Selective A3 adenosine receptor antagonists: Water-soluble 3,5-diacyl- 1,2,4-trialkylpyridinium salts and their oxidative generation from dihydropyridine precursors

Article abstract of DOI:10.1021/jm990234x

A₃ adenosine receptor antagonists are sought for their potential antiinflammatory, antiasthmatic, and antiischemic properties. We have found that 3,5-diacyl-1,2,4-trialkyl-6-phenylpyridinium derivatives constitute a novel class of selective A

Full text of DOI:10.1021/jm990234x

4232
J . Med. Chem. 1999, 42, 4232-4238
Selective A3 Ad en osin e Recep tor An ta gon ists: Wa ter -Solu ble
3,5-Dia cyl-1,2,4-tr ia lk ylp yr id in iu m Sa lts a n d Th eir Oxid a tive Gen er a tion fr om
Dih yd r op yr id in e P r ecu r sor s
Rongyuan Xie,† An-Hu Li,† Xiao-Duo J i,† 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 May 11, 1999
A3 adenosine receptor antagonists are sought for their potential antiinflammatory, antiasth-
matic, and antiischemic properties. We have found that 3,5-diacyl-1,2,4-trialkyl-6-phenyl-
pyridinium derivatives constitute a novel class of selective A3 adenosine receptor antagonists.
The structure-activity relationships of this class of antagonists, incorporating the 3-thioester,
have been explored. The most potent analogue in this group was 2,4-diethyl-1-methyl-3-
(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridinium iodide (11), which had an
equilibrium inhibition constant (Ki) value of 219 nM at human A3 receptors (binding of [125I]-
AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine)) expressed in Chinese
hamster ovary (CHO) cells and >10 µM at rat brain A1 and A2A receptors and at recombinant
human A2B receptors. Compound 11 could be generated through oxidation of the corresponding
3,5-diacyl-1,2,4-trialkyl-6-phenyl-1,4-dihydropyridine, 24, with iodine or in the presence of rat
brain homogenates. A 6-cyclopentyl analogue was shown to increase affinity at human A3
receptors upon oxidation from the 1-methyl-1,4-dihydropyridine analogue, 25, to the corre-
sponding pyridinium derivative, 23 (Ki 695 nM), suggesting a prodrug scheme. Homologation
of the N-methylpyridinium derivatives to N-ethyl and N-propyl at the 1-position caused a
progressive reduction in the affinity at A3 receptors. Modifications of the alkyl groups at the
2-, 3-, 4-, and 5-positions failed to improve potency in binding at A3 receptors. The pyridinium
antagonists are not as potent as other recently reported, selective A3 receptor antagonists;
however, they display uniquely high water solubility (43 mM for 11). Compound 11 antagonized
the inhibition of adenylate cyclase elicited by IB-MECA in CHO cells expressing the human
A3 adenosine receptor, with a KB value of 399 nM, and did not act as an agonist, demonstrating
that the pyridinium salts are pure antagonists.
In tr od u ction
for novel receptor ligands (Figure 1). The 4-phenyleth-
ynyl-6-phenyl-1,4-dihydropyridine (1)13,14 was >1300-
fold selective for human A3 receptors versus other
subtypes of adenosine receptors, and a dihydropyridine
of this class was shown to be specific for adenosine
receptors versus other receptors and ion channels.
The A3 adenosine receptor occurs in the inflammatory/
immune system,1,2 the cardiovascular system,3,4 the
renal system,5 and the central nervous system.6,7 Acti-
vation of the receptor is coupled to the inhibition of
adenylyl cyclase and the stimulation of phospholipase
C. A3 adenosine receptor antagonists are sought as
potential antiinflammatory, antiasthmatic, or anti-
ischemic agents.1,2,10
The classical adenosine antagonists, xanthines, while
potent and/or selective for A1/A2A/A2B receptors, have not
provided fruitful leads for A3 receptor-selective antago-
nists.11 Among the first selective antagonists reported
for adenosine A3 receptors were 1,4-dihydropyridine
derivatives.12 The substituents on the structures of
common calcium channel blockers of this chemical class
were modified to achieve A3 receptor selectivity and
eliminate binding to L-type calcium channels, thus the
1,4-dihydropyridine structure was used as a template
The template approach was further extended with the
report that not only could the substituent groups of the
dihydropyridine be substituted with resulting increases
in affinity but the dihydropyridine ring could be oxi-
dized, leading to pyridine derivatives as novel A3 recep-
tor selective antagonists.15 A 3-thioester group proved
beneficial for affinity of pyridines at the A3 receptor.
There are also pronounced species differences in A3
antagonist affinity,16,17 and the pyridine antagonists
tended to have higher affinity and selectivity at rat A3
receptors than did other classes of antagonists.15 For
example, 2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyl-
oxylcarbonyl-6-phenylpyridine (2) was >100-fold selec-
tive for rat A3 versus A1 receptors. A three-dimensional
quantitative structure-activity relationship (3D-QSAR)
for 1,3-diacylpyridines as human A3 receptor antago-
nists was obtained by applying comparative molecular
field analysis (CoMFA)18 and a previously reported
* Address correspondence to Dr. K. A. J acobson, Chief, Molecular
Recognition Section, LBC, NIDDK, NIH, Bldg. 8A, Rm. B1A-19,
Bethesda, MD 20892-0810. Tel: (301) 496-9024. Fax: (301) 480-8422.
E-mail: kajacobs@helix.nih.gov.
† National Institutes of Health.
‡ Duke University Medical Center.
10.1021/jm990234x This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society
Published on Web 09/16/1999

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4233
F igu r e 1. Sequential steps in the design of key A3 adenosine receptor selective antagonists. In screening of chemical libraries
for adenosine receptor affinity, 1,4-dihydropyridines used as L-type Ca2+ channel blockers were found to be weak, nonselective
adenosine antagonists. Subsequently, derived 1,4-dihydropyridines (such as MRS 1191, 1) and 3,5-diacylpyridines (such as 2)
were optimized for A3 adenosine receptor affinity using a chemical template approach. The present study introduces pyridinium
salts that are selective A3 adenosine receptor antagonists of moderate potency and selectivity.
general A3 receptor pharmacophore model.19 A model
Sch em e 1. Synthesis of Substituted
1,4-Dihydropyridines Using the Hantzsch Reaction and
of the entire transmembrane region of the human A3
receptor, containing a docked pyridine reference ligand
2, was reported.18 Other antagonists for the human A3
receptor have been described.11,20,21
Oxidation to Pyridine Derivativesa,b
In the present study, we report a conceptual extension
of the template approach (Figure 1), which allows charge
properties and lipophilicity to be altered. The pyridine
ring may be quaternized as the 1-alkyl-pyridinium salts,
resulting in decreased affinity at adenosine receptors
compared to the pyridine derivatives such as 2, but with
retention of A3 receptor selectivity. The pyridine an-
tagonists of A3 receptors have the deficiency of low water
solubility, which is overcome in the present study
involving permanently charged pyridinium ligands.
Moreover, the corresponding 1-methyl dihydropyridine
derivatives, which may be oxidized biochemically to the
pyridinium antagonists, may be considered prodrugs.
The oxidation occurs relatively rapidly in the presence
of rat brain homogenates.
Resu lts
Syn th esis. As in the previous studies,15,18 pyridine
analogues, 7, were prepared from the corresponding
dihydropyridines, 6 (Scheme 1), which were prepared
through condensation of three components (3-5). The
3-thioester group, shown previously to enhance A3
receptor affinity in the pyridine series,15 was incorpo-
rated in the present set of pyridinium analogues. Two
routes may be used to prepare the pyridinium salts,
either the quaternization of 7 or the oxidation of the
1-alkyl-dihydropyridine derivative, 8. Yields and char-
acterization of the pyridinium salts synthesized and
tested as A3 receptor antagonists are described in Table
1. The affinities in radioligand binding assays22-24 at
adenosine receptors of the 3,5-diacylpyridinium deriva-
tives prepared (10-23) are shown in Table 2.
a
Structures 6, 7, and 9 are chemical families that include
selective A3 receptor antagonists, while structure 8 may be
considered a precursor “prodrug” for in vivo conversion. This
prodrug generates 9 under oxidative chemical or biochemical
conditions. Compounds 24 and 25 are examples of general
structure 8, and compounds 10-23 are examples of general
structure 9. b Reagents: (a) EtOH, 90 °C, overnight; (b) tetrachloro-
benzoquinone, THF; (c) I2/nitromethane or brain homogenate; (d)
NaH, THF, R1-I, 0 °C; (e) nitromethane, R1-I, 90 °C, 2 days.
The reduced precursors corresponding to 11 and 23,
the 1-methyl-1,4-dihydropyridine derivatives, 24 and 25,
respectively, were synthesized. Compounds 24 and 25
were prepared by the condensation of three components,
3-5 (Scheme 1), followed by alkylation of the dihydro-
pyridine, 6, using methyl iodide in THF.
P h a r m a cology. Ki values were determined in the
binding of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-
5′-N-methylcarbamoyladenosine)24 to recombinant hu-
man A3 adenosine receptors expressed in Chinese
hamster ovary (CHO) cells. The pyridinium salts gener-
ally only weakly displaced binding at A1 and A2A
receptors in rat brain membranes, using the radio-
ligands [3H]R-PIA22 and [3H]CGS 21680,23 respectively.
Compound 11, a 1-methyl-2,3,4,5-tetraethyl pyri-
dinium analogue, was the most potent in the present
set of compounds at displacing radioligand binding at
human A3 receptors (Table 2). A Ki value of 219 nM was
determined; thus, the selectivity for human A3 receptors
versus rat A1 and A2A receptors was ∼50-fold. In binding
to rat A3 receptors, a Ki value of 7.54 ( 1.07 µM was

4234 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Xie et al.
Ta ble 1. Characterization of Pyridinium (Compounds 10-23)
and Dihydropyridine (Compounds 24 and 25) Derivatives
only slightly diminished A3 receptor affinity. Introduc-
tion of cycloalkyl groups at the 2- and 6- positions
diminshed affinity by 6- and 3-fold, respectively, in
compounds 21 and 23.
Introduction of a long chain at the 2-position, in 22,
did not decrease the human A3 receptor affinity.
The affinity of 11 at recombinant human A2B recep-
tors expressed in human embryonic kidney (HEK-293)
cells was measured. At 30 µM, the degree of displace-
ment of specific binding of the radiolabeled antagonist
[3H]ZM24138525 was only 40%. Thus, this pyridinium
salt is selective in binding to human A3 versus A2B
receptors.
Compound 11 effectively antagonized the effects of an
agonist in a functional A3 receptor assay, i.e. inhibition
of adenylate cyclase in CHO cells expressing cloned
human A3 receptors.8,14 In this functional assay (Figure
2), IB-MECA inhibited adenylate cyclase via human A3
receptors with an IC50 of 41.4 ( 14.9 nM (n ) 3). In the
presence of 10 µM of 11, the concentration response
curve was shifted 26-fold to the right, with an IC50 of
1.08 ( 0.19 µM (n ) 3). From a Schild analysis30 a KB
value obtained for antagonism by 11 was 399 nM, i.e.
approximately 1.8 times the Ki value obtained in binding
to human A3 receptors.
Pyridinium salt 11 could be generated through oxida-
tion of the corresponding reduced precursor, 1-methyl-
2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-
6-phenyl-1,4-dihydropyridine, 24. The conversion of 24
to 11 by chemical means (iodine in nitromethane) and
during incubation at 37 °C with rat brain membranes,
to simulate in vivo conditions, was studied. The chemi-
cal conversion occurred readily, and the time course of
the biochemical oxidation was recorded (Figure 3). At
regular time points, aliquots were removed from the
incubation mixture, extracted with ether, and both the
compda
formula
analysis
yield (%)
10
11
12b
13c
14
15
16
17
18
19
20
21
22
23
24d
25d
C
C
21H26INO3S‚0.85I2
22H28INO3S‚0.55I2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
HRMS
52
49
31
25
56
52
22
48
49
27
51
40
24
47
71
34
C23H30INO3S‚1.64H2O
C24H32INO3S‚0.98H2O
C
C
C
C
C
C
C
C
C
C
22H27FINO3S‚0.90I2
23H30INO3S‚1.80I2
24H32INO3S‚0.73I2
25H33FINO3S‚1.80I2
25H32INO4S2‚0.75I2
30H31IN2O5S‚1.00I2
24H32INO3S‚0.50I2
24H30INO3S‚0.70I2
31H38INO4S‚1.15I2
21H32INO3S‚0.80I2
C22H29NO3S
C21H33NO3S
a
Unless otherwise noted, the R1I used in the preparation of
b
pyridinium salts (compounds 10-23) is MeI. The R1I used is EtI.
c The R1I used is 1-PrI. Compounds 24 and 25 are 1-methyl-1,4-
d
dihydropyridines.
determined. The corresponding 2-methyl derivative, 10,
was slightly less potent at the three adenosine receptor
subtypes. Homologation of the 1-position substituent to
ethyl, 12, and propyl, 13, caused a progressive reduction
in the affinity at A3 receptors. Similarly, homologation
at the 3-, 4-, and 5-positions and/or the introduction of
fluorine atoms at the terminal carbons of the ester
groups, i.e. compounds 14-17, failed to improve potency
in binding at A3 receptors.
At the 4-position, introduction of a thioester group (cf.
18 vs 15) had no effect on the A3 receptor affinity, while
steric bulk in the form of a phthaloylamino group, in
compound 19, increased affinity at A1 and A2A receptors,
thus diminishing selectivity. Homologation, as in 20, or
introduction of steric bulk at the 2-position, as in 15,
Ta ble 2. Affinities of Pyridinium Derivatives in Radioligand Binding Assays at A1, A2A, and A3 Receptorsa-d
Ki (nM) or % displacement
a
b
c
compd
R2
R3
R4
CH2CH3
CH2CH3
CH2CH3
R5
R6
rat A1
rat A2A
human A3
10
11
12
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
Ph
Ph
Ph
22 ( 12% (10-4
31900 ( 8700
43700 ( 12400
)
20800 ( 9500
11600 ( 2900
25 ( 1% (10-4
379 ( 94
219 ( 59
577 ( 64
CH2CH3
CH2CH3
)
)
(R1 ) Et)
13
CH2CH3
CH2CH3
CH2CH3
CH2CH3
Ph
Ph
76900 ( 12700
28 ( 1% (10-4
1350 ( 490
(R1 ) n-Pr)
14
15
16
17
18
19
20
21
22
23
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
(CH2)3CH3
cyclobutyl
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH2CH3
CH2CH2F
30 ( 2% (10-4
d (10-4
)
)
56600 ( 11200 364 ( 107
CH2CH2CH3 Ph
CH2CH2CH3 Ph
CH2CH2CH3 Ph
)
18700 ( 6200
25% (10-4
7370 ( 1010
483 ( 154
2020 ( 1190
465 ( 99
d (10-4)
)
CH2CH2CH2F CH2CH2CH3
d (10-4
)
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH2SCOCH3 CH2CH2CH3 Ph
CH2CH2N-Pth
CH2CH3
CH2CH3
CH2CH2CH3
CH2CH3
45 ( 3% (10-4
1530 ( 130
14500 ( 5900
53200 ( 23900 538 ( 106
2150 ( 410
6020 ( 1670
33200 ( 7400
11900 ( 2700
7720 ( 2800
CH2CH3
CH2CH3
CH2CH3
Ph
Ph
Ph
1250 ( 200
436 ( 152
1410 ( 440
348 ( 126
695 ( 198
88000 ( 31700
CH2CH2OCH2Ph CH2CH3
CH2CH3 CH2CH3
CH2CH2CH3 Ph
CH2CH3 cyclopentyl 11500 ( 1800
21 ( 8% (10-4
)
a
Displacement of specific [3H]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). Displacement of specific [3H]CGS 21680 binding in rat striatal membranes,
b
expressed as Ki ( SEM in µM (n ) 3-6) or as a percentage of specific binding displaced at the indicated concentration (M). 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 )
d
3-4). Displacement of e10% of specific binding at the indicated concentration (M).

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4235
5-ethyloxycarbonyl-6-cyclopentyl-1,4-dihydropyridine (25),
was shown to increase affinity at human A3 receptors
upon oxidation to the corresponding pyridinium salt
(23). Compound 25 had a Ki value at human A3
receptors of 6.40 ( 0.78 µM, while 23 was an order of
magnitude more potent (Ki of 695 nM), suggesting a
prodrug scheme. At rat A1 receptors, both the precursor,
compound 25 (34 ( 1% displacement at 100 µM), and
the oxidized product, 23 (Ki of 11.5 µM), bound only
weakly.
Discu ssion
The role of A3 receptors in the central nervous system
is under investigation. It appears that an A3 receptor
antagonist is cerebroprotective in a model of global
ischemia in gerbils.10 Furthermore, A3 receptor antago-
nists may have a general function in inflammation and
asthma.1,2 Thus, any advance in the design of A3
antagonists that may enhance the bioavailability or
increase the specificity of the compound within the body
is of potential therapeutic interest.
F igu r e 2. Inhibition of adenylate cyclase by N6-(4-amino-3-
iodobenzyl)-5′-N-methylcarbamoyladenosine (IB-MECA) in
membranes from Chinese hamster ovary (CHO) cells stably
transfected with human A3 receptors and its antagonism by
the pyridinium derivative 11. The assay was carried out as
described in the Experimental Section in the presence of 5 µM
forskolin, 20 µM Ro 20-1724, and 25 mM NaCl. Each data
point is shown as mean (SEM for three determinations.
Responses are shown for agonist alone (O) or in combination
with the A3 adenosine antagonist 11 (10 µM, 9). IC50 values
were 41.4 ( 14.9 nM (IB-MECA alone), 1.08 ( 0.19 µM (+
11).
The pyridinium antagonists are not as potent as other
recently reported selective A3 receptor antagonists;
however, they display uniquely high water solubility (43
mM for compound 11) as well as the ability to be
extracted readily into ether. Moreover, the present set
of pyridinium antagonists provides a means for oxida-
tive generation in vivo from prodrugs (1-methyl-1,4-
dihydropyridine derivatives). In the present study, one
reduced precursor, 25, was found to be clearly less
potent than the corresponding pyridinium salt, 23, in
binding to adenosine receptors, especially the A3 sub-
type, and another precursor, 24, was found to have the
identical human A3 receptor affinity before and after
oxidation. Thus, depending on the structure of the
pyridine substitutents, one can determine the degree
to which the antagonist requires preactivation of a
prodrug form in order to acheive antagonism of A3
receptors.
A3 antagonists have been proposed for use in inflam-
mation.1,2 In inflammation, oxidation in tissue may
occur more readily due to the enhanced presence of
various enzymes.26 In ischemic tissue, reactive oxygen
species are generated. Thus, it is conceivable that the
activation of the prodrug may be enhanced in affected
tissues, thus potentially providing site-selective activa-
tion of the A3 antagonist.
F igu r e 3. Biochemical oxidation of a 1-methyl-1,4-dihydro-
pyridine derivative, 24, as a prodrug for the selective A3
receptor antagonist 11. Concentrations of the prodrug (0) and
pyridinium salt (b) are expressed as percent of total. Incuba-
tion was carried out at 37 °C. The initial concentration of 24
was 1.27 mM, and the pH was 7.4.
The use of dihydropyridines as prodrugs has been
thoroughly studied by Bodor and co-workers.27,28 By the
Bodor approach, an N-methyl dihydropyridine moiety
is temporarily attached to a drug. Following passage
across the blood-brain barrier, an oxidation occurs and
the resultant 1-Me pyridinium derivative is restricted
from leaving the brain. The pyridinium moiety gradu-
ally is cleaved from the active drug moiety in the brain
compartment. In the present approach, the pyridinium
species, itself, is the biologicallly active drug and thus
is immediately available to act following oxidation. The
in vivo properties of the 1-methyldihydropyridine and
pyridinium derivatives in the present study, including
the ability to cross biological membranes such as the
blood-brain barrier, have yet to be explored.
precursor and the pyridinium salt were assayed in the
evaporated organic phase using HPLC. The oxidation
occurred cleanly with a t1/2 of approximately 47 min.
The 1-methyl dihydropyridine 24 was found to bind
selectively to human A3 adenosine receptors. At human
A3 receptors, the Ki value was 379 ( 122 nM (n ) 4).
The Ki value at rat A1 receptors was 28.4 ( 2.5 µM (n
) 3). At rat A2A receptors, the percent displacement of
specific radioligand binding was 48 ( 7% at 100 µM.
Therefore, to demonstrate the prodrug principle, i.e. an
inactive prodrug that could be converted to a selective
adenosine antagonist, the corresponding 6-cyclopentyl
dihydropyridine 25 was prepared. The 6-cyclopentyl
analogue, 1-methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-

4236 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Xie et al.
2 H), 4.30-4.40 (m, 2 H), 7.47-7.50 (m, 3 H), 7.64-7.67 (m, 2
H). MS (CI/NH3): m/z 390 (MH+-Me-I).
Exp er im en ta l Section
Ch em ica l Syn th esis. Ma ter ia ls. Iodomethane was pur-
chased from Fluka (Buchs, Switzerland). Iodoethane and
1-iodopropane were purchased from Aldrich (Milwaukee, WI).
PBS (1× pH 7.4) was purchased from Biofluids, Inc. (Rockville,
MD). Starting 3,5-diacyl-2,4-dialkylpyridine and dihydropy-
ridine derivatives were described previously.15,18 All other
materials were obtained from commercial sources.
1-Met h yl-2-et h yl-4-et h yl-3-(et h ylsu lfa n ylca r b on yl)-5-
1
p r op yloxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (15). H
NMR δ: 0.68 (t, J ) 7.8 Hz, 3 H), 1.25 (t, J ) 7.8 Hz, 3 H),
1.39 (m, 2 H), 1.49 (t, J ) 7.8 Hz, 3 H), 1.57 (t, J ) 7.8 Hz, 3
H), 2.97 (q, J ) 7.8 Hz, 2 H), 3.28 (q, J ) 7.8 Hz, 2 H), 3.38 (q,
J ) 7.8 Hz, 2 H), 4.07 (t, J ) 6.9 Hz, 2 H), 4.21 (s, 3 H), 7.69-
7.76 (m, 5 H). MS (CI/NH3): m/z 386 (MH+-Me-I).
Proton nuclear magnetic resonance spectroscopy was per-
formed on a Varian GEMINI-300 spectrometer, and all spectra
were obtained in CDCl3. Chemical shifts (δ) relative to
tetramethylsilane are given. Chemical-ionization (CI) mass
spectrometry was performed with a Finnigan 4600 mass
spectrometer, and electron-impact (EI) mass spectrometry was
performed with a VG7070F mass spectrometer at 6 kV.
Elemental analysis was performed by Galbraith Laboratories,
Inc. (Knoxville, TN) and/or Atlantic Microlab, Inc. (Norcross,
GA).
Gen er a l P r oced u r e for P r ep a r a tion of P yr id in iu m
Sa lt (10, 11, 14-23) by Qu a ter n a r y Am in a tion of 3,5-
Diacyl-2,4-Dialkylpyr idin e Der ivatives with Iodom eth an e
(Sch em e 1). A mixture of 2 (14 mg, 0.038 mmol) and
iodomethane (59 mg, 0.38 mmol) in 2 mL of anhydrous
nitromethane was sealed in a Pyrex tube and was heated at
80 °C for 2 days. After the mixture cooled to room temperature,
the solvent and excess MeI were removed under reduced
pressure to leave a yellow oil. It was applied to TLC separation
[ethyl acetate:petroleum ether ) 1:4 (v/v) for the first develop-
ment; methanol:chloroform ) 1:5 (v/v) for a second develop-
ment and ethyl acetate: petroleum ether ) 1:1 (v/v) for a third
development] and 9.5 mg of the desired product (11) was
afforded as a yellow solid (yield: 49%). If methanol or acetone
was used as the solvent, the yield was much lower than with
nitromethane.
1-Meth yl-2-eth yl-4-p r op yl-3-(eth ylsu lfa n ylca r bon yl)-5-
p r op yloxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (16). H
1
NMR δ: 0.67 (t, J ) 7.8 Hz, 3 H), 1.04 (t, J ) 7.8 Hz, 3 H),
1.40 (m, 2 H), 1.48 (t, J ) 7.8 Hz, 3 H), 1.55 (t, J ) 7.8 Hz, 3
H), 1.74 (m, 2 H), 2.87 (t, J ) 7.8 Hz, 2 H), 3.27 (q, J ) 7.8 Hz,
2 H), 3.42 (q, J ) 7.8 Hz, 2 H), 4.05 (t, J ) 7.8 Hz, 2 H), 4.20
(s, 3 H), 7.62-7.74 (m, 5 H). MS (CI/NH3): m/z 558 (M++NH4),
525 (M+-1-Me), 414 (M+-I), 369 (M+-1-I-Me-Et).
1-Met h yl-2-et h yl-4-p r op yl-3-(3-flu or op r op ylsu lfa n yl-
ca r bon yl)-5-p r op yloxyca r bon yl-6-p h en ylp yr id in iu m Io-
1
d id e (17). H NMR δ: 0.68 (t, J ) 7.8 Hz, 3 H), 1.04 (t, J )
7.8 Hz, 3 H), 1.41 (m, 2 H), 1.55 (t, J ) 7.8 Hz, 3 H), 1.73 (m,
2 H), 2.16 (m, 2 H), 2.88 (m, 2 H), 3.35 (q, J ) 7.8 Hz, 2 H),
3.41 (t, J ) 6.9 Hz, 2 H), 4.06 (t, J ) 6.9 Hz, 2 H), 4.22 (s, 3
H), 4.55 (t, J ) 6.0 Hz, 1 H), 4.71 (t, J ) 6.0 Hz, 1 H), 7.68-
7.75 (m, 5 H). MS (CI/NH3): m/z 432 (MH+-Me-I).
1-Meth yl-2-eth yl-4-(2-a cetylth ioeth yl)-3-(eth ylsu lfa n yl-
ca r bon yl)-5-p r op yloxyca r bon yl-6-p h en ylp yr id in iu m Io-
d id e (18). 1H NMR δ: 0.66 (t, J ) 7.5 Hz, 3 H), 1.37 (m, 2 H),
1.39 (t, J ) 7.8 Hz, 3 H), 1.45 (t, J ) 7.2 Hz, 3 H), 2.35 (s, 3
H), 2.89-3.02 (m, 4 H), 3.11 (m, 2 H), 3.28 (q, J ) 7.2 Hz, 2
H), 4.00 (t, J ) 6.9 Hz, 2 H), 4.23 (s, 3 H), 7.53-7.55 (m, 3 H),
7.67-7.70 (m, 2 H). MS (CI/NH3): m/z 460 (MH+-Me-I).
1-Meth yl-2-eth yl-4-(2-p h th a lim id oeth yl)-3-(eth ylsu lfa -
n ylcar bon yl)-5-eth yloxycar bon yl-6-ph en ylpyr idin iu m Io-
1
d id e (19). H NMR δ: 0.94 (t, J ) 6.9 Hz, 3 H), 1.34 (t, J )
HPLC results showed that 11 is free of the starting 2. The
mobile phase used for the analysis consisted of methanol,
acetonitrile, and water (45:45:10). At a flow rate of 1.0 mL/
min with a 4.6 × 250 mm (internal diameter) reverse-phase
300 Å C-18 column operated at ambient temperature, 11 had
a retention time of 2.3 min (purity >99%). CHN analysis of
11: Calcd for C22H28INO3S: C, 51.47%; H, 5.50%; N, 2.73%.
Found: C, 51.42%; H, 5.14%; N, 2.43%. HR-MS (FAB, m-b):
Calcd for C22H28NO3S (M+-I): 386.1790. Found: 386.1776. UV
spectra was measured using a Beckman DU 640 spectropho-
7.2 Hz, 3 H), 1.48 (t, J ) 7.2 Hz, 3 H), 2.94 (q, J ) 6.9 Hz, 2
H), 3.15 (t, J ) 7.8 Hz, 2 H), 3.24 (q, J ) 7.2 Hz, 2 H), 4.01 (t,
J ) 7.8 Hz, 2 H), 4.21 (s, 3 H), 4.25 (q, J ) 7.2 Hz, 2 H), 7.51
(m, 3 H), 7.65 (m, 2 H), 7.78 (m, 2 H), 7.89 (m, 2 H). MS (CI/
NH3): m/z 517 (MH+-Me-I).
1-Meth yl-2-bu tyl-4-eth yl-3-(eth ylsu lfa n ylca r bon yl)-5-
eth yloxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (20). 1H
NMR δ: 0.90 (t, J ) 7.5 Hz, 3 H), 1.04 (t, J ) 7.5 Hz, 3 H),
1.29 (t, J ) 7.5 Hz, 3 H), 1.34-1.43 (m, 2 H), 1.46 (t, J ) 7.5
Hz, 3 H), 1.84 (m, 2 H), 2.76 (q, J ) 7.5 Hz, 2 H), 2.88 (t, J )
7.5 Hz, 2 H), 3.16 (q, J ) 7.5 Hz, 2 H), 4.05 (q, J ) 7.5 Hz, 2
H), 4.26 (s, 3 H), 7.52-7.55 (m, 3 H), 7.69-7.72 (m, 2 H). MS
(CI/NH3): m/z 400 (MH+-Me-I).
tometer. In methanol at ambient temperature, 11 had λmax
)
203 nm, ꢀmax ) 6.65 × 104 L mol-1 cm-1; λmax ) 224 nm, ꢀmax
) 3.30 × 104 L mol-1 cm-1; λmax ) 288 nm, ꢀmax ) 9.62 × 103
L mol-1 cm-1
.
1-Meth yl-2-cyclobu tyl-4-eth yl-3-(eth ylsu lfan ylcar bon yl)-
5-eth yloxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (21). 1H
NMR δ: 0.99 (t, J ) 7.5 Hz, 3 H), 1.29 (t, J ) 7.5 Hz, 3 H),
1.45 (t, J ) 7.5 Hz, 3 H), 1.88-1.97 (m, 1 H), 1.97-2.07 (m, 1
H), 2.18-2.32 (m, 2 H), 2.53-2.65 (m, 2 H), 2.71 (q, J ) 7.5
Hz, 2 H), 3.13 (q, J ) 7.5 Hz, 2 H), 3.81 (m, 1 H), 4.01 (q, J )
7.5 Hz, 2 H), 4.23 (s, 3 H), 7.54-7.56 (m, 3 H), 7.73-7.75 (m,
2 H). MS(CI/NH3): m/z 398 (MH+-Me-I).
1-Met h yl-2-(2-b en zyloxylet h yl)-4-p r op yl-3-(et h ylsu lf-
a n ylca r bon yl)-5-p r op yloxyca r bon yl-6-p h en ylp yr id in iu m
Iod id e (22). 1H NMR δ: 0.69 (t, J ) 7.2 Hz, 3 H), 1.02 (t, J )
7.2 Hz, 3 H), 1.44 (t, J ) 7.2 Hz, 3 H), 1.45 (m, 2 H), 1.68 (m,
2 H), 2.71 (m, 2 H), 3.17 (q, J ) 7.2 Hz, 2 H), 3.24 (t, J ) 7.2
Hz, 2 H), 3.99 (t, J ) 7.2 Hz, 2 H), 4.02 (q, J ) 7.2 Hz, 2 H),
4.25 (s, 3 H), 4.58 (s, 2 H), 7.29-7.35 (m, 5 H), 7.53-7.56 (m,
3 H), 7.68-7.71 (m, 2 H). MS (CI/NH3): m/z 648 (MH+), 506
(MH+-Me-I), 445 (MH+-Me-I-SEt).
1-Meth yl-2,4-d ieth yl-3-(eth ylsu lfa n ylca r bon yl)-5-eth yl-
oxyca r bon yl-6-cyclop en tylp yr id in iu m Iod id e (23). 1H
NMR δ: 1.10 (t, J ) 7.5 Hz, 3 H), 1.32 (t, J ) 7.5 Hz, 3 H),
1.41 (t, J ) 7.5 Hz, 3 H), 1.44 (t, J ) 7.5 Hz, 3 H), 1.66 (m, 2
H), 1.95 (m, 7 H), 2.62 (q, J ) 7.5 Hz, 2 H), 2.81 (q, J ) 7.5
Hz, 2 H), 3.97 (q, J ) 7.5 Hz, 2 H), 4.28 (s, 3 H), 4.40 (q, J )
7.5 Hz, 2 H). MS(CI/NH3): m/z 364 (MH+-Me-I).
The water solubility of 11 was measured by the following
method. Deionized water (100 µL) was saturated with 5 mg of
11 with heating. After cooling to room temperature and the
disappearance of turbidity, 50 µL of the clear supernatant was
withdrawn and lyophilized to give 1.1 mg of 11. The water
solubility of 11 was calculated to be 42.8 mM at room
temperature.
1-Meth yl-2-m eth yl-4-eth yl-3-(eth ylsu lfa n ylca r bon yl)-
5-eth yloxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (10). 1H
NMR δ: 0.88 (t, J ) 6.9 Hz, 3 H), 1.26 (t, J ) 7.5 Hz, 3 H),
1.33 (t, J ) 7.5 Hz, 3 H), 2.69 (s, 3 H), 2.79 (q, J ) 7.5 Hz, 2
H), 3.24 (q, J ) 7.5 Hz, 2 H), 4.01 (q, J ) 6.9 Hz, 2 H), 4.17 (s,
3 H), 7.49-7.53 (m, 3 H), 7.65-7.68 (m, 2 H). MS (CI/NH3):
m/z 500 (MH+), 358 (MH+-Me-I), 297 (MH+-Me-I-SEt).
1-Meth yl-2,4-d ieth yl-3-(eth ylsu lfa n ylca r bon yl)-5-eth yl-
oxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (11). 1H NMR
δ: 0.86 (t, J ) 7.2 Hz, 3 H), 1.32 (t, J ) 7.8 Hz, 3 H), 1.41 (t,
J ) 7.8 Hz, 3 H), 1.44 (t, J ) 7.8 Hz, 3 H), 2.84 (q, J ) 7.8 Hz,
2 H), 3.22 (q, J ) 7.8 Hz, 2 H), 3.44 (q, J ) 7.8 Hz, 2 H), 3.98
(q, J ) 7.2 Hz, 2 H), 4.22 (s, 3 H), 7.56-7.62 (m, 3 H), 7.72-
7.75 (m, 2 H). MS (CI): m/z 514 (MH+), 372 (MH+-Me-I).
1-Met h yl-2,4-d iet h yl-3-(et h ylsu lfa n ylca r b on yl)-5-(2-
flu or oeth yloxycar bon yl)-6-ph en ylpyr idin iu m Iodide (14).
1H NMR δ: 1.20 (t, J ) 7.5 Hz, 3 H), 1.39 (t, J ) 7.5 Hz, 3 H),
1.46 (t, J ) 7.5 Hz, 3 H), 2.79 (q, J ) 7.5 Hz, 2 H), 2.99 (q, J
) 7.5 Hz, 2 H), 3.27 (q, J ) 7.5 Hz, 2 H), 4.20 (s, 3 H), 4.28 (m,
P r ep a r a tion of P yr id in iu m Sa lt 12 by Qu a ter n a r y
Am in a t ion of 2,4-Diet h yl-3-(et h ylsu lfa n ylca r b on yl)-5-

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4237
applied to TLC separation [ethyl acetate:petroleum ether )
eth yloxyca r bon yl-6-p h en ylp yr id in e (2) w ith Iod oeth a n e
(Sch em e 1). A mixture of 2 (14 mg, 0.038 mmol) and
iodoethane (59 mg, 0.38 mmol) in 2 mL of anhydrous ni-
tromethane was sealed in a Pyrex tube and was heated at 90
°C for 2 days. After the mixture cooled to room temperature,
the solvent and excess EtI were removed under reduced
pressure to leave a yellow oil. It was applied to TLC separation
[ethyl acetate:petroleum ether ) 1:4 (v/v) for the first develop-
ment; methanol:chloroform ) 1:5 (v/v) for a second develop-
ment and ethyl acetate:petroleum ether ) 1:1 (v/v) for a third
development], and 6 mg of the desired product (12) was
afforded (yield: 31%).
1-Eth yl-2,4-d ieth yl-3-(eth ylsu lfa n ylca r bon yl)-5-eth yl-
oxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (12). 1H NMR
δ: 0.89 (t, J ) 7.5 Hz, 3 H), 1.28 (t, J ) 7.5 Hz, 3 H), 1.39 (t,
J ) 7.5 Hz, 3 H), 1.44 (t, J ) 7.2 Hz, 3 H), 2.79 (q, J ) 7.5 Hz,
2 H), 2.94 (t, J ) 7.5 Hz, 3 H), 3.11 (q, J ) 7.5 Hz, 2 H), 3.34
(q, J ) 7.5 Hz, 2 H), 4.02 (q, J ) 7.2 Hz, 2 H), 5.20 (q, J ) 7.5
Hz, 2 H), 7.52-7.58 (m, 3 H), 7.68-7.71 (m, 2 H). MS (CI/
NH3): m/z 372 (MH+-Et-I).
P r ep a r a tion of P yr id in iu m Sa lt 13 by Qu a ter n a r y
Am in a t ion of 2,4-Diet h yl-3-(et h ylsu lfa n ylca r b on yl)-5-
eth yloxyca r bon yl-6-p h en ylp yr id in e (2) w ith 1-Iod op r o-
p a n e (Sch em e 1). A mixture of 2 (14 mg, 0.038 mmol) and
1-iodopropane (65 mg, 0.38 mmol) in 2 mL of anhydrous
nitromethane was sealed in a Pyrex tube and was heated at
100 °C for 2 days. After the mixture cooled to room temper-
ature, the solvent and excess PrI were removed under reduced
pressure to leave a yellow oil. It was applied to TLC separation
[ethyl acetate:petroleum ether ) 1:4 v/v for the first develop-
ment then 1:1 for a second development], and 5 mg of the
desired product (13) was afforded (yield: 25%).
1-P r op yl-2,4-d ieth yl-3-(eth ylsu lfa n ylca r bon yl)-5-eth yl-
oxyca r bon yl-6-p h en ylp yr id in iu m Iod id e (13). 1H NMR
δ: 0.91 (m, J ) 7.2 Hz, 6 H), 1.29 (t, J ) 7.5 Hz, 3 H), 1.39 (t,
J ) 7.5 Hz, 3 H), 1.43 (t, J ) 7.2 Hz, 3 H), 2.44 (t, J ) 7.2 Hz,
3 H), 2.79 (q, J ) 7.2 Hz, 2 H), 3.07 (q, J ) 7.5 Hz, 2 H), 3.38
(q, J ) 7.2 Hz, 2 H), 4.01 (q, J ) 7.2 Hz, 2 H), 4.29 (q, J ) 7.2
Hz, 2 H), 5.22 (q, J ) 7.2 Hz, 2 H), 7.51-7.54 (m, 3 H), 7.68-
7.71 (m, 2 H). MS (CI/NH3): m/z 372 (MH+-Pr-I).
1:4 v/v for the first development then 1:1 for
a second
development], and 2 mg of a yellow solid was obtained (yield:
30%), with 1H NMR and MS data consistent with those of
compound 11.
Oxid a tion of a 1-Meth yl-1,4-d ih yd r op yr id in e Der iva -
tive (24) in th e P r esen ce of Ra t Br a in Hom ogen a te.27,28
The rat brain homogenate was prepared by the following
method. One rat (1 kg) was killed, and the brain (weighing
2.26 g) was removed and homogenized in 12 mL of PBS
(Biofluids, Inc., 1× pH 7.4). The homogenate was centrifuged
at 12000g rpm, and the supernatant was used. Then 5 mg of
24 dissolved in 0.2 mL of DMSO was mixed with 10 mL of
brain homogenate (initial concentration of 1.27 mM), which
was previously equilibrated to 37 °C in a water bath incubator,
and shaking was continued at that temperature. Aliquots of
500 µL were withdrawn at 2, 4, 8, 16, 32, 64, 128, 256, and
768 min (2× each) from the test medium, added immediately
to 3 mL of ice-cold ethyl ether, shaken vigorously, and placed
in a freezer. When all samples had been collected, the ether
layer of each sample was separated. After evaporation of the
ether, each residue was dissolved in 200 µL of methanol,
filtered through Whatman No. 1 filter paper, and analyzed by
HPLC.
HP LC Meth od for th e Stu d y of Oxid a tion of a 1-Meth -
yl-1,4-d ih yd r op yr id in e Der iva tive (24) in th e P r esen ce
of Ra t Br a in Hom ogen a te. The chromatographic analysis
was performed on a Hewlett-Packard Series 1100 HPLC
system consisting of a G1311A QuatPump, G1315A DAD,
G1322A Degasser, G1328A Manlnj, and HP Kayak PC work-
stations operated at HP ChemStation. A 4.6 × 250 mm
(internal diameter) reverse-phase 300 Å C-18 column (What-
man, Inc.), operated at ambient temperature, was used for all
analyses. The mobile phase used for the analysis consisted of
methanol, acetonitrile, and water (45:45:10). At a flow rate of
1.0 mL/min, 24 had a retention time of 4.4 min, while its
oxidation product had a retention time of 2.3 min. The mean
value (in percentage of total) of two samples at each time point
was calculated and plotted against time of incubation.
P h a r m a cology. Ad en yla te Cycla se Assa y. Adenylate
cyclase assays were performed with membranes prepared from
Chinese hamster ovary (CHO) cells stably expressing either
the human A1 receptor or human A3 receptor by the method
of Salomon et al.,29 as described previously with the following
modifications.14 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidi-
none (Ro 20-1724, 20 µM, Calbiochem, San Diego, CA) was
employed to inhibit phosphodiesterases rather than papaver-
ine, and the NaCl concentration in the assay was 25 mM.
Membranes were pretreated with 2 units/mL adenosine deam-
inase, and the antagonist 11 (10 µM) at 30 °C for 5 min prior
to initiation of the adenylate cyclase assay. Adenylate cyclase
was stimulated with forskolin (1 µM). Concentration-response
data for the inhibition of adenylate cyclase activity by IB-
MECA (human A3 receptor) were obtained. Maximal inhibition
of adenylate cyclase by IB-MECA at the human A3 receptor
correlated to ∼60% of total stimulation, respectively. IC50
values were calculated using InPlot (GraphPad, San Diego,
CA). KB values were calculated as described.30
R a d ioliga n d Bin d in g St u d ies. Binding of [3H]R-N6-
phenylisopropyladenosine ([3H]R-PIA) to A1 receptors from rat
cerebral cortex membranes and of [3H]-2-[4-[(2-carboxyethyl)-
phenyl]ethylamino]-5′-N-ethylcarbamoyladenosine ([3H]CGS
21680) to A2A receptors from rat striatal membranes was
performed as described previously.22,23 Adenosine deaminase
(3 units/mL) was present during the preparation of the brain
membranes, in a preincubation of 30 min at 30 °C, and during
the incubation with the radioligands. Nonspecific binding was
determined in the presence of 10 µM (A1 receptors) or 20 µM
(A2A receptors) 2-chloroadenosine.
Gen er a l P r oced u r e for P r ep a r a tion of 1-Meth yl Di-
h yd r op yr id in es (24 a n d 25) by Alk yla tion of 3,5-Dia cyl-
2,4-d ia lk yl-1,4-d ih yd r op yr id in e Der iva t ives w it h Io-
d om eth a n e (Sch em e 1). A solution of the appropriate DHP
(2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-
phenyl-1,4-dihydropyridine, 15 mg, 0.04 mmol) in 2 mL of
anhydrous THF was treated with NaH (60%, 2 mg, 0.08 mmol)
at room temperature under stirring for 5 min. Then io-
domethane (28 mg, 0.2 mmol) was added, and the reaction
mixture was stirred for another 10 min (monitor by TLC). At
completion the reaction mixture was applied to TLC separation
[ethyl acetate:petroleum ether ) 1:19 v/v], and 11 mg of 24
was obtained (yield: 71%).
1-Meth yl-2,4-d ieth yl-3-(eth ylsu lfa n ylca r bon yl)-5-eth yl-
oxyca r bon yl-6-p h en yl-1,4-d ih yd r op yr id in e (24). 1H NMR
δ: 0.85 (t, J ) 7.2 Hz, 3 H), 0.93 (t, J ) 7.2 Hz, 3 H), 1.20 (t,
J ) 7.2 Hz, 3 H), 1.29 (t, J ) 7.2 Hz, 3 H), 2.74 (q, J ) 7.2 Hz,
2 H), 2.85 (s, 3 H), 2.92 (q, J ) 7.2 Hz, 2 H), 3.07 (q, J ) 7.2
Hz, 2 H), 3.87 (q, J ) 7.2 Hz, 2 H), 4.02 (t, J ) 7.2 Hz, 1 H),
7.20 (m, 2 H), 7.38-7.41 (m, 3 H). MS (CI/NH3): m/z 388
(MH+).
1-Meth yl-2,4-d ieth yl-3-(eth ylsu lfa n ylca r bon yl)-5-eth yl-
oxyca r bon yl-6-cyclop en tyl-1,4-d ih yd r op yr id in e (25). 1H
NMR δ: 0.78 (t, J ) 7.8 Hz, 3 H), 1.11 (t, J ) 7.8 Hz, 3 H),
1.28 (t, J ) 7.8 Hz, 3 H), 1.32 (t, J ) 7.8 Hz, 3 H), 1.46 (m,
2H), 1.60-1.78 (m, 7H), 2.10 (m, 2 H), 2.60-2.67 (m, 1 H),
2.89 (q, J ) 7.8 Hz, 2 H), 3.01-3.13 (m, 1 H), 3.15 (s, 3 H),
4.10 (t, J ) 6.0 Hz, 1 H), 4.13-4.26 (m, 2 H). MS (CI/NH3):
m/z 380 (MH+), 318 (M+-SEt, base).
Ch em ica l Tr a n sfor m a tion of 24 to 11 th r ou gh Oxid a -
tion w ith Iod in e (Sch em e 1). A solution of 24 (5 mg, 0.013
mmol) in 0.5 mL of dry nitromethane was treated with iodine
(10 mg, 0.040 mmol) at room temperature with stirring for 1
day (monitor by TLC). At completion the reaction mixture was
Binding of [125I]N6-(4-amino-3-iodobenzyl)-5′-N-methylcar-
bamoyladenosine ([125I]AB-MECA)24 to membranes prepared
from human embryonic kidney (HEK-293) cells stably express-
ing the human A3 receptor16 or clone HS-21a (Receptor Biology,
Inc., Beltsville, MD) or to membranes prepared from Chinese

4238 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Xie et al.
(13) J iang, J .-l.; van Rhee, A. M.; Chang, L.; Patchornik, A.; Evans,
P.; Melman, N.; J acobson, K. A. Structure activity relationships
of 4-phenylethynyl-6-phenyl-1,4-dihydropyridines as highly se-
lective A3 adenosine receptor antagonists. J . Med. Chem. 1997,
40, 2596-2608.
(14) J acobson, K. A.; Park, K. S.; J iang, J .-l.; Kim, Y.-C.; Olah, M.
E.; Stiles, G. L.; J i, X. d. Pharmacological characterization of
novel A3 adenosine receptor-selective antagonists. Neurophar-
macology 1997, 36, 1157-1165.
(15) Li, A.-H.; Moro, S.; Melman, N.; J i, X.-d.; J acobson, K. A.
Structure activity relationships and molecular modeling of 3,5-
diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine
receptor antagonists. J . Med. Chem. 1998, 41, 3186-3201.
(16) Salvatore, C. A.; J acobson, M. A.; Taylor, H. E.; Linden, J .;
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hamster ovary (CHO) cells stably expressing the rat A3
receptor8 was performed as described at 4 °C.24 The assay
medium consisted of a buffer containing 10 mM Mg2+, 50 mM
Tris, 3 units/mL adenosine deaminase, and 1 mM EDTA, at
pH 8.0 (4 °C). The glass incubation tubes contained 100 µL of
the membrane suspension (0.3 mg protein/mL, stored at -80
°C in the same buffer), 50 µL of [125I]AB-MECA (final concen-
tration 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-phenylisopropyladenosine (NECA).
All nonradioactive compounds were initially dissolved in
DMSO and diluted with buffer to the final concentration,
where the amount of DMSO never exceeded 1%.
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 equation31 and Kd values
of 1.0 nM ([3H]R-PIA); 15.5 nM ([3H]CGS 21680); 0.59 and 1.46
nM ([125I]AB-MECA at human and rat A3 receptors, respec-
tively).
(18) Li, A.-H.; Moro, S.; Forsyth, N.; Melman, N.; J i, X.-d.; J acobson,
K. A. Synthesis, CoMFA analysis, and receptor docking of 3,5-
diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine
receptor antagonists. J . Med. Chem. 1999, 42, 706-721.
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1239-1248.
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Novel selective non-xanthine selective A3 adenosine receptor
antagonists. Drug Dev. Res. 1996, 37, 131.
Ack n ow led gm en t. We thank Kristie Calvert for
technical assistance.
(21) Van Muijlwijk-Koezen, J . E.; Timmerman, H.; Link, R.; Van der
Goot, H.; IJ zerman, A. P. A novel class of adenosine A3 receptor
ligands. II. Structure affinity profile of a series of isoquinoline
and quinazolinecompounds. J . Med. Chem. 1998, 41, 3994-4000.
(22) Schwabe, U.; Trost, T. Characterization of adenosine receptors
in rat brain by (-) [3H]N6-phenylisopropyladenosine. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1980, 313, 179-187.
(23) J arvis, M. F.; Schutz, R.; Hutchison, A. J .; Do, E.; Sills, M. A.;
Williams, M. [3H]CGS 21680, an A2 selective adenosine receptor
agonist directly labels A2 receptors in rat brain tissue. J .
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