Synthesis and biological evaluation of the enantiomers of the potent and selective A1-adenosine antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbonyl)]- xanthine

Article abstract of DOI:10.1021/jm970013w

The individual enantiomers 8 and 12 of the potent and highly selective racemic A₁-adenosine antagonist 1,3-dipropyl-8-[2-(5,6- epoxynorbornyl)]xanthine (ENX, 4) were synthesized utilizing asymmetric Diels-Alder cycloadditions for the construction of the norbornane moieties. The absolute configuration of 12 was determined by X-ray crystallography of the 4-bromobenzoate 14, which was derived from the bridged secondary alcohol 13. The latter was obtained from 12 by an acid-catalyzed intramolecular rearrangement. The binding affinities of the enantiomers 8 and 12 and the racemate 4 at guinea pig, rat, and cloned human A₁- and A(2a)-adenosine receptor subtypes were determined. The S-enantiomer 12 (CVT-124) appears to be one of the more potent and clearly the most A₁-selective antagonist reported to date, with K(i) values of 0.67 and 0.45 nM, respectively, at the rat and cloned human A₁-receptors and with 1800-fold (rat) and 2400-fold (human) subtype selectivity. Both enantiomers, administered intravenously to saline-loaded rats, induced diuresis via antagonism of renal A₁-adenosine receptors.

Full text of DOI:10.1021/jm970013w

J . Med. Chem. 1997, 40, 1773-1778
1773
Expedited Articles
Syn th esis a n d Biologica l Eva lu a tion of th e En a n tiom er s of th e P oten t a n d
Selective A1-Ad en osin e An ta gon ist 1,3-Dip r op yl-8-[2-(5,6-ep oxyn or bon yl)]-
xa n th in e
J u¨rg R. Pfister,*,† Luiz Belardinelli,‡,§ Gavin Lee,† Robert T. Lum, Peter Milner,| William C. Stanley,
J oel Linden,∇ Stephen P. Baker,‡ and George Schreiner#
CV Therapeutics, Palo Alto, California 94304, Departments of Medicine and Pharmacology, College of Medicine,
University of Florida, Gainesville, Florida 32610, and University of Virginia School of Medicine,
Charlottesville, Virginia 22908
Received J anuary 8, 1997X
The individual enantiomers 8 and 12 of the potent and highly selective racemic A1-adenosine
antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine (ENX, 4) were synthesized utilizing
asymmetric Diels-Alder cycloadditions for the construction of the norbornane moieties. The
absolute configuration of 12 was determined by X-ray crystallography of the 4-bromobenzoate
14, which was derived from the bridged secondary alcohol 13. The latter was obtained from
12 by an acid-catalyzed intramolecular rearrangement. The binding affinities of the enanti-
omers 8 and 12 and the racemate 4 at guinea pig, rat, and cloned human A1- and A2a-adenosine
receptor subtypes were determined. The S-enantiomer 12 (CVT-124) appears to be one of the
more potent and clearly the most A1-selective antagonist reported to date, with Ki values of
0.67 and 0.45 nM, respectively, at the rat and cloned human A1-receptors and with 1800-fold
(rat) and 2400-fold (human) subtype selectivity. Both enantiomers, administered intravenously
to saline-loaded rats, induced diuresis via antagonism of renal A1-adenosine receptors.
In tr od u ction
Despite extensive efforts over several decades in both
academic and industrial laboratories,1 the only approved
adenosinergic agent is the nonselective endogenous
agonist adenosine itself, which is used for the treatment
and diagnosis of cardiac arrhythmias (Adenocard)2 and,
together with radionuclide imaging, for the diagnosis
of ischemic heart disease (Adenoscan).3 The recent
availability of cloned A1-, A2a-, A2b-, and A3-adenosine
receptors4 has facilitated the pharmacological screening
of ligands for these receptors and has contributed to a
new impetus for the search for more potent and selective
agonists and antagonists. Among the latter, A1-selec-
tive antagonists related to 8-cycloalkyl-substituted 1,3-
dipropylxanthines5 have recently received particular
attention. Representatives of this group are KF-15372
(1) and KW-3902 (NAX, 2, Figure 1), for which diuretic
activity and protective effects against acute renal failure
have been described.6,7 In contrast, the chiral oxocy-
clopentyl derivative KFM 19 (3, Figure 1) appears to
have therapeutic potential for dementia and related
cognitive deficits.8 Because adenosine receptors are
widely distributed both in the vasculature9 and in the
CNS,10 acute versus chronic administration of one of
F igu r e 1. Newer, selective A1-adenosine antagonists: KF-
15372 (1), KW-3902 (NAX, 2), KFM 19 (3), and ENX (4).
these fairly lipophilic dipropylxanthines could be im-
portant in terms of separating peripheral from central
effects.11
Recently, we described the biological activity of the
potent and highly selective A1-adenosine receptor an-
tagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine12
(ENX, 4, Figure 1). In this compound, the xanthine ring
and the epoxide are respectively situated endo and exo
to the norbornane moiety, and the latter has an asym-
metric center at C-2. Due to the marked stereochemical
requirements for adenosine receptor affinity exhibited
by both agonist and antagonist asymmetric ligands,13
it became of interest to synthesize the individual enan-
tiomers of 4 and to evaluate their biological activity.
* Address for correspondence: Dr. J . Pfister, Roche Bioscience R6-
201, 3401 Hillview Ave, Palo Alto, CA 94304-1397. E-mail: jurg.
pfister@roche.com.
† Current address: Roche Bioscience, Palo Alto, CA 94304.
‡ Department of Pharmacology, University of Florida.
§ Department of Medicine, University of Florida.
| Current address: ARYx Therapeutics, Los Altos Hills, CA 94022.
Current address: Case Western Reserve University, Cleveland,
OH 44106.
∇ University of Viginia School of Medicine.
#
Current address: Scios Inc., Sunnyvale, CA 94086.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(97)00013-7 CCC: $14.00 © 1997 American Chemical Society

1774 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Pfister et al.
Sch em e 1a
a
(a) NaOH; (b) oxalyl chloride; (c) 5,6-diamino-1,3-dipropyluracil, cat. DMAP, pyridine; (d) 2 N NaOH, dioxane; (e) MMPP, aqueous
i-PrOH.
Sch em e 2a
a
(a) NaOH; (b) oxalyl chloride; (c) 5,6-diamino-1,3-dipropyluracil, cat. DMAP, pyridine; (d) 2 N NaOH, dioxane; (e) MMPP, aqueous
i-PrOH.
Sch em e 3a
a
(a) 2 N HCl, EtOH; (b) 4-bromobenzoyl chloride, pyridine.
Ch em istr y
moiety in these compounds should render the endo-face
of the norbornenyl group even less sterically accessible.
In a transformation mechanistically related to the
iodolactonization of endo-5-norbornene-2-carboxylic acid,19
(S)-epoxynorbornylxanthine 12 underwent an acid-
catalyzed intramolecular cyclization reaction on treat-
ment with HCl in ethanol (Scheme 3). The resulting
secondary alcohol 13 was converted into the 4-bro-
mobenzoate 14, which was subjected to X-ray crystal
structure analysis (Figure 2). This conclusively proved
that the absolute configuration at C-2 of the norbornane
group in 12 is S and that the epoxide and the xanthine
are respectively situated exo and endo to the carbocycle.
From the many known norbornene syntheses based
on asymmetric Diels-Alder cycloadditions with dieno-
philes containing removable chiral auxiliaries,14 those
utilizing (S)-2-hydroxy-N-methylsuccinimde15 and pan-
tolactone16 were chosen. They provided the required
optically active precursors 5 and 9, respectively, which
were treated with aqueous NaOH to remove the chiral
auxiliaries (Schemes 1 and 2). The resulting crude
carboxylic acids were directly converted into the corre-
sponding acid chlorides, which produced amides 6 and
10 on reaction with 1,3-dipropyluracil-5,6-diamine. Base-
induced ring closure provided the endo-norbornenyl-
substituted xanthines 7 and 11. The chromatographi-
cally distinct exo-analog5c was not observed, indicating
that the cyclization step had occurred without epimer-
ization.
Epoxidation of 7 and 11 with magnesium monoper-
oxyphthalate17 resulted stereoselectively in the forma-
tion of the desired target exo-epoxides 8 and 12. Since
the peracid oxidation of norbornene itself is already
highly exo-selective18 (exo:endo ratio ca. 700:1), it is
unlikely that any endo epoxidation could take place with
either 7 or 11, because the presence of the xanthine
Resu lts a n d Discu ssion
The binding affinities of 4 (ENX, racemate) and its
R- and S-enantiomers 8 and 12 were determined at
guinea pig, rat, and cloned human A1- and A2a-adenos-
ine receptors (see the Experimental Section). While
there was little differentiation between the enantiomers
at guinea pig and human A1 receptors, 12 (Ki ca. 0.5
nM at both rat and human receptors) appeared to be
approximately 8-fold more potent than 8 in the rat
(Table 1). Affinities at the A2a receptor from all three

A1-Adenosine Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1775
Ta ble 1. Binding Affinities of Enantiomers 8 and 12, Racemate 4, and Other Alkylxanthines at A1- and A2a-Adenosine Receptors
A1K1 (nM)
A2aKi (nM)
compd
guinea piga
rata
humanb
guinea pigc
ratc
humand
8
3.17 ( 0.005
3.15 ( 0.19
2.56 ( 0.23
1.10 ( 0.59e
2.93 ( 0.89e
5.12 ( 1.63
1.43 ( 0.20
0.67 ( 0.14
12.60 ( 2.90
3.77 ( 0.45
0.80 ( 0.09
0.82 ( 0.05
0.45 ( 0.04
0.72 ( 0.12
2.52 ( 0.05
250 ( 38
465 ( 197
930 ( 30
230 ( 75e
320 ( 87e
551 ( 173
309 ( 61
201 ( 24
475 ( 66
1100 ( 318
108 ( 15
157 ( 53
4 (ENX)
12
2 (KW-3902)
CPX
1250 ( 262
510 ( 10
260 ( 40
a
b
Inhibition of radioligand binding to membranes from guinea pig and rat forebrain. Inhibition of binding to membranes from HEK-
293 cells stably transfected with recombinant human A1 AdoRs. c Inhibition of binding to membranes from guinea pig and rat striatal
d
tissue. Inhibition of binding to membranes from HEK-293 cells stably transfected with recombinant human A2a AdoRs. Radioligands
(see the Experimental Section) were used at or below their Kd values. e Reference 12.
Ta ble 3. Diuretic Effects of (Epoxynorbornyl)xanthines in
Saline-Loaded Ratsa
urine output
(mL/kg
per 4 h)
Na+ output
(mmol/kg
per 4 h)
K+ output
(mmol/kg
per 4 h)
dose
(mg/kg, iv)
compd
vehicle
8
20.3 ( 2.8
26.8 ( 3.3
31.3 ( 2.3
44.1 ( 4.2*
23.2 ( 2.6
33.0 ( 2.0
2.90 ( 0.30
3.80 ( 0.30
4.56 ( 0.16* 0.89 ( 0.09
5.99 ( 0.47* 1.84 ( 0.14*
0.89 ( 0.09
0.97 ( 0.10
0.01
0.1
1.0
0.01
0.1
1.0
0.01
0.1
1.0
4 (ENX)
3.54 ( 0.34
5.14 ( 0.28
1.10 ( 0.09
1.22 ( 0.05
43.2 ( 1.3** 6.54 ( 0.08* 1.42 ( 0.16
12
21.7 ( 2.3
35.1 ( 2.3*
37.1 ( 1.9*
3.02 ( 0.25
0.75 ( 0.15
5.29 ( 0.23* 1.34 ( 0.19
5.86 ( 0.35* 1.09 ( 0.07
a
Compounds were administered intravenously to rats (n ) six
or seven per dose), and urine was collected for 4 h; see the
Experimental Section for details. Values are expressed as means
( SEM of vehicle control and drug treatment values, respectively.
*p < 0.01. **p < 0.05.
binding modes proposed for 8-substituted xanthines
with respect to the corresponding N6-substituted ad-
enosine analogs,21 these observations decisively support
the so-called “N6-C8” model first postulated by Peet et
al.,13c according to which xanthines bind to the receptor
in a flipped and rotated orientation with respect to
adenosines.
In accordance with the finding that introduction of
bulky substituents into the 8-position of 1,3-dialkyl-
xanthines attenuates phosphodiesterase (PDE) inhibi-
tory activity,22 relatively high concentrations of 12 (EC50
values 10-300 µM; data not shown) were required to
inhibit PDE isoenzymes types I-V.
F igu r e 2. X-ray crystal structure of 4-bromobenzoyl deriva-
tive 14. The atom numbering system used is not conventional.
Ta ble 2. Selectivity Ratios for Enantiomers 8 and 12 and
Other Alkylxanthines
Ki ratio A2a/A1
compd
stereochem
guinea pig
rat
human
The diuretic activity of the test compounds was
assessed intravenously in saline-loaded rats (Table 3).
Both enantiomers and the racemate increased urine and
sodium output in a dose-dependent manner. In keeping
with the relatively small affinity difference (<10-fold)
between 8 and 12 for the rat A1-receptor, there was
virtually no difference in their potency in vivo.
The S-enantiomer 12 (CVT-124) recently completed
a phase I study in humans, demonstrating a diuresis
profile characterized by dose-dependent increases in the
excretion of sodium ion, chloride, and uric acid.23
Because concomitant increases in potassium ion excre-
tion were not observed, this agent could be useful as a
potent and potassium-sparing diuretic for the treatment
of edema associated with congestive heart failure.
8
12
R
S
80
360
210
110
110
1800
40
250
2400
150
60
2 (KW-3902)
CPX
70
species were much lower (Ki values 250-1250 nM). Both
enantiomers thus showed good A1-selectivity across the
three species (Table 2), with the S-enantiomer 12 being
the more selective, particularly at the rat (1800-fold) and
the human (2400-fold) adenosine receptors. In com-
parison, the selectivity of both the noradamantyl de-
rivative 2 and the well-known antagonist CPX (8-
cyclopentyl-1,3-dipropylxanthine) was more than 10-fold
lower and of the same order of magnitude as that of the
racemic endo-norbornyl analog lacking the epoxide
functionality.7a On the basis of these data, the S-
enantiomer 12 appears to be one of the more potent and
clearly the most A1-selective adenosine receptor antago-
nist reported to date.
Exp er im en ta l Section
Ch em istr y: Gen er a l. Melting points were determined on
a Mel-Temp II apparatus and are uncorrected. Proton and
carbon NMR spectra were recorded in deuteriochloroform on
a Varian Gemini 400 spectrometer. Chemical shifts are
reported in ppm downfield from tetramethylsilane (internal
standard) and coupling constants (J ) in hertz (Hz). Mass
Recently, it was reported that the structurally analo-
gous S-enantiomer of N6-epoxynorbornyladenosine was
10-12-fold more potent as an A1-agonist than the
corresponding R-enantiomer.20 Of the three different

1776 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Pfister et al.
spectra were recorded at the University of Kansas on a
Nermag R10-10 quadrupole GC/MS system with SPECTRAL
30 data system. Optical rotations were measured on a J ASCO
DIP-370 digital polarimeter in chloroform. Elemental analyses
were performed by Galbraith Laboratories, Inc., Knoxville, TN.
Analytical thin-layer chromatography (TLC) was conducted on
Analtech silica gel GF plates. Compounds were visualized
with UV light, iodine vapor, or ammonium molybdate/sulfuric
acid spray. Centrifugally accelerated preparative thin-layer
chromatography was performed on a Chromatotron model
7924T (Harrison Research, Palo Alto, CA). Chiral purity of
the target compounds was determined on a Waters HPLC
system fitted with a Chiralcel OD column under isocratic
conditions with a hexane/2-propanol (98:2) mobile phase.
Racemic 1,3-dipropyl-8-(5,6-exo-epoxy-2-endo-norbornyl)xan-
thine was prepared as described previously.12
1,3-Dipr opyl-8-[(1S,2S)-5-n or bor n en -2-yl]xan th in e (11).
The procedure for preparing 7 was followed, except that 100
mL of a THF/MeOH/water mixture (3:3:4) was used for the
hydrolytic removal of the chiral auxiliary. Starting with 7.87
g (31.5 mmol) of 9,16 6.5 g (63%) of recrystallized 11 were
obtained in this manner: mp 140-141 °C; [R]20D ) -142.3° (c
1
) 2.0); H NMR δ 0.98 (6H, m), 1.46 (1H, d, J ) 8 Hz), 1.57
(1H, d, J ) 8 Hz), 1.72 (3H, m), 1.81 (2H, m), 2.25 (1H, m),
3.04 (1H, s), 3.46 (1H, s), 3.56 (1H, m), 4.03 (2H, m), 4.10 (2H,
m), 5.84 (1H, m), 6.27 (1H, m); 13C NMR δ 11.17, 11.38, 21.35,
31.22, 38.73, 42.92, 43.25, 45.15, 47.52, 49.78, 132.63, 138.13,
151.05, 155.20, 158.05. Anal. (C18H24N4O2) C, H, N.
1,3-Dip r op yl-8-[5,6-exo-ep oxy-(1S,2S)-n or b or n -2-yl]-
xa n th in e (12). Epoxidation of 11 (8.4 g, 25.6 mmol) with
MMPP (15.8 g) as described for the preparation of 8 afforded
5.8 g (66%) of 12 as a white solid: mp 143-145 °C (ether/
hexane); [R]20 ) -67.25° (c ) 2.0); ee ) 97.2%; 1H NMR δ
1,3-Dip r op yl-8-[(1R,2R)-5-n or bor n en -2-yl]xa n th in e (7).
To a solution of 10.7 g (43 mmol) of the norbornenecarboxylic
ester 515 in 100 mL of methanol was added a solution of 8.6 g
of NaOH (215 mmol) in 30 mL of water. After being stirred
at room temperature for 4 h, the solution was concentrated
on a rotary evaporator, cooled in an ice bath, and acidified with
2 N HCl. The resulting mixture was extracted with dichlo-
romethane (3 × 200 mL), and the combined extracts were
washed with brine. After being dried over MgSO4, the extracts
were filtered and concentrated to ca. 150 mL. DMF (3 drops)
was added, followed by a mixture of oxalyl chloride (4.7 mL)
and dichloromethane (20 mL) dropwise with stirring. After
being stirred at room temperature for an additional 2.5 h, the
reaction mixture was evaporated to dryness and then coevapo-
rated twice with dichloromethane (50 mL) to remove traces of
oxalyl chloride. The residue was cooled in an ice bath under
dry nitrogen for 15 min and then dissolved in pyridine (75 mL).
DMAP (500 mg) and 5,6-diamino-1,3-dipropyluracil24 (10.7 g)
were added, and the resulting mixture was stirred at ambient
temperature for 2 h, then cooled in an ice bath, and acidified
with 6 N HCl. After extraction with AcOEt (2 × 300 mL),
and evaporation to dryness, the resulting amide 6 was heated
under reflux with 2 N aqueous NaOH (150 mL) in dioxane
(150 mL) for 4 h. Concentration on a rotary evaporator to
remove most of the dioxane, followed by neutralization with 2
N HCl and extraction with AcOEt, afforded crude 7, which
was purified by filtration through a short column of silica gel
(3:1 hexanes/AcOEt) followed by crystallization from CH2Cl2/
hexanes (final volume ca. 80 mL). White needles of 7 (10.46
D
0.96 (3H, t, J ) 7.5 Hz), 0.98 (3H, t, J ) 7.5 Hz), 1.00 (1H, m),
1.54 (1H, m), 1.72 (2H, m), 1.79 (2H, m), 2.06 (2H, m), 2.67
(1H, m), 3.08 (1H, m), 3.10 (1H, m), 3.25 (1H, m), 3.43 (1H,
m), 4.02 (2H, t, J ) 7.5 Hz), 4.11 (2H, t, J ) 7.5 Hz); 13C NMR
δ 11.22, 11.44, 21.38, 27.68, 29.60, 37.36, 39.99, 41.68, 43.46,
45.28, 49.40, 51.09, 106.92, 149.01, 150.87, 155.68, 156.08; MS
m/e 344 (M+). Anal. (C18H24N4O3) C, H, N.
Rea r r a n gem en t P r od u ct 13. A solution of 12 (850 mg,
2.47 mmol) in EtOH (17.5 mL) containing 6 N HCl (2.5 mL)
was stirred at room temperature for 30 h and then concen-
trated to a small volume under vacuum. After dilution with
brine (10 mL), the mixture was extracted twice with AcOEt
(30 mL). The combined extracts were washed with brine, dried
over MgSO4, filtered, and evaporated. The residue was
purified on the chromatotron (5% MeOH in CH2Cl2) to give
710 mg (83.5%) of 13 as a white foam. An analytical sample
was obtained by recrystallization from AcOEt/i-Pr2O: mp 131-
1
133 °C; [R]20 ) -30.8° (c ) 2.0); H NMR δ 0.93 (3H, t, J )
D
7.5 Hz), 1.02 (3H, t, J ) 7.5 Hz), 1.40 (1H, d, J ) 13 Hz), 1.66
(2H, m), 1.78 (2H, m), 1.82 (2H, m), 2.04 (1H, m), 2.21 (1H, d,
J ) 11 Hz), 2.51 (1H, s), 3.04 (1H, m), 3.62 (1H, t, J ) 4 Hz),
3.71 (1H, s), 3.80 (1H, m), 3.95 (2H, t, J ) 7.5 Hz), 4.15 (1H,
m), 4.23 (1H, d, J ) 5 Hz); 13C NMR δ 10.77, 11.31, 21.15,
22.27, 33.45, 34.09, 43.27, 45.94, 46.06, 53.60, 67.17, 77.69,
119.38, 136.91, 150.43, 157.28, 158.52; MS m/e 344 (M+). Anal.
(C18H24N4O3) C, H, N.
4-Br om oben zoyl Der iva tive 14. A solution of 13 (440 mg,
1.28 mmol), 4-bromobenzoyl chloride (420 mg, 1.92 mmol), and
DMAP (25 mg) in pyridine (10 mL) was stirred under dry N2
at 50 °C for 20 h. Water (10 mL) was added, and the mixture
was stirred at room temperature for 4 h in order to hydrolyze
unreacted acyl chloride. After acidification (2 N HCl), the
mixture was extracted twice with ethyl acetate (30 mL). The
extracts were washed with 2 N HCl, water, aqueous, NaHCO3,
and brine. Evaporation of the dried (MgSO4), filtered extract
left an oil (585 mg, 86.8%) which was crystallized from
g, 74%) with a mp of 140-141 °C were thus obtained: [R]20
D
) +140.8° (c ) 2.0); 1H NMR δ 0.97 (6H, m), 1.46 (1H, d, J )
8 Hz), 1.56 (1H, d, J ) 8 Hz), 1.70 (3H, m), 1.81 (2H, m), 2.25
(1H, m), 3.04 (1H, s), 3.45 (1H, s), 3.57 (1H, m), 4.01 (2H, m),
4.08 (2H, m), 5.85 (1H, m), 6.27 (1H, m); 13C NMR δ 11.18,
11.38, 21.34, 31.43, 42.92, 43.23, 45.15, 47.54, 49.82, 106.65,
132.62, 138.25, 148.68, 151.05, 157.97. Anal. (C18H24N4O2)
C, H, N.
AcOEt/i-Pr2O: mp 140 °C; [R]20 ) -65.3° (c ) 2.0); 1H NMR
1,3-Dip r op yl-8-[5,6-exo-ep oxy-(1R,2R)-n or b or n -2-yl]-
xa n th in e (8). To a stirred suspension of 7 (3.6 g, 11 mmol)
in 2-propanol (30 mL) and water (15 mL) was added magne-
sium monoperoxyphthalate (ca. 80%, 7.5 g, ca. 1.1 equiv) in
one portion. After the mixture was stirred for 20 h at room
temperature, excess peracid was destroyed by successive
additions of KI and Na2S2O3 to the clear solution, which
thereafter was diluted with water (150 mL). The resulting
suspension was extracted with AcOEt (2 × 150 mL), and the
combined extracts were washed with water, aqueous NaHCO3,
and brine. The dried (MgSO4), filtered extracts were evapo-
rated to dryness, and the residue was filtered through a short
column of neutral alumina (activity III, CH2Cl2). Evaporation
of the eluate gave an oil which was crystallized from ether/
hexane to afford 2.20 g (58.3%) of 8 as a white solid: mp 143.5-
D
δ 0.81 (3H, m), 0.95 (3H, m), 1.13 (2H, m), 1.67 (3H, m), 1.83
(1H, d, J ) 12 Hz), 2.18 (2H, m), 2.77 (1H, m), 3.19 (1H, m),
3.73 (1H, m), 3.78 (1H, m), 3.99 (2H, m), 4.10 (1H, m), 4.41
(1H, d, J ) 4 Hz), 4.88 (1H, s), 7.64 (2H, m), 7.89 (2H, m).
Anal. (C25H27BrN4O4) C, H, N.
X-r a y Str u ctu r a l An a lysis of 14. A flat monoclinic crystal
of 14 was used for the data collection performed on a R3m
Siemens four-circle diffractometer using graphite-monochro-
mated Cu KR radiation. The cell parameters were obtained
by a least-squares fit of 25 reflections with 2θ range 35-45°.
A total of 2500 reflections were measured using the ω/2θ scan
with variable scan speed (2-29°/min) to a maximum 2θ ) 110°.
Three standard reflections were monitored every 97 reflections.
No systematic intensity variations were found. No absorption
correction was applied. The structure was solved by the direct
method (SHELX-86). Hydrogen atoms were introduced geo-
metrically. The structure was refined on F2 using the full-
matrix least-squares technique (SHELX-93) with anisotropic
144.5 °C; [R]20 ) +66.4° (c ) 2.0); 1H NMR δ 0.97 (3H, t, J )
D
7.5 Hz), 0.99 (3H, t, J ) 7.5 Hz), 1.01 (1H, m), 1.56 (1H, m),
1.72 (2H, m), 1.81 (2H, m), 2.07 (2H, m), 2.68 (1H, m), 3.09
(1H, m), 3.11 (1H, m), 3.26 (1H, m), 3.44 (1H, m), 4.03 (2H, t,
J ) 7.5 Hz), 4.12 (2H, t, J ) 7.5 Hz); 13C NMR δ 11.22, 11.44,
21.36, 27.66, 29.58, 37.34, 39.98, 41.66, 43.46, 45.27, 49.40,
51.10, 106.91, 148.97, 150.86, 155.65, 156.04. Anal.
(C18H24N4O3) C, H, N.
thermal parameters for all non-hydrogen atoms. Final R1
)
6.2% for 1910 F0 > 4σ(F0). No peaks greater than 0.37 e/Å3
were found in the final difference Fourier map. The absolute
structure parameter ø converged at -0.06(5).

A1-Adenosine Antagonist
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1777
Ra d ioliga n d Bin d in g Assa ys. Binding affinities (Ki) at
the A1- and A2a-adenosine receptors were determined by
measurement of the displacement of the binding of the A1
AdoR antagonist [3H]CPX or the A2a AdoR agonist [3H]CGS-
21680 to membranes prepared from guinea pig and rat
forebrain (A1) or striatal tissue (A2a) as described previously12
and from HEK-293 cells stably transfected with a vector
containing the cDNA of the human A1 or A2a AdoR as described
below. Values given are mean ( SEM of triplicate determina-
tions in each of four preparations. All Hill slopes were close
to unity.
(Coromed, Inc., Troy, NY) for the diuresis data, and Dr.
Mitch Rosner and Allan Encarnacion for analytical
support.
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J M970013W