Intracellular Ca2+-mobilizing adenine nucleotides. Synthesis and biological activity of cyclic ADP-carbocyclic-ribose and C-glycosidic analog of adenophostin A

Article abstract of DOI:10.1081/NCN-100002320

We designed novel Ca²⁺-mobilizing purine nucleotides, cyclic ADP-carbocyclicribose 4, and its inosine congener 5, and C-glycosidic adenophostin A 6. In the synthesis of cADPR analogs, the intramolecular condensation to form the pyrophosphate linkage should be the key step. We developed an efficient method for forming such an intramolecular pyrophosphate linkage by the activation of the phenylthiophosphate group with I₂ or AgNO₃. Using this method, we achieved to synthesize the target compounds 4 and 5. The synthesis of C-glycosidic analog 6 of adenophostin A was achieved using a temporary silicon-tethered radical coupling reaction for constructing (3′α, 1″α)-C-glycosidic structure as the key step.

Full text of DOI:10.1081/NCN-100002320

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INTRACELLULAR Ca2+-MOBILIZING ADENINE
NUCLEOTIDES. SYNTHESIS AND BIOLOGICAL ACTIVITY
OF CYCLIC ADP-CARBOCYCLIC-RIBOSE AND C-
GLYCOSIDIC ANALOG OF ADENOPHOSTIN A
Satoshi Shuto a , Masayoshi Fukuoka a , Hiroshi Abe a & Akira Matsuda a
a Graduate School of Pharmaceutical Sciences, Hokkaido University , Kita-12, Nishi-6,
Kita-ku, Sapporo, 060-0812, Japan
Published online: 07 Feb 2007.
To cite this article: Satoshi Shuto , Masayoshi Fukuoka , Hiroshi Abe & Akira Matsuda (2001) INTRACELLULAR Ca2+-
MOBILIZING ADENINE NUCLEOTIDES. SYNTHESIS AND BIOLOGICAL ACTIVITY OF CYCLIC ADP-CARBOCYCLIC-RIBOSE AND C-
GLYCOSIDIC ANALOG OF ADENOPHOSTIN A, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 461-470, DOI: 10.1081/
NCN-100002320
To link to this article: http://dx.doi.org/10.1081/NCN-100002320
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 461–470 (2001)
INTRACELLULAR Ca2+-MOBILIZING
ADENINE NUCLEOTIDES. SYNTHESIS AND
BIOLOGICAL ACTIVITY OF CYCLIC
ADP-CARBOCYCLIC-RIBOSE AND
C-GLYCOSIDIC ANALOG OF ADENOPHOSTIN A
Satoshi Shuto,∗ Masayoshi Fukuoka, Hiroshi Abe,
and Akira Matsuda
Graduate School of Pharmaceutical Sciences, Hokkaido University,
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
ABSTRACT
We designed novel Ca2+-mobilizing purine nucleotides, cyclic ADP-carbocycl-
icribose 4, and its inosine congener 5, and C-glycosidic adenophostin A 6.
In the synthesis of cADPR analogs, the intramolecular condensation to form
the pyrophosphate linkage should be the key step. We developed an efficient
method for forming such an intramolecular pyrophosphate linkage by the acti-
vation of the phenylthiophosphate group with I2 or AgNO3. Using this method,
we achieved to synthesize the target compounds 4 and 5. The synthesis of
C-glycosidic analog 6 of adenophostin A was achieved using a temporary
silicon-tethered radical coupling reaction for constructing (3ꢀα, 1ꢀꢀα)-C-glycosi-
dic structure as the key step.
An elevation in intracellular levels of Ca2+ is known to be a key signaling
event coupling cell activation by extracellular stimuli to a characteristic physi-
ological response. These intracellular Ca2+ releases are mediated by two sec-
ond messengers; one is myo-inositol trisphosphate (IP3, 1) (1) and the other is
cyclic ADP-ribose (cADPR, 2) (2). Adenophostin A (3), a 3ꢀ-O-glucosyladenosine
∗ Corresponding author. E-mail: shu@pharm.hokudai.ac.jp
461
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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SHUTO ET AL.
Figure 1.
trisphosphate, recently isolated from Penicillium brevicompactum, was identified
as the strongest IP3 receptor ligand yet known (3). We designed and synthesized
novel Ca2+-mobilizing purine nucleotides, cyclic ADP-carbocyclic-ribose 4, and
its inosine congener 5, and C-glycosidic adenophostin A 6, the structures of which
are shown in Figure 1, based on the structural feature of cADPR and adenophostin
A, since specific ligands for the receptors of the Ca2+-mobilizing second mes-
sengers are very useful for proving the mechanism of Ca2+-mobilizing signaling
pathways.
THE STABLE MIMICS OF CADPR
Design of cADPcR and cIDPcR
cADPR (2) is a eighteen-membered cyclic pyrophosphate of N-1-ribosyl-
adenosine (2b), and its Ca2+-mobilizing activity was found by Lee in 1987 (2a). In
cells, cADPR is synthesized from NAD+ by ADP-ribosylcyclase and acts as a sec-
ond messenger; it is hydrolyzed promptly by cADPR hydrolase to give ADP-ribose

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463
and inactivated under physiological conditions (2c,d). cADPR is also known to be
readily hydrolyzed non-enzymatically at the unstable N-1-glycosidic linkage of
its adenine moiety to give ADP-ribose, even in neutral aqueous solution (2e). Al-
though further intensive studies of cADPR are needed because of its biological
importance, this biological as well as chemical instability of cADPR limits stud-
ies of its physiological role, at least to some extent. Therefore, stable analogs of
cADPR that exhibit a Ca2+-mobilizing activity in cells similar to that of cADPR
are urgently required.
WedesignedcyclicADP-carbocyclic-ribose(cADPcR, 4)andthecorrespond-
ing inosine congener, cIDP-carbocyclic-ribose (cIDPcR, 5) as stable mimics of
cADPR, in which an oxygen atom in the ribose ring of cADPR is replaced by a
methylene group (4). The mimics 4 and 5 should be resistant to both enzymatic
and chemical hydrolysis, since they lack the unstable N-1-glycosidic linkage of
cADPR. These analogs preserve all of the functional groups of cADPR, except
for this ring oxygen, and should have a conformation similar to that of cADPR.
Therefore, we expected that these analogs would effectively mobilize intracellular
Ca2+, like cADPR, so that they could be used as pharmacological tools for studying
the cADPR-modulated Ca2+ signaling pathways.
The First Synthesis of cIDPcR
We first tried to synthesize 5, in which the intramolecular condensation of
bisphosphate 7 to form the pyrophosphate linkage was a key step, but was diffi-
cult (Fig. 2) (4a,b,d). The difficulty of forming such an intramolecular pyrophos-
phate linkage has also been experienced by other groups (5). Sih reported that
Figure 2.

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SHUTO ET AL.
Figure 3.
condensation between the two phosphate groups of N-1-phosphoribosyl-AMP (9)
with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was
unsuccessful (5a). Later, ring-closure of bisphosphate 10 through the formation
of a pyrophosphate linkage was examined by Fortt and Potter, but they also failed
(5b).
We presumed that the conformation of the molecule determines whether or
not intramolecular condensation occurs. The syn-anti conformation is an impor-
tant determinant of the three-dimensional structure of nucleosides (6). As shown
in Figure 3, in the syn-conformation, the two phosphate moieties are close to each
other, however, the anti-conformer is predominant over the syn-conformers in usual
nucleosides (6). This may be why these intramolecular condensations were unsuc-
cessful.
It is well known that introducing a bulky substituent into the 8-position of
purine nucleosides restricts the conformation in a syn-form through steric repulsion
(6). We therefore designed the 8-bromo-substrate 11. The 8-bromo group would
restrict the conformation to the syn-form to facilitate the desired intramolecular
condensation reaction.
An optically active carbocyclic trifrate 12 was prepared from cyclopenta-
diene by the previously reported method (4a). SN2 reaction between the carbo-
cyclic trifrate 12 and this sugar-protected 8-bromoinosine 13 was performed with
potassium bicarbonate in DME to give the desired N-1-substituted 14 in 44% yield,
along with the corresponding O6-substituted isomer in 9% yield. After removal of
thesilylprotectinggroups, thedesiredbisphosphate11wassuccessfullyobtainedby
a phosphoramidite method with (2-cyanoethoxy)(N,N-diisopropylamino)chlorop-
hosphine.
The intramolecular condensation reaction was investigated with EDC in
N-methylpyrrolidone (NMP). Whenthebisphosphatewas heatedwithEDCinNMP
at 50◦C, the desired cyclization product 15 was obtained in 23% yield. The
8-bromo group was removed reductively by catalytic hydrogenation. Finally, acidic
treatment gave the target cIDPcR (5), as shown in Scheme 1 (4a).

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465
Scheme 1.
The Improved Synthesis of cIDPcR.
Although, as described above, we achieved synthesis of cIDPcR, which was
the first total synthesis of a cADPR-related compound, the overall yield was very
low. Therefore, we need to develop more efficient general methods for preparing
various cADPR analogs. Especially, the development of both an efficient con-
densation to form the intramolecular pyrophosphate linkage and a straightforward
construction of the N-1-carbocyclic structure were needed.
The improved synthetic plan is summarized in Scheme 2. Formation of the
intramolecular pyrophosphate linkage is investigated by treating a 5ꢀ-phenylthio-
phosphate 16 as a substrate with silver nitrate or iodine (7). The N-1-carbocyclic-
ribosyl-structure is constructed from N-1-(2,4-dinitrophenyl)inosine derivative 17

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SHUTO ET AL.
Scheme 2.
and optically active carbocyclic amine 18, which is readily prepared from a com-
mercially available bicyclic lactam 20 (8).
This N-1-dinitrophenylinosine derivative 17, prepared according to Piccialli’s
method(9), washeatedwithanexcessofthecarbocyclicamine18at50◦CinDMFto
give the ring-cleaved product 21. After the 5ꢀꢀ-hydroxyl protection and bromination
at the 2-position, the resulting 23 was heated with potassium carbonate at 50◦C
in DMF to give the desired ring-closed 24 in high yield. This method is clearly
superior to our previous one using an SN2-type reaction. The compound 24 was
converted into the thiophenylphosphate 16 in five steps.
The intramolecular condensation reaction of the substrate 16 was investigated
under various conditions. We found that the desired cyclization product 15 was
quantitatively obtained when 16 was treated with I2 in the presence of molecular
sieves 3A (MS 3A) in pyridine at room temperature (4c).
Scheme 3.

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467
Wenextexaminedtheuseofthe8-unsubstitutedsubstrate25intheintramolec-
ular condensation reaction to investigate whether or not the 8-bromo substituent
at the purine moiety actually facilitates intramolecular condensation. Thus, this
8-unsubstituted substrate 25, prepared from 21 in several steps, was treated under
the best condensation conditions described above to give the desired cyclization
product 8 in 81%-isolated yield (4e).
Scheme 4.
These results suggest that the 8-bromo group facilitates the intramolecular
condensation, at least to some extent, due to conformational restriction of the sub-
strate in a syn-form.
In the similar way, the corresponding adenosine congener, cADPcR (4), was
successfully synthesized (10).
C-GLUCOSIDE TRISPHOSPHATES AS IP3 RECEPTOR LIGANDS
Design of C-Glycosidic Analog of Adenophostin A
Adenophostin A (3), the strongest IP3 receptor agonist known so far, is a
trisphosphate of 3ꢀ-O-α-glucosyladenosine. These interesting biological and struc-
tural features have prompted us (11) as well as other groups (12) to perform syn-
thetic studies of novel IP3 receptor ligands. We synthesized, for an example, the
two compounds shown in Figure 4; one is an adenophostin analog 26 (11a-c) lack-
ing the adenine moiety, and the other is a C-glucoside trisphosphate 27 (11g), a
Figure 4.

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SHUTO ET AL.
structurally simplified analog of adenophostin A. These analogs showed a potent
binding affinity for the receptor with IC50 values of 25 and 36 nmol, respectively,
which are comparable to IP3 (11b, c). However, their activities are weaker than that
of adenophostin A.
These results suggested that the α-D-glucopyranose structure is a good
bioisostere of the myo-inositol backbone of IP3; that the adenine moiety signif-
icantly enhances activity; and also that the O-glucosidic bond can be replaced
with a C-glucosidic bond. Based on these results and considerations, we chose the
C-glycosidic analog 6 of adenophostin A as a next target.
Synthesis of C-Glycosidic Analog of Adenophostin A
The synthesis is summarized in Scheme 5. The construction of the (3ꢀα, 1ꢀꢀα)-
C-glycosidic structure was considered to be the most important step. We planned
to construct it by a temporary silicon-tethered radical coupling reaction.
Phenyl seleno-β-D-glucopyranoside 26 and 3-exomethylene-ribose derivative
27, prepared from D-glucose and D-xylose, respectively, were connected with a
dimethylsilyl tether to give the radical coupling reaction substrate 28. Thus, the
Scheme 5.

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469
substrate 28 was successively treated with Bu3SnH/AIBN in benzene and TBAF
in THF to give the coupling product 30 with the desired (3α,1ꢀα)-configuration as
the major product.
The target compounds 6 was synthesized from 30 via the introduction of
adenine base by the Vorbru¨ggen method and phosphorylation of the hydroxyls by
the phosphoramidite method (11d,f).
ACKNOWLEDGMENT
We are grateful to Ms. T. Iizawa and Drs. M. Takahashi and H. Hotoda,
Sankyou Co. Ltd, for the biological evaluation, and to Dr. A. M. Riley and Prof. B.
V. L. Potter, University of Bath, for useful discussions.
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