Synthesis of potent agonists of the D-myo-inositol 1,4,5-trisphosphate receptor based on clustered disaccharide polyphosphate analogues of adenophostin A

Article abstract of DOI:10.1021/jm000957c

Clustered disaccharide analogues of adenophostin A (2), i.e. mono-, di-, and tetravalent derivatives 6-8, respectively, were synthesized and evaluated as novel ligands for the tetrameric D-myo-inositol 1,4,5-trisphosphate receptor (IP₃R). The s

Full text of DOI:10.1021/jm000957c

J . Med. Chem. 2000, 43, 3295-3303
3295
Syn th esis of P oten t Agon ists of th e D-m yo-In ositol 1,4,5-Tr isp h osp h a te Recep tor
Ba sed on Clu ster ed Disa cch a r id e P olyp h osp h a te An a logu es of Ad en op h ostin A
Martin de Kort,† Vanessa Correa,‡ A. Rob P. M. Valentijn,† Gijs A. van der Marel,† Barry V. L. Potter,*,§
Colin W. Taylor,*,‡ and J acques H. van Boom*,†
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands,
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ , U.K., and
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath BA2 7AY, U.K.
Received March 28, 2000
Clustered disaccharide analogues of adenophostin A (2), i.e. mono-, di-, and tetravalent
derivatives 6-8, respectively, were synthesized and evaluated as novel ligands for the tetrameric
D-myo-inositol 1,4,5-trisphosphate receptor (IP3R). The synthesis was accomplished via
Sonogashira coupling of propargyl 2-O-acetyl-5-O-benzyl-3-O-(3,4-di-O-acetyl-2,6-di-O-benzyl-
R-D-glucopyranosyl)-â-D-ribofuranoside (16) with iodobenzene 18, 22, or 25, followed by
deacetylation, phosphorylation, and deprotection. The abilities of the target compounds 6-8,
as well as ribophostin 4, propylphostin 5, and IP3 (1), to evoke Ca2+ release from permeabilized
hepatocytes or displacement of [3H]IP3 from its receptor in hepatic membranes were compared.
Although the binding affinities of 4-8 were similar, there were modest though significant
differences in their potencies in Ca2+ release assays: tetraphostin 8 > IP3 ∼ diphostin 7 >
phenylphostin 6 > ribophostin 4 ∼ propylphostin 5.
In tr od u ction
Channel opening appears to require binding of IP3 to
at least three, and possibly all four, subunits of the
IP3R15-17 followed by binding of Ca2+ to at least one site
on either the receptor or an associated protein.15,17,18
Inhibition of IP3Rs by cytosolic Ca2+ is regulated by IP3,
with IP3 binding controlling whether stimulatory or
inhibitory Ca2+-binding sites are accessible.19 Finally,
IP3 binding also causes a slower partial inactivation of
the receptor.20 It is important to resolve whether IP3
binding to a single subunit within the tetrameric
receptor is sufficient to cause inactivation and whether
IP3 binding regulates the Ca2+-binding sites of only the
subunit to which IP3 is bound or Ca2+ binding to each
subunit of the tetramer.15,19
It occurred to us that these IP3-binding events to the
tetrameric receptor could be studied in more detail by
using multivalent ligands. The latter class of molecules
has been used to explore both carbohydrate-protein
interactions21 and the interactions between cyclic GMP
and cation channels22 and can give rise to enhanced
binding affinity22,23 relative to their monovalent coun-
terparts. However, it might be expected that the func-
tionalization of IP3 (1) necessary for coupling to a central
scaffold would result in loss of potency. In this respect,
the metabolically resistant adenophostins A and B (2
and 3, Figure 1)24 as highly potent,25 full agonists of
IP3R provide attractive alternatives. More importantly,
analogues26-28 lacking the adeninyl moiety (e.g. ribo-
phostin 4)29 are still equipotent to IP3.30 The latter
implies that analogues of adenophostin A (2) in which
the adenine is replaced by an appropriate spacer might
be used as an acceptable substitute for a corresponding
IP3 derivative.
The intracellular messenger D-myo-inositol 1,4,5-
trisphosphate (IP3, 1) is released after activation of
receptors that stimulate phospholipase C activity1,2 and
then binds to IP3 receptors (IP3R) within intracellular
stores, causing the large conductance channel of the
IP3R to open.3 Ca2+ flowing through the open channel
causes a local increase in cytosolic [Ca2+] that can both
regulate intracellular events4 and control further release
of intracellular Ca2+ stores to produce more widespread
increases in cytosolic [Ca2+].5
In mammals, the three closely related subtypes of
IP3R (types 1-3)6 assemble to form tetrameric Ca2+
channels which may be either homo- or heteromeric6
and which cryo-electron microscopy studies have re-
vealed as large particles with 4-fold symmetry and a
width of about 12 nm.7 For all three receptor subtypes,
the Ca2+ channel is formed by membrane-spanning
regions close to the C-terminal, while the IP3-binding
domain lies within the N-terminal.8,9
More detailed studies of type 1 IP3Rs indicate that
the pore of the channel is formed by the fifth and sixth
(final) transmembrane regions10 and the intervening
loop,10 while the IP3-binding site is formed by two
distinct domains within the N-terminus.11 Although the
different IP3R subtypes are differentially expressed in
different tissues12 and differ in some aspects of their
regulation,6 the physiological significance of the receptor
diversity is unclear.13,14
Activation, inhibition, and inactivation of IP3R are
complex processes regulated by both IP3 and Ca2+
.
* To whom correspondence should be addressed. For J .H.v.B.:
Phone: (+31) 71 527 4374. Fax: (+31) 71 527 4307. E-mail: j.boom@
chem.leidenuniv.nl.
We report here in full detail31 the synthesis and
biological evaluation of the novel mono-, di-, and tetra-
valent adenophostin A analogues: ‘propylphostin’ 5,
‘phenylphostin’ 6, ‘diphostin’ 7, and ‘tetraphostin’ 8.
† Leiden University.
‡ University of Cambridge.
§ University of Bath.
10.1021/jm000957c CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/28/2000

3296 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
de Kort et al.
F igu r e 1. Structures of D-myo-inositol 1,4,5-trisphosphate (IP3, 1), adenophostin A (2) and B (3), ribophostin 4, propylphostin 5,
phenylphostin 6, diphostin 7, and tetraphostin 8.
Sch em e 1. Construction of Acetylene Derivative 16 and Synthesis of Propylphostin 5a
a
Reagents and conditions: (i) butane-2,3-dione (1.1 equiv), CH(OCH3)3, cat. CSA, CH3OH, reflux, 3 h, 86% (2 regioisomers); (ii) BnBr,
NaH, DMF, 1 h, 98%; (iii) NIS/cat. TfOH, Et2O, 45 min, 83%; (iv) a. TBAF (1 M in THF)/1,4-dioxane, 1/4, v/v, 50 °C, 8 h, b. BnBr, NaH,
DMF, 92%; (v) a. HOAc/H2O/(HOCH2)2, 14/6/3, v/v/v, reflux, 75 min, b. Ac2O, pyr, 16 h, 86%; (vi) propargyl alcohol (2 equiv), TMSOTf,
(CH2Cl)2, 30 min, 81%; (vii) a. NaOMe, MeOH, then Dowex H+, b. dibenzyl (N,N-diisopropyl)phosphoramidite, 1H-tetrazole, (CH2Cl)2/
CH3CN, 3/1, v/v, 30 min, then t-BuOOH, 0 °C, 30 min, 80%; (viii) Pd/C, H2 (1 atm), NaOAc, 1,4-dioxane/i-PrOH/H2O, 4/2/1, v/v/v, 16 h,
85%.
Resu lts a n d Discu ssion
glucopyranoside (9) with trimethyl orthoformate and
butane-2,3-dione36,37 in the presence of a catalytic
amount of camphorsulfonic acid (CSA) gave, after
separation of two regioisomers, 3,4-O-butane-2,3-di-
acetal (BDA) derivative 10. Benzylation of 10 with
benzyl bromide (BnBr) and sodium hydride (NaH)
yielded fully protected glucosyl donor 11.
Glycosylation of known34 ribose acceptor 12 with 11
in the presence of the promotor N-iodosuccinimide (NIS)
and a catalytic amount of trifluoromethanesulfonic acid
(TfOH) proceeded in a stereoselective fashion to afford
R-linked disaccharide 13 in 83% yield. Conversion of 13
into the more acid-stable derivative 14 was effected by
desilylation of 13 with n-tetrabutylammonium fluoride
(TBAF) and subsequent benzylation of the resulting
primary hydroxyl function. Concomitant removal of the
BDA and isopropylidene groups in 14 under mild acidic
conditions34 followed by acetylation of the resulting
hydroxyl functions afforded the fully protected dimer
Syn th esis. Target compounds 6-8 (Figure 1) are
composed of a phenyl core that is mono-, di-, or tetra-
substituted with phosphorylated â-O-propyl-(3-O-R-glu-
cosyl)ribosyl units, respectively. Retrosynthetic analysis
indicates that these molecules can be assembled via
Sonogashira coupling32,33 of iodobenzene (18), 1,4-di-
iodobenzene (22), or 1,2,4,5-tetraiodobenzene (25) with
the common building block propargyl 2-O-acetyl-3-O-
(3,4-di-O-acetyl-2,6-di-O-benzyl-R-D-glucopyranosyl)-5-
O-benzyl-â-D-ribofuranoside (16). The R-glucosidic link-
age in key disaccharide 16 can in principle be introduced
by condensing, as reported34 for the synthesis of adeno-
phostin A, the ribose unit 12 (see Scheme 1) with ethyl
3,4,6-tri-O-acetyl-2-O-benzyl-1-thio-â-D-glucopyrano-
side. In our route, the latter glucosyl donor is replaced
by the more easily accessible thioglucoside 11, which is
prepared by the following two-step procedure (see
Scheme 1). Protection of known35 ethyl 1-thio-â-D-

Potent Agonists of IP3R
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3297
competition binding experiments with [3H]IP3, and
45Ca2+-flux measurements. The results of these experi-
ments were compared with those obtained for IP3 (1),
ribophostin 4,29,30 and propylphostin 5. As can be seen
in Table 1, a maximally effective concentration of either
IP3 (1) or the analogues (4-8) releases about the same
fraction of the intracellular Ca2+ stores (∼35%) and that
combined applications of 10 µM IP3 with 10 µM of any
of the analogues releases no more Ca2+ than a maximal
concentration of either agonist alone (Table 1, penulti-
mate column). Each analogue (4-8) binds with similar
affinity to liver IP3Rs (∼2-fold less than the affinity of
IP3), and none are more than 2-fold less potent than IP3
in stimulating Ca2+ release. The steep Hill coefficients
(h > 2) in the functional analysis of each agonist (1, 4-8)
and the unitary Hill slope for IP3 binding are consistent
with previous suggestions that at least three of the four
IP3 receptor subunits must independently bind IP3
before the Ca2+ channel opens.15,16 The unitary Hill
coefficients of the binding curves for the monomeric
ligands 4-6 are as expected, but the similar unitary
coefficients for diphostin 7 and tetraphostin 8 suggest
that it is unlikely that more than one of their possible
ligands binds simultaneously. It is, however, noteworthy
that the potency of the ligands increases with the
number of phosphorylated disaccharide within the
cluster: tetraphostin 8 (which is actually slightly more
potent than IP3) > diphostin 7 > monovalent ligands
4-6. Because it is difficult to define the number of
disaccharide units available for binding within the
clusters (binding of one unit may restrict binding of
another within the cluster), it is not yet clear whether
the greater potency of polyvalent ligands simply reflects
an increase in effective concentration of the agonist.
However, closer inspection of Table 1 suggests that
diphostin 7 and tetraphostin 8 achieve greater potency
(lower EC50) without an increase in their apparent
affinity (Kd): the EC50/Kd ratio is significantly lower for
these ligands than for the monovalent ligands. This
observation is difficult to reconcile with the increased
potency of the polyvalent ligands resulting solely from
an increase in the effective concentration of the agonist
and instead suggests that they may be more efficacious
than their monovalent counterparts (i.e. more effectively
activate the IP3 receptor once they have bound).
Sch em e 2. Synthesis of Mono-, Di-, and Tetravalent
Analogues 6-8 by Sonogashira Coupling of 16 with
Mono-, Di-, or Tetraiodobenzene (18, 22, or 25), respec-
tively, followed by Phosphorylation and Deprotectiona
a
Reagents and conditions: (i) 5 mol % PdCl2(PPh3)2, 10 mol %
CuI, Et3N/DMF, 1/20, v/v, 16 h, 19: 74%; 23: 100%; 26: 52%; (ii)
a. NaOMe, MeOH, then Dowex H+, b. dibenzyl (N,N-diisopropyl)-
phosphoramidite, 1H-tetrazole, (CH2Cl)2/CH3CN, 3/1, v/v, 30 min,
then t-BuOOH, 0 °C, 1 h; 20: 88%; 24: 88%; 27: 65%; (iii) Pd/C,
H2 (1 atm), NaOAc, 1,4-dioxane/i-PrOH/H2O, 4/2/1, v/v/v, 16 h, 6:
80%; 7: 42%; 8: 42%.
15 as a mixture of anomers. Glycosidation of 15 with
propargyl alcohol under the agency of a catalytic amount
of trimethylsilyl triflate (TMSOTf) gave building block
16 in 53% yield based on 12. Deacetylation of 16 and
subsequent phosphitylation with dibenzyl N,N-diiso-
propylphosphoramidite38 followed by in situ oxidation
of the resulting phosphite triesters afforded trisphos-
phate 17 in 80% over the three steps. Debenzylation and
concomitant reduction of the acetylene moiety was
effected by hydrogenation to give, after purification by
HW-40 gel filtration, propylphostin 5 (Na+ salt).
The assembly of phenyl derivatives 6-8 as presented
in Scheme 2 commences with Sonogashira coupling9 of
terminal acetylene 16 with monoiodobenzene (18) under
the influence of PdCl2(PPh3)/CuI/Et3N to give the ex-
pected phenylacetylene derivative 19 and the homo-
coupled byproduct 21 in a 2:1 ratio. Formation of the
latter compound could be suppressed by slow addition
(∼1 h) of acetylene 16 to the iodobenzene/catalyst
solution. In a similar way, the di- and tetravalent
derivatives 23 and 26 were readily available starting
from 1,4-diiodobenzene (22) and 1,2,4,5-tetraiodoben-
zene39 (25), respectively. Phosphorylation and depro-
tection of 19, 23, and 26, as described for the prepara-
tion of compound 5, furnished phenylphostin 6, diphostin
7, and tetraphostin 8, the identity and homogeneity of
The structural basis for the superpotent activity of
the adenophostins is still unclear. It has recently been
demonstrated that the phosphate charge distribution on
adenophostin A (2) is very similar to that observed in
IP3 (1),41 and it now seems that a specific interaction
of the adenine base moiety with the receptor (and an
associated interplay with the conformationally mobile
disaccharide unit) is most likely the prime factor
underlying the unusual potency.42-45 It is thus of
interest to evaluate analogues with modified bases and
their surrogates.42,46 In this context, it is useful to
compare the relative potencies of the series ribophostin
4, propylphostin 5, and phenylphostin 6. Ribophostin 4
and propylphostin 5 are essentially identical in activity,
and the extra length of the hydrophobic chain of 5 over
4 apparently confers no relative advantage, nor adeno-
phostin-like potency. In particular 6, which possesses
both a flexible chain and a flat aromatic hydrophobic
motif, exhibits only a relatively marginal improvement
1
which were fully ascertained by H, 13C, and 31P NMR
spectroscopy as well as ES mass spectrometry.
Biologica l Eva lu a tion . Preliminary evaluation of
the mobilizing ability of the novel compounds was made
using a sea urchin homogenate assay system40 coupled
with fluorescence techniques. All of the compounds were
found to release Ca2+ with a roughly similar potency
when compared to IP3 (data not shown). With this in
hand, the accurate binding affinity and Ca2+-releasing
ability of phenylphostin 6, diphostin 7, and tetraphostin
8 at rat liver IP3Rs were determined using equilibrium

3298 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
de Kort et al.
Ta ble 1. 45Ca2+ Release EC50 Values and Kd Values of Ribophostin 4, Propylphostin 5, Phenylphostin 6, Diphostin 7, and
Tetraphostin 8 Obtained by Equilibrium Competition Binding with [3H]IP3
Ca2+ releaseb
equilibrium bindinga
max response
release with 10 µM IP3
compd
IP3, 1
ribophostin 4
propylphostin 5
phenylphostin 6
diphostin 7
Kd (nM)
h
EC5 0 (nM)a
h
(% Ca2+ stores)
(% IP3-sensitive Ca2+ stores)c
EC50/Kd
1.7 ( 0.3
4.4 ( 0.8
4.7 ( 1.2
3.1 ( 0.8
3.6 ( 1.1
4.3 ( 0.7
0.88 ( 0.15
0.99 ( 0.05
0.97 ( 0.17
1.16 ( 0.08
0.95 ( 0.14
1.04 ( 0.18
201 ( 20
484 ( 20
491 ( 36
381 ( 10
215 ( 15
161 ( 8
2.9 ( 0.5
3.4 ( 1.4
2.1 ( 0.3
3.9 ( 1.7
2.2 ( 0.3
2.1 ( 0.3
32 ( 4
33 ( 3
42 ( 6
34 ( 7
31 ( 2
42 ( 5
120 ( 28
110 ( 20
104 ( 28
123 ( 32
60 ( 19
37 ( 6
89 ( 6
87 ( 7
87 ( 6
92 ( 8
101 ( 5
tetraphostin 8
a
b
Results are means ( SEM; n ) 3 (7 for 1). Results are means ( SEM; n ) 3 (5 for 1). c Fraction of the IP3-sensitive Ca2+ stores
released by 10 µM ligand in combination with 10 µM IP3.
Exp er im en ta l Section
Gen er a l Meth od s. 1H, 13C, and 31P NMR spectra were
recorded with a J EOL J NM-FX-200 (200/50.1/80.7 MHz), a
Bruker WM-300 (300/75.1/121 MHz), or a Bruker DMX-600
1
spectrometer (600/150/242 MHz). H and 13C chemical shifts
are given in ppm (δ) relative to tetramethylsilane as internal
standard and 31P chemical shifts relative to 85% H3PO4 as
external standard. Mass spectra were recorded with a Finni-
gan MAT TSQ70 triple-quadrupole mass spectrometer equipped
with a custom-made electrospray interface (ES). HRMS (ES)
spectra were measured in the negative mode with a MAT 900
double-focusing mass spectrometer equipped with an electro-
spray interface. The samples were prepared in a mixture of
2-propanol/H2O (80/20, v/v) containing 1.0 × 10-4 M NaOAc,
the clusters of which were used as internal standards. Optical
F igu r e 2. Schematic representation for IP3R activation with
tetraphostin 8: A, cooperative mode of binding of tetraphostin
8; B, noncooperative binding of four tetraphostin 8 molecules.
rotations were determined with a Propol automatic polarim-
eter. Melting points were determined with a Bu¨chi (Flawil,
Switzerland) melting point apparatus. Elemental analysis was
performed with a Perkin-Elmer Series II CHNS/O analyzer
2400. Toluene, CH2Cl2 and pyridine were boiled under reflux
for 3 h with P2O5 and stored over molecular sieves (4 Å). Et2O
was freshly distilled from LiAlH4. 1,2-Dichloroethane (Biosolve;
HPLC grade), 2-propanol, DMF and 1,4-dioxane (Baker; p.a.)
were stored over molecular sieves (4 Å). MeOH (Rathburn;
HPLC grade) was stored over molecular sieves (3 Å). Column
chromatography was performed on Baker silica gel (0.063-
0.200 mm) and TLC analysis on DC-fertigfolien (Schleicher &
Schu¨ll F1500, LS 254) with detection by UV absorption (254
nm) and charring with 20% H2SO4 in EtOH or ammonium
in potency. We conclude, therefore, that the phenylpro-
poxy group of 6 is unable to access the presumed
hydrophobic receptor motif or is too flexible to allow
subsequent correct positioning of the 2′-phosphate group
for adenophostin-like activity. In this regard, it would
clearly be worthwhile to evaluate biologically the less
flexible corresponding ribofuranosyl 1′-O-benzyl or phen-
yl derivatives, since adenophostin analogues with sur-
rogate bases are still able to exhibit potent activity.46
molybdate (25 g‚L-1) and ceric ammonium sulfate (10 g‚L-1
)
in 10% aqueous H2SO4, followed by charring at 140 °C.
Reactions were carried out at ambient temperature, unless
otherwise stated. Prior to reactions that required anhydrous
conditions, traces of water were removed by coevaporation with
toluene or pyridine. Butanedione, propargyl alcohol, N-iodo-
succinimide, Dowex 50XW4, bis(triphenylphosphine)palla-
dium(II) chloride, camphor sulfonic acid, copper(I) iodide,
trifluoromethanesulfonic acid, trimethyl orthoformate, tetra-
butylammonium fluoride (1.0 M in THF) (Acros), benzyl
bromide, ethylene glycol, sodium hydride (60%), t-BuOOH
(80% in di-tert-butyl peroxide) (Merck), trimethylsilyl triflate,
iodobenzene, 1,4-diiodobenzene, 1H-tetrazole (Aldrich) and
acetic anhydride (Baker) were all used as received. Imino
diacetate resin (Chelex; Na+ form) was purchased from Sigma.
Purification of the target compounds was performed by gel
filtration with a Fractogel column (HW 40 (s), 26/60) with
triethylammonium bicarbonate buffer (0.15 M) as eluent (1.5
mL‚min-1). The collected product was analyzed with strong
anion-exchange HPLC on a Pharmacia MonoQ column by
elution with a mixture of buffers (pH 12) A: 0.01 N NaOH
and B: 0.01 N NaOH in 1.2 N NaCl (gradient 0 f 50% B).
45Ca 2+ Relea se. For the preliminary sea urchin homogenate
assay L. Pictus egg homogenate (2.5% v/v, prepared as previ-
ously described40) was incubated at 17 °C in intracellular-like
medium (IM) containing an ATP-regenerating system, mito-
chondrial inhibitors and Fluo-3 (3 µM), and extra-microsomal
Ca2+ was measured by monitoring Fluo-3 fluoresence (excita-
tion 490 nm and emission 535 nm) using a Perkin-Elmer
LS50-B luminescence spectrophotometer controlled by FL-
Con clu sion s
The results indicate that the mono-, di-, and tetra-
valent adenophostin A analogues 6-8 do not exhibit
cooperative binding to the tetravalent IP3R, excluding
the (rather unlikely) possibility that activation of IP3R
occurs after binding of a single tetravalent ligand as
illustrated in Figure 2A. Instead, we suggest that the
four IP3-binding sites of the receptor are capable of
accommodating four molecules of tetraphostin 8 (Figure
2B), which is, of course, much larger than the natural
ligand, IP3 (1), implying that the IP3-binding sites are
either exposed at the surface of the receptor or at least
accessible to bulky negatively charged ligands.
In summary, the mono-, di-, and tetravalent adeno-
phostin A analogues 6-8 present a novel class of ligands
for IP3R, with the polyvalent compounds not showing
the cooperative binding we had anticipated but rather
an apparent increase in their efficacy. When the location
of the IP3-binding sites on the receptor becomes estab-
lished, it may be possible to exploit methods similar to
those reported herein to synthesize polyvalent adeno-
phostin analogues with the size and geometry of the
central scaffold more precisely matched to the dimen-
sions of the binding sites.

Potent Agonists of IP3R
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3299
Winlab. Homogenate (0.5 mL) was placed in a cuvette and
additions of test compound (dissolved in IM with 10 µM EGTA)
were made. Estimates of the amount of Ca2+ released were
based upon calibrations with known amounts of Ca2+ added
to the assay homogenate as described previously.40 For the
quantitative work, hepatocytes (106 cells‚mL-1) permeabilized
with saponin (10 µg‚mL-1) were loaded to steady state with
45Ca2+ (20 µCi‚mL-1) at 37 °C in a cytosol-like medium (CLM)
with a free [Ca2+] of 200 nM.30 After 5 min, cells were diluted
5-fold into similar medium at 37 °C but without ATP and
supplemented with thapsigargin (1.25 µM) to inhibit the Ca2+
pumps of the intracellular Ca2+ stores. After 15 s, appropriate
concentrations of the ligand were added, and after a further
60 s, the 45Ca2+ contents of the intracellular stores were
determined after rapid filtration using a Brandel receptor-
binding harvester.30 CLM had the following composition: 140
mM KCl, 20 mM NaCl, 2 mM MgCl2, 1 mM ethylene glycol
bis(â-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), 300
µM CaCl2 (free [Ca2+] ) 200 nM), 10 µM carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (FCCP), 7.5 mM ATP,
20 mM 1,4-piperazinediethanesulfonic acid (PIPES), pH 7.0
at 37 °C.
[3H]IP 3 Bin d in g. Hepatic membranes were prepared by a
method modified from that described by Prpic et al.47 The livers
of two male Wistar rats (250-300 g) were perfused through
the portal vein with cold saline (50 mL: 118 mM NaCl, 5.4
mM KCl, 0.8 mM MgSO4, 0.96 mM NaH2PO4, 25 mM NaHCO3,
11 mM glucose, 1 mM EGTA, pH 7.4 at 4 °C); the livers were
then removed and homogenized in cold buffered sucrose (50
mL: 250 mM sucrose, 1 mM EGTA, 5 mM Hepes, pH 7.4 at 4
°C) in a Dounce homogenizer with 10 passes of a loose-fitting
plunger and 5 passes of a tight plunger. The homogenate from
both livers (in 100 mL of buffered sucrose) was filtered through
gauze and centrifuged (10 min, 2500g), and the pellet resus-
pended in buffered sucrose (96 mL) supplemented with Percoll
(12.9 mL, 1.13 g‚mL-1). After centrifugation (30 min, 35000g)
membranes were harvested as a discrete layer near the top of
the tube, resuspended in cold hypoosmotic buffer (100 mL: 1
mM EGTA, 5 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethane-
sulfonic acid) (Hepes), pH 7.4), and recentrifuged (10 min,
48000g). The pellet was resuspended in binding medium (5
mL: 50 mM Tris, 1 mM EGTA, pH 8.3) to give a final protein
concentration of about 3 mg protein‚mL-1 and then frozen in
liquid nitrogen before storage at -80 °C. For equilibrium
competition binding assays, membranes in binding buffer (500
µL containing 0.1 mg membrane protein) were incubated for
5 min at 4 °C with [3H]IP3 (typically, 45 nCi, 40 Ci/mmol) and
appropriate concentrations of competing ligands. Incubations
were terminated by centrifugation (5 min, 20000g, 4 °C), and
the pellets resuspended for liquid scintillation counting.
Da ta An a lysis. Ca 2+ r elea se: Concentration-response
relationships were fitted to logistic equations using Kaleide-
graph software (Synergy Software, PA) from which the maxi-
mal response, half-maximally effective agonist concentration
(EC50), and Hill slope (h) were determined. All results are
expressed as means ( SEM. Binding parameters were deter-
mined by curve-fitting to logistic equations as previously
described.30 Nonspecific binding was typically 10-15% of total
binding. All experiments were performed in triplicate (n ) 3;
n ) 5 - 7 for IP3), and the combined results are expressed as
means ( SEM.
(2′S,3′S)-E t h yl 3,4-Di-O-(2′,3′-d im et h oxyb u t a n e-2′,3′-
d iyl)-1-th io-â-D-glu cop yr a n osid e (10) a n d (2′R,3′R)-Eth yl
2,3-Di-O-(2′,3′-d im eth oxybu ta n e-2′,3′-d iyl)-1-th io-â-D-glu -
cop yr a n osid e. Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-â-D-glu-
copyranoside (33.2 g, 84.7 mmol) was deacylated with NaOMe
(0.22 g, 4.20 mmol) in MeOH (300 mL). When TLC analysis
showed complete conversion of starting material the reaction
mixture was neutralized with Dowex 50WX4 H+ and filtered.
Trimethyl orthoformate (27.2 mL, 280 mmol), butane-2,3-dione
(7.37 mL, 93.2 mmol) and camphorsulfonic acid (1.93 g, 9.3
mmol) were added and the mixture was heated until reflux.
After 3 h triethylamine (1.42 mL, 10.0 mmol) was added and
the reaction mixture was concentrated. The crude oil was
purified by column chromatography (Et2O/light petroleum, 1/1
f 1/0, v/v) which afforded 10 and its regioisomer as white
foams in a 1:1 ratio. Combined yield: 24.6 g (72.8 mmol, 86%).
1
3,4-BDA isom er : Rf 0.59 (EtOAc); H NMR (CDCl3) δ 4.41
(d, 1 H, H-1, J 1,2 9.5 Hz), 3.88 (ddd, 1 H, H-5, J 5,6a 12.1 Hz,
J 5,6b 2.7 Hz, J 4,5 5.6 Hz), 3.80-3.52 (m, 5 H, H-2, H-3, H-4,
H-6), 3.32, 3.26 (2× s, 6 H, 2× OCH3), 2.75 (q, 2 H, CH2 of
SEt, J 7.4 Hz), 2.53 (d, 1 H, OH-2, J 1.8 Hz), 2.04 (dd, 1 H,
OH-6, J 7.4 Hz, J 5.6 Hz), 1.35, 1.31 (2× s, 6 H, 2× CH3), 1.29
(t, 3 H, CH3 of SEt); 13C{1H} NMR (CDCl3) δ 99.5, 99.2 (2×
Cq, BDA), 86.2 (C-1), 77.8, 73.2, 69.6, 65.3 (C-4, C-5, C-3, C-2),
60.9 (C-6), 47.7 (2× CH3 OMe), 24.2 (CH2 of SEt), 17.4, 17.3
(2× CH3, BDA), 15.0 (CH3 of SEt). 2,3-BDA isom er : Rf 0.44
(EtOAc); 1H NMR (CDCl3) δ 4.63 (d, 1 H, H-1, J 1,2 9.8 Hz),
3.88-3.82 (m, 4 H, H-3, H-4, H-5, H-2), 3.53-3.46 (m, 2 H,
H-6), 3.30, 3.29 (2× s, 6 H, 2× OCH3), 2.72 (q, 2 H, CH2 of
SEt, J 7.5 Hz), 1.34, 1.33 (2× s, 6 H, 2× CH3), 1.31 (t, 3 H,
CH3 of SEt); 13C{1H} NMR (CDCl3) δ 99.5, 99.1 (2× Cq, BDA),
82.4 (C-1), 79.7, 73.8, 68.5, 66.6 (C-3, C-5, C-2, C-4), 61.5 (C-
6), 47.6 (2× CH3 OMe), 24.2 (CH2 of SEt), 17.2 (2× CH3, BDA),
14.6 (CH3 of SEt). Anal. (C14H26O7S) C, H.
(2′S,3′S)-E t h yl 2,6-Di-O-b en zyl-3,4-d i-O-(2′,3′-d im et h -
oxybu ta n e-2′,3′-d iyl)-1-th io-â-D-glu cop yr a n osid e (11). So-
dium hydride (60%, 1.74 g, 50.8 mmol) was added to a cooled
(0 °C) solution of 10 (4.9 g, 14.5 mmol) in DMF (75 mL). After
strirring for 15 min, benzyl bromide (3.77 mL, 31.9 mmol) was
added. When TLC analysis showed complete conversion of
starting material into a more lipophilic product, the excess of
sodium hydride was destroyed with MeOH and the reaction
mixture was diluted with Et2O (100 mL) and H2O (20 mL).
The layers were separated and the DMF/H2O mixture was
extracted with Et2O (2 × 50 mL). The combined organic layers
were washed with H2O, dried (MgSO4) and concentrated in
vacuo. Purification by column chromatography (Et2O/light
petroleum, 3/1 f 1/1, v/v) gave 11 as a white solid: yield 7.24
g (14.2 mmol, 98%); Rf 0.91(Et2O/light petroleum, 2/1, v/v); 1H
NMR (CDCl3) δ 7.45-7.23 (m, 10H, CH arom), 4.83 (AB, 2 H,
CH2 Bn, J -12.1 Hz), 4.58 (AB, 2 H, CH2 Bn, J -12.3 Hz), 4.46
(d, 1 H, H-1, J 1,2 9.3 Hz), 3.86 (t, 1 H, H-3, J 2,3 ) J 3,4 9.4 Hz),
3.80-3.57 (m, 4 H, H-4, H-5, H-6), 3.48 (t, 1 H, H-2), 3.29,
3.20 (2× s, 6 H, 2× OCH3), 2.82-2.64 (m, 2 H, CH2 of SEt),
1.35, 1.29 (2× s, 6 H, 2× CH3), 1.30 (t, 3 H, CH3 of SEt, J 7.5
Hz); 13C{1H} NMR (CDCl3) δ 138.4 (2× Cq Bn), 128.8, 127.6,
127.4 (CH Bn), 99.5 (2× Cq, BDA), 85.0 (C-1), 78.3, 77.5, 74.8,
65.9 (C-2, C-3, C-4, C-5), 75.2, 73.3 (2× CH2 Bn), 68.5 (C-6),
47.8, 47.7 (2× CH3 OMe), 24.4 (CH2 of SEt), 17.8, 17.7 (2×
CH3, BDA), 15.3 (CH3 of SEt); mp 78-80 °C. Anal. (C28H38O7)
C, H.
(2′′S ,3′′S )-3-O-[2′,6′-Di-O-b e n zyl-3′,4′-d i-O-(2′′,3′′-d i-
m eth oxybu tan e-2′′,3′′-diyl)-r-D-glu copyr an osyl]-1,2-O-iso-
p r op ylid en e-5-O-ter t-bu tyld ip h en ylsilyl-R-D-r ibofu r a n o-
sid e (13). 1,2-O-Isopropylidene-5-O-tert-butyldiphenylsilyl-R-
D-ribofuranose (12) (1.71 g, 3.99 mmol) and compound 11 (2.27
g, 4.38 mmol) were dried by coevaporation with 1,2-dichloro-
ethane. The remaining oil was dissolved in Et2O (20 mL) and
stirred with powdered molecular sieves (4 Å) under an inert
atmosphere. After 5 min N-iodosuccinimide (0.99 g, 4.38 mmol)
and trifluoromethanesulfonic acid (60 µL, 0.44 mmol) were
added. The resulting mixture was allowed to stir at room
temperature for 45 min after which TLC analysis revealed
complete disappearance of glucosyl donor and acceptor. The
molecular sieves were filtered off over Hyflo and rinsed with
Et2O. The filtrate was washed with aqueous Na2S2O3 (10%),
aqueous NaHCO3 (10%), H2O and dried (MgSO4). The crude
product was purified by column chromatography (Et2O/light
petroleum, 1/6 f 1/3, v/v) to afford dimer 13 in a yield of 2.93
g (3.31 mmol, 83%) as a colorless oil: Rf 0.60 (Et2O/light
petroleum, 2/1, v/v); 1H NMR (300 MHz, CDCl3, HH-COSY) δ
7.70-7.62 (m, 4 H, CH arom Ph), 7.41-7.21 (m, 16 H, CH
arom), 5.79 (d, 1 H, H-1, J 1,2 3.7 Hz), 5.25 (d, 1 H, H-1′, J 1′,2′
4.0 Hz), 4.78-4.73 (m, 3 H, H-2, CH2 6′-O-Bn), 4.47 (AB, 2 H,
CH2 Bn, J -12.1 Hz), 4.32 (d, 1 H, H-3, J 2,3 4.3 Hz, J 3,4 8.8
Hz,), 4.23-4.12 (m, 2 H, H-4, H-3′), 3.98 (dd, 1 H, H-5a, J 5a,5b
-11.9 Hz, J 4,5a 1.2 Hz), 3.87-3.81 (m, 3 H, H-4′, H-5′, H-5b),

3300 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
de Kort et al.
3.67-3.60 (m, 2 H, H-2′, H-6a′), 3.51 (dd, 1 H, H-6b′, J 6a′,
appropriate fractions afforded tetraacetate 15 as a white
foam: yield 2.33 g (86%, 2 steps, R:â ) 1:7); Rf 0.56 (EtOAc/
light petroleum, 1/1, v/v); 1H NMR (CDCl3) δ 7.29-7.26 (m,
15 H, CH arom), 6.43 (d, 0.13 H, H-1R, J 1,2 4.4 Hz), 6.12 (s,
0.87 H, H-1â), 5.42-5.30 (m, 6 H, 2× CH2 Bn, H-1′, H-3′),
5.10-5.00 (m, 3 H, CH2 Bn, H-4′), 4.68-4.10 (m, 4 H, H-2,
H-3, H-4, H-5′), 3.71-3.30 (m, 5 H, H-5, H-6, H-2′), 1.96, 1.93,
1.87, 1.86 (4 × s, 4 × CH3 Ac); 13C{1H} NMR (CDCl3) δ 170.0,
169.9, 169.4, 169.0 (4 × C(O) Ac), 137.9, 137.6, 137.3 (3× Cq
Bn), 128.2-127.1 (CH arom), 98.3 (C-1), 96.2 (C-1′), 81.0, 76.4,
73.7, 73.4, 71.7, 68.7, 68.8 (C-2′, C-3′, C-4′, C-5′, C-2, C-3, C-4),
73.2, 73.0 (3× CH2 Bn), 69.1, 67.4 (C-6′, C-5), 20.8, 20.7, 20.5,
20.3 (4 × CH3 Ac); ES-MS m/z 691 [M - OAc]+, 773 [M + Na]+,
790 [M + K]+. Anal. (C40H46O14) C, H.
6b′
-11.0 Hz, J 5′,6b′ 1.9 Hz), 3.29, 3.17 (2× s, 6 H, 2× OCH3), 1.52
(s, 3 H, CH3 isoprop), 1.35 (2× s, 6 H, CH3, BDA, CH3 isoprop),
1.30 (s, 3 H, CH3, BDA), 1.01 (s, 9 H, t-Bu TBDPS); 13C{1H}
NMR (CDCl3) δ 138.8, 138.7, 135.5, 135.3 (4× Cq arom),
129.4-127.0 (CH arom), 112.7 (Cq isoprop), 104.1 (C-1), 99.4,
99.3 (2× Cq, BDA), 95.8 (C-1′), 79.2, 76.9, 75.9, 72.7, 69.3, 65.5
(C-2′, C-3′, C-4′, C-5′, C-2, C-3, C-4), 73.4, 71.8 (2× CH2 Bn),
67.4 (C-6′), 61.5 (C-5), 47.8, 47.7 (2× CH3 OMe), 26.7, 26.6
(CH3 t-Bu TBDPS, isoprop), 19.1 (Cq t-Bu TBDPS), 17.8, 17.6
(2× CH3, BDA), J H-1′, C-1′ 171.4 Hz: R-glucoside; J H-1, C-1 181.7
Hz; ES-MS m/z 908 [Na]+, 924 [M + K]+. Anal. (C50H64O12Si)
C, H.
(2′′S,3′′S)-5-O-Ben zyl-3-O-[2′,6′-d i-O-b en zyl-3′,4′-d i-O-
(2′′,3′′-d im eth oxybu ta n e-2′′,3′′-d iyl)-r-D-glu cop yr a n osyl]-
1,2-O-isop r op ylid en e-r-D-r ibofu r a n osid e (14). Compound
13 (3.51 g, 3.97 mmol) was stirred at 50 °C in a mixture of
dioxane (26 mL) and TBAF (1.0 M in THF, 6.13 mL). After 8
h TLC analysis revealed the reaction to be complete and the
mixture was concentrated under reduced pressure. The residue
was dissolved in EtOAc (100 mL), washed with brine (2 × 20
mL), H2O (10 mL) and dried (MgSO4). Purification by column
chromatography (Et2O/light petroleum, 1/1 f 1/0, v/v) gave
the desilylated disaccharide: yield of 2.37 g; Rf 0.47 (Et2O);
1H NMR (CDCl3) δ 7.42-7.26 (m, 10 H, CH arom), 5.76 (d, 1
H, H-1, J 1,2 3.6 Hz), 5.15 (d, 1 H, H-1′, J 1′,2′ 3.8 Hz), 4.77 (AB,
2 H, CH2 Bn, J -11.9 Hz), 4.68 (dd, 1 H, H-2, J 2,3 3.9 Hz), 4.56
(AB, 2 H, CH2 Bn, J -12.2 Hz), 4.13 (m, 3 H, H-3, H-4, H-3′),
3.86 (m, 2 H, H-4′, H-5′), 3.65 (m, 5 H, H-2′, H-5, H-6′), 3.30,
3.17 (2× s, 6 H, 2× OCH3), 1.53 (s, 3 H, CH3 isoprop), 1.34
(2× s, 6 H, CH3, BDA, CH3 isoprop), 1.28 (s, 3 H, CH3, BDA);
13C{1H} NMR (CDCl3) δ 138.7, 138.5 (2× Cq Bn), 128.1-127.1
(CH arom), 112.8 (Cq isoprop), 104.0 (C-1), 99.5, 99.3 (2× Cq,
BDA), 95.6 (C-1′), 78.4, 76.5, 75.9, 72.3, 69.2, 69.1, 66.0 (C-2′,
C-3′, C-4′, C-5′, C-2, C-3, C-4), 73.3, 71.8 (2× CH2 Bn), 67.8
(C-6′), 59.8 (C-5), 48.0, 47.7 (2× CH3 OMe), 26.6, 26.5 (2× CH3
isoprop), 17.7, 17.5 (2× CH3, BDA).
[2′-O-Acetyl-3′-O-(3′′,4′′-d i-O-a cetyl-2′′,6′′-d i-O-ben zyl-r-
D-glu copyr an osyl)-5′-O-ben zyl-â-D-r ibofu r an osyloxy]pr op-
3-yn e (16). A solution of compound 15 (0.75 g, 1.00 mmol) in
dichloroethane (10 mL) was stirred with powdered molecular
sieves 4 Å (0.5 g) under a nitrogen atmosphere. Propargyl
alcohol (0.15 mL, 2.50 mmol) was added via syringe, followed
by the addition of TMSOTf (0.05 mL, 0.25 mmol). The mixture
was stirred for 30 min at room temperature, after which TLC
analysis showed the appearance of a higher-running product.
The reaction was quenched by the addition of triethylamine
(0.10 mL, 0.70 mmol), filtered over Hyflo, and the solvents
were removed in vacuo. Purification was performed by column
chromatography (EtOAc/light petroleum, 1/4 f 1/1, v/v) to give
acetylene derivative 16 as a colorless oil: yield 0.25 g (81%) +
0.09 g (12%) of recovered R-15; Rf 0.33 (acetone/CH2Cl2, 2/98,
1
v/v); H NMR (600 MHz, CDCl3, HH-COSY) δ 7.43-7.23 (m,
15 H, CH arom), 5.38 (t, 1 H, H-3′′, J 2′′3′′ ) J 3′′,4′′ 9.8 Hz), 5.31
(d, 1 H, H-2′, J 2′,3′ 4.6 Hz), 5.19 (s, 1 H, H-1′), 5.03 (t, 1 H,
H-4′′, J 4′′,5′′ 9.8 Hz), 5.00 (d, 1 H, H-1′′, J 1′′,2′′ 3.5 Hz), 4.61 (AB,
2 H, CH2 Bn, J -12.2 Hz), 4.55 (dd, 1 H, H-3′, J 3′,4′ 7.4 Hz),
4.50 (AB, 2 H, CH2 Bn, J -12.0 Hz), 4.39 (AB, 2 H, CH2 Bn, J
4
-12.1 Hz), 4.37 (m, 1 H, H-4′), 4.20 (ABX, 2 H, H-1, J 1,3 2.4
Hz, J 1a,1b -15.3 Hz), 3.85 (m, 1 H, H-5"), 3.69 (dd, 1 H, H-5a′,
J 5a′,5b′ -10.9 Hz), 3.57 (dd, 1 H, H-5b′, J 4′,5b′ 5.1 Hz), 3.54 (dd, 1
H, H-2"), 3.33 (dd, 1 H, H-6a", J 6a′′,6b′′ -10.8 Hz, J 5′′,6a′′ 2.6 Hz),
3.27 (dd, 1 H, H-6b", J 5“,6b” 3.9 Hz), 2.43 (t, 1 H, H-3), 1.92,
1.87, 1.86 (3 × s, 9H, 3 × CH3 Ac); 13C{1H} NMR (CDCl3) δ
170.2, 170.1, 169.6, (3 × C(O) Ac), 138.0, 137.7, 137.6 (3× Cq
Bn), 128.3-127.4 (CH arom), 103.2 (C-1′), 96.3 (C-1"), 80.4,
76.6, 74.7, 73.5, 71.9, 69.0, 68.8 (C-2′′, C-3′′, C-4′′, C-5′′, C-2′,
C-3′, C-4′, C-3), 74.2 (C-2), 72.4, 72.3 72.1 (3× CH2 Bn), 69.4,
Benzylation was performed as described earlier for 11.
Purification was effected by column chromatography (Et2O/
light petroleum, 1/5 f 1/2, v/v) to give fully protected 14 as a
white solid: yield 2.66 g (92%, 2 steps); Rf 0.65 (Et2O/light
petroleum, 2/1, v/v); 1H NMR (300 MHz, CDCl3, HH-COSY) δ
7.42-7.23 (m, 15 H, CH arom), 5.79 (d, 1 H, H-1, J 1,2 3.7 Hz),
5.19 (d, 1 H, H-1′, J 1′,2′ 4.0 Hz), 4.76 (AB, 2 H, CH2 Bn, J -12.4
Hz), 4.70 (dd, 1 H, H-2, J 2,3 3.8 Hz), 4.56 (AB, 2 H, CH2 Bn, J
-12.1 Hz), 4.50 (AB, 2 H, CH2 Bn, J -12.1 Hz), 4.32 (ddd, 1 H,
H-4, J 3,4 9.3 Hz, J 4,5a 1.8 Hz, J 4,5b 3.9 Hz), 4.14-4.07 (m, 2 H,
H-3, H-3′), 3.82-3.76 (m, 3 H, H-5b, H-2′, H-4′), 3.56 (dd, 1 H,
H-6b′, J 6a′,6b′ -10.2 Hz, J 5′,6b′ 1.5 Hz), 3.30, 3.18 (2× s, 6 H, 2×
OCH3), 1.53 (s, 3 H, CH3 isoprop), 1.35 (2× s, 6 H, CH3, BDA,
CH3 isoprop), 1.31 (s, 3 H, CH3, BDA); 13C{1H} NMR (CDCl3)
δ 138.8, 138.1, 137.9 (3× Cq Bn), 128.2-127.2 (CH arom),
112.8 (Cq isoprop), 104.1 (C-1), 99.5, 99.4 (2× Cq, BDA), 95.7
(C-1′), 77.8, 76.5, 76.0, 69.4, 69.2, 65.9 (C-2′, C-3′, C-4′, C-5′,
C-2, C-3, C-4), 73.4, 71.9 (3× CH2 Bn), 68.1, 67.7 (C-6′, C-5),
48.0, 47.9 (2× CH3 OMe), 26.7, 26.6 (2× CH3 isoprop), 17.9,
20
66.7 (C-6′′, C-5′), 53.0 (C-1), 19.8, 19.6, 19.5 (3 × CH3 Ac); [R]D
+74.4 (c 2.0, CHCl3); ES-MS m/z 769 [M + Na]+. Anal.
(C41H46O13) C, H.
[5′-O-ben zyl-3′-O-(2′′,6′′-di-O-ben zyl-r-D-glu copyr an osyl)-
â-D-r ibofu r a n osyloxy]p r op -3-yn e 2′,3′′,4′′-Tr is(d iben zyl
p h osp h a te) (17). Triacetate 16 (63 mg, 84 µmol) was dissolved
in a mixture of sodium methoxide (3 mg, 50 µmol) in dry MeOH
(5 mL) and stirred for 1.5 h. The solution was neutralized with
Dowex H+, filtered and repeatedly concentrated under reduced
pressure. The residue was concentrated with 1,2-dichloro-
ethane (2 × 5 mL), dissolved in CH2Cl2 (5 mL) and subse-
quently dibenzyl (N,N-diisopropyl)phosphoramidite38 (0.10 mL,
0.29 mmol) and a solution of 1H-tetrazole (52 mg, 0.70 mmol)
in CH3CN (2 mL) were added. The solution was stirred under
an inert atmosphere for 30 min after which TLC analysis
(toluene/EtOAc, 9/1, v/v) revealed complete conversion into a
higher running product (Rf 0.82). The mixture was cooled (0
°C) and t-BuOOH (0.15 mL, 85% solution in di-tert-butyl
peroxide, 1.3 mmol) was added. After 30 min at room temper-
ature the solution was diluted with EtOAc (20 mL), washed
with H2O, dried (MgSO4) and concentrated. The product was
purified by column chromatography (EtOAc/light petroleum,
1/4 f 1/0, v/v) to give 17 as a colorless oil: yield 93 mg (80%,
2 steps); Rf 0.68 (toluene/EtOAc/MeOH, 180/50/5, v/v/v); 1H
NMR (CDCl3) δ 7.42-7.01 (m, 45 H, CH arom), 5.33 (s, 1 H,
H-1′), 5.08 (d, 1 H, H-1", J 1′′,2′′ 3.4 Hz), 5.01-4.82 (m, 14 H,
H-2′, H-3′′, 6× CH2 Bn), 4.69 (AB, 2 H, CH2 Bn, J -12.1 Hz),
4.54-4.45 (m, 5 H, CH2 Bn, H-3′, H-4′′, H-4′), 4.35 (AB, 2 H,
20
17.6 (2× CH3, BDA); [R]D +150.8 (c 1.0, CHCl3); mp 93-94
°C; ES-MS m/z 754 [M + NH4]+, 759 [M + Na]+, 775 [M +
K]+. Anal. (C41H52O12) C, H.
1,2-Di-O-a cetyl-3-O-(3′,4′-d i-O-a cetyl-2′,6′-d i-O-ben zyl-
r-D-glu cop yr a n osyl)-5-O-b en zyl-D-r ib ofu r a n osid e (15).
Compound 14 (2.66 g, 3.61 mmol) was heated until reflux in
a mixture of acetic acid/H2O/ethylene glycol (75 mL, 14/6/3,
v/v/v). After 75 min the reaction mixture was cooled (0 °C) and
quenched with aqueous NaHCO3 (10%, 125 mL). The resulting
suspension was extracted with EtOAc (3 × 100 mL). The
organic layer was washed with H2O (3 × 50 mL), dried
(MgSO4) and concentrated. The crude product (Rf 0.53, MeOH/
EtOAc, 8/92, v/v) was dissolved in a mixture of acetic anhydride/
pyridine (50 mL, 3/7, v/v) and stirred for 16 h at room
temperature. The mixture was diluted with toluene and
concentrated under reduced pressure (3×). The oily product
was applied onto a column of silica gel which was eluted with
Et2O/light petroleum (1/9 f 1/1, v/v). Concentration of the
4
CH2 Bn, J -11.6 Hz), 4.13 (d, 2 H, H-1, J 1,3 2.3 Hz), 3.81 (m,

Potent Agonists of IP3R
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3301
1 H, H-5′′), 3.67-3.45 (m, 5 H, H-5′, H-2′′, H-6′′), 2.30 (t, 1 H,
H-3); 13C{1H} NMR (CDCl3) δ 138.1, 137.9, 137.6 (3× Cq Bn),
136.2, 136.0, 135.9, 135.8, 135.6, 135.5 (6× Cq Bn), 128.7-
127.4 (CH Bn), 102.9 (C-1′), 95.1 (C-1′′), 80.0, 78.1, 77.3, 77.2,
76.9, 74.3, 73.8, 73.6 (C-2“, C-3”, C-4“, C-5”, C-2′, C-3′, C-4′,
C-3), 74.8, 73.1, 72.0 (3× CH2 Bn), 70.1, 69.7, 69.6, 69.5, 69.3,
69.2, 69.0, 68.9, 67.9 (6× CH2 Bn, C-6′′, C-5′, C-2), 54.3 (C-1);
H-4′), 3.85 (dt, 2 H, H-5′′, J 5′′,6′′ 3.1 Hz, J 4′′,5′′ 9.9 Hz), 3.68 (dd,
2 H, H-5a′, J 5a′,5b′ -10.8 Hz, J 4′,5a′ 3.4 Hz), 3.56 (dd, 2 H, H-5b′,
J 4′,5b′ 4.7 Hz), 3.53 (dd, 2 H, H-2′′), 3.29 (ABX, 4 H, H-6′′, J 6a′′,6b′′
-10.8 Hz), 1.92, 1.88, 1.86 (3× s, 18 H, CH3 Ac); 13C{1H} NMR
(CDCl3) δ 170.1, 170.0, 169.5, (3× C(O) Ac), 137.9, 137.7, 137.5
(3× Cq Bn), 128.3-127.4 (CH Bn), 103.2 (C-1′), 96.2 (C-1′′),
80.4, 76.5, 74.5, 73.4, 71.9, 69.0, 68.7 (C-2′′, C-3′′, C-4′′, C-5′′,
C-2′, C-3′, C-4′), 74.4 (C-2), 73.4, 73.3 73.1 (3× CH2 Bn), 70.5
(C-3), 70.3, 67.6 (C-6′′, C-5′), 54.3 (C-1), 20.8, 20.6, 20.5 (3×
CH3 Ac); ES-MS m/z 1508 [M + NH4]+, 1513 [M + Na] +, 1529
[M + K]+. Anal. (C82H90O26) C, H.
31P{1H} NMR (CDCl3) δ -0.74, -1.34, -1.69; [R]D +21.2 (c
20
0.5, CHCl3); ES-MS m/z 1401 [M + H]+, 1423 [M + Na]+. Anal.
(C77H79O19P3) C, H.
[2′-O-Acetyl-5′-O-ben zyl-3′-O-(3′′,4′′-d i-O-a cetyl-2′′,6′′-d i-
O-ben zyl-r-D-glu copyr an osyl)-â-D-r ibofu r an osyloxy]pr op-
3-yn ylben zen e (19). A solution of iodobenzene (18; 48 µL,
0.43 mmol) in DMF (4 mL) was degassed and stirred under
an inert atmosphere. Et3N (0.5 mL), Pd(PPh3)2Cl2 (15 mg, 5
mol %) and CuI (8 mg, 10 mol %) were added and the mixture
was degassed again. Acetylene derivative 16 (0.32 g, 0.43
mmol) in DMF (6 mL) was added via syringe during a 1 h
period. The reaction mixture was stirred until, after 16 h,
complete conversion of the acetylene (Rf 0.68) into a higher-
running product was observed (16 h) with TLC analysis. The
reaction mixture was diluted with Et2O, washed with brine,
aqueous NaHCO3 (10%) and H2O. The organic layer was dried
with MgSO4 and concentrated. The brown residue was sub-
jected to column chromatography (EtOAc/light petroleum, 1/3
f 2/3, v/v) to give phenyl acetylene derivative 19 as a brownish
foam: yield 0.26 g (74%); Rf 0.78 (toluene/EtOAc/MeOH, 90/
25/2.5, v/v/v); 1H NMR (HH-COSY, 300 MHz, CDCl3) δ 7.62-
7.26 (m, 20 H, CH arom), 5.39 (t, 1 H, H-3′′, J 2′′,3′′ ) J 3′′,4′′ 9.6
Hz), 5.35 (d, 1 H, H-2′, J 2′,3′ 4.5 Hz), 5.27 (s, 1 H, H-1′), 5.03 (t,
1 H, H-4′′, J 4′′,5′′ 9.7 Hz), 4.99 (d, 1 H, H-1′′, J 1′′,2′′ 3.3 Hz), 4.56
(dd, 1 H, H-3′, J 3′,4′ 7.9 Hz), 4.54 (AB, 2 H, CH2 Bn, J -12.1
Hz), 4.45 (m, 4 H, CH2 Bn, H-1), 4.38 (m, 3 H, CH2 Bn, H-4′),
3.86 (dt, 1 H, H-5′′, J 5′′,6′′ 3.0 Hz, J 4′′,5′′ 7.4 Hz), 3.72 (dd, 1 H,
H-5a′, J 5a′,5b′ -10.5 Hz, J 4′,5a′ 3.4 Hz), 3.55 (dd, 1 H, H-5b′, J 4′,5b′
4.9 Hz), 3.54 (dd, 1 H, H-2′′), 3.31 (ABX, 2 H, H-6′′, J 6a′′,6b′′
-10.9 Hz), 1.92, 1.87, 1.86 (3× s, 9 H, CH3 Ac); 13C{1H} NMR
(CDCl3) δ 170.0, 169.9, 169.4, (3 × C(O) Ac), 137.9, 137.6, 137.4
(3× Cq Bn), 131.6, 128.3-127.4 (CH arom), 122.2 (Cq Ph),
103.1 (C-1′), 96.1 (C-1′′), 86.4, 83.8 (C-2, C-3 alkyne), 80.2, 76.5,
74.6, 73.4, 71.8, 68.9, 68.6 (C-2′′, C-3′′, C-4′′, C-5′′, C-2′, C-3′,
C-4′), 73.3, 73.2 73.0 (3× CH2 Bn), 70.4, 67.5 (C-6′′, C-5′), 54.7
(C-1), 20.7, 20.6, 20.4 (3 × CH3 Ac); [R]D20 +54.8 (c 1.0, CHCl3);
ES-MS m/z 840 [M + NH4]+, 845 [M + Na] +, 861 [M + K]+.
Anal. (C47H50O13) C, H.
1,4-B i s {[2′-O -a c e t y l-5′-O -b e n z y l-3′-O -(3′′,4′′-d i -O -
a cet yl-2′′,6′′-d i-O-b en zyl-r-D-glu cop yr a n osyl)-â-D-r ib o-
fu r a n osyloxy]p r op -3-yn yl}ben zen e (23). 1,4-Diiodobenzene
(22; 60 mg, 0.19 mmol) was treated with acetylene 16 (0.35 g,
0.47 mmol) as described for for the preparation of 19. TLC
analysis revealed complete conversion into a lower-running
product after 2 h. Column chromatography (EtOAc/light
petroleum, 2/3, v/v) afforded disubstituted benzene derivative
23 as a brownish foam: Rf 0.24 (toluene/EtOAc/MeOH, 180/
25/2.5, v/v/v); yield 0.29 g (100%); 1H NMR (HH-COSY, 300
MHz, CDCl3) δ 7.36 (s, 4 H, CH-arom Ph), 7.34-7.26 (m, 30
H, CH-arom Bn), 5.38 (t, 2 H, H-3′′, J 2′′,3′′ ) J 3′′,4′′ 9.7 Hz), 5.36
(d, 2 H, H-2′, J 2′,3′ 4.8 Hz), 5.27 (s, 2 H, H-1′), 5.03 (t, 2 H,
H-4′′, J 4′′,5′′ 9.6 Hz), 5.01 (d, 2 H, H-1′′, J 1′′,2′′ 3.1 Hz), 4.58 (dd,
2 H, H-3′, J 3′,4′ 8.2 Hz), 4.51 (AB, 4 H, CH2 Bn, J -12.2 Hz),
4.48 (m, 8 H, CH2 Bn, H-1), 4.38 (AB, 4 H, CH2 Bn, J -12.1
Hz), 4.36 (m, 2 H, H-4′), 3.85 (dt, 2 H, H-5′′, J 5′′,6′′ 2.9 Hz, J 4′′,5′′
7.3 Hz), 3.70 (dd, 2 H, H-5a′, J 5a′,5b′ -10.7 Hz, J 4′,5a′ 3.4 Hz),
3.57 (dd, 2 H, H-5b′, J 4′,5b′ 4.8 Hz), 3.54 (dd, 2 H, H-2′′), 3.28
(ABX, 4 H, H-6′′, J 6a′′,6b′′ -10.6 Hz), 1.92, 1.87, 1.86 (3× s, 18
H, CH3 Ac); 13C{1H} NMR (CDCl3) δ 170.1, 169.5, (3 × C(O)
Ac), 137.6, 137.5, 137.4 (3× Cq Bn), 133.1 (CH Ph), 128.2-
127.3 (CH arom), 122.4 (Cq Ph), 103.2 (C-1′), 96.1 (C-1′′), 85.8
(C-2, C-3 alkyne), 80.3, 76.5, 74.6, 73.4, 71.8, 68.9, 68.6 (C-2′′,
C-3′′, C-4′′, C-5′′, C-2′, C-3′, C-4′), 73.2, 73.1 73.0 (3× CH2 Bn),
20
70.4, 67.5 (C-6′′, C-5′), 54.7 (C-1), 20.6, 20.4 (3 × CH3 Ac); [R]D
+56.5 (c 0.87, CHCl3); ES-MS m/z 1584 [M + NH4]+, 1589 [M
+ Na] +, 1605 [M + K]+. Anal. (C88H94O26) C, H.
1,4-Bis{[5′-O-ben zyl-3′-O-(2′′,6′′-d i-O-ben zyl-r-D-glu co-
p yr a n osyl)-â-D-r ib ofu r a n osyloxy]p r op -3-yn yl 2′,3′′,4′′-
tr is(d iben zyl p h osp h a te)}ben zen e (24). Deacetylation and
phosphorylation of 23 were performed as described for the
synthesis of 17. Purification was established by column
chromatography (EtOAc/light petroleum, 1/3 f 1/0, v/v) to give
24 as a colorless oil: yield 0.15 g (88%, 2 steps); Rf 0.50 (EtOAc/
light petroleum, 2/1, v/v); 1H NMR (CDCl3) δ 7.41-7.02 (m,
94 H, CH arom), 5.38 (s, 2 H, H-1′), 5.08 (d, 2 H, H-1′′, J 1′′,2′′
3.7 Hz), 5.03-4.81 (m, 28 H, H-2′, H-3′′, 12× CH2 Bn), 4.69
(AB, 4 H, CH2 Bn, J -11.8 Hz), 4.57-4.26 (m, 18 H, 2× CH2
Bn, H-3′, H-4′′, H-4′, H-1), 3.83 (m, 2 H, H-5′′), 3.71-3.48 (m,
10H, H-5′, H-2′′, H-6′′); 13C{1H} NMR (CDCl3) δ 138.0, 137.5,
135.8, 135.6, 135.5, 135.4 (9× Cq Bn), 131.6 (CH Ph), 128.4-
127.4 (CH Bn), 122.4 (Cq Ph), 103.2 (C-1′), 95.1 (C-1′′), 85.9
(C-2, C-3 alkyne), 80.0, 78.1, 77.4, 76.9, 74.3, 73.7, 73.6 (C-2′′,
C-3′′, C-4′′, C-5′′, C-2′, C-3′, C-4′), 73.1, 72.0, 70.1, 69.7, 69.3,
67.9 (9× CH2 Bn, C-6", C-5′), 55.2 (C-1); 31P{1H} NMR (CDCl3)
δ -0.75, -1.42, -1.74. Anal. (C160H160O38P6) C, H.
[5′-O-Ben zyl-3′-O-(2′′,6′′-d i-O-ben zyl-r-D-glu cop yr a n o-
syl)-â-D-r ibofu r a n osyloxy]p r op -3-yn ylb en zen e 2′,3′′,4′′-
Tr is(d iben zyl p h osp h a te) (20). Deacetylation and phosphor-
ylation of phenylacetylene derivative 19 were performed as
described for the synthesis of 17. The product was purified by
column chromatography (EtOAc/light petroleum, 1/3 f 1/1,
v/v) to give 20 as a colorless oil: yield 0.33 g (88%, 2 steps); Rf
0.75 (EtOAc/light petroleum, 1/1, v/v); 1H NMR (CDCl3) δ
7.52-7.08 (m, 50 H, CH arom), 5.46 (s, 1 H, H-1′), 5.13 (d, 1
H, H-1′′, J 1′′,2′′ 2.9 Hz), 5.08-4.85 (m, 14 H, H-2′, H-3′′, 6× CH2
Bn), 4.73 (AB, 2 H, CH2 Bn, J -12.2 Hz), 4.59-4.47 (m, 5 H,
CH2 Bn, H-3′, H-4′′, H-4′), 4.39 (m, 4 H, CH2 Bn, H-1), 3.89
(m, 1 H, H-5′′), 3.72-3.48 (m, 5 H, H-5′, H-2′′, H-6′′); 13C{1H}
NMR (CDCl3) δ 137.9, 137.4 (3× Cq Bn), 136.0, 135.9, 135.7,
135.6, 135.4, 135.3 (6× Cq Bn), 131.6 (CH Ph), 129.7-126.8
(CH Bn), 122.2 (Cq Ph), 103.0 (C-1′), 95.0 (C-1′′), 86.4, 83.9
(C-2, C-3 alkyne), 79.9, 78.0, 77.2, 76.8, 76.8, 74.1, 73.6, 73.6
(C-2′′, C-3′′, C-4′′, C-5′′, C-2′, C-3′, C-4′), 74.1, 73.6, 73.0, 71.9,
70.0, 69.6, 69.2, 68.9, 67.8 (9× CH2 Bn, C-6′′, C-5′), 55.1 (C-1);
1,2,4,5-Tetr a k is{[2′-O-a cetyl-5′-O-ben zyl-3′-O-(3′′,4′′-d i-
O-a cetyl-2′′,6′′-d i-O-ben zyl-r-D-glu cop yr a n osyl)-â-D-r ibo-
fu r a n osyloxy]p r op -3-yn yl}ben zen e (26). 1,2,4,5-Tetraiodo-
benzene39 (25; 53 mg, 91 µmol) was treated with acetylene 16
(0.30 g, 0.40 mmol) as described for the preparation of 19. After
4 h TLC analysis (toluene/EtOAc/MeOH, 90/15/2, v/v/v) re-
vealed complete conversion of 16 (Rf 0.54) into a lower-running
product. Column chromatography (EtOAc/light petroleum, 2/3
f 3/2, v/v) afforded tetramer 26 as a brown foam: Rf 0.26;
31P{1H} NMR (CDCl3) δ -0.74, -1.36, -1.70. Anal. (C83H83
O19P3) C, H.
-
1,6-Bis[2′-O-a cetyl-5′-O-ben zyl-3′-O-(3′′,4′′-d i-O-a cetyl-
2′′,6′′-d i-O-b en zyl-r-D-glu cop yr a n osyl)-â-D-r ib ofu r a n o-
syloxy]-2,4-h exa d iyn e (21): colorless oil; Rf 0.41 (toluene/
EtOAc/MeOH, 90/15/2, v/v/v); 1H NMR (300 MHz, CDCl3, HH-
COSY) δ 7.38-7.17 (m, 30H, CH arom), 5.37 (t, 2 H, H-3′′,
J 2′′,3′′ ) J 3′′,4′′ 9.7 Hz), 5.30 (d, 2 H, H-2′, J 2′,3′ 4.6 Hz), 5.15 (s,
2 H, H-1′), 5.02 (t, 2H, H-4′′, J 4′′,5′′ 9.7 Hz), 5.00 (d, 2 H, H-1′′,
J 1′′,2′′ 3.1 Hz), 4.68-4.44 (m, 20 H, H-3′, 6× CH2 Bn, H-1, H-6,
1
yield 0.14 g (52%); H NMR (300 MHz, CDCl3, HH-COSY) δ
7.48 (s, 2 H, H-2 and H-5 Ph) 7.34-7.19 (m, 60 H, CH arom),
5.37 (t, 4 H, H-3′′, J 2′′,3′′ ) J 3′′,4′′ 9.7 Hz), 5.35 (d, 4 H, H-2′, J 2′,3′
4.7 Hz), 5.26 (s, 4 H, H-1′), 5.03 (t, 4 H, H-4′′, J 4′′,5′′ 9.7 Hz),
5.03 (d, 4 H, H-1′′, J 1′′,2′′ 3.0 Hz), 4.58 (dd, 4 H, H-3′, J 3′,4′ 8.2
Hz), 4.51 (AB, 8 H, CH2 Bn, J -12.2 Hz), 4.44 (m, 16 H, CH2
Bn, H-1), 4.38 (AB, 8 H, CH2 Bn, J -11.9 Hz), 4.37 (m, 4 H,

3302 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
de Kort et al.
H-4′), 3.85 (dt, 4 H, H-5′′, J 5′′,6′′ 3.0 Hz, J 4′′,5′′ 7.4 Hz), 3.69 (dd,
4 H, H-5a′, J 5a′,5b′ -10.9 Hz, J 4′,5a′ 3.3 Hz), 3.57 (dd, 4 H, H-5b′,
J 4′,5b′ 4.9 Hz), 3.56 (dd, 4 H, H-2′′), 3.28 (ABX, 8 H, H-6′′, J 6a′′,6b′′
-10.8 Hz), 1.91, 1.85 (2 × s, 36 H, 3 × CH3 Ac); 13C{1H} NMR
(CDCl3) δ 170.2, 170.0, 169.6, (3 × C(O) Ac), 138.0, 137.8, 137.6
(3× Cq Bn), 135.4 (CH Ph), 128.3-127.4 (CH Bn), 124.9 (Cq
Ph), 103.0 (C-1′), 96.1 (C-1′′), 90.2, 83.9 (C-2, C-3 alkyne), 80.3,
76.6, 74.5, 73.5, 72.0, 69.1, 68.7 (C-2′′, C-3′′, C-4′′, C-5′′, C-2′,
C-3′, C-4′), 73.4, 73.3 73.1 (3× CH2 Bn), 70.4, 67.6 (C-6′′, C-5′),
Hz), 4.37 (q, 1 H, H-3′′, J 2′′,3′′ ) J 3′′,4′′ ) 3J 3′′,P 9.2 Hz), 4.25 (dd,
1 H, H-3′, J 3′,4′ 7.6 Hz), 4.20 (m, 1 H, H-4′), 3.97 (q, 1 H, H-4′′,
J 4′′,5′′ )3J 4′′,P 9.6 Hz), 3.89 (dd, 1 H, H-6a′′, J 6a′′,6b′′ -9.6 Hz, J 5′′,6a′′
3.7 Hz), 3.83-3.61 (m, 5 H, H-5a′, H-6b′′, H-5′′, H-2′′, H-1a),
3.58-3.47 (m, 2 H, H-5b′, H-1b), 2.68 (t, 2 H, H-3, J 7.5 Hz),
1.88 (m, 2 H, H-2); 13C{1H} NMR (D2O, CH-COSY, 75 MHz) δ
143.1 (Cq Ph), 129.5, 129.4, 126.8 (CH Ph), 107.2 (C-1′), 97.7
(C-1′′), 81.6 (C-4′), 77.9 (C-3′′), 74.7 (C-2′, C-3′), 72.7 (C-4′′,
C-2′′), 71.7 (C-5′′), 68.3 (C-1), 64.0 (C-5′), 61.0 (C-6′′), 32.3 (C-
3), 31.2 (C-2); 31P{1H} NMR (PH-COSY, 121 MHz, D2O) δ 3.37
(P-4′′), 3.13 (P-2′), 2.17 (P-3′′); ES-MS m/z 669 [M - H]-, 691
[M - 2H + Na]-, 713 [M - 3H + 2Na]-; HR-MS C20H32O19P3
[M - H]-calcd 669.0751, found 669.0747 ((0.0033).
20
54.5 (C-1), 20.8, 20.6, 20.5 (3 × CH3 Ac); [R]D +52.0 (c 0.4,
CHCl3). Anal. (C170H182O52) C, H.
1,2,4,5-Tet r a k is{[5′-O-b en zyl-3′-O-(2′′,6′′-d i-O-b en zyl-
r-D-glu cop yr a n osyl)-â-D-r ib ofu r a n osyloxy]p r op -3-yn yl-
2′,3′′,4′′-tr is(d iben zyl p h osp h a te)}ben zen e (27). Deacety-
lation and phosphorylation of 26 were performed as described
for the synthesis of 17. Purification was established by column
chromatography (EtOAc/light petroleum, 1/2 f 1/0, v/v) to
afford 27 as a slightly yellowish oil: yield 77 mg (65%, 2 steps);
Rf 0.54 (toluene/EtOAc/MeOH, 25/10/1, v/v/v); 1H NMR (CDCl3)
δ 7.39-6.99 (m, 182 H, CH arom), 5.58 (s, 4 H, H-1′), 5.08 (d,
2 H, H-1′′, J 1′′,2′′ 3.7 Hz), 5.03-4.81 (m, 28 H, H-2′, H-3′′, 12×
CH2 Bn), 4.69 (AB, 4 H, CH2 Bn, J -11.8 Hz), 4.57-4.26 (m,
18 H, 2× CH2 Bn, H-3′, H-4′′, H-4′, H-1), 3.83 (m, 2 H, H-5′′),
3.71-3.48 (m, 10 H, H-5′, H-2′′, H-6′′); 13C{1H} NMR (CDCl3)
δ 138.0, 137.4 (3× Cq Bn), 136.0, 135.8, 135.7, 135.6, 135.5,
135.3 (6× Cq Bn), 135.4 (CH Ph), 128.5-127.4 (CH Bn), 124.9
(CH Ph), 103.0 (C-1′), 95.1 (C-1′′), 90.4 (C-2), 83.5 (C-3), 79.9,
78.3, 77.4, 76.9, 74.3, 73.8, 73.7 (C-2′′, C-3′′, C-4′′, C-5′′, C-2′,
C-3′, C-4′), 74.6, 73.2, 72.1 (3× CH2 Bn), 69.8, 69.7, 69.6, 69.5,
69.3, 69.2, 69.1, 67.8 (6× CH2 Bn, C-6′′, C-5′), 55.0 (C-1);
1,4-Bis{[3′-O-(r-D-glu copyr an osyl)-â-D-r ibofu r an osyloxy]-
p r op -3-yl 2′,3′′,4′′-t r isp h osp h a t e}b en zen e (Na + Sa lt ),
‘Dip h ostin ’ (7). Deprotection of 24 and purification as de-
scribed for the synthesis of 5 afforded dimer 7 as a white
hygroscopic powder. Release from HW40 fractogel column at
t ) 69 min: yield 42% (18 mg, 27 µmol); anion-exchange HPLC
elution at 30% buffer B; 1H NMR (300 MHz, D2O, HH-COSY)
δ 7.24 (s, 4 H, CH Ph), 5.34 (s, 2 H, H-1′), 5.15 (d, 2 H, H-1′′,
3
J 1′′,2′′ 3.9 Hz), 4.52 (dd, 2 H, H-2′, J 2′,3′ 3.4 Hz, J 2′,P 6.9 Hz),
4.33-4.22 (m, 6 H, H-3′′,H-3′, H-4′), 3.99 (q, 2 H, H-4′′, J 4′′,5′′
)3J 4′′,P 9.4 Hz), 3.93 (dd, 2 H, H-6a′′, J 6a′′,6b′′ -10.3 Hz, J 5′′,6a′′
3.6 Hz), 3.87-3.71 (m, 10 H, H-5a′, H-6b′′, H-5′′, H-2′′, H-1a),
3.68-3.52 (m, 4 H, H-5b′, H-1b), 2.65 (t, 4 H, H-3, J 7.7 Hz),
1.88 (m, 4 H, H-2); 13C{1H} NMR (D2O) δ 140.3 (Cq Ph), 130.4
(CH Ph), 107.3 (C-1′), 97.4 (C-1′′), 81.8 (C-4′), 77.5 (C-3′′), 74.5,
74.2 (C-2′, C-3′), 73.3, 72.8 (C-4′′, C-2′′), 71.9 (C-5′′), 68.7 (C-
1), 64.1 (C-5′), 61.2 (C-6′′), 32.8 (C-3), 31.0 (C-2); 31P{1H} NMR
(PH-COSY, 121 MHz, D2O) δ 4.17 (broad, P-4′′, P-2′, P-3′′);
ES-MS m/z 630 [M - 2H]2-, 641 [M - 3H + Na]2-; HR-MS
31P{1H} NMR (CDCl3) δ -0.74, -1.53, -1.83; [R]D +8.4 (c
20
0.5, CHCl3). Anal. (C314H314O76P12) C, H.
P r op yl 3′-O-(r-D-Glu cop yr a n osyl)-â-D-r ibofu r a n osid e
2′,3′′,4′′-Tr isp h osp h a te (Na + Sa lt), ‘P r op ylp h ostin ’ (5).
Compound 17 (64 mg, 46 µmol) and NaOAc (45 mg, 4 equiv/
phosphate) were dissolved in a mixture of dioxane/2-propanol/
H2O (7 mL, 4/2/1, v/v/v) and the solution was degassed.
Palladium on carbon (10%, 50 mg) was added and the reaction
mixture was stirred under an atmosphere of hydrogen gas.
After 16 h the catalyst was removed by filtration over glass
Fiber (GF/2A, Whatman). The filtrate was concentrated under
reduced pressure and the product was purified by gel-filtration
over a Fractogel HW-40 column by elution with a triethyl-
ammonium bicarbonate buffer (0.15 M). Concentration and
repeated concentration (MeOH/H2O, 4/1, v/v, 3 × 15 mL) of
the appropriate fractions (at t ) 85 min), followed by lyo-
philization gave trisphosphate 5 in pure form. The product was
converted to the Na+ form by ion-exchange with Dowex 50WX4
(Na+-form) and imino diacetate resin (Chelex, Na+ form),
followed by lyophylization: yield 28 mg (85%, 39 µmol); anion-
exchange HPLC elution at 24% buffer B; 1H NMR (300 MHz,
D2O, HH-COSY) δ 5.32 (s, 1 H, H-1′), 5.11 (d, 1 H, H-1′′, J 1′′,2′′
C
34H58O38P6 [M - 2H]2- calcd 630.0515, found 630.0508
((0.0025).
1,2,4,5-Tet r a k is{[3′-O-(r-D-glu cop yr a n osyl)-â-D-r ib o-
fu r a n osyloxy]p r op -3-yl 2′,3′′,4′′-t r isp h osp h a t e}b en zen e
(Na + Sa lt), ‘Tetr a p h ostin ’ (8). Deprotection of 26 and
purification were performed as described for the synthesis of
5 to give tetramer 8 as a highly hygroscopic solid. Release from
HW40 fractogel column at t ) 65 min: yield 42% (15 mg, 6.1
µmol); anion-exchange HPLC elution at 36% buffer B; 1H NMR
(600 MHz, D2O, HH-COSY) δ 7.10 (s, 2 H, CH Ph), 5.32 (s, 4
H, H-1′), 5.13 (d, 4 H, H-1′′, J 1′′,2′′ 4.0 Hz), 4.51 (dd, 4 H, H-2′,
J
2′,3′ 4.5 Hz, 3J 2′,P 7.3 Hz), 4.25 (m, 12 H, H-3′′, H-4′, H-3′), 3.96
(m, 8 H, H-4′′, H-6a′′), 3.78 (m, 12 H, H-5a′, H-2′′, H-1a), 3.70
(dd, 4 H, H-6b′′, J 6a′′,6b′′ -13.3 Hz, J 5′′,6b′′ 2.1 Hz), 3.66-3.57
(m, 12 H, H-5b′, H-5′′, H-1b), 2.61 (t, 8 H, H-3, J 8.0 Hz), 1.84
(m, 8 H, H-2); 13C{1H} NMR (D2O, CH-COSY, 75 MHz) δ 138.8
(Cq Ph), 131.1 (CH Ph), 107.5 (C-1′), 96.9 (C-1′′), 81.9 (C-4′),
77.0 (C-3′′), 74.4 (C-2′), 73.8 (C-3′), 72.9 (C-5′′, C-2′′), 72.0 (C-
4′′), 69.2 (C-1), 64.2 (C-5′), 61.1 (C-6′′), 31.3 (C-3), 28.8 (C-2);
31P{1H} NMR (PH-COSY, 121 MHz, D2O) δ 4.57 (P-3′′), 4.34
(P-4′′), 4.25 (P-2′); ES-MS m/z 611 [M - 4H]4-, 616 [M - 5H
+ Na]4-, 622 [M - 6H + 2Na]4-, 627 [M - 7H + 3Na]4-, 633
[M - 8H + 4Na]4-, 822 [M - 4H + Na]3-, 830 [M - 5H +
2Na]3-, 837 [M - 6H + 3Na]3-, 845 [M - 7H + 4Na]3-, 852
[M - 8H + 5Na]3-, 859 [M - 9H + 6Na]3-; HR-MS
3
3.9 Hz), 4.48 (dd, 1 H, H-2′, J 2′,3′ 3.2 Hz, J 2′,P 6.8 Hz), 4.24-
4.19 (m, 3 H, H-3′′,H-3′, H-4′), 3.97 (dd, 1 H, H-5a′, J 5a′,5b′ -10.1
Hz, J 4′,5a′ 3.0 Hz), 3.91 (q, 1 H, H-4′′, J 3′′,4′′ ) 3J 4",P 9.0 Hz), 3.81-
3.73 (m, 2 H, H-5′′, H-5b′), 3.71-3.55 (m, 4 H, H-6′′, H-2′′,
H-1a), 3.48 (m, 1 H, H-1b), 1.55 (m, 2 H, H-2), 0.86 (t, 3 H,
H-3, J 7.4 Hz); 13C{1H} NMR (D2O, CH-COSY, 75 MHz) δ
107.1 (C-1′), 96.4 (C-1′′), 81.6 (C-4′), 76.6 (C-3′′), 74.2 (C-2′),
73.4 (C-3′), 73.3 (C-5′′), 73.1 (C-2′′), 73.0 (C-4′′), 70.6 (C-1), 63.9
(C-6′′), 60.9 (C-5′), 22.7 (C-2), 10.5 (C-3); 31P{1H} NMR (PH-
COSY, D2O, 121 MHz) δ 5.1 (P-3′′), 4.5 (P-4′′), 4.2 (P-2′).; ES-
MS m/z 593 [M - H]-, 615 [M - 2H + Na]-, 637 [M - 3H +
2Na]-; HR-MS C14H28O19P3 [M - H]- calcd 593.0437, found
593.0434 ((0.0028).
C
62H110O76P12 [M - 4H]4- calcd 610.5398, found 610.5405
((0.0021).
Ack n ow led gm en t. These investigations were sup-
ported by The Netherlands Foundation for Chemical
Research (CW) with financial aid from The Netherlands
Organization for Scientific Research (NWO). We thank
H. van den Elst and N. Meeuwenoord for assistance in
purification of the target compounds and C. Erkelens
and F. Lefeber for recording NMR spectra. We thank
the Wellcone Trust for program grant support (Grant
045491 to B.V.L.P. and Grant 039662 to C.W.T.), Mr.
R. Pederick for preliminary studies using sea urchin
homogenates, and Dr. A. M. Riley for useful discussions.
[3′-O-(r-D -Glu cop yr a n osyl)-â-D -r ib ofu r a n osyloxy]-
p r op -3-ylben zen e 2′,3′′,4′′-Tr isp h osp h a te (Na + Sa lt),
‘P h en ylp h ostin ’ (6). Deprotection of 20 and purification as
described for the synthesis of 5 gave phenyl derivative 6 as a
white fluffy solid. Release from HW40 fractogel column at t )
110 min: yield 80% (49 mg, 76 µmol); anion-exchange HPLC
elution at 26% buffer B; 1H NMR (300 MHz, D2O, HH-COSY)
δ 7.37-7.20 (m, 5 H, CH Ph), 5.20 (s, 1 H, H-1′), 5.15 (d, 1 H,
3
H-1′′, J 1′′,2′′ 3.9 Hz), 4.53 (dd, 1 H, H-2′, J 2′,3′ 4.1 Hz, J 2′,P 8.4

Potent Agonists of IP3R
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