Synthesis and biological data of 4-amino-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid ethyl esters, a new series of A1-adenosine receptor (A1AR) ligands

Article abstract of DOI:10.1016/S0960-894X(01)00483-8

The synthesis of a new family of A₁-adenosine receptor (A₁AR) ligands 3a-n has been performed in a straightforward way. Affinity data at A₁AR, A_2AAR and A₃AR in bovine membranes show that these new co

Full text of DOI:10.1016/S0960-894X(01)00483-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2529–2531
Synthesis and Biological Data of 4-Amino-1-(2-chloro-2-
phenylethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic Acid Ethyl
Esters, a New Series of A1-Adenosine Receptor (A1AR) Ligands
Silvia Schenone,a,* Olga Bruno,a Paola Fossa,a Angelo Ranise,a Giulia Menozzi,a
Luisa Mosti,a Francesco Bondavalli,a Claudia Martinib and Letizia Trincavellib
a
` `
Dipartimento di Scienze Farmaceutiche, Facolta di Farmacia dell’Universita degli Studi di Genova,
Viale Benedetto XV, 3, 16132, Genoa, Italy
` `
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Facolta di Farmacia dell’Universita degli Studi di Pisa,
b
Via Bonanno 6, 56126, Pisa, Italy
Received 28 March 2001; revised 18 June 2001; accepted 10 July 2001
Abstract—The synthesis of a new family of A1-adenosine receptor (A1AR) ligands 3a–n has been performed in a straightforward
way. Affinity data at A1AR, A2AAR and A3AR in bovine membranes show that these new compounds bind the A1AR in a selective
way over A2AAR and A3AR and one of them (3j) presents a very high affinity, probably due to the phenethylamine substituent at
C-4. # 2001 Elsevier Science Ltd. All rights reserved.
Adenosine, whose physiological function has been
defined and reported in recent years,1 is a neuro-
transmitter distributed through a wide variety of tissues
in mammal.2 This nucleoside modulates its effects
through interaction with four subtype receptors desig-
nated as A1, A2A, A2B and A3.3 They belong to the big
family of the G-protein coupled receptors which consist
of seven trans-membrane helices. Researching for selec-
tive agonists and/or antagonists for each type of recep-
tor and also for different tissue is an expanding target in
medicinal chemistry development.
studied as antagonists at A1 and A2ARs; they are gen-
erally flat molecules, aromatic, nitrogen-containing het-
erocycles, often 6,5-fused.2,6 The pyrazolo[3,4-b]pyridines
Tracazolate 1a and Etazolate 1b (Fig. 1) are among the
first non-xanthine antagonists which have been found to
inhibit A1-adenosine brain receptor binding.8,9
A series of pyrazolo[3,4-b]pyridine-5-carboxylic acid
derivatives 2 (Fig. 1), which show an interesting affinity
to A1 and A2 receptors, have subsequently been repor-
ted,10 while many other products with similar activity
have recently been patented.11
Adenosine interacts in many aspects of the brain func-
tion by mediating central inhibitory effects.4,5 It is
believed that A1-adenosine receptor (A1AR) antago-
nists, which stimulate the activity of the central nervous
system, will, in future, be used in the treatment of var-
ious forms of dementia, such as Alzheimer’s disease.6
We are going to report in this communication the
synthesis and preliminary biological data of a new
family of potent and selective A1AR ligands 3 (Fig. 1)
which are structurally related to 1 and 2, but bear a 2-
chloro-2-phenylethyl chain in order to improve the
lipophilic character of the substituent in position 1, in
comparison with the simple methyl or ethyl groups
usually present in their analogues 1 and 2.
Xanthines (caffeine and theophilline) are prototypic
adenosine receptor antagonists, but are non-selective
and of moderate potency.7 Many structurally different
non-xanthine derivatives have been synthesized and
The synthesis of compounds 312 is outlined in Scheme 1.
Reaction of 2-hydrazino-1-phenylethanol 4, pre-
pared following
a
literature procedure,13 with
ethylethoxymethylenecyanoacetate 5 gave ethyl ester
*Corresponding author. Tel.: +39-010-353-8866; fax: +39-010-353-
8358; e-mail: schensil@unige.it
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00483-8

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S. Schenone et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2529–2531
of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-
4-carboxylic acid 6 in a very good yield. Basic hydrolysis
of 6, followed by thermal decarboxylation,10 quantitatively
afforded 2-(5-amino-pyrazol-1-yl)-1-phenylethanol 7.
Compounds were tested for their ability to displace
[3H]N6-cyclohexyladenosine ([3H]CHA) to A1 adenosine
receptors, [3H]-2-[[4-(2-carboxyethyl)phenethyl]-amino]-
5-(N-ethyl-carbamoyl)adenosine ([3H]CGS21680) to A2A
adenosine receptors, and, for the most active compounds,
Condensation of the latter with diethyl ethoxy-
methylenemalonate gave the intermediate 8, which upon
treatment with POCl3 at reflux (36 h) underwent cycli-
zation to the pyrazolo-pyridine nucleus with a con-
current chlorination of the hydroxylic side chain to 9 in
yields of 50% after chromatographic purification.
[
125I]-N-(3-iodo-4-aminobenzyl)-50-N-methylcarbox-
amidoadenosine ([125I]AB-MECA) to A3 adenosine
receptors in bovine membranes following a previously
reported procedure.15 The A1, A2A, A3 receptor-binding
affinities, expressed as Ki or % of inhibition binding, for
compounds 3a–n are reported in Table 1.
Regioselective substitution of chlorine at C-4, with an
excess of various amines, afforded the desired products
3 in good yield.
The data show that this family of compounds is very
selective against the A1 adenosine receptor. In fact, in
the range of concentrations used for the tests, most
compounds show moderate to high affinity for A1 and
no affinity for A2A and A3 adenosine receptors.
It is interesting to point out that, in all of the syntheses
performed, the chlorine atom at the side chain has never
been substituted by the amine in spite of its benzylic
Compound 3j showed the highest affinity value against
A1AR (50 nM), thus being the best of the series and
comparable to the more active compounds reported in
literature,16 while compounds 3a, 3b, 3e, 3f and 3i
1
position, as shown by the H NMR chemical shifts of
the CH2–CH side-chain protons, which give an ABX
complex pattern owing to their non equivalence.14
Figure 1.
Scheme 1.

S. Schenone et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2529–2531
2531
Table 1. Affinity of 3a–n derivatives at bovine brain A1, A2A and
A3ARs
bon atom dramatically drops the affinity (see Table 1),
the only exception being 3f, which still maintains a sig-
nificant affinity value (98.2nM). The length of the chain
(see affinity of 3c in comparison with 3a) as well as its
bulkiness are also very important (see 3d). So as to bet-
ter understand the SAR in this A1AR antagonist family,
a molecular modeling study is in progress and will be
reported in a future paper.
Ki (nM) or % inhibitiona
b
c
d
Compounds
R
bA1
bA2A
bA3
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
NH(CH2)2CH3
NH cyclopropyl
NH(CH2)3CH3
NHC(CH3)3
NH(CH2)2OC2H5
1-Pyrrolidino
4-Morpholino
NHC6H5
NHCH2C6H5
NHCH2CH2C6H5
1-Piperidino
100ꢀ8.4 11%
112ꢀ9.6 10%
23%
—
4100ꢀ2
3
3%
0%
—
—
4800ꢀ3219%
—
151ꢀ10
2%
98.2ꢀ7.3 17%
—
0%
470ꢀ2
9
348ꢀ21 11%
—
—
34%
—
—
—
Acknowledgements
139ꢀ10
50ꢀ3.7
41%
35%
0%
0%
4%
23%
22%
6%
Financial support from Italian MURST (Cofin 2000) is
gratefully acknowledged.
1-Hexahydroazepino
1-Piperazino
1-(4-Methyl)piperazino
22%
2140ꢀ112—
aThe Ki values are meansꢀSEM of three separate assays, each per-
References and Notes
formed in triplicate.
bDisplacement of specific [3H]CHA binding in bovine cortical mem-
branes or percentage of inhibition of specific binding at 10 mM con-
centration.
1. Jacobson, K. A.; Van Galen, P. J. M.; Williams, M. J.
Med. Chem. 1992, 35, 407.
2. Daly, J. W. J. Med. Chem. 1982, 25, 197.
3. Fredholm, B.; Abbracchio, M. P.; Burnstock, G.; Daly,
J. W.; Harden, T. K.; Jacobson, K. A.; Leff, P.; Williams, M.
Pharmacol. Rev. 1994, 46, 143.
cDisplacement of specific [3H]CGS21680 binding in bovine striatal
membranes or percentage of inhibition of specific binding at 10 mM
concentration.
dDisplacement of specific [125I]AB-MECA binding in bovine cortical
membranes or percentage of inhibition of specific binding at 10 mM
concentration. Only compounds 3a, 3f and 3j were tested.
4. Williams, M. Neurochem. Inter. 1989, 14, 249.
5. Erfurth, A. CNS Drugs 1994, 2, 184.
6. Poulsen, S. A.; Quinn, S. A. Bioorg. Med. Chem. 1998, 6,
619 and references cited therein.
showed good affinity data (in a range of 98.2–152 nM).
Among the other compounds, 3g and 3h were also
effective although with lower affinity (470 and 348 nM);
compounds 3l, 3m and 3n did not show any detectable
affinity.
7. Camaioni, A.; Costanzi, S.; Vittori, S.; Volpini, R.; Klotz,
K. N.; Cristalli, G. Bioorg. Med. Chem. 1998, 6, 523.
8. Murphy, K. M.; Synder, S. H. Life Sci. 1981, 28, 917.
9. Williams, M.; Risley, E. A.; Huff, J. R. Can. J. Phys.
Pharmacol. 1981, 59, 897.
10. Daly, W. J.; Hutchinson, K. D.; Secunda, S. I.; Shi, D.;
Padgett, W. L.; Shamin, M. T. Med. Chem. Res. 1994, 4, 293.
11. Akane, A.; Kuroda, S.; Itani, H.; Shimizu, Y. Patent NO
WO9803507, 29-1-1998, CA 128:154090. Akane, A.; Nishi-
mura, S.; Kurota, S.; Itani, H. Patent NO JP10182643, 7-7-
1998 CA 129:144876.
The structural requirements for high affinity in these
pyrazolo[3,4-b]pyridine compounds appear to be an N-1
2-chloro-2-phenylethyl side chain together with a phen-
ethylamine substituent at C-4 carbon atom. Many other
differently N-1 substituted compounds have been syn-
thesized and biologically tested, the data of which will
be reported in full in due course. It is, however, worth
pointing out that compounds with the N-1 2-chloro-2-
phenylethyl side chain always showed higher affinity
values.
12. All the synthesized compounds gave correct spectroscopic
data and elemental analyses (range ꢀ0.3%).
13. Benoit, G. Bull. Soc. Chim. Fr. 1939, 6, 708.
14. 1H NMR of CH2–CH group of compound 9: d 4.87 and
4.93 (dd, 1H of CH2), 5.08 and 5.15 (dd, 1H of CH2), 5.54 and
5.60 (dd, 1H, CHCl).
15. Da Settimo, F.; Primofiore, G.; Taliani, S.; Marini, A. M.;
La Motta, C.; Novellino, E.; Lavecchia, A.; Trincavelli, L.;
Martini, C. J. Med. Chem. 2001, 44, 316.
16. Muller, C. E.; Stein, B. Curr. Pharm. Des. 1996, 2, 501 and
references cited therein.
The C-4 substitution plays a very important role in
modulating the affinity of this class of compounds to the
A1 adenosine receptor. Variation of the nature of the
amine function (from secondary to tertiary) at C-4 car-