Design, Synthesis, and Biological Evaluation of New 8-Heterocyclic Xanthine Derivatives as Highly Potent and Selective Human A2B Adenosine Receptor Antagonists

Article abstract of DOI:10.1021/jm0309654

Here we report the synthesis of 8-heterocycle-substituted xanthines as potent and selective A_2B adenosine receptor antagonists. The structure-activity relationships (SAR) of the xanthines synthesized in binding to recombinant human A_2B adenosine receptors (ARs) in HEK-293 cells (HEK-A_2B) and at other AR subtypes were explored. The synthesized compounds showed A_2B adenosine receptor affinity in the nanomolar range and good levels of selectivity evaluated in radioligand binding assays at human (h) A₁, A_2A, A_2B, and A₃ ARs. We introduced several heterocycles, such as pyrazole, isoxazole, pyridine, and pyridazine, at the 8-position of the xanthine nucleus and we have also investigated different spacers (substituted acetamide, oxyacetamide, and urea moieties) on the heterocycle introduced. Various groups at the 3- and 4-positions of phenylacetamide moiety were studied. This study allowed us to identify the derivatives 2-(3,4-dimethoxyphenyl)-N-[5-(2,6-dioxo-1,3-dipropyl-2,3,6, 7-tetrahydro-1H-purin8-yl)-1-methyl-1H-pyrazol-3-yl] acetamide (29b, MRE2028F20) [K_i(hA_2B) = 38 nM, K_i(hA₁,hA _2A,-hA₃) >1000 nM], N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6, 7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy] acetamide (62b, MRE2029F20) [K_i(hA_2B) = 5.5 nM, K_i(hA ₁,hA_2A,hA₃) > 1000 nM], and N-(3,4-dimethoxyphenyl)-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6, 7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy] acetamide (72b, MRE2030F20) [K_i(hA_2B = 12 nM, K_i(hA ₁,hA_2A, hA₃) > 1000 nM], which showed high affinity at the A_2B receptor subtype and very good selectivity vs the other ARs. Substitution of the acetamide with an urea moiety afforded bioisosteric xanthines with good affinity and selectivity comparable to the acetamide derivatives. Substitution at the para-position of a 4-benzyloxy group of the phenylacetamido chain enhanced affinity at the A_2B receptor [compound 30b (K_i(hA_2B) = 13 nM) vs compound 21b (K _i(hA_2B = 56 nM)] but did not favor selectivity. The derivatives with higher affinity at human A_2B AR proved to be antagonists, in the cyclic AMP assay, capable of inhibiting the stimulatory effect of NECA (100 nM) with IC₅₀ values in the nanomolar range, a trend similar to that observed in the binding assay (62b, IC₅₀ = 38 nM; 72b, IC₅₀ = 46 nM). In conclusion, the 8-pyrazolo-1,3-dipropyl-1H-purine-2,6-dione derivatives described herein represent a new family of selective antagonists for the adenosine A _2B receptor.

Full text of DOI:10.1021/jm0309654

1434
J . Med. Chem. 2004, 47, 1434-1447
Design , Syn th esis, a n d Biologica l Eva lu a tion of New 8-Heter ocyclic Xa n th in e
Der iva tives a s High ly P oten t a n d Selective Hu m a n A2B Ad en osin e Recep tor
An ta gon ists
Pier Giovanni Baraldi,*,† Mojgan Aghazadeh Tabrizi,† Delia Preti,† Andrea Bovero,† Romeo Romagnoli,†
Francesca Fruttarolo,† Naser Abdel Zaid,† Allan R. Moorman,‡ 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` di Ferrara, 44100 Ferrara Italy, and King Pharmaceutical R & D, 4000 Centre-Green Way, Suite 300,
Cary, North Carolina 27513
Received J uly 21, 2003
Here we report the synthesis of 8-heterocycle-substituted xanthines as potent and selective
A2B adenosine receptor antagonists. The structure-activity relationships (SAR) of the xanthines
synthesized in binding to recombinant human A2B adenosine receptors (ARs) in HEK-293 cells
(HEK-A2B) and at other AR subtypes were explored. The synthesized compounds showed A2B
adenosine receptor affinity in the nanomolar range and good levels of selectivity evaluated in
radioligand binding assays at human (h) A1, A2A, A2B, and A3 ARs. We introduced several
heterocycles, such as pyrazole, isoxazole, pyridine, and pyridazine, at the 8-position of the
xanthine nucleus and we have also investigated different spacers (substituted acetamide,
oxyacetamide, and urea moieties) on the heterocycle introduced. Various groups at the 3- and
4-positions of phenylacetamide moiety were studied. This study allowed us to identify the
derivatives 2-(3,4-dimethoxyphenyl)-N-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-1-methyl-1H-pyrazol-3-yl]acetamide (29b, MRE2028F20) [Ki(hA2B) ) 38 nM, Ki(hA1,hA2A,-
hA3) >1000 nM], N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-
purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide (62b, MRE2029F20) [Ki(hA2B) ) 5.5 nM,
Ki(hA1,hA2A,hA3) > 1000 nM], and N-(3,4-dimethoxyphenyl)-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-
tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]acetamide (72b, MRE2030F20) [Ki(hA2B
) 12 nM, Ki(hA1,hA2A, hA3) > 1000 nM], which showed high affinity at the A2B receptor subtype
and very good selectivity vs the other ARs. Substitution of the acetamide with an urea moiety
afforded bioisosteric xanthines with good affinity and selectivity comparable to the acetamide
derivatives. Substitution at the para-position of a 4-benzyloxy group of the phenylacetamido
chain enhanced affinity at the A2B receptor [compound 30b (Ki(hA2B) ) 13 nM) vs compound
21b (Ki(hA2B ) 56 nM)] but did not favor selectivity. The derivatives with higher affinity at
human A2B AR proved to be antagonists, in the cyclic AMP assay, capable of inhibiting the
stimulatory effect of NECA (100 nM) with IC50 values in the nanomolar range, a trend similar
to that observed in the binding assay (62b, IC50 ) 38 nM; 72b, IC50 ) 46 nM). In conclusion,
the 8-pyrazolo-1,3-dipropyl-1H-purine-2,6-dione derivatives described herein represent a new
family of selective antagonists for the adenosine A2B receptor.
In tr od u ction
sive family of G-protein coupled receptors.1,2 Adenosine
activates four subtypes of receptors (ARs): A1, A2A, A3,
In the past few years, significant advancement has
been made in the understanding of the molecular
pharmacology and physiology of A2B adenosine recep-
tors, but due to the lack of highly potent and selective
ligands for this subtype, many questions about the
pathophysiological role of A2B receptors have not yet
been answered.1,2 The biological activity of adenosine
occurs through the activation of specific receptors
located on cell membranes and belonging to the exten-
1,3
and A2B
.
The adenosine receptors are associated with different
messenger systems: A1 and A3 mediate adenylate
cyclase inhibition, whereas A2A and A2B stimulate the
adenylate cyclase activity controlling intracellular cyclic
AMP levels.4 Both A2A and A2B ARs are positively
coupled to adenylate cyclase via Gs. However, coupling
to phospholipase C via Gq, resulting in mobilization of
intracellular calcium and direct coupling to calcium
channels, has also been described.1,2
* Corresponding author. Address: Dipartimento di Scienze Farma-
ceutiche, Universita` di Ferrara, Via Fossato di Mortara 17-19, 44100
Ferrara, Italy. Tel: +39-0532-291293. Fax: +39-0532-291296. E-
mail: pgb@dns.unife.it.
Functional A2B receptors have been found in mast
cells.5 Its expression and activation is associated with
control of gene expression,6 cell growth,7 intestinal
function,8 neurosecretion,9 vascular tone,10 andasthma.2,11,12
The alkylxanthine theophylline, I (Figure 1), is a weak,
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ King Pharmaceutical R & D.
§ Dipartimento di Medicina Clinica
Farmacologia, Universita` di Ferrara.
e Sperimentale-Sezione di
10.1021/jm0309654 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/17/2004

New 8-Heterocyclic Xanthines as Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1435
F igu r e 1. Representative structures of nonxanthine and xanthines that act as antagonists at A2B receptors.
nonselective AR antagonist used therapeutically for the
treatment of asthma.13,14 The use of theophylline has
been associated with unpleasant side effects, such as
insomnia and diuresis. While the A1, A2A, and A3
adenosine receptors have been pharmacologically char-
acterized through the use of highly potent and selective
agonists and/or antagonists, the study of the A2B recep-
tor has been precluded due to the lack of selective
ligands15,16 and the absence of an appropriate radioli-
gand binding assay. Only recently, [3H]ZM241385, II,
has been proposed as a useful radioligand for studying
the A2B adenosine receptor subtype.17 In this field of
research, J acobson and co-workers have reported some
xanthine derivatives endowed with good affinity but
limited significant selectivity for the human A2B ad-
enosine receptor subtype.18,19
An evolution of this study led to the synthesis of
8-phenylxanthinecarboxylic acid congeners, which proved
to be potent and selective A2B antagonists. In particular,
the derivative named MRS-1754, III20 ([N-(4-cyanophe-
nyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-
purin-8-yl)phenoxy]acetamide], proved to be the most
potent and selective A2B adenosine receptor antagonist
ever reported. This result led to the synthesis and
characterization of the tritium-labeled form of MRS-
1754 as the first radioligand for the A2B receptor.21
Different nonxanthine structures displaying affinity for
the A2B subtype in binding assays were recently re-
ported.22,23
In recent years, we have investigated a series of
pyrazolo[4,3-e]1,2,4-triazolo-[1,5-c]pyrimidine deriva-
tives, modifying the chain at either the N7 or N8
pyrazole nitrogen and introducing different chains at
the amino function in the 5-position,24-27 with the aim
of studying the affinity of this class of compounds for
the human adenosine receptor subtypes. In particular,
the derivatives with the free amino group at the
5-position and the compound with a â-phenylethyl chain
at the N-8 pyrazole nitrogen showed good affinity for
A2B adenosine receptors.28
Mu¨ller and co-workers have synthesized 1,8-disub-
stituted xanthine derivatives bearing polar substituents
to obtain water-soluble derivatives that appeared to be
less potent than MRS-1754.29 More recently, J acobson
and co-workers have reported xanthine derivatives
substituted at the 1-, 3-, 7-, and 8-positions.30
nists for adenosine receptors.32 As demonstrated by
J acobson and co-workers, the introduction at the 8-posi-
tion of the xanthine nucleus of a phenyl group func-
tionalized in the para-position with oxymethylene chains
is associated with increased affinity at A2B receptors.
In our initial attempts, we prepared several different
compounds maintaining the xanthine nucleus and in-
troducing at the 8-position a differently substituted
pyrazole ring. Subsequently, we synthesized and evalu-
ated a series of 8-pyrazolylxanthine derivatives, linking
a substituted phenylacetamido moiety on the pyrazole
5-amino function. We also prepared several bioisosteric
xanthines containing phenyl-substituted urea moieties.
In some cases, we have prepared the corresponding
8-phenylxanthine derivatives for comparison of biologi-
cal data. From these initial studies we have identified
a new class of xanthine derivatives with high affinity
and selectivity at human A2B receptors. Compound 29b
[2-(3,4-dimethoxyphenyl)-N-[5-(2,6-dioxo-1,3-dipropyl-
2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-
3-yl]acetamide, MRE2028F20] in this series showed
high affinity (38 nM) for human A2B adenosine receptors
and good selectivity vs human A1, A2A, and A3 adenosine
subtypes.
To further understand the structure-activity rela-
tionships of the most potent A2B antagonists discovered
in our initial search and to explore structural modifica-
tions that might lead to enhanced affinity and selectiv-
ity, we designed a new series of 8-heterocyclic xanthine
derivatives. In this series we have maintained the
8-pyrazole xanthine nucleus and, as recently reported
by J acobson and co-workers in the XAC IV20 (Figure 1)
series, we have introduced an oxyacetamide chain on
the 3-position of the pyrazole ring. Furthermore, we
replaced the pyrazole nucleus with different hetero-
cycles, such as pyridine, pyridazine, and isoxazole, in
order to verify the importance of the nature of the
heterocycle at this position. To obtain water-soluble
compounds, we synthesized derivatives with a pipera-
zine nucleus on the lateral chain. The synthesized
compounds have been evaluated using binding experi-
ments to CHO cells transfected with A1, A2A, and A3
human adenosine receptor subtypes. Moreover, compe-
tition experiments were performed to study the affinity
(Ki) of the new compounds to the A2B adenosine receptor
subtype using [3H]DPCPX as radioligand.33
Suzuki and co-workers evaluated previously the
8-polycycloalkyl-1,3-dipropylxanthines as potent and
selective antagonists for the A1 adenosine receptors.31
Recently, Volpini et al. have reported the para-substi-
tuted 1,3-dialkyl-8-phenylxanthines as classical antago-
Ch em istr y
Synthesis of the intermediates 8-aminopyrazolo- and
8-aminophenylxanthines 7a , 8b-10b was performed by
the classical method starting from 1,3-disubsituted-5,6-

1436 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Baraldi et al.
Sch em e 1
diaminouracils.34,35 The preparation of the target com-
pounds was achieved by a two-step sequence involving
reaction between 1,3-disubsituted-5,6-diaminouracils
2a ,b and the corresponding pyrazole carboxylic acids 3
and 436 or phenyl carboxylic acid 5 in methanol solution
using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide
hydrochloride (EDAC) as condensing agent, followed by
ring closure in the presence of sodium hydroxide at 70
°C. The diamino uracils 2a ,b were obtained by reduction
of the nitroso uracils 1a ,b using sodium dithionite (see
Scheme 1).
5-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-2-methyl-2H-pyrazole-3-carboxylic acid, 12b, was
synthesized by condensation of 2b and 1-methyl-1H-
pyrazole-3,5-dicarboxylic acid 5-ethyl ester, 6,36 under
basic conditions. The methylation of 8-(aminophenyl)-
xanthine 10b in the 7-position led to xanthine 11b.in
good yields. Derivatives 13b-16b were prepared start-
ing from 9b, by condensation with appropriate carboxy-
lic acids.
Xanthine amide derivatives 17b-37b, 38a , and 39b-
41b were prepared by a coupling reaction among the
amine derivatives 7a , 8b-10b, and the appropriate
phenylacetic acid chloride derivatives obtained, in turn,
by reaction of the corresponding phenylacetic acid with
thionyl chloride and triethylamine in dichloromethane.
(Scheme 2).
The urea derivatives 42b-47b were synthesized by
condensation of the intermediates 9b and 10b with the
appropriate isocyanates at room temperature in anhy-
drous dioxane (see Scheme 2).
Oxyacetamide xanthine derivatives (60b-73b, 74c,
75c, 80b-83b, 86d , 76b, 77b, 78c, 79c, 88b-91b) were
synthesized through the condensation of 5,6-diamino-
uracils, 2b-d , with the appropriate carboxylic acid
intermediates (48, 49, or 52), to yield 8-substituted
xanthines containing the oxyacetic acid chain (50b-d ,
51b,c, 87b). Subsequently, condensation with meta- or
para-substituted anilines or N-substituted piperazine
in the presence of EDAC and HOBt (1-hydroxybenzo-
trizole) in DMF at room temperature yielded the target
compounds. The piperazinyl amides 80b and 82b were
transformed into the corresponding hydrochlorides 84b
and 85b by treatment with a saturated methanolic HCl
solution. 8-(6-Hydroxypyridin-3-yl)-1,3-dipropyl-3,7-di-
hydropurine-2,6-dione and 8-(6-hydroxypyridazin-3-yl)-
1,3-dipropyl-3,7-dihydropurine-2,6-dione (55b, 56b) were
prepared by condensation of 2b with 6-hydroxypyridine-
3-carboxylic acid or 6-hydroxypyridazine-3-carboxylic
acid (53),37 respectively (Scheme 3). These intermediates
were alkylated with R-bromo-4-iodoacetanilide (57)38 in
the presence of TEA and DMF at room temperature, to
yield the final compounds 58b and 59b.
The preparation of intermediate derivatives 48, 49,
52 is described in Scheme 4. Alkylation of hydroxypyra-
zolecarboxylic acid methyl esters 92 and 9339,40 and
hydroxyisoxazolecarboxylic acid methyl esters 10041,42
with ethyl bromoacetate and subsequent alkaline hy-
drolysis afforded the corresponding dicarboxylic acids
derivatives 96, 97, and 102. These were transformed
into derivatives 48, 49, and 52 by a selective esterifi-

New 8-Heterocyclic Xanthines as Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1437
Sch em e 2
cation of the aliphatic carboxylic acids in the presence
of toluene-4-sulfonic acid at room temperature.
The chemical characterization of all the final com-
pounds are reported in Tables 1-7.
binding parameters of compounds 17b and 41b show
that the affinities for A2B are essentially identical, while
the greater selectivity is with compound 17b and not
for compound 41b.
Comparison of compound 7a vs compound 9b suggests
that smaller substitution (methyl vs n-propyl) at N-1
and N-3 of the purine nucleus leads to enhanced affinity
and greatly enhanced selectivity. But comparison of
compounds 17b and 38a shows that it is not the case
when the amine has been acylated with phenylacetic
acid. This observation prompted us to prepare the series
of compounds 18b-37b.
The enhanced affinity of compound 29b (Ki(hA2B) )
38 nM) and compound 17b (Ki(hA2B) ) 35 nM) with
respect to compound 40b (Ki(hA2B) ) 95 nM) and
compound 39b (Ki(hA2B) ) 649 nM) confirms the
importance of replacing the phenyl ring by the pyrazolo
nucleus.
Resu lts
Affinities of xanthine derivatives in radioligand bind-
ing assays at A1, A2A, A2B, and A3 receptors are reported
in Table 8. Receptor binding assays were performed
using Chinese hamster ovary cells (CHO) transfected
with A1, A2A, A2B, and A3 adenosine receptors and
human embryonic kidney cells (HEK-293) stably trans-
fected with A2B adenosine receptors. The derivatives
with higher affinity at human A2B adenosine receptors
were antagonists capable of inhibiting the cAMP pro-
duction induced by 100 nM NECA in CHO cells trans-
fected with human (h) A2B adenosine receptor. Binding
parameters (Ki, nM) and functional data (IC50, nM) are
listed in Table 9.
From our preliminary studies, we have inferred that
the introduction of an N1- or N2-methylpyrazole-3-yl ring
in place of the phenyl ring in MRS1754 gave compounds
7a , 8b, and 9b, showing moderate A2B adenosine recep-
tor affinity. Comparison of biological data for compound
8b vs compound 9b suggests that increased affinity and
selectivity for A2B should be achieved with the substitu-
tion pattern of compound 8b. On the contrary, the
Comparison of 8b and 9b vs 12b suggests the
importance of an amine function (positive charge) on
the pyrazole, rather than a negatively charged carboxy-
late group. Trying to enhance this interaction by acy-
lation with 4-aminobutyric acid to provide compound
14b decreased the affinity for both A1 and A2B receptors
and decreased selectivity for A2B receptors. Comparison
of compounds 13b and 14b, designed following J acob-
son’s findings,27 further confirms the need for a posi-

1438 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Baraldi et al.
Sch em e 3
tively charged group rather than a negatively charged
group, as suggested by the comparison of 9b vs 12b.
Further substitution (o-CF3, m-CF3, p-NO2) of the
4-benzyloxy group at the para-position of compound 21b
produced compounds 30b, 31b, 32b. Compound 30b is
more potent (Ki(hA2B) ) 13 nM) than 21b (Ki(hA2B) )
56 nM), but less selective versus the A1 adenosine
receptor.
Bulky substituents, such as a 3,4-dimethoxy group,
at the 3- and 4-positions on the phenyl ring of the
phenylacetamide chain led to a potent and selective A2B
adenosine receptor antagonist (compound 29b, MRE-
2028F20), whereas 3,4,5-trimethoxy substitution (com-
pound 35b) showed no affinity.
All of the urea derivatives show good affinity and
rather high selectivity as A2B adenosine receptor an-
tagonists. In some cases, compounds 42b and 43b, the
introduction of the bioisosteric urea moiety in place of
the acetamido chain (compounds 27b and 24b) increases
the affinity at the A2B adenosine receptor.
The derivatives 17b, 21b, 26b, 30b (Ki ) 35, 56, 50,
13 nM, respectively) are antagonists at the human A2B
adenosine receptor, capable of inhibiting cAMP produc-
tion induced by 100 nM NECA, with IC50 values in the
nanomolar range (93-185 nM) (Figure 2). Interestingly,
in the cyclic AMP assay the compounds examined
exhibited a rank order of potency very close to that
observed in binding experiments (Figure 3). The Spear-
man’s rank correlation coefficient between IC50 values
in the cyclic AMP assay and receptor affinity values (Ki)
showed a highly significant positive correlation.
From these initial results, it is evident that introduc-
tion of the pyrazole nucleus at the 8-position of xanthine
is fundamental for the A2B subtype in binding assays.
Identifying a new lead (MRE2028F20) for this receptor,
we wished to explore the structural modifications that
might lead to enhanced activity and/or selectivity. The
purpose of our investigations was to synthesize a new
series of 8-substituted xanthine derivatives in which the
xanthine nucleus was linked to a various substituted
oxyacetamide chains by a heterocyclic spacer (pyrazole,
isoxazole, pyridine, pyridazine) and to determinate the
effects of such substitutions on the affinity and selectiv-
ity of these compounds as A2B adenosine receptor
antagonists. MRE2028F20 was the reference compound
for the biological data comparison.
The results reported provide interesting information
about the effect of substitution at the 8-position of the
xanthine nucleus. Many of the tested compounds ap-
peared to be potent A2B receptor ligands (Ki in the

New 8-Heterocyclic Xanthines as Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1439
Sch em e 4
heterocyclic spacer ring, in addition to steric factors,
plays a fundamental role in influencing A2B binding
parameters.
Further important information was furnished by the
introduction of different moieties on the side chain of
the heterocyclic spacer. In particular, we investigated
the effect of several chemically different substitutions
at the 3- and/or 4-position of the phenyl ring of the
phenylcarbamoyl moiety (60b-73b, 76b, 77b, 74c, 75c,
78c, 79c, 88b-91b). The modifications performed (4-
F, 4-Br, 3,4-methylenedioxy, 4-sec-butyl, 4-N-morpho-
line, 3,4-dimethoxy, 3,4-dimethyl) differed in electronic,
steric, and lipophilic features. Small hydrophobic resi-
dues in the 3- and/or 4-position of the phenyl ring
seemed to increase affinity toward the A2B receptor (60b,
61b, 62b, 66b, 70b-72b). In particular, the presence
of electron-donating groups, such as methoxy or 3,4-
methylenedioxy (72b, 62b), increased affinity. An ad-
ditional aspect that appeared important was the capa-
bility of these moieties (-OR) to form a hydrogen bond
with the A2B adenosine receptor interaction site. Bulky
lipophilic groups, such as 4-sec-butyl chain, led to a loss
of affinity (65b). Introduction of substituents containing
a carbonyl function (64b, 68b, 69b) yielded a slight loss
of affinity but an improvement of selectivity versus other
adenosine receptors subtypes, in particular versus the
A1 adenosine receptors. One of the most interesting
compounds in terms of affinity was 62b (MRE2029F20,
Ki(hA2B) ) 5.5 nM), containing the 3,4-methylenedioxy
function on the phenyl ring, while the corresponding 3,4-
dimethoxy derivative 72b (MRE2030F20, Ki(hA2B) ) 12
nM) retained good affinity with improved selectivity for
the A2B receptor. In compounds 63b and 73b, the phenyl
ring of the anilide moiety was replaced by a pyridine
ring. This modification led to a substantial loss of
affinity toward the A2B adenosine receptors, especially
when a small lipophilic function such as -CH3 was
introduced on the heterocycle (63b).
nanomolar range) with different degrees of selectivity.
Among them, several derivatives also showed good
potency as A2B antagonists in terms of IC50 in the
functional cAMP assay. Derivatives 68b, 69b, 73b, 81b,
88b, 89b, and 91b were comparable to MRE2028F20
as A2B ligands, whereas several compounds appeared
to have more A2B receptor affinity than the reference
compound (60b, 62b, 70b-72b, 80b, 84b).
In particular, the presence of a pyrazole ring at the
8-position of the xanthine moiety was confirmed to be
an important structural requirement for A2B adenosine
receptor binding. In fact, all compounds containing such
a heterocyclic ring (60b-73b, 76b, 77b, 80b-85b, 74c,
75c, 77c, 78c, 86d ) showed high affinity toward A2B
receptors. Among them, derivatives 76b, 77b, 78c, and
79c, bearing the N2-methylpyrazol-3-yl isomer, exhib-
ited lower affinity than the corresponding N1-meth-
ylpyrazol-3-yl analogues. In addition, it was observed
that N2-methyl derivatives demonstrated a global in-
crease of affinity toward A1 receptor, with consequent
loss of selectivity.
Compounds 88b-91b, bearing the isoxazole nucleus
at the 8-position, showed lower affinity at the A2B
receptor than the corresponding 8-pyrazole derivatives.
However, replacing the pyrazole ring with an isoxazole
enhanced selectivity versus the A1 adenosine receptor.
Another result that supported the fundamental impor-
tance of the pyrazole is the complete loss of affinity
produced by substitution of the pyrazole with pyridine
or pyridazine rings (58b, 59b). Therefore, it seemed that
replacing the five-membered heterocycle with a six-
members heterocycle determined the loss of important
interactions between the examined molecules and theA2B
adenosine receptor. Although pyridine and pyridazine
were shown to be detrimental in terms of affinity, the
pyridine derivative seemed to interact with the A2B
receptor better than the pyridazine derivative. This
suggests that the electronic and lipophilic nature of this
To obtain water-soluble compounds, we replaced the
phenyl ring in the side chain with a 4-substituted
piperazine moiety (80b-83b, 86d ) and prepared the
corresponding hydrochloride derivatives (84b, 85b).
Introducing different kinds of substituents at the N-4
position of piperazine, we obtained the series of com-
pounds shown in Table 5. The choice of a 4-fluorophenyl
ring appeared to be the most interesting modification
in terms of both affinity and selectivity (81b). In
particular, the presence of the fluorine moiety in the
para-position seemed to confer selectivity toward A2B
versus A1 adenosine receptors (see compounds 80b and
81b). 8-{2-Methyl-5-[2-oxo-2-(4-phenylpiperazin-1-yl)-
ethoxy]-2H-pyrazol-3-yl}-1,3-dipropyl-3,7-dihydropurine-
2,6-dione (80b) and its hydrochloride salt (84b) showed
high affinity and good selectivity for the A2B receptor
(respectively Ki(hA2B) ) 15 nM, Ki(hA2B) ) 12 nM). For
the same reason, we synthesized compound 69b bearing
a free carboxylic function at the para-position of the
anilide moiety (Ki(hA2B) ) 36 nM).
In our SAR study, we have also investigated the
importance of the alkyl substitution at the 1,3-positions
of the xanthine structure. Introduction of an isobutyl
chain produced a marked decrease of affinity toward A2B
adenosine receptors (compare 60b and 61b versus 74c
and 75c). Another interesting finding was that replace-

1440 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Baraldi et al.
Ta ble 1. Chemical Characterization of 8-Substituted Xanthine Derivatives 7a and 8b-16b
ment of the propyl groups at the 1- and 3-positions of
the xanthine nucleus with allyl chains produced an
increase of selectivity (versus A1 receptor) with a slight
decrease of affinity at A2B adenosine receptors (80b vs
86d ). The most interesting compounds of this series in
binding assays were evaluated in the cAMP assay on
CHO cells. The IC50 values thus obtained are reported
in Table 9. The most potent derivatives showed an IC50
value of about 40 nM (62b, 70b, 72b). Very interest-
ingly, the compounds that show an high A2B affinity
were also the most potent in functional assays.
tained by introducing n-propyl chains on the 1- and
3-positions of the xanthine nucleus and an N1-meth-
ylpyrazole spacer on the 8-position linked to an oxyacet-
amide moiety in the side chain bearing small lipophilic
electron-releasing functions in the meta- and/or para-
position of an aromatic ring (62b, 72b). Replacement
of the pyrazole spacer with other heterocycles led, in
some cases (88b-91b), to an increase of selectivity
toward A2B receptors. The introduction of a piperazine
moiety between the oxyacetyl and aryl groups allowed
us to improve the water solubility of derivative com-
pounds, especially for hydrochloride salts (84b, 85b),
while receptor affinity was maintained.
Con clu sion s
From our study on this new class of 8-substitued
xanthine derivatives emerged a series of important
considerations. The most significant results were ob-
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

New 8-Heterocyclic Xanthines as Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1441
Ta ble 2. Chemical Characterization of Phenyl and Pyrazolo Acetamide Xanthine Derivatives 17b-37b, 38a , and 39b-41b
F245 Merck plates). Products were visualized with iodine or
potassium permanganate solution. 1H NMR spectra 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 Hz. Light petroleum ether
refers to the fractions boiling at 40-60 °C. Melting points were
determined on a Buchi-Tottoli instrument and are uncorrected.
Chromatography was performed using Merck 60-200 mesh
silica gel. All products reported showed 1H NMR spectra in
agreement with the assigned structures. Organic solutions
were dried over anhydrous magnesium sulfate. Elemental
analyses were performed by the microanalytical laboratory of
Dipartimento di Chimica, University of Ferrara, and were
within (0.4% of the theoretical values for C, H, and N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Am in op y-
r a zolo Der iva tives 7a , 8b, 9b, Am in op h en yl Der iva tive
10b, a n d P yr a zolo Ca r boxylic Acid Der iva tive 12b.
To a solution of 1,3-disubstituted-5,6-diaminouracil 2a or
2b (2.2 mmol), in methanol (10 mL), was added an equimolar
amount of the appropriate carboxylic acid (3-6) (protected as
carbamic acid benzyl ester on the amino group for compounds
7a , 8b, 9b, 10b and as ethyl ester on carboxylic function for
compound 12b) and EDAC (2.21 mmol). The reaction mixture
was stirred at room temperature for 4-5 h with being
monitored by TLC. The solvent was concentrated in vacuo and

1442 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Baraldi et al.
Ta ble 3. Chemical Characterization of Urea Xanthine
Derivatives (42b-47b)
acid (0.45 mmol) in 10 mL of thionyl chloride was refluxed for
1 h. The excess of thionyl chloride was removed by evaporation.
The residue was dissolved in CH2Cl2 (5 mL) and added to a
solution of the desired amine derivative (0.30 mmol) and
triethylamine (0.08 mL) in CH2Cl2. The reaction mixture was
stirred at room temperature for 4 h and subsequently evapo-
rated to dryness under reduced pressure.The residue was
taken up in H2O (10 mL) and the aqueous layer was extracted
with EtOAc (2 × 20 mL). The organic phase was dried on
anhydrous MgSO4, filtered, and concentrated at reduced
pressure to afford the amide derivatives. The purification of
compounds was achieved by flash chromatography eluting with
a suitable mixture of solvents (ethyl acetate-hexane).
Ger n er a l P r oced u r e for th e P r ep a r a tion of Ur ea
Der iva tives 42b-47b. A solution of amino derivatives 9b and
10b (3 mmol) and the appropriate isocyanate derivatives (3.2
mmol) in 10 mL of anhydrous dioxane was stirred at room
temperature for 2 h with monitoring by TLC. The reaction
mixture was concentrated under reduced pressure and the
residue was purified by flash chromatography on silica gel
eluting with a suitable mixture of solvents to afford the desired
products.
Gen er a l P r oced u r e for th e P r ep a r a tion of P yr a zolo
Oxya cetic Acid Xa n th in es 50b -d a n d 51b,c, Isoxa zole
Oxya cet ic a cid Xa n t h in e 87b , a n d Hyd r oxyp yr id in /
p yr id a zin e Xa n th in es 55b a n d 56b. To a solution of 1,3-
disubstituted-5,6-diaminouracil 2b-d (2.2 mmol), in methanol,
was added an equimolar amount of the appropriate carboxylic
acid derivative, and then a slightly excessive amount of EDAC
(N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochlo-
ride) was added. The reaction mixture was stirred at room
temperature for 4-5 h with monitoring by TLC. Addition of
water caused precipitation, and the solid was filtered off. The
solid was taken up with 20 mL of 10% aqueous NaOH solution
and stirred at 70 °C for 0.5 h. The reaction mixture was
allowed to cool and acidified with 10% HCl to pH 5, and the
product was collected by filtration.
compd
R1
R2
mp (°C)
MW
formula
anal
42b
43b Cl
44b OCH3
45b
46b Cl
47b OCH3
H
N(CH3)2 266-268 dec 509.60
C
C
C
C
C
C
24H31N9O3
22H25ClN8O3 C, H, N
23H28N8O4
23H28N8O4
24H25ClN6O3 C, H, N
25H28N6O4 C, H, N
C, H, N
H
H
>300
>300
>300
>300
>300
500.98
496.56
496.56
480.95
476.53
C, H, N
C, H, N
H
OCH3
H
H
the amidic intermediate derivative was precipitated by the
addition of water. After filtration, the solid was dissolved in
methanol (10 mL) and NaOH (2.5 N, 15 mL) and stirred at
70-80 °C for 12 h. The methanol was distilled off, and the
residue was taken up in H2O and acidified with HCl to pH
4-5. The precipitate was filtered off, washed with water, and
purified by flash chromatography by elution with different
mixtures of ethyl acetate-petroleum ether.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 2-[6-(2,6-
Dioxo-1,3-d ip r op yl-2,3,6,7-tetr a h yd r o-1H-p u r in -8-yl)-p y-
r id a zin -3-yloxy]-N-(4-iod op h en yl)a ceta m id e (58b) a n d
2-[5-(2,6-Dioxo-1,3-d ip r op yl-2,3,6,7-tetr a h yd r o-1H-p u r in -
8-yl)p yr id in -2-yloxy]-N-(4-iod op h en yl)a ceta m id e (59b).
To a solution of hydroxypyridazine xanthine derivative 56b
or 8-hydroxypyridine 55b (0.20 mmol) in anhydrous DMF (10
mL) was added an equimolar amount of triethylamine (TEA),
and the reaction mixture was stirred at room temperature for
10 min. 2-Bromo-N-(4-iodophenyl)acetamide 57 (0.2 mmol) was
added, and the mixture was stirred at room temperature for
10 h. The solvent was distilled off, the residue was taken up
in cold water, and the precipitate was filtered off. The product
was purified by column chromatography on silica gel eluting
with a suitable mixture of CH2Cl2-CH3OH.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-[(Eth oxy-
ca r b on yl)m et h oxy)]-2-m et h yl-2H -p yr a zole-3-ca r b oxyl-
ic Acid (48), 5-[(Eth oxyca r bon yl)m eth oxy]-1-m eth yl-1H-
p yr a zole-3-ca r boxylic Acid (49), a n d 3-[(Eth oxyca r bon -
yl)m eth oxy]isoxa zole-5-ca r boxylic Acid (52). The acid
derivative 96, 97, or 102 (4 mmol) was dissolved in ethanol
(50 mL), and to the solution was added a catalytic amount of
toluene-4-sulfonic acid (100 mg). The reaction mixture was
stirred overnight at room temperature, ethanol was distilled
off, and the residue was crystallized by ethanol and water.
P r ep a r a tion of 8-(4-Am in op h en yl)-7-m eth yl-1,3-d ip r o-
p yl-3,7-d ih yd r op u r in e-2,6-d ion e (11b). To a solution of
compound 10b (0.73 mmol) in anhydrous DMF (10 mL) were
added K2CO3 (0.88 mmol) and dimethyl sulfate (0.88 mmol,
0.08 mL). The reaction mixture was stirred at room temper-
ature for 1 h, DMF was removed in vacuo. To the residue was
added water and the mixture was extracted with ethyl acetate
(30 mL). After evaporation in vacuo, the residual solid was
purified by silica gel column chromatography by elution with
petroleum ether-ethyl acetate (1:1).
P r ep a r a tion of N-[5-(2,6-Dioxo-1,3-d ip r op yl-2,3,6,7-tet-
r a h yd r o-1H-p u r in -8-yl)-1-m eth yl-1H-p yr a zol-3-yl]su cci-
n a m ic Acid (13b). To a solution of compound 9b (60 mg, 0.18
mmol) in dioxane (20 mL) was added succinic anhydride (18
mg, 0.18 mmol). The mixture was stirred at 80-90 °C for 5 h.
The solvent was evaporated to obtain 13b as a crude solid,
which was suspended in water, collected by filtration, and
crystallized from ethanol.
P r ep a r a tion of 4-Am in o-N-[5-(2,6-d ioxo-1,3-d ip r op yl-
2,3,6,7-tetr a h yd r o-1H-p u r in -8-yl)-1-m eth yl-1H-p yr a zol-3-
yl]bu tyr a m id e (Hyd r och lor id e) (14b). A solution of com-
pound 9b (50 mg, 0.15 mmol), 4-tert-butoxycarbonylamino
butyric acid (0.9 mmol), EDAC (0.9 mmol), TEA (0.3 mL, 2.25
mmol), and a catalytic amount of DMAP in anhydrous DMF
(7 mL) was stirred at room temperature for 4 h. The mixture
was evaporated to dryness under reduced pressure, and the
residue was purified by column chromatography on silica gel
eluting with ethyl acetate-petroleum ether (3:2) to yield 40
mg of N-Boc derivative as a white solid (mp 215-216 °C),
which was stirred in a saturated solution of ethyl acetate/HCl-
(g) (5 mL) for 1 h at room temperature. The product was
filtered off and the white solid was crystallized by methanol-
diethyl ether.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-(Ca r -
boxym eth oxy)-2-m eth yl-2H-p yr a zole-3-ca r boxylic Acid
(96), 5-(Ca r boxym et h oxy)-1-m et h yl-1H-p yr a zole-3-ca r -
boxylic Acid (97), 3-(Ca r boxym eth oxy)isoxa zole-5-ca r -
boxylic Acid (102). 94, 95, or 101 (4.0 mmol) was refluxed
in a mixture of methanol (60 mL) and 5% aq NaOH solution
(20 mL) for 1 h at 100 °C. Methanol was distilled off, and the
residue was taken up in water and acidified with HCl to pH
4. The precipitate was filtered off and washed with cold water.
Gen er a l P r oced u r e for th e P r ep a r a tion of P yr a zolo
P r op ion a m id e Der iva tives 15b a n d 16b a n d P yr a zolo
(P h en yl) Aceta m id e Xa n th in es Der iva tives 17b-37b,
38a , a n d 39b-41b. A solution of the appropriate carboxylic
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-[(Eth oxy-
ca r b on yl)m et h oxy]-2-m et h yl-2H -p yr a zole-3-ca r b oxylic
Acid Meth yl Ester (94), 5-[(Eth oxyca r bon yl)m eth oxy]-

New 8-Heterocyclic Xanthines as Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1443
Ta ble 4. Analytical Data of N1/N2-Methylpyrazole Oxyacetamide Derivatives
compd
R
R1
mp (°C)
MW
formula
anal.
60b
61b
62b
63b
64b
65b
66b
67b
68b
69b
70b
71b
72b
73b
74c
75c
76b
77b
78c
79c
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
i-C4H9
i-C4H9
n-C3H7
n-C3H7
i-C4H9
i-C4H9
4-F-phenyl
4-Br-phenyl
3,4-(methylenedioxy)phenyl
5-methylpyridin-2-yl
4-acethylphenyl
4-s-Bu-phenyl
264
280
483.50
544.40
509.51
480.52
507.54
521.61
479.53
550,61
537.57
509.51
493.56
534.39
525.56
466.49
511.55
572.45
544.40
483.50
572.45
511.55
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
23H26FN7O4
23H26BrN7O4
24H27N7O6
23H28N8O4
25H29N7O5
27H35N7O4
24H29N7O4
27H34N8O5
26H31N7O6
24H27N7O6
25H31N7O4
23H25Cl2N7O4
25H31N7O6
22H26N8O4
25H30FN7O4
25H30BrN7O4
23H26BrN7O4
23H26FN7O4
25H30BrN7O4
25H30FN7O4
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
272-273
278-280
273-275
240-242
256-257
296-298
294
>300
264-265
258
291-293
248-251
227-230
258-260
242
4-tolyl
4-N-morpholinylphenyl
4-(ethoxycarbonyl)phenyl
4-carboxyphenyl
3,4-dimethylphenyl
3,4-dichlorophenyl
3,4-dimethoxyphenyl
pyridin-4-yl
4-F-phenyl
4-Br-phenyl
4-Br-phenyl
4-F-phenyl
262
234
251-252
4-Br-phenyl
4-F-phenyl
Ta ble 5. Analytical Data of Pyrazolo Piperazinamide Derivatives 80b-85b and 86d
compd
R
R1
mp (°C)
MW
formula
anal.
80b
81b
82b
83b
84b
85b
86d
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
CH2CHdCH2
phenyl
4-F-phenyl
CH3
benzyl
phenyl
CH3
273-274
235-237
192-194
236-237
290
273
289-290
507.24
552.60
472.54
548.64
534.61
509.24
530.58
C
C
C
C
C
C
C
27H34N8O4
27H33 FN8O4
22H32N8O4
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
28H36N8O4
27H34N8O4‚HCl
22H32N8O4‚HCl
27H30N8O4
phenyl
Ta ble 6. Analytical Data of Isoxazole Oxyacetmide Derivatives
88b-91b
Ta ble 7. Analytical Data of Pyridine/Pyridazine Oxyacetamide
Derivatives 58b and 59b
compd
R1
R2
mp (°C) MW
formula
anal.
88b OCH2O
278-280 496.47 C23H24N6O7 C, H, N
compd
X
mp (°C)
MW
formula
anal.
89b OCH3 OCH3 253-255 512.52 C24H28N6O7 C, H, N
90b
91b
H
H
F
287
470.45 C22H23FN6O5 C, H, N
58b
59b
N
CH
310
293
589.09
588.10
C
C
23H24IN7O4
24H25IN6O4
C, H, N
C, H, N
OCH3 285-287 482.49 C23H26N6O6 C, H, N
1-m eth yl-1H-p yr a zole-3-ca r boxylic Acid Meth yl Ester
(95), 3-[(Eth oxyca r bon yl)m eth oxy]-isoxa zole-5-ca r boxy-
lic Acid Meth yl Ester (101). To a solution of 92, 93, or 100
(7.14 mmol) in anhydrous acetone (30 mL) was added 1.20 g
(8.6 mmol) of K2CO3 and an equimolar amount of ethyl
bromoacetate. The reaction mixture was refluxed for 2 h with
monitoring by TLC. Acetone was removed in vacuo, and the
residue was taken up with water and extracted with ethyl
acetate (50 mL). The organic layer was anhydrified on MgSO4
and evaporated, affording the desired alkylated derivatives 94,
95, 101.
CH2Cl2 (30 mL) were added R-bromoacethyl bromide (1.4 mL)
and TEA (15.0 mmol) at 0 °C. The reaction mixture was stirred
at room temperature for 1 h. Then the solvent was removed
and the residue was treated with 5% HCl (30 mL). The
aqueous layer was extracted with ethyl acetate. The organic
extract was washed with brine, dried with Na2SO4, and
evaporated. The solid residue was purified by crystallization
from ethyl acetate.
Gen er a l P r oced u r e for th e P r ep a r a tion of Oxya ceta -
m id e Der iva tives 60b-73b, 74c, 75c, 76b, 77b, 78c, 79c,
80b-83b, 86d , a n d 88b-91b. To a solution of the carboxylic
acid derivatives 50b-d , 51b, 51c, or 87b (1.80 mmol) in 30
mL of anhydrous DMF (dimethylformamide) was added EDAC
P r ep a r a t ion of 2-Br om o-N-(4-iod op h en yl)a cet a m id e
(57). To a solution of 4-iodoaniline (14.5 mmol) in anhydrous

1444 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Baraldi et al.
Ta ble 8. Adenosine Receptor Affinities and A2B Selectivities of Synthesized Xanthine Derivatives
Ki (nM)
a
b
c
d
compd
hA1
hA2A
hA2B
hA3
h
A1/hA2B
hA1/hA2B
hA1/hA2B
7a
8b
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
175 (134-229)
58 (45-74)
235 (209-264)
9 (7-12)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>6
2.4
0.8
7.2
>2
-
>6
>18
>4
>6
>18
>4
140 (123-159)
201 (172-236)
65 (56-75)
>1000
9b
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
31b
32b
33b
34b
35b
36b
37b
38a
39b
40b
41b
42b
43b
44b
45b
46b
47b
58b
59b
60b
61b
62b
63b
64b
65b
66b
67b
68b
69b
70b
71b
72b
73b
74c
75c
76b
77b
78c
79c
80b
81b
82b
83b
84b
85b
86d
88b
89b
90b
91b
>100
>2
>100
516 (455-585)
>1000
2
>1000
>1000
-
-
-
>1000
-
-
548 (464-648)
>1000
2065 (1866-2284)
>1000
0.3
-
-
26
>11
-
>0.5
-
>0.5
-
>1000
>1000
-
-
900 (811-996)
>1000
35 (27-45)
96 (80-114)
>1000
>29
>11
-
>29
>11
-
>1000
>1000
78 (63-96)
56 (42-77)
103 (79-136)
88 (84-92)
100 (83-120)
160 (142-179)
50 (41-60)
1628 (1374-1930)
28 (23-33)
38 (33-43)
13 (11-16)
90 (73-110)
111 (100-124)
130 (113-150)
78 (63-96)
>1000
>13
>18
>10
2.3
8.5
28
>13
>18
>10
>12
>10
>6
>13
>18
>10
>12
>10
>6
>1000
>1000
200
850 (762-946)
4481 (3650-5501)
3227 (2799-3720)
>1000
65
>20
>0.6
>36
>27
>77
>11
>9
>20
>0.6
>36
>27
>77
>11
>9
>0.6
520 (484-558)
>1000
19
>27
4.3
1.1
1.5
3.9
15
56 (47-67)
100 (83-120)
163 (137-193)
746 (659-843)
1898 (1723-2091)
>1000
>6
>6
>8
>8
-
31
>3
>2
>2
18
-
-
566 (516-621)
>1000
>1000
>1000
18 (12-27)
342 (274-426)
569 (506-640)
649 (563-747)
95 (86-105)
34 (26-46)
59 (44-81)
39 (33-46)
90 (73-110)
81 (70-110)
110 (93-130)
58 (53-64)
>1000
69
>56
>3
5.1
>2
>2
>2
>2
1725 (1374-2165)
55 (46-65)
2410 (1760-3301 )
448 (365-550)
1993 (1658-2397)
1440 (1250-2211)
1361 (1103-1679)
1175 (1103-1679)
>1000
>11
>30
>17
>26
>11
>13
>9
>11
>30
>17
>26
>11
>13
>9
1.6
41
11
22
18
12.3
20
>17
-
>10
>84
>50
>182
>1
>17
-
>10
>84
>50
>182
>1
-
>1000
108 (75-155)
12 (7-21)
>10
5.4
7.5
40
65 (48-86)
150 (132-170)
200 (180-226)
>1000
20 (16-25)
5.5 (4.6-6.5)
1012 (819-1250)
86 (77-96)
74 (67-81)
19 (12-29)
86 (78-93)
32 (22-45)
36 (27-47)
10 (8-13)
>1
>12
14
>1000
>12
>14
>53
>12
>32
>28
>100
>63
>84
>25
>4
>12
>14
>53
>12
>32
>28
>100
>63
>84
>25
>4
1005 (916-1103)
79 (72-86)
>1000
4
>12
>32
>28
70
>1000
>1000
700 (650-760)
300 (240-380)
>1000
16 (12-20)
12 (8-17)
19
>84
955 (896-1017)
467 (400-546)
2427 (2067-2850)
168 (140-201)
181 (127-258)
49 (34-72)
72 (45-114)
250 (181-348)
>1000
41 (35-48)
303 (260-352)
132 (98-178)
93 (82-105)
185 (163-210)
66 (38-116)
207 (162-265)
15 (10-21)
55 (46-65)
122 (108-136)
85 (66-95)
12 (7-19)
23
1.5
18
>8
>8
1.8
1
0.7
0.4
17
>19
>9
9.5
22
>14
>42
>22
>20
>15
>19
>11
>6
>11
>6
>16
>5
>16
>5
>67
>19
>9
>67
>19
>9
>1000
810 (763-859)
260 (232-287)
>1000
>12
>84
>14
>42
>22
>20
>15
>19
>12
>84
>14
>42
>22
>20
>15
>19
76 (67-86)
24 (18-32)
47 (43-52)
51 (44-58)
70 (61-80)
53 (40-69)
>1000
>1000
>1000
>1000
>1000
a
b
Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells (n ) 3-6). Displacement of specific
[3H]ZM241385 binding at human A2A receptors expressed in CHO cells (n ) 3-6). c Displacement of specific [3H] DPCPX binding at
human A2B receptors expressed in HEK293 cells (n ) 3-6). Displacement of specific [3H]MRE3008-F20 binding at human A3 receptors
d
expressed in CHO cells (n ) 3-6). Data are expressed as geometric means with 95% confidence limits.

New 8-Heterocyclic Xanthines as Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1445
Ta ble 9. Binding and Functional Data of Selected A2B
Adenosine Compoundsa
[3H]DPCPX binding in
HEK-293 membranes
expressing human
A2B adenosine
cAMP assay in
CHO cells expressing
human A2B adenosine
receptors IC50 (nM)
compd
receptors Ki (nM)
17b
21b
26b
28b
29bb
30b
36b
41b
59b
60b
61b
62bc
66b
68b
69b
70b
71b
72bd
80b
84b
85b
86d
88b
35 (27-45)
56 (42-77)
50 (41-60)
28 (23-33)
38 (33-43)
13 (11-16)
18 (12-27)
34 (26-46)
108 (75-155)
12 (7-21)
20 (16-25)
5.5 (4.6-6.5)
19 (12-29)
32 (22-45)
36 (27-47)
10 (8-13)
16 (12-20)
12 (8-17)
15 (10-21)
12 (7-19)
76 (67-86)
24 (18-32)
47 (43-52)
103 (92-115)
185 (154-198)
160 (146-188)
128 (114-144)
120 (103-140)
93 (84-102)
95 (90-101)
152 (136-170)
477 (420-510)
88 (82-95)
108 (91-129)
38 (29-51)
75 (66-89)
120 (103-140)
115 (74-89)
40 (32-50)
60 (52-69)
46 (35-61)
65 (52-78)
70 (64-78)
322 (280-348)
81 (74-89)
F igu r e 2. Inhibition curves representing the capability of the
antagonists to block the effect of 100 nM NECA on adenylyl
cyclase assays of human A2B adenosine receptors. Values are
the means and vertical lines show the SE of the mean.
Formation of cyclic AMP was detected in the absence of stimuli
(basal levels ) 15 ( 3 pmol cAMP/106 cells) and upon
stimulation of 100 nM NECA (stimulated levels ) 90 ( 10
pmol cAMP/106 cells).
140 (124-165)
a
Each value is the geometric mean (with 95% confidence limits
in parentheses) of at least three experiments performed in
duplicate. MRE2028F20. c MRE2029F20 MRE2030F20.
b
d
(2.16 mmol), (HOBt) 1-hydroxy-benzotrizole (2.05 mmol), and
at last the appropriate amine derivative (1.80 mmol). The
reaction mixture was stirred at room temperature for 4-5 h
with monitoring by TLC. The solvent was removed by evapo-
ration under reduced pressure, and the residue was chromato-
graphed on silica gel (ethyl acetate:petroleum ether).
Biology Exp er im en ts. CHO Mem br a n es P r ep a r a tion .
The human A1, A2A, A2B, and A3 receptors were transfected in
CHO cells according with the method described by Klotz et
al.43 The cells were grown adherently and maintained in
Dulbecco’s Modified Eagles Medium with nutrient mixture F12
(DMEM/F12) without nucleosides, containing 10% fetal calf
serum, penicillin (100 U/mL), streptomycin (100 µg/mL),
L-glutamine (2 mM), and Geneticin (G418, 0,2 mg/mL) at 37
°C in 5% CO2/95% air. CHO cells were split two or three times
weekly at a ratio between 1:5 and 1:20. For membrane
preparation, the culture medium was removed and the cells
were washed with PBS and scraped off T75 flasks in ice-cold
hypotonic buffer (5 mM Tris HCl, 2 mM EDTA, pH 7.4). The
cell suspension was homogenized with a Polytron and the
homogenate was spun for 10 min at 1000g. The supernatant
was then centrifuged for 30 min at 100 000g. The membrane
pellet was suspended in 50 mM Tris HCl buffer, pH 7.4 (for
A3 adenosine receptors, 50 mM Tris HCl, 10 mM MgCl2, 1 mM
EDTA) and incubated with 3 UI/mL of adenosine deaminase
for 30 min at 37 °C. Then the suspension was frozen at -80
°C. HEK 293 cells transfected with the human recombinant
A2B adenosine receptor were obtained from Receptor Biology,
Inc. (Beltsville, MD).
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. All synthesized compounds have been
tested for their affinity at human A1, A2A, A2B, and A3
adenosine receptors. Displacement experiments of [3H]DPCPX
to CHO cells transfected with the human recombinant A1
adenosine receptor were performed for 120 min at 25 °C in
0.2 mL of 50 mM Tris HCl buffer, pH 7.4, containing 1 nM
[3H]DPCPX, diluted membranes (50 µg of protein/assay), and
at least six to eight different concentrations of antagonists
studied. Nonspecific binding was determined in the presence
of 10 µM of CHA and this was always e10% of the total
binding.
F igu r e 3. Correlation of 23 compounds studied for affinity
and potency versus human A2B adenosine receptors (Table 9).
Comparison between affinity values (Ki) of [3H]DPCPX binding
and IC50 obtained in cAMP assays of human A2B adenosine
receptors.
Binding of [3H]ZM 241385 to CHO cells transfected with
the human recombinant A2A adenosine receptors (50 µg of
protein/assay) was performed using 0.2 mL of 50 mM Tris HCl
buffer, 10 mM MgCl2, pH 7.4, and at least six to eight different
concentrations of antagonists studied for an incubation time
of 60 min at 4 °C. Nonspecific binding was determined in the
presence of 1 µM ZM 241385 and was about 20% of total
binding.
Competition experiments of [3H]DPCPX to HEK-293 cells
transfected with the human recombinant A2B adenosine recep-
tor were performed 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 contain-
ing 40 nM [3H]DPCPX, diluted membranes (20 µg of protein/
assay), and at least six to eight different concentrations of
selected compounds. Nonspecific binding was determined in

1446 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Baraldi et al.
(2) Feoktistov, I.; Palosa, R.; Holgate, S. T.; Biaggioni, I. Adenosine
A2B receptors: A novel target in asthma. Trends Pharmacol. Sci.
1998, 19, 148-153.
the presence of 100 µM of NECA and was always e30% of the
total binding.
Binding of [3H]MRE 3008F20 to CHO cells transfected with
the human recombinant A3 adenosine receptors was previously
performed.33 Competition experiments were carried out in
duplicate in a final volume of 250 µL in test tubes containing
1 nM [3H]MRE 3008F20, 50 mM Tris HCl buffer, 10 mM
MgCl2, 1 mM EDTA, pH 7.4, 100 µL of diluted membranes
(50 µg of protein/assay), and at least six to eight different
concentrations of examined ligands for 120 min at 4 °C.
Nonspecific binding was defined as binding in the presence of
1 µM MRE 3008F20 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 (ef-
ficiency 57%) with Micro-Scint 20.
Ad en yla te Cycla se Assa y. Mea su r em en t of Cyclic AMP
Levels in CHO Cells Tr a n sfected w ith Hu m a n A2B Ad -
en osin e Recep tor s. CHO cells transfected with human A2B
adenosine receptors were washed with phosphate-buffered
saline and diluted tripsine and centrifuged for 10 min at 200g.
The pellet containing the CHO cells (1 × 106 cells/assay) was
suspended in 0.5 mL of incubation mixture [(mM) NaCl 15,
KCl 0.27, NaH2PO4 0.037, MgSO4 0.1, CaCl2 0.1, Hepes 0.01,
MgCl2 1, glucose 0.5, pH 7.4 at 37 °C], 2 IU/mL adenosine
deaminase, and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidi-
none (Ro 20-1724) as phosphodiesterase inhibitor and prein-
cubated for 10 min in a shaking bath at 37 °C. The potencies
of antagonists studied were determined by antagonism of
NECA (100 nM)-induced stimulation of cyclic AMP levels. The
reaction was terminated by the addition of cold 6% thrichlo-
roacetic acid (TCA). The TCA suspension was centrifuged at
2000g for 10 min at 4 °C and the supernatant was extracted
four times with water-saturated diethyl ether. The final
aqueous solution was tested for cyclic AMP levels by a
competition protein binding assay. Samples of cyclic AMP
standard (0-10 pmol) were added to each test tube containing
the incubation buffer (0.1 M trizma base, 8.0 mM aminophyl-
line, 6.0 mM 2 mercaptoethanol, 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, samples were centrifuged at 2000g for 10 min. The
clear supernatant was counted in a Beckman scintillation
counter.
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concentration of the radioligand and KD* is its dissociation
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Ack n ow led gm en t. We thank King Pharmaceutical
R & D, for financial support, and, in particular, Dr.
Eddie Leung for useful discussion. We also thank Prof.
Karl-Norbert Klotz for cDNA encoding the human
adenosine receptors.
Su p p or tin g In for m a tion Ava ila ble: Selected 1H NMR
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