Fluorosulfonyl-substituted xanthines as selective irreversible antagonists for the A1-adenosine receptor

Article abstract of DOI:10.1021/jm000181f

FSCPX (1) has been reported to be a potent, selective, and irreversible antagonist for the A₁-adenosine receptor (AR). To obtain an irreversible A₁AR antagonist with potentially better stability and to further elucidate the effects o

Full text of DOI:10.1021/jm000181f

J . Med. Chem. 2000, 43, 4973-4980
4973
F lu or osu lfon yl-Su bstitu ted Xa n th in es a s Selective Ir r ever sible An ta gon ists for
th e A1-Ad en osin e Recep tor
Anthony R. Beauglehole,† Stephen P. Baker,§ and Peter J . Scammells*,†
Centre for Chiral and Molecular Technologies, Deakin University, Geelong, Victoria 3217, Australia, and Department of
Pharmacology and Therapeutics, University of Florida, P.O. Box 100267, Gainesville, Florida 32610
Received April 26, 2000
FSCPX (1) has been reported to be a potent, selective, and irreversible antagonist for the A1-
adenosine receptor (AR). To obtain an irreversible A1AR antagonist with potentially better
stability and to further elucidate the effects of linker structure on the pharmacological
characteristics, several new analogues were targeted in which the labile ester linkage of 1 was
replaced by more stable functionalities. In particular, alkyl and amide linkers between the
xanthine pharmacophore and the reactive 4-fluorosulfonylphenyl group were explored. The
data showed that the chemical composition of the linker affects the affinity and apparent
irreversible binding to the A1AR. Overall, compound 23b appeared to have the most
advantageous characteristics as a potential irreversible ligand for the A1AR. These include
relatively high affinity for the A1AR as compared to the A2AAR, concentration-dependent and
selective apparent irreversible binding to the A1AR, and ease of removal of unbound ligand
from biological membranes. These properties indicate that 23b has the potential to be a useful
tool for further study of the structure and function of the A1AR.
In tr od u ction
other hand, 8-cyclopentyl-3-(3-((4-fluorosulfonylbenzoyl)-
oxy)propyl)-1-propylxanthine (FSCPX, 1),8,9 incorporat-
Adenosine receptors (AR) are distributed throughout
the body where they mediate the many physiological
functions of the autocoid adenosine.1-3 These effects
include inhibition of neuronal firing, release of neuro-
transmitters, inhibition of platelet aggregation, cardiac
depression, and vasodilation, vasoconstriction, and im-
munosuppressant effects. For the study of receptor
structure and function, chemoreactive and photoreactive
irreversible antagonists are useful tools. Desirable
characteristics of a chemoreactive antagonist include a
pharmacophore that provides affinity and selectivity for
the receptor and a reactive moiety that provides cova-
lent incorporation into the receptor allowing for the
control of receptor concentrations over a wide range.
These characteristics have made chemoreactive antago-
nists useful in a variety of studies including the
identification and mapping of ligand-binding sites,4 the
physiological function of a receptor and its subtypes,5
the relationship between receptor occupancy and a
tissue or cellular response,6 as well as the kinetics of
the turnover and cellular processing of receptors.7
In 1994, a series of compounds were synthesized in
which several reactive moieties were attached to the N-3
position of the xanthine ring in order to develop ir-
reversible antagonists for the A1AR subtype.8 The
apparent irreversible binding of these ligands is de-
pendent upon the reactive moiety and its location. For
example, the 3-bromoacetoxypropyl ester and 3-fluoro-
sulfonylphenyl derivatives were found to have relatively
high affinity for the A1AR but showed little or no
apparent irreversible binding to the receptor. On the
ing the chemoreactive 4-fluorosulfonylphenyl moiety,
was found to have high affinity and selectivity for the
A1AR as compared to the A2AAR. This compound also
produced a concentration-dependent reduction in the
maximal binding of [3H]-8-cyclopentyl-1,3-dipropylxan-
thine ([3H]DPCPX) to the A1AR of DDT1 MF-2 cells
indicating that it is an irreversible ligand. The utility
of 1 to study the A1AR is indicated by recent reports
where the apparent irreversible nature of this compound
was used to determine the relationship between receptor
number and a tissue response.10,11 Although compound
1 binds to the A1AR in an apparent irreversible manner
using in vitro biological preparations, the linkage
between the 4-fluorosulfonylphenyl moiety and the
xanthine ring contains an ester group which has the
potential to be cleaved by esterases either in vivo or in
preparations containing significant enzyme activity.
Cleavage at the ester linkage would render the ligand
inactive in terms of potential irreversible receptor
binding. To obtain an irreversible A1AR antagonist with
potentially better stability and to further elucidate the
effects of linker structure on the pharmacological char-
acteristics, several analogues of 1 were targeted in
which this labile ester linkage was replaced by more
* To whom correspondence should be addressed. Tel: +61 3 5227
1439. Fax: +61 3 5227 1040. E-mail: scam@deakin.edu.au.
† Deakin University.
§ University of Florida.
10.1021/jm000181f CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/22/2000

4974 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Beauglehole et al.
Sch em e 1a
Sch em e 2
while intramolecular hydrogen bonding may have low-
ered the reactivity of the hydroxyl group of 4.
Because of the low reactivity of the 4-fluorosulfonyl-
benzyl bromide toward nucleophilic attack by the alkox-
ide formed from 4, an alternative approach was adopted
where the alkoxide was formed on the 4-fluorosulfonyl-
benzyl moiety. Bromination of 8-cyclopentyl-3-(3-hy-
droxypropyl)-1-propylxanthine (2a ) was attempted us-
ing phosphorus tribromide in chloroform (Scheme 2).
While bromination occurred the product, 3-(3-bromo-
propyl)-8-cyclohexyl-1-propylxanthine (6), underwent a
facile intramolecular substitution to afford the cyclized
product 7.
Rather than pursue this target further, it was decided
to adopt an alternative synthetic strategy in which the
4-fluorosulfonylphenyl linker unit was prepared sepa-
rately and attached directly to N-3. This approach offers
the flexibility to accommodate a variety of linkers and
offers the advantage of a more convergent approach. The
latter is particularly valuable as it avoids the need to
optimize the attachment of the 4-fluorosulfonylphenyl
group at the end of a long linear synthesis (over 10 steps
from allylurea).
Alk a n e Lin k er . Scheme 3 depicts a revised synthetic
strategy to target compounds 17-19 containing one-,
three-, and five-methylene spacers where the reactive
4-fluorosulfonylphenyl moiety is assembled separate
from the xanthine heterocycle prior to attachment and
subsequent deprotection. Debenzylation of compound 9
initially proved difficult. Attempts to N-3 debenzylate
with either activated Pd-C catalyst or Pearlmans
catalyst [Pd(OH)2] with hydrogen at 50 psi were unsuc-
cessful. The desired target was eventually synthesized
using activated palladium on carbon in methanol with
ammonium formate16 as the hydrogen source. This
reaction worked best when the reagents were thor-
oughly mixed (by dissolving in methanol and then
evaporating the methanol) and then heated in the melt
at 140 °C. It should be noted that this reaction gave
little or no product on some attempts, possibly due to
some contaminant (undetectable by NMR) poisoning the
catalyst.
a
Reagents: (i) NaH or KH, BrCH2C6H4SO2F, Bu4NI, DMF; (ii)
NaH, SEM-Cl, DMF; (iii) aq HCl.
stable functionalities. Synthetic routes to ether, alkyl,
and amide linkers between the xanthine pharmacophore
and the reactive 4-fluorosulfonylphenyl group were
explored. All target xanthines possessed 1-propyl sub-
stitution and either 8-cyclopentyl or 8-cyclohexyl sub-
stitution to enhance pharmacophore affinity for the
A1AR. All new compounds were characterized for affin-
ity and apparent irreversible binding at the A1AR and
A
2AAR subtypes.
Ch em istr y
Eth er Lin k er . Scheme 1 depicts the synthetic route
to analogues of 1 with an ether linkage between a
4-fluorosulfonylbenzyl moiety and the xanthine. At-
tempts to selectively O-alkylate 8-cyclopentyl-3-(3-hy-
droxypropyl)-1-propylxanthine (2) with 4-fluorosulfonyl-
benzyl bromide12 proved unsuccessful as N-alkylation
in the 7-position occurred in preference to O-alkylation
under a range of conditions. To overcome this problem
it was decided to protect N-7 prior to formation of the
ether linkage. Although the protecting groups trialled
initially (acetyl and tritylthio) blocked the hydroxyl
group, selective protection of N-7 was eventually achieved
using the [2-(trimethylsilyl)ethoxy]methyl protecting
group.13-15 Reaction of 3-(3-hydroxypropyl)xanthine (2a )
with [2-(trimethylsilyl)ethoxy]methyl chloride in the
presence of sodium hydride in DMF gave a 43% yield
of the desired product. This process was later optimized
using 2b (potassium carbonate proved to be a superior
base) to afford a 98% yield of the N-7-protected xan-
thine.
Formation of an ether linkage between 4-fluorosul-
fonylbenzyl bromide to the N-7-protected 3-(3-hydroxy-
propyl)xanthine 4 under typical Williamson conditions
(sodium or potassium hydride in DMF or THF) proved
unsuccessful, returning starting material. Addition of
tetrabutylammonium iodide to generate 4-fluorosul-
fonylbenzyl iodide in situ had no effect. The reduced
reactivity of the alkyl halide may be explained by the
electron-withdrawing effect of the fluorosulfonyl group,
The alkyl linker with a one-methyl spacer (compound
11) was synthesized by literature methods.12 Construc-
tion of the alkyl linkers with the three- and five-
methylene spacers (compounds 12 and 13, respectively)
was achieved by chlorosulfonation of 1-phenylpropyl
bromide and 1-phenylpentyl bromide with chlorosulfonic
acid, followed by halogen exchange with potassium

Fluorosulfonyl-Substituted Xanthines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4975
Sch em e 3a
acid (24) and 3-bromopropylamine hydrobromide with
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)
activation and DIPEA afforded 25 in 63% yield. Initial
attempts focused on coupling 4-fluorosulfonylbenzoic
acid with 3-amino-1-propanol, followed by bromine
exchange with the alcohol. Amide coupling using EEDQ
activation with DIPEA yielded the alcohol in 83% yield,
which was then brominated in 59% yield with N-
bromosuccinimide, triphenylphosphine, and pyridine. It
was later found that this unit could be prepared more
efficiently by coupling 4-fluorosulfonylbenzoic acid with
3-bromopropylamine hydrobromide directly. Alkylation
of N-(3-bromopropyl)-4-fluorosulfonylbenzamide (25) by
the xanthine 10 produced the desired 3-substituted
xanthine 22 in 87% yield. Subsequent removal of the
SEM protecting group with aqueous HCl in ethanol
produced the target compound 23 in 96% yield.
P h a r m a cology
The reactive xanthine derivatives were tested for
affinity and apparent irreversible binding at the A1AR
in DDT cells and A2AAR in PC-12 cells. Affinity was
determined as the concentration of derivative that
inhibited radioligand binding to the receptor by 50%
(IC50). Apparent irreversible binding was determined by
incubating the derivatives with the cells followed by cell
washing to remove unbound ligand and then determin-
ing the KD and Bmax for radioligand binding in cell
membranes. Similar to that previously reported, com-
pound 1 inhibited [3H]DPCPX binding to the A1AR in
the low-nanomolar range and reduced the receptor
content after pretreatment with 5 nM (Table 1). This is
consistent with the compound binding irreversibly to the
A1AR. However, preincubation with a higher concentra-
tion of 1 (100 nM) reduced the receptor content further
but decreased the KD for the radioligand to the receptors
remaining. This suggests that under the standard
preincubation and cell washing conditions that residual
ligand was retained by the membranes. The C-8 cyclo-
hexylxanthine with a single-methylene linker between
the N-3 of the xanthine ring and the 4-fluorosulfo-
nylphenyl moiety (17) has an IC50 for inhibition of
[3H]DPCPX binding of 165 nM. Increasing the number
of methylenes in the linker to three (18) or five (19) did
not significantly change the IC50 as compared to 17.
Derivative 17 did not show apparent irreversible bind-
ing to the A1AR because the Bmax for [3H]DPCPX
binding did not change after preincubation with this
compound at a relatively high concentration followed
by cell washing. However, pretreatment of cells with 200
nM 18 or 19 reduced the receptor content by <15% and
<50%, respectively indicating that apparent irreversible
binding of these two derivatives is relatively weak. Since
the five-methylene spacer of compound 19 should allow
the fluorosulfonyl group to access the same part of the
receptor as the corresponding group of compound 1, we
believe that the reduced apparent irreversible binding
may have resulted from the electron-releasing linker
deactivating the fluorosulfonyl group to nucleophilic
attack. It is also possible that the ester carbonyl of 1
may be involved in some intermolecular interaction with
the receptor that holds the fluorosulfonyl group in a
favorable position for a reaction to take place. To
increase the reactivity of the fluorosulfonylphenyl group,
a
Reagents: (i) K2CO3, SEM-Cl, DMF; (ii) Pd-C, NH4CO2,
MeOH; (iii) K2CO3, Br(CH2)nC6H4SO2F (11 n ) 1, 12 n ) 3, 13 n
) 5), DMF; (iv) aq HCl.
fluoride in refluxing dioxane. The five-methylene spacer
required initial bromination of 5-phenyl-1-pentanol with
phosphorus tribromide as this bromide was not com-
mercially available. Attachment of the reactive moieties
to the SEM-protected species 10 produced the penulti-
mate compounds 14-16. This conversion was effected
using potassium carbonate in DMF with the corre-
sponding alkyl bromide with yields between 39% and
51%. Subsequent deprotection with aqueous HCl in
ethanol produced the target compounds 17-19 in
respectable yields (66-82%).
Am id e Lin k er . A similar synthetic approach to the
one used for the synthesis of 18 was initially pursued
to prepare analogues with an amide linker. It was
thought that only slight modifications to this approach
would allow the preparation of 3-(3-aminopropyl)xan-
thine that could be reacted with 4-fluorosulfonylbenzoyl
chloride to give the desired target. However, hydro-
boration-amination of 3-allyl-8-cyclopentyl-1-propyl-
xanthine returned a complex mixture of products, and
attempts to convert the hydroxyl group of 2a to the
corresponding amine were also unsuccessful.
An alternative synthesis was devised, whereby 1-
phthalimidopropyl bromide was attached directly to the
SEM-protected xanthine 10, to afford the 3-substituted
xanthine 20 (Scheme 4). Compound 20 was heated in a
pressure bomb at 50 °C with methylamine for 14 h to
afford the desired amine in 56% yield. This conversion
was also achieved in identical yield by refluxing 20 in
hydrazine monohydrate for 15 h. Coupling of 4-fluoro-
sulfonylbenzoic acid to the amine 21 using N,N-diiso-
propylethylamine (DIPEA) and the water-soluble 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI) for activation afforded the penultimate com-
pound 22a , which was deprotected to give the desired
target 23a .
A more convergent synthesis with construction of the
reactive 3-(4-fluorosulfonylbenzamido)propyl group sepa-
rate from the xanthine heterocycle was also investigated
(Scheme 4). Amide coupling of 4-fluorosulfonylbenzoic

4976 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Beauglehole et al.
Sch em e 4a
a
Reagents: (i) K2CO3, Br(CH2)3Phth, DMF; (ii) method A - MeNH2, MeOH, method B - NH2NH2‚H2O; (iii) HO2CC6H4SO2F, EDCI,
DIPEA; (iv) Br(CH2)3NHC(O)C6H4SO2F, K2CO3, DMF; (v) aq HCl.
Ta ble 2. Ligand Binding to the A2A AR
Sch em e 5
IC50 vs
[3H]ZM214385,
µM
% loss of specific KD [3H]ZM241385
[3H]ZM241385
binding (concn)a
binding post-
no.
xanthine, nMb
1
17
18
19
23a
23b
1.2 ( 0.2
>10
44 ( 11 (0.2)
ND
ND
ND
63 ( 5 (0.2)
65 ( 7 (0.2)
0.23 ( 0.08
ND
ND
ND
0.27 ( 0.05
0.34 ( 0.03
>10
>10
0.42 ( 0.05
2.8 ( 0.4
a
PC-12 cells were pretreated with the indicated xanthine
derivative at the concentration (µM) in parentheses. The cells were
then washed 8 times and the membranes were assayed for Bmax
and KD as described in the Experimental Section. Control Bmax
was 3.4 ( 0.3 pmol/mg protein. Control KD was 0.25 ( 0.03 nM.
Data are the mean ( SE, n ) 3-4. ND, not determined.
Ta ble 1. Ligand Binding to the A1AR
IC50 vs
[3H]DPCPX,
nM
% loss of specific
KD [3H]DPCPX
binding post-
xanthine, nMb
b
[3H]DPCPX
no.
binding (concn)a
moiety, inhibited [3H]DPCPX binding to the A1AR with
an IC50 of 24.9 nM. Interestingly, compound 23a only
differs from 19 by the exchange of two methylenes for
an amide in the linker, and this resulted in a 4-fold
decrease in the IC50 for 23a . This suggests that the
amide may provide additional contact points with the
receptor to increase the affinity. Pretreatment of DDT
cells with 23a resulted in a concentration-dependent
decrease in the A1AR content indicating that 23a is an
irreversible ligand (Table 1). Furthermore, at the high-
est pretreatment concentration used (100 nM), the KD
for [3H]DPCPX binding did not change for the receptors
remaining suggesting that, unlike 1, compound 23a
easily washed out of the membrane preparation. Re-
placing the C-8 cyclohexyl group of 23a with a cyclo-
pentyl (23b) did not alter the IC50 of the latter for the
A1AR, although its IC50 for the A2AAR was 6.6-fold
higher as compared to that for 23a (Table 2). Similar
to 23a , 23b produced a concentration-dependent de-
crease in the A1AR content and it appeared to wash
out the membrane preparation relatively easily. This
was indicated by the lack of change in KD value for
[3H]DPCPX binding to the receptors remaining after cell
pretreatment with a high concentration (100 nM) of 23b.
The binding of 17-19 to the A2AAR was rela-
1
11.8 ( 3.2
67 ( 3 (5)
0.33 ( 0.05
0.71 ( 0.04
0.32 ( 0.04
0.37 ( 0.08
0.81 ( 0.09
0.28 ( 0.02
0.27 ( 0.03
0.37 ( 0.03
0.34 ( 0.05
0.26 ( 0.03
0.24 ( 0.03
0.36 ( 0.06
0.31 ( 0.09
77 ( 4 (100)
0 (200)
17
18
19
23a
165 ( 35
112 ( 10
101 ( 37
24.9 ( 7.6
11 ( 6 (200)
43 ( 0.8 (200)
16 ( 5 (0.05)
43 ( 5 (0.1)
64 ( 2 (5)
82 ( 2 (100)
33 ( 4 (0.05)
64 ( 3 (0.1)
74 ( 4 (5)
23b
21 ( 7
83 ( 3 (100)
a
DDT1 MF-2 cells were pretreated with the indicated xanthine
derivative at the concentration (nM) in parentheses, and after 10
cell wash cycles membranes were assayed for Bmax and KD as
described in the Experimental Section. Control Bmax from indi-
vidual experiments ranged from 326 to 571 fmol/mg protein.
b
Control KD was 0.34 ( 0.03 nM. Data are the mean ( SE, n )
3-6.
an electron-withdrawing amide linker was targeted. It
was predicted that the electron-withdrawing effects of
the amide would be more similar to those of compound
1 and would increase the reactivity of the fluorosulfonyl
moiety.
Xanthine derivative 23a , which contains an amide
linkage between the xanthine ring and the reactive

Fluorosulfonyl-Substituted Xanthines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4977
NMR (DMSO-d6) δ 0.81 (t, 3H, CH2CH2CH3), 1.47-1.86 (m,
12H, CH2CH2CH3, CH2CH2CH2OH, 4 × cyclopentyl CH2), 3.27
(m, 1H, cyclopentyl CH), 3.45 (dt, 2H, CH2CH2CH2OH), 3.77
(t, 2H, CH2CH2CH3), 4.04 (t, 2H, CH2CH2CH2OH), 4.53 (t, 1H,
OH), 5.76 (s, 2H, CH2Ar), 7.50, 8.12 (2 × d, 4H, phenyl); 13C
NMR (DMSO-d6) δ 11.6, 20.9, 25.7, 31.4, 32.1, 36.1, 42.3, 47.2,
58.9, 106.4, 128.6, 129.4, 131.0 (d), 146.8, 148.0, 150.8, 154.5,
157.7.
tively weak as the IC50 values for the inhibition of
[3H]ZM241385 binding were greater than 10 µM (Table
2). Because of the high IC50 values of these compounds
for the A2AAR, they were not tested for irreversible
binding. In contrast, the IC50 of 23a for the A2AAR was
in the mid-nanomolar range, whereas the IC50 values
for 1 and 23b were in the low-micromolar range. Upon
the basis of the ratio of the IC50 values for binding to
the A1AR and A2AAR, compounds 23a , 23b, and 1 are
17-, 132-, and 105-fold more selective for the A1AR over
the A2AAR, respectively. Pretreatment of PC-12 cells
with 200 nM 23a , 23b, or 1 reduced the Bmax of
[3H]ZM241385 binding without a change in the KD value
of the radioligand for the receptors remaining. This
indicated that these three compounds bound to the
A2AAR in an irreversible manner. However, in keeping
with their IC50 selectivity for the A1AR, a 40-fold higher
concentration of 23a , 23b, and 1 was required to
produce similar apparent irreversible binding to the
8-Cycloh exyl-3-(3-h yd r oxyp r op yl)-1-p r op yl-7-(2-(t r i-
m eth ylsilyl)eth oxym eth yl)xa n th in e (4). Anhydrous potas-
sium carbonate (206 mg, 1.5 mmol) was added to 8-cyclohexyl-
3-(3-hydroxypropyl)-1-propylxanthine (391 mg, 1.2 mmol) in
DMF (12 mL) at room temperature and the mixture stirred
for 1 h. 2-(Trimethylsilyl)ethoxymethyl chloride (230 µL, 1.3
mmol) was added dropwise via a syringe and the mixture
stirred for 30 min at room temperature. The mixture was
filtered and evaporated to dryness, affording a viscous yellow
1
oil: yield 535 mg, 98%; H NMR (CDCl3) δ -0.04 (s, 9H, Si-
(CH3)3), 0.88-0.96 (m, 5H, CH2CH2CH3, CH2CH2Si), 1.24-1.96
(m, 14H, CH2CH2CH3, CH2CH2CH2OH, 5 × cyclohexyl CH2),
2.85 (m, 1H, CH), 3.46 (t, 2H, CH2CH2CH2OH), 3.66 (t, 2H,
CH2CH2Si), 3.95 (t, 2H, CH2CH2CH3), 4.25 (t, 2H, CH2CH2-
CH2OH), 5.73 (s, 2H, NCH2O); 13C NMR (CDCl3) δ -1.5, 11.3,
17.8, 21.2, 25.4, 25.9, 31.0, 31.5, 36.0, 39.5, 42.9, 57.5, 66.7,
72.6, 106.0, 148.0, 151.2, 154.9, 159.8. In the DMSO spectra:
4.53 (br s, 1H, CH2CH2CH2OH).
A2AAR as 5 nM did for the A1AR. Therefore, 23a , 23b,
and 1 selectively bind to the A1AR in an apparent
irreversible manner.
In summary, a series of new N-3 xanthine derivatives
were synthesized and tested for affinity and apparent
irreversible binding at the A1AR and A2AAR. The data
indicated that the chemical composition of the linker
between the N-3 position of the xanthine ring and the
4-fluorosulfonylphenyl moiety contributed to the affinity
and apparent irreversible binding to the A1AR. Fur-
thermore, the selectivity of apparent irreversible bind-
ing to the A1AR as compared to the A2AAR was en-
hanced by substitution of a cyclohexyl ring for a
cyclopentyl moiety at the C-8 position of the xanthine
ring. Overall, compound 23b appeared to have the most
advantageous characteristics as an apparent irrevers-
ible ligand for the A1AR. These include relatively high
affinity and selectivity for the A1AR as compared to the
Br om in a tion of 8-Cyclop en tyl-3-(3-h yd r oxyp r op yl)-1-
p r op ylxa n th in e (6). To a solution of 98% PPh3 (376 mg, 1.4
mmol) and 99% NBS (280 mg, 1.6 mmol) in anhydrous
dichloromethane (8 mL) at 0 °C was added a solution of
8-cyclopentyl-3-(3-hydroxypropyl)xanthine (104 mg, 0.32 mmol)
in anhydrous dichloromethane (8 mL). The mixture was
allowed to warm to room temperature with stirring for 20 h.
Ethyl acetate (150 mL) was added and the mixture washed
with a solution of saturated NaHCO3 (2 × 100 mL) and brine
(100 mL). The organic fraction was dried (MgSO4), filtered and
the solvent removed under reduced pressure to afford the crude
as a brown solid. The crude mixture was purified by column
chromatography using ethyl acetate/petroleum ether (3:2) to
afford the pure product: yield 107 mg, 86%; 1H NMR (DMSO-
d6) 0.84 (t, 3H, CH2CH2CH3), 1.47-2.03 (m, 10H, CH2CH2CH3,
4 × cyclopentyl CH2), 2.21 (m, 2H, CH2CH2CH2Br), 3.12 (m,
1H, CH), 3.53 (m, 2H, 2 × CH2CH2CH2Br), 3.81 (t, 2H, CH2-
CH2CH3), 4.08 (t, 2H, CH2CH2CH2Br); 13C NMR (DMSO-d6) δ
11.0, 20.1, 20.7, 25.1, 30.6, 35.3, 38.7, 41.0, 42.2, 107.0, 138.2,
149.1, 151.6, 154.3; ES/MS (+) 383.1. The cyclized compound
7 was also isolated: 1H NMR (DMSO-d6) 0.84 (t, 3H, CH2-
CH2CH3), 1.45-2.02 (m, 10H, CH2CH2CH3, 4 × cyclopentyl
CH2), 2.19 (m, 2H, (O)CNCH2CH2CH2N), 3.20 (m, 1H, CH),
3.81 (m, 4H, CH2CH2CH3, (O)CNCH2CH2CH2N), 4.02 (t, 2H,
(O)CNCH2CH2CH2N); 13C NMR (DMSO-d6) δ 11.1, 20.6, 20.9,
25.1, 30.5, 35.9, 38.7, 39.9, 41.7, 111.6, 138.7, 150.0, 150.3,
156.5; ES/MS (+) 303.3.
A2AAR, concentration-dependent and selective apparent
irreversible binding to the A1AR, and ease of removal
(washout) of unbound ligand from biological mem-
branes. As a result, compound 23b has the potential to
be a useful tool to further study the structure and
function of the A1AR.
Exp er im en ta l Section
Merck Kieselgel 60 and 60 F254 were used for column and
thin-layer chromatography. Melting points were determined
on Electrothermal melting point apparatus and are uncor-
rected. 1H and 13C NMR spectra were recorded on a Varian
300-MHz Unity Plus spectrometer using TMS as an internal
standard. Electrospray mass spectral data was obtained on a
Fisons VG Micromass Platform II spectromter. 8-Cyclopentyl-
3-(3-hydroxypropyl)-1-propylxanthine (2a ), 8-Cyclopentyl-3-(3-
hydroxypropyl)-1-propylxanthine (2b), and 4-fluorosulfonyl-
benzyl bromide (11) were prepared using literature method-
ology.8,12
Rep r esen ta tive P r oced u r e for SEM P r otection . Anhy-
drous potassium carbonate (5.7 mmol) was added to the N-7
free xanthine (4.6 mmol) in DMF (30 mL), and the mixture
stirred for 2 h at room temperature. 2-(Trimethylsilyl)-
ethoxymethyl chloride (5.5 mmol) was added dropwise via a
syringe and the mixture stirred for 0.5-20 h. The mixture was
filtered to remove excess potassium carbonate and evaporated
down. The residue was dissolved in methanol, adhered to silica
and purified by column chromatography using petroleum
ether/ethyl acetate (6:1).
8-Cyclop en t yl-7-((4-flu or osu lfon ylb en zoyl)oxy)-3-(3-
h yd r oxyp r op yl)-1-p r op ylxa n th in e (3). 8-Cyclopentyl-3-(3-
hydroxypropyl)-1-propylxanthine (100 mg, 0.31 mmol) was
dissolved in dry DMF (3 mL). Sodium hydride (15 mg, 0.62
mmol) in dry DMF (2 mL) was added dropwise and the solution
was stirred at ambient temperature for 15 min. 4-Fluorosul-
fonylbenzyl bromide (87 mg, 0.34 mmol) was added and
stirring was continued for a further 4 h. The reaction was
quenched with water (20 mL) and extracted with chloroform
(3 × 20 mL). Column chromatography using ethyl acetate/
hexane (1:1) as an eluent afforded pure 3 in 67% yield: 1H
3-Ben zyl-8-cycloh exyl-1-p r op yl-7-(2-(t r im et h ylsilyl)-
eth oxym eth yl)xa n th in e (9a ). The title compound (white
solid) was prepared in 97% yield using the representative
procedure for SEM protection: mp 96-97 °C; 1H NMR (DMSO-
d6) δ -0.10 (s, 9H, Si(CH3)3), 0.78-0.84 (m, 5H, CH2CH2CH3,
CH2CH2Si), 1.22-1.86 (m, 12H, CH2CH2CH3, 5 × cyclohexyl
CH2), 2.92 (m, 1H, CH), 3.58 (t, 2H, CH2CH2Si), 3.82 (t, 2H,
CH2CH2CH3), 5.15 (s, 2H, CH2Ph), 5.70 (s, 2H, NCH2O), 7.23-
7.34 (m, 5H, phenyl); 13C NMR (DMSO-d6) δ -1.5, 11.1, 17.3,
20.8, 25.2, 25.3, 31.2, 34.9, 42.0, 45.7, 65.5, 72.1, 105.6, 127.4,
127.7, 128.4, 136.9, 147.3, 150.3, 154.1, 159.5.

4978 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Beauglehole et al.
3-Ben zyl-8-cyclop en tyl-1-p r op yl-7-(2-(tr im eth ylsilyl)-
eth oxym eth yl)xa n th in e (9b). The title compound (colorless
oil) was prepared in 93% yield using the representative
procedure for SEM protection: 1H NMR (CDCl3) δ -0.03 (s,
9H, Si(CH3)3), 0.89-0.96 (m, 5H, CH2CH2CH3, CH2CH2Si),
1.62-1.74, 1.91-2.09 (2 × m, 10H, CH2CH2CH3, 4 × cyclo-
pentyl CH2), 3.29 (m, 1H, CH), 3.66 (t, 2H, CH2CH2Si), 3.95
(t, 2H, CH2CH2CH3), 5.26 (s, 2H, CH2Ph), 5.72 (s, 2H, NCH2O),
7.25-7.33, 7.55-7.58 (2 × m, 5H, phenyl); 13C NMR (CDCl3)
δ -1.5, 11.2, 17.8, 21.2, 25.7, 32.5, 36.7, 42.8, 46.3, 66.4, 72.5,
106.7, 127.6, 128.3, 129.2, 136.8, 147.7, 151.0, 154.8, 159.7.
2H, PhCH2CH2CH2CH2CH2), 7.18-7.32 (m, 5H, phenyl); 13C
NMR (CDCl3) δ 27.8, 30.6, 32.7, 33.8, 35.7, 125.7, 128.3, 128.4,
142.3.
5-Phenylpentyl bromide (1.3 g, 5.8 mmol) was dissolved in
dry chloroform (25 mL) and chlorosulfonic acid (4.0 mL) added
dropwise. The mixture was stirred at room temperature for
16 h and poured over ice (100 mL). Water (100 mL) was added
and the aqueous layer extracted with ethyl acetate (3 × 100
mL). The organic layer was dried (MgSO4), filtered and
evaporated down to afford the 4-chlorosulfonyl intermediate
(1.9 g, 5.8 mmol) as a brown oil. The residue was dissolved in
1,4-dioxane (20 mL) and refluxed for 3 h with potassium
fluoride (1.3 g, 23 mmol). The mixture was allowed to cool to
room temperature and poured over ice (250 mL). Water was
added and the aqueous phase extracted with chloroform (3 ×
100 mL). The organic extracts were dried (MgSO4), filtered
and purified by column chromatography using petroleum
ether/ethyl acetate (6:1): yield 1.5 g, 82%; 1H NMR (CDCl3) δ
1.48-1.56, 1.64-1.75, 1.86-1.95 (3 × m, 6H, CH2), 2.76 (t,
2H, PhCH2CH2CH2CH2CH2), 3.41 (t, 2H, PhCH2CH2CH2-
CH2CH2), 7.43, 7.93 (2 × d, 4H, phenyl); 13C NMR (CDCl3) δ
27.6, 30.0, 32.4, 33.5, 35.8, 128.6, 129.6, 151.2.
Rep r esen ta tive P r oced u r e for N-3 Alk yla tion . The N-3
free xanthine (0.75 mmol) was stirred in dry DMF (10 mL)
with potassium carbonate (1.0 mmol) for 1 h. A solution of the
alkyl bromide (0.86 mmol) in dry DMF (5 mL) was added and
the mixture stirred for 1 h - 3 days, while being monitored
by TLC. The reaction mixture was filtered, adhered to silica
and purified by column chromatography using petroleum
ether/ethyl acetate (4:1).
8-Cycloh exyl-3-(4-flu or osu lfon ylben zyl)-1-p r op yl-7-(2-
(tr im eth ylsilyl)eth oxym eth yl)xa n th in e (14). The title com-
pound was prepared in 51% yield using the representative
procedure for N-3 alkylation: 1H NMR (DMSO-d6) δ -0.09 (s,
9H, Si(CH3)3), 0.82 (t, 5H, CH2CH2CH3, CH2CH2Si), 1.18-1.84
(m, 12H, CH2CH2CH3, 5 × cyclohexyl CH2), 2.92 (m, 1H, CH),
3.59 (t, 2H, CH2CH2Si), 3.82 (t, 2H, CH2CH2CH3), 5.30 (s, 2H,
CH2Ph), 5.71 (s, 2H, NCH2O), 7.69, 8.10 (2 × d, 4H, phenyl);
13C NMR (DMSO-d6) δ -1.5, 11.1, 17.2, 20.8, 25.2, 25.3, 31.2,
34.9, 42.1, 45.4, 65.6, 72.2, 105.7, 128.7, 129.2, 130.4 (d), 146.1,
147.1, 150.4, 154.1, 159.5.
Rep r esen ta tive P r oced u r e for Deben zyla tion . The
3-benzylxanthine (1.8 mmol) was refluxed in dry methanol (30
mL) with ammonium formate (18.6 mmol) and activated 10%
Pd-C under an atmosphere of argon for 2 h. The reflux
condenser was removed and the solvent vapor allowed to
escape while under a stream of nitrogen. The residue was then
heated at 140 °C for 1.5 h. The residue was dissolved in
methanol and filtered over a Celite pad. The filtrate was then
concentrated down to afford a pure white solid.
8-Cycloh exyl-1-pr opyl-7-(2-(tr im eth ylsilyl)eth oxym eth -
yl)xa n th in e (10a ). The title compound (white solid) was
prepared in 89% yield using the representative procedure for
N-3 debenzylation: mp 138-139 °C; 1H NMR (DMSO-d6) δ
-0.10 (s, 9H, Si(CH3)3), 0.78-0.85 (m, 5H, CH2CH2CH3,
CH2CH2Si), 1.18-1.84 (m, 12H, CH2CH2CH3, 5 × cyclohexyl
CH2), 2.87 (m, 1H, CH), 3.55 (t, 2H, CH2CH2Si), 3.76 (t, 2H,
CH2CH2CH3), 5.66 (s, 2H, NCH2O), 11.83 (s, 1H, NH); 13C
NMR (DMSO-d6) δ -1.5, 11.1, 17.2, 20.8, 25.3, 25.4, 31.3, 34.9,
41.1, 65.4, 71.8, 105.4, 146.9, 150.7, 154.9, 159.5.
8-Cyclopen tyl-1-pr opyl-7-(2-(tr im eth ylsilyl)eth oxym eth -
yl)xa n th in e (10b). The title compound (colorless oil) was
prepared in 67% yield using the representative procedure for
N-3 debenzylation: 1H NMR (DMSO-d6) δ -0.08 (s, 9H, Si-
(CH3)3), 0.84-0.92 (m, 5H, CH2CH2CH3, CH2CH2Si), 1.61-2.08
(2 × m, 10H, CH2CH2CH3, 4 × cyclopentyl CH2), 3.23 (m, 1H,
CH), 3.61 (t, 2H, CH2CH2Si), 3.93 (t, 2H, CH2CH2CH3), 5.70
(s, 2H, NCH2O), 11.71 (br s, 1H, NH); 13C NMR (CDCl3) δ -1.6,
11.1, 17.7, 21.1, 25.5, 32.4, 36.7, 42.1, 66.4, 72.4, 107.9, 146.2,
151.2, 155.3, 159.9.
8-Cycloh exyl-3-(3-(4-flu or osu lfon ylp h en yl)p r op yl)-1-
p r op yl-7-(2-(tr im eth ylsilyl)eth oxym eth yl)xa n th in e (15).
The title compound was prepared in 28% yield using the
representative procedure for N-3 alkylation: 1H NMR (DMSO-
d6) δ -0.10 (s, 9H, Si(CH3)3), 0.81 (t, 5H, CH2CH2CH3, CH2CH2-
Si), 1.19-1.85 (m, 12H, CH2CH2CH3, 5 × cyclohexyl CH2), 2.09
(m, 2H, CH2CH2CH2Ph), 2.79 (t, 2H, CH2CH2CH2Ph), 2.90 (m,
1H, CH), 3.56 (t, 2H, CH2CH2Si), 3.76 (t, 2H, CH2CH2CH3),
4.02 (t, 2H, CH2CH2CH2Ph), 5.66 (s, 2H, NCH2O), 7.54, 7.94
(2 × d, 4H, phenyl); 13C NMR (DMSO-d6) δ -1.6, 11.1, 17.2,
20.7, 25.2, 25.3, 27.6, 31.2, 32.0, 34.8, 41.8, 42.0, 65.4, 72.0,
105.5, 128.1, 128.8 (d), 129.0, 129.9, 147.2, 150.2, 151.1, 154.0,
159.2.
8-Cycloh exyl-3-(5-(4-flu or osu lfon ylp h en yl)p en t yl)-1-
p r op yl-7-(2-(tr im eth ylsilyl)eth oxym eth yl)xa n th in e (16).
The title compound was prepared in 45% yield using the
representative procedure for N-3 alkylation: 1H NMR (DMSO-
d6) δ -0.10 (s, 9H, Si(CH3)3), 0.78-0.84 (m, 5H, CH2CH2CH3,
CH2CH2Si), 1.13-1.78 (m, 18H, CH2CH2CH3, CH2CH2CH2CH2-
CH2Ph, CH2CH2CH2CH2CH2Ph, CH2CH2CH2CH2CH2Ph, 5 ×
cyclohexyl CH2), 2.71 (t, 2H, CH2CH2CH2CH2CH2Ph), 2.90 (m,
1H, CH), 3.56 (t, 2H, CH2CH2Si), 3.81 (t, 2H, CH2CH2CH3),
3.98 (t, 2H, CH2CH2CH2CH2CH2Ph), 5.69 (s, 2H, NCH2O),
7.57, 8.00 (2 × d, 4H, phenyl); 13C NMR (DMSO-d6) δ -1.5,
11.1, 17.2, 20.8, 25.2, 25.3, 26.9, 29.5, 31.2, 34.7, 34.8, 41.8,
42.2, 65.5, 72.0, 105.5, 128.4, 128.6 (d), 130.2, 147.4, 150.3,
152.2, 154.1, 159.4.
3-(4-F lu or osu lfon ylp h en yl)p r op yl Br om id e (12). 3-
Phenylpropyl bromide (497 mg, 2.5 mmol) was dissolved in
dry chloroform (10 mL) and chlorosulfonic acid (1.7 mL) added
dropwise. The mixture was stirred at room temperature for
20 h and poured over ice (100 mL). Water (100 mL) was added
and the aqueous layer extracted with ethyl acetate (3 × 40
mL). The organic layer was dried (MgSO4), filtered and
evaporated down to afford the 4-chlorosulfonyl intermediate
(666 mg, 2.2 mmol) as a pale yellow oil. The residue was
dissolved in 1,4-dioxane (10 mL) and refluxed for 3 h with
potassium fluoride (487 mg, 8.4 mmol). The mixture was
allowed to cool to room temperature and poured over ice (100
mL). Water (50 mL) was added and the aqueous phase
extracted with chloroform (3 × 40 mL). The organic extracts
were dried (MgSO4), filtered and the solvent removed under
vacuum to afford the crude: yield 537 mg, 77%; 1H NMR
(DMSO-d6) δ 2.09-2.18 (m, 2H, PhCH2CH2CH2), 2.87 (m, 2H,
PhCH2CH2CH2), 3.51 (m, 2H, PhCH2CH2CH2), 7.64, 8.04 (2 ×
d, 4H, phenyl); 13C NMR (CDCl3) δ 33.1, 33.5, 34.0, 128.6, 129.2
(d), 130.4, 150.6.
5-(4-Flu or osu lfon ylph en yl)pen tyl Br om ide (13). 5-Phen-
yl-1-pentanol (99%, 965 mg, 5.8 mmol) was cooled on an ice
bath. Phosphorus tribromide (170 µL, 1.8 mmol) was added
and the mixture was stirred on ice for 15 min, then at room
temperature for 2 h, and at 60 °C for 26.5 h. Ice water (50
mL) and brine (50 mL) were added and the aqueous layer
extracted with ether (100 mL). The ether extract was washed
with brine (50 mL), dried (MgSO4), filtered and purified by
column chromatography using petroleum ether with 2% ethyl
acetate, affording the alkyl bromide as a clear liquid: yield
Rep r esen ta tive P r oced u r e for SEM Dep r otection . The
SEM-protected compound (0.28 mmol) was refluxed in a 120
°C oil bath with 1 M HCl (3.0 mL) and ethanol (6 mL) for 5.5
h. The mixture was evaporated down under reduced pressure,
partitioned between water (pH adjusted to 7 with NaOH) and
chloroform and further extracting with chloroform. The organic
1
1.2 g, 89%; H NMR (CDCl3) δ 1.46-1.54, 1.62-1.69, 1.90 (3
× m, 6H, CH2), 2.64 (t, 2H, PhCH2CH2CH2CH2CH2), 3.42 (t,

Fluorosulfonyl-Substituted Xanthines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 4979
extracts were dried (MgSO4), filtered and evaporated down,
affording a creamy solid which was recrystallized from ethanol
and water.
leum ether/isopropyl alcohol (80:20), plus 1.5% NH4OH: yield
48 mg, 56%.
8-Cycloh exyl-3-(3-(4-flu or osu lfon ylben za m ido)p r op yl)-
1-pr opyl-7-(2-(tr im eth ylsilyl)eth oxym eth yl)xan th in e (22a).
Meth od A. A solution of 3-(3-aminopropyl)-8-cyclohexyl-1-
propyl-7-(2-(trimethylsilyl)ethoxymethylxanthine (55 mg, 0.12
mmol) and 4-fluorosulfonylbenzoic acid (95%, 27 mg, 0.13
mmol) in dry DMF (7.5 mL) was cooled over ice. EDCI (25 mg,
0.13 mmol) was added and the mixture stirred on ice for 1.5
h, then at room temperature for 2.5 h. The mixture was cooled
on ice again and N,N-diisopropylethylamine (22 µL, 0.13 mmol)
was added. Stirring was continued on ice for 1 h then at room
temperature for 16.5 h. Ethyl acetate (30 mL) was added and
the solution washed with H2O (3 × 25 mL). The organic phase
was dried (MgSO4), filtered and the solvent removed under
vacuum to afford a clear yellow oil which was purified by
column chromatography using petroleum ether/ethyl acetate
8-Cycloh exyl-3-(4-flu or osu lfon ylben zyl)-1-p r op ylxa n -
th in e (17). The title compound was prepared in 73% yield
using the representative procedure for SEM deprotection: mp
254-255 °C; 1H NMR (DMSO-d6) δ 0.83 (t, 3H, CH2CH2CH3,),
1.15-1.90 (m, 12H, CH2CH2CH3, 5 × cyclohexyl CH2), 2.70
(m, 1H, CH), 3.81 (t, 2H, CH2CH2CH3), 5.28 (s, 2H, CH2Ph),
7.67, 8.08 (2 × d, 4H, phenyl), 13.21 (s, 1H, NH); 13C NMR
(DMSO-d6) δ 11.2, 20.8, 25.3, 25.3, 30.9, 37.6, 42.2, 45.6, 106.1,
128.7, 129.0, 130.3 (d), 146.4, 147.2, 150.8, 153.9, 158.4.
8-Cycloh exyl-3-(3-(4-flu or osu lfon ylp h en yl)p r op yl)-1-
p r op ylxa n th in e (18). The title compound was prepared in
82% yield using the representative procedure for SEM depro-
1
tection: mp 182-183 °C; H NMR (DMSO-d6) δ 0.83 (t, 3H,
CH2CH2CH3), 1.18-1.89 (m, 12H, CH2CH2CH3, 5 × cyclohexyl
CH2), 2.08 (m, 2H, CH2CH2CH2Ph), 2.70 (tt, 1H, CH), 2.80 (t,
2H, CH2CH2CH2Ph), 3.76 (t, 2H, CH2CH2CH3), 4.00 (t, 2H,
CH2CH2CH2Ph), 7.56, 7.95 (2 × d, 4H, phenyl), 13.03 (s, 1H,
NH); 13C NMR (DMSO-d6) δ 11.2, 20.8, 25.3, 27.8, 31.0, 32.2,
37.5, 42.0, 42.3, 106.0, 128.2, 128.8 (d), 130.1, 147.4, 150.6,
151.2, 153.8, 158.1.
1
(3:2): yield 22 mg, 29%; mp 98-103 °C; H NMR (DMSO-d6)
δ -0.09 (s, 9H, Si(CH3)3), 0.78-0.85 (m, 5H, CH2CH2CH3,
CH2CH2Si), 0.92-1.08, 1.18-1.76 (2 × m, 12H, CH2CH2CH3,
5 × cyclohexyl CH2), 1.95 (m, 2H, CH2CH2CH2NHCO), 2.84
(m, 1H, CH), 3.27-3.34 (m, 2H, CH2CH2CH2NHCO), 3.56 (t,
2H, CH2CH2Si), 3.81 (t, 2H, CH2CH2CH3), 4.08 (t, 2H, CH2-
CH2CH2NHCO), 5.67 (s, 2H, NCH2O), 8.16, 8.26 (2 × d, 4H,
phenyl), 8.88 (t, 1H, NHCO); 13C NMR (DMSO-d6) δ -1.5, 11.1,
17.2, 20.8, 25.1, 25.3, 27.4, 31.1, 34.8, 36.8, 40.6, 41.9, 65.5,
72.0, 105.6, 128.6, 129.0, 133.4 (d), 141.6, 147.3, 150.3, 154.2,
159.3, 164.2.
8-Cycloh exyl-3-(5-(4-flu or osu lfon ylp h en yl)p en t yl)-1-
p r op ylxa n th in e (19). The title compound was prepared in
66% yield using the representative procedure for SEM depro-
1
tection: mp 147-148 °C; H NMR (DMSO-d6) δ 0.82 (t, 3H,
CH2CH2CH3), 1.14-1.84 (m, 18H, CH2CH2CH3, CH2CH2CH2-
CH2CH2Ph, CH2CH2CH2CH2CH2Ph, CH2CH2CH2CH2CH2Ph,
5 × cyclohexyl CH2), 2.64-2.74 (m, 3H, CH2CH2CH2CH2CH2-
Ph, CH), 3.80 (t, 2H, CH2CH2CH3), 3.95 (t, 2H, CH2CH2CH2-
CH2CH2Ph), 7.57, 8.00 (2 × d, 4H, phenyl), 13.06 (s, 1H, NH);
13C NMR (DMSO-d6) δ 11.1, 20.8, 25.3, 25.3, 27.0, 29.6, 31.0,
34.8, 37.5, 41.9, 42.4, 106.0, 128.4, 128.7 (d), 130.2, 147.6,
150.6, 152.2, 153.9, 158.2.
Meth od B. The title compound was prepared in 87% using
the representative procedure for N-3 alkylation: 1H NMR
(DMSO-d6) δ -0.09 (s, 9H, Si(CH3)3), 0.78-0.85 (m, 5H, CH2-
CH2CH3, CH2CH2Si), 0.92-1.08, 1.18-1.76 (2 × m, 12H,
CH2CH2CH3, 5 × cyclohexyl CH2), 1.95 (m, 2H, CH2CH2CH2-
NHCO), 2.84 (m, 1H, CH), 3.27-3.34(m, 2H, CH2CH2CH2-
NHCO), 3.56 (t, 2H, CH2CH2Si), 3.81 (t, 2H, CH2CH2CH3), 4.08
(t, 2H, CH2CH2CH2NHCO), 5.67 (s, 2H, NCH2O), 8.16, 8.26
(2 × d, 4H, phenyl), 8.88 (t, 1H, NHCO); 13C NMR (DMSO-d6)
δ -1.5, 11.1, 17.2, 20.8, 25.1, 25.3, 27.4, 31.1, 34.8, 36.8, 40.6,
41.9, 65.5, 72.0, 105.6, 128.6, 129.0, 133.4 (d), 141.6, 147.3,
150.3, 154.2, 159.3, 164.2.
8-Cyclopen tyl-3-(3-(4-flu or osu lfon ylben zam ido)pr opyl)-
1-pr opyl-7-(2-(tr im eth ylsilyl)eth oxym eth yl)xan th in e (22b).
The title compound was prepared in 56% yield using repre-
sentative procedure for N-3 alkylation: 1H NMR (CDCl3) δ
-0.03 (s, 9H, Si(CH3)3), 0.89-0.97 (m, 5H, CH2CH2CH3,
CH2CH2Si), 1.60-2.14 (m, 12H, CH2CH2CH3, CH2CH2CH2-
NHCO, 4 × cyclopentyl CH2), 3.24 (m, 1H, CH), 3.42 (m, 2H,
CH2CH2CH2NHCO), 3.66 (t, 2H, CH2CH2Si), 3.99 (t, 2H, CH2-
CH2CH3), 4.26 (t, 2H, CH2CH2CH2NHCO), 5.73 (s, 2H, NCH2O),
8.10-8.16 (m, 4H, phenyl); 13C NMR (CDCl3) δ -1.5, 11.3, 17.8,
21.3, 25.5, 27.8, 32.7, 35.8, 36.8, 40.2, 42.9, 66.6, 72.6, 106.8,
128.4, 128.7, 135.1 (d), 141.6, 147.5, 151.8, 154.6, 159.9, 165.0.
8-Cycloh exyl-3-(3-(4-flu or osu lfon ylben za m id o)p r op yl)-
1-p r op ylxa n th in e (23a ). The title compound was prepared
in 96% yield using the general procedure for SEM deprotec-
tion: mp 258-259 °C; 1H NMR (DMSO-d6) δ 0.84 (t, 3H, CH2-
CH2CH3), 1.04-1.80 (m, 12H, CH2CH2CH3, 5 × cyclohexyl
CH2), 1.95 (m, 2H, CH2CH2CH2NHCO), 2.60 (tt, 1H, CH),
3.26-3.32 (m, 2H, CH2CH2CH2NHCO), 3.81 (t, 2H, CH2CH2-
CH3), 4.06 (t, 2H, CH2CH2CH2NHCO), 8.15, 8.25 (2 × d, 4H,
phenyl), 8.87 (t, 1H, NHCO), 13.03 (s, 1H, NH); 13C NMR
(DMSO-d6) δ 11.2, 20.8, 25.2, 25.3, 27.4, 30.9, 36.8, 37.5, 40.7,
42.0, 106.0, 128.6, 129.0, 133.4 (d), 141.6, 147.5, 150.6, 153.9,
158.1, 164.3.
8-Cyclopen tyl-3-(3-(4-flu or osu lfon ylben zam ido)pr opyl)-
1-p r op ylxa n th in e (23b). The title compound was prepared
in 71% yield using the general procedure for SEM deprotec-
tion: 1H NMR (CDCl3 + CD3OD) δ 0.87 (m, 3H, CH2CH2CH3),
1.55-1.71 (m, 10H, CH2CH2CH3, CH2CH2CH2NHCO, 4 ×
cyclopentyl CH2), 3.09 (m, 1H, CH), 3.38 (m, 2H, CH2CH2CH2-
NHCO), 3.89 (t, 2H, CH2CH2CH3), 4.16 (t, 2H, CH2CH2CH2-
NHCO), 8.06 (m, 4H, phenyl); 13C NMR (CDCl3 + CD3OD) δ
11.0, 21.1, 25.1, 27.2, 32.2, 36.3, 39.4, 41.0, 43.1, 106.6, 128.4,
128.5, 134.9 (d), 141.1, 147.6, 151.4, 154.7, 158.3, 165.5.
8-Cycloh exyl-3-(3-p h t h a lim id op r op yl)-1-p r op yl-7-(2-
(tr im eth ylsilyl)eth oxym eth yl)xa n th in e (20). The title com-
pound was prepared in quantitative yield using the represen-
1
tative procedure for N-3 alkylation: mp 96-97 °C; H NMR
(DMSO-d6) δ -0.11 (s, 9H, Si(CH3)3), 0.81 (m, 5H, CH2CH2CH3,
CH2CH2Si), 1.02-1.87 (m, 12H, CH2CH2CH3, 5 × cyclohexyl
CH2), 2.03 (m, 2H, CH2CH2CH2Phth), 2.81 (m, 1H, CH), 3.54
(t, 2H, CH2CH2Si), 3.61 (t, 2H, CH2CH2CH2Phth), 3.77 (t, 2H,
CH2CH2CH3), 4.00 (t, 2H, CH2CH2CH2Phth), 5.62 (s, 2H,
NCH2O), 7.83 (s, 4H, phenyl); 13C NMR (DMSO-d6) δ -1.5,
11.1, 17.2, 20.7, 25.1, 25.2, 26.3, 31.0, 34.8, 35.1, 41.8, 65.4,
72.0, 105.6, 122.9, 131.5, 134.3, 147.1, 150.3, 154.1, 159.3,
167.8.
3-(3-Am in opr op yl)-8-cycloh exyl-1-p r opyl-7-(2-(tr im eth -
ylsilyl)eth oxym eth yl)xa n th in e (21). Meth od A. A solution
of 8-cyclohexyl-3-(3-phthalimidopropyl)-1-propyl-7-(2-(trimeth-
ylsilyl)ethoxymethylxanthine (109 mg, 0.18 mmol) in dry
methanol (1.5 mL) and methylamine (40% in methanol, 1.0
mL, 13 mmol) was heated in a pressure apparatus in an oil
bath at 50 °C for 14 h. The solvent was removed under vacuum
to afford the crude as a yellow oil which was purified by column
chromatography using petroleum ether/isopropyl alcohol/am-
1
monia (80:20:1.5): yield 48 mg, 56%; H NMR (DMSO-d6) δ
-0.10 (s, 9H, Si(CH3)3), 0.78-0.85 (m, 5H, CH2CH2CH3,
CH2CH2Si), 1.19-1.85 (m, 14H, CH2CH2CH3, CH2CH2CH2-
NH2, 5 × cyclohexyl CH2), 2.48-2.52 (m, 2H, CH2CH2CH2-
NH2), 2.91 (m, 1H, CH), 3.57 (t, 2H, CH2CH2Si), 3.82 (t, 2H,
CH2CH2CH3), 4.03 (t, 2H, CH2CH2CH2NH2), 5.70 (s, 2H,
NCH2O); 13C NMR (DMSO-d6) δ -1.5, 11.1, 17.2, 20.8, 25.2,
25.3, 30.9, 31.2, 34.9, 38.2, 40.2, 65.5, 72.0, 105.5, 147.4, 150.4,
154.2, 159.4; ES/MS (+) 464.4.
Meth od B. A solution of 8-cyclohexyl-3-(3-phthalimidopro-
pyl)-1-propyl-7-(2-(trimethylsilyl)ethoxymethylxanthine (109
mg, 0.18 mmol) in dry methanol (1.5 mL) and hydrazine
monohydrate (1.0 mL, 21 mmol) was refluxed for 15 h. NaOH
(3 M, 20 mL) was added and the mixture extracted with ethyl
acetate (3 × 20 mL). The organic fractions were dried (MgSO4),
filtered and the solvent removed under reduced pressure. The
crude was purified by column chromatography using petro-

4980 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Beauglehole et al.
N-(3-Br om op r op yl)-4-flu or osu lfon ylben za m id e (25). A
solution of 4-(fluorosulfonyl)benzoic acid (95%, 565 mg, 2.6
mmol), 3-bromopropylamine hydrobromide (98%, 758 mg, 3.4
mmol) and EEDQ (735 mg, 3.0 mmol) in anhydrous DMF (20
mL) was cooled to 0 °C on an ice bath. N,N-Diisopropylethyl-
amine (920 µL, 5.3 mmol) was added slowly and the mixture
stirred a further 8 h on ice and 8 h at room temperature. Water
(50 mL) was added and the mixture extracted with ethyl
acetate (3 × 50 mL). The organic extracts were washed with
HCl (2%, 3 × 25 mL), dried (MgSO4), filtered and purified by
column chromatography using petroleum ether/ethyl acetate
(2:1): yield 534 mg, 63%; 1H NMR (CDCl3) δ 2.24 (m, 2H,
NHCH2CH2CH2), 3.51 (t, 2H, NHCH2CH2CH2), 3.67 (m, 2H,
NHCH2CH2CH2), 6.58 (br s, 1H, NH), 8.06 (m, 4H, phenyl);
13C NMR (CDCl3) δ 30.8, 31.7, 39.1, 128.2, 128.8, 135.5 (d),
140.9, 165.4.
Su p p or tin g In for m a tion Ava ila ble: Microanalyses for
compounds 17-19 and 23a ,b. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Bioch em ica l Meth od s. Solu tion s. Stock solutions of the
xanthine derivatives were prepared by dissolving in DMSO
to give a concentration of 10 mM. On the day of use, the
compounds were diluted to the desired concentration with
Hank’s balanced salt solution (HBSS). Control incubations
contained the same final concentration of DMSO.
Cell Cu ltu r e, P r etr ea tm en ts, a n d Mem br a n e Isola tion .
DDT1 MF-2 and PC-12 cells were grown as monolayers as
described previously.8 Cells were subcultured weekly and used
in experiments at 1 day preconfluence. Cell pretreatments
were performed by washing the monolayers twice with HBSS
(2 × 10 mL) and incubation of the cells with 10 mL of HBSS
containing the indicated concentration of xanthine derivative
at 37 °C for 20 min. At the end of the incubation, the HBSS
was aspirated and replaced with 10 mL of ligand-free HBSS.
After 5 min at room temperature, the HBSS was replaced with
another 10 mL of HBSS. This washing procedure was per-
formed 10 times with DDT cells and 8 times with PC-12 cells.
At the end of the washing procedure, cell membranes were
prepared as previously described8 and the protein content was
determined by the Bradford17 method using bovine serum
albumin as standard.
(11) Morey, T. E.; Belardinelli, L.; Dennis, D. M. Validation of
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Adenosine Receptor Reserve in Guinea Pig Atrium. Br. J .
Pharmacol. 1998, 123, 1425-1433.
Ra d ioliga n d Bin d in g Assa ys. The A1AR content of DDT
cell membranes was determined by specific [3H]-8-cyclopentyl-
1,3-dipropylxanthine as previously reported.18 The A2AAR of
PC-12 membranes was determined by a modification of a
previously reported procedure.19 Briefly, cell membranes (15-
25 µg protein) were incubated in a total volume of 0.25 mL
containing 50 mM Tris-HCl buffer at pH 7.4, 5 mM MgCl2, 2
units/mL adenosine deaminase, 0.1-3 nM [3H]ZM241385 with
and without 50 µM 5′-N-ethylcarboxamidoadenosine (NECA)
for 120 min at 25 °C. At the end of the incubation, each
suspension was diluted with 3 mL of ice-cold 10 mM Tris-HCl
buffer at pH 7.4, and the membranes with bound radioligand
were collected by filtration under reduced pressure. The filters
were washed with an additional 6 mL of ice-cold buffer and
placed in a vial with 3 mL of Scinti-Verse BD, and the
radioactivity was determined using a liquid scintillation
counter. Specific [3H]ZM241385 binding to the A2AAR was
calculated as the difference between total binding in the
absence of NECA and the nonspecific binding determined in
the presence of NECA. In displacement of [3H]ZM241385
experiments using the xanthine derivatives, the concentration
of the radioligand was 1.5 nM. All binding assays were
performed in triplicate.
(12) Baker, B. R.; Lourens, G. J . Irreversible Enzyme Inhibitors.
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(18) Zhang, J .; Belardinelli, L.; J acobson, K. A.; Otero, D. H.; Baker,
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and guinea pig heart. Mol. Pharmacol. 1997, 52, 491-498.
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G Protein Coupling, and Efficiency of Facilitatory Adenosine A2A
Receptors in the Limbic Cortex, but not Striatum, of Aged Rats.
J . Neurochem. 1999, 73, 1733-1738.
The concentration of compounds which inhibited specific
radioligand binding by 50% (IC50) was determined by nonlinear
regression analysis of the data using the GraphPad Inplot
program (GraphPad Software, San Diego, CA). The dissocia-
tion constant (KD) and maximal radioligand binding (Bmax
)
were determined by nonlinear regression analysis of saturation
data plotted by the method of Scatchard.20
(20) Scatchard, G. The Attraction of Protein for Small Molecules and
Ions. Ann. N. Y. Acad. Sci. 1949, 51, 660-672.
Ack n ow led gm en t. The authors thank the Austra-
lian Research Council for financial support.
J M000181F