Preparation, Properties, Reactions, and Adenosine Receptor Affinities of Sulfophenylxanthine Nitrophenyl Esters: Toward the Development of Sulfonic Acid Prodrugs with Peroral Bioavailability

Article abstract of DOI:10.1021/jm0310030

Many currently known antagonists for P2 purinergic receptors are anionic molecules bearing one or several phenylsulfonate groups. Among the P1 (adenosine) receptor antagonists, the xanthine phenylsulfonates are a potent class of compounds. Due to their high acidity, phenylsulfonates are negatively charged at physiologic pH values and do not easily penetrate cell membranes. The present study was aimed at developing lipophilic, perorally bioavailable prodrugs of sulfonates by converting them into chemically stable nitrophenyl esters. Initial stability tests at different pH values using nitrophenyl tosylates as model compounds showed that m-nitrophenyl esters were stable over a wide pH range, while the ortho and para isomers were less stable under strongly acidic or basic conditions. A series of m- and p-nitrophenyl esters of p-sulfophenylxanthine derivatives were synthesized as model compounds. The target xanthine derivatives were obtained in high yields by condensation of the appropriate 5,6-diaminouracils with 4-(nitrophenoxysulfonyl)benzoic acids in the presence of a carbodiimide, followed by ring closure with polyphosphoric acid trimethylsilyl ester. The chemical and enzymatic stability of the m-nitrophenyl esters was investigated in vitro by means of capillary electrophoresis. High stability in aqueous solution, in artificial gastric acid, and in serum was observed. However, compound 5d, used as a prototypic xanthine m-nitrophenylsulfonate, was hydrolyzed by rat liver homogenate indicating an enzymatic pathway of hydrolysis. Thus, nitrophenyl esters of sulfonic acids have a potential as peroral prodrugs of drugs bearing a sulfonate group. The nitrophenyl esters of sulfophenylxanthines were additionally investigated for their adenosine receptor affinities. They showed high affinity at A ₂, A_2A, and A_2B, but not at A₃ ARs. One of the most potent compounds was 1-propyl-8-[4-[[p-nitrophenoxy]sulfonyl]-phenyl]xanthine (9d), a mixed A ₁/A_2B antagonist (K_iA₁ 3.6 nM, K_iA_2B 5.4 nM) selective versus the other subtypes. As a further result of this study, the m-nitrophenoxy group was found to be a suitable protecting group for sulfonates in organic synthesis due to its high lipophilicity and stability; it can be split off under strongly basic conditions. This new protection strategy allowed for the upscaling of the synthesis of 1-propyl-8-p-sulfophenylxanthine (PSB-1115), a selective A _2B antagonist.

Full text of DOI:10.1021/jm0310030

J . Med. Chem. 2004, 47, 1031-1043
1031
P r ep a r a tion , P r op er ties, Rea ction s, a n d Ad en osin e Recep tor Affin ities of
Su lfop h en ylxa n th in e Nitr op h en yl Ester s: Tow a r d th e Develop m en t of Su lfon ic
Acid P r od r u gs w ith P er or a l Bioa va ila bility
Luo Yan and Christa E. Mu¨ller*
University of Bonn, Pharmaceutical Chemistry Poppelsdorf, Pharmaceutical Sciences, Bonn, Germany
Received August 11, 2003
Many currently known antagonists for P2 purinergic receptors are anionic molecules bearing
one or several phenylsulfonate groups. Among the P1 (adenosine) receptor antagonists, the
xanthine phenylsulfonates are a potent class of compounds. Due to their high acidity,
phenylsulfonates are negatively charged at physiologic pH values and do not easily penetrate
cell membranes. The present study was aimed at developing lipophilic, perorally bioavailable
prodrugs of sulfonates by converting them into chemically stable nitrophenyl esters. Initial
stability tests at different pH values using nitrophenyl tosylates as model compounds showed
that m-nitrophenyl esters were stable over a wide pH range, while the ortho and para isomers
were less stable under strongly acidic or basic conditions. A series of m- and p-nitrophenyl
esters of p-sulfophenylxanthine derivatives were synthesized as model compounds. The target
xanthine derivatives were obtained in high yields by condensation of the appropriate
5,6-diaminouracils with 4-(nitrophenoxysulfonyl)benzoic acids in the presence of a carbodiimide,
followed by ring closure with polyphosphoric acid trimethylsilyl ester. The chemical and
enzymatic stability of the m-nitrophenyl esters was investigated in vitro by means of capillary
electrophoresis. High stability in aqueous solution, in artificial gastric acid, and in serum was
observed. However, compound 5d , used as a prototypic xanthine m-nitrophenylsulfonate, was
hydrolyzed by rat liver homogenate indicating an enzymatic pathway of hydrolysis. Thus,
nitrophenyl esters of sulfonic acids have a potential as peroral prodrugs of drugs bearing a
sulfonate group. The nitrophenyl esters of sulfophenylxanthines were additionally investigated
for their adenosine receptor affinities. They showed high affinity at A1, A2A, and A2B, but not
at A3 ARs. One of the most potent compounds was 1-propyl-8-[4-[[p-nitrophenoxy]sulfonyl]-
phenyl]xanthine (9d ), a mixed A1/A2B antagonist (KiA1 3.6 nM, KiA2B 5.4 nM) selective versus
the other subtypes. As a further result of this study, the m-nitrophenoxy group was found to
be a suitable protecting group for sulfonates in organic synthesis due to its high lipophilicity
and stability; it can be split off under strongly basic conditions. This new protection strategy
allowed for the upscaling of the synthesis of 1-propyl-8-p-sulfophenylxanthine (PSB-1115), a
selective A2B antagonist.
In tr od u ction
Since the late 1970s, when Burnstock proposed the
subclassification of purinergic receptors into P1 and P2
families,1 the development of potent and selective
antagonists for these two classes of receptors as phar-
macological tools and as potential drugs has been an
active area of research.2,3 P2 receptors are activated by
nucleotides such as ATP, ADP, UTP, or UDP, which
bear phosphate groups that are negatively charged at
physiological pH values.3 The vast majority of P2
receptor antagonists known to date are also anionic
molecules, e.g. suramin, Reactive Blue 2, PPADS, and
derivatives thereof3 (Figure 1). All of them share a
common partial structure containing one or several
phenylsulfonate moieties. The negatively charged phos-
phate groups of P2 agonists and the negatively charged
* Address correspondence to Dr. Christa E. Mu¨ller, Pharmazeu-
tisches Institut, Pharmaceutical Sciences Bonn, Pharmazeutische
Chemie Poppelsdorf, Kreuzbergweg 26, D-53115 Bonn, Ger-
many. Phone: +49-228-73-2301. Fax: +49-228-73-2567. E-mail:
christa.mueller@uni-bonn.de.
F igu r e 1. Examples of P2 receptor antagonists with phenyl-
sulfonate structure.
10.1021/jm0310030 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/10/2004

1032 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Yan and Mu¨ller
F igu r e 2. Examples of P1 (adenosine) receptor antagonists
with phenylsulfonate structure: 8-p-sulfophenylxanthine de-
rivatives.
F igu r e 3. Structure of the P2X7 receptor antagonist KN62.
acid esters are used in synthetic organic chemistry (e.g.
mesylates, tosylates) and as anticancer drugs (busul-
fane, treosulfane) due to their alkylating properties.15
However the stability of sulfonic acid esters greatly
depends on the substitution pattern. Highly stable
esters can be obtained if suitable, electron-withdrawing,
deactivating substituents are introduced. Examples of
obviously stable sulfonic acid esters are the 1-N,O-bis-
1,5-isoquinolinesulfonyl-N-methyl-L-tyrosyl-4-phenylpip-
erazine (KN 62) (Figure 3), which was introduced as a
P2X7 antagonist, and related compounds.16 In the
present study we synthesized nitrophenyl esters of a
series of sulfophenylxanthine derivatives in order to test
our hypothesis that chemically stable sulfonic acid
esters may be suitable sulfonate prodrugs. The stability,
reactivity, and enzymatic hydrolysis of the sulfonate
esters was investigated, and adenosine receptor affini-
ties of the new xanthine derivatives were also deter-
mined.
sulfonate groups of P2 antagonists are believed to bind
to positively charged amino acid residues which are
typically found in the ligand binding pocket of P2
receptors.3,4
In the P1 antagonist field, sulfophenyl derivatives
have also been described, including 8-p-sulfophenyl-
theophylline (SPT) and 1,3-dipropyl-8-p-sulfophenyl-
xanthine (DPSPX), which were introduced by Daly et
al. because of their high water-solubility.5,6 Recently,
1-propyl-8-p-sulfophenylxanthine (PSB-1115) was de-
veloped as an A2B-selective adenosine receptor (AR)
antagonist7 (Figure 2). The sulfonic acid group appears
to contribute to the high selectivity of the compound at
A2B ARs versus the other AR subtypes, especially versus
A1 ARs.7
Due to the low pKa value of free sulfonic acid groups
(pKa < 1),8 sulfophenylxanthine derivatives and P2
antagonists with phenylsulfonate structure are depro-
tonated under physiological conditions. This means that
they do not penetrate into the central nervous system
(CNS) and thus are only peripherally active.9-11 How-
ever, to be able to investigate CNS effects in vivo, e.g.
a potential neuroprotective effect of P2 antagonists,12
penetration into the brain is essential. Furthermore,
because of their polar character, sulfonates are not
readily able to cross cell membranes and thus can
hardly be absorbed from the gut if they are perorally
applied. Therefore, they are only parenterally ap-
plicable, if a systemic effect is desired. However, the
preferred way of drug application is peroral.
Resu lts a n d Discu ssion
Ch em istr y. Nitrophenyl esters were selected as
potential lipophilic prodrugs of sulfonic acid drugs due
to their generally high chemical stability.17-22 Initial
stability studies were performed with nitrophenyl esters
of tosylate, namely o-, m-, and p-nitrophenyl tosylate
as model compounds, which were synthesized as previ-
ously described.23 Subsequently, nitrophenyl esters of
p-sulfophenylxanthine derivatives, potent adenosine
receptor antagonists, were prepared.
Our approach to overcome these obstacles was the
development of prodrugs of these pharmacologically
active aryl sulfonic acid derivatives. The prodrugs
should be more lipophilic than their parent drugs in
order to be able to cross cell membranes and to be
absorbed from the gastrointestinal tract. The com-
pounds should be stable in the strongly acidic medium
of the stomach and stay intact before being absorbed
and reaching the target cells. They should release the
parent active drug in vivo, preferably by an enzymatic
mechanism.
Ester prodrugs of compounds bearing acidic groups
have been described. Such prodrugs are well-known for
carboxylic and phosphoric acids, but not for sulfonic
acids.13,14 Two kinds of sulfonic acid derivatives are
conceivable as potential prodrugs: amides of sulfonic
acids (sulfonamides) or sulfonic acid esters. Sulfona-
mides are highly stable in vivo and therefore are
unsuitable as sulfonate prodrugs.14 In contrast, sulfonic
acid esters are relatively unstable since they may react
with nucleophiles in vitro and in vivo. Reactive sulfonic
Syn th esis of Xa n th in e Su lfon ic Acid Nitr op h en -
yl Ester s. 1,3-Dimethyl-8-p-sulfophenylxanthine was
prepared according to published procedures.5,6,24 5,6-
Diamino-1,3-dimethyluracil 1a was used as a starting
compound (Scheme 1). For the preparation of the
carboxybenzamidouracil 2a , the diaminouracil 1a was
reacted with p-sulfobenzoic acid potassium salt using
the water-soluble N-(3-dimethylaminopropyl)-N′-ethyl-
carbodiimide hydrochloride (EDC) as a condensing
agent. Ring closure of 2a to xanthine 2b was performed
in aqueous sodium hydroxide solution. Methylation
yielded the corresponding caffeine derivative 2d as
previously described.24 Esterification of 2b was initially
attempted by transformation of the p-sulfophenylxan-
thine to the corresponding chlorosulfonylphenylxan-
thine derivative using thionyl chloride, followed by
esterification with nitrophenol under basic conditions.
In fact, chlorination of the p-sulfophenyl xanthine could
successfully be performed by refluxing compound 2b
with thionyl chloride, as confirmed by mass spectrom-
etry of product 2c (Scheme 1). However, the subsequent

Sulfophenylxanthine Nitrophenyl Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1033
Sch em e 1. Initial Strategy for the Synthesis of m-Nitrophenyl Ester Prodrugs of p-Sulfophenyltheophyllinea
a
Reagents: (a) potassium p-sulfobenzoate, EDC, H2O, rt, 1 h; (b) 2.5 N NaOH, 70 °C, 10 min; (c) SOCl2, reflux, 2 h; (d) MeI, DMF,
K2CO3, rt, overnight; (e) m-nitrophenol or sodium m-nitrophenoxide.
esterification of the chlorosulfonylphenylxanthine with
an excess of m-nitrophenol or sodium m-nitrophenoxide
could not be achieved under various reaction conditions,
e.g. in the presence of triethylamine or pyridine as a
base at different temperatures ranging from -30 to 30
°C.
large, multigram scale. Monosubstituted carboxamido-
uracil derivatives possess low reactivity, and the ring
closure to obtain 1-substituted 8-p-sulfophenylxanthines
requires harsh reaction conditions, e.g. reflux with
hexamethyldisilazane (HMDS) for more than 50 h.7 If
the reported PPSE method was applied for ring closure,
isolation of the desired products was difficult, since
PPSE as well as the products are well soluble in water
and methanol. 1-Substituted 8-p-sulfophenylxanthines
have considerable pharmaceutical potential because of
their good water-solubility and their high selectivity for
A2B ARs.7 Therefore gram amounts were required for
extended pharmacological investigations including ani-
mal experiments.30 Since large amounts were difficult
to obtain by the reported methods, we tried to develop
an improved synthesis using the nitrophenyl esters as
protecting groups. m-Nitrophenyl esters of 1-methyl-
and 1-propyl-8-p-sulfophenylxanthine (compounds 5c
and 5d ) could easily be obtained, isolated, and purified
in multigram amounts (Scheme 2). Subsequent alkaline
hydrolysis using 2 N aqueous sodium hydroxide solution
yielded the desired sulfophenylxanthine derivatives 6c
and 6d within 30 min in almost quantitative yields,
isolated yields ranging between 80 and 90% (Scheme
3). The products were recrystallized from H2O as
reported.7 Alternatively, preparative HPLC was applied,
which proved to be an excellent method for the purifica-
tion even of large amounts (several grams) of 1-substi-
tuted-8-p-sulfophenylxanthines. For the quick deter-
mination of the purity of the synthesized xanthine
derivatives 6c and 6d , a fast, convenient capillary
electrophoresis ultraviolet (CE-UV) method was devel-
oped. Figure 4 shows a typical electropherogram of
compound 6d , confirming the high purity of 1-propy-8-
p-sulfophenylxanthine.
As a consequence, an alternative synthetic pathway
was developed (Scheme 2). First p-sulfobenzoic acid
potassium salt was chlorinated at low temperature
using chlorosulfonic acid.26,27 The resulting sulfonyl
chloride was converted to 4-(m-nitrophenoxysulfonyl)-
benzoic acid 3, or 4-(p-nitrophenoxysulfonyl)benzoic acid
7, respectively, under Schotten-Baumann conditions.28
Compounds 3 and 7 were condensed with 5,6-diamino-
uracils 1a -e using N-(3-dimethylaminopropyl)-N′-ethyl-
carbodiimide hydrochloride (EDC) as condensing agent
to obtain 6-amino-5-[4-[[m-nitrophenoxy]sulfonyl]benz-
amido]uracil derivatives 4a -e or 6-amino-5-[4-[[p-nitro-
phenoxy]sulfonyl]benzamido]uracil derivatives 8a -e.
Ring closure of 4a -e and 8a -e to 8-[4-[[m-nitrophen-
oxy]sulfonyl]phenyl]xanthine derivatives 5a -e and the
corresponding p-nitrophenyl-substituted derivatives 9a-e
were achieved by heating of the compounds with poly-
phosphoric acid trimethylsilyl ester (PPSE), a powerful
but mild condensing agent, at 170 °C for 1.5-2 h25,29
(Scheme 2).
Since the nitrophenoxysulfonylphenylxanthine de-
rivatives could easily be obtained, isolated, and purified
in large quantities by this method, we subsequently
tested whether the nitrophenoxy group might be used
as a protecting group in the synthesis of phenylsulfonic
acid derivatives.
m-Nitr oph en oxy as a P r otectin g Gr ou p for P h en -
ylsu lfon ic Acid s: New , Im p r oved Syn th esis of
1-Su b st it u t ed 8-p -Su lfop h en ylxa n t h in e Der iva -
tives. The synthesis of 1-substituted 8-p-sulfophenyl-
xanthine derivatives, such as 1-propyl- or 1-butyl-8-p-
sulfophenylxanthine, has been reported.7,25 However,
the described syntheses are difficult to perform on a
Thus, hydrolysis of m-nitrophenyl esters of 1-substi-
tuted sulfophenylxanthine derivatives is a convenient
method to obtain 1-substituted 8-p-sulfophenylxanthine
derivatives, especially when gram amounts of compound

1034 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Yan and Mu¨ller
Sch em e 2. Synthesis of Nitrophenyl Ester Prodrugs of p-Sulfophenylxanthine Derivativesa
a
Reagents: (a) 1. chlorosulfonic acid, rt overnight, 2. chipped ice; (b) m-nitrophenol or p-nitrophenol, THF, pH ) 8-9, 4 h; (c) 3 or 7,
EDC, MeOH, rt; (d) PPSE, 120 °C, 10 min, 170 °C, 1.5-2 h. 6c: R1 ) methyl. 6d : R1 ) propyl.
Sch em e 3. Preparation of 1-Substituted 8-p-Sulfophenylxanthine Derivatives by Hydrolysis of the m-Nitrophenyl
Estersa
a
Reagent: (a) 2 N NaOH; 70 °C, 30 min.
are needed. On the other hand, the hydrolysis reaction
showed that these sulfonic acid esters are relatively
stable since complete hydrolysis lasted for about 20 min
at 70 °C in 2 N aqueous sodium hydroxide solution. A
considerable amount of starting compound was still
detectable after 10 min of hydrolysis under these
conditions. Due to the balanced stability-reactivity
profile of m-nitrophenyl esters of phenylsulfonic acids,
m-nitrophenyl is a suitable protecting group for sulfonic
acid derivatives cleavable under strongly basic condi-
tions.
An a lyt ica l Da t a . In Table 1, yields, melting
points, and analytical data of all final and most inter-
mediate products are collected. 1H and 13C NMR
data of all intermediate and final products were re-
corded; they were in accordance with the proposed
structures. Complete NMR data are available as
Supporting Information.
Stability of Sim ple Su lfon ic Acid Ester s as Model
Com p ou n d s. Investigations of the hydrolysis and
stability of different sulfonic acid esters have previously
been performed.17-22 Nitrophenoxy sulfonates belong to
the most stable esters among differently substituted
sulfonic acid esters. However, there are no further
reports on a comparison of the hydrolysis constants of
o-, m-, and p-nitrophenyl benzene sulfonates at different
pH values. Therefore syntheses and stability tests of
model compounds, i.e., o-, m-, and p-nitrophenyl tosy-
late, were initially performed.
Since these esters are not water-soluble, acetonitrile
was added for solubilization, the ratio of acetonitrile:
buffer being 10:90 or 5:95, respectively. Different aceto-

Sulfophenylxanthine Nitrophenyl Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1035
Ta ble 1. Yields, Melting Points, and Analytical Data of the Synthesized Compounds
compd
formula
MW
anal.a
yield [%]
mp [°C]
2b
2d
3
C
13H12N4O5S‚2H2O
372.37
395.41
323.28
323.28
484.45
540.57
470.42
489.47
512.50
457.42
522.54
450.61
480.46
485.48
389.33
417.39
475.44
531.55
461.41
489.47
503.49
475.44
522.54
470.43
471.45
485.48
C, H, N
C, H,b N
C, H, N
C, H, N
C,c H, N
C,d H, Ne
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C,f H, N
65
35
45
41
60
55
55
57
42
40
36
65
50
88
87
81
71
68
60
57
66
38
31
93
65
76
>300 (lit. mp > 300)7
C14H14N4O5S‚2.5H2O
>300
213.5
218-219
239
245
268
C
C
C
C
C
C
C
C
C
C
C
C
C
C
13H9NO7S
13H9NO7S
19H17N5O8S‚0.5H2O
23H25N5O8S ‚0.5H2O
18H15N5O8S‚0.5H2O
20H19N5O8S
21H21N5O8S‚0.5H2O
19H15N5O7S
23H23N5O7S‚0.5H2O
18H13N5O7S ‚0.4H2O
20H17N5O7S‚0.5H2O
21H19N5O7S
7
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6c
6d
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
252
253
>300
>300
>300
>300
>300
>300
>300 (lit. mp > 300)7
>250
>250
>250
>250
>250
>300
>300
>300
>300
>300
12H9N4O2SNa‚2.5H2O
14H13N4O5SNa‚2.5H2O
C,g H,h
C, H, N
n.d.i
N
C19H17N5O8S
C
C
C
C
C
C
C
C
C
23H25N5O8S‚0.5H2O
18H15N5O8S
20H19N5O8S
21H21N5O8S
19H15N5O7S‚H2O
23H23N5O7S ‚0.5H2O
18H13N5O7S‚1.5H2O
20H17N5O7S
n.d.
n.d.
n.d.
n.d.
C, H, N
C, H, N
C, H, N
C,j H,k N
C, H, N
21H19N5O7S
a
b
Elemental analyses were within (0.4% of calculated values, unless otherwise noted. Calcd, 4.81; found, 4.3. c Calcd, 47.06; found
47.52. Calcd, 51.06; found 51.55. e Calcd, 12.95; found, 12.45. f Calc, 51.96; found, 52.57. g Calcd, 36.98; found, 36.44. Calcd, 3.59; found,
2.90. n.d, not determined (intermediate products). Calcd, 50.95; found, 50.31. Calcd, 3.63; found, 4.39.
d
h
i
j
k
F igu r e 5. Hydrolysis of o-, m-, and p-nitrophenyl tosylate in
acetonitrile: buffer (5:95) as a function of pH at 25 °C (K )
hydrolysis rate constant).
F igu r e 4. Purity determination of compound 6d by capillary
electrophoresis; running buffer: phosphate 20 mM (pH 7.4),
voltage: 10 kV.
p-nitrophenyl tosylate was the least stable ester among
these three differently substituted sulfonate esters.
m-Nitrophenyl tosylate was quite stable over a large pH
range, while o- and p-nitrophenyl tosylate exhibited
lower stability at basic (pH 9.8) and particularly at
strongly acidic (pH 1) pH values. Therefore, m-nitro-
phenyl esters of xanthine phenylsulfonic acids were
selected as potential prodrugs.
Sta bility Stu d ies of Su lfop h en ylxa n th in e m -
Nitr op h en yl Ester s a s P oten tia l Su lfon a te P r o-
d r u gs. An effective oral prodrug must be (i) lipophilic
enough to be absorbed from the gastrointestinal tract,
(ii) stable enough toward any hydrolysis that might
occur before reaching the bloodstream, and (iii) capable
of releasing the active parent drug in the body e.g.
through enzymatic biotransformation. Therefore, it was
necessary to study the chemical and enzymatic in vitro
stability of the synthesized putative prodrugs.
nitrile concentrations were used in order to investigate
whether the organic solvent had any influence on the
hydrolysis rate. Hydrolysis at five different pH values
was investigated, i.e., pH 1, 6, 7, 8, and 9.8, ranging
from strongly acidic to alkaline conditions. Hydrolysis
constants (K) were measured spectrophotometrically by
detecting the absorption of released o-, m-, or p-
nitrophenol. It was found that all hydrolysis reactions
followed a pseudo-first-order kinetic. Plots of log(At -
A0) (At: absorption at a certain time; A0: absorption at
zero time) versus time were linear for three half-lives,
the slopes yielding the rate constants. Figure 5 shows
the hydrolysis rate constants (K) of the three sulfonate
esters at five different pH values in acetonitrile: buffer
(5:95). We found that the addition of acetonitrile did not
have significant effects on the hydrolysis of sulfonic acid
esters, since the K values were almost identical in the
presence of 5% acetonitrile and 10% acetonitrile. m-
Nitrophenyl tosylate was the most stable ester, while
The most frequently used analytical method for
stability tests is HPLC. However, because the synthe-
sized xanthine sulfonic acid esters are very lipophilic,

1036 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Yan and Mu¨ller
Ta ble 2. Stability of Sulfophenylxanthine m-Nitrophenyl
Esters 5a-e toward 0.001 N Aqueous Sodium Hydroxide
Solution at 37 °C Determined by Capillary Electrophoresis with
UV Detection at 254 nm
a
compd
R1
R2
t1/2 (h)a
K (s-1
)
5a
5b
5c
5d
5e
methyl
propyl
methyl
propyl
butyl
methyl
propyl
H
H
H
20.8
21.2
20.2
23.8
19.1
9.2 × 10-6
9.1 × 10-6
9.6 × 10-6
8.1 × 10-6
10.1 × 10-6
a
Values represent means of two to three separate experiments.
the UV spectra of the compounds formed. Figure 6
shows the CE electropherograms of compound 5d before
incubation and after 26 h of incubation in 0.001 N
aqueous sodium hydroxide solution at 37 °C. The
hydrolysis products 1-propyl-8-p-sulfophenylxanthine
and m-nitrophenol could clearly be detected showing
migration times of 5.3 and 4.9 min, respectively. It was
found that the chemical hydrolysis by hydroxide ions
of these sulfonic acid esters followed a first-order kinetic.
In Table 2, the hydrolysis constants (K) and the half-
lives (t1/2) of the esters are collected. It was shown that
the xanthine sulfonate esters were very stable toward
chemical hydrolysis by 0.001 N aqueous sodium hydrox-
ide solution with half-lives of about 20 h.
Because the chemical structure of all investigated
m-nitrophenyl sulfonate esters is similar, their stability
should be comparable. This was confirmed by the
chemical stability tests, in which no significant differ-
ences between the half-lives of esters 5a -e were
observed. Therefore only one representative compound
was selected for performing further in vitro stability
tests.
Compound 5d , 1-propyl-8-[4-[[m-nitrophenoxy]sulfon-
yl]phenyl]xanthine, was selected as an example to
investigate its biological stability at 37 °C in (i) fetal
calf serum, (ii) simulated gastric acid, and (iii) rat liver
homogenate. These various media were thought to be
valid in vitro models for the conditions that may affect
the potential prodrugs during oral application.
For the biological in vitro stability tests, different CE
separation conditions were applied than for the chemical
stability tests because of the protein content in the
biological samples, which does not impede CE analysis
but influences the migration and separation by CE.
Thus, voltage and SDS concentration had to be adjusted.
Detection in biological samples was at 320 nm in order
to exclude interference from biological molecules, some
of which show absorption at 254 nm. The internal
standard had to be chosen accordingly (diclofenac
sodium salt for the chemical hydrolysis experiments,
p-aminosalicylate sodium salt for the biological experi-
ments). Table 3 summarizes the separation conditions
used in chemical and biological stability tests.
F igu r e 6. Electropherograms of 1-propyl-8[4-[[m-nitrophen-
oxy]sulfonyl]phenyl]xanthine 5d before incubation (A) and
after 26 h of incubation in 0.001 N NaOH (B). CE conditions:
voltage, 10 kV; running buffer, 100 mM borate buffer, 50 mM
SDS (pH 8); detection at 320 nm; a: m-Nitrophenol, 4.9 min;
b: Internal standard (diclofenac-Na), 5.1 min; c: 1-Propyl-8-
(p-sulfophenyl)xanthine, 5.3 min; d: 1-Propyl-8-[4-[[[m-nitro-
phenoxy]sulfonyl]phenyl]xanthine, 8.8 min.
they are insoluble in most common HPLC solvents, such
as methanol, acetonitrile, H2O, etc. Therefore, capillary
electrophoresis (CE) with a diode array detector (DAD)
was used for the analyses. Compared with HPLC, CE
is advantageous in many regards including speed,
versatility, low running costs, high separation efficiency,
and the requirement of only very small sample volumes.
The application field of CE comprises not only the
separation of proteins, DNA fragments, and carbohy-
drates, but also drug metabolism studies.31 In biological
applications CE has the advantage of not requiring
sample pretreatment; the incubation solution can di-
rectly be injected without prior removal of proteins.
At first, the chemical stability of the m-nitrophenyl
esters was investigated. Stock solutions (2 mM) in
dimethyl sulfoxide (DMSO) were prepared, since the
esters were very well soluble in DMSO, but less soluble
in water. To reduce or eliminate the influence of DMSO,
stock solutions were diluted into aqueous buffer solu-
tions at a ratio of 1:100. The decomposition of the esters
5a -e in 0.001 N aqueous sodium hydroxide solution (pH
11) was studied at 37 °C in order to determine the
chemical stability of the compounds. Since we know that
the esters are less stable in alkaline media than in
neutral solutions, it is likely that, if they are stable in
alkaline medium, they should also be stable at neutral
pH value.
The stability test in simulated gastric acid was
performed for 4 h since drugs usually do not stay in the
stomach any longer. Compound 5d was found to be very
stable under these conditions and degradation was
negligible. After 4 h of incubation in simulated gastric
acid, 94% of 5d could still be detected.
Expected hydrolysis products of these sulfonic acid
esters are the respective 8-p-sulfophenylxanthine de-
rivatives and m-nitrophenol. This could be confirmed
by CE/UV analyses based on the migration times and

Sulfophenylxanthine Nitrophenyl Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1037
Ta ble 3. Separation Conditions in Capillary Electrophoresis for the Investigation of Hydrolysis of Compound 5d
incubation medium
ce buffer
voltage (kV)
internal standard
wavelength (nm)
0.001 N aq NaOH solution 100 mM borate buffer + 50 mM SDS (pH 8)
10
15
15
12
diclofenac sodium salt
254
320
320
320
fetal calf serum
100 mM borate buffer + 100 mM SDS (pH 8)
100 mM borate buffer + 100 mM SDS (pH 8)
100 mM borate buffer + 150 mM SDS (pH 8)
p-aminosalicylate sodium salt
p-aminosalicylate sodium salt
p-aminosalicylate sodium salt
simulated gastric acid
rat liver homogenate
unknown which enzyme(s) may be responsible for the
metabolism of the sulfonic acid m-nitrophenyl esters
Thus, the ester was very stable in simulated gastric
acid as well as in serum. However, it was readily
hydrolyzed by incubation with rat liver homogenate,
indicating an enzymatic pathway of hydrolysis.
In contrast to the chemical stability tests (see above),
hydrolysis products could not be detected in the elec-
tropherograms of the biological in vitro stability tests.
The only change that could be observed was the de-
crease of the peak area of the ester with increased
incubation time. The reason no hydrolyzed product could
be detected by CE analyses is not clear. One possible
explanation would be that both hydrolysis products
interacted with the proteins in the biological media
forming neutral, insoluble compounds; thus they were
not detectable by CE anymore. As a matter of fact we
observed the precipitation of insoluble material during
incubation with rat liver homogenate. To prove that the
ester was actually cleaved to release the parent drug
by enzymatic hydrolysis, thin-layer chromatography
(TLC) was used as a supporting analytical method. It
was found that after ca. 2 h of incubation of 5d in rat
liver homogenate at 37 °C, a large amount of 1-propyl-
8-p-sulfophenylxanthine could be detected by TLC on
silica gel using dichloromethane:methanol (4:1) as the
mobile phase, while the educt had almost quantitatively
disappeared. Thus it was proven that the active parent
drug was actually released presumably by enzymatic
biotransformation.
F igu r e 7. Stability test of 1-propyl-8-[4-[[m-nitrophenoxy]-
sulfonyl]phenyl]xanthine 5d in fetal calf serum at 37 °C (C )
concentration of compound, C0 ) concentration of compound
at time zero).
From the chemical and biological in vitro stability
tests, it can be concluded that the synthesized sulfophen-
ylxanthine m-nitrophenyl esters are relatively stable,
since their half-lives in 0.001 N aqueous sodium hy-
droxide solution solution at 37 °C reach 20 h on average.
In biological media, the prototypic compound 5d was
not degraded by simulated gastric acid within 4 h of
incubation at 37 °C. This means that it will be stable
in the gastrointestinal tract and should cross the
gastrointestinal barrier reaching the blood stream
without prior degradation. The compound will remain
as an intact ester in the circulation since its half-life in
fetal calf serum was ca. 16 h. Finally, the ester will be
hydrolyzed in the liver by enzymatic biotransformation.
F igu r e 8. Hydrolysis of 1-propyl-8-[4-[[m-nitrophenoxy]sul-
fonyl]phenyl]xanthine 5d by rat liver homogenate (protein
concentration: 16 mg/mL, concentration of 5d : 2 × 10-5 M)
at 37 °C; t1/2 ) 41 min (C ) concentration of compound, C0 )
concentration of compound at time zero).
Ad en osin e Recep tor Affin ity. To investigate the
effects of esterification of 8-(p-sulfophenyl)xanthine
derivatives with nitrophenols on adenosine receptor
affinity, all compounds were investigated in radioligand
binding studies at rat brain A1, A2A, and human A3 ARs.
[3H]-2-Chloro-N6-cyclopentyladenosine (CCPA), [3H]-3-
(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-pro-
pargylxanthine (MSX-2), and [3H]-2-phenyl-8-ethyl-4-
methyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-
5-one (PSB-11), respectively, were used as A1, A2A, and
A3 radioligands. Selected compounds were also tested
at human A2B ARs using [3H]8-((4-(2-hydroxyethyl-
amino)-2-oxo-ethoxy)phenyl)-1-propylxanthine (PSB-
In the next step, hydrolysis of 5d in fetal calf serum
was measured as an indicator for the stability of the
m-nitrophenyl sulfonate esters in blood serum. The
hydrolysis of 5d in fetal calf serum was also very slow
with a half-life (t1/2) of 16 h following a zero-order kinetic
(Figure 7).
Finally, metabolism of 5d by liver enzymes was
investigated. The hydrolysis of compound 5d in freshly
prepared rat liver homogenate could be described as a
first-order kinetic reaction (Figure 8). Its half-life (t1/2
)
was 40 min under the applied conditions. It is currently

1038 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Yan and Mu¨ller
Ta ble 4. Adenosine Receptor Affinities of 8-Phenylxanthines, 8-p-Sulfophenylxanthines and Corresponding Sulfonic Acid Nitrophenyl
Esters
Ki ( SEM [nM] (or % inhibition of radioligand binding at 10 µM)
A1-affinity rat
A2A-affinity rat
A2B-affinity human
A3-affinity human
compd
R1
R2
[3H]CCPA (n ) 3)
[3H]MSX-2 (n ) 3)
[3H]PSB-298 (n ) 2)
[3H]PBS-11 (n ) 2)
m-Nitrophenyl Esters of Sulfophenylxanthines
5a
5b
methyl
propyl
methyl
propyl
21 ( 3
117 ( 22
28 ( 29
n.d.a
43 ( 3.5
(25% ( 1)
4300 ( 230
(66% ( 3)
(11% ( 4)
(14% ( 3)
(21% ( 1)
4.4 ( 0.3
5c
5d
5e
methyl
propyl
butyl
H
H
H
18 ( 2
6.5 ( 0.9
5.7 ( 0.1
63 ( 29
54 ( 5.2
21 ( 2.6
n.d.
13 ( 3.6
23 ( 6
p-Nitrophenyl Esters of Sulfophenylxanthines
9a
9b
9c
9d
9e
methyl
propyl
methyl
propyl
butyl
methyl
propyl
H
H
H
7.7 ( 0.5
2.7 ( 0.1
6.3 ( 7
3.6 ( 0.03
2.6 ( 0.3
99 ( 21
16 ( 1.5
34 ( 12
74 ( 2
n.d.
n.d.
n.d.
5.4 ( 4.3
26 ( 2.5
(16% ( 9)
(44% ( 2)
(5% ( 3)
(47% ( 9)f
(9% ( 9)
78 ( 18
8-Phenylxanthines
10a
10b
10c
10d
10e
methyl
propyl
methyl
propyl
butyl
methyl
propyl
H
H
H
8932,b
1032,b
26025,b
6725,b
407
85032,b
19032,b
220025,b
190025,b
6427
41533,d
18.933,d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d
4.77,e
11.87,e
8-p-Sulfophenylxanthines
6a (SPT)
6b (DPSPX)
6c (PSB-1115)
6d
methyl
propyl
methyl
propyl
butyl
methyl
propyl
H
H
H
1400032,b
1400032,b
140032,b
11800
133033,d
25035
n.d.
1100034,c
18334,c
n.d.
21032,b
1238
220025,b
4757
2400025,b
80707
53.47,e
707,e
14%7
6e
39%7
a
b
d
n.d.: not determined. [3H]PIA was used as A1-radioligand and [3H]NECA as A2A-radioligand. c Sheep A3 receptor.
[
125I]ABOPX
binding at human HEK-293 A2B receptor. e [3H]ZM241385 binding to recombinant human A2B receptor. f N ) 3.
298). Native brain tissues could be used for A1 (cortex)
and A2A (striatum) assays due to the high density of
those receptor subtypes in the brain. A2B and A3
receptors are expressed in much lower density, and thus
artificial recombinant systems had to be used. It has
previously been shown that species differences for rat
and human A1, A2A, and A2B ARs are moderate, only
for the A3 ARs large species differences have been
described, antagonists being in most cases considerably
less potent at rat as compared to human A3 ARs.2
Therefore we have chosen to determine the affinity at
human A3 ARs as being more relevant for drug develop-
ment. However, rat data for A1 and A2A affinity were
thought to be sufficient for obtaining a rough estimation
of the compounds’ selectivity even though data from
different species were compared. Determined affinities
of the synthesized nitrophenyl esters are collected in
Table 4. Data of 1,3-substituted 8-phenylxanthines
(10a -e) and 1,3-substituted 8-p-sulfophenylxanthines
(6a -e) are given for comparison.
have now found that esterification with m- or p-
nitrophenol not only restored but even increased A1 and
A2A affinity in comparison with the corresponding
8-phenylxanthine derivatives. Thus, the esterified com-
pounds are no prodrugs in a classical sense since they
show enhanced affinity. A similar trend, but less
pronounced, was observed for the A2B AR with a selected
set of compounds (Table 4). All nitrophenyl esters
showed weak to negligible binding to human A3 ARs.
The most potent compound at A3 ARs was the 1,3-
dipropyl-substituted m-nitrophenyl ester 5b with a Ki
value of 4.3 µM confirming the previously observed
favorable effects of 1,3-dipropyl substitution of xan-
thines on A3 affinity.7 However, all compounds, includ-
ing 5b, were much more potent at the other AR subtypes
than at A3 receptors.
All investigated nitrophenyl esters were more potent
at A1 than at A2A ARs (3.5-30-fold). 1-Propyl- and
1-butyl-substituted p-nitrophenyl esters (9d , 9e) showed
the highest A1 versus A2A selectivity (20- and 30-fold)
respectively, which was, however, lower versus the A2B
receptor subtype.
At A1 ARs p-nitro-substitution (9a -e) was superior
to a m-nitro substitution (5a -e, see Table 4) resulting
in about 2-fold lower Ki values ranging from 2.6 to
7.7 nM. At A2A ARs, 1,3-disubstituted and 1-methyl-
substituted xanthine p-nitrophenyl sulfonates also ex-
hibited higher affinities than the corresponding xan-
thine m-nitrophenyl sulfonates, but the 1-propyl- and
The nitrophenyl esters of p-sulfophenylxanthine de-
rivatives (5a -e, 9a -e) showed high affinities for A1,
A2A, and A2B adenosine receptors. Figure 9 illustrates
the effects on A1 and A2A AR affinity of the esters in
comparison with the corresponding 8-phenylxanthine
and 8-p-sulfophenylxanthine derivatives. It had been
observed earlier that a p-sulfonate group results in a
decrease in A1 and A2A AR affinity as compared with
the corresponding 8-phenylxanthine derivatives.24,25 We

Sulfophenylxanthine Nitrophenyl Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1039
F igu r e 9. Effects of sulfonate and nitrophenoxysulfonate groups in 8-phenylxanthine derivatives on rat A1 (A) and A2A (B)
adenosine receptors. Affinities determined in radioligand binding assays are given as pKi values (-log Ki values) [M].
1-butyl-substituted xanthine derivatives showed the
opposite effect. The influence of the 1,3-substitution
pattern on A1 and A2A affinity was altogether moderate.
N1-Monoalkylated compounds were similarly potent as
1,3-disubstituted derivatives. At A1 ARs the rank order
of potency in the p- and m-nitrophenyl series was very
similar: 1,3-dipropyl ≈ 1-butyl ≈ 1-propyl > 1-methyl
≈ 1,3-dimethyl. At A2A ARs the rank order of potency
was similar as above for the m-nitrophenyl esters, but
somewhat different for the p-nitrophenyl derivatives; in
the latter series, the 1-methyl-substituted compound
was surprisingly potent with the following rank order
of potency: 1,3-dipropyl > 1-methyl > 1-propyl ≈
1-butyl > 1,3-dimethyl.
The 1,3-dipropyl-substituted p-nitrophenyl ester 9b
belonged to the most potent A1 and A2A ligands of the
present series, with a Ki value of 2.7 nM at A1 and 16
nM at A2A ARs, whereas the 1,3-dimethyl-substituted
m-nitrophenyl ester 5a had the lowest affinity with a
Ki value of 21 nM at A1 and 117 nM at A2A ARs. Thus,
it can be concluded that the introduction of a second
propyl group (in the 3-position) of 1-propylxanthine
ARs (compare 5b/5d ). This appears to be a general trend
and is also true for 8-phenyl-, 8-p-sulfophenyl-, and
8-cycloalkylxanthine derivatives.7 The most potent A2B
antagonist of the present series was 1-propylxanthine
p-nitrophenylsulfonate 9d with a Ki value of 5.4 nM at
human A2B ARs; the compound showed similarly high
affinity for A1 ARs and therefore was nonselective.
Compared with the corresponding 8-p-sulfophenylxan-
thines, it can be observed that the introduction of a
nitrophenyl substituent increased A2B affinity greatly,
but the selectivity of A2B versus other AR subtypes
tended to decrease. Compared with the corresponding
8-phenylxanthines, the introduction of a bulky nitro-
phenoxysulfonyl group decreased the A2B affinity slightly,
and A2B selectivity decreased as well.
Con clu sion s
In conclusion, nitrophenyl esters of sulfophenyl xan-
thine derivatives, that are potent adenosine receptor
antagonists, have been synthesized. The chemical and
enzymatic stability of the esters was determined in
vitro. The compounds showed high stability in aqueous
solution, in artificial gastric acid, and in serum. How-
ever,compound1-propyl-8-[4-[[m-nitrophenoxy]sulfonyl]-
phenyl]xanthine 5d , investigated as a prototypic ester,
was readily cleaved by incubation with rat liver homo-
genate. Thus, nitrophenyl esters of sulfonic acids have
a potential as peroral prodrugs of drugs bearing a
sulfonate group. Besides, the nitrophenyl esters of 8-p-
sulfophenylxanthine derivatives showed high potency
at A1, A2A, and A2B ARs. 1-Butyl-8-[4-[[p-nitrophenoxy]-
sulfonyl]phenyl]xanthine (9e) was the most potent and
selective A1 AR antagonist of the present series with a
Ki value of 2.6 nM at rat A1 ARs and 30-fold selectivity
nitrophenyl sulfonates increased the affinity to A1, A2A
,
and A3 ARs, while the introduction of a second methyl
group into the 3-position of 1-methylxanthine nitrophen-
yl sulfonates decreased the affinity to A1 and A2A ARs.
Compounds 5b, 5d , 5e, 9d , and 9e were selected for
additional testing at A2B ARs. Table 4 shows that all
compounds investigated exhibited relatively high affin-
ity for A2B ARs. There is not much difference between
p- and m-nitrophenyl-substituted derivatives (compare
5d /9d and 5e/9e). 3-Unsubstituted xanthines which had
been somewhat less potent or similarly potent to 1,3-
dipropyl-substituted derivatives at A1 and A2A ARs
(compare 5b/5d and 9b/9d ) were more potent at A2B

1040 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Yan and Mu¨ller
nitrophenol (1.99 g, 0.014 mmol) was dissolved in 50 mL of
THF and 50 mL of Tris-HCl buffer (50 mM, pH 9). A solution
of 4-chlorosulfonylbenzoic acid (3.4 g, 0.014 mol) in 50 mL of
THF was added slowly to the above solution. The pH value of
the mixture was kept at 8-9 by the addition of 2.5 N NaOH.
After stirring for about 4 h at rt, no more phenol could be
detected by TLC. Then the solution was brought to pH 7 by
the addition of 1 N HCl, and the THF was removed in vacuo.
The product precipitated when the aqueous solution was
acidified with 1 N HCl to pH 1. The formed product was
recrystallized from acetone/cyclohexane.
Compound 3: Yield 45% (calcd. from p-sulfobenzoic acid);
mp 213.5 °C; 1H NMR (acetone-d6) δ (ppm): 7.56 (m, 1H,
aromatic), 7.74 (t, 1H, J ) 8.35 Hz, aromatic), 7.93 (t, 1H, J )
2.2 Hz, aromatic), 8.09 (d, 2H, J ) 8.51 Hz, aromatic), 8.25
(m, 1H, aromatic), 8.3 (d, 2H, J ) 8.51 Hz, aromatic). 13C NMR
(acetone-d6) δ (ppm): 118.57, 123.32, 129.7, 129.76, 131.67,
132.19, 137.27, 139.38, 150.38 (aromatic), 165.99 (COOH).
Anal. (C13H9NO7S) C, H, N.
versus rat A2A, and 10-fold versus human A2B receptors,
the compound was inactive at human A3 ARs. The
corresponding 1-propylxanthine derivative (9d ) was a
very potent mixed A1/A2B antagonist (KiA1 3.6 nM, KiA2B
5.4 nM). m-Nitrophenol has been shown to be a suitable
protecting group for phenylsulfonic acids, which can be
cleaved off in alkaline solution.
Exp er im en ta l Section
Ch em ica l Syn th esis. 1H and 13C NMR spectra were
performed on a Bruker Avance 500 MHz spectrometer. The
chemical shifts of the remaining protons of the deuterated
solvent served as internal standards for spectra recorded in
DMSO-d6: δ 1H: 2.49, δ 13C: 39.1. Tetramethylsilane was used
as internal standard for spectra recorded in acetone-d6 and
D2O. All compounds were checked for purity by TLC on silica
gel 60 F254 (Merck) aluminum plates, using dichloromethane:
methanol (4:1) or dichloromethane:methanol (8:1) as the
mobile phase. Melting points were determined on a Buechi 530
melting point apparatus and were uncorrected. Elemental
microanalyses were performed at the Pharmaceutical Insti-
tute, Pharmaceutical Chemistry Endenich, University of Bonn.
Mass spectra were recorded on an MS-50 spectrometer at the
Chemical Institute, University of Bonn.
o-, m-, and p-Nitrophenyl tosylates were synthesized by
reacting toluenesulfonyl chloride with o-, m-, or p-nitrophenol
in dichloromethane in the presence of triethylamine (TEA) as
described.23
1,3-Disubstituted-5,6-diaminouracils (1a , 1b) were prepared
from 1,3-disubstituted urea and cyanoacetic acid followed by
ring closure, nitrosation and reduction as described.36,37 3-Sub-
stituted 5,6-diaminouracils 1c-e were prepared from 6-amino-
uracil via regioselective alkylation followed by nitrosation and
reduction as described.38,39
6-Am in o-1,3-d im eth yl-5-(4-su lfoben za m id o)u r a cil (2a ).
A solution of 0.9 g (5.3 mmol) of 1,3-dimethyl-5,6-diamino-
uracil, 1.3 g (5.4 mmol) of p-sulfobenzoic acid potassium salt,
and 1.0 g (5.2 mmol) of N-(3-(dimethylamino)propyl)-N-ethyl-
carbodiimide hydrochloride (EDC) in 20 mL of water was
stirred for 1 h at rt. The solvent was removed by evaporation,
a small amount of methanol was added, and the residue was
collected by filtration. The filtrate was cooled to complete the
precipitation of further product.
1,3-Dim eth yl-8-p-su lfop h en ylxa n th in e (2b). Carboxa-
mide derivative 2a (0.91 g, 0.26 mmol) was dissolved in 25
mL of 2.5 N NaOH and heated at 70 °C for 10 min. After
cooling to 0 °C, the product was precipitated by the addition
of concd HCl to pH 6. The precipitate was washed with cold 1
N HCl. The product was recrystallized by dissolution in
aqueous NaOH solution followed by acidification with aqueous
HCl solution.
1,3-Dim eth yl-8-(p-ch lor osu lfon ylp h en yl)xa n th in e (2c).
Compound 2b (59 mg, 1.16 mmol) was refluxed with 10 mL of
thionyl chloride for 2 h, and then remaining thionyl chloride
was completely removed in vacuo. A white solid was obtained,
which did not have to be further purified.
8-p-Su lfop h en yl-1,3,7-tr im eth ylxa n th in e (2d ). 1,3-Di-
methyl-8-p-sulfophenylxanthine 2b (100 mg, 0.3 mmol) was
dissolved in 10 mL of DMF, K2CO3 (8.2 mg, 0.6 mmol) and
MeI (0.37 mL, 6 mmol) were added, and the mixture was
stirred at rt overnight. The solution was filtered and diethyl
ether (20 mL) was added to the filtrate to precipitate the
product. The white precipitation was filtered off and washed
with diethyl ether.
Compound 7: Yield 41% (calcd. from p-sulfobenzoic acid);
mp 218-219 °C; 1H NMR (DMSO-d6) δ (ppm): 7.36 (d, 2H, J
) 9.14 Hz, armatic), 8.02 (d, 2H, J ) 8.82 Hz, aromatic), 8.18
(d, 2H, J ) 8.82 Hz, aromatic), 8.26 (d, 2H, J ) 9.14 Hz,
aromatic). 13C NMR (DMSO-d6) δ (ppm): 123.81, 126.26,
129.06, 131.03, 137.12, 137.54, 146.62, 153.3 (aromatic), 166.11
(COOH). Anal. (C13H9NO7S) C, H, N.
6-Amino-1,3-dimethyl-5-[4-[[m-nitrophenoxy]sulfonyl]benza-
mido]uracil (4a ), 6-amino-1,3-dipropyl-5-[4-[[m-nitrophenoxy]-
sulfonyl]benzamido]uracil (4b ), 6-amino-1-methyl-5-[4-[[m-
nitrophenoxy]sulfonyl]benzamido]uracil (4c), 6-amino-1-propyl-
5-[4-[[m-nitrophenoxy] sulfonyl]benzamido]uracil (4d), 6-amino-
1-butyl-5-[4-[[m-nitrophenoxy]sulfonyl] benzamido]uracil (4e),
6-amino-1,3-dimethyl-5-[4-[[p-nitrophenoxy]sulfonyl]benzami-
do] uracil (8a ), 6-amino-1,3-dipropyl-5-[4-[[p-nitrophenoxy]-
sulfonyl]benzamido]uracil (8b ), 6-amino-1-methyl-5-[4-[[p-
nitrophenoxy]sulfonyl]benzamido]uracil (8c), 6-amino-1-propyl-
5-[4-[[p-nitrophenoxy]sulfonyl]benzamido]uracil (8d ), 6-amino-
1-butyl-5-[4-[[p-nitro- phenoxy]sulfonyl]benzamido]uracil (8e).
Gen er a l P r oced u r e. To a suspension of 1,3-substituted-5,6-
diaminouracil 1a -e (7.2 mmol) in methanol was added an
equimolar amount of 4-[[m-nitrophenoxy]sulfonyl]benzoic acid
3 or 4-[[p-nitrophenoxy]sulfonyl]benzoic acid 7, and an equi-
molar or slightly excessive amount of EDC. The mixture was
stirred at rt. The color of the solution turned yellow, and a
precipitate began to form. The reaction was stopped when no
more diaminouracil could be detected by TLC after several
hours. The precipitate was filtered off and washed with cold
water. Products were recrystallized by dissolving them in DMF
followed by dropwise addition of water.
1,3-Dimethyl-8-[4-[[m-nitrophenoxy]sulfonyl]phenyl]xan-
thine (5a ), 1,3-dipropyl-8-[4-[[m-nitrophenoxy]sulfonyl]phenyl]-
xanthine (5b), 1-methyl-8-[4-[[m-nitrophenoxy]sulfonyl] phenyl]-
xanthine (5c), 1-propyl-8-[4-[[m-nitrophenoxy]sulfonyl]phenyl]-
xanthine (5d ), 1-butyl-8-[4-[[m-nitrophenoxy]sulfonyl]phenyl]-
xanthine (5e), 1,3-dimethyl-8-[4-[[p-nitrophenoxy]sulfonyl]phen-
yl]xanthine (9a ), 1,3-dipropyl-8-[4-[[p-nitrophenoxy]sulfo-
nyl]phenyl]xanthine (9b ), 1-methyl-8-[4-[[p-nitrophenoxy]-
sulfonyl]phenyl]xanthine (9c), 1-propyl-8-[4-[[p-nitrophenoxy]-
sulfonyl]phenyl]xanthine (9d ), 1-butyl-8-[4-[[p-nitrophenoxy]
sulfonyl]phenyl]xanthine (9e). Gen er a l P r oced u r e. A mix-
ture of carboxamido derivative 4a -e or 8a -e, (0.32 mmol) and
4 g of polyphosphoric acid trimethylsilyl ester (PPSE) was first
heated at 120 °C for 10 min and then at 170 °C for 1.5-2 h.
After cooling, 20 mL of methanol was added to the mixture
and the formed precipitate was collected by filtration and
washed with methanol. The products were finally recrystal-
lized by dissolving them in DMF and subsequent dropwise
addition of water.
4-[[m -Nit r op h en oxy]su lfon yl]b en zoic Acid (3), 4-[[p -
Nitr op h en oxy]su lfon yl]ben zoic Acid (7). Gen er a l P r o-
ced u r e. Chlorosulfonic acid (50 mL) was slowly added to 5 g
(0.021 mol) of p-sulfobenzoic acid potassium salt, while the
temperature was kept below 30 °C. The mixture was stirred
overnight. A crystalline precipitate was obtained when chipped
ice was carefully added to the above mixture. After filtration,
the residue was washed with cold water and dried to
yield 4-chlorosulfonylbenzoic acid.26,27 m-Nitrophenol or p-
1-Meth yl-8-p-su lfop h en ylxa n th in e (6c), 1-P r op yl-8-p-
su lfop h en ylxa n th in e (6d ). Gen er a l P r oced u r e. Compound
5c or 5d (6 mmol) was dissolved in 60 mL of 2 N NaOH. The
solution was heated at 70 °C for 30 min. After cooling to rt,
the pH was carefully adjusted to 7 by concd HCl, and the
solution was extracted with CH2Cl2 serveral times to remove

Sulfophenylxanthine Nitrophenyl Esters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1041
the hydrolyzed m-nitrophenol. The product was precipitated
by careful acidification to pH 4 with HCl, filtered off, and
recrystallized from water or purified by preparative HPLC (see
below).
analysis by capillary electrophoresis. The initial concentration
of the test compound in the analytical sample was 2 × 10-5 M.
Biologica l Sta bility. A stock solution of 5d (30 µL, 50 mM
in DMSO; 150 µL, 2 mM in DMSO; 10 µL, 20 mM in DMSO)
was added to fetal calf serum (1470 µL), simulated gastric acid
(9850 µL), and rat liver homogenate (9850 µL), respectively.
The mixture was incubated in a water bath at 37 °C with a
rotation speed of 900 rpm. At certain time intervals, 100 µL
of fetal calf serum incubation solution was withdrawn and
diluted with 4.7 mL of H2O and 50 µL of a solution of
p-aminosalicylate sodium salt (1 mg/mL) as internal standard;
400 µL of simulated gastric acid incubation solution was
withdrawn and mixed with 100 µL of a solution of p-amino-
salicylate sodium salt (0.1 mg/mL) as internal standard; 50
µL of rat liver homogenate incubation solution was withdrawn
and added to 100 µL of 0.1% TFA, 250 µL of Dulbecco’s
phosphate-buffered saline (DPBS), and 100 µL of a solution
of p-aminosalicylate sodium salt (0.1 mg/mL) as internal
standard. The solutions were analyzed by CE after short
vortexing. The initial concentration of the test compound was
2 × 10-5 M.
Hyd r olysis Mea su r em en ts of Mod el Com p ou n d s. Hy-
drolysis rates of o-, m-, and p-nitrophenyl tosylates were
measured by following UV absorbance changes using an HP
8452A spectrophotometer equipped with a diode array detec-
tor. All reactions were carried out under pseudo-first-order
conditions while the buffer concentration was maintained in
large excess over that of the esters. Acetonitrile was used as
a cosolvent (10% v/v or 5% v/v) to keep esters in solution. Five
buffer solutions were used: 50 mM KCl-HCl buffer (pH 1),
50 mM KH2PO4 buffer (pH 6, pH 7, pH 8), and 50 mM H3-
BO3-KCl buffer (pH 9.8). Rate constants were measured by
following the appearance of the product continuously at fixed
wavelengths. Absorbance readings were made at the λmax of
the products: p-nitrophenol (400 nm), m-nitrophenol (376 nm),
o-nitrophenol (400 nm). Hydrolysis rate constants (K) were
calculated as the slope of a plot of log(At - A0) versus time.
P r ep a r a tive HP LC. The HPLC system consisted of a
WellChrom Preparative HPLC pump K-1800 (Knauer GmbH,
Berlin, Germany), a spectrophotometer K-2600 (Knauer GmbH,
Berlin, Germany), and a computer equipped with the Euro-
chrom 3.05 preparative software (Knauer GmbH, Berlin,
Germany). The preparative column and precolumn used were
Eurospher 100, C18, 10 µm, 250 × 20 mm and Eurospher 100,
During the stability experiment in simulated gastric acid,
the migration time of the prodrug was delayed for ca. 2-3 min
after each test, probably due to proteins absorbed to the
capillary. Since an alkaline borate buffer (pH 8) was applied
as the running buffer, pepsin in simulated gastric acid may
have precipitated in the capillary, inducing a change of the
capillary’s surface. The resulting prolongation of the migration
time of the compounds did not influence the results.
C
18, 5 µm, 30 × 20 mm, respectively. The elution system was
as follows: eluent A, 35% methanol and 65% water; eluent B,
water. Sample (10 mg/mL, 10 mL) was injected into the HPLC
system at a speed of 10 mL/min. Then a linear gradient at
the speed of 20 mL/min from 50% eluent A + 50% eluent B to
100% eluent A over 20 min was used. The retention time of
compound 6d was 11 min.
The half-lives were determined by the curve fitting program
GraphPad Prism, version 2.0 (GraphPad, San Diego, CA).
Ra d ioliga n d Bin d in g Assa ys. Ma ter ia ls. Radioligands
were obtained from the following sources: [3H]CCPA was from
NEN Life Sciences (54.9 Ci/mmol), [3H]MSX-2 (85 Ci/mmol),
[3H]PSB-11 (53 Ci/mmol), and [3H]PSB-298 (124 Ci/mmol)
were from Amersham. The nonradioactive precursors of [3H]-
MSX-2 (MSX-1), [3H]PSB-11 (PSB-10), and [3H]PSB-298 (PSB-
297) were synthesized in our laboratory.7,44,45
Mem br a n e P r ep a r a tion s. Frozen rat brains obtained from
Pel Freez, Rogers, AR, were dissected to obtain cortical
membrane preparations for A1 assays, and striatal membrane
preparations for A2A assays, as described.46,47 Membranes from
Chinese hamster ovary (CHO) cells stably transfected with the
human A2B and A3 AR were prepared as described.48
Ca p illa r y Electr op h or esis. Ap p a r a tu s. A P/ACE 5500
CE instrument (Beckman Coulter Instruments, Fullerton, CA)
equipped with a DAD-UV detection system using a fused silica
capillary (30/37 cm long; 75 µm diameter) was used for the
purity, stability, and degradation studies. An electrokinetic
injection for 5 s was applied for introducing the sample. The
capillary temperature was kept constant at 25 °C.
P u r it y Det er m in a t ion b y Ca p illa r y E lect r op h or esis
(CE). The buffer used for the purity determination was 20 mM
phosphate buffer (pH 7.4) at a voltage of 10 kV. The migration
time of compound 6d was 3.2 min.
Ra d ioliga n d Bin d in g Assa ys. Stock solutions of the
compounds were prepared in dimethyl sulfoxide (DMSO); the
final concentration of DMSO in the assay was 2.5%. The
radioligands and their concentrations were as follows: [3H]-
CCPA,49 0.5 nM (rat A1), [3H]MSX-2,44 1 nM (rat A1), [3H]PSB-
298 (manuscript in preparation), 5 nM (human A2B); [3H]PSB-
11,45 0.5 nM (human A3). Binding assays were performed
essentially as described.44,45,48,49 About 70 µg/mL of protein was
used in the A1 and A2A assays; ca. 50-100 µg/mL of protein
was used in the human recombinant A2B and A3 assay,
respectively. Membranes were preincubated for 20 min with
0.12-0.22 IU/mL of adenosine deaminase in order to remove
endogenous adenosine. Curves were determined using 6-7
different concentrations of test compounds spanning 3 orders
of magnitude. At least two to three separate experiments were
performed, each in duplicate or triplicate. Data were analyzed
using GraphPad Prism, version 2.0 (GraphPad, San Diego,
CA). For nonlinear regression analysis, the Cheng-Prusoff
equation and KD values of 0.2 nM (rat A1) for [3H]CCPA, 8
nM (rat A2A) for [3H]MSX-2, 56 nM (human A2B) for [3H]PSB-
298, and 4.9 nM (human A3) for [3H]PSB-11 were used to
calculate Ki values from IC50 values.
St a b ilit y a n d Degr a d a t ion St u d ies w it h Ca p illa r y
Electr op h or esis. Ma ter ia ls. Fetal calf serum was bought
from Sigma (F 7524). Simulated gastric acid was made
according to lit:40 To 3.20 g of pepsin, 2.0 g of NaCl, and 80
mL of HCl (1 mol/L) was added distilled water to obtain 1000
mL, and this solution was stored at 4 °C. Rat liver homogenate
was prepared as described with slight modifications:41,42 Fresh
rat liver (6.5 g) was homogenized in Dulbecco’s phosphate-
buffered saline (DPBS) (consisting of 132.5 mg of CaCl2‚2H2O,
100 mg of MgCl2‚6H2O, 200 mg of KCl, 200 mg of KH2PO4,
8000 mg of NaCl, and 1500 mg of Na2HPO4 in a total volume
of 1000 mL, pH 7.2, freshly prepared) and centrifuged at 9000
× g for 30 min at 4 °C. The supernatant was carefully decanted
to obtain the rat liver homogenate. The protein concentration
was 16 mg/mL as determined by the method of Bradford.43
The rat liver homogenate was kept at -80 °C before use.
Meth od a n d Con d ition s. Borate buffer (pH 8.0, 100 mM),
containing various amounts of SDS, and different voltages
were used according to the different biological media. In
degradation studies with 0.001 N NaOH solution, 10 kV
voltage was applied and 50 mM SDS was used, while in studies
with fetal calf serum and simulated gastric acid media, 5 kV
voltage and 100 mM SDS gave better results; in studies with
rat liver homogenate, 12 kV and 150 mM SDS was used.
Ch em ica l Sta bility. Stock solutions (1 mL, 2 mM in
DMSO) of 5a -e were incubated with 99 mL of 0.001 N NaOH
at 37 °C in a water bath. At certain time intervals, 4.8 mL of
incubation solution was withdrawn and mixed with 0.1 mL of
1 mg/mL diclofenac sodium salt as internal standard for
Ack n ow led gm en t. We thank Sonja Hinz for per-
forming the A3 radioligand binding assays, and Birgit
Preiss for running some of the A2B binding assays. Luo
Yan is grateful for a STIBET scholarship for foreign
students by the DAAD (Deutscher Akademischer Aus-
tauschdienst). C. E. Mu¨ller thanks the Fonds der

1042 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Yan and Mu¨ller
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