Novel 3-aralkyl-7(amino-substituted)-1,2,3-triazolo[4,5-d]pyrimidines with high affinity toward A1 adenosine receptors

Article abstract of DOI:10.1021/jm9701334

Three series of several 1,2,3-triazolo[4,5-d]pyrimidine derivatives bearing various amino substituents at the 7 position and one of three lipophilic substituents at the 3 position (benzyl, phenethyl, or 2- chlorobenzyl) were prepared starting from the corresponding 7-chloro compounds, by nucleophilic substitution by the appropriate amine. Radioligand binding assays at bovine brain adenosine A₁ and A(2A) receptors showed that some compounds possessed a high affinity and selectivity for the A₁ receptor subtype. In particular the biological results suggested the compounds bearing cycloalkylamino (cyclopentyl- and cyclohexylamino) or aralkylamino (α- methylbenzyl- and 1-methyl-2-phenylethylamino or amphetamino) substituents at the 7 position were the most active derivatives. The best lipophilic substituent at the 3 position was the 2-chlorobenzyl (A₁ affinity K(i) < 50 nM) followed by the benzyl and then the phenethyl groups. This pattern of structure-activity relationship (SAR) was similar to that previously reported for analogous 1,2,3-triazolopyridazino derivatives (Biagi et al., 1994, 1995, 1996) except for the compounds bearing substituted aromatic amines which presented a generalized and strong decrease of the A₁ receptor affinity. These facts allowed us to attribute to these molecules a binding mode within the A₁ adenosine receptor analogous to that of the corresponding triazolopyridazines.

Full text of DOI:10.1021/jm9701334

668
J . Med. Chem. 1998, 41, 668-673
Novel 3-Ar a lk yl-7-(a m in o-su bstitu ted )-1,2,3-tr ia zolo[4,5-d ]p yr im id in es w ith High
Affin ity tow a r d A1 Ad en osin e Recep tor s
Laura Betti,§ Giuliana Biagi,† Gino Giannaccini,§ Irene Giorgi,† Oreste Livi,*,† Antonio Lucacchini,§
Clementina Manera,† and Valerio Scartoni†
Dipartimento di Scienze Farmaceutiche and Istituto Policattedra di Discipline Biologiche, Facolta` di Farmacia,
Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy
Received March 4, 1997
Three series of several 1,2,3-triazolo[4,5-d]pyrimidine derivatives bearing various amino
substituents at the 7 position and one of three lipophilic substituents at the 3 position (benzyl,
phenethyl, or 2-chlorobenzyl) were prepared starting from the corresponding 7-chloro com-
pounds, by nucleophilic substitution by the appropriate amine. Radioligand binding assays at
bovine brain adenosine A1 and A2A receptors showed that some compounds possessed a high
affinity and selectivity for the A1 receptor subtype. In particular the biological results suggested
the compounds bearing cycloalkylamino (cyclopentyl- and cyclohexylamino) or aralkylamino
(R-methylbenzyl- and 1-methyl-2-phenylethylamino or amphetamino) substituents at the 7
position were the most active derivatives. The best lipophilic substituent at the 3 position
was the 2-chlorobenzyl (A1 affinity Ki < 50 nM) followed by the benzyl and then the phenethyl
groups. This pattern of structure-activity relationship (SAR) was similar to that previously
reported for analogous 1,2,3-triazolopyridazino derivatives (Biagi et al., 1994, 1995, 1996) except
for the compounds bearing substituted aromatic amines which presented a generalized and
strong decrease of the A1 receptor affinity. These facts allowed us to attribute to these molecules
a binding mode within the A1 adenosine receptor analogous to that of the corresponding
triazolopyridazines.
In tr od u ction
In the past, with the aim of obtaining potent A1
antagonists, we synthesized several 8-azaadenines (1,2,3-
triazolo[4,5-d]pyrimidines) easily prepared providing
flexibility of substitution at C2 , N6, and N9 positions.18-23
This rational study, aimed at discovering the best
substituents in the three different positions, led to the
preparation, among others, of 2-phenyl-N6-cyclohexyl-
N9-benzyl-8-azaadenine (Ki 1.6 nM)24 and was inter-
preted as evidence for the presence of three lipophilic
pockets in the A1 receptor site.25-30 The phenyl group
on C2, the substitution of the ribosyl moiety on N9 with
a lipophilic group (e.g., benzyl group), and a lipophilic
group on N6 concurred to increase A1 affinity and
selectivity among 8-azaadenines as antagonists. Ac-
cording to this finding we hypothesized the possible dual
mode of binding of the bound exogenous molecule inside
the A1 receptor, sterically related by rotation around an
ideal C2-N7 axis of the 8-azaadenine nucleus.25 Other
similar results concerning the arrangement of some
antagonist molecules in the A1 receptor subtype have
been also reported separately by Mu¨ller et al.31
A wide variety of actions of the local modulator
adenosine in the nervous, cardiovascular, renal, im-
mune, and other systems is mediated by adenosine
receptors.1 Up to now four subtypes of adenosine
receptors (A1, A2A, A2B, A3) have been defined on the
basis of pharmacological distinctions and on molecular
cloning and characterization.2-6
The receptor subtype A2A exhibits high affinity for
adenosine in the low-nanomolar range, while the sub-
type A2B is a low-affinity receptor in the low-micromolar
range. Activation of A1 and A3 adenosine receptors can
lead to an inhibition of adenylate cyclase activity, while
on the contrary the cyclase can be stimulated by the
activation of A2A and A2B adenosine receptors.
All of the adenosine receptor agonists synthesized
thus far are structurally related to adenosine itself.1,7,8
In fact it is known that compounds which present the
ribose moiety mainly intact are agonist,1 whereas the
substitution of the ribose moiety with a lipophilic group
leads to products which act as antagonists.9-12
Recent studies concerning 7-hydroxy-1,2,3-triazolo-
[4,5-d]pyridazines, bearing lipophilic substituents in the
1 position and amino substituents in the 4 position of
the heterocycle, showed high affinity and selectivity
toward the adenosine A1 receptor.32-35 As in the N6-
substituted adenosines,36 the structure-activity rela-
tionship (SAR) analysis of these compounds required a
hydrogen atom on N4 together with a lipophilic sub-
stituent such as an unsubstituted cycloalkyl, meta- or
para-monosubstituted aryl, R-methylbenzyl, or 1-meth-
ylphenethyl one. Compounds bearing a chiral substitu-
ent on N4 had shown a stereoselective effect. The R
Many selective agonists or antagonists have shown
promise as potential therapeutic agents for the treat-
ment of cognitive disease, renal failure, Alzheimer’s
disease, and cardiac arrhythmias (A1) or Parkinson’s
disease,13-15 Huntington’s chorea, schizophrenia, my-
asthenia gravis, and myastenic syndromes (A2A).16 Few
selective and/or high-affinity antagonists for A2B and A3
receptor subtypes have been reported.17
* Author for correspondence and reprint requests.
† Dipartimento di Scienze Farmaceutiche.
§ Istituto Policattedra di Discipline Biologiche.
S0022-2623(97)00133-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/30/1998

3-Aralkyl-7-(amino-substituted)-1,2,3-triazolo[4,5-d]pyrimidines
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5 669
Sch em e 1a
1,2,3-triazolo[4,5-d]pyridazines which had been found
previously to be biologically active. In addition some
other new derivatives, bearing the same substituents
in the 3 position but previously unknown groups in the
7 position, were also prepared.
The 7-(amino-substituted)-3-benzyl-1,2,3-triazolo[4,5-
d]pyrimidine derivatives are reported in the Supporting
Information (Table 1); the 1,2,3-triazole 1a 38 and the
triazolopyrimidine intermediates, 7-hydroxy 2a 39 and
7-chloro 3a ,40 have already been described in the
literature. The 4a -17a derivatives were obtained ac-
cording to the general procedure, starting from the
chloroazapurine 3a and the appropriate primary amine;
compounds 18a and 19a were obtained by reduction of
the corresponding nitro derivatives 16a and 17a with
hydrazine hydrate. 3-Benzyl-substituted compounds
(series a ) 4-6, 8-10, and 12-15 were analogues of the
triazolopyridazines previously described.
Fewer than in the series a , 3-phenethyl-1,2,3-triazolo-
[4,5-d]pyrimidine derivatives (Table 2 in Supporting
Information) were prepared, because of the low biologi-
cal activity of the 1-phenethyl-substituted triazolopy-
ridazines. The 1-phenethyl-4-carbamoyl-5-amino-1H-
1,2,3-triazole (1b) has been described in the literature,41
while the 8-azapurines 2b and 3b were prepared in the
usual manner. The 3-phenethyl-substituted compounds
(series b) 4, 6, 10, and 11 were analogues of the
previously reported triazolopyridazines.34
The 3-(2-chlorobenzyl)-1,2,3-triazolo[4,5-d]pyrimidines
(series c), analogous to the 1-(2-chlorobenzyl)-1,2,3-
triazolo[4,5-d]pyridazines35 which were previously found
to provide the highest affinity, are reported in the
Supporting Information (Table 3). Thus, starting from
2-chlorobenzyl azide,42 according to the usual synthetic
route (Scheme 1), the 1,2,3-triazole compound 1c43 and
the triazolopyrimidine intermediates 2c and 3c were
prepared in good yield.
a
(a) CNCONH2; (b) HCONH2; (c) SOCl2, CHCl3, DMF; (d)
R1NH2, HMDS; (e) R1NH2, TEA, EtOH.
configuration both in N4-R-methylbenzyl and in N4-1-
methyl-2-phenylethyl led to the more effective stereo-
isomer, as in the selective A1 agonist R-PIA.36b,c The
best lipophilic substituent at the 1 position was the
2-chlorobenzyl, which assured the greatest affinity
compared with the benzyl and phenethyl ones. These
results confirmed the presence inside the adenosine A1
receptor of a lipophilic pocket with well-defined dimen-
sions facing the N1 position of the 1,2,3-triazolo[4,5-d]-
pyridazine nucleus. To investigate the mode of binding
of these compounds with the A1 receptor, we undertook
the synthesis and biological evaluation of new 1,2,3-
triazolo[4,5-d]pyrimidines (8-azapurines) as analogues
of the 1,2,3-triazolo[4,5-d]pyridazines quoted above. The
comparison of the SAR analysis of such compounds
should allow the understanding of the similarities
between the possible mode of binding with the receptor.
The structures of all the newly prepared compounds
were confirmed by analytical and spectroscopic data. 1H
NMR data of some selected compounds are reported in
the Supporting Information (Table 4).
Ch em istr y
Biologica l Eva lu a tion
Synthesis of the 1,2,3-triazolo[4,5-d]pyrimidine ring
and its 7-amino-substituted derivatives is illustrated in
Scheme 1. The appropriate azide, by ionic 1,3-dipolar
cycloaddition reaction with cyanacetamide, provided the
corresponding 1-substituted-4-carbamoyl-5-amino-1H-
1,2,3-triazoles 1a -c. These compounds, by heating in
an excess of formamide, were cyclized to the triazolopy-
rimidines 2a -c, which were then converted by thionyl
chloride to the corresponding reactive chloro derivatives
3a -c. Then the halogen atom was easily replaced by
nucleophilic displacement with primary amines, even
if weakly basic, in the presence of triethylamine, to
obtain the final products 4-19.
The silylation-amination reaction of aromatic hy-
droxy N-heterocycles37 using hexamethyldisilazane and
6-hydroxy-8-azahypoxanthine (2) gave unsatisfactory
results, contrary to the results obtained with 1,2,3-
triazolo[4,5-d]pyridazines.33-35
The choice of amines, used for the nucleophilic
displacement reaction, and that of azides, for the
cycloaddition reaction, to introduce the lipophilic groups
on C7 and C3, respectively, were based in part on the
The 7-(amino-substituted)-1,2,3-triazolo[4,5-d]pyrim-
idines 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-(2-Phenyliso-
propyl)adenosine (R-PIA) was used as the A1 radioligand
and [3H]-2-{[[p-(2-carboxyethyl)phenyl]ethyl]amino}-5′-
(N-ethylcarbamoyl) adenosine (CGS 21680) as the A2A
radioligand.
Resu lts a n d Discu ssion
In Table 1 are reported only the results of the A1
adenosine receptor binding assay, expressed as inhibi-
tion constants (Ki, nM), for the three series [a , 3-benzyl;
b, 3-(2-phenylethyl); c, 3-(2-chlorobenzyl)] of 7-(amino-
substituted)-1,2,3-triazolo[4,5-d]pyrimidine derivatives.
In fact the binding assay at adenosine A2A receptors
showed that these compounds presented very low
inhibition percentages at 1 µM, so that the correspond-
ing Ki values were not calculated for the majority of the
compounds. The more effective compound toward A2A

670 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Betti et al.
Ta ble 1. A1 Adenosine Receptor Binding [Ki (nM) ( SEM] of
1,2,3-Triazolo[ 4,5-d]pyrimidines 4a -19a , 4b-13b, and
4c-18ca
mixture (14c: Ki ) 73 nM). For 15c and 14c the
receptor selectivity (Ki A2A/Ki A1) was >250 and 46,
respectively.
The 3-benzyl derivatives 16a -19a and the 3-(2-
chlorobenzyl) derivatives 16c-18c possessed low recep-
tor affinity. Instead the 3-(2-phenylethyl) derivative
13b showed an appreciable affinity (Ki ) 245 nM).
Compounds 8a -11a , 6b-9b, and 8c-11c, bearing an
aromatic amino substituent at C7, were less potent at
A1 receptors than the corresponding triazolopyridazine
derivatives.32-35 In addition, contrary to these last
compounds, the 7-toluidino-substituted triazolopyrim-
idines showed that the para substitution on the aro-
matic ring (compounds 8a , 6b, and 8c) appeared clearly
more effective than substitution at the meta position
(compounds 9a , 7b, and 9c). But the contribution to
the effectiveness of the molecules shown by the lipophilic
substituents on N3 followed the known trend: 2-chlo-
robenzyl > benzyl > 2-phenylethyl, only for the 7-p-
toluidino derivatives 8a , 6b, and 8c. Considering the
previous triazolopyridazine derivatives, we had pre-
ferred, at the start of this work, the meta substitution,
so that other new derivatives (10a , 11a , 8b, 9b, 10c,
and 11c) with a m-nitro- or m-chloro-substituted anilino
group in the 7 position were prepared. In these cases
the best lipophilic substituent in the 3 position of the
heterocycle was the benzylic group.
The observed change in affinity among compounds
bearing C7 meta-substituted anilino groups would ap-
pear to depend on inductive and/or mesomeric effects;
in fact in the benzyl (a ) and 2-phenylethyl (b) series the
nitro group (10a and 8b) was slightly better than the
chloro atom (11a and 9b), which was in turn better than
the methyl group (9a and 7b). Finally introduction of
a phenylhydrazino substituent in the 7 position (12a ,
10b, and 12c) caused a strong decrease of the receptor
binding affinity.
a
The tests were carried out dissolving the compounds in DMSO
(DMSO/buffer, 2%) unless otherwise indicated. The Ki values are
means ( SEM of four separate assays, each performed in triplicate.
receptors was 13a , which showed an affinity constant
of Ki ) 1750 nM.
Compounds 4 and 5 had low Ki values with a moder-
ate preference for the cyclopentyl (5a -c) over the
cyclohexyl (4a -c) group; the most potent compounds
were 5c (Ki ) 21 nM) and 4c (Ki ) 43 nM), with a
receptor selectivity (Ki A2A/Ki A1) corresponding to 215
and 230, respectively. A comparison of these results
showed that the A1 affinity increased with changes in
the lipophilic substituent at the 3 position of the
heterocycle, according to the sequence: 2-chlorobenzyl
> benzyl > 2-phenylethyl.
Introduction of a methyl group at the 3 position of
the cyclohexyl ring (compounds 6a ,c), chosen by analogy
to the m-toluidino substituent of the active triazolopy-
ridazine derivatives,34,35 caused a decrease in receptor
affinity, which appeared more considerable when the
cyclohexyl ring was more distant from the nitrogen atom
(compounds 7a ,c).
The aralkylamino derivatives with the (R,S)-R-meth-
ylbenzylamino substituent (13a , 11b, and 13c) and the
(R,S)-amphetamino substituent (14a , 12b, and 14c)
presented the same A1 affinity trend as for compounds
4 and 5 regarding the substituent at the 3 position of
the heterocycle. Compared with compounds bearing the
R-methylbenzyl group, the 2-chlorobenzyl derivatives
bearing the amphetamino substituent (14c and 15c)
presented the highest affinity, and the R enantiomer
(15c: Ki ) 39 nM) was more potent than the racemic
Su m m a r y
In conclusion, the best agreement between triazolo-
pyridazines and triazolopyrimidines was found with the
cyclohexylamino, cyclopentylamino, and amphetamino
groups on C4 and C7, respectively, in the two series a
and c. In addition, we observed the same enantiose-
lective effect regarding the amphetamino derivatives:
the R stereoisomer (15a ,c) was more active than the
racemic mixture (14a ,c). Our data regarding the ster-
eochemical requirement of the site facing the N7 posi-
tion when engaged by a 1-methyl-2-phenylethyl group
were in accordance with the results obtained with
R-PIA.36
The important similarities of stereoselectivity to-
gether with those concerned with the compounds bear-
ing cycloalkyl substituents, neglecting some minor
behavior differences among less active products, induced
us to consider that both triazolopyridazines and triaz-
olopyrimidines could bind to the same site in the A1
adenosine receptor.
Exp er im en ta l Section
Ch em istr y. 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 spectra were recorded with a Varian CFT-20 spectrom-
eter in δ units from TMS as an internal standard. Mass

3-Aralkyl-7-(amino-substituted)-1,2,3-triazolo[4,5-d]pyrimidines
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5 671
spectra were performed with a Hewlett-Packard MS/System
Compounds 16a and 17a crystallized from the reaction
mixture, were collected by filtration, and were triturated with
boiling EtOH (Table 1 in Supporting Information).
5988. Elemental analyses (C,H,N) were within (0.4% of the
theoretical values and were performed on
a Carlo Erba
elemental analyzer model 1106 apparatus. Optical rotations
were measured with a Violet AA-5 polarimeter.
3-P h en et h yl-7-(su b st it u t ed a m in o)-1,2,3-t r ia zolo[4,5-
d ]p yr im id in es 4b-13b. To a stirred suspension of 3-phen-
ethyl-7-chloro-1,2,3-triazolo[4,5-d]pyrimidine (3b) (0.400 g,
1.54 mmol) in 10 mL of absolute EtOH were added 0.23 mL
(1.70 mmol) of TEA and 1.70 mmol of the suitable amine (for
compound 13b, 3.40 mmol of TEA, because p-nitrobenzylamine
hydrochloride was employed). The mixture was heated under
reflux for the time reported in Table 2 (Supporting Informa-
tion). After one night the crystallized precipitate was collected
by filtration, washed with H2O and EtOH, and eventually
recrystallized (Table 2 in Supporting Information).
3-P h e n e t h yl-7-h yd r oxy-1,2,3-t r ia zolo[4,5-d ]p yr im i-
d in e (2b). A solution of 5.08 g (22.0 mmol) of 1-phenethyl-
4-carbamoyl-5-amino-1H-1,2,3-triazole (1b)41 in 24 mL of
formamide was refluxed for 2 h. After cooling the solution was
diluted with H2O, and the precipitated solid was collected by
filtration and washed with H2O: 4.73 g, yield 89%; mp 262-
264 °C (EtOH); MS 241 (M+), 150 (base peak). Anal.
(C12H11N5O) C, H, N.
3-(2-Ch lor ob en zyl)-7-h yd r oxy-1,2,3-t r ia zolo[4,5-d ]p y-
r im id in e (2c). A solution of 1.0 g (3.97 mmol) of 1-(2-
chlorobenzyl)-4-carbamoyl-5-amino-1H-1,2,3-triazole (1c)43 in
4 mL of formamide was worked up as described for the
preparation of 2b: 1.02 g, 98% yield; mp 282-285 °C (EtOH).
Anal. (C11H8N5OCl) C, H, N.
3-P h en et h yl-7-ch lor o-1,2,3-t r ia zolo[4,5-d ]p yr im id in e
(3b). To a suspension of 2b (2.0 g, 8.3 mmol) in 40 mL of
boiling anhydrous CHCl3 were added 1.5 mL of DMF and 7.0
mL of SOCl2. The reaction mixture was refluxed for 2 h, the
solvent was evaporated in vacuo (temperature < 35 °C), and
the residue, after cooling at 0 °C, was triturated with crushed
ice. The solid formed was collected by filtration, dried, and
extracted repeatedly with boiling 60-80 °C petroleum ether.
The combined extracts were evaporated in vacuo to give 3b
as a white solid: 1.64 g, 76% yield; mp 94-95 °C; MS 259 (M+),
104 (base peak). Anal. (C12H10N5Cl) C, H, N.
3-(2-Ch lor ob e n zyl)-7-ch lor o-1,2,3-t r ia zolo[4,5-d ]p y-
r im id in e (3c). To a suspension of 2c (2.26 g, 8.67 mmol) in
40 mL of boiling anhydrous CHCl3 were added 1.5 mL of DMF
and 8.2 mL of SOCl2. The reaction mixture was worked up
as described for the preparation of 3b: 1.74 g, 72% yield; mp
112-115 °C; MS 226 (M+), 125 (base peak). Anal. (C11H7N5-
Cl2) C, H, N.
3-(2-Ch lor oben zyl)-7-(cycloa lk yla m in o)-1,2,3-tr ia zolo-
[4,5-d ]p yr im id in es 4c-7c. To a stirred suspension of 3-(2-
chlorobenzyl)-7-chloro-1,2,3-triazolo[4,5-d]pyrimidine (3c) (0.400
g, 1.43 mmol) in 10 mL of absolute EtOH were added TEA
(0.18 mL, 1.70 mmol) and the suitable amine (1.70 mmol for
4c and 5c; 5.10 mmol for 6c and 7c), and the mixture was
refluxed for the time reported in Table 3 (Supporting Informa-
tion). For compounds 4c, 5c, and 6c the reaction mixture was
evaporated in vacuo, the liquid residue was treated with H2O
and 10% HCl (pH = 3), and the solid formed was collected
and crystallized (Table 3 in Supporting Information). Com-
pound 7c crystallized from reaction mixture and was collected
after one night (Table 3 in Supporting Information).
3-(2-Ch lor oben zyl)-7-(a r yla m in o)-1,2,3-tr ia zolo[4,5-d ]-
p yr im id in es 8c-12c. To a stirred suspension of 3c (0.400
g, 1.43 mmol) in 10 mL of absolute EtOH were added TEA
(0.18 mL, 1.70 mmol, for 8c, 10c, and 11c; 0.54 mL, 5.10 mmol,
for 9c and 12c) and the suitable amine (5.10 mmol for 8c and
11c; 3.40 mmol for 9c, 10c, and 12c). The mixture was
refluxed for the time reported in Table 3 (Supporting Informa-
tion). The title compounds crystallized from the reaction
mixture and were collected after one night (Table 3 in
Supporting Information).
3-Ben zyl-7-(cycloa lk yla m in o)-1,2,3-t r ia zolo[4,5-d ]p y-
r im id in es 4a -7a . A suspension of 3-benzyl-7-chloro-1,2,3-
triazolo[4,5-d]pyrimidine (3a )40 (0.400 g, 1.63 mmol), TEA (0.25
mL, 1.80 mmol), and 1.80 mmol of the appropriate cycloalkyl-
amine in 10 mL of absolute EtOH was heated under reflux
for the time reported in Table 1 (Supporting Information). For
compounds 4a , 5a , and 6a , the reaction mixture was evapo-
rated in vacuo and the residue was triturated with H2O and
10% HCl (pH = 4), collected by filtration, washed with EtOH,
and crystallized (Table 1 in Supporting Information). Com-
pound 7a crystallized from the reaction mixture (Table 1 in
Supporting Information).
3-Be n zyl-7-(a r yla m in o)-1,2,3-t r ia zolo[4,5-d ]p yr im -
id in es 8a -12a . A suspension of 3a (0.400 g, 1.63 mmol), TEA
(0.25 mL, 1.80 mmol), and 1.80 mmol of the appropriate
arylamine in 10 mL of absolute EtOH was refluxed for the
time reported in Table 1 (Supporting Information). The title
compounds crystallized from the reaction mixture, were col-
lected by filtration, were washed with H2O and EtOH, and
eventually recrystallized (Table 1 in Supporting Information).
3-Ben zyl-7-(a r a lk yla m in o)-1,2,3-tr ia zolo[4,5-d ]p yr im -
id in es 13a -17a . A suspension of 3a (0.400 g, 1.63 mmol),
TEA (0.25 mL, 1.80 mmol, for 13a -15a ; 0.50 mL, 3.60 mmol,
for 16a and 17a , because amino hydrochlorides were used),
and 1.80 mmol of the appropriate aralkylamine in 10 mL of
absolute EtOH was refluxed for the time reported in Table 1
(Supporting Information). For compounds 13a -15a the reac-
tion mixture was evaporated in vacuo to give an oily residue.
For 13a the residue was dissolved in CHCl3, and the solution,
after washing with 10% HCl and H2O, was evaporated to give
13a as an oil (77% yield) which was converted to solid
hydrochloride (Table 1 in Supporting Information). For 14a
the residue was treated with 10% HCl (pH = 4), and the solid
formed was collected by filtration and crystallized (Table 1 in
Supporting Information). For 15a the residue was dissolved
in AcOEt/60-80 °C petroleum ether mixture and converted
to solid hydrochloride (Table 1 in Supporting Information).
3-(2-Ch lor oben zyl)-7-(a r a lk yla m in o)-1,2,3-tr ia zolo[4,5-
d ]p yr im id in es 13c-17c. To a stirred suspension of 3c (0.400
g, 1.43 mmol) in 10 mL of absolute EtOH were added TEA
(0.18 mL,1.70 mmol, for 13c, 14c, and 15c; 0.54 mL, 5.10
mmol, for 16c and 17c) and the suitable amine (5.10 mmol
for 13c; 3.40 mmol for 16c and 17c as hydrochlorides; 1.70
mmol for 14c and 15c). The mixture was refluxed for the time
reported in Table 3 (Supporting Information). Compounds
13c, 16c, and 17c crystallized from the reaction mixture and
therefore were collected and recrystallized (Table 3 in Sup-
porting Information). For compounds 14c and 15c the reaction
mixture was evaporated in vacuo, and the residue was
triturated with H2O and 10% HCl (pH ) 3-4); the acid
solution was decanted and the residue washed with H2O and
dried. For 14c the residue was dissolved in AcOEt, 60-80 °C
petroleum ether was added, and the mixture cooled at -20
°C; the precipitated solid was collected and recrystallized
(Table 3 in Supporting Information). For 15c the residue was
extracted with portions of boiling 60-80 °C petroleum ether,
and the combined extracts were evaporated. The oily residue
was purified by flash chromatography (230-400 mesh silica
gel column, 14 × 2 cm) eluting with 1:3 AcOEt/40-60 °C
petroleum ether mixture. Compound 15c was isolated as a
viscous oil which was converted to solid hydrochloride (Table
3 in Supporting Information).
3-Ben zyl-7-[(p-a m in oben zyl)a m in o]-1,2,3-tr ia zolo[4,5-
d ]p yr im id in e (18a ) a n d 3-Ben zyl-7-[(p-a m in op h en eth yl)-
a m in o]-1,2,3-tr ia zolo[4,5-d ]p yr im id in e (19a ). To a stirred
and boiling solution of 0.70 mmol of the nitro derivative 16a
or 17a in 5 or 10 mL of EtOH, respectively, was added 50-
100 mg of Raney nickel, and successively a solution of 99%
hydrazine hydrate (0.15 mL, 3.20 mmol) in 3 mL of EtOH was
dripped. The refluxing was continued for 1 h; then the catalyst
was filtered off and washed with boiling EtOH. The combined
filtrates were concentrated in vacuo until the title compounds
crystallized (Table 1 in Supporting Information).

672 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Betti et al.
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3-(2-Ch lor oben zyl)-7-[(p-a m in oben zyl)a m in o]-1,2,3-tr i-
a zolo[4,5-d ]p yr im id in e (18c). A stirred suspension of 16c
(0.505 g, 1.27 mmol) and 99% hydrazine hydrate (0.18 mL,
1.27 mmol) in 10 mL of 1,2-dichloroethane-EtOH (1:1)
mixture was heated at 30 °C for 20 min; =100 mg of Raney
nickel was added and the suspension heated at 40 °C for 10
h. The catalyst was filtered off, and the filtrate was evapo-
rated in vacuo. The crude solid residue (0.340 g) was purified
by flash chromatography (230-400 mesh silica gel column, 14
× 2 cm) eluting with AcOEt/40-60 °C petroleum ether (2:3)
mixture (Table 3 in Supporting Information).
Bioch em ica l Assa ys: A1 Recep tor Bin d in g. Bovine
cerebral cortex was homogenized in ice-cold 0.32 M sucrose
containing protease inhibitors, as previously described.44 The
homogenate was centrifuged at 1000g for 10 min at 4 °C and
the supernatant again centrifuged at 48000g for 15 min at 4
°C. The final pellet was dispersed in 10 volumes of fresh
buffer, incubated with adenosine deaminase (2 units/mL) to
remove endogenous adenosine at 37 °C for 60 min, and then
recentrifuged at 48000g for 15 min at 4 °C. The 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 (0.2-0.3 mg of
protein) at 25 °C for 45 min in 0.5 mL of Tris-HCl, pH ) 7.7,
containing 2 mM MgCl2, with approximately 1.2 nM [3H]CHA.
Nonspecific binding was defined in the presence of 50 µM
R-PIA. The assay was completed by filtration through What-
man GF/C glass microfiber filters under suction and washing
twice with 5 mL of ice-cold buffer.
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of 2-substituted 9-(2′-hydroxy-3′-aminopropyl)-8-azahypoxan-
thines and 8-azaadenines (5-substituted 3-(2′-hydroxy-3′-ami-
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A
2A Recep tor Bin d in g. Bovine striatum was homogenized
in 20 volumes of ice-cold 50 mM Tris-HCl, pH ) 7.5, containing
10 mM MgCl2 and protease inhibitors. The membrane homo-
genate was centrifuged at 48000g for 10 min at 4 °C. The
resulting pellet was resuspended in buffer containing 2 units/
mL of adenosine deaminase and incubated at 37 °C for 30 min.
The membrane homogenate was centrifuged, and the final
pellet was frozen at -80 °C. Routine assays were performed
in triplicate by incubating an aliquot of striatal membranes
(0.2-0.3 mg of protein) in 50 mM Tris-HCl, 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 at 25 °C for 90 min. Nonspecific binding was defined in
the presence of 50 µM 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 the
binding. The concentrations of the tested compounds to
produce 50% inhibition of specific [3H]CHA or [3H]CGS 21680
binding (IC50) were determined from semilog plots of data from
experiments of binding inhibition. The Ki values were calcu-
lated from the IC50 values using the equation IC50/(L/Kd).45 For
[3H]CHA Kd )10.5 nM and L ) 1.2 nM; for [3H]CGS 21680 Kd
) 1 nM and L ) 5 nM. Protein estimation was based on the
method reported,46 using bovine serum albumin as standard.
(20) Barili, P. L.; Biagi, G.; Livi, O.; Scartoni, V. A method for the
synthesis of racemic and optically active 2-substituted 9-(2′,3′-
dihydroxypropyl)-8-azahypoxanthines and 8-azadenines. J . Het-
erocycl. Chem. 1991, 28, 1351-1355.
Ack n ow led gm en t. We wish to thank the Consiglio
Nazionale delle Ricerche (CNR) for financial support.
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inhibitory activity of new 2-aryl-8-azadenosines. VIII. Farmaco
1992, 47, 537-550.
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Evaluation of the quantitative contribution of an aryl group on
C(2) of 8-azaadenines to binding with adenosine deaminase: a
new synthesis of 8-azaadenosines. XI. Farmaco 1992, 47, 1457-
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Su p p or tin g In for m a tion Ava ila ble: Tables containing
physicochemical data of compounds 4a -19a (Table 1), 4b-
13b (Table 2), and 4c-18c (Table 3) and 1H NMR spectral
data (δ) of some selected compounds (Table 4) (4 pages). See
any current masthead page for ordering information.
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