Synthesis and biological evaluation of cyclophostin: A 5',6-tethered analog of adenophostin A

Article abstract of DOI:10.1016/S0040-4020(00)00480-4

The synthesis, conformational analysis and biological evaluation of 5'-6_-tethered adenophostin A, so-called cyclophostin 14, and its de-adeninylated analog 15 are described. They are prepared via ring-closing metathesis of diolefin 28, consecutive coupling of the central building block 33 to 6-N-benzoyladenine or propargyl alcohol, respectively, followed by phosphorylation and deprotection. NMR spectroscopy and a molecular dynamics simulation indicated that the 5'-6_-tether induces a conformational change from 2'-endolsyn in 1 to 3'-endo/anti in 14. The unexpected small loss of Ca²⁺-releasing potency of cyclophostin 14, which is reflected by the low EC₅₀/IC₅₀ ratio in comparison with cycloribophostin 15, suggests that the interaction of the adenine with IP₃R plays a decisive role in determining the high activity of adenophostin A (1). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00480-4

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5915±5928
Synthesis and Biological Evaluation of Cyclophostin:
A 50,600-Tethered Analog of Adenophostin A
Martin de Kort,a Anouk D. Regenbogen,a Herman S. Overkleeft,a R. A. John Challiss,b
Yoriko Iwata,c Shuichi Miyamoto,c Gijs A. van der Marela and Jacques H. van Booma,
*
aLeiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
bDepartment of Cell Physiology & Pharmacology, Maurice Shock Medical Sciences Building, University of Leicester,
University Road, Leicester, LE1 9HN, UK
cExploratory Chemistry Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 25 February 2000; revised 9 May 2000; accepted 25 May 2000
AbstractÐThe synthesis, conformational analysis and biological evaluation of 50-600-tethered adenophostin A, so-called cyclophostin 14,
and its de-adeninylated analog 15 are described. They are prepared via ring-closing metathesis of diole®n 28, consecutive coupling of the
central building block 33 to 6-N-benzoyladenine or propargyl alcohol, respectively, followed by phosphorylation and deprotection. NMR
spectroscopy and a molecular dynamics simulation indicated that the 50-600-tether induces a conformational change from 20-endo/syn in 1 to
30-endo/anti in 14. The unexpected small loss of Ca21-releasing potency of cyclophostin 14, which is re¯ected by the low EC50/IC50 ratio in
comparison with cycloribophostin 15, suggests that the interaction of the adenine with IP3R plays a decisive role in determining the high
activity of adenophostin A (1). q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
than the corresponding 1-phosphate in IP3 (4). We recently
reported8 that the ,20-fold decreased activities of the rigid
analogs spirophostin (3R)-12 and (3S)-13 relative to IP3 (4)
can be ascribed to an improper spatial orientation of the
non-vicinal 3-phosphates. The aforementioned observations
imply that a cooperative effect of the adenine moiety and the
20-phosphate may be responsible for the enhanced activity
of adenophostin A (1). It was envisaged that a non-de-
adeninylated analog in which the conformational freedom
of the isolated 20-phosphate function is restricted would be
of value in probing the possible existence of the proposed
cooperative effect of the adenine.
The gluconucleosides adenophostin A and B (1 and 2)
exhibit ,10±100 times higher Ca21-mobilizing potencies
in comparison with the natural second messenger d-myo-
inositol 1,4,5-trisphosphate (IP3, 4).1 Despite many
synthetic efforts in determining the minimal structural
requirements for the high activities of 1 and 2, no simpli®ed
analogs displaying a higher activity than IP3 (4) have been
reported. The observed1c ,1000-fold reduced binding
af®nity of the 20-dephosphorylated derivative 3 and the
IP3-like potencies found for the de-adeninylated analogs
5±72 show that the extremely high potency of adenophostin
A (1) is mainly governed by the adenine and the 20-phos-
phate moieties. Additional studies have revealed that the
Ca21-releasing activity of conformationally ¯exible analogs
of 1 is signi®cantly reduced. For instance, the hydroxyethyl
glucosides 93 and 104 exhibit ,10-fold diminished
potencies relative to 5, whereas acyclophostin 115 is a pH-
dependent partial agonist. It has been proposed that the
tripodal arrangement of the phosphates in adenophostin A
(1) may stabilize a long-range interaction with the IP3 recep-
tor (IP3R).6 The latter is endorsed by molecular modeling7,3b
studies, which showed that the 20-phosphate in 1 occupies a
more remote position from the trans-300,400-bisphosphate
In this paper, we describe the synthesis of the conformation-
ally restricted adenophostin 14, so-called cyclophostin, and
its de-adeninylated analog 15. In addition, the biological
activity of both analogs in terms of conformational behavior
is assessed by NMR spectroscopy and molecular modeling
(Fig. 1).
Results and Discussion
Comparison of the biological activities of furanophostin 79
and ribophostin 6,2 as well as those of xylose-derivative 10
and glucopyranose 9,3 shows that HO-50 and HO-600 do not
contribute to the activity of 1. Moreover, a recent molecular
modeling study7 of adenophostin A (1) revealed that
both hydroxyls are in close proximity, inferring that the
rotational freedom of 1 can be limited by anchoring either
Keywords: adenophostin A; gluconucleoside; ring-closing metathesis;
calcium release; molecular modeling.
* Corresponding author. Tel.: 131-71-527-4375; fax: 131-71-527-4307;
e-mail: j.boom@chem.leidenuniv.nl
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00480-4

5916
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
Figure 1. Structures of adenophostin A (1) and B (2), IP3 (4), ribophostins 5±8, acyclophostin 11, spirophostins 12 and 13, and cyclophostins 14 and 15.
HO-50 to HO-600 or CH2-50 to CH2-600 with an appropriate
alkyl spacer. To this end, attention was focused on the
synthesis of disaccharides 26±28 (see Scheme 1) which,
after ring-closing metathesis10 (RCM), would lead to
the respective eight-, eleven- and fourteen-membered ring
derivatives 29±31.
acceptor 2117 with donor 19, prepared by silylation and
benzylation of 17, gave dimer 24 which, in turn, was readily
transformed into diene 28 by desilylation (!25) and allyl-
ation in 72% yield over the last three steps. Gratifyingly,
subjection of 28 to RCM proceeded uneventfully to give 31
as a mixture of E/Z isomers. Selective hydrogenation
(Scheme 2) of the ole®n bond in 31 with H2/PtO2 gave
homogeneous 32, the structure of which was fully ascer-
tained by NMR spectroscopy and mass spectrometry.
Acid-mediated deprotection13 of 32 and subsequent acetyl-
ation afforded key building block 33. VorbruÈggen-type
condensation of 33 with silylated N6-benzoyladenine in
the presence of trimethylsilyl tri¯uoromethanesulfonate
(TMSOTf) gave the tethered adenosine derivative 34 as a
single isomer. The introduction of the three phosphate
groups was performed by the following three-step sequence
of reactions.17 Selective deacetylation of 34 by short
treatment with potassium t-butoxide in MeOH, followed
by 1H-tetrazole-assisted phosphitylation of the alcohol
functions with N,N-diisopropyl-bis-[2-(methylsulfonyl)-
ethyl] (MSE) phosphoramidite,18 and in situ oxidation of
the intermediate phosphite triesters with t-butylhydroperox-
ide, gave trisphosphate 35. Deprotection of the latter was
affected in two consecutive steps involving removal of the
base-labile groups with Tesser's base,19 and hydrogenolysis
of the 20-O-benzyl ether to afford, after puri®cation by
HW-40 gel ®ltration and Dowex-ion exchange chromato-
graphy, homogeneous cyclophostin 14 (Na1-salt). The de-
adeninylated analog of 14, i.e. cycloribophostin 15, was
prepared via deacetylation of propargyl glycoside 36,
obtained by glycosylation of propargyl alcohol with tetra-
acetate 33, and phosphitylation of the intermediate triol with
N,N-diisopropyl-dibenzyl phosphoramidite,20 to furnish
trisphosphate 37. Debenzylation of 37 by hydrogenolysis,
followed by puri®cation as described earlier, gave homo-
geneous target compound 15. The identity of 14 and 15 was
fully ascertained by 1H, 13C and 31P NMR spectroscopy, as
well as by high resolution mass spectrometry.
The synthesis of key dienes 26 and 27 commenced with
N-iodosuccinimide (NIS)-mediated11 coupling of known
ole®n 1612 with thioglucopyranoside 18, prepared by trityl-
ation and subsequent benzylation of known 17,16 to afford
the a-linked disaccharide 22 in 86% yield. Detritylation of
22 under the in¯uence of p-toluenesulfonic acid gave disac-
charide 23, which was readily converted to diene 26 by
Swern oxidation of the primary hydroxyl group and subse-
quent Wittig ole®nation. Alternatively, allylation of 23 gave
the 60-O-allyl derivative 27. Having the dienes 26 and 27 in
hand, the formation of the eight- and eleven-membered
rings 29 and 30 by RCM using the Grubbs' catalyst14
(PCy3)2Cl2RuˆCHPh was undertaken. It was established
that the bis-vinyl substrate 26 failed to give the eight-
membered ring 29 under a variety of reaction conditions.
On the other hand, RCM of the 60-O-allyl-4-vinyl derivative
27 led to the exclusive formation of the unwanted
60-tethered dimer in 69% yield. The outcome of these
experiments may be explained by the steric congestion of
the vinyl functions15 and an indomitable ring strain.16 In
order to overcome the problems encountered in the above
cyclizations, the sterically less demanding 5,60-di-O-allyl
derivative 28 was used for the construction of the four-
teen-membered macrocycle 31 (see Scheme 1). Thus,
coupling of 5-O-trityl-1,2-isopropylidene-a-d-ribofurano-
side26 (20) with donor 18 gave the a-linked dimer 24
(RˆTr). However, ensuing detritylation of 24 (RˆTr)
under the in¯uence of 1% p-TsOH in CH2Cl2/MeOH (1/1,
v/v) was accompanied by concomitant cleavage of the
glucosidic bond. The latter disadvantage could be circum-
vented as follows. Condensation of partially protected

M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
5917
Scheme 1. Reagents and conditions: (i) a. TrCl, pyridine, 16 h, 85%; b. NaH, BnBr, DMF, 2 h, 93%. (ii) a. TBDPSCl, pyridine, 16 h, 78%; b. NaH, BnBr,
Ê
DMF, 2 h, 91%. (iii) NIS, TfOH, Ms 4 A, Et2O or toluene/1,4-dioxane, 1/3, v/v, 1 h, 22: 86%, 24: 85%. (iv) 1% p-TsOH, CH2Cl2/MeOH, 1/1, v/v, 4 h, 84%. (v)
a. (C(O)Cl)2, Et3N, DMSO, CH2Cl2, 2608C, 2 h; b. MePPh3Br, n-BuLi, THF, 08C, 2 h, 71%, 2 steps. (vi) NaH, allylbromide, DMF, 3 h, 27: 70%; 28: 89%.
(vii) 1 M TBAF, THF, 508C, 16 h, 95%. (viii) 5 mol% RuCl2(PCy3)2CHPh, 0.025 mM in toluene, 16 h, 29: 0%, 30: 0% (69% of 60-O-allyl dimer formed), 31:
75%.
Scheme 2. Reagents and conditions: (i) PtO2, H2, 2 h, quant. (ii) a. HOAc/H2O/(HOCH2)2, 14/6/3, v/v/v, re¯ux, 90 min.; b. Ac2O/pyridine, 4 h, 92%, 2 steps.
(iii) a. HMDS, pyridine, 6-N-benzoyladenine, 7 h; b. TMSOTf, 1,2-dichloroethane, re¯ux, 16 h, 82%. (iv) propargyl alcohol, TMSOTf, (CH2Cl)2, 6 h, 82%.
(v) a. t-BuOK (1 M in MeOH), 1 min; b. (MSEO)2PN(i-Pr)2, 1H-tetrazole, 30 min; c. t-BuOOH, 30 min. (vi) a. NaOH (4 M)/1,4-dioxane/MeOH, 1/14/5, v/v/v,
16 h; b) H2, Pd-black, H2O, 16 h, 56% (based on 34). (vii) a. NaOMe, MeOH, 1 h; b. (BnO)2PN(i-Pr)2, 1H-tetrazole, 30 min; c. t-BuOOH 30 min, 60%. (viii)
H2, 10% Pd/C, 1,4-dioxane/i-PrOH/H2O (4/2/1, v/v/v), 16 h, 84%.

5918
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
Table 1. Coupling constants (in Hz) of adenophostin A (1), cyclophostins 14 and 15 and ribophostin 8
0
J1 ,2
0
0
J2 ,3
0
0
J3 ,4
0
Compound
Conformation
Ribose
Adenine
Adenophostin A (1)a
Cyclophostin (14)c
Ribophostin (8)d
6.6
3.0
,0
,0
5.1
4.8
3.2
4.4
2.9
6.7
9.8
9.7
C-20-endo (71%)b
C-30-endo (73%)b
C-30-endo (,100%)e
C-30-endo (,100%)e
syn
anti
±
Cycloribophostin (15)c
±
a See Ref. 7.
b Calculated with the conformational analysis program Pseurot v6.3.30
c Measured at 600 MHz in 50 mM phosphate buffer at p2H 6.8.
d Measured at 300 MHz in D2O.
2
21,22
e Based on the assumption that 10£ J1 ,2 represents the percentage of 20-endo (S-type) conformer.
0
0
Conformational Analysis
phate (P-20) and the conformational rigidity was investi-
gated by a molecular dynamics simulation of cyclophostin
14. The data of the global minimum conformation,
presented in Fig. 4A and Table 2 (entry 3), clearly indicate
that the distances between the trans-300,400-bisphosphate and
P-20 do not deviate substantially from those observed7 in
adenophostin A (1). The different values of tc and x (see
Table 2 and Fig. 3) of the global minimum 30-endo/anti
conformation of cyclophostin 14 are in accord with its
NMR-spectroscopic data (see Table 1). Superimposition
of cyclophostin 14 and adenophostin A (1), as illustrated
in Fig. 4A, showed that the distance between the individual
1
In the ®rst instance, the H NMR spectroscopic data of
cyclophostin 14 and its de-adeninylated analog 15 were
compared with those of adenophostin A (1).7 It turned out
that the glucose moiety in 14 and 15 adopts, based on the
coupling constants of the glucose ring protons (see Experi-
mental section), a 4C1 conformation as in 1. In contrast, the
coupling constants of the ribose protons (see Table 1)
clearly show21,22 that the C-30-endo conformation in both
14 and 15 prevails over the C-20-endo conformation of
adenophostin A (1). The same holds, gauged by the absence
0
Ê
of a vicinal coupling between H-10 and H-20 (J1 ,2 ,0), for
2 -phosphates is 1.4 A. Furthermore, it can be seen that the
triangular arrangement of the phosphates in 14 is slightly
smaller than the phosphorous triad in 1 and that the
0
0
ribophostin 8.2c,13
0
00
Ê
distances between C-5 and C-6 are the same (i.e. 4.4 A,
see entries 2 and 3 in Table 2). It can also be derived from
the superimposition of the four lowest energy conformers of
14 (see Fig. 4B) that the conformation of the tethered
disaccharide is constrained, whereas the adenine nucleobase
adopts several preferred positions in the anti conformation.
Apart from this, the presence of three conformers of
cyclophostin 14 having a 20-endo conformation similar to
As can be seen in Fig. 2, the NOEs between H-8 of the
adenine and H-10, H-20 and H-30 of the ribose ring clearly
show that the nucleobase in cyclophostin 14 adopts an anti
conformation. Moreover, the earlier assigned preference of
14 in adopting the C-30-endo conformation is endorsed by
the absence of a NOE signal between H-10 and H-40.
The effect of the 50,600-tether on the position of the 20-phos-
Figure 2. Part of the 600 MHz NOESY spectrum of cyclophostin 14 (10 mM) in 50 mM phosphate buffer in D2O at p2H 6.8.

M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
5919
x
t
t
t
t

5920
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
cyclophostin 14 (entry 3), respectively. Moreover, the 50,600-
tethered ligands 14 and 15 display a consistent ,15-fold
diminished binding af®nity in comparison with adeno-
phostin A (1) and ribophostin 8.
A functional response to cyclophostins 14 and 15 was
studied by measuring the 45Ca21-release from intracellular
stores upon binding to IP3Rs in permeabilized SH-SY5Y
neuroblastoma cells24 in comparison with the activities of
adenophostin A (1), IP3 (4) and ribophostin 8 (see Table 4).
Perusal of Table 4 reveals that the relative potencies and
slope (h) of the concentration±response curves of all
analogs are in agreement with the binding data presented
in Table 3. Interestingly, the Ca21-mobilizing potency of
cyclophostin 14 (entry 3) is now only ,5-fold decreased
in comparison with adenophostin A (1), whereas the teth-
ered ribophostin derivative 15 is ,13-fold less effective
than the corresponding ribophostin 8.
Figure 3. Torsion angles t and x assigned in molecular modeling of
adenophostin A (1, RˆH) and cyclophostin 14 [R,Rˆ(CH2)4].
adenophostin A (1) was established (entries 4±6). However,
the latter conformers are less stable (i.e. ,17 kJ mol21) than
the energy-minimized 30-endo-puckering structure (entry 3).
The respective binding and Ca21-release data in Tables 3
and 4 were compared by calculating the EC50/IC50 ratios
(see Table 4), which give an indication of the ef®cacy of
each of the ligands. The relatively high value for IP3 (4)
(entry 1) can perhaps be ascribed, despite the short (30 s)
incubation period, to partial metabolism of IP3 in the
45Ca21-assay. The metabolically stable adenophostin A (1,
entry 2) and its de-adeninylated analog 8 (entry 4) have
similar EC50/IC50 ratios. Despite the substantial loss of
biological activity, the ef®cacy of the tethered cycloribo-
phostin 15 (entry 5) is entirely predictable. In contrast, the
EC50/IC50 ratio of cyclophostin 14 (entry 3) is ,3-fold
reduced, illustrating that the Ca21-releasing potency of 14
is less affected than the binding af®nity for IP3R.
In summary, based on the molecular modeling results it may
be concluded that the 30-O-a-glucosyl ribose moiety in
cyclophostin 14 closely resembles the geometry of the
glucosidic bond in adenophostin A (1) and that the distance
between the C-50 and C-600 positions is preserved. On the
other hand, the adenosine moiety undergoes a confor-
mational change from 20-endo/syn in 1 to 30-endo/anti in 14.
Biological Evaluation
The IC50 values (see Table 3) of cyclophostin 14 and cyclo-
ribophostin 15, as well as those of adenophostin A (1), IP3
3
(4) and ribophostin 82c,13 were determined in H-IP3 dis-
placement binding experiments using bovine adrenal cortex
membranes.23 The relative displacing potencies of adeno-
phostin A (1) and IP3 (4) (entries 1 and 2) are consistent with
earlier reported data while those of cyclophostin 14 (entry 3)
and ribophostin 8 (entry 4) are similar to IP3. The de-
adeninylated analogs ribophostin 8 and cycloribophostin
15 (entry 5) both exhibit a ,20-fold decreased binding af®nity
to IP3R in comparison with adenophostin A (1, entry 2) and
In summary, the binding af®nity of cyclophostin 14 and
cycloribophostin 15 is in both cases ,15-fold diminished
in comparison with adenophostin A (1) and ribophostin 8,
respectively. However, the Ca21-releasing activity of 14 is
only ,5-fold lower than that observed for 1 (still ,5-fold
higher than IP3), which is in contrast to the ,13-fold
reduced potency of 15, relative to 8.
Figure 4. Graphical representation of the low-energy conformers with superimposition of the six-membered rings: A. global energy minimized conformations
of cyclophostin 14 (blue) and adenophostin A (1); B. superposition of the four lowest-energy conformers of cyclophostin 14.

M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
5921
3
Table 3. H-IP3-displacement/binding IC50 values of cyclophostins 14 and 15 in comparison with IP3 (4), adenophostin A (1) and ribophostin 8 (values are
shown as ^s.e. mean for the concentration which causes 50% of speci®c 3H-IP3 displacement (IC50), with h as the slope of the concentration-response curve,
for n experiments)
Entry
Compound
IP3 (4)
Adenophostin A (1)
Cyclophostin (14)
Ribophostin (8)
2log IC50
IC50 value (nM)
Ratio
h
n
1
2
3
4
5
8.13^0.05
9.39^0.07
8.23^0.05
8.08^0.09
6.94^0.04
7.4
0.41
5.9
8.3
114
18
1
14
20
277
0.99^0.01
1.51^0.13
1.20^0.05
0.79^0.03
0.80^0.02
5
4
4
4
6
Cycloribophostin (15)
Table 4. 45Ca21-release EC50 values of cyclophostins 14 and 15 in comparison with IP3 (4), adenophostin A (1) and ribophostin 8 (values are shown as ^s.e.
mean for the concentration which causes 50% of maximal 45Ca21 release (EC50), with h as the slope of the concentration-response curve, the % release is
relative to ionomycin-induced Ca21 release, for n experiments)
Entry
Compound
IP3 (4)
Adenophostin A (1)
Cyclophostin (14)
Ribophostin (8)
2log EC50
EC50 value (nM)
Ratio
Release (%)
h
EC50/IC50 ratio
n
1
2
3
4
5
6.73^0.09
8.15^0.04
7.42^0.08
6.84^0.09
5.73^0.13
188
7.0
38.2
145
1858
27
1
5
21
265
75.7^5.8
80.3^5.4
79.9^4.7
76.6^3.9
74.0^6.9
0.98^0.05
1.51^0.08
1.44^0.10
1.17^0.07
0.91^0.09
25
17
6.5
18
16
5
4
4
4
4
Cycloribophostin (15)
Conclusion
used as received. Allyl bromide, butane-2,3-dione, camphor-
sulfonic acid, ethylene glycol, tri¯uoromethane-sulfonic
acid, oxalyl chloride, triphenylmethyl chloride, sodium
hydride, t-butyldiphenylsilyl chloride, tetrabutylammonium
¯uoride and propargyl alcohol (Acros), Dowexw 50WX4,
methyltriphenylphosphonium bromide and di-t-butyl perox-
ide (Fluka), N-iodosuccinimide, trimethylsilyl tri¯uoro-
methanesulfonate and 10% palladium on charcoal were
purchased from Aldrich. Benzyl bromide, imidazole and
p-toluenesulfonic acid (Merck) were used as received.
N,N-Diisopropyl-bis-[2-(methylsulfonyl)ethyl] phosphor-
amidite18 and N,N-diisopropyl-dibenzyl phosphoramidite20
were prepared as described. All experiments were per-
formed under anhydrous conditions at room temperature
unless stated otherwise. Reactions were followed by TLC
analysis conducted at Schleicher and SchuÈll DC Fertigfolien
(F 1500 LS 254). Compounds were visualized by UV light
and by spraying with 20% sulfuric acid in MeOH followed
by charring at 1408C. Column chromatography was per-
formed on silica gel 60, 0.063±0.200 mm (Baker). NMR
spectra were recorded with a Bruker WM-200 (1H and 13C
at 200 and 50.1 MHz, respectively) and a Bruker WM-300
spectrometer (1H at 300 MHz). 1H- and 13C-chemical shifts
are given in ppm (d) relative to tetramethylsilane as an
internal standard. Mass spectra were recorded on a Finnigan
MAT TSQ-70 or PE-SIEX API 165 mass spectrometer
equipped with an electrospray inonization (ES) interface.
HRMS (ES) spectra were measured with a Finnigan MAT
900 double focusing mass spectrometer equipped with an
ES interface. The samples of the target compounds 14 and
15 were prepared in a mixture of isopropanol/H2O (80/20, v/
v) containing 1.0£1024 M NaOAc, the clusters of which
were used as internal standards in the negative ion detection
mode.
The synthesis of the fourteen-membered macrocycle cyclo-
phostin 14, as well as its de-adeninylated analog 15, has been
accomplished via ring-closing metathesis of carbohydrate
diene 28. NMR spectroscopy and molecular modeling
studies of these conformationally restricted analogs of
adenophostin A (1) revealed that the 50,600-O-n-butyl tether
in 14 and 15 induces a C-30-endo/anti conformation, rather
than a C-20-endo/syn conformation as in 1. Although the
mode of binding is at present not fully understood, the
,5-fold higher Ca21-releasing potency relative to IP3 (4)
indicates that cyclophostin 14, despite the presence of a
conformational restraint, closely resembles the bioactive
conformation of adenophostin A (1). Moreover, the unex-
pected small loss of Ca21-mobilizing potency of cyclo-
phostin 14, which is re¯ected by the low EC50/IC50 ratio
in comparison with cycloribophostin 15, shows that the
loss of activity caused by the 50,600-tether can still be
compensated by the adenine nucleobase. The latter implies
that the adenine may have a more decisive in¯uence on the
enhanced activity of adenophostin A (1) than the orientation
of the 20-phosphate.25 The cooperative effect of the adenine
and the 20-phosphate may be studied in more detail by vary-
ing the length of the 4C-tether in 14 or limiting the confor-
mational freedom of the nucleobase by replacing the
adenine with 8-bromoadenine.
Experimental
General methods and materials
CH2Cl2 and toluene were dried by distillation from P2O5
(5 g L21). Et3N and pyridine were re¯uxed for 2 h in the
presence of CaH2 (5 g L21) and subsequently distilled.
1,2-dichloroethane (p.a. Rathburn), 1,4-dioxane (p.a.
Baker), i-propanol (p.a. Baker), DMF (p.a. Baker), DMSO
(p.a. Baker), THF (Acros) were stored over molecular sieves
4 A. CH3CN (p.a. Rathburn) and MeOH (HPLC-grade,
Ê
Rathburn) were stored over molecular sieves 3 A. Acetic
acid (p.a. Baker) and acetic anhydride (p.a. Baker) were
(20S,30S) Ethyl 2-O-benzyl-3,4-di-O-(20,30-dimethoxybu-
tane-20,30-diyl)-1-thio-6-O-trityl-b-d-glucopyranoside (18).
Trityl chloride (5.19 g, 18.5 mmol) was added to a solution
of 1713 (5.71 g, 16.8 mmol) in pyridine (25 mL) and the
mixture was stirred overnight at 508C. The reaction mixture
was quenched with MeOH (1.0 mL) and concentrated. The
residue was dissolved in EtOAc (100 mL) and washed with
Ê

5922
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
sat. aq. solution of NaHCO3 (2£25 mL) and H2O (25 mL).
The organic layer was dried over MgSO4, ®ltered and
concentrated. Traces of pyridine were removed by coevapo-
ration with toluene (2£10 mL). The residue was applied
onto a column of silica gel (Et2O/light petroleum/Et3N,
66/33/1, v/v/v) to yield the tritylated product as a white
solid (6.69 g, 11.6 mmol). Mp 748C. Rf 0.68 (Et2O/light
petroleum, 2/1, v/v). 13C{1H} NMR (CDCl3): d 143.5 (Cq
Ph), 128.4±125.0 (CH arom), 99.3, 99.0 (2£Cq BDA), 85.8
(C-1), 77.0, 73.7, 69.5, 65.2 (C-2, C-3, C-4, C-5), 61.6
(C-6), 47.6 (2£OCH3 BDA), 23.5 (CH2 SEt), 17.4, 17.2
(2£CH3 BDA), 15.3 (CH3 SEt). Sodium hydride (60% wt,
0.69 g, 17.3 mmol) was added to a cooled (08C) solution of
tritylated 17 (6.69 g, 11.6 mmol) in DMF (60 mL), followed
by the addition of benzyl bromide (1.50 mL, 12.7 mmol).
When TLC analysis showed complete conversion of starting
material, excess sodium hydride was destroyed with MeOH
(0.5 mL) and the reaction mixture was diluted with Et2O
(50 mL). H2O (15 mL) was added and the DMF/H2O layer
was extracted with Et2O. The organic layer was rinsed with
H2O (2£15 mL), dried over MgSO4, ®ltered and concen-
trated in vacuo. Compound 18 was obtained as a white
solid by crystallization from Et2O/light petroleum. Yield
7.42 g (11.0 mmol, 65% over two steps). Mp 698C. Rf
Jˆ27.5 Hz), 1.04 (s, 9H, CH3 t-Bu Si); 13C{1H} NMR
(CDCl3): d 138.1 (Cq Bn), 135.5, 135.3 (Cq TBDPS),
135.6, 129.5±126.8 (CH arom), 101.1, 101.0 (2£Cq
BDA), 84.1 (C-1), 79.8, 79.0, 77.1, 69.0 (C-2, C-3, C-4,
C-5), 74.5 (CH2 Bn), 63.3 (C-6), 47.8 (2£OCH3 BDA),
26.6 (t-Bu Si), 24.5 (CH2 SEt), 19.2 (Cq t-Bu Si), 17.8,
17.6 (2£CH3 BDA), 15.1 (CH3 SEt). ES-MS: 689
[M1Na]1. HRMS (ES): Calcd C37H51O7SSi [M1H]1
667.3135, found 667.3139 (^ 0.0023).
1,2-O-Isopropylidene-5-O-trityl-a-d-ribofuranoside (20).
1,2-O-Isopropylidene-a-d-ribofuranoside26 (4.8 g, 25.0
mmol) was tritylated as described for the synthesis of 18.
Puri®cation of the product was accomplished by column
chromatography (Et2O/light petroleum/Et3N, 14/85/1, v/v/
v) to give 18 as a white solid (9.13 g, 21.3 mmol, 86%). Rf
0.94 (Et2O). Mp 1018C. 1H NMR (CDCl3): d 7.58±7.18 (m,
15H, H arom), 5.89 (d, 1H, H-1, J1,2ˆ4.4 Hz), 4.59 (t, 1H,
H-2, J2,3ˆ3.7 Hz), 4.17±4.07 (m, 1H, H-4), 4.04±3.87 (m,
1H, H-3), 3.63±3.24 (m, 2H, H-5a, H-5b), 2.30 (d, 1H, OH),
1.57, 1.38 (2£s, 6H, 2£CH3 isoprop). 13C{1H} NMR
(CDCl3): d 143.5 (Cq Ph), 128.4±126.7 (CH arom), 112.1
(Cq isoprop), 103.8 (C-1), 86.3 (Cq Tr), 79.4, 78.3, 71.8 (C-
2, C-3, C-4), 62.7 (C-5), 26.2 (2£CH3 isoprop). C27H28O5
(432): Calcd C 74.98, H 6.52; found C 75.14, H 6.59.
1
0.85 (Et2O/light petroleum, 1/1, v/v). H NMR (300 MHz,
H±H-COSY, CDCl3): d 7.52±7.18 (m, 20H, H arom), 4.87
(s, 2H, CH2 Bn), 4.54 (d, 1H, H-1, J1,2ˆ9.5 Hz), 3.95 (t, 1H,
H-2, J2,3ˆ9.5 Hz), 3.84 (t, 1H, H-3, J3,4ˆ8.8 Hz), 3.65±3.41
(m, 3H, H-4, H-5, H-6a), 3.28 (s, 3H, OCH3 BDA), 3.13±
3.08 (m, 1H, H-6b), 3.06 (s, 3H, OCH3 BDA), 2.99±2.70
(m, 2H, CH2 SEt), 1.42±1.27 (m, 3H, CH3 SEt), 1.36, 1.16
(2£s, 6H, 2£CH3 BDA); 13C{1H} NMR (CDCl3): d 143.7
(Cq Tr), 138.0 (Cq Bn), 128.4±126.5 (CH arom), 99.2, 99.0
(2£Cq BDA), 85.9 (Cq Tr), 84.3 (C-1), 78.1, 76.7, 74.6,
65.3 (C-2, C-3, C-4, C-5), 75.0 (CH2 Bn), 61.4 (C-6),
47.8, 47.4 (2£OCH3 BDA), 23.8 (CH2 SEt), 17.5, 17.1
(2£CH3 BDA), 15.0 (CH3 SEt). C40H46O7S (671): Calcd C
71.62, H 6.91; found C 71.73, H 6.96.
General glycosylation procedure
A mixture of acceptor 16, 20 or 21 (1.00 mmol) and donor
18 or 19 (1.00±1.25 mmol) was dried by coevaporation with
1,4-dioxane (3£5 mL) and was stirred for 15 min in the
appropriate solvent mixture (10 mL) with powdered molec-
Ê
ular sieves (4 A) under an atmosphere of argon. NIS (0.27 g,
1.20 mmol) and a catalytic amount of TfOH (9 mL,
0.11 mmol) were subsequently added. After completion of
the reaction (^30 min) as judged by TLC analysis, the reac-
tion mixture was ®ltered and diluted with EtOAc (15 mL).
The ®ltrate was washed with aq. Na2S2O3 (1 M, 5 mL) and
sat. aq. NaHCO3 (5 mL), dried over MgSO4 and concen-
trated in vacuo to afford the crude product.
(20S,30S) Ethyl 2-O-benzyl-6-O-t-butyldiphenylsilyl-3,4-
di-O-(20,30-dimethoxybutane-20,30-diyl)-1-thio-b-d-gluco-
pyranoside (19). t-Butyldiphenylsilyl chloride (2.30 mL,
8.87 mmol) was added to a solution of 1713 (2.51 g,
7.39 mmol) in pyridine (15 mL) and the mixture was stirred
for 2 h. MeOH (0.4 mL) was added and the mixture was
concentrated. The residue was dissolved in EtOAc
(50 mL) and washed with sat. aq. solution of NaHCO3
(15 mL) and H2O (15 mL). The organic layer was dried
over MgSO4, ®ltered and concentrated. Traces of pyridine
were removed by coevaporation with dry toluene
(2£10 mL). The crude product (Rf 0.67, Et2O) was benzyl-
ated as described for 18. Compound 19 was puri®ed by
column chromatography (EtOAc/light petroleum, 1/10!1/
1, v/v) and obtained as a colorless oil. Yield 4.40 g
(6.58 mmol, 89% over two steps). Rf 0.79 (Et2O/light petro-
leum, 1/1, v/v). 1H NMR (300 MHz, H±H-COSY, CDCl3):
d 7.75±7.66 (m, 4H, H arom), 7.47±7.21 (m, 11H, H arom),
4.87 (AB, 2H, CH2 Bn, Jˆ212.3 Hz), 4.47 (d, 1H, H-1,
J1,2ˆ9.4 Hz), 4.16 (dd, 1H, H-3, J2,3ˆ8.9 Hz, J2,3ˆ
(200S,300S) 3-O-(20-O-Benzyl-30,40-di-O-(200,300-dimethoxy-
butane-200,300-diyl)-6-O-trityl-a-d-glucopyranosyl)-5,6-di-
deoxy-1,2-O-isopropylidene-a-d-allo-hex-5-enofuranoside
(22). Glycosylation of 5,6-dideoxy-1,2-O-isopropylidene-
a-d-allo-hex-5-enofuranoside (16, 0.93 g, 5.0 mmol) with
thioglucoside 17 (4.01 g, 6.0 mmol) was carried out in a
mixture of Et2O/CH2Cl2 (50 mL, 10/1, v/v) as described in
the general procedure. The product was puri®ed by column
chromatography (Et2O/light petroleum, 1/4!1/1, v/v) to
give dimer 22 as a white foam. Yield 3.28 g (4.3 mmol,
1
86%). Rf 0.31 (Et2O/light petroleum, 1/1, v/v). H NMR
(CDCl3): d 7.47±7.18 (m, 20H, H arom), 6.01±5.87 (m,
1H, H-5), 5.83 (d, 1H, H-1, J1,2ˆ3.7 Hz), 5.47 (d, 1H,
H-6a, J5,6aˆ16.8 Hz), 5.29 (d, 1H, H-10, J1 ,2 ˆ3.7 Hz),
5.23 (d, 1H, H-6b, J5,6bˆ10.2 Hz), 4.82 (s, 2H, CH2 Bn),
0
0
4.76 (t, 1H, H-2, J2,3ˆ3.9 Hz), 4.62 (m, 1H, H-3), 4.12 (t,
1H, H-30, J2 3 ˆJ3 4 ˆ9.4 Hz), 3.93±3.67 (m, 4H, H-4, H-2 ,
H-40, H-50), 3.32 (m, 1H, H-6a0), 3.29 (s, 3H, OCH3 BDA),
3.04 (m, 1H, H-6b0), 3.01 (s, 3H, OCH3 BDA), 1.57, 1.39,
1.32, 1.16 (4£s, 12H, 4£CH3 isoprop, BDA); 13C{1H} NMR
(CDCl3): d 143.5 (Cq Tr), 138.7 (Cq Bn), 134.3 (C-5),
128.3±126.6 (CH arom), 117.5 (C-6), 112.4 (Cq isoprop),
0
0
0
0 0
9.9 Hz), 4.00 (dd, 1H, H-6, J6a,6bˆ211.4 Hz, J5,6a
ˆ
1.9 Hz), 3.83 (m, 2H, H-4, H-5), 3.36 (m, 2H, H-2, H-6b),
3.20, 3.18 (2£s, 6H, OCH3 BDA), 2.74 (m, 2H, CH2 SEt),
1.36, 1.28 (2£s, 6H, 2£CH3 BDA), 1.33 (t, 3H, CH3 SEt,

M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
5923
103.7 (C-1), 99.0 (2£Cq BDA), 95.2 (C-10), 86.0 (Cq Tr),
78.4, 77.9, 76.4, 76.0, 69.3, 69.1, 66.2 (C-2, C-3, C-4, C-20,
C-30, C-40, C-50), 71.5 (CH2 Bn), 61.5 (C-60), 47.6 (2£OCH3
BDA), 27.3 (2£CH3 isoprop), 17.5, 17.2 (2£CH3 BDA).
ES-MS: 818 [M1Na]1. C47H54O11 (795): Calcd C 71.01,
H 6.85; found C 70.93, H 6.79.
1.15 mmol) was accomplished in a mixture of toluene and
1,4-dioxane (10 mL, 3/1, v/v) according to the general
glycosylation procedure. Puri®cation was performed by
column chromatography (light petroleum/EtOAc, 6/1!1/
1, v/v) to afford bis-silylated dimer 24 in 74% yield
(0.76 g, 0.74 mmol), as a white foam. Rf 0.57 (light petro-
1
leum/EtOAc, 5/1, v/v). H NMR (300 MHz, H±H-COSY,
CDCl3): d 7.76±7.53 (m, 8H, H-arom Ph-Si), 7.43±7.18 (m,
17H, arom Bn, Ph-Si), 5.76 (d, 1H, H-1, J1,2ˆ3.9 Hz), 5.24
(200S,300S) 3-O-(20-O-Benzyl-30,40-di-O-(200,300-dimethoxy-
butane-200,300-diyl)-a-d-glucopyranosyl)-5,6-dideoxy-1,2-
(d, 1H, H-10, J1 ,2 ˆ3.9 Hz), 4.80 (s, 2H, CH2 Bn), 4.64 (t,
1H, H-2, J2,3ˆ4.0 Hz), 4.24 (ddd, 1H, H-4, J3,4ˆ4.2 Hz,
0
0
O-isopropylidene-a-d-allo-hex-5-enofuranoside
(23).
Dimer 22 (0.30 g, 0.38 mmol) was dissolved in a solution
of 1% p-TsOH in MeOH/CH2Cl2 (7.5 mL, 1/1, v/v) and
stirred for 4 h. The mixture was neutralized with Et3N and
concentrated. Puri®cation of the crude product by column
chromatography (light petroleum/EtOAc, 1/1, v/v) gave
alcohol 23 (0.18 g, 0.32 mmol, 84%) as a white foam. Rf
J4,5aˆ2.5 Hz, J4,5bˆ9.2 Hz), 4.15 (t, 1H, H-30, J3 ,4
ˆ
ˆ
0
0
0
0
J2 ,3 ˆ9.9 Hz), 4.13 (dd, 1H, H-3), 3.92 (dd, H-5a, J5a,5b
29.8 Hz), 3.88 (t, 1H, H-40, J4 ,5 ˆ9.8 Hz), 3.82±3.66 (m,
4H, H-5b, H-50, H-60), 3.68 (dd, 1H, H-20), 3.29, 3.26 (2£s,
6H, 2£OCH3 BDA), 1.52, 1.39, 1.38, 1.33 (4£s, 4£CH3
isoprop, BDA), 0.99, 0.91 (2£s, 18H, CH3 t-Bu Si);
13C{1H} NMR (CDCl3): d 139.1 (Cq Bn), 135.9, 135.7,
135.5, 135.4 (CH arom Ph-Si), 133.8, 133.4, 133.0 (Cq
Ph-Si), 129.6±127.2 (CH arom Bn, Ph-Si), 112.9 (Cq
isoprop), 104.3 (C-1), 99.5 (2£Cq BDA), 95.3 (C-10),
79.5, 77.2, 76.6, 72.7, 70.4, 69.5, 65.5 (C-2, C-3, C-4, C-
20, C-30, C-40, C-50), 72.0 (CH2 Bn), 62.0, 61.5 (C-5, C-60),
48.1, 48.0 (2£OCH3 BDA), 26.9, 26.8 (2£CH3 isoprop, CH3
t-Bu Si), 19.3, 19.2 (2£Cq t-Bu Si), 18.1, 17.8 (2£CH3
BDA). ES-MS: 1056 [M1Na]1. [a]D20ˆ191.08 (c 1.0
CHCl3). C59H76O12Si2 (1033): Calcd C 68.57, H 7.41;
found C 68.75, H 7.49.
0
0
1
0.15 (Et2O/light petroleum, 2/3, v/v); H NMR (CDCl3): d
7.43±7.26 (m, 5H, H arom), 5.86±5.78 (m, 1H, H-5), 5.81
(d, 1H, H-1, J1,2ˆ3.7 Hz), 5.51±5.25 (m, 2H, H-6a, H-6b),
5.13 (d, 1H, H-10, J1 ,2 ˆ3.7 Hz), 4.78 (AB, 2H, CH2 Bn,
Jˆ212.4 Hz), 4.69 (t, 1H, H-2, J2,3ˆ4.0 Hz), 4.56 (m, 1H,
0
0
H-3), 4.15 (t, 1H, H-30, J2 3 ˆJ3 4 ˆ9.6 Hz), 3.77±3.57 (m,
6H, H-4, H-20, H-40, H-50, H-6a0, H-6b0), 3.32, 3.27 (2£s,
6H, 2£OCH3 BDA), 2.05 (s, 1H, OH), 1.55, 1.35, 1.32 (3£s,
12H, 4£CH3 isoprop, BDA); 13C{1H} NMR (CDCl3): d
138.9 (Cq Ph), 134.3 (C-5), 128.5±127.1 (CH arom),
118.7 (C-6), 112.9 (Cq isoprop), 103.8 (C-1), 99.6, 99.4
(2£Cq BDA), 95.9 (C-10), 78.9, 78.2, 76.5, 76.2, 69.7,
69.0, 66.1 (C-2, C-3, C-4, C-20, C-30, C-40, C-50), 71.9
(CH2 Bn), 60.9 (C-60), 48.1, 47.8 (2£OCH3 BDA), 26.6
(2£CH3 isoprop), 17.8, 17.6 (2£CH3 BDA).
0
0
0 0
(200S,300S) 3-O-(20-O-Benzyl-6,7-dideoxy-30,40-di-O-(200,300-
dimethoxybutane-200,300-diyl)-a-d-gluco-hept-6-enopyrano-
syl)-5,6-dideoxy-1,2-O-isopropylidene-a-d-allo-hex-5-eno-
furanoside (26). To a cooled (2608C) solution of oxalyl
chloride (0.38 mL, 4.3 mmol) in CH2Cl2 (6.7 mL) under a
N2 atmosphere was added dropwise a solution of DMSO
(0.61 mL, 8.3 mmol) in CH2Cl2 (3.4 mL). After stirring
for 2 min, compound 23 (0.88 g, 1.60 mmol) in CH2Cl2
(5 mL) was added dropwise and the reaction mixture was
stirred at 2608C for 30 min. Et3N (2.6 mL, 18.4 mmol) was
added and after 10 min the solution was allowed to warm to
RT. The mixture was diluted with CH2Cl2 (30 mL) and
washed with sat. aq. NaCl (2£10 mL). The organic layer
was dried over MgSO4, ®ltered and concentrated under
reduced pressure. The crude aldehyde was immediately
used without further puri®cation. To a solution of methyl-
triphenylphosphonium bromide (0.86 g, 2.4 mmol) in THF
(18 mL) under a nitrogen atmosphere was added n-butyl-
lithium (1.5 mL, 1.6 M in hexanes). After stirring for 1 h, a
solution of the aldehyde (1.6 mmol) in CH2Cl2 (3.5 mL) was
added dropwise at 08C to the yellow suspension. When TLC
analysis showed complete conversion into a more lipophilic
product the reaction mixture was ®ltered over silica gel,
concentrated and puri®ed by column chromatography
(Et2O/light petroleum, 1/4, v/v) to afford disaccharide 26
as a colorless oil. Yield 0.62 g (1.12 mmol, 70%). Rf 0.63
(200S,300S) 3-O-(20-O-Benzyl-30,40-di-O-(200,300-dimethoxy-
butane-200,300-diyl)-60-O-trityl-a-d-glucopyranosyl)-1,2-
O-isopropylidene-5-O-trityl-a-d-ribofuranoside (24 RˆTr).
Condensation of trityl derivative 20 (0.76 g, 1.1 mmol) with
18 (0.47 g, 1.1 mmol) was performed in a mixture of Et2O/
CH2Cl2 (10 mL, 10/1, v/v) as described in the general glyco-
sylation procedure. The disaccharide was puri®ed by
column chromatography (Et2O/light petroleum, 1/4, v/v)
to give the title compound as a white foam. Yield 0.85 g
(0.82 mmol, 75%). Rf 0.68 (Et2O/light petroleum, 1/1, v/v);
1H NMR (CDCl3): d 7.41±7.05 (H arom), 6.04 (d, 1H, H-1,
J1,2ˆ3.7 Hz), 5.31 (d, 1H, H-10, J1 ,2 ˆ3.7 Hz), 4.85 (t, 1H,
H-2, J2,3ˆ3.8 Hz), 4.76 (s, 2H, CH2 Bn), 4.36 (dd, 1H, H-3,
J3,4ˆ9.5 Hz), 4.27 (d, 1H, H-4), 3.87±3.70 (m, 4H, H-20, H-
30, H-40, H-50), 3.57, 3.39 (m, 2H, H-5), 3.27 (m, 1H, H-6a0),
3.15, 3.18 (2£s, 6H, 2£OCH3 BDA), 2.84 (m, 1H, H-6b0),
1.56, 1.43, 1.30, 1.11 (4£s, 4£CH3 isoprop, BDA); 13C{1H}
NMR (CDCl3): d 143.5 (Cq Tr), 138.7 (Cq Bn), 112.5 (Cq
isoprop), 104.2 (C-1), 99.0 (2£Cq BDA), 94.6 (C-10), 85.8,
85.7 (2£Cq Tr), 77.4, 76.2, 69.5, 68.4, 59.8 (C-2, C-3, C-4,
C-20, C-30, C-40, C-50), 71.7 (CH2 Bn), 65.5, 59.8 (C-5, C-
60), 47.9, 47.6 (2£OCH3 BDA), 26.5 (2£CH3 isoprop), 17.8,
17.3 (2£CH3 BDA). ES-MS: 1042 [M1H]1, 1064
[M1Na]1. HRMS (ES): Calcd C65H69O12 [M1H]1
1041.4799, found 1041.4794 (^ 0.0029).
0
0
1
(light petroleum/EtOAc, 3/1, v/v); H NMR (CDCl3): d
7.44±7.22 (m, 5H, H arom), 5.96±5.76 (m, 2H, H-5, H-
60), 5.80 (d, 1H, H-1, J1,2ˆ3.7 Hz), 5.52±5.22 (m, 4H, H-
(200S,300S)
3-O-(20-O-Benzyl-6-O-t-butyldiphenylsilyl-
6, H-70), 5.14 (d, 1H, H-10, J1 ,2 ˆ3.7 Hz), 4.79 (AB, 2H,
CH2 Bn, Jˆ212.4 Hz), 4.71 (dd, 1H, H-2, J2,3ˆ4.4 Hz),
4.57 (dd, 1H, H-3, J3,4ˆ9.5 Hz), 4.14 (m, 2H, H-40, H-30),
0
0
30,40-di-O-(200,300-dimethoxybutane-200,300-diyl)-a-d-gluco-
pyranosyl)-1,2-O-isopropylidene-5-O-t-butyldiphenylsilyl-
a-d-ribofuranoside (24). Condensation of compound 21
(0.43 g, 1.00 mmol) with thioglucoside 19 (0.76 g,
3.72 (dd, 1H, H-50, J5 ,6 ˆ4.4 Hz, J4 ,5 ˆ9.5 Hz), 3.62 (dd,
0
0
0
0
1H, H-20, J2 ,3 ˆ9.3 Hz), 3.42 (t, 1H, H-4 , J3 ,4 ˆ9.5 Hz),
0
0
0
0
0

5924
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
3.32, 3.21 (2£s, 6H, 2£OCH3 BDA), 1.56, 1.36, 1.35, 1.31
(4£s, 12H, 4£CH3 isoprop, BDA); 13C{1H} NMR (CDCl3):
d 138.9 (Cq Ph), 134.3 (C-5), 133.6 (C-60), 128.0±127.2
(CH arom), 118.6 (C-70), 118.0 (C-6), 112.9 (Cq isoprop),
103.8 (C-1), 99.6, 99.3 (2£Cq BDA), 95.7 (C-10), 78.8,
78.2, 76.6, 76.2, 70.4, 69.8, 69.2 (C-2, C-3, C-4, C-20,
C-30, C-40, C-50), 71.5 (CH2 Bn), 48.1, 47.7 (2£OCH3
BDA), 26.6 (2£CH3 isoprop), 17.8, 17.6 (2£CH3 BDA);
ES-MS: 566 [M1NH4]1, 571 [M1Na]1, 587 [M1K]1.
HRMS (ES): Calcd C29H41O10 [M1H]1 549.2710, found
549.2714 (^ 0.0019).
colorless oil. Yield 0.59 g (0.93 mmol, 89%). Rf 0.90
(EtOAc); 1H NMR (300 MHz, H±H-COSY, CDCl3): d
7.42±7.24 (H arom), 5.97±5.83 (m, 2H, 2£CH allyl), 5.80
(d, 1H, H-1, J1,2ˆ3.7 Hz), 5.31±5.13 (m, 4H, 2£CH2CH
allyl), 5.18 (d, 1H, H-10, J1 ,2 ˆ4.4 Hz), 4.77 (AB, 2H,
CH2 Bn, Jˆ212.4 Hz), 4.70 (t, 1H, H-2, J2,3ˆ4.0 Hz),
4.29 (ddd, H-4, J3,4ˆ9.3 Hz, J4,5aˆ1.9 Hz, J4,5bˆ4.0 Hz),
4.18±3.92 (m, 8H, H-3, H-30, H-40, H-50, 2£OCH2 allyl),
3.81 (m, 1H, H-6a0), 3.75 (dd, 1H, H-5a, J5a,5bˆ211.2 Hz),
3.65±3.51 (m, 3H, H-20, H-6b0, H-5b), 3.32, 3.25 (2£s, 6H,
2£OCH3 BDA), 1.53, 1.35, 1.31 (3£s, 12H, 4£CH3 isoprop,
BDA). 13C{1H} NMR (CDCl3): d 138.7 (Cq Bn), 134.4,
134.2 (2£CH allyl), 127.8, 127.1 (CH arom Bn), 116.9
(2£CH2vCH allyl), 112.7 (Cq isoprop), 104.0 (C-1),
99.4, 99.3 (2£Cq BDA), 95.6 (C-10), 77.4, 76.3, 75.9,
73.1, 69.2, 68.9, 65.5 (C-2, C-3, C-4, C-20, C-30, C-40,
C-50), 72.3, 71.8 (CH2 Bn, OCH2 allyl), 67.8, 67.2 (C-60,
C-5), 48.0, 47.7 (2£OCH3 BDA), 26.5 (2£CH3 isoprop),
17.5, 17.5 (2£CH3 BDA); ES-MS: 659 [M1Na]1. [a]D20ˆ
1135.48 (c 2.0 CHCl3). HRMS (ES): Calcd C33H49O12
[M1H]1 637.3223, found 637.3219 (^ 0.0027). C33H48O12
(636): Calcd C 62.25, H 7.60; found C 62.45, H 7.64.
0
0
(200S,300S) 3-O-(60-O-Allyl-20-O-benzyl-30,40-di-O-(200,300-
dimethoxybutane-200,300-diyl)-a-d-glucopyranosyl)-5,6-
dideoxy-1,2-O-isopropylidene-a-d-allo-hex-5-enofurano-
side (27). To a cooled solution (08C) of dimer 23 (0.20 g,
0.36 mmol) in DMF (3 mL) was added sodium hydride
(60% wt, 22 mg, 0.54 mmol). After stirring for 15 min,
allyl bromide (34 mL, 0.40 mmol) was added and the reac-
tion mixture allowed to stir for 3 h after which it was
quenched with MeOH (0.3 mL). The mixture was diluted
with Et2O (5 mL) H2O (15 mL) was added and the DMF/
H2O layer was extracted with Et2O. The organic layer was
rinsed with H2O (2£5 mL), dried over MgSO4, ®ltered and
concentrated in vacuo. After puri®cation by column
chromatography (Et2O/light petroleum, 1/3, v/v) compound
27 was obtained as a white foam. Yield 0.15 g (0.25 mmol,
General procedure for ring-closing metathesis
Residual H2O was removed from the diene (1.0 mmol) by
coevaporation with dry toluene (3£5 mL), after which it
was dissolved in dry toluene (40 mL). The solution was
degassed by bubbling through with argon for 20 min. Cata-
lyst 29 (41 mg, 5 mol%) was added and degassing was
continued for 20 min, after which the solution was stirred
overnight under an argon atmosphere. When TLC analysis
indicated termination of the reaction, the reaction mixture
was concentrated and the product was puri®ed by column
chromatography (light petroleum/Et2O, 8/1!1/1, v/v).
1
70%). Rf 0.41 (Et2O/light petroleum, 1/1, v/v); H NMR
(CDCl3): d 7.43±7.24 (m, 5H, H arom), 5.95±5.73 (m,
2H, H-5, CH allyl), 5.79 (d, 1H, H-1, J1,2ˆ3.7 Hz), 5.51±
5.18 (m, 4H, H-6, CH2vCH allyl), 5.14 (d, 1H, H-10,
0
0
J1 ,2 ˆ4.4 Hz), 4.78 (AB, 2H, CH2 Bn, Jˆ212.4 Hz), 4.70
(t, 1H, H-2, J2,3,ˆ3.9 Hz), 4.55 (m, 1H, H-3), 4.17±3.97 (m,
3H, H-4, H-30, OCH2 allyl), 3.80±3.60 (m, 6H, H-20, H-40,
H-50, H-60, OCH2 allyl), 3.31, 3.25 (2£s, 6H, 2£OCH3
BDA), 1.55, 1.35, 1.33, 1.31 (4£s, 12H, 4£CH3 isoprop,
BDA); 13C{1H} NMR (CDCl3): d 138.9 (Cq Bn), 134.4,
134.3 (C-5, OCH2 allyl), 128.0, 127.2 (CH arom Bn),
118.5 (C-6), 116.9 (CH2vCH allyl), 112.8 (Cq isoprop),
103.8 (C-1), 99.5, 99.3 (2£Cq BDA), 95.8 (C-10), 78.7,
78.2, 76.5, 76.0, 69.2, 69.0, 65.8 (C-2, C-3, C-4, C-20,
C-30, C-40, C-50), 72.4, 71.9 (CH2 Bn, CH allyl), 67.5
(C-60), 48.0, 47.8 (2£OCH3 BDA), 26.6 (2£CH3 isoprop),
17.8, 17.6 (2£CH3 BDA); ES-MS: 594 [M1H]1, 516
[M1Na]1. HRMS (ES): Calcd C31H45O11 [M1H]1
593.2972, found 593.2969 (^ 0.0017).
Dimer of 27. Attempted ring-closing metathesis of diene 27
(100 mg, 0.17 mmol) according to the general procedure
yielded a single isomer of dimer 30 as a brownish oil.
1
Yield 66 mg (0.12 mmol, 69%). Rf 0.83 (Et2O); H NMR
(CDCl3): d 7.43±7.27 (m, 10H, H arom Bn), 5.89±5.73 (m,
4H, H-5, CH allyl), 5.80 (d, 2H, H-1, J1,2ˆ3.7 Hz), 5.50±
5.23 (m, 4H, H-6a, H-6b), 5.14 (d, 2H, H-10, J1 ,2 ˆ3.7 Hz),
4.77 (AB, 4H, CH2 Bn, Jˆ212.4 Hz), 4.69 (t, 2H, H-2,
J2,3ˆ3.9 Hz), 4.57 (dd, 2H, H-3), 4.09 (t, 2H, H-30,
0
0
0
0
0
0
J3 ,4 ˆJ2 ,3 ˆ9.7 Hz), 4.00±3.98 (m, 4H, H-4, OCH2 allyl),
3.78±3.43 (m, 12H, H-20, H-40, H-50, H-6a0, H-6b0, OCH2
allyl), 3.31, 3.23 (2£s, 12H, 2£OCH3 BDA), 1.55, 1.35,
1.34, 1.30 (4£s, 24H, CH3 isoprop, BDA); 13C{1H} NMR
(CDCl3): d 138.9 (Cq Ph), 134.4 (C-5, CH allyl), 128.0,
127.6, 127.2 (CH arom Bn), 118.6 (C-6), 112.9 (Cq
isoprop), 103.8 (C-1), 99.6, 99.3 (2£Cq BDA), 95.9 (C-
10), 79.6, 78.7, 76.5, 76.0, 69.2, 69.0, 65.8 (C-2, C-3, C-4,
C-20, C-30, C-40, C-50), 72.4 (OCH2 allyl), 71.3 (CH2 Bn),
67.6 (C-60), 48.1, 47.8 (2£OCH3 BDA), 26.6 (2£CH3
isoprop), 17.8, 17.7 (2£CH3 BDA). ES-MS: 1158
[M1H]1, 1170 [M1Na]1. HRMS (ES): Calcd C60H85O22
[M1H]1 1157.5542, found 1157.5549 (^ 0.0028).
(200S,300S) 5-O-Allyl-3-O-(60-O-allyl-20-O-benzyl-30,40-di-
O-(200,300-dimethoxybutane-200,300-diyl)-a-d-glucopyrano-
syl)-1,2-O-isopropylidene-a-d-ribofuranoside (28). Com-
pound 24 (1.14 g, 1.10 mmol) was dissolved in a mixture of
1,4-dioxane (15 mL) and a 1.0 M solution of TBAF in THF
(3.29 mL). After stirring overnight at 508C the mixture was
concentrated and the oily residue was dissolved in EtOAc
(25 mL). The solution was rinsed with sat. aq. NaCl
(3£10 mL) and H2O (10 mL). The organic layer was dried
(MgSO4) and concentrated. Puri®cation was effected by
column chromatography (EtOAc/MeOH, 1/0!98/2, v/v)
to give diol 25 as a white foam. Yield 0.59 g (1.04 mmol,
95%). Rf 0.18 (EtOAc). Allylation of compound 25 (0.57 g,
1.02 mmol) was executed as described for the preparation of
27. The crude product was puri®ed by column chroma-
tography (Et2O/light petroleum, 1/6, v/v), to afford 28 as a
(E/Z, 200S,300S) 5,60-Di-O-but-2-en-1,4-diyl-3-O-(20-O-
benzyl-30,40-di-O-(200,300-dimethoxybutane-200,300-diyl)-a-
d-glucopyranosyl)-1,2-O-isopropylidene-a-d-ribofurano-
side (31). Ring-closing metathesis of diene 28 (0.30 g,

M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
5925
0.47 mmol) was effected according to the general procedure
to give a 2:1 mixture of E/Z-isomers of 14-membered
macrocycle 31. The light brownish oily product was used
in the next reaction without puri®cation. Combined yields
0.21 g (0.32 mmol, 75%). Rf 0.53 and 0.42 (Et2O). The
higher-running product was the major isomer: H NMR
(600 MHz, H±H-COSY, CDCl3): d 7.44±7.23 (m, 5H, H
arom Bn), 5.89 (m, 2H, H-b, H-c), 5.78 (d, 1H, H-1,
1,2-Di-O-acetyl-3-O-(30,40-di-O-acetyl-20-O-benzyl-b-d-
glucopyranosyl)-5,60-di-O-(butane-1,4-diyl)-d-ribofurano-
side (33). Compound 32 (0.15 g, 0.24 mmol) was dissolved
in a solution of AcOH/H2O/(CH2OH)2, 14/6/3, v/v/v (7 mL)
and was re¯uxed for 90 min. TLC analysis (MeOH/EtOAc,
8/92, v/v) indicated the appearance of one product (Rf 0.42).
The reaction mixture was concentrated under reduced
pressure, coevaporated with toluene (3£5 mL) and stirred
in a mixture of in pyridine/acetic anhydride (8 mL, 2/1, v/v).
The mixture was concentrated, coevaporated with toluene
(3£5 mL) and subjected to column chromatography (light
petroleum/EtOAc, 1/0!1/1, v/v) to yield 33 (138 mg,
0.22 mmol, 92%, a:b,1:9) as a white foam. Rf 0.15
(EtOAc/light petroleum, 1/1, v/v). 13C{1H} NMR
(CDCl3): d 169.9 (CˆO Ac), 138.5 (Cq Bn), 128.2, 127.7,
127.4 (CH arom Bn), 97.7 (C-1), 94.9 (C-10), 80.7, 76.7,
71.6, 70.9, 70.3, 69.6, 68.7 (C-2, C-3, C-4, C-20, C-30, C-40,
C-50), 77.6, 76.4, 72.3, 69.9, 76.6 (2£OCH2 n-Bu, C-5, C-60,
CH2 Bn), 26.3 (2£CH2 n-Bu), 20.6 (4£CH3 Ac). ES-MS:
565 [M2OAc]1, 647 [M1Na]1, 1272 [2M2Na]1. HRMS
(ES): Calcd C30H41O14 [M1H]1 625.2506, found 625.2508
(^ 0.0016).
1
J1,2ˆ3.7 Hz), 5.12 (d, 1H, H-10, J1 ,2 ˆ4.2 Hz), 5.78 (AB,
2H, CH2 Bn, Jˆ212.1 Hz), 4.69 (t, 1H, H-20, J2,3ˆ4.0 Hz),
4.52 (dd, 1H, H-3, J3,4ˆ9.1 Hz), 4.27 (m, 2H, H-4, H-a),
0
0
4.17 (m, 1H, H-d), 4.14 (t, 1H, H-30, J2 ,3 ˆJ3 ,4 ˆ9.8 Hz),
0
0
0
0
4.06 (dt, 1H, H-50, J4 ,5 ˆJ5 ,6b ˆ9.9 Hz, J5 ,6a 1.5 Hz), 3.95
0
0
0
0
0
0
(dd, 1H, H-d0, Jc,d ˆ5.3 Hz, Jd,d ˆ210.2 Hz), 3.89 (dd, 1H,
0
0
H-a0, Ja ,bˆ5.3 Hz, Ja,a ˆ29.9 Hz), 3.83 (d, 1H, H-5a,
J5a,5bˆ211.2 Hz), 3.75 (dd, 1H, H-6a0, J6a,6bˆ212.0 Hz),
3.67 (dd, 1H, H-5b, J4,5bˆ1.8 Hz), 3.64 (dd, 1H, H-20),
3.59 (dd, 1H, H-6b0), 3.42 (t, 1H, H-40), 3.33, 3.21 (2£s,
6H, 2£OCH3 BDA), 1.50, 1.35, 1.34, 1.32 (4£s, 12H,
4£CH3 isoprop, BDA); 13C{1H} NMR (CDCl3): d 139.0
(Cq Ph), 130.5, 129.9 (C-b, C-c), 128.0±127.0 (CH arom),
112.8 (Cq isoprop), 104.2 (C-1), 99.7, 99.4 (2£Cq BDA),
95.4 (C-10), 77.6, 76.2, 71.9, 69.5, 67.7, 67.4 (C-2, C-3, C-4,
C-20, C-30, C-40, C-50), 71.8, 69.4, 67.2, 66.4, 66.0 (C-a,
C-d, C-5, C-60, CH2 Bn), 48.3, 47.8 (2£OCH3 BDA),
26.8, 26.6 (2£CH3 isoprop), 17.9, 17.7 (2£CH3 BDA);
ES-MS: 632 [M1Na]1. C31H44O12 (609): Calcd C 61.17,
H 7.29; found C 61.11, H 7.35.
0
0
20-O-Acetyl-6-N-benzoyl-50,600-di-O-(n-butane-1,4-diyl)-
30-O-(300,400-di-O-acetyl-200-O-benzyl-a-d-glucopyranosyl)-
adenosine (34). A suspension of 6-N-benzoyladenine
(0.14 g, 0.60 mmol) in 1,1,1,3,3,3-hexamethyldisilazane
(1.1 mL) and pyridine (0.5 mL) was re¯uxed for 7 h under
an Ar atmosphere. The reaction mixture was cooled, diluted
with toluene (5 mL) and concentrated in vacuo under
careful exclusion of H2O. The residual oil was diluted
with toluene (3£5 mL) and concentrated in vacuo to remove
excess 1,1,1,3,3,3-hexamethyldisilazane. Tetraacetate 33
(0.13 g, 0.20 mmol) in (CH2Cl)2 (5 mL) and a catalytic
amount of TMSOTf (8 mL, 25 mol%) were added to the
silylated 6-N-benzoyladenine. After stirring for 16 h at
re¯ux temperature TLC analysis showed conversion of 33
into a lower-running product. The reaction mixture was
quenched with Et3N (0.25 mL), diluted with CH2Cl2
(10 mL) and poured into sat. aq. NaHCO3 (5 mL). The
organic phase was washed with H2O (5 mL), dried
(MgSO4), ®ltered and concentrated under reduced pressure.
The crude product was puri®ed by column chromatography
(CH2Cl2/MeOH, 1/0 to 95/5, v/v). Concentration of the
appropiate fractions yielded 34 as a yellowish foam. Yield
0.13 g (0.16 mmol, 82%); Rf 0.65 (EtOAc/MeOH, 95/5,
(200S,300S) 3-O-(20-O-Benzyl-30,40-di-O-(200,300-dimethoxy-
butane-200,300-diyl)-a-d-glucopyranosyl)-5,60-di-O-(butane-
1,4-diyl)-1,2-O-isopropylidene-a-d-ribofuranoside (32).
A mixture of E/Z isomers of 31 (0.21 g, 0.32 mmol) was
dissolved in EtOAc (10 mL) and the solution was degassed
and placed under a blanket of N2. A catalytic amount of
PtO2 (7 mg, 10 mol%) was added, the mixture was degassed
once more and stirred under a H2-atmosphere. TLC analysis
revealed complete conversion of 31 after 25 min into a
single higher-running product. The reaction mixture was
®ltered over Glass Fiber (GF/2A, Whatmanw) and concen-
trated in vacuo to give 32 in quantitative yield (0.21 g,
1
0.32 mmol). Rf 0.64 (Et2O) as a brownish oil. H NMR
(CDCl3, 300 MHz, H±H-COSY): d 7.43±7.22 (m, 5H, H
arom), 5.83 (d, 1H, H-1, J1,2ˆ3.8 Hz), 5.17 (d, 1H, H-10,
0
0
J1 ,2 ˆ4.2 Hz), 4.79 (AB, 2H, CH2 Bn, Jˆ212.4 Hz), 4.74
(dd, 1H, H-2, J2,3ˆ4.5 Hz), 4.52 (dd, 1H, H-3, J3,4ˆ9.1 Hz),
4.25 (dd, 1H, H-4, J4,5aˆ1.4 Hz), 4.15±4.04 (m, 3H, H-30,
H-50), 3.86 (dd, 1H, H-5a, J5a,5bˆ211.6 Hz), 3.74 (dd, 1H,
1
v/v); H NMR (CDCl3, 600 MHz, H±H-COSY): d 9.05 (s,
1H, NH), 8.81 (s, 1H, H-2), 8.63 (s, 1H, H-8), 8.02, (d, 2H,
H-6a0, J6a ,6b ˆ211.7 Hz, J5 ,6b ˆ1.6 Hz), 3.65 (dd, 1H,
arom Bz, Jˆ7.4 Hz), 7.60 (t, 1H, arom Bz, Jˆ7.4 Hz), 7.52
0
0
0
0
H-20, J2 ,3 ˆ10.1 Hz), 3.61±3.41 (m, 7H, H-5b, H-4 ,
H-6b0, OCH2 n-Bu), 3.33, 3.21 (2£s, 6H, 2£OCH3 BDA),
1.88, 1.63 (m, 4H, CH2 n-Bu), 1.49, 1.35, 1.32 (4£s, 12H,
4£CH3 isoprop, BDA); 13C{1H} NMR (CDCl3): d 138.8
(Cq Ph), 127.9±126.9 (CH arom), 112.7 (Cq isoprop),
104.0 (C-1), 99.5, 99.3 (2£Cq BDA), 95.2 (C-10), 78.0,
76.2, 71.9, 69.6, 67.3 (C-2, C-3, C-4, C-20, C-30, C-40,
C-50), 71.8, 70.0, 69.1, 68.7, 67.0 (2£OCH2 n-Bu, C-5,
C-60, CH2 Bn), 48.1, 47.7 (2£OCH3 BDA), 26.6 (2£CH3
isoprop), 25.8, 25.5 (2£CH2 n-Bu), 17.7, 17.5 (2£CH3
BDA). [a]2D0ˆ1138.68 (c 1.0 CHCl3). ES-MS: 634 [M1
Na]1. HRMS (ES): Calcd C31H47O12 [M1H]1 611.3067,
found 611.3072 (^ 0.0019). Calcd C 57.69, H 6.45; found
C 57.82, H 6.53.
(t, 2H, arom Bz, Jˆ7.8 Hz), 7.38±7.28 (m, 5H, H arom Bn),
0
0
0
0
6.27 (d, 1H, H-10, J1 ,2 ˆ1.8 Hz), 5.88 (dd, 1H, H-2 , J2 ,3
ˆ
0
0
0
0
4.7 Hz), 5.44 (t, 1H, H-300, J2 ,3 ˆJ3 ,4 ˆ9.6 Hz), 5.07 (d,
00 00
00 00
1H, H-100, J1 ,2 ˆ3.7 Hz), 4.86 (dd, 1H, H-3 , J3 ,4
ˆ
0
00 00
0
0
7.9 Hz), 4.71 (t, 1H, H-400, J4 ,5 ˆ9.8 Hz), 4.60 (AB, 2H,
00 00
CH2 Bn, Jˆ211.9 Hz), 4.48 (m, 1H, H-40), 4.01 (dd, 1H,
H-5a0, J5a ,5b ˆ211.2 Hz, J4 ,5a ˆ2.7 Hz), 3.97 (dt, 1H,
0
0
0
0
H-500, J5 ,6 ˆ2.0 Hz), 3.71 (dd, 1H, H-5b , J4 ,5b ˆ2.7 Hz),
0
00 00
0
0
3.69±3.67, 3.64±3.60 (2£m, 2H, H-a/d), 3.55 (1H, dd,
H-200, J2 ,3 ˆ10.0 Hz), 3.50±3.42 (m, 2H, H-6a , H-a/d),
00
00 00
3.39 (1H, dd, H-6b00, J6a ,6b ˆ211.0 Hz), 2.04, 1.92, 1.87
(3£s, 9H, CH3 Ac), 1.94±1.82, 1.74±1.69 (2£m, 4H, H-b,
H-c); 13C{1H} NMR (150 MHz, C±H-COSY, CDCl3): d
170.1, 170.0, 168.8 (3£CvO Ac), 164.6 (CvO Bz),
00
00

5926
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
00 00
152.3, 151.1, 149.4, 141.8 (C-4, C-6, C-2, C-8), 137.6 (Cq
Bn), 133.7 (Cq Bz), 132.7 (CH arom Bz), 128.3, 128.2,
127.9, 127.6 (CH arom), 123.5 (C-5), 95.9 (C-100), 87.8
(C-10), 81.3 (C-40), 76.9 (C-200), 73.8 (CH2 Bn), 72.8 (C-
20), 71.7 (C-300), 71.4 (C-30), 71.1, 70.7 (OCH2 n-Bu), 70.0
(C-600), 69.9 (C-500), 69.8 (C-400), 68.0 (C-50), 26.7, 26.3
(CH2 n-Bu), 20.8, 20.7, 20.4 (3£CH3 Ac); ES-MS: 827
[M1Na]1, 805 [M1H]1, 565 [M2ABz]1, 240 [ABz1
H]1. [a]D20ˆ143.88 (c 1.0 CHCl3). HRMS (ES): Calcd
C40H46N5O13 [M1H]1 804.3091, found 804.3082 (^
0.0029). Calcd C 59.77, H 5.64; found C 59.85, H 7.58.
(d, 1H, H-6a00, Jˆ211.7 Hz), 4.00 (t, 1H, H-500, J4 ,5
ˆ
00
00
00
00
J5 ,6b ˆ9.8 Hz), 3.86±3.81 (m, 2H, H-4 , H-2 ), 3.77 (dd,
1H, H-5a0, J4 ,5a ˆ3.9 Hz, J5a ,5b ˆ211.7 Hz), 3.69±3.61 (m,
3H, OCH2 n-Bu, H-6b00, H-5b00), 3.57±3.51, 3.47±3.44
(2£m, 3H, OCH2 n-Bu), 1.68±1.63, 1.56±1.52, 1.50±1.42
(3£m, 4H, 2£CH2 n-Bu); 13C{1H} NMR (D2O): d 148.1 (C-
4/C-6), 153.6 (C-2), 120.1 (C-5), 96.4 (C-100), 88.2 (C-10),
81.9 (C-40), 77.3, 74.0, 73.3, 72.8, 71.1 (C-20, C-30, C-200, C-
300, C-400, C-500), 70.7, 70.3, 70.0, 68.4, 69.5 (2£OCH2 n-Bu,
C-50, C-600), 26.3, 25.9 (2£CH2 n-Bu); 31P NMR (D2O,
242 MHz, P±H-COSY): d 4.44 (P-300, P-20), 2.92 (P-400);
0
0
0
0
ES-MS: 722 [M2H]2, 360.5 [M22H]22
,
691
50,600-Di-O-(n-butane-1,4-diyl)-30-O-(a-d-glucopyranosyl)-
adenosine 20,300,400-trisphosphateÐCyclophostin (14). To
a solution of glucopyranosyl adenosine 34 (83 mg,
0.10 mmol) in 1,4-dioxane (3 mL) was added a solution of
KO-tBu in MeOH (1 M, 4 mL). After stirring vigorously for
1 min, the reaction mixture was neutralized by the addition
of AcOH (0.23 mL, 4.0 mmol). The solution was poured
into sat. aq. NaHCO3 (5 mL) and the resulting mixture
which was extracted with CH2Cl2 (2£10 mL). The organic
phase was washed with H2O (3 mL), dried (MgSO4), ®ltered
and concentrated in vacuo. The crude deacetylated dimer
was used without puri®cation. Rf 0.43 (EtOAc/MeOH, 9/1,
v/v). ES-MS: 679 [M1H]1, 701 [M1Na]1. A mixture of
the triol (70 mg, 0.10 mmol) and N,N-diisopropylamino-
bis-[2-(methylsulfonyl)ethyl]-phosphine (0.23 g, 0.62
mmol) was coevaporated with 1,4-dioxane (2£5 mL). The
mixture was dissolved in CH2Cl2 (3 mL) and a solution of
1H-tetrazole (56 mg, 0.80 mmol) in CH3CN (3 mL) was
added. TLC analysis indicated the formation of a single
product (Rf 0.43, CH2Cl2/MeOH, 9/1, v/v) which was
complete after 30 min. Oxidation of the intermediate phos-
phite triesters was effected by the addition of t-BuOOH
(0.80 mL) at 08C. After 30 min the mixture was diluted
with CH2Cl2 (10 mL), washed with H2O (5 mL) and dried
(MgSO4). Concentration of the organic phase gave tris-
phosphate 35. Rf 0.14 (CH2Cl2/MeOH, 9/1, v/v). 31P{1H}
NMR (CDCl3): d 22.00, 22.39, 22.45. Crude 35 was
dissolved in a mixture of NaOH (4 M)/1,4-dioxane/MeOH
(1/14/5, v/v/v, 10 mL) and stirred for 16 h. The mixture was
neutralized with AcOH (0.06 mL), concentrated and the
product was puri®ed by gel-®ltration over a Fractogel
HW-40 column (elution: 0.15 M triethyl ammonium
carbonate). Concentration, coevaporation with MeOH/H2O
(3£5 mL, 4/1, v/v) and lyophilization of the appropriate
fractions afforded the 20-O-benzyl derivative of cyclo-
phostin in pure form. Yield 82 mg (56% over the three
steps from 34). Debenzylation was accomplished by dis-
solving the compound in H2O (10 mL) and stirring the solu-
tion in the presence of Pd-black (50 mg) and AcOH (2
drops) under a H2 atmosphere. After 16 h the catalyst was
removed by ®ltration over Glass Fiber (GF/2A, Whatmanw).
The ®ltrate was concentrated and lyophilized to give cyclo-
phostin 14 which was converted into the Na1-form by ion-
exchange with Dowexw 50Wx4 (Na1-form) and imino
diacetate resin (Chelexw, Na1 form). Lyophilization gave
14 as a white ¯uffy foam. Yield 45 mg (48 mmol). 1H NMR
(D2O, 600 MHz, H±H-COSY): d 8.39 (H-2), 8.25 (H-80)0,
[M23H12Na]2, 713 [M24H13Na]2. HRMS (ES):
Calcd C20H31N5O18P3 [M2H]2722.0877, found 722.0865
(^ 0.0025).
Propargyl 20-O-acetyl-30-O-(300,400-di-O-acetyl-200-O-ben-
zyl-a-d-glucopyranosyl)-50,600-di-O-(butane-1,4-diyl)-b-
d-ribofuranoside (36). Compound 33 (0.13 g, 0.21 mmol)
was coevaporated with (CH2Cl)2 and dissolved in (CH2Cl)2
(3 mL). Propargyl alcohol (18 mL, 0.31 mmol) and
Ê
activated molecular sieves (4 A) were added and the
mixture was stirred for 15 min under a blanket of N2.
TMSOTf (8 mL, 25 mol%) was added and after 6 h the
reaction mixture was ®ltered over Hy¯ow, diluted with
Et2O (10 mL) and washed with sat. aq. NaHCO3. The
organic layer was dried over MgSO4 and concentrated in
vacuo. The crude residue was puri®ed by column chroma-
tography (light petroleum/EtOAc, 3/1!0/1, v/v) to afford
acetylene derivative 34 as
a colorless oil (70 mg,
0.11 mmol, 54%). Rf 0.38 (EtOAc/light petroleum, 1/1,
v/v). H NMR (CDCl3): d 7.33±7.29 (m, 5H, H-arom Bn),
1
0
00
5.39 (t, 1H, H-300, J2 ,3 ˆJ3 ,4 ˆ9.5 Hz), 5.36 (d, 1H, H-2 ,
00 00
00 00
0
0
0
J2 ,3 ˆ4.4 Hz), 5.16 (s, 1H, H-1 ), 5.02 (d, 1H, H-1 ,
00
00
00 00
J1 ,2 ˆ3.6 Hz), 4.76±4.69 (m, 2H, H-4 , H-5 ), 4.59 (AB,
2H, CH2 Bn, Jˆ211.4 Hz), 4.32±4.22 (m, 3H, H-30, H-1
propargyl), 3.97 (ddd, 1H, H-40, J4 ,5 ˆ8.6 Hz, J4 ,5b
ˆ
0
0
0
0
1.1 Hz), 3.77 (dd, 1H, H-5a0, J5a ,5b ˆ211.7 Hz,
0
0
0
0
0
J4 ,5a ˆ2.9 Hz), 3.69±3.30 (m, 7H, H-5b , 2£OCH2 n-Bu,
H-600), 2.03, 1.92, 1.88 (3£s, 9H, CH3 Ac). 1.95±1.51 (m,
4H, 2£CH2 n-Bu); 13C{1H} NMR (CDCl3): d 170.0
(3£C(O) Ac), 137.5 (Cq Ph), 128.2±127.5 (CH arom),
102.2 (C-10), 95.5 (C-100), 80.3, 76.7, 73.2, 71.6, 69.8,
69.2 (C-20, C-30, C-40, C-200, C-300, C-400, C-500, CH pro-
pargyl), 73.1, 70.8, 70.6, 70.1, 68.6 (2£OCH2 n-Bu, C-50,
C-600, CH2 Bn, Cq propargyl), 53.3 (CH2 propargyl), 26.5,
25.9 (2£CH2 n-Bu), 20.6 (3£CH3 Ac). [a]2D0ˆ161.38 (c 1.0
CHCl3). HRMS (ES): Calcd C31H41O13 [M1H]1 621.2547,
found 621.2543 (^ 0.0021). Calcd C 59.99, H 6.50; found C
60.08, H 6.57.
n-Propyl 50,600-di-O-(n-butane-1,4-diyl)-30-O-(a-d-gluco-
pyranosyl)-b-d-ribofuranoside 20,300,400-trisphosphate
(15). Triacetate 36 (70 mg, 0.11 mmol) was dissolved in a
0.1 M solution of NaOMe in MeOH. After 1 h the mixture
was neutralized with Dowexw H1 which was subsequently
removed by ®ltration. The ®ltrate was concentrated and the
residual oil was dried by coevaparation with 1,4-dioxane
(3£5 mL). The thus obtained triol (56 mg, 0.11 mmol) and
dibenzyl N,N-diisopropyl phosphoramidite (0.14 mL,
0.68 mmol) were dissolved in (CH2Cl)2 (4 mL) and a solu-
tion of 1H-tetrazole (65 mg, 0.96 mmol) in CH3CN (2 mL)
was added under N2. After strirring for 30 min TLC analysis
6.44 (d, 1H, H-10, J1 ,2 ˆ2.3 Hz), 5.32 (d, 1H, H-1 ,
0
0
J1 ,2 ˆ4.1 Hz), 5.29 (ddd, 1H, H-2 , J2 ,3 ˆ5.0 Hz, 3JPˆ
0
00 00
0
0
7.9 Hz), 4.95 (dd, 1H, H-30, J3 ,4 ˆ5.9 Hz), 4.47 (m, 1H,
0
0
3
H-40), 4.28 (q, 1H, H-300, J2 ,3 ˆJ3 ,4 ˆ JPˆ9.2 Hz), 4.11
00 00
00 00

M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
5927
(Et2O/light petroleum, 1/1, v/v) showed complete con-
version of starting material into a higher-running product
(Rf 0.56, Et2O/light petroleum 1/1, v/v). The reaction
mixture was cooled (08C), t-butyl hydroperoxide (0.26
mL) was added and strirring was continued for 30 min
after which TLC analysis revealed complete disappearance
of the phosphite triester intermediate into a lower-running
product (Rf 0.24, Et2O). The reaction mixture was diluted
with EtOAc (10 mL), washed with H2O, dried over MgSO4,
®ltered and concentrated in vacuo. Compound 37 was
obtained as a colorless oil after puri®cation by column
chromatography (light petroleum/EtOAc, 3/1!0/1, v/v) in
a 60% yield (86 mg, 68 mmol). 31P{1H} NMR (CDCl3): d
23.2, 23.7, 23.9; ES-MS: 1275 [M1H]1, 1297 [M1
21 Ci mmol21; NEN) and adrenal cortex membranes;
incubations were stopped after 30 min at 48C by rapid
vacuum ®ltration.23 Non-speci®c binding was de®ned in
the presence of 10 mM IP3. The displacement isotherm of
each of the ligands (not shown) was used to obtain an
estimate of the IC50 values (see Table 3) using GraphPad
Prism and are given as 2log IC50 values (^s.e. mean).
45Ca21-Release experiments
Assays were performed using SH-SY5Y human neuro-
blastoma cells (passage 20±30) essentially as described
previously24 with certain modi®cations. Con¯uent mono-
layers of SH-SY5Y cells were washed and harvested
using 10 mM HEPES, 0.9% NaCl, 0.02% EDTA, pH 7.4
and recovered by centrifugation (400£g, 3 min). Cells were
resuspended in an `intracellular-like' buffer (ICB: 20 mM
HEPES, 135 mM KCl, 2.5 mM MgCl2, 2 mM ATP, 20 mM
CaCl2, pH 7.1; the free [Ca21] was buffered to 100±150 nM
by addition of EGTA) and centrifuged (400£g, 3 min), this
latter step was repeated and the ®nal cell pellet was gently
resuspended in ICB supplemented with an ATP regener-
ating system (10 mM phosphocreatine, 10 U mL21 creatine
phosphokinase) and permeabilization was achieved by
addition of 50 mg mL21 a-escin. After 2 min 1 mCi mL21
45Ca21 (1000 Ci mmol21, Amersham, Little Chalfont, UK)
was added and the permeabilized cell suspension was added
to ICB containing different concentrations of adenophostin
A (1), IP3 (4), cyclophostin 14, ribophostin 8 or cycloribo-
phostin 15. Incubations were continued for 2 min (IP3, 30 s)
and samples were then centrifuged (13,000£g, 3 min). A
silicone oil mixture (300 mL of Dow-Corning 556/550,
1:1, v/v) was then added to each tube and the samples
were recentrifuged (13,000£g, 3 min). The ICB and oil
were then aspirated, tubes inverted and allowed to drain
for $60 min before addition of 1.1 mL FloScint IV scintil-
lation cocktail (Packard Bioscience BV, Groningen, the
Netherlands). Samples were stored overnight in the dark
13
Na]1; C{1H} NMR (CDCl3): d 137.3 (Cq Bn-200), 136.4,
135.8, 135.7, 135.4 (6£Cq Bn), 128.7±127.6 (CH Bn),
102.3 (C-10), 94.5 (C-100), 80.4, 80.2, 79.1, 78.2, 77.1,
74.9, 72.7, 72.6 (C-200, C-300, C-400, C-500, C-20, C-30, C-40,
C-3 propargyl), 74.5, 71.9, 70.6 (C-2 propargyl, 2£OCH2
n-Bu), 69.9, 69.7, 69.6, 69.2, 69.1, 68.9 (7£CH2 Bn), 65.7,
65.4 (C-600, C-50), 53.1 (C-1 propargyl), 26.9, 25.7 (2£CH2
n-Bu). Trisphosphate 37 (86 mg, 68 mmol) was dissolved in
a mixture of 1,4-dioxane (5 mL), i-propanol (2.5 mL) and
H2O (1.25 mL) containing NaOAc (66 mg, 0.80 mmol). The
mixture was degassed and 10% Pd/C (50 mg), was added
and stirred under an atmosphere of H2 for 16 h. The catalyst
was removed by ®ltration over Glass Fiber (GF/2A,
Whatmanw) and the ®ltrate was concentrated. Puri®cation
was performed as described for the synthesis of cyclo-
phostin 14 to give derivative 15 as a white ¯uffy foam.
1
Yield 52 mg (57 mmol, 84%, Na1-form). H NMR (D2O,
600 MHz, H±H-COSY): d 5.15 (s, 1H, H-10), 5.11 (d, 1H,
0
H-100, J1 ,2 ˆ3.9 Hz), 4.55 (dd, 1H, H-2 , J2 ,3 ˆ4.4 Hz,
00 00
0
0
3
JPˆ8.5 Hz), 4.46 (dd, 1H, H-30, J3 ,4 ˆ9.7 Hz), 4.34 (q,
0
0
3
1H, H-300, J2 ,3 ˆJ3 ,4 ˆ JPˆ9.7 Hz), 4.27 (m, 1H, H-4 ),
0
00 00
00 00
4.02 (t, 1H, H-500, J4 ,5 ˆJ5 ,6b ˆ9.2 Hz), 3.91 (d, 1H,
00 00
00
00
H-6a00, Jˆ211.8 Hz), 3.89 (d, 1H, H-5a0, J5a ,5b
ˆ
0
0
211.6 Hz), 3.80 (q, 1H, H-400 3JPˆ9.6 Hz), 3.69 (dd, 1H,
before scintillation counting. The total releaseable 45Ca21
-
H-200), 3.67±3.56 (m, 6H, H-6b00, 2£OCH2 n-Bu, H-1a
n-Pr), 3.51 (dd, 1H, H-5b0, J4 ,5b ˆ4.5 Hz), 3.51 (m, 1H,
H-1b n-Pr), 1.79±1.74; 1.64±1.58 (2£m, 4H, 2£CH2
n-Bu), 1.55 (m, 2H, H-2 n-Pr), 0.86 (t, 3H, H-3 n-Pr,
0
0
pool was de®ned as that released by addition of 10 mM
ionomycin. Each release isotherm was used to obtain
estimates of the EC50 value, the slope factor (h) and the
maximum obtainable release (expressed as a % of the
total ionomycin-releaseable pool) using GraphPad Prism.
Jˆ7.7 Hz); C{1H} NMR (D2O): d 106.7 (C-10), 98.5
13
(C-100), 80.5 (C-40), 77.7, 75.8, 75.2, 71.8 (C-20, C-30,
C-200, C-300, C-400, C-500), 71.0, 70.9, 70.3, 69.9, 69.5
(2£OCH2 n-Bu, C-1 n-Pr, C-50, C-600), 26.2, 25.3 (CH2
n-Bu), 22.9 (C-2 n-Pr), 22.9 (C-3 n-Pr); 31P NMR (D2O,
242 MHz, P±H-COSY): d 2.35 (P-300), 1.35 (P-20), 1.11
(P-400); ES-MS: 647 [M2H]2, 669 [M22H1Na]2, 691
[M23H12Na]2, 713 [M24H13Na]2. HRMS (ES):
Calcd C19H38O19P3 [M2H]2 663.1219, found 663.1208
(^ 0.0023).
Molecular modeling
All calculations were run on IRIS workstations according to
Hotoda et al.7 Full details on the molecular dynamics
simulation will be published elsewhere.29
Acknowledgements
Biological Evaluation
The authors like to thank Rajendra Mistry (Department of
Cell Physiology & Pharmacology, University of Leicester)
for his excellent technical contribution to the IP3R binding
experiments and Cees Erkelens and Fons Lefeber for
recording NMR spectra. We thank Prof. Dr C. Altona for
helpful discussions. This research has been ®nancially
supported by the Council for Chemical Sciences of the
Netherlands Organization for Scienti®c Research (CW-
NWO).
3H-IP3 Displacement binding experiments
A `P2' fraction of bovine adrenal cortex was prepared as
described previously.27 Increasing concentrations of adeno-
phostin A (1),28 IP3 (4), cyclophostin 14, ribophostin 82c,13
and cycloribophostin 15 were incubated with a constant
3
amount of H-IP3 (approx. 9000 d.p.m. per assay; stock:

5928
M. de Kort et al. / Tetrahedron 56 (2000) 5915±5928
11. Demchenko, A.; Stauch, T.; Boons, G.-J. Synlett 1997, 818±
820.
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