Potent and orally bioavailable 8-bicyclo[2.2.2]octylxanthines as adenosine A1 receptor antagonists

Article abstract of DOI:10.1021/jm0605381

In the search for a selective adenosine A₁ receptor antagonist with greater aqueous solubility than the compounds currently in clinical trials as diuretics, a series of 1,4-substituted 8-cyclohexyl and 8-bicyclo-[2.2.2] octylxanthines were inve

Full text of DOI:10.1021/jm0605381

J. Med. Chem. 2006, 49, 7119-7131
7119
Potent and Orally Bioavailable 8-Bicyclo[2.2.2]octylxanthines as Adenosine A1 Receptor
Antagonists
William F. Kiesman,*,† Jin Zhao,† Patrick R. Conlon,† James E. Dowling,† Russell C. Petter,† Frank Lutterodt,‡ Xiaowei Jin,‡
Glenn Smits,‡ Mary Fure,§ Andrew Jayaraj,| John Kim,| Gail Sullivan, and Joel Linden
Departments of Chemistry, Pharmacology, Pharmaceutical DeVelopment, and Preclinical DeVelopment, Biogen Idec, Inc., 14 Cambridge
Center, Cambridge, Massachusetts 02142, and Departments of Medicine (Cardiology) and Pharmacology, UniVersity of Virginia Medical
Center, CharlottesVille, Virginia 22901
ReceiVed May 9, 2006
In the search for a selective adenosine A1 receptor antagonist with greater aqueous solubility than the
compounds currently in clinical trials as diuretics, a series of 1,4-substituted 8-cyclohexyl and 8-bicyclo-
[2.2.2]octylxanthines were investigated. The binding affinities of a variety of cyclohexyl and bicyclo[2.2.2]-
octylxanthines for the rat and human adenosine A1, A2A, A2B, and A3 receptors are presented.
Bicyclo[2.2.2]octylxanthine 16 exhibited good pharmaceutical properties and in vivo activity in a rat diuresis
model (ED50 ) 0.3 mg/kg po). Optimization of the bridgehead substituent led to propionic acid 29 (BG9928),
which retained high potency (hA1, Ki ) 7 nM) and selectivity for the adenosine A1 receptor (915-fold
versus adenosine A2A receptor; 12-fold versus adenosine A2B receptor) with improved oral efficacy in the
rat diuresis model (ED50 ) 0.01 mg/kg) as well as high oral bioavailability in rat, dog, and cynomolgus
monkey.
Introduction
Adenosine, a metabolite of ATP with a variety of intra- and
extracellular signaling functions, is released from cells under
ischemic or hypoxic conditions.1 Although a transient signaling
molecule with a plasma half-life under a few seconds,2 adenosine
exerts a plethora of pharmacologic effects via four G-protein
coupled adenosine receptors: A1, A2A, A2B, A3. The adenosine
receptor subtypes belong to a family of rhodopsin-like receptors
that contain seven transmembrane helical domains linked by
three intracellular and three extracellular loops.3 The alpha-
helices of the adenosine A1 receptor designated HI through HVII
form a ligand binding pocket.4 Site-directed mutagenesis studies
have postulated direct interaction of adenosine with transmem-
brane domains, while the third intracellular loop and the
Figure 1. Adenosine A1 receptor antagonists containing bulky lipo-
philic substitution at the 8-position of 1,3-dipropylxanthine.
carboxyl terminus interact with Gi proteins.5 The adenosine A1
receptors in nervous tissues, heart, and kidney modulate
neurotransmitter release, heart rate, and renal hemodynamics,
respectively.6 Antagonists have been examined clinically as renal
protective agents and also as possible treatments for congestive
heart failure.7
There are many examples of potent adenosine A1 antagonists
that contain bulky lipophilic substitution at the 8-position of
1,3-dipropylxanthines (Figure 1).8,9 The highest affinity xan-
thine-based molecules pictured in Figure 1 lack appreciably
polar substituents. The utility of most of these compounds for
intravenous administration in the treatment of acutely decom-
pensated congestive heart failure patients in the clinic, however,
may be limited because of their low water solubility. An
exception to the general property of low solubility among potent
Figure 2. Xanthine amine congener (XAC), cyclohexylxanthine-, and
bicyclo[2.2.2]octylxanthine targets with linear substitution patterns.
A1 antagonists is the 8-aryl-substituted xanthine amine congener
(XAC) first described by Jacobson et al.10a XAC has long been
used in the elucidation of the pharmacologic actions of adenosine
A1 receptors in living systems and possesses moderate aqueous
solubility (90 µM in 0.1 M sodium phosphate at pH 7.2; Figure
2). Numerous reports describe a variety of XAC derivatives with
linear substitution patterns (i.e., 1,4-disubstitution on the aryl
ring attached to the 8-position of the xanthine) and their effects
on the binding affinities and selectivities.11 Olah et al. suggested
* To whom correspondence should be addressed: Tel.: 617-679-2790.
Fax: 617-679-3635. E-mail: william.kiesman@biogenidec.com.
† Department of Chemistry, Biogen Idec, Inc.
‡ Department of Pharmacology, Biogen Idec, Inc.
§ Department of Pharmaceutical Development, Biogen Idec, Inc.
| Department of Preclinical Development, Biogen Idec, Inc.
University of Virginia Medical Center.
10.1021/jm0605381 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/02/2006

7120 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Kiesman et al.
Scheme 1. Synthesis of 8-Cyclohexylxanthinesa
that amino acids in an 11-residue segment of the second
extracellular loop of the adenosine A1 receptor may directly
interact with antagonist ligands and lead to the high binding
affinities.5d In addition, covalent attachment of XAC-related
compounds to the receptor through reactive functional groups10d,12
and a number of modeling studies13 suggested that the xanthine
portion of the antagonist binds deep within the receptor
transmembrane binding cleft and that the 8-position on the
xanthine ring system is oriented outward toward the membrane
surface. This binding mode suggests that tethered polar sub-
stituents might be introduced without greatly affecting binding
affinity. Despite this promising lead in the 8-aryl series,
relatively little work has been done to examine in a systematic
way the effects of linear substitution on saturated carbocycles
at the xanthine 8-position. One notable exception is the
examination of binding affinities and adenylate cyclase activity
of a small group of cyclopentyl- and cyclohexyl-substituted
xanthines by Wells and colleagues.14 We have expanded the
examination of this class of ligands and herein describe the SAR
of 8-cyclohexyl and 8-bicyclo[2.2.2]octylxanthines that contain
linear substitution patterns (Figure 2). Also presented are data
regarding in vivo efficacy and bioavailability of some of the
most potent of these adenosine A1 receptor antagonists.
Chemistry
The targeted 8-cyclohexyl-substituted xanthines were prepared
in the classical 2-step procedure outlined in Scheme 1. trans-
Cyclohexane-1,4-dicarboxylic acid monomethyl ester (2)15 was
coupled with 5,6-diamino-1,3-dipropyl-1H-pyrimidine-2,4-dione
(1)16 via a HATU-mediated (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate) amidation. Subse-
quent ring closure and dehydration to form xanthine 3 occurred
in hot 1 N KOH/isopropyl alcohol. Yields for the 1,3-
dipropylxanthines ranged from 40 to 95% overall for the two-
step process. Acid 3 was subjected to a second coupling reaction
to produce amides 6-9. Reduction of 3 with BH3-THF gave
alcohol 4, which was then capped by reaction with benzyl
bromide under basic conditions to give benzyl ether 5. A similar
coupling-cyclization sequence with the protected cyclohexyl
amino acid 10 and 1 was followed to produce compounds 11
and 12 as a mixture of cis and trans isomers. Hydrogenation of
11 with in situ trapping with acetic anhydride and chromato-
graphic separation gave acetyl derivatives 13 and 14.
The analogous 8-bicyclo[2.2.2]octyl-substituted xanthine 16
was prepared in the same manner to the cyclohexyl acid 3
(Scheme 2.). Bicyclo[2.2.2]octane-1,4-dicarboxylic acid monom-
ethyl ester (15) was obtained commercially and also synthesized
by literature procedures.17 Again, a subsequent coupling reaction
of 16 with a variety of amines gave amides 17-22. Coupling
of 1 with the pentyl-substituted acid 23 and base-induced
cyclization gave compound 24. Esterification of acid 16 with
acidic MeOH gave ester 25, which underwent a clean LiBH4
reduction to alcohol 26 (Scheme 3). Treatment of 26 with Dess-
Martin periodinane (DMPI) produced aldehyde 27, which served
as a common starting material for a series of homologs of acid
16. Wittig-type olefinations with a series of phosphonates added
one, two, or three carbon atoms between the bridgehead position
and the carboxylic acid in compounds 28, 29, 31, and 33.
a Reagents and conditions: (a) acid 2, NEt3, HATU, CH3CN; (b) 1 N
KOH, i-PrOH/H2O (1:1), reflux; (c) amine, NEt3, HATU, CH3CN; (d) 1.0
M BH3-THF, 40 °C, THF; (e) 1.0 M t-BuOK, THF, BnBr; (f) acid 10,
NEt3, HATU, CH3CN; (g) 1 N KOH, i-PrOH:H2O (1:1), reflux; (h) H2,
5% Pd/C, MeOH, Ac2O (isomers separated by prep HPLC).
antagonists and employed binding affinity determinations with
a mix of rat-derived A1 receptors and human platelet-derived
A2A receptors, which were available at the time. We chose to
examine the binding affinities of the target compounds with
the four available cloned human adenosine receptors (hA1, hA2A,
hA2B, and hA3). The biological activities of the antagonists were
evaluated by the following procedures. The primary screen
consisted of a single-point assay performed in duplicate on
membranes derived from stably transfected HEK (hA2A, hA2B,
and hA3) or CHO-K1 (hA1 receptors) cells expressing one of
the four human adenosine receptor subtypes (hA1, hA2A, hA2B,
and hA3).18 Membranes were incubated at room temperature
for 2 h with 125I-labeled radioligands, competing antagonists,
and 1 U/mL adenosine deaminase, filtered over glass fiber filters,
and retained radioactivity counted in a γ-counter. Nonspecific
binding was measured in the presence of 50 µM XAC or 10
µM BW-1433 (hA3). Data are presented as percent (%) of
Results and Discussion
Our attention was initially drawn to the 8-cyclohexyl deriva-
tives by an article published by Wells and co-workers.14b His
work described a series of 1,4-substituted cyclohexanes that
showed some promise as moderately selective adenosine A1

8-Bicyclo[2.2.2]octylxanthines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7121
Scheme 2. Synthesis of Bicyclo[2.2.2]octylxanthinesa
a Reagents and conditions: (a) acid 15, NEt3, HATU, CH3CN, 25 °C; (b) 1 N KOH, i-PrOH/H2O (1:1), reflux; (c) amine, NEt3, HATU, CH3CN, 25 °C;
(d) acid 23, NEt3, HATU, CH3CN, 25 °C; (e) 1 N KOH, i-PrOH/H2O (1:1), reflux.
Scheme 3. Synthesis of Higher Homologs of Carboxylic Acid 16a
a Reagents and conditions: (a) H2SO4, MeOH, 25 °C; (b) LiBH4, MeOH, THF, reflux; (c) DMPI, CH2Cl2, 25 °C; (d) methoxymethyl triphenylphosphonium
chloride, KHMDS, toluene/THF, -78 °C, then hydrolysis with 1 N HCl at 25 °C; (e) t-BuOH, 2-methyl-2-butene, NaClO2, 0 °C to 25 °C; (f) trimethyl
phosphonoacetate, KHMDS, toluene, 0 °C; (g) KOH, MeOH/H2O, reflux; (h) H2, Pd/C, MeOH/H2O; (i) methoxymethyl triphenylphosphonium chloride,
KHMDS, toluene/THF, -78 °C; (j) H2, Pd/C, EtOH; (k) 1 M LiOH, THF, 25 °C.
3
radioligand bound in the presence of target compound relative
to control. Compounds that displayed good hA1 binding activity
in the single-point assays were further evaluated to determine
IC50 values and inhibition constants (Ki values).19 Duplicate full
binding curves were derived from antagonist concentrations that
ranged from 10-11-10-5 M. The binding affinities for rat A1
(rA1) and A2A (rA2A) receptors were also determined for specific
compounds that exhibited high human adenosine A1 receptor
binding affinity. Compounds were incubated with either H-
labeled radioligand (DPX or ZM241385) and aliquots of crude
membrane suspensions prepared from either rat brain cortex (for
rA1) or rat brain striatum (for rA2A). Values of Ki were
determined from concentration-response relationships for each
compound to displace binding of specific radioligands.18
Our testing of the trans-4-carboxylic acid 3 gave a low
binding affinity (31% in the single-point assay; estimated Ki >

7122 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Kiesman et al.
Table 1. Adenosine Receptor Binding Affinities for Cyclohexyl-Substituted Derivatives of 1,3-Dipropylxanthine
a All Ki values were calculated from binding curves generated from the mean of four determinations per concentration (seven antagonist concentrations),
with the variation in individual values of <15%. b Data are presented as percent (%) of radioligand bound in the presence of target compound relative to
control.
500 nM) relative to the result reported by Wells and co-workers
(rA1 ) 59 nM). This result was not surprising because it has
been our experience that, in general, the same antagonist can
have a 10-fold higher affinity for the rA1 receptor versus the
hA1 receptor. Jacobson et al., in examining a series of 8-arylx-
anthines, also observed a loss in hA1 activity with the introduc-
tion of a 4-carboxyl group.10a Alcohol 4 had increased hA1 and
hA2A affinity when compared to the acid (Table 1). Capping
with a benzyl group decreased hA1 affinity by about 4-fold but
had a greater negative effect on hA2A affinity (8-fold loss). This
result suggested that the hA2A receptor was less able to
accommodate the nonpolar benzyl group in the outer region of
the receptor (i.e., near the membrane surface). Examples of
tertiary amides in the 4-position, compounds 8 and 9, showed
poor binding. The n-butyl amide 6, with an N-H in the
analogous position to the O-H in 4, had about the same affinity
for the hA1 receptor as alcohol 4 but was more selective versus
hA2A (hA2A/hA1 ratio ) 49 vs 8). Substitution with the basic
N,N-dimethylethylenediamine gave 7, the most potent hA1
antagonist of this series, with a hA1 Ki ) 12 nM and significant
activity against the hA2A receptor (Ki ) 168 nM). This
observation mirrors Wells’ results in the cyclohexyl series and
indicated that amino substitution at the terminus was well
received by both hA1 and hA2A receptors. Acylamino substitu-
tion on the cyclohexyl ring (with cis or trans stereochemistry)
produced compounds with low affinities (13, 14). The trans
benzylcarbamate 12 had modest hA1 activity (109 nM) and
selectivity against all of the human receptors similar to that of
benzyl ether 5 (see Table 1).
Addition of a two-carbon bridge linking the 1- and 4-positions
across the cyclohexane ring gave bicyclo[2.2.2]octane deriva-
tives with added steric bulk at the 8-position and no stereo-
chemical complexity (Figure 2). The first example, 16, despite
bearing a bridgehead carboxylic acid, demonstrated surprisingly

8-Bicyclo[2.2.2]octylxanthines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7123
Table 2. Adenosine Receptor Binding Affinities for Bicyclo[2.2.2]octyl-Substituted Derivatives of 1,3-Dipropylxanthine
a All Ki values were calculated from binding curves generated from the mean of four determinations per concentration (seven antagonist concentrations),
with the variation in individual values of <15%. b Data are presented as percent (%) of radioligand bound in the presence of target compound relative to
control.
good hA1 affinity (33 nM; Table 2), a marked improvement
over its cyclohexyl congener 3 (estimated Ki > 500 nM), and
was in direct contrast to the negative effects of acid substitution
noted with 8-arylxanthines.10a To probe the area beyond the
bicycle for polar binding interactions that would differentiate
between the hA1 and the hA2A receptors, a series of amides
were prepared that tethered amines, acids, and esters with a
variety of methylene spacers. The N,N-dimethylethylenediamine
amide 17 had a hA1 binding affinity similar to the cyclohexyl
variant 7 and also remarkably similar hA2A affinity (132 nM
vs 168 nM). The terminal amine substitution offered no
selectivity enhancement, so a series of carboxylic acids were
then examined. The glycine methyl ester analog 18 maintained
hA1 affinity (8 nM) and had a hA2A/hA1 selectivity ratio ) 85,
similar to that of the bridgehead methyl ester 25; hA2A/hA1 )
100. Methylation of the amide nitrogen (20) or installation of
piperidine-4-carboxylate (22) led to >10-fold hA1 affinity losses.
Insertion of a methylene spacer gave 21 and led to a 3-fold
loss in hA1 potency and had a smaller effect on hA2A binding
affinity. The glycine-free-acid analog (19) on the other hand
gave the most hA1-selective example with the hA2A/hA1
selectivity ratio of 160. It appeared that proper placement of
the carboxylate in 8-bicyclo[2.2.2]octyl xanthines markedly
decreased hA2A affinities and generally maintained the hA1
binding properties. Other less-polar examples, alcohol 26 and
aldehyde 27, had better hA1 affinities, but had selectivity ratios
similar to acid 16. Replacement of the carboxylic acid with a
pentyl chain (24) dramatically diminished the hA1 binding
affinity (>500 nM). This result suggested that there were either
polar pockets within the adenosine receptors in regions between
the xanthine binding domain and the cell surface or that acid
16 had a significantly different binding mode.
Bicyclo[2.2.2]octyl structures (sans amide linkages) with
carboxylic acids of various lengths attached to the bridgehead
position were investigated (Table 3). The addition of a meth-
ylene spacer between the bridgehead position and the carboxylic
acid in 16 led to a 3-fold loss in rA1 affinity (31). Insertion of
a trans-double bond, compound 28, increased A1 affinity to
single-digit nanomolar and imparted a 10-fold increase in hA1
selectivity over hA2A: ratio ) 333. The saturated propionic acid
analog 29 had similar hA1 affinity (7.4 nM) but extraordinary
selectivity (915-fold) over the hA2A receptor. Evidently, the hA2A
receptor was unable to accommodate the modest increase in
bulk of the alkyl linker. Further elongation of the chain with
an additional methylene spacer gave butyrate 33 that had binding
affinities and selectivities similar to acid 16. All of the acids

7124 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Kiesman et al.
Table 3. Carboxylic Acid Substitution at the Bridgehead Position of Bicyclo[2.2.2]octylxanthines
a All Ki values were calculated from binding curves generated from the mean of four determinations per concentration (seven antagonist concentrations),
with the variation in individual values of <15%. b Data are presented as percent (%) of radioligand bound in the presence of target compound relative to
control. c Ki values were determined from concentration-response relationships for each compound to displace binding of radioligand to rat brain cortex (for
rA1) or rat brain striatum (for rA2A). d (S)-ENX: (1,3-dipropyl-8-[2-(5,6-exo-epoxy-(1S,2S)-norborn-2-yl)]-xanthine) rat values from ref 9. e NAX :
3-noradamantyl-1,3-dipropylxanthine.
Figure 3. Rat oral efficacy screen: measurement of UNaV in µEq/h
(mean ( SEM), in a 4-hour period, of a 0.3 mg/kg oral dose of
Figure 4. Reversal effect (inhibition) of increasing concentrations of
compound 29 on CPA (130 nM) suppression of the isoproterenol-
stimulated (30 nM) heart rate in beating, isolated rat atria. (5 atria/
dose group).
antagonist in a 0.5% CMC suspension.
exhibited virtually no adenosine hA3 receptor binding at
concentrations up to 1 µM. The most potent hA1 antagonists,
28, 29, and 33, all had some cross activity (∼100 nM) against
the hA2B receptor. Antagonism of this ubiquitously expressed
low-affinity adenosine receptor is thought to play a beneficial
role in ischemic preconditioning of the heart under hypoxic
conditions and modulation of mast cell degranulation in
asthmatics.20 Propionic acid 29 had similar hA1 affinity when
compared to previous clinical compounds, (S)-ENX and NAX,
but better hA2A selectivity (>7-fold higher).
Table 4. Pharmacokinetic Parameters Following a Single Oral Dose of
Selected Adenosine A1 Receptor Antagonistsa
F
(%)
t1/2
(h)
CL
Vds
(L/kg)
compd
species
(mL/min/kg)
16
29
rat (2 mg dose)
rat (1 mg/kg)
dog (1 mg/kg)
cyno (1 mg/kg)
rat (1 mg/kg)
97
99
78
94
48
2.14 ( 0.87 2.26 ( 0.41
3.14 ( 0.14 1.56 ( 0.26
0.57 ( 0.03
0.32 ( 0.02
2.64 ( 1.29
4.25 ( 0.70
1.16 ( 0.20
6.40 ( 4.0
11.1 ( 4.2
11.8 ( 0.6
5.82 ( 0.45
33
2.04 ( 0.65 7.10 ( 2.58
Biological evaluations of the most potent hA1 antagonists in
both the cyclohexyl and bicyclo[2.2.2]octyl series were per-
formed. Oral activity was assessed in a rat diuresis model at a
fixed dose of 0.3 mg/kg. The test article was delivered by gavage
as a 0.5% carboxymethylcellulose (CMC) suspension to rats
housed in metabolic cages. Over a 4-hour period, urine was
collected and Na and K excretions (determined as microequiva-
lents) were measured by FIS. The results for selected compounds
appear in Figure 3. Despite a 10-fold difference in rA1 binding
a n ) 3 male rats, 4 male dogs, and 4 male cynomolgus monkeys.
affinity between the bridgehead carboxylate 16 and the (S)-
ENX, the rat urinary sodium excretion (UNaV) values were
similar. The N,N-dimethylethylenediamine amide 17 also showed
good in vivo activity, in contrast to the amides that possessed
a terminal carboxylic acid or ester (18, 20). It is noteworthy
that the bridgehead carboxylate 16, some 5-fold less-active in
vitro than amine 17, exhibited better in vivo efficacy. Propi-

8-Bicyclo[2.2.2]octylxanthines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7125
Figure 5. Blockade by 0.3, 3.0, or 30.0 nM compound 29 or vehicle
control (DMSO) of the inhibitory effect of increasing concentrations
of CPA on isoproterenol-stimulated rat atria in vitro (5-6 atria/group).
In Schild analysis: slope ) -0.865; intercept ) 8.49; and pA2 ) 9.8.
See Supporting Information for plot.
Figure 8. Dose response for UKV in µEq/h (mean ( SEM) over 4 h,
following single oral doses of vehicle (n ) 3) or compound 29, ranging
from 0.001 to 3 mg/kg in rats (0.001 mg/kg, 0.003 mg/kg, 0.01 mg/
kg, each n ) 4; 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, each n ) 5; 1.0
mg/kg, 3.0 mg/kg, each n ) 3).
Table 5. Solubility Profiles of Adenosine A1 Antagonists in Various
Solvent Systemsa
compd
16
WFIb
0.9% saline
D5Wc
EtOH
51.0
octanol
60.0
18.1
24.4
0.5
0.46
<0.01
29.8
25.4
63.0
12.4
29
33
(S)-ENXd
NAXe
0.43
0.25
85.3
24.0
4.6
82.3
a All solubility values were measured in units of mg/mL and calculated
from duplicate determinations in each solvent, with the variation in
individual values of <5%. b Sterile water for injection. c Dextrose 5% in
water. d 1,3-Dipropyl-8-[2-(5,6-exo-epoxy-(1S,2S)-norborn-2-yl)]-xan-
thine. e 3-Noradamantyl-1,3-dipropylxanthine.
Figure 6. Dose response for urine volume in mL (mean ( SEM) over
4 h, following single oral doses of vehicle (n ) 3) or compound 29
ranging from 0.001 to 3 mg/kg in rats (0.001 mg/kg, 0.003 mg/kg,
0.01 mg/kg, each n ) 4; 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, each n )
5; 1.0 mg/kg, 3.0 mg/kg, each n ) 3).
a larger volume of distribution. The pharmacokinetics of 29 was
further evaluated in the dog and cynomolgus monkey and
demonstrated rapid absorption and widespread distribution,
followed by bi-phasic disposition. Bioavailability was nearly
complete in the cynomolgus monkey and slightly lower in the
dog. Exposure was highest in the rat, followed by the monkey
and dog, with correspondingly increased clearance with increas-
ing body weight. Elimination half-lives following a 1 mg/kg
dose are relatively similar in the rat and dog at 3 and 6 h,
respectively, and longer in the monkey at approximately 11 h.
Further work explored the functional activity of compound
29. The sinoatrial (S-A) node is a small crescent strip of
specialized muscle located in the posterior wall of the right
atrium immediately beneath and medial to the opening of the
superior vena cava. Any action potential that begins in the S-A
node spreads immediately to the atrium, providing the atrium
with automatic rhythmicity that allows it to beat independently
when isolated from the heart. Decreases or increases in heart
rate, as assessed in rat atria, have been used to quantify responses
mediated by the adenosine A1 receptor. Pharmacological potency
of a series of reference adenosine analogues possessing selectiv-
ity for the adenosine A1 receptor has been used to define the
profile for A1 adenosine receptor antagonists.21 The following
study was designed to evaluate the ability of 29 to block the
negative chronotropic effects of activation of A1 receptors by
N6-cyclopentyl adenosine (CPA) in the isolated rat atrium and,
thus, to assess its potency as an adenosine A1 receptor
antagonist. After atrial beat rate stabilization, 130 nM CPA was
added to baths to cause a 75% reduction in atrial beating rate
(zero point). Increasing concentrations of 29 were then added
until the rate was restored to maximum (Figure 4). The mean
EC50 of 29 in the CPA dose reversal experiment was 16.1 (
7.7 nM. The next set of experiments was used to determine the
affinity of 29 for its receptor. Atrial rate was recorded in the
Figure 7. Dose response for UNaV in µEq/h (mean ( SEM) over 4
h, following single oral doses of vehicle (n ) 3) or compound 29,
ranging from 0.001 to 3 mg/kg in rats (0.001 mg/kg, 0.003 mg/kg,
0.01 mg/kg, each n ) 4; 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, each n )
5; 1.0 mg/kg, 3.0 mg/kg, each n ) 3).
onates 28 and 29 demonstrated superiority over the other
compounds, with sodium output almost twice that of any of
the other compounds tested.
The pharmacokinetic parameters of 16, 29, and 33, admin-
istered as a single oral dose to male Sprague-Dawley rats,
beagle dogs, and cynomolgus monkeys, are presented in Table
4. In the rat, 16 and 29 had excellent bioavailability, 97 and
99%, respectively, and exhibited relatively low clearance and
volume of distribution. The half-life of 29 was also quite good
in the rat (>3 h). In contrast, the higher homolog 33 had about
half the bioavailability of the other acids, higher clearance, and

7126 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Kiesman et al.
was also used for preparative purposes (LiChroprep C-18, 310 ×
25 mm). 1H and 13C NMR spectra were obtained using Bruker 300,
400, and 500 MHz NMR spectrometers. High-resolution mass
spectroscopic data were obtained on a Thermo Electron LTQ FTMS.
The data were acquired at the positive, full scan, and SIM scan FT
MS mode, protonated molecular ion designated as MH+. All
chemicals and reagents were supplied by Aldrich Chemical Co.,
Inc, Milwaukee, WI, unless otherwise indicated.
presence of 30 nM isoproterenol and compound 29 (0, 0.3, 3.0,
and 30 nM). In the continued presence of isoproterenol and 29,
increasing concentrations of CPA from 1 nM to 30 µM were
added cumulatively until the atrial rate was lowered to zero.
The EC50 was determined for the vehicle control and each of
the antagonist concentrations. Schild analysis was used to
calculate the affinity of 29, the competitive antagonist, for its
receptor (pA2). Parallel rightward shifts in CPA inhibition curves
were seen with increasing concentrations of compound 29,
indicative of competitive antagonism (Figure 5). The pA2 for
compound 29 was calculated to be 9.8.
The oral activity of 29 was investigated in a series of
experiments that examined, in a dose-related fashion, diuresis
(Figure 6), natriuresis (Figure 7), and kaliuresis (Figure 8).
Compound 29 caused a dose-related diuretic and natriuretic
effect, which reached a maximum for both with a dose between
0.3 and 1 mg/kg. The half-maximal effect (ED50) was ap-
proximately 15 µg/kg. The diuretic and natriuretic effects were
associated with a neutral effect on potassium excretion (UKV),
that is, UKV increased in proportion to volume. Potassium
concentration in urine remained constant or slightly decreased
across the dose ranges (Figure 8).
As the product was targeted for delivery to acutely decom-
pensated congestive heart failure patients in the hospital setting,
the solubilities of 16, 29, and 33 were evaluated in comparison
to the earlier clinical candidate, (S)-ENX, and another adenosine
A1 antagonist, NAX, in prototype solutions suitable for intra-
venous administration. These results are shown in Table 5. Acids
16 and 29 had acceptable solubilities in most of the preformu-
lation solutions tested and were significantly better than the other
clinical candidates.
Compound 29 was designated a clinical development can-
didate, given the product code BG9928, and put into a series
of preclinical toxicity studies where it was well tolerated when
administered intravenously or orally to rats and cynomolgus
monkeys for periods up to 3 months. The clinical candidate
has been examined in IV and oral studies in healthy volunteers
and stable congestive heart failure patients and is the subject
of ongoing longer term human clinical trials.22
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
cyclohexanecarboxylic acid, 3. To a stirred mixture of 2.00 g (10.7
mmol) of trans-cyclohexane-1,4-dicarboxylic acid monomethyl ester
(2),15 2.82 g (10.7 mmol) of 5,6-diamino-1,3-dipropyl-1H-pyrimi-
dine-2,4-dione hydrochloride (1),16 4.52 mL (32.2 mmol) of NEt3,
and 50 mL anhydrous acetonitrile were added 4.25 g (11.2 mmol)
of HATU. The reaction solution was stirred at rt for 30 min. The
reaction mixture was concentrated in vacuo and combined with 40
mL of EtOAc and 40 mL of 10% citric acid. The aqueous layer
was separated and washed twice with 40-mL portions of EtOAc.
The combined organic fractions were washed with 20-mL portions
of satd NaHCO3 and brine and concentrated in vacuo. The resultant
oil was combined, in a 200-mL round-bottom flask equipped with
a condenser, with a mixture of 30 mL of i-PrOH and 32.2 mL of
1 N KOH (32.2 mmol) and heated to reflux. After heating for 1 h,
the reaction solution was concentrated in vacuo, taken up in 40
mL of water, chilled in an ice bath, and acidified with concentrated
HCl. The resultant precipitate was collected by suction filtration,
washed with water, and dried to give 2.44 g (63% yield) of an
1
off-white solid. H NMR (300 MHz, DMSO-d6) δ 0.85 (m, 6H),
1.64 (m, 8H), 1.96 (m, 4H), 2.29 (dt, 1H), 2.71 (dt, 1H), 3.81 (dd,
2H), 3.91 (dd, 2H), 8.98 (s, 1H); 13C NMR (125 MHz, DMSO-d6)
δ 11.0, 11.1, 20.8, 28.1, 30.0, 37.0, 41.6, 41.9, 44.2, 106.0, 147.5,
150.6, 153.9, 157.7, 176.5; HRMS m/z ) 363.20279 (MH+), calcd
) 363.20268; tR ) 3.97 min.
8-(4-Hydroxymethyl-cyclohexyl)-1,3-dipropyl-3,7-dihydro-pu-
rine-2,6-dione, 4. To a stirred suspension of 3 (0.200 g, 0.55 mmol)
in 10 mL THF at 40 °C was added 1.50 mL of 1.0 M BH3-THF.
The mixture resolved into a clear solution over 1 h, which was
then treated with 2.00 mL of 50% glacial acetic acid and stirred
for an additional 3 h. The solution was concentrated in vacuo, and
the residue was washed with water and dried to give 0.160 g of a
1
white solid (83% yield). H NMR (300 MHz, CDCl3) δ 0.90 (m,
6H), 1.11 (m, 2H), 1.65 (m, 8H), 1.98 (m, 2H), 2.09 (m, 2H), 2.80
(dt, 1H), 3.47 (d, 2H), 4.03 (m, 4H); MS (MH+) ) 349.25; 13C
NMR (125 MHz, CDCl3) δ 11.2, 11.2, 11.5, 21.4, 21.4, 28.7, 29.0,
30.9, 38.7, 40.0, 43.3, 45.4, 68.1, 106.6, 148.6, 151.0, 155.6, 158.9;
HRMS m/z ) 349.22357 (MH+), calcd ) 349.22342; tR ) 3.90
min.
Conclusion
In summary, we have prepared a novel series of xanthine-
based adenosine A1 receptor antagonists. Bicyclo[2.2.2]octyl
substitution at the 8-position produced antagonists with high
potencies toward the adenosine A1 receptor. Optimization of
the lead molecule, 16, by the linear extension of an acidic
bridgehead side chain gave 29, which possessed remarkable hA1/
hA2A selectivity, 915-fold, and moderate selectivity over the
hA2B receptor (Ki ) 90 nM, 12-fold versus hA1). The in vivo
activity (ED50 ) 0.01 mg/kg-rat) and pharmacokinetics of 29
in animal studies support its use either as a potential once-daily
oral therapy or in an IV formulation for acute use. Single-dose
and multiple-dose studies in healthy volunteers and congestive
heart failure patients are ongoing and will be reported in due
time.
8-(4-Benzyloxymethyl-cyclohexyl)-1,3-dipropyl-3,7-dihydro-
purine-2,6-dione, 5. To a stirred solution of 4 (0.040 g, 0.11 mmol)
in 6 mL of THF was added 0.13 mL of 1.0 M t-BuOK in THF.
After 30 min, 0.014 mL (0.12 mmol) of benzyl bromide was added.
The solution was kept under reflux for another 3 h and then
concentrated in vacuo. The residue was purified by flash chroma-
tography (ethyl acetate/hexane, 4:1) to give 0.044 g (87%) of a
white solid. 1H NMR (300 MHz, CDCl3) δ 0.90 (m, 6H), 1.10 (m,
2H), 1.70 (m, 8H), 1.95 (m, 2H), 2.07 (m, 2H), 2.80 (dt, 1H), 3.35
(d, 2H), 4.00 (m, 4H), 4.45 (s, 2H), 7.33 (m, 5H); 13C NMR (125
MHz, CDCl3) δ 11.3, 11.4, 21.4, 28.4, 30.0, 30.8, 31.3, 35.8, 37.3,
42.7, 44.7, 73.0, 75.7, 107.1, 127.6, 127.8, 138.6, 148.0, 151.2,
155.5, 157.6; HRMS m/z ) 439.2706 (MH+), calcd ) 439.27037;
tR ) 6.94 min.
8-[4-(Morpholine-4-carbonyl)-cyclohexyl]-1,3-dipropyl-3,7-di-
hydro-purine-2,6-dione, 8. To a stirred solution of 0.050 g (0.138
mmol) of 3 in 1 mL of acetonitrile and 58 µL (0.414 mmol) of
NEt3 and morpholine (12 µL; 0.138 mmol) was added 0.055 g
(0.144 mmol) of HATU. The reaction solution was stirred at rt for
30 min. The reaction mixture was concentrated in vacuo and
purified by preparative HPLC (acetonitrile/water gradient 10-90%;
C18 stationary phase, over 30 min) to give a white solid. 1H NMR
(300 MHz, CDCl3) δ 1.00 (m, 6H), 1.80 (m, 8H), 2.00 (m, 2H),
Experimental Section
Unless otherwise stated, reactions were carried out under nitrogen
in oven-dried glassware. The HPLC method used to determine
purity was performed on an HP1100 system, YMC-ODS-AM C18
reversed-phase column (4.6 × 100 mm), guard column YMC-ODS-
AM S-5 120A (direct connect); 20-100% CH3CN/H2O gradient
over 8 min, buffered with 0.1% TFA at 1.5 mL/min flow rate,
detector set at dual wavelength 214 and 254 nm. The purity of all
compounds listed were >95A% at 254 nm. Reversed-phase HPLC

8-Bicyclo[2.2.2]octylxanthines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7127
2.10 (m, 1H), 2.30 (m, 2H), 2.65 (m, 1H), 3.00 (m, 1H), 3.65 (s,
2H), 3.80 (m, 4H), 4.10 (m, 4H); MS m/z ) 432.25 (MH+); tR )
3.99 min.
mmol) of NEt3, and 30 mL of anhydrous acetonitrile was added
3.76 g (9.89 mmol) of HATU. The reaction solution was stirred at
rt for 1 h. The reaction mixture was concentrated in vacuo and
combined with 40 mL of EtOAc and 40 mL of 10% citric acid.
The aqueous layer was separated and washed twice with 40-mL
portions of EtOAc. The combined organic fractions were washed
with 20-mL portions of satd NaHCO3 and brine and concentrated
in vacuo. The resultant solid was combined, in a 200-mL round-
bottom flask equipped with a condenser, with a mixture of 35 mL
of i-PrOH and 35 mL of 1 N KOH (35 mmol) and heated to reflux.
After heating for 1 h, the reaction solution was concentrated in
vacuo, taken up in 40 mL of water, and washed twice with 30-mL
portions of CH2Cl2. The aqueous layer was acidified with concen-
trated HCl, and the resultant precipitate was collected by suction
filtration to give 3.00 g (87% yield) of an off-white solid. 1H NMR
(500 MHz, CDCl3) δ 0.95 (two triplets partially obscured, 6H),
1.69 (q, 2H), 1.80 (q, 2H), 2.05 (m, 12 H), 4.00 (q, 2H), 4.11 (q,
2H), 12.70 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 11.5, 11.6,
21.6, 21.7, 28.5, 30.2, 34.2, 39.0, 43.7, 45.7, 106.9, 149.7, 151.3,
156.8, 161.8, 182.6; HRMS m/z ) 389.21850 (MH+), calcd )
389.21833; tR ) 4.62 min.
The following compounds were made in an analogous manner.
8-(4-Pentyl-bicyclo[2.2.2]oct-1-yl)-1,3-dipropyl-3,7-dihydro-
purine-2,6-dione, 24. 1H NMR (300 MHz, CDCl3) δ 0.90 (m, 9H),
1.10 (m, 2H), 1.20 (m, 4H), 1.30 (m, 2H), 1.40 (m, 6H), 1.50 (dq,
2H), 1.75 (dq, 2H), 1.85 (m, 6H), 3.85 (dd, 2H), 3.95 (dd, 2H),
12.80 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 11.6, 11.9, 14.5,
21.7, 23.0, 23.8, 31.0, 31.1, 31.17, 33.2, 34.1, 41.9, 43.5, 45.5,
107.2, 149.3, 151.5, 155.7, 162.7; Anal. (C24H38N4O2) C, H, N;
HRMS m/z ) 415.30689 (MH+), calcd ) 415.30675; tR ) 8.52
min.
The following compounds were made in an analogous manner.
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
cyclohexanecarboxylic Acid Butylamide, 6. 1H NMR (300 MHz,
CDCl3) δ 0.90 (m, 9H), 1.30 (m, 2H), 1.43 (m, 2H), 1.63 (m, 8H),
2.09 (m, 4H), 2.74 (m, 1H), 2.94 (s, 1H), 3.19 (dt, 2H), 4.00 (m,
4H), 5.37 (t, 1H); MS (MH+ ) 418.31); 13C NMR (125 MHz,
DMSO-d6) δ 11.1, 11.2, 13.7, 19.5, 20.9, 21.0, 28.9, 30.7, 31.3,
37.9, 42.0, 43.4, 44.2, 148.2, 150.8, 154.9, 159.2, 163.3, 165.3,
174.8; HRMS m/z ) 418.28143 (MH+), calcd ) 418.28127; tR )
4.78 min.
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
cyclohexanecarboxylic Acid (2-Dimethylamino-ethyl)-amide, 7.
1H NMR (300 MHz, DMSO-d6) δ 0.85 (m, 6H), 1.53 (m, 6H),
1.67 (dt, 2H), 1.81 (m, 2H), 1.94 (m, 3H), 2.14 (s, 6H), 2.26 (t,
2H), 2.70 (m, 1H), 3.13 (q, 2H), 3.82 (dd, 2H), 3.92 (dd, 2H), 7.70
(t, 1H); MS (MH+ ) 433.25); 13C NMR (125 MHz, CDCl3) δ 11.2,
11.5, 21.4, 21.4, 29.1, 30.5, 36.5, 37.9, 43.3, 44.6, 45.1, 45.3, 57.7,
106.6, 149.1, 151.0, 155.7, 158.9, 175.4; HRMS m/z ) 433.29218
(MH+), calcd ) 433.29217; tR ) 3.04 min.
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
cyclohexanecarboxylic Acid Diethylamide, 9. 1H NMR (300
MHz, CDCl3) δ 0.90 (t, 6H), 1.05 (t, 3H), 1.15 (t, 3H), 1.75 (m,
10H), 2.15 (m, 2H), 2.50 (m, 1H), 2.95 (m, 1H), 3.30 (dd, 2H),
3.35 (dd, 2H), 3.90 (dd, 2H), 4.05 (dd, 2H); 13C NMR (125 MHz,
CDCl3) δ 11.2, 11.5, 13.2, 15.1, 21.4, 29.2, 30.5, 37.8, 39.7, 40.5,
41.9, 43.2, 45.4, 106.5, 151.1, 155.4, 158.5, 174.8; MS m/z )
418.90 (MH+); tR ) 4.97 min.
[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
cyclohexyl]-carbamic Acid Benzyl Ester, 11. See the procedure
to make compound 3, starting material cis-trans mixture of
4-benzyloxycarbonylamino-cyclohexanecarboxylic acid (10) and
gave a white solid (68% yield). HRMS m/z ) 468.26067 (MH+),
calcd ) 468.26053; tR ) 5.62 min (cis) and 5.74 min (trans).
trans-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-cyclohexyl]-carbamic Acid Benzyl Ester, 12. See procedure
to make compound 3, starting material all-trans 4-benzyloxycar-
bonylamino-cyclohexanecarboxylic acid and gave a white solid.
13C NMR (125 MHz, CDCl3) δ 11.2, 11.5, 21.4, 21.4, 30.1, 32.9,
37.8, 43.3, 45.3, 49.5, 66.7, 106.7, 127.8, 128.2, 128.6, 136.5, 149.0,
151.0, 155.7, 156.4, 158.5; Anal. (C25H33N5O4) C, H, N; HRMS
m/z ) 468.26067 (MH+), calcd ) 468.26053.
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]octane-1-carboxylic Acid Methyl Ester, 25. Acid
16 (1.50 g, 3.86 mmol) was combined with 60 mL of MeOH and
10 drops of concentrated H2SO4. The reaction solution was brought
to reflux until consumption of starting material ceased. Saturated
NaHCO3 was then added until neutral pH, and the reaction mixture
was concentrated in vacuo. The residue was taken up in EtOAc
and washed with satd NaHCO3 and brine and dried over Na2SO4.
The EtOAc solution was concentrated in vacuo to give 1.51 g (97%
1
yield) of a white solid. H NMR (300 MHz, CDCl3) δ 0.90 (m,
6H), 1.58 -1.80 (m, 4H), 1.90 (m, 6H), 1.98 (m, 6H), 3.6 (s, 3H),
4.00 (m, 4H), 12.00 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 11.6,
11.8, 21.7, 28.1, 28.5, 30.2, 34.1, 39.2, 52.2, 107.3, 149.3, 151.5,
155.9, 161.6, 178.1; MS m/z ) 403.13 (MH+); tR ) 5.33 min.
8-(4-Hydroxymethyl-bicyclo[2.2.2]oct-1-yl)-1,3-dipropyl-3,7-
dihydro-purine-2,6-dione, 26. Ester 25 (1.40 g, 3.48 mmol) was
combined with LiBH4 (0.379 g, 17.4 mmol), MeOH (0.141 mL,
3.48 mmol), and 100 mL of THF, and the resultant mixture was
brought to reflux for 18 h. After cooling to rt, 50 mL of 1 M HCl
were added, and the mixture was concentrated in vacuo. The residue
was dissolved in EtOAc and washed with 1 M HCl, satd NaHCO3,
and brine and dried over Na2SO4. The EtOAc solution was
concentrated in vacuo to give 1.15 g (88% yield) of a white solid.
1H NMR (300 MHz, CDCl3) δ 0.89 (m, 6H), 1.50 (m, 6H), 1.55-
1.80 (m, 4H), 1.93 (m, 6H), 3.28 (s, 2H), 3.95 (dd, 4H), 4.05 (dd,
4H); 13C NMR (125 MHz, CDCl3) δ 11.2, 11.5, 21.4, 27.7, 30.2,
30.3, 34.3, 39.2, 43.2, 45.2, 71.2, 106.8, 148.8, 151.1, 155.4, 161.8;
Anal. (C20H30N4O3) C, H, N; HRMS m/z ) 375.23916 (MH+), calcd
) 375.23907; tR ) 4.34 min.
trans-N-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-pu-
rin-8-yl)-cyclohexyl]-acetamide, 14. Palladium on carbon (10%;
0.015 g) was added to a solution of 11 (0.120 g, 0.257 mmol) in 3
mL of MeOH and 48 µL of Ac2O. The vessel was flushed with
nitrogen and charged with hydrogen (∼15 psi). The mixture was
stirred for 3 h and concentrated in vacuo, and the residue was
redissolved in 10 mL of EtOAc, filtered, and washed with 10-mL
portions of satd NaHCO3 and brine. The organic layer was dried
over Na2SO4 and concd to give 0.059 g (60% yield) of a mixture
of cis and trans isomers. The isomers were separated by preparative
HPLC (acetonitrile/water, 30 min gradient, 10-90% ACN; C18
stationary phase) to give a white solid. 1H NMR (300 MHz, MeOD)
δ 0.85 (m, 6H), 1.20 (m, 2H), 1.25 (m, 1H), 1.50-1.70 (m, 7H),
1.85 (s, 3H), 1.95 (m, 4H), 2.95 (m, 1H), 3.85 (dd, 2H), 3.95 (dd,
2H); HRMS m/z ) 376.23445 (MH+), calcd ) 376.23432; tR
3.64 min.
)
cis-N-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-cyclohexyl]-acetamide, 13. Prepared according to the pro-
cedure for 14. 1H NMR (300 MHz, CDCl3) δ 0.95 (m, 6H), 1.60-
2.00 (m, 9H), 2.05 (s, 3H), 1.95 (m, 4H), 3.05 (m, 1H), 4.00 (dd,
2H), 4.10 (dd, 2H), 6.40 (d, 1H); MS m/z ) 376.25 (MH+); tR )
3.71 min.
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]octane-1-carboxylic Acid, 16. To a stirred mixture
of 2.00 g (8.84 mmol) of bicyclo[2.2.2]octane-1,4-dicarboxylic acid
monomethyl ester (15),17 2.60 g (9.89 mmol) of 5,6-diamino-1,3-
dipropyl-1H-pyrimidine-2,4-dione hydrochloride (1), 5.32 mL (38.1
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]octane-1-carbaldehyde, 27. To a solution of 0.092
g (0.246 mmol) of 26 in 5 mL of CH2Cl2 was added 0.125 g (0.295
mmol) Dess-Martin periodinane. The reaction mixture was stirred
at rt until the oxidation was complete. The reaction solution was
filtered through a plug of basic alumina, washed with satd NaHCO3,
and brine and dried over Na2SO4. The CH2Cl2 solution was
concentrated in vacuo to give 0.057 g (62% yield) of an off-white
1
solid. H NMR (300 MHz, CDCl3) δ 0.90 (m, 6H), 1.60 -1.80
(m, 10H), 2.05 (m, 6H), 4.00 (m, 4H), 9.50 (s, 1H), 12.00 (s, 1H);
13C NMR (100 MHz, CDCl3) δ 10.2, 10.5, 20.3, 23.7, 24.4, 28.4,

7128 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Kiesman et al.
33.5, 42.6, 105.9, 147.9, 150.1, 154.5, 159.8, 204.1; HRMS m/z )
373.22358 (MH+), calcd ) 373.22342; tR ) 4.86 min.
partitioned between satd aqueous NH4Cl (100 mL) and EtOAc (100
mL), and the aqueous phase was extracted with EtOAc (50 mL).
The combined organic extracts were washed with satd aqueous NaCl
(100 mL), concentrated in vacuo, redissolved in THF, and
concentrated to a volume of approximately 20 mL. To the solution
was added an equal volume of 1 N HCl, and the mixture was stirred
overnight. The mixture was diluted with EtOAc (20 mL), and the
aqueous phase was separated and extracted with EtOAc (10 mL).
The combined organic phases were then washed with saturated
aqueous NaCl (2 × 25 mL), dried (MgSO4), filtered, and concen-
trated in vacuo. The resulting orange oil was purified in batches
by radial chromatography (2 mm plate) using 3% MeOH and 3%
THF in CH2Cl2 as eluent. Product-containing fractions were
combined and concentrated to afford 290 mg (75%) of a white solid.
1H NMR (400 MHz, CDCl3) δ 0.91 (t, 3H), 0.93 (t, 3H), 1.63 (m,
2H), 1.77 (m, 2H, partially obscured), 1.82 (m, 6H), 2.01 (m, 6H),
2.32 (s, 2H), 3.95 (m, 2H), 4.07 (m, 2H), 12.74 (s, 1H); MS m/z )
387.37 (MH+); tR ) 7.46 min.
2-((4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)bicyclo[2.2.2]octan-1-yl)(methyl)amino)acetic Acid, 20. To a
stirred mixture of 0.100 g (0.257 mmol) of 16, 0.039 g (0.257 mmol)
of sarcosine hydrochloride, 0.143 mL (1.03 mmol) of NEt3, and 2
mL of anhydrous acetonitrile was added 0.103 g (0.270 mmol) of
HATU. The reaction solution was stirred at rt for 16 h. The reaction
mixture was concentrated in vacuo and combined with 10 mL of
EtOAc and 10 mL of 10% citric acid. The aqueous layer was
separated and washed twice with 10-mL portions of EtOAc. The
combined organic fractions were washed with 10-mL portions of
satd NaHCO3 and brine and concentrated in vacuo. The resultant
solid was dissolved in a mixture of 5 mL of MeOH and 5 mL of
1 N NaOH and stirred for 16 h. The reaction solution was
concentrated in vacuo, taken up in 10 mL of water, and washed
twice with 10-mL portions of CH2Cl2. The aqueous layer was
acidified with concentrated HCl, and the resultant precipitate was
collected by suction filtration to give 0.094 g (77% yield) of an
off-white solid. 1H NMR (300 MHz, CDCl3) δ 0.90 (m, 6H), 1.65
(m, 4H), 1.75 (dt, 2H), 1.95 (s, 3H), 2.00 (m, 12H), 3.20 (s, 3H),
3.95 (dd, 2H), 4.00 (dd, 2H), 4.10 (s, 2H), 12.05 (s, 1H); 13C NMR
(100 MHz, CDCl3) δ 2.3, 11.6, 11.7, 28.2, 30.2, 34.2, 38.9, 40.1,
151.1, 173.9, 176.9; MS m/z ) 460.18 (MH+); tR ) 3.91 min.
The following compounds were made in an analogous manner.
4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]octane-1-carboxylic Acid (2-Dimethylamino-ethyl)-
amide, 17. 1H NMR (300 MHz, CDCl3) δ 0.95 (m, 6H), 1.68 (dt,
2H), 1.75 (dt, 2H), 1.90 (m, 6H), 2.00 (m, 6H), 2.95 (s, 6H), 3.30
(m, 2H), 3.65 (m, 2H), 3.98 (dd, 2H), 4.08 (dd, 2H), 10.40 (s, 1H);
MS m/z ) 459.17 (MH+); tR ) 3.41 min.
[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]oct-1-yl]-acetic Acid, 31. To a solution of 30 (170
mg, 0.440 mmol) in t-BuOH (10 mL) and 2-methyl-2-butene (10
equiv, 4.4 mmol, 470 µL), cooled with the aid of an ice bath, was
added NaClO2 (1.5 equiv, 0.66 mmol). The resulting yellow solution
was allowed to reach ambient temperature over a period of 14 h
and then concentrated in vacuo. The resulting oily residue was
partitioned between water (10 mL) and CH2Cl2 (10 mL). The
aqueous phase was acidified by the dropwise addition of concen-
trated HCl, and the resulting precipitate was collected, washed with
1
water, and dried to afford 105 mg (59%) as a white powder. H
NMR (400 MHz, CDCl3) δ 0.91 (t, 3H), 0.93 (t, 3H), 1.63 (m,
2H), 1.77 (m, 2H, partially obscured), 1.82 (m, 6H), 2.01 (m, 6H),
2.32 (s, 2H), 3.95 (m, 2H), 4.07 (m, 2H), 12.74 (s, 1H); 13C NMR
(125 MHz, CDCl3) δ 11.2, 11.3, 21.3, 21.3, 30.1, 30.6, 33.6, 43.0,
43.1, 43.3, 45.3, 107.1, 148.8, 151.0, 156.3, 161.9, 176.5; HRMS
m/z ) 403.23414 (MH+), calcd ) 403.23398.
{[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]octane-1-carbonyl]-amino}-acetic Acid Methyl Es-
1
ter, 18. H NMR (300 MHz, CDCl3) δ 0.97 (m, 6H), 1.68-1.84
(m, 4H), 1.98 (m, 6H), 2.06 (m, 6H), 3.78 (s, 3H), 4.06 (s, 6H),
6.25 (t, 1H); 13C NMR (125 MHz, DMSO-d6) δ 11.0, 11.2, 20.8,
20.8, 27.7, 29.6, 33.2, 37.9, 41.9, 44.2, 60.0, 72.2, 106.4, 147.3,
150.6, 153.9, 160.3, 171.3, 176.5; MS m/z ) 460.30 (MH+); tR )
4.26 min.
(E)-3-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-bicyclo[2.2.2]oct-1-yl]-acrylic Acid, 28. Trimethylphosphono
acetate (0.161 g, 0.886 mmol) was dissolved in 12 mL of toluene
and cooled to between 0 and 5 °C. KHMDS (0.5 M in toluene;
3.54 mL) was added dropwise while stirring over a period of 5
min. After an additional 30 min at 0-5 °C, 0.300 g (0.805 mmol)
of 27 was added, and the reaction was allowed to warm to rt and
stirred for 16 h. The reaction mixture was concentrated in vacuo.
To the dissolved crude material in 25 mL of MeOH and 10 mL of
water was added 0.150 g LiOH, and the mixture was stirred at rt
overnight, concentrated in vacuo, and redissolved in 15 mL of water.
The water layer was extracted thrice with 20-mL portions of EtOAc
and acidified with concentrated HCl, and the precipitate was
collected by suction filtration to give 0.190 g (57% yield) of the
trans-acrylic acid product. 1H NMR (500 MHz, CDCl3) δ 0.78 (2
t partially obscured, 6H), 1.50 (m, 2H), 1.52 (m, 6H), 1.88 (m,
6H), 3.83 (dd, 1H), 3.93 (dd, 2H), 5.67 (d, 1H), 6.85 (d, 1H), 12.27
(s, 1H); 13C NMR (125 MHz, CDCl3) δ 11.2, 11.4, 21.3, 21.3,
30.0, 30.2, 34.0, 34.2, 43.4, 45.3, 106.5, 118.2, 149.2, 150.9, 156.2,
158.1, 161.7, 170.6; Anal. (C22H30N4O4) C, H, N; HRMS m/z )
415.23414 (MH+), calcd ) 415.23398; tR ) 4.80 min.
{[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]octane-1-carbonyl]-amino}-acetic Acid, 19. 1H
NMR (500 MHz, CDCl3) δ 0.87 (t, 3H), 0.90 (t, 3H partially
obscured), 1.59 (q, 2H), 1.72 (q, 2H), 1.92 (m, 6H), 1.99 (m, 6H),
3.94 (t, 2H), 4.03 (t, 2H partially obscured), 4.07 (m, 2H), 6.06 (s,
1H), 12.18 (s, 1H), 13.55 (br s, 1H); 13C NMR (125 MHz, CDCl3)
δ 11.2, 11.3, 21.2, 21.3, 28.4, 29.8, 33.8, 38.9, 40.9, 43.5, 45.5,
106.1, 149.5, 150.6, 156.3, 161.4, 174.5, 177.0; MS m/z ) 446.06
(MH+).
3-{[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)-bicyclo[2.2.2]octane-1-carbonyl]-amino}-propionic Acid Meth-
1
yl Ester, 21. H NMR (300 MHz, CDCl3) δ 0.96 (m, 6H), 1.63-
1.83 (m, 4H), 1.83-2.07 (m, 12H), 2.56 (t, 2H), 3.55 (dt, 2H),
3.72 (s, 3H), 4.02 (dt, 2H), 4.10 (dt, 2H), 6.53 (t, 1H); MS m/z )
474.40 (MH+); tR ) 4.32 min.
1-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)-bicyclo[2.2.2]octane-1-carbonyl]-piperidine-4-carboxylic Acid,
22. 1H NMR (400 MHz, CDCl3) δ 0.84 (t, 3H), 0.085 (t, 3H), 1.50-
1.68 (m, 6H), 1.84-1.92 (m, 14H), 2.44 (m, 1H), 2.86 (m, 2H),
3.78 (t, 2H), 3.91 (t, 2H), 4.15 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 11.9, 12.0, 22.8, 22.8, 29.6, 30.1, 31.3, 35.4, 41.5, 42.4,
46.5, 79.2, 79.6, 79.8, 80.0, 122.6, 130.4, 149.9, 152.6, 153.3, 156.5,
162.6, 177.5, 178.4; tR ) 4.20 min.
[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-purin-8-yl)-
bicyclo[2.2.2]oct-1-yl]-acetaldehyde, 30. To a stirred suspension
of methoxymethyl triphenylphosphonium chloride (1.1 g, 3.2 mmol)
in THF (60 mL) at -78 °C was added a solution of KHMDS (0.5
M in toluene, 10 mL, 5 mmol). The resulting yellow mixture was
stirred at this temperature for 1.5 h, and a solution of 27 (372 mg,
1.0 mmol) in THF (12 mL) was added over a period of 20 min.
The mixture was held at -78 °C for 6 h and allowed to reach
ambient temperature overnight (12 h). The reaction mixture was
3-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)-bicyclo[2.2.2]oct-1-yl]-propionic Acid, 29. Acrylic acid 28
(0.050 g) was dissolved in 5 mL of MeOH and combined with
0.005 g of 10% Pd/C. The reaction vessel was purged three times
with N2 and then placed under a balloon of H2 gas. After 2 h, the
reaction mixture was filtered and concd to give 0.037 g (74% yield)
1
of a white solid. H NMR (400 MHz, DMSO-d6) δ 0.598 (t, 3H),
0.604 (t, 3H), 1.14 (m, 8H), 1.28 (tq, 2H), 1.41 (tq, 2H), 1.59 (m,
6H), 1.86 (dd, 2H), 3.57 (t, 2H), 3.67 (t, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 11.4, 11.5, 21.2, 29.0, 30.2, 30.2, 30.4, 33.6, 36.0,
42.3, 44.5, 106.7, 147.7, 151.0, 154.3, 161.0, 175.3; mp 278 °C;
Anal. (C22H32N4O4) C, H, N; HRMS m/z ) 417.24976 (MH+), calcd
) 417.24963; tR ) 4.90 min.
4-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)-bicyclo[2.2.2]oct-1-yl]-butyric Acid Methyl Ester, 32. A

8-Bicyclo[2.2.2]octylxanthines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7129
solution of 30 (233 mg, 0.604 mmol) and (triphenyl-phospha-
nylidene)-acetic acid methyl ester (242 mg, 0.725 mmol) in THF
(25 mL) was heated at 75 °C for 6 h. The reaction mixture was
allowed to cool to rt and concentrated in vacuo to afford an oil
that was purified by radial chromatography (2 mm plate) using
2-5% MeOH in CH2Cl2 as eluent. The resulting mixture of cis/
trans-olefins was dissolved in EtOH (6 mL) and hydrogenated using
Pd on carbon (10 mol %) and a balloon of hydrogen affixed to a
3-way stopcock/ground glass adapter. After stirring overnight, the
mixture was degassed, filtered through Celite, and concentrated in
The right atrium was dissected and cleaned of surrounding
myocardial and vascular tissue. Two lengths of thread were attached
at opposite ends of the atrium. One thread anchored the tissue to a
glass rod, and the other was connected to an isometric force
transducer. The tissue was suspended in a water-jacketed reservoir
warmed to 37 °C and bubbled with 95% O2/5% CO2. A preload
tension of 2 grams (g) was applied using a precalibrated Gould
recorder. Hung tissue was washed with warm, oxygenated Krebs
buffer, while maintaining 2 g of tension. Baseline atrial beat rate
was measured on Ponemah software from Gould Instruments
(Valley View, Ohio).
1
vacuo to give a brittle foam (140 mg, 54%). H NMR (400 MHz,
CDCl3) δ 0.93 (m, 6H), 1.11-1.15 (m, 2H), 1.47-1.68 (m, 10H),
1.74 (dd, 2H), 1.92-1.96 (m, 6H), 2.65 (dd, 2H), 3.64 (s, 3H),
3.99 (dd, 2H), 4.06 (dd, 2H), 11.55 (s, 1H); Anal. (C24H36N4O4)
C, H, N; HRMS m/z ) 445.28102 (MH+), calcd ) 445.28093; tR
) 8.93 min.
Determination of EC50 of 29 Using the CPA Dose Reversal
Paradigm. Isoproterenol (30 nM) was added to all baths containing
atria to increase the baseline atrial rate to between 350 and 400
beats per minute (bpm). Following rate stabilization, 130 nM CPA
was added to baths to cause a 75% reduction in atrial beating rate
(control 0). Increasing concentrations of 27 were then added to the
baths until the rate was restored to maximum and the effective
concentration at which 50% response was obtained (EC50) was
determined. Five atria were used in this experiment. The effects of
compound 29 were fully reversible (in the presence of CPA) after
washout of the compound from the isolated atria.
Blockade Paradigm: Schild Plot (pA2) Analysis. Isoproterenol
(30 nM) was added to all baths containing atria to increase the
baseline atrial rate to between 350 and 400 bpm. Varying
concentrations of 29 (0.3 nM, 3.0 nM, and 30.0 nM; or vehicle
control (dimethylsulfoxide [DMSO])) were then added to isolated
tissue baths with beating atria, and 5 min was allowed to ensure
stabilization (control 0). Increasing concentrations of CPA from 1
nM to 30 µM were added cumulatively until the atrial rate was
lowered to zero. The EC50 was determined for the vehicle control
and each of the 29 concentrations. Schild analysis was used to
calculate the affinity of 29, the competitive antagonist, for its
receptor (pA2). Five or six atria were used for each 29 concentration
and the vehicle control.
Statistical Analysis. In the dose reversal experiment, the mean
and standard error of the mean (SEM) of the atrial beating rate
(bpm) were calculated at baseline and following the addition of
isoproterenol, CPA, and each dose of 29. The mean ((SEM)
percent change from baseline for the control (0) was calculated as
(CPA bpm at baseline - CPA bpm at baseline)/(isoproterenol bpm
at baseline - CPA bpm at baseline) × 100. The mean ((SEM)
percent change from baseline for each 29 dose was calculated as
(29 dose bpm - CPA baseline bpm)/(isoproterenol bpm at baseline
- CPA bpm at baseline) × 100. The EC50 was determined as the
effective concentration of 29 at which a 50% response was obtained.
In the blockade experiment, atrial rates were recorded at baseline
and following addition of isoproterenol, 29, or vehicle control
(control 0) and each CPA dose. The percent change was calculated
as (CPA dose bpm - 29 bpm [control])/(29 bpm [control] bpm) ×
100. Using the data from the blockade experiment, a Schild analysis
was performed to determine the affinity of 29, the antagonist, for
its receptor with CPA as the agonist (pA2).
4-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-
yl)-bicyclo[2.2.2]oct-1-yl]-butyric Acid, 33. A solution of ester
32 (45 mg, 100 µmol) in THF (4 mL) was treated with 1 M LiOH
(2 mL), and the resulting turbid solution was stirred at rt overnight.
The solution was concentrated in vacuo, diluted with water (2 mL),
and acidified by the dropwise addition of concentrated HCl. The
resulting precipitate was collected, washed with water, and dried
to afford a white powder (35 mg, 81%). 1H NMR (500 MHz,
CDCl3) δ 0.96 (t, 3H), 1.00 (t, 3H), 1.28 (dd, 2H), 1.60 (m, 8H),
1.68 (q, 2H), 1.82 (q, 2H), 2.05 (m, 6H), 2.48 (t, 2H), 4.01 (t, 2H),
4.19 (t, 2H), 12.59 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 11.1,
11.3, 18.1, 21.2, 21.4, 30.5, 30.6, 32.8, 34.1, 40.6, 43.5, 45.7, 106.2,
148.8, 150.7, 156.1, 162.1, 178.2; HRMS m/z ) 431.26540 (MH+),
calcd ) 431.26528; tR ) 7.52 min.
Human Adenosine Receptor Screening: Initial screening was
of a solution of the antagonist (1 µM) incubated with membranes
in 50 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl2, and 1
U/mL adenosine deaminase. DMSO was included in all assays
except hA3 at a final concentration of 5%. Radioligands consisted
of the following: hA1, 0.3 nM 125I-aminobenzyladenosine (125I-
ABA); hA2A, 0.7 nM 125I-ZM241385; hA2B, 0.5 nM 125I-3-(4-
aminobenzyl)-8-phenyloxyacetate-1-propyl-xanthine; and hA3, 0.6
nM 125I-ABA. Nonspecific binding was measured in the presence
of 50 µM xanthine amine congener or 10 µM BW-1433 (hA3).
Rat Adenosine Receptor Screening: Compounds were incu-
bated at room temperature for 90 min with radioligand (2 nM 3H-
CPX for rA1; 0.5-1.2 nM 3H-ZM241385 for rA2A), 50 mM Tris-
HCl buffer (pH 7.4), adenosine deaminase (2 U/mL), and 100-µL
aliquots of crude membrane suspensions (10-20 µg protein)
prepared from either rat brain cortex (for rA1) or rat brain striatum
(for rA2A). Incubations were terminated by the addition of ice-cold
50 mM Tris-HCl buffer and the collection of membranes was done
on Whatman GF/C glass fiber filters by vacuum filtration.
Membrane-bound radioactivity was quantified by liquid scintillation
counting. Values of Ki were determined from concentration-
response relationships for each compound to displace binding of
radioligand, using GraphPad Prism (GraphPad, San Diego, CA).
Rat Oral Efficacy Screen: Rats were placed into metabolic
cages and dosed by gavage with various doses of 29. The doses
and group sizes were: vehicle (0.5% CMC; n ) 3); 29, 0.001 mg/
kg (n ) 4), 0.003 mg/kg (n ) 4), 0.01 mg/kg (n ) 4), 0.03 mg/kg
(n ) 5), 0.1 mg/kg (n ) 5), 0.3 mg/kg (n ) 5), 1.0 mg/kg (n ) 3),
and 3.0 mg/kg (n ) 3). Urine was collected for 4 h after dosing.
Urine volume was measured gravimetrically, and sodium and
potassium concentrations were determined by flame photometry.
Urine flow, UNaV, and UKV were calculated and are shown as
units per hour as an average for the 4-hour collection period.
All experiments were conducted in accordance with the NIH
Guide for the Care and Use of Laboratory Animals, and the
protocols were approved by the Institutional Animal Care and Use
Committee.
Male Sprague-Dawley rats were purchased from Charles River
Laboratories (Raleigh, NC) and housed in the Biogen virus-free
laboratory animal facility in ventilated isolator cage racks. Animals
were allowed to acclimatize for 4 days prior to the beginning of
the study. Rats had ad libitum access to irradiated standard chow
(LabDiet Prolab 5P75 Isopro RMH 3000) and sterile water
throughout the acclimatization and experimental period.
Isolation of Atria From Rat Heart. Hearts were removed from
the rats and placed in petri dishes containing Krebs Henseleit
(Krebs) buffer prewarmed to 37 °C and bubbled with 95% O2/5%
CO2. The composition of Krebs buffer was 118 mM NaCl, 4.7 mM
KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM
CaCl2, and 11 mM glucose, pH 7.4.
Acknowledgment. We thank, Gnanasambandam Kumaravel,
Hexi Chang, Carol Ensinger, and Eric Whalley for their
numerous contributions to the adenosine A1 anatagonist project.
We also acknowledge Xiaopeng Hronowski for HRMS analyses.
Supporting Information Available: Experimental details (de-
tails of HPLC, MS, and elemental analyses of compounds) and

7130 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Kiesman et al.
(9) Pfister, J. R.; Belardinelli, L.; Lee, G.; Lum, R. T.; Milner, P.; Stanley,
W. C.; Linden, J.; Baker, S. P.; Schreiner, G. Synthesis and biological
evaluation of the enantiomers of the potent and selective A1-
adenosine antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]-xan-
thine. J. Med. Chem. 1997, 40, 1773-1778.
data for pA2 determination and statistical analysis. This material is
available free of charge via the Internet at http://pubs.acs.org.
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