Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at human A(2B) adenosine receptors

Article abstract of DOI:10.1021/jm990421v

No highly selective antagonists of the A(2B) adenosine receptor (AR) have been reported; however such antagonists have therapeutic potential as antiasthmatic agents. Here we report the synthesis of potent and selective A(2B) receptor antagonists. The stru

Full text of DOI:10.1021/jm990421v

J . Med. Chem. 2000, 43, 1165-1172
1165
An ilid e Der iva tives of a n 8-P h en ylxa n th in e Ca r boxylic Con gen er Ar e High ly
P oten t a n d Selective An ta gon ists a t Hu m a n A2B Ad en osin e Recep tor s
Yong-Chul Kim,† Xiao-duo J i,† Neli Melman,† J oel Linden,‡ and Kenneth A. J acobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, and Department of Internal Medicine and Molecular
Physiology & Biological Physics, University of Virginia, Box MR4 6012, Health Science Center, Charlottesville, Virginia 22908
Received August 19, 1999
No highly selective antagonists of the A2B adenosine receptor (AR) have been reported; however
such antagonists have therapeutic potential as antiasthmatic agents. Here we report the
synthesis of potent and selective A2B receptor antagonists. The structure-activity relationships
(SAR) of 8-phenyl-1,3-di-(n-propyl)xanthine derivatives in binding to recombinant human A2B
ARs in HEK-293 cells (HEK-A2B) and at other AR subtypes were explored. Various amide
derivatives of 8-[4-[[carboxymethyl]oxy]phenyl]-1,3-di-(n-propyl)xanthine, 4a , were synthesized.
A comparison of aryl, alkyl, and aralkyl amides demonstrated that simple anilides, particularly
those substituted in the para-position with electron-withdrawing groups, such as nitro, cyano,
and acetyl, bind selectively to human A2B receptors in the range of 1-3 nM. The unsubstituted
anilide 12 had a Ki value at A2B receptors of 1.48 nM but was only moderately selective versus
human A1/A2A receptors and nonselective versus rat A1 receptors. Highly potent and selective
A2B antagonists were a p-aminoacetophenone derivative 20 (Ki value 1.39 nM) and a
p-cyanoanilide 27 (Ki value 1.97 nM). Compound 27 was 400-, 245-, and 123-fold selective for
human A2B receptors versus human A1/A2A/A3 receptors, respectively, and 8.5- and 310-fold
selective versus rat A1/A2A receptors, respectively. Substitution of the 1,3-dipropyl groups with
1,3-diethyl offered no disadvantage for selectivity, and high affinities at A2B receptors were
maintained. Substitution of the p-carboxymethyloxy group of 4a and its amides with acrylic
acid decreased affinity at A2B receptors while increasing affinity at A1 receptors. 1,3-Di-
(cyclohexylmethyl) groups greatly reduced affinity at ARs, although the p-carboxymethyloxy
derivative 9 was moderately selective for A2B receptors. Several selective A2B antagonists
inhibited NECA-stimulated calcium mobilization in HEK-A2B cells.
In tr od u ction
The alkylxanthine theophylline, 1 (Figure 1), a weak
nonselective adenosine antagonist,1 is effective when
used therapeutically for the treatment of asthma, but
its use is associated with unpleasant side effects, such
as insomnia and diuresis.2 In recent years, use of
theophylline as a bronchodilator for relief of asthma has
been supplanted by drugs of other classes, i.e. selective
â2-adrenergic agonists, corticosteroids, and recently
leukotriene antagonists.3 All of these compounds have
F igu r e 1. Structures of various xanthines that act as
limitations, so the development of a theophylline-like
antagonists at A2B receptors.
drug with reduced side effects is still desirable. The
mechanism of action of theophylline in asthma has long
sine-induced contraction,5 the adenosine antagonist
properties of theophylline had been doubted as an
explanation of the therapeutic properties,6 until rela-
tively recently. One reason was that enprofylline, 3-pro-
pylxanthine, 3, formerly used clinically as an anti-
asthmatic in Europe, is much weaker than theophylline
as an antagonist of two AR subtypes previously well-
defined pharmacologically, i.e. A1/A2A ARs. With the
recent focus on A2B ARs,7 it has been noted that
therapeutic concentrations of enprofylline block human
A2B receptors and proposed that antagonists selective
for this subtype may have potential use as anti-
asthmatic agents.8,9 Enprofylline has a Ki value of 7 µM
and is somewhat selective in binding to human A2B
ARs.9,10 A2B ARs are expressed in some mast cells, such
as canine BR mastocytoma cells, in which they appear
been the subject of controversy. It is recognized that at
therapeutically relevant doses, theophylline and its
closely related analogue caffeine block endogenous
adenosine acting as a local modulator in the brain and
other organs. Adenosine activates four subtypes of G
protein-coupled adenosine receptors (ARs): A1/A2A/A2B
/
A3.4 In comparison to the other known actions of
theophylline, e.g. inhibition of phosphodiesterases, it is
more potent in antagonism of ARs. Although lung tissue
from asthmatic patients is hyperresponsive in adeno-
* Correspondence to: Dr. K. A. J acobson, Bldg. 8A, Rm. B1A-19,
NIH, NIDDK, LBC, Bethesda, MD 20892-0810. Tel: (301) 496-9024.
Fax: (301) 480-8422. E-mail: kajacobs@helix.nih.gov.
† National Institute of Diabetes, Digestive and Kidney Diseases.
‡ University of Virginia.
10.1021/jm990421v CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/26/2000

1166 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Kim et al.
to be responsible for triggering acute Ca2+ mobilization
and degranulation.11,12 A2B ARs also trigger Ca2+ mo-
bilization10 and participate in a delayed IL8 release from
human HMC-1 mast cells.13 Other functions associated
with the A2B AR are the control of cell growth and gene
expression,14 endothelial-dependent vasodilation,15 and
fluid secretion from intestinal epithelia.16 Adenosine
acting through A2B ARs can stimulate chloride perme-
ability in cells expressing the cystic fibrosis transport
regulator.17
Sch em e 1. Synthesis of Amide Derivatives of Xanthine
Carboxylic Acid Congeners as Potentially Selective A2B
AR Antagonistsa
Although AR subtype-selective probes are available
for the A1/A2A/A3 ARs,18 only few weakly selective
antagonists19,20 and no selective agonists21,22 are known
for the A2B receptor. Recently radioligand binding
assays9,10,23 have been reported which will aid in the
identification of selective antagonists. Although there
are several classes of non-xanthine adenosine antag-
onists24-26 that have been found to be potent and
slightly selective A2B receptor antagonists, we have
selected xanthines as a suitable lead for the develop-
ment of structure-activity relationships (SAR). Among
xanthines, an 8-phenyl group is associated with in-
creased affinity at A2B receptors.7,27 The 8-phenyl ana-
logue of theophylline, 2, displayed a 22-fold enhance-
ment of binding affinity at A2B receptors.19 Leads for
achieving moderate selectivity (at least 20-fold versus
A1/A2A/A3 ARs) have recently been identified among
derivatives of 8-[4-[(carboxymethyl)oxy]phenyl]-1,3-
dipropylxanthine,19,20,27 4a . Compound 4b (MRS 1204,
N-hydroxysuccinimide ester of 4a ) displayed approxi-
mately 20-fold selectivity for human A2B receptors
versus A1/A2A/A3 ARs.19 A 1,2-dimethylmaleimide de-
rivative, 4c (MRS 1595), bound to human A2B receptors
with a Ki of 19 nM and was selective versus human A1/
a
Reagents: (1) EDAC, DMAP, amines in DMF/CH2Cl2; (2) BOP-
Cl, triethylamine, amines in CH2Cl2; or (3) SOCl2, amines in
pyridine/CH2Cl2.
(Tables 1-3) and a functional assay (Figure 2). Ki values
of xanthine derivatives were determined in displace-
ment of binding of two nonselective radioligands: [3H]-
ZM241385 (4-(2-[7-amino-2-furyl[1,2,4]triazolo[2,3-a]-
[1,3,5]triazin-5-yl]aminoethyl)phenol)23 and [125I]IABOPX
(3-(4-amino-3-iodobenzyl)-8-phenyloxyacetate-1-propyl-
xanthine),10 at human A2B receptors stably expressed
in HEK-293 cell membranes.10 Results with these two
radioligands were identical. To determine selectivity,
the xanthines were evaluated using standard binding
assays at A1/A2A/A3 receptors. The initial screening
utilized rat brain A1/A2A receptors (with radioligands
[3H]R-PIA and [3H]CGS21680), and selected compounds
were examined at the recombinant human subtypes,
using [3H]CPX (8-cyclopentyl-1,3-dipropylxanthine) (A1)30
and [125I]ZM241385 (A2A).31 Affinity at cloned human
A3 receptors expressed in HEK-293 cells was deter-
mined using [125I]IABA (N6-(4-amino-3-[125I]iodobenzyl)-
adenosine)32 or [125I]IAB-MECA (N6-(4-amino-3-iodo-
benzyl)adenosine-5′-N-methyluronamide).33
A series of xanthine carboxylic acid derivatives 4a -
10 allowed comparison of the effects of substitutions at
the 1- and 3-positions and variation of the linkage
between the carboxylate group and the 8-phenyl ring.
Among 8-(4-carboxymethyloxyphenyl) derivatives dif-
fering only in the 1- and 3-substitutents, 4a -7, affinity
at A2B receptors decreased in the order: 1,3-dipropyl G
1,3-di-n-butyl > 1,3-diallyl > 1,3-dibenzyl. The diallyl
derivative was more A2B receptor-selective but less
potent than the dipropyl derivative. Thus, 4a was
selected for further derivatization as amides.
8-(4-Phenylacrylic) acid derivatives 8-10 tended to
be more potent at A1 receptors and less potent at A2B
receptors than the 8-(4-carboxymethyloxyphenyl) de-
rivatives. The 1,3-dicyclohexylmethyl derivative 9 was
25-fold selective for A2B receptors. A primary carbox-
amide 11 was more potent than the carboxylic acid 4a
at A1 (3-fold) and A2A (29-fold) receptors and equipotent
at A2B receptors.
The AR affinities of aryl, 12 and 18-33, alkyl, 17,
and aralkyl, 13-16, amides of 4a were compared. A
benzyl amide 13 and simple anilides had the highest
affinity of binding, in the nanomolar range, to human
A2B receptors. Selectivities for the human A2B versus
rat A1 receptors ranged from 1-fold (30) to 27-fold (20),
A2A/A3 receptors by 160-, 100-, and 35-fold, respec-
tively.20 The A2B receptor selectivity of enprofylline was
lost in its 8-aryl-substituted analogues.20 Aryl amide
derivatives were previously reported to be highly potent
antagonists at A2A receptors in human platelets.28 In
the present study, these and additional amide deriva-
tives of 4a were synthesized, to identify novel, selective
A2B receptor antagonists.
Resu lts
The structures of the xanthine derivatives 4a -40
tested for affinity in radioligand binding assays at ARs
are shown in Tables 1-3. Most of the xanthines are
derivatives of the carboxylic congener 4a 27 in which the
carboxylic acid group has been condensed to various
amines through amide coupling reactions shown in
Scheme 1. This approach was taken based on the high
potency in the A2B receptor binding assay of an N-
hydroxysuccinimide ester 4b (Ki value of 9.75 nM)19 and
related acyl hydrazides,20 such as 4c.
Besides 4a , various other analogues of xanthine
carboxylic acid congeners, 7-10, were synthesized ac-
cording to established synthetic procedures of xan-
thines,29 and their corresponding amide or acyl hy-
drazide conjugates, 34-38, were also prepared and
tested for comparison. The yields and chemical charac-
terization of all new compounds are reported in Table
4.
The potency of the xanthine derivatives at human A2B
receptors was evaluated using two binding assays

Highly Potent and Selective AR Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1167
Ta ble 1. Affinities or Antagonistic Activities of Xanthine Carboxylic Acid Derivatives in Radioligand Binding Assays at A1/A2A/A2B/A3
Receptors
Ki (nM)
hA1/ hA2A
hA2B hA2B hA2B
/
hA3/
a
b
c
d
compd
R1
n-Pr
X
R2
rA1
rA2A
hA2B
hA3
4a
OCH2
OH
58
2200
40 ( 4
3910 ( 2140
75700 ( 6500 (r)
227
4.4 15
98
25
175 ( 57 (h)
153
11.1 ( 2.4
595 ( 128 (h)
127
126 ( 41
4b
4c
n-Pr
n-Pr
OCH2
OCH2
O-succinimide
NHN-
9.75 ( 4.80
26.6 ( 4.0
670 ( 154
110
0.9
12
3.1 53
74
2.4
17
dimethylmaleyl 3030 ( 1110 (h) 1970 ( 550 (h)
4d
5
n-Pr
OCH2
OCH2
OCH2
OCH2
NH(CH2)2NH2
1.2
63
7.75 ( 0.14
141 ( 29 (h)
48.0 ( 16.9
25.6 ( 5.0
3.3
6.82 ( 1.57 (h)
756 ( 147
1660 ( 580 (h)
43.1 ( 9.9
149 ( 83 (h)
679 ( 190
15
140 ( 3 (h)
602 ( 24
4890 ( 530 (h)
201 ( 23
18.4 ( 0.03 (h)
4290 ( 570
2370 ( 290 (h)
874 ( 107
2540 ( 1250 (h)
25 ( 3% (10-4
800
allyl
OH
OH
OH
816 ( 91
173000 ( 18000 (r)
90.3 ( 14.2
5.8
1.9
6
n-butyl
27500 ( 2500 (r)
e
7
8
Bn
n-Pr
)
1760 ( 110
60 ( 2
CHdCH OH
30 ( 14
15000 ( 1700 (r)
922 ( 399
2.3
25
3.2
7.6
0.5
4.6
190 ( 71 (h)
e
9
c-HexMe CHdCH OH
<10% (10-5
)
199 ( 52
469 ( 23
1518 ( 980 (h)
4450 ( 1230
10
Bn CHdCH OH
a
Displacement of specific binding in rat brain membranes ([3H]R-PIA) or recombinant human A1 receptors in HEK-293 cells ([125I]IABA),
expressed as Ki ( SEM (n ) 3-5). Displacement of specific binding in rat striatal membranes ([3H]CGS21680) or recombinant human
b
A2A receptors in HEK-293 cells ([125I]iodo-ZM241385), expressed as Ki ( SEM (n ) 3-5). c Displacement of specific [3H]ZM241385 or
d
[
125I]IABOPX binding at human A2B receptors expressed in HEK-293 cells, in membranes, expressed as Ki ( SEM (n ) 3-4). Displacement
of specific [125I]IAB-MECA or [125]IABA binding at human A3 receptors expressed in HEK cells, in membranes, expressed as Ki ( SEM
(n ) 3-4). e % Displacement of specific binding at the designated molar concentration.
Ta ble 2. Affinities or Antagonistic Activities of Xanthine Amide Derivatives in Radioligand Binding Assaysa at A1/A2A/A2B/A3
Receptors
Ki (nM)
hA1/ hA2A
/
hA3/
compd
R
rA1
rA2A
hA1
hA2A
hA2B
hA3
hA2B hA2B hA2B
11
12
13
14
15
16
17
NH2
NH-Ph
NH-CH2Ph
NH-CH(Ph)2
N(CH2Ph)2
N(CH3)Ph
20.0 ( 3.8
76.3 ( 14.0
16.3 ( 4.2
4.22 ( 0.88 45.6 ( 1.4
5.02 ( 0.55 25.9 ( 7.6
40.1 ( 5.1
25.8 ( 4.5
1.48 ( 0.63
137 ( 54
27
17
12
93
39
54.7 ( 21.2 23.8 ( 5.71 2.04 ( 0.17
79.2 ( 17.8 27
120 ( 21
167 ( 49
218 ( 80
26.8 ( 2.4
27.9 ( 1.6
439 ( 111
37.6 ( 4.0
38.4 ( 3.9
10.2 ( 2.5
20 ( 8% (10-6
) 33.7 ( 17.0
2750 ( 800
497 ( 250
999 (144
434 ( 129
949 ( 394
548 ( 183
541 ( 128
683 ( 167
98.1 ( 49.6
220 ( 79
690 ( 98
642 ( 198 9.88 ( 1.05
5.42 ( 1.71
284 ( 14
70
65
29
N(CH2COOEt)2
43.4 ( 8.4
18b NH-Ph(2-COCH3)
335 ( 64
234 ( 28
157 ( 8
225 ( 9
431 ( 176 2.74 ( 1.01
61.9 ( 3.4 120
160
12
81
23
72
170
92
19
NH-Ph(3-COCH3)
58.9 ( 7.1
112 ( 37
4.92 ( 0.55
1.39 ( 0.30
352 ( 69
230 ( 23
363 ( 148
48
110
57
20b NH-Ph(4-COCH3)
21
22
23
24
25
26
NH-Ph(4-COOCH3)
NH-Ph(4-CONH2)
NH-Ph(4-CONHCH3) 24.8 ( 1.8
NH-Ph(4-COOH)
NH-Ph(4-CH3)
NH-Ph(4-OH)
3100 ( 1540 3.93 ( 1.35
7.75 ( 1.11
790
3.34 ( 0.51
145 ( 28
17.5 ( 5.0
5.88 ( 1.06 63.3 ( 20.4
16.8 ( 3.6
13.1 ( 3.9 1180 ( 360
44.6 ( 6.5 917 ( 258
161 ( 53
39.0 ( 21.5 16.1 ( 4.7 >5000
1.88 ( 0.76
10
2.4 >300
126 ( 38
3.71 ( 0.76
503 ( 10.8 1.97 ( 0.31
70.0 ( 10.7 1.52 ( 0.24
27b NH-Ph(4-CN)
612 ( 287
403 ( 194
57.0 ( 3.1
61.2 ( 8.2
17.9 ( 4.5
570 ( 184 210
138 ( 17.1 38
213 ( 94
391 ( 147
260
46
110
7.5
400
700
2400
290
91
28
29
30
31
32
33
NH-Ph(4-NO2)
NH-Ph(4-CF3)
NH-Ph(4-F)
NH-Ph(4-Cl)
NH-Ph(4-Br)
NH-Ph(4-I)
238 ( 28
16.6 ( 3.6
2.14 ( 0.47
2.22 ( 0.19
2.47 ( 0.71 1870 ( 370
29
8.1
20
31
100
176
760
980
600
2.72 ( 0.51 988 ( 518
6.35 ( 1.47 995 ( 550
7.46 ( 2.66 221 ( 36
49.7 ( 14.2 187 ( 38
73.5 ( 23.3 1640 ( 660 2.35 ( 0.01 2300 ( 420
15.7 ( 4.2
152 ( 47
293 ( 67
5140 ( 540 2.13 ( 0.12 1270 ( 130 140
a
b
The methods of each binding assay are shown in Table 1. 18, MRS 1668; 20, MRS 1706; 27, MRS 1754.
while comparisons within the same species (human)
generally led to greater selectivities. Anilides substi-
tuted in the para-position with groups such as nitro,
cyano, and acetyl displayed the highest selectivity. An
N-methylanilide of 4a , 16, was 40- and 92-fold selective
for human A2B receptors versus rat A1/A2A receptors;
thus the N-methylation reduced affinity by 3.7-fold but
increased selectivity. An ortho-substituted acetophenone

1168 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Kim et al.
Ta ble 3. Affinities or Antagonistic Activities of Miscellaneous Xanthine Derivatives in Radioligand Binding Assaysa at A1/A2A/A2B/A3
Receptors
Ki (nM)
compd
R1
n-Pr
X
R2
rA1
rA2A
hA2B
hA3
34
CHdCH
NHN-dimethylmaleyl
3.94 ( 1.20
105 ( 5 (h)
7.67 ( 2.20
406 ( 105
223 ( 55 (h)
143 ( 50
16.7 ( 3.0
31.0 ( 3.1
35
36
37
38
39
40
n-Pr
c-HexMe
Bn
Bn
Et
CHdCH
CHdCH
CHdCH
OCH2
OCH2
OCH2
NH-Ph(2-COCH3)
NH-Ph(2-COCH3)
NH-Ph(2-COCH3)
NH-Ph(2-COCH3)
3.65 ( 0.98
121 ( 138
b (10-5
34300
)
b (10-5
b (10-5
b (10-5
)
)
)
b (10-5
b (10-5
b (10-5
)
)
)
b (10-5
)
NH-Ph(4-CH3)
34.9 ( 0.3
65.0 ( 15.4
71.1 ( 7.7
1370 ( 490
1.78 ( 0.43
15.2 ( 6.8
b (10-6
)
Et
NH-Ph(4-CH2CONH(CH2)2NH2)
a
The methods of each binding assay are shown in Table 1. b <10% Displacement of specific binding at the designated molar concentration.
Ta ble 4. Yields and Chemical Characterization of Xanthine Derivatives
compd
% yield
mp (°C)
MS
formula
27H22N4O5
C28H22N4O4
anal.
7
10
12
13
14
15
16
17
18
19
20
21
22
23
24
27
28
29
30
31
32
33
4c
34
35
36
37
38
13
40
71
41
46
55
49
13
68
29
29
56
19
45
51
44
31
44
48
31
36
13
79
49
42
33
76
18
>310
>310
301-302
268
269-270
230
215
225
294
269-270
309-310
>310
>310
>310
>310
>310
307
>310
298
309
>310
>310
>310
302-303
281-283
305
EI: 482
FAB: 479
CI: 462
CI: 476
EI: 551
EI: 565
FAB: 476
CI: 558
EI: 503
EI: 503
EI: 503
CI: 520
CI: 505
FAB: 519
FAB: 506
CI: 487
CI: 507
CI: 530
CI: 480
CI: 496
CI: 540
CI: 588
FAB: 509
EI: 504
CI: 500
FAB: 608
CI: 596
EI: 599
C
HRMSa
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
HRMSa
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
HRMSa
C,H,N
HRMSa
C
C
C
C
C
C
C
C
25H27N5O4
26H29N5O4
32H33N5O4
33H35N5O4
26H29N5O4
27H35N5O8
27H29N5O5
27H29N5O5
C27H29N5O5‚0.23H2O
C
C
C
C
C
C
C
C
C
C
C
C
C
27H29N5O6
26H28N6O5
27H30N6O5‚1.8CH2Cl2
26H27N5O6‚0.60CH2Cl2
26H26N6O4
25H26N6O6‚0.43CH3OH
26H26F3N5O4‚0.26CH3OH
25H26FN5O4
25H26ClN5O4‚0.26(CH3)2CO
25H26BrN5O4
25H26IN5O4‚0.60CH3OH
25H28N6O6
26H28N6O5
C28H29N5O4‚0.27MeOH
36H41N5O4
C36H29N5O4‚0.60H2O
35H29N5O5
C
308-309
284
C
a
High-resolution mass in EI or FAB+ mode (m/z) determined to be within acceptable limits: 7: calcd, 482.1590; found, 482.1597; 19:
calcd, 503.2169; found, 503.2169; 36: calcd, 608.3237; found, 608.3251; 38: calcd, 599.2169; found, 599.2171. HPLC demonstrated >95%
purity, retention times (mobile phase, min): 7: A, 9.94; B, 10.14; 19: A, 14.76; B, 17.52; 36: A, 23.97; B, 24.79; 38: A, 17.58; B, 24.20.
Mobile phases consisted of: (A) 0.1 M TEAA (pH ) 5.0)/CH3CN, 80:20 to 20:80, in 30 min; and (B) H2O/MeOH, 80:20 to 20:80, in 30 min,
both with a flow rate of 1 mL/min.
18 was 120-, 160-, and 23-fold selective for human A2B
receptors versus human A1/A2A/A3 receptors and 10- and
160-fold selective versus rat A1/A2A receptors. The para-
substituted acetophenone 20 was more potent at A2B
receptors than the corresponding ortho- and meta-
isomers. Other highly potent and moderately selective
A1/A2A/A3 receptors. A p-toluide of 4a , 25, was reported
previously27 and displayed a Ki value at human A2B
receptors of 1.88 nM.
The introduction of the dimethylmaleimido group20
in 34 and in the p-acryloyl derivative 35 resulted in
moderate selectivity for A2B receptors versus other
human but not rat ARs.
A2B antagonists were a p-trifluoromethyl derivative, 29
(Ki value 2.14 nM), and a p-cyanoanilide, 27 (Ki value
1.97 nM), which was highly selective versus the other
human subtypes but only 8.5-fold selective versus rat
A1 receptors. A p-nitro derivative, 28, bound to human
A2B receptors with a Ki of 1.52 nM but was only 35-fold
selective versus human A1 receptors. A p-iodo deriva-
tive, 33 (Ki value 2.13 nM), was 140-, 2400-, and 600-
fold selective for human A2B receptors versus human
Introduction of a p-acrylic acid group in an anilide
derivative, e.g. 36 versus 18, decreased selectivity.
Bulky 1,3-di(cyclohexylmethyl) groups or 1,3-dibenzyl
groups in anilide derivatives 37 and 38, respectively,
greatly diminished binding to ARs. Substitution of the
1,3-dipropyl groups with ethyl, as in 40 and 41, offered
no disadvantage for selectivity, and high affinities were
maintained.

Highly Potent and Selective AR Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1169
High-affinity compounds, such as 18 and 27, may be
the first selective pharmacological probes needed to
investigate the physiological role of this AR subtype and
in tritiated form may be suitable as selective radio-
ligands for the A2B receptor. There is evidence that both
A2B/A3 ARs may play a role in asthma.12 The A3 AR
mediates the degranulation of rat RBL mast-like cells34
and is present in high density in human blood eosino-
phils.35 The availability of antagonists selective for the
A2B receptor should provide an opportunity to explore
the importance of these two receptor subtypes in
asthma.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. Compounds 4a , 11, 25, and 26 were synthesized
as reported.27 Compounds 5-7 and 10 were synthesized as
reported.29 Compounds 39 and 40 were synthesized as re-
ported.28 Compounds 8 and 9 were obtained from Dr. Susan
Daluge, Glaxo, Research Triangle, NC. R-PIA, and 2-chloro-
adenosine were purchased from Research Biochemicals Inter-
national (Natick, MA). All other agents were purchased from
Aldrich (St. Louis, MO).
Syn th esis. Proton nuclear magnetic resonance spectroscopy
was performed on a Varian GEMINI-300 spectrometer and
spectra were taken in DMSO-d6 or CDCl3. Unless noted,
chemical shifts are expressed as ppm downfield from tetra-
methylsilane or relative ppm from DMSO (2.5 ppm). Chemical
ionization (CI) mass spectrometry was performed with a
Finnigan 4600 mass spectrometer and electron impact (EI)
mass spectrometry with a VG7070F mass spectrometer at 6
kV for high-resolution mass. FAB (fast atom bombardment)
mass spectrometry was performed with a J EOL SX102 spec-
trometer using 6-kV Xe atoms. Elemental analysis ((0.4%
acceptable) was performed by Atlantic Microlab Inc. (Norcross,
GA). All melting points were determined with a Unimelt
capillary melting point apparatus (Arthur H. Thomas Co., PA)
and were uncorrected. All xanthine derivatives were homoge-
neous as judged using TLC (MK6F silica, 0.25 mm, glass
backed; Whatman Inc., Clifton, NJ ). Where needed, evaluation
of purity was done on a Hewlett-Packard 1090 HPLC system
using an OD-5-60 C18 analytical column (150 mm × 4.6 mm;
Separation Methods Technologies, Inc., Newark, DE) in two
different linear gradient solvent systems, at a flow rate of 1
mL/min. One solvent system (A) was 0.1 M TEAA (pH ) 5.0)/
CH3CN, 80:20 to 20:80, in 30 min, and the other (B) was H2O/
MeOH, 80:20 to 20:80, in 30 min. Peaks were detected by UV
absorption using a diode array detector.
F igu r e 2. Inhibition by several selective A2B AR antagonists
of NECA-stimulated calcium mobilization in HEK-A2B cells.
Cells were loaded with Indo-1 for 1 h: (A) calcium mobilization
in response to 10 nM and 1 µM NECA added at the arrow; (B)
calcium mobilization in response to 10 nM NECA added at
the arrow in cells pretreated for 2 min with 1% DMSO (control)
or with 100 nM of the indicated antagonists. The results are
typical of replicate experiments.
The functional effects of several selective A2B antago-
nists in inhibiting the effects of NECA in HEK-A2B cells
were examined (Figure 2). Several selective A2B AR
antagonists at 100 nM nearly completely inhibited
NECA-stimulated calcium mobilization. In comparison,
XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]-
phenyl]-1,3-dipropylxanthine), which has a Ki value of
12.3 nM in binding to human A2B ARs,21 inhibited the
effect by roughly one-half. Thus, the potency of the
xanthines in the functional assay was parallel to results
from the binding assay.
Gen er a l P r oced u r e for th e P r ep a r a tion of Am id e
Der iva tives of 4a a n d 7-10. Meth od A (Ca r bod iim id e).
A solution of a 4a analogue (0.0517 mmol), the desired amine
compound (0.103 mmol), EDAC (20 mg, 0.103 mmol), and
DMAP (4 mg, 0.032 mmol) in 2 mL of anhydrous DMF/CH2Cl2
(1:1 v/v) was stirred at room temperature for 24 h. The mixture
was evaporated to dryness under reduced pressure, and the
residue was purified by preparative silica gel TLC (CHCl3:
MeOH ) 20:1) and crystallization in MeOH/ether or MeOH/
CH2Cl2 to afford the desired compounds (12-14, 18, 36).
Meth od B (BOP -Cl). A solution of a 4a analogue (0.0517
mmol), the desired amine compound (0.103 mmol), BOP-Cl (14
mg, 0.0517 mmol), and triethylamine (20 µL, 0.206 mmol) in
2 mL of anhydrous CH2Cl2 was stirred at room temperature
for 24 h. The mixture was treated with the same procedure
as method A for purification of the desired compounds (15,
17, 19, 20, 38).
Meth od C (Acid Ch lor id e). A solution of a 4a analogue
(0.0517 mmol) in 1 mL of thionyl chloride was stirred at 70
°C for 4 h and the excess thionyl chloride was removed by
nitrogen stream. To the residue was added a solution of the
desired amine compound (0.103 mmol) in 1 mL of anhydrous
pyridine and 1 mL of anhydrous CH2Cl2. The mixture was
stirred at room temperature for 24 h, then subjected to the
same procedure as method A for purification of the desired
compounds (16, 21, 22, 27-35, 37).
Discu ssion
We have identified amide derivatives of 4a , such as
20, as adenosine antagonists which are potent and
selective for human A2B receptors. High affinity for the
receptor has been achieved through the formation of
anilides and a benzyl amide of 4a . To increase selectiv-
ity, substitution of the aryl ring in the anilide deriva-
tives was carried out. It appears that electron-with-
drawing substituents at the 2- or 4-position are best
suited for A2B receptor selectivity. Further SAR studies
are in progress to enhance the pharmacological profile
of these xanthine derivatives as A2B receptor antago-
nists. Although this study reveals xanthines having high
selectivity for the human subtypes, there is still a need
for improving A2B receptor selectivity in the rat.
There is also a need to enhance water solubility in
potent and selective antagonists such as 18, 27, and 33,
which are highly hydrophobic. An attempt to introduce
the charged p-carboxylate group, in 24, resulted in lower
affinity, although selectivity was maintained.

1170 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Kim et al.
8-[4-[(Ca r b oxym et h yl)oxy]p h en yl]-1,3-d ib en zylxa n -
th in e (7): 1H NMR (DMSO-d6) 4.23 (s, 2H, -OCH2-), 5.10 (s,
2H, -NCH2-), 5.23 (s, 2H, -NCH2-), 6.88 (d, 2H, J ) 8.8
Hz, Ar), 7.22-7.41 (m, 10H, 2×-Ph), 8.01 (d, 2H, J ) 8.8 Hz,
Ar).
8-[4-[2-(2-Acetylph en yl)car bam oyl-tr a n s-vin yl]ph en yl]-
1,3-d iben zylxa n th in e (37): 1H NMR (DMSO-d6) 4.79 (d, 3H,
J ) 5.8 Hz, -COCH3), 5.13 (s, 2H, -NCH2-), 5.27 (s, 2H,
-NCH2-), 6.93 (d, 1H, J ) 15.6 Hz, -CHd), 7.24-7.41 (m,
10H, 2×-Ph), 7.46 (d, 1H, J ) 15.6 Hz, -CHd), 7.57 (t, 1H, J
) 8.8 Hz, Ar), 7.69 (t, 1H, J ) 7.8 Hz, Ar), 7.75 (d, 2H, J ) 8.8
Hz, Ar), 8.03 (d, 2H, J ) 7.8 Hz, Ar), 8.20 (d, 2H, J ) 7.8 Hz,
Ar), 8.54 (t, 1H, J ) 5.8 Hz, -NH-).
8-[4-[[(2-Acetylp h en yl)ca r ba m oylm eth yl]oxy]p h en yl]-
1,3-d iben zylxa n th in e (38): 1H NMR (DMSO-d6) 4.68 (s, 3H,
-COCH3), 4.71 (s, 2H, -OCH2-), 5.12 (s, 2H, -NCH2-), 5.26
(s, 2H, -NCH2-), 7.15 (d, 2H, J ) 8.8 Hz, Ar), 7.23-7.42 (m,
10H, 2×-Ph), 7.55 (t, 1H, J ) 7.8 Hz, Ar), 7.68 (t, 1H, J ) 7.8
Hz, Ar), 8.02 (d, 2H, J ) 7.8 Hz, Ar), 8.11 (d, 2H, J ) 8.8 Hz,
Ar), 8.48 (t, 1H, J ) 4.8 Hz, -NH-).
P h a r m a cology. The human A2B receptor cDNA was sub-
cloned into the expression plasmid pDoubleTrouble.36 The
plasmid was amplified in competent J M109 cells and plasmid
DNA isolated using Wizard Megaprep columns (Promega
Corp., Madison, WI). A2B ARs were introduced into HEK-293
cells by means of Lipofectin.37
Cell Cu ltu r e. Transfected HEK cells were grown under 5%
CO2/95% O2 humidified atmosphere at a temperature of 37
°C. Colonies were selected by growth of cells in 0.6 mg/mL
G418. Transfected cells were maintained in DMEM supple-
mented with Hams F12 nutrient mixture (1/1), 10% newborn
calf serum, 2 mM glutamine, and containing 50 IU/mL
penicillin, 50 µg/mL streptomycin, and 0.2 mg/mL Geneticin
(G418, Boehringer Mannheim). Cells were cultured in 10- cm
diameter round plates and subcultured when grown confluent
(approximately after 72 h).
Ra d ioliga n d Bin d in g Stu d ies. At A2B r ecep tor s: Con-
fluent monolayers of HEK-A2B cells were washed with PBS
followed by ice-cold buffer A (10 mM HEPES, 10 mM EDTA,
pH 7.4) with protease inhibitors (10 mg/mL benzamidine, 100
mM phenylmethanesulfonyl fluoride, and 2 mg/mL of each
aprotinin, pepstatin, and leupeptin). The cells were homog-
enized in a polytron (Brinkmann) for 20 s and centrifuged at
30000g and the pellets washed twice with buffer HE (10 mM
HEPES, 1 mM EDTA, pH 7.4 with protease inhibitors). The
final pellet was resuspended in buffer HE, supplemented with
10% sucrose, and frozen in aliquots at -80 °C. For binding
assays membranes were thawed and diluted 5-10-fold with
HE to a final protein concentration of approximately 1 mg/
mL. To determine protein concentrations, membranes and
bovine serum albumin standards were dissolved in 0.2%
NaOH/0.01% SDS and protein was determined using fluores-
camine fluorescence.38
To prepare [125I]IABOPX, 10 mL of 1 mM ABOPX in
methanol/1 M NaOH (20:1) was added to 50 mL of 100 mM
phosphate buffer, pH 7.3. One or 2 mCi of Na125I was added,
followed by 10 mL of 1 mg/mL chloramine-T in water. After a
20-min incubation at room temperature, 50 mL of 10 mg/mL
Na-metabisulfite in water was added to quench the reaction.
The reaction mixture was applied to a C18 HPLC column,
eluting with a mixture of methanol and 4 mM phosphate, pH
6.0. After 5 min at 35% methanol, the methanol concentration
was ramped to 100% over 15 min. Unreacted ABOPX eluted
in 11-12 min; [125I]IABOPX eluted at 18-19 min in a yield of
50-60% with respect to the initial 125I.
8-(4-(2-Ca r boxy-tr a n s-vin yl)p h en yl)-1,3-d iben zylxa n -
1
th in e (10): H NMR (DMSO-d6) 5.12 (s, 2H, -NCH2-), 5.26
(s, 2H, -NCH2-), 6.63 (d, 1H, J ) 15.6 Hz, -CHd), 7.22-
7.43 (m, 10H, 2×-Ph), 7.63 (d, 1H, J ) 15.6 Hz, -CHd), 7.84
(d, 2H, J ) 8.8 Hz, Ar), 8.17 (d, 2H, J ) 8.8 Hz, Ar).
8-[4-[(P h en ylca r b a m oylm et h yl)oxy]p h en yl]-1,3-d i(n -
1
p r op yl)xa n th in e (12): H NMR (DMSO-d6) 0.89 (2t, 6H, J
) 7.8 Hz, 2×-CH3), 1.58 and 1.74 (2m, 4H, 2×-CH2-), 3.87
and 4.02 (2t, 4H, J ) 6.8 Hz, 2×-NCH2-), 4.80 (s, 2H, -OCH2-
), 7.06-7.12 (m, 1H, -Ph), 7.14 (d, 2H, J ) 8.8 Hz, Ar), 7.33
(t, 2H, J ) 7.8 Hz, -Ph), 7.64 (d, 2H, J ) 7.8 Hz, -Ph), 8.09
(d, 2H, J ) 8.8 Hz, Ar), 10.13 (s, 1H, -NH).
8-[4-[((4-Acetylp h en yl)ca r ba m oylm eth yl)oxy]p h en yl]-
1,3-d i(n -p r op yl)xa n th in e (20): 1H NMR (DMSO-d6) 0.89 (2t,
6H, J ) 7.8 Hz, 2×-CH3), 1.58 and 1.74 (2m, 4H, 2×-CH2-),
2.54 (s, 3H, -COCH3), 3.87 and 4.02 (2t, 4H, J ) 6.8 Hz, 2×-
NCH2-), 4.85 (s, 2H, -OCH2-), 7.15 (d, 2H, J ) 8.8 Hz, Ar),
7.79 (d, 2H, J ) 7.8 Hz, Ar), 7.96 (d, 2H, J ) 7.8 Hz, Ar), 8.09
(d, 2H, J ) 8.8 Hz, Ar), 10.48 (s, 1H, -NH-).
8-[4-[((4-Meth ylca r ba m oyl)p h en ylca r ba m oylm eth yl)-
oxy]p h en yl]-1,3-d i(n -p r op yl)xa n th in e (23). A solution of
20 mg of 21 (0.0358 mmol) in 1 mL of 40% aqueous methyl-
amine was stirred at room temperature for 1 h. The mixture
was evaporated to dryness under reduced pressure, and the
residue was purified by preparative silica gel TLC (CHCl3:
MeOH ) 20:1) and crystallization in MeOH/CH2Cl2 to give 9
mg of 23: 1H NMR (DMSO-d6) 0.89 (2t, 6H, J ) 7.8 Hz, 2×-
CH3), 1.58 and 1.73 (2m, 4H, 2×-CH2-), 2.76 (s, 3H, -NHCH3),
3.86 and 4.01 (2t, 4H, J ) 6.8 Hz, 2×-NCH2-), 4.82 (s, 2H,
-OCH2-), 7.14 (d, 2H, J ) 8.8 Hz, Ar), 7.71 (d, 2H, J ) 7.8
Hz, Ar), 7.81 (d, 2H, J ) 7.8 Hz, Ar), 8.09 (d, 2H, J ) 8.8 Hz,
Ar), 8.33 (m, 1H, -NHCH3), 10.34 (s, 1H, -NH-).
8-[4-[((4-Car boxyph en yl)car bam oylm eth yl)oxy]ph en yl]-
1,3-d i(n -p r op yl)xa n th in e (24). A suspension of 20 mg of 21
(0.0385 mmol) in 1 mL of 1 N NaOH solution was stirred for
2 h to turn to a clear solution. The mixture was neutralized
by adding 1 mL of 1 N HCl. The precipitate was collected by
filtration and purified by low-pressure C18 column chroma-
tography using linear gradient elution of 1 M triethylammo-
niumacetate buffer (pH ) 7.0) and CH3CN (90/10 to 40/60) to
give 10 mg of 24: 1H NMR (DMSO-d6) 0.89 (2t, 6H, J ) 7.8
Hz, 2×-CH3), 1.58 and 1.74 (2m, 4H, 2×-CH2-), 3.87 and 4.01
(2t, 4H, J ) 6.8 Hz, 2×-NCH2-), 4.84 (s, 2H, -OCH2-), 7.14
(d, 2H, J ) 8.8 Hz, Ar), 7.77 (d, 2H, J ) 8.8 Hz, Ar), 7.92 (d,
2H, J ) 8.8 Hz, Ar), 8.09 (d, 2H, J ) 8.8 Hz, Ar), 10.45 (s, 1H,
-NH-).
8-[4-[((4-Cya n op h en yl)ca r ba m oylm eth yl)oxy]p h en yl]-
1,3-d i(n -p r op yl)xa n th in e (27): 1H NMR (DMSO-d6) 0.89 (2t,
6H, J ) 7.8 Hz, 2×-CH3), 1.58 and 1.74 (2m, 4H, 2×-CH2-),
3.86 and 4.01 (2t, 4H, J ) 6.8 Hz, 2×-NCH2-), 4.85 (s, 2H,
-OCH2-), 7.13 (d, 2H, J ) 8.8 Hz, Ar), 7.80 (d, 2H, J ) 7.8
Hz, Ar), 7.84 (d, 2H, J ) 7.8 Hz, Ar), 8.09 (d, 2H, J ) 8.8 Hz,
Ar), 10.58 (s, 1H, -NH-).
8-(4-(2-Ca r b oxy-tr a n s-vin yl)p h en yl)-1,3-d i(n -p r op yl)-
xa n th in e N′,N′-(1,2-d im eth ylm a leyl)h yd r izid e (34): 1H
NMR (CDCl3) 1.01 (2t, 6H, J ) 7.8 Hz, 2×-CH3), 1.72 and 1.89
(2m, 4H, 2×-CH2-), 2.05 (s, 6H, 2×-CH3), 4.02 and 4.17 (2t,
4H, J ) 6.8 Hz, 2×-NCH2-), 6.67 (d, 1H, J ) 15.6 Hz, -CHd),
7.63 (d, 2H, J ) 8.8 Hz, Ar), 7.74 (d, 1H, J ) 15.6 Hz, -CHd),
8.09 (d, 2H, J ) 8.8 Hz, Ar), 9.43 (s, 1H, -NH-).
8-[4-[2-(2-Acetylph en yl)car bam oyl-tr a n s-vin yl]ph en yl]-
1,3-d i(n -p r op yl)xa n th in e (35): 1H NMR (DMSO-d6) 0.89 (2t,
6H, J ) 7.8 Hz, 2×-CH3), 1.59 and 1.76 (2m, 4H, 2×-CH2-),
3.88 and 4.04 (2t, 4H, J ) 6.8 Hz, 2×-NCH2-), 4.79 (d, 3H, J
) 4.9 Hz, -COCH3), 6.93 (d, 1H, J ) 15.6 Hz, -CHd), 7.51
(d, 1H, J ) 15.6 Hz, -CHd), 7.57 (t, 1H, J ) 7.8 Hz, Ar), 7.69
(t, 1H, J ) 7.8 Hz, Ar), 7.75 (d, 2H, J ) 7.8 Hz, Ar), 8.03 (d,
2H, J ) 7.8 Hz, Ar), 8.18 (d, 2H, J ) 7.8 Hz, Ar), 8.53 (t, 1H,
J ) 5.8 Hz, -NH-).
Saturation binding assays for human A2B ARs were per-
formed with [3H]ZM214385 (17 Ci/mmol; Tocris Cookson,
Bristol, U.K.)23 or [125I]IABOPX (2200 Ci/mmol).
In equilibrium binding assays the ratio of [127I/125I]ABOPX
was 10-20/1. Radioligand binding experiments were per-
formed in triplicate with 20-25 µg of membrane protein in a
total volume of 0.1 mL of HE buffer supplemented with 1 U/mL
adenosine deaminase and 5 mM MgCl2. The incubation time
was 3 h at 21 °C. Nonspecific binding was measured in the
presence of 100 µM NECA. Competition experiments were
carried out using 0.6 nM [125I]IABOPX. Membranes were
filtered on Whatman GF/C filters using a Brandel cell har-
vester (Gaithersburg, MD) and washed three times during 15-
20 s with ice-cold buffer (10 mM Tris, 1 mM MgCl2, pH 7.4).

Highly Potent and Selective AR Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1171
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Bmax and KD values were calculated by Marquardt’s nonlinear
least-squares interpolation for single-site binding models.39 Ki
values for different compounds were derived from IC50 values
as described, assuming a KD value for [125I]IABOPX of 36 nM.40
Data from replicate experiments are tabulated as means (
SEM.
At oth er ARs: [3H]CPX,30 125I]iodo-ZM241385, and [125I]-
[
IABA were utilized in radioligand binding assays to mem-
branes derived from HEK-293 cells expressing recombinant
human A1/A2A/A3 ARs, respectively. Binding of [3H]R-N6-
phenylisopropyladenosine41 ([3H]R-PIA; Amersham, Chicago,
IL) to A1 receptors from rat cerebral cortical membranes and
of [3H]CGS2168042 (NEN, Boston, MA) to A2A receptors from
rat striatal membranes was performed as described. Adenosine
deaminase (3 units/mL) was present during the preparation
of the brain membranes, in a preincubation of 30 min at 30
°C, and during the incubation with the radioligands. All
nonradioactive compounds were initially dissolved in DMSO
and diluted with buffer to the final concentration, where the
amount of DMSO never exceeded 2%. Incubations were
terminated by rapid filtration over Whatman GF/B filters,
using a Brandell cell harvester (Brandell, Gaithersburg, MD).
The tubes were rinsed three times with 3 mL of buffer each.
At least six different concentrations of competitor, spanning
3 orders of magnitude adjusted appropriately for the IC50 of
each compound, were used. IC50 values, calculated with the
nonlinear regression method implemented in Graph-Pad
(Prism, San Diego, CA), were converted to apparent Ki values
as described.40 Hill coefficients of the tested compounds were
in the range of 0.8-1.1.
F u n ction a l a ssa y: HEK-A2B cells from one confluent T75
flask were rinsed with Ca2+- and Mg2+-free Dulbecco’s phos-
phate-buffered saline (PBS) and then incubated in Ca2+- and
Mg2+-free HBSS with 0.05% trypsin and 0.53 mM EDTA until
the cells detached. The cells were rinsed twice by centrifuga-
tion at 250g in PBS and resuspended in 10 mL of HBSS
composed of 137 mM NaCl, 5 mM KCl, 0.9 mM MgSO4, 1.4
mM CaCl2, 3 mM NaHCO3, 0.6 mM Na2HPO4, 0.4 mM
KH3PO4, 5.6 mM glucose, and 10 mM HEPES, pH 7.4, and
the Ca2+-sensitive fluorescent dye Indo-1-AM (5 µM) 37 °C for
60 min. The cells were rinsed once and resuspended in 25 mL
dye-free HBSS supplemented with 1 U/mL adenosine deami-
nase and held at room temperature. Adenosine receptor
antagonists prepared as 100× stocks in DMSO or vehicle was
added and the cells were transferred to a 37 °C bath for 2 min.
Then the cells (1 million in 2 mL) were transferred to a stirred
cuvette maintained at 37 °C within an Aminco SLM 8000
spectrofluorometer (SML Instruments, Urbana IL). The ratios
of Indo-1 fluorescence obtained at 400 and 485 nm (excitation,
332 nm) was recorded using a slit width of 4 nm. NECA was
added after a 100-s equilibration period.
(21) de Zwart, M.; Link, R.; von Frijtag Drabbe Ku¨nzel, J . K.;
Cristalli, G.; J acobson, K. A.; Townsend-Nicholson, A.; IJ zerman,
A. P. A screening of adenosine analogues on the human adeno-
sine A2B receptor as part of a search for potent and selective
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recombinant adenosine receptors. Drug Dev. Res. 1998, 45, 176-
181.
Ack n ow led gm en t. We thank Melissa Marshall for
technical assistance with the binding assays and ac-
knowledge the grant support from NIH HL37942 and
HL56111.
Su p p or tin g In for m a tion Ava ila ble: Characterization of
xanthine derivates by proton NMR and elemental analyses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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