Synthesis, biological activity, and molecular modeling investigation of new pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as human A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm0109614

A new series of pyrazolotriazolopyrimidines bearing different substitutions on the phenylcarbamoyl moieties at the N5 position, being highly potent and selective human A₃ adenosine receptor antagonists, is described. The compounds represent an extension and an improvement of our previous work on this class of compounds (J. Med. Chem. 1999, 42, 4473-4478; J. Med. Chem. 2000, 43, 4768-4780). All the synthesized compounds showed A₃ adenosine receptor affinity in the subnanomolar range and high levels of selectivity in radioligand binding assays at the human A₁, A_2A, A_2B, and A₃ adenosine receptors. In particular, the effect of the substitution and its position on the phenyl ring have been studied. From binding data, it is evident that the unsubstituted derivatives on the phenyl ring (e.g., compound 59, hA₃ = 0.16 nM, hA₁/hA₃ = 3713, hA_2A/hA₃ = 2381, hA_2B/hA₃ = 1388) showed the best profile in terms of affinity and selectivity at the human A₃ adenosine receptors. The introduction of a sulfonic acid moiety at the para position on the phenyl ring was attempted in order to design water soluble derivatives. However, this substitution led to a dramatic decrease of affinity at all four adenosine receptor subtypes. A computer-generated model of the human A₃ receptor was built and analyzed to better interpret these results, demonstrating that steric control, in particular at the para position on the phenyl ring, plays a fundamental role in the receptor interaction. Some of the synthesized 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 with IC₅₀ values in the nanomolar range, with a statistically significative linear relationship with the binding data.

Full text of DOI:10.1021/jm0109614

770
J . Med. Chem. 2002, 45, 770-780
Syn th esis, Biologica l Activity, a n d Molecu la r Mod elin g In vestiga tion of New
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 Hu m a n A3
Ad en osin e Recep tor An ta gon ists
Pier Giovanni Baraldi,*,† Barbara Cacciari,† Stefano Moro,*,‡ Giampiero Spalluto,*,§ Giorgia Pastorin,§
Tatiana Da Ros,§ Karl-Norbert Klotz,|| Katia Varani, Stefania Gessi, 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; and Institut fu¨r
Pharmakologie, Universita¨t Wu¨rzburg, Versbacher Str.9, D-97078 Wu¨rzburg, Germany
Received J une 21, 2001
A new series of pyrazolotriazolopyrimidines bearing different substitutions on the phenylcar-
bamoyl moieties at the N5 position, being highly potent and selective human A3 adenosine
receptor antagonists, is described. The compounds represent an extension and an improvement
of our previous work on this class of compounds (J . Med. Chem. 1999, 42, 4473-4478; J . Med.
Chem. 2000, 43, 4768-4780). All the synthesized compounds showed A3 adenosine receptor
affinity in the subnanomolar range and high levels of selectivity in radioligand binding assays
at the human A1, A2A, A2B, and A3 adenosine receptors. In particular, the effect of the
substitution and its position on the phenyl ring have been studied. From binding data, it is
evident that the unsubstituted derivatives on the phenyl ring (e.g., compound 59, hA3 ) 0.16
nM, hA1/hA3 ) 3713, hA2A/hA3 ) 2381, hA2B/hA3 ) 1388) showed the best profile in terms of
affinity and selectivity at the human A3 adenosine receptors. The introduction of a sulfonic
acid moiety at the para position on the phenyl ring was attempted in order to design water
soluble derivatives. However, this substitution led to a dramatic decrease of affinity at all four
adenosine receptor subtypes. A computer-generated model of the human A3 receptor was built
and analyzed to better interpret these results, demonstrating that steric control, in particular
at the para position on the phenyl ring, plays a fundamental role in the receptor interaction.
Some of the synthesized 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 with IC50 values in the nanomolar range,
with a statistically significative linear relationship with the binding data.
In tr od u ction
receptor agonists and antagonists for the pharmacologi-
cal characterization of this family of G-protein coupled
receptors. Although potent and selective ligands have
been identified for the A1, A2A, and A3 receptors, there
is still a need for such ligands for the A2B subtype.1,3
The A3 adenosine receptor subtype, recently cloned
from different species (e.g., rat, human, dog, sheep),4-9
is coupled to the modulation of at least two second
messenger systems: inhibition of adenylate cyclase3 and
stimulation of phospholipase C10 and D.11 Activation of
A3 adenosine receptors also causes the release of
inflammatory mediators such as histamine from mast
cells, in a rat model.12 In human, A3 receptors have been
found in several organs, such as lung, liver, kidney, and
heart, with a lower density in the brain.13 This receptor
subtype is under examination in relation to its potential
therapeutic applications. In particular, antagonists for
A3 receptors seem to be useful for the treatment of
inflammation14 and regulation of cell growth.15,16
In the last 5 years, many efforts have been conducted
for searching potent and selective human A3 adenosine
antagonists. In this field many different classes of
compounds have been proposed,17 possessing good af-
finty (nM range) but without significant selectivity.17a,b
Many physiological functions may be regulated by
interaction of adenosine with a set of specific receptors,
classified as A1, A2A, A2B, and A3. For this reason,
adenosine receptors could be considered as potential
targets in the treatment of different pathophysiologic
conditions. Adenosine receptor subtypes belong to the
family of seven transmembrane domain receptor coupled
G proteins and exert their physiogical role by activation
or inhibition of different second messenger systems. In
particular, the modulation of adenylate cyclase activity
could be considered to be the prominent signal mediated
by these receptors subtypes.1,2
In the last 20 years, intense medicinal chemistry
efforts led to the synthesis of a variety of adenosine
* 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` di Padova.
§ Universita` degli Studi di Trieste.
|| Universita¨t Wu¨rzburg.
Dipartimento di Medicina Clinica
Farmacologia, Universita` degli Studi di Ferrara.
e Sperimentale-Sezione di
10.1021/jm0109614 CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/11/2002

Human A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 771
Ta ble 1. Structures and Binding Affinity at hA1, hA2A, hA2B, and hA3 Adenosine Receptors of the Most Interesting Compounds
Previously Reported
compd
R
R1
hA1 (Ki, nM)a
hA2A (Ki, nM)b
hA2B (Ki, nM)c
hA3 (Ki, nM)d
hA1/hA 3
5485
hA2A/hA3
6950
hA2B/hA3
1305
1
CH3
4-OMe
1097
(928-1297)
350
1390
(1220-1590)
1195
261
(226-301)
205
0.20
(0.17-0.24)
0.40
2
3
CH3
3-Cl
4-OMe
3-Cl
875
1710
155
1496
33
2987
1733
112
175
1340
250
158
50
512
408
93.7
2570
62.6
946
116
158
157
(323-378)
(1024-1396)
(157-268)
(0.24-0.64)
C2H5
1026
1040
245
0.60
(785-1341)
249
(830-1310)
180
(188-320)
150
(0.51-0.70)
1.60
4
C2H5
(215-289)
(160-210)
(107-210)
(1.42-1.79)
5
n-C3H7
n-C3H7
n-C4H9
n-C4H9
4-OMe
3-Cl
1197
140
2056
0.80
(1027-1396)
30
(23-40)
296
(269-326)
245
(120-155)
1220
(1637-2582)
57
(50-63)
303
(260-352)
70
(0.63-0.1.00)
0.91
6
(880-1682)
(0.85-0.98)
7
4-OMe
3-Cl
80
0.32
925
408
43
(65-92)
95
(0.27-0.34)
0.60
8
(213-281)
(70-130)
(56-89)
(0.52-0.68)
9
4-OMe
3-Cl
7.6
9.4
22
0.14
(6.5-8.7)
8.1
(8.7-10.2)
9.5
(19-27)
30
(0.08-0.24)
0.19
10
54
67
(6.9-9.7)
(8.7-10.5)
(23-40)
(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]SCH58261 binding at human A2A receptors expressed in CHO cells (n ) 3-6). c Displacement of specific [3H]DPCPX binding at human
d
A
2B receptors expressed in HEK-293 cells (n ) 3-6). Displacement of specific [3]MRE-3008F20 binding at human A3 receptors expressed
in CHO cells (n ) 3-6). Data are expressed as geometric means, with 95% confidence limits.
Recently, our group proposed a series of pyrazolo-
triazolopyrimidines bearing, at the amino group in 5
position, the 3-chlorophenylcarbamoyl or the 4-meth-
oxyphenylcarbamoyl residues, respectively, as highly
potent and selective human A3 adenosine receptor
antagonists.18,19 (Table 1)
Table 1 briefly summarizes the most interesting
compounds (1-10) previously synthesized as human A3
adenosine receptor antagonists.19 It is clearly evident
that the 4-methoxy group on the phenyl ring confers
more affinity versus the human A3 receptors if compared
with the 3-chloro substitution. In addition, the optimal
affinity and selectivity for the human A3 adenosine
receptors was obtained with the presence of a small
alkyl chain at the N8 position. In fact, the combination
of the 4-methoxyphenylcarbamoyl moiety at the N5
position with the methyl group at the N8 pyrazole
nitrogen resulted in compound (1), which shows the best
binding profile both in terms of affinity and selectivity
F igu r e 1. General modifications of designed compounds.
or methoxy substituents, introduced in the previous
series,18,19 has been also evaluated (Figure 1).
As previously reported, we also demonstrated the
importance of the pyrazole nitrogen for both affinity and
selectivity. In fact, derivatives 9 and 10, derived from
the triazoloquinazoline CGS 15943 substituted at the
N5 position with phenyl carbamoyl moieties, showed
good affinity for the A3 adenosine receptor, but a
significant loss of selectivity vs other receptor subtypes
was observed.19 With the aim to better investigate all
the structural requirements necessary for the human
A3 adenosine receptor interaction, we replaced the
arylcarbamoyl function with the phenylacetyl group
characteristic of the nonselective human A3 antagonist
5-phenylacetylamino-9-chloro-2-(2-furyl)[1,2,4]triazolo-
[1,5-c]quinazoline (MRS1220, 67), reported by J acobson
and co-workers20 (Figure 2).
(hA3 ) 0.2 nM, hA1/hA3 ) 5485; hA2A/hA3 ) 6950; hA2B
hA3 ) 1305) ever reported.
/
Starting from these experimental observations, we
decided to investigate the steric and the electronic
properties of the substituents on the phenyl ring neces-
sary for the receptor recognition. For this purpose,
different groups were introduced at the 4 position on
the phenyl ring, and at the same time, small alkyl
chains (methyl, ethyl, n-propyl, n-butyl) were main-
tained at the N8 position. The effect of the position of
the substitution (ortho, meta, and para) of the chloro

772 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Baraldi et al.
Ta ble 2. Structures and Physicochemical Parameters of Synthesized Compounds
compd
R
R1
mp (°C)
MW
compd
R
R1
mp (°C)
MW
59
63
11
12
13
14
15
16
17
18
19
20
21
60
64
22
23
24
25
26
27
28
29
30
31
32
61
65
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph-NH
165-167
255
204
118-120
180
195-197
>300
>300
203
>300
205
384.20
450.08
442.04
418.11
419.10
388.13
453.26
392.35
442.36
404.39
404.39
408.80
408.80
388.13
468.09
456.06
432.12
433.12
402.15
467.28
406.38
456.39
418.41
418.41
422.83
422.83
402.15
482.11
33
34
35
36
37
38
39
40
41
42
43
62
66
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
CH3
3,4-Cl2-Ph-NH
3,4-OCH2O-Ph-NH
4-NO2-Ph-NH
4-CH3-Ph-NH
4-Br-Ph-NH
4-F-Ph-NH
4-CF3-Ph-NH
2-OMe-Ph-NH
3-OMe-Ph-NH
2-Cl-Ph-NH
4-Cl-Ph-NH
Ph-NH
4-SO3H-Ph-NH
3,4-Cl2-Ph-NH
3,4-OCH2O-Ph-NH
4-NO2-Ph-NH
4-CH3-Ph-NH
4-Br-Ph-NH
4-F-Ph-NH
4-CF3-Ph-NH
2-OMe-Ph-NH
3-OMe-Ph-NH
2-Cl-Ph-NH
4-Cl-Ph-NH
Ph-CH2
208
111-113
190
181-184
225
193
202
195
175
185
216
145
240-241
205-206
115-117
195
175-177
227
207
221
186
185
185
210
198-200
106-108
101-103
101
470.07
446.14
447.14
416.16
481.31
420.40
470.41
432.44
432.44
436.86
436.86
416.17
496.12
484.09
460.16
461.15
430.18
495.34
434.43
484.44
446.47
446.47
450.89
450.89
373.12
387.14
401.16
415.17
4-SO3H-Ph-NH
3,4-Cl2-Ph-NH
3,4-OCH2O-Ph-NH
4-NO2-Ph-NH
4-CH3-Ph-NH
4-Br-Ph-NH
4-F-Ph-NH
4-CF3-Ph-NH
2-OMe-Ph-NH
3-OMe-Ph-NH
2-Cl-Ph-NH
4-Cl-Ph-NH
Ph-NH
4-SO3H-Ph-NH
3,4-Cl2-Ph-NH
3,4-OCH2O-Ph-NH
4-NO2-Ph-NH
4-CH3-Ph-NH
4-Br-Ph-NH
a
>300
225
180
CH3
a
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
225
185-188
124-126
185
175-178
220
200
180
128
178
127
220
155
235
4-F-Ph-NH
4-CF3-Ph-NH
2-OMe-Ph-NH
3-OMe-Ph-NH
2-Cl-Ph-NH
4-Cl-Ph-NH
Ph-NH
C2H5
n-C3H7
n-C4H9
Ph-CH2
Ph-CH2
Ph-CH2
a
n-C3H7
n-C3H7
4-SO3H-Ph-NH
a
See Baraldi et al. J .Med.Chem. 2001, 44, 2735-2742.
68-71 with the commercially available isocyanates 72-
8019 (Scheme 1).
When not commercially available, the isocyanates (81,
82) were prepared by reacting the corresponding sub-
stituted anilines with trichloromethylchloroformate, as
described in the literature.21
The sulfonic acid derivatives 63-66 were prepared
starting from the already reported phenyl carbamoyl
derivatives 59-62,22 by reaction with chlorosulfonic acid
at 0 °C. Subsequent hydrolysis of the chlorosulfonyl
derivative in dioxane (2 mL) and 10% aqueous HCl
afforded desired final compounds 63-66, which were
purified by preparative HPLC using a reverse phase
column (Scheme 2).
The latest compounds of this series (55-58), structur-
ally related to MRS 1220 (67), were prepared by
acylation with phenylacetyl chloride (1.3 equiv) of the
unsubstituted derivatives 68-71 in dry THF at reflux
(18 h) in the presence of trietylamine (1.3 equiv)
(Scheme 3).
F igu r e 2. Rational design of hybrid derivatives (55-58)
between MRS 1220 (67) and the pyrazolotriazolopyrimidine
series.
Resu lts a n d Discu ssion
Table 3 summarizes the receptor binding affinities of
Ch em istr y
compounds 11-66 determined at the human A1, A2A
,
A
2B, and A3 receptors expressed in CHO (A1, A2A, A3)
Compounds 11-66 (Table 2) were prepared following
the general synthetic strategy summarized in Schemes
1 and 3.
The final compounds (11-54) were obtained by a
coupling reaction of the previously reported derivatives
and HEK-293 (A2B) cells.
[3H]-1,3-dipropyl-8-cyclopentyl xanthine ([3H]DPC-
PX)23,24 (A1 and A2B), [3H]-5-amino-7-(2-phenylethyl)-2-
(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-pyrimidine

Human A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 773
Sch em e 1
Reagents: (i) ClCOOCCl3, dioxane, reflux; (ii) dry THF, reflux, 18 h.
Sch em e 2
stituted derivatives on the phenyl ring, which display
a slight improvement of potency and a good retention
of selectivity at the human A3 adenosine receptors also
with respect to the previously substituted-phenyl coun-
terparts (see Table 1) (e.g., compound 59, hA3 ) 0.16
nM, hA1/hA3 ) 3713, hA2A/hA3 ) 2381, hA2B/hA3 ) 1388
vs compund 1, hA3 ) 0.20 nM, hA1/hA3 ) 5485, hA2A
/
hA3 ) 6950, hA2B/hA3 ) 1305). Accordingly, a careful
check of binding data of derivatives bearing a substitu-
ent at the para or meta positions on the phenyl ring do
not permit us to find any classical QSAR correlations.
In particular, we have tried to correlate, withouth any
success, electronic parameters (measured by σp or σm
costants),26 steric properties (calculated by the corre-
sponding molecular volumes),26 and lipophilic (measured
by calculated log P) descriptors26 with the affinity value
at the human A3 adenosine receptors.
Reagents: (i) chlorosulfonic acid, 0 °C, 4 h; (ii) dioxane, 10 M
HCl, rt, 2 days, then prepatative HPLC.
Sch em e 3
A molecular modeling investigation has been per-
formed to elucidate the hypothetical binding mode of
these new A3 antagonists and, in particular, to better
rationalize the correlation between their chemical struc-
tures and the corresponding binding results. Unfortu-
nately, little structural information is available con-
cerning the details of ligand/GPCR interactions. However,
very recently a 2.8 Å resolution crystal structure of
bovine rhodopsin was published.27 Using a homology
approach, we have built an improved model of the
transmembrane region of the human A3 receptor, using
the crystal structure of the rhodopsin as a template,
which can be considered a further refinement in build-
ing the hypothetical binding site of the A3 receptor
antagonists already proposed.28 Details of the model
building are given in the Experimental Section.
Reagents: (i) dry THF, Et3N, reflux, 18 h.
([3H]-SCH 58261) (A2A),25 and [3H]-5-(4-methoxyphenyl-
carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine ([3H]-MRE3008-F20) (A3)24 have
been used as radioligands in binding assays.
All the synthesized compounds showed affinity in the
subnanomolar range for the human A3 adenosine recep-
tors with different degrees of selectivity versus the other
receptor subtypes, confirming, as previously observed,
that small alkyl substituents at the N8 pyrazole nitrogen
are indispensable for having good affinity and selectivity
at the hA3 adenosine receptors.18,19
From a preliminary analysis of the data summarized
in Table 3, affinities to the hA3 receptors seem to
correlate more with the position of the substituent than
with the nature of the substitution on the phenyl ring.
From a careful examination of the binding affinities, it
appears evident that the most interesting compounds
in terms of both affinity and selectivity are the unsub-
We have selected derivative 59, corresponding to the
unsubstituted derivative on the phenyl ring, as refer-
ence ligand in our docking studies. However, the most
stable docked conformation is very similar for all the
analyzed compounds. As shown in Figure 3, after the
Monte Carlo/annealing sampling, we identified the
hypothetical binding site of 59 surrounded by TM3,
TM5, TM6, and TM7 with the furan ring pointing
toward the extracellular environment. Interestingly, a
very similar conformation has been already reported for
another potent but not selective human A3 adenosine
receptor antagonist as well CGS 15943.28

774 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Baraldi et al.
Ta ble 3. Binding Affinity at hA1, hA2A, hA2B, and hA3 Adenosine Receptors of Synthesized Compounds
compd
hA1 (Ki, nM)a
hA2A (Ki, nM)b
hA2B (Ki, nM)c
hA3 (Ki, nM)d
hA1/hA3
3713
hA2A/hA3
2381
hA2B/hA3
1388
59
594
(436-810)
>10000
(436-810)
392
381
(351-415)
594
222
(181-273)
>10000
0.16
(0.13-0.20)
25
(19-34)
3.4
63
11
12
13
14
15
16
17
18
19
20
21
60
64
22
23
24
25
26
27
28
29
30
31
32
61
65
33
34
35
36
37
38
39
40
41
42
>400
115
24
42
>400
34
143
(114-179)
680
116
(91-148)
142
(324-475)
(3.05-3.78)
0.24
1015
4229
2593
2358
1304
2059
1014
643
2833
1616
355
217
353
189
257
200
220
621
222
6
592
419
974
393
665
386
319
313
111
441
489
>250
13
(885-1163)
1115
(552-839)
(128-156)
(0.21-0.28)
695
180
0.43
(984-1263)
(534-906)
110
(159-204)
302
(0.38-0.48)
0.31
731
(601-888)
600
(93-130)
(244-373)
(0.25-0.38)
100
181
0.46
(512-698)
(83-120)
120
(152-216)
226
(0.39-0.54)
0.34
700
(602-814)
750
(103-140)
(183-278)
(0.28-0.41)
140
286
0.74
(654-856)
(112-177)
180
(235-347)
223
(0.64-0.86)
0.70
450
(379-535)
500
(146-220)
(187-265)
(0.59-0.83)
160
251
0.80
625
(406-616)
(140-186)
200
(213-296)
101
(0.71-0.91)
0.91
400
440
(332-479)
430
(174-230)
(89-116)
(0.82-1.01)
180
128
0.29
1483
1811
>250
175
(351-526)
(162-200)
40
(33-48)
249
(199-311)
352
(115-143)
88
(67-115)
>10000
(0.23-0.36)
0.18
326
(275-386)
>10000
(0.13-0.23)
40
(31-50)
3.0
(2.7-3.4)
0.27
(0.22-0.34)
0.65
526
(453-609)
802
40
(32-51)
48
(37-63)
226
117
2133
945
214
108
70
(300-414)
576
2970
1745
1871
1351
700
178
348
943
800
286
242
350
250
323
425
1606
> 333
12
(724-889)
(511-648)
614
1134
(967-1329)
262
(490-768)
(192-267)
132
(0.56-0.75)
30
0.14
(248-276)
(23-40)
40
(118-146)
(0.11-0.18)
0.37
500
296
(389-640)
602
(30-54)
(261-336)
246
(0.29-0.47)
60
0.86
(509-712)
(48-78)
53
(51-55)
133
(108-163)
140
(211-287)
(0.77-0.97)
0.97
450
235
464
57
(346-586)
300
(178-310)
196
(0.86-1.09)
0.56
536
238
163
500
800
413
10
(216-412)
(162-237)
(0.49-0.64)
0.86
400
215
465
(345-465)
348
(114-170)
(184-250)
97
(0.77-0.96)
150
0.30
1160
2000
1173
>333
244
(267-453)
(122-187)
160
(67-141)
(0.23-0.40)
0.20
400
85
(330-486)
176
(150-207)
>10000
(134-190)
(68-105)
241
(206-282)
>10000
(0.15-0.28)
62
0.15
(40-96)
305
(0.10-0.18)
30
(22-41)
2.5
(2.0-3.1)
0.30
(240-388)
611
(552-676)
842
401
31
(25-40)
53
(40-69)
305
(240-388)
46
(34-62)
150
160
2223
1377
30
(362-445)
667
2807
1499
755
177
377
115
333
555
267
485
438
86
(738-961)
(543-820)
(0.22-0.40)
1214
1115
0.81
(1033-1425)
302
(984-1263)
12
(8-18)
50
(40-62)
42
(0.63-1.04)
0.40
(237-384)
(0.31-0.50)
350
0.45
778
111
145
67
(290-418)
376
(124-180)
161
(0.37-0.56)
0.29
1297
784
(294-480)
(34-50)
(140-186)
(0.21-0.40)
400
34
136
0.51
(330-478)
250
(24-48)
100
(115-161)
165
(0.42-0.62)
0.34
735
294
283
170
(180-345)
(73-138)
(125-218)
(0.27-0.43)
275
113
175
0.40
688
(198-382)
300
(96-133)
121
(149-204)
61
(0.32-0.50)
0.71
423
(245-370)
(103-142)
(49-77)
(0.61-0.83)

Human A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 775
Ta ble 3 (Continued)
compd
hA1 (Ki, nM)a
hA2A (Ki, nM)b
hA2B (Ki, nM)c
hA3 (Ki, nM)d
hA1/hA3
956
hA2A/hA3
412
hA2B/hA3
224
43
325
140
(123-162)
148
76
0.34
(0.24-0.48)
0.21
(265-400)
(56-103)
62
66
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
409
50
1948
> 213
191
705
7.49
134
752
915
114
73
238
> 213
9.19
156
96
(358-468)
>10000
(102-215)
(43-58)
> 10000
(0.12-0.37)
352
47
(300-414)
495
(39-55)
3.7
(3.2-4.3)
0.50
(0.40-0.62)
0.55
708
(598-838)
410
34
(26-45)
78
(402-608)
376
820
(314-536)
(332-426)
503
(57-105)
584
52
1062
1338
330
(511-667)
281
(442-571)
(45-60)
161
(0.46-0.64)
24
0.21
767
133
170
181
219
250
91
(210-376)
(18-33)
66
(127-203)
(0.16-0.28)
0.91
300
121
(222-400)
318
(47-93)
(88-165)
136
(0.82-1.02)
50
0.80
398
63
(253-401)
(36-70)
31
(107-172)
(0.70-0.92)
0.72
350
130
486
43
(264-463)
200
(25-40)
(105-170)
125
(0.61-0.84)
91
0.57
351
160
158
116
258
522
345
303
360
(156-258)
(71-11))
95
(116-134)
(0.46-0.70)
0.60
248
150
413
(184-333)
280
(78-114)
(124-180)
78
(0.53-0.68)
100
0.86
326
(245-319)
(83-120)
111
(65-94)
(0.76-0.98)
0.43
300
87
698
202
204
156
142
91
(240-368)
702
(91-137)
(76-98)
165
(0.37-0.50)
423
0.81
867
(624-790)
(379-471)
335
(139-156)
(0.69-0.97)
1.03
714
161
693
(589-866)
351
(289-437)
(132-196)
143
(0.79-1.34)
306
1.01
348
(269-458)
(255-366)
400
(118-173)
(0.65-1.58)
1.11
602
101
542
(525-691)
(312-515)
(89-116)
(0.74-1.67)
a
b
Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells (n ) 3-6). Displacement of specific
[3H]SCH58261 binding at human A2A receptors expressed in HEK-293 cells. 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 expressed
d
in HEK-293 cells. Data are expressed as geometric means, with 95% confidence limits.
In this model, the lowest energy conformation of 59
presents the carbamoyl moiety in the 5-position of the
pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine structure
surrounded by four polar amino acids: Ser242 (TM6),
Ser271 (TM7), His274, and Ser275 (TM7). This region
seems to be very critical for the recognition of the
antagonist structures. According to the J acobson’s site-
directed mutagenesis results for the human A2A recep-
tor, the Ser281, corresponding to Ser275 in the A3
receptors, was found to be crucial for the binding of both
agonists and antagonists.29
The strong interactions between the carbamoyl moiety
and these polar amino acids orient the carbamoyl phenyl
ring in the middle of TM6 and TM7, as clearly indicated
in Figure 4. The receptor region around the para postion
of the phenyl ring is mostly hydrophobic [Val235 (TM6),
Leu236 (TM6), and Asn278 (TM7)]. This environment
could justify why polar substituents at the para position
are not very well tolerated, as in the case of the sulfonic
acid group.
In fact, when a sulfonic acid group was introduced at
the para position on the phenyl ring, a very interesting
group of water-soluble derivatives were obtained, but a
significant reduction of affinity for all four adenosine
receptor subtypes was observed. In fact, compounds 63-
66 lost about 150-300-fold of their affinity vs the hA3
receptor and became almost inactive at the hA1 and
hA2B subtypes (>10 mM). However, these compounds
displayed affinity for the hA2A subtype (250-600 nM)
receptors with a consequent decrease of hA2A/hA3 se-
lectivity, ranging from 6 to 24.
Evaluation of the ligand binding pocket of the receptor
reveals that very limited empty space is present be-
tween TM6 and TM7, and consequently, steric control
seems to be taking place around the para position of
the phenyl ring. In fact, substituents larger than
fluorine, chlorine, methoxy, or methyl are not well
tolerated (see entries 13, 24, 35, 46 for p-NO2, 17, 28,
39, 50 for p-CF3, or 15, 26, 37, 48 for p-Br in Table 3).
All these substituents induce a decrease of affinity at
the human A3 adenosine receptors of about 2-5-fold
with respect to the unsubstituted derivatives (59-62)
or to the previously reported compounds (Table 1), in
which an electron donor group is present at the para
position (1, 3, 5, 7).
The same very rigid steric control is also active when
substituents larger than hydrogen are present at the
meta position of the phenyl ring. In this case a strong
steric repulsion among substituents, at the meta posi-
tion, and amino acid side chains of TM6 and TM7 could
drastically reduce the affinity at the hA3 receptor. On
the contrary, substituents at the ortho position seem to
occupy an empty region of the binding cavity. According
with this model, in the chloro-substituted series the
change of the position (2- or 4-substitution) does not
seem to influence affinity at the hA1, hA2A, and hA2B

776 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Baraldi et al.
F igu r e 3. Stereoview of human A3 transmembrane helical bundle model viewed along the helical axes from the extracellular
end (on the right) and perpendicular to the helical axes (on the left). The docked antagonist compound 59 is shown. See Experimental
Section for details.
F igu r e 4. Side view of the A3-59 complex model. The side chains of the important residues in proximity (e5 Å) to the docked 59
molecule are highlighted and labeled.
receptors compared to the 3-chloro-substituted com-
pounds 2, 4, 6, 8, maintaning it in the high nanomolar
range (100-500 nM), while significant differences ap-
peared in binding affinity to the hA3 adenosine recep-
tors. In particular, it is evident that the best position
of substitution is the para position; in fact, a higher
affinity in comparison with the previously reported
compounds is observed. (e.g., compound 43, hA3 ) 0.34
nM, hA1/hA3 ) 956, hA2A/hA3 ) 412, hA2B/hA3 ) 224,
vs compund 6, hA3 ) 0.91 nM, hA1/hA3 ) 33, hA2A/hA3

Human A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 777
Ta ble 4. Inhibition of Agonist-Mediated cAMP Production by
Compounds with an N8-methyl Substituenta
) 1340, hA2B/hA3 ) 63). In contrast, introduction of a
chloro atom at the ortho position does not seem to alter
significantly the biological profile with respect to the
reference compounds, in terms of affinity, but a modest
increase of selectivity is obtained (e.g., compound 42,
compd
IC50 (nM)
compd
IC50 (nM)
11
12
13
14
15
16
17
26 (15-44)
18
19
20
21
55
59
63
5.6 (3.0-10.8)
7.8 (5.5-10.9)
8.2 (4.4-15.2)
2.4 (1.2-4.9)
6.4 (4.4-9.3)
2.0 (1.4-2.9)
190 (130-279)
3.2 (1.8-5.6)
5.5 (4.3-7.0)
4.6 (2.7-7.8)
4.2 (2.7-6.6)
3.0 (2.0-4.3)
6.6 (3.7-11.7)
hA3 ) 0.71 nM, hA1/hA3 ) 423, hA2A/hA3 ) 170, hA2B
/
hA3 ) 86, vs compund 6, hA3 ) 0.91 nM, hA1/hA3 ) 33,
hA2A/hA3 ) 1,340, hA2B/hA3 ) 63). A similar behavior
was observed also for the methoxy series, but due to
the high levels of affinity, the substitution at the para
positon was not always the best in terms of potency. In
fact, only when the substituent at the N8 of the pyrazole
nitrogen is methyl or butyl is the 4-methoxy substitution
(compounds 1 and 7, Table 1) optimal in terms of both
affinity and selectivity versus the human A3 adenosine
receptors (e.g., compound 18, hA3 ) 0.7 nM hA1/hA3 )
643, hA2A/hA3 ) 257, hA2B/hA3 ) 319, vs compund 1
a
hA3 adenosine receptors were stimulated with 100 nM IB-
MECA, and the results are shown as IC50 values (nM). Values are
the means of at least three experiments and in parentheses the
95% confidence limits are shown.
retical model suggests that the hydrophilic environment
created by Ser243 (TM6) and Ser271 (TM7) might be
responsible for the fact that NH of the phenylcarbamoyl
derivatives is better at accommodating the hypothetical
binding site than CH2 of the phenylacetyl derivatives.
These results seem to suggest, in agreement with
previous observations,19 that the pyrazole nitrogens
could modulate the selectivity, while the urea group is
able to confer potency versus this receptor subtype.
The synthesized compounds bearing at the N8 position
the methyl group (compounds 11-21, 55, 59, 63) were
also tested for inhibition of cAMP-generation by IB-
MECA, in membranes of CHO cells stably transfected
with the human A3 receptor, confirming their antago-
nistic properties (Table 4).24
This result was in accordance with data from previ-
ously synthesized compounds.19 The potency of the
antagonists in the cAMP assay strictly correlated with
that observed in binding assays. In fact, comparing the
binding data with IC50 values of functional assays
resulted in a linear relationship between binding affinity
and antagonistic potency, as clearly indicated by the
following equation:
hA3 ) 0.2 nM, hA1/hA3 ) 5485, hA2A/hA3 ) 6950, hA2B
/
hA3 ) 1305). If an ethyl or propyl chain is linked to the
pyrazole nitrogen, the 2-methoxy substitution gives the
best results in terms of affinity when compared to
reference compounds (e.g., compound 5, hA3 ) 0.80 nM
hA1/hA3 ) 1496, hA2A/hA3 ) 175, hA2B/hA3 ) 2570, vs
compund 40, hA3 ) 0.34 nM, hA1/hA3 ) 735, hA2A/hA3
) 294, hA2B/hA3 ) 485).
Another important interaction, probably π-π interac-
tion, is predicted between the CONH2 group of Asn250
(TM6) and the furan ring of 59. Also, this asparagine
residue, conserved among all adenosine receptor sub-
types, was found to be important for ligand binding.30
Moreover, the hydrophobic pocket, delimited by apolar
amino acids, such as Leu90 (TM3), Phe182 (TM5),
Ile186 (TM5), and Leu190 (TM5), is also present in the
binding site model. The amino acids corresponding to
Leu90 and Phe182 in the human A2A receptor were
found to be essential for the binding of both agonists
and antagonists.29,30 The methyl group at the N8 posi-
tion of 59 is located within this region of the receptor
according to our binding hypothesis. No direct interac-
tions are predicted between the antagonist structure
and the two polar amino acids Thr94 (TM3) and Ser97
(TM3). As previously reported, the corresponding two
amino acids in the A1 and A2A receptors, respectively,
are important for the coordination of agonists, but not
for antagonists.29,30
y ) 0.88((0.03)x + 0.97((0.02) (r ) 0.990)
We found another example demonstrating that the
inibition of the human A3 receptor activity is strictly
correlated with the modality of the recognition between
antagonist and receptor binding domain.31
Con clu sion s
Another interesting aspect studied in this work is
inherent to the evaluation of derivatives bearing at the
N5 position the phenylacetic chain (55-59), character-
istic of MRS 1220 (67). In a previous paper,19 we have
also synthesized two derivatives stucturally related to
MRS 1220 (67)20 by substituting the phenylacetic chain
with the urea moieties (derivatives 9, 10; Table 1),
demonstrating that the phenylcarbamoyl moiety and the
pyrazole nitrogens play an important role both for
potency and selectivity at the human A3 adenosine
receptor subtypes.
In conclusion, the present study provides useful
information concerning the optimal structural require-
ments necessary for antagonist recognition of the A3
adenosine receptor. It demonstrates that the unsubsti-
tuted phenylcarbamoyl moiety confers a slightly better
affinity for the A3 adenosine receptor subtype with
respect to the para-substituted derivatives (e.g., com-
pound 59, hA3 ) 0.16 nM, hA1/hA3 ) 3713, hA2A/hA3 )
2381, hA2B/hA3 ) 1388, vs compund 1, hA3 ) 0.20 nM,
hA1/hA3 ) 5485, hA2A/hA3 ) 6950, hA2B/hA3 ) 1305).
By the use of molecular modeling studies, it has been
demonstrated that the steric factors at the para position
on the phenyl ring play a fundamental role for affinity
at the human A3 receptors. In fact, when a sulfonic acid
group was introduced in the para position on the phenyl
ring, to obtain water-soluble derivatives, a significant
reduction of affinity (150-300-fold) at the hA3 receptor
was observed, with a consequent decrease of selectivity
versus the other receptor subtypes. In addition, all the
Interestingly, as summarized in Table 3, the synthe-
sized compounds (55-58) resulted to be about 4-8-fold
less potent at the human A3 adenosine receptors com-
pared to the parent urea derivatives, with a consequent
significant loss of selectivity (e.g., compound 55, hA3 )
0.81 nM, hA1/hA3 ) 867, hA2A/hA3 ) 522, hA2B/hA3 )
204, vs compund 1, hA3 ) 0.2 nM, hA1/hA3 ) 5485, hA2A
/
hA3 ) 6950, hA2B/hA3 ) 1305). Accordingly, our theo-

778 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Baraldi et al.
for 18 h. Then the solvent was removed under reduced
pressure and the residue was dissolved in EtOAc (30 mL) and
washed twice with water (15 mL). The organic phase was dried
on Na2SO4 and concentrated undr reduced pressure. The
residue was purified by flash chromatography (EtOAc-light
petroleum 4:6) to afford the desired compounds as solids.
Biology.CHO Mem br a n es P r ep a r a tion . The expression
of the human A1, A2A, and A3 receptors in CHO cells has been
previously described.32 The cells were grown adherently and
maintained in Dulbecco’s modified Eagle’s medium with nutri-
ent mixture F12 without nucleosides, containing 10% fetal calf
serum, penicillin (100 U/mL), streptomycin (100 µg/mL),
l-glutamine 2 mM, and Geneticin (0.2 mg/mL) 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 prepara-
tions. The cells were washed with phosphate-buffered saline
and scraped off flasks in ice-cold hypotonic buffer (5 mM Tris
HCl, 2 mM EDTA, pH 7.4). The cell suspension was homog-
enized 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; and in 50 mM Tris HCl, 10 mM MgCl2, 1 mM EDTA
at pH 7.4 for A3 adenosine receptors, and the resuspensions
were utilized for binding and adenylate cyclase assays.
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.24
studies performed clearly demonstrated interactions
involved in the binding of this class of compounds, such
as Ile186 (TM5), Leu190 (TM5), Val235 (TM6), Leu237
(TM6), Ser243 (TM6), Asp250 (TM6), Ser271 (TM7),
Ser275 (TM7), and Asp278 (TM7). Thus, this study
provides new information about the structural require-
ments necessary for antagonist-receptor recognition.
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 hertz. 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. HPLC separations were conducted with a Waters Delta
Prep 3000 A reversed-phase column (30 × 3 cm, 15 mm). The
compounds were eluted with a gradient of 0-60% B in 25 min
at a flow rate of 30 mL/min, and the mobile phases were
solvent A (10%, v/v, acetonitrile in 0.1% TFA) and solvent B
(60%, v/v, acetonitrile in 0.1% TFA). The pure products were
converted into the corresponding hydrochloride forms by
addition of 0.1 N HCl aqueous solution to the mobile phase
containing the pure product. Analytical HPLC analyses were
performed on a Bruker liquid chromatography LC 21-C instru-
ment using a Vydac 218 TP 5415 C18 column (250 × 4 mm, 5
mm particle size) and equipped with a Bruker LC 313 UV
variable-wavelength detector. Recording and quantification
were accomplished with a chromatographic data processor
coupled to an Epson computer system (QX-10). 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.
Gen er a l P r oced u r es for th e P r ep a r a tion of 5-[[(Su b-
stitu ted-ph en yl)am in o]car bon yl]am in o-8-alkyl-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 (11-54).
Amino compound (68-71) (10 mmol) was dissolved in freshly
distilled THF (15 mL), and the appropriate isocyanate (72-
82) (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
11-54.
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
six to eight different concentrations of examined compounds.
Nonspecific binding was determined in the presence of 10 mM
of CHA, and this was always e10% of the total binding.
Binding of [3H]-SCH58261 to CHO cells transfected with
the human recombinant A2A adenosine receptors (50 µg of
protein/assay) was performed according to the method of
Varani et al.33 In competition studies, at least six to eight
different concentrations of compounds were used, and non-
specific binding was determined in the presence of 50 mM
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
A
2B adenosine receptors was performed as already described.24
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 pH 7.4, 2 IU/mL adenosine deaminase
containing 40 nM [3H]DPCPX, diluted membranes (20 mg of
protein/assay), and at least six to eight different concentration
of tested compounds. Nonspecific binding was determined in
the presence of 100 mM of 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 the method of Varani et al.24 Competition experi-
ments were carried out in duplicate in a final volume of 250
µL in test tubes containing 1 nM [3H] MRE3008-F20, 50 mM
Tris HCl buffer, 10 mM MgCl2 (pH 7.4), 100 µL of diluted
membranes (50 µg protein/assay), and at least six to eight
different concentrations of examined ligands. Incubation time
was 120 min at 4 °C, according to the results of previous time-
course experiments.24 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 instrument (efficiency 57%) with
Micro-Scint 20. The protein concentration was determined
according to the Bio-Rad method34 with bovine albumin as
reference standard.
Gen er a l P r oced u r es for th e P r ep a r a tion of 4-[3-(2-
F u r a n -2-yl-8-a lk yl-8H-p yr a zolo[4,3-e][1,2,4]tr ia zolo[1,5-
c]p yr im id in -5-yl)u r eid o]ben zen esu lfon ic Acid Der iva -
tives (63-66). To a cooled 0 °C solution of chlorosulfonic acid
(2 mL) was added the phenyl urea derivative (59-62) (0.35
mmol) in small portions in 20 min. The resulting mixture was
stirred at 0 °C for 4 h, then water (15 mL) was added slowly
to keep the temperature at 0 °C. The white precipitate
(chlorosulfonyl derivative) was filtered and dissolved in diox-
ane (2 mL) and 10% acqueous HCl, and the solution was
stirred at room temperature for 2 days. Then the solvent was
removed under reduced pressure, and the residue was purified
by preparative HPLC in 25 min at a flow rate of 30 mL/min
(reversed phase column; gradient elution 0-60% solvent B),
to afford the desired compounds 63-66 as solids.
Gen er a l P r oced u r es for th e P r ep a r a tion of 5-[(Ben -
zyl)ca r b on yl]a m in o-8-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 e (55-58). Amino compound
(68-71) (10 mmol) was dissolved in freshly distilled THF (15
mL), and phenylacetyl chloride (13 mmol) and triethylamine
(13 mmol) were added. The mixture was refluxed under argon
Ad en yla te Cycla se Assa y. Membranes were suspended
in 0.5 mL of incubation mixture (50 mM tris HCl, MgCl2 10

Human A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 779
mM, EDTA 1 mM, pH 7.4) containing GTP 5 µM, 0.5 mM 4-(3-
buthoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as
phosphodiesterase inhibitor, and 2.0 IU/mL adenosine deami-
nase and preincubated for 10 min in a shaking bath at 37 °C.
Then IB-MECA or antagonists examined plus ATP (1 mM) and
forskolin (10 mM) was added to the mixture and the incubation
continued for an additional 10 min. The potencies of antago-
nists were determined by antagonism of the inhibition oc
cyclic-AMP production induced by IB-MECA (100 nM). The
reaction was terminated by transfer to a boiling water bath.
After 2 min at boiling temperature the tubes were cooled to 4
°C and centrifuged at 2000g for 10 min. Supernatants (100
µL) were used in a protein competition assayfor c-AMP carried
out essentially according to the method of Varani et al.24
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-fu-
ranyl)[1,2,4]triazolo[1,5-c]quinazoline; CHA, N6-cyclohexyladen-
osine; DPCPX, 1,3-dipropyl-8-cytclopentylxanthine; DMSO,
dimethyl sulfoxide; THF, tetrahydrofuran; CHO cells, Chinese
hamster ovary cells; EDTA, ethylenediaminotetraacetate;
SCH58261, 5-amino-2-(2-furyl)-7-(2-phenylethyl)pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidine; 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]pyrimi-
dine; GTP, guanosine-5′-triphosphate; cAMP, cyclic adenosine-
5′-monophosphate; ATP, adenosin-5′-triphosphate; TLC, thin-
layer chromatography; HCl, hydrochloric acid; mp, melting
point; EtOAc, ethyl acetate; HPLC, high performance liquid
chromatography; IR, infrared spectra; NMR, nuclear magnetic
resonance; CDCl3, deuterated chloroform.
Samples of cyclic AMP standards (0-10 pmol) were added
to each test tube containing the incubation buffer (trizma base,
0.1 M; aminophylline, 8.0 mM; 2-mercaptoethanol, 6.0 mM;
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
and, after the addition of charcoal, the samples were centrifu-
gated 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.
Ack n ow led gm en t. We wish to thank Dr. Kenneth
A. J acobson, NIH, Bethesda, MD, for the use of its
human A3 adenosine receptor model in the molecular
modeling studies, and Regione Friuli Venezia Giulia
(Fondo 2000), for partial financial support.
Com p u ta tion a l Ap p r oa ch . All calculations were per-
formed on a Silicon Graphics Octane R12000 workstation.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This material is available free of charge via the Internet
at http://pubs.acs.org.
The human A3 receptor model was built and optimized using
the MOE (2000.02) modeling package35 based on the approach
described by Moro et al.36 Briefly, transmembrane domains
were identified with the aid of Kyte-Doolittle hydrophobicity
and Emin surface probability parameters.37 Transmembrane
helices were built from the sequences and minimized individu-
ally. The minimized helices were then grouped together to form
a helical bundle matching the overall characteristics of the
recently published crystal structure of bovine rhodopsin (PDB
ID: 1F88). The helical bundle was minimized using the
Amber94 all-atoms force field,38 until the rms value of the
conjugate gradient (CG) was <0.01 kcal/mol/Å. A fixed dielec-
tric constant ) 4.0 was used throughout these calculations.
All pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives
were fully optimized without geometry constraints using RHF/
AM1 semiempirical calculations.39 Vibrational frequency analy-
ses were used to characterize the minimal stationary points
(zero imaginary frequencies). The software package Gaussian-
98 was utilized for all quantum mechanical calculations.40
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