New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful for studying the interaction with the HBp-3 area (hydrogen bond point area) in the benzodiazepine site on the γ-aminobutyric acid type A (GABAA) receptor

Article abstract of DOI:10.1016/j.bmc.2011.04.009

The pharmacophoric model using ADLR procedure, based on pyrazolo[5,1-c][1,2,4]benzotriazine system, studied in our laboratory, allowed us to identify the essential interaction points (HBp-1, HBp-2, and Lp-1) and the important areas for affinity modulation (HBp-3 and Lp-2) for binding recognition at benzodiazepine (Bzs) site of GABA_A receptors (GABA_A-Rs). In this work ADLR method is used to rationalize the affinity data of 23 new compounds and to improve the knowledge on HBp-3 area, hydrogen bond area. Among these new compounds emerge the pyrrolo derivatives (18, 25, 28, 34, and 37) for their good affinity value (14.9 > K _i(nM) > 63.0). In the orientations proposed by ADLR, the NH moiety of the pyrrole ring, independently of the position in the pyrazolobenzotriazine core, fits in HBp-3 area and points out the acceptor feature of this hydrogen bond area, already known as donor area. Unexpectedly, the oxygen atom of the furane ring does not form efficient hydrogen bond with the same area, probably for an imperfect distance. The size of substituent at position 8 is important but not necessary for the receptor recognition, in fact the interdependence between the features of the 3- and 8-substituent's is again verified, (i.e., compound 20 vs 32).

Full text of DOI:10.1016/j.bmc.2011.04.009

Bioorganic & Medicinal Chemistry 19 (2011) 3074–3085
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful
for studying the interaction with the HBp-3 area (hydrogen bond point area)
in the benzodiazepine site on the c-aminobutyric acid type A (GABAA) receptor
Gabriella Guerrini a, , Giovanna Ciciani a, Fabrizio Bruni a, Silvia Selleri a, Fabrizio Melani b, Simona Daniele c,
⇑
Claudia Martini c, Annarella Costanzo a
a Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente attivi (HeteroBioLab) Università degli Studi di Firenze,
Via U. Schiff, 6, 50019 Polo Scientifico, Sesto Fiorentino—Firenze, Italy
b Dipartimento di Scienze Farmaceutiche, Laboratorio di Molecular Modeling, Cheminformatics and QSAR, Università degli Studi di Firenze, Via U. Schiff, 6, 50019 Polo Scientifico,
Sesto Fiorentino—Firenze, Italy
c Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università degli Studi di Pisa, via Bonanno, 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The pharmacophoric model using ADLR procedure, based on pyrazolo[5,1-c][1,2,4]benzotriazine system,
studied in our laboratory, allowed us to identify the essential interaction points (HBp-1, HBp-2, and Lp-1)
and the important areas for affinity modulation (HBp-3 and Lp-2) for binding recognition at benzodiaz-
epine (Bzs) site of GABAA receptors (GABAA-Rs). In this work ADLR method is used to rationalize the affin-
ity data of 23 new compounds and to improve the knowledge on HBp-3 area, hydrogen bond area. Among
these new compounds emerge the pyrrolo derivatives (18, 25, 28, 34, and 37) for their good affinity value
(14.9 > Ki(nM) > 63.0). In the orientations proposed by ADLR, the NH moiety of the pyrrole ring, indepen-
dently of the position in the pyrazolobenzotriazine core, fits in HBp-3 area and points out the acceptor
feature of this hydrogen bond area, already known as donor area. Unexpectedly, the oxygen atom of
the furane ring does not form efficient hydrogen bond with the same area, probably for an imperfect dis-
tance. The size of substituent at position 8 is important but not necessary for the receptor recognition, in
fact the interdependence between the features of the 3- and 8-substituent’s is again verified, (i.e., com-
pound 20 vs 32).
Received 20 January 2011
Revised 1 April 2011
Accepted 5 April 2011
Available online 12 April 2011
Keywords:
Pyrazolo[5,1-c][1,2,4]benzotriazine system
Benzodiazepine binding site
GABAA-receptors
Ligand-based pharmacophoric model
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Subunit b, although needed to generate a functional GABA gated
ion channel, does not affect the sensitivity to the Bz site. Using
The
c
-aminobutyric (GABA)-ergic system is the most important
genetically engineered mice and comparing drug-induced behav-
ioral responses in mutated and wild-type mice, with respect to
diazepam,4–6 GABAA-R subtype functions have been identified.
inhibitory mechanism in the adult mammalian brain.1 The GABA
neurotransmitter binds to GABAA receptors (GABAA-Rs), members
of the Cys-loop family of ligand gated ion channels (LGICs) and
to metabotropic G-protein-coupled GABAB receptors.2 GABAA-Rs
are pentameric trans-membrane proteins formed by a combination
The
a1b2c2 receptors mediate the sedative, anterograde amnesic
and anticonvulsant actions of diazepam.7–9 The anxiolytic-like ef-
fect and the muscle relaxant activities are mediated by the
of 19 different subunits (
with the most common composition being of two single alpha sub-
units, two single beta subunits, and one gamma subunit
2b2c 3
The combination of the isoforms 1–3, 5, b2/3, and 2 form a
subset of GABAA-Rs ( 1b2 2, 2b3 2, 3b3 2, and 5b3 2)
a
1–6, b1–3,
c
1–3, d,
e
, h,
p
, and
q
1–3)
a c2 and a3bc2 receptors even if the a3-containing GABAA-Rs
2b
seem to be involved in the inhibitory input for the dopaminergic
system.10–12 The
5b 2 receptors, that are preferentially localized
a
.
a c
a
c
in the hippocampus, seem to influence learning and memory.1
Currently, the unified pharmacophore/receptor model for li-
gands at the Bzs site on the GABAA-Rs developed by Cook et al.,13
has been updated14 and correlated with a comparative model15
to individuate residues relative to descriptors of the pharmaco-
phore/receptor model.
a
c
a
c
a
c
a
c
modulated by benzodiazepines (Bzs) and by non-benzodiazepine
ligands belonging to several chemical classes. The Bz binding site
is located at the interface of an
a and a c subunit and its pharma-
cology is mainly determined by the isoforms of these two subunits.
To increase our understanding of the requirements of the Bzs
site on GABAA-R, we have developed a pharmacophoric model
based on pyrazolo[5,1-c][1,2,4]benzotriazine ligands,16 using ADLR
procedure. This procedure allowed us to identify the essential
⇑
Corresponding author. Tel.: +39 055 4573766; fax: +39 055 4573671.
E-mail address: gabriella.guerrini@unifi.it (G. Guerrini).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.009

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
3075
At the same time, we elaborated structure–activity/affinity rela-
tionships for the pyrazolobenzotriazine system 3-, 8-disubstituted,
identifying the heteroaryl groups, the thienylmethoxycarbonyl
group17 the iodine atom,18 and the furoyl group19 as better 3-sub-
stituents,20 and, as 8-substituents, the groups endowed with differ-
ent electronic/steric features (alkyl, thioalkyl, alkyloxy, halogen,
aryloxy, and difluoromethoxy).16,21–23 Among these compounds,
it was very intriguing to modify the 8-aryloxy derivatives for
which it was hypothesized that the 8-oxygen atom engaged a
hydrogen bond interaction with the HBp-3 area.16 In this paper
we have decided to investigate the 8-aryl/heteroaryl derivatives,16
maintaining at position 3 the better previously identified substitu-
ents. These modifications (introduction at position 8 of aryl/het-
eroaryl rings) permit us to evaluate whether the HBp-3 area is
involved or if other receptor interactions can occur (p–p stacking,
hydrophobic interaction. . .) and to measure the size and shape of
lipophilic regions Lp-1 and Lp-2.
Figure 1. Overlapping of ligand conformations following the ADLR procedure: the
figure shows the essential interaction points for binding recognition (yellow circles)
and the important areas for affinity modulation (blue circles).
2. Chemistry
interaction points for binding recognition (HBp-1, HBp-2, and Lp-1)
and the important areas for affinity modulation (HBp-3 and Lp-2)
of the pyrazolobenzotriazine system, Figure 1.
All compounds described here are listed in Table 1 (chemical
data).
Table 1
Chemical data for new synthesized compounds
N
N
R8
N
R3
N
COOEt
O
N
N+
O-
NH2
NO2
3-28, 30, 32-37, 40-42
39
Compda
R3
R8
Yield (%)
Mp °C (recryst. solvent)
3
4
5
6
7
H
H
H
H
H
H
H
I
I
I
I
I
I
I
I
I
2-OMePh
4-OMePh
4-Py
2-Thienyl
3-Thienyl
2-Furyl
1-Boc-2-pyrrolyl
2-OMePh
2-OHPh
4-OMePh
4-Py
2-Thienyl
3-Thienyl
2-Furyl
1-Boc-2-pyrrolyl
(1H)-2-Pyrrolyl
Ph
4-OMePh
2-Thienyl
3-Thienyl
2-Furyl
1-Boc-2-pyrrolyl
(1H)-2-Pyrrolyl
3-Thienyl
3-Thienyl
3-Thienyl
I
4-OMePh
1-Boc-2-pyrrolyl
(1H)-2-Pyrrolyl
4-OMePh
1-Boc-2-pyrrolyl
(1H)-2-Pyrrolyl
2-furyl
60
54
52
65
73
67
70
40
35
45
27
54
45
50
45
30
25
56
30
25
84
45
77
37
57
72
30
21
80
69
21
73
78
90
58
23
35
190–192 (ethanol)
196–197 (ethanol)
220–222 (ethanol)
200–202 (ethanol)
206–207 (ethanol)
201–202 (ethanol)
196–198 (ethanol)
212–214 (ethanol)
250–251 (ethanol)
234–236 (ethanol)
267–269 (ethanol)
247–248 (ethanol)
258–259 (methoxyethanol)
215–216 (ethanol)
207–208 (ethanol)
>300 (ethanol)
237–238 (ethanol)
250–252 dec (chromatography)
229–230 (methoxyethanol)
233–234 (methoxyethanol)
240–241 (ethanol)
196–198 dec (ethanol)
>300 (ethanol)
249–250 (chromatography)
194–195 dec (ethanol)
219–220 (ethanol)
233–234 (chromatography)
200–202 (chromatography)
138–140 dec (ethanol)
>300 (ethanol)
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
30
32
33
34
35
36
37
39
40
41
42
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
2-Furyl
1-Boc-2-pyrrolyl
(1H)-2-Pyrrolyl
CO-2-furyl
COOCH2-2-thienyl
COOCH2-2-thienyl
COOCH3
CO-2-furyl
CO-2-furyl
CO-2-furyl
COOEt
COOH
COOCH2-2-thienyl
CO-2-furyl
258–259 (chromatography)
184–186 dec (ethanol)
>300 (ethanol)
140–142 (ethanol)
279–280 (ethanol)
2-furyl
2-Furyl
2-Furyl
215–216 (ethanol)
230–233 dec (ethanol)
a
Compound 1–2, 29, 31, and 38 known, see chemical section for references. Compound 39, ethyl 1-(2-nitro-5-fur-2-ylphenyl)-5-aminopyrazole-4-carboxylate.

3076
G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
The Suzuki coupling reaction on suitable iodinate starting
derivatives 19–24. The deprotection of 24 was realized in the same
previously described conditions,27 and compound 25 was obtained.
To obtain the 8-(3-thienyl)-3-heteroaryl substituted deriva-
tives, 26–27 (Scheme 2), compound 15 was used as starting mate-
rial for the Suzuki coupling. The deprotection of 27 afforded the
pyrrole derivative 28.
Scheme 3 depicts the synthetic pathway for obtaining deriva-
tives 32–37. The starting material is 3-carboxy-8-iodopyrazol-
o[5,1-c][1,2,4]benzotriazine 5-oxide, 29,22 that was reacted by a
Friedel–Craft reaction with thionyl chloride, SnCl4, and furane19
to obtain the new 3-(fur-2-ylcarbonyl)-8-iodopyrazolo[5,1-c]-
[1,2,4]benzotriazine 5-oxide, 30.
Following a described procedure, compound 29 was also used
to obtain 3-(2-thienylmethoxycarbonyl)-8-iodopyrazolo[5,1-c]-
[1,2,4]benzotriazine 5-oxide, 31.22 Both compounds 30 and 31
were subjected to Suzuki coupling and derivatives 32, 33, 35, and
36 were synthesized. The treatment of derivative 33 with
NaOCH3/CH3OH caused, at the same time, pyrrole deprotection
and trans-esterification, producing derivative 34. In the same
material was used for the synthesis of all 3-aryl-, 8-aryl-, and
3,8-diarylderivatives. As reported in Scheme 1, the 8-iodopyrazol-
o[5,1-c][1,2,4]benzotriazine 5-oxide,18 1, was used to obtain the
8-aryl derivatives 3–9, using the suitable arylboronic acid.
The reaction with 2-furylboronic acid produced several prob-
lems, and for the synthesis of compound 8, the most versatile
and stable potassium 2-furantrifluoroborate in presence of the pal-
ladium catalyst, palladiumchloride (diphenylphosphine-ferrocene)
PdCl2(dppf)ꢀCH2Cl2 was used as the coupling partner.24,25 (All the
3- and/or 8-(2-furyl) derivatives were synthesized following this
procedure). Compounds 3–9 were in turn iodinated at position 3
with ceric ammonium nitrate (CAN) and iodine26 to give deriva-
tives 10, 12–17 which are useful for structure–affinity relation-
ships. The demethylation of derivative 10 with CH2Cl2/BBr3 gave
the corresponding 2-hydroxyphenyl derivative 11, while the
deprotection of 17 was realized with NaOCH3/CH3OH in THF,27 to
give 18. The starting material 3-(3-thienyl)-8-iodopyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide,22 2, was instead used to obtain
N°
8-Ar/Het
N°
3
4
5
6
7
8
9
8-Ar/Het
10 2-OMe-Ph
11 2-OH-Ph
12 4-OMe-Ph
13 4-Py
14 2-thienyl
15 3-thienyl
16 2-furyl
2-OMe-Ph
4-OMe-Ph
4-Py
2-thienyl
3-thienyl
2-furyl
iii
iv
1-Boc-2-pyrrolyl
17 1-Boc-2-pyrrolyl
18 1(H)-2-pyrrolyl
N
N
N
Ar/Het
H
N
N
I
H
C
3
ii
1, R3=H
I
N
N
CH
3
R3
N+
i
O-
10-18
N°
N
3-9
N+
O-
8-Ar/Het
19
20
21
22
Ph
N
4-OMe-Ph
2-thienyl
3-thienyl
2, R3=3-thienyl
1-2 a
Ar/Het
N
S
23
2- furyl
N
24
25
1-Boc-2-pyrrolyl
1(H)-2-pyrrolyl
N+
O-
iv
19-25
Scheme 1. For compound 1 see,18 for compound 2 see22 Reagents: (i) Suzuki conditions, Arylboronic acids, ethanol abs, (PPh3)4Pd tetrakis(triphenylphosphine)palladium (0),
sodium carbonate 2 M; for synthesis of derivatives 8 and 23, potassium 2-furyltrifluoroborate, n-propanol, TEA, PdCl2(dppf); (ii) I2/CAN/CH3CN; (iii) for hydrolysis of
compound 10, CH2Cl2/BBr3; (iv) for deprotection of N-Boc derivatives (17 and 24) THF/MeONa.
N
N
S
S
N
I
N
Ar/Het
i
N
N+
O-
N
N+
O-
15
N°
26
27
28
3-Ar/Het
2-furyl
1-Boc-2-pyrrolyl
2-pyrrolyl
ii
Scheme 2. Reagents: (i) Suzuki conditions, potassium 2-furyltrifluoroborate, n-propanol, TEA, PdCl2(dppf) and N-(Boc)pyrrole-2-boronic acid, ethanol abs, (PPh3)4Pd
tetrakis(triphenylphosphine)palladium (0), sodium carbonate 2 M; (ii) for deprotection of N-Boc derivative (27) THF/MeONa.

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
3077
41
42
R3= COOCH2(2-thienyl)
R3=CO-2furyl
N
N
O
R3
N
N+
O-
iii
N
N
N
I
N
I
Ar/Het
N
N
COOH
R3
R3
N
N
N
N+
O-
N+
O-
ii
N+
O-
i
29a
N° R3
8-Ar/Het
30 R3=CO-2furyl
31a
R3= COOCH2(2-thienyl)
32 COOCH2-2-thienyl 4-OMe-Ph
33 COOCH2-2-thienyl 1-Boc-2-pyrrolyl
iv
iv
34 COOCH3
35 CO-2furyl
36 CO-2furyl
37 CO-2furyl
1(H)-2-pyrrolyl
4-OMe-Ph
1-Boc-2-pyrrolyl
1(H)-2-pyrrolyl
Scheme 3. For compound 29 and 31 see22: Reagents: (i) Friedel–Craft reaction on 29, 3-carboxy-8-iodopyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide,22 to obtain 30: thionyl
chloride/CH2Cl2/SnCl4/furane; starting from 29, following a procedure described in22 to obtain 31; (ii) Starting material 30 or 31,22 reacted in Suzuki conditions: 4-
methoxyphenylboronic acid for compound 32 and 35, N-Boc-2-pyrrolylboronic acid for compounds 33 and 36, ethanol abs, tetrakis, sodium carbonate 2 M; (iii) potassium 2-
furyltrifluoroborate, n-propanol, TEA, PdCl2(dppf); (iv) for deprotection of N-Boc derivatives (33 and 36) THF/MeONa. In case of derivative 33 in this reaction condition a
trans-esterification occurred, obtained compound 34.
N
N
I
N
COOEt
N
COOEt
O
NH2
NO2
i
NH2
38a
NO2
39
N
ii
N
O
O
S
N
N
O
N
N+
O-
41
O
COOH
iii
N
N+
O-
iv
40
N
N
O
O
O
N
N+
O-
42
Scheme 4. For 38.22 Reagents: (i) potassium 2-furantrifluoroborate, PdCl2(dppf), TEA; (ii) 10% NaOH solution, diglyme; (iii) NEt3, i-butylchloroformate, 2-thiofenemethanol;
(iv) CH2Cl2, Cl3CCN, PPh3, SnCl4, and furane.
reaction conditions, in derivative 36, the removal of N-Boc group
was selective and derivative 37 was obtained. The reaction of cou-
pling of the furyl ring at position 8 on compounds 30 and 31, with
potassium 2-furantrifluoroborate gave very low yields and with a
heavy work-up, thus the synthesis of compounds 41–42 was made
with an alternative synthetic way, Scheme 4. The intermediate
ethyl
1-(2-nitro-5-iodophenyl)-5-aminopyrazole-4-carboxylate
38,22 was used as starting material to introduce the 2-furyl group,

3078
G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
Table 2
using again potassium 2-furantrifluoroborate and palladium chlo-
ride(diphenylphosphine ferrocene) PdCl2(dppf)ꢀCH2Cl2 and 39
was obtained in good yield.
BZR ligand affinity of new 8-aryl-/heteroarylpyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide 3-substituted
The next cyclization in alkali medium28 gave the 3-carboxy-8-
(2-furyl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 40, that was
esterified by the mixed anhydride method (triethyl amine/i-butyl-
chloroformate and 2-thiophenmethanol), to yield compound 41.
The same compound 40 was transformed into corresponding acid
chloride in mild conditions, by treatment with trichloroacetonitrile
and triphenylphosphine in methylene chloride29; this acyl chloride
was not isolated, but subjected to a Friedel–Craft reaction with
SnCl4 and furane to obtain derivative 42.
N
R8
N
R3
N
N+
O-
a
N°
R3
R8
Ki (nM)
A27b
10
11
12
13
14
15
16
18
19
20
21
22
23
25
26
28
32
33
34
41
35
37
42
I
I
I
I
I
I
I
I
Ph
2-OMePh
2-OHPh
4-OMePh
4-Py
2-Thienyl
3-Thienyl
2-Furyl
(1H)-2-Pyrrolyl
Ph
4-OMePh
2-Thienyl
3-Thienyl
2-Furyl
(1H)-2-Pyrrolyl
3-Thienyl
3-Thienyl
4-OMePh
1-Boc-2-pyrrolyl
(1H)-2-Pyrrolyl
2-Furyl
4-OMePh
(1H)-2-Pyrrolyl
2-Furyl
966 95
305 29.3
2480 120
241 23.2
2380 190
202 20
56 4.9
190 22
25 2.4
642 62.6
817.0 16.7
370 35.7
357 33.9
209 20
19.4 2.8
26.9 2.5
48.3 6.1
29.4 1.8
395.4 39
14.9 1.1
54.8 4.1
238.67 18
63.0 5.5
856.9 4.2
3. Results and discussion
3.1. Biological results
I
The affinity at the Bz site on GABAA-R of the new synthesized
compounds was evaluated by their ability to displace [3H]flu-
mazenil (Ro 15-1788) from its specific binding in bovine brain
membrane.19 The affinity was expressed as Ki only for those com-
pounds inhibiting radioligand binding by more than 80% at fixed
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
2-Furyl
concentrations of 10 lM.
From the binding results reported in Table 2, some observations
arise. The new tested compounds show receptor recognition with a
Ki range of 14.9–2480 nM. Compounds 3–9 as well as compound
40, (Table 1) were not tested because previous reports indicate that
these types of derivatives have no receptor recognition.21
Among the 3-iodopyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides,
bearing various aryl/heteroaryl rings at position 8, 10–18, the fea-
tures of ring moiety are very important. In fact, the optimization of
the 8-phenyl derivative, A27,16 (Ki = 966 nM), that is, compounds
(1H)-2-pyrrolyl
COOCH2-2-thienyl
COOCH2-2-thienyl
COOMe
COOCH2-2-thienyl
CO-2-furyl
CO-2-furyl
CO-2-furyl
a
Ki value are means SEM of five determinations.
See Ref. 16.
b
10 and 12, allow
a threefold higher binding affinity: 10,
Ki = 305 nM and 12 Ki = 241 nM. These compounds have a methoxy
group in the phenyl ring (ortho- and para-position, respectively)
that could reinforce the interaction with receptor protein.16
A more hydrophilic substituent at position 8 is detrimental for
the binding as shown for compound 11 (Ki = 2480 nM), obtained
from demethylation of 10 and for the 8-(4-pyridyl)-derivative
(Ki = 2380 nM), compound 13. The isosteric replacement of the
8-phenyl ring with five-membered heteroaryl rings 2-, 3-thienyl,
2-furyl and (1H)-2-pyrrolyl gave compounds 14–16 and 18. The
best affinity was shown by derivative (1H)-2-pyrrolyl, 18
(Ki = 25 nM), that contains a hydrogen bond donor atom (NH).
Among the other heteroaryl rings, only the 8-(3-thienyl) derivative
15 is endowed with good binding affinity (Ki = 56 nM) compared to
the 8-(2-thienyl), 14, Ki = 202 nM and the 8-(2-furyl), 16,
Ki = 190 nM. These results suggest that the smaller size of the 8-
substituents versus phenyl ring (Ki = 966 nM) permits better recep-
tor interaction.
In the series of the 3-thienyl derivatives (19–23, 25) the intro-
duction at position 8 of aromatic/heteroaromatic rings gave useful
information. The 8-phenyl derivative 19 shows a poor binding
affinity (Ki = 642 nM), as well as the 8-(4-OCH3phenyl) derivative,
20, Ki = 817 nM. When in position 8 was introduced a five-mem-
bered ring, 2-, 3-thienyl, 2-furyl and (1H)-2-pyrrolyl, 21–23 and
25, the receptor recognition falls in the range of 370–19.4 Ki(nM),
confirming again the importance of ring size. In particular, the
need of hydrogen bond donor moiety (NH pyrrole) is evidenced
as for compound 25 (Ki = 19.4 nM), while the presence of an accep-
tor moiety (furane oxygen) gave weak affinity (23, Ki = 209 nM).
We thought that it might be useful to invert the substituents of
positions 3 and 8 of compounds 23 and 25 to evaluate the influence
of their interchangeability in receptor protein recognition, thus
obtaining compounds 26 and 28. The various orientations that
the pyrazolobenzotriazine scaffold assumes, when selected by
ADLR model, show that the substituents at position 3 and 8 can
interact indiscriminately with the pharmacophoric points Lp-1 or
Lp-2 in case of lipophilic moiety and HBp-1, HBp-2, or HBp-3 in
case of a moiety able to form a hydrogen bond interaction.16,23 This
would explain the good affinity shown by 3-(2-furane derivative),
26 (Ki = 26.9 nM) compared to its isomer 23 (8-(2-furane deriva-
tive, Ki = 209 nM), that probably has less interaction strength. The
two pyrrole derivative isomers, instead, have good affinity: 3-
(1(H)-2-pyrrol-) derivative 28, Ki = 48.3 nM and 8-(1(H)-2-pyrrol-
) derivative 25, Ki = 19.4 nM. (We are going to rationalize these re-
sults in the next molecular modeling section).
The (hetero)aryl rings at position 8 in the 3-ester derivatives,
32–34, 41, were chosen considering the influence, positive or neg-
ative, which they have in the two previous series. In the 3-ester
series the negligible influence of the ring size at position 8 was
seen, in fact the affinity value is in the same order of magnitude
(14.9 6 Ki(nM) 6 54.8) for all compounds, except derivative 33,
(Ki = 395.4 nM). The presence of a six-membered ring (para-
methoxyphenyl), as in derivative 32, or five-membered ring, com-
pound 41, confers quite comparable affinity values, 32 Ki = 29.4 nM
and 41 Ki = 54.8 nM. When position 8 was substituted with a 2-
(1H)-pyrrolyl, as in the case of the 3-methoxycarbonyl derivative
34, the affinity value was the best, Ki = 14.9 nM. The NH pyrrole
protection, as in compound 33, reduced the affinity value confirm-
ing the importance of the NH pyrrole in the binding. Thus, the
importance of anchorage of the 3-ester group is again confirmed17
and the affinity modulation due to substituent at position 8 is
emphasized.16
The importance of the type of 8-substituent in the 3-furoyl
derivatives, 35, 37, 42 that show binding affinity values in the

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
3079
range of 63 6 Ki(nM) 6 856 is again verified. Compound 37, the 8-
(1H)-2-pyrrolyl emerges for its good affinity, Ki = 63 nM.
From binding results it is possible assert that compounds bear-
ing the (1H)-2-pyrrolyl moiety are very good ligands (18, 25, 28,
34, and 37) in each series, suggesting the critical role of a hydrogen
bond donor (pyrrole NH) in receptor recognition. Good affinity
values were also identified for compounds bearing acceptor atom
moiety (26, 32, and 41) if only by having the suitable substituent
in the complementary position. Moreover, the size of the substitu-
ent at position 8 is very important only in the 3-iodine-, 3-hetero-
aryl-, and 3-furoylderivative, while it is negligible in the 3-ester
series.
fits in the HBp-3 area permitting the NH to form hydrogen bond
interaction, independently of the position of the pyrrole moiety
in the pyrazolobenzotriazine core (see Fig. 2).
When the pyrrole ring is replaced with furane (16, 23, 42, 41,
and 26, Figs. 3 and 4) bearing a potential hydrogen bond acceptor
atom (O), ADLR oriented the furane oxygen mainly toward the
HBp-3 area. As depicted in Figure 3,compounds 16, 23, and 42
present the furane moiety in this mode, but the lower affinity value
of compounds (Ki = 190, 209, and 856 nM, respectively) could be
due to a reduced hydrogen bond force or no efficient interaction
with LP-1 area (for 16 and 23) and steric hindrance for 42. Unex-
pectedly, compound 26 (isomer of compound 23) presents a differ-
ent orientation proposed by ADLR (Fig. 4).
3.2. Molecular modeling
The thiophene ring efficiently interacts with the lipophilic area
Lp-1, justifying the best affinity (Ki = 26.9 nM). Compound 41 bear-
ing an ester group (the 2-thienylmethoxycarbonyl group) at posi-
tion 3 shows a good affinity value (Ki = 54.8 nM) according to the
fact that the ester group contributes significantly to receptor
anchorage as previously stated,16 interacting with the lipophilic
area Lp-1 or Lp-2 depending on the orientation (A or B), see Figure 5.
The ADLR method16,23 was used here to rationalize the affinity
data and to improve our knowledge of the HBp-3 area. Among
the compounds endowed with better binding affinity, emerge
derivatives with the pyrrole moiety in position 8 or 3 (18, 25, 34,
37, and 28). In the overlapping performed by ADLR, the pyrrole ring
Figure 2. Compounds 18, 25, 28, 34, and 37 in the pharmacophoric model performed by ADLR. The pyrrole ring fits in the HBp-3 area permitting the NH moiety to form
hydrogen bond interaction, independently of the position of the pyrrole moiety in the pyrazolobenzotriazine core.

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G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
Figure 3. Compounds 16, 23, 42 in the pharmacophoric model performed by ADLR. The furane ring is oriented toward the HBp-3, but a reduced hydrogen bond force in the
HBp-3 area or no efficient interaction with Lp-1 area (16 and 23) and a steric hindrance (42) are responsible of a low affinity (16, Ki = 190 nM; 23, Ki = 209 nM; 42,
Ki = 856 nM).
(16, 23, and 42) do not have a good binding, despite the ability
to form a hydrogen bond interaction through the oxygen atom,
could be due to an imperfect distance from hydrogen bond donor
in the HBp-3 area.
In a preliminary study (data not published) derivatives bearing
a phenylethylamine moiety (-NHCH2Ph) at position 8 of pyra-
zolobenzotriazine system were endowed with very good binding
affinity (in a range of 0.1 nM), thus the synthesis of 8-NH isoster
of 8-OAr16 will be the object of our next research project.
5. Experimental
5.1. Chemistry
Figure 4. Compounds 26 in the pharmacophoric model performed by ADLR:
unexpected orientation of the furane ring; the driving force of the binding is due to
lipophilic interaction of thiophene ring.
Melting points were determined with a Gallenkamp apparatus
and were uncorrected. Silica gel plates (Merck F254) and silica gel
60 (Merck 70–230 mesh) were used for analytical and column
The importance of the anchorage by the ester group is also evi-
denced in compound 32 (Ki = 29.4 nM), in which the presence of a
large size group at position 8 is not binding for affinity, Figure 5.
chromatography, respectively. The structures of all compounds
were supported by their IR spectra (KBr pellets in nujol mulls, Per-
kin–Elmer 1420 spectrophotometer) and 1H NMR data (measured
with a Bruker 400 MHz). Chemical shifts were expressed in d
ppm, using DMSO-d6 or CDCl3 as solvent. The chemical and physi-
cal data of new compounds are shown in Tables 1; all new com-
pounds possess a purity P95%: microanalyses were performed
with a Perkin–Elmer 260 analyzer for C, H, N.
4. Conclusion
In previous studies it was hypothesized that the 8-oxygen atom
of the 8-aryloxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide deriv-
atives engaged hydrogen bond interaction with the HBp-3 area.16
On the other hand, the idea/hypothesis that this HBp-3 was a
bifunctional area arose when the CGS 9698 compound, used to val-
idate our pharmacophoric model with respect to Cook’s ‘unified
pharmacophore/receptor model’, showed hydrogen bond interac-
tion with the same area. In this study, this hypothesis is confirmed
since the NH group of the pyrrole ring engages a strong hydrogen
bond interaction with HBp-3. The fact that the furane derivatives
5.2. General procedure for the synthesis of3–7, 9, 19–22, 24, 27,
32–33, 35–36
Tetrakis(triphenylphosphine)palladium (0) (20 mg, 0.017 mmol)
and the suitable iodopyrazolo benzotriazine (0.15 mmol) were
combined in anhydrous tetrahydrofurane (5.0 mL). The starting
materials were: compound 1,18 for the synthesis of derivatives

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
3081
Figure 5. Compounds 41 (in two orientations A, B) and compound 32 in the pharmacophoric model performed by ADLR. Contribute of ester group to receptor anchorage.
3–7, 9; compound 2,22 for the synthesis of derivatives 19–22, 24;
compound 15 (see below) for the synthesis of derivative 27; com-
pound 30 for the synthesis of 35 and 36; compound 31,22 for the
synthesis of derivatives 32 and 33. The solution of the suitable
arylboronic acid (0.23 mmol, 1:1.5) in absolute ethanol and aque-
ous sodium carbonate (2 M, 2 mL) were added and the reaction
was maintained at 25–70 °C (see the exact condition in each com-
pound) and monitored by TLC. The final suspension was treated
with water, filtrated to crude final product and purified by recrys-
tallization by suitable solvent.
H-7); 7.70 (m, 2H, H-3 and H-5 pyridine); 6.82 (d, 1H, H-3). Anal.
C, H, N.
5.2.4. 8-(Thien-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(6)
From 1,18 and 2-thiophen boronic acid, 3 h, 70 °C. Yellow crys-
tals; TLC eluent: diisopropyl ether/cyclohexane 8:3 v/v; 1H NMR
(CDCl3) d 8.59 (d, 1H, H-9); 8.57 (d, 2H, H-6); 8.14 (d, 1H, H-2);
7.87 (dd, 1H, H-7); 7.69 (d, 1H, H-5 thiophene); 7.57 (d, 1H, H-3
thiophene); 7.22 (m, 1H, H-4 thiophene); 6.79 (d, 1H, H-3). Anal.
C, H, N.
5.2.1. 8-(2-Methoxyphenyl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (3)
5.2.5. 8-(Thien-3-yl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(7)
From 1,18 and 2-methoxy phenylboronic acid, 4 h, room
temperature. Yellow crystals; TLC eluent: diisopropyl ether/cyclo-
hexane 8:3 v/v; 1H NMR (DMSO-d6) d 8.48 (m, 2H, H-6 and H-9);
8.29 (d, 1H, H-2); 7.87 (dd, 1H, H-7); 7.53 (m, 2H, H-4 and H-6
phenyl); 7.25 (d, 1H, H-3 phenyl); 7.16 (t, 1H, H-5 phenyl); 6.92
(d, 1H, H-3); 3.85 (s, 3H, OCH3). Anal. C, H, N.
From 1,18 and 3-thiophen boronic acid, 1.5 h, room tempera-
ture. Yellow crystals; TLC eluent: diisopropyl ether/cyclohexane
8:3 v/v; 1H NMR (CDCl3) d 8.49 (m, 2H, H-6 and H-9); 8.05 (d,
1H, H-2); 7.78 (m, 2H, H-7 and H-2 thiophene); 7.51 (m, 1H, H-4
thiophene); 7.45 (m, 1H, H-5 thiophene); 6.69 (d, 1H, H-3). Anal.
C, H, N.
5.2.2. 8-(4-Methoxyphenyl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (4)
5.2.6. 8-(N-Boc-pyrrol-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (9)
From 1,18 and 4-methoxyphenylboronic acid, 3 h, room temper-
ature. Yellow crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.48 (m, 2H, H-6 and H-9); 8.31
(d, 1H, H-2); 8.05 (dd, 1H, H-7); 7.92 (d, 2H, H-2 and H-6 phenyl);
7.15 (d, 2H, H-3 and H-5 phenyl); 6.92 (d, 1H, H-3); 3.85 (s, 3H,
OCH3). Anal. C, H, N.
From 1,18 and N(Boc)pyrrole-2-boronic acid 3 h, 60–70 °C, in
toluene. Red crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; 1H NMR (CDCl3) d 8.45 (m, 2H, H-6 and H-9); 8.02 (d,
1H, H-2); 7.73 (dd, 1H, H-7); 7.40 (m, 1H, H-3 pyrrole); 6.65 (d, 1H,
H-3); 6.50 (m, 1H, H-5, pyrrole); 6.30 (m, 1H, H-4 pyrrole); 1.40 (s,
9H, (CH3)3). Anal. C, H, N.
5.2.3. 8-(4-Pyridyl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(5)
5.2.7. 3-(Thien-3-yl)-8-phenylpyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (19)
From 1,18 and 4-pyridin boronic acid, 3 h, reflux temperature.
Yellow crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.85 (m, 2H, H-2 and H-6 pyri-
dine); 8.70 (m, 2H, H-6 and H-9); 8.18 (d, 1H, H-2); 7.90 (dd, 1H,
From 2,22 and phenylboronic acid, 8 h, 50–60 °C. Red crystals;
TLC eluent: diisopropyl ether/cyclohexane 8:3 v/v; 1H NMR (CDCl3)
d 8.62 (d, 1H, H-6); 8.59 (d, 1H, H-9); 8.36 (s, 1H, H-2); 7.93 (d, 1H,

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G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
H-2 thiophene); 7.88 (dd, 1H, H-7); 7.80 (d, 2H, H-2 and H-6 phe-
nyl); 7.68 (d, 1H, H-4 thiophene); 7.55 (m, 3H, H-3, H-4 and H-5
phenyl); 7.46 (m, 1H, H-5 thiophene). Anal. C, H, N.
5.2.14. 3-(2-Thienylmethoxycarbonyl)-8-(N-Boc-pyrrol-2-yl)-
pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (33)
From 31,22 and N(Boc)pyrrole-2-boronic acid, 3 h, refluxing
temperature, in toluene. Yellow crystals; TLC eluent: toluene/ethyl
acetate/acetic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.67 (s, 1H, H-
2); 8.40 (d, 1H, H-6), 8.23 (d, 1H, H-9); 7.82 (dd, 1H, H-7); 7.60 (m,
1H, H-5 thiophene); 7.55 (m, 1H, H-3 pyrrole); 7.30 (m, 1H, H-3, 3-
thiophene); 7.01 (m, 1H, H-4 thiophene); 6.71 (m, 1H, H-5, pyr-
role); 6.42 (m, 1H, H-4 pyrrole); 5.51 (s, 2H, CH2); 1.40 (s, 9H,
(CH3)3). Anal. C, H, N.
5.2.8. 3-(Thien-3-yl)-8-(4-Methoxyphenyl)pyrazolo[5,1-
c][1,2,4]-benzotriazine 5-oxide (20)
From 2,22 and 4-methoxyphenylboronic acid, 4 h, 60–70 °C. Red
crystals; TLC eluent: diisopropyl ether/cyclohexane 8:3 v/v; 1H
NMR (DMSO-d6) d 8.75 (s, 1H, H-2); 8.47 (m, 2H, H-6 and H-9);
8.05 (dd, 1H, H-7); 8.00 (d, 1H, H-2 thiophene); 7.93 (d, 2H, H-20
and H-60 phenyl); 7.79 (d, 1H, H-4 thiophene); 7.72 (m, 1H, H-5 thi-
ophene); 7.15 (d, 2H, H-30 and H-50 phenyl). Anal. C, H, N.
5.2.15. 3-(Fur-2-ylcarbonyl)-8-(4-methoxyphenyl)pyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (35)
From 30 (see below), and 4-methoxyphenylboronic acid, 12 h,
room temperature. Yellow crystals; TLC eluent: toluene/ethyl ace-
tate 8:3 v/v; 1H NMR (CDCl3) d 8.91 (s, 1H, H-2); 8.55 (d, 1H, H-9);
8.40 (d, 1H, H-6); 8.18 (m, 2H, H-7 and H-5 furane); 7.98 (d, 2H, H-
2 and H-6 phenyl); 7.61 (d, 1H, H-3 furane); 7.18 (d, 2H, H-3 and H-
5 phenyl); 6.82 (m, 1H, H-4 furane); 3.80 (s, 3H, CH3). Anal. C, H, N.
5.2.9. 3-(Thien-3-yl)-8-(thien-2-yl)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (21)
From 2,22 and 2-thiophenboronic acid, 2 h, room temperature.
Red crystals; TLC eluent: diisopropyl ether/cyclohexane 8:3 v/v;
1H NMR (CDCl3) d 8.58 (m, 2H, H-6 and H-9); 8.36 (s, 1H, H-2);
7.93 (m, 1H, H-2, 3-thiophene); 7.85 (dd, 1H, H-7); 7.68 (m, 2H,
H-4, 3-thiophene and H-3, 8-thiophene); 7.58 (dd, 1H, H-5, 8-thio-
phene); 7.46 (dd, 1H, H-5, 3-thiophene); 7.21 (dd, 1H, H-4, 8-thio-
phene). Anal. C, H, N.
5.2.16. 3-(Fur-2-ylcarbonyl)-8-(N-Boc-pyrrol-2-yl)pyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (36)
From 30 and N(Boc)pyrrole-2-boronic acid, 3 h, refluxing tem-
perature, in tetrahydrofurane. Yellow crystals; TLC eluent: tolu-
ene/ethyl acetate/acetic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d
8.91 (s, 1H, H-2); 8.42 (d, 1H, H-6), 8.30 (d, 1H, H-9); 8.12 (d, 1H,
H-5 furane); 7.82 (dd, 1H, H-7); 7.62 (m, 1H, H-3 furane); 7.58
(m, 1H, H-3 pyrrole); 6.82 (m, 1H, H-4, furane); 6.72 (m, 1H, H-5,
pyrrole); 6.42 (m, 1H, H-4 pyrrole); 1.40 (s, 9H, (CH3)3). Anal. C,
H, N.
5.2.10. 3-(Thien-3-yl)-8-(thien-3-yl)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (22)
From 2,22 and 3-thiophenboronic acid, 3 h, 80 °C, in toluene. Red
crystals; TLC eluent: diisopropyl ether/cyclohexane 8:3 v/v; 1H
NMR (CDCl3) d 8.59 (m, 2H, H-6 and H-9); 8.36 (s, 1H, H-2); 7.93
(m, 1H, H-2, 3-thiophene); 7.86 (m, 2H, H-7 and H-2, 8-thiophene);
7.69 (dd, 1H, H-4, 3-thiophene); 7.61 (dd, 1H, H-4, 8-thiophene);
7.54 (dd, 1H, H-5, 8-thiophene); 7.46 (dd, 1H, H-5, 3-thiophene).
Anal. C, H, N.
5.3. 3-(Fur-2-ylcarbonyl)-8-iodopyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (30)
The starting material 29,22 (0.22 mmol) was reacted with
2.5 mL of thionyl chloride and maintained at refluxing temperature
for 1 h. The final solution was evaporated in vacuo and the corre-
sponding 3-carbonylchloride intermediate was utilized as that in
the next Friedel–Craft reaction. Dichloromethane (10 mL), SnCl4
(0.2 mL), and furane (0.2 mL) were added to the carbonylchloride
residue and the reaction was heated at 40–50 °C, monitoring by
TLC (eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v). The
reaction was quenched with HCl 1:1, diluted with dichlorometh-
ane and the organic phase separated. The dichloromethane phase
was washed with a saturated solution of sodium bicarbonate, dried
over sodium sulfate anhydrous and evaporated. The final residue
was purified by chromatography column (eluent: toluene/ethyl
acetate/acetic acid 8:2:1 v/v/v) and 30 was eluted as the fast band.
Yellow crystals; 1H NMR (DMSO-d6) d 8.91 (s, 1H, H-2); 8.78 (d, 1H,
H-9); 8.18 (m, 3H, H-7, H-6 and H-5 furane); 7.61 (d, 1H, H-3 fur-
ane); 6.82 (m, 1H, H-4 furane).
5.2.11. 3-(Thien-3-yl)-8-(N-Boc-pyrrol-2-yl)pyrazolo[5,1-c]-
[1,2,4]benzotriazine 5-oxide (24)
From 2,22 and N(Boc)pyrrole-2-boronic acid, 3 h, 60–70 °C, in
toluene. Red crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.73 (s, 1H, H-2); 8.40 (d, 1H, H-
6), 8.21 (d, 1H, H-9); 7.99 (m, 1H, H-2, 3-thiophene); 7.75 (m, 3H,
H-7 and H-5, 3-thiophene and H-3 pyrrole); 7.57 (m, 1H, H-4, 3-
thiophene); 6.70 (m, 1H, H-5, pyrrole); 6.51 (m, 1H, H-4 pyrrole);
1.40 (s, 9H, (CH3)3). Anal. C, H, N.
5.2.12. 3-(N-Boc-pyrrol-2-yl)-8-(thien-3-yl)pyrazolo[5,1-c]-
[1,2,4]benzotriazine 5-oxide (27)
From 15 (see below) and N(Boc)pyrrole-2-boronic acid, 3 h,
60–70 °C, in toluene. Red crystals; TLC eluent: toluene/ethyl
acetate/acetic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.51 (d, 1H,
H-9); 8.38 (m, 1H, H-2 thiophene); 8.36 (d, 1H, H-6); 8.32 (s, 1H,
H-2); 8.18 (dd, 1H, H-7); 7.80 (m, 1H, H-5, thiophene); 7.70 (m,
1H, H-4 thiophene); 7.39 (m, 1H, H-3 pyrrole); 6.40 (m, 1H, H-5,
pyrrole); 6.30 (m, 1H, H-4 pyrrole); 1.30 (s, 9H, (CH3)3). Anal. C,
H, N.
5.4. General procedure for the synthesis of derivatives 8, 23, 26,
and 39
5.2.13. 3-(2-Thienylmethoxycarbonyl)-8-(4-methoxy-
phenyl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (32)
From 31,22 and 4-methoxyphenylboronic acid, 8 h, room tem-
perature. Yellow crystals; TLC eluent: toluene/ethyl acetate 8:3 v/
v; 1H NMR (CDCl3) d 8.68 (s, 1H, H-2); 8.51 (d, 1H, H-9); 8.49 (d,
1H, H-6); 8.12 (dd, 1H, H-7); 7.95 (d, 2H, H-2 and H-6 phenyl);
7.60 (d, 1H, H-5 thiophene); 7.30 (d, 1H, H-3 thiophene); 7.60 (d,
2H, H-3 and H-5 phenyl); 7.08 (m, 1H, H-5 thiophene); 5.52 (s,
2H, CH2); 3.80 (s, 3H, CH3). Anal. C, H, N.
A
solution of potassium 2-furantrifluoroborate (60 mg,
0.34 mmol), PdCl2(dppf)ꢀCH2Cl2 (20 mg), triethylamine (0.1 mL)
and the suitable starting materials: 1,18 2,22, 15, 38,22 (0.2 mmol)
in n-propyl alcohol (10 mL) was stirred at reflux for 3 h. The reac-
tion was monitored by TLC (eluent: toluene/ethyl acetate/acetic
acid 8:2:1 v/v/v) and when the staring material disappeared, was
cooled to room temperature, and diluted with water (15 mL). The
appropriate work up of the final suspension gave the desired final
products 8, 23, 26, and 39.

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
3083
5.4.1. 8-(Fur-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide
(8)
5.5.3. 3-Iodo-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide (13)
From 1,18 the final suspension was extracted with ethyl acetate
and dried over sodium sulfate anhydrous. Evaporation of solution
gave orange residue. 1H NMR (DMSO-d6) d 8.45 (m, 2H, H-6 and
H-9); 8.20 (d, 1H, H-2); 8.08 (m, 2H, H-7 and H-5 furane); 7.58
(m, 1H, H-3 furane); 6.92 (d, 1H, H-3); 6.80 (m, 1H, H-4 furane).
Anal. C, H, N.
From 5. Orange crystals; TLC eluent: toluene/ethyl acetate/ace-
tic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.79 (d, 2H, H-2 and H-6
pyridine); 8.64 (d, 1H, H-9); 8.55 (d, 1H, H-6); 8.44 (s, 1H, H-2);
8.15 (dd, 1H, H-7); 7.98 (m, 2H, H-3 and H-5 pyridine). Anal. C,
H, N.
5.5.4. 3-Iodo-8-(thien-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (14)
5.4.2. 3-(Thien-3-yl)-8-(fur-2-yl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (23)
From 6. Orange crystals; TLC eluent: diisopropyl ether/cyclo-
hexane 8:3 v/v; 1H NMR (CDCl3) d 8.58 (m, 2H, H-6 and H-9);
8.14 (s, 1H, H-2); 7.88 (dd, 1H, H-7); 7.69 (d, 1H, H-5 thiophene);
7.57 (d, 1H, H-3 thiophene); 7.22 (m, 1H, H-4 thiophene). Anal.
C, H, N.
From 2,22 the final suspension was extracted with ethyl
acetate (10 mL ꢁ 3), dried over sodium sulfate anhydrous and
evaporated in vacuo and the residue recrystallized. Red crystals;
1H NMR (DMSO-d6) d 8.78 (s, 1H, H-2); 8.49 (m, 2H, H-6 and
H-9); 8.08 (d, 1H, H-7); 8.02 (s, 1H, H-2, thiophene); 8.00 (d,
1H, H-5 furane); 7.77 (m, 1H, H-4, thiophene); 7.72 (m, 1H,
H-5, thiophene); 7.58 (d, 1H, H-3 furane); 6.78 (m, 1H, H-4
furane). Anal. C, H, N.
5.5.5. 3-Iodo-8-(thien-3-yl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (15)
From 7. Orange crystals; TLC eluent: diisopropyl ether/cyclo-
hexane 8:3 v/v; 1H NMR (CDCl3) d 8.57 (m, 2H, H-6 and H-9);
8.14 (s, 1H, H-2); 7.88 (m, 2H, H-7 and H-2 thiophene); 7.59 (m,
1H, H-4 thiophene); 7.45 (m, 1H, H-5 thiophene). Anal. C, H, N.
5.4.3. 3-(Fur-2-yl)-8-(thien-3-yl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (26)
From 15. The precipitate was filtered and purified by silica gel
chromatography (eluting with toluene/ethyl acetate/acetic acid
8:2:1 v/v/v). Red crystals; 1H NMR (DMSO-d6) d 8.60 (s, 1H, H-2);
8.58 (d, 1H, H-9); 8.45 (m, 2H, H-6 and H-2 thiophene); 8.12 (dd,
1H, H-7); 7.88 (m, 1H, H-5, thiophene); 7.80 (m, 2H, H-4 thiophene
and H-5 furane); 6.85 (d, 1H, H-3 furane); 6.68 (m, 1H, H-4 furane).
Anal. C, H, N.
5.5.6. 3-Iodo-8-(Fur-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide (16)
From 8. Orange crystals; TLC eluent: diisopropyl ether/cyclo-
hexane 8:3 v/v; 1H NMR (DMSO-d6) d 8.47 (m, 3H, H-6, H-2, and
H-9); 8.05 (dd, 1H, H-7); 7.98 (m, 1H, H-5 furane); 7.52 (m, 1H,
H-3 furane); 6.79 (m, 1H, H-4 furane). Anal. C, H, N.
5.5.7. 3-Iodo-8-(N-Boc-pyrrol-2-yl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (17)
5.4.4. Ethyl 1-(2-nitro-5-(fur-2-yl)phenyl)-5-aminopyrazol-4-
carboxylate (39)
From 9. Orange crystals; TLC eluent: toluene/ethyl acetate/ace-
tic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.44 (m, 3H, H-2, H-6 and
H-9); 7.85 (d, 1H, H-7); 7.79 (m, 1H, H-3 pyrrole); 6.72 (m, 1H, H-5,
pyrrole); 6.42 (m, 1H, H-4 pyrrole); 1.40 (s, 9H, (CH3)3). Anal. C,
H, N.
From 38,22 dark-yellow crystals; 1H NMR (DMSO-d6) d 8.21 (d,
1H, H-30); 8.01 (dd, 1H, H-40); 7.97 (d, 1H, H-60); 7.93 (s, 1H, H-5
furane); 7.70 (s, 1H, H-3); 7.41 (d, 1H, H-3 furane); 6.73 (m, 1H,
H-4 furane); 6.57 (br s, 2H, NH2, exch.); 4.22 (q, 2H, CH2); 1.35 (t,
3H, CH3). Anal. C, H, N.
5.6. 3-Iodo-8-(2-hydroxyphenyl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (11)
5.5. General procedure for the synthesis of derivatives 10 and
12–17
A solution of 10 (0.24 mmol) in dichloromethane (15 mL) was
reacted with BBr3 (0.3 mL) at refluxing temperature until the start-
ing material disappeared in TLC (eluent: toluene/ethyl acetate/ace-
tic acid 8:2:1). Then the final solution was treated with a 10%
sodium hydroxide solution and the organic layer eliminated. The
aqueous phase, after acidification (HCl 1:1) was extracted with
ethyl acetate, dried and evaporated dryness obtained a yellow res-
idue that was purified by recrystallization. 1H NMR (DMSO-d6) d
10.2 (br s, 1H, OH); 8.55 (d, 1H, H-9); 8.46 (d, 1H, H-6); 8.34 (s,
1H, H-2); 7.95 (dd, 1H, H-7); 7.50 (d, 1H, H-3 phenyl); 7.35 (t,
1H, H-5 phenyl); 7.01 (d, 1H, H-6 phenyl); 7.00 (t, 1H, H-4 phenyl).
Anal. C, H, N.
A solution of appropriate starting material 3–9 (0.30 mmol) in
acetonitrile (10 mL) was added of iodine (0.18 mmol) and cerium
ammonium nitrate (CAN, 0.18 mmol) and maintained at room
temperature for 45 min. The final red solution was evaporated
under reduced pressure and the residue was washed with a 10% so-
dium hydroxide solution, the precipitate filtered and recrystallized
by suitable solvent.
5.5.1. 3-Iodo-8-(2-methoxyphenyl)pyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (10)
From 3. Orange crystals; TLC eluent: toluene/ethyl acetate/ace-
tic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.46 (d, 1H, H-6); 8.41 (d,
1H, H-9); 8.34 (s, 1H, H-2); 7.87 (dd, 1H, H-7); 7.53 (m, 2H, H-4 and
H-6 phenyl); 7.25 (d, 1H, H-3 phenyl); 7.16 (t, 1H, H-5 phenyl);
3.85 (s, 3H, OCH3). Anal. C, H, N.
5.7. General procedure for the synthesis of derivatives18, 25, 28,
34 and 37
To a mixture of starting material 17, 24, 27, 33, and 36
(0.15 mmol) and anhydrous THF (2 mL) was added a solution of so-
dium methoxide in methanol (2 mL of a 0.43 M solution). The mix-
ture was allowed to stir 1 h at room temperature and then treated
with water. The resulting suspension was worked up and the row
product recrystallized by suitable solvent.
5.5.2. 3-Iodo-8-(4-methoxyphenyl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (12)
From 4. Orange crystals; TLC eluent: toluene/ethyl acetate/ace-
tic acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.46 (m, 2H, H-2 and H-
6); 8.40 (d, 1H, H-9); 8.05 (dd, 1H, H-7); 7.92 (d, 2H, H-2 and H-6
phenyl); 7.13 (d, 2H, H-3 and H-5 phenyl); 3.85 (s, 3H, OCH3). Anal.
C, H, N.
In case of starting material 33 a trans-esterification occurred
and the final product was the 3-methyl ester derivative 34.

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G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 3074–3085
5.7.1. 3-Iodo-8-(pyrrol-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (18)
to permit the anhydride to form. The 2-thiophen methanol was
added in excess (0.2 mL) and the reaction was monitored by TLC
(eluent: toluene/ethyl acetate 8:2). The final suspension was di-
luted with water and extracted with ethyl acetate which was in
turn washed with sodium hydrogen carbonate and, after the nor-
mal work up, the residue was purified by chromatography column.
Yellow crystals. 1H NMR (DMSO-d6) d 8.68 (s, 1H, H-2); 8.52 (d, 1H,
H-9); 8.49 (d, 1H, H-6); 8.14 (dd, 1H, H-7); 8.03 (d, 1H, H-5 furane);
7.60 (m, 2H, H-5 thiophene and H-3 furane); 7.30 (m, 1H, H-3, 3-
thiophene); 7.06 (m, 1H, H-4 thiophene); 6.80 (m, 1H, H-4 furane);
5.55 (s, 2H, CH2). Anal. C, H, N.
From 17. Red crystals; TLC eluent: toluene/ethyl acetate/acetic
acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 12.00 (br s, 1H, NH, exch.);
8.43 (s, 1H, H-2); 8.40 (d, 1H, H-9); 8.25 (d, 1H, H-6); 8.00 (dd, 1H,
H-7); 7.10 (m, 1H, H-3 pyrrole); 7.05 (m, 1H, H-5, pyrrole); 6.30 (m,
1H, H-4 pyrrole). Anal. C, H, N.
5.7.2. 3-(Thien-3-yl)-8-(pyrrol-2-yl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (25)
From 24. Red crystals; TLC eluent: toluene/ethyl acetate/acetic
acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 12.00 (br s, 1H, NH, exch.);
8.73 (s, 1H, H-2); 8.50 (d, 1H, H-9); 8.35 (d, 1H, H-6); 8.00 (m, 2H,
H-7 and H-2, 3-thiophene); 7.78 (m, 1H, H-5, 3-thiophene); 7.70
(m, 1H, H-5, 3-thiophene); 7.12 (m, 1H, H-5, pyrrole); 7.01 (m,
1H, H-4 pyrrole); 6.30 (m, 1H, H-3 pyrrole). Anal. C, H, N.
5.10. 3-(Fur-2-ylcarbonyl)-8-(fur-2-yl)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (42)
To a mixture of acid 40 (0.20 mmol) and trichloroacetonitrile
(0.60 mmol) in dichloromethane (10 mL) was added triphenyl-
phosphine (0.60 mmol) in dichloromethane (1.0 mL) dropwise at
room temperature. The reaction mixture was stirred at room tem-
perature for 2 h until the starting material disappeared in TLC (elu-
ent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v). The reaction
was then treated with SnCl4 (0.1 mL) followed furane (0.1 mL).
After 300 the solution was treated with water and the organic layer
was separated, dried and evaporated to dryness. The residue was
recovered by diisopropyl ether and recrystallized by suitable sol-
vent. Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.91 (s, 1H, H-2); 8.58 (d, 1H, H-
9); 8.50 (d, 1H, H-6), 8.16 (dd, 1H, H-7); 8.12 (m, 1H, H-5, 2-furoyl);
8.06 (m, 1H, H-5 furane); 7.62 (m, 2H, H-3, 2-furoyl and furane);
6.83 (m, 2H, H-4, 2-furoyl and furane). Anal. C, H, N.
5.7.3. 3-(Pyrrol-2-yl)-8-(thien-3-yl)pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (28)
From 27. Red crystals; TLC eluent: toluene/ethyl acetate/acetic
acid 8:2:1 v/v/v; 1H NMR (DMSO-d6) d 11.35 (br s, 1H, NH, exch.);
8.51 (m, 2H, H-6 and H-2); 8.42 (m, 2H, H-9 and H-2 thiophene);
8.01 (dd, 1H, H-7); 7.83 (m, 1H, H-5, thiophene); 7.70 (m, 1H, H-
4 thiophene); 6.90 (m, 1H, H-3 pyrrole); 6.60 (m, 1H, H-5, pyrrole);
6.18 (m, 1H, H-4 pyrrole). Anal. C, H, N.
5.7.4. 3-Methoxycarbonyl-8-(pyrrol-2-yl)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (34)
From 33, in reaction conditions a trans-esterification occurred.
Red crystals; TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1
v/v/v; 1H NMR (DMSO-d6) d 12.1 (br s, 1H, NH, exch.); 8.67 (s,
1H, H-2); 8.55 (d, 1H, H-9); 8.38 (d, 1H, H-6), 8.08 (dd, 1H, H-7);
7.18 (m, 1H, H-3 pyrrole); 7.08 (m, 1H, H-5, pyrrole); 6.30 (m,
1H, H-4 pyrrole); 3.90 (s, 3H, CH3). Anal. C, H, N.
5.11. Radioligand binding assay
[3H]Ro15-1788 (specific activity 78.8 Ci/mmol) was obtained
from Perkin–Elmer.30,31 All the other chemicals, which were of re-
agent grade, were obtained from commercial suppliers.
Bovine cerebral cortex membranes were prepared as previously
described. The membrane preparations were diluted with 50 mM
Tris–citrate buffer pH 7.4, and used in the binding assay. Protein
concentration was assayed using the method of Lowry et al.32
[3H]Ro 15-1788 binding studies were performed as previously re-
ported.20 At least six different concentrations of each compound
were used. The data of n = 5 experiments carried out in triplicate
were analyzed by means of an iterative curve-fitting procedure
5.7.5. 3-(Fur-2-ylcarbonyl)-8-(pyrrol-2-yl)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (37)
From 36. TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
v; 1H NMR (DMSO-d6) d 12.00 (br s, 1H, NH, exch.); 8.91 (s, 1H, H-
2); 8.60 (d, 1H, H-9); 8.38 (d, 1H, H-6), 8.12 (m, 2H, H-7 and H-5
furane); 7.62 (m, 1H, H-3 furane); 7.15 (m, 1H, H-3 pyrrole); 7.08
(m, 1H, H-5, pyrrole); 6.80 (m, 1H, H-4 furane); 6.30 (m, 1H, H-4
pyrrole). Anal. C, H, N.
5.8. 3-Carboxy-8-(fur-2-yl)pyrazolo[5,1-c][1,2,4]benzotriazine
(program Prism, GraphPad, San Diego, CA), which provided IC50,
5-oxide (40)
Ki, and SEM values for tested compounds, the Ki values being calcu-
lated from the Cheng and Prusoff equation.33
A solution of 39 (0.5 mmol) in diglyme (5 mL) was added of
25 mL of 10% sodium hydroxide solution and was kept at 50–
60 °C until the starting material disappeared, monitoring by TLC.
The final suspension was treated with hydrochloric acid and the fil-
tration of the precipitate afforded to the acid derivative 40. The
crude products were then recrystallized by suitable solvent. Yellow
crystals; TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v;
1H NMR (DMSO-d6) d 12.9 (br s, 1H, OH, exch.); 8.61 (s, 1H, H-3);
8.51 (d, 1H, H-9); 8.48 (d, 1H, H-6); 8.15 (dd, 1H, H-7); 8.03 (d,
1H, H-5 furane); 7.60 (d, 1H, H-3 furane); 6.80 (m, 1H, H-4 furane).
Anal. C, H, N.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.04.009.
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