well known that neurosteroids influence activity
and plasticity of neurons and glial cells in their early
development and continue to exert trophic effects in the
*Corresponding author. Tel.: +39-55-275-7291x296; fax: +39-55-
240776; e-mail: silvia.selleri@unifi.it
0968-0896/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00192-4

2662
S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
adult CNS.27 Moreover, PBR density alters in several
neurological dysfunctions (hepatic encephalopathy, cer-
ebral ischemia, multiple sclerosis, sciatic nerve injury,
epilepsy and Alzheimer’s disease).28 The new studies to
evaluate the changes in PBR density and expression
related to neurological disorders and post-traumatic
injury highlight the importance to develop novel
strategies for the diagnosis and therapy of the above-
mentioned pathologies using PBR ligands.
to both peripheral and central benzodiazepine receptor
(PBR Ki=0.5–7 nM; CBR Ki=1–28 nM).38 Interest-
ingly, extensive SAR studies on the acetamido[1,2-
a]pyrimidine nucleus of Alpidem have suggested that
the introduction of suitable substituents and proper
modifications of this nucleus lead to compounds with
improved affinity and the PBR versus CBR
selectivity.4,5,39ꢀ41 In this paper, we report synthesis,
biological evaluations and preliminary SAR studies for
a new class of N,N-diethyl-(2-arylpyrazolo[1,5-a]pyr-
imidin-3-yl)acetamides (Fig. 1) which bind with both
high affinity and selectivity to the PBR.
The PBR is characterized by its interaction with iso-
quinolines such as PK 11195,29 benzodiazepines as Ro
5-4864,30 imidazopyridines (Alpidem),31 indole deriva-
tives (FGIN-1)4,5 and two potent and selective ligands,
DAA1097 and DAA1106, recently identified32 (Fig. 1).
This receptor appears to be a heteromeric complex of at
least three different subunits, including an isoquinoline
binding subunit (18 kDa), a voltage-dependent anion
channel VDAC (32 kDa) and an adenine nucleotide
carrier (30 kDa).33 The isoquinolines that bind specifi-
cally to PBR interact with the 18 kDa subunit, whereas
the BZ recognition site is supposed to be constituted by
an association of 18 kDa and VDAC components34 or,
alternatively, on the 18 kDa subunit in a restrictive
conformational state.35,36 However, the complex is
made up of a not yet well-established mitochondrial
structure of the mitochondrial permeability transition
called (MPT)-pore, on whose opening the mitochondrial
permeability depends.37
Our attention was focused mainly on the 5-, 6-, and 7-
positions to evaluate their influence on selectivity
toward the PBR; moreover the effect of para-substitu-
tions on the 2-phenyl ring was investigated. However,
we maintained in all the series the tertiary acetamide
moiety at the 3-position, whose hydrogen bond interac-
tion with the receptor protein is reported to be of para-
mount importance for the binding to the PBR.4,5,39ꢀ42
In fact, it is known that the substitution of the acet-
amide moiety with an ester function and the modifica-
tion of the length of the carbon chain between the amide
and the heterocyclic nucleus resulted in a marked loss of
affinity.40 The models proposed in the literature suggest
that a hydrogen bond-donating function (d1) and some
lipophilic pockets (PAR, LA, FRA) are the essential
pharmacophoric elements for the interaction between
ligands and receptor protein. The schematic representa-
tion of the interaction models of Alpidem with CBR
and PBR proposed by Bourguignon was the hypothesis
most frequently reported38,39,43 (Fig. 2).
In order to gain further information as to the function
of the PBR, the synthesis of potent and selective mole-
cules would appear to be an invaluable tool for investi-
gating the physiological role of the PBR in human
tissues. In designing compounds that might exhibit high
affinity and selectivity for the PBR, we have thought it
interesting to synthesize compounds structurally closely
related to Alpidem, which binds with nanomolar affinity
Recently, a unique 3-D interaction model has been
described for endogenous and synthetic PBR ligands44
which confirmed two lipophilic regions (L1and L3 cor-
responding to PAR and FRA) and one polar group (H2
related to d1), as essential elements of the PBR interac-
tion, and a lipophilic region modulating the receptor
binding (L4 corresponding to LA) into which H2 is
expressed.
A subset of the synthesized compounds showing high
affinity was also tested for their ability to modulate
steroid biosynthesis in C6 glioma cells, since this test is
considered so far a reliable method to prove the affinity
and/or intrinsic activity of PBR ligands. Some of the
Figure 2. Comparison between the schematic representation of the
interaction models of Alpidem with CBR and PBR: PAR, planar
aromatic region; FRA, freely rotating aromatic ring region; OPR, out-
plane region; LA, lipophilic area; d1, electron-rich zone.
Figure 1. Ligands of peripheral benzodiazepine receptor (PBR).

S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
2663
tested compounds were found to stimulate pregneno-
lone formation with potency comparable to Ro 5-4864
and PK 11195.
b-diketones and b-ketoaldehydes or their acetals led
generally to regioisomeric mixtures of pyrazolopyr-
imidines. A simple H NMR method, suggested in our
1
previous papers47,48 for distinguishing isomeric 7-methyl
and 5-methyl derivatives, has been used for ascertaining
the structure of regioisomers 3f, 3g; 3n, 3o; 3p, 3q and
3s, 3t. Convincing data for the structural assignment of
the methyl group on the pyrimidine moiety came from
Chemistry
The synthesis of compounds 3f–y, shown in Table 1,
was accomplished via the general strategy presented in
Scheme 1. The new N,N-diethyl-(2-arylpyrazolo[1,5-
a]pyrimidin-3-yl)acetamides were prepared in a three-
step procedure starting from the appropriate aroylace-
tonitriles, which were synthesized following methods
previously reported.45,46 The achievement of the expec-
ted N,N-diethylbutanamides (1a–e) was performed by
exploiting the reactivity of the methylene of aroylaceto-
nitriles which were reacted in alkaline medium with
N,N-diethylchloroacetamide. Unfortunately, following
this procedure, the expected N,N-diethylbutanamides
(1a–e) were obtained together with tars; therefore, a
thorough purification by means of column chromato-
graphy was mandatory. In the second step, the inter-
mediates 1a–e were reacted at reflux in ethanol with
hydrazine hydrate, in the presence of acetic acid, to give
the corresponding N,N-diethyl-(3-amino-5-arylpyrazol-
4-yl)acetamides (2a–e) in good yield.
1
the H NMR spectra where diagnostic different values
are shown by 5- or 7-methyl compounds (d 2.58–2.63
versus 2.81–2.90 ppm, respectively). Moreover, the cou-
pling constant measured for the H6 with its vicinal proton
(4.5 Hz<JCHꢀCH<5.0 Hz) appears to be in accordance
with a ¼CH–CH¼ pattern, so that the 7-substitution is
ascertained even for 3y.
Finally, the condensation of 2a–e with the suitable elec-
trophilic reagents, reported in Table 1, led to the closure
of the pyrimidine ring, affording the expected pyr-
azolo[1,5-a]pyrimidin-3-yl acetamides 3f–y. The con-
densation of aminopyrazoles with electrophiles such as
Scheme 1. (i) N,N-diethylchloroacetamide; (ii) hydrazine hydrate; (iii)
reagents are reported in Table 1.
Table 1. Experimental data for 2-Arylpyrazolo[1,5-a]pyrimidin-3-ylacetamides
Compd
R
R1
R2
R3
Formula (MW)
Analysisa
Reagentb
3f
H
H
H
H
Cl
F
CH3
OCH3
F
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
C19H22N4O (322.41)
C19H22N4O (322.41)
C18H20N4O (308.38)
C20H24N4O (336.44)
C20H23N4OCl (370.88)
C20H23N4OF (354.43)
C21H26N4O (350.46)
C21H26N4O (366.46)
C19H21N4OF (340.40)
C19H21N4OCl (356.85)
C21H23N4OF3 (404.43)
C21H23N4OF3 (404.43)
C21H20N4O2F6 (474.41)
C25H25N4OCl (432.95)
C25H25N4OCl (432.95)
C21H25N4OCl (384.91)
C23H27N4O3Cl (442.94)
C22H26N4O3 (394.47)
C25H26N4O (398.51)
C24H24N4O (384.48)
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
A
A
B
C
C
C
C
C
A
A
D
D
E
F
F
G
H
I
3g
3h
3i
3j
3k
3l
3m
3n
3ꢁ
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CF3
CF3
CH3
Ph
CH3
CH3
H
H
H
CH3
CH3
CH3
CH3
CH3
H
CH3
CF3
CH3
CF3
Ph
CH3
CH3
CH3
CH3
H
Cl
CH3
CH3
OCH3
Cl
Cl
Cl
Cl
H
CH3
H
CH3
COOEt
COOEt
Ph
H
J
K
Ph
aAll the compounds were analyzed within ꢂ0.4% of the theoretical values.
bA=4,4-dimethoxy-2-butanone, B=1,1,3,3-tetraethoxypropane, C=2,4-pentanedione, D=1-trifluoromethyl-2,4-pentanedione, E=1,5-trifluoro-
methyl-2,4-pentanedione, F=1-phenyl-1,3-butanedione, G=3-methyl-2,4-pentanedione, H=ethyl 2-acetyl-3-oxo butanoate, I=ethyl 2-acetyl-3-
ethoxyacrylate, J=phenylmalondialdehyde, K=1-phenyl-3-dimethylamino-2-propen-1-one.

2664
S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
Biological Studies
effect of Ro 5-4864 has been reported as five times lower
at 25 ꢁC than at 4 ꢁC. In the present preliminary study,
the inhibitory effect of both the new ligands and PK
11195 on [3H]PK 11195 binding was almost the same at
4 and 25 ꢁC, showing that the binding properties of the
new pyrazolopyrimidine ligands and PK 11195 are
thermodynamically similar.
The ability of the compounds 3f–y to interact with the
peripheral benzodiazepine receptor (PBR) was investi-
gated by means of a binding assay using [3H]PK 11195
and [3H]Ro 5-4864 as radioligands and membranes
from rat kidney tissues as receptor source.40,49 More-
over, in order to examine the PBR versus CBR selec-
tivity of 3f–y, binding studies were carried out by using
[3H]Ro 15-1788 as specific radioligand for benzodiaze-
pine binding site on GABAA receptor complex and
membranes from rat brain tissues as receptor
source.50ꢀ52 Binding data for all new pyrazolo[1,5-
a]pyrimidin-3-yl acetamides are reported in Table 2 and
the affinity values of PK 11195, Ro 5-4864 and Alpidem
(as reference compounds) are also included. The binding
data are expressed as Ki values, which were calculated
from the corresponding IC50 values according to the
equation of Cheng and Prusoff.53 Most of the synthe-
sized compounds 3f–y exhibited high affinity and PBR
selectivity.
Furthermore, a subset of the synthesized compounds
showing high affinity for the PBR was examined for the
compound’s ability to stimulate pregnenolone forma-
tion from the mitochondria of C6 glioma cells.49 All the
selected ligands and reference compounds, PK 11195,
Ro 5-4864 and Clonazepam, were tested at the same
concentration (40 mM) in order to directly compare the
efficacies. The references PK 11195 and Ro 5-4864 are
known to stimulate steroidogenesis in a concentration-
dependent way, whereas the selective CBR ligand, Clo-
nazepam, does not elicit any stimulatory effect at this
concentration (Fig. 3).
Therefore, with the aim of evaluating thermodynamic
similarities with the reference compounds, we per-
formed the binding assay of N,N-diethyl-(2-arylpyr-
azolo[1,5-a]pyrimidin-3-yl)acetamides (3f–y) at two
different temperatures. As reported in literature, the Ro
5-4864 binding values are temperature dependent,
whereas the PK 11195 values are not.54,55 The inhibitory
Results and Discussion
It appears from the results reported in Table 2 that most
of the synthesized compounds display high affinity and
selectivity for the PBR; only compounds 3f–h, 3n and 3o
show a significant affinity for both CBR and PBR. In
particular, compound 3h, devoid of any substituent on
Table 2. Affinities of compounds 3f–y for PBR and CBR
Ki (nM)a PBR
Ki (nM)a CBR
Compd
R
R1
R2
R3
[3H]PK 11195
[3H]Ro 5-4864
[3H]Ro 15-1788
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
PK 11195
Ro 5-4864
Alpidemc
H
H
H
H
Cl
F
CH3
OCH3
F
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
40.0ꢂ4.0
155.0ꢂ16.0
NTb
21.0ꢂ1.0
67.0ꢂ3.0
1200.0ꢂ100.0
9.2ꢂ0.2
1.4ꢂ0.2
4.3ꢂ0.2
ꢂ0.2
92.6ꢂ7.0
518.0ꢂ20.0
251.0ꢂ18.0
>10,00
>10,00
>10,00
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CF3
CF3
CH3
Ph
CH3
CH3
H
H
H
CH3
CH3
CH3
CH3
CH3
H
CH3
CF3
CH3
CF3
Ph
CH3
CH3
CH3
CH3
H
29.0ꢂ3.0
2.4ꢂ0.2
9.2ꢂ1.0
0.8ꢂ0.11
4.7ꢂ0.4
25.0ꢂ2.0
8.1ꢂ0.3
0.9ꢂ0.12.3
1.0ꢂ0.11
16.0ꢂ1.0
2.4ꢂ0.2
3.4ꢂ0.2
6.1ꢂ0.3
8.4ꢂ0.4
NTb
.7
.8
>10,00
>10,00
3.1ꢂ0.2
67.0ꢂ3.0
2.9ꢂ 0.2
ꢂ0.2
85.0ꢂ3.0
207.0ꢂ10.0
5800.0ꢂ145.0
ꢂ170.0
Cl
CH3
CH3
OCH3
Cl
Cl
Cl
Cl
H
CH3
H
ꢂ0.16900.0
3.4ꢂ0.2
2.5ꢂ0.2
6260.0ꢂ250.0
>10,00
2.7ꢂ0.2
>10,00
>10,00
>10,00
NTb
>10,00
>10,00
>10,00
>10,00
1–28
CH3
COOEt
COOEt
Ph
H
5.2ꢂ0.3
8.0ꢂ0.5
942.0ꢂ5.0
45.0ꢂ3.0
160.0ꢂ10.0
57.0ꢂ5.0
Ph
NTb
9.3ꢂ0.5
23.0ꢂ3.0
0.5–7
aKi values represent the mean SEM of three determinations.
bNot tested.

S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
2665
IC50=29 nM) or to N7 of imidazo[4,5-b]pyridines,39
does not seem to affect PBR binding potency, while
its role in CBR versus PBR selectivity remains to be
studied in detail.
Encouraged by these preliminary results, we turned our
attention to the 6-position of the pyrimidine moiety,
corresponding to the 7-position in the imidazopyridine
nucleus, that has not been investigated yet. Compounds
3u–x, bearing different substituents at the 6-position,
retained the affinity and selectivity for the PBR. This
result could suggest that, unlike CBR, a bulky sub-
stituent at this position is well tolerated by the periph-
eral receptor protein. The significant loss of affinity of
3w is probably due to the spatial orientation of ethox-
ycarbonyl group with regard to the planar pyr-
azolopyrimidine nucleus. In fact the lack of substituent
at the 5-position might lead to an unfavorable orienta-
tion of the ethoxycarbonyl group for the interaction
between the ligand and the receptor protein (see affinity
values for 3w and 3v). As regards the p-substitutions at
the 2-phenyl ring (3i–m) a general improvement in PBR
affinity values is observed in comparison to the refer-
ence compound 3i; in addition, the chemical features
(electrophilicity, lipophilicity and steric hindrance) did
not seem to affect either PBR affinity or selectivity.
However, further investigations about the position of
the substituents on the phenyl ring are required.
Figure 3. The effect of N,N-diethyl-(2-arylpyrazolo[1,5-a]pyrimidin-3-
yl)acetamides on the pregnenolone accumulation in C6 glioma rat
cells. All the compounds were used at the same concentration (40 mM);
at the end of incubation period (2 h), the amount of pregnenolone was
quantified by radio immunoassay (RIA), using an antibody obtained
from ICN Biochemical Inc., CA, USA. The values are the mean-
ꢂSEM of at least three determinations. (see Experimental).
the pyrimidine moiety, exhibits the ratio value CBR
versus PBR for the CBR, while a marked improvement
of PBR affinity is observed for 3f, 3g, 3n and 3o (3g ꢃ
3n < 3f < 3o), although their PBR versus CBR selec-
tivity is still modest and comparable to Alpidem. The
binding data of the unselective compounds 3f, 3n and
3g, 3o, bearing a methyl group at the 5- or 7-positions
respectively, suggest that the title compounds could be
well tolerated into the lipophilic pockets of both per-
ipheral and central receptor proteins probably because
of their small lipophilic substituents. Moreover, taking
into account the low affinity value of compound 3h, it
seems reasonable to hypothesize that the substitution on
the pyrimidine moiety could be the key to promoting
the PBR selectivity. This hypothesis is supported by the
PBR selectivity of 3y, bearing the phenyl ring at the 7-
position, in comparison to the 7-methyl derivative 3g. In
addition, compounds 3i–m and 3p–t, bearing sub-
stituents at the 5- and 7-positions, display high affinity
and selectivity for the PBR. On the other hand, as
appears in Table 2, the effect of substituents at the 5-
and 7-positions, which are endowed with increasing
lipophilic nature and steric bulk (3p–t), produces gen-
erally constant or improved affinity values for PBR in
comparison to compounds 3i–m. The findings obtained
in this study seem to confirm similar results observed in
the imidazopyrimidine series, where the substitution at
the 6- and 8-positions, corresponding to the 5- and 7-
positions of pyrazolopyrimidine series, led to com-
pounds more than 1000-fold selective for PBR versus
CBR.40,41 Therefore, the replacement of imidazo[1,2-
a]pyridine nucleus of Alpidem with the pyrazolo[1,5-
a]pyrimidine one appears as a bioisosteric modification.
However, the pyrimidine nitrogen (N4), which could be
related to pyrazine nitrogen of Zopiclone (CBR ligand
Some of the title compounds (3i–m, 3o–r, 3t–v) have
been examined for their ability to increase steroid bio-
synthesis (Fig. 3) and some of these stimulate ster-
oidogenesis with potency similar to Ro 5-4864 and PK
11195. Surprisingly, these results show that there is no
correlation between PBR affinity and steroidogenic
activity. In fact, compounds 3i, 3k–m, 3p and 3t,
although showing high affinity, do not produce any
effect on the pregnenolone level. A moderate increase in
pregnenolone formation can be observed for the 5-tri-
fluoromethyl-7-methyl derivative, 3q, and the 5,7-tri-
fluoromethyl derivative, 3r, in comparison to the
corresponding 5,7-dimethyl-substituted compounds, 3l
and 3m. An analogous moderate increase is observed
for the 5, 6, 7-substituted 3u and 3v; while the most
steroidogenic active compounds appear to be 3j and 3o,
bearing a p-chlorine atom on the 2-phenyl ring. In a
preliminary evaluation, PK 11195, the partial agonist of
the steroidogenic action of PBR ligands,5 was used in
the presence of the most active ligand, 3j; it moderately
inhibits the increase in pregnenolone accumulation eli-
cited by compound 3j per se (PK 11195+3j=24.3ꢂ0.2
pregnenolone ng/mg protein; PK 11195=28.0ꢂ1.5
pregnenolone ng/mg protein). Moreover, compound 3i,
endowed with PBR affinity but devoid of steroidogenic
action, was used together with Ro 5-4864, a well-known
modulator in steroid biosynthesis. The inefficiency of 3i
to have any effect on the Ro 5-4864 activity could sug-
gest that this compound fails to antagonize the ster-
oidogenic action of PBR ligand in C6 glioma cells (Ro
5-4864+3i=29.5ꢂ0.6 pregnenolone ng/mg protein; Ro
5-4864=26.0ꢂ3.1pregnenolone ng/mg protein). Fur-
ther investigations and more detailed study are required
to define if tissue-specificity of PBR ligands exist and/or

2666
S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
if PBR ligands display different behavioral profiles in
different tissues.
samples was determined by means of TLC, which was
performed using Machery-Nagel Duren, Alugram silica
gel plates. Silica gel 60 (Merck 70–230 mesh) was used
for column chromatography.
Conclusions
In this paper, we report synthesis and biological eva-
luation of a novel class of high affinity and selective
PBR ligands, N,N-diethyl-(2-arylpyrazolo[1,5-a]pyr-
imidin-3-ylacetamides (3f–y). The preliminary SAR
study suggests that the new pyrazolopyrimidines are
bioisteres of imidazopyridine derivatives40,41 and, in
particular, substitutions on the pyrimidine moiety seem
to be the key factor promoting the PBR versus CBR
selectivity. In addition, introduction of substituents at
the para position of the 2-phenyl ring is able to increase
the affinity values for the PBR. To clarify the role of the
amide moiety at the 3-position and the main structural
features concerning the number and length of the alkyl
substituents on the amide nitrogen, a targeted study is
currently in progress. Some of the highest affinity com-
pounds (3i–m, 3o–r, 3t–v) were also tested for their
ability to stimulate steroid biosynthesis in C6 glioma rat
cells, and some of these increased the pregnenolone
formation with potency similar to Ro 5-4864 and PK
11195. The effects of these compounds on steroid pro-
duction take on further interest in the light of recent
reports showing that neuroactive steroids seem to elicit
anxiolytic, anticonvulsant, sedative, hypnotic, and even
memory modulating effects.56,57 On the other hand, the
results of our preliminary steroidogenic assay show that
there is no correlation between PBR affinity and ster-
oidogenic activity; in fact, some of the tested com-
pounds, although showing high affinity, did not produce
any effect on pregnenolone level. This observation
prompted us to continue our investigations since no
PBR ligands with antagonistic properties on ster-
oidogenesis have been described yet. The availability of
such molecules related to Alpidem structure and
endowed with different intrinsic activities may afford
new opportunities to understand the PBR function, its
localization, and the proposed existence of PBR sub-
types, in addition to giving new tools for evaluating
PBR pathological alterations. Further studies of the
biological properties of these compounds, together with
an expansion of their SAR and molecular modeling
efforts, are underway.
General procedure for synthesis of compounds 1a–e. To a
solution of NaOH (10 mmol, 0.39 g) in 80% EtOH
(80 mL) the suitable aroylacetonitrile (10 mmol) and
N,N-diethylchloroacetamide (10 mmol, 1.37 mL), with
NaI (30 mmol, 4.49 g), were added under magnetic stir-
ring. The reaction mixture was stirred at reflux for 8 h.
After cooling the suspension was filtered to eliminate
the inorganic materials and the filtrate was con-
centrated; the residue was then purified by silica gel
column chromatography (toluene/EtOAc, 8:3 v/v, as
eluent) to give the corresponding N,N-diethylamides.
N,N-Diethyl-(3-cyano-4-oxo-4-phenyl)butanamide (1a).
This compound was obtained from 3-oxo-3-phenylpro-
panenitrile45 as yellow liquid; yield 12%; 1H NMR
(CDCl3) d 1.09 (t, 3H, N(CH2CH3)2), 1.22 (t, 3H,
N(CH2CH3)2), 2.90 (dd, 1H, CH2), 3.25–3.49 (m, 5H:
4H, N(CH2CH3)2; 1H, CH2), 5.01–5.1 (m, 1H, CH),
7.48–7.52 (m, 3H, Ph), 8.01–8.12 (m, 2H, Ph).
N,N-Diethyl-[3-cyano-4-(4-chlorophenyl)-4-oxo]butana-
mide (1b). This compound was obtained from 3-(4-
chlorophenyl)-3-oxopropanenitrile45 as yellow liquid;
yield 20%; 1H NMR (CDCl3) d 1.09–1.24 (m, 6H,
N(CH2CH3)2), 2.88 (dd, 1H, CH2), 3.22–3.52 (m, 5H:
4H, N(CH2CH3)2; 1H, CH2), 4.92–5.01(m, 1H, CH),
7.52 (d, 2H, Ph), 8.09 (d, 2H, Ph).
N,N-Diethyl-[3-cyano-4-(4-fluorophenyl)-4-oxo]butana-
mide (1c). This compound was obtained from 3-(4-
fluorophenyl)-3-oxopropanenitrile46 as yellow liquid;
yield 13%; 1H NMR (CDCl3) d: 1.1–1.35 (m, 6H,
N(CH2CH3)2), 2.89 (dd, 1H, CH2), 3.22–3.52 (m, 5H:
4H, N(CH2CH3)2; 1H, CH2), 4.98–5.10 (m, 1H, CH),
7.19–7.30 (m, 2H, Ph), 8.03–8.09 (m, 2H, Ph).
N,N-Diethyl-[3-cyano-4-(4-methylphenyl)-4-oxo]butana-
mide (1d). This compound was obtained from 3-(4-
methylphenyl)-3-oxopropanenitrile45 as yellow liquid;
yield 42%; 1H NMR (CDCl3) d 1.14–1.31 (m, 6H,
N(CH2CH3)2), 2.42 (s, 3H, CH3), 2.86 (dd, 1H, CH2),
3.28–3.48 (m, 5H: 4H, N(CH2CH3)2; 1H, CH2), 4.99–
5.12 (m, 1H, CH), 7.31 (d, 2H, Ph), 7.98 (d, 2H, Ph).
Experimental
The structures of all compounds were supported by
their 1H NMR data (measured with a Varian Gemini at
200 MHz, chemical shifts expressed in d (ppm) using
DMSO-d6 or CDCl3 as solvent). The following abbre-
viations are used: b=broad, d=doublet, m=multiplet,
q=quartet, t=triplet and s=singlet. Melting points
were determined with a Gallenkamp apparatus and
were uncorrected. Elemental analyses were performed
by the laboratories of Dipartimento Farmaco-Chimico-
Tecnologico of Universita di Siena, Italy, with a Perkin-
Elmer model 240C, elemental analyzer, and their results
are within ꢂ0.4% of theoretical values. The purity of
N,N-Diethyl-[3-cyano-4-(4-methoxyphenyl)-4-oxo]buta-
namide (1e). This compound was obtained from 3-(4-
methoxyphenyl)-3-oxopropanenitrile45 as yellow liquid;
yield 10%; 1H NMR (CDCl3) d 1.03–1.371 (m, 6H,
N(CH2CH3)2), 2.85 (dd, 1H, CH2), 3.22–3.52 (m, 5H:
4H, N(CH2CH3)2; 1H, CH2), 3.98 (s, 3H, OCH3 ), 4.96–
5.04 (m, 1H, CH), 7.00 (d, 2H, Ph), 8.03 (d, 2H, Ph).
General procedure for synthesis of compounds 2a–e. To a
solution of 1a–e (10 mmol) in EtOH (50 mL) hydrazine
hydrate (20 mmol, 0.97 mL) and acetic acid (1mL) were
added and the reaction mixture was refluxed for 6 h.

S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
2667
After cooling, evaporation of the solvent, under reduced
pressure, gave a residue that was purified by silica gel
column chromatography (CHCl3/MeOH, 10:1 v/v, as
eluent).
2.58 (s, 3H, 5-CH3), 3.36–3.57 (m, 4H, N(CH2CH3)2),
3.92 (s, 2H, CH2), 6.62 (d, JH6ꢀH7=6.9 Hz, 1H, H-6),
7.42–7.49 (m, 3H, Ph), 7.76–7.81(m, 2H, Ph), 8.49 (d,
JH7ꢀH6=6.9 Hz, 1H, H-7).
N,N-Diethyl-(3-amino-5-phenylpyrazol-4-yl)acetamide (2a).
Ivory crystals obtained from compound 1a (10 mmol,
2.58 g); yield 18%; mp 157–158 ꢁC; 1H NMR (CDCl3) d:
0.90 (t, 3H, N(CH2CH3)2), 1.07 (t, 3H, N(CH2CH3)2),
3.05 (q, 2H, N(CH2CH3)2), 3.33 (q, 2H, N(CH2CH3)2),
3.52 (s, 2H, CH2), 5.65 (bs, 2H, NH2 exchangeable),
7.34–7.41(m, 5H, Ph).
N,N-Diethyl-(2-phenyl-7-methylpyrazolo[1,5-a]pyrimidin-
3-yl)acetamide (3g). From 2a (1mmol, 272 mg) and of
4,4-dimethoxy-2-butanone (1.1 mmol, 0.15 mL) in the
presence of 37% HCl (0.3 mL); the reaction mixture was
refluxed under magnetic stirring for 30 min, poured into
ice-water and extracted with Et2O (3ꢄ5 mL). The com-
bined extracts were washed with water (3ꢄ10 mL), dried
over sodium sulfate, and concentrated under reduced
pressure. Purification of the residue was accomplished
by column chromatography (CHCl3/MeOH, 10:1 v/v,
as eluent). Ivory crystals; yield 45%; mp 127–128 ꢁC, 1H
NMR (CDCl3) d 1.12–1.31 (m, 6H, N(CH2CH3)2), 2.82
(s, 3H, CH3) 3.39–3.58 (m, 4H, N(CH2CH3)2) 4.02 (s,
2H, CH2), 6.72 (d, JH6ꢀH5=4.8 Hz, 1H, H-6), 7.38–7.50
(m, 3H, Ph), 7.80–7.83 (m, 2H, Ph), 8.39 (d,
JH5ꢀH6=4.8 Hz, 1H, H-5).
N,N-Diethyl-[3-amino-5-(4-chlorophenyl)pyrazol-4-yl]-
acetamide (2b). Ivory crystals obtained from compound
1b (10 mmol, 2.92 g); yield 38%; mp 180–181 ꢁC; 1H
NMR (CDCl3) d 0.96 (t, 3H, N(CH2CH3)2), 1.09 (t, 3H,
N(CH2CH3)2), 3.1(q, 2H, N(CH CH3)2), 3.33 (q, 2H,
2
N(CH2CH3)2), 3.49 (s, 2H, CH2), 4.82 (bs, 2H, NH2
exchangeable), 7.34–7.42 (m, 4H, Ph).
N,N-Diethyl-[3-amino-5-(4-fluorophenyl)pyrazol-4-yl]-
acetamide (2c). Ivory crystals obtained from compound
1c (10 mmol, 2.76 g); yield 40%; mp 137–138 ꢁC; 1H
NMR (CDCl3) d 0.90 (t, 3H, N(CH2CH3)2), 1.12 (t, 3H,
N(CH2CH3)2), 3.10 (q, 2H, N(CH2CH3)2), 3.40 (q, 2H,
N,N-Diethyl-(2-phenylpyrazolo[1,5-a]pyrimidin-3-yl)ace-
tamide (3h). From 2a (1 mmol, 272 mg) and 1,1,3,3 tet-
raethoxypropane (1.1 mmol, 0.32 mL) in the presence of
37% HCl (0.2 mL); yellow crystals purified by silica gel
column chromatography (CHCl3/MeOH, 10:1 v/v, as
eluent); yield 30%; mp 133–134 ꢁC; 1H NMR (CDCl3) d
1.10–1.30 (m, 6H, N(CH2CH3)2), 3.30–3.61(m, 4H,
N(CH2CH3)2), 4.04 (s, 2H, CH2), 6.88–6.92 (m, 1H, H-
6), 7.26–7.50 (m, 3H, Ph), 7.77–7.82 (m, 2H, Ph), 8.50–
8.53 (m, 1H, H-5), 8.84 (dd, 1H, H-7).
N(CH2CH3)2), 3.53 (s, 2H, CH2), 6.1(bs, 2H, NH
exchangeable), 7.01–7.20 (m, 2H, Ph), 7.35–7.43 (m, 2H,
Ph).
2
N,N-Diethyl-[3-amino-5-(4-methylphenyl)pyrazol-4-yl]a-
cetamide (2d). Light yellow crystals obtained from
compound 1d (10 mmol, 2.72 g) and recrystallized from
80% EtOH; yield 44%; mp 210–211 ꢁC; 1H NMR
(CDCl3) d 0.96 (t, 3H, N(CH2CH3)2), 1.12 (t, 3H,
N(CH2CH3)2), 2.40 (s, 3H, CH3), 3.1(q, 2H,
N(CH2CH3)2), 3.33 (q, 2H, N(CH2CH3)2), 3.52 (s, 2H,
N,N-Diethyl-(2-phenyl-5,7-dimethylpyrazolo[1,5-a]pyri-
midin-3-yl)acetamide (3i). From 2a (1mmol, 272 mg)
and 2,4-pentanedione (1mmol, 0.1mL); light yellow
crystals purified by column chromatography (CHCl3/
MeOH, 20:1v/v, as eluent); yield 30%; mp 143 ꢁC; H
1
CH2), 4.41(bs, 2H, NH exchangeable), 7.27–7.40 (m,
2
4H, Ph).
NMR (CDCl3) d 1.15–1.28 (m, 6H, N(CH2CH3)2), 2.55
(s, 3H, 5-CH3), 2.75 (s, 3H, 7-CH3), 3.48–3.62 (m, 4H,
N(CH2CH3)2), 3.93 (s, 2H, CH2), 6.52 (s, 1H, H-6),
7.41–7.45 (m, 3H, Ph), 7.80–7.84 (m, 2H, Ph).
N,N-Diethyl-[3-amino-5-(4-methoxyphenyl)pyrazol-4-yl]-
acetamide (2e). Yellow crystals obtained from com-
pound 1e (10 mmol, 2.88 g); yield 72%; mp 140–141 ꢁC;
1H NMR (CDCl3) d 0.86–1.18 (m, 6H, N(CH2CH3)2),
3.18–3.23 (m, 4H, N(CH2CH3)2), 3.5 (s, 2H, CH2), 3.83
(s, 3H, OCH3), 6.99 (d, 2H, Ph), 7.31(d, 2H, Ph).
N,N-Diethyl-[2-(4-chlorophenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl]acetamide (3j). From 2b (1mmol,
306 mg) and 2,4-pentanedione (1mmol, 0.1mL); light
yellow crystals purified by column chromatography
(CH2Cl2/MeOH, 20:1v/v, as eluent); yield 34%; mp
129–130 ꢁC; 1H NMR (CDCl3) d 1.05–1.30 (m, 6H,
N(CH2CH3)2), 2.57 (s, 3H, 5-CH3), 2.78 (s, 3H, 7-
CH3), 3.38–3.61(m, 4H, N(CH 2CH3)2) 3.99 (s, 2H,
CH2), 6.58 (s, 1H, H-6), 7.41(d, 2H, Ph), 7.81(d, 2H,
Ph).
General procedure for synthesis of compounds 3f–y. To a
solution of the suitable N,N-diethyl-(2-arylpyrazolo[1,5-
a]pyrimidin-3-yl)acetamide (2a–e) (1mmol) in EtOH
(5 mL) the appropriate electrophilic reagent (1mmol)
was added and the mixture was refluxed under stirring
for 4 h. The progress of reaction was monitored by
TLC. The solvent was evaporated under reduced pres-
sure and the residue was purified by silica gel column
chromatography to give the amides 3f–y.
N,N-Diethyl-[2-(4-fluorophenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl]acetamide (3k). From 2c (1mmol,
290 mg) and 2,4-pentanedione (1mmol, 0.1mL); white
crystals purified by column chromatography (CH2Cl2/
MeOH, 20:1 v/v, as eluent); yield 33%; mp 113–114 ꢁC;
1H NMR (CDCl3) d 1.11–1.31 (m, 6H, N(CH2CH3)2),
2.58 (s, 3H, 5-CH3), 2.80 (s, 3H, 7-CH3), 3.40–3.62 (m,
4H, N(CH2CH3)2), 3.98 (s, 2H, CH2), 6.58 (s, 1H, H-6),
7.10–7.21 (m, 2H, Ph), 7.80–7.88 (m, 2H, Ph).
N,N-Diethyl-(2-phenyl-5-methylpyrazolo[1,5-a]pyrimidin-
3-yl)acetamide (3f). From 2a (1mmol, 272 mg) and 4,4-
dimethoxy-2-butanone (1mmol, 0.14 mL); ivory crystals
purified by silica gel column chromatography (CHCl3/
MeOH, 20:1 v/v, as eluent); yield 32%; mp 135–136 ꢁC;
1H NMR (CDCl3) d 1.08–1.26 (m, 6H, N(CH2CH3)2),

2668
S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
N,N-Diethyl-[2-(4-methylphenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl]acetamide (3l). From 2d (1mmol,
286 mg) and 2,4-pentanedione (1mmol, 0.1mL); white
crystals purified by column chromatography (CH2Cl2/
N(CH2CH3)2), 3.99 (s, 2H, CH2), 6.97 (s,1H, H-6), 7.28
(d, 2H, Ph), 7.82 (d, 2H, Ph).
N,N-Diethyl-[2-(4-methoxyphenyl)-5,7-trifluoromethyl-
pyrazolo[1,5-a]pyrimidin-3-yl] acetamide (3r). From 2e
(1mmol, 302 mg) and 1,5-trifluoromethyl-2,4-pentane-
dione (1mmol, 0.14 mL) in absolute EtOH (5 mL); yel-
low crystals purified by column chromatography with
CHCl3/MeOH, 10:1 v/v, as eluent; yield 10%; mp
MeOH, 20:1v/v, as eluent); yield 80%; mp 120 ꢁC; H
1
NMR (CDCl3) d: 1.10–1.25 (m, 6H, N(CH2CH3)2), 2.41
(s, 3H, CH3), 2.60 (s, 3H, 5-CH3), 2.79 (s, 3H, 7-CH3),
3.38–3.58 (m, 4H, N(CH2CH3)2) 3.98 (s, 2H, CH2), 6.55
(s, 1H, H-6), 7.25 (d, 2H, Ph), 7.80–7.88 (d, 2H, Ph).
ꢁ
1
157 C; H NMR (CDCl3) d 1.18 (t, 3H N(CH2CH3)2),
1.36 (t, 3H, N(CH2CH3)2), 3.44 (q, 2H, N(CH2CH3)2)
3.60 (q, 2H, N(CH2CH3)2) 3.88 (s, 3H, OCH3), 4.04 (s,
2H, CH2), 7.02 (d, 2H, Ph), 7.40 (s,1H, H-6), 7.89 (d,
2H, Ph).
N,N-Diethyl-[2-(4-metoxyphenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl]acetamide (3m). From 2e (1mmol,
302 mg) and 2,4-pentanedione (1mmol, 0.1mL); ivory
crystals purified by column chromatography (CH2Cl2/
MeOH, 20:1 v/v, as eluent); yield 44%; mp 212–213 ꢁC;
1H NMR (CDCl3) d 1.04–1.23 (m, 6H, N(CH2CH3)2),
2.58 (s, 3H, 5-CH3), 2.78 (s, 3H, 7-CH3), 3.40–3.56 (m, 4H,
N(CH2CH3)2), 3.83 (s, 2H, CH2), 3.97 (s, 3H, OCH3), 6.50
(s, 1 H, H-6), 6.99 (d, 2H, Ph), 7.80 (d, 2H, Ph).
N,N-Diethyl-[2-(4-chlorophenyl)-5-methyl-7-phenylpyra-
zolo[1,5-a]pyrimidin-3-yl] acetamide (3s) and N,N-diethyl
-[2-(4-chlorophenyl)-5-phenyl-7-methylpyrazolo[1,5-a]pyr-
imidin-3-yl]acetamide (3t). These compounds were
obtained from 2b (1 mmol, 306 mg) and 1-phenyl-1,3-
butanedione (1mmol, 156 mg) following the general
procedure. After cooling, compound 3s was obtained as
a yellow solid in 40% yield by filtration and recrys-
tallized from EtOH. Otherwise the evaporation of the
filtrate gave a residue mainly consisting of compound
3t, which was purified by column chromatography with
toluene/EtOAc, 8:3 v/v, as eluent.
N,N-Diethyl-[2-(4-fluorophenyl)-5-methylpyrazolo[1,5-a]-
pyrimidin-3-yl]acetamide (3n). From 2c (1mmol,
290 mg) and 4,4-dimethoxy-2-butanone (1mmol,
0.14 mL); ivory crystals purified by column chromato-
graphy (CHCl3/MeOH, 10:1 v/v, as eluent); yield 34%;
ꢁ
1
mp 103–104 C; H NMR (CDCl3) d 1.05–1.25 (m, 6H,
N(CH2CH3)2), 2.60 (s, 3H, 5-CH3), 3.38–3.60 (m, 4H,
N(CH2CH3)2), 3.95 (s, 2H, CH2), 6.64 (d,
JH6ꢀH7=7.0 Hz, 1H, H-6), 7.10–7.24 (m, 2H, Ph), 7.78–
7.95 (m, 2H, Ph), 8.55 (d, JH7ꢀH6=7.0 Hz, 1H, H-7).
3s: yellow crystals; yield 40%; mp 185 ꢁC; 1H NMR
(CDCl3) d 1.16 (t, 3H, N(CH2CH3)2), 1.24 (t, 3H,
N(CH2CH3)2), 2.62 (s, 3H, 5-CH3), 3.48–3.52 (m, 4H,
N(CH2CH3)2), 3.99 (s, 2H, CH2), 6.79 (s,1H, H-6), 7.4
(d, 2H, Ph), 7.58–7.61(m, 2H, Ph), 7.81(d, 2H, Ph),
8.1-8.21 (m, 3H, Ph).
N,N-Diethyl-[2-(4-chlorophenyl)-7-methylpyrazolo[1,5-a]-
pyrimidin-3-yl]acetamide (3o). Starting from 2b
(1mmol, 306 mg) the title compound was prepared as
described for 3g. Purification by column chromato-
graphy (CHCl3/MeOH, 20:1v/v, as eluent) gave pure 3o
3t: yellow crystals; yield 18%; mp 177–178 ꢁC; 1H NMR
(CDCl3) d 1.15 (t, 3H, N(CH2CH3)2), 1.22 (t, 3H,
N(CH2CH3)2), 2.82 (s, 3H, 7-CH3), 3.41(q, 2H,
N(CH2CH3)2), 3.62 (q, 2H, N(CH2CH3)2), 4.05 (s, 2H,
CH2), 7.09 (s,1H, H-6), 7.41–7.58 (m, 5H, Ph), 7.93 (d,
2H, Ph), 8.1–8.20 (m, 2H, Ph).
as white crystals; yield 32%; mp 126–127 ꢁC; H NMR
1
(CDCl3) d 1.10–1.32 (m, 6H, N(CH2CH3)2), 2.81(s, 3H,
7-CH3) 3.38–3.60 (m, 4H, N(CH2CH3)2) 3.99 (s, 2H,
CH2), 6.70 (d, JH6ꢀH5=4.8 Hz, 1H, H-6), 7.41 (d, 2H,
Ph), 7.80 (d, 2H, Ph), 8.39 (d, JH5ꢀH6=4.8 Hz, 1H, H-5).
N,N-Diethyl-[2-(4-methylphenyl)-5-methyl-7-trifluorometh-
ylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide (3p) and N,N-
diethyl-[2-(4-methylphenyl)-5-trifluoromethyl-7-methylpy-
razolo[1,5-a]pyrimidin-3-yl]acetamide (3q). These com-
pounds were obtained from 2d (1mmol, 286 mg) and 1-
trifluoromethyl-2,4-pentanedione (1mmol, 0.12 mL)
following the general procedure. The isomeric com-
pounds 3p and 3q were separated by column chromato-
graphy with toluene/EtOAc, 4:1v/v as eluent; in
particular the first material eluted was compound 3p
and the second 3q.
N,N-Diethyl-[2-(4-chlorophenyl)-5,6,7-trimethylpyrazolo[1,5-
a]pyrimidin-3-yl]acetamide (3u). From 2b (1mmol,
306 mg) and 3-methyl-2,4-pentanedione (1mmol,
0.14 mL); ivory crystals purified by silica gel column
chromatography (CHCl3/MeOH, 10:1 v/v, as eluent);
yield 58%; mp 146–147 ꢁC; H NMR (CDCl3) d 1.05–
1
1.29 (m, 6H, N(CH2CH3)2), 2.30 (s, 3H, 6-CH3), 2.59 (s,
3H, 5-CH3), 2.81(s, 3H, 7-CH 3), 4.48–5.52 (m, 4H,
N(CH2CH3)2) 3.98 (s, 2H, CH2), 7.44 (d, 2H, Ph), 7.81
(d, 2H, Ph).
N,N-Diethyl-[2-(4-chlorophenyl)-5,7-methyl-6-ethoxycar-
bonylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide (3v). From
2b (1mmol, 306 mg) and ethyl 2-acetyl-3-oxo butanoate
(1mmol, 172 mg); ivory crystals purified by silica gel
column chromatography (toluene/EtOAc, 8:3 v/v, as
eluent); yield 70%; mp 116–117 ꢁC; 1H NMR (CDCl3) d
1.05–1.25 (m, 6H, N(CH2CH3)2), 1.42 (t, 2H,
OCH2CH3), 2.53 (s, 3H, 5-CH3), 2.90 (s, 3H, 7-CH3),
3.38–3.60 (m, 4H, N(CH2CH3)2) 3.93 (s, 2H, CH2), 4.42
(q, 2H, OCH2CH3 ), 7.42 (d, 2H, Ph), 7.82 (d, 2H, Ph).
3p: yellow crystals; yield 57%; mp 170 ꢁC, 1H NMR
(CDCl3) d 1.08–1.31 (m, 6H, N(CH2CH3)2), 2.41(s, 3H,
CH3), 2.63 (s, 3H, 5-CH3), 3.42–3.48 (m, 4H,
N(CH2CH3)2), 3.98 (s, 2H, CH2), 6.99 (s,1H, H-6), 7.24
(d, 2H, Ph), 7.68 (d, 2H, Ph).
3q: ivory crystals; yield 18%; mp 138 ꢁC, 1H NMR
(CDCl3) d 1.04–1.37 (m, 6H, N(CH2CH3)2), 2.42 (s, 3H,
CH3), 2.9 (s, 3H, 7-CH3), 3.47–3.52 (m, 4H,

S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
2669
N,N-Diethyl-(2-phenyl-6-ethoxycarbonyl-7-methylpyra-
zolo[1,5-a]pyrimidin-3-yl)acetamide (3w). From 2a
(1mmol, 272 mg) and ethyl 2-acetyl-3-ethoxyacrylate
(1mmol, 200 mg); white crystals purified by silica gel
column chromatography (toluene/EtOAc, 4:1v/v, as
eluent); yield 16%; mp 85–87 C; H NMR (CDCl3) d
1.07–1.29 (m, 6H, N(CH2CH3)2), 1.44 (t, 2H,
OCH2CH3), 3.22 (s, 3H, 7-CH3), 3.40–3.46 (m, 4H,
N(CH2CH3)2) 3.93 (s, 2H, CH2), 4.42 (d, 2H,
OCH2CH3), 7.41–7.51 (m, 3H, Ph), 7.82–7.90 (m, 2H,
Ph), 8.97 (s, 1H, 5-H).
with minor modification, from kidneys of male Wistar
rats killed by cervical dislocation. Kidneys were homo-
genized in 20 volumes of ice-cold 50 mM Tris–HCl, pH
7.4, 0.32 M sucrose, 1mM EDTA (buffer A), containing
protease inhibitors (160 mg/mL benzamidine, 200 mg/mL
bacitracine and 20 mg/mL soybean trypsin inhibitor)
and centrifuged at 600g for 10 min at 4 ꢁC. The resulting
supernatant was centrifuged at 10,000g for 10 min at
4 ꢁC. The pellet was then suspended, homogenized in 20
volumes of ice-cold 50 mM Tris–HCl, pH 7.4 (buffer B),
and centrifuged at 10,000g for 10 min at 4 ꢁC. The crude
mitochondrial pellet was frozen at ꢀ20 ꢁC until the time
of assay or incubated with either 0.6 nM [3H]PK11195
or 1nM [ 3H]Ro 5-4864 in buffer B, with a range of
concentrations of the tested compounds (0.1nM–
10 mM) in a total volume of 0.5 mL for 90 min at 4 ꢁC.
The incubation was terminated by dilution to 5 mL with
ice-cold buffer B, followed immediately by rapid filtra-
tion through glass fiber Whatman GF/C filters. The fil-
ters were then washed (2ꢄ5 mL) with buffer B and the
amount of radioactivity retained on the filters was
determined by Packard 1600 TR liquid scintillation
counter at 66% efficiency. Non-specific binding was
estimated in each case in the presence, respectively, of
unlabeled 1 mM PK 11195 or Ro 5-4864. For the active
compounds, the IC50 values were determined and Ki
values were derived according to the equation of Cheng
and Prusoff.53 Protein concentration was estimated by
the method of Lowry et al.,60 with bovine serum as
standard.
ꢁ
1
N,N-Diethyl-[2-(4-methylphenyl)-6-phenylpyrazolo[1,5-a]-
pyrimidin-3-yl]acetamide (3x). This compound was
obtained from 2d (1mmol, 286 mg) and phenylma-
58
londialdehyde (1mmol, 161mg)
in glacial acetic acid
(2 mL) following the general procedure. After cooling,
concentration of the solvent under reduced pressure
gave a residue that was diluted with H2O (5 mL) and
then extracted with Et2O (2ꢄ5 mL). The extracts were
dried over sodium sulfate and evaporated under
reduced pressure to give compound 3x. Ivory crystals;
ꢁ
1
yield 89%; mp 141–142 C; H NMR (CDCl3) d 1.04–
1.29 (m, 6H, N(CH2CH3)2), 2.41(s, 3H, CH3), 3.41–3.58
(m, 4H, N(CH2CH3)2) 4.01(s, 2H, CH2), 7.30 (d, 2H,
Ph), 7.48–7.61(m, 5H, Ph), 7.72 (d, 2H, Ph), 8.72 (s,
1H, 5-H), 8.84 (s, 1H, 7-H).
N,N-Diethyl-(2,7-phenylpyrazolo[1,5-a]pyrimidin-3-yl)-
acetamide (3y). Following the general procedure, this
compound was obtained from 2a (1mmol, 272 mg) and
1-phenyl-3-dimethylamino-2-propen-1-one
(1 mmol,
[3H]Ro15-1788 binding to rat cerebral cortex mem-
branes. Rat cerebral cortex membranes were prepared
as previously described.50 After differential centrifuga-
tion, the obtained crude membrane fraction was sub-
jected to washing procedures to remove endogenous
GABA.51 The washed membranes were incubated with
0.2 nM [3H]Ro 15-1788 for 90 min at 0 ꢁC in 500 mL of
buffer 50 mM Tris–citrate buffer pH 7,4, as previously
described.52
166 mg), synthesized according to a procedure described
for a similar compound,59 in glacial acetic acid (3 mL)
as solvent. Compound 3y was purified by column chro-
matography (toluene/EtOAc/AcOH, 8:2:1v/v/v, as elu-
ent). Yellow crystals; yield 43%; mp 131–132 ꢁC; 1H
NMR (CDCl3) d: 1.08–1.31 (m, 6H, N(CH2CH3)2),
3.38–3.59 (m, 4H, N(CH2CH3)2) 4.03 (s, 2H, CH2), 6.89
(d, JH6ꢀH5=4.5 Hz, 1H, 6-H), 7.40–7.53 (m, 2H, Ph),
7.54–7.60 (m, 3H, Ph), 7.78–7.86 (m, 2H, Ph), 8.10–8.22
(m, 3H, Ph), 8.50 (d, JH5ꢀH6=4.5 Hz, 1H, 5-H).
Cell culture. Rat glioma C6 cells were cultured in Dul-
becco’s Modified Eagles Medium supplemented with
10% FBS, 2 mM l-glutamine, 100 units/mL penicillin,
and 100 mg/mL streptomycin. Cultures were maintained
in a humidified atmosphere of 5% CO2/95% air at
37 ꢁC.
Biological methods
Materials. [3H]PK 11195 (sp.act. 87.0 Ci/mmol),
[3H]Ro5-4864 (sp.act. 84.5 Ci/mmol) and [3H]Ro15-
1788 (sp.act. 84.2 Ci/mmol) were purchased from
Dupont-New England Nuclear (Boston, MA, USA).
Culture medium, Fetal Bovine Serum (FBS), l-gluta-
mine and antibiotics were purchased from Bio-Whit-
taker Italia. PK11195, Ro 5-4864 and clonazepam were
obtained from Sigma. 1,2,3,4-tetrahydro-4-oxo-7-
chloro-2-naphthylpyridine (SU10603) and 2a,4a,
5a,17b-4,5epoxy-17-hydroxy-3-oxoandrostane-2-carbo-
nitrile (trilostane) were gifts from Novartis Farma S.p.a.
and Dr. D. Zister, University of Dublin, respectively.
All other reagents were obtained from commercial sup-
pliers.
Steroid biosynthesis. C6 cells were seeded in 24-well
plates at a density of ꢅ1ꢄ106 cells/well in a final
volume of 1mL. Prior to measurement of pregnenolone
production, the cells were washed three times with a
simple salts medium consisting of 140 mM NaCl, 5 mM
KCl, 1.8 mM CaCl2, 1mM MgSO 4, 10 mM glucose,
10 mM HEPES/NaOH, pH 7.4, plus 0.1% BSA. During
experiments, cells were incubated with this simple salts
medium in air incubator at 37 ꢁC. In order to measure
pregnenolone secreted into the medium, its further
metabolism was blocked by the addition of trilostane
(25 mM) and SU 10603 (10 mM) (inhibitors of 3b-hydro-
xysteroid dehydrogenase and 17a-hydroxylase, respec-
tively) to the simple salts medium, as previously
described.49 The addition of the novel compounds and
[3H]PK 11195 and [3H]Ro 5-4864 binding to rat kidney
mitochondrial membranes. For binding studies, mito-
chondria were prepared as previously described40,49

2670
S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
of PK 11195, Ro 5-4864, or clonazepam to the C6 cells
was made by the complete change of the simple salts
medium to medium containing the appropriate con-
centration (40 mM) of compound. The final concentra-
tion of ethanol was constant for all the wells within each
experiment and did not exceed 0.5% (v/v), a concentra-
tion, which on its own had no effect on steroid produc-
tion. At the end of the incubation period (2 h) the cell
medium was retained and centrifuged at 1500g for
10 min. The amount of pregnenolone secreted into the
medium was quantified by radio immunoassay (RIA),
using an antibody obtained from ICN Biochemical Inc.,
CA, USA, under the conditions recommended by the
supplier. Cell protein concentration was measured
according to the method of Lowry et al.60
24. Curran, T.; Morgan, J. I. Science 1985, 229, 1265.
25. Brown, R. C.; Cascio, C.; Papadopoulos, V. J. Neu-
rochem. 2000, 74, 847.
26. Serra, M.; Madau, P.; Chessa, M. F.; Caddeo, M.; Sanna,
E.; Trapani, G.; Franco, M.; Liso, G.; Purdy, R. H.; Barbaccia,
M. L.; Biggio, G. Br. J. Pharmacol. 1999, 127, 177.
27. Schumacher, M.; Akwa, Y.; Guennoun, R.; Robert, F.;
Labombarda, F.; Desarnaud, F.; Robel, P.; De Nicola, A. F.;
Baulieu, E. E. J. Neurocytol 2000, 29, 307.
28. Rao, V. L. R.; Dogan, A.; Bowen, K. K.; Dempsey, R. J.
Exp. Neurol. 2000, 161, 102.
29. Le Fur, G.; Perrier, M. L.; Vaucer, N.; Imbault, F.; Fla-
mier, A.; Benavides, J.; Uzan, A.; Renault, C.; Dubroeucq,
M. C.; Gueremy, C. Life Sci. 1983, 32, 1839.
30. Marangos, P. J.; Patel, J.; Boulenger, J. P.; Clark-Rosen-
berg, R. Mol. Pharmacol. 1982, 22, 26.
31. Langer, S. Z.; Arbilla, S.; Benavides, J.; Scatton, B. Adv.
Biochem. Psychopharmacol. 1990, 46, 61 .
32. Okuyama, S.; Chaki, S.; Yoshikawa, R.; Ogawa, S.;
Suzuki, Y.; Okubo, T.; Nakazato, A.; Nagamine, M.; Tomi-
sawa, K. Life Sci. 1999, 64, 1455.
References and Notes
33. McEnery, M. W.; Snowman, A. M.; Trifiletti, R. R.;
Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3170.
34. Garnier, M.; Dimchev, A. B.; Boujrad, N.; Price, J. M.;
Musto, N. A.; Papadopoulos, V. Mol. Pharmacol. 1994, 45, 201 .
35. Joseph-Liauzun, E.; Farges, R.; Delmas, P.; Ferrara, P.;
Loison, G. J. Biol. Chem. 1997, 272, 28102.
36. Joseph-Liauzun, E.; Delmas, P.; Shire, D.; Ferrara, P. J.
Biol. Chem. 1998, 273, 2146.
37. Salvetti, F.; Chelli, B.; Gesi, M.; Pellegrini, A.; Gian-
naccini, G.; Lucacchini, A.; Martini, C. Life Sci. 2000, 66,
1165.
38. Bourguignon, J. J. In Peripheral Benzodiazepine Receptors;
Giesen-Crouse, E., Ed.; Academic: London, 1993; pp 59–85.
39. Anzini, M.; Cappelli, A.; Vomero, S.; Giorgi, G.; Langer,
T.; Bruni, G.; Romeo, M. R.; Basile, A. S. J. Med. Chem.
1996, 39, 4275.
40. Trapani, G.; Franco, M.; Ricciardi, L.; Latrofa, A.; Gen-
chi, G.; Sanna, E.; Tuveri, F.; Cagetti, E.; Biggio, G.; Liso, G.
J. Med. Chem. 1997, 40, 3109.
1. Braestrup, C.; Squires, R. F. Proc. Natl. Acad. Sci. U.S.A.
1977, 74, 3805.
2. Anholt, R. R.; Pedersen, P. L.; De Souza, E. B.; Snyder,
S. H. J. Biol. Chem. 1986, 261, 576.
3. Hirsch, J. D.; Beyer, C. F.; Malkowitz, L.; Loullis, C. C.;
Blume, A. J. Mol. Pharmacol. 1988, 34, 164.
4. Romeo, E.; Auta, J.; Kozikowski, A. P.; Ma, D.; Papado-
poulos, V.; Puia, G.; Costa, E.; Guidotti, A. J. Pharmacol.
Exp. Ther. 1992, 262, 971.
5. Kozikowski, A. P.; Ma, D.; Brewer, J.; Sun, S.; Costa, E.;
Romeo, E.; Guidotti, A. J. Med. Chem. 1993, 36, 2908.
6. Langer, S. Z.; Arbilla, S. Pharmacol. Biochem. Behav. 1988,
29, 763.
7. Basile, A. S.; Skolnick, P. J. Neurochem. 1986, 46, 305.
8. Taupin, V.; Toulmond, S.; Serrano, A.; Benavides, J.;
Zavala, F. J. Neuroimmunol. 1993, 42, 177.
9. Costa, E.; Guidotti, A. Annu. Rev. Pharmacol. Toxicol.
1979, 19, 531.
10. Gallager, D. W.; Mallorga, P.; Oertel, W.; Henneberry,
R.; Tallman, J. F. J. Neurosci. 1981, 1, 218.
41. Trapani, G.; Franco, M.; Latrofa, A.; Ricciardi, L.; Car-
otti, A.; Serra, M.; Sanna, E.; Biggio, G.; Liso, G. J. Med.
Chem. 1999, 42, 3934.
42. Cappelli, A.; Anzini, M.; Vomero, S.; De Benedetti, P. G.;
Menziani, M. C.; Giorgi, G.; Manzoni, C. J. Med. Chem.
1997, 40, 2910.
43. Lentini, G.; Bourguignon, J. J.; Wermuth, C. G. In
QSAR: Rational Approaches to the Design of Bioactive Com-
pounds; Silipo, C., Vittoria, A., Eds.; Elsevier Science:
Amsterdam, 1991; pp 257–260.
11. Syapin, P. J.; Skolnick, P. J. Neurochem. 1979, 32, 1047.
12. Papadopoulos, V. Endocr. Rev. 1993, 14, 222.
13. Saano, V. Acta Pharmacol. Toxicol. 1986, 58, 333.
14. Olson, J. M.; Ciliax, B. J.; Mancini, W. R.; Young, A. B.
Eur. J. Pharmacol. 1988, 152, 47.
15. Woods, M. J.; Williams, D. C. Biochem. Pharmacol. 1996,
52, 1805.
16. Krueger, K. E. Biochim. Biophys. Acta 1995, 1241, 453.
17. Gavish, M.; Bachman, I.; Shoukrun, R.; Katz, Y.; Veen-
man, L.; Weisinger, G.; Weizman, A. Pharmacol. Rev. 1999,
51, 629.
18. Culty, M.; Li, H.; Boujrad, N.; Amri, H.; Vidic, B.; Ber-
nassau, J. M.; Revers, J. L.; Papadopoulos, V. J. Steroid Bio-
chem. Mol. Biol. 1999, 69, 123.
19. Mestre, M.; Carriot, T.; Belin, C.; Uzan, A.; Renault, C.;
Dubroeucq, M. C.; Gueremy, C.; Doble, A.; Le Fur, G. Life
Sci. 1985, 36, 391.
44. Cinone, N.; Holtje, H. D.; Carotti, A. J. Comput.-Aided
¨
Mol. Des. 2000, 14, 753.
45. Long, R. S. J. Am. Chem. Soc. 1947, 69, 990.
46. Kuthan, J.; Paleeek, J.; Valihrach, J. Collect. Czech.
Chem. Commun. 1981, 46, 748.
47. Chimichi, S.; Cosimelli, B.; Bruni, F.; Selleri, S. Can. J.
Chem. 1992, 70, 1093.
48. Chimichi, S.; Cosimelli, B.; Bruni, F.; Selleri, S. Magn.
Reson. Chem. 1992, 30, 1117.
20. Miccoli, L.; Poirson-Bichat, F.; Sureau, F.; Bras Gon-
c¸ alves, R.; Bourgeois, Y.; Dutrillaux, B.; Poupon, M. F.;
Oudard, S. J. Natl. Cancer Inst. 1998, 90, 1400.
21. Torres, S. R. R.; Nardi, G. M.; Ferrara, P.; Ribeiro-do-
Valle, R. M.; Farges, R. C. Eur. J. Pharmacol. 1999, 385, R1 .
22. Zorov, D. B.; Krasnikov, B. F.; Kuzminova, A. E.; Vyso-
kikh, M. Y.; Zorova, L. D. Biosci. Rep 1997, 17, 507.
49. Campiani, G.; Nacci, V.; Fiorini, I.; De Filippis, M. P.;
Garofalo, A.; Ciani, S. M.; Greco, G.; Novellino, E.; Wil-
liams, D. C.; Zisterer, D. M.; Woods, M. J.; Mihai, C.; Man-
zoni, C.; Mennini, T. J. Med. Chem. 1996, 39, 3435.
50. Squires, R. F.; Braestrup, C. Nature 1997, 266, 732.
51. Karobath, M.; Sperk, G. Proc. Natl. Acad. Sci. U.S.A.
1979, 76, 1004.
23. Moreno-Sa
´
nchez, R.; Houge, B. A.; Bravo, C.; Newman,
52. Costanzo, A.; Guerrini, G.; Ciciani, G.; Bruni, F.; Selleri,
S.; Costa, B.; Martini, C.; Lucacchini, A.; Aiello, P. M.;
Ipponi, A. J. Med. Chem. 1999, 42, 2218.
A. H.; Basile, A. S.; Chang, P. K. Biochem. Pharmacol. 1991,
41, 1479.

S. Selleri et al. / Bioorg. Med. Chem. 9 (2001) 2661–2671
2671
53. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
54. Le Fur, G.; Vaucher, N.; Perrier, M. L.; Flamier, A.;
Benavides, J.; Renault, C.; Dubroeucq, M. C.; Gueremy, C.;
Uzan, A. Life Sci. 1983, 33, 449.
55. Chaki, S.; Funakoshi, T.; Yoshikawa, R.; Okuyama, S.;
Okubo, T.; Nakazato, A.; Nagamine, M.; Tomisawa, K. Eur.
J. Pharmacol. 1999, 371, 197.
56. Rupprecht, R.; Holsboer, F. Steroids 1999, 64, 83.
57. Tsutsui, K.; Ukena, K.; Usui, M.; Sakamoto, H.; Takase,
M. Neurosci. Res. 2000, 36, 261.
58. Coppola, G. M.; Hardtmann, G. E.; Huegi, B. S. J. Het-
erocyclic Chem. 1974, 11, 51 .
59. Zaleplon Drugs Future 1996, 21, 37.
60. Lowry, O. H.; Rosenburg, N. J.; Farr, A. L.; Randall,
R. J. J. Biol. Chem. 1951, 193, 265.