Synthesis and pharmacology of pyrido[2,3-d]pyrimidinediones bearing polar substituents as adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2005.12.008

Amino-substituted pyrido[2,3-d]pyrimidinediones have previously been found to bind to adenosine A₁ and A_2A receptors in micromolar concentrations. The present study was aimed at studying the structure-activity relationships of this c

Full text of DOI:10.1016/j.bmc.2005.12.008

Bioorganic & Medicinal Chemistry 14 (2006) 2837–2849
Synthesis and pharmacology
of pyrido[2,3-d]pyrimidinediones bearing polar
substituents as adenosine receptor antagonists
Jacek Bulicz,a Daniela C. G. Bertarelli,b Dieter Baumert,b Friederike Fulle,b
¨
Christa E. Mullerb and Dieter Hebera,*
¨
aDepartment of Pharmaceutical Chemistry, Pharmaceutical Institute, University of Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany
bPharmaceutical Chemistry Poppelsdorf, Pharmaceutical Institute, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany
Received 27 September 2005; revised 28 November 2005; accepted 2 December 2005
Available online 11 January 2006
Abstract—Amino-substituted pyrido[2,3-d]pyrimidinediones have previously been found to bind to adenosine A1 and A2A receptors
in micromolar concentrations. The present study was aimed at studying the structure–activity relationships of this class of
compounds in more detail. Most of the investigated compounds were provided with polar substituents, such as ethoxycarbonyl
groups and basic amino functions, in order to improve their water-solubility. The compounds were synthesized starting from 6-ami-
no-1,3-dimethyluracil via different reaction sequences involving (cyano)acetylation, Vilsmeier formylation, or reaction with diethyl
ethoxymethylenemalonate (EMME). The most potent and selective compound of the present series was 6-carbethoxy-1,2,3,4-tetra-
hydro-1,3-dimethyl-5-(2-naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (11c) with a Ki value of 5 nM at rat and 25 nM at
human A1 receptors. The compound was more than 60-fold selective versus A3 and more than 300-fold selective versus A2A recep-
tors. It showed an over 300-fold improvement with respect to the lead compound. In GTPcS binding studies at membranes of Chi-
nese hamster ovary cells recombinantly expressing the human adenosine A1 receptor, 11c behaved as an antagonist with inverse
agonistic activity. A regioisomer of 11c, 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-(2- naphthylmethyl)aminopyrido[2,3-d]py-
rimidine-2,4-dione (7a) in which the 2-naphthylmethylamino substituent at position 5 of 11c was moved to the 7-position, was a
relatively potent (Ki = 226 nM) and selective (>20-fold) A3 ligand. In the series of compounds lacking an electron-withdrawing eth-
oxycarbonyl or cyano substituent in the 6-position, compounds with high affinity for adenosine A2A receptors were identified, such
as 1,2,3,4-tetrahydro-1,3-dimethyl-5-(1-naphthyl)aminopyrido[2,3-d]pyrimidine-2,4-dione 16b (Ki human A2A = 81.3 nM, Ki human
A1 = 153 nM, and Ki human A3 > 10,000 nM).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
at adenosine receptors modulates a variety of important
physiological processes and exhibits central nervous sys-
tem (CNS) depressant, cardiodepressant, antidiuretic,
and immunomodulatory effects, among others.1,2 The
AR subtypes A1 and A2A are Ôhigh-affinity subtypesÕ
since they are usually activated by low, nanomolar con-
centrations of adenosine.2–4 In many tissues, for exam-
ple, in certain brain areas, A1 and A2A receptors
appear to be tonically activated. Selective antagonists
for the AR subtypes are being developed as potential
new drugs.3–9 Selective A1 antagonists may be useful
as kidney-protective diuretics, for the treatment of con-
gestive heart failure due to their diuretic and positive
inotropic effects, and have potential for the treatment
Adenosine is formed within cells of all mammalian
organisms by hydrolysis of adenine nucleotides or from
S-adenosylhomocysteine and can be released to act at
specific receptors which belong to the family of G-pro-
tein-coupled receptors.1 Extracellular metabolism of
adenine nucleotides can also produce adenosine. Four
adenosine receptor (AR) subtypes have been cloned
and characterized from various species including hu-
mans and rat: A1, A2A, A2B, and A3.2 Adenosine acting
Keywords: Adenosine receptor antagonists; Pyrido[2,3-d]pyrimidines;
Inverse agonist; A1-selectivity; Structure–activity relationships;
Gould–Jacobs reaction; Vilsmeier formylation.
of brain diseases, such as depression or dementia.5,6
A
large body of evidence has been accumulated that selec-
tive A2A antagonists can be very effective in the symp-
tomatic treatment of ParkinsonÕs disease and may
*
Corresponding author. Tel.: +49 431 880 1118; fax: +49 431 880
1352; e-mail: dheber@pharmazie.uni-kiel.de
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.008

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J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
exhibit neuroprotective effects.7,8 A3 AR antagonists
might be therapeutically useful for the acute treatment
of stroke, for glaucoma, and also as antiasthmatic and
antiallergic drugs.9
2,4-diones were identified that showed affinity and selec-
tivity for A1 ARs.24,25 Based on those preliminary find-
ings, we have now synthesized a larger series of
derivatives with variations in the substitution pattern of
the pyridine ring. The new compounds were investigated
in radioligand binding assays at adenosine A1, A2A, and
A3 receptors in order to get more insight into the
structure–activity relationships of pyrido[2,3-d]pyrimid-
inediones, taking N5-butyl-6-cyano-1,3-dimethyl-pyri-
A large number of AR antagonists have been developed
during the past 30 years, initially based on the xanthine
derivatives theophylline and caffeine as lead structures,
and later on from various families of heterocyclic com-
pounds many of which are structurally more or less
related to adenine.5,10 Despite considerable effort by
pharmaceutical companies, no selective adenosine recep-
tor antagonist has made it to the market as yet. After
initial unsuccessful clinical trials with first-generation
A1 AR antagonists, there is now a renewed high interest
in this class of compounds.11–21 Up to now, the non-se-
lective AR antagonists theophylline and caffeine are still
the only AR antagonists that are used as drugs. Reasons
for the slow progress in the development of drugs that
block ARs may be unfavorable pharmacokinetic prop-
erties. One major problem is the low water-solubility
of many of these mostly highly lipophilic compounds
featuring flat, aromatic ring systems, which limits the
absorption of the compounds from the gut.5,10,22 In
addition, intermolecular clustering via hydrogen bond-
ing and p-stacking results in water-insoluble compounds
which exhibit poor brain penetration. In the present
study, we combined features of the xanthine derivatives
theophylline and caffeine (the dimethylpyrimidinedione
ring) with an amino-substituted heterocycle typical for
non-xanthine AR antagonists, in our case 2-amino-
pyrimidine, or 4-aminopyrimidine, respectively. The
structural features of the resulting aminopyrido[2,
3-d]pyrimidine-2,4-diones include a planar nitrogen-
containing hetero-bicyclic ring system with a monosub-
stituted exocyclic amino function which is characteristic
of several classes of potent non-xanthine adenosine
receptor antagonists1,2,5,10,22,23 (Fig. 1).
do[2,3-d]pyrimidine-2,4-dione as
a
lead structure
(Fig. 1). Particular emphasis was laid on the introduction
of polar groups, such as ethoxycarbonyl and moderately
basic amino functions, which would increase water-solu-
bility but at the same time still allow brain penetration.
2. Results and discussion
2.1. Chemistry
For the preparation of our target compounds we devel-
oped different approaches commonly applied to the syn-
thesis of pyrido[2,3-d]pyrimidine derivatives. Starting
from 6-amino-1,3-dimethyluracil (1) 7-aminopyrido[2,3-
d]pyrimidines 3 were prepared using a two-step procedure
via Vilsmeier formylation, nucleophilic substitution with
methylamine, and subsequent condensation of the pyrim-
idine 2 with acetonitrile derivatives (Fig. 2).31
O
O
1.) DMF/POCl3
2.) MeNH2
H3C
O
H3C
O
CH3
N
N
N
H
NH
N
NH2
N
CH3
CH3
1
2
3
a
R
O
Earlier work in our laboratories involved the synthesis of
5-aminopyrido[2,3-d]pyrimidine-2,4-diones and their
preliminary screening at ARs.24,25 Background of those
studies had been the observation that some members of
this class of compounds produced positive inotropic ef-
fects as determined in guinea pig left atria.26–30 In order
to elucidate their molecular mechanism of action, we
had investigated their affinity for adenosine receptors.
R-CH2-CN/
EtONa
OCH3
H3C
R
N
OCH3
O
N
N
NH2
CH3
b
c
CO2C2H5
CN
3a - 3c
Figure 2. Synthesis of 7-amino-substituted pyrido[2,3-d]pyrimidine-
2,4-diones.
A
few 5-amino-1,3-dimethylpyrido[2,3-d]pyrimidine-
CH3
H
H
N
H
O
N
N
O
H
N
N
CN
CH3
O
N
H3C
O
N
N
N
N
N
N
N
CH3
CH3
Rib
theophylline
(xanthine derivative)
pyrido[2,3-d]pyrimidinedione
lead structure
adenosine
Rib = 1-β-D-ribofuranosyl
Ki (rat A1) = 1.8 μM
Ki (rat A2A) = 18.9 μM
Figure 1. Lead structure and its structural relationship to xanthine and adenine derivatives.

J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
2839
Another synthetic route involved the condensation of 1
and diethyl ethoxymethylenemalonate (EMME) 4 to
give the anellated pyridone 5 followed by Vilsmeier
reaction for introduction of chlorine in position 7 and
subsequent nucleophilic substitution to yield the
7-aminopyrido[2,3-d]pyrimidines 7 (Fig. 3).28
of an aralkyl group with optional hydroxyl on the alkyl
portion. The 5-amino substituent turned out to be of
great importance for high AR affinity of the present ser-
ies of compounds. Furthermore, we wanted to obtain
more information about the role of the electron-with-
drawing group in the 6-position and decided to employ
a hydrogen atom or cyano group. Recently, we had de-
scribed the synthetic pathway outlined in (Fig. 5).28
Introduction of the keto function in position 5 (com-
pounds 1235 and 1336) was followed by ring closure
using Vilsmeier formylation (compounds 14 and 1537)
and subsequent introduction of amino groups yielded
the desired pyrido[2,3-d]pyrimidines 16.
The reaction of 6-aminopyrimidine 1 with EMME has
been widely used for the synthesis of 6-carbethoxy-5-
oxopyrido[2,3-d]pyrimidines32,33 and further quinolone
antimicrobial agents (Fig. 4).34 Thus, it has been shown
that the condensation of 1 and EMME in ethanol con-
taining one equivalent of sodium ethoxide gave in the
normal Gould–Jacobs manner the desired enamine 8,
thermal cyclization of which afforded in refluxing
diphenyl ether the pyrido[2,3-d]pyrimidine 9 in excellent
yield.28
2.2. Biological activity
The compounds were investigated in radioligand bind-
ing studies at A1 ARs of rat brain cortical membranes
using the A1-selective radioligand [3H]2-chloro-N6-
cyclopentyladenosine ([3H]CCPA]38 and at A2A ARs
of rat brain striatal membranes using the A2A-selective
radioligand [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-meth-
oxystyryl)-1-propargylxanthine ([3H]MSX-2).39 Addition-
al experiments were performed at human recombinant
A1, A2A, and A3 adenosine receptors stably expressed
in Chinese hamster ovary (CHO) cells. For A3 binding
studies the A3-selective radioligand [3H]8-ethyl-4-meth-
yl-2-phenyl(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-pur-
in-5-one40 ([3H]PSB-11) was applied. Functional properties
of the most potent compounds were investigated in
[35S]GTPcS binding studies at human recombinant A1
receptors expressed in CHO cells.
Quinolones can frequently be chlorinated using phos-
phorus oxychloride without any solvent;30 unfortunate-
ly, this procedure failed when applied to 9. But we found
that treatment with POCl3 in DMF under Vilsmeier
conditions provided the desired 5-chloro derivative
10.28 As expected, nucleophilic displacement of the hal-
ogen atom by the appropriate amine generally occurred
in high yield after 30 min of reaction in refluxing ethanol
yielding the 5-aminopyrido[2,3-d]pyrimidines 11. The
relative ease of displacement is presumably due to the ef-
fect of the carboxylic acid ester together with the carbon
amide group adjacent to the reaction center. The alkyl
side chain was selected on the basis of results previously
obtained in the 4-aminoquinoline series30 and consisted
O
O
H3C
CO2C2H5
CO2C2H5
H3C
O
CO2C2H5
O
N
O
N
+
O
N
NH2
H3C
N
N
H
CH3
CH3
4
5
1
O
H3C
CO2C2H5
N
DMF/POCl3
Amine/EtOH
O
N
N
Cl
CH3
6
7
R
O
H3C
O
CO2C2H5
R
HN
N
a
N
N
CH3
b
c
N
N
N
N
CH2
CH2
7
NO2
Figure 3. Synthesis of N7-substituted 7-aminopyrido[2,3-d]pyrimidine-2,4-diones.

2840
J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
O
O
H3C
O
H3C
O
H5C2O2C
CO2C2H5
OC2H5
N
N
+
CO2C2H5
CO2C2H5
N
NH2
N
N
H
CH3
CH3
4
8
1
O
OH
N
O
Cl
N
Diphenyl-
ether
258 C
H3C
O
H3C
O
CO2C2H5
CO2C2H5
DMF/POCl3
o
N
N
N
N
CH3
CH3
9
10
11
a
R1
R2
O
NR1R2
H3C
O
CO2C2H5
N
R1R2NH/EtOH
H
H
H
N
N
CH3
b
c
d
11
H
H
OH
CH3
OH
e
CH3
CH3
Figure 4. Synthesis of 5-amino-substituted 6-carbethoxypyrido[2,3-d]pyrimidine-2,4-dione.
2.2.1. Affinity in different species: rat and human aden-
osine receptors. For initial screening of the compounds
we used native rat brain tissues expressing the A1 or
A2A AR in high density. Rats and mice are frequently
used animals for in vivo testing of drugs and therefore
it is essential to know the affinity of new ligands for ro-
dent receptors. Since the ultimate target of new ligands
are the human receptors, either as pharmacological tools
used in human tissues or cell lines, or for diagnostic or
therapeutic purposes in patients, the affinity at human
receptors is also important to know. Only moderate spe-
cies differences have been reported for A1 and A2A ARs
between rat and humans due to the high degree of se-
quence homology.2,5,22 Only the A3 AR usually shows
larger species differences.2,9 Because rat brain and most
other rat tissues express only a very low density of A3
ARs, only human recombinant A3 ARs were employed.
Depending on the affinity at rat A1 and A2A receptors,
selected compounds were additionally investigated at
human recombinant A1 and A2A ARs. In fact, the set
of pyridopyrimidinediones assayed at rat and human
ARs showed only moderate species differences. Ki values
were in all cases in a similar range. The compounds ap-
peared to be somewhat less potent at human A1 as com-
pared to rat A1 ARs (ca. 3- to 5-fold) and slightly more
potent at human A2A as compared to rat A2A ARs (e.g.,
no significant difference for 7b, ca. 3-fold difference for
16b).
2.2.2. Structure–activity relationships. Pyridopyrimidine
derivatives 3a–3c and 7a–7c (Table 1) contain an amino
group in the 7-position. Compounds 3b–3c bearing an
unsubstituted amino group with small, electron-with-
drawing substituents in the neighboring 6-position (eth-
oxycarbonyl, 3b; cyano, 3c) exhibited affinity for A1 and
A2A receptors at low micromolar concentrations, but
showed only very low affinity for A3 ARs. Both com-
pounds were slightly more potent at A1 than at A2A
receptors. The ethoxycarbonyl substituent (compound
3b) was superior to a cyano group (compound 3c). How-
ever, the introduction of a bulky, lipophilic 3,4-dimeth-
oxyphenyl group in the 6-position was not well tolerated

J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
2841
O
O
O
H3C
O
H3C
CN
AcOAc
N
N
N
N
+
NC-CH2-COOH
N
NH2
O
N
NH2
CH3
CH3
12
1
AcOAc/glacial AcOH
DMF/POCl3
O
N
O
O
Cl
H3C
O
H3C
O
CN
CH3
NH2
N
N
N
CH3
CH3
13
14
DMF/POCl3
Amine/EtOH
R
O
Cl
N
O
H3C
O
H3C
O
Y
Amine/EtOH
N
N
N
N
CH3
CH3
15
16
R
16
a
Y
HN
H
b
c
N
N
CH2
CH3
H
H
HN
HN
CN
CN
H
d
e
f
NH2
HN
g
H
O
N
Figure 5. Synthesis of 5-amino-substituted 6-unsubstituted or 6-cyano-substituted pyrido[2,3-d]pyrimidine-2,4-diones.
by the receptors (compound 3a). In the next step, the
ethoxycarbonyl group in position 6 was combined with
various substituents on the 7-amino group (7a–7c). A
bulky 2-naphthylmethyl substituent was tolerated by
the A1 AR but resulted in a less potent compound (7a)
compared to the corresponding unsubstituted 3b. How-
ever, the 2-naphthylmethyl group increased A3 affinity
to a large extent. Compound 7a exhibited a Ki value
of 230 nM at human A3 ARs and was more than 20-fold
selective versus the other AR subtypes. Despite its polar

Table 1. Affinities of amino-substituted pyrido[2,3-d]pyrimidinediones and standard ligands for adenosine receptors
Compound
R
A1 Ki SEM (lM)
versus [3H]CCPA
rat cortex (n = 3)a
A1 Ki SEM (lM)
versus [3H]CCPA
hA1-CHO (n = 3)a
A2A Ki SEM (lM)
versus [3H]MSX-2 rat
striatum (n = 3)a
A2A Ki SEM (lM) A3 Ki SEM (lM)
versus [3H]MSX-2
hA2A-CHO
versus [3H]PSB-11
hA3-CHO membranes
membranes (n = 3)a (n = 3)a
O
O
H
O
CH3
H3C
O
N
N
H3C
R
4
N
N
5
N3
2
6
7
8
1
N
N
N
N
N
O
O
N
NH2
CH3
CH3
Caffeine
DPCPX
3a-c
Caffeine
DPCPX
3a
—
—
18.8 5.6
0.0005 0.0002
44.9 6.2
0.0030 0.0008
32.5 8.0
0.157 0.006
23.4 7.1b
0.060 0.045b
n.d.
n.d.
0.243 0.056
3,4-Dimethoxyphenyl
Ethoxycarbonyl
Cyano
>1.0 (19 1% inhibition)c n.d.
ꢀ1.0 (1 1% inhibition)c
ꢀ1.0 (0% inhibition)c
>10 (29 2% inhibition)c
>10 (15 2% inhibition)c
3b
3c
1.23 0.13
4.07 0.35
n.d.
n.d.
5.50 2.04
ca. 10 (46 6% inhibition)c n.d.
n.d.
O
N
O
O
O
H3C
H3C
O
O
CH3
N
O
CH3
O
N
N
NHR
N
N
N
CH3
CH3
N
R
7a
7b-c
7a
7b
7c
2-Naphthylmethyl
Phenyl
p-Nitrophenyl
5.2 2.5d
2.50 0.17
1.60 0.27
>10 (29% inhibition)c >10 (23 5% inhibition)c
n.d.
2.00 1.02
n.d.
0.226 0.110
1.83 0.68
0.793 0.029
n.d.
n.d.
2.23 0.22
6.10 7.30d
H3C
R
O
NHR
O
O
N
N
O
H3C
O
H3C
O
N
O
CH3
N
O
CH3
N
N
N
CH3
CH3
11a-d
11e

11a
11b
11c
11d
11e
H
Benzyl
2-Naphthylmethyl
5.25 0.15
P1.0 (46 3% inhibition)c n.d.
0.00497 0.00082 0.0251 0.0041
n.d.
3.98 0.95
n.d.
n.d.
n.d.
n.d.
n.d.
P10 (37 15% inhibition)c
4.65 0.44
1.54 0.58
>1.0 (23 3% inhibition)c
>10 (35 2% inhibition)c
P10 (41 5% inhibition)c
>10 (13 3% inhibition)c
(1RS,2RS)1-Methyl-2-hydroxy-2-phenylethyl 1.90 0.25
(1RS,2RS)1-Methyl-2-hydroxy-2-phenylethyl 5.83 0.26
n.d.
n.d.
P10 (43 1% inhibition)c
P10 (34 1% inhibition)c
O
N
O
NHR
O
NHR
N
O
N
H3C
O
CN
H3C
O
O
N
N
H3C
N
N
N
H3C
O
N
N
N
N
O
N
N
CH3
16d,e
CH3
N
CH3
16a-c
CH3 16f
16g
16a
16b
16c
16d
16e
16f
16g
Benzyl
1-Naphthyl
(RS)-Phenylisopropyl
>1.0 (27 3% inhibition)c n.d.
>1.0 (10 2% inhibition)c
0.217 0.037
4.13 2.24
n.d.
0.0813 0.0303
9.18 2.43
>10 (26 6% inhibition)c
4.50 0.28
0.0383 0.0088
0.300 0.025
0.153 0.009
1.063 0.182
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
H
Phenethyl
5.20 0.10d
0.540 0.066
>10 (23 5% inhibition)c
6.03 2.08
P10 (38 5% inhibition)c
4.30 1.16
1.44 0.16
n.d.
n.d.
—
—
ꢀ1.0 (3% inhibition)c
ꢀ1.0 (0% inhibition)c
ꢀ1.0 (8 4% inhibition)c
ꢀ1.0 (0% inhibition)c
ꢀ1.0 (1% inhibition)c
ꢀ1.0 (0% inhibition)c
a Unless otherwise noted.
b Determined versus [3H]CGS21680 as radioligand; data for antagonists determined with [3H]CGS21680 have been shown to be similar to those determined with [3H]MSX-2.39
c Percent inhibition of radioligand binding at the indicated concentration (1 or 10 lM) depending on the solubility of the compound.
d n = 2.

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J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
ethoxycarbonyl substituent, compound 7a showed only
moderate water-solubility. In order to increase water-
solubility, piperazinyl-substituted compounds 7b and
7c were prepared containing a basic, protonatable nitro-
gen atom. Both compounds, the benzylpiperazinyl (7b)
and the p-nitrobenzylpiperazinyl derivative (7c), showed
A1, A2A, and A3 affinity in the low micromolar concen-
tration range; they were of similar potency to the unsub-
stituted parent compound 3b at A1 and A2A ARs;
however, they exhibited a higher affinity for A3 recep-
tors. Thus, the substitution of the 7-amino group gave
no advantage with respect to A1 or A2A AR affinity,
but it increased A3 affinity and even led to A3-selective
compounds, such as 7a.
cyano group did not alter A1 and A3 affinity but reduced
A2A affinity (compare 16d/11a). Additional substitution
on the amino group with a bulky phenethyl residue (16e)
led to a moderately potent (Ki rat A1 = 540 nM, human
A1 = 1.44 lM), only weakly A1-selective AR antagonist.
In the series of 6-unsubstituted pyridopyrimidinediones,
substitution of the 5-position by a benzylpiperazinyl
(16f) or a morpholinyl residue (16g) resulted in totally
inactive compounds. Both compounds are lacking a free
NH group that may be required in this position for
hydrogen bonding with the receptor. As seen with the
previous series of 6-ethoxycarbonyl derivatives, a 5-ben-
zylamino residue was not well tolerated by the receptors
(16a, compare with 11b), it was best tolerated by the A3
AR (Ki = 9.18 lM). However, phenylisopropyl (2-meth-
yl-2-phenylethyl) substitution of the 5-amino group, a
substituent that had resulted in high A1 affinity and
selectivity when attached to the exocyclic amino group
of adenosine—a standard A1 agonist is (R)-phenyliso-
propyladenosine (R-PIA)1–4—was much better tolerat-
ed: compound 16c had a Ki value of 300 nM at rat A1,
1.06 lM at human A1, 4.13 lM at rat A2A, and
4.50 lM at human A3 ARs (Table 1). The best com-
pound in this series of 6-unsubstituted 5-amino-pyrido-
pyrimidines was 16b bearing a 1-naphthyl substituent
at the 5-amino group, which was also the only arylami-
no-substituted compound in this series. It showed high
A1 affinity (Ki 38 nM rat, 103 nM human) and was also
relatively potent at A2A receptors (Ki 217 nM rat, 80 nM
human). The compound was virtually inactive at human
A3 ARs. Thus, 16b is a balanced A1/A2A AR antagonist
and could serve as a lead structure for the development
of A2A-selective compounds, which are of high interest
as novel drugs for ParkinsonÕs disease.7 Thus, an aryl
substituent at the exocyclic amino group may be advan-
tageous for A1 as well as A2A receptor affinity.
A series of isomeric derivatives was investigated in
which the amino group occupies the 5-position instead
of the 7-position (compounds 11a–11e). These com-
pounds were analogs of the previously investigated
derivatives24,25 (Fig. 1). Compound 11a, the isomer of
the 7-amino derivative 3b featuring an unsubstituted 5-
amino group, was similar in potency to 3b at all three
receptor subtypes. Substitution of the amino group with
a 2-naphthylmethyl residue, however, had a dramatic ef-
fect: compound 11c was a very potent A1 antagonist
(vide infra) with a Ki value of 4.97 nM at the rat A1
receptor and 25 nM at the human A1 AR. The com-
pound showed very high selectivity versus the rat A2A
AR (>400-fold) and the A3 AR (>60-fold); it was more
than 1000-fold more potent at the A1 AR than its regio-
isomer 7a. All analogs bearing bulky substituents at N5
showed low affinity for the A2A AR. A benzyl (11b) or a
1-methyl-2-hydroxy-2-phenylethyl substituent (11d)
resulted in much weaker A1 antagonists than the 2-
naphthylmethyl substitution in 11c. N-Methylation of
11d yielding 11e, which lacks the hydrogen bond donor
function of the 5-amino group, further decreased AR
affinity. The human A3 AR best tolerated a 2-naph-
thylmethyl or a benzyl substituent at the 5-amino group,
but the affinities were in the low micromolar range at
best (11c: Ki = 1.54 lM).
The structure–activity relationships are in accordance
with A1 AR pharmacophore models5,11,23 that postulate
the requirement of a hydrogen bond donor in proximity
to a hydrogen bond acceptor, in addition to a hydropho-
bic substituent. Figure 6 shows a possible binding mode
of a prototypical A1-selective AR antagonist (1,3-dipro-
pyl-8-cyclopentylxanthine, DPCPX) and a prototypical
A1 agonist N6-cyclopentyladenosine (CPA) in compari-
son with that of compound 11c. The lipophilic cyclopen-
Subsequently, a further series of 5-amino-substituted
pyridopyrimidinediones was investigated, in which the
6-position remained unsubstituted (16a–16c, 16f, 16g)
or was substituted by a cyano group (16d, 16e; see
Table 1). Replacement of the ethoxycarbonyl group by a
HO
O
H
CH3
H
H3C
O
N
O
N
N
N
N
H
N
O
N
N
N
N
HO
O
N
H3C
N
HO
N
O
6
DPCPX
11c
N -cyclopentyladenosine (CPA)
CH3
O
CH3
Hydrogen bond acceptor
Hydrogen bond donor
Figure 6. Putative binding mode of pyrido[2,3-d]pyrimidindione derivative 11c with respect to the A1-selective xanthine antagonist DPCPX and the
A1-selective agonist CPA.

J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
2845
tyl group in DPCPX and CPA may correspond to the 2-
naphthylmethyl residue in 11c. Surprisingly, replace-
ment of the 2-naphthylmethyl by a benzyl substituent
(in 11b) abolished affinity for the A1 receptor.
As previously described, the inverse agonist DPCPX
showed a decrease in [35S]GTPcS binding with an
EC50 value of 2.4 nM consistent with its affinity for hu-
man A1 ARs (Ki = 3.9 nM).44 Both investigated pyrido-
pyrimidinediones, 11c and 16b, did not enhance
[35S]GTPcS binding but led to a significant inhibition.
The maximal effect was lower than that of DPCPX
(ca. 70% of the DPCPX effect), which is one of the most
efficacious inverse agonists of A1 ARs described to
date.41 These experiments clearly show that the pyrido-
pyrimidinediones are antagonists at A1 ARs with inverse
agonistic potency.
2.2.3. [35S]GTPcS binding studies. The two most potent
A1 AR ligands of the present series of pyridopyrimidin-
ediones, 11c and 16b, were investigated in [35S]GTPcS
binding studies at membrane preparations of CHO cells
recombinantly expressing the human A1 AR in order to
obtain information about their functional properties.
The results are shown in Figure 7. The A1-selective
AR agonist N6-cyclopentyladenosine (CPA) and the
A1-selective AR antagonist DPCPX, that has previously
been shown to act as an inverse agonist,41 were included
as standard compounds for comparison. The binding of
agonists to Gi protein-coupled receptors (GPCR) can be
modulated by GTP upon its binding to the G-protein.
The addition of GTP, for example, has been shown to
cause a rightward shift of the competition curve for ago-
nists but not for antagonists at the Gi-coupled A1 AR.42
Conversely, A1 AR agonists can also modulate GTP
binding to the G-protein. This may be measured in radi-
oligand binding assays using [35S]GTPcS, a radiola-
beled, stable analog of GTP.43 The A1 AR agonist
CPA caused a concentration-dependent increase in
[35S]GTPcS binding with an EC50 value of 3.5 nM
(Fig. 7), which corresponds well with the Ki value of
CPA obtained in radioligand binding experiments at hu-
man A1 ARs (Ki = 2.3 nM).44 So-called neutral antago-
nists would not have any influence on [35S]GTPcS
binding, while inverse agonists lead to a reduction in
[35S]GTPcS binding.41 While agonists are believed to
bind preferentially to the active receptor conforma-
tion(s), neutral antagonists are thought to have no
preference for either conformation and inverse agonists
to bind preferentially to inactive conformation(s).41
3. Conclusions
In conclusion, we have studied the structure–activity
relationships for pyrido[2,3-d]pyrimidinediones as li-
gands of adenosine receptors and identified a novel
highly potent and selective A1 antagonist, 6-carbeth-
oxy-1,2,3,4-tetrahydro-1,3-dimethyl-5-(2-naphthyl)meth-
ylaminopyrido[2,3-d]pyrimidine-2,4-dione (11c). It is
360-fold more potent than our lead compound identified
in a previous study24 (Fig. 1). Furthermore, a regioiso-
mer of 11c, 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimeth-
yl-7-(2-naphthyl)methylaminopyrido[2,3-d]pyrimidine-2,
4-dione (7a) in which the 2-naphthylmethylamino-sub-
stituent at position 5 of 11c was moved to the 7-position,
was a relatively potent (Ki = 226 nM) and selective (>20-
fold) A3 AR ligand. Both compounds have an ethoxy-
carbonyl residue in the 6-position. In the series of
compounds lacking a substituent in that position,
compounds with good affinity for the A2A AR were
identified, such as the non-selective 1,2,3,4-tetrahydro-
1,3-dimethyl-5-(1-naphthyl)aminopyrido[2,3-d]pyrimidine-
2,4-dione (16b, Ki human A2A = 81.3 nM, Ki human
A1 = 153 nM, Ki human A3 > 10,000 nM). Compound
150
100
50
CPA
DPCPX
16b
11c
0
-50
-100
10-15
10-13
10-11
10-9
10-7
10-5
compound, [M]
Figure 7. [35S]GTPcS binding studies of selected pyridopyrimidinediones 11c and 16b at human recombinant adenosine A1 receptors expressed in
Chinese hamster ovary cells (normalized data with respect to DPCPX as full inverse agonist set at À100% = maximal inhibition). The maximal
stimulation by the full agonist CPA was set at +100%. Data points are means of at least three independent experiments SEM.

2846
J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
16b could serve as a new lead structure for the develop-
ment of A2A-selective AR antagonists.
4.1.4. Amino-substituted 1,3-dimethyl-1,2,3,4-tetrahydro-
pyrido[2,3-d]pyrimidine-2,4-diones 7, 11, 16 (general pro-
cedure). A solution of 2 mmol of the chloro derivatives
6, 10, 14, and 15, respectively, and 20 mmol of the
appropriate amine in 40 ml of ethanol was refluxed for
30 min. After cooling at 0 ꢂC, the crude product was col-
lected by filtration, washed with ice-cold ethanol, and
recrystallized from ethanol.
4. Experimental
4.1. Synthetic procedures
4.1.1. General procedures. Elemental analyses were per-
formed at the Microanalytical Laboratory of the Insti-
tute of Inorganic Chemistry, University of Kiel.
Melting points are uncorrected and were recorded with
4.1.5. 6-Carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-
(2-naphthyl)methylaminopyrido[2,3-d]pyrimidine-2,4-dione
(7a). 1 mmol of the educt was used. Yield: 334 mg,
80%; mp: 197 ꢂC; IR (KBr, m cmÀ1): 1667 (–CO–, lac-
tam), 1688 (–CO–, lactam), 1707 (–CO–, ester), 3325
a Buchi 510 melting point apparatus. IR spectra (KBr
¨
pellet) were measured on a Perkin-Elmer FT-IR 16 PC
1
spectrometer. The H NMR spectra (internal standard:
1
(NH). H NMR (CDCl3, d ppm): 1.32 (t, J = 7.0 Hz,
Me4Si) were recorded in DMSO-d6 on a Bruker ARX
300 spectrometer (d given in ppm, J in Hz). Macherey-
Nagel Polygramꢁ SIL G/UV254 on plastic sheets was
used for TLC monitoring.
3H, CH3), 3.33 (s, 3H, N-CH3), 3.60 (s, 3H, N-CH3),
4.25 (q, J = 7.0 Hz, 2H, CH2), 5.17 (t, J = 7.0 Hz,
2H, CH2), 7.10–7.35 (m, 1H-arom), 7.37–7.50 (m, 4
H-arom), 7.74–7.85 (d, 2H-arom), 7.99 (d, J = 7.0 Hz,
1H-arom), 8.80 (s, 1H, H-5), 8.96–9.08 (br s, 1H, NH).
Compounds 2, 3b, and 3c,31 5,33 8, 9,32 10, 11a, 11b,
14, and 16e,28 12,35 13,36 1537 are described in the
literature.
C23H22N4O4 (418.45): Calcd C, 66.08; H, 5.26; N, 13.40.
Found: C, 66.96; H, 5.48; N, 12.75.
4.1.2.
7-Amino-6-(3,4-dimethoxyphenyl)-1,3-dimethyl-
4.1.6. 6-Carbethoxy-7-benzylpiperazino-1,2,3,4-tetrahy-
dro-1,3-dimethylpyrido[2,3-d]pyrimidine-2,4-dione (7b).
1 mmol of the educt was used. Yield: 174 mg, 40%;
mp: 95 ꢂC; IR (KBr, m cmÀ1): 1661 (–CO–, lactam),
1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-2,4-dione (3a).
To a solution of 5,4 g of Na in 800 ml of ethanol were
added 113.7 g (0.58 mol) of the pyrimidine 2 and
113.4 g (0.64 mol) of 3,4-dimethoxyphenylacetonitrile.
The resulting slurry was heated at reflux for 3 h, during
which time a homogeneous solution slowly was formed.
Toward the end of the period a yellow precipitate began
to separate. The mixture was cooled and the precipitate
of crude 3a was filtered and washed with diethyl ether.
After this has been recrystallized from 95% ethanol,
109.8 g (61%) of 3a, mp 272 ꢂC was obtained. IR
(KBr, m cmÀ1): 1675 (–CO–, lactam), 1730 (–CO–, lac-
tam), 1755 (–CO–, ester). 1H NMR (DMSO-d6, d
ppm): 3.25 (s, 3H, N-CH3), 3.50 (s, 3H, N-CH3), 3.79
(s, 6H, 2 x OCH3), 6.94 (m, 3H-arom, 2H, NH2), 7.71
(s, 1H, H-5).
1
1707 (–CO–, ester). H NMR (CDCl3, d ppm): 1.32 (t,
J = 7.0 Hz, 3H, CH3), 3.23 (s, 3H, N-CH3), 3.31 (s,
4H, 2· CH2), 3.44 (s, 3H, N-CH3), 3.55 (m, 6H, 3·
CH2), 4.25 (q, J = 7.0 Hz, 2H, CH2), 7.32 (s, 5H-arom),
8.32 (s, 1H, H-5).
C23H27N5O4 (437.42): Calcd C, 63.21; H, 6.18; N, 16.02.
Found: C, 62.27; H, 6.18; N, 15.88.
4.1.7. 6-Carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-
(p-nitrobenzyl)piperazinopyrido[2,3-d]pyrimidine-2,4-dione
(7c). Yield: 501 mg, 52%; mp: 164 ꢂC; IR (KBr, m cmÀ1):
1653 (–CO–, lactam), 1695 (–CO–, lactam), 1700 (–CO–,
C17H18N4O4 (342.70): Calcd C, 59.70; H, 5.30; N, 16.38.
Found: C, 57.92; H, 5.44; N, 15.58.
1
ester). H NMR (CDCl3, d ppm): 1.32 (t, J = 7.0 Hz,
3H, CH3), 3.23 (s, 3H, N-CH3), 3.45 (s, 3H, N-CH3),
3.58 (m, 4H, 2· CH2), 3.73 (s, 2H, CH2), 4.27 (q,
J = 7.0 Hz, 2H, CH2), 7.63 (d, 2H-arom), 8.20 (d, 2H-
arom), 8.34 (s, 1H, H-5).
4.1.3. 6-Carbethoxy-7-chloro-1,3-dimethyl-1,2,3,4-tetra-
hydropyrido[2,3-d]pyrimidine-2,4-dione (6). To DMF
(5.0 ml) at 0 ꢂC was added phosphorus oxychloride
(15.0 ml) dropwise with stirring. To this solution was
added the pyrido[2,3-d]pyrimidine 5 (2.0 g, 7.2 mmol)
and after 5 min the mixture was heated at 65 ꢂC for
3 h. On pouring the reaction mixture into ice water,
the crude product precipitated was collected by filtration
and washed with petroleum ether. Recrystallization
from ethanol afforded compound 6 (0.7 g, 60%). Mp:
133.8 ꢂC (ethanol), IR (KBr, m cmÀ1): 1650 (–CO–, lac-
tam), 1720 (–CO–, lactam), 1755 (–CO–, ester). 1H
NMR (DMSO-d6, d ppm): 1.37 (t, J = 6.0 Hz, 3H,
CH3), 3.38 (s, 3H, N-CH3), 3.65 (s, 3H, N-CH3), 4.39
(q, J = 7.0 Hz, 2H, CH2), 9.05 (s, 1H, H-5).
C23H26N6O6 (482.87): Calcd C, 57.31; H, 5.39; N, 17.42.
Found: C, 57.09; H, 5.49; N, 17.60.
4.1.8. 6-Carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-5-
(2-naphthyl)methylaminopyrido[2,3-d]pyrimidine-2,4-dione
(11c). Yield 454mg, 54%; mp: 145 ꢂC; IR (KBr, m cmÀ1):
1645 (–CO–, lactam), 1702 (–CO–, lactam), 1710 (–CO–,
ester). 1H NMR (CDCl3, d ppm): 1.21 (t, 3H, CH3), 3.17
(s, 3H, N-CH3), 3.46 (s, 3H, N-CH3), 4.17 (q, 2H, CH2),
4.79 (d, 2H, CH2), 7.45–7.57 (m, 4H-arom), 7.86–7.99 (m,
3H-arom), 8,45 (s, 1H-arom), 10,20 (s, 1H, NH).
C12H12ClN3O4 (297.70): Calcd C, 48.53; H, 4.07; N,
14.15. Found: C, 48.79; H, 4.12; N, 14.75.
C23H22N4O4 (418.85): Calcd C, 66.08; H, 5.26; N, 13.14.
Found: C, 65.94; H, 5.33; N, 13.45.

J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
2847
4.1.9. 6-Carbethoxy-1,2,3,4-tetrahydro-5-(1-hydroxy-1-
phenylprop-2-yl)amino-1,3-dimethylpyrido[2,3-d]pyrimi-
dine-2,4-dione (11d). 1 mmol of the educt was used.
Yield: 191 mg, 45%; mp: 146 ꢂC; IR (KBr, m cmÀ1):
1644 (–CO–, lactam), 1690 (–CO–, lactam), 1705
CH), 6.30 (d, J = 6.0 Hz, 1H, H-6), 7.06–7.31 (m, 5H-
arom), 7.97 (d, J = 6.0 Hz, 1H, H-7), 9.58 (d, 1H, NH).
C17H18N4O2 (310.21): Calcd C, 65.86; H, 5.85; N, 18.07.
Found: C, 65.68; H, 5.87; N, 18.26.
1
(–CO–, ester), 3440 (NH). H NMR (CDCl3, d ppm):
1.12 (d, 3H, CH3), 1.38 (t, J = 7.0 Hz, 3H, CH3), 2,40
(s, 1H, OH), 3.43 (s, 3H, N-CH3), 3.64 (s, 3H, N-
CH3), 3.88–4.07 (m, 1H, CH), 4.29–4.45 (m, 2H,
CH2), 4.95 (s, 1H, CH), 7.24–7.35 (m, 5H-arom), 8.50
(s, 1H, H-7), 10.30 (d, 1H, NH).
4.1.14. 5-Amino-6-cyano-1,2,3,4-tetrahydro-1,3-dimethyl-
pyrido[2,3-d]pyrimidine-2,4-dione (16d). Yield: 189 mg,
41%; mp: 151 ꢂC; IR (KBr, m cmÀ1): 1670 (–CO–, lactam),
1690 (–CO–, lactam), 1720 (–CO–, ester), 3330 (NH). 1H
NMR (CDCl3, d ppm): 3.24 (m, J = 7 Hz, 3H, N-CH3,
CH2), 3.48 (s, 3H, N-CH3), 7.86 (s, J = 7.0 Hz, 1H, H-
7), 8,52 (br s, 1H, NH), 9.02 (s, 1H, NH).
C22H36N4O5 (426.52): Calcd C, 62.02; H, 6.10; N, 13.14.
Found: C, 60.11; H, 5.76; N, 13.43.
C10H9N5O2 (231.46): Calcd C, 64.48; H, 5.07; N, 20.89.
Found: C, 63.57; H, 5.18; N, 20.59.
4.1.10. 6-Carbethoxy-1,2,3,4-tetrahydro-5-[(1-hydroxy-1-
phenylprop-2-yl)methyl]amino-1,3-dimethylpyrido[2,3-
d]pyrimidine-2,4-dione (11e). 1 mmol of the educt was
used. Yield: 127 mg, 30%; mp: 122 ꢂC; IR (KBr, m
cmÀ1): 1651 (–CO–, lactam), 1690 (–CO–, lactam),
4.1.15.
6-Cyano-1,2,3,4-tetrahydro-1,3-dimethyl-5-(2-
phenylethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (16e).
Yield: 509 mg, 76%; mp: 151 ꢂC; IR (KBr, cmÀ1): 2999
(w, C@C, aromatic), 2219 (s-m, CN), 1704 (–CO–, lac-
tam), 1647 (–CO–, lactam), 1599 (–CO–, ester). 1H
NMR (CDCl3, ppm): 3.25-3.29 (m, J = 7.0 Hz, 5H, N-
CH3, CH2), 3.38 (s, 3H, N-CH3), 4.56 (m, J = 7.0 Hz,
2H, CH2), 6.45 (d, J = 7.0 Hz, 1H, H-6), 7.29–7.34 (m,
5H-arom), 9.54 (br s, 1H, NH).
1
1708 (–CO–, ester), 3419 (NH). H NMR (CDCl3, d
ppm): 0.97 (d, 3H, CH3), 1.39 (t, J = 7.0 Hz, 3H,
CH3), 3.12 (s, 3H, N-CH3), 3.48 (s, 3H, N-CH3),
3.70–3.76 (m, 4H, CH, N-CH3), 4.31–4.46 (m, 2H,
OCH2), 4.86 (s, 1H, OH), 5.00 (s, 1H, OCH), 7.20–
7.30 (m, 5H-arom), 8.72 (s, 1H, H-7).
C21H24N4O5 (426.52): Calcd C, 61.21; H, 6.82; N, 13.59.
Found: C, 62.08; H, 6.25; N, 12.97.
C18H17N5O2 (335.41): Calcd C, 64.48; H, 5.07; N, 20.89.
Found: C, 63.57; H, 5.18; N, 20.59.
4.1.11. 5-Benzylamino-1,2,3,4-tetrahydro-1,3-dimethyl-
pyrido[2,3-d]pyrimidine-2,4-dione (16a). 1 mmol of the
educt was used. Yield: 133 mg, 45%; mp: 121 ꢂC; IR
(KBr, m cmÀ1): 1670 (–CO–, lactam), 1690 (–CO–, lac-
tam), 1720 (–CO–, ester), 3330 (NH). 1H NMR (CDCl3,
d ppm): 3.21 (s, 3H, N-CH3), 3.46 (s, 3H, N-CH3), 3.30–
3.69 (m, 4H, (CH2)2), 6.52 (d, J = 6.0 Hz, 1H, H-6),
7.23–7.31 (m, 5H-arom), 8.06 (s, J = 6.0 Hz, 1H, H-7),
9.14 (br s, 1H, NH).
4.1.16. 5-Benzylpiperazino-1,2,3,4-tetrahydro-1,3-dimethyl-
pyrido[2,3-d]pyrimidine-2,4-dione (16f). Yield: 365 mg, 50%;
mp: 130 ꢂC; IR (KBr, m cmÀ1): 2940 (w, C@C, arom), 2813
(w, C@C, arom), 1691 (s, –CO–, lactam), 1651 (s, –CO–, lac-
1
tam). H NMR (CDCl3, d ppm): 3.21 (s, J = 7.0 Hz 5H,
N-CH3, N-CH2), 3.30 (s, 5H, N-CH3, N-CH2), 3.49 (m,
4H, N-(CH2)2), 6.73 (d, J = 7.0 Hz, 1H, H-6), 7.24–7.30
(m, 5H-arom), 8.19 (d, J = 7.0 Hz, 1 H, H-7).
C20H23N5O2 (365.52): Calcd C, 65.93; H, 6.13; N, 19.51.
Found: C, 68.85; H, 6.04; N, 19.23.
C16H16N4O2 (296.41): Calcd C, 64.92; H, 5.44; N, 18.92.
Found: C, 63.35; H, 4.96; N, 19.45.
4.1.17.
1,2,3,4-Tetrahydro-1,3-dimethyl-5-morpholino-
4.1.12. 1,2,3,4-Tetrahydro-1,3-dimethyl-5-(1-naphthyl)amino-
pyrido[2,3-d]pyrimidine-2,4-dione (16b). 1 mmol of the educt
was used. Yield: 104 mg, 30%; mp: 232 ꢂC; IR (KBr, m
cmÀ1): 1670 (–CO–, lactam), 3330 (NH). 1H NMR (CDCl3,
d ppm): 3.22 (s, 3H, N-CH3), 3.53 (s, 3H, N-CH3), 4.96 (d,
J = 7.0 Hz, 2H, CH2), 6.60 (d, J = 6.0 Hz, 1H, H-6), 7.42–
7.49 (m, 2H-arom), 7.55–7.63 (m, 2H-arom), 7.88–7.91 (m,
1H-arom), 7.97–8.00 (dd, J = 7.0 Hz, 1H-arom), 8.08–8.12
(m, 2H, H-7, 1H-arom), 9.55 (t; 1H, NH).
pyrido[2,3-d]pyrimidine-2,4-dione (16g). Yield: 176 mg,
32%; mp: 184 ꢂC; IR (KBr, m cmÀ1): 1670 (–CO–, lac-
tam), 1690 (–CO–, lactam), 1720 (–CO–, ester), 3330
1
(NH). H NMR (CDCl3, d ppm): 3.25 (s, 7H, N-CH3,
N(CH2)2), 3.57 (s, 3H, N-CH3), 3.78 (t, J = 7.0 Hz,
4H, (CH2)2), 6.78 (d, J = 6.0 Hz, 1 H, H-6), 8.26 (s,
J = 6.0 Hz, 1H, H-7).
C13H16N4O3 (276.42): Calcd C, 56.57; H, 5.84; N, 20.30.
Found: C, 55.92; H, 6.08; N, 20.22.
C20H18N4O2 (346.41): Calcd C, 68.67; H, 4.82; N, 16.87.
Found: C, 69.66; H, 5.35; N, 15.87.
4.2. Receptor radioligand binding studies
4.1.13.
1,2,3,4-Tetrahydro-1,3-dimethyl-5-(2-phenylpro-
4.2.1. Materials. [3H]CCPA (54.9 Ci/mmol) and
[35S]GTPcS (1250 Ci/mmol) were from NEN Life Sci-
ences, [3H]MSX-2 (85 Ci/mmol) and [3H]PSB-11
(53 Ci/mmol) were obtained from Amersham. The
non-radioactive precursors of [3H]MSX-2 (MSX-1)45
and [3H]PSB-11 (PSB-10)46 were synthesized in our
laboratory.
pyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (16c). 1 mmol
of the educt was used. Yield: 155 mg, 50%; mp: 131,7 ꢂC;
IR (KBr, m cmÀ1): 1640 (–CO–, lactam), 1689 (–CO–, lac-
tam), 1720 (–CO–, ester), 3362 (NH). H NMR (CDCl3,
d ppm): 1.62 (d, J = 7.0 Hz, 3H, CH3), 3.27 (s, 3H, N-
CH3), 3.45 (s, 3H, N-CH3), 4.83 (quintet, J = 7.0 Hz, 1H,
1

2848
J. Bulicz et al. / Bioorg. Med. Chem. 14 (2006) 2837–2849
4.2.2. Membrane preparations. Frozen rat brains ob-
tained from Pel Freezꢁ, Rogers, AR, USA, were dissect-
ed to obtain cortical membrane preparations for A1
assays and striatal membrane preparations for A2A as-
says as described.46,47 CHO cells recombinantly express-
ing the human adenosine A1, A2A, and A3 receptors
were a gift from Dr. K.-N. Klotz and grown as de-
scribed.44 Membrane preparations were obtained as pre-
viously described.44
nant cell lines expressing the human adenosine receptor
subtypes and Sonja Hinz for expert technical assistance.
References and notes
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¨
4.2.3. Adenosine receptor binding assays. Stock solutions
of the compounds were prepared in dimethylsulfoxide
(DMSO), the final concentration of DMSO in the assays
being 2.5%. The radioligands and their concentrations
were as follows: [3H]CCPA, 0.5 nM (A1), [3H]MSX-2,
1 nM (A2A), and [3H]PSB-11, 0.5 nM (A3). Binding assays
were performed essentially as described.39,47 Membranes
(ca. 70 lg protein/mL) were preincubated for 20 min with
0.12–0.22 IU/mL of adenosine deaminase in order to re-
move endogenous adenosine. Curves were determined
using 6–7 different concentrations of test compounds
spanning 3 orders of magnitude. At least three separate
experiments were performed, each in triplicate. For
non-linear regression analysis, the Cheng–Prusoff equa-
tion and KD values of 0.2 nM (rat A1) and 0.6 nM (human
A1), respectively, for [3H]CCPA,38 8 nM (rat A2A) and
7 nM (human A2A), respectively, for [3H]MSX-2,39 and
4.9 nM (human A3) for [3H]PSB-1140 were used to calcu-
late Ki values from IC50 values.
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assays were performed as previously described43 by
incubating membrane preparations of recombinant
CHO cells expressing the human A1 AR (5 lg per tube)
with 0.5 nM [35S]GTPcS in a total volume of 200 ll in
50 mM Tris–HCl buffer, pH 7.4, containing 1 mM
EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 10 lM
GDP, 100 mM NaCl, 2 IU/ml adenosine deaminase,
0.5% bovine serum albumin, and different concentra-
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determined in the presence of 10 lM of unlabeled
GTPcS. After 1 h of incubation at room temperature,
the assay was filtered through glass-fiber filters (What-
man GF/B), which had been presoaked for 30 min in
ice-cold filtration buffer, on a Brandel cell harvester,
followed by three washing steps with ice-cold 50 mM
Tris–HCl containing 5 mM MgCl2, pH 7.4. The
punched-out filters were immediately transferred to
mini-vials and incubated with 2.5 ml of Ultima Goldꢁ
scintillation cocktail (Canberra Packard) for 9 h before
counting in a liquid scintillation counter Tricarb
2100TR (Canberra Packard). Data were analyzed using
GraphPad Prismꢁ, version 3.0 (GraphPad, San Diego,
California, USA). Experiments were carried out in trip-
licate in at least three independent experiments.
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This work was supported by the Deutsche Forschungs-
gemeinschaft (GRK 677, scholarship for D. Bertarelli).
We thank K. N. Klotz, University of Wurzburg,
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