Phosphate Prodrugs of 1-Propargyl-8-styrylxanthines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 447
mined at λmax ) 282 nm. The concentration of the solution was
calculated using a standard calibration curve (five single
concentrations from 10-100 µM, ꢀ ) 11 800 L‚cm-1‚mol-1).
Results are mean values from three separate experiments. The
water solubility of 9b (MSX-3) was determined to be 9 mg/mL
(17 mM).
Ra d ioliga n d Bin d in g Assa ys. The compounds were tested
in radioligand binding assays for affinity to A1 and A2A ARs
in rat cortical membrane and rat striatal membrane prepara-
tions, respectively. The A1-selective agonist [3H]-N6-cyclohexyl-
of 0.003, 0.01, 0.03, 0.1, 0.3, and 1 µM. The same dilutions
were prepared for a solution of 9b which had not been
pretreated with AP. The Ki values were determined as
described above.
Ack n ow led gm en t. We thank Dr. J u¨rgen Deckert
and Prof. Dr. Peter Riederer, Klinische Neurochemie,
Klinik und Poliklinik fu¨r Psychiatrie der Universita¨t
Wu¨rzburg, Fu¨chsleinstrasse 15, D-97080 Wu¨rzburg,
Germany, for providing human brain tissues. C.E.M. is
grateful for support by the Fonds der Chemischen
Industrie.
adenosine (CHA; 1 nM) was used as A1 ligand and the A2A
-
selective agonist [3H]-2-[[[4-(carboxyethyl)phenyl]ethyl]amino]-
5′-(N-ethylcarbonylamino)adenosine (CGS21680; 5 nM) as A2A
ligand as previously described.26-28 Inhibition of receptor-
radioligand binding was determined by a range of five to six
concentrations of the compounds in triplicate in at least three
separate experiments. The Cheng-Prusoff equation and KD
values of 1 nM for [3H]CHA and 14 nM for [3H]CGS21680 were
used to calculate the Ki values from IC50 values, determined
by the nonlinear curve-fitting program GraphPad Prism,
version 2.0 (GraphPad, San Diego, CA).
Su p p or tin g In for m a tion Ava ila ble: Selected 1H NMR
data of synthesized compounds. This material is available free
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A1 and A3 AR affinities to human recombinant receptors
expressed in CHO cell membranes were determined as de-
scribed using [3H]-2-chloro-N6-cyclopentyladenosine (CCPA) as
A1, [3H]CGS21680 as A2A, and [3H]-5′-(N-ethylcarbonylamino)-
adenosine (NECA) as A3 radioligands.29 A2A AR binding to
human recombinant cells expressed in CHO cells was per-
formed analogously using 5 nM [3H]CGS21680.29 The following
Kd values were used to calculate Ki values: [3H]CCPA, A1 AR,
0.6 nM; [3H]CGS21680, A2A AR, 32 nM; [3H]NECA, A3 AR, 6
nM.
P r ep a r a tion of P ost-Mor tem Br a in (ca p u t n u clei ca u -
d a ti) Mem br a n e P r ep a r a tion . The brains of three people
(2 female, 1 male, ages: 84, 71, and 73), who had no history
of CNS disorders (“control brains”), were dissected 12-54 h
post-mortem and stored at -80 °C. Crude membranes for
radioligand binding experiments were prepared as follows:
Frozen caput nuclei caudati were thawed and cut into smaller
pieces. The tissue from the three brains was pooled and
homogenized on ice (polytron, 10 s with high speed) in ice-
cold HEPES buffer (consisting of 15 mM HEPES, 120 mM
NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, pH 7.4,
freshly prepared). Then the homogenate was centrifuged for
20 min at 4 °C, 40000g. The membrane pellet was resuspended
in HEPES, washed twice, and then stored in aliquots at -80
°C with a protein concentration of 7.1 mg/mL. J ust before the
experiment the tissue was washed once again with TRIS buffer
(pH 7.4, 50 mM) for 20 min at 4 °C and 40000g.
Ra d ioliga n d Bin d in g Assa ys a t Hu m a n P ost-Mor tem
Br a in A2A ARs. Radioligand binding assays were performed
essentially as described.28 3[H]CGS21680 was used as A2A
ligand in a concentration of 5 nM. The protein concentration
in the assays was 50 µg/mL. A Kd value of 22 nM30 was used
to calculate Ki values.
The nonspecific binding amounted to ca. 40% of total
binding, while it was somewhat higher (ca. 50%) in assays
using recombinant A2A receptors.
Ad en yla te Cycla se Assa ys. Inhibition of NECA-induced
stimulation of adenylate cyclase by 5e was determined at
human recombinant A2B receptors as described.29
Effects of P h osp h a ta se on AR Bin d in g of P h osp h a te
P r od r u g 9b (MSX-3). For these experiments, [3H]CCPA31 (0.5
nM) was used as A1-selective radioligand and [3H]-3-(3-
hydroxypropyl)-8-(m-methoxystyryl)-1-propargylxanthine([3H]-
MSX-2, 1 nM) as A2A-selective radioligand.1,31 Binding assays
were performed essentially as described.32,33
A 0.1 mM solution of 9b in TRIS-HCl buffer (pH 7.4) was
incubated at 37 °C, alkaline phosphatase (AP) (3.5 µg protein/
mL) was added, and the solution was gently shaken while
incubated for 1 h. In a separate experiment it was confirmed
that 9b hydrolyzed quantitatively under these conditions as
detected by TLC with UV detection (eluent: 2-propanol:NH3:
H2O ) 6:3:1). Aliquots from the incubated solution were taken
and diluted with TRIS-HCl buffer to the final concentrations
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of xanthines with variable substituents in the 1-, 3-, 7- and
8-positions. Synthesis 1995, 1295-1299.