Synthesis of theophylline derivatives and study of their activity as antagonists at adenosine receptors

Article abstract of DOI:10.1016/j.bmc.2010.02.014

The synthesis of oligo(ethylene glycol)-alkene substituted theophyllines in positions 7 and/or 8 is described. The binding activity at adenosine receptors of selected derivatives was studied. Compound 2 showed high affinity for human A_2B receptor (K_i = 4.16 nM) with a selectivity K_iA2A/K_iA2B of 24.1, and a solubility in water of 1 mM. The alkenyl substituent in some of the theophylline derivatives allows for covalent attachment of them onto hydrogen-terminated silicon substrate surfaces via hydrosilylation. Alternatively, an azido group was incorporated to an oligo(ethylene glycol)theophylline derivative as an anchor for tethering the molecules on ethynyl presenting surfaces via click reaction.

Full text of DOI:10.1016/j.bmc.2010.02.014

Bioorganic & Medicinal Chemistry 18 (2010) 2081–2088
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of theophylline derivatives and study of their activity as antagonists
at adenosine receptors
a
b
b
Jesús Hierrezuelo a, J. Manuel López-Romero a, , Rodrigo Rico , José Brea , M. Isabel Loza ,
*
Chengzhi Cai c, Manuel Algarra d
a Dept. de Química Orgánica, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain
b Dept. de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
c Department of Chemistry and Center for Materials Chemistry, University of Houston, Houston, TX 77204-5003, USA
d Centro de Geologia do Porto, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of oligo(ethylene glycol)-alkene substituted theophyllines in positions 7 and/or 8 is
described. The binding activity at adenosine receptors of selected derivatives was studied. Compound
2 showed high affinity for human A2B receptor (Ki = 4.16 nM) with a selectivity KiA2A/KiA2B of 24.1, and
a solubility in water of 1 mM. The alkenyl substituent in some of the theophylline derivatives allows
for covalent attachment of them onto hydrogen-terminated silicon substrate surfaces via hydrosilylation.
Alternatively, an azido group was incorporated to an oligo(ethylene glycol)theophylline derivative as an
anchor for tethering the molecules on ethynyl presenting surfaces via click reaction.
Received 19 November 2009
Revised 8 February 2010
Accepted 10 February 2010
Available online 15 February 2010
Keywords:
Heterocyclic compound
Schiff bases
Ó 2010 Elsevier Ltd. All rights reserved.
Azides
Caffeine derivatives
Oligo(ethylene glycol)
Silicon surface
1. Introduction
It has been found that substitution at position 8 of theophylline
(i.e., 1b, 1e) plays an important role in the antagonistic activities.5,6
The methylxanthine theophylline (1a, Fig. 1) and its derivatives
(i.e., 1b) have been extensively studied. For example, they have
been used (i.e., 1c) as templates in molecularly imprinting related
research. Due to its multivalency with various hydrogen-bonding
donor and acceptor sites, this base can be recognized by the bind-
ing sites of molecularly imprinted polymers (MIPs) with specific
structures.1 Also, theophylline and its derivatives (i.e., 1d) have
been used for the conjugation with different high-molecular
weight poly(ethylene glycol)s.2 Among the xanthine derivatives,
theophylline shows greater binding efficacy with DNA than theo-
bromine and caffeine. It has been demonstrated that theophylline
forms complexes with DNA through hydrogen bonds, while serving
as a strong antioxidant that prevents DNA damage.3
The biological effect of theophylline is associated with its affin-
ity with a family of adenosine receptors known as A1, A2A, A2B, and
A3.4 These adenosine receptors (or P1 receptors) are a class of puri-
nergic receptors, G-protein coupled receptors with adenosine as
endogenous ligand. In the last few years several groups designed
and studied the structure–activity relationship of xanthine deriva-
tives in searching for more potent and highly A2B-selective ligands.
For example, compound 1e presents an A2B Ki of 1.39 nM.7 Re-
cently, several 1,3-dialkyl deazaxanthines have been reported as
potent A2B adenosine receptor antagonists.8
Through the binding with the above adenosine receptors, the-
ophylline derivatives are well known to influence neuronal activi-
ties.9 We are interested in immobilizing them on silicon substrate
surfaces and studying their interactions with and triggered activi-
ties of neuron cells immobilized on the adjacent regions. Therefore,
in this work we also introduce handles for attaching the theophyl-
line derivatives on silicon substrate surfaces. Specifically, we incor-
porate a vinyl group to the molecules, allowing them to be
attached via hydrosilylation forming Si–C bonds with hydrogen-
terminated silicon surfaces.10,11 In addition, we also incorporate
an azido group for tethering the molecules onto alkynyl-presenting
monolayers on silicon surfaces. Furthermore, an oligo(ethylene
glycol) spacer is used to connect the theophylline moiety with
the handles. This oligo(ethylene glycol) spacer allows the theoph-
ylline to stand above the surface of a monolayer of oligo(ethylene
glycol)-terminated thin film used to render the silicon substrate
surface resistant to non-specific adsorption of proteins,12–14 which
is a key requirement for the applications. In this paper, we report
the synthesis of several theophylline-oligo(ethylene glycol) based
anchors. We also demonstrate that some of the compounds possess
* Corresponding author.
E-mail address: jmromero@uma.es (J. Manuel López-Romero).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.014

2082
J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
R4
N
1a, R1=R2= H, R3=R4= CH3, Theophylline
1b, R1= Ph, R2= H, R3=R4= CH3, 8-Phenyltheophylline
1c, R1= H, R2= CH2CH2OH, R3=R4= CH3
1d, R1= H, R2= CH2OH, R3=R4= CH3
O
N
N
R1
N
R3
R2
1
2
3
4
1e
, R = p-(C6H4)O(CH2)CONH(p-C6H4)COCH3), R = H, R =R = (CH2)2CH3
O
Figure 1. Several active theophylline derivatives.
substantial activities at adenosine receptors, and thus are ready to
be tested on silicon surfaces.
We also report the preparation of 7-substituted theophyllines
10a–c shown in Scheme 4. The derivative 10a was obtained from
the sodium salt of 1a in good yield (85%). 8-Phenyltheophylline
(1b) was prepared from 7 by construction of the imidazole ring
using benzaldehyde and DIAD as imine cyclization agent.15 How-
ever, coupling of 1b with 4 using 60% NaH as the base19 afforded
10b (<5%) in poor yields, probably due to the presence of the bulky
phenyl group at position 8 of theophylline. Finally, reaction of 11-
bromoundec-1-yl with 1a in the presence of Et3N gave compound
10c in 20% yield.20
In addition to the direct attachment of the alkenyl-substituted
theophylline derivatives on silicon surfaces via hydrosilylation,
we are also interested in tethering theophylline via click chemistry
based on Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction be-
tween azides and alkynes.21–26 Recently we modified the copper
catalysts reported by Chan et al.27 to render them water soluble,
and highly efficient for surface conjugation in aqueous solutions
and in a microarray formate.28
2. Results and discussion
2.1. Synthesis
The fact that substitution at the 8 position of theophylline,
mainly with phenyl or cycloalkyl groups,5,6 increases the activity
at adenosine receptors prompted us to the synthesis of theophyl-
lines modified at this position by introduction of a phenyl-
tetra(ethylene glycol) terminated in alkene moiety in compound
2 and a di(ethylene glycol) substituent in derivative 3 (Fig. 2).
For the synthesis of 2, starting material tosyl tetra(ethylene gly-
col) monoundec-9-enyl ether (4) has been prepared by treatment
of tetra(ethylene glycol) with 11-bromoundec-1-yl to afford 5 in
83%15 yield followed by tosylation to give 4 in 81% yield (Scheme
1).16,17
The approach based on click chemistry in microarray format is
highly efficient for optimizing the spacer and the density of the-
ophylline derivatives on silicon surfaces for interaction with neu-
rons. For this purpose, we need to introduce azido groups to the
theophylline derivatives. The synthesis of the theophylline deriva-
tive 10e with an azido handle is outlined in Scheme 5. Treatment of
1a with tetra(ethylene glycol)ditosylate in the presence of 60% NaH
afforded the 7-substituted theophylline 10d in 50% yield. This
product was converted to the azide 10e in 33% yield by treatment
with excess of NaN3 in anhydrous DMF.
The preparation of 2 has been accomplished as shown in
Scheme 2 with a good overall yield (20%). Condensation of the
aldehyde 6 with 4,5-diamino-1,3-dimethyl uracil (7) gave the
imine 8 which was oxidatively cyclized by treatment with diiso-
propyl azodicarboxylate (DIAD) instead of DEAD as reported in
the literature for similar compounds,18 to give the xanthine 2 in
77% yield (Scheme 2).
Compound 3 was synthesized in two steps. First, amide 9 was
obtained by treating the diaminouracil 7 with 2-[(2-(2-methoxy-
ethoxy)ethoxy]acetic acid in the presence of N0-(3-dimethylamino-
propyl)-N-ethylcarbodiimide hydrochloride (EDC), and used in the
next step without purification. Second, the 8-diethylene glycol
derivative of theophylline 3 was obtained by ring closure reaction
of the amide 9 with sodium methoxide in refluxing methanol in
52% yield (Scheme 3).9
2.2. Binding assays
We report here the study on radioligand binding at adenosine
receptors of some of the synthesized theophylline derivatives.
Compounds 2, 3, 10a and 10e were assayed at human recombinant
Me
Me
O
N
O
N
O
Me
N
N
O
O
O
9
N
N
2
4
N
H
N
H
Me
Me
O
O
2
3
Figure 2. 8-Substituted theophylline derivatives.
(i)
(ii)
Ts
O
O
H
O
O
5
H
O
O
H
9
9
4
4
4
4
(i) 1) 50% NaOH, 100
°C, 30 min; 2) Br
, 100°C, 2h. (ii) TsCl, DMAP/Et3N, CH2Cl2, 20°C, 12h.
9
Scheme 1. Preparation of the tetra(ethylene glycol) derivative 4.

J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
2083
(i)
OHC
OH
OHC
O
O
9
4
6
Me
N
O
NH2
NH2
Me
N
O
N
NH2
N
Me
(iii)
O
7
2
N
Me
(ii)
O
O
O
9
4
8
(i) 4, K2CO3/CH3CN, reflux, 1h. (ii) 7, EtOH/AcOH, reflux, 12h. (iii) DIAD, DME, reflux, 12h.
Scheme 2. Synthesis of the 8-substituted theophylline derivative 2.
Me
N
O
O Me
2
HO
O
NH2
O
NaMeO
7
3
O
O Me
2
N
MeOH
Reflux, 4h
EDC, MeOH
20°C, 24h
Me
N
H
O
O
9
Scheme 3. Synthesis of the theophylline-di(ethylene glycol) derivative 3.
Me
N
activity analysis, since compounds 2 and 3 present an ethylene gly-
col derivative substituent at position 8 of theophylline, while com-
pounds 10a and 10e present the substitution over the N7 of the
base. Radioligand assays were performed in vitro at such receptors
by measuring the percent inhibition of specific binding at a single
Me
N
O
O
N
N
N
(i)
R1
R1
N
N
N
H
Me
Me
R2
O
concentration (10 lM) and for compounds showing percentage
O
values over 50% concentration–response curves in the range of
0.1 nM–1 mM for the affinity (Ki) calculation (Fig. 3).
The affinity results are summarized in Table 1. Compounds 3
and 10e show percent inhibition values around or below 50% at
10 lM. We may therefore assume that these compounds have a
low affinity for these receptors. However, compound 10a shows
some affinity for A1 receptors and A2B, for which the Ki could be cal-
culated, and especially compound 2 shows a high affinity and
selectivity for A2B receptors, presenting a Ki in the nanomolar scale
(4.16 nM) and it can be defined as a high affinity and selective A2B
receptor ligand. Selectivity of 2 between A2A and A2B receptors
1a, R1= H
1b, R1= Ph
1
2
1a, 60% NaH, 4, DMF.
1b, 60% NaH, 4, DMF.
10a
(i)
, R = H, R =
O
9
4
(i)
10b, R1= Ph,R2=
10c, R1=H, R2=
, CH2Cl2.
O
9
4
(i) 1a, Et3N, Br
9
9
Scheme 4. Substitution at the theophylline N7.
reach the ratio 24.1 for KiA2A/KiA2B
.
As it can be seen in Table 1, low affinity for A3 receptors has
been found for these compounds, which do not present substitu-
tion at 1 and 3 positions of the base. This result was expected, since
A1, A2A, A2B and A3 receptors as described in the experimental sec-
tion.29 We choose these compounds for comparative structure–
Me
Me
O
N
O
N
O
N
N
(i)
N
N
(ii)
Ts
Ts
O
O
Ts
N
N
4
Me
Me
O
O
O
N3
4
3
10e
10d
(i) 1) 1a, 60% NaH, DMF; 2) Tetra(ethylene glycol)ditosylate , DMF, 20°C, 12h. (ii) NaN3, DMF, 60°C, 12h.
Scheme 5. Preparation of the azide derivative 10e.

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J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
A
B
150
100
50
150
100
50
0
0
-11 -10
-9
-8
-7
-6
-5
-4
-11 -10
-9
-8
-7
-6
-5
-4
log [compound 2] (M)
log [compound 2] (M)
C
150
100
50
0
-10 -9
-8
-7
-6
-5
-4
-3
-2
log [compound 10a] (M)
Figure 3. Binding competition experiments at cloned hA1 hA2A and hA2B receptors. (A) Concentration–response curve for compound 2 at hA2A receptors labeled with 3 nM
[3H]ZM241385. (B) Concentration–response curve for compound 2 at hA2B receptors labeled with 25 nM [3H]DPCPX. (C) Concentration–response curve for compound 10a at
hA1 receptors labeled with 2 nM [3H]DPCPX. Values represent the mean sem (vertical bars) of three independent experiments.
Table 1
Affinity (Ki and percent displacement of specific binding) at A1, A2A, A2B and A3 receptors exhibited by the theophylline derivatives
Compound
A1
Ki (nM)
A2A
Ki (nM)
A2B
Ki (nM)
A3
Ki (nM)
% (10
—
l
M)
% (10
l
M)
% (10
l
M)
% (10
—
lM)
1a
2
3
10a
10e
(10.1 2.1) 103
—
—
—
(18.5 0.1) 103
100.4 6.5
—
—
—
—
—
(2.7 0.2) 103
4.16 0.71
—
—
—
(85.1 1.5) 103
38
37
53
24
1
5
4
3
7
1
3
2
—
—
—
—
—
20
18
17
2
2
3
10
47
3
3
3
1
(5.0 0.7) 103
—
19
2
5
1
Values show the mean sem of three independent assays with duplicate determinations.
the 1,3 unsubstituted caffeine or theophylline derivatives exhibit
dramatically reduced affinity for the A3 receptor in comparison,
for example, with 1,3-dipropyl substituted derivatives.30
A
2B-selective ligands, and in the last few years some very potent
and highly A2B-selective xanthines have been discovered.7 The
affinities of these compounds are similar to that of the xanthine
derivative 2.
Chemical structure of compound 2 presents a para-3,6,9,12-tet-
raoxatricos-22-enyloxy substituted phenyl ring at position 8 of
theophylline. The presence of this phenyl ring seems to be deter-
minant for the affinity of this compound over A2A and even more
over A2B receptors since the homologue compound 3, lacking this
ring, showed no affinity at those receptor subtypes. Structure–
activity relationship (SAR) regarding the substitution at position
8 of theophylline derivatives and other xanthine bases has been
extensively studied.5,6,31,32 These studies show that the presence
of a phenyl or cycloalkyl group at this position of theophylline in-
creases the relative affinity at adenosine receptors, and mainly at
the A2B receptor.31
On the other hand, the presence of substituents at position N7
of the base in compounds 10a and 10e results in the reduction of
its affinity for the receptors in comparison to that of 2. This fact
can be attributed to the hypothesized activity of this nitrogen atom
as a hydrogen-bond acceptor at the binding pocket, which it is not
allowed in tertiary N7.5 However, compound 10a still shows higher
affinity at A2B and A1 receptors than 3 and 10e (Table 1). This may
be related to a favourable distal electronic interaction with the
receptor due to the combination in 10a of a long tetra(ethylene
glycol) chain with a terminal electron-rich alkene group, similar
to that in 2.6,32
Theophylline and 3-N-(propyl)xanthine showed to be selective,
albeit weak, antagonists at the A2B adenosine receptors.33 These
findings prompted several groups to design and test a large num-
ber of xanthine derivatives in search for new, more potent and
Finally, solubility in water and hydrophilic solvents is necessary
for biological applications of active compounds. This physicochem-
ical property has been a major issue with xanthine and non-xan-
thine antagonists of adenosine receptors, since the 8-phenyl

J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
2085
substitution in the xanthine pharmacophore increases receptor
blocking activity, it also markedly decreases solubility.34 In order
to analyze this point, we measured the solubility of the oligo(eth-
ylene glycol)-substituted theophyllines, compounds 2 and 3, in
water, di(ethylene glycol) and glycerin. While the solubility of both
compounds in glycerin is poor, in water and di(ethylene glycol) is
remarkably better to that of theophylline (1a) and 8-phenylthe-
ophylline (1b). For instance, while 1a and 1b are not soluble in
di(ethylene glycol), derivatives 2 and 3 displayed a solubility of
0.1 mM and 10 mM, respectively, in this solvent. Compound 3
reach 14 mM in water. In this sense, it is important to remark that
the presence of the oligo(ethylene glycol) chain in compounds 2
and 3 increase the water solubility, and consequently the ratio sol-
ubility to receptor affinity.34
cooled to 20 °C and extracted with cyclohexane (3 ꢀ 20 mL), the or-
ganic phase was washed with brine, dried over MgSO4 and concen-
trated to dryness. The residue was purified by column
chromatography (cc) eluting with EtOAc/cyclohexane (1:1) to ob-
tain 5 as a yellowishliquid (3.45 g, 83%);15 1H NMR (400 MHz, CDCl3)
d: 5.83–5.73 (m, 1H; @CH), 4.98–4.88 (m, 2H; @CH2), 3.69 (d,
J = 4.4 Hz, 2H; CH2OH), 3.64–3.54 (m, 14H; 7 ꢀ OCH2), 3.41 (t,
J = 6.8 Hz, 2H; CH2), 2.01–1.99 (m, 2H; @CH–CH2), 1.56–1.53 (m,
2H; CH2), 1.33–1.21 (m, 12H; 6 ꢀ CH2); 13C NMR (100 MHz, CDCl3)
d: 139.1 ppm (@CH), 114.0 (@CH2), 72.5, 71.4, 70.46, 70.44, 70.42,
70.39, 70.1, 69.9, 61.57, 33.7, 29.42, 29.40, 29.31, 29.29, 29.0, 28.8,
25.9; IR (KBr) m .
: 3466, 2923, 2854, 1104, 909 cmꢁ1
4.2.2. 3,6,9,12-Tetraoxatricos-22-enyl 4-tosylate (4)
Under argon atmosphere, over a cooled (0 °C) solution of 5
(2.93 g, 8.47 mmol), Et3N (5.9 mL, 42.4 mmol) and DMAP (15 mg)
in dry CH2Cl2 (80 mL) was added a solution of tosyl chloride
(3.38 g, 17.8 mmol) dry CH2Cl2 (20 mL). The reaction mixture was
stirred at 20 °C for 12 h. After this period, the mixture was poured
over ice-water (20 mL) and then extracted with CH2Cl2
(3 ꢀ 20 mL). The organic solution was washed with 10% NH4Cl
(20 mL), brine (20 mL), dried over MgSO4 and concentrated to dry-
ness. The residue was separated by cc eluting with EtOAc/cyclo-
hexane 1:1) to afford compound 4 as a yellowish solid (3.44 g,
81%); mp 42–43 °C; 1H NMR (400 MHz, CDCl3) d: 7.77 ppm (d,
J = 8.4 Hz, 2H; 2 ꢀ ArH), 7.32 (d, J = 8 Hz, 2H; 2 ꢀ ArH), 5.84–5.73
(m, 1H; @CH), 4.99–4.89 (m, 1H; @CH2), 4.15 (dd, J = 5.2, 6 Hz,
2H; CH2OTs), 3.66 (t, J = 4.8 Hz, 2H; CH2), 3.61–3.54 (m, 12H;
12 ꢀ OCH2), 3.41 (t, J = 6.8 Hz, 2H; OCH2), 2.42 (s, 3H; CH3), 2.02
(m, 2H; CH2), 1.54 (t, J = 7.2 Hz, 2H; CH2), 1.36–1.25 (m, 12H;
6 ꢀ CH2); 13C NMR (100 MHz, CDCl3) d: 144.7 ppm (C), 139.2
(@CH), 132.9 (C), 129.8 (2 ꢀ CH), 127.9 (2 ꢀ CH), 114.0 (@CH2),
71.5, 70.7, 70.6, 70.51, 70.46, 70.0, 69.2, 68.6, 33.7, 29.6, 29.5,
3. Conclusions
In summary, we have designed and synthesized two kinds of
theophylline derivatives with a handle (vinyl or azido group) and
an oligo(ethylene glycol) spacer for immobilization onto silicon
substrate surfaces. The method for the synthesis of theophylline-
oligo(ethylene glycol)-alkene derivatives is versatile, allowing for
efficient preparation of the 7 and 8 substituted derivatives. We
have also identified the theophylline derivative
2 with an
oligo(ethylene glycol) side chain as adenosine antagonist which
is potent and highly selective antagonist for human A2B receptors,
and also more soluble than the primary pharmacophore 8-phenyl-
theophylline. Unfortunately, the azido-modified theophylline
derivative 10e exhibited a low affinity to the receptors. However,
we expect that introducing a phenylene unit between the ethylene
glycol spacer and the theophylline, similar to 2, should greatly im-
prove the binding affinity. We are currently pursuing the synthesis
of these compounds. The attachment of the alkenyl- and azido-
substituted theophylline derivatives to silicon substrate surfaces
at various densities and the study of the response of neuron cells
to the immobilized neurotransmitters that are relevant to the field
of machine brain interface35 will be reported in due course.
29.41, 29.37, 29.1, 28.9, 26.8, 21.6, 26.0 (CH3); IR (KBr)
m:
2924 cmꢁ1, 2854, 1357, 1176, 1097, 913; MS (EI, 70 eV) m/z: 500
(1) [M+], 287 (7), 243 (9), 199 (100), 155 (22), 91 (17); HRMS m/
z: 500.2808 (calcd for C26H44O7S: 500.2808).
4.2.3. 4-(3,6,9,12-Tetraoxatricos-22-enyloxy)benzaldehyde (6)
A solution of p-hydroxybenzaldehyde (488 mg, 4 mmol), K2CO3
(553 mg, 4 mmol) and 4 (2.0 g, 4 mmol) in acetonitrile (30 mL) was
refluxed under stirring for 1 h. After this period, the reaction mix-
ture was cooled and 5% HCl (5 mL), followed by EtOAc (20 mL)
were added. The organic phase was washed with water, dried over
Na2SO4 and concentrated under vacuum to dryness. The residue
was separated by cc eluting with cyclohexane/EtOAc 5:5 to obtain
6 as a yellowish syrup (1.06 g, 59%); 1H NMR (400 MHz, CDCl3) d:
9.86 ppm (s, 1H; CHO), 7.81 (d, J = 9.2 Hz, 2H; 2 ꢀ ArH), 7.00 (d,
J = 8.8 Hz, 2H; 2 ꢀ ArH), 5.82–5.75 (m, 1H; @CH), 4.99–4.89 (m,
2H; @CH2), 4.19 (t, J = 4.8 Hz, 2H; OCH2), 3.87 (t, 2H, J = 4.8 Hz,
OCH2), 3.72–3.40 (m, 12H; OCH2), 3.41 (t, J = 6.4 Hz, 2H; OCH2),
2.01 (dd, J = 6.8, 14 Hz, 2H; CH2), 1.54 (q, J = 6.8 Hz, 2H; CH2),
1.39–1.15 (m, 12H; 6 ꢀ CH2); 13C NMR (100 MHz, CDCl3) d:
190.8 ppm (C@O), 163.8 (C), 139.2 (@CH), 131.9 (2 ꢀ CH), 129.8
(C), 114.8 (2 ꢀ CH), 114.1 (@CH2), 71.5, 70.9, 70.6, 70.5, 70.0,
69.4, 69.2, 68.6, 67.7 (OCH2), 33.8, 29.6, 29.5, 29.43, 29.39, 29.1,
4. Experimental
4.1. General
Melting points were determined with a Gallenkamp instrument
and are given uncorrected. UV spectra were recorded with a Hew-
lett–Packard8452Aspectrophotometer, andIRspectrawithBeckman
Aculab IV and Perkin–Elmer 883 spectrophotometers. Mass spec-
trometry was carried out with a Thermo Finnigan instrument, using
the direct injection and electron-impact (EI) modes. HRMS were re-
corded with a Micromass (Autospec-Q) spectrometer. 1H NMR and
13C NMR spectra were recorded on 400 MHz ARX 400 Bruker spec-
trometer, using the residual solvent peak in CDCl3 (dH 7.24 ppm for
1H and dC 77.0 ppm for 13C), or CD3SOCD3 (dH 2.50 ppm for 1H). Chem-
ical shifts are given in ppm, and J values in hertz. TLC analyses were
performed on Merck Silica Gel 60 F 254 plates, and column chroma-
tography on Silica Gel 60 (0.040–0.063 mm).
28.9, 26.0; IR (KBr) m
: 2924 cmꢁ1, 2854, 1687, 1602, 1584, 1286,
4.2. Synthesis of 8-(4-(3,6,9,12-tetraoxatricos-22-
enyloxy)phenyl)-theophylline (2)
1155, 1099, 836; UV (MeOH) kmax (log e): 284 nm (3.97), 222
(3.43), 204 (2.11); MS (EI, 70 eV) m/z (%): 450 (6) [M+], 254 (17),
224 (30), 210 (23), 192 (8), 148 (100), 121 (38), 97 (22), 83 (32);
HRMS m/z: 450.2988 (calcd for C26H42O6: 450.2981).
4.2.1. 3,6,9,12-Tetraoxatricos-22-en-1-ol (5)
Under an Ar atmosphere, a mixture of 50% sodium hydroxide
(1 mL) and tetra(ethylene glycol) (20.8 mL, 120 mmol) was stirred
at 100 °C for 30 min. After this period, 11-bromoundec-1-yl
(2.62 mL, 12.0 mmol) was added. The reaction was followed by tlc
(EtOAc/cyclohexane 1:1) to completion. The reaction mixture was
4.2.4. 5-(4-(3,6,9,12-Tetraoxatricos-22-enyloxy)benzylideneami
no)-6-amino-1,3-dimethyluracil (8)
Under an Ar atmosphere, a mixture of 6 (1.06 g, 2.4 mmol), 4,5-
diamino-1,3-dimethyl uracil (7, 0.36 g, 2.1 mmol) and acetic acid

2086
J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
(0.1 mL) in ethanol (15 mL) was refluxed for 12 h. After this period,
the reaction mixture was cooled at 20 °C, CH2Cl2 (20 mL) was
added, and the solution was washed with water (20 mL) and brine
(15 mL), dried over MgSO4 and the solvent eliminated in vacuum.
The crude of reaction was separated by cc eluting with EtOAc to
obtain 8 as a yellowish syrup (0.55 g, 43%); 1H NMR (400 MHz,
CDCl3) d: 9.73 ppm (s, 1H; C@NH), 7.69 (d, J = 8.6 Hz, 2H; 2 ꢀ ArH),
6.92 (d, J = 8.6 Hz, 2H; 2 ꢀ ArH), 5.82–5.74 (m, 1H; @CH), 4.99–
4.89 (m, 2H; @CH2), 4.15 (t, J = 4.8 Hz, 2H; OCH2), 3.85 (t,
J = 4.8 Hz, 2H; OCH2), 3.72–3.55 (m, 12H; 6 ꢀ OCH2), 3.52 (s, 3H;
NCH3), 3.43 (t, J = 6.8 Hz, 2H; OCH2), 3.37 (s, 3H; NCH3), 2.03 (q,
J = 7.2 Hz, 2H; CH2), 1.55 (m, 2H; CH2), 1.38 (m, 12H; 6 ꢀ CH2);
13C NMR (100 MHz, CDCl3) d: 172.4 ppm (C@N), 160.2 (C), 158.2
(C@O), 152.9, 151.8 (C@O), 150.4 (@CNH2), 139.2 (@CH), 131.4
(C), 128.8 (2 ꢀ CH), 114.6 (2 ꢀ CH), 114.1 (@CH2), 71.5, 70.8, 70.6
(3 ꢀ CH2), 70.5, 70.0, 69.6, 67.4, 33.8, 29.6, 29.5, 29.44, 29.40,
and water (5 mL) was added. The acidity was adjusted to acidic
pH with 6 M HCl. The solid was filtered, the solution was concen-
trated to dryness under vacuum, and redissolved in CHCl3 (40 mL).
The organic solution was dried over MgSO4 and concentrated to
dryness. The solid residue was separated by cc eluting with
CH2Cl2/MeOH 10:0.1 to obtain 3 as a yellowish solid (624 mg,
52%), mp 95–97 °C; 1H NMR (400 MHz, CDCl3) d: 3.76–3.72 ppm
(m, 10H; 5 ꢀ OCH2), 3.56 (s, 3H; OCH3), 3.45 (s, 3H; NCH3), 3.40
(s, 3H; NCH3); 13C NMR (100 MHz, CDCl3) d: 154.8 ppm (C@O),
151.9 (C@O), 151.0 (C-4), 148.8 (C-8), 107.0 (C-5), 71.7, 70.7,
70.6, 70.5, 66.4, 58.9, 30.0 (NCH3), 28.1 (NCH3); IR (KBr)
m:
3148 cmꢁ1, 1702, 1649, 1104, 993, 746; UV (MeOH) kmax (log
e):
274 nm (1.82), 208 (3.81); MS (EI, 70 eV) m/z (%): 312 (18) [M+],
209 (100), 193 (30); HRMS m/z: 312.1434 (calcd for C13H20N4O5:
312.1434).
29.1, 28.9, 27.6, 26.0; IR (KBr)
m
: 3164 cmꢁ1, 2923, 2854, 1688,
): 282 nm (3.48), 204 (3.62);
4.4. Synthesis of 7-(3,6,9,12-tetraoxatricos-22-
enyl)theophylline (10a)
1649, 1106; UV (MeOH) kmax (log
e
MS (EI, 70 eV) m/z (%): 603 (2) [M+], 450 (12), 407 (7), 254 (20),
224 (39), 210 (27), 148 (100), 121 (28), 89 (31), 83 (40); HRMS
m/z: 602.3672 (calcd for C35H50N4O7: 602.3679).
60% NaH (84 mg, 2.1 mmol) was added over a solution of the-
ophylline (1a, 342 mg, 1.9 mmol) in DMF (20 mL). After H2 evolu-
tion ceased, a solution of 4 (1.90 g 3.8 mmol) in DMF (15 mL)
was added, and the mixture of reaction was stirred at 20 °C for
12 h. After this period, the reaction was diluted with CH2Cl2
(40 mL), washed with water (3 ꢀ 30 mL) and brine (2 ꢀ 50 mL).
The organic phase was dried over MgSO4 and concentrated to dry-
ness under vacuum. Compound 10a was isolated by cc eluting with
CH2Cl2/methanol 99:1 as a yellowish syrup (820 mg, 85%); 1H NMR
(400 MHz, CDCl3) d: 7.70 ppm (s, 1H; H-8), 5.81–5.74 (m, 1H;
@CH), 4.98–4.88 (m, 2H; @CH2), 4.74 (t, J = 4.6 Hz, 2H; OCH2),
3.78 (t, J = 5 Hz, 2H; OCH2), 3.62–3.53 (m, 12H; 6 ꢀ CH2), 3.57 (s,
3H; NCH3), 3.41 (t, J = 6.8 Hz, 2H; OCH2), 3.37 (s, 3H; NCH3), 2.01
(q, J = 6.8, 6.8 Hz, 2H; CH2), 1.53 (m, 2H; CH2), 1.29–1.24 (m,
12H; 6 ꢀ CH2); 13C NMR (100 MHz, CDCl3) d: 152.0 ppm (C@O),
148.8 (C@O), 142.6 (C-8), 140.1 (C), 139.2 (@CH), 114.1 (@CH2),
106.7 (C), 71.5, 70.59 (2 ꢀ CH2), 70.57, 70.54, 70.4, 70.0, 69.4,
46.8, 33.8, 29.7, 29.6, 29.5, 29.43, 29.40, 29.1, 28.9, 27.9, 26.0; IR
4.2.5. 8-(4-(3,6,9,12-Tetraoxatricos-22-enyloxy)phenyl)-
theophylline (2)
Imine 8 (0.50 g, 0.83 mmol), was dissolved by refluxing in DME
(10 mL). Then, DIAD (0.21 mL, 1.1 mmol) was added, and the mix-
ture refluxed for 24 h. Then the mixture was cooled at 20 °C and
the product 2 was obtained by crystallization from the reaction
crude by addition of ethanol (5 mL) as a white solid (0.38 g, 77%),
mp 195–196 °C; 1H NMR (400 MHz, CDCl3) d: 8.18 ppm (d,
J = 8.8 Hz, 2H; 2 ꢀ ArH), 6.97 (d, J = 8.8 Hz, 2H; 2 ꢀ ArH), 5.81–
5.73 (m, 1H; @CH), 4.98–4.88 (m, 2H; @CH2), 4.18 (t, J = 4.8 Hz,
2H; OCH2), 3.88 (t, J = 4.6 Hz, 2H; OCH2), 3.68 (s, 3H; NCH3),
3.73–3.52 (m, 12H; 6 ꢀ OCH2), 3.50 (s, 3H; NCH3), 3.42 (t,
J = 6.8 Hz, 2H; OCH2), 2.00 (q, J = 7.2 Hz, 2H; CH2), 1.54 (m, 2H;
CH2), 1.25 (m, 12H; 6 ꢀ CH2); 13C NMR (100 MHz, CDCl3) d:
160.8 ppm (C), 155.5 (C@N), 151.8 (C-4), 151.5 (C@O), 149.9
(C@O), 139.2 (@CH), 128.5 (2 ꢀ CH), 121.4 (C), 114.8 (2 ꢀ CH),
114.0 (@CH2), 107.4 (C-5), 71.5, 70.9, 70.61 (2 ꢀ OCH2), 70.59,
70.56, 70.0, 69.6, 67.5, 33.8, 30.2, 29.6, 29.5, 29.43, 29.40, 29.1,
(KBr)
(log
m
: 2924 cmꢁ1, 2855, 1703, 1661, 1108; UV (MeOH) kmax
e
): 274 nm (1.25), 208 (3.98); MS (EI, 70 eV) m/z (%): 508
(14) [M+], 312 (42), 282 (17), 250 (45), 224 (52), 207 (100), 180
(95); HRMS m/z: 508.2818 (calcd for C26H44N4O6: 508.2808).
28.9, 28.4, 26.0; IR (KBr)
m
: 3163 cmꢁ1, 2924, 2854, 1687, 1646,
): 316 nm (3.20), 250
1483, 1247, 1107; UV (MeOH) kmax (log
e
(2.04); MS (EI, 70 eV) m/z (%): 599 (2) [M+], 355, 338, 323, 281,
238, 207; HRMS m/z: 623.3420 (calcd for C32H48N4NaO7:
623.3421).
4.5. Synthesis of 8-phenyl-7-(3,6,9,12-tetraoxatricos-22-
enyl)theophylline (10b)
4.5.1. 8-Phenyltheophylline (1b)
4.3. Synthesis of 8-((2-(2-methoxyethoxy)ethoxy)methyl)theo
phylline (3)
Under an Ar atmosphere, a mixture of benzaldehyde (2.57 g,
24.2 mmol), 4,5-diamino-1,3-dimethyl uracil (7, 3.74 g, 22.0 mmol)
and acetic acid (1 mL) in ethanol (60 mL) was refluxed for 12 h. After
this period, the reaction mixture was cooled at 20 °C and a yellow so-
lid appeared (needles). The solid was filtered off, washed with etha-
nol (2 ꢀ 5 mL) and ethyl ether (2 ꢀ 5 mL), and identified as 6-amino-
5-(benzylideneamino)-1,3-dimethyluracil (4.61 g, 81%),18 which
was used in the next step without more purification; 1H NMR
(400 MHz, CDCl3) d: 9.78 ppm (s, 1H; N@CH imine), 7.75 (d,
J = 6.4 Hz, 2H; ArH), 7.38–7.36 (m, 3H; ArH), 5.71 (br s, 2H; NH2),
3.50 (s, 3H; NCH3), 3.37 (s, 3H; NCH3); MS (EI, 70 eV) m/z (%): 258
(100) [M+], 181 (17), 155 (32). Imine (1.24 g, 4.81 mmol) was dis-
solved by refluxing in DME (20 mL). Then, DIAD (1.24 mL,
6.25 mmol) was added, and after 5 min a white solid appeared.
The reaction mixture was refluxed for 25 min more. After cooling
at room temperature the solid was filtered and washed with cold
EtOH (2 ꢀ 5 mL) and ethyl ether (2 ꢀ 5 mL) to obtain 1b as a white
solid (1.09 g, 82%);14 1H NMR (400 MHz, DMSO-d6) d: 8.14 ppm (d,
J = 6.4 Hz, 2H; ArH), 7.52–7.50 (m, 3H; ArH), 3.51 (s, 3H; NCH3),
3.27 (s, 3H; NCH3); MS (EI, 70 eV) m/z (%): 256 (100) [M+].
4.3.1. N-(6-Amino-1,3-dimethyl-uracil-5-yl)-2-(2-(2-
methoxyethoxy)ethoxy)acetamide (9)
Over
a suspension of 4,5-diamino-1,3-dimethyl uracil (7,
715 mg, 4.2 mmol) in methanol (30 mL) was added 2-[2-(2-
methoxyethoxy)ethoxy]acetic acid (0.69 mL, 4.5 mmol). The mix-
ture of reaction was stirred at 20 °C for 30 min, and then N0-(3-
dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC,
0.9 g, 4.7 mmol) was added and the mixture was stirred at 20 °C
for 24 h. After this period, the solvent was removed under vacuum
to obtain 9 (2.95 g, orange syrup). This compound was used in the
next reaction without further purification; MS (EI, 70 eV) m/z (%):
330 (64) [M+], 227 (30), 197 (100), 169 (85).
4.3.2. 8-((2-(2-Methoxyethoxy)ethoxy)methyl)theophylline (3)
A freshly prepared methanolic solution of MeONa in MeOH (Na
3.41 g, 148.1 mmol) was added to the oily amide 9. The reaction
mixture was kept at reflux for 4 h. It was then cooled to 40 °C,

J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
2087
4.5.2. 8-Phenyl-7-(3,6,9,12-tetraoxatricos-22-enyl)theophylline
(10b)
274 nm (1.48), 208 (3.95); HRMS m/z: 533.1700 (calcd for
22H30N4NaO8S: 533.1682).
C
The reaction was carried out as above for 10a by using 1b
(0.90 g, 3.5 mmol), 60% NaH (0.16 mg, 3.9 mmol),
4
(5.27 g,
4.7.2. 7-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)theophyl
line (10e)
10.6 mmol) and DMF (70 mL). The reaction crude was purified by
cc, eluting with CH2Cl2/methanol 99:1 to afford 10b as a yellowish
syrup (108.4 mg, 5%); 1H NMR (400 MHz, CDCl3) d: 7.83–7.80 ppm
(m, 2H; ArH), 7.49–7.47 (m, 3H; ArH), 5.83–5.73 (m, 1H; @CH),
4.99–4.89 (m, 2H; @CH2), 4.47 (t, J = 5.2 Hz, 2H; OCH2), 3.92 (t,
J = 5.2 Hz, 2H; OCH2), 3.62 (s, 3H; NCH3), 3.64–3.50 (m, 12H;
OCH2), 3.42 (s, 3H; NCH3), 3.40 (t, J = 6.8 Hz, 2H; OCH2), 2.01 (q,
J = 7.2 Hz, 2H; CH2), 1.55–1.52 (m, 2H; CH2), 1.36–1.24 (m, 12H;
6 ꢀ CH2); 13C NMR (100 MHz, CDCl3) d: 155.2 ppm (C@N), 153.3
(C@O), 151.7 (C@O), 148.7 (C-4), 139.2 (@CH), 130.2 (CH), 129.9
(2 ꢀ CH), 128.7 (2 ꢀ CH), 128.6 (C), 114.1 (@CH2), 107.6 (C), 71.4,
70.55, 70.54, 70.3, 70.1, 70.0, 69.9, 46.3, 33.7, 29.8, 29.55, 29.47,
A solution of 10d (1.4 g, 2.75 mmol), NaN3 (0.218 g, 3.4 mmol)
in DMF (30 mL) was stirred at 60 °C for 12 h. After this period,
the reaction mixture concentrated to dryness under vacuum, and
then CH2Cl2 (100 mL) was added. The solution was washed with
water (3 ꢀ 100 mL) and brine (100 mL), dried over MgSO4, filtered
and the solvent removed in vacuum. The residue was purified by cc
eluting with EtOAc, to obtain 10e as a yellowish syrup (0.34 g,
33%); 1H NMR (400 MHz, CDCl3) d: 7.69 ppm (s, 1H; H-8), 4.49–
3.58 (m, 16H; 8 ꢀ OCH2), 3.62 (s, 3H; NCH3), 3.39 (s, 3H; NCH3);
13C NMR (100 MHz, CDCl3) d: 155.4 ppm (C@O), 151.7 (C@O),
148.8, 142.5 (C-8), 106.4 (C-5), 70.7, 70.6, 70.53, 70.49, 70.0, 69.4
(6 ꢀ CH2), 50.6 (CH2N), 46.8 (CH2N3), 29.8 (NCH3), 27.9 (NCH3);
29.40, 29.37, 29.1, 28.0, 26.0 (CH2, NCH3); IR (KBr)
m
: 2924 cmꢁ1
): 292 nm (2.64),
,
2854, 1700, 1660, 1106; UV (MeOH) kmax (log
e
IR (KBr) m e):
: 2869 cmꢁ1, 2099, 1701, 1654; UV (MeOH) kmax (log
228 (3.91), 208 (2.01); MS (EI, 70 eV) m/z (%): 585 (0.1) [M+], 583
(0.5), 283 (15), 256 (38), 83 (79), 69 (85), 55 (100); HRMS m/z:
607.3476 (calcd for C32H48N4O6Na: 607.3472).
272 (4.26), 224 nm (3.88); MS (EI, 70 eV) m/z (%): 381 (11) [M+],
339 (60), 160 (100); HRMS m/z: 404.1667 (calcd for C15H23N7NaO5:
404.1658).
4.8. Radioligand binding competition assays
4.6. Synthesis of 7-(undec-10-enyl)theophylline (10c)
4.8.1. Human A1 receptors
A solution of 1a (1.80 g, 10 mmol), Et3N (1.4 mL, 10 mmol) in
dry CH2Cl2 (20 mL) was stirred under argon atmosphere at 20 °C
for 20 min. Then 11-bromoundec-1-yl (2.4 mL, 11 mmol) was
added dropwise. The mixture was refluxed for 4 h. After this peri-
od, dichloromethane (40 mL) was added, and the solution was
washed successively with 1 M HCl (5 mL), satd NaHCO3 (5 mL)
and water (3 ꢀ 5 mL). Then the solution was dried over MgSO4, fil-
tered and the solvent eliminated in vacuum. The crude residue was
separated by cc eluting with cyclohexane/CH2Cl2 6:4 and then
CH2Cl2/MeOH 9.5:0.5, to obtain 10c as a white solid (310 mg,
20%), mp 50–52 °C; 1H NMR (400 MHz, CDCl3) d: 7.50 ppm (s,
1H; H-8), 5.82–5.72 (m, 1H; @CH), 4.98–4.88 (m, 2H; @CH2),
4.25 (t, J = 7.0 Hz, 2H; NCH2), 3.56 (s, 3H; NCH3), 3.38 (s, 3H;
NCH3), 2.00 (q, J = 6.4, 6.4 Hz, 2H; CH2), 1.84 (t, J = 6.4 Hz, 2H,
CH2), 1.28–1.23 (m, 12H; 6 ꢀ CH2); 13C NMR (100 MHz, CDCl3) d:
154.8 ppm (C@O), 151.8 (C@O), 151.0 (C-8), 148.6 (C-4), 139.2
(@CH), 114.1 (@CH2), 107.0 (C-5), 71.7, 70.66, 70.61, 70.5, 66.4,
Adenosine A1 receptor competition binding experiments were
carried out in membranes from CHO-A1 cells (Euroscreen, Goss-
elies, Belgium). On the day of assay, membranes were defrosted
and re-suspended in incubation buffer 20 mM Hepes, 100 mM
NaCl, 10 mM MgCl2, 2 units/mL adenosine deaminase (pH 7.4).
Each reaction well of a GF/C multiscreen plate (Millipore, Ma-
drid, Spain), prepared in duplicate, contained 15
2 nM [3H]DPCPX and test compound. Non-specific binding was
determined in the presence of 10 M (R)-PIA. The reaction mix-
ture was incubated at 25 °C for 60 min, after which samples
were filtered and measured in a microplate beta scintillation
counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain). A con-
centration–response curve of xanthin amino congener (XAC)
was carried out in all the assays as an internal control
(Ki = 23.2 3 nM)
lg of protein,
l
4.8.2. Human A2A receptors
58.9, 30.0 (NCH3), 28.1 (NCH3); IR (KBr)
m
: 3100 cmꢁ1, 2919,
e): 274 nm (1.52), 212 (4.00);
Adenosine A2A receptor competition binding experiments were
carried out in membranes from HeLa-A2A cells. On the day of assay,
membranes were defrosted and re-suspended in incubation buffer
50 mM Tris–HCl, 1 mM EDTA, 10 mM MgCl2 and 2 UI/mL adeno-
sine deaminase (pH 7.4). Each reaction well of a GF/C multiscreen
plate (Millipore, Madrid, Spain), prepared in duplicate, contained
2850, 1644; UV (MeOH) kmax (log
MS (EI, 70 eV) m/z (%): 332 (100) [M+], 221 (20), 207 (24), 194
(34), 180 (90); HRMS m/z: 332.2210 (calcd for C18H28N4O2:
332.2212).
lg
of protein, 3 nM [3H]ZM241385 and test compound
4.7. Synthesis of 7-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)
ethyl)theophylline (10e)
10
C0036E08. Non-specific binding was determined in the presence
of 50 M NECA. The reaction mixture was incubated at 25 °C for
l
4.7.1. 7-(2-(2-(2-(2-Tosylethoxy)ethoxy)ethoxy)ethyl)
theophylline (10d)
30 min, after which samples were filtered and measured in a
microplate beta scintillation counter (Microbeta Trilux, Perkin El-
Compound 10d was obtained by following the same procedure
described for 10a by using 60% NaH (0.244 g, 6.1 mmol), theophyl-
line (1.0 g, 5.6 mmol) in DMF (55 mL) and tetra(ethylene gly-
col)ditosylate (5.0 g, 27.8 mmol) in DMF (70 mL). Compound 10d
was isolated by cc eluting with EtOAc/cyclohexanes 2:1 as a brown
syrup (1.41 g, 50%); 1H NMR (400 MHz, CDCl3) d: 7.77 ppm (d,
J = 8.0 Hz, 2H; 2 ꢀ ArH), 7.69 (s, 1H; H-8), 7.31 (d, J = 8.0 Hz, 2H;
2 ꢀ ArH), 4.47–3.54 (m, 16H; 8 ꢀ OCH2), 3.53 (s, 3H; NCH3), 3.37
(s, 3H; NCH3), 2.41 (s, 3H; CH3); 13C NMR (100 MHz, CDCl3) d:
155.7 ppm (C@O), 151.5 (C@O), 149.1 (C), 145.1, 142.9 (C-8),
142.0 (C), 130.1, 128.3 (CH), 106.8 (C-5), 71.0, 70.8, 70.7, 69.71,
69.69, 69.0.4 (CH2), 47.1 (CH2N), 30.1 (NCH3), 28.2 (NCH3), 21.9
mer, Madrid, Spain).
CGS15943 was carried out in all the assays as an internal control
(Ki = 2.27 0.5 nM).
A
concentration–response curve of
4.8.3. Human A2B receptors
Adenosine A2B receptor competition binding experiments were
carried out in membranes from HEK-293-A2B cells (Euroscreen,
Gosselies, Belgium) prepared following the provider’s protocol.
On the day of assay, membranes were defrosted and re-suspended
in incubation buffer 50 mM Tris–HCl, 1 mM EDTA, 10 mM MgCl2,
0.1 mM benzamidine, 10
deaminase (pH 6.5). Each reaction well prepared in duplicate, con-
tained 18
g of protein, 35 nM [3H]DPCPX and test compound.
lg/mL bacitracine and 2 UI/mL adenosine
(CH3); IR (KBr) m e):
: 2869 cmꢁ1, 1701, 1656; UV (MeOH) kmax (log
l

2088
J. Hierrezuelo et al. / Bioorg. Med. Chem. 18 (2010) 2081–2088
Non-specific binding was determined in the presence of 400 lM
References and notes
NECA. The reaction mixture was incubated at 25 °C for 30 min,
after which samples were filtered through a multiscreen GF/C
microplate and measured in a microplate beta scintillation counter
(Microbeta Trilux, Perkin Elmer, Madrid, Spain). A concentration–
response curve of ZM241385 was carried out in all the assays as
an internal control (Ki = 19.3 2.4 nM).
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l
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Acknowledgements
This work was funded by the Spain’s Nanotechnology Research
Project # NAN04-09312 C03-02 and the Ministerio de Educación y
Ciencia Project
Foundation.
# CTQ2007-62386-PPQ, and by the Welch