Interaction of 1,4-dihydropyridine and pyridine derivatives with adenosine receptors: Selectivity for A3 receptors

Article abstract of DOI:10.1021/jm9600205

1,4-Dihydropyridine and pyridine derivatives bound to three subtypes of adenosine receptors in the micromolar range. Affinity was determined in radioligand binding assays at rat brain A₁ and A(2A) receptors using [³H]- (R)-PIA [[

Full text of DOI:10.1021/jm9600205

2980
J . Med. Chem. 1996, 39, 2980-2989
In ter a ction of 1,4-Dih yd r op yr id in e a n d P yr id in e Der iva tives w ith Ad en osin e
Recep tor s: Selectivity for A3 Recep tor s
A. Michiel van Rhee,† J i-long J iang,† Neli Melman,† Mark E. Olah,‡ Gary L. Stiles,‡ 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 Departments of Medicine and Pharmacology,
Duke University Medical Center, Durham, North Carolina 27710
Received J anuary 9, 1996X
1,4-Dihydropyridine and pyridine derivatives bound to three subtypes of adenosine receptors
in the micromolar range. Affinity was determined in radioligand binding assays at rat brain
A1 and A2A 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], respec-
tively. 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 at
adenosine receptors indicated that sterically bulky groups at the 4-, 5-, and 6-positions are
tolerated. (R,S)-Nicardipine, 12, displayed Ki values of 19.6 and 63.8 µM at rat A1 and A2A
receptors, respectively, and 3.25 µM at human A3 receptors. Similarly, (R)-niguldipine, 14,
displayed Ki values of 41.3 and 1.90 µM at A1 and A3 receptors, respectively, and was inactive
at A2A receptors. A preference for the R- vs the S-enantiomer was observed for several
dihydropyridines at adenosine receptors, in contrast with the selectivity at L-type Ca2+ channels.
A 4-trans-â-styryl derivative, 24, with a Ki value of 0.670 µM at A3 receptors, was 24-fold
selective vs A1 receptors (Ki ) 16.1 µM) and 74-fold vs A2A receptors (Ki ) 49.3 µM). The affinity
of 24 at L-type Ca2+ channels, measured in rat brain membranes using [3H]isradipine, indicated
a Ki value of 0.694 µM, and the compound is thus nonselective between A3 receptors and L-type
Ca2+ channels. Inclusion of a 6-phenyl group enhanced A3 receptor selectivity: Compound 28
(MRS1097; 3,5-diethyl 2-methyl-6-phenyl-4-(trans-2-phenylvinyl)-1,4(R,S)-dihydro-pyridine-3,5-
dicarboxylate) was 55-fold selective vs A1 receptors, 44-fold selective vs A2A receptors, and over
1000-fold selective vs L-type Ca2+ channels. In addition, compound 28 attenuated the A3
agonist-elicited inhibitory effect on adenylyl cyclase. Furthermore, whereas nicardipine, 12,
displaced radioligand from the Na+-independent adenosine transporter with an apparent affinity
of 5.36 ( 1.51 µM, compound 28 displaced less than 10% of total binding at a concentration of
100 µM. Pyridine derivatives, when bearing a 4-alkyl but not a 4-phenyl group, maintained
affinity for adenosine receptors. These findings indicate that the dihydropyridines may provide
leads for the development of novel, selective A3 adenosine antagonists.
The 1,4-dihydropyridines have been developed exten-
sively as potent blockers and activators of L-type
calcium channels.1 A number of these channel blockers,
such as nifedipine (Figure 1, 9) and nicardipine (Figure
1, 12), are used therapeutically in the treatment of
cardiovascular disorders, especially hypertension and
coronary heart disease.2
Dihydropyridines appear to be “privileged structures”
in medicinal chemistry and pharmacology, i.e., they
display affinity for many diverse binding sites.3 This
adaptability of dihydropyridines has been utilized to
optimize affinity in binding to R1a-adrenergic receptors
(e.g., the antagonist SNAP 5089, Figure 1),4 to platelet
activating factor (PAF, 1-O-hexadecyl/octadecyl-2-O-
acetyl-sn-glycero-3-phosphorylcholine) receptors,5 and at
other receptor targets.6 Thus, by careful structural
modification, it has been possible to select for affinity
at sites other than Ca2+ channels. For example, a
dihydropyridine derivative, 3-ethyl 5-[2-(phenylthio)-
ethyl] 2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarbox-
ylate (Figure 1, 4), has been found to inhibit PAF
binding with an affinity of 69 nM, while the same
derivative was inactive as a calcium channel blocker.5
Several dihydropyridines were also found to bind to A1
adenosine receptors in rat brain.7,8 In the present study
we have used the 1,4-dihydropyridine nucleus as a
template for probing structure-activity relationships
(SAR) at several subtypes of central adenosine receptors
(A1, A2A, and A3), in an effort to design new selective
antagonists. The classical (A1 and A2A) adenosine
receptor antagonists are xanthines,9 but it would be
desirable to expand the list of diverse chemical struc-
tures known to bind to adenosine receptors. There is
already a large number of non-xanthine antagonists
known for the A1 receptor.9-13 Although selective
agents have been reported for both A1 and A2A subtypes
of adenosine receptors,9 the development of ligands for
the recently discovered A3 receptor is lagging behind the
other subtypes.14 Activation of the A3 receptor results
in hypotension and promotion of release of inflammatory
mediators from mast cells.15 A3 adenosine receptor
antagonists are being sought as potential antiasth-
matic,15 antiinflammatory,15 and cerebroprotective
agents.16 There may also be an involvement of A3
receptors in cancer17 and apoptosis.18 We have intro-
duced the first selective agonists for the A3 adenosine
receptor subtype, including N6-(3-iodobenzyl)-5′-(N-
* Address 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.
† NIH.
‡ Duke University Medical Center.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0022-2623(96)00020-9 CCC: $12.00 © 1996 American Chemical Society

Dihydropyridine Interaction with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2981
F igu r e 1. Structures of 1,4-dihydropyridines as potent calcium channel antagonists (nifedipine, 9, and nicardipine, 12) or as
ligands at other receptor sites (SNAP 5089, an adrenoceptor antagonist, and 3-ethyl 5-[2-(phenylthio)ethyl] 2,4,6-trimethyl-1,4-
dihydropyridine-3,5-dicarboxylate, 4, a PAF antagonist).
Sch em e 1. General Procedure for the Synthesis of
1,4-Dihydropyridine Derivatives
methylcarbamoyl)adenosine (IB-MECA),19,20 but studies
of the SAR of xanthines at this subtype have thus far
failed to identify principles of achieving selectivity.21
Furthermore, at A3 receptors the affinity of xanthines
is generally much weaker than at the A1 or A2A
subtypes,21,22 and considerable species variability in
their affinity has been noted.23-25 A screen of >100
cyclic compounds of diverse structure has provided
several unexplored leads for A3 selectivity, but potencies
were generally low.11 A recent study has identified
additional natural product leads having moderate af-
finity, but the degree of selectivity was still minimal.13
Thus, the design of potent and selective A3 antagonists
has remained an unfulfilled challenge.14
Resu lts
Syn th esis. The structures of the 1,4-dihydropy-
ridines and related derivatives tested for affinity in
radioligand binding assays at adenosine receptors are
shown in Table 1. Some of the derivatives, i.e., both
well-known Ca2+ channel ligands (nifedipine, 9; nitren-
dipine, 10; nicardipine, 12; nimodipine, 13; niguldipine,
14 and 15; Bay K 8644, 18 and 19) and a PAF
antagonist (4),5 were obtained from commercial sources.
Other compounds were synthesized using standard
methodology (Scheme 1),26-28 in fair yield, as reported
in Table 2. The synthesis consisted of the Hantzsch
condensation of a 3-amino-2-butenoate ester, 33a ,b, an
aldehyde, 34a -o, and a 3-ketopropionate ester deriva-
tive, 35a -e, that were dissolved in ethanol and heated
to 100 °C. In order to obtain substitution at the
4-position, the aldehyde component, 34a -o, was varied.
Substitution at the 6-position was achieved by varying
the 3-ketopropionate ester component, 35a -e (Scheme
1).
Oxidation of 1,4-dihydropyridines (1 and 27) to the
corresponding pyridine derivatives (29 and 30, respec-
tively) was carried out using tetrachloro-1,4-benzo-
quinone (chloranil, 37) in tetrahydrofuran (Scheme 2).29
Most of the dihydropyridines examined, except for two
pairs of pure enantiomers, 14 and 15, 18 and 19, and
the nonchiral compounds 9 and 11, were racemic
mixtures at position C-4.
Bin d in g a t Ad en osin e Recep tor s. Ki values at A1
and A2A receptors were determined in radioligand
binding assays in rat brain membranes vs [3H]-(R)-PIA
[[3H]-(R)-N6-(phenylisopropyl)adenosine] or [3H]CGS
21680 [[3H]-2-[[4-[(2-carboxyethyl)phenyl]ethylamino]-
5′-(N-ethylcarbamoyl)adenosine], respectively.30,31 Af-
finity at cloned human A3 receptors expressed in HEK-
293 cells23 was determined using [125I]AB-MECA [N6-
(4-amino-3-[125I]iodobenzyl)-5′-(N-methylcarbamoyl)-
adenosine].13,32
For compound 26, the precursor butyl â-keto ester
(35e) was prepared from the condensation of the enolate
lithium salt of ethyl acetate and valeryl chloride.28
Good yields of the 6-phenyl-1,4-dihydropyridines were
obtained using a 72 h reaction time.
SAR analysis at adenosine receptors indicated that
sterically bulky groups are tolerated at the 4-, 5-, and
6-positions (Table 1). R5 was varied from ethoxycarbo-

2982 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
van Rhee et al.
Ta ble 1. Affinities of Dihydropyridine and Pyridine Derivatives in Radioligand Binding Assays at A1, A2A, and A3 Receptorsa-d
R4
R3
R2
R5
R6
N
H
Ki (mM) or % inhibitionc
compd
R2
R3
R4
R5
CO2CH2CH3
R6
rA1
rA2A
hA3
1
2
CH3 CO2CH3
CH3 CO2CH3
CH3
CH3
CH3
CH3
32.6 ( 6.3
49.2 ( 0.7
46.1 ( 6.8 32.3 ( 5.1
37 ( 18% 62.3 ( 16.7
(10-4
CO2(CH2)2OCH3
)
3
4
5
6
7
CH3 CO2CH3
CH3 CO2CH2CH3 CH3
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH3
CH3
CO2CH2Ph
CH3
CH3
CH3
CH3
CH3
6.45 ( 1.47
6.50 ( 0.47
7.52 ( 2.79
8.17 ( 1.58
9.10 ( 2.90
9.72 ( 0.63 2.78 ( 0.89
7.10 ( 2.46 5.56 ( 1.36
9.56 ( 2.69 13.6 ( 2.0
11.5 ( 3.8 6.51 ( 0.74
23.1 ( 8.6 7.90 ( 0.88
CO2(CH2)2SPh
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CH2CH3
(CH2)2CH3
CH2CHCH3(CH2)2-
CHdC(CH3)2 (R,S)
Ph
2-NO2Ph
3-NO2Ph
8
CH3 CO2CH3
CH3 CO2CH3
CO2CH2CH3
CO2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH2N(CH3)CH2Ph CH3
CO2CH2CH2OCH3
CH3
CH3
CH3
CH3
11.0 ( 1.6
2.89 ( 0.23
8.96 ( 2.06
3.34 ( 2.17
19.6 ( 1.9
20.1 ( 1.7
41.3 ( 3.5
2.74 ( 0.85 12.0 ( 3.3
18.2 ( 2.51 8.29 ( 2.41
23.0 ( 3.7 8.30 ( 1.41
18.2 ( 7.9 2.51 ( 0.15
63.8 ( 4.2 3.25 ( 0.26
44.3 ( 14.4 8.47 ( 2.75
9 (nifedipine)
10 (nitrendipine) CH3 CO2CH3
11
12 (nicardipine) CH3 CO2CH3
13 (nimodipine) CH3 CO2CH(CH3)2 3-NO2Ph
14 ((R)-
CH3 CO2CH2CH3 3-NO2Ph
3-NO2Ph
CH3
CH3
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH3
3-NO2Ph
3-NO2Ph
4-NO2Ph
d (10-4
)
1.90 ( 0.40
Ph
niguldipine)
CO2(CH2)3N
Ph
Ph
Ph
15 ((S)-
niguldipine)
CH3
d (10-4
)
d (10-4
)
2.80 ( 0.35
CO2(CH2)3N
16
CO2CH2CH3
CH3
37 ( 14%
(10-4
6.68 ( 2.37
35.6 ( 1.9 5.90 ( 1.65
20.7 ( 2.8 11.6 ( 1.7
)
17
18 ((R)-
CH3 CO2CH3
CH3 CO2CH3
2-CF3Ph
2-CF3Ph
CO2CH2CH3
NO2
CH3
CH3
0.785 ( 0.113 35.1 ( 10.1 2.77 ( 0.34
BayK8644)
19 ((S)-
BayK8644)
CH3 CO2CH3
2-CF3Ph
NO2
CH3
6.66 ( 1.89
86.3 ( 23.4 23.5 ( 0.6
20
21
22
23
24
25
26
27
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH3
CH3 CO2CH2CH3 CH3
CH3 CO2CH2CH3 CH3
CH3 CO2CH2CH3 Ph-CHdCH-(trans)
4-CH3OPh
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
CO2CH3
CH3
CH3
CH3
CH3
CH3
CH3
2.75 ( 0.35
51.0 ( 3.7
3.66 ( 0.61
8.81 ( 0.92
16.1 ( 0.5
5.39 ( 0.33
12.7 ( 3.8 4.10 ( 0.14
56.8 ( 1.9 32.1 ( 9.2
5.27 ( 1.97 4.58 ( 1.11
6.71 ( 2.06 2.30 ( 0.70
49.3 ( 12.5 0.670 ( 0.195
38.3 ( 7.9 0.940 ( 0.070
38.0 ( 10.6 47.1 ( 10.8
35.9 ( 15.3 7.24 ( 2.13
4.77 ( 0.29 0.108 ( 0.012
8.96 ( 0.93 29.5 ( 2.1
28.4 ( 9.1 4.47 ( 0.46
3-CH3O-4-OHPh
3,4-OCH2OPh
PhCH2CH2
Ph-CHdCH-(trans)
Ph-CtC-
(CH2)3CH3 10.8 ( 3.52
Ph
Ph
CH3
Ph
25.9 ( 7.3
5.93 ( 0.27
6.95 ( 2.66
7.41 ( 1.29
28 (MRS1097)
29
30
31
32
CH3 CO2CH3
CH3 CO2CH2CH3 CH3
CH3 CO2CH3 2-NO2Ph
4-CH3OPh
CH3
CH3
H
d (10-4
)
d (10-4
71 ( 29
)
d (10-4
d (10-4
)
)
H
H
H
44.5 ( 1.0
a
b
Displacement of specific [3H]-(R)-PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5). Displacement of
specific [3H]CGS 21680 binding in rat striatal membranes, expressed as Ki ( SEM in µM (n ) 3-6). c Displacement of specific [125I]AB-
MECA binding at human A3 receptors expressed in HEK cells, in membranes, expressed as Ki ( SEM in µM (n ) 2-3), or as a percentage
d
of specific binding displaced at 10 µM. Displacement of e 10% of specific binding at the indicated concentration. nd, not determined.
nyl to larger ester groups in a homologous series (1 vs
2, 3, and 4), all having a methyl group at the 4-position,
resulting in a general enhancement of affinity at all
three adenosine receptor subtypes. Compound 4 is a
potent PAF-acether antagonist5 and not a calcium
channel antagonist; thus there is an uncoupling of the
SAR in this series for interaction with both PAF and
adenosine receptors on the one hand and L-type calcium
channels on the other hand.
Another means of enhancing adenosine receptor af-
finity (relative to compound 1) was to enlarge the R4
substituent. In the series of ethyl, n-propyl, and even
larger alkyl substituents at the 4-position (5, 6, and 7)
it was demonstrated that steric bulk at this position is
tolerated in adenosine receptor binding and favored at
the A3 subtype and not detrimental at the A1 subtype.
The affinity at the A2A subtype is generally decreased
with increasing chain length of the alkyl substituent.
Consecutively, 4-aryl substituents, typical of the potent
Ca2+ channel blockers in clinical use, were also exam-
ined. In general, the affinity and receptor subtype
selectivity of 4-aryl analogues was highly dependent on
the substituents of the phenyl ring. For example, the
unsubstituted 4-phenyl compound 8 was more potent
(4-fold) at A2A receptors than at either A1 or A3 recep-
tors. Of the nitrophenyl derivatives, compound 10,
bearing an o-nitro group, was 3-fold selective for A1 vs
A3, and 6-fold for A1 vs A2A receptors, whereas the
m-nitro derivative 10 was about equipotent at A1 and
A3 receptors, but 3-fold less potent at A2A receptors than
at either A1 or A3 receptors. To complete the series, we
also synthesized the p-nitro-substituted compound 16,
which displayed a >17-fold selectivity for A3 receptors
vs A1 receptors and 6-fold selectivity for A3 receptors
vs A2A receptors. In addition, we prepared compounds
with different substituents. Compound 17, bearing a
o-trifluoromethyl group behaved similarly to the o-nitro-
substituted compound 10 and was 2-fold selective for
A1 vs A3 receptors. The p-methoxy derivative 20 was
almost equipotent at A1 (Ki ) 2.75 µM), and A3 receptors
(Ki ) 4.10 µM), but slightly less potent at A2A receptors
(Ki ) 12.7 µM). Affinity at A3 receptors did not appear

Dihydropyridine Interaction with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2983
Ta ble 2. Characterization of Dihydropyridine and Pyridine Derivatives
no.
Tm (°C)
formula
C13H19NO4
MS
analysis
yield (%)
purification
1
2
3
5
6
125-126
88-89
oil
83-84
95-96
glass
121-122
165-166
151-152
114-115
112-113
190
253 (CI)
283 (CI)
315 (CI)
267 (CI)
281 (CI)
363 (CI)
315 (CI)
374 (EI)
360 (CI)
383 (CI)
345 (CI)
361 (CI)
359 (CI)
343 (CI)
341 (CI)
339 (EI)
309 (CI)
329 (CI)
417 (CI)
251 (EI)
327 (CI)
C,H,N
C,H,N
C,H,N
C,H,N
H,Nb
c
C,H,N
C,H,N
C,H,N
C,Hd
C,H,N
C,H,N
C,H,N
e
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
f
98.2
53.0
70.5
50.5
73.3
33.4
78.6
35.5
68.1
45.6
61.8
79.6
63.0
71.9
87.9
43.3
58.8
53.0
34.7
57.9
48.9
1; MeOH
2; TLC
2; TLC
1; MeOH
1; MeOH
2; TLC
1; MeOH
1; EtOAc
1; MeOH
2; TLC
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
14H21NO5‚0.25H2O
18H21NO4‚0.50H2O
14H21NO4
15H23NO4
21H33NO4
18H21NO4
19H22N2O6
18H20N2O6
19H20F3NO4
19H23NO5‚0.21EtOAc
19H23NO6
19H21NO6‚0.32EtOAc
20H25NO4
20H23NO4
20H21NO4
17H27NO4
19H23NO4
7
8
11
16
17
20
21
22
23
24
25
26
27
28
29
30
2; TLC
1; MeOH
2; TLC
oil
87-88
135-136
176-177
oil
123-124
oil
2; TLC
1; MeOH
1; EtOH
2; TLC
1; pe 35-60
2; column
2; TLC
26H27NO4
13H17NO4
19H21NO4‚0.20EtOH
oil
oil
C,H,N
2; TLC
aPurification was achieved by either (1) (re)crystallization from the solvent specified or (2) chromatography by the specified method,
using EtOAc/pe, 20:80 (v/v), eluent. MeOH, methanol; EtOH, ethanol; EtOAc, ethyl acetate; pe, petroleum ether 35-60 °C fraction; TLC,
preparative thin layer chromatography, silica 60, 1000 µm layer thickness; column, preparative column chromatography, silica 60, 220-
b
440 mesh. 6 (C15H23NO4) H, N; C: calcd, 64.03; found, 64.75. EI: calcd, 281.1627; found, 281.1630. c 7 (C21H33NO4). This compound
decomposes upon prolonged standing. Biological data and mass spectrometry data were obtained with the freshly purified compound,
but extensive drying and shipping proved detrimental for the elemental analysis. C, H, N: calcd, 69.39, 9.15, 3.85; found, 61.65, 7.27,
d
4.70. EI: calcd, 363.2410; found, 363.2398. 17 (C19H20F3NO4) C, H; N: calcd, 3.65; found, 4.17. EI: calcd, 383.1344; found, 383.1346.
e 23 (C20H25NO4) EI: calcd, 343.1784; found, 343.1791. f 29 (C13H17NO4) EI: calcd, 251.1158; found, 251.1162.
Sch em e 2. General Procedure for the Oxidation of
1,4-Dihydropyridine Derivatives (Compounds 29 and 30)
analogue, 25, was nearly as potent at A3 receptors, but
not as selective as 24 for A3 vs A1 receptors (only 6-fold).
Substitution at the 3- and 5-positions of the dihydro-
pyridine moiety also had a large effect on affinity at
adenosine receptors. Changing an asymmetrical mixed
methyl and ethyl ester of nitrendipine, 10, to the
corresponding symmetrical diethyl ester, 11, resulted
in a 3.3-fold enhancement of A3 affinity. At A1 and A2A
receptors a similar effect was observed, and subtype
selectivity was therefore not affected. Thus, compound
11 displayed Ki values of 3.34 and 18.2 µM at rat A1
and A2A receptors, respectively, and 2.51 µM at human
A3 receptors. Nicardipine,33 12, differing from nitren-
dipine,34 10, in the presence of a sterically bulky ester
group at the 5-position, displayed a moderate enhance-
ment of affinity at human A3 receptors of 2.6-fold, while
at rat A1 and A2A receptors affinity was diminished 2-3
fold. Even larger ester substituents (i.e., 14, and 15)
were well tolerated at human A3 receptors, while affinity
at A1 and A2A receptors was drastically diminished.
to be a direct function of electron density of the phenyl
substituents; i.e. compounds containing an electron-
withdrawing p-nitro group (16) and an electron-donat-
ing p-methoxy group (20) were equipotent. Remarkably,
the introduction of a hydroxy substituent on the para
position of the 4-phenyl ring (in addition to a m-methoxy
substituent) led to a general decrease in affinity (21; Ki
) 51.0 µM at A1, 56.8 µM at A2A, and 32.1 µM at A3
receptors). Thus, steric factors surrounding the phenyl
ring may be more important than electronic factors. The
piperonal derivative 22 had a generally increased af-
finity at A1 and A3 adenosine receptor subtypes (vs the
unsubstituted phenyl compound 8), but did not display
subtype selectivity.
Affinities of enantiomeric pairs, i.e., of Bay K 8644
(18 and 19, R and S, a Ca2+ channel blocker and
activator, respectively) and the Ca2+ channel blocker
niguldipine (14 and 15, R and S, respectively), indicated
a general preference for the R- over the S-enantiomer
at all of the receptor subtypes. This is in contrast to
the affinity at L-type calcium channels [(R)-(-)-nigul-
dipine, Ki ) 8.1 nM; (S)-(+)-niguldipine, Ki ) 0.18 nM;
or 45-fold stereoselectivity]35 and at R1a adrenoceptors
[(R)-(-)-niguldipine, Ki ) 4.7 nM; (S)-(+)-niguldipine,
Ki ) 0.16 nM; or 29-fold stereoselectivity],4 at which the
(S)-(+)-enantiomer is preferred. Both niguldipine enan-
tiomers tended particularly towards selectivity for the
A3 subtype (vs A1 and A2A receptors, but not vs Ca2+
channels). Thus, (R)-niguldipine, 14, displayed Ki val-
ues of 41.3 and >100 µM at rat A1 and A2A receptors,
respectively, and 1.90 µM at human A3 receptors.
The relatively high affinity of a 4-aryl group larger
than phenyl (22), an arylalkyl group (23), and dehydro
analogues thereof (24, 25) further indicated a bulk
tolerance at this position. The 4-trans-â-styryl deriva-
tive, 24, was particularly potent (Ki ) 0.670 µM) and
selective (24- and 74- fold vs rat A1 and A2A, respec-
tively) at human A3 receptors. The phenylacetylenic

2984 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
van Rhee et al.
Ta ble 4. Effects of Dihydropyridine Derivatives at a
Concentration of 10 µM on the Inhibition of Adenylyl Cyclase
Elicited by the A3 Agonist IB-MECA
Ta ble 3. Inhibition of Specific Binding of [3H]Isradipine
Binding at L-Type Calcium Channels by Various
Dihydropyridine and Pyridine Derivatives in Rat Brain
Membranes and the Selectivity Ratio vs Affinity at Cloned
Human A3 Receptors
% inhibitiona
compd
IB-MECA (10-7 M)
n
IB-MECA (10-6 M)
n
a
compd
Ki (rCa2+
)
ratio of Ki (rCa2+)/Ki (hA3)
control
12
18
31.4 ( 4.0
7.94 ( 3.15b
19.4 ( 0.6b
5
5
2
40.2 ( 3.3
27.2 ( 8.5
27.8 ( 7.8
4
4
2
3
14
23
24
25
27
28
30
1.17 ( 0.08
0.42
0.031
0.40
0.0597 ( 0.0001
0.910 ( 0.207
0.694 ( 0.165
a
1.04
Assayed in membranes from CHO cells stably expressing the
cloned rat A3 receptor using a previously reported method in the
presence of 1 µM forskolin.15,28 b Statistically significant (p < 0.02)
vs the control value.
23 ( 3% (10-4
)
>106
>14
>1000
>2.2
17 ( 5% (10-4)
<10% (10-4
<10% (10-5
)
)
a
Expressed in µM as Ki ( SEM or percent displacement of
specific binding at the indicated concentration (M) for three
determinations, each performed in duplicate. Rat brain mem-
branes (ca. 100 µg of protein/tube) were incubated for 1 h at 25 °C
with 0.1 nM [3H]isradipine and varying concentrations of the
dihydropyridine or pyridine derivative in a 50 mM Tris buffer,
pH 7.4 in a total volume of 0.5 mL. Nonspecific binding was
determined in the presence of 10 µM nitrendipine. Ki values were
calculated using the Cheng-Prusoff equation43 assuming a Kd
value of 0.13 nM for [3H]isradipine.
µM at Ca2+ channels and 2.78 ( 0.89 µM at A3
receptors) and the 4-(2-phenylethyl) analogue 23 (Ki )
0.910 ( 0.207 µM at Ca2+ channels and 2.30 ( 0.70 µM
at A3 receptors) were about 2.5-fold selective for Ca2+
channels compared to human A3 receptors.
F u n ction a l Assa ys a t A3-Ad en osin e Recep tor s.
We examined the antagonist properties of two dihydro-
pyridine derivatives in a functional assay utilizing
cloned rat A3 adenosine receptors expressed in CHO
cells. Forskolin-stimulated adenylyl cyclase was inhib-
ited by IB-MECA [N6-(3-iodobenzyl)-5′-(N-methylcar-
bamoyl)adenosine; 10-9-10-4 M) in CHO cells trans-
fected with rat A3 adenosine receptors (maximum degree
of inhibition was 40-50%).22 The concentration of IB-
MECA used to test prospective antagonists was between
10 nM and 1.0 µM, i.e., respectively 0.1- and 10-fold the
IC50 of rat A3 receptor mediated inhibition of adenylyl
cyclase. Both nicardipine, 12, and (R)-Bay K 8644, 18,
at a concentration of 10 µM showed a significant (p <
0.02) effect on the agonist-mediated inhibition of ade-
nylyl cyclase (Table 4). The inhibition of adenylyl
cyclase induced by 10-6 M IB-MECA (40.2 ( 3.3%) was
attenuated to 27.2 ( 8.5% by 10 µM nicardipine, 12,
and the inhibition of adenylyl cyclase activity caused
by 10-7 M IB-MECA was nearly completely reversed.
A similar, but not as extensive, effect could be observed
for (R)-Bay K 8644, 18, even though the affinity of these
compounds at human A3 adenosine receptors was virtu-
ally identical. Compound 28 at a concentration of 50
µM attenuated the IB-MECA induced inhibition of
adenylyl cyclase activity at all concentrations tested
(Figure 2). This suggests that compound 28 is not only
an effective displacer of the A3-selective radioligand
At the 6-position, n-butyl substitution (26) was toler-
ated, and phenyl substitution (27) enhanced A3 adeno-
sine receptor binding (4.5-fold vs 1), and lead to slight
subtype selectivity. Combination of 6-phenyl and 4-trans-
â-styryl substituents greatly enhanced both affinity and
selectivity. Consequently, compound 28 was 55-fold
selective for A3 vs A1 receptors, and 44-fold selective for
A3 vs A2A receptors. Two other blockers of L-type Ca2+
channels, verapamil and diltiazem, which are structur-
ally unrelated to the dihydropyridines, failed to bind
appreciably to adenosine receptors.
Pyridine derivatives maintained affinity for adenosine
receptors, particularly when bearing a 4-alkyl group,
e.g., the oxidized compound 29, displayed a Ki value of
29.5 µM at human A3 receptors, whereas the related
dihydropyridine 1 displayed a Ki value of 32.3 µM at
the same receptor subtype. Similarly, a comparison of
the 6-phenyldihydropyridine 27 and the corresponding
pyridine 30 shows only minor differences in affinity. In
contrast, the nifedipine metabolite 31 no longer bound
to adenosine receptors. The distantly related pyridine
derivative 32 demonstrated that affinity of 4-arylpyri-
dine analogues for adenosine receptors may be rescued
to some extent by careful manipulation of the other
substituents.
[
125I]AB-MECA but also an effective functional antago-
nist at this receptor subtype.
Bin d in g a t L-Typ e Ca lciu m Ch a n n els. Affinity at
L-type Ca2+ channels, measured in rat brain mem-
branes using [3H]isradipine36 (Table 3), indicated a Ki
value of 0.694 ( 0.165 µM for the styryl derivative 24
(cf. 0.670 µM at human A3 receptors, and therefore not
selective). In the same assay, (R)-niguldipine, 14, had
a Ki value of 59.7 ( 0.1 nM (cf. 8.1 nM at guinea pig
skeletal muscle35 and 1.90 µM at human A3 receptors,
and therefore at least 32-fold selective for Ca2+ chan-
nels). Compound 30 at a concentration of 10 µM (Ki )
4.47 µM at A3 receptors) or 28 (Ki ) 0.108 µM at human
A3 receptors, and therefore at least 1000-fold A3 selective
vs Ca2+ channels) at a concentration of 100 µM displaced
<10% of specific binding. Slightly more potent in Ca2+
channel binding were 27 (17% displacement at 100 µM;
Ki ) 7.24 µM at human A3 receptors, and thus >14-
fold A3 selective) and 25 (23% displacement at 100 µM;
Ki ) 0.94 µM at human A3 receptors, and thus >100-
fold A3 selective). The 5-benzyl ester 3 (Ki ) 1.17 ( 0.08
In ter a ction of 1,4-Dih yd r op yr id in es w ith th e
NBI-Sen sitive Nu cleosid e Tr a n sp or ter P r otein . It
was shown previously that the nucleoside uptake in-
hibitor NBI (S-(4-nitrobenzyl)-6-thioinosine) binds to
1,4-dihydropyridine ([3H]nimodipine) binding sites on
human red blood cell membranes.37 We therefore
investigated the possibility that 1,4-dihydropyridines
may bind to the NBI-sensitive nucleoside transporter
protein. Both the commercial Ca2+ channel blocker
nicardipine, 12, and the A3 adenosine receptor selective
ligand 24 displaced [3H]NBI from its binding site in rat
forebrain membranes, with Ki values of 5.36 ( 1.51 and
1.20 ( 0.11 µM, respectively. In contrast, the A3-
selective antagonist 28 displaced less than 10% of
specific binding at a concentration of 100 µM. Com-
pound 28 is therefore >900-fold selective for A3 recep-
tors compared to the NBI-sensitive nucleoside trans-
porter.

Dihydropyridine Interaction with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2985
Ta ble 5. Affinity of and Selectivity Ratio for Selected
Dihydropyridine Derivatives at Rat A3 vs Human A3, Rat A1,
and Rat A2A Adenosine Receptors, Respectively
selectivity ratio
compd
Ki (µM)a
rA3/hA3
rA1/rA3
rA2A/rA3
12
18
24
28
11.0 ( 7.3
6.20 ( 2.12
12.1 ( 2.6
4.16 ( 1.55
3.38
2.24
18.1
38.6
1.78
0.13
1.33
1.43
5.80
5.66
4.07
1.15
a
Assayed in membranes from CHO cells stably expressing the
cloned rat A3 receptor using [125I]-N6-(4-amino-3-iodobenzyl)-5′-
(N-methylcarbamoyl)adenosine, as described previously.13,28
receptor, being more susceptible to displacement of a
selective radioligand by competing compounds, therefore
allowed for a better comparison between those com-
pounds.25 The comparison was justified by the similar-
ity of affinity of dihydropyridines at rat and human non-
A3 adenosine receptors. For example, the calcium
channel blocker nifedipine (9) displayed a Ki value of
5.6 ( 0.3 µM at human A2A adenosine receptors (un-
published data) and a Ki value of 18 ( 3 µM at rat A2A
receptors (this manuscript), but the affinities of 1,4-
dihydropyridines at rat A3 receptors differed consider-
ably from their affinity at human A3 receptors (Table
5). Least affected by the species difference was (R)-Bay
K 8644 (18) with Ki values of 2.77 ( 0.34 and 6.20 (
2.12 µM at human and rat A3 receptors, respectively.
Thus, the species selectivity ratio for (R)-Bay K 8644
(18) is only 2.2-fold, but this value increases to 38.6-
fold for our most potent and selective compound (28),
which displayed a Ki value of 4.16 ( 1.55 µM at rat A3
receptors. Consequently, the receptor subtype selectiv-
ity of the test compounds for A3 vs either A1 or A2A
adenosine receptors was disproportionately affected
(Table 5).
F igu r e 2. Attenuation of A3 agonist elicited inhibition of
forskolin stimulated adenylyl cyclase activity via cloned rat
A3 adenosine receptors. Adenylyl cyclase activity was deter-
mined in membranes from CHO cells stably expressing the
rat A3
activity was stimulated with 1 µM forskolin, and this stimula-
tion was inhibited by the A3 selective agonist IB-MECA
(shaded bars). Addition of 50 µM MRS1097, 28, attenuated
the IB-MECA induced inhibition of adenylyl cyclase activity
(closed bars). The asterisk (*) denotes a statistically significant
effect (p < 0.02) vs the control experiment.
receptor, as reported previously.28 Adenylyl cyclase
Affin ity a t Oth er Bin d in g Sites. Since compound
28 was the most potent and (A3 adenosine receptor)
selective compound of the series, its affinity at non-
adenosine receptor binding sites was examined in a
battery of radioligand binding assays (NovaScreen,
Division of Oceanix Biosciences, Hanover, MD).38 At a
concentration of 10-5 M, there was no significant (0 (
30%) displacement of radioligand from adrenergic (R1,
R2, â), cholinergic (nicotinic and muscarinic M1, M2, M3),
dopaminergic (D1 and D2), histaminergic (H1 and H2),
serotoninergic (5-HT1, 5-HT2, and 5-HT3), C5a comple-
ment, central benzodiazepine (RO 151788), GABAA
(muscimol), GABAB (baclofen), NMDA (kainate, quis-
qualate, and phencyclidine), glycine (strychnine sensi-
tive and insensitive), σ (MK-801), angiotensin (AT-II),
vasopressin V1, neuropeptide Y, cholecystokinin (CCK-
B), neurotensin, somatostatin, ANF1, and EGF recep-
tors. There was also no significant displacement of
binding of radioligand from second messenger sites
(forskolin, phorbol ester, and inositol trisphosphate) and
ion channels (N-type calcium channels, chloride chan-
nels, and low conductance potassium channels). Sig-
nificant displacement of radioligand was observed for
the CCK-A (40%) and VIP (45%) binding sites, but the
(estimated) IC50 for these compounds was still higher
than 10 µM.
Discu ssion
In the present study, we have demonstrated that
structural modification and careful examination of the
SAR of 1,4-dihydropyridines resulted in the develop-
ment of one of the first A3 adenosine receptor-selective
non-xanthine ligands. Compound 28 [3,5-diethyl 2-meth-
yl-6-phenyl-4-(trans-2-phenylvinyl)-1,4(R,S)-dihydropy-
ridine-3,5-dicarboxylate; MRS1097] displayed a good
(submicromolar) affinity in a radioligand binding assay
(Ki ) 0.108 ( 0.012 µM). In addition, we have shown
that dihydropyridines can be effective in attenuating the
IB-MECA elicited inhibition of adenylyl cyclase in CHO
cells expressing the cloned rat A3 adenosine receptor.
Compound 28 was 55-fold selective vs A1 receptors, 44-
fold selective vs A2A receptors, and over 1000-fold
selective vs L-type Ca2+ channels.
It has been suggested that 1,4-dihydropyridines may
bind to the nucleoside transporter protein of human red
blood cells and human brain.37,43 We therefore mea-
sured the ability of some 1,4-dihydropyridines to dis-
place [3H](nitrobenzyl)thioinosine ([3H]NBI) from the
Na+-independent adenosine transporter in rat forebrain.
Whereas the commercial compound nicardipine (12) and
the novel compound MRS1045 (24) displaced [3H]NBI
with affinities similar to their affinity at human A3
receptors (5.36 ( 1.51 and 1.20 ( 0.11 µM, respectively),
the most selective compound of this series (MRS1097,
28) displaced less than 10% of total [3H]NBI binding at
a concentration of 10-4 M.
Th e E ffect of Sp ecies Differ en ces on Liga n d
Selectivity. Ordinarily, a comparison of A1, A2A, and
A3 affinities within one species would be preferred. We
chose the human A3 receptor for our study instead of
the rat A3 receptor for the following reasons: Whereas
the affinity of many ligands varies slightly between rat
and human A1 and A2A adenosine receptors, the affinity
of most known adenosine receptor antagonists is only
marginal at rat A3 receptors.11,12,21,25 The human A3

2986 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
van Rhee et al.
a
Unimelt capillary melting point apparatus (Arthur H.
In this study we have shown that the affinity of
dihydropyridines can be optimized selectively for ad-
enosine receptors vs L-type calcium channels and for
A3 vs A1 and A2A receptors. Hu et al. previously found
no effect of nifedipine binding on A1 receptor mediated
adenylyl cyclase inhibition,7 but current work with the
more recently cloned A3 receptor22 demonstrates that
IB-MECA-induced inhibition of adenylyl cyclase was
attenuated by several compounds of the 1,4-dihydropy-
ridine class (Table 4). Preliminary tests show that both
nicardipine, 12, and (R)-Bay K 8644, 18 (both Ca2+
channel blockers), functionally antagonize the inhibitory
effects of an A3 selective agonist on adenylyl cyclase.
Furthermore, we have shown that MRS1097, 28, sig-
nificantly (p < 0.02) attenuates the effects of IB-MECA
on adenylyl cyclase activity (Figure 2). Further phar-
macological characterization will be required to deter-
mine if this is due to a competitive interaction with
adenosine A3 receptors or at some other stage of the
functional cascade. Thus, we have discovered dihydro-
pyridines as a promising lead in the search for A3
adenosine receptor antagonists.
L-type calcium channel blockers such as nimodipine,
13, have been examined as cerebroprotective agents in
experimental models of neurotoxicity in cell culture and
in vivo.39 Adenosine agonists and antagonists have also
been evaluated in models of brain ischemia. It has also
been postulated that an adenosine A3 receptor antago-
nist would have cerebroprotective properties, based on
the in vivo effects of the A3 receptor agonist IB-MECA
administered to gerbils in a chronic regimen.16 Thus, a
compound that would display a dual action in vivo might
be even more useful in treating stroke than either an
L-type calcium channel blocker or A3 receptor antago-
nist alone. Therefore, some of the compounds found in
the present study to have affinity at both sites may be
clinically useful as combined therapeutic agents.
Thomas Co., Philadelphia, PA) and were uncorrected.
Gen er a l P r oced u r e for P r ep a r a tion of 1,4-Dih yd r o-
p yr id in e-3,5-d ica r boxyla te Ester s (36; 1-3, 5-8, 11, 16,
17, 20-28). Equimolar amounts (0.5 mmol) of the appropriate
3-amino-2-butenoate ester (33a ,b), aldehyde (34a -o), and
3-ketopropionate ester (35a -e) derivative were dissolved in
5 mL of absolute ethanol. The solution was sealed in a glass
tube and heated to 100 °C for at least 24 h, and at most 72 h.
The solvent was then evaporated, and products were purified
either by crystallization, column chromatography (silica 60;
220-440 mesh; Fluka, CH; 20% ethyl acetate-80% petroleum
ether 35-60) or preparative TLC (silica 60; 1000 µm; Analtech,
DE; 20% ethyl acetate-80% petroleum ether 35-60). From
the moment the reactants were sealed into the glass tube, all
procedures were performed under nitrogen and low-light
conditions to prevent oxidation of the products. The products
were shown to be homogeneous by TLC.
3-Meth yl 5-eth yl 2,4,6-tr im eth yl-1,4(R,S)-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (1): 1H NMR (CHCl3-d) δ 0.98 (d,
3H, 4-CH3, J ) 6.7 Hz), 1.31 (t, 3H, 5-methyl, J ) 7.2 Hz),
2.28 (s, 6H, 2- and 6-CH3), 3.73 (s, 3H, 3-methyl), 3.83 (q, 1H,
H-4, J ) 6.4 Hz); 4.13-4.26 (m, 2H, 5-methylene), 5.51 (wide,
1H, H-1).
3-Meth yl 5-(2-m eth oxyeth yl) 2,4,6-tr im eth yl-1,4(R,S)-
d ih yd r op yr id in e-3,5-d ica r boxyla te (2): 1H NMR (CHCl3-
d) δ 0.99 (d, 3H, 4-CH3, J ) 6.7 Hz), 2.28 (s, 6H, 2- and 6-CH3),
3.41 (s, 3H, 5-methoxy), 3.66 (t, 2H, 5-(2-methylene), J ) 4.9
Hz), 3.73 (s, 3H, 3-methyl), 3.85 (q, 1H, H-4, J ) 6.5 Hz), 4.22-
4.38 (m, 2H, 5-(1-methylene)), 5.54 (wide, 1H, H-1).
3-Meth yl 5-ben zyl 2,4,6-tr im eth yl-1,4(R,S)-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (3): 1H NMR (CHCl3-d) δ 0.98 (d,
3H, 4-CH3, J ) 5.0 Hz), 2.28 (s, 6H, 2- and 6-CH3), 3.72 (s,
3H, 3-methyl), 3.89 (q, 1H, H-4, J ) 6.5 Hz), 5.21 (q, 2H,
5-methylene, J ) 14.8 Hz), 5.54 (wide, 1H, H-1), 7.30-7.41
(m, 5H, 5-phenyl).
3-Met h yl 5-et h yl 2,6-d im et h yl-4-et h yl-1,4(R,S)-d ih y-
d r op yr id in e-3,5-d ica r boxyla te (5): 1H NMR (DMSO-d6) δ
0.63 (t, 3H, 4-methyl, J ) 7.4 Hz), 1.18 (t, 3H, 5-methyl, J )
7.1 Hz), 2.19 (s, 6H, 2- and 6-CH3), 3.58 (s, 3H, 3-methyl), 3.73
(t, 1H, H-4, J ) 5.4 Hz), 4.00-4.13 (m, 4H, 4- and 5-methyl-
ene).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-p r op yl-1,4(R,S)-d ih y-
d r op yr id in e-3,5-d ica r boxyla te (6): 1H NMR (DMSO-d6) δ
0.77 (t, 3H, 4-methyl, J ) 6.7 Hz), 1.12 (wide, 4H, 4a- and
4b-methylene), 1.19 (t, 3H, 5-methyl, J ) 7.2 Hz), 2.19 (s, 6H,
2- and 6-CH3), 3.59 (s, 3H, 3-methyl), 3.76 (t, 1H, H-4, J ) 5.2
Hz), 4.07 (q, 2H, 5-methylene, J ) 6.6 Hz).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(2(R,S),6-d im eth ylh ex-
en -5-yl)-1,4(R,S)-d ih yd r op yr id in e-3,5-d ica r boxyla te (7):
1H NMR (CHCl3-d) δ 0.91 (double d, 3H, 4-(2-CH3), J 1 ) 5.8
Hz, J 2 ) 24.0 Hz), 1.30 (m, 7H), 1.63 (d, 6H, 4-(6- and 7-CH3),
J ) 25.4 Hz), 2.28 (d, 6H, 2- and 6- CH3, J ) 7.8 Hz), 3.71 (s,
3H, 3-methyl), 3.97 (t, 1H, H-4, J ) 7.2 Hz), 4.11-4.24 (m,
2H, 5-methylene), 5.07 (t, 1H, 4-(2-methyne), J ) 6.5 Hz), 5.71
(wide, 1H, H-1).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-p h en yl-1,4(R,S)-d ih y-
d r op yr id in e-3,5-d ica r boxyla te (8): 1H NMR (DMSO-d6) δ
1.11 (t, 3H, 5-methyl, J ) 6.4 Hz), 2.23 (s, 6H, 2- and 6-CH3),
3.51 (s, 3H, 3-methyl), 3.97 (m, 2H, 5-methylene), 4.84 (s, 1H,
H-4), 7.08-7.20 (m, 5H, 4-phenyl).
Exp er im en ta l Section
Ma ter ia ls. (R)-(+)- and (S)-(-)-Bay K 8644, (R)-(-)- and
(S)-(+)-niguldipine, nicardipine, nifedipine, nimodipine, and
oxidized nifedipine were purchased from Research Biochemi-
cals International (Natick, MA). Ethyl acetoacetate (35a ),
acetaldehyde (34a ), propionaldehyde (34b), butyraldehyde
(34c), benzaldehyde (34d ), anisaldehyde (34e), vanillin (34f),
methyl 3-aminocrotonate (33a ), and trans-cinnamaldehyde
(34g) were obtained from Fluka (Ronkonoma, NY). o-Ni-
trobenzaldehyde (34h ), m-nitrobenzaldehyde (34i), p-nitroben-
zaldehyde (34j), ethyl 3-aminocrotonate (33b), phenylpropar-
gylaldehyde (34k ), R,R,R-trifloro-o-tolualdehyde (34l), benzyl
acetoacetate (35b), 2-methoxyethyl acetoacetate (35c), piper-
onal (34m ), tetrachloro-1,4-benzoquinone (37), valeryl chloride,
and ethyl benzoylacetate (35d ) were from Aldrich (St. Louis,
MO). Hydrocinnamaldehyde (34n ) was from Eastman (Roch-
ester, NY), and (()-citronellal (34o) was from ICN (Plainview,
NY). Compound 4 was from Tocris-Cookson (St. Louis, MO),
and (S)-(4-nitrobenzyl)-6-thioguanosine was from Sigma (St.
Louis, MO). [3H]PN200,110 (isradipine) was from DuPont
NEN (Boston, MA). Compound 32 was prepared according to
the literature.40 All other materials were obtained from
commercial sources.
3,5-Dieth yl 2,6-d im eth yl-4-(3-n itr op h en yl)-1,4(R,S)-d i-
h yd r op yr id in e-3,5-d ica r boxyla te (11): 1H NMR (CHCl3-
d) δ 1.23 (t, 6H, 3- and 5-methyl, J ) 6.9 Hz), 2.38 (s, 6H, 2-
and 6-CH3), 4.02-4.18 (m, 4H, 3- and 5-methylene), 5.10 (s,
1H, H-4), 5.68 (wide, 1H, H-1), 7.38 (t, 1H, H-5′, J ) 8.0 Hz),
7.65 (d, 1H, H-6′, J ) 7.8 Hz), 8.02 (d, 1H, H-4′, J ) 7.4 Hz),
8.14 (s, 1H, H-2′).
3-Met h yl 5-et h yl 2,6-d im et h yl-4-(4-n it r op h en yl)-1,4-
(R,S)-d ih yd r op yr id in e-3,5-d ica r b oxyla t e (16): 1H NMR
(DMSO-d6) δ 1.11 (t, 3H, 5-methyl, J ) 7.2 Hz), 2.26 (s, 6H, 2-
and 6-CH3), 3.52 (s, 3H, 3-methyl), 4.00 (m, 2H, 5-methylene),
4.96 (s, 1H, H-1), 7.39 (d, 2H, H-2′ and H-6′, J ) 8.7 Hz), 8.09
(d, 2H, H-3′ and H-5′, J ) 8.7 Hz).
Syn th esis. Proton nuclear magnetic resonance spectros-
copy was performed on a Varian GEMINI-300 spectrometer,
and spectra were taken in DMSO-d6 or CHCl3-d. 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. Elemental analysis was performed by Atlantic Microlab
Inc. (Norcross, GA). All melting points were determined with
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(2-R,R,R-tr iflu or om eth -
ylp h en yl)-1,4(R,S)-d ih yd r op yr id in e-3,5-d ica r b oxyla t e

Dihydropyridine Interaction with Adenosine Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2987
(17): 1H NMR (DMSO-d6) δ 1.05 (m, 3H, 5-methyl), 2.21 (s,
6H, 2- and 6-CH3), 3.44 (s, 3H, 3-methyl), 3.84-4.10 (m, 2H,
5-methylene), 5.38 (s, 1H, H-4), 7.31 (t, 1H, J ) 7.3 Hz), 7.51
(m, 3H, 4-phenyl).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(4-m eth oxyp h en yl)-
1,4(R,S)-d ih yd r op yr id in e-3,5-d ica r boxyla te (20): 1H NMR
(CHCl3-d) δ 1.23 (t, 3H, 5-methyl, J ) 7.8 Hz), 2.34 (s, 6H, 2-
and 6-CH3), 3.65 (s, 3H, 4′-OCH3), 3.76 (s, 3H, 3-methyl), 4.07-
4.14 (m, 2H, 5-methylene), 4.94 (s, 1H, H-4), 5.57 (wide, 1H,
H-1), 6.76 (d, 2H, H-2′ and H-6′, J ) 8.5 Hz), 7.20 (d, 2H, H-3′
and H-5′, J ) 8.6 Hz).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(4-h yd r oxy-3-m eth ox-
yp h e n yl)-1,4(R ,S )-d ih yd r op yr id in e -3,5-d ica r b oxyla t e
(21): 1H NMR (DMSO-d6) δ 1.13 (t, 3H, 5-methyl, J ) 7.4 Hz),
2.21 (s, 6H, 2- and 6-CH3), 3.52 (s, 3H, 3′-OCH3), 3.66 (s, 3H,
3-methyl), 3.96-4.01 (m, 2H, 5-methylene), 4.74 (s, 1H, H-4),
6.49 (t, 1H, H-6′, J ) 3.9 Hz), 6.58 (d, 1H, H-5′, J ) 7.9 Hz),
6.66 (s, 1H, H-2′).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-[3,4-(m eth ylen ed ioxy)-
p h e n yl]-1,4(R ,S )-d ih yd r op yr id in e -3,5-d ica r b oxyla t e
(22): 1H NMR (CHCl3-d) δ 1.24 (t, 3H, 5-methyl, J ) 7.3 Hz),
2.33 (s, 6H, 2- and 6-CH3), 3.66 (s, 3H, 3-methyl), 3.73 (s, 1H,
H-4), 4.09-4.22 (m, 2H, 5-methylene), 4.92 (s, 2H, 3′,4′-
methylenedioxy), 5.57 (wide, 1H, H-1), 5.89 (s, 1H, H-2′), 6.66
(d, 1H, H-5′, J ) 8.1 Hz), 6.75 (d, 1H, H-6′, J ) 7.9 Hz).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(2-p h en yl)eth yl-1,4-
(R,S)-d ih yd r op yr id in e-3,5-d ica r b oxyla t e (23): 1H NMR
(CHCl3-d) δ 1.25-1.55 (m, 3H, 5-methyl), 1.63-1.71 (m, 2H,
4-(R-methylene)), 2.31 (s, 6H, 2- and 6-CH3), 2.51-2.57 (m, 2H,
4-(â-methylene)), 3.73 (s, 3H, 3-methyl), 4.05 (t, 1H, H-4, J )
5.6 Hz), 4.12-4.27 (m, 2H, 5-methylene), 5.58 (wide, 1H, H-1),
7.15 (d, 2H, H-2′ and H-6′, J ) 6.4 Hz), 7.24 (t, 3H, H-3′, and
H-4′ and H-5′, J ) 6.8 Hz).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(tr a n s-2-p h en ylvin yl)-
1,4(R,S)-d ih yd r op yr id in e-3,5-d ica r boxyla te (24): 1H NMR
(CHCl3-d) δ 1.31 (t, 3H, 5-methyl, J ) 7.1 Hz), 2.34 (s, 6H, 2-
and 6-CH3), 3.74 (s, 3H, 3-methyl), 4.14-4.28 (m, 2H, 5-meth-
ylene), 4.63 (d, 1H, H-4, J ) 5.4 Hz), 5.60 (wide, 1H, H-1),
6.19 (t, 1H, 4-(H-1 vinylidene), J ) 6.0 Hz), 7.18 (d, 1H, 4-(H-2
vinylidene), J ) 6.6 Hz), 7.24-7.34 (m, 5H, 4-phenyl).
3-Meth yl 5-eth yl 2,6-d im eth yl-4-(p h en yleth yn yl)-1,4-
(R,S)-d ih yd r op yr id in e-3,5-d ica r boxyla t e (25): 1H NMR
(CHCl3-d) δ 1.35 (t, 3H, 5-methyl, J ) 7.1 Hz), 2.36 (s, 6H, 2-
and 6-CH3), 3.80 (s, 3H, 3-methyl), 4.23-4.31 (m, 2H, 5-meth-
ylene), 4.99 (s, 1H, H-4), 5.71 (wide, 1H, H-1), 7.24 (t, 3H, H-3′,
and H-4′, and H-5′, J ) 3.2 Hz), 7.36 (d, 2H, H-2′ and H-6′, J
) 3.6 Hz).
3,5-Diet h yl 2,4-d im et h yl-6-b u t yl-1,4(R,S)-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (26): 1H NMR (CHCl3-d) δ 0.92-
0.98 (m, overlap, 6H, 6-(4-CH3) and 4-CH3, J ) 7.0 Hz), 1.27
(t, 6H, 3- and 5-methyl, J ) 7.0 Hz), 1.51 (m, 4H, 6-(2- and
3-CH2)), 2.30 (s, 3H, 2-CH3), 2.52-2.76 (m, 2H, 6-(1-CH2)), 3.85
(q, 1H, H-4, J ) 7.0 Hz), 4.18 (m, 4H, 3- and 5-methylene),
5.53 (wide, 1H, H-1).
Hz), 2.16 (s, 3H, 4-CH3), 2.39 (s, 6H, 2- and 6-CH3), 3.87 (s,
3H, 3-methyl), 4.36 (q, 2H, 5-methylene, J ) 7.4 Hz).
3,5-Dieth yl 2,4-d im eth yl-6-p h en yl-p yr id in e-3,5-d ica r -
boxyla te (30): 1H NMR (CHCl3-d) δ 1.00 (t, 3H, 3-methyl, J
) 7.4 Hz), 1.43 (t, 3H, 5-methyl, J ) 7.5 Hz), 2.37 (s, 3H,4-
CH3), 2.62 (s, 3H, 2-CH3), 4.10 (q, 2H, 3-methylene, J ) 7.4
Hz), 4.47 (q, 2H, 5-methylene, J ) 7.4 Hz), 7.40-7.58 (m, 5H,
C6H5).
Eth yl Va ler yla ceta te (35e). A solution of n-butyllithium
(6.3 mL, 1.6 M in hexane) was slowly added to a solution of
N-isopropylcyclohexylamine (1.41 g, 10 mmol) in dry THF (20
mL) at -5 °C. The mixture was stirred for 15 min and then
cooled to -78 °C. Ethyl acetate (440 mg, 5 mmol) was added
dropwise over a period of 5 min, followed by valeryl chloride
(600 mg, 5 mmol). The reaction mixture was allowed to stir
an additional 10 min, at which point it was quenched with 5
mL of 20% HCl in water. The organic layer was separated
off, and the aqueous layer was extracted with 10 mL of diethyl
ether twice. The combined organic phases were washed with
a saturated sodium bicarbonate solution (10 mL × 2) and brine
(5 mL × 2) and dried over anhydrous sodium sulfate. The
solvent was evaporated and the residue purified by column
chromatography (silica gel 60, eluted with ethyl acetate/
petroleum ether, 1:9) to yield ethyl valerylacetate (772 mg,
90%): 1H NMR (CHCl3-d) δ 0.90 (t, 3H, CH3, J ) 7.0 Hz),
1.20-1.65 (m, 7H, CH3 and 2 × CH2), 2.30 (t, 2H, COCH2, J
) 7.0 Hz), 2.65 (s, 2H, COCH2CO), 4.15 (q, 2H, COOCH2, J )
7.0 Hz); MS (EI) 85 [CH3(CH2)3CO]+, 172 (base), 57 [CH3-
(CH2)3]+. The ethyl valerylacetate (35e) was then used in the
procedure described above for the preparation of compound
26.
P h a r m a cology: Ra d ioliga n d Bin d in g Stu d ies. Binding
of [3H]-(R)-N6-(phenylisopropyl)adenosine ([3H]-(R)-PIA) to A1
receptors from rat cerebral cortex membranes and of [3H]-2-
[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-N-(ethylcarbamoyl)-
adenosine ([3H]CGS 21680) to A2A receptors from rat striatal
membranes was performed as described previously.30,31 Ad-
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.
Binding of [125I]-N6-(4-amino-3-iodobenzyl)-5′-(N-methylcar-
bamoyl)adenosine ([125I]AB-MECA) to membranes prepared
from HEK-293 cells stably expressing the human A3 receptor
(Receptor Biology, Inc., Baltimore, MD) or to membranes
prepared from CHO cells stably expressing the rat A3 receptor
was performed as described.13,32 The assay medium consisted
of buffer containing 50 mM Tris, 10 mM MgCl2, 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 [125I]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 100 µM N6-(phenylisopropyl)adenosine (R-PIA).
Binding of [3H]isradipine to rat cerebral cortex membranes
was performed essentially as described in Moody et al.36
Briefly, an aliquot of a membrane suspension corresponding
to 100 µg of protein was incubated in a total volume of 0.5 mL
of Tris buffer (50 mM, pH 7.4, 25 °C) for 1 h in the presence
of 0.1 nM [3H]isradipine and varying concentrations of the
dihydropyridine or pyridine derivatives. Nonspecific binding
was determined in the presence of 10 µM nitrendipine.
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
3 mL of buffer each.
At least five 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 the InPlot pro-
gram (Graph-PAD, San Diego, CA), were converted to apparent
Ki values using the Cheng-Prusoff equation41 and Kd values
of 1.0, 14, and 0.13 nM for [3H]-(R)-PIA, [3H]CGS 21680, and
3,5-Dieth yl 2,4-d im eth yl-6-p h en yl-1,4(R,S)-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (27): 1H NMR (CHCl3-d) δ 0.89 (t,
3H, 3-methyl, J ) 7.4 Hz), 1.14 (d, 3H, 5-methyl, J ) 7.4 Hz),
1.31 (t, 3H, J ) 7.0 Hz), 2.3 (s, 3H, CH3), 3.94 (m, 3H), 4.23
(m, 2H), 7.28-7.41 (m, 5H, C6H5).
3,5-Dieth yl 2-m eth yl-4-(tr a n s-2-p h en ylvin yl)-6-p h en yl-
1,4(R,S)-d ih yd r op yr id in e-3,5-d ica r boxyla te (28): 1H NMR
(CHCl3-d) δ 0.92 (t, 3H, 5-methyl, J ) 7.0 Hz), 1.32 (t, 3H,
3-methyl, J ) 7.0 Hz), 2.37 (s, 3H, 2-CH3), 3.94 (q, 3H,
5-methylene, J ) 6.7 Hz), 4.22, (q, 3H, 3-methylene, J ) 6.7
Hz), 4.76 (d, 1H, H-4, J ) 6.2 Hz), 5.78 (wide, 1H, H-1), 6.30-
6.38 (m, 2H, CHdCH), 7.20-7.44 (m, 10H, 2 x C6H5).
Gen er a l P r oced u r e for Oxid a tion of 1,4-Dih yd r op yr i-
d in e-3,5-d ica r boxyla te Ester s (38; 29, 30). Equimolar
amounts (0.25 mmol) of the 1,4-dihydropyridine-3,5-dicarboxy-
late ester (36; 1, 27) and tetrachloro-1,4-benzoquinone (37) in
tetrahydrofuran (2 mL) were mixed and refluxed for up to 4
h. The solvent was then evaporated, and products were
purified by preparative TLC (silica 60; 1000 µm; Analtech, DE;
20% ethyl acetate-80% petroleum ether 35-60).
3-Meth yl 5-eth yl 2,4,6-tr im eth ylp yr id in e-3,5-d ica r box-
yla te (29): 1H NMR (DMSO-d6) δ 1.27 (t, 3H, 5-methyl, J 6.9

2988 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
van Rhee et al.
[3H]isradipine, respectively, and 0.59 nM for binding of [125I]-
AB-MECA at human A3 receptors, respectively.
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Interaction of dihydropyridine calcium channel agonists and
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693-718.
In h ibition of Ad en ylyl Cycla se Activity. Adenylyl cy-
clase activity was determined in membranes from CHO cells
stably expressing the rat A3 receptor, prepared as reported
previously.32 [R-32P]ATP was added to a membrane suspension
containing 1 µM forskolin to stimulate adenylyl cyclase. The
reaction was terminated by addition of a stop buffer containing
20 000 cpm/mL [3H]cyclic AMP. Cyclic AMP was isolated by
consecutive chromatography over columns of Dowex 50 ion
exchange resin and alumina. Maximal stimulation of adenylyl
cyclase activity was obtained in the presence of 1 µM forskolin
(circa 6-8-fold), and maximal inhibition of forskolin-stimulated
adenylyl cyclase activity (40-50% residual activity from the
stimulated level) was obtained in the presence of 10 µM IB-
MECA. Statistical significance was calculated in a t-test, with
4 degrees of freedom at a significance level of 98%.
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In ter a ction of 1,4-Dih yd r op yr id in es w ith th e NBI-
Sen sitive Nu cleosid e Tr a n sp or ter P r otein . Competition
of 1,4-dihydropyridines for binding of [3H]NBI to the NBI-
sensitive nucleoside transporter protein was carried out by the
slightly modified procedure of Marangos et al.42 Rat forebrain
membranes were incubated for 30 min at 23 °C with 0.7 nM
[3H]NBI and varying concentrations of competitor in Tris
buffer (50 mM, pH 7.4) in a total of 500 µL. Then 5 µM (S)-
(4-nitrobenzyl)-6-thioguanosine was added to determine non-
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total binding. A Kd value of 0.15 nM was used for the
calculation of the Ki values.42
Abbr evia tion s: [125I]AB-MECA, [125I]-N6-(4-amino-3-iodo-
benzyl)-5′-(N-methylcarbamoyl)adenosine; Bay K 8644, 3-
methyl 2,6-dimethyl-5-nitro-4-(2-(R,R,R-trifluoromethyl)phen-
yl)-1,4-dihydropyridine-3-carboxylate; CGS 21680, 2-[[4-(2-
ca r boxyet h yl)ph en yl]et h yla m in o]-5′-(N-et h ylca r ba m oyl)-
adenosine; CHA, N6-cyclohexyladenosine; CHO cells, Chinese
hamster ovary cells; DMSO, dimethyl sulfoxide; HEK cells,
human embryonic kidney cells; IB-MECA, N6-(3-iodobenzyl)-
5′-(N-methylcarbamoyl)adenosine; Ki, equilibrium inhibition
constant; NBI, S-(4-nitrobenzyl)-6-thioinosine; NECA, (N-
ethylcarbamoyl)adenosine; PAF, 1-O-hexadecyl/octadecyl-2-O-
acetyl-sn-glycero-3-phosphorylcholine, platelet activating fac-
tor; (R)-PIA, (R)-N6-(phenylisopropyl)adenosine; SAR, structure-
activity relationship; Tris, tris(hydroxymethyl)aminomethane;
XAC, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-
1,3-dipropylxanthine, xanthine amine congener.
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J acobson, K. A. Structure-activity relationships of 1,3-dialkyl-
xanthine derivatives at rat A3 adenosine receptors. J . Med.
Chem. 1994, 37, 3373-3382.
(22) Zhou, Q. Y.; Li, C. Y.; Olah, M. E.; J ohnson, R. A.; Stiles, G. L.;
Civelli, O. Molecular cloning and characterization of an adeno-
sine receptor - The A3 adenosine receptor. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 7432-7436.
(23) Salvatore, C. A.; J acobson, M. A.; Taylor, H. E.; Linden, J .;
J ohnson, R. G. Molecular cloning and characterization of the
human A3 adenosine receptor. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 10365-10369.
Ack n ow led gm en t. We thank Gilead Sciences (Fos-
ter City, CA) and the Cystic Fibrosis Foundation (Silver
Spring, MD) for financial support and Dr. Bernhard
Witkop (NIH) for helpful discussions. We thank Dr. Pat
Towers of Amersham (Cardiff, U.K.) and Dr. Garth
Brown of DuPont NEN (North Billerica, MA) for provid-
ing samples of [125I]AB-MECA.
(24) Linden, J .; Taylor, H. E.; Robeva, A. S.; Tucker, A. L.; Stehle, J .
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