1,8-Disubstituted xanthine derivatives: Synthesis of potent A2B-selective adenosine receptor antagonists

Article abstract of DOI:10.1021/jm011049y

3-Unsubstituted xanthine derivatives bearing a cyclopentyl or a phenyl residue in the 8-position were synthesized and developed as A_2B adenosine receptor antagonists. Compounds bearing polar substituents were prepared to obtain water-soluble derivatives. 1-Alkyl-8-phenylxanthine derivatives were found to exhibit high affinity for A_2B adenosine receptors (ARs). 1,8-Disubstituted xanthine derivatives were equipotent to or more potent than 1,3,8-trisubstituted xanthines at A_2B ARs, but generally less potent at A₁ and A_2A, and much less potent at A₃ ARs. Thus, the new compounds exhibited increased A_2B selectivity versus all other AR subtypes. 9-Deazaxanthines (pyrrolo[2,3-d]pyrimidindiones) appeared to be less potent at A_2B ARs than the corresponding xanthine derivatives. 1-Propyl-8-p-sulfophenylxanthine (17) was the most selective compound of the present series, exhibiting a K_i value of 53 nM at human A_2B ARs and showing greater than 180-fold selectivity versus human A₁ ARs. Compound 17 was also highly selective versus rat A₁ ARs (41-fold) and versus the other human AR subtypes (A_2A > 400-fold and A₃ > 180-fold). The compound is highly water-soluble due to its sulfonate function. 1-Butyl-8-p-carboxyphenylxanthine (10), another polar analogue bearing a carboxylate function, exhibited a K_i value of 24 nM for A_2B ARs, 49-fold selectivity versus human and 20-fold selectivity versus rat A₁ ARs, and greater than 150-fold selectivity versus human A_2A and A₃ ARs. 8-[4-(2-Hydroxyethylamino)-2-oxoethoxy)phenyl]-1-propylxanthine (29) and 1-butyl-8-[4-(4-benzyl)piperazino-2-oxoethoxy)phenyl]xanthine (35) were among the most potent A_2B antagonists showing K_i values at A_2B ARs of 1 nM, 57-fold (29) and 94-fold (35) selectivity versus human A₁, & ca. 30-fold selectivity versus rat A₁, and greater than 400-fold selectivity versus human A_2A and A₃ ARs. The new potent, selective, water-soluble A_2B antagonists may be useful research tools for investigating A_2B receptor function.

Full text of DOI:10.1021/jm011049y

1500
J . Med. Chem. 2002, 45, 1500-1510
1,8-Disu bstitu ted Xa n th in e Der iva tives: Syn th esis of P oten t A2B-Selective
Ad en osin e Recep tor An ta gon ists
Alaa M. Hayallah,†,| J esu´s Sandoval-Ram´ırez,‡, Ulrike Reith,† Ulrike Schobert,‡ Birgit Preiss,†
Britta Schumacher,† J ohn W. Daly,§ and Christa E. Mu¨ller*,†,‡
University of Bonn, Pharmaceutical Institute Poppelsdorf, Bonn, Germany, University of Wu¨rzburg, Institute of Pharmacy and
Food Chemistry, Wu¨rzburg, Germany, and Laboratory of Bioorganic Chemistry, National Institutes of Health,
Bethesda, Maryland
Received October 4, 2001
3-Unsubstituted xanthine derivatives bearing a cyclopentyl or a phenyl residue in the 8-position
were synthesized and developed as A2B adenosine receptor antagonists. Compounds bearing
polar substituents were prepared to obtain water-soluble derivatives. 1-Alkyl-8-phenylxanthine
derivatives were found to exhibit high affinity for A2B adenosine receptors (ARs). 1,8-
Disubstituted xanthine derivatives were equipotent to or more potent than 1,3,8-trisubstituted
xanthines at A2B ARs, but generally less potent at A1 and A2A, and much less potent at A3 ARs.
Thus, the new compounds exhibited increased A2B selectivity versus all other AR subtypes.
9-Deazaxanthines (pyrrolo[2,3-d]pyrimidindiones) appeared to be less potent at A2B ARs than
the corresponding xanthine derivatives. 1-Propyl-8-p-sulfophenylxanthine (17) was the most
selective compound of the present series, exhibiting a Ki value of 53 nM at human A2B ARs
and showing greater than 180-fold selectivity versus human A1 ARs. Compound 17 was also
highly selective versus rat A1 ARs (41-fold) and versus the other human AR subtypes (A2A
>
400-fold and A3 > 180-fold). The compound is highly water-soluble due to its sulfonate function.
1-Butyl-8-p-carboxyphenylxanthine (10), another polar analogue bearing a carboxylate function,
exhibited a Ki value of 24 nM for A2B ARs, 49-fold selectivity versus human and 20-fold
selectivity versus rat A1 ARs, and greater than 150-fold selectivity versus human A2A and A3
ARs. 8-[4-(2-Hydroxyethylamino)-2-oxoethoxy)phenyl]-1-propylxanthine (29) and 1-butyl-8-[4-
(4-benzyl)piperazino-2-oxoethoxy)phenyl]xanthine (35) were among the most potent A2B
antagonists showing Ki values at A2B ARs of 1 nM, 57-fold (29) and 94-fold (35) selectivity
versus human A1, ca. 30-fold selectivity versus rat A1, and greater than 400-fold selectivity
versus human A2A and A3 ARs. The new potent, selective, water-soluble A2B antagonists may
be useful research tools for investigating A2B receptor function.
In tr od u ction
massive cell death, or as a consequence of inflammatory
processes.5,6
The nucleoside adenosine plays an important physi-
ological role acting via four different subtypes of G-
protein-coupled receptors (GPCR), A1, A2A, A2B, and A3.1
A1 and A2A adenosine receptors (ARs) are stimulated
by low (submicromolar to low nanomolar) adenosine
concentrations, while higher adenosine levels (micro-
molar concentrations) are required for the activation of
A2B and A3 ARs in the body.2 In artificial systems with
high adenosine receptor expression, A1, A2A, and A3 ARs
have been shown to be stimulated by low adenosine
concentrations, but the A2B AR still requires micromolar
adenosine concentrations.3,4 Increased levels of adeno-
sine, which might be sufficient to stimulate low affinity
ARs, are found under pathophysiological conditions, e.g.,
as a result of hypoxic or ischemic conditions, after
The existence in brain of high and low affinity A2 ARs
was demonstrated by Daly and co-workers in 1983.7
Such receptors were later named A2A and A2B ARs,
respectively. The low affinity A2B AR has now been
cloned from various species including rat, mouse, and
human.1,8 The homology of the amino acid sequences of
rodent and human A2B receptors is 86-87%, while it is
much higher if rat and mouse sequences are being
compared (96%).8 The A2B ARs show a ubiquitous
distribution, with highest levels being found in the large
intestine, mast cells, and hematopoietic cells, while
lower levels are detected in other organs, such as brain
and liver.8
A2B ARs, like A2A ARs, are positively coupled to
adenylate cyclase via Gs; however, coupling to phospho-
lipase C via Gq resulting in mobilization of intracellular
calcium and direct coupling to calcium channels (stimu-
lation of calcium influx) have also been described.1,8
Adenosine may cause mast cell degranulation,9
vasodilation,10 chloride secretion in epithelial cells,11,12
growth inhibition of smooth muscle cells,13 enhanced
synthesis of cytokines in astrocytes,14 and stimulation
of glucose production in rat hepatocytes15 via A2B
adenosine receptors. Potential therapeutic applications
* Address correspondence to Dr. Christa Mu¨ller, Pharmazeutisches
Institut, Pharmazeutische Chemie Poppelsdorf, Kreuzbergweg 26,
D-53115 Bonn, Germany. Phone: +49-228-73-2301. Fax: +49-228-
73-2567. E-mail: christa.mueller@uni-bonn.de.
† Pharmaceutical Institute, Bonn.
| A.H. was on leave from the Faculty of Pharmacy, University of
Assiut, Egypt.
J .S.R. was on leave from the Faculty of Chemical Sciences, BUAP,
Puebla, Mexico.
‡ Institute of Pharmacy and Food Chemistry, Wu¨rzburg.
§ Laboratory of Bioorganic Chemistry, NIH.
10.1021/jm011049y CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/14/2002

1,8-Disubstituted Xanthine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1501
been described.26 3-Substituted-5,6-diaminouracils 3a -
3c and 1,3-dipropyl-5,6-diaminouracil 3d were used as
starting compounds (Schemes 1 and 2). For the prepa-
ration of benzylidene derivatives 4a a , 4ba , 4a b, 4bb,
4cb, 4bc, 4a d , 4be, and 4a f, diaminouracils 3a -3c
were reacted with different (unsubstituted or p-substi-
tuted) aldehydes in ethanol at reflux temperature.26
Subsequent ring closure of these derivatives was per-
formed either by stirring the imines in thionyl chloride
overnight, followed by refluxing for 1 h,27 or by reflux
in ethanol in the presence of anhydrous ferric chloride
for 3 h affording 1,8-disubstituted xanthine derivatives
5-13. Compound 14 was prepared by heating of 4a d
in thionyl chloride for 30 min to afford the acid chloride
derivative of 11, which was subsequently converted to
the methyl ester 14 by refluxing it with methanol for
30 min.28
Ch a r t 1. Nonselective Standard Xanthine Derivative
XAC (1) and A2B-Selective Analogue 2a
a
h ) human, r ) rat.
Xanthine 15 was prepared by hydrolysis of methyl
ester 13 in dimethylformamide in the presence of 0.1 N
aqueous sodium carbonate solution upon heating.29
Carboxamide derivatives 16a g, 16bg, 16d b, and 16d h
were prepared by the reaction of 5,6-diaminouracils 3a ,
3b, or 3d with p-substituted benzoic acid derivatives,
using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC) as condensing agent (Scheme 2).30
Ring closure to xanthines 17-18 was achieved either
by heating of 16a g or 16bg, respectively, in hexameth-
yldisilazane in the presence of a catalytic amount of
ammonium sulfate for 50 h at 140 °C31 or by refluxing
in trimethylsilylpolyphosphate at 160-180 °C for 1 h.26
Xanthine derivatives 19 and 20 were obtained by
heating 16d b or 16d h in methanol in the presence of
10% sodium hydroxide at 70 °C for 30 min.32
postulated for A2B AR antagonists include asthma and
chronic obstructive pulmonary disease,6,16,17 type II
diabetes,15 cystic fibrosis,11 secretory diarrhoea associ-
ated with inflammation,12 and Morbus Alzheimer.14
A thorough investigation of the (patho)physiological
roles of A2B ARs, however, has so far been hampered
by a lack of selective tools. Only a few structure-activity
relationship studies of A2B receptor ligands have been
published.2,4,15,18-22 Neither potent nor selective A2B
agonists are available.4,23 Only recently, the first selec-
tive A2B antagonists have been described.18 A series of
1,3-disubstituted 8-phenylxanthines derived from the
xanthine amine congener XAC (1) proved to be potent
A2B antagonists (see Chart 1). One of the best com-
pounds was MRS-1754 (2), exhibiting a Ki value of 1.97
nM at human A2B ARs and selectivity versus the other
human AR subtypes. However, the compound was only
moderately selective versus rat A1 ARs (8.5-fold).18 In
addition, it is highly lipophilic, possessing very low
water solubility. Compound 1 has been prepared in
tritiated form for radioligand binding studies at recom-
binant A2B receptors.24 Furthermore, 2-alkynyl-9-meth-
yladenine derivatives have been described as potent, but
nonselective A2B antagonists that were orally active in
a mouse model of diabetes.15
The present study was aimed at identifying and
developing potent A2B antagonists with high selectivity
versus the other AR subtypes combined with good water
solubility. Starting point was a study published by
Bruns in 1980,25 in which a large series of compounds,
including many xanthine derivatives, were investigated
in functional studies in a human fibroblast cell line
expressing A2B ARs. Analysis of the stucture-activity
relationships (SAR) revealed that the 1-substituent but
not the 3-substituent of xanthine derivatives might be
important for high A2B affinity.2 An 8-phenyl substituent
largely increased A2B affinity.2,25 We have now synthe-
sized and evaluated a series of 3-unsubstituted xanthine
derivatives, most of which are bearing polar substitu-
ents in the 8-position to increase water solubility. In a
few cases the corresponding 1,3-disubstituted xanthines
and 9-deazaxanthines (pyrrolo[2,3-d]pyrimidindiones)
were investigated for comparison.
Compound 21 was prepared by condensation of 5,6-
diamino-1-propyluracil 3a with 2,6-naphthalene dicar-
boxylic acid in dimethylformamide in the presense of
N-methylmorpholine and isobutylformate.33 Ring clo-
sure of 21 was achieved by refluxing it in hexamethyl-
disilazane in the presence of trimethylchlorosilane
(TMSCl) and p-toluenesulfonic acid for 36 h affording
compound 22 (Scheme 2).
An alternative procedure was used for the preparation
of compound 27, since neither p-carboxymethylbenzoic
acid nor p-carboxymethylbenzaldehyde were commer-
cially available (Scheme 3). Thus, p-chloromethylbenzoic
acid 23 was treated with sodium cyanide in the presence
of sodium carbonate in tetrahydrofurane to afford
p-cyanomethylbenzoic acid 24.34,35 Compound 24 was
condensed with 5,6-diamino-1-butyluracil 3b as above
affording the carboxamide derivative 25, which subse-
quently underwent ring closure in hexamethyldisilazane
to give compound 26. Compound 26 was hydrolyzed by
aqueous sulfuric acid under reflux for 6 h affording
phenylacetic acid derivative 27.34
Xanthine amide derivatives 28-37 were prepared by
three different methods (Scheme 4). Amide derivatives
31 and 37 were prepared by direct condensation of 12
with the appropriate piperazine derivatives at room
temperature in anhydrous dimethylformamide/dichlo-
romethane (1:1) in the presence of N-(3-dimethylami-
nopropyl)-N′-ethyl-carbodiimide hydrochloride and 4-(di-
methylamino)pyridine. Amide derivatives 30 and 32-
36 were prepared by conversion of 12 to its acid chloride
derivative by refluxing it in thionyl chloride for 4 h at
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of some of the investigated
3-unsubstituted xanthine derivatives has previously

1502 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hayallah et al.
Sch em e 1. Synthesis of 1,8-Disubstituted Xanthine Derivativesa
a
(a) 4-(Un)substituted benzaldehyde, ethanol, reflux; (b) 4-(un)substituted benzaldehyde, ethanol, acetic acid, rt or reflux; (c) 1. SOCl2
at 0 °C, 2. reflux, 3. stirring overnight at room temperature; (d) FeCl3, ethanol, reflux; (e) 1. SOCl2, reflux, 2. methanol, reflux; (f) DMF,
aq Na2CO3, steam bath.
Sch em e 2. Synthesis of 1,8-Di- and 1,3,8-Trisubstituted Xanthine Derivativesa
a
(a) 4-Substituted benzoic acid, MeOH or MeOH/H2O (1:1), EDC, 24 h, rt; (b) N-methylmorpholine, isobutylformate, 2,6-naphthalene
dicarboxylic acid, DMF; (c) 1. HMDS, (NH4)2SO4, reflux, 2. MeOH; (d) 1. PPSE, 160-180 °C, 2. MeOH; (e) 1. MeOH, aq NaOH, 70 °C, 2.
aq HCl; (f) 1. HMDS, TMSCl, p-toluene sulfonic acid, reflux 36 h, 2. H2O, ∆T.
70 °C. After distillation of excess thionyl chloride, the
residue was dissolved in a mixture of anhydrous pyri-
dine and dichloromethane, then the appropriate amine
derivative was added.18 In some cases, the yields

1,8-Disubstituted Xanthine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1503
Sch em e 3. Synthesis of 1-Butyl-8-(4-carboxymethyl)phenylxanthinea
a
(a) NaCN, aq NaHCO3, THF, 20-25 °C, 48 h; (b) 3-butyl-5,6-diaminouracil, MeOH/H2O (1:1), EDC, rt, overnight; (c) 1. HMDS,
(NH4)2SO4, 140 °C, 50 h, 2. MeOH/H2O; (d) 1. concd H2SO4, H2O, reflux, 6 h, 2. H2O, 3. NaOH/HCl.
Sch em e 4. Synthesis of Amide Derivatives of
evaluated in binding assays at human recombinant A1
and A2A ARs stably expressed in Chinese hamster ovary
(CHO) cells in order to assess selectivity in humans. The
compounds were evaluated for A2B affinity in radiolig-
and binding assays at human recombinant receptors
using [3H]4-[2-[[7-amino-2-(furyl)1,2,4-triazolo[2,3-a]1,3,5-
triazin-5-yl]amino]ethyl]phenol (ZM-241385) as radio-
ligand. Selected compounds were additionally investi-
gated in binding assays at human recombinant A3 ARs
with the A3-selective antagonist radioligand [3H]2-
phenyl-8-ethyl-4-methyl-(8R)-4,5,7,8-tetrahydro-1H-im-
idazo[2,1-i]purin-5-one (PSB-11).
3-Unsubstituted 8-Phenylxanthinesa
Str u ctu r e-Activity Rela tion sh ip s. Determined af-
finities of 3-unsubstituted 1,8-disubstituted xanthine
derivatives are collected in Tables 2 and 3. Data of a
few corresponding 1,3,8-trisubstituted xanthines, in-
cluding the standard AR antagonists 8-cyclopentyl-1,3-
dipropylxanthine (DPCPX, 39) and 1,3-dipropyl-8-p-
sulfophenylxanthine (DPSPX, 42), are given for com-
parison.
8-Cyclopentyl-1,3-dipropylxanthine (DPCPX or CPX,
39) is a standard antagonist for A1 ARs. It also exhibits
considerable affinity for A2B receptors (Ki ) 51 nM) and
has been used in tritiated form as a radioligand for A2B
AR binding assays.19 8-Cyclopentyl-1-propylxanthine
(38) retained A2B affinity but was much less potent at
A1 ARs than its 3-propyl derivative 39.
a
(a) Amine derivative, EDC, DMAP, DMF/CH2Cl2, rt, 24-48
h; (b) amine derivative, pyridine/CH2Cl2, rt, 24-48 h; (c) amine
derivative, DMF, ∆T, 24-48 h.
8-Cyclopentyl substitution is known to be favorable
for high A1 affinity only. 8-Phenyl substitution, however,
appears to generally increase affinity of xanthine de-
rivatives for all AR subtypes.2,25 Therefore most of the
new compounds were 8-phenylxanthine derivatives.
8-Phenylxanthine (40) itself was more potent at A2B
(Ki ) 810 nM) than at A1 and A2A ARs and exhibited
3-fold selectivity versus A1 ARs. The introduction of a
1-alkyl substituent led to a large increase in AR affinity,
which was most pronounced at A2B receptors. The order
obtained by this method were better than that obtained
by the carbodiimide method, and also the side products
were less, resulting in an easier purification procedure.
Amide derivatives 28 and 29 were prepared by direct
reaction of xanthine methyl ester 13 with amine deriva-
tives in hot dimethylformamide.28
In Table 1, yields, melting points, and analytical data
for intermediate and final products are collected. 1H and
13C NMR data of all final compounds and most inter-
mediate products were recorded and were in accordance
with the proposed structures. NMR data are available
as Supporting Information.
Biologica l Eva lu a tion . All compounds were inves-
tigated in radioligand binding studies at rat brain A1
and A2A ARs using [3H]2-chloro-N6-cyclopentyladenosine
(CCPA) and [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-
methoxystyryl)-1-propargylxanthine (MSX-2), respec-
tively, as radioligands. Selected compounds were also
of potency was 1-propyl g 1-butyl > 1-ethyl for A1, A2A
,
and A2B ARs. The A2B selectivity of 1-alkyl-8-phenyl-
xanthines 41, 5, and 6 was 3- to 8-fold (versus rat A1
ARs) and much higher versus the other AR subtypes.
Introduction of a bromine atom into the para-position
of the phenyl ring (compounds 7, 8) further increased
AR affinity. The increase was similar for A1, A2A, and
A2B receptors. Therefore, A2B selectivity was not im-
proved. 1-Propargyl-8-p-bromophenylxanthine (9) was
somewhat less potent than the corresponding 1-propyl

1504 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hayallah et al.
Ta ble 1. Yields, Melting Points, and Analytical Data of the Intermediate and Final Products
comp
formula
14H16N4O2
15H18N4O2
14H15N4O2Br
15H17N4O2Br
14H11N4O2Br‚H2O
16H18N4O4
17H18N4O4‚0.5C2H5OH‚H2O
17H20N4O5
17H20N4O5‚0.5H2O
14H14N4O2
C15H16N4O2
14H13N4O2Br
15H15N4O2Br
14H9N4O2Br‚H2O
16H16N4O4‚0.25H2O
17H16N4O4‚0.5H2O
17H18N4O5‚0.5H2O
17H18N4O5
18H18N4O4
16H16N4O5‚1.5H2O
14H15N4O6SK
MW
anal.a
yield [%]
mp [°C]
4a a
4ba
4a b
4bb
4cb
4bc
4a d
4be
4a f
5
6
7
8
9
10
11
12
13
C
C
C
C
C
C
C
C
C
C
272.31
286.34
351.20
365.23
365.19
330.35
383.41
360.37
369.38
270.29
284.32
349.19
363.22
363.18
332.84
349.35
369.17
358.35
354.36
371.39
406.46
420.49
427.30
474.30
415.52
429.50
391.27
438.27
382.38
373.37
341.37
336.88
349.57
395.42
387.39
533.97
488.50
519.48
549.50
536.55
542.09
575.07
594.58
ndb
80
86
82
69
79
88
80
82
95
80
82
37
71
79
80
91
89
90
70
83
75
72
79
87
53
50
84
68
70
90
78
69
76
77
83
42
38
38
41
55
45
40
42
204-205
165-166
250
C,c H, N
C, H, N
C, H, N
234
C, H,d
nd
N
237-238
269-270
230 (dec)
172-173
230
C, H, N
nd
C, H, N
nd
>300 (lit. mp >300)54
351-352
>250
C,e H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
HRMSf
C, H,g N
nd
C
C
C
C
C
C
C
C
C
C
>270
322-323
310-311
>250
302-303
>250
14
15
>250
>250
16a g
16bg
16d b
16d h
17
18
19
20
21
22
25
26
27
28
29
30
31
>300 (lit. mp >300)26
>300
C15H17N4O6SK
nd
C
C
C
17H21N4O3Br‚H2O
C, H, N
C, H, N
C, H, N
193
211
17H21N4O3I‚H2O
14H13N4O5SK‚1.5H2O
>300 (lit. mp >300)26
>300
C15H15N4O5SK‚1.5H2O
C, H,h
N
C
C
C
C
17H19N4O2Br
17H19N4O2I
C, H, N
C, H, N
nd
C, H, N
nd
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H,i N
C, H, N
C, H, Nj
C, H, Nk
C, H, N
C, H, N
C, H, N
>270
>270
19H18N4O5
19H16N4O4‚0.5H2O
C17H19N5O3
17H17N5O2
17H18N4O4
18H22N6O4‚0.5H2O
18H21N5O5
25H25N5O6‚0.5CH2Cl2
23H30N6O4‚0.4CH2Cl2
23H28N6O5‚0.6CH2Cl2
24H30N6O6‚0.6CH2Cl2
27H30N6O4‚0.4CH2Cl2
28H32N6O4‚0.3CH2Cl2
28H32N6O5‚0.5CH2Cl2
29H32N6O6‚0.4CH2Cl2
>250
>250
283-285 (dec)
>300
C
C
C
C
C
C
C
C
C
C
C
C
>300
>250
>250
288-289
276-277
294-295
281-283
280-281
262-263
269-270
266-267
32
33
34
35
36
37
a
b
Analyses were within (0.4% of calculated values unless otherwise noted. nd) not determined (intermediate products). c Calcd, 62.92;
found, 62.30. Calcd, 3.60; found, 3.12. e Calcd, 63.37; found, 62.83. f High-resolution mass in EI mode (m/z) determined to be within
d
acceptable limits: calcd, 354.1328; found, 354.1327. g Calcd, 5.17; found, 4.50. Calcd, 4.23; found, 4.76. Calcd, 6.37; found, 6.82. Calcd,
h
i
j
k
15.30; found, 14.83. Calcd, 15.67; found, 16.18.
derivative 7, but it showed improved selectivity versus
A1 ARs. The 1-alkyl-8-p-bromophenylxanthines 7 and
8 were investigated at human A3 ARs. They were
considerably less potent at A3 receptors than at A2B ARs.
1-Propyl substitution appeared to be optimal for high
A3 affinity. The order of potency was 1-propyl > 1-pro-
pargyl > 1-butyl at A3 ARs. 1,3-Dipropyl-8-phenylxan-
thine derivatives with halogen substitution in the
p-phenyl position (19, 20) were prepared for comparison.
1,3-Dipropyl-8-p-bromophenylxanthine (19) exhibited
about the same A2B affinity as the corresponding 3-un-
disubstituted xanthine 7 (19, Ki ) 3.8 nM; 7, Ki ) 2.7
nM). However, the additional 3-propyl residue in 19 led
to an increase in A2A and A1 affinity, resulting in
decreased selectivity for A2B ARs. Replacement of
bromine (in 19) for iodine (in 20) resulted in an increase
in affinity for A1, A2A, and A2B ARs, which was more
pronounced at A1 and A2A (3-fold) than at A2B receptors
(2-fold).
A2A receptors as compared to xanthine derivatives.36 We
have now investigated 9-deaza analogues 44 and 45 of
3-unsubstituted 1-methyl- and 1-propyl-8-phenylxan-
thine. Both compounds exhibited lower A2B affinity than
the corresponding xanthine derivatives (compare 1-meth-
yl-8-phenylxanthine25 and 44: 3.5-fold difference;
5/45: 9-fold difference). Thus, 9-deazaxanthines exhib-
36
ited increased A1 selectivity not only versus A2A but
also versus A2B ARs. Interestingly, N-propyl-substituted
deazaxanthine 45 was relatively potent at human A3
ARs (Ki ) 380 nM).
To increase water solubility of the highly lipophilic,
insoluble 8-phenylxanthine derivatives, polar groups,
e.g., acidic or basic functions, were introduced into the
para-position of the phenyl ring. A carboxylate group
(compound 10) in 1-butyl-8-phenylxanthine (6) was well
tolerated by A2B ARs but not by the other AR subtypes.
Thus, benzoic acid derivative 10 was a potent (Ki ) 24
nM) and selective A2B antagonist (49-fold versus human
A1, 20-fold versus rat A1, 158-fold versus rat A2A, 193-
fold versus human A3). The corresponding phenyl acetic
9-Deazaxanthine derivatives (pyrrolo[2,3-d]pyrimidin-
diones) had earlier been found to be potent AR antago-
nists exhibiting increased selectivity for A1 ARs versus
acid derivative 27 was slightly more potent at A1, A2B
,

1,8-Disubstituted Xanthine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1505
Ta ble 2. Adenosine Receptor Affinities and A2B Selectivities of Xanthine and 9-Deazaxanthine Derivatives
Ki ( SEM [nM] or % inhibition of
radioligand binding at 10 µM
rat
rat
human
A2B vs
human
A3 vs
(or human)
A1 vs
(or human)
A
2A vs
[3H]ZM-
241385
(n ) 2)
[3H]PSB-
11
A2B selectivity
A1/A2B A2A/A2B A3/A2B
[3H]CCPA
(n ) 3)
[3H]MSX-2
compd
R1
R3
R8
(n ) 3)
(n ) 2)
8-Mono-, 1,8-Di-, and 1,3,8-Trisubstituted Xanthine Derivatives
38
propyl
propyl
H
ethyl
propyl
butyl
propyl
butyl
propargyl
propyl
propyl
butyl
H
cyclopentyl
1426
58026
34.4 ( 10.3 nd
5150
795 ( 139 0.02
810 ( 150 nd
0.4
169
9
nd
16
nd
50
nd
nd
43
>1000
70
nd
nd
39 (DPCPX)
propyl cyclopentyl
0.952
47052
40
41
5
6
7
8
9
19
20
10
H
H
H
H
H
H
H
phenyl
phenyl
phenyl
phenyl
p-bromophenyl
p-bromophenyl
p-bromophenyl
250026
2100026
3
8
7
3
5
5
9
2
1
26
95
97
54
84
21
29
10
6
15026
180026
19 ( 1.5
4.7 ( 3.3
950 ( 32
31 ( 2.7 6726
40 ( 22
18 ( 5
458 ( 71 190026
642 ( 151
336 ( 80
57 ( 38
nd
11.8 ( 1.2 nd
4.4 ( 0.38 173 ( 9
13 ( 9
2.7 ( 1.6
6.8 ( 0.2
45% ( 9
477 ( 1
60 ( 8.8
5.7 ( 1.6
2.1 ( 1.1
481 ( 83.1
(1181 ( 88)a
199 ( 4.6
39 ( 7
14 ( 6
3800 ( 458
(55% ( 11)a
5905 ( 1258
propyl p-bromophenyl
propyl p-iodophenyl
3.8 ( 0.57 nd
2.5 ( 0.55 nd
H
H
H
H
p-carboxyphenyl
24 ( 6
15.1 ( 4.3 2857 ( 16 14
53.4 ( 18.2 14% ( 20
4622 ( 323 20 (49)a
158
193
27
butyl
p-(carboxymethyl)- 207 ( 105
phenyl
p-sulfophenyl
391
189
17 (PSB-1115) propyl
220026
(35% ( 6)a
2400026
41 (>180)a 453
>180
18
butyl
propyl
propyl
p-sulfophenyl
475 ( 34
8070 ( 1850
140052
70 ( 12
25055
13 ( 1.8
39% ( 8
7
1
8
115
6
>700
>140
1
91
42 (DPSPX)
22
propyl p-sulfophenyl
H
21052
183 (s)b,56
1184 ( 169
6-carboxy-2-
naphthyl
110 ( 9
43% ( 10
(55% ( 4)a
8-(Phenylacrylic acid)xanthine Derivatives
11
propyl
propyl
propyl
H
COOH
68 ( 11
13% ( 15
14.5 ( 8.2 1217 ( 231
5
0.25
>500
13
>500
84
0.5
43
4318
14
propyl COOH
H
1518
80018
6018
3018
COOCH3
91 ( 13
30% ( 10
26 ( 21
1119 ( 147 3.5
8-(4-Carboxymethyloxyphenyl)xanthine Derivatives
15
12
13
propyl
butyl
propyl
H
H
H
COOH
COOH
COOCH3
145 ( 3
81 ( 43
15 ( 6
4% ( 13
1877 ( 908
709 ( 71
372 ( 51
nd
0.4
7
4
>1000 nd
10.8 ( 8.3 1192 ( 147
171
177
108
3.7 ( 0.3
520 ( 78
80.6 ( 10.9
22
9-Deazaxanthine Derivatives
9736 200036
3936 (45 ( 2)a
44
45
methyl
propyl
H
H
phenyl
phenyl
2098 ( 299 0.2
4
29
4
9
120036 (58% ( 11)a 42 ( 0.8
380 ( 177 1 (1)a
a
b
Human recombinant receptors (n ) 2). Sheep A3 receptor.
and A3 ARs and somewhat less potent at A2A ARs,
retaining high affinity and some selectivity for A2B ARs.
A para-sulfonate group in 1-propyl- and 1-butyl-8-
phenylxanthine (compounds 17 and 18) was less well
tolerated than a carboxylate group. However, it was
again better tolerated by the A2B than by the other AR
subtypes yielding A2B antagonists with improved selec-
tivity. 1-Propyl-8-p-sulfophenylxanthine (17) was the
compound with the highest selectivity of the present
series. It showed a Ki value at human A2B receptors of
53 nM and was greater than 180-fold selective versus
human A1, 41-fold selective versus rat A1, and more
than 180-fold selective versus rat A2A and human A3
ARs. The corresponding 1-butyl-8-p-sulfophenylxan-
thine (18) was less potent at A2B ARs (Ki ) 70 nM) but
more potent at A1 and A2A ARs, thus exhibiting reduced
selectivity. Data for 1,3-dipropyl-8-p-sulfophenylxan-
thine (DPSPX, 42), a nonselective, water-soluble stan-
dard antagonist, were included (compare 42 and 17).
The additional propyl group in 42 results in largely
increased A1 (11-fold), A2A (17-fold), and particularly A3
affinity (>50-fold). In contrast, A2B affinity is reduced
in 42 (5-fold) compared to the 1,8-disubstituted xanthine
17. It can be concluded from these and other data (see
below) that the 3-substituent in xanthines is important
for A1, A2A, and particularly for A3 affinity but not for
affinity to A2B ARs. Sulfophenylxanthine derivatives,
such as 42 and 8-p-sulfophenyltheophylline, do not
penetrate into the central nervous system (CNS) and
are only peripherally active.37,38 Thus, 1-propyl-8-p-
sulfophenylxanthine (17) is expected to be a peripheral
A2B antagonist without effects on the CNS. Perorally
applied 17 is probably not being absorbed due to its
highly polar character and may exert its actions only
locally in the intestine, where a high density of A2B ARs
is known to exist. 1-Propyl-8-p-sulfophenylxanthine (17)
has been investigated in a functional assay at human
recombinant A2B ARs.39 It was shown to inhibit NECA-
induced stimulation of adenylate cyclase with a Ki value
of 820 nM.39 An X-ray structure of 17 revealed a nearly
coplanar conformation of the xanthine ring system on
the 8-phenyl substituent.40

1506 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hayallah et al.
Ta ble 3. Adenosine Receptor Affinities and A2B Selectivities of Amide Derivatives of 3-Unsubstituted 8-Phenylxanthines
Ki ( SEM [nM] or % inhibition of
radioligand binding at 10 µM
rat
rat
human
A2B vs
human
A3 vs
(or human)
A1 vs
(or human)
A2A vs
A2B selectivity
[3H]ZM-
241385
[3H]PSB-
11
compd
R1
R2
[3H]CCPA
[3H]MSX-2
A1/A2B A2A/A2B A3/A2B
Amides of 8-(4-Carboxymethyloxyphenyl)xanthines
28
29
30
propyl 2-aminoethyl
propyl 2-hydroxyethyl
butyl p-carboxymethylphenyl 41 ( 4.1
24 ( 4
365 ( 30
2139 ( 1278
479 ( 97
10 ( 0.8
nd
2
37
nd
422
128
35 ( 6 (68 ( 35)a
1.2 ( 0.5 422 ( 35 29 (57)a 2139
5.3 ( 0.2 676 ( 224
8
90
Piperazinyl Amides of 8-(4-Carboxymethyloxyphenyl)xanthines
31
32
33
34
35
36
37
butyl ethyl
butyl acetyl
butyl ethyloxycarbonyl
butyl phenyl
butyl benzyl
18 ( 8.5
30 ( 16
20 ( 9.6
17 ( 8.7
290 ( 170
450 ( 292
900 ( 485
43% ( 11
5.5 ( 0.4 nd
6.5 ( 0.3 nd
6.1 ( 0.3 nd
14.7 ( 0.4 nd
3
5
3
1
53
69
nd
nd
nd
nd
365
102
nd
148
>500
37 ( 11 (122 ( 54)a 550 ( 65 (55% ( 10)a 1.3 ( 0.2 475 ( 114 28 (94)a 423
butyl 2-methoxyphenyl
butyl benzyloxycarbonyl
24 ( 6
15 ( 1.7
320 ( 224
130 ( 133
1.2 ( 0.4 102 ( 2
2.9 ( 1.4 nd
20
5
267
45
a
Human recombinant receptors (n ) 2).
In a previous study,39 1-propargyl-3-methyl-8-(6-car-
boxy-2-naphthyl)xanthine, originally developed as an
A2A antagonist,33 had been found to be somewhat
selective for A2B ARs. We have now synthesized the
corresponding 1-propyl-8-(6-carboxy-2-naphthyl)xan-
thine (22). As expected, the compound was very potent
at A2B ARs (Ki ) 13 nM). Selectivity versus A2A and A3
ARs was high, but it was much lower versus rat A1 ARs
(8-fold). A structurally related compound, the phenyl-
acrylic acid derivative 11, showed nearly the same Ki
values as the naphthyl carboxylic acid 22. Data for the
1,3-dipropyl analogue (43) of 11 have been published.18
A comparison of 11 and 43 again showed that the
3-propyl residue leads to an increase in A1 (4.5-fold) and
A2A (>12-fold) affinity and a dramatic increase in A3
affinity (41-fold), but reduces A2B affinity (4-fold). While
the dipropyl derivative 43 was slightly A1-selective, the
3-unsubstituted 11 was A2B-selective. Methylation of the
carboxylate of 11 leading to 14 had no major effects on
affinity at all AR subtypessas seen with compounds 10
and 27sconfirming that the acidic group is not required
for high AR affinity.
potent A2B antagonist (13, Ki ) 3.7 nM) with moderate
selectivity versus A1 and A3 and high selectivity versus
A2A ARs.
A second series of xanthine derivatives was prepared
in which the carboxylic acid derivatives 11, 12, and 15
were coupled with amines bearing polar groups in order
to increase water solubility (Table 3). All 3-unsubsti-
tuted xanthine amides exhibited high A2B affinity with
Ki values at low nanomolar concentrations. The hy-
droxyethylamide 29 of 1-propyl-8-(4-carboxymethyloxy-
phenyl)xanthine (15) exhibited a similarly high A2B
affinity (Ki ) 1.2 nM) but was more selective versus A1
ARs (59-fold versus human, 29-fold versus rat A1 ARs).
In comparison, the corresponding aminoethylamide 28,
in which the terminal hydroxy group in 29 was formally
replaced by an amino group, was 10-fold less potent at
A2B receptors but showed increased A1 and A2A affinity.
Compound 28 is a 3-unsubstituted analogue of the 1,3-
dipropylxanthine XAC (1). In comparison with 1, 28
exhibited decreased A1 (20-fold) and A2A (6-fold) affinity
and increased A2B affinity. This result confirms that
3-unsubstituted xanthines exhibit increased A2B selec-
tivity. Amide 30 bearing a terminal carboxylate was well
tolerated by A2B ARs and was quite selective versus the
other AR subtypes.
Carboxymethyloxyphenyl derivatives (12, 13, 15) can
be envisaged as bioisosteric analogues of carboxynaph-
thyl (22) and phenyl acrylic acid derivatives (11, 14).
1-Propyl-(8-(4-carboxymethyloxy)phenyl)xanthine (15),
however, exhibited considerably (>20-fold) lower A2B
affinity than the bioisosteric compounds 22 and 11. At
A1 ARs, 15 was only slightly weaker than 11 and 22
and was therefore not selective for A2B ARs. Replace-
ment of the 1-propyl substituent (in 15) by 1-butyl
(compound 12) resulted in a 34-fold increase in A2B
affinity, while A1 affinity was increased only by 2-fold.
The corresponding 1,3-dibutyl derivative described by
J acobson et al.18 was more potent at A1, A2A, and
particularly A3 ARs. However, it was less potent at A2B
ARs than its 3-unsubstituted analogue 12. Methylation
of the carboxylate 15 increased affinity, leading to a
A series of seven piperazinyl amides was prepared
(compounds 31-37). The N-alkyl- and N-aryl-pipera-
zinylamides contained a basic nitrogen atom, which can
be protonated leading to increased water solubility. All
piperazinylamides exhibited high affinity for A2B ARs.
Their Ki values ranged from 1 to 15 nM (Table 3). Large
substituents, e.g., benzyl, 2-methoxyphenyl, and ben-
zyloxycarbonyl, were well tolerated. There was not much
difference in affinities of ethyl (31) and acetyl (32) or
benzyl (35) and benzyloxycarbonyl (37) derivatives. The
most potent compound of the series was the N-ben-
zylpiperazine derivative 35 of 1-butyl-8-(4-carboxymeth-
yloxyphenyl)xanthine, exhibiting a Ki value of 1.3 nM

1,8-Disubstituted Xanthine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1507
an equimolar or slightly excessive amount of N-(3-(dimethyl-
amino)-propyl)-N′-ethylcarbodiimide hydrochloride (EDC) was
added. The reaction mixture was stirred overnight at room
temperature. The product was separated either by evaporation
of the solvent in vacuo followed by suspension of the residue
in a small amount of methanol and subsequent filtration, or
by precipitation of the product by the addition of water followed
by filtration.
at human A2B ARs and high selectivity versus the other
AR subtypes.
In conclusion, 3-unsubstituted xanthine derivatives
bearing a cyclopentyl or (substituted) phenyl residue in
the 8-position were found to exhibit high potency at A2B
ARs and increased selectivity in comparison with xan-
thines bearing a 3-substituent. Some of the new A2B
antagonists were not only highly selective versus human
but also versus rat A1 ARs. A2B-selective AR antagonists
with high water solubility were obtained, which may
be useful research tools to investigate the (patho)-
physiology and pharmacology of A2B receptors.
6-Am in o-5-(6-car boxy)n aph th -2-oylam in o-3-pr opylu r a-
cil (21). 2,6-Naphthalenedicarboxylic acid (0.22 g, 1 mmol) was
dissolved in 15 mL of DMF, then 0.1 g (1 mmol) of N-
methylmorpholine was added, and the reaction mixture was
cooled in an ice bath. Isobutylchloroformate (0.14 g, 1 mmol)
was added, and 25 min later 5,6-diamino-3-propyluracil (3a ,
0.19 g, 1 mmol) was added. A precipitate was formed within
10-20 min, which was filtered off and washed with water.
8-P h en yl-1-p r op ylxa n th in e (5), 1-Bu tyl-8-p h en ylxa n -
th in e (6), 8-(4-Br om op h en yl)-1-p r op a r gylxa n th in e (9),
1-Bu tyl-8-(4-ca r boxy)p h en ylxa n th in e (10), 8-Cin n a m yl-
1-p r op ylxa n th in e (11), 1-Bu tyl-8-(4-ca r boxym eth yloxy)-
ph en ylxan th in e (12), an d 8-(4-Meth oxycar bon ylm eth oxy)-
p h en yl-1-p r op ylxa n th in e (13). Gen er a l P r oced u r e. 3-Sub-
stituted 6-amino-5-benzylidenaminouracil 4a a , 4ba , 4cb, 4bc,
4a d , 4be, or 4a f (4.4 mmol) was dissolved at 0 °C in 120 mL
of thionyl chloride. The reaction mixture was refluxed for 1 h,
and then the mixture was stirred at room temperature
overnight. In the case of 4a d , the solution was refluxed for 30
min and subsequently stirred for 2 h. Compound 4a f was
stirred for 2 h at 70 °C only. Thionyl chloride was distilled
off, the residue was suspended in iced water and then filtered,
and the residue was washed with water affording the expected
products, which were recrystallized by dissolving them in DMF
followed by dropwise addition of water.
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. DMSO-
d6 was used as solvent. The chemical shifts of the deuterated
solvent served as internal standard: δ 1H: 2.50; 13C: 39.1.
The mass spectra were performed on an MS-50 A.E.I. mass
spectrometer at the Institute of Organic Chemistry, University
of Bonn. Purity of the prepared compounds was checked by
TLC on silica gel 60 F254 (Merck) aluminum plates, using
dichloromethane:methanol (9:1) or dichloromethane:methanol
(3:1) as the mobile phase. Melting points were determined on
a Bu¨chi 530 melting point apparatus and are uncorrected.
Elemental microanalyses were performed at the Pharmaceuti-
cal Institute, University of Bonn.
8-Cyclopentyl-1-propylxanthine (38),26 8-phenylxanthine
(40),41 1-ethyl-8-phenylxanthine (41),26 3-methyl-6-phenyl-1,5-
dihydropyrrolo[3,2-d]pyrimidin-2,4-dione (43),36 and 6-phenyl-
3-propyl-1,5-dihydropyrrolo[3,2-d]pyrimidin-2,4-dione (44)36
were prepared as described. 8-Cyclopentyl-1,3-dipropylxan-
thine (DPCPX 39) was obtained from commercial sources.
3-Substituted 5,6-diaminouracils (3a -3c) were prepared from
6-aminouracil via regioselective alkylation followed by nitro-
sation and reduction as described.42,43 1,3-Disubstituted 6-ami-
nouracils were prepared from urea derivatives as described
followed by nitrosation and reduction to obtain 1,3-disubsti-
tuted 5,6-diaminouracils.44
8-(4-Br om op h en yl)-1-p r op ylxa n th in e (7), 8-(4-Br om o-
p h en yl)-1-bu tylxa n th in e (8). Benzylidene derivatives 4a b
or 4bb (1.71 mmol) and 2.07 mmol of anhydrous ferric chloride
were refluxed for 3 h in 15 mL of methanol. The reaction
mixture was cooled, 30 mL of water was added, and the formed
precipitate was collected by filtration. The products were
recrystallized by dissolving them in DMF followed by dropwise
addition of water.
1-P r op yl-8-(4-su lfop h en yl)xa n th in e (17) a n d 1-Bu tyl-
8-(4-su lfop h en yl)xa n th in e (18). HMDS Meth od . Carbox-
amide derivative 16a g or 16bg (2.71 mmol) was dissolved in
50 mL of hexamethyldisilazane (HMDS) in the presence of a
catalytic amount of (NH4)2SO4. The reaction mixture was
refluxed at 140 °C for 50 h. HMDS was distilled off in vacuo,
and the residue was treated with 10 mL of methanol and 10
mL of water. The formed precipitate was filtered off and
recrystallized from water.
P P SE Meth od . Carboxamide derivative 16a g or 16bg (2.71
mmol) was refluxed in 8 mL of polyphosphoric acid trimeth-
ylsilyl ester (PPSE) at 160-180 °C for 1 h. After cooling, the
reaction mixture was treated with 20 mL of methanol and the
formed precipitate was filtered off and recrystallized from
water.
8-(4-Br om op h en yl)-1,3-d ip r op ylxa n th in e (19) a n d 8-(4-
Iod op h en yl)-1,3-d ip r op ylxa n th in e (20). Carboxamide de-
rivative 16d b or 16d h (0.977 mmol) was refluxed in a mixture
of 10 mL of methanol and 10 mL of 10% aqueous NaOH
solution for 30 min at 70 °C. The reaction mixture was filtered
while hot. The methanol was distilled off, and the residue was
taken up in H2O and acidified with HCl to pH 4. The
precipitate was filtered off and washed with 20 mL of water.
8-(6-Ca r boxyn a p h th -2-yl)-1-p r op ylxa n th in e (22). Com-
pound 21 (0.1 g, 0.26 mmol) was dissolved in 20 mL of HMDS,
and 0.1 mL of trimethylchlorosilane (TMSCl) and 20 mg of
p-toluenesulfonic acid were added. The reaction mixture was
refluxed for 36 h. HMDS was removed in vacuo, then water
was added, and the mixture was boiled for 10 min. The
obtained suspension was filtered after cooling.
6-Am in o-5-ben zylid en a m in o-3-p r op ylu r a cil (4a a ), 6-
Am in o-5-ben zylid en a m in o-3-bu tylu r a cil (4ba ), 6-Am in o-
5-(4-br om oben zylid en )a m in o-3-p r op a r gylu r a cil (4cb), 6-
Am in o-3-bu tyl-5-(4-car boxyben zyliden )am in ou r acil (4bc),
6-Am in o-5-cin n a m ylid en a m in o-3-p r op ylu r a cil (4a d ), a n d
6-Am in o-3-bu tyl-5-(4-ca r boxym eth yloxyben zylid en )a m i-
n ou r a cil (4be). Gen er a l P r oced u r e (Meth od A). To a
solution of 3-substituted 5,6-diaminouracil (2.8 mmol) 3a , 3b,
or 3c, in ethanol, was added an equimolar amount of the
appropriate aldehyde. The reaction mixture was refluxed for
4-5 h with monitoring by TLC. The reaction mixture was
allowed to cool, and the reaction product was collected by
filtration, dried, and recrystallized from ethanol.
6-Am in o-5-(4-b r om ob en zylid en a m in o)-3-p r op ylu r a -
cil (4a b), 6-Am in o-5-(4-br om oben zylid en a m in o-3-bu tyl-
u r acil (4bb), an d 6-Am in o-5-(4-m eth oxycar bon ylm eth oxy-
ben zylid en a m in o)-3-p r op ylu r a cil (4a f). Gen er a l P r oce-
d u r e (Meth od B). To a solution of 3-substituted 5,6-diami-
nouracil (2.8 mmol) 3a , 3b, or 3c, in ethanol, was added an
equimolar amount of the appropriate aldehyde followed by a
few drops of acetic acid. The reaction mixture was stirred at
room temperature for 1 h and then precipitated by the addition
of water to yield 4a b and 4bb. In case of 4a f, the starting
compounds were refluxed for 0.5 h and then cooled, and the
product was collected by filtration.
6-Am in o-3-p r op yl-5-(4-su lfop h en yl)ca r b oxa m id ou r a -
cil (16a g), 6-Am in o-3-bu tyl-5-(4-su lfop h en yl)ca r boxa m i-
dou r acil (16bg), 6-Am in o-5-(4-br om oph en yl)car boxam ido-
1,3-d ip r op ylu r a cil (16d b), a n d 6-Am in o-1,3-d ip r op yl-5-
(4-iodoph en yl)car boxam idou r acil (16dh ). Gen er al P r oce-
d u r e. To a solution of 5,6-diaminouracil (2.8 mmol) 3a , 3b, or
3d , in methanol or methanol:water (1:1) was added an
equimolar amount of the appropriate acid derivative, and then
1-Bu tyl-8-[4-(ca r boxym eth yl)p h en yl]xa n th in e (27). (a )
P r ep a r a tion of 4-Cya n om eth ylben zoic Acid (24). A solu-
tion of 4-chloromethylbenzoic acid (23, 3 g, 17.6 mmol) in THF

1508 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hayallah et al.
was carefully added to a saturated solution of NaHCO3 (15
mL). NaCN (5.08 g, 103.6 mmol) was added followed by H2O
(16.5 mL). The reaction mixture was kept at 20-25 °C for 48
h. Then it was cooled in an ice bath and acidified with
concentrated HCl to pH 4 (with caution). THF was removed
by evaporation under reduced pressure (caution!), and then
the solution was acidified with HCl to pH 2. The brown solid
which had formed was collected, washed with H2O, and then
dissolved in 1 N NaOH (30 mL). Acidification of the charcoal-
treated and filtered solution afforded the expected product 24
which was collected by filtration.
(b) P r ep a r a tion of 6-Am in o-3-bu tyl-5-(4-cya n om eth yl-
ben zoyl)a m id ou r a cil (25). Compound 3b (0.93 g, 4.7 mmol),
an equimolar amount of 24 (0.76 g, 4.7 mmol), and EDC (0.9
g, 4.7 mmol) were dissolved in 40 mL of CH3OH:H2O (1:1, v/v).
The reaction mixture was stirred at room temperature over-
night, the solvent was evaporated in vacuo, and the residue
was suspended in a small amount of methanol and then
filtered to afford the expected product 25 which was recrystal-
lized from methanol.
mmol) was dissolved at 150 °C in DMF, then the solution was
cooled to 60 °C, and 67 mg (1.116 mmol) of ethylenediamine
was added. After stirring for 2 days at room temperature, the
reaction mixture was concentrated producing a precipitate
which was filtered off and washed with water and methanol
affording the expected product 28.
8-[4-((2-H yd r oxyet h yla m in o)-2-oxoet h oxy)p h en yl]-1-
p r op ylxa n th in e (29). Compound 13 (100 mg, 0.279 mmol)
was dissolved in hot DMF, 17 mg (0.279 mmol) of ethanol-
amine was added at 40 °C, and the mixture was stirred
overnight at room temperature. The formed precipitate was
filtered off and washed with methanol affording the expected
product 29.
8-(4-Met h ylca r b oxyet h ylid en e)p h en yl-3-p r op ylxa n -
th in e (14). Compound 4g (0.2 g, 0.58 mmol) was suspended
in 10 mL of SOCl2, and the mixture was refluxed for 30 min.
Then the excess of SOCl2 was distilled off. The solid residue
was cooled in an ice bath, and 15 mL of methanol was added.
The suspension was refluxed for 30 min. The cream-colored
solid was filtered off and washed with methanol.
(c) P r ep a r a tion of 1-Bu tyl-8-[4-(cya n om eth yl)p h en yl]-
xa n th in e (26). Compound 25 (0.5 g, 1.46 mmol) was dissolved
in 50 mL of HMDS in the presence of a catalytic amount of
(NH4)2SO4. The reaction mixture was refluxed for 50 h. The
reaction was monitored by TLC until the starting compound
had completely disappeared. HMDS was distilled off in vacuo,
and to the residue were added 10 mL of CH3OH and 10 mL of
H2O. Then the precipitate was filtered off and recrystallized
by dissolving it in DMF and subsequent dropwise addition of
H2O.
(d ) P r ep a r a tion of 1-Bu tyl-8-[4-(ca r boxym eth yl)p h en -
yl]xa n th in e (27). Compound 26 (0.25 g, 0.77 mmol) was
dissolved in 3.4 mL of H2O and 3 mL of H2SO4 and refluxed
for 6 h. Then the reaction mixture was cooled to room
temperature, 10 mL of H2O was added, and the mixture was
left in the refrigerator overnight. After filtration, the solid
product was dissolved in aqueous NaOH solution, reprecipi-
tated by addition of aqueous HCl (to pH 4), then filtered off,
and recrystallized by dissolving it in DMF followed by the
dropwise addition of H2O.
Syn th esis of Am id e Der iva tives 28-37. 1-Bu tyl-8-[4-
((4-eth ylpiper azin -1-yl)-2-oxo-eth oxy)ph en yl]xan th in e (31)
a n d 1-Bu t yl-8-[4-((4-b en zyloxyca r bon ylp ip er a zin -1-yl)-
2-oxoeth oxy)p h en yl]xa n th in e (37). Meth od A (Ca r bod i-
im id e Meth od ). A solution of 12 (0.558 mmol), the desired
amine derivatives (1.116 mmol), EDC (1.116 mmol), and
4-(dimethylamino)pyridine (DMAP, 0.346 mmol) in 22 mL of
anhydrous DMF:CH2Cl2 (1:1, v/v) was stirred at room tem-
perature for 48 h (31) or for 3 days (37) with follow up by TLC.
The mixture was evaporated to dryness under reduced pres-
sure, and the residue was dissolved in 40 mL of CH2Cl2 and a
few drops of MeOH and left for precipitation. The precipitate
was filtered off and recrystallized from CH2Cl2:CH3OH (8:2)
affording the desired products 31 and 37.
1-Bu t yl-8-[4-((4-ca r b oxym et h yl)p h en yla m in o-2-oxo-
eth oxy)p h en yl]xa n th in e(30), 1-Bu tyl-8-[4-((4-a cetyl-p i-
p er a zin -1-yl)-2-oxoeth oxy)p h en yl]xa n th in e (32), 1-Bu tyl-
8-[4-((4-eth oxycar bon yl-piper azin -1-yl)-2-oxo-eth oxy)ph en -
yl]xa n th in e (33), 1-Bu tyl-8-[4-((4-p h en ylp ip er a zin -1-yl)-
2-oxoeth oxy)ph en yl]xan th in e (34), 1-Bu tyl-8-[4-((4-ben zyl-
p ip er a zin -1-yl)-2-oxoeth oxy)p h en yl]xa n th in e (35), a n d
1-Bu t yl-8-[4-((4-(2-m et oxyp h en yl-p ip er a zin -1-yl)-2-oxo-
eth oxy)p h en yl]xa n th in e (36). Meth od B (Acyl Ch lor id e
Meth od ). A solution of 12 (0.558 mmol) in 11 mL of thionyl
chloride was stirred at 70°C for 4 h. Then the excess of thionyl
chloride was removed by evaporation. To the residue was
added a solution of the desired amine derivative (1.116 mmol)
in 22 mL of anhydrous pyridine:CH2Cl2 (1:1, v/v), and the
mixture was stirred at room temperature for 24-48 h and
subsequently evaporated to dryness under reduced pressure.
Isolation and purification of compounds 30 and 32-36 was
achieved as described above for compounds 31 and 37.
8-(4-Ca r boxym eth yloxyp h en yl)-1-p r op ylxa n th in e (15).
Compound 13 (0.36 g 1 mmol) was dissolved in 5 mL of DMF,
and 5 mL of 0.1 N aqueous Na2CO3 solution was added. The
reaction mixture was heated in a steam bath for 30 min. Then
the solvent was concentrated, and the mixture was filtered.
The filtrate was acidified (to pH 3) with concentrated HCl, and
the formed precipitate was filtered off.
Biologica l Assa ys. Ma ter ia ls. Radioligands were obtained
from the following sources: [3H]CCPA from NEN Life Science
(54.9 Ci/mmol), [3H]MSX-2 from Amersham (85 Ci/mmol), [3H]-
ZM241385 from Tocris (17 Ci/mmol), and [3H]PSB-11 from
Amersham (53 Ci/mmol). The nonradioactive precursors of [3H]-
MSX-2 and [3H]PSB-11 were synthesized in our laboratory.
Mem br a n e P r ep a r a tion s. Membranes from Chinese ham-
ster ovary (CHO) cells stably transfected with the human A1,
the human A2A, or the human A3 AR were prepared as
described.45 For A2B adenosine receptor assays, commercially
available membrane preparations containing the human A2B
AR were obtained from Biotrend (Cologne, Germany).
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
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 A2B assays was 1%, and in the
other assays not more than 2.5%. The radioligand concentra-
tions were as follows: [3H]CCPA:48 0.5 nM (rat or human A1);
[3H]MSX-2:49 1.0 nM (rat or human A2A); [3H]ZM241385:50
5
nM (human A2B); [3H]PSB-11:51 0.5 nM (human A3). Binding
assays were performed essentially as described.45,48-50 The A3
binding assay is described below. About 40-70 µg/mL of
protein were used in the assays. Membranes were preincu-
bated for 30 min with 0.12 IU/mL of adenosine deaminase in
order to remove endogenous adenosine. Curves were deter-
mined 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 (human recep-
tors) or triplicate (rat receptors).
A3 Bin d in g Assa ys. Binding assays were performed using
[3H]PSB-1151 in 50 mM TRIS-HCl buffer at pH 7.4. Assays
were incubated on a shaking water bath at 23 °C for 30 min.
Nonspecific binding was determined in the presence of 100 µM
of R-PIA and amounted to less than 5% of total binding. Total
binding was determined in the presence of 2% DMSO, and ca.
50 µg of protein per tube (containing a final volume of 0.5 mL)
was added to start the reaction. Termination of the incubation
was performed by rapid filtration through GF/B glass fiber
filters, presoaked in rinse buffer, using a Brandel 48 channel
harvester. Filters were washed three times with 2 mL of ice-
cold rinse buffer each. Radioactivity of the punched-out wet
filters was counted after 9 h of preincubation with 3 mL of
Ultima Gold scintillation cocktail (Canberra Packard, Dreieich,
Germany).
8-[4-((2-Am in oeth yla m in o)-2-oxoeth oxy)p h en yl]-1-p r o-
p ylxa n th in e (28). Meth od C. Compound 13 (200 mg, 0.558

1,8-Disubstituted Xanthine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1509
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Da ta An a lysis. Data were analyzed using Graph Pad
PRISM Version 3.0 (San Diego, CA). For non-linear regression
analysis, the Cheng-Prusoff equation and KD values of 0.5 nM
(rat A1) and 0.61 nM (human A1) for [3H]CCPA, 8.0 nM (rat
A2A) and 7.3 nM (human A2A) for [3H]MSX-2, 33 nM for
[3H]ZM241385, and 4.9 nM for [3H]PSB-11 were used to
calculate Ki values from IC50 values.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
data of synthesized compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. A.M.H. thanks the Egyptian
government for a Ph.D. scholarship. J .S.-R. was sup-
ported by the Deutscher Akademischer Austauschdienst
(DAAD). U.R. obtained a scholarship by the Hilmer
foundation, and B.S. obtained a scholarship by the
Deutsche Forschungsgemeinschaft (DFG, Graduierten-
kolleg GRK-677). C.E.M. is grateful for support by the
Fonds der Chemischen Industrie and the Bundesmin-
isterium fu¨r Bildung und Forschung (BMBF).
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