Chemical synthesis and calcium release activity of N1-ether strand substituted cADPR mimic

Article abstract of DOI:10.1016/S0960-894X(02)00033-1

8-Chloro cyclic inosine 5′-diphosphate ethoxymethyl ether 3 was synthesized by means of chemical method from protected inosine via phenylthio-type biphosphate substrate. The detection of Ca²⁺ release activity shows that 3 is a potent agonist of cADPR and has activity in intact Hela cells.

Full text of DOI:10.1016/S0960-894X(02)00033-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 887–889
Chemical Synthesis and Calcium Release Activity of
N1-Ether Strand Substituted cADPR Mimic
Li-Jun Huang,a Yong-Yuan Zhao,b Lan Yuan,c Ji-Mei Mina and Li-He Zhanga,*
aNational Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, PR China
bDepartment of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
cHealth and Drug Analytical Center, Peking University, Beijing 100083, PR China
Received 9 November 2001; accepted 8 January 2002
Abstract—8-Chloro cyclic inosine 50-diphosphate ethoxymethyl ether 3 was synthesized by means of chemical method from pro-
tected inosine via phenylthio-type biphosphate substrate. The detection of Ca2+ release activity shows that 3 is a potent agonist of
cADPR and has activity in intact Hela cells. # 2002 Elsevier Science Ltd. All rights reserved.
Cyclic ADP-ribose (cADPR, 1), a metabolite of NAD+,
is of great interest because of its significant biological
importance as a physiological modulator of ryanodine
receptor.1 Accordingly, many cADPR analogues have
been synthesized from NAD+ analogues by the ADP-
ribosyl cyclase (ADPR-cyclase) and used for the studies
on the mechanism of cADPR mediated Ca2+ release
pathway.2 The most structural modifications of cADPR
focused on the adenosine moiety and pyrophosphate
unit.3 Due to the inherent substrate specificity of
ADPR-cyclase, differentgroups have worked on hte
chemical synthesis of cADPR analogues (Fig. 1).4,5
synthesis of the N1-ribose ring modification, that is an
N1-acyclic ether chain analogue, and it was found that
the N1-acyclic mimic 3 showed some Ca2+ release
activity in Hela cells.
According to the synthetic strategy of cADPR, N-1
substitution and intramolecular cyclization are the key
steps. To date, the most conventional preparations of
acyclic nucleoside involved an N-9 substitution of purine
with 2-chloromethoxyethyl acetate in the presence of a
base such as K2CO3 or NaH as well as in situ substitu-
tion with trimethylsilyl iodide and 1,3-dioxolane.13,14
Unfortunately, the N-1 substituted derivative 5 was not
obtained by the described methods due to the weak
nucleophilicity of this position. Interestingly, the reac-
tion of protected inosine 46a with 2-chloromethoxyethyl
The only effort related to the N1-ribosyl-modified cADPR
analogue, a carbocyclic mimic of cADPR 2a and 2b,
was reported by Matsuda’s group.6 The biological data
of 2b showed that 2b caused a significantrelease of
Ca2+ in sea urchin eggs via micro-injection. Among the
analogues of cADPR, none of the 8-substituted analo-
gues synthesized has Ca2+ release activity in sea urchin
egg homogenates, but most of them block cADPR from
releasing Ca2+.7 On the contrary, cyclic aristeromycin-
diphosphate-ribose, cyclic ATP-ribose and 7-deaza-
cADPR have been shown to be agonistic analogues of
cADPR.8ꢀ10 Some analogues were found to be cell per-
meant and resistant to both heat and enzymatic hydro-
lysis.11,12 To investigate the relationships between the
structure and biological activities of cADPR, it would
be interesting to synthesize the N1-ribosyl-modified
cADPR analogues.The present study introduced a facile
Figure 1. Structure of cADPR 1, cyclic IDP-carbocyclic ribose 2a,
cyclic ADP-carbocyclic ribose 2b and N1-ether strand analogue 3.
*Corresponding author. E-mail: zdszlh@mail.bjmu.edu.cn
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00033-1

888
L.-J. Huang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 887–889
Figure 2. Effectof 3 and cADPR on intracellular Ca2+ dynamic
changes in intact Hela cells. The data shown are typical curves for at
least two experiments carried out in duplicate using different Hela cell
preparations.
zole in pyridine in 48% yield over two steps. However,
MALDI-TOF MS of 9 showed that the bromo group
was replaced with chloro group during the phosphor-
ylation reaction (Scheme 1).
The successive removal of diphenylamino group and
phenylthio group of 9 with isoamyl nitrite in a mixed
solventof pyridine–AcOH–Ac O and H3PO2 gave 10
2
for intramolecular condensation in 67% yield. The
intramolecular cyclization of 10 was performed under
the promotion of I2 and 3A MS in pyridine via a syringe
pump according to Matsuda’s method.16 The desired
cyclic product 1117 as a triethylammonium salt was
obtained after purification by HPLC in 43% yield. The
cyclic structure of 11 was characterized by the data of
HR-FABMS and 31P NMR. Deprotection of cyclic
product 11 provided the compound 318 in 45% yield in
60% HCO2H solution.
In human Hela cells transfected with CD 38, a type II
transmembrane glycoprotein, cADPR is shown to play
a role in regulating the cell doubling time.19 Itis inetr-
esting to know if the cellular Ca2+ level correlates with
the cADPR level in Hela cells. We measured the cellular
Ca2+ level in Hela cells by cofocal system after incuba-
tion with cADPR and analogue 3, respectively. In this
study, the calcium level was represented by relative
fluorescence intensity of Fluo-3. The suspension at a
density of 2ꢁ105 cells/mL was attached to a 35-mm dish
coated glass coverslips for 18 h. The packed cells were
incubated in HEPES buffer solution containing (mM):
125 NaCl, 1.2 KH2PO4, 1.2 MgCl2, 2 CaCl2, 6 glucose,
25 HEPES [pH 7.4 containing 20 mM Fluo-3 AM
Scheme 1. Reagents and conditions: (i) DBU, ClCH2OCH2CH2OAc,
CH2Cl2, rt; (ii) TBAF in THF, rt; (iii) (PhNH)2POCl, Py, tetrazole, rt;
(iv) CH3ONa, CH3OH, rt; (v) PSS, TPS, tetrazole, Py, rt; (vi) (a)
isoamyl nitrite, Py/AcOH/Ac2O (2:1:1), rt; (b) H3PO2, Et3N, Py, rt;
(vii) I2, 3A MS, Py, rt; (viii) 60% aqueous HCO2H, rt.
ꢂ
(Molecular Probe)] at37 C in dark for 30 min. The cells
acetate in the presence of excess DBU afforded regiose-
lectively the desired 5 in 82% yield. The structure of N1-
substituted ether strand was determined by the spectra
of UV and 1H NMR. The tert-butyldimethylsilyl
(TBDMS) group of 5 was selectively removed and the
resulting 50-primary hydroxy was reacted with
(PhNH)2POCl to give phosphoroamide 6 in 84% yield.
Deacetylation of 7 with CH3ONa gave the mono-
hydroxy compound 8, which was transformed into the
corresponding biphosphate 915 by treatment with cyclo-
hexylammonium S,S-diphenylphosphorodithioate (PSS),
triisopropylbenzenesulfonyl chloride (TPS) and tetra-
were washed twice and then incubated for another 30
min in a dye-free-HEPES solution. The Fluo-3 fluores-
cence measurements were carried out using a Leica TCS
NT cofocal laser-scanning microscope. The results
showed that compound 3 could cause the abrupt fluor-
escence increase in Hela cells after exposure to 100 mM 3
for about30 s. ButcADPR cannotinduce an efficient
increase in Fluo-3 fluorescence within 10 min under the
same concentration (Fig. 2). By combining both the
modifications on the N1 and 8-position of cADPR, we
found that compound 3 is a potent agonist of cADPR
and has activity in intact Hela cells. This is the first study

L.-J. Huang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 887–889
889
to implicate that the N1-ribosyl may nothave a crucial
role for the Ca2+ release activity of cADPR analogues
and 3 should be a useful tool to investigate the cADPR
signaling pathway.
13. Beauchamp, L. M.; Dolmath, B. L.; Schaeffer, H. J.; Col-
lins, P.; Baue, D. J.; Keller, P. M. J. Med. Chem. 1985, 28, 982.
14. Barrio, J. R.; Bryant, J. D.; Keyser, G. E. J. Med. Chem.
1980, 23, 572.
1
15. 9: H NMR (300 MHz, DMSO-d6): d 1.26, 1.51 (each 3H,
each s, (CH3)2C–), 3.81 (2H, m, H-50), 4.06–4.36 (5H, m,
–OCH2CH2O–, H-40), 5.06 (1H, m, H-30), 5.31 (1H, m, H-20),
5.42–5.53 (2H, m, –OCH2O–), 6.03 (1H, d, J=1.8 Hz, H-10),
6.72–7.52 (20H, m, arom H), 8.02 (1H, d, J=9.9 Hz, NH),
8.10 (1H, d, J=9.9 Hz, NH), 8.30 (1H, s, H-2). 13C NMR
(75 MHz, DMSO-d6): d 25.20, 26.00, 64.17, 67.04, 68.14, 75.12,
80.92, 82.87, 85.60, 90.62, 113.86, 117.26, 120.50, 123.75,
124.36, 125.60, 126.40, 127.26, 127.69, 128.35, 128.67, 128.82,
129.57, 129.76, 133.34, 135.12, 140.93, 141.08, 147.96,
149.12, 154.78. 31P NMR (121 MHz, DMSO-d6): d 2.65 (s),
51.58 (s); m/z 933 (M+Na)+, 949 (M+K)+. Anal. calcd for
C40H41ClN6O9P2S2: C, 52.72; H, 4.53; N, 9.22. Found: C,
53.12; H, 4.72; N, 9.26.
Acknowledgements
We are grateful for the support of the National Natural
Science Foundation of China.
References and Notes
1. Galine, A.; Lee, H. C.; Busa, W. B. Science 1991, 253, 1143.
2. Walseth, T. F.; Lee, H. C. Biochim. Biophys. Acta 1993,
1178, 235.
3. Zhang, F. J.; Gu, Q. M.; Sih, C. J. Bioorg. Med. Chem.
1999, 7, 653.
4. Fortt, S. M.; Potter, B. V. L. Tetrahedron Lett. 1997, 38,
5371.
5. Hutchinson, E. J.; Tayler, B. F.; Blackburn, G. M. J.
Chem. Soc. Chem. Commun. 1997, 1859.
6. (a) Shuto, S.; Shirato, M.; Sumita, Y.; Veno, Y.; Matsuda,
A. J. Org. Chem. 1998, 63, 1986. (b) Shuto, S.; Fukuoka, M.;
Manikowasky, A.; Ueno, Y.; Nakano, T.; Kuroda, R.; Kur-
oda, H.; Matsuda, A. J. Am. Chem. Soc. 2001, 123, 8750.
7. Lee, H. C. Physiol. Rev. 1997, 77, 1133.
8. Bailey, V. C.; Fortt, S. M.; Summerhill, R. J.; Galione, A.;
Potter, B. V. L. FEBS Lett. 1996, 379, 227.
9. Zhang, F. J.; Yawada, S.; Qu, Q. M.; Sih, C. J. Bioorg.
Med. Chem. Lett. 1996, 6, 1203.
16. Fukuota, M.; Shuto, S.; Shirato, M.; Sumita, Y.; Veno,
Y.; Matsuda, A. J. Org. Chem. 2000, 65, 5238.
17. 11: 1H NMR (500 MHz, D2O): d 1.28, 1.45 (each 3H, each
s, CH3ꢁ2), 3.61 (1H, m, H-50a), 3.70 (2H, m, OCH2CH2O–),
3.75 (3H, m, CH2, H-50b), 4.40 (1H, m, H-40), 5.08 (1H, d,
J=11 Hz, –OCHN–), 5.30 (1H, dd, J=2.5, 6.5 Hz, H-30), 5.71
(1H, d, J=11 Hz, –OCHN–), 5.81 (1H, d, J=2.5 Hz, H-20),
6.22 (1H, s, H-10). 31P NMR (D2O, 121 MHz): d ꢀ9.04 (d,
J=15.7 Hz), ꢀ9.86 (d, J=15.7 Hz); high resolution m/z calcd
for C16H19ClN4O12P2: 557.0247 (MꢀH)ꢀ. Found: 557.0254.
18. 3: 1H NMR (500 MHz, D2O): d 3.69 (3H, m, H-50a, CH2),
3.80 (2H, m, CH2), 3.92 (1H, dd, J=3.0, 9.0 Hz, H-50b), 4.18
(2H, m, CH2), 4.64 (1H, m, H-40), 4.75 (1H, m, H-30), 5.18
(1H, d, J=11 Hz, –OCHN–), 5.52 (1H, t, J=5.0 Hz, H-20),
5.85 (1H, d, J 11 Hz, –OCHN–), 5.98 (1H, d, J=5.0 Hz, H-10).
31P NMR (D2O, 121 MHz): d ꢀ10.44, ꢀ10.79; high resolution
m/z calcd for C13H15ClN4O12P2: 5169934 (MꢀH)ꢀ. Found:
516.9925.
10. Bailey, V. C.; Sethi, J. K.; Fortt, S. M.; Galione, A.; Pot-
ter, B. V. L. Chem. Biol. 1997, 4, 51.
11. Wong, L.; Aarhus, R.; Lee, H. C.; Walseth, T. F. Biochim.
Biophys. Acta 1999, 1472, 555.
12. Bailey, V. C.; Sethi, J. K.; Galione, A.; Potter, B. V. L. J.
Chem. Soc. Chem. Commun. 1997, 695.
19. Zocchi, E.; Franco, L.; Daga, A.; Usai, C.; Franco, L.;
Guida, L.; Bruzzone, S.; Costa, A.; Marchetti, C.; Deflora, A.
J. Biol. Chem. 1998, 273, 8017.