Multigram-Scale Syntheses, Stability, and Photoreactions of A2A Adenosine Receptor Antagonists with 8-Styrylxanthine Structure: Potential Drugs for Parkinson's Disease

Article abstract of DOI:10.1021/jo0358574

The improved multigram-scale syntheses of the important 8-styrylxanthine A_2A adenosine receptor antagonist MSX-2 (8), its water-soluble prodrug MXS-3 (9), and KW-6002 (16) are described. N-Alkylation reactions at different positions of uracil derivatives were optimized. Two different methods for xanthine formation from 6-amino-5-cinnainoylaminouracil precursors were investigated, (a) the elimination of water by alkaline catalysis and (b) hexamethyldisilazane as a condensing agent; the latter was found to be superior. The photosensitivity of 8-styrylxanthines was studied. The (E)-configurated stryrylxanthine MSX-2 (8) isomerized in diluted solution, and the resulting (Z)-isomer (10a) was isolated and characterized. Furthermore, we describe for the first time that solid 8-styrylxanthines can dimerize upon exposition to daylight or irradiation with UV light. The resulting cyclobutane derivatives with head-to-tail (syn) configuration exhibited a considerably lower A _2A adenosine receptor affinity than their parent compounds. The dimerization product of MSX-2 was a moderately potent nonselective A ₁ and A_2A antagonist (K_i(A_i) = 273 nM, K_i(A_2A) = 175 nM) while the dimer of the related compound KW-6002 was inactive at A₁ and only weakly active at A _2A adenosine receptors (K_i = 1.57 μM). The light sensitivity of 8-styrylxanthine derivatives, not only in solution, but also in the solid state, has to be considered when using those compounds as pharmacological tools or drugs.

Full text of DOI:10.1021/jo0358574

Mu ltigr a m -Sca le Syn th eses, Sta bility, a n d P h otor ea ction s of A2A
Ad en osin e Recep tor An ta gon ists w ith 8-Styr ylxa n th in e Str u ctu r e:
P oten tia l Dr u gs for P a r k in son ’s Disea se
J o¨rg Hockemeyer, J oachim C. Burbiel, and Christa E. Mu¨ller*
University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry Poppelsdorf, Kreuzbergweg 26,
D-53115 Bonn, Germany
christa.mueller@uni-bonn.de
Received December 22, 2003
The improved multigram-scale syntheses of the important 8-styrylxanthine A2A adenosine receptor
antagonist MSX-2 (8), its water-soluble prodrug MXS-3 (9), and KW-6002 (16) are described.
N-Alkylation reactions at different positions of uracil derivatives were optimized. Two different
methods for xanthine formation from 6-amino-5-cinnamoylaminouracil precursors were investigated,
(a) the elimination of water by alkaline catalysis and (b) hexamethyldisilazane as a condensing
agent; the latter was found to be superior. The photosensitivity of 8-styrylxanthines was studied.
The (E)-configurated stryrylxanthine MSX-2 (8) isomerized in diluted solution, and the resulting
(Z)-isomer (10a ) was isolated and characterized. Furthermore, we describe for the first time that
solid 8-styrylxanthines can dimerize upon exposition to daylight or irradiation with UV light. The
resulting cyclobutane derivatives with head-to-tail (syn) configuration exhibited a considerably lower
A2A adenosine receptor affinity than their parent compounds. The dimerization product of MSX-2
was a moderately potent nonselective A1 and A2A antagonist (Ki(A1) ) 273 nM, Ki (A2A) ) 175 nM)
while the dimer of the related compound KW-6002 was inactive at A1 and only weakly active at
A2A adenosine receptors (Ki ) 1.57 µM). The light sensitivity of 8-styrylxanthine derivatives, not
only in solution, but also in the solid state, has to be considered when using those compounds as
pharmacological tools or drugs.
In tr od u ction
in addition, they have been shown to exhibit neuropro-
tective properties in contrast to most currently used anti-
Parkinsonian drugs, thus opening up new opportunities
in therapy.3-6 The breakthrough in the discovery of truly
A2A-selective antagonists was achieved with the synthesis
of 7-methyl-8-styrylxanthine derivatives.7-9 In the past
years, our group has contributed to the design of potent
and selective A2A adenosine receptor antagonists result-
ing in the development of (E)-3-(3-hydroxypropyl)-8-[2-
(3-methoxyphenyl)vinyl]-7-methyl-1-prop-2-ynyl-3,7-di-
hydropurine-2,6-dione (MSX-2, 8)10 and its water-soluble
prodrug (E)-phosphoric acid mono-[3-[8-[2-(3-methox-
yphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-
tetrahydropurin-3-yl]propyl] ester disodium salt (MSX-
3).10 MSX-2 is used in its tritiated form for radioligand
binding studies in tissues expressing A2A receptors in
In addition to its intracellular physiological functions,
the nucleoside adenosine is also an important extracel-
lular signaling molecule.1 Adenosine activates specific G
protein-coupled receptors in cell membranes, whereas the
well-known xanthine alkaloid caffeine mediates most of
its effects by a blockade of adenosine receptors.2 The fact
that four subtypes of adenosine receptors have been
identified opens up the possibility of evoking differential
pharmacological effects by designing subtype-specific
agonists or antagonists. The A2A subtype of adenosine
receptors, one of the most dominant adenosine receptors
in the brain, is characterized by a restricted distribution,
with high levels found in the caudate-putamen, nucleus
accumbens, and olfactory tubercle.3
Adenosine receptor antagonists, in particular those
with selectivity for the A2A subtype, have been proposed
as novel therapeutics for Parkinson’s disease. This neu-
rodegenerative disorder is characterized by a chronically
proceeding degeneration of certain brain areas, including
dopaminergic neurons of the basal ganglia.3 A2A antago-
nists ameliorate symptoms of Parkinson’s disease, and
(4) Ikeda, K.; Kurakowa, M.; Aoyama, S.; Kuwana, Y. J . Neurochem.
2002, 80, 262-270.
(5) Blum, D.; Gals, M. C.; Pintor, A.; Brouillet, E.; Ledent, C.; Mu¨ller,
C. E.; Bantubungi, K.; Galluzzo, M.; Gall, D.; Cuvelier, L.; Rolland, A.
S.; Popoli, P.; Schiffmann, S. N. J . Neurosci. 2003, 23, 5361-5369.
(6) Abbracchio, M. P.; Cattabeni, F. Ann. NY Acad. Sci. 1999, 890,
79-92.
(7) Erickson, S. H.; Hiner, R. N.; Feeney, S. W.; et al. J . Med. Chem.
1991, 34, 1431-1435.
* To whom correspondence should be addressed. Phone: +49-228-
73-2301. Fax: +49-228-73-2567.
(1) Mu¨ller, C. E.; Scior, T. Pharm. Acta Helv. 1993, 68, 77-111.
(2) Daly, J . W. J . Auton. Nerv. Syst. 2000, 81, 44-52.
(3) Mu¨ller, C. E. Drugs of the Future 2000, 25, 1043-1052 and
references therein.
(8) Shimada, J .; Suzuki, F.; Nonaka, H.; Ishii, A.; Ichikawa, S. J .
Med. Chem. 1992, 35, 2342-2345.
(9) J acobson, K. A.; Gallo-Rodriguez, C.; Melman, N.et al. J . Med.
Chem. 1993, 36, 1333-1342.
(10) Sauer, R.; Maurinsh, J .; Reith, U.; Fu¨lle, F.; Klotz, K.-N.;
Mu¨ller, C. E. J . Med. Chem. 2000, 43, 440-448.
10.1021/jo0358574 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/16/2004
3308
J . Org. Chem. 2004, 69, 3308-3318

A2A Adenosine Receptor Antagonists with 8-Styrylxanthine Structure
vitro.11 It has also been labeled by carbon-11, a positron-
emitting nuclide, and was shown to exhibit a high degree
of brain penetration in mice.12 The phosphate prodrug
of MSX-2, MSX-3, exhibits high water solubility (9 mg/
mL)10 and therefore has found broad application as a
pharmacological tool for in vivo studies. It has shown
activity in a number of animal models of Parkinson’s
disease, both after systemic and local application. Due
to its water-solubility, MSX-3 can be used for intracere-
bral application by injecting small volumes of drug
solution directly into specific brain areas in order to study
the mechanism of the anti-Parkinsonian action of A2A
antagonists.13 A related 8-styrylxanthine derivate (E)-8-
[2-(3,4-dimethoxyphenyl)vinyl]-1,3-diethyl-7-methyl-3,7-
dihydropurine-2,6-dione (KW-6002, 16),14 which has been
developed by Kyowa Hakko Co., J apan, is a promising
new drug currently undergoing phase IIb clinical trials
for Parkinson’s disease and depression in the United
States and J apan.15 MSX-2, MSX-3, and KW-6002 have
become standard A2A antagonists and are frequently used
in in vitro and in vivo studies.14,16a-e
formed showed significantly lower adenosine receptor
affinities than their monomeric precursors.
The goals of the present study were to improve and
upscale the syntheses of the potent and selective A2A
antagonists MSX-2, MSX-3, and KW-6002 in order to
make these compounds available as pharmacological tools
on a multigram level for extended in vitro and especially
for in vivo studies. Furthermore, the photochemical
stability and reactivity of 8-styrylxanthines was thor-
oughly investigated.
Ch em istr y
The synthesis of MSX-2 ((E)-3-(3-hydroxypropyl)-8-[2-
(3-methoxyphenyl)vinyl]-7-methyl-1-prop-2-ynyl-3,7-di-
hydropurine-2,6-dione, 8), which is substituted at the N1-
position by a 3-hydroxypropyl group and at the N3-
position by a propargyl group, is more complex than the
synthesis of KW-6002 ((E)-8-[2-(3,4-dimethoxyphen-
yl)vinyl]-1,3-diethyl-7-methyl-3,7-dihydro-purine-2,6-di-
one, 16), which is substituted by two ethyl groups in the
corresponding positions of the xanthine nucleus.
However, photoinduced E/Z-isomerization of various
bioactive 8-styrylxanthines, including CSC (8-(m-chlo-
rostyryl)caffeine),17 CS-DMPX (8-[2-(3-chlorophenyl)vi-
nyl]-3,7-dimethyl-1-prop-2-ynyl-3,7-dihydropurine-2,6-
dione),18 and KW-6002,19,20 has been described to occur
in diluted solutions.17-19,21 Mixtures containing (Z)-con-
figurated 8-styrylxanthine derivatives have been shown
to possess much lower A2A adenosine receptor affinity in
comparison with their corresponding (E)-configurated
isomers.18,19 On the other hand, it had been observed that
animal studies with MSX-3 afforded variable results
when the solid compound had been exposed to light prior
to its application.22 The resulting compounds of the solid-
state photochemical reaction have now been character-
ized as [2 + 2]-cycloaddition products. To the best of our
knowledge, this kind of reaction has not been described
for styrylxanthines so far. The cyclobutane derivatives
Op tim ized , La r ge-Sca le Syn th esis of MSX-3
Commercially available 6-aminouracil (6-amino-1H-
pyrimidine-2,4-dione, 1) was used as a starting material
for the synthesis of 9 (MSX-3).10 Regioselective 3-alky-
lation of 1 with 3-bromopropyne was based on the method
developed by Mu¨ller via a silylated intermediate, which
was formed by ammonium sulfate-catalyzed reaction of
1 with HMDS (1,1,1,3,3,3-hexamethyldisilazane) under
reflux conditions.23 It was observed that a larger excess
(1.7 equiv) of alkylation reagent led to a more complete
conversion to 6-amino-3-prop-2-ynyl-1H-pyrimidine-2,4-
dione 2. The formation of a small amount of the 3,5-
dialkylated byproduct 6-amino-3,5-diprop-2-ynyl-1H-
pyrimidine-2,4-dione 2a , which was isolated by column
chromatography and completely characterized, does not
impede the process in its subsequent stages. Since the
C5-position was blocked by an additional propargyl
group, 2a was not nitrosated in the following step, and
in contrast to the 5-nitroso derivate 6-amino-5-nitroso-
3-prop-2-ynyl-1H-pyrimidine-2,4-dione 3,24 it remained in
solution under the applied reaction conditions (sodium
nitrite in aqueous acetic acid). If the nitrosation was
carried out at 50-60 °C, a lower temperature than cited
in the original literature,24 nitrosation of 2 resulted in a
better yield of 3, with smaller amounts of darker colored
byproducts. The poorly soluble 3 was precipitated from
the reaction solution and isolated by filtration.
(11) Mu¨ller, C. E.; Maurinsh, Y.; Sauer, R. Eur. J . Pharm. Sci. 2000,
10, 259-265.
(12) Holschbach, M.; Mu¨ller, C. E.; Wutz, W.; Schu¨ller, M.; Coenen,
H. H. Nucl. Med. 2000, 39, Abst P154.
(13) Hauber, W.; Nagel, J .; Sauer, R.; Mu¨ller, C. E. Neuroreport
1998, 9, 1803-1806.
(14) Rabasseda, L. A.; Mart´ın, L.; Leeson, P. A.; Castan˜er, J . Drugs
Future 2001, 26, 20-24.
(15) Knutsen, L. J .; Weiss, S. M. Curr. Opin. Invest. Drugs 2001, 2,
668-673.
(16) a) J ustinova, Z.; Ferre´, S.; Segal, P. N.; Antoniou, K.; Solinas,
M.; Pappas, L. A.; Hoghkin, J . L.; Hockemeyer, J .; Munzar, P.;
Goldberg, S. R. J . Pharmacol. Exp. Ther. 2003, in press. (b) Nagel, J .;
Schladebach, H.; Koch, M.; Schwienbacher, I.; Mu¨ller, C. E.; Hauber,
W. Synapse 2003, 49, 279-286. (c) Karcz-Kubicha, M.; Antoniou, K.;
Terasmaa, A.; Quarta, D.; Solinas, M.; J ustinova, Z.; Pezzola, A.;
Reggio, R.; Mu¨ller, C. E.; Fuxe, K.; Goldberg, S. R..; Popoli, P.; Ferre´,
S. Neuropharmacology 2003, 28, 1281-1291. (d) Hauber, W.; Neus-
cheler, P.; Nagel, J .; Mu¨ller, C. E. Eur. J . Neurosci. 2001, 14, 1287-
1293. (e) Stromberg, I.; Popoli, P.; Mu¨ller, C. E.; Ferre´, S.; Fuxe, K. Eur.
J . Neurosi. 2002, 11, 4033-4037.
Reduction of 3 with sodium dithionite in aqueous
ammonia yielded 5,6-diamino-3-prop-2-ynyl-1H-pyrimi-
dine-2,4-dione 4,24,25 a compound considered to be sensi-
tive to oxygen, humidity, and light. After removal of
reactive byproducts by recrystallization from water, and
subsequent drying under vacuum, pure 4 was found to
remain stable for several weeks at room temperature.
Coupling of 4 with (E)-3-methoxycinnamic acid, result-
ing in the formation of an amide bond, has previously
been performed by using the water-soluble (3-dimethyl-
(17) Kim, H. O.; J i, X.-D.; Melman, N.; Olah, M. E.; Stiles, G. L.;
J acobson, K. A. J . Med. Chem. 1994, 37, 3373-3382.
(18) Mu¨ller, C. E.; Geis, U.; Hipp, J .; Schobert, U.; Frobenius, W.;
Pawlowski, M.; Suzuki, F.; Sandoval-Ram´ırez, J . J . Med. Chem. 1997,
40, 4396-4405.
(19) Nonaka, Y.; Shimada, J .; Nonaka, H.; Koike, N.; Aoki, N.;
Kobayashi, H.; Kase, H.; Suzuki, F. J . Med. Chem. 1993, 36, 3731-
3733.
(20) Petzer, J . P.; Steyn, S.; Castagnoli, K. P.; Chen, J .-F.; Schwarz-
schild, M. A.; Van der Schyf, C. J .; Castagnoli, N. Bioorg. Med. Chem.
2003, 11, 1299-1310.
(21) Philip, J .; Szulczewski, H. D. J . Pharm. Sci. 1973, 1885-1887.
(22) Hauber, W. Personal communication.
(23) Mu¨ller, C. E. Tetrahedron Lett. 1991, 32, 6539.
(24) Mu¨ller, C. E. Synthesis 1993, 125.
(25) Mu¨ller, C. E.; Sandoval-Ram´ırez, J . Synthesis 1995, 1295-1299.
J . Org. Chem, Vol. 69, No. 10, 2004 3309

Hockemeyer et al.
SCHEME 1. Syn th esis of (E)-N-[6-Am in o-1-(3-h yd r oxyp r op yl)-2,4-d ioxo-3-p r op -2-yn yl-1,2,3,4-
tetr a h yd r op yr im id in -5-yl]-3-(3-m eth oxyp h en yl)a cr yla m id e (6) a n d Its Acetic Acid Ester (17)a
a
Reagents, conditions and yields: (a) 2.1 equiv of HMDS, reflux, 60-70 °C, 1.7 equiv of 3-bromopropyne, 120 °C, 2 h, H2O (75%); (b)
aq AcOH, HNO2, 50-60 °C (72%); (c) sodium dithionite/NH3/H2O, 70 °C (67%); (d) (E)-3-methoxycinnamic acid chloride, pyridine, ethyl
acetate, rt (80%); (e) 1.8 equiv 3-iodopropan-1-ol, DMF, K2CO3, rt (90%); (f) 1.7 equiv of acetic acid 3-iodopropyl ester, DMF, K2CO3.
SCHEME 2. Rin g Closu r e of Ur a cil Cin n a m oyl Am id e Der iva tives to th e Cor r esp on d in g Xa n th in esa
a
Reagents, conditions and yields: (a) HMDS, reflux, methanol/H2O (85%, 18); (b) KOH/methanol/H2O, H+ (max 92%).
aminopropyl)ethylcarbodiimide hydrochloride (EDC) as
a condensing agent.10 In view of the high costs of the
reagent EDC, we used (E)-3-methoxycinnamic acid chlo-
ride and pyridine as an acid acceptor in ethyl acetate
suspension to obtain (E)-N-(6-amino-2,4-dioxo-3-prop-2-
ynyl-1,2,3,4-tetrahydropyrimidin-5-yl)-3-(3-methoxyphen-
yl)acrylamide 5 resulting in a simplified isolation proce-
dure and an improved yield (80%). Compound 5 is poorly
soluble in common solvents but could be recrystallized
from hot DMF by the addition of water.
The 3-hydroxypropyl group could be introduced directly
at the N1-atom of the uracil derivate 5 either with
3-bromopropan-1-ol at elevated temperature (60 °C)10 or
with 3-iodopropan-1-ol at room temperature, in DMF in
the presence of a base.
The drawback of direct preparation of the N-hydroxy-
propyl-substituted uracil derivative 6 was that the
required large excess of alkylation reagent impeded the
subsequent crystallization. Due to the low solubility of
6, classical extractive isolation of the compound could
only be achieved with large quantities of solvent. The
subsequent ring-closure reaction of 6 with an excess of
HMDS produced xanthine derivate 7 (MSX-1)10 as well
as compound 7a , which was silylated at the free hydroxy-
propyl group, in roughly equal proportions. However,
alkaline hydrolysis of this mixture led directly to the
formation of pure 7.
A simple solution to the problems mentioned above was
to convert the hydroxyl group of 3-iodopropan-1-ol to its
acetyl ester. Having thus protected it, a small excess of
alkylation reagent was brought to reaction with 5 in DMF
in the presence of potassium carbonate. After 24 h at
room temperature, an excellent yield of the resulting 17,
(E)-acetic acid 3-[6-amino-5-[3-(3-methoxyphenyl)acryl-
oylamino]-2,4-dioxo-3-prop-2-ynyl-3,4-dihydro-2H-pyrimidin-
1-yl]propyl ester, was obtained and isolated by precipitat-
ing the product with water. Further purification was
obtained by subsequent recrystallization from methanol/
water (Scheme 1).
Ring-closure reaction of 6 and 17 under elimination of
water to yield the xanthine ring system could be per-
formed in alkaline aqueous solution or with HMDS,
respectively.10 The disadvantage of the former method
was that hydrolysis of the amide bond as a side reaction
is not only inevitable but increases drastically with the
rise of temperature in the presence of alkali and water.
Reaction of 17 with an excess of HMDS in the presence
of ammonium sulfate under reflux conditions produced
exclusively the desired xanthine derivative 18 (Scheme
2). Treatment of the reaction mixture with methanol/
water under neutral conditions allowed the isolation of
the compound. Splitting off the ester group by hydrolysis
of the same mixture with alkali in methanol at increased
temperature and subsequent precipitation of the product
3310 J . Org. Chem., Vol. 69, No. 10, 2004

A2A Adenosine Receptor Antagonists with 8-Styrylxanthine Structure
SCHEME 3. Syn th esis of 7-Meth ylxa n th in e Der iva tivesa
a
Reagents, conditions, and yields: (a) MeI, DMF, K2CO3 (93%, 8; 98%, 19); (b) KOH/methanol, rt, 0.5 h (93%); (c) PO(OMe)3/POCl3, rt,
1 h, H2O (82%, 9).
with water after acidification with hydrochloric acid (to
pH 2) proved to be a straightforward method of yielding
7 (Scheme 2).
Methylation in the N7-position of 7 and 18 was
achieved almost quantitatively using methyl iodide in
DMF and potassium carbonate as a base following
standard procedures.10
After a reaction time of 2 h at room temperature the
methylation of 18 was completed and the acetic acid (E)-
3-[8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-
2-ynyl-1,2,6,7-tetrahydropurin-3-yl]propyl ester 19 could
be precipitated by adding water to the reaction mixture
(Scheme 3).
To split off the acetyl protecting group and produce
MSX-2 (8),10 19 was treated for 0.5 h with a methanolic
solution of potassium hydroxide. Once the reaction was
concluded, pure 8 could be isolated in a high yield after
the addition of water and filtering off the pale yellow
precipitate.
The varying solubility of the different xanthine deriva-
tives and their corresponding acrylamides is particularly
striking. The xanthine precursor 17 was better soluble
in halogenated hydrocarbons than 6, but both compounds
could easily be recrystallized from methanol/water.
In contrast to their precursors 6 and 17, the N7-
unsubstituted xanthine 7 was scarcely soluble, while 18
was poorly soluble in common organic solvents. NMR
spectra of these compounds could therefore only be
measured in DMSO-d6. However, methylation of the N7-
position of the xanthine system led to a high solubility
of the derivatives 8 and 19 in halogenated hydrocarbons,
an effect which is well-known for caffeine (1,3,7-trimeth-
ylxanthine) versus theophylline (1,3-dimethylxanthine).
The phosphoric acid ester 9 (MSX-3 free acid)10 was
obtained by direct phosphorylation of 8 using phosphoryl
chloride in phosphoric acid trimethyl ester as a phos-
phorylating agent.26 Although the reaction of aliphatic
alcohols with phosphoryl chloride generally yields phos-
phoric acid esters, a possible side reaction is the conver-
sion to the corresponding alkyl chloride. Indeed, the
reaction of 8 with phosphoryl chloride in phosphoric acid
trimethyl ester as a solvent at room temperature led to
(E)-phosphoric acid mono[3-[8-[2-(3-methoxyphenyl)-
vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydro-
purin-3-yl]propyl] ester 9, contaminated with variable
amounts of the chlorinated product 9a , depending on the
reaction conditions (Scheme 3). Side product 9a could
easily be separated by column chromatography and was
completely characterized. To minimize its formation, a
large excess of the solvent phosphoric acid trimethyl ester
was used, in addition to a small excess of phosphoryl
chloride (molar ratio 8/phosphoryl chloride ) 1:4). The
reaction time was reduced to 1 h, and a temperature of
0 °C was maintained. Subsequently, 9 was precipitated
by adding a ca. -20 °C mixture of ice, sodium chloride,
and water. After filtration and drying at 70 °C, no further
purification steps were required for obtaining a purity
of at least 95%. Removal of 9a and other nonpolar
byproducts was achieved by dissolving crude 9 in diluted
aqueous sodium hydroxide solution, followed by filtration
and reprecipitation of 9 by acidification with hydrochloric
acid. The disodium salt of 9 was prepared by dissolving
the crude product in a minimal volume of an aqueous
solution containing 2 equiv of NaOH. After precipitation
by adding brine, the yellow disodium salt (MSX-3) was
isolated by filtration and subsequently dried at 70 °C.
In view of the photosensitivity of 8 as well as its
xanthine precursors and derivatives, it is recommended
that compounds should not be exposed to light. To
minimize photolytic degradation reactions, the disodium
salt (MSX-3) should be prepared in situ from the free acid
9 just before application. The preparation of a 5 mM
aqueous solution of MSX-3 is described in the Experi-
mental Section.
Op tim ized , La r ge-Sca le Syn th esis of KW-6002
The starting material for the synthesis of 16 (KW-
6002),14,20,28 6-aminouracil-1,3-diethyl-1H-pyrimidine-2,4-
(26) Vallgarda, J .; Hacksell, U. Tetrahedron Lett. 1991, 32, 5625-
5628.
(27) Slotin, L. A. Synthesis 1976, 737-752 and references therein.
J . Org. Chem, Vol. 69, No. 10, 2004 3311

Hockemeyer et al.
SCHEME 4. Syn th esis of KW-6002a
a
Reagents, conditions, and yields: (a) aq AcOH, HNO2, 50-60 °C (74%); (b) sodium dithionite/NH3/H2O, 60 °C (80%); (c) (E)-3,4-
dimethoxycinnamic acid chloride, pyridine, CH2Cl2, 16 h, rt (65%); (d) HMDS, ammonium sulfate, glass pressure tube, 170-180 °C, 4 h
(92%); (e) 1.4 equiv of MeI, DMF, potassium carbonate (95%).
dione 11, was obtained by direct reaction of the dialkyl
urea with cyanoacetic acid following the protocol of
Papesch and Schro¨der.29 Subsequent introduction of the
nitroso group in the 5-position to yield 6-amino-1,3-
diethyl-5-nitroso-1H-pyrimidine-2,4-dione 12 was achieved
in high yields analogously to the synthesis of 3.30,31
The reaction of 12 to the 5,6-diamino-1,3-diethyl-1H-
pyrimidine-2,4-dione 1330,31 was achieved, as described
above, in aqueous ammonia with sodium dithionite as a
reducing agent. Due to its high solubility, 13 could not
be isolated by simple concentration of the reaction
mixture and subsequent precipitation of the compound
in a satisfactory yield. A better result was obtained by
extraction of the aqueous reaction mixture with dichlo-
romethane, classical separation, and recrystallization
from a mixture of dichloromethane/diethyl ether.
To avoid expensive water-soluble carbodiimide con-
densing agents,28,32,33 coupling of the diaminouracil 13
with (E)-3,4-dimethoxycinnamic acid chloride was per-
formed in dichloromethane solution in the presence of
pyridine, leading to (E)-N-(6-amino-1,3-diethyl-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-5-yl)-3-(3,4-dimethoxyphen-
yl)acrylamide 14.35 After aqueous extraction of byprod-
ucts and concentration of the organic phase, recrystalli-
zation of the oily residue from methanol/water afforded
compound 14 as colorless crystals.
described by Suzuki et al.34 showed that a mixture of
equivalent volumes of dioxane and a 1 M aqueous
solution of sodium hydroxide yielded (E)-8-[2-(3,4-
dimethoxyphenyl)vinyl]-1,3-diethyl-3,7-dihydropurine-
2,6-dione 15 after a reaction time of 10 min under reflux
conditions. After dilution of the reaction mixture with
water, the product was precipitated by acidification with
hydrochloric acid, filtered, and washed with water. The
described procedure led to a very pure product with a
maximal yield of 62% only. This observation, confirmed
by detection of (E)-3,4-dimethoxycinnamic acid in the
crude reaction mixture by TLC analysis, pointed again
to alkaline hydrolysis of the amide bond as a side
reaction.
To avoid this problem, an alternative ring-closure
reaction was performed using HMDS as a condensing
agent. In fact, elimination of water could be achieved at
170-180 °C in the presence of ammonium sulfate under
elevated pressure. After hydrolysis with methanol and
filtration, 15 was obtained in a yield of 92%.
An alternative synthesis of compound 15 starting from
13 using the conventional method34 has recently been
described.20 The reported yield of 35% over two steps is
significantly lower as compared to the yields which were
obtained by the method described above.
The up-scaled methylation of the N7-position of 15 to
16 was achieved in analogy to the preparation of 8 in
DMF using methyl iodide in the presence of potassium
carbonate.10 After a reaction time of less than 5 h at room
temperature, KW-6002 (16) could be precipitated by
adding water and a high yield of the pure, light-yellow
product was isolated (Scheme 4).20,28,32,33,35
The subsequent ring-closure reaction of the amide
precursor 14 to the corresponding xanthine has been
described by Shimada et al. without giving the exact
experimental proportions and product isolation protocol.28
An investigation of the conditions for related reactions
(28) Shimada, J .; Koike, N.; Nonaka, H. Bioorg. Med. Chem. Lett.
1997, 7, 2349-2352.
P h otoch em ica l Rea ction s
(29) Papesch, V.; Schroeder, E. F. J . Org. Chem. 1951, 16, 1879-
1890.
The preparation of compounds 8 and 9 must be carried
out under strict exclusion of light. When stored in normal
glass vials and exposed to daylight, within a few days
the pale yellow crystals were observed to turn into a
colorless solid. TLC investigation of the obtained material
indicated a decrease in 8 and the appearance of an
additional spot, which could subsequently be identified
as a photoreaction product.
(30) Blicke, F. F.; Godt, H. C., J r. J . Am. Chem. Soc. 1954, 76, 2798-
2800.
(31) Speer, R. J . Am. Chem. Soc. 1953, 75, 114.
(32) Suzuki, F.; Shimada, J .; Koike, N.; Nakamura, J .; Shoizaki, S.;
Ichikawa, S. (Kyowa Hakko Kogyo Co., Ltd.) EP 0590919, J P
1994211856, US 5484920.
(33) Suzuki, F.; Shimada, J .; Ishii, A.; Nakamura, J .; Ichikawa, S.;
Kitamurs, S.; Koike, N. (Kyowa Hakko Kogyo Co., Ltd.) EP 0628311,
J P 1994502746, WO 9401114.
(34) Suzuki, F.; Shimada, J .; Shiozaki, S.; Ichikawa, S.; Ishii, A.;
Nakamura, J .; Nonaka, H.; Kobayashi, H.; Fuse, E. J . Med. Chem.
1993, 36, 2508-2518.
(35) Miwa, K.; Ito, K.; Kato, N.; Kuge, Y.; Kousai, M.; Tomioka, S.
(Kyowa Hakko Kogyo Co., Ltd.) J P 1997040652.
1
The H NMR spectrum of the colorless solid, isolated
by column chromatography, gave a first indication of the
formation of a cyclobutane ring system by a [2 + 2]-
3312 J . Org. Chem., Vol. 69, No. 10, 2004

A2A Adenosine Receptor Antagonists with 8-Styrylxanthine Structure
SCHEME 5. Solid -Sta te P h otor ea ction s of
8-Styr ylxa n th in e Der iva tives
tives, which do not dimerize, since the half-life of their
first excited singlet state is too short.38 Apart from that,
solid-state photoreaction of (E)-configurated ethylenes
forces trans-trans dimerization, as the fixed positions in
the crystal-lattice do not permit conversion to the (Z)-
isomer.
Having excluded all other possibilities, only two ht
isomers had to be considered in the case of compound
10: the cis-trans-cis (syn) and the all-trans (anti) con-
figurations.
With heterostilbenes, which exhibit a strong asym-
metry of charge, ht orientations are to be expected and,
additionally, along with dipole components perpendicular
to the main axis, even syn-arrangements.36 Further
differentiation within the two ht isomers should be
obtained from specific NMR spectral features. All de-
scribed ht cyclobutanes with syn-configuration derived
from asymmetrically substituted (E)-stilbenes or from
heterocyclic analogues exhibited a symmetric AA′BB′-
system for cyclobutane ring protons, consisting of two
half-spectra with a lack of fine structure, so that coupling
constants could not be easily extracted.36,39,40 Each half-
spectrum generally exhibits four lines. We found similar
multiplets for the cyclobutane proton AA′BB′ systems in
all of the NMR spectra of cyclic dimers described in this
paper. Only a few examples for ht cyclobutanes with anti-
configuration are described in the literature. On the basis
of molecular symmetry (C2v), a theoretical A2B2 system
with a half-spectrum of seven lines would be expected
for the aliphatic cyclobutane hydrogen atoms of these
isomers.40-42 However, superimposition of lines makes it
impossible to distinguish the spin systems clearly.
An additional reference to the syn-configuration of the
isolated cyclobutanes gave the chemical shift of the four
protons directly bonded to the cyclobutane ring. The so-
called midpoint method, developed by Ben-Efraim and
Green,43 is useful for the rapid and straightforward
assignment of isomer structures. It is reported that the
center of the cyclobutane proton signals of aryl-substi-
tuted derivatives in general appears around 4.5 ppm for
the cis-trans-cis (syn), and around 3.7 ppm for the all-
trans (anti) cyclobutanes.40,42-44 The center of the cy-
clobutane multiplets presented for 10 as well as for the
cyclobutane derivatives 7b, 9b, and 16b, described below
as photoreaction products of 8-styrylxanthines 7, 9a , and
16, appeared between 4.56 and 4.73 ppm. In accordance
with theory, ht syn-configuration for these compounds is
proposed. With a cyclobutane multiplet centered at 4.15
ppm derivative 18b, described below as photoreaction
product of 18, constituted an exception. After splitting
off the acetyl groups by alkaline hydrolysis, the resulting
cycloaddition reaction of two ethylenic bonds. A sym-
metrical AA′BB′-spin system, typical of 1,2,3,4-tetrasub-
stituted cyclobutanes, appeared around 4.69 ppm. Cor-
respondingly, the signals for the ethylenic (E)-hydrogen
atoms of 8 had disappeared.
The mass spectrum of 10 not only confirmed the
suggested [2 + 2]-cycloaddition of 8 with a molecular ion
M
+ 788 (EI, 35) for the product but also gave some clues
to elucidate the possible constitution of the cyclobutane
derivative that had been formed. The base peak at m/z
394 M+/2 (EI, 100) indicated a symmetrical [2 + 2]-rever-
sion of the dimerization product and hence the formation
of a h(ead)-to-t(ail) cyclobutane isomer (Scheme 5). Since
fragments characteristic of asymmetrical reversion were
present neither in the mass spectra of 10 nor in those of
all cyclobutanes described in this paper, the formation
of hh dimers could be excluded.36,37
With regard to E/Z-isomerization of the starting com-
pound 8, which represents an asymmetrically substituted
ethylene, and the hypothetical existence of both isomers
in the reaction mixture, theoretically one can expect
eleven stereoisomers formed by trans-trans, trans-cis, and
cis-cis dimerization in an hh and ht modus. According to
the mass spectral data, the number of possible structures
could be reduced to five, because six of the stereoisomers
correspond with an hh dimerization and could therefore
be excluded.
1
product showed exactly the same H NMR spectrum as
that obtained for 7b, which also indicated the likelihood
of a ht syn-configuration for 18b (Scheme 5).
(38) Stegemeyer, H. Chimia 1965, 19, 536.
(39) Andresen, G.; Eriksen, A. B.; Dalhus, B.; Gundersen, L.-L.; Rise,
F. J . Chem. Soc., Perkin Trans. 1 2001, 1662-1672.
(40) Montaudo, G.; Caccamese, S.; Librando, V. Org. Magn. Reson.
1974, 6, 534-536.
(41) Gu¨nther, H. Angew. Chem., Int. Ed. Engl. 1972, 11, 861.
(42) Andreani, F.; Andrisano, R.; Salvadori, G.; Tramotini, M. J .
Chem. Soc., Perkin Trans. 1 1977, 1737-1742.
(43) Ben-Efraim, D. A.; Green, B. S. Tetrahedron 1974, 30, 2357-
2364.
Three of the remaining five ht isomers are formed
theoretically including (Z)-configurated ethylene deriva-
(36) Kaupp, G.; Frey, H.; Behmann, G. Chem. Ber. 1988, 121, 2135-
2146.
(37) Sˇkoric´, I.; Zˇeljko; M.; Sˇindler-Kulyk, M. Heterocycles 2000, 53,
55-68.
(44) Ito, Y.; Kajita, T.; Kunimoto, K.; Matsuura, T. J . Org. Chem.
1989, 54, 587-591 and references therein.
J . Org. Chem, Vol. 69, No. 10, 2004 3313

Hockemeyer et al.
TABLE 1. Ad en osin e Recep tor Affin ities of
Dim er iza tion P r od u cts of P oten t, Selective
2A-Ad en osin e Recep tor An ta gon ists w ith
8-Styr ylxa n th in e Str u ctu r e
SCHEME 6. P h otor ea ction of MSX-2 in Dilu ted
Dich lor om eth a n e Solu tion
A
Ki ( SEM (nM), n ) 3
2A affinity
rat brain cortical rat brain striatal
A1 affinity
A
A
2A selectivity
vs A1
membranes
membranes
compd
vs [3H]CCPA
vs [3H]MSX-2
(Ki A1/Ki A2A
1.6
>7
)
10
273 ( 34
175 ( 15
1570 ( 33
16b
>10 000 (7%a)
The photochemical properties of compound 8, as here
described, should applysin varying degreessto all xan-
thine derivates substituted in the 8-position by a styryl
group. It proved extremely difficult to predict how easily
each of the xanthines described in this study would
undergo solid-state photodimerization. In the case of the
less sensitive xanthines, it took several weeks to observe
evidence of photolysis caused by daylight. In contrast,
the light-yellow compounds 8 and 9a behaved quite
differently. When exposed to daylight, in the form of a
thin crystalline film in a normal glass vial, they decol-
orized completely within ca. 8 h and were quantitatively
transformed into their corresponding photodimers 10 and
9b, respectively. The 8-styrylxanthine 16 (KW-6002)
proved considerably more stable: Even after 6 months
of storage under diffuse daylight, the compound exhibited
virtually no sign of photolytic reaction. However, brief
irradiation of solid 16 with ultraviolet light led almost
quantitatively to the formation of photodimer 16b (Scheme
5). The colorless xanthine derivatives 7 and 18 are
characterized by a free NH-function in the 7-position.
They showed a moderate degree of photosensitivity.
Exposed to daylight, a thin crystal film of the compounds
photolyzed almost completely within ca. 4 weeks to form
the corresponding cyclobutanes 7b and 18b.
a
Displacement at 10 µM, n ) 2.
region for cis-arranged protons. The extreme high field
shift of ca. 0.4 ppm for the signal of the 7-N-methyl group
was further evidence of a reaction to the (Z)-isomer 10a .
1
The H NMR spectra of all obtained fractions showed no
typical signals of cyclobutane protons; hence, when
MSX-2 was photolyzed in diluted solution there was no
evidence of [2 + 2]-cycloaddition products. The 13C NMR
experiment served to confirm these observations: Be-
tween 40 and 45 ppm, no typical signals of cyclobutane
C-atoms were observed. The mass spectrum of 10a did
not differ from that of the starting compound. These
observations do not exclude the possibility that the
irradiation of more concentrated solutions of 8-styryl-
xanthines could also lead to the formation of cyclobu-
tanes.
Biologica l Activity
The new cyclobutane derivatives 10 and 16b, dimer-
ization products of the potent A2A-selective adenosine
receptor antagonists 8 and 16, were investigated in
radioligand binding studies at A1 adenosine receptors of
rat brain cortical membranes using the A1-selective
radioligand [3H]-2-chloro-N6-cyclopentyladenosine ([3H]-
CCPA) and at A2A adenosine receptors of rat brain
striatal membranes using the A2A-selective radioligand
[3H]MSX-2. Data of dimerization products were compared
Since it was not possible to prepare an appropriate
crystal film of 7, due to its poor solubility in volatile
organic solvents, the results of UV irradiation were
unsatisfactory. A brief period of irradiation produced very
poorly soluble, obviously polymeric material. This could
point to a competing radical reaction in addition to the
[2 + 2]-cyclodimerization.
with those of the monomeric compounds. The A2A
-
selective adenosine receptor antagonists MSX-2 and KW-
6002 were similarly potent A2A antagonists with Ki values
in the low nanomolar range.10,28 Both compounds were
considerably less active at A1 receptors. Dimerization
reduced the A2A affinity of both compounds dramatically,
but the decrease was more pronounced for the KW-6002
dimer (16b) as compared to the MSX-2 dimer (10, see
Table 1).
At A1 adenosine receptors, dimerization of KW-6002
resulted in a complete loss of affinity. In contrast, the
dimerization product of MSX-2 retained some A1 affinity
and was a moderately potent, nonselective adenosine
receptor antagonist (10, Ki(A1) ) 273 nM, Ki(A2A) ) 175
nM). It is quite surprising that the receptors can accom-
modate such a bulky dimeric molecule. However, subtle
structural differences in the dimers, which did not play
a noteworthy role in the monomers, resulted in large
differences in A2A (9-fold) and particularly in A1 affinity
(>37-fold) between the dimeric cyclobutane derivatives
10 and 16b. These structure-activity relationships will
be useful for molecular modeling studies of adenosine
receptors and their complexes with ligands and may be
helpful in future drug design studies.
Several photolytic reactions of 8-styrylxanthines in
diluted solutions have been described, and it was con-
sidered that irradiation leads to the rapid establishment
of a photostationary state, an equilibrium of (E) and (Z)
isomers.17-19,21 On no occasion so far has photodimeriza-
tion been observed. To support these findings and
distinguish them from solid-state photolysis, an 8.5 mM
solution of MSX-2 in dichloromethane was irradiated.
After 10 min reaction time, analysis by TLC indicated
the formation of a reaction product close to the spot of
the starting material. The irradiation was stopped after
1.5 h, and the concentrated oily residue was separated
by column chromatography. Two main fractions were
obtained in this way and analyzed by NMR and mass
1
spectroscopy. The H NMR spectrum of the first fraction
indicated an enrichment of the starting compound 8. Its
(Z)-isomer 10a , which was slightly contaminated with 8,
could be detected in the second fraction (Scheme 6).
An important evidence of the (Z)-isomer was the
coupling constant for the ethylenic protons, which was
found to be ca. 3 Hz lower for 10a (12.4 Hz) as compared
with the (E)-isomer 8 (15.8 Hz), hence, in the expected
3314 J . Org. Chem., Vol. 69, No. 10, 2004

A2A Adenosine Receptor Antagonists with 8-Styrylxanthine Structure
mmol) of 5, anhydrous K2CO3 (4.1 g, 30 mmol), and 11.4 g (50
Con clu sion s
mmol) of acetic acid 3-iodopropyl ester in DMF (75 mL) was
stirred for at least 24 h at rt until a clear solution was obtained
and no further starting material could be detected by TLC
(eluent: CH2Cl2/methanol, 7:1). The product was precipitated
by the addition of water (75 mL), filtered off, and washed with
water (150 mL). The colorless needles, obtained by recrystal-
lization with methanol/water (1:1), were filtered off under
reduced pressure and washed with diethyl ether (100 mL).
After drying at 70 °C, a pale yellow solid was obtained.
Compound 17 could also be isolated by extraction with CH2-
Cl2 as described for compound 7 (92% yield): mp 129.4 °C; 1H
NMR (500 MHz, CDCl3) δ 2.01 (m, 2H), 2.06 (s, 3H), 2.19 (t, J
) 2.2 Hz, 1H), 3.81 (s, 3H), 3.96 (m, 2H), 4.11 (m, 2H), 4.63
(d, J ) 2.2 Hz, 2H), 5.98 (s, 2H), 6.67 (d, J ) 15.4 Hz, 1H),
8-Styrylxanthine derivatives are an important class of
A2A adenosine receptor antagonists. For two of the most
frequently used derivatives of this structural class of
compounds, MSX-3 (9) and KW-6002 (16), improved, up-
scaled syntheses have been developed. The overall yield
of 9 has been increased (ca. 20%, starting from the uracil
derivative 1), and multigram quantities could be ob-
tained. In the case of 16, an overall yield of about 30%
(starting from 11) has been reached. For a better under-
standing of the reactions, byproducts were isolated and
characterized, ultimately leading to subtle but consider-
able improvements in reaction strategies.
6.89-7.27 (m, 4H), 7.56 (d, J ) 15.4 Hz, 1H), 7.82 (s, 1H); 13
C
The photosensitivity of the 8-styrylxanthines was
investigated. In addition to the well-known E/Z-isomer-
ization that occurs in diluted solutions, it was found that
in the solid state some derivatives are even more light-
sensitive. Instead of E/Z-isomerization, [2 + 2]-cycload-
dition was observed when the crystals were irradiated
by normal daylight or by a mercury lamp at 200-600
nm. No simple correlation could be found between the
substitution pattern and the degree of photosensitivity.
Generally, N7-unsubstituted 8-styrylxanthines appeared
to be less photoreactive than their N7-methylated deriva-
tives. The amide precursors of 8-styrylxanthines did not
show any photosensitivity at all. It was proven by NMR
sprectroscopy that all solid-state photoreactions described
led to head-to-tail cis-trans-cis (syn) cyclobutane deriva-
tives.
NMR (125 MHz, CDCl3) δ 20.9, 27.2, 30.8, 40.6, 55.3, 61.6,
70.9, 78.4, 91.6, 113.1, 115.9, 120.0, 120.6, 135.7, 142.3, 135.7,
149.0, 149.5, 159.1, 159.9, 165.9, 170.8; EIMS (m/z) 440 (M+,
100).
(E)-Acetic Acid 3-[8-[2-(3-Meth oxyp h en yl)vin yl]-2,6-
d ioxo-1-p r op -2-yn yl-1,2,6,7-t e t r a h yd r op u r in -3-yl]p r o-
p yl Ester (18). A mixture of 3 g (6.8 mmol) of 17, (NH4)2SO4
(0.25 g), and HMDS (30 mL) was heated under vigorous reflux
and vigorous stirring for 16 h. The reaction was monitored by
TLC (eluent: CH2Cl2/methanol, 9:1). The end of the reaction
was reached when the molten, poorly soluble starting material
had been transformed to a fine suspension of colorless product
in a clear yellow solution. After hydrolysis by adding methanol
and cooling to rt, the colorless solid was filtered off under
reduced pressure, washed with methanol (100 mL) and
subsequently with diethyl ether (75 mL), and dried at 70 °C
(85% yield): mp 219.6 °C; 1H NMR (500 MHz, DMSO-d6) δ
1.91 (s, 3H), 2.02 (m, 2H), 3.04 (t, J ) 2,2 Hz, 1H), 3.73 (s,
3H), 4.01 (m, 2H), 4.09 (m, 2H), 4.58 (d, J ) 2.2 Hz), 7.01 (d,
J ) 16.4 Hz, 1H), 6.89-7.31 (m, 4H), 7.58 (d, J ) 16.4 Hz,
1H), 13.62 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 21.1, 27.2,
30.7, 41.1, 55.7, 62.3, 73.3, 80.2, 112.5, 115.7, 116.5, 120.2,
130.5, 136.0, 137.3, 170.7, 107.5, 149.1, 150.4, 150.6, 153.4,
160.2, 170.7; EIMS (m/z) 422 (M+, 100).
Exp er im en ta l Section
Compounds 2-523-25 and 810 as well as compounds 12,
13,25,30,31 and 1620,28,32,33,35 were synthesized according to
described procedures with only slight modifications; for details
also see the Supporting Information.
(E)-3-(3-Hydr oxyp r opyl)-8-[2-(3-m eth oxyph en yl)vin yl]-
1-p r op -2-yn yl-3,7-d ih yd r op u r in e-2,6-d ion e (7, MSX-1).10
A suspension of 5 g of 17 (11.4 mmol) or alternatively 5 g (12.6
mmol) of 6, 0.25 g of (NH4)2SO4, and HMDS (50 mL) was
heated under vigorous reflux and stirred for 16 h until the
molten, poorly soluble starting compound had been trans-
formed to a fine suspension of the colorless product in a clear
yellow solution.
To remove the acetyl group of the intermediate 18 and the
silyl group of the O-silylated byproduct 7a , the reaction
mixture was treated with a solution of KOH (2 g) in methanol
(50 mL). After being heated at reflux for 0.5 h, the mixture
was concentrated under vacuum, and the oily residue was
diluted with a mixture of methanol (50 mL) and water 150
mL). The colorless product was precipitated by adding concd
HCl dropwise (f pH = 2), filtered off, washed with water (250
mL), and dried at 70 °C (92% yield): mp 250 °C; 13C NMR
(125 MHz, DMSO-d6) δ 30.3, 31.1, 41.1, 55.3, 58.6, 72.9, 79.9,
112.1, 115.3, 116.1, 119.8, 130.1, 135.3, 136.1, 107.5, 148.8,
150.2, 150.2, 153.2, 159.8; EIMS (m/z) 380 (M+, 100).
(E)-8-[2-(3-Met h oxyp h en yl)vin yl]-1-p r op -2-yn yl-3-(3-
t r im e t h y ls ila n y lo x y p r o p y l)-3,7-d ih y d r o p u r in e -2,6-
d ion e (7a ). For separation of compound 7a , the reaction
mixture which was obtained from the reaction of 6 with HMDS
was hydrolyzed under almost neutral conditions with an excess
of methanol. After evaporation of the solvents, the residue was
purified by column chromatography on silica gel (eluent: CH2-
Cl2/methanol, 7:1), yielding 7 and 7a as colorless solids in
varying proportions. Data for 7a : mp > 205 °C; 1H NMR (500
MHz, CDCl3) δ 0.09 (s, 9H), 2.07 (m, 2H), 2.17 (t, J ) 2.6 Hz,
1H), 3.72 (m, 2H), 3.83 (s, 3H), 4.29 (m, 2H), 4.88 (d, J ) 2.6
Hz, 1H), 7.11 (d, J ) 16.1 Hz, 1H), 6.87-7.30 (m, 4H), 7.77 (d,
6-Am in o-3,5-dipr op-2-yn yl-1H-pyr im idin e-2,4-dion e (2a).
Compound 2a was separated from crude 2 by column chro-
matography on silica gel (eluent: CH2Cl2/methanol, 3:1).
Subsequent recrystallization from ethanol/diethyl ether (1:2)
yielded 2a as slightly reddish crystals in varying proportions,
depending on its content in the crude 2. Data for 2a : mp 220.5
1
°C; H NMR (500 MHz, DMSO-d6) δ 2.61 (t, J ) 2.6 Hz, 1H),
2.98 (t, J ) 2.6 Hz, 1H), 3.12 (d, J ) 2.6 Hz, 2H), 4.41 (d, J )
2.6 Hz, 2H), 6.27 (s, 2H), 10.45 (s, 1H); 13C NMR (500 MHz,
DMSO-d6) δ 12.4, 28.9, 69.5, 72.4, 80.3, 80.6, 83.2, 149.5, 150.7,
161.4; EIMS (m/z) 203 (M+, 100).
(E)-N-[6-Am in o-1-(3-h yd r oxyp r op yl)-2,4-d ioxo-3-p r op -
2-yn yl-1,2,3,4-tetr ah ydr opyr im idin -5-yl]-3-(3-m eth oxyph e-
n yl)a cr yla m id e (6).10 A mixture of 10 g (29.4 mmol) of 5, 4.1
g of anhydrous K2CO3 (30 mmol), and 10 g (54 mmol) of
3-iodopropan-1-ol in DMF (75 mL) was stirred for 16 h at rt.
The reaction mixture was diluted with 1000 mL of water and
extracted four times with 250 mL of CH2Cl2 each. The
combined organic extracts were dried over MgSO4, filtered, and
concentrated under vacuum, particularly in order to remove
traces of DMF. The oily residue was dissolved in a small
amount of methanol and then treated with a mixture of diethyl
ether and petroleum ether (1:1) to crystallize the product as
colorless needles. After filtration and washing with a mixture
of diethyl ether and petroleum ether (1:1), the solid was dried
at 70 °C (90% yield): mp 123 °C; 13C NMR (125 MHz, DMSO-
d6) δ 30.0, 30.9, 55.3, 58.1, 72.6, 80.1, 87.5, 112.9, 115.3, 119.9,
123.1, 130.2, 136.6, 138.9, 149.8, 152.1, 158.2, 159.8, 165.5;
EIMS (m/z) 398 (M+, 100).
(E)-Acet ic Acid 3-[6-Am in o-5-[3-(3-m et h oxyp h en yl)-
a cr yloyla m in o]-2,4-d ioxo-3-p r op -2-yn yl-3,4-d ih yd r o-2H-
p yr im id in -1-yl]p r op yl Ester (17). A mixture of 10 g (29.4
J . Org. Chem, Vol. 69, No. 10, 2004 3315

Hockemeyer et al.
J ) 16.1 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 0.1, 30.8, 31.0,
41.8, 55.3, 60.3, 71.0, 78.9, 112.4, 115.2, 115.4, 120.2, 129.8,
136.9, 137.5, 107.1, 150.3, 150.3, 151.7, 154.9, 160.0; EIMS
(m/z) 452 (M+, 30).
and/or sonication. The yellow solution was filtered, and the
product was precipitated by addiion of brine (ca. 50 mL). After
filtration and drying at 70 °C, a light-sensitive, pale yellow
solid was obtained (75% yield): 13C NMR (125 MHz, D2O) δ
31.7, 31.8, 31.8, 33.4, 37.8, 58.2, 64.5 (d, J ) 2.7 Hz), 110.4,
114.0, 116.8, 122.1, 132.3, 138.4, 139.8, 150.4, 153.2, 153.5,
156.3, 161.6.
P r ep a r a tion of a n Isoton ic Solu tion of (E)-P h osp h or ic
Acid Mon o[3-[8-[2-(3-m eth oxyp h en yl)vin yl]-7-m eth yl-
2,6-d ioxo-1-p r op -2-yn yl-1,2,6,7-t e t r a h yd r op u r in -3-yl]-
p r op yl] Ester Disod iu m Sa lt (MSX-3) fr om 9. In a solution
of 4.5 mg (0.11 mmol) of NaOH in 10 mL of 0.9% saline was
dissolved 25 mg (ca. 0.053 mmol) of crude 9 under exclusion
of light. After stirring and/or sonication for about 5 min, the
slightly turbid solution was filtered off through a 0.45 µm
membrane filter. A clear yellow 5 mM solution of MSX-3
disodium salt was obtained.10
(E)-Acetic Acid 3-[8-[2-(3-Meth oxyph en yl)vin yl]-7-m eth -
yl-2,6-d ioxo-1-p r op -2-yn yl-1,2,6,7-tetr a h yd r op u r in -3-yl]-
p r op yl Ester (19). A suspension of 0.2 g (0.47 mmol) of 18,
K2CO3 (0.14 g, 1 mmol), and 0.14 g (1 mmol) of methyl iodide
in DMF (5 mL) was stirred at rt for 5 h under exclusion of
light (TLC control, eluent: CH2Cl2/methanol, 9.5:0.5). The pale
yellow, light-sensitive product was precipitated by addition of
water (30 mL), filtered off under reduced pressure, washed
with water (70 mL), and dried at 70 °C (98% yield): mp 158.4
°C; 1H NMR (500 MHz, CDCl3) δ 2.03 (s, 3H), 2.16 (t, J ) 2.2
Hz, 1H), 2.17 (m, 2H), 3.84 (s, 3H), 4.03 (s, 3H), 4.15 (m, 2H),
4.26 (m, 2H), 4.77 (d, J ) 2.2 Hz, 2H), 6.86 (d, J ) 15.5 Hz,
1H) 6.89-7.32 (m, 4H), 7.73 (d, J ) 15.5 Hz, 1H); 13C NMR
(125 MHz, CDCl3) δ 20.6, 27.1, 30.4, 31.5, 40.6, 55.3, 62.0, 70.4,
78.8, 111.4, 112.7, 115.1, 120.0, 129.9, 136.7, 138.6, 107.8,
148.6, 150.3, 150.5, 154.1, 160.0, 171.0; EIMS (m/z) 436 (M+,
100).
Hyd r olysis of 19. To a solution of 0.5 g KOH in methanol
(30 mL), 3 g (7.1 mmol) of 19 were added. The suspension was
stirred for 0.5 h at rt. After the complete disappearance of 19
(TLC control, eluent: CH2Cl2/methanol, 9.5:0.5), 8 (MSX-2)
was precipitated by dilution with water (70 mL), filtered off
under reduced pressure, washed with water (200 mL), and
dried at 70 °C (93% yield).
P h otor ea ction s
Solid -Sta te P h otor ea ction of 8 (MSX-2) to (1r,2r,3â,
4â)-1,3-Bis[3-(3-h yd r oxyp r op yl)-7-m eth yl-1-p r op -2-yn yl-
3,7-dih ydr opu r in e-2,6-dion e-8-yl]-2,4-bis(3-m eth oxy)ph en -
ylcyclobu ta n e (10). A crystal film of 100 mg (0.25 mmol) of
8 was prepared by dissolving the yellowish solid in a minimum
of CH2Cl2 and subsequently leaving the solvent to evaporate
on the surface of a photoreactor. After 90 min of irradiation,
the essentially decolorized product was dissolved in CH2Cl2,
filtered to remove oligomeric material, and recrystallized by
the addition of diethyl ether to yield colorless crystals (63
(E)-P h osp h or ic Acid Mon o[3-[8-[2-(3-m eth oxyp h en yl)-
vin yl]-7-m et h yl-2,6-d ioxo-1-p r op -2-yn yl-1,2,6,7-t et r a h y-
1
d r op u r in -3-yl]p r op yl] Ester (9, MSX-3 F r ee Acid ).10
A
mg): mp 252 °C dec; H NMR (500 MHz, CDCl3) δ 1.96 (m,
4H), 2.14 (t, J ) 2.2 Hz, 2H), 3.51 (m, 4H), 3.64 (s, 6H), 3.71
(s, 6H), 4.24 (m, 4H), 4.44-4.47 and 4.90-4.93 (AA′BB′, 4H),
4.70 (m, 4H), 6.70-7.15 (m, 8H); 13C NMR (125 MHz, CDCl3)
δ 30.5, 31.0, 31.8, 39.7, 40.7, 44.5, 55.2, 57.6, 70.5, 78.5, 112.5,
114.2, 119.7, 130.0, 138.9, 107.6, 147.8, 150.6, 152.0, 153.9,
159.8; EIMS (m/z) 788 (M+, 38). TLC (eluent: ethyl acetate,
Rf ) 0.45).
P h otolysis of 8 in Solu tion . A 0.85 mM solution of MSX-
2, prepared by dissolving 250 mg (0.63 mmol) of 8 in CH2Cl2
(850 mL), was transferred into a photoreactor and irradiated
with a medium-pressure mercury lamp. The reaction was
monitored by TLC. After ca. 10 min, an additional spot was
detected by TLC, indicating a photoreaction product (eluent:
ethyl acetate, 8, Rf ) 0.68; 10a , Rf ) 0.38). After 1.5 h the
reaction was stopped, and the solvent was evaporated. The
residue was separated by column chromatography (eluent:
CH2Cl2/ethanol, 9.5:0.5). Two main fractions were obtained:
one enriched with the starting material (E)-MSX-2, the other
enriched with the (Z)-isomer of MSX-2. After evaporation of
the solvents, the crude residues were analyzed by mass and
NMR spectroscopy.
(Z)-3-(3-H yd r o x yp r o p yl)-8-[-2-(3-m e t h oxy p h e n y l)-
v i n y l]-7-m e t h y l-1-p r o p -2-y n y l-3,7-d i h y d r o p u r i n e -
2,6-d ion e (10a ): 1H NMR (500 MHz, CDCl3) δ 1.88 (m, 2H),
2.15 (t, J ) 2.5 Hz, 1H), 3.44 (m, 2H), 3.63 (s, 3H), 3.69 (s,
3H), 4.17 (m, 2H), 4.73 (d, J ) 2.5 Hz, 2H), 6.31 (d, J ) 12.4
Hz, 1H), 6.75-7.91 (m, 4H.), 6.99 (d, J ) 12.4 Hz, 1H); 13C
NMR (125 MHz, CDCl3) δ 30.4, 30.9, 32.4, 39.8, 55.1, 57.9,
70.5, 78.5, 114.2, 114.2, 114.6, 121.0, 129.6, 136.4, 139.8, 107.1,
148.3, 149.2, 150.7, 154.0, 159.5; EIMS (m/z) 394 (M+, 100).
Solid -St a t e P h ot or ea ct ion of (E)-P h osp h or ic Acid
Mon o[3-[8-[2-(3-m eth oxyph en yl)vin yl]-7-m eth yl-2,6-dioxo-
1-p r op -2-yn yl-1,2,6,7-tetr a h yd r op u r in -3-yl]p r op yl] Ester
Disod iu m Sa lt (9). An analytical sample of 9 disodium salt
was exposed to daylight for 4 days. The crude 9c was analyzed
by mass spectrometry: ESI negQ1MS m/z 947 (M4- + 3H+),
969 (M4- + 2H+ + Na+).
solution of 1.0 g (2.5 mmol) of 8 in phosphoric acid trimethyl
ester (10 mL) was prepared by gentle heating. After cooling
to 0 °C, 1.0 mL (10 mmol) of phosphoryl chloride was added
to the stirred solution. The mixture was stirred at rt under
exclusion of light for 0.5 h and subsequently hydrolyzed with
a mixture of ice and NaCl at about -20 °C. The formed yellow,
light-sensitive precipitate was filtered off, washed with water
(350 mL), and dried at 70 °C (g95% yield): mp 193-195 °C;
1H NMR (500 MHz, DMSO-d6) δ 2.03 (m, 2H), 3.06 (t, J ) 2.2
Hz, 1H), 3.81 (s, 3H), 3.93 (m, 2H), 4.03 (s, 3H), 4.11 (m, 2H),
4.59 (d, J ) 2.2 Hz, 2H), 6.92-7.31 (m, 5H), 7.66 (d, J ) 15.8
Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 28.86 (d, J ) 7 Hz),
30.11, 31.68, 40.28, 55.39, 63.39 (d, J ) 5 Hz), 72.9, 79.8, 112.7,
113.1, 115.4, 120.4, 129.9, 137.1, 137.3, 107.4, 148.0, 150.0,
150.2, 153.4, 159.8; 31P NMR (202 MHz, DMSO-d6) δ -0.29;
ESI negQ1 m/z 473 (M - H+). TLC control: (methanol, Rf )
0.71, spraying reagent for phosphate derivatives: 0.1% FeCl3
and 7% 5′-sulfosalicylic acid in 75% aq ethanol).
Isola tion of 9a . When the reaction of 8 to 9 was performed
using a larger excess of phosphoryl chloride (molar ratio
8/POCl3, 1:10) combined with hydrolysis at higher temperature
(rt), chlorination of the hydroxyl group became predominant.
The byproduct (E)-3-(3-chloropropyl)-8-[2-(3-methoxyphenyl)-
vinyl]-7-methyl-1-prop-2-ynyl-3,7-dihydropurine-2,6-dione 9a
was isolated by extracting the reaction mixture with CH2Cl2
after hydrolysis. The organic extract was dried over MgSO4,
filtered, and concentrated in vacuo. The residue was crystal-
lized from CH2Cl2/petroleum ether (1:1) to yield 9a as a pale
1
yellow solid: mp 223.2 °C dec; H NMR (500 MHz, CDCl3) δ
2.16 (t, J ) 2.2 Hz, 1H), 2.30 (m, 2H), 3.63 (m, 2H), 3.84 (s,
3H), 4.04 (s, 3H), 4.30 (m, 2H), 4.77 (d, J ) 2.2 Hz, 2H), 6.86
(d, J ) 15.8 Hz, 1H), 6.91-7.32 (m, 4H), 7.76 (d, J ) 15.8 Hz,
1H); 13C NMR (125 MHz, CDCl3) δ 30.5, 31.2, 31.6, 41.3, 55.4,
42.3, 70.4, 78.7, 111.3, 112.8, 115.2, 120.0, 129.9, 136.8, 138.8,
107.9, 148.5, 150.3, 150.6, 154.1, 160.0; EIMS (m/z) 412 (M+,
100).
(E)-P h osp h or ic Acid Mon o[3-[8-[2-(3-m eth oxyp h en yl)-
vin yl]-7-m et h yl-2,6-d ioxo-1-p r op -2-yn yl-1,2,6,7-t et r a h y-
d r op u r in -3-yl]p r op yl] Ester Disod iu m Sa lt (MSX-3).10 In
a solution of 90 mg (2.3 mmol) of NaOH in water (ca. 80 mL)
was dissolved 500 mg (1.05 mmol) of crude 9 under stirring
Solid -St a t e P h ot or ea ct ion of 9a t o (1r,2r,3â,4â)-
1,3-Bis[3-(3-ch lor op r op yl)-7-m eth yl-1-p r op -2-yn yl-3,7-d i-
h ydr opu r in e-2,6-dion -8-yl]-2,4-bis(3-m eth oxy)ph en ylcyclo-
bu ta n e (9b). A thin crystal film of 50 mg (0.12 mmol) of 9a
3316 J . Org. Chem., Vol. 69, No. 10, 2004

A2A Adenosine Receptor Antagonists with 8-Styrylxanthine Structure
was prepared by dissolving the yellow solid in a minimum of
CH2Cl2 and subsequently leaving the solvent to evaporate on
the inner surface of a round-bottomed flask. After 8 h under
daylight conditions, the formed colorless product was rerys-
tallized from diethyl ether (30 mg yield): mp 243.8 °C dec; 1H
NMR (500 MHz, CDCl3) δ 2.14 (t, J ) 2.5 Hz, 2H), 2.23 (m,
4H), 3.58 (m, 4H), 3.65 (s, 6H), 3.70 (s, 6H), 4.28 (m, 4H), 4.43-
4.46 and 5.00-5.04 (AA′BB′, 4H), 4.69 (m, 4H), 6.68-7.13 (m,
8H); 13C NMR (125 MHz, CDCl3) δ 30.4, 31.3, 31.6, 40.9, 42.5,
40.7, 44.6, 55.2, 70.3, 78.7, 112.0, 114.1, 119.8, 129.7, 139.8,
107.7, 147.6, 150.5, 152.6, 154.0, 159.7; EIMS (m/z) 825 (M+,
22).
(500 MHz, CDCl3) δ 1.16 (t, J ) 7.25 Hz, 3H), 1.28 (t, J ) 7.25
Hz, 3H), 3.86 (s, 3H), 3.87 (s, 3H), 3.94 (m, 4H), 5.72 (s, 2H),
6.54 (d, J ) 15.4 Hz, 1H), 6.79 (d, J ) 8.5 Hz, 1H), 6.97 (d, J
) 1.6 Hz, 1H.), 7.02 (dd, J ) 8.5 (1.6) Hz, 1H.), 7.51 (d, J )
15.4 Hz, 1H), 7.81 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 13.1,
13.3, 36.9, 38.3, 55.8, 55.9, 92.4, 111.0, 117.6, 122.2, 127.5,
142.0, 149.1, 149.7, 150.8, 159.8, 165.8; EIMS (m/z) 388 (M+,
100).
(E)-8-[2-(3,4-Dim et h oxyp h en yl)vin yl]-1,3-d iet h yl-3,7-
d ih yd r op u r in e-2,6-d ion e (15).20,28,32,33,35 Method A: To a
solution of 1 g (2.6 mmol) of 14 in dioxane (30 mL) was added
30 mL of a 1 M solution of NaOH. The clear yellowish solution
was heated under reflux for 10 min, diluted with 250 mL of
water, and acidified by dropwise addition of concd HCl (f pH
= 2). The precipitate was filtered off, washed with 300 mL of
water, and dried at 70 °C (62% yield).
Method B: A mixture of 1.18 g (3 mmol)of 14, (NH4)2SO4
(0.2 g) and HMDS (10 mL) was heated in a glass pressure tube
at 170-180 °C for 4 h. The reaction was monitored by TLC
(ethyl acetate/methanol, 1:1). The end of the reaction was
reached when the molten, poorly soluble starting material had
been transformed to a fine suspension of colorless product in
a clear yellow solution. After hydrolysis by addition of metha-
nol (20 mL), the mixture was concentrated under reduced
pressure with cooling, and the obtained residue was treated
with ethyl acetate (40 mL) to dissolve unconverted starting
material. The colorless product was filtered under reduced
pressure, washed with ethyl acetate (40 mL), and dried at 70
°C (92% yield): mp 264.7 °C; 1H NMR (500 MHz, CDCl3) δ
1.30 (t, J ) 7.3 Hz, 3H), 1.39 (t, J ) 7.3 Hz, 3H), 3.91 (s, 6H),
4.22 (m, 4H), 6.94 (d, J ) 16.2 Hz, 1H) 6.86-7.12 (m, 3H),
7.73 (d, J ) 16.2 Hz, 1H), 13.00 (s, 1H); 13C NMR (125 MHz,
CDCl3) δ 13.4, 13.5, 36.9, 39.0, 55.8, 56.0, 107.3, 109.1, 111.2,
113.4, 121.2, 128.6, 136.8, 149.3, 149.7, 150.4, 150.5, 151.7,
155.6; EIMS (m/z) 370 (M+, 100).
Recep tor Bin d in g Stu d ies. Radioligand competition ex-
periments were performed as previously described using rat
brain cortical membrane preparations as a source of A1, and
a rat brain striatal membrane preparation for A2A adenosine
receptor assays.46 [3H]-2-Chloro-N6-cyclopentyladenosine ([3H]-
CCPA) was used as A1 radioligand and [3H]-(E)-3-(3-hydroxy-
propyl)-8-[2-(3-methoxyphenyl)vinyl]-7-methyl-1-prop-2-ynyl-3,7-di-
hydropurine-2,6-dione ([3H]MSX-2) as A2A ligand.
The frontal cortex of rat brains was dissected as A1 receptor
source, and right and left striata were dissected for A2A
adenosine receptor studies. Tissues were homogenized in 50
mM Tris-HCl buffer (pH 7.4) which was used as buffer in all
following steps, unless otherwise noted. Membrane fractions
were purified by a series of centrifugation steps as described.47-49
For each assay, 1 mL of membrane suspension containing
about 70 µg/mL protein was preincubated with adenosine
deaminase (ADA) to convert endogenous adenosine into inac-
tive inosine. To obtain inhibition curves, six to seven different
concentrations of the tested compound in DMSO were pre-
pared, spanning 3 orders of magnitude. At least three separate
experiments were performed, each in triplicate.
Solid -Sta te P h otor ea ction of 16 to (1r, 2r, 3â, 4â)-1,3-
Bis[1,3-d iet h yl-7-m et h yl-3,7-d ih yd r op u r in e-2,6-d ion -8-
yl]-2,4-bis(3,4-dim eth oxy)ph en ylcyclobu tan e (16b). A crys-
tal film of 40 mg (0.1 mmol) of 16 was prepared by dissolving
the yellow solid in a minimum of CH2Cl2 and subsequently
leaving the solvent to evaporate on the inner surface of a
photoreactor. After 100 min of irradiation, the essentially
decolorized product was dissolved in CH2Cl2, filtered to remove
oligomeric material, and recrystallized by addition of diethyl
1
ether to yield colorless crystals (12 mg): mp 259 °C dec; H
NMR (500 MHz, CDCl3) δ 1.18 (t, 6H, J ) 7.3 Hz), 1.33 (t, 6H,
J ) 7.3 Hz), 3.57 (s, 6H), 3.68 (s, 6H), 3.78 (s, 6H), 3.99 (q, J
) 7.3 Hz, 4H), 4.18 (q, J ) 7.3 Hz, 4H), 4.26-4.29 and 5.01-
5.05 (AA′BB′, 4H), 6.63 (d, J ) 1.9 Hz, 2H), 6.68 (d, J ) 8.2
Hz, 2H), 6.83 (dd, J ) 8.2 (1.9) Hz, 2H); 13C NMR (125 MHz,
CDCl3) δ 13.3, 13.5, 31.4, 36.3, 38.3, 42.0, 43.9, 55.8, 55.8,
108.1, 110.5, 110.9, 120.5, 131.1, 147.1, 148.5, 148.9, 150.6,
152.1, 154.9; EIMS (m/z) 769 (M+, 58).
Solid -Sta te P h otor ea ction of 7 (MSX-1) a n d 18. A
crystal film of 7 or 18, respectively, was prepared by dissolving
ca. 50 mg of the compound in a minimum of THF and
subsequently leaving the solvent to evaporate on the inner
surface of a round-bottomed flask. After 4 weeks under
daylight conditions the crude colorless products were inves-
tigated by NMR and mass spectroscopy.
(1r, 2r, 3â, 4â)-1,3-Bis[3-(3-h yd r oxyp r op yl)-1-p r op -2-
yn yl-3,7-d ih yd r op u r in e-2,6-d ion e-8-yl]-2,4-b is(3-m et h -
oxy)p h en ylcyclobu ta n e (7b): 1H NMR (500 MHz, DMSO-
d6) δ 1.72 (m, 4H), 3.02 (t, 2H, J ) 2.2 Hz), 3.40 (m, 4H), 3.63
(s, 6H), 4.00 (m, 4H), 4.47 (br s, 2H), 4.60-4.64 and 4.69-
4.72 (AA′BB′, 4H), 4.53 (d, J ) 2.2 Hz, 4H), 6.66-7.17 (m, 8H),
13.5 (br s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 30.2, 31.0,
40.8, 41.0, 44.7, 55.0, 58.5, 72.8, 78.8, 112.3, 113.3, 119.7, 129.2,
140.3, 107.3, 148.8, 150.0, 150.1, 152.9, 159.1; EIMS (m/z) 760
(M+, 25).
(1r, 2r, 3â, 4â)-1,3-Bis[3-(3-a cetoxyp r op yl)-1-p r op -2-
yn yl-3,7-d ih yd r op u r in e-2,6-d ion -8-yl]-2,4-bis(3-m eth oxy)-
p h en ylcyclobu ta n e (18b): 1H NMR (500 MHz, CDCl3) δ 2.00
(m, 4H), 2.04 (s, 6H), 2.27 (t, J ) 2.2 Hz, 2H), 3.59 (s, 6H),
4.13 (m, 4H), 4.05-4.10 and 4.18-4.24 (AA′BB′, 4H), 4.76 (m,
4H), 4.79 (s, 4H), 6.55-7.03 (m, 8H), 12.16 (s, 2H); 13C NMR
(125 MHz, CDCl3) δ 21.0, 27.2, 31.0, 41.0, 41.3, 45.4, 55.1, 62.1,
71.2, 78.7, 106.5, 111.5, 113.9, 119.7, 129.3, 140.0, 149.2, 150.2,
154.5, 154.7, 159.5, 171.2; EIMS (m/z) 844 (M+, 18).
(E )-N -(6-Am in o-1,3-d ie t h y l-2,4-d iox o -1,2,3,4-t e t r a -
h y d r o p y r i m i d i n -5 -y l )-3 -(3 ,4 -d i m e t h o x y p h e n y l )-
a cr yla m id e (14).20,28,32,33,35 To a solution of 3 g (15.1 mmol)
of 13 and pyridine (6 mL) in CH2Cl2 (150 mL) was added 5 g
(22 mmol) of (E)-3,4-dimethoxycinnamic acid chloride45 at 20
°C under vigorous stirring. After 16 h of stirring at rt, the
mixture was diluted with additional CH2Cl2 (100 mL) and
washed three times with 100 mL of 5% NaHCO3 solution each.
The organic phase was dried over MgSO4, filtered over silica
gel, and concentrated in vacuo. The oily residue was recrystal-
lized from methanol/water (1:5) to form colorless needles. After
removal of crystal water at 120 °C, a yellowish solid was
obtained (65% yield): mp 135-140 °C (sublimation); 1H NMR
For radioligand binding to rat brain cortical membranes,
the solution of the test compound was diluted with buffer.
Subsequently a diluted stock solution of the radioligand was
added in order to obtain a final concentration of 0.5 nM of [3H]-
CCPA. Finally, the cortical membrane suspension was added
and the samples were incubated on a shaking water bath at
23 °C for 90 min.
(46) Mu¨ller, C. E.; Thorand, M.; Qurishi, R.; Dieckmann, M.;
J acobson, K. A.; Padgett, W. L.; Daly, J . W. J . Med. Chem. 2002, 45,
3440.
(47) Bruns, R. F.; Lu, G. H.; Pugsley, T. A. Mol. Pharmacol. 1986,
29, 331-346.
(48) J acobson, K. A.; Ukena, D.; Kirk, K. L.; Daly, J . W. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 4089-4093.
(45) Matulenko, M. A.; Meyers, A. I. J . Org. Chem. 1996, 61, 2, 573-
580.
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J . Org. Chem, Vol. 69, No. 10, 2004 3317

Hockemeyer et al.
Incubation was terminated by rapid filtration using a cell
harvester through GF/B glass fiber filters, which had been
presoaked with buffer. Filters were rinsed three times with 2
mL each of the buffer. Nonspecific binding was defined using
10 µM of 2-chloroadenosine and amounted to less than 5% of
total binding.
For radioligand binding to rat brain striatal membranes the
solution of the test compound was diluted with buffer and
treated with a diluted stock solution of the radioligand in order
to obtain a 1 nM final concentration of [3H]MSX-2. After
addition of the striatal membrane suspension, incubation (30
min) and termination by rapid filtration were performed as
described above. The filters were presoaked with 0.5% aqueous
polyethylenimine solution for 45 min prior to filtration.
Nonspecific binding was defined using 50 µM 5′-(N-ethylcar-
boxamido)adenosine (NECA) and amounted to less than 25%
of total binding.
in a liquid scintillation counter. Data were analyzed using
Graph Pad PRISM version 3.0 (San Diego, CA).
Ack n ow led gm en t. We gratefully acknowledge sup-
port for our studies by the Fonds der Chemischen
Industrie and the Bundesministerium fu¨r Bildung und
Forschung. We thank Mr. Alaa M. Hayallah for isolating
compound 2a and Ms. Sonja Hinz and Mr. Dieter
Baumert for performing the radioligand binding studies.
Su p p or tin g In for m a tion Ava ila ble: A detailed Experi-
mental Section is given, including improved syntheses, spec-
troscopic data of all intermediates, and final products, together
with assignments of NMR data. Copies of 1H NMR spectra
and microanalytical data of all new compounds reported in
this manuscript are available. This material is available free
of charge via the Internet at http://pubs.acs.org.
Radioactivity of the punched-out wet filters was counted
after 9 h of preincubation with 3 mL of scintillation cocktail
J O0358574
3318 J . Org. Chem., Vol. 69, No. 10, 2004