Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and L-α-threofuranosyl ring systems: Interactions with P2Y receptors

Article abstract of DOI:10.1016/j.bmc.2004.07.067

We have synthesized nucleotide analogues substituted with two novel ring systems and examined them as P2Y receptor ligands: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) l-α-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol- containing P2Y₁ receptor antagonist MRS2283. The ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y ₁ receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y₂ and P2Y₄ receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) l-α-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y₁ receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a K_i value of 22.5 nM in binding to the human P2Y₁ receptor, and antagonized the stimulation of PLC by the potent P2Y₁ receptor agonist 2-methylthio-ADP (30 nM) with an IC₅₀ of 650 nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A ₃). Thus, this ring system afforded some P2Y receptor selectivity. A l-α-threofuranosyl bisphosphate derivative 9 displayed an IC₅₀ of 15.3 u?M for inhibition of 2-methylthio-ADP-stimulated PLC activity. l-α-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y₂ (EC₅₀ = 9.9 u?M) versus P2Y₄ receptors. The P2Y₁ receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking.

Full text of DOI:10.1016/j.bmc.2004.07.067

Bioorganic & Medicinal Chemistry 12 (2004) 5619–5630
Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and
L-a-threofuranosyl ring systems: interactions with P2Yreceptors
Michihiro Ohno,a Stefano Costanzi,a Hak Sung Kim,a,c Veerle Kempeneers,d
Karen Vastmans,d Piet Herdewijn,d Savitri Maddileti,b Zhan-Guo Gao,a
T. Kendall Hardenb and Kenneth A. Jacobsona,
*
aMolecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health,
Department of Health and Human Services, Bethesda, MD 20892-0810, USA
bDepartment of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599-7365, USA
cCollege of Pharmacy and Medicinal Resources Research Center, Wonkwang University, Iksan, 570-749 Chonbuk, South Korea
dLaboratory of Pharmaceutical Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 24 March 2004; revised 30 July 2004; accepted 30 July 2004
Available online 9 September 2004
Abstract—The ribose moiety of adenine nucleotide 30,50-bisphosphate antagonists of the P2Y1 receptor has been successfully sub-
stituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in recep-
tor binding. Similarly, at P2Y2 and P2Y4 receptors, nucleotides constrained in the (N) conformation interact equipotently with the
corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two
novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) L-a-
threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known
anhydrohexitol-containing P2Y1 receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a Ki value
of 22.5nM in binding to the human P2Y1 receptor, and antagonized the stimulation of PLC by the potent P2Y1 receptor agonist
2-methylthio-ADP (30nM) with an IC50 of 650nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A3).
Thus, this ring system afforded some P2Y receptor selectivity. A L-a-threofuranosyl bisphosphate derivative 9 displayed an IC50 of
15.3lM for inhibition of 2-methylthio-ADP-stimulated PLC activity. L-a-Threofuranosyl-UTP 13 was a P2Y receptor agonist with
a preference for P2Y2 (EC50 = 9.9lM) versus P2Y4 receptors. The P2Y1 receptor binding modes, including rotational angles, were
estimated using molecular modeling and receptor docking.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Various ribose ring substitutions have been incorpo-
rated into biologically active nucleosides and nucleic
acids. A methanocarba modification introduced by
Marquez and co-workers2,3 has been used to constrain
the pseudosugar (cyclopentane) ring of carbocyclic
nucleosides in various systems. The methanocarba ring
system consists of a fused cyclopropane and cyclopen-
tane rings, to attain either a (N) or (S) conformation
depending on the position of fusion. Methanocarba ana-
logues helped to define the role of sugar puckering in
stabilizing the active bound conformation at both pur-
ine and pyrimidine receptors and thereby allowed iden-
tification of a favored conformation.4–6 Using such
conformationally constrained analogues, a preference
for the (N) conformation of ribose at the P2Y1 recep-
tor5,6 was deduced. The bisphosphate derivatives 1
(MRS2279) and 2 (MRS2500), which are locked in a
The ribose rings of nucleosides and nucleotides exist in
an equilibrium covering a range of conformations,
which has been described as a pseudorotational cycle.1
The North ((N), 20-exo, 30-endo) and South ((S), 20-endo,
30-exo) conformations are the most relevant to the bio-
logical activities observed for nucleosides and nucleo-
tides in association with DNA, RNA, and various
enzymes.1–6
Keywords: Nucleoside; Purine; Pyrimidine; G protein-coupled recep-
tor; Carbocyclic; Phospholipase C; Radioligand binding; Molecular
model.
*
Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480
8422; e-mail: kajacobs@helix.nih.gov
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.067

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M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
(N) conformation by the bicyclo[3.1.0]hexane ring sys-
tem, are the most potent known antagonists of the
P2Y1 receptor (Chart 1).6–9 Subsequently we discovered
that P2Y2, P2Y4, and P2Y11 receptors can recognize
(N)-methanocarba nucleoside 50-triphosphates 3 and 4
roughly as potently as the corresponding ribosides.5
However, in the case of P2Y6 receptors, the (N)-metha-
nocarba equivalent 5 of the natural nucleotide activator,
UDP, was inactive.5
different pseudorotational angle from that of the
(N)-methanocarba ring system.
Another novel ribose substitution adapted to P2Y
receptor ligands in the present study is the L-a-threofu-
ranosyl ring. Eschenmoser and colleagues have demon-
strated that Watson–Crick pairing in RNA is not
exclusively dependent on the ribofuranosyl ring sys-
tem.18,19 The unnatural threofuranosyl ring system
(leading to threofuranosyl nucleic acids, TNAs, for
example, 8), which is lacking one carbon in comparison
to the ribosyl moiety, is capable of base pairing to RNA
strands with a high strength.
In the present study, aimed at further investigating the
conformational preferences of P2Y receptors, we have
explored additional substitutions of the ribose moiety
in P2Y receptor agonists and antagonists.
Here we report the first synthesis of bisphosphate antag-
onists of the P2Y1 receptor 21 and 9, derived from car-
bocyclic locked adenine 7 and L-a-threofuranosyl
adenine 22, respectively. These novel ring structures
are compared both in biological assays and in P2Y1
receptor model20 docking with 9-riboside analogues,
for example, 10 and 11, and with a previously reported
anhydrohexitol bisphosphate antagonist 12 of the
P2Y1 receptor.7 L-a-Threofuranosyl-UTP 13 was also
synthesized and assayed at P2Y2, P2Y4, and P2Y6
receptors.
One such substitution is based on the Ôlocked nucleic
acidsÕ (LNAs), for example, containing monomer 6,
which were introduced several years ago10–15 and which
hybridize effectively to natural RNA. Recently, we
reported the synthesis of a novel ring system corre-
sponding to the ring methylene equivalent of the LNAs,
that is, carbocyclic LNAs (cLNAs), for example, 7.16
cLNAs, which contain the oxabicyclo[2.2.1]heptane ring
system, are expected to be more stable than oxygen
LNAs because of their nonglycosidic nature, as has been
discussed for methanocarba ribose in comparison with
natural ribose.4,5,17 The cLNA would prefer an approx-
imate (N)-conformation,16 but is expected to adopt a
Since the A3 adenosine receptor (AR)4 also showed a
preference for the (N) conformation of the ribose, we
Chart 1.

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
5621
tested the nucleoside precursors of 2, 9, and 21 in bind-
ing studies at the ARs.
ers.21 Treatment of 15b with methylamine gave 2-iodo-
N6-methyladenine 16 in good yield.
The key step of this synthesis was the coupling of the
nucleobase 16 with a triflate intermediate16 17 to provide
18. In a previous study of cLNAs,16 we tried several cou-
pling procedures, but most were unsuccessful because
the leaving group was at the concave face of the cLNA
scaffold, which decreased the reactivity. Only the triflate
ester 17 could be applied to this coupling reaction. Also,
the coupling step required the use of well-dried potas-
sium carbonate. In the course of this synthesis, the tri-
flate 17 was crystallized from ethyl acetate/petroleum
ether. This enabled a large-scale preparation and storage
of the triflate substrate as a common intermediate,
which is an important advantage in synthetic nucleoside
chemistry and in parallel synthesis.
2. Results
2.1. Chemical synthesis
We prepared cLNA analogues of bisphosphate deriva-
tives (Scheme 1), in which the bicyclo[2.2.1]heptane ring
system fixed the pseudoribose moiety in a rigid (N)-enve-
lope conformation. Identification of compounds was
confirmed by NMR (1H and 31P) and by high-resolution
mass spectrometry (HRMS), and purity was demon-
strated with high-performance liquid chromatography
(HPLC) in two different solvent systems.
In a previous publication,16 we reported an efficient syn-
thetic procedure for formation of the bicyclo[2.2.1]hep-
tane system. Generally, the formation of carbocyclic
nucleosides requires more synthetic steps than does the
preparation of the corresponding natural nucleosides.
In the reported LNA synthesis10,11 the ribose ring was
cyclized after introduction of the base. This required
more synthetic steps and also made it difficult to synthe-
size numerous analogues. The coupling of sugar and
base moieties at a later stage (Scheme 1) provided great-
er versatility in the efficient preparation of nucleoside
and nucleotide derivatives.
The benzoyl groups of the resulting 18 were hydrolyzed
to give 19. Compound 19 was phosphorylated by the
phosphoramidite method to give 20. The tert-butyl pro-
tecting group of 20 was removed using trifluoroacetic
exchange resin chromatography, to obtain the target
compound 21.
The TNA bisphosphate 9 was prepared as shown in
Scheme 2. The synthesis of the UTP analogue 13 was
by standard methods of triphosphate formation.34
2.2. Pharmacological activity
An important aspect in the preparation was the nucleo-
base synthesis. In this synthesis, we applied a method
featuring N9-protection, as described previously for the
synthesis of 2-iodo-6-chloroadenine.8 N9-Protection of
the adenine precursor 14 by the pivaloyloxymethyl
group, which improved the solubility, enabled various
transformation reactions. The 2-iodination of 15a was
then performed by the method of Nair and co-work-
We recently developed [3H]MRS2279 1 as a high-affinity
and selective radioligand for quantification of the P2Y1
receptor.9 Therefore, the human P2Y1 receptor can be
expressed from a baculovirus to high levels in Sf9 insect
cells, and membranes prepared from these cells can be
used with [3H]1 to directly assess the affinity of newly
synthesized molecules at the P2Y1 receptor. Human
Scheme 1.

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M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
Figure 1. Activation of the human P2Y1 receptor (A) and competitive
antagonism (B) of 2-MeSADP-promoted activation. PLC activity was
measured as described in Section 4 in 1321N1 human astrocytoma cells
stably expressing the human P2Y1 receptor. Assays were in the
presence of the indicated concentrations of the agonist 2-MeSADP
alone or in the presence of 100nM–100lM MRS2457 9. The data are
the means of triplicate determinations and are representative of results
obtained in three separate experiments.
Scheme 2.
P2Y1 receptor-expressing membranes were incubated
with approximately 20nM [3H]1 and a wide range of
concentrations of 2-substituted (N)-methanocarba bis-
phosphate analogues. The novel cLNA adenine nucleo-
tide MRS2584 21 interacted with the P2Y1 receptor, as
shown by its capacity to inhibit [3H]1 binding, and this
inhibition occurred with kinetics consistent with interac-
tion at a single site by the law of mass action kinetics.
IC50 values were determined from each competition
curve, and a Ki value was calculated as 22.5nM accord-
ing to the relationship Ki = IC50/1 + [[3H]1]/Kd of [3H]1.
The Ki value for compounds 9, 10, and 12 were also
determined, indicating that the riboside 10, bound more
potently than the anhydrohexitol derivative 12 and the
of the molecules to increase inositol phosphate accumu-
lation by activating the phospholipase C (PLC)-coupled
P2Y1 receptor, and antagonist activity at the P2Y1
receptor was assessed by measuring the capacity of these
molecules to inhibit 2-MeSADP (30nM)-stimulated
inositol phosphate accumulation. None of the bisphos-
phate analogues exhibited agonist activity. The cLNA
bisphosphate derivative 21 antagonized the P2Y1 recep-
tor with an IC50 value of 650nM. The TNA bisphos-
phate 9 only weakly antagonized the human P2Y1
receptor with an IC50 value of 15.3lM (Fig. 1). Antag-
onist potencies at the P2Y1 receptor for the previously
reported7 antagonists 10–12 are presented in Table 1
for comparison. Included among the archival com-
pounds was the known anhydrohexitol-containing
P2Y1 receptor antagonist MRS2283 12.7
TNA bisphosphate 9, by factors of
respectively.
5 and 77,
We also tested the activities of bisphosphate analogues
as agonists and antagonists at the human P2Y1 receptor
stably expressed in 1321N1 human astrocytoma cells.
Agonist activity was tested by measuring the capacity
Table 1. In vitro pharmacological data at P2Y1 receptors in binding (human receptor expressed in Sf9 cells)9and for inhibition of PLC activity
stimulated by 30nM 2-MeSADP (human receptor expressed in 1321N1 astrocytoma cells or in turkey erythrocytes)
Compound
R = (2-position)
R0 = (N6-position)
Binding, Ki, nMa
Antagonism, IC50, nMb
1 MRS2279c
2 MRS2500c
9 MRS 2457
10 MRS 2216
11 MRS 2246
12 MRS 2283
21 MRS 2584
Cl
I
CH3
CH3
H
2.5 0.4
0.78 0.08
666 333
8.6 2.6
ND
51.6 0.8 (t)
8.4 0.8 (h)
H
Cl
I
15,300 300 (h)
206 53 (t)
CH3
CH3
CH3
CH3
891 233 (t)
566 224 (t)c
650 100 (h)
Cl
I
43 10
22.5 10.4
Mean s.e.m. is given for three separate determinations. None of the compounds displayed agonist effects.
a The affinities were determined by using [3H]1 in a radioligand binding assay, as recently described.9 The human P2Y1 receptor was expressed to high
levels in Sf9 insect cells with a recombinant baculovirus. Membranes prepared from these cells were incubated for 30min at 4ꢁC in the presence of
ꢀ20nM [3H]1.
b Antagonist IC50 values represent the concentration needed to inhibit by 50% the effect elicited by 30nM 2-MeSADP. n = 3, unless otherwise
indicated in parentheses. t = turkey, h = human.
c Values from Refs. [7–9,35]. ND not determined.

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
5623
Activity of the UTP analogue in the TNA series, com-
pound 13, was also examined at the PLC-coupled
human P2Y2 and P2Y4 receptors stably expressed
in 1321N1 human astrocytoma cells. The EC50 values
for receptor activation by 13 were found to be
9.9 2.1lM (n = 3) and 26lM (n = 2) at human P2Y2
and P2Y4 receptors, respectively (Fig. 2). The corre-
sponding EC50 values for the riboside, UTP, were 8
and 49nM, respectively.5 Although 13 was a full agonist
at the P2Y4 receptor, it was completely inactive at the
human P2Y6 receptor.
(MRS3210), that is, the unsubstituted cLNA adeno-
sine, at 10lM had no effect in binding at two ARs
(hA1, hA2A) and displaced ꢀ30% binding at the hA3AR.
We also tested the corresponding 2-iodo-N6-methyl
nucleoside analogue 19 (MRS3342) in binding assays,
and obtained a Ki value of 4.90lM at the hA3AR.
The threofuranosyl adenine nucleoside 22 (which was
the precursor of 9) showed no measurable affinity
at 10lM in binding to the hA1, A2A, A3ARs or in
functional agonism of the hA2BAR. However, the (N)-
methanocarba nucleosides typically bind more readily
to adenosine receptors and have already been shown
to favor selectivity of binding to the A3AR over other
subtypes. (N)-Methanocarba-2-chloro-N6-methyladeno-
sine, which may be considered the 20-hydroxy equivalent
of the nucleoside precursor of 1, and thus an (N)-meth-
anocarba analogue of 19 (MRS3342), had a Ki value of
23nM at the hA3AR and bound more weakly to the
rA1AR.22 Even the parent (N)-methanocarba-adeno-
sine, which was comparable to 7 (MRS3210), had a Ki
value of 404nM in binding to the hA3AR.4
The adenine nucleosides precursors of 2, 9, and 21 were
examined in binding to human ARs. Compound 7
2.3. Molecular modeling
With the goal of understanding the molecular features
important for the binding of MRS2279 1, we studied
with computational and molecular biology tools the
interactions of 1 with the P2Y1 receptor.20,23 As already
stated in the introduction, MRS2279 1 is a potent and
selective P2Y1 receptor antagonist. We also studied by
means of computational techniques the interactions be-
tween our rhodopsin-based P2Y1 model20 and the
nonriboside antagonists 2, 9, 12, and 21. Like 1, all of
these P2Y1 antagonists may be regarded as analogues
of the 20-deoxyadenosine-30,50-bisphosphate in which
the 20-deoxyribose is replaced by various pseudosugar
moieties.
The ligands were first fully optimized by means of
quenched molecular dynamics and energy minimization.
Then they were flexibly superimposed, taking into
account both steric and electronic features, to the bound
conformation of 1, as derived from our docking experi-
ments.20 Starting from these ligand/receptor complexes,
we carried out automatic docking experiments at the
P2Y1 receptor, to explore the interactions between ami-
no acids in the receptor binding pocket and the various
antagonists (Fig. 3). The docking was based on a Monte
Carlo/simulated annealing approach, During the dock-
ing procedure the ligands and the binding site were trea-
ted as fully flexible. In this way we allowed both the
receptor and the ligands to undergo the conformational
changes necessary for mutual adaptation.
To estimate the conformational changes that the ligands
had to go through in order to bind to the receptor, we
also studied the sugar puckering of the nucleotides
before and after docking at the P2Y1 receptor by calcu-
lating the puckering coordinates of the pseudosugars.
The puckering coordinates can be derived from the tor-
sional angles of the rings and furnish a complete descrip-
tion of the puckering. All of the torsional angles
could be calculated from the puckering coordinates by
Figure 2. Activation of the human P2Y2 (A), P2Y4 (B), and P2Y6 (C)
receptors by the uracil nucleotides UDP and UTP and by the L-a-
threofuranosyl analogue of UTP (MRS2488 13). PLC activity was
measured as described in Section 4 in 1321N1 human astrocytoma cells
stably expressing the human P2Y receptors. The data are the means of
triplicate determinations and are representative of results obtained in
three separate experiments.

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M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
Figure 3. P2Y1 receptor complexes with the ligands MRS2500 2 (a), MRS2584 21 (b), MRS2283 12 (c), and MRS2457 9 (d). The residues of the P2Y1
binding pocket are colored according to the following scheme: cyan (TM1), orange (TM2), green (TM3), red (TM6), gray (TM7), white (EL2).
applying the inverse formulae. For a description of the
puckering of the five-membered rings we used the coordi-
nates P (phase angle of pseudorotation) and hm (pucker-
ing amplitude) as defined by Altona and
Sundaralingham,1 while for the six-membered ring we
used the coordinates H (phase angle of symmetrical inter-
conversion), P2 (phase angle of pseudorotation), and Q
(total puckering amplitude) as defined by Haasnoot.24
and antagonists at the P2Y1 receptor.25,26 The N6-Me
and 2-halo substituents, when present, interacted with
Y58(1.39) and Y100(2.53), respectively.
In 2 (Fig. 3a), as in 1, the 2-deoxyribose was substituted
by a methanocarba ring system constrained in the (N)
conformation by a (bicyclo[3.1.0]hexane) ring system.
The bound conformation of 2 showed a pseudorota-
tional angle (P) of À17ꢁ, which indicated an almost pure
C20-exo conformation (P = À18ꢁ). This result was in
excellent agreement with the crystal structures of various
methanocarba nucleosides developed and analyzed by
Marquez and co-workers during the past few years.27
According to the biological data, the (N)-methanocarba
ring system confers to the nucleotides the ideal confor-
mation for the interaction with the P2Y1 receptor.
The special disposition and steric features of the pseudo-
sugar and the phosphate moieties were studied after the
superimposition of the adenine moieties of all the
docked ligands. This involved analyzing the conforma-
tional similarities to the most potent compound 2 (Fig.
4).
As previously observed for 1, the P2Y1 receptor binding
pocket was identified within the upper regions of the
TMs (transmembrane domains) 3, 6, and 7 and the
EL2 (the second extracellular loop), with the phosphate
moieties coordinated by the essential cationic residues
R128(3.29), K280(6.55), and K310(7.39). As in previous
models,20,23 Q307 (7.36) always acted as a hydrogen
bond acceptor from the exocyclic amino group, while
S314(7.43) donated a hydrogen bond to N-1 of the ade-
nine moiety. Mutagenesis studies outlined the impor-
tance of these five residues for the binding of agonists
The bound conformation of 2 showed only minimal
deviation from the free ligand optimized in vacuo, indi-
cating that the ligand required small conformational
adjustment to fit into binding site of the P2Y1 receptor
and to establish the optimal interactions with the three
fundamental cationic residues.
In the case of the cLNA analogue 21, the ribose moiety
was replaced by a oxabicyclo[2.2.1]heptane ring system.
Like the methanocarba analogues, compound 21 was

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
5625
explanation of the diminished affinity of 21 with respect
to its (N)-methanocarba analogue 2.
The six-membered pseudosugar moiety of the anhydro-
hexitol derivative 12, which in the free ligand showed a
clear preference for an almost pure chair conformation
with a H of about 0ꢁ, after binding to P2Y1 receptor
(Fig. 3c) adopted a O50-exo/C30-exo screw–boat confor-
mation (H = 77ꢁ, P2 = À84ꢁ, Q = 57ꢁ). The superimposi-
tion of the bound conformations of 12 and 2 showed a
good overlapping of the phosphates and of the pseudo-
sugar moieties, indicating that this anhydrohexitol
derivative has the capability to resemble the conforma-
tion of the potent (N)-methanocarba analogues (Fig.
4b). The conformational differences that we detected be-
tween the P2Y1 receptor-bound form of 12 and its free
form, optimized in vacuo, could be one of the reasons
for lower affinity of this compound compared to the
(N)-methanocarba analogues.
However, in good agreement with the biological data,
the threofuranosyl moiety of 9 did not allow a complete
overlapping of the phosphate moieties with those of the
most active compounds (1 and 2); thus, the interactions
with the key cationic residues were not optimal (Fig.
3d). Being unsubstituted at N6 and 2 positions, the com-
pound lacked also the favorable hydrophobic interac-
tions with the two Y residues in TM1 and TM2.20
With respect to the conformation of the pseudosugar
moiety, we detected a substantial difference between
the puckering of the free form of 9 optimized in vacuo
(P = 215, hmax = 44), and its P2Y1 receptor-bound con-
formation (P = 260, hmax = 44). Nonetheless, the bound
compound 9 did not resemble the bound conformation
of 2, but showed significant divergence in the position
of the phosphate moieties and in the overall shape of
the pseudosugar (Fig. 4c).
Figure 4. Superimpositions of the bound conformation of MRS2584
21 (a), MRS2283 12 (b), and MRS2457 9 (c) with MRS2500 2,
performed by overlapping the adenine moieties. The anhydrohexitol
derivative 12 can resemble the bound conformation of the most active
compound 2.
3. Discussion
locked in a (N) conformation by this bicyclic system.
However, its bound conformation showed a P of 21ꢁ,
very close to a pure C30-endo conformation (P = 18ꢁ).
Besides having different P angles, the pseudosugar moi-
eties of the two molecules showed other dissimilarities in
overall geometry (Fig. 4). The five-membered ring of 2
tended to be relatively flat, with a hm of 31ꢁ; In contrast,
in the case of 21 it tended to fold more markedly, show-
ing a hm of 60ꢁ. Despite these differences, our docking
studies suggested that 21 still established interactions
with the fundamental cationic residues of TM3, TM6,
and TM7 (Fig. 3b). Also in the case of 21, we detected
limited conformational changes between the free ligand
optimized in vacuo and the ligand docked into the
receptor. The superimposition of the bound conforma-
tions of 21 and the potent methanocarba derivative 2
showed a good overlapping of the 50-phosphate moie-
ties, while greater divergence was found in the position
of the 30-phosphates and in the overall shape of the
pseudosugars (Fig. 4a). In particular, the steric hin-
drance exerted by the bulky 20-cyclic alkoxy group of
21 on the nearby residues of TM3 could be a possible
The success of conformationally constrained antagonists
in enhancing affinity at the P2Y1 receptor and the lack
of success so far in applying this approach to other
P2Y subtypes,5–8 prompted us to search for other (N)-
like ring systems, especially those that may adopt a
slightly different conformation according to the pseudo-
rotational cycle and therefore may have an altered phar-
macological profile.
These potential advantages encouraged us to synthesize
ring-constrained carbocyclic-type analogues for im-
proved ligands for the G protein-coupled P2Y receptors
by using both novel cLNA units of the (N) conforma-
tion and L-a-threofuranosyl substitution of ribose as a
test of the conformational requirements at the putative
binding sites.
According to the biological data and our docking exper-
iments, the (N)-methanocarba pseudosugar moiety
seemed to be endowed with a puckering that could en-
sure an optimal fit of the ligand in the P2Y1 binding
pocket, together with the ideal orientation of the

5626
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
bisphosphates for the interactions with the cationic res-
idues of the binding site. Also, the cLNA analogue 21
was locked within a preferred conformation range of
the pseudoribose for binding to the P2Y1 receptor.
calculated to be +21ꢁ and its conformation belonged
to the (N)–(3E) category. This cLNA bisphosphate
derivative displayed potent binding affinity at the hu-
man P2Y1 receptor. In contrast, the cLNA nucleoside
bound only weakly to the hA3AR receptor and did
not bind appreciably to other adenosine receptors. Thus,
this ring system contributes to selectivity for the P2Y1
receptor. Thus, the order or suitability of various non-
ribose ring systems to bind to P2Y1 receptors was:
bicyclo[3.1.0]hexane > 2-oxa-bicyclo[2.2.1]heptane > L-a-
threofuranose. The L-a-threofuranosyl analogue of
UTP maintains a preference for activating the P2Y2 in
comparison to the P2Y4 receptor.
The cLNA analogue 21 (MRS2584) was an antagonist at
the hP2Y1 receptor and displayed a high binding affinity
indicated by a Ki value of 22.5nM. Compound 21 was
roughly equipotent with the 2-iodo ribose analogue 11,
which had an IC50 value of 891nM in a functional assay
at the turkey P2Y1 receptor. The nucleotide 21 was 3-fold
less potent in binding than the corresponding 2-chloro
ribose analogue 10, which had a Ki value of 8.6nM. In
comparison to the methanocarba analogue 2, the potency
of 21 was somewhat diminished. Compound 21 was 29-
fold less potent than 2 (MRS2500), which had a Ki value
of 0.78nM in binding to the hP2Y1 receptor. Compound
21 was 9-fold less potent than 1 (MRS2279), which had a
Ki value of 2.5nM in binding to the hP2Y1 receptor.
4. Experimental
4.1. Chemical synthesis
Nucleosides and synthetic reagents were purchased from
Sigma Chemical Co. (St. Louis, MO) and Aldrich (Mil-
waukee, WI). Compound 12 was synthesized as described7.
Previous data indicated that the presence of a ribose 20-
OMe group decreased antagonist potency at the P2Y1
receptor.35 A 20-ether group is present in 21, and the
effect of this 20-cyclic alkoxy group on antagonist po-
tency is unknown. It was also suggested from molecular
modeling and docking in the human P2Y1 receptor (Fig.
3b) that amino acids from TM3, that is, F131(3.32),
H132(3.33), and L135(3.36) may have steric interactions
with the 20-substituents. Furthermore, the substitution
of the ribose with the oxabicyclo[2.2.1]heptane ring did
not allow the optimal orientation of the 30-phosphate
group for interaction with the essential cationic residues.
1H NMR spectra were obtained with a Varian Gemini-
300 spectrometer (300MHz) with D2O, CDCl3,
CD3OD, and DMSO-d6 as a solvent. 31P NMR spectra
were recorded at room temperature with a Varian XL-
300 spectrometer (121.42MHz); orthophosphoric acid
(85%) was used as an external standard.
Purity of compounds was checked with a Hewlett–Pack-
ard 1090 HPLC apparatus equipped with an SMT
OD-5-60 RP-C18 analytical column (250 · 4.6mm;
Separation Methods Technologies, Inc., Newark, DE)
in two solvent systems. System A: linear gradient solvent
system: 0.1M TEAA/CH3CN from 95/5 to 40/60 in
20min; the flow rate was 1mL/min. System B: linear gra-
dient solvent system: 5mM TBAP/CH3CN from 80/20
to 40/60 in 20min; the flow rate was 1mL/min. System
C: linear gradient solvent system: H2O/CH3CN from
95/5 to 0/100 in 30min; the flow rate was 1mL/min.
Peaks were detected by UV absorption with a diode ar-
ray detector. All derivatives tested for biological activity
showed P 97% purity in the HPLC systems. Low-reso-
lution CI-NH3 (chemical ionization) mass spectra were
measured with a Finnigan 4600 mass spectrometer and
high-resolution EI (electron impact) mass spectrometry
was performed with a VG7070F mass spectrometer at
6kV. High-resolution FAB (fast atom bombardment)
mass spectrometry was performed with a JEOL SX102
spectrometer with 6kV Xe atoms following desorption
from a glycerol matrix. Purification of the nucleotide
analogues for biological testing was carried out on
Sephadex-DEAE-A-25 resin columns with a linear gra-
dient (0.01–0.5M) of 0.5M ammonium bicarbonate.
The adenosine receptor binding results for 19 showed
that a cLNA scaffold does not fit in the binding site of
the hA3AR as effectively as does the (N)-methanocarba
ring system. Thus, the cLNA, containing the oxabicy-
clo[2.2.1]heptane ring system, apparently is a P2 recep-
tor-selective scaffold. The molecular modeling results
of the hA3AR binding site model also indicated a direct
interaction of the 20-OH and 30-OH groups with
Q167(EL2) and H272(7.43). Furthermore, the models
suggest that 20-substitutions may exhibit steric interfer-
ence with L90(3.32) and L91(3.33) of hA3AR.28,29 On
the other hand the (N)-methanocarba moiety exhibited
an optimal puckering for interaction with the A3AR
(and to a greater degree than other AR subtypes). Com-
pound 22 (precursor of 9) was also inactive at adenosine
receptors. However, since TNAs only weakly activated
the P2Y receptors, a statement about the receptor selec-
tivity of this ring system would not be justified.
The two threofuranosyl derivatives examined in this
study only weakly antagonized (9 at P2Y1) or activated
(13 at P2Y2, and P2Y4 receptors) the P2Y receptors. In
good agreement with the biological data, our molecular
modeling studies emphasized that the threofuranosyl
moiety does not confer to the ligands the steric features
necessary for recognition by the P2Y1 receptor.
4.1.1. (20R,30R,40S)-Phosphoric acid mono-[2-(6-amino-
purin-9-yl)-4-phosphonooxy-tetrahydro-furan-3-yl] ester
(9). Compound 23 (10mg, 0.013mmol) was dissolved
in a mixture of methanol (2mL) and water (1mL) and
hydrogenated over a 10% Pd/C (10mg) at room temper-
ature for 2d. The catalyst was removed by filtration and
the methanol was evaporated. The residue was treated
In conclusion, we have synthesized a novel (N)-locked
carbocyclic bisphosphate using a cLNA scaffold. The
pseudorotational P value of the cLNA compound was

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
5627
with ammonium bicarbonate solution and subsequently
frozen and lyophilized. Purification of the obtained res-
idue was performed on an ion-exchange column packed
with Sephadex-DEAE A-25 resin, a linear gradient
(0.01–0.5M) of 0.5M ammonium bicarbonate was ap-
plied as the mobile phase, and UV and HPLC were used
to monitor the elution, which furnished 9 (1.8mg, 30%).
1H NMR (D2O) d 8.41 (s, 1H), 8.31 (s, 1H), 6.33 (s, 1H),
5.10 (d, 1H, J = 8.7Hz), 4.93–4.90 (br, 1H), 4.51 (d, 1H,
J = 10.3Hz), 4.41(dd, 1H, J = 3.3, 10.3Hz); 31P NMR
(D2O) d À0.29 (br s); MS (m/e) (negative-FAB) 396
stirring for 10min at 0ꢁC, the reaction mixture was trea-
ted with sat NaHCO3 (2mL) and extracted with chloro-
form. The organic layer was separated and washed with
brine, dried over anhydrous sodium sulfate, filtered, and
concentrated. The residue was purified by column chro-
matography (silica gel, AcOEt/petroleum ether = 5/1–3/
1) to give triflate 17 (73.6mg, 99%). 1H NMR (CDCl3) d
8.05–8.01 (m, 2H), 7.99–7.94 (m, 2H), 7.63–7.54 (m,
2H), 7.47–7.39 (m, 4H), 5.33 (ddd, 1H, J = 2.2, 2.7,
9.9Hz), 5.13 (s, 1H), 4.63 (d, 1H, J = 2.2Hz), 4.50 (AB
quartet, 2H, J = 12.1Hz), 4.32 (dd, 1H, J = 3.3,
7.4Hz), 4.05 (d, 1H, J = 7.4Hz), 2.56 (ddd, 1H,
J = 3.3, 10.1, 14.6Hz), 2.01 (dd, 1H, J = 3.0, 14.6Hz).
MS (m/e) (positive-FAB) 501 (M++H).
(M+ÀH).
HRMS
(negative-FAB)
calcd
for
C9H12N5O9P2 396.0110. Found 396.0119; HPLC
16.0min (99%) (system B), 2.7min (99%) (system C).
4.1.2. 2-Amino-6-chloropurin-9-yl-methyl 2,2-dimethyl-
propionate (15a). 2-Amino-6-chloropurine 14 (1.00g,
5.90mmol) was dissolved in DMSO (5.0mL) under
heating. DMF (20.0mL), chloromethyl pivalate
(1.0mL, 6.94mmol) and K2CO3 (990mg, 7.16mmol)
were added and the mixture was stirred at room temper-
ature for 3d. The reaction mixture was filtered and the
filtrate was evaporated. The obtained residue was puri-
fied by flash chromatography (AcOEt/petroleum
ether = 2/1) and recrystallized from AcOEt–petroleum
ether, which furnished the desired compound 15a
4.1.6. (10S,40R,60S,70S)-Benzoic acid 4-benzoyloxy-
methyl-6-(2-iodo-6-methylaminopurin-9-yl)-2-oxa-bicy-
clo[2.2.1]-hept-7-yl ester (18). (10S,40R,60S,70S)-Benzoic
acid 4-benzoyloxymethyl-6-trifluoromethanesulfonyl-
oxy-2-oxa-bicyclo[2.2.1]hept-7-yl ester 17 (20mg,
0.040mmol), 2-iodo-6-methylaminopurine 16 (25mg,
0.091mmol), potassium carbonate (18mg, 0.130mmol)
and 18-crown-6 (4mg, 0.015mmol) in DMF (0.30mL)
was stirred at 60ꢁC for 3h. The resulting mixture was
dried up under vacuum, and the residue was purified
by silica gel column chromatography (AcOEt) and pre-
parative TLC (silica gel, AcOEt/petroleum ether = 1/1),
to give dibenzoyl adenine derivative 18 (23mg, 91%).
1H NMR (CDCl3) d 8.06–7.96 (m, 4H), 7.90 (s, 1H),
7.58 (m, 2H), 7.42 (m, 4H), 5.88 (br s, 1H), 5.64 (s,
1H), 4.80 (dd, 1H, J = 5.1, 9.6Hz), 4.71 (s, 1H), 4.68
(d, 1H, J = 11.7Hz), 4.61 (d, 1H, J = 11.7Hz), 4.28
(dd, 1H, J = 3.0, 7.2Hz), 3.97 (d, 1H, J = 7.2Hz), 3.17
(br s, 3H), 2.70 (dd, 1H, J = 9.6, 13.8Hz), 2.33 (m,
1H); MS (m/e) (positive-FAB) 626 (M++H).
1
(1.33g, 79 %). H NMR (CDCl3) d 8.01 (s, 1H), 6.00
(s, 2H), 5.20 (br s, 2H), 1.18 (s, 9H); MS (m/e) (posi-
tive-FAB) 284, 286 (peak height ratio is 3:1) (M++H).
4.1.3. 6-Chloro-2-iodopurin-9-yl-methyl 2,2-dimethyl-
propionate (15b). To a solution of 2-amino-6-chloropu-
rin-9-yl-methy 2,2-dimethylpropionate 15a (704mg,
2.48mmol) in MeCN (2.0mL) was added diiodometh-
ane (8.0mL) and t-butylnitrite (0.90mL, 9.99mmol)
and oxygen was purged by N2 bubbling. The tube was
sealed and stirred at 80ꢁC for 2.5h. The solvent was
removed under vacuum and the obtained residue
was purified by flash chromatography (AcOEt/petro-
leum ether = 1/2), which furnished 15b (561mg, 57 %).
1H NMR (CDCl3) 8.29 (s, 1H), 6.14 (s, 2H), 1.19 (s,
9H); MS (m/e) (positive-FAB) 395, 397 (peak height
ratio is 3:1) (M++H).
4.1.7. (10S,40R,60S,70S)-6-(2-Iodo-6-methylaminopurin-9-
yl)-4-hydroxymethyl-2-oxa-bicyclo[2.2.1]hept-7-ol (19).
A mixture of dibenzoyl adenine derivative 18 (21mg,
0.034mmol), potassium carbonate (17mg, 0.123mmol)
in THF (0.5mL) and methanol (1.0mL) was stirred at
room temperature for 25h. The solvent was removed
by nitrogen purging and the residue was purified by sil-
ica gel column chromatography (MeOH/CHCl3 = 1/5),
to give the cLNA adenine derivative 19 (12.5mg, 89
4.1.4. 2-Iodo-6-methylamino-9H-purine (16). 2,2-Di-
methyl-propionic acid 6-chloro-2-iodopurin-9-ylmethyl
ester 15b (148mg, 0.375mmol) was dissolved in THF
(2.0mL) and i-PrOH (5.0mL) in sealed tube. 40%
MeNH2 in water (1.0mL) was added, and the solution
stirred at 60ꢁC for 18h. The precipitate, which formed
was filtered and washed with small amount of water to
furnish 2-iodo-6-methylaminopurine 16 (102mg, 99%).
1H NMR (CDCl3) d 7.99 (s, 1H), 7.86 (br s, 1H), 2.88
(br s, 3H); MS (m/e) (positive-FAB) 276 (M++H).
1
%). H NMR (CD3OD) d 8.03 (s, 1H), 4.63 (dd, 1H,
J = 6.0, 9.3Hz), 4.32 (s, 1H), 4.15 (s, 1H), 3.90 (dd,
1H, J = 2.1, 6.6Hz), 3.85 (d, 1H, J = 11.1Hz), 3.73 (d,
1H, J = 11.1Hz), 3.68 (d, 1H, J = 6.6Hz), 3.04 (br
s, 3H), 2.41–2.26 (m, 2H); MS (m/e) (positive-FAB)
418 (M++H), HR-MS (positive-FAB) calcd for
C13H17N5O3I 418.0376. Found 418.0363.
4.1.8. (10S,40R,60S,70S)-6-(2-Iodo-6-methylaminopurin-9-
yl)-4-[(di-t-butyl-phosphato)-methyl]-7-(di-t-butyl-phos-
phato)-2-oxa-bicyclo[2.2.1]heptane (20). The cLNA ade-
nine derivative 19 (8.2mg, 0.020mmol) and 1H-
tetrazole (20mg, 0.285mmol) were dissolved in 1.0mL
of anhydrous THF. Di-t-butyl diethylphosphoramidite
(0.055mL, 0.198mmol) was added, and the mixture
was stirred for 3h at room temperature. The reaction
mixture was cooled to À78ꢁC and treated with a solu-
tion of m-CPBA (70% max, 55mg) in CH2Cl2
4.1.5. (10S,40R,60S,70S)-Benzoic acid 4-benzoxymethyl-6-
trifluoromethanesulfonyloxy-2-oxa-bicyclo[2.2.1]hept-7-
yl ester (17). To a stirred solution of (10S,40R,60S,70S)-
benzoic acid 4-benzoxymethyl-6-hydroxy-2-oxa-bicy-
clo[2.2.1]hept-7-yl ester16 (54mg, 0.147mmol) and
4-dimethylaminopyridine (195mg, 1.60mmol) in meth-
ylene chloride (6.0mL) was added trifluoromethanesulf-
onic anhydride (75lL, 0.446mmol) at 0ꢁC. After

5628
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
(2.5mL). The resulting mixture was warmed to room
temperature and 5% NaHSO3 (2.0mL) was added. The
reaction mixture was stirred another 30min at room
temperature and extracted with AcOEt. The organic
phase was subsequently washed with saturated aqueous
NaHCO3 and brine, and then dried over Na2SO4 and fil-
tered. The solvent was removed under reduced pressure.
The residue obtained was purified by silica gel column
chromatography (MeOH/CHCl3 = 1/5), which fur-
4.2. Pharmacology
2-MeSADP and GTP were purchased from Sigma (St.
Louis, MO). Myo-[3H]inositol (20Ci/mmol) was ob-
tained from American Radiolabeled Chemicals (St.
Louis, MO).
P2Y receptor-promoted stimulation of inositol phos-
phate formation was measured at human P2Y1, P2Y2,
P2Y4, or P2Y6 receptors stably expressed in 1321N1 hu-
man astrocytoma cells as previously described.5,30,31
The IC50 values were averaged from 3 to 8 independ-
ently determined concentration–effect curves for each
compound. Briefly, cells plated in 24-well dishes were
labeled in inositol-free medium (DMEM; Gibco, Gai-
thersburg MD) containing 1.0lCi of 2-[3H]myo-inositol
(20Ci/mmol; American Radiolabeled Chemicals, Inc.,
St. Louis MO) for 18–24h in a humidified atmosphere
of 95% air/5% CO2 at 37ꢁC. PLC activity was measured
the following day by quantitating [3H]inositol phos-
phate accumulation after a 10min incubation at 37ꢁC
in the presence of 10mM LiCl. Total [3H]inositol phos-
phates were quantified by anion exchange chromatogra-
phy as previously described.30,31 The affinities of
bisphosphate analogues for the human P2Y1 receptor
were directly determined by using [3H]1 in a radioligand
binding assay, as we recently described in detail.9 Bind-
ing and functional parameters were estimated using
GRAPHPAD PRISM software (GraphPAD, San Diego,
CA, USA).
1
nished 20 (10mg, 64%). H NMR (CDCl3) d 8.06 (s,
1H), 6.20 (br s, 1H), 4.84 (d, 1H, J = 4.8Hz), 4.67 (m,
2H), 4.21 (m, 2H), 4.00 (m, 1H), 3.82 (d, 1H,
J = 6.9Hz), 3.16 (br s, 3H), 2.60–3.50 (m, 1H), 2.40–
2.30 (m, 1H), 1.48 (s · 2, 18H), 1.47 (s · 2, 18H); MS
(m/e) (positive-FAB) 802 (M++H), 824 (M++Na).
4.1.9. (10S,40R,60S,70S)-Phosphoric acid mono-[6-(2-iodo-
6-methylaminopurin-9-yl)-4-phosphonooxymethyl-2-oxa-
bicyclo[2.2.1]hept-7-yl] ester (21). A solution of 20
(8.0mg, 0.011mmol) in 5% TFA/CH2Cl2 (1.0mL) was
stirred for 2h at 25ꢁC. The solvent was removed under
reduced pressure and the residue was quenched by addi-
tion of 5.0mL of triethylammonium bicarbonate buffer
(1.0M). The mixture was subsequently frozen and
lyophilized. Purification of the obtained residue was per-
formed on an ion-exchange column packed with Sepha-
dex-DEAE A-25 resin, a linear gradient (0.01–0.5M) of
0.5M ammonium bicarbonate was applied as the mobile
phase, and UV and HPLC were used to monitor the elu-
tion, which furnished 21 (5.3mg, 73%). 1H NMR (D2O)
d 8.23 (s, 1H), 4.64 (m, 1H), 4.47 (br s, 2H), 4.12 (m,
1H), 3.96 (m, 2H), 3.83 (d, 1H, J = 6.9Hz), 3.09 (br s,
3H), 2.53–2.32 (m, 2H); 31P NMR (D2O) d 2.22, 1.81
(2s); MS (m/e) (negative-FAB) 576 (M+ÀH); HRMS
(negative-FAB) calcd for C13H17N5O9P2I 575.9546.
Found 575.9557; HPLC 9.1min (99%) (system A),
15.8min (99%) (system B).
4.3. Molecular modeling
Molecular mechanics calculations have been carried out
by means of the Discover3 module of INSIGHTII,32 using
the CFF91 forcefield.33 The P2Y1 model used for the
docking experiments was the rhodopsin based homology
model previously constructed by us.20 Before perform-
ing the docking experiments, the ligands have been
optimized by means of a quenched molecular dynamics
simulation followed by energy minimization.
4.1.10. (20R,30R,40S)-Phosphoric acid 2-(6-aminopurin-9-
yl)-4-(bis-benzyloxy-phosphoryloxy)-tetrahydro-furan-3-
yl ester dibenzyl ester (23). To a stirred solution
of (20R,30R,40S)-2-(6-aminopurin-9-yl)-tetrahydro-furan-
3,4-diol 22 (5mg, 0.021mmol) and 1H-tetrazole
(13mg, 0.186mmol) in anhydrous THF (2.0mL) was
added dibenzyl diisopropylphosphoramidite (22mg,
0.069mmol). After stirring for 3h at room temperature,
the reaction mixture was cooled to À78ꢁC and was trea-
ted with a solution of m-CPBA (70% max, 25mg) in
CH2Cl2 (1mL). The resulting mixture was warmed to
room temperature, treated with 5% NaHSO3 (2.0mL)
for another 30min at room temperature and extracted
with AcOEt. The organic phase was washed with satd
NaHCO3 aq and brine and dried over Na2SO4 and fil-
tered. The solvent was removed under reduced pressure.
The residue obtained was purified by column chroma-
tography (silica gel, eluent: CHCl3/MeOH = 10/1), to
give 23 (10mg, 63%). 1H NMR (CDCl3) d 8.18 (s,
1H), 7.87 (s, 1H), 7.36–7.19 (m, 20H), 6.51 (br s, 2H),
6.18 (d, 1H, J = 1.5Hz), 5.33 (d, 1H, J = 7.5Hz), 5.07
(m, 4H), 5.00 (br, 1H), 4.91 (m, 4H), 4.31 (d, 1H,
J = 10.8Hz), 4.10(dd, 1H, J = 3.9, 10.8Hz); MS (m/e)
(negative-CI) 756 (M+ÀH).
An
NVT
(constant-volume/constant-temperature)
molecular dynamic simulation was carried out at con-
stant temperature of 300K for 10ps, using a time step
of 1fs. During the simulation, snapshots of the system
were taken at regular intervals of 1ps. The structures
in each snapshot were energy minimized (BFGS Newton
method) until an RMS (root mean squared) of
˚
0.00001kcal/mol/A was reached and the one showing
the lowest energy was used as starting point for the
docking experiments.
The flexible superimpositions have been carried out by
means of the FIELDFIT program of Search Compare
module of INSIGHTII,32 giving equal weight to the steric
and the electrostatic factors. The bound conformation
of MRS2279 was kept rigid during the calculation,
while the structures to be superimposed were fully flex-
ible. An alignment of the molecules based on their di-
pole and quadripole moments was used as the starting
position.

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 5619–5630
5629
The automatic docking experiments were carried out by
means of the Monte Carlo minimization/simulated
annealing approach implemented in the Affinity module
of INSIGHTII. The binding site was defined as all the res-
spectral measurements were carried out by Dr. Victor
Livengood and NMR by Wesley White (NIDDK). We
thank Prof. A. Eschenmoser and Dr. R. Krishnamurthy
of the Scripps Res. Inst. (San Diego, CA) for helpful
discussions and for the gift of L-a-threofuranosyl-
adenine.
˚
idues within 6A distance from the ligands. Full flexibil-
ity was granted to the ligands and to the residues of the
binding site. All the atoms of the ligands and the key res-
idues of the binding pocket (R128, K280, R310, Q307,
S314) capable of donating or accepting hydrogen bonds
were manually marked. During the Monte Carlo simula-
tion the scaling factor for the van der Waals term was
set at 0.1, while the coulombic and hydrogen bond terms
were set at 1. The maximum movement in each random
References and notes
1. Altona, C.; Sundaralingham, M. J. Am. Chem. Soc. 1972,
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2. Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.;
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Chem. 1996, 39, 3739.
˚
translation and rotation of the ligand were set at 0.1A
3. Shin, K. J.; Moon, H. R.; George, C.; Marquez, V. E.
J. Org. Chem. 2000, 65, 2172.
4. Jacobson, K. A.; Ji, X.-D; Li, A. H.; Melman, N.; Siddiqi,
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Chem. 2000, 43, 2196.
5. Kim, H. S.; Ravi, R. G.; Marquez, V. E.; Maddileti, S.;
Wihlborg, A.-K.; Erlinge, D.; Malmsjo¨, M.; Harden, T.
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and 1ꢁ, respectively. Fifty stages of simulated annealing
were performed after the Monte Carlo simulation, set-
ting the initial temperature at 500K, the final tempera-
ture at 300K, the final van der Waals and coulombic
scaling factors at 1.0 and the final hydrogen bonds scal-
ing factor at 0.0. In other words the van der Waals and
coulombic factors were fully considered during the sim-
ulated annealing and the hydrogen bond external biases
were gradually removed. After the docking procedure,
the ligand/receptor complexes were energy minimized
˚
until reaching an RMS of 0.05kcal/mol/A.
The puckering coordinates P, hm, P2, Q, and H were cal-
culated by means of the formulae:
8. Kim, H. S.; Ohno, M.; Xu, B.; Kim, H. O.; Choi, Y.; Ji, X.
D.; Maddileti, S.; Marquez, V. E.; Harden, T. K.;
Jacobson, K. A. J. Med. Chem. 2003, 46, 4974.
9. Waldo, G. L.; Corbitt, J.; Boyer, J. L.; Ravi, G.; Kim, H.
S.; Ji, X.-D.; Lacy, J.; Jacobson, K. A.; Harden, T. K.
Mol. Pharmacol. 2002, 62, 1249.
P ¼ atanf½ðm4 þ m1Þ À ðm3 þ m0ފ=½2m2ðsin p=5
þ sin 2p=5ފg
10. Obika, S.; Nanbu, D.; Fari, Y.; Morio, K.-I.; In, Y.;
Ishida, T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735.
11. (a) Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen,
J. P. J. Am. Chem. Soc. 2002, 124, 5974; (b) Petersen, M.;
Wengel, J. Trends Biotechnol. 2003, 21, 74.
hm ¼ m2= cos P
h ꢀ
ꢁ.ꢀ
ꢁi
X
X
5
5
P2 ¼ atan À
mj sin 2pj=3
mj cos 2pj=3
j¼0
j¼0
12. Koshkin, A. A.; Fensholdt, J.; Pfundheller, H. M.;
Lomholt, C. J. Org. Chem. 2001, 66, 8504.
13. Hodgson, D. M.; Gibbs, A. R.; Drew, M. G. B. J. Chem.
Soc., Perkin Trans. 1 1999, 24, 3579.
14. (a) Alex, G.; Kruger, A. W.; Meyers, A. I. Tetrahedron
Lett. 2001, 42, 4305; (b) Arrington, M. P.; Meyers, A. I.
Chem. Commun. 1999, 15, 1371.
ꢂ
2
hꢀ
ꢁ.
i
X
5
Q ¼
mj cos 2pj=3 ð3 cos P2Þ
j¼0
ꢃ
1=2
hꢀ
ꢁ
i
X
2
5
15. Obike, S.; Morio, K.-I.; Nanbu, D.; Hari, Y.; Itoh, H.;
Imanishi, T. Tetrahedron 2002, 58, 3039.
þ
mj cos pj =6
j¼0
16. Kim, H. S.; Jacobson, K. A. Org. Lett. 2003, 5, 1665.
17. Ravi, R. G.; Kim, H. S.; Servos, J.; Zimmermann, H.; Lee,
K.; Maddileti, S.; Boyer, J. L.; Harden, T. K.; Jacobson,
K. A. J. Med. Chem. 2002, 45, 2090.
18. Scho¨ning, K.; Scholz, P.; Guntha, S.; Wu, X.; Krishna-
murthy, R.; Eschenmoser, A. Science 2000, 290, 1347.
19. Scho¨ning, K.; Scholz, P.; Wu, X.; Guntha, S.; Delgado,
G.; Krishnamurthy, R.; Eschenmoser, A. Helv. Chim.
Acta 2002, 85, 4111.
20. Costanzi, S.; Mamedova, L.; Gao, Z. G.; Jacobson, K. A.
J. Med. Chem. (in press).
21. (a) Nair, V.; Fasbender, A. J. Tetrahedron 1993, 49,
2169–2184; (b) Nair, V.; Richardson, S. G. J. Org. Chem.
1980, 45, 3969; (c) Nair, V.; Richardson, S. G. Synthesis
1982, 670.
n ꢀ
ꢁ.
X
X
5
H ¼ atan 2
mj cos 2pj=3
j¼0
5
h
ꢀ
ꢁio
cos P2
mj cos pj
j¼0
which we derived from the works of Altona and Sunda-
ralingham1 and of Haasnoot.24 The pseudosugar endo-
cyclic torsion angles mj are numbered according to the
IUPAC-IUB recommendations for the conformation
of polysaccharide chains (www.chem.qmul.ac.uk/iupac/
misc/psac.html). All the angles are expressed in radians.
Acknowledgements
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MO is on sabbatical from Toray Industries (Kamakura,
Japan) and thanks them for financial support. Mass

5630
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