New 8-substituted xanthiene derivatives as potent bronchodilators

Article abstract of DOI:10.1016/j.farmac.2005.08.011

The synthesis and structure determination of 8-aryl/alkyl aryl 1, 3-dimethyl-3, 7-dihydropurin-2, 6-dione derivatives (1-13), was carried out in this study. Bronchodilator activity is investigated using isolated guinea-pig tracheal strips, pre-contracted

Full text of DOI:10.1016/j.farmac.2005.08.011

Il Farmaco 60 (2005) 974–980
http://france.elsevier.com/direct/FARMAC/
Original article
New 8-substituted xanthiene derivatives as potent bronchodilators
Barkın Berk a, Hülya Akgün a,*, Kevser Erol b, Bas¸ar Sırmagül b,
Zhan-Guo Gao c, Kenneth A. Jacobson c
a Yeditepe University, Faculty of Pharmacy, 26 Ag˘ustos Yerles¸imi, 34755, Kayıs¸dag˘ı, Istanbul, Turkey
b Osman Gazi University, Faculty of Medicine, Department of Pharmacology, 26480 Eskis¸ehir, Turkey
c National Institute of Diabetes and Digestive and Kidney Diseases, Molecular Recognition Section, Laboratory of Bioorganic Chemistry,
National Institutes of Health. Bldg. 8 A, Rm. B1 A-19, MD 20892-0810 Bethesda, USA
Received 10 August 2005; received in revised form 28 August 2005; accepted 29 August 2005
Abstract
The synthesis and structure determination of 8-aryl /alkyl aryl 1, 3-dimethyl-3, 7-dihydropurin-2, 6-dione derivatives (1-13), was carried
out in this study. Bronchodilator activity is investigated using isolated guinea-pig tracheal strips, pre-contracted by acetylcholine and hista-
mine. Spasmolytic activity of the compounds was compared to theophylline. Synthesized compounds (1-13) did not inhibit the acetylcholine-
induced pre-contractions except compound (8) at 10−5 M concentration. In contrast, some of the compounds, especially (7), (11), (12) at
10−5 M and (3), (4), (9) and (11) in 10−4 M displayed inhibitory activity on the tracheal strips pre-contracted by histamine. The potency of the
compounds at human adenosine receptors was evaluated using radioligand binding assay and a cyclic AMP functional assay in CHO cells
expressing these receptors. Compound (11) displayed the greatest activity against radioligand binding of specific agonists to A2 A and A2B
receptors. The compounds were relatively selective for bothA2 A andA2B compared withA1 andA3 receptors.All compounds were also tested
for the inhibition of NECA-induced cAMP accumulation mediated by the A2B adenosine receptor and compound (11) was found to be the
most effective. Our results showed that these compounds are acting as selective adenosine antagonists, especially for adenosineA2B receptors,
and are promising as potent anti-inflammatory agents rather than bronchodilator for the treatment of asthma.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Xanthine derivatives; Adenosine receptor subtypes; Adenosine antagonist; Bronchodilator activity; Radioligand binding
1. Introduction
action of theophylline is not completely understood, it is
known to be a non-selective inhibitor for phosphodiesterases
(PDE) and a non-selective adenosine receptor antagonist (A2B
adenosine receptor Ki = 7059 nm, selectivityA1 0.6:A2A 0.6:
A3 14) [11]. Its use is limited with narrow therapeutic index
and several cardiac, central nervous side effects [12–14].
Therefore the synthesis of potent and/or selective analogues
of 1, 3-dialkylxanthine derivatives as antagonists for adenos-
ine receptors remains a challenge.
Studies on structure activity relationships of xanthines
showed that the introduction of aryl or cycloaryl group at eight
position of xanthine ring increases antagonist activity and
alkyl groups at 1- and 3-position increase affinity for both
adenosine receptor subtypes [15–20].
Adenosine is an endogenous agonist that can activate all
four adenosine receptor subtypes—A1, A2A, A2B, and A3 [1].
These subtypes have been identified on the basis of inhibi-
tion or stimulation of adenylate cyclase by adenosine and ana-
logues [2–5]. Activation of the A2B adenosine receptors on
mast cells may play a putative role in asthma by enhancing
mast cell degranulation and releasing inflammatory cytok-
ines (e.g. interleukin-4, 8, and 13) [6]. Furthermore, activa-
tion of A2B receptors on bronchial smooth muscle cells has
been demonstrated to lead to the release of interleukin-6 and
monocytic chemotactic peptide-1 [7]. Xanthine derivatives,
like theophylline, are used for enteral treatment of acute and
chronic asthma attacks [8–10]. Although the mechanism of
In general, to achieve high affinity at adenosine receptors
the following criteria have to be fulfilled: adenosine receptor
antagonists should be flat, aromatic or P-electron rich, and
nitrogen-containing heterocycles [16].
* Corresponding author. Tel.: +90 0216 5780839; fax: +90 216 5780068.
E-mail address: hakgun@yeditepe.edu.tr (H. Akgün).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.08.011

B. Berk et al. / Il Farmaco 60 (2005) 974–980
975
A functionalized congener approach to xanthine antago-
nists and determination of bronchodilator activity are the main
aims of the present study. Therefore, some 8-alkylaryl/ary/-
1,3-dimethyl-3,7-dihydropurin-2,6-dione and 8-(4-substitute-
darylcarbonyl)phenyl]-1,3-dimethylxanthine derivatives were
synthesized. Their structures were proven with IR, 1H-NMR,
13C-NMR and elemental analysis data. The antagonistic effect
of synthesized compounds was evaluated in human CHO cells
by using specific radioligands of A1, A2 and A3 receptors. In
addition, all compounds were tested for the inhibition of
NECA-induced cAMP accumulation mediated by the adenos-
ine A2B receptor. Their bronchodilator activity was evaluated
using isolated guinea-pig tracheal strips.
applied at 10−4–10−5 M concentrations, respectively. The
results of testing for bronchodilator activity on the isolated
guinea-pig tracheal strips are shown in Table 1.
3.1.2. Radioligand binding and antagonist activity
of compounds at adenosine receptors
The binding experiment was carried out using standard
agonist radioligands with methods previously described.
[3H]R-PIA, [3H]CGS21680 and [125I] I-AB-MECA were used
forA1, A2A, andA3 adenosine receptors, respectively, to bind
to membranes of transfected CHO cells [26]. The competi-
tive binding of all compounds was tested according to the
procedure described by Gao et al. [27]. All compounds were
tested for the inhibition of agonist-induced cyclicAMP accu-
mulation mediated by the A2B adenosine receptor. Results
are shown in Table 2.
2. Chemistry
The new compounds were synthesized, using an earlier
developed procedure from uracil and corresponding aryl car-
boxylic acids or aryl aldehydes [18,20–24] as fallows; 5,6-
diamino-1,3-dimethyluracil was reacted either with appropri-
ate aldehydes in acetic acid and methanol to form an imine
intermediates (1a) and (2a), or with appropriate carboxylic
or benzoic acid derivatives in the presence of N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimid in methanol to
form an amide intermediates. The xanthine ring cyclisation
was performed using sodium periodate or sodium hydroxide
to form (1-8) like the literature for (2) [17] respectively. The
same cyclisation was also tried using polyphosphoric acid
and ferric chloride as stated in previous studies, but overall
yields were very low and some side products were accom-
plished by the cleavage of 5,6-diamino-1,3-dimethyluracil
structure [25]. Compounds (9-13) were synthesized by using
8-(4-carboxyphenyl)-1,3-dimethyl-3,7-dihydropurin-2,6-
dione (2), N-N’-dicyclohexylcarbodi imid (DCC) and vari-
ous amines as indicated Scheme 1 and Table 1.
3.2. Discussion
In the present study, a series of 8-(aryl/alkylaryl)-1,3-
dimethyl-3,7-dihydropurin-2,6-dione (1-8) and 8-[4-
(substituedcarbamoyl)phenyl]-1,3-dimethyl-3,7-dihydro-
purin-2,6-dione (9-13) were synthesized in two stages. In the
first step commercially available 5,6-diamino-1,3- dimethyl
uracil and approriate aldehydes or carboxylic acids were
reacted to obtain intermediate product 6-amino-5-[substituted
methylenamino]-1,3-dimethyl-1H-pyrimidine-2,4-dione (1a,
2a) and 6-amino-5-(substituedcarbonylamino)-1,3-dimethyl-
1H-pyrimidine- 2,4-dione (3a-8a) respectively, in good yields.
In the next step, these intermediates were converted into
8-aryl–1,3-dimethyl-3,7-dihydropurin-2,6-dions (1-8) under
the action of sodium periodate and dimethylformamide as sol-
vent or NaOH solution, respectively. Then (2) [17] was con-
verted into compounds (9-13) via a modified Kim’s method
[17] (Scheme 1). The compounds were not synthesized in a
serial manner because reactions proceeded in low yields for
different substituents. Compounds (9-13) showed better water
solubility and better pharmacologic activity then (2) [22]. The
amide moiety and electron-rich functional groups enhanced
hydrophilicity of the molecules, therefore in generally a
decrease in activity is expected, but in a balance with aroma-
ticity, number of P electrons and nitrogen and/or heteroaro-
matic containing functionals are overcome this proposal.
3. Results and Discussion
3.1. Biological results
3.1.1. Screening of bronchodilator activity
on guinea-pig tracheal strips
Isolated tracheal strips pre-contracted by acetylcholine or
histamine and either test compounds or theophylline were
Scheme 1. General Synthesis.

976
B. Berk et al. / Il Farmaco 60 (2005) 974–980
Table 1
Table 2
Relaxation of Isolated Guinea Pigs Trachea Strips
The binding affinity of 8-substituted xanthine derivatives to human A1, A2 A
and A3 receptors, and their potency to block the human A2B receptors in a
cAMP functional assay
No.
1
3
4
5
6
7
8
9
10
11
12
13
A1
A2A
A2B
A3
> 100
> 100
> 100
> 100
> 100
> 100
> 100
~10
3.5 1.2
2.3 0.6
> 10
9.4 1.9
> 10
~10
> 10
> 10
> 100
> 10
> 100
~10
> 10
1.5 0.3
10.3 2.4
5.6 1.3
3.2 0.8
4.9 1.7
2.4 0.3
1.6 0.1
3.0 0.6
2.2 0.4
> 10
> 10
> 10
4.1 0.7
2.8 1.6
2.7 0.3
1.7 0.3
9.0 2.2
3.0 0.8
> 10
> 10
> 10
> 10
>100
> 100
> 10
> 100
> 100
Results are expressed as IC50 values (µM) from radioligand binding assays
or a cyclic AMP functional assay (N = 3). Inhibition of radioligand binding
was measured at hA1, hA2 A, and hA3 receptors. The concentrations of the
radioligands used in binding assays were approximated to their Kd values.
Inhibition of NECA (100 nM)-induced accumulation of cyclicAMP measu-
red at hA2B receptors.
A2B among them confirming that p-substituted phenyl amid
derivatives even in cyclic structure also enhance A2 selectiv-
ity. It has been shown that distal structural changes on a chain
attached through the 8-(p-substitutedphenyl) position of the
xanthines can modulate the potency and selectivity of the
drugs, perhaps through interactions at receptor sites distal to
the binding site for the pharmacophore [21].
During the experiments conducted on tracheal strips, to
determine the bronchodilator activity, the synthesized com-
pounds did not show considerable inhibitory effect on
acetylcholine-induced precontractions except 8-[1-(4-
benzoylphenyl)ethyl]-1,3-dimethyl-3,7-dihydropurin-2,6-
dione (8) which had a similar response to the standard theo-
phylline. In addition, the compounds 3 and 11 displayed
significant inhibitory activity on the tracheal strips precon-
tracted by histamine. In this respect, compound (7) and (11)
showed similar activity as theophylline whereas compound
(12) showed better relaxation than theophylline in 10−5 M.
At 10−4 M concentrations; compounds (3) and (11) had quite
impressive results, on the other hand, compounds (4) and (9)
induced similar inhibitory effect compared to the standard,
theophylline.
In the present study, some of the synthesized compounds
displayed significant bronchodilator activity on the tracheal
strips pre-contracted by histamine or acetylcholine and inhibi-
tory activity over NECA-induced accumulation of cAMP.
These results implied that synthesized compounds might have
an inhibitory effect independent from cAMP accumulation
on smooth muscle contraction induced by histamine and ace-
tylcholine. On the other hand, antagonistic activities of syn-
thesized compounds on adenosineA2B receptors were clearly
confirmed by radioligand binding and cAMP functional
assays. This is a promising result in terms of asthma treat-
ment, because, in recent years, anti-inflammatory agents
gained considerable popularity for asthma therapy. Espe-
The potencies of some of these compounds are tested with
radioligand binding at adenosine receptors at micromolar
range.Although these compounds are generally not so potent,
some are relatively selective for A2A or both A2A and A2B
compared with A1 and A3. 8-[4-[4-(2-Hydroxyethyl)
piperidinocarbonyl]phenyl]-1,3-dimethyl-3,7-dihydropurin-
2,6-dione (11) displayed the greatest selectivity to A2A and

B. Berk et al. / Il Farmaco 60 (2005) 974–980
977
cially, after discovery of that adenosine A2B receptor activa-
tion played a putative role in asthma by enhancing mast cell
degranulation and releasing inflammatory cytokines, efforts
are concentrated to discover selective A2B adenosine recep-
tor antagonists [11]. In conclusion, these synthesized com-
pounds might be additional cornerstones to discover specific
anti-asthmatic medicines.
400 MHz): d (ppm) = 3.10 (3H; s; CH3-N), 3.40 (3H; s; CH3-
N), 7.00 (2H; s; NH2), 7.90 (4H; m; Ar-CH), 9.00 (1H; s;
-N=CH-).
4.2.2. Synthesis of 6-amino-5-(substituecarbonylamino)-
1,3-dimethyl-1H-pyrimidine-2,4-dione (3a-8a)
A suspension of 29 mmol of appropriate carboxylic acid,
29 mmol of 5,6-diamino-1,3-dimethyluracil and 32 mmol of
N-(3-dimethyaminopropyl)-N’-ethylcarbodi-imid (EDAC) in
40 ml methanol were stirred at room temperature for about
24 h. Then 60 ml of water was added to the mixture and stirred
for an additional 3 h. The precipitate was filtered and crystal-
lized from dimethylformamide -water.
4. Experimental Procedure
4.1. Chemistry
All Chemicals were supplied by Aldrich (Milwaukee,
USA) and Sigma (St. Louis, MO,USA).
4.2.2.1. 6-Amino-5-[[5-(2-chlorophenyl)-2-furoil)]amino]-
1,3-dimethyl-1H-pyrimi-dine-2,6-dione (3a). Yield. 2.99 g
(79.80%), m.p. > 300 °C. IR (KBr) : 3342, 3201, 3025, 2915,
1702, 1640, 1618, 1476, 1095 cm−1. 1H-NMR (DMSO-[D6],
400 MHz): d (ppm) = 3.05 (3H; s; CH3- N), 3.40 (3H; s; CH3-
N), 7.00 (3H; s; NH2 and furan HC=CH), 7.75 (6H; m; furan
HC=CH andAr), 9.45 (1H; s; -NH-). Anal. (C17H15ClN4O4),
Calc. C: 54.48, H: 4.03, N: 14.94 Found: C: 54.44, H: 4.29,
N: 14.87.
Melting points were determined on a Thomas Hoover Cap-
illary Melting Point Apparatus (Philadelphia, PA: USA) and
uncorrected. Infrared spectra were recorded on a Perkin Elmer
1720 X (Beaconsfield, UK) FT IR. 1H and 13C NMR spectra
were acquired in DMSO-d6 on a BrukerAC 80 and 400 MHz
FT NMR Instruments (Karlsruhe, Germany). Chemical shifts
values were reported as d (ppm) values. Elemental analyses
were performed by Scientific and Technical Research Coun-
cil of Turkey, Instrumental Analysis Laboratories, Ankara,
Turkey, and were within 0.4% of the theoretical values.
4.2.2.2. 6-Amino-5-[[5-(2-nitro-4-chlorophenyl)-2-furoil-
]amino]-1,3-dimethyl-1H-pyrimidine-2,6-dione (4a). Yield:
3.30 g (78.70%), m.p. > 300 °C. IR (KBr) :3329, 3205, 2968,
4.2. Synthesis
1
2953, 1710, 1667, 1602, 1508, 1463, 1113 cm−1. H-NMR
4.2.1. Synthesis of 6-amino-5-[substituemethylenamino]-
1,3-dimethyl-1H-pyrimidine-2,4-diones (1a-2a)
(DMSO-[D6], 80 MHz): d (ppm) = 3.10 (3H; s; CH3-N), 3.80
(3H; s; CH3-N), 6.80 (3H; s; NH2 and furan HC=CH), 7.40
(1H; s; furan HC=CH), 8.00 (3H, m, Ar-CH) 9.00 ppm (1H;
s; -NH-). Anal. (C17H14ClN5O6), Calc. C: 48.59, H: 3.36, N:
16.67 Found: C: 48.47, H: 3.49, N: 16.76.
A
suspension of 29 mmol of 5,6-diamino-1,3-
dimethyluracil (I) in 20 ml of methanol was added slowly to
the mixture of 29 mmol of appropriate aldehydes in 35 ml
methanol and 5 ml of acetic acid, was heated up to 45 °C and
stirred for 2 h in this temperature. Then 50 ml of water was
added. The mixture was stirred for an additional 1 h. The
mixture was then filtered and crystallized from
dimethylformamide-water.
4.2.2.3. 6-Amino-5-[[5-(2-chloro-5-trifluorophenyl)-2-
furoil]amino]-1,3-dimethyl-1H-pyrimidine-2,6-dione
(5a). Yield. 3.28 g (72.60%), m.p. > 300 °C. IR (KBr): 3439,
3349, 3000, 2900, 1775, 1651, 1627, 1480, 1140 cm−1.
1H-NMR (DMSO-[D6], 80 MHz): d (ppm) = 3.10 (3H; s;
CH3-N), 3.30 (3H; s; CH3- N), 6.90 (2H; s; NH2), 7.20 (1H,
d, J = 7.00 Hz furan HC=CH), 7.40 (1H; d; furan HC=CH),
7.70-8.60 (3H, m,Ar-CH) 9.20 and (1H; s; -NH-) . 13C-NMR
(DMSO-[D6], 75 MHz): d (ppm) = 159.70, 158.49, 158.16,
152.99, 151.61, 150.98, 149.12, 148.82, 132.69, 132.61,
132.46, 128.82, 126.20, 125.40, 109.09, 52.48, 30.41 and
27.93. Anal. (C18H14ClF3N4O4. 1/2 H2O), Calc. C: 47.80, H:
13.32, N: 12.39 Found: C: 47.42, H: 13.70, N:12.18.
4.2.1.1. 6-Amino-5-[[5-(4-bromophenyl)-2-furyl)methylen]a-
mino]-1,3-dimethyl-1H-pyrimidine-2,4-dione (1a). Yield:
3.4 g (83.70%), m.p. > 300 °C. IR (KBr):3427, 3021, 2990,
1695,1598, 1474, 1074 cm−1. 1H-NMR (DMSO-[D6],
400 MHz): d(ppm) = 3.30 (3H; s; CH3-N),3.60 (3H; s; CH3-
N), 7.30 (2H; s; NH2), 7.50 (2H; d; J = 6.5 Hz, furan CH),
7.80 (4H; m; Ar-CH), 9.70 ppm (1H; s; -N=CH-). 13C-NMR
(DMSO-d6, 75 MHz) d :178.32, 157.53, 154.85,153.76,
152.78, 152.21, 150.00, 138.03, 132.56, 132.20, 129.50,
128.18, 127.26, 125.98,109.56, 30.74 and 27.41. Anal.
(C17H15BrN4O3 1/2 H20), Calc. C: 49.47, H: 3.88, N: 13.58.
Found; C: 49.61, H: 4.12, N: 13.76.
4.2.2.4. 6-Amino-5–[2-(4-isobutylphenyl)propanoil]amino-
1,3-dimethyl-1H-pyrimidine-2,6-dione (6a). Yield, 2.55 g
(71.30%), m.p. > 300 °C. IR (KBr): 3440, 3156, 3095, 2975,
1717, 1649, 1607 cm−1. 1H-NMR (DMSO-[D6], 80 MHz): d
(ppm) = 0.70 (6H; d; J = 7 Hz, CH3-CH), 1.40 (3H; d;
J = 7 Hz, CH3-CH), 1.8 (1H; m; -CH-), 2.40 (2H; d;
J = 6.5 Hz, -CH2-), 3.05 (3H; s; CH3-N), 3.40 (3H; s; CH3-
N), 3.80 (1H; q; CH3-CH), 6.40 (2H; s; NH2), 7.05 (4H, m,
4.2.1.2. 6-Amino-5-[(4-carboxybenzyliden)amino]-1,3-
dimethyl-1H-pyrimidine-2,4-dione (2a). Yield: 2.82 g
(93.70%), m.p. > 300 °C. m.p. IR (KBr): 3418, 3307, 3017,
2980, 1693, 1633, 1677, 1554 cm−1. 1H-NMR (DMSO-[D6],

978
B. Berk et al. / Il Farmaco 60 (2005) 974–980
Ar-CH), 8.40 (1H; s; -NH-). Anal. (C19H26N4O3), Calc.C:
63.67, H: 7.31, N: 15.63, Found: C: 63.38, H: 7.07, N: 15.43.
reached 2. The precipitates were filtered and recrystalized
from 4 N NaOH/ HCl mixture.
4.2.2.5. 6-Amino-5–[2-(6-methoxy-2-naphtyl)propanoy-
l]amino-1,3-dimethyl-1H-pyrimidine -2,6-dione (7a). Yield,
2.05 g (53.70%), m.p. > 300 °C. IR (KBr): 3314, 3200, 3050,
2950, 1703, 1644, 1602, 1039 cm−1. 1H-NMR (DMSO-[D6],
80 MHz): d (ppm) = 1.40 (3H; d; J = 6.7 Hz, CH-CH3), 3.05
(3H; s; CH3-N), 3.40 (3H; s; CH3-N), 3.80 (3H; s; CH3-O),
3.60 (1H; q; CH-CH3), 6.35 (2H; s; NH2), 7.40 (6H, m,
Ar-CH), 8.50 (1H; s; -NH-). Anal. (C20H22N4O4), Calc. C:
62.82, H: 5.80, N: 14.65, Found: C: 62.95 H: 5.58, N: 14.21.
4.2.4.1. 8-[5-(2-Chlorophenyl)-2-furyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (3). Yield: 2.72
g (72.60%)
m.p. > 300 °C. IR (KBr): 3342, 3024, 2951, 1690, 1640, 1486,
1095 cm−1. 1H-NMR (DMSO-[D6], 80 MHz): d (ppm) = 3.05
(3H; s; CH3-N), 3.60 (3H; s; CH3-N), 6.90 (1H, d, furan,
J = 7 Hz, HC=CH), 7.20 (1H; d; furan, J = 7 Hz, HC=CH),
7.90 (4H, m, Ar- CH), 9.00 (1H; s; -NH-). Anal.
(C17H13ClN4O3. H20), Calc. C: 54.43, H: 4.00, N:14.94,
Found: C: 54.48, H: 3.74, N: 14.40.
4.2.2.6. 6-Amino-5–[2-(4-benzoylphenyl)propanoyl]amino-
1,3-dimethyl-1H-pyrimidine-2,6-dione (8a). Yield, 2.30 g
(54.30%), m.p. > 300 °C. IR (KBr): 3342, 3187, 2950, 2900,
1712, 1679, 1603 cm−1. 1H-NMR (DMSO-[D6], 80 MHz): d
(ppm) = 1.40 (3H; d; J = 6.8 Hz, CHCH3), 3.05 (3H; s; CH3-
N), 3.30 (3H; s; CH3-N), 3.60 (1H; q; CH-CH3), 6.04 (2H; s;
NH2), 7.45 (9H, m, Ar-CH), 8.40 ppm (1H; s; -NH-). Anal.
(C22H22N4O4 . H2O), Calc. C: 62.20, H: 5.65, N: 13.19,
Found: C: 62.44, H: 5.37, N: 13.15.
4.2.4.2. 8-[5-(2-Nitro-4-chlorophenyl)-2-furyl]-1,3-dimethyl-
3,7-dihydropurin-2,6-dione (4). Yield: 2.94 g (73.40%), m.p.
>300 °C. IR (KBr): 3117, 2984, 2893, 1692, 1586, 1537,
1461, 1117 cm-1. 1H-NMR (DMSO-[D6], 80 MHz): d
(ppm) = 3.1 (3H; s; CH3-N), 3.5 (3H; s; CH3-N), 6.9 (1H, d,
furan, J = 7 Hz, HC=CH), 7.3 (1H; d; furan, J = 7 Hz,
HC=CH), 8.0 (2H, m, Ar-CH), 14 (1H; s; Ar-CH) . Anal.
(C17H12ClN5O5), Calc. C: 50.82, H: 3.01, N: 17.43, Found:
C: 50.45, H: 2.60, N: 17.49.
4.2.4.3. 8-[5-(2-Chloro-5-trifluoromethylphenyl)-2-furyl]-
1,3-dimethyl-3,7-dihydro purin- 2,6-dione (5)
4.2.3. Synthesis of 8-(aryl)-1,3-dimethyl-3,7-dihydropurin-
2,6-diones (1-2)
Yield: 3.28 g (74.30%), m.p. > 300 °C. IR (KBr): 3507,
3060, 2956, 1698, 1650, 1537, 1128 cm-1. 1H-NMR (DMSO-
[D6], 80 MHz): d (ppm) = 3.10 (3H; s; CH3-N), 3.50 (3H; s;
CH3-N), 7.20 (1H, d, furan, J = 7 Hz, HC=CH), 7.40 (1H; d;
furan, J = 7 Hz, HC=CH), 7.60 (3H, m, Ar-CH), 8.40 (1H; s;
-NH-).Anal. (C18H12ClF3N4O3 .H20) Calc. C: 48.78, H: 3.16,
N: 12.64, Found: C: 48.35, H: 2.81, N: 12.25.
22 mmol’s of (1a-2a) were dissolved in 50 ml of dimeth-
ylformamide and heated up 150 °C. 22 mmol of sodium perio-
date was added to this mixture and refluxed for 8 h at the
same temperature. 40 ml of water was added; the precipitate
was filtered and dried.
4.2.3.1. 8-[5-(4-Bromophenyl)-2-furyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (1). Yield, 2.98 g (74.50%), m.p.
>300 °C. IR (KBr): 3407, 3124, 2981, 1710, 1642, 1476,
1074 cm−1. 1HNMR (DMSO-[D6], 80 MHz): d (ppm) = 3.20
(3H; s; CH3-N), 3.40 (3H; s; CH3-N), 7.20
(1H, d, J = 7 Hz, furan HC=CH), 7.30 (1H; d; J = 7 Hz,
furan HC=CH), 7.60-7.80 (4H, m, Ar- CH) 9.50 ppm (1H; s;
-NH-) . Anal. (C17H13BrN4O3) Calc. C: 50.89, H: 3.27, N:
13.96, Found: C: 50.43, H: 3.17, N:13.52.
4.2.4.4. 8-[1-(4-Isobutylphenyl)ethyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (6). Yield: 2.29
g (67.40%),
m.p. > 300 °C. IR (KBr): 3446, 3148, 2954, 1717, 1649 cm−1.
1H-NMR (DMSO-[D6], 80 MHz): d (ppm) = 0.65 (6H; d;
(CH3)2-CH), 1.30 (1H; m; (CH3)2-CH), 1.40 (3H; d; CH3-
CH), 2,20 (2H;d; -CH2-), 3.05 (3H; s; CH3-N), 3.40 (3H; s;
CH3-N), 4.00 (1H; q; CH3-CH), 7.00 (4H, m, Ar-CH), 7.90
(1H; s; -NH-). Anal. (C19H24N4O2) Calc. C: 67.04, H: 7.11,
N: 16.46, Found: C: 67.42, H: 7.28, N: 15.95.
4.2.3.2. 8-(4-Carboxyphenyl)-1,3-dimethyl-3,7-dihydropurin-
2,6-dione (2). Yield: 2.68 g (89.55%), m.p. > 300 °C. m.p.
IR (KBr): 3152, 3014, 2956, 1704, 1660, 1614 cm−1. 1HNMR
(DMSO-[D6], 400 MHz): d (ppm) = 3.25 (3H; s; CH3-N),
3.50 (3H; s; CH3-N), 8.00-8.20 (4H, m, Ar-C-H) 13.40 (1H;
s; Ar-COOH) . 13C-NMR (DMSO-[D6], 75 MHz): d
(ppm) = 167.11, 154.65, 151,49, 148.80, 132.17, 130.16,
128.49, 127.56, 105.98, 30.13 and 28.17.
4.2.4.5. 8-[1-(6-Methoxy-2-naphthyl)ethyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (7). Yield, 2.30
m.p. > 300 °C. IR (KBr): 3325, 3166, 2937, 1705, 1653,
1031 cm−1. 1H-NMR (DMSO-[D6], 400 MHz):
g (63.30%),
d
(ppm) = 1.75 (3H; d; J = 6.8 Hz, CH-CH3), 3.25 (3H; s; CH3-
N), 3.50 (3H; s; CH3-N), 3.90 (3H; s; CH3-O), 4.50 (1H; q;
CH-CH3), 7.00-7.09 (6H, m, Ar-CH), 13.40 ppm (1H; s;
-NH-) . Anal. (C20H20N4O3), Calc. C: 65.92, H: 5.53, N:
15.38, Found: C: 65.93 H: 5.80, N: 15.29.
4.2.4.6. 8-[1-(4-Benzoylphenyl)ethyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (8)
Yield:2.50 g (64.40%), m.p. > 300 °C. IR (KBr): 3158,
3100, 2981, 1702, 1650, 1598 cm−1. 1H-NMR (DMSO-[D6],
400 MHz): d (ppm) = 1.80 (3H; d; J = 6.7 Hz, CH-CH3), 3.20
4.2.4. Synthesis of 8-[aryl/alkylaryl]-1,3-dimethyl-3,7-
dihydropurin-2,6-diones (3-8)
22 mmol’s of (3a-8a) were suspended in 20 ml of
methanol-water (8:1 v/v). 40 ml of 10% sodium hydroxide
was added to the mixture and heated at 65 °C for 3 h. Then
concentrated HCl was added to the mixture until the pH

B. Berk et al. / Il Farmaco 60 (2005) 974–980
979
(3H; s; CH3-N), 3.45 (3H; s; CH3-N), 4.40 (1H; q; CH-CH3),
7.10-8.00 (9H, m, Ar-CH). Anal. (C22H20N4O3), Calc. C:
68.03, H: 5.19, N: 14.42, Found: C: 68.25, H: 5.62, N: 13.99.
1707, 1649, 1603 cm−1. 1H-NMR (DMSO-[D6], 400 MHz):
d (ppm) = 2.12 (3H; s; -CH3), 3.24 (4H; m; -N- CH2-CH2-
N-), 3.24 (3H; s; N-CH3), 3.76 (3H; s; N-CH3), 7.9-8.11 (4H;
m; Ar-CH), 14 (1H;s; NH). Anal. (C18H18N6O3.H2O), Calc.
C: 56.19, H: 5.20, N: 21.85, Found: C: 56.23, H: 5.56, N:
21.55.
4.2.5. Synthesis of 8-[4-(substituedcarbomoyl)phenyl]-1,3-
dimethyl-3,7-dihydro purin-2,6-dione (9-13)
22 mmol of 8-[4-(carboxy)phenyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (2) was dissolved in 20 ml of hot dim-
ethylformamide and cooled to the room temperature.
220 mmol of corresponding amines and 25 mmol of N-N’-
dicyclohexylcarbodiimid (DCC) were added to this mixture.
The mixtures were stirred at the room temperature for 8 h.
After the filtration of the formed white precipitates, dimeth-
ylformamide was evaporated under vacuum; the residues were
crystallized from diethylether-methanol.
4.3. Biological materials and methods
4.3.1. Materials
Swiss albino male or female guinea pigs were used in the
study. They were housed in temperature and humidity con-
trolled facilities atAnimal Care Facilities of OsmanGazi Uni-
versity, Eskisehir, Turkey with a 12 h cycle of light and dark.
They were fed with water and pellated food. All results were
recorded by with an isometric transducer (TB- 65IT: Nihon-
Kohden Recorder system, Tokyo, Japan). [125I]N6-(4-
Amino-3- iodobenzyl)adenosine-50-N6-methyluronamide
([125I]I-AB-MECA; 2000 Ci/mmol), [3H]R-PIA (R-N6 [phe-
nyl isopropyl]adenosine, 34 Ci/mmol), [3H]CGS21680 (2-[p-
(2-carboxyethyl)phenylethylamino]-50-N6-
4.2.5.1. 8-[4-[4-(2-Hydroxyethyl)piperazinocarbonyl]phenyl]
-1,3-dimethyl-3,7-di hydropurin-2,6-dione (9). Yield: 2.66 g
(61.80%), m.p. > 300 °C. IR (KBr): 3394, 3154, 2949, 1705,
1
1651, 1598 cm−1. H-NMR (DMSO-[D6], 400 MHz): d
(ppm) = 2.56 (6H; s; CH2-N-(CH2)2), 3.05 (4H; s; CO-N-
(CH2)2), 3.32 (3H; s; N-CH3), 3.56 (5H; s; N-CH3 and
HO-CH2), 8.0-8.30 (4H; m;Ar-CH), 10.0 (1H; s; -NH).Anal.
(C20H24N6O4.H20), Calc. C: 55.75, H: 6.04, N: 19.51, Found:
C: 55.70, H: 6.26, N: 18.97.
ethylcarboxamido-adenosine, 47 Ci/ mmol) and [3H]cyclic
AMP (40 Ci/mmol) were obtained from Amersham Pharma-
cia Biotech (Buckinghamshire, UK).
4.2.5.2. 8-[4-[4-(2-Hydroxyethyl)piperidinocarbonyl]phenyl]-
1,3-dimethyl-3,7-di hydropurin-2,6-dione (10). Yield: 2.52 g
(58.70%), m.p. > 300 °C. IR (KBr): 3350, 3149, 2929, 2833,
1705, 1645, 1591 cm−1. 1H-NMR (DMSO-[D6], 400 MHz):
d (ppm) = 1.0-1.3 (5H; m; (CH2)2-CH-), 1.5 (2H; m; -CH-
CH2), 3.07 (4H; t; N-(CH2)2), 3.25 (3H; s; N-CH3), 3.27 (2H;
t; HO-CH2), 3.31 (3H; s; N-CH3), 7.7-8.00 (4H; m; Ar-CH).
Anal. (C21H25N5O4. H2O), Calc. C: 58.67, H: 6.28, N:
16.29, Found: C: 58.63, H: 6.67, N: 16.15.
4.3.2. General Scanning for the bronchodilator activity
in isolated guinea pig tracheas
The animals were sacrificed by cervical dislocation, and
trachea was immediately removed. The tracheal strips were
mounted vertically in a 10 ml organ bath containing Krebs -
Henseleit solution of the following composition in mg/l: NaCl,
118.3, KCl 4.73, CaCl2 2.54, NaHPO4 1.19, MgSO41.18,
NaHCO3 26.2, glucose 11.09. The bath contents were main-
tained at 37 °C and aerated by 95% O2 and 5% CO2. The
preparations were allowed to equilibrate for at least 80 min-
utes, with regular washes in every 15 minutes. Contractions
were induced by 10uM. acetylcholine or histamine. The
10uM. and 10−4 M. concentrations of standard aminophyl-
line and the compounds, diluted in dimethylsulfoxide were
added to the bath in volumes of 0.1 ml. The responses of all
were subtracted from those of DMSO. The concentration-
response curves were obtained and the data were expressed
as mean S.E.
4.2.5.3. 8-[4-(2-Hidroxymethylpiperidinocarbonyl)phenyl]
-1,3-dimethyl-3,7-di hydropurin-2,6 -dione (11). Yield:
2.58 g(62.30%), m.p. > 300 °C. IR (KBr): 3277, 3163, 3040,
2950-2863, 1707, 1644, 1593 cm−1. 1H-NMR (DMSO-[D6],
400 MHz): d (ppm) = 1.5-2.0 (7H; m; piperidine CH2), 2.71
(2H; t; CO-N-CH2), 3.27 (3H; s; N-CH3), 3.50 (2H; t;
HO-CH2), 3.71 (3H; s; N-CH3), 8.0-8.4 (4H;m;Ar-CH).Anal.,
(C20H23N5O4.H2O), Calc. C: 57.77, H: 6.01, N: 16.85, Found:
C: 57.65, H: 5.84, N: 16.63.
4.3.3. Cell culture and membrane preparation
4.2.5.4. 8-[4-(Morfolinocarbonyl)phenyl]-1,3-dimethyl-3,7-
dihydropurin-2,6-dione (12). Yield: 2.79 g (75.70%),
m.p. > 300 °C. IR (KBr): 3450, 3156, 2952, 1708, 1600 cm−1.
1H-NMR (DMSO-[D6], 400 MHz): d (ppm) = 3.3 (3H; s;
N-CH3), 3.4 (4H; m; CH2-N-CH2), 3.6 (3H; s; N-CH3), 3.9
(4H; m; -CH2-O-CH2-), 8.0-8.4 (4H; m; Ar-CH), 14 (1H; s;
NH). Anal. (C18H19N5O4), Calc. C: 58.53, H: 5.18, N: 18.96,
Found: C: 58.48, H: 5.15, N: 18.63.
The CHO cells stably expressing recombinant human
adenosine receptors were cultured in DMEM and F12 (1:1)
supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin and 2 µmol/ml glutamine.
After harvest and homogenization, cells were centrifuged at
500 X g for 10 min, and the pellet was re-suspended in 50 mM
Tris–HCl buffer (pH 7.4) containing 10 mM MgCl2, 1 mM
EDTA. The suspension was homogenized with an electric
homogenizer for 10 s, and was then re-centrifuged at 20,000 X
g for 20 min at 4 °C. The resultant pellets were resuspended
in buffer in the presence of 3 units/ml adenosine deaminase,
4.2.5.5. 8-[4-(2-Methyl-4,5-dihydroimidazolylcarbonyl)phe-
nyl]-1,3-dimethyl-3,7-dihydropurin-2,6-dione (13). Yield:
3.01 g (78.40%), m.p. > 300 °C. IR (KBr): 3156, 3050, 2944,

980
B. Berk et al. / Il Farmaco 60 (2005) 974–980
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4.3.4. Binding assays and inhibition of adenyl cyclase
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