1,2,3-Triazolo[4,5-d]pyridazines. Part VI. New 1-substituted-4-amino derivatives and their affinity towards A1 and A(2A) adenosine receptors

Article abstract of DOI:10.1016/S0014-827X(99)00072-5

Starting from the appropriate azides (4-chlorobenzyl-, 2-thiophenemethyl-, 2-fluorobenzyl-, and 4-fluorobenzylazides) agreeing with the substituent determining four series of derivatives (a-d), some 4-amino-substituted 1,2,3-triazolo[4,5-d]pyridazines (4a-d) corresponding to previously prepared derivatives were obtained by a well experimented synthetic route. Other new derivatives (6c,e) which were different from 4a-d because a chlorine atom had substituted the hydroxyl or the tautomeric oxamido group in the 7 position of the triazolopyridazine ring, were prepared from the suitable azides (2-fluorobenzyl and 2-chlorobenzyl), which similarly determine the series c and e, respectively, via the 4,7-dichloro derivatives 5. The radioligand binding assays at bovine brain adenosine A₁ and A(2A) receptors showed that some compounds 4 possessed high affinity and selectivity for the A₁ receptor subtype whilst binding affinity decreased in compounds 6 indicating the importance of a hydrogen bond donor in the 7 position of the triazolopyridazine ring. It is worth noting that compounds bearing the new lipophilic substituents 2-fluorobenzyl and 2-thiophenemethyl in the 1 position of the triazolopyridazine ring were the most active in the series. Copyright (C) 1999 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(99)00072-5

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 615–623
1,2,3-Triazolo[4,5-d]pyridazines
Part VI. New 1-substituted-4-amino derivatives and their affinity
towards A1 and A2A adenosine receptors
Giuliana Biagi a, Irene Giorgi a, Oreste Livi a,*, Clementina Manera a, Valerio Scartoni a,
Laura Betti b, Gino Giannaccini b , Antonio Lucacchini b
a Dipartimento di Scienze Farmaceutiche, Facolta` di Farmacia, Uni6ersita` di Pisa, 6ia Bonanno 6, I-56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Uni6ersita` di Pisa, 6ia Bonanno 6, I-56126 Pisa, Italy
Received 20 December 1998; accepted 21 May 1999
Abstract
Starting from the appropriate azides (4-chlorobenzyl-, 2-thiophenemethyl-, 2-fluorobenzyl-, and 4-fluorobenzylazides) agreeing
with the substituent determining four series of derivatives (a–d), some 4-amino-substituted 1,2,3-triazolo[4,5-d]pyridazines (4a–d)
corresponding to previously prepared derivatives were obtained by a well experimented synthetic route. Other new derivatives
(6c,e) which were different from 4a–d because a chlorine atom had substituted the hydroxyl or the tautomeric oxamido group in
the 7 position of the triazolopyridazine ring, were prepared from the suitable azides (2-fluorobenzyl and 2-chlorobenzyl), which
similarly determine the series c and e, respectively, via the 4,7-dichloro derivatives 5. The radioligand binding assays at bovine
brain adenosine A1 and A2A receptors showed that some compounds 4 possessed high affinity and selectivity for the A1 receptor
subtype whilst binding affinity decreased in compounds 6 indicating the importance of a hydrogen bond donor in the 7 position
of the triazolopyridazine ring. It is worth noting that compounds bearing the new lipophilic substituents 2-fluorobenzyl and
2-thiophenemethyl in the 1 position of the triazolopyridazine ring were the most active in the series. © 1999 Elsevier Science S.A.
All rights reserved.
Keywords: 1,2,3-Triazolo[4,5-d]pyridazines; Adenosine receptor binding
On the basis of our previous results, the investiga-
tions into these structures were continued in order to
1. Introduction
study the mode of binding of these compounds with the
Our previous studies on 4-amino-substituted 1,2,3-
A1-receptors by comparison with the SAR analysis.
triazolopyridazines [1–4] reported that some com-
Therefore we report the synthesis and biological evalua-
pounds, bearing a benzyl or 2-chlorobenzyl group as a
tion of some series of 1,2,3-triazolo[4,5-d]pyridazines
lipophilic substituent on the 1 position, showed high
(4a–d), amino-substituted analogues of derivatives pre-
affinity and selectivity toward the A1 receptor subtype.
These and our other studies on 1,2,3-triazolopyrim-
idines [5–10], according to the actual receptor binding
site models for adenosine agonists and antagonists [11],
showed three lipophilic binding regions (corresponding
to the N-6, C-2 and N-9 positions of the azapurine
ring) in the A1 adenosine receptors, arranged as a
viously reported [1–4] characterized by new benzyl-type
substituents (a: R=4-chlorobenzyl; b: R=2-thenyl; c:
R=2-fluorobenzyl; d: R=4-fluorobenzyl).
In addition, in order to examine the eventual involve-
ment of the hydroxylic and/or of the tautomeric oxam-
ido group in the 7 position of the triazolopyridazine
ring in the binding toward the A1 adenosine receptors,
fan-shape with respect to the NH function which was
two series of 4-amino-substituted triazolopyridazines
engaged as a hydrogen bond donor.
(6c: R=2-fluorobenzyl; and 6e: R=2-chlorobenzyl),
bearing a chlorine atom in the 7 position, were pre-
* Corresponding author. Fax: +39-050 40517.
pared and tested.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00072-5

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G. Biagi et al. / Il Farmaco 54 (1999) 615–623
The binding affinities of these compounds have been
route (see Scheme 1). All the azides are reported in the
literature (4-chlorobenzylazide [12], 2-thenylazide [13],
2-fluorobenzylazide [14] and 4-fluorobenzylazide [15])
but preparation of 2-thenylazide is not described.
For the preparation of the triazolopyridazine deriva-
tives 4a–d (Scheme 1), the suitable azide was reacted
with methylacetylenedicarboxylate to give the triazole
diesters 1a–d which were converted into the corre-
sponding dicarboxyhydrazides 2a–d by treatment with
excess of 99% hydrazine hydrate. Compounds 2a,c,d,
by heating with 10% HCl, underwent an intramolecular
cyclization with the elimination of hydrazine to give the
compared to those of the corresponding analogous
derivatives 4c and 1-(2-chlorobenzyl)-substituted previ-
ously prepared and tested [4], as well as to those of the
corresponding 8-azapurine (or triazolopyrimidine)
derivatives [5] bearing a nuclear nitrogen atom in that
position of the ring.
In the 4 position of the triazolopyridazine ring the
amino substituents which conferred biological activity
to previously prepared molecules (cyclopentyl- and cy-
clohexylamino, meta- and para-toluidino, (9)-a-
methylbenzylamino, (9)-a-methylphenethylamino or
amphetamino) and the 3-pentylamino, a substituent
corresponding to the opening of the cyclopentane ring ,
have been introduced.
expected
1-substituted-4,7-dihydroxy-1,2,3-triazolo-
[4,5-d]pyridazines (3a,c,d) in good yield; cyclization of
2b to 3b was accomplished by heating in boiling
Dowtherm A to avoid polymerization of thiophene in
acid medium.
Some derivatives bearing cycloalkylamino or aralky-
lamino substituents on the 4 position were prepared
from 3a–d by the procedure based upon the HMDS/
(NH4)2SO4 reagent [16]. On the contrary, with the less
basic primary aromatic amines, the P2O5/TEA · HCl
2. Chemistry
Compounds 4 were prepared starting from the ap-
propriate azide, which determined the R substituent
(series a–d) and following a well experimented synthetic
Scheme 1. (i) MeOOCCꢀCCOOMe; (ii) N2H4H2O; (iii) 10% HCl; (iv) R1NH2, HMDS; (v) POCl3; (vi) R1NH2.

G. Biagi et al. / Il Farmaco 54 (1999) 615–623
617
3. Biological evaluation
The 4-(amino-substituted)-1,2,3-triazolo[4,5-d]pyrid-
azines were tested in radioligand binding assays for
affinity at A1 and A2A adenosine receptors in bovine
brain cortical membranes and in bovine brain striatal
membranes, respectively. [3H]R-(−)-N6-cyclohexy-
ladenosine (CHA) was used as the A1 radioligand and
[3H]-2-{[[p-(2-carboxyethyl)phenyl]ethyl]amino}-5%-(N-
ethylcarbamoyl)adenosine (CGS 21680) as the A2A ra-
dioligand. The experimental details of the receptor
binding assays have been reported in a previous paper
[5].
Fig. 1. Monochloro isomers.
reagent had to be employed [17], but this reagent gave
unsatisfactory results with 3a and 3c. However, the
meta-toluidino derivative 4c.4 (Scheme 1) was prepared
via the 4-monochloro derivative 7c, in its turn obtained
together with the 7-monochloro derivative isomer
8c (Fig. 1) by chlorination of 3c and isolated by
column chromatography. The tedious separation of the
7c and 8c isomers led us to prepare only the 4c.4 as
an example of N(4)-aryl-substituted compound. The
physico-chemical data of synthesized derivatives 4,
corresponding to the four series a, b, c, d (a: R=
4-chlorobenzyl; b: R=2-thenyl; c: R=2-fluoro-
benzyl; d: R=4-fluorobenzyl) are reported in Table
1.
For the preparation of the two series of the 7-chloro-
4-amino derivatives 6, the 2-fluorobenzyl azide [14]
(series c) and the 2-chlorobenzyl azide [12] (series e)
were employed by the reaction sequence described in
Scheme 1 to give the corresponding 4,7-dichloro-1,2,3-
triazolo[4,5-d]pyridazines 5c and 5e [4] via chlorination
with phosphorus oxychloride of 3c and 3e, respectively.
The intermediates 1e, 2e and 3e of the 2-chlorobenzyl
series have been described in the literature [4]. The
desired derivatives 6c and 6e (Table 2) were obtained
by reaction in a closed tube at 120°C in toluene and
160°C in DMF, respectively, of the dichloro derivatives
5c and 5e with an equimolar amount of the suitable
primary amine (cyclopentyl, cyclohexyl, (9)-a-methyl-
benzyl, (9)-a-methylphenetyl or amphetamine, m-
toluidine, p-toluidine or 3-pentyl amine) in the
presence of triethylamine. The physico-chemical data
of compounds 6 corresponding to the series c (R=2-
fluorobenzyl) and e (R=2-chlorobenzyl) are reported
in Table 1.
4. Results and discussion
The results of the A1 and A2A adenosine receptor
binding assays, expressed as inhibition constants (Ki,
nM) reported in Table 2, show that introduction of the
p-chlorobenzyl substituent on the triazole ring in the 1
position (compounds 4a.1,2) reduces the A1 adenosine
receptor binding compared to the 2-chlorobenzyl sub-
stituent (corresponding compounds have Ki=70 and
200 nM, respectively) [4]. Only the triazolopyridazine
4a.2 (racemic a-methylbenzylamino derivative) showed
a moderate affinity toward A1 (Ki=800 nM) as well as
A2A (Ki=900 nM) receptors, therefore it was lacking
in selectivity. Thus, moving the chlorine atom from the
ortho to the para position suggested a moderate and
well-defined extent of the lipophilic region which re-
ceives the benzyl substituent, whereas the ortho-chlo-
rine atom on the benzyl group increased the affinity to
A1 receptors [4]. This fact could signify that the para-
chloro substitution generally caused a steric repulsion
within the receptor, owing to the limited depth of the
lipophilic site.
The 2-thiophenemethyl (2-thenyl) substituent (series
4b) had been chosen because it could partially imitate
the 2-chlorobenzyl substituent for its steric and elec-
tronic characteristics. In fact, this substituent appeared
actively involved in the A1 adenosine receptor binding:
the triazolopyridazine derivatives 4b showed high A1
adenosine receptor affinity (KiB100 nM): 4b.1 (cyclo-
pentylamino, Ki=47 nM), 4b.3 (racemic amphet-
amino, Ki=56 nM) and 4b.2 (cyclohexylamino, Ki=
75 nM).
The structures of all the new compounds were as-
signed on the basis of the well-known reaction mecha-
nisms, already largely experimented in our laboratory
[1–4,18]: 1,3-dipolar cycloaddition of azides to alkynes,
formation of a pyridazine ring, chlorination reaction
and nucleophilic displacement of the halogen by
amines or O-silylation–amination reaction of amides.
The structures were also confirmed by analytical and
spectroscopic data.
The selection of the fluorobenzyl substituents to be
compared with the corresponding chlorobenzyl sub-
stituents [4] was based upon the consideration that the
strong electronegativity of the fluorine able to accept a
hydrogen bond, could allow the evaluation of the ef-
fect of the eventual electronic factors, as opposed to
steric factors, on the receptor binding.

618
G. Biagi et al. / Il Farmaco 54 (1999) 615–623
Table 1
Physico-chemical data of triazolopyridazines 4, 6 and intermediates
Comp.
Yield (%)
Crystall. solvent
M.p. (°C)
Analyses (C, H, N)
MS m/z
M+
Base peak
125
1a
1b
1c
1d
65
85
92
59
EtOH
83–84
C13H12N3O4Cl
C13H12N3O4F
278 (M+−31)
a
a
a
a
EtOH
45–48
262 (M+−31)
109
2a
2b
2c
2d
93
85
84
90
EtOH
EtOH
EtOH
EtOH
185–186
158–159
167–170
175–178
C11H12N7O2Cl
C9H11N7O2S
C11H12N7O2F
C11H12N7O2F
309
281
293
293
125
97
109
109
3a
3b
3c
3d
75
40
88
94
H2O
300–301
232–234
269–271
300–303
C11H8N5O2S
C9H7N5O2Cl
C11H8N5O2F
C11H8N5O2F
248 (M+−29)
249
125
97
109
109
EtOH
EtOH
H2O
261
232 (M+−29)
4a.1
4a.2
10
29
EtOH
EtOH
199–201
195–197
C16H17N6OCl
C19H17N6OCl
344
380
125
125
b
b
b
4b.1
4b.2
4b.3
12
18
14
190–192
186–188
174–176
C14H16N6OS
C15H18N6OS
C18H18N6OS
316
330
366
97
97
97
b
4c.1
4c.2
4c.3
4c.4
8
12
77
20
194–195
179–181
166–169
251–254
C16H17N6OF
C17H19N6OF
C20H19N6OF
328
342
378
350
109
109
109
109
MeOH
EtOH
b
C18H15N6OF
b
4d.1
4d.2
10
15
176–180
207–209
C16H17N6OF
C17H19N6OF
328
342
109
109
EtOH
4e.1
13
MeOH
202–206
C16H19N6OCl
346
125
6c.1
6c.2
6c.3
6c.4
6c.5
6c.6
48
39
51
32
50
15
EtOH
EtOH–H2O
MeOH
MeOH
EtOH
132–136
68–72
144–146
126–130
172–175
90–93
C16H16N6ClF
C17H18N6ClF
C19H16N6ClF
C20H18N6ClF
C18H14N6ClF
C18H14N6ClF
346
361
382
396
367
367
109
109
109
305
109
109
EtOH–H2O
6e.1
6e.2
6e.3
6e.4
6e.5
6e.6
6e.7
33
28
36
37
11
10
23
EtOH
EtOH
MeOH
167–170
144–146
151–155
147–150
107–110
148–152
112–116
C16H16N6Cl2
363
377
398
412
383
383
365
125
125
125
321
125
125
125
C
17H18N6Cl2
C19H16N6Cl2
C20H18N6Cl2
C18H14N6Cl2
C18H14N6Cl2
C16H18N6Cl2
EtOH
b
b
b
a Liquid compound, uncharacterized.
b Isolated by flash-chromatography.
With regard to the 2-fluorobenzyltriazolopyridazine
derivatives 4c.1–4, the most effective compound and the
only one with a Ki value B100 nM was the meta-tolu-
idino derivative 4c.4 (Ki=31.5 nM, and high selectivity).
The results of the 4c series agree with those of previous
analogous triazolopyridazines, either for the aromatic
meta substitution [3,4] or for the lower receptor affinity
of the cycloaliphatic amino derivatives [1,2]. Contrary to
what occurred with the triazolopyrimidine derivatives [5],
the results of the A1 adenosine receptor binding in the
triazolopyridazine derivatives indicated that the 2-fluoro-
benzyl substituent is less effective than the 2-chloroben-
zyl one [4]. Similarly, the two compounds (4d.1 and 4d.2)
bearing the 4-fluorobenzyl substituent showed a moder-
ate affinity, the cyclohexylamino derivative 4d.2 being
more effective than the corresponding cyclopentylamino
4d.1.
A molecular modelling study based upon a model of
the trans-membrane portion of the human A1 adenosine
receptor [19] confirmed that the high affinity shown by
the 2-methylthiophene derivatives might be supported by
a stacking interaction with the 278-histidine present in

G. Biagi et al. / Il Farmaco 54 (1999) 615–623
619
Table 2
A1 and A2A adenosine receptor binding of derivatives 4 and 6

620
G. Biagi et al. / Il Farmaco 54 (1999) 615–623
Table 2 (continued)
the VII trans-membrane domain analogously to that
previously observed for the benzyl group in tria-
zolopyrimidines [19].
The triazolopyridazino derivatives 6 (Table 2), bear-
ing a chlorine atom in the 7 position, show a decrease
of the receptor binding (Ki\100 nM). The compari-
son of the binding values of the 2-fluorobenzyl deriva-
tives 6c.1, 6c.2, 6c.4 and 6c.6 with the corresponding
derivatives 4c shows a gradual and increasing reduc-
tion of the binding affinity, starting from the cy-
clopentyl derivative 6c.1 (Ki value similar to 4c.1), the
cyclohexylamino derivative 6c.2 and the (9)-am-
phetamino derivative 6c.4 up to the m-toluidino
derivative 6c.5 (Ki value:30-fold higher than 4c.4).

G. Biagi et al. / Il Farmaco 54 (1999) 615–623
621
The comparison of the binding values of the 6e.1–6
derivatives with the corresponding 2-chlorobenzyl-tria-
zolopyridazines previously prepared [4] shows a much
more marked affinity decrease. The 3-pentylamino
derivative 6e.7 with a good affinity value (Ki=91 nM) is
an exception. The corresponding 7-hydroxy derivative
4e.1 was prepared and it showed a binding affinity
(Ki=59 nM) higher than the chloro derivative 6e.7.
Therefore this result confirms that the 7 or 6 position of
the triazolopyridazine ring can be involved in the receptor
binding as a hydrogen bond donor and that the aliphatic
3-pentylamino substituent in the 4 position is biologically
effective.
5.1.3. 1-Substituted-4,5-dicarboxyhydrazide-1H-1,2,3-
triazoles (2a–d)
To a solution of 13 mmol of the appropriate triazole
diester (1a, 1b, 1c or 1d) in 25 ml of EtOH, 99% hydrazine
hydrate (6.4 ml, $130 mmol) was added and the mixture
was refluxed for 1 h. The title compounds were crystal-
lized by cooling the reaction mixture and were collected
by filtration (Table 1).
5.1.4. 1-Substituted-4,7-dihydroxy-1,2,3-triazolo[4,5-d]-
pyridazines (3a,c,d)
A mixture of 10 mmol of the appropriate dicarboxyhy-
drazide (2a, 2c or 2d) in 30 ml of 10% HCl was heated
under reflux for 2 h. The solid precipitated after cooling
was collected and recrystallized (Table 1).
5. Experimental
5.1.5. 1-(2-Thenyl)-4,7-dihydroxy-1,2,3-triazolo[4,5-d]-
pyridazine (3b)
5.1. Chemistry
A solution of 2b (0.500 g, 1.77 mmol) in 10 ml of
Dowtherm was refluxed ($220°C) for 1 h. After cooling,
40–60°C petroleum ether was added to the suspension
and the solid material was collected by filtration and
washed with petroleum ether. The solid was stirred with
10% HCl for 30 min, collected and washed with H2O
(Table 1).
Melting points were determined on a Kofler hot-stage
and are uncorrected. IR spectra in Nujol mulls were
recorded on a Perkin–Elmer Model 1310 spectrometer.
1H NMR and 13C NMR spectra were recorded with a
Bruker AC 200 spectrometer in l units from TMS as an
internal standard. Mass spectra were performed with a
Hewlett Packard MS/System 5988. The TLC data were
5.1.6. 1-Substituted-4-cyclopentylamino-7-hydroxy-
1,2,3-triazolo[4,5-d]pyridazine deri6ati6es (4a.1, 4c.1
and 4d.1)
obtained with a Riedel de Haen, 37360 DC-Karten F254
,
0.2 mm, eluting with ethyl acetate/40–60°C petroleum
ether, 1:3 mixture. Elemental analyses (C, H, N) were
within 90.4% of the theoretical values and were per-
formed on a Carlo Erba Elemental Analyzer Model 1106
apparatus.
A mixture of the suitable dihydroxytriazolopyridazine
(3.0 mmol of 3a, 3c or 3d), HMDS (5 ml, $24 mmol),
ammonium sulfate (0.132 g, 1.0 mmol) and cyclopenty-
lamine (0.5 ml, 5.0 mmol) was heated at 120°C for 24 h.
The reaction mixture was dissolved in CHCl3 and the
solution was washed with 10% HCl, 10% NaOH and H2O,
dried and evaporated. The crude residue obtained under-
went a flash-chromatography on silica gel (eluting mix-
ture AcOEt/40–60°C petroleum ether, 1:2) to give the
title compounds generally in very poor yields (Table 1).
5.1.1. 2-Azidomethyl thiophene
To a solution of 2-chloromethyl thiophene (5.24 g, 39.5
mmol) in 25 ml of MeOH and 4 ml of H2O, NaN3 (7.68
g, 118 mmol) was added and the suspension was stirred at
room temperature for 24 h. The inorganic precipitate was
removed by filtration and the filtrate was concentrated,
treated with H2O and extracted with CHCl3. The chloro-
form extract was dried and evaporated in vacuo to give
the title compound as a yellow oil which was used without
purification: 5.19 g, 94.5% yield. The azide was distilled in
a tubular oven at 40–45°C/1.8 mmHg; IR (m): 4.75 (N3).
5.1.7. 1-Substituted-4-amino-substituted-7-hydroxy-
1,2,3-triazolo[4,5-d]pyridazine deri6ati6es
(4a.2, 4b.1–3, 4c.2,3 and 4d.2)
A mixture of the suitable dihydroxytriazolopyridazine
(3.0 mmol of 3a, 3b, 3c or 3d), in HMDS (5 ml, $24
mmol) was heated at 140°C for 3 h. After cooling,
ammonium sulfate (0.132 g, 1.0 mmol) and 5 mmol of the
appropriate amine (cyclohexyl-, (9)-a-methylbenzyl-,
(9)-a-methylphenethyl-, or cyclopentyl-amine) were
added and heating at 140°C was continued for 20 h. The
reaction mixture was dissolved in CHCl3 and the chloro-
form extract was washed with 10% HCl, 10% NaOH and
H2O, dried and evaporated. The crude residue of 4a.2 and
4d.2 was purified by crystallization (Table 1), all other
derivatives underwent a flash-chromatography on silica
gel (eluting mixture AcOEt/40–60°C petroleum ether,
1:2) to give the title compounds (Table 1).
5.1.2. 1-Substituted-4,5-dicarbomethoxy-1H-1,2,3-
triazoles (1a–d)
A solution of the suitable azide (13.15 mmol) and
dimethylacetylenedicarboxylate (2.75 ml, 22.4 mmol) in
20 ml of anhydrous benzene was heated under reflux for
22 h. The reaction mixture was evaporated in vacuo to
give a liquid residue consisting of the title compounds.
The residues of 1a and 1d triturated with EtOH and
MeOH, respectively, converted to the corresponding solid
diester; 1b and 1c diesters were used without purification
(Table 1).

622
G. Biagi et al. / Il Farmaco 54 (1999) 615–623
5.1.8. 1-(2-Fluorobenzyl)-4,7-dichloro-1,2,3-triazolo-
[4,5-d]pyridazine (5c)
5.1.11. 1-(2-Fluorobenzyl)-4-amino-substituted-7-chloro-
1,2,3-triazolo[4,5-d]pyridazine deri6ati6es (6c.1–6)
To a solution of the dichloro derivative 5c (0.300 g,
1.00 mmol) in 5 ml of toluene and 3 ml of triethy-
lamine, 1.00 mmol of the suitable amine (cyclopentyl,
cyclohexyl, (9)-a-methylbenzyl, (9)-a-methylphenetyl
amine, m-toluidine or p-toluidine) was added and the
mixture was heated in a closed tube at 120°C for 12 h.
The mixture was evaporated in vacuo, the residue
treated with CHCl3 and the organic layer washed with
10% HCl, 10% NaOH and H2O. The solution was dried
and evaporated and the residue crystallized to give the
title compounds (Table 1).
A solution of 3c (1.0 g, 3.80 mmol) in 5 ml of POCl3
and 1 ml of N,N-diethylaniline was refluxed for 8 h.
The reaction mixture poured into crushed ice and the
precipitated solid was collected by filtration and treated
with 10% NaOH. The insoluble material collected and
washed with H2O consisted of 5c: 0.876 g, 77%; m.p.
118–120°C (MeOH); MS 298 (M+), 109 (base peak).
Anal. (C, H, N) for C11H6N5Cl2F.
5.1.9. 1-(2-Fluorobenzyl)-4-chloro-7-hydroxy-1,2,3-
triazolo[4,5-d]pyridazine (7c) and 1-(2-fluorobenzyl)-
4-hydroxy-7-chloro-1,2,3-triazolo[4,5-d]pyridazine (8c)
A solution of the dihydroxytriazolopyridazine (3c)
(2.80 g, 10.6 mmol) in 20 ml of POCl3 was refluxed for
4 h. The reaction mixture was poured into crushed ice
and the solid precipitated was collected by filtration,
washed (H2O) and stirred in 5% NaHCO3 to solubilize
the starting material. The solid residue collected and
dried (1.450 g) was extracted with CHCl3 and the
soluble material ($0.950 g) underwent a flash-chro-
matography on silica gel (eluting mixture AcOEt/40–
60°C petroleum ether, 1:3). After elution of a small
amount of the 4,7-dichloro derivative 5c (Rf 0.70), the
4-monochloro derivative 7c (Rf 0.36) and its isomer
7-monochloro 8c (Rf 0.11) were eluted.
5.1.12. 1-(2-Chlorobenzyl)-4-amino-substituted-7-chloro-
1,2,3-triazolo[4,5-d]pyridazine deri6ati6es (6e.1–7)
To a solution of the dichloro derivative 5e [4]
(0.300 g, 0.95 mmol) in 1.5 ml of DMF and 3 ml of
triethylamine, 0.95 mmol of the suitable amine (cy-
clopentyl, cyclohexyl, (9)-a-methylbenzyl, (9)-a-
methylphenetyl amine, m-toluidine, p-toluidine or
3-pentylamine) were added and the mixture was heated
in a closed tube at 160°C for 12 h. The mixture was
worked up as described above (Table 1). Compounds
6e.5–7 were isolated by flash-chromatography through
silica gel eluting with an ethyl acetate/40–60° petro-
leum ether/diethylamine, 1.5:10:0.5 mixture (Table 1).
7c: 0.490 g, 16.5%; m.p. 203–207°C; MS 279 (M+),
109 (base peak). 1H NMR (DMSO) l: 6.09 (s, 2H,
CH2); 7.16–7.45 (m, 4H, Ar); 13.30 (brs, 1H, NH). 13C
NMR (DMSO) l: 47.02 JCF 3.5 Hz (CH2); 115.63 JCF
20.8 Hz (C-3%); 121.87 JCF 14.4 Hz (C-1%); 124.85 JCF
3.2 Hz (C-5%); 128.64 (C-3a); 130.74 JCF 3.0 Hz (C-4%);
131.00 JCF 8.3 Hz (C-6%); 131.10 (C-7a); 141.42 (C-4);
153.51 (C-7); 160.05 JCF 247.4 Hz (C-2%). Anal. (C, H,
N) for C11H7N5OClF.
5.1.13. 1-(2-Chlorobenzyl)-4-(3-pentylamino)-7-hydroxy-
1,2,3-triazolo[4,5-d]pyridazine (4e.1)
A solution of 0.300 g (1.08 mmol) of 4,7-dihydroxy-
triazolopyridazine 3e [4] in 1.2 ml (6.02 mmol) of
HMDS was heated at 140°C for 1 h. After cooling,
0.050 g of (NH4)2SO4 and 0.70 ml (6.02 mmol) of
3-pentylamine were added and the mixture was heated
again at 140°C for 20 h. The reaction mixture was
treated with CHCl3 and the chloroform was washed
with 10% HCl, 10% NaOH and H2O, then dried and
evaporated to give a residue which was crystallized
(Table 1).
8c: 0.250 g, 8.5%; m.p. 220–222°C; MS 279 (M+),
109 (base peak). 1H NMR (DMSO) l: 6.13 (s, 2H,
CH2); 7.16–7.46 (m, 4H, Ar); 13.26 (brs, 1H, NH). 13C
NMR (DMSO) l: 46.78 JCF 3.5 Hz (CH2); 115.63 JCF
20.8 Hz (C-3%); 122.10 JCF 14.3 Hz (C-1%); 124.67 (C-
3a); 124.94 JCF 3.0 Hz (C-5%); 129.70 JCF 2.9 Hz (C-4%);
130.77 JCF 8.2 Hz (C-6%); 131.41 (C-7a); 139.64 (C-7);
155.67 (C-4). 159.64 JCF 246.4 Hz (C-2%). Anal. (C, H,
N) for C11H7N5OClF.
Acknowledgements
We wish to thank the Consiglio Nazionale delle
Ricerche (CNR) for financial support.
5.1.10. 1-(2-Fluorobenzyl)-4-(m-toluidino)-7-
hydroxy-1,2,3-triazolo[4,5-d]pyridazine (4c.4)
References
A mixture of the 4-monochloro derivative 7c (0.250
g, 0.954 mmol) and m-toluidine (0.20 ml, 1.90 mmol)
was heated in a closed tube at 140°C for 12 h. The
reaction mixture was treated with H2O and extracted
with CHCl3; the chloroform layer was washed with 5%
HCl, H2O, dried and evaporated. The crude residue
was purified by flash-chromatography through silica gel
[1] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, C. Martini, P. Tacchi,
S. Merlino, M. Pasero, 1,2,3-Triazolo[4,5-d]pyridazines. II. New
derivatives tested on adenosine receptors, Farmaco 49 (1994)
175–181.
[2] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, A. Lucacchini, G.
Senatore, P.L. Barili, 1,2,3-Triazolo[4,5-d]pyridazines. III. Syn-
thesis of new 4-amino derivatives and their affinity toward
adenosine receptors, Farmaco 49 (1994) 357–362.
eluting with CHCl3/Et2NH/MeOH, 10:0.1:0.1 (Table 1).
.

G. Biagi et al. / Il Farmaco 54 (1999) 615–623
623
[3] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, S. Velo, C. Martini, G.
Senatore, P.L. Barili, 1,2,3-Triazolo[4,5-d]pyridazines. IV. Prepa-
ration and adenosine receptor binding of new 4- and/or 7-amino
derivatives, Farmaco 50 (1995) 99–105.
[4] G. Biagi, I. Giorgi, O. Livi, C. Manera, V. Scartoni, A. Lucac-
chini, G. Senatore, 1,2,3-Triazolo[4,5-d]pyridazines. V. Prepara-
tion and adenosine receptor binding of new 4-amino derivatives,
Farmaco 51 (1996) 601–608.
substituted 2-phenyl-N(9)-benzyl-8-azaadenines, Farmaco 51
(1996) 395–399.
[11] M.W. Dudley, N. Peet, D. Demeter, H.J.R. Weintraub, Ad.P.
Ijzerman, G. Nordvall, P.J.M. van Galen, K.A. Jacobson,
Adenosine A1 receptor and ligand molecular modeling, Drug
Dev. Res. 28 (1993) 237–243.
[12] W. Ried, J. Laoutidis, Synthesis neuer stickstoffreicher heterocy-
clen, Chem. Ztg. 114 (1990) 246–248.
[5] L. Betti, G. Biagi, G. Giannaccini, I. Giorgi, O. Livi, A. Lucac-
chini, C. Manera, V. Scartoni, Novel 3-aralkyl-7-(amino-substi-
tuted)-1,2,3-triazolo[4,5-d]pyrimidines with high affinity toward
A1 adenosine receptors, J. Med. Chem. 41 (1998) 668–673.
[6] G. Biagi, I. Giorgi, O. Livi, A. Lucacchini, C. Martini, V.
Scartoni, P. Tacchi, N(6)-Substituted 2-n-butyl-9-benzyl-8-aza-
adenines. Affinity for adenosine A1 and A2 receptors. IV, Far-
maco 49 (1994) 183–186.
[7] G. Biagi, I. Giorgi, O. Livi, A. Lucacchini, C. Martini, V.
Scartoni, P. Tacchi, N(6)-Substituted 2-phenyl-9-benzyl-8-
azaadenines. Affinity for adenosine A1 and A2 receptors. A
comparison with 2-n-butyl analogous derivatives. V, Farmaco 49
(1994) 187–191.
[13] T. Aubert, M. Farnier, R. Guilard, New method for synthesis of
isoindolinones and isoquinolines by condensation of iminophos-
phoranes with o-phthalaldehyde: reactions, mechanisms and
structural study, Can. J. Chem. 68 (1990) 842–851.
[14] R. Kreher, U. Bergman, Nitrogen eliminations by electrophiles
— Part 9 — Investigations on the formation of cyclic N-diazo-
nium ions, Tetrahedron Lett. 47 (1976) 4259–4262.
[15] P.G. Baraldi, S. Manfredini, D. Simoni, L. Zappaterra, C.
Zocchi, S. Dionisotti, E. Ongini, Synthesis of new pyrazolo[4,3-
e]1,2,4-triazolo[1,5-c]pyrimidine and 1,2,3-triazolo[4,5-e]1,2,4-tri-
azolo[1,5-c]pyrimidine displaying potent and selective activity as
A2A adenosine receptor antagonists, Bioorg. Med. Chem. Lett. 4
(1994) 2539–2544.
[8] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, A. Lucacchini, C.
Martini, P. Tacchi, Synthesis of new N(6)-substituted 2-phenyl-
8-azaadenosines. Their affinity for adenosine A1 and A2 recep-
[16] H. Vorbruggen, K. Krolikiewicz, Silylation-amination of hy-
droxy N-heterocycles, Chem. Ber. 117 (1984) 1523–1541.
[17] L. Andersen, F.E. Nielsen, E.B. Pedersen, Synthesis of new
1,2,3-triazolo[4,5-d]pyridazine derivatives, Chem. Scr. 29 (1989)
45–49.
[18] G. Biagi, I. Giorgi, O. Livi, C. Manera, V. Scartoni, 1,2,3-Tria-
zolo[4,5-d]-1,2,4-triazolo[4,3-b]pyridazines, J. Heterocycl. Chem.
34 (1997) 65–69.
[19] A.M. Bianucci, M.U. Bigi, G. Biagi, I. Giorgi, O. Livi, V.
Scartoni, A 3D model of the human A1 adenosine receptor. An
evaluation of the binding free-energy with ligands, Drug Des.
Discovery 15 (1998) 149–156.
tors.
A
comparison with the corresponding 2-phenyl-9-
benzyl-8-azaadenines, Farmaco 50 (1995) 13–19.
[9] G. Biagi, C. Breschi, I. Giorgi, O. Livi, C. Martini, V. Scartoni,
R. Scatizzi, N(6)- or N(9)-Substituted 2-phenyl-8-azaadenines:
affinity for A1 adenosine receptors. VII, Farmaco 50 (1995)
659–667.
[10] G. Biagi, I. Giorgi, O. Livi, A. Lucacchini, V. Scartoni, N(9)-
Substituted 2-phenyl-N(6)-benzyl-8-azaadenines: A1 adenosine
receptor affinity. A comparison with the corresponding N(6)-
.