N6-cycloalkyl-2-phenyl-3-deaza-8-azaadenines: a new class of A1 adenosine receptor ligands. A comparison with the corresponding adenines and 8-azaadenines.

Article abstract of DOI:10.1016/j.ejmech.2003.09.003

Several 9-benzyl-N6-cycloalkyl-2-phenyladenines, 9-benzyl-N6-cycloalkyl-2-phenyl-8-azaadenines and 4-cycloalkylamino-1-benzyl-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridines were prepared and assayed as A1 adenosine receptor ligands. The 1H-1,2,3-triazolo[4,5-

Full text of DOI:10.1016/j.ejmech.2003.09.003

European Journal of Medicinal Chemistry 38 (2003) 983Á990
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Original article
N6-Cycloalkyl-2-phenyl-3-deaza-8-azaadenines: a new class of A1
adenosine receptor ligands. A comparison with the corresponding
adenines and 8-azaadenines
Giuliana Biagi a, Irene Giorgi a,*, Oreste Livi a, Antonio Nardi a, Federica Pacchini a,
Valerio Scartoni a, Antonio Lucacchini b
a Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno, 6-56126Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, via Bonanno, 6-56126 Pisa, Italy
Received 10 June 2003; received in revised form 12 September 2003; accepted 12 September 2003
Abstract
Several 9-benzyl-N6-cycloalkyl-2-phenyladenines, 9-benzyl-N6-cycloalkyl-2-phenyl-8-azaadenines and 4-cycloalkylamino-1-ben-
zyl-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridines were prepared and assayed as A1 adenosine receptor ligands. The 1H-1,2,3-
triazolo[4,5-c]pyridines were obtained starting from N,N-diethyl-1-benzyl-4-carboxyamido-5-methyl-1H-1,2,3-triazole by lithiation
in anhydrous tetrahydrofurane in the presence of benzonitrile. The usual work up afforded the isolation of 1-benzyl-6-phenyl-1H-
1,2,3-triazolo[4,5-c]pyridin-4-one which was treated with phosphorous oxychloride and cycloalkylamines. Some compounds showed
high affinity and selectivity and the trend of Ki values corresponds to the series of 9-benzyl-N6-cycloalkyl-2-phenyladenines and 9-
benzyl-N6-cycloalkyl-2-phenyl-8-azaadenines, therefore they can be considered bioisosteres. The affinity data permitted us to
ascertain the role and the importance of the N(3) in the adenine or 8-azaadenine moiety in the receptor binding and to study the
dimension of the receptor lipophilic pocket which is filled by the N6 substituent of adenosine derivatives.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: A1 Adenosine receptor ligands; 4-Cycloalkylamino-1-benzyl-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridines; 9-Benzyl-N6-cycloalkyl-2-
phenyladenines; 9-Benzyl-N6-cycloalkyl-2-phenyl-8-azaadenines; Bioisosterism
1. Introduction
renin secretion, diuresis, natriuresis and other effects [1].
A great number of physiological effects have been
demonstrated for A1 receptors. For example, hundreds
of agonists and antagonists have been assayed to obtain
some compounds useful in therapy. A number of A1
antagonists have been studied as novel potassium
sparing diuretics with kidney-protection properties, in
dementia, Alzheimer’s disease, cardiac therapy and
kidney disease [2].
In the course of our studies directed toward synthesis
of A1 adenosine receptor antagonists, we had occasion
to prepare and test a number of 8-azaadenines and
adenines having three lipophilic substituents in the
positions 2, 9 and N6 [3]. Initially, we synthesised 2-,
N6- and 9-substituted-8-azaadenines by a facile two-
stage method [4]. This procedure afforded a large
number of compounds with good affinity towards A1
adenosine receptors. These had high selectivity A1/A2
Adenosine is an endogenous nucleoside which med-
iates a variety of important physiological effects by its
specific receptors: A1 receptors were the first subtype to
be identified. They are widely distributed in the central
nervous system and in peripheral tissues and mediate
diverse biological effects. The role of adenosine as a
neuroprotective agent during hypoxia and ischemic
conditions seems to be mediated by the A1 receptors;
these receptors have been involved in sedative, anti-
seizure and anxiolytic effects. A1 receptors mediate
cardiac depression through negative chronotropic, dro-
motropic and inotropic effects. In the kidney, activation
of A1 receptors causes vasocostriction, inhibition of
* Corresponding author.
E-mail address: igiorgi@farm.unipi.it (I. Giorgi).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.09.003

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G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á990
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and some were very effective having a Ki in the
nanomolar range. The most active compounds were 9-
benzyl-2-phenyl-8-azaadenines N6-cyclopentyl and N6-
cyclohexyl substituted (general formula I, Fig. 1), having
Ki 11 [3] and 1.6 nM [5], respectively. Thus, we have
completed the series, by synthesising all the N6-
cycloalkyl derivatives from three to eight carbons. We
also prepared the corresponding adenine derivatives
(general formula II, Fig. 1) in order to compare their
affinity with the analogous 8-azaadenines (I), and to
confirm the bioisosterism of the two nuclei, which we
demonstrated recently [6].
three series of compounds (2-phenyl-9-benzyl-N6-cy-
cloalkyl substituted adenines, 8-azadenines and 3-
deaza-8-azaadenines) was also useful to explore the
steric tolerance of the A1 receptor lipophilic pocket in
front of N6 of adenosine which is able to hold a
cyclopentyl or a cyclohexyl moiety in potent agonists
(as cyclohexyladenosine or cyclopentyladenosine) or
antagonists as N6-cyclohexyl- or cyclopentyl-2-phenyl-
9-benzyl-8-azaadenines.
2. Chemistry
In an effort to further explore other structural
modifications of the purine nucleus, we sought to verify
the importance of N(3) in the binding of ligands with A1
adenosine receptors. In the past, some studies regarding
the relative importance of nuclear nitrogen atoms in
several deaza analogs of adenosine were synthesised [7].
More recently, N-cycloalkyl derivatives of adenosine
and 1-deazaadenosine, were compared regarding the
importance both of N-1 atom and of the dimension of
the cycloalkyl inserted on N6 [8]. Similar analogous
studies on adenine derivatives have not been reported in
the literature.
First we planned to prepare 9-benzyl-2-phenyl-3-
deaza-8-azaadenines (general formula III, Fig. 1) and
9-benzyl-2-phenyl-3-deazaadenines (general formula IV,
Fig. 1) by cyclisation of 3,4-diamino-2-chloro-6-phenyl-
pyridine by classical methods, but this first idea was
rejected because these compounds are not easy to obtain
by this synthetic pathway. In fact in the literature the
preparation of N4-substituted-2-chloro-3,4-diamino-
pyridine [9] or 2-hydroxy-3,4-diamino-pyridine [10] is
reported, but their 6-phenylsubstituted analogs are not.
So we studied a new synthetic route which permitted us
to prepare, starting from 1-benzyl-5-methyl-1H-1,2,3-
triazole-4-carboxylic acid, 4-cycloalkyl derivatives of 7-
The 9-benzyl-2-phenyladenine (4) and its N6-cy-
cloalkyl derivatives (5Á11) were obtained from 9-ben-
/
zyl-6-chloro-2-phenylpurine (3) according to described
procedures [6] (Fig. 2); also the 9-benzyl-N6-cycloalkyl-
2-phenyl-8-azaadenines (14Á
viously reported [11] (Fig. 3).
Compounds 27Á34 were obtained by a new method
/18) were prepared as pre-
/
(Fig. 4). In fact, up to now, 1,2,3-triazolo[4,5-c]pyridines
have been prepared from the corresponding 3,4-diami-
nopyridines by diazotation reaction [9,12]. In this paper
we present a synthetic route starting from 1-benzyl-5-
methyl-1H-1,2,3-triazole-4-carboxylic acid ethyl ester
(19), obtained as described previously in the literature
[14] by regiospecific and base-catalysed cycloaddition of
benzylazide and ethyl acetoacetate under mild condi-
tions; 19 was hydrolysed with sodium hydroxide to
obtain the corresponding acid (compound 20) [15]
which, in turn, was converted into chloride 21 by
treatment with thionyl chloride at reflux: this was
converted in four stages into 1-benzyl-4-aminosubsti-
tuted-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridines. This
synthetic method is based on a lithiation reaction: a
first attempt was carried out on 1-benzyl-5-methyl-1H-
1,2,3-triazole-4-(N-methyl)-carboxamide (22) in the pre-
sence of benzonitrile and lithium diisopropyl amide
(LDA). This reaction was used by Poindexter [15] who
transformed 1-methyl-2-(N-methyl)-carboxamido ben-
zene into an isochinoline derivative. The chloride 21 was
reacted with methyl amine to obtain the corresponding
amide 22. A treatment of 22 with benzonitrile and a 2 M
phenyl-1-benzyl-1H-1,2,3-triazolo[4,5-c]pyridine
(2-
phenyl-9-benzyl-3-deaza-8-azaadenines) which, up to
now, have not been reported. Comparing the capability
of these compounds to bind A1 adenosine receptors with
the corresponding adenine and 8-azaadenine derivatives,
we were able to study the importance of the purine N(3)
atom in the binding with A1 receptors. Synthesis of the
solution of LDA at ꢀ74 8C, did not give the expected 1-
/
Fig. 1. General formulas of N6-substituted 9-benzyl-2-phenyl-8-azaadenines (I), N6-substituted 9-benzyl-2-phenyladenines (II), N6-substituted 9-
benzyl-2-phenyl-3-deaza-8-azaadenines (III) N6-substituted 9-benzyl-2-phenyl-3-deazaadenines (IV).

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á
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985
obtain compound 25 by heating 23 in basic or acid
conditions failed. Only the starting material, sometimes
contaminated with decomposition products, was recov-
ered. A good yield (60%) of 25 was obtained employing
the lithiation reaction on 1-benzyl-5-methyl-1H-1,2,3-
triazole-4-(N,N-diethyl)-carboxamide (24), prepared
from 21 by reaction with diethylamine. Examples of
cyclisation by lithiation reaction using diethylamides
were described by Mitsuaki et al. in 1984 [16]. 1-Benzyl-
6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridine-4-one (25) was
transformed into the corresponding chloride 26 by
reaction with phosphorous oxychloride. Finally, the 4-
aminosubstituted compounds 27Á
treatment of the chloride with the appropriated amine at
140Á150 8C in a sealed tube.
/34 were obtained by
/
3. Biological evaluation
Compounds 4Á11, 14Á
/
/
18 and 27Á34 were tested in
/
radioligand binding assays for affinity at A1 and A2A
adenosine receptors in bovine brain cortical membranes
and bovine striatum respectively. In Table 1 the results
of A1 adenosine receptor binding assays are reported; in
A2A adenosine receptor binding assays, all the com-
pounds showed inhibition values B60% at 1 mM, and
/
are considered inactive.
Fig. 2. Synthesis of N6-substituted 2-phenyl-9-benzyladenines (4Á
11).
/
4. Results and discussion
All synthesised compounds, assayed as A1 and A2A
adenosine receptor ligands, showed good affinity and
selectivity for A1 subtype. As can be seen in the
histogram of Table 1, trends of Ki values in series I
and II are very similar; by increasing the size of the N6-
cycloalkyl substituent, affinity increased up to five or six
carbon atoms, then affinity decreased for the com-
pounds with N6-cycloheptyl or -cyclooctyl substituents.
The biological results (Table 1) also indicate that the
two series would bind to the receptor in the same site
with the same orientation; the receptor pocket in front
of N6 of adenosine would be very large and capable of
binding either little or large cycloalkyl substituents. The
different size of the cycloalkyl substituent modulated the
affinity, and cyclopentyl, cyclohexyl or norbornyl
groups conferred the highest affinity to the molecules.
The comparison of the values of Ki of adenines (Table
1) with those of the corresponding 8-azaadenines (Table
Fig. 3. Synthesis of 9-benzyl-2-phenyl-6-cycloalkylamino-8-azaade-
nines (14Á18).
/
1, newly developed compounds 14Á
/
18 and compounds
AÁ
/
C already reported in the literature [3,5]) showed that
they are very close, either for N6-unsubstituted as for
N6-cycloalkylsubstituted compounds. This strong simi-
larity of affinity permits us to consider the two nuclei as
bioisosteric: the N(8) did not contribute to the binding
benzyl-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridine-4-one
(25). From the reaction mixture, only the open imino
intermediate adduct 23, formed between lithiated tria-
zole and benzonitrile, was isolated. All attempts to

986
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á990
/
Fig. 4. Synthesis of 1-benzyl-4-amino-6-phenyl-1H-1,2,3-triazol[4,5-c]pyridine (27) and 1-benzyl-4-cycloalkylamino-6-phenyl-1H-1,2,3-triazol[4,5-
c]pyridines (28Á34).
/
of these molecules towards the receptor protein in a
different manner from the C(8).
tant, but not crucial, to stabilise the complex between
these ligands and the A1 receptors.
Regarding triazolopyridines (series III), biological
results (Table 1) indicated a general lowering of A1
affinity with respect to the analogous compounds
having nitrogen in the position 3 (series I and II):
most of the triazolopyridine derivatives (compounds 27,
5. Experimental
5.1. Chemistry
28, 32, 33, 34) have a Kiꢀ100 nM, and only compounds
/
29, 30 and 31 are very potent A1 ligands, having Ki
values of 44, 14 and 11 nM, respectively. The trend of Ki
values in series III is similar to that of series I and II for
Melting points were determined on a Kofler hot-stage
1
apparatus and are uncorrected. H-NMR spectra were
recorded on a Varian Gemini 200 spectrometer in d
units using TMS as an internal standard; the com-
pounds were dissolved in CDCl3. Mass spectra were
the first five compounds (compounds 27Á
/
31 vs. 4Á7 and
/
A, 14, 15, B, 16) but it is different for the other ones, in
which we can observe an abrupt lowering of activity. So,
in the triazolopyridines, the size of the cycloalkyl
substituent was more critical, because affinity decreased
quickly when the cycloalkyl moiety was different from
cyclobutyl, cyclopentyl or norbornyl group. In conclu-
sion, from the biological data we may presume that
nitrogen in the position 3 of 2-phenyl-8-azaadenines (I)
and their bioisoster 2-phenyladenines (II) was impor-
performed on a HewlettÁ
5988A. TLC was performed on precoated silica gel
254 plates (Merck). Flash-column chromatography was
performed using Merck Kieselgel 60 (230Á400 mesh).
/Packard GC/MS System
F
/
Microanalyses (C H N) were carried out on a Carlo
Erba elemental analyser (model 1106) and were within
9
/
0.4% of the theoretical values. PetroleumÁ
/ether
corresponds to fraction boiling at 40Á60 8C.
/

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á
/990
987
Table 1
A1 adenosine receptor binding results and related histogram
Table 2
Reaction and physico-chemical data
Compound Amine
Yield
(%)
M.p.
(8C)
Analysis
(C, H, N)
4
NH3
85
210
173
225
C18H15N5
C21H19N5
C22H21N5
5
cyclopropylamine 62
cyclobutylamine 58
cyclopentylamine 80
norbornylamine 47
cyclohexylamine 80
cycloheptylamine 39
cyclooctylamine 79
cyclopropylamine 63
cyclobutylamine 70
norbornylamine 58
cycloheptylamine 58
cyclooctylamine 62
6
7
165Á
150
170
/
167 C23H23N5
8
C25H25N5
C24H25N5
C25H27N5
9
10
11
14
15
16
17
18
27
28
29
30
31
32
33
34
140
137Á
193Á
170
185
/
139 C26H29N5
195 C20H18N6
C21H20N6
/
C24H24N6
C24H26N6
144
176Á
145Á
156Á
136
147
/
178 C25H28N6
146 C18H15N5
157 C21H19N5
C22H21N5
NH3
68
/
cyclopropylamine 93
cyclobutylamine 95
cyclopentylamine 92
norbornylamine 44
cyclohexylamine 90
cycloheptylamine 83
cyclooctylamine 87
/
C23H23N5
140Á
138Á
/
143 C25H25N5
139 C24H25N5
C25H27N5
/
121
106Á
/
107 C26H29N5
2) (6 mmol) and N,N-diethylaniline (0.1 g, 0.67 mmol)
was refluxed for 12 h. Then the solution was evaporated
at reduced pressure, diluted with chloroform and
washed with 10% HCl and water. After evaporation of
the organic layer, the residue was crystallised from
isopropanol (Tables 2 and 3).
§
See Fig. 1.
* Compound 8 is not enough soluble at the assay conditions.
a
Ref. [3]; b Ref. [5].
5.1.3. 1-Benzyl-5-methyl-1H-1,2,3-triazol-4-carboxylic
acid ethyl ester (19)
Title compound was obtained as described in the
5.1.1. General synthesis of N6-substituted 2-phenyl-9-
benzyladenines (4Á11)
/
literature [13]. M.p. 78Á
/
79 8C (lit. 76Á78 8C).
/
9-Benzyl-6-chloro-2-phenylpurine 3 [6] (0.15 g, 0.47
mmol), the suitable amine (Table 2) (6.60 mmol) and
some drops of N,N-diethylaniline were dissolved in
absolute ethanol (3 mL), and refluxed for 12 h. After
evaporation, the mixture was diluted with chloroform,
washed with 10% HCl and 5% NaHCO3 and H2O,
5.1.4. 1-Benzyl-5-methyl-1H-1,2,3-triazol-4-carboxylic
acid (20)
A mixture of 19 and 30 mL of 10% NaOH was
refluxed for 2 h; after cooling, the title compound,
precipitated by acidification to pH of 2, was filtered,
evaporated and crystallised from diethyl etherÁhexane
/
washed with water and crystallised from chloroformÁ
hexane. M.p. 168Á169 8C (lit. 168Á169 8C [15]). MS (m/
e): 217 [Mꢁ], 188, 91, 65. 1H-NMR (CDCl3): 7.38Á
7.16
/
to give the title compounds (Tables 2 and 3).
/
/
/
5.1.2. General synthesis of 9-benzyl-2-phenyl-6-
(m, 5H); 5.51 (s, 2H); 2.48 (s, 3H).
cycloalkylamino-8-azaadenines (14Á18)
/
To a mixture of 2-phenyl-9-benzyl-8-azahypoxanthine
(12) [17] (1.0 g, 3.31 mmol) and N,N-diethylaniline (0.8
g, 5.36 mmol), phosphorous oxychloride (6 mL, 65.1
mmol) was added and heated at 90 8C for 4 h. Then the
mixture was evaporated at reduced pressure and the
resulting 6-chloro-2-phenyl-9-benzyl-8-azaadenine (13)
[18] was used without purification for the substitution
reactions. In a well-stoppered flask a mixture of 13 (0.2
g, 0.62 mmol), ethanol (5 mL), the suitable amine (Table
5.1.5. 1-Benzyl-5-methyl-1H-1,2,3-triazol-4-carbonyl
chloride (21)
1-Benzyl-5-methyl-1H-1,2,3-triazol-4-carboxylic acid
(20) (3.0 g, 13.8 mmol) was refluxed in SOCl2 for 3 h.
By evaporation under reduced pressure a solid was
obtained (3.1 g, 13.3 mmol, 96% yield) which was
crystallised from chloroformÁ
/
hexane. M.p. 94Á95 8C.
/
MS (m/e): 235 [Mꢁ], 206, 200, 178, 91, 65. H-NMR
1

988
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á990
/
Table 3
1H-NMR and MS spectral data of some synthesised compounds
Compound 1H-NMR
MS (m/z, %)
Aromatic H
Benzylic
H
Aliphatic H
Exchang.
H
4
7.52Á
8.43 (m 2H)
7.56Á7.36 (m 8H), 7.84 (s 1), 8.58Á
8.53 (m 2H)
/
7.32 (m 8H), 7.77 (s 1), 8.46Á
/
5.45 (m
2H)
Á
/
5.62 (2H) 301 ([Mꢁ] 28), 91 (100)
341 ([Mꢁ] 3.5), 91 (100)
5
/
/
5.45 (m
2H)
1.02Á
2.51Á
2.16Á
1.67Á
1.67Á
/
0.86 (m 4H), 3.45 (m 1H)
1.21 (m 7H), 5.30 (m 1H)
1.73 (m 8H), 4.95 (m 1H)
0.89 (m 10H), 5.30 (m 1H)
0.89 (m 10H), 5.86 (m 1H)
Á/
6
7.58Á
2H)
7.63Á
8.53Á
7.53Á
8.49 (m 2H)
/
7.38 (m 8H), 7.89 (s 1), 8.56 (m 5.46 (m
/
5.30 (1H) 355 ([Mꢁ] 2), 300 (2) 236
(11), 91 (100)
2H)
7.43 (m 8H), 5.39 (m
7
/8.41 (s 1H), 7.60Á
/
/
5.7 (1H)
369 ([Mꢁ] 5), 300(1) 333 (13),
91(100)
/8.41 (m 2H)
2H)
5.44 (m
2H)
8
/7.32 (m 8H), 7.72(s 1H), 8.54Á
/
/
5.86 (1H) 395 ([Mꢁ] 11), 301 (16), 91
(100)
9
7.83 (m 1H), 7.58Á/7.50(s 8H), 8.54Á
/
5.44 (m
2H)
/
6.15 (1H) 395 ([Mꢁ] 11), 301 (16), 91
(100)
8.49 (m 2H)
10
11
14
15
16
17
18
27
28
29
30
31
32
33
34
7.51Á
8.52 (m 2H)
8.55Á8.50 (m 2H), 7.71 (s 1H), 7.46Á
7.36 (m 8H)
8.59Á8.54 (m 2H), 7.58Á
7.31 (m 3H)
8.55Á8.52 (m 2H), 7.53Á
7.37Á7.34 (m 3H)
8.54Á8Á51 (m 2H), 7.51Á
/7.37 (m 8H), 7.75 (s 1H), 8.56Á
/
5.44 (m
2H)
1.65 (m 10H), 2.19Á
(m 1H)
/
2.13 (m 2H), 4.60 4.91 (1H) 397 ([Mꢁ] 5), 301 (32), 91
(100)
2.03 (m 2 H), 4.64 5.75 (1H) 411([Mꢁ] 3), 301(25), 91
(100)
/
/
5.43 (m
2H)
1.66 (m 12H), 2.13Á
(m 1H)
/
/
/
7.53 (m 5H), 5.84 (s
1.16Á
/
0.80 (m 4H) 3.68 (m 1H)
6.30 (1H) 342 ([Mꢁ] 17), 315 (2), 91
(100)
2H)
7.50 (m 5H), 5.81 (s
2H)
/
/
1.94 (m 2H), 2.20 (m 2H), 2.60 (m 2H), 6.32 (1H) 328 ([Mꢁ]ꢀ
4.97 (m 1H) 91 (100)
/28 49), 300 (5.6),
/
/
/
/
7.30 (m 8H) 5.80 (s
2H)
2.80 (m 1H), 2.34 (m 2H), 1.65Á
(m 7H), 4.66 (m 1H)
/
1.00 6.25 (1H) 396 ([Mꢁ] 10), 339 (4), 273
(15), 91 (100)
1.58 (m 10H), 2.20 (m 2H), 4.560 6.21 (1H) 398 ([Mꢁ] 2.8), 302 (18) 273
8.56Á
7.33Á
8.60Á
/
8.53 (m 2H), 7.52Á
7.31 (m 3H)
8.57 (m 2H), 7.64Á
/
7.49 (m 5H), 5.81 (s
1.75Á
(m 1H)
1.70Á1.55 (m 12H), 2.10Á
5.10 (m 1H)
/
/
2H)
7.37 (m 8H) 5.83 (s
(36), 91 (100)
2.04 (m 2H), 6.22 (1H) 412 ([Mꢁ] 10), 302 (19) 273
(42), 91 (100)
/
/
/
/
2H)
7.31 (m 8H), 5.80 (s
2H)
7.91Á
6.93 (s 1H)
8.03Á7.99 (m 2H), 7.48Á
6.91 (s 1H)
7.99Á7.95 (m 2H), 7.48Á
6.87 (s 1H)
8.01Á7.96 (m 2H), 7.44Á
6.87 (s 1H)
7.99Á7.94 (m 2H), 7.49Á
6.86 (s 1H)
7.99Á7.94 (m 2H), 7.50Á
6.86 (s 1H)
8.00Á7.97 (m 2H), 7.48Á
6.86 (s 1H)
8.00Á7.97 (m 2H), 7.47Á
6.86 (s 1H)
/
7.85 (m 2H), 7.51Á
/
5.69 (1H) 301 ([Mꢁ] 10), 272 (5),
91(100)
/
/
7.30 (m 8H) 5.78 (s
3.22 (m 1H), 0.95 (m 2H), 0.75 (m 2H) 6.38 (1H) 341([Mꢁ] 7), 91(100), 77(13)
2H)
7.30 (m 8H) 5.77 (s
2H)
/
/
4.97 (m 1H), 2.54 (m 2H), 2.06 (m 2H), 6.01 (1H) 355 ([Mꢁ] 2), 326 (23),
1.84 (m 2H)
91(100)
1.67 5.94 (1H) 369 ([Mꢁ] 2), 301(10) 272(5),
91(100)
/
/
7.27 (m 8H), 5.77 (s
2H)
7.30 (m 8H) 5.77 (s
4.78 (m 1H), 2.21 (m 2H), 1.79Á
(m 6H)
/
/
/
4.64 (m 1H), 2.32Á
/
0.85 (m 10H)
5.96 (1H) 395 ([Mꢁ] 15.4), 366(12)
301(30), 91(100)
2H)
7.30 (m 8H) 5.77 (s
2H)
/
/
4.35 (m 1H), 2.21 (m 2H), 1.87Á
(m 8H)
/
1.26 5.81 (1H) 383 ([Mꢁ] 6), 301(72)
272(28.8) 91(100)
/
/
7.30 (m 8H) 5.77 (s
2H)
4.57 (m 1H), 2.19 (m 2H), 1.67Á
(m 10H)
/
1.60 5.82 (1H) 397([Mꢁ] 7), 301(81) 272(22),
91(100)
/
/
7.30 (m 8H) 5.77 (s
2H)
4.61 (m 1H), 2.18Á
/
1.57 (m 14H)
5.82 (1H) 411([Mꢁ] 2), 301(14) 272(4),
91(100) 77(6)
(CDCl3): 7.38Á
Anal.: C,H,N.
/
7.16 (m, 5H); 5.52 (s, 2H); 2.45 (s, 3H).
mmol, 92% yield). M.p. 134Á
/
135 8C. MS (m/e): 230
[Mꢁ], 201, 91, 41. H-NMR (CDCl3): 7.30Á
7.12 (m,
5Hꢁ1H exchangeable); 5.47 (s, 2H); 2.97 (d, 3H); 2.49
(s, 3H). Anal.: C,H,N.
1
/
/
5.1.6. 1-Benzyl-5-methyl-4-(N-methyl)-carboxamido-
1H-1,2,3-triazole (22)
To an iced solution of 21 (2.9 g, 12.7 mmol) in THF
(50 mL), 40% aqueous methylamine (25 mL) was added
slowly. After stirring for a day at room temperature
(r.t.), the mixture was concentrated at reduced pressure,
then filtered and the solid washed with water and
5.1.7. 1-Benzyl-4-(N-methyl)-carboxamido-5(1-phenyl-
1-iminoethyl)-1H-1,2,3-triazole (23)
To a stirring solution of 22 (2.0 g, 8.68 mmol) and
benzonitrile (1.1 g, 0.01 mol) in anhydrous THF (50
mL), under nitrogen and cooled to ꢀ
2M LDA was added. Stirring was maintained for 30 min
at ꢀ74 8C, then the mixture was allowed to reach r.t.
/
74 8C, 10 mL of
crystallised from chloroformÁ
/
hexane. The pure title
compound was obtained with high yield (2.7 g, 11.8
/

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á
/990
989
After evaporation at reduced pressure, the residue was
dissolved in CH2Cl2 and the solution was washed with
saturated NH4Cl, H2O and evaporated. The orange oil,
5.1.11. 1-Benzyl-4-amino-6-phenyl-1H-1,2,3-triazol[4,5-
c]pyridine (27)
An iced solution of 26 (0.1 g, 0.31 mmol) in a mixture
treated at reflux with petroleum ether 40Á
/
60 to dissolve
the unreacted benzonitrile, was repeatedly crystallised
of 1,2-dimethoxyethaneÁH2O (1:1) was saturated with
/
gaseous NH3, then heated in a steel bomb at 140 8C for
36 h. After cooling, 5% HCl was added dropwise to the
mixture. The solid obtained was filtered, washed with
from toluene and finally from ethanol to give pure 23
(0.43 g, 1.35 mmol, 15% yield). M.p. 183Á
/
185 8C. MS
(m/e): 375 [Mꢁ], 234. H-NMR (CDCl3): 8.00 (s, 1H,
exc.); 7.97 (s, 1H, exc.); 7.61Á7.11 (m, 10H); 5.62 (s, 2H);
1
H2O and crystallised from 1,2-dimethoxyethaneÁH2O
/
/
to give 0.63 g (0.21 mmol, 67% yield) of the title
compound (Tables 2 and 3).
4.72 (s, 2H); 2.98 (d, 3H). Anal.: C,H,N.
5.1.12. General synthesis of 1-benzyl-4-cycloalkylamino-
6-phenyl-1H-1,2,3-triazol[4,5-c]pyridine (28Á34)
/
5.1.8. 1-Benzyl-5-methyl-1H-4-(N,N-diethyl)-
carboxamido-1,2,3-triazole (24)
To a solution of 26 (0.31 mmol) in 1,2-dimethox-
yethane, the suitable amine (Table 2) (2.0 mmol) was
added, then heated in a steel bomb at150 8C for 24 h.
After cooling, 20 mL of 5% HCl was added dropwise to
the mixture. The solid obtained was filtered, washed
To an iced solution of 21 (3.0 g, 12.7 mmol) in
CH2Cl2, N,N-diethylamine (5.0 g, 33.7 mmol) was
slowly added. The mixture was refluxed for 2 h, then
cooled and repeatedly washed with 5% HCl. The
organic layer was evaporated to give an oil which was
with H2O and crystallised from 1,2-dimethoxyethaneÁ
H2O (Tables 2 and 3).
/
flash-chromatographed using as eluent CHCl3ÁMeOH
/
99:1. The pure title compound was obtained as an oil
(2.96 g, 10.9 mmol, 85% yield). MS (m/e): 272 [Mꢁ],
5.2. Biochemistry
1
201, 91. H-NMR (CDCl3): 7.41Á/7.17 (m, 5H); 5.50 (s,
5.2.1. A1 receptor binding
2H); 3.84 (q, 2H); 3.53 (q, 2H); 2.42 (s, 3H); 1.32 (t, 3H);
1.24 (t, 3H). Anal.: C,H,N.
Bovine cerebral cortex was homogenised in ice-cold
0.32 M sucrose containing protease inhibitors, as
previously described [19]. The homogenate was centri-
fuged at 1000ꢂ
again centrifuged at 48 000ꢂ
final pellet was dispersed in 10 volumes of fresh buffer,
incubated with adenosine deaminase (2 U mLꢀ1) to
remove endogenous adenosine at 37 8C for 60 min and
/
g for 10 min at 4 8C and the supernatant
g for 15 min at 4 8C. The
5.1.9. 1-Benzyl-6-phenyl-1H-1,2,3-triazol[4,5-c]pyridin-
4-oxo (25)
To a stirring solution of 24 (3.0 g, 11.0 mmol) and
benzonitrile (1.25 g, 12.1 mmol) in anhydrous THF (20
/
mL), under nitrogen and cooled to ꢀ70 8C, 8 mL of 2
/
then recentrifuged at 48 000ꢂg for 15 min at 4 8C. The
/
M LDA was added. Then the mixture was allowed to
reach r.t. and stirring was maintained overnight. After
evaporation at reduced pressure, the oily residue was
dissolved in CH2Cl2 (200 mL) and the solution was
washed with saturated NH4Cl and concentrated to 15
mL, obtaining a solid which was filtered (2.05 g, 6.78
pellet was suspended in buffer and used in the binding
assay.
The [3H]CHA binding assay was performed in
triplicate by incubating aliquots of the membrane
fraction (0.2Á
0.5 mL of TrisÁ
/
0.3 mg of protein) at 25 8C for 45 min in
HCl, pH 7.7, containing 2 mM MgCl2,
/
with approximately 1.2 nM [3H]CHA. Nonspecific
binding was defined in the presence of 50 mM R-PIA.
Binding reactions were terminated by filtration through
Whatman GF/C filters under reduced pressure. Filters
were washed three times with 5 mL of ice-cold buffer.
mol, 62% yield) and crystallised from ethanol. M.p.
1
240Á
NMR (CDCl3): 9.43 (s, 1H, exc.); 7.47Á
6.30 (s, 1H); 5.71 (s, 2H). Anal.: C,H,N.
/
242 8C. MS (m/e): 302 [Mꢁ], 273, 91, 77, 65. H-
/
7.22 (m, 10H);
5.1.10. 1-Benzyl-4-chloro-6-phenyl-1H-1,2,3-triazol[4,5-
c]pyridine (26)
5.2.2. A2A receptor binding
Bovine striatum was homogenised in 20 volumes of
A solution of 25 (0.2 g, 0.66 mmol) and POCl3 (1 mL)
was refluxed for 30 min. To the mixture, iced and
stirred, ethanol (15 mL) and then H2O (30 mL) were
slowly added. The solid obtained was filtered, washed
ice-cold 50 mM TrisÁ
MgCl2 and protease inhibitors. The membrane homo-
genate was centrifuged at 48 000ꢂg for 10 min at 4 8C.
The resulting pellet was resuspended in buffer contain-
ing 2 U mLꢀ1 of adenosine deaminase and incubated at
37 8C for 30 min. The membrane homogenate was
/
HCl, pH 7.5, containing 10mM
/
with H2O and crystallised from 1,2-dimethoxyethaneÁ
H2O to give 0.19 g (0.61 mmol, 92% yield) of the title
compound. M.p. 173Á
174 8C. MS (m/e): 320 [Mꢁ], 291,
257, 91, 77, 65. 1H-NMR(CDCl3): 7.97 (d, 1H); 7.93 (d,
1H); 7.49Á7.31 (m, 9H); 5.91 (s, 2H). Anal.: C,H,N.
/
/
centrifuged, and the final pellet frozen at ꢀ
Routine assays were performed in triplicate by incuba-
tion an aliquot of striatal membranes (0.2Á0.3 mg of
/
80 8C.
/
/

990
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 983Á990
/
[3] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, C. Martini, R. Scatizzi,
Farmaco 50 (1995) 659Á667.
[4] G. Biagi, M. Franchi, I. Giorgi, O. Livi, V. Scartoni, J.
Heterocyclic Chem. 26 (1989) 39Á43.
[5] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, A. Lucacchini, Farmaco
51 (1996) 395Á399.
protein) in cold 50 mM TrisÁHCl, pH 7.5, containing 10
/
mM MgCl2 with approximately 5 nM [3H]CGS 21680 in
a final volume of 0.5 mL. Incubation was carried out for
90 min at 25 8C. Non-specific binding was defined in the
presence of 50 mM CGS 21680. Binding reactions were
terminated by filtration through Whatman GF/C filters
under reduced pressure. Filters were washed three times
with 5 mL of ice-cold buffer and placed in scintillation
vials. The radioactivity was counted in a 4 mL Beckman
Ready-Protein scintillation cocktail in a scintillation
counter. The compounds were dissolved in DMSO and
added to the assay mixture to make a final volume of 0.5
mL. Blank experiments were carried out to determine
the effect of the solvent (2%) on binding. The concen-
trations of the tested compounds to produce 50%
inhibition of specific [3H]CHA or [3H]CGS 21 680
binding (IC50) were determined from semilog plots of
data from experiments of binding inhibition. The Ki
values were calculated from the IC50 values using the
/
/
/
[6] A.M. Bianucci, G. Biagi, A. Coi, I. Giorgi, O. Livi, F. Pacchini,
V. Scartoni, A. Lucacchini, B. Costa, Drug Dev. Res. 54 (2001)
52Á
[7] G. Cristalli, M. Grifantini, S. Vittori, W. Balduini, F. Cattabeni,
Nucleosides Nucleotides 4 (1985) 625Á639.
/
65.
/
[8] S. Vittori, A. Lorenzen, C. Stannek, S. Costanzi, R. Volpini, A.P.
Ijzerman, J.K. Von Frijtag Drabbe Kunzel, G. Cristalli, J. Med.
Chem. 43 (2000) 250Á
[9] J.L. Kelley, C.S. Koble, R.G. Davis, E.W. McLean, F.E. Soroko,
B.R. Cooper, J. Med. Chem. 38 (1995) 4131Á4134.
[10] C.A. Salemink, G.M. van der Want, Recl. Trav. Chim: Pays-Bas
68 (1949) 1013Á1029.
[11] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, A. Lucacchini, C.
Martini, P. Tacchi, Farmaco 49 (1994) 187Á191.
[12] O. Bremer, Justus Liebigs Ann. Chim. 539 (1939) 276Á
[13] I.F. Cottrel, D. Hands, P.G. Houtghton, G.R. Humphrey, S.H.B.
Wright, J. Heterocyclic Chem. 28 (1991) 301Á304.
[14] L. von Wolff, R. Kruche, Justus Liebigs Ann. Chem. 394 (1912)
/260.
/
/
/
/
296.
/
equation IC50/(L/Kd) [20] ([3H]CHA Kdꢃ
/
10.5 nM and
5 nM);
¨
Lꢃ 1 nM and Lꢃ
/
1.2 nM; [3H]CGS 21 680 Kdꢃ
/
/
48Á
[15] G.S. Pointdexter, J. Org. Chem. 47 (1982) 3787Á
[16] W. Mitsuaki, S. Masanori, K. Masaki, F. Snao, R.J. Billedeau, V.
Snieckus, J. Org. Chem. 49 (1984) 742Á747.
[17] P.L. Barili, G. Biagi, O. Livi, V. Scartoni, J. Heterocyclic Chem.
22 (1984) 1607Á1610.
[18] G. Biagi, I. Giorgi, F. Pacchini, O. Livi, V. Scartoni, Farmaco 56
(2001) 929Á931.
[19] C. Martini, M. Poli, A. Lucacchini, Bull. Mol. Biol. Med. 11
(1986) 1Á10.
[20] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099Á
/
59.
/3788.
protein estimation was based on the method
reported [21] using bovine serum albumin as
standard.
/
/
/
References
/
[1] A.K. Dhalla, J.C. Shryock, R. Shreeniwas, L. Belardinelli, Curr.
/
Top. Med. Chem. 3 (2003) 369Á
[2] B.B. Fredholm, I.J.zerman A.P. Jacobson, K.A. Klotz, K.N.
Linden, J. Pharmacol. Rev. 53 (2001) 527Á552.
/
385.
3108.
[21] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, J. Biol.
/
Chem. 193 (1951) 265Á275.
/