Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A3 adenosine receptor antagonists: Influence of the chain at the N8 pyrazole nitrogen

Article abstract of DOI:10.1021/jm001047y

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A₃ adenosine receptor antagonists is described. The synthesized compounds showed A₃ adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A₁, A(2A), A(2B), and A₃ adenosine receptors. In particular, the effect of the chain at the N⁸ pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N⁸ pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N⁵ position as the compound with the best binding profile in terms of both affinity and selectivity (hA₃ = 0.2 nM, hA₁/hA₃ = 5485, hA₂A/hA₃ = 6950, hA(2B)/hA₃ = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A₃ receptor. The new compounds are among the most potent and selective A₃ antagonists so far described. The derivatives with higher affinity at human A₃ adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC₅₀ values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N⁸ pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

Full text of DOI:10.1021/jm001047y

4768
J . Med. Chem. 2000, 43, 4768-4780
P yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e Der iva tives a s High ly P oten t a n d
Selective Hu m a n A3 Ad en osin e Recep tor An ta gon ists: In flu en ce of th e Ch a in a t
th e N8 P yr a zole Nitr ogen
Pier Giovanni Baraldi,*,§ Barbara Cacciari,§ Romeo Romagnoli,§ Giampiero Spalluto,† Stefano Moro,#
Karl-Norbert Klotz, Edward Leung,| Katia Varani,‡ Stefania Gessi,‡ Stefania Merighi,‡ and Pier Andrea Borea‡
Dipartimento di Scienze Farmaceutiche and Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia,
Universita` degli Studi di Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy; Dipartimento di Scienze
Farmaceutiche, Universita` degli Studi di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy; Molecular Modeling Section,
Dipartimento di Scienze Farmaceutiche, Universita` di Padova, Via Marzolo 5, I-35131 Padova, Italy; Institut fu¨r
Pharmakologie, Universita¨t Wu¨rzburg, Versbacher Strasse 9, D-97078 Wu¨rzburg, Germany; and Medco Research,
Research Triangle Park, North Carolina 27709
Received J uly 31, 2000
An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form
(Baraldi et al. J . Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A3
adenosine receptor antagonists is described. The synthesized compounds showed A3 adenosine
receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in
radioligand binding assays at human A1, A2A, A2B, and A3 adenosine receptors. In particular,
the effect of the chain at the N8 pyrazole nitrogen was analyzed. This study allowed us to
identify the derivative with the methyl group at the N8 pyrazole combined with the
4-methoxyphenylcarbamoyl moiety at the N5 position as the compound with the best binding
profile in terms of both affinity and selectivity (hA3 ) 0.2 nM, hA1/hA3 ) 5485, hA2A/hA3 )
6950, hA2B/hA3 ) 1305). All the compounds proved to be full antagonists in a specific functional
model where the inhibition of cAMP generation by IB-MECA was measured in membranes of
CHO cells stably transfected with the human A3 receptor. The new compounds are among the
most potent and selective A3 antagonists so far described. The derivatives with higher affinity
at human A3 adenosine receptors proved to be antagonists, in the cAMP assay, capable of
inhibiting the effect of IB-MECA with IC50 values in the nanomolar range, with a trend strictly
similar to that observed in the binding assay. Also a molecular modeling study was carried
out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled
structure-activity relationship was found for the N8 pyrazole substituted derivatives, showing
a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-
pyrimidine derivatives and their experimental Ki values.
In tr od u ction
(72%) for the A3 receptors have been observed between
species.10,11 The rat A3 adenosine receptor in particular
behaves anomalously in ligand binding assays. For
these reasons, the hypothesis of the existence of two
different adenosine A3 receptor subtypes has been
proposed, but the question has not yet been answered.12
The actions of adenosine are mediated through G-
coupled receptors that are classified into four different
subtypes, named A1, A2A, A2B, and A3, based on phar-
macological criteria and coupling to adenylyl cyclase.1,2
While the adenosine A1 and A2A receptor subtypes have
been pharmacologically characterized through the use
of selective ligands,3 the A3 and A2B subtypes are still
under investigation.
The adenosine A3 receptor has been recently cloned
from a rat testis cDNA library4 and is the subject of
intensive pharmacological characterization. The adenos-
ine A3 receptors have now also been cloned from several
other species such as human,5,6 sheep,7 mouse,8 and
rabbit.9 Significant differences in sequence homology
Activation of adenosine A3 receptors has been shown
to stimulate phospholipase C13 and D14 and to inhibit
adenylate cyclase.3 Activation of A3 adenosine receptors
in the rat results in hypotension, by promotion of release
of inflammatory mediators from mast cells.15 It has also
been suggested that the A3 receptor plays an important
role in brain ischemia,16,17 immunosuppression,18 and
bronchospasm in several animal models.19a On these
pharmacological observations, highly selective A3 ad-
enosine receptor antagonists are being sought as po-
tential antiasthmatic,19b antiinflammatory, and cere-
broprotective agents.10,20 In the past few years, different
classes of compounds with nonxanthinic structures have
been reported to be A3 adenosine receptor antago-
nists:21 dihydropyridine and pyridine analogues,22-26
flavonoid,27,28 isoquinoline,29-31 and triazoloquinazoline
derivatives.32-33
* To whom correspondence should be addressed. Phone: +39-(0)-
532-291293. Fax: +39-(0)532-291296. E-mail: pgb@dns.unife.it.
§ Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Ferrara.
‡ Dipartimento di Medicina Clinica
e Sperimentale-Sezione di
Farmacologia, Universita` degli Studi di Ferrara.
† Universita` degli Studi di Trieste.
#
Universita` di Padova.
Universita¨t Wu¨rzburg.
| Medco Research.
10.1021/jm001047y CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/21/2000

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4769
Sch em e 1a
a
Reagents: (i) NaH, DMF, RX; (ii) HC(OEt)3, reflux; (iii) 2-furoic hydrazide, MeO(CH2)2OH; (iv) Ph2O, 260 °C, flash chromatography;
(v) HCl, reflux; (vi) NH2CN, 1-methyl-2-pyrrolidone, pTsOH, 140 °C.
Sch em e 2a
In this field of research, our group has recently
reported, in preliminary form, a new series of pyrazolo-
[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly
potent and selective human A3 adenosine receptor
antagonists.34 In particular two of these compounds,
5-[[(4-methoxyphenyl)amino]carbonyl]amino-8-ethyl-2-
(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine (1)
and 5-[[(4-methoxyphenyl)amino]carbonyl]amino-8-n-
propyl-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyri-
midine (2), proved to be the most potent and selective
adenosine A3 antagonists in humans ever reported,
while proving ineffective in a rat model. The design of
these derivatives was based on the formation of hybrid
molecules between the 3-chloro- or 4-methoxyphenyl-
carbamoyl moieties present in A3 agonists35,36 and the
nucleus of A2A antagonists37-40 previously reported by
a
Reagents: (i) 99% HCOOH, 130 °C, 48 h.
our group.
the chain at the N8 position has been modified in order
to optimize the steric and lipophilic characteristics of
this moiety for the recognition of the human A3 adenos-
ine receptor subtype. In addition, on the basis of the
studies previously reported by J acobson and co-workers,
we investigated the effect of a phenylcarbamoyl chain
on the interaction with A3 adenosine receptors, intro-
ducing it on the amino function present in the 5-amino-
9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazoline (CGS-
15943) which possesses affinity for the human A3
adenosine receptor (Ki hA3 90 nM).32
Ch em istr y
Compounds 1-40 were prepared following the gen-
eral synthetic strategy summarized in Schemes 1 and
3. Compounds 1-40 were synthesized according to a
well-known procedure for the synthesis of the pyrazolo-
[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines, previously re-
ported39 (Table 1)
Alkylation of 5-amino-4-cyanopyrazole 41 with the
appropriate alkyl halide in dry dimethylformamide led
to an approximately 1:4 mixture of the N1 and N2
regioisomers 42-52 as an inseparable mixture, used for
the following steps without any further purification34
In this fully detailed version, we wish to report all
the synthesized compounds (1-40) of this series, in
which the substituent on the phenyl ring (3-chloro or
4-methoxy) at the 5-position has been mantained while
(Scheme 1). Pyrazoles 42-52 were transformed into the
corresponding imidates 53-63 through refluxing in
triethyl orthoformate. The imino ethers 53-63 were
reacted with 2-furoic hydrazide in refluxing 2-methoxy-

4770 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Baraldi et al.
Sch em e 3a
ethanol to provide the pyrazolo[3,4-d]pyrimidine inter-
mediates. These were converted through a thermally
induced cyclization in diphenyl ether to the desired
derivatives 64-74 in good overall yield (50-63%), after
separation of N7 (minor product) and N8 (major product)
regioisomers by flash chromatography.34 Hydrolysis
with aqueous 10% HCl afforded the aminotriazoles 75-
85, which were in turn converted into the 5-amino-8-
(substituted)-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-
c]pyrimidine derivatives 3-13.
The unsubstituted (14) and N8-tert-butyl (15) deriva-
tives were prepared by treatment of the corresponding
N7-tert-butyl derivative 86, by treatment with 99%
formic acid at 130 °C for 48 h. These recaction conditions
afforded a separable mixture of 14 and 1537 (Scheme
2).
The urea derivatives 1, 2, and 16-40 were obtained
by a coupling reaction of derivatives 3-15 with the
a
Reagents: (i) THF, reflux, 12 h.
Ta ble 1. Structures and Physicochemical Parameters of the Synthesized Compounds
compd
R
R1
mp (°C)
245 dec
MW
formula
10H7N7O
18H14N8O3
17H11N8O2Cl
anal.
14
16
17
3
18
19
4
1
20
5
2
21
6
22
7
23
24
15
25
26
8
27
28
9
29
30
10
31
32
11
33
34
12
35
36
13
37
38
39
40
H
H
H
H
241.07
390.11
394.06
255.24
404.39
408.80
269.26
418.41
422.83
283.29
432.44
436.86
281.27
430.15
297.32
446.47
450.89
297.13
446.18
450.13
311.34
460.49
464.91
309.33
458.48
462.90
345.36
494.51
498.93
359.39
508.54
512.96
578.86
727.91
731.86
397.16
546.21
551.01
434.84
439.26
C
C
C
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
249-250
276-277
167-168
193-195
142-145
249-250
200-201
204-205
209-210
146-148
138-139
207-209
180
200-203
197-198
210-212
108-110
198-203 dec
203-206
212-213
192-193
199-200
178-179
198-199
204-205
183-185
180-181
186-187
168-170
174-175
183-184
145-146
155
CH3
CH3
CH3
C2H5
C2H5
C2H5
n-C3H7
n-C3H7
n-C3H7
C11H9N7O
C19H16N8O3
C18H13N8O2Cl
C12H11N7O
C20H18N8O3
C19H15N8O2Cl
C13H13N7O
C21H20N8O3
C20H17N8O2Cl
C13H11N7O
C21H18N8O3
C14H15N7O
C22H22N8O3
C21H19N8O2Cl
C14H15N7O
C22H22N8O3
C21H19N8O2Cl
C15H17N7O
C23H24N8O3
C22H21N8O2Cl
C15H15N7O
C23H22N8O3
C22H19N8O2Cl
C18H15N7O
C26H22N8O3
C25H19N8O2Cl
C19H17N7O
C27H24N8O3
C26H21N8O2Cl
C18H12N7OBr3
C26H19N8O3Br3
C25H16N8O2Br3Cl
22H19N7O
30H26N8O3
29H23N8O2Cl
21H15N6O3Cl
20H12N6O2Cl2
CH2-CHdCH2
CH2-CHdCH2
n-C4H9
n-C4H9
n-C4H9
t-C4H9
t-C4H9
t-C4H9
(CH3)2CH-CH2-CH2
(CH3)2CH-CH2-CH2
(CH3)2CH-CH2-CH2
(CH3)2CdCH-CH2
(CH3)2CdCH-CH2
(CH3)2CdCH-CH2
Ph-CH2-CH2
Ph-CH2-CH2
Ph-CH2-CH2
Ph-CH2-CH2-CH2
Ph-CH2-CH2-CH2
Ph-CH2-CH2-CH2
2,4,5-Br3-Ph-CH2-CH2
2,4,5-Br3-Ph-CH2-CH2
2,4,5-Br3-Ph-CH2-CH2
2-(R-naphthyl)ethyl
2-(R-naphthyl)ethyl
2-(R-naphthyl)ethyl
158-160
175-177
187-188
195-196
216-217
212-213
C
C
C
C
C
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4771
commercially available isocyanates 87 and 8835,36
(Scheme 3).
the best compounds in term of both affinity and selec-
tivity. In fact the N8 unsubstituted (16), methyl (18),
ethyl (1), propyl (2), and butyl (23) derivatives showed
affinity values in the sub-nanomolar range (0.14-0.8
nM) and high selectivity versus the other receptor
subtype (200-7000). From these results, it seems
evident that small chains, in particular methyl substi-
tution, together with the 4-methoxyphenylcarbamoyl
moiety are indispensable for obtaining derivatives with
high potency and selectivity. In fact, compound 18
resulted as not only one of the most potent but also the
most selective compounds (hA3 ) 0.2 nM, hA1/hA3 )
5485, hA2A/hA3 ) 6950, hA2B/hA3 ) 1305) ever reported.
Also comparing this derivative with the previously
reported compound 1,34 an improvement in both affinity
and selectivity was obtained.
Resu lts a n d Discu ssion
Table 2 gives the receptor binding affinities of com-
pounds 1, 2, and 16-40 and the corresponding N5
unsubstituted derivatives 3-15, determined at human
A1, A2A, A2B, and A3 receptors expressed in CHO (A1,
A
2A, A3) or HEK-293 (A2B) cells.
[3H]-1,3-Dipropyl-8-cyclopentylxanthine ([3H]DPC-
PX)41,42 (A1, A2B), [3H]-5-amino-7-(2-phenylethyl)-2-(2-
furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine ([3H]-
SCH 58261) (A2A),43 and [3H]-5-(4-methoxyphenylcar-
bamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidine ([3H]MRE3008-F20) (A3)42 were
used as radioligands in binding assays.
As previously obseved, it is clearly evident that
compounds with the free amino group at the N5 position
(3-5, 7-15) show high affinity for both A1 and A2A
receptors and surprisingly also for the human A2B
subtype with different degrees of selectivity.34 In par-
ticular compounds with a long alkyl chain (8, 9) or a
phenylethyl moiety (10) showed the best binding profile
at human A2B receptors (5-12 nM) but, unfortunately,
resulted ineffective at human A3 adenosine receptors
(micromolar range) while having high affinity for A1 and
A2A subtypes (0.3-5 nM).
The compounds with the 2-phenylethyl (10) and
3-phenylpropyl (11) chains at the pyrazole nitrogen
show the best profile at the human A2A adenosine
receptor (0.16-0.34 nM) but low selectivity versus A1
and A2B receptor subtypes. These two derivatives are
the regioisomers of two of the most potent and selective
A2A antagonists, named SCH 58261 and SCH 63390,
previously reported.39 These data confirm the impor-
tance of the position of the chain in discriminating the
affinities at different receptor subtypes.
A different biological profile was observed when the
phenylethyl (31, 32) and phenylpropyl (33, 34) chains
were introduced; in fact, a reduction in affinity of about
10-100-fold is evident (e.g. compound 2: hA3 ) 0.80
nM vs compound 33: hA3 ) 40 nM). This effect could
be attributed to both the steric and lipophilic contribu-
tions of the chain.
When branched chains such as isopentyl (27, 28) or
2-isopentenyl (29, 30) were introduced on the pyrazole
nitrogen, a significant loss in both affinity and selectiv-
ity was observed. But, when the steric bulk is close to
the pyrazole nitrogen, as in the case of the tert-butyl
moiety (25, 26) affinity and selectivity were well re-
tained. These results suggest that the steric factor (in
particular when the bulky group is far from the pyrazole
nitrogen) is more important than the lipophilic one. To
support this hypothesis, we synthesized derivatives 35-
38, in which the 2-(R-naphthyl)ethyl and 2,4,5-tribro-
mophenylethyl moieties were introduced on the pyrazole
nitrogen. From the data reported in Table 2 it appears
evident that the compounds with the 2-(R-naphthyl)-
ethyl chain (37, 38), in which the steric factor is
unaltered while the lipophilic character is increased,
have a slight decrease in affinity but retention of good
selectivity. On the contrary, for derivatives 35 and 36
in which both steric and lipophilic factors are increased,
a dramatic reduction of affinity and selectivity was
observed. Another interesting aspect, the rigidity of the
chain at the N8 position, was investigated by introducing
unsaturated chains such as allyl (22) or 2-isopentenyl
(29, 30). As summarized in Table 2 it is evident that
more rigid chains do not improve both affinity and
selectivity toward human A3 adenosine receptors with
respect to saturated chains (e.g. 27, 28 vs 29, 30 and/or
2 vs 22). On the other hand, these derivatives represent
useful precursors for the preparation of tritiated radio-
ligands, as recently reported by our group for derivative
22. Reduction of the double bond in 22 with tritium gas
resulted in the tritiated form of compound 2, well-
characterized as the first radioligand antagonist for
human A3 adenosine receptors, named [3H]MRE3008-
F20.42,44 With the aim to better investigate the effect of
the phenylcarbamoyl chain on affinity toward human
A3 adenosine receptors, we introduced these chains on
the amino group of CGS15943 (compounds 39, 40)
following the same rationale suggested by J acobson and
co-workers for the synthesis of 9-chloro-2-(2-furyl)-5-
These N5 unsubstituted derivatives, in general, were
ineffective at the human A3 adenosine receptors, but
interestingly, compounds with small alkyl or aralkyl
chains (e.g. compounds 3-5, 10, 11) or hydrogen (14)
at the pyrazole nucleus showed slightly better affinity
toward human A3 receptors (300-400 nM) with respect
to compounds with a bulky aralkyl chain (e.g. com-
pounds 12, 13: hA3 > 3 µM).
As previously observed, when the substituted phe-
nylcarbamoyl chain is present at the N5 position all the
synthesized derivatives 1, 2, and 16-40 show affinities
in the nanomolar range toward human A3 receptors
with a different degree of selectivity versus the other
receptor subtypes.34 In particular in all the examples
shown in Table 2, it is evident that the 4-methoxyphe-
nylcarbamoyl moiety (e.g. compounds 1, 2, 16, 18, 25)
confers higher affinity to the human A3 receptor than
the 3-chlorophenylcarbamoyl chain (e.g. compounds 20,
21, 17, 19, 26) with a difference of about 2-10-fold. On
the contrary, in some cases, the presence of 3-chloro
substitution on the phenyl ring (e.g. compounds 17, 21,
24) seems to produce an increased affinity toward
human A2B receptors of about 5-40-fold with respect
to the 4-methoxy substituted derivatives (e.g. com-
pounds 16, 18, 2, 23), with a concomitant decrease in
selectivity for the human A3 adenosine receptor subtype.
Analyzing this enlarged series of compounds, it is
evident that small chains on N8 of the pyrazole produce

4772 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Baraldi et al.
Ta ble 2. Binding Affinity at hA1, hA2A, hA2B, and hA3 Adenosine Receptors of the Synthesized Compounds
Ki (nM)
a
b
c
d
compd
14
hA1
hA2A
hA2B
hA3
hA1/hA3
0.40
hA2A/hA3
0.06
hA2B/hA3
0.15
140
(123-160)
448
20
51
348
(267-453)
0.14
(17-24)
520
(44-58)
350
16
17
3
3200
476
0.33
5485
875
0.015
1710
155
0.024
1496
33
3715
496
2500
140
(365-550)
238
(484-558)
248
(332-369)
70
(0.08-0.27)
0.50
(179-317)
(218-283)
(61-80)
(0.34-0.73)
101
2.80
90
300
0.009
6950
2987
0.005
1733
112
0.30
1305
512
(81-127)
1097
(2.40-3.55)
1390
(81-101)
261
(226-301)
(265-339)
0.20
18
19
4
(928-1297)
(1220-1590)
(0.17-0.24)
350
1195
205
0.40
(323-378)
5.00
(1024-1396)
1.95
(157-268)
65
(56-75)
(0.24-0.64)
331
0.19
408
(4.05-6.20)
(1.70-2.10)
(285-385)
1
1026
1040
245
0.60
(785-1341)
249
(830-1310)
180
(188-320)
150
(107-210)
(0.51-0.70)
1.60
20
5
93.7
0.095
2570
62.6
4229
0.09
946
(215-289)
(160-210)
(1.42-1.79)
10
2.51
39
408
0.006
175
(7-14)
1197
(1027-1396)
(1.90-3.37)
140
(35-45)
2056
(1637-2582)
(364-460)
0.80
2
(120-155)
(0.63-0.1.00)
21
22
7
30
1220
57
0.91
1340
366
(23-40)
1531
(1354-1372)
(880-1682)
176
(50-63)
2030
(1770-2327)
(0.85-0.98)
0.48
3189
0.02
925
408
0.15
995
365
0.002
5.8
(150-207)
(0.32-0.74)
14
1.60
53
600
0.002
250
(11-17)
296
(1.4-2.1)
80
(40-69)
303
(260-352)
(525-691)
0.32
23
24
15
25
26
8
(269-326)
(65-92)
(0.27-0.34)
245
95
70
0.60
158
116
(213-281)
175
(70-130)
45
(36-55)
(56-89)
400
(0.52-0.68)
1149
0.04
681
0.35
2144
162
(134-229)
(343-468)
(1061-1245)
796
545
1715
0.80
(632-1003)
1015
(461-645)
796
(1531-1920)
450
(0.63-1.00)
2.78
286
(885-1163)
(632-1003)
(432-469)
(2.13-3.62)
2.00
0.78
9.1
700
0.001
2.16
2.87
0.0009
25.6
2.0
0.013
1.80
1.5
(1.72-2.36)
175
(0.60-1.00)
65
(7.4-11.3)
54
(45-64)
(664-738)
30
27
28
9
(149-204)
(47-78)
(23-40)
100
115
61
40
2.5
(83-120)
5.42
(85-164)
0.80
(55-68)
12
(8-18)
(33-48)
811
(691-952)
0.006
7.5
0.014
1.45
1.47
0.0017
82.6
11.2
0.011
20
(4.72-6.23)
(0.65-1.06)
29
30
10
31
32
11
33
34
12
35
36
13
37
38
CGS15943
39
40
300
1025
58
40
(254-356)
352
(850-1230)
110
(52-65)
81
(70-94)
(35-47)
55
6.4
(329-375)
(85-140)
(50-61)
1.00
0.34
5.1
280
0.003
205
6.3
0.001
122
(0.66-1.51)
201
(0.22-0.44)
120
(3.9-6.7)
81
(70-94)
(247-317)
0.98
(172-236)
(100-145)
(1.22-1.78)
251
105
90
8.0
13
(229-276)
3.00
(90-125)
0.16
(81-101)
49
(38-63)
(7.2-8.9)
430
(378-488)
0.007
6.27
25
0.0004
25.2
3.3
(2.65-3.39)
(0.06-0.48)
251
1010
802
40
(221-286)
1500
(880-1160)
200
(719-895)
226
(33-48)
60
3.7
(1325-1693)
(185-210)
(183-278)
(51-70)
247
348
1113
4481
0.06
10.2
5.50
0.10
154
62
0.08
81.2
51
0.25
>400
26
(170-358)
255
(267-453)
2030
(934-1327)
>10000
(3650-5501)
25
(191-339)
(1770-2327)
(17-36)
389
3600
1816
(1681-1961)
4294
71
(327-462)
348
(3420-3788)
896
(65-78)
3416
(3228-3614)
0.26
204
1.26
>625
>196
0.27
158
(267-453)
(730-1100)
(3828-4817)
2466
3260
>10000
16
(2362-2575)
3164
(2826-3761)
>10000
(14-19)
51
(34-76)
>10000
>196
0.005
50
(2894-3460)
4.4
0.43
(0.37-0.49)
9.4
(8.7-10.2)
23
85
0.05
43
(4.1-4.7)
7.6
(6.5-8.7)
(18-29)
(76-95)
0.14
22
(19-27)
30
(23-40)
(0.08-0.24)
8.1
9.5
0.19
54
67
157
(6.9-9.7)
(8.7-10.5)
(0.13-0.27)
a
b
Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells (n ) 3-6). Displacement of specific
[3H]SCH 58261 binding at human A2A receptors expressed in CHO cells (n ) 3-6). c Displacement of specific [3H]DPCPX binding at
human A2B receptors expressed in HEK-293 cells (n ) 3-6). Displacement of specific [3H]MRE3008-F20 binding at human A3 receptors
d
expressed in CHO cells (n ) 3-6). Data are expressed as geometric means with 95% confidence limits.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4773
[(phenylacetyl)amino][1,2,4]triazolo[1,5-c]quinazoline
(MRS1220).32
The binding data of these derivatives followed the
same profile observed for the pyrazolic series. In fact
the introduction of substituted-phenylcarbamoyl chains
at the N5 position induced an increase of potency at the
human A3 adenosine receptors (hA3 ) 0.14-0.19 nM)
with respect to the unsubstituted compound CGS15943
(hA3 ) 85 nM), on the contrary the synthesized com-
pounds showed low selectivity versus other receptor
subtypes if compared with the pyrazolic series. These
results allow us to hypothesize that, while the substi-
tuted-phenylcarbamoyl moiety confers potency at the
human A3 adenosine receptors, small chains on the
pyrazole nucleus and probably the nitrogens present on
the heterocycle permit to discriminate the affinity to
human A3 adenosine receptors versus other receptor
subtypes. In fact, from a comparison between compound
16 (hA3 ) 0.14 nM, hA1/hA3 ) 3200, hA2A/hA3 ) 3715,
hA2B/hA3 ) 2500) or 18 (hA3 ) 0.2 nM, hA1/hA3 ) 5485,
hA2A/hA3 ) 6950, hA2B/hA3 ) 1305) with compound 39
F igu r e 1. Relationship between molecular volume values and
human A3 affinities (Ki, nM) for the tested pyrazolo[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidine derivatives.
mentioned, a small hydrophobic pocket is present
around the N8 pyrazole position (Leu90, TM3; Phe182,
TM5), and consequently the dimension of the N8 pyra-
zole substituent has to fit very well with the shape of
the receptor pocket. In fact, as clearly shown in Table
2, bulky substituents at the N8 position are not well-
tolerated in the pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyri-
midine series. The pharmacophore maps can be sum-
marized as shown in Figure 2. This pharmacophore
model fits well with the binding site model in the
transmembrane region, based on a variety of human A3
receptor antagonists, that has been recently proposed.
All of the synthesized compounds were also found to
be functional antagonists in a specific functional model
where the inhibition of cAMP generation by IB-MECA
was measured in membranes of CHO cells stably
transfected with the human A3 receptor (Table 3).46
As expected, all the derivatives are full antagonists
with different degrees of potency. Compounds 3-5 and
7-15, with a free amino group at the 5-position, as
observed in binding studies, showed poor activity,
inhibiting the effect of 100 nM IB-MECA in a range of
3-28% at 1 µM concentration. On the contrary, the N5
substituted compounds (1, 2, 16-40) were more potent
in the functional assay than the unsubstituted deriva-
tives (3-5, 7-15).
The derivatives with higher affinity at human A3
adenosine receptors (e.g. compounds 16, 18) proved to
be antagonists capable of inhibiting the effect of IB-
MECA, with a potency strictly corresponding to that
observed in the binding assay. In particular, the deriva-
tives with a binding affinity close to or less than 1 nM
inhibit the effect of 100 nM IB-MECA at 1 µM concen-
tration from 68% to 98%, displaying IC50 values in the
nanomolar range (1.8-14 nM) as observed by inhibition
curves for compounds 16, 18, 21, 22, 2, and 31 (Figure
3). On the contrary, derivatives with binding affinities
higher than 2 nM at human A3 adenosine receptors (e.g.
compounds 15, 26-28) inhibit the effect of 100 nM IB-
MECA in a range of 32-70% at 1 µM concentration.
(hA3 ) 0.14 nM, hA1/hA3 ) 43, hA2A/hA3 ) 50, hA2B
/
hA3 ) 158) it is clearly evident these compounds cover
the same range of affinity at human A3 adenosine
receptors but differ significantly in terms of selectivity
in favor of pyrazolic derivatives. From these experimen-
tal observations, it is possible to hypothesize that not
only small chains possess the ideal, steric and probably
lipophilic, characteristics for interaction with the human
adenosine A3 receptor subtype but also the nitrogens
present on the pyrazole nucleus play an important role
for receptor interaction. To better rationalize these
experimental observations, a molecular modeling study
was carried out to identify a possible pharmacophore
map. Moro et al. recently developed a model of the
human A3 receptor, using rhodopsin as a template, by
adapting a facile method to simulate the reorganization
of the native receptor structure induced by the ligand
coordination (cross-docking procedure).45 As already
reported, the recognition of classic A3 antagonists ap-
pears to occur in the upper region of the transmembrane
helical bundle.45 TMs 3, 5, 6, and 7 seem to be crucial
for the recognition of both agonists and antagonists.45
Accordingly, the pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]py-
rimidine nucleus should be most favorably oriented
perpendicular to the plane of the lipid bilayer, with the
carbamoyl moiety at the N5 position in proximity to
Ser275 (TM7) and the oxygen atom of the 2-(2-furyl)
group close to Asn250 (TM6). Two important hydrophilic
interactions, probably hydrogen-bonding interactions,
seem to be involved between the two ester group and
these two polar amino acids. In addition, two hydro-
phobic pockets are likely present around the N8 pyrazole
position where an alkyl/aryl side chain would bind
(Leu90, TM3; Phe182, TM5) and around the 2-position
where the furyl ring would bind (Leu90, TM3; Ile186,
TM5). Moreover, a sterically controlled structure-
activity relationship (SAR) was found for the N8 pyra-
zole substituted derivatives. As shown in Figure 1, there
is a significtive correlation between the calculated
molecular volume of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-
pyrimidine derivatives and their experimental Ki values.
Con clu sion s
The present study provides important information
concerning the structural requirements necessary for
the recognition of the human A3 adenosine receptor. It
In particular A3 affinities decrease with the increasing
molecular volume values at the N8 position. As already

4774 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Baraldi et al.
F igu r e 2. Pharmacophore map for binding at the human A3 receptor for the tested pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine
derivatives.
Ta ble 3. Functional Assaya
compd
% inhibition
IC50 (nM)
compd
% inhibition
IC50 (nM)
compd
11
33
34
12
35
36
13
% inhibition
IC50 (nM)
14
16
17
3
18
19
4
1
20
5
24 (16-35)
98 (92-105)
88 (77-100)
26 (14-48)
94 (79-112)
92 (81-104)
25 (15-41)
86 (63-116)
68 (51-91)
23 (15-35)
90 (79-106)
80 (63-100)
90 (73-110)
15 (10-21)
23
24
15
25
26
8
27
28
9
29
30
10
31
32
93 (85-101)
85 (70-103)
9 (5-15)
84 (63-110)
70 (62-68)
14 (8-23)
48 (35-65)
45 (37-54)
12 (7-19)
42 (33-52)
38 (26-54)
28 (16-51)
78 (64-94)
58 (49-69)
4.1 (2.9-6.0)
4.5 (3.0-6.9)
18 (15-22)
49 (38-63)
39 (28-55)
3 (1-9)
50 (34-73)
32 (18-59)
4 (1-14)
52 (38-71)
40 (32-49)
30 (17-52)
97 (92-102)
95 (84-107)
1.8 (0.9-3.7)
5.3 (3.8-7.6)
6.8 (5.1-8.9)
2.7 (1.7-4.2)
4.8 (2.8-8.1)
4.8 (2.5-9.3)
14.8 (10.8-21.5)
37
38
CGS15943
39
40
406 (354-465)
2.0 (1.1-3.8)
2.4 (1.8-3.3)
2
5.2 (4.4-6.5)
5.5 (4.0-7.5)
5.1 (3.5-7.6)
21
22
7
13.9 (10.7-21.5)
a
Percentage of blockade by 1 µM of each compound (1-40) of the inhibition by 100 nM IB-MECA-inhibited cAMP accumulation in
CHO cells expressing human A3 adenosine receptors. For the most potent compounds (1, 2, 16-31, 39, 40) and reference compound
(CGS15943) IC50 values are also shown. Values are the means of at least three experiments, and in parentheses the 95% confidence
limits are shown.
confirms, as previously observed,34 that a substituted
phenylcarbamoyl moiety confers affinity and selectivity
for the human A3 adenosine receptor subtype at the
pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine nucleus,
while the N5 unsubstituted derivatives lacks both af-
finity and selectivity for human A3 receptors, showing
high affinity for the A1, A2A, and A2B receptor subtypes.
An interesting correlation between the calculated mo-
lecular volume of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]py-
rimidine derivatives and their experimental Ki values
has been found. In fact it has been observed that the
A3 affinities decrease with the increasing molecular
volume values at the N8 position. All these observations
suggest that small alkyl groups at the N8 pyrazole
nitrogen combined with the N5-(4-methoxyphenyl)car-
bamoyl substitution afford the best compounds in terms
of affinity and selectivity at the human A3 adenosine
receptors. In particular, when the N8-methyl and N5-
(4-methoxyphenyl)carbamoyl substitutions were com-
bined, the most potent and selective human A3 adenos-
ine antagonist (18: hA3 ) 0.2 nM, hA1/hA3 ) 5485,
hA2A/hA3 ) 6950, hA2B/hA3 ) 1305) was obtained.
All these experimental findings were further vali-
dated by molecular modeling studies, which demon-
strated that bulky substituents at the N8 position are
not well-tolerated in the pyrazolo[4,3-e]1,2,4-triazolo-
[1,5-c]pyrimidine series and that this class of compounds
interact perfectly with the human A3 receptor model
proposed by Moro et al.45 identifying a possible phar-
macophore map.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4775
to warm to 25 °C and stirred for 3-5 h (TLC: EtOAc 1:1).
The reaction was quenched by addition of H2O (80 mL), and
the aqueous layer was extracted with EtOAc (5 × 25 mL). The
organic layers were recombined, dried (Na2SO4), filtered and
concentrated at reduced pressure, to afford the alkylated
pyrazoles 42-52 as an inseparable mixture of N1 and N2
isomers (ratio approximately 1:4). This mixture of N1 and N2
substituted 4-cyano-5-aminopyrazoles 42-52 was then dis-
solved in triethyl orthoformate (60 mL) and the solution was
refluxed under nitrogen for 8 h. Then the solvent was removed
under vacuum, the oily residue constituted by the mixture of
imidates 53-63 was dissolved in 2-methoxyethanol (50 mL),
and 2-furoic acid hydrazide (13 mmol) was added. The mixture
was refluxed for 5-10 h, then, after cooling, the solvent was
removed under reduced pressure and the dark oily residue was
cyclized further any other purification in diphenyl ether (50
mL) at 260 °C using a Dean-Stark for the azeotropic elimina-
tion of water produced in the reaction. After 1.5 h, the mixture
was poured onto hexane (300 mL) and cooled. The precipitate
was filtered off and purified by chromatography (EtOAc-
hexane 1:1). In this way, the major product (N8 alkylated) (64-
74) was obtained in good overall yield.
F igu r e 3. Inhibition curves of cAMP accumulation in human
A3 adenosine receptors by adenosine antagonists blocking the
effect of 100 nM IB-MECA. Curves are representative of a
single experiment from a series of three independent experi-
ments.
From the results presented herein, it is possible to
hypothesize that also the pyrazole nitrogens are indis-
pensable for selectivity at the human A3 adenosine
receptors. In fact, by introducing phenylcarbamoyl
moieties on the amino group at the 5-position of
CGS15943, retention of affinity but significant loss of
selectivity were observed.
8-Meth yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (64). Yield: 45%; yellow solid; mp 155-156 °C
(EtOAc-light petroleum). IR (KBr): 1615, 1510 cm-1. 1H NMR
(DMSO-d6): δ 4.1 (s, 3H); 6.32 (m, 1H); 7.25 (m, 1H); 8.06 (m,
1H); 8.86 (s, 1H); 9.38 (s, 1H). Anal. (C11H8N6O) C, H, N.
8-E t h yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]-
p yr im id in e (65). Yield: 50%; pale yellow solid; mp 188-189
1
°C (EtOAc-light petroleum). IR (KBr): 1620, 1500 cm-1. H
NMR (DMSO-d6): δ 1.67 (t, 3H, J ) 7); 4.53 (q, 2H, J ) 7);
6.59 (m, 1H); 7.23 (m, 1H); 7.64 (s, 1H); 8.34 (s, 1H); 9.10 (s,
1H). Anal. (C12H10N6O) C, H, N.
8-P r op yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (66). Yield: 60%; yellow solid; mp 189-190 °C
(EtOAc-light petroleum). IR (KBr): 1600, 1505 cm-1. 1H NMR
(DMSO-d6): δ 0.98 (t, 3H, J ) 7); 2.03-2.10 (m, 2H); 4.41 (q,
2H, J ) 7); 6.60 (m, 1H); 7.24 (m, 1H); 7.64 (s, 1H); 8.32 (s,
1H); 9.10 (s, 1H). Anal. (C13H12N6O) C, H, N.
Exp er im en ta l Section
Ch em istr y. Gen er a l. Reactions were routinely monitored
by thin-layer chromatography (TLC) on silica gel (precoated
F254 Merck plates) and products visualized with iodine or
potassium permanganate solution. Infrared spectra (IR) were
1
measured on a Perkin-Elmer 257 instrument. H NMR were
determined in CDCl3 or DMSO-d6 solutions with a Bruker AC
200 spectrometer, peaks positions are given in parts per
million (δ) downfield from tetramethylsilane as internal
standard, and J values are given in Hz. Light petroleum ether
refers to the fractions boiling at 40-60 °C. Melting points were
determined on a Buchi-Tottoli instrument and are uncorrected.
Chromatographies were performed using Merck 60-200 mesh
silica gel. All products reported showed IR and 1H NMR
spectra in agreement with the assigned structures. Organic
solutions were dried over anhydrous magnesium sulfate.
Elemental analyses were performed by the microanalytical
laboratory of Dipartimento di Chimica, University of Ferrara,
and were within (0.4% of the theoretical values for C, H, and
N.
Abbr evia tion s: CGS15943, 5-amino-9-chloro-2-(2-furyl)-
[1,2,4]triazolo[1,5-c]quinazoline; MRS1220, 9-chloro-2-(2-fur-
anyl)[1,2,4]triazolo[1,5-c]quinazoline; CHA, N6-cyclohexylad-
enosine; DPCPX, 1,3-dipropyl-8-cytclopentylxanthine; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; THF, tetrahy-
drofuran; CHO cells, Chinese hamstery ovary cells; EDTA,
ethylenediaminetetraacetate; SCH 58261, 5-amino-2-(2-furyl)-
7-(2-phenylethyl)pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimi-
dine; IB-MECA, 3-iodobenzyl-5′-(N-ethylcarbamoyl)adenosine;
NECA, 5′-(N-ethylcarbamoyl)adenosine; HEK cells, human
embryonic kidney cells; MRE3008-F20, 5-[[(4-methoxyphenyl)-
amino]carbonyl]amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidine; GTP, guanosine-5′-triphosphate;
cAMP, cyclic adenosine-5′-monophosphate; SCH 63390, 5-amino-
2-(2-furyl)-7-(3-phenylpropyl)pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidine; TLC, thin-layer chromatography; EtOH, ethanol;
mp, melting point; MeOH, methanol; NaH, sodium hydride,
EtOAc, ethyl acetate.
8-(2-P r open -1-yl)-2-(2-fu r yl)pyr azolo[4,3-e]1,2,4-tr iazolo-
[1,5-c]p yr im id in e (67). Yield: 53%; yellow solid; mp 130-
131 °C (EtOAc-light petroleum). IR (KBr): 1620, 1515 cm-1
.
1H NMR (DMSO-d6): δ 4.51-4.54 (m, 1H); 5.04-5.23 (m, 3H);
6.01-6.09 (m, 1H); 6.73 (dd, 1H, J ) 2, J ) 4); 7.27 (d, 1H, J
) 4); 7.96 (d, 1H, J ) 2); 8.52 (s, 1H); 9.60 (s, 1H). Anal.
(C13H10N6O) C, H, N.
8-n -Bu tyl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (68). Yield: 50%; pale yellow solid; mp 245-247
1
°C (EtOAc-light petroleum). IR (KBr): 1610, 1500 cm-1. H
NMR (DMSO-d6): δ 0.9 (m, 3H); 1.3 (m, 2H); 1.9 (m, 2H); 4.5
(t, 2H, J ) 7.2); 6.2 (m, 1H); 7.3 (m, 1H); 8.0 (m, 1H); 8.9 (s,
1H); 9.4 (s, 1H. Anal. (C14H14N6O) C, H, N.
8-(3-Meth yl-1-bu tyl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr i-
a zolo[1,5-c]p yr im id in e (69). Yield: 54%; pale yellow solid;
mp 235-237 °C (EtOAc-light petroleum). IR (KBr): 1635,
1
1510, 1450 cm-1. H NMR (DMSO-d6): δ 1.0 (d, 6H, J ) 6.2);
1.5-1.9 (m, 3H); 4.6 (t, 2H, J ) 7.4); 6.6 (m, 1H); 7.3 (m, 1H);
7.7 (m, 1H); 8.8 (s, 1H); 9.1 (s, 1H). Anal. (C15H16N6O) C, H,
N.
8-(3-Meth yl-2-bu ten -1-yl)-2-(2-fu r yl)pyr azolo[4,3-e]1,2,4-
tr ia zolo[1,5-c]p yr im id in e (70). Yield: 48%; yellow solid; mp
210-212 °C (EtOAc-light petroleum). IR (KBr): 1625, 1500,
1
1430 cm-1. H NMR (DMSO-d6): δ 1.79 (s, 3H); 1.87 (s, 3H);
5.05 (d, 2H, J ) 6); 5.55-5.63 (m, 1H); 6.60 (m, 1H); 7.24 (m,
1H); 7.64 (s, 1H); 8.34 (s, 1H); 9.10 (s, 1H). Anal. (C15H14N6O)
C, H, N.
8-(2-P h en yleth yl)-2-(2-fu r yl)pyr azolo[4,3-e]1,2,4-tr iazolo-
[1,5-c]p yr im id in e (71). Yield: 56%; yellow solid; mp 268-
270 °C (EtOAc-light petroleum). IR (KBr): 1660, 1510, 1450
cm-1. 1H NMR (DMSO-d6): δ 3.32 (t, 2H, J ) 6.7); 4.72 (t, 2H,
J ) 6.7); 6.73 (s, 1H); 7.23 (m, 5H); 7.95 (s, 1H); 8.8 (s, 1H);
9.41 (s, 1H). Anal. (C18H14N6O) C, H, N.
Gen er a l P r oced u r e for P r ep a r a tion of 8-(Ar )a lk yl-2-
(2-fu r yl)pyr azolo[4,3-e]1,2,4-tr iazolo[1,5-c]pyr im idin es 64-
74. A solution of 41 (10 mmol) in 40 mL of DMF cooled to 0 °C
was treated with NaH (60% in oil, 12 mmol) in several portions
over 10 min. After 45 min, the appropriate (ar)alkyl halide
(12 mmol) was added and the reaction mixture was allowed
8-(3-P h en ylp r op yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia -
zolo[1,5-c]p yr im id in e (72). Yield: 63%; yellow solid; mp

4776 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Baraldi et al.
165-166 °C (EtOAc-light petroleum). IR (KBr): 1630, 1500,
1440 cm-1. 1H NMR (DMSO-d6): δ 2.34-2.48 (m, 2H); 2.67 (t,
2H, J ) 7.5); 4.43 (t, 2H, J ) 7.5); 6.61 (m, 1H); 7.16-7.32 (m,
6H); 7.64 (d, 1H, J ) 2); 8.29 (s, 1H); 9.02 (s, 1H). Anal.
(C19H16N6O) C, H, N.
8-[2-(2′,4′,5′-Tr ibr om oph en yl)eth yl]-2-(2-fu r yl)pyr azolo-
[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (73). Yield: 49%; yel-
low solid; mp 215-218 °C (EtOAc-light petroleum). IR
(KBr): 1650, 1520, 1455 cm-1. 1H NMR (DMSO-d6): δ 3.39 (t,
2H, J ) 6.7); 4.79 (t, 2H, J ) 6.7); 6.73-6.75 (m, 1H); 7.03 (s,
1H); 7.27 (d, 1H, J ) 4); 7.42 (s, 1H); 7.91 (d, 1H, J ) 2); 8.45
(s, 1H); 9.31 (s, 1H). Anal. (C18H11N6OBr3) C, H, N.
5-Am in o-8-(3-m eth yl-2-bu ten -1-yl)-2-(2-fu r yl)p yr a zolo-
[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (9). Yield: 58%; pale
yellow solid. IR (KBr): 3520-2950, 1665, 1640, 1610, 1555,
1
1450 cm-1. H NMR (DMSO-d6): δ 1.74 (s, 3H); 1.77 (s, 3H);
4.87 (d, 2H, J ) 7); 5.43-5.46 (m, 1H); 6.72 (m, 1H); 7.18 (m,
1H); 7.62 (bs, 2H); 7.93 (s, 1H); 8.55 (s, 1H).
5-Am in o-8-(2-p h e n yle t h yl)-2-(2-fu r yl)p yr a zolo[4,3-
e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (10). Yield: 45%; white
solid. IR (KBr): 3500-2900, 1670, 1645, 1620, 1530, 1455
cm-1. 1H NMR (DMSO-d6): δ 3.21 (t, 2H, J ) 6.4); 4.53 (t, 2H,
J ) 6.4); 6.7 (s, 1H); 7.1-7.4 (m, 6H); 7.65 (bs, 2H); 7.93 (s,
1H); 8.45 (s, 1H).
5-Am in o-8-(3-p h en ylp r op yl)-2-(2-fu r yl)p yr a zolo[4,3-
e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (11). Yield: 57%; yellow
solid. IR (KBr): 3510-2950, 1665, 1640, 1615, 1520, 1455
8-[2-(R-Na p h th yl)eth yl]-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-
tr ia zolo[1,5-c]p yr im id in e (74). Yield: 53%; yellow solid; mp
182-183 °C (EtOAc-light petroleum). IR (KBr): 1635, 1515,
1
cm-1. H NMR (DMSO-d6): δ 2.14-2.21 (m, 2H); 2.54 (t, 2H,
1
1450 cm-1. H NMR (DMSO-d6): δ 3.31 (t, 2H, J ) 6.7); 4.53
J ) 7); 4.29 (t, 2H, J ) 6.4); 6.72 (s, 1H); 7.14-7.32 (m, 6H);
7.64 (bs, 2H); 7.93 (s, 1H); 8.64 (s, 1H).
5-Am in o-8-[2-(2′,4′,5′-tr ibr om op h en yl)eth yl]-2-(2-fu r yl)-
(t, 2H, J ) 6.7); 6.61 (dd, 1H, J ) 2, J ) 4); 7.23-7.48 (m,
5H); 7.53-7.73 (m, 3H); 7.83 (d, 1H, J ) 2); 8.45 (s, 1H); 9.27
(s, 1H). Anal. (C22H16N6O) C, H, N.
p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr im id in e
(12).
Gen er a l P r oced u r e for P r ep a r a tion of 5-Am in o-8-(a r )-
a lk yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im -
id in es 3-13. A solution of the mixture of triazolo pirimidine
64-74 (10 mmol) in aqueous 10% HCl (50 mL) was refluxed
for 3 h. Then the solution was cooled and neutralized with a
saturated solution of NaHCO3 at 0 °C. The compounds 75-
85 were extracted with EtOAc (3 × 20 mL); the organic layers
were dried with Na2SO4 and evaporated under vacuum. The
obtained crude amine 75-85 was dissolved in N-methylpyr-
rolidone (40 mL), cyanamide (60 mmol) and p-toluenesulfonic
acid (15 mmol) were added, and the mixture was heated at
160 °C for 4 h. Then cyanamide (60 mmol) was added again,
and the solution was heated overnight. Then the solution was
diluted with EtOAc (80 mL), and the precipitate (excess of
cyanamide) was filtered off; the filtrate was concentrated
under reduced pressure and washed with water (3 × 30 mL).
The organic layer was dried (Na2SO4) and evaporated under
vacuum. The residue was purified by flash chromatography
(EtOAc-light petroleum 2:1) to afford the desired product (3-
13) as a solid.
Yield: 49%; yellow solid. IR (KBr): 3300-2900, 1655, 1520,
1
1455 cm-1. H NMR (DMSO-d6): δ 3.41 (t, 2H, J ) 6.7); 4.73
(t, 2H, J ) 6.7); 6.70-6.75 (m, 1H); 7.10 (s, 1H); 7.25 (d, 1H,
J ) 4); 7.38 (s, 1H); 7.65 (bs, 2H); 7.87 (d, 1H, J ) 2); 8.25 (s,
1H).
5-Am in o-8-[2-(R-n a p h t h yl)et h yl]-2-(2-fu r yl)p yr a zolo-
[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (13). Yield: 53%; yel-
low solid. IR (KBr): 3345-2980, 1630, 1520, 1450 cm-1 1H
.
NMR (DMSO-d6): δ 3.37 (t, 2H, J ) 6.5); 4.41 (t, 2H, J ) 6.5);
6.65 (dd, 1H, J ) 2, J ) 4); 7.33-7.51 (m, 5H); 7.61 (bs, 2H);
7.65-7.73 (m, 3H); 7.87 (d, 1H, J ) 2); 8.23 (s, 1H).
P r ep a r a tion of 5-Am in o-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-
tr ia zolo[1,5-c]p yr im id in e (14) a n d 5-Am in o-8-ter t-bu tyl-
2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e
(15). A solution of 86 (297 mg, 1 mmol) in formic acid 98% (15
mL) was heated at 130 °C for 48 h. Then the solvent was
removed at reduced pressure and the residue purified by
chromatography (EtOAc-MeOH 7:3) to afford 14 and 15,
respectively, as yellow solids.
14: yield 43%; yellow solid. IR (KBr): 3345-2990, 1620,
5-Am in o-8-m eth yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia -
zolo[1,5-c]p yr im id in e (3). Yield: 53%; yellow solid. IR
1
1515, 1450 cm-1. H NMR (DMSO-d6): δ 6.71 (dd, 1H, J ) 2,
J ) 4); 7.22 (d, 1H, J ) 4); 7.92 (d, 1H, J ) 2); 7.98 (bs, 2H);
8.17 (s, 1H); 11.34 (bs, 1H).
(KBr): 3500-2950, 1680, 1645, 1610, 1560, 1455 cm-1 1H
.
NMR (DMSO-d6): δ 4.12 (s, 3H); 6.70 (m, 1H); 6.99 (bs, 2H);
15: yield 38%; pale yellow solid. IR (KBr): 3350-2950,
7.18 (m, 1H); 7.81 (s, 1H); 8.42 (s, 1H).
1615, 1520, 1450 cm-1 1H NMR (DMSO-d6): δ 1.61 (s, 9H);
.
5-Am in o-8-eth yl-2-(2-fu r yl)pyr azolo[4,3-e]1,2,4-tr iazolo-
[1,5-c] p yr im id in e (4). Yield: 65%; yellow solid. IR (KBr):
6.72 (dd, 1H, J ) 2, J ) 4); 7.16 (d, 1H, J ) 4); 7.59 (bs, 2H);
7.93 (d, 1H, J ) 2); 8.78 (s, 1H).
3430-2950, 1680, 1655, 1620, 1550, 1450 cm-1 1H NMR
.
Gen er al P r ocedu r e for P r epar ation of 5-[[(Su bstitu ted-
ph en yl)am in o]car bon yl]am in o-8-(ar )alkyl-2-(2-fu r yl)pyr a-
zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in es 1, 2, a n d 16-
38 a n d 5-[[(Su bstitu ted -p h en yl)a m in o]ca r bon yl]a m in o-
9-ch lor o-2-(2-fu r yl)[1,2,4]t r ia zolo[1,5-c]q u in a zolin es 39
a n d 40. Amino compound 3-13 (CGS15943) (10 mmol) was
dissolved in freshly distilled THF (15 mL) and the appropriate
isocyanate (87, 88) (13 mmol) was added. The mixture was
refluxed under argon for 18 h. Then the solvent was removed
under reduced pressure and the residue was purified by flash
chromatography (EtOAc-light petroleum 4:6) to afford the
desired compounds 1, 2, and 16-40.
(DMSO-d6): δ 1.46 (t, 3H, J ) 7); 4.30 (d, 2H, J ) 7); 6.72 (m,
1H); 7.18 (m, 1H); 7.93 (bs, 2H); 7.93 (s, 1H); 8.62 (s, 1H).
5-Am in o-8-pr opyl-2-(2-fu r yl)pyr azolo[4,3-e]1,2,4-tr iazolo-
[1,5-c]p yr im id in e (5). Yield: 57%; pale yellow solid. IR
(KBr): 3400-2900, 1660, 1645, 1610, 1545, 1430 cm-1 1H
.
NMR (DMSO-d6): δ 0.83 (t, 3H, J ) 7); 1.81-1.91 (m, 2H);
4.22 (d, 2H, J ) 7); 6.71 (m, 1H); 7.19 (m, 1H); 7.63 (bs, 2H);
7.93 (s, 1H); 8.61 (s, 1H).
5-Am in o-8-(2-pr open -1-yl)-2-(2-fu r yl)pyr azolo[4,3-e]1,2,4-
tr ia zolo[1,5-c]p yr im id in e (6). Yield: 48%; yellow solid. IR
1
(KBr): 3350-2970, 1625, 1510 cm-1. H NMR (DMSO-d6): δ
4.93 (d, 2H, J ) 6); 5.33-5.42 (m, 2H); 6.01-6.09 (m, 1H);
6.47 (bs, 1H); 6.58 (dd, 1H, J ) 2, J ) 4); 7.20 (d, 1H, J ) 4);
7.62 (d, 1H, J ) 2); 8.17 (s, 1H).
5-[[(4-Meth oxyp h en yl)a m in o]ca r bon yl]a m in o-2-(2-fu -
r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr im id in e (16).
Yield: 84%; pale yellow solid. IR (KBr): 3245-2980, 1685,
1615, 1520, 1450 cm-1 1H NMR (DMSO-d6): δ 3.78 (s, 3H);
.
5-Am in o-8-n -bu tyl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia -
zolo[1,5-c]p yr im id in e (7). Yield: 47%; white solid. IR
6.54 (dd, 2H, J ) 2, J ) 4); 7.35(d, 1H, J ) 4); 7.50 (d, 2H, J
) 9); 7.83 (d, 2H, J ) 9); 8.01 (d, 1H, J ) 2); 8.03 (s, 1H); 9.45
(bs, 1H); 10.81 (bs, 1H); 11.09 (bs, 1H).
(KBr): 3500-2900, 1685, 1640, 1620, 1550, 1450 cm-1 1H
.
NMR (DMSO-d6): δ 0.9 (t, 3H); 1.2 (m, 2H); 1.8 (m, 2H); 4.2
(t, 2H); 6.7 (m, 1H); 7.2 (m, 2H); 7.6 (s, 1H); 8.0 (s, 1H); 8.6 (s,
1H).
5-[[(3-Ch lor oph en yl)am in o]car bon yl]am in o-2-(2-fu r yl)-
p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr im id in e
(17).
Yield: 78%; pale yellow solid. IR (KBr): 3240-2980, 1680,
5-Am in o-8-(3-m et h yl-1-b u t yl)-2-(2-fu r yl)p yr a zolo[4,3-
e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (8). Yield: 60%; off-white
solid. IR (KBr): 3500-2850, 1670, 1650, 1615, 1560, 1455
1
1625, 1530, 1435 cm-1. H NMR (DMSO-d6): δ 6.80 (dd, 2H,
J ) 2, J ) 4); 7.26-7.45 (m, 4H); 7.75-8.02 (m, 3H); 8.35 (s,
1H); 9.48 (bs, 1H); 11.11 (bs, 1H); 11.42 (bs, 1H).
5-[[(4-Meth oxyp h en yl)a m in o]ca r bon yl]a m in o-8-m eth -
yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr im i-
1
cm-1. H NMR (DMSO-d6): δ 0.96 (d, 6H, J ) 6.4); 1.59 (m,-
1H); 1.86 (m, 2H); 4.32 (t, 2H, J ) 6.4); 6.58 (m, 1H); 6.72 (bs,
2H); 7.21 (d, 1H, J ) 4.2); 7.63 (d, 1H, J ) 1.2); 8.10 (s, 1H).

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4777
d in e (18). Yield: 99%; yellow solid. IR (KBr): 3200-2900,
1664, 1625, 1600, 1500 cm-1. 1H NMR (CDCl3): δ 3.81 (s, 3H);
4.20 (s, 3H); 6.61 (m, 1H); 6.85 (d, 2H, J ) 9); 7.26 (m, 1H);
7.55 (d, 2H, J ) 9); 7.65 (s, 1H); 8.21 (s, 1H); 8.59 (bs, 1H);
10.96 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-m eth yl-
2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e
(19). Yield: 98%; pale yellow solid. IR (KBr): 3210-2930,
1660, 1630, 1610, 1500 cm-1. 1H NMR (CDCl3): δ 4.21 (s, 3H);
6.60 (m, 1H); 7.11 (d, 1H, J ) 8); 7.13-7.28 (m, 2H); 7.55 (d,
1H, J ) 8); 7.65 (s, 1H); 7.78 (d, 1H, J ) 2); 8.22 (s, 1H); 8.61
(bs, 1H); 11.24 (bs, 1H).
(dd, 1H, J ) 2, J ) 4); 7.14-7.34 (m, 3H); 7.55-7.64 (m, 2H);
7.82 (d, 1H, J ) 2); 8.39 (s, 1H); 8.62 (bs, 1H); 11.25 (bs, 1H).
5-[[(4-Meth oxyph en yl)am in o]car bon yl]am in o-8-(3-m eth -
yl-1-bu tyl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (27). Yield: 98%; white solid. IR (KBr): 3230-
1
2970, 1660, 1615, 1600, 1500 cm-1. H NMR (CDCl3): δ 0.99
(d, 6H, J ) 7.5); 1.58-1.22 (m, 1H); 1.87-1.97 (m, 2H); 3.82
(s, 3H); 4.40 (t, 2H, J ) 7); 6.62 (m, 1H); 6.91 (d, 2H, J ) 9);
7.23 (m, 1H); 7.58 (d, 2H, J ) 9); 7.66 (s, 1H); 8.23 (s, 1H);
8.59 (bs, 1H); 10.94 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-(3-m eth -
yl-1-bu tyl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (28). Yield: 97%; pale yellow solid. IR (KBr):
5-[[(4-Meth oxyp h en yl)a m in o]ca r bon yl]a m in o-8-eth yl-
2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e
(1). Yield: 99%; pale yellow solid. IR (KBr): 3250-2950, 1665,
1
3230-2950, 1655, 1600, 1510 cm-1. H NMR (CDCl3): δ 1.01
(d, 6H, J ) 7.5); 1.49-1.51 (m, 1H); 1.88-2.03 (m, 2H); 4.42
(t, 2H, J ) 7); 6.62 (m, 1H); 7.13 (d, 1H, J ) 8); 7.34 (m, 1H);
7.57 (d, 1H, J ) 8); 7.67 (s, 1H); 7.80 (s, 1H); 8.24 (s, 1H); 8.63
(bs, 1H); 11.25 (bs, 1H).
1
1620, 1610, 1520 cm-1. H NMR (CDCl3): δ 1.71 (t, 3H, J )
7); 3.85 (s, 3H); 4.49 (q, 2H, J ) 7); 6.65 (m, 1H); 6.88 (d, 2H,
J ) 9); 7.26 (m, 1H); 7.58 (d, 2H, J ) 9); 7.69 (s, 1H); 8.28 (s,
1H); 8.63 (bs, 1H); 10.99 (bs, 1H).
5-[[(4-Meth oxyph en yl)am in o]car bon yl]am in o-8-(3-m eth -
yl-2-b u t en -1-yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo-
[1,5-c]p yr im id in e (29). Yield: 96%; pale yellow solid. IR
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-eth yl-2-
(2-fu r yl)pyr azolo[4,3-e]1,2,4-tr iazolo[1,5-c]pyr im idin e (20).
Yield: 98%; pale yellow solid. IR (KBr): 3220-2930, 1660,
(KBr): 3235-2950, 1665, 1620, 1600, 1510 cm-1 1H NMR
.
1
1620, 1600, 1500 cm-1. H NMR (CDCl3): δ 1.71 (t, 3H, J )
(CDCl3): δ 1.83 (s, 3H); 1.87 (s, 3H); 3.81 (s, 3H); 4.97 (d, 2H,
J ) 7); 5.57 (m, 1H); 6.61 (m, 1H); 6.93 (d, 2H, J ) 9); 7.24
(m, 1H); 7.54 (d, 2H, J ) 9); 7.66 (s, 1H); 8.25 (s, 1H); 8.58 (bs,
1H); 10.96 (bs, 1H).
7); 4.50 (q, 2H, J ) 7); 6.67 (m, 1H); 7.20 (d, 1H, J ) 8); 7.31
(m, 1H); 7.61 (d, 1H, J ) 8); 7.70 (s, 1H); 7.84 (s, 1H); 8.30 (s,
1H); 8.67 (bs, 1H); 11.30 (bs, 1H).
5-[[(4-Meth oxyph en yl)am in o]car bon yl]am in o-8-pr opyl-
2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e
(2). Yield: 98%; pale yellow solid. IR (KBr): 3230-2950, 1660,
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-(3-m eth -
yl-2-b u t en -1-yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo-
[1,5-c]p yr im id in e (30). Yield: 99%; white solid. IR (KBr):
1
1
1620, 1600, 1530 cm-1. H NMR (CDCl3): δ 0.98 (t, 3H, J )
3245-2960, 1650, 1600, 1510 cm-1. H NMR (CDCl3): δ 1.84
7); 2.04-2.08 (m, 2H); 3.82 (s, 3H); 4.35 (t, 2H, J ) 7); 6.61
(m, 1H); 6.89 (d, 2H, J ) 9); 7.25 (m, 1H); 7.56 (d, 2H, J ) 9);
7.65 (s, 1H); 8.23 (s, 1H); 8.59 (bs, 1H); 10.95 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-p r op yl-
2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e
(21). Yield: 95%; white solid. IR (KBr): 3210-2920, 1655,
(s, 3H); 1.88 (s, 3H); 5.01 (d, 2H, J ) 8); 5.57 (m, 1H); 6.62 (m,
1H); 7.12 (d, 1H, J ) 8); 7.29 (m, 1H); 7.56 (d, 1H, J ) 8); 7.66
(s, 1H); 7.80 (s, 1H); 8.26 (s, 1H); 8.60 (bs, 1H); 11.26 (bs, 1H).
5-[[(4-Meth oxyph en yl)am in o]car bon yl]am in o-8-(2-ph e-
n yleth yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p y-
r im id in e (31). Yield: 99%; white solid. IR (KBr): 3245-2960,
1
1
1615, 1600, 1510 cm-1. H NMR (CDCl3): δ 1.71 (t, 3H, J )
1660, 1615, 1600, 1500 cm-1. H NMR (CDCl3): δ 3.42 (t, 2H,
7); 2.04 (m, 2H); 4.36 (q, 2H, J ) 7); 6.62 (m, 1H); 7.12 (d, 1H,
J ) 8); 7.27 (m, 1H); 7.56 (d, 1H, J ) 8); 7.66 (s, 1H); 7.80 (s,
1H); 8.24 (s, 1H); 8.62 (bs, 1H); 11.08 (bs, 1H).
J ) 7); 3.82 (s, 3H); 4.60 (t, 2H, J ) 7); 6.60 (m, 1H); 6.93 (d,
2H, J ) 9); 7.09 (m, 2H); 7.20-7.28 (m, 4H); 7.56 (d, 2H, J )
8); 7.60 (s, 1H); 7.89 (s, 1H); 8.59 (bs, 1H); 10.96 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-(2-p h e-
n yleth yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p y-
r im id in e (32). Yield: 98%; white solid. IR (KBr): 3250-2970,
5-[[(4-Meth oxyp h en yl)a m in o]ca r bon yl]a m in o-8-(2-p r o-
p en -1-yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p y-
r im id in e (22). Yield: 90%; white solid. IR (KBr): 3210-2920,
1665, 1620, 1600, 1520 cm-1. 1H NMR (CDCl3): δ 3.83 (s, 3H);
5.01 (d, 2H, J ) 7); 5.36-5.48 (m, 2H); 6.03-6.16 (m, 1H);
6.62(dd, 1H, J ) 2, J ) 4); 6.91(d, 2H, J ) 9); 7.25 (d, 1H, J
) 4); 7.56 (d, 1H, J ) 9); 7.67 (d, 1H, J ) 2); 8.28 (s, 1H); 8.65
(bs, 1H); 10.94 (bs, 1H).
1
1660, 1610, 1515 cm-1. H NMR (CDCl3): δ 3.33 (t, 2H, J )
7); 4.62 (t, 2H, J ) 7); 6.60 (m, 1H); 7.19-7.35 (m, 7H); 7.57
(d, 1H, J ) 8); 7.61 (s, 1H); 7.81 (s, 1H); 7.89 (s, 1H); 8.63 (bs,
1H); 11.27 (bs, 1H).
5-[[(4-Meth oxyph en yl)am in o]car bon yl]am in o-8-(3-ph e-
n ylp r op yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (33). Yield: 98%; white solid. IR (KBr): 3240-
5-[[(4-Meth oxyp h en yl)a m in o]ca r bon yl]a m in o-8-n -bu -
t yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr im i-
d in e (23). Yield: 96%; white solid. IR (KBr): 3250-2960,
1
2950, 1665, 1615, 1600, 1510 cm-1. H NMR (CDCl3): δ 2.46
1
1665, 1610, 1600, 1520 cm-1. H NMR (CDCl3): δ 0.98 (t, 3H,
(m, 2H); 2.73 (t, 2H, J ) 7); 4.42 (t, 2H, J ) 7); 6.67 (m, 1H);
6.96 (d, 2H, J ) 9); 7.22-7.41 (m, 6H); 7.60 (d, 2H, J ) 8);
7.64 (s, 1H); 8.25 (s, 1H); 8.65 (bs, 1H); 11.16 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-(3-p h e-
n ylp r op yl)-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (34). Yield: 99%; pale yellow solid. IR (KBr):
J ) 7); 1.38-1.42 (m, 2H); 2.02-2.05 (m, 2H); 3.82 (s, 3H);
4.39 (t, 2H, J ) 7); 6.63 (m, 1H); 6.92 (d, 2H, J ) 9); 7.25 (m,
1H); 7.57 (d, 2H, J ) 9); 7.67 (s, 1H); 8.23 (s, 1H); 8.60 (bs,
1H); 10.95 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-n -bu tyl-
2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e
(24). Yield: 97%; white solid. IR (KBr): 3240-2970, 1650,
1610, 1510 cm-1. 1H NMR (CDCl3): δ 1.00 (t, 3H, J ) 7); 1.39-
1.41 (m, 2H); 1.99-2.03 (m, 2H); 4.41 (q, 2H, J ) 7); 6.63 (m,
1H); 7.14 (d, 1H, J ) 8); 7.29 (m, 1H); 7.56 (d, 1H, J ) 8); 7.67
(s, 1H); 7.80 (s, 1H); 8.25 (s, 1H); 8.63 (bs, 1H); 11.26 (bs, 1H).
5-[[(4-Met h oxyp h en yl)a m in o]ca r b on yl]a m in o-8-ter t-
b u t yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr i-
m id in e (25). Yield: 97%; white solid. IR (KBr): 3220-2990,
1
3245-2960, 1665, 1610, 1515 cm-1. H NMR (CDCl3): δ 2.46
(m, 2H); 2.73 (t, 2H, J ) 7); 4.43 (t, 2H, J ) 7); 6.66 (m, 1H);
7.19-7.40 (m, 8H); 7.59 (d, 1H, J ) 8); 7.64 (s, 1H); 7.85 (m,
1H); 8.25 (s, 1H); 8.67 (bs, 1H); 11.30 (bs, 1H).
5-[[(4-Me t h oxyp h e n yl)a m in o]ca r b on yl]a m in o-8-[2-
(2′,4′,5′-t r ib r om op h en yl)et h yl]-2-(2-fu r yl)p yr a zolo[4,3-
e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (35). Yield: 79%; yellow
1
solid. IR (KBr): 3230-2960, 1665, 1615, 1600, 1500 cm-1. H
NMR (CDCl3): δ 3.39 (t, 2H, J ) 7); 3.80 (s, 3H); 4.57 (t, 2H,
J ) 7); 6.63 (dd, 1H, J ) 2, J ) 4); 7.09 (s, 1H); 7.27 (d, 1H, J
) 4); 7.43 (s, 1H); 7.62 (d, 1H, J ) 2); 8.01 (s, 1H); 8.63 (bs,
1H); 11.11 (bs, 1H).
1
1658, 1615, 1512 cm-1. H NMR (CDCl3): δ 1.75 (s, 9H); 3.81
(s, 3H); 6.59 (dd, 1H, J ) 2, J ) 4); 6.90 (d, 2H, J ) 8); 7.24 (d,
1H, J ) 4); 7.55 (d, 2H, J ) 9); 7.64 (d, 1H, J ) 2); 8.37 (s,
1H); 8.59 (bs, 1H); 10.93 (bs, 1H).
5-[[(3-Ch lor oph en yl)am in o]car bon yl]am in o-8-[2-(2′,4′,5′-
tr ibr om op h en yl)eth yl]-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr i-
a zolo[1,5-c]p yr im id in e (36). Yield: 83%; yellow solid. IR
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-8-ter t-bu -
t yl-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]p yr im i-
d in e (26). Yield: 93%; white solid. IR (KBr): 3220-2950,
(KBr): 3250-2930, 1670, 1620, 1600, 1500 cm-1 1H NMR
.
1
1665, 1620, 1500 cm-1. H NMR (CDCl3): δ 1.76 (s, 9H); 6.60
(CDCl3): δ 3.41 (t, 2H, J ) 7); 4.63 (t, 2H, J ) 7); 6.63 (dd,

4778 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Baraldi et al.
1H, J ) 2, J ) 4); 7.05- -7.48 (m, 5H); 7.51-7.78 (m, 3H); 8.06
(s, 1H); 8.65 (bs, 1H); 11.25 (bs, 1H).
protein/assay) was performed according to Varani et al.46 In
competition studies, at least 6-8 different concentrations of
compounds were used and nonspecific binding was determined
in the presence of 50 µM NECA for an incubation time of 60
min at 25 °C.
5-[[(4-Meth oxyp h en yl)a m in o]ca r bon yl]a m in o-8-[2-(R-
n a p h th yl)eth yl]-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo-
[1,5-c]p yr im id in e (37). Yield: 68%; yellow solid. IR (KBr):
Binding of [3H]DPCPX to HEK-293 cells (Receptor Biology
Inc., Beltsville, MD) transfected with the human recombinant
3285-2970, 1665, 1610, 1520, 1450 cm-1 1H NMR (CDCl3):
.
δ 3.35 (t, 2H, J ) 6.5); 3.84 (s, 3H); 4.41 (t, 2H, J ) 6.5); 6.63
(dd, 1H, J ) 2, J ) 4); 7.19-7.55 (m, 7H); 7.61-7.90 (m, 6H);
8.05 (s, 1H); 8.97 (bs, 1H); 10.57 (bs, 1H).
A
2B adenosine receptors was performed as already described.42
In particular, assays were carried out for 60 min at 25 °C in
0.1 mL of 50 mM Tris HCl buffer, 10 mM MgCl2, 1 mM EDTA,
0.1 mM benzamidine at pH 7.4, 2 IU/mL adenosine deaminase
containing 40 nM [3H]DPCPX, diluted membranes (20 µg of
protein/assay) and at least 6-8 different concentration of
tested compounds. Nonspecific binding was determined in the
presence of 100 µM of NECA and was always e30% of the total
binding.
5-[[(3-Ch lor op h en yl)a m in o]ca r b on yl]a m in o-8-[2-(R-
n a p h th yl)eth yl]-2-(2-fu r yl)p yr a zolo[4,3-e]1,2,4-tr ia zolo-
[1,5-c]p yr im id in e (38). Yield: 68%; yellow solid. IR (KBr):
3300-2975, 1660, 1615, 1515, 1450 cm-1 1H NMR (CDCl3):
.
δ 3.37 (t, 2H, J ) 6.5); 4.38 (t, 2H, J ) 6.5); 6.62 (dd, 1H, J )
2, J ) 4); 7.08-7.46 (m, 7H); 7.57-7.89 (m, 6H); 8.02 (s, 1H);
9.45 (bs, 1H); 11.34 (bs, 1H).
Binding of [3H]MRE3008-F20 to CHO cells transfected with
the human recombinant A3 adenosine receptors was performed
according to Varani et al.42 Competition experiments were
carried out in duplicate in a finale volume of 250 µL in test
tubes containing 1 nM [3H]MRE3008-F20, 50 mM Tris HCl
buffer, 10 mM MgCl2 at pH 7.4, 100 µL of diluted membranes
(50 µg protein/assay) and at least 6-8 different concentrations
of examined ligands. Incubation time was 120 min at 4 °C,
according to the results of previos time course experiments.42
Nonspecific binding was defined as binding in the presence of
1 µM of MRE3008-F20 and was about 25% of total binding.
Bound and free radioactivity were separated by filtering the
assay mixture through Whatman GF/B glass fiber filters using
a Micro-Mate 196 cell harvester (Packard Instrument Co.). The
filter bound radioactivity was counted on a Top Count (ef-
ficiency 57%) with Micro-Scint 20. The protein concentration
was determined according to the Bio-Rad method55 with bovine
albumin as the reference standard.
Ad en yla te Cycla se Assa y. Membrane preparation was
suspended in 0.5 mL of incubation mixture (50 mM Tris HCl,
10 mM MgCl2, 1 mM EDTA, pH 7.4) containing 5 µM GTP,
0.5 mM 4-(3-buthoxy-4-methoxybenzyl)-2-imidazolidinone (Ro
20-1274) as phosphodiesterase inhibitor, 2.0 IU/mL adenosine
deaminase and preincubated for 10 min in a shaking bath at
37 °C. Then IB-MECA or the antagonists examined plus ATP
(1 mM) and forskolin 10 µM were added to the mixture and
the incubation continued for a further 10 min. The potencies
of antagonists were determined by antagonism of the IB-
MECA (100 nM)-induced inhibition of cyclic AMP production.
The reaction was terminated by transferring to a boiling water
bath. Boiling was for 2 min, and then the tubes were cooled to
room temperature and centrifuged at 2000g for 10 min at 4
°C. Supernatants (100 µL) were used in a competition protein
binding assy carried out essentially according to Varani et al.42
Samples of cyclic AMP standards (0-10 pmol) were added
to each test tube containing the incubation buffer (0.1 M trizma
base, 8.0 mM aminophylline, 6.0 mM 2-mercaptoethanol, pH
7.4) and [3H]cyclic AMP in a total volume of 0.5 mL. The
binding protein, previously prepared from beef adrenals, was
added to the samples previously incubated at 4 °C for 150 min;
after addition of charcoal the samples were centrifugated at
2000g for 10 min. The clear supernatant (0.2 mL) was mixed
with 4 mL of Atomlight in a LS-1800 Beckman scintillation
counter.
5-[[(4-Meth oxyph en yl)am in o]car bon yl]am in o-9-ch lor o-
2-(2-fu r yl)[1,2,4]tr iazolo[1,5-c]qu in azolin e (39). Yield: 88%;
pale yellow solid. IR (KBr): 3250-2975, 1665, 1610, 1515, 1450
1
cm-1. H NMR (CDCl3): δ 3.80 (s, 3H); 6.65 (dd, 1H, J ) 2, J
) 4); 6.87 (d, 1H, J ) 8); 6.95 (d, 2H, J ) 11); 7.27 (d, 2H, J
) 11); 7.53 (d, 1H, J ) 4); 7.56 (d, 1H, J ) 8); 7.70 (d, 1H, J
) 2); 7.79 (s, 1H); 8.53 (bs, 1H); 11.18 (bs, 1H).
5-[[(3-Ch lor op h en yl)a m in o]ca r bon yl]a m in o-9-ch lor o-
2-(2-fu r yl)[1,2,4]tr iazolo[1,5-c]qu in azolin e (40). Yield: 92%;
pale yellow solid. IR (KBr): 3280-2955, 1668, 1615, 1510, 1450
1
cm-1. H NMR (CDCl3): δ 6.66 (dd, 1H, J ) 2, J ) 4); 6.91-
7.39 (m, 4H); 7.65 (s, 1H); 7.78-7.89 (m, 3H); 8.06 (d, 1H, J )
2); 8.75 (bs, 1H); 10.98 (bs, 1H).
Com p u ta tion a l Meth od ologies. Calculations were per-
formed on a Silicon Graphics O2 R10000 workstation. All the
pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives were
constructed using the Molecule Builder module of Molecular
Operating Environment (MOE 1998.10).47 These structures
were minimized using the MMFF94 force field,48-51 until the
rms value of the truncated Newton method (TN) was <0.001
kcal/mol/Å. The optimized geometries of all pyrazolo[4,3-
e]1,2,4-triazolo[1,5-c]pyrimidine structures were fully mini-
mized using the RHF/AM1//RHF/3-21G(*) ab initio level of
Gaussian 98.52 Atomic charges were calculated by fitting to
electrostatic potential maps (CHELPG method).53 Molecular
volume and log P values (log of the octanol/water partition
coefficient), a hydrophobicity indicator, were empirically cal-
culated using the atom fragment method implemented in the
QuaSAR-Descriptor module of MOE.47
Biology. CHO Mem br a n es P r ep a r a tion . The expression
of human A1, A2A and A3 receptors in CHO cells has been
previously described.54 The cells were grown adherently and
maintained in Dulbecco’s modified Eagle’s medium with nutri-
ent mixture F12 without nucleosides at 37 °C in 5% CO2/95%
air. Cells were split two or three times weekly and then the
culture medium was removed for membrane preparations. The
cells were washed with phosphate-buffered saline and scrapped
off into flasks in ice-cold hypotonic buffer (5 mM Tris HCl, 2
mM EDTA, pH 7.4). The cell suspension was homogenized with
a Polytron and the homogenate was centrifuged for 30 min at
48000g. The membrane pellet was resuspended in 50 mM Tris
HCl buffer at pH 7.4 for A1 adenosine receptors, in 50 mM
Tris HCl, 10 mM MgCl2 at pH 7.4 for A2A adenosine receptors,
in 50 mM Tris HCl, and 10 mM MgCl2, 1 mM EDTA at pH
7.4 for A3 adenosine receptors and were utilized for binding
and adenylate cyclase assays.
Ack n ow led gm en t. We thank Medco Research, Tri-
angle Park, NC, for financial support, Dr. Kenneth A.
J acobson, NIH, Bethesda, MD, for use of the human A3
adenosine receptor model in the creation of the phar-
macophore maps, and Dr. Ennio Ongini, San Raffaele
Scientific Park, Milan, Italy, for providing us with [3H]-
SCH 58261.
Hu m a n Clon ed A1, A2A, A2B, a n d A3 Ad en osin e Recep -
tor Bin d in g Assa y. Binding of [3H]DPCPX to CHO cells
transfected with the human recombinant A1 adenosine recep-
tor was performed as previously described.42
Displacement experiments were performed for 120 min at
25 °C in 0.20 mL of buffer containing 1 nM [3H]DPCPX, 20
µL of diluted membranes (50 µg of protein/assay) and at least
6-8 different concentrations of examined compounds. Non-
specific binding was determined in the presence of 10 µM of
CHA and this was always e10% of the total binding.
Binding of [3H]SCH 58261 to CHO cells transfected with
the human recombinant A2A adenosine receptors (50 µg of
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