Synthesis and binding activity of some pyrazolo[1,5-c]quinazolines as tools to verify an optional binding site of a benzodiazepine receptor ligand

Article abstract of DOI:10.1021/jm9509206

The synthesis and binding activity at the benzodiazepine receptor of some 2-substituted pyrazolo[1,5-c]quinazolines are reported. The structure- activity relationships and in vitro efficacy of the title compounds, which are devoid of the proton acceptor atom at position 1, are similar to those of some previously reported tricyclic heteroaromatic compounds. This suggests that a proton acceptor at position 1 is an optional binding site of a benzodiazepine receptor ligand which only affects potency.

Full text of DOI:10.1021/jm9509206

J . Med. Chem. 1996, 39, 2915-2921
2915
Syn th esis a n d Bin d in g Activity of Som e P yr a zolo[1,5-c]qu in a zolin es a s Tools To
Ver ify a n Op tion a l Bin d in g Site of a Ben zod ia zep in e Recep tor Liga n d
Vittoria Colotta, Daniela Catarzi, Flavia Varano, Guido Filacchioni, and Lucia Cecchi*
Dipartimento di Scienze Farmaceutiche, Universita’ di Firenze, Via Gino Capponi 9, 50121 Firenze, Italy
Alessandro Galli and Chiara Costagli
Dipartimento di Farmacologia Preclinica e Clinica, Universita’ di Firenze, Viale G.B. Morgagni 65, 50134 Firenze, Italy
Received December 15, 1995X
The synthesis and binding activity at the benzodiazepine receptor of some 2-substituted
pyrazolo[1,5-c]quinazolines are reported. The structure-activity relationships and in vitro
efficacy of the title compounds, which are devoid of the proton acceptor atom at position 1, are
similar to those of some previously reported tricyclic heteroaromatic compounds. This suggests
that a proton acceptor at position 1 is an optional binding site of a benzodiazepine receptor
ligand which only affects potency.
In tr od u ction
Interaction of γ-aminobutyric acid (GABA) with its
GABAA receptor is responsible for inhibitory neuro-
transmission in the vertebrates. The inhibitory effect
of GABA can be modulated by the interaction of diverse
chemical structures with allosteric sites. Allosteric
GABAA receptor modulators, such as benzodiazepines
which interact with the so-called benzodiazepine recep-
F igu r e 1. Schematic representation of the essential and
optional pharmacophoric descriptors of a BZR ligand. Essential
pharmacophoric descriptors: L1 and L2, lipophilic areas; a2,
tor (BZR), are widely used as therapeutic agents. The
BZR ligands are functionally classified as agonists
(positive modulators like the benzodiazepines in clinical
use), inverse agonists (negative modulators), and neu-
tral antagonists (binding but without functional conse-
quences). Agonists, antagonists, and inverse agonists
bind to the same domain of the receptor protein or at
least with different states of the same domain.1 It
follows that all BZR ligands should have certain com-
mon characteristics that allow for recognition regardless
of the type of efficacy.
An enormous amount of structure-activity relation-
ship (SAR) data available for a large number of diverse
structural classes of ligands has resulted in the formu-
lation of several models of the pharamacophore for the
BZR binding.2 The common feature of these models is
the attempt to explain ligand efficacy as a function of
ligand-receptor interaction at the molecular level, on
the basis of changes in the conformation of the receptor
from its unoccupied resting state.3-12
Recently we proposed a schematic representation for
the binding to the BZR of some 6,6,5-tricyclic heteroaro-
matic compounds in which some essential and optional
pharmacophoric descriptors (see Figure 1) were
identified.13-16 The essential pharmacophoric descrip-
tors were thought to be two lipophilic substituents called
L1 and L2 and a proton acceptor atom designated a2,
while the optional sites, which were not necessary for
receptor-ligand interaction but only affect the potency
of a ligand, were a proton acceptor site called a1 and a
proton donor site called d. The optional hydrogen-
bonding sites a1 and d are identical with H2 and A2 sites,
respectively, of Cook’s model.11,12 The hypothesis of the
optionality of the proton donor site d is well supported
proton acceptor site. Due to the lone pair orientation of the
nitrogen and the carbonyl oxygen in the a2 proton acceptor
area, a favorable three-centered hydrogen bond with a proton
donor of the receptor site could be engaged. Optional binding
sites: d, proton donor area; a1, proton acceptor region. The
model is in agreement with that proposed by Cook11,12 (d, a1,
and a2 coincide with Cook’s A2, H2, and H1, respectively).
by the synthesis and binding activity of compounds
devoid of the proton donor d.14,15,17-20 In agreement
with Cook’s report,11,12 the interaction at point d appears
to be important for potent inverse agonist activity but
is not a requirement for high affinity in vitro. On the
contrary, only a few examples13,16 of 6,6,5-tricyclic
compounds not bearing the proton acceptor atom a1 are
known. Thus to verify the hypothesis that the proton
acceptor site a1 is optional, a hypothesis which cannot
be based on the binding activity of just a few com-
pounds,13,16 the synthesis and BZR binding activity of
further 1-deaza 6,6,5-tricyclic derivatives, namely, pyra-
zolo[1,5-c]quinazolin-5-ones, are reported.
Ch em istr y
The synthesis of ethyl 5,6-dihydro-5-oxopyrazolo[1,5-
c]quinazolin-2-carboxylate (1),13 its 9-chloro analog 2,21
and 5,6-dihydro-2-phenylpyrazolo[1,5-c]quinazolin-5-one
(3)13 is reported elsewere.
The synthesis of 2-arylpyrazolo[1,5-c]quinazolin-5-
ones 4-11 is shown in Schemes 1-4. In Scheme 1 the
two methods to prepare the 1,3-diaryl-2-propenones 12-
19,22,23 which with hydrazine hydrate yielded the 4,5-
dihydro-3,5-diarylpyrazoles 20-27, are illustrated. The
choice of method depended on the availability of the
starting materials.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0022-2623(95)00920-4 CCC: $12.00 © 1996 American Chemical Society

2916 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Colotta et al.
Sch em e 1a
Sch em e 3a
a
(a) H2/Pd/C, AcOEt; (b) (CCl3O)2CO, Et3N, THF.
Sch em e 4a
a
(a) RCOMe/BF3‚2MeCOOH, MeCOOH; (b) N2H4‚H2O, EtOH;
(c) RCHO/KOH, MeOH/H2O.
Sch em e 2a
a
(a) H2/PtO2, AcOEt; (b) (CCl3O)2CO, Et3N, THF; (c) Pb(AcO)4,
MeCOOH.
Sch em e 5a
a
(a) Method A: Pb(AcO)4/HCl (concd), EtOH at 60 °C; (b)
Method B: Pb(AcO)4/SiO2 at 10 °C.
a
(a) MeI or EtI, NaH, DMF.
The 4,5-dihydropyrazoles 20, 21, and 24-27 were
dehydrogenated with lead tetraacetate (see Scheme 2).
The crude materials were treated either with concen-
trated hydrochloric acid and ethanol at 60 °C (method
A) or with silica gel at 10 °C (method B) to give the
pyrazoles 28-33. It has to be noted that when in 4,5-
dihydropyrazoles 20-27 the substituent R is a hetero-
cyclic moiety, the dehydrogenation to pyrazoles procedes
with low yields (see in Table 3 compounds 32-33). In
the case of 4,5-dihydropyrazoles 22 and 23, only traces
of the corresponding (2-nitrophenyl)pyrazoles could be
detected in the 1H NMR spectra of the reaction mixture.
Thus the final tricyclic derivatives 6 and 7 were
obtained following a different procedure. Catalytic
reduction (Pd/C) of the (2-nitrophenyl)pyrazoles 28-33
afforded the (2-aminophenyl)pyrazoles 34-39 which
were cyclized with triphosgene to the tricyclic deriva-
tives 4, 5, 8-11 (see Scheme 3).
The preparation of the 2-pyridyl- and 2-furylpyrazolo-
[1,5-c]quinazolin-5-ones 6 and 7 was achieved as shown
in Scheme 4. Briefly, the 4,5-dihydro-(2-nitrophenyl)-
pyrazoles 22 and 23 were catalytically reduced (PtO2)
to the corresponding 4,5-dihydro-(2-aminophenyl)pyra-
zoles 40 and 41 which with triphosgene yielded the
1,5,6,10b-tetrahydroderivatives 42 and 43. The latter
were dehydrogenated with lead tetraacetate to the final
compounds 6 and 7. Alkylation of compounds 1 and 3
with alkyl iodides yielded the 6-N-alkyl derivatives 44-
4621 (see Scheme 5).
Ester 1 was hydrolyzed to the corresponding acid 47.21
The latter, upon treatment with thionyl chloride and
alcohol or sodium phenoxide, afforded the esters 48-
5021 (see Scheme 6). On the other hand, reaction of 1
with anhydrous hydrazine gave the hydrazide 51 which
with imidates provided the oxadiazolyl derivatives 52

Synthesis of Pyrazolo[1,5-c]quinazolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2917
Ta ble 1. Binding Activities and GABA Ratiosa
Sch em e 6a
compd
R
R1 R2
X
Ki (nM)b
GR
1c
2
COOEt
COOEt
Ph
H
Cl
H
Cl
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
Me
H
H
H
O
28 ( 4.9
50 ( 3.0
59 ( 3.5
105 ( 7.0
67 ( 14
1.2
1.16
0.99
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
3c
4
Ph
5
6
7
8
2-FC6H4
2-pyridyl
2-furyl
4-MeC6H4
4-ClC6H4
2-furyl
2-thienyl
COOEt
COOEt
Ph
53 ( 4.1
41 ( 2.1
9500 ( 800
5300 ( 630
16 ( 1.4
7.4 ( 0.3
>10 000
>10 000
>10 000
>10 000
22 ( 1.1
434 ( 36
3147 ( 419
NTd
0.73
0.94
9
10
11
44
45
46
47
48
49
50
52
0.97
1.08
COOH
COOMe
COOCH(Me)2
COOPh
1.07
a
(a) HCl/AcOH; (b) SOCl2, R2OH or PhONa, benzene; (c) N2H4,
H
H
EtOH; (d) R2C(NH)OEt‚HCl, pyridine.
Sch em e 7a
53
H
H
O
4592 ( 815
54
55
56
57
58
59
COOEt
Ph
COOEt
Ph
COOEt
Ph
H
H
H
H
H
H
H
H
H
H
S
S
3287 ( 419
>10 000
H2 70 ( 5.6
H2 6573 ( 419
>10 000
0.91
>10 000
diazepam
â-CCE
Ro 15-1788
10.5 ( 1.4
4.5 ( 0.7
1.50
0.67
0.96
2.5 ( 0.3
a
GABA ratio (GR): IC50(compound)/IC50(compound + 0.1 mM
b
GABA). Ki values are means ( SEM of three to five separate
determinations. c Reference 13. NT ) not tested because of the
d
insolubility of the compound.
present in all the cyclic derivatives bearing the NH
group.
Bioch em istr y
Compounds 2, 4-11, 44-50, and 52-59 were tested
for their ability to displace [3H]flunitrazepam (1 nM, KD
) 2.3 nM) from its specific binding sites in rat brain
cortical membranes. The BZR affinities of the tested
compounds, expressed as Ki, are listed in Table 1
together with the in vitro efficacy trends of the most
active ones (Ki < 100 nM), expressed by the GABA ratio
(GR). In Table 1 the Ki and GR of 2-carbethoxy 1 and
2-phenylpyrazoloquinazolin-5-one 313 are also reported
together with the Ki and GR of diazepam, ethyl â-car-
boline-3-carboxylate (â-CCE), and flumazenil (Ro 15-
1788) as representative of an agonist, an inverse
agonist, and an antagonist, respectively.
a
(a) CS2, pyridine/H2O; (b) (CH2O)n, benzene; (c) HCOOH.
and 53. Finally, treatment of the previously reported
ethyl 5-(2-aminophenyl)pyrazole-3-carboxylate13 or
2-phenyl-5-(2-aminophenyl)pyrazole13 with either car-
bon disulfide, paraformaldehyde, or formic acid yielded
compounds 54-59, respectively (see Scheme 7).
The chemical structures of the reported compounds
have been attributed on the basis of H NMR and IR
spectra. In particular the 5-thione structure of deriva-
tives 54 and 55 is due to the presence in the IR spectra
of the strong stretching bands at 1550 cm-1 (CdS) and
3200 cm-1 (NH). The presence of the NH is confirmed
1
Resu lts a n d Con clu sion s
The binding results on the fundamental pyrazolo[1,5-
c]quinazolin-5-ones 1-11 show that these 1-deaza tri-
cyclic derivatives display good BZR affinity with the
exception of the 2-(p-substituted-phenyl) derivatives 8
1
in the H NMR by the singlet at about δ 13 which is

2918 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Colotta et al.
Ta ble 2. Physical Properties of 1,3-Diarylpropenones
and 9. Comparison of the BZR affinity of the lead
compounds 1 and 3 with those of their 9-chloro analogs
2 and 4 reveals that the 9-chloro substituent halves the
binding potency of these series of compounds to the BZR.
This behavior is confirmed by the decreased binding
activity of the 9-chloro derivative 7 with respect to that
of its 9-unsubstituted analog 10. A nonadditive 9-sub-
stituent effect has already been observed.15,16
compd
R
R1
mp (°C)
solva
yield (%)
Also the importance of the position of the substituent
on the 2-phenyl ring is in agreement with previous
findings.15 In fact, the para-substitution drammatically
affects the BZR affinity (see compouds 8 and 9), while
the presence of a fluorine atom in the ortho-position (5)
or a 2-heteroaryl moiety (6, 7, 10, and 11) resulted in
an unchanged or increased binding activity. The favor-
able effect of the ortho-substituent and 2-heteroaryl
moiety is also in accordance with previous data.16 This
effect may be explained by the existence of the hydro-
philic “pocket 5” proposed by Crippen.24 This hydro-
philic pocket, according to Cook’s most plausible align-
ment of his inclusive model,11 would accommodate the
o-fluoro or the heteroatom of the heterocyclic ring. No
meta-substituent was introduced on the 2-phenyl moiety
since previous data16 indicate that the m-fluoro deriva-
tive was less potent than its o-fluoro isomer.
12
Ph
Cl
H
H
Cl
H
H
H
H
115-120
125-130
142-143
155-158
128-130
125-127
99-100
94-95
A
B
A
C
A
A
A
D
54
82
68
30
93
91
70
78
13
2-FC6H4
2-pyridyl
2-furyl
4-MeC6H4
4-ClC6H4
2-furyl
14b
15
16c
17d
18
19
2-thienyl
a
Recrystallization solvents: A ) ethanol, B ) ethyl acetate, C
b
) glacial acetic acid, D ) methanol. Reference 22: mp 141 °C.
c Reference 23: mp 134-135 °C. Reference 23: mp 123-124 °C.
d
sions confirm that in the BZR recognition site there is
the optional H2 hydrogen-bonding site proposed by
Cook.11 In fact, Cook’s H2 acceptor site corresponds to
our a1, and likewise it is found to be unnecessary for
inverse agonist/antagonist activity.11
Comparison of the BZR affinities of 2-carboxymethyl
48, 2-carboxyethyl 1, 2-carboxyisopropyl 49, and 2-car-
boxyphenyl 50 indicates that the smaller the ester group
the better the binding activity. The BZR affinity of the
bulky bioisoster of the carboxylic ester, i.e., 5-methyl-
1,3,4-oxadiazol-2-yl derivative 53, supports this sugges-
tion. The inactivity of the 6-N-alkylated derivatives
44-46 and that of the 2-carboxylic acid 47 are also in
accordance with previous data.16,25
Exp er im en ta l Section
Ch em istr y. Silica gel plates (Merck F254) and silica gel 60
(Merck; 70-230 mesh) were used for analytical and column
chromatography, respectively. All melting points were deter-
mined on a Gallenkamp melting point apparatus. Microanaly-
ses were performed with
a Perkin-Elmer 260 elemental
analyzer for C, H, N, and the results are within (0.4% of the
theoretical values. The IR spectra were recorded with a
Perkin-Elmer 1420 spectrometer in Nujol mull and are ex-
pressed in cm-1. The 1H NMR spectra were obtained with a
Varian Gemini 200 instrument at 200 MHz. The chemical
shifts are reported in δ (ppm) and are relative to the central
peak of the solvent. The following abbreviations are used, s
) singlet, d ) doublet, dd ) double doublet, t ) triplet, td )
triple doublet, q ) quartet, m ) multiplet, br ) broad, and ar
) aromatic protons. The physical data of the newly synthe-
sized compounds are shown in Tables 2-4.
1,3-Dia r yl-2-p r op en on es 12-19. Meth od A. A solution
of equimolar amounts (16 mmol) of the suitable 2-nitroaryl
aldehyde and aryl methyl ketone in glacial acetic acid (8 mL)
was treated with the acetic acid complex of boron trifluoride
(48 mmol). The mixture was stirred at room temperature for
5 days. The resulting solid was collected, washed with water,
and recrystallized. Following this method, compounds 12-
1522 were prepared. Compound 12 displayed the following:
1H NMR (CDCl3) 7.34 (d, 1H, H-2, J ) 15.7 Hz), 7.50-7.70
(m, 5H, ar), 8.01-8.15 (m, 4H, 3H ar + H-3).
The chemical modifications performed in the lead
compounds 1 and 3 at the level of the carbonyl oxygen
at position 5, i.e., compounds 54, 55, 57, and 59, show
the paramount importance of the three-centered hydro-
gen-bonding formation between the nitrogen at position
3 and the carbonyl oxygen at position 5 with the proton
donor of the receptor site. The discrepancy of the BZR
affinity of compound 56 devoid of the carbonyl oxygen
and with a BZR affinity only 2-fold lower than that of
the parent compound 3 seems to be the only exception
to this rule.
In Table 1 the GRs of the most active compounds are
also shown. This in vitro classification method is useful
for roughly estimating the trend of efficacy of test
compounds.26-28 The GR values (close to or below 1.0)
show that the efficacy trend of the tested pyrazolo-
quinazolines is that of antagonists with only one po-
tential partial inverse agonist (6).
Meth od B. Compounds 16-1923 were obtained from equi-
molar amounts (15 mmol) of 2-nitroacetophenone and the
suitable aryl aldehyde following the procedure described23 to
prepare 16 and 17. Compound 19 displayed the following: 1H
NMR (CDCl3) 6.79 (d, 1H H-2, J ) 15.8 Hz), 7.03-7.08 (m,
1H, thienyl proton), 7.24-7.51 (m, 4H, 3H ar + H-3), 7.60-
7.79 (m, 2H, ar), 8.17 (dd, 1H, ar, J ) 8.1, 1.1 Hz).
4,5-Dih yd r o-3-a r yl-5-(2-n itr oa r yl)p yr a zoles 20-23 a n d
4,5-Dih yd r o-3-(2-n itr op h en yl)-5-a r ylp yr a zoles 24-27. Hy-
drazine hydrate (55%, 9.6 mmol) was added to a suspension
of 12-19 (8 mmol) in ethanol (60 mL). The mixture was
refluxed for 1-2 h. The reaction was monitored by TLC.
Compounds 20-22, which became solid upon cooling, were
collected and recrystallized. Compound 23 was obtained by
evaporation at reduced pressure of the solvent, and the
resulting oily residue became solid upon treatment with
diethyl ether. In the case of 24-27, evaporation of the solvent
at reduced pressure yielded an oil which was purified by
In conclusion, the SAR and the GRs on pyrazolo-
quinazolines 1-11, 44-50, and 52-59 are very similar
to those of the previously reported 1,2,4-triazolo-qui-
noxalines16 and 1,2,4-triazolobenzoxazines.15 It follows
that compounds 1-11, 44-50, and 52-59 bind to the
BZR in a similar way and that the pharmacophoric
descriptors shown in Figure 1 should be the same. Since
the compounds reported in this paper are devoid of the
proton acceptor a1, our hypotheses are confirmed as
follows: (i) that the proton acceptor a1 is not essential
for the anchoring of inverse agonists/antagonists to the
BZR and (ii) that the proton acceptor a1 is an optional
binding site which only affects potency. These conclu-

Synthesis of Pyrazolo[1,5-c]quinazolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2919
Ta ble 4. Physical Properties of Pyrazolo[1,5-c]quinazolines
Ta ble 3. Physical Properties of 3,5-Diarylpyrazoles
yield
(%)
compd
R
R1
R2
mp (°C)
solva yield (%)
compd
R
R1 R2
X
mp (°C)
solva
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Ph
Cl NO2 142-145
A
A
A
B
C
C
D
E
F
G
H
I
90
87
87
73
47
37
47
55
47
27
64
35
25
19
94
97
98
96
80
87
62
40
2b
4
5
6
7
COOEt
Ph
Cl
Cl
H
H
Cl
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
Me
H
H
H
O
298-300
A
B
B
A
C
B
B
B
B
D
E
40
42
74
48
55
98
77
61
76
91
79
90
45
40
93
68
49
50
67
71
2-FC6H4
2-pyridyl
2-furyl
4-MeC6H4
4-ClC6H4
2-furyl
2-thienyl
Ph
2-FC6H4
4-MeC6H4
4-ClC6H4
2-furyl
2-thienyl
Ph
2-FC6H4
4-MeC6H4
4-ClC6H4
2-furyl
2-thienyl
2-pyridyl
2-furyl
H
H
NO2
86-87
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
>310
NO2 152-154
2-FC6H4
2-pyridyl
2-furyl
4-MeC6H4
4-ClC6H4
2-furyl
2-thienyl
2-pyridyl
2-furyl
COOEt
COOEt
Ph
290-292
301-304
300-302
295-297
Cl NO2 103-105
H
H
H
H
NO2 139 decb
NO2 140-142b
NO2 142-144b
NO2 138 decb
8
9
>310
10
11
42
43
44b
45
46
47b
48c
49
50
51
52d
304-305
299-300
303-305
297 dec
Cl NO2 158-160
H
H
H
H
H
NO2 161-163
NO2 153-155
NO2 161-163
NO2 121 dec
NO2 105-106
184-185
163-165
244-245
282 dec
295-297
258-259
298-300
F
C
E
J
A
D
G
H
B
B
D
Cl NH2 172-174
COOH
H
H
H
H
H
H
NH2 142-144
NH2 179-180
NH2 224-225
NH2 127-128
NH2 176-178
NH2 140-141
J
COOMe
COOCH(Me)2
COOPh
CONHNH2
K
A
J
J
A
L
H
H
H
>310
>310
Cl NH2 125-128
53
H
H
O
>310
D
36
a
Recrystallization solvents: A ) ethanol, B ) diethyl ether, C
) column chromatography, eluting system cyclohexane/ethyl
acetate (7:3), D ) column chromatography, eluting system chlo-
roform/ethyl acetate (8:2), E ) column chromatography, eluting
system chloroform/tetrahydrofuran/cyclohexane (8:1:1), F ) col-
umn chromatography, eluting system cyclohexane/ethyl acetate
(8:2), G ) column chromatography, eluting system chloroform/
ethyl acetate (9:1), H ) column chromatography, eluting system
chloroform/ethyl acetate (4:6), I ) column chromatography, eluting
system cyclohexane/ethyl acetate (1:1), J ) benzene, K ) cyclo-
54
55
56
57
58
59
COOEt
Ph
COOEt
Ph
COOEt
Ph
H
H
H
H
H
H
H
H
S
S
H2
H2
258 dec
B
B
I
I
A
H
85
72
55
20
80
53
309-311
116-117
120-212
155-156
131-132
a
Recrystallization solvents: A ) ethanol, B ) glacial acetic acid,
C ) tetrahydrofuran, D ) dimethylformamide, E ) diethyl ether/
ethanol, F ) ethyl acetate, G ) glacial acetic acid/water, H )
b
hexane/ethyl acetate, L ) diethyl ether/ethanol. Picrate melting
b
point.
methanol, I ) cyclohexane. Reference 21: mp not reported.
c Reference 21: mp 295-297 °C with softening at 290 °C. Due
d
to its insolubility, the compound was not recrystallized. The mp
refers to the crude product.
column chromatography. By treating 24-27 with picric acid,
the solid picrates were obtained. Compounds 20 and 27
displayed the following 1H NMR spectra: 20 (CDCl3) 2.97 (dd,
1H, H-4, J ) 16.8, 10.5 Hz), 3.86 (dd, 1H, H-4, J ) 16.8, 10.9
Hz), 5.46 (td, 1H, H-5, J ) 10.7, 4.3 Hz), 6.01 (d, 1H, NH, J )
4.3 Hz), 7.36-7.44 (m, 4H, ar), 7.65-7.70 (m, 2H, ar), 7.96-
8.05 (m, 2H, ar); 27 (CDCl3) 3.01 (dd, 1H, H-4, J ) 16.2, 9.1
Hz), 3.38 (dd, 1H, H-4, J ) 16.2, 10.1 Hz), 5.27 (td, 1H, H-5,
J ) 9.6, 1.8 Hz), 6.27 (d, 1H, NH, J ) 1.8 Hz), 6.95-7.05 (m,
2H, ar), 7.22.7.27 (m, 1H, ar), 7.48-7.68 (m, 3H, ar), 7.81 (d,
1H, ar, J ) 7.4 Hz).
3-Ar yl-5-(2-n itr oa r yl)p yr a zoles 28-33. To a solution of
20, 21, and 24-27 (4.5 mmol) in anhydrous dichloromethane
(25 mL) was added dropwise a solution of equimolar amount
of lead tetraacetate in anhydrous dichloromethane (25 mL).
The mixture was stirred at room temperature for 15 min.
Addition of water (70 mL) and elimination of the dark solid
gave two layers. The organic layer was washed four times with
water (30 mL each time) and dried (sodium sulfate).
Meth od A. Evaporation at reduced pressure of the solvent
yielded an oily residue which was treated with concentrated
hydrochloric acid (0.11 mL) and ethanol (20 mL). The mixture
became a solution by heating it at 60 °C for 10 min. Chloro-
form (80 mL) was added to the cooled solution. The solution
was washed once with a 2.5% solution of sodium hydrogen
carbonate (50 mL) and three times with water (30 mL each
time), dried (sodium sulfate), and evaporated at reduced
pressure. The resulting oily residue became solid upon stand-
ing. Following this method, compounds 30 and 31 were
prepared. Compound 30 displayed the following: 1H NMR
(CDCl3) 2.38 (s, 3H, CH3), 6.66 (s, 1H, pyrazole proton), 7.19-
7.26 (m, 2H, ar), 7.47-7.65 (m, 4H, ar), 7.68-7.77 (m, 2H, ar).
Meth od B. Silica gel (30 g) was added to the organic layer.
The mixture was allowed to stand in a refrigerating apparatus
(10 °C) for 15-60 h. The reaction was monitored by 1H NMR.
The silica gel was filtered off and washed several times with
ethyl acetate. Evaporation of the solvent at reduced pressure
and at a temperature not higher than 30-40 °C yielded an
oily residue which was purified by column chromatography.
Following this method, compounds 28, and 29, 32, and 33 were
prepared. Compound 28 displayed the following: 1H NMR
(CDCl3) 6.70 (s, 1H, pyrazole proton), 7.43-7.48 (m, 4H, ar),
7.55-7.62 (m, 2H, ar), 7.72-7.78 (m, 2H, ar).
3-Ar yl-5-(2-a m in oa r yl)p yr a zoles 34-39. A mixture of
28-33 (1.5 mmol) and 30% (w/w) Pd/C (10%) in ethyl acetate
(100 mL) was hydrogenated in a Parr apparatus at 30 psi for
6 h. Elimination of the catalyst and evaporation at reduced
pressure of the solvent yielded a residue which was recrystal-
lized. Compound 34 displayed the following: 1H NMR (DMSO-
d6) 6.5 (br s, 2H, NH2), 6.77 (d, 1H, ar), 7.04 (dd, 1H, ar, J )
8.5, 2.2 Hz), 7.31-7.54 (m, 4H, ar), 7.62 (d, 1H, ar, J ) 2.2
Hz), 7.85 (d, 1H, ar, J ) 7.0 Hz), 13.4 (br s, 1H, NH).
5,6-Dih yd r o-2-a r ylp yr a zolo[1,5-c]qu in a zolin -5-on es 4,
5, 8, a n d 11. Triphosgene (0.56 mmol) and triethylamine (3.4
mmol) were successively added to a solution of 34-39 (1.40

2920 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Colotta et al.
mmol) in anhydrous tetrahydrofuran (20 mL). The mixture
was stirred at room temperature for 30 min and then diluted
with water (50 mL). The resulting solid was collected and
recrystallized. Compound 4 displayed the following spectral
data: 1H NMR (DMSO-d6) 7.33-7.60 (m, 5H, ar), 7.86 (s, 1H,
H-1), 7.98-8.02 (m, 2H, ar), 8.18-8.21 (m, 1H, ar), 12.0 (br s,
1H, NH); IR 3200, 1740.
4,5-Dih yd r o-3-a r yl-5-(2-a m in oa r yl)p yr a zoles 40 a n d 41.
A mixture of 22 and 23 (1.4 mmol) and 10% (w/w) platinum
dioxide monohydrate in ethyl acetate (60 mL) was hydroge-
nated in a Parr apparatus at 25 psi for 18 h. Elimination of
the catalyst and evaporation at reduced pressure of the solvent
yielded a residue which was recrystallized. Compound 40
displayed the following: 1H NMR (CDCl3) 3.48 (d, 2H, H-4, J
) 11.9 Hz), 4.3 (br s, 2H, NH2), 4.97 (td, 1H, H-5, J ) 11.9,
3.7 Hz), 6.08 (d, 1H, NH, J ) 3.7 Hz), 6.68-6.76 (m, 2H, ar),
7.09-7.26 (m, 3H, ar), 7.69 (td, 1H, ar, J ) 7.9, 1.7 Hz), 7.92
(d, 1H, ar, J ) 8.0 Hz), 8.59 (d, 1H, ar, J ) 4.8 Hz).
1,5,6,10b-Tetr a h yd r o-2-a r ylp yr a zolo[1,5-c]qu in a zolin -
5-on es 42 a n d 43. The title compounds were obtained from
40 and 41 (1.40 mmol) and triphosgene (0.56 mmol) following
the procedure described above to prepare 4, 5, 8, and 11.
Compound 42 displayed the following spectral data: 1H NMR
(DMSO-d6) 3.54 (dd, 1H, H-1, J ) 17.1, 14.3 Hz), 3.99 (dd,
1H, H-1, J ) 17.1, 10.6 Hz), 5.29 (dd, 1H, H-10b, J ) 14.3,
10.6 Hz), 6.95-7.07 (m, 2H, ar), 7.23-7.32 (m, 2H, ar), 7.45-
7.51 (m, 1H, ar), 7.85-8.07 (m, 2H, ar), 8.68 (d, 1H, ar, J )
4.8 Hz), 9.7 (br s, 1H, NH); IR 3240, 3160, 3100, 1700.
4,5-Dih yd r o-2-a r ylp yr a zolo[1,5-c]qu in a zolin -5-on es 6
a n d 7. Lead tetraacetate (1.38 mmol) was added to a warm
(60 °C) solution of 42 and 43 (0.69 mmol) in glacial acetic acid
(10 mL). The mixture was heated at 60 °C for 30 min in the
case of 42 and for 3 h in the case of 43. The solid obtained
upon cooling was collected. A second crop of the title com-
pounds was obtained as follows: The mother solution was
diluted with water (15 mL) and extracted with ethyl acetate
(20 mL). The organic layer was washed with a 0.1 M solution
of hydrochloric acid (20 mL) and water until neutralization,
dried (sodium sulfate), and evaporated at reduced pressure.
The oily residue became solid upon treatment with diethyl
ether. Compound 6 displayed the following spectral data: 1H
NMR (DMSO-d6) 7.30-7.39 (m, 2H, ar), 7.45-7.60 (m, 2H, ar),
7.82 (s, 1H, H-1), 7.99 (td, 1H, ar, J ) 7.9, 1.7 Hz), 8.21 (d,
2H, ar, J ) 7.9 Hz), 8.72 (d, 1H, ar, J ) 4.8 Hz), 11.95 (s, 1H,
NH); IR 3240, 3180, 3060, 1740.
5,6-Dih yd r o-6-N -a lk ylp yr a zolo[1,5-c]q u in a zolin -5-
on es 44-46.21 Alkyl iodide (1.97 mmol) and sodium hydride
(80%, 2.32 mmol) were added to a solution of 1 and 3 (1.16
mmol) in anhydrous dimethylformamide (5 mL). The mixture
was stirred at room temperature for 1-2 h. The reaction was
monitored by TLC. Ice and water (20 mL) were added, and
the mixture was extracted three times with chloroform (30 mL
each time). The organic layer was washed three times with
water (30 mL each time), dried (sodium sulfate), and evapo-
rated at reduced pressure to afford a residue which was
recrystallized. Compound 46 displayed the following spectral
data: 1H NMR (DMSO-d6) 3.73 (s, 3H, CH3), 7.43-7.66 (m,
6H, ar), 7.77 (s, 1H, H-1), 8.04 (d, 2H, ar, J ) 7.8 Hz), 8.15 (d,
1H, ar, J ) 7.6 Hz); IR 3140, 1700.
played the following spectral data: 1H NMR (DMSO-d6) 1.37
(d, 6H, 2CH3, J ) 6.2 Hz), 5.23 (q, 1H, CH, J ) 6.2 Hz), 7.28-
7.39 (m, 2H, ar), 7.52-7.60 (m, 1H, ar), 7.68 (s, 1H, H-1), 8.17
(d, 1H, ar, J ) 7.8 Hz); IR 3140, 1760, 1730.
5,6-Dih yd r o-5-oxop yr a zolo[1,5-c]q u in a zolin e -2-h y-
d r a zid e (51). Anhydrous hydrazine (5.8 mmol) was added
to a boiling suspension of 1 (1.9 mmol) in absolute ethanol (15
mL). The mixture was refluxed for 1 h. The resulting solid
was collected and recrystallized. Compound 51 displayed the
following spectral data: 1H NMR (DMSO-d6) 4.57 (br s, 2H,
NH2), 7.27-7.38 (m, 2H, ar), 7.51-7.58 (m, 2H, 1H ar + H-1),
8.11 (d, 1H, ar, J ) 7.41 Hz), 8.5 (br s, 1H, NH), 12.0 (br s,
1H, NH); IR 3380, 3320, 3100, 1750, 1720.
5,6-Dih ydr o-2-(1,3,4-oxadiazolyl)pyr azolo[1,5-c]qu in azo-
lin -5-on es 52 a n d 53. The suitable ethyl imidate hydrochlo-
ride (2.88 mmol) was added to a mixture of 51 (1.44 mmol) in
pyridine (5 mL). The mixture was refluxed for 2-3 h. The
solid was collected and, in the case of 53, recrystallized.
Because of its insolubility, compound 52 could not be recrys-
tallized; however, it was pure enough to be characterized.
Compound 53 displayed the following: 1H NMR (DMSO-d6)
2.65 (s, 3H, CH3), 7.31-7.41 (m, 2H, ar), 7.55-7.62 (m, 1H,
ar), 7.68 (s, 1H, H-1), 8.22 (d, 1H, ar, J ) 7.8 Hz), 12.0 (br s,
1H, NH).
Eth yl 5,6-Dih yd r o-5-th ioxop yr a zolo[1,5-c]qu in a zolin e-
2-ca r boxyla te (54) a n d 5,6-Dih yd r o-2-p h en ylp yr a zolo-
[1,5-c]qu in a zolin e-5-th ion e (55). Carbon disulfide (4 mL)
was added to a solution of ethyl 5-(2-aminophenyl)pyrazole-
2-carboxylate13 or 3-phenyl-5-(2-aminophenyl)pyrazole13 (0.92
mmol) in water (1 mL) and pyridine (5 mL). The mixture was
refluxed for 3-5 h. The reaction was monitored by TLC, and
the heating was continued until the starting material had
disappeared. Evaporation at reduced pressure of the solvent
yielded a residue which was collected, washed with diethyl
ether (15 mL), and recrystallized. Compound 54 displayed the
following spectral data: 1H NMR (DMSO-d6) 1.39 (t, 3H, CH3,
J ) 7.1 Hz), 4.43 (q, 2H, CH2, J ) 7.1 Hz), 7.41-7.50 (m, 1H,
ar), 7.63-7.69 (m, 2H, ar), 7.86 (s, 1H, H-1), 8.26 (d, 1H, ar, J
) 8.5 Hz), 13.53 (s, 1H, NH); IR 3200, 3140, 1720, 1550.
E t h yl 5,6-Dih yd r op yr a zolo[1,5-c]q u in a zolin e-2-ca r -
boxyla te (56) a n d 5,6-Dih yd r o-2-p h en ylp yr a zolo[1,5-c]-
qu in a zolin e (57). A mixture of the suitable (2-aminophenyl)-
pyrazole13 (1.3 mmol) and paraformaldehyde (1.45 mmol) in
benzene (5 mL) was refluxed for 4-5 h. Evaporation of the
solvent at reduced pressure yielded a residue which was
purified by column chromatography, eluting system chloroform/
acetonitrile, 8:2. Evaporation of the second eluates at reduced
pressure afforded a residue which was recrystallized. Com-
pound 56 displayed the following spectral data: 1H NMR
(DMSO-d6) 1.32 (t, 3H, CH3, J ) 7.1 Hz), 4.30 (q, 2H, CH2, J
) 7.1 Hz), 5.42 (s, 2H, CH2), 6.75-6.90 (m, 3H, 2H ar + NH),
7.14-7.21 (m, 2H, 1H ar + H-1), 7.61 (d, 1H, ar, J ) 7.6 Hz);
IR 3380, 1740.
Eth yl P yr a zolo[1,5-c]qu in a zolin e-2-ca r boxyla te (58)
a n d 2-P h en ylp yr a zolo[1,5-c]qu in a zolin e (59). A solution
of the suitable (2-aminophenyl)pyrazole13 (1.3 mmol) in formic
acid (2 mL) was refluxed for 30 min. Addition of water (30
mL) to the cooled solution yielded a solid which was collected
and recrystallized. Compound 58 displayed the following
spectral data: 1H NMR (DMSO-d6) 1.38 (t, 3H, CH3, J ) 7.1
Hz), 4.41 (q, 2H, CH2, J ) 7.1 Hz), 7.70-7.85 (m, 3H, 2H ar +
H-1), 7.92-7.99 (m, 1H, ar), 8.43 (d, 1H, ar, J ) 8.0 Hz), 9.49
(s, 1H, H-5); IR 3140, 3060, 1740.
Bioch em istr y. Crude synaptic membranes were prepared
from cerebral cortices of male Sprague-Dawley rats (170-
250 g) according to Zukin et al.29 Tissue was homogenized in
15 vol of ice-cold 0.32 M sucrose, containing 20 µg/mL phen-
ylmethanesulfonyl fluoride, using a glass-Teflon homogenizer
(clearance ) 0.15-0.23 mm). The homogenate was centrifuged
at 1000g for 10 min and the resulting supernatant further
centrifuged at 20000g for 20 min. The final pellet was
resuspended in 15 vol of ice-cold distilled water, dispersed with
an Ultra-Turrax sonicator (30% of maximum speed) for 30 s,
and centrifuged at 8000g for 20 min. The supernatant and
the soft upper layer of the pellet were collected together and
centrifuged at 48000g for 20 min. The membranes were
5,6-Dih yd r o-5-oxop yr a zolo[1,5-c]qu in a zolin e-2-ca r box-
ylic Acid 47.21 The title compound was obained by hydrolysis
of 1 (2.56 mmol) as described.21 Compound 47 displayed the
following: 1H NMR (DMSO-d6) 7.33-7.40 (m, 2H, ar), 7.35-
7.57 (m, 1H, ar), 7.65 (s, 1H, H-1), 8.16 (d, 1H, ar, J ) 7.5
Hz), 12.1 (br s, 1H, NH), 13.4 (br s, 1H, OH).
5,6-Dih yd r o-5-oxop yr a zolo[1,5-c]qu in a zolin e-2-ca r box-
yla tes 48 a n d 50.21 A mixture of 47 (1.74 mmol) in thionyl
chloride (10 mL) was refluxed for 5 h. The excess of thionyl
chloride was distilled off. The residue acyl chloride was
washed with cyclohexane (1 mL). The suitable alcohol (10 mL)
or a suspension of sodium phenoxide (1.74 mmol) in anhydrous
benzene (20 mL) was then added to the intermediate acyl
chloride. The mixture was refluxed. The esterification reac-
tion was monitored by TLC, and heating was continued until
the starting acid had disappeared. The cooled mixture af-
forded a solid which was recrystallized. Compound 49 dis-

Synthesis of Pyrazolo[1,5-c]quinazolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2921
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Cecchi, L.; Galli, A.; Costagli, C. Benzodiazepine binding activity
of some tricyclic heteroaromatic systems to define the hydrogen
bonding strength of the proton donors of the receptor site.
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novel benzodiazepine receptor ligand. Farmaco 1994, 49, 89-
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Martini, C.; Giusti, L.; Lucacchini, A. Synthesis of some 2-aryl-
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essential pharmacophoric descriptors of a benzodiazepine recep-
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[1]Benzopyranopyrrol-4-ones and [1]benzopyrano1,2,3-triazol-4-
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resuspended once more in distilled water, centrifuged, and
frozen at -70 °C.
On the day of the experiment, appropriate amounts of
membranes were thawed at room temperature, resuspended
(0.5 mg of protein/mL) in 0.05 M Tris-HCl buffer, at pH 7.4,
containing 0.01% (v/v) Triton X-100, incubated at 37 °C for 60
min, and centrifuged at 48000g for 20 min. The membranes
were then washed with two additional resuspension and
centrifugation cycles and finally resuspended in cold Tris-HCl
buffer to yield 0.2-0.3 mg of protein/assay tube. [3H]Fluni-
trazepam (83.4 Ci/mmol) binding assays were carried out in
ice for 60 min at 1 nM ligand concentration in a total 0.5 mL
volume. Bound radioactivity was separated by rapid filtration
through Whatman GF/B filters using a Brandel cell harvester.
Nonspecific binding was determined in the presence of 10 µM
diazepam. The IC50 values were calculated from displacement
curves based on four to six scalar concentrations of the test
compounds in triplicate, using the ALLFIT computer pro-
gram,30 and converted to Ki values by application of the
Cheng-Prusoff equation.31 The GABA ratios26 of the com-
pounds with the lowest Ki values were calculated by measur-
ing, in the same experiment, the IC50 value of each compound
in the absence and presence of 0.1 mM GABA. A stock 1 mM
solution of the test compounds was prepared in 50% ethanol.
Subsequent dilutions were accomplished in buffer. Ethanol
up to a final 5% concentration was seen to affect [3H]-
flunitrazepam binding negligibly (e3%).
Ack n ow led gm en t. Valuable assistance in animal
handling was provided by M. G. Giovannini.
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