Design and synthesis of 4″,6″-unsaturated cyclic ADP-carbocyclic ribose as a Ca2+-mobilizing agent

Article abstract of DOI:10.1016/j.tetlet.2008.04.100

4″,6″-Didehydro-cADPcR (3), an unsaturated carbocyclic ribose analog of a Ca²⁺-mobilizing second messenger cyclic ADP-ribose (cADPR), was designed and successfully synthesized using a key intramolecular condensation reaction forming the 18-memb

Full text of DOI:10.1016/j.tetlet.2008.04.100

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3976–3979
Design and synthesis of 400,600-unsaturated cyclic ADP-carbocyclic
ribose as a Ca2+-mobilizing agent
Takashi Kudoh a, Karin Weber b, Andreas H. Guse b, Barry V. L. Potter c, Minako Hashii d,
Haruhiro Higashida d, Mitsuhiro Arisawa a, Akira Matsuda a, Satoshi Shuto a,*
a Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
b The Calcium Signalling Group, University Medical Center Hamburg-Eppendorf, Center of Experimental Medicine,
Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistr. 52, 20246 Hamburg, Germany
c Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
d Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan
Received 1 April 2008; revised 14 April 2008; accepted 17 April 2008
Available online 22 April 2008
Abstract
400,600-Didehydro-cADPcR (3), an unsaturated carbocyclic ribose analog of a Ca2+-mobilizing second messenger cyclic ADP-ribose
(cADPR), was designed and successfully synthesized using a key intramolecular condensation reaction forming the 18-membered pyro-
phosphate ring structure with a S-phenyl phosphorothioate-type substrate. Biological evaluation showed that 400,600-didehydro-cADPcR
is a potent Ca2+-mobilizing agent in T cells.
Ó 2008 Elsevier Ltd. All rights reserved.
NH2
Cyclic ADP-ribose (cADPR, 1, Fig. 1) is a general medi-
ator involved in Ca2+ signaling,1 and synthesis of its ana-
logs has been extensively investigated, since these can be
used in investigating the mechanism of cADPR-mediated
Ca2+ signaling pathways and are also expected to be leads
for the development of potential drug candidates.2–5 In the
synthesis of cADPR and its analogs, construction of
the large 18-membered pyrophosphate ring structure is
the key step, and we have developed an efficient method
for forming the 18-membered ring by activating the
S-phenyl phosphorothioate-type substrates with iodine or
AgNO3.4
cADPR is in a zwitterionic form with a positive charge
around the N(1)–C(6)–N6 moiety, making the molecule
unstable, since the charged adenine moiety attached to
the anomeric carbon of the N1-linked ribose can be an effi-
cient leaving group to cleave the N1-ribosyl linkage. We
previously designed and synthesized cyclic ADP-carbo-
HO
NH2
HO
2"
6
1"
6"
N
N
HO
HO
N
N
1'
N
N1
3"
8
4"
X
2
9
N
N
O
5"
O
P
O
O
O
P
O
O
2'
O
4'
OH
OH
O
O
O
O
O
O
3'
P
P
5'
OH
OH
O
O
1: X = O (cADPR)
2: X = CH2 (cADPcR)
3: 4",6"-didehydro-cADPcR
Fig. 1. cADPR (1), cADPcR (2), and 400,600-didehydro-cADPcR (3).
cyclic-ribose (cADPcR, 2) as a stable mimic of cADPR,
in which the oxygen atom in the N1-ribose ring of cADPR
is replaced by a methylene group. cADPcR was shown to
be actually resistant to both enzymatic and chemical
hydrolysis, and it, like cADPR, effectively mobilizes intra-
cellular Ca2+ in sea urchin eggs.4c
Our attention has been focused on developing cell-type
selective cADPR analogs, which can be useful as biological
tools and/or potential drug leads. Thus, we found that
although cADPcR and its 30-deoxy derivative are more
*
Corresponding author. Tel./fax: +81 11 706 3769.
E-mail address: shu@pharm.hokudai.ac.jp (S. Shuto).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.100

T. Kudoh et al. / Tetrahedron Letters 49 (2008) 3976–3979
3977
NH2
N
NH2
N
be constructed by condensation between the optically active
cyclopentenyl amine 6 with a known imidazole nucleoside
7.4c
N
N
N
N
HO
N
N
HO
The chiral cyclopentenyl amine 6 was synthesized from a
cyclopentenone 8, which was prepared by the method
reported by Jeong and co-workers10 (Scheme 2). 1,2-
Reduction of the enone system of 8 with NaBH4/CeCl3
in MeOH stereoselectively gave the allylic a-alcohol 9, of
which mesylation and subsequent treatment with LiN3 in
HMPA/DMSO11 afforded the b-azide 10. Removal of the
TBDPS group of 10 followed by reduction of the azido
group with Ph3P in aqueous THF gave the desired cyclo-
pentenyl amine 6.
The target 400,600-didehydro-cADPcR (3) was success-
fully synthesized from the chiral cyclopentenyl amine 6 as
shown in Scheme 3. The 400,600-didehydro-N1-carbocyclic-
ribosyl adenosine derivative 5 was obtained in 58% yield
by the treatment of a mixture of the amine 6 and the imid-
azole nucleoside 74c with K2CO3 in MeOH at room tem-
perature. The 500-hydroxy group of 5 was protected with
a dimethoxytrityl (DMTr) group, and the 50-O-TBS group
of the product was removed with TBAF to give 11. Treat-
ment of 11 with an S,S0-diphenyl phosphorodithioate
(PSS)/2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl)/
pyridine system12 gave the 50-bis(phenylthio)phosphate
12. After removal of 50-O-DMTr group of 12 with aqueous
AcOH, a phosphoryl group was introduced at the resulting
500-primary hydroxyl by Yoshikawa’s method with POCl3/
(EtO)3PO,13 followed by treatment of the product with
H3PO2 and Et3N14 in the presence of N-methylmaleimide
(NMM) in pyridine,4c to afford the 500-O-phosphoryl S-
phenyl phosphorothioate 4, the substrate for the key intra-
molecular condensation.
HO OH
HO OH
neplanocin A
aristeromycin
Fig. 2. Naturally occurring carbocyclic adenine nucleosides.
potent than cADPR in neuronal cells,7 they are almost
inactive in T cells, while cADPR works effectively as a
Ca2+-mobilizing second messenger both in T cell and in
neuronal cell systems.5a–c These very important findings
indicate that the target proteins and/or the mechanism of
action of cADPR in T cells and neuronal cells are
different.5d
Carbocyclic nucleosides are known as effective mimics
of natural nucleosides.6 An antibiotic aristeromycin (Fig.
2) is a naturally occurring mimic of adenosine,7 in which
the ribose of adenosine is replaced by a carbocyclic-ribose,
and has antiviral and antitumor activities, due to its com-
petitive inhibitory effect to adenosine against S-adenosyl-
homocysteine (AdoHcy) hydrolase.8 Another nucleosidic
antibiotic neplanocin A (Fig. 2), the structure of which
corresponds to the 40,60-unsaturated derivative of aristero-
mycin, has significant antiviral and also antitumor effects,
which clearly surpass those of aristeromycin.8,9 Therefore,
since an unsaturated carbocyclic-ribose may mimic the
ribose more precisely than the corresponding saturated
congener, we newly designed the unsaturated carbocyclic
ribose analog of cADPR, that is, 400,600-didehydro-cADPcR
(3). We describe here synthesis and biological effects of 3 to
show that it is significantly active in T cells.
We planned to synthesize the target 400,600-didehydro-
cADPcR (3) by a route, as summarized in Scheme 1, in
which intramolecular condensation reaction with a S-phe-
nyl phosphorothioate-type substrate 4 forming the 18-mem-
bered pyrophosphate structure was employed. It was key
point whether the condensation could occur, since the
18-membered pyrophosphate structure containing an
unsaturated carbocyclic ribose seemed to be strained
compared with the corresponding saturated congener.
Substrate 4 could be converted from the N1-unsaturated-
carbocyclic-ribosyl adenosine derivative 5, which would
O
OH
NaBH4, CeCl3
MeOH, 0 °C
TBDPSO
TBDPSO
O
O
O
O
8
9
1. MsCl, Et3N
CH2Cl2, 0 °C
2. LiN3
N3
1. TBAF, THF
2. Ph3P, aq. THF
TBDPSO
HMPA/DMSO
O
O
6
10
89%
90% from 8
Scheme 2.
NH2
HO
O
O
O
O
O
O
NH
NH2
N
6
N
N
+
N
N
N
N
O
O
PhS P
O
CN
OH
N
N
TBSO
HO P
O
O
O
N
O
O
3
TBSO
N
CH(OMe)
O
O
O
O
O
4
5
O
O
7
Scheme 1.

3978
T. Kudoh et al. / Tetrahedron Letters 49 (2008) 3976–3979
O
O
O
O
O
O
NH
NH
NH
N
N
N
N
N
N
N
N
N
O
ODMTr
d
OH
b
OH
6
+
7
TBSO
RO
N
(PhS)2P O
N
N
O
O
O
a
O
O
O
O
O
O
13
5
c
11: R = H
12: R = PO(SPh)2
OH
O
O
NH2
O
O
NH2
HO
N
NH2
N
N
N
N
N
N
N
O
N
N
O
O
N
O
O
O
P
O
P
O
P
O
P
O
P
PhS
P
O
N
HO
O
O
O
O
O
g
e
f
O
O
O
OH
O
O
O
O
O
O
HO
O
O
4
14
3
Scheme 3. Reagents and conditions: (a) K2CO3, MeOH, rt, 58%; (b) (1) DMTrCl, pyridine, rt; (2) TBAF, THF, AcOH, rt, quant; (c) PSS, TPSCI,
˚
pyridine, rt, 74%; (d) aq 60% AcOH, rt, 80%; (e) (1) POCl3, (EtO)3PO, 0 °C; (2) H3PO2, Et3N, NMM, pyridine, 0 °C to rt, 28%; (f) AgNO3, MS 3 A, Et3N,
pyridine, rt, 65%; (g) aq HCO2H, rt; (2) aq NH3, 86%.
When a solution of 4 in pyridine was added slowly to a
only a weak agonist for Ca2+ release in permeabilized
Jurkat T cells, 400,600-didehydro-cADPcR (3) is much more
potent than cADPcR (2) (Fig. 3). At 100 lM 400,600-didehy-
dro-cADPcR induced a robust response while almost no
effect of cADPcR was obtained. Interestingly, at 500 lM
400,600-didehydro-cADPcR a significantly higher amplitude
was observed as compared to the natural second messenger
cADPR. It is an important finding that, although the
saturated carbocyclic analog cADPcR (2) is inactive, the
corresponding 400,600-unsaturated analog 3 has a significant
Ca2+-mobilizing potency in T cells.
In conclusion, 400,600-didehydro-cADPcR (3) designed as
a novel stable mimic of cADPR was successfully synthe-
sized using the key intramolecular condensation reaction
with the S-phenyl phosphorothioate-type substrate.
Evaluation of Ca2+-mobilizing potency using permeabi-
lized Jurkat T-lymphocytes showed that 400,600-didehydro-
cADPcR is significantly active in T cells and therefore it
may be useful as a biological tool and/or drug lead because
of the biologically and chemically stable carbocyclic
structure.16
mixture of a large excess of AgNO3 and Et3N in the pres-
4,15
˚
ence of MS 3 A in pyridine at room temperature,
the
desired cyclization product 14 was successfully obtained
in 65% yield, in spite of the strained 18-membered struc-
ture. Finally, removal of the isopropylidene groups of 14
with aqueous HCO2H followed by treatment with aqueous
ammonia furnished 400,600-didehydro-cADPcR (3).17
The Ca2+-mobilizing effect of 400,600-didehydro-cADPcR
(3) was evaluated in permeabilized Jurkat T cells. cADPR
(1) released Ca2+ from intracellular stores as reported pre-
viously.3d While cADPcR (2) as compared to cADPR (1) is
References and notes
1. Cyclic ADP-ribose ADP-Ribose and NAADP: Structures, Metabo-
lism and Functions; Lee, H. C., Ed.; Kluwer Academic: Dordrecht,
2002.
2. Reviews on cADPR analogs: (a) Zhang, F.-J.; Gu, Q.-M.; Sih, C. J.
Bioorg. Med. Chem. 1999, 7, 653–664; (b) Shuto, S.; Matsuda, A.
Curr. Med. Chem. 2004, 11, 827–845; (c) Potter, B. V. L.; Walseth, T.
F. Curr. Mol. Med. 2004, 4, 303–311.
3. For examples: (a) Bailey, V. C.; Fortt, S. M.; Summerhill, R. J.;
Galione, A.; Potter, B. V. L. FEBS Lett. 1996, 379, 227–230; (b)
Galeone, A.; Mayol, L.; Oliviero, G.; Piccialli, G.; Varra, M.
Tetrahedron 2002, 58, 363–368; (c) Xu, J.; Yang, Z.; Dammermann,
W.; Zhang, L.; Guse, A. H.; Zhang, L.-H. J. Med. Chem. 2006, 5501–
Fig. 3. Effects of cADPR (1), cADPcR (2), and 400,600-didehydro-cADPcR
(3) in permeabilized Jurkat T cells. Jurkat T cells were permeabilized by
saponin. Ca2+ stores were refilled by addition of ATP and ATP-
regenerating system. [Ca2+] was determined fluorimetrically using fura2/
free acid. Compounds were added at the concentrations indicated. Data
are differences between the Ca2+ concentration upon addition and before
addition of compound. Mean SEM (n = 3–6).

T. Kudoh et al. / Tetrahedron Letters 49 (2008) 3976–3979
3979
5512; (d) Wagner, G. K.; Guse, A. H.; Potter, B. V. L. J. Org. Chem.
2005, 70, 4810–4819.
N.; Niizuma, S.; Kim, S.-H. ; Wataya, Y.; Matsuda, A. J. Med. Chem.
2002, 45, 748–751 and references cited therein.
4. (a) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda, A. J. Org.
Chem. 1998, 63, 1986–1994; (b) Fukuoka, M.; Shuto, S.; Minakawa,
N.; Ueno, Y.; Matsuda, A. J. Org. Chem. 2000, 65, 5238–5248; (c)
Shuto, S.; Fukuoka, M.; Manikowsky, M.; Ueno, T.; Nakano, T.;
Kuroda, R.; Kuroda, H.; Matsuda, A. J. Am. Chem. Soc. 2001, 123,
8750–8759.
5. (a) Guse, A. H.; Cakir-Kiefer, C.; Fukuoka, M.; Shuto, S.; Weber, K.;
Matsuda, A.; Mayer, G. W.; Oppenheimer, N.; Schuber, F.; Potter, B.
V. L. Biochemistry 2002, 41, 6744–6751; (b) Shuto, S.; Fukuoka, M.;
Kudoh, T.; Garnham, C.; Galione, A.; Potter, B. V. L.; Matsuda, A.
J. Med. Chem. 2003, 46, 4741–4749; (c) Hashii, M.; Shuto, S.;
Fukuoka, M.; Kudoh, T.; Matsuda, A.; Higashida, H. J. Neurochem.
2005, 94, 316–323; (d) Kudoh, T.; Fukuoka, M.; Ichikawa, S.;
Murayama, T.; Ogawa, Y.; Hashii, M.; Higashida, H.; Kunerth, S.;
Weber, K.; Guse, A. H.; Potter, B. V. L.; Matsuda, A.; Shuto, S. J.
Am. Chem. Soc. 2005, 127, 8846–8855; (e) Kudoh, T.; Murayama, T.;
Ogawa, Y.; Matsuda, A.; Shuto, S. Nucleosides, Nucleotides, Nucleic
Acids 2006, 25, 583–599; (f) Kudoh, T.; Murayama, T.; Matsuda, A.;
Shuto, S. Bioorg. Med. Chem. 2007, 15, 3032–3040.
10. Choi, W. J.; Moon, H. R.; Kim, H. O.; Too, B. N.; Lee, J. A.; Shin,
D. H.; Jeong, L. S. J. Org. Chem. 2004, 69, 2634–2636.
11. Marquez, V. E.; Lim, M.-I.; Tseng, C. K.-H.; Markovac, A.; Priest,
M. A.; Khan, M. S.; Kaskar, B. J. Org. Chem. 1988, 53, 5709–5714.
12. (a) Sekine, M.; Nishiyama, S.; Kamimura, T.; Osaki, Y.; Hata, T.
Bull. Chem. Soc. Jpn. 1985, 58, 850–860; (b) Sekine, M.; Hata, T. Cur.
Org. Chem. 1993, 3, 25–66.
13. Yoshikawa, M.; Kato, T.; Takenishi, T. Bull. Chem. Soc. Jpn. 1969,
42, 3505–3508.
14. Hata, T.; Kamimura, T.; Urakami, K.; Kohno, K.; Sekine, M.;
Kumagai, I.; Shinozaki, K.; Miura, K. Chem. Lett. 1987, 117–120.
15. The intermolecular condensation between a S-phenyl phosphorothio-
ate and a phosphomonoester is effectively promoted by I2 or AgNO3
to give the corresponding pyrophosphate compound: see Ref. 12.
16. In NG 108-15 neuronal cells,3 did not show apparent Ca2+
-
mobilizing activity, which suggests that 3 is selectively active in T
cells.
17. Physical data of
3
(triethylammonium salt): 1H NMR (D2O,
500 MHz, K+ salt) d 8.63 (s, 1H), 8.49 (s, 1H), 6.31 (m, 1H), 6.07
(d, 1H, J = 5.7 Hz), 5.44 (m, 1H), 5.29 (m, 1H), 4.91 (m, 2H), 4.67 (m,
1H), 4.62 (m, 2H), 4.34 (m, 2H), 4.07 (m, 1H); 13C NMR (D2O,
125 MHz) d 153.1, 151.6, 147.4, 146.0, 143.3, 124.4, 121.0, 91.3, 85.0,
74.1, 73.6, 73.2, 71.0, 69.0, 64.7, 61.7; 31P NMR (D2O, 202 MHz) d
ꢀ10.0 (d, J = 15.3 Hz), ꢀ10.6 (d, J = 15.3 Hz); HRMS (FAB,
negative) calcd for C16H20N5O12P2 536.0589 [(MꢀH)ꢀ], found
536.0577; UV (H2O) kmax 260 nm.
6. Agrofoglio, L. A.; Challand, S. R. Acyclic, Carbocyclic and L-
Nucleosides; . Kluwer Academic: Dordrecht, 1998.
7. Kusaka, T.; Yamamoto, H.; Shibata, M.; Muroi, M.; Kishi, T. J.
Antibiot. 1968, 21, 255–263.
8. Wolfe, M. S.; Borchardt, R. T. J. Med. Chem. 1991, 34, 1521–1530.
9. (a) Yaginuma, S.; Muto, N.; Tsujino, M.; Sudate, Y.; Hayashi, M.;
Otani, M. . J. Antibiot. 1981, 34, 359–366; (b) Shuto, S.; Minakawa,