High affinity central benzodiazepine receptor ligands: Synthesis and structure-activity relationship studies of a new series of pyrazolo[4,3- c]quinolin-3-ones

Article abstract of DOI:10.1016/S0968-0896(97)10039-6

A large series of 2-aryl(heteroaryl)-2,5-dihydropyrazolo[4,3-c]quinolin- 3-(3H)-ones, carrying appropriate substituents at the quinoline and N₂- phenyl rings, were prepared and tested as central benzodiazepine receptor ligands. Results from structure-affinity relationship studies were in full agreement with previously proposed pharmacophore models and, in addition, quantitative structure-activity analysis gave further significant insight into the main molecular determinants of high benzodiazepine receptor affinity. The intrinsic activity of some active ligands was also determined and preliminary discussed.

Full text of DOI:10.1016/S0968-0896(97)10039-6

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 389±399
High Anity Central Benzodiazepine Receptor Ligands:
Synthesis and Structure±Activity Relationship Studies of a New
Series of Pyrazolo[4,3-c]quinolin-3-ones
L. Savini,a P. Massarelli,a C. Nencini,a C. Pellerano,a G. Biggio,b
A. Maciocco,b G. Tuligi,b A. Carrieri,c N. Cinonec and A. Carottic,*
a
Á
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi, via Banchi di Sotto 55, I-53100 Siena, Italy
b
Á
Dipartimento di Biologia Sperimentale, Universita degli Studi, via Palabanda 12, I-09123 Cagliari, Italy
c
Á
Dipartimento Farmaco Chimico, Universita degli Studi, via E. Orabona 4, I-70125 Bari, Italy
Received 17 July 1997; accepted 18 October 1997
AbstractÐA large series of 2-aryl(heteroaryl)-2,5-dihydropyrazolo[4,3-c]quinolin-3-(3H)-ones, carrying appropriate
substituents at the quinoline and N2-phenyl rings, were prepared and tested as central benzodiazepine receptor ligands.
Results from structure±anity relationship studies were in full agreement with previously proposed pharmacophore
models and, in addition, quantitative structure±activity analysis gave further signi®cant insight into the main molecular
determinants of high benzodiazepine receptor anity. The intrinsic activity of some active ligands was also determined
and preliminary discussed. # 1998 Published by Elsevier Science Ltd. All rights reserved.
Introduction
Cl channel. Most functional subtypes of GABAA
Ligands that interact with the benzodiazepine receptor
(BzR) and allosterically modulate the action of GABA
on neuronal chloride ion ¯ux have a continuum of
intrinsic activity, ranging from full agonists (anxiolytic,
hypnotic, and anticonvulsant agents), through antago-
nists (nil ecacy), to inverse agonists (proconvulsant
and anxiogenic agents).1±3 Diazepam, b-CCM, and
Ro15-1788 are typical representative of an agonist, an
inverse agonist and an antagonist, respectively.4 The
heterogeneity, and the functional and structural com-
plexity of the GABAA receptor/Cl ionophore com-
plexes have strongly hampered up to now a clear
detection and de®nition of the stereoelectronic require-
ments necessary for eliciting a speci®c intrinsic activity.
A total of at least 14 subunits (a1 to a6, b1 to b3, g1 to
g3, d and E)5,6 of the GABAA receptor have been iden-
ti®ed by molecular cloning, and these subunits are
taught to assemble into a pentameric structure to form a
receptors contain a, b, and g subunits, with di€erent
subtypes showing high sensitivity to di€erent benzodia-
zepine receptor ligands.7±9 Unfortunately, the key
structural and dynamic properties of the BzR cannot be
directly measured, or modeled, because of the limited
current data and methodologies and therefore, several
indirect studies have been carried out to detect the
common structural features of diverse classes of BzR
ligands most likely responsible for high anity and
possibly for speci®c intrinsic activity and de®nite phar-
macological pro®le.10±17
Despite the recent signi®cant advances in the structural
and functional studies of BzR,6 and in the molecular
pharmacology and physiopathology of anxiety disor-
ders,3 much remains to be achieved for the design and
development of truly innovative drugs, lacking the
numerous side e€ects of benzodiazepines.18
Key words: Central benzodiazepine receptor ligands; pyra-
zolo[4,3-c]quino lin-3-ones; QSAR; 35S-TBPS binding; phar-
macophore re®nement.
*Corresponding author. Tel: 39 80 544 2782; Fax: 39 80 544
2724; E-mail: carotti@ippo.uniba.it
From a medicinal chemistry view point, the recent years
have seen the proposal and re®nement of several phar-
macophore models,10±17 which may constitute an useful
guide for the design of new potent ligands. However,
0968-0896/98/$19.00 # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(97)10039-6

390
L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
they have not achieved yet the ability to correctly pre-
dict the intrinsic activity.
Chemistry
The pyrazolo[4,3-c]quinolin-3-ones (4) were prepared
through the synthetic pathway shown in Scheme 1,
according to reported literature procedures;21,29 thus,
properly substituted anilines (1) were reacted with di-
ethyl(ethoxymethylene) malonate (EMME) in re¯uxing
Dowtherm A to give ethyl 4-hydroxyquinoline-3-car-
boxylates (2) via thermal ring closure. Treatment of (2)
with phosphorus oxychloride led to the corresponding
chloroderivatives (3), which were cyclized with the
appropriate hydrazines (hydrazine hydrate 85%, R0-
substituted phenylhydrazines, and a-N-heterocyclic-
hydrazines to a€ord PQs (4).
An interesting contribution in this ®eld came also from
some recent 2-D and 3-D QSAR studies19,20 of a new
class of BzR ligands, namely the 2-aryl-2,5-dihydropyr-
idazino[4,3-b]indol-3-(3H)-ones (PIs), which are struc-
turally related to the well known 2-aryl-2,5-
dihydropyrazolo[4,3-c]quinoline-3-(3H)ones (PQs).
Since the discovery of PQs as high anity BzR ligands21
interest in their peculiar pharmacological activity has
continued to grow. The lack of some unwanted side
e€ects found in classical benzodiazepines as well as the
net shift of intrinsic activity caused by small structural
changes, render the PQs a fascinating and intriguing
class of BzR ligands. It is very surprising therefore that
only few papers dealing with SAR studies of PQs had
appeared in the literature.22±26
All the synthesized PQs were isolated in satisfactory
yields (40±60%) and their chemical structures were
1
con®rmed by means of IR and H NMR data.
For all compounds (4), a strong band between 1620 and
1
Inasmuch as PQ ligands show higher anity than the
corresponding PI analogs, and moreover small struc-
tural modi®cations lead to a complete span of the
intrinsic activity (from agonists to inverse agonists), we
decided to carry out an extensive structure±activity
relationships study by designing and testing a large ser-
ies of new PQ analogs. Thus, the molecular skeleton of
PQs was properly substituted aiming at the identi®ca-
tion of the primary ligand±receptor interactions all
around the quinoline and the N2-phenyl rings responsi-
ble for recognition and di€erent activation of BzR. To
reach this important objective, a high number of PQ
analogs (listed in Table 1) have been synthesized and
their BzR anity measured by radioligand binding
assay. In particular, several mono and polysubstituted
derivatives on the quinoline ring and monosubstituted
ortho, meta, and para congeners at the N2-phenyl ring
were prepared. The aryl substituents at position 2 were
replaced also by aza-heterocycles. The overall structural
modi®cations were such that the substituent physico-
chemical space resulted more completely explored for
meta and para congeners and for the positions 6 and 8
at the quinoline moiety.
1680 cm and a weak band at 3400 to 3100 cm 1, due
to the stretching vibration of the carbonyl and the NH
groups, respectively, were observed in the IR spectra. In
1
the H NMR spectra the characteristic signals for com-
pounds (4), in good accordance with the literature
data,21 were a singlet between 8.31 and 9.52 d, attribu-
table to H-4 proton, and a broad singlet, exchangeable
with D2O, between 12.3 and 13.2 d due to the N5-H
proton.
1
In Table 3, the IR and H NMR spectral data of some
representative PQ 4 have been reported.
OH
COOC H
2
5
i
R
R
∆
N
ii
NH
2
2
1
∆
X
N
N
N
Cl
The synthesis and BzR anity of some PQs listed in
Table 1 have been already reported by some of us.27,28
Unfortunately, due to an undetected and not systematic
error in the experimental procedure of the binding
assay, those data were wrong and therefore they have
been correctly redetermined for the present study.
COOC H
2
5
O
iii
R
R
N
4 H
3
R'
, 2'-pyridyl, 2'-pyrimidyl, and 2'-pyrazinyl
X =
With few exceptions, the biological data listed in Table 1
come from the present work and constitute a very
homogeneous and well suited data set for SAR and QSAR
studies. Literature data used in the actual study were nor-
malized with respect to internal standards (as shown).
R and R' = as reported in Table 1
Scheme 1. (i) Diethyl(ethoxymethylene)malonate, Dowtherm A;
(ii) POCl3; (iii) hydrazine, R0-phenylhydrazine (ethanol re¯ux);
a-(N)-heterocyclichydrazines (rt).

L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
391
Table 1. Physicochemical and binding data of pyrazolo[4,3-c]quinolin-3-ones(41±68
)
R'
4'
3'
2'
N
N
9
6
O
8
7
R
I
N
H
0
0
0
c
Compound
R6
R7
R8
R9
R2
R3
R4
mp(ꢀC)a
Crystallization
solventb
pIC50
Intrinsic
efficacyd
e
41
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
unsubstituted at N2
>300
Ð
A
6.30
9.35
9.00
9.17
6.84
6.75
8.16
7.18
5.73
5.66
9.54
8.70
9.26
9.48
9.34
9.37
9.17
8.85
9.00
5.93
9.50
8.77
9.22
8.35
5.55
8.60
8.36
8.16
7.75
6.94
9.15
9.40
8.60
8.47
7.46
8.20
7.62
9.40
7.20
9.62
7.82
8.79
7.90
9.00
f,g
42
H
H
H
H
Cl
f,g
43
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ð
f,g
44
H
H
H
OCH3
H
Ð
g,h
45
H
H
H
Ð
g,h
46
H
H
H
Cl
Ð
47
F
H
H
H
>300
>300
294±296
288±290
>300
>300
>300
290(dec)
>300
Ð
Am
A
k
48
CH3
CF3
OCH3
H
H
H
H
49
H
H
H
B
k
410
H
H
H
A
l
411
F
H
H
A
Dm
412
413
H
F
NO2
NH2
H
H
H
F
H
A
l
414
H
F
OCH3
OH
H
A
Am
415
H
F
H
f,g
416
H
Cl
H
f,g
417
H
OCH3
OC2H5
n.C4H9
n.C4H9
n.C4H9
n.C4H9
n.C4H9
cC6H11
cC6H11
cC6H11
cC6H11
cC6H11
OBn
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
H
H
Ð
418
H
H
H
>300
Ð
B
i
419
H
H
H
420
421
422
423
H
H
COOH
>300
263±267
>300
270(dec)
Ð
E
H
2-pyrid-20-yl
2-pyrimid-20-yl
2-pyrazin-20-yl
Bm
Am
Bm
H
H
i
424
H
H
H
H
H
425
426
427
428
H
H
COOH
>300
>300
>300
>300
Ð
Am
A
H
2-pyrid-20-yl
2-pyrimid-20-yl
2-pyrazin-20-yl
AG
AG
H
Cm
B
H
g
429
H
H
H
H
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
H
unsubstituted at N2
>300
>300
268±271
291±294
290±292
>300
295(dec)
>300
>300
>300
>300
>300
>300
>300
>300
Bm
A
F
H
H
F
H
H
H
H
H
H
Cl
CH3
H
H
H
Bm
H
H
H
B
H
Br
CH3
Cl
H
A
B
H
H
H
H
H
H
A
A
D
B
H
H
F
H
H
H
NO2
NH2
H
H
H
H
H
H
H
Br
CH3
Cl
F
B
H
H
H
A
A
A
H
H
H
AG
H
H
H

392
L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
Table 1Ðcontd
0
0
0
c
Compound
R6
R7
R8
R9
R2
R3
R4
mp(ꢀC)a
Crystallization
solventb
pIC50
Intrinsic
efficacyd
445
446
447
448
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
OCF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
NO2
NH2
OCH3
OH
H
>300
>300
>300
284±287
Ð
C
7.40
9.10
9.22
9.63
9.62
8.84
7.87
8.02
6.79
8.12
7.82
6.47
6.94
8.43
7.70
7.15
7.13
7.68
8.14
8.90
5.52
8.50
7.24
8.94
OCF3
Bm
Bm
Am
OCF3
OCF3
H
H
AG
H
g
449
OH
OCH3
H
g
450
H
H
Ð
j
451
F
H
Ð
j
452
j
F
F
H
H
Ð
453
F
F
H
H
Br
Ð
j
454
F
F
H
H
OCH3
Ð
455
456
457
F
F
H
2-pyrid-20-yl
2-pyrimid-20-yl
2-pyrazin-20-yl
>300
>300
>300
Ð
A
IA
IA
F
F
H
Am
Am
F
F
H
j
458
H
F
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
459
460
461
462
463
464
465
466
467
468
F
H
CH3
Cl
>300
>300
>300
>300
>300
298±301
>300
>300
297(dec)
267(dec)
A
Am
A
F
F
F
H
F
F
F
H
F
F
F
H
F
A
F
F
F
H
OCH3
H
A
H
H
H
H
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
A
Dm
COOH
2-pyrid-20-yl
2-pyrimid-20-yl
2-pyrazin-20-yl
A
Am
Am
IA
aReported only for the newly synthesized compounds and for compounds already described but lacking those data.
bCrystallization solvent(s): A, EtOH; B, EtOH+H2O; C, DMF; D, DMF+H2O; E, MeOH; F, AcOEt.
cBinding data from the displacement of [3H]-¯unitrazepam.
dIntrinsic ecacy from [35S]TBPS binding assay (see text and Figure 2). AG, agonist; IA, inverse agonist.
eRef 30.
fRef 25; pIC50 has been redeterminated for molecules 42 and 43. Compounds 42, 43, and 44 are better known as CGS-8216, CGS-9896,
and CGS-9895, respectively.
gRef 24.
hN5-Methyl derivatives.
iRef 27.
jRef 28.
kRef 31.
lRef 32.
mCrystallized with n-water molecules; n was determined by Karl Fischer method.
The known compounds CGS-8216 (42) and CGS-9896
(43) were also resynthesized as biological standards and
their physical and spectral properties are consistent with
literature values.21
Biochemistry
The compounds listed in Table 1 were tested for their
ability to displace [3H]¯unitrazepam binding from rat
brain membranes. The pIC50 values reported in Table 1
have been normalized using CGS-8216 (42) and
CGS-9896 (43) as reference compounds. Also literature
data (Table 1) were normalized by the same method.
The intrinsic activity of some selected compounds in
enhancing GABAA receptor function was evaluated by
measuring their ecacy in reducing 35S-t-butylbicyclo-
phosphorotionate (35S-TBPS) binding in unwashed
membranes (where GABA is present) from rat cerebral
The hydroxyderivatives (415), and (448) were conveni-
ently obtained from the corresponding methoxy-
derivatives (414), and (447) respectively, by hydrolysis
with 48% HBr in glacial acetic acid.
Catalytic reduction of compounds (412), (439) and (445)
a€orded the target aminoderivatives (413), (440) and
(446), respectively.

L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
393
cortex. In fact, by measuring 35S-TBPS binding in the
above membrane preparation it is possible to charac-
terize the pharmacological pro®le (agonist, inverse ago-
nist, antagonist) as well as the ecacy of di€erent
benzodiazepine receptor ligands at the GABAA receptor
level.33±35
as recently hypothesized in other regions for simi-
lar BzR ligands.37
.
.
Para-substitution at the 2-phenyl ring a€orded
congeners more active than the corresponding
meta analogs with two clear exceptions, the amino
(compare 446 versus 440) and the ¯uoro (compare
444 versus 438) derivatives. The para-carboxy sub-
stituent always caused a drastic decrease of activ-
ity (see 420, 425, and 465).
The replacement of the 2-phenyl ring with aza-
heterocycles gave interesting results; higher anity
arise for the 8-cC6H11 and 8-nC4H9 substituted
congeners (compare 426 versus 424 and 419 versus
423 and 421) whereas a lower anity was detected
for the 7,8,9-trimethoxy substituted congeners
(compare 464 versus 466 and 467). The 2-pyridyl
congeners were generally more active than 2-pyri-
midyl and 2-pyrazinyl ones.
Substitution at position 6 was less tolerated than
in positions 7, 8 and 9. Most likely, the formation
of an ecient hydrogen bond of the N5-H group
with an HB acceptor group in the receptor (A2)
might be prevented or limited by the presence of
bulky groups at position 6. A similar e€ect was
very recently reported at the same topological
position for imidazo[1,5]quinoxalin-4-ones.38
Mono substitution at the quinoline ring a€orded
ligands more active than those deriving from
multiple substitutions. The substitution at posi-
tion 9, in particular, gave the most satisfactory
results.
Results and discussion
In agreement with and as integration of previous ®nd-
ings (see Figure 1 for the sake of clarity) the following
general remarks on SAR can be made:
.
The presence of an aryl or heteroaryl substituent
at N2 (occupation of the L1±L2 regions) was cru-
cial for a high anity (see the low activity of
compds 41 and 430).
.
.
The substitution at N5 with a methyl group
strongly diminished the anity (compare 42 and
43 versus 45 and 46: elimination of an HB with A2)
Ortho-substitution at the 2-phenyl ring reduced
the activity (partial occupation of the `sterically
unaccessible' S1 region) with the notable exception
of the ¯uoro derivative 432, which, in contrast,
presented an activity slightly higher than the
unsubstituted congener 431. This result may be
explained considering both the small dimensions
of the ¯uorine atom and its possible involvement
in a three-centered hydrogen bonding with the N1
atom and a hydrogen bond donor in the receptor,
.
.
Quantitative structure±activity relationship (QSAR)
studies
L3
L2
In order to gain useful indications at a quantitative level
on the topography of the BzR all around the molecular
skeleton of PQ, a QSAR study by the classical Hansch
approach39 was carried out. Recently, an extensive
review on the quantitative structure±activity relation-
ships of several classes of BzR ligands, not inclusive of
PQ compounds, was carried out by Hansch himself.40 In
our case, the lack of parameters for some substituent in
the data set and the presence of vicinal polisubstituted
congeners, quite dicult to correctly parametrize, pre-
L1
H2
N
N
S1
O
N
H1
H
vented
a safe application of the classical QSAR
A2
approach to the overall data set. The Hansch approach,
on the other hand, is well suited for congeneric series
and for this reason our QSAR analysis was limited to
the 8 and 6 substituted 2-aryl congeners and to the meta
and para substituted congeners of the 8-OCF3 derivative
431. A multiple regression analysis with cross-validation
was performed on the binding data of Table 1 using the
chemical descriptors listed in Table 2. The electronic and
hydrophobic e€ects of substitutents were assessed by the
Hammett (s) and Hansch (p) substituent constants,
Figure 1. Proposed pharmacophore model17 for BzR. The
main binding sites for pyrazoloquinoline CGS-8216 (42) are
indicated as H1 and H2 (HB donor sites), A2 (HB acceptor site),
L1 and L2 (hydrophobic sites). L3 is another lipophilic region
reached by the 5-phenyl ring of classical benzodiazepines. S1
represents a sterically unaccessible region. Binding to H2 and
A2 is not necessary for inverse agonist activity.36

394
L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
Table 2. Substituent parameters used for the derivation of regression eqs (1)±(3)a
Substituent
ꢀb
ꢁ
MRc
vW
L
B1
B5
H
0.00
0.35
0.16
0.22
0.00
1.04
2.13
2.51
0.71
0.02
1.66
0.67
0.14
0.38
0.56
0.88
0.86
0.28
1.23
0.10
0.79
1.96
2.67
0.60
0.79
3.17
0.28
0.09
1.25
0.57
0.50
0.89
0.74
0.54
0.08
1.60
3.12
4.26
1.07
1.49
4.95
0.49
0.36
2.42
1.01
1.11
1.32
1.20
0.67
2.06
4.57
6.17
6.17
3.52
3.98
8.20d
2.74
2.65
4.80
2.87
3.30
3.82
3.44
2.78
1.00
1.35
1.52
1.91
1.80
1.35
1.61d
1.35
1.35
1.35
1.52
1.99
1.95
1.70
1.35
1.00
3.61
4.54
3.49
1.80
3.07
4.44d
1.93
1.35
3.36
2.04
2.61
1.95
2.44
1.97
OCF3
n.C4H9
cC6H11
Cl
0.23 (0.37)
0.27
0.23
OCH3
OBn
OH
0.37
0.06 (0.34)
0.24
F
OC2H5
CH3
CF3
0.17 ( 0.07)
0.54
Br
0.23 (0.39)
0.78 (0.71)
0.66 ( 0.16)
NO2
NH2
aData taken from ref 41. See text for the signi®cance of parameter symbols.
bValues for meta substituents are reported in parentheses.
cScaled by 0.1.
dReferring to the benzyloxy group in a planar conformation.
whereas the molar refractivity (MR), the van der Waals
volume (vW) and the STERIMOL Verloop parameters
L, B1, and B5 were employed to model bulkiness and
polarizability e€ects.41
gests a stronger steric e€ect in position 6, which may
limit the formation of an HB of the N5-H group. The
negative sign with s and MR in eq (3) indicates that the
interaction of the 2-phenylsubstituted ring at the L1 (L2)
receptor regions is favoured by small electron donor
substituents at both meta and para positions. An equa-
tion of comparable statistical value can be obtained by
substituting MR with vW.
The following equations were derived:
8 substituted; 2 phenyl congeners
pIC50
n ˆ 9; r2 ˆ 0:883; Q2 ˆ 0:745; s ˆ 0:208; F ˆ 52:77
ˆ
0:32ꢀÆ0:10†vW ‡ 9:63ꢀÆ0:28†
The lipophilic character of the substituents on the phe-
nyl ring seems to play no or marginal role in the recep-
tor-ligand interaction and this casts some doubt about
the lipophilic nature assigned in several pharmacophore
models to the L2 receptor regions.
ꢀ1†
6 substituted; 2 phenyl congeners
pIC50
ˆ
2:63ꢀÆ0:47†vW ‡ 9:35ꢀÆ0:45†
In addition eq (3) evidences a limited steric accessibility
of the L2 region, as noticed also in our previous
study.20 Finally, eq (3) suggests that a p±p stacking
interaction,42 in which the substituted phenyl ring acts
as p electron donor, might take place at the L1 receptor
region.
n ˆ 5; r2 ˆ 0:912; Q2 ˆ 0:801; s ˆ 0:543; F ˆ 31:23
ꢀ2†
8
OCF3; 2 phenyl substituted congeners
pIC50
n ˆ 15; r2 ˆ 0:803; Q2 ˆ 0:723; s ˆ 0:418; F ˆ 24:51
ˆ
1:40ꢀÆ0:29†ꢀ 1:55ꢀÆ0:42†MR ‡ 9:46ꢀÆ0:24†
Structure±ecacy relationships: preliminary results
ꢀ3†
The most used in vitro methods to predict the intrinsic
activity of a BzR ligand are the GABA ratio (IC50
without GABA/IC50 with GABA) and the binding assay
with [35S]TBPS.
In eqs (1)±(3), n is the number of compounds, r and Q
are the correlation coecient and the cross-validated
correlation coecient, respectively; s is the standard
deviation; F is the F statistic. Eqs (1)±(3) present good
statistics both in terms of ®tting and predictive power.
Eqs (1) and (2) point out a signi®cant steric hindrance
on both 8 and 6 positions. The negative coecient with
vW is much higher in eq (2) than in eq (1) and this sug-
To estimate the antagonist or inverse agonist activity of
BzR ligands 4, we decided to use the binding assay with
[35]TBPS which is considered one of the best biochem-
ical tool, sometimes superior to GABA shift,43,44 to

L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
395
Conclusions
150
125
100
75
Despite the relatively high number of synthesized com-
pounds, only few of them displayed an anity higher
than the parent compound CGS-8216 (42). In fact, as
clearly evidenced in the SAR and QSAR analyses dis-
cussed herein, most of the substitutions, both on quino-
line and 2-phenyl rings, were not tolerated primarily
because of steric reasons. Evidently, the lead compound
CGS-8216 (42) had already an ideal chemical structure,
in terms of spatial disposition of the putative pharma-
cophoric elements and overall physicochemical proper-
ties, to engage an ecient, strong interaction with the
BzR binding sites. However, PQ derivatives bearing
small substituents in de®nite positions, such as the
¯uoro derivatives 411 and 432, the hydroxy derivatives
415, 448, and 449 and the amino derivative 440, showed an
outstanding anity.
50
25
0
-8
-7
-6
-5
-4
10
31
10
10
10
10
Log [DRUG], M
4 Abecarnil
68
4
4
4
4
4
4
47
26
57
43
55
Figure 2. E€ects of modulation of the indicated PQ 4 and
Abecarnil on [35S]TBPS binding in rat cerebral cortex.
evaluate the intrinsic activity of both positive and nega-
tive modulator of GABAA receptor complex.45 Accord-
ingly, GABA agonists decrease the total number of [35S]
TBPS binding sites in a dose-dependent manner whereas
an opposite e€ect can be seen for GABA inverse ago-
nists.33±35,41 No signi®cant changes have been observed
for antagonists.
It is worth noting that the very active 8-OCF3 congeners
440 and 448 as well as congener 415 contain highly
hydrophilic substituents (OH and NH2) and this cast
some doubt on the supposedly lipophilic character of
the L2 pocket of the BzR receptors as claimed so far.
Moreover, that pocket should have a limited steric
accessibility as clearly pointed out by eq (3), in good
agreement with a previous QSAR study on PI.20 The
strong substituent electronic e€ect on the BzR anity is
also indicated by eq (3): electrondonor substitutents on
the 2-phenyl ring favor the ligand-receptor binding which
might take place trough a p±p stacking interaction.42
In this preliminary phase of our study on structure±e-
cacy relationships we limited our analysis to seven
compounds, which were chosen to test mainly the e€ect
of the para-substitution at the 2-phenyl ring (as origin-
ally made with CGS congeners)21 and of the replace-
ment of the phenyl ring with nitrogen heterocycles. The
results obtained are listed on the last column of Table 1
and represented graphically in Figure 2.
Conversely, no electronic e€ect has been detected in the
QSAR analysis of PI analogs and this could be due also
to some di€erent orientation of the phenyl ring in PQ
and PI, as found by molecular modelling studies.20
Unlike what has been found in the original PQ series
(compds 42, 43 and 44)21, no change in the functional
e€ects on BzR was observed for the corresponding 8-
OCF3 congeners 431, 443, and 447, all eliciting a clear
agonist activity.
A preliminary analysis of the structure±intrinsic ecacy
relationships, based at the moment only on very few
ligands, indicated that small structural modi®cations on
PQ may evoke a diverse BzR activation. However,
compds 431, 443, and 447 bearing H, Cl, and OCH3 sub-
stituents at the para position of 2-phenyl ring, all elici-
ted an agonistic activity, unlike the corresponding
congeners of the PQ and PI series, for which a di€erent
intrinsic activity has been found.19±21 These unexpected
results deserve further investigation. A study on the
in¯uence of the 8-OCF3 group on the peculiar molecular
properties of compds 431, 443, and 447 compared to the
corresponding 8-unsubstituted PQ and PI congeners, is
underway and should help clarifying this point.
That was unexpected since also in our recent investiga-
tion on the BzR activity of pyridazino-indolones, a net
in¯uence of the nature of para-substituents on the
intrinsic activity has been detected.19,20
With the only exception of compound 426, the presence
of an heterocyclic substituent at position 2 (compds 455,
457, and 468) elicited an inverse agonist activity. These
®ndings are quite interesting and in good agreement
with those obtained for 2-thienyl substituted PQs.22,23
Further investigations to evaluate whether the presence
of a nitrogen heterocycles at position 2 may induce an
inverse agonist activity and how such an activity may be
modulated or reversed by the nature (and position?) of
other substituents in the quinoline ring are warranted.
An inverse agonist pro®le was instead observed for
compounds bearing a nitrogen heterocyclic substituent
at position 2. This structural feature however seems
necessary, though not sucient, to evoke such an activity

396
L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
since compd 426 which does bear a 2-heterocyclic sub-
stituent, was a BzR agonist. The testing of further, and
eventually newly designed PQs, has been planned to
fully evaluate the in¯uence of the nature and position of
the substituents in the quinoline ring on the intrinsic
activity of congeners bearing a 2-heterocyclic substitu-
ent. Also in this case, theoretical and a molecular mod-
elling studies based on quantomechanical and 3-D
QSAR methods47 should lead to a better interpretation
of the structure±ecacy relationships data.
Chemical Experimental
Melting points were determined on a Buchi 510 capil-
lary melting point apparatus and are uncorrected.
Elemental analyses were performed on a Perkin±Elmer
elemental analyzer Mod. 240 and the data for C, H, N
are within ‹0.40% of calculated values. Spectral ana-
lyses (IR and 1H NMR) are consistent with the chemical
structures indicated. IR spectra were determined in
nujol mull on a Perkin±Elmer 398 or a Perkin±Elmer
Table 3. 1H NMR and IR spectral data of some representative PQ 4
1
Compound
1H NMR(d, ppm, J=Hz)
IR(cm
)
41
(DMSO-d6) d 7.44±7.67 (m, 3H, H-(7,8,9)), 8.08 (u dd, 1H, H-6, J6±7=7.8 Hz), 8.51 (s, 1H,
H-4), 11.37 (s, 1H, NH, D2O exchangeable), 12.43 (br s, 1H, NH, D2O exchangeable)
(DMSO-d6): 4.00 (s, 3H, OCH3), 7.14 (t, 1H), 7.27 (d, 1H, Jorto=8.0 Hz), 7.38±7.51 (m,
3H), 7.75 (d, 1H, Jorto=8.0 Hz), 8.18 (d, 2H), 8.31 (s, 1H, H-4), 12.24 (br s, 1H, NH, D2O
exchangeable).
1654 (CO)
1634 (CO)
410
412
413
(DMSO-d6) d 7.60 (td, 1H), 7.71±7.85 (m, 2H), 7.95 and 8.03 (2dd, 2H), 8.67 (u dd, 1H),
8.83 (s, 1H, H-4), 9.12 (t, 1H, H-20), 13.07 (br s, 1H, NH, D2O exchangeable).
(DMSO-d6) d 5.13 (br s, 2H, NH2, D2O exchangeable), 6.36 (u dd, 1H), 7.03 (t, 1H, H-50),
7.33 (u dd, 1H), 7.43 (t, 1H, H-20), 7.53 (td, 1H, H-7), 7.71±7.85 (m, 2H, H-(6,9)), 8.67 (s,
1H, H-4), 12.78 (br s, 1H, NH, D2O exchangeable).
1654 (CO)
1654 (CO)
418
(DMSO-d6) d 2.47 (t, 3H, CH3±CH2O), 4.16 (q, 2H, CH3±CH2O), 7.14 (t, 1PhH), 7.25 (dd,
1H, H-7, J7±6=9.0 Hz, J7±9=2.7 Hz), 7.42 (t, 2PhH), 7.55 (d, 1H, H-9, J9±7=2.7 Hz), 7.64
(d, 1H, H-6, J6±7=9.0 Hz), 8.21 (dd, 2PhH), 8.61 (s, 1H, H-4), 12.75 (br s, 1H, NH, D2O
exchangeable).
1625 (CO)
1670 (CO)
421
(DMSO-d6) d 0.94 (t, 3H, CH3±CH2±CH2±CH2±), 1.36 (st, 2H, CH3±CH2±CH2±CH2±),
1.66 (qt, 2H, CH3±CH2±CH2±CH2±), 2.77 (t, 2H, CH3±CH2±CH2±CH2±), 7.31 (td, 1H, H-5
pyridyl), 7.56 (u dd, 1H, H-7, J7±6=8.7 Hz), 7.69 (d, 1H, H-6, J6±7=8.7 Hz), 7.95±8.05 (m,
2H, H-9 and H-4 pyridyl), 8.34 (u dd, 1H, H-3 pyridyl, J3±4=8.4 Hz), 8.56 (u dd, 1H, H-6
pyridyl), 8.75 (s, 1H, H-4), 12.70 (br s, 1H, NH, D2O exchangeable).
428
(DMSO-d6) d 1.36±1.54 and 1.73±1.88 (2m, 10H, 5CH2 cyclohexyl), 2.53±2.72 (m, 1H, CH
cyclohexyl), 7.58±7.70 (m, 2H, H-(6,7)), 8.05 (d, 1H, H-9, J9±7=1.3 Hz), 8.48 and 8.60 (2d,
2H, H-(5,6)pyrazinyl, Jorto=2.6 Hz), 8.77 (s, 1H, H-4), 9.57 (u d, 1H, H-3 pyrazinyl), 12.89
(br s, 1H, NH, D2O exchangeable).
1665 (CO)
432
435
(DMSO-d6) d 7.36±7.74 (m, 5H, H-7 and 4PhH), 7.88 (d, 1H, H-6, J6±7=9.2 Hz), 7.99 (u d,
1H, H-9), 8.85 (s, 1H,H-4), 12.95 (br s, 1H, NH, D2O exchangeable).
(DMSO-d6) d 7.37±7.49 (m, 2PhH), 7.73 (dd, 1H, H-7, J7±6=9.1 Hz, J7±9=2.3 Hz), 7.88 (d,
1H, H-6, J6±7=9.1 Hz), 8.13 (u d, 1H, H-9), 8.28 (dt, 1H, H-60), 8.50 (s, 1PhH), 8.85 (s, 1H,
H-4), 13.00 (br s, 1H, NH, D2O exchangeable).
1625 (CO)
1633 (CO)
445
(DMSO-d6) d 7.69 (dd, 1H, H-7), 7.82 (d, 1H, H-6, J6±7=9.0 Hz), 8.03 (u d, 1H, H-9), 8.30
and 8.47 (2d, 4PhH, Jorto=9.5 Hz), 8.85 (s, 1H, H-4), 13.11 (br s, 1H, NH, D2O
exchangeable).
1620 (CO)
1654 (CO)
446
(DMSO-d6) d 5.03 (br s, 2H, NH2, D2O exchangeable), 6.61 (d, 2PhH, Jorto=8.8 Hz), 7.62
(dd, 1H, H-7, J7±6=9.1 Hz, J7±9=2.4 Hz), 7.73 (d, 2PhH, Jorto=8.8 Hz), 7.80 (d, 1H, H-6,
J6±7=9.1 Hz), 7.99 (u d, 1H, H-9), 8.69 (s, 1H, H-4), 12.85 (br s, 1H, NH, D2O exchange-
able).
456
463
465
(DMSO-d6) d 7.38 (u d, 1H, H-7), 7.72 (t, 2H, H-9 and H-5 pyrimidinyl), 8.51 (s, 1H, H-4),
8.86 (u d, 2H, H-(4,6) pyrimidinyl), 12.91 (br s, 1H, NH, D2O exchangeable).
(DMSO-d6) d 3.77 (s, 3H, OCH3), 7.01 (d, 2PhH, Jorto=9.1 Hz), 7.97±8.04 (m, 3H, H-9 and
2PhH), 8.56 (s, 1H, H-4), 13.15 (br s, 1H, NH, D2O exchangeable).
1655 (CO)
1635 (CO)
1660 (CO)
(DMSO-d6) d 3.83, 3.88, and 3.97 (3s, 9H, 3OCH3), 7.03 (s, 1H, H-6), 7.99 (d, 2PhH,
Jorto=8.7 Hz), 8.37 (d, 2PhH, Jorto=8.7 Hz), 8.60 (s, 1H, H-4), 12.60 (br s, 2H, NH and
COOH, D2O exchangeable).
468
(DMSO-d6) d 3.82, 3.88, and 3.93 (3s, 9H, 3OCH3), 7.04 (s, 1H, H-6), 8.42 and 8.55 (2d,
2H, H-(5,6)pyrazinyl, Jorto=2.5 Hz), 8.62 (s, 1H, H-4), 9.36 (u d, 1H, H-3 pyrazinyl), 12.61
(br s, 1H, NH, D2O exchangeable).
1660 (CO)

L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
397
FT-IR 1600 spectrophotometers. 1H NMR spectra were
recorded on a Varian XL-200 or AC 200 Bruker instru-
ments. The chemical shifts (d) are relative to Me4Si used
as internal standard. The following abbreviations were
used: s=singlet, d=doublet, t=triplet, dd=double
doublet, q=quartet, qt=quintet, st=sextet, m=multi-
plet, dt=doublet of triplets, td=triplet of doublets,
u=unresolved, br=broad. The coupling constants J
poured onto ice water and made basic with sodium
carbonate. The resulting precipitate was ®ltered o€ and
puri®ed by steam distillation (mp 58±60ꢀ C). Anal. calcd
.
for C13H9ClF3NO3 H2O: C, 48.18; H, 2.95; N, 4.32.
1
Found: C, 48.19; H, 2.76; N, 4.29. H NMR (CDCl3) d
1.46 (t, 3H, CH3±CH2O), 4.51 (q, 2H, CH3±CH2O),
7.70 (dd, 1H, H-7, J7-8=9.5, J7-5=2.8), 8.20 (d, 2H, H-
(5,8), J8-7=9.5), 9.21 (s,1H, H-2).
1
were in hertz. IR and H NMR spectral data of some
representative PQ derivatives 4 are listed in Table 3.
Ethyl 6,7,8-tri¯uoro-4-chloroquinoline-3-carboxylate (3b).
Compound 3b was obtained from the 6,7,8-tri¯uoro-4-
hydroxyquinoline-3-carboxylate and POCl3 under the
same conditions described above for compound 3a. It
was crystallized from ethanol (mp 106±108ꢀ C). Anal.
calcd for C12H7ClF3NO2: C, 49.76; H, 2.45; N, 4.84.
TLC on silica-gel plates (Merck, 60, F254) was used for
purity check and reaction monitoring. Column chroma-
tography on silica-gel (Merck, 70±230 mesh) was
applied, when necessary, to isolate and purify the dif-
ferent reaction products.
1
Found: C, 49.64; H, 2.40; N, 4.82. H NMR (CDCl3) d
1.46 (t, 3H, CH3±CH2O), 4.51 (q, 2H, CH3±CH2O),
7.94±8.05 (m, 1H, H-5), 9.21 (s, 1H, H-2).
Some unknown ethyl 4-chloroquinoline-3-carboxylates
(3a, R=8-OCF3; and 3b, R=6,7,8-F3), and two other
intermediate compounds (A; and 2a, R=8-OCF3), are
also described in this section.
General procedure for the preparation of 2,5-dihydro-
pyrazolo[4,3-c]quinolin-3-(3H)-ones (41, 430) and 2-aryl-
2,5-dihydropyrazolo[4,3-c]quinolin-3-(3H)-ones (47±12, 414,
418, 420, 425, 430±39, 441±45, 447, 459±65). Title compounds
were prepared from ethyl 4-chloroquinoline-3-carboxyl-
ates (3) and hydrazine hydrate 85% or the appropriate
arylhydrazines by the general method described in ref 26
(Table 1).
Physicochemical properties of the newly synthesized
PQs are summarized in Table 1.
Diethyl [(4-tri¯uoromethoxyanilino)methylene]malonate
(A). A mixture of equimolar amounts of 4-(tri¯uoro-
methoxy)aniline and diethyl (ethoxymethylene)malo-
nate was heated on a steam bath for 48 h. After cooling
at room temperature a solid was obtained, which was
puꢀri®ed by crystallization from EtOH±H2O, mp 76±
General procedure for the preparation of 2-hetero-
arylpyrazolo[4,3-c] quinolin-3-(3H)-ones (421±23, 426±28
,
455±57, 466±68). To a solution of ethyl 4-chloroquinoline-
3-carboxylate (3) (4 mmol) in absolute ethanol, the
appropriate a-(N)-heterocyclichydrazine (4.4 mmol) was
added. The reaction mixture was stirred at room tem-
perature for a variable time (1±12 h) until a yellow solid
precipitated. It was ®ltered o€ and puri®ed by crystalli-
zation from a suitable solvent (Table 1).
.
78 C. Anal. calcd for C15H16F3NO5 H2O: C, 50.56; H,
4.81; N, 3.93. Found: C, 50.20; H, 4.42; N, 3.81. 1H
NMR (CDCl3) d 1.28±1.40 (2t, 6H, 2CH3±CH2O), 4.18±
4.35 (2q, 4H, 2CH3±CH2O), 7.11±7.25 (m, 4PhH), 8.45
(d, 1H, CH=C, JCH=NH=13.1); 11.03 (d, 1H, NH,
JNH=CH=13.1, D2O exchangeable).
Ethyl 6-tri¯uoromethoxy-4-hydroxyquinoline-3-carboxylate
(2a). A (3 g, 8.6 mmol) was re¯uxed in Dowtherm A
(30 mL) at 260±280 ꢀC for 40 min. After cooling to room
temperature, the reaction mixture was diluted with Et2O
to give a white precipitate, which was ®ltered, washed
with Et2O, and puri®ed by crystallization from DMF,
mp>300ꢀ C. Anal. calcd for C13H10F3NO4: C, 51.83; H,
3.35; N, 4.65. Found: C, 51.79; H, 3.24, N, 4.50. 1H
NMR (DMSO-d6) d 1.26 (t, 3H, CH3±CH2O), 4.20 (q,
2H, CH3±CH2O), 7.67±7.78 (m, 2H, H-(7,8)), 7.97 (u d,
1H, H-5), 8.59 (s, 1H, H-2), 11.78 (br s, 1H, NH/OH,
D2O exchangeable).
In some instances (compds 421±23, 427, and 456), this
reaction a€orded the uncyclized intermediates, ethyl 4-
N,N0-heteroarylhydrazinoquinolin-3-carboxylates, whose
1
chemical structures were con®rmed by H NMR. They
were cyclized to the corresponding pyrazoloquino-
linones (4), without further puri®cation, by adding an
aqueous solution of 1 N sodium hydroxyde (2.2 mmol)
to a suspension of compound (2 mmol) in ethanol. After
1 h stirring at room temperature, the solution was acid-
i®ed with diluted acetic acid and the resulting precipitate
was ®ltered o€, washed with water and puri®ed by
crystallization (Table 1).
Ethyl 6-tri¯uoromethoxy-4-chloroquinoline-3-carboxylate
(3a). To ethyl 6-tri¯uoromethoxy-4-hydroxyquinoline-
3-carboxylate (2a) (10 g, 46 mmol) was added POCl3
(25 mL). The mixture was heated in a sand bath at
135ꢀ C for 45 min. After cooling, the solution was
General procedure for the preparation of 2-(amino-
phenyl)pyrazolo[4,3-c]quinolin-3-(3H)-ones (413, 440, 446).
To a suspension of the appropriate 2-(nitrophenyl)pyr-
azolo[4,3-c]quinolin-3-(3H)-one (412, 439, 445) (150 mg)
in absolute ethanol (150 mL), 10% Pd/C was added. The

398
L. Savini et al./Bioorg. Med. Chem. 6 (1998) 389±399
mixture was hydrogenated in a Parr apparatus at 40 psi at
room temperature. After 2±4 h the catalyst was ®ltered
o€ and the solution taken to dryness gave a solid, which
was crystallized from a suitable solvent (Table 1).
aliquots. After 60 min incubation at 4ꢀ C, the assay was
terminated by rapid ®ltration through glass-®ber ®lter
strips (Whatman GF/B). The ®lters were rinsed with
2Â4 mL ice-cold 50 mM Tris-HCl bu€er with a cell
Harvester ®ltration manifold (Model M-24, brandel)
and transferred in plastic minivials with 3 mL scintilla-
tion ¯uid (AtomLight, New England Nuclear). Six to
eight concentrations of the samples in triplicate were
used to determine the IC50 values. Nonspeci®c binding
was determined as the binding in the presence of 5 mM
diazepam and was 85 to 90% of the total binding.
General procedure for the preparation of 2-(40-hydro-
xyphenyl)pyrazolo[4,3-c]quinolin-3-(3H)-ones (415, 448).
A mixture of the appropriate methoxy-pyrazoloquinoli-
none (414, 447) (0.8 mmol) in glacial acetic acid (ꢁ4 mL),
was stirred under nitrogen for 10 min and then heated
until an homogenous solution was obtained. Aqueous
hydrogen bromide (48%, 10 mL) was added and the
reaction mixture was re¯uxed on an oil bath, and mon-
itored to completeness by TLC (chloroform:methanol,
18:2 as eluent) for a variable time [3 h for (415); 1 h for
(448)]. After cooling under nitrogen, the reaction mix-
ture was diluted with water (ꢁ70 mL) to give a precipi-
tate, which was collected, washed with water, and
crystallized (Table 1).
[35S]TBPS binding
Cerebral cortices were homogenized with a polytron PT
10 (setting 5, for 20 s) in 50 volumes of ice-cold 50 mM
Tris-citrate bu€er (pH 7.4 at 25ꢀ C) containing 100 mM
NaCl. The homogenate was centrifuged at 20,000 g for
20 min and resuspended in 50 volumes of 50 mM Tris-
citrate bu€er without NaCl. The [35S]TBPS was deter-
mined in a ®nal volume of 1000 mL consisting of 400 mL
tissue homogenate (400±500 mg protein), 100 mL 2 nM
[35S]TBPS, 100 mL 0.2 M NaCl, 5 mL drugs dissolved as
describe above or solvent (total and nonspeci®c sam-
ples). The incubation (25ꢀ C) were started by the addi-
tion of tissue homogenate and terminated by rapid
®ltration through glass-®ber ®lter strips (Whatman GF/
B) with a cell Harvester ®ltration manifold (Model M-
24, brandel). The ®lters were rinsed with 2Â4 mL ice-
cold 50 mM Tris-citrate bu€er. Non-speci®c binding
was de®ned as binding in the presence of 100 mM picro-
toxin, and represented about 10% of total binding.
Biochemical Experimental
Chemicals
[3H]Flunitrazepam (New England Nuclear, Boston,
U.S.A.) had a speci®c activity of 84.3 Ci/mmol and a
radiochemical purity >74.6%. [35S]TBPS (New England
Nuclear, Boston) had a speci®c activity of 72.4 Ci/mmol.
Animals
Male Sprague±Dawley rats (Charles River, Como, Italy)
with body weights of 150 to 200 g were kept under a 12 h
light/dark cycle at a temperature of 23‹2 ꢀC and 65%
humidity. Upon arrival at the animals facilities there
was a minimum of seven days acclimatization, during
which the animals had a free access to food and water.
The animals were killed by decapitation and the brains
were rapidly removed, the cerebral cortex was dissected
out and was used for the measurement of [3H]Flunitra-
zepam and [35S]TBPS binding.
Acknowledgments
The authors are thankful to C.N.R. (Rome) and
M.U.R.S.T. (Rome) for ®nancial support.
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