Synthesis, biological activity, and molecular modeling of ribose- modified deoxyadenosine bisphosphate analogues as P2Y1 receptor ligands

Article abstract of DOI:10.1021/jm990249v

The structure-activity relationships of adenosine-3',5'-bisphosphates as P2Y₁ receptor antagonists have been explored, revealing the potency- enhancing effects of the N⁶-methyl group and the ability to substitute the ribose moiety (Nandanan et al. J. Med. Chem. 1999, 42, 1625-1638). We have introduced constrained carbocyclic rings (to explore the role of sugar puckering), non-glycosyl bonds to the adenine moiety, and a phosphate group shift. The biological activity of each analogue at P2Y₁ receptors was characterized by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit its stimulation elicited by 30 nM 2-methylthioadenosine-5'-diphosphate (antagonist effect). Addition of the N₆-methyl group in several cases converted pure agonists to antagonists. A carbocyclic N⁶-methyl-2'-deoxyadenosine bisphosphate analogue was a pure P2Y₁ receptor antagonist and equipotent to the ribose analogue (MRS 2179). In the series of ring-constrained methanocarba derivatives where a fused cyclopropane moiety constrained the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle, the 6-NH₂ (N)-analogue was a pure agonist of EC₅₀ 155 nM and 86-fold more potent than the corresponding (S)-isomer. The 2-chloro-N⁶-methyl-(N)-methanocarba analogue was an antagonist of IC₅₀ 51.6 nM. Thus, the ribose ring (N)-conformation appeared to be favored in recognition at P2Y₁ receptors. A cyclobutyl analogue was an antagonist with IC₅₀ of 805 nM, while morpholine ring-containing analogues were nearly inactive. Anhydrohexitol ring-modified bisphosphate derivatives displayed micromolar potency as agonists (6-NH₂) or antagonists (N⁶-methyl). A molecular model of the energy-minimized structures of the potent antagonists suggested that the two phosphate groups may occupy common regions. The (N)- and (S)-methanocarba agonist analogues were docked into the putative binding site of the previously reported P2Y₁ receptor model.

Full text of DOI:10.1021/jm990249v

J . Med. Chem. 2000, 43, 829-842
829
Syn th esis, Biologica l Activity, a n d Molecu la r Mod elin g of Ribose-Mod ified
Deoxya d en osin e Bisp h osp h a te An a logu es a s P 2Y1 Recep tor Liga n d s
Erathodiyil Nandanan,§ Soo-Yeon J ang,§ Stefano Moro,† Hea Ok Kim,# Maqbool A. Siddiqui, Pamela Russ,
Victor E. Marquez, Roger Busson,| Piet Herdewijn,| 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, Department of Pharmacology, University of North
Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7365, Laboratory of Medicinal Chemistry, National Cancer
Institute, Bethesda, Maryland 20892, Laboratory of Pharmaceutical Chemistry, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, Molecular Modeling Section,
Pharmaceutical Science Department, University of Padova, Via Marzolo, 5, 35131 Padova, Italy, and
College of Medicine, Yonsei University, Seoul 120-752, Korea
Received May 19, 1999
The structure-activity relationships of adenosine-3′,5′-bisphosphates as P2Y1 receptor an-
tagonists have been explored, revealing the potency-enhancing effects of the N6-methyl group
and the ability to substitute the ribose moiety (Nandanan et al. J . Med. Chem. 1999, 42, 1625-
1638). We have introduced constrained carbocyclic rings (to explore the role of sugar puckering),
non-glycosyl bonds to the adenine moiety, and a phosphate group shift. The biological activity
of each analogue at P2Y1 receptors was characterized by measuring its capacity to stimulate
phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit its stimulation
elicited by 30 nM 2-methylthioadenosine-5′-diphosphate (antagonist effect). Addition of the
N6-methyl group in several cases converted pure agonists to antagonists. A carbocyclic N6-
methyl-2′-deoxyadenosine bisphosphate analogue was a pure P2Y1 receptor antagonist and
equipotent to the ribose analogue (MRS 2179). In the series of ring-constrained methanocarba
derivatives where a fused cyclopropane moiety constrained the pseudosugar ring of the
nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the
pseudorotational cycle, the 6-NH2 (N)-analogue was a pure agonist of EC50 155 nM and 86-fold
more potent than the corresponding (S)-isomer. The 2-chloro-N6-methyl-(N)-methanocarba
analogue was an antagonist of IC50 51.6 nM. Thus, the ribose ring (N)-conformation appeared
to be favored in recognition at P2Y1 receptors. A cyclobutyl analogue was an antagonist with
IC50 of 805 nM, while morpholine ring-containing analogues were nearly inactive. Anhydro-
hexitol ring-modified bisphosphate derivatives displayed micromolar potency as agonists (6-
NH2) or antagonists (N6-methyl). A molecular model of the energy-minimized structures of
the potent antagonists suggested that the two phosphate groups may occupy common regions.
The (N)- and (S)-methanocarba agonist analogues were docked into the putative binding site
of the previously reported P2Y1 receptor model.
In tr od u ction
Nucleotide agonists binding at P2Y1 receptors induce
activation of phospholipase C (PLC), which generates
inositol phosphates and diacylglycerol from phospha-
tidylinositol (4,5)bisphosphate,8 leading to a rise in
intracellular calcium. A P2Y1 receptor in platelets is
involved in ADP-promoted aggregation.9-11 Thus, a
selective P2Y1 receptor antagonist may have potential
as an antithrombotic agent, while a selective P2Y1
receptor agonist may have potential as an antihyper-
tensive or antidiabetic agent.12,13
P2 receptors, which are activated by purine and/or
pyrimidine nucleotides, consist of two families:
G
protein-coupled receptors termed P2Y, of which five
mammalian subtypes have been cloned, and ligand-
gated cation channels termed P2X, of which seven
mammalian subtypes have been cloned.1,2 The P2Y1
receptor, which is present in the heart, skeletal and
various smooth muscles, prostate, ovary, and brain,3
was the first P2 subtype to be cloned.4 The nomenclature
of P2 receptors and their various ligand specificities
have been reviewed.5-7
Recently, progress in the synthesis of selective P2
receptor antagonists has occurred.11,14-21 Adenosine-
3′,5′- and -2′,5′-bisphosphates were recently shown to
be selective antagonists or partial agonists at P2Y1
receptors.19,20 Other classes of P2 antagonists include
pyridoxal phosphate derivatives,16,21 isoquinolines,15
large aromatic sulfonates related to the trypanocidal
drug suramin22 and various dyestuffs,4 and 2′,3′-nitro-
phenyl nucleotide derivatives.14 SAR (structure-activity
relationship) studies of analogues of adenosine bis-
* 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.
§ NIH.
‡ University of North Carolina.
NCI.
| Rega Institute for Medical Research.
† University of Padova.
#
Yonsei University.
10.1021/jm990249v CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/23/2000

830 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Nandanan et al.
Sch em e 1. Synthesis of
N6-Methyl-2′-deoxyaristeromcyin Derivativesa
a
Reagents: (a) (1) MeI, DMF, (2) MH4OH, 90 °C; (b) DCTIDS/
imidazole, DMF; (c) PhOCSCl/DMAP, CH3CN; (d) n-Bu3SnH/
AIBN, toluene; (e) n-Bu4NF, THF; (f) LDA/TBPP; (g) (1) H2/Pd-
C, (2) NH4HCO3.
anhydrohexitol27 modifications of the ribose ring (Figure
1) are tolerated at P2Y1 receptors. We have further
explored the SAR of these two series and introduced
other major modifications of the ribose moiety. These
modifications include fixing the ring pucker conforma-
tion in the carbocyclic series using a bridging cyclopro-
pane ring,28,29 ring enlargement with introduction of a
nitrogen atom,30 and ring contraction.31
F igu r e 1. Structures of ribose-modified nucleotide analogues
synthesized as ligands for the P2Y1 receptor. The correspond-
ing ammonium salts were synthesized and tested for biological
activity. Rigid compounds 4 and 5 are in the E (N) and E (S)
2
3
conformations, respectively, as defined by the pseudorotational
cycle.29 The anti-conformation (not shown) of 4 is favored.
phosphates17,18 have resulted in N6-methyl-2′-deoxy-
adenosine-3′,5′-bisphosphate (1a , MRS 2179; Figure 1),
a competitive antagonist at human and turkey P2Y1
receptors, with a KB value of approximately 100 nM.18
The presence of an N6-methyl group and the absence of
a 2′-hydroxyl group both enhanced affinity and de-
creased agonist efficacy, thus resulting in a pure an-
tagonist at both turkey and human P2Y1 receptors. The
corresponding 2-Cl analogue (1b)19 was slightly more
potent than 1a as an antagonist at turkey P2Y1 recep-
tors, with an IC50 value of 0.22 µM in blocking the effects
of 10 nM 2-methylthioadenosine-5′-diphosphate (2-
MeSADP). Compound 1a was inactive at P2Y2, P2Y4,
and P2Y6 subtypes, at the adenylyl cyclase-linked P2Y
receptor in C6 glioma cells (J . Boyer, personal com-
munication),23 at a novel avian P2Y receptor that
inhibits adenylyl cyclase,24 and at the canine P2Y11
receptor (B. Torres, A. Zambon, and P. Insel, unpub-
lished observations). However, the selectivity of certain
nucleotide antagonists for the P2Y1 receptor is not
absolute, since 1a also displayed considerable antago-
nist affinity at P2X1 receptors (IC50 1.15 µM) and at
P2X3 receptors (IC50 12.9 µM), but not at P2X2 and P2X4
receptors.25
Resu lts
Ch em ica l Syn th esis. We have explored structural
modifications (Figure 1) of N6-methyl-2′-deoxyadeno-
sine-3′,5′-bisphosphate (1a ) in order to increase P2Y1
receptor affinity and selectivity and to probe the rela-
tionship between ribose structure and agonist efficacy.
2-Position adenine modifications (e.g., chloro, 1b; amino,
1c; and methylthio, 1d ) known to be favorable for
antagonist potency19 have been included for comparison.
Synthetic routes are shown in Schemes 1-7, and
characterization and yields of the nucleotide derivatives
prepared are summarized in Table 1.
An isomer of 1a , in which a phosphate group is shifted
from the 5′- to the 2′-position, 2, was synthesized by
phosphorylation of 5′-deoxyadenosine by the tetrabenzyl
pyrophosphate method,32,33 followed by hydrogenation
to remove the benzyl groups. We have also prepared 2′-
deoxyadenosine bisphosphate analogues containing four-
and five-membered carbocyclic ring (3-6) and six-
membered ring (7 and 8) modifications of the ribose
moiety.
Some of the nucleosides utilized for phosphorylation
were obtained commercially, such as the 5′-deoxy ana-
logue leading to compound 2. The carbocyclic 2′-deoxy-
adenosine bisphosphate 3a was reported in the previous
study. The N6-methyl group in 3b and 3c was intro-
duced in the carbocyclic series by the Dimroth re-
arrangement (Scheme 1), starting with aristeromycin,
10. The 2′-hydroxyl group was removed selectively using
To move away from the nucleotide structure of 1a and
thereby increase biological stability and selectivity for
the receptors in the present study, further structural
modifications of the ribose moiety have been carried out.
In a previous study19 it was found that carbocyclic26 and

Ribose Deoxyadenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 831
Sch em e 2. Synthesis of N6-Methyl-(N)-
methanocarbaadenosine-3′,5′-bisphosphatea
Sch em e 4. Synthesis of a 9-Cyclobutyladenine
Bisphosphate Derivativea
a
Reagents: (a) (1) MeI, DMF, (2) NH4OH, 90 °C; (b) LDA/TBPP/
THF; (c) (1) H2/Pd-C, (2) NH4HCO3.
Sch em e 3. Synthesis of 2-Chloro-N6-methyl-(N)-
methanocarbaadenosine-3′,5′-bisphosphatea
a
Reagents: (a) KOt-BU, heat; (b) HCC-COOEt, CH2Cl2, 50 °C;
(c) DBU, DMF, rt; (d) LiAlH4, THF, 0 °C; (e) acetone, HCl, rt,
NaBH4, MeOH, 0 °C; (f) POCl3, Proton Sponge, (CH3O)3PO, 0 °C;
(g) NH4HCO3.
A deoxyanhydrohexitol adenine nucleoside was pre-
pared by the method of Verheggen et al.27 as the
precursor of the unsubstituted bisphosphate 7a , re-
ported previously.19 The corresponding N6-methyl ana-
logue 7c was synthesized as shown in Scheme 5, leading
also to compound 7b, a triphosphate derivative. A
2-chloro analogue, 7d was also prepared. Displacement
by 2-chloro-N6-methyladenine, 9b, of a tosyl derivative,
9a , of the protected sugar provided the nucleoside
starting material for the bisphosphate derivative 7d
(Scheme 6).
Morpholine rings were prepared by periodate oxida-
tive opening of the ribose ring of adenosine-5′-mono-
phosphate,30 followed by reductive amination with
sodium cyanoborohydride (Scheme 7). A phosphonate
group could be introduced directly in the reductive
amination step using aminoethylphosphonic acid.
Biologica l Activity. Adenine nucleotides markedly
stimulate inositol lipid hydrolysis by phospholipase C
in turkey erythrocyte membranes,36 through activation
of a P2Y1 receptor.37 The agonist used in screening these
analogues, 2-MeSADP, has a higher potency than the
corresponding triphosphate53 for stimulation of inositol
phosphate accumulation in membranes isolated from
[3H]inositol-labeled turkey erythrocytes.
As in our previous studies,17-19 the deoxyadenosine
bisphosphate nucleotide analogues prepared in the
present study were tested separately for agonist and
antagonist activity in the PLC assay at the P2Y1
receptor in turkey erythrocyte membranes, and the
results are reported in Table 2. Concentration-response
curves were obtained for each compound alone and in
combination with 30 nM 2-MeSADP. Concentration-
response curves for representative compounds are shown
in Figure 2.
a
Reagents: (a) DEAD, Ph3P, THF; (b) MeNH2; (c) BCl3; (d) (1)
LDA/TBPP, (2) BCl3.
tributyltin hydride and 2,2′-azobis[isobutyronitrile]
(AIBN) on the 3′,5′-protected nucleoside 13. Compound
13 was deprotected and phosphorylated using tetraben-
zyl pyrophosphate, leading to 3a .
The methanocarbocyclic 2′-deoxyadenosine analogues
in which the fused cyclopropane ring fixes the confor-
mation of the carbocyclic nucleoside into a rigid North-
ern (N) or Southern (S) envelope conformation, as
defined in the pseudorotational cycle, were synthesized
as precursors of nucleotides 4 and 5 by the general
approach of Marquez and co-workers.28,29,34,35 Again, the
N6-methyl group was introduced by the Dimroth re-
arrangement (Scheme 2). 2-Position adenine modifica-
tions were further introduced in the (N)-conformation
series as shown in Scheme 3. The 9-cyclobutyladenine
analogue 29 (Scheme 4) was synthesized by an adapta-
tion of the method of Schneller et al.31 and phosphor-
ylated by the phosphorus oxychloride method,19 leading
to compound 6.

832 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Nandanan et al.
Sch em e 5. Synthesis of an 9-Anhydrohexitol Adenine
Sch em e 7. Synthesis of Phosphonate Derivatives of
Bisphosphate Derivativea
Adeninylmorpholinea
a
Reagents: (a) NaIO4, H2O; (b) NH2(CH2)2PO3H2; (c) NABH3CN;
(d) NH4HCO3.
Ta ble 1. Synthetic Data for Nucleotide Derivatives, Including
Structural Verification Using HRMS and Purity Verification
Using HPLC
FAB (M - H+)
calcd found
HPLC (rt; min)a
sys A sys B yield (%)b
method,
no.
formula
2
C10H15O9N5P2
410.0267 410.0269 3.53
422.0631 422.0664 3.41
420.0474 420.0482 3.92
434.0631 434.0622 5.91
10.72
8.21
7.30
7.83
8.54
6.84
6.82
12.74
9.36
9.97
8.78
9.23
10.02
B, 21.7
B, 8.0
A, 5.5
B, 8.3
B, 2.3
A, 7.5
A, 24.3
A, 1.8
B, 50.1
B, 31.3
8.0
3b
4a
4b
4c
5
C
C
C
C
C
C
C
C
C
C
C
C
12H19O8N5P2
12H17O8N5P2
13H19O8N5P2
13H18O8N5P2Cl 468.0241 468.0239 8.05
12H17O8N5P2 420.0474 420.0481 4.02
11H16O8N5P2Cl 442.0084 442.0070 6.67
12H20O12N5P3 518.0237 518.0243 4.98
12H19O9N5P2
12H18O9N5P2Cl 472.0201 472.0190 5.67
12H20O8N6P2 437.0740 437.0721 2.37
6
7b
7c
7d
8a
8b
8c
438.0580 438.0580 4.63
12H21O11N6P3 517.0403 517.0404 2.42
12H22O14N6P4 597.0066 597.0053 2.96
7.2
4.0
a
Reagents: (a) (1) MeI, DMF, (2) NH4OH, 90 °C; (b) POCl3,
a
Purity of each derivative was g95%, as determined using
(MeO)3PO, Proton Sponge; (c) LDA/TBPP, THF; (d) (1) H2/Pd-C,
HPLC with two different mobile phases: system A, gradient of
0.1 M TEAA/CH3CN from 95/5 to 40/60, and system B, gradient
of 5 mM TBAP/CH3CN from 80/20 to 40/60. Phosphorylation
methods: method A refers to use of phosphorus oxychloride, and
method B refers to use of tetrabenzyl pyrophosphate/lithium
diisopropylamide followed by hydrogenation. The percent yields
refer to overall yield for each phosphorylation sequence. For the
method of synthesis of 8 refer to Experimental Section.
(2) NH4HCO3.
b
Sch em e 6. Synthesis of an 9-Anhydrohexitol
2-Chloro-N6-methyladenine Bisphosphate Nucleoside
Intermediatea
Compound 3b was equipotent to the ribose analogue 1a .
Curiously, the concurrent presence of a 2-amino group,
in 3c, entirely canceled the effect of the N6-methyl
group, resulting in a mixed agonist/antagonist.
Marquez and co-workers28,29 have introduced the
concept of ring-constrained carbocyclic nucleoside ana-
logues, based on cyclopentane rings constrained in the
(N)- and (S)-conformations by fusion with a cyclopropane
(methanocarba) ring. In the series of ring-constrained
(N)-methanocarba derivatives, the 6-NH2 analogue 4a
was a pure agonist of EC50 155 nM and 86-fold more
potent than the corresponding (S)-isomer 5, also an
agonist. Thus, the ribose ring (N)-conformation ap-
peared to be favored in recognition at P2Y1 receptors.
The N6-methyl- and 2-chloro-N6-methyl-(N)-methano-
carba analogues 4b and 4c were antagonists having IC50
values of 276 and 53 nM, respectively. A 2-chloro-N6-
methyl-9-cyclobutyl analogue 6 was a pure antagonist
of IC50 805 nM.
a
Reagents: (a) LiH, DMF; (b) 80% acetic acid, 60 °C.
For comparison purposes the previously reported N6-
methyl analogues 1a -d are shown in Table 2. All are
antagonists, with the potency of 2-substituted analogues
decreasing in the order Cl > SCH3 > NH2. The analogue
in which formalistically a phosphate group of the
previous bisphosphates has been shifted from the 5′- to
the 2′-position, 2, was a very weak antagonist at P2Y1
receptors.
Analogues lacking a glycosyl bond, 3, 4, 6, 7, and 9,
demonstrated that numerous non-ribose structures are
recognized at the P2Y1 receptor binding site. Addition
of the N6-methyl group in several cases converted pure
agonists to pure antagonists (cf. 4a and 4b; 7a and 7c).
Among simple carbocyclic adenosine analogues 3a -c,
the presence of the N6-methyl group in several cases
converted a partial agonist, e.g., 3a , to a pure antago-
nist, 3b , and at the same time increased potency.
Six-membered ring anhydrohexitol bisphosphate ana-
logues displayed micromolar potency at P2Y1 receptors,
as either agonists (7a , 6-NH2) or antagonists (7c, N6-
methyl). The triphosphate analogue 7b was principally
an antagonist of moderate potency, with an IC50 of 2.37
µM. The 2-chloro-N6-methyl analogue 7d was a more

Ribose Deoxyadenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 833
Ta ble 2. In Vitro Pharmacological Data for Stimulation of
PLC at Turkey Erythrocyte P2Y1 Receptors (agonist effect) and
Inhibition of PLC Stimulation Elicited by 30 nM 2-MeSADP
(antagonist effect), for at Least Three Separate Determinations
agonist
effect, % of
maximal
increasea
antagonist
effect, % of
maximal
inhibitionb
no.
EC50, µMa
IC50, µMb (n)
1a c,e
1bc,e
1ce
1d e
2
3a e
3b
3ce
4a
NE
NE
4
6 ( 2
NE
27 ( 11
NE
26 ( 3
92 ( 5
NE
NE
50 ( 4
NE
100
18 ( 5
NE
99 ( 1
95 ( 1
96 ( 2
94 ( 2
47 ( 2f
73 ( 11
100
0.331 ( 0.059 (5)
0.206 ( 0.053
1.85 ( 0.74
0.362 ( 0.119
small decrease (4)
2.53 ( 0.57
d
d
7.21 ( 4.40
6.51 ( 2.75
0.148 ( 0.069 (5)
5.42 ( 2.13
74 ( 3
0.155 ( 0.021 NE
F igu r e 2. Effects of deoxyadenosine bisphosphate derivatives
on P2Y1 receptor-activated phospholipase C activity in turkey
erythrocyte membranes: concentration-dependent stimulation
of inositol phosphate formation by 2-MeSADP (0), compound
4a (3), and compound 5 (O) and its inhibition in the presence
of 30 nM 2-MeSADP by compound 4c (2) and compound 5 (b).
Membranes from [3H]inositol-labeled erythrocytes were incu-
bated for 5 min at 30 °C in the presence of the indicated
concentrations of 2-MeSADP or of test compound, either alone
or in combination with 30 nM 2-MeSADP. The data shown
are typical curves for at least three experiments carried out
in duplicate using different membrane preparations.
4b
4c
5
100
100
42 ( 10%f
100
NE
0.157 ( 0.060
0.0516 ( 0.0008
∼40
18.9 ( 5.7
6
0.805 ( 0.349 (5)
7a e
7b
7c
2.99 ( 0.35
9.35 ( 3.43
82 ( 5
100
2.37 ( 0.54
1.64 ( 0.43
7d
8a
8b
8c
NE
NE
11 ( 1
NE
100
0.566 ( 0.224
small decrease
37 ( 6f
d
37 ( 6f
NE
41 ( 12%f
small decrease
a
Agonist potencies were calculated using a four-parameter
logistic equation and the GraphPad softaware 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
respectively, were docked into the putative binding site
of our previously reported P2Y1 receptor model. Accord-
ing to their structural similarity, the cross-docking
procedure demonstrated that the receptor architecture
found for binding of ATP and 1a was energetically
appropriate also for the binding of both 4a and 5.
However, the (N)-methanocarba/P2Y1 complex appeared
more stable than the (S)-methanocarba/P2Y1 complex
by approximately 20 kcal/mol (does not include entropic
and solvation effects). Figure 3A represents the lowest
energy docked complex of (N)-methanocarba agonist in
the proposed ligand binding cavity. In this model, the
side chain of Gln307(TM7) is within hydrogen-bonding
distance of the N6 atom at 1.8 Å and the side chain of
Ser314(TM7) is positioned at 2.0 Å from the N1 atom
and at 3.4 Å from the N6 of the purine ring. As already
reported, another three amino acids are important for
the coordination of the phosphate groups in this an-
tagonist: Arg128(TM3), Lys280(TM6), and Arg310-
(TM7). As shown in Figure 3A Lys280 may interact
directly with both 3′- and 5′-phosphates (1.7 Å, O3′; and
1.7 Å, O5′), whereas Arg128(TM3) and Arg310(TM7) are
within ionic coupling range to both the O2 and O3 atoms
of the 5′-phosphate. As shown in Figure 3B, a poor
superimposition (rms ) 1.447) between the (N)- and (S)-
methanocarba agonist analogues has been found inside
the receptor binding domain. In particular, the adenine
moiety and 5′-phosphate of the (S)-methanocarba de-
rivative are shifted out of position with respect to those
presented by the (N)-methanocarba isomer, decreasing
the stability of the (S)-methanocarba/P2Y1 complex. This
fact might be correlated with the difference of their
biological activity (see Table 3).
b
in the same experiment. Antagonist IC50 values (mean ( stan-
dard 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 concentration. c 1a , MRS 2179; 1b, MRS
2216; 3b, MRS 2267; 4a , MRS 2268; 4c, MRS 2279; 5, MRS 2266;
d
6, MRS 2264; 7a , MRS 2255; 7c, MRS 2269; 7d , MRS 2283. EC50
was not calculated for increases of e15% at 100 µM. e Values from
refs 17, 19. NE, no effect at 100 µM.
potent antagonist activity, with an IC50 of 0.57 µM.
Another set of six-membered rings, morpholino (mono-,
di-, or triphosphate) analogues 8a -c containing an
aminophosphonic acid, was nearly inactive at P2Y1
receptors.
Molecu la r Mod elin g. To better understand the role
of the sugar puckering on the human P2Y1 agonist and
antagonist activities, we carried out a molecular model-
ing study of this new generation of ribose-modified
ligands. Such modifications include cyclopentyl rings
constrained in the (N)- and (S)-conformations with
cyclopropyl (methanocarba) groups, six-membered rings
(morpholino and anhydrohexitol analogues), and cyclo-
butyl nucleotides. We have recently developed a model
of the human P2Y1 receptor, using rhodopsin as a
template, by adapting a facile method to simulate the
reorganization of the native receptor structure induced
by the ligand coordination (cross-docking procedure).38
Details of the model building are given in the Experi-
mental Section. We have also reported the hypothetical
molecular basis for recognition by human P2Y1 receptors
of the natural ligand ATP and the new potent, competi-
tive antagonist 2′-deoxy-N6-methyladenosine-3′,5′-bis-
phosphate (1a ). Both ATP and 1a are present in the
hypothetical binding site with a (N)-sugar ring confor-
mation. In the present work, the sterically constrained
(N)- and (S)-methanocarba agonist analogues 4a and 5,
Using the information that a common binding site
could be hypothesized among these deoxyadenosine
bisphosphate analogues, a superimposition analysis of
the energy minimization of the more potent antagonists
has been performed. In this analysis we have used 1a
as a reference compound, and we have defined three

834 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Nandanan et al.
F igu r e 4. (A) Superposition of docked P2Y1 antagonist
structures, all of which contain an N6-Me group. The trans-
membrane helical bundle is not highlighted, but it conserves
the same arrangement shown in Figure 3A: red ) 1a ; magenta
) 4b; yellow ) 6; blue ) 7c. (B) Alignment of 4b (magenta)
generated by “Fit Atoms” analysis using 1a (red) as reference
structure.
F igu r e 3. (A) Side view of the 4a /P2Y1 receptor complex
model. The side chains of the important residues in proximity
to the docked 4a molecule are highlighted and labeled.
Residues in proximity (e5 Å) to the docked 4a molecule:
Arg128(TM3), Tyr136(TM3), His277(TM6), Lys280(TM6),
Gln307(TM7), Arg310(TM7), and Ser314(TM7). (B) Alignments
generated by “Fit Atoms” analysis between 4a (green) and 5
(red).
phosphate groups, to carry out the superimposition
analysis. As reported in Table 3, acceptable rms values
have been obtained for all the antagonists compared
with the 1a structure. As shown in Figure 4A, this
superimposition study suggested that the two phosphate
groups may occupy common receptor regions, and a
general pharmacophore model for bisphosphate antago-
nists binding to the human P2Y1 receptor can be
extrapolated (see Figure 4B). The model defines ap-
proximate distances between phosphate groups (3.8 Å)
and between 5′- and 3′-phosphates and the exocyclic
amine (7.6 and 9.0 Å, respectively).
Ta ble 3. Superimposition rms Values of Bisphosphate Agonist
and Antagonist Derivatives (agonist reference structure: 4a ;
antagonist reference structure: 1a )
no.
rms, Å
EC50, µMa
Agonists
0.152
13.3
IC50, µMb
4a
5
1.447
Discu ssion
Antagonists
1a
4b
6
7c
c
0.331
0.157
0.805
1.64
In conclusion, the present study has identified new
pharmacological probes of P2Y1 receptors, including full
agonists, partial agonists, and antagonists. Such probes
may be useful, for example, in characterizing anti-
thrombotic effects in platelets, in which at least three
P2 receptors coexist.9-11 The selectivity of the present
compounds for P2Y1 receptors is being explored. While
compound 1a was moderately potent at P2X1 receptors,
antagonists 1b, 3b, and 7c did not block P2X1 or P2X3
receptors at 10 µM.25
0.359
0.777
0.799
0.829
1.60
See footnote a of Table 2. See footnote b of Table 2. c 2-[2-
a
b
(6-Methylaminopurin-9-yl)ethyl]propane-1,3-bisoxy(diammoni-
um phosphate).41
matching pairs of atoms, corresponding to the N6 atom
of the purine ring and the P atoms of both 3′- and 5′-

Ribose Deoxyadenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 835
The SAR of 1a indicates that the ribose ring oxygen
may be readily substituted with carbon, as in 3.
Furthermore, analogues of constrained conformation,
e.g., the (N)-methanocarba analogues 4, displayed en-
hanced receptor affinity. Additional 2-chloro and N6-
methyl substitution is favorable for affinity at P2Y1
receptors, and nearly pure antagonism is maintained
provided that the N6-methyl group is present. Thus, in
this respect, the carbocyclic analogues behaved similar
to the ribose series of P2Y1 receptor antagonists,19 in
which 2-substitution tended to increase the potency.
Similarly, in the agonist series of 5′-AMP and 5′-ATP
derivatives,39,45 2-thioether substitution increased po-
tency at P2Y1 receptors. The 2-amino analogue 3c was
a mixed agonist/antagonist.
Interestingly, we have already reported that these
electrostatic contacts are also crucial for the recognition
of bisphosphate antagonists. Using superimposition
analysis, a general pharmacophore model for the bis-
phosphate antagonists binding to the P2Y1 receptor has
been proposed (see Figure 4B). According to the phar-
macophore map, recognition of all bisphosphate antago-
nists at a common region inside the receptor binding
site and, consequently, a common electrostatic potential
profile is possible. As well as for the agonists, the (N)-
conformation seems to be essential to maximize the
electrostatic interactions between the negatively charged
phosphates and the positively charged amino acids
present in the receptor binding cleft. As we predicted
using the previously reported P2Y1 receptor model,38 the
sugar moiety does not seem to be crucial for the ligand
recognition process. This is indicated by the affinity of
carbocyclic antagonists 4b and 6 (Figure 4A) and acyclic
nucleotides which are P2Y1 antagonists, such as 2-[2-
(6-methylaminopurin-9-yl)ethyl]propane-1,3-bisoxy(di-
ammonium phosphate).41 In our pharmacophore model,
the sugar moiety plays the role of appropriate spacer
between the N6 position of the purine system and the
two phosphate groups (3′ and 5′, respectively). Conse-
quently, the sugar ring can be replaced without drasti-
cally losing biological activities.
Among anhydrohexitol adenine bisphosphate deriva-
tives, 7a was a pure agonist of potency similar to ATP,
while the N6-methyl analogues 7c and 7d were pure
antagonists. The triphosphate 7b displayed both agonist
and antagonist properties. Thus, the biological potency
and efficacy of this series of bisphosphates appeared to
be highly dependent on subtle conformational factors,
which would influence the orientation of the phosphate
groups within the receptor binding site.
The sugar moiety of nucleosides and nucleotides in
solution is known to exist in a rapid, dynamic equilib-
rium between extreme (N)-(2′-exo/3′-endo) and (S)-(2′-
endo/3′-exo) conformations29,40 as defined in the pseudo-
rotational cycle. While the energy gap between (N)- and
(S)-conformations is in the neighborhood of 4 kcal/mol,
such a disparity can explain the difference between
micromolar and nanomolar binding affinities. Using a
molecular modeling approach, we have analyzed the
sugar conformational requirements for a new class of
bisphosphate ligands binding to the human P2Y1 recep-
tor. As experimentally shown, the ribose ring (N)-
conformation appeared to be favored in recognition at
the human P2Y1 receptor (see Table 3). We have found
new support to our recently presented hypothesis in
which three important recognition regions are present
in the bisphosphate molecular structures: the N1 atom
of the purine ring and the P atoms of both 3′- and 5′-
phosphate groups. The (N)-conformation seems to be
essential to maximize the electrostatic interactions
between the negatively charged phosphates and the
positively charged amino acids present in the receptor
binding cleft, as well as Arg128(TM3), Lys280(TM6),
and Arg310(TM7), as shown in Figure 3A.
As described above, the simple addition of the N6-
methyl group in several cases converted pure agonists
to antagonists. From a pharmacological point of view,
this is really a unique situation. Generally, how agonist
binding transforms a resting GPCR into its active form
and the microscopic basis of binding site blockade by
an antagonist are still unclear. Also in this specific case,
there are not enough data to appropriately answer the
question. We know that Gln307(TM7) and Ser314(TM7)
are positioned, in our model, in the vicinity of the N6-
amine of the adenine moiety. We speculate that these
two amino acids may be involved in the recognition of
the nucleotide base in the agonist structure. The
important role of HN6 of the adenine moiety, putatively
through Gln307 and Ser314, as a hydrogen-bond accep-
tor has been demonstrated using a doubly alkylated N6-
derivative of ATP, for which no agonist activity was
observed.17,42 Moreover, a markedly reduced response
of 2-MeSADP was observed for the Q307A and S314A
mutant receptors compared with the wild-type recep-
tor.38 Rotations and translations of the TM domains are
crucial factors in the ligand recognition and activation
process in different GPCRs, as recently described from
Moro et al.38 We hypothesize that the N6-amine of the
adenine moiety could simultaneously make a double
hydrogen-bonding interaction with both Gln307(TM7)
and Ser314(TM7), and this double hydrogen-bonding
interaction could be the trigger of the activation process.
With the addition of the N6-methyl group it is not
possible to have a double hydrogen-bonding interaction,
and consequently, the activation pathway is blocked.
However, for all the N6-methyl antagonists the possibil-
ity to participate in at least one of the two possible
hydrogen bonds appears to be very important for the
increase in affinity at the P2Y1 receptor.
Our hypothesis is that the conformationally rigid
pseudosugar of 4 serves as a scaffold to position the
phosphate groups in the correct orientation relative to
the adenine ring for optimal interaction. Other differ-
ences between 4 and 5 that may in principle contribute
to the (N)-methanocarba effect on receptor affinity
include the barrier to pseudoglycosyl bond rotation ((N)-
conformation favors an anti-conformation at the glyco-
sidic bond54) and the steric and hydrophobic properties
of the cyclopropane methylene group. In this regard, it
is to be noted that the methylene carbon does not
protrude outside the region of the five-membered ring
into the binding regions of the base or hydroxyl groups
due to the constraints of the pseudoboat shape of the
bicyclo[3.1.0]hexane (Figure 1).
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

836 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Nandanan et al.
Aldrich (St. Louis, MO). 6-Chloro-2′-deoxypurine riboside was
obtained from Sigma. Compound 1a was synthesized in our
laboratory as described.17 Several 2′-deoxy nucleosides, includ-
ing an anhydrohexitol adenine nucleoside25 and 2′-deoxyaris-
teromycin,19 were synthesized as reported.
1H NMR spectra were obtained with a Varian Gemini-300
spectrometer using D2O as a solvent. 31P NMR spectra were
recorded at room temperature by use of a Varian XL-300
spectrometer (121.42 MHz); orthophosphoric acid (85%) was
used as an external standard.
5′ -Deoxya d en osin e-2′,3′-bis(d ia m m on iu m p h osp h a te)
(2). 24.3 mg (0.0967 mmol) of 5′-deoxyadenosine reacted with
tetrabenzyl pyrophosphate following the general procedure B
to give 26.9 mg (0.0348 mmol, 36.0% yield) of the desired
compound, 5′-deoxyadenosine-2′,3′-bis(dibenzyl phosphate): 1H
NMR (CD3OD) δ 1.35 (3H, d, J ) 6.8 Hz, 4′-CH3), 4.22 (1H,
m, H-4′), 4.98 (1H, m, H-2′), 5.10 (2H, d. J ) 9.8 Hz, CH2),
5.75 (1H, m, H-3′), 6.10 (1H, d, J ) 4.9 Hz, H-1′), 7.24 (5H, m,
C6H5), 8.12 (1H, bs, H-2, H-8); 31P NMR (CD3OD) δ -1.15 (bs).
For final deprotection of the phosphate groups, 20.0 mg
(0.0259 mmol) of the tetrabenzyl intermediate was converted
to the corresponding phosphoric acid analogue by catalytic
hydrogenation as described in the general procedure B to give
7.5 mg (0.0156 mmol, 60.4% yield) of the desired compound:
1H NMR (D2O) δ 1.41 (3H, d, J ) 6.8 Hz, 4′-CH3), 4.44 (1H,
m, H-4′), 4.62 (1H, m, H-2′), 5.17 (1H, m, H-3′), 6.20 (1H, d, J
) 4.9 Hz, H-1′), 8.25 (1H, s, H-2), 8.42 (1H, s, H-8); 31P NMR
(D2O) δ 2.78, 3.06 (2s, 2′-P, 3′-P).
Ca r bocyclic N6-Meth yl-2′-d eoxya d en osin e-3′,5′-bis(d i-
a m m on iu m p h osp h a t e) (3b) [N6-Met h yl-2′-d eoxya r is-
ter om ycin -3′,5′-bis(d ia m m on iu m p h osp h a te)]. 17.9 mg
(0.0228 mmol) of compound 16 was converted to the corre-
sponding phosphoric acid analogue using hydrogenation fol-
lowing the general procedure B. Purification was performed
on an ion-exchange column packed with Sephadex-DEAE A-25
resin, linear gradient (0.01 to 0.5 M) of 0.5 M ammonium
bicarbonate was applied as the eluent to give 3.0 mg (0.0061
mmol, 26.8% yield) of the desired compound:1H NMR (D2O) δ
1.85-2.61 (1H, m, H-4′, 2H, m, CH2-2′, 2H, m, CH2-4′), 3.07
(3H, s, N6-CH3), 3.94 (2H, m, CH2-5′), 4.67 (1H, m, H-3′), 5.06-
5.11 (1H, m, H-1′), 8.22 (1H, s, H-2), 8.27 (1H, s, H-8); 31P NMR
(D2O) δ 1.20, 2.34 (2s, 3′-P, 5′-P).
(N)-Met h a n oca r b a -2′-d eoxya d en osin e-3′,5′-b is(d ia m -
m on iu m p h osp h a te) (4a ) [(1R,2S,4S,5S)-1-[(P h osp h a to)-
m eth yl]-4-(6-am in opu r in -9-yl)bicyclo[3.1.0]h exan e-2-ph os-
p h a t e Tet r a a m m on iu m Sa lt ]. Starting from 16 mg (0.06
mmol) of (N)-methanocarba-2′-deoxyadenosine and following
the general phosphorylation procedure A we obtained 1.8 mg
(0.0037 mmol, 5.5% yield) of the desired compound: 1H NMR
(D2O) δ 0.90 (1H, m, CH2-6′), 1.10 (1H, m, CH2-6′), 1.82 (1H,
m, CH-5′), 1.91 (1H, m, CH2-3′) 2.23 (1H, m, CH2-3′), 3.49 (1H,
d, J ) 11.7 Hz, CH2O), 4.16 (1H, d, J ) 11.7 Hz, CH2O), 4.90-
4.97 (1H, m, CH-4′), 5.12 (1H, d, J ) 6.9 Hz, CH-2′), 8.39 (1H,
s, H-2), 8.54 (1H, s, H-8); 31P NMR (D2O) δ 0.43 (s, 5′-P), -0.19
(s, 3′-P).
(N)-Meth a n oca r ba -N6-m eth yl-2′-d eoxya d en osin e-3′,5′-
bis(diam m on iu m ph osph ate) (4b) [(1R,2S,4S,5S)-1-[(P h os-
ph ato)m eth yl]-4-(6-m eth ylam in opu r in -9-yl)bicyclo[3.1.0]-
h exa n e-2-p h osp h a te Tetr a a m m on iu m Sa lt]. Compound 18
(13.5 mg, 0.0170 mmol) was converted to the corresponding
phosphoric acid analogue using hydrogenation following the
general procedure B. Purification was performed on an ion-
exchange column packed with Sephadex-DEAE A-25 resin,
eluting with a linear gradient of ammonium bicarbonate (0.01
M to 0.5 M) to give 3.0 mg (0.0060 mmol, 35.3% yield) of the
desired compound: 1H NMR (D2O) δ 0.93-0.98 (1H, m, CH2-
6′), 1.17 (1H, m, CH2-6′), 1.86-1.88 (1H, m, CH-5′), 1.94-1.98
(1H, m, CH2-3′), 2.23-2.31 (1H, m, CH2-3′), 3.09 (3H, bs, N6-
CH3), 3.61-3.64 (1H, m, CH2O), 4.51-4.55 (1H, m, CH2O),
5.01-5.03 (1H, m, CH-4′), 5.19-5.21 (1H, m, CH-2′), 8.22 (1H,
s, H-2), 8.51 (1H, s, H-8); 31P NMR (D2O) δ 1.26, 1.92 (2s, 3′-P,
5′-P).
(N)-Meth a n oca r ba -N6-m eth yl-2-ch lor o-2′-d eoxya d en o-
sin e-3′,5′-b is(d ia m m on iu m p h osp h a t e) (4c) [(1R,2S,4S,
5S)-1-[(P h osp h a t o)m et h yl]-4-(2-ch lor o-6-a m in op u r in -9-
yl)b icyclo[3.1.0]h exa n e-2-p h osp h a t e Tet r a a m m on iu m
Sa lt]. The nucleoside, compound 23, reacted with tetrabenzyl
pyrophosphate, as in method B, followed by an alternative
deprotection procedure. Starting from 10 mg (0.0323 mmol)
of (N)-methanocarba-N6-methyl-2-chloro-2′-deoxyadenosine and
following the general phosphorylation procedure (method B)
we obtained 9.5 mg (0.0114 mmol, 35% yield) of the desired
compound, (N)-methanocarba-N6-methyl-2-chloro-2′-deoxy-
adenosine-3′,5′-bis(dibenzyl phosphate): 1H NMR (CDCl3) δ
Purity of compounds was checked using a Hewlett-Packard
1090 HPLC apparatus equipped with an SMT OD-5-60 RP-
C18 analytical column (250 × 4.6 mm; Separation Methods
Technologies, Inc., Newark, DE) in two solvent systems.
System A: linear gradient solvent system: 0.1 M TEAA/
CH3CN from 95/5 to 40/60 in 20 min and the flow rate was 1
mL/min. System B: linear gradient solvent system: 5 mM
TBAP/CH3CN from 80/20 to 40/60 in 20 min and the flow rate
was 1 mL/min. Peaks were detected by UV absorption using
a diode array detector. All derivatives showed more than 95%
purity in the HPLC systems.
Low-resolution CI-NH3 (chemical ionization) mass spectra
were carried out with Finnigan 4600 mass spectrometer and
high-resolution EI (electron impact) mass spectrometry with
a VG7070F mass spectrometry at 6 kV. High-resolution FAB
(fast atom bombardment) mass spectrometry was performed
with
a J EOL SX102 spectrometer using 6-kV Xe atoms
following desorption from a glycerol matrix.
Purification of most of the nucleotide analogues for biological
testing was carried out on DEAE-A25 Sephadex columns as
described above. However, compounds 7b and 8a -c required
HPLC purification (system A, semipreparative C18 column)
of the reaction mixtures.
Gen er a l P r oced u r e of P h osp h or yla tion . Meth od A:
The nucleoside (0.1 mmol) and Proton Sponge (107 mg, 0.5
mmol) were dried for several hours in high vacuum at room
temperature and then suspended in 2 mL of trimethyl phos-
phate. Phosphorus oxychloride (Aldrich; 37 µL, 0.4 mmol) was
added, and the mixture was stirred for 1 h at 0 °C. The reaction
was monitored by analytical HPLC (eluting with a gradient
consisting of buffer:CH3CN in the ratio 95:5 to 40:60, in which
the buffer was 0.1 M triethylammonium acetate (TEAA);
elution time was 20 min; flow rate was 1 mL/min; column was
SMT OD-5-60 RP-C18; detector was by UV in the Emax range
of 260-300 nm). The reaction was quenched upon addition of
2 mL of triethylammonium bicarbonate buffer and 3 mL of
water. The mixture was subsequently frozen and lyophilized.
Purification was performed on an ion-exchange column packed
with Sephadex-DEAE A-25 resin, a linear gradient (0.01 to
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. All nucleotide bisphosphates were collected, frozen and
lyophilized as the ammonium salts. All synthesized compounds
gave correct molecular mass (high-resolution FAB) and showed
more than 95% purity (HPLC, retention times are reported in
Table 1).
Meth od B: Nucleoside (0.1 mmol) dried for several hours
in high vacuum at room temperature was dissolved in 2 mL
of dry THF. Lithium diisopropylamide solution (Aldrich; 2.0
M in THF, 0.4 mmol) was added slowly at -78 °C. After 15
min, tetrabenzyl pyrophosphate (Aldrich; 0.4 mmol) was added
and the mixture was stirred for 30-60 min at -78 °C. The
reaction mixture was warmed to 0 °C to room temperature
and stirred for an additional period ranging from 2 to 24 h.
Chromatographic purification (pTLC, CHCl3:CH3OH, 10:1)
gave the tetrabenzyl phosphorylated compound. This com-
pound (20 mg) was dissolved in a mixture of methanol (2 mL)
and water (1 mL) and hydrogenated over a 10% Pd-on-C
catalyst (10 mg) at room temperature for 62 h. The catalyst
was removed by filtration and the methanol was evaporated.
The residue was treated with ammonium bicarbonate solution
and subsequently frozen and lyophilized. Purification, if neces-
sary, was by the same procedure as in method A.

Ribose Deoxyadenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 837
0.75-0.81 (1H, m, CH2-6′), 1.03-1.08 (1H, m, CH2-6′), 1.49-
1.51 (1H, m, CH-5′), 1.84-1.94 (1H, m, CH2-3′), 1.99-2.10 (1H,
m, CH2-3′), 3.12 (3H, bs, N6-CH3), 4.11-4.20 (1H, m, CH2O),
4.50-4.55 (1H, m, CH2O), 4.90-4.98 (8H, m, -OCH2), 4.99-
5.01 (1H, m, CH-4′), 5.23-5.30 (1H, m, CH-2′), 5.90 (1H, bs,
NH), 7.20-7.29 (20H, m, C6H5), 7.82 (1H, s, H-8); 31P NMR
(D2O) δ -0.58 (s, 5′-P); -1.06 (s, 3′-P); MS (CI-NH3) (M + 1)
830; HRMS (FAB-) (M + Cs) calcd 962.1252, found 962.1252.
The tetrabenzyl-protected intermediate (9.5 mg, 0.0114
mmol) was added to dry CH2Cl2 (1.0 mL) and cooled to -78
°C under argon. The mixture was treated with 100 µL of boron
trichloride solution (1 M in CH2Cl2) and 100 µL of anisole.43
The reaction mixture was stirred for 24 h at 0 °C, allowed to
warm to room temperature, and quenched with 1 M triethyl-
ammonium bicarbonate solution (Sigma). The CH2Cl2 was
removed in vacuo, and the aqueous residue was lyophilized.
Purification was performed on an ion-exchange column packed
with Sephadex-DEAE A-25 resin, eluting with a linear gradi-
ent of 0.01 to 0.5 M ammonium bicarbonate to give 0.4 mg
(0.0007 mmol, 7% yield) of the desired compound, 4c: 1H NMR
(D2O) δ 0.91-0.96 (1H, m, CH2-6′), 1.12-1.16 (1H, m, CH2-
6′), 1.80-1.84 (1H, m, CH-5′), 1.85-1.98 (1H, m, CH2-3′), 2.20-
2.50 (1H, m, CH2-3′), 3.08 (3H, bs, N6-CH3), 3.57-3.60 (1H,
m, CH2OH), 4.52-4.67 (1H, m, CH2OH), 4.94-4.96 (1H, m,
CH-4′), 5.18-5.21 (1H, m, CH-2′), 8.52 (1H, s, H-8); 31P NMR
(D2O) δ 1.82, 2.52 (2s, 3′-P, 5′P).
(S)-Met h a n oca r b a -2′-d eoxya d en osin e-3′,5′-b is(d ia m -
m on iu m p h osp h a te) (5) [(1S,3S,4R,5S)-4-[(P h osp h a to)-
m eth yl]-1-(6-am in opu r in -9-yl)bicyclo[3.1.0]h exan e-3-ph os-
p h a t e Tet r a a m m on iu m Sa lt ]. Starting from 16 mg (0.06
mmol) of (S)-methanocarba-2′-deoxyadenosine and following
the general phosphorylation procedure A, we obtained 2.1 mg
(0.0043 mmol, 7.5% yield) of the desired compound 5: 1H NMR
(D2O) δ 1.36 (1H, m, CH2-6′), 1.53 (1H, t, J ) 4.8 Hz, CH2-6′),
2.05 (1H, m, CH2-5′), 2.30 (1H, m, CH-4′), 2.46 (2H, m, CH2-
2′), 3.97 (2H, m, CH2OH), 4.45 (1H, d, J ) 6.6 Hz, CH-3′), 8.16
(1H, s, H-2), 8.30 (1H, s, H-8); 31P NMR (D2O) δ 0.85 (bs, 5′-
P), 0.31 (bs, 3′-P).
2′-(2-Ch lor o-6-m eth ylam in opu r in -9-yl)cyclobu tan e-1′,5′-
bis(d ia m m on iu m p h osp h a te) (6). Starting from 15 mg
(0.052 mmol) of 2′-(2-chloro-6-methylaminopurin-9-yl)cyclo-
butane, 29, and following the general phosphorylation proce-
dure A, we obtained 5.6 mg (0.0126 mmol, 24.3% yield) of the
desired compound: 1H NMR (D2O) δ 2.55 (1H, m, H-2′), 3.07
(3H, s, NHCH3), 3.33 (2H, m, CH2-4′), 4.10 (2H, t, J ) 5.1 Hz,
CH2-5′), 4.48 (2H, m, H-1′ and H-3′), 8.29 (1H, s, H-8); 31P NMR
(D2O) δ 0.99 (s, 4′-P), -0.53 (d, J ) 2.4 Hz, 2′-P).
from 15.0 mg (0.048 mmol) of 9d and following the general
phosphorylation procedure of method B, we obtained 30.0 mg
(0.036 mmol, 75.0% yield) of the desired compound, 1,5-
anhydro-2-(2-chloro-N6-methyladenin-9-yl)-2,3-dideoxy-D-ara-
bino-hexitol-4,6-(dibenzyl phosphate): 1H NMR (CDCl3) δ
1.99-2.09 (1H, m, H-3′), 2.60-2.64 (1H, m, H-3′), 3.20 (3H, s,
N6-CH3), 3.55-3.60 (1H, m, H-5′), 3.83 (1H, dd, J ) 2.0,12.7,
H-1′), 4.07-4.13 (1H, m, H-4′), 4.18 (1H, d, J ) 12.7, H-1′),
4.32-4.38 (2H, m, CH2-6′), 4.98 (8H, 4d, J ) 7.9 Hz, O-CH2),
6.11 (1H, bs, H-2′), 7.03 (1H, bs, NH), 7.30 (20H, m, C6H5),
8.08 (1H, s, H-8); 31P NMR (CD3Cl) -0.76, -1.77 (2s, 4′-P, 6′-
P).
30.0 mg (0.036 mmol) of the tetrabenzyl-protected interme-
diate added to dry CH2Cl2 (1.0 mL) was cooled to -78 °C under
argon and treated with 400 µL of boron trichloride solution (1
M in CH2Cl2) and 400 µL of anisole. The reaction mixture was
stirred for 12 h at 0 °C to room temperature and added with
triethylammonium bicarbonate (1.0 mL) in ice bath. After the
removal of CH2Cl2 under nitrogen stream, the reaction mixture
was lyophilized. Purification was performed on Sephadex ion-
exchange column chromatography described in general pro-
cedure A to afford 8.0 mg (0.015 mmol, 41.7% yield) of the
desired compound, 1,5-anhydro-2-(2-chloro-N6-methyladenin-
9-yl)-2,3-dideoxy-D-arabino-hexitol-4,6-bis(diammonium phos-
phate): 1H NMR (D2O) δ 2.13-2.23 (1H, m, H-3′), 2.65-2.69
(1H, m, H-3′), 3.06 (3H, s, N6-CH3), 3.68-3.72 (1H, m, H-5′),
3.98-4.06 (1H, m, H-1′) 4.09-4.11 (2H, m, CH2-6′), 4.17-4.20
(1H, m, H-4′), 4.30 (1H, d, J ) 12.7, H-1′), 4.82 (1H, m, H-2′),
8.40 (1H, s, H-8); 31P NMR (D2O) δ 3.61, 2.07 (2s, 4′-P, 6′-P);
HRMS (FAB-) calcd 472.0201, found 472.0190; HPLC 5.67
min (sys A).
P h osp h or ic Acid Mon o[(2S)-6-(6-a m in op u r in -9-yl)-4-
(2′-p h osp h on oeth yl)m or p h olin -2-ylm eth yl] Ester (8a ).
Adenosine 5′-monophosphate sodium salt (240.0 mg, 0.481
mmol), sodium periodate (102.8 mg, 0.481 mmol) and 2-amino-
ethylphosphonic acid (72.2 mg, 0.577 mmol) were dissolved in
2.0 mL of water. The reaction mixture was stirred at room
temperature for 1.5 h. Sodium cyanoborohydride (71.1 mg,
0.962 mmol) was added, and the reaction mixture was stirred
for an additional 30 min and passed through Amberlite CG50
(H+ form) with an elution of water. The acidic fractions were
collected, neutralized with triethylamine, purified by Sephadex
ion-exchange column chromatography and semipreparative
HPLC described in general procedure A to afford 19.5 mg
(0.038 mmol, 8.0% yield) of the desired compound: 1H NMR
(D2O) δ 2.03-2.11 (2H, m, CH2), 3.16 (1H, t, J ) 11.7 Hz,
5-CH2), 3.82 (1H, d, J ) 11.7 Hz, 5-CH2), 3.39-3.43 (2H, m,
CH2), 3.62-3.70 (2H, m, 3-CH2), 4.08-4.10 (2H, m, 2-CH2),
4.42-4.45 (1H, m, H-2), 6.18-6.22 (1H, d, J ) 10.7 Hz, H-6),
8.25 (1H, s, H-2), 8.33 (1H, s, H-8); 31P NMR (D2O) δ 19.93,
0.84 (2s, 2-P, 4-P).
P h osp h or ic Acid Di[(2S)-6-(6-a m in op u r in -9-yl)-4-(2′-
p h osp h on oeth yl)m or p h olin -2-ylm eth yl] Ester (8b). Ad-
enosine 5′-diphosphate monopotassium salt (140.2 mg, 0.280
mmol), sodium periodate (59.8 mg, 0.280 mmol) and 2-amino-
ethylphosphonic acid (42.0 mg, 0.336 mmol) were dissolved in
2.0 mL of water. The reaction mixture was stirred at room
temperature for 1.5h. Sodium cyanoborohydride (41.3 mg,
0.559 mmol) was added, and the reaction mixture was stirred
for an additional 30 min and passed through Amberlite CG50
(H+ form) with an elution of water. The acidic fractions were
collected, neutralized with triethylamine, purified by Sephadex
ion-exchange column chromatography and semipreparative
HPLC described in general procedure A to afford 12.2 mg
(0.0202 mmol, 7.2% yield) of the desired compound:1H NMR
(D2O) δ 1.94-2.06 (2H, m, CH2), 2.90 (1H, t, J ) 11.7 Hz,
5-CH2), 3.59-3.62 (1H, d, J ) 11.7 Hz, 5-CH2), 3.17-3.25 (2H,
m, CH2), 3.35-3.52 (2H, m, 3-CH2), 4.12-4.18 (2H, m, 2-CH2),
4.41-4.45 (1H, m, H-2), 6.13 (1H, dd, J ) 10.7, 2.9 Hz, H-6),
8.19 (1H, s, H-2), 8.26 (1H, s, H-8); 31P NMR (D2O) δ 19.54 (t,
J ) 7.3 Hz, 4-P), -10.05,-10.90 (2d, J ) 20.6 Hz, 2-POP).
N6-Met h yl-1,5-a n h yd r o-2-(a d en in -9-yl)-2,3-d id eoxy-D-
a r a bin o-h exit ol-6-t r ip h osp h a t e Tet r a a m m on iu m Sa lt
(7b). Starting from 26.6 mg (0.0952 mmol) of 31, and following
the general phosphorylation procedure A. Purification was
performed by semipreparative HPLC using system A, and we
obtained 1.0 mg (0.0017 mmol, 1.8% yield) of the desired
compound: 1H NMR (D2O) δ 2.09 (1H, m, H-3′), 2.51 (1H, m,
H-3′), 3.09 (3H, s, N6-CH3), 3.63 (1H, m, H-5′), 3.88 (1H, m,
H-4′), 4.10-4.30 (1H, m, H-1′), 4.27 (2H, m, H-6′), 8.25 (1H, s,
H-2), 8.43 (1H, s, H-8); 31P NMR (D2O) δ -7.96 (m), -10.39
(m), -22.22 (m).
N6-Met h yl-1,5-a n h yd r o-2-(a d en in -9-yl)-2,3-d id eoxy-D-
a r a bin o-h exit ol-4,6-b is(d ia m m on iu m p h osp h a t e) (7c).
Compound 32 (25.0 mg, 0.0312 mmol) was converted to the
corresponding phosphoric acid analogue using hydrogenation
following the general procedure B to give 14.2 mg (0.0280
mmol, 89.7% yield) of the desired compound: 1H NMR (D2O)
δ 2.12-2.22 (1H, m, H-3′), 2.64-2.68 (1H, m, H-3′), 3.04 (3H,
s, N6-CH3), 3.66-3.75 (1H, m, H-5′), 3.88-3.96 (1H, m, H-4′),
4.08-4.09 (2H, m, CH2-6′), 3.96-4.30 (2H, m, H-1′), 4.86 (1H,
bs, H-2′), 8.17 (1H, s, H-2), 8.39 (1H, s, H-8); 31P NMR (D2O)
δ 3.97, 2.55 (2s, 3′-P, 5′-P).
1,5-An h yd r o-2-(2-ch lor o-N 6-m e t h yla d e n in -9-yl)-2,3-
d id eoxy-D-a r a bin o-h exit ol-4,6-b is(d ia m m on iu m p h os-
p h a te) (7d ). The 2-chloro-N6-methylanhydrohexitol nucleo-
side, 9d , reacted with tetrabenzyl pyrophosphate, as in method
B, followed by an alternative deprotection procedure. Starting
P h osp h or ic Acid Tr i[(2S)-6-(6-a m in op u r in -9-yl)-4-(2′-
p h osp h on oeth yl)m or p h olin -2-ylm eth yl] Ester (8c). Ad-
enosine 5′-triphosphate disodium salt (100.0 mg, 0.181 mmol),

838 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Nandanan et al.
sodium periodate (38.8 mg, 0.181 mmol) and 2-aminoethyl-
phosphonic acid (27.2 mg, 0.218 mmol) were dissolved in 2.0
mL of water. The reaction mixture was stirred at room
temperature for 1.5 h. Sodium cyanoborohydride (26.8 mg,
0.363 mmol) was added, and the reaction mixture was stirred
for an additional 30 min and passed through Amberlite CG50
(H+ form) with an elution of water. The acidic fractions were
collected, neutralized with triethylamine, purified by Sephadex
ion-exchange column chromatography and semipreparative
HPLC described in general procedure A to afford 5.0 mg
(0.0071 mmol, 4.0% yield) of the desired compound: 1H NMR
(D2O) δ 1.82-1.93 (2H, m, CH2), 2.53 (1H, t, J ) 10.8, 5-CH2)
3.36-3.40 (1H, d, J ) 10.8 Hz, 5-CH2), 2.90-3.24 (4H, m, CH2,
3-CH2), 4.11-44.13 (2H, m, 2-CH2), 4.27-4.31 (1H, m, H-2),
6.01 (1H, d, J ) 8.8 Hz, H-6), 8.25 (1H, s, H-2), 8.37 (1H, s,
H-8); 31P NMR (D2O) δ 21.28 (pt, 4-P), -8.68 (d, J ) 18.9 Hz),
-10.83 (d, J ) 19.6 Hz), -22.34 (t, J ) 20.2 Hz).
CH2-5′), 4.29-4.32 (1H, m, H-2′), 4.53-4.59 (1H, m, H-3′),
4.60-4.65 (1H, m, H-1′), 6.84 (1H, d, J ) 4.9 Hz, 2′-OH), 7.73
(1H, s, H-2), 8.20 (1H, s, H-8); MS (CI-NH3) 522 (M+ + 1);
HRMS (FAB-) calcd 521.2853, found 521.2850.
Carbocyclic N6-Methyl-2′-O-[phenoxy(thiocarbonyl)]-3′,5′-O-
(tetraisopropyldisiloxa-1,3-diyl)adenosine (13). A suspension
of compound 12 (400 mg, 0.766 mmol) and 4-(dimethylamino)-
pyridine (280 mg, 2.29 mmol) in dry acetonitrile (2.0 mL) was
treated with phenyl chlorothionoformate (127 µL, 0.918 mmol).
The suspended solid slowly went into solution when stirred
at 24 °C under nitrogen. After 12 h, the solvent was evaporated
under reduced pressure, and the residue was purified by pTLC
using MeOH:CHCl3 (1:15) to afford a yellowish liquid (450 mg,
0.684 mmol, 89.3%): 1H NMR (CDCl3) δ 0.85-1.09 (28H, m,
CH(CH3)2), 2.15-2.22 (3H, m, H-4′, 4′-CH2), 3.13 (3H, bs, N6-
CH3), 3.90 (2H, dd, J ) 61.1, 11.7 Hz, CH2-5′), 4.80-4.85 (1H,
m, H-3′), 5.04-5.09 (1H, m, H-1′), 5.86 (1H, bs, NH), 5.93-
5.95 (1H, m, H-2′), 7.01-7.35 (5H, m, C6H5), 7.66 (1H, s, H-2),
8.24 (1H, s, H-8); MS (CI-NH3) 658 (M+ + 1); HRMS (FAB-)
calcd 657.2836, found 657.2812.
1,5-An h yd r o-2-(2-ch lor o-6-m eth yla m in op u r in -9-yl)-2,3-
d id eoxy-D-a r a bin o-h exitol (9d ). A mixture of 2-chloro-6-
methylaminopurine (9b; 37 mg, 0.2 mmol), lithium hydride
(1.6 mg, 0.2 mmol), and 1,5-anhydro-4,6-O-benzylidene-3-
deoxy-2-O-(p-tolylsulfonyl)-D-ribo-hexitol (9a ; 78 mg, 0.2 mmol)
in DMF (4 mL) was stirred at 80 °C for 48 h. A second amount
of 1,5-anhydro-4,6-O-benzylidene-3-deoxy-2-O-(p-tolylsulfonyl)-
D-ribo-hexitol (78 mg, 0.2 mmol) was added and the mixture
was further heated for 48 h at 80 °C. After addition of water
(0.5 mL), the reaction mixture was evaporated, diluted with
CH2Cl2 (20 mL), filtered and washed with water (20 mL). The
organic layer was dried and purified by column chromatogra-
phy on silica (CH2Cl2:CH3OH, 97.5:2.5). The resulting com-
pound (70 mg, 87%) was directly deprotected by treatment
with 80% of acetic acid at 60 °C for 6 h. Evaporation, followed
by preparation thin-layer chromatography (CH2Cl2-CH3OH
90:10) yielded 20 mg (65%) of the title compound: MS (LSIMS)
m/z: 314 (M + H+); 13C NMR (DMSO-d6) δ 27.2 (CH3), 35.9
(C3′), 50.4 (C2′), 60,5 (C6′), 60.7 (C4′), 67.9 (C1′), 83.0 (C5′),
Car bocyclic N6-Meth yl-2′-deoxy-3′,5′-O-(tetr aisopr opyl-
d isiloxa -1,3-d iyl)a d en osin e (14). Compound 13 (450 mg,
0.684 mmol) was dissolved in dry toluene (0.2 mL). After
degassing with oxygen-free argon for 20 min, tributyltin
hydride (368 µL, 1.37 mmol) and azobis[isobutyronitrile] (40.1
mg, 0.244 mmol) were added. The reaction mixture under
argon was heated at reflux for 3 h. The solvent was evaporated
under reduced pressure, and the residue was purified by pTLC
using MeOH:CHCl3 (1:10) to afford a yellowish liquid (284 mg,
0.562 mmol, 82.1%):1H NMR (CDCl3) δ 0.90-1.05 (28H, m,
CH(CH3)2), 0.78-1.28 (2H, m, H-2′), 1.88-2.04 (2H, m, 4′-CH2),
2.25-2.33 (1H, m, H-4′), 3.12 (3H, bs, N6-CH3), 3.70 (1H, dd,
J ) 11.7, 4.9 Hz, CH2-5′), 3.97 (1H, dd, J ) 11.7, 2.9 Hz, CH2-
5′), 4.56-4.64 (1H, m, H-3′), 4.95-5.01 (1H, m, H-1′), 7.71 (1H,
s, H-2), 8.31 (1H, s, H-8); MS (CI-NH3) 506 (M+ + 1); HRMS
(FAB-) calcd 505.2904, found 505.2880.
Ca r bocyclic N6-Meth yl-2′-d eoxya d en osin e (15). A solu-
tion of compound 14 (280 mg, 0.554 mmol) in dry tetrahydro-
furan (2.5 mL) was treated with tetrabutylammonium fluoride
(210 mg, 0.664 mmol). The reaction mixture was stirred at
room temperature for 30 min. The solvent was evaporated
under reduced pressure, and the residue was purified by pTLC
using MeOH:CHCl3 (1:10) to afford a yellowish liquid (90.2
mg, 0.343 mmol, 61.9%): 1H NMR (CD3OD) δ 1.89-1.96 (1H,
m, 2′-CH2), 2.20-2.27 (2H, m, 4′-CH2), 2.39-2.46 (1H, m, 2′-
CH2), 2.52-2.56 (1H, m, H-4′), 3.12 (3H, bs, N6-CH3 ), 3.35-
3.78 (2H, m, CH2-5′), 3.78-4.33 (1H, m, H-3′), 5.10-5.20 (1H,
m, H-1′), 8.17 (1H, s, H-2), 8.25 (1H, s, H-8); MS (EI) 263 (M+);
HRMS (FAB-) calcd 263.1382, found 263.1375.
Ca r bocyclic N6-Meth yl-2′-d eoxya d en osin e-3′,5′-bis(d i-
b en zyl p h osp h a t e) (16). Carbocyclic N6-methyl-2′-deoxy-
adenosine (15; 20.0 mg, 0.0759 mmol) was phosphorylated
following the general procedure B to give 17.9 mg (0.0228
mmol, 30.0% yield) of the desired compound 16: 1H NMR
(CD3OD) δ 1.71-2.38 (2H, m, 2′-CH2), 3.03 (3H, s, N6-CH3),
3.69-3.73 (1H, m, H-4′), 4.00-4.04 (2H, m, 5′-CH2), 4.83-4.86
(1H, m, H-3′), 4.96 (2H, d, J ) 8.8 Hz, CH2), 7.24 (5H, m, C6H5),
7.91 (1H, s, H-2), 7.98 (1H, s, H-8), 8.12 (1H, m, H-1′); 31P NMR
(CD3OD) 0.36, -0.60 (2s, 3′-P, 5′-P); HRMS (FAB-) calcd
783.2586, found 783.2568.
(1R ,2S ,4S ,5S )-1-[(H yd r oxy)m e t h yl]-2-h yd r oxy-4-(6-
m eth yla m in op u r in -9-yl)bicyclo[3.1.0]h exa n e (17b). The
Dimroth rearrangement (Scheme 2) was carried out on (N)-
methanocarba-2′-deoxyadenosine. Specifically, the (N)-meth-
anocarba-2′-deoxyadenosine (17a ; 50.0 mg, 0.191 mmol) was
heated at 40 °C with methyl iodide (71.5 µL, 1.15 mmol) in
dry DMF (2.0 mL) for 48 h. The solvent was evaporated under
reduced pressure, and the residue was heated at 90 °C with
ammonium hydroxide (4.0 mL) for 4 h. The water was
evaporated, and the residue was purified by pTLC using
MeOH:CHCl3 (1:9) to afford compound 17b as a colorless solid
(40 mg, 0.15 mmol, 76%): 1H NMR (CD3OD) δ 0.77-0.81 (1H,
m, CH2-6′), 1.03-1.07 (1H, m, CH2-6′), 1.68-1.72 (1H, m, CH-
5′), 1.79-1.89 (1H, m, CH2-3′), 2.00-2.07 (1H, m, CH2-3′), 3.12
1
117.9 (C5), 140.0 (C8), 149.4 (C4), 153.2 (C2), 155.6 (C6); H
NMR (DMSO-d6) δ 1.89 (ddd, J ) 4.4, 11.4 and 13.4 Hz, H-3′a),
2.26 (br d, J ) 13.2 Hz, H-3′e), 2.93 (d, J ) 4.2 Hz, CH3), 3.20
(ddd, J ) 2.2, 4.4 and 9.0 Hz, H-5′), 3.52 (tt, J ) 5.1 and 10.2
Hz, H-4′), 3.60 (dt, J ) 5.8 and 11.7 Hz, H-6′a), 3.69 (ddd, J )
2.0, 5.1 and 11.7 Hz, H-6′e), 3.85 (dd, J ) 2.7 and 12.7 Hz,
H-1′a), 4.17 (d pst, J ) 2.3 and 12.7 Hz, H-1′e), 4.62 (dd, J )
6.4 and 5.6 Hz, 6′-OH), 4.73 (br s, H-2′), 4.89 (d, J ) 5.6 Hz,
4′-OH), 8.19 (br, NH), 8.28 (s, H-8). Anal. (C12H16N5O3Cl) Calcd
for (MW 313.74) C, 45.94; H, 5.14; N, 22.32. Found: C, 45.71;
H, 5.03; N, 22.17.
Ca r bocyclic N6-Meth yla d en osin e (N6-Meth yla r ister o-
m ycin ) (11). The Dimroth rearrangement (Scheme 1) of
carbocyclic adenosine (10; 450 mg, 1.81 mmol) gave compound
11 as a light yellowish solid (400 mg, 1.52 mmol, 84.0%).
Specifically carbocyclic adenosine was heated at 40 °C with
methyl iodide (672 µL, 10.8 mmol) in dry DMF (4.0 mL) for
48 h. The solvent was evaporated under reduced pressure, and
the residue was heated at 90 °C with ammonium hydroxide
(4.0 mL) for 4 h. The water was evaporated, and the residue
was purified by pTLC using MeOH:CHCl3 (1:9): 1H NMR
(CD3OD) δ 1.87-2.50 (3H, m, H-4′, 4′-CH2), 3.10 (3H, bs, N6-
CH3), (1H, m, H-4′), 3.30 (1H, d, J ) 2.0 Hz, CH2-5′), 3.69 (2H,
d, J ) 4.9 Hz, CH2-5′), 4.05 (1H, m, H-2′), 4.48-4.52 (1H, m,
H-1′), 8.15 (1H, s, H-2), 8.22 (1H, s, H-8); MS (CI-NH3) 280
(M+ + 1).
Ca r bocyclic N6-Meth yl-3′,5′-O-(tetr a isop r op yld isiloxa -
1,3-d iyl)a d en osin e (12). A solution of carbocyclic N6-methyl-
adenosine (11; 350 mg, 1.25 mmol) in dry DMF (2.0 mL) was
treated with imidazole (340 mg, 4.99 mmol) followed by 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (481 µL, 1.50 mmol).
The reaction mixture was stirred at 24 °C under nitrogen for
24 h. The solvent was evaporated under reduced pressure, and
the residue was purified by pTLC using MeOH:CHCl3 (1:10)
to afford a yellowish liquid (409 mg, 0.783 mmol, 62.5%): 1H
NMR (CDCl3) δ 0.76-1.06 (28H, m, CH(CH3)2), 1.96-2.19 (2H,
m, 4′-CH2), 2.22 (1H, m, H-4′), 3.07 (3H, bs, N6-CH3), 3.75 (1H,
dd, J ) 11.7, 4.9 Hz, CH2-5′), 3.94 (1H, dd, J ) 11.7, 2.9 Hz,

Ribose Deoxyadenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 839
(3H, bs, N6-CH3), 3.33 (1H, d, J ) 11.7 Hz, CH2OH), 4.29 (1H,
d, J ) 11.7 Hz, CH2OH), 4.89-4.92 (1H, m, CH-4′), 5.02 (1H,
d, J ) 6.9 Hz, CH-2′), 8.24 (1H, s, H-2), 8.49 (1H, s, H-8); MS
(CI-NH3) 276 (M + 1); HRMS (FAB-) calcd 275.1382, found
275.1389.
was removed from reaction mixture by distillation in vacuo
at 65-70 °C (bath temperature 110 °C). The bath temperature
was raised to 120-140 °C, and product was distilled out from
the reaction mixture at 85-95 °C in vacuo. Exposure to
moisture in the air was avoided, since the product was highly
reactive and polymerized to a white solid: 1H NMR (CDCl3) δ
1.18-1.35 (m, 6 H, 2 x CH3), 3.09 (s, 2 H, CH2d), 3.83 (q, 4 H,
2 x CH2).
3,3-Dieth oxy-2-(eth oxyca r bon yl)cyclobu ten e (26). A
mixture of ketene diethyl acetal (3.28 g, 28.2 mmol) and ethyl
propiolate (3 mL, 30.1 mmol) in dry dichloromethane (25 mL)
was heated at 50 °C under a nitrogen atmosphere for 28 h.
After the reaction mixture was concentrated to dryness, the
residue was purified by quick short distillation to give com-
pound 25 as a colorless liquid at 51 °C/0.09 Torr (lit.44 bp 40
°C/0.003 Torr): 1H NMR (CDCl3) δ 1.22 (t, J ) 7.1 Hz, 3 H,
OCH2CH3), 1.29 (t, J ) 7.1 Hz, 3 H, OCH2CH3), 2.67 (d, J )
1.3 Hz, 2 H, -CH2-), 3.67-3.78 (m, 4 H, 2 x OCH2CH3), 4.22
(q, J ) 7.1 Hz, 2 H, COOCH2CH3), 7.15 (t, J ) 1.3 Hz, CHd);
MS (CI) m/e 215 (MH+).
(()-2-Ch lor o-9-[(1r,2â)-3,3-d ieth oxy-2-(eth oxyca r bon -
yl)cyclobu tyl]-N6-m eth yla d en in e (27). 2-Chloro-N6-meth-
yladenine was prepared as follows: A solution of 2,6-dichloro-
purine (1.38 g, 7.32 mmol) in 40% methylamine in water (12
mL) was heated at 100 °C for 1 h in a sealed bottle. Solid
precipitate was filtered, washed with cold water, and dried to
give the title compound (1.25 g, 93.5%): 1H NMR (DMSO-d6)
δ 2.91 (br s, 3 H, CH3), 8.09 (s, 1 H, H-8), 7.90 (br s, 1 H, NH),
13.07 (br s, 1 H, NH).
(N)-Meth a n oca r ba -N6-m eth yl-2′-d eoxya d en osin e-3′,5′-
bis(d iben zyl p h osp h a te) (18) [(1R,2S,4S,5S)-1-[(Diben -
zylp h osp h a t o)m e t h yl]-4-(6-m e t h yla m in op u r in -9-yl)-
bicyclo[3.1.0]h exa n e-2-(d iben zyl p h osp h a te)]. Starting
from 20.0 mg (0.0726 mmol) of (N)-methanocarba-N6-methyl-
2′-deoxyadenosine, 17b, and following the general phosphor-
ylation procedure (method B) we obtained 13.5 mg (0.0170
mmol, 23.4% yield) of the desired protected intermediate 18
(Scheme 2): 1H NMR (CDCl3) δ 0.73-0.78 (1H, m, CH2-6′),
0.94-0.98 (1H, m, CH2-6′), 1.53-1.54 (1H, m, CH-5′), 1.81-
1.91 (1H, m, CH2-3′), 2.05-2.13 (1H, m, CH2-3′), 3.15 (3H, bs,
N6-CH3), 3.70-3.83 (1H, m, CH2OP), 4.49-4.55 (1H, m,
CH2OP), 4.89-5.00 (8H, m, -OCH2), 5.02-5.06 (1H, m, CH-
4′), 5.27-5.32 (1H, m, CH-2′), 5.86 (1H, bs, NH), 7.21-7.23
(20H, m, C6H5), 7.86 (1H, s, H-2), 8.31 (1H, s, H-8); 31P NMR
(D2O) δ -0.56, -1.05 (2s, 3′-P, 5′-P); HRMS (FAB-) (M + Cs)
calcd 928.1641; found 928.1700.
(1R,2S,4S,5S)-1-[(Ben zyloxy)m eth yl]-2-ben zyloxy-4-(2,6-
d ich lor op u r in -9-yl)bicyclo[3.1.0]h exa n e (21). To an ice-
cold solution of triphenylphosphine (278 mg, 1.06 mmol) in
dry THF (2 mL) was added diethyl azadicarboxylate (170 µL,
1.06 mmol) dropwise under a nitrogen atmosphere, and the
mixture was stirred for 20 min until the solution turned red
orange (Scheme 3). This mixture was added dropwise to a cold
stirred mixture of the starting alcohol (135 mg, 0.417 mmol)
and 2,6-dichloropurine (157 mg, 0.883 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 pTLC using
EtOAc:petroleum ether (1:1) to afford a thick liquid (132 mg,
0.263 mmol, 64%):1H NMR (CD3OD) δ 0.85 (m, 1H), 1.13 (m,
1H), 1.59 (m, 1H), 1.68 (m, 1H), 2.06 (m, 1H), 3.17 (d, J )
10.8 Hz, 1H), 4.11-4.57 (m, 5H), 5.20 (d, J ) 6.9 Hz, 1H), 6.6
(bs, 1H), 7.23-7.37 (m, 10H), 8.98 (s, 1H); MS (EI) 494 (M+).
(1R,2S,4S,5S)-1-[(Ben zyloxy)m eth yl]-2-ben zyloxy-4-(2-
ch lor o-6-m eth ylam in opu r in -9-yl)bicyclo[3.1.0]h exan e (22).
Compound 21 (100 mg, 0.202 mmol) was dissolved in methyl-
amine in methanol (30% solution, 3 mL) and was stirred at
room temperature for 12 h under a nitrogen atmosphere. The
solvent was evaporated, and the crude product was purified
by pTLC using EtOAc:petroleum ether (6:4) to afford 22 as a
light yellow solid (86 mg, 0.176 mmol, 88%): 1H NMR (CD3OD)
δ 0.70 (m, 1H), 1.06 (m, 1H), 1.50 (m, 1H), 1.76 (m, 1H), 1.96
(m, 1H), 3.01 (s, 3H), 3.08 (m, 2H), 4.03 (m, 4H), 4.45 (bs, 1H),
5.02 (bs, 1H), 8.38 (s, 1H); MS (CI) 490 (M + 1).
(1R,2S,4S,5S)-1-[(Hydr oxy)m eth yl]-2-h ydr oxy-4-(2-ch lo-
r o-6-m et h yla m in op u r in -9-yl)b icyclo[3.1.0]h exa n e (23).
Compound 22 (40 mg, 0.0816 mmol) was dissolved in dry
CH2Cl2 (1.0 mL), and hydrogenated using BCl3 (1 M in CH2Cl2,
175 µL) for 50 min at -78 °C under argon. The solvent was
evaporated, and the crude product was purified by pTLC using
CHCl3:MeOH (10:1) to afford 23 as a light yellow solid (10.0
mg, 0.0323 mmol, 39.6%): 1H NMR (CD3OD) δ 0.77-0.81 (1H,
m, CH2-6′), 1.02-1.05 (1H, m, CH2-6′), 1.65-1.68 (1H, m, CH-
5′), 1.78-1.91 (1H, m, CH2-3′), 1.99-2.07 (1H, m, CH2-3′), 3.08
(3H, bs, N6-CH3), 3.37 (1H, d, J ) 11.7 Hz, CH2OH), 4.27 (1H,
d, J ) 11.7 Hz, CH2OH), 4.89-4.91 (1H, m, CH-4′), 4.97 (1H,
d, J ) 6.8 Hz, CH-2′), 8.46 (1H, s, H-8); MS (CI-NH3) 310 (M
+ 1); HRMS (FAB-) calcd 309.0992, found 309.0991.
To a mixture of 3,3-diethoxy-2-(ethoxycarbonyl)cyclobutene
(26; 1.07 g, 5.0 mmol) and 2-chloro-N6-methyladenine (0.658
g, 3.58 mmol) in anhydrous DMF (21 mL) was added DBU
(0.536 mL, 3.58 mmol) at 0 °C under a nitrogen atmosphere.
After the reaction mixture was stirred at room temperature
for 18 h, DMF was evaporated by rotary evaporation. The
residue was dissolved in chloroform (30 mL) and washed with
saturated NaHCO3, brine, dried over anhydrous Na2SO4,
filtered, and concentrated to dryness. The residue was purified
by silica gel column chromatography (hexanes:EtOAc, 5:1f3:
1f1:1f1:3) to give compound 27 (1.1 g, 77%) as a white
solid: Rf ) 0.24 (hexanes:EtOAc, 1:1), 0.27 (CHCl3:MeOH, 20:
1
1); H NMR (CDCl3) δ 1.19 (t, J ) 6.8 Hz, 3 H, CH3), 1.26 (t,
J ) 6.8 Hz, 3 H, CH3), 1.28 (t, J ) 6.8 Hz, 3 H, CH3), 2.79 (dd,
J ) 12.4, 8.8 Hz, 1 H, -CH2a-), 2.96 (dd, J ) 12.4, 8.8 Hz, 1 H,
-CH2b-), 3.18 (br s, 3 H, NHCH3), 3.50 (m, 2 H), 3.67 (m, 1H),
3.82 (m, 1 H), 3.98 (d, J ) 7.8 Hz, 1 H), 4.20 (m, 2 H), 5.15 (dt,
J ) 8.8 Hz, 1 H), 6.00 (br s, 1 H, NH), 7.81 (s, 1 H, H-8); low-
resolution MS (CI) m/e 398 (MH+).
(()-2-Ch lor o-9-[(1r,2â)-3,3-d iet h oxy-2-(h yd r oxym et h -
yl)cyclobu tyl]-N6-m eth yla d en in e (28). To a solution of
compound 27 (1.036 g, 2.6 mmol) in anhydrous THF (15 mL)
was added 1.0 M LiAlH4 in THF (4.03 mL, 4.03 mmol) at 0 °C
under a nitrogen atmosphere. The reaction mixture was stirred
at 0 °C for 1 h and 20 min and quenched by the addition of
H2O (0.2 mL), 5 M NaOH (0.2 mL), and H2O (0.5 mL). After
the mixture was stirred vigorously for 20 min, a solid was
removed by filtration, and the filtrate was concentrated to
dryness. The white solid was dissolved in H2O (0.5 mL) and
chloroform (30 mL), and the aqueous layer was extracted with
chloroform (30 mL × 3). The combined organic layer was
washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated to dryness. The residue was purified by silica gel
column chromatography (CHCl3:MeOH, 20:1) to give com-
pound 28 (833 mg, 90%) as a white solid: Rf ) 0.21 (CHCl3:
MeOH, 20:1); 1H NMR (DMSO-d6) δ 1.15 (t, J ) 6.0 Hz, 3 H),
1.17 (t, J ) 6.0 Hz, 3 H), 2.49 (m, 1 H), 2.81 (dd, J ) 11.7, 8.8
Hz, 1 H), 2.91 (d, J ) 2.9 Hz, 3 H, NHCH3), 3.10 (m, 1 H),
3.33-3.61 (m, 5 H), 3.72 (m, 1 H), 4.34 (dt, J ) 8.8 Hz, 1 H),
8.21 (br d, J ) 2.9 Hz, 1 H), 8.29 (s, 1 H, H-8); 1H NMR (CDCl3)
δ 1.22 (t, J ) 7.1 Hz, 3 H, CH3), 1.24 (t, J ) 7.1 Hz, 3 H, CH3),
2.57 (ddd, J ) 12.7, 7.8, 1.5 Hz, 1 H), 3.01 (m, 2 H), 3.2 (br s,
3 H, NHCH3), 3.44-3.61 (m, 4 H), 3.74 (br s, 1 H), 3.91 (m, 1
H), 4.00 (m, 1H), 4.67 (dt, 8.5, 7.8 Hz, 1 H), 6.03 (br s, 1 H,
NH), 7.84 (s, 1 H, H-8); low-resolution MS (CI) m/e 356 (MH+).
Keten e Dieth yla ceta l (25). Ketene diethylacetal (Scheme
4) was prepared from bromoacetaldehyde diethylacetal (24)
(Aldrich Chemical Co.) and potassium tert-butoxide (Aldrich
Chemical Co.) by direct mixing and distillation at a 110 °C
bath temperature. Specifically, in a 100 mL two-neck round-
bottomed flask equipped with a Vigreux column distillation
apparatus were added bromoacetaldehyde diethyl acetal (19.71
g, 0.1 mol) and potassium tert-butoxide (11.2 g, 0.1 mol) under
a nitrogen atmosphere at room temperature. tert-Butyl alcohol

840 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Nandanan et al.
(()-2-Ch lor o-9-[(1r,2â,3r)-3-h yd r oxy-2-(h yd r oxym eth -
yl)cyclobu tyl]-N6-m eth yla d en in e (29). To a solution of
compound 28 (833 mg, 2.34 mmol) in acetone (117 mL) was
added 1 N HCl (22 mL) slowly. The reaction mixture was
stirred at room temperature for 2 days. After acetone was
removed by rotary evaporation, the residue was treated with
5 N NaOH to neutral. It was extracted with EtOAc (3 × 30
mL), and the combined organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated to
dryness to give the crude ketone as a white solid: Rf ) 0.09
(CHCl3:MeOH, 20:1); 1H NMR (DMSO-d6) δ 2.5 (m, 1 H), 2.92
(s, 3 H, NHCH3), 3.46 (ddd, J ) 17.8, 8.8, 2.2 Hz, 1 H), 3.61-
3.79 (m, 2 H), 4.16 (m, 1 H), 5.05 (t, J ) 4.4 Hz, exchangeable
with D2O, 1 H, OH), 5.20 (dt, J ) 8.6, 6.8 Hz, 1 H), 8.30 (br s,
1 H, NH), 8.41 (s, 1 H, H-8); low-resolution MS (CI) m/e 282
(MH+).
The above crude product was dissolved in anhydrous MeOH
(45 mL) and NaBH4 (177 mg, 4.68 mmol) was added in three
portions at 0 °C. The reaction mixture was stirred at that
temperature for 1 h before the reaction was quenched by the
addition of acetone (2 mL). After the mixture was stirred for
another 20 min, it was concentrated to dryness. The residue
was purified by silica gel column chromatography (CHCl3:
MeOH, 10:1) to give compound 29 (350 mg, 52.7%) as a white
solid: 1H NMR (DMSO-d6) δ 2.20 (m, 1 H), 2.72 (dt, J ) 10.8,
7.3 Hz, 1 H), 2.82 (m, 1 H), 2.90 (s, 3 H, NHCH3), 3.55 (m, 2
H), 3.82 (m, 1 H), 4.26 (dt, J ) 8.1, 8.8 Hz, 1 H), 4.69 (t, J )
4.0 Hz, exchangeable with D2O, 1 H, OH), 5.29 (d, J ) 6.4 Hz,
exchangeable with D2O, 1 H, OH), 8.18 (br s, exchangeable
with D2O, 1 H, NH), 8.27 (s, 1 H, H-8); low-resolution MS (CI)
m/e 284 (MH+).
N6-Met h yl-1,5-a n h yd r o-2-(a d en in -9-yl)-2,3-d id eoxy-D-
a r a bin o-h exitol (31). The Dimroth rearrangement (Scheme
5) of 1,5-anhydro-2-(adenin-9-yl)-2,3-dideoxy-D-arabino-hexitol
(40.0 mg, 0.151 mmol) gave compound 31 as a colorless solid
(30.0 mg, 0.107 mmol, 71.2%). Specifically, 1,5-anhydro-2-
(adenin-9-yl)-2,3-dideoxy-D-arabino-hexitol the was heated at
40 °C with methyl iodide (56.3 µL, 0.905 mmol) in dry DMF
(2.0 mL) for 48 h. The solvent was evaporated under reduced
pressure, and the residue was heated at 90 °C with ammonium
hydroxide (4.0 mL) for 4 h. The water was evaporated, and
the residue was purified by pTLC using MeOH:CHCl3 (1:9):
1H NMR (CD3OD): δ 1.96-2.07 (1H, m, H-3′), 2.39-2.49 (1H,
m, H-3′), 3.14 (3H, bs, N6-CH3), 3.36 (1H, m, H-5′), 3.58-3.67
(1H, m, H-4′), 3.78 (1H, dd, J ) 11.7, 4.9 Hz, H-6′), 3.90 (1H,
dd, J ) 11.7, 2.0 Hz, H-6′), 4.04 (1H, d, J ) 13.4 Hz, H-1′),
4.37 (1H, d, J ) 13.4 Hz, H-1′), 4.79 (1H, m, H-2′), 8.29 (1H,
s, 2-H), 8.46 (1H, s, 8-H); MS (CI-NH3) 280 (M+ + 1); HRMS
(FAB-) calcd 279.1331, found 279.1319.
1,5-An h yd r o-2-(a d en in -9-yl)-N6-m eth yl-2,3-d id eoxy-D-
a r a bin o-h exitol-4,6-bis(d iben zyl p h osp h a te) (32). Start-
ing from 23.0 mg (0.0726 mmol) of 1,5-anhydro-2-(adenin-9-
yl)-N6-methyl-2,3-dideoxy-D-arabino-hexitol and following the
general phosphorylation procedure (method B) we obtained
32.4 mg (0.0405 mmol, 55.8% yield) of the desired compound:
1H NMR (CD Cl3) δ 2.04-2.17 (1H, m, H-3′), 2.70-2.75 (1H,
m, H-3′), 3.23 (3H, s, N6-CH3), 3.58-3.63 (1H, m, H-5′), 3.87
(1H, dd, J ) 12.7, 2.9 Hz, H-1′), 4.25 (1H, d, J ) 12.7 Hz, H-1′),
4.08-4.16 (1H, m, H-4′), 4.32-4.38 (2H, m, CH2-6′), 4.72 (8H,
4d, J ) 7.9 Hz, CH2), 5.95 (1H, bs, H-2′), 7.00 (1H, bs, NH),
7.30 (20H, m, C6H5), 8.10 (1H, s, H-2), 8.38 (1H, s, H-8); 31P
NMR (CD3Cl) δ -0.76, -1.78 (2s, 3′-P, 5′-P); MS (SIMS) 800
(M+ + 1); HRMS (FAB-) (M + Cs) calcd 932.1590, found
932.1608.
°C. Erythrocyte ghosts were prepared by rapid lysis in hypo-
tonic buffer (5 mM sodium phosphate, pH 7.4, 5 mM MgCl2, 1
mM EGTA) as described.36 Phospholipase C activity was
measured in 25 µL of [3H]inositol-labeled ghosts (approxi-
mately 175 µg of protein, 200-500000 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 the indicated
concentrations of nucleotide analogues. Ghosts were incubated
at 30 °C for 5 min, and total [3H]inositol phosphates were
quantified by anion-exchange chromatography as previously
described.8,36
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 inhibition is
equal to 100 minus the residual fraction of stimulation at the
highest antagonist concentration.
All concentration-effect curves were repeated in at least
three separate experiments carried out with different mem-
brane preparations using duplicate or triplicate assays.
Molecu la r Mod elin g. All calculations were performed on
a Silicon Graphics Indigo 2 R8000 workstation. All the ligand
structures were constructed using the “Sketch Molecule” of
SYBYL 6.5.46 Semiempirical molecular orbital calculations
were done using the AM1 Hamiltonian47 as implemented in
MOPAC 6.048 (keywords: PREC, GNORM ) 0.1, EF, MMOK
if necessary).
Relative to the published X-ray structures of the parent
methanocarba nucleosides,35,49 bisphosphate analogues 4a and
5 retained their characteristic (N) (P ) 342) and (S) (P ) 198)
orientations, respectively, in the pseudorotational cycle.54
However, the maximum degree of puckering, νmax, which
defines the extent that the ring deviates from planarity showed
that the bisphosphate analogues were significantly more
planar (νmax ) 10) than the parent compounds (νmax ) 28).49
Whether this flattening of the ring is a direct consequence of
the attraction between the two phosphate groups and/or is a
consequence of the semiempirical methodology used, perhaps
in combination with the lack of aqueous environment in the
calculation, will be the subject of a separate investigation.
Superimposition of these geometry-optimized ligand struc-
tures was carried out using the “Fit Atoms” method imple-
mented in SYBYL. The quality of the fit is represented by the
rms value computed for the matched atoms.
The three-dimensional human P2Y1 receptor model was
built and optimized using SYBYL 6.5 and Macromodel 6.0,50
respectively, based on the approach described by Moro et al.38
Briefly, the seven-transmembrane helical domains were iden-
tified with the aid of Kyte-Doolittle hydrophobicity51 and
51
Emini surface probability parameters. The helices were built
and energy-minimized for each transmembrane sequence. The
minimized helices were then grouped together to form a helical
bundle matching the overall characteristics of the electron
density map of rhodopsin. The helical bundle was energy-
minimized using the AMBER52 all-atom force field, 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.
The structures of 4a and 5 were rigidly docked into the
helical bundle using graphical manipulation with continuous
energy monitoring (Dock module of SYBYL). Both local energy-
minimized receptor-ligand complexes were subjected to an
additional CG minimization run of 300 steps. Partial atomic
charges for the ligands were taken from the MOPAC output
files. We have recently introduced the cross-docking procedure
to obtain energetically refined structures of GPCR/ligand
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.8,36 The K0.5 values were averaged from
3-8 independently determined concentration-effect curves for
each compound. Briefly, 1 mL of washed turkey erythrocytes
was incubated in inositol-free medium (DMEM; Gibco, Gaith-
ersburg, MD) with 0.5 mCi of 2-[3H]myo-inositol (20 Ci/mmol;
American Radiolabelled Chemicals, Inc., St. Louis, MO) for
18-24 h in a humidified atmosphere of 95% air/5% CO2 at 37

Ribose Deoxyadenosine Bisphosphate Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 841
complexes.38 We applied this technique to predict the structure
of both 4a /P2Y1 and 5/P2Y1 receptor complexes. Cross-docking
allows possible ligand-induced rearrangements of the 7TM
bundle to be explored by sampling 7TM conformations in the
presence of the docked ligands. Small translations and rota-
tions were applied to each helix relative to its original position
until a new lower energy geometry was obtained. These
manual adjustments were followed by 25 ps of molecular
dynamics (MD module of Macromodel) performed at a constant
temperature of 300 K using a time step of 0.001 ps and a
dielectric constant ) 4.0. This procedure was followed by
another sequence of CG energy minimization to a gradient
threshold of <0.1 kcal/mol/Å. Energy minimization of the
complexes was performed using the AMBER all-atom force
field in MacroModel.
The interaction energy values were calculated as follows:
E(complex) ) E(complex) - (E(L) + E(receptor)). These ener-
gies are not rigorous thermodynamic quantities, but can only
be used to compare the relative stabilities of the complexes.
Consequently, these interaction energy values cannot be used
to calculate binding affinities since changes in entropy and
solvation taken into account.
(11) Fagura, M. S.; Dainty, I. A.; McKay, G. D.; Kirk I. P.; Humphries,
R. G.; Robertson, M. J .; Dougall, I. G.; Leff, P. P2Y1-receptors
in human platelets which are pharmacologically distinct from
P2YADP-receptors. Br. J . Pharmacol. 1998, 124, 157-164.
(12) Crowley, M. R. Oxygen-induced pulmonary vasodilation is
mediated by adenosine triphosphate in newborn lambs. J .
Cardiovasc. Pharmacol. 1997, 30, 102-109.
(13) Loubatie`res-Mariani, M.-M.; Hillaire-Buys, D.; Chapal, J .; Ber-
trand, G.; Petit, P. P2 purinoceptor agonists: New insulin
secretagogues potentially useful in the treatment of noninsulin-
dependent diabetes mellitus. Purinergic Approaches in Experi-
mental Therapeutics; Wiley: New York, 1997; pp 253-260.
(14) Virginio, C.; Robertson, G.; Surprenant, A.; North, R. A. Trinitro-
phenyl-substituted nucleotides are potent antagonists selective
for P2X1, P2X3, and heteromeric P2X2/3 receptors. Mol. Phar-
macol. 1998, 53, 969-973.
(15) Humphreys, B. D.; Virginio, C.; Surprenant, A.; Rice, J .; Dubyak,
G. R. Isoquinolines as antagonists of the P2X7 nucleotide
receptor: high selectivity for the human versus rat receptor
homologues. Mol. Pharmacol. 1998, 54, 22-32.
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F.; Wildman, S. S.; Rychkov, A.; Yoburn, J .; Kim, H.; Mohanram,
A.; Harden, T. K.; Boyer, J . L.; Burnstock, G.; J acobson, K. A.
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Abbr evia tion s: AIBN, 2,2′-azobis[isobutyronitrile]; ATP,
adenosine-5′-triphosphate; CG, conjugate gradient; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DCTIDS, 1,3-dichlorotetraiso-
propyl-1,1,3,3-disiloxane; DEAD, diethyl azadicarboxylate;
DEAE, diethylaminoethyl; DMAP, 4-(dimethylamino)pyridine;
DMF, dimethylformamide, DMSO, dimethyl sulfoxide; FAB,
fast atom bombardment (mass spectroscopy); HPLC, high-
pressure liquid chromatography; MS, mass spectroscopy;
HRMS, high-resolution mass spectroscopy; LDA, lithium di-
isopropylamide; 2-MeSADP, 2-methylthioadenosine-5′-diphos-
phate; TBAP, tetrabutylammonium phosphate; TBPP, tetra-
benzyl pyrophosphate; TEAA, triethylammonium acetate;
THF, tetrahydrofuran; pTLC, preparative thin-layer chroma-
tography.
(17) Camaioni, E.; Boyer, J . L.; Mohanram, A.; Harden, T. K.;
J acobson, K. A. Deoxyadenosine-bisphosphate derivatives as
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Ack n ow led gm en t. We thank Gilead Sciences (Fos-
ter City, CA) for financial support to E.N. and for the
gift of aristeromycin. This work was supported by
USPHS Grants GM38213 and HL54889.
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