Acyclic and cyclopropyl analogues of adenosine bisphosphate antagonists of the P2Y1 receptor: Structure-activity relationships and receptor docking

Article abstract of DOI:10.1021/jm010082h

The activation of P2Y₁ receptors in platelets contributes to platelet aggregation, and selective antagonists are sought as potential antithrombotic agents. We reported (Kim et al. J. Med. Chem. 2000, 43, 746-755) that acyclic analogues of adeni

Full text of DOI:10.1021/jm010082h

3092
J . Med. Chem. 2001, 44, 3092-3108
Acyclic a n d Cyclop r op yl An a logu es of Ad en osin e Bisp h osp h a te An ta gon ists of
th e P 2Y1 Recep tor : Str u ctu r e-Activity Rela tion sh ip s a n d Recep tor Dock in g
Hak Sung Kim,‡ Dov Barak,‡,X T. Kendall Harden,† J ose´ L. Boyer,† and Kenneth A. J acobson*,‡
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, and Department of Pharmacology,
University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7365
Received February 21, 2001
The activation of P2Y1 receptors in platelets contributes to platelet aggregation, and selective
antagonists are sought as potential antithrombotic agents. We reported (Kim et al. J . Med.
Chem. 2000, 43, 746-755) that acyclic analogues of adenine nucleotides, containing two
phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of
adenine, are moderately potent P2Y1 receptor antagonists. In this study we have varied the
chain structure, to include asymmetric substitution, olefinic, and cyclopropyl groups. These
antagonists inhibited the stimulation of phospholipase C in turkey erythrocyte membranes
induced by 30 nM 2-MeS-ADP in the micromolar range. In the series of symmetrically branched
aliphatic groups substituted with two phosphate groups, the optimal antagonist potency
occurred with the 2-methylpropyl group. A 2-chloro-N6-methyladenine derivative, 2-[2-(2-chloro-
6-methylaminopurin-9-yl)methyl]propane-1,3-bisoxy(diammoniumphosphate) (7), was a full
antagonist at the P2Y1 receptor with an IC50 value of 0.48 µM. Esterification of one of the
phosphate groups or substitution with O-acetyl greatly reduced the antagonist potency at the
P2Y1 receptor. Removal of a methylene group of 7 or inclusion of an olefinic or cyclopropyl
group also reduced potency. A pair of enantiomeric glycerol derivatives demonstrated a 5-fold
stereoselectivity for the S-isomer. Stereoisomerically defined analogues of 7 containing a
cyclopropyl group in place of the branched carbon were less potent than 7 as antagonists, with
IC50 values of 2-3 µM. No agonist activity was observed for these analogues. A new rhodopsin-
based molecular model of the P2Y1 receptor indicated that the optimal docked orientation of
the two monophosphate moieties relative to the adenine N6 (compared to a rigid, bicyclic
analogue) was consistent with the dependence of antagonist potency on chain length. The 3′-
phosphate was predicted to occupy a restricted space, deeper in the binding cleft than the
5′-phosphate location. In summary, modification of the flexible spacer chain linking bisphosphate
groups to the adenine moiety provided many moderately potent antagonists.
In tr od u ction
reported,13,14 and the absence of the P2Y1 receptors in
platelets of these mice interfered with ADP-promoted
aggregation.
P2 receptors, which are activated by purine and/or
pyrimidine nucleotides, consist of two families:
G
We have developed selective P2Y1 receptor agonists
and antagonists of high affinity for use as pharmaco-
logical probes.15-20 With simplified pharmacophores
we are exploring the steric and electronic constraints
of the receptor binding site and the structural basis of
receptor activation. Various naturally occurring bispho-
sphates of adenosine (e.g., adenosine 3′,5′-bisphosphate)
act as competitive antagonists at the P2Y1 receptor,15
and N6-methyl and 2-chloro groups on the adenine
moiety have been found to increase the potency and
selectivity of the bisphosphate derivatives acting as
antagonists.16 Among ribose modifications, both rigidi-
fying (e.g., bicyclic carbocycles)19 and adding flexibility
(e.g., acyclic modifications)20 have led to effective P2Y1
receptor ligands. Rigid rings in the bicyclic “methano-
carba” series (Figure 1) have defined a preference at the
receptor binding site for the northern (N) conformation.
Compound 1 ((1R,2S,4S,5S)-1-[(phosphato)methyl]-4-(6-
aminopurin-9-yl)bicyclo [3.1.0]-hexane-2-phosphate), an
(N)-methanocarba analogue, is a potent agonist (EC50
) 155 nM), 120-fold more potent than the corresponding
protein-coupled receptors, termed P2Y, and ligand-gated
cation channels, termed P2X. Numerous subtypes have
been cloned within each family regulating a diverse
range of functions in the central and peripheral nervous
systems, the cardiovascular system, the endocrine sys-
tem, lungs, intestines, muscle, and the immune sys-
tem.1-3
The P2Y1 receptor has been cloned from chick, turkey,
human, rat, and mouse.4-6 ADP induces aggregation of
human platelets by acting at both P2Y1 and P2Y12
receptors.7-10 The P2Y1 receptor antagonist N6-methyl-
2′-deoxyadenosine 3′,5′-bisphosphate has been shown to
inhibit ADP-induced platelet aggregation.11,12 A mouse
line lacking expression of the P2Y1 receptor has been
* Address correspondence to Dr. Kenneth A. J acobson, Chief,
Molecular Recognition Section, Bldg. 8A, Rm. B1A-19, NIH, NIDDK,
LBC, Bethesda, MD 20892-0810. Tel: (301) 496-9024. Fax: (301) 480-
8422. E-mail: kajacobs@helix.nih.gov.
‡ National Institute of Diabetes, Digestive and Kidney Diseases,
NIH.
† University of North Carolina, School of Medicine.
X On leave from Israel Institute for Biological Research, Ness-Ziona,
Israel.
10.1021/jm010082h CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/22/2001

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3093
Sch em e 2. Synthesis of Isobutyl Bisphosphates,
Compounds 6 and 7a
F igu r e 1. Structures of rigid and acyclic nucleotide analogues
found to be receptor agonist (1) or antagonists (2-4).
southern (S) isomer. Compound 2, the corresponding
2-Cl-N6-methyl analogue, is a potent P2Y1 receptor
antagonist (IC50 ) 52 nM).
The present study explores the SAR of nucleotide
analogues containing acyclic aliphatic chains, such as
3 and 4, which act as P2Y1 receptor antagonists.20
Compound 3 (2-[2-(6-methylaminopurin-9-yl)ethyl]-
propane-1,3-bisphosphate) and its 2-chloro- analogue
were found to be selective antagonists of the P2Y1
receptor with EC50 values of 1.60 and 0.84 µM, respec-
tively. Compound 4 was shown to be inactive at the rat
P2X1 receptor.21 We have introduced shorter chains than
in the previous study20 and also elements of rigidity,
a
Reaction conditions: (i) TBSCl, TEA, DMAP, CH2Cl2, rt,
overnight, 99%; (ii) BH3, THF, rt, overnight, then H2O2, NaOH,
60%; (iii) MsCl, TEA, CH2Cl2, rt, 1 h; (iv) 2-chloro- N6-methylami-
nopurine (for 32) or N6-methylaminopurine (for 31), K2CO3, 18-
crown-6, DMF, 60% two-step yield for 31, 72% two-step yield for
32; (v) AcOH:H2O:THF ) 3:2:2, rt, 2 d, 79% for 33, 93% for 34;
(vi) tetrabenzylpyrophosphate, NaH, THF, rt, 1 h, 81% for 35, 53%
for 36; (vii) BCl3, CH2Cl2, 5 °C, 2 d, 75% for 6, 41% for 7.
such as olefinic and cyclopropyl groups. Molecular
modeling of the receptor and its docked ligands, by
homology to the recently reported high-resolution struc-
ture of rhodopsin,22 was carried out. In general, modify-
ing the flexible spacer chain linking bisphosphate
groups to the adenine moiety, within spatial require-
ments as defined by the docked positions of the phos-
phates relative to the adenine N6, provided many
moderately potent antagonists.
Sch em e 1. Synthesis of Isopropyl Bisphosphate,
Compound 5a
Resu lts
Ch em ica l Syn th esis. Acyclic and cyclopropyl ana-
logues 5-20 of adenosine bisphosphates were prepared
in the ammonium salt form and tested biologically as
antagonists at the P2Y1 receptor (Table 1). The nucleo-
tide analogues were characterized by high-resolution
mass spectrometry and by HPLC in two different
solvent systems (Table 2).
The synthesis of these nucleotide analogues and the
corresponding nucleoside precursors are shown in
Schemes 1-10. Symmetrical (Schemes 1, 2, and 4) and
chiral acyclic aliphatic (Scheme 5), olefinic (Scheme 6),
and cyclopropyl moieties (Schemes 7-10) were included.
In general, introduction of the 2-chloro-N6-methyl- (or
2-H) adenine moiety was accomplished either by direct
alkylation of a mesylate with 2-chloro-N6-methylamino-
purine or by a Mitsunobu reaction utilizing 2,6-dichlo-
ropurine, followed by displacement of 6-chloro with
methylamine. During the displacement, the diols were
protected by silylation (e.g., TBS, tert-butyldimethyl-
silyl) or masked as a precursor olefin (Scheme 4).
Subsequently, phosphorylation of each nucleoside
analogue (yields in Table 2) was achieved in two steps.
First, benzyl- or tert-butyl-protected phosphate groups
a
Reaction conditions: (i) NaBH4, EtOH, 0 °C, 30 min, 91%; (ii)
2,6-dichloropurine, DEAD, PPh3, THF, rt, overnight, 68%; (iii)
CH3NH2, THF, rt, 4 h, 88%; (iv) Dowex 50x-8 200, MeOH, reflux,
overnight, 75%; (v) Et2NP(OtBu)2, tetrazole, THF, rt, 20 min, then
MCPBA, 73%; (vi) Dowex 50x-8 200, MeOH:H2O ) 1:1, 80 °C, 16
h, 63%.

3094 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Kim et al.
Ta ble 1. In Vitro Pharmacological Data for Antagonism of Turkey Erythrocyte P2Y1 Receptors, Indicated by the Inhibition of PLC
Stimulation Elicited by 30 nM 2-MeSADP, for at Least Three Separate Determinations. “P” ) Phosphate
a
At 100 µM, none of the acyclic adenosine bisphosphate nucleotide analogues displayed agonist activity at the P2Y1 receptor in turkey
erythrocyte membranes.

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3095
Ta ble 2. Synthetic Data for Nucleotide Derivatives, Including Structural Verification Using High Resolution Mass Spectroscopy and
Purity Verification Using HPLC
HPLC (rt; min)a
FAB (M - H+)
system A
(purity %)
system B
(purity %)
no.
formula
C9H13O8N5ClP2
calcd
found
yield (%)b
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
415.9928
396.0474
430.0084
350.0421
458.0397
392.0527
509.9748
458.0397
415.9928
415.9928
442.0084
458.0397
458.0397
456.0241
442.0084
456.0241
415.9940
396.0482
430.0094
350.0423
458.0416
392.0517
509.9751
458.0385
415.9945
415.9938
442.0086
458.0392
458.0415
456.0256
442.0089
456.0251
4.981 (97)
7.779 (99)
6.655 (95)
8.963 (97)
13.579 (95)
7.452 (99)
6.062 (99)
7.865 (99)
5.960 (100)
5.314 (99)
5.279 (99)
10.648 (96)
10.391 (99)
7.882 (100)
5.036 (96)
7.211 (98)
8.900 (98)
6.639 (98)
6.453 (97)
2.942 (99)
5.534 (99)
4.206 (99)
7.970 (96)
6.205 (100)
8.466 (99)
7.610 (100)
8.565 (99)
8.564 (98)
8.457 (99)
6.176 (100)
8.592 (98)
6.522 (100)
63
70
41
79
76
59
72
69
44
55
63
77
57
71
61
55
C
C
C
C
C
C
C
10H16O8N5P2
10H15O8N5ClP2
10H14O5N5ClP
12H19O8N5ClP2
12H16O6N5ClP
10H16O11N5ClP3
12H19O8N5ClP2
C9H13O8N5ClP2
C9H13O8N5ClP2
C
C
C
C
C
C
11H15O8N5ClP2
12H19O8N5ClP2 (FAB positive)
12H19O8N5ClP2 (FAB positive)
12H17O8N5ClP2
11H15O8N5ClP2
12H17O8N5ClP2
a
Purity of each derivative was g95%, as determined using HPLC with two different mobile phases. System A: gradient of 0.1 M
b
TEAA/CH3CN from 95/5 to 40/60. System B: gradient of 5 mM TBAP/CH3CN from 80/20 to 40/60. The percent yields refer to yield for
deprotection sequence.
Sch em e 3. Synthesis of Asymmetrically Phosphorylated Isobutyl Derivatives, 8-11a
a
Part A, reaction conditions: (i) TBSCl, imidazole, DMF, rt, overnight, 38%; (ii) Et2NP(OtBu)2, tetrazole, THF, rt, 20 min, then MCPBA,
83%; (iii) TBAF, THF, rt, 10 min, 76%; (iv) 5% TFA in CH2Cl2, rt, 2 h, 79%; (v) (MeO)2P(O)Cl, NaH, THF, rt, 1 h, 31%; (vi) 5% TFA in
CH2Cl2, rt, 2 h, 76%. Part 3B, reaction conditions: (i) Ac2O, TEA, CH2Cl2, rt, 2 h, 96%; (ii) 5% TFA in CH2Cl2, rt, 30 min, 59%; (iii)
iPr2NP(OBn)2, tetrazole, THF, rt, 20 min, then MCPBA, 45%; (iv) 5% TFA in CH2Cl2, rt, 30 min, 89%; (v) C(O)(imi)2, NEt3‚H3PO4, DMF,
rt, 3 d, 74%; (vi) TMSBr, rt, 10 min, 72%.
were incorporated, thus facilitating product purification
at this intermediate stage. Benzyl phosphates were
introduced by the treatment of diols with NaH and
tetrabenzylpyrophosphate (Schemes 2, 3B, and 7).20 The
yields for this method of phosphorylation were variable,
being limited by the solubility of the diols in THF. In
such cases, use of DMF as solvent failed to give the
desired product. Alternatively, bisphosphates were made
via the tert-butyl phosphate esters,23 which were syn-
thesized by treatment of diols with di-tert-butyl dieth-
ylphosphoramidite and tetrazole in THF, followed by
oxidation with m-chloroperoxybenzoic acid (MCPBA,
Schemes 1, 3A, 4-6, and 8-10). The two-step synthesis
of tert-butyl phosphates had the following advantage
over the tetrabenzylpyrophosphate method: limited
solubility of the diols in THF could be overcome since
these conditions allowed use of excess phosphoramidite
and tetrazole reagents with overnight stirring, which
either slowly or rapidly resulted in a homogeneous
reaction mixture without significant side reactions.
To synthesize compound 11, containing both mono-
phosphate and diphosphate groups, it was necessary to

3096 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Sch em e 4. Synthesis of Compound 12a
Kim et al.
Sch em e 6. Synthesis of Isopentenyl Bisphosphate,
Compound 10a
a
Reaction conditions: (i) MsCl, TEA, CH2Cl2, rt, 1 h; (ii)
2-chloro-N6-methylaminopurine, K2CO3, 18-crown-6, DMF, 60-
70 °C, overnight, 60% two-step yield; (iii) (a) OsO4, NaIO4, acetone/
H2O ) 4:1, rt, 4 h, (b) NaBH4, EtOH, 0 °C, 2 h, 32% two-step yield;
(iv) Et2NP(OtBu)2, tetrazole, THF, rt, 1 h then MCPBA, 73%; (v)
5% TFA in CH2Cl2, rt, 30 min, 69%.
Sch em e 5. Synthesis of R- and S-Glyceryl
Bisphosphate Derivatives, Compounds 13 and 14a
a
Reaction conditions: (i) EtO2CCH2P(O)(OEt)2, NaH, THF, rt,
2 h, 85%; (ii) DIBAL, toluene, -78 °C to rt, 1 h, 74%; (iii) 2,6-
chloropurine, DEAD, PPh3, THF, rt, 2 h, 57%; (iv) CH3NH2, THF,
rt, 6 h, 85%; (v) AcOH:H2O:THF ) 1:2:2, 40 °C, 5 h, 86%; (vi)
Et2NP(OtBu)2, tetrazole, THF, rt, 1 h then MCPBA, 35%; (v)
Dowex 50x-8 200, MeOH/H2O ) 1:1, 50 °C, 3 h, 63%.
(Schemes 3A,B), which was smoothly achieved in four
steps from 34. The tert-butyl group was readily depro-
tected selectively in the presence of a dibenzyl phos-
phate ester by treatment with 5% trifluoroacetic acid
in methylene chloride. The conversion of monophos-
phate of 43 to the diphosphate in moderate yield utilized
a phosphoryl imidazolide intermediate, which reacted
with triethylammonium phosphate.24 Preparation of
analogues 16 and 17 (Scheme 7) required the synthesis
of an intermediate, 68, using a modification of a
literature procedure, which also permitted the assign-
ment of the stereochemical identity of each isomer.25
meso-cis- (minor), 71, and meso-trans-cyclopropylmethyl
(major), 70, isomers were separated after condensation
of the mesylate with adenine moieties, using silica gel
column chromatography. The preparation of a racemic
cyclopropylmethyl moiety, leading to 18, was accom-
a
Reaction conditions: (i) MsCl, TEA, CH2Cl2, rt, 1 h; (ii)
2-chloro-N6-methylaminopurine, K2CO3, 18-crown-6, DMF, 80-
90 °C, 12 h, 50% two-steps yield for 54, 54% two-steps yield for
55; (iii) Dowex 50x-8 200, MeOH, 40 °C, 4 h, 88% for 56, 85% for
57; (iv) Et2NP(OtBu)2, tetrazole, THF, rt, overnight then MCPBA,
56% for 58, 69% for 59; (v) 5% TFA in CH2Cl2, rt, 1 h, 44% for 13,
55% for 14.
distinguish between the two diols present. This was
accomplished through the synthesis of 42, containing
di-tert-butyl- and dibenzyl-protected phosphate groups

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3097
Sch em e 7. Synthesis of meso-cis- and
meso-trans-Cyclopropyl Bisphosphate Derivatives,
Compounds 16 and 17a
Sch em e 8. Synthesis of Racemic Cyclopropyl
Bisphosphate Derivative, Compound 18a
a
Reaction conditions: (i) CH3OCH2Cl, Hu¨nig base, CH2Cl2, rt,
4 h, 93%; (ii) TBAF, THF, rt, 3 h, 95%; (iii) TBDPSCl (1 equiv),
DMAP (1 equiv), CH2Cl2, rt, 3 d, 70%; (iv) MsCl, TEA, CH2Cl2, rt,
1 h, 95%; (v) 2-chloro-N6-methylaminopurine, K2CO3, DMF, 70 °C,
overnight, 71%; (vi) c-HCl:MeOH ) 1:3, 80 °C, 4 h, 65%; (vii)
Et2NP(OtBu)2, tetrazole, THF, rt, 20 min then MCPBA, 62%; (viii)
5% TFA in CH2Cl2, rt, 30 min, 71%.
a
Reaction conditions: (i) N2CH2CO2Et, Rh2(OAc)4, CH2Cl2, rt,
17 h, 61%; (ii) LiBH4, ethyl ether, toluene, 100 °C, 1 h, 93%; (iii)
2,6-chloropurine, DEAD, PPh3, THF, rt, overnight, 59%; (iv)
CH3NH2, THF, rt, 5 min then separation, total 75%, 70:71 ) 2.5:
1; (v) AcOH:THF:H2O ) 3:1:1, rt, 4 h; (vi) ((BnO)2(O)P)2O, LDA,
THF, -78 °C to rt, overnight, 46% two-step yield for 74, 33% two-
step yield for 75; (vii) BCl3, CH2Cl2, -78 °C for 1 h and 6-7 °C for
2 d, 77% for 16, 57% for 17.
Sch em e 9. Synthesis of Cyclopropyl Bisphosphate
Derivative, Compound 19a
plished through a mesylate displacement (Scheme 8),
utilizing TBDPS- (tert-butyl-diphenylsilyl) and MOM-
(methoxymethyl) protected intermediates. Another cy-
clopropyl derivative, 19, was synthesized through dis-
placement of a bromo diester, 83, with ring closure,
followed by diester reduction to generate the diol group
(Scheme 9).26
Biologica l Activity. Adenine nucleotides activate a
P2Y1 receptor in turkey erythrocyte membranes to
markedly stimulate inositol lipid hydrolysis by phos-
pholipase C.27-29 The analogues were screened for
activation or inhibition of P2Y1 receptor promoted-
effects on phospholipase C, as indicated by the ac-
cumulation of inositol phosphates in membranes iso-
lated from [3H]inositol-labeled turkey erythrocytes (Table
1). As in our previous study,20 none of the acyclic
adenosine bisphosphate nucleotide analogues displayed
agonist activity at the P2Y1 receptor in turkey eryth-
rocyte membranes. Concentration-response curves were
obtained for each compound for inhibition of the effects
of a fixed concentration (30 nM) of the agonist
2-MeSADP.
a
Reaction conditions: (i) 2-chloro-N6-methylaminopurine, K2CO3,
DMF, 35 °C, 2 d, 41%; (ii) DIBAL, CH2Cl2, -78 °C to rt, 2 h, 70%;
(iii) Et2NP(OtBu)2, tetrazole, THF, rt, 20 min then MCPBA, 85%;
(iv) 5% TFA in CH2Cl2, rt, 20 min, 61%.
Replacement of one of the phosphate groups of 7 with
an uncharged moiety (i.e., racemic alcohol, 8, phospho-
triester, 9, or O-acetyl ester, 10) resulted in a 500- to
1000-fold loss of potency. Replacement of a phosphate
group with diphosphate, 11, increased potency.
Methylene homologation at the phosphate ester posi-
tions of 7, resulting in compound 12, decreased potency
at the P2Y1 receptor 4-fold. Removal of a phosphate-
linked methylene group of 7, resulting in an enantio-
meric pair of glycerol derivatives, 13 and 14, demon-
strated a 5-fold stereoselectivity of binding at the P2Y1
receptor. The S-isomer, 14, was favored with an IC50
An acyclic 2-chloro-N6-methyladenine derivative, 7,
containing a symmetric isobutyl bisphosphate moiety,
was a potent P2Y1 receptor antagonist, with an IC50
value of 0.48 µM. The corresponding 2-H derivative, 6,
displayed 2-fold weaker antagonist potency. Thus, an-
tagonists 6 and 7 were roughly twice as potent as the
next higher homologues, 3 and 4, respectively. The lower
homologue, the isopropyl derivative, 5, was nearly an
order of magnitude less potent than 7.

3098 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Kim et al.
Sch em e 10. Synthesis of Cyclopropyl Bisphosphate
Derivative, Compound 20a
F igu r e 2. Structural correspondence of ATP (orange) and 2
(red), superimposed in the putative receptor binding site. TMs
(magenta) and EL2 (cyan), joining TM4 and 5, are shown. The
adenine moieties occupy the same binding subsite.
fied, by mutagenesis studies,30 and by possible modes
of superposition of the two ligand structures.
The energy-optimized structures of P2Y1 receptor
complexes with ATP and 2 were practically superim-
posable with respect to the protein backbone atoms. The
structural correspondence of the two ligands (Figure 2)
indicated that in both cases the adenine moieties
occupied the same binding subsite. According to this
model, the 5′-phosphate of 2 overlapped the â-phosphate
moiety of ATP, and the 3′-phosphate of 2 had no
structural equivalent in the bound ATP structure. As
in the previous model,30 neither the ribose ring of ATP
nor the methanocarba ring of 2 participated in binding
interactions with the receptor.
Using the receptor-2 complex as a template, the
various acyclic bisphosphates were docked into the
binding site and the models reoptimized. The structural
correspondence between the receptor-bound conformer
of 4 and that of 2 is shown in Figure 3. This figure also
depicts the various residues involved in specific interac-
tions with the ligands, including Lys280 and Arg310,
which interacted with both phosphate groups of the
ligands,30 and other residues. The residues Lys196,
Tyr306, and Asp204 apparently were involved in polar
interactions with the phosphate group corresponding to
the 5′-phosphate of 2. The residues Phe226, Tyr273, and
His277 formed a sterically restricted pocket to accom-
modate the 3′-phosphate of 2. The residues Tyr59, Tyr
273, and Ser314 interacted with the adjacent adenine
nitrogen atoms (Figure 3). The similarity in positioning
of the binding elements for 2 and 4 with respect to the
modeled receptor manifold (see Figure 3) was consistent
with the more potent inhibition of the P2Y1 receptor by
the latter (see Table 1). Furthermore, ligands homolo-
gous to 4 (having 0 to 3 methylenes adjacent to adenine
N-9), representing a >100-fold span of IC50 values,
displayed varying degrees of correspondence to this
pharmacophoric arrangement based on 2. An overall
comparison of the bound conformations of 4 and the
three homologues is shown in Figure 4, while specific
a
Reaction conditions: (i) Et2Zn, CH2I2, CH2Cl2, rt, overnight,
96%; (ii) MsCl, TEA, CH2Cl2, rt, 1 h, 93%; (iii) 2-chloro-N6-
methylaminopurine, K2CO3, 18-crown-6, DMF, 60 °C, overnight,
62%; (iv) AcOH:THF:H2O ) 1:2:2, 40 °C, overnight, 55%; (v)
Et2NP(OtBu)2, tetrazole, THF, rt, 30 min then MCPBA, 40%; (v)
5% TFA:CH2Cl2:THF ) 10:20:1, rt, 30 min, 55%.
value of 1.47 µM. Introduction of an olefinic group, i.e.,
compound 15, decreased antagonist potency compared
to 7 at the P2Y1 receptor 4-fold. meso-trans-, 16, and
meso-cis-, 17, isomers of a cyclopropylmethyl derivative
(two of the four possible stereoisomers) failed to display
stereoselectivity of binding, and the IC50 values were
approximately 2 µM. The racemate of the remaining two
isomers, 18, as well as 19 and 20, had a similar potency.
Thus, the antagonistic potency among cyclopropyl de-
rivatives was nearly invariable.
Molecu la r Mod elin g. To better understand the
affinities of the acyclic bisphosphate adenine analogues
and, in particular, the correspondence between the
receptor bound conformers of these compounds and the
rigid, high affinity analogues, we carried out molecular
modeling of the ligand-receptor complexes. The human
P2Y1 receptor model used for this purpose included the
seven transmembrane helical domains (TMs) and the
second extracellular loop (EL2) and was built in homol-
ogy to the recently published X-ray structure of bovine
rhodopsin.22 In this X-ray structure EL2, but not EL1
and 3, appears to interact directly with retinal.
Using this construct, docking experiments were car-
ried out to establish a binding site for both prototypical
agonists ATP or ADP and an antagonist, 2.19 These
experiments were guided by our previous modeling
experiments,30,31 in which parts of the binding pocket
accommodating the adenine moiety have been identi-

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3099
F igu r e 3. Structural correspondence between the receptor bound conformer of 4 (magenta) and that of 2 (yellow), depicting the
residues involved in specific interactions with the ligands. Lys280 and Arg310 interact with both phosphate groups of the ligands.
Ta ble 3. Distances in Angstroms between Pharmacophoric
Points of P2Y1 Docked Antagonists and Template Compound 2a
compd
3′-P
5′-P
6-N
92
93
5
7
4
94
12
13
14
15
16
17
18
0.35
0.28
0.42
0.38
0.42
0.33
0.44
0.45
0.45
0.16
0.50
0.27
0.78
0.27
0.10
0.15
0.26
0.08
0.36
1.88
0.79
0.50
1.08
2.22
1.78
0.83
1.40
0.42
0.86
1.60
0.72
1.31
0.81
0.83
0.63
0.54
0.05
0.20
1.27
0.27
0.49
0.7
0.43
1.24
0.46
0.25
0.21
0.30
0.50
0.27
0.15
0.21
0.18
0.10
isomer 1
isomer 2
R-config
S-config
R-config
S-config
19
20
F igu r e 4. Comparison of the bound conformations of 4 (white)
and three homologous derivatives, 5 (orange), 7 (magenta), and
the analogue containing an n-propyl spacer 94 (green).
a
Compound 2 (MRS 2279) is (1R,2S,4S,5S)-1-[(phosphato)-
methyl]-4-(2-chloro-6-methylaminopurin-9-yl)bicyclo [3.1.0]-hex-
ane-2-phosphate. Compounds are grouped as cyclic template
compounds (92, 93), acyclic bisphosphates of variable chain length
(0-3 methylenes) (5, 7 (MRS 2298), 4, 94), other acyclic compounds
(12-15), and cyclopropyl derivatives (16-20). Compounds 92 (2′-
deoxy-2-chloro-N6-methyladenosine-3′,5′-bisphosphate (MRS 2216),
IC50 0.206 µM), 93 (phosphoric acid mono-[3-(2-chloro-6-methyl-
aminopurin-9-yl)-2-phosphonooxymethylcyclobutyl] ester (MRS
2264), IC50 0.805 µM), and 94 (2-[3-(6-methylaminopurin-9-yl)-
propyl]propane-1,3-bisphosphate, IC50 >30 µM) were reported
previously.19,20
deviations of atoms corresponding to 3′-P, 5′-P, and 6-N
of 2 are listed in Table 3.
Table 3 compares the receptor-bound conformations
of rigid and acyclic P2Y1 antagonists. A relatively high
affinity was maintained, as long as the position of a
given antagonist did not deviate significantly with
respect to 6-N and 3′-P of the template. In contrast, the
binding pocket, including the relatively flexible EL2,
provided significantly more room for the group bearing
the 5′-phosphate. To further examine this suggestion,
the docked conformations of three potent, cyclic P2Y1
antagonists,18,19 92 (2′-deoxy-2-chloro-N6-methyladeno-
sine-3′,5′-bisphosphate), 2, and 93 (phosphoric acid
mono-[3-(2-chloro-6-methylaminopurin-9-yl)-2-phosphono-
oxymethylcyclobutyl] ester), have been compared (Fig-
ure 5). Indeed, the requirements with respect to posi-
tioning the 5′-phosphate in the receptor appeared not
to be very stringent, since the location of the corre-
sponding phosphate of 93 deviated considerably from
that of 2. Such a model is also consistent with the high
activity of 11 (see Table 1), where the adenine and
monophosphate binding elements could be expected to
occupy receptor subsites analogous to those of 7. The
flexible chain bearing the diphosphate moiety allows for
its accommodation in a similar way to that of the R-
and â-phosphates in ADP or ATP. The relative orienta-
tion of the three pharmacophoric elements in the

3100 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Kim et al.
template structure of 2 is presented (Figure 5). The
resulting distances differ somewhat from those cited in
our previous publication30 due to the refined receptor
model used in the present study.
F igu r e 5. Docked conformations of three relatively rigid
potent P2Y1 antagonists (see Table 3 for chemical names): 92
(cyan, a riboside), 2 (yellow), and 93 (white, a cyclobutyl
analogue). Also shown is the relative orientation of the three
pharmacophoric elements in the template structure of 2. The
requirements with respect to positioning the 5′-phosphate in
the receptor appeared not to be very stringent, since the
location of the corresponding phosphate of 93 deviated con-
siderably from that of 2.
Discu ssion
We have improved upon the affinity of 4 and found
the optimal length of the spacer chain to be one
methylene, i.e., 7. Numerous cyclopropyl analogues were
designed to add an element of rigidity, by analogy to
antagonists of metabotropic glutamate receptors in
which selectivity was achieved specifically depending
on the stereochemistry.32 However, all of the cyclopropyl
analogues displayed Ki values in the 1-2 µM range.
Although the lead bisphosphate structure, upon which
these analogues were based, i.e., 4, was selective for the
P2Y1 subtype,33 it will nevertheless be useful to examine
the acyclic compounds as agonists and/or antagonists
at other subtypes of P2 receptors.
We have refined our previous modeling of the P2Y1
receptor30 utilizing the high-resolution template struc-
ture of rhodopsin now available.22 In general, the two
receptor models are similar with respect to the overall
juxtaposition of the side chains forming the nucleotide
binding pocket (Figure 3), while differing in the length
of the individual helices and in their mutual orientation
within the helical bundle. In addition, a portion of EL2
bridging TM4 and TM5 is deeply embedded in the TM-
manifold, much like the â4 strand of the antiparallel
â-sheet of EL2 in rhodopsin.22 Thus, while both P2Y1
receptor models30 predict proximity of EL2 to the path
of approach of the ligand toward the TM binding site,
the present model indicates participation of certain
residues of this loop in coordination of the ligand bound
within the TM domain (Figure 2).
quirements: differentially accommodating (a) the con-
formationally constrained agonists ATP and ADP; (b)
rigid antagonists such as 2 (Figure 2); (c) flexible, acyclic
antagonists reported here (Figure 4).
Initially, basic residues needed to accommodate the
phosphate moieties of the ligands were identified (Lys128,
Lys196, Lys280, Arg310). The model suggested that the
â-phosphate moieties of ATP and ADP, as well as the
5′-phosphate substituent of 2, may occupy a position
adjacent to the antiparallel â-sheet of EL2. This inter-
pretation was based on the mutual orientation of these
basic side chains, as well as the juxtaposition of the
main chain amide nitrogens of residues Asp204 and
Thr205. In this position the 5′-phosphate moiety did not
seem to be sterically constrained, and its main interac-
tions were with the flexible lysine and arginine side
chains. Interestingly, according to this ligand orienta-
tion, the γ-phosphate of ATP should not contribute to
affinity due to its location above the â4 strand of the
EL2 â-sheet.
In contrast to the relaxed steric requirements for
accommodation of the 5′-phosphate moiety of 2, the
receptor seemed to present a restricted binding pocket
for the 3′-phosphate substituent. Consequently, it ap-
peared that the ligand binding environment of the P2Y1
receptor consisted of rigidly defined binding subsites for
the adenine and the 3′-phosphate moieties (using ligand
2 as structural template) while positioning of the 5′-
phosphate was less stringent. Such notion is consistent
with the affinities of the different P2Y1 ligands in which
these three pharmacophoric elements are held together
by various spacers, such as carbocyclic and heterocyclic
rings or carbon chains. In all these cases, the ligand-
bound conformation seemed to conform to the proposed
model (Table 3). We note that compound 11, where the
monophosphate and the diphosphate substituents mimic
Previous modeling studies19,30 suggested that agonists
and antagonists may share a common binding site
within the receptor. In particular cases, substitution of
a single N6-methyl group converted a pure P2Y1 agonist
into an antagonist, apparently without affecting affinity.
The first modeling experiment carried out with this
P2Y1 receptor model was the docking of the prototypical
agonists ATP and ADP, aimed to rationalize the appar-
ently superior binding of the latter to the receptor.31 The
binding site model satisfied the following binding re-

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3101
mixed and dried by high vacuum pump for about 30 min. The
mixture was dissolved in anhydrous tetrahydrofuran (5 mL)
and cooled to 0 °C. To this solution was dropwise added
diethylazodicarboxylate (149 mg, 0.855 mmol) via syringe.
After the mixture was stirred for 2-4 h at room temperature
(the progress of the reaction was monitored by thin-layer
chromatography, and if necessary it was overnight stirred),
tetrahydrofuran was removed in vacuo. The residue was
purified by silica gel column chromatography (hexanes/ethyl
acetate ) 5:1) to give a desired 2,6-dichloropurine analogue
23 (260 mg, 68%).
The 2,6-dichloropurine analogue 23 was treated with 2 M
methylamine in tetrahydrofuran (5 mL, 10 mmol), stirred for
5 h, filtrated through silica gel (10 g), and washed with
hexanes/ethyl acetate (1:2). Evaporation of the filtrate gave a
desired 6-amino-2-chloropurine analogue 24 (225 mg, 88%).
the 3′-substituent of 2 and the 5′-substituent of ADP,
respectively, was the most potent of the acyclic P2Y1
antagonists reported here.
In conclusion, two binding pockets for monophosphate
moieties were defined. The flexibility of the spacer chain
linking bisphosphate groups to the adenine moiety, in
general, allowed a relaxation of spatial requirements,
although the more potent antagonists in the acyclic
series conformed to a three-point model of the docked
positions of the phosphates relative to the adenine N6.
This was especially true for the series of symmetrically
substituted homologues 3-7 and 94 (Table 3). Elonga-
tion of the ethylene group of 4 was previously shown to
greatly decrease potency at the P2Y1 receptor.20 We
have identified the optimal chain length for P2Y1
antagonism to be one methylene group, in 7. We have
now achieved potency in an acyclic derivative compa-
rable to the best antagonists in the ribose series.16 Thus,
both approaches of rigidifying ribose19 and ring open-
ing20 have proven successful design approaches for P2Y1
antagonists.
9-[2-(t er t -Bu t yld im e t h ylsila n yloxy)-1-(t er t -b u t yld i-
m et h ylsila n yloxym et h yl)et h yl]-2,6-d ich lor o-9H -p u r in e
1
(23). H NMR (CDCl3) δ 0.008 (s, 6H), 0.014 (s, 6H), 0.84 (s,
18H), 3.99 (dd, 2H, J ) 5.2, 10.6 Hz), 4.08 (dd, 2H, J ) 5.4,
10.6 Hz), 4.81 (m, 1H). 8.34 (s, 1H). MS (CI/NH3) m/z 491 (M
+ H+)+.
{9-[2-(ter t-Bu t yld im et h ylsila n yloxy)-1-(ter t-b u t yld i-
m eth ylsila n yloxym eth yl)eth yl]-2-ch lor o-9H-p u r in -6-yl}-
m eth yla m in e (24). 1H NMR (CDCl3) δ 0.008 (s, 6H), 0.014
(s, 6H), 0.84 (s, 18H), 3.94 (dd, 2H, J ) 5.4, 10.3 Hz), 4.01 (dd,
2H, J ) 5.1, 10.3 Hz), 4.70 (m, 1H). 6.04 (bs, 1H), 7.91 (s, 1H).
MS (CI/NH3) m/z 486 (M + H+)+.
Meth od B. Hyd r olysis of TBS Gr ou p or Aceton id e
Gr ou p w ith Acid ic Dow ex Resin . A mixture of disilyl ether
24 (215 mg, 0.442 mmol), Dowex 50x-8 200 (500 mg), and
methanol (5 mL) was stirred overnight at refluxing temper-
ature. The resin was removed by filtration of the resulting
mixture through glass filter (10-20 µm, pore size). The filtrate
was evaportated in vacuo to give diol 25 (85 mg, 75%). (This
method was also used in deprotection of acetonide group of
54 and 55).
Exp er im en ta l Section
Ch em ica l Syn th esis. Ma ter ia ls a n d In str u m en ta tion .
Nucleosides and synthetic reagents were purchased from
Sigma Chemical Co. (St. Louis, MO) and Aldrich (Milwaukee,
WI).
1H NMR spectra were obtained with a Varian Gemini-300
spectrometer using CDCl3, DMSO-d6, or D2O as a solvent. The
chemical shifts are expressed as ppm downfield from tetra-
methylsilane or as relative ppm from DMSO (2.5 ppm) or HOD
peaks (4.78 ppm). 31P NMR spectra were recorded at room
temperature by use of Varian XL-300 spectrometer (121.42
MHz); orthophosphoric acid (85%) was used as an external
standard. Low-resolution CI-NH3 (chemical ionization) mass
spectra were carried out with a Finnigan 4600 mass spec-
trometer and high-resolution EI (electron impact) mass spec-
trometry with a VG7070F mass spectrometer at 6 kV. High-
resolution FAB (fast atom bombardment) mass spectrometry
was performed with J EOL SX102 spectrometer using 6-kV Xe
atoms following desorption from a glycerol matrix.
2-(2-Ch lor o-6-m eth yla m in op u r in -9-yl)p r op a n e-1,3-d i-
ol (25). 1H NMR (DMSO-d6) δ 2.91 (bs, 3H), 3.76 (dd, 2H, J )
5.4, 11.4 Hz), 3.83 (dd, 2H, J ) 7.0, 11.4 Hz), 4.46 (m, 1H),
8.15 (s, 1H). MS (CI/NH3) m/z 258 (M + H+)+.
Meth od C. P r ep a r a tion of Bis(d i-ter t-bu tyl p h osp h a te)
fr om Diol. To a stirred suspension of diol 25 (22 mg, 0.0854
mmol) and diethyl di-tert-butylphosphoramidite (53 mg, 0.213
mmol) in anhydrous tetrahydrofuran (5 mL) at room temper-
ature was added solid tetrazole (36 mg, 0.513 mmol) in one
pot. The reaction mixture was stirred at room temperature
for 20 min (sometimes overnight) and then cooled to -78 °C.
To this cooled reaction mixture was added solid MCPBA (57-
85%, calculated by 57%, 64 mg), and the resulting mixture
was stirred at -78 °C for 30 min and at room temperature for
10 min. After removal of tetrahydrofuran in vacuo, the residue
was directly purified by preparative thin-layer chromatogra-
phy (CHCl3/MeOH ) 20/1) to give 26 (40 mg, 73%).
The determinations of purity were performed with a Hewlett-
Packard 1090 HPLC system using an SMT OD-5-60 C18
analytical column (250 mm × 4.6 mm, Separation Methods
Technologies, Inc., Newark, DE) in two different linear gradi-
ent solvent systems. One solvent system (A) was 0.1 M
triethylammonium acetate buffer:CH3CN in ratios of 95:5 to
40:60 for 20 min with flow rate 1 mL/min. The other (B) was
5 mM tetrabutylammonium phosphate buffer:CH3CN, 80:20
to 40:60, in 20 min with flow rate 1 mL/min. Peaks were
detected by UV absorption using a diode array detector. All
phosphate derivatives showed more than 95% purity as
determined using HPLC.
1,3-Bis-(ter t-bu tyld im eth ylsila n yloxy)p r op a n -2-ol (22).
To a cooled (0 °C) and stirred solution of disilyl ketone 21 (1.20
g, 3.77 mmol) in ethyl alcohol (6 mL) was added a solid sodium
borohydride (80 mg, 2.11 mmol) in one pot. After the mixture
was stirred at 0 °C for 30 min, water (10 mL) was added, and
solid ammonium bicarbonate (5 g) was added at the same
temperature to quench excess residual sodium borohydride.
The resulting mixture was partitioned between ethyl acetate
(50 mL) and brine (20 mL). The organic layer was washed with
brine/water (1:1, 10 mL × 2), dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo to give a crude
disilyl alcohol 22 (1.10 g, 91%). 1H NMR (CDCl3) δ 0.07 (s,
12H), 0.90 (s, 18H), 3.54-3.72 (m, 5H).
P h osp h or ic Acid Di-ter t-b u t yl E st er 2-(2-Ch lor o-6-
m eth ylam in opu r in -9-yl)-3-(di-ter t-bu toxyph osph or yoxy)-
1
p r op yl Ester (26). H NMR (CDCl3) δ 1.39 (s, 18H), 1.41 (s,
18H), 3.17 (bs, 3H), 4.34 (dd, 2H, J ) 5.4, 11.0, 11.0 Hz), 4.47
(dd, 2H, J ) 6.6, 11.0, 11.0 Hz), 5.00 (m, 1H), 6.16 (bs, 1H),
7.88 (s, 1H). MS (CI/NH3) m/z 642 (M + H)+, 659 (M + NH4+)+.
3-(ter t-Bu tyld im eth ylsila n yloxy)-2-(ter t-bu tyld im eth -
ylsila n yloxym eth yl)p r op en e (28). To a stirred solution of
propenediol 27 (3.0 g, 34.1 mmol), triethylamine (6.90 g, 68.2
mmol), and 4-(dimethylamino)pyridine (20 mg, 0.264 mmol)
in methylene chloride (30 mL) at room temperature was added
solid tert-butyldimethylsilyl chloride (10.7 g, 71.5 mmol). After
being stirred overnight at room temperature, the resulting
mixture was diluted with ethyl acetate (100 mL). The organic
layer was washed with 1 N hydrochloric acid (20 mL),
saturated aqueous sodium bicarbonate (30 mL × 2), brine/
water (1:1, 20 mL × 2), dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo to dryness to give crude
28 (10.7 g, 99%). 1H NMR (CDCl3) δ 0.07 (s, 12H), 0.92 (s, 18H),
Meth od A. Mitsu n obu Rea ction a n d Su bstitu tion of
6-Ch lor o Gr ou p by Meth yla m in e To In tr od u ce N6-Meth -
yla m in o-2-ch lor o- (or h yd r o-)p u r in e Moiety. The alcohol
22 (250 mg, 0.78 mmol), triphenylphosphine (307 mg, 1.17
mmol), and 2,6-dichloropurine (189 mg, 0.858 mmol) were

3102 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Kim et al.
4.17 (t, 4H, J ) 1.5 Hz), 5.09 (t, 2H, J ) 1.5 Hz). MS (CI/NH3)
m/z 317 (M + H+)+.
2-(6-Met h yla m in op u r in -9-ylm et h yl)p r op a n e-1,3-d iol
(33). Meth od E. Compound 33 (106 mg, 79%) was obtained
1
from 31 (260 mg, 0.993 mmol). H NMR (CD3OD) δ 2.23 (m,
3-(ter t-Bu tyld im eth ylsila n yloxy)-2-(ter t-bu tyld im eth -
ylsila n yloxym eth yl)p r op a n -1-ol (29). A 1 M solution of
borane (in tetrahydrofuran, 34.2 mL, 34.2 mmol) was added
dropwise to a cooled (0 °C), stirred solution of 28 (3.6 g, 11.37
mmol) in anhydrous tetrahydrofuran (10 mL). After being
stirred at the same temperature, the reaction mixture was
warmed to room temperature and stirred overnight. The
resulting mixture was treated with potassium carbonate (10
g, 72.4 mmol) and 30% hydrogen peroxide (20 mL) at 0 °C,
stirred at room temperature for 2 h, and partitioned between
ethyl acetate (100 mL) and water (50 mL). The organic layer
was separated, washed with brine/water (1:1, 20 mL × 3),
dried over anhydrous sodium sulfate, filtered, concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (hexanes/ethyl acetate ) 2:1) to give 29 (2.28 g, 60%).
1H NMR (CDCl3) δ 0.07 (s, 12H), 0.90 (s, 18H), 1.90 (m, 1H),
3.73 (d, 2H, J ) 6.0 Hz), 3.75 (d, 2H, J ) 6.0 Hz), 3.78 (d, 2H,
J ) 5.1 Hz). MS (CI/NH3) m/z 335 (M + H+)+.
Meth od D. Mesyla tion a n d N-Alk yla tion To In tr od u ce
N6-Meth yla m in o-2-ch lor o- (or 2-h yd r o)p u r in e Moiety. To
a cooled (0 °C), stirred solution of alcohol 29 (1.47 g, 4.40 mmol)
and triethylamine (1.34 g, 13.2 mmol) in methylene chloride
(10 mL) was dropwise and neatly added methanesulfonyl
chloride (756 mg, 6.60 mmol). After being stirred at 0 °C for
10 min and at room temperature for 1 h, the resulting mixture
was partitioned between ethyl acetate (50 mL) and brine (20
mL). The organic layer was separated and washed with
saturated sodium bicarbonate (20 mL) and brine/water (1:1,
10 mL × 2), dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give a crude mesylate 30 (1.90 g).
The mesylate 30 was used in the following N-alkylation
without further purification.
1H), 3.12 (bs, 3H), 3.54 (m, 4H), 4.33 (d, 2H, J ) 6.7 Hz), 8.05
(s, 1H), 8.26 (s, 1H). MS (CI/NH3) m/z 238 (M + H+)+.
2-(2-C h lo r o -6-m e t h y la m in o p u r in -9-y lm e t h y l)p r o -
1
p a n e-1,3-d iol (34). Meth od E. H NMR (CD3OD) δ 2.23 (m,
1H), 3.06 (bs, 3H), 3.54 (m, 4H), 4.27 (d, 2H, J ) 6.7 Hz), 8.00
(s, 1H). MS (CI/NH3) m/z 272 (M + H+)+.
Meth od F . P r ep a r a tion of Tetr a ben zylbisp h osp h a tes
fr om Diol. To a cooled (0 °C), stirred of diol 34 (50 mg, 0.184
mmol) and tetrabenzylpyrophosphate (247 mg, 0.423 mmol)
in anhydrous tetrahydrofuran (5 mL) was added solid sodium
hydride (80 mg, 50% in mineral oil, ∼1.67 mmol) in one pot.
The reaction mixture was stirred at 0 °C for 10 min and at
room temperature for 30 min. The resulting mixture was
poured into ice-water (20 mL) solution, and the aqueous layer
was extracted with ethyl acetate (20 mL × 3). The combined
organic phase was washed with brine (10 mL), dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(ethyl acetate/methanol ) 15:1) to afford a desired bis(dibenzyl
phosphate) 36 (82 mg, 53%). (In the case of cyclopropanediols
73 and 72, lithium diethylamide was used as a base as
substitute for sodium hydride.)
P h osp h or ic Acid Diben zyl Ester 3-(Bisben zyloxyp h os-
p h oyloxy)-2-(6-m eth yla m in op u r in -9-ylm eth yl)p r op yl Es-
ter (35). Meth od F . Compound 35 (59 mg, 81%) was obtained
from 33 (23 mg, 0.097 mmol). 1H NMR (CDCl3) δ 2.57 (m, 1H),
3.19 (bs, 3H), 3.90 (m, 4H), 4.03 (d, 2H, J ) 6.9 Hz), 5.01 (m,
8H), 5.99 (q, 1H, J ) 5.1 Hz), 7.22-7.38 (m, 20H), 7.61 (s, 1H),
8.34 (s, 1H). 31P NMR (D2O) δ -22.363. MS (positive-ion FAB)
m/z 758 (M + H+)+.
P h osp h or ic Acid Diben zyl Ester 3-(Bisben zyloxyp h os-
p h oyloxy)-2-(2-ch lor o-6-m eth yla m in op u r in -9-ylm eth yl)-
p r op yl Ester (36). Meth od F . 1H NMR (CDCl3) δ 2.49 (m,
1H), 3.17 (bs, 3H), 3.86 (m, 4H), 3.97 (d, 2H, J ) 7.0 Hz), 4.89-
5.08 (m, 8H), 5.99 (bs, 1H), 7.15-7.0.38 (m, 20H), 7.57 (s, 1H).
MS (CI/NH3) m/z 792 (M + H+)+.
A mixture of crude mesylate 30 (1.9 g), 2-chloro-6-methyl-
aminopurine (808 mg, 4.40 mmol), potassium carbonate (912
mg, 6.60 mmol), 18-crown-6 (10 mg, mmol), and dimethylfor-
mamide (10 mL) was stirred overnight at 40-60 °C. The
resulting mixture was partitioned between ethyl acetate (150
mL) and brine/water (40 mL:20 mL). The organic layer was
separated, washed with brine (20 mL × 4), and dried over
anhydrous sodium sulfate, filtered, concentrated to dryness.
The residue was purified by silica gel column chromatography
(hexanes/ethyl acetate ) 1:1) to give 32 (1.47 g, 72% two steps
yield).
(()-3-(t er t -Bu t yld im e t h ylsila n yloxy)-2-(2-ch lor o-6-
m eth yla m in op u r in -9-ylm eth yl)p r op a n -1-ol ((()-37). To a
stirred solution of diol 34 (370 mg, 1.34 mmol), imidazole (140
mg, 2.06 mmol), in dimethylformamide at room temperature
was added solid tert-butyldimethylsilyl chloride (226 mg, 1.50
mmol) in one pot. The reaction mixture was overnight stirred
at room temperature. Dimethylformamide was removed in
vacuo, and the residue was purified by preparative thin-layer
chromatography (chloroform/methanol ) 10:1) to give monosi-
Meth an esu lfon ic Acid 3-(ter t-Bu tyldim eth ylsilan yloxy)-
2-(ter t-bu tyld im eth ylsila n yloxym eth yl)p r op yl Ester (30).
1H NMR (CDCl3) δ 0.06 (s, 12H), 0.89 (s, 18H), 2.10 (m, 1H),
3.00 (s, 3H), 3.63 (dd, 2H, J ) 5.2, 10.2 Hz), 3.68 (dd, 2H, J )
5.8, 10.2 Hz), 4.30 (d, 2H, J ) 5.8 Hz). MS (CI/NH3) m/z 413
(M + H+)+.
1
lyl derivative, 37 (200 mg, 38%). H NMR (CDCl3) δ 0.05 (s,
6H), 0.89 (s, 9H), 2.22 (m, 1H), 3.15 (bs, 3H), 3.35-3.52 (m,
2H), 3.51 (dd, 1H, J ) 7.1, 10.4 Hz), 3.61 (dd, 1H, J ) 5.0,
10.4 Hz), 4.32 (m, 2H), 6.60 (bs, 1H), 7.71 (s, 1H). MS (positive-
ion FAB) 386 (M + H+)+.
{9-[3-(ter t-Bu t yld im et h ylsila n yloxy)-1-(ter t-b u t yld i-
m eth ylsila n yloxym eth yl)-pr op yl]-9H-p u r in -6-yl}-m eth yl-
a m in e (31). Meth od D. Compound 31 (280 mg, 60% two steps
(()-P h osp h or ic Acid Di-ter t-bu tyl Ester 3-(ter t-Bu -
tyld im eth ylsila n yloxy)-2-(2-ch lor o-6-m eth yla m in op u r in -
9-ylm et h yl)p r op yl E st er (38). Met h od C. Compound 38
1
yield) was obtained from 30 (410 mg, 0.993 mmol). H NMR
(CDCl3) δ 0.04 (s, 12H), 0.90 (s, 18H), 2.32 (m, 1H), 3.22 (bs,
3H), 3.54 (dd, 2H, J ) 5.9, 10.2 Hz), 3.65 (dd, 2H, J ) 4.8,
10.2 Hz), 4.22 (d, 2H, J ) 7.2 Hz), 5.79 (bs, 1H), 7.75 (s, 1H),
8.41 (s, 1H). MS (CI/NH3) m/z 466 (M + H+)+.
{9-[3-(ter t-Bu t yld im et h ylsila n yloxy)-1-(ter t-b u t yld i-
m eth ylsilan yloxym eth yl)pr opyl]-2-ch lor o-9H-pu r in -6-yl}-
m eth yla m in e (32). Meth od D. 1H NMR (CDCl3) δ 0.04 (s,
12H), 0.90 (s, 18H), 2.29 (m, 1H), 3.20 (bs, 3H), 3.52 (dd, 2H,
J ) 5.9, 10.2 Hz), 3.64 (dd, 2H, J ) 5.0, 10.2 Hz), 4.19 (d, 2H,
J ) 7.0 Hz), 5.93 (bs, 1H), 7.71 (s, 1H). MS (CI/NH3) m/z 500
(M + H+)+.
Meth od E. Dep r otection of Disilyl Eth er a n d Ac-
eton id e Gr ou p s w ith Acetic Acid /THF /Wa ter . A solution
of disilyl ether 32 (300 mg, 0.644 mmol) in acetic acid/
tetrahydofuran/water (3 mL:2 mL:2 mL) was stirred at 50 °C
for 1 day. All volatile material was removed in vacuo, and the
residue was washed with hexanes/ethyl ether (1:1) to give diol
34 (163 mg, 93%).
1
(250 mg, 83%) was obtained from 37 (200 mg, 0.52 mmol). H
NMR (CDCl3) δ 0.04 (s, 6H), 0.90 (s, 9H), 1.49 (s, 18H), 2.49
(m, 1H), 3.19 (bs, 3H), 3.56 (dd, 1H, J ) 5.4, 10.5 Hz), 3.61
(dd, 1H, J ) 4.8, 10.5 Hz), 3.88-4.06 (m, 2H), 4.24 (d, 2H, J
) 6.9 Hz), 6.05 (bs, 1H), 7.77 (s, 1H). MS (positive-ion FAB)
578 (M + H+)+.
(()-P h osp h or ic Acid Di-ter t-bu tyl Ester 2-(2-Ch lor o-
6-m eth yla m in op u r in -9-ylm eth yl)-3-h yd r oxy-p r op yl Es-
ter (39). A solution of silyl phosphate 38 (250 mg, 0.432 mmol)
in tetrahydrofuran (10 mL) at room temperature was treated
with tetrabutylammonium fluoride (0.52 mL, 0.52 mmol, 1 M
solution in tetrahydrofuran). The reaction mixture was stirred
for 10 min and applied directly to preparative thin-layer
chromatography (chloroform/methanol ) 10:1) to give 39 (190
1
mg, 76%). H NMR (CDCl3) δ 1.51 (s, 9H), 1.52 (s, 9H), 2.41
(m, 1H), 3.19 (bs, 3H), 3.40-3.56 (m, 2H), 3.84-4.11 (m, 2H),
4.23-4.40 (m, 2H), 6.34 (bs, 1H), 7.81 (s, 1H). MS (positive-
ion FAB) 464 (M + H+)+.

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3103
(()-P h osp h or ic Acid Di-ter t-bu tyl Ester 2-(2-Ch lor o-
6-m eth ylam in opu r in -9-ylm eth yl)-3-(dim eth yoxyph osph o-
r yloxy)p r op yl Ester (40). To a stirred solution of 39 (10 mg,
0.0216 mmol) and dimethylphosphoryl chloride (12 mg, 0.083
mmol) in anhydrous tetrahydrofuran at room temperature was
added solid sodium hydride (8.6 mg, 0. 215 mmol, 60% in
mineral oil) in one pot. The reaction mixture was stirred for 1
h at the same temperature, followed by quenching with water
under water-ice bath. The mixture was applied to prepatative
thin-layer chromatography (chloroform/methanol ) 10:1) to
Meth a n esu lfon ic Acid 2-Allylp en t-4-en yl Ester (46)
1
Meth od D. H NMR (CDCl3) δ 1.96 (m, 1H), 2.17 (t, 4H, J )
7.0 Hz), 3.15 (s, 3H), 4.14 (d, 2H, J ) 5.5 Hz), 5.03-5.16 (m,
4H), 5.76 (m 2H).
[9-(2-Allyl-p en t-4-en yl)-2-ch lor o-9H-p u r in -6-yl]m eth yl-
a m in e (47). Meth od D. Compound 47 (69 mg, 60% two steps
yield) obtained from 45 (50 mg, 0.396 mmol). 1H NMR (CDCl3)
δ 2.07 (t, 4H, J ) 6.6 Hz), 2.23 (m, 1H), 3.18 (s, 3H), 4.06 (d,
2H, J ) 7.2 Hz), 5.01-5.11 (m, 4H), 5.75 (m 2H). MS (positive-
ion FAB) 292 (M + H+)+.
1
3-(2-Ch lor o-6-m eth yla m in op u r in -9-ylm eth yl)p en ta n e-
1,5-d iol (48). A mixture of diene 47 (39 mg, 0.134 mmol),
osmium tetroxide (0.3 mL of stock solution, 1 g OsO4/100 mL
H2O), sodium metaperiodate (340 mg, 1.59 mmol), and a mixed
solvent (5 mL, acetone/water ) 4:1) was stirred at room
temperature for 4 h. All volatile materials were removed in
vacuo to dryness. The residue was stirred in a mixed solvent
(ethyl acetate/methanol ) 10:1) and filtrated through a short
column packed with silica gel (20 g). The filtrate was collected
and evaporated, and the residue was dissolved in ethyl alcohol
in an ice-water bath. Solid sodium borohydride was added to
this solution, and the reaction mixture was stirred for 2 h at
room temperature, followed by quenching excess sodium
borohydride with 1 N hydrochloric acid. After removal of all
volatile materials in vacuo to dryness, the residue was purified
by preparative thin-layer chromatography (chloroform/metha-
give 40 (3.8 mg, 31%). H NMR (CDCl3) δ 1.497 (s, 9H), 1.500
(s, 9H), 2.73 (m, 1H), 3.18 (bs, 3H), 3.80 (d, 3H, J ) 11.1 Hz),
3.82 (d, 3H, J ) 11.1 Hz), 3.86-4.19 (m, 4H), 4.21-4.38 (m,
2H), 6.17 (bs, 1H), 7.85 (s, 1H). MS (positive-ion FAB) (M +
H+)+.
(()-Acetic Acid 2-(2-Ch lor o-6-m eth yla m in op u r in -9-yl-
m eth yl)-3-(di-ter t-bu toxyph osph or yloxy)pr opyl Ester (41).
A mixture of 39 (11.5 mg, 0.0248 mmol), triethylamine (9.4
mg, 0.0933 mmol), and acetic anhydride (3.80 mg, 0.0372
mmol) was stirred for 2 h at room temperature. The resulting
mixture was purified by preparative thin-layer chromatogra-
phy (chloroform/methanol ) 15:1) to give 41 (12 mg, 96%). 1H
NMR (CDCl3) δ 1.497 (s, 9H), 1.502 (s, 9H), 2.07 (s, 3H), 2.70
(m, 1H), 3.18 (bs, 3H), 3.86-4.05 (m, 2H), 4.09 (d, 2H, J ) 5.9
Hz), 4.29 (d, 2H, J ) 7.0 Hz), 6.08 (bs, 1H), 7.79 (s, 1H). MS
(positive-ion FAB) 506 (M + H+)+.
(()-P h osp h or ic Acid Diben zyl Ester 2-(2-Ch lor o-6-
m et h yla m in op u r in -9-ylm et h yl)-3-(d i-ter t-b u t oxyp h os-
p h or yloxy)p r op yl Ester (42). To a stirred suspension of 39
(13 mg, 0.028 mmol) and diisopropyl dibenzylphosphoramidite
(29 mg, 0.084 mmol) in anhydrous tetrahydrofuran (2 mL) at
room temperature was added solid tetrazole (11.7 mg, 0.167
mmol) in one pot. The reaction mixture was stirred at room
temperature for 20 min and cooled to -78 °C. To this cooled
reaction mixture was added solid MCPBA (57-85%, calculated
by 57%, 25 mg), and the resulting mixture was stirred at -78
°C for 30 min and at room temperature for 10 min. After
removal of tetrahydrofuran in vacuo, the residue was directly
purified by preparative thin-layer chromatography (CHCl3/
MeOH ) 20/1) to give 42 (9.2 mg, 45%). 1H NMR (CDCl3) δ
1.46 (s, 18H), 2.61 (m, 1H), 3.18 (bs, 3H), 3.79-4.08 (m, 4H),
4.14 (d, 2H, J ) 7.0 Hz), 4.97-5.13 (m, 4H), 6.04 (bs, 1H),
7.28-7.42 (m, 10H), 7.74 (s, 1H). MS (positive-ion FAB) 724
(M + H+)+.
1
nol ) 10:1) to give 48 (13 mg, 32%). H NMR (CD3OD) δ 1.43
- 1.65 (m, 4H), 2.27 (m, 1H), 3.07 (bs, 3H), 3.51-3.71 (m, 4H),
4.18 (d, 2H, J ) 7.2 Hz), 8.05 (s, 1H). MS (positive-ion FAB)
300 (M + H+)+.
P h osp h or ic Acid Di-ter t-bu tyl Ester 3-(2-Ch lor o-6-
m et h yla m in op u r in -9-ylm et h yl)-5-(d i-ter t-b u t oxyp h os-
p h or yloxy)-p en tyl Ester (49). Meth od C. Compound 49 (20
mg, 73%) obtained from 48 (12 mg, 0.040 mmol). 1H NMR
(CDCl3) δ 1.47 (s, 36H), 1.65-1.75 (m, 4H), 2.29 (m, 1H), 3.18
(bs, 3H), 3.93-4.16 (m, 4H), 4.21 (d, 2H, J ) 6.7 Hz), 6.12 (bs,
1H), 7.84 (s, 1H). MS (positive-ion FAB) 684 (M + H+)+.
(4S)-Meth a n esu lfon ic Acid 2,2-Dim eth yl-[1,3]d ioxola n -
4-ylm eth yl Ester (52) (fr om R-Isom er Alcoh ol 50). Meth od
D. Crude 52 (700 mg) was obtained from 50 (500 mg, 3.78
mmol).1H NMR (CDCl3) δ 1.36 (s, 3H), 1.44 (s, 3H), 3.06 (s,
3H), 3.82 (dd, 1H, J ) 5.5, 8.8 Hz), 4.10 (dd, 1H, J ) 6.5, 8.8
Hz), 4.22 (d, 2H, J ) 5.2 Hz), 4.37 (m, 1H). MS (CI/NH3) m/z
211 (M + H+)+, 228 (M + NH4+)+.
(4R)-Meth a n esu lfon ic Acid 2,2-Dim eth yl-[1,3]d ioxola n -
4-ylm eth yl Ester (53) (fr om S-Isom er Alcoh ol 51). Meth od
D. Crude 53 (850 mg) was obtained from 51 (500 mg, 3.78
mmol), and it showed the identical proton NMR spectrum to
52. MS (CI/NH3) m/z 228 (M + NH4)+.
(R)-[2-Ch lor o-9-(2,2-dim eth yl-[1,3]dioxolan -4-ylm eth yl)-
9H-p u r in -6-yl]-m eth yl-a m in e (54) (Or igin a ted fr om R-
Isom er 50). Meth od D. Compound 54 (567 mg, 50% two steps
yield) was obtained from 50 (500 mg, 3.78 mmol). 1H NMR
(CDCl3) δ 1.33 (s, 3H), 1.39 (s, 3H), 3.19 (bs, 3H), 3.69 (dd,
1H, J ) 5.8, 8.8 Hz), 4.14 (dd, 1H, J ) 6.6, 8.8 Hz), 4.20 (dd,
1H, J ) 6.4, 14.4 Hz), 4.27 (dd, 1H, J ) 3.2, 14.4 Hz), 4.46 (m,
1H). MS (CI/NH3) 298 m/z (M + H+)+.
(S)-[2-Ch lor o-9-(2,2-dim eth yl-[1,3]dioxolan -4-ylm eth yl)-
9H-p u r in -6-yl]-m eth yl-a m in e (55) (Or igin a ted fr om S-
Isom er 51). Meth od D. Compound 55 (611 mg, 54% two steps
yield) was obtained from 51 (500 mg, 3.78 mmol), and it
showed the identical proton NMR spectrum and mass spec-
trum to 54. MS (CI/NH3) 298 m/z (M + H+)+.
(R)-3-(2-Ch lor o-6-m eth yla m in op u r in -9-yl)p r op a n e-1,2-
d iol (56) (Or igin a t ed fr om R-Isom er 50). Met h od B.
Compound 56 (114 mg, 88%) was obtained from 54 (150 mg,
0.504 mmol).1H NMR (DMSO-d6) δ (d, 3H, J ) 3.6 Hz), 3.32
(dd, 1H, J ) 6.2, 11.1 Hz), 3.40 (dd, 1H, J ) 5.1, 11.1 Hz),
3.82 (m, 1H), 3.95 (dd, 1H, J ) 8.5, 13.9 Hz), 4.25 (dd, 1H, J
) 3.4, 13.9 Hz), 8.04 (s, 1H), 8.16 (m, 1H). MS (CI/NH3) m/z
258 (M + H+)+.
(()-P h osp h or ic Acid Diben zyl Ester 2-(2-Ch lor o-6-
m eth yla m in op u r in -9-ylm eth yl)-3-p h osp h on ooxyp r op yl
Ester (43). Meth od I. Compound 43 (8 mg, 89%) was obtained
from 42 (9.1 mg, 0.0126 mmol) as a triethylammonium salt.
1H NMR (CD3OD) δ 2.65 (m, 1H), 3.07 (bs, 3H), 3.93-4.13 (m,
4H), 4.24 (d, 2H, J ) 7.0 Hz), 5.03 (d, 4H, J ) 8.9 Hz), 7.34 (s,
10H). 8.07 (s, 1H). HPLC 16.372 min (purity >98%) in solvent
system A, 13.449 min in system B.
(()-P h osp h or ic Acid Diben zyl Ester 2-(2-Ch lor o-6-
m et h yla m in op u r in -9-ylm et h yl)-3-p h osp h on op h osp h o-
n ooxyp r op yl Ester (44). Monotriethylamine salt 43 (8 mg,
0.0112 mmol) was dissolved in anhydrous dimethylformamide
(1 mL), followed by an addition of carbonyldiimidazole (5.46
mg, 0.0337 mmol). The mixture was stirred for 20 min at room
temperature and then triethylammonium phosphate (13.4 mg,
0.067 mmol) in anhydrous dimethylformamide (0.5 mL) was
added. The reaction mixture was stirred for 3 days at room
temperature. All volatile material was removed by nitrogen
stream. The residue was purified with an ion-exchange column
chromatography using Sephadex-DEAE-25 resin with a linear
gradient (0.01 to 1.0 M) of 1.0 M ammonium bicarbonate as
the mobile phase to give phosphate 44 (6 mg, 74%) as
1
ammonium salt. H NMR (D2O) δ 2.66 (m, 1H), 2.87 (bs, 3H),
3.91-4.19 (m, 4H), 4.20-4.41 (m, 2H), 4.65-4.90 (m, 2H),
7.06-7.25 (m, 5H), 7.30-7.41 (m, 5H). 8.08 (s, 1H). High-
resolution MS (negative-ion FAB) calcd for C24H28O11N5ClP3
[M - H+]- 690.0687, found 690.0684.
(S)-3-(2-Ch lor o-6-m eth yla m in op u r in -9-yl)p r op a n e-1,2-
d iol (57) (Or igin a ted fr om S-Isom er 51) Meth od B.
Compound 57 (110 mg, 85%) was obtained from 55 (150 mg,

3104 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Kim et al.
0.504 mmol) and it showed the identical proton NMR spectrum
and mass spectrum to 56.
2H, J ) 7.4 Hz), 5.60 (t, 1H, J ) 7.4 Hz), 8.11 (s, 1H), 8.20
(bs, 1H). MS (CI/NH3) m/z 284 (M + H+)+.
(R)-P h osp h or ic Acid Di-ter t-bu tyl Ester 1-(2-Ch lor o-
6-m eth yla m in op u r in -9-ylm eth yl)-2-(d i-ter t-bu toxyp h os-
p h or yloxy)eth yl Ester (58) (Or igin a ted fr om R-Isom er
50). Meth od C. Compound 58 (42 mg, 56%) was obtained from
P h osp h or ic Acid Di-ter t-b u t yl E st er 4-(2-Ch lor o-6-
m et h yla m in op u r in -9-yl)-2-(d i-ter t-b u t oxyp h osp h or yl-
oxym eth yl)bu t-2-en yl Ester (65). Meth od C. Compound 65
1
(40 mg, 34%) was obtained from 64 (50 mg, 0.176 mmol). H
1
56 (30 mg, 0.116 mmol). H NMR (CDCl3) δ 1.34 (s, 9H), 1.44
NMR (CDCl3) δ 1.45 (s, 18H), 1.49 (s, 18H), 3.18 (bs, 3H), 4.52
(d, 2H, J ) 6.7 Hz), 4.70 (d, 2H, J ) 9.1 Hz), 4.96 (d, 2H, J )
7.1 Hz), 5.95 (t, 1H, J ) 7.3 Hz), 6.17 (bs, 1H), 7.87 (s, 1H).
MS (CI/NH3) m/z 668 (M + H+)+.
(s, 9H), 1.507 (s, 9H), 1.513 (s, 9H), 3.18 (bs, 3H), 4.04-4.27
(m, 2H), 4.34-4.55 (m, 2H), 4.78 (m, 1H), 6.50 (bs, 1H), 7.88
(s, 1H). MS (CI/NH3) m/z 642 (M + H+)+.
(S)-P h osp h or ic Acid Di-ter t-bu tyl Ester 1-(2-Ch lor o-
6-m eth yla m in op u r in -9-ylm eth yl)-2-(d i-ter t-bu toxyp h os-
p h or yloxy)eth yl Ester (59) (Or igin a ted fr om R-Isom er
50). Meth od C. Compound 59 (43 mg, 69%) was obtained from
2,3-Bis-(t er t -b u t yld im e t h ylsila n yloxym e t h yl)cyclo-
p r op a n eca r boxylic Acid E t h yl E st er (67). A solution of
ethyl diazoacetate (containing <10% methylene chloride, 9 mL)
at room temperature was added dropwise over 18 h, using a
syringe pump, to a stirred solution of (Z)-1,4-bis[(tertbutyldi-
methylsilyl)oxy]-2-butene 66 (25 g, 78.95 mmol) and rhodium-
(II) acetate dimer (410 mg, 0.928 mmol) in methylene chloride
(50 mL). After the mixture was stirred for an additional 6 h,
the solvent was removed in vacuo, and the residue was
chromatographed on silica gel eluting 0.5-2% ethyl acetate
in hexanes to give a mixture 67 of meso-cis and meso-trans
isomers (19.27 g, 61%, meso-cis:meso-trans ) 7.36 g: 11.91 g).
67: 1H NMR (CDCl3) δ 0.01-0.08 (m, 12H), 0.80-0.99 (m,
18H), 1.20-1.35 (m, 3H), 1.52-1.88 (m, 3H), 3.65-4.26 (m,
6H).
1
57 (25 mg, 0.097 mmol). H NMR (CDCl3) δ 1.34 (s, 9H), 1.44
(s, 9H), 1.507 (s, 9H), 1.513 (s, 9H), 3.18 (bs, 3H), 4.04-4.27
(m, 2H), 4.34-4.55 (m, 2H), 4.78 (m, 1H), 6.21 (bs, 1H), 7.85
(s, 1H). MS (CI/NH3) m/z 642 (M + H+)+.
4-(ter t-Bu tyld im eth ylsila n yloxy)-3-(ter t-bu tyld im eth -
ylsila n yloxylm eth yl)bu t-2-en oic Acid Eth yl Ester (60).
Neat ethyl (diethoxy)phosphoacetate (3.10 g, 13.8 mmol) was
added dropwise to a cooled (0 °C), stirred suspension of sodium
hydride (50%, 662 mg, 13.8 mmol) in anhydrous tetrahydro-
furan (60 mL). After being stirred at room temperature for 1
h until the reaction mixture became homogeneous, the reaction
mixture was cooled to 0 °C, and to this solution was added a
solution of 21 (4.0 g, 12.6 mmol) in anhydrous tetrahydrofuran
(10 mL) at the same temperature. The reaction mixture was
stirred at room temperature for 2 h and poured into ethyl
acetate (100 mL) and water (50 mL). The organic layer was
separated, washed with brine/water (1:1, 20 mL × 3), dried
over anhydrous sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (hexanes/ethyl acetate ) 15:1) to give 60 (4.17 g,
85%). 1H NMR (CDCl3) δ 0.07 (s, 6H), 0.10 (s, 6H), 0.90 (s,
9H), 0.93 (s, 9H), 1.29 (t, 3H, J ) 7.2 Hz), 4.16 (q, 2H, J ) 7.2
Hz), 4.44 (m, 2H), 4.87 (m, 2H), 5.98 (m, 1H). MS (CI/NH3)
m/z 389 (M + H+)+.
[2,3-Bis-(ter t-b u t yld im et h ylsila n yloxym et h yl)cyclo-
p r op yl]m eth a n ol (68). To a stirred solution of the mixture
67 (400 mg, 0.99 mmol) in ether (5 mL) and toluene (2.7 mL)
at room temperature was added solid lithium borohydride (22
mg, 1.01 mmol), and the reaction mixture was stirred at 100
°C with continuous removal of ethyl ether. More lithium
borohydride (40 mg, 2.02 mmol) was added. After the mixture
was stirred for 1 h at the same temperature, ice (20 g) was
added in one pot with a rigorous stirring to decompose excess
lithium borohydride, and the resulting mixture was stirred for
30 min. The aqueous layer was extracted with ethyl acetate
(50 mL), and the organic layer was washed with water (30
mL × 2), dried over anhydrous sodium sulfate, filtered,
concentrated in vacuo to give a crude mixture of meso-cis and
4-(ter t-Bu tyld im eth ylsila n yloxy)-3-(ter t-bu tyld im eth -
ylsila n yloxylm eth yl)bu t-2-en -1-ol (61). To a cooled (-78
°C), stirred solution of 60 (1.3 g, 3.34 mmol) in toluene (20
mL) was added a 1 M solution of DIBAL (in tetrahydrofuran,
8.0 mL, 8.0 mmol). After being stirred for 2 h at -20 °C, the
mixture was warmed to room temperature, stirred for 1 h, and
cooled to 0 °C, followed by an addition of methanol (7 mL).
The resulting mixture was partitioned between 10% Rochelle
salt solution (100 mL) and ethyl acetate (100 mL). This two-
phase solution was stirred until the two phases were clearly
separated with more addition of Rochelle salt. The organic
layer was separated, washed with brine/water (1:1, 20 mL ×
2), dried over anhydrous sodium sulfate, filtered, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexanes/ethyl acetate ) 1:1) to give 61 (860
1
meso-trans cyclopropanol isomers 68 (320 mg, 1:1.6, 93%). H
NMR (CDCl3) δ 0.02-0.11 (m, 12H), 0.87-0.95 (m, 18H), 1.01-
1.54 (m, 3H), 3.46-3.95 (m, 6H). MS (CI/NH3) m/z 361 (M +
H+)+.
9-[2,3-Bis-(ter t-bu tyld im eth ylsila n yloxym eth yl)cyclo-
p r op ylm eth yl]-2,6-d ich lor o-9H-p u r in e (69). Meth od A. A
mixture of two isomers 69 (147 mg, 59%) was obtained from a
1
mixture 68 (160 mg, 0.464 mmol). H NMR (CDCl3) δ 0.01-
0.20 (m, 12H), 0.61-0.89 (m, 18H), 1.42-1.55 (m, 3H), 3.54-
4.48 (m, 6H). 8.59 and 8.61 (two singlets, 1H).
m eso-1,2-tr a n s-{9-[2,3-Bis-(ter t-b u t yld im et h ylsila n yl-
oxym eth yl)cyclop r op ylm eth yl]-2-ch lor o-9H-p u r in -6-yl}-
m eth yla m in e (70) (m eso-tr a n s). Meth od A. Compound 70
(78 mg) and 71 (31 mg) were obtained from 69 (147 mg, 0.276
mmol) and separated by silica gel column chromatography. 1H
NMR (CDCl3) δ 0.03 (s, 12H), 0.87 (s, 18H), 1.20-1.35 (m, 3H),
3.18 (bs, 3H), 3.62 (dd, 2H, J ) 5.9, 10.3 Hz), 3.78 (dd, 2H, J
) 4.4, 10.3 Hz), 4.05 (d, 2H, J ) 7.3 Hz), 6.11 (bs, 1H), 8.19 (s,
1H). MS (CI/NH3) m/z 526 (M + H+)+.
m eso-1,2-cis-{9-[2,3-Bis-(ter t-bu tyld im eth ylsila n yloxy-
m e t h yl)cyclop r op ylm e t h yl]-2-ch lor o-9H -p u r in -6-yl}-
m eth yla m in e (71) (m eso-cis). Meth od A. Compound 70 (78
mg) and 71 (31 mg) were obtained from 69 (147 mg, 0.276
mmol) and separated by silica gel column chromatography. 1H
NMR (CDCl3) δ 0.06 (s, 6H), 0.07 (s, 6H), 0.90 (s, 18H), 1.35-
1.55 (m, 3H), 3.19 (bs, 3H), 3.78 (dd, 2H, J ) 8.8, 11.7 Hz),
3.92 (dd, 2H, J ) 5.9, 11.7 Hz), 4.32 (d, 2H, J ) 5.9 Hz), 6.07
(bs, 1H), 8.18 (s, 1H). MS (CI/NH3) m/z 526 (M + H+)+.
1
mg, 74%). H NMR (CDCl3) δ 0.08 (s, 6H), 0.09 (s, 6H), 0.91
(s, 9H), 0.92 (s, 9H), 4.10-4.28 (m, 6H), 5.82 (m, 1H). MS (CI/
NH3) m/z 347 (M + H+)+.
9-[4-(t er t -Bu t yld im e t h ylsila n yloxy)-3-(t er t -b u t yld i-
m et h ylsila n yloxylm et h yl)b u t -2-en yl]-2,6-d ich lor o-9H -
p u r in e (62). Meth od A. Crude 62 (230 mg, 57%) was obtained
from 61 (270 mg, 0.779 mmol). 1H NMR (CDCl3) δ 0.05 (s, 6H),
0.11 (s, 6H), 0.89 (s, 9H), 0.91(s, 9H), 4.19 (s, 2H), 4.25 (s, 2H),
5.03 (d, 2H, J ) 7.5 Hz), 5.78 (t, 1H, J ) 7.5 Hz), 8.17 (s, 1H).
{9-[4-(ter t-Bu t yld im et h ylsila n yloxy)-3-(ter t-b u t yld i-
m eth ylsilan yloxylm eth yl)bu t-2-en yl]-2-ch lor o-9H-pu r in -6-
yl}-m eth yla m in e (63). Meth od A. Compound 63 (168 mg,
85%) was obtained from crude 62 (200 mg, 0.386 mmol). 1H
NMR (CDCl3) δ 0.05 (s, 6H), 0.09 (s, 6H), 0.89 (s, 9H), 0.90 (s,
9H), 3.19 (bs, 3H), 4.20 (s, 2H), 4.33 (s, 2H), 4.89 (d, 2H, J )
7.4 Hz), 5.76 (t, 1H, J ) 7.4 Hz), 7.74 (s, 1H). MS (CI/NH3)
m/z 512 (M + H+)+.
m eso-1,3-tr a n s-[2-(2-Ch lor o-6-m eth yla m in op u r in -9-yl-
m et h yl)-3-h yd r oxym et h ylcyclop r op yl]m et h a n ol
(72)
(m eso-tr a n s). Meth od E. Crude 72 (50 mg) was obtained
from 70 (78 mg, 0.148 mmol). 1H NMR (CDCl3) δ 1.24 (m, 1H),
1.38(m, 2H), 3.05 (bs, 3H), 3.48-3.71 (m, 2H), 4.08 (d, 2H, J
) 6.9 Hz), 8.13 (s, 1H). MS (CI/NH3) m/z 298 (M + H+)+.
2-[2-(2-Ch lor o-6-m eth yla m in op u r in -9-yl)-eth ylid en e]-
p r op a n e-1,3-d iol (64). Meth od E. Compound 64 (100 mg,
86%) was obtained from 63 (210 mg, 0.410 mmol). 1H NMR
(DMSO-d6) δ 2.91 (s, 3H), 3.96 (s, 2H), 4.12 (s, 2H), 4.85 (d,

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3105
Meth od D. Compound (()-80 (430 mg, 71%) was obtained
m eso-1,3-cis-[2-(2-Ch lor o-6-m e t h yla m in op u r in -9-yl-
m eth yl)-3-h yd r oxym eth ylcyclop r op yl]m eth a n ol
1
(73)
from (()-79 (520 mg, 1.05 mmol). H NMR (CDCl3) δ 1.05 (s,
(m eso-cis). Meth od E. Crude 73 (20 mg) was obtained
9H), 1.04-1.46 (m, 3H), 3.18 (bs, 3H), 3.28 (s, 3H), 3.24-3.45
(m, 2H), 3.50 (dd, 1H, J ) 6.0, 10.4 Hz), 3.70 (dd, 1H, J ) 8.0,
11.4 Hz), 3.98 (dd, 1H, J ) 5.1, 11.4 Hz), 4.28 (dd, 1H, J )
7.0, 14.6 Hz), 4.54 (m, 2H), 6.18 (bs, 1H), 7.33-7.49 (m, 6H),
7.61-7.74 (m, 4H), 8.05 (s, 1H). MS (positive-ion FAB) 580
(M + H+)+.
1
from 71 (31 mg, 0.059 mmol). H NMR (CD3OD) δ 1.38-1.63
(m, 3H), 3.07 (bs, 3H), 3.72-3.93 (m, 4H), 4.36 (d, 2H, J ) 7.1
Hz), 8.16 (s, 1H). MS (CI/NH3) m/z 298 (M + H+)+.
m eso-1,3-tr a n s-P h osp h or ic Acid Diben zyl Ester 2-(bis-
ben zyloxyph osph oyloxym eth yl)-3-(2-ch lor o-6-m eth ylam i-
n op u r in -9-ylm eth yl)cyclop r op ylm eth yl Ester (74) (m eso-
tr a n s). Meth od F . Compound 74 (56 mg, 46% two steps yield
from 70) was obtained from crude 72 (50 mg).1H NMR (CDCl3)
δ 1.14-1.43 (m, 3H), 3.10 (bs, 3H), 3.82-3.97 (m, 6H), 4.90-
5.06 (m, 8H), 5.99 (bs, 1H), 7.21-7.39(m, 20H), 7.68 (bs, 1H).
(()-1,2-cis-1,3-tr a n s-[2-(2-Ch lor o-6-m eth yla m in op u r in -
9-ylm eth yl)-3-h yd r oxym eth ylcyclop r op yl]m eth a n ol ((()-
81). A mixture of silyl ether (()-80 (230 mg, 0.467 mmol),
c-HCl (2 mL), and methanol (6 mL) was stirred at 80 °C for 4
h. All volatile materials were removed by nitrogen purging.
The residue was dissolved in methanol (20 mL) and treated
with potassium carbonate (100 mg). The mixture was stirred
at room temperature for 4 h, filtered, and evaporated. The
residue was purified by PTLC (chloroform/methanol ) 4:1) to
31P NMR (CDCl3) δ -22.458. MS (CI/NH3) m/z 818 (M + H) +
.
High-resolution MS (positive-ion FAB) calcd for C40H42N5O8-
+
ClP2Cs [M2- + H+ + Cs2+
] 950.1252, found 950.1229.
m eso-1,3-cis-P h osp h or ic Acid Diben zyl Ester 2-(bis-
ben zyloxyph osph oyloxym eth yl)-3-(2-ch lor o-6-m eth ylam i-
n op u r in -9-ylm eth yl)cyclop r op ylm eth yl Ester (75) (m eso-
cis). Meth od F . Compound 75 (17 mg, 33% two steps yield
from 71) was obtained from crude 73 (20 mg). 1H NMR (CDCl3)
δ 1.46 (m, 2H), 1.61 (m, 2H), 3.15 (bs, 3H), 3.97 (dd, 2H, J )
7.8, 10.8 Hz), 4.04 (d, 2H, J ) 6.9 Hz), 4.18 (dd, 2H, J ) 6.6,
10.8 Hz), 4.90-5.19 (m, 8H), 5.99 (bs, 1H), 7.23-7.40(m, 20H),
7.70 (bs, 1H). 31P NMR (CDCl3) δ -22.338. MS (CI/NH3) m/z
1
give diol (()-81 (90 mg, 65%). H NMR (CD3OD) δ 1.07-1.43
(m, 3H), 3.08 (bs, 3H), 3.32-3.66 (m, 3H), 3.92 (dd, 1H, J )
5.8, 11.8 Hz), 4.20-4.34 (m, 2H), 8.20 (s, 1H). MS (positive-
ion FAB) 298 (M + H+)+.
(()-1,2-cis-1,3-tr a n s-P h osp h or ic Acid Di-ter t-bu tyl Es-
t er 2-(2-Ch lor o-6-m et h yla m in op u r in 9-yl)-3-(d i-ter t-b u -
toxyph osph or yloxym eth yl)cyclopr opylm eth yl Ester ((()-
82). Meth od C. Compound (()-82 (46 mg, 62%) was obtained
from diol (()-81 (32 mg, 0.108 mmol). 1H NMR (CDCl3) δ 1.43
(s, 9H), 1.45 (s, 9H), 1.49 (s, 18H), 3.18 (s, 3H), 3.73 3.98 (m,
3H), 4.15-4.42 (m, 3H), 6.19 (bs. 1H), 7.93 (s, 1H). MS
(positive-ion FAB) 682 (M + H+)+.
(()-2-(2-Ch lor o-6-m eth ylam in opu r in -9-yl)cyclopr opan e-
1,1-d ica r boxylic Acid Dieth yl Ester ((()-84). A mixture of
allyl bromide 83 (115 mg, 0.435 mmol), potassium carbonate
(71 mg, 0.52 mmol), N6-methyl-2-chloropurine (80 mg, 0.435
mmol), and dimethylformamide (3 mL) was stirred for 2 days
at 30-35 °C. After the usual aqueous workup using ethyl
acetate (40 mL) and 5% NaCl solution (20 mL × 3), the organic
layer was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography (hexanes/ethyl acetate ) 1:5) to give (()-84
(68 mg, 41%). 1H NMR (CDCl3) δ 1.01 (t, 3H, J ) 7.1 Hz), 1.32
(t, 3H, J ) 7.1 Hz), 2.06 (dd, 1H, J ) 6.6, 8.2 Hz), 2.74 (t, 1H,
J ) 6.3 Hz), 3.15 (bs, 3H), 3.87-4.06 (m, 2H), 4.19-4.39 (m,
3H), 6.19 (bd, 1H, J ) 3.6 Hz), 7.68 (s, 1H). MS (positive-ion
FAB) 368 (M + H+)+.
818 (M + H+)+. High-resolution MS (positive-ion FAB) calcd
+
for C40H42N5O8ClP2Cs [M2- + H+ + Cs2+
950.1298.
]
950.1252, found
1,2-Bis-(ter t-bu tyld im eth ylsila n yloxym eth yl)-3-m eth -
oxym et h oxym et h ylcyclop r op a n e (76). Chloromethyl-
methyl ether (234 mg, 2.90 mmol) was added dropwise by
syringe over 2 min to a stirred solution of cyclopropyl alcohol
68a (1.0 g, 2.90 mmol) and diisopropylethylamine (563 mg,
4.36 mmol) in methylene chloride (15 mL) cooled in an ice-
water bath. The reaction mixture was stirred for 1 h at the
same temperature, for 4 h at room temperature. Ice (10 g) was
added, the mixture was stirred for 10 min, and water (30 mL)
was added. The aqueous layer was extracted with ethyl acetate
(150 mL), and the organic layer was washed with brine/water
(30 mL × 2, 1/1), dried over sodium sulfate, filtered, and
concentrated to give 76 (1.05 g, 93%). 1H NMR (CDCl3) δ 0.05
(s, 6H), 0.90 (s, 9H), 0.94-1.16 (m, 3H), 3.36 (s, 3H), 3.45 (d,
1H, J ) 5.9 Hz), 3.66-3.76 (m 4H), 4.66 (s, 2H). MS (CI/NH3)
m/z 405 (M + H+)+
(2-Hydr oxym eth yl-3-m eth oxym eth oxym eth ylcyclopr o-
p yl)m eth a n ol (77). A mixture of disilyl ether 76 (640 mg,
1.65 mmol), tetrabutylammonium fluoride (2.47 mL, 2.47
mmol, 1 M solution in tetrahydrofuran), and tetrahydrofuran
(5 mL) was stirred for 3 h at room temperature. After removal
of volatile materials in vacuo, the residue was purified by
column chromatography (ethyl acetate/methanol ) 10:1) to
give diol 77 (275 mg, 95%). MS (CI/NH3) m/z 194 (M + NH4+)+.
(()-1,2-cis-1,3-tr a n s-[2-(ter t-Bu tyld ip h en ylsila n yloxy-
m eth yl)-3-m eth oxym eth oxym eth ylcyclopr opyl]m eth an ol
((()-78). Neat tert-butyldiphenylsilyl chloride (498 mg, 1.56
mmol) was added to a stirred solution of diol 77 (275 mg, 1.56
mmol) and (dimethylamino)pyridine (190 mg, 1.56 mmol) in
methylene chloride (7 mL). The reaction mixture was stirred
for 3 days at room temperature. Methylene chloride was
removed in vacuo, and the residue was purified by column
chromatography (hexanes/ethyl acetate ) 1:1) to give 78 (479
(()-[2-(2-Ch lor o-6-m eth yla m in op u r in -9-yl)-1-h yd r oxy-
m eth ylcyclop r op yl]m eth a n ol ((()-85). To a cooled (-78
°C), stirred solution of diester (()-84 (33 mg, 0.0860 mmol) in
methylene chloride was added 1 M DIBAL (in hexanes, 0.52
mL, 0.52 mmol). The reaction mixture was stirred for 20 min
at the same temperature and for 1 h at room temperature.
After quenching with methanol (0.5 mL), the resulting solution
was directly purified by PTLC (chloroform/methanol ) 4:1) to
1
give (()-85 (17 mg, 70%). H NMR (CDCl3 0.5 mL + CD3OD
30 µL) δ 1.14 (dd, 1H, J ) 4.5, 6.8 Hz), 1.32 (t, 1H, J ) 6.3,
6.7 Hz), 3.07 (bs, 3H), 3.11 (d, 1H, J ) 12.4 Hz), 3.29 (dd, 1H,
J ) 4.3, 7.7 Hz), 3.60 (d, 1H, J ) 12.2 Hz), 3.70 (ABq, 1H, J
) 12.0 Hz), 7.09 (bs, 1H), 7.74 (s, 1H). MS (positive-ion FAB)
284 (M + H+)+.
(()-P h osp h or ic Acid Di-ter t-bu tyl Ester 2-(2-Ch lor o-
6-m eth yla m in op u r in -9-yl)-1-(d i-ter t-bu toxyp h osp h or yl-
oxym eth yl)cyclop r op ylm eth yl Ester ((()-86). Meth od C.
(()-86 (30 mg, 85%) was obtained from (()-85 (15 mg, 0.053
1
mg, 70%). H NMR (CDCl3) δ 0.93 (m, 1H), 1.07 (s, 9H), 1.21
1
(m, 1H), 1.41 (m, 1H), 3.30 (s, 3H), 3.34-3.49 (m, 4H), 4.02
(dd, 1H, J ) 5.4, 12.0 Hz), 4.11 (dd, 1H, J ) 5.4, 11.5 Hz),
4.57 (s, 2H), 7.36-7.50 (m, 6H), 7.65-7.76 (m, 4H). MS (CI/
NH3) m/z 415 (M + H+)+.
(()-1,2-cis-1,3-tr a n s-Meth a n esu lfon ic Acid 2-(ter t-Bu -
tyld ip h en ylsila n yloxym eth yl)-3-m eth oxym eth oxym eth -
ylcyclop r op ylm eth yl Ester ((()-79). Meth od D. Crude (()-
79 (538 mg, 95%) was obtained from (()-78 (479 mg, 1.15
mmol).
mmol). H NMR (CDCl3) δ 1.39 (s, 9H), 1.41 (s, 9H), 1.51 (s,
9H), 1.52 (s, 9H), 1.63 (t, 1H, J ) 7.3 Hz), 1.78 (dd, 1H, J )
4.9, 7.0 Hz), 3.17 (bs, 3H), 3.58 (dd, 1H, J ) 5.0, 7.7 Hz), 3.74-
3.87 (m, 2H), 4.07-4.21 (m, 2H), 6.10 (bs, 1H), 7.79 (s, 1H).
MS (positive-ion FAB) 668 (M + H+)+.
(()-[2,2-Bis-(t er t -b u t yld im e t h ylsila n yloxym e t h yl)-
cyclop r op yl]m eth a n ol ((()-87). To a stirred, cooled (0 °C)
solution of allyl alcohol 61 (220 mg, 0.635 mmol) in methylene
chloride (8 mL) was added diethylzinc (1 M in hexane, 4.45
mL, 4.45 mmol), followed by an addition of diiodomethane (510
mg, 1.91 mmol). After 20 min at room temperature, the
mixture was allowed to reach rt and stirred overnight. Aqueous
(()-1,2-cis-1,3-tr a n s-{9-[2-(ter t-Bu t yld ip h en ylsila n yl-
o x y m e t h y l)-3-m e t h o x y m e t h o x y m e t h y l-c y c lo p r o p y l-
m et h yl]-2-ch lor o-9H -p u r in -6-yl}-m et h yla m in e ((()-80).

3106 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Kim et al.
saturated ammonium chloride was carefully added, and the
resulting mixture was extracted with ether (30 mL), washed
successively with saturated sodium bicarbonate (20 mL), brine
(20 mL), dried over sodium sulfate, filtered, evaporated. The
residue was purified by silica gel column chromatography
(hexanes/ethyl acetate ) 4:1) to give 87 (220 mg, 96%). 1H
NMR (CDCl3) δ 0.04 (s, 6H), 0.11 (s, 3H), 0.12 (s, 3H), 0.35 (t,
1H, J ) 4.1 Hz), 0.70 (dd, 1H, J ) 5.0, 8.4 Hz), 0.90 (s, 9H),
0.92 (s, 9H), 1.19 (m, 1H), 2.87 (d, 1H, J ) 10.3 Hz), 3.25 (d,
1H, J ) 11.0 Hz), 3.26 (t, 1H, J ) 12.1 Hz), 3.96 (dd, 1H, J )
5.4, 12.1 Hz), 4.13 (d, 1H, J ) 10.3 Hz), 4.29 (d, 1H, J ) 11.0
Hz). MS (positive-ion FAB) 361 (M + H+)+.
to 0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase
to give bisphosphate 7 (9.6 mg, 41%).
P h osp h or ic Acid Mon o-[2-(6-m eth yla m in op u r in -9-yl-
m eth yl)-3-p h osp h on ooxy-p r op yl] Ester , Am m on iu m Sa lt
(6). Meth od H. Compound 6 (12.0 mg, 70%) was obtained from
1
35 (28 mg, 0.369 mmol). H NMR (D2O) δ 2.57 (m, 1H), 3.11
(bs, 3H), 3.87 (m, 4H), 4.36 (d, 2H, J ) 7.1 Hz), 8.17 (s, 1H),
8.22 (s, 1H). 31P NMR (D2O) -21.005. MS (negative-ion FAB)
396 (M - H+)-.
P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-m eth yla m in op u -
r in -9-ylm eth yl)-3-p h osp h on ooxyp r op yl] Ester , Am m o-
n iu m Sa lt (7). Meth od H. Compound 7 (9.6 mg, 41%) was
obtained from 36 (37 mg, 0.0467 mmol). 1H NMR (D2O) δ 2.56
(m, 1H), 3.04 (bs, 3H), 3.87 (m, 4H), 4.28 (d, 2H, J ) 7.3 Hz),
8.09 (s, 1H). 31P NMR (D2O) -20.975 (s).
Meth od I. Dep r otection of ter t-Bu tylp h osp h a tes w ith
Tr iflu or oa cetic Acid . A solution of tert-butylphosphate 39
(5 mg, 0.011 mmol) 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 using
ion-exchange column chromatography on Sephadex-DEAE-25
resin and a linear gradient (0.01 to 0.5 M) of 0.5 M ammonium
bicarbonate as the mobile phase to give monophosphate 8 (3.3
mg, 79%) as an ammonium salt.
(()-P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-m eth yla m i-
n op u r in -9-ylm et h yl)-3-h yd r oxyp r op yl] E st er , Am m o-
n iu m Sa lt ((()-8). Meth od I. Compound 8 (3.3 mg, 79%) was
obtained from (5.0 mg, 0.0107 mmol). 1H NMR (D2O) δ 2.55
(m, 1H), 3.11 (s, 3H), 3.69 (d, 2H, J ) 5.9 Hz), 3.82-4.02 (m,
2H), 4.44 (d, 2H, J ) 7.8 Hz), 8.87 (s, 1H). 31P NMR (D2O)
1.292 (s).
(()-P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-m eth yla m i-
n op u r in -9-ylm et h yl)-3-(d im et h oxyp h osp h or yloxy)p r o-
p yl] Ester , Am m on iu m Sa lt ((()-9). Meth od I. Compound
9 (2.5 mg, 76%) was obtained from 40 (3.8 mg, 0.00664 mmol).
1H NMR (D2O) δ 2.73 (m, 1H), 3.09 (bs, 3H), 3.67 (d, 3H, J )
3.2 Hz), 3.71 (d, 3H, J ) 3.2 Hz), 3.96 (m, 2H), 4.16 (m, 2H),
4.38 (d, 2H, J ) 6.0 Hz), 8.14 (s, 1H). 31P NMR (D2O) 0.664
(bs), 1.538 (s).
(()-Meth a n esu lfon ic Acid 2,2-bis-(ter t-bu tyld im eth yl-
sila n yloxym et h yl)cyclop r op ylm et h yl E st er ((()-88).
Meth od D. Crude (()-88 (250 mg, 93%) was obtained from
(()-87 (220 mg, 0.610 mmol).
(()-{9-[2,2-Bis-(ter t-b u t yld im et h ylsila n yloxym et h yl)-
c yc lop r o p ylm e t h y l]-2-c h lor o -9H -p u r in -6-y l}m e t h yl-
a m in e ((()-89). Meth od D. Compound (()-89 (185 mg, 62%)
was obtained from crude (()-88 (250 mg, 0.570 mmol). 1H
NMR (CDCl3) δ 0.02 (s, 6H), 0.04 (s, 3H), 0.06 (s, 3H), 0.0.54
(t, 1H, J ) 5.0 Hz), 0.79 (dd, 1H, J ) 5.0, 8.7 Hz), 0.88 (s, 9H),
0.89 (s, 9H), 1.31 (m, 1H), 3.20 (bs, 3H), 3.42 (d, 1H, J ) 10.2
Hz), 3.62 (d, 1H, J ) 11.1 Hz), 3.66 (d, 1H, J ) 10.3 Hz), 3.97
(d, 1H, J ) 11.0 Hz), 4.04 (dd, 1H, J ) 9.1, 14.3 Hz), 4.42 (dd,
1H, J ) 6.0, 14.4 Hz), 8.08 (s, 1H). MS (positive-ion FAB) 526
(M + H+)+.
(()-[2-(2-Ch lor o-6-m et h yla m in op u r in -9-ylm et h yl)-1-
h yd r oxym eth ylcyclop r op yl]m eth a n ol ((()-90). Meth od
E. Compound (()-90 (56 mg, 55%) was obtained from (()-89
(180 mg, 0.342 mmol). 1H NMR (CD3OD) δ 0.60 (t, 1H, J )
5.4 Hz), 0.77 (dd, 1H, J ) 5.1, 8.7 Hz), 1.37 (m, 1H), 3.08 (bs,
3H), 3.41 (d, 1H, J ) 11.4 Hz), 3.62 (d, 1H, J ) 11.1 Hz), 3.65
(d, 1H, J ) 11.8 Hz), 3.99 (d, 1H, J ) 12.0 Hz), 4.28 (d, 2H, J
) 7.3 Hz), 8.18 (s, 1H). MS (positive-ion FAB) 298 (M + H+)+.
(()-P h osp h or ic Acid Di-ter t-bu tyl Ester 2-(2-ch lor o-6-
m et h yla m in op u r in -9-ylm et h yl)-1-(d i-ter t-b u t oxyp h os-
p h or yloxym et h yl)cyclop r op ylm et h yl E st er ((()-91).
Meth od C. Compound (()-91 (23 mg, 40%) was obtained from
1
(()-90 (25 mg, 0.0840 mmol). H NMR (CDCl3) δ 0.77 (t, 1H,
(()-Acetic Acid 2-(2-Ch lor o-6-m eth yla m in op u r in -9-yl-
m eth yl)-3-p h osp h or yloxyp r op yl Ester , Am m on iu m Sa lt
((()-10). Meth od I. Compound 10 (6 mg, 59%) obtained from
J ) 5.7 Hz), 1.03 (dd, 1H, J ) 5.7, 8.4 Hz), 1.21-1.41 (m, 1H),
1.44 (s, 9H), 1.45 (s, 9H), 1.49 (s, 18H), 3.17 (bs, 3H), 3.74 (dd,
1H, J ) 5.9, 10.4 Hz), 3.95 (dd, 1H, J ) 5.4, 11.5 Hz), 4.04
(dd, 1H, J ) 6.6, 10.6 Hz), 4.27 (d, 2H, J ) 6.8 Hz), 4.38 (dd,
1H, J ) 4.5, 11.4 Hz), 6.66 (bs, 1H), 7.95 (s, 1H). MS (positive-
ion FAB) 682 (M + H+)+.
1
41 (12 mg, 0.0237 mmol). H NMR (D2O) δ 1.99 (s, 3H), 2.73
(m, 1H), 3.09 (bs, 3H), 3.98 (m, 2H), 4.12 (d, 2H, J ) 5.1 Hz),
4.44 (d, 2H, J ) 6.3 Hz), 8.60 (s, 1H). 31P NMR (D2O) 0.352
(s).
Meth od G. Dep r otection of ter t-Bu tylp h osp h a tes w ith
Dow ex 50 Resin . A mixture of bis(di-tert-butyl phosphate)
26 (20 mg, mmol) and Dowex 50 resin (100 mg) and methanol/
water (1:1, 5 mL) was stirred at 80 °C for 16 h. The resulting
mixture was filtered off through glass filter and washed with
water (5 mL × 2), the combined filtrate was lyophilized, and
the residue was purified with an ion-exchange column chro-
matography using Sephadex-DEAE A-25 resin with a linear
gradient (0.01 to 0.5 M) of 0.5 M ammonium bicarbonate as
the mobile phase to give bisphosphate 5 (9.0 mg, 63%) as an
ammonium salt.
(()-Diph osph or ic Acid Mon o-[2-(2-ch lor o-6-m eth ylam i-
n op u r in -9-ylm eth yl)-3-p h osp h on ooxyp r op yl] Ester , Am -
m on iu m Sa lt ((()-11). Compound 44 (6 mg, 0.00808 mmol)
was dissolved in methylene chloride (1 mL), and trimethylsilyl
bromide (60 µL, 0.455 mmol) was added. The reaction mixture
was stirred at room temperature for 10 min, followed by
quenching with 0.1 M triethylammonium bicarbonate (5 mL).
Methylene chloride was removed in vacuo, and the residue was
lyophilized and purified using ion-exchange column chroma-
tography on Sephadex-DEAE-25 resin with a linear gradient
(0.01-1.0 M) of 1.0 M ammonium bicarbonate as the mobile
phase to give bisphosphate 11 (3.5 mg, 72%) as an ammonium
P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-m eth yla m in op u -
r in -9-yl)-3-p h osp h on ooxyp r op yl] Ester , Am m on iu m Sa lt
1
salt. H NMR (D2O) δ 2.59 (m, 1H), 3.05 (bs, 3H), 3.74-4.09
(m, 4H), 4.31 (d, 2H, J ) 7.1 Hz), 8.12 (s, 1H). 31P NMR (D2O)
-10.457 (bs), 0.774 (s).
1
(5). H NMR (D2O) δ 3.03 (s, 3H), 4.35 (m, 2H), 4.46 (m, 2H),
5.16 (m, 1H), 8.97 (s, 1H). 31P NMR (D2O) 4.107. MS (negative-
ion FAB) 416 (M - H+)-.
P h osp h or ic Acid Mon o-[3-(2-ch lor o-6-m eth yla m in op u -
r in -9-ylm eth yl)-5-p h osp h on ooxyp en tyl] Ester , Am m o-
n iu m Sa lt (12). Meth od I. Compound 12 (8.0 mg, 69%) was
obtained from 49 (15 mg, 0.0219 mmol) as an ammonium salt.
1H NMR (D2O) δ 1.61-1.85 (m, 4H), 2.41 (m, 1H), 3.08 (s, 3H),
3.80-4.09 (m, 4H), 4.33 (d, 2H, J ) 7.3 Hz), 8.95 (s, 1H). 31P
NMR (D2O) 0.689 (s).
Meth od H. Dep r otection of Ben zyl P h osp h a tes w ith
Bor on Tr ich lor id e. Bis(dibenzyl phosphate) 36 (37 mg,
0.0442 mmol) was dissolved in anhydrous methylene chloride
(3 mL) and cooled to 0 °C. To this solution was added 1 M
boron trichloride (in dichloromethane, 0.442 mL, 0.442 mmol),
and the reaction mixture was stirred at 5 °C for 2 d (the
progress of the reaction was monitored by HPLC). After the
deprotection was done, the resulting mixture was quenched
by 1 M triethylammonium acetate (5 mL), followed by addition
of water (5 mL). This mixture was lyophilized, and the residue
was purified with an ion-exchange column chromatography
using Sephadex-DEAE-25 resin with a linear gradient (0.01
(R)-P h osp h or ic Acid Mon o-[1-(2-ch lor o-6-m eth yla m i-
n op u r in -9-ylm eth yl)-2-p h osp h on ooxyeth yl] Ester , Am -
m on iu m Sa lt (13). Meth od I. Compound 13 (5.0 mg, 44%)
was obtained from 58 (15 mg, 0.0234 mmol) as an ammonium
1
salt. H NMR (D2O) δ 3.07 (bs, 3H), 3.97 (m, 2H), 4.32-4.60
(m, 3H), 8.10 (s, 1H). 31P NMR (D2O) -0.446, -1.673.

SAR of Adenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3107
(S)-P h osp h or ic Acid Mon o-[1-(2-ch lor o-6-m eth yla m i-
n op u r in -9-ylm eth yl)-2-p h osp h on ooxyeth yl] Ester , Am -
m on iu m Sa lt (14). Meth od I. Compound 14 (8.3 mg, 55%)
was obtained from 59 (20 mg, 0.0311 mmol) as an ammonium
the indicated concentrations of nucleotide analogues. Ghosts
were incubated at 30 °C for 5 min, and total [3H]inositol
phosphates were quantitated by anion exchange chromatog-
raphy as previously described.27,28
1
salt. H NMR (D2O) δ 3.06 (bs, 3H), 3.78 (t, 2H, J ) 4.5 Hz),
Da ta An a lysis. Agonist potencies were calculated using a
four-parameter logistic equation and the GraphPad software
package (GraphPad, San Diego, CA). EC50 values (mean (
standard error) represent the concentration at which 50% of
the maximal effect is achieved. Relative efficacies (%) were
determined by comparison with the effect produced by a
maximal effective concentration of 2-MeSADP in the same
experiment. 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 inhibi-
tion is equal to 100 minus the residual fraction of stimulation
at the highest antagonist concentration. All concentration-
effect curves were replicated in at least three separate experi-
ments, carried out with different membrane preparations
using duplicate or triplicate assays.
Com p u ta tion a l Meth od s. A molecular model of the hu-
man P2Y1 receptor was built and optimized using Sybyl 6.634
modeling package, following the homology modeling approach
described in our previous studies30,35 and using the recently
reported X-ray structure of bovine rhodopsin22 as a structural
template. All calculations were performed on a Silicon Graph-
ics Octane R12000 workstation. Briefly, sequences of the P2Y1
transmembrane domains, identified in our previously pub-
lished model, were amended by comparison to the correspond-
ing domains of rhodopsin, according to a published sequence
alignment.36 Transmembrane P2Y1 helices were built from
these sequences, in homology to the corresponding helices of
rhodopsin, and minimized individually. The minimized helices
were then grouped by adding one at a time until a helical
bundle (TM region), matching the overall characteristics of the
crystallographic structure of rhodopsin, was obtained. The TM
region was further modified by adding the amino acid residues
189-213, which comprise EL2. The conformation of EL2 was
initially modeled according to the corresponding domain in
rhodopsin including the Cys124-Cys202 disulfide bond. At
each step the structures were minimized using the Tripos force
field with Amber37 all-atom force parameters until the rms
value of the conjugate gradient (CG) was <0.1 kcal/mol/Å. A
fixed dielectric constant ) 4.0 was used throughout these
calculations. A model of adenosine 5′-triphosphate (ATP) was
constructed from the crystallographic coordinates of ATP-
phosphoglycerate kinase.38 Models of 92 and other nucleotides
were constructed from ATP using the “Sketch Molecule”
module of Sybyl. The ligands were minimized in Sybyl (using
MOPAC calculated partial atomic charges) and were rigidly
docked into the helical bundle using graphical manipulation
coupled to continuous energy monitoring (Dock module of
Sybyl). Whenever a final position was reached, consistent with
a local energy minimum, the complexes of receptor and ligand
were subjected to an additional CG minimization run of up to
1500 steps.
4.30-4.55 (m, 3H), 8.17 (s, 1H). 31P NMR (D2O) 3.851, 2.745.
MS (negative-ion FAB) 416 (M - H+)-.
P h osp h or ic Acid 4-(2-Ch lor o-6-m eth yla m in op u r in -9-
yl)-2-p h osp h on ooxym eth ylbu t-2-en yl Ester , Am m on iu m
Sa lt (15). Meth od I. Compound 15 (9.7 mg, 63%) was
obtained from 65 (20 mg, 0.0299 mmol) as an ammonium salt.
1H NMR (D2O) δ 3.03 (bs, 3H), 4.53 (d, 2H, J ) 7.0 Hz), 4.67
(d, 2H, J ) 7.0 Hz), 5.01 (d, 2H, J ) 6.6 Hz), 6.03 (m, 1H),
8.61 (s, 1H). 31P NMR (D2O) 0.342.
m eso-1,3-tr a n s-P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-
m eth yla m in op u r in -9-ylm eth yl)-3-p h osp h on ooxym eth yl-
cyclop r op ylm eth yl] Ester , Am m on iu m Sa lt (16). Meth od
H. Compound 16 (28.0 mg, 77%) was obtained from 74 (56
mg, 0.068 mmol) as an ammonium salt.1H NMR (D2O) δ 1.42-
1.52 (m, 3H), 3.05 (bs, 3H), 3.93 (m, 4H), 4.13 (d, 2H, J ) 6.6
Hz), 8.32 (s, 1H). 31P NMR (D2O) -21.217. MS (positive-ion
FAB) 458 (M + H+)+.
m eso-1,3-cis-P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-
m eth yla m in op u r in -9-ylm eth yl)-3-p h osp h on ooxym eth yl-
cyclop r op ylm eth yl] Ester , Am m on iu m Sa lt (17). Meth od
H. Compound 17 (6.2 mg, 57%) was obtained from 75 (17.0
mg, 0.0208 mmol) as an ammonium salt. 1H NMR (D2O) δ 1.67
(m, 2H), 1.82 (m, 1H), 3.07 (bs, 3H), 4.05 (m, 2H), 4.22 (m,
2H), 4.40 (d, 2H, J ) 6.9 Hz), 8.28 (s, 1H). 31P NMR (D2O)
+
-21.287. MS (positive-ion FAB) 458 (M + H+)
.
(()-1,2-cis-1,3-tr a n s-P h osph or ic Acid Mon o-[2-(2-ch lor o-
6-m eth yla m in op u r in -9-ylm eth yl)-3-p h osp h on ooxym eth -
ylcyclop r op ylm eth yl] Ester , Am m on iu m Sa lt ((()-18).
Meth od H. Compound ((()-18) (11.0 mg, 71%) was obtained
1
from (()-82 (20 mg, 0.0293 mmol) as an ammonium salt. H
NMR (D2O) δ 1.41 (m, 1H), 1.49-1.67 (m, 2H), 2.99 (bs, 3H),
3.62-3.97 (m, 4H), 4.15-4.28 (m, 4H), 8.48 (s, 1H). 31P NMR
(D2O) 0.583.
(()-P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-m eth yl-
a m in o p u r in -9-y l)-1-p h o s p h o n o o x y m e t h y lc y c lo p r o -
p ylm eth yl] Ester ((()-19). Meth od I. Compound (()-19 (7.0
mg, 61%) was obtained from (()-86 (15 mg, 0.0225 mmol) as
1
an ammonium salt. H NMR (D2O) δ 1.69 (t, 1H, J ) 7.8 Hz),
1.77 (dd, 1H, J ) 4.6, 7.2 Hz), 3.06 (bs, 3H), 3.49 (dd, 1H, J )
4.4, 11.3 Hz), 3.79 (dd, 1H, J ) 4.6, 7.8 Hz), 3.91 (dd, 1H, J )
6.4, 11.0 Hz), 4.10 (dd, 1H, J ) 4.4, 11.3 Hz), 4.35 (dd, 1H, J
) 6.7, 11.0 Hz), 8.67 (s, 1H). 31P NMR (D2O) 0.183, 0.644 (s).
(()-P h osp h or ic Acid Mon o-[2-(2-ch lor o-6-m eth yla m i-
n op u r in -9-ylm eth yl)-1-p h osp h on ooxym eth ylcyclop r op y-
lm eth yl] Ester ((()-20). Meth od I. Compound (()-20 (5.0
mg, 55%) was obtained from (()-91 (12 mg, 0.0176 mmol) as
1
an ammonium salt. H NMR (D2O) δ 0.85 (t, 1H, J ) 5.5 Hz),
1.06 (dd, 1H, J ) 5.8, 8.7 Hz), 1.63 (m, 1H), 3.08 (bs, 3H), 3.67
(dd, 1H, J ) 4.8, 10.5 Hz), 3.86 (dd, 1H, J ) 3.6, 11.3 Hz),
4.01 (dd, 1H, J ) 5.5, 10.6 Hz), 4.20-4.40 (m, 3H), 8.39 (s,
1H). 31P NMR (D2O) 0.548 (s).
Ack n ow led gm en t. This work was supported by
USPHS Grants GM38213 and HL54889. D.B. is grateful
for financial support from Gilead Sciences (Foster City,
CA).
P h ar m acological An alyses. P2Y1 receptor promoted stimu-
lation of inositol phosphate formation by adenine nucleotide
analogues was measured in turkey erythrocyte membranes as
previously described.27,28 The K0.5 values were averaged from
3 to 8 independently determined concentration-effect curves
for each compound. Briefly, 1 mL of washed turkey erythro-
cytes was incubated in inositol-free medium (DMEM; Gibco,
Gaithersburg MD) with 0.5 mCi 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. Erythrocyte ghosts were prepared by rapid lysis in
hypotonic buffer (5 mM sodium phosphate, pH 7.4, 5 mM
MgCl2, 1 mM EGTA) as described.27 Phospholipase C activity
was measured in 25 µL of [3H]inositol-labeled ghosts (ap-
proximately 175 µg of protein, 200-500,000 cpm/assay) in a
medium containing 424 µM CaCl2, 0.91 mM MgSO4, 2 mM
EGTA, 115 mM KCl, 5 mM KH2PO4, and 10 mM Hepes, pH
7.0. Assays (200 µL final volume) contained 1 µM GTPγS, and
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