Syntheses and calcium-mobilizing evaluations of N1-glycosyl-substituted stable mimics of cyclic ADP-ribose

Article abstract of DOI:10.1021/jm010530l

Cyclic ADP-ribose (cADPR) is not only a potent endogenous calcium modulator but also a second messenger. However, studies on the mechanism of cADPR action were limited due to its instability and lack of available structural modifications in the N¹-glyosyl unit of cADPR. In the present work, a series of N¹-glycosyl mimics with different configurational glycosyls or an ether strand were designed and synthesized mimicking the furanose ring. S_N2 substitutions were carried out between the protected inosine and glycosyl triflates to form the N¹-glycosylinosine derivatives, accompanied with some O⁶-glycosyl-substituted as side products. The intramolecular cyclization was followed the strategy described by Matsuda et al. It was found that the 8-unsubstituted substrate could also be used to construct the intramolecular cyclic pyrophosphate. The activities of N¹-glycosyl-substituted cADPR mimics were evaluated by induced Ca²⁺ release in rat brain microsomes and HeLa cells. It was found that the configuration of the N¹-glycosyl moiety in cADPR is not a critical structural factor for retaining the activity of mobilizing Ca²⁺ release. More interestingly, the N¹-acyclic analogue 6 exhibited strong activity by inducing Ca²⁺ release in both rat brain microsomes and HeLa cells. It constitutes a useful tool for further studies.

Full text of DOI:10.1021/jm010530l

5340
J . Med. Chem. 2002, 45, 5340-5352
Syn th eses a n d Ca lciu m -Mobilizin g Eva lu a tion s of N1-Glycosyl-Su bstitu ted
Sta ble Mim ics of Cyclic ADP -Ribose
Li-J un Huang,§ Yong-Yuan Zhao,‡ Lan Yuan,† J i-Mei Min,§ and Li-He Zhang*,§
National Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, P. R. China, 100083,
‡Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing, P. R. China, 100083, and
†Health and Drug Analytical Centre, Peking University, Beijing, P. R. China, 100083
Received November 19, 2001
Cyclic ADP-ribose (cADPR) is not only a potent endogenous calcium modulator but also a second
messenger. However, studies on the mechanism of cADPR action were limited due to its
instability and lack of available structural modifications in the N1-glyosyl unit of cADPR. In
the present work, a series of N1-glycosyl mimics with different configurational glycosyls or an
ether strand were designed and synthesized mimicking the furanose ring. SN2 substitutions
were carried out between the protected inosine and glycosyl triflates to form the N1-
glycosylinosine derivatives, accompanied with some O6-glycosyl-substituted as side products.
The intramolecular cyclization was followed the strategy described by Matsuda et al. It was
found that the 8-unsubstituted substrate could also be used to construct the intramolecular
cyclic pyrophosphate. The activities of N1-glycosyl-substituted cADPR mimics were evaluated
by induced Ca2+ release in rat brain microsomes and HeLa cells. It was found that the
configuration of the N1-glycosyl moiety in cADPR is not a critical structural factor for retaining
the activity of mobilizing Ca2+ release. More interestingly, the N1-acyclic analogue 6 exhibited
strong activity by inducing Ca2+ release in both rat brain microsomes and HeLa cells. It
constitutes a useful tool for further studies.
In tr od u ction
Ryanodine receptors, together with IP3 receptors,
represent a major pathway of Ca2+-release from intra-
cellular stores and have been shown to be involved in
many physiological and pathological processes.1 Cyclic
ADP-ribose (cADPR, 1), a novel cyclic nucleotide known
since 1987,2 has attracted worldwide attention in recent
years because of its potent calcium-mobilizing activities
in many systems, especially as a potential second
messenger in cellular Ca2+ homeostasis.3
Due to their biological importance, many analogues
with modifications at the adenosine unit have been
synthesized from NAD+ analogues using ADP-ribosyl
cyclase and served as valuable research tools in the
elucidation of the mechanism of cADPR action.4 How-
ever, the analogues that can be obtained by enzymatic
and chemoenzymatic methods are limited by the inher-
ent substrate specificity of the enzyme. In fact, there
are few analogues modified at the N1-ribosyl moiety of
cADPR in contrast to many adenosine-modified cADPR
analogues prepared by an enzymatic method. Further-
more, in some cases, the newly formed glycosylic bond
is attached to the N-7 of the purine ring instead of the
desired N-1 position.5 In addition, cADPR can be readily
hydrolyzed at the unstable N1-glycosylic linkage to give
ADP-ribose.6 Therefore, to build up a structure-activity
profile for the Ca2+-releasing ability of cADPR, stable
F igu r e 1. Structures of cADPR and its analogues.
N1-substituted cADPR mimics are urgently needed and
have to be made by means of chemical synthesis.7
Recently, Matsuda and co-workers first described the
synthesis of cyclic IDP-carbocyclic-ribose (cIDP-carbori-
bose, 2a ), in which the 4′-oxygen8 in the N1-ribosyl
moiety is substituted by a methylene group.9 In the
present study, we designed a novel class of stable cIDPR
mimics (3, 4a , 4b, 5a , 5b, 6), in which the N1-ribosyl
moiety is replaced by different configurational glycosyl
and the N1-glycosyl linkage is shifted from N1-C1′′ to the
N1-C2′′ position of furanose in order to improve the
stabilization (Figure 1).10 The 3′′-O-allyl and 8-bromo
modifications have been included due to their antici-
pated increased cell permeability.11 Furthermore, to
explore the role of N1-glycosyl linkage in structural
* To whom correspondence should be addressed. E-mail: zdszlh@
mail.bjmu.edu.cn.
§ National Key Laboratory of Natural and Biomimetic Drugs, Peking
University.
‡ Department of Chemical Biology, School of Pharmaceutical Sci-
ences, Peking University.
† Health and Drug Analytical Centre, Peking University.
10.1021/jm010530l CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/18/2002

N1-Glycosyl-Substituted Mimics of Cyclic ADP-Ribose
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5341
Sch em e 1a
a
Reagents and conditions: (i) (CF3SO2)2O, Py, CH2Cl2, -15 °C; (ii) K2CO3, 18-crown-6, THF, 45 °C; (iii) TBAF, THF, rt; (iv) (PhNH)2POCl,
tetrazole, Py, rt; (v) 80% AcOH, 70 °C; (vi) a) MMTrCl, Py, rt; b) Ac2O, rt; (vii) 5% Cl3CCOOH, CH2Cl2, rt; (viii). PSS, TPSCl, Py, tetrazole.
modifications, an N1-acyclic analogue has been synthe-
sized in this study. These new cIDPR analogues will
provide a greater understanding of ryanodine receptor
function and could lead to novel therapeutic agents.
prepared from corresponding sugar. Starting from 17,
5-O-benzoyl-3-O-allyl-1,2-O-isopropylidene-R-D-xylose 19
was attained in a 65% yield over four steps using the
same method (Scheme 2).14 To obtain compound 20,
deprotection of 1,2-O-isopropylidene furanoside 19 could
be completed using the Et3SiH/TMSOTf system in CH2-
Cl2.15 However, the yield was only moderate even under
conditions such as prolonging reaction time and use of
4-fold excess of catalyst. Alternatively, the use of Et3-
SiH/BF3-Et2O in CH2Cl2 led to 20 in excellent yield.16
Compound 20 was converted into 21 by debenzoylation
and subsequent reaction with MMTrCl in 79% overall
yield. By means of a similar method, the key N1-
glycosyl-substituted inosine derivative 23a was pre-
pared by SN2 substitution with triflate 22 in 34% yield,
accompanied by the O6-substituted compound 23b in
6.8% yield. The bisphosphate compound 27 was obtained
in four steps from 23a . The stereochemistry at C-2′′ was
as expected according to the NOESY of compound 26,
in which there is a cross-peak between H-5a′′ and H-2.
From the same starting material 17, compound 28 can
be obtained by known procedures and a conventional
route was chosen to synthesize protected 1,4-anhydro-
D-ribitol 31 from 28 (Scheme 3).14,16,17 Reaction of 31
with trifluoromethanesulfonic anhydride gave triflate
32, which was directly used for substitution with
protected inosine 9 without isolation at room temper-
ature. The N1-glycosyl inosine derivative 33a was
formed in 26.8% yield, together with the O6-substituted
compound 33b in 21.5% yield. The corresponding bis-
phosphate 39 was prepared according to a similar
sequence described in Scheme 2. The configuration of
C-2′′ was also assigned from the NOESY spectrum of
compound 33a , in which a significant NOE was ob-
served between the H-2 and H-5a′′.
Resu lts a n d Discu ssion
Ch em istr y: (1) Th e Syn th eses of N1-Glycosyl-
Su bstitu ted In osin e Bisp h osp h a te Der iva tives.
The key steps for the synthesis of cIDPR analogues
involve substitution at N-1 position of inosine and
intramolecular cyclization of corresponding bisphos-
phate. Starting with protected L-xylitol 7,12 triflate 8
was prepared by the known method.9 8 was reacted with
protected 8-bromoinosine 9 in the presence of K2CO3
and 18-crown-6 to give the key intermediate, 5′-O-
TBDMS-2′,3′-di-O-acetyl-N1-(2′′-deoxy-1′′,4′′-anhydro-
3′′,5′′-O-benzylidene-L-lyxitol-2′′-yl)-8-bromoinosine 10a
in 28% yield, accompanied by O6-substituted derivative
10b in 8% yield. (Scheme 1) After treatment of TBAF
in THF, the 5′-hydroxyl derivative 11 was obtained in
high yield and 11 was phosphorylated by (PhNH)2POCl
and tetrazole in pyridine to afford 12 in 89% yield. The
structure of 12 was identified by X-ray crystallographic
analysis.13 The 3′′,5′′-O-benzylidene group of 12 was
hydrolyzed and an attempt to direct coupling of 13 with
cyclohexylammonium S,S-diphenyl phosphorodithioate
(PSS) failed. Thus, compound 13 was selectively pro-
tected by reaction with p-anisylchlorodiphenylmethane
(MMTrCl) and then followed by acetylation to give the
intermediate 14 in 70% overall yield. Deprotection of
14 was carried out by treatment with 5% Cl3CCOOH
(TCA) in CH2Cl2 to produce the 5′′-hydroxyl derivative
15. The resulting 5′′-primary hydroxyl of the lyxitol
moiety was phosphorylated with PSS, triisopropylben-
zenesulfonyl chloride (TPSCl), and tetrazole in pyridine
to afford 16 in 91% yield.
The synthesis of the N1-acyclic sugar analogue 41 is
depicted in Scheme 4. The reaction of protected inosine
409 with excess 2-chloromethoxyethyl acetate afforded
Employing the same strategy, the other two N1-
glycosyl inosine derivatives, 27 and 39, were also

5342 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Huang et al.
Sch em e 2a
a
Reagents and conditions: (i) TrCl, Py, rt; (ii) 80% NaH, allyl bromide, DMF; (iii) 5% Cl3CCOOH, CH2Cl2, rt; (iv) BzCl, Py, rt; (v)
Et3SiH, BF3Et2O, CH2Cl2, rt; (vi) CH3ONa, CH3OH, rt; (vii) MMTrCl, Py, rt; (viii) (CF3SO2)2O, Py, -15 °C; (ix) 9, K2CO3, 18-crown-6,
THF; (x) TBAF, THF, rt; (xi) (PhNH)2POCl, tetrazole, Py; (xii) PSS, TPSCl, tetrazole, Py, rt.
Sch em e 3a
a
Reagents and conditions: (i) Reference 17; (ii) CH3ONa, CH3OH, rt; (iii) TrCl, Py, rt; (iv) 80% NaH, allyl bromide, DMF; (v) 5%
Cl3CCOOH, CH2Cl2, rt; (vi). BzCl, Py, rt; (vii). Et3SiH, BF3Et2O, CH2Cl2, rt; (viii) (CF3SO2)2O, Py, -15 °C; (ix) 9, K2CO3, 18-crown-6,
THF; (x) TBAF, THF, rt; (xi) (PhNH)2POCl, tetrazole, Py; (xii) (a) MMTrCl, Py, rt; (b) Ac2O, rt; (xiii) PSS, TPSCl, tetrazole, Py, rt.
the desired N1-acyclic compound 41 regioselectively in
82% yield using DBU as base.18 The bisphosphate 45
was produced as described for compounds 16, 27, and
39. However, MALDI-TOF MS of 45 showed that the

N1-Glycosyl-Substituted Mimics of Cyclic ADP-Ribose
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5343
Sch em e 4a
Sch em e 5a
a
Reagents and conditions: (i) DBU, ClCH2OCH2CH2OAc, DCM,
rt; (ii) TBAF, THF, rt; (iii) (PhNH)2POCl, tetrazole, Py; (iv)
CH3ONa, CH3OH, rt; (v) PSS, TPSCl, tetrazole, Py, rt.
bromine at the 8-position of inosine had been replaced
by chlorine during the phosphorylation. Further inves-
tigation indicated that the excess of TPSCl in that
reaction caused the substitution of bromine at the
8-position of inosine.
(2) Th e In tr a m olecu la r Cycliza tion . Matsuda’s
group developed an effective strategy for the cyclization
of cADPR analogues. They found that introducing a
bulky group into the 8-position of purine nucleosides
could restrict the conformation to a syn-form in which
the two phosphate moieties are near each other and
facilitate intramolecular condensation in the presence
of EDC.9 Using this method, Capua et al. reported that
they failed to obtain the N1-glucosyl substituted cADPR
analogue due to the absence of the bulky group at the
8-position.19 Matsuda et al. also reported that the
cyclization was completed by I2/MS 3 Å as a promoter
in pyridine in quantitative yield,20 and they synthesized
cyclic ADP-carbocyclic-ribose by using a similar method.21
We followed the Matsuda strategy to synthesize the N1-
glycosyl substituted mimics (3, 4a , 4b, 5a , 5b, and 6).
a
Reagents and conditions: (i) (a) isoamyl nitrite, Py:AcOH:
(AcO)2O (2:1:1); (b) anhydrous H3PO2, Py, Et3N; (ii) I2, 3 Å MS,
Py, rt; (iii) CH3ONa, CH3OH, rt. (iv) 60% HCOOH, rt.
phate linkage from 50. Interestingly, a debrominated
cyclic product 51a was obtained in addition to the
normal product 51b during the cyclization. Deacetyla-
tion of 51a and 51b gave the targets 4a and 4b. Their
structures were characterized by the 1H NMR, 31P NMR,
and HR FAB mass spectra (Scheme 6). Debromination
was also observed in the selective deprotection of
phosphorodithioate derivative 39, affording the 8-bromo-
substituted 52b accompanied by 8-unsubstituted de-
rivative 52a . The debromination was investigated under
various conditions. The result showed that the ratio of
H3PO2 and triethylamine was the key condition and that
increased H3PO2 can lead to the abnormal debromina-
tion or other side reactions. On the basis of the same
method, we synthesized two cyclic products 53a and 53b
starting from precursors 52a , 52b, respectively. Deacet-
ylation gave the desired targets 5a and 5b, which were
Treatment of 16 with isoamyl nitrite in a mixed
solvent of pyridine-AcOH-Ac2O and H3PO2 gave 46,
the cyclic precursor for the intramolecular condensation,
in 60.7% yield as a triethylammonium salt. The in-
tramolecular cyclization was performed by adding a
solution of 46 slowly over 20h, using a syringe pump,
to a large excess of promoter (I2/MS 3 Å) in pyridine at
room temperature. The cyclic product 47 was purified
as its triethylammonium salt by HPLC in 28% yield.
The structure of 47 was identified by HR FABMS and
NMR. The corresponding peak [M - 2 × (acetyl)]+ was
observed at 644.9647 m/z in a HR FAB spectrum, which
characterized by the H NMR, 31P NMR, and HR FAB
mass spectra.
1
1
was consistent with H NMR results. Further support
for the cyclic product comes from the 31P NMR at -9.37
and -10.16 ppm that are typical shifts for a pyrophos-
phate moiety.22 Deprotection of 47 gave the target
molecule 3 in 27.9% yield (Scheme 5). Similarly, the
desired cyclic product 49 was also obtained as a tri-
ethylammonium salt after purification by HPLC in
42.6% yield. The cyclic structure was characterized by
HR FABMS and 31P NMR. Deprotection of cyclic product
49 provided the target molecule 6 in 44.6% yield in 60%
Ca lciu m Relea se Assa y. A series of cADPR ana-
logues with substitutions at the 8-position of the adenine
ring have been synthesized and found to be antagonists
of cADPR.23 The order of potency is 8-amino > 8-azido
. 8-Br > 8-I-cADPR. In particular, 8-amino-cADPR has
been shown to be effective not only in sea urchin egg
microsomes but also intact sea urchin eggs24 as well as
intact and permeabilized mammalian cells.25,26 Ad-
ditionally, several agonists of cADPR have also been
synthesized from NAD+ analogues. For example, 2′-
deoxy-cADPR and cADPR-2′-phosphate are similar to
cADPR in inducing Ca2+ release, however, 3′-O-methyl
or 3′-deoxy-cADPR has not the same activity. It was
suggested that the 3′-OH was essential for the calcium
releasing activity.27
18
HCOOH solution.
The selective deprotection of bisphosphate 27 was
carried out under a similar condition as above. The
phenylthio substrate 50 was obtained in 70% yield. An
intramolecular condensation was carried out by promo-
tion of I2 and 3 Å MS to construct the cyclic pyrophos-

5344 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Huang et al.
Sch em e 6a
F igu r e 2. Ca2+ release induced by cADPR, 3, 4a , 4b, 5a , 5b,
and 6 in rat brain microsomes measured fluorometrically with
Fluo-3. Microsomes were loaded with Fluo-3 and then assayed
as described in the Experimental Section. The data shown are
typical curves for at least two experiments carried out in
duplicate using different microsomal preparations.
a similar method. It is known that fluo-3 has a higher
affinity for Ca2+ than other divalent cations.33 The
increase in fluorescence intensity indicates more com-
plex formation between fluorescence dye and Ca2+. In
this study, the changes in the fluorescence intensity
were used to monitor the changes in free Ca2+ concen-
trations in rat brain microsomes and intact HeLa cells.
The data shown are typical curves for at least two
experiments carried out in duplicate using different rat
brain microsomes and HeLa cells preparations. The
concentrations of samples are measured according to the
extinction coefficients of their UV spectra.
Figure 2 describes the fluo-3 fluorescence changes
when cADPR or the synthetic sample (3, 4a , 4b, 5a , 5b,
and 6) was added into the microsome homogenates.
cADPR or the synthetic samples induced Ca2+ release
from brain microsomes in a concentration-determination
with a threshold concentration of less than 15 µM under
these experimental conditions. Ca2+ was soon reseques-
tered into the microsomes (<400 s). An abrupt fluores-
cence increase was observed when compounds 5a or N1-
acyclic mimic 6 in 1-5 µL aliquot was added to 100 µL
of rat brain microsomes at 15 µM, respectively, and then
the fluorescence signals quickly declined after about 30
s. The other synthetic compounds (4a , 4b, 3, 5b) and
cADPR are not capable of stimulating obvious fluo 3
fluorescence increase at the same concentration (15 µM).
At a concentration of 30 µM, compounds 4a , 5b, and
cADPR can initiate similar fluorescence spikes while the
amplitude of the fluorescence spike induced by com-
pound 4a is higher than cADPR and 5b. A low fluores-
cence spike was triggered by compound 3 at 60 µM. 4b
was not able to induce fluorescence increase even at a
concentration of 60 µM. According to the changes in fluo-
3 fluorescence, we can compare the relative Ca2+ release
activities of these compounds in rat brain microsomes
homogenates under these experimental conditions. Syn-
thetic compounds 4a , 5a , and 6 have more potent
activity in inducing [Ca2+]i increase than authentic
cADPR. It is evident that the 8-nonsubstituted ana-
logues (4a , 5a ) have more potent calcium modulating
activity than the corresponding 8-bromo cADPR mimics
(4b, 5b). It seems that the configuration of N1-glycosyl
of cADPR is not a critical structural factor for retaining
the activity of induced Ca2+ release.
a
Reagents and conditions: (i) (a) isoamyl nitrite, Py:AcOH:
(AcO)2O (2:1:1); (b) anhydrous H3PO2, Py, Et3N; (ii) I2, 3 Å MS,
Py, rt; (iii) CH3ONa, CH3OH, rt.
The first non-hydrolysable mimic of cADPR is cyclic
aristeromycin-diphosphate-ribose, which had a similar
calcium release profile to that of cADPR.28 Another
agonist of cADPR, 3-deaza-cADPR, is 70-fold more
potent than cADPR.29 A further structural modification
of cADPR includes the pyrophosphate moiety, which has
been replaced by triphosphate30 or 1-(2-nitrophenyl)-
ethyl pyrophosphate.31 The cyclic adenosine triphos-
phate ribose is more potent in inducing calcium release.
However, little emphasis has placed on the Ca2+-release
activities of cADPR analogues modified at the N1-ribosyl
unit. Recently, Shuto et al. reported that the stable
mimic of cADPR, cADPcR (2b), caused a significant
release of Ca2+ in sea urchin eggs.21
Rat brain microsomes were one of the first mam-
malian cell preparations in which cADPR and IP3 were
shown to trigger Ca2+ release independently of each
other.32 The six novel cyclic nucleotide analogues (3, 4a ,
4b, 5a , 5b, and 6) were tested for their abilities to
release calcium from rat brain microsomes and com-
pared to authentic cADPR by using a confocal laser-
scanning microscope (CLSM). The dilution and all
experiments were conducted at 17 °C. The cross-
membrane property was tested with HeLa cells through
Lee has reviewed 23 different cellular systems re-
sponsive to cADPR.34 The widespread responses to

N1-Glycosyl-Substituted Mimics of Cyclic ADP-Ribose
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5345
acyclic analogue mimicking the furanose ring. SN2
substitution was carried out between the protected
inosine and glycosyl triflates to form the N1-glycosyl
inosine derivatives, accompanied with some O6-glycosyl
substituted as side products. The intramolecular cy-
clization was based on the strategy described by Mat-
suda et al. It was found that successful cyclization
depended on the reaction conditions, and that the
8-unsubstituted substrate can also be used to construct
the intramolecular cyclic pyrophosphate. The activities
of N1-glycosyl-substituted cADPR mimics were evalu-
ated by induced Ca2+ release in rat brain microsomes
and HeLa cells. It was found that the configuration of
N1-glycosyl moiety in cADPR is not a critical structural
factor for retaining the activity of induced Ca2+ release.
More interestingly, the N1-acyclic sugar analogue 6
exhibited strong activity by induced Ca2+ release in both
rat brain microsomes and HeLa cells. These analogues
should prove to be useful tools for further studies on
the mechanism of cADPR action.
F igu r e 3. Effects of cADPR, and different N1-glycosyl sub-
stituted cADPR analogues (3, 4a , 4b, 5a , 5b, and 6) on intra-
cellular calcium levels of intact HeLa cells. Cells were loaded
with Fluo-3 and then assayed as described in the Experimental
Section. The data shown are typical curves for at least two
experiments carried out in duplicate using different HeLa cell
preparations.
cADPR among species indicate it regulates a general
Ca2+-releasing mechanism in cells. To date, cell prepa-
rations include intact cells accessed by microinjection,
patch clamping or detergent permeabilization, and
isolated organelles. To determine the cross-membrane
property of the analogues, intact cells were chosen to
evaluate the Ca2+ release. In human HeLa cells trans-
fected with CD 38, a lymphocyte antigen, cADPR is
shown to play a role in regulating the cell doubling
time.35 It would be interesting to know if the cellular
Ca2+ level correlates with the cADPR level in HeLa cells.
Our results are shown in Figure 3. cADPR (200 µM)
exhibited no Ca2+ -releasing activity, presumably be-
cause it cannot cross the cell membrane. However, when
challenged with 200 µM 3, 4b, and 5b, the [Ca2+]i of
about one-half recorded cells promptly displayed a sharp
increase in fluorescence intensity which soon declined.
Acyclic compound 6 causes a [Ca2+]i increase at a
concentration of 100 µM. The 8-unsubstituted analogue
5a was not able to provoke calcium release at the same
concentration and only elicited calcium release in less
than 20% of cells at concentrations as high as 400 µM
while the 8-unsubstituted mimic 4a exhibited no Ca2+
release activity even at 400 µM concentration.
Exp er im en ta l Section
Ch em istr y. Anhydrous H3PO2 was prepared by coevapo-
rating 50% aqueous H3PO2 with dry pyridine until the total
weight was not reduced further. All solvents were dried and
distilled prior to use. Unless otherwise noted, materials were
obtained from commercial suppliers and were used as pro-
vided. Thin-layer chromatography was performed on silica gel
GF-254 (Qing-Dao Chemical Co., China) plates. Column chro-
matography was done on silica gel (200-300 mesh; Qing-Dao
Chemical Co.) Melting points were determined on an XT-4A
melting point apparatus and are uncorrected. Optical rotation
was determined with Perkin-Elmer 243B polarimeter. UV
spectra were recorded on
a Varian DMS200 UV-visible
spectrophotometer. Mass spectra were obtained on either VG-
ZAB-HS or Bruker APEX. High-resolution FAB (fast atom
bombardment) mass and HR EIMS (electrospray ionization)
were performed with Bruker BIFLEX III. 1H NMR and 13C
NMR were recorded with a J EOL AL300 or a Varian VXR-
500 spectrometer using CDCl3, DMSO-d6, or D2O as a solvent.
Chemical shifts are reported in parts per million downfield
from TMS (1H and 13C). 31P NMR spectra were recorded at
room temperature by use of Bruker Avance 300 spectrometer
(121.42 MHz). Orthophosphoric acid (85%) was used as an
external standard. Evaporations were carried out under
reduced pressure with a bath temperature <30 °C. Compounds
(3, 4a , 4b, 5a , 5b, and 6) can be purified on C18 reverse phase
column by two different developing buffer systems: MeCN/
TEAA (pH 7.0) or MeCN/TEAB (pH 7.0).
These results are consistent with the findings de-
scribed above that mimics with different configurations
at the N1-glycosyl moiety retained the activities of
induced Ca2+ release and 8-substituted mimics (4b, 5b)
facilitate the permeability of cell membrane. More
interestingly, the N1-acyclic analogue 6 exhibited a
strong potency to induce Ca2+ release in both of rat
brain microsomes and intact HeLa cells. The data
obtained from the experiments on HeLa cells showed
that the extracellular cADPR mimics can increase the
level of Ca2+ in intact cells, but it is still not well
understood if the cellular Ca2+ level correlates with the
level of cADPR mimics in cells or the cellular Ca2+ level
is elevated from other unknown mechanisms. Although
the mechanism of their activity is not clear, the N1-
glycosyl substituted mimics will be used as tools for
further investigation.
8-Br om o-N 1-(1′′,4′′-a n h yd r o-2′′-d e oxy-3′′,5′′-O-b e n z-
ylid en e-L-lyxitol-2′′-yl)-5′-O-TBDMS-2′,3′-d i-O-a cetyl-in o-
sin e (10a ) a n d 8-Br om o-O6-(1′′,4′′-a n h yd r o-2′′-d eoxy-3′′,5′′-
O-b e n zylid e n e -L-lyxit ol-2′′-yl)-5′-O-TBDMS-2′,3′-d i-O-
a cet yl-in osin e (10b). Trifluoromethansulfonic anhydride
(0.22 mL, 0.13 mmol) was added dropwise at -15 °C to a
solution of compound 7 (220 mg, 1 mmol) in dry CH2Cl2 (5
mL) and pyridine (0.5 mL). The mixture was stirred for 1 h
and poured into 2.5 mL of cool saturated NaHCO3 solution.
The organic phase was separated and the aqueous layer
extracted with CH2Cl2 (5 mL × 2). Combined organic phase
was dried over MgSO4, and solvent was concentrated to give
triflate 8, which was used for the next reaction without
purification. A mixture of 9 (544 mg, 1 mmol), K2CO3 (278 mg,
2 mmol), and catalytic amount 18-Crown-6 in anhydrous THF
(6 mL) was refluxed for 1 h. After cooling to room temperature,
triflate 8 was added and the resulting mixture stirred for 24
h at 45 °C. After removal of the solvent, the residue was
purified by column chromatography (SiO2, petroleum ether-
EtOAc) to afford 10a as a white solid (209 mg, 27.9%) and
In conclusion, a series of N1-glycosyl modified mimics
of cADPR have been synthesized. The N1-glycosyl mim-
ics were designed with different configurations or an
10b a solid (60 mg, 8%). 10a : mp 106-108 °C. [R]25 -89.4 (c
D
MeOH
0.71, CH3OH), UV λmax
208.2 (3.7), 232.4 (4.04), FAB-MS-

5346 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Huang et al.
(m/z): 749 [(M + 1)+]. 1H NMR (300 MHz, CDCl3) δ -0.01,
in vacuo. The resulting residue was purified by silica gel
0.01(each s, each 3H, (CH3)2Si), 0.82 (9H, (CH3)3C), 2.02, 2.13
column chromatography (CH2Cl2-MeOH) to afford 13 (106 mg,
MeOH
(each s, each 3H, 2 × AcO), 3.80 (dd, 1H, J 5′b
,
) 11.1 Hz,
73.7%) as a solid: mp 148-150 °C. UV λmax
202.5 (4.28),
5′a
J 5′b
,
) 4.8 Hz, H5′b), 3.90 (dd, 1H, J 5′a
,
) 11.1 Hz, J 5′a
,
)
251.2 (4.27); MALDI-TOF (m/z): 799 [(M + Na)+], 815 [(M +
K)+]. 1H NMR (300 MHz, DMSO) δ 2.07(s, 6H, 2 × AcO), 3.56-
3.67 (m, 2H, 2 × H5′), 3.81-3.97 (m, 2H, 2 × H5′′), 4.18 (m,
1H, H4′′), 4.22 (m, 1H, H3′′), 4.28-4.41 (m, 3H, H4′, 2 × H1′′),
4.60 (t, 1H, J ) 5.4 Hz, H3′), 5.32 (m, 1H, J ) 5.4 Hz, H2′′),
5.42 (m, 1H, 3′′-OH, exchangeable), 5.72 (m, 1H, exchangeable,
5′′-OH), 6.01-6.02 (m, 2H, H1′, H2′), 6.74-7.04 (m, 10H, Ar
H), 8.02, 8.03 (each d, each 1H, J P,H ) 3.3 Hz, exchangeable,
NH × 2), 8.01 (s, 1H, H2). 13C NMR (75 MHz, DMSO) δ 20.6,
55.4, 59.9, 63.7, 69.1, 69.7, 72., 80.3, 80.4, 83.9, 88.7, 114.2,
114.3, 117.2, 117.5, 117.6, 120.6, 120.7, 124.1, 125.6, 128.9,
147.8, 148.0, 169.7, 169.9. Anal. (C31H34BrN6O11P) C, H, N.
4′
5′b
4′
4.5 Hz, H5′a), 3.97 (m, 1H, H4′′), 4.06-4.22 (m, 2H, H4′, H1′′),
4.27-4.34 (m, 2H, H5′,H5′′), 4.42-4.47 (m, 1H, H1′′), 4.60 (m,
1H, H3′′), 5.50 (s, 1H, PhCH), 5.70 (t, 1H, J ) 5.4 Hz, H3′),
5.97-6.05 (m, 2H, H1′, H2′′), 6.27 (t, 1H, J ) 5.7 Hz, H2′), 7.37
(m, 5H, C6H5), 8.37 (s, 1H, H2). 13C NMR (75 MHz, CDCl3): δ
-5.4, -5.5, 18.3, 20.3, 20.6, 25.7, 54.3, 62.3, 67.4, 69.8, 70.3,
71.6, 73.8, 74.2, 83.1, 88.0, 99.1, 124.6, 125.7, 126.2, 128.5,
129.1, 136.9, 146.8, 148.1, 155.5, 169.1, 169.4. Anal. (C32H41
-
BrN4O10Si) C, H, N. 10b: mp 80-81 oC. [R]25 -51.9 (c 0.9,
D
MeOH
CH3OH), UV λmax
210.7 (4.16), 256.8(4.17); MALDI-TOF
1
(m/z): 772 [(M + Na)+], 788 [(M + K)+]. H NMR (500 MHz,
CDCl3) δ -0.04, -0.03, (each s, each 3H, (CH3)2Si), 0.83 (s,
9H, (CH3)3C), 2.06, 2.14 (each s, each 3H, 2 × AcO), 3.84 (dd,
1H, J ) 4.5, 11.0 Hz, H5′a), 3.95 (dd, 1H, J ) 5.0 Hz, 11 Hz,
H5′b), 4.02 (m, 1H, H4′′), 4.15 (dd, J ) 1.5, 13 Hz, H1′′a), 4.23
(dd, J ) 5.0, 10 Hz, H4′), 4.38 (m, 1H, H1b′′), 4.43 (m, 1H, H5′′a),
4.49 (m, 1H, H5′′b), 4.96 (dd, 1H, J ) 2.0, 5.0 Hz, H3′′), 5.69
(, dt, J ) 4.0, 9.0, 9.0 Hz, H2′′), 5.86 (t, 1H, J ) 5.5 Hz, H3′),
6.10 (d, 1H, J ) 5.5 Hz, H1′), 6.50 (t, J ) 5.5 Hz, H2′), 7.30 (m,
3H, Ar H), 7.45 (m, 2H, Ar H), 8.48 (s, 1H, H2). 13C NMR (75
MHz, DMSO) δ 11.26, 20.41, 20.61, 25.75, 62.42, 67.86, 69.09,
70.58, 71.54, 72.72, 73.86, 83.02, 88.31, 99.01, 126.32, 128.09,
128.86, 130.32, 130.42, 137.63, 151.92, 152.90, 158.64, 169.56,
169.37. Anal. (C32H41BrN4O10Si) C, H, N.
8-Br om o-N1-(5′′-O-MMTr -1′′,4′′-a n h yd r o-3′′-O-a cetyl-2′′-
d eoxy-L-lyxitol-2′′-yl)-5′-O-d ia n ilin op h osp h or yl-2′,3′-d i-O-
a cetyl-in osin e (14). The mixture of 13 (413 mg, 0.53 mmol)
and MMTrCl (245 mg, 0.795 mmol) in anhydrous pyridine (5
mL) was stirred for 48 h at room temperature, and then acetic
anhydride (270 mg, 2.65 mmol) was added to the resulting
mixture. The mixture was stirred overnight at room temper-
ature, and the solvent was concentrated in vacuo. CH2Cl2 (30
mL) and water (15 mL) were added, and the resulting mixture
was partitioned. The organic layer was separated and washed
in sequence with 1 M H2SO4 (10 mL), saturated NaHCO3
solution (10 mL), and brine (10 mL) and dried (MgSO4). The
solvent was concentrated and the residue was purified by silica
gel column chromatography (CH2Cl2-MeOH) to give 14 (406
8-Br om o-N 1-(1′′,4′′-a n h yd r o-2′′-d e oxy-3′′,5′′-O-b e n z-
ylid en e-L-lyxitol-2′′-yl)- 2′,3′-d i-O-a cetyl-in osin e (11). A
mixture of 10a (143 mg, 0.19 mmol) and TBAF (1 M in THF,
0.38 mL, 0.38 mmol) in THF (2 mL) was stirred for 6 h at
room temperature under neutral conditions. The resulting
mixture was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography
mg, 70%) as a white solid: mp 145-147 °C. [R]25D -4.3 (c 0.58,
MeOH
CH3OH); UV λmax
205.6 (4.7), 232.8 (4.53); MALDI-TOF
(m/z): 1113 [(M + Na)+], 1129 [(M + K)+]. 1H NMR (300 MHz,
DMSO) δ 1.50, 2.03, 2.10 (each s, each 3H, 3 × AcO), 3.00 (m,
1H, H5a′), 3.26 (m, 1H, H5′b), 3.74 (s, 3H, CH3O), 4.11-4.44
(m, 6H, 2 × H5′′, 2 × H1′′, H4′, H4′′), 5.34 (m, 1H, H3′′), 5.73 (m,
1H, H3′), 5.76 (m, 1H, H2′′), 5.98 (d, 1H, J ) 5.4 Hz, H1′), 6.12
(m, 1H, H2′), 6.7-7.35 (m, 24H, Ar H), 7.84 (s, 1H, H2), 8.05,
8.06 (each d, each 1H, 2H, J P,H ) 5.4 Hz, exchangeable, NH ×
2). 13C NMR (75 MHz, DMSO) δ 19.52, 20.17, 20.27, 53.59,
55.06, 60.63, 63.36, 69.03, 69.65, 71.14, 79.41, 80.35, 85.82,
88.07, 113.25, 117.24, 120.37, 123.46, 125.92, 126.99, 127.89,
128.53, 128.58, 129.94, 134.69, 140.69, 140.91, 140.97, 143.83,
144.05, 146.69, 147.29, 154.14, 158.27, 168.40, 169.30, 169.38.
Anal. (C53H52BrN6O13P) C, H, N.
(CH2Cl2-MeOH) to give 11 (109 mg, 90%): mp 126-128 °C.
[R]25 -8.3 (c 0.46, CH3OH); UV λmax
203.6 (4.41), 233.1
MeOH
D
(4.06); MALDI-TOF (m/z): 635 [(M + 1)+], 657[(M + Na)+],
673 [(M + K)+]. 1H NMR (300 MHz, DMSO) δ 2.04, 2.09 (each
s, each 3H, 2 × AcO), 3.57-3.74 (m, 2H, 2 × H5′′), 3.94 (m,
1H, H4′′), 4.07-4.24 (m, 4H, H1′′a, 2 × H5′, H4′), 4.45 (m, 1H,
H1′′b), 4.68 (m, 1H, H3′′), 5.13 (t, J ) 5.7 Hz, exchangeable, 5′-
OH), 5.60 (s, 1H, PhCH<), 5.62 (t, 1H, J ) 5.7 Hz, H3′), 5.74-
(m, 1H, H2′′), 6.02 (d, 1H, J ) 5.7 Hz, H1′), 6.09 (t, 1H, J ) 5.7
Hz, H2′), 7.29 (m, 5H, C6H5), 8.43(s, 1H, H2). 13C NMR (75 MHz,
DMSO) δ 20.23, 20.44, 55.46, 60.83, 66.76, 69.24, 70.10, 71.50,
73.10, 73.50, 83.06, 88.04, 98.07, 123.71, 126.00, 128.15,
128.82, 137.91, 147.73, 148.00, 154.80, 169.41, 169.55. Anal.
(C26H27BrN4O10) C, H, N.
8-Br om o-N 1-(1′′,4′′-a n h yd r o-3′′-O-a ce t yl-2′′-d e oxy-L-
lyxitol-2′′-yl)-5′-O-d ia n ilin op h osp h or yl-2′,3′-d i-O-a cetyl-
in osin e (15). The solution of 14 (380 mg, 0.35 mmol) in 5%
(w/v) TCA (6 mL) was stirred for 30 min and then poured into
cool saturated NaHCO3 solution (5 mL). The organic phase
was separated and dried over MgSO4. After removal of the
solvent, the residue was purified by silica gel column chroma-
tography (CH2Cl2-MeOH) to give compound 15 (190 mg,
8-Br om o-N 1-(1′′,4′′-a n h yd r o-2′′-d e oxy-3′′,5′′-O-b e n z-
yliden e-L-lyxitol-2′′-yl)-5′-O-(dian ilin oph osph or yl)-2′,3′-di-
O-a cetyl-in osin e (12). To solution of 11 (66 mg, 0.104 mmol)
and tetrazole (36 mg, 0.52 mmol) in anhydrous pyridine (2 mL)
was added (PhNH)2POCl (139 mg, 0.52 mmol) at room tem-
perature, and the mixture was stirred for 48 h. The solvent
was evaporated to dryness, and the residue was purified by
silica gel column chromatography (CH2Cl2-MeOH) to afford
12 as a white solid (80 mg, 89%). The crystal needles were
obtained from 95% ethanol: mp 146-148°. [R]25D -47.2 (c 0.33,
CH3OH); UV λmaxMeOH 235.6 (4.00); MALDI-TOF (m/z): 887 [(M
66.8%): mp 137-139 °C. [R]25 -5.6 (c 0.36, CH3OH), UV
D
MeOH
λmax
203.1 (4.29), 232.4 (4.01) MALDI-TOF (m/z): 841 [(M
+ Na)+], 857 [(M + K)+]. H NMR (300 MHz, DMSO) δ 2.01,
2.03, 2.08 (each s, each 3H, 3 × AcO), 3.92-4.01 (m, 2H, 2 ×
H5′), 4.07-4.43 (m, 7H, 2 × H5′′, 2 × H1′′, H4′, H4′′), 5.43 (m,
1H, H3′), 5.56 (m, 1H, H2′′), 5.76 (m, 1H, exchangeable, 5′′-OH),
6.02 (m, 2H, H1′, H2′), 6.74-7.05 (m, 10H, Ar H), 8.02-8.06
(m, 3H, 2 × NH, exchangeable, H2). 13C NMR (75 MHz, DMSO)
δ 20.62, 21.08, 55.14, 55.39, 63.39, 63.65, 69.30, 69.46, 69.63,
72.39, 80.27, 80.40, 88.82, 117.60, 117.66, 121.05, 124.19,
125.95, 129.05, 140.94, 141.00, 147.92, 155.28, 170.05, 170.25,
171.10. Anal. (C33H36BrN6O12P) C, H, N.
1
1
+ Na)+], 903 [(M + K)+]. H NMR (500 MHz, DMSO), δ 2.08,
2.09 (each s, each 3H, 2 × AcO), 3.88 (m, 1H, H4′′), 4.09 (t, J
) 10.5 Hz, 1H, H1′′), 4.17-4.26 (m, 4H, H1′′, H5′, 2H5′′), 4.33
(m, 1H, H5′), 4.44 (dt, J 4′,5′a ) 6.5 Hz, J 4′,5′b ) 3.5 Hz, J 4′,3′
)
8-Br om o-N1-(1′′,4′′-an h ydr o-5′′-O-[bis-(ph en ylth io)ph os-
p h or yl]-3′′-O-a cetyl-2′′-d eoxy-L-lyxitol-2′′-yl)-5′-O-d ia n ili-
n op h osp h or yl-2′,3′-d i-O-a cetyl-in osin e (16). To a solution
of 15 (150 mg, 0.183 mmol), PSS (244 mg, 0.64 mmol), and
tetrazole (44 mg, 0.64 mmol) in anhydrous pyridine (2 mL)
was added TPSC (110 mg, 0.37 mmol). The resulting mixture
was stirred for 72 h, and then the solvent was evaporated. The
residue was partitioned between CH2Cl2 (20 mL) and water
(10 mL), and the organic phase was washed with aqueous
saturated NaHCO3 (10 mL) and brine (10 mL), dried (Na2SO4),
6.5 Hz, 1H, H4′), 4.63 (dd, J 3′′,4′′ ) 5 Hz, J 3′′,2′′ ) 2.0 Hz, 1H,
H3′′), 5.58 (s, 1H, PhCH), 5.72 (m, 1H, H2′′), 5.80 (t, J ) 6.5
Hz, 1H, H3′), 6.03 (dd, J 1′,2′ ) 4.0 Hz, 1H, H1′), 6.05 (m, 1H,
H2′), 7.33-6.7 (m, 15H, Ar H), 8.06 (d, J P,H ) 3.0 Hz, 1H, NH)
8.04 (d, J P,H ) 3.5 Hz, 1H, NH), 8.22 (s, 1H, H2). Anal.(C38H38
-
BrN6O11P) C, H, N.
8-Br om o-N1-(1′′,4′′-a n h yd r o-2′′-d eoxy-L-lyxitol-2′′-yl)-5′-
O-d ia n ilin op h osp h or yl-2′,3′-d i-O-a cetyl-in osin e (13). The
solution of 12 (160 mg, 0.185 mmol) in 80% HOAc (2 mL) was
stirred for 4 h at 70 °C, and then the mixture was evaporated

N1-Glycosyl-Substituted Mimics of Cyclic ADP-Ribose
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5347
and evaporated under reduced pressure. The residue was
H), 8.27 (s, 1H, H2). 13C NMR (125 MHz, DMSO) δ -5.48, -5.4,
18.31, 20.4, 20.56, 25.81, 53.86, 55.22, 61.13, 62.84, 70.57,
71.51, 71.77, 77.66, 78.57, 82.18, 83.02, 88.28, 113.17, 118.6,
124.4, 126.15, 126.93, 127.14, 127.81, 127.86, 128.36, 129.18,
130.24, 132.52, 139.18, 147.06, 147.15, 148.11, 155.41, 158.64,
169.31, 169.54. Anal. (C48H57BrN4O11Si) C, H, N. 23b: 1H NMR
(300 MHz, CDCl3) δ: -0.02, -0.03 (each s, each 3H, (CH3)2-
Si), 0.833 (s, 9H, 3 × CH3), 2.04, 2.12 (each s, 2 × AcO), 3.52
(m, 2H, 2 × H5′), 3.75 (s, 3H, OCH3), 3.8-4.0 (m, 7H, OCH2,
2H1′′, H5′′a, H4′, H4′′), 4.20 (m, 2H, H3′′, H5′′b), 4.56 (m, 1H, H3′),
5.10 (m, 2H, CH2d), 5.69 (m, 1H, CHd), 5.84 (m, 1H, H2′′),
6.10 (d, 1H, J ) 4.8 Hz, H1′), 6.50 (m, 1H, H2′), 6.80-7.45 (m,
purified by silica gel column chromatography (CH2Cl2-MeOH)
to give compound 16 (188 mg, 95%): mp 111-113 °C. [R]25
D
MeOH
-57.1 (c 0.80, CH3OH), UV λmax
203.8 (4.54), 232.4 (4.54);
MALDI-TOF (m/z): 1105 [(M + Na)+], 1121 [(M + K)+]. 1H
NMR (300 MHz, DMSO) δ 1.95, 2.01, 2.08 (each s, each 3H, 3
× AcO), 3.96-4.09 (m, 3H, 2 × H5′, H4′), 4.20-4.24 (m, 2H, 2
× H5′′), 4.30-4.37 (m, 2H, 2 × H1′′), 4.45 (m, 1H, H4′′), 5.38 (m,
1H, H3′′), 5.71-5.76 (m, 2H, H3′, H2′′), 6.02 (d, 1H, J ) 4.5 Hz,
H1′), 6.09 (m, 1H, H2′), 6.72-7.42 (m, 20H, Ar H), 7.88 (s, 1H,
H2), 8.05 (m, 2H, exchangeable, 2 × NH). 13C NMR (75 MHz,
DMSO) δ: 20.02, 20.22, 20.58, 53.96, 61.81, 63.55, 68.31, 69.45,
71.63, 76.59, 78.64, 80.29, 80.40, 88.00,117.18, 117.26, 117.34,
120.37, 124.18, 124.96, 125.62, 128.51, 128.59, 129.52, 129.62,
129.87, 135.07, 135.15, 135.23, 140.88, 140.97, 146.78, 147.29,
154.54, 169.22, 169.28, 169.87. 31P NMR (121 MHz, DMSO) δ
3.93(s), 51.26(s). Anal. (C45H45BrN6O13P2S2) C, H, N.
14H, Ar H), 8.45(s, 1H, H2). HRMS (ESI, positive) for C48H57
-
BrN4O11SiNa: Calcd, 995.2868 [(M + Na)+]; Found, 955.2855.
8-Br om o-N1-(5′′-O-MMTr -1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-O-
a llyl-D-lyxitol-2′′-yl)-2′,3′-d i-O-a cetyl-in osin e (24). Starting
from 23a (430 mg, 0.44 mmol) with the same procedure shown
in the preparation of 11, 24 (310 mg) was obtained as a white
5-O-Ben zoyl-3-O-a llyl-1,4-a n h yd r o-D-xylitol (20). To a
solution of 19 (6.4 g, 19.2 mmol) in 75 mL of anhydrous CH2-
Cl2 were added 28.4 mL of Et3SiH (192 mmol) and 22.6 mL of
BF3‚Et2O solution at -5 °C under nitrogen atmosphere. After
the mixture was stirred at room temperature overnight, 100
mL saturated NaHCO3 solution was slowly added to the
mixture to destroy the excess of BF3-Et2O at 0 °C. The organic
layer was separated, and the aqueous phase was extracted
with CH2Cl2 (70 mL × 2). Combined organic phase was dried
over MgSO4, and the solvent was evaporated under reduced
pressure. The residue was purified using silica gel column
chromatography (petroleum ether-EtOAc) to yield white solid
20 (4.8 g, 90%). FAB-MS (m/z): 278 [M + 1]. 1H NMR (300
MHz, DMSO-d6): δ 3.57 (dd, 1H, J ) 1.5 Hz, 12 Hz, H1b), 3.88
(m, 1H H2), 4.00 (m, 2H, 2 × H5), 4.18 (m, 1H, H3), 4.31 (m,
2H, OCHa, H4), 4.38 (m, 1H, H1a), 4.45 (dd, 1H, J ) 4.2, 10.8,
OCHb), 5.12-5.28 (m, 2H, CH2d), 5.29 (d, 1H, J ) 2.4 Hz,
2-OH exchangeable), 5.89 (m, 1H, dCH), 7.55 (m, 2H, Ar H),
7.67 (m, 1H, Ar H), 7.97 (m, 2H, Ar H). 13C NMR (75 MHz,
DMSO): δ 63.5, 70.05, 73.3, 77.26, 84.13, 116.54, 128.78,
129.17, 129.63, 133.39, 134.96, 165.63. Anal. (C15H15O5) C, H,
N.
solid (86%): mp 75-77 °C. [R]25D +0.033 (c 3.15, CH3OH); UV
MeOH
λmax
204.2 (4.45); MALDI-TOF (m/z): 881 [(M + Na)+], 897
[(M + K)+]. H NMR (500 MHz, DMSO) δ 2.04, 2.12 (each s,
each 3H, 2 × AcO), 3.08 (dd, 1H, J ) 5.5, 9.5 Hz, H5′a), 3.31
(m, 1H, H5′b), 3.39 (m, 1H, OCHa), 3.38-3.64 (m, 2H, OCHb,
1
H
5′′a), 3.69 (m, 1H, H5′′b), 3.98 (s, 1H, CH3O), 4.02 (m, 1H, H1′′a),
4.17-4.20 (m, 3H, H4′′, H4′, H3′′), 4.25 (m, 1H, H1′′b), 4.78 (m,
2H, CH2d), 5.07 (t, J ) 5.5 Hz, exchangeable, 5′-OH), 5.26 (m,
1H, CHd), 5.56 (t, J ) 5.5, H3′), 5.66 (m, 1H, H2′′), 6.02 (d, J
) 5.5 Hz, H1′), 6.09 (t, 1H, J ) 5.5 Hz, H2′), 6.09-6.92 (m, 2H,
Ar H), 7.26-7.43 (m, 12H, Ar H), 8.093 (s, 1H, H2). 13C NMR
(125 MHz, DMSO) δ 20.22, 20.38, 54.22, 55.05, 59.77, 60.90,
61.61, 69.52, 70.13, 71.41, 71.90, 77.54, 80.89, 83.07, 85.89,
87.90, 113.23, 116.88, 123.60, 125.85, 126.92, 127.94, 129.97,
133.41, 134.95, 144.04, 144.36, 147.17, 147.63, 154.72, 158.22,
162.31, 169.43, 169.49. Anal. (C42H43BrN4O11) C, H, N.
8-Br om o-N1-(5′′-O-MMTr -1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-O-
a llyl-D-lyxitol-2′′-yl)-5′-O-(d ia n ilin op h osp h or yl)-2′,3′-d i-O-
a cetyl-in osin e (25). Starting from 24 (150 mg, 0.175 mmol)
with the same procedure shown in the preparation of 12, 180
mg of 25 was obtained as a white solid (95%): mp 218-220
MeOH
°C. [R]25 +4.26 (c 0.63, CH3OH); UV λmax
204.6 (5.03),
D
5-O-MMTr -3-O-a llyl-1,4-a n h yd r o-D-xylitol (21). Com-
pound 20 (4.8 g, 17.2 mmol) was dissolved in a mixture of
methanol (50 mL) and sodium methoxide(1.39 g, 25.8 mmol)
and stirred overnight. The mixture was neutralized with HOAc
and concentrated to dryness in vacuo. The resulting residue
was coevaporated with dry pyridine (3.5 mL) and used in the
next reaction without further purification. To a solution of
crude compound in anhydrous pyridine (20 mL) was added
MMTrCl (8.0 g, 25.8 mmol) under nitrogen atmosphere. The
mixture was stirred for 48 h and evaporated to dryness. The
residue was applied to a silica gel column and eluted (petro-
leum ether-EtOAc) to yield 21 (6.05 g, 78.8%). FAB-MS(m/
235.9 (5.23); MALDI-TOF (m/z): 1112 [(M + Na + 1)+], 1128
[(M + K + 1)+]. 1H NMR (500 MHz, DMSO-d6) δ 2.05, 2.07
(each s, each 3H, 2 × AcO), 3.07 (dd, 1H, J ) 3.3, 5.4 Hz, H5′a),
3.32 (m, 2H, H5′b, CHaO), 3.51 (dd, 1H, J ) 3.3 Hz, 12.5 Hz,
CHbO), 3.72 (s, 3H, CH3O), 4.04-4.12 (m, 2H, H1′′a, H3′′), 4.21-
4.28 (m, 3H, H5′′a, H4′, H4′′), 4.32 (m, 1H, H5′′b), 4.41 (dd, 1H, J
) 6.5, 10.5 Hz, H1′′b), 4.72 (m, 2H, CH2d), 5.20 (m, 1H, >CHd),
5.64 (m, 2H, H2′′, H3′), 5.97 (d, 1H, J ) 4.0, 6.0 Hz, H2′), 6.02
(d, J ) 4.0 Hz, H1′), 6.77-7.43 (m, 24H, Ar H), 8.03 (s, 1H,
H2), 8.11 (t, 2H, J P,H ) 10 Hz, 2 × NH, exchangeable).
8-B r o m o -N 1-(1′′,4′′-a n h y d r o -2′′-d e o x y -3′′-O -a lly l-D -
lyxitol-2′′-yl)-5′-O-(d ia n ilin op h osp h or yl)-2′,3′-d i-O-a cetyl-
in osin e (26). Starting from 25 (210 mg, 0.193 mmol) with the
same procedure shown in the preparation of 15, 125 mg of 26
1
z): 446 [M + ]. H NMR (300 MHz, DMSO):δ 3.09(m, 2H, 2 ×
H5), 3.49 (m, 1H, H1a), 3.74 (s, 3H, CH3O), 3.86 (m, 4H, H1b
,
H2, H3, OCHa), 4.14 (m, 2H, H4, OCHb), 5.06 (m, 2H, CH2d),
5.20 (m, 1H, exchangeable, 2-OH), 5.70 (m, 1H, -CHd), 6.89-
7.39 (m, 14H, Ar H). Anal. (C28H30O5) C, H, N.
8-Br om o-N1-(5′′-O-MMTr -1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-O-
a lly l-D -ly x i t o l-2′′-y l)-5′-O -T B D MS -2′,3′-d i -O -a c e t y l-
in osin e (23a) an d 8-Br om o-O6-(5′′-O-MMTr -1′′,4′′-an h ydr o-
2′′-d eoxy-3′′-O-a llyl-D-lyxitol-2′′-yl)-5′-O-TBDMS-2′,3′-d i-O-
a cetyl-in osin e (23b). Compound 23a (solid, 426 mg, 34%) and
the corresponding O6-regioisomer 23b (syrup, 85 mg, 6.8%)
were obtained from 700 mg of 9 (1.29 mmol) as described for
was obtained as a white solid (79.6%): mp 116-118 °C. [R]25
D
MeOH
+20.8 (c 0.59, CH3OH); UV λmax
204.3 (4.33), 231.6 (4.36);
MALDI-TOF (m/z): 839 [(M + Na)+], 855 [(M + K)+]. 1H NMR
(500 MHz, DMSO) δ 2.07, 2.07 (each s, each 3H, 2 × AcO),
3.41 (dd, 1H, J ) 5.5, 13 Hz, H1′′a), 3.60-3.65 (m, 2H, 2 × H5′),
3.76 (dd, 1H, J ) 5.0, 13 Hz, H1′′b), 3.88 (dd, 1H, J ) 5.0, 11
Hz, H4′), 3.93 (dd, 1H, J ) 8.0, 10 Hz, CHaO), 4.08 (m, 1H,
H
5a′′), 4.17 (dd, 1H, J ) 4.5, 10 Hz, H5b′′), 4.2 (dd, 1H, J ) 5.0,
6.0 Hz, H3′′), 4.35 (m, 1H, CHbO), 4.42 (m, 1H, H4′′), 4.77 (bs,
1H, exchangeable, 5′′-OH), 5.41 (m, 1H, CHd), 5.66 (m, 2H,
H2′′, H3′), 6.02 (m, 2H, H1′, H2′), 6.78-7.15 (m, 10H, Ar H), 8.13
(s, 1H, H2), 8.12 (t, 2H, J P,H ) 10 Hz, exchangeable NH × 2).
Anal. (C34H38BrN6O11P) C, H, N.
the preparation of 10, with 22 instead of 8. 23a : mp 88-91
MeOH
°C. [R]25 -1.15 (c 0.35, CH3OH); UV λmax
206.9 (4.66),
D
232.8 (4.53); MALDI-TOF (m/z): 995 [(M + Na)+], 1011 [(M +
K)+]. H NMR (500 MHz, CDCl3) δ 0.05, 0.06 (each 3H, each
1
3H, (CH3)2Si), 0.90 (s, 9H, (CH3)3C), 2.09, 2.15, (each 3H, each
s, 2 × AcO), 3.65,3.77 (m, 2H, 2 × H5′), 3.81 (s, 3H, CH3O),
3.82-4.08 (m, 6H,2 × H1′′, OCH2, H4′, H5′′a), 4.17 (dd, 1H, J )
1.8 Hz, 6.6 Hz, H5′′b), 4.23 (m, 1H, H4′′), 4.42 (dd, 1H, J ) 3.3
Hz, 3.9 Hz, H3′′), 5.05 (m, 2H, CH2d), 5.53 (m, 1H, CHd), 5.72
(t, 1H, J ) 3.3 Hz, H3′), 5.88 (m, 1H, H2′′), 6.06 (d, 1H, J ) 3.3
Hz, H1′), 6.29 (t, 1H, H2′, J ) 3.3 Hz), 7.01-7.48 (m, 14H, Ar
8-Br om o-N1-[5′′-O-[bis-(p h en ylth io)p h osp h or yl]-1′′,4′′-
a n h yd r o-2′′-d eoxy-3′′-O-a llyl-D-lyxitol-2′′-yl]-5′-O-(d ia n ili-
n op h osp h or yl)-2′,3′-d i-O-a cetyl-in osin e (27). Starting from
26 (200 mg, 0.29 mmol) with the same procedure shown in
the preparation of 16, 255 mg of 27 was obtained as a white
solid (96.3%): mp 88-90 °C. [R]25 +22.3 (c 0.45, CH3OH);
D
MeOH
UV λmax
207.1 (4.86), 229.4 (4.69); MALDI-TOF (m/z): 1103

5348 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Huang et al.
1
[(M + Na)+], 1116 [(M + K)+]. H NMR (300 MHz, DMSO-d6)
4.59 (m, 2H, 2 × H1′′), 5.04-5.18 (m, 2H, CH2d), 5.21-5.23
(m, 2H, H3′, H3′′), 5.55 (t, 1H, J ) 5.7 Hz, 5′-OH, exchangeable),
5.84 (m, 1H, CHd), 6.02 (d, 1H, J ) 5.4 Hz, H1′), 6.08 (t, 1H,
J ) 5.4 Hz, H2′), 7.48 (t, 2H, J ) 4.5 Hz, Ar H), 7.64 (t, 2H, J
) 7.5 Hz, Ar H), 7.91 (d, 1H, J ) 7.2 Hz, Ar H), 8.39 (s, 1H,
H2). Anal. (C29H31BrN4O11) C, H, N.
8-Br om o-N1-(5′′-O-Ben zoyl-1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-
O-a llyl-D-r ibitol-2′′-yl)-5′-O-(d ia n ilin op h osp h or yl)-2′,3′-d i-
O-a cetyl-in osin e (35). Starting from 34 (0.8 g, 1.16 mmol)
with the same procedure shown in the preparation of 12, 1 g
δ 2.04, 2.04 (each s, each 3H, 2 × AcO), 3.29 (dd, 1H, J ) 4.8,
12.6 Hz, H1′′a), 3.67 (dd, 1H, J ) 5.1, 12.6 Hz, H1′′b), 3.95-4.13
(m, 4H, OCH2, 2 × H5′′), 4.22-4.45 (m, 5H, 2 × H5′, H4′, H4′′
,
H
3′′), 4.8 (m, 2H, CH2d), 5.34 (m, 1H, CHd), 5.63-5.67 (m,
2H, H2′′, H3′), 5.98-6.01 (m, 2H, H1′, H2′), 6.81-7.55 (m, 20H,
Ar H), 8.11 (s, 1H, H2), 8.11-8.14 (m, 2H, exchangeable, NH
× 2). 13C NMR (75 MHz, DMSO) δ 20.21, 54.05, 63.96, 65.96,
69.69, 69.92, 71.62, 71.82, 77.15, 80.03, 88.08, 117.02, 117.03,
120.43, 123.49, 125.48, 125.58, 125.67, 128.74, 129.75, 129.87,
133.12, 135.09, 135.14, 141.00, 147.70, 147.87, 154.71, 169.29,
of 35 was obtained as a white solid (93%): mp 110-112 °C.
169.42. 31P NMR δ: 4.01 (s), 50.45 (s). Anal. (C46H47
-
[R]25 +17.8 (c 0.39, CH3OH); UV λmax
203.3 (5.07), 229.9
MeOH
D
(5.01); MALDI-TOF (m/z): 943 [(M + Na)+], 959 [(M + K)+].
1H NMR (500 MHz, DMSO-d6) δ 2.07, 2.08 (each s, each 3H,
2 × AcO), 3.97 (dd, 1H, J ) 4.5,11.5 Hz, H5′a), 4.10-4.18 (m,
4H, OCH2, H5′b, H1′′a), 4.25-4.31 (m, 2H, 2 × H5′′), 4.43 (m,
1H, H4′), 4.48-4.58 (m, 2H, H4′′, H3′′), 4.50 (dd, 1H, J ) 4.0, 12
Hz, H1′′b), 5.02-5.15 (m, 2H, CH2d), 5.20 (m, 1H, H2′′), 5.64
(m, 1H, H3′), 5.79 (m, 1H, CHd), 6.04-6.05 (m, 2H, H1′, H2′),
6.78 (m, 2H, Ar H), 6.97-7.11 (m, 8H, Ar H), 7.49 (m, 2H, Ar
BrN6O12P2S2) C, H, N.
5-O-Ben zoyl-3-O-a llyl-1,4-a n h yd r o-D-r ibitol (31). Start-
ing from 30 (4.9 g, 14.7 mmol) with the same procedure shown
in the preparation of 20, 3.4 g of 31 was obtained as pale yellow
syrup (83%). FAB-MS (m/z): 279 [M + H] +. 1H NMR (300 MHz,
DMSO-d6) δ 3.63 (dd, 1H, J ) 2.7, 9.3 Hz, H5a), 3.79 (dd, 1H,
J ) 4.8, 7.2 Hz, H4), 3.92 (dd, 1H, J ) 4.5, 9.3 Hz, H5b), 3.40-
4.07 (m, 2H, 2 × H1), 4.13-4.30 (m, 3H, OCHa, H2, H3), 4.43
(dd, 1H, J ) 3.0 Hz, 8.7 Hz, OCHb), 4.10 (d, 1H, J ) 4.8 Hz,
2-OH, exchangeable), 5.09-5.29 (m, 2H, CH2d), 5.88 (m, 1H,
CHd), 7.49-7.97 (m, 5H, Ar H). 13C NMR (75 MHz, DMSO) δ
64.94, 68.73, 70.26, 73.02, 77.47, 79.60, 116.61, 128.76, 129.17,
129.55, 133.40, 135.16, 165.57. Anal. (C15H18O5) C, H, N.
H), 7.64 (m, 1H, Ar H), 7.93 (m, 2H, Ar H), 8.07 (t, 2H, J P,H
)
6.0 Hz, exchangeable, 2 × NH), 8.03 (s, 1H, H2). Anal. (C41H42
-
BrN6O12P) C, H, N.
8-Br om o-N1-(1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-O-a llyl-D-r ibi-
tol-2′′-yl)-5′-O-(d ia n ilin op h osp h or yl)-in osin e (36). To solu-
tion of 35 (0.9 g, 0.98 mmol) in methanol (10 mL) was added
CH3ONa (190 mg, 3.5 mmol), and the reaction mixture was
stirred overnight. The resulting mixture was evaporated under
reduced pressure and purified by silica gel column chroma-
tography (CH3OH-CH2Cl2) to yield white solid 36 (676 mg,
95%): mp 130-132 °C. MALDI-TOF (m/z): 755 [(M + Na)+].
1H NMR (500 MHz, DMSO-d6) δ 3.58 (m, 1H, H5′a), 3.68 (m,
1H, H5′b), 3.77 (dd, 1H, J ) 4.0, 8.5 Hz, H5′′a), 3.99 (dd, 1H, J
) 6.0, 13.5 Hz, OCHa), 4.06-4.32 (m, 8H, OCHb, 2 × H1′′, H4′,
8-Br om o-N1-(5′′-O-ben zoyl-1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-
O-a llyl-D -r ib it ol-2′′-yl)-5′-O-TBDMS-2′,3′-d i-O-a ce t yl-
in osin e (33a) an d 8-br om o-O6-(5′′-O-ben zoyl-1′′,4′′-an h ydr o-
2′′-d eoxy-3′′-O-a llyl-D-r ibitol-2′′-yl)-5′-O-TBDMS-2′,3′-d i-O-
a cetyl-in osin e (33b). Starting from 31 (1.62 g, 5.8 mmol),
with the same procedure shown in the preparation of 10, 33a
(1.25 g, 26.8%) was obtained as white solid, accompanied by
the O6-regioisomer 33b (1 g, 21.5%). 33a : mp 106-107 °C.
[R]25 +19.7 (c 0.21, CH3OH); UV λmax
209.7 (4.41), 250.7
MeOH
D
H
4′′, H3′′, H3′, H5′′b), 4.97 (m, 1H, exchangeable, 3′-OH), 5.08-
(4.00); MALDI-TOF (m/z): 827 [(M + Na)+], 843 [(M + K)+].
1H NMR (500 MHz, CDCl3) δ -0.01,0.01 (each s, each 3H,
(CH3)2Si), 0.85 (s, 9H, (CH3)3C), 2.08, 2.15 (each s, each 3H, 2
× AcO), 3.75 (dd, 1H, J ) 5.0, 11.5 Hz, H5′a), 3.85 (dd, 1H, J
) 5.0, 11.5 Hz, H5′b), 3.96 (m, 1H, H4′), 4.09 (dd, 1H, J ) 6.5,
13 Hz, OCHa), 4.18-4.23 (m, 1H, H4′′, H3′′), 4.23-4.35 (m, 2H,
2 × H1′′), 4.40 (dd, 1H, J ) 5.0, 13 Hz, OCHb), 4.52 (dd, 1H, J
) 6.0, 12 Hz H5′′a), 4.56 (dd, 1H, J ) 4.0, 12 Hz, H5′′b), 5.13-
5.27 (m, 2H, CH2d), 5.47 (m, 1H, H2′′), 5.68 (t, 1H, J ) 5.5 Hz,
H3′), 6.82 (m, 1H, CHd), 6.05 (d, 1H, J ) 5.5 Hz, H1′), 6.27 (t,
1H, J ) 5.5 Hz, H2′), 7.39 (t, 2H, J ) 9.0 Hz, Ar H), 7.54 (m,
1H, Ar H), 7.94 (m, 2H, Ar H), 8.33 (s, 1H, H2). 13C NMR (75
MHz, CDCl3) δ -5.45, -3.70, 20.58, 20.77, 20.41, 25.62, 25.76,
59.58, 62.39, 63.97, 70.37, 71.60,71.66, 71.79, 82.88, 83.30,
87.00, 88.10, 118.37, 128.45, 129.42, 129.60, 133.23, 133.43,
133.49, 144.79, 145.27, 148.04, 154.07, 166.12, 169.50, 169.54.
5.11 (m, 2H, H2′, H2′′), 5.17-5.25 (m, 2H, CH2d), 5.47 (d, J )
5.0 Hz, exchangeable, 2′-OH), 5.63 (m, exchangeable, 5′′-OH),
5.82-5.87 (m, 2H, CHd, H1′), 6.74-7.15 (m, 10H, Ar H), 8.04,
8.09 (each d, 2H, J P,H ) 10 Hz, exchangeable, 2 × NH), 8.37
(s, 1H, H2). Anal. (C30H34BrN6O9P) C, H, N.
8-Br om o-N1-(5′′-O-MMTr -1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-O-
a llyl-D-r ibitol-2′′-yl)-5′-O-(d ia n ilin op h osp h or yl)-2′,3′-d i-O-
a cetyl-in osin e (37). Starting from 36 (600 mg, 0.82 mmol)
with the same procedure shown in the preparation of 14, 520
mg of 37 was obtained as a white solid (58.3%). MALDI-TOF
(m/z): 1111 [(M + Na)+], 1127 [(M + K)+]. 1H NMR (300 MHz,
CDCl3) δ 2.05, 2.08 (each s, each 3H, 2 × AcO), 3.58 (m, 1H,
H
5′a), 3.75 (s, 3H, CH3O), 3.82-4.00 (m, 3H, H5′b, 2 × H5′′),
4.09-4.20 (m, 6H, OCH2, 2 × H1′′, H4′, H4′′), 4.57 (m, 1H, H3′′),
5.12-5.30 (m, 3H, CH2d, H2′′), 5.73-5.84 (m, 2H, H3′, CHd),
6.05 (d, 1H, J ) 5.4 Hz, H1′), 6.36 (t, 1H, J ) 5.4 Hz, H2′),
6.69-7.24 (m, 12H, Ar H), 8.54 (s, 1H, H2). Anal. (C54H54
BrN6O12P) C, H, N.
Anal. (C35H45BrN4O11Si) C, H, N. 33b: mp 49 °C. [R]25D -34.2
202.3 (4.37), 257.9 (4.10). 1H
MeOH
-
(c 0.87, CH3OH); UV λmax
NMR (300 MHz, CDCl3) δ: -0.01,0.03 (each s, each 3H, (CH3)2-
Si), 0.831 (s, 9H, (CH3)3C), 2.06, 2.13 (each s, each 3H, 2 ×
AcO), 3.85 (dd, 1H, J ) 5.1, 11.1 Hz, H5a′), 3.96 (dd, 1H, J )
8-Br om o-N1-(1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-O-a llyl-D-r ibi-
t ol-2′′-yl)-5′-O-(d ia n ilin op h osp h or yl)-2′,3′-d i-O-a ce t yl-
in osin e (38). Starting from 37 (480 mg, 0.44 mmol) with the
same procedure shown in the preparation of 15, 180 mg of 38
was obtained as a white solid (50%) and 80 mg 37 was
4.8, 11.1 Hz, H5b′), 4.13-4.27 (m, 7H, 2 × H1′′, H4′, H4′′, H3′′
,
OCH2), 4.50 (m, 2H, 2 × H5′′), 5.24 (m, 2H, CH2d), 5.68 (m,
1H, H2′′), 5.81 (m, 1H, H3′), 5.89 (m, 1H, CHd), 6.10 (d, 1H, J
) 4.8 Hz, H1′), 6.48 (dd, 1H, J ) 4.8, 12 Hz, H2′), 7.41 (m, 3H,
Ar H), 8.03 (m, 2H, Ar H), 8.47 (s, 1H, H2). 13C NMR (75 MHz,
CDCl3) δ: -5.57, -5.59, 18.26, 20.39, 20.55, 25.73, 62.37, 64.35,
70.58, 71.26, 71.61, 72.01, 76.58, 77.00, 77.43, 81.19, 81.85,
82.99, 84.07,88.38, 117.77, 122.19, 128.32, 129.32, 129.74,
130.38, 132.95, 133.81, 151.83, 152.93, 158.36, 166.31, 169.49.
Anal. C35H45BrN4O11Si) C, H, N.
recovered: mp 119-121 °C. [R]25 +13.0 (c 0.33 CH3OH);
D
MALDI-TOF (m/z): 839 [(M + Na)+], 855 [(M + K)+]. 1H NMR
(300 MHz, CDCl3) δ 2.07, 2.10 (each s, each 3H, 2 × AcO),
3.35 (bs, 1H, OH, exchangeable), 3.59 (dd, 1H, J ) 2.4, 12.3
Hz, H5′a), 3.81-3.87 (m, 3H, 2 × H5′′, H5′b), 4.10-4.23 (m, 2H,
OCH2), 4.30-4.44 (m, 4H, 2 × H1′′, H4′, H4′′), 4.63 (m, 1H, H3′′),
5.10-5.31 (m, 3H, CH2d, H2′′), 5.72-5.88 (m, 2H, CHd, H3′),
6.08 (d, 1H, J ) 5.4, H1′), 6.38 (t, 1H, J ) 5.4 Hz, H2′), 6.71-
8-Br om o-N1-(5′′-O-Ben zoyl-1′′,4′′-a n h yd r o-2′′-d eoxy-3′′-
O-allyl-D-r ibitol-2′′-yl) -2′,3′-di-O-acetyl-in osin e (34). Start-
ing from 33a (1.16 g, 1.4 mmol) with the same procedure
shown in the preparation of 11, 0.9 g of 34 was obtained as a
7.24 (m,12H, Ar H, 2 × NH), 8.55 (1, 1H, H2). Anal. (C34H38
-
BrN6O11P) C, H, N.
8-Br om o-N1-[5′′-O-[bis-(p h en ylth io)p h osp h or yl]-1′′,4′′-
a n h yd r o-2′′-d eoxy-3′′-O-a llyl-D-r ibitol-2′′-yl]-5′-O-(d ia n ili-
n op h osp h or yl)-2′,3′-d i-O-a cetyl-in osin e (39). Starting from
38 (150 mg, 0.183 mmol) with the same procedure shown in
white solid (90%): mp 89-91 °C. [R]25D +8.1 (c 1.22, CH3OH);
MeOH
UV λmax
203.3 (4.34), 257.5 (4.02); MALDI-TOF (m/z): 713
[(M + Na)+]. H NMR (300 MHz, DMSO) δ 2.30, 2.12 (each s,
each 3H, 2 × AcO), 3.51 (m, 1H, H5′a), 3.66 (m, 1H, H5′b), 4.15-
4.20 (m, 4H, OCH2, 2 × H5′′), 4.32-4.34 (m, 2H, H4′, H4′′), 4.52-
1
the preparation of 16, 190 mg of 39 was obtained as a white
solid (96%). [R]25 +45.6 (c 0.17, CH3OH); UV λmax
236.1
MeOH
D

N1-Glycosyl-Substituted Mimics of Cyclic ADP-Ribose
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5349
(4.00); MALDI-TOF (m/z):1104 [(M + Na + 1)+], 1120 [(M +
K + 1)+]. 1H NMR (300 MHz, CDCl3) δ: 2.03, 2.05 (each s, each
3H, 2 × AcO), 3.92 (m, 1H, H5′a), 3.94-4.25 (m, 7H, H5′b, 2 ×
pyridine (254 mg, ca. 2.69 mmol), Et3N (179 µL, 1.29 mmol),
and pyridine (3 mL), and the resulting solution was stirred
for 11 h at room temperature. After the solvent was evaporated
to dryness under reduced pressure, the residue was partitioned
between CHCl3 and H2O. The aqueous layer was evaporated
(<30 °C) with pyridine (5 mL), and the residue was dissolved
in TEAA (0.1 M, pH 7.0, 10 mL). The solution was purified by
a C18 reverse phase column (1.8 × 15 cm), and the column
was developed using a linear gradient of 0-40% CH3CN in
TEAA buffer (0.1 M, pH 7.0, 600 mL). Appropriate fractions
were evaporated, and excess TEAA was removed by C18
reverse phase column chromatography (1.8 × 13 cm, eluted
was 20% aqueous CH3CN) to give 46 (80 mg, yield 60.7%) as
H
5′′, OCH2, H3′′, H4′′), 4.42 (m, 3H, 2 × H1′′, H4′), 5.03-5.11 (m,
2H, CH2d), 5.16 (m, 1H, H3′), 5.61 (m, 1H, H2′′), 5.81 (m, 1H,
CHd), 6.02 (m, 2H, H1′, H2′), 6.74-7.56 (m, 20H, AromH), 8.06
(m, 2H, exchangeable, 2 × NH), 8.24 (s, 1H, H2). 31P NMR (121
MHz, CDCl3) δ 3.05 (s), 49.91(s). Anal. (C46H47BrN6O12PS2) C,
H, N.
8-Br om o-N1-[(2-a cetoxyeth oxy)-m eth yl]-5′-O-TBDMS-
2′,3′-O-isop r op ylid en e-in osin e (41). To the mixture of 40
(500 mg, 1 mmol) and DBU (0.76 g, 0.5 mmol) in CH2Cl2 (10
mL) was added ClCH2OCH2CH2OAc (1.52 g, 10 mmol) at 0
°C. After stirring for 30 min, the solvent was evaporated in
vacuo and the residue was purified by silica gel column
chromatography (CH2Cl2-MeOH) to give compound 41 (605
a triethylammonium salt. [R]25 -41.6 (c 0.69, H2O); UV
D
λmax O 243.3 (4.30). 1H NMR (500 MHz, D2O) δ 2.01, 2.02, 2.09
H
2
(each s, each 3H, 3 × CH3), 3.84 (m, 1H, H5′a), 3.97 (m, 1H,
1
H
5′b), 4.09 (m, 1H, H4′′), 4.21 (m, 2H, H4′, H1′′a), 4.34-4.41 (m,
mg, 82%). H NMR (300 MHz, DMSO) δ -0.01, 0.00 (each s,
3H, H1′′b, 2 × H5′′), 5.26 (m, 1H, H3′′), 5.64-5.74 (m, 3H, H2′,
H3′, H2′′), 6.11 (d, 1H, J ) 3.0 Hz, H1′), 6.96-7.05 (m, 5H, Ar
H), 8.23 (s, 1H, H2). 31P NMR (D2O 121 MHz, decoupled with
1H) δ 1.08 (s), 15.39 (s). HRMS (FAB, negative) for C27H30N4-
each 3H, (CH3)2Si), 0.88 (s, 9H, (CH3)3C), 1.44, 1.63 (each s,
each 3H, (CH3)2C), 2.09 (s, 3H, AcO), 3.60-3.85 (m, 4H, OCH2,
2 × H5′), 3.42-4.27 (m, 3H, H4′, CH2OAc), 5.09 (dd, 1H, J )
3.6, 6.3 Hz, H3′), 5.57 (dd, 2H, J ) 10.5,17.1, OCH2N), 5.71
(m, 1H, H2′), 6.15 (d, J ) 1.8 Hz), 8.61 (s, 1H, H2).
O
16P2SBr: Calcd, 839.0041 [(M - 1)-]; Found, 839.0049.
N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-O-a cetyl-L-lyxitol-2′′-
8-Br om o-N 1-[(2-a ce t oxye t h oxy)-m e t h yl]-2′,3′-O-iso-
p r op ylid en e-in osin e (42). Starting from 41 (143 mg, 0.23
mmol) with the same procedure shown in the preparation of
11, 119 mg of 42 was obtained as a white solid (90%). 1H NMR
(300 MHz, DMSO) δ 1.24, 1.33 (each s, each 3H, (CH3)2C), 1.98
(s, 3H, AcO), 3.76 (m, 2H, 2 × H5′), 3.78 (m, 2H, OCH2), 4.14
(m, 3H, CH2OAc, H4′), 4.96 (m, 2H, H3′, OH, exchangeable),
5.01 (m, 2H, OCH2N), 5.56 (dd, 1H, J ) 2.1, 6.3 Hz, H2′), 6.05
(d, 1H, 2.1 Hz), 8.55 (s, 1H, H2).
yl]-8-br om o-5′-O-ph osph or yl-2′,3′-di-O-acetyl-in osin e 5′,5′′-
cyclicp yr op h osp h a te (47). A solution of 46 (32 mg, 31.3 µm)
in pyridine (10 mL) was added slowly over 20 h, using a
syringe pump, to a mixture of I2 (170 mg, 646 µmol) and MS
3 Å (2 g), in pyridine (40 mL) at room temperature in the dark.
The MS 3 Å was filtered off with Celite and washed with H2O.
The combined filtrate was evaporated, and the residue was
partitioned between CHCl3 and H2O. The aqueous layer was
evaporated, and the residue was dissolved in 0.1 M TEAA
buffer (5.0 mL), which was applied to C18 reverse phase
column (1.5 × 11 cm). The column was developed using a linear
gradient of 0-40% CH3CN in TEAA buffer (0.1 M, pH 7.0, 300
mL). Appropriate fractions were evaporated under reduced
pressure, and excess of TEAA was removed by C18 reversed
phase column chromatography (1.5 × 11 cm, eluted with 20%
aqueous CH3CN) to afford 47 (8 mg, yield 28.1%) as a
triethylammonium salt. 1H NMR (500 MHz, D2O) δ 2.02, 2.05,
2.11 (each s, each 3H, 3 × AcO), 3.89 (m, 2H, 2 × H5′), 4.02 (t,
1H, J ) 10 Hz, H4′′), 4.18 (m, 1H, H5′′a), 4.24 (t, 1H, J ) 10 Hz,
H1′′a), 4.38 (m, 1H, H5′′b), 4.46 (m, 1H, H1′′b), 4.76 (m, 1H, H3′′),
5.18 (m, 1H, H3′), 6.25 (s, 1H, H1′), 6.34 (m,1H, H2′′), 6.39 (d,
1H, 5.0 Hz, H2′), 8.20 (s, 1H, H2). 31P NMR (D2O 121 MHz,
decoupled with 1H) δ -7.9, -10.1. HRMS (FAB, negative) for
C17H20N4O14P2Br Calcd, 644.9640 [(M - 1)-]; Found, 644.9647
(removal of two acetyl group).
8-Br om o-N1-[(2-a cetoxyeth oxy)-m eth yl]-5′-O-d ia n ilin o-
p h osp h or yl-2′,3′-O-isop r op ylid en e-in osin e (43). Starting
from 42 (66 mg, 0.135 mmol) with the same procedure shown
in the preparation of 12, 84 mg of 43 was obtained as a white
MeOH
solid (84.5%). UV λmax
203.8 (4.16), 256.8 (4.17) MALDI-
TOF (m/z): 755 [(M + Na)+], 771 [(M + K)+]. 1H NMR (300
MHz, DMSO) δ 1.20, 1.53 (each s, each 3H, CH3 × 2), 1.96 (s,
3H, AcO), 3.74 (t, 2H, J ) 4.5 Hz, 2 × H5′), 4.10 (t, 2H, J ) 4.5
Hz, OCH2), 4.23 (m, 2H, OCH2OAc), 4.37 (m, 1H, H4′), 5.08
(dd, 1H, J ) 3.6, 6.0 Hz, H3′), 5.33 (m, 1H, H2′), 5.48 (m,
OCH2N), 6.05 (d, 1H, J ) 1.8 Hz, H1′), 6.73-7.12 (m, 10H, Ar
H), 8.02, 8.03 (each d, each1H, J P,H ) 9.9 Hz, exchangeable,
NH × 2), 8.32 (s, 1H, H2). 13C NMR (75 MHz, DMSO) δ 20.62,
25.18, 26.96, 62.91, 64.15, 67.22, 75.06, 80.88, 82.87, 85.51,
90.58, 113.81, 117.21, 120.41, 124.29, 125.58, 128.62, 128.77,
140.93, 141.07, 147.94, 149.10, 154.70, 170.32. HRMS (ESI,
positive) for C30H35BrN6O9P: Calcd, 733.1381 [(M + 1)+];
Found, 733.1383.
N1-[5′′-O-P h osp h or yl-2′′-d eoxy-L-lyxitol-2′′-yl]-8-br om o-
5′-O-p h osp h or yl-in osin e 5′, 5′′-cyclicp yr op h osp h a te (3).
To the solution of compound 47 (75 OD254) in methanol (5 mL)
were added CH3ONa (5 mg) and the mixture was stirred for 3
h at room temperature. The solvent was evaporated in vacuo
and the residue was dissolved in 0.1 M TEAA buffer (5.0 mL),
which was applied to C18 reverse phase column (1.5 × 11 cm).
The column was developed using a linear gradient of 10-40%
CH3CN in TEAA buffer (0.1 M, pH 7.0, 200 mL). Appropriate
fractions were evaporated under reduced pressure, and excess
of TEAA was removed by C18 reverse phase column chroma-
tography (1.5 × 11 cm, eluted with 20% aqueous CH3CN) to
8-Ch lor o-N1-[[[bis(ph en ylth io)ph osph or yl]oxy-eth oxy]-
m et h yl]-5′-O-(d ia n ilin op h osp h or yl)-2′,3′-O-isop r op yli-
d en ein osin e (45). To the mixture of 43 (73 mg, 0.1 mmol) in
2.5 mL of methanol was added CH3ONa (10 mg, 0.19 mmol),
and the resulting solution was stirred overnight. After removal
of the solvent, the residue was purified by silica gel column
chromatography (CH2Cl2-MeOH) to give 50 mg of 44, which
was dissolved in anhydrous pyridine (3 mL) and then were
added TPSCl (70 mg, 0.232 mmol), PSS (44 mg, 0.116 mmol),
and tetrazole (8 mg, 0.116 mmol). The resulting mixture was
stirred for 24 h at room temperature. After removal of the
solvent, the residue was partitioned between CH2Cl2 (20 mL)
and water (10 mL), and the organic phase was washed with
aqueous saturated NaHCO3 (10 mL) and brine (10 mL), dried
(Na2SO4), and evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (CH2Cl2-
afford (48 OD254, yield 48%) as a triethylammonium salt. [R]25
D
H
O
1
2
-39.6 (c 0.114, H2O); UV λmax
205.5 (4.20), 250.5 (3.71). H
NMR (500 MHz, D2O) δ: 4.00 (m, 2H, 2 × H5′), 4.06 (m, 1H,
H4′′), 4.16 (m, 1H, J ) 5.0, 10 Hz, H5′′a), 4.24 (m, 1H, H1′′a),
4.32 (m, 1H, H5′′b), 4.45 (m, 1H, H1′′b), 4.55 (m, 2H, H3′′, H4′),
4.86 (dd, 1H, J ) 2.0, 5.0 Hz, H3′), 5.19 (t, 1H, J ) 5.0 Hz,
H2′), 5.76 (m, 1H, H2′′), 5.98 (d, 1H, J ) 5.0 Hz, H1′), 8.27 (s,
1H, H2). HRMS (FAB, negative) for C15H18N4O13P2Br: 602.9534
[(M - 1)-]; Found, 602.9536.
MeOH) to give compound 4518 (44 mg, 48.4%). [R]25 +1.46 (c
D
MeOH
0.27, CH3OH), UV λmax
202.5 (4.63).
8-Br om o-N1-[5′′-O-(p h en ylt h io)p h osp h or yl-2′′-d eoxy-
3′′-O-a ce t yl-L-lyxit ol-2′′-yl]-5′-O-p h osp h or yl-2′,3′-d i-O-
a cetyl-in osin e (46). A mixture of 16 (140 mg, 0.129 mmol)
and isoamyl nitrite (261 µL, 1.94 mmol) in pyridine-AcOH-
Ac2O (2:1:1, v/v, 4 mL) was stirred at room temperature for 8
h. After the reaction mixture was evaporated (at <30 °C), the
residue was dissolved in a mixture of H3PO2 solution in
8-Ch lor o-N1-[[[2-(p h en ylth io)p h osp h or yl]oxy-eth oxy]-
m et h yl]-5′-O-p h osp h or yl-2′,3′-O-isop r op ylid en ein osin e
(48). Starting from 45 (120 mg, 0.132 mmol) with the same
procedure shown in the preparation of 46, 75 mg of 48 was
obtained as a triethylammonium salt (67%). [R]25 +0.54 (c
D

5350 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Huang et al.
H
O
1
H
2
2
0.18, H2O); UV λmax
246.0 (4.00). H NMR (300 MHz, D2O)
UV λmax O 206.4 (4.02), 251.5 (3.62). 1H NMR (500 MHz, D2O)
δ: 3.63 (1H, m, OCHa), 3.69 (dd, 1H, J ) 5.0, 13 Hz, H5′a), 3.86
(m, 2H, OCHb, H5′b), 4.05 (m, 3H, 2 × H5′′, H4′′), 4.24-4.35 (m,
4H, 2 × H1′′, H4′, H3′′,), 4.50 (m, 1H, H2′′), 4.82 (m, 2H, CH2d),
5.36 (t, 1H J ) 5.5 Hz, H3′), 5.55 (m, 1H, CHd), 5.59 (m, 1H,
H2′), 5.84 (d, 1H, J ) 5.5 Hz, H1′), 8.05 (s, 1H, H8), 8.66 (s, 1H,
H2). 31P NMR (D2O 121 MHz, decoupled with 1H) δ -9.93,
-10.83. HRMS (FAB, negative) for C18H23N4O13SP2: Calcd,
565.0742 [(M - 1)-]; Found, 565.0742. 4b: [R]25D +34.9 (c 0.18,
δ 1.50,1.76 (each s, each 3H, (CH3)2C), 3.77-3.83 (m, 4H,
POCH2-, 2 × H5′), 3.97 (m, 2H, OCH2), 4.32 (m, 1H, H4′), 5.11
(m, 1H, H3′), 5.24 (d, 1H, J ) 10.8 Hz, -NCHaO), 5.38 (m, 1H,
H2′), 5.68 (d,1H, J ) 10.8 Hz, -NCHbO), 6.13 (d, 1H, H1′), 7.08-
7.25 (m, 5H, Ar H), 8.25 (s, 1H, H2). 31P NMR (D2O 121 MHz,
1
decoupled with H) δ 18.68 (s), 2.80 (s). HRMS (FAB, nagative)
for C22H27N4O12P2SCl: Calcd, 667.0437 [(M - 1)-]; Found
667.0433.
H
O
1
N1-[(2-O-P h osp h or yl-et h oxy)-m et h yl]-8-ch lor o-5′-O-
p h osp h or yl-2′,3′-O-isop r op ylid en e-in osin e 5′,5′′-cyclic-
p yr op h osp h a te (49). Starting from 41 (26 mg, 0.23 mmol)
with the same procedure shown in the preparation of 47, 12
mg of 49 was obtained as a triethylammonium salt (46.2%). 18
2
H2O), UV λmax
207 (4.23), 256.2 (3.93). H NMR (300 MHz,
D2O) δ 3.69 (dd, 1H, J ) 5.0, 13 Hz, H5′a), 3.82 (m, 2H, OCH2),
3.87 (dd, 1H, J ) 5.5, 13 Hz, H5′b), 4.02 (m, 3H, 2H5′′, H4′),
4.22 (m, 3H, H4′′ H3′, H1′′), 4.34 (m, 1H, H1′′), 4.50 (m, 1H, H3′′),
4.79 (m, 2H, CH2d), 5.49 (m, 1H, H2′′), 5.53 (m, 1H, CHd),
5.57 (1H, m, H2′), 5.98 (d, 1H, 6.5 Hz). 31P NMR (D2O 224 MHz,
N1-[(2-O-P h osp h or yl-et h oxy)-m et h yl]-8-ch lor o-5′-O-
p h osp h or yl-in osin e 5′, 5′′-cyclicp yr op h osp h a te (6). The
mixture of 49 (150 OD254) in 60% HCOOH (3 mL) was stirred
for 5 h and then evaporated under reduced pressure. The
purification of the residue was performed at the same proce-
dure as above by C18 reverse column to give the target
1
decoupled with H) δ: -10.11, -10.95; HRMS (FAB, negative)
for C18H22N4O13P2Br: Calcd, 642.9847 [(M - 1)-]; Found,
642.9849.
N1-[5′′-O-(P h en ylth io)p h osp h or yl-2′′-d eoxy-3′′-O-a llyl-
D -r i b i t o l-2′′-y l]-5′-O -P h o s p h o r y l-2′,3′-d i -O -a c e t y l-
in osin e (52a ) a n d 8-Br om o-N-1-[5′′-O-(p h en ylth io)p h os-
ph or yl- 2′′-deoxy-3′′-O-allyl-D-r ibitol-2′′-yl]-5′-O-ph osph or yl-
2′,3′-d i-O-a cetyl-in osin e (52b). Starting from 39 (100 mg,
92.6 µm) with the same procedure shown in the preparation
of 46, only the ratio between H3PO2 and Et3N (3:1), 52a (25
mg, 28.7%) and 52b (27 mg, 28.7%) were obtained as triethyl-
molecule 6. 18 (67 OD254, 44.6%) [R]25 +18.7(c 0.21, H2O); UV
D
H
O
1
2
λmax
201.6 (4.05). H NMR (500 MHz, D2O) δ 3.69 (3H, m,
H5′a, -OCH2), 3.80 (2H, m, POCH2O), 3.92 (1H, dd, J ) 3.0
Hz, 9.0 Hz, H5′b), 4.18 (2H, m, CH2), 4.64 (1H, m, H4′), 4.75
(1H, m, H3′), 5.18 (1H, d, J ) 11 Hz, -OCHaN), 5.52 (1H, t, J
) 5.0 Hz, H2′), 5.85 (1H, d, J ) 11 Hz, -OCHbN), 5.98(1H, d,
J ) 5.0 Hz H1′).13P NMR δP (D2O 121 MHz, decoupled with
1H): -10.44, -10.79. HRMS (FAB-) for C13H16N4O12P2Cl:
516.9934 [(M-H)-]; Found 516.9925.
ammonium salts. 52a : [R]25 11.6 (c 0.36, H2O); UV λmax
H
O
2
D
202.5 (4.39), 244.2 (3.97). 1H NMR (300 MHz, D2O) δ: 1.83,2.01
(each s, each 3H, 2 × AcO), 3.48 (m, 1H, H5′a), 3.69 (m, 1H,
N1-[5′′-O-(P h en ylth io)p h osp h or yl-2′′-d eoxy-3′′-O-a llyl-
D-lyxitol-2′′-yl]-5′-O-p h osp h or yl-2′, 3′-d i-O-a cetyl-in osin e
(50). Starting from 27 (68 mg, 62.9 µm) with the same
procedure shown in the preparation of 46, 45 mg of 50 was
obtained as a white solid (70%). 1H NMR (300 MHz, D2O) δ
1.94, 1.11 (each s, each 3H, 2 × CH3), 3.27 (m, 1H, H5′a), 3.68
H
5′b), 3.86-4.13 (m, 8H, OCH2, 2 × H5′′, H4′, 2 × H1′′, H4′′), 4.42
(m, 1H, H3′′), 4.93 (m, 2H, CH2d), 5.03 (m, 1H, H2′′), 5.46 (m,
1H, H3′), 5.63 (m, 1H, H2′), 5.69 (m, 1H, CHd), 6.18 (d, 1H, J
) 6.0 Hz, H1′), 6.75-6.83 (m, 3H, Ar H), 7.10-7.13 (m, 2H, Ar
H), 8.12 (s, 1H, H2), 8.36 (s, 1H, H8). 31P NMR (D2O 121 MHz,
1
decoupled with H) δ: 1.80 (s), 18.02 (s). HRMS (FAB, negative)
Calcd, C28H33N4O15SP2 759.1144 [(M - 1)-], Found, 759.1144.
(m, 1H, H5′b), 3.94-4.18 (m, 8H, 2 × H1′′, OCH2, H4′′, 2 × H5′′
,
H4′), 4.37 (m, 1H, H3′′), 4.64 (m, 2H, CH2d), 5.19 (m, 1H, CHd
), 5.58 (m, 2H, H2′′, H3′), 5.92 (m, 1H, H1′), 6.17 (m, 1H, H2′),
7.20-7.45 (m, 5H, C6H5), 8.13 (s, 1H, H2). 31P NMR (D2O 121
52b: [R]25D +18.2 (c 0.4, H2O); UV λmax O 248.2 (4.06). 1H NMR
H
2
(300 MHz, D2O) δ: 1.97, 2.05 (each s, each 3H, 2 × AcO), 3.75
(dd, 1H, m, H5′a,), 3.83 (dd, 1H, J ) 5.7, 13.6 Hz, H5′b), 3.95-
4.10 (m, 6H, OCH2, 2 × H5′′, H4′, H1′′a), 4.16 (m, 2H, H1′′b, H4′′),
4.42 (m, 1H, H3′′), 5.07 (m, 2H, CH2d), 5.13 (m, 1H, H2′′), 5.62
(m, 1H, H3′), 5.72 (m, 1H, CHd), 5.95 (dd, 1H, J ) 4.5, 6.6 Hz,
H2′), 6.21 (d, 1H, J ) 4.5 Hz, H1′), 6.96 (m, 3H, Ar H), 7.30 (m,
2H, Ar H), 8.23 (s, 1H, H2). 31P NMR (D2O 121 MHz, decoupled
with 1H) δ: 4.47 (s), 17.46 (s). HRMS (FAB, negative) for
1
MHz, decoupled with H) δ 5.39, 18.93. HRMS (FAB, negative)
for (C28H32N4O15SP2Br): 837.0249 [(M - 1)-]; Found 837.0267.
N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-O-a llyl-D-lyxit ol-2′′-
yl]-5′-O-ph osph or yl-2′,3′-di-O-acetyl-in osin e 5′,5′′-cyclicpy-
r op h osp h a te (51a ) a n d N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-
O-a llyl-D-lyxitol-2′′-yl]-8-br om o-5′-O-p h osp h or yl-2′,3′-d i-
O-acetyl-in osin e 5′,5′′-cyclicpyr oph osph ate (51b). Starting
from 50 (41 mg, 40 µm) with the same procedure shown in
the preparation of 47, 4 mg of 51a was obtained (12%),
accompanied by 12 mg of 51b (33%) as triethylammonium
salts. 51a : 1H NMR (500 MHz, D2O) δ 1.89,2.04 (each s, each
3H, 2 × CH3), 3.67 (dd, 1H, J ) 4.5, 13.5 Hz, H5′a), 3.75 (1H,
m, OCHa), 3.83 (1H, dd, J ) 5.5, 13.5 Hz, H5′b), 3.94 (1H, m,
OCHb), 4.00 (dd, 1H, J ) 5.5, 12 Hz, H5′′a), 4.07 (m, 1H, H1′′a),
4.22 (m, 1H, H4′′), 4.31 (m, 2H, H1′′b, H4′), 4.40 (m, 1H, H3′′),
4.61 (t, 1H, J ) 9.0 Hz, H3′′), 4.79 (m, 1H, CH2d), 5.45 (m, 1H,
CHd), 5.52 (1H, m, H2′′), 5.67 (m, 1H, H3′), 6.11 (d, J ) 6.0
Hz, H1′), 6.28 (m, 1H, H2′), 7.95 (s, 1H, H8), 8.62 (s, 1H, H2).
31P NMR (D2O 121 MHz, decoupled with 1H) δ -8.64, -9.50.
HRMS (FAB, negative) for C22H27N4O15P2: Calcd, 649.0953 [(M
- 1)-]; Found, 649.0968. 51b: 1H NMR (300 MHz, D2O)
δ2.04,2.92 (each s, each 3H, 2 × AcO), 3.72-4.39 (m, 11H, 2
× H5′, 2 × H5′′, H4′′, CH2, 2 × H1′′, H4′, H3′′), 4.61 (2H, m, CH2d
), 5.50 (m, 2H, H2′′, CHd), 5.71 (m, H3′, 1H,), 6.16 (d, J ) 5.4
Hz, H1′), 6.29 (t, 1H, J ) 5.4 Hz, H2′), 8.60 (s, 1H, H2). HRMS
(FAB, negative) for C22H26N4O15P2Br: Calcd, 727.0059 [(M -
1)-]; Found, 727.0067.
C
28H32N4O15SP2Br: Calcd, 837.0249 [(M - 1)-]; Found,
837.0232.
N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-O-a llyl-D-r ib it ol-2′′-
yl]-5′-O-p h osp h or yl-2′,3′-d i-O-a cet yl-in osin e 5′, 5′′-cy-
clicp yr op h osp h a te (53a ). Starting from 52a (15 mg, 15.9
µmol) with the same procedure shown in the preparation of
47, 8 mg of 53a was obtained as a white solid (60.6%). 1H NMR
(500 MHz, D2O) δ: 1.91,2.04 (each s, each 3H, 2 × AcO), 3.90
(dd, 1H, J ) 5.0, 12 Hz, H5′a), 3.95-4.03 (m, 3H, H5′b, 2 × H5′′),
4.07 (m, 2H, H4′, H4′′), 4.10-4.22 (m, 2H, OCH2), 4.32 (m, 2H,
2 × H1′′), 4.41 (1H, m, H3′′), 5.06-5.19 (m, 2H, CH2d), 5.31
(m, 1H, H2′′), 5.71 (dd, J ) 5.5, 10.5 Hz, 1H, H3′), 5.82 (m, 1H,
CHd), 6.12 (d, 1H, J ) 5.5 Hz, H1′), 6.26 (t, 1H, J ) 5.5 Hz,
H2′), 7.97 (s, 1H, H8), 8.46 (s, 1H, H2). 31P NMR (D2O 121 MHz,
decoupled with 1H); δ: -8.07, -9.43. HRMS (FAB, negative)
for C22H27N4O15P2: Calcd, 649.0953 [(M - 1)-]; Found, 649.0951.
N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-O-a llyl-D-r ib it ol-2′′-
yl]-5′-O-p h osp h or yl-in osin e 5′,5′′-cyclicp yr op h osp h a t e
(5a ). Starting from 53a (89 OD254) with the same procedure
shown in the preparation of 3, 5a (33 OD254, 37%) was obtained
as triethylammonium salt. [R]25D 28.3 (c 0.22, H2O), UV λmax
H
O
N1-[5′′-O-p h osp h or yl-2′′-d eoxy-3′′-O-a llyl-D-lyxit ol-2′′-
yl]-5′-O-ph osph or yl-in osin e 5′,5′′-cyclicpyr oph osph ate (4a)
a n d N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-O-a llyl-D-lyxitol-2′′-
yl]-8-b r om o-5′-O-p h osp h or yl-in osin e 5′,5′′-cyclicp yr o-
p h osp h a te (4b). Starting from 51a (60 OD254) and 51b (40
OD254) with the same procedure shown in the preparation of
3, 4a (28 OD254, 46.7%) and 4b (15 OD254, 37.5%) were obtained
2
207.1 (4.18), 251.2 (3.75). 1H NMR (500 MHz, D2O) δ: 3.96
(m, 2H, 2 × H5′), 4.05 (m, 2H, 2 × H5′′), 4.16 (m, 2H, H4′, H4′′),
4.26 (m, 2H, OCH2, H1′′a), 4.32 (m, 1H, H1′′b), 4.38 (m, 1H, H3′′),
4.56 (m, 1H, H2′′), 5.11-5.28 (m, 2H, OCH2d), 5.32 (t, 1H, J )
5.5 Hz, H3′), 5.38 (m, 1H, H2′), 5.86 (m, 1H, CHd), 5.88 (d, 1H,
J ) 5.5, H1′), 8.07 (s, 1H, H8), 8.49(s, 1H, H2). 31P NMR (D2O
121 MHz, decoupled with 1H) δ: -8.07, -9.43. HRMS (FAB,
as triethylammonium salts. 4a : [R]25 +18.9. (c 0.20, H2O),
D

N1-Glycosyl-Substituted Mimics of Cyclic ADP-Ribose
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5351
negative) for C18H23N4O13P2: Calcd, 565.0742 [(M - 1)-];
Found, 565.0743.
resolution; CLSM, confocal laser-scanning microscope;
HPLC, high performance liquid chromatography; MS,
mass spectrometry; IP3, inositol triphosphate, 3 Å MS,
3 Å molecular sieve, EDC, 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride; THF, tetrahydro-
furan; DMSO, dimethyl sulfoxide; TsCl, tosyl chloride;
Py, pyridine; PDC, pyridinium dichromate; TPSCl,
triisopropylbenzenesulfonyl chloride; MMTrCl, p-anis-
ylchlorodiphenylmethane; DCM, dichloromethane; TCA,
trichloroacetic acid; PSS, cyclohexylammonium S,S-
diphenyl phosphorodithioate; TBDMS, tert-butyldi-
methylsilyl; TEAA, triethylammonium acetate; TEAB,
triethylammonium bicarbonate; TBAF, tetrabutyl-
ammonium fluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-
7-ene; TrCl, triphenylmethyl chloride.
N1-[5′′-O-P h osp h or yl-2′′-d eoxy-3′′-O-a llyl-D-r ib it ol-2′′-
yl]-8-b r om o-5′-O-p h osp h or yl-in osin e 5′,5′′-cyclicp yr o-
p h osp h a te (5b). Starting from 52b (15 mg, 14.7 µm) with
the same procedure shown in the preparation of 47, compound
53b was obtained, accompanied by minor of 53a as a triethyl-
ammonium salt. Compound 53b was directly used to deacety-
lation without removal of excess of TEAA. After neutralizing
the mixture, the residue was evaporated to dryness in vacuo
and purified by C18 reverse column to give the target product
5b: [R]25 +18.7 (c 0.13, H2O), UV λmax
206.4 (4.23), 253.1
H
O
2
D
(3.48). 1H NMR (500 MHz, D2O) δ: 3.95 (m, 2H, 2 × H5′), 4.05
(m, 2H, 2 × H5′′), 4.13-4.32 (m, 7H, 2 × H1′′, OCH2, H4′′, H4′,
H
3′′), 4.64 (m, 1H, H2′′), 5.11-5.34 (m, 2H, CH2d), 5.35 (m, 1H,
H3′), 5.42 (t, J ) 5.5 Hz, H2′), 5.83 (m, 1H, CHd), 6.00 (d, 1H,
J ) 5.5 Hz, H1′), 8.46 (s, 1H, H2).31P NMR (D2O 121 MHz,
decoupled with 1H) δ: -8.27 (d, J ) 12 Hz), -9.53 (d, J ) 12
Hz). HRMS (FAB, negative) for C18H22N4O13P2Br: Calcd,
642.9847 [(M - 1)-]; Found, 642.9858.
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Biologica l Activity Assa ys. (1) Preparations of rat brain
microsomes homogenates and HeLa cells culture. Microsomes
were prepared from Sprague-Dawley rat brain described
previously.32 The homogenate medium was 250 mM N-meth-
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concentration of 1 mg/mL for the release experiment.
Aliquots (10-20 mL) of HeLa Cells from RPMI 1640
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17 °C using 500 µL of cell suspension and microsomes
homogenate after loading fluorescence indicator on CLSM
(Lecia TCS-NT, Germany). Free [Ca2+]i variation was mea-
sured by monitoring fluorescence at excitation and emission
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Ack n ow led gm en t. We thank National Natural
Science Foundation of China for financial support. We
also thank Professor G. M. Blackburn, the University
of Sheffield, for a helpful discussion.
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