Synthesis and biological evaluation of novel N6-[4-(substituted) sulfonamidophenylcarbamoyl]adenosine-5′-uronamides as A3 adenosine receptor agonists

Article abstract of DOI:10.1021/jm0408161

A new series of 1-deoxy-1-[(6-(4-(substituted-aminosulfonyl)phenyl)amino) carbonylamino-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamides (83-102) have been synthesized and tested at the human A₃ adenosine receptor subtype. All the derivatives des

Full text of DOI:10.1021/jm0408161

J . Med. Chem. 2004, 47, 5535-5540
5535
Syn th esis a n d Biologica l Eva lu a tion of Novel
N6-[4-(Su bstitu ted )su lfon a m id op h en ylca r ba m oyl]a d en osin e-5′-u r on a m id es a s A3
Ad en osin e Recep tor Agon ists
Pier Giovanni Baraldi,*† Francesca Fruttarolo,† Mojgan Aghazadeh Tabrizi,† Romeo Romagnoli,† Delia Preti,†
Andrea Bovero,† Maria J ose` Pineda de Las Infantas,‡ Allan Moorman,§ Katia Varani,| and Pier Andrea Borea|
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, 44100 Ferrara, Italy, Departamento de Quimica
Organica y Farmaceutica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/ n,
18071 Granada, Spain, King Pharmaceutical, 7001 Weston Parkway Suite 300, Cary, North Carolina 27513, and
Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universita` di Ferrara, 4100 Ferrara, Italy
Received March 24, 2004
A new series of 1-deoxy-1-[(6-(4-(substituted-aminosulfonyl)phenyl)amino)carbonylamino-9H-
purin-9-yl]-N-ethyl-â-D-ribofuranuronamides (83-102) have been synthesized and tested at
the human A3 adenosine receptor subtype. All the derivatives described in this work displayed
affinity versus this receptor in the nanomolar range and good selectivity versus A1 adenosine
receptor subtype, confirming that the p-sulfonamido moiety positively affected the activity of
the molecules. The best substituents at the sulfonamido nucleus were found to be small alkyl
groups, like methyl, isopropyl, ethyl, or allyl moieties (compounds 96-100), whereas mono-
substitution at the amino group led to a decrease in A3 affinity values. The selectivity versus
A1 adenosine receptor subtype is increased when the amino group in the sulfonamido core is
represented by a hydrogenated heterocyclic ring like piperidine, morpholine, or pyrroline. Bulky
groups, like adamantane and alkyl chains with more than four carbon atoms, are detrimental
for the affinity and the selectivity of the A3 adenosine receptor agonists described here.
In tr od u ction
to the discovery of N6-(3-iodo-benzyl)adenosine-5′-N-
methyluronamide (IB-MECA) as the first potent and
selective agonist for rat A3 adenosine receptors. IB-
MECA is 50-fold selective for A3 versus either A1 or A2A
receptors. Starting from this observation, a selective
radioligand, the [125I]-N6-(4-amino-3-iodobenzyl)adenos-
ine-5′-N-methyluronamide ([125I]-AB-MECA), was de-
veloped.12 This compound, endowed with high affinity
but low selectivity, is still considered the standard
agonist for the A3 adenosine receptor. In an evolution
of this work, the effect of substitution at the C-2 position
was studied.13 It was observed that substitutions at the
2-position with halogen, methylamino, or thiomethyl
groups increase both affinity and selectivity at the A3
receptor subtype. Other studies have demonstrated that
substituents at the 3-position on the benzyl group are
well tolerated. These studies suggest that both affinity
and selectivity for the A3 receptor subtype appears to
be related more to the type of substitution on the phenyl
ring than to the position of the substituent. A large
series of purine and ribose-modified adenosine ana-
logues have been studied for their affinity at rat A3
adenosine receptors, but none of these compounds shows
a better profile with respect to the reference compounds.
Several modifications utilized14 have shown that (i)
deaza derivatives are well tolerated at the A3 receptor;
(ii) substitutions at the 8-position are detrimental, in
terms of affinity; (iii) carbocyclic nucleosides exhibit, in
general, weak affinity at all receptor subtypes; (iv)
replacement of the 6-NHCH2 linkage with hydroxy-
lamino or hydrazino moieties is well tolerated; and (v)
substitution of the 4′-hydrogen with a methyl group
retains agonist activity and selectivity at rat A3 adenos-
Adenosine exerts a number of physiological functions
through activation of four cell membrane receptors
classified as A1, A2A, A2B, and A3.1 Although all adenos-
ine subclasses belong to the G protein-coupled receptors,
they are associated with different second messenger
systems.1 The A3 receptor has been cloned from a rat
brain cDNA library,2 and this subtype has a character-
istic second messenger profile, in that it has been shown
to mediate adenylyl cyclase inhibition and phospholi-
pase C activation. Several studies indicate that the
adenosine A3 receptor may play a basic role in the
modulation of cerebral ischemia,3 inflammation,4 hy-
potension,5 ischemic heart preconditioning,6 and asthma.7
These and other findings make the A3 receptor a
promising therapeutic target for regulation of cell
growth,7 apoptosis, and study in leukemic J urkat T
cells,8 the human malignant melanoma A375 cell line,
and human neutrophils.9
The cloned human A3 adenosine receptor was first
characterized with N6-(4-amino-3-[125I]iodobenzyl)ad-
enosine.10 In a preliminary study, it was observed that
the presence of the N6-benzyl group in the adenosine
structure determines a significant increase in A3 recep-
tor affinity and selectivity.11 Additionally, it was shown
that a methyl uronamide moiety at the 5′-position
confers superior affinity and selectivity at the A3 ad-
enosine receptor. This combination of substitutions led
* To whom correspondence should be addressed. Tel: +39-0532-
291293. Fax: +39-0532-291296. E-mail: pgb@dns.unife.it.
† Universita` di Ferrara.
‡ Universidad de Granada.
§ King Pharmaceutical.
| Universita` di Ferrara.
10.1021/jm0408161 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/17/2004

5536 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Baraldi et al.
Ta ble 1. New A3 Adenosine Receptor Agonists Synthesized
ine receptors. Recently, our group has proposed that the
N6-benzylamino chain can be successfully replaced by
n urea moiety.15,16 While N6-alkylcarbamoyl derivatives
have been reported to exhibit low affinity and selectivity
at all adenosine receptor subtypes, interesting results
can be obtained with the N6-arylcarbamoyl substituted
analogues. Among this new series of compounds, the
1-deoxy-1-[6-[[[4-sulfonamido-phenyl)-amino]carbonyl]-
amino]-9H-purin-9-yl]-N-ethyl-â-D-ribofuranurona-
mide (compound A)16 showed an interesting increase in
its selectivity versus A1 receptor [Ki (rA1) ) 453 nM; Ki
(rA3) ) 9.73 nM], whereas substitution of the amino
group of the sulfonamido radical with some heteroaryl
groups, like pyridine, pyrimidine, or isoxazole, led to
derivatives with modest affinity at rat A3 adenosine
receptor subtype.16
(83-102)
Starting from these observations, our group designed
an enlarged series of substituted-p-sulfonamido phe-
nylcarbamoyl adenosine analogues with a conserved
ethyl uronamide group at the 5′-position as A3 adenosine
receptor agonists. The new adenosine agonists were
prepared (compounds 83-102) as sulfonamido deriva-
tives substituted with aliphatic groups (cyclic or linear)
or aromatic radicals on the sulfonamido nitrogen in
order to better evaluate the importance of this position
and the structure-activity relationships of A3 adenosine
receptor agonists. The structures of these derivatives
are indicated in Table 1. These compounds, including
the parent compound A, have been pharmacologically
characterized, determining their binding affinity and
selectivity at CHO cells transfected with human A1, A2A
,
and A3 adenosine receptors using agonists as radioli-
gands that preferentially recognize the high affinity
state in order to better evaluate the pharmacological
characteristics of the compounds analyzed.
anhydrous dioxane as solvent at reflux afforded the
adducts 63-82 in a quite good yield. These intermedi-
ates were converted into the final desired compounds
83-102 by deprotection of the ribosyl moiety in aqueous
1 N HCl and dioxane at 65 °C.
Ch em istr y
The preparation of the N-substituted-sulfonamidophe-
nyl derivatives of NECA, compounds 83-102, was
performed following the general synthetic strategy
depicted in Scheme 1. The amino group at the 6-position
of NECA is not very reactive; therefore, it was necessary
to protect the hydroxyl groups of the ribose moiety
during the nucleophilic reaction with the appropriate
isocyanate. This was achieved by protection of the 2′-
and 3′-hydroxyl groups as the isopropylidene derivative
(1a ). The isocyanates were prepared by reacting the
corresponding substituted anilines (23-42) using trichlo-
romethylchloroformate as previously described17 and
reported in Scheme 2. Reaction of the 2′,3′-O-isopropy-
lidene-protected NECA 1a with the isocyanates in
Resu lts a n d Discu ssion
All of the synthesized compounds were evaluated in
radioligand binding assays for affinity at the human A1,
A2A, and A3 adenosine receptors. The results are sum-
marized in Table 2. The NECA derivative N-6 substi-
tuted with a simple phenylsulfonamido function (com-
pound A, see Table 2) exhibits good affinity for the hA3
adenosine receptor subtype but, differently from the
data reported from the rat receptors, low selectivity
(hA1/hA3 ) 2.03 and hA2A/hA3 ) 6.43). The new syn-
thesized compounds reported in this work are all
functionalized at the 6-position of the purine moiety
with a mono- or bis-substituted sulfonamidopheny-

Novel Uronamides as A3 Adenosine Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5537
Sch em e 1a
Ta ble 2. Affinities of Synthesized Derivatives (83-102) in
Radioligand Binding Assays at Human A1, A2A, and A3
Adenosine Receptors
b
Ki (A2A
)
or
compound
IB-MECA
Ki (A1)a
% inhibition
Ki (A3)c
A1/A3
54 (49-59)
56 (48-64)
1.1 (0.8-1.4)
0.33 (0.3-0.4)
49
2500
Cl-IB-MECA 823 (778-870) 448 (365-550)
NECA
A
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
18.2 (14.2-23.4) 12.2 (8.4-17.7)
30.1 (22.9-39.6) 95.2 (86.4-105.0) 14.8 (10.2-21.5) 2.03
34.8 (26.7-45.3) 0.52
750 (664-849) >1000 (15%)
725 (620-849) >1000 (20%)
250 (179-348) >1000 (34%)
300 (226-398) >1000 (30%)
445 (369-537) >1000 (32%)
280 (192-409) >1000 (34%)
290 (272-309) >1000 (31%)
700 (614-799) >1000 (23%)
730 (602-886) >1000 (27%)
250 (166-374) >1000 (32%)
240 (193-297) >1000 (31%)
350 (333-434) >1000 (25%)
415 (304-568) >1000 (19%)
17 (13-22)
20 (12-34)
23 (17-32)
24 (18-33)
80 (66-97)
23 (15-34)
25 (17-36)
22 (17-29)
23 (17-32)
20 (11-36)
35 (28-43)
23 (18-28)
24 (16-36)
10 (6-16)
8 (6-11)
44
36.25
10.86
12.5
5.56
12.17
11.6
31.8
31.74
12.5
7.5
15.22
17.3
20.6
40.6
23.33
38
206(179-238)
>1000 (10%)
325 (282-374) >1000 (13%)
280 (230-342) >1000 (21%)
380 (321-451) >1000 (15%)
350 (307-398) >1000 (28%)
250 (171-364) >1000 (17%)
350 (288-426) >1000 (15%)
98
99
100
101
102
12 (10-16)
10 (5-17)
9 (4-18)
38.89
13.16
25
19 (14-26)
14 (10-21)
a
Displacement of [3H]CHA binding to hA1 adenosine receptors
b
expressed in CHO cells. Ki values in nM. Displacement of
[3H]CGS21680 binding to hA2A adenosine receptors expressed in
CHO cells. Ki values in nM. c Displacement of [125I]-AB-MECA
binding to hA3 adenosine receptors expressed in CHO cells. Ki
values in nM.
heterocyclic rings like morpholine, piperidine, or pyr-
roline. All of the newly synthesized compounds showed
affinity at the human A3 adenosine receptor in the
nanomolar range and high selectivity versus the hA1
adenosine receptor subtype demonstrating that the
sulfonamido functions positively influence the affinity
and substitutions at the sulfonamido moiety increase
the selectivity versus the A1 adenosine receptor subtype.
From an analysis of the data, it seems that the presence
of an N-hydrogen on the sulfonamido moiety is quite
detrimental for the A3 affinity. When the monosubsti-
tution at the sulfonamido moiety is a carbon chain
where the number of carbon atoms is more than four,
affinity toward the A3 adenosine receptor subtype is
diminished (compound 87). In general, disubstitution
of the amino group provides better values in terms of
A3 interaction. The best results in terms of A3 affinity
are achieved when the two substituents on the sulfona-
mido nitrogen moiety are short alkyl chains (compounds
96-100) like methyl, allyl, ethyl, and isopropyl groups.
Selectivity versus the A1 adenosine receptor subtype is
increased when the amino group of the sulfonamido
moiety is substituted by a reduced heterocycle like
piperidine, morpholine, or pyrroline (compounds 83, 84,
90) or when halogen atoms are present on the alkyl
chains (compound 91). The presence of a basic nitrogen
at the sulfonamido group seems to be very important
for the A3 interaction. Compounds 83 and 90, which
respectively hold radicals like the piperidine and pyr-
roline moieties, showed a better affinity than the
corresponding N-monosubstituted compounds 94 and
95. The introduction of a sterically bulky substituent,
such as the adamantyl group (compound 93), proved
detrimental for the interaction with the A3 adenosine
receptor binding site.
a
Reagents: (i) dioxane, reflux, 18 h; (ii) 1 N hydrochloric acid,
65 °C.
Sch em e 2a
a
Reagents: (i) HSO3Cl, 100 °C; (ii) substituted amines,
dioxane; (iii) aqueous 20% HCl; (iv) Cl3COCOCl, dioxane.17
lamino function. The substituents introduced are aro-
matic functions, small alkyl chains like methyl, ethyl,
allyl, or isopropyl, cycloalkyl functions, or hydrogenated

5538 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Baraldi et al.
Ta ble 3. Functional Assay. Inhibitory Effect of Selected
Agonists on cAMP Production Stimulated by Forskolin 10 µM
in hA3 CHO Cells
(0.572 mmol) and a catalytic amount of TEA (two drops) were
added. The mixture was refluxed under argon for 18 h. Next
the solvent was removed under reduced pressure, and the
residue was purified by flash chromatography (EtOAc/light
petroleum 8:2) to afford the desired compounds 63-82.
1-Deoxy-1-[6-[[[(4-(p ip er id in -1-ylsu lfon yl)p h en yl)a m i-
n o]ca r bon yl]a m in o]-9H-p u r in -9-yl]-N-eth yl-2,3-O-(1-m e-
th yleth ylid en e)-â-D-r ibofu r a n u r on a m id e (63): yield 46%;
compounds
IC50 (nM)
Cl-IB-MECA
A
96
97
99
100
2.2 (1.9-2.6)
81 (69-97)
42 (33-52)
55 (46-65)
65 (56-74)
50 (42-61)
1
white solid; mp 195-197 °C; H NMR (CD3OD) δ 0.62 (t, 3H,
J ) 7.2), 1.21 (s, 3H), 1.32 (s, 3H), 1.42 (m, 5H), 1.59 (m, 8H),
1.68 (s, 1H), 2.79-2.81 (m, 2H), 5.55 (m, 1H), 5.68 (d, 1H),
6.45 (s, 1H), 7.71-7.74 (d, 2H, J ) 9), 7.86-7.89 (d, 2H, J )
9), 10.52 (bs, 1H), 12.10 (bs, 1H).
The ability of some of the compounds examined to
inhibit forskolin-stimulated 10 µM cAMP production via
the adenosine A3 receptor was also studied. In particu-
lar, a functional assay based on the evaluation of cyclic
AMP levels in hCHOA3 cells indicated that some of the
new ligands were true A3 adenosine agonists and were
able to inhibit cyclic AMP accumulation showing IC50
values in the nanomolar range (Table 3). The com-
pounds tested showed almost partial inhibition of the
cAMP production comparable with the reference full
agonist Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-5′-
methylcarboxamidoadenosine). All the adenosine de-
rivatives showed IC50 values in the range 42-80 nM
and a maximal inhibition in the range 55-65% at the
concentration 1 µM. Under the same conditions, 1 µM
Cl-IB-MECA caused an inhibition of cyclic AMP levels
of 88%.
1-Deoxy-1-[6-[[[(4-(m or p h olin -1-yl-su lfon yl)p h en yl)-
a m in o]ca r bon yl]a m in o]-9H-p u r in -9-yl]-N-eth yl-2,3-O-(1-
m eth yleth ylid en e)-â-D-r ibofu r a n u r on a m id e (64): yield
1
49%; white solid; mp 211 °C; H NMR (CD3OD) δ 0.58-0.63
(t, 3H, J ) 7.3), 1.42 (s, 3H), 1.59 (s, 3H), 2.80 (m, 3H), 2.95-
2.98 (m, 4H, J ) 5), 3.70-3.72 (m, 4H, J ) 4), 4.68 (s, 1H),
5.53 (m, 1H), 5.68 (d, 1H, J ) 4), 6.48 (s, 1H), 7.78 (m, 2H),
7.91 (m, 2H), 8.46 (s, 1H), 8.65 (s, 1H), 10.52 (bs, 1H), 12.10
(bs, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-Deoxy-1-
[6-[[[(4-(su b st it u t ed -a m in osu lfon yl)p h en yl)a m in o]ca r -
bon yl]am in o]-9H-pu r in -9-yl]-N-eth yl-â-D-r ibofu r an u r on a-
m id e 83-102. A solution of the isopropylidene derivatives 63-
82 (0.09 mmol) in aqueous 1 N HCl (5 mL) and dioxane (5
mL) was stirred at 65 °C for 1 h. The solvent was then removed
at reduced pressure, and the residue was purified by crystal-
lization from ethanol to afford the desired compounds 83-102
as solids.
1-Deoxy-1-[6-[[[(4-(p ip er id in -1-ylsu lfon yl)p h en yl)a m i-
n o]ca r bon yl]a m in o]-9H-p u r in -9-yl]-N-eth yl-â-D-r ibofu r a -
n u r on a m id e (83): yield 98%; white solid; mp 176-178 °C;
1H NMR (DMSO-d6) d 1.07 (t, 3H, J ) 7), 1.35-1.37 (m, 2H),
1.48-1.52 (m, 4H), 2.85.2.88 (m, 4H), 3.16-3.26 (m, 2H), 4.23
(bs, 1H), 4.36 (s, 1H), 4.66-4.69 (m, 1H), 5.65-5.79 (m, 2H),
6.10 (m, 1H), 7.70 (d, 2H, J ) 8), 7.88 (d, 2H, J ) 8), 8.45 (m,
1H), 8.49 (s, 1H), 8.73 (s, 1H), 10.50 (bs, 1H), 12.05 (bs, 1H).
Anal. (C24H30N8O7S) C, H, N.
1-Deoxy-1-[6-[[[(4-(m or ph olin -1-ylsu lfon yl)p h en yl)a m i-
n o]ca r bon yl]a m in o]-9H-p u r in -9-yl]-N-eth yl-â-D-r ibofu r a -
n u r on a m id e (84): yield 97%; white solid; mp 204-205 °C;
1H NMR (DMSO-d6) d 1.06 (t, 3H, J ) 7); 2.84-2.86 (m, 4H);
3.16-3.26 (m, 2H); 3.61-3.64 (m, 4H); 4.23 (bs, 1H); 4.36 (s,
1H); 4.66-4.69 (m, 1H); 5.65-5.79 (m, 2H); 6.09 (d, 1H, J )
7); 7.76 (d, 2H, J ) 8); 7.88 (d, 2H, J ) 8); 8.46-8.47 (m, 1H);
8.74 (s, 1H); 8.81 (s, 1H); 10.55 (bs, 1H); 12.1 (bs, 1H). Anal.
(C23H28N8O8S) C, H, N.
Con clu sion
In the present study we have described the affinity
at human A1, A2A, and A3 adenosine receptor subtypes
of a new series of N6-[4-(substituted)sulfonamidophe-
nylcarbamoyl]adenosine-5′-uronamides. All these mol-
ecules displayed high affinity versus the hA3 receptor
in the nanomolar range and an increase of selectivity
versus the hA1 subtype with respect to the parent
compound A. Both affinity and selectivity were in-
creased when the substituents on the sulfonamido
moiety were represented by small alkyl groups or by
hydrogenated heterocyclic rings.
Exp er im en ta l Section
Biology Exp er im en ts. All synthesized compounds have
been tested for their affinity at human A1, A2A, and A3
adenosine receptor subtypes cloned in CHO cells. Some of these
compounds have been studied to evaluate their potency versus
the hA3 CHO cells.
Ch em istr y. Reaction courses and product mixtures were
routinely monitored by thin-layer chromatography (TLC) on
silica gel (precoated F254 Merck plates) and visualized with
aqueous potassium permanganate or ethanolic ninhydrin
solutions. Infrared spectra (IR) were measured on a Perkin-
1
Elmer 257 instrument. H NMR were determined in CD3OD
Hu m a n Clon ed A1, A2A, a n d A3 Ad en osin e Recep tor
Bin d in g Assa y. The expression of the human A1, A2A, and A3
receptors in CHO cells has been previously described.18 The
cells were grown adherently and mantained in Dulbecco’s
modified Eagles medium with nutrient mixture F12 (DMEM/
F12) without nucleosides, containing 10% fetal calf serum,
penicillin (100 units/mL), streptomycin (100 µg/mL), L-
glutamine (2 mM), and Geneticin (G418, 0.2 mg/mL) at 37 °C
in 5% CO2/95% air. Cells were split 2 or 3 times weekly at a
ratio between 1:5 and 1:20. For membrane preparation the
culture medium was removed and the cells were washed with
PBS and scraped off T75 flasks in ice-cold hypotonic buffer (5
mM Tris HCl, 2 mM EDTA, pH 7.4). The cell suspension was
homogenized with a Polytron, and the homogenate was spun
for 10 min at 1000g. The supernatant was then centrifuged
for 30 min at 100000g. The membrane pellet was resuspended
in 50 mM Tris HCl buffer pH 7.4 (for A3 adenosine receptors:
50 mM Tris HCl, 10 mM MgCl2, 1 mM EDTA) and incubated
with 3 IU/mL of adenosine deaminase for 30 min at 37 °C.
Then the suspension was frozen at -80 °C. HEK 293 cells
or DMSO-d6 solutions with a Bruker AC 200 spectrometer.
Peak positions are given in parts per million (δ) downfield from
tetramethylsilane as internal standard, and J values are given
in hertz. Light petroleum refers to the fractions boiling at 40-
60 °C. Melting points were determined on a Buchi-Tottoli
instrument and are uncorrected. Chromatography was per-
formed with Merck 60-200 mesh silica gel. All products
reported showed 1H NMR spectra in agreement with the
assigned structures. Organic solutions were dried over anhy-
drous sodium sulfate. Elemental analyses were performed by
the microanalytical laboratory of Dipartimento di Chimica,
University of Ferrara, and were within (0.4% of the theoreti-
cal values for C, H, and N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-Deoxy-1-
[6-[[[(4-(su b st it u t ed -a m in osu lfon yl)p h en yl)a m in o]ca r -
bon yl]a m in o]-9H-p u r in -9-yl]-N-eth yl-2,3-O-(1-m eth yleth -
ylid en e)-â-D-r ib ofu r a n u r on a m id e 63-82. 2′,3′-O-Isopro-
pylidene-NECA 1a (0.286 mmol) was dissolved in freshly
distilled dioxane (5 mL), and then the appropriate isocyanate

Novel Uronamides as A3 Adenosine Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5539
transfected with the human recombinant A2A adenosine recep-
tor were obtained from Receptor Biology, Inc. (Beltsville, MD).
Binding of [3H]CHA to CHO cells transfected with the human
recombinant A1 adenosine receptor was performed according
to the method previously described by Klotz et al.19 Displace-
ment experiments were performed for 120 min at 25 °C in 0.2
mL of 50 mM Tris HCl buffer pH 7.4 containing 1 nM [3H]-
CHA, diluted membranes (50 µg of protein/assay), and at least
6-8 different concentrations of agonists studied. Nonspecific
binding was determined in the presence of 10 µM CHA, and
this was always e10% of the total binding. Binding of [3H]-
CGS21680 to CHO cells transfected with the human recom-
binant A2A adenosine receptors (50 µg of protein/assay) was
performed using 0.2 mL of 50 mM Tris HCl buffer, 10 mM
MgCl2 pH 7.4, and at least 6-8 different concentrations of
agonists studied for an incubation time of 30 min at 25 °C.
Nonspecific binding was determined in the presence of 50 µM
NECA and was about 20% of total binding. Binding of [125I]-
AB-MECA to CHO cells transfected with the human recom-
binant A3 adenosine receptors was performed according to
Varani et al.8 Competition experiments were carried out in
duplicate in a final volume of 100 µL in test tubes containing
1 nM [125I]-AB-MECA, 50 mM Tris HCl buffer, 10 mM MgCl2,
1 mM EDTA pH 7.4 and 100 µL of diluted membranes (50 µg
of protein/assay) and at least 8-10 different concentrations
of examined agonists. Incubation time was 120 min at 4 °C,
according to the results of previous time-course experiments.
Ack n ow led gm en t. We thank the King Pharmaceu-
tical R&D for financial support and, in particular, Dr.
Eddie Leung for the useful discussion.
Su p p or tin g In for m a tion Ava ila ble: Selected yields, mp,
and 1H NMR data of synthesized compounds. Elemental
analyses of 83-102. This material is available free of charge
via Internet at http://pubs.acs.org.
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