Convergent Synthesis and Unexpected Ca2+-Mobilizing Activity of 8-Substituted Analogues of Cyclic ADP-Carbocyclic-Ribose, a Stable Mimic of the Ca2+-Mobilizing Second Messenger Cyclic ADP-Ribose

Article abstract of DOI:10.1021/jm030227f

Cyclic ADP-carbocyclic-ribose (cADPcR, 2) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, 1), a Ca²⁺-mobilizing second messenger. In this study, a series of 8-substituted analogues of cADPcR, namely the 8-chloro

Full text of DOI:10.1021/jm030227f

J . Med. Chem. 2003, 46, 4741-4749
4741
Con ver gen t Syn th esis a n d Un exp ected Ca 2+-Mobilizin g Activity of 8-Su bstitu ted
An a logu es of Cyclic ADP -Ca r bocyclic-Ribose, a Sta ble Mim ic of th e
Ca 2+-Mobilizin g Secon d Messen ger Cyclic ADP -Ribose
Satoshi Shuto,*,† Masayoshi Fukuoka,† Takashi Kudoh,† Clive Garnham,‡ Antony Galione,‡
Barry V. L. Potter,§ and Akira Matsuda†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, J apan,
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K., and Wolfson Laboratory for
Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, U.K.
Received May 13, 2003
Cyclic ADP-carbocyclic-ribose (cADPcR, 2) is a biologically and chemically stable equivalent of
cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger. In this study, a series of
8-substituted analogues of cADPcR, namely the 8-chloro analogue 6 (8-Cl-cADPcR), the 8-azido
analogue 7 (8-N3-cADPcR), the 8-amino analogue 8 (8-NH2-cADPcR), and the 8-phenylthio
analogue 9 (8-SPh-cADPcR), were designed as effective pharmacological tools for studies on
cADPR-modulated Ca2+ signaling pathways. These target compounds were synthesized by a
convergent route via 8-Cl-cADPcR bisacetonide (14) as the common intermediate, in which a
method for forming the intramolecular pyrophosphate linkage by activation of the phenylthio-
phosphate type substrate 15 with AgNO3 to produce 14 was used as the key step. The carbocyclic
analogues were tested for activity in the sea urchin egg homogenate system. Compounds were
assessed for their calcium-mobilizing effects and their ability to cross-desensitize with calcium
release induced by a normally maximal concentration of cADPR, as well as cADPR antagonism
of cADPR-evoked calcium release. While cADPcR was 3-4 times more potent than cADPR,
the 8-substituted analogues were less efficacious, with 8-SPh-cADPcR largely acting as a
competitive antagonist. Most surprisingly, given that 8-N3-cADPR and 8-NH2-cADPR are known
as potent antagonists, 8-N3-cADPcR and 8-NH2-cADPcR were full agonists, but ca. 80 and 2
times less potent than cADPR, respectively. These data contribute to developing structure-
activity relationships for the interaction of cADPR with its receptor.
In tr od u ction
Considerable attention has been focused on cyclic
ADP-ribose (cADPR, 1), an intracellular Ca2+-mobilizing
adenine nucleotide.2 cADPR has been shown to mobilize
intracellular Ca2+ in various cells, such as sea urchin
eggs, pancreatic beta cells, smooth muscles, cardiac
muscles, T-lymphocytes, and cerebella neurons, indicat-
ing that it is a general mediator involved in Ca2+
signaling.3 The structure of cADPR has been investi-
gated4 and was confirmed by X-ray crystallographic
analysis as shown in Figure 1.4c
F igu r e 1. Structures of cADPR (1), cADPcR (2), and their
8-substituted analogues.
In cells, cADPR is synthesized from NAD+ by ADP-
ribosyl cyclases and acts as a second messenger; it is
hydrolyzed rapidly by cADPR hydrolases to give the
non-Ca2+ mobilizing metabolite ADP-ribose under physi-
ological conditions.3 cADPR is also known to be readily
hydrolyzed nonenzymatically at the unstable N-1-gly-
cosidic linkage of its adenine moiety to give ADP-ribose,
even in neutral aqueous solution.5 Consequently, the
biological as well as chemical instability of cADPR
limits, to some extent, further studies of its physiological
role. Therefore, stable analogues of cADPR exhibiting
a Ca2+-mobilizing activity in cells similar to that of
cADPR are very useful in biological studies.
We designed cyclic ADP-carbocyclic-ribose (cADPcR,
2) as a stable mimic of cADPR,6 in which the oxygen
atom in the N-1-ribose ring of cADPR is replaced by a
methylene group. We hypothesized that (1) the mimic
should be resistant to both enzymatic and chemical
hydrolysis, since it has a chemically and biologically
stable N-alkyl linkage instead of the unstable N-1-
glycosidic linkage of cADPR, and that (2) the mimic, like
cADPR, would effectively mobilize intracellular Ca2+
since it preserves all of the functional groups of cADPR
except for the ring oxygen of the N-1-linked ribose and
should have a conformation similar to that of cADPR.
We recently accomplished the total synthesis of the
mimic 2, showing that it is actually chemically and
biologically stable.6f The preliminary evaluation of 2
injected into sea urchin eggs suggested that it seems to
* Corresponding author. Tel: 81-11-706-3229. Fax: 81-11-706-
4980. E-mail: shu@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ University of Oxford.
§ University of Bath.
10.1021/jm030227f CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/23/2003

4742 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Shuto et al.
have very potent Ca2+-mobilizing activity.6f Therefore,
studies on the mechanism of cADPR-modulated Ca2+
signaling pathways using the mimic 2 are now in
progress.
Sch em e 1
cADPR and its analogues have been synthesized by
enzymatic or chemoenzymatic methods using ADP-
ribosyl cyclase from Aplysia californica, which mediates
the intramolecular ribosylation of NAD+ and some
modified NAD+ (prepared chemically or enzymatically)
at the N-1-position of the purine moiety, to yield cADPR
or the corresponding analogues.7 These studies disclosed
that some 8-substituted analogues of cADPR, such as
8-Br-cADPR (3), 8-N3-cADPR (4), or 8-NH2-cADPR (5),
are antagonists of cADPR at its intracellular receptor,7a
and these analogues have been effectively used as
pharmacological tools for the studies on cADPR-modu-
lated Ca2+ signaling pathways.3
Encouraged by these results, we decided to synthesize
the target 8-substituted cADPcR analogues 6-9 by a
convergent route, as summarized in Scheme 2, using
8-Cl-cADPcR bis-acetonide (14) as the common inter-
mediate. Acidic deprotection of 14 would readily provide
8-Cl-cADPcR (6). The other three targets 7, 8, and 9
would be obtained by nucleophilic addition-elimination
reaction at the 8-position of 14 followed by deprotection.
The intramolecular pyrophosphate linkage could be
constructed according to the method described above:
treatment of 8-chloro-5′-phenylthiophosphate substrate
15 with AgNO3/MS 3A as a promoter6c,e,f was expected
to give the cyclized 14. The substrate 15 would be
provided from the 2-chloroimidazole nucleoside deriva-
tive 16 and the optically active carbocyclic amine 17,
which could be prepared from 5-aminoimidazole-4-
carboxamide riboside (AICAR, 18) and commercially
available (1R)-(-)-2-azabicyclo[2.2.1]hept-5-en-3-one
(19).8c
These findings prompted us to synthesize the 8-modi-
fied analogues of cADPcR, which were expected to
possess the properties of both the carbocyclic analogue
2 and the 8-substituted cADPR analogues. For example,
8-NH2-cADPcR (8) was expected to be a chemically and
biologically stable potent antagonist of cADPR, and
8-N3-cADPcR (7) might be used as an efficient photo-
affinity labeling probe for cADPR-related proteins.
In this report, we describe the convergent synthesis
of a series of 8-substituted analogues of cADPcR, namely
the 8-chloro analogue 6 (8-Cl-cADPcR), the 8-azido
analogue 7 (8-N3-cADPcR), the 8-amino analogue 8
(8-NH2-cADPcR), and the 8-phenylthio analogue 9
(8-SPh-cADPcR) (Figure 1), and their effects on Ca2+
-
Con str u ction of th e N-1-Ca r bocyclic-Ribosyla d e-
n osin e Str u ctu r e. An efficient method for constructing
the N-1-carbocyclic-ribosyladenosine structure was es-
sential for completing the synthesis of the targets. We
previously found that treatment of the imidazole nu-
cleoside 22 and the chiral carbocyclic amine 17 under
mild basic conditions with K2CO3 provided the N-1-
carbocyclic-ribosyladenosine derivative 23 in high yield
(Scheme 3).6f Thus, construction of the 8-chloro-N-1-
carbocyclic-ribosyl derivative 24 was investigated ac-
cording to a similar procedure. A chloro group was
introduced at the imidazole 2-position of the known
imidazole nucleoside 208c by treatment with N-chloro-
succinimide (NCS) in THF to give 21. The 2-chloroimi-
dazole nucleoside 21 was heated in HC(OMe)3 under
reflux in the presence of a catalytic amount of TFA to
give the methoxymethylene derivative 16, which was
next subjected to the pyrimidine ring-closure reaction.
When a mixture of 16 and 17 (1.4 equiv) was treated
with K2CO3 in DMF at room temperature, the ring-
closure reaction proceeded to give the desired product
24 in 89% yield. The N-1-carbocyclic-ribosyladenine
structure of 24 was confirmed by an NOE experiment;
a correlation (19.6%) between the H-2 of the adenine
and the C-1′′ of the cyclopentane ring was observed.
Syn th esis of 8-Cl-cADP cR. 8-Cl-cADPcR (6) was
successfully synthesized from the N-1-carbocyclic-ribo-
syl-adenosine derivative 24, as shown in Scheme 4.
After protection of the 5′′-hydroxyl of 24 with a
monomethoxytrityl (MMTr) group, the 5′-O-TBS group
of the product 25 was removed with TBAF to give 26.
Treatment of 26 with an S,S′-diphenylphosphorodi-
thioate (PSS)/2,4,6-triisopropylbenzenesulfonyl chloride
(TPSCl)/pyridine system9 gave the 5′-bis(phenylthio)-
phosphate 27 in 67% yield. After removal of the 5′′-O-
mobilization in the sea urchin homogenate system. The
detailed Ca2+-mobilizing activity of cADPcR is also
described. The carbocyclic analogues were tested for
activity in sea urchin egg homogenate, a robust assay
for calcium mobilization, and the system in which the
calcium mobilizing properties of cADPR were first
discovered.4a
Resu lts a n d Discu ssion
Syn th etic P la n . Most of the previous cADPR ana-
logues have been synthesized by enzymatic or chemoen-
zymatic methods with ADP-ribosyl cyclase from A.
californica as described above.7 Although the specificity
of ADP-ribosyl cyclase is somewhat loose, the analogues
obtained by this method are limited by the substrate
specificity of the enzyme.7p
On the other hand, in the chemical synthesis of
cADPR and its analogues, the key intramolecular
condensation step to form the pyrophosphate linkage
has proved difficult, preventing completion of the chemi-
cal synthesis of the target cADPR analogues.8 However,
in recent years, we have developed an efficient method
for forming the intramolecular pyrophosphate linkage
by activation of phenylthiophosphate type substrates,
such as 10 or 11, with I2 or AgNO3 in the presence of
molecular sieves (MS) 3A in pyridine, where the cy-
clization products 12 and 13 were obtained in 93% and
81% yield, respectively (Scheme 1).6 Using this method
we have successfully synthesized cADPcR (2) and its
inosine congener.6e,f This represents a general method
for synthesizing these types of biologically important
cyclic nucleotides and particularly those chemically
modified in the N-1-linked ribose moiety, which are not
expected to be accessible by the chemoenzymatic route.6g

Unexpected Agonism by 8-Substituted cADPcR
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4743
Sch em e 2
Sch em e 3a
MMTr group of 27, a phosphoryl group was introduced
at the resulting 5′′-primary hydroxyl of 28 using Yoshika-
wa’s method with POCl3/(EtO)3PO,10 followed by treat-
ment of the product with H3PO2 and Et3N11 in the
presence of N-methylmaleimide (NMM) in pyridine,12
affording 5′-phenylthiophosphate 15 in 47% yield as the
triethylammonium salt. When a solution of 15 in
pyridine was added slowly to a mixture of a large excess
of AgNO3 and Et3N in the presence of MS 3A in pyridine
at room temperature,6c,e,f the desired cyclization product
14 was obtained in 88% yield. Finally, removal of the
isopropylidene groups of 14 was carried out with aque-
ous HCO2H to furnish 8-Cl-cADPcR (6).
Syn th esis of th e 8-Azid o-, 8-Am in o-, a n d 8-P h e-
n ylth io-cADP cR An a logu es. The other three 8-sub-
stituted cADPcR analogues, 7, 8, and 9, were synthe-
sized from the isopropylidene-protected 8-Cl-cADPcR 14
via a nucleophilic addition-elimination reaction at the
8-position. Thus, treatment of 14 with LiN3 or PhSH in
pyridine at room temperature produced the correspond-
ing 8-azido and 8-phenylthio derivatives 29 and 31 in
excellent yields (Scheme 5). The azido group of 29 was
readily reduced under usual catalytic hydrogenation
conditions with Pd-C to give the 8-amino derivative 30.
The isopropylidene groups of 29, 30, and 31 were
removed with aqueous HCO2H to provide the target
compounds, 8-N3-cADPcR (7), 8-NH2-cADPcR (8), and
8-SPh-cADPcR (9), respectively.
The results of this study as well as the previous
syntheses of cADPcR (2)7f and its inosine congener7c,e
clearly demonstrate that the strategy using a phenyl-
thiophosphate-type substrate in the key intramolecular
condensation reaction forming the pyrophosphate link-
age is very efficient for the total syntheses of cADPR-
related compounds.13
Biology. In the sea urchin egg homogenate, cADPR
(1) mobilizes sequestered Ca2+ from microsomal vesicles
in a concentration-dependent fashion (Figure 2a).14 Such
Ca2+ release exhibits the property of self-induced de-
sensitization,15 in that a subsequent challenge by a
normally maximum concentration of cADPR (250 nM)
is reduced depending on the initial concentration of the
first cADPR challenge. We demonstrated the biological
properties of cADPcR (2) by showing that it was a potent
Ca2+-mobilizing molecule and that after sequestration
of mobilized calcium it cross-desensitized with calcium
release by the subsequent addition of a normally
maximal concentration of cADPR (250 nM) in a concen-
tration-dependent manner as for cADPR (Figure 2b).
a
Reagents and conditions: (a) NCS, THF, rt, 84%; (b) HC(OMe)3,
cat. CF3CO2H, reflux, 95% (16), quant % (22); (c)17, K2CO3, MeOH
or DMF, rt, 83% (23), 89% (24).
Interestingly, cADPcR, with an EC50 value of 14.6 nM,
was ca. 3-4 times more potent than cADPR (EC50 ) 50
nM). These qualitative data, showing that cADPcR is
an even more potent Ca2+-mobilizing agent than cAD-
PR, are in accord with the preliminary results by the
injection of the compounds into sea urchin eggs.6f
Next, the effects of a series of 8-substituted carbocyclic
analogues of cADPR were examined. Surprisingly, both
8-N3-cADPcR (7) and 8-NH2-cADPcR (8) were full ago-
nists, with EC50 values of 3.9 µM (Figure 2c) and 80
nM, respectively (Figure 2d), and were therefore ca. 80
and 2 times less potent than cADPR in mobilizing
intracellular Ca2+. This is in contrast to the effects of
the corresponding non-carbocyclic analogues, 8-N3-
cADPR and 8-NH2-cADPR, which are both antagonists.7a
In addition, the 8-substituted carbocyclic analogues 7
and 8 also exhibited the property of cross-desensitiza-
tion with cADPR-induced Ca2+ release.
Further 8-substituted analogues were assessed. 8-Cl-
cADPcR (6) mobilized Ca2+ with a steep concentration-
dependence (Figure 2e) with an EC50 of 19 µM, i.e., ca.
400-fold less potent than cADPR. This compound,
however, did not fully cross-desensitize with cADPR for
reasons that are not immediately apparent. Increasing
the size and hydrophobicity of the 8-substituted group
in 8-SPh-cADPcR (9) dramatically altered its pharma-
cological properties. This compound had minimal Ca2+
mobilizing efficacy, while at high concentrations it
behaved as a competitive cADPR antagonist (Figure 2f).

4744 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Shuto et al.
Sch em e 4a
a
Reagents and conditions: (a) MMTrCl, pyridine, rt, 79%; (b) TBAF, THF, AcOH, rt, 88%; (c) PSS, TPSCl, py, rt, 67%; (d) aq 80%
AcOH, rt, 85%; (e) 1) POCl3, (EtO)3PO,0 °C, (2) H3PO2, Et3N, NMM, pyridine, rt, 47%; (g) AgNO3, MS 3A, Et3N, py, rt, 88%; (h) aq 60%
HCO2H, rt, 98%.
Sch em e 5a
speculate at this time as to the reasons behind this, but
clearly deletion of the “northern” ribofuranosyl oxygen
atom removes a structural motif in an extremely sensi-
tive area that determines the fundamental activity.
Interestingly, when cADPcR (2) and its 8-amino deriva-
tive 8 were examined for Ca2+-mobilizing activity in
another well characterized biological system, J urkat
T-lymphocytes, both compounds were found to exhibit
weak agonism, being about 5-10-fold less potent than
cADPR.6g Thus, in a mammalian cell system, cADPcR
behaves, in terms of potency, differently to in the
invertebrate sea urchin system. However, 8-NH2-cAD-
PcR, while weak, does also exhibit agonist rather than
antagonist properties, which is in qualitative agreement
with the data reported here. No further interpretation
on these relative aspects can be entered into at the
present time, except insofar as it is known that these
two receptors exhibit different recognition characteris-
tics toward some cADPR analogues,3h pointing to not
surprising potentially wider differences in protein struc-
ture and function.3 It is worth also noting that recently
diverse analogues have been synthesized with more
radical changes in the adenine ring and also in the
“northern” ribose than those reported here.13 Interest-
ingly, some of these compounds, although substituted
at the 8-position, like most of the compounds reported
here, also appear in intact HeLa cells and rat brain
microsomes to exhibit agonist rather than antagonist
properties.13b,c
a
Reagents and conditions: (a) PhSH, py, rt, 86% (29); (b) LiN3,
py, 50 °C, 81%; (c) H2, Pd-C, H2O, rt, 67%; (d) aq 60% HCO2H, rt,
97% (7), 83% (8), 88% (9).
cADPcR is a highly potent agonist in sea urchin
homogenates and indeed even more potent than cADPR
itself. This is due either to lack of hydrolysis during the
assay, or perhaps to a more favorable interaction of the
modified N-1-attached “northern ribose”6g with the
receptor, which would be rather surprising given that
a potential H-bond acceptor has been deleted.
The simple expectation for the activity of the 8-sub-
stituted analogues reported here was that they would
retain significantly the activity of their parent 8-sub-
stituted cADPR analogues, perhaps with some modula-
tion of Ca2+-mobilizing potency, but that the nature of
the activity agonist vs antagonist would not be affected.
If we leave aside the activity of the 8-phenylthio
analogue 9, which only begins to be apparent at high
concentrations, it is very striking that all three of the
other analogues 6, 7, and 8 that possess the 8-substitu-
tions, previously shown to confer antagonist behavior
to various degrees in the cADPR series, become agonists
as their cADPcR counterparts. Nowhere is this more
apparent than with 8-NH2-cADPcR (8), which has a
potency very close to cADPR itself. It is only possible to
Although the factors that control agonist/antagonist
behavior in cADPR are not well understood, the present
work provides new insight into them in analogues
particularly close in structure to cADPR itself. It seems
unlikely however that one unique motif is all pervading
in this respect and more so that a global conformation
is adopted in either mode, that may be perturbed by
point changes in the molecule, for example by disruption
of hydrogen bonds or changes in the conformation of the

Unexpected Agonism by 8-Substituted cADPcR
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4745
F igu r e 2. Ca2+ release by cADPR (1) and its carbocyclic analogues in the sea urchin egg homogenate: (a) cADPcR (1), (b) cADPcR
(2), (c) 8-N3-cADPcR (7), (d) 3-NH2-cADPcR (8), (e) 8-Cl-cADPcR (6), and (f) 8-SPh-cADPcR (9).
1
ribose groups. The previous preliminary H NMR analy-
sis suggested that an overall change in the conformation
of cADPcR compared with natural cADPR is unlikely.6f
Further detailed studies to clarify the precise conforma-
tion of the molecules are required, particularly in
relation to the biological activity. There is no doubt that
this antagonist/agonist switch induced by a rather
conservative carbocycle introduction is a novel effect
that will stimulate further work toward this goal.
agonist. Elucidation of the structural basis for this effect
will be of significant future interest.
Exp er im en ta l Section
Chemical shifts are reported in ppm downfield from tet-
1
ramethylsilane (1H and 13C) or H3PO4 (31P). All of the H NMR
assignments described were in agreement with COSY spectra.
Thin-layer chromatography was done on Merck coated plate
60F254. Silica gel chromatography was done on Merck silica
gel 5715. Reactions were carried out under an argon atmos-
phere.
Con clu sion . A series of 8-substituted analogues of
cADPcR was synthesized by a convergent route via 8-Cl-
cADPcR bis-acetonide as the common intermediate, in
which a method for forming the intramolecular pyro-
phosphate linkage by activation of the phenylthiophos-
phate moiety with AgNO3 was used as the key step. We
2-Ch lor o-5-a m in o-1-[5-O-(ter t-bu tyld im eth ylsilyl)-2,3-
O-(isop r op ylid e n e )-â-D-r ib ofu r a n osyl]im id a zole -4-n i-
tr ile (21). A mixture of 20 (5.9 g, 15.0 mmol) and NCS (2.4 g,
18.0 mmol) in THF (150 mL) was stirred at room temperature
for 10.5 h. After addition of saturated aqueous NaHCO3 (10
mL), the resulting solution was stirred at room temperature
for 10 min and then evaporated. The residue was partitioned
between EtOAc and H2O, and the organic layer was washed
with brine, dried (Na2SO4), and evaporated. The residue was
purified by column chromatography (SiO2, 20% AcOEt in
hexane) to give 21 (5.4 g, 84%) as a yellow solid: 1H NMR
(CDCl3, 500 MHz) δ 5.84 (d, 1 H, H-1′, J 1′,2′ ) 4.0 Hz), 5.04 (s,
2 H, NH2), 4.97 (dd, 1 H, H-2′, J 2′,1′ ) 4.0, J 2′,3′ ) 6.8 Hz), 4.89
(dd, 1 H, H-3′, J 3′,2′ ) 6.8, J 3′,4′ ) 4.3 Hz), 4.14 (ddd, 1 H, H-4′,
J 4′,3′ ) 4.3, J 4′,5′a ) 1.7, J 4′,5′b ) 0.9 Hz), 4.02 (dd, 1 H, H-5′a,
J 5′a,4′ ) 1.7, J 5′a,5′b ) 11.7 Hz), 3.90 (dd, 1 H, H-5′b, J 5′b,4′ ) 0.9,
J 5′b,5′a ) 11.7 Hz), 1.59, 1.36 (each s, each 3 H, isopropyl CH3),
have characterized quantitatively the more potent Ca2+
-
mobilizing activity of cADPcR than the natural second
messenger cADPR. Additionally, to contribute toward
the emerging SAR of cADPR, we have evaluated the
8-substituted analogues, most of which possess nonad-
ditive and unexpected biological activity as agonists
rather than antagonists. One, in particular, by means
of a single substitution illustrates a dramatic switch
from a highly potent antagonist to an almost equipotent

4746 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Shuto et al.
0.93 (s, 9H, tert-butyl), 0.14, 0.13 (each s, each 3H, dimethyl);
13C NMR (CDCl3, 125 MHz) δ 147.3, 125.6, 115.6, 114.6, 93.5,
93.5, 90.6, 84.5, 82.1, 78.8, 62.4, 27.3, 26.0, 25.4, 18.7, -5.4;
HRMS (FAB, positive) calcd for C18H30ClN4O4Si 429.1725
1.28 (each s, each 3 H, isopropyl CH3), 0.88 (s, 9 H, tert-butyl),
0.02, 0.01 (each s, each 3 H, dimethyl); 13C NMR (CDCl3, 67.8
MHz) δ 158.5, 152.8, 146.4, 144.6, 144.5, 141.1, 135.7, 135.0,
130.4, 128.4, 127.7, 126.8, 122.8, 114.2, 113.1, 113.0, 90.0, 87.6,
86.2, 83.1, 82.4, 81.7, 64.8, 64.2, 63.0, 55.2, 44.9, 33.2, 27.7,
27.2, 25.9, 25.4, 25.3, 18.4, -5.3, -5.4; HRMS (FAB, positive)
calcd for C48H61ClN5O8Si 898.3978 (MH+), found 898.3979; UV
(MeOH) λmax 263 nm, sh 301 nm. Anal. (C48H60ClN5O8Si) C,
H, Cl, N.
(MH+), found 429.1683; UV (MeOH) λmax 250 nm. Anal. (C18H29
-
ClN4O4Si) C, H, Cl, N.
2-Ch lor o-5-(m eth oxym eth ylen ea m in o)-1-[5-O-(ter t-bu -
tyld im eth ylsilyl)-2,3-O-(isop r op ylid en e)-â-D-r ibofu r a n o-
syl]im id a zole-4-n itr ile (16). A mixture of 21 (4.7 g, 11 mmol)
and TFA (43 µL, 550 µmol) in (MeO)3CH (23.5 mL, 220 mmol)
was stirred at 50 °C for 10 min. The mixture was evaporated,
and the residue was purified by column chromatography (SiO2,
20% AcOEt in hexane) to give 16 (4.9 g, 95%) as a yellow
solid: 1H NMR (CDCl3, 500 MHz) δ 8.34 (s, 1 H, NdCH), 5.94
(d, 1 H, H-1′, J 1′,2′ ) 3.2 Hz), 5.23 (dd, 1 H, H-2′, J 2′,1′ ) 3.2,
J 2′,3′ ) 6.8 Hz), 4.79 (dd, 1 H, H-3′, J 3′,2′ ) 6.8, J 3′,4′ ) 5.1 Hz),
4.05 (m, 1 H, H-4′), 3.98 (s, 3 H, OCH3), 3.83-3.76 (m, 2 H,
H-5′), 1.58, 1.36 (each s, each 3 H, isopropyl CH3), 0.89 (s, 9
H, tert-butyl), 0.06, 0.05 (each s, each 3 H, dimethyl); 13C NMR
(CDCl3, 67.8 MHz) δ 160.9, 145.7, 130.5, 115.3, 114.4, 98.9,
89.0, 85.7, 82.4, 80.3, 62.8, 55.2, 27.3, 25.9, 25.5, 18.4, -5.4;
HRMS (FAB, positive) calcd for C20H32ClN4O5Si 471.1830
8-Ch lor o-N-1-[(1R,2S,3R,4R)-2,3-(isopr opyliden edioxy)-
4-[(5-m on om eth oxytr ityl)oxym eth yl]cyclop en tyl]-2′,3′-O-
isop r op ylid en ea d en osin e (26). A mixture of 25 (6.1 g, 6.8
mmol), TBAF (1.0 M in THF, 15 mL, 15 mmol), and AcOH
(440 µL, 6.9 mmol) in THF (10 mL) was stirred at room
temperature for 1 h and then evaporated. The residue was
purified by column chromatography (SiO2, 60% AcOEt in
hexane) to give 26 (4.7 g, 88%) as a yellow foam: 1H NMR
(CDCl3, 500 MHz) δ 7.69 (s, 1 H, H-2), 7.44-6.81 (m, 15 H,
NH, Ar-H), 6.00 (d, 1 H, H-1′, J 1′,2′ ) 5.0 Hz), 5.28 (m, 1 H,
5′-OH), 5.11-5.09 (m, 2 H, H-2′, H-2′′), 5.02 (dd, 1 H, H-3′,
J 3′,2′ ) 5.8, J 3′,4′ ) 1.0 Hz), 4.91 (m, 1 H, H-1′′), 4.53 (m, 1 H,
H-3′′), 4.45 (m, 1 H, H-4′), 3.91 (m, 1 H, H-5′a), 3.79 (s, 3 H,
OCH3), 3.75 (m, 1 H, H-5′b), 3.34 (dd, 1 H, H-5′′a, J 5′′a,4′′ ) 3.5,
(MH+), found 471.1805; UV (MeOH) λmax 277 nm. Anal.(C20H31
ClN4O5Si C18H29ClN4O4Si) C, H, Cl, N.
-
J 5′′a,5′′b ) 8.8 Hz), 3.18 (dd, 1 H, H-5′′b, J 5′′b,4′′ ) 5.6, J 5′′b,5′′a )
8.8 Hz), 2.45-2.38 (m, 3 H, H-4′′, H-6′′), 1.64, 1.52, 1.38, 1.27
(each s, each 3 H, isopropyl CH3); 13C NMR (CDCl3, 67.8 MHz)
δ 158.5, 152.5, 146.7, 144.5, 144.5, 140.3, 135.7, 134.5, 130.3,
128.4, 127.7, 126.8, 123.4, 114.2, 113.3, 113.0, 92.2, 86.1, 85.5,
83.1, 82.2, 81.5, 81.2, 64.8, 64.2, 63.1, 55.2, 44.7, 33.4, 27.7,
27.5, 25.3, 25.3; HRMS (FAB, positive) calcd for C42H47ClN5O8
784.3113 (MH+), found 784.3090; UV (MeOH) λmax 264 nm,
sh 307 nm. Anal. (C42H46ClN5O8) C, H, Cl, N.
8-Ch lor o-N-1-[(1R,2S,3R,4R)-2,3-(isopr opyliden edioxy)-
4-(h yd r oxym eth yl)cyclop en tyl]-5′-O-(ter t-bu tyld im eth yl-
silyl)-2′,3′-O-isop r op ylid en ea d en osin e (24). A mixture of
16 (4.7 g, 10 mmol), 17 (2.7 g, 14 mmol), and K2CO3 (20 mg,
0.15 mmol) in DMF (65 mL) was stirred at room temperature
for 20 h and then evaporated. The residue was partitioned
between EtOAc and H2O, and the organic layer was washed
with brine, dried (Na2SO4), and evaporated. The residue was
purified by column chromatography (SiO2, 90% AcOEt in
hexane) to give 24 (5.6 g, 89%) as a white foam: 1H NMR
(CDCl3, 500 MHz) δ 7.61 (s, 1 H, H-2), 7.17 (br s, 1 H, NH),
8-Ch lor o-N-1-[(1R,2S,3R,4R)-2,3-(isopr opyliden edioxy)-
4-[(5-m on om et h oxyt r it yl)oxym et h yl]cyclop en t yl]-5′-O-
[bis(p h en ylth io)p h osp h or yl]-2′,3′-O-isop r op ylid en ea d e-
n osin e (27). After stirring a mixture of PSS (7.6 g, 20 mmol)
and TPSCl (6.1 g, 20 mmol) in pyridine (40 mL) at room
temperature for 2 h, 26 (4.7 g, 5.9 mmol) was added, and the
resulting mixture was stirred at room temperature for further
2 h. The mixture was evaporated, and the residue was
partitioned between CHCl3 and H2O. The organic layer was
washed with brine, dried (Na2SO4), and evaporated, and the
residue was purified by column chromatography (SiO2, 60%
AcOEt in hexane) to give 27 (4.1 g, 67%) as a yellow foam: 1H
NMR (CDCl3, 500 MHz) δ 7.66 (s, 1 H, H-2), 7.48-6.81 (m, 25
H, NH, Ar-H), 6.13 (d, 1 H, H-1′, J 1′,2′ ) 1.3 Hz), 5.44 (dd, 1
H, H-2′, J 2′,1′ ) 1.3, J 2′,3′ ) 6.3 Hz), 5.09 (m, 1 H, H-2′′), 5.08
(dd, 1 H, H-3′, J 3′,2′ ) 6.3, J 3,4′ ) 3.4 Hz), 4.92 (m, 1 H, H-1′′),
4.50 (m, 1 H, H-3′′), 4.42-4.33 (m, 3 H, H-4′, H-5′), 3.78 (s, 3
6.07 (d, 1 H, H-1′, J 1′,2′ ) 2.0 Hz), 5.48 (dd, 1 H, H-2′, J 2′,1′
)
2.0, J 2′,3′ ) 6.3 Hz), 5.32 (dd, 1 H, H-2′′, J 2′′ 1′′ ) 5.2, J 2′′,3′′ ) 5.8
Hz), 4.99 (dd, 1 H, H-3′, J 3′,2′ ) 6.3, J 3′,4′ ),3.8 Hz), 4.74 (dd, 1
H, H-3′′, J 3′′,2′′ ) 5.8, J 3′′,4′′ ) 2.8 Hz), 4.59 (br s, 1 H, 5′-OH),
4.52 (ddd, 1 H, H-1′′, J 1′′,2′′ ) 5.2, J 1′′,6′′a ) 9.8, J 1′′,6′′b ) 9.7 Hz),
4.20 (ddd, 1 H, H-4′, J 4′,3′ ) 3.8, J 4′,5′a ) 6.1, J 4′,5′b ) 6.1 Hz),
3.80 (dd, 1 H, H-5′′a, J 5′′a,4′′ ) 3.8, J 5′′a,5′′b ) 10.8 Hz), 3.74-
3.68 (m, 3 H, H-5′, H-5′′b), 2.63 (m, 1 H, H-6′′a), 2.54 (m, 1 H,
H-4′′), 2.45 (m, 1 H, H-6′′b), 1.59, 1.56, 1.37, 1.31 (each s, each
3 H, isopropyl CH3), 0.85 (s, 9 H, tert-butyl), 0.00, -0.02 (each
s, each 3 H, dimethyl); NOE (CDCl3, 400 MHz) irradiated H-2,
observed H-1′′ (19.6%); 13C NMR (CDCl3, 67.8 MHz) δ 152.5,
147.4, 141.5, 135.6, 122.8, 114.3, 111.9, 90.0, 87.6, 83.5, 83.2,
82.0, 81.5, 71.0, 64.7, 63.0, 44.9, 30.2, 28.1, 27.2, 25.9, 25.4,
25.3, 18.4, -5.3, -5.4; HRMS (FAB, positive) calcd for C28H45
-
H, OCH3), 3.33 (m, 1 H, H-5′′a), 3.16 (dd, 1 H, H-5′′b, J 5′′b,4′′ )
ClN5O7Si 626.2777 (MH+), found 626.2747; UV (MeOH) λmax
263 nm, sh 300 nm (ꢀ ) 14860 at λmax ) 263 nm (pH 2.0));
Anal. (C28H44ClN5O7Si) C, H, Cl, N.
4.0, J 5′′b,5′′a ) 8.6 Hz), 2.40 (m, 3 H, H-4′′, H-6′′), 1.61, 1.52 1.38,
1.22 (each s, each 3 H, isopropyl CH3); 13C NMR (CDCl3, 67.8
MHz) δ 158.5, 152.6, 146.7, 144.5, 144.5, 135.6, 135.3, 135.3,
135.1, 135.0, 130.3, 129.5, 129.3, 128.4, 127.7, 126.8, 125.9,
114.6, 113.2, 113.0, 90.1, 86.2, 85.7, 85.6, 83.7, 82.5, 81.6, 81.3,
66.2, 66.1, 64.8, 64.2, 55.2, 44.8, 33.4, 27.7, 27.1, 25.3; 31P NMR
(CDCl3, 202 MHz, decoupled with 1H) δ 50.9; HRMS (FAB,
positive) calcd for C54H56ClN5O9PS2 1048.2945 (MH+), found
8-Ch lor o-N-1-[(1R,2S,3R,4R)-2,3-(isopr opyliden edioxy)-
4-[(5-m on om et h oxyt r it yl)oxym et h yl]cyclop en t yl]-5′-O-
(ter t-b u t yld im et h ylsilyl)-2′,3′-O-isop r op ylid en ea d en o-
sin e (25). A mixture of 24 (5.5 g, 8.8 mmol) and MMTrCl (5.4
g, 17.6 mmol) in pyridine (50 mL) was stirred at room
temperature for 1.5 h and then evaporated. The residue was
partitioned between EtOAc and H2O, and the organic layer
was washed with brine, dried (Na2SO4), and evaporated. The
residue was purified by column chromatography (SiO2, 30%
AcOEt in hexane) to give 25 (6.2 g, 79%) as a yellow foam: 1H
NMR (CDCl3, 500 MHz) δ 7.63 (s, 1 H, H-2), 7.45-6.82 (m, 15
H, NH, Ar-H), 6.08 (d, 1 H, H-1′, J 1′,2′ ) 2.0 Hz), 5.52 (dd, 1
1048.2950; UV (MeOH) λmax 254 nm, sh 305 nm. Anal. (C54H55
ClN5O9PS2) C, H, Cl, N.
-
8-Ch lor o-N-1-[(1R,2S,3R,4R)-2,3-(isopr opyliden edioxy)-
4-(h ydr oxym eth yl)cyclopen tyl]-5′-O-[bis(ph en ylth io)ph os-
p h or yl]-2′,3′-O-isop r op ylid en ea d en osin e (28). A solution
of 27 (3.9 g, 3.7 mmol) in 80% aqueous AcOH (40 mL) was
stirred at room temperature for 6 h and then evaporated. The
residue was partitioned between EtOAc and aqueous saturated
NaHCO3, and the organic layer was washed with H2O and
then with brine, dried (Na2SO4), and evaporated. The residue
was purified by column chromatography (SiO2, 3% MeOH in
CHCl3) to give 28 (2.4 g, 85%) as a white foam: 1H NMR
(CDCl3, 500 MHz) δ 7.66 (br s, 1 H, H-2), 7.49-7.27 (m, 11 H,
NH, Ar-H), 6.13 (d, 1 H, H-1′, J 1′,2′ ) 1.0 Hz), 5.43 (dd, 1 H,
H, H-2′, J 2′,1′ ) 2.0, J 2′,3′ ) 6.3 Hz), 5.13 (dd, 1 H, H-2′′, J 2′′,1′′
)
4.8, J 2′′,3′′ ) 6.8 Hz), 5.01 (dd, 1 H, H-3′, J 3′,2′ ) 6.3, J 3′,4′ ) 3.6
Hz), 4.89 (m, 1 H, H-1′′), 4.56 (dd, 1 H, H-3′′, J 3′′,2′′ ) 6.8, J 3′′,4′′
) 6.8 Hz), 4.22 (ddd, 1 H, H-4′, J 4′,3′ ) 3.6, J 4′,5′a ) 6.5, J 4′,5′b
6.1 Hz), 3.79 (s, 3 H, OCH3), 3.73 (dd, 1 H, H-5′a, J 5′a,4′ ) 6.5,
J 5′a,5′b ) 10.7 Hz), 3.70 (dd, 1 H, H-5′b, J 5′b,4′ ) 6.1, J 5′b,5′a
)
)
10.7 Hz), 3.34 (dd, 1 H, H-5′′a, J 5′′a,4′′ ) 4.5, J 5′′a,5′′b ) 9.0 Hz),
3.19 (dd, 1 H, H-5′′b, J 5′′b,4′′ ) 6.5, J 5′′b,5′′a ) 9.0 Hz), 2.52 (m, 1
H, H-6′′a), 2.42-2.38 (m, 2 H, H-4′′, H-6′′b), 1.60, 1.53, 1.39,
H-2′, J 2′,1′ ) 1.0, J 2′,3′ ) 6.2 Hz), 5.25 (dd, 1 H, H-2′′, J 2′′,1′′
)
5.4, J 2′′,3′′ ) 5.5 Hz), 5.07 (dd, 1 H, H-3′, J 3′,2′ ) 6.2, J 3′,4′ ) 3.0

Unexpected Agonism by 8-Substituted cADPcR
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4747
Hz), 4.68 (dd, 1 H, H-3′′, J 3′′,2′′ ) 5.5, J 3′′,4′′ ) 1.7 Hz), 4.54 (m,
1 H, H-1′′), 4.41-4.31 (m, 3 H, H-4′, H-5′), 3.73 (dd, 1 H, H-5′′a,
calculated using ꢀ ) 14860 at λmax ) 263 nm of 24 (pH 2.0)] as
a triethylammonium salt: 1H NMR (D2O, 500 MHz) δ 8.75 (s,
1 H, H-2), 6.39 (br s, 1 H, H-1′), 5.86 (d, 1 H, H-2′, J 2′,3′ ) 5.6
Hz), 5.47 (dd, 1 H, H-3′, J 3′,2′ ) 5.6, J 3′,4′ ) 2.3 Hz), 4.83-4.81
(m, 3 H, H-1′′, H-2′′, H-3′′), 4.55 (m, 1 H, H-4′), 4.15 (m, 1 H,
H-5′′a), 4.04 (m, 2 H, H-5′a, H-5′′b), 3.87 (m, 1 H, H-5′b), 3.16
(q, 6 H, -CH2N, J ) 7.3 Hz), 3.12 (m, 1 H, H-6′′a), 2.87 (m, 1
H, H-4′′), 2.75 (m, 1 H, H-6′′b), 1.60, 1.59, 1.42, 1.38 (each s,
each 3 H, isopropyl CH3), 1.23 (t, 9 H, CH3CH2N, J ) 7.3 Hz);
13C NMR (D2O, 125 MHz) δ 152.8, 150.0, 146.6, 144.8, 121.1,
117.3, 115.2, 113.0, 93.7, 89.9, 89.8, 89.6, 87.5, 86.1, 84.2, 72.6,
69.3, 67.1, 49.5, 46.7, 46.6, 30.9, 28.9, 28.8, 27.2, 26.9, 11.0;
J 5′′a,4′′ ) 3.7, J 5′′a,5′′b ) 10.8 Hz), 3.69 (dd, 1 H, H-5′′b, J 5′′b,4′′
)
2.6, J 5′′b,5′′a ) 10.8 Hz), 2.53 (m, 2 H, H-4′′, H-6′′a), 2.40 (m, 1
H, H-6′′b), 1.61, 1.55 1.38, 1.30 (each s, each 3 H, isopropyl
CH3); 13C NMR (CDCl3, 67.8 MHz) δ 152.5, 147.7, 141.3, 135.3,
135.3, 135.2, 135.0, 135.0, 130.2, 129.6, 129.6, 129.5, 129.5,
129.3, 129.3, 128.0, 126.0, 125.9, 125.7, 122.7, 114.5, 111.8,
90.2, 86.8, 85.7, 83.8, 83.3, 82.3, 81.4, 70.4, 66.2, 66.1, 64.5,
44.8, 30.1, 28.0, 27.1, 25.2; 31P NMR (CDCl3, 202 MHz,
decoupled with 1H) δ 51.1; HRMS (FAB, positive) calcd for
C34H40ClN5O8PS2 776.1744 (MH+), found 776.1733; UV (MeOH)
1
31P NMR (D2O, 202 MHz, decoupled with H) δ -10.56 (d, J )
λmax 256 nm, sh 304 nm. Anal. (C34H39ClN5O8PS2) C, H, Cl, N.
16.5 Hz), -10.71 (d, J ) 16.5 Hz); HRMS (FAB, negative) calcd
for C22H29ClN5O12P2 652.0976 [(M - H)-], found 652.0963; UV
(H2O) λmax ) 263 nm.
8-Ch lor o-N-1-[(1R,2S,3R,4R)-2,3-(isopr opyliden edioxy)-
4-(p h osp h on oxym eth yl)cyclop en tyl]-5′-O-[(p h en ylth io)-
p h osp h or yl]-2′,3′-O-isop r op ylid en ea d en osin e (15). A mix-
ture of POCl3 (93 µL, 1.0 mmol) and 28 (78 mg, 0.10 mmol) in
PO(OMe)3 (2.0 mL) was stirred at 0 °C for 2 h. After addition
of aqueous saturated NaHCO3 (3.0 mL), the resulting mixture
was stirred at 0 °C for 10 min. To the mixture was added
triethylammonium acetate (TEAA, 2.0 M, pH 7.0, 1.0 mL)
buffer and H2O (4.0 mL), and the resulting solution was
applied to a C18 reversed phase column (1.1 × 11 cm). The
column was developed using a linear gradient of 0-65% MeCN
in TEAA buffer (0.1 M, pH 7.0, 400 mL). Appropriate fractions
were evaporated, and excess TEAA was removed by C18
reversed phase column chromatography (1.1 × 11 cm, eluted
with 60% aqueous MeCN). Appropriate fractions were evapo-
rated, and the residue was coevaporated with pyridine (1.0
mL x 3). A mixture of the residue, NMM (68 mg, 0.6 mmol),
H3PO2 (62 µL, 1.2 mmol), and Et3N (85 µL, 0.60 mmol) was
stirred at room temperature for 3.5 h. After addition of TEAA
buffer (2.0 M, pH 7.0, 0.5 mL), the resulting mixture was
evaporated. The residue was partitioned between EtOAc and
H2O, and the aqueous layer diluted with TEAA buffer (2.0 M,
pH 7.0, 0.5 mL) was evaporated. A solution of the residue in
H2O (5.0 mL) was applied to a C18 reversed phase column (1.1
× 11 cm), and the column was developed using a linear
gradient of 0-40% MeCN in TEAA buffer (0.1 M, pH 7.0, 400
mL). Appropriate fractions were evaporated, and excess TEAA
was removed by C18 reversed phase column chromatography
(1.1 × 18 cm, eluted with 40% aqueous MeCN) to give 15 (41
mg, 47%) as a triethylammonium salt: 1H NMR (D2O, 500
MHz) δ 8.67 (s, 1 H, H-2), 7.19-7.10 (m, 5 H, Ar-H), 6.37 (s,
1 H, H-1′), 5.78 (d, 1 H, H-2′, J 2′,3′ ) 6.1 Hz), 5.23 (dd, 1 H,
H-3′, J 3′,2′ ) 6.1, J 3′,4′ ) 2.5 Hz), 4.91-4.82 (m, 3 H, H-1′′, H-2′′,
H-3′′), 4.65 (m, 1 H, H-4′), 4.21 (m, 1 H, H-5′a), 4.06 (m, 1 H,
H-5′b), 4.00 (m, 2 H, H-5′′), 3.14 (q, 6 H, -CH2N, J ) 7.3 Hz),
2.59-2.54 (m, 2 H, H-4′′, H-6′′a), 2.39 (m, 1 H, H-6′′b), 1.62,
1.61, 1.40, 1.40 (each s, each 3 H, isopropyl CH3), 1.23 (t, 9 H,
CH3CH2N, J ) 7.3 Hz); 13C NMR (D2O, 125 MHz) δ 152.7,
149.1, 147.4, 144.8, 133.7, 133.7, 132.7, 131.7, 130.1, 120.8,
118.5, 117.7, 93.9, 89.9, 89.8, 86.4, 86.0, 83.9, 83.2, 68.1, 67.9,
49.4, 46.2, 46.2, 35.5, 28.7, 38.6, 26.9, 26.8, 11.0; 31P NMR (D2O,
8-Ch lor o-cyclic ADP -ca r bocyclic-r ibose (6). A solution
of 14 (98.3 OD263 units) in 60% aqueous HCO2H (1 mL) was
stirred at room temperature for 3.5 h and then evaporated.
After the residue was coevaporation with H2O (2 mL × 3), the
resulting residue was dissolved in TEAB buffer (0.1 M, pH
7.0, 30 µL) and H2O (2 mL), and the solution was lyophilized
to give 6 (96.8 OD263 units, 98%) as a triethylammonium salt:
1H NMR (D2O, 500 MHz) δ 9.13 (s, 1 H, H-2), 6.13 (d, 1 H,
H-1′, J 1′,2′ ) 6.3 Hz), 5.16 (dd, 1 H, H-2′, J 2′,1′ ) 6.3, J 2′,3′ ) 4.8
Hz), 4.94 (m, 1 H, H-1′′), 4.62 (dd, 1 H, H-3′, J 3′,2′ ) 4.8, J 3′,4′
) 2.4 Hz), 4.51 (m, 1 H, H-5′a), 4.38-4.35 (m, 2 H, H-2′′, H-4′),
4.21 (dd, 1 H, H-3′′, J 3′′,2′′ ) 4.4, J 3′′,4′′ ) 4.4 Hz), 4.17 (m, 2 H,
H-5′′), 4.07 (m, 1 H, H-5′b), 3.16 (q, 6 H, -CH2N, J ) 7.3 Hz),
3.02 (m, 1 H, H-6′′a), 2.51 (m, 1 H, H-4′′), 2.37 (m, 1 H, H-6′′b),
1.23 (t, 9 H, CH3CH2N, J ) 7.3 Hz); 13C NMR (potassium salt,
D2O, 125 MHz) δ 151.8, 147.6, 145.8, 142.5, 119.7, 91.0, 86.1,
79.6, 74.7, 74.2, 71.5, 66.3, 65.7, 64.7, 43.7, 29.0; 31P NMR (D2O,
1
202 MHz, decoupled with H) δ -9.29 (d, J ) 11.4 Hz), -10.31
(d, J ) 11.4 Hz); HRMS (FAB, negative) calcd for C16H21
-
ClN5O12P2 572.0350 [(M - H)-], found 572.0359; UV (H2O) λmax
) 263 nm. The absolute amount of 6 was calculated using ꢀ )
14860 at λmax ) 263 nm of 24 (pH 2.0)
8-Azido-cyclic ADP -car bocyclic-r ibose Diaceton ide (29).
A mixture of 14 (9.0 mg, 12 µmol) and LiN3 (23 mg, 48 µmol)
in pyridine (5.0 mL) was stirred at 50 °C for 4 days. After
addition of TEAA buffer (2.0 M, pH 7.0, 0.5 mL), the resulting
solution was evaporated, and the residue was partitioned
between AcOEt and H2O. The aqueous layer was evaporated,
and a solution of the residue in H2O (5.0 mL) was applied to
a C18 reverse phase column (1.1 × 12 cm). The column was
developed using a linear gradient of 0-40% MeCN in TEAA
buffer (0.1 M, pH 7.0, 300 mL). Appropriate fractions were
evaporated, and excess TEAA was removed by C18 reverse
phase column chromatography (1.1 × 11 cm, eluted with 30%
aqueous Me3CN) to give 29 (7.4 mg, 81%) as a triethylammo-
nium salt: 1H NMR (D2O, 500 MHz) δ 8.71 (s, 1 H, H-2), 6.16
(d, 1 H, H-1′, J 1′,2′ ) 1.2 Hz), 5.81 (dd, 1 H, H-2′, J 2′,1′ ) 1.2,
J 2′,3′ ) 5.9 Hz), 5.45 (dd, 1 H, H-3′, J 3′,2′ ) 5.9, J 3′,4′ ) 2.2 Hz),
4.89-4.85 (m, 3 H, H-1′′, H-2′′, H-3′′), 4.58 (m, 1 H, H-4′), 4.17
(m, 1 H, H-5′′a), 4.08 (m, 2 H, H-5′a, H-5′′b), 3.94 (m, 1 H,
H-5′b), 3.20 (q, 6 H, CH2N, J ) 7.3 Hz), 3.14 (m, 1 H, H-6′′a),
2.91 (m, 1 H, H-4′′), 2.76 (m, 1 H, H-6′′b), 1.63, 1.63, 1.45, 1.43
(each s, each 3 H, isopropyl CH3), 1.28 (t, 9 H, CH3CH2N, J )
7.3 Hz); 13C NMR (D2O, 125 MHz) δ 152.1, 151.8, 149.6, 145.6,
120.6, 117.2, 115.2, 92.4, 89.6, 89.4, 87.4, 86.0, 84.2, 72.4, 69.2,
67.2, 49.5, 46.8, 31.1, 28.9, 28.8, 27.1, 26.9, 11.0; 31P NMR (D2O,
202 MHz, decoupled with 1H) δ -10.57 (d, J ) 15.3 Hz),
-10.69 (d, J ) 15.3 Hz); HRMS (FAB, negative) calcd for
1
202 MHz, decoupled with H) δ 1.08 (s), 17.2 (s); HRMS (FAB,
negative) calcd for C28H35ClN5O12P2S 762.1167 [(M - H)-],
found 762.1165; UV (H2O) λmax ) 263 nm.
8-Ch lor o-cyclic ADP -ca r bocyclic-r ibose Dia ceton id e
(14). To a mixture of AgNO3 (34 mg, 200 µmol), Et3N (27 µL,
200 µmol), and MS 3A (820 mg) in pyridine (7 mL) was added
a solution of 14 (8.1 mg, 9.4 µmol) in pyridine (7 mL) slowly
over 15 h, using a syringe-pump, at room temperature in the
dark. The MS 3A was filtered off with Celite and washed with
H2O. To the combined filtrate and washings was added TEAA
buffer (2.0 M, pH 7.0, 1 mL), and the resulting solution was
evaporated. The residue was partitioned between EtOAc and
H2O, and the aqueous layer was evaporated. A solution of the
residue in TEAA buffer (0.1 M, pH 7.0, 5 mL) was applied to
a C18 reverse phase column (1.1 × 11 cm), and the column was
developed using a linear gradient of 0-40% MeCN in TEAA
buffer (0.1 M, pH 7.0, 200 mL). Appropriate fractions were
evaporated, and excess TEAA was removed by C18 reverse
phase column chromatography (1.1 × 11 cm, eluted with 20%
aqueous MeCN) to give 14 [123.5 OD263 units (ꢀ ) 14860), 88%,
C
22H29N8O12P2 659.1380 [(M - H)-], found 659.1345; UV (H2O)
λmax ) 284 nm.
8-Am in o-cyclic ADP -ca r bocyclic-r ibose Dia ceton id e
(30). A mixture of 29 (181 OD284 units, 9.9 µmol) and 10%
Pd-C (1.1 mg) in H2O (1.0 mL) was stirred under atmospheric
pressure of H2 at 50 °C for 30 min. The Pd-C was filtered off
with Celite and washed with H2O, and the combined filtrate
and washing was evaporated to give 30 (139 OD277 units, 87%)
as a triethylammonium salt: 1H NMR (D2O, 500 MHz) δ 8.60
(s, 1 H, H-2), 6.16 (s, 1 H, H-1′), 5.93 (d, 1 H, H-2′, J 2′,3′ ) 5.7

4748 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Shuto et al.
Hz), 5.48 (d, 1 H, H-3′, J 3′,2′ ) 5.7 Hz), 4.83 (m, 3 H, H-1′′,
H-2′′, H-3′′), 4.55 (m, 1 H, H-4′), 4.18 (m, 1 H, H-5′′a), 4.04 (m,
2 H, H-5′a, H-5′′b), 3.88 (m, 1 H, H-5′b), 3.20 (q, 6 H, -CH2N,
J ) 7.3 Hz), 3.13 (m, 1 H, H-6′′a), 2.91 (m, 1 H, H-4′′), 2.79
(m, 1 H, H-6′′b), 1.64, 1.63, 1.47, 1.42 (each s, each 3 H,
isopropyl CH3), 1.28 (t, 9 H, CH3CH2N, J ) 7.3 Hz); 13C NMR
(D2O, 125 MHz) δ 157.9, 149.6, 117.1, 115.2, 91.9, 89.8, 89.3,
87.3, 85.9, 84.3, 72.2, 69.0, 67.1, 49.5, 46.5, 30.8, 28.9, 28.7,
27.1, 26.9, 11.0; 31P NMR (D2O, 202 MHz, decoupled with 1H)
δ -10.38 (d, J ) 15.3 Hz), -10.64 (d, J ) 15.3 Hz); HRMS (FAB,
negative) calcd for C22H31N6O12P2 633.1475 [(M - H)-], found
633.1480; UV (H2O) λmax ) 277 nm.
λmax ) 277 nm (pH 7.0). The absolute amount of 6 was
calculated using ꢀ ) 16000 at λmax ) 274 nm of 8-amino-cADPR
(5).7a
8-P h en ylth io-cyclic ADP -ca r bocyclic-r ibose (9). A solu-
tion of 31 (117 OD286 units) in aqueous 60% HCO2H (1.0 mL)
was stirred at room temperature for 1.5 h and then evaporated.
A solution of the residue in TEAA buffer (0.1 M, pH 7.0, 5.0
mL) was applied to a C18 reverse phase column (1.1 × 15 cm),
and the column was developed using a linear gradient of
0-35% MeCN in TEAA buffer (0.1 M, pH 7.0, 200 mL).
Appropriate fractions were evaporated under the reduced
pressure, and excess TEAA was removed by C18 reverse phase
column chromatography (1.1 × 11 cm, eluted with 30%
aqueous MeCN) to give 9 (104 OD286 units, 89%) as a
triethylammonium salt: 1H NMR (D2O, 500 MHz) δ 9.11 (s, 1
8-P h en ylth io-cyclic ADP -ca r bocyclic-r ibose Dia ceto-
n id e (31). A mixture of 14 (9.0 mg, 12 µmol) and PhSH (49
µL, 48 µmol) in pyridine (1.0 mL) was stirred at room
temperature for 1.5 h. After addition of TEAA buffer (2.0 M,
pH 7.0, 0.5 mL), the resulting solution was evaporated. The
residue was partitioned between AcOEt and H2O, and the
aqueous layer was evaporated. A solution of the residue in H2O
(5.0 mL) was applied to a C18 reverse phase column (1.1 × 11
cm), and the column was developed using a linear gradient of
0-50% MeCN in TEAA buffer (0.1 M, pH 7.0, 200 mL).
Appropriate fractions were evaporated, and excess TEAA was
removed by C18 reverse phase column chromatography (1.1 ×
11 cm, eluted with 30% aqueous MeCN) to give 31 (8.5 mg,
119 OD286 units, 86%) as a triethylammonium salt: 1H NMR
(D2O, 500 MHz) δ 8.73 (s, 1 H, H-2), 7.66-7.51 (m, 5 H, Ar-
H), 6.41 (d, 1 H, H-1′, J 1′,2′ ) 1.2 Hz), 5.91 (dd, 1 H, H-2′, J 2′,1′
H, H-2), 7.67-7.40 (m, 5 H, Ar-H), 6.21 (d, 1 H, H-1′, J 1′,2′
)
6.4 Hz), 5.20 (dd, 1 H, H-2′, J 2′,1′ ) 6.4, J 2′,3′ ) 5.9 Hz), 4.92
(m, 1 H, H-1′′), 4.64 (m, 1 H, H-3′), 4.56 (m, 1 H, H-5′a), 4.41
(m, 1 H, H-4′), 4.36 (m, 1 H, H-2′′), 4.22 (m, 1 H, H-3′′), 4.19
(m, 2 H, H-5′′), 4.11 (m, 1 H, H-5′b), 3.20 (q, 6 H, CH2N, J )
7.3 Hz), 3.04 (m, 1 H, H-6′′a), 2.55 (m, 1 H, H-4′′), 2.40 (m, 1
H, H-6′′b), 1.27 (t, 9 H, CH3CH2N, J ) 7.3 Hz); 13C NMR (D2O,
125 MHz) δ 157.1, 152.5, 150.8, 146.7, 136.9, 133.1, 133.0,
130.0, 122.5, 92.7, 87.8, 81.6, 76.7, 76.0, 73.4, 68.2, 67.5, 66.9,
49.5, 45.7, 31.0, 11.0; 31P NMR (D2O, 202 MHz, decoupled with
1H) δ -9.27 (d, J ) 11.4 Hz), -10.24 (d, J ) 11.4 Hz); HRMS
(FAB, negative) calcd for C22H26N5O12P2S 646.0774 [(M - H)-],
found 646.0762; UV (H2O) λmax ) 285 nm. The absolute amount
of 9 was calculated using ꢀ ) 15265 at λmax ) 285 nm based
on the total phosphate analysis using KH2PO4 as a standard.
Biologica l Assa y. Sea urchin eggs from Lytechinus pictus
(Marinus, Long Beach, CA) were obtained by intracoelomic
injection of 0.5 M KCl, shed into artificial seawater (in mM,
NaCl 435, MgCl2 40, MgSO4 15, CaCl2 11, KCl 10, NaHCO3
2.5, EDTA 1, and [pH 8]), dejellied by passing through 90 µm
nylon mesh, and then washed twice by centrifugation. Homo-
genates of sea urchin eggs were prepared as described previ-
ously.2 Briefly, eggs were disrupted in an intracellular-like
medium consisting of 250 mM potassium gluconate, 250 mM
N-methylglucamine, 20 mM HEPES, and 1 mM MgCl2, [pH
7.2] supplemented with 1 mM ATP, 10 U/mL creatine kinase,
and 10 mM phosphocreatine and protease inhibitors. Ca2+
concentrations were measured with fluo-3 (3 µM) at 17 °C,
using 500 µL of continuously stirred homogenate in a fluo-
rimeter (Perkin-Elmer LS-50B) at 506 nm excitation and 526
nm emission.
) 1.2, J 2′,3′ ) 5.9 Hz), 5.48 (dd, 1 H, H-3′, J 3′,2′ ) 5.9, J 3′,4′
)
2.6 Hz), 4.83-4.78 (m, 3 H, H-1′′, H-2′′, H-3′′), 4.57 (m, 1 H,
H-4′), 4.19 (m, 1 H, H-5′′a), 4.12-4.08 (m, 2 H, H-5′a, H-5′′b),
3.91 (m, 1 H, H-5′b), 3.20 (q, 6 H, CH2N, J ) 7.3 Hz), 3.13 (m,
1 H, H-6′′a), 2.91 (m, 1 H, H-4′′), 2.78 (m, 1 H, H-6′′b), 1.64,
1.61, 1.46, 1.41 (each s, each 3 H, isopropyl CH3), 1.28 (t, 9 H,
CH3CH2N, J ) 7.3 Hz); 13C NMR (D2O, 125 MHz) δ 156.7,
152.2, 151.0, 145.9, 136.8, 133.1, 133.0, 129.9, 122.3, 117.3,
115.2, 113.0, 93.4, 89.7, 89.5, 89.5, 87.5, 85.9, 84.1, 72.5, 69.3,
67.2, 49.5, 46.6, 46.6, 30.9, 28.9, 27.2, 26.9, 11.0; 31P NMR (D2O,
1
202 MHz, decoupled with H) δ -10.78 (d, J ) 15.3 Hz), -10.94
(d, J ) 15.3 Hz); HRMS (FAB, negative) calcd for C28H34N5-
O
12P2S 726.1400[(M - H)-], found 724.1427; UV (H2O) λmax
)
286 nm.
8-Azid o-cyclic ADP -ca r bocyclic-r ibose (7). Compound
7 (63.3 OD283 units, 97%) was obtained from 29 (65.0 OD282
units) as described for the synthesis of 6 as a triethylammo-
nium salt: 1H NMR (D2O, 500 MHz) δ 9.13 (s, 1 H, H-2), 5.93
(d, 1 H, H-1′, J 1′,2′ ) 6.2 Hz), 5.17 (dd, 1 H, H-2′, J 2′,1′ ) 6.2,
J 2′,3′ ) 5.7 Hz), 4.99 (m, 1 H, H-1′′), 4.66 (m, 1 H, H-3′), 4.55
(m, 1 H, H-5′a), 4.41 (m, 2 H, H-2′′, H-4′), 4.27 (m, 1 H, H-3′′),
4.22 (m, 2 H, H-5′′), 4.12 (m, 1 H, H-5′b), 3.22 (m, 6 H, CH2N),
3.09 (m, 1 H, H-6′′a), 2.58 (m, 1 H, H-4′′), 2.46 (m, 1 H, H-6′′b),
1.30 (m, 9 H, CH3CH2N); 13C NMR (D2O, 125 MHz) δ 152.4,
149.6, 146.4, 139.6, 120.7, 91.2, 87.6, 81.5, 74.6, 75.8, 73.4, 68.1,
67.6, 67.1, 49.5, 45.7, 30.9, 11.0; 31P NMR (D2O, 202 MHz,
decoupled with 1H) δ -9.30 (d, J ) 11.4 Hz), -10.26 (d, J )
11.4 Hz); HRMS (FAB, negative) calcd for C16H21N8O12P2
579.0754 [(M - H)-], found 579.0792; UV (H2O) λmax ) 283
nm. The absolute amount of 7 was calculated using ꢀ ) 18215
at λmax ) 281 nm of 8-azido-cADPR (4).7a
Ack n ow led gm en t . This investigation was sup-
ported by Grant-in-Aid for Creative Scientific Research
(13NP0401) and Research Fellowships for Young Sci-
entists (M.F.) from the J apan Society for the Promotion
of Science and The Wellcome Trust (A.G.) and Wellcome
Trust Project Grant No. 55709 (B.V.L.P and A.G). We
are grateful to Ms. H. Matsumoto, A. Maeda, S. Oka,
and N. Hazama (Center for Instrumental Analysis,
Hokkaido University) for technical assistance with
NMR, MS, and elemental analysis.
Su p p or tin g In for m a tion Ava ila ble: HPLC charts of the
final compounds 6-9. This material is available free of charge
via the Internet at http://pubs.acs.org.
8-Am in o-cyclic ADP -ca r bocyclic-r ibose (8). Compound
8 (19.8 OD277 units, 83%) was obtained from 30 (23.9 OD277
units) as described for the synthesis of 6 as a triethylammo-
nium salt: 1H NMR (D2O, 500 MHz) δ 8.96 (s, 1 H, H-2), 5.90
(d, 1 H, H-1′, J 1′,2′ ) 6.3 Hz), 5.24 (dd, 1 H, H-2′, J 2′,1′ ) 6.3,
J 2′,3′ ) 5.7 Hz), 4.92 (m, 1 H, H-1′′), 4.63 (m, 1 H, H-3′), 4.52
(m, 1 H, H-5′a), 4.36 (m, 2 H, H-2′′, H-4′), 4.23-4.19 (m, 3 H,
H-3′′, H-5′′), 4.09 (m, 1 H, H-5′b), 3.19 (q, 6 H, CH2N, J ) 7.3
Hz), 3.03 (m, 1 H, H-6′′a), 2.54 (m, 1 H, H-4′′), 2.43 (m, 1 H,
H-6′′b), 1.27 (t, 9 H, CH3CH2N, J ) 7.3 Hz); 13C NMR (D2O,
125 MHz) δ 158.3, 150.4, 149.6, 144.3, 120.6, 90.8, 87.4, 81.5,
76.5, 75.4, 73.4, 68.1, 67.6, 66.8, 49.5, 45.6, 30.8, 11.0; 31P NMR
(D2O, 202 MHz, decoupled with 1H) δ -9.32 (d, J ) 11.4 Hz),
-10.19 (d, J ) 11.4 Hz); HRMS (FAB, negative) calcd for
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(12) NMM was used as a scavenger of PhSH produced in the reaction.
When the reaction performed without NMM, the yield of 15 was
low.
(13) The method has been successfully employed for the synthesis
of cADPR analogues by other groups: (a) 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. (b) Huang, L.-J .; Zhao, Y.-Y.;
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