Structure-activity relationship of (N)-methanocarba phosphonate analogues of 5′-AMP as cardioprotective agents acting through a cardiac P2X receptor

Article abstract of DOI:10.1021/jm9018542

P2X receptor activation protects in heart failure models. MRS2339 3, a 2-chloro-AMP derivative containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system, activates this cardioprotective channel. Michaelis-Arbuzov and Wittig reactions provided phosphonate analogues of 3, expected to be stable in vivo due to the C-P bond. After chronic administration via a mini-osmotic pump (Alzet), some analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice (genetic model of heart failure) compared to vehicle-infused mice (all inactive at the vasodilatory P2Y₁ receptor). Two phosphonates,(1′S,2′R,3′S,4′R,5′S)- 4′-(6-amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy) -1′-(phosphonomethylene)-bicyclo[3.1.0]hexane, 4 (MRS2775), and its homologue 9 (MRS2935), both 5′-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening (20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 5′-extended phosphonates, all 2-H analogues, and a CH₃-phosphonate were inactive. Thus, chronic administration of nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor activation. Thus, we have greatly expanded the range of carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure.

Full text of DOI:10.1021/jm9018542

2562 J. Med. Chem. 2010, 53, 2562–2576
DOI: 10.1021/jm9018542
Structure-Activity Relationship of (N)-Methanocarba Phosphonate Analogues of 50-AMP
as Cardioprotective Agents Acting Through a Cardiac P2X Receptor
T. Santhosh Kumar,† Si-Yuan Zhou,‡ Bhalchandra V. Joshi,†,§ Ramachandran Balasubramanian,† Tiehong Yang,‡
Bruce T. Liang,‡ and Kenneth A. Jacobson*,†
†Molecular Recognition Section, National Institutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892, and
‡Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, Connecticut 06030.
Present address: Nektar Therapeutics, Huntsville, Alabama 35801.
§
Received December 15, 2009
P2X receptor activation protects in heart failure models. MRS2339 3, a 2-chloro-AMP derivative
containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system, activates this cardioprotective channel.
Michaelis-Arbuzov and Wittig reactions provided phosphonate analogues of 3, expected to be stable in
vivo due to the C-P bond. After chronic administration via a mini-osmotic pump (Alzet), some
analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice
(genetic model of heart failure) compared to vehicle-infused mice (all inactive at the vasodilatory P2Y1
receptor). Two phosphonates, (10S,20R,30S,40R,50S)-40-(6-amino-2-chloropurin-9-yl)-20,30-(dihydroxy)-
10-(phosphonomethylene)-bicyclo[3.1.0]hexane, 4 (MRS2775), and its homologue 9 (MRS2935), both
50-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening
(20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 50-extended phos-
phonates, all 2-H analogues, and a CH3-phosphonate were inactive. Thus, chronic administration of
nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor
activation. Thus, we have greatly expanded the range of carbocyclic nucleotide analogues that represent
potential candidates for the treatment of heart failure.
Introduction
native cardiac P2X receptor, which mediates ionic current
induced by extracellular ATP.4 This P2X current was
up-regulated in cardiac ventricular myocytes of the CSQ
hearts. Furthermore, cardiac myocyte-specific overex-
pression of the P2X4 receptor can mimic the beneficial
effects following chronic infusion of a P2X agonist ana-
logue. This analysis suggested that regulation of this
cardiac P2X receptor is protective in cardiac hypertrophy
or failure.
(10S,20R,30S,40R,50S)-4-(6-Amino-2-chloro-9H-purin-9-yl)-
1-[phosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol
(MRS2339, 3) is an (N)-methanocarba monophosphate deri-
vative of 2-chloro-AMP 2 that contains a rigid bicyclic ring
system (bicyclo[3.1.0]hexane) in place of ribose that we re-
ported to to be effective in an in vivo model of cardiomyo-
pathy.8 This ring system impedes hydrolysis of the 50-phosphate
in a model compound by its nucleotidase.9 Compound 3
induced a current in the CSQ myocyte similar to that by
compound 1, characteristic of the action of the P2X4 receptor.8
Chronically administered compound 3 rescued the hyper-
trophic and heart failure phenotype in the CSQ-overexpressing
mouse.7 When administered via an Alzet mini-osmotic pump,
it significantly increased longevity as compared to vehicle-
injected mice. The improvement in survival was associated with
decreases in heart weight/body weight ratio and in cross-section
area of the cardiac myocytes. Compound 3 was devoid of any
vasodilator action in mouse aorta ring preparations, indicating
that its salutary effect in heart failure was not due to any
vascular unloading.
The actions of extracellular nucleotides in cell signaling are
mediated by two classes of cell surface purinergic receptors:
P2X receptors are ligand-gated ion channels activated by
extracellular ATP, and P2Y receptors are G protein-coupled
receptors activated by both adenine and uracil nucleotides.1,2
In the heart, a variety of P2 receptors are expressed.3
Cardiac P2X receptors represent a novel and potentially
important therapeutic target for the treatment of heart failure.
A P2X receptor on the cardiomyocyte mediates cardioprotec-
tion and is activated by ATP or its potent analogue 2-MeSATP,
1 (Chart 1), as demonstrated using the calsequestrin (CSQa)
model of cardiomyopathy. Extracellular ATP can cause an
ionic current in mouse, rat, and guinea pig cardiac ventricular
myocytes.4-6 The P2X4 receptor is an important subunit of the
*To whom correspondence should be addressed. Phone: 301-496-
9024. Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov. Correspond-
ing author: Laboratory of Bioorganic Chemistry, National Institutes of
Diabetes and Digestive and Kidney Diseases, NIH, Bldg. 8A, Room
B1A-19, Bethesda, MD 20892-0810.
aAbbreviations: 50-AMP, adenosine 50-monophosphate; CSQ, calse-
questrin; DIBAL-H, diisobutylaluminium hydride; DMEM, Dulbec-
co’s modified Eagle medium; FS, fractional shortening; HEPES, N-2-
hydroxyethylpiperazine-N0-2-ethanesulfonic acid; HPLC, high perfor-
mance liquid chromatography; HRMS, high resolution mass spectros-
copy; LV, left ventricular; LVEDD, left ventricular end-diastolic
diameter; LVESD, left ventricular end-systolic diameter; NS, normal
saline; PLC, phospholipase C; SAR, structure-activity relationship;
TBAF, tetrabutylammonium fluoride; TBAP, tetrabutylammonium
dihydrogenphosphate; TBDPS-Cl, tert-butyl(chloro)diphenylsilane;
THF, tetrahydrofuran; TEAA, triethylammonium acetate.
r
pubs.acs.org/jmc
Published on Web 03/01/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2563
Chart 1. Representative Adenine Nucleotide Derivatives that
Table 1. Phosphonate Analogues: Structure and Effects on in Vivo
Heart Function as Determined by Echocardiography-Derived FS in
CSQ Heart Failure Mice
Display Activity at Various P2 Receptorsa
a Compound 3 served as the lead compound for the present set of
phosphonate analogues.
Activation of this myocyte P2X receptor leads to the open-
ing of a nonselective cation channel permeable to Naþ, Kþ,
and Ca2þ. The current is inward at negative membrane
potentials, reverses near 0 mV, and becomes outward at
positive potentials.4 The continuous activation of this recep-
tor channel under the resting or negative membrane potentials
would produce an inward current while its activation during
depolarized portions of the action potential should lead to an
outward current. These ionic currents represent a possible
ionic mechanism by which the cardiomyocyte P2X channel
achieves its protective effect.
We have explored the structure activity relationship (SAR)
of phosphonate analoguesof3 ina model of cardioprotection.
Although a (N)-methanocarba nucleoside 50-monophosphate
was shown to be a poor substrate of 50-nucleotidase (CD73),9
replacement of the phosphoester group of 3 with a phos-
phonate would be expected to further increase the in vivo
half-life because of the stability of the C-P bond. Phos-
phonate analogues of nucleotides and other known ligands,
in some cases, have been shown to display activity at P2
receptors.14-17
a At 3.3 μM. The vehicle control mice displayed a %FS of 13.78 (
1.19% (n = 16). ND: not determined.
Results
Chemical Synthesis. The phosphonate derivatives on var-
ied carbon skeletons (Table 1) were synthesized by the
methods shown in Schemes 1-6. In some cases, the phos-
phorus atom was bonded directly to the 50 carbon atom
(Schemes 1, 2), while in other cases, a carbon atom was added
at that position to form either a saturated or unsaturated
nucleotide analogue (Schemes 3-5). Alternately, a methyl-
phosphonate group was included in compounds 11 and 12,
which were otherwise equivalent to 4 and 5, respectively
(Scheme 6). The 2 position contained either hydrogen (in
compounds 5, 8, 10, 12) or Cl (as in the known active
compound 3 and the novel analogues 4, 7, 9, 11). The
reference compound 3 was synthesized by a modification
of the reported method,13 which led to improved yields
(Supporting Information).
iodo group at the 50 position, a classical two-step procedure
was implemented. This involved the initial activation of the
50-alcohol as a mesylate followed by an SN2 nucleophilic
attack of iodide on the activated 50-position, resulting in the
50-iodo compound 17 in 95% yield. The iodo compound 17
was subjected to classical Michaelis-Arbuzov reaction con-
ditions with excess triethylphosphite and heating up to 110 °C
for 17 h to provide a phosphonate diester 18 in excellent 94%
yield (Scheme 1). Desilylation using TBAF resulted in alcohol
19, which was a suitable substrate for Mitsunobu base coupling
reactions.35
The alcohol 19 was used as a common key intermediate to
synthesize phosphonates 4 and 5 (Scheme 2). Synthetic proce-
dures for phosphonates 4 and 5 involved initial Mitsunobu
base coupling reaction using triphenylphosphine, diisopropyl
azodicarboxylate, and the corresponding purine base followed
by amination at the 6 position of the purine ring using 2 M NH3
in isopropyl alcohol (Scheme 2). Finally, the simultaneous
deprotection of both the phosphonate diester and the acetonide
Known alcohol 1313 was protected as a O-tert-butyl-
dimethylsilyl ether using TBDPS-Cl, imidazole and DMAP
to get compound 14, followed by the reduction of the ethyl
ester using DIBAL-H in anhydrous THF, resulting in com-
pound 15 in very good yield (Scheme 1). To introduce an

2564 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Kumar et al.
Scheme 1a
a Reagents and conditions: (a) tert-butylchlorodiphenylsilane, imidazole, DMAP, anhyd CH2Cl2, 93%; (b) DIBAL-H, anhyd THF, 82%;
(c) methanesulfonyl chloride, triethylamine, anhyd CH2Cl2, 96%; (d) NaI, 65 °C, anhyd 1,4-dioxane, 95%; (e) triethylphosphite, 110 °C, 94%;
(f) TBAF, THF, 88%.
Scheme 2a
a Reagents and conditions: (a) triphenylphosphine, 2,6-dichloropurine, diisopropyl azodicarboxylate, anhyd THF, 75%; (b) 2 M NH3 in i-PrOH,
70 °C, 80% for 9, 79% for 11; (c) Iodotrimethylsilane, anhyd CH2Cl2, 23% for 4, and 27% for 5; (d) triphenylphosphine, 6-chloropurine, diisopropyl
azodicarboxylate, anhyd THF, 87%.
of 21 and 23 was achieved upon treatment with freshly opened
iodotrimethylsilane to get target phosphonates 4 and 5, respec-
tively (Scheme 2). Our efforts to use alternative relatively milder
reagents, bromotrimethylsilane and Dowex-50 ion-exchange
resin, resulted in partial deprotection (results not shown).
The synthetic routes to the elongated saturated and un-
saturated phosphonate derivatives 7-10 are shown in
Schemes 3-5. The saturated phosphonate 10 was synthe-
sized by oxidation of known alcohol 2413 to the 50-aldehyde
25 in 80% yield. It is noteworthy that not even a small
amount of decomposition of the aldehyde was observed
after storage at room temperature (rt) for several days. The
R,β-unsaturated alkyl phosphonate ester 26 was prepared
from aldehyde 25 in a Wittig-type reaction using tetraiso-
propyl methylenediphosphonate and sodium hydride in
anhydrous THF.22,23 The E-configuration of the resulting
alkene could be inferred from the large coupling constant
(3J = 17.1 Hz). Amination followed by hydrolysis of pho-
phonate diester and acetonide resulted in R,β-unsaturated
alkyl phosphonate 7 (Scheme 3).
Catalytic hydrogenation of 27 in the presence of H2 (3 bar),
palladium on carbon, and MeOH:2 M aq NaOH (1:1, v/v)24,25
resulted in the expected olefin reduction and dechlorination to
give the corresponding saturated phosphonate diester 28.
Compound 28 was converted to the long chain saturated
alkyl phosphonate 10 using the previously described iodo-
trimethylsilane deprotection reaction conditions. To our
surprise, our various efforts to synthesize 36 (Scheme 5)
from 27 by olefin reduction, running the reaction at atmos-
pheric pressure and using less weight percent of catalyst,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2565
Scheme 3a
a Reagents and conditions: (a) Dess-Martin periodinane, anhyd CH2Cl2, 80%; (b) tetraisopropyl methylenediphosphonate, NaH, anhyd THF, 83%;
(c) 2 M NH3 in i-PrOH, 70 °C, 80%; (d) 10% Pd/C, H2 (3 bar), MeOH:2M aq NaOH (1:1, v/v), 79%; (e) iodotrimethylsilane, anhyd CH2Cl2, 47% for 10,
and 28% for 7.
Scheme 4a
a Reagents and conditions: (a) Dess-Martin periodinane, anhyd CH2Cl2, 72%; (b) tetraisopropyl methylenediphosphonate, NaH, anhyd THF, 48%;
(c) TBAF, THF, 88%; (d) triphenylphosphine, 6-chloropurine, diisopropyl azodicarboxylate, anhyd THF, 85%; (e) 2 M NH3 in i-PrOH, 70 °C, 85%;
(f) iodotrimethylsilane, anhyd CH2Cl2, 78%.
either resulted in an incomplete reaction or generated an
inseparable mixture of dehalogenated and halogenated
products (results not shown).
Hence, to synthesize phosphonates 8 and 9, we decided
to install the phosphonate diester before the Mitsunobu
base coupling reaction, as described in Schemes 4 and 5.

2566 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Kumar et al.
Scheme 5a
a Reagents and conditions: (a) 10% Pd/C, H2 (3 bar), MeOH, 72%; (b) triphenylphosphine, 2,6-dichloropurine, diisopropyl azodicarboxylate, anhyd
THF, 40%; (c) 2 M NH3 in i-PrOH, 70 °C, 71%; (d) iodotrimethylsilane, anhyd CH2Cl2, 45% combined yield.
Scheme 6a
a Reagents and Conditions: (a) CBr4, triphenylphosphine, triethylamine, 81%; (b) diethylmethylphosphite, 110 °C, 95%; (c) TBAF, THF, 91%,
(d) triphenylphosphine, 2,6-dichloropurine, diisopropyl azodicarboxylate, anhyd THF, 75%; (e) 2 M NH3 in i-PrOH, 70 °C, 60%; (f) iodotrimethylsi-
lane, anhyd CH2Cl2, 20% combined yield.
The 50-alcohol of compound 15 was oxidized using Dess-
Martin periodinane reaction to get aldehyde 29. Similar to
the aldehyde 25, aldehyde 29 also displayed considerable
stability at rt. The R,β-unsaturated alkyl phosphonate
diester 30 was obtained using the previously described
Wittig-type reaction conditions. Desilylation under stan-
dard conditions resulted in the formation of alcohol 31,
which served as the key intermediate for the synthesis of
long chain unsaturated and saturated alkyl phosphonates 8
and 9, respectively.
the phophonate diester and acetonide resulted in formation
of long chain R,β-unsaturated alkyl phosphonate 8. Sequen-
tial catalytic hydrogenation of the resulting vinyl phospho-
nate diester 31 in the presence of palladium on carbon, a
Mitsunobu base coupling reaction, and amination provided
the long chain saturated phosphonate diester 36. Simulta-
neous deprotection of the phosphonate diester and the
acetonide using iodotrimethylsilane resulted in the desired
phosphonate 9 along with the formation of corresponding
dehalogenated product phosphonate 10.
A Mitsunobu base coupling reaction on compound 31
using triphenylphosphine, 6-chloropurine, and diisopropyl
azodicarboxylate followed by amination and hydrolysis of
The synthetic approach to methylphosphonates 11 and
12 is shown in Scheme 6. It involved initial 50-bromination
using CBr4 and treatment with triphenylphosphine and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2567
Figure 1. Beneficial effects of 2-Cl substituted phosphonate deri-
vatives of (N)-methanocarba AMP in heart failure mice. Various
derivatives of phosphonates were dissolved in sterile NS at 3.3 μM
and were infused subcutaneously individually via an Alzet mini-
pump in CSQ mice as described in General Methods. After 28 days
of infusion, the in vivo heart function was assessed using echocar-
diography-derived FS. (A) The 2-Cl substituted 50-phosphonate
derivative 4 was able to improve in vivo cardiac contractile perfor-
mance in heart failure mice as compared to normal saline-treated
heart failure animals (one-way ANOVA with posttest comparison,
P < 0.05) while the unsubstituted 5 was ineffective (P > 0.05). (B)
Similarly, 2-Cl substituted higher homologue 9 was able to enhance
cardiac contractile function (P < 0.05), while the parent unsub-
stituted 10 did not improve the contractile function (P > 0.05).
Data were mean ( SE.
Figure 2. Chronic infusion of compound 4 resulted in improved
echocardiographically derived FS in CSQ heart failure mice. Fol-
lowing chronic subcutaneous infusion of NS or compound 4, two-
dimensional directed M-mode echocardiography was carried out as
described in General Methods. The heart rate (HR) is indicated on
each figure. Representative M-mode echocardiography was shown
for a CSQ animal infused with NS (A) and for a CSQ mouse infused
with compound 4 (B). A heart from the NS-infused mice showed less
shortening of both septum and LV free wall than did compound
4-infused mice.
one OH group of the phosphonate has been replaced with
CH3. This reduces the overall charge on the molecule and is
intended to make it more bioavailable. Evidently, the
binding site of the receptor requires both oxygens for the
most favorable improvement in FS. Therefore, as summa-
rized in Figure 1, the most significant improvement in FS
was associated with the saturated homologues containing a
2-Cl substitution and an unmodified phosphonate group,
4 and 9.
triethylamine to result in 50-bromosugar 37 in 81% yield.
A subsequent Michaelis-Arbuzov reaction using diethyl
methylphosphite, followed by desilylation with TBAF,
gave the 50-methylphosphonate monoester 38 with 1-alcohol
39 as an inseparable mixture of diastereomers. A further
Mitsunobu base coupling reaction with 2,6-dichloropurine,
followed by amination and final deprotection, gave the
desired methylphosphonate 11 along with corresponding
dehalogenated methylphosphonate 12.
Biological Evaluation. Various phosphonate derivatives
were infused subcutaneously individually via an Alzet mini-
pump in CSQ mice. After 28 days of infusion, the in vivo
heart function was assessed using echocardiography-derived
fractional shortening (FS), which is the ratio of the change in
the diameter of the left ventricle between the contracted and
relaxed states. Thus, a lower percentage represents a decrease
in function. Two of the phosphonates, 4 and 9, were able to
cause an improved FS as compared to vehicle (Figure 1,
Table 1) and in comparison to the reference nucleotide 3.
Other analogues tested in this model (2-H analogues 5, 8, and
10, and 2-Cl analogues 7 and 11) had lower FS values.
Compounds 7, 8, and 10 were not protective at this dose,
i.e., FS in vehicle control-treated CSQ mice was similar to
that from mice treated chronically with these nucleotides.
Thus, in the saturated phosphonate series, the orientation of
the phosphorus relative to the methanocarba ring was some-
what structurally permissive, although inclusion of an olefin
in the spacer prevented the cardioprotective action. In 11,
An echocardiographic image of compound 4-treated versus
normal saline (NS)-infused CSQ hearts is shown in Figure 2.
An increased shortening of both the septum and left ventricular
(LV) free wall was evident in the heart from mice treated with 4
in comparison to that from vehicle (NS)-infused mice.
We also evaluated the ability of the nucleotide analogues
to activate the human P2Y1 receptor (Table 1). This receptor
is vasodilatory, and agonist action at this subtype would be
expected to be relevant to the observed cardiac effects.
Compound 3 was previously reported to activate phospho-
lipase C (PLC) mediated by the human P2Y1 receptor.9 The
phosphonate derivatives were tested in a FLIPR assay of
calcium flux induced in 1321N1 astrocytoma cells stably
expressing the human P2Y1 receptor. The known P2Y1 recep-
tor agonist 2-MeSADP induced a Ca2þ flux with an EC50 of
10.3 ( 0.4 nM (n = 3) in the transfected cells (Figure 3), but in
control 1321N1 astrocytoma cells, there was no change in
intracellular Ca2þ in response to 10 μM 2-MeSADP. At
concentrations up to 10 μM, the phosphonate analogues
4-11 produced no effect in the same assay. However, com-
pound 3 was active in this assay as an agonist, with an EC50 of
722 ( 55 nM (n = 3). The maximal effect of 3 was ∼80-90%
of that of the full agonist 2-MeSADP.

2568 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Kumar et al.
C has a common intermediate 26 for the synthesis of long
chain saturated and unsaturated 50-phosphonates 7-10, we
have decided to explore the synthesis of these phosphonates
by the shorter synthetic route C.
Biological Analysis. There is increasing evidence that
chronic activation of the native cardiac P2X receptor by
nucleotide analogues protects against the progression of
heart failure. The P2X4 receptor is an essential subunit of
this native receptor, but we do not know what other P2X
subtypes are present. The cardiac myoctye receptor is not
identical to the vascular P2X4 receptor, which has a key role
in the response of endothelial cells to changes in blood
flow.27
The synthetic nucleotide analogue 3 activates the native
cardiac P2X receptor, as indicated in electrophysiological
experiments with normal cardiac myocytes and those that
overexpress CSQ and based on its in vivo ability to improve
the heart failure phenotype of these animals. The rigid
carbocyclic ring system contained in this derivative stabilizes
nucleotides toward the action of nucleotidases. Therefore,
compound 3 is expected to be more stable than the corre-
sponding riboside. In the present study, we have synthesized
fully hydrolysis-resistant adenosine monophosphate deriva-
tives based on phosphonate linkages. The C-O-P bond of 3
was found to be stable over 24 h in aqueous medium at pH
1.5 to simulate the acidity of the stomach, however incuba-
tion at 37 °C in the presence of mammalian cell membranes
(1321N1 astrocytoma cells) resulted in considerable hydro-
lysis of the 50-phosphate of 3 (data not shown). Therefore, a
more stable structural alternate to the 50-phosphate linkage
was sought.
An in vivo screen of cardiac function was used to test the
novel analogues. Thus, the results of this chronic study likely
reflect both pharmacodynamic and pharmacokinetic fac-
tors. Several of the novel phosphonate analogues displayed
the same agonist activity as 3 in protecting the heart muscle
when chronically administered in the CSQ model. The SAR
analysis showed that considerable cadioprotection was
associated with specific structural features of the phosphonate
derivatives. The variation in the chain length and saturation
at the 50 carbon provided consistent results in the in vivo
screen. Two of the phosphonates, 4 and 9, both saturated
homologues containing a 2-Cl substitution, improved FS,
while the unsaturated phosphonates and 2-H analogues were
inactive. The most favorable FS (20.25%, compared to
13.78% in controls) was observed for (10S,20R,30S,40R,50S)-
40-(6-amino-2-chloropurin-9-yl)-20,30-(dihydroxy)-10-(phos-
phonomethylene)-bicyclo[3.1.0]hexane 4, which is the equi-
valent of 3 in which the 50-O has been excised. The higher
homologue 9 displayed a FS of 19.26%. Thus, it is possible to
extend the SAR around compound 3 for chronic activation
of the cardiac P2X receptor, leading to a beneficial effect in
heart failure.
The activity measured here does not reflect direct measure-
ments at a P2X receptor, and the likely association with this
mechanism is due to the close resemblance to compound 3,
which clearly activates a P2X4 receptor-dependent ion flux
that has been shown to protect in this model of cardiomyo-
pathy. It is not feasible to study the analogues at a recom-
binant homotrimeric P2X4 receptor system12 because the
endogenous cardiac P2X receptor is thought to be composed
of P2X4 receptor subunits in heteromeric association with a
yet unidentified P2X subtype. The P2X4 receptor is known
to associate with other P2X receptor subtypes, and these
Figure 3. Changes in intracellular calcium in 1321N1 human astro-
cytoma cells stably expressing the hP2Y1 receptor. Fluorescence
in response to a known hP2Y1 receptor agonist 2-MeSADP (EC50
10.3 ( 0.4 nM), compound 3 (EC50 722 ( 55 nM), or the phospho-
nate analogues (compounds 4-11, all inactive at 10 μM) was
quantified using a FLIPR-Tetra.
Discussion
Retrosynthetic Analysis. We set out to synthesize 50-phos-
phonate and 50-methyl phosphonate derivatives of (N)-
methanocarba-adenosine 5 and 12 or the corresponding
2-Cl derivatives 4 and 11 using the previously reported
intermediate 13.13 At least two synthetic pathways leading
to these target molecules can be envisioned viz routes A and
B (Scheme 7A) based on the installation of phosphonate
group. Route A involves phosphonate installation at the
nucleoside level to generate key intermediate 42, which could
be used as common intermediate for the generation of both
phosphonates and methyl phosphonates 43 using Michaelis-
Arbuzov reaction conditions. Route B involves phosphonate
installation at the sugar level on halogenated intermediates 17
and 37 to get intermediates 18 and 38, respectively. Route B
does not have a common intermediate, like route A, and as a
result, it involves a longer and more laborious synthetic
sequence. Generally, the Michaelis-Arbuzov reaction condi-
tions to generate phosphonate derivatives require long reac-
tion times (24-48 h) at elevated temperatures (120-180 °C).
Moreover, removal of trialkyl phosphite reagent needs high
temperatures and high vacuum. Because of these harsh con-
ditions, a Michaelis-Arbuzov reaction at the nucleoside level
generallyresultsinverypoor yields (lessthan25%yield) ofthe
desired phosphonates29 along with the formation of a dark-
colored thermal degraded products of the nucleosides. On the
other hand, Michaelis-Arbuzov reactions at the sugar level
generally result in very good yields.29 Hence, although route B
is time-consuming and contains more synthetic steps than
route A, we have decided to obtain these phosphonate
derivatives via route B, believing that it would be reliable with
good yields.
Long chain saturated and unsaturated phosphonates of
(N)-methanocarba adenine or 2-Cl adenine derivatives 7-10
could be achieved from the same starting compound 13 as for
the phosphonates 4, 5, 11, and 12. Similar to the synthesis of
phosphonates 4, 5, 11, and 12, these phosphonates could
possibly be obtained via two synthetic routes viz, routes C
and D (Scheme 7B), based on the installation of the phos-
phonate group. The long chain unsaturated phospho-
nate could be introduced by oxidation of the 50-alcohol of
either nucleoside 24 or compound 15 followed by the Wittig-
type reaction using tetraisopropyl methylenediphosphonate
and NaH to provide phosphonate diester 26 or 30, res-
pectively.22,23 One could expect this reaction to proceed
smoothly at both nucleoside and sugar stages. Because route

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2569
Scheme 7a
a (A) Retrosynthetic analysis of 50-phosphonate and 50-methyl phosphonates of (N)-methanocarba adenine or 2-Cl adenine derivatives. Use of route
B, in which the phosphonate was introduced prior to the nucleobase, proved successful. (B) Retrosynthetic analysis of saturated and unsaturated long
chain 50-phosphonates of (N)-methanocarba adenine or 2-Cl adenine derivatives. Use of the shorter synthetic route C, in which the nucleobase was
introduced prior to the phosphonate, proved successful.
heterotrimers, for example heteromers with P2X1 subunits,
are pharmacologically distinct from P2X4 homotrimers.26
More recently, the existence of P2X4/P2X6 and P2X4/P2X7
heteromers has also been described.31,34
The P2X4 receptor also is distributed widely through the
central and peripheral nervous systems, the epithelial layers
of ducted glands and airways, bladder smooth muscle,
gastrointestinal tract, uterus, and fat cells.32 Its activation
in spinal microglial cells participates in the pathogenesis of
chronic pain.33 Might the effects of overactivation of P2X4
receptors outside the myocardium complicate the use of such
a phosphonate derivative for treatment of cardiac failure
and cardiomyopathy? We have no evidence that these mono-
phosphate analogues activate a P2X4 receptor channel at any
other location. This is a topic for further investigation.
Another site of action of adenine nucleotides in cardiac
tissue is the metabotropic P2Y1 receptor, which causes a
nitric oxide-dependent relaxation of the vascular smooth
muscle. Therefore, we tested the nucleotides as P2Y1 recep-
tor agonists to account for the possibility that the observed
cadiovascular effects of the phosphonate derivatives were
a result of activation of an endothelial P2Y1 receptor.

2570 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Kumar et al.
Compound 3 was initially characterized in assays of PLC as a
weak hP2Y1 receptor agonist (EC50 1.89 μM)9 and that
conclusion is consistent with the potency obseved here in
inducing calcium transients in the same cell line. All of the
phosphonate derivatives tested were inactive at the P2Y1
receptor. This suggests the use of these compounds as more
selective pharmacological probes of the endogenous cardiac
P2X receptor than compound 3. However, it is worth noting
that the cardioprotection provided by 3 was shown to be
independent of the P2Y1 receptor by its inability to dilate aortic
rings and by use of a P2Y1-selective antagonist MRS2500. This
antagonist could not block the membrane current evoked by 3
under voltage clamp in mouse cardiac myocytes.8
In conclusion, we have greatly expanded the range of
carbocyclic nucleotide analogues that represent potential
candidates for the treatment of heart failure. A more chemi-
cally and biologically stable linkage than the phosphate
group in compound 3 has been introduced in the form of
the phosphonate groups,30 which in several cases preserve
heart contractile function in a genetic model of heart failure.
Facile routes for the synthesis of phosphonate analogues
of 3 in the conformationally constrained (N)-methanocarba
series were developed using Michaelis-Arbuzov and
Wittig reactions. A further advantage of the phosphonate
linkage is that the undesired activity as agonist of the P2Y1
receptor has been eliminated. The beneficial effects of these
nucleotidase-resistant agonists can now be explored in
additional models of cardiac failure and cardiomyopathy.
Purification of the nucelotide derivatives for biological test-
ing was performed by HPLC with a Luna 5 μ RP-C18(2)
semipreparative column (250 mm ꢀ 10.0 mm; Phenomenex,
Torrance, CA) under the following conditions: flow rate of
2 mL/min, 10 mM triethylammonium acetate (TEAA)-CH3CN
from 100:0 (v/v) to 70:30 (v/v) in 30 min and isolated in the
triethylammonium salt form. Analytical purity of compounds
was checked using a Hewlett-Packard 1100 HPLC equipped
with Zorbax SB-Aq 5 μm analytical column (50 mm ꢀ 4.6 mm;
Agilent Technologies Inc., Palo Alto, CA). Mobile phase:
linear gradient solvent system, 5 mM TBAP (tetrabutyl-
ammonium dihydrogenphosphate)-CH3CN from 80:20 to
40:60 in 13 min; the flow rate was 0.5 mL/min. Peaks were
detected by UV absorption with a diode array detector at
254, 275, and 280 nm. All derivatives tested for biological
activity showed >99% purity by HPLC analysis (detection
at 254 nm).
(10S,20R,30S,40R,50S)-40-(6-Amino-2-chloropurin-9-yl)-20,30-
(dihydroxy)-10-(phosphonomethylene)-bicyclo[3.1.0]hexane (4).
Nucleoside 21 (30 mg, 0.064 mmol) was coevaporated with
anhydrous toluene (3 ꢀ 3 mL) and dissolved in anhydrous
CH2Cl2 (3 mL). To this soution was added iodotrimethylsilane
(91 μL, 0.64 mmol). After stirring for 17 h, the reaction mixture
was cooled to 0 °C followed by the addition of ice-cold H2O
(25 mL) and CH2Cl2 (25 mL). The phases were separated, and the
aqueous phasewashed withCH2Cl2 (1 ꢀ 35mL) anddiethyl ether
(3 ꢀ 35 mL). The resulting aqueous phase evaporated to dryness
and purified by HPLC (retention time: 19.1 min) to afford 4
(8.5 mg, 23%) as a white solid material. ESI-HRMS m/z 374.0397
([M - H]-, C12H14ClN5O5P-: calcd 374.0421). 1H NMR (D2O)
δ 8.21 (s, 1H), 4.71 (s, 1H), 4.57 (d, 1H, J = 6.6 Hz), 4.01 (d, 1H,
J = 6.6 Hz), 3.19 (q, 24H, J = 7.2 Hz), 2.23 (t, 1H, 15.5 Hz),
1.63-1.77 (m, 2H), 1.42-1.49 (m, 1H), 1.26 (t, 36H), 0.96-1.04
(m, 1H). 31P NMR (D2O) δ 23.68. Purity >99% by HPLC
(retention time: 4.51 min).
(10S,20R,30S,40R,50S)-40-(6-Aminopurin-9-yl)-20,30-(dihydroxy)-
10-(phosphonomethylene)-bicyclo[3.1.0]hexane (5). Nucleoside 23
(25 mg, 0.057 mmol) was coevaporated with anhydrous toluene
(3 ꢀ 3 mL) and dissolved in anhydrous CH2Cl2 (3 mL). Iodo-
trimethylsilane (83 μL, 0.57 mmol) was added. After stirring for
15 h, the reaction mixture was cooled to 0 °C followed by the
addition of ice-cold H2O (25 mL) and CH2Cl2 (25 mL). The
phases were separated, and the aqueous phase was washed with
CH2Cl2 (1 ꢀ 35 mL) and diethyl ether (3 ꢀ 35 mL). The resulting
aqueous phase was evaporated to dryness and purified by HPLC
(retention time: 17.5 min) to afford 5 (6.8 mg, 27%) as a white solid
material. ESI-HRMS m/z 340.0817 ([M - H]-, C12H15N5O5P-:
calcd 340.0811). 1H NMR (D2O) δ 8.36 (s, 1H), 8.20 (s, 1H) 4.75 (s,
1H), 4.63 (d, 1H, J = 6.1 Hz), 4.09 (d, 1H, J = 6.1 Hz), 3.19 (q, 6H,
J = 7.2 Hz), 1.95-2.18 (m,2H), 1.75-1.84 (m, 1H), 1.40-1.46 (m,
1H), 1.26 (t, 9H, J=7.2 Hz), 0.92-1.02 (m, 1H). 31PNMR(D2O) δ
25.36. Purity >99% by HPLC (retention time: 2.9 min).
(10S,20R,30S,40R,50S)-40-(6-Amino-2-chloropurin-9-yl)-20,30-
(dihydroxy)-10-[(E)-phosphonoethenyl]-bicyclo[3.1.0]-hexane (7).
Nucleoside 27 (12 mg, 0.023 mmol) was coevaporated with
anhydrous toluene (3 ꢀ 2 mL) and dissolved in anhydrous
CH2Cl2 (2 mL). Iodotrimethylsilane (35 μL, 0.24 mmol) was
added. After stirring for 18 h, the reaction mixture was cooled to
0 °C, followed by the addition of ice-cold H2O (15 mL) and
CH2Cl2 (15 mL). The phases were separated, and the aqueous
phase was washed with CH2Cl2 (1 ꢀ 25 mL) and diethyl ether
(3 ꢀ 35 mL). The resulting aqueous phase was evaporated to
dryness and purified by HPLC (retention time: 22.8 min) to
afford 7 (2.5 mg, 28%) as a white solid material. ESI-HRMS m/z
386.0403 ([M - H]-, C13H14N5ClO5P-: calcd 386.0421). 1H
NMR (D2O) δ 7.99 (s, 1H), 6.21-6.36 (m, 1H), 6.06 (t, 1H, J =
17.5 Hz), 4.84-4.89 (m,1H), 4.06 (d, 1H, J = 6.6 Hz), 3.22 (q,
3H, J = 7.2 Hz), 1.99-2.06 (m,1H), 1.78-1.87 (m, 1H), 1.29 (t,
6H, J = 7.2 Hz), 1.21-1.26 (m, 1H). 31P NMR (D2O) δ 14.68.
Purity >99% by HPLC (retention time: 4.3 min).
Experimental Procedures
General Methods. Compound 13 was either synthesized as
reported13 or obtained as a custom synthesis from Natland
International Corporation (Research Triangle Park, NC). All
other reagents and solvents (regular and anhydrous) were of
analytical grade and obtained from commercial suppliers and
used without further purification. Reactions were conducted
under an atmosphere of nitrogen whenever anhydrous solvents
were used. All reactions were monitored by thin-layer chroma-
tography (TLC) using silica gel coated plates with a fluorescence
indicator which were visualized: (a) under UV light, (b) by
dipping in 5% conc H2SO4 in absolute ethanol (v/v) followed
by heating, or (c) by dipping in a solution of anisaldehyde:
H2SO4 (1:2, v/v) in MeOH followed by heating. Silica gel
column chromatography was performed with silica gel (SiO2,
˚
200-400 mesh, 60 A) using moderate air pressure. Evaporation
of solvents was carried out under reduced pressure at a tem-
perature below 50 °C. After column chromatography, appro-
priate fractions were pooled, evaporated, and dried at high
vacuum for at least 12 h to give the obtained products in high
purity. 1H NMR and 31P NMR ascertained sample purity. No
corrections in yields were made for solvent of crystallization.
1H NMR and 31P NMR spectra were recorded at 300 and
121.5 MHz, respectively. Chemical shifts are reported in parts
per million (ppm) relative to tetramethylsilane or deuterated
solvent as the internal standard (dH: CDCl3 7.26 ppm). For
compounds 38-41, the integral of the H30-signal of the least
predominant isomer was set to 1.0. Systematic compound
names for bicyclic nucleosides are given according to the von
Baeyer nomenclature.21 High resolution mass spectroscopic
(HRMS) measurements were performed on a proteomics opti-
mized Q-TOF-2 (Micromass-Waters) using external calibration
with polyalanine. Observed mass accuracies are those expected
on the basis of known performance of the instrument as well as
the trends in masses of standard compounds observed at inter-
vals during the series of measurements. Reported masses are
observed masses uncorrected for this time-dependent drift in
mass accuracy.

Article
(10S,20R,30S,40R,50S)-40-(6-Aminopurin-9-yl)-20,30-(dihydroxy)-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2571
Imidazole (0.69 g, 10.20 mmol), DMAP (0.04 g, 0.34 mmol), and
tert-butylchlorodiphenylsilane (1.74 mL, 6.81 mmol) were added.
After stirring at rt for 16 h, the reaction mixture was diluted with
CH2Cl2 (50 mL) and washed with satd aq NaHCO3 (1 ꢀ 30 mL).
The aqueous phase was back-extracted with CH2Cl2 (2 ꢀ 50 mL).
The combined organic phase was evaporated to dryness, and the
resulting crude residue was purified by silica gel column chroma-
tography (0-8% EtOAc in petroleum ether, v/v) to afford
compound 14 (1.52 mg, 93%) as a colorless oil. Rf = 0.3 (10%
EtOAc in CH2Cl2, v/v). ESI-HRMS m/z 519.1981 ([M þ K]þ,
10-[(E)-phosphonoethenyl]-bicyclo[3.1.0]hexane (8). Nucleoside 33
(20 mg, 0.042 mmol) was coevaporated with anhydrous toluene
(3 ꢀ 5 mL) and dissolved in anhydrous CH2Cl2 (5 mL). Iodo-
trimethylsilane (60 μL, 0.42 mmol) was added. After stirring for
17 h, the reaction mixture was cooled to 0 °C, followed by the
addition of ice-cold H2O (25 mL) and CH2Cl2 (25 mL). The
phases were separated and the aqueous phase was washed with
CH2Cl2 (1 ꢀ 35 mL) and diethyl ether (3 ꢀ 35 mL). The resulting
aqueous phase was evaporated to dryness and purified by HPLC
(retention time: 16.5 min) to afford 8 (11.8 mg, 78%) as a white solid
C28H36O5Si Kþ: calcd 519.1969). 1H NMR (CDCl3) δ 7.69-7.80
3
material. ESI-HRMS m/z 352.0821 ([M - H]-, C13H15N5O5P þ
:
(m, 4H, Ph), 7.32-7.45 (m, 6H, Ph), 5.11 (d, 1H, J = 6.6 Hz), 4.42
(t, 1H, J = 6.1 Hz), 3.99-4.18 (m, 3H), 2.17-2.25 (m, 1H), 1.91 (t,
1H, J = 5.5 Hz), 1.57 (s, 3H), 1.45-1.53 (m, 1H), 1.18-1.24 (m,
6H), 1.07 (s, 9H).
1
calcd 352.0811). H NMR (D2O) δ 8.30 (s, 1H), 8.06 (s, 1H),
6.30-6.44 (m, 1H), 6.07 (t, 1H, J = 17.5), 4.97 (s, 1H), 4.89 (d, 1H,
J = 7.2 Hz), 4.09 (d, 1H, J = 7.2 Hz), 3.21 (q, 3H, J = 7.2 Hz),
2.03-2.10 (m, 2H), 1.84-1.89 (m, 1H), 1.29 (t, 7H, J = 7.2 Hz).
31P NMR (D2O) δ 15.71. Purity >99% by HPLC (retention time:
3.5 min).
(1S,2R,3S,4S,5S)-1-Hydroxymethyl-2,3-O-(isopropylidene)-
4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane (15). Com-
pound 14 (0.98 g, 2.04 mmol) was coevaporated with anhy-
drous toluene (2 ꢀ 20 mL), dissolved in anhydrous THF
(30 mL), and cooled to -70 °C. DIBAL-H (1.5 M in toluene
10.8 mL, 16.32 mmol) was added slowly to this solution over
20 min. After stirring at -70 °C for 3 h, the reaction was
quenched with the very careful addition of ice-cold MeOH
(20 mL), followed by warming the reaction mixture to rt. Then
1 M cold H2SO4 (20 mL) was added to the mixture and it was
stirred for 1 h, followed by addition of CH2Cl2 (100 mL). The
phases were separated, and the aqueous phase was extracted
with CH2Cl2 (3 ꢀ 35 mL). The combined organic phase was
evaporated to dryness, and the resulting residue was purified by
silica gel column chromatography (0-45% EtOAc in petro-
leum ether, v/v) to afford compound 15 (0.74 g, 82%) as a
colorless oil. Rf = 0.4 (50% EtOAc in petroleum ether, v/v).
(10S,20R,30S,40R,50S)-40-(6-Aminopurin-9-yl)-20,30-(dihydroxy)-
10-(phosphonoethenyl)-bicyclo[3.1.0]hexane (10). Nucleoside 28
(15 mg, 0.032 mmol) was coevaporated with anhydrous toluene
(3 ꢀ 2 mL) and dissolved in anhydrous CH2Cl2 (2 mL). Iodo-
trimethylsilane (45 μL, 0.32 mmol) was added. After stirring for
15 h, the reaction mixture was cooled to 0 °C followed by the
addition of ice-cold H2O (15 mL) and CH2Cl2 (15 mL). The
phases were separated, and the aqueous phase was washed with
CH2Cl2 (1 ꢀ 25 mL) and diethyl ether (3 ꢀ 35 mL). The resulting
aqueous phase was evaporated to dryness and purified by HPLC
(retention time: 16.6 min) to afford 10 (6.7 mg, 47%) as a white solid
material. ESI-HRMS m/z 354.0970 ([M - H]-, C13H17N5O5P-:
1
calcd 354.0967). H NMR (CDCl3) δ 8.20 (s, 1H), 8.14 (s, 1H),
4.76 (s, 1H), 4.58 (d, 1H, J = 6.1 Hz), 4.09 (d, 1H, J = 6.1 Hz),
3.21 (q, 3H, J = 7.2 Hz), 1.69-2.14 (m, 4H), 1.59-1.69 (m, 1H),
1.39-1.349 (m, 1H), 1.29 (t, 6H, J = 7.2 Hz), 0.81-0.92 (m, 1H).
31P NMR (D2O) δ 27.95. Purity >99% by HPLC (retention time:
2.91 min).
ESI-HRMS m/z 461.2112 ([M þ Na]þ, C26H34O4Si Naþ: calcd
3
461.2124). 1H NMR (CDCl3) δ 7.71-7.78 (m, 4H, Ph),
7.31-7.44 (m, 6H, Ph), 4.73 (d, 1H, J = 6.5 Hz), 4.44 (t, 1H,
J = 6.5 Hz), 4.09 (t, 1H, J = 6.5 Hz), 3.59-3.67 (m, 1H),
3.41-3.49 (m, 1H), 1.57-1.59 (m, 4H), 1.33 (t,1H, J = 5.5 Hz),
1.20 (s, 3H), 1.07 (s, 9H), 0.56-0.68 (m, 1H).
(10S,20R,30S,40R,50S)-40-(6-Amino-2-chloropurin-9-yl)-20,30-
dihydroxy-10-(methylphosphonicacid)-bicyclo[3.1.0]hexane (11)
and (10S,20R,30S,40R,50S)-40-(6-Aminopurin-9-yl)-20,30-dihydroxy-
10-(methylphosphonicacid)-bicyclo[3.1.0]hexane (12). Nucleoside
29 (15 mg, 0.034 mmol) was coevaporated with anhydrous
toluene (3 ꢀ 3 mL) and dissolved in anhydrous CH2Cl2 (4 mL).
Iodotrimethylsilane (91 μL, 0.33 mmol) was added. After stirring
for 19 h, the reaction mixture was cooled to 0 °C, followed by the
addition of ice-cold H2O (25 mL) and CH2Cl2 (25 mL). The
phases were separated and the aqueous phase was washed with
CH2Cl2 (1 ꢀ 35 mL) and diethyl ether (3 ꢀ 35 mL). The resulting
aqueous phase was evaporated to dryness and purified by HPLC
(retention time: 16.8 min) to afford 11 (1.3 mg, 11%) and 12
(0.8 mg, 20%, combined yield) as a white solid material.
Analytical Data of Compound 11. ESI-HRMS m/z 372.0625
[M - H]-, C13H16ClN5O4P-: calcd 372.0628. 1H NMR (D2O) δ
8.23 (s, 1H), 4.76-4.79 (m, 1H), 4.63 (d, 1H, J = 6.2 Hz), 4.12
(d, 1H, J = 6.2 Hz), 3.21 (q, 2H, J = 7.2 Hz), 2.34 (t, 1H, J =
15.3 Hz), 1.75-1.86 (m, 1H), 1.68-1.75 (m, 1H), 1.51-1.56 (m,
1H) 1.35 (d, 3H, J = 13.2 Hz), 1.26 (t, 1H, J = 7.2 Hz)
0.96-1.04 (m, 1H). 31P NMR (D2O) δ 46.01. Purity >99% by
HPLC (retention time: 4.19 min).
(1S,2R,3S,4S,5S)-2,3-O-(Isopropylidene)-1-methanesulfonyl-
oxymethyl-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane (16).
Compound 15 (0.59 g, 1.36 mmol) was coevaporated with anhy-
drous toluene (2 ꢀ 20 mL), dissolved in anhydrous CH2Cl2
(30 mL), and cooled to 0 °C. Triethylamine (0.95 mL, 6.79 mmol)
and methanesulfonyl chloride (0.22 mL, 2.72 mol) were added at
0 °C over 10 min. After warming the reaction mixture to rt, it was
stirred for 17 h. Then, ice-cold H2O (25 mL) was added, and the
mixture was extracted with EtOAc (2 ꢀ 45 mL). The combined
organic phase was washed with satd aq NaHCO3 (2 ꢀ 35 mL) and
evaporated to dryness. The resulting residue was purified by silica
gel column chromatography (0-50% EtOAc in petroleum ether,
v/v) to afford compound 16 (0.68 g, 96%) as a colorless oil.
Rf = 0.5 (50% EtOAc in petroleum ether, v/v). ESI-HRMS m/z
555.1623 ([M þ K]þ, C27H36O6SSi Kþ: calcd 555.1639). 1H
3
NMR (CDCl3) δ 7.69-7.77 (m, 4H, Ph), 7.31-7.45 (m, 6H,
Ph), 4.69 (d, 1H, J = 6.5 Hz), 4.47 (t, 1H, J = 6.5 Hz), 4.37-4.43
(dd, 1H, J = 10.9 Hz), 4.07 (t, 1H, J = 6.5 Hz), 3.90-3.95 (dd,
1H, J = 10.9 Hz), 2.99 (s, 3H), 1.67-1.72 (m, 2H), 1.55 (s, 3H),
1.20 (s, 3H), 1.08 (s, 9H), 0.72-0.79 (m, 1H).
Analytical Data of Compound 12. ESI-HRMS m/z 338.1016
[M - H]-, C13H17N5O4P-: calcd 338.1018. 1H NMR (D2O) δ
8.26 (s, 1H), 8.25 (s, 1H), 4.76-4.79 (m, 1H), 4.63 (d, 1H, J = 6.2
Hz), 4.12 (d, 1H, J = 6.2 Hz), 3.21 (q, 2H, J = 7.2 Hz), 2.34 (t,
1H, J = 15.3 Hz), 1.75-1.86 (m, 1H), 1.68-1.75 (m, 1H),
1.51-1.56 (m, 1H) 1.35 (d, 3H, J = 13.2 Hz), 1.26 (t, 1H, J = 7.2
Hz) 0.96-1.04 (m, 1H). 31P NMR (D2O) δ 46.0. Purity >99%
by HPLC (retention time: 5.91 min).
Ethyl-(1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-O-(tert-butyl-
dimethylsilyl)-bicyclo[3.1.0]hexanecarboxylate (14). Known alco-
hol 13 (0.83 g, 3.40 mmol) was coevaporated with anhydrous
toluene (2 ꢀ 10 mL) and dissolved in anhydrous CH2Cl2 (25 mL).
(1S,2R,3S,4S,5S)-1-Iodomethyl-2,3-O-(isopropylidene)-4-
O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane (17). Com-
pound 16 (0.68 g, 1.32 mmol) was coevaporated with anhy-
drous toluene (2 ꢀ 20 mL), and the residue dissolved in
anhydrous 1,4-dioxane (25 mL). NaI (0.59 g, 3.94 mol) was
added to the mixture, and it was heated to 65 °C. After
stirring for 17 h, the reaction mixture was cooled to rt and
diluted with H2O (25 mL) and CH2Cl2 (75 mL). The phases
were separated, and the aqueous phase was extracted with
CH2Cl2 (3 ꢀ 35 mL). The combined organic phase was
evaporated to dryness, and the resulting residue was purified
by silica gel column chromatography (0-20% EtOAc in

2572 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Kumar et al.
petroleum ether, v/v) to afford compound 17 (0.69 g, 95%) as
a colorless oil. Rf = 0.5 (20% EtOAc in petroleum ether, v/v).
Diethyl-(10S,20R,30S,40R,50S)-40-(6-chloropurin-9-yl)-20,30-
O-(isopropylidene)-bicyclo[3.1.0]hexane Phosphonate (22).
Diisopropyl azodicarboxylate (100 μL, 0.50 mmol) was
added at rt to a mixture of triphenylphosphine (133 mg,
0.50 mmol) and 6-chloropurine (96 mg, 0.50 mmol) in anhy-
drous THF (3 mL). After stirring the mixture for 30 min, a
solution of compound 19 (81 mg, 0.26 mmol) in THF (3 mL)
was added. After stirring for 17 h, the reaction mixture was
evaporated to dryness. The resulting residue was purified by
silica gel column chromatography (0-4% MeOH in EtOAc, v/v)
to afford nucleoside 22 (100 mg, 87%) as a white solid material.
Rf = 0.5 (5% MeOH in EtOAc, v/v). ESI-HRMS m/z 457.1417
ESI-HRMS m/z 549.1322 ([M þ H]þ, C26H33IO3Si Hþ: calcd
3
549.1322). 1H NMR (CDCl3) δ 7.68-7.77 (m, 4H, Ph), 7.32-
7.46 (m, 6H, Ph), 4.69 (d, 1H, J = 6.5 Hz), 4.43 (t, 1H, J = 6.5 Hz),
4.09 (t, 1H, J = 6.5 Hz), 3.55-3.60 (dd, 1H, J = 10.5 Hz),
3.97-4.02 (dd, 1H, J = 10.5 Hz), 2.02 (t, 1H, J = 4.9 Hz),
1.54-1.57 (s, 4H), 1.20 (s, 3H), 1.07 (s, 9H), 0.83-0.90 (m, 1H).
Diethyl-(1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-O-(tert-
butyldimethylsilyl)-bicyclo[3.1.0]hexane Phosphonate (18). Com-
pound 17 (0.68 g, 1.23 mmol) was dissolved in triethylphosphite
(17 mL), and the mixture was heated to 110 °C. After stirring for
17 h, the reaction mixture was cooled to rt and evaporated to
dryness. The resulting residue was purified by silica gel column
chromatography (0-90% EtOAc in petroleum ether, v/v) to afford
compound 18 (0.65 g, 94%) as a colorless oil. Rf = 0.3 (EtOAc).
([M þ H]þ, C19H26ClN4O5P Hþ: calcd 457.1408). 1H NMR
3
(CDCl3) δ 8.84 (s, 1H), 8.78 (s, 1H), 5.39 (d, 1H, J = 6.5 Hz),
5.15 (s, 1H), 4.62 (d, 1H, J = 6.5 Hz), 4.07-4.21 (m, 4H), 2.44
(t, 1H, J = 16.5 Hz), 1.94-2.18 (m,1H), 1.83-1.90 (m, 1H), 1.58
(s, 3H), 1.33 (t, 3H, J = 7.2 Hz), 1.24-1.30 (m, 4H), 0.97-1.06
(m, 1H).
ESI-HRMS m/z 559.2665 ([M þ H]þ, C30H43O6PSi Hþ: calcd
3
559.2645). 1H NMR (CDCl3) δ 7.71-7.77 (m, 4H, Ph), 7.30-7.43
(m, 6H, Ph), 4.80 (d, 1H, J = 6.5 Hz), 4.47 (t, 1H, J = 6.5 Hz), 4.10
(t, 1H, J = 6.5 Hz), 3.91-4.05 (m, 4H), 2.22 (t, 1H, J = 16.5 Hz),
1.63-1.71 (m,1H), 1.57-1.61 (m, 2H), 1.55 (s, 3H), 1.22 (t, 3H, J=
7.2 Hz), 1.20 (t, 3H, J = 7.2 Hz), 1.19 (s, 3H), 1.07 (s, 9H), 0.53-
0.60 (m, 1H). 31P NMR (CDCl3) δ 29.93.
Diethyl-(10S,20R,30S,40R,50S)-40-(6-aminopurin-9-yl)-20,30-
O-(isopropylidene)-bicyclo[3.1.0]hexane Phosphonate (23).
Nucleoside 22 (100 mg, 0.22 mmol) was treated with 2 M
NH3 in i-PrOH (5 mL) and heated up to 70 °C. After stirring
for 19 h, the reaction mixture was evaporated to dryness. The
resulting residue was purified by silica gel column chroma-
tography (0-6% MeOH in CH2Cl2, v/v) to afford nucleoside
23 (75 mg, 79%) as a white solid material. Rf = 0.4 (5%
MeOH in CH2Cl2, v/v). ESI-HRMS m/z 438.1912 ([M þ H]þ,
Diethyl-(1S,2R,3S,4S,5S)-4-hydroxy-2,3-O-(isopropylidene)-
bicyclo[3.1.0]hexane Phosphonate (19). Compound 18 (0.65 g,
1.16 mmol) was dissolved in a mixture of THF (20 mL) and
tetrabutylammonium fluoride (1 M in THF, 2.91 mL, 2.91
mmol). After stirring for 17 h, the reaction mixture was
evaporated to dryness. The resulting residue was purified by
silica gel column chromatography (0-7% MeOH in EtOAc,
v/v) to afford compound 19 (0.33 g, 88%) as a colorless oil. Rf =
0.3 (5% MeOH in EtOAc, v/v). ESI-HRMS m/z 321.1466 ([M þ
C19H28N5O5P Hþ: calcd 438.1906). 1H NMR (CDCl3)
3
δ 8.38 (s, 1H), 8.36 (s, 1H), 5.54 (s, 2H), 5.36 (d, 1H, J =
7.2 Hz), 5.03 (s, 1H), 4.63 (d, 1H, J = 7.2 Hz), 4.06-4.20 (m,
4H), 2.38 (t, 1H, J = 16.5 Hz), 1.97-2.11 (m, 1H), 1.78-1.85
(m,1H), 1.68 (s, 3H), 1.32 (t, 3H, J = 7.2 Hz), 1.27 (t, 3H, J =
7.2 Hz), 1.23 (s, 3H),1.18-1.21 (m, 1H), 0.95-1.02 (m, 1H).
(10S,20R,30S,40R,50S)-40-(2,6-Dichloropurin-9-yl)-10-formyl-
2,3-O-(isopropylidine)-bicyclo[3.1.0]hexane (25). Known nucleo-
side13 24 (150 mg, 0.41 mmol) was coevaporated with anhydrous
toluene (2 ꢀ 8 mL) and dissolved in anhydrous CH2Cl2 (8 mL).
Dess-Martin periodinane (257 mg, 0.61 mmol) was added. After
stirring for 1 h, the reaction mixture was diluted with EtOAc
(50 mL) and washed with an aqueous mixture of Na2S2O3 and
NaHCO3 (3 ꢀ 35 mL). The aqueous phase was then extracted
with EtOAc (2 ꢀ 35 mL). The combined organic phase was
evaporated to dryness, and the resulting residue was purified by
silica gel column chromatography (0-100% EtOAc in petroleum
ether, v/v) to afford compound 25 (120 mg, 80%) as a white
solid material. Rf = 0.6 (EtOAc). ESI-HRMS m/z 369.0527
H]þ, C14H25O6P Hþ: calcd 321.1467). 1H NMR (CDCl3) δ 5.02
3
(d, 1H, J = 6.1 Hz), 4.50-4.58 (m, 2H), 4.02-4.17 (m, 4H),
2.32-2.37 (m, 1H), 2.26 (t, 1H, J = 16.5 Hz), 1.88-1.96 (m,1H),
1.61-1.74 (m, 1H), 1.54 (s, 3H), 1.32 (t, 6H, J = 7.2 Hz), 1.28 (s,
3H),1.21-1.27 (m, 1H), 0.60-0.67 (m, 1H). 31P NMR (CDCl3)
δ 29.01.
Diethyl-(10S,20R,30S,40R,50S)-40-(2,6-dichloropurin-9-yl)-20,30-
O-(isopropylidene)-bicyclo[3.1.0]hexane Phosphonate (20). Di-
isopropyl azodicarboxylate (97 μL, 0.49 mmol) was added at
rt to a mixture of triphenylphosphine (128 mg, 0.49 mmol) and
2,6-dichloropurine (92 mg, 0.49 mmol) in anhydrous THF
(3 mL). After stirring for 30 min, a solution of compound 19
(78 mg, 0.25 mmol) in THF (3 mL) was added to the mixture.
After stirring for 51 h, the reaction mixture was evaporated
to dryness. The resulting residue was purified by silica gel
column chromatography (0-4% MeOH in EtOAc, v/v) to
afford nucleoside 20 (90 mg, 75%) as a white solid material.
Rf = 0.5 (5% MeOH in EtOAc, v/v). ESI-HRMS m/z 491.1013
([M þ H]þ, C15H14Cl2N4O3 Hþ: calcd 369.0521). 1H NMR
3
(CDCl3) δ 9.62 (s, 1H), 8.05 (s, 1H), 5.94 (d, 1H, J = 7.2 Hz),
4.97 (s, 1H), 4.83 (d, 1H, J = 7.2 Hz), 2.22-2.29 (m, 1H), 1.73
(t, 1H, J = 6.1 Hz), 1.57 (s, 3H), 1.30 (s, 3H).
1
([M þ H]þ, C19H25Cl2N4O5P Hþ: calcd 491.1018). H NMR
(10S,20R,30S,40R,50S)-40-(2,6-Dichloropurin-9-yl)-10-[diisopropyl-
(E)-ethenylphosphonate]-20,30-O-(isopropylidine)-bicyclo[3.1.0]hexane
(26). Tetraisopropyl methylenediphosphonate (165 μL, 0.51 mmol)
was added to a suspension of NaH (60% dispersion in mineral oil,
25 mg, 1.02 mmol) in anhydrous THF (2 mL) at 0 °C. After H2
evolution ceased, a solution of aldehyde 25 (125 mg, 0.34 mmol) in
anhydrous THF (3 mL) was added dropwise carefully at 0 °C. After
stirring at 0 °C for 1 h, the mixture was warmed to rt. After stirring at
rt for 1 h, the reaction mixture was cooled to 0 °C, and ice-cold H2O
(20 mL) was added. The phases were separated, and the aqueous
phase was extracted with EtOAc (3 ꢀ 35 mL). The combined organic
phase was evaporated to dryness, and the resulting residue was
purified by silica gel column chromatography (0-4% MeOH in
EtOAc, v/v) to afford nucleoside 26 (150 mg, 83%) as a white solid
material. Rf = 0.3 (EtOAc). ESI-HRMS m/z 531.1313 ([M þ H]þ,
3
(CDCl3) δ 8.82 (s, 1H), 5.39 (d, 1H, J = 6.5 Hz), 5.10 (s, 1H),
4.61 (d, 1H, J = 6.5 Hz), 4.02-4.21 (m, 4H), 2.46 (t, 1H, J = 16.5
Hz), 1.91-2.06 (m,1H), 1.74-1.82 (m, 1H), 1.54 (s, 3H), 1.32 (t,
3H, J = 7.2 Hz), 1.26 (t, 3H, J = 7.2 Hz), 1.24 (s, 3H),1.08-1.21
(m, 1H), 0.97-1.06 (m, 1H).
Diethyl-(10S,20R,30S,40R,50S)-40-(6-amino-2-chloropurin-9-yl)-
20,30-O-(isopropylidene)-bicyclo[3.1.0]hexane Phosphonate (21).
Nucleoside 20 (90 mg, 0.19 mmol) was treated with 2 M NH3
in i-PrOH (5 mL), and the mixture was heated to 70 °C and
stirred for 17 h. The reaction mixture was evaporated to dryness,
and the resulting residue was purified by silica gel column chroma-
tography (0-5% MeOH in CH2Cl2, v/v) to afford nucleoside 21 (70
mg, 80%) as a white solid material. Rf =0.5(5%MeOHinCH2Cl2,
v/v). ESI-HRMS m/z 472.1519 ([M þ H]þ, C19H27ClN5O5P Hþ:
3
C22H29Cl2N4O5P Hþ: calcd 531.1331). 1H NMR (CDCl3) δ 8.04 (s,
calcd 472.1517). 1H NMR (CDCl3) δ 8.31 (s, 1H), 5.98 (s, 2H), 5.36
(d, 1H, J = 7.1 Hz), 4.97 (s, 1H), 4.61 (d, 1H, J = 6.5 Hz),
4.03-4.19 (m, 4H), 2.39 (t, 1H, J = 16.5 Hz), 2.03-2.17 (m, 1H),
1.70-1.77 (m, 1H), 1.52 (s, 3H), 1.32 (t, 3H, J= 7.2 Hz), 1.25 (t, 3H,
J = 7.2 Hz), 1.23 (s, 3H), 1.18-1.21 (m, 1H), 0.96-1.04 (m, 1H).
3
1H), 6.50-6.65 (m, 1H), 5.97 (t, 1H, J = 17.1 Hz), 5.53 (d, 1H, J =
7.2 Hz), 4.98 (s, 1H), 4.77 (d, 1H, J = 7.2 Hz), 4.60-4.74 (m, 2H),
1.82-1.90 (m, 1H), 1.59 (s, 3H), 1.22-1.38 (m, 16H), 0.83-0.90
(m, 1H). 31P NMR (CDCl3) δ 16.64.

Article
(10S,20R,30S,40R,50S)-40-(6-Amino-2-chloropurin-9-yl)-10-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2573
(t, 1H, J = 6.5 Hz), 4.11 (t, 1H, J = 6.5 Hz), 1.93-1.99 (m, 1H),
1.78-1.85 (m, 1H), 1.57 (s, 3H), 1.19-1.32 (m, 15H), 1.07 (s, 9H),
0.93-1.01 (m, 1H).
[diisopropyl-(E)-ethenylphosphonate]-2,3-O-(isopropylidine)-
bicyclo-[3.1.0]-hexane (27). Nucleoside 26 (100 mg, 0.19 mmol)
was treated with 2 M NH3 in i-PrOH (5 mL) and heated to 70 °C.
After stirring for 16 h, the reaction mixture was evaporated to
dryness. The resulting residue was purified by silica gel column
chromatography (0-8% MeOH in CH2Cl2, v/v) to afford nu-
cleoside 27 (85 mg, 88%) as a white solid material. Rf = 0.3 (5%
MeoH in EtOAc, v/v). ESI-HRMS m/z 512.1821 ([M þ H]þ,
(1S,2R,3S,4S,5S)-1-[Diisopropyl-(E)-phosphonoethenyl]-4-
(hydroxy)-2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane (31).
Compound 30 (0.45 g, 0.76 mmol) was dissolved in THF
(10 mL), and tetrabutylammonium fluoride (1.0 M in THF,
2.3 mL, 2.3 mmol) was added. After stirring for 13 h, the
reaction mixture was evaporated to dryness. The resulting
residue was purified by silica gel column chromatography
(0-7% MeOH in EtOAc, v/v) to afford compound 31 (0.26 g,
98%) as a colorless oil. Rf = 0.3 (EtOAc). ESI-HRMS m/z
C22H31ClN5O5P Hþ: calcd 512.1830). 1H NMR (CDCl3) δ 7.69
3
(s, 1H), 6.52-6.68 (m, 1H), 5.94 (t, 1H, J = 17.5 Hz), 5.75 (s, 2H),
5.51 (d, 1H, J = 7.2 Hz), 4.91 (s, 1H), 4.76 (d, 1H, J = 7.2 Hz),
4.59-4.73 (m, 2H), 1.80-1.89 (m, 1H), 1.61 (s, 3H), 1.21-1.37
(m, 15H), 1.08-1.17 (m, 1H), 0.77-0.95 (m, 1H).
361.1790 ([M þ H]þ, C17H29O6P Hþ: calcd 361.1780). 1H
3
NMR (CDCl3) δ 6.34-6.49 (m, 1H), 5.74 (t, 1H, J = 17.5 Hz),
4.99 (d, 1H, J = 6.5 Hz), 4.47-4.69 (m, 4H), 2.40 (d, 1H, J =
9.5 Hz), 2.07-2.15 (M, 1H), 1.59-1.63 (m, 1H), 1.58 (s, 3H),
1.22-1.34 (m, 15H), 0.93-1.07 (m, 1H).
(10S,20R,30S,40R,50S)-40-(6-Aminopurin-9-yl)-10-(diisopropyl-
phosphonoethenyl)-20,30-O-(isopropylidine)-bicyclo[3.1.0]hexane
(28). Nucleoside 27 (20 mg, 0.04 mmol) was dissolved in a
mixture of MeOH and aqueous 2 M NaOH (3 mL, 2:1, v/v).
Then 10% Pd/C (20 mg) and H2 (3 bar) were added to this
solution. After stirring the mixture for 19 h, the catalyst was
removed by filtration through a celite pad, which was washed
with MeOH (40 mL), and the filtrate was evaporated to dryness.
The resulting residue was purified by silica gel column chroma-
tography (0-10% MeOH in EtOAc, v/v) to afford nucleoside
28 (15 mg, 79%) as white solid material. Rf = 0.5 (15% MeOH
in EtOAc, v/v). ESI-HRMS m/z 480.2385 ([M þ H]þ, C22H34-
(10S,20R,30S,40R,50S)-40-(6-Chloropurin-9-yl)-10-[diisopropyl-
(E)-phosphonoethenyl]-20,30-O-(isopropylidene)-bicyclo[3.1.0]hexane
(32). Diisopropyl azodicarboxylate (90 μL, 0.45 mmol) was added
at rt to a mixture of triphenylphosphine (117 mg, 0.45 mmol) and
6-chloropurine (70 mg, 0.45 mmol) in anhydrous THF (5 mL).
After stirring for 30 min, a solution of the compound 31 (80 mg,
0.23 mmol) in THF (5 mL) was added. After stirring for 60 h, the
reaction mixture was evaporated to dryness. The resulting residue
was purified by silica gel column chromatography (0-55% acetone
in petroleum ether, v/v) to afford nucleoside 32 (92 mg, 85%) as
a white solid material. Rf = 0.4 (60% acetone in petroleum ether,
N5O5P Hþ: calcd 480.2376). 1H NMR (CDCl3) δ 8.32 (s, 1H),
3
7.79 (s, 1H), 5.80 (s, 2H), 5.19 (d, 1H, J = 7.2 Hz), 4.83 (s, 1H),
4.74 (d, 1H, J = 7.2 Hz), 4.63-4.73 (m, 2H), 1.60-2.35 (m, 4H),
1.52 (s, 3H), 1.44-1.51 (m, 1H), 1.33 (s, 6H), 1.31 (s, 6H), 1.23
(s, 3H), 1.04-1.09 (m, 1H), 0.76-0.83 (m, 1H). 31P NMR
(CDCl3) 30.04.
v/v). ESI-HRMS m/z519.1532 ([M þ Na]þ,C22H30ClN4O5P Naþ:
3
1
calcd 519.1540). H NMR (CDCl3) δ 8.71 (s, 1H), 8.07 (s, 1H),
6.49-6.63 (m, 1H), 5.96 (t, 1H, J = 17.5), 5.53 (d, 1H, J = 6.5 Hz),
5.02 (s, 1H), 4.79 (d, 1H, J=6.5Hz), 4.61-4.73(m, 2H), 1.86-1.92
(m, 1H), 1.58-1.63 (m, 1H), 1.54 (s, 3H), 1.22-1.38 (m, 16H).
(10S,20R,30S,40R,50S)-40-(6-Aminopurin-9-yl)-10-[diisopropyl-
(E)-phosphonoethenyl]-20,30-O-(isopropylidene)-bicyclo[3.1.0]hexane
(33). Nucleoside 32 (90 mg, 0.19 mmol) was treated with 2 M NH3
in i-PrOH (7 mL) and heated to 70 °C. After stirring for 17 h, the
reaction mixture was evaporated to dryness. The resulting residue
was purified by silica gel column chromatography (0-12% MeOH
in CH2Cl2, v/v) to afford nucleoside 33 (74 mg, 85%) as a white
solid material. Rf = 0.2 (10% MeOH in EtOAc, v/v). ESI-HRMS
(1S,2R,3S,4S,5S)-1-Formyl-2,3-O-(isopropylidene)-4-O-(tert-
butyldimethylsilyl)-bicyclo[3.1.0]hexane (29). Compound 15
(0.63 g, 1.43 mmol) was coevaporated with anhydrous toluene
(2 ꢀ 25 mL) and dissolved in anhydrous CH2Cl2 (25 mL).
Dess-Martin periodinane (0.91 g, 2.13 mmol) was added to
this solution. After stirring for 4 h, the reaction mixture was
diluted with EtOAc (50 mL) and washed with an aqueous
mixture of Na2S2O3 and NaHCO3 (3 ꢀ 50 mL). The aqueous
phase was extracted with EtOAc (2 ꢀ 50 mL). The combined
organic phase was evaporated to dryness, and the resulting
residue was purified by silica gel column chromatography
(0-25% EtOAc in petroleum ether, v/v) to afford aldehyde 29
(452 mg, 73%) as a colorless oil. Rf = 0.6 (50% EtOAc in
petroleum ether, v/v). ESI-HRMS m/z 459.1986 ([M þ Na]þ,
m/z 478.2198 ([M þ H]þ, C22H32N5O5P Hþ: calcd 478.2219). 1H
3
NMR (CDCl3) δ 8.30 (s, 1H), 7.73 (s, 1H), 6.51-6.66 (m, 1H), 5.96
(t, 1H, J = 17.5), 5.47-5.54 (m, 3H), 4.95 (s, 1H), 4.78 (d, 1H, J =
6.5 Hz), 4.60-4.72(m, 2H), 1.86-1.94 (m, 1H), 1.53-1.57(m, 4H),
1.24-1.37 (m, 16H).
C26H32O4Si Naþ: calcd 459.1968). 1H NMR (CDCl3) δ 8.92 (s,
(1S,2R,3S,4S,5S)-1-(Diisopropyl-phosphonoethenyl)-4-(hydroxy)-
2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane (34). Compound 31
(30 mg, 0.083 mmol) was dissolved in MeOH (3 mL), and 10%
Pd/C (25 mg) and H2 (3 bar) was added. After stirring the
mixture for 17 h, the catalyst was removed by filtration through
a celite pad, which was washed with MeOH (40 mL), and the
filtrate was evaporated to dryness. The resulting residue was
purified by silica gel column chromatography (0-90% acetone in
petroleum ether, v/v) to afford nucleoside 34 (22 mg, 72%) as white
solid material. Rf = 0.3 (5% MeOH in EtOAc, v/v). ESI-HRMS
3
1H), 7.68-7.78 (m, 4H, Ph), 7.31-7.48 (m, 6H, Ph), 5.13 (d, 1H,
J = 6.5 Hz), 4.41 (t, 1H, J = 6.5 Hz), 4.16 (t, 1H, J = 6.5 Hz),
2.19-2.28 (m, 1H), 2.10-2.18 (m, 1H), 1.55 (s, 3H), 1.43-1.51
(m, 1H), 1.23 (s, 3H), 1.09 (s, 9H).
(1S,2R,3S,4S,5S)-1-[Diisopropyl-(E)-phosphonoethenyl]-2,3-
O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane
(30). Tetraisopropyl methylenediphosphonate (475 μL, 1.47 mmol)
was added to a suspension of sodium hydride (71 mg, 2.95 mmol,
60% dispersion in mineral oil) in anhydrous THF (6 mL) at 0 °C.
After H2 evolution ceased, a solution of aldehyde 29 (0.43 g,
0.98 mmol) in anhydrous THF (4 mL) was added dropwise carefully
at 0 °C. After stirring at 0 °C for 1 h, the mixture was warmed to rt.
After stirring at rt for 1 h, the mixture was cooled to 0 °C, and ice-
cold H2O (20 mL) was added. The phases were separated, and the
aqueous phase was extracted with EtOAc (3 ꢀ 35 mL). The com-
bined organic phase was evaporated to dryness, and the resulting
residue was purified by silica gel column chromatography (0-70%
EtOAc in petroleum ether, v/v) to afford nucleoside 30 (0.24 mg,
48%) as a white solid material. Rf = 0.4 (70% EtOAc in petroleum
1
m/z 363.1933 ([M þ H]þ, C17H31O6P Hþ: calcd 363.1937). H
3
NMR (CDCl3) δ 4.61-4.76 (m, 2H), 4.43-4.54 (m, 2H),
4.17-4.35 (m, 1H), 2.32 (d, J = 9.8 Hz, 1H), 1.43-1.93 (m, 8H),
1.22-1.37 (m, 15H), 1.07-1.14 (m,1H), 0.47-0.56 (m, 1H).
(10S,20R,30S,40R,50S)-40-(2,6-Dichloropurin-9-yl)-10-(diisopropyl-
phosphonoethenyl)-20,30-O-(isopropylidene)-bicyclo[3.1.0]hexane
(35). Diisopropyl azodicarboxylate (93 μL, 0.47 mmol) was added
at rt to a mixture of triphenylphosphine (123 mg, 0.47 mmol) and
2,6-dichloropurine (89 mg, 0.47 mmol) in anhydrous THF (4 mL).
After stirring for 30 min, a solution of the compound 34 (85 mg,
0.24 mmol) in THF (4 mL) was added. After stirring for 65 h, the
reaction mixture was evaporated to dryness. The resulting residue
was purified by silica gel column chromatography (0-5% MeOH
in EtOAc, v/v) to afford nucleoside 35 (50 mg, 40%) as a white
ether, v/v). ESI-HRMS m/z599.2938 ([M þ H]þ,C33H47O6PSi Hþ:
3
calcd 599.2958). 1H NMR (CDCl3) δ 7.69-7.77 (m, 4H,
Ph), 7.31-7.45 (m, 6H, Ph), 6.24-6.40 (m, 1H), 5.66 (t, 1H,
J = 17.5 Hz), 4.75 (d, 1H, J = 6.5 Hz), 4.52-4.64 (m, 2H) 4.42

2574 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Kumar et al.
solid material. Rf = 0.4 (5% MeOH in EtOAc, v/v). ESI-HRMS
(CDCl3) δ7.68-7.77 (m, 6.8H, Ph), 7.29-7.46 (m, 10.2H, Ph), 4.75
(d, 0.7H, J = 6.5 Hz), 4.68 (d, 1H, J = 6.5 Hz), 4.43-4.49 (m,
1.7H), 3.87-4.14 (m, 5.1 H), 1.73-1.92 (m, 3.4H), 1.62 (s, 5.1H),
1.57-1.60 (m,1.7H), 1.48 (d, 3H, J = 3.4 Hz), 1.44 (d, 2.1H, J =
3.4 Hz), 1.26 (t, 3H, J = 7.2 Hz), 1.22 (t, 2.1H, J = 7.2 Hz), 1.19 (s,
2.1H), 1.18 (s, 3H), 1.09-1.13 (m, 1.7H), 1.07 (s, 15.3H), 0.52-0.69
(m, 1H).
m/z 533.1497 ([M þ H]þ, C22H31Cl2N4O5P Hþ: calcd 533.1487).
3
1H NMR (CDCl3) δ 8.09 (s, 1H), 5.20 (d, 1H, J = 7.2 Hz), 4.86 (s,
1H), 4.73 (d, 1H, J = 7.2 Hz), 4.63-4.73 (m, 2H), 2.25-2.44 (m,
1H), 1.79-2.09 (m, 2H), 1.58-1.70 (m, 1H), 1.52 (s, 3H),
1.43-1.52 (m, 1H), 1.28-1.35 (m, 12H), 1.24 (s, 3H), 1.04-1.11
(m,1H), 0.78-0.87 (m, 1H).
(10S,20R,30S,40R,50S)-40-(6-Amino-2-chloropurin-9-yl)-10-(diiso-
propyl-phosphonoethenyl)-20,30-O-(isopropylidene)-bicyclo[3.1.0]-
hexane (36). Nucleoside 35 (50 mg, 0.094 mmol) was treated with
2 M NH3 in i-PrOH (5 mL) and heated to 70 °C. After stirring for
19 h, the reaction mixture was evaporated to dryness. The
resulting residue was purified by silica gel column chromato-
graphy (0-10% MeOH in CH2Cl2, v/v) to afford nucleoside 36
(34 mg, 71%) as a white solid material. Rf = 0.4 (8% MeOH in
EtOAc, v/v). ESI-HRMS m/z 514.1978 ([M þ H]þ, C22H33ClN5-
(1S,2R,3S,4S,5S)-1-C-(Ethoxymethylphosphinyl)-4-hydroxy-
2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane (39). Compound 38
(0.30 g, 0.57 mmol) was dissolved in THF (10 mL) and tetra-
butylammonium fluoride (1.0 M in THF, 1.70 mL, 1.70 mmol)
was added. After stirring for 21 h, the reaction mixture was
evaporated to dryness. The resulting residue was purified by silica
gel column chromatography (0-15% MeOH in EtOAc, v/v) to
afford an inseparable diastereomeric mixture of compound 39
(0.15 g, 91%) as a colorless oil. Rf = 0.2 (15% MeOH in CH2Cl2,
v/v). ESI-HRMSm/z 291.1366([M þ H]þ, C13H23O5P Hþ: calcd
O5P Hþ: calcd 514.1986). 1H NMR (CDCl3) δ 7.73 (s, 1H), 5.84
3
3
1
291.1361). H NMR (CDCl3) δ 4.89-5.03 (m, 2H), 4.50-4.60
(s, 2H), 5.20 (d, 1H, J = 6.5 Hz), 4.76 (s, 1H), 4.72 (d, 1H, J =
6.5 Hz), 4.62-4.71 (m, 2H), 2.24-2.40 (m, 1H), 1.75-2.08 (m,
1H), 1.56-1.74 (m, 5H), 1.40-1.47(m, 1H), 1.28-1.35 (m, 12H),
1.24 (s, 3H), 1.01-1.06 (m,1H), 0.74-0.82 (m, 1H).
(m, 4H), 3.97-4.14 (m, 4H), 2.34-2.40 (m, 2H), 1.95 (t, 2H, J =
7.7 Hz), 1.90 (t, 2H, J = 7.7 Hz),1.78-1.87 (m, 2H), 1.66 (s, 6H),
1.55 (d, 3H, J = 3.9 Hz), 1.50 (d, 3H, J = 3.9 Hz), 1.29-1.35 (m,
6H), 1.28 (s, 6H), 1.23-1.26 (m, 2H), 0.60-0.71 (m, 2H).
(10S,20R,30S,40R,50S)-4-(6-Amino-2-chloropurin-9-yl)-20,30-
(dihydroxy)-10-(phosphonoethenyl)-bicyclo[3.1.0]hexane (9).
Nucleoside 23 (25 mg, 0.049 mmol) was coevaporated with
anhydrous toluene (3 ꢀ 4 mL) and dissolved in anhydrous
CH2Cl2 (4 mL). Iodotrimethylsilane (70 μL, 0.49 mmol) was
added. After stirring for 19 h, the reaction mixture was
cooled to 0 °C followed by the addition of ice-cold H2O
(25 mL) and CH2Cl2 (25 mL). The phases were separated,
and the aqueous phase was washed with CH2Cl2 (1 ꢀ 35 mL)
and diethyl ether (3 ꢀ 35 mL). The resulting aqueous phase
was evaporated to dryness and purified by HPLC (retention
time: 21.5 min) to afford 9 (8.5 mg) and 10 (1.3 mg, 53%,
combined yield) as a white solid materials. ESI-HRMS m/z
388.0574 ([M - H]-, C13H16ClN5O5P-: calcd 388.0578). 1H NMR
(D2O) δ 8.15 (s, 1H), 4.74 (s, 1H), 4.61 (d, 1H, J = 7.2 Hz), 4.11 (d,
1H, J = 7.2 Hz), 3.21 (q, 3H, J = 7.2 Hz), 1.70-2.11 (m, 4H),
1.63-1.71 (m, 1H), 1.33-1.38 (m, 1H), 1.29 (t, 3H, J = 7.2 Hz),
0.81-0.91 (m, 1H). 31P NMR (D2O) δ 28.26. Purity >99% by
HPLC (retention time: 4.6 min).
(10S,20R,30S,40R,50S)-10-C-(Ethoxymethylphosphinyl)-40-(2,6-
dichloropurin-9-yl)-20,30-O-(isopropylidene)-bicyclo[3.1.0]hexane
(40). Diisopropyl azodicarboxylate (360 μL, 1.82 mmol) was
added at rt to a mixture of triphenylphosphine (0.48 g, 1.82
mmol) and 2,6-dichloropurine (0.35 g, 1.82 mmol) in anhydrous
THF (5 mL). After stirring for 30 min, a solution of the
compound 39 (0.27 g, 0.91 mmol) in THF (5 mL) was added.
After stirring for 60 h, the reaction mixture was evaporated to
dryness. The resulting residue was purified by silica gel column
chromatography (0-10% MeOH in EtOAc, v/v) to afford in-
separable diastereomeric mixture of nucleoside 40 (0.25 mg, 60%)
as a white solid material. Rf = 0.2 (10% MeOH in EtOAc, v/v).
ESI-HRMS m/z 461.0899 ([M þ H]þ, C18H23Cl2N4O4P Hþ:
3
calcd 461.0912). 1H NMR (CDCl3) δ 8.79 (s, 0.5H), 8.51 (s,
1H), 5.45 (d, 0.5H, J = 7.2 Hz), 5.32 (d, 1H, J = 7.2 Hz), 5.06 (s,
0.5H), 4.96 (s, 1H), 4.66 (d, 1.5H, J = 7.2 Hz), 3.98-4.21 (m, 3H),
2.40-2.51 (m, 0.5H), 2.15-2.24 (m, 1H), 1.93-2.11 (m, 1.5H),
1.63-1.75 (m, 0.5H), 1.61 (s, 5.5H), 1.59 (d, 3H, J = 3.9 Hz), 1.55
(d, 1.5H, J = 3.9 Hz), 1.35 (t, 3H, J = 7.2 Hz), 1.25 (t, 1.5H, J =
7.2 Hz), 1.24 (s, 4.5H), 1.18-1.23 (m, 1.5H), 0.95-1.13 (m, 1.5H).
(10S,20R,30S,40R,50S)-40-(6-Amino-2-chloropurin-9-yl)-10-
C-(ethoxymethylphosphinyl)-20,30-O-(isopropylidene)-bicyclo[3.1.0]-
hexane (41).Nucleoside 40 (0.20 g, 0.44 mmol) was treated with 2 M
NH3 in i-PrOH (8 mL) and heated to 70 °C. After stirring for 15 h,
the reaction mixture was evaporated to dryness. The resulting
residue was purified by silica gel column chromatography (0-7%
MeOH in CH2Cl2, v/v) to afford nucleoside 41 (150 mg, 79%) as a
white solid material. Rf = 0.4 (10% MeOH in CH2Cl2, v/v). ESI-
(1S,2R,3S,4S,5S)-1-Bromomethyl-2,3-O-(isopropylidene)-4-
O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane (37). Compound
15 (0.30 g, 0.69 mmol) was coevaporated with anhydrous toluene
(3 ꢀ 10 mL) and dissolved in anhydrous CH2Cl2 (8 mL). CBr4
(0.46 g, 1.36 mmol), triphenylphosphine (0.36 g, 1.36 mmol), and
triethylamine (0.3 mL, 2.07 mmol) were added. After stirring for
17 h, the reaction mixture was diluted with CH2Cl2 (50 mL) and
satd aq NaCl (25 mL). The phases were separated and aqueous
phase was extracted with CH2Cl2 (3 ꢀ 25 mL). The combined
organic phase was evaporated to dryness, and the resulting
residue was purified by silica gel column chromatography
(0-20% EtOAc in petroleum ether, v/v) to afford compound
37 (0.28 g, 81%) as a colorless oil. Rf = 0.8 (50% EtOAc in
petroleum ether, v/v). ESI-HRMS m/z 523.1296 ([M þ Na]þ,
HRMS m/z 442.1416 ([M þ H]þ, C18H26ClN5O4P Hþ: calcd
3
1
442.1411). H NMR (CDCl3) δ 8.21 (s, 0.5H), 8.00 (s, 1H), 5.96
(s, 3H), 5.40 (d, 0.5H, J = 6.5 Hz), 5.30 (d, 1H, J = 6.5 Hz), 4.91 (s,
0.5H), 4.81 (s, 1H), 4.62-4.71 (d, 1.5H, J = 7.2 Hz), 4.10-4.22 (m,
2H), 3.98-4.09 (m, 1H), 3.60-3.80 (m, 1H), 2.64 (t, 1H, J = 15.3
Hz), 2.35 (t, 0.5H, J = 15.3 Hz), 2.01-2.17 (m, 0.5H), 1.87 (t, 1.5H,
J = 15.8 Hz), 1.75 (s, 4.5H), 1.55-1.69 (m, 4.5H), 1.36 (t, 3H, J =
7.2 Hz), 1.25 (t, 1.5H, J = 7.2 Hz), 1.24 (s, 4.5H), 1.17-1.22 (m,
1.5H), 0.96-1.07 (m, 1.5H).
C26H33BrO3Si Naþ: calcd 523.1280). 1H NMR (CDCl3) δ
3
7.67.77 (m, 4H, Ph), 7.30-7.45 (m, 6H, Ph), 4.77 (d, 1H, J =
6.5 Hz), 4.44 (t, 1H, J = 6.5 Hz), 4.08 (t, 1H, J = 6.5 Hz), 3.76 (d,
1H, J = 10.5 Hz), 3.13 (d, 1H, J = 10.5 Hz), 1.80-1.87 (m, 1H),
1.60-1.70 (m, 1H), 1.55 (s, 3H), 1.21 (s, 3H), 1.05 (s, 9H),
0.71-0.86 (m, 1H).
Biological Evaluation. CSQ Mice and Compound Administra-
tion. Mice displaying the CSQ model of severe cardiomyopathy
and heart failure were bred and maintained according to a
previously described method.7 The CSQ transgenic (TG) mice
were originally provided by Dr. Larry Jones10,11 and developed
hypertrophy followed by a lethal heart failure phenotype with
death near the age of 3 months.
Compound 3 and its analogues were dissolved in phosphate-
buffered saline, pH = 7.4 at 3.3 μM (200 μL total volume),
filtered for sterility for in vivo administration at 6 μL per day for
28 days via a mini-osmotic pump (Alzet) in the CSQ mice. Intact
(1S,2R,3S,4S,5S)-1-C-(Ethoxymethylphosphinyl)-2,3-O-(iso-
propylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane (38).
Compound 37 (0.28 g, 0.56 mmol) was dissolved in diethylmethyl-
phosphite (4 mL) and heated up to 110 °C. After stirring for 17 h,
the reaction mixture was cooled to rt and evaporated to dryness.
The resulting residue was purified by silica gel column chromato-
graphy (0-90% EtOAc in petroleum ether, v/v) to afford insepar-
able diastereomeric mixture of compound 38 (0.28 g, 95%) as a
colorless oil. Rf = 0.6 (5% MeOH in EtOAc, v/v). ESI-HRMSm/z
529.2532 ([M þ H]þ, C29H41O5PSi Hþ: calcd 529.2539). 1H NMR
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2575
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Mouse Echocardiography. Transthoracic echocardiography
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described methods.18,19 Two dimensional-targeted M-mode echo-
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Activation of Human P2Y1 Receptors. Activity at the hP2Y1
receptor was quantified in 1321N1 human astrocytoma cells
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calcium-4 assay kit (Molecular Devices, Sunnyvale, CA) was
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40 mL of dye with probenecid in each well and incubated for 1 h
at rt. The compound plate was prepared with dilutions of
various compounds in Hank’s Buffer at pH 7.2. Samples were
run in duplicate with a FLIPR-Tetra (Molecular Devices) at rt.
Cell fluorescence (excitation = 485 nm; emission = 525 nm)
was monitored following exposure to a compound. Increases in
intracellular calcium are reported as the maximum fluorescence
value after exposure minus the basal fluorescence value before
exposure.
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Synthesis of ethyl (1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-
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tile chiral synthon for preparation of adenosine and P2 receptor
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5079.
Data Analysis. Unless otherwise indicated, data were pro-
vided as mean ( standard error of the mean. For analysis of
multiple groups, one-way ANOVA and posttest comparison
were used. Student’s t test for paired or unpaired samples
was used to evaluate the effects of experimental interventions;
P < 0.05 was taken as statistically significant.
(15) Cosyn, L.; Van Calenbergh, S.; Joshi, B. V.; Ko, H.; Carter, R. L.;
Harden, T. K.; Jacobson, K. A. Synthesis and P2Y receptor activity
of nucleotide 50-phosphonate derivatives. Bioorg. Med. Chem. Lett.
2009, 19, 3002–3005.
(16) Xu, B.; Stephens, A.; Kirschenheuter, G.; Greslin, A. F.; Cheng,
X.; Sennelo, J.; Cattaneo, M.; Zighetti, M. L.; Chen, A.; Kim, S. A.;
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Acknowledgment. Mass spectral measurements were carried
out by Dr. John Lloyd and Dr. Noel Whittaker (NIDDK). We
thank Dr. T. Kendall Harden (University of North Carolina,
Chapel Hill, NC) for the gift of tranfected 1321N1 astrocyoma
cells and Dr. William Trenkle (NIDDK) for helpful discussion.
This research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and Diges-
tive and Kidney Diseases. This work was supported in part by
RO1-HL48225 and a Ray Neag Distinguished Professorship to
Bruce T. Liang.
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deling after myocardial infarction in mice. Circulation 2005, 111,
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Supporting Information Available: Updated synthetic methods
for compound 3 and NMR spectral data and HPLC traces of
selected nucleotide derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
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