Characterization of 'mini-nucleotides' as P2X receptor agonists in rat cardiomyocyte cultures. An integrated synthetic, biochemical, and theoretical study

Article abstract of DOI:10.1021/jm990085i

The design and synthesis of 'mini-nucleotides', based on a xanthine- alkyl phosphate scaffold, are described. The physiological effects of the new compounds were evaluated in rat cardiac cell culture regarding Ca²⁺ elevation and contractility. The results indicate biochemical and physiological profiles similar to those of ATP, although at higher concentrations. The biological target molecules of these 'mini-nucleotides' were identified by using selective P2-R and A₁-R antagonists and P2-R subtype selective agonists. On the basis of these results and of experiments in Ca²⁺ free medium, in which [Ca²⁺](i) elevation was not observed, we concluded that interaction of the analogues is likely with P2X receptor subtypes, which causes Ca²⁺ influx. Theoretical calculations analyzing electronic effects within the series of xanthine-alkyl phosphates were performed on reduced models at quantum mechanical levels. Calculated dipole moment vectors, electrostatic potential maps, and volume parameters suggest an explanation for the activity or inactivity of the synthesized derivatives and predict a putative binding site environment for the active agonists. Xanthine-alkyl phosphate analogues proved to be selective agents for activation of P2X-R subtypes, whereas ATP activated all P2-R subtypes in cardiac cells. Therefore, these analogues may serve as prototypes of selective drugs aiming at cardiac disorders mediated through P2X receptors.

Full text of DOI:10.1021/jm990085i

J . Med. Chem. 1999, 42, 2685-2696
2685
Ch a r a cter iza tion of “Min i-Nu cleotid es” a s P 2X Recep tor Agon ists in Ra t
Ca r d iom yocyte Cu ltu r es. An In tegr a ted Syn th etic, Bioch em ica l, a n d Th eor etica l
Stu d y
Bilha Fischer,*† Revital Yefidoff,† Dan T. Major,† Irit Rutman-Halili,‡ Valadimir Shneyvays,‡ Tova Zinman,‡
Kenneth A. J acobson,§ and Asher Shainberg‡
Department of Chemistry and Faculty of Life Sciences, Gonda-Goldschmied Medical Research Center, Bar-Ilan University,
Ramat-Gan 52900, Israel, and NIH, NIDDK, LBC, Molecular Recognition Section, Bethesda, Maryland 20892-0810
Received February 22, 1999
The design and synthesis of “mini-nucleotides”, based on a xanthine-alkyl phosphate scaffold,
are described. The physiological effects of the new compounds were evaluated in rat cardiac
cell culture regarding Ca2+ elevation and contractility. The results indicate biochemical and
physiological profiles similar to those of ATP, although at higher concentrations. The biological
target molecules of these “mini-nucleotides” were identified by using selective P2-R and A1-R
antagonists and P2-R subtype selective agonists. On the basis of these results and of
experiments in Ca2+ free medium, in which [Ca2+]i elevation was not observed, we concluded
that interaction of the analogues is likely with P2X receptor subtypes, which causes Ca2+ influx.
Theoretical calculations analyzing electronic effects within the series of xanthine-alkyl
phosphates were performed on reduced models at quantum mechanical levels. Calculated dipole
moment vectors, electrostatic potential maps, and volume parameters suggest an explanation
for the activity or inactivity of the synthesized derivatives and predict a putative binding site
environment for the active agonists. Xanthine-alkyl phosphate analogues proved to be selective
agents for activation of P2X-R subtypes, whereas ATP activated all P2-R subtypes in cardiac
cells. Therefore, these analogues may serve as prototypes of selective drugs aiming at cardiac
disorders mediated through P2X receptors.
In tr od u ction
ring, sugar moiety, or the triphosphate chain.6 Synthetic
analogues circumvent some of the problems inherent in
ATP, e.g., lack of enzymatic stability, relatively low
affinity at P2-Rs, and lack of receptor subtype selectiv-
ity.6 However, as nucleotides they still may potentially
interact with the multitude of other ATP binding and
metabolizing proteins ubiquitous in mammalian physi-
ology.
Intracellular ATP plays a fundamental and ubiqui-
tous role in energy metabolism, nucleic acid synthesis,
pump activities, and enzyme regulation. There is also
widespread evidence that extracellular adenine nu-
cleotides exert significant biological actions on various
tissues,1 which are mediated via membrane ATP recep-
2
tor (P2-R) subtypes termed P2X and P2Y.
Our goal was, therefore, to design novel selective P2-R
agonists based on a “mini-nucleotide” scaffold, namely,
a structure lacking a ribose moiety and having a
modified purine. Further, we aimed for the evaluation
of these agonists as prototypes of selective drugs for
cardiac disorders mediated by P2 receptors.
In this paper we report the first synthesis of non-ATP-
based P2X-R agonists, their biochemical and physiologi-
cal evaluation on cultured rat cardiac cells, interpreta-
tion of the structure-activity relationship (SAR), as well
as prediction of the putative binding site of these ligands
based on theoretical calculations.
It has been shown that cardiac cells have P2 receptors
that modulate transmembrane potential and cytosolic
Ca2+ concentrations.3 Extracellular ATP in micromolar
concentrations causes Ca2+ elevation in cardiac cells
grown in culture, which is explained as a result of
production of 1,4,5-inositol triphosphate.4 ATP is also
known to cause complex changes in the heart function
when infused through the circulation.5 These changes
are difficult to interpret from a mechanistic or cellular
perspective because of the variety of cell types present,
the complexity of the cardiac function, and the rapid
degradation of ATP to adenosine. Use of neonatal rat
cardiac myocyte cultures obviates many of the difficul-
ties of the whole heart system and permits the use of
the fluorescent indicators (e.g., indo-1) in intracellular
Ca2+ measurements.
Resu lts
Ch em istr y. Design of “Min i-Nu cleotid es”. The
guidelines for the design of new P2-R ligands, which are
structurally different from ATP, were obtained by
studying various protein-nucleotide complexes avail-
able in the Protein Data Bank (PDB). Assuming that
binding interactions with small molecules may be of the
same type in both enzymes and receptors, the structures
of the complexes were probed for possible electrostatic
interactions (up to 10 Å between heavy atoms), hydro-
Currently, all identified P2-R agonists are compounds
based on a nucleotide skeleton modified at the purine
* Address correspondence to Bilha Fischer, Department of Chem-
istry, Bar-Ilan University, Ramat-Gan 52900, Israel. Tel: 972-3-
5138303. Fax: 972-3-5351250. E-mail: bfischer@mail.biu.ac.il.
† Department of Chemistry, Bar-Ilan University.
‡ Faculty of Life Sciences, Bar-Ilan University.
§ NIH.
10.1021/jm990085i CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/26/1999

2686 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Fischer et al.
Sch em e 1. Synthesis of Xanthine Alkyl Phosphate
Analogues, Compounds 6-7 and 11-12
A major interaction common in all cases was the binding
of the triphosphate moiety to positively charged amino
acid residues (Lys or Arg) present in the binding site.
Ribose 2′- and/or 3′-hydroxyls were typically hydrogen-
bonded to Glu or Asp residues or to amide functions in
the protein backbone. The adenine moiety was typically
bound via N6 H-bonding with Gln, Arg, or Lys or via
π-stacking interactions, e.g., with Tyr.
On the basis of the PDB data, it was suggested that
a new structure hypothesized to occupy a P2-R binding
site might contain a heterocyclic planar skeleton (ca-
pable of forming H-bonds as well as π-stacking or
hydrophobic interactions) and a negatively charged
moiety tethered to it.
Recent reports have indicated that adenosine-based
agonists and xanthine-based antagonists binding to the
adenosine A1 receptor share similar electrostatic prop-
erties.7 The electrostatic features which dictate binding
of both adenosine and xanthine ligands to the same site
in the A1 receptor might also hold true for the interac-
tions of other recognition sites, namely, P2-Rs, with
adenosine 5′-triphosphate and possibly with an ap-
propriately modified xanthine.
Therefore, as a reasonable starting point for identify-
ing new P2-R ligands, we chose xanthine derivatives
(theobromine and theophylline) which share several
structural and electrostatic similarities with adenine.
To complete the analogy to ATP we substituted these
compounds with an alkyl spacer bearing a mono- or
triphosphate moiety, thus obtaining a “mini-nucleotide”
structure (Schemes 1-3).
gen bonds (up to 5 Å between heavy atoms), and
aromatic interactions (up to 10 Å between heavy atoms).
Syn th esis of N-Alk yl P h osp h a te Th eobr om in e
a n d Th eop h yllin e Der iva tives. Theophylline (1,3-
Sch em e 2. Synthesis of 8-Substituted Xanthine-Alkyl Phosphate Analogues, Compounds 13-17

Characterization of “Mini-Nucleotides”
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2687
ester 810 or by treatment of the bromo derivative 9 with
bis(tributyltin)oxide/silver nitrate followed by hydroly-
sis.11
Finally, products 5a ,b and 10a ,b were subjected to a
one-pot phosphorylation with phosphorus oxychloride12
followed by addition of bis(tributylammonium)-pyro-
phosphate, to produce triphosphate derivatives 6b, 7b,
11b, and 12b. The corresponding monophosphate de-
rivatives were isolated as side products.12
Syn t h esis of C-8 Su b st it u t ed N-Alk yl P h os-
p h a tes Xa n th in e Der iva tives. Next, various elec-
tronic effects on the xanthine ring system that might
affect binding affinity to P2-Rs were studied. The scope
of the search for “mini-nucleotides” as potential P2-R
ligands was therefore extended to C-8 substituted alkyl
phosphate xanthines 13-17 and 21. The C-8 position
in 6a ,b and 11a ,b was substituted with either electron-
withdrawing atoms (Cl, Br) or electron-donating groups
(OEt, SPh) (Scheme 2).
F igu r e 1. ORTEP drawing of 3b from X-ray analysis.
Sch em e 3. Synthesis of 8-Cl-7-(2-Phosphate-ethylene)-
theophylline Derivatives, Compounds 21a ,b
Thus, 8-chloro-7-(2-monophosphate-ethylene)-theo-
phylline and the corresponding triphosphate derivative,
21a ,b, were obtained from 8-chloro-theophylline, 19, by
treatment with NaH followed by addition of 2-bromo-
ethanol and then one-pot triphosphorylation (Scheme
3). 8-Bromo derivatives were prepared from 1-(2-hy-
droxyethylene)-theobromine, 10a , and 7-(2-hydroxyeth-
ylene)-theophylline, 5a, treated with Br2 in nitrobenzene/
CCl4 solution.13 8-Bromo-1-(2-hydroxyethylene)-theo-
bromine, 14a , and 8-bromo-7-(2-hydroxyethylene)-theo-
phylline, 13a , were used also as starting materials for
the preparation of the corresponding 8-ethoxy and
8-thiophenyl derivatives 15-17 by treating the former
ones with sodium ethoxide/EtOH or sodium thiopheno-
late/DMF, respectively (Scheme 2). Treatment of 13a
with EtONa led, in addition to 17a , to a tricyclic
product, 18 (35% yield), formed by intramolecular
cyclization. To reduce the amount of side product 18,
the reaction time was shortened to 5 min. Derivatives
15a -17a were finally phosphorylated as described
before.
dimethyl-xanthine), 1, and theobromine (3,7-dimethyl-
xanthine), 2, were used as starting materials for the
synthesis of a series of N-7 or N-1 alkyl phosphate
derivatives (Scheme 1). The new compounds were
obtained as follows: (a) isolation of dimethyl-xanthine
sodium salt; (b) alkylation with an ethyl haloester or a
dihaloalkane; (c) formation of a primary alcohol at the
end of the side chain; (d) one-pot phosphorylation of the
primary alcohol.
For SAR studies, the N-alkyl phosphate xanthine
derivatives 6-7 and 11-12 were modified with respect
to the following parameters: (1) alkyl phosphate posi-
tion, namely, N-1 substitution (on theobromine) or N-7
substitution (on theophylline); (2) length of the spacer,
namely, the alkyl tether linking the phosphate to the
xanthine scaffold, which may consist of two or three
methylenes; (3) length of the phosphate chain, which
may consist of one or three phosphate residues. These
variations were expected to shed light on the stereo-
electronic requirements of the receptor binding site.
N-7 alkylation of theophylline, 1, was performed by
treatment with NaH/DMF followed by dibromoalkane
addition at 60 °C. In addition to product 3b, dimer 4
was isolated in 28% yield, even though the alkylating
agent was present in excess. Alkylation took place
selectively at N-7 as clearly shown in the X-ray crystal
structure of 3b (Figure 1).
Bioch em ica l a n d P h ysiologica l Eva lu a tion . The
novel derivatives were evaluated on rat cardiomyocytes
as potential P2-R agonists. Their pharmacological effect
was evaluated by measurements of intracellular Ca2+
elevation and contractility of the cells. These effects
were compared with those of standard P2-R agonists
(ATP, 2-MeS-ATP, and R,â-CH2-ATP). In our series of
experiments we explored the effect of the new com-
pounds on the heart and characterized them as P2-R
ligands. Moreover, we studied their selectivity to certain
P2-R subtypes and their possible interaction with other
receptors, e.g., A1-R, ryanodine-R. Next, the structure-
activity relationship of these ligands and their possible
use as protoypes for selective drugs for treatment of
cardiac disorders involving P2-Rs were analyzed.
In tr a cellu la r Ca 2+ Eleva tion . To evaluate the new
compounds as potential P2-R ligands, their effect on
intracellular Ca2+ elevation was measured and com-
N-1 alkylation of theobromine, 2, was carried out by
NaOH addition and isolation of the dry sodium salt
followed by treatment with ethyl halocarboxylate8 in the
presence of a catalytic amount of NaI or by treatment
with dihaloalkane (Scheme 1). No O-alkylation products
were obtained under these conditions.9
Hydroxyl formation at the end of the side chain was
achieved either by NaBH4/t-BuOH/MeOH reduction of

2688 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Fischer et al.
Ta ble 1. Effect of Xanthine Alkyl Phosphate Derivatives on
Contractility and [Ca2+]i Elevationa
reductn of
Ca2+
heart rate elevatn
reductn of
heart rate elevatn
Ca2+
compd
(%)
(nM)
compd
(%)
(nM)
Part A
6b
6a
11b
11a
13c
64
16
3
10
32
87 ((8)
80 ((7)
54 ((5)
61 ((9)
37 ((7)
13b
14c
14b
21a
21b
17
50
NA
95
28
54 ((8)
94 ((7)
80 ((7)
49 ((5)
29 ((3)
Part B
7b
7a
12b
12a
17c
ND
ND
ND
ND
ND
NA
NA
60
NA
NA
17b
16b
16c
15b
ND
ND
ND
ND
NA
NA
NA
NA
a
Cardiac cells 3-5 days in vitro were exposed to the above
ligands at 100 µM concentration. Basal concentration of Ca2+ is
100 nM. Data of 2-3 experiments are given. [Ca2+]i elevation was
determined with indo-1 as previously described.36 The rate of heart
contraction was determined 1 min after drug application using
motion detector system.36 Data of representative experiments are
given. NAsnot active, NDsnot determined.
those for 20 µM ATP were obtained with respect to rate
and magnitude of Ca2+ transients and contractions
(Figure 2B, Table 1A). Treatment of the cardiac cells
with other compounds, such as 6b and 11a (100 µM),
brought about responses similar to that of ATP (10 µM),
immediately causing Ca2+ elevation of ca. 70 nM. A
moderate response was obtained with compounds such
as 14b and 11a (Figure 2C). A third group of agents
(Table 1B) were inactive regarding Ca2+ elevation.
Ca r d ia c Con tr a ction s. The effect of the new com-
pounds on cardiomyocytes contractility was measured
and compared with that of ATP. ATP at 10 µM, and the
new derivatives at a 100 µM concentration, accelerated
the rate of heart contractions immediately upon ap-
plication. However, measurement of contractions per-
formed 1, 5, and 30 min after application showed a
reduction of heart rate (Table 1). ATP caused a 72%
inhibition in cardiac contraction rate, whereas 21a
reduced the rate by 95% (from 150 to 8 bpm) after 1
min. Compound 6b was less effective and reduced heart
rate by 64% after 1 min. Compound 14c was also
effective and reduced heart rate by 50% after 1 min.
Other xanthine phosphate analogues, e.g., 11b and 13c,
were less effective in reducing contracting rate (3-32%
reduction, Table 1). Compounds 11a and 6b, in addition
to reducing heart rate, caused arrhythmia.
Ch a r a cter iza tion of th e New Der iva tives a s P 2X
Recep tor Liga n d s. To identify the P2-R subtypes,
present in cardiac cells that might bind the newly
synthesized ligands, standard P2Y-R and P2X-R ago-
nists were applied and [Ca2+]i was determined. Thus,
when 2-methylthio-ATP (2-MeS-ATP), a potent and
selective agonist for several P2Y-Rs and P2X-Rs,6 was
applied at 10 µM, a large Ca2+ elevation (170 nM) was
observed. Ca2+ elevation was dose-dependent, and an
increase up to 275 nM was measured upon treatment
with 100 µM 2-MeS-ATP. Furthermore, at 100 µM, R,â-
CH2-ATP (a selective P2X1-R and P2X3-R agonist)14 also
increased calcium level (50 nM). The following order of
activity was observed: 2-MeS-ATP > ATP > R,â-CH2-
ATP, indicative of the presence of P2X-R in addition to
P2Y-R subtypes. Moreover, when the cells were trans-
ferred to Ca2+-free PBS containing 1 mM EGTA and
F igu r e 2. Effect of ATP and xanthine alkyl phosphate
derivatives on intracellular Ca2+ concentration in cultured
cardiomyocytes. Each beat was accompanied by an increase
in fluorescence emission at 405 nm and a decrease in emission
at 495 nm wavelength of indo-1. The ratio of 405/495 is
proportional to Ca2+ concentration. The time of ligand addition
is marked by a line (1) perpendicular to the x-axis. (A)
Intracellular Ca2+ elevation associated with very rapid and
short lasting Ca2+ transients and contractions caused by 10
µM ATP. (B) Changes caused by 100 µM 14c were comparable
to those caused by 20 µM ATP with respect to rate and
magnitude of Ca2+ transients and contractions. (C) a moderate
response was obtained with 100 µM 11a .
pared with that of ATP. ATP at a 10 µM concentration
caused intracellular Ca2+ elevation associated with very
rapid and short lasting (20 s) Ca2+ transients and
contractions (Figure 2A). However, upon application of
a higher dose (20 µM), Ca2+ elevation was faster with a
higher amplitude. As a result of such high concentration
of intracellular Ca2+, the cardiac cells became paralyzed.
Later, after 30-60 s, the level of Ca2+ returned to the
basal level and the rate of Ca2+ transients was reduced,
as was observed for 10 µM ATP. When 14c (100 µM)
was given to the cardiac cells, results comparable to

Characterization of “Mini-Nucleotides”
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2689
Ta ble 2. Antagonism by CPX and PPADS of the Effects of
ence between triphosphate derivatives and the corre-
sponding monophosphate derivatives with regard to
[Ca2+]i elevation.
Xanthine Alkyl Phosphate Derivatives on Ca2+ Elevationa
inhibition of
inhibition of
Ca2+ elevation by
Ca2+ elevation by
SAR In ter p r eta tion a n d Bin d in g Site P r ed ic-
tion sTh eor etica l Ca lcu la tion s. A Red u ced Mod el
for th e Ca lcu la tion s. The structural difference be-
tween class A ligands (Table 1A) and class B ligands
(Table 1B) implies that the stereoelectronic effects of
the C-8 substituent may play a major role in determin-
ing activity in this set of compounds. Therefore, elec-
tronic and steric effects in the xanthine series were
analyzed using molecular orbital (MO) quantum me-
chanical (QM) methods. The calculations were used to
interpretate the SAR results and to evaluate the ste-
reoelectronic features of the ligand that dictate molec-
ular recognition by the receptor. Moreover, the calcu-
lations’ results were used for predicting a binding site
for class A ligands. Theoretical analysis of the set of
compounds required the use of a reduced model suitable
for calculation at semiempirical and ab initio MO QM
levels. SAR analysis indicates that the activity of the
compounds does not depend on the position of the
N-alkyl phosphate substitution on the xanthine, either
on N-1 or N-7 (e.g., 6a and 11a ). Furthermore, certain
xanthines bearing an alkyl phosphate substitution on
either N-1 or N-7, are not active at all (e.g., 15b and
16b). This indicates that the alkyl phosphate substitu-
tion does not solely determine activity, whereas the
nature of C-8 substituents affects activity (e.g., 13b,c
and 14b,c vs 17b,c and 15b,c). N-1- and N-7 alkyl
phosphate xanthines possibly occupy the same confor-
mational space in the binding pocket due to the flex-
ibility of the alkyl phosphate group. Moreover, since the
alkyl phosphate moiety has no effect on the electronic
features of the xanthine, a reduced model based on
caffeine (1,3,7-trimethylxanthine), 22a , was applied. In
compd
CPX
PPADS
compd
CPX
PPADS
6b
6a
11b
11a
14b
-
+
+
+
-
+
+
-
+
+
14c
21a
21b
ATP
R,â-CH2-ATP
ND
-
+
ND
+
-
-
+/-b
+
-
a
Xanthine alkyl phosphate analogues at 100 µM concentration
were added to cells incubated for 5 min with 1 µM CPX (A1-R
antagonist) or 100 µM PPADS (P2-R antagonist). Complete
inhibition is designated by +. No inhibition is designated by -.
b
ND ) not determined. ATP was only 60% inhibited.
treated with ATP (20 µM) or with 11b, at 100 µM, no
Ca2+ elevation occurred, suggesting that the rise is from
calcium influx, i.e., involving P2X-R subtypes. Pyri-
doxal-phosphate-6-azophenyl-2′,4′-disulfonic acid tetra-
sodium (PPADS), which is a selective antagonist at
certain P2-R subtypes,15 was applied for distinguishing
between P2 receptors and the A1 receptor as possible
sites of action of the new derivatives. Ca2+ elevation was
completely prevented upon addition of certain ana-
logues, e.g., 14c and 6b (100 µM), to cells pretreated
with 100 µM PPADS cells (Table 2). However, there was
still some Ca2+ elevation upon addition of ATP (10 µM)
to PPADS (100 µM)-treated cells. The results suggest
that these ligands bind to specific P2-R subtype(s),
which may be blocked by PPADS, whereas ATP acti-
vates all P2-R subtypes, including those that may or
may not be blocked by PPADS. To study the possible
involvement of the A1-R, an A1-R antagonist, 8-cyclo-
pentyl-1,3-dimethylxanthine (CPX), was added to the
cardiac cells. When ATP, R,â-CH2-ATP, or certain
analogues, e.g., 6b, were administered to CPX-treated
cells, Ca2+ elevation was still observed, which implies
that the involvement of the A1-R is less likely. In most
cases, the activity of the ligand was not blocked by CPX
(Table 2). These results might imply that the new
ligands bind to a P2X receptor, rather than to a P2Y-
receptor or A1-R.
Str u ctu r e-Activity Rela tion sh ip . The novel xan-
thine phosphate derivatives can be classified into two
groups regarding their physiological activity (Table
1A,B). Class A, which is usually characterized by [Ca2+
]
i
elevation and reduction of contractility, consists of active
xanthine-alkyl phosphate derivatives bearing hydrogen
or halogen on C-8 (e.g., 6b, 14c, Table 1A). Class B
includes inactive compounds, where the loss of activity
is due to substitution of C-8 by an electron-donating
group (e.g., 15c, 17c) or due to elongation of the alkyl
spacer, tethering the phosphate to the xanthine by an
additional methylene unit (e.g., 7b, 12b, Table 1B). In
general, Class A ligands at 100 µM concentration
mimicked the activity of 10 µM ATP, although with
various values of [Ca2+]i elevation and reduction of
contractility. Class A ligands elevate [Ca2+]i levels in
the range of 30-95 nM. The percentage of heart rate
reduction ranges from 3% to 95% (Table 1A). Within
class A, there is no significant difference in activity due
to the change of the position of the alkyl phosphate
moiety on either N-1 or N-7, namely, between phospho-
rylated theobromine or theophylline derivatives (e.g., 6a
and 11a ). In class A, there was usually a small differ-
this model, the alkyl phosphate moiety was replaced by
a methyl group, and the C-8 position was substituted
by either a hydrogen (22a ), an electron-withdrawing
group (22b,c), or an electron-donating group (22d ,e).
According to the same guidelines, ethoxy and thiophe-
noxy groups were replaced by methoxy and thiomethyl,
respectively. Such changes make almost no difference
in resonance or inductive effects. This simplified model,
which bears no conformational or tautomeric complica-
tions, was used for the description of the electronic
nature of the modified xanthines by calculating dipole
moment vectors and electrostatic potential maps and
for the description of steric effects by calculating the
volume of the modified xanthines. The former helps to
determine factors such as potential H-binding and
complementarity of the xanthine moiety with the pro-
tein binding cavity.

2690 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Fischer et al.
Ta ble 3. Calculated Dipole Moment Vectors for a Series of Caffeine Derivatives Using HF/3-21G* and HF/6-31G*
dipole moment
HF/3-21G*
angle (deg)a
HF/6-31G*
angle (deg)a
compd
caffeine
8-bromo-caffeine
8-chloro-caffeine
8-methoxy-caffeine
8-thiomethyl-caffeine
8-thiophenyl-caffeine
value (D)
value (D)
expt
4.70 ( 0.05 (benzene)b
4.1518
3.0690
2.7465
5.0449
4.0823
4.4109
75.13
69.06
65.80
105.47
102.52
106.90
4.2697
2.9524
2.7877
5.1264
4.3010
4.8501
77.60
73.06
70.42
103.71
104.85
112.28
a
The dipole moment vector angle was defined as the clockwise angle between the axis from C-4 to C-5 and the dipole moment vector.
Reference 19.
b
Ta ble 4. Minima in the Electrostatic Potential in the Vicinity of Hydrogen-Bonding Atoms at the 0.002 electron/au3 Isodensity
Surfacea
Vmin(r) (kcal/mol)
HF/3-21G*
O-6
HF/6-31G*
O-6
compd
caffeine
8-bromo-caffeine
8-chloro-caffeine
8-methoxy-caffeine
8-thiomethyl-caffeine
O-2
N-9
O-2
N-9
-55.084
-52.953
-52.473
-56.279
-55.017
-48.495
-46.472
-45.896
-50.925
-50.298
-47.929
-45.418
-45.764
-39.496
-38.870
-47.041
NDb
-44.162
-47.824
-45.822
-41.775
NDb
-38.424
-45.121
-42.200
-39.151
NDb
-36.138
-31.291
-30.184
a
b
Wave functions were found using HF/3-21G* and HF/6-31G*. Not determined as the 6-31G* basis set for Br is not included in PC
Spartan 1.0.
Va lid a tion of Ca lcu la tion Meth od . The electronic
characteristics of the ligand, represented by the reduced
models 22a -e, were analyzed using MO QM calcula-
tions. The appropriate calculation method was selected
by conducting first AM116 followed by ab initio (HF/3-
21G and HF/6-31G*17) calculations on the reduced
models and comparing the calculated geometry and
dipole moment with experimental values. Thus, HF/3-
21G* bond lengths and bond angles were calculated for
caffeine, 22a , and compared with the X-ray crystal
structure of 3b (because of the lack of X-ray structure
for caffeine). Average absolute errors in bond lengths
and bond angles were 0.015 Å and 1.1°, respectively.
For the HF/6-31G* calculation, the values were 0.020
Å and 1.0°. These average absolute errors in bond
lengths and bond angles are within the acceptable
range, as discussed by Hehre.18 Therefore, HF-SCF
methods with small basis sets (3-21G* and 6-31G*) were
considered reliable for further calculations. Indeed, the
dipole moment calculated for caffeine using HF/6-31G*
(0.43 D) was the closest to the experimental value, Table
3.19 For analogues 22d ,e geometry optimizations were
performed on the most stable conformer in the gas
phase.
Dip ole Mom en t. The dipole moment vector describes
the charge distribution in a molecule and may thus
account for electronic effects involved in molecular
recognition. Therefore, dipole moment values were
calculated for 22a -e at both AM1 (results not shown)
and ab initio levels (HF/3-21G* and HF/6-31G*). Table
3 describes dipole moment values and vector direction,
expressed by the clockwise angle between the axis from
C-4 to C-5 and the dipole moment vector. The dipole
size and angle are smaller in the electron-poor deriva-
tives (22b,c) than in the electron-rich derivatives (22d,e).
This is explained in terms of known properties of the
substituted groups. The derivatives bearing electron-
donating groups (22d ,e) have larger electron densities
at O-6, in accordance with a possible resonance struc-
ture, as indicated by a large dipole directed toward O-6
In 22b,c, the dipole is smaller and pointing more toward
O-2. These results suggest that the series of caffeine
model derivatives divides to two distinct classes, based
on the vector direction. Caffeine (8-H), as well as 8-Br-
and 8-Cl-caffeine, form one group defined by an angle
range of 70.42-77.60, whereas 8-MeO-, 8-MeS-, and
8-SPh-caffeine form a second class with an angle range
of 103.1-112.28.
Molecu la r Electr osta tic P oten tia ls. H-binding is
one of the factors dictating binding of a small molecule
to a protein. Therefore, the electrostatic potential of
H-bond acceptors of the xanthine scaffold, e.g., O-2, O-6,
and N-9 was explored as a function of various C-8
substituents. The molecular electrostatic potential
(MEP)20 represents the first perturbation order interac-
tion energy between a molecule and a proton. Since
H-bonds are mainly electrostatic in nature,21 the MEP
may be used to model potential H-binding sites. Specif-
ically, the minimum in the MEP, V(r)min, is used to
predict sites vulnerable to electrophilic attacks and
possible H-bond acceptors.22
The MEP was calculated at the 0.002 electrons/au3
electron isodensity surface,23 as this procedure has been
successfully applied to biological recognition processes.24
The maps for 22b,c were electron-poor and the 22d ,e
maps were electron-rich relative to caffeine. All deriva-
tives showed three clear minima in V(r) in the vicinity
of O-2, O-6, and N-9 (Table 4). For 22d ,e the two
oxygens, O-2 and O-6, were more electronegative than
in caffeine. Models 22b,c showed less attraction for the
point positive charge with O-2 and O-6. Furthermore,
both electron-donating groups caused a larger decrease
in the MEP value for O-6 relative to O-2, which is in
accordance with a resonance structure for these electron-
donating groups. A possible electronic requirement for
a P2X-R agonist of this series is a dipole pointing more
toward O-2, namely, an angle in the range of 70.5-77.6°
(Table 4).

Characterization of “Mini-Nucleotides”
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2691
Ta ble 5. Molecular Volumes within the 0.002 electron/au3
Isodensity Surface Using HF/3-21G* and HF/6-31G*
PPADS blocked the response to some of the potent
ligands (e.g., 14c and 6b, Table 2), whereas ATP under
the same conditions was not completely blocked, indi-
cated that the synthetic ligands may be more specific
than ATP in activating certain PPADS sensitive P2-
receptor subtype(s). To determine the P2-R subtype(s)
potentially involved in binding, 2-MeS-ATP, an agonist
which is selective for certain P2Y-Rs and P2X-Rs,14 was
applied and caused a large Ca2+ elevation. Furthermore,
R,â-CH2-ATP (a P2X1-R and P2X3-R agonist14) also
increased the calcium level. The following order of
activity was observed: 2-MeS-ATP > ATP > R,â-CH2-
ATP, indicative of the presence of P2X1-R or P2X3-R, in
addition to P2Y-R subtypes.14 Moreover, when the cells
were transferred to Ca2+-free PBS containing 1 mM
EGTA and treated with ATP or 11b, no Ca2+ elevation
occurred, suggesting that the ligands activated an ionic
channel of P2X-R subtype rather than a P2Y receptor.
Indeed, a recent report describes the localization of P2X1
and P2X2 receptors in rat heart,26 in addition to
P2Y1,2,4,6-Rs.27 P2X1,3,4-Rs were shown to be present also
in the human foetal heart together with P2Y2,4,6-Rs.3d
A1-R is the major adenosine receptor in the heart.28
To exclude the possibility that the new ligands or their
metabolites caused their physiological effect through the
A1-R, the cardiac cells were pretreated with CPX, an
A1-R antagonist. For most of the active new derivatives,
CPX treatment did not block Ca2+ elevation (Table 2),
thus the effect was not mediated by the A1-R. However,
in some cases, e.g., 6a , the activity of the ligand was
blocked by CPX, which indicates A1-R activation; on the
other hand, this ligand was also blocked by PPADS,
which indicates P2-R activation (Table 2). This apparent
contradiction might imply unselectivity of these ana-
logues for P2-Rs.
volume (Å3)
compd
caffeine
8-bromo-caffeine
8-chloro-caffeine
8-methoxy-caffeine
8-thiomethyl-caffeine
HF/3-21G*
HF/6-31G*
176.82
201.17
196.14
203.44
219.07
178.77
NDa
196.44
205.23
219.13
a
Not determined as the 6-31G* basis set for Br is not included
in PC Spartan 1.0.
Within the series of model compounds, N-1, N-3, and
N-7 show relatively large V(r) values, as is expected,
since their lone pairs are delocalized. However, 22a -c,
exhibit lower V(r) values for N-9 (Table 4). In addition,
N-9 in 22d ,e is significantly less negative, but the N-9
position seems blocked to H-bonds by the methyl group
in 22d ,e. Recent studies of the H-binding capabilities
of oxygen atoms covalently bound to an sp2 hybridized
atom showed that these are often considerably weaker
H-acceptors than equivalent nitrogen atoms.25 There-
fore, it is likely that H-binding of the two carbonyls is
not the only factor in molecular recognition of the
theobromine and theophylline moieties at the receptor
site and that N-9 possibly participates in H-binding with
the receptor. Moreover, the low negativity of N-9 in
models of the inactive derivatives (22d ,e, Table 4)
suggests that the role of N-9 as an H-acceptor could be
crucial for binding to the protein.
Ster ic F a ctor s. Electronic factors do not solely
determine binding interactions with the protein, and
steric factors, as well as the presence of hydrophobic
moieties, could play a role. Table 5 presents the calcu-
lated volume for the series of caffeine derivatives. The
larger volume of 22d ,f derivatives relative to caffeine
and 22b,c suggests an additional explanation to the loss
for activity in those derivatives.
The structures of the synthesized analogues resemble
that of caffeine. The latter is known to release Ca2+ from
the sarcoplasmic reticulum (SR) through ryanodine
receptors.29 However, it is unlikely that the novel
analogues activate ryanodine receptors due to their
highly charged structure, which prevents membrane
permeability, and antagonism by PPADS.
Discu ssion
A novel series of “mini-nucleotides”, based on a
xanthine-alkyl phosphate scaffold (Schemes 1-3), was
evaluated for effects on Ca2+ elevation and contractility
in rat cardiac cell cultures. The novel derivatives could
be classified into two groups according to agonist activity
or lack of activity (Table 1): Class A, generally charac-
terized by [Ca2+]i elevation and reduction of contractil-
ity, consisted of active xanthine-alkyl phosphate deriva-
tives bearing hydrogen or halogen on C-8 (Table 1A);
whereas class B included inactive compounds, the
inactivity of which could be attributed to substitution
of C-8 by electron-donating groups or to elongation of
the alkyl spacer (Table 1B). Class A compounds exhib-
ited a biochemical profile similar to that of ATP (al-
though in higher concentrations), which might imply the
involvement of certain P2-R subtypes. To test this
hypothesis and to rule out the involvement of A1-R or
ryanodine-R, a series of experiments were performed.
The effect of the active agonists on [Ca2+]i elevation
in heart cells was tested in the presence of PPADS, a
potent and subtype selective P2-R antagonist.15 PPADS,
which is effective at P2Y1,2,6 receptor subtypes15 as well
as on P2X1,2,3,7 subtypes,15 was applied for distinguish-
ing between P2 receptors and the A1 receptor as possible
binding proteins of the new derivatives. The fact that
SAR analysis indicates that the optimal spacer link-
ing the phosphate to the xanthine scaffold is ethylene
rather than propylene (Table 1 A,B). A P2X-R putative
phosphate binding site adjacent to transmembrane helix
2 (TM2) was suggested recently.30 It is possible that the
new ligands bind to this phosphate binding site while
the xanthine ring system binds to the upper part of
TM2. If the linker between the phosphate and the
xanthine is more than two methylenes long, it may shift
the xanthine from its binding site and turn these
derivatives (Table 1B) inactive.
Theoretical calculations, at the MO QM level, char-
acterized the electronic features of the active derivatives
vs the inactive ones and predicted the xanthine-
phosphate binding site within the P2 receptor. We
predict that next to the xanthine N-9 there is an H-bond
donor amino acid residue. Furthermore, the low nega-
tivity of N-9 in models of the inactive derivatives (22d ,e)
suggests that the role of N-9 as an H-acceptor could be
crucial for binding to the protein, although steric factors
at the C-8 position may also account for inactivity of
these compounds. The latter might imply a limited space

2692 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Fischer et al.
white solid, mp 163 °C (0.99 g, 67%).1H NMR (CDCl3, 200
MHz) δ: 7.54 (s, 1H, H-8), 4.76 (s, 2H, N1-CH2), 4.23 (q, J )
7.14 Hz, 2H, CH2O), 3.98 (s, 3H, N7-Me), 3.59 (s, 3H, N3-
Me), 1.30 (t, J ) 7.10 Hz, 3H, Me) ppm. 13C NMR (CDCl3, 200
MHz) δ: 168.37 (CO), 154.71 (C-6), 151.30 (C-2), 149.28 (C-
4), 141.77 (C-8), 107.51 (C-5), 61.55 (CH2O), 42.08 (N1-CH2),
33.57 (N7-Me), 29.80 (N3-Me), 14.13 (Me) ppm. HRMS (DCI,
CH4): calcd for C11H15N4O4 (MH+) 267.1093, found 267.1060.
within the binding cavity, next to C-8. Dipole moments
of models of the active derivatives, 22a -c, are smaller
than those of the electron-rich analogues, 22d ,e, and
point more toward O-2. This might suggest a comple-
mentary positive binding site, or an H-bond donor in
the vicinity of O-2. Thus, derivatives with a dipole
moment pointing to O-6 may not fit the electronic
demands of the binding site at this position and would
likely be inactive.
In conclusion, several xanthine-alkyl phosphate ana-
logues (e.g., 6a ,b, 14b,c) proved to be useful selective
agents for activation of P2X-R subtypes, whereas ATP
activates all P2-R subtypes in cardiac cells. These novel
“mini-nucleotides” are expected, therefore, to serve as
prototypes for selective drugs aiming at cardiac disor-
ders mediated through P2X receptors.
1-(2-H yd r oxyet h yl)-3,7-d im et h yl-3,7-d ih yd r op u r in e-
2,6-d ion e (10). To a suspension of 8a (0.12 g, 0.45 mmol) and
NaBH4 (0.205 g, 12 equiv) in hot t-BuOH (5.7 mL) at 90 °C
was added slowly methanol (3.7 mL) over a 1.25 h period. The
solvent was removed under vacuum, and the residue was
separated on a silica gel column using a linear gradient of
EtOAc:EtOH 4:1 as the eluent. The product was obtained as
a white solid after crystallization from EtOH, mp 191 °C (0.057
1
g, 57%). H NMR (CDCl3, 200 MHz) δ: 7.53 (s, 1H, H-8), 4.30
(t, J ) 4.83 Hz, 2H, N1-CH2), 3.99 (s, 3H, N7-Me), 3.90 (q,
J ) 4.88 Hz, 2H, CH2OH), 3.59 (s, 3H, N3-Me), 2.67 (t, J )
4.38 Hz, 1H, OH) ppm. 13C NMR (CDCl3, 300 MHz) δ: 155.91
(C-6), 152.38 (C-2), 149.01 (C-4), 141.83 (C-8), 107.71 (C-5),
62.04 (CH2OH), 43.79 (N1-CH2), 33.65 (N7-Me), 29.87 (N3-
Me) ppm. HRMS (DCI, CH4): calcd for C9H13N4O3 (MH+)
225.0987, found 225.0992.
Ma ter ia ls a n d Meth od s
Ch em istr y. Gen er a l. New compounds were characterized
and resonances assigned by proton and carbon nuclear mag-
netic resonance using a Bruker AC-200, DPX-300, or DMX-
600 NMR spectrometers. Tetramethylsilane was used as an
internal standard while, for samples in D2O, HOD signal was
used as a reference at 4.78 ppm. Nucleotides were character-
ized also by 31P NMR in D2O using 85% H3PO4 as an external
reference. Samples were treated with CHELEX-100 (BioRad,
Richmond, CA) prior to spectral measurement. Xanthine
derivatives were characterized on a AutoSpec-E fision VG mass
spectrometer by chemical ionization MS and HRMS. Phos-
phorylated xanthines were desorbed from a glycerol matrix
under FAB and HRFAB negative conditions using 6 kV Xe
atoms. 2-Hydroxyethyl theophylline and 8-Cl-theophylline
were obtained from Sigma Co. The preparation of tri-n-
butylammonium pyrophosphate for the triphosphate synthesis
as well as the preparation of TEAB (triethylammonium
bicarbonate) buffer has been described.6a Silica gel (Merck
9385) was used for chromatography. Purification of phospho-
rylated xanthines was achieved on an Isco UA-6 LC system
Gen er a l P h osp h or yla tion P r oced u r e. The phosphoryl-
ation procedure was adapted from Kovacs and O¨ tvo¨s12a and
Moffat.12b The reaction was carried out on 2 (12 mg, 0.054
mmol). Separation was performed on Sephadex DEAE A-25
column (5 cm × 1 cm) using 0-0.4 M NH4HCO3 linear gradient
(350 mL of each). The product was obtained in 39% yield (9.6
mg) in 99% purity after further HPLC purification on a
semipreprative column applying a linear gradient of TEAA:
CH3CN 80:20 to 40:60 in 20 min (3 mL/min). Rt ) 4.08 min.
1-(2-Mon op h osp h a te-eth yl)-3,7-d im eth yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (11a ) a n d 1-(2-Tr ip h osp h a te-eth yl)-3,7-
d im eth yl-3,7-d ih yd r o-p u r in e-2,6-d ion e (11b). A solution of
2 (33 mg, 0.147 mmol) in dry trimethyl phosphate (0.8 mL),
obtained upon heating, was cooled slowly in an ice bath under
a nitrogen atmosphere. Proton sponge (47 mg) was added
followed by a dropwise addition of POCl3 (29.5 µL). After 20
min at 0 °C, TLC (propanol:NH4OH:H2O 11:2:7) indicated the
formation of a polar product. TEAB (0.2 M, 7.35 mL) was
added, and the ice bath was removed. After being stirred at
room temperature for 0.75 h, the reaction solution was freeze-
dried and separated on a Sephadex DEAE A-25 column
applying a linear gradient of 0-0.2 M NH4HCO3 (250 mL of
each). The appropriate fractions were freeze-dried 3 times
sequentially. Product 11a was obtained as a white powder
(38% yield) after HPLC purification (Rt ) 4.6 min (flow rate
3 mL/min, isocratic conditions TEAA:CH3CN 50:50) and 3.58
min (in solvent system III)). Product 11b was obtained as a
white powder (35% yield, 90% pure) after HPLC purification
(Rt ) 7.1 min (flow rate 3 mL/min, isocratic conditions TEAA:
CH3CN 50:50) and 4.55 min (in solvent system III)). 11a : 1H
NMR (D2O, 300 MHz) δ: 7.87 (s, 1H, H-8), 4.23 (t, J ) 5.64
Hz, 2H, N1-CH2), 4.04 (q, J ) 6.48, 5.65 Hz, 2H, CH2OP),
3.93 (s, 3H, N7-Me), 3.50 (s, 3H, N3-Me) ppm. 31P NMR (D2O,
200 MHz, pH 5.0) δ: 1.16 (s) ppm. HRFAB: calcd for
C9H12N4O6P 303.0494, found 303.0500. UV: λmax 273.9 nm.
11b: 1H NMR (D2O, 300 MHz) δ: 7.86 (s, 1H, H-8), 4.28 (t, J
) 5.29 Hz, 2H, N1-CH2), 4.17 (q, J ) 6.12, 5.47 Hz, 2H, CH2-
OP), 3.94 (s, 3H, N7-Me), 3.52 (s, 3H, N3-Me) ppm. 31P NMR
(D2O, 200 MHz, pH 9.5) δ: -5.45 (d), -9.99 (d), -21.54 (t)
ppm. HRFAB: calcd for C9H13N4O12P 461.9742, found 461.9663.
UV: λmax 273.9 nm.
-
using DEAE A-25 Sephadex (HCO3 form) columns and a
linear gradient of 0-0.4 or 0.6 M NH4HCO3. Peaks were
detected by UV absorption at 280 nm using a UV detector.
Their final purification was done on a Merck-Hitachi HPLC
system using a semipreparative nucleoside/nucleotide 7U (1
× 25 cm, Alltech Associates, Inc., Deerfield, IL) and a linear
gradient of acetonitrile (A):0.1 M triethylammonium acetate
buffer (TEAA, pH 7.5) (B) (B:A) 90:10-30:70 (solvent system
I) or 90:10-20:80 (solvent system II) in 20 min and with a
flow rate of 5 mL/min. The purity of the “mini-nucleotides”
described below was evaluated in two different solvent sys-
tems, solvent system I or II and solvent system III. Solvent
system III was a linear gradient of 5 mM TBAP in MeOH (A):
60 mM ammonium phosphate and 5 mM TBAP in 90% water/
10% MeOH (B) (A:B) 25:75-75:25 in 20 min with a flow rate
of 1 mL/min on LiChroCART Lichrospher 60 RP-select B
column (0.46 × 25 cm, Merck Co., Darmstadt, Germany). A
diode array detector was used, and peaks absorbing at 274
nm were collected.
(3,7-Dim et h yl-2,6-d ioxo-2,3,6,7-t et r a h yd r o-p u r in e-1-
yl) Acetic Acid Eth yl Ester (8a ). A suspension of theobro-
mine 2 (1 g, 5.56 mmol) and NaOH (0.22 g) in H2O (15 mL)
and EtOH (28 mL) was heated under reflux for 30 min to give
a clear solution. The solvent was removed under high vacuum
and heating to give theobromine sodium salt. A suspension of
ethyl chloroacetate (3.4 g, 5 equiv) and NaI (4.16 g, 5 equiv)
in dry DMF (16 mL) was stirred at room temperature for 2 h
and added to a solution of theobromine sodium salt in DMF.
After the mixture was stirred at 110 °C for 4.5 h, the solvent
was removed under high vacuum. The solid residue was
dissolved in CHCl3, and the salts were filtered. The filtrate
was extracted with water, dried over Na2SO4, and evaporated.
The yellowish residue was crystallized from EtOH to give a
7-(2-Mon op h osp h a te-eth yl)-1,3-d im eth yl-1,3-d ih yd r o-
p u r in e-2,6-d ion e (6a ) a n d 7-(2-Tr ip h osp h a te-eth yl)-1,3-
d im eth yl-1,3-d ih yd r o-p u r in e-2,6-d ion e (6b). Product 6a
was obtained as a white powder (33% yield) after HPLC
purification (Rt ) 4.2 min (solvent system I) and 3.32 min (in
solvent system III)). The product was >95% pure. 6a : 1H NMR
(D2O, 300 MHz) δ: 8.03 (s, 1H, H-8), 4.52 (t, J ) 5.10 Hz, 2H,
N7-CH2), 4.06 (q, J ) 6.60, 5.40 Hz, 2H, CH2OP), 3.52 (s, 3H,

Characterization of “Mini-Nucleotides”
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2693
N3-Me), 3.33 (s, 3H, N1-Me) ppm. 31P NMR (D2O, 200 MHz,
pH 6.0) δ: 3.20 (s) ppm. HRMS: calcd for C9H13N4O6P
304.0572, found 304.0486. UV: λmax at 273.9 nm. Product
6a was obtained in 42% yield after HPLC purification (Rt )
3.0 min (solvent system I )). The product was >92% pure. 6b:
1H NMR (D2O, 200 MHz) δ: 8.08 (s, 1H, H-8), 4.61 (t, J )
4.58 Hz, 2H, N7-CH2), 4.31 (q, J ) 4.84 Hz, 2H, CH2OP), 3.54
(s, 3H, N3-Me), 3.35 (s, 3H, N1-Me) ppm. 31P NMR (D2O,
200 MHz, pH 6.5) δ: -9.81 (d), -10.79 (d), -22.52 (t) ppm.
HRMS: calcd for C9H14N4O12P3 462.9821, found 462.9368.
UV: λmax 273.9 nm.
1-(3-Br om o-p r op yl)-3,7-d im et h yl-3,7-d ih yd r o-p u r in e-
2,6-d ion e (9). A suspension of 2 (0.5 g, 2.78 mmol) and NaOH
in water:EtOH (0.11 g of NaOH in 7.5:14 mL, respectively)
was heated under reflux for 30 min to yield a clear solution.
Evaporation of the solvent to dryness led to the theobromine
sodium salt which was dissolved in DMF (14 mL) and 1,3-
dibromopropane (5 mL, 10 equiv). The solution was heated to
140 °C for 2.5 h. The solvent was removed under high vacuum,
and the residue was dissolved in diethyl ether and washed
twice with water. The organic phase was dried over Na2SO4,
and the solvent was removed. The residue was triturated with
hexane and then crystallized from EtOH. Product 9 was
obtained as a white solid in 23% (0.19 g), mp 142 °C. 1H NMR
(CDCl3, 200 MHz) δ: 7.52 (s, 1H, H-8), 4.16 (t, J ) 6.84 Hz,
2H, N1-CH2), 3.99 (s, 3H, N7-Me), 3.58 (s, 3H, N3-Me), 3.45
(t, J ) 6.90 Hz, 2H, CH2Br), 2.25 (qu, J ) 7.01 Hz, 2H, CH2)
ppm. 13C NMR (CDCl3, 300 MHz) δ: 155.20 (C-6), 151.47 (C-
2), 148.88 (C-4), 141.58 (C-8), 107.59 (C-5), 40.27 (N1-CH2),
33.63 (N7-Me), 31.34 (CH2Br), 30.46 (CH2), 29.73 (N3-Me)
ppm. HRMS (DCI, CH4): calcd for C10H13N4O2Br 300.0221,
302.0201; found 300.0183, 302.0214.
turned clear within several min. 1,3-Dibromopropane (1.97
mL, 3.5 equiv) was added, and the solution was stirred at 60
°C for 2.5 h. The solvent was evaporated, and the residue was
separated on a silica gel column (MeOH:EtOAC 1:6). Product
3 was obtained in 63% yield after crystallization from EtOH,
1
mp 132 °C. H NMR (CDCl3, 200 MHz) δ: 7.65 (s, 1H, H-8),
4.47 (t, J ) 6.43 Hz, 2H, N7-CH2), 3.60 (s, 3H, N3-Me), 3.41
(s, 3H, N1-Me), 3.33 (t, J ) 6.21 Hz, 2H, CH2Br), 2.45 (qu, J
) 6.29 Hz, 2H, CH2) ppm. 13C NMR (CDCl3, 200 MHz) δ:
155.15 (C-6), 151.66 (C-2), 149.33 (C-4), 141.53 (C-8), 106.79
(C-5), 45.27 (N7-CH2), 32.65 (CH2Br), 29.83 (N3-Me), 29.41
(CH2), 28.04 (N1-Me) ppm. HRMS (DCI, CH4): calcd for
C
10H13N4O2Br 300.0221, 302.0201; found 300.0242, 302.0278.
1
Side product 4: H NMR (CDCl3, 200 MHz) δ: 7.60 (s, H-8),
4.37 (t, J ) 7.05 Hz, N7-CH2), 3.59 (s, N3-Me), 3.39 (s, N1-
Me), 2.55 (qu, J ) 7.08 Hz, CH2) ppm. MS (CI/NH3) 401 (MH+).
7-(3-Hyd r oxy-p r op yl)-1,3-d im eth yl-3,7-d ih yd r o-p u r in e-
2,6-d ion e (5b). Product 5b was prepared as described for 10b
and was obtained in 46% yield after crystallization from EtOH,
1
mp 133 °C. H NMR (CDCl3, 200 MHz) δ: 7.61 (s, 1H, H-8),
4.47 (t, J ) 6.33 Hz, 2H, N7-CH2), 3.60 (s, 3H, N3-Me), 3.58
(t, J ) 5.73 Hz, 2H, CH2OH), 3.42 (s, 3H, N1-Me), 2.07 (qu,
J ) 5.25 Hz, 2H, CH2) ppm. 13C NMR (CDCl3, 600 MHz) δ:
155.78 (C-6), 151.51 (C-2), 149.05 (C-4), 141.62 (C-8), 107.22
(C-5), 58.04 (CH2OH), 43.15 (N1-CH2), 34.00 (CH2), 29.86
(N3-Me), 28.15 (N1-Me) ppm. HRMS (DCI, CH4): calcd for
C
10H15N4O3 (MH+) 239.1144, found 239.1120.
7-(3-Mon op h osp h a te-p r op yl)-1,3-d im eth yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (7a ) a n d 7-(3-Tr ip h osp h a te-p r op yl)-1,3-
d im eth yl-3,7-d ih yd r o-p u r in e-2,6-d ion e (7b). Product 7a
was obtained in 54% yield after HPLC purification (Rt ) 4.7
min (solvent system I) and 3.76 min (in solvent system III)).
The product was >95% pure. 7a : 1H NMR (D2O, 200 MHz) δ:
7.98 (s, 1H, H-8), 4.38 (t, J ) 5.97 Hz, 2H, N7-CH2), 3.73 (q,
J ) 4.68 Hz, 2H, CH2OP), 3.48 (s, 3H, N3-Me), 3.30 (s, 3H,
N1-Me), 2.08 (m, 2H, CH2) ppm. 31P NMR (D2O, 200 MHz,
pH 6.0) δ: 2.63 (s) ppm. FAB (negative): 316.99 (M). UV:
λmax 273.9 nm. Product 7b was obtained in 22% yield after
HPLC purification (Rt ) 4.13 min (solvent system I) and 3.19
min (in solvent system III)). The product was 93% pure. 7b:
1H NMR (D2O, 300 MHz) δ: 8.06 (s, 1H, H-8), 4.45 (t, 2H, N7-
CH2), 3.93 (q, J ) 4.76 Hz, 2H, CH2OP), 3.53 (s, 3H, N3-Me),
3.34 (s, 3H, N1-Me), 2.18 (m, 2H, CH2) ppm. 31P NMR (D2O,
200 MHz, pH 8.0) δ: -5.37 (d), -10.11 (d), -21.44 (t) ppm.
HRFAB: calcd for C10H16N4O12P3 476.9977, found 476.9890.
UV: λmax 273.9 nm.
8-Ch lor o-7-(2-h yd r oxy-eth yl)-1,3-d im eth yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (20). NaH (57 mg, 2.38 mmol) was added
to a suspension of 8-chlorotheophylline (0.51 g, 2.38 mmol) in
dry DMF (17 mL). When a clear solution was attained,
2-bromoethanol (6 mL, 6 equiv) was added. The reaction
mixture was heated at 110 °C for 7 days. The solvent was
removed under high vacuum, and the residue was separated
on a silica gel column (EtOAc). Product 20 was obtained in
20% yield (0.11 g) after crystallization from EtOH, mp 159
°C.1H NMR (CDCl3, 200 MHz) δ: 4.51 (t, J ) 5.48 Hz, 2H,
N7-CH2), 3.99 (q, J ) 5.06 Hz, 2H, CH2OH), 3.55 (s, 3H, N3-
Me), 3.39 (s, 3H, N1-Me), 2.74 (t, J ) 5.77 Hz, 1H, OH) ppm.
13C NMR (CDCl3, 300 MHz) δ: 155.04 (C-6), 151.20 (C-2),
147.45 (C-4), 139.38 (C-8), 108.31 (C-5), 61.34 (CH2OH), 48.41
(N7-CH2), 29.93 (N3-Me), 28.16 (N1-Me) ppm. HRMS (DCI,
CH4): calcd for C9H12N4O3Cl (MH+) 259.0597, found 259.0617.
1-(3-Hyd r oxy-p r op yl)-3,7-d im eth yl-3,7-d ih yd r o-p u r in e-
2,6-d ion e (10b). To a clear solution of 9 (0.2 g, 0.67 mmol) in
DMF (9 mL) was added AgNO3 (0.22 g, 2 equiv) and bis-
(tributyltin)oxide (0.74 mL, 2.2 equiv). The reaction mixture
was stirred at room temperature for 5 days, and its color
turned black. Water (20 mL) was added to the mixture. Silver
salts were filtered, the solvent was removed under high
vacuum, and the residue was separated on a silica gel column
(MeOH:EtOAc 1:6). Product 10b was obtained in 46% yield
after crystallization from EtOH, mp 138-9 °C. 1H NMR
(CDCl3, 200 MHz) δ: 7.56 (s, 1H, H-8), 4.18 (t, J ) 6.15 Hz,
2H, N1-CH2), 4.00 (s, 3H, N7-Me), 3.59 (s, 3H, N3-Me), 3.53
(t, J ) 4.98 Hz, 2H, CH2OH), 1.91 (qu, J ) 5.10 Hz, 2H, CH2)
ppm. 13C NMR (CDCl3, 300 MHz) δ: 155.90 (C-6), 151.93 (C-
2), 148.99 (C-4), 141.88 (C-8), 107.54 (C-5), 58.53 (CH2OH),
37.80 (N1-CH2), 33.67 (N7-Me), 30.83 (CH2), 29.87 (N3-Me)
ppm. HRMS (DCI, CH4) calcd for C10H15N4O3 (MH+) 239.1144,
found 239.1139.
1-(3-Mon op h osp h a te-p r op yl)-3,7-d im eth yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (12a ) a n d 1-(3-Tr ip h osp h a te-p r op yl)-
3,7-d im eth yl-3,7-d ih yd r o-p u r in e-2,6-d ion e (12b). Product
12a was obtained as a white powder (23% yield) after HPLC
purification (Rt ) 4.6 min (solvent system I) and 3.58 min (in
solvent system III)). The product was >98% pure. 12a : 1H
NMR (D2O, 300 MHz) δ: 7.86 (s, 1H, H-8), 4.05 (t, J ) 7.20
Hz, 2H, N1-CH2), 3.92 (s, 3H, N7-Me), 3.90 (q, J ) 6.60, 3.60
Hz, 2H, CH2OP), 3.49 (s, 3H, N3-Me), 1.92 (qu, J ) 7.00 Hz,
2H, CH2) ppm. 31P NMR (D2O, 200 MHz, pH 6.0) δ: 1.38 (s)
ppm. HRFAB: calcd for C10H14N4O6P 317.0650, found 317.0740.
UV: λmax 273.9 nm. Product 12b was obtained as a white
powder (49% yield) after HPLC purification (Rt ) 4.3 min
(solvent system I) and 4.64 min (in solvent system III)). The
product was 90% pure. 12b: 1H NMR (D2O, 200 MHz) δ: 7.82
(s, 1H, H-8), 4.01 (qu, J ) 7.28 Hz, 4H, N1-CH2 + CH2OP),
3.90(s, 3H, N7-Me), 3.47 (s, 3H, N3-Me), 1.93 (qu, J ) 7.02
Hz, 2H, CH2) ppm. 31P NMR (D2O, 200 MHz, pH 6.5) δ: -9.26
(d), -10.26 (d), -22.40 (t) ppm. HRFAB: calcd for C10H16N4-
8-Ch lor o-7-(2-m on op h osp h a te-eth yl)-1,3-d im eth yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (21a ) a n d 8-Ch lor o-7-(2-t r i-
p h osp h a t e-et h yl)-1,3-d im et h yl-3,7-d ih yd r o-p u r in e-2,6-
d ion e (21b). Product 21a was obtained in 28% yield after
HPLC purification (Rt ) 4.8 min (solvent system I) and 4.21
min (in solvent system III)). The product was >98% pure. 21a :
1H NMR (D2O, 300 MHz) δ: 4.56 (t, J ) 5.21 Hz, 2H, N7-
CH2), 4.10 (q, J ) 6.14, 5.47 Hz, 2H, CH2OP), 3.49 (s, 3H, N3-
Me), 3.33 (s, 3H, N1-Me) ppm. 31P NMR (D2O, 200 MHz, pH
6.0) δ: 2.56 (s). HRFAB: calcd for C9H11N4O6PCl 337.0104,
found 337.0060. UV: λmax 278.8 nm. Product 21b was
O
12P3 476.9977, found 476.9970. UV: λmax 273.9 nm.
7-(3-Br om o-p r op yl)-1,3-d im et h yl-3,7-d ih yd r o-p u r in e-
2,6-d ion e (3). To theophylline 1 (1 g, 5.56 mmol) in dry DMF
(10 mL) was added NaH (0.135 g, 1.1 equiv). The solution

2694 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
obtained in 39% yield after HPLC purification (Rt ) 5.1 min
Fischer et al.
system III)). The product was >98% pure. 16c: 1H NMR (D2O,
300 MHz) δ: 7.49-7.38 (m, 5H, Ph), 4.72 (t, J ) 4.38 Hz, 2H,
N7-CH2), 4.31 (qu, J ) 7.76, 4.33 Hz, 2H, CH2OP), 3.44 (s,
3H, N3-Me), 3.34 (s, 3H, N1-Me) ppm. 31P NMR (D2O, 200
MHz, pH 5.5) δ: -9.08 (d), -11.23 (d), -22.49 (t) ppm.
HRFAB: calcd for C15H18N4O12P3S 411.0528, found 411.0400.
UV: λmax 297.5 nm.
1
(solvent system I)). 21b: H NMR (D2O, 200 MHz) δ: 4.60 (t,
J ) 3.90 Hz, 2H, N7-CH2), 4.31 (q, J ) 5.71 Hz, 2H, CH2OP),
3.48 (s, 3H, N3-Me), 3.32 (s, 3H, N1-Me) ppm. 31P NMR (D2O,
200 MHz, pH 6.0) δ: -10.28 (d), -11.00 (d), -22.72 (t) ppm.
HRFAB: calcd for C9H13N4O12P3Cl 496.9431, found 496.9480.
UV: λmax 278.8 nm.
8-Br om o-7-(2-h yd r oxy-eth yl)-1,3-d im eth yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (13a ). Br2 (0.75 mL, 1.7 equiv) in nitroben-
zene (1.2 mL) was added dropwise to a suspension of 2-hy-
droxyethyl theophylline (1.87 g, 8.34 mmol) in nitrobenzene
(8.8 mL) and CCl4 (2.85 mL). The reaction solution was heated
at 60 °C, with an efficient ice-water cooling, for 4.5 h.
Unreacted starting material was removed by filtration. Ni-
trobenzene was removed from the filtrate by distillation, and
the residue was separated on a silica gel column (MeOH:CHCl3
1:6). Product 14a was obtained as a yellowish solid (0.45 g,
18%) after crystallization from EtOH, mp 172 °C. 1H NMR
(CDCl3, 200 MHz) δ: 4.52 (t, J ) 5.37 Hz, 2H, N7-CH2), 3.98
(q, J ) 5.28 Hz, 2H, CH2OH), 3.56 (s, 3H, N3-Me), 3.39 (s,
3H, N1-Me), 2.85 (t, J ) 5.85 Hz, 1H, OH) ppm. 13C NMR
(CDCl3, 300 MHz) δ: 155.07 (C-6), 151.09 (C-2), 148.39 (C-4),
128.29 (C-8), 109.49 (C-5), 61.51 (CH2OH), 49.36 (N7-CH2),
29.95 (N3-Me), 28.20 (N1-Me) ppm. HRMS (DCI, CH4): calcd
for C9H12N4O3Br (MH+) 303.0092, found 303.0090.
8-Br om o-7-(2-m on op h osp h a te-eth yl)-1,3-d im eth yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (13b ) a n d 8-Br om o-7-(2-t r i-
p h osp h a te-eth yl)-1,3-d im eth yl-3,7-d ih yd r o-p u r in e-2,6-d i-
on e (13c). Product 13b was obtained 53% yield after HPLC
purification (Rt ) 5.7 min (solvent system I) and 4.32 min (in
solvent system III)). The product was >98% pure. 13b: 1H
NMR (D2O, 300 MHz) δ: 4.53 (t, J ) 5.40 Hz, 2H, N7-CH2),
4.31 (q, J ) 6.30 Hz, 2H, CH2OP), 3.47 (s, 3H, N3-Me), 3.31
(s, 3H, N1-Me) ppm. 31P NMR (D2O, 200 MHz, pH 6.5) δ: 3.16
(s) ppm. HRFAB: calcd for C9H11N4O6PBr 380.9599, found
380.9600. UV: λmax 280 nm. Product 13c was obtained 37%
yield after HPLC purification (Rt ) 5.3 min (solvent system
I) and 3.33 min (in solvent system III)). The product was >98%
pure. 13c: 1H NMR (D2O, 300 MHz) δ: 4.60 (t, J ) 4.54 Hz,
2H, N7-CH2), 4.30 (qu, J ) 8.43, 4.95 Hz, 2H, CH2OP), 3.48
(s, 3H, N3-Me), 3.31 (s, 3H, N1-Me) ppm. 31P NMR (D2O,
200 MHz, pH 5.5) δ: -10.23 (d), -11.01 (d), -22.68 (t) ppm.
HRFAB: calcd for C9H15N4O12P3Br 542.9082, found 542.9050.
UV: λmax 280 nm.
8-Eth oxy-1-(2-m on op h osp h a te-eth yl)-1,3-d im eth yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (17a ). To a solution of 13a (173
mg, 0.57 mmol) in absolute EtOH (15 mL) was added sodium
ethoxide solution (14 mg, in 1.4 mL EtOH). After 5 min, the
solvent was evaporated and the residue was separated on a
silica gel column (MeOH:EtOAc 1:6). Product 17a was obtained
as white needles in 52% (80 mg) after crystallization from
1
EtOH/CHCl3, mp 176 °C. H NMR (CDCl3, 200 MHz) δ: 4.54
(q, J ) 7.09 Hz, 2H, CH2), 4.24 (t, J ) 5.46 Hz, 2H, N7-CH2),
3.89 (t, J ) 5.44 Hz, 2H, CH2OH), 3.51 (s, 3H, N3-Me), 3.36
(s, 3H, N1-Me), 1.45 (t, J ) 7.08 Hz, 3H, Me) ppm. 13C NMR
(CDCl3, 300 MHz) δ: 155.88 (C-8), 155.45 (C-6), 151.47 (C-2),
146.92 (C-4), 103.53 (C-5), 67.47 (CH2), 61.75 (CH2OH), 45.45
(N7-CH2), 29.48 (N3-Me), 27.94 (N1-Me), 14.62 (Me) ppm.
HRMS (DCI, CH4): calcd for C11H17N4O4 (MH+) 269.1249,
found 269.1233. Side product 18: 1H NMR (CDCl3, 200 MHz)
δ: 5.15 (t, J ) 7.80 Hz, 2H, CH2O), 4.44 (t, J ) 7.80 Hz, 2H,
N7-CH2), 3.52 (s, 3H, N3-Me), 3.39 (s, 3H, N1-Me) ppm.
13C NMR (CDCl3, 300 MHz) δ: 154.13 (C-6), 151.66 (C-2),
162.95 (C-8), 145.12 (C-4), 102.39 (C-5), 75.14 (CH2O), 43.53
(N7-CH2), 29.71 (N3-Me), 27.51 (N1-Me) ppm.
8-Eth oxy-1-(2-m on op h osp h a te-eth yl)-1,3-d im eth yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (17b) a n d 8-Eth oxy-1-(2-tr i-
p h osp h a t e-et h yl)-1,3-d im et h yl-3,7-d ih yd r o-p u r in e-2,6-
d ion e (17c). Product 17b was obtained in 28% yield after
HPLC purification (Rt ) 6.0 min (solvent system II) and 6.62
min (in solvent system III)). The product was 85% pure. 17b:
1H NMR (D2O, 300 MHz) δ: 4.53 (q, J ) 7.11 Hz, 2H, CH2),
4.29 (t, J ) 4.94 Hz, 2H, N7-CH2), 4.08 (q, J ) 6.50, 5.36 Hz,
2H, CH2OP), 3.47 (s, 3H, N3-Me), 3.29 (s, 3H, N1-Me), 1.42
(t, J ) 7.10 Hz, 3H, Me) ppm. 31P NMR (D2O, 200 MHz, pH
5.5) δ: 0.81 (s). HRFAB: calcd for C11H16N4O7P 347.0756,
found 347.0800. UV: λmax 281.7 nm. Product 17c was
obtained in 43% yield after HPLC purification (Rt ) 5.5 min
(solvent system II) and 8.01 min (in solvent system III)). The
product was 90% pure. 17c: 1H NMR (D2O, 300 MHz) δ: 4.56
(q, J ) 7.20 Hz, 2H, CH2), 4.35 (m, 2H, N7-CH2), 4.25 (m,
2H, CH2OP), 3.50 (s, 3H, N3-Me), 3.32 (s, 3H, N1-Me), 1.44
(t, J ) 7.20 Hz, 3H, Me) ppm. 31P NMR (D2O, 200 MHz, pH
5.5) δ: -10.30 (d), -10.99 (d), -22.72 (t) ppm. HRFAB: calcd
for C11H18N4O13P3 507.0083, found 507.0140. UV: λmax 281.7
nm.
7-(2-Hyd r oxy-eth yl)-1,3-d im eth yl-8-p h en ylsu lfa n yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (16a ). To a solution of thiophenol
(6 µL, 1.1 equiv) in dry DMF (10 mL) was added NaH (14 mg,
1.1 equiv), and a clear solution was attained within several
min. Compound 13a (160 mg, 0.52 mmol) in dry DMF (4 mL)
was added to the thiophenolate solution. The reaction mixture
was heated at 50 °C for 2 h. The solvent was removed under
high vacuum, and the residue was separated on a silica gel
column (MeOH:EtOAc 1:6). The product was obtained as a
yellowish solid in 71% yield (0.125 mg) after crystallization
8-Br om o-1-(2-h yd r oxy-eth yl)-3,7-d im eth yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (14a ). Product 14a was prepared from 10a
as described for 13a and was obtained in 17% yield after
1
crystallization from EtOH, mp 199 °C. H NMR (CDCl3, 200
MHz) δ: 4.28 (t, J ) 5.37 Hz, 2H, N1-CH2), 3.95 (s, 3H, N7-
Me), 3.88 (q, J ) 5.14 Hz, 2H, CH2OH), 3.55 (s, 3H, N3-Me),
2.56 (t, J ) 5.53 Hz, 1H, OH) ppm. 13C NMR (CDCl3, 200 MHz)
δ: 154.95 (C-6), 151.95 (C-2), 148.39 (C-4), 128.68 (C-8), 109.53
(C-5), 61.91 (CH2OH), 43.88 (N1-CH2), 34.05 (N7-Me), 29.95
(N3-Me) ppm. HRMS (DCI, CH4): calcd for C9H12N4O3Br
(MH+) 303.0092, found 303.0010; calcd for C9H10N4O2Br
(MH+-H2O) 284.9987, found 285.0018.
8-Br om o-1-(2-m on op h osp h a te-eth yl)-1,3-d im eth yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (14b ) a n d 8-Br om o-1-(2-t r i-
p h osp h a te-eth yl)-1,3-d im eth yl-3,7-d ih yd r o-p u r in e-2,6-d i-
on e Tetr a k is-tr ieth yla m m on iu m sa lt (14c). Product 14b
was obtained in 26% yield after HPLC purification (Rt ) 6.5
min (solvent system I) and 4.82 min (in solvent system III)).
The product was 93% pure. 14b: 1H NMR (D2O, 300 MHz) δ:
4.23 (t, J ) 5.84 Hz, 2H, N1-CH2), 4.03 (q, J ) 6.27, 5.83 Hz,
2H, CH2OP), 3.90 (s, 3H, N7-Me), 3.46 (s, 3H, N3-Me) ppm.
31P NMR (D2O, 200 MHz, pH 5.5) δ: 0.92 (s) ppm. HRFAB:
calcd for C9H11N4O6PBr 380.9599, found 380.9610. UV: λmax
280.4 nm. Product 14c was obtained in 38% yield after HPLC
purification (Rt ) 5.5 min (solvent system I)). 14c: 1H NMR
1
from EtOH/CHCl3 mp 137 °C. H NMR (CDCl3, 200 MHz) δ:
7.45-7.32 (m, 5H, Ph), 4.58 (t, J ) 5.14 Hz, 2H, N7-CH2),
3.83 (q, J ) 5.22 Hz, 2H, CH2OH), 3.53 (s, 3H, N3-Me), 3.38
(s, 3H, N1-Me), 3.00 (t, J ) 5.92 Hz, 1H, OH) ppm. 13C NMR
(CDCl3, 300 MHz) δ: 155.61 (C-6), 151.21 (C-2), 148.54 (C-4),
147.45 (C-8), 131.06 (C-H), 130.77 (C-ipso), 129.59 (C-H),
128.53 (C-H), 109.49 (C-5), 61.87 (CH2OH), 48.60 (N7-CH2),
29.97 (N3-Me), 28.18 (N1-Me) ppm. HRMS (DCI, CH4): calcd
for C15H17N4O3S (MH+) 333.1021, found 333.1015.
7-(2-Mon op h osp h a te-eth yl)-1,3-d im eth yl-8-p h en ylsu l-
fa n yl-3,7-d ih yd r o-p u r in e-2,6-d ion e (16b). Product 16b was
obtained in 33% yield after HPLC purification (Rt ) 9.3 min
(solvent system I) and 14.12 min (in solvent system III)). The
product was 98% pure. 16b: 1H NMR (D2O, 300 MHz) δ:
7.50-7.35 (m, 5H, Ph), 4.66 (m, 2H, N7-CH2), 4.07 (m, 2H,
CH2OP), 3.43 (s, 3H, N3-Me), 3.33 (s, 3H, N1-Me) ppm. 31P
NMR (D2O, 200 MHz, pH 5.5) δ: 3.63 (s). HRFAB: calcd for
C15H16N4O6PS 411.0528, found 411.0630. UV: λmax 297.5 nm.
Product 16c was obtained in 44% yield after HPLC purification
(Rt ) 4.6 min (solvent system I) and 12.64 min (in solvent

Characterization of “Mini-Nucleotides”
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2695
(D2O, 300 MHz) δ: 4.27 (m, 2H, N1-CH2), 4.17 (m, 2H, CH2-
OP), 3.91 (s, 3H, N7-Me), 3.48 (s, 3H, N3-Me) ppm. 31P NMR
(D2O, 200 MHz, pH 9.0) δ: -5.31, -10.45, -21.92 ppm.
HRFAB: calcd for C9H15N4O12P3Br 542.9082, found 542.9110.
UV: λmax 280.4 nm.
enabling the display of the dipole moment vectors and the
calculation and display of molecular electrostatic potential
(MEP) maps. The 8-Br-caffeine HF/6-31G* MEP map was not
calculated since the PC Spartan package does not include the
6-31G* basis set for Br. Calculations with Gaussian 94 were
performed on an IBM RISC/6000 computer with the AIX 4
operating system.
1-(2-Hyd r oxy-eth yl)-3,7-d im eth yl-8-p h en ylsu lfa n yl-3,7-
d ih yd r o-p u r in e-2,6-d ion e (15a ). Product 15a was prepared
from 14a as described for 16a and was obtained in 68% yield
1
after crystallization from EtOH/CHCl3, mp 157 °C. H NMR
Ack n ow led gm en t. The authors thank the Israeli
Ministry of Science and the “Marcus center for phar-
maceutical and medicinal chemistry” for financial sup-
port. The authors also thank Ms. A. Laxer for the
synthesis of one of the derivatives.
(CDCl3, 200 MHz) δ: 7.34 (br s, 5H, Ph), 4.28 (t, J ) 5.00 Hz,
2H, N1-CH2), 3.90 (s, 3H, N7-Me), 3.87 (t, J ) 5.02 Hz, 2H,
CH2OH), 3.54 (s, 3H, N3-Me), ppm. 13C NMR (CDCl3, 300
MHz) δ: 155.44 (C-6), 152.11 (C-2), 148.39 (C-4), 147.24 (C-
8), 130.73 (C-H), 130.68 (C-ipso), 129.67 (C-H), 128.40 (C-
H), 109.63 (C-5), 62.10 (CH2OH), 43.85 (N1-CH2), 33.17 (N7-
Me), 29.97 (N3-Me) ppm. HRMS (DCI, CH4): calcd for
C15H17N4O3S (MH+) 333.1021, found 303.1010.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
1-(2-Mon op h osp h a te-eth yl)-3,7-d im eth yl-8-p h en ylsu l-
fa n yl-3,7-d ih yd r o-p u r in e-2,6-d ion e Bistr ieth yla m m on i-
u m Sa lt (15b). Product 15b was obtained in 28% yield after
HPLC purification (Rt ) 8.8 min (solvent system II)). The
Refer en ces
(1) (a) Burnstock, G. Purinergic nerves. Pharmacol. Rev. 1972, 24,
509-81. (b) King, B. F.; Townsend-Nicholson, A.; Burnstock, G.
Metabotropic receptors for ATP and UTP: exploring the cor-
respondence between native and recombinant nucleotide recep-
tors. Trends Pharmacol. Sci. 1998, 19, 506-514. (c) Chan, C.
M.; Unwin, R. J .; Burnstock, G. Potential functional roles of
extracellular ATP in kidney and urinary tract. Exp. Nephrol.
1998, 6, 200-207. (d) Boaeder, M. R.; Hourani, S. M. O. The
regulation of vascular function by P2 receptors: multiple site
and multiple receptors. Trends Pharmacol. Sci. 1998, 19, 99-
107. (e) Barnard, E. A.; Simon, J .; Webb, T. E. Nucleotide
receptors in the nervous system- An abundant component using
diverse transduction mechanisms. Mol. Neurobiol. 1997, 15,
103-129. (f) Inoue, K. The function of ATP receptors in the
hippocampus. Pharmacol. Res. 1998, 38, 323-331. (g) Abbrachio,
M. P. P1 and P2 receptors in cell growth and differentiation.
Drug Dev. Res. 1996, 39, 393-406.
(2) (a) Spedding, M.; Williams, M. Development in purine and
pyrimidine receptors based therapeutics. Drug Dev. Res. 1996,
39, 436-441. (b) Abbracchio, M. P.; Burnstock, G. Purinocep-
tors: are there families of P2X and P2Y Purinoceptors? Phar-
macol. Ther. 1994, 64, 445-475. (c) Barnard, E. A.; Burnstock,
G.; Webb, T. E. The G-protein coupled receptors for ATP and
other nucleotides: a new receptor family. Trends Pharmacol.
Sci. 1994, 15, 67-70.
(3) (a) Christie, A.; Sharma, V. K.; Sheu, S. S. Mechanism of
extracellular ATP-induced increase of cytosolic Ca2+ concentra-
tion in isolated rat ventricular myocytes. J . Physiol 1992, 445,
369-388. (b) De Young, M. B.; Scarpa, A. Extracellular ATP
induces Ca2+ transients in cardiac myocytes which are potenti-
ated by norepinephrine. FEBS Lett. 1987, 223, 53-58. (c) Zheng,
J . S.; Christie, A.; Levi, M. N.; Scarpa, A. Ca2+ mobilization by
extracellular ATP in rat cardiac myocytes: regulation by protein
kinase C and A. Am. J . Physiol. 1992, 263, C933-C940. (d)
Bogdanov, Y.; Rubino, A.; Burnstock, G. Characterisation of
subtypes of the P2X and P2Y families of ATP receptors in the
foetal human heart. Life Sci. 1998, 62, 697-703.
(4) Bjornsson, O. G.; Monck, J . R.; Williamson, J . R. Identification
of P2Y purinoceptors associated with voltage-activated cation
channels from cardiac ventricular myocytes of the rat. J .
Biochem. 1989, 186, 395-404.
(5) Hohl, C. M.; Hearse, D. J . Vascular and contractile responses
to extracellular ATP: studies in the isolated rat heart. Can. J .
Cardiol. 1985, 1, 207-216.
(6) (a) Fischer, B.; Boyer, J . L.; Hoyle, C. H. V.; Ziganshin, A. V.;
Brizzolara, A. L.; Knight, G. E.; Zimmet, J .; Burnstock, G.;
Harden, T. K.; J acobson, K. A. Identification of potent, selective
P2Y-purinoceptor agonists: structure-activity relationships for
2-thioether derivatives of adenosine 5′-triphosphate. J . Med.
Chem. 1993, 36, 3937. (b) Burnstock, G.; Fischer, B.; Hoyle, C.
H. V.; Maillard, M.; Ziganshin, A. V.; Brizzolara, A. L.; von
Isakovics, A.; Boyer, J . L.; Harden, T. K.; J acobson, K. A.
Structure activity relationship for derivatives of adenosine 5′-
triphosphate as agonists at P2-purinoceptors: heterogeneity
within P2X and P2Y subtypes. Drug Dev. Res. 1994, 31, 206. (c)
Boyer, J . L.; O’Tuel, J . W.; Fischer, B.; J acobson, K. A.; Harden,
T. K. 2-Thioether derivatives of adenine nucleotides are excep-
tionally potent agonists at adenylyl cyclase-linked P2Y-pu-
rinoceptors. Br. J . Pharmacol. 1995, 116, 2611.
1
product was 93% pure. H NMR (D2O, 300 MHz) δ: 7.39 (br
s, 5H, Ph), 4.22 (t, J ) 5.40 Hz, 2H, N1-CH2), 4.04 (q, J )
6.19, 5.74 Hz, 2H, CH2OP), 3.92 (s, 3H, N7-Me), 3.46 (s, 3H,
N3-Me) ppm. 31P NMR (D2O, 200 MHz, pH 5.5) δ: 0.99 (s)
ppm. HRFAB: calcd for
C15H16N4O6PS 411.0528, found
411.0400. UV: λmax 300.0 nm.
P h a r m a cologica l Eva lu a tion . Cell Cu ltu r e. Rat hearts
(1-2 days old) were removed under sterile conditions and
washed 3 times in phosphate buffered saline (PBS) to remove
excess blood cells. The hearts were minced to small fragments
and then agitated gently in a solution of proteolytic enzymes,
RDB (Biological Institute, Ness-Ziona, Israel), which was
prepared from a fig tree extract. The RDB was diluted 1:50 in
Ca2+ and Mg2+-free PBS at 25 °C for a few cycles of 10 min
each, as described previously.31 Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% horse serum (Biological
Industries, Kibbutz Beit Haemek, Israel) was added to super-
natant suspensions containing dissociated cells. The mixture
was centrifuged at 300g for 5 min. The supernatant phase was
discarded, and the cells were suspended again. The suspension
of the cells was diluted to 1.0 × 106 cells/mL, and 1.5 mL were
placed in 35 mm plastic culture dishes on collagen/gelatin-
coated coverglasses. The cultures were incubated in a humidi-
fied atmosphere of 5% CO2/95% air at 37 °C. Confluent
monolayers exhibiting spontaneous contractions were devel-
oped in culture within 2 days. The growth medium was
replaced after 24 h and then every 3 days.
Mea su r em en t of Con tr a ctility. Culture dishes were
placed into a specially designed Plexiglas chamber exposed to
a 95% air/5% CO2 atmosphere at 37 °C. The chamber was
placed on the stage of an inverted phase contrast microscope
(Olympus). The contractile state of the cardiomyocytes at
baseline and in response to interventions was conducted using
a video motion detector system, analogous to that described
by Zangen and Shainberg.32 Analogue tracing was recorded
using a oscilloscope joined through
a specially designed
interface to an IBM computer, and kinetic data were analyzed
using Microsoft Excel.
In tr a cellu la r Ca 2+ Mea su r em en ts. Intracellular free
calcium concentration, [Ca2+]i, was estimated from indo-1
fluorescence using the ratio method described by Grynkiewicz
33
et al. The cardiac cells grown on a coverglass were trans-
ferred to a chamber on the stage of a Zeiss inverted microscope
filtered with UV epifluorescence illumination. Indo-1 was
excited at 355 nm, and the emitted light was then split by a
dichroic mirror to two photomultipliers (Hamamatsu, J apan)
with input filters at 405 and 495 nm. The fluorescence ratio
of 405/495 nm, which is proportional to [Ca2+]i, was monitored.
Th eor etica l Ca lcu la tion s. All geometry optimizations
were first performed using Gaussian 94,34 and the AM1, HF/
3-21G*, and HF/6-31G* optimized coordinates were imported
into PC Spartan 1.0.35 Using PC Spartan, single-point calcula-
tions were performed to generate the wave functions, thus
(7) van der Wenden, E. M.; Price, S. L.; Apaya, R. P.; Ijzerman, A.
P.; Soudijn W. Relative binding orientations of adenosine A1
receptor ligands- a test case for distributed multipole analysis
in medicinal chemistry. J . Comput. Aided Mol. Des. 1995, 9, 44-
54 and references therein.

2696 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Fischer et al.
(8) (a) Marzilli, L. G.; Epps, L. A.; Sorrell, T.; Kistenmacher, T.
Reaction of coordinated purines. A facile, high yield synthetic
route to N7-alkylated xanthines and hypoxanthines. The struc-
ture of [bis(dimethylglyoximato) (xanthinato)-(tri-n-butylphos-
phine) cobalt (III)] and the trans influence in cobalt (III)
chemistry. J . Am. Chem. Soc. 1975, 97, 3351. (b) Daly, J . W.;
Hide, I.; Mueller, C. E.; Shamim, M. Caffeine analogues:
Structure-activity relationships at adenosine receptors. Phar-
macology 1991, 42, 309-321.
(9) (a) Lister, J . H. In Fused pyrimidines Part II: purines; Wiley
Interscience: New York, 1971. (b) Cottam, H. B.; Shih, H.;
Tehrani, L. R.; Wasson, D. B.; Carson, D. Substituted xanthines,
pteridinediones, and related compounds as potential antiinflam-
matory agents. Synthesis and biological evaluation of inhibitors
of tumor necrosis factor R. J . Med. Chem. 1996, 39, 2-9.
(10) (a) Mandal, S. B.; Giri, V. S.; Pakrashi, S. C. Sodium borohydride
desulfurization of thiolactams to amines. Heterocycles, 1988, 27,
11. (b) Soai, K.; Oyamada, H.; Ookawa, A. Sodium borohydride
tert-butyl alcohol-methanol as an efficient system for the selec-
tive reduction of esters. Synth. Commun. 1982, 12, 463-467.
(11) Gingras, M.; Chan, T. H. Silver-assisted reactions of organotin
oxides. A mild, neutral and anhydrous one-step conversion of
primary organic halides to alcohols. Tetrahedron Lett. 1989, 30,
279-282.
1982, 104, 2797-2803. (c) Pietro, W. J .; Francl, M. M.; Hehre,
W. J .; DeFrees, D. J .; Pople, J . A.; Binkley, J . S. Self-consistent
molecular orbital methods. 24. Supplemented small split-valence
basis sets for second-row elements. J . Am. Chem. Soc. 1982, 104,
5039-5048.
(18) Hehre, W. J .; Radom, L.; Schleyer, P. V. R.; Pople, J . A. Ab Initio
molecular orbital theory; Wiley: New York, 1986; Chapter 6.
(19) Weiler-Feilchenfeld, H.; Bergmann, E. D. The dipole moments
of some purine and pyrimidine derivatives. Israel J . Chem. 1968,
6, 823-826.
(20) (a) Naray-Szabo, G.; Ferenczy, G. G. Molecular Electrostatics.
Chem. Rev. 1995, 95, 829-847. (b) Doucet, J .-P.; Weber, J .
Computer-Aided Molecular Design: Theory and Applications;
Academic Press Inc.: San Diego, CA, 1996; Chapter 10.
(21) Pauling, L. Proc. Natl. Acad. Sci. U.S.A. 1928, 14, 359.
(22) Politzer, P., Murray, J . S., Lipkowitz, K. B., Boyd, D. B., Eds.
Molecular electrostatic potentials and chemical reactivity. Re-
views in Computational Chemistry; VCH: New York 1991: Vol.
2, Chapter 7, pp 273-312.
(23) Kahn, S. D.; Pau, C. F.; Hehre, W. J . Models for chemical
reactivity: Mapping of intermolecular potentials onto electron
density surfaces. Int. J . Quantum Chem., Quantum Chem. Symp.
1988, 22, 575-591.
(24) Sjoberg, P.; Murray, J . S.; Brinck, T.; Evans, P.; Politzer, P. The
use of the electrostatic potential at the molecular surface in
recognition interactions: dibenzo-p-dioxins and related Systems.
J . Mol. Graphics 1990, 8, 81-85.
(25) Nobeli, I.; Price, S. L.; Lommerse, J . P. M.; Taylor, R. Hydrogen
bonding properties of oxygen and nitrogen acceptors in aromatic
heterocycles. J . Comput. Chem. 1997, 18, 2060-2074.
(26) Hansen, M. A.; Balcar, V. J .; Barden, J . A.; Bennett, M. R.
Localisation of P2X subtypes in heart, artery and bladder. Drug
Dev. Res. (Special issue, 6th International Symposium on
Adenosine and Adenine Nucleotides.) 1998, 43, 5.
(27) Webb, T. E.; Boluyt, M. O.; Barnard, E. A. Molecular biology of
P2Y purinoceptors: Expression in rat heart. J . Auton. Pharma-
col. 1996, 16, 303-307.
(28) Shryock, J . C.; Belardinelli, L. Adenosine and adenosine recep-
tors in the cardiovascular system: Biochemistry, physiology and
pharmacology. Am. J . Cardiol. 1997, 79, 2-10.
(29) Hasenfuss G. Alterations of calcium-regulatory proteins in heart
failure. Cardiovasc. Res. 1998, 37, 279-289.
(12) (a) Kovacs, T.; Otvos, L. Simple synthesis of 5-vinyl- and
5-ethenyl-2′-deoxyuridine-5′-triphosphates. Tetrahedron Lett.
1988, 29, 4525-4528. (b) Moffat, J . G. A general synthesis of
nucleoside 5′-triphosphates. Can. J . Chem. 1964, 42, 599.
(13) Kopel, H. C.; Springer, R. H.; Robins, R. K.; Schneider, F. H.;
Cheng, C. C. Synthesis of some 8-substituted bis(â-chloroethyl)-
amino derivatives of naturally occurring N-methylated purines.
J . Org. Chem. 1962, 27, 2173.
(14) Fischer, B. Therapeutic applications of ATP-(P2)-receptors ago-
nists and antagonists. Expert. Opin. Ther. Pat. 1999, 9, 385-
399.
(15) (a) Lambrecht, G.; Ardanuy, U.; Baumert, H. G.; Bo, X.; Hoyle,
C. H. V.; Nickel, P.; Pfaff, O.; Ralevic, V.; Windschief, U.;
Ziganshin, A. U.; Ziyal, R.; Mutscheler, E.; Burnstock, G. Design
and Pharmacological characterization of selective P2-purinocep-
tor antagonists. In Perspectives in Receptor Research; Pharmaco
Chemistry Library, Giardina, D., Piergentili, S., Pigini, M., Eds.;
Elsevier Science Publishers: Amsterdam, 1996; pp 337-350. (b)
Boyer, J . L.; Zohn, I. E.; J acobson, K. A.; Harden, T. K.
Differential effects of P2-purinergic receptor antagonists on
phospholipase C and adenylyl cyclase-coupled P2Y purinergic
receptors. Br. J . Pharmacol. 1994, 113, 614-620. (c) Harden,
T. K.; Nicholas, R. A.; Schachter, J . B.; Lazarowski, E.; Boyer,
J . L. Pharmacological selectivities of molecularly defined sub-
types of P2Y receptors. In The P2 Nucleotide Receptors; Turner,
J . T., Weisman, G., Fedan, J ., Eds.; The Receptors; Humana
Press: Clifton, NJ , 1997; Chapter 5, pp 109-134. (d) Evans, R.
J .; Surprenant, A.; North, R. A. P2X receptors. In The P2
Nucleotide Receptors; Turner, J . T., Weisman, G., Fedan, J ., Eds.;
The receptors; Humana Press: Clifton, NJ , 1997; Chapter 2, pp
43-61.
(16) Dewar, M. J . S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J . J . P.
AM1: A new general purpose quantum mechanical molecular
model. J . Am. Chem. Soc. 1985, 107, 3902-3909.
(17) (a) Binkley, J . S.; Pople, J . A.; Hehre, W. J . Self-consistent
molecular orbital methods. 21. Small split-valence basis sets for
first-row elements. J . Am. Chem. Soc. 1980, 102, 939-947. (b)
Gordon, M. S.; Binkley, J . S.; Pople, J . A.; Pietro, W. J .; Hehre,
W. J . Self-consistent molecular orbital methods. 22. Small split-
valence basis sets for second-row elements. J . Am. Chem. Soc.
(30) Hansen, M. A.; Barden, J . A.; Balcar, V. J .; Keay, K. A.; Bennett,
M. R. Structural motif and characteristics of the extracellular
domain of P2X receptors. Biochem. Biophys. Res. Commun. 1997,
236, 670-675.
(31) El-Ani, D.; J acobson, K. A.; Shainberg, A. Regulation of A1
adenosine receptors by amiodarone and electrical stimulation
in rat myocardial cells in vitro. Biochem. Pharmacol. 1997, 54,
583-587.
(32) Zangen, A.; Shainberg, A. Thiamine deficiency in cardiac cells
in culture. Biochem. Pharmacol. 1997, 54, 575-582.
(33) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of
Ca2+ indicators with greatly improved fluorescence properties.
J . Biol. Chem. 1985, 260, 3440-3450.
(34) Gaussian 94, Gaussian Inc.; Pittsburgh, PA.
(35) PC Spartan 1.0, Wavefunction Inc., Irvine, CA.
(36) Manoach, M.; Varon, D.; Shainberg, A.; Zinman, T.; Isaack, A.;
Halili-Rutman, I.; Kaplan D.; Tribulova N. The protective effect
of class III antiarrhythmic agents against calcium overload in
cultured myocytes. Life Sci. 1997, 61, PL 227-234.
J M990085I