6-phenyl-1,4-dihydropyridine derivatives as potent and selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm960457c

An approach to designing dihydropyridines that bind to adenosine receptors without binding to L-type calcium channels has been described. 1,4- Dihydropyridine derivatives substituted with β-styryl or phenylethynyl groups at the 4-position and aryl groups at the 6-position were synthesized and found to be selective for human A₃ receptors. Combinations of methyl, ethyl, and benzyl esters were included at the 3- and 5-positions. Affinity was determined in radioligand binding assays at rat brain A₁ and A(2A) receptors using [³H]-(R)-PIA [[³H]-(R)-N⁶(phenylisopropyl)adenosine] and [³H]CGS 21680 [[³H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]5'-(N- ethylcarbamoyl)adenosine], respectively. Affinity was determined at cloned human and rat A₃ receptors using [¹²⁵I]AB-MECA [N⁶-(4-amino-3- iodobenzyl)-5'-(N-methylcarbamoyl)-adenosine]. Structure-activity analysis indicated that substitution of the phenyl ring of the β-styryl group but not of the 6-phenyl substituent was tolerated in A₃ receptor selective agents. Replacement of the 6-phenyl ring with a 3-thienyl or 3-furyl group reduced the affinity at A₃ receptors by 4- and 9-fold, respectively. A 5-benzyl ester 4-trans-β-styryl derivative, 26, with a K(i) value of 58.3 nM at A₃ receptors, was > 1700-fold selective vs either A₁ receptors or A(2A) receptors. Shifting the benzyl ester to the 3-position lowered the affinity at A₃ receptors 3-fold. A 5-benzyl, 3-ethyl ester 4-phenylethynyl derivative, 28, displayed a K(i) value of 31.4 nM at A₃ receptors and 1300- fold selectivity vs A₁ receptors. The isomeric 3-benzyl, 5-ethyl diester was >600-fold selective for A₃ receptors. Oxidation of 28 to the corresponding pyridine derivative reduced affinity at A₃ receptors by 88-fold and slightly increased affinity at A₁ receptors.

Full text of DOI:10.1021/jm960457c

J . Med. Chem. 1996, 39, 4667-4675
4667
6
3
-P h en yl-1,4-d ih yd r op yr id in e Der iva tives a s P oten t a n d Selective A Ad en osin e
Recep tor An ta gon ists
J i-long J iang, A. Michiel van Rhee, Neli Melman, Xiao-duo J i, 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
X
Received J une 25, 1996
An approach to designing dihydropyridines that bind to adenosine receptors without binding
to L-type calcium channels has been described. 1,4-Dihydropyridine derivatives substituted
with â-styryl or phenylethynyl groups at the 4-position and aryl groups at the 6-position were
synthesized and found to be selective for human A
3
receptors. Combinations of methyl, ethyl,
and benzyl esters were included at the 3- and 5-positions. Affinity was determined in
3
3
6
1
radioligand binding assays at rat brain A and A2A receptors using [ H]-(R)-PIA [[ H]-(R)-N -
(
3
3
phenylisopropyl)adenosine] and [ H]CGS 21680 [[ H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-
5
′-(N-ethylcarbamoyl)adenosine], respectively. Affinity was determined at cloned human and
receptors using [1 I]AB-MECA [N -(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)-
25
6
rat A
3
adenosine]. Structure-activity analysis indicated that substitution of the phenyl ring of the
â-styryl group but not of the 6-phenyl substituent was tolerated in A receptor selective agents.
Replacement of the 6-phenyl ring with a 3-thienyl or 3-furyl group reduced the affinity at A
receptors by 4- and 9-fold, respectively. A 5-benzyl ester 4-trans-â-styryl derivative, 26, with
3
3
a K
receptors. Shifting the benzyl ester to the 3-position lowered the affinity at A
A 5-benzyl, 3-ethyl ester 4-phenylethynyl derivative, 28, displayed a K
receptors and 1300-fold selectivity vs A receptors. The isomeric 3-benzyl, 5-ethyl diester was
600-fold selective for A receptors. Oxidation of 28 to the corresponding pyridine derivative
reduced affinity at A
i
value of 58.3 nM at A
3
receptors, was >1700-fold selective vs either A
1
receptors or A2A
receptors 3-fold.
3
i
value of 31.4 nM at A
3
1
>
3
3
1
receptors by 88-fold and slightly increased affinity at A receptors.
The physiological role of A3 adenosine receptors,1
the most recently cloned receptors of the adenosine,
-4
enantiomer of niguldipine, 3, which is the more potent
enantiomer at L-type channels, binds to human A3
adenosine receptors with a Ki value of 2.8 µM and is
totally inactive at A1 and A2A receptors. Thus, with
respect to adenosine receptors alone it is highly specific
3
5
,6
is under investigation, and potent and selective antago-
nists are needed as pharmacological probes and as
potential therapeutic agents. Activation of the A3
7
receptor in the rat results in hypotension by promotion
for the A subtype.
8
of release of inflammatory mediators from mast cells.
We have recently described approaches to designing
4
IB-MECA, a selective A3 receptor agonist, was shown
to inhibit the release of TNF-R in macrophages. A3
adenosine receptor antagonists are being sought as
potential antiasthmatic, antiinflammatory, and cere-
broprotective agents.11 Selective A3 receptor agonists
at high concentrations were found to induce apoptosis
in a HL-60 human leukemia cell line.12 There may also
be an involvement of A3 receptors in the cytolytic
activity of antitumor killer lymphocytes.1
1
,4-dihydropyridines that bind to adenosine receptors
9
16
without binding to L-type calcium channels. Further-
more, the moderate affinity for A3 adenosine receptors
of readily available 1,4-dihydropyridines has provided
leads for novel antagonists for that subtype. Inclusion
of 4-trans-â-styryl and 6-phenyl substituents enhanced
A3 receptor selectivity in an additive fashion and
completely abolished recognition at L-type channels.
Compound 4 (3,5-diethyl 2-methyl-6-phenyl-4-[2-phenyl-
1
0
10
3
1
,4-Dihydropyridine blockers of L-type calcium chan-
(E)-vinyl]-1,4-(()-dihydropyridine-3,5-dicarboxylate, MRS
nels14 are used extensively in the treatment of cardio-
vascular disorders as dilators of coronary arteries. We
have found that, in addition to binding to calcium
channels, this class of 1,4-dihydropyridines tends to bind
to three subtypes of adenosine receptors, i.e. A1, A2A,
1
097) was 55-fold selective for human A3 receptors vs
rat A1 receptors and 44-fold selective vs rat A2A recep-
tors. In a functional assay, compound 4 attenuated the
A3 agonist-elicited inhibitory effect on adenylyl cyclase.
Furthermore, whereas nicardipine, 2, displaced radio-
1
5,16
and A3.
For example, nifedipine (Figure 1, 1) is
3
ligand ([ H]-S-(4-nitrobenzyl)-6-thioinosine) from the
bound by these three adenosine receptors with micro-
molar affinity, although it is much more potent at L-type
channels. A newer generation calcium channel blocker,
nicardipine, 2, within the family of adenosine receptors
is actually selective for the A3 subtype. The (S)-
+
Na -independent adenosine transporter with an appar-
ent affinity of 5.4 µM, compound 4 displaced less than
1
0% of total binding at a concentration of 100 µM. The
1
7
selectivity of compound 4 for adenosine receptors was
further demonstrated through its inactivity in binding
assays at other receptors, ion channels, and second
messenger components. The present study examines
combinations and compatibility of dihydropyridine sub-
stituents found previously to enhance affinity at A3
*
Address correspondence to: Dr. K. A. J acobson, Bldg 8A, Rm B1A-
9, NIH, NIDDK, LBC, Bethesda, MD 20892-0810. Tel.: (301) 496-
024. Fax: (301) 480-8422. E-mail: kajacobs@helix.nih.gov.
1
9
X
Abstract published in Advance ACS Abstracts, October 15, 1996.
S0022-2623(96)00457-8
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

4
668 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
J iang et al.
F igu r e 1. Structures of 1,4-dihydropyridines as potent calcium channel antagonists (1-3) and an adenosine receptor antagonist
4). K values (µM) are shown for rat brain A receptors (rA ) and for cloned human A receptors (hA ).
(
i
1
1
3
3
receptors. New derivatives of exceptionally high po-
tency and selectivity have been discovered.
tively) was carried out using tetrachloro-1,4-benzo-
quinone (chloranil, 45) in tetrahydrofuran (Scheme 5).23
Bin d in g a t Ad en osin e Recep tor s. Ki values at A1
and A2A receptors were determined in radioligand
Resu lts
3
Syn th esis. The structures of the 1,4-dihydropy-
ridines (4-31) and related pyridine derivatives (32-
binding assays in rat brain membranes vs [ H]-(R)-PIA
3
6
3
[[ H]-(R)-N -(phenylisopropyl)adenosine] or [ H]CGS
3
3
4) tested for affinity in radioligand binding assays at
21680 [[ H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-
(N-ethylcarbamoyl)adenosine], respectively.
2
4,25
adenosine receptors are shown in Table 1. New dihy-
dropyridine derivatives were synthesized using stan-
dard methodology (Scheme 1),18-20 as reported in Table
Affinity
at cloned human A3 receptors expressed in HEK-293
26
125
6
cells was determined using [ I]AB-MECA [N -(4-
1
25
2
. The synthesis consisted of the Hantzsch condensa-
tion of a 3-amino-2-butenoate ester, 35a ,b, an aldehyde,
6a -h , and a 3-ketopropionate ester derivative, 37a -
amino-3-[ I]iodobenzyl)-5′-(N-methylcarbamoyl)ade-
nosine].
2
7,28
3
Structure-activity relationship (SAR) analysis of 1,4-
dihydropyridine derivatives at adenosine receptors has
indicated that increasing the size of the substituents
from small alkyl either at the C-4 position or at the
5-ester position tends to increase binding affinity at A1,
i, that were dissolved in ethanol and refluxed under N2.
In order to obtain substitution at the 4-position the
aldehyde component, 36a -h , was varied. Substitution
at the 6-position was achieved by varying the 3-keto-
propionate ester component, 37e-g,i (Scheme 2), pre-
1
6
A2A, and A3 subtypes (Table 1). This was illustrated
2
1
16
pared according to method of Straley and Adams. In
with the 5-benzyl ester, 6, and the 4-ethyl analogue, 7.
the synthesis of compounds 29-31, the precursor, ethyl
The benzyl ester, 6, vs compound 5 provided a 12-fold
enhancement of affinity at A3 receptors. The 4-â-styryl
substituent in 8 provided an even greater enhancement
of A3 receptor affinity (48-fold vs 5) and selectivity. The
combination of the 5-benzyl ester and 4-â-styryl sub-
stituents in 10 was compatible in its affinity enhance-
ment at A1 and A3, but not at A2A, subtypes.
3
-aminocinnamate was prepared from the imidate hy-
2
2
drochloride and Meldrum’s acid (Scheme 3).
The
yields of the 6-phenyl-1,4-dihydropyridines obtained
using a e72 h reaction time varied between 6 and 63%.
All of the dihydropyridines examined in this study were
racemic mixtures at position C-4.
An aromatic nitro group on the C-4 side chain of a
dihydropyridine could be reduced selectively using zinc/
acetic acid (Scheme 4). This was desired as a precursor
for the potential radioiodination reaction for the prepa-
ration of an A3 receptor radioligand, as has been carried
out for an agonist.27
The 6-phenyl substituent enhanced affinity particu-
larly at A3 receptors, thus compound 11 was slightly
A3 selective. Extending the 4-methyl group to ethyl in
12 did not result in an enhancement of affinity, as it
did in the 6-methyl case (compound 7 vs 5). The
combination of the 5-benzyl ester and 6-phenyl substit-
uents in 13 was additive in affinity enhancement at A2A
and A3, but not at A1, subtypes. The ratios of affinity
Oxidation of 1,4-dihydropyridines (4 and 28) to the
corresponding pyridine derivatives (33 and 34, respec-

A
3
Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4669
Ta ble 1. Affinities of Dihydropyridine and Pyridine Derivatives in Radioligand Binding Assays at A1, A2a, and A3 Receptorsa-f
Ki (µM) or % inhibitiond
compd
R3
CH3
CH3
CH3
CH3
R4
R5
R6
rA1a
rA2Ab
hA3c
rA1/hA3
e
5
6
7
8
CH3
CH3
CH2CH3
CH2CH3 CH3
CH2Ph
CH2CH3 CH3
CH2CH3 CH3
CH2CH3 CH3
CH2Ph
CH2CH3 Ph
CH2CH3 Ph
CH2Ph
CH2CH3 Ph
CH2CH3 4-CH3Ph
32.6 ( 6.3
6.45 ( 1.47
7.52 ( 2.79
16.1 ( 0.5
4.65 ( 1.21
13.7 ( 2.6
25.9 ( 7.3
21.5 ( 2.7
26.0 ( 8.7
5.93 ( 0.27
14.9 ( 4.9
46.1 ( 6.8
32.3 ( 5.1
2.78 ( 0.89
13.6 ( 2.0
1.0
2.3
0.53
24
5.2
4.4
3.6
2.5
15
e
CH3
9.72 ( 0.63
7.89 ( 2.87
49.3 ( 12.5
9.23 ( 3.60
14 ( 4% (10-4)
35.9 ( 15.3
14.5 ( 3.5
e
e
PhCHdCH-(trans)
0.670 ( 0.195
0.887 ( 0.138
3.13 ( 0.51
7.24 ( 2.13
8.49 ( 1.74
1.75 ( 0.47
0.108 ( 0.012
9.13 ( 2.43
1.43 ( 0.37
0.785 ( 0.272
4.14 ( 0.51
0.907 ( 0.307
0.407 ( 0.066
0.334 ( 0.059
0.109(0.017
9
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
CH2CH3 CH3
CH2CH3 CH2CH3
CH2CH3 CH3
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
CH2CH3 PhCHdCH-(trans)
1
1
1
1
0
1
2
3
4
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
CH3
e
e
Ph
3.15 ( 0.96
4.77 ( 0.29
37 ( 2% (10-4)
55
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
1.6
6.6
42
3.2
6.1
18
-
4
CH2CH3 4-OCH3Ph 9.49 ( 1.99
d (10 )
-
-
-
4
4
4
CH2CH3 4-ClPh
CH2CH3 4-NO2Ph
CH2CH3 3-furyl
CH2CH3 3-thienyl
33.0 ( 7.5
13.1 ( 1.6
5.49 ( 0.25
7.52 ( 1.38
25.3 ( 2.4
6.03 ( 1.39
23 ( 9% (10
31 ( 3% (10
1.20 ( 0.14
11.0 ( 0.1
35 ( 3% (10
33 ( 1% (10
40.1 ( 7.5
12 ( 7% (10
40 ( 3% (10
35 ( 6% (10
)
)
)
-
4
d (10
)
CH2CH3 2-OCH3PhCHdCH-(trans) CH2CH3 Ph
17 ( 12% (10-4)
76
55
-
4
CH2CH3 2-NO2PhCHdCH-(trans)
CH2CH3 4-NO2PhCHdCH-(trans)
CH2CH3 4-NH2PhCHdCH-(trans)
CH2CH3 (Ph)2CdCH-
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
CH2Ph
CH2Ph
CH2Ph
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
CH2CH3
d (10
)
-
-
4
4
33% (10-4)
26 ( 6% (10
12 ( 6% (10
)
)
0.0585 ( 0.0164 >1700
-4
)
)
0.198 ( 0.047
1.42 ( 0.23
>500
0.84
140
-
4
26 ( 12% (10 4)
-
0.0766 ( 0.0151
CH2CH3 PhCtC-
-
-
4
4
-4
CH2CH3 PhCHdCH-(trans)
CH2CH3 4-NO2PhCHdCH-(trans)
CH2CH3 PhCtC-
CH2Ph
CH2Ph
CH2Ph
Ph
Ph
Ph
)
)
15 ( 3% (10
)
0.0583 ( 0.0124 >1700
-4
d (10
d (10
)
)
0.0724 ( 0.0377 >1300
-
4
f
0.0314 ( 0.0028
0.142 ( 0.047
0.286 ( 0.038
0.169 ( 0.026
4.47 ( 0.46
1300
>700
>400
>600
-
-
4
4
16 ( 11% (10-4)
PhCHdCH-(trans)
4-NO2PhCHdCH-(trans)
PhCtC-
d (10
d (10
)
)
-4
d (10
)
)
24 ( 4% (10-4) d (10
-4
e
CH3
7.41 ( 1.29
2.49 ( 0.47
11.6 ( 4.8
28.4 ( 9.1
1.7
0.85
4.2
PhCHdCH-(trans)
PhCtC-
CH2CH3
CH2Ph
2.40 ( 0.22
2.80 ( 1.78
2.75 ( 0.78
43 ( 2% (10-4)
a
3
Displacement of specific [ H]-(R)-PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5), or as a percentage
of specific binding displaced at the indicated concentration (M). b Displacement of specific [ H]CGS 21680 binding in rat striatal membranes,
3
c
expressed as Ki ( SEM in µM (n ) 3-6), or as a percentage of specific binding displaced at the indicated concentration (M). Displacement
1
25
of specific [ I]AB-MECA binding at human A3 receptors expressed in HEK cells, in membranes, expressed as Ki ( SEM in µM (n )
d
e
16 f
3
-4). Displacement of e10% of specific binding at the indicated concentration (M). Values taken from van Rhee et al.
Ki value at
1
rat A3 receptors stably expressed in CHO cells found to be 3.53 ( 0.61 µM.
Sch em e 1. General Procedure for the Synthesis of 1,4-Dihydropyridine Derivatives 9-22 and 24-31
of compound 13 vs the corresponding 5-ethyl ester, 11,
were 11- and 4.1-fold at A2A and A3 receptors, respec-
tively.
We have previously demonstrated that compound 4
is 55-fold selective for human A3 vs rat A1 receptors;
thus the combination of the 4-â-styryl and 6-phenyl
substituents is well tolerated at the A3 receptor.16 The
compatibility of the 4-â-styryl group with other aromatic
substituents at the 6-position was examined in the
current study. In compounds 14-17, the 6-phenyl ring

4
670 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
J iang et al.
Ta ble 2. Characterization of Dihydropyridine and Pyridine Derivatives
no.
Tm (°C)
formula
MS
analysis
yielda (%)
9
148-149
115-117
106-108
112-114
129-131
137-139
149-151
oil
C21H25NO4
355(CI)
417 (CI)
343 (CI)
391 (EI)
431 (CI)
447 (CI)
451 (EI)
462 (EI)
407 (CI)
423 (EI)
447 (EI)
462 (EI)
462 (EI)
432 (EI)
491 (EI)
415 (EI)
479 (EI)
524 (EI)
477 (CI)
479 (EI)
524 (EI)
477 (CI)
415 (EI)
475 (EI)
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
b
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
c
C,H,N
C,H,N
C,H,N
d
C,H,N
C,H,N
e
16.3
69.5
25.5
62.9
32.2
6.1
31.2
15.3
25.5
15.1
12.0
8.0
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
0
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
4
C26H27NO4
C20H25NO4
C24H25NO4
C27H29NO4
C21H33NO4
C26H26NClO4
C26H26N2O6
C24H25NO5
C24H25NSO4‚0.50EtOH
C27H29NO5
C26H26N2O6
C26H26N2O6
C26H28N2O4
C32H31NO4
C26H25NO4
C31H29NO4
C31H28N2O6
C31H27NO4
C31H29NO4
C31H28N2O6
C31H27NO4
C26H25NO4
C31H25NO4
114-116
oil
130-132
175-176
195-196
oil
164-165
132-134
161-163
oil
156-158
149-151
oil
124-125
oil
58.2
24.1
8.2
36.4
27.9
26.2
31.0
41.5
32.9
29.3
29.8
74.0
C,H,N
f
g
oil
a
Purification was achieved by thin layer chromatography, silica 60, 1000 µm layer thickness, using EtOAc-petroleum ether (pe) 35-
b
6
0, 20:80 (v/v) as eluent. 17 pure on analytical TLC (silica 60, 250 µm) EtOAc-pe ) 20:80 (v/v), Rf ) 0.21; CH2Cl2-MeOH ) 40:1 (v/v),
c
Rf ) 0.46; EI calcd 462.1806, found 462.1791. 23 (C26H28N2O4) insufficient quantity for CHN, pure on analytical TLC (silica 60, 250 µm)
d
EtOAc-pe ) 20:80 (v/v), Rf ) 0.06; CHCl3-MeOH-CH3COOH ) 94:6:1 (v/v/v), Rf ) 0.47; EI calcd 432.2049, found 432.2042. 27 pure
on analytical TLC (silica 60, 250 µm) EtOAc-pe ) 20:80 (v/v), Rf ) 0.23; CH2Cl2-MeOH ) 40:1 (v/v), Rf ) 0.68; EI calcd 524.1938, found
e
5
24.1947. 30 pure on analytical TLC (silica 60, 250 µm) EtOAc-pe ) 20:80 (v/v), Rf ) 0.21; CH2Cl2-MeOH ) 40:1 (v/v), Rf ) 0.71; EI
f
calcd 524.1938, found 524.1928. 33 pure on analytical TLC (silica 60, 250 µm) EtOAc-pe ) 20:80 (v/v), Rf ) 0.62; CH2Cl2-MeOH ) 40:1
g
(
v/v), Rf ) 0.69; EI calcd 415.1772, found 415.1783. 34 pure on analytical TLC (silica 60, 250 µm) EtOAc-pe ) 20:80 (v/v), Rf ) 0.52;
CH2Cl2-MeOH ) 40:1 (v/v), Rf ) 0.75; EI calcd 475.1776, found 475.1783.
Sch em e 2. General Procedure for the Synthesis of
â-Keto Esters
Sch em e 3. Procedure for the Synthesis of Ethyl
3-Aminocinnamate
A3 receptors than 4. Phenyl substitution at the â-ole-
finic carbon in 24 resulted in relatively weak affinity
at adenosine receptors (A1, A3 > A2A). Compound 24
was 5-fold more potent than 4 at A1 receptors and 13-
fold less potent at A3 receptors. The affinity of the
dehydro equivalent (4-phenylethynyl substituent) of 4,
i.e. compound 25, was very similar to its affinity at A1
and A3 receptors.
Triple substitutions of the simple dihydropyridines
were also probed in compounds 26-31. All contained
the 6-phenyl substituent and either a 4-â-styryl or a
4-phenylethynyl group. Benzyl esters at the 3- (29-
31) or 5-positions (26-28) were included. A 5-benzyl
ester 4-trans-â-styryl derivative, 26, with a Ki value of
58.3 nM at A3 receptors, was >1700-fold selective vs
either A1 receptors or A2A receptors. The enhanced
selectivity was mainly the result of loss of affinity at
A1 receptors. Shifting the benzyl ester from the 5- to
was substituted at the para position with methyl,
methoxy, chloro, and nitro groups. All substitutions
were less potent than 6-phenyl at A3 receptors, although
the 4-chloro derivative, 16, retained 42-fold selectivity
vs A1 receptors. The 4-methoxy derivative, 15, was 85-
fold less potent than 4 at A3 receptors. Affinity at A2A
receptors was particularly sensitive to substitution of
the 6-phenyl ring. Substitution of the 6-phenyl ring
with 3-furyl, 18, or 3-thienyl, 19, groups reduced the
affinity at A3 receptors by 9- and 4-fold, respectively.
Substitutions of the phenyl ring of the â-styryl group,
2
0-23, suggested much more freedom in this region in
binding at A1 and A3, but not A2A, subtypes. The 4-nitro
analogue, 22 (MRS 1222), was slightly more potent at

A
3
Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4671
Sch em e 4. Method for Reduction of the Nitro Group on
,4-Dihydropyridines
indicated that sterically bulky groups in 1,4-dihydro-
pyridines are tolerated at the 3-, 4-, 5-, and 6-positions.
In the present study simultaneous substitution at two
or three, but not all four, of these positions was carried
out. In this study large substituents at the 4-(â-styryl
or phenylethynyl) and 6-positions (phenyl) were com-
bined with bulky esters at either the 3- or 5-position,
and a beneficial effect on selectivity for human A3
receptors was observed. In combination with the 4- and
1
6
-position substitutions mentioned above, a benzyl ester
group preserved affinity (vs 4) at the A3 receptors to a
greater extent when located at the 5-position (e.g. 26)
than at the 3-position (e.g. 29). Nevertheless, it is
surprising that combinations of bulky substituents at
the 3-, 4-, and 6-positions result in receptor affinity
profiles not radically different from those combinations
at the 4-, 5-, and 6-positions. This suggests that the A3
receptor binding site is not highly sterically constrained.
Also, similar enhancements of affinity at A3 receptors
by either a 4-trans-â-styryl or a 4-phenylethynyl group
point to the same conclusion. Both of these groups are
highly rigid, yet occupy nonidentical spatial regions.
Sch em e 5. General Procedure for the Oxidation of
,4-Dihydropyridine Derivatives Using
Tetrachlorobenzoquinone, 45
1
A series of 8-styrylxanthines30,31 were found to be
subject to photoisomerization about the olefinic bond,
which reduces their utility as pharmacological probes.
One might expect a similar complication with the
4
-styryldihydropyridine derivatives. Incorporation of a
linear phenylethynyl group instead of the styryl group
avoids this potential problem of isomerization.
Pyridine derivatives, which have a distinctly more
planar geometry than that of the corresponding dihy-
dropyridines, lost affinity for A3 adenosine receptors but,
remarkably, gained affinity for A1 receptors. Numerous
classes of adenosine receptor non-xanthine antagonists
have been found for the A1 receptor, and all tend to have
the 3-position as in 29 lowered the affinity at A3
receptors 3-fold. The presence of a 4-nitro group on the
â-styryl substituent, as in 27 and 30, had a negligible
effect on affinity at any of the receptors studied. 3-Ethyl
5
-benzyl 2-methyl-4-(phenylethynyl)-6-phenyl-1,4-(()-
2
9
a planar geometry. It is possible that the A3 receptor
does not share this preference for planar antagonists.
dihydropyridine-3,5-dicarboxylate, 28 (MRS 1191),
displayed a Ki value of 31.4 nM at A3 receptors and
There appears to be more flexibility of substitution
of the phenyl ring of 4-â-styryl derivatives (e.g. com-
pounds 20-23) than of the 6-phenyl substituent in A3
receptor selective agents. Substitution of the 6-phenyl
ring in the para position with a variety of electron-
donating (methyl, methoxy) or -withdrawing (nitro,
chloro) groups or its replacement with 3-thienyl or
1
300-fold selectivity vs A1 receptors. Relative to the
corresponding 5-ethyl ester derivative, 25, compound 28
was 2.4-fold more potent at A3 receptors and less potent
at A1 receptors. A derivative isomeric to compound 28,
the corresponding 3-benzyl 5-ethyl diester, 31, was
>
600-fold selective for A3 receptors. Thus, bulky groups
at combinations of either 3-, 4-, and 6-positions or 4-,
-, and 6-positions still resulted in high A3 receptor
3
-furyl groups (e.g. compounds 14-19) all reduced the
5
affinity at A3 receptors.
selectivity as a result of very low affinity at A1 and A2A
receptors.
It will now be necessary to investigate the regio- (and
stereo-) selectivity of the ligand receptor interaction
more fully. Many of the dihydropyridines examined in
Oxidation of a simple 6-phenyl-1,4-dihydropyridine,
1, to the corresponding pyridine derivative increased
1
1
6
affinity at A1 but not A3 receptors. The presence of
additional bulky substituents, e.g. at the 4- and 5-posi-
tions, caused the effects of oxidation to be less tolerated
in binding, at A3 receptors in particular. Oxidation of
our previous study are asymmetric only on account of
nonequivalent ester substitutions at the 3- and 5-posi-
tions. One such pair of enantiomers, (R)- and (S)-
niguldipine, was studied at adenosine receptors. The
difference in affinity at human A3 receptors between (R)-
and (S)-niguldipine, 3, was insignificant (Ki values of
1.9 and 2.8 µM, respectively). Thus, by analogy to the
present set of mixed ethyl and benzyl esters, it is likely
that there is no dramatic difference in A3 receptor
affinity between (R)- and (S)-enantiomers of 3,4- or 4,
2
8 to the corresponding pyridine derivative, 34, reduced
affinity at A3 receptors by 88-fold, while affinity at A1
receptors increased 4-fold.
Discu ssion
Among the most selective ligands in the present study
were compounds 26-28, i.e. 5-benzyl esters having
g1300-fold selectivity for A3 vs A1 adenosine receptors,
and compounds 29-31, i.e. 3-benzyl esters having g
5
-disubstituted dihydropyridines (i.e. in which methyl
groups occur at both the 2- and 6-positions). The effects
of 6-aryl substitution on stereoselectivity of binding at
adenosine receptors have yet to be explored.
4
00-fold selectivity. The para-substituted 4-styryl-6-
phenyl derivatives 22 and 23 were also highly selective.
Structure-activity analysis at A3 adenosine receptors
At the rat A3 receptor, the affinity of 1,4-dihydropy-
ridines is considerably lower than at human A3 recep-

4
672 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
J iang et al.
tors.16 For example the Ki value of 28 at the rat A3
receptor was found to be 3.53 µM, while at the human
A3 receptor the Ki value is only 31 nM. Although
compound 28 is still somewhat selective for A3 receptors
in the rat (11-fold), it is 110-fold less potent than at
human A3 receptors. This species dependence of A3
receptor affinity has also been demonstrated for xan-
20% ethyl acetate-80% petroleum ether 35-60). All proce-
dures were performed under nitrogen and low-light conditions
to prevent oxidation of the products. The products were shown
to be homogeneous by analytical TLC.
3
,5-Dieth yl 2,6-Dim eth yl-4-[2-p h en yl-(E)-vin yl]-1,4-(()-
1
d ih yd r op yr id in e-3,5-d ica r b oxyla t e
CDCl ): δ 1.29 (t, 6H, J ) 6.8 Hz, 3 and 5-CH
H, 2 and 6-CH ), 4.16 (m, 4H, 3 and 5-OCH ), 4.61 (d, 1H, J
) 5.8 Hz, 4-H), 5.60 (br, 1H, NH), 6.17 (dd, 1H, J ) 5.9, 16.6
Hz, C
(9).
H
NMR
(
6
3
CH ), 2.33 (s,
2 3
3
2
2
,4
thines
and other classes of adenosine antagonists,
such as flavonoids,28 which tend to bind with lower Ki
6
H
5
CdCH), 6.25 (d, 1H, J ) 16.6 Hz, C
.33 (m, 5H, C ). MS (CI/NH ): m/ z 356 (MH) , 252 (M -
CHdCH) , base.
-Eth yl 5-Ben zyl 2,6-Dim eth yl-4-[2-p h en yl-(E)-vin yl]-
-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (10).
6
H
5
CHdC), 7.16-
+
7
C
6
+
H
5
3
values at human vs rat A3 receptors.
6 5
H
It is expected that all of the 4-(arylalkyl)-6-phenyl-
3
1
,4-dihydropyridines prepared in the present study are
6
2
+
selective for adenosine receptors vs L-type Ca chan-
nels, since this principle was demonstrated in a previous
study for compound 4.16 Moreover, compound 4 was
1
H NMR (CDCl ), 2.34
s, 3H, 2-CH ), 2.38 (s, 3H, 6-CH ), 4.70
(d, 1H, J ) 5.8 Hz, 4-H), 5.20 (AB, 2H, J ) 12.7 Hz, 5-OCH ),
6.16 (dd, 1H, J ) 5.9, 15.8 Hz, C H CdCH), 6.21 (d, 1H, J )
6 5
15.8 Hz, C
CI/NH
3
): δ 1.29 (t, 3H, J ) 7.0 Hz, 3-CH
2
CH
3
(
3
3
), 4.22 (m, 2H, 3-OCH
2
2
also shown to have negligible affinity for the adenosine
transporter.16 Furthermore, it was demonstrated that
6
H
5
CHdC), 7.20-7.44 (m, 10H, 2 × C
6 5
H ). MS
+
(
3
): m/ z 418 (MH ), base.
dihydropyridines such as compound 4 effectively at-
tenuate the IB-MECA elicited inhibition of adenylyl
cyclase in CHO cells expressing the cloned rat A3
3
,5-Dieth yl 2-Meth yl-4-eth yl-6-p h en yl-1,4-(()-d ih yd r o-
1
p yr id in e-3,5-d ica r boxyla te (12). H NMR (CDCl
2t, 6H, J ) 6.8 Hz, 3 and 5-CH CH ), 1.32 (t, 3H, J ) 7.0 Hz,
-CH CH ), 1.55 (m, 2H, 4-CH CH ), 2.33 (s, 3H, 2-CH ), 3.90
m, 2H, 3-OCH ), 4.06 (t, 1H, J ) 6.5 Hz, 4-H), 4.22 (m, 2H,
5-OCH ), 5.63 (br, 1H, NH), 7.29-7.41 (m, 5H, 6-C ). MS
(CI/NH
3-Eth yl 5-Ben zyl 2,4-Dim eth yl,6-p h en yl-1,4-(()-d ih y-
3
): δ 0.88
(
4
(
2
3
1
6
adenosine receptor. Thus, the selective ligands intro-
duced here should be useful as antagonists in probing
the role of A3 receptors, especially for the human
homologue of the receptor. These selective agents are
now suitable for study in functional assays and in
investigations of the physiological role of A3 receptors.
With slight additional improvement of affinity, dihy-
dropyridines may also provide affinity probes such as
radioligands for human A3 receptors, although it has
not been determined if the binding is competitive.
2
3
2
3
3
2
2
6 5
H
+
+
3
): m/ z 361 (M + NH ), base, 344 (MH ).
4
1
d r op yr id in e-3,5-d ica r boxyla te (13). H NMR (CDCl
3
): δ
), 1.32 (t, 3H, J ) 7.0 Hz,
), 4.02 (q, 1H, J ) 6.7 Hz, 4-H),
), 4.95 (AB, 2H, J ) 12.7 Hz, 5-OCH ),
). MS (EI):
1
3
4
5
.15 (d, 3H, J ) 6.6 Hz, 4-CH
-CH CH ), 2.31 (s, 3H, 2-CH
.21 (m, 2H, 3-OCH
3
2
3
3
2
2
.70 (br, 1H, NH), 6.95-7.36 (m, 10H, 2 × C
6 5
H
+
+
m/ z 376 (M - CH
3
) , 91 (C
6
H
5
CH
2
), base.
3
,5-Dieth yl 2-Meth yl-4-[2-p h en yl-(E)-vin yl]-6-(4-tolu yl)-
1
1
(
,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (14). H NMR
DMSO-d ): δ 0.78 (t, 3H, J ) 7.0 Hz, 3CH CH ), 1.20 (t, 3H,
J ) 7.0 Hz, 5-CH CH ), 2.28 (s, 3H, toluyl-CH ), 2.34 (s, 3H,
), 3.78 (q, 2H, J ) 6.7 Hz, 3-OCH ), 4.10 (m, 2H, 5-OCH ),
.49 (d, 1H, J ) 7.3 Hz, 4-H), 6.20 (m, 2H, CHdCH), 7.18-
.36 (m, 9H, C and C ), 9.01 (br, 1H, NH). MS (CI/
): m/ e 432 (MH ), 328 (M - C
Exp er im en ta l Section
6
2
3
2
3
3
2
4
7
-CH
3
2
2
Ma ter ia ls. Ethyl acetoacetate (37a ), acetaldehyde (36a ),
propionaldehyde (36b), ethyl 3-aminocrotonate (35a ), and
trans-cinnamaldehyde (36c) were obtained from Fluka
6
H
4
6 5
H
+
+
NH
3
6 5
H CHdCH ), base.
(
Ronkonoma, NY). Ethyl 3-aminocrotonate (35a ), 4-nitro-
trans-cinnamaldehyde (36d ), 2-nitro-trans-cinnamaldehyde
36e), 2-methoxy-trans-cinnamaldehyde (36f), â-phenylcinna-
3,5-Dieth yl 2-Meth yl-4-[2-p h en yl-(E)-vin yl]-6-(4-m eth -
oxyp h e n yl)-1,4-(R ,S )-d ih yd r op yr id in e -3,5-d ica r b oxy-
(
1
maldehyde (36g), phenyl propargylaldehyde (36h ), benzyl
acetoacetate (37b), ethyl benzoylacetate (37c), and tetrachloro-
la te (15). H NMR (CDCl
3
): δ 1.00 (t, 3H, J ) 7.0 Hz,
CH ), 2.38 (s, 3H,
), 4.00 (q, 2H, J ) 6.7 Hz, 3-OCH ),
), 4.74 (d, 1H, J ) 6.1 Hz, 4-H), 5.76 (br,
1H, NH), 6.32 (dd, 1H, J ) 6.6, 15.8 Hz, C CdCH), 6.41 (d,
1H, J ) 15.8 Hz, C CHdC), 6.94 (d, 2H, J ) 8.7 Hz, 2′- and
), 7.37 (d, 2 H, J ) 7.3 Hz, 3′-
3-CH
2-CH
2
CH
3
), 1.33 (t, 3H, J ) 7.0 Hz, 5-CH
2
3
1
,4-benzoquinone (45) were from Aldrich (St. Louis, MO).
3
), 3.85 (s, 3H, 4′-OCH
3
2
Compounds 4-8 and 32 were prepared as described in van
4.22 (m, 2H, 5-OCH
2
1
6
Rhee et al. (R)-PIA and 2-chloroadenosine were purchased
from Research Biochemicals International (Natick, MA). All
other materials were obtained from commercial sources.
6 5
H
6
H
5
6 5
6′-H), 7.19-7.31 (m, 5H, C H
+
and 6′-H). MS (CI/NH
3
6 5
): m/ z 448 (MH) , 344 (M - C H -
Syn th esis. Proton nuclear magnetic resonance spectros-
copy was performed on a Varian GEMINI-300 spectrometer,
+
CHdCH) , base.
and spectra were taken in DMSO-d
6
or CHCl
3
-d. Chemical-
3,5-Dieth yl 2-Meth yl-4-[2-p h en yl-(E)-vin yl]-6-(4-ch lo-
r oph en yl)-1,4-(()-dih ydr opyr idin e-3,5-dicar boxylate (16).
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. Elemental analysis 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.
1
H NMR (CDCl
3
): δ 0.99 (t, 3H, J ) 7.2 Hz, 3-CH
CH ), 2.37 (s, 3H, 2-CH
), 4.22 (m, 2H, 5-OCH ), 4.75 (d, 1H,
J ) 6.3 Hz, 4-H), 5.71 (br, 1H, NH), 6.28 (dd, 1H, J ) 6.6,
15.8 Hz, C CdCH), 6.40 (d, 1H, J ) 15.8 Hz, C CHdC),
7.20-7.41 (m, 9H, C and 6-C ). MS (EI): m/ z 451 (M) ,
) , 378 (M - CO
2
CH
3
), 1.29
(t, 3H, J ) 7.2 Hz, 5-CH
2
3
3
), 3.97 (q,
2H, J ) 7.0 Hz, 3-OCH
2
2
6
H
5
6 5
H
+
6
H
4
6 5
H
+
+
4
22 (M - C
2
H
5
2
C
2
H
5
) , base, 348 (M - C
6
H
5
-
Gen er a l P r oced u r e for th e P r ep a r a tion of 1,4-Dih y-
d r op yr id in e-3,5-d ica r b oxyla t e E st er s (9-22, 24-31).
Equimolar amounts (0.5 mmol) of the appropriate 3-amino-2-
propenoate ester (35a ,b), aldehyde (36a -h ), and 3-ketopro-
pionate ester (37a -i) derivative were dissolved in 5 mL of
absolute ethanol. The solution was sealed in a glass tube and
heated to 100 °C (for volatile aldehydes) or was refluxed under
+
CHdCH) .
3,5-Dieth yl 2-Meth yl-4-[2-p h en yl-(E)-vin yl]-6-(4-n itr o-
p h en yl)-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (17).
1
H NMR (CDCl
3
): δ 1.21 (t, 3H, J ) 6.8 Hz, 3-CH
CH ), 2.38 (s, 3H, 2-CH
), 4.25 (m, 2H, 5-OCH ), 4.77 (d, 1H,
J ) 5.9 Hz, 4-H), 5.67 (br, 1H, NH), 6.26 (dd, 1H, J ) 6.6,
15.8 Hz, C CdCH), 6.37 (d, 1H, J ) 15.8 Hz, C CHdC),
7.21-7.54 (m, 7H, 3′,5′-H and 6-C ), 8.28 (m, 2H, 2′ and 6′-
2
CH
3
), 1.28
(t, 3H, J ) 7.0 Hz, 5-CH
2H, J ) 7.0 Hz, 3-OCH
2
3
3
), 4.13 (q,
2
2
2
N for at least 24 h, and at most 72h. The solvent was then
evaporated, and products were purified either by crystalliza-
tion, column chromatography (silica 60; 220-440 mesh; Fluka,
Buchs, CH; 20% ethyl acetate-80% petroleum ether 35-60),
or preparative TLC (silica 60; 1000 µm; Analtech, Newark, DE;
6
H
5
6 5
H
6
H
5
+
+
H). MS (EI): m/ z 462 (M) , 433 (M - C H ) , 389 (M -
CO
2 5
+
+
2
C
2
H
5
6 5
) , base, 359 (M - C H CHdCH) .

A
3
Adenosine Receptor Antagonists
,5-Dieth yl 2-Meth yl-4-[2-p h en yl-(E)-vin yl]-6-(3-fu r yl)-
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4673
3
(silica 60; petroleum ether 35-60-EtOAc, 80:20, R
f
) 0.06;
) 0.47). H NMR (CDCl ):
CH ), 1.31 (t, 3H, J ) 6.8 Hz,
), 3.67 (br, 2H, 4′-NH ), 3.92 (m,
), 4.17 (m, 2H, 5-OCH ), 4.70 (d, 1H, J ) 6.8 Hz,
4-H), 5.78 (br, 1H, NH), 6.08 (dd, 1H, J ) 6.8, 15.6 Hz,
CdCH), 6.30 (d, 1H, J ) 15.6 Hz, C CHdC), 6.60 (d,
H, J ) 8.8 Hz, 3′- and 5′-H), 7.18 (d, 2H, J ) 8.8 Hz, 2′- and
1
1
1
,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (18). H NMR
δ 1.13 (t, 3H, J ) 7.0 Hz, 3-CH CH ), 1.31 (t, 3H, J ) 7.0 Hz,
-CH CH ), 2.37 (s, 3H, 2-CH ), 3.85 (s, 3H, 4′-OCH ), 4.08
m, 2H, 3-OCH ), 4.21 (q, J ) 7.1 Hz, 2H, 5-OCH ), 4.73 (d,
H, J ) 6.3 Hz, 4-H), 5.73 (br, 1H, NH), 6.24 (dd, 1H, J ) 6.7,
5.8 Hz, C CdCH), 6.35 (d, 1H, J ) 15.8 Hz, C CHdC),
.52 (m, 1H, 3′-H), 7.19-7.37 (m, 5H, C ), 7.45 (m, 1 H, 2′-
): m/ z 408 (MH) , 304 (M
CHCl
δ 0.91 (t, 3H, J ) 6.8 Hz, 3-CH
5-CH CH ), 2.34 (s, 3H, 2-CH
2H, 3-OCH
3
-MEOH-HOAc, 94:6:1, R
f
3
2
3
2
3
5
(
1
1
6
2
3
3
3
2
3
3
2
2
2
2
2
C
6
H
5
6 5
H
6
H
5
6 5
H
2
6
H
5
+
+
6 5
6′-H), 7.32-7.42 (m, 5H, 6-C H ). MS (EI): m/ z 432 (M) , 387
H), 7.65 (s, 1H, 5′-H). MS (CI/NH
3
+
+
2 2 5
C H ) .
+
(M - OC
2
H
5
) , 359 (M - CO
-
C
6
H
5
CHdCH) , base.
3
,5-Diet h yl 2-Met h yl-4-(2,2-d ip h en ylvin yl)-6-p h en yl-
3
,5-Dieth yl 2-Meth yl-4-[2-p h en yl-(E)-vin yl]-6-(3-th ie-
1
1
(
)
3
1
,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24). H NMR
CDCl ): δ 0.84 (t, 3H, J ) 7.0 Hz, 3-CH CH ), 1.10 (t, 3H, J
7.2 Hz, 5-CH CH ), 2.32 (s, 3H, 2-CH ), 3.70-4.18 (m, 4H,
and 5-OCH ), 4.95 (d, 1H, J ) 9.9 Hz, 4-H), 5.82 (br,
H, NH), 6.02 (d, 1H, J ) 10.3 Hz, C CdCH), 7.17-7.46
and 4-C and 6-C ). MS (EI): m/ z 491
) , 418 (M - CO
1
n yl)-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (19).
NMR (CDCl ): δ 1.05 (t, 3H, J ) 7.0 Hz, 3-CH CH ), 1.27 (t,
H, J ) 7.0 Hz, 5-CH CH ), 2.38 (s, 3H, 2-CH ), 4.05 (m, 2H,
-OCH ), 4.22 (m, 2H, 5-OCH ), 4.73 (d, 1H, J ) 6.2 Hz, 4-H),
.88 (br, 1H, NH), 6.30 (dd, 1H, J ) 6.5, 15.8 Hz, C CdCH),
.38 (d, 1H, J ) 15.8 Hz, C CHdC), 7.06 (m, 1H, 4′-H),
.18-7.31 (m, 5H, C ), 7.35 (d, 1H, J ) 5.2 Hz, 3′-H), 7.40
H
3
2
3
3
2
3
2
3
3
3
3
5
6
7
2
3
3
-OCH
2
2
2
2
6
H
5
6
H
5
(m, 15H, 4-C
6
H
5
6
H
5
6
H
5
6
H
5
+
+
+
5
(M) , 446 (M - OC
2
H
5
2
2
C H ) .
6
H
5
3
,5-Diet h yl 2-Met h yl-4-(p h en ylet h yn yl)-6-p h en yl-1,4-
+
(
d, 1H, J ) 4.8 Hz, 5′-H). MS (EI): m/ z 423 (M) , 350 (M -
1
(
(
()-d ih yd r op yr id in e-3,5-d ica r b oxyla t e (25).
CDCl ): δ 0.95 (t, 3H, J ) 7.0 Hz, 3-CH CH ), 1.32 (t, 3H, J
CH ), 2.37 (s, 3H, 2-CH ), 3.98 (m, 2H,
), 4.27 (m, 2H, 3-OCH ), 5.12 (s, 1H, 4-H), 5.92 (br, 1H,
H NMR
+
CO
2
CH
,5-Dieth yl 2-Meth yl-4-[2-(2-m eth oxyph en yl)-(E)-vin yl]-
-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (20).
2
CH
3
), base, 320 (M - C
6 5
H CHdCH) .
3
2
3
3
) 6.8 Hz, 3-CH
3-OCH
2
3
3
6
2
2
1
+
H NMR (CDCl
t, 3H, J ) 6.8 Hz, 5-CH
H, 2′-OCH
3
): δ 0.93 (t, 3H, J ) 7.3 Hz, 3-CH
CH ), 2.35 (s, 3H, 2-CH
), 3.92 (m, 2H, 3-OCH ), 4.20 (m, 2H, 5-OCH
.75 (d, 1H, J ) 5.9 Hz, 4-H), 5.76 (br, 1H, NH), 6.26 (dd, 1H,
CdCH), 6.84 (d, 1H, J ) 16.6 Hz, C
CHdC), 7.15-7.48 (m, 9H, 4-C and 6-C ). MS (EI): m/ z
) , 374 (M - CO
,5-Dieth yl 2-Meth yl-4-[2-(2-n itr op h en yl)-(E)-vin yl]-6-
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (21).
2
CH
), 3.82 (s,
),
3
), 1.33
NH), 7.23-7.43 (m, 10H, 2 × C
6 5
H ). MS (EI): m/ z 415 (M) ,
+
+
(
3
4
2
3
3
386 (M - C
2
H
5
) , 342 (M - CO
2
C
2
H
5
) , base.
3-E t h yl 5-Ben zyl 2-m et h yl-4-[2-p h en yl-(E)-vin yl]-6-
3
2
2
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (26).
1
J ) 5.9, 15.6 Hz, C
6
H
5
6
H
5
-
H NMR (CDCl
3
): δ 1.28 (t, 3H, J ) 6.8 Hz, 3-CH
2
CH
), 4.78 (d, 1H, J ) 6.2 Hz,
4-H), 4.99 (AB, J ) 12.7 Hz, 5-OCH ), 5.76 (br, 1H, NH), 6.30
3
), 2.38
H
4
6
H
2
5
3 2
(s, 3H, 2-CH ), 4.22 (m, 2H, 3-OCH
6
+
+
+
5
4
47 (M) , 402 (M - OC
2
H
5
C
2
H
) .
2
(
dd, 1H, J ) 6.5, 16.2 Hz, C
6 5
H CdCH), 6.38 (d, 1H, J ) 16.2
3
Hz, C CHdC), 6.98 (m, 2H, 2′- and 6′-H), 7.21-7.38 (m, 13H,
2
-
CH
6
H
5
+
× C
6
H
5
, 3′-, 4′-, and 5′-H). MS (EI): m/ z 479 (M) , 388 (M
1
H NMR (CDCl
t, 3H, J ) 7.3 Hz, 5-CH
H, J ) 7.2 Hz, 3-OCH
3
): δ 0.91 (t, 3H, J ) 7.3 Hz, 3-CH
CH ), 2.37 (s, 3H, 2-CH
), 4.27 (q, 2H, J ) 7.2 Hz, 5-OCH
.78 (d, 1H, J ) 5.9 Hz, 4-H), 5.93 (br, 1H, NH), 6.34 (dd, 1H,
CdCH), 6.90 (d, 1H, J ) 16.6 Hz, C
CHdC), 7.30-7.48 (m, 8H, 4-C and 6-C ), 7.90 (d, 1H, J
9.8 Hz, 3′-H). MS (EI): m/ z 462 (M) , 417 (M - OC
2
CH
), 3.96 (q,
),
3
), 1.33
+
+
C
6
H
5
CH
2
) , 376 (M - C
6
H
5
2 2
CHdCH) , 344 (M - CO CH -
(
2
4
2
3
3
+
3
), 91 (C
6
H
5
CH
2
) , base.
2
2
3
-Eth yl 5-Ben zyl 2-Meth yl-4-[2-(4-n itr oph en yl)-E)-vin yl]-
6
-ph en yl-1,4-(R,S)-dih ydr opyr idin e-3,5-dicar boxylate (27).
J ) 5.9, 15.6 Hz, C
6
H
5
6
H
5
-
1
H NMR (CDCl
s, 3H, 2-CH ), 4.24 (m, 2H, 3-OCH
-H), 5.02 (AB, J ) 12.7 Hz, 5-OCH
3
): δ 1.31 (t, 3H, J ) 6.9 Hz, 3-CH
), 4.84 (d, 1H, J ) 5.8 Hz,
), 5.82 (br, 1H, NH), 6.43
CdCH), 6.45 (d, 1H, J )
16.0 Hz, NO C H CHdC), 6.96 (dd, 2H, J ) 2.0, 7.7 Hz, 3′-
2 3
CH ), 2.38
6
H
3
6 5
H
(
4
3
2
+
+
)
2
H
5
) ,
2
+
2 2 5
C H ) .
3
89 (M - CO
,5-Dieth yl 2-Meth yl-4-[2-(4-n itr op h en yl)-(E)-vin yl]-6-
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (22).
2 6 4
(dd, 1H, J ) 6.5, 16.0 Hz, NO C H
3
2
6
4
and 5′-H), 7.19-7.39 (m, 5H, C H ), 8.13 (d, 2H, J ) 8.8 Hz,
6
5
1
+
H NMR (CDCl
3
): δ 0.89 (t, 3H, J ) 7.3 Hz, 3-CH
CH ), 2.39 (s, 3H, 2-CH
), 4.24 (q, 2H, J ) 6.8 Hz, 5-OCH
.80 (d, 1H, J ) 4.9 Hz, 4-H), 5.82 (br, 1H, NH), 6.47 (dd, 1H,
CdCH), 7.32-7.51 (m, 8H, C CHdC
), 8.15 (d, 2H, J ) 8.8 Hz, 3′- and 5′-
2
CH
), 3.94 (q,
),
3
), 1.32
2 6 5
2′- and 6′-H). MS (EI): m/ z 524 (M) , 389 (M - CO C H -
+
+
+
2
(
2
4
t, 3H, J ) 7.3 Hz, 5-CH
2
3
3
CH
2
2 6 5 6 5
) , 376 (M - NO C H CHdCH) , 91 (C H CH ) , base.
H, J ) 6.8 Hz, 3-OCH
2
2
3-Eth yl 5-Ben zyl 2-m eth yl-4-p h en yleth yn yl-6-p h en yl-
1
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (28). H NMR
J ) 3.9, 16.1 Hz, C
and 4-C and 6-C
H). MS (EI): m/ z 462 (M) , 417 (M - OC
6
H
5
6
H
5
(CDCl
2-CH ), 4.27 (m, 2H, 3-OCH
5.19 (s, 1H, 4-H), 5.87 (br, 1H, NH), 7.08-7.39 (m, 15H, 3 ×
C H ). MS (CI/NH ): m/ z 478 (MH) , 376 (M - C H CdC)
3
): δ 1.35 (t, 3H, J ) 6.8 Hz, 3-CH
2
CH
3
), 2.37 (s, 3H,
H
2
6
H
5
3
2
), 5.10 (AB, J ) 12.7 Hz, 5-OCH
2
),
6
+
+
5
H ) , 389 (M -
2
+
+
+
,
CO
2
C
2
H
5
) .
,5-Dieth yl 2-Meth yl-4-[2-(4-a m in op h en yl)-(E)-vin yl]-
-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (23).
6
5
3
6
5
+
2
42 (M - CO
-Ben zyl 5-E t h yl 2-Met h yl-4-[2-p h en yl-(E)-vin yl]-6-
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (29).
C H CH ) , base.
2 6 5 2
3
3
6
Compound 23 was prepared by the catalytic reduction of
compound 22 with zinc and acetic acid as described previ-
1
H NMR (CDCl
3
): δ 0.91 (t, 3H, J ) 7.0 Hz, 5-CH
), 4.81 (d, 1H,
), 5.78
CdCH), 6.36
2 3
CH ), 2.38
(
s, 3H, 2-CH ), 3.92 (q, 2H, J ) 6.7 Hz, 5-OCH
3
2
3
0
ously. Compound 22 (23 mg, 0.05 mmol) was dissolved in
.5 mL of glacial acetic acid. Zn powder (0.15 mmol, 10 mg)
J ) 5.9 Hz, 4-H), 5.18 (AB, J ) 12.7 Hz, 2H, 3-OCH
2
1
(
(
C
6 5
br, 1H, NH), 6.31 (dd, 1H, J ) 5.9, 16.0 Hz, C H
was added to the solution, and the reaction mixture was stirred
with a magnetic stirring bar at room temperature. Six hours
after the start of the reaction, another batch of zinc powder
was added. At 9 h reaction time, TLC (silica 60; petroleum
ether 35-60-EtOAc ) 80:20) analysis of the reaction mixture
indicated that all starting material had been converted. The
reaction mixture was diluted with 30 mL of water and
d, 1H, J ) 16.0 Hz, C
6
H
5
CHdC), 7.20-7.42 (m, 10H, 2 ×
+
6
H
5
). MS (EI): m/ z 406 (M - CO
2
CH
2
) , 376 (M - C
6
H
5
-
+
+
CHdCH) , 91 (C
-Ben zyl 5-E t h yl 2-Met h yl-4-[2-(4-n it r op h en yl)-(E)-
vin yl]-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r b oxy-
6 5 2
H CH ) , base.
3
1
la te (30). H NMR (CDCl
3
): δ 0.88 (t, 3H, J ) 6.8 Hz,
), 2.39 (s, 3H, 2-CH ), 4.09 (q, 2H, J ) 6.8 Hz,
), 4.84 (d, 1H, J ) 5.8 Hz, 4-H), 5.30 (AB, J ) 12.2 Hz,
), 5.87 (br, 1H, NH), 6.40 (d, 1H, J ) 16.0 Hz, NO
CdCH), 6.96
dd, 2H, J ) 2.0, 7.7 Hz, 3′- and 5′-H), 7.19-7.39 (m, 5H, C ),
8.14 (d, 2H, J ) 7.8 Hz, 2′- and 6′-H). MS (EI): m/ z 524 (M)
5
3
3
-CH
2
CH
3
3
3
neutralized with saturated NaHCO solution. The aqueous
-OCH
-OCH
2
solution was extracted three times with 15 mL of chloroform,
and the organic phase was washed once with 20 mL of water.
The organic phase was separated and dried over anhydrous
2
2 6 4
C H -
2 6 4
CHdC), 6.50 (dd, 1H, J ) 5.9, 16.6 Hz, NO C H
(
6 5
H
MgSO
µm of silica 60; petroleum ether 35-60-EtOAc, 90:10) to yield
.2 mg (24%) of a slightly yellow oil, which was shown to be
4
. The product was purified by preparative TLC (1000
+
,
+
+
389 (M - CO C H CH ) , 376 (M - NO C H CHdCH) , 91-
(C H CH ) , base.
6 5 2
2
6
5
2
2
6
5
+
5
pure by HPLC (OD-5-60, C-18 column, Separation Methods
Technologies, Inc, Newark, DE; 0.1 M triethylammonium
3-Ben zyl 5-Eth yl 2-Meth yl-4-(p h en yleth yn yl)-6-p h en yl-
1
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (31). H NMR
acetate buffer-CH
3
CN, 40:60 gradient to 10:90 in 20 min; 1
(CDCl
2-CH
3
): δ 0.96 (t, 3H, J ) 6.8 Hz, 5-CH
2
CH
3
), 2.38 (s, 3H,
mL/min flow; retention time ) 4.45 min), and analytical TLC
3
), 4.00 (q, 2H, J ) 6.8 Hz, 5-OCH
2
), 5.21 (s, 1H, 4-H),

4
674 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
J iang et al.
5
.30 (AB, J ) 13.6 Hz, 3-OCH
2
), 5.87 (br, 1H, NH), 7.30-7.49
3
Spring, MD) for financial support. We thank Prof. Gary
L. Stiles and Dr. Mark Olah of Duke University School
of Medicine (Durham, NC) for providing samples of [ I]-
+
(
C
m, 15H, 3 × C
6
H
5
). MS (CI/NH ): m/ z 478 (MH) , 376 (M -
+
6
H
5
CdC) , base.
125
P r oced u r e for Oxid a tion of 1,4-Dih yd r op yr id in e-3,5-
AB-MECA.
d ica r boxyla te Ester s (33-34). Equimolar amounts (0.25
mmol) of the 1,4-dihydropyridine-3,5-dicarboxylate ester (4,
2
8) and tetrachloro-1,4-benzoquinone (45) in tetrahydrofuran
Refer en ces
(2 mL) were mixed and refluxed for up to 4 h. The solvent
(
1) Zhou, Q. Y.; Li, C. Y.; Olah, M. E.; J ohnson, R. A.; Stiles, G. L.;
was then evaporated, and products were purified by prepara-
tive TLC (silica 60; 1000 µm; Analtech, Newark, DE; 20% ethyl
acetate-80% petroleum ether 35-60).
Civelli, O. Molecular cloning and characterization of an adenos-
ine receptor - The A adenosine receptor. Proc. Natl. Acad. Sci.
3
U.S.A. 1992, 89, 7432-7436.
3
,5-Dieth yl 2-Meth yl-4-[2-ph en yl-(E)-vin yl]-6-ph en ylpy-
3
(2) Linden, J . Cloned adenosine A receptors - pharmacological
1
properties, species -differences and receptor functions. Trends
Pharmacol. Sci. 1994, 15, 298-306.
3) J acobson, K. A; Nikodijevic, O.; Shi, D.; Gallo-Rodriguez, C.;
r id in e-3,5-d ica r boxyla te (33). H NMR (CDCl
3
): δ 0.96 (t,
), 1.26 (t, 3H, J ) 6.8 Hz, 5-CH CH ),
), 4.07 (q, 2H, J ) 6.8 Hz, 3-OCH ), 4.35 (q,
H, J ) 6.8 Hz, 5-OCH ), 6.88 (d, 1H, J ) 16.6 Hz, C CdCH),
.88 (d, 1H, J ) 16.6 Hz, C CHdC), 7.31-7.61 (2m, 10H, 2
). MS (EI): m/ e 415 (M) , base, 370 (M - OC
3
2
2
6
H, J ) 6.9 Hz, 3-CH
2
CH
3
2
3
(
.67 (s, 3H, 2-CH
3
2
Olah, M. E.; Stiles, G. L.; Daly, J . W. A role for central
2
6
H
5
A -adenosine receptors: Mediation of behavioral depressant
3
6
H
5
effects. FEBS Lett. 1993, 336, 57-60.
+
+
2 5
H ) ,
(4) J acobson, K. A.; Kim, H. O.; Siddiqi, S. M.; Olah, M. E.; Stiles,
×
C
6
H
5
+
2 2
dCH ) .
G.; von Lubitz, D. K. J . E. A
selective ligands and therapeutic prospects. Drugs Future 1995,
3
adenosine receptors: design of
3
58 (386 - CH
3
-Eth yl 5-Ben zyl 2-Meth yl-4-(p h en yleth yn yl)-6-p h e-
2
0, 689-699.
1
n ylp yr id in e-3,5-d ica r boxyla te (34). H NMR (CDCl
CH ), 2.68 (s, 3H, 2-CH ), 4.48
), 5.17 (s, 2H, 5- OCH ), 7.10-7.60
3
): δ
(
5) Stiles, G. L. Adenosine receptor subtypes: New insights from
1
.42 (t, 3H, J ) 6.8 Hz, 3-CH
2
3
3
cloning and functional studies. Wiley: New York, in press.
(
(
q, 2H, J ) 6.8 Hz, 3-OCH
2
2
+
(6) J acobson, K. A.; van Galen, P. J . M.; Williams, M. Adenosine
receptors - pharmacology, structure activity relationships, and
therapeutic potential. J . Med. Chem. 1992, 35, 407-422.
4m, 15H, 3 × C
6 5
H ). MS (EI): m/ e 475 (M) , 438 (M -
+
+
+
2
OC
2
H
5
) , 384 (M - C
6
H
5
CH
2
2
) , 356 (M - CH dCH ) , base.
(
7) Hannon, J . P.; Pfannkuche, H. J .; Fozard, J . R. A role for mast-
cells in adenosine A
receptor-mediated hypotension in the rat.
Br. J . Pharmacol. 1995, 115, 945-952.
(8) Ramkumar, V.; Stiles, G. L.; Beaven, M. A.; Ali, H. The A
P h a r m a cology. Ra d ioliga n d Bin d in g Stu d ies. Binding
3
3
6
3
of [ H]-(R)-N -(phenylisopropyl)adenosine ([ H]-(R)-PIA) to A
1
3
receptors from rat cerebral cortex membranes and of [ H]-2-
[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)-
3
[
adenosine receptor is the unique adenosine receptor which
facilitates release of allergic mediators in mast cells. J . Biol.
Chem. 1993, 268, 16887-16890.
3
adenosine ([ H]CGS 21680) to A2A receptors from rat striatal
membranes was performed as described previously.2
4,25
Ad-
(
9) Sajjadi, F. G.; Takabayashi, K.; Foster, A. C.; Domingo, R. C.;
Firestein, G. S. Inhibition of TNF-R expression by adenosine. J .
Immunol. 1996, 156, 3435-3442.
enosine 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.
(10) Beaven, M. A.; Ramkumar, V.; Ali, H. Adenosine-A receptors
3
1
25
6
Binding of [ I]-N -(4-amino-3-iodobenzyl)-5′-(N-methylcar-
in mast-cells. Trends Pharmacol. Sci. 1994, 15, 13-14.
(11) von Lubitz, D. K. J . E.; Lin, R. C. S.; Popik, P.; Carter, M. F.;
1
25
bamoyl)adenosine ([ I]AB-MECA) to membranes prepared
from HEK-293 cells stably expressing the human A receptor
Receptor Biology, Inc., Baltimore, MD) or to membranes
prepared from CHO cells stably expressing the rat A receptor
The assay medium consisted
3
J acobson, K. A. Adenosine A receptor stimulation and cerebral
3
ischemia. Eur. J . Pharmacol. 1994, 263, 59-67.
(
(
12) Kohno, Y.; Sei, Y.; Koshiba, M.; Kim, H. O.; J acobson, K. A.
Induction of apoptosis in HL-60 human promyelocytic leukemia
cells by selective adenosine A3 receptor agonists. Biochem.
Biophys. Res. Commun. 1996, 219, 904-910.
3
2
7,28
was performed as described.
2
+
of a buffer containing 50 mM Tris, 10 mM Mg , and 1 mM
EDTA, at pH 8.0. The glass incubation tubes contained 100
µL of the membrane suspension (0.3 mg of protein/mL, stored
at -80 °C in the same buffer), 50 µL of [ I]AB-MECA (final
concentration 0.3 nM), and 50 µL of a solution of the proposed
antagonist. Nonspecific binding was determined in the pres-
ence of 200 µM NECA.
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 What-
man GF/B filters, using a Brandell cell harvester (Brandell,
Gaithersburg, MD). The tubes were rinsed three times with
(
13) MacKenzie, W. M.; Hoskin, D. W.; Blay, J . Adenosine inhibits
the adhesion of anti-CD3-activated killer lymphocytes to adeno-
carcinoma cells through an A
3
receptor. Cancer Res. 1994, 54,
1
25
3
521-3526.
(
14) Triggle, D. J . Drugs acting on ion channels and membranes. In
Comprehensive Medicinal Chemistry; Emmett, J . C., Ed.; Per-
gamon Press: London, 1985; Vol. 3, pp 1047-1099.
15) Hu, P. S.; Lindgren, E.; J acobson, K. A.; Fredholm, B. B.
Interaction of dihydropyridine calcium channel agonists and
antagonists with adenosine receptors. Pharmacol. Toxicol. 1987,
(
6
1, 121-125.
(
16) van Rhee, A. M.; J iang, J .-l.; Melman, N.; Olah, M. E.; Stiles,
G. L.; J acobson, K. A. Interaction of 1,4-dihydropyridine and
pyridine derivatives with adenosine receptors: selectivity for A
3
receptors. J . Med. Chem. 1996, 39, 2980-2989.
3
mL of buffer each.
At least five different concentrations of competitor, spanning
orders of magnitude adjusted appropriately for the IC50 of
(
17) Sorkin, E. M.; Clissold, S. P. Nicardipine: A review of its
pharmacological and pharmacokinetic properties and therapeu-
tic efficacy, in the treatment of angina pectoris, hypertension,
and related cardiovascular disorders. Drugs 1987, 33, 296-345.
3
each compound, were used. IC50 values, calculated with the
nonlinear regression method implemented in the InPlot pro-
gram (Graph-PAD, San Diego, CA), were converted to apparent
(18) Stout, D. M.; Myers, A. I. Recent advances in the chemistry of
dihydropyridines. Chem. Rev. 1982, 82, 223-243.
(19) Singer, A.; McElvain, S. M. 2,6-Dimethylpyridine. Org. Synth.
1934, 14, 30-33.
3
2
K
i
values using the Cheng-Prusoff equation and K
d
values
3
3
of 1.0 and 14 nM for [ H]-(R)-PIA and [ H]CGS 21680,
respectively, and 0.59 nM for binding of [ I]AB-MECA at
human A receptors, respectively.
Abbr evia tion s: [ I]AB-MECA, [ I]-N -(4-amino-3-iodo-
benzyl)-5′-(N-methylcarbamoyl)adenosine; CGS 21680, 2-[[4-
(
20) Rathke, M. W.; Deitch, J . The reaction of lithium ester enolates
with acid chlorides. A convenient procedure for the preparation
of â-keto esters. Tetrahedron Lett. 1971, 31, 2953-2956.
1
25
3
1
25
125
6
(21) Straley, J . M.; Adams, A. C. Ethyl benzoylacetate. Organic
Syntheses; Wiley: New York, 1963; Collect. Vol. IV, pp 415-
4
17.
(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)ad-
(
22) Celerier, J .-P.; Deloisy, E.; Kapron, P.; Lhommet, G.; Maitte, P.
Imidate Chemistry. Synthesis 1981, 130-133.
enosine; CHO cells, Chinese hamster ovary cells; DMSO,
dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid;
HEK cells, human embryonic kidney cells; IB-MECA, N -(3-
iodobenzyl)-5′-(N-methylcarbamoyl)adenosine; K
inhibition constant; NECA, 5′-(N-ethylcarbamoyl)adenosine;
(
23) Braude, E. A.; Hannah, J .; Linstead, R. Hydrogen transfer. Part
XVI. Dihydrides of nitrogenous heterocycles as hydrogen donors.
J . Chem. Soc. 1960, 3249-3257.
6
i
, equilibrium
(24) Schwabe, U.; Trost, T. Characterization of adenosine receptors
H]N -phenylisopropyladenosine. Naunyn-
6
in rat brain by (-) [
3
6
(
R)-PIA, (R)-N -(phenylisopropyl)adenosine; SAR, structure-
Schmiedeberg's Arch. Pharmacol. 1980, 313, 179-187.
activity relationship; Tris, tris(hydroxymethyl)aminomethane.
(
25) J arvis, M. F.; Schutz, R.; Hutchison, A. J .; Do, E.; Sills, M. A.;
3
Williams, M. [ H]CGS 21680, an A
2
selective adenosine receptor
receptors in rat brain tissue. J .
Pharmacol. Exp. Ther. 1989, 251, 888-893.
Ack n ow led gm en t. We thank Gilead Sciences (Fos-
agonist directly labels A
2
ter City, CA) and the Cystic Fibrosis Foundation (Silver

A
3
Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4675
(
26) Salvatore, C. A.; J acobson, M. A.; Taylor, H. E.; Linden, J .;
J ohnson, R. G. Molecular cloning and characterization of the
human A adenosine receptor. Proc. Natl. Acad. Sci. U.S.A. 1993,
0, 10365-10369.
27) Olah, M. E.; Gallo-Rodriguez, C.; J acobson, K. A.; Stiles, G. L.
(30) J acobson, K. A.; Gallo-Rodriguez, C.; Melman, N.; Fischer, B.;
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