Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human a3 adenosine receptor antagonists

Full text of DOI:10.1021/jm991114s

© Copyright 1999 by the American Chemical Society
Volume 42, Number 22
November 4, 1999
Communications to the Editor
observed responses to chronic agonist and antagonist
P yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]-
p yr im id in e Der iva tives a s High ly P oten t
a n d Selective Hu m a n A3 Ad en osin e
Recep tor An ta gon ists
exposure are the exact opposite to the predicted effects
based on short-term exposure to these agents.12
Activation of adenosine A3 receptors has been shown
to stimulate phospholipase C13 and D14 and to inhibit
adenylate cyclase.3 Activation of A3 adenosine receptors
also causes the release of inflammatory mediators such
as histamine from mast cells.11,15 These mediators are
responsible for processes such as inflammation10 and
hypotension.11 It has also been suggested that the A3
receptor plays an important role in brain ischemia,16,17
immunosuppression,18 and bronchospasm in several
animal models.19
Pier Giovanni Baraldi,*,§ Barbara Cacciari,§
Romeo Romagnoli,§ Giampiero Spalluto,†
Karl-Norbert Klotz,| Edward Leung,∇ Katia Varani,‡
Stefania Gessi,‡ Stefania Merighi,‡ and
Pier Andrea Borea‡
Dipartimento di Scienze Farmaceutiche and
Dipartimento di Medicina Clinica e Sperimentale-Sezione di
Farmacologia, Universita` degli Studi di Ferrara,
Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy,
Dipartimento di Scienze Farmaceutiche, Universita` degli
Studi di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy,
Institut fu¨r Pharmakologie, Universita¨t of Wu¨rzburg,
D-97078 Wu¨rzburg, Germany, and Medco Research,
Research Triangle Park, North Carolina 27709
Taking into account these results, highly selective A3
adenosine receptor antagonists have been indicated as
potential drugs for the treatment of asthma and inflam-
mation.10,20
In the past few years, different classes of compounds
have been reported to be A3 adenosine receptor antago-
nists. Four classes of compounds with non-xanthine
structures have been synthesized: dihydropyridine and
pyridine analogues,21-24 flavonoid,25,26 isoquinoline,27-28
and triazoloquinazoline derivatives.29-30
In the latter class of compounds, J acobson and co-
workers,29 started from the experimental observation
that the 5-amino-9-chloro-2-2-furyl[1,2,4]triazolo[1,5-
c]quinazoline (CGS15943) possesses affinity for the
human A3 adenosine receptor (Ki hA3 14 nM). In fact,
by its acylation with a phenylacetyl group at the amino
function at the 5-position was obtained MRS 1220 (1),
the most potent but not highly selective A3 adenosine
receptor antagonist reported in the literature.
Received J uly 1, 1999
In tr od u ction . Adenosine modulates many physi-
ological functions through activation of four known
receptor subtypes, classified as A1, A2A, A2B, and A3.1,2
The adenosine A1 and A2A receptor subtypes have been
pharmacologically characterized through the use of
selective ligands.3
The adenosine A3 receptor was initially cloned from
a rat testis cDNA library.4 Subsequently, A3 receptors
have been cloned from human,5,6 sheep,7 mouse,8 and
rabbit.9 Considerable differences in sequence homology
(72%) for the A3 receptors have been observed between
species.10,11 Affinities for antagonist ligands also showed
differences between species. For these reasons, the
hypothesis of the existence of two different adenosine
A3 receptor subtypes has been proposed. However
different A3 receptor subtypes within a single species
have not yet been demonstrated.12
The adenosine A3 receptor appears to be responsible
for many physiological effects, but in some cases the
* Correspondence to: Prof. Pier Giovanni Baraldi. Phone: +39-(0)-
532-291293. Fax: +39-(0)532-291296. E-mail: pgb@dns.unife.it.
§ Dipartimento di Scienze Farmaceutiche, Ferrara.
† Universita` degli Studi di Trieste.
| Universita¨t of Wu¨zburg.
In the past few years, we have synthesized a large
series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine
∇ Medco Research.
‡ Dipartimento di Medicina Clinica e Sperimentale, Ferrara.
10.1021/jm991114s CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/08/1999

4474 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Communications to the Editor
Ch a r t 1. Rational Design of hA3 Adenosine Receptor
Antagonists
with 2-furoic hydrazide in refluxing 2-methoxyethanol
to provide the pyrazolo[3,4-d]pyrimidine intermediates.
These were converted through a thermally induced
cyclization in diphenyl ether to the desired derivatives
22-25 in good overall yield (50-63%), after separation
of N7 (minor product) and N8 (major product) regioiso-
mers by flash chromatography.32
Hydrolysis with aqueous 10% HCl afforded the ami-
notriazoles 26-29, which were in turn converted into
the 5-amino-8-(substituted)-2-(2-furyl)pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidine derivatives 30-33. The
final compounds 5-12 were obtained by a coupling
reaction of derivatives 30-33 with the commercially
available isocyanates 34 and 35 (Scheme 2).35
The coupling reaction with isocyanates was successful
using N8 alkylated compounds, but it failed in the case
of the N7 pattern of substitution (classic A2A antagonists;
e.g. 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-
e]-1,2,4-triazolo[1,5-c]pyrimidine, SCH 58261), probably
due to the less nucleophilic character of the amino group
at the 5-position. In fact, the nitrogen lone pair of the
5-amino group in N7 derivatives is highly delocalized
on the heterocyclic ring. This does not happen in the
case of the N8 derivatives, because the relative meso-
meric structures do not allow the same degree of
delocalization.
Resu lts a n d Discu ssion . Table 1 gives the receptor
binding affinities of compounds 5-12 and the corre-
sponding N5-unsubstituted derivatives 30-33 deter-
mined at rat A1 and A2A receptors and human A3
receptors expressed in HEK-293 cells, using [3H]-1,3-
dipropyl-8-cyclopentylxanthine ([3H]DPCPX),37 [3H]-5-
amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine ([3H]SCH 58261),38 and [125I]-
N6-(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)-
adenosine ([125I]AB-MECA)39 as radioligands, respec-
tively.
Compounds lacking the phenylcarbamoyl moiety at
the N5 position show high affinity for the A2A receptor
with low selectivity versus the A1 receptor and are
inactive at human A3 adenosine receptor subtypes. As
expected, compounds with the greatest affinity at the
A2A receptors, 32 and 33, have the 2-phenylethyl and
3-phenylpropyl chains, respectively, at the pyrazole
nitrogen, typical of A2A antagonists. These derivatives
are the regioisomers of two of the most potent previously
reported A2A antagonists: SCH 58261 and 5-amino-7-
(3-phenylpropyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-tria-
zolo[1,5-c]pyrimidine (SCH 63390).33
On the contrary, when the substituted phenylcar-
bamoyl chain is present at the N5 position, all the
synthesized derivatives 5-12 show high affinity to
human A3 receptors with a high degree of selectivity
versus the other receptor subtypes. In particular the
4-methoxyphenylcarbamoyl moiety (compounds 5, 7, 9,
11) appears to confer higher affinity to the human A3
receptor than the 3-chlorophenylcarbamoyl chain (com-
pounds 6, 8, 10, 12) with a difference of about 2-10
orders of magnitude.
It is evident that small chains such as ethyl and
propyl on N8 of the pyrazole afford compounds with high
affinity and selectivity. In particular, compound 5, with
the ethyl group at the N8 pyrazole combined with the
4-methoxyphenylcarbamoyl moiety at the N5-position,
derivatives of general formula 2, structurally related to
CGS15943, which turned out to be potent and selective
A2A antagonists.31-34 In addition, several N6-(substituted
phenylcarbamoyl)adenosine-5′-uronamides of general
formula 3 have been reported to act as potent agonists
for the rat A3 adenosine receptor subtype (Chart 1).35,36
Specifically, we observed that the introduction of the
3-chlorophenyl and 4-methoxyphenyl moieties gave the
best results in terms of affinity at rat A3 receptors (Ki
4.4 and 6.6 nM, respectively).
On this basis, we decided to link the amino group at
the 5-position, of compounds of general formula 2, with
the two bulky substituents (3-chlorophenylcarbamoyl
and 4-methoxyphenylcarbamoyl moieties), which dis-
played the best results in the field of A3 agonists,35,36
in an attempt to modulate the affinity and the selectiv-
ity at the human A3 receptors. (Chart 1). Maintaining
the substituents on the phenyl ring (3-chloro or 4-meth-
oxy) at the 5-position, the effects of the lipophilic groups,
such as small alkyl and aralkyl moieties, on the pyrazole
nitrogen of the synthesized hybrid molecules of general
formula 4 have been evaluated.
Ch em istr y. Compounds 5-12 were prepared follow-
ing the general synthetic strategy summarized in
Schemes 1 and 2. Compounds 5-12 were synthesized
according to a well-known procedure for the synthesis
of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines.32
Alkylation of 5-amino-4-cyanopyrazole (13) with the
appropriate alkyl halide in dry dimethylformamide led
to an approximately 1:4 mixture of N1 and N2 regioiso-
mers (14-17) as an inseparable mixture, used for the
following steps without any further purification (Scheme
1).32
Pyrazoles 14-17 were transformed into the corre-
sponding imidates 18-21 through refluxing in triethyl
orthoformate. The imino ethers 18-21 were reacted

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4475
Sch em e 1a
a
Reagents: (i) NaH, DMF, RX; (ii) HC(OEt)3, reflux; (iii) 2-furoic hydrazide, MeO(CH2)2OH; (iv) Ph2O, 260 °C, flash chromatography;
(v) HCl, reflux; (vi) NH2CN, 1-methyl-2-pyrrolidone, pTsOH, 140 °C.
Sch em e 2a
a
Reagents: (i) THF, reflux, 12 h.
shows the best binding profile with a high human A3
affinity (Ki 0.28 nM) and selectivity versus A1 and A2A
receptors, higher than 35 000. This derivative could be
considered, at present, the most potent and selective hA3
antagonist ever synthesized. The same degree of affin-
ity, even with a decrease of selectivity, has been
observed in compounds with a propyl substituent at N8
of selectivity. In particular, the most potent compound
at human A3 receptors of this series (5) showed again a
high level of selectivity versus other receptor subtypes
(>3 000). On the contrary, in a rat model (Table 3), all
the compounds proved to be almost inactive showing
different percentages of inhibition (1-40%) of specific
binding at a concentration of 10 µM. These results are
in accordance with the low degree of sequence homology
(72%) of the A3 adenosine receptor subtypes.10,11 All the
synthesized compounds were also found to be functional
antagonists in a specific functional model where the
inhibition of cAMP generation by IB-MECA was mea-
sured in membranes of CHO cells stably transfected
with the human A3 receptor (Table 4).43
As expected, all the derivatives are antagonists with
different degrees of potency. Compounds 30-33, with
the free amino group at the 5-position, as observed in
binding studies, showed poor activity, inhibiting the
effect of 100 nM IB-MECA in a range of 3-40% at 1
µM concentration. On the contrary, the N5-substituted
compounds (5-12) were more potent in the functional
assay than unsubstituted derivatives (30-33). In par-
ticular, the most potent derivatives in binding studies
inhibit the effect of IB-MECA at 1 µM concentration
from 50% to 88%, displaying IC50 values in the nano-
molar range (4.5-15.1 nM).
(e.g. compound 7 hA3 0.29 nM, rA1/hA3 >34 000, rA2A
/
hA3 6 872). Significant differences have been observed
when the phenylethyl and phenylpropyl chains were
introduced; in fact, a reduction of affinity of about 10-
100-fold is evident (e.g. compound 7 hA3 0.29 nM vs
compound 11 hA3 19.81 nM).
Earlier experiments using cloned A1 and A2A human
adenosine receptors have shown a significant decrease
in selectivity for many ligands whith respect to rat A1
and A2A receptors.40 In this study, all the synthesized
compounds have been tested on human A1,41 human
A2A,
38 and rat A325a,42 adenosine receptors, to verify the
selectivity in the same species (Tables 2, 3).
It is clearly evident, from the results shown in Table
2, that in the human species, these compounds show
an increase in affinity of about 10-fold at human A1 and
A2A receptors, with a consequent decrease of selectivity
for A3 receptors. Nevertheless, all the N5-substituted
compounds (5-12) retain good A1/A3 and A2A/A3 ratios

4476 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Communications to the Editor
Ta ble 1. Binding Affinity at rA1, rA2A and hA3 Adenosine Receptors of Compounds 5-12 and 30-33
compd
R
R1
rA1 (Ki, nM)a
rA2A (Ki, nM)b
hA3 (Ki, nM)c
rA1/hA3
rA2A/hA3
1, MRS 1220d
30
52.7 ( 11.8
95.09
10.3 ( 3.7
11.15
0.65 ( 0.25
3579
81
0.03
16
0.003
C2H5
H
(86.76-104.22)
(9.84-12.63)
(3376-3793)
5
6
C2H5
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
>10000
>10000
0.28
>35714
1291
0.22
>35714
1339
0.03
(0.25-0.32)
2.09
(1.9-2.31)
613
(582-646
0.29
(0.27-0.32)
0.49
(0.47-0.52)
2785
(2463-3149)
1.47
(1.22-1.78)
13.28
(10.87-16.23)
2666
C2H5
2699
(2521-2889)
139
(107-181)
>10000
2799
(2621-2989)
20.23
(16.14-25.36) )
1993
(1658-2397)
>10000
31
7
n-C3H7
n-C3H7
n-C3H7
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
>34482
3228
0.0007
872
6872
>20408
0.0002
951
8
1582
(1447-1730)
2.16
(1.9-2.47)
1282
(1148-1432)
1049
(961-1145)
11.13
(9.34-13.27)
1514
(1332-1721)
>10000
32
9
Ph-CH2-CH2
0.70
(0.53-0.91)
1398
(1225-1594)
1698
(1524-1892)
0.59
Ph-CH2-CH2
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
H
10
33
11
12
Ph-CH2-CH2
79
128
Ph-CH2-CH2-CH2
Ph-CH2-CH2-CH2
Ph-CH2-CH2-CH2
0.004
76.4
0.0002
>504
75
(0.44-0.81)
>10000
(2533-2805)
19.81
(17.61-22.27)
42.65
4-MeO-Ph-NHCO
3-Cl-Ph-NHCO
3200
(3025-3385)
>234
(39.92-45.57)
a
b
Displacement of specific [3H]DPCPX binding (A1) in rat brain membranes (n ) 3-6). Displacement of specific [3H]SCH 58261 binding
(A2A) in rat striatal membranes. c Displacement of specific [125I]AB-MECA binding at human A3 receptors expressed in HEK-293 cells.
Data are expressed as geometric means, with 95% confidence limits. Values taken from ref 30.
d
Ta ble 2. Binding Affinity at hA1, hA2A, and hA3 Adenosine Receptors of Compounds 5-12 and 30-33
compd
hA1 (Ki, nM)a
hA2A (Ki, nM)b
hA3 (Ki, nM)c
hA1/hA3
0.0014
hA2A/hA3
0.0005
30
5.00
1.90
3579
(4.05-6.20)
(1.72-2.09)
(3376-3793)
5
6
1026
1045
0.28
3664
119
3732
88.5
(785-1341)
249
(834-1309)
185
(0.25-0.32)
2.09
(215-289)
(159-214)
(1.9-2.31)
31
7
10
2.55
613
0.016
4127
61
0.004
486
(7-14)
1197
(1027-1396)
30
(23-40)
1.00
(0.66-1.51)
201
(1.91-3.39)
141
(123-161)
1221
(882-1689)
0.31
(0.20-0.48)
124
(582-646)
0.29
(0.27-0.32)
0.49
(0.47-0.52)
2785
2463-3149)
1.47
8
2491
0.0001
84.3
32
9
0.0003
136
(172-236)
251
(229-276)
3.00
(2.65-3.39)
251
(221-286)
1500
(100-155)
110
(93-130)
0.18
(0.07-0.50)
1015
(885-1163)
202
(1.22-1.78)
13.28
(10.87-16.23)
2666
(2533-2805)
19.81
(17.61-22.27)
42.65
10
33
11
12
19
8.2
0.001
12.6
35.1
0.00006
51.2
4.7
(1325-1693)
(11-216)
(39.92-45.57)
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 [125I]AB-MECA binding at human
A3 receptors expressed in HEK-293 cells. Data are expressed as geometric means, with 95% confidence limits.
From these experimental observations, it is possible
to hypothesize that the characteristic aralkyl chains of
Con clu sion s. The present study provides useful
information concerning the structural requirements
necessary for recognition by the A3 adenosine receptor.
It confirms that a substituted phenylcarbamoyl moiety
confers affinity and selectivity for the A3 adenosine
receptor subtype at the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-
A2A antagonists are not optimal for A3 receptor interac-
tion, while small chains present the ideal, steric and
probably lipophilic, characteristics for the interaction
with the human adenosine A3 receptor subtype.

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4477
modifications of the substituents at N5-, N7-, and N8-
positions could modulate both affinity and selectivity
for the different adenosine receptor subtypes.
Ta ble 3. Binding Affinity at hA3 and rA3 Adenosine Receptors
of Compounds 5-12 and 30-33
a
compd
rA3 (% inhibn)
1
hA3 (Ki, nM)b
30
3579
(3376-3793)
Ack n ow led gm en t. We thank Medco Research, Tri-
angle Park, NC, for financial support and Dr. Ken A.
J acobson, NIH, Bethesda, MD, for a preliminary binding
assay of a couple of reference compounds on hA3
adenosine receptors.
5
6
39
36
2
0.28
(0.25-0.32)
2.09
(1.9-2.31)
613
(582-646)
0.29
(0.27-0.32)
0.49
(0.47-0.52)
2785
(2463-3149)
1.47
(1.22-1.78)
13.28
(10.87-16.23)
2666
31
7
37
20
12
37
30
32
11
20
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This information is available free of charge via the
Internet at http://pubs.acs.org.
8
32
9
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10
33
11
12
(2533-2805)
19.81
(17.61-22.27)
42.65
(39.92-45.57)
Displacement of specific [125I]AB-MECA binding at rat A3
a
receptors expressed in CHO cells; data are expressed as percentage
of inhibition of specific binding at a concentration of 10 mM.
Displacement of specific [125I]AB-MECA binding at human A3
b
receptors expressed in HEK-293 cells; data are expressed as
geometric means, with 95% confidence limits.
Ta ble 4. Functional Assay: Percentage of Blockade by 1 µM of
Each Compound 30-33 and 5-12 of the Inhibition by 100 nM
IB-MECA-Inhibited cAMP Accumulation in CHO Cells
Expressing hA3 Adenosine Receptorsa
compd
% inhibn
IC50 (nM)
30
5
6
31
7
8
32
9
10
33
11
12
3 (2-7)
88 (79-97)
71 (61-81)
43 (30-61)
89 (79-100)
87 (76-99)
5 (3-7)
6.5 (3.7-11.2)
15.1 (9.9-23.0)
40.2 (32.7-49.3)
4.5 (3.7-5.5)
5.3 (3.2-8.9)
81 (75-89)
62 (43-75)
6 (3-10)
56 (37-7.1)
50 (34-72)
12.4 (8.0-19.2)
a
For compounds 5-8 and 31, IC50 values are also shown. Values
are the means of at least three experiments, and in parentheses
the 95% confidence limits are shown.
c]pyrimidine nucleus, while the N5-unsubstituted de-
rivatives lacks both affinity and selectivity for human
A3 receptors, showing high affinity for A1 and/or A2A
receptor subtypes. When the pyrazolo[4,3-e]-1,2,4-tria-
zolo[1,5-c]pyrimidine nucleus is substituted at the N8-
position with small alkyl groups, higher affinity and
selectivity at the human A3 receptors was observed.
When the N8-ethyl and N5-4-methoxyphenylcarbamoyl
substitutions were combined, the most potent and
selective human A3 adenosine antagonist (5) was ob-
tained (Ki ) 0.28 nM, rA1/hA3 > 35 000, rA2A/hA3 >
35 000, hA1/hA3 > 3 600, hA2A/hA3 > 3 600).
In addition, the results herein presented and our
previous studies31-33 allow us to propose the pyrazolo-
[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus as a pos-
sible template for generating adenosine receptor subtype-
selective ligands. In fact, it is quite evident that

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