2-Substitution of Adenine Nucleotide Analogues containing a Bicyclo[3.1.0]hexane Ring System Locked in a Northern Conformation: Enhanced Potency as P2Y1 Receptor Antagonists

Article abstract of DOI:10.1021/jm030127+

Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of P2Y ₁ receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose su

Full text of DOI:10.1021/jm030127+

4974
J . Med. Chem. 2003, 46, 4974-4987
2-Su bstitu tion of Ad en in e Nu cleotid e An a logu es Con ta in in g a
Bicyclo[3.1.0]h exa n e Rin g System Lock ed in a Nor th er n Con for m a tion :
En h a n ced P oten cy a s P 2Y1 Recep tor An ta gon ists
Hak Sung Kim,†,‡ Michihiro Ohno,† Bin Xu,† Hea Ok Kim,§ Yongseok Choi,| Xiao D. J i,† Savitri Maddileti,∧
Victor E. Marquez,| T. Kendall Harden,∧ and Kenneth A. J acobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of
Health and Human Services, Bethesda, Maryland 20892-0810, Division of Chemistry and Molecular Engineering, Seoul
National University, Seoul 151-742, Korea, Laboratory of Medicinal Chemistry, Center for Cancer Research, NCIsFrederick,
National Institutes of Health, Frederick, Maryland 21702, and Department of Pharmacology, School of Medicine,
University of North Carolina, Chapel Hill, North Carolina 27599-7365
Received March 18, 2003
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide
3′,5′-bisphosphate antagonists of P2Y1 receptors was established by using a ring-constrained
methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J . Med.
Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba
modification with other functionalities at the 2-position of the adenine moiety. A new synthetic
route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation
of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly
synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-
stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the
recombinant human P2Y1 receptor and by using the radiolabeled antagonist [3H]2-chloro-N6-
methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5 in a newly developed binding
assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule
at the hP2Y1 receptor was an (N)-methanocarba N6-methyl-2-iodo analogue 12, which displayed
a Ki value in competition for binding of [3H]5 of 0.79 nM and a KB value of 1.74 nM for inhibition
of PLC. Thus, 12 is the most potent antagonist selective for the P2Y1 receptor yet reported.
The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route
for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-
2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less
well, exhibiting micromolar affinity at P2Y1 receptors. An enzymatic method of synthesis of
the 3′,5′-bisphosphate from the corresponding 3′-monophosphate, suitable for the preparation
of a radiophosphorylated analogue, was explored.
In tr od u ction
The P2Y1 receptor is distributed in the heart, skeletal,
and various smooth muscles and the prostate, ovary,
brain, and circulating blood cells.8 This receptor was
first cloned from chick brain;9 its regional brain distri-
bution and presence in glial cells have been de-
scribed,10-12 and abnormalities in P2Y1 receptor levels
may exist in the brains of persons with Alzheimer’s
disease.13 Activation of this receptor by ADP mediates
relaxation in the guinea pig aorta.14 The P2Y1 receptor
plays a major physiological role in platelet aggrega-
tion.15
P2Y receptor ligands are being explored for thera-
peutic applications in the cardiovascular, endocrine, and
other systems. The P2Y1 and P2Y12 receptors are the
furthest advanced in the P2Y family with respect to
selective agonists and antagonists.16-26 3′,5′-Bisphos-
phate nucleotides have been developed as selective
antagonists of the P2Y1 receptor, and high receptor
affinities have been achieved in this series.20 The 2′-
deoxyribose moiety of widely used P2Y1 receptor an-
tagonists such as N6-methyl-2′-deoxyadenosine 3′,5′-
bisphosphate 1 may be replaced with carbocyclics,
smaller and larger rings, acyclics, and conformationally
constrained rings,17-19 resulting in retention or enhance-
Two families of P2 nucleotide receptors exist:
G
protein-coupled P2Y receptors, of which eight mam-
malian subtypes (P2Y1,2,4,6,11-14) are currently sequence-
defined, and P2X ligand-gated cation channels, of which
seven mammalian subtypes (P2X1-7) have been cloned.1-4
Adenine nucleotides are required for activation of the
P2Y1, P2Y11, P2Y12, and P2Y13 subtypes, and uracil
nucleotides activate P2Y2, P2Y4, and P2Y6, subtypes.
The P2Y2 receptor is equipotently activated by UTP and
ATP. The most recently identified subtypes are the
P2Y13 receptor, which is present in the immune system,5
and the P2Y14 receptor, which is activated by UDP-
glucose.6 There is evidence that multiple P2Y receptors
may be involved in the activation of eosinophils and
neutrophils.7
* Corresponding author. Tel: (301) 496-9024. Fax: (301) 480-8422.
E-mail: kajacobs@helix.nih.gov.
† NIDDK, National Institutes of Health.
‡ Present address: College of Pharmacy and Medicinal Resources
Research Center, Wonkwang University, Iksan, 570-749, Chonbuk,
South Korea.
§ Seoul National University.
| NCI-Frederick, National Institutes of Health.
∧ University of North Carolina.
10.1021/jm030127+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/14/2003

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4975
Ta ble 1. In Vitro Pharmacological Data at Human P2Y1
Receptors for Inhibition of PLC Elicited by 30 nM 2-MeSADP
(receptor expressed in 1321N astrocytoma cells) and in Binding
(receptor expressed in Sf9 cells)31 d
ment of affinity for the receptor. A 2-chloro group was
found to enhance potency and selectivity in the case of
nucleotide 2 and the acyclic bisphosphate analogue
3.19,27 An acyclic bisphosphonate analogue 4 was also
shown to be a P2Y1 receptor antagonist.19
binding,
antagonism,
IC50, nMb
compd
R
R′
Ki, nMa
7
8
9
I
H
H
12.6 ( 0.3
863 ( 400
17.6 ( 2.7
45 ( 21
87 ( 1
367 ( 194
157 ( 60
356 ( 122
52 ( 1
SCH3
H
F
Cl
Br
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
10
5c
2.5 ( 0.4
5.1 ( 0.6
0.78 ( 0.08
91 ( 12
11
12c
13
14
15
16
17
37 ( 6
I
8.4 ( 0.8
221 ( 30
377 ( 154
49 ( 1
452 ( 221
1870 ( 590
SCH3
SeCH3
CH3
(CH2)5CH3
trans-CHdCH-
(CH2)3CH3
35 ( 4
3.6 ( 0.7
80 ( 10
330 ( 70
18
CtC-(CH2)3CH3
CH3
430 ( 200
2400 ( 600
a
The affinities were determined by using [3H]5 in a radioligand
binding assay, as recently described.31 The human P2Y1 receptor
was expressed to high levels in Sf9 insect cells with a recombinant
baculovirus. Membranes prepared from these cells were incubated
b
for 30 min at 4 °C in the presence of ∼20 nM [3H]5. Antagonist
IC50 values (mean ( standard error) represent the concentration
needed to inhibit by 50% the effect elicited by 30 nM 2-MeSADP.
The percent of maximal inhibition is equal to 100 minus the
residual fraction of stimulation at the highest antagonist concen-
tration. N ) 3, unless otherwise indicated in parentheses. c 5, MRS
d
2279; 12, MRS 2500. Mean ( SEM given for three separate
determinations. None of the compounds displayed agonist effects.
R and R′ are defined in Scheme 1.
The most recently introduced P2Y1 receptor antago-
nists contain novel substitutions of the ribose moiety
or of the adenine ring system. In solution, the conforma-
tion of the ribose ring of nucleosides and nucleotides
exists in a rapid, dynamic two-state equilibrium be-
tween a (N) (northern, 2′-exo) or (S) (southern, 2′-endo)
conformation, although their target receptors probably
prefer specific conformations. We have replaced the
ribose moiety for P2Y1 antagonists with a carbocyclic
ring locked in a preferred conformation, a fusion of
cyclopropane and cyclopentane rings known as the
methanocarba modification.28-30 Two structural varia-
tions, depending on the position of the cyclopropane
ring, restrict the ring pucker, i.e., hold the riboselike
ring (pseudosugar) in either a (N)- or (S)-envelope
conformation, as defined in the pseudorotational cycle.
A prototypical ring-constrained P2Y1 antagonist is the
highly potent (N)-methanocarba derivative 2-chloro-N6-
methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bis-
phosphate 5.18,20 This molecule was also prepared in
[3H]-labeled form and provides the first broadly reliable
high-affinity antagonist for quantitation of a P2Y recep-
tor in a radioligand binding assay.31 The recently
reported C-nucleoside pyrazolo[1,5-a]-1,3,5-triazine bis-
phosphate 6 is also a potent P2Y1 antagonist.25
The 2-iodo substituent could also be introduced at
various stages of the synthesis.
Resu lts
Ch em ica l Syn th esis. We prepared methanocarbo-
cyclic analogues of various bisphosphate derivatives
(Table 1) in which fused cyclopropane and cyclopentane
rings were fixed the pseudoribose moiety in a rigid (N)-
envelope conformation. Synthetic intermediates in-
cluded O-benzyl-protected phosphoesters, carbocyclic
derivatives, adenine derivatives, and O-tert-butyl-
protected phosphoesters (Schemes 1-5). The nucleotide
analogues 7-18 were prepared in the ammonium salt
form according to the methods shown in Schemes 1 and
4 and tested biologically as antagonists at the P2Y1
receptor (Table 1). Identity was confirmed by NMR (1H
and 31P) and high-resolution mass spectrometry (HRMS),
and purity was demonstrated with high-performance
liquid chromatography (HPLC) in two different solvent
systems.
To construct the pseudoribose ring leading to the 2′-
deoxy-(N)-methanocarba-2′-deoxyadenosine precursors,
a bicyclo[3.1.0]hexane intermediate 19 was prepared by
the new, optimized approach of Yoshimura et al.,32
which used simple starting materials and enzymatic
transformations. Scheme 1 illustrates the usual syn-
thetic route to the bisphosphate derivatives, in which
the phosphorylation was carried out on the preformed
nucleoside precursor. To prepare that nucleoside, a
commercially available 2-substituted adenine interme-
diate, 6-chloro-2-aminopurine 20, was condensed with
the protected bicyclohexane derivative 19 via a Mit-
sunobu reaction to give 21, which was then deprotected
to give diol 22. The 3′- and 5′-hydroxyl groups then were
phosphorylated by the phosphoramidite method33 using
In the present study, we combined the ring-con-
strained (N)-methanocarba modification of adenine
nucleotides with other adenine 2-position functionalities
to prepare novel analogues, which were evaluated for
affinity at the human P2Y1 receptor. In one case, a
2-iodo substitution conferred a significant increase in
antagonist potency. New synthetic routes to antagonists
in this series, including methods for introduction of
phosphate groups as either the terminal step or prior
to the coupling of sugar and nucleobase, were explored.

4976 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Kim et al.
Sch em e 1a
a
Reagents: (i) DIAD, Ph3P, rt, 2-amino-6-chloropurine; (ii) BCl3, CH2Cl2, -40 °C; (iii) 1H-tetrazole then m-CPBA; (iv) CH2I2, or MeSSMe,
or MeSeSeMe, t-BuONO, MeCN; (v) MeNH2/THF; (vi) BCl3, CH2Cl2, 5 °C; (vii) NH3/H2O, 70 °C, 2 h; (viii) MeNH2/H2O.
dibenzyl diisopropyl phosphoramidite 23 to give the
benzyl-protected derivative 24. The 2-amino group of
24 was replaced with an iodo, methylthio, or methylse-
leno group (protected intermediates 25-27) with the use
of a neutral purinyl radical, which was generated
transiently from the thermal homolysis of the 2-diazo-
nium intermediate. This was accomplished by using the
approach introduced by Nair and Fasbender34 for the
preparation of 2-methylthio and other 2-derivatives of
adenosine. Selenium was selected for inclusion because
of its similarity to sulfur and because of its known use
in X-ray crystallography of proteins, when incorporated
into ligands as a heavy atom.35 To introduce an amino
or methylamino group at the 6-position, it was necessary
to substitute 6-Cl with the corresponding amine, which
could be accomplished either before (to give 13) or
following (to give 7, 8, 12, or 14) deprotection of the
phosphate groups. First we synthesized the N6-methyl-
2-methylthio derivative 13, following the procedure in
Scheme 1. However, we were unable to apply this route
for the corresponding N6-amino derivative 8 because
treatment of 26 with ammonia to replace the 6-chloro
group caused side reactions. Therefore, we carried out
an ammonia replacement reaction following deprotec-

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4977
Sch em e 2a
Sch em e 4a
a
Reagents: (i) Ac2O, cat. DMAP, TEA, CH2Cl2, rt, 4 h; (ii) wet
5% Pd/C, H2, MeOH, rt, 4 h; (iii) (t-BuO)2PNEt, tetrazole, THF,
rt, 1 h then m-CPBA, -78 to 0 °C, 20 min; (iv) K2CO3, MeOH, rt,
2 h.
Sch em e 3a
a
Reagents: (i) SbBr3/CH2Br2, t-Bu nitrite; (ii) HF/pyridine, t-Bu
nitrite; (iii) CH3NH2/THF or H2O.
tion of the benzyl groups to obtain the N6-amino
derivative 8. We used similar procedures of phospho-
ester deprotection followed by replacement of the 6-chlo-
ro group for the preparation of the 2-methylseleno
derivative 14.
a
Reagents: (i) 2-substituted (iodo, methyl, 2-hexenyl) 6-me-
thylaminopurine or 2-iodo-6-chloropurine, Ph3P, DEAD, THF, rt,
2 h; (ii) 2-substituted (fluoro, bromo, or 2-hexynyl) 6-methyl-
aminopurine, Ph3P, DIAD, THF; (iii) 5% Pd/C, H2, MeOH, rt, 2 h;
(iv) 5% TFA in CH2Cl2, rt, 2 h; (v) PdCl2(PPh3)2, Me3SnSnMe3,
dioxane, 60 °C; (vi) I2, THF, rt.
As an alternate synthetic route to the bisphosphate
derivatives, phosphate groups were introduced into a
versatile phosporylated bicyclo[3.1.0]hexane precursor
34, protected with tert-butyl ester groups, prior to the
Mitsunobu coupling (Scheme 2).
Sch em e 5a
We first attempted to synthesize 2-fluoro 10 and
2-bromo 11 derivatives by the approach shown in
Scheme 1, which used the phosphoramidite route to
phosphorylate the corresponding nucleoside diol deriva-
tive.18 However, only traces of the products were ob-
tained due to side reactions, which seemed to result
from the high nucleophilicity of the 6-amino group.
Thus, an alternate route was needed. For substitution
of the 2-position of the purine ring with bromo or fluoro,
the corresponding adenine precursors 37 and 38 were
prepared (Scheme 3). We used 20, a commercially
available material, to prepare the dihaloadenines 35 and
36 by a method from the literature.36 Subsequent
methylamination of the 6-position gave 37 and 38,
which were coupled with the methanocarba moiety 34
by a Mitsunobu reaction. 2-Iodo-N6-methyladenine 39
was prepared with the method of Nair and Fasbender.34
The two 2-alkene/alkyne-substitituted adenines 41 and
42 were prepared as previously reported.37 These 2-ha-
loadenine derivatives and several 2-C-substituted ana-
logues were incorporated into the nucleoside structure
as shown in Scheme 4, via the protected bicyclo[3.1.0]-
hexane precursor 34. Following the Mitsunobu reaction,
the hexynyl intermediate 50 was reduced to the corre-
sponding alkane to give 48. Deprotection of the tert-
butyl groups was carried out with TFA to give the
bisphosphate derivatives 10-12 and 15-18.
a
Reagents: (i) K2CO3, DMSO-DMF, rt, 3 d; (ii) CH2I2, t-
BuONO, acetonitrile, 60 °C, 2.5 h; (iii) PdCl2(PPh3)2, Me3SnSnMe3,
dioxane, 100 °C, 5 h; (iv) Pd2(dba)3, P(o-tol)3, CuBr, K2CO3, MeI,
DMF, 60 °C, 3 h; (v) CH3NH2/THF, rt, 22 h; (vi) 5N NaOH, i-PrOH,
rt, 3 d.
The preparation of additional 2-substituted adenine
intermediates, including 6-chloro-2-iodopurine 43 used
in Scheme 4, required a separate synthetic strategy

4978 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Kim et al.
Sch em e 6a
(Scheme 5). The adenine precursor 20 dissolved in polar
organic solvents, e.g. pyridine or DMSO, only to a
limited extent, which made the selection of transforma-
tion conditions difficult. To proceed further, we applied
a protecting group at the N-9 position, expecting an
improvement in solubility. The pivaloyloxymethyl group
was chosen as the protecting group, and 54 was pre-
pared with the method of Rasmussen and Leonard.41
Compound 54 dissolved well in the usual organic
solvents, allowing the 2-iodination of 54 to be performed
by the method of Nair and Fasbender.34
With the success of [3H]5 as a selective radioligand
for P2Y1 receptor characterization,31 we sought to design
new antagonists in this series as high-affinity receptor
probes. As a route to a novel iodinated analogue that
could be potentially radiolabeled, we explored the
substitution of the iodo group of 12 (Scheme 4) and its
adenine precursors (Scheme 5) with trimethyltin.38,39
This provides a means of rapidly obtaining the iodinated
ligand 12. Similar methodology based on trialkyl tin
precursors has been used to prepare radioiodinated
ligands for SPECT (single photon emission computed
tomography).39,40 The 2-iodo product 55 was converted
to the 2-trimethylstannyl derivative 56 by the method
of Baranowska-Kortylewicz et al.42 Compound 56 was
a useful intermediate because it could serve as a
suitable substrate for Stille coupling; thus, we decided
to prepare the 2-methyl derivative. Compound 56
reacted with methyl iodide under the conditions of
Suzuki et al.43 The product 57 was treated with methyl-
amine to make 40, which was coupled with the metha-
nocarba moiety by a Mitsunobu reaction. Deprotection
was carried out with TFA to give the bisphosphate
derivative 15.
a
Reagents: (i) polynucleotide kinase, ATP; (ii) polynucleotide
kinase, alone.
F igu r e 1. Inhibition of [3H]5 binding to human P2Y1 receptors
by 2-substituted analogues. [3H]5 binding assays were carried
out as described in the Experimental Section using Sf9
membranes expressing recombinant human P2Y1 receptor.
The data are presented as percent of total radioligand binding
measured in the presence of the indicated concentrations of
2-chloro (5), 2-fluoro (10), 2-bromo (11), 2-iodo (12), and
2-methylthio (13) N6-methyl substituted analogues. The data
are the mean of triplicate determinations, and the results are
consistent with those obtained in three separate experiments.
As an alternate route to introduce iodine in the
antagonist 12, we also attempted stannylation of 46
with the method of Baranowska-Kortylewicz et al., to
be followed by reiodination.42 However, we obtained only
the 2-H derivative, which formed upon replacement of
the trimethylstannyl group by hydrogen, which was
readily available from an acidic proton such as the
6-NH(Me). As an alternative (Scheme 4), we tried
stannylation using a 6-chloro derivative 51. This inter-
mediate was synthesized from 43, which was obtained
by hydrolysis of 55, and was coupled with the metha-
nocarba moiety by a Mitsunobu reaction. Intermediate
51 was successfully stannylated with the method of
Baranowska-Kortylewicz et al.42 The trimethyl stannyl
derivative 52 reacted with iodine (which could be
generated rapidly from iodide) and re-formed 51. By
mass spectral analysis of the iodination reaction at
various time points (1-40 min), an optimal reaction
time was determined to be 10 min. Deprotection of 51
on a small scale with TFA was accomplished to give the
bisphosphate derivative 29, which was then converted
to the antagonist 12 (Scheme 1).
with polynucleotide kinase (EC 2.7.1.78).44 The 3′-
monophosphorylated intermediate 5845 was treated with
the enzyme and ATP as the phosphate source. Following
incubation for 60 min, the product 5 was detected in
∼40% yield, as judged by using HPLC. The intermediate
58 was found to be chemically stable under the incuba-
tion conditions. A side reaction, removal of the 3′-
phosphate group of 58 to yield the nucleoside analogue
59, occurred to a significant degree only in the absence
of ATP.
P h a r m a cologica l Activity. We recently developed
[3H]5 as a high-affinity and selective radioligand for
quantification of the P2Y1 receptor.31 Therefore, the
human P2Y1 receptor can be expressed from a baculo-
virus to high levels in Sf9 insect cells, and membranes
prepared from these cells can be labeled specifically with
[3H]5 to directly assess the affinity of newly synthesized
molecules at the P2Y1 receptor. Therefore, human P2Y1
receptor-expressing membranes were incubated with 10
nM [3H]5 and a wide range of concentrations of 2-sub-
stituted (N)-methanocarba bisphosphate analogues. As
illustrated in Figure 1, all of the bisphosphate deriva-
tives tested interacted with the P2Y1 receptor, as shown
by their capacity to inhibit [3H]5 binding, and this
We have explored a synthetic methodology that would
allow a phosphate group to be incorporated in a potent
P2Y1 receptor antagonist in the final synthetic step.
Although chemical phosphorylation methods were used
until now to prepare P2Y1 receptor antagonists in this
series, we devised an enzymatic method for the phos-
phorylation at the 5′-position, leading to compound 5.
This method (Scheme 6) is based on phosphorylation

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4979
inhibition occurred with kinetics consistent with inter-
action by the law of mass action kinetics at a single site.
IC50 values were determined (Table 1) from each
competition curve, and a Ki value was calculated for
each analogue according to the relationship Ki ) IC50/1
+ [[3H]5]/Kd of [3H]5.
We also tested the functional activities of (N)-metha-
nocarba bisphosphate analogues as agonists and an-
tagonists at the human P2Y1 receptor stably expressed
in 1321N1 human astrocytoma cells (Table 1). Agonist
activity was tested by measuring the capacity of mol-
ecules to increase inositol phosphate accumulation by
activating the phospholipase C-coupled P2Y1 receptor,
and antagonist activity at the P2Y1 receptor was as-
sessed by measuring the capacity of these molecules to
inhibit 2-methylthio-adenosine 5′-diphosphate (2Me-
SADP)-stimulated accumulation of inositol phosphates.
None of these 2-substituted molecules exhibited ago-
nist activity in the 1321N1 cell-test system, but all
displayed a capacity to inhibit 2MeSADP-stimulated
inositol phosphate accumulation, with an IC50 value
consistent with their Ki values determined in binding
experiments. Within the series of 2-halo analogues, the
most potent antagonist in both functional and binding
models was a (N)-methanocarba-2-iodo analogue 12, and
all other 2-halo derivatives were clearly less potent. This
derivative displayed a Ki value in competition for
binding of [3H]5 at the hP2Y1 receptor of 0.79 nM. The
N6-methyl derivative 12 was 16-fold more potent in
binding than the corresponding 6-amino analogue 7. The
corresponding N6-methyl 2-bromo and 2-chloro deriva-
tives 11 and 5 exhibited similar Ki values and affinities
that were 5-10-fold less than that for 12, and the fluoro
derivative 10 exhibited a much lower affinity (Figure
1). The (N)-methanocarba-2-methylthio-, 2-methylse-
leno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues
exhibited lower affinities than did most of the 2-halo
derivatives, with Ki values in the micromolar range
observed for these compounds at P2Y1 receptors. No
significant difference was observed between the alkene
and alkyne derivatives 17 and 18, whereas the corre-
sponding alkane 16 exhibited a 4-5-fold-lower affinity,
with a Ki value in binding calculated to be 80 nM.
However, the 2-methyl derivative 15 was nearly as
potent as the corresponding 2-chloro analogue 5.
Schild analysis of antagonism by 12 of inositol phos-
phate accumulation in 1321N1 human astrocytoma cells
stably expressing the human P2Y1 receptor, stimulated
by 2-MeS-ADP, was carried out (Figure 2). A KB value
of 1.74 nM was determined, in close agreement with
binding data for this compound.
F igu r e 2. Competitive antagonism of 2MeSADP-promoted
activation of the human P2Y1 receptor. Phospholipase C
activity was measured as described in the Experimental
Section in 1321N1 human astrocytoma cells stably expressing
the human P2Y1 receptor. Top panel: assays were in the
presence of the indicated concentrations of the agonist 2Me-
SADP alone or in the presence of the potent antagonist 12 at
3, 10, 30, 100, and 300 nM. The data are the means of triplicate
determinations and are representative of results obtained in
four separate experiments. Lower panel: Schild analysis of
antagonism by 12 of inositol phosphate accumulation in
1321N1 human astrocytoma cells stably expressing the human
P2Y1 receptor, stimulated by 2-MeS-ADP.
F igu r e 3. Correlation of binding affinities at the human P2Y1
receptor expressed in Sf9 cells and functional potencies as P2Y1
receptor antagonists (measuring inhibition of inositol phos-
phate accumulation in 1321N1 human astrocytoma cells stably
expressing the human P2Y1 receptor, stimulated by 30 nM
2-MeS-ADP).
A correlation of binding affinities (expressed as Ki
values) and functional antagonistic potencies (expressed
as IC50 values) at the human P2Y1 receptor was
examined for nine compounds (Figure 3). The degree of
correlation was high, with an r2 value of 0.98. Thus, as
we showed recently in radioligand binding assays with
[3H]5 and previously studied bisphosphate analogues,31
the radioligand binding assay is a readily applicable
predictor of the functional effects of the P2Y1 receptor
antagonists.
system provides an enhancement of affinity at the P2Y1
receptor. We strengthened this conclusion in the present
study. For example, the compounds homologous to 8,
12, and 13 were reported in the ribose series,17 and the
IC50 values in the same phospholipase C assay in turkey
erythrocytes expressing an endogenous P2Y1 receptor
were 1890, 891, and 362 µM, respectively. This is to be
compared to the values for 8, 12, and 13 of 367, 8.4,
and 221 nM, respectively. Thus, the enhancement
achieved upon conformationally constraining the ribose-
like ring in the preferred (N)-conformation was present
in all three cases. However, the degree of enhancement
Discu ssion
In previous studies,18,20 we concluded that substitu-
tion of the ribose moiety with the (N)-methanocarba ring

4980 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Kim et al.
ranged from slight, in the case of the N6-methyl-2-
methylthio analogue 13, to dramatic, i.e., 106-fold, in
the case of the N6-methyl-2-iodo analogue 12.
was performed with a J EOL SX102 spectrometer with 6-kV
Xe atoms following desorption from a glycerol matrix.
Purification of the nucleotide analogues for biological testing
was carried out on Sephadex-DEAE-A-25 resin columns with
a linear gradient (0.01-0.5 M) of 0.5 M ammonium bicarbonate
applied as the mobile phase.
(1′R,2′S,4′S,5′S)-4-(6-Am in o-2-iod o-9H -p u r in -9-yl)-1-
[(p h o s p h a t o )m e t h y l]-2-(p h o s p h a t o )b i c y c lo [3.1.0]-
h exa n e Tetr a a m m on iu m Sa lt (7). Compound 29 (1.48 mg,
0.0023 mmol) was dissolved in 30% NH3 in water (0.50 mL)
and stirred for 6.5 h at 60 °C in a sealed tube. The solvent
was removed under reduced pressure.
Purification of the obtained residue was performed on an
ion-exchange column packed with Sephadex-DEAE A-25 resin.
A linear gradient (0.01-0.5 M) of 0.5 M ammonium bicarbon-
ate was applied as the mobile phase, and UV and HPLC were
used to monitor the elution, which furnished 7 (0.77 mg,
54%): 1H NMR (D2O) δ 8.58 (s, 1H), 5.20-4.80 (m, 2H), 4.60-
4.40 (m, 1H), 3.60-3.56 (m, 1H), 2.35-2.20 (m, 1H), 2.00-
1.88 (m, 1H), 1.88-1.80 (m, 1H), 1.18-1.12 (m, 1H), 0.99-
0.91 (m, 1H); 31P NMR (D2O) δ 0.17, -0.25 (2s, 3′-P, 5′-P); MS
(m/e) (negative-FAB) 546 (M+ - 1); HRMS (negative-FAB)
calcd for C12H15N5O8P2I 545.9441, found 545.9454; HPLC 8.3
min (99%) (system A), 14.3 min (99%) (system B).
(1′R,2′S,4′S,5′S)-4-(6-Am in o-2-m et h ylt h io-9H -p u r in -9-
yl)-1-[(p h osp h a t o)m et h yl]-2-(p h osp h a t o)b icyclo[3.1.0]-
h exa n e Tetr a a m m on iu m Sa lt (8). To a solution of 26 (1.70
mg, 0.0020 mmol) in CH2Cl2 (0.30 mL) was added 1.0 M BCl3
in CH2Cl2 (0.50 mL) and the mixture stirred for 2 d at 5 °C.
The reaction was quenched upon addition of 5.0 mL of
triethylammonium bicarbonate buffer (1.0 M). The mixture
was subsequently frozen and lyophilized, which furnished
crude 8 (1.90 mg): MS (m/e) (negative-FAB) 485, 487 (peak
height ratio ) 3:1) (M+ - 1); HPLC 15.6 min (99%) (system
B).
The crude sample of 8 (1.90 mg) was dissolved in 30% NH3
in water (3.0 mL) and stirred for 2 h at 70 °C in a sealed tube.
The solvent was removed under reduced pressure, the obtained
residue was purified on an ion-exchange column packed with
Sephadex-DEAE A-25 resin, a linear gradient (0.01-0.5 M)
of 0.5 M ammonium bicarbonate was applied as the mobile
phase, and UV and HPLC were used to monitor the elution,
which furnished homogeneous 8 (1.90 mg, 94%): 1H NMR
(D2O) δ 8.44 (s, 1H), 5.30-5.20 (m, 1H), 5.09 (d, 1H, J ) 6.5
Hz), 4.90-4.75 (m, 1H), 3.70-3.55 (m, 1H), 2.60 (s, 3H), 2.30-
2.20 (m, 1H), 2.00-1.94 (m, 1H), 1.85-1.80 (m, 1H), 1.17-
1.14 (m, 1H), 0.96-0.94 (m, 1H); 31P NMR (D2O) δ 2.03, 1.36
(2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 466 (M+ - 1); HRMS
(negative-FAB) calcd for C13H18N5O8P2S 466.0374, found
466.0363; HPLC 8.7 min (99%) in solvent system A, 14.1 min
(99%) in a solvent system B.
(1′R,2′S,4′S,5′S)-4-(2-F lu or o-6-m eth yla m in o-9H-p u r in -
9-yl)-1-[(p h osp h a to)m eth yl]-2-(p h osp h a to)bicyclo[3.1.0]-
h exa n e Tetr a a m m on iu m Sa lt (10). The mixture of 44 (5
mg, 0.0074 mmol) in 5% TFA/CH2Cl2 (5 mL) was stirred at
room temperature for 3 h. After solvent was removed, the
residue was treated with triethylammonium bicarbonate buffer
(1 M, 0.5 mL) and stirred for 30 min. The aqueous layer was
washed twice with CH2Cl2 and evaporated to dryness under
reduced pressure. The residue was purified with ion-exchange
column chromatography with the use of Sephadex-DEAE-A-
25 resin with a linear gradient (0.01-0.5 M) of 0.5 M NH4-
HCO3 as the mobile phase. After lyophilization, 10 (1.38 mg,
36%) was obtained as a white solid: 1H NMR (D2O) δ 8.25 (s,
1H), 5.25-5.1 (m, 2H), 3.8-3.65 (m, 2H), 3.05 (s, 3H), 2.4-
2.1 (1H), 2.1-1.9 (m, 2H), 1.1-0.9 (1H); 31P NMR (D2O) 0.878
(s), 1.2 (s); HRMS (m/e) (negative-FAB) calcd forC13H18FN5O8P2
452.0536, found 452.0548; HPLC 8.2 (99%) min in solvent
system A, 16.1 min (99%) in solvent system B.
In the present study, we focused on optimization of
the 2-position substitution. Halo substitution demon-
strated a potency order of I > Br, Cl > F, which did not
correspond to the relative effects of these substitutions
in the ribose series.17 The 2-alkylthio substitution, which
was found to greatly enhance potency in the agonist
series, i.e., 5′-mono-, di-, and triphosphates,21-23 did not
enhance antagonist potency in the present series. This
observation is similar to previous findings with other
nucleotide antagonists of the P2Y1 receptor.17 In con-
trast with C-substitution at the 2-position of adenine
reported previously24 (i.e., 2-methyl in the ribose series),
we did not find an enhancement of affinity in the alkene
and alkyne analogues synthesized here.
The methods explored in this study for incorporation
of iodo and phosphate groups from simple chemical
precursors might be suitable for the preparation of
radioligands (containing 125I, 33P, etc.) of high affinity
for the P2Y1 receptor.
In conclusion, we have enhanced the potency of ring-
constrained P2Y1 receptor antagonists by 2-position
substitution. The structure-activity relationship (SAR)
patterns were not entirely in parallel to the SAR
observed in the ribose series. Thus, subtle differences
in the mode of receptor binding probably occur between
ribose-containing and (N)-methanocarba nucleotides. A
key finding in this study is that compound 12 is the
most potent antagonist selective for the P2Y1 receptor
yet reported. These findings promise to be useful in
defining the microscopic determinants of the binding
sites on these receptors and in designing novel phar-
macological probes, including radioligands, and/or thera-
peutic agents.
Exp er im en ta l Section
Ch em ica l Syn th esis. Nucleosides and synthetic reagents
were purchased from Sigma Chemical Co. (St. Louis, MO) and
Aldrich (Milwaukee, WI). 2,6-Dichloropurine was obtained
from Sigma. The protected intermediate 19 was synthesized
in our laboratory as described.45 5 and 9 were prepared as
described.18 37 and 38 were prepared as described.37
1H NMR spectra were obtained with a Varian Gemini-300
spectrometer (300 MHz) with D2O, CDCl3, CD3OD, and DMSO-
d6 as a solvent. Tetramethylsilane (TMS) was the external
standard. 31P NMR spectra were recorded at room temperature
with a Varian XL-300 spectrometer (121.42 MHz); orthophos-
phoric acid (85%) was used as an external standard.
Purity of compounds was checked with a Hewlett-Packard
1100 HPLC apparatus equipped with a Luna 5µ C18(2)
analytical column (250 × 4.6 mm, Phenomenex, Torrance, CA)
in two solvent systems.
System A: linear gradient solvent system, 0.1 M TEAA/CH3-
CN from 95/5 to 40/60 in 20 min; the flow rate was 1 mL/min.
System B: linear gradient solvent system, 5 mM TBAP/CH3-
CN from 80/20 to 40/60 in 20 min; the flow rate was 1 mL/
min.
Peaks were detected by UV absorption with a diode array
detector. All derivatives tested for biological activity showed
g97% purity in the HPLC systems.
Low-resolution CI-NH3 (chemical ionization) mass spectra
were measured with a Finnigan 4600 mass spectrometer, and
high-resolution EI (electron impact) mass spectrometry was
performed with a VG7070F mass spectrometer at 6 kV. High-
resolution FAB (fast atom bombardment) mass spectrometry
(1′R,2′S,4′S,5′S)-[4-(2-Br om o-6-m eth yla m in o-9H-p u r in -
9-yl)-1-[(p h osp h a to)m eth yl]-2-(p h osp h a to)bicyclo[3.1.0]-
h exa n e Tetr a a m m on iu m Sa lt (11). The mixture of 45 (3.0
mg, 0.0041 mmol) in 5% TFA/CH2Cl2 (5 mL) was stirred at
room temperature for 3 h. After removal of the solvent, the

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4981
residue was treated with triethylammonium bicarbonate buffer
(1 M, 0.5 mL) and stirred for 30 min. The aqueous layer was
washed twice with CH2Cl2 and evaporated to dryness. The
residue was purified with ion-exchange column chromatogra-
phy with the use of Sephadex-DEAE-A-25 resin with a linear
gradient (0.01-0.5 M) of 0.5 M NH4HCO3 as the mobile phase.
After lyophilization, 11 (1.36 mg, 58%) was obtained as a white
solid: 1H NMR (D2O) δ 8.42 (s, 1H), 5.4-5.1 (m, 2H), 3.8-3.6
(m, 2H), 3.1 (s, 3H), 2.4-2.1 (m, 2H), 2.1-1.8 (m, 2H), 1.1-
0.9 (1H); 31P NMR (D2O) δ 0.4 (s), 0.9 (s); HRMS (m/e)
(negative-FAB) calcd for C13H18BrN5O8P2 511.9736, found
511.9763; HPLC 9.3 min (99%) in solvent system A, 14.5 min
(99%) in solvent system B.
(1′R,2′S,4′S,5′S)-4-(2-Iod o-6-m eth yla m in op u r in -9-yl)-1-
[(p h o s p h a t o )m e t h y l]-2-(p h o s p h a t o )b i c y c lo [3.1.0]-
h exa n e (12). Meth od A. Compound 29 (3.61 mg, 0.0057
mmol) was dissolved in 40% MeNH2 in water (2.0 mL) and
stirred for 26 h at room temperature. The solvent was removed
under reduced pressure. Purification of the obtained residue
was performed on an ion-exchange column packed with
Sephadex-DEAE A-25 resin, a linear gradient (0.01-0.5 M)
of 0.5 M ammonium bicarbonate was applied as the mobile
phase, and UV and HPLC were used to monitor the elution,
which furnished 12 (0.73 mg, 20%).
Meth od B. A solution of the 2-iodo bis(di-tert-butyl phos-
phate) derivative 46 (7.1 mg, 9.03 µmol) in 5% trifluoroacetic
acid in methylene chloride was stirred for 2 h at room
temperature. All volatile material was removed in vacuo. The
residue was purified by using ion-exchange column chroma-
tography on Sephadex-DEAE-25 resin and a linear gradient
(0-0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase
to give the 2-iodo bisphosphate derivative 12 (2.9 mg, 55%) as
an ammonium salt: 1H NMR (D2O) δ 8.54 (bs, 1H), 5.19 (m,
1H), 5.01 (d, 1H, J ) 6.9 Hz), 4.58 (dd, 1H, J ) 4.7, 11.3 Hz),
3.73 (dd, 1H, J ) 4.4, 11.0 Hz), 3.07 (bs, 3H), 2.28 (dd, 1H, J
) 7.7, 14.6 Hz), 1.92-2.09 (m, 2H), 1.26 (dd, 1H, J ) 4.1, 6.1
Hz), 1.06 (dd, 1H, J ) 9.7, 16.3 Hz); 31P NMR (D2O) 0.651 (s);
HRMS (negative-ion FAB) calcd for C13H17N5O8P2I 559.9597,
found 559.9604; HPLC 9.8 min (97%) in solvent system A, 15.4
min (97%) in system B.
(1′R,2′S,4′S,5′S)-4-(6-Meth yla m in o-2-m eth ylth io-9H-p u -
r in -9-yl)-1-[(p h osp h a t o)m et h yl]-2-(p h osp h a t o)b icyclo-
[3.1.0]h exa n e Tetr a a m m on iu m Sa lt (13). To a solution of
28 (4.2 mg, 0.0050 mmol) in CH2Cl2 (0.30 mL) was added 1.0
M BCl3 in CH2Cl2 (0.60 mL) and the mixture stirred for 2 d at
5 °C. The reaction was quenched upon the addition of 5.0 mL
of triethylammonium bicarbonate buffer (1.0 M). The mixture
was subsequently frozen and lyophilized.
1H), 2.05-1.85 (m, 1H), 1.85-1.75 (m, 1H), 1.25-1.14 (m, 1H),
1.00-0.95 (m, 1H); 31P NMR (D2O) δ 3.00, 3.53 (2s, 3′-P, 5′-P);
MS (m/e) (negative-FAB) 528 (M+ - 1), HRMS (negative-FAB)
calcd for C14H20N5O8P2Se 527.9948, found 527.9950; HPLC
10.8 min (99%) in solvent system A, 16.8 min (99%) in solvent
system B.
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid [4-(2-Meth yl-6-m e-
th ylam in opu r in -9-yl)-1-ph osph on ooxym eth ylbicyclo[3.1.0]-
h ex-2-yl] Mon oester (15). A solution of 47 (8.0 mg, 0.012
mmol) in 5% TFA/CH2Cl2 (2.0 mL) was stirred for 3 h at 25
°C. The solvent was removed under reduced pressure and the
residue was quenched by addition of 5.0 mL of triethylammo-
nium bicarbonate buffer (1.0 M). The mixture was subse-
quently frozen and lyophilized. Purification of the obtained
residue was performed on an ion-exchange column packed with
Sephadex-DEAE A-25 resin, a linear gradient (0.01-0.5 M)
of 0.5 M ammonium bicarbonate was applied as the mobile
phase, and UV and HPLC were used to monitor the elution,
which furnished 15 (4.5 mg, 73%): 1H NMR (D2O) δ 8.45 (s,
1H), 5.18 (m, 1H), 5.00 (d, 1H, J ) 5.4 Hz), 4.57-4.50 (m, 1H),
3.69-3.62 (m, 1H), 3.13 (s, 3H), 2.53 (s, 3H), 2.30-2.20 (m,
1H), 2.24-1.92 (m, 1H), 1.88 (m, 1H), 1.19 (m, 1H), 0.98 (m,
1H); 31P NMR (D2O) δ 1.05, 0.65 (2s, 3′-P, 5′-P); MS (m/e)
(negative-FAB) 448 (M+ - 1); HRMS (negative-FAB) calcd for
C
14H20N5O8P2 448.0787, found 448.0807; HPLC 7.3 min (99%)
(system A), 14.2 min (99%) (system B).
(1′R,2′S,4′S,5′S)-4-(2-Hexyl-6-m eth yla m in op u r in -9-yl)-
1-[(p h o s p h a t o )m e t h y l]-2-(p h o s p h a t o )b ic y c lo [3.1.0]-
h exa n e (16). The 2-hexanyl bisphosphate derivative 16 (5.6
mg, 69%) was prepared by the same method as above for 11,
except the 2-hexanyl bis(di-tert-butyl phosphate) derivative 48
(11.2 mg, 0.015 mmol) was used as the starting material: 1H
NMR (D2O) δ 8.54 (bs, 1H), 5.23 (m, 1H), 5.10 (d, 1H, J ) 6.6
Hz), 4.57 (dd, 1H, J ) 5.2, 11.3 Hz), 3.70 (m, 1H), 3.25 (bs,
3H), 2.85 (dd, 2H, J ) 7.4, 7.7 Hz), 2.29 (dd, 1H, J ) 7.7, 15.1
Hz), 2.03 (dt, 1H, J ) 6.6, 16.8 Hz), 1.92 (dd, 1H, J ) 3.8, 9.1
Hz), 1.773 (m, 2H), 1.13-1.43 (m, 9H), 1.03 (dd, 1H, J ) 6.6,
7.7 Hz), 0.85 (m, 3H); 31P NMR (D2O) 0.827 (s), 0.375 (s);
HRMS (negative-ion FAB) calcd for C19H30N5O8P2 518.1570,
found 518.1544; HPLC 13.2 min (98%) in solvent system A,
16.6 min (97%) in system B.
(1′R,2′S,4′S,5′S)-4-(2-Hex-1-yn yl-6-m eth yla m in op u r in -
9-yl)-1-[(p h osp h a to)m eth yl]-2-(p h osp h a to)bicyclo[3.1.0]-
h exa n e (17). The 2-hexenyl bisphosphate derivative 17 (3.2
mg, 68%) was prepared by the same method as above for 11,
except the 2-hexenyl bis(di-tert-butyl phosphate) derivative 49
(6.0 mg, 8.08 µmol) was used as the starting material: 1H NMR
(D2O) δ 8.54 (s, 1H), 7.18 (dt, 1H, J ) 7.1, 15.4 Hz), 6.45 (m,
1H), 5.22 (m, 1H), 5.08 (d, 1H, J ) 6.3 Hz), 4.57 (dd, 1H, J )
4.7, 11.0 Hz), 3.71 (dd, 1H, J ) 5.0, 10.7 Hz), 3.22 (bs, 3H),
2.23-2.44 (m, 3H), 2.03 (m, 1H), 1.95 (dd, 1H, J ) 3.7, 8.8
Hz), 1.32-1.61 (m, 4H), 1.24 (dd, 1H, J ) 4.1, 5.5 Hz), 1.05
(dd, 1H, J ) 6.3, 8.2 Hz), 0.94 (m, 3H); 31P NMR (D2O) 0.601
(s), 0.299 (s); HRMS (negative-ion FAB) calcd for C19H28N5O8P2
516.1413, found 516.1403; HPLC 13.5 min (99%) in solvent
system A, 16.9 min (99%) in solvent system B.
(1′R,2′S,4′S,5′S)-4-(2-Hex-1-en yl-6-m eth yla m in op u r in -
9-yl)-1-[(p h osp h a to)m eth yl]-2-(p h osp h a to)bicyclo[3.1.0]-
h exa n e (18). The 2-hexynyl bisphosphate derivative 18 (4.0
mg, 73%) was prepared by the same method as above for 11,
except the 2-hexynyl bis(di-tert-butyl phosphate) derivative 50
(7.0 mg, 9.46 µmol) was used as the starting material: 1H NMR
(D2O) δ 8.46 (s, 1H), 5.16 (m, 1H), 4.97 (d, 1H, J ) 6.3 Hz),
4.56 (dd, 1H, J ) 4.7, 11.3 Hz), 3.70 (dd, 1H, J ) 6.3, 11.3
Hz), 3.07 (bs, 3H), 2.50 (t, 2H, J ) 7.1 Hz), 2.24 (dd, 1H, J )
7.4, 14.6 Hz), 1.91-2.05 (m, 2H), 1.42-1.71 (m, 4H), 1.23 (dd,
1H, J ) 4.1, 5.8 Hz), 1.03 (dd, 1H, J ) 6.1, 8.2 Hz), 0.95 (t,
3H, J ) 7.2 Hz); 31P NMR (D2O) 0.777 (s), 0.375 (s); HRMS
(negative-ion FAB) calcd for C19H26N5O8P2 514.1257, found
514.1234; HPLC 12.4 min (98%) in solvent system A, 12.3 min
(99%) in solvent system B.
Purification of the obtained residue was performed on an
ion-exchange column packed with Sephadex-DEAE A-25 resin,
a linear gradient (0.01-0.5 M) of 0.5 M ammonium bicarbonate
was applied as the mobile phase, and UV and HPLC were used
to monitor the elution, which furnished 13 (1.69 mg, 81%): 1H
NMR (D2O) δ 8.45 (s, 1H), 5.25-5.15 (m, 1H), 5.08-5.02 (m,
1H), 4.65-4.60 (m, 1H), 3.65-3.55 (m, 1H), 3.13 (bs, 3H), 2.62
(s, 3H), 2.30-2.20 (m, 1H), 2.00-1.85 (m, 1H), 1.85-1.75 (m,
1H), 1.15-1.10 (m, 1H), 1.00-0.95 (m, 1H); 31P NMR (D2O) δ
2.21, 2.90 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 480 (M+
-
1); HRMS (negative-FAB) calcd for C14H20N5O8P2S 480.0488,
found 480.0498; HPLC 10.3 min (99%) in solvent system A,
16.4 min (99%) in solvent system B.
(1′R,2′S,4′S,5′S)-4-(6-Met h yla m in o-2-m et h ylselen op u -
r in -9-yl)-1-[(p h osp h a t o)m et h yl]-2-(p h osp h a t o)b icyclo-
[3.1.0]h exa n e Tetr a a m m on iu m Sa lt (14). Compound 30
(0.38 mg, 0.63 µmol) was dissolved in 40% MeNH2 in water
(3.0 mL) and stirred for 8 h at 80 °C. The solvent was removed
under reduced pressure. Purification of the obtained residue
was performed on an ion-exchange column packed with
Sephadex-DEAE A-25 resin, a linear gradient (0.01-0.5 M)
of 0.5 M ammonium bicarbonate was applied as the mobile
phase, and UV and HPLC were used to monitor the elution,
which furnished 14 (0.30 mg, 79%): 1H NMR (D2O) δ 8.51 (s,
1H), 5.10-5.00 (m, 1H), 5.00-4.90 (m, 1H), 4.74-4.72 (m, 1H),
3.65-3.50 (m, 1H), 3.14 (bs, 3H), 2.53 (s, 3H), 2.40-2.18 (m,
Gen er a l (P h osp h or yla tion ) P r oced u r e: Syn th esis of
(N)-Meth an ocar baaden osin e Der ivatives (21-57).(1′R,2′S,-
4′S,5′S)-4-(2-Amino-6-chloro-9H-purin-9-yl)-1-(benzyloxymethyl)-

4982 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Kim et al.
2-ben zyloxybicyclo[3.1.0]h exa n e (21). To a solution of tri-
phenylphosphine (0.20 g, 0.76 mmol) in anhydrous tetrehy-
drofuran (THF, 1.0 mL) was added dropwise diisopropyl
azodicarboxylate (DIAD, 0.15 mL, 0.76 mmol) at -20 °C with
stirring for 0.5 h. Compound 19 (76 mg, 0.23 mmol) and
2-amino-6-chloropurine 20 (92 mg, 0.54 mmol) in THF (5.0 mL)
were added to the reaction mixture and it stirred for 18.5 h at
room temperature. The solvent was removed under vacuum,
and the residue obtained was purified by using flash chroma-
tography with 1/1 petroleum ether/ethyl acetate to furnish 21
(103 mg, 92%): 1H NMR (CDCl3) δ 8.58 (s, 1H), 7.40-7.21 (m,
10H), 5.01 (bs, 1H), 4.97 (d, 1H, J ) 6.6 Hz), 4.66 (t, 1H, J )
8.25 Hz), 4.54 (s, 2H), 4.45 (d, 1H, J ) 11.7 Hz), 4.38 (d, 1H,
J ) 11.7 Hz), 4.09 (d, 1H, J ) 9.8 Hz), 3.14 (d, 1H, J ) 9.8
Hz), 2.09-2.00 (m, 1H), 1.85-1.74 (m, 1H), 1.59-1.54 (m, 1H),
1.11-1.06 (m, 1H), 0.81-0.75 (m, 1H); MS (m/e) (positive-FAB)
476, 478 (peak height ratio ) 3:1) (M+ + 1).
(1′R,2′S,4′S,5′S)-4-(2-Am in o-6-ch lor o-9H-p u r in -9-yl)-1-
(h ydr oxym eth yl)-2-h ydr oxybicyclo[3.1.0]h exan e (22). Com-
pound 21 (169 mg, 0.353 mmol) was dissolved in CH2Cl2 (5.0
mL) and treated with BCl3 (1 M in CH2Cl2, 1.50 mL) for 1 h
at -40 °C under argon. i-PrOH (0.40 mL) was added to the
reaction mixture and it was warmed to room temperature. The
solvent was evaporated, and the crude product was purified
by flash column chromatography with AcOEt/i-PrOH (1/1) to
yield 22 as a white solid (72 mg, 69%): 1H NMR (CD3OD) δ
8.54 (s, 1H), 4.94 (d, 1H, J ) 6.6 Hz), 4.26 (d, 1H, J ) 11.7
Hz), 4.03 (s, 1H), 3.33 (d, 1H, J ) 11.7 Hz), 2.09-1.98 (m, 1H),
1.84-1.73 (m, 1H), 1.67-1.62 (m, 1H), 1.04-0.99 (m, 1H),
0.79-0.74 (m, 1H); MS (m/e) (negative-FAB) 294, 296 (peak
height ratio ) 3:1) (M+ - 1).
(1′R,2′S,4′S,5′S)-4-(2-Am in o-6-ch lor o-9H-p u r in -9-yl)-1-
[(d ib en zylp h osp h a t o)m et h yl]-2-(d ib en zylp h osp h a t o)-
bicyclo[3.1.0]h exa n e (24). To a stirred solution of 22 (56 mg,
0.190 mmol) and 1H-tetrazole (130 mg, 1.86 mmol) in 3.0 mL
of anhydrous THF was added dibenzyl diisopropylphosphora-
midite (23, 0.30 mL, 0.89 mmol), and the mixture was stirred
for 4.5 h at room temperature. The reaction mixture was cooled
to -78 °C and treated with a solution of m-chloroperbenzoic
acid (m-CPBA, 70% max, 350 mg) in CH2Cl2 (5.0 mL). The
resulting mixture was warmed to room temperature, and the
solvent was removed under reduced pressure. The residue
obtained was purified by flash column chromatography (1/1
) AcOEt/CH2Cl2), which furnished 24 (52 mg, 35%): 1H NMR
(CDCl3) δ 7.82 (s, 1H), 7.33-7.25 (m, 20H), 5.41 (dd, 1H, J )
7.8, 14.1 Hz), 5.27 (bs, 2H), 5.05-4.91 (m, 8H), 4.74 (d, 1H, J
) 6.9 Hz), 4.54 (dd, 1H, J ) 5.7, 11.1 Hz), 3.94 (dd, 1H, J )
6.0, 11.1 Hz), 2.14-2.04 (m, 1H), 1.98-1.87 (m, 1H), 1.62-
1.52 (m, 1H), 1.00-0.93 (m, 1H), 0.87-0.80 (m, 1H); MS (m/e)
(positive-FAB) 816, 818 (peak height ratio ) 3:1) (M+ + 1).
(1′R,2′S,4′S,5′S)-4-(6-Ch lor o-2-iod o-9H -p u r in -9-yl)-1-
[(d ib en zylp h osp h a t o)m et h yl]-2-(d ib en zylp h osp h a t o)-
bicyclo[3.1.0]h exa n e (25). To a solution of 24 (62.1 mg,
0.0761 mmol) in acetonitrile (0.50 mL) were added di-
iodomethane (2.0 mL) and tert-butyl nitrite (0.10 mL, 1.11
mmol) were added, and oxygen was purged by N2 bubbling.
The tube was sealed and stirred at 85 °C for 3.0 h. Di-
iodomethane was removed upon passage though a short
column (silica gel, eluting with CHCl3, then AcOEt). The
residue obtained was purified by preparative thin-layer chro-
matography (AcOEt), which furnished 25 (25.8 mg, 37%): 1H
NMR (CDCl3) δ 8.19 (s, 1H), 7.36-7.25 (m, 20H), 5.29 (q, 1H,
J ) 6.9 Hz), 5.10-4.92 (m, 9H), 4.61 (dd, 1H, J ) 6.3, 12.0
Hz), 3.84 (dd, 1H, J ) 6.6, 12.0 Hz), 2.05-1.88 (m, 2H), 1.62-
1.52 (m, 1H), 1.02 (m, 1H), 0.95-0.84 (m, 1H); MS (m/e)
(positive-FAB) 928, 930 (peak height ratio ) 3:1) (M+ + 1).
preparative thin-layer chromatography (2/1)AcOEt/CH2Cl2),
which furnished 26 (3.0 mg, 64%): 1H NMR (CDCl3) δ 8.15 (s,
1H), 7.33-7.25 (m, 20H), 5.30 (dd, 1H, J ) 7.8, 14.7 Hz), 5.07-
4.91 (m, 9H), 4.62 (dd, 1H, J ) 6.0, 11.5 Hz), 3.74 (dd, 1H, J
) 6.6, 11.5 Hz), 2.61 (s, 3H), 2.14-2.04 (m, 1H), 1.98-1.87
(m, 1H), 1.63-1.56 (m, 1H), 1.07-1.03 (m, 1H), 0.88-0.81 (m,
1H); MS (m/e) (positive-FAB) 847, 849 (peak height ratio )
3:1) (M+ + 1).
(1′R ,2′S ,4′S ,5′S )-4-(6-C h lo r o -2-m e t h y ls e le n o -9H -
p u r in -9-yl)-1-[(d iben zylp h osp h a to)m eth yl]-2-(d iben zyl-
p h osp h a to)bicyclo[3.1.0]h exa n e (27). To a solution of 24
(16.0 mg, 0.0196 mmol) in MeCN (0.30 mL) were added
dimethyldiselenide (0.10 mL, 1.06 mmol) and tert-butyl nitrite
(0.100 mL, 1.11 mmol), and oxygen was purged by nitrogen
bubbling. The tube was sealed and stirred at 65 °C for 20 h.
The solvent was removed under reduced pressure. The residue
obtained was purified by preparative thin-layer chromatog-
raphy (1/1 ) AcOEt/CH2Cl2), which furnished 27 (6.7 mg,
38%): 1H NMR (CDCl3) δ 8.16 (s, 1H), 7.40-7.25 (m, 20H),
5.28 (dd, 1H, J ) 8.4, 14.4 Hz), 5.10-4.90 (m, 9H), 4.62 (dd,
1H, J ) 6.0, 11.4 Hz), 3.74 (dd, 1H, J ) 6.6, 14.4 Hz), 2.53 (s,
3H), 2.15-2.00 (m, 1H), 2.00-1.85 (m, 1H), 1.75-1.65 (m, 1H),
1.45-1.35 (m, 1H), 1.10-1.00 (m, 1H); MS (m/e) (positive-FAB)
895, 897 (peak height ratio ) 3:1) (M+ + 1).
(1′R,2′S,4′S,5′S)-4-(6-Met h yla m in o-2-m et h ylt h io-9H -
p u r in -9-yl)-1-[(d iben zylp h osp h a to)-m eth yl]-2-(d iben zyl-
p h osp h a to)bicyclo[3.1.0]h exa n e (28). Compound 26 (3.0
mg, 0.0035 mmol) was dissolved in 2.0 M MeNH2 in THF (0.50
mL) and stirred for 2.5 h at room temperature. The solvent
was removed under reduced pressure. The residue obtained
was purified by preparative thin-layer chromatography (AcO-
Et), which furnished 28 (2.7 mg, 91%): 1H NMR (CDCl3) δ
7.77 (s, 1H), 7.30-7.25 (m, 20H), 5.72 (bs, 1H), 5.38 (dd, 1H,
J ) 7.5, 14.1 Hz), 5.03-4.91 (m, 9H), 4.59 (dd, 1H, J ) 6.3,
11.4 Hz), 3.78 (dd, 1H, J ) 5.1, 11.4 Hz), 3.19 (bs, 3H), 2.57
(s, 3H), 2.19-2.10 (m, 1H), 1.96-1.82 (m, 1H), 1.60-1.55 (m,
1H), 1.04-1.00 (m, 1H), 0.88-0.78 (m, 1H); MS (m/e) (positive-
FAB) 842 (M+ + 1).
(1′R,2′S,4′S,5′S)-4-(6-Ch lor o-2-iod o-9H -p u r in -9-yl)-1-
[(p h o s p h a t o )m e t h y l]-2-(p h o s p h a t o )b i c y c lo [3.1.0]-
h exa n e Tetr a a m m on iu m Sa lt (29). To a solution of 25 (13.6
mg, 0.015 mmol) in CH2Cl2 (0.80 mL) was added 1.0 M BCl3
in CH2Cl2 (0.80 mL) and the mixture stirred for 24 h at 5 °C.
The reaction was quenched upon addition of 3.0 mL of
triethylammonium bicarbonate buffer (1.0 M). The mixture
was subsequently frozen and lyophilized. Purification of the
residue obtained was performed on an ion-exchange column
packed with Sephadex-DEAE A-25 resin. A linear gradient
(0.01-0.5 M) of 0.5 M ammonium bicarbonate was applied as
the mobile phase, and UV and HPLC were used to monitor
the elution, which furnished 29 (5.56 mg, 60%): 1H NMR (D2O)
δ 8.83 (s, 1H), 5.30-5.20 (m, 1H), 5.16 (d, 1H, J ) 6.3 Hz),
4.60-4.50 (m, 1H), 3.75-3.65 (m, 1H), 2.40-2.20 (m, 1H),
2.10-1.95 (m, 1H), 1.95-1.90 (m, 1H), 1.25-1.20 (m, 1H),
1.05-1.00 (m, 1H); 31P NMR (D2O) δ 2.02, 1.40 (2s, 3′-P, 5′-P);
MS (m/e) (negative-FAB) 565, 567 (peak height ratio ) 3:1)
(M+ - 1); HPLC 9.8 min (98%) in solvent system A, 16.0 min
(98%) in solvent system B.
(1′R,2′S,4′S,5′S)-4-(6-Ch lor o-2-m eth ylselen o-9H-p u r in -
9-yl)-1-[(p h osp h a to)m eth yl]-2-(p h osp h a to)bicyclo[3.1.0]-
h exa n e Tetr a a m m on iu m Sa lt (30). To a solution of 27 (6.7
mg, 0.0075 mmol) in CH2Cl2 (0.50 mL) was added 1.0 M BCl3
in CH2Cl2 (0.80 mL) and the mixture stirred for 2 d at 5 °C.
The reaction was quenched upon addition of 5.0 mL of
triethylammonium bicarbonate buffer (1.0 M). The mixture
was subsequently frozen and lyophilized. Purification of the
obtained residue was performed on an ion-exchange column
packed with Sephadex-DEAE A-25 resin, a linear gradient
(0.01-0.5 M) of 0.5 M ammonium bicarbonate was applied as
the mobile phase, and UV and HPLC were used to monitor
the elution, which furnished 30 (1.28 mg, 28%): 1H NMR (D2O)
δ 8.78 (s, 1H), 5.35-5.22 (m, 2H), 4.60-4.50 (m, 1H), 3.70-
3.65 (m, 1H), 2.61 (s, 3H), 2.45-2.30 (m, 1H), 2.15-1.95 (m,
1H), 1.95-1.85 (m, 1H), 1.25-1.18 (m, 1H), 1.05-0.95 (m, 1H);
(1′R,2′S,4′S,5′S)-4-(6-Ch lor o-2-m eth ylth io-9H-p u r in -9-
yl)-1-[(diben zylph osph ato)m eth yl]-2-(diben zylph osph ato)-
bicyclo[3.1.0]h exa n e (26). To a solution of 24 (4.5 mg, 0.0056
mmol) in MeCN (0.25 mL) were added dimethyl disulfide (0.15
mL, 1.26 mmol) and tert-butylnitrite (0.050 mL, 0.56 mmol),
and oxygen was purged by nitrogen bubbling. The tube was
sealed and stirred at 70 °C for 5.5 h. The solvent was removed
under reduced pressure. The residue obtained was purified by

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4983
31P NMR (D2O) δ 1.67, 1.09 (2s, 3′-P, 5′-P); MS (m/e) (negative-
FAB) 533, 535 (peak height ratio ) 3:1) (M+ - 1), HRMS
(negative-FAB) calcd for C13H16N4O8P2Se 532.9293, found
532.9295; HPLC 11.0 min (99%) in solvent system A, 15.5 min
(99%) in solvent system B.
to provide 35 (0.404 g, 87%): 1H NMR (DMSO-d6) δ 8.72 (s,
1H); MS (m/e) (positive-FAB) 233 (M+ + H).
6-Ch lor o-2-flu or o-9H-p u r in e (36). Compound 20 (100 mg,
0.572 mmol) was added to 60% HF-pyridine (which was
prepared from 1.5 g of 70% HF-pyridine diluted with 0.25 g
of pyridine) at -50 °C with stirring. The temperature was
allowed to rise from -30 °C, and 0.1 mL of tert-butyl nitrite
(0.1 mL) was added dropwise. Stirring was continued for 10
min, and the reaction mixture was poured into ice water. The
aqueous layer was extracted with CHCl3 and ethyl acetate.
The combined organic layer was washed with H2O, dried over
Na2SO4, and filtered, and the filtrate was evaporated. The
given residue was purified on a flash column chromatography
(MeOH/CHCl3 ) 1/10) to give 36 (90 mg, 91%) as a white
solid: 1H NMR (CD3OD) δ 8.5 (s, 1H); MS (m/e) (positive-FAB)
173 (M+ + H).
2-F lu or o-6-m eth yla m in o-9H-p u r in e (37). The mixture of
36 (56 mg, 0.324 mmol) in CH3NH2 (2.0 M in THF, 3 mL) was
stirred at room temperature overnight. After removal of the
solvent, the residue was purified by preparative thin-layer
chromatography (MeOH/CHCl3 ) 1/10) to yield 37 (43 mg,
80%): 1H NMR (DMSO-d6) δ 8.12 (br, 1H), 8.06 (s, 1H), 3.30
(s, 1H), 2.91 (s, 3H); MS (m/ e) (positive-FAB) 168 (M+ + H).
2-Br om o-6-m eth yla m in o-9H-p u r in e (38). The mixture of
35 (130 mg, 0.557 mmol) in CH3NH2 (40% in H2O, 2 mL) was
heated to 70 °C and stirred at this temperature for 6 h. The
reaction mixture was cooled to room temperature, and the
formed white precipitate was filtered and washed with a small
amount of water/MeOH. The precipitate was dried in a vacuum
to give 38 (118 mg, 93%): 1H NMR (DMSO-d6) δ 8.08 (s, 1H),
8.06 (bs, 1H), 3.30 (s, 1H), 2.91 (s, 3H); MS (m/e) (positive-
FAB) 228 (M+ + H).
(1′R,2′S,4′S,5′S)-4-(2-F lu or o-6-m eth yla m in o-9H-p u r in -
9-yl)-1-(di-ter t-bu toxyph osph or yloxym eth yl)bicyclo[3.1.0]-
h ex-2-yl-p h osp h or ic Acid Di-ter t-bu tyl Ester (44). To an
ice-cold solution of triphenylphosphine (23.6 mg, 0.09 mmol)
in dry THF (0.5 mL) was added DIAD (18.7 µL, 0.09 mmol)
dropwise under a nitrogen atmosphere, and the mixture was
stirred for 20 min until the solution turned red. This mixture
was added dropwise to a cold, stirred mixture of 34 (14.4 mg,
0.027 mmol) and 37 (15.03 mg, 0.09 mmol) under a nitrogen
atmosphere. The reaction mixture was stirred in an ice bath
for 30 min and then allowed to warm to room temperature,
and stirring continued for 12 h. Solvent was removed by
nitrogen purge, and the residue was purified by preparative
thin-layer chromatography (MeOH/CHCl3 ) 1/20) to produce
44 (8.61 mg, 47%) as a thick liquid: 1H NMR (CDCl3) δ 8.08
(s, 1H), 6.18 (br, 1H), 5.45-5.25 (m, 1H), 5.10-4.9 (m, 1H),
4.75-4.55 (m, 1H), 4.0-3.8 (m, 1H), 3.17 (s, 3H), 2.45-2.2 (m,
1H), 2.14-1.95 (m, 1H), 1.86-1.75 (m, 1H), 1.49 (s, 18H), 1.47
(s, 18H), 1.2-1.05 (m, 1H), 1.05-0.9 (m, 1H); MS (m/e)
(positive-FAB) 678 (M+ + H).
(1′R,2′S,4′S,5′S)-4-(2-Br om o-6-m eth yla m in o-9H-p u r in -
9-yl)-1-(di-ter t-bu toxyph osph or yloxym eth yl)bicyclo[3.1.0]-
h ex-2-yl-p h osp h or ic Acid Di-ter t-bu tyl Ester (45). To a
solution of triphenylphosphine (15 mg, 0.057 mmol) in anhy-
drous THF (0.5 mL) was added DIAD (12 µL, 0.057 mmol)
dropwise at 0 °C under a nitrogen atmosphere, and the
mixture was stirred at this temperature for 20 min until the
solution turned red. This reaction mixture was added slowly
to a cold stirred solution of alcohol 34 (9.2 mg, 0.0174 mmol)
and 38 (13.1 mg, 0.057 mmol) under a nitrogen atmosphere.
The reaction mixture was stirred in an ice bath for 30 min
and then allowed to warm to room temperature while stirring
continued for 12 h. Solvent was removed by nitrogen purge,
and the residue was purified by preparative thin-layer chro-
matography (MeOH/CHCl3 ) 1/20) to produce 45 (3.1 mg, 23%)
as a thick liquid: 1H NMR (CDCl3) δ 8.05 (s, 1H), 5.92 (br,
1H), 5.45-5.25 (m, 1H), 5.15-5.0 (m, 1H), 4.75-4.55 (m, 1H),
3.95-3.80 (m, 1H), 3.18 (s, 3H), 2.4-2.2 (m, 1H), 2.20-2.0 (m,
1H), 1.85-1.7 (m, 1H), 1.58 (s, 18H), 1.49 (s, 18H), 1.2-1.05
(m, 1H), 1.05-0.9 (m, 1H); MS (m/e) (positive-FAB) 738 (M+
+ H).
(1′R,2′S,4′R,5′S)-Acetic Acid 4-Hydr oxy-5-h ydr oxym eth -
ylbicyclo[3.1.0]h ex-2-yl Ester (32). A mixture of dibenzyl
alcohol 19 (137 mg, 0.422 mmol), acetic anhydride (140.4 mg,
1.38 mmol), triethylamine (436 mg, 4.3 mmol), 4-(dimethy-
lamino)pyridine (10 mg, 0.2 mmol), and dichloromethane (5
mL) was stirred at room temperature for 4 h. The resulting
mixture was partitioned between ethyl acetate (50 mL) and
water/brine (1/1, 20 mL). The organic layer was washed with
saturated aqueous sodium bicarbonate (10 mL) and brine (10
mL), dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was used in the next reaction without
further purification. The dibenzyl acetate 31 (crude 164 mg)
was dissolved in methanol (5 mL). Five percent Pd/C (150 mg)
was added. The reaction flask was applied to a vacuum and
filled with hydrogen several times. The reaction mixture was
stirred for 4 h at room temperature under a hydrogen balloon.
The resulting mixture was filtrated through a pad of Celite
and washed with methanol (10 mL × 3). The residue was
purified by flash column chromatography (chloroform/metha-
nol ) 10/1) to give a diol 32 (76 mg, 97% for two steps yield):
1H NMR (CDCl3) δ 5.13 (m, 1H), 4.45 (t, 1H, J ) 8.2 Hz), 3.96
(d, 1H, J ) 11.8 Hz), 3.24 (d, 1H, J ) 11.8 Hz), 2.33 (dt, 1H,
J ) 8.0, 13.2 Hz), 2.01 (s, 3H), 1.66 (m, 1H), 1.27 (dt, 1H, J )
8.8, 13.5 Hz), 1.09 (dd, 1H, J ) 3.8, 5.5 Hz), 0.55 (dd, 1H, J )
5.5, 8.0 Hz).
(1′R,2′S,4′R,5′S)-Acet ic Acid 4-(Di-ter t-b u t oxyp h os-
p h or yloxy)-5-(d i-t er t -b u t oxyp h osp h or yloxym e t h yl)-
bicyclo[3.1.0]h ex-2-yl Ester (33). To a stirred suspension
of diol 32 (25 mg, 0.134 mmol) and diethyl di-tert-butylphos-
phoramidite (100.5 mg, 0.402 mmol) in anhydrous tetrahy-
drofuran (4 mL) at room temperature was added solid tetrazole
(94 mg, 1.34 mmol) in one pot. The reaction mixture was
stirred at room temperature for 1 h and then cooled to -78
°C. To this cooled reaction mixture was added solid m-CPBA
(57-85%, calculated by 57%, 240 mg), and the resulting
mixture was stirred at -78 °C for 20 min and at room
temperature for 10 min. After removal of tetrahydrofuran in
vacuo, the residue was directly purified by flash column
chromatography (CHCl3/MeOH ) 10/1) to give 33 (71 mg,
93%): 1H NMR (CDCl3) δ 4.87 (dd, 1H, J ) 8.0, 14.6 Hz), 4.41
(m, 1H), 4.40 (dd, 1H, J ) 7.7, 11.0 Hz), 3.69 (dd, 1H, J ) 6.9,
11.0 Hz), 2.46 (dt, 1H, J ) 7.7, 13.5 Hz), 1.78 (m, 1H), 1.485
(s, 9H), 1.478 (s, 9H), 1.469 (s, 9H), 1.457 (s, 9H), 1.39 (m,
1H), 1.25 (dd, 1H, J ) 5.0, 5.2 Hz), 0.67 (dd, 1H, J ) 6.3, 7.4
Hz).
(1′R,2′S,4′R,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-b u t oxyp h osp h or yloxym e t h yl)-4-h yd r oxyb i-
cyclo[3.1.0]h ex-2-yl Ester (34). Acetate 33 (70 mg, 0.122
mmol) was dissolved in methanol (3 mL), and then solid
potassium carbonate (20 mg, 0.145 mmol) was added. After
the reaction mixture was stirred for 2 h, methanol was
removed in vacuo and the residue was purified by flash column
chromatography (CHCl3/MeOH ) 30/1) to produce the bis-
phosphate alcohol 34 (51 mg, 79%): 1H NMR (CDCl3) δ 5.13
(m, 1H), 4.45 (t, 1H, J ) 8.2 Hz), 3.96 (d, 1H, J ) 11.8 Hz),
3.24 (d, 1H, J ) 11.8 Hz), 2.33 (dt, 1H, J ) 8.0, 13.2 Hz), 2.01
(s, 3H), 1.66 (m, 1H), 1.27 (dt, 1H, J ) 8.8, 13.5 Hz), 1.09 (dd,
1H, J ) 3.8, 5.5 Hz), 0.55 (dd, 1H, J ) 5.5, 8.0 Hz); MS (m/e)
(positive-FAB) 529.35 (M+ + 1), 551.31 (M+ + Na + 1).
2-Br om o-6-ch lor o-9H-p u r in e (35). To a stirred solution
of 20 (0.35 g, 2 mmol) in CH2Br2 (5 mL) was added a solution
of SbBr3 in CH2Br2 (5 mL) at -10 °C. This solution was stirred
at this temperature for 10 min, and tert-butyl nitrite (4.5 mL)
was added. The reaction mixture was stirred for 2.5 h at -10
°C and then was poured into a stirred mixture of 2.4 g of
NaHCO3 in 100 mL of water and crushed ice. This mixture
was filtered, and the filtrate was extracted with CHCl3 and
dried over Na2SO4. After removal of the solvent, the residue
was purified by flash chromatography (MeOH/CHCl3 ) 1/10)
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-b u t yl E st er

4984 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Kim et al.
1-(Di-ter t-bu toxyp h osp h or yloxym eth yl)-4-(2-iod o-6-m e-
th ylam in opu r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester (46). The
alcohol 34 (10 mg, 0.0189 mmol), triphenylphosphine (20 mg,
0.0756 mmol), and 2-iodo-6-methylaminopurine 39 (10.4 mg,
0.0378 mmol) were mixed and dried under high vacuum for
∼30 min. The residue was dissolved in anhydrous tetrahydro-
furan (1 mL) and cooled to 0 °C. Diethyl azodicarboxylate
(DEAD, 9.9 mg, 0.0567 mmol) was added dropwise via syringe
to this solution. After the solution was stirred for 2 h at room
temperature (the progress of the reaction was monitored by
thin-layer chromatography), tetrahydrofuran was removed in
vacuo. The residue was purified by preparative thin-layer
chromatography (CHCl3/MeOH ) 20/1) to give the desired
2-iodo bis(di-tert-butyl phosphate) derivative 46 (7.1 mg,
48%): 1H NMR (CDCl3) δ 7.98 (s, 1H), 5.85 (bs, 1H, -NH),
5.35 (dd, 1H, J ) 7.7, 14.3 Hz), 5.07 (d, 1H, J ) 6.9 Hz), 4.66
(dd, 1H, J ) 4.7, 11.0 Hz), 3.91 (dd, 1H, J ) 6.6, 11.3 Hz),
3.17 (bs, 3H), 2.31 (dd, 1H, J ) 8.0, 15.4 Hz), 2.09 (dt, 1H, J
) 8.2, 14.8 Hz), 1.76 (dd, 1H, J ) 4.4, 8.5 Hz), 1.32 (s, 18H),
1.29 (s, 9H), 1.26 (s, 9H), 1.10 (dd, 1H, J ) 4.1, 6.0 Hz), 0.99
(m, 1H); MS (m/e) (positive-FAB) 786.3 (M+ + 1).
2-Am in o-6-ch lor op u r in -9-ylm eth yl 2,2-Dim eth ylp r op i-
on a te (54). 2-Amino-6-chloropurine 20 (1.00 g, 5.90 mmol) was
dissolved in DMSO (5.0 mL) under heating. To this solution
were added DMF (20.0 mL), chloromethyl pivalate 53 (1.0 mL,
6.94 mmol), and K2CO3 (990 mg, 7.16 mmol), and the mixture
was stirred at room temperature for 3 days. The reaction
mixture was filtered and the filtrate was evaporated. The
obtained residue was purified by flash chromatography (AcOEt/
petroleum ether ) 2/1) and recrystallized from AcOEt/
petroleum ether, which furnished 54 (1.33 g, 79%): 1H NMR
(CDCl3) δ 8.01 (s, 1H), 6.00 (s, 2H), 5.20 (bs, 2H), 1.18 (s, 9H);
MS (m/e) (positive-FAB) 284, 286 (peak height ratio 3:1)
(M+ + 1).
1.09-1.03 (m, 1H); MS (m/e) (positive-FAB) 791, 793 (peak
height ratio 3:1) (M+ + 1).
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-bu toxyp h osp h or yloxym eth yl)-4-(2-iod o-6-m e-
th yla m in op u r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester (46). To
a solution of 6-chloro-2-methylpurin-9-ylmethyl 2,2-dimethyl-
propionate 51 (13.0 mg, 0.016 mmol) in THF (0.10 mL) was
added 2.0 N MeNH2 in THF (1.0 mL) and the mixture stirred
for 3 h. The solvent was removed under reduced pressure and
the obtained residue was purified by flash chromatography
(CHCl3/MeOH ) 5/1), which furnished 46 (6.1 mg, 47%).
6-Ch lor o-2-t r im et h ylst a n n ylp u r in -9-ylm et h yl 2,2-Di-
m eth ylp r op ion a te (56). To a solution of 6-chloro-2-iodopurin-
9-ylmethyl 2,2-dimethylpropionate 55 (323 mg, 0.819 mmol)
and PdCl2(PPh3)2 (30 mg, 0.043 mmol) in dioxane (0.75 mL)
was added Me3SnSnMe3 (820 mg, 2.50 mmol) in dioxane (2.0
mL) and the mixture stirred at 100 °C for 5 h. The solvent
was removed under vacuum and the residue obtained was
purified by flash chromatography (AcOEt/petroleum ether )
1/2), which furnished 56 (310 mg, 88%): 1H NMR (CDCl3) δ
8.26 (s, 1H), 6.21 (s, 2H), 1.18 (s, 9H), 0.45 (s, 9H); MS (m/e)
(positive-FAB) 431(M+ + 1).
6-Ch lor o-2-m eth ylp u r in -9-ylm eth yl 2,2-Dim eth ylp r o-
p ion a t e (57). A mixture of tris(dibenzylideneacetone)di-
palladium(0) chloroform adduct (Pd2(dba)3, 16 mg, 0.031
mmol), tri o-tolylphosphine (P(o-Tol)3, 31 mg, 0.102 mmol),
CuBr (19 mg, 0.135 mmol), and K2CO3 (26 mg, 0.188 mmol)
in a flask was dried under reduced pressure, and the vacuum
was replaced by dry nitrogen. A solution of 6-chloro-2-tri-
methylstannylpurin-9-ylmethyl 2,2-dimethylpropionate 56 (109
mg, 0.253 mmol) in DMF (2.0 mL) and MeI (0.15 mL, 2.41
mmol) was added, and the mixture was stirred at 60 °C for 3
h. The resulting reaction mixture was directly pass through a
short column (SiO2, 2.0 mL, eluent AcOEt) and evaporated.
The residue obtained was purified by flash chromatography
(AcOEt/petroleum ether ) 1/2), which furnished 57 (45 mg,
62%): 1H NMR (CDCl3) δ 8.29 (s, 1H), 6.16 (s, 2H), 2.83 (s,
3H), 1.18 (s, 9H); MS (m/e) (positive-FAB) 283, 285 (peak
height ratio 3:1) (M+ + 1).
6-Meth yla m in o-2-m eth ylp u r in e (40). To a solution of
6-chloro-2-methylpurin-9-yl-methyl 2,2-dimethylpropionate 57
(34 mg, 0.120 mmol) in THF (1.0 mL) was added 2.0 N MeNH2
in THF (2.0 mL) and the mixture stirred for 22 h. The solvent
was removed under reduced pressure and the obtained residue
was purified by preparative thin-layer chromatography (first,
AcOEt; second, CHCl3/MeOH ) 5/1), which also furnished
6-methylamino-2-methylpurine 40 (27 mg, 81%) and 6-methy-
lamino-2-methylpurin-9-ylmethyl 2,2-dimethylpropionate (4
mg, 12%): 1H NMR (DMSO) δ 7.96 (s, 1H), 7.40 (bs, 1H), 2.94
(s, 3H), 2.40 (s, 3H); MS (m/e) (positive-FAB) 164 (M+ + 1).
6-Meth yla m in o-2-m eth ylp u r in -9-yl-m eth yl 2,2-d im eth -
ylp r op ion a te: 1H NMR (DMSO) δ 8.11 (s, 1H), 7.65 (bs, 1H),
6.10 (s, 2H), 2.94 (s, 3H), 2.44 (s, 3H), 1.10 (s, 9H); MS (m/e)
(positive-FAB) 278 (M++1).
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-b u t oxyp h osp h or yloxym et h yl)-4-(2-m et h yl-6-
m eth yla m in op u r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester (47).
To a solution of triphenylphosphine (52 mg, 0.198 mmol) in
anhydrous THF (0.40 mL) was added DEAD (0.030 mL, 0.191
mmol) dropwise at -20 °C with stirring for 1.0 h. Compound
34 (16.0 mg, 0.030 mmol) and 6-methylamino-2-methylpurine
40 (27 mg, 0.0975 mmol) in THF (1.50 mL) were added to the
reaction mixture, which stirred at room temperature for 3 d.
The solvent was removed under vacuum and the residue
obtained was purified by preparative thin-layer chromatog-
raphy (AcOEt), which furnished 47 (10.0 mg, 49%): 1H NMR
(CDCl3) δ 7.99 (s, 1H), 5.62 (br, 1H), 5.38 (m, 1H), 5.09 (d, 1H,
J ) 6.9 Hz), 4.65 (dd, 1H, J ) 4.5, 11.4 Hz), 3.92 (dd, 1H, J )
6.3, 11.4 Hz), 3.22 (s, 3H), 2.60 (s, 3H), 2.40-2.29 (m, 1H),
2.18-2.05 (m, 1H), 1.80 (m, 1H), 1.49 (s, 18H), 1.47 (s, 9H),
1.45 (s, 9H), 1.13-1.08 (m, 1H), 1.01-0.95 (m, 1H).
6-Ch lor o-2-iod op u r in -9-ylm eth yl 2,2-Dim eth ylp r op i-
on a te (55). To a solution of 2-amino-6-chloropurin-9-yl-methyl
2,2-dimethylpropionate 54 (704 mg, 2.48 mmol) in MeCN (2.0
mL) were added diiodomethane (8.0 mL) and tert-butylnitrite
(0.90 mL, 9.99 mmol), and oxygen was purged by N2 bubbling.
The tube was sealed and the contents stirred at 80 °C for 2.5
h. The solvent was removed under vacuum and the obtained
residue was purified by flash chromatography (AcOEt/
petroleum ether ) 1/2), which furnished 55 (561 mg, 57%):
1H NMR (CDCl3) 8.29 (s, 1H), 6.14 (s, 2H), 1.19 (s, 9H); MS
(m/e) (positive-FAB) 395, 397 (peak height ratio 3:1) (M+
1).
+
6-Ch lor o-2-iod op u r in e (43). To a solution of 6-chloro-2-
iodopurin-9-ylmethyl 2,2-dimethylpropionate 55 (125 mg,
0.317 mmol) in i-PrOH (5.0 mL) and H2O (0.500 mL) was
added 5 N aq NaOH (0.200 mL) and the mixture stirred at
room temperature for 3 days. The solvent was removed under
reduced pressure, the residue was poured into saturated aq
NaHCO3 and extracted with THF-AcOEt. The organic layer
was washed with brine, dried over Na2SO4, and evaporated.
The residue obtained was purified by flash chromatography
(AcOEt), which furnished 43 (42 mg, 48%): 1H NMR (DMSO)
δ 8.63 (s, 1H); MS (m/e) (positive-FAB) 281, 283 (peak height
ratio 3:1) (M++1).
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-b u t oxyp h osp h or yloxym et h yl)-4-(6-ch lor o-2-
iod op u r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester (51). To a solu-
tion of triphenylphosphine (70 mg, 0.26 mmol) in anhydrous
THF (0.50 mL) was added DEAD (0.035 mL, 0.22 mmol)
dropwise at -20 °C with stirring for 1.0 h. Compound 34 (18
mg, 0.035 mmol) and 6-chloro-2-iodopurine 43 (65 mg, 0.23
mmol) in THF (1.50 mL) were added to the reaction mixture
and it stirred at room temperature for 3 days. The solvent was
removed under vacuum and the residue obtained was purified
by preparative thin-layer chromatography (AcOEt), which
furnished 51 (15.5 mg, 57%): 1H NMR (CDCl3) δ 8.44 (s, 1H),
5.34 (dd, 1H, J ) 8.1, 15.0 Hz), 5.16 (d, 1H, J ) 6.9 Hz), 4.69
(dd, 1H, J ) 5.1, 11.4 Hz), 3.94 (dd, 1H, J ) 6.6, 11.4 Hz),
2.40-2.30 (m, 1H), 2.22-2.10 (m, 1H), 1.85-1.80 (m, 1H), 1.50
(s, 9H), 1.49 (s × 2, 18H), 1.48 (s, 9H), 1.18-1.14 (m, 1H),
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-bu toxyp h osp h or yloxym eth yl)-4-(2-h exyl-6-m e-
t h yla m in op u r in -9-yl)b icyclo[3.1.0]h ex-2-yl E st er (48).

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4985
2-Hexynyl bis(di-tert-butyl phosphate) derivative 50 (13 mg,
0.0176 mmol) was dissolved in methanol (5 mL), and 5% Pd/C
(150 mg) was added. The reaction flask was applied to a
vacuum and filled with hydrogen several times. The reaction
mixture was stirred for 2 h at room temperature under a
hydrogen balloon. The resulting mixture was filtrated through
a pad of Celite and washed with methanol (5 mL × 3). The
residue was purified by preparative thin-layer chromatography
(CHCl3/MeOH ) 20/1) to give the desired 2-hexanyl bis(di-
tert-butyl phosphate) derivative 48 (11.5 mg, 88%): 1H NMR
(CDCl3) δ 8.02 (s, 1H), 5.62 (bs, 1H, -NH), 5.35 (dd, 1H, J )
7.7, 14.6 Hz), 5.12 (d, 1H, J ) 6.9 Hz), 4.64 (dd, 1H, J ) 4.4,
11.0 Hz), 3.90 (dd, 1H, J ) 6.3, 11.0 Hz), 3.23 (d, 3H, J ) 4.9
Hz), 2.81 (dd, 2H, J ) 7.7, 8.0 Hz), 2.34 (dd, 1H, J ) 7.7, 15.1
Hz), 2.09 (dt, 1H, J ) 8.0, 15.7 Hz), 1.23-1.88 (m, 9H), 1.497
(s, 9H), 1.489 (s, 9H), 1.479 (s, 9H), 1.455 (s, 9H), 1.13 (dd,
1H, J ) 4.4, 5.8 Hz), 0.98 (dd, 1H, J ) 6.3, 8.5 Hz), 0.89 (m,
3H); MS (m/e) (positive-FAB) 744.5 (M+ + 1).
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-bu toxy-p h osp h or yloxym eth yl)-4-(2-h ex-1-en yl-
6-m eth ylam in opu r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester (49).
2-Hexenyl bis(di-tert-butyl phosphate) derivative 49 (6.1 mg,
44%) was prepared by a method similar to that for 46, except
2-hexenyl-6-methylaminopurine 41 (10 mg, 0.0189 mmol) was
used as the starting material: 1H NMR (CDCl3) δ 8.01 (s, 1H),
7.11 (dt, 1H, J ) 6.9, 15.4 Hz), 6.48 (dt, 1H, J ) 1.4, 15.6 Hz),
5.62 (bq, 1H, -NH), 5.38 (dd, 1H, J ) 7.7, 14.3 Hz), 5.14 (d,
1H, J ) 6.9 Hz), 4.65 (dd, 1H, J ) 4.7, 11.3 Hz), 3.91 (dd, 1H,
J ) 6.3, 11.3 Hz), 3.24 (d, 3H, J ) 5.0 Hz), 2.35 (dd, 1H, J )
8.5, 15.4 Hz), 2.29 (m, 2H), 2.11 (dt, 1H, J ) 7.7, 15.4 Hz),
1.80 (dd, 1H, J ) 4.1, 8.5 Hz), 1.33-1.61 (m, 4H), 1.496 (s,
9H), 1.490 (s, 9H), 1.482 (s, 9H), 1.446 (s, 9H), 1.13 (dd, 1H, J
) 4.4, 5.8 Hz), 0.98 (dd, 1H, J ) 6.3, 8.5 Hz), 0.94 (m, 3H);
MS (m/e) (positive-FAB) 742.5 (M+ + 1).
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-bu toxyp h osp h or yloxym eth yl)-4-(2-h ex-1-yn yl-
6-m eth ylam in opu r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester (50).
2-Hexynyl bis(di-tert-butyl phosphate) derivative 50 (20.1 mg,
72%) was prepared by a method similar to that for 44, except
2-hexynyl-6-methylaminopurine 42 (20 mg, 0.0378 mmol) was
used as the starting material: 1H NMR (CDCl3) δ 8.14 (s, 1H),
5.77 (bs, 1H), 5.33 (dd, 1H, J ) 8.2, 14.8 Hz), 5.21 (d, 1H, J )
6.9 Hz), 4.67 (dd, 1H, J ) 4.4, 11.3 Hz), 3.83 (dd, 1H, J ) 6.6,
11.3 Hz), 3.25 (bs, 3H), 2.46 (t, 2H, J ) 7.1 Hz), 2.29 (dd, 1H,
J ) 8.0, 15.4 Hz), 2.08 (m, 1H), 1.60-1.82 (m, 5H), 1.499 (s,
9H), 1.488 (s, 9H), 1.472 (s, 9H), 1.462 (s, 9H), 1.12 (dd, 1H, J
) 4.4, 5.8 Hz), 0.9 (m, 1H), 0.94 (t, 3H, J ) 7.1 Hz); MS (m/e)
(positive-FAB) 740.5 (M+ + 1).
(1′R,2′S,4′S,5′S)-P h osp h or ic Acid Di-ter t-bu tyl Ester
1-(Di-ter t-b u t oxy-p h osp h or yloxym et h yl)-4-(6-ch lor o-2-
tr im eth ylsta n n ylp u r in -9-yl)bicyclo[3.1.0]h ex-2-yl Ester
(52). To a solution of 51 (2.7 mg, 0.0034 mmol) and PdCl2-
(PPh3)2 (3.5 mg, 0.0050 mmol) in dioxane (0.30 mL) was added
Me3SnSnMe3 (20 mg, 0.061 mmol) and the mixture stirred at
60 °C for 3.5 h. The solvent was removed under vacuum and
the residue obtained was purified by preparative TLC (AcOEt),
which furnished 52 (0.7 mg, 25%): 1H NMR (CDCl3) δ 8.42 (s,
1H), 5.40-5.20 (m, 2H), 4.65 (m, 1H), 3.90 (m, 1H), 2.40-2.25
(m, 1H), 2.25-2.10 (m, 1H), 1.90-1.80 (m, 1H), 1.50 (s, 9H),
1.48 (s, 18H), 1.44 (s, 9H), 1.25-1.20 (m, 1H), 1.05-1.00 (m,
1H), 0.43 (s, 9H); MS (m/ e) (positive-FAB) 827 (M++1), 849
(M++Na).
29) was dissolved in 40% MeNH2 in water (0.50 mL) and
stirred for 3 h at room temperature. The solvent was removed
under reduced pressure. The residue (crude 12) was purified
by HPLC (system A or system B).
En zym a tic Syn th esis of 5. ATP (20 nmol, 2 µL of a 10-
mM solution in water) and polynucleotide kinase (Sigma, 10
IU) were added to 20 µL of a buffer consisting of 50 mM Tris
HCl, 10 mM MgCl2, 5 mM dithiothreitol, 0.1 mM spermidine,
and 0.1 mM EDTA. The nucleotide substrate 5841 (1 µL of a
10-mM solution in water) was then added, and the mixture
was mixed with vortexing and incubated at 37 °C for the time
indicated (18 h incubation was found to be optimal). Aliquots
(10 µL) were injected for HPLC analysis. A C18 Zorbax column
(Agilent) was used, with the flow rate 1 mL/min and the mobile
phase consisting of a gradient of acetonitrile/0.1 M triethy-
lammonium acetate (aq) of 10/90 to 15/85 in 10 min, then to
30/70 in 10 min. The product of the enzymatic transformation
(5) was detected in ∼40% yield, as judged with HPLC. The
retention time of the product was 6.2 min, compared with the
starting material, which had a retention time of 8.1 min. These
three individual peaks were collected and lyophilized. Mass
spectrometry (FAB) confirmed the identity of the starting
material (m/z 387, M - 1) and product 5 (m/z 468, M - 1).
The side product at retention time 11.6 min displayed an m/z
of 310, consistent with the corresponding nucleoside (M + 1).
An unidentified impurity present in the starting material was
also detected at 7.7 min.
Compound 58 was found to be chemically stable. The HPLC
profile of a solution in H2O/acetonitrile/buffer was unchanged
following 60 h incubation at 37 °C. However, when enzyme
was added in the absence of ATP, ∼20% of the compound was
dephosphorylated during an incubation of 17 h to the nucleo-
side 59 (HPLC retention time ) 11.6 min). Under the same
incubation conditions in the presence of the enzyme, 5 was
stable.
P h a r m a cologica l An a lyses. The affinities of 2-substituted
(N)-methanocarbo bisphosphate analogues for the human P2Y1
receptor were directly determined by using [3H]5 in a radio-
ligand binding assay, as we recently described in detail.31
Briefly, the human P2Y1 receptor was expressed to high levels
in Sf9 insect cells with a recombinant baculovirus. Membranes
prepared from these cells were incubated for 30 min at 4 °C
in the presence of ∼20 nM [3H]5 and a wide range of
concentrations of the newly synthesized analogues. Binding
reactions were terminated by the addition of ice-cold tris-
(hydroxymethyl)aminomethane (Tris) wash buffer (10 mM
Tris, pH 7.5, and 145 mM NaCl) to the samples followed by
rapid filtration over GF/A glass-fiber filters. Each filter was
washed with an additional 4 mL of wash buffer, and radio-
activity retained by the filters was quantified by liquid
scintillation spectrometry. All assays were carried out in
triplicate, and competition curves for all molecules were
generated in three separate experiments. IC50 values were
determined from each competition curve, and a Ki value was
calculated for each analogue according to the relationship Ki
) IC50/1 + [[3H]5]/Kd of [3H]5, where the Kd of [3H]5 determined
in separate experiments was 8 nM.
P2Y receptor-promoted stimulation of inositol phosphate
formation was measured at human P2Y1 receptors stably
expressed in 1321N1 human astrocytoma cells as previously
described.19,42,43 The K0.5 values were averaged from three to
eight independently determined concentration-effect curves for
each compound. Briefly, cells plated in 24-well dishes were
labeled in inositol-free medium (DMEM; Gibco, Gaithersburg,
MD) containing 1.0 µCi of 2-[3H]myo-inositol (20 Ci/mmol;
American Radiolabeled Chemicals, Inc., St. Louis, MO) for 18-
24 h in a humidified atmosphere of 95% air/5% CO2 at 37 °C.
Phospholipase C activity was measured the following day by
quantitating [3H]inositol phosphate accumulation after a 10-
min incubation at 37 °C in the presence of 10 mM LiCl. Total
[3H]inositol phosphates were quantified by anion-exchange
chromatography as previously described.42,43
Sm a ll-Sca le Con ver sion of Com p ou n d 52 in to Com -
p ou n d 12. A 20 µL (0.35 µmol) portion of an iodine solution
(4.5 mg of I2 in 1.00 mL of AcOEt) was added to a solution of
52 (0.15 mg, 0.18 µmol) in AcOEt (0.100 mL) and the mixture
stirred at room temperature for 10 min. Then 0.01 mL of 5%
aqueous NaHSO3 was added to stop the reaction, and the
solvent was removed under vacuum. The residue (crude 29)
was dissolved in 5% TFA/CH2Cl2 (0.50 mL) and stirred for 3 h
at 25 °C. The solvent was removed under reduced pressure,
and the residue was quenched by addition of 5.0 mL of
triethylammonium bicarbonate buffer (1.0 M). The mixture
was subsequently frozen and lyophilized. The residue (crude
Da ta An a lysis. IC50 values obtained in radioligand binding
assays and in assays of inhibition of 2MeSADP-stimulated

4986 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Kim et al.
(17) Nandanan, E.; Camaioni, E.; J ang, S. Y.; Kim, Y.-C.; Cristalli,
G.; Herdewijn, P.; Secrist, J . A.; Tiwari, K. N.; Mohanram, A.;
Harden, T. K.; Boyer, J . L.; J acobson, K. A. Structure activity
relationships of bisphosphate nucleotide derivatives as P2Y1
receptor antagonists and partial agonists. J . Med. Chem. 1999,
42, 1625-1638.
(18) Nandanan, E.; J ang, S. Y.; Moro, S.; Kim, H.; Siddiqi, M. A.;
Russ, P.; Marquez, V. E.; Busson, R.; Herdewijn, P.; Harden, T.
K.; Boyer, J . L.; J acobson, K. A. Synthesis, biological activity,
and molecular modeling of ribose-modified adenosine bisphos-
phate analogues as P2Y1 receptor ligands. J . Med. Chem. 2000,
43, 829-842.
(19) Kim, H. S.; Barak, D.; Harden, T. K.; Boyer, J . L.; J acobson, K.
A. Acyclic and cyclopropyl anlaogues of adenosine bisphosphate
antagonists of the P2Y1 receptor: Structure activity relation-
ships and receptor docking. J . Med. Chem. 2001, 44, 3092-3108.
(20) Boyer, J . L.; Adams, M.; Ravi, R. G.; J acobson, K. A.; Harden,
T. K. 2-Chloro-N6-methyl-(N)-methanocarba-2-deoxyadenosine-
inositol phosphate accumulation were calculated by a four-
parameter logistic equation and the GraphPad software pack-
age (GraphPad, San Diego, CA). All concentration-effect
curves were repeated in at least three separate experiments,
carried out in duplicate or triplicate with different membrane
preparations or different 1321N1 cell cultures.
Ack n ow led gm en t. This work was supported by
USPHS grants GM38213 and HL54889 (to T.K.H.).
M.O. is on sabbatical from Toray Industries (Kamakura,
J apan) and thanks them for financial support. Mass
spectral measurements were carried out by Victor
Livengood and NMR by Wesley White (NIDDK). We
thank Ariel Gross (NIDDK) for proofreading the manu-
script.
3,5-bisphosphate is
a selective high affinity P2Y1 receptor
antagonist. Br. J . Pharmacol. 2002, 135, 2004-2010.
(21) Fischer, B.; Boyer, J . L.; Hoyle, C. H. V.; Ziganshin, A. U.;
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, 3937-3946.
(22) Boyer, J . L.; Siddiqi, S.; Fischer, B.; Romera-Avila, T.; J acobson,
K. A.; Harden, T. K. Identification of potent P2Y purinoceptor
agonists that are derivatives of adenosine 5′-monophosphate. Br.
J . Pharmacol. 1996, 118, 1959-1964.
Refer en ces
(1) von Ku¨gelgen, I.; Wetter, A. Molecular pharmacology of P2Y-
receptors.Naunyn Schmiedebergs Arch. Pharmacol. 2000, 362,
310-323.
(2) Khakh, B. S.; Burnstock, G.; Kennedy, C.; King, B. F.; North,
R. A.; Seguela, P.; Voigt, M.; Humphrey, P. P. International
union of pharmacology. XXIV. Current status of the nomencla-
ture and properties of P2X receptors and their subunits. Phar-
macol. Rev. 2001, 53, 107-118.
(23) Fischer, B.; Chulkin, A.; Boyer, J . L.; Harden, T. K.; Gendron,
F. P.; Beaudoin, A. R.; Chapal, J .; Hillaire-Buys, D.; Petit, P.
2-Thioether 5′-O-(1-thiotriphosphate)adenosine derivatives as
new insulin secretagogues acting through P2Y-Receptors. J .
Med. Chem. 1999, 42, 3636-3646.
(24) Raboisson, P.; Baurand, A.; Cazenave, J . P.; Gachet, C.; Retat,
M.; Spiess, B.; Bourguignon, J . J . Novel antagonists acting at
the P2Y1 purinergic receptor: Synthesis and conformational
analysis using potentiometric and nuclear magnetic resonance
titration techniques. J . Med. Chem. 2002, 45, 962-972.
(25) Raboisson, P.; Baurand, A.; Cazenave, J . P.; Gachet, C.; Schultz,
D.; Spiess, B.; Bourguignon, J . J . A general approach toward
the synthesis of C-nucleoside pyrazolo[1,5-a]-1,3,5-triazines and
their 3′,5′-bisphosphate C-nucleotide analogues as the first
reported in vivo stable P2Y1-receptor antagonists. J . Org. Chem.
2002, 67, 8063-8071.
(26) Ingall, A. H.; Dixon, J .; Bailey, A.; Coombs, M. E.; Cox, D.;
McInally, J . I.; Hunt, S. F.; Kindon, N. D.; Theobald, B. J .; Willis,
P. A.; Humphries, R. G.; Leff, P.; Clegg, J . A.; Smith, J . A.;
Tomlinson, W. Antagonists of the platelet P2T receptor: A novel
approach to antithrombotic therapy. J . Med. Chem. 1999, 42,
213-220.
(27) Brown, S. G.; King, B. F.; Kim, Y.-C.; Burnstock, G.; J acobson,
K. A. Activity of novel adenine nucleotide derivatives as agonists
and antagonists at recombinant rat P2X receptors. Drug Devel.
Res. 2000, 49, 253-259.
(28) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang,
J .; Wagner, R. W.; Matteucci, M. D. Nucleosides with a twist.
Can fixed forms of sugar ring pucker influence biological activity
in nucleosides and oligonucleotides? J . Med. Chem. 1996, 39,
3739-3747.
(29) Ezzitouni, A.; Russ, P.; Marquez, V. E. (1S,2R)-[(Benzyloxy)-
methyl]cyclopent-3-enol. A versatile synthon for the preparation
of 4′,1′a-methano and 1′,1′a-methano carbocyclic nucleosides. J .
Org. Chem. 1997, 62, 4870-4873.
(30) Siddiqui, M. A.; Ford, H., J r.; George, C.; Marquez, V. E.
Synthesis, conformational analysis, and biological activity of a
rigid carbocyclic analogue of 2′-deoxyaristeromycin built on a
bicyclo[3.1.0]hexane template. Nucleosides Nucleotides 1996, 15,
235-250.
(31) Waldo, G. L.; Corbitt, J .; Boyer, J . L.; Ravi, G.; Kim, H. S.; J i,
X. D.; Lacy, J .; J acobson, K. A.; Harden, T. K. Quantitation of
the P2Y1 receptor with a high affinity radiolabeled antagonist.
Mol. Pharmacol. 2002, 62, 1249-1257.
(3) Hollopeter, G.; J antzen, H. M.; Vincent, D.; Li, G.; England, L.;
Ramakrishnan, V.; Yang, R. B.; Nurden, P.; Nurden, A.; J ulius,
D.; Conley, P. B. Identification of the platelet ADP receptor
targeted by antithrombotic drugs. Nature 2001, 409, 202-207.
(4) Communi, D.; Suarez Gonzalez, N.; Detheux, M.; Bre´zillon, S.;
Lannoy, V.; Parmentier, M.; Boeynaems, J .-M. Identification of
a novel human ADP receptor coupled to Gi. J . Biol. Chem. 2001,
276, 41479-41485.
(5) Abbracchio, M. P.; Boeynaems, J .-M.; Barnard, E. A.; Boyer, J .
L.; Kennedy, C.; Miras-Portugal, M. T.; King, B. F.; Gachet, C.;
J acobson, K. A.; Weisman, G. A.; Burnstock, G. The UDP-glucose
receptor renamed the P2Y14 receptor. Trends Pharmacol. Sci.
2003, 24, 52-55.
(6) Chambers, J . K.; Macdonald, L. E.; Sarau, H. M.; Ames, R. S.;
Freeman, K.; Foley, J . J .; Zhu, Y.; McLaughlin, M. M.; Murdock,
P.; McMillan, L.; Trill, J .; Swift, A.; Aiyar, N.; Taylor, P.; Vawter,
L.; Naheed, S.; Szekeres, P.; Hervieu, G.; Scott, C.; Watson, J .
M.; Murphy, A. J .; Duzic, E.; Klein, C.; Bergsma, D. J .; Wilson,
S.; Livi, G. P. A G protein-coupled receptor for UDP-glucose. J .
Biol. Chem. 2000, 275, 10767-10771.
(7) Mohanty, J . G.; Raible, D. G.; McDermott, L. J .; Pelleg, A.;
Schulman, E. S. Effects of purine and pyrimidine nucleotides
on intracellular Ca2+ in human eosinophils: Activation of
purinergic P2Y receptors. J . Allergy Clin. Immunol. 2001, 107,
849-855.
(8) J anssens, R.; Communi, D.; Pirotton, S.; Samson, M.; Parmen-
tier, M.; Boeynaems, J . M. Cloning and tissue distribution of
the human P2Y1 receptor. Biochem. Biophys. Res. Commun.
1996, 221, 588-593.
(9) Webb, T. E.; Simon, J .; Krishek, B. J .; Bateson, A. N.; Smart, T.
G.; King, B. F.; Burnstock, G.; Barnard, E. A. Cloning and
functional expression of a brain G-protein-coupled ATP receptor.
FEBS Lett. 1993, 324, 219-225.
(10) Moore, D.; Chambers, J .; Waldvogel, H.; Faull, R.; Emson, P. J .
Regional and cellular distribution of the P2Y1 purinergic receptor
in the human brain: Striking neuronal localisation. Comput.
Neurol. 2000, 421, 374-384.
(11) Franke, H.; Krugel, U.; Schmidt, R.; Grosche, J .; Reichenbach,
A.; Illes, P. P2 receptor-types involved in astrogliosis in vivo.
Br. J . Pharmacol. 2001, 134, 1180-1189.
(12) Czajkowski, R.; Lei, L.; Sabala, P.; Baranska, J . ADP-evoked
phospholipase C stimulation and adenylyl cyclase inhibition in
glioma C6 cells occur through two distinct nucleotide receptors,
P2Y1 and P2Y12. FEBS Lett. 2002, 513, 179-183.
(13) Moore, D.; Iritani, S.; Chambers, J .; Emson, P. Immunohis-
tochemical localization of the P2Y1 purinergic receptor in Alzhe-
imer’s disease. Neuroreport 2000, 11, 3799-3803.
(14) Kaiser, R. A.; Buxton, I. L. Nucleotide-mediated relaxation in
guinea-pig aorta: Selective inhibition by MRS2179. Br. J .
Pharmacol. 2002, 135, 537-545.
(15) Eckly, A.; Gendrault, J . L.; Hechler, B.; Cazenave, J . P.; Gachet,
C. Differential involvement of the P2Y1 and P2YT receptors in
the morphological changes of platelet aggregation. Thromb.
Haemost. 2001, 85, 694-701.
(16) J acobson, K. A. Purine and pyrimidine nucleotide (P2) receptors.
Ann. Rep. Med. Chem. 2002, 37, 75-84.
(32) Yoshimura, Y.; Moon, H. R.; Choi, Y.; Marquez, V. E. Enantio-
selective synthesis of bicyclo[3.1.0]hexane carbocyclic nucleosides
via a lipase-catalyzed asymmetric acetylation. Characterization
of an unusual acetal byproduct. J . Org. Chem. 2002, 67, 5938-
5945.
(33) Perich, J . W.; J ohns, R. B. Di-tert-butyl N, N-diethylphosphora-
midite. A new phosphitylating agent for the efficient phospho-
rylation of alcohols. Synthesis 1988, 2, 142-144.
(34) Nair, V.; Fasbender, A. J . C-2 Functionalized N6-cyclosubstituted
adenosines: Highly selective agonists for the adenosine A1
receptor. Tetrahedron 1993, 49, 2169-2184.

2-Substitution of Adenine Nucleotide Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4987
(35) Wilds, C. J .; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M.
Selenium-assisted nucleic acid crystallography: Use of phos-
phoroselenoates for MAD phasing of a DNA structure. J . Am.
Chem. Soc. 2002, 124, 14910-14916.
ing kit/generator for 5-radiohalogenated uridines J ournal of
Labeled Compounds and Radiopharceuticals 1994, 34, 513-522.
(43) Suzuki, M.; Doi, H.; Bjorkman, M.; Andersson, Y.; Langstom,
B.; Watanabe, Y.; Noyori, R. Rapid coupling of methyl iodide
with aryltributylstannanes mediated by palladium (0) com-
plexes: A general protocol for the synthesis of 11CH3-labeled PET
tracers Chem. Eur. J . 1997, 3, 2039-2042.
(44) Chaconas, G.; v. d. Sande, J . H. 5′-32P labeling of RNA and DNA
restriction fragments. Methods Enzymol. 1980, 65, 75-85.
(45) Xu, B.; Stephens, A.; Kirschenheuter, G.; Greslin, A. F.; Cheng,
X.; Sennelo, J .; Cattaneo, M.; Zighetti, M. L.; Chen, A.; Kim, S.
A.; Kim, H. S.; Bischofberger, N.; Cook, G.; J acobson, K. A.
Acyclic analogues of adenosine bisphosphates as P2Y receptor
antagonists: Phosphate substitution leads to multiple pathways
of inhibition of platelet aggregation. J . Med. Chem. 2002, 45,
5694-5709.
(46) Lee, K.; Cass, C.; J acobson, K. A. Synthesis using ring closure
metathesis and effect on nucleoside transport of a (N)-metha-
nocarba S-(4-nitrobenzyl)thioinosine derivative. Org. Lett. 2001,
3, 597-599.
(47) Harden, T. K.; Hawkins, P. T.; Stephens, L.; Boyer, J . L.;
Downes, P. Phosphoinositide hydrolysis by guanosine 5′-[gamma-
thio]triphosphate-activated phospholipase C of turkey erythro-
cyte membranes. Biochem. J . 1988, 252, 583-593.
(36) Robins, M. J .; Uznanski, B. Nucleic acid related compounds. 34.
Nonaqueous diazotization of tert-butyl nitrite. Introduction of
fluorine, chlorine, and bromine at C-2 of purine nucleosides. Can.
J . Chem. 1981, 59, 2608-2611.
(37) Kim, H. O. Synthesis of 2-alkyl substituted-N6-methyladenine
derivatives as potential adenosine receptor ligand. Arch. Pharm.
Res. 2001, 6, 508-513.
(38) Coenen, H. H.; Moerlein, S. M.; Stocklin, G. No-Carrier-Added
Radiohalogenation Methods with Heavy Halogens. Radiochimica
Acta 1983, 34, 47-68.
(39) Wilbur, D. S.; Hamlin, D. K.; Srivastava, R. R.; Burns, H. D.
Synthesis and radioiodination of N-Boc-p-(tri-butylstannyl)-l-
phenylalanine tetrafluorophenyl ester: Preparation of a radio-
labeled phenylalanine derivative for peptide synthesis. Biocon-
jugate Chem. 1993, 4, 574-580.
(40) Foged, C.; Halldin, C.; Hiltunen, J .; Braestrup, C.; Thomsen, C.;
Hansen, H. C.; Suhara, T.; Pauli, S.; Swahn, C. G.; Karlsson,
P.; Larsson, S.; Farde, L. Development of 123I-labeled NNC 13-
8241 as a radioligand for SPECT visualization of benzodiazepine
receptor binding. Nucl. Med. Biol. 1996, 23, 201-209.
(41) Rasmussen, M.; Leonard, N. J . The synthesis of 3-(2′-Deoxy-D-
ribofuranosyl)adenine. Application of a new protecting group,
Pivaloyloxymethyl (Pom) J . Am. Chem. Soc. 1967, 89, 9 (21),
5439-5445.
(48) Boyer, J . L.; Downes, C. P.; Harden, T. K. Kinetics of activation
of phospholipase C by P2Y purinergic receptor agonists and
guanine nucleotides. J . Biol. Chem. 1989, 264, 884-890.
(42) Baranowska-Kortylewicz, J .; Helseth, L. D.; Lai, J .; Schneider-
man, M. H.; Schneiderman, G. S.; Dalrymple, G. V. Radiolabel-
J M030127+