8-(Sulfostyryl)xanthines: Water-soluble A(2A)-selective adenosine receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(98)00025-X

8-(Sulfostyryl)xanthine derivatives were synthesized as water-soluble A(2A)-selective adenosine receptor (AR) antagonists. meta- and para-sulfostyryl-DMPX (3,7-dimethyl-1-propargylxanthine) derivatives 11a and 11b exhibited high affinity to rat A(2A)-AR in submicromolar concentrations, and were 20- to 30-fold selective versus rat A₁-AR. Styryl-DMPX derivatives were inactive at human A(2B)- and A₃-AR. 1,3-Dipropyl-8-p-sulfostyrylxanthine (13) and its 7-methyl derivative (14) showed similar (13) or higher (14) A(2A) affinity than 11a and 11b but showed no (13) or only a low degree (14) of selectivity versus A₁-, A(2B)-, and A₃-AR. The A(2A)-selective sulfostyryl-DMPX derivatives exhibit high water-solubility and may be useful research tools for in vivo studies. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00025-X

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 707±719
8-(Sulfostyryl)xanthines: Water-soluble A2A-Selective
Adenosine Receptor Antagonistsy
Christa E. Muller,a,* Jesus Sandoval-Ramõrez,a,{ Ulrike Schobert,a Uli Geis,a
Wolfram Frobeniusa and Karl-Norbert Klotzb
aInstitut fur Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universitat Wurzburg, Pharmazeutische Chemie, Am Hubland,
D-97074 Wurzburg, Germany
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È
È
È
bInstitut fur Pharmakologie und Toxikologie, Julius-Maximilians-UniversitaÈt Wurzburg, Versbacher Str. 9, D-97078 Wurzburg, Germany
È
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Received 22 September 1997; accepted 24 November 1997
AbstractÐ8-(Sulfostyryl)xanthine derivatives were synthesized as water-soluble A2A-selective adenosine receptor (AR)
antagonists. meta- and para-sulfostyryl-DMPX (3,7-dimethyl-1-propargylxanthine) derivatives 11a and 11b exhibited
high anity to rat A2A-AR in submicromolar concentrations, and were 20- to 30-fold selective versus rat A1-AR.
Styryl-DMPX derivatives were inactive at human A2B- and A3-AR. 1,3-Dipropyl-8-p-sulfostyrylxanthine (13) and its
7-methyl derivative (14) showed similar (13) or higher (14) A2A anity than 11a and 11b but showed no (13) or only a
low degree (14) of selectivity versus A1-, A2B-, and A3-AR. The A2A-selective sulfostyryl-DMPX derivatives exhibit
high water-solubility and may be useful research tools for in vivo studies. # 1998 Elsevier Science Ltd. All rights
reserved.
Introduction
selectivity for the A1-AR is most advanced as compared
Cell membrane receptors for the physiological nucleo-
side adenosine are found in many organs and tissues.1
Four di€erent types of adenosine receptors (AR) have
been identi®ed and cloned, namely the high-anity
subtypes A1 and A2A and the low anity subtypes A2B
and A3.2 All AR agonists known so far are derivatives
of adenosine, the ribose moiety of the nucleoside being
essential for agonism of the compounds.3 The main
classes of potent AR antagonists at least for the high
anity receptor subtypes A1 and A2A are (i) adenine
derivatives and adenine analog, and (ii) xanthine deri-
vatives.4 The development of AR-antagonists with
to other subtypes, several compounds being currently
evaluated in clinical studies for the treatment of senile
dementias, such as Alzheimer's disease, and for the pre-
vention of acute renal failure.5 A2A-selective AR
antagonists have potential as novel therapeutic agents
for the treatment of Parkinson's disease. Further pro-
posed indications include, for example, hypotension and
acute ischemias.6 The ®rst AR antagonist described in
the literature as A2A-selective was 3,7-dimethyl-1-prop-
argylxanthine (DMPX, 1), a compound of low AR-
anity and low A2A-selectivity versus A1, however no
selectivity versus A2B-AR.4,7 Nevertheless DMPX is still
widely used as A2A-antagonist in in vivo studies due to
its good water-solubility and high bioavailability. Dur-
ing the past years potent and selective A2A-AR antago-
nists have been developed, including 8-styrylxanthine
derivatives (2±5);8±10 among the most potent and most
selective compounds are the 8-styryl-DMPX derivatives,
such as 4 and 5.10 Further potent A2A-selective AR
antagonists include compounds that can be envisaged
as adenine analogs, namely the pyrazolo-[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidines,11 (e.g. SCH58261, and the
Key words: Xanthines; sulfonic acids; A1-, A2A-, A2B-, A3-ade-
nosine receptors; A2A-antagonists; water-solubility.
*Corresponding author. Tel: +49-(0)931-888-5440; Fax: +49-
(0)931-888-5462; E-mail: mueller@pharmazie.uni-wuerzburg.de
yPreliminary results were presented at the International Sym-
posium ``Purines '96'' in Milan, Italy; abstract published in
Drug Dev. Res. 1996, 37, 112.
{J. S.-R. was on leave from the Facultad de Ciencias Quõmicas,
Benemerita Universidad Autonoma de Puebla (BUAP),
Puebla, Mexico, with support from Deutscher Akademischer
Austauschdienst (DAAD).
triazolo-[1,5-a][1,3,5]triazine ZM241385.12
A major
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00025-X

708
C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
problem associated with potent selective A2A-antago-
nists is their typically low water-solubility, which limits
their in vivo applicability. So far, only few e€orts have
1 and 2). p-Sulfocinnamic acid 7a was prepared by sul-
fonation of cinnamic acid with fuming sulfuric acid
according to a procedure published in 1927.16 Sulfona-
tion yielded the p-substituted isomer 7a as the main
product. m-Sulfocinnamic acid was obtained as a by-
product. Both isomers were separated by fractionating
crystallization. The meta-sulfonated cinnamic acid deri-
vative 7b could alternatively be obtained by nucleophilic
substitution of m-bromocinnamic acid with sodium sul-
®te as described (Method B).16 The latter method is
preferable for the preparation of 7b resulting in higher
yields. Isomers 7a and 7b could easily be distinguished
been undertaken to improve water-solubility of A2A
-
antagonists. Jacobson et al. synthesized a congener of 8-
(m-aminostyryl)ca€eine, a succinic acid amide, bearing
a carboxylic function.9 Solubility of that compound in
basic solution (0.1 M K2HPO4) was high (19 mM/L),
but may be lower at physiological pH values. AR
antagonists with extraordinarily high water-solubility
over a wide range of pH values are the 8-sulfophenyl-
xanthines SPT (15) and DPSPX (18), compounds
derived from the A1-selective antagonists 8-phenyltheo-
phylline and 1,3-dipropyl-8-phenylxanthine.13,14 Despite
lacking receptor subtype selectivity, these compounds
have become important pharmacological tools due to
their excellent water-solubility. Since sulfonic acids are
deprotonated under physiological conditions, such
compounds do not penetrate into the brain and are only
peripherally active.13,15
1
by their H and 13C NMR spectra (see Experimental).
1-Propargylxanthine derivatives were prepared starting
from 5,6-diamino-3-propargyluracil 617,18 by condensation
with para- or meta-sulfocinnamic acid 7a, or 7b, respec-
tively. Amides 8a and 8b were methylated in the 3-posi-
tion in analogy to described procedures.18 Direct ring
closure of 8a and 8b is not successful, since deprotona-
tion in alkaline solution reduces the nucleophilicity of
the 6-amino function. Ring closure in acidic media,
however, leads to side reactions of the propargyl
group.19 Treatment of the methylated compounds 9a
and 9b with a solution of sodium hydroxide in a mixture
of methanol and water yielded xanthines 10a and 10b,
which were methylated in the 7-position to yield the
target compounds 11a and 11b (Scheme 1).
In the present study, we prepared sulfo-derivatives of
A2A-selective styrylxanthines and evaluated their AR
anity and selectivity pro®le. Our goal was to obtain
A
2A-selective antagonists with high water-solubility at
physiological pH values useful as pharmacological tools
and potential drugs without central stimulatory e€ects.
For the preparation of 1,3-dipropylxanthine derivatives
the standard procedure was applied.13,14 Thus, 5,6-di-
amino-1,3-dipropylxanthine (12) was condensed with
sulfocinnamic acid 7a, followed by intramolecular con-
densation in alkaline solution to xanthine 13, which was
methylated to 14 (Scheme 2).
Results and Discussion
Chemistry
8-Sulfostyrylxanthines were synthesized from 5,6-di-
aminouracil derivatives and sulfocinnamic acids (Schemes
Chart 1. A2A-Adenosine receptor antagonists with xanthine structure.

C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
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709
Scheme 1. Synthesis of 8-sulfostyryl-3,7-dimethyl-1-propargylxanthine (8-sulfostyryl-DMPX derivatives).
1H and 13C NMR spectral data for intermediate and
®nal products con®rmed the proposed structures
(Tables 1±3). (E)-con®guration of the double bond is
retained as shown by the coupling constant of about
16 Hz for the vinylic protons of the sulfostyryl derivatives
(Tables 1 and 2). For (Z)-con®gurated isomers a coupling
constant of ca. 10±13 Hz would have to be expected.20,21
including water, dimethylsulfoxide (DMSO) and
chloroform. Sulfostyrylxanthines, however, are well
soluble in water and DMSO, and 13C NMR spectra can
be routinely recorded (Table 3).
Styrylxanthine derivatives had been observed to exhibit
photo-induced isomerization in dilute solution.10,20 We
investigated E/Z-isomerization for one representative
example of sulfostyrylxanthines, p-sulfostyryl-DMPX
(11a), by measurement of the decrease in UV absorption
at 356 nm, induced by exposure to normal daylight. Our
It had been dicult to obtain 13C NMR spectra of 8-
styrylxanthine derivatives investigated so far due to
their low solubility in polar as well as nonpolar solvents,

710
C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
Scheme 2. Synthesis of 1,3-dipropyl-8-sulfostyrylxanthine derivatives.
Table 1. 1H NMR data of intermediate carboxamidouracil derivatives
d (ppm) in DMSO-d6, J (Hz)
R5
Compd
R1
R3
6-NH2
8a
10.67 (br s, 1H) 3.05 (t, J=2.3, 2H), 6.83 (d, J=15.8, 1H, Hvinylic), 7.45 (d, J=15.8, 1H,
4.45 (d J=2.3, 2H) vinylic), 7.56 (d, J=8.3, 2H, Haromatic), 7.67 (d, J=8.3,
2H, Haromatic), 8.74 (s, 1H, NH)
10.62 (br s, 1 H) 3.05 (t, J=2.1, 1H), 6.85 (d, J=16.0, 1H, Hvinylic), 7.36±7.86 (m, 5H,
4.42 (d, J=2.2, 2H) vinylic, Haromatic), 8.75 (s, 1H, NH)
3.04 (t, J=2.1, 1H), 6.82 (d, J=15.8, 1H, Hvinylic), 7.45 (d, J=15.8, 1H,
4.50 (d, J=2.1, 2H) vinylic), 7.55 (d, J=8.2, 2H, Haromatic), 7.65 (d, J=6.5,
2H, Haromatic), 8.72 (s, 1H, NH)
3.04 (t, J=2.0, 1H), 6.82 (d, J=15.7, 1H, Hvinylic), 7.40±7.66 (m, 5H,
4.48 (d, J=2.1, 2H) Hvinylic, Haromatic
6.26 (br s, 2H)
H
8b
9a
6.23 (br s, 2H)
6.84 (br s, 2H)
H
3.35 (s, 3H)
3.32 (s, 3H)
H
9b
6.86 (br s, 2H)
)

C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
711
Table 2. 1H NMR data of sulfostyrylxanthine derivatives
d (ppm) in DMSO-d6, J (Hz)
R3 R7
Compd
R1
R8
a
10a
2.94 (t, J=2.3, 1H), 4.58 (d, J=3.43 (s, 3H)
2.3, 2H)
7.00 (d, J=16.2, 1H, Hvinylic), 7.19 (d,
J=16.2, 1H, Hvinylic), 7.45 (d, J=8.3,
2H, Haromatic), 7.45 (d, J=8.3, 2H,
Haromatic
7.01 (d, J=16.1, 1H, Hvinylic), 7.18±
7.75 (m, 5H, Haromatic, Hvinylic
)
a
10b
11a
2.95 (t, J=1.8, 1H), 4.58 (d, J=3.42 (s, 3H)
1.9, 2H)
)
3.05 (t, J=2.3, 1H), 4.57 (d, J=3.46 (s, 3H)
2.3, 2H)
4.01 (s, 3H)
7.31 (d, J=15.8, 1H, Hvinylic), 7.67 (d,
J=15.8, 1H, Hvinylic), 7.62 (d, J=8.0,
2H, Haromatic), 7.73 (d, J=8.0, 2H,
Haromatic
7.06 (d, J=15.9, 1H, Hvinylic), 7.36±
7.79 (m, 5H, Hvinylic, Haromatic
)
11b
13
3.08 (t, 1H), 4.57 (d, 2H)
3.45 (s, 3H)
4.03 (s, 3H)
)
a
0.82 (q, J=12.8, J=5.5, 6H,
CH3), 1.54 (sext., J=15.0, J=
7.5, 2H, CH2-CH3), 3.82(t, J=
7.1, 2H, N-CH2)
0.82 (q, J=12.8, J=5.5, 6H,
CH3, 1.69 (sext., J=14.6, J=
7.5, 2H, CH2-CH3), 3.95 (t, J=
6.7, 2H, N-CH2)
7.04 (d, J=16.4, 1H, CH=CH), 7.58
(d, J=8.4, 2H, Haromatic), 7.62 (d, J=
16.3, 1H, CH=CH), 7.66 (d, J=8.4,
2H, Haromatic
)
14
0.86, (q, J=15.0, J=7.4, 3H,
CH3), 1.54 (sext., J=15.0, J=
0.86 (q, J=15.0, J=7.4, 3H,
CH3), 1.71 (sext., J=14.6, J=
4.01 (s, 3H)
7.36 (d, J=17.7, 1H, CH=CH), 7.61
(d, J=8.6, 2H, Haromatic) 7.63 (d, J=
15.2, 1H, CH=CH), 7.74 (d, J=8.5,
7.5, 2H, CH2-CH3), 3.80 (t, J= 7.5, 2H, CH2-CH3), 3. (t, J=
7.5, 2H, N-CH2) 7.6, 2H, N-CH2)
2H, Haromatic
)
aN7-H could not be detected due to rapid exchange.
Table 3. 13C NMR data of selected sulfostyrylxanthine derivatives
d (ppm)in DMSO-d6
Compd
C-2
C-4
C-5
C-6
C-8
R1
R3
R7
R8
11a
150.11 148.85* 107.09 153.16 149.97 29.47,
72.70,
29.92
31.47 112.96 (CH=CH), 126.00, 127.04, 135.50,
136.33, 138.27, 148.27 (Caromatic, CH=CH)
79.65
150.86 148.58 107.45 154.07 149.61 11.23,
13
14
11.41,
21.08,
44.61
11.35,
21.02,
44.22
Ð
116.50 (CH=CH), 126.45, 126.84, 134.70,
135.85, 148.58 (Caromatic, CH=CH)
21.08,
42.34
150.86 148.80 107.50 154.38 149.77 11.20,
20.96,
42.07
31.57 113.45 (CH=CH), 126.18, 127.27, 135.85,
136.15, 147.92 (Caromatic, CH=CH)
*arbitrary assignment

712
C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
results for 11a were in accordance with results for other
8-styrylxanthine derivatives.10,20 Thus, isomerization
was much faster in dilute solutions (ꢀ 0.1 mM) as com-
pared to more concentrated solutions. Isomerization
was faster in methanol, or in aqueous bu€er solution,
A2A-AR. No data were available of their A2B- and A3-
AR activity.
The 3-unsubstituted 1-propyl-8-p-sulfophenylxanthine
(17) exhibits similar anity as SPT (15) for A1-, A2A-,
and A2B-AR. Only at the A3-AR anity of 17 is several-
fold higher compared to 15 demonstrating the impor-
tance of a 1-propyl substituent for high A3-anity of
xanthine derivatives.
respectively, than in DMSO. After ca. 30 min,
a
0.01 mM solution in aqueous bu€er reached its equili-
brium (data not shown). It is concluded that under test
conditions, stable mixtures of E- and Z-isomers are
present in the dilute test solutions.
One of the most potent and selective A2A-AR antago-
nists is 8-m-bromostyryl-DMPX (5).10 Compound 5 has
now been investigated at human A2B- and A3-AR. It
was found that 5 exhibits no anity for these `low a-
nity AR' and is highly selective for A2A versus all other
AR subtypes.
Pharmacology
The new compounds were tested in radioligand binding
assays for anity to A1- and A2A-adenosine receptors in
rat cortical membrane, and rat striatal membrane pre-
parations, respectively. The A1-selective agonist [3H]N6-
cyclohexyladenosine ([3H]CHA) was used as A1-ligand,
and the A2A-selective agonist [3H]2-[4-[carboxyethyl)-
phenylethylamino]-50-N-ethylcarboxamidoadenosine ([3H]
Introduction of a para- or meta-sulfo substituent into
8-styryl-DMPX (Ki A1=1.1 mM, A2A=0.027 mM)10
decreases AR-anity of the compound ®ve to eightfold at
A1-AR, and 9- to 11-fold at A2A-AR. The resulting sulfo-
styryl-DMPX derivatives 11a and 11b retain A2A-selec-
tivity, which is 20-fold for 11a, and 30-fold for 11b versus
CGS21680) as A2A ligand. Since a radioligand for A2B
-
AR was not available, the inhibition of N-ethylcarbox-
amidoadenosine- (NECA-) stimulated adenylate cyclase
by test compounds was measured using human recom-
binant A2B-AR expressed in chinese hamster ovary
(CHO) cells.22 A3-AR anity was determined using
human recombinant A3-AR, expressed in CHO cells
with [3H]NECA as radioligand.
A1-AR. The compounds are virtually inactive at A2B
and A3-AR, like 5, another 8-styryl-DMPX derivative.
-
In order to investigate which structural parameters are
responsible for the high selectivity of compounds 11a,
11b, and 5 versus A2B- and A3-AR, 1,3-dipropyl-8-p-
sulfostyrylxanthines were synthesized and compared
with sulfostyryl-DMPX derivatives.
Structure±activity relationships
8-p-Sulfophenylxanthines had been introduced as non-
selective AR-antagonists, which are freely soluble in
water at physiological pH values.13,14 The sulfonic acid
group exhibits a pKa value of <1 (e.g. benzenesulfonic
acid: pKa= 6.5)23 and is deprotonated under physiologic
conditions. The introduction of a p-sulfo substituent in
the A1-selective 8-phenyltheophylline or its 1,3-dipropyl
homolog had resulted in a large decrease in A1-anity,
however it reduced A2A-anity to a lower degree.13
Therefore, we investigated the introduction of a p-sulfo
substituent into A2A-selective xanthine derivatives.
The 7-unsubstituted 1,3-dipropyl derivative 13 showed
similar potency at all AR subtypes (150±670 nM) and
therefore was virtually nonselective. As expected, 7-
methylation resulted in a decrease in A1-anity (25-fold)
and an increase in A2A-anity (®vefold). Compound 14
is the most potent, highly water-soluble A2A-AR antago-
nist of the present series. Selectivity, however, is moderate
(sevenfold versus A1). 7-Methylation of 13 to 14 resulted
in a slight decrease in A2B- and A3-anity (twofold).
Since acidic functions on the 8-substituent of xanthine
derivatives can enhance A3-AR anity,24 and because
acidic groups are also well tolerated by A2B-AR (e.g.
13,14) we investigated a carboxy-substituted 8-(2-naph-
thyl)xanthine derivative (20) at A2B- and A3-AR. The
compound had been investigated as a sterically ®xed
analog of 8-styryl-DMPX.26 Indeed, 20 was relatively
potent at A2B- and A3-AR. This result is particularly
surprising, since 20 is an 8-substituted DMPX deriva-
tive, which usually show very low activity at A2B- and
A3-AR (e.g. 5, 11a, and 11b). Compound 20 is about
threefold selective for A2B-AR versus A1 and A2A and
ca. ®vefold selective versus A3 and thus could be a new
lead for the development of A2B-AR antagonists.
8-p-Sulfophenyltheophylline (SPT, 15) exhibits low a-
nity for A1-, A2A-, A2B-, and A3-AR in the micromolar
concentration range (see Table 4). The compound is
somewhat more potent at A1- and A2B- as compared to
A
2A- and A3-AR. The 1,3-dipropyl analog of 15,
1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, 18) is
more potent at all AR subtypes, ca. 20-fold at A1,
10-fold at A2A, ®vefold at A2B- and ca. 60-fold at A3-AR
(see Table 4). Thus, DPSPX (18) is equipotent at A1-,
A
2B- and A3-AR and about ®ve to eightfold less potent
at A2A-AR. The corresponding 7-methylated xanthine
derivatives 16 and 19 are considerably less potent than
the 7-unsubstituted compounds 15 and 18 at A1- and

C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
713

714
C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
In conclusion, 8-styryl-DMPX derivatives, including the
water-soluble 8-sulfostyryl-DMPX derivatives 11a and
11b, exhibit selectivity for A2A-AR versus A1, and
extraordinarily high selectivity versus A2B- and A3-AR,
at which these compounds are virtually inactive. In
contrast, 1,3-dipropyl-(7-methyl)-8-styrylxanthine deri-
vatives (13 and 14) retain activity at A2B- and A3-AR.
Propyl substitution in the 1- and 3-position is particu-
larly important for high A3-AR anity.
Some of these `second generation A1-AR antagonists'
are now under clinical development as drugs.5 Solubility
of the standard A2A-antagonist KF17837 (2) has been
reported to be as low as 0.06 g/mL (0.146 mM/L).20
DMSO has to be used for in vivo studies to dissolve the
compound, and solubility was reported to be a major
limitation for the in vivo use of 2.28,29 Dionisotti et al.
found the compound to be inactive in vivo in their
experimental setting.30
Water solubilities of other A2A-selective xanthine deri-
vatives have not been reported. For chlorostyrylca€eine
(CSC, 3), another standard A2A-antagonist, dissolution
in 45% aqueous 2-hydroxypropyl-b-cyclodextrin solu-
tion is recommended, and solubility in that mixture is
given to be less than 0.3 g/L (<900 mM/L).31
Water solubility
A certain degree of water-solubility is a prerequisite for
in vivo activity of a drug. Bruns and Fergus had postu-
lated that the solubility over AR-anity ratio of a
compound (the so-called Bruns±Fergus or BF index)
has to be greater than 100 in order to exhibit good in
vivo activity.27 For experimental drugs it is often desir-
able that they are highly water-soluble (e.g. in order to
allow for parenteral application).
Potent non-xanthine A2A-antagonists also exhibit low
water-solubility. ZM241385, for example, had to be
intravenously administered in a solution of 50% poly-
ethylenglycol (PEG 400) and 50% aqueous NaOH
solution (0.1 M) prepared by sonication.32
A
major problem associated with AR-antagonists
developed by in vitro screening methods is their gen-
erally low water-solubility, which limits their usefulness,
especially for in vivo studies. PurineÐincluding xan-
thineÐderivatives, in particular, are often poorly solu-
ble in water due to base-stacking and the formation of
intermolecular hydrogen bonds.
We investigated the solubilities and determined solubi-
-
lity over A2A-AR anity ratios for the potent, A2A
selective xanthine derivatives 3 (CSC) and 4 (CS-
DMPX), and for the new water-soluble sulfostyryl-
xanthine derivative 11a (Table 5). These data were
compared with values for the standard A2A antagonist
KF17837 (2), the therapeutically used xanthine deriva-
tives theophylline (21) and ca€eine (22), and the low-
anity A2-AR antagonist DMPX (1). DMPX shows
reasonable water-solubility (5 mM/L), the main reason,
E€orts have been undertaken to improve the water-
solubility of potent A1-selective AR-antagonists by
introducing hydrophilic substituents into the molecules
at positions where they are tolerated by the receptor.5
Table 5. Water solubilities of A2A-selective xanthine derivatives and reference compounds
Compd
K1 A2A [mM] rat brain
A2A-selectivity versus
A1 rat brain [3H]CHA
Solubility [mM]‹SEM
at rt in buffer pH 7.4
containing 1% DMSO
Solubility over A2A
affinity ratio
(Bruns±Fergus index)
[3H]CGS21680
11a p-SS-DMPX
2.4
20
6,400‹1,750
(7,800)a
26,830
1 DMPX
4 CS-DMPX
8.610
0.013
1.410 b
100
,
5000c
580
23
0.296‹0.070
(0.476‹0.085)d
0.14620
2 KF-17837
3 CSC
0.008510
0.03610
(0.054)9
9.410
3110
>2810
(540)9
17
5
0.179‹0.014
(0.211‹0.030)d
44,400e
21 Theophylline
22 Ca€eine
1.810
110
4720
4910
2210
108,000e
aSolubility in water at room temperature determined by UV photometry (single determination).
bExhibits somewhat higher A2A-selectivity in a comparison of data from other test systems, (e.g ®vefold selectivity versus [3H]PIA
binding to rat A1-AR).42
cSolubility in water.31
dSolubility [mM] at room temperature in bu€er pH 7.4 containing 2.5% DMSO.
eSolubility in water at 25 ꢁC.43

C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
715
why it is still widely used for in vivo studies despite its
low potency and low selectivity towards A1- and A2B
AR. Theophylline and ca€eine are both well soluble in
water in millimolar concentrations. Solubility over A2A
anity ratio for both compounds is almost 5000.
DMPX is somewhat less soluble, but still exhibits a
exothermic reaction below 35 ꢁC. After all of the cin-
namic acid had been added the reaction was stirred at rt
for 30 min. The thick light-brown solution was carefully
poured on 200 mL of ice water and left standing over-
night at rt. The crystalline product that separated was
collected by ®ltration using a sintered ®lter and washed
with 100 mL of diluted sulfuric acid (25%) and subse-
quently with 100 mL of diethyl ether. After drying 42 g
of p-sulfocinnamic acid were obtained as slightly orange
crystals. A second crop (2 g) could be ®ltered o€ after
cooling the ®ltrate.
-
-
Bruns-Fergus index of 580. The high anity A2A
-
antagonists, styrylxanthine derivatives 2, 3, and 4, how-
ever, show very low water-solubility in submicromolar
concentrations. As a consequence, their solubility over
anity ratio is only between 5 for CSC (3) and 23 for
CS-DMPX (4), which is far below the required 100. In
contrast, p-sulfostyryl-DMPX (11a) exhibits very high
solubility of ca. 6 mM/L and a BF-index of more than
26,000. Solubility for 11a was determined by the same
radioreceptor assay that was used for the other com-
pounds; an additional determination was made using
UV-photometry. The values obtained with the two dif-
ferent methods were similar (Table 5).
p-Sulfocinnamic acid. Yield: 57%; mp 82 ꢁC. H NMR
1
(D2O): d 6.25 (d, J=16.1 Hz, 1H, 2-H), 7.40 (d,
J=16.1 Hz, 1H, 3-H), 7.43 (d, J=8.3 Hz, 2H, 20-H, 60-
H), 7.61 (d, J=8.3 Hz, 2H, 30-H, 50-H). 13C NMR (D2O):
d 121.90 (C-2), 128.57 (C-30, C-50), 131.24 (C-20, C-60),
139.25 (C-10), 146.48 (C-40), 147.29 (C-3), 172.79 (C-1).
p-Sulfocinnamic acid potassium salt (7a). The p-sulfocin-
namic acid was converted to its potassium salt by treat-
ment with KOH, or K2CO3, respectively. Mp >260 ꢁC.
1H NMR (D2O): d 6.54 (d, J=16.1 Hz, 1H, 2-H), 7.68
(d, J=16.1 Hz, 1H, 3-H), 7.69 (d, J=8.3 Hz, 2H, 20-H,
60-H), 7.81 (s, J=8.3 Hz, 2H, 30-H, 50-H). 13C NMR
(D2O): d 122.12 (C-2), 128.58 (C-20, C-60), 131.28 (C-30,
C-50), 139.41 (C-10), 146.41 (C-40), 147.27 (C-3), 173.04
(C-1)
In conclusion, we have developed A2A-selective AR
antagonists, which exhibit high water-solubility and
may be useful research tools for in vivo studies.
Experimental
Chemistry
NMR spectra were performed on a Bruker WP-80 (1H:
80 MHz, 13C: 20 MHz), or a Bruker AC-250 spectro-
meter (1H: 250 MHz, 13C: 60 MHz), respectively.
DMSO-d6 or D2O, respectively, was used as solvent.
The chemical shifts of the remaining protons of the
deuterated solvent served as internal standard. IR spec-
tra were measured on a Perkin-Elmer 1750 spectro-
meter. All compounds were checked for purity by TLC
on 0.2 mm aluminum sheets with silica gel 60 F254
(Merck); as eluent dichloromethane:methanol (9:1, or
99:1, respectively) was used. Melting points were taken
on a Buchi 510 melting point apparatus and are uncor-
rected. Elemental analyses were performed by the Insti-
tute of Chemistry, University of Tubingen, or the
Institute of Inorganic Chemistry, University of Wurz-
burg, respectively. Satisfactory microanalyses were
obtained for ®nal products: C‹0.4, H‹0.32, N‹0.35,
unless otherwise noted.
m-Sulfocinnamic acid and its potassium salt (7b).
Method A:16 Sulfonation of cinnamic acid yields a mix-
ture of p- and m-sulfocinnamic acid. After the main
product, p-sulfocinnamic acid, had been ®ltered o€ (see
above, preparation of 7a) the aq ®ltrate was diluted to
1 L and 400 g of BaCO3 was added with vigorous stir-
ring. The formed precipitate of BaSO4 was ®ltered o€
and washed with 200 mL of H2O. The combined solu-
tions were evaporated to dryness and the residue was
dissolved in MeOH. K2CO3 was added in small portions
until e€ervescence ceased. The precipitate was ®ltered
o€ and the ®ltrate was concentrated to precipitate 7b,
which was collected by ®ltration. Puri®cation was
achieved by several recrystallizations from MeOH.
Yield: 10.3 g (11%); mp >260 ꢁC. 13C NMR (D2O): d
121.65 (C-2), 127.57 (C-20), 129.83 (C-40), 132.30 (C-50),
133.53 (C-60), 137.38 (C-10), 145.82 (C-30), 147.49 (C-3),
173.16 (C-1)
The syntheses of compounds 5, 17, and 20 have been
described.10,26,33
m-Sulfocinnamic acid. Free m-sulfocinnamic acid was
obtained by treatment of 7b with sulfuric acid furnishing
orange crystals. 1H NMR (DMSO-d6): d 6.52 (d,
J=16.1 Hz, 1H, 2-H), 7.36 7.85 (m, 5H, 3-H, Haromat.).
Method B:16 To a boiling solution of 3.7 g (44.0 mmol)
of sodium bicarbonate in 25 mL of water, 10 g
(44.0 mmol) of m-bromocinnamic acid and 5.8 g of an
aq sodium bisul®te solution (92%, 51.0 mmol) was
p-Sulfocinnamic acid and its potassium salt (7a).16 Finely
ground cinnamic acid (50.0 g, 337 mmol) was dissolved
in small portions in 75 mL of fuming sulfuric acid (con-
taining 30% of SO3) under vigorous stirring and cooling
with an ice-bath, maintaining the temperature of the

716
C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
added and the mixture was re¯uxed until a sample gave
no precipitate when acidi®ed. Then, 7.0 g of an aq
sodium bisul®te solution (92%, 52.7 mmol) and 0.5 g
(3.13 mmol) of CuSO4 were added and the mixture was
heated in a sealed tube at 175 ꢁC for 45 h. After remov-
ing the formed precipitate, the colorless ®ltrate was
treated with 5 g (29.2 mmol) of Ba(OH)2 to precipitate
sulfate and excess sul®te, which were removed by ®ltra-
tion. To the ®ltrate another 15 g of Ba(OH)2
(87.5 mmol) was added, the solution was concentrated
to 20 mL and then heated to re¯ux until no more bar-
ium sul®te precipitated. The precipitate formed was
removed from time to time by ®ltration. The ®ltrate was
then cooled in a mixture of ice and NaCl=3:1 (w/w)
and ®ltered again. The ®ltrate was saturated with CO2
and ®ltered one more time. The ®ltrate was then acid-
i®ed with 6 N HCl to a pH value of 0. Some of the
unreacted m-bromocinnamic acid precipitated, further
bromocinnamic acid was extracted with diethyl ether.
The aq layer was separated, heated to boiling and trea-
ted with 6 g (28.8 mmol) of BaCl2 in 25 mL of water. On
cooling, a precipitate of the barium salt of m-sulfocin-
namic acid formed which was ®ltered o€ and recrys-
tallized from water; yield: 73%. The free acid was
obtained by treatment with sulfuric acid; yield: 97%; mp
74 ꢁC. The potassium salt 7b can be prepared from the
acid as described above (Method A)
3-Methyl-1-propargyl-8-(p-sulfostyryl)xanthine potassium
salt (10a). Compound 9a (3.0 g, 6.78 mmol) was sus-
pended in 100 mL of MeOH, and then 2 mL of a 20%
aq solution of NaOH was added. The reaction mixture
was heated to re¯ux for 3 h. After cooling to rt 80 mL of
diethyl ether were poured into the ¯ask producing a
precipitate, which was ®ltered o€, washed with diethyl
ether, and dried in an oven at 90 ꢁC. Yield: 2.14 g (74%);
mp >300 ꢁC. C17H13KN4O5S*2 H2O: C, H, N. IR,
cm 1: 3425, 3271, 2125, 1674, 1628, 1593.
3,7-Dimethyl-1-propargyl-8-(p-sulfostyryl)xanthine pot-
assium salt (11a). Compound 10a (0.93 g, 2.19 mmol)
and K2CO3 (1.5 g, 11 mmol) were suspended in 20 mL of
DMF and 0.58 mL (9.3 mmol) of methyl iodide was
added. The reaction mixture was stirred at rt overnight.
The precipitate formed was collected by ®ltration and
washed with DMF. Filtrate and washings were com-
bined and concentrated nearly to dryness in vacuo.
Then 20 mL of diethyl ether was added generating a
white precipitate, which was ®ltered o€ and washed with
diethyl ether. Puri®cation was achieved by dissolution in
hot methanol, ®ltration, and subsequent concentration
of the methanol solution. The slightly yellowish solid
was dried in the oven. Yield: 83%; mp >300 ꢁC.
C18H15KN4O5S*2 H2O: C, H, N. IR, cm 1: 2125, 1706,
1661, 1598, 1545, 1483.
6-Amino-3-propargyl-5-(p-sulfocinnamoyl)aminouracil
potassium salt (8a). A suspension of 2.0 g (11.1 mmol)
of 5,6-diamino-3-propargyluracil (6),17,18 2.13 g
(11.1 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride (EDC) and 2.96 g (11.1 mmol) of
the monopotassium salt of p-sulfocinnamic acid (7a) in
60 mL of water and 1 mL of MeOH was stirred vigor-
ously at rt overnight. After that period of time no more
diaminouracil could be detected by TLC. The reaction
mixture was concentrated in vacuo and the precipitate
was collected by ®ltration. The solid was washed with
MeOH and dried at 90 ꢁC a€ording 3.69 g (77.6%) of a
white solid. C16H13KN4O6S*H2O: C, calcd 43.04;
found, 42.53; H, N. Mp > 300 ꢁC. IR, cm 1: 3470,
3271, 3214, 2125, 1737, 1676, 1651, 1626.
6-Amino-3-propargyl-5-(3-sulfostyrylcarboxamido)uracil
(8b). To
a suspension of 0.477 g (2.09 mmol) of
3-sulfocinnamic acid (7b) and 0.370 g (2.05 mmol) of
5,6-diamino-3-propargyluracil17,18 in 17 mL of MeOH
0.402 g (2.09 mmol) of EDC was added and the mix-
ture was stirred at rt for 24 h. The mixture was
concentrated in vacuo and the precipitate collected by
®ltration and washed with MeOH. Yield: 0.50 g (63%);
mp >250 ꢁC.
6-Amino-1-methyl-3-propargyl-5-(3-sulfostyrylcarboxamido)
uracil (9b). Compound 8b (0.400 g, 1.03 mmol) was dis-
solved in 10 mL of DMF. Methyl iodide (1.49 g,
10.3 mmol, 642 mL) and K2CO3 (0.313 g, 2.27 mmol)
were added and the mixture was stirred at rt for 48 h.
The solvent was removed in vacuo and the residue was
dissolved in 25% aq HCl solution. Solid impurities were
removed by ®ltration. The ®ltrate was evaporated to
dryness and the residue suspended in MeOH. The light-
yellow product was collected by ®ltration. Yield: 0.190 g
(46%); mp >250 ꢁC.
6-Amino-1-methyl-3-propargyl-5-(p-sulfocinnamoyl)amino-
uracil potassium salt (9a). To a suspension of 2.14 g
(5 mmol) of 8a and 1.38 g (10 mmol) of K2CO3 in 20 mL
of DMF 0.4 mL (6 mmol) of methyl iodide was added.
The reaction mixture was stirred for 8 h at rt. Then
water (30 mL) was added. The precipitate formed was
®ltered o€ and washed with water, then with MeOH,
and ®nally with diethyl ether. After drying, 2.04 g (92%)
of a white solid was obtained. C18H13KN4O6S*3H2O:
C, calcd 41.12; found, 39.40; H, N. Mp 301±302 ꢁC
(decomp.). IR, cm 1: 3464, 3253, 3011, 2125, 1703,
1671, 1632, 1591.
3-Methyl-1-propargyl-8-p-sulfostyrylxanthine monosodium
salt (10b). Compound 9b (0.240 g, 0.595 mmol) was
dissolved in a mixture of 40.0 mL of MeOH and 6 mL of
water. After the addition of 17.0 mL 20% aq NaOH
solution the mixture was heated to 65 ꢁC for 3 h. After
cooling, the volume was reduced in vacuo, and the

C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
717
formed precipitate was collected by ®ltration. Yield:
0.180 g (74%); mp >250 ꢁC.
Pharmacological Methods
Materials
3,7-Dimethyl-1-propargyl-8-(3-sulfostyryl)xanthine pot-
assium salt (11b). Compound 10b (0.100 g, 0.242 mmol),
K2CO3 (0.185 g, 1.35 mmol) and methyl iodide (0.148 g,
1.14 mmol, 71.3 mL) were suspended in 2.5 mL of DMF
and stirred at rt for 48 h. The formed precipitate was
®ltered o€ and washed with DMF. The ®ltrate was
evaporated to dryness, and the residue was taken up in
5 mL of MeOH. After the addition of 30 mL of diethyl
ether a light-yellow precipitate was obtained which was
collected by ®ltration. Yield: 0.070 g (66%); mp 255 ꢁC.
C18H15KN4O5S*3 H2O: C, H, N.
Radiolabelled compounds were from NEN Life Sci-
ences, Dreieich, Germany. All other materials were from
sources as described earlier.22,34
A1- and A2A-Adenosine receptor radioligand binding as-
says. Inhibition of binding of [3H]N6-cyclohexyladeno-
sine ([3H]CHA) to A1-adenosine receptors of rat cerebral
cortical membranes and inhibition of [3H]2-[4-(carboxy-
ethyl)phenylethylamino]-50 -N-ethylcarboxamido-adeno-
sine ([3H]CGS21680) to A2A-adenosine receptors of rat
striatal membranes were assayed as described.34 Inhibi-
tion of the receptor-radioligand binding was determined
by a range of 5 to 6 concentrations of the compounds in
triplicate in at least two to three separate experiments.
The Cheng Pruso€ equation35 and KD values of 1 nM
for [3H]CHA and 14 nM for [3H]CGS21680 were used
to calculate the Ki values from the IC50 values, deter-
mined by the nonlinear curve ®tting program Prism
(GraphPad, San Diego, California, USA).
1,3-Dipropyl-8-p-sulfostyrylxanthine
monopotassium
salt (13). 5,6-Diamino-1,3-dipropyluracil (12, 0.919 g,
4.06 mmol) was dissolved in a mixture of 10 mL of water
and 10 mL of MeOH. 4-Sulfocinnamic acid mono-
potassium salt (7a, 1.13 g, 4.26 mmol) and EDC (0.814 g,
4.26 mmol) were added. The mixture was stirred at rt for
24 h. Then the solvent was removed in vacuo and the
residue was suspended in a small amount of MeOH.
Diethyl ether was added to precipitate 6-amino-1,3-
dipropyl-5-(p-sulfostyrylcarboxamido)uracil, which was
collected by ®ltration.
Transfected cells and membranes containing human A2B
-
and A3-AR. CHO cells were stably transfected with the
human A2B- and A3-adenosine receptors as described.22
Cells were grown on petri dishes (é 140 mm) to con-
¯uency. Then cells were washed and used immediately
for membrane preparation for measurement of AC
activity (A2B), or frozen in the dishes and kept at 25 ꢁC
until membranes were prepared for binding studies (A3).
6-Amino-1,3-dipropyl-5-(p-sulfostyrylcarboxamido)uracil
monopotassiun salt. 1H NMR (DMSO-d6) d 0.88 (m,
6H, 2ÂCH3), 1.60 (m, 4H, 2ÂCH2-CH3), 3.71 (t, 2H, N-
CH2), 3.81 (t, 2H, N-CH2), 6.71 (s, 2H, NH2), 6.82 (d,
J=16.1, 1H, CH=CH), 7.42 (d, J=15.9, 1H, CH=CH),
7.53 (d, J=7.9, 2H, Haromatic), 7.63 (d, J=8.0, 2H,
For the preparation of crude membranes for A3-recep-
tor binding frozen cells were thawed and then scraped
o€ the petridishes in hypotonic bu€er (5 mM Tris/HCl,
2 mM EDTA, pH 7.4). The cell suspension was homo-
genized (Ultra-Turrax, 2Â15 s at full speed) and the
homogenate was spun for 10 min at 1,000 g. The super-
natant was then centrifuged for 40 min at 50,000 g. The
membrane pellet was resuspended in 50 mM Tris/HCl,
pH 8.25 containing 1 mM EDTA and 10 mM MgCl2,
frozen in liquid nitrogen at a protein concentration of
1±3 mg/mL and stored at 80 ꢁC.
H
aromatic), 8.68 (s, 1H, NH). 13C NMR (DMSO-d6) d
10.93 (CH3), 11.38 (CH3), 15.84 (2ÂCH2-CH3), 42.55
(N-CH2), 44.72 (N-CH2), 74.90 (C-5), 111.83
(CH=CH), 126.50 (2ÂCaromatic), 127.12 (2ÂCaromatic),
135.21, 149.28 (Caromatic, CH=CH), 150.46 (C-2), 151.49
(C-6), 159.20 (C-4), 165.44 (C=O).
Ring closure to the xanthine 13 was achieved by dis-
solution of the uracil derivative in 20 mL of 10% aq
NaOH solution, heating for 20 min at 60 ꢁC, ®ltration of
the hot solution, and precipitation of the product by
acidi®cation of the ®ltrate with concd HCl solution to a
pH value of 4. Yield: 0.870 g (49%); mp >300 ꢁC.
C19H21KN4O5S* H2O: C, H, N.
For the measurement of adenylate cyclase activity (A2B
)
a slightly modi®ed protocol with only one centrifugation
step was used. Fresh cells were homogenized and the
homogenate was sedimented for 30 min at 54,000 g. The
resulting pellet was resuspended in 50 mM Tris/HCl pH
7.4 and used for the adenylate cyclase assay immediately.
1,3-Dipropyl-7-methyl-8-p-sulfostyrylxanthine (14). Com-
pound 13 (0.200 g, 0.438 mmol) and K2CO3 (0.303 g,
2.19 mmol) were suspended in DMF. Methyl iodide
(120 mL, 1.93 mmol) was added and the mixture was
stirred for 2 days at rt. The solvent was removed in vacuo
and the residue was taken up in water, precipitated by the
addition of concd HCl, and collected by ®ltration. Yield:
0.120 g (63%); mp >250 ꢁC. C20H24N4O5S* H2O: C, H, N.
Adenylate cyclase activity
The procedure was carried out as described previously36
with minor modi®cations. Membranes were incubated
with about 150,000 cpm of [a-32P]ATP for 20 min in the

718
C. E. Muller et al./Bioorg. Med. Chem. 6 (1998) 707±719
È
incubation mixture as described36 without EGTA and
NaCl. IC50 values for concentration-dependent inhibi-
tion of NECA-stimulated adenylate cyclase (5 mM
NECA) caused by the antagonists under investigation
were calculated with the Hill equation. Hill coecients
in all experiments were near unity. Dissociation con-
stants (Ki) for antagonist at A2B-adenosine receptors
were then calculated with the Cheng±Pruso€ equation.35
2. Collis, M. G.; Hourani, S. M. O. Trends Pharmacol. Sci.
1993, 14, 360.
3. Muller, C. E.; Scior, T. Pharm. Acta Helv. 1993, 68, 77.
4. Muller, C. E.; Stein, B. Current Pharm. Design 1996, 2, 501.
5. Muller, C. E. Exp. Opin. Ther. Patents 1997, 7, 419.
6. Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Borioni, A.;
Viziano, M.; Dionisotti, S.; Ongini, E. Curr. Med. Chem. 1995,
2, 707.
7. Seale, T. W.; Abla, K. A.; Shamim, M. T.; Carney, J. M.;
Daly, J.W. Life Sci. 1988, 43, 1671.
A3-AR radioligand binding assay
8. Shimada, J.; Suzuki, F.; Nonaka, H.; Ishii, A.; Ichikawa, S.
J. Med. Chem. 1992, 35, 2342.
Dissociation constants (Ki values) for the antagonists at
A3-adenosine receptors were determined in radioligand
competition experiments. The nonselective agonist
[3H]NECA was used as the radioligand at a concentra-
tion of 10 nM. Binding was carried out in a total volume
of 200 mL in 96-well ®lter-bottom microplates (Millipore
MultiScreen MAFC) with 20±25 mg of membrane pro-
tein in 50 mM Tris/HCl with 1 mM EDTA and 10 mM
MgCl2, pH 8.25. Samples were incubated for 3 h at
25 ꢁC, ®ltered and washed as described.22 Data were
analyzed by nonlinear curve-®tting with the the pro-
gram SCTFIT.37
9. Jacobson, K. A.; Gallo-Rodriguez, C.; Melman, N.; Fischer,
B.; Maillard, M.; van Bergen, A.; van Galen, P. J. M.; Karton,
Y. J. Med. Chem. 1993, 36, 1333.
10. Muller, C. E.; Geis, U.; Hipp, J.; Schobert, U.; Frobenius,
W; Pawlowski, M.; Suzuki, F.; Sandoval-Ramõrez, J. J. Med.
Chem. 1997, 40, 4396.
11. Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Pineda de las
Infantas y Villatoro, M. J.; Zocchi, C.; Dionisotti, S.; Ongini,
E. J. Med. Chem. 1996, 39, 1164.
12. Poucher, S. M.; Keddie, J. R.; Singh, P.; Stoggall, S. M.;
Caulkett, P. W. R.; Jones, G.; Collis, M. G. Br. J. Pharmacol.
1995, 115, 1096.
13. Daly, J. W.; Padgett, W.; Shamim, M. T.; Butts-Lamb, P.;
Waters, J. J. Med. Chem. 1985, 28, 487.
Solubility determination
14. Hamilton, H. W.; Ortwine, D. F.; Worth, D. F.; Badger,
E.W.; Bristol, J. A.; Bruns, R. F.; Haleen, S.J.; Ste€en, R. P. J.
Med. Chem. 1985, 28, 1071.
Solubilities of compounds were determined according to
the method of Bruns and Fergus.27 A 10 mM solution of
compounds in DMSO was prepared, diluted 1:100, or
1:40, respectively, in Tris±HCl bu€er, 50 mM, pH 7.4,
and allowed to reach equilibrium with shaking for 24 h
at rt in the dark. After centrifugation, the supernatant
was ®ltered through cotton. Several dilutions of these
saturated stock solutions were made in the bu€er and an
15. Baumgold, J.; Nikodijevic, O.; Jacobson, K. A. Biochem.
Pharmacol. 1992, 43, 889.
16. Moore, F. J.; Tucker, G. R. J. Am. Chem. Soc. 1927, 49,
258.
17. Muller, C. E. Synthesis 1993, 125±128.
18. Muller, C. E.; Sandoval-Ramõrez, J.A. Synthesis 1995, 1295.
19. Muller, C. E. J. Org. Chem. 1994, 59, 1928.
A
2A-AR binding assay was performed (see above). The
solubility of each compound was then calculated by
dividing the IC50 value of a compound, determined
separately, by the fold dilution of the saturated solution
required to give 50% inhibition of radioligand binding.
20. Nonaka, Y.; Shimada, J.; Nonaka, H.; Koike, N.; Aoki,
N.; Kobayashi, H.; Kase, H.; Yamaguchi, K.; Suzuki, F. J. J.
Med. Chem. 1993, 36, 3731.
21. Friebolin, H. Ein- und zweidimensionale NMR-Spektros-
kopie (One- and two-dimensional NMR spectroscopy), 2nd ed.
1992, VCH Weinheim, 1992; p. 99 €.
Solubility of the highly water-soluble sulfostyryl xanthine
11a was additionally determined by UV-spectroscopy.
22. Klotz, K.-N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.;
Fredholm, B. B.; Lohse, M. J. Naunyn-Schmiedeberg's Arch.
Pharmacol. 1998, 357, 1.
Acknowledgements
23. Cerfontain, H.; Koeberg-Felder, A.; Kruk, C. Tetrahedron
Lett. 1975, 3639.
The expert technical assistance of Ms. Sonja Kachler is
gratefully acknowledged. J.S.-R. was supported by a
grant from the Deutscher Akademischer Austausch-
dienst (DAAD). C.E.M. is grateful for support by the
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