Substitution at the 8-position of 3″-deoxy-cyclic ADP-carbocyclic-ribose, a highly potent Ca2+-mobilizing agent, provides partial agonists1

Article abstract of DOI:10.1016/j.bmc.2007.02.001

We previously showed that 3″-deoxy-cyclic ADP-carbocyclic-ribose (3″-deoxy-cADPcR, 4) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca²⁺-mobilizing second messenger. From these results, we designed and synthesized ot

Full text of DOI:10.1016/j.bmc.2007.02.001

Bioorganic & Medicinal Chemistry 15 (2007) 3032–3040
Substitution at the 8-position of 300-deoxy-cyclic
ADP-carbocyclic-ribose, a highly potent Ca2+-mobilizing agent,
provides partial agonists1
Takashi Kudoh,a Takashi Murayama,b Akira Matsudaa and Satoshi Shutoa,*
aFaculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
bDepartment of Pharmacology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
Received 10 January 2007; revised 1 February 2007; accepted 2 February 2007
Available online 4 February 2007
Abstract—We previously showed that 300-deoxy-cyclic ADP-carbocyclic-ribose (300-deoxy-cADPcR, 4) is a stable and highly potent
analogue of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger. From these results, we designed and synthesized
other 300-modified analogues of cADPcR having a substituent at the 8-position and found that this modification at the 8-position
made them partial agonists. Among these compounds, 8-NH2-300-deoxy-cADPcR (10) was identified as a potent partial agonist with
an EC50 value of 17 nM.
ꢀ 2007 Published by Elsevier Ltd.
NH2
HO
NH2
HO
1. Introduction
N
N
Y
N
N
HO
N
O
N
X
CH2
O
Cyclic ADP-ribose (cADPR, 1, Fig. 1), a naturally
occurring metabolite of NAD+,2 has been shown to
mobilize intracellular Ca2+ in various cells, and is now
recognized as a second messenger involved in Ca2+ sig-
naling.3 Under neutral conditions, cADPR is in a zwit-
terionic form with a positive charge around the
N1–C6–N6 moiety of its adenine ring, making the mol-
ecule unstable. Since the charged adenine moiety
attached to the anomeric carbon of the N1-ribose can
be an efficient leaving group, cADPR is readily hydro-
lyzed at the unstable N1-ribosyl linkage to produce
ADP-ribose (ADPR), even in neutral aqueous solution.4
Under physiological conditions, cADPR is also hydro-
lyzed at the N1-ribosyl linkage by cADPR hydrolase
to give the inactive ADPR.4
N
O
N
O
O
O
O
O
P
O
P
P
O
OH
OH
O
O
O
O
P
OH
OH
O
O
O
1: X = H (cADPR)
2: X = NH2 (8-NH2-cADPR)
3: Y = OH (cADPcR)
4: Y = H (3"-deoxy-cADPcR)
NH2
HO
N
N
HO
N
X
CH2
N
O
O
5: X = Cl (8-Cl-cADPcR)
6: X = N3 (8-N3-cADPcR)
7: X = NH2 (8-NH2-cADPcR)
O
P
O
P
OH
O
O
OH
O
O
cADPR analogues can be used to prove the mechanism
of Ca2+ signaling pathways mediated by cADPR and
may be lead structures for the development of clinically
useful drugs, since cADPR plays a variety of important
roles in physiological processes.3a,c The synthesis of
NH2
HO
N
N
N
X
CH2
N
O
O
P
8: X = Cl (8-Cl-3"-deoxy-cADPcR)
9: X = N3 (8-N3-3"-deoxy-cADPcR)
10: X = NH2 (8-NH2-3"-deoxy-cADPcR)
11: X = NHCH2CH2CH3
O
O
P
OH
O
O
Keywords: Calcium; Carbocyclic-ribose; Cyclic ADP-ribose; Partial
agonist; Second messenger.
OH
(8-propylamino-3"-deoxy-cADPcR)
O
O
*
Corresponding author. Tel./fax: +81 11 706 3769; e-mail:
shu@pharm.hokudai.ac.jp
Figure 1. cADPR and its analogues.
0968-0896/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.02.001

T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
3033
cADPR analogues has been extensively investigated by
enzymatic and chemo-enzymatic methods using ADP-ri-
bosyl cyclase-catalyzed cyclization.3b–e For instance, a
series of 8-substituted analogues of cADPR were syn-
thesized by the chemo-enzymatic method to show that
these analogues were antagonists of cADPR. Among
them, 8-NH2-cADPR (2) was identified as the most po-
tent antagonist5 and has been used effectively as a bio-
logical tool.3 However, the analogues obtained by
these methods are limited due to the substrate-specificity
of the ADP-ribosyl cyclase.3d
most potent agonist 300-deoxy-cADPcR, namely whether
these analogues are agonists or antagonists? In this
report, we describe the synthesis and Ca2+-mobilizing
effect of a series of 300-deoxy-cADPcR analogues having
a substituent at the 8-position, which are the 8-Cl deriv-
ative 8, the 8-N3 derivative 9, the 8-NH2 derivative 10,
and the 8-propylamino derivative 11, respectively.
2. Results and discussion
2.1. Synthetic plan
We developed an efficient method for forming the back-
bone 18-membered pyrophosphate ring structure
employing phenylthiophosphate-type substrates, which
is the key step in the chemical synthesis of cADPR
and its analogues.6 When these substrates were activated
by AgNO3 or I2 in the presence of molecular sieves in
pyridine, the corresponding 18-membered pyrophos-
phate ring products were obtained in high yields.6b,c
Using this method, we successfully synthesized cyclic
ADP-carbocyclic-ribose (cADPcR, 3),6c designed as a
stable mimic of cADPR, in which the oxygen atom in
the N-1-ribose ring of cADPR is replaced by a methy-
lene group. Biological evaluation of cADPcR showed
that it actually acts as a biologically and chemically sta-
ble equivalent of cADPR.6c
We planned to synthesize the target 8-substituted
cADPcR analogues 8–11 by the route summarized in
Scheme 1, in which the O-protected 8-Cl-300-deoxy-cAD-
PcR derivative 12 is used as the common intermediate.
Acidic deprotection of 12 would readily provide 8-Cl-
300-deoxy-cADPcR (8). The other three targets 9, 10,
and 11 would be obtained via nucleophilic addition–
elimination at the 8-position of 12. The key intramolec-
ular pyrophosphate linkage formation could be achieved
according to the method described above: treatment of
8-chloro-50-phenylthiophosphate substrate 13 with
AgNO3/MS 3A as a promoter.6b,c The substrate 13
would be derived from the two previously reported
units, the 2-chloroimidazole nucleoside derivative 147a
and the optically active carbocyclic amine 15.7c
While investigating a structure–activity relationship
study of cADPcR7 with a sea urchin egg homogenate as-
say system, we found 300-deoxy-cADPcR (4) to be the
most potent Ca2+-mobilizing compound in the series.7c
On the other hand, 8-substituted cADPcR analogues
were designed and synthesized to possess specifically
the properties of both the stable carbocyclic analogue
3 and the 8-substituted cADPR analogues; for example,
we expected that 8-NH2-cADPcR (7) might be a
chemically and biologically stable potent antagonist of
cADPR. However, their biological evaluation disclosed
that the 8-substituted cADPcR analogues 5–7 were ago-
nists.7a Thus, only a subtle change, that is, ‘–O–’ into
‘–CH2–’, can bring about a dramatic switch from a high-
ly potent antagonist 8-NH2-cADPcR (2) to an almost
equipotent agonist 8-NH2-cADPcR (7).
2.2. Synthesis
The synthesis of the key common intermediate 12 is
shown in Scheme 2. When a mixture of 14 and 15 was
treated with K2CO3 in DMF at room temperature, the
desired N1-substituted adenosine derivative 16 was
obtained in 90% yield. After protection of the 500-hydroxyl
of 16 with a dimethoxytrityl (DMTr) group, the 50-O-TBS
group of the product was removed with TBAF to give 17.
Treatment of 17 with an S,S0-diphenylphosphorodithio-
ate (PSS)/2,4,6-triisopropylbenzenesulfonyl chloride
(TPSCl)/pyridine system8 gave the 50-bis(phenyl-
thio)phosphate 18 in 55% yield. After removal of the
500-O-DMTr group of 18, a phosphoryl group was intro-
duced at the resulting 500-primary hydroxyl of the resulting
19 with POCl3/(EtO)3PO,9 followed by treatment of the
product with H3PO2, Et3N10, and N-methylmaleimide
(NMM)7a in pyridine, affording 50-phenylthiophosphate
With these results in mind, we were interested in the bio-
logical activity of the 8-substituted derivatives of the
CN
N
Cl
OMOM
NH2
N
TBSO
OMOM
N
CH(OMe)
O
O
+
O
P
N
N
O
NH
N
N
O
Cl
O
O
O
P
14
N
N
O
O
P
O
N
O
AgNO3
Cl
N
O
PhS P
O
O
8—11
O
O
O
O
O
NH2
O
O
O
HO
12
13
15
OMOM
Scheme 1.

3034
T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
OMOM
OMOM
OMOM
DMTrO
DMTrO
NH
NH
N
HO
NH
N
N
N
N
N
O
N
N
Cl
Cl
N
N
N
(PhS)2P
O
Cl
N
HO
O
TBSO
O
a
O
b
c
14 + 15
O
O
O
O
O
O
18
17
16
OMOM
HO
NH
N
N
O
N
Cl
N
(PhS)2P
O
e
d
O
f
12
13
O
O
19
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, rt, 90%; (b) 1—DMTrCl, pyridine, rt; 2—TBAF, THF, AcOH, rt, 99%; (c) PSS, TPSCl, py, rt,
55%; (d) aq 60% AcOH, rt, 82%; (e) 1—POCl3, (EtO)3PO, 0 ꢁC; 2—H3PO2, Et3N, NMM, pyridine, 0 ꢁC, rt, 52%; (f) AgNO3, MS 3A, Et3N, py, rt, 85%.
13 in 52% yield as a triethylammonium salt. When a solu-
tion of 13 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,6b,c the desired intra-
molecular condensation effectively occurred to give the
cyclized common intermediate 12 in 85% yield.
cADPcR analogues showed Ca2+-mobilizing activity.
Figure 2a shows a comparison of dose-dependent
Ca2+-release curves of the 8-substituted analogues 8, 9,
and 10 with those of cADPcR (3) and 300-deoxy-cAD-
PcR (4). cADPcR released Ca2+ from the homogenate
in a dose-dependent manner (EC50 = 79 nM) and 300-de-
oxy cADPcR (EC50 = 17 nM) showed more potent
activity than cADPcR with the same maximal activity.
All of the target compounds were synthesized from 12 as
shown in Scheme 3. Treatment of 12 with aqueous
HCO2H and then NH4OH furnished 8-Cl-300-deoxy-
cADPcR (8). The other three target compounds, 9, 10,
and 11, were synthesized via a nucleophilic addition–
elimination reaction at the 8-position. Thus, treatment
of 12 with LiN3 or propylamine at room temperature
produced the corresponding 8-azido and 8-propylamino
derivatives 20 and 22, respectively. The azido group of
20 was reduced under catalytic hydrogenation condi-
tions with Pd–C to give the 8-amino derivative 21.
Finally, acidic deprotection of 20 and 21 gave the target
8-N3-300-deoxy-cADPcR (9) and 8-NH2-300-deoxy-cAD-
PcR (10), respectively. Although similar acidic
treatment of 22 did not produce the desired 8-propyla-
mino-300-deoxy-cADPcR (11) but rather the glycosidic
bond-cleaved product 23, the desired 11 was obtained
via reaction of the 8-Cl-cADPcR (8) and propylamine
in 39% yield along with a glycosidic bond-cleaved
product 24.
OMOM
NH2
N
N
X
O
N
N
a or b
O P O
12
O
O
P
O
O
O
O
d
O
20: X = N3
21: X = NH2
22: X = NHCH2CH2CH3
c
e
8
11
NH2
HO
N
N
O
d
NH
CH2
N
H
N
O
2.3. Ca2+-mobilizing activity in sea urchin egg
homogenate
O
Y
P
O
P
O
9
or 10
O
OH
O
The Ca2+-mobilizing ability of the newly synthesized
8-substituted 300-deoxy-cADPcR analogues 8–11 was
evaluated by fluorometrically monitoring Ca2+ with
Hemicentrotus pulcherrimus sea urchin egg homoge-
nate,7c,11 and the results are shown in Figure 2 and Ta-
ble 1. Although the 8-propylamino derivative 11 was
completely inactive, the other 8-substituted 300-deoxy-
O
HO
23: Y = OH
24: Y = NHCH2CH2CH3
Scheme 3. Reagents and conditions: (a) LiN3, py, rt, 76% (20); (b)
propylamine, rt, 74% (22); (c) H2, Pd–C, H2O, rt, 89%; (d) aq 80%
HCO2H, then 28% NH4OH, rt, 54% (8), 56% (9), 43% (10); (e)
propylamine, rt, 39% (11), 24% (23).

T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
3035
Table 1. Ca2+-mobilizing activity of 8-substituted 300-deoxy-cADPcR
analogues 8–11 in sea urchin egg homogenate
This is consistent with previous reports.7c,d The 8-substi-
tuted 300-deoxy-cADPcR analogues 8, 9, and 10 released
Ca2+ from the homogenate in a dose-dependent manner,
but the attainable maximal activity was significantly
lower than that of cADPcR; the average maximal level
was 14%, 16%, and 21% of the cADPcR for the
8-NH2 derivative 10, the 8-N3 derivative 9, and the
8-Cl derivative 8, respectively. The 8-NH2 derivative
10 was more potent (EC50 = 17 nM) than the 8-Cl and
the 8-N3 derivatives (EC50 = 0.19 lM (8) and 0.49 lM
(9), respectively). These results suggest that the 8-substi-
tuted analogues of 300-deoxy-cADPcR are partial ago-
nists, in contrast to their full agonistic lead
compounds cADPcR and 300-deoxy-cADPcR.
Compound
EC50 (nM)
Maximal activity (%)a
cADPcR (3)
300-Deoxy-cADPcR (4)
79
17
100
100
14
8
190
490
9
16
21
10
11
17
Inactive
—
a Maximal Ca2+-release level relative to that of cADPcR (100%).
44 nM, 0.41 lM, and 1.1 lM for the 8-NH2 derivative
10, the 8-Cl derivative 8, and the 8-N3 derivative 9,
respectively, and the rank order was well correlated
with that of the EC50 value (Fig. 2a). These findings
provide evidence that these 8-substituted analogues of
300-deoxy-cADPcR are partial agonists.
The cADPR-induced Ca2+ release exhibits the property
of self-induced desensitization, in which the effect of a
subsequent challenge by a normally maximal concentra-
tion of cADPR is reduced depending on the concentra-
tion of the first cADPR challenge.3a Such
desensitization for the natural agonist cADPR is also
observed with cADPcR,6c 300-deoxy cADPcR,7c and
some 8-substituted cADPcR analogues.7a We therefore
tested whether the 8-substituted 300-deoxy-cADPcR ana-
logues show such desensitization. As shown in Figure
2b, all three compounds cross-desensitized for the
Ca2+ release of the homogenate with Ca2+ release by
the maximal concentration of cADPcR (1 lM). The
IC50 values for the cADPcR-induced Ca2+ release were
The 8-substituted analogues of cADPcR were shown to
release Ca2+ from Lytechinus species sea urchin egg
homogenate.7a Because deletion of the hydroxyl group
at the 300-position greatly increases the potency for
Ca2+ release of cADPcR (see Fig. 2a), it would be inter-
esting to compare the Ca2+-release activity of the
8-substituted cADPcR analogues having the 300-hydrox-
yl with that of the corresponding 8-substituted ana-
logues lacking the 300-hydroxyl. Figure 2c shows dose-
dependent Ca2+ release by the 8-substituted cADPcR
analogues 5, 6, and 7, having the 300 hydroxyl group.
Figure 2. Ca2+-mobilizing activity of compounds in sea urchin egg homogenate. (a and c) dose-dependent Ca2+-mobilizing activity of cADPcR and
its analogues; the activity of each compound was expressed as a percent change in fura-2 fluorescence ratio (F340/F380) relative to 1 lM cADPcR.
(b and d) dose-dependent desensitization by cADPcR analogues; desensitization of each compound was expressed as a percent inhibition of Ca2+
release induced by 1 lM cADPcR after treatment of homogenate with the compound.

3036
T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
8-NH2-cADPcR (7) exhibited a Ca2+ release with an
apparent saturation of the maximal activity at 33% of
the cADPcR, indicating that it is a partial agonist. This
is inconsistent with the previous report showing it to be
a full agonist.7a The reason still remains unclear, but dif-
ferent sea urchin species (Lytechinus species in the previ-
ous work7a vs Hemicentrotus species in this study) or
assay conditions might cause this difference. The poten-
cy for Ca2+ release (EC50 = 89 nM) was comparable to
that of cADPcR (EC50 = 79 nM), but significantly lower
than that of 8-NH2-300-deoxy-cADPcR (EC50 = 17 nM).
ring is replaced by a methylene, behaved as full agonists,
even when the adenine ring was modified.7a Here, we
show that deletion of the 300-hydroxyl of the N1-ribose
moiety shifted the compound toward becoming more
antagonistic, since a partial agonist is regarded as an
intermediate between a full agonist and an antagonist.
Thus, the N1-ribose moiety may also be critically impor-
tant in agonist/antagonist switching of the cADPR
activity. Replacement of the oxygen atom with a meth-
ylene may shift the compound toward agonistic, and
deletion of the 300-hydroxyl may reverse it.
8-Cl- and 8-N3-cADPcR (5 and 6, respectively) were
much less potent and we could not see saturation even
at 30 lM. In the previous report, they were shown to
be full agonists.7a Assuming that the two compounds
are full agonists, the EC50 values for them would be
21 lM (5) and 47 lM (6), respectively. This is in marked
contrast to the results with the corresponding 300-deoxy
congeners 8 and 9 which showed partial agonist activity
with lower EC50 values. All three of the analogues 5, 6,
and 7 showed cross-desensitization with cADPcR
(Fig. 2d). 8-NH2-cADPcR (7) was much more potent
(IC50 = 96 nM) than the 8-Cl (5, IC50 = 3.0 lM) or the
8-N3 (6, IC50 = 4.2 lM) derivatives, and the rank order
of desensitization correlated with the potency of Ca2+
release.
As described, this study presents substantial results to
understand the SAR of cADPR and its analogues at
the 8- and the 300-positions, especially as regards ago-
nist/antagonist switching.
3. Experimental
3.1. General methods
Chemical shifts are reported in ppm downfield from
Me4Si (1H in CDCl3), HDO (1H in D2O), MeCN (13C
in D2O), CDCl3 (13C in CDCl3), or H3PO4 (31P).
All of the 1H-NMR assignments described were in
agreement with COSY spectra. Thin-layer chromatogra-
phy was done on Merck coated plate 60F254. Silica gel
chromatography was done on Merck silica gel 5715.
Reactions were carried out under an argon atmosphere.
The present work demonstrated the important role of
the 300-hydroxyl in the structure–activity relationships
of cADPcR. It has been reported that 300-deoxy-cAD-
PcR is more potent than cADPcR (Fig. 2a).7c Here,
we showed that the 8-substituted 300-deoxy-cADPcR
analogues were more potent (lower EC50 values) in
Ca2+ release from the sea urchin homogenate than the
corresponding 8-substituted cADPcR analogues having
the 300-hydroxyl (Figs. 2a and c). This is also the case
with dose-dependency for desensitization (Figs. 2b and
d). Thus, deletion of the 300-hydroxyl may increase the
affinity for the putative receptor irrespective of modifica-
tion of the 8-position of adenine ring.
3.2. 8-Chloro-N-1-[(1R,2R,4S)-2-(methoxymethyloxy)-4-
(hydroxymethyl)cyclopentyl]-50-O-(tert-butyldimethylsi-
lyl)-20,30-O-isopropylideneadenosine (16)
A mixture of 14 (370 mg, 0.786 mmol), 15 (176 mg,
1.00 mmol), and K2CO3 (5 mg, 39 lmol) in DMF
(8 mL) was stirred at room temperature for 16 h, and
then the solvent was evaporated. The residue was parti-
tioned between EtOAc and H2O, and the organic layer
was washed with brine, dried (Na2SO4), and evaporated.
The residue was purified by column chromatography
(SiO2, 10% MeOH in EtOAc) to give 16 (435 mg,
90%) as a pale yellow foam: 1H-NMR (CDCl3,
500 MHz) d 7.65 (s, 1H, H-2), 6.07 (d, 1H, H-10,
In addition, the 300-hydroxyl may also affect the agonistic
activity. Deletion of the 300-hydroxyl of the full agonistic
8-Cl- and 8-N3-cADPcR converted them into partial
agonists. Even for 8-NH2 derivatives which showed par-
tial agonistic activity in spite of the presence or absence
the 300-hydroxyl, the maximal level of Ca2+ release was
significantly lower in the 300-deoxy-type compound 10.
These findings suggest that deletion of the 300-hydroxyl
of the N1-ribose may reduce the maximal Ca2+ releasing
activity of the compounds.
J1 ;2 ¼ 2:2 Hz), 5.53 (dd, 1H, H-20, J2 ;1 ¼ 2:2 Hz,
0
0
0
0
J2 ;3 ¼ 6:4 Hz), 5.00 (m, 2H, H-100, H-30), 4.58 (d, 1H,
MOM-CH2, J = 6.7 Hz), 4.55 (d, 1H, MOM-CH2,
J = 6.7 Hz), 4.50 (m, 1H, H-200), 4.21 (m, 1H, H-40),
0
0
3.72 (dd, 1H, H-50a, J5 a;5 b ¼ 10:8 Hz, J5 a;4 ¼ 6:3 Hz),
0
0
0
0
3.68 (m, 2H, H-50b, H-500a), 3.59 (dd, 1H, H-500b,
00
00
00
00
J5 b;5 a ¼ 10:8 Hz, J5 b;4 ¼ 3:7 Hz), 3.21 (s, 3H,
MOM-CH3), 2.43 (m, 1H, H-400), 2.32 (m, 2H, H-300a,
H-300b), 2.20 (m, 1H, H-600a), 1.85 (m, 1H, H-600b),
1.59, 1.38 (each s, each 3H, isopropylidene), 0.86 (s,
9H, tert-butyl), 0.001, ꢀ0.015 (each s, each 3H, Si-
CH3); 13C-NMR (CDCl3, 125 MHz) d 152.9, 147.6,
141.4, 135.4, 122.6, 114.3, 96.4, 90.1, 87.5, 83.0, 81.6,
78.5, 68.9, 67.16, 63.0, 55.4, 36.5, 34.3, 28.4, 27.2, 25.9,
25.5, 18.4, ꢀ5.4, ꢀ5.4; FAB-MS m/z 614 (MH)+; UV
(MeOH) kmax 263 nm; Anal. Calcd for C27H44ClN5O7Si:
C, 52.80; H, 7.22; N, 11.40. Found: C, 52.77; H, 7.17; N,
11.30.
It is known that modification of cADPR at the 8-posi-
tion of the adenine ring with NH2, N3, or halogen sub-
stituent converts it from agonist to antagonist.3 In
addition, 7-deaza-cADPR, in which the N7 atom of
the adenine ring is replaced by a methine, has been
shown to behave as a partial agonist.12 Thus, there is
no doubt that the adenine ring plays an important role
in the agonist/antagonist switch of the cADPR and its
analogues.3 On the other hand, 8-substituted cADPcR
analogues, in which the oxygen atom in the N1-ribose

T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
3037
3.3. 8-Chloro-N-1-[(1R,2R,4S)-2-(methoxymethyloxy)-4-
(dimethoxytrityloxymethyl)cyclopentyl]- 20,30- O-isopro-
pylideneadenosine (17)
145.1, 141.0, 136.2, 135.3, 135.2, 135.0, 135.0, 134.6,
130.0, 129.5, 129.5, 129.5, 129.3, 128.1, 127.7, 126.6,
126.1, 126.0, 125.9, 125.8, 122.5, 114.5, 113.0, 95.78,
90.1, 85.7, 85.6, 83.6, 81.4, 79.9, 66.4, 66.3, 63.5, 55.5,
55.1, 35.9, 34.46, 33.5, 27.1, 25.3; 31P-NMR (CDCl3,
202 MHz) d 50.61 (s); FAB-MS m/z 1066 (MH)+; UV
(MeOH) kmax 295 (sh) nm; Anal. Calcd for C54H57ClN5
O10PS2: C, 60.81; H, 5.39; N, 6.57. Found: C, 60.64; H,
5.47; N, 6.31.
A mixture of 16 (1.51 g, 2.45 mmol) and DMTrCl
(2.49 g, 7.35 mmol) in pyridine (25 mL) was stirred at
room temperature for 1 h. After addition of MeOH
(20 mL), the resulting mixture was evaporated. The res-
idue was partitioned between EtOAc and H2O, and the
organic layer was washed with brine, dried (Na2SO4),
and evaporated. A mixture of the residue, TBAF
(1.0 M in THF, 4.90 mL, 4.90 mmol), and AcOH
(140 lL, 2.45 mmol) in THF (15 mL) was stirred at
room temperature for 3 h, and then the solvent was
evaporated. The residue was purified by column chro-
matography (SiO2, 75% EtOAc in hexane) to give 17
(1.94 g, 99%) as a white foam: 1H-NMR (CDCl3,
500 MHz) d 7.68 (s, 1H, H-2), 6.82–7.43 (m, 13H,
3.5. 8-Chloro-N-1-[(1R,2R,4S)-2-(methoxymethyloxy)-4-
(hydroxymethyl)cyclopentyl]-50-O-[bis(phenylthio)phos-
phoryl]-20,30-O-isopropylideneadenosine (19)
A solution of 18 (1.32 g, 1.24 mmol) in aqueous 60%
AcOH (10 mL) was stirred at room temperature for
1 h and then evaporated. The residue was partitioned
between EtOAc and aqueous saturated NaHCO3, and
the organic layer was washed with H2O and brine, dried
(Na2SO4), and evaporated. The residue was purified by
column chromatography (SiO2, 10% MeOH in EtOAc)
DMTr), 6.00 (d, 1H, H-10, J1 ;2 ¼ 5:1 Hz), 5.22 (m,
0
0
1H, H-100), 5.11 (dd, 1H, H-20, J2 ;1 ¼ 5:1 Hz,
0
0
J2 ;3 ¼ 5:7 Hz), 5.01 (dd, 1H, H-30, J3 ;2 ¼ 5:7 Hz,
0
0
0
0
1
0
0
J3 ;4 ¼ 0:9 Hz), 4.60 (s, 2H, MOM-CH2), 4.46 (m, 2H,
H-200, H-40), 3.91 (m, 1H, H-50a), 3.79 (s, 6H, DMTr-
OMe · 2), 3.74 (m, 1H, H-50b), 3.25 (s, 3H, MOM-
CH3), 3.10 (m, 1H, H-500a), 3.06 (m, 1H, H-500b), 2.54
(m, 1H, H-400), 2.44 (m, 1H, H-600a), 1.94 (m, 2H, H-
300a, H-300b), 1.68 (m, 1H, H-600b), 1.64, 1.37 (each s, each
3H, isopropylidene); 13C-NMR (CDCl3, 125 MHz) d
171.0, 167.7, 158.4, 153.0, 145.9, 145.0, 140.4, 136.3,
134.5, 132.4, 130.8, 130.0, 128.8, 128.1, 127.7, 126.7,
123.1, 114.2, 113.0, 95.6, 92.3, 85.7, 85.5, 83.1, 81.2,
79.6, 68.1, 66.5, 63.1, 60.3, 55.5, 55.2, 38.7, 35.7, 34.3,
33.8, 30.3, 28.9, 27.6, 25.4; HRMS (FAB, positive) calcd
for C42H49ClN5O9 802.3219 (MH)+, found 802.3214;
MeOH kmax 263 nm.
to give 19 (775 mg, 82%) as a white foam: H-NMR
(CDCl3, 500 MHz) d 7.76 (s, 1H, H-2), 7.30–7.50 (m,
10H, SPh · 2), 6.13 (d, 1H, H-10, J1 ;2 ¼ 1:8 Hz), 5.48
0
0
(dd, 1H, H-20, J2 ;1 ¼ 1:8 Hz, J2 ;3 ¼ 6:3 Hz), 5.06 (dd,
0
0
0
0
1H, H-30, J3 ;2 ¼ 6:3 Hz, J3 ;4 ¼ 3:1 Hz), 4.91 (m, 1H,
H-100), 4.58 (d, 1H, MOM-CH2, J = 6.8 Hz), 4.56 (d,
1H, MOM-CH2, J = 6.8 Hz), 4.51 (m, 1H, H-200), 4.42
(m, 2H, H-40, H-50a), 4.31 (m, 1H, H-50b), 3.67 (dd,
0
0
0
0
1H, H-500a, J5 a;5 b ¼ 11:0 Hz, J5 a;4 ¼ 2:7 Hz), 3.57
00
00
00
00
(dd, 1H, H-500b, J5 b;5 a ¼ 11:0 Hz, J5 b;4 ¼ 2:6 Hz),
3.21 (s, 3H, MOM-CH3), 2.41 (m, 1H, H-400), 2.29 (m,
2H, H-300a, H-300b), 2.16 (m, 1H, H-600a), 1.80 (m, 1H,
H-600b), 1.61, 1.39 (each s, each 3H, isopropylidene);
13C-NMR (CDCl3, 125 MHz) d 152.8, 148.1, 141.3,
135.4, 135.4, 135.1, 135.0, 129.6, 129.6, 129.6, 129.4,
126.0, 126.0, 125.8, 125.7, 122.5, 114.6, 96.4, 90.3,
85.7, 85.7, 83.7, 81.5, 78.8, 66.8, 66.2, 66.2, 55.4, 36.8,
34.1, 28.1, 27.1, 25.4; 31P-NMR (CDCl3, 202 MHz) d
50.82 (s); FAB-MS m/z 764 (MH)+; UV (MeOH) kmax
262, 295 (sh) nm; Anal. Calcd for C33H39ClN5O8PS2:
C, 51.86; H, 5.14; N, 9.16. Found: C, 51.96; H, 5.18;
N, 8.88.
00
00
00
00
3.4. 8-Chloro-N-1-[(1R,2R,4S)-2-(methoxymethyloxy)-4-
(dimethoxytrityloxymethyl)cyclopentyl]- 50-O-[bis(phenyl-
thio)phosphoryl]-20,30-O-isopropylideneadenosine (18)
A mixture of PSS (2.70 g, 7.07 mmol) and TPSCl
(1.93 g, 6.36 mmol) in pyridine (23 mL) was stirred at
room temperature for 1 h. To the resulting mixture
was added 17 (1.89 g, 2.36 mmol), and the mixture was
stirred at room temperature for further 90 min. The sol-
vent was evaporated, and the residue was partitioned be-
tween EtOAc and H2O. The organic layer was washed
with brine, dried (Na2SO4), and evaporated. The residue
was purified by column chromatography (SiO2, 75%
EtOAc in hexane) to give 18 (1.38 g, 55%) as a white
3.6. 8-Chloro-N-1-[(1R,2R,4S)-2-(methoxymethyloxy)-4-
(phosphonoxymethyl)cyclopentyl]-50- O-[(phenylthio)phos-
phoryl]-20,30-O-isopropylideneadenosine (13)
A mixture of POCl3 (186 lL, 2.00 mmol) and 19
(153 mg, 0.200 mmol) in PO(OEt)3 (2.0 mL) was stirred
at 0 ꢁC for 2 h. After addition of aqueous saturated
NaHCO3 (3 mL), the resulting mixture was stirred at
0 ꢁC for 10 min. To the mixture were added triethylam-
monium acetate (TEAA) buffer (2.0 M, pH 7.0, 1 mL)
and H2O (5 mL), and the resulting solution was applied
to a C18 reversed phase column (1.1 · 17 cm). The col-
umn was developed using a linear gradient of 0–70%
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 chro-
matography (1.1 · 17 cm, eluted with 70% aqueous
MeCN). Appropriate fractions were evaporated, and
the residue was co-evaporated with pyridine
1
foam: H-NMR (CDCl3, 500 MHz) d 7.66 (s, 1H, H-
2), 6.81-7.49 (m, 23H, DMTr, SPh · 2), 6.13 (d, 1H,
H-10,
J1 ;2 ¼ 1:7 Hz),
5.46
(dd,
1H,
H-20,
0
0
J2 ;1 ¼ 1:7 Hz, J2 ;3 ¼ 6:3 Hz), 5.14 (m, 1H, H-100),
0
0
0
0
5.04 (dd, 1H, H-30, J3 ;2 ¼ 6:3 Hz, J3 ;4 ¼ 3:1 Hz), 4.58
(d, 1H, MOM-CH2, J = 6.8 Hz), 4.55 (d, 1H, MOM-
CH2, J= 6.8 Hz), 4.50 (m, 1H, H-200), 4.41 (m, 2H, H-
40, H-50a), 4.31 (m, 1H, H-50b), 3.78 (s, 6H, DMTr-
OMe · 2), 3.22 (s, 3H, MOM-CH3), 3.12 (m, 1H, H-
500a), 3.09 (m, 1H, H-500b), 2.53 (m, 1H, H-400), 2.38
(m, 1H, H-600a), 1.94 (m, 2H, H-300a, H-300b), 1.74 (m,
1H, H-600b), 1.61, 1.38 (each s, each 3H, isopropylidene);
13C-NMR (CDCl3, 125 MHz) d 158.4, 153.2, 146.1,
0
0
0
0

3038
T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
(2.0 mL · 3). A mixture of the residue, NMM (169 mg,
1.53 mmol), H3PO2 (155 lL, 3.05 mmol), and Et3N
(213 lL, 1.53 mmol) was stirred at 0 ꢁC for 4 h under
shading. After addition of TEAA buffer (1.0 M, pH
7.0, 2 mL), the resulting mixture was evaporated. The
residue was partitioned between EtOAc and H2O, and
the aqueous layer was evaporated. A solution of the res-
idue in H2O (5 mL) was applied to a C18 reversed phase
column (1.1 · 17 cm), and the column was developed
using a linear gradient of 0–35% 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 · 17 cm,
eluted with 50% aqueous MeCN). Appropriate fractions
were evaporated, and the residue was lyophilized to give
1H, MOM-CH2, J = 7.1 Hz), 4.58 (m, 1H, H-40), 4.34
(m, 1H, H-200), 4.24 (m, 1H, H-500a), 4.14 (m, 1H, H-
500b), 4.08 (m, 1H, H-50a), 3.97 (m, 1H, H-50b), 3.45 (s,
3H, MOM-CH3), 3.19 (q, 6H, Et3NH–CH2 · 3,
J = 7.3 Hz), 2.97 (m, 1H, H-600a), 2.81 (m, 1H,
H-400), 2.51 (m, 1H, H-600b), 2.15 (m, 1H, H-300a),
2.05 (m, 1H, H-300b), 1.64, 1.44 (each s, each 3H, isopro-
pylidene), 1.27 (t, 9H, Et3NH–CH3 · 3, J = 7.3 Hz);
13C-NMR (D2O, 125 MHz) d 150.7, 146.9, 145.3,
141.9, 118.8, 115.1, 97.2, 91.7, 87.2, 85.5, 84.8, 82.2,
67.7, 66.5, 64.3, 56.2, 47.1, 37.2, 33.4, 30.3, 26.3, 24.7,
8.6; 31P-NMR (D2O, 202 MHz)
d
ꢀ8.86 (d,
J = 11.4 Hz), ꢀ10.08 (d, J = 11.4 Hz); HRMS (FAB,
positive) calcd for C21H31ClN5O12P2 642.1127 (MH+),
found 642.1136; UV (H2O) kmax 263 nm.
1
13 (89 mg, 52%) as a triethylammonium salt: H-NMR
(D2O, 500 MHz) d 8.76 (s, 1H, H-2), 7.14–7.24 (m,
3.8. 8-Chloro-300-deoxy-cyclic ADP-carbocyclic-ribose (8)
5H, SPh), 6.39 (br s, 1H, H-10), 5.78 (d, 1H, H-20,
J2 ;3 ¼ 6:3 Hz), 5.25 (dd, 1H, H-30, J3 ;2 ¼ 6:3 Hz,
A solution of 12 (19 mg, 25 lmol) in aqueous 80%
HCO2H (1 mL) was stirred at room temperature for
49 h and then evaporated. After co-evaporation of
the residue with H2O (2 mL · 3), aqueous 28%
NH4OH (1 mL) was added, and the mixture was stir-
red at room temperature for 2 h. The solvent was evap-
orated, and the residue was co-evaporated with H2O
(2 mL · 3). The resulting residue was dissolved in
TEAB buffer (0.1 M, pH 7.0, 786 lL), and the solution
was lyophilized to give 8 (8 mg, 54%) as a triethylam-
monium salt: 1H-NMR (D2O, 500 MHz, potassium
0
0
0
0
J3 ;4 ¼ 2:7 Hz), 4.84 (m, 1H, H-100), 4.77 (d, 1H,
MOM-CH2, J = 7.1 Hz), 4.73 (d, 1H, MOM-CH2,
J = 7.1 Hz), 4.68 (m, 2H, H-200, H-40), 4.22 (m, 1H, H-
50a), 4.10 (m, 1H, H-50b), 3.88 (m, 2H, H-500 · 2), 3.34
(s, 3H, MOM-CH3), 3.17 (q, 6H, Et3NH–CH2 · 3,
J = 7.3 Hz), 2.66 (m, 1H, H-400), 2.58 (m, 1H, H-600a),
2.20 (m, 1H, H-300a), 2.07 (m, 1H, H-300b), 1.88 (m,
1H, H-600b), 1.63, 1.42 (each s, each 3H, isopropylidene),
1.26 (t, 9H, Et3NH–CH3 · 3, J = 7.3 Hz); 13C-NMR
(D2O, 125 MHz) d 150.4, 146.7, 145.3, 142.4, 131.4,
130.5, 129.4, 127.8, 118.6, 115.4, 96.8, 91.4, 87.6, 87.6,
83.8, 81.8, 81.7, 68.3, 66.2, 65.9, 56.3, 47.1, 34.8, 34.7,
32.8, 32.0, 26.3, 24.6, 8.7; 31P-NMR (D2O, 202 MHz)
d 17.31 (s), 1.43 (s); HRMS (FAB, negative) calcd for
C27H35ClN5O12P2S 750.1167 [(M ꢀ H)ꢀ], found
750.1141; UV (H2O) kmax 263 nm.
0
0
salt)
d
9.09 (s, 1H, H-2), 6.18 (d, 1H, H-10,
J1 ;2 ¼ 6:2 Hz), 5.25 (m, 1H, H-20), 4.87 (m, 1H,
H-100), 4.67 (m, 1H, H-30), 4.51 (m, 2H, H-200, H-500a),
4.42 (m, 1H, H-40), 4.20 (m, 1H, H-500b), 4.11 (m,
1H, H-50b), 4.04 (m, 1H, H-500b), 2.91 (m, 1H,
H-600a), 2.79 (m, 1H, H-400), 2.51 (m, 1H, H-600b),
2.17 (m, 1H, H-300a), 1.98 (m, 1H, H-300b); 13C-NMR
(D2O, 125 MHz) d 150.9, 147.6, 145.0, 142.5, 119.0,
90.4, 85.5, 78.6, 73.5, 71.0, 68.2, 68.0, 65.2, 37.5,
35.9, 30.7; 31P-NMR (D2O, 202 MHz) d ꢀ9.72 (d,
J = 11.4 Hz), ꢀ10.62 (d, J = 11.4 Hz); HRMS (FAB,
positive) calcd for C16H23ClN5O11P2 558.0552 (MH+),
found 558.0561; UV (H2O) kmax 263 nm.
0
0
3.7. 8-Chloro-200-O-methoxymethyl-300-deoxy-cyclic ADP-
carbocyclic-ribose 20,30-O-acetonide (12)
To a mixture of AgNO3 (412 mg, 2.43 mmol), Et3N
(338 lL, 2.43 mmol), and MS 3A (powder, 500 mg) in
pyridine (80 mL), a solution of 13 (99 mg, 0.12 mmol)
in pyridine (80 mL) was added slowly over 15 h, using
a syringe-pump, at room temperature under shading.
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, 2 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 H2O (5 mL)
3.9. 8-Azido-200-O-methoxymethyl-300-deoxy-cyclic ADP-
carbocyclic-ribose 20,30-O-acetonide (20)
A mixture of 12 (46 mg, 61 lmol) and LiN3 (60 mg,
1.2 mmol) in pyridine (2.0 mL) was stirred at room tem-
perature for 4 days. To the mixture were added TEAA
buffer (2.0 M, pH 7.0, 1 mL) and H2O (5 mL), and the
resulting solution was applied to a C18 reversed phase
column (1.1 · 17 cm). The column was developed using
a linear gradient of 0–30% MeCN in TEAA buffer
(0.1 M, pH 7.0, 400 mL). Appropriate fractions were
evaporated, and excess TEAA was removed by C18 re-
versed phase column chromatography (1.1 · 17 cm,
eluted with 50% aqueous MeCN). Appropriate fractions
were evaporated, and the residue was lyophilized to give
was applied to
a
C18 reversed phase column
(1.1 · 17 cm), and the column was developed using a lin-
ear gradient of 0–25% MeCN in TEAA buffer (0.1 M,
pH 7.0, 400 mL). Appropriate fractions were evaporat-
ed, and excess TEAA was removed by C18 reversed
phase column chromatography (1.1 · 17 cm, eluted with
40% aqueous MeCN). Appropriate fractions were evap-
orated, and the residue was lyophilized to give 12
(73 mg, 85%) as a triethylammonium salt: 1H-NMR
(D2O, 500 MHz) d 9.19 (s, 1H, H-2), 6.40 (br s, 1H,
1
20 (35 mg, 76%) as a triethylammonium salt: H-NMR
(D2O, 500 MHz) d 9.12 (s, 1H, H-2), 6.14 (br s, 1H,
H-10), 5.63 (d, 1H, H-20, J2 ;3 ¼ 6:2 Hz), 5.57 (dd, 1H,
H-10), 5.57 (d, 1H, H-20, J2 ;3 ¼ 6:1 Hz), 5.50 (d, 1H,
0
0
0
0
H-30, J3 ;2 ¼ 6:2 Hz, J3 ;4 ¼ 2:5 Hz), 4.95 (m, 1H,
H-30, J3 ;2 ¼ 6:1 Hz), 4.94 (m, 1H, H-100), 4.90 (d, 1H,
0
0
0
0
0
0
H-100), 4.89 (d, 1H, MOM-CH2, J = 7.1 Hz), 4.78 (d,
MOM-CH2, J = 7.2 Hz), 4.79 (d, 1H, MOM-CH2,

T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
3039
J = 7.2 Hz), 4.57 (m, 1H, H-40), 4.41 (m, 1H, H-200), 4.21
(m, 1H, H-500a), 4.10 (m, 2H, H-500b, H-50a), 3.96 (m,
1H, H-50b), 3.46 (s, 3H, MOM-CH3), 3.20 (q, 6H,
Et3NH–CH2 · 3, J = 7.3 Hz), 2.97 (m, 1H, H-600a),
2.81 (m, 1H, H-400), 2.49 (m, 1H, H-600b), 2.17 (m, 1H,
H-300a), 2.05 (m, 1H, H-300b), 1.62, 1.43 (each s, each
3H, isopropylidene), 1.28 (t, 9H, Et3NH–CH3 · 3,
J = 7.3 Hz); 13C-NMR (D2O, 125 MHz) d 149.5, 149.4,
146.9, 143.9, 118.3, 114.9, 97.1, 90.5, 87.0, 85.4, 84.8,
82.3, 67.6, 66.8, 64.4, 56.2, 47.1, 37.2, 33.5, 30.5, 26.3,
24.7, 8.7; 31P-NMR (D2O, 202 MHz) d ꢀ9.43 (d,
J = 11.4 Hz), ꢀ10.77 (d, J = 11.4 Hz); HRMS (FAB,
2.93 (m, 1H, H-600a), 2.79 (m, 1H, H-400), 2.53 (m, 1H,
H-600b), 2.12 (m, 1H, H-300a), 2.00 (m, 1H, H-300b),
1.62, 1.43 (each s, each 3H, isopropylidene), 1.26 (t,
9H, Et3NH–CH3 · 3, J = 7.3 Hz); 13C-NMR (D2O,
125 MHz) d 155.3, 147.70, 146.8, 141.9, 118.2, 114.9,
97.2, 89.7, 86.9, 85.8, 84.7, 82.4, 67.5, 66.5, 64.3, 56.2,
47.1, 37.3, 33.1, 30.1, 26.3, 24.7, 8.7; 31P-NMR (D2O,
202 MHz)
d
ꢀ9.46 (d, J = 15.3 Hz), ꢀ10.61 (d,
J = 15.3 Hz); HRMS (FAB, negative) calcd for
C21H31N6O12P2 621.1481 [(M ꢀ H)ꢀ], found 621.1462;
UV (H2O) kmax 277 nm.
negative)
calcd
for
C21H29N8O12P2
647.1386
3.12. 8-Amino-300-deoxy-cyclic ADP-carbocyclic-ribose
(10)
[(M ꢀ H)ꢀ], found 647.1389; UV (H2O) kmax 284 nm.
3.10. 8-Azido-300-deoxy-cyclic ADP-carbocyclic-ribose (9)
Compound 10 (6.0 mg, 43%) was obtained from 21
(15 mg, 21 lmol) as described for the synthesis of 8:
1H-NMR (D2O, 500 MHz) d 8.88 (s, 1H, H-2), 5.91
Compound 9 (11 mg, 56%) was obtained from 20
(22 mg, 29 lmol) as described for the synthesis of 8:
1H-NMR (D2O, 500 MHz) d 9.02 (s, 1H, H-2), 5.89
(d, 1H, H-10, J1 ;2 ¼ 6:3 Hz), 5.31 (dd, 1H, H-20,
0
0
J2 ;1 ¼ 6:3 Hz, J2 ;3 ¼ 4:9 Hz), 4.82 (m, 1H, H-100),
0
0
0
0
(d, 1H, H-10, J1 ;2 ¼ 6:2 Hz), 5.17 (m, 1H, H-20),
4.84 (m, 1H, H-100), 4.62 (m, 1H, H-30), 4.49 (m,
1H, H-200), 4.47 (m, 1H, H-50a), 4.36 (m, 1H, H-40),
4.19 (m, 1H, H-500a), 4.08 (m, 1H, H-50b), 4.02 (m,
1H, H-500b), 3.18 (q, 6H, Et3NH–CH2 · 3,
J = 7.3 Hz), 2.90 (m, 1H, H-600a), 2.77 (m, 1H,
H-400), 2.52 (m, 1H, H-600b), 2.16 (m, 1H, H-300a),
1.97 (m, 1H, H-300b), 1.26 (t, 9H, Et3NH–CH3 · 3,
4.64 (dd, 1H, H-30, J3 ;2 ¼ 4:9 Hz, J3 ;4 ¼ 2:3 Hz),
4.49 (m, 2H, H-50a, H-200), 4.37 (m, 1H, H-40), 4.19
(m, 1H, H-500a), 4.10 (m, 1H, H-50b), 4.01 (m, 1H,
H-500b), 3.20 (q, 6H, Et3NH–CH2 · 3, J = 7.3 Hz),
2.89 (m, 1H, H-600a), 2.77 (m, 1H, H-400), 2.53 (m,
1H, H-600b), 2.16 (m, 1H, H-300a), 1.96 (m, 1H, H-
300b), 1.28 (t, 9H, Et3NH–CH3 · 3, J = 7.3 Hz); 13C-
NMR (D2O, 125 MHz) d 156.0, 148.0, 147.4, 118.3,
88.4, 85.1, 85.0, 78.7, 72.8, 71.0, 68.0, 67.9, 65.2,
47.2, 37.6, 35.9, 30.6, 8.7; 31P-NMR (D2O,
0
0
0
0
0
0
J = 7.3 Hz); 13C-NMR (D2O, 125 MHz)
d 150.1,
149.8, 147.4, 143.9, 118.4, 88.8, 85.3, 78.6, 73.3,
71.0, 68.3, 67.9, 65.2, 47.1, 37.6, 35.9, 30.7, 8.7;
31P-NMR (D2O, 202 MHz) d ꢀ9.64 (d, J = 11.4 Hz),
ꢀ10.48 (d, J = 11.4 Hz); HRMS (FAB, positive) calcd
for C16H23N8O11P2 565.0956 (MH+), found 565.0968;
UV (H2O) kmax 282 nm.
202 MHz)
d
ꢀ9.38 (d, J = 11.4 Hz), ꢀ10.11 (d,
J = 11.4 Hz); HRMS (FAB, positive) calcd for
C16H25N6O11P2 539.1051 (MH)+, found 539.1052;
UV (H2O) kmax 276 nm.
3.13. 8-Propylmino-200-O-methoxymethyl-300-deoxy-cyclic
ADP-carbocyclic-ribose 20,30-O-acetonide (22)
3.11. 8-Amino-200-O-methoxymethyl-300-deoxy-cyclic
ADP-carbocyclic-ribose 20,30-O-acetonide (21)
A solution of 12 (15 mg, 20 lmol) in propylamine
(1.0 mL) was stirred at room temperature for 69 h and
then evaporated. To the residue were added TEAA buffer
(2.0 M, pH 7.0, 500 lL) and H2O (3 mL), and the result-
ing solution was applied to a C18 reversed phase column
(1.1 · 17 cm). 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 col-
umn chromatography (1.1 · 17 cm, eluted with 50%
aqueous MeCN). Appropriate fractions were evaporated,
and the residue was lyophilized to give 22 (13 mg, 74%) as
A mixture of 20 (12 mg, 16 lmol) and 10% Pd–C
(10 mg) in H2O (1.0 mL) was stirred under atmospheric
pressure of H2 at room temperature for 1 h. The Pd–C
was filtered off with Celite and washed with H2O, and
the combined filtrate and washing were evaporated. To
the residue were added TEAA buffer (2.0 M, pH 7.0,
500 lL) and H2O (3 mL), and the resulting solution
was applied to
a
C18 reversed phase column
(1.1 · 17 cm). The column was developed using a linear
gradient of 0–25% MeCN in TEAA buffer (0.1 M, pH
7.0, 300 mL). Appropriate fractions were evaporated,
and excess TEAA was removed by C18 reversed phase
column chromatography (1.1 · 17 cm, eluted with 40%
aqueous MeCN). Appropriate fractions were evaporat-
ed, and the residue was lyophilized to give 21 (10 mg,
89%) as a triethylammonium salt: 1H-NMR (D2O,
500 MHz) d 9.06 (s, 1H, H-2), 6.16 (br s, 1H, H-10),
1
a triethylammonium salt: H-NMR (D2O, 500 MHz) d
8.03 (s, 1H, H-2), 6.39 (d, 1H, H-20, J2 ;3 ¼ 5:7 Hz),
0
0
5.87 (s, 1H, H-10), 5.06 (d, 1H, H-30, J3 ;2 ¼ 5:7 Hz),
4.85 (d, 1H, MOM-CH2, J = 7.0 Hz), 4.82 (d, 1H,
MOM-CH2, J = 7.0 Hz), 4.41 (m, 1H, H-40), 4.37 (m,
1H, H-100), 4.21 (m, 1H, H-200), 3.74 (m, 1H, H-500a),
3.45 (s, 3H, MOM-CH3), 3.42 (m, 1H, H-50a), 3.36 (m,
1H, NH–CH2–CH2CH3), 3.28 (m, 2H, H-500b, NH–
CH2–CH2CH3), 3.19 (q, 12H, Et3NH–CH2 · 6,
J = 7.3 Hz), 2.93 (m, 1H, H-50b), 2.51 (m, 1H, H-400),
2.09 (m, 4H, H-600a, H-600b, H-300a, H-300b), 1.68 (m, 2H,
NH–CH2–CH2–CH3), 1.60, 1.51 (each s, each 3H, isopro-
pylidene), 1.27 (t, 18H, Et3NH–CH3 · 6, J = 7.3 Hz),
0
0
5.65 (d, 1H, H-20, J2 ;3 ¼ 6:2 Hz), 5.58 (dd, 1 H, H-30,
0
0
J3 ;2 ¼ 6:2 Hz, J3 ;4 ¼ 2:3 Hz), 4.91 (m, 1H, H-100),
4.88 (d, 1H, MOM-CH2, J = 7.2 Hz), 4.76 (d, 1H,
MOM-CH2, J = 7.2 Hz), 4.50 (m, 1H, H-40), 4.29 (m,
1H, H-200), 4.23 (m, 1H, H-500a), 4.12 (m, 1H, H-500b),
4.02 (m, 1H, H-50a), 3.88 (m, 1H, H-50b), 3.45 (s, 3H,
MOM-CH3), 3.18 (q, 6H, Et3NH–CH2 · 3, J = 7.3 Hz),
0
0
0
0

3040
T. Kudoh et al. / Bioorg. Med. Chem. 15 (2007) 3032–3040
0.97 (t, 3H, NH–CH2CH2–CH3, J = 7.4 Hz); 13C-NMR
(D2O, 125 MHz) d 152.8, 149.6, 148.9, 148.0, 117.1,
113.9, 95.3, 89.9, 87.5, 83.2, 82.3, 69.8, 65.3, 58.0, 55.8,
47.1, 45.2, 36.2, 36.1, 32.6, 31.3, 26.0, 24.6, 22.3, 11.2,
3.15. Ca2+-mobilizing activity in sea urchin egg
homogenate
The assay was carried out as described previously.7c
8.7; 31P-NMR (D2O, 202 MHz)
d
-10.00 (d,
J = 11.4 Hz), ꢀ11.00 (d, J = 11.4 Hz); HRMS (FAB, po-
sitive) calcd for C24H39N6O12P2 665.2101 (MH)+, found
665.2108; UV (H2O) kmax 284 nm.
Acknowledgments
We wish to acknowledge financial support from Minis-
try of Education, Culture, Sports, Science and Technol-
ogy, Japan, for Scientific Research 17659027 and
17390027.
3.14. 8-Propylamino-300-deoxy-cyclic ADP-carbocyclic-
ribose (11)
A solution of 8 (7.0 mg, 12 lmol) in propylamine
(1.0 mL) was stirred at room temperature for 11 days
and then evaporated. To the residue were added TEAA
buffer (2.0 M, pH 7.0, 500 lL) and H2O (3 mL), and the
resulting solution was applied to a C18 reversed phase
column (1.1 · 17 cm). The column was developed using
a linear gradient of 0–25% MeCN in TEAA buffer
(0.1 M, pH 7.0, 300 mL). Appropriate fractions for 11
and for 24 were evaporated, respectively. Excess TEAA
of each fraction was removed by C18 reversed phase col-
umn chromatography (1.1 · 17 cm, eluted with 40%
aqueous MeCN). The residues were lyophilized to give
11 (4.0 mg, 39%) and 24 (2.0 mg, 24%) as triethylammo-
References and notes
1. This report constitutes Part 247 of Nucleosides and
Nucleotides. Part 246: Hirano, S.; Ichikawa, S.; Matsuda,
A. Submitted for publication.
2. Clapper, D. L.; Walseth, T. F.; Dargie, P. J.; Lee, H. C.
J. Biol. Chem. 1987, 262, 9561–9568.
3. (a) Lee, H. C. Physiol. Rev. 1997, 77, 1133–1164; (b) Zhang,
F.-J.; Gu, Q.-M.; Sih, C. J. Bioorg. Med. Chem. 1999, 7,
653–664; (c)Cyclic ADP-ribose and NAADP: Structures,
Metabolism and Functions; Lee, H. C., Ed.; Kluwer
Academic Publishers: Dordrecht, 2002; (d) Shuto, S.;
Matsuda, A. Curr. Med. Chem. 2004, 11, 827–845; (e)
Potter, B. V. L.; Walseth, T. F. Curr. Mol. Med. 2004, 4,
303–311.
1
nium salts, respectively: 11: H-NMR (D2O, 500 MHz)
d 8.01 (s, 1H, H-2), 5.72 (d, 1H, H-20, J2 ;3 ¼ 4:3 Hz),
5.63 (br s, 1H, H-10), 4.71 (m, 1H, H-30), 4.39 (m, 1H,
H-100), 4.13 (m, 1H, H-40), 4.01 (m, 1H, H-200), 3.80
(m, 1H, H-50a), 3.73 (m, 1H, H-500a), 3.64 (m, 1H, H-
50b), 3.44 (m, 1H, H-500b), 3.33 (m, 1H, NH–CH2–
CH2CH3), 3.28 (m, 1H, NH–CH2–CH2CH3), 3.17 (q,
12H, Et3NH–CH2 · 6, J = 7.3 Hz), 2.54 (m, 1H, H-400),
2.13 (m, 1H, H-300a), 2.01 (m, 1H, H-600a), 1.98 (m,
1H, H-300b), 1.95 (m, 1H, H-600b), 1.68 (m, 2H, NH–
CH2–CH2–CH3), 1.25 (t, 18H, Et3NH–CH3 · 6,
J = 7.3 Hz), 0.96 (t, 3H, NH–CH2CH2–CH3,
J = 7.3 Hz); 13C-NMR (D2O, 125 MHz) d 152.4, 148.8,
147.2, 90.0, 82.7, 77.3, 72.5, 70.3, 69.4, 63.8, 59.8, 47.1,
45.3, 36.0, 34.3, 31.3, 22.1, 11.1, 8.7; 31P-NMR (D2O,
202 MHz) d ꢀ9.19 (s), ꢀ9.37 (s); HRMS (FAB, nega-
tive) calcd for C19H29N6O11P2 579.1375 [(M ꢀ H)ꢀ],
0
0
4. Lee, H. C.; Aarhus, R. Biochem. Biophys. Acta 1993, 1164,
68–74.
5. Walseth, T. F.; Lee, H. C. Biochem. Biophys. Acta 1993,
1178, 235–242.
6. (a) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda,
A. J. Org. Chem. 1998, 63, 1986–1994; (b) Fukuoka, M.;
Shuto, S.; Minakawa, N.; Ueno, Y.; Matsuda, A. J. Org.
Chem. 2000, 65, 5238–5248; (c) Shuto, S.; Fukuoka, M.;
Manikowsky, A.; Ueno, Y.; Nakano, T.; Kuroda, R.;
Kuroda, H.; Matsuda, A. J. Am. Chem. Soc. 2001, 123,
8750–8759.
7. (a) Shuto, S.; Fukuoka, M.; Kudoh, T.; Garnham, C.;
Galione, A.; Potter, B. V. L.; Matsuda, A. J. Med. Chem.
2003, 46, 4741–4749; (b) Hashii, M.; Shuto, S.; Fukuoka,
M.; Kudoh, T.; Matsuda, A.; Higashida, H. J. Neurochem.
2005, 94, 316–323; (c) Kudoh, T.; Fukuoka, M.; Ichikawa,
S.; Murayama, T.; Ogawa, Y.; Hashii, M.; Higashida, H.;
Kunerth, S.; Weber, K.; Guse, A. H.; Potter, B. V. L.;
Matsuda, A.; Shuto, S. J. Am. Chem. Soc. 2005, 127,
8846–8855; (d) Kudoh, T.; Murayama, T.; Ogawa, Y.;
Matsuda, A.; Shuto, S. Nucleosides, Nucleotides, Nucleic
Acids 2006, 25, 583–599.
8. Sekine, M.; Nishiyama, S.; Kamimura, T.; Osaki,
Y.; Hata, T. Bull. Chem. Soc. Jpn. 1985, 58, 850–
860.
9. Yoshikawa, M.; Kato, T.; Takenishi, T. Bull, Chem. Soc.
Jpn. 1969, 42, 3505–3508.
10. Hata, T.; Kamimura, T.; Urakami, K.; Kohno, K.;
Sekine, M.; Kumagai, I.; Shinozaki, K.; Miura, K. Chem.
Lett. 1987, 117–120.
11. Shiwa, S.; Murayama, T.; Ogawa, Y. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 2002, 282,
R727–R737.
1
found 579.1376; UV (H2O) kmax 282 nm; 24: H-NMR
(D2O, 500 MHz) d 8.01 (s, 1H, H-2), 5.98 (d, 1H, H-
10, J1 ;2 ¼ 7:6 Hz), 4.76 (dd, 1H, H-20, J2 ;1 ¼ 7:6 Hz,
0
0
0
0
J2 ;3 ¼ 5:7 Hz), 4.49 (dd, 1H, H-30, J3 ;2 ¼ 5:7 Hz,
0
0
0
0
J3 ;4 ¼ 2:6 Hz), 4.34 (m, 1H, H-40), 4.30 (m, 1H, H-
50a), 4.25 (m, 1H, H-100), 4.21 (m, 1H, H-50b), 3.88 (m,
1H, H-500 · 2), 3.45 (m, 1H, H-200), 3.41 (m, 4H, NH–
CH2–CH2CH3 · 2), 3.18 (q, 6H, Et3NH–CH2 · 3,
J = 7.3 Hz), 2.50 (m, 1H, H-400), 2.34 (m, 1H, H-300a),
1.96 (m, 1H, H-600a), 1.79 (m, 1H, H-600b), 1.67 (m,
4H, NH–CH2–CH2–CH3 · 2), 1.61 (m, 1H, H-300b),
1.26 (t, 9H, Et3NH–CH3 · 3, J = 7.3 Hz), 0.96 (m, 6H,
NH–CH2CH2–CH3 · 2); 13C-NMR (D2O, 125 MHz) d
152.4, 151.0, 149.0, 148.3, 116.5, 86.8, 84.6, 74.8, 70.9,
70.4, 69.2, 65.9, 58.3, 47.1, 44.7, 43.1, 35.0, 34.5, 30.6,
22.5, 22.3, 11.1, 11.0, 8.7; 31P-NMR (D2O, 202 MHz)
d ꢀ10.43 (d, J = 19.1 Hz), ꢀ11.64 (d, J = 19.1 Hz);
FAB-MS m/z 638 [(M ꢀ H)ꢀ]; UV (H2O) kmax 283 nm.
0
0
12. Bailey, V. C.; Sethi, J. K.; Fortt, S. M.; Galione, A.;
Potter, B. V. L. Chem. Biol. 1997, 4, 51–61.