Synthesis and biological evaluation of novel membrane-permeant cyclic ADP-ribose mimics: N1-[(5″-O-phosphorylethoxy)methyl]-5′- O-phosphorylinosine 5′,5″-cyclicpyrophosphate (cIDPRE) and 8-substituted derivatives

Article abstract of DOI:10.1021/jm040092t

N¹-[(5″-O-Phosphorylethoxy)methyl]-5′-O- phosphorylinosine 5′,5″-cyclicpyrophosphate (cIDPRE 2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e) were synthesized from N¹-[(5″-acetoxyethoxy)methy

Full text of DOI:10.1021/jm040092t

5674
J. Med. Chem. 2004, 47, 5674-5682
Synthesis and Biological Evaluation of Novel Membrane-Permeant Cyclic
ADP-Ribose Mimics:
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphorylinosine
5′,5′′-Cyclicpyrophosphate (cIDPRE) and 8-Substituted Derivatives
Xianfeng Gu,† Zhenjun Yang,† Liangren Zhang,† Svenja Kunerth,‡ Ralf Fliegert,‡ Karin Weber,‡
Andreas H. Guse,‡ and Lihe Zhang*,†
National Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Beijing 100083, China, and University Hospital Hamburg-Eppendorf, Center of Experimental Medicine,
Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistrasse 52,
20246 Hamburg, Germany
Received May 7, 2004
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphorylinosine 5′,5′′-cyclicpyrophosphate (cIDPRE
2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e)
were synthesized from N1-[(5′′-acetoxyethoxy)methyl]-2′,3′-O-isopropylideneinosine (5) in good
yields. The pharmacological activities of cIDPRE and the 8-substituted derivatives (2a-e) were
analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicate that
cIDPRE permeates the plasma membrane, releases Ca2+ from an intracellular, cADPR-sensitive
Ca2+ store, and subsequently initiates Ca2+ release-activated Ca2+ entry. The Ca2+-releasing
activity of cIDPRE was confirmed directly in permeabilized cells. Using time-resolved confocal
Ca2+ imaging at the single cell level, the development of global Ca2+ signals starting from
local small Ca2+ signals evoked by cIDPRE was observed. 8-N3-cIDPRE 2c and 8-NH2-cIDPRE
2d were similarly effective in their agonistic activity as compared to cIDPRE 2a, showing almost
indistinguishable concentration-response curves for 2a, 2c, and 2d and very similar kinetics
of Ca2+ signaling. In contrast, the halogenated derivatives 8-Br- and 8-Cl-cIDPRE (2b and 2e)
did not significantly elevate [Ca2+]i. Therefore, cIDPRE 2a, 8-N3-cIDPRE 2c, and 8-NH2-cIDPRE
2d are novel membrane permeant cADPR mimic and may provide important novel tools to
study cADPR-mediated Ca2+ signaling in intact cells.
Introduction
Ryanodine receptors and D-myo-inositol 1,4,5-trispho-
sphate receptors represent the major pathways of Ca2+
-
release from intracellular stores and have been shown
to be involved in many physiological and pathological
processes.1-4 Cyclic ADP-ribose (cADPR, 1) (Figure 1)
is an endogenous nucleotide synthesized from NAD+ by
ADP-ribosyl cyclase in cells.5 The structure of cADPR
was identified by X-ray crystallography in 1994.6 cADPR
has been attracting much attention in recent years
because of its potent calcium-mobilizing activities in
many cellular systems, especially as a potential second
messenger in cellular Ca2+ homeostasis in insulin
secretion7 and in T-lymphocyte activation.8
Many derivatives of cADPR have been synthesized
from the corresponding NAD+ analogues using ADP-
ribosyl cyclase and served as valuable research tools in
the elucidation of the mechanism of cADPR action,9 e.g.
labeling of binding proteins10 or antagonizing Ca2+
mobilization by cADPR.11 Specifically, cADPR analogues
with substitutions at the 8-position of the adenine ring
were found to be antagonists of cADPR.11 8-Amino-
cADPR has been shown to be effective not only in sea
urchin egg microsomes, but also in intact sea urchin
Figure 1. The structures of cADPR and 8-substituted cI-
DPRE.
eggs12 as well as intact and permeabilized mammalian
cells.13,14 Another possibility to turn cADPR into an
antagonist is by replacing the 3′-OH group by an 3′-O-
methyl group.15 Regarding modifications in the 2′-OH
group significant differences between lower and higher
eukaryotic systems were described: while 2′-phospho-
cADPR was fully active in higher eukaryotic cells,16,17
no Ca2+ release was observed in the sea urchin egg
system.18
The first nonhydrolyzable mimic of cADPR is cyclic
aristeromycin-diphospho-ribose which retained a similar
calcium release profile to that of cADPR.19 Some struc-
tural modifications on adenine moiety of cADPR make
the mimic more potent than its parent, e.g. 3-deaza-
* Corresponding author. Tel: 86-10-82801700; Fax: 86-10-82802724,
e-mail: zdszlh@bjmu.edu.cn.
† Peking University.
‡ University Hospital Hamburg-Eppendorf.
10.1021/jm040092t CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/07/2004

Membrane-Permeant Cyclic ADP-Ribose Mimics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5675
cADPR is 70-fold more potent than cADPR.20 Additional
structural modification of cADPR includes the pyro-
phosphate moiety, which has been replaced by triphos-
phate21 and the caged compound 2-(nitrophenyl-1)ethyl
pyrophosphate-cADPR;22 unexpectedly, cyclic adenosine
triphosphate ribose was more potent in inducing Ca2+
release as compared to cADPR. The N1-pentyl cyclic
inosine diphosphate ribose and N1-carbocyclic, N9-butyl
analogue of cADPR were also reported.23,24
Shuto and co-workers first described the chemical
synthesis of cyclic IDP-carbocyclic-ribose (cIDP-carbori-
bose), in which the 4′′-oxo in furanose was substituted
by a methylene group25 and reported that the corre-
sponding stable mimic of cADPR, cADPcR, caused a
significant release of Ca2+ in sea urchin eggs;26 however,
in mammalian cells cADPcR acted much weaker as
compared to the sea urchin system.27 Recently, a series
of 8-substituted analogues of cADPcR were synthesized.
In the sea urchin egg system, it was found that cADPcR
was 3-4 times more potent than cADPR while the
8-substituted analogues were less efficacious.28
of excess DBU to afford 4a in 69% yield. The O6-
substituted side product was obtained in 19% yield. The
structure of the N1-substituted inosine 4a was confirmed
1
by UV, H NMR, and 13C NMR. Compared to the UV
spectrum of the known compound N1-methylinosine,
compound 4a showed a similar spectrum at 245 nm
(λmax) and a shoulder around 270 nm, whereas the UV
spectrum of the O6-substituted side product was differ-
ent. The 1H NMR spectrum of 4a showed two doublets
at 5.51 and 5.55 ppm (H1”a, H1′′b, JH1′′a, H1′′b ) 10.5 Hz),
which coupled with C2 in the HMBC spectrum. How-
ever, only a single peak was observed at 5.86 ppm in
1H NMR spectrum of the O6-substituted product, and
no coupling peak was found in the HMBC spectrum. The
above spectra data support strongly that the structure
of compound 4a is the N1-substituted inosine derivative.
After deprotection of the 5′-tert-butyldimethylsilyl (TB-
DMS) group in compound 4a, the resulting 5′-hydroxyl
of intermediate 5a was reacted with (PhNH)2POCl to
give phosphoroamide 6a in 91% yield. 31P NMR (3.06
ppm, s) indicated the signal of phosphoroamide group
in compound 6a. Deacetylation of 6a with catalytic
amount CH3ONa gave the monohydroxy compound 7a
in 95% yield. Compound 7a was phosphorylated with
cyclohexylammonium S,S-diphenylphosphorodithioate
(PSS) in the presence of triisopropylbenzenesulfonyl
chloride (TPSCl) and tetrazole in pyridine to give 8a in
79% yield. The presence of two different phosphate
groups in the molecule was supported by 31P NMR (3.21
ppm, s; 49.39 ppm, s).
The intramolecular cyclization of 9a was completed
by using Matsuda’s strategy.25,26 The selective removal
of diphenylamino group in compound 8a was carried out
with isoamyl nitrite in a mixed solvent of pyridine-
AcOH-Ac2O and H3PO2 to give 9a, the precursor for
the intramolecular condensation reaction, in 73% yield
as a triethylammonium salt. Matsuda’s group suggested
that introducing a bulky group into the 8-position of
purine nucleosides could restrict the conformation to
syn-form in which the two phosphate moieties are near
each other and facilitate intramolecular condensation
for the synthesis of cADPR.25,26 Recently, they reported
that in the synthesis of 8-unsubstituted cADPcR, the
cyclization was performed by I2/3 Å MS (3 Å molecular
sieve) or AgNO3/3 Å MS in high yield.25,26 In the present
case, the intramolecular cyclization was performed in
the presence of excess I2 and 3 Å MS in pyridine by
adding a solution of compound 9a slowly over 20 h using
a syringe pump. The cyclic product 10a was purified by
HPLC as its triethylammonium salt in 71% yield.
Compared to the yields of the cyclization in the synthe-
ses of 8-Br-cIDPRE 10b (87%) and 8-N3-cIDPRE 10c
(87%), a more flexible N1-ether strand in compound 9a
resulted in a larger spatial distance of the two phos-
phate groups resulting in a lower yield of cyclization.
The cyclic structure of 10a was confirmed by the data
of HR-FABMS (523.0645, [M - 1]-) and 31P NMR (-9.69
and -10.61 ppm of pyrophosphate group). Finally,
removal of the isopropylidene group of 10a was carried
out with aqueous HCOOH at room temperature for 8 h
to furnish cIDPRE 2a. cIDPRE 2a was purified by two
subsequent HPLC steps and characterized by 1H NMR,
31P NMR (-9.69 ppm, d, J ) 13.4 Hz; -10.38 ppm, d, J
) 13.4 Hz) and HR-FAB MS (483.0324, [M - 1]-).
A series of N1-glycosyl-substituted cyclic IDP-ribose
derivatives were reported in our laboratory.29,30 It was
found that mimics with different configurations at the
N1-glycosyl moiety of cyclic IDP-ribose retained the
cADPR activities to induce Ca2+ release. More interest-
ingly, the N1-ethoxymethyl-8-chloro-cyclic IDP-ribose
analogue exhibited a strong potency to induce Ca2+
release in both rat brain microsomes and intact HeLa
cells. This is the first mimic of cADPR with complete
replacement of the terminal ribose with an ether strand,
and the analogue not only remained active, but also
appears to be cell permeant. Therefore, the N1-ribosyl
moiety and the 6-amino group in cADPR are not critical
structural factors for retaining the biological activity.
However, the relationship between the structure and
agonist/antagonist behavior in cADPR and its mimics
is not well understood. In contrast to N1-ethoxymethyl-
8-chloro-cyclic IDP-ribose30 all 8-substituted cADPR
analogues prepared so far were antagonists of cADPR-
mediated Ca2+ release. However, in the case of cADPcR,
8-azido- and 8-amino-cADPcR were full agonists, al-
though approximately 80 and 2 times less potent than
cADPR, respectively.26
To further investigate the Ca2+ release activity of
cyclic IDP-ribose derivatives in which the terminal
ribose is replaced by an ether strand, we report here
the syntheses of N1-[(5′′-O-phosphorylethoxy)methyl]-
5′-O-phosphorylinosine 5′,5′′-cyclicpyrophosphate (2a;
abbreviated cIDPRE) (Figure 1) and some 8-substituted
derivatives (2a-e) and their pharmacological charac-
terizations in Jurkat T cells.
In this paper, the structural positions of cIDPRE and
its derivatives are numbered as follows: a single prime
numbering scheme is used for the position of the N9-
ribosyl moiety and a double prime numbering scheme
is used for the position of the N1-substitution (Figure
1).
Results and Discussion
Chemistry. The syntheses of 8-substituted cIDPRE
2a-2e are summarized in Scheme 1. An N1 substitution
was carried out regioselectively on the protected inosine
3a with 2-chloromethoxyethyl acetate in the presence

5676 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Gu et al.
Scheme 1a
a Reagents and conditions: (a) DBU, ClCH2OCH2CH2OAc, CH2Cl2, rt; (b) TBAF, THF, rt; (c) (PhNH)2POCl, tetrazole, Py, rt; (d) CH3ONa,
CH3OH, rt; (e) PSS, TPSCl, tetrazole, Py, rt; (f) i. isoamyl nitrite, Py:AcOH:Ac2O (2:1:1), ii. H3PO2, Et3N, Py; (g) I2, 3 Å MS, Py, rt; (h) 60%
HCOOH, rt; (i) NaN3, DMSO, 80 °C; (j) H2, 10% Pd/C, C2H5OH, rt.
To synthesize the 8-substituted cIDPRE, protected
8-bromoinosine 3b was used as a starting material and
the known procedure was followed to prepare the pre-
cursor 7b 29 (Scheme 1). It was reported from our lab-
oratory that the excess of TPSCl in the phosphorylation
caused the substitution of bromo group at the 8-position
of 7b by chloro group.29 Therefore, 7b was phosphory-
lated with PSS, TPSCl and tetrazole in pyridine with
the molecular ratio of 7b:TPSCl to 1: 2 to afford 8b in
87% yield. The selective deprotection of 8b and intramo-
lecular cyclization were carried out under a similar con-
dition as above to give the 10b in 88% yield. After depro-
tection, compound 2b was obtained in 95% yield. 8-Bromo
group of 6b was substituted by azido group in the pres-
ence of NaN3 in DMSO to give 6c in 90% yield. Using
the same strategy for the synthesis of cIDPRE 2a, 10c
was obtained in 89% yield and 2c was in 93% yield from
10c. 10c was hydrogenated under the catalyst of 10%
Pd/C to give 10d in 99% yield. After removing the
protecting group, 2d was obtained in 90% yield.
Pharmacology. The pharmacological activities of
cIDPRE 2a and 8-Br- 2b, 8-N3- 2c, 8-NH2- 2d, 8-Cl-
cIDPRE 2e30 were analyzed in intact and permeabilized
human Jurkat T-lymphocytes. Addition of cIDPRE 2a
to intact cells in the presence of extracellular Ca2+ acti-
vated a concentration-dependent biphasic pattern of Ca2+
mobilization, starting with a small rise that was then
followed by a much larger increase (Figure 2A,B). The
intracellular Ca2+ concentration ([Ca2+]i) remained eleva-
ted for at least 15 min. Since even at 1 mM cIDPRE 2a
saturation of the response was not achieved (Figure 2B),
no EC50 can be given. Using a Ca2+ free/Ca2+ reintroduc-
tion protocol, cIDPRE 2a activated a transient Ca2+ re-
lease from intracellular Ca2+ stores (Figure 2C). Readdi-
tion of Ca2+ after the signal had returned to the baseline
rapidly activated Ca2+ entry from the extracellular space
resulting in a sustained elevation of [Ca2+]i (Figure 2C).
These results indicate that cIDPRE 2a permeates the
plasma membrane, releases Ca2+ from an intracellular,
cADPR-sensitive Ca2+ store, and subsequently initiates
Ca2+ release-activated Ca2+ entry. The Ca2+ releasing
activity of cIDPRE 2a was confirmed directly in per-
meabilized cells (Figure 2D, lower panel); quantitative
analysis resulted in a lower efficacy of cIDPRE 2a as
compared to cADPR 1 (Figure 2D, upper panel).
One of the hallmarks of Ca2+ signaling is the develop-
ment of global Ca2+ signals starting from local subcel-
lular pacemaker signals. Using confocal Ca2+ imaging
at the single cell level, upon extracellular addition of
cIDPRE 2a, we demonstrate the induction of localized
small Ca2+ signals close to the cell border (Figure 2E;
image at 138 s; arrow). With time isolated neighboring
Ca2+ signals merge into larger signals reflecting recruit-
ment of additional Ca2+ release microdomains (Figure
2E; image at 147 and 148 s). Finally, individual signals
are almost indistinguishable from each other: the signal
becomes global (Figure 2E; image at 153 s).
Modification of cIDPRE at C8 of inosine had strikingly
differential effects. 8-N3-cIDPRE 2c (Figure 3B) and
8-NH2-cIDPRE 2d (Figure 3C) were similarly effective

Membrane-Permeant Cyclic ADP-Ribose Mimics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5677
Figure 2. Effect of the cADPR mimic cIDPRE on Ca2+ signaling in T cells. Intact Jurkat T cells were loaded with Fura2/AM
(A-C, E), and [Ca2+]i was determined fluorimetrically in cell suspension (A-C) or by confocal Ca2+ imaging on the single cell
level (E) as described in the Experiment Section. A: cIDPRE was added as indicated (final concentration 0.5 mM); a representative
tracing out of five experiments is displayed. B: Concentration-response data are given as mean ( SEM (n ) 3 to 5; error bars
are partially not visible because the values are smaller than the symbol size). C: Cells were kept in a nominal Ca2+ free buffer
in the first part of the experiment; cIDPRE was added as indicated. Then, CaCl2 was readded. The dashed line shows a control
experiment where buffer was added instead of cIDPRE. D: Cells were permeabilized as described in the experiment section.
Vehicle (H2O) and cIDPRE (100 µM) were added as indicated (lower panel). Upper panel: data are mean ( SEM (n g 3; error
bars are partially not visible because the values are smaller than the symbol size). E: Characteristic confocal ratiometric pseudocolor
images of a single Jurkat cell and magnifications of a defined subcellular region are shown. Basal phase (before addition of 0.5
mM cIDPRE [final concentration]), pacemaker phase and global phase are indicated. White bar corresponds to 3 µm in the upper
panel and to 1 µm in the lower panel. In total 15 cells were analyzed.
in their agonistic activity as compared to cIDPRE 2a
(Figure 3A-C); note the quantitatively almost indistin-
guishable concentration-response curves for 2a, 2c and
2d (Figure 3D) and the very similar kinetics of Ca2+
signaling (Figure 3A-C). In contrast, the halogenated
derivatives 8-Br- and 8-Cl-cIDPRE (compounds 2b and
2e) did not significantly elevate [Ca2+]i (Figure 3D).
These data indicate that the amino or the azido group
at the 8-position of the inosine ring much better
complies with the structural requirements for an agonist
as compared to a halogen group.
cIDPR,31 the compounds described in the present study,
cIDPRE 2a and 8-NH2- and N3-cIDPRE (compounds 2d
and 2c), are novel and important tools similarly suitable
for studying the structure-activity relationship of
cADPR and to be used as a membrane permeant cADPR
mimics in intact cells. The cADPR/Ca2+ signaling path-
way has been demonstrated in many different cell types
and tissues (see Table 1 in ref 4); however, in most of
the cases either cADPR had to be delivered into intact
cells by microinjection32 or infusion via a patch-clamp
pipet,33 or broken cell preparations such as permeabi-
lized cells13 or cell homogenates34 were used. Clearly,
the advantage of a membrane-permeant mimic is that
In addition to the previously described N1-glycosyl-
substituted cyclic IDP-ribose derivatives29,30 and 8-Br-

5678 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Gu et al.
Figure 3. Effect of the cIDPRE and 8-substituted cIDPRE analogues on Ca2+ signaling in T cells. Intact Jurkat T cells were
loaded with Fura2/AM, and [Ca2+]i was determined fluorimetrically in cell suspension as described in the Experiment Section.
Different concentrations of cIDPRE (A), 8-N3-cIDPRE (B) and 8-NH2-cIDPRE (C) were added (arrow) as indicated. Mean values
( SEM (n ) 3 to 5) are displayed as concentration-response curve in D.
the cells remain intact and mechanically untouched.
This is especially important in cell types where me-
chanical influences would activate (or inhibit) Ca2+
signaling on their own. Thus, time-resolved subcellular
Ca2+ imaging is technically less difficult using a mem-
brane permeant cADPR mimic simply added to the
extracellular medium as compared to microinjection of
the natural compound cADPR. A further advantage is
that the involvement of the cADPR/Ca2+ signaling
system can be studied in a larger number of cells, e.g.
in cell suspension, thus providing a more accurate mean
of the cellular response.
Comparison of the different cIDPRE derivatives tested
here revealed a remarkable structure-activity relation-
ship: while any functional group larger than the natural
H-atom at C8 in cADPR turned the molecule into a
cADPR antagonist, e.g. 8-NH2-cADPR,11,13 8-Br-cAD-
PR,11,13 8-OCH3-cADPR,8 8-CH3-cADPR,17 in the case
of cyclic IDP-ribose mimics, different functional groups
at the 8-position resulted in completely different biologi-
cal activities. In our case, the electronegative action of
halogen atoms may either directly prevent binding at
the cADPR binding pocket or may influence the overall
conformation of the whole molecule in a way that it does
not resemble cADPR any more. The latter may be due
to a greater flexibility of the whole molecule introduced
by replacement of the terminal ribose by the ether
strand. In contrast, the 8-N3- and 8-NH2-analogues
(compounds 2c and 2d) either still fit into the binding
site or the functional groups support a conformation
much more like cADPR. Specifically, the amino ligand
at the 8-position of the inosine ring of cIDPRE may
contribute as a proton donor for the formation of a
hydrogen bond with the neighboring amino acid residue
of the receptor. The longer lasting activity of 8-N3-
cIDPRE 2c as compared to cIDPRE or 8-NH2-cIDPRE
(Figure 3B vs Figures 3A and 3C) may, in addition, be
due to UV-induced formation of covalent bonds between
this ligand and its receptor protein.
Taken together, chemical synthesis of cIDPRE and
its analogues resulted in important novel tools to study
cADPR-mediated Ca2+ signaling in intact cells and new
and unexpected insights into the structure-activity
relationship of cADPR.
Experiment Section
Chemistry. UV spectra were recorded on a Varian DMS200
UV-visible spectrophotometer. Mass spectra were obtained
on either VG-ZAB-HS or Bruker APEX. High-resolution FAB
(fast atom bombardment) mass and HR EIMS (electrospray
ionization) were performed with Bruker BIFLEX III. 1H NMR
and 13C NMR were recorded with a JEOL AL300 or a Varian
VXR-500 spectrometer using DMSO-d6 or D2O as a solvent.
Chemical shifts are reported in parts per million downfield
from TMS (1H and 13C). 31P NMR spectra were recorded at
room temperature by use of Bruker Avance 300 spectrometer
(121.42 MHz); Orthophosphoric acid (85%) was used as an
external standard. Compounds (2a-d, 9a-c and 10a-d) were
purified on Alltech preparative C18 reversed phase column
(2.2 × 25 cm) with Gilson HPLC by two different buffer
systems: MeCN/TEAA (pH 7.0) or MeCN/TEAB (pH 7.5).
N1-[(5′′-Acetoxyethoxy)methyl]-5′-O-TBDMS-2′,3′-O-iso-
propylideneinosine (4a). To the solution of 3a (422 mg, 1
mmol) and DBU (1.4 mL, 10 mmol) in CH2Cl2 (10 mL) was
added ClCH2OCH2CH2OAc (0.7 mL, 5 mmol) at 0°C. After
being stirred for 30 min, the solvent was evaporated in vacuo
and the residue was purified by silica gel column chromatog-
raphy (hexane-acetone) to give compound 4a (371 mg, 69%).
1H NMR (300 MHz, DMSO) δ 0.00 (s, 6H, (CH3)2Si), 0.82 (s,
9H, (CH3)3C-), 1.31, 1.52 (each s, each 3H, (CH3)2C), 1.90 (s,
3H, AcO), 3.77-3.87 (m, 4H, OCH2, 2 × H5′), 4.09-4.13 (m,
2H, CH2OAc), 4.28-4.29 (m, 1H, H4′), 4.99 (dd, 1H, J H3′,H4′
3.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.28 (dd, 2H, JH1′,H2′ ) 2.5, JH2′,H3′
)
)
6.0 Hz, H2′), 5.51 (d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′a), 5.55 (d,
1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′b), 6.14 (d, 1H, JH2′,H1′ ) 2.5 Hz,
H1′), 8.15, 8.36 (each s, each 1H, H2, H8); 13C NMR (75 MHz,
DMSO) δ 170.21, 155.98, 148.90, 146.95, 139.38, 123.89,
113.12, 89.75, 86.85, 83.94, 81.01, 74.68, 67.00, 63.12, 62.89,

Membrane-Permeant Cyclic ADP-Ribose Mimics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5679
JH1′′a,H1′′b ) 10.2 Hz, H1′′a), 5.48 (d, 1H, JH1′′a,H1′′b ) 10.2 Hz,
26.92, 25.69, 25.14, 20.54, 17.92, -5.57; UV (MeOH) λmax 207
nm, 245 nm, sh 270 nm. Anal. (C24H37N4O8Si) C, H, N.
H1′′b), 6.19 (d, 1H, JH1′,H2′ ) 2.4 Hz, H1′), 6.80-7.57 (m, 20H,
N1-[(5′′-Acetoxyethoxy)methyl]-2′,3′-O-isopropylide-
neinosine (5a). A solution of 4a (360 mg, 0.67 mmol), and
TBAF (1 M in THF, 1.3 mL, 1.3 mmol) in THF (5 mL) was
stirred for 2 h at room temperature under neutral conditions.
The resulting mixture was evaporated under reduced pressure,
and the residue was purified by silica gel column chromatog-
raphy (hexane-acetone) to give 5a (256 mg, 90%): 1H NMR
(300 MHz, DMSO) δ 1.32, 1.54 (each s, each 3H, (CH3)2C-),
1.94 (s, 3H, AcO), 3.53-3.56 (m, 2H, 2 × H5′), 3.74-3.77 (m,
2H, OCH2), 4.08-4.11 (m, 2H, CH2OAc,), 4.23-4.24 (m, 1H,
H4′), 4.94 (dd, 1H, JH3′,H4′ ) 2.1 Hz, JH2′,H3′ ) 6.0 Hz, H3′), 5.12
Ar H), 8.13, 8.16 (each d, each1H, J
) 6.3 Hz, NH ×
NHa,NHb
2), 8.33, 8.43 (each s, each 1H, H8,H2); 31P NMR (DMSO, 81
MHz, decoupled with 1H) 3.21 ppm (s); 49.39 ppm (s). ESI-
TOF+-MS: m/z ) 877.2373 [(M + 1)+]; Anal. (C40H42N6O9P2S2‚
1/2hexane) C, H, N.
N1-[[[5′′-(Phenylthio)phosphoryl]oxyethoxy]methyl]-
5′-O-phosphoryl-2′,3′-O- isopropylideneinosine (9a). A
solution of 8a (120 mg, 0.137 mmol) and isoamyl nitrite (277
µL, 2.05 mmol) in pyridine-AcOH-Ac2O (2:1:1, v/v, 4 mL) was
stirred at room temperature for 8 h. After the reaction mixture
was evaporated (at <30 °C), the residue was dissolved with
H3PO2 (140 µL, 2.74 mmol) and Et3N (190 µL, 1.37 mmol) in
pyridine (3 mL), and the resulting solution was stirred for 11
h at room temperature. After the solvent was evaporated to
dryness under reduced pressure, the residue was partitioned
between CHCl3 and H2O. The aqueous layer was coevaporated
(<30 °C) with pyridine (5 mL), and the residue was dissolved
in TEAA (0.1 M, pH 7.0, 5 mL). The solution was purified by
a C18 reverse phase column (2.2 × 25 cm) using a linear
gradient of 0-40% CH3CN in TEAA buffer (0.1 M, pH 7.0)
within 30 min to give 9a as a triethylammonium salt. The
compound was purified again using same column eluting with
MeCN/TEAB buffer (pH 7.5) (84 mg, 73%). 1H NMR (500 MHz,
D2O) δ 1.45, 1.67 (each s, each 3H, 2 × CH3), 3.87-3.88 (m,
2H, H5′), 4.05-4.10 (m, 2H, CH2O), 4.11-4.13 (m, 3H, H4′,
CH2OP), 5.15 (dd, 1H, JH3′,H4′ ) 2.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.31
(dd, 1H, JH2′,H1′ ) 3.0, JH3′,H2′ ) 6.0 Hz, H2′), 5.50 (d, 1H,
JH1′′a,H1′′b ) 10.5 Hz, H1′′a), 5.56 (d, 1H, JH1′′a,H1′′b ) 10.5 Hz,
H1′′b), 6.21 (d, 1H, JH1′,H2′ ) 3.0 Hz, H1′), 7.18-7.43 (m, 5H, Ar
H), 8.38, 8.43 (each s, each1H, H2, H8); 31P NMR (D2O 81 MHz,
decoupled with 1H) δ 2.06(s), 17.79(s). HRMS (TOF, negative)
for C22H28N4O12P2S: Calcd, 633.0827 [(M - 1)-]; found,
633.0818.
N1-[(5′′-O-Phosphorylethoxy)methyl]-2′,3′-O-isopropy-
lidene 5′-O-phosphorylinosine 5′,5′′-cyclicpyrophosphate
(10a). A solution of 9a (30 mg, 36 µmol) in pyridine (5 mL)
was added slowly over 20 h, using a syringe pump, to a mixture
of I2 (190 mg) and MS 3 Å (1.9 g), in pyridine (50 mL) at room
temperature in the dark. The MS 3 Å was filtered off with
Celite and washed with H2O. The combined filtrate was
evaporated, and the residue was partitioned between CHCl3
and H2O. The aqueous layer was evaporated, and the residue
was dissolved in 0.1 M TEAA buffer (1.0 mL), which was
applied to C18 reversed phase column (2.2 × 25 cm). The
column was developed using a linear gradient of 0-80%
CH3CN in TEAA buffer (0.1 M, pH 7.0) within 30 min to give
10a as a triethylammonium salt. The compound was purified
again using same column eluting with MeCN/TEAB buffer (pH
7.5) (18.5 mg, 71%). 1H NMR (500 MHz, D2O) δ 1.47, 1.63 (each
s, each 3H, 2 × CH3), 3.72-3.77 (m, 1H, H5a′), 3.86-3.89 (m,
2H, CH2O), 3.92-3.97 (m, 3H, H5′b, CH2OP), 4.60 (m, 1H, H4′),
5.35 (dd, 1H, JH3′,H4′ ) 2.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.49 (d, 1H,
JH1′′a,H1′′b ) 11.5, H1′′a), 5.88 (d, 1H, JH1′′a,H1′′b ) 11.5, H1′′b), 5.92
(d, 1H, JH2′,H3′ ) 6.0, H2′), 6.33 (s,1H, H1′), 8.22, 8.49 (each s,
each 1H, H2, H8); 31P NMR (D2O 81 MHz, decoupled with 1H)
δ -9.69, -10.61. HRMS (FAB, negative) for C16H22N4O12P2
Calcd, 523.0637 [(M - 1)-]; found, 523.0645.
(t, 1H, JOH,H5′ ) 5.1, OH), 5.27 (dd, 1H, JH1′,H2′ ) 2.1, JH3′,H2′
)
6.0 Hz, H2′), 5.46 (s, 2H, H1′′), 6.11 (d, 1H, JH1′,H2′ ) 2.1 Hz,
H1′), 8.36, 8.51; (each s, each 1H, H2, H8); 13C NMR (75 MHz,
DMSO) δ 170.27, 156.00, 149.01, 147.11, 139.46, 123.72,
113.11, 89.62, 86.77, 83.86, 81.25, 74.73, 67.05, 62.94, 61.41,
26.99, 25.13, 20.55; Anal. (C18H29N4O8) C, H, N.
N1-[(5′′-Acetoxyethoxy)methyl]-5′-O-dianilinophospho-
ryl-2′,3′-O-isopropylideneinosine (6a). To a solution of 5a
(350 mg, 0.825 mmol) and tetrazole (231 mg, 3.302 mmol) in
dry pyridine (5 mL) was added (PhNH)2POCl (880 mg, 3.302
mmol) at room temperature, and the mixture was stirred for
48 h. The solvent was evaporated to dryness, and the residue
was purified by silica gel column chromatography (CH2Cl2-
1
MeOH) to afford 6a (487 mg, 91%) as a white foam. H NMR
(300 MHz, DMSO) δ 1.29, 1.52 (each s, each 3H, CH3 × 2),
1.94 (s, 3H, AcO), 3.73-3.77 (m, 2H, 2 × H5′), 4.07-4.19 (m,
4H, CH2OAc, OCH2), 4.43 (m, 1H, H4′), 4.99 (dd, 1H, JH3′,H4′
3.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.25 (dd, 1H, JH2′,H1′ ) 2.4, JH2′,H3′
)
)
6.0 Hz, H2′), 5.40 (d, 1H, JH1′′a,H1′′b ) 10.4 Hz, H1′′a), 5.46 (d,
1H, JH1′′a,H1′′b ) 10.4 Hz, H1′′b) 6.15 (d, 1H, JH1′,H2′ ) 2.4 Hz,
H1′), 6.75-7.16 (m, 10H, Ar H), 8.07, 8.11 (each d, each1H,
JNHa,NHb ) 9.9 Hz, NH × 2), 8.28, 8.39 (each s, each 1H, H8,
H2). 13C NMR (75 MHz, DMSO) δ 170.32, 156.00,148.95,
146.98, 141.04, 140.98, 139.55, 128.81, 128.76, 123.96, 120.52,
117.35, 117.25, 113.67, 113.61, 89.35, 84.47, 83.55, 80.93,
74.82, 67.13, 64.44, 62.96, 26.93, 25.13, 20.58; 31P NMR
(DMSO, 81 MHz, decoupled with 1H) δ 3.06 (s). Anal.
(C30H36N6O9P‚2CH3OH) C, H, N.
N1-(5′′-Hydroxylethoxymethyl)-5′-O-(dianilinophospho-
ryl)-2′,3′-O-isopropylideneinosine (7a). Compound 6a (460
mg, 0.703 mmol) was dissolved in 10 mL of methanol. To the
solution was added CH3ONa (8 mg, 0.14 mmol) and stirred
for 2 h. Dowex-50W acid cation-exchange resin was added to
adjust pH to 7 and then filtered, and solvent was removed
under reduced pressure to give 7a (409 mg, 95%): 1H NMR
(300 MHz, DMSO) δ 1.28,1.51 (each s, each 3H, CH3 × 2),
3.46-3.49 (m, 2H, 2 × H5′), 3.54-3.57 (m, 2H, OCH2-), 4.07-
4.22 (m, 2H, CH2OH), 4.43 (m, 1H, H4′), 4.99 (dd, 1H, JH3′,H4′
) 3.0, JH3′.H2′ ) 6.0 Hz, H3′), 5.25 (dd, 1H, JH1′,H2′ ) 2.5, JH2′,H3′
) 6.0 Hz, H2′), 5.39 (d, 1H, JH1′′a,H1′′b ) 10.4 Hz, H1′′a), 5.46 (d,
1H, JH1′′a,H1′′b ) 10.4, H1′′b), 6.15 (d, 1H, JH1′,H2′ ) 2.5 Hz, H1′),
6.75-7.15 (m, 10H, Ar H), 8.06, 8.09 (each d, each1H, J NHa,NHb
) 6.3 Hz, NH × 2), 8.27, 8.37 (each s, each 1H, H8, H2); 13C
NMR (75 MHz, DMSO) δ 155.95, 148.89, 146.95, 141.00,
140.96, 139.50, 128.81, 128.75, 123.95, 120.52, 117.34, 117.25,
113.58, 89.34, 84.48, 83.54, 80.91, 75.00, 70.93, 64.51, 60.05,
26.93, 25.13; 31P NMR (DMSO, 81 MHz, decoupled with 1H) δ
3.17. Anal. (C28H33N6O8P) C, H, N.
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphoryli-
nosine 5′,5′′-Cyclicpyrophosphate (cIDPRE 2a). The solu-
tion of 10a (18.5 mg, 25.5 µmol) in 60% HCOOH (5 mL) was
stirred for 8 h and then evaporated under reduced pressure.
The purification of the residue was performed at the same
procedure as compound 10a by twice HPLC on C18 reverse
column eluting with MeCN/TEAA (pH 7.0) and MeCN/TEAB
(pH 7.5) buffer, respectively, to give the target molecule 2a
(16 mg. 91%): 1H NMR (500 MHz, D2O) δ 3.75-3.86 (m, 3H,
H5a′, CH2O-), 3.88-3.96 (m, 2H, CH2OP), 4.04-4.06 (m, 1H,
H5′b), 4.19-4.23 (m, 1H, H4′), 4.30-4.32 (1H, m, H3′), 5.43 (t,
1H, JH1′,H2′ ) 4.0 Hz, JH3′,H2′ ) 4.0 Hz, H2′), 5.48 (d, 1H, JH1′′a,H1′′b
) 11.5 Hz, H1′′a), 5.80 (d, 1H, JH1′′a,H1′′b ) 11.5 Hz, H1′′b), 6.05
(d, 1H, JH1′,H2′ ) 4.0 Hz, H1′), 8.19, 8.50 (each s, each 1H, H2,
N1-[[[Bis-5′′-(phenylthio)phosphoryl]oxyethoxy]methyl]-
5′-O-(dianilinophosphoryl)-2′,3′-O-isopropylideneinos-
ine (8a) Compound 7a (400 mg, 0.653 mmol) was dissolved
in dry pyridine (7 mL) and then added TPSCl (395 mg, 1.31
mmol), PSS (742 mg, 1.96 mmol) and tetrazole (137 mg, 1.96
mmol). The resulting solution was stirred for 12 h at room
temperature. After removed of the solvent, the residue was
purified by silica gel column chromatography (hexane-acetone)
to give compound 8a (452 mg, 79%). 1H NMR (300 MHz,
DMSO) δ 1.31,1.56 (each s, each 3H, CH3 × 2), 3.85 (m, 2H, 2
× H5′), 4.15-4.23 (m, 2H, OCH2-), 4.35 (m, 2H, CH2OP), 4.47
(m, 1H, H4′), 5.03 (dd, 1H, JH3′,H4′ ) 3.0, JH2′,H3′ ) 6.0 Hz, H3′),
5.27 (dd, 1H, JH1′,H2′ ) 2.4, JH2′,H3′ ) 6.0 Hz, H2′), 5.38 (d, 1H,
1
H8); 31P NMR (D2O 81 MHz, decoupled with H) δ -9.69 (d,

5680 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Gu et al.
JP,P ) 13.4 Hz), -10.38 (d, JP,P ) 13.4 Hz); HRMS (FAB,
negative) for C13H18N4O12P2 Calcd, 483.0324 [(M - 1)-]; found,
483.0324.
The purification of the residue was performed at the same
procedure as compound 2a by twice HPLC on C18 reverse
column eluting with MeCN/TEAB (pH 7.5) and MeCN/TEAA
(pH 7.0) buffer respectively to give the target molecule 2b (8.5
mg. 89%). 1H NMR (500 MHz, D2O) δ 3.77-3.85 (m, 2H,
CH2O-), 3.93-4.00 (m, 2H, OCH2OP), 4.03-4.09 (m, 1H, H5a′),
4.30-4.32 (2H, m, H5b′, H4′), 4.85 (m, 1H, H3′), 5.25 (d, 1H,
JH1′′a,H1′′b ) 11.0 Hz, H1′′a), 5.68(t, 1H, JH1′,H2′ ) 5.0 Hz, JH3′,H2′
) 5.0 Hz, H2′), 5.99 (d, 1H, JH1′′a,H1′′b ) 11.0 Hz, H1′′b), 6.11(d,
1H, JH1′,H2′ ) 5.0 Hz, H1′), 8.51 (s, 1H, H2); 31P NMR (D2O 81
N1-[[[Bis-5′′-(phenylthio)phosphoryl]oxyethoxy]methyl]-
5′-O-(dianilinophosphoryl)-2′,3′-O-isopropylidene-8-bro-
moinosine (8b). Compound 6b (200 mg, 0.27 mmol)25 was
dissolved in 10 mL of methanol. To the solution was added
CH3ONa (4 mg, 0.07 mmol) and stirred at room temperature
for 2h. Dowex-50W acid cation-exchange resin was added to
adjust the solution to pH 7, and then filtered and removed of
the solvent under reduce pressure to give 7b. Compound 7b
was dissolved in dry pyridine (15 mL) and then added TPSCl
(164 mg, 0.54 mmol), PSS (308 mg, 0.81 mmol) and tetrazole
(57 mg, 0.81 mmol). The resulting solution was stirred for 12
h at room temperature. After removed of the solvent, the
residue was purified by silica gel column chromatography
(hexane-acetone) to give compound 8b (224 mg, 87%). 1H NMR
(300 MHz, DMSO) δ 1.51,1.29 (each s, each 3H, CH3 × 2), 3.80
(m, 2H, 2 × H5′), 4.07-4.37 (m, 5H, OCH2-, CH2OP, H4′), 5.06
(dd, 1H, JH3′,H4′ ) 3.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.30 (d, 1H,
1
MHz, decoupled with H) δ -9.70 (d, JP,P ) 10.0 Hz), -10.35
(d, JP,P ) 10.0 Hz); HRMS (TOF, negative) for C13H17BrN4O12P2
Calcd, 560.9502 [(M - 1)-]; found, 560.9449.
N1-[[[Bis-5′′-(phenylthio)phosphoryl]oxyethoxy]methyl]-
5′-O-(dianilinophosphoryl)-2′,3′-O-isopropylidene-8-azi-
doinosine (8c). Compound 6b (350 mg, 0.48 mmol) and NaN3
(100 mg, 1.52 mmol) were dissolved in 5 mL of anhydrous
DMSO, the mixture was reacted at 80 °C for 12 h under argon.
After the removal of DMSO, the residue was extracted with
CH2Cl2 (3 mL x 3). The extract was purified on silica gel
column (petroleum ether: acetone ) 3:1) to give 300 mg of 6c
in 90% yield. Compound 6c (200 mg, 0.29 mmol) was dissolved
in 10 mL of methanol. To the solution was added CH3ONa (4
mg, 0.07 mmol) and stirred at room temperature for 2h.
Dowex-50W acid cation-exchange resin was added to adjust
pH to 7, and then filtered and removed of the solvent under
reduced pressure to give 7c. Compound 7c was dissolved in
dry pyridine (10 mL) and then added TPSCl (179 mg, 0.59
mmol), PSS (354 mg, 0.93 mmol) and tetrazole (65 mg, 0.93
mmol). The resulting solution was stirred for 12 h at room
temperature. After removal of the solvent, the residue was
purified by silica gel column chromatography (hexane-acetone)
to give compound 8c (237 mg, 80%). 1H NMR (300 MHz,
DMSO) δ 1.26,1.49 (each s, each 3H, CH3 × 2), 3.82 (m, 2H, 2
× H5′), 4.00-4.33 (m, 5H, OCH2-, CH2OP, H4′), 5.05 (dd, 1H,
JH3′,H4′ ) 3.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.32 (dd, 1H, JH1′,H2′ ) 2.4,
JH1′′a,H1′′b ) 10.2 Hz, H1′′a), 5.45 (dd, 1H, JH1′,H2′ ) 2.4, JH2′,H3′
)
6.0 Hz, H2′), 5.49 (d, 1H, JH1′′a,H1′′b ) 10.2 Hz, H1′′b), 6.02 (d,
1H, JH1′,H2′ ) 2.4 Hz, H1′), 6.72-7.53 (m, 20H, Ar H), 8.01, 8.06
(each d, each1H, J
) 12.6 Hz, NH × 2), 8.30 (s, 1H,
NHa,NHb
1
H2); 31P NMR (DMSO, 81 MHz, decoupled with H) 2.80 ppm
(s); 51.08 ppm (s). ESI-TOF+-MS: m/z ) 956.2284 [(M + 1)+];
Anal. (C40H41N6O9BrP2S2‚2acetone) C, H, N.
N1-[[[5′′-(Phenylthio)phosphoryl]oxyethoxy]methyl]-
5′-O-phosphoryl-2′,3′-O- isopropylidene-8-bromoinosine
(9b). A solution of 8b (115 mg, 0.12 mmol) and isoamyl nitrite
(243 µL, 1.80 mmol) in pyridine-AcOH-Ac2O (2:1:1, v/v, 4 mL)
was stirred at room temperature for 8 h. After the reaction
mixture was evaporated (at <30 °C), the residue was dissolved
with H3PO2 (123 µL, 2.4 mmol) and Et3N (167 µL, 1.2 mmol)
in pyridine (3 mL), and the resulting solution was stirred for
11 h at room temperature. After the solvent was evaporated
to dryness under reduced pressure, the residue was partitioned
between CHCl3 and H2O. The aqueous layer was coevaporated
(<30 °C) with pyridine (5 mL), and the residue was purified
with the same procedure shown in the preparation of 9a to
give 9b as a triethylammonium salt. The compound was
purified again using same column eluting with MeCN/TEAA
buffer (pH 7.0) (93 mg, 85%). 1H NMR (500 MHz, D2O) δ 1.45,
1.65 (each s, each 3H, 2 × CH3), 3.70 (m,1H, H5′a), 3.92 (m,
3H, CH2O, H5′b), 4.08-4.12 (m, 3H, H4′, CH2OP), 5.26 (dd, 1H,
JH2′,H3′ ) 6.0 Hz, H2′), 5.38 (d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′a
)
5.45 (d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′b), 5.89 (d, 1H, JH1′,H2′
)
2.4 Hz, H1′), 6.76-7.52 (m, 20H, Ar H), 8.08 (t, J NHa,NHb ) 9.9
Hz, NH × 2), 8.34 (s, 1H, H2); 13C NMR (75 MHz,DMSO) δ
154.75, 148.56, 146.82, 143.42, 141.07, 135.07, 135.01, 129.70,
128.74, 128.62, 125.67, 125.58, 121.50, 120.38, 117.25, 117.17,
117.08, 113.68, 87.68, 85.18, 82.71, 80.64, 74.97, 68.11, 66.97,
64.17, 26.92, 25.17. 31P NMR (DMSO, 81 MHz, decoupled with
1H) 3.21 ppm (s); 49.37 ppm (s). ESI-TOF+-MS: m/z ) 918.21
[(M + 1)+]; Anal. (C40H41N9O9P2S2‚2acetone) C, H, N.
JH3′,H4′ ) 2.0, JH2′,H3′ ) 6.0 Hz, H3′), 5.39 (d, 1H, JH1′′a,H1′′b
)
N1-[[[5′′-(Phenylthio)phosphoryl]oxyethoxy]methyl]-
5′-O-phosphoryl-2′,3′-O-isopropylidene-8-azidoinosine (9c).
A solution of 8c (120 mg, 0.13 mmol) and isoamyl nitrite (263
µL, 1.95 mmol) in pyridine-AcOH-Ac2O (2:1:1, v/v, 4 mL) was
stirred at room temperature for 8 h. After the reaction mixture
was evaporated (at <30 °C), the residue was dissolved with
H3PO2 (133 µL, 2.6 mmol) and Et3N (181 µL, 1.3 mmol) in
pyridine (3 mL), and the resulting solution was stirred for 11
h at room temperature. The purification of 9c was same as
the shown in 9a and 9c was offered as a triethylammonium
salt. The compound was purified again using same column
eluting with MeCN/TEAA buffer (pH 7.0) (96 mg, 84%). 1H
NMR (500 MHz, D2O) δ 1.42, 1.65 (each s, each 3H, 2 × CH3),
3.84 (m, 2H, H5′), 4.11 (m, 4H, CH2O, CH2OP), 4.51 (m, 1H,
H4′) 5.18 (dd, 1H, JH3′,H4′ ) 3.0, JH2′,H3′ ) 6.5 Hz, H3′), 5.32 (dd,
1H, JH2′,H1′ ) 4.5, JH3′,H2′ ) 6.5 Hz, H2′), 5.42 (d, 1H, JH1′′a,H1′′b
) 10.5 Hz, H1′′a), 5.55 (d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′b), 6.09
(d, 1H, JH1′,H2′ ) 4.5 Hz, H1′), 7.20-7.42 (m, 5H, Ar H), 8.19 (s,
1H, H2); 31P NMR (D2O 81 MHz, decoupled with 1H) δ 3.01(s),
17.65(s). HRMS (TOF, negative) for C22H27N7O12P2S: Calcd,
675.0914 [(M - 1)-]; found, 675.0956.
10.5 Hz, H1′′a), 5.51 (dd, 1H, JH2′,H1′ ) 3.0, JH3′,H2′ ) 6.0 Hz,
H2′), 5.67(d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′b), 6.25 (d, 1H, JH1′,H2′
) 3.0 Hz, H1′), 7.18-7.52 (m, 5H, Ar H), 8.38 (s, 1H, H2); 31P
NMR (D2O 81 MHz, decoupled with 1H) δ 2.84(s), 17.69(s).
HRMS (TOF, negative) for C22H27N4O12BrP2S: Calcd, 712.3780-
[(M - 1)-]; found, 711.9582.
N1-[(5′′-O-Phosphorylethoxy)methyl]-2′,3′-O-isopropy-
lidene-5′-O-phosphoryl-8-bromoinosine 5′,5′′-Cyclicpy-
rophosphate (10b). A solution of 9b (25 mg, 27 µm) in
pyridine (5 mL) was added slowly over 20 h, using a syringe
pump, to a mixture of I2 (144 mg) and MS 3 Å (1.44 g), in
pyridine (40 mL) at room temperature in the dark. The
purification of 10b was same as the preparation of 10a and
10b was obtained as a triethylammonium salt. The compound
was purified again using same column eluting with MeCN/
TEAB buffer (pH 7.5) (19 mg, 87% yield). 1H NMR (500 MHz,
D2O) δ 1.46, 1.63 (each s, each 3H, 2 × CH3), 3.79-3.86 (m,
3H, CH2O, H5a′), 3.92-3.95 (m, 3H, H5′b, CH2OP), 4.59 (m, 1H,
H4′), 5.25 (d, 1H, JH1′′a,H1′′b ) 11.5 Hz, H1′′a) 5.49 (d, 1H, JH2′,H3′
) 4.0 Hz, H3′), 5.98-6.04 (m, 2H, H1′′b, H2′), 6.33 (s,1H, H1′),
1
8.52, (s, 1H, H2); 31P NMR (D2O 81 MHz, decoupled with H)
N1-[(5′′-O-Phosphorylethoxy)methyl]-2′,3′-O-isopropy-
lidene-5′-O-phosphoryl-8-azidoinosine 5′,5′′-Cyclicpyro-
phosphate (10c). A solution of 9c (29 mg, 33 µm) in pyridine
(5 mL) was added slowly over 20 h, using a syringe pump, to
a mixture of I2 (175 mg) and MS 3 Å (1.75 g), in pyridine (50
mL) at room temperature in the dark. After purification as
described in 10a, 10c was obtained as a triethylammonium
δ -9.65(d, JP,P ) 10.0 Hz), -10.35(d, JP,P ) 10.0 Hz). HRMS
(FAB, negative) for C16H21BrN4O12P2 Calcd, 600.9815 [(M -
1)-]; found, 600.9735.
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphoryl-
8- bromoinosine 5′,5′′-Cyclicpyrophosphate (2b). The
solution of 10b (10 mg, 12 µmol) in 60% HCOOH (4 mL) was
stirred for 8 h, and then evaporated under reduced pressure.

Membrane-Permeant Cyclic ADP-Ribose Mimics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5681
salt. The compound was purified again using same column
eluting with MeCN/TEAB buffer (pH 7.5) (22 mg, 87%). 1H
NMR (500 MHz, D2O) δ 1.45, 1.61 (each s, each 3H, 2 × CH3),
3.76-3.94 (m, 6H, CH2O, H5′, CH2OP), 4.55 (m, 1H, H4′), 5.30
(d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′a) 5.41 (dd, 1H, JH4′,H3′ ) 2.0
Hz, JH2′,H3′ ) 6.0 Hz, H3′), 5.89 (d, 1H, JH2′,H3′ ) 6.0 Hz,H2′),
5.94 (d, 1H, JH1′′a,H1′′b ) 10.5 Hz, H1′′b), 6.13 (s,1H, H1′), 8.45 (s,
Changes in Fura2 fluorescence were measured using a Hitachi
F-2000 spectrofluorometer (alternating excitation at 340 and
380, emission 495 nm). Intracellular [Ca2+] was calculated
after calibration using 0.1% Triton X-100 to obtain the
maximal ratio and EGTA/Tris (8 mM/60 mM) to obtain the
minimal ratio.
Determination of [Ca2+]i in Permeabilized Cells. Jur-
kat T cells were permeabilized by saponin and experiments
were done exactly as described.27 In brief, cells were exposed
to saponin in an intracellular buffer (20 mM HEPES, 110 mM
KCl, 2 mM MgCl2, 5 mM KH2PO4, 10 mM NaCl, pH 7.2) in
the absence of extracellular Ca2+. Then, saponin was removed
by repeated wash procedures, and the cells were finally
resuspended in intracellular buffer. After a 2 h period on ice
to allow for resealing of intracellular stores, the stores of
permeabilized cells were reloaded upon addition of ATP, and
an ATP-regenerating system consisting of creatine phosphate
and creatine kinase.35 [Ca2+] was monitored fluorimetrically
by addition of Fura2/free acid as described above for intact
cells. When the Ca2+ stores were refilled, cIDPRE or cADPR
were added. Each experiment was calibrated using excess
CaCl2 and EGTA/Tris.
1
1H, H2); 31P NMR (D2O 81 MHz, decoupled with H) δ -9.99
(d, JP,P ) 10.0 Hz), -10.72(d, JP,P ) 10.0 Hz). HRMS (TOF,
negative) for C16H21N7O12P2 Calcd, 564.0723 [(M - 1)-]; found,
564.0637.
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphoryl-
8-azidoinosine 5′,5′′-Cyclicpyrophosphate (2c). The same
procedure for the deprotection of 10a was applied to 10c (12
mg, 15.6 µmol) in 60% HCOOH (5 mL) to give the target
molecule 2c (10.5 mg) in 93% yield. 1H NMR (500 MHz, D2O)
δ 3.75-3.85 (m, 2H, CH2O), 3.93-9.98 (m, 2H, CH2OP), 4.02-
4.05 (m, 1H, H5a′), 4.27-4.31(m, 2H, H5b′, H4′), 4.85 (1H, m,
H3′), 5.26 (d, 1H, JH1′′a,H1′′b ) 11.5 Hz, H1′′a), 5.68(dd, 1H, JH1′,H2′
) 4.5 Hz, JH3′,H2′ ) 5.0 Hz, H2′), 5.87(d, 1H, JH1′,H2′ ) 4.5 Hz,
H1′), 5.98 (d, 1H, JH1′′a,H1′′b ) 11.5 Hz, H1′′b), 8.45 (s, 1H, H2);
31P NMR (D2O 81 MHz, decoupled with 1H) δ -9.72 (d, JP,P
)
Ratiometric Ca2+ Imaging. All procedures were carried
out exactly as described in detail in previous publications.36,38,39
In brief, we used an Improvision imaging system (Heidelberg,
Germany) consisting of a monochromator system (Polychro-
mator IV, TILL Photonics, Graefelfing, Germany) and a gray
scale CCD camera (type C4742-95-12ER; Hamamatsu, Enfield,
United Kingdom; operated in 8-bit mode) built around a Leica
DM IRB2 microscope at 100-fold magnification. Digital con-
focal images were obtained by mathematical deconvolution
based on the point-spread function using the nearest-neighbor
algorithm (Openlab software v3.0.9, Improvision, Heidelberg).
10.1 Hz), -10.10 (d, JP,P ) 10.1 Hz); HRMS (TOF, negative)
for C13H17N7O12P2 Calcd, 524.0410 [(M - 1)-]; found, 524.0344.
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphoryl-
8-aminoinosine 5′,5′′-Cyclicpyrophosphate (2d). The solu-
tion of 10c (10 mg, 13 µmol) in ethanol (5 mL) was stirred
hydrogen in the presence of 10% Pd/C at room temperature
for 8 h, then the catalyst was filtered and the solution was
evaporated under reduced pressure. The purification of the
residue was performed at the same procedure as compound
10c to give 10d in 99% yield (9.5 mg). Compound 10d was
deprotected with 60% formic acid solution at room temperature
and purified by twice HPLC on C18 reverse column eluting
with MeCN/TEAA (pH 7.0) and MeCN/TEAB (pH 7.5) buffer,
Acknowledgment. This study was supported by the
National Natural Science Foundation of China and the
Deutsche Foschungsgemeinschaft (grant no. GU 260/
2-4 and 2-5 to A.H.G), the Werner-Otto-Foundation (to
A.H.G), Research Funds of the Faculty of Medicine
(grant no. F-408-1 to A.H.G), and the Deutsche Akade-
mische Austauschdienst (grant no. 314-vigoni-dr to
A.H.G). This article is based in part on doctoral studies
by X.F.Gu in the School of Pharmaceutical Sciences,
Peking University, and R.Fliegert in the Faculty of
Biology, University of Hamburg.
1
respectively, to give the target molecule 2d (8 mg. 90%.) H
NMR (500 MHz, D2O) δ 3.74-3.81 (m, 2H, CH2O-), 3.91-9.93
(m, 2H, CH2OP), 4.02-4.04 (m, 1H, H5a′), 4.27-4.31 (m, 2H,
H5b′, H4′), 4.85 (1H, m, H3′), 5.23 (d, 1H, JH1′′a,H1′′b ) 11.0 Hz,
H
1′′a), 5.70 (t, 1H, JH1′,H2′ ) 5.0 Hz, JH3′,H2′ ) 5.0 Hz, H2′), 5.84
(d, 1H, JH1′,H2′ ) 5.0 Hz, H1′), 5.98 (d, 1H, JH1′′a,H1′′b ) 11.0 Hz,
H
1′′b), 8.34 (s, 1H, H2); 31P NMR (D2O 81 MHz, decoupled with
1H) δ -9.75 (d, JP,P ) 10.5 Hz), -9.90 (d, JP,P ) 10.5 Hz);
HRMS (TOF, positive) for C13H19N5O12P2 Calcd, 500.0505 [(M
+ 1)-]; found, 500.0834.
N1-[(5′′-O-Phosphorylethoxy)methyl]-5′-O-phosphoryl-
8- chloroinosine 5′,5′′-Cyclicpyrophosphate (2e) was pre-
pared by followed the known procedure.30
Appendix
Abbreviations: Ac, acetyl; cADPR, cyclic adenosine
5′-diphosphoribose; cIDPR, cyclic inosine 5′-diphospho-
ribose; cADPcR, cyclic adenosine 5′-diphosphocarbori-
bose; cIDPRE, N1-[(5′′-O-phosphorylethoxy)methyl]-5′-
O-phosphorylinosine 5′,5′′-cyclicpyrophosphate; NAD+,
nicotinamide adenosine dinucleotide; DBU, 1,8-diaza-
bicyclo[5.4.0]undec-7-ene; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; 1H NMR, proton nuclear
magnetic resonance; 13C NMR, carbon-13 nuclear mag-
netic resonance; 31P NMR, phosphorus nuclear magnetic
resonance; HMBC, heteronuclear multiple bond cor-
relation; HRFAB MS, high-resolution fast atom bom-
bardment mass spectrometry; MALDI, matrix-assisted
laser desorption/ionization; ESI, electrospray ionization;
TOF, time-of-flight; HPLC, high performance liquid
chromatography; InsP3, inositol trisphosphate, 3 Å MS,
3 Å molecular sieve, THF, tetrahydrofuran; Py, pyridine;
TPSCl, triisopropylbenzenesulfonyl chloride; PSS, cy-
clohexylammonium S,S-diphenyl phosphorodithioate;
TBDMSCl, tert-butyldimethylsilyl chloride; TEAA, tri-
ethylammonium acetate; TEAB, triethylammonium bi-
carbonate; TBAF, tetrabutylammonium fluoride; TMS,
tetramethyl silicane; UV, ultraviolet spectrometry.
Pharmacology. Cell Culture. Jurkat T-lymphocytes (clone
JMP and #E2) were cultured as described.35,36 In brief, cells
were cultured in RPMI 1640 supplemented with Glutamax I,
25 mM HEPES, 100 units/mL penicillin, 50 µg/mL strepto-
mycin (both clones), 7.5% (v/v) newborn calf serum (clone
JMP), 10% (v/v) fetal calf serum, 1 mM sodium pyruvate, 50
µg/mL hygromycin and 400 µg/mL G418-Sulfat (clone #E2).
Loading of Cells with Fura2/AM. The cells were loaded
with Fura-2/AM as described32 and kept in the dark at room
temperature until use.
Determination of [Ca2+]i in Intact Cells. [Ca2+]i was
measured in the presence of 1 mM extracellular Ca2+ in Jurkat
T-lymphocytes using Fura2/AM as described35 except that a
F-2000 spectrofluorimeter (Hitachi) was used in the ratio
mode. A modified version of the Ca2+ free/Ca2+ readdition
protocol37 was used. For the latter, 2.4 × 107 Jurkat cells were
pelleted for 5 min at 500g, and the supernatant was removed.
Cells were washed in extracellular solution composed of 140
mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM
glucose, 0.1% bovine serum albumin, 15 mM HEPES, pH 7.4,
and resuspended in 1 mL of extracellular solution supple-
mented with 4 µM Fura2/AM. Cells were incubated for 30 min
at 37°C, washed once and resuspended in extracellular solution
at 2 × 106 cells/mL. Aliquots of 2 mL were kept in the dark at
room temperature until use. Before each measurement, cells
were washed twice in extracellular solution without CaCl2.

5682 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Gu et al.
(23) Galeone, A.; Mayol, L.; Oliviero, G.; Piccialli, G.; Varra, M.
Synthesis of a new N1-pentyl analogue of cyclic inosine diphos-
phate ribose (cIDPR) as a stable potential mimic of cyclic ADP
ribose (cADPR). Eur. J. Org. Chem. 2002, 4234-4238
(24) Galeone, A.; Mayol, L.; Oliviero, G.; Piccialli, G.; Varra, M.
Synthesis of a novel N-1 carbocyclic, N-9 butyl analogue of cyclic
ADP ribose (cADPR). Tetrahedron 2002, 58, 363-368
(25) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda A.
Nucleosides and nucleotides 173. Synthesis of cyclic IDP-
carbocyclic-ribose, a stable mimic of cyclic ADP-ribose, significant
facilitation of the intramolecular condensation reaction of N-1-
(carbocyclic-ribosyl) inosine 5′,6′-diphosphate derivatives by an
8-bromo-substitution at the hypoxyanthine moiety. J. Org.
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(26) Shuto, S.; Fukuoka, M.; Manikowasky, A.; Ueno, Y.; Nakano,
T.; Kuroda, R.; Kuroda, H.; Matsuda, A. Total synthesis of cyclic
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(27) Guse, A. H.; Cakir-Kiefer, C.; Fukuoka, M.; Shuto, S.; Weber,
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Supporting Information Available: Elemental analyses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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