7-Substituted 5-amino-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as A2A adenosine receptor antagonists: A study on the importance of modifications at the side chain on the activity and solubility

Article abstract of DOI:10.1021/jm010924c

It was demonstrated in the early 1990s that adenosine exerts many physiological functions through the interaction with four different receptors, named A₁, A_2A, A_2B, and A₃. In the past few years, our group has been involved in the development of A_2A antagonists, which led to the synthesis of SCH 58261 (1), the first potent and selective adenosine A_2A antagonist, which has been widely used as a reference compound. In this paper, we present an extended series of pyrazolotriazolopyrimidines synthesized with the aim to investigate the influence of the substitutions on the pyrazole ring. The choice of the substituents was based on their capability to improve water solubility while retaining high affinity and selectivity at the human A_2A adenosine receptor subtype. In this series, some structural characteristics that are important for activity, i.e., tricyclic structure, free amino group at 5-position, furan ring, and substituent at 7-position on the pyrazole moiety, have been maintained. We focused our attention on the nature of the phenyl ring substituent to improve water solubility. Following this strategy, we developed new compounds with good affinity and selectivity for A_2A adenosine receptors, such as 8d (K_i 0.12 nM; hA₁/hA_2A ratio = 1025; Rm = 2.8), 8h (K_i 0.22; hA₁/hA_2A ratio = 9818; R_m = 3.4), 8i (K_i 0.18 nM; hA₁/hA_2A ratio = 994; R_m = 2.8), 8k (K_i 0.13 nM; hA₁/hA_2A ratio = 4430; R_m = 3.6), and 14b (K_i 0.19 nM; hA₁/hA_2A ratio = 2273; R_m = 2.7). All the new synthesized compounds have no significant interaction with either A_2B or A₃ receptor subtypes. This new series of compounds deeply enlightens some structural requirements to display high affinity and selectivity for the A_2A adenosine receptor subtype, although our goal of identifying new compounds with increased water solubility was not completely achieved. On this basis, other strategies will be devised to improve this class of compounds with a profile that appears to be promising for treatment of neurodegenerative disorders, such as Parkinson's disease.

Full text of DOI:10.1021/jm010924c

J . Med. Chem. 2002, 45, 115-126
115
7-Su bstitu ted 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 es
a s A2A Ad en osin e Recep tor An ta gon ists: A Stu d y on th e Im p or ta n ce of
Mod ifica tion s a t th e Sid e Ch a in on th e Activity a n d Solu bility
Pier Giovanni Baraldi,*,† Barbara Cacciari,† Romeo Romagnoli,† Giampiero Spalluto,‡ Angela Monopoli,§
Ennio Ongini,§ Katia Varani,| and Pier Andrea Borea|
Dipartimento di Scienze Farmaceutiche, 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, Centro Ricerche Schering-Plough, Parco Scientifico S. Raffaele, Via Olgettina 58, I-20132 Milano, Italy,
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
Received May 9, 2001
It was demonstrated in the early 1990s that adenosine exerts many physiological functions
through the interaction with four different receptors, named A1, A2A, A2B, and A3. In the past
few years, our group has been involved in the development of A2A antagonists, which led to the
synthesis of SCH 58261 (1), the first potent and selective adenosine A2A antagonist, which has
been widely used as a reference compound. In this paper, we present an extended series of
pyrazolotriazolopyrimidines synthesized with the aim to investigate the influence of the
substitutions on the pyrazole ring. The choice of the substituents was based on their capability
to improve water solubility while retaining high affinity and selectivity at the human A2A
adenosine receptor subtype. In this series, some structural characteristics that are important
for activity, i.e., tricyclic structure, free amino group at 5-position, furan ring, and substituent
at 7-position on the pyrazole moiety, have been maintained. We focused our attention on the
nature of the phenyl ring substituent to improve water solubility. Following this strategy, we
developed new compounds with good affinity and selectivity for A2A adenosine receptors, such
as 8d (Ki 0.12 nM; hA1/hA2A ratio ) 1025; Rm ) 2.8), 8h (Ki 0.22; hA1/hA2A ratio ) 9818; Rm
)
3.4), 8i (Ki 0.18 nM; hA1/hA2A ratio ) 994; Rm ) 2.8), 8k (Ki 0.13 nM; hA1/hA2A ratio ) 4430;
Rm ) 3.6), and 14b (Ki 0.19 nM; hA1/hA2A ratio ) 2273; Rm ) 2.7). All the new synthesized
compounds have no significant interaction with either A2B or A3 receptor subtypes. This new
series of compounds deeply enlightens some structural requirements to display high affinity
and selectivity for the A2A adenosine receptor subtype, although our goal of identifying new
compounds with increased water solubility was not completely achieved. On this basis, other
strategies will be devised to improve this class of compounds with a profile that appears to be
promising for treatment of neurodegenerative disorders, such as Parkinson’s disease.
In tr od u ction
field, there are a number of compounds belonging to
different chemical classes possessing a common feature,
namely, high A2A receptor affinity combined with weak
interactions with other adenosine receptors.5,6
Part of the biological activity of adenosine occurs
through the activation of specific receptors located on
cell membranes and belonging to the extensive family
of G-protein coupled receptors.1,2 Currently, four ad-
enosine receptors have been cloned and characterized
pharmacologically, namely, A1, A2A, A2B, and A3. The
adenosine receptors are associated with different second
messenger systems: A1 and A3 mediate adenylate
cyclase inhibition, whereas A2A and A2B stimulate the
adenylate cyclase activity controlling intracellular cyclic
AMP levels.1
One of the most important reference compound re-
ported is the pyrazolotriazolopyrimidine SCH 58261 (1),
(5-amino-7-(2-phenyl)ethyl-2-(2-furyl)pyrazolo[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidine), which has been widely char-
acterized in a variety of binding and functional assays.7
From the initial work on SCH 58261 (1), we have
prepared several different compounds series, bearing
different substitution at the pyrazole nitrogen, such as
SCH 63390 (2) and their oxygenated derivatives (3-6)
(Chart 1).
The result of this effort has been an improvement of
A2A receptor affinity, separation from other receptors,
and improved biological activity in pharmacological
assays.8,9 This synthetic strategy is relevant for the
identification of compounds possessing a suitable phar-
macological profile in models of neurodegenerative
disorders for diagnostic10 and therapeutic uses, a target
area of medical research where A2A receptor antagonists
can have a highly interesting perspective.11,2,3
Recently, important progress has been made with the
development of selective A2A receptor antagonists hav-
ing an interesting pharmacological profile.3,4 In this
* Corresponding author 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.
‡ Universita` degli Studi di Trieste.
§ Centro Ricerche Schering-Plough.
| Dipartimento di Medicina Clinica e Sperimentale-Sezione di Far-
macologia, Universita` degli Studi di Ferrara.
10.1021/jm010924c CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/06/2001

116 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Baraldi et al.
Ch a r t 1
Ch a r t 2
Some characteristics of the reference compounds have
to be considered important from a structure-activity
relationship point of view; mainly, the tricyclic structure
of the pyrazolotriazolopyrimidine, the presence of the
furan ring, the free amino group at the 5-position, and
the arylalkyl substituent on the nitrogen at the 7-posi-
tion seem to be essential for both affinity and selectivity.
Thus, we decided to investigate the modifications on the
phenyl ring of the side chain using different polar
moieties, based upon the good results obtained with the
oxygenated derivatives 3-6 (Chart 2).7-9
In particular, we conjectured to introduce different
substituents, like basic or acidic moieties suitable for
the preparation of salts or possible prodrugs, with the
aim to modify the physicochemical properties of the
compounds, maintaining the same affinity and selectiv-
ity for the adenosine A2A receptor subtype. These
modifications should be able to improve the water
solubility of compounds that could be proposed as
possible candidates for further development. In addition,
a full biological characterization at all four cloned
an enlarged series of derivatives has been prepared
(Table 1).
With this aim in mind, a common key intermediate
that, after alkylation with an appropriate alkyl halide,
could afford the desired final compounds has been
prepared. This common intermediate has been identified
in 2-furan-2-yl-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-ylamine (7), which has been synthesized as
previously reported.8 This derivative could be easily
alkylated, using anhydrous potassium carbonate as base
in dry DMF, on the pyrazole nitrogen without competi-
tion by the amino group on the pyrimidine ring to afford
a series of alkylated compounds. The two regioisomers
at the 7- and the 8-positions of the pyrazolotriazolo-
pyrimidine nucleus (ratio 3:1) were purified by flash
chromatography, and only the alkylated derivatives at
the 7-position were tested in biological assays (8a -g,
Scheme 1). When the appropriate alkyl halides were not
commercially available, they were synthesized by stand-
ard methods (Schemes 3 and 4).12-15 All the other
compounds 8h -n were obtained by modifications of the
former analogues (Scheme 2).16
human adenosine receptor subtypes (hA1, hA2A, hA2B
hA3) of the synthesized derivatives is reported.
,
A different chemical pathway was utilized for the
synthesis of the sulfo analogues of SCH 58261 (1) and
its homologue SCH 63390 (2) due to the presence of the
sulfonic moiety on the phenyl ring, which hampers the
alkylation of 7 under basic conditions. To avoid these
problems, the chlorosulfonic group was introduced
directly onto the SCH 58261 (1) and SCH 63390 (2)
Ch em istr y. In previous papers, we reported the
synthesis of compounds structurally related to SCH
58261 starting from the most appropriate hydrazines
in order to obtain only the 7-substituted derivatives.8,9
As mentioned above, to better evaluate the importance
of the substituents on the phenyl ring in the side chain,

Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 117
Ta ble 1. Structures and Physicochemical Parameters of Synthesized Compounds
skeletons by chlorosulfonic acid to afford the corre-
sponding derivatives 9 and 10, respectively, in very good
yield.17 The latter could be easily transformed in the
corresponding sulfonamido analogues 11b -f through
the reaction with the appropriate amines and the
sulfonic acids 11a and 12 by hydrolysis (Scheme 5).
) 658, hA3/hA2A ratio > 3000) and for the presence of a
biologically common substituent on the pyrazole ring.9
For these reasons, we decided to modify this compound
to improve its affinity and, in particular, its water
solubility (Rm ) 3.6). The goal was achieved by the intro-
duction of a sulfonic moiety to furnish 13. A further im-
provement was performed by the introduction of func-
tions, such as carboxylic and alcoholic moieties, starting
from the dihydroxy derivative 69 by reaction with
diethyl dibromomalonate and subsequent reduction to
the alcohol or decarboxylation (14a -e) (Scheme 6).
In a previous paper, we reported 5-amino-7-[3-[3,4-
(methylenedioxy)phenyl]propyl]-2-(2-furyl)pyrazolo[4,3-
e]1,2,4-triazolo[1,5-c]pyrimidine (5), which was of inter-
est for its affinity and selectivity at the human A2A
adenosine receptors (hA2A Ki ) 3.3 nM, hA1/hA2A ratio

118 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Baraldi et al.
Sch em e 1
Sch em e 2
Resu lts a n d Discu ssion
e]1,2,4triazolo[1,5-c]pyrimidine ([3H]-SCH 58261) (A2A),20
and [3H]-5-(4-methoxyphenylcarbamoyl)amino-8-propyl-
2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimi-
dine ([3H]-MRE3008-F20) (A3)19 have been used as
radioligands in binding assays.
The binding data of the new series of pyrazolotria-
zolopyrimidines and their water solubility measured as
Rm values are reported in Tables 2 and 3, respectively.
Table 2 summarizes the receptor binding affinities of
8a -n , 11a -f, 12, 13, and 14a -e determined at the
human A1, A2A, A2B, and A3 receptors expressed in CHO
(A1, A3) or HEK-293 (A2A, A2B) cells. [3H]-1,3-Dipropyl-
8-cyclopentyl xanthine ([3H]-DPCPX)18,19 (A1 and A2B),
[3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-
All the new compounds show a good affinity and
selectivity for hA2A adenosine receptor with a different
degree of selectivity versus hA1. In contrast, all the
synthesized compounds resulted to be almost inactive
or inactive in binding hA2B and hA3 receptors. This

Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 119
Sch em e 3
Sch em e 4
nM; hA1/hA2A ratio ) 2000; Rm ) 2.5; 11c hA2A Ki )
0.8 nM; hA1/hA2A ratio ) 3118, Rm ) 2.7). These
findings were not so surprisingly in view of our hypoth-
esis previously reported.9 Recently, on the basis of the
thermodynamic studies, we hypothesized the presence
of a lipophilic pocket where the side chain can take place
and be able to form a hydrogen bond. Probably the
lipophilic properties of this pocket hampered the adjust-
ment of the substituent, possibly due to the anionic form
of the sulfonic moieties at the physiological pH. The
parameters of water solubility and receptor affinity
suggest that compounds 11b and c are the best com-
pounds in terms of human A2A affinity in the series of
the sulfonic derivatives. Moreover, they support the
hypothesis of a hydrogen bond, during the adjustment
of the side chain in this pocket. In this family of
compounds, 11e was prepared in analogy with nitrogen
mustards with the aim to obtain an irreversible A2A
antagonist to more deeply investigate the biochemical
characteristics of this receptor subtype.22 Surprisingly,
11e showed a reversible antagonism similar to that of
the other reported compounds, displaying an interesting
affinity and selectivity for the human A2A adenosine
receptor (11e hA2A Ki ) 0.59 nM; hA1/hA2A ratio ) 8977;
Rm ) 3.8), although the water solubility, measured by
the Rm value, was shown to be poor as expected.
experimental observation further validates the efficiency
and selectivity of pyrazolotriazolopyrimidine derivatives
in comparison to the structurally related reference
compound CGS 15943 (5-amino-9-chloro-2-(2-furyl)-
[1,2,4]triazolo[1,5-c]quinazoline), which turned out to be
nonselective showing binding in the nanomolar range
of hA1 (Ki ) 4.4 nM), hA2A (Ki ) 0.43 nM), hA2B (Ki )
23 nM), and hA3 (Ki ) 85 nM) receptors.21 This finding
strongly supports, as previously observed,21 that the
pyrazole nitrogens also play a fundamental role in
receptor recognition and discrimination and that the
substitutions on the pyrazolotriazolopyrimidine nucleus
modulate affinity and selectivity vs adenosine receptor
subtypes.
Unfortunately, our main goal, water solubility, was
not completely achieved; in fact, the two sulfonic ana-
logues 11a and 12 was shown to be completely water-
soluble, but a significant reduction of both the affinity
and the selectivity was observed (11a hA2A Ki ) 100 nM;
hA1/hA2A ratio ) 1.9; Rm ) 0.78; 12 hA2A Ki ) 140; hA1/
hA2A ratio ) 1; Rm ) 0.77). The other sulfo derivatives
(11b-d , f) are quite water-soluble, as indicated by the
Rm values (Table 3). A good retention of the affinity for
To retain the water solubility of 11a and 12 and to
restore the affinity and selectivity for the A2A receptor
subtype, a weaker acidic function on the phenyl ring,
such as the carboxylic moiety or its ethyl ester, was
introduced. The affinity of the 8m for human A2A
adenosine receptor was acceptable and comparable to
the corresponding unsubstituted reference compound
SCH 63390 (2), but the water solubility was lower than
that of the sulfonic derivative 12, although the Rm value
was better than that of reference compound 2 (8m hA2A
Ki ) 4.4 nM; hA1/hA2A ) 1128; Rm ) 2.6; SCH 63390,
2, hA2A Ki ) 1.2 nM; hA1/hA2A ) 291; Rm ) 3.6; 12 hA2A
Ki ) 140 nM; hA1/hA2A ) 1; Rm ) 0.77). Moreover, the
carboxylic function, when introduced on the skeleton of
5, was successful in terms of the water solubility as
shown by the Rm values of its analogues, i.e., the
dicarboxylic derivative 14c (Rm ) 0.18) and its sodium
salt 14e (Rm ) 0.16), but the affinity for the human A2A
A2A adenosine receptor was detected (11b hA2A Ki ) 1.31

120 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Baraldi et al.
Sch em e 5
Sch em e 6
adenosine receptor strongly decreased (14c hA2A Ki )
120 nM; hA1/hA2A ) 78; 14e hA2A Ki ) 120 nM; hA1/
hA2A ) 79), displaying Ki values comparable to the
sulfonic derivatives 11a and 12. The monocarboxylated
compound 14d and mainly its ethyl ester 14a showed
a good affinity for human A2A adenosine receptor, but

Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 121
Ta ble 2. Binding Affinity at hA1, hA2A, hA2B, and hA3 Adenosine Receptors of Synthesized Compounds
Ki (nM)
a
b
c
d
compd
hA1
hA2A
hA2B
hA3
hA1/hA2A
hA2B/hA2A
hA3/hA2A
1 SCH 58261
2 SCH 63390
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
11a
11b
11c
11d
11e
11f
12
549 (322-987)
1.1 (0.75-1.6)
1.2 (1.03-1.4)
1.00 (0.94-1.27)
4.8 (4.58-5.03)
1.1 (0.9-1.3)
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
499
291
1026
87
507
1025
75.5
1123
9760
9818
994
10
4430
1.36
1128
1064
1.9
2 007
3 118
97
>9 090
>8 333
>10 000
>2 083
>9 090
.10 000
>116
>2 500
.10 000
.10 000
.10 000
>1 666
.10 000
>181
>9 090
>8 333
>10 000
>2 083
>9 090
.10 000
>116
>2 500
.10 000
.10 000
.10 000
>1 666
.10 000
>181
350 (332-370)
1026 (785-1341)
419 (374-470)
558 (467-667)
123 (98-154)
0.12 (0.1-0.16)
86 (77-96)
6496 (6024-7006)
4494 (4040-5000)
4197 (3844-4581)
2160 (1816-2569)
179 (146-220)
4.0 (3.6-4.5)
0.43 (0.4-0.47)
0.22 (0.16-0.31)
0.18 (0.15-0.22)
6.0 (5.2-7.6)
60 (55-70)
576 (532-625)
0.13 (0.10-0.17)
55 (49-61)
4.4 (4.1-4.7)
75 (66-85)
4965 (4691-5255)
4927 (4392-5528)
190 (172-209)
>2 272
>2 160
>100
>2 272
>2 160
>100
4.63 (4.28-5.01)
100 (89-112)
1.31 (1.18-1.43)
0.80 (0.69-0.92)
3.8 (3.4-4.2)
0.59 (0.49-0.70)
50.0 (48.9-52.4)
140 (129-152)
75 (66-85)
2630 (2366-2924)
2495 (2153-2891)
369 (324-420)
5297 (4943-5678)
9330 (8783-9911)
139 (107-181)
6392 (5697-7171)
1793 (1460-2201)
432 (363-514)
9399 (9038-9773)
3599 (3420-3788)
9533 (9257-9817)
>7 633
.10 000
>2 631
.10 000
>200
>7 633
.10 000
>2 631
.10 000
>200
8 977
186
1
>714
>133
>714
>133
13
85
14a
14b
14c
14d
14e
5.48 (4.95-6.08)
0.19 (0.16-0.21)
120 (98-144)
59 (49-72)
327
2 273
78
>1 824
.10 000
>83
>1 824
.10 000
>83
61
79
>169
>83
>169
>83
120 (98-144)
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 HEK-293 cells. c Displacement of specific [3H]-DPCPX binding at human
d
A
2B receptors expressed in HEK-293 cells (n ) 3-6). Displacement of specific [3H]-MRE3008-F20 binding at human A3 receptors expressed
in CHO cells. Data are expressed as geometric means, with 95% confidence limits.
Ta ble 3. Rm Values of Synthesized Compounds Measured at
pH 7.0 by TLC in Methanol-Water System
receptor were significantly reduced (13 hA2A Ki ) 75
nM; hA1/hA2A ratio ) 85; Rm ) 1.2).
compd
Rm (0)a
compd
Rm (0)a
The cyano derivative 8e showed a poor affinity for the
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
3.9 ( 0.2
2.7 ( 0.2
2.4 ( 0.2
2.8 ( 0.2
2.9 ( 0.2
3.0 ( 0.1
3.3 ( 0.1
3.4 ( 0.2
2.8 ( 0.2
2.5 ( 0.2
3.6 ( 0.2
2.9 ( 0.2
2.6 ( 0.2
2.1 ( 0.2
11a
11b
11c
11d
11e
11f
12
0.78 ( 0.1
2.5 ( 0.1
2.7 ( 0.2
1.9 ( 0.2
3.8 ( 0.1
2.2 ( 0.2
0.77 ( 0.2
1.2 ( 0.1
3.4 ( 0.2
2.7 ( 0.1
0.18 ( 0.1
2.1 ( 0.1
0.16 ( 0.1
A2A receptor subtype, but it was a useful key-intermedi-
ate for obtaining the aminomethyl analogue 8j, the
N-hydroxyamidine 8i and the amidine 8l, which showed
a restored affinity for this receptor subtype. In particu-
lar, the compound 8i displayed a good affinity for the
A2A receptor and a better water solubility with respect
to the reference compound SCH 63390 (2) (8i hA2A Ki
) 0.18 nM hA1/hA2A ratio ) 994; Rm ) 2.8; SCH 63390,
2, Rm ) 3.6).
Considering the importance of a moiety on the phenyl
ring of the side chain capable of a hydrogen bond, we
also investigated the modification of the hydroxy group
of 3 previously described.9 In particular, we synthesized
8g and 8n structurally related to the selective A2A
agonist CGS 21680 (2-[4-(2-carboxyethyl)phenethyl-
amino]-5′-N-ethylcarboxamido adenosine).23 8g showed
a high affinity but a low water solubility differently from
the derivative 8n , which had exactly an opposite be-
havior, confirming a common trend (8g hA2A Ki ) 0.43
nM; hA1/hA2A ratio ) 9760; Rm ) 3.3; 8n hA2A Ki ) 4.63
nM; hA1/hA2A ratio ) 1064; Rm ) 2.1).
13
14a
14b
14c
14d
14e
a
The Rm values were measured with a mobile phase of different
concentrations of CH3OH/H2O. Rm values are reported as theoreti-
cal at 0% organic solvent in the mobile phase (Rm(0)).
their Rm values were not encouraging (14d hA2A Ki )
59 nM, hA1/hA2A ) 61; Rm ) 2.1; 14a hA2A Ki ) 5.48
nM; hA1/hA2A ) 327; Rm ) 3.4). On the whole, none of
these derivatives showed an interesting profile at the
human A2A adenosine receptor, strongly supporting the
negative influence of acidic moieties for the hA2A affin-
ity, which seems strictly correlated to the nature of the
function on the phenyl ring. Better results were ob-
tained with the corresponding dihydroxy derivative 14b,
which shows again a good affinity but lower water
solubility (14b hA2A Ki ) 0.19 nM; hA1/hA2A ratio )
2,273; Rm ) 2.7). Also in this case, we tried to introduce
the sulfonic moiety on 5 but, as expected, both the
affinity and the selectivity at the human A2A adenosine
Finally, we decided to investigate the modification of
the amino group on the phenyl ring, with the aim to
obtain soluble salts, even though the basicity of an
aromatic amino group is not significantly high. This
choice was fruitful mainly in regards of affinity for the
adenosine A2A receptor subtype, as shown in Table 2.
The two derivatives 8h and 8k possess high affinity and
selectivity for the human A2A adenosine receptor, even

122 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Baraldi et al.
mL) to the cooled reaction mixture and separating the layers.
The organic layer was washed with water, 5% aqueous
NaHCO3 solution, and saturated NaCl solution; dried; and
though the water solubility was not significantly in-
creased (8h hA2A Ki ) 0.22 nM; hA1/hA2A ratio ) 9818;
Rm ) 3.4; 8k hA2A Ki ) 0.13 nM; hA1/hA2A ) 4430; Rm
) 3.6). However, from these results, the latter com-
pounds appear to be more promising for further phar-
macological studies, possibly as prodrugs.
1
evaporated to obtain 17 as an oil (0.19 g, 76% yield). H NMR
(CDCl3): 1.39 (t, 3H, J ) 8), 2.06-2.13 (m, 2H), 2.80-2.88
(m, 2H), 3.31 (t, 2H, J ) 6), 4.36 (q, 2H, J ) 8), 7.25-7.29 (m,
3H), 7.97 (d, 2H, J ) 6). IR (neat): 3415, 1715. Anal. (C12H16O3)
C, H.
Con clu sion s
Syn th esis of 2-(4-Hyd r oxyp h en yl)eth a n ol (19). 4-Hy-
droxyphenylacetic acid (18, 5 g, 0.032 mol) was refluxed in
ethanol (50 mL) in the presence of H2SO4 (50 µL) for 3 h, then
the solvent was removed, and the residue (5.58 g, 0.031 mol)
was dissolved in dry THF (15 mL). This solution was added
dropwise to a suspension of LiAlH4 (1.76 g, 0.046 mol) in dry
THF (30 mL) cooled at 0 °C. The mixture was allowed to reach
room temperature and was stirred for 3 h. The solution was
cooled again to 0 °C, and water was added slowly to precipitate
salts that were filtered off. The filtrate was dried and
evaporated to furnish the desired compound as an oil (4.2 g,
95% yield). 1H NMR (CDCl3): 2.74 (t, 2H, J ) 8), 3.79 (bs,
1H), 3.86 (t, 2H, J ) 8), 6.68 (d, 2H, J ) 6), 6.95 (d, 2H, J )
6), 10.51 (bs, 1H). IR (neat): 3435. Anal. (C8H10O2) C, H.
Gen er a l P r oced u r e for th e Syn th esis of Ch lor o De-
r iva tives (20-22). A solution of alcohol (0.066 mol) and
pyridine (5.5 mL) in dry benzene (40 mL) cooled at 0 °C was
added dropwise of a solution of thionyl chloride (9.5 mL) in
dry benzene (20 mL) and stirred at room temperature for 2 h.
At the end of this time, the mixture was cooled again at 0 °C,
and water (50 mL) was added. The organic layer was first
washed twice with saturated NaHCO3 (30 mL), then dried,
and evaporated. The residue was purified by flash chroma-
tography (EtOAc/light petroleum 20%).
In conclusion, the present study confirmed the im-
portance of some common structural parameters re-
quired to maintain affinity and selectivity for the human
A2A adenosine receptor in the SCH 58261 series. There-
fore, the series of compounds (retaining the pyrazolo-
triazolopyrimidine nucleus, a free amino group at the
5-position, the furan ring, and the substituent at the
7-position) still displays good affinity and selectivity for
this receptor subtype. Unfortunately, our rationale of
designing a new compound endowed with both water
solubility and affinity was not achieved, which can be
explained in part by Lipinski’s rules.24 Anyway, the
study herein presented provides new useful information
about the structural requirements necessary for A2A
antagonist receptor recognition.
Exp er im en ta l Section
Ch em istr y. Reaction courses and product mixtures were
routinely monitored by thin-layer chromatography (TLC) on
silica gel (precoated F254 Merck plates) and visualized with
iodine or aqueous potassium permanganate. Infrared spectra
(IR) were measured on a Perkin-Elmer 257 instruments. 1H
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 hertz. Light
petroleum refers to the fractions boiling at 40-60 °C. Melting
points were determined on a Buchi-Tottoli instrument and are
uncorrected. Chromatography was performed with Merck
230-400 mesh silica gel. All products reported showed IR and
1H NMR spectra in agreement with the assigned structures.
Organic solutions were dried over anhydrous sodium 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.
Syn th esis of 3-(4-Cya n op h en yl)p r op a n ol (16). 4-Cyano-
benzaldehyde (15, 5 g, 0.038 mol) and triphenylphosphor-
anylidene acetic acid ethyl ester (16 g, 0.045 mol) were
dissolved in dry benzene (70 mL), and the solution was stirred
at room temperature for 12 h. Then the solvent was removed
under vacuum, and the residue was taken up with light
petroleum; the solid that formed was removed by filtration,
and the organic phase was evaporated to give 3-(4-cyano-
phenyl)-3-propenoic acid ethyl ester as a yellow oil. The oil
(4.59 g, 0.022 mol), without any further purification, was
dissolved in absolute ethanol (100 mL) and added dropwise to
a suspension of sodium borohydride (8.55 g, 0.22 mol) in
ethanol (100 mL) cooled at 0 °C. The mixture was allowed to
reach room temperature, and stirring was maintained for 18
h after which time the mixture was poured onto water and
extracted with ethyl acetate several times. The combined
organic solutions were washed twice with aqueous 2 N HCl
(20 mL) and finally once with water (30 mL). The organic
phase was dried, and subsequent evaporation of the solvent
gave 16 as a light yellow oil (3.22 g, 53% yield from 15). 1H
NMR (CDCl3): 1.86-1.93 (m, 2H), 2.76 (t, 2H, J ) 8), 3.65-
3.71 (m, 2H), 7.24-7.28 (m, 3H), 7.96 (d, 2H, J ) 8). IR
(neat): 3403, 2228, 1607. Anal. (C10H11NO) C, H, N.
1
3-(4-Cya n op h en yl)p r op yl Ch lor id e (20). 89% yield; H
NMR (CDCl3): 2.05-2.15 (m, 2H), 2.84 (t, 2H, J ) 7), 3.52 (t,
2H, J ) 7), 7.32 (d, 2H, J ) 8), 7.56 (d, 2H, J ) 8). IR (neat):
2261. Anal. (C10H10ClN) C, H, N.
E t h yl 4-(3-Ch lor op r op yl)b en zoa t e (21). 91% yield; 1H
NMR (CDCl3): 1.38 (t, 3H, J ) 8), 2.05-2.12 (m, 2H), 2.83 (t,
2H, J ) 8), 3.51 (t, 2H, J ) 8), 4.36 (q, 2H, J ) 8), 7.27 (d, 2H,
J ) 8), 7.96 (d, 2H, J ) 8). IR (neat): 1716. Anal. (C12H15
ClO2) C, H.
-
2-(4-Hyd r oxyp h en yl)eth yl Ch lor id e (22). 88% yield; 1H
NMR (CDCl3): 3.01 (t, 2H, J ) 7), 3.67 (t, 2H, J ) 7), 6.86 (d,
2H, J ) 8), 7.14 (d, 2H, J ) 8), 10.3 (bs, 1H). IR (neat): 3355.
Anal. (C8H9ClO) C, H.
Syn th esis of Eth yl 4-(2-Ch lor oeth yl)p h en oxya ceta te
(23). Compound 22 (0.86 g, 5.5 mmol) and K2CO3 (0.76 g, 5.5
mmol) were suspended in dry DMF (10 mL), stirred at room
temperature for 20 min, and then ethyl bromoacetate (0.6 mL,
5.5 mmol) was added. The mixture was stirred at room
temperature for 12 h, then the solvent was removed under
reduced pressure, and the residue was suspended in water (20
mL) and extracted with EtOAc (20 × 3 mL). The organic layer,
dried and evaporated, afforded 23 as an oil (1.23 g, 92% yield).
1H NMR (CDCl3): 1.3 (t, 3H, J ) 7), 3.0 (t, 2H, J ) 8), 3.67 (t,
2H, J ) 8), 4.26 (q, 2H, J ) 7), 4.6 (s, 2H), 6.87 (d, 2H, J ) 8),
7.14 (d, 2H, J ) 8). IR (neat): 1752. Anal. (C12H15ClO3) C, H.
Syn th esis of 2-(4-Nitr op h en yl)eth yl Ch lor id e a n d 3-(4-
Nitr op h en yl)p r op yl Ch lor id e (26, 27). The chloro deriva-
tives 24 or 25 (0.076 mol) were cooled at 0 °C, and 90% HNO3
(20 mL) was added dropwise, and the mixture was stirred at
the same temperature for 20 min. Then water (100 mL) was
added slowly and extracted with CH2Cl2 (40 × 3 mL). The
organic layer was dried and evaporated. The residue was
purified by flash chromatography (Et2O/light petroleum 15%;
30% ortho and 70% para nitro derivatives).
2-(4-Nitr op h en yl)eth yl Ch lor id e (26). Yellow solid (68%
yield), mp 44-45 °C. 1H NMR (CDCl3): 3.21 (t, 2H, J ) 7),
3.8 (t, 2H, J ) 7), 7.42 (d, 2H, J ) 8), 8.19 (t, 2H, J ) 8). IR
(neat): 1495, 1324, 839. Anal. (C8H8ClNO2) C, H.
Syn th esis of Eth yl 4-(3-Hyd r oxyp r op yl)ben zoa te (17).
A stirred solution of 16 (0.2 g, 1.2 mmol) in 5 mL of 95%
ethanol and concentrated H2SO4 (30 µL) was refluxed for 24
h. Workup consisted of adding CH2Cl2 (5 mL) and water (3
3-(4-Nitr op h en yl)p r op yl Ch lor id e (27). Yellow oil (67%
1
yield); H NMR (CDCl3): 2.31-2.36 (m, 2H), 3.2 (t, 2H, J )
7), 3.81 (t, 2H, J ) 7), 7.42 (d, 2H, J ) 8), 8.22 (d, 2H, J ) 8).
IR (neat): 1505, 1335, 840. Anal. (C9H10ClNO2) C, H.

Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 123
Syn t h esis of 2-(4-Am in op h en yl)et h yl Ch lor id e (28).
2-(4-Nitrophenyl)ethyl chloride 26 (1.24 g, 6.7 mmol) in CH3OH
(30 mL) in the presence of 10% Pd/C (65 mg) was reducted in
a Parr apparatus under pressure (40 psi) for 10 min. The
catalyst was removed by filtration on a pad of Celite, and the
filtrate was evaporated to give 28 (1 g, 96% yield) as an oil,
which was used in the next step without further purification.
Syn th esis of 2-[(4-Acetyla m in o)p h en yl]eth yl Ch lor id e
(29). The 2-(4-aminophenyl)ethyl chloride 28 (1 g, 6.45 mmol)
in pyridine (20 mL) and acetic anhydride (2.45 mL, 25.8 mmol)
were stirred at room temperature for 24 h. Then, the solvent
was removed under reduced pressure, and the residue was
suspended in water (70 mL) and extracted with EtOAc (20 ×
3 mL). The organic phase, dried and evaporated, furnished the
compound as white solid (0.8 g, 68% yield); mp 117-119 °C.
1H NMR (CDCl3): 2.16 (s, 3H), 3.02 (t, 2H, J ) 7), 3.68 (t, 2H,
J ) 7), 7.16 (d, 2H, J ) 8), 7.43 (bs, 1H), 7.47 (d, 2H, J ) 8).
IR (KBr): 3425, 1654. Anal. (C10H12ClO) C, H.
7.94 (s, 1H), 8.08 (bs, 2H), 8.16 (s, 1H). IR (KBr): 3470, 3378,
729.
8d . White solid (63% yield); H NMR (DMSO-d6): 3.01 (t,
1
2H, J ) 8), 3.31-3.35 (m, 4H), 3.47-3.49 (m, 4H), 4.39 (t, 2H,
J ) 8), 4.72 (bs, 2H), 6.56 (d, 2H, J ) 8), 6.71-6.74 (m, 1H),
6.95 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4), 7.94 (s, 1H), 8.08 (bs,
2H), 8.16 (s, 1H). IR (KBr): 3451, 3354, 814.
8e. Off-white solid (65% yield); 1H NMR (DMSO-d6): 2.22-
2.29 (m, 2H), 2.71 (t, 2H, J ) 8), 4.33 (t, 2H, J ) 8), 6.62-6.65
(m, 1H), 7.2 (d, 1H, J ) 4), 7.38 (d, 2H, J ) 8), 7.61 (d, 2H, J
) 8), 7.7 (bs, 2H), 7.91 (s, 1H), 8.07 (s, 1H). IR (KBr): 3351,
2262, 842.
8f. Off-white solid (56% yield); 1H NMR (CDCl3): 1.37 (t,
3H, J ) 7), 2.27-2.31 (m, 2H), 2.72 (t, 2H, J ) 8), 4.33-4.42
(m, 4H), 5.93 (bs, 2H), 6.59-6.62 (m, 1H), 7.23-7.27 (m, 3H),
7.64 (s, 1H), 7.94 (d, 2H, J ) 8), 8.21 (s, 1H). IR (KBr): 3346,
1758, 789.
8g. White solid (58% yield); 1H NMR (DMSO-d6): 3.1 (t, 2H,
J ) 8), 4.43 (t, 2H, J ) 8), 4.71 (s, 2H), 6.71-6.72 (m, 1H),
6.79 (d, 2H, J ) 8), 7.07 (d, 2H, J ) 8), 7.21 (d, 1H, J ) 4),
7.93 (s, 1H), 8.08 (bs, 2H), 8.15 (s, 1H). IR (KBr): 3458, 1747,
1112, 616.
Syn th esis of 2-[(4-N,N-Bis-â-h yd r oxyeth yla m in o)p h en -
yl]eth yl Ch lor id e (30). To a suspension of 2-(4-aminophenyl)-
ethyl chloride 28 (1 g, 6.4 mmol) in 25% CH3COOH (15 mL),
ethylene oxide (3.6 mL) was added with swirling. The mixture
was stirred at room temperature for 24 h, after which the clear
solution was made slightly basic with NaHCO3 and extracted
with EtOAc (30 × 3 mL). After being dried, the solvent was
removed in a vacuum to afford 30 as an oil (1.48 g, 95% yield).
1H NMR (CDCl3): 2.95 (t, 2H, J ) 8), 3.52 (t, 4H, J ) 4), 3.65
(t, 2H, J ) 8), 3.79 (t, 4H, J ) 4), 4.05 (bs, 2H), 6.61 (d, 2H, J
Syn th esis of 7-[3-(4-Am in op h en yl)p r op yl]-2-fu r a n -2-yl-
7H -p y r a zo lo [4,3-e][1,2,4]t r ia zo lo [1,5-c]p y r im id in -5-
yla m in e (8h ). Compound 8a (40 mg, 0.1 mmol) was suspended
in dry dioxane (3 mL) and 10% Pd/C (3 mg) and hydrazine
monohydrate (0.3 mL, 6.17 mmol) were added. The mixture
was refluxed for 45 min, then the catalyst was removed by
filtration over a bed of Celite, and the filtrate was dried and
evaporated to afford the compound in quantitative yield as a
) 8), 7.1 (d, 2H, J ) 8). IR (neat): 3413, 786. Anal. (C12H18
ClNO2) C, H, N.
-
1
Syn th esis of 3-(4-Am in op h en yl)p r op yl Ch lor id e (31).
3-(4-Nitrophenyl)propyl chloride 27 (1 g, 5 mmol) in CH3OH
(25 mL), in the presence of 10% Pd/C (50 mg), was reducted
in a Parr apparatus under pressure (40 psi) for 10 min. The
catalyst was removed by filtration on a pad of Celite, and the
filtrate was evaporated to give the compound as an oil, which
was used in the next step without further purification (0.82
g, 97% yield).
Syn th esis of 3-[(4-N,N-Bis-â-h yd r oxyeth yla m in o)p h en -
yl]p r op yl Ch lor id e (32). To a suspension of 3-(4-amino-
phenyl)propyl chloride 31 (0.5 g 2.9 mmol) in 25% CH3COOH
(7 mL), ethylene oxide (1.8 mL) was added with swirling. The
mixture was stirred at room temperature for 24 h, after which
the clear solution was made slightly basic with NaHCO3 and
extracted with EtOAc (15 × 3 mL). After being dried, the
solvent was removed in a vacuum to afford 32 as an oil (0.69
g, 92% yield). 1H NMR (CDCl3): 1.98-2.06 (m, 2H), 2.67 (t,
2H, J ) 8), 3.48-3.55 (m, 6H), 3.79 (t, 4H, J ) 4), 3.91 (bs,
2H), 6.62 (d, 2H, J ) 8), 7.5 (d, 2H, J ) 8). IR (neat): 3388,
851. Anal. (C13H20ClNO2) C, H, N.
white solid. H NMR (DMSO-d6): 2.03-2.07 (m, 2H), 2.42 (t,
2H, J ) 8), 4.23 (t, 2H, J ) 8), 4.84 (d, 2H, J ) 4), 6.47 (d, 2H,
J ) 8), 6.72-6.74 (m, 1H), 6.86 (d, 2H, J ) 8), 7.22 (d, 1H, J
) 4), 7.94 (s, 1H), 8.09 (bs, 2H), 8.16 (s, 1H). IR (KBr): 3435,
3379, 757.
Syn t h esis of 7-[3-(4-Am in op h en yl)et h yl]-2-fu r a n -2-
yl-7H -p yr a zolo[4,3-e][1,2,4]t r ia zolo[1,5-c]p yr im id in -5-
yla m in e (8k ). Compound 8b (60 mg, 0.145 mmol) was
dissolved in a mixture of dioxane (10 mL) and aqueous 10%
HCl (8 mL) and refluxed for 2 h. The solution was concentrated
under reduced pressure, the pH was adjusted around 7 with
aqueous 5% NaOH, and it was extracted with EtOAc (10 × 3
mL). The organic layer was dried and evaporated to give the
1
compound as a pale yellow solid (50 mg, 96% yield). H NMR
(DMSO-d6): 2.97 (t, 2H, J ) 8), 4.38 (t, 2H, J ) 8), 4.86 (bs,
2H), 6.44 (d, 2H, J ) 8), 6.72-6.74 (m, 1H), 6.62 (d, 2H, J )
8), 7.21 (d, 1H, J ) 4), 7.94 (s, 1H), 8.04 (bs, 2H), 8.15 (s, 1H).
IR (KBr): 3415, 3386, 754.
Gen er a l P r oced u r e for Syn th esis of {4-[3-(5-Am in o-2-
fu r a n -2-ylp yr a zolo[4,3-e][1,2,4]tr ia zolo[1,5-c]p yr im id in -
7-yl)eth yl]p h en oxy}a cetic Acid (8n ) a n d 4-[3-(5-Am in o-
2-fu r an -2-ylpyr azolo[4,3-e][1,2,4]tr iazolo[1,5-c]pyr im idin -
7-yl)p r op yl]ben zoic Acid (8m ). The ester, 8f or 8g, (0.082
mmol) was suspended in aqueous 10% NaOH (4 mL) and
refluxed for 30 min. The solution was cooled, and the pH was
adjusted to around 7 with aqueous 5% HCl. The precipitate
was filtered and dried under vacuum.
8n . White solid (98% yield); 1H NMR (DMSO-d6): 3.1 (t, 2H,
J ) 8), 4.44 (t, 2H, J ) 8), 4.58 (s, 2H), 6.72-6.74 (m, 1H),
6.78 (d, 2H, J ) 8), 7.08 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4),
7.94 (s, 1H), 8.08 (bs, 2H), 8.16 (s, 1H), 13.01 (bs, 1H). IR
(KBr): 3437, 1702, 616.
8m . White solid (97% yield); 1H NMR (DMSO-d6): 2.27-
2.31 (m, 2H), 2.73 (t, 2H, J ) 8), 4.32 (t, 2H, J ) 8), 6.59-6.72
(m, 1H), 7.23-7.27 (m, 3H), 7.64 (s, 1H), 7.94 (d, 2H, J ) 8),
8.07 (bs, 2H), 8.15 (s, 1H), 12.56 (bs, 1H). IR (KBr): 3478, 1712,
658.
Gen er a l P r oced u r e for Alk yla tion of 2-F u r a n -2-yl-
7H -p yr a zolo[4,3-e][1,2,4]t r ia zolo[1,5-c]p yr im id in -5-yl-
a m in e (8a -g). To a mixture of 78 (100 mg, 0.41 mmol) and
K2CO3 (62 mg, 0.45 mol) in dry DMF (10 mL), the opportune
halide (0.45 mmol) was added, and the mixture was heated at
100 °C for 6-8 h. Then the solvent was removed under reduced
pressure, and the residue was suspended in water (20 mL)
and extracted with EtOAc (15 × 3 mL). The organic phase was
dried, evaporated, and purified by flash chromatography
(EtOAc/light petroleum 50-80%).
8a . Light yellow solid (56% yield); 1H NMR (DMSO-d6):
2.03-2.07 (m, 2H), 2.42 (t, 2H, J ) 8), 4.23 (t, 2H, J ) 8), 6.49
(d, 2H, J ) 8), 6.72-6.74 (m, 1H), 6.88 (d, 2H, J ) 8), 7.22 (d,
1H, J ) 4), 7.94 (s, 1H), 8.09 (bs, 2H), 8.16 (s, 1H). IR (KBr):
3397, 1551, 1349, 723.
1
8b. White solid (61% yield); H NMR (DMSO-d6): 1.99 (s,
3H), 3.11 (t, 2H, J ) 8), 4.45 (t, 2H, J ) 8), 6.72-6.74 (m,
1H), 7.07 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4), 7.43 (d, 2H, J )
8), 7.94 (s, 1H), 8.09 (bs, 2H), 8.16 (s, 1H), 9.85 (bs, 1H). IR
(KBr): 3423, 3354, 1655, 678.
8c. White solid (61% yield); 1H NMR (DMSO-d6): 2.05-2.09
(m, 2H), 2.46 (t, 2H, J ) 8), 3.34 (t, 4H, J ) 6), 3.49 (t, 4H, J
) 6), 4.25 (t, 2H, J ) 8), 4.72 (t, 2H, J ) 6), 6.58 (d, 2H, J )
8), 6.72-6.74 (m, 1H), 6.98 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4),
Syn t h esis of 7-[3-(4-Am in om et h ylp h en yl)p r op yl]-2-
fu r a n -2-yl-7H-p yr a zolo[4,3-e][1,2,4]tr ia zolo[1,5-c]p yr im i-
d in -5-yla m in e Hyd r och lor id e (8j). The hydrogenation of 8e
(10 mg, 0.026 mmol), dissolved in EtOH (20 mL) and CHCl3
(5 mL), was carried out on a Parr apparatus (PtO2 0.65 mg,
45 psi, 2.5 h) followed by filtration through Celite and
evaporation of the solvent under reduced pressure. The residue

124 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Baraldi et al.
was crystallized from MeOH to give the compound as a white
solid (95% yield). 1H NMR (DMSO-d6): 2.08-2.16 (m, 2H), 2.61
(t, 2H, J ) 8), 3.95 (bs, 2H), 4.27 (t, 2H, J ) 8), 6.72-6.74 (m,
1H), 7.21-7.22 (m, 1H), 7.24 (d, 2H, J ) 8), 7.38 (d, 2H, J )
8), 7.95 (s, 1H), 8.1 (bs, 2H), 8.16 (s, 1H), 8.39 (bs, 3H). IR
(KBr): 3402, 2924, 1596, 764.
2H, J ) 8), 7.69 (d, 2H, J ) 8), 7.94 (bs, 3H), 8.15 (s, 1H). IR
(KBr): 3339, 1667, 1330, 1157.
Syn th esis of 4-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]tr ia zolo[1,5-c]p yr im id in -7-yl)eth yl]-N,N-bis-(2-h y-
d r oxyet h yl)b en zen esu lfon a m id e (11c). The diethanol-
amine (47 mg, 0.45 mmol) was added to a solution of the crude
9 (0.38 mmol) in dry dioxane (10 mL), and the mixture was
stirred at room temperature for 3 h. Then the solvent was
removed under reduced pressure, and the residue was purified
by flash chromatography (EtOAc 100%) to furnish 11c as a
Syn th esis of 4-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]tr iazolo[1,5-c]pyr im idin -7-yl)eth yl]-N-h ydr oxyben z-
a m id in e (8i). A suspension of 8e (20 mg, 0.052 mmol),
hydroxylamine hydrochloride (18 mg, 0.26 mmol), and triethyl-
amine (0.036 mL, 0.26 mmol) in methanol (3 mL) was stirred
with heating at 50 °C for 18 h. The right compound was filtered
after precipitation from the cooled mixture as a white solid
1
white solid (65% yield). H NMR (DMSO-d6): 3.04 (t, 4H, J )
6), 3.27 (t, 2H, J ) 6.6), 3.41-3.50 (m, 4H), 4.54 (t, 2H, J )
6.6), 4.81 (t, 2H, J ) 6), 6.72-6.73 (m, 1H), 7.21 (d, 1H, J )
4), 7.35 (d, 2H, J ) 8), 7.64 (d, 2H, J ) 8), 7.94 (s, 1H), 8.08
(bs, 2H), 8.16 (s,1H). IR (KBr): 3200, 1667, 1340, 1160.
1
(MeOH, 96% yield). H NMR (DMSO-d6): 2.12-2.21 (m, 2H),
2.61 (t, 2H, J ) 8), 4.27 (t, 2H, J ) 8), 5.76 (bs, 2H), 6.72-
6.74 (m, 1H), 7.2-7.24 (m, 3H), 7.58 (d, 2H, J ) 8), 7.94 (s,
1H), 8.08 (bs, 2H), 8.16 (s, 1H), 9.56 (s, 1H). IR (KBr): 3457,
3245, 1678, 689.
Syn th esis of 2-F u r a n -2-yl-7-{3-[4-(4-m eth ylp ip er a zin e-
1-su lfon yl)ph en yleth yl}-7H-pyr azolo[4,3-e][1,2,4]tr iazolo-
[1,5-c]p yr im id in -5-yla m in e (11d ). The crude 9 (0.52 mmol)
in dry dioxane (10 mL) at 0 °C was added to 1-methylpiper-
azine (104 mg, 1.04 mmol, 2 equiv), and the mixture was
stirred at room temperature for 2 h. Then the solvent was
removed, and the residue was purified by flash chromatogra-
phy (EtOAc 100%) to give 11d as a pale yellow solid (64%
yield). 1H NMR (DMSO-d6): 2.24 (s, 3H), 2.39 (t, 4H, J ) 4.2),
2.79 (t, 4H, J ) 4.2), 3.3 (t, 2H, J ) 6), 4.59 (t, 2H, J ) 6),
6.62-6.63 (m, 1H), 7.17 (d, 1H, J ) 3.2), 7.23 (d, 2H, J ) 8),
7.5 (d, 2H, J ) 8), 7.63 (bs, 2H), 7.93 (s, 1H), 8.08 (s, 1H). IR
(KBr): 3265, 1667, 1350, 1160.
Syn th esis of 4-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b e n za m i-
d in e Acetic Acid Sa lt (8l). To a solution of 8i (20 mg, 0.048
mmol) in acetic acid (10 mL), acetic anhydride (5 µL, 1.1 equiv)
and 10% Pd/C (0.5 mg) were added, and the mixture was
poured in a Parr apparatus (40 psi, 24 h). Then the catalyst
was removed by filtration through a bed of Celite, and the
filtrate was evaporated under reduced pressure to afford the
desired compound as a white solid (MeOH, 69% yield). 1H NMR
(DMSO d6): 1.81 (s, 3H), 2.01-2.03 (m, 2H), 2.5 (t, 2H, J )
8), 3.39 (bs, 1H), 4.18-4.28 (m, 3H), 6.72-6.74 (m, 1H), 7.22
(d, 1H, J ) 4), 7.44 (d, 2H, J ) 8), 7.71-7.73 (m, 4H), 7.94 (s,
1H), 8.19 (bs, 3H). IR (KBr): 3342, 2989, 1678, 685.
Gen er a l P r oced u r e for Syn th esis of 4-[3-(5-Am in o-2-
fu r a n -2-ylp yr a zolo[4,3-e][1,2,4]tr ia zolo[1,5-c]p yr im id in -
7-yl)et h yl]b en zen esu lfon yl Ch lor id e (9) a n d 4-[3-(5-
Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e][1,2,4]t r ia zolo[1,5-
c]p yr im id in -7-yl)p r op yl]ben zen esu lfon yl Ch lor id e (10).
The tricyclic derivative, SCH 58261 1 or SCH 63390 2 (0.35
mmol), was added in small portions to the chlorosulfonic acid
(2 mL) cooled at 0 °C. The mixture was stirred at this
temperature for 1 h, and then water (15 mL) was added
carefully keeping the temperature at 0 °C. The white precipi-
tate was filtered off and immediately used for the next step
(98% yield).
Syn th esis of 4-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]tr iazolo[1,5-c]pyr im idin -7-yl)eth yl]-N,N-bis-(2-ch lo-
r oeth yl)ben zen esu lfon a m id e (11e). To a solution of crude
9 (0.38 mmol) and TEA (73 µL, 0.53 mmol, 1.4 equiv) in dry
dioxane (10 mL) cooled at 0 °C, bis(2-chloroethyl)amine
hydrochloride (80 mg, 0.45 mmol, 1.2 equiv) was added, and
the mixture was stirred at room temperature for 3 h. Then
the solvent was removed under vacuum, and the residue was
purified by flash chromatography (EtOAc/light petroleum 50%)
1
to afford the desired 11e as an off-white solid (68% yield). H
NMR (DMSO-d6): 3.32-3.41 (m, 6H), 3.64 (t, 4H, J ) 6.8),
4.54 (t, 2H, J ) 6), 6.71-6.72 (m, 1H), 7.20 (d, 1H, J ) 3),
7.34 (d, 2H, J ) 8), 7.68 (d, 2H, J ) 8), 7.93 (s, 1H), 8.05 (bs,
2H), 8.15 (s, 1H). IR (KBr): 3210, 1667, 1355, 1170.
Syn th esis of {4-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-
e][1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zen e-
su lfon yla m in o}a cetic Acid (11f). A solution of crude 9 (0.5
mmol) in dry dioxane (10 mL) was added slowly to a solution
of glycine ethyl ester hydrochloride (69 mg, 0.5 mmol) and TEA
(0.2 mL, 1.5 mmol) in dry dioxane (4 mL), and the mixture
was stirred at room temperature for 2 h. Then the solvent was
removed, and the residue was dissolved in EtOAc (15 mL) and
washed with saturated NaCl solution (2 × 5 mL). The organic
phase was dried and evaporated. The residue was dissolved
in a mixture of THF/CH3OH/H2O (4:1:1, 3 mL), and lithium
hydroxide monohydrate (10.8 mg) was added. The mixture was
stirred for 12 h at room temperature, and then it was washed
with CH2Cl2 (2 × 3 mL). The aqueous layer was acidified to
pH 3 with 10% HCl, and the precipitate was filtered off. The
compound was crystallized (dioxane/Et2O) to afford a pale
yellow solid (63% yield, two steps). 1H NMR (DMSO-d6): 3.07
(t, 2H, J ) 7), 3.49-3.51 (m, 3H), 4.52 (t,2H, J ) 7), 6.72-
6.74 (m, 1H), 7.21 (d, 1H, J ) 3.6), 7.37 (d, 2H, J ) 8), 7.67 (d,
2H, J ) 8), 7.95 (bs, 2H), 8.1 (bs, 2H), 8.16 (s, 1H). IR (KBr):
3265, 1667, 1350, 1155.
Syn th esis of 5-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zo[1,3]-
d ioxole 2,2-Dica r boxylic Acid Eth yl Ester (14f). To a
suspension of 6 (100 mg, 0.25 mmol) and K2CO3 (120 mg, 3.5
equiv) in acetone (4 mL), diethyl dibromomalonate (80 mg, 1
equiv) in acetone (2 mL) was added dropwise, and the mixture
was stirred at room temperature for 24 h. Then EtOAc (20
mL) was added and washed with water (2 × 10 mL); the
organic phase was dried and evaporated under vacuum to
furnish 14f as off-white solid (72% yield; acetone/DMF). 1H
NMR (CDCl3): 1.34 (t, 6H, J ) 7), 2.19-2.28 (m, 2H), 2.57 (t,
Gen er a l P r oced u r e for Syn th esis of 4-[3-(5-Am in o-2-
fu r a n -2-ylp yr a zolo[4,3-e][1,2,4]tr ia zolo[1,5-c]p yr im id in -
7-yl)eth yl]ben zen esu lfon ic a cid (11a ) a n d 4-[3-(5-Am in o-
2-fu r an -2-ylpyr azolo[4,3-e][1,2,4]tr iazolo[1,5-c]pyr im idin -
7-yl)p r op yl]ben zen esu lfon ic Acid (12). To a solution of the
chlorosulfonyl derivative, 9 or 10, (0.32 mmol) in dioxane (2
mL), aqueous 10% HCl (2 mL) was added. The mixture was
stirred at room temperature for 2 days. Then the solvent was
removed under reduced pressure, and the residue was taken
up several times with benzene and methanol. 11a or 12 were
purified by preparative HPLC in 25 min at a flow rate of 30
mL/min (reversed phase column; gradient elution 0-60%
solvent B, where solvent A is 10% (v/v) acetonitrile in 0.1%
TFA and solvent B is 60% (v/v) acetonitrile in 0.1% TFA).
11a . White solid (60% yield); 1H NMR (D2O): 3.01 (t, 2H, J
) 6), 4.14 (t, 2H, J ) 6), 6.36-6.37 (m, 1H), 6.75-6.76 (m,
1H), 7.12 (d, 2H, J ) 8), 7.39 (s, 1H), 7.58 (d, 2H, J ) 8), 7.69
(s, 1H). IR (KBr): 3040, 1667, 1350, 1165.
12. White solid (65% yield); 1H NMR (D2O): 2.19-2.22 (m,
2H), 2.66 (t, 2H, J ) 6), 4.16 (t, 2H, J ) 6), 6.64-6.65 (m,
1H), 7.09-7.11 (m, 1H), 7.32 (d, 2H, J ) 8), 7.68 (d, 2H, J )
8), 7.71 (s, 1H), 8.0 (s, 1H). IR (KBr): 3050, 1667, 1352, 1160.
Syn th esis of 4-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]tr ia zolo[1,5-c]p yr im id in -7-yl)eth yl]ben zen esu lfon -
a m id e (11b). In a solution of the crude chlorosulfonyl deriva-
tive 9 (0.338 mmol) in dry dioxane (20 mL) cooled at 0 °C was
bubbled NH3 till saturation of the solvent. The precipitate that
formed was filtered off, and the filtrate was purified by flash
chromatography (EtOAc 100%) to afford 11b as a white solid
1
(yield 63%). H NMR (DMSO-d6): 3.27-3.39 (m, 4H), 4.53 (t,
2H, J ) 6), 6.71-6.73 (m, 1H), 7.21-7.23 (m, 1H), 7.35 (d,

Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 125
2H, J ) 7.4), 4.27 (t, 2H, J ) 7.4), 4.35 (q, 4H, J ) 7), 6.15 (bs,
2H), 6.59-6.61 (m, 1H), 6.7-6.86 (m, 3H), 7.24 (d, 1H, J ) 5),
7.64 (s, 1H), 8.2 (s, 1H). IR (KBr): 3278, 1745, 677.
tated by the addition of light petroleum (15 mL). Aqueous 10%
HCl (0.5 mL) was added to the precipitate suspended in
dioxane (2 mL), and the mixture was stirred at room temper-
ature for 3 d. The solvent was removed under vacuum, and
the residue was purified by flash chromatography (MeOH/
Syn th esis of 5-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zo[1,3]-
d ioxole 2-Ca r boxylic Acid Eth yl Ester (14a ). To a solution
of 14f (0.09 mmol) in DMSO (2 mL) and 1 drop of H2O, NaCl
(5 mg, 1 equiv) was added, and the mixture was heated at 160
°C for 12 h. Then water was added (20 mL), and the solution
was extracted with EtOAc (4 × 10 mL). The organic phase
was dried and evaporated under reduced pressure to give 14a .
1
EtOAc 20%) to give 13 as a white solid (52% yield). H NMR
(DMSO-d6): 2.14 (m, 2H), 2.95 (t, 2H, J ) 7), 4.25 (t, 2H, J )
7), 5.97 (s, 2H), 6.70 (s, 1H), 6.71-6.72 (m, 1H), 7.22 (s, 1H),
7.23 (d, 1H, J ) 4), 7.94 (s, 1H), 8.15 (bs, 2H), 8.16 (s, 1H). IR
(KBr): 3432, 1668, 1178, 629.
Deter m in a tion of Rm Va lu es by C18 RP -HP TLC. The
1
HPTLC determinations were carried out on Whatman KC18
F
Off-white solid (42% yield; EtOAc/light petroleum); H NMR
plates as previously described.25 Solvent mixtures of methanol-
water buffer at pH 7.0 were used as mobile phase. The
methanol concentration ranged from 60% to 90%. The solutes
were detected under UV 254-nm light.
(CDCl3): 1.34 (t, 3H, J ) 7), 2.17-2.27 (m, 2H), 2.58 (t, 2H, J
) 8), 4.31-4.37 (m, 4H), 6.03 (bs, 2H), 6.27 (s, 1H), 6.61-6.62
(m, 1H), 6.69-6.75 (m, 3H), 7.25 (d, 1H, J ) 3.4), 7.63 (s, 1H),
8.2 (s, 1H). IR (KBr): 3220, 1745, 667.
Biology. CHO Mem br a n es P r ep a r a tion . The expression
of the human adenosine receptors in CHO cells is described
elsewhere.26,27 The cells were grown adherently and main-
tained in Dulbecco’s modified Eagle’s medium with nutrient
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 flasks in ice cold hypotonic buffer (5 mM Tris-HCl, 2 mM
EDTA, pH 7.4). The cell suspension was homogenized with
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 and 10 mM MgCl2 at pH 7.4 for A2A adenosine
receptors; and in 50 mM Tris-HCl, 10 mM MgCl2, and 1 mM
EDTA at pH 7.4 for A3 adenosine receptors. They were utilized
for binding assay. HEK-293 cells transfected with the human
recombinant A2B adenosine receptor were obtained from
Receptor Biology, Inc. (Beltsville, MD).
Bin d in g Assa ys of th e Hu m a n Clon ed A1, A2A, A2B, a n d
A3 Ad en osin e Recep tor . Recep tor Bin d in g Assa ys. Bind-
ing of [3H]-DPCPX to CHO cells transfected with the human
recombinant A1 adenosine receptor was performed according
to the method previously described by Lohse et al.18
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 is always e10% of the total binding.
Binding of [3H]-SCH58261 to HEK-293 cells transfected
with the human recombinant A2A adenosine receptors (Re-
search Biomedical International, Natick, MA) (20 µg of protein/
assay) was performed according to Zocchi et al.20 In competi-
tion 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.
Binding of [3H]-DPCPX to HEK-293 cells (Receptor Biology
Inc., Beltsville, MD) transfected with the human recombinant
A2B adenosine receptors was performed as already described
by Varani et al.19 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, and 0.1 mM benzamidine, 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 bind-
ing was determined in the presence of 100 µM NECA and was
always e30% of the total binding.
Binding of [3H]-MRE3008-F20 to CHO cells transfected with
the human recombinant A3 adenosine receptors was performed
according to Varani et al.19 Competition experiments were
carried out in duplicate in a finale volume of 100 µL in test
tubes containing 1 nM [3H]-MRE3008-F20, 50 mM Tris-HCl
buffer, 10 mM MgCl2, pH 7.4, and 100 µL of diluted mem-
branes (50 µg of protein/mL) and at least 6-8 different
concentrations of examined ligands. Incubation time was 120
min at 4 °C, according to the results of previous time-course
Syn th esis of 5-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zo[1,3]-
d ioxole 2,2-Dica r boxylic Acid (14c). To a solution of 14f
(50 mg, 0.09 mmol) in dioxane (5 mL), aqueous 2 N KOH (1
mL) was added, and the mixture was refluxed for 6 h. Then
the pH was adjusted to around 3 with aqueous 10% HCl, and
the solution was extracted with EtOAc (2 × 10 mL). The
organic layer was dried and evaporated under reduced pres-
sure to give 14c as a pale yellow solid (89% yield; EtOAc/light
1
petroleum). H NMR (DMSO-d6): 1.98-2.07 (m, 2H), 2.54 (t,
2H, J ) 6), 3.52 (bbs, 2H), 4.28 (t, 2H, J ) 6), 6.71-6.75 (m,
2H), 6.9-6.94 (m, 2H), 7.16 (d, 1H, J ) 4.2), 7.94 (s, 1H), 8.15
(bs, 2H), 8.17 (s, 1H). IR (KBr): 3189, 1698, 677.
Syn th esis of 5-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zo[1,3]-
d ioxole 2,2-Dica r boxylic Disod iu m Sa lt (14e). The com-
pound 14c (20 mg, 0.04 mmol) was dissolved in aqueous 0.04
M NaOH (2 mL), and the mixture was stirred at room
temperature for 12 h. Then the solvent was removed in vacuo
to afford 14e as white solid (97% yield). 1H NMR (D2O): 2.01-
2.11 (m, 2H), 2.45 (t, 2H, J ) 6), 4.06 (t, 2H, J ) 6), 6.62-6.72
(m, 4H), 7.06 (d, 1H, J ) 5), 7.79 (s, 1H), 7.83 (s, 1H). IR
(KBr): 2540-3350, 1640, 667.
Syn th esis of 5-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zo[1,3]-
d ioxole 2-Ca r boxylic Acid (14d ). Aqueous 1 N NaOH (2 mL)
was added to a solution of 16a (20 mg, 0.042 mmol) in THF (2
mL), and the mixture was refluxed for 30 min. Then the
solution was cooled, and the pH was adjusted to around 3 with
acqueous 1 N HCl and extracted with EtOAc (4 × 10 mL). The
organic layer was dried and evaporated under reduced pres-
sure to afford 14d . Pale yellow solid (98% yield; EtOAc/light
petroleum); 1H NMR (DMSO-d6): 2.11 (t, 2H, J ) 6), 3.34 (bs,
1H), 4.25 (t, 2H, J ) 6), 6.45 (s, 1H), 6.69-6.72 (m, 2H), 6.83-
6.87 (m, 2H), 7.22 (d, 1H, J ) 3.2), 7.94 (s, 1H), 8.09 (bs, 2H),
8.17 (s, 1H). IR (KBr): 3230, 1710, 687.
Syn th esis of {5-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-
e][1,2,4]tr ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]-2-h yd r oxy-
m eth ylben zo[1,3]d ioxol-2-yl}m eth a n ol (14b). To a solution
of 14f (50 mg, 0.09 mmol) in distilled THF (10 mL), NaBH4
(70 mg, 20 equiv) was added in small portions, and the mixture
was stirred at room temperature for 2 h. Then EtOAc (15 mL)
and water (10 mL) were added, and the organic phase was
separated and washed with water (2 × 10 mL) and saturated
NaCl solution. The organic layer was dried and evaporated to
give 14b as white solid (70% yield). 1H NMR (DMSO-d6):
2.18-2.25 (m, 2H), 2.49 (t, 2H, J ) 7), 3.86 (d, 4H, J ) 6.4),
4.2 (t, 2H, J ) 6.4), 4.32 (t, 2H, J ) 7), 6.40-6.53 (m, 4H),
6.73 (bs, 2H), 7.24 (d, 1H, J ) 5), 7.64 (s, 1H), 8.15 (s, 1H). IR
(KBr): 3345, 3256, 1610, 677.
Syn th esis of 6-[3-(5-Am in o-2-fu r a n -2-ylp yr a zolo[4,3-e]-
[1,2,4]t r ia zolo[1,5-c]p yr im id in -7-yl)p r op yl]b en zo[1,3]-
d ioxole 5-Su lfon ic Acid (13). To a suspension of 5 (50 mg,
0.12 mmol) in distilled CH2Cl2 (10 mL) cooled at 0 °C was
added chlorosulfonic acid (3 drops), and the mixture was
stirred at this temperature for 1 h (till complete dissolution).
The solvent was removed, and the residue was taken up with
EtOAc (7 mL), and the chlorosulfonic derivative was precipi-

126 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Baraldi et al.
experiments.19 Nonspecific binding was defined as binding in
the presence of 1 µM 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 Instru-
ment Company). The filter-bound radioactivity was counted
on Top Count (efficiency 57%) with Micro-Scint 20. The protein
concentration was determined according to a Bio-Rad method28
with bovine albumin as reference standard.
A weighted nonlinear least-squares curve-fitting program
LIGAND was used for computer analysis of inhibition experi-
ments.29 Inhibitory binding constant, Ki, values were calculated
from the IC50 values according to the Cheng and Prusoff
equation, Ki ) IC50/(1 + [C*]/Kd*), where [C*] is the concentra-
tion of the radioligand and Kd* is its dissociation constant.30
triglyceride analogs as potential computed tomography imaging
agents for the liver. J . Med. Chem. 1995, 38, 636-646.
(13) Akaboshi, S.; Ikegami, S. Synthesis of the heterocyclic com-
pounds by Pschorr cyclization. III. Synthesis of 10,11-methyl-
enedioxy-7,8-dihydro-6H-benzo[c]pyrid[1,2-a]azepinium salts and
their derivatives. Chem. Pharm. Bull. 1966, 14, 622-627.
(14) Meschino, J . A.; Bond, C. H. 2-Amino-5,6-dihydro-1,3-oxazines.
The reduction of carboxylic esters with sodium borohydride. J .
Org. Chem. 1963, 28, 3129-3134.
(15) Anzalone, L.; Hirsch, J . A. Substituent effects on hydrogenation
of aromatic rings: hydrogenation vs. hydrogenolysis in cyclic
analogues of benzyl ethers. J . Org. Chem. 1985, 50, 2128-2133.
(16) Baraldi, P. G.; Guarneri, M.; Moroder, F.; Simoni, D.; Vicentini,
C. B. Synthesis of 3-methyl-6-phenylamino-substituted-1H-
pyrazolo[4,3-d]pyrimidine-7(6H)-ones. Synthesis 1982, 70-71.
(17) Baker, B. R.; Cory, M. Irreversible enzyme inhibitors. 180.
Irreversible inhibitors of the C′1a component of complement
derived from m-(phenoxypropoxy)benzamidine and phenoxy-
acetamide. J . Med. Chem. 1971, 14, 119-125.
(18) Lohse, M. J .; Klotz, K.-N.; Lindernborn-Fotinos, J .; Reddington,
M.; Schwabe, U.; Olsson, R. A. 8-Cyclopentyl 1,3-dipropyl-
xanthine DPCPX a selective high affinity antagonist radioligand
for A1 adenosine receptors. Naunyn-Schmiedeberg’s Arch. Phar-
macol. 1987, 336, 204-210.
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