Spirophostins: Conformationally restricted analogues of adenophostin A

Article abstract of DOI:10.1002/1521-3765(20000804)6:15<2696::AID-CHEM2696>3.0.CO;2-Y

The synthesis, biological evaluation, and molecular modeling of two conformationally restricted analogues of adenophostin A (1), denominated as spirophostin (3R)-10 and (3S)-11, as novel ligands for the D-myo-inositol 1,4,5-trisphosphate receptor (IP

Full text of DOI:10.1002/1521-3765(20000804)6:15<2696::AID-CHEM2696>3.0.CO;2-Y

FULL PAPER
Spirophostins: Conformationally Restricted Analogues of AdenophostinA
Martin deKort,[a] Anouk D. Regenbogen,[a] A. RobP. M. Valentijn, [a]
R. A. John Challiss,[b] Yoriko Iwata,[c] Shuichi Miyamoto,[c] Gijs A. vanderMarel,[a] and
Jacques H. vanBoom*[a]
Abstract: The synthesis, biological evaluation, and molecular modeling of two
conformationally restricted analogues of adenophostinA (1), denominated as
spirophostin (3R)-10 and (3S)-11, as novel ligands for the d-myo-inositol 1,4,5-
trisphosphate receptor (IP3R), is presented. These diastereoisomeric spiroketals are
synthesized by spiroketalization of d-glucose derivatives (2S)-15 and (2R)-16,
separation of the protected isomers (3R)-19 and (3S)-20, followed by phosphorylation
and deprotection. The spirophostins (3R)-10 and (3S)-11 display comparable
Keywords: adenophostinA ´ calci-
um release ´ carbohydrates ´ mo-
lecular modeling ´ spiro compounds
3
biological activity, with a H-IP3-displacing and Ca2‡-releasing potency less than IP3
and adenophostinA.
6 are full agonists of IP3 with ꢀtenfold reduced activity in
comparison with IP3 (4). On the other hand, the correspond-
ing adenine-containing acyclophostin[5] 7 is a pH-dependent
partial agonist and exhibits a binding affinity in the range of
IP3. These results indicate that the conformationally more
flexible analogue 7 cannot counterbalance the loss of Ca2‡-
releasing potency and that the orientation of the phosphate at
the 2'-position is of crucial importance for optimal binding of
1. This assumption is also endorsed by the observation that
ribophostin[6] 8, and the recently reported[7] furanophostin 9,
are ten times more potent than 5 and 6. It may therefore be
concluded that the high activity of adenophostinA (1) can be
ascribed to an optimal spatial arrangement of the 2'-phos-
phate and/or an additional cooperative interaction of the
adeninyl moiety with a region in the vicinity of the IP3 binding
site.[8] In order to study in detail the effect of the spatial
orientation of the 2'-phosphate on the biological activity, it
would be of interest to prepare a deadeninylated analogue of
adenophostinA in which the 2'-phosphate is part of a
constrained spiro[4.5]decane constellation.[9]
Introduction
The fungal metabolites adenophostinA and B (1 and 2,
Figure 1),[1] which are full agonists of the d-myo-inositol 1,4,5-
trisphosphate receptor (IP3R), show potencies ꢀ10 ± 100
times higher than IP3 (4). Interestingly, the observed 1000-
fold reduced binding affinity[1c] of the 2'-dephosphorylated
derivative 3, indicates that the 2'-phosphate contributes
significantly to the activity of adenophostinA (1). Moreover,
molecular modeling studies[2, 3] showed that this phosphate
occupies a slightly more remote position from the trans-3'',4''-
bisphosphate than in IP3 (4).
In order to get a better insight into the precise role of the 2'-
phosphate and the adenine function in adenophostinA (1),
several analogues have been synthesized and evaluated.[4±7]
The studies revealed that the hydroxyethyl glucosides[4] 5 and
[a] Prof. Dr. J. H. vanBoom, Dr. M. deKort,
A. D. Regenbogen, Dr. A. R. P. M. Valentijn, Dr. G. A. vanderMarel
Leiden Institute of Chemistry, Gorlaeus Laboratories Leiden Univer-
sity, P.O. Box 9502, 2300 RA Leiden
In this paper, we describe the synthesis of the diastereoiso-
meric spirophostins (3R)-10 and (3S)-11. In addition, the biolog-
ical activity of both analogues in terms of stereochemistry and
conformational properties is assessed by molecular modeling.
(The Netherlands)
Fax : (‡31)71-527-4307
E-mail: j.boom@chem.leidenuniv.nl
[b] Dr. R. A. J. Challiss
Department of Cell Physiology & Pharmacology
Maurice Shock Medical Sciences Building
University of Leicester, University Road, Leicester
LE1 9HN (UK)
Results and Discussion
The construction of the precursors of the target spiroketals
(3R)-10 and (3S)-11 from the known[10] ethyl (2'S,3'S)-2,6-
di-O-benzyl-3,4-di-O-(2',3'-dimethoxybutane-2',3'-diyl)-1-thio-
b-d-glucopyranoside (12) is presented in Scheme 1. Hydrol-
[c] Y. Iwata, Dr. S. Miyamoto
Exploratory Chemistry Research Laboratories
Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku
Tokyo 140-8710 (Japan)
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Chem. Eur. J. 2000, 6, No. 15

2696±2704
NH2
N
N
N
R1O
(HO)2(O)PO4"
(HO)2(O)PO 3"
HO
O
R1
OH
N
OH
4
R2
O
O
(HO)2(O)PO
(HO)2(O)PO
(HO)2(O)PO
OP(O)(OH)2
2'
OR2
(HO)2(O)PO
5
HO
HO 1
HO
OP(O)(OH)2
O
O
5 R1= R2= H
6 R1= CH2OH, R2= H,
7 R1= CH2OH, R2= CH2-adenin-9-yl
1 R1= H, R2= P(O)(OH)2
2 R1= Ac, R2= P(O)(OH)2
3 R1= R2 = H
4 IP3
R2
R1
HO
1
HO
R1
O
(HO)2(O)PO8
(HO)2(O)PO
O
O
O 5
HO
R2
(HO)2(O)PO
(HO)2(O)PO
9
OH
3
3
R2
(HO)2(O)PO
(HO)2
9
R1
O
HO
(O)PO
OP(O)(OH)2
8 HO
O
1O
(3R)-10 R1= OP(O)(OH)2, R2= H
(3S)-11 R1= H, R2= OP(O)(OH)2
8 R1= CH2OH, R2= OCH3
9 R1= R2= H
Alternative orientation
of (3R)-10 and (3S)-11
Figure 1. Structures of adenophostinA (1), analogues 3 ± 9, d-myo-inositol 1,4,5-trisphosphate IP3 (4), and the spirophostins (3R)-10 and (3S)-11.
Scheme 1. Reagents and conditions: i) 1. 0.1m NIS, TfOH CH2Cl2/THF/H2O, 75:1:1, v/v/v, 88%; 2. DMSO/Ac2O, 2 :1, v/v, 2 h, 708C, quant.;
ii) Allylmagnesium bromide (1.05 equiv), THF, À788C, 86 % (a:b ˆ 9:1); iii) cat. K2OsO4 ´ 2H2O, NMO (2.0 equiv), acetone/H2O, 4 :1, v/v, 1 h quant.
(a:b ˆ 9:1, (2S)-15:(2R)-16 ˆ 1:1).
Table 1. Spiroketalization of ketosugars (2S)-15 and (2R)-16.
ysis of 12 under the influence of N-iodosuccinimide (NIS) and
subsequent oxidation of the anomeric hydroxy group under
Entry Conditions
17 ‡ 18 [%] 19 ‡ 20 [%] a:b [a] 21 [%]
Albright ± Goldman conditions[11] afforded lactone 13 in 88%
yield over the two steps. Allylation of 13 with allylmagnesium
bromide gave ketoglycoside 14 as a mixture of diastereoisom-
ers (a:b, 9:1).[12] Treatment of the resulting olefin 14 with a
catalytic amount of OsO4 in the presence of N-methylmor-
pholine N-oxide led to an inseparable mixture of the
protected nonulosides (2S)-15 and (2R)-16 (ratio ˆ 1:1;
a:b ˆ 9:1) in a quantitative yield.[13]
1
HOAc/H2O, 70/30,
63
0
9:1
0
reflux, 2 h
2
3
4
CSA/CH2Cl2, 4 h
CSA/MeOH, 16 h
TfOH/MeOH, reflux, 6 h
0
0
0
34
86
60
1:1 36
1:1
0
0
19:1
[a] a ˆ 5R; b ˆ 5S ; all entries ratio 3R:3S ˆ 1:1.
At this stage, it was of interest to find out whether (2S)-15
and (2R)-16 could be converted under acidic conditions[14]
into the suitably protected triols (3R)-17a and (3S)-18a,
required for the introduction of the phosphate groups. It can
be seen (entry 1 in Table 1) that treatment of (2S)-15 and
(2R)-16 with aqueous acetic acid led to spiroketalization and
concomitant removal of the butane 3,4-diacetal (BDA)
function, to give an intractable mixture of (3R)-17a and
(3S)-18a. Interestingly, subjection of (2S)-15 and (2R)-16 to a
catalytic amount of camphorsulfonic acid (CSA) under
anhydrous conditions in CH2Cl2 afforded the BDA-protected
isomers (3R)-19 and (3S)-20 and the unexpected furan[15]
derivative 21 in near equal amounts (entry 2, Table 1).
Gratifyingly, execution of the same spiroketalization in
MeOH as the solvent provided a separable mixture of the
diastereoisomers (3R)-19 and (3S)-20 in good yield (entry 3,
Table 1). In addition, the a-anomers of (3R)-19 and (3S)-20
were preferentially formed by executing the reaction at
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J. H. van Boom et al.
FULL PAPER
elevated temperature and using a more acidic catalyst (entry
4, Table 1). The identity of the individual spiroketals (3R)-19a
and (3S)-20a was firmly established by mass spectrometry as
well as 1H NMR-NOESY spectroscopy. In addition, the
configuration of HO-3 could be assigned unambiguously
by spectroscopic analysis (Scheme 2) of the corresponding
(R)- and (S)-Mosher ester (MTPA)[16] derivatives. Thus,
the Dd (ppm) values of the a-protons in the respective
MTPA-derivatives were in full accordance with those pre-
dicted on the basis of the observed nuclear Overhauser
effects.
were identified by 1H, 13C, and 31P NMR spectroscopy, as well
as ES mass spectrometry.
Biological evaluation: The binding affinities of spirophostin
(3R)-10 and (3S)-11 were compared with those of adeno-
phostinA (1) and IP3 (4) in 3H-IP3 displacement binding
experiments using bovine adrenal cortex membranes.[18] The
displacement curves and the estimated IC50 values of the four
compounds are displayed in Figure 2 and Table 2, respective-
Removal of the BDA protecting group (Scheme 3) in
spiro[4.5]decanes (3R)-19a and (3R)-19b with aqueous tri-
fluoroacetic acid (TFA) proceeded with concomitant isomer-
ization of the spirocenter to give (3R)-17a as a single
diastereoisomer. Similarly, conversion of the 3S isomers 20a
and 20b led also to the exclusive formation of (3S)-18a. The
three hydroxy functions in 17 and 18 were phosphitylated with
the monofunctional reagent dibenzyloxy-(N,N-diisopropyl-
amino)phosphine[17] (21), followed by in situ oxidation of the
resulting phosphite triesters with tert-butyl hydroperoxide, to
yield the fully benzylated trisphosphates 22 and 23. Purifica-
tion of the debenzylated products by HW-40 gel filtration and
Dowex-ion exchange chromatography gave the homogeneous
Figure 2. 3H-IP3 displacement isotherms for spirophostin (3R)-10 and (3S)-
11 using bovine adrenal cortex membrane IP3Rs. Data are expressed as
3
percentage displacements of specific H-IP3 binding (Æs. e. mean for four
‡
spiroketals (3R)-10 and (3S)-11 (Na -salt) (Scheme 3), which
experiments each performed in triplicate).
Scheme 2. Observed NOEs in spiroketals (3R)-19a and (3S)-20a and Dd ˆ values of the corresponding MTPA esters. Reagents and conditions: i) (R)-(À)-
or (S)-(‡)-2-methoxy-2-(trifluoromethyl)phenylacetic acid chloride (2.5 equiv), (CH2Cl)2/pyridine, mol. sieves (3 Š), 3 h; Dd ˆ dR À dS (ppm).
Scheme 3. Reagents and conditions: i) TFA/H2O, 95:5, v/v, 2 h, 73% for 17a, 69% for 18a; ii) 1. 21, 1H-tetrazole, (CH2Cl)2/CH3CN, 3:1, v/v, 30 min; 2. tBuOOH,
08C, 30 min, 60% for 22, 65% for 23; iii) 10% Pd/C, H2 (1 atm), NaOAc, 1,4-dioxane/propan-2-ol/H2O, 4 :2 :1, v/v/v, 16 h, 87% for (3R)-10, 57% for (3S)-11.
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Chem. Eur. J. 2000, 6, No. 15

Spirophostins
2696±2704
Table 2. Spirophostin (3R)-10 and (3S)-11 3H-IP3 displacement binding
IC50 values with respect to adenophostinA (1) and IP3 (4).[a]
Entry Compound
Àlog IC50
IC50 [nm]
h
n
1
2
3
4
adenophostinA (1)
IP3 (4)
9.409 Æ 0.072
8.075 Æ 0.062
0.39
8.4
1.51 Æ 0.13
0.99 Æ 0.04
0.82 Æ 0.01
0.90 Æ 0.03
3
8
4
4
spirophostin (3R)-10 6.626 Æ 0.022 237
spirophostin (3S)-11 6.848 Æ 0.012 142
[a] Values are shown as Æs. e. mean for the concentration which causes
50% of specific 3H-IP3 displacement (IC50), h the slope of the concen-
tration-response curve, for n experiments.
ly. These data clearly show that the spirophostins are
approximately 20-fold less effective than IP3, while the
relative displacing potencies of adenophostinA and IP3 are
consistent with earlier reported data. It is also evident (see
Table 2) that the IC50 for the S isomer 11 is roughly two times
lower than for the R isomer 10.
A functional response to the spirophostins was studied by
measuring 45Ca2‡ release from intracellular stores upon bind-
ing to IP3Rs in permeabilized SH-SY5Y neuroblastoma cells
in comparison with the activities of adenophostinA (1) and
IP3 (4). The potency (Table 3) and slope (h) of the concen-
tration-response curves (Figure 3) of (3R)-10 and (3S)-11 are
again quite similar to each other in agreement with the
Figure 3. 45Ca2‡-Release response curves for spirophostin (3R)-10 and
(3S)-11 using permeabilized SH-SY5Y neuroblastoma cells. Data are
expressed as percentage 45Ca2‡ release (Æs. e. mean for four experiments
each performed in duplicate).
binding data presented in Table 2 (see entries 3 and 4).
Interestingly however, the Ca2‡-releasing potency of spiro-
phostin (3R)-10 is higher than that of the corresponding
isomer (3S)-11, and the potency order is opposite to that
observed in the displacement experiments.
In summary, spirophostin (3R)-10 and (3S)-11 are approx-
imately equipotent with respect to their IP3R-binding and
Ca2‡-releasing properties, and both are significantly less
potent than IP3 (4) and adenophostinA (1) with respect to
their biological activity.
Table 3. Spirophostin (3R)-10 and (3S)-11 45Ca2‡-release EC50 values compared
with those of adenophostinA (1) and IP3 (4).[a]
Entry Compound
Àlog EC50 EC50
[nm]
h
% Release
n
Molecular modeling: In order to rationalize the outcome of
the biological experiments with respect to the three-dimen-
sional structure of both spirophostins, a comparative molec-
ular modeling study was undertaken. To this end, the energy-
minimized conformers of (3R)-10 and (3S)-11, as defined by
Hotoda et al.,[3] were compared with the structures of IP3 (4)
and adenophostinA (1).
1
2
3
4
adenophostinA (1) 8.15 Æ 0.04
7.03 1.51 Æ 0.08 80.3 Æ 5.4
4
4
4
4
IP3 (4)
6.68 Æ 0.09 208
0.99 Æ 0.04 74.7 Æ 6.9
1.10 Æ 0.11 73.8 Æ 4.3
1.02 Æ 0.14 74.8 Æ 3.6
spirophostin (3R)-10 5.88 Æ 0.06 1306
spirophostin (3S)-11 5.59 Æ 0.05 2594
[a] Values are shown as Æs. e. mean for the concentration which causes 50% of
maximal 45Ca2‡ release (EC50), with h as the slope of the concentration-response
curve, the% release is relative to ionomycin-induced Ca2‡ release, for n
experiments.
The conformational data as presented in Table 4 and
Figure 4 reveal that the distance between the trans-8,9-
H
H
H
O
P
O
O
O
O
H
H
H
4"
N
O
O
P
O
P
H
H
O
N
O
O
O
O
NH2
O
O
O
4
8
O
O
τb
O
O
P
3"
O
O
O
O
τc
τd
O
τ
O
H
O
H
O
aO
N
P
3
O
O
O
O
O
O
τa
τb
N
P
H
O
1
2'
τd
5
9
P
O
P
O
O
H
O
O
O
H
τc
O
O
H
O
O
P
H
H
O
O
H
O
θab
θbc
IP3
spirophostins
adenophostin A
Figure 4. Torsion angles t and directional angles qab and qbc assigned in the molecular modeling.
Table 4. Conformational data for adenophostinA (1), IP3 (4), spirophostin (3R)-10 and (3S)-11.
Distance between P atoms [Š]
Compound[a]
ta [8]
tb [8]
tc [8]
td [8]
1 ± 4 (2'-4ª)[b](3 ± 8)[c]
1 ± 5 (2'-3ª)[b](3 ± 9)[c]
4 ± 5 (3ª-4º)[b](8 ± 9)[c]
IP3 (4)
±
59
76
81
±
100
26
±
±
8.1
9.6
10.2
10.2
6.9
8.2
9.5
9.5
4.1
4.1
4.1
4.1
adenophostinA (1)
spirophostin (3R)-10
spirophostin (3S)-11
À 23
À 127
116
122
39
À 147
À 121
[a] Modeling of IP3 and adenophostinA: see ref. [3]; for spirophostins the conformers R-1 and S-1 were used (see Experimental Section). [b] Numbering for
adenophostinA. [c] Numbering for spirophostins.
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J. H. van Boom et al.
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Table 5. Distances [Š] and the directional angles q between P-3, P-1, and P-2' in
superimpositioned models of (3R)-10 and (3S)-11 with IP3 (4) and adenophostinA
(1).
bisphosphate moiety and the 3-phosphate (P-3) in both
spirophostins is increased in comparison with adenophostinA
and IP3. Superimposition of (3R)-10 with (3S)-11 (see Fig-
ure 5A) shows that the interplay between different puckering
of the five-membered ring in the spiroketals (i.e. C-5 exo in
(3R)-10 and O-1 endo/C-2 exo in (3S)-11) and an opposite
value of the torsion angles td results in nearly the same
arrangement of the phosphorous atoms, in which P-3 is at a
distance of 10.2 and 9.5 Š from P-8 and P-9, respectively (see
Table 4).
[b]
Compound
Superposition[a] P P distance [Š]
Direction of P-3
qbc [8][c]
À
3 ± 1
3 ± 2'
qab [8][c]
spirophostin (3R)-10 straight
2.7
4.6
2.8
4.5
1.8
4.7
2.0
4.6
61
37
30
74
36
37
37
51
alternative
spirophostin (3S)-11 straight
alternative
[a] Superimposition of six-membered rings, see Figure 5. [b] Left column: super-
imposition with IP3; right column: superimposition with AdenophostinA. [c] See
Figure 4.
angle td. On the other hand, the direction of the P-3 in (3S)-11,
À
as defined by the angle between the vectors along the O P
bonds (qab and qbc in Figure 4 and Table 5), is more parallel to
the direction of P-1 in IP3 than in the case of (3R)-10.
It has been suggested[19] that the side of adenophostinA (1)
À
which results from a 1808 rotation around the C-3'' C-4'' bond
in the glucose moiety would be responsible for binding to
IP3R. This alternative mode of binding is also not excluded for
the spirophostins (see Figure 1), the more so, as the spiro-
center mimics the axial HO-2 of IP3 which tolerates bulky
modifications.[20] However, alternative superimpositioning of
(3R)-10 and (3S)-11 with the straight binding mode of IP3
(Figure 5D) or adenophostinA reveals that in all cases the
distance between P-3 and P-1 or P-2' (see Table 5) is now
approximately twofold increased, and indicates that this
alternative mode of binding is highly unlikely.
In summary, based on the molecular modeling results it may
be concluded that by virtue of the opposite geometry of their
spiro[4.5]decane units, the phosphorous atoms at the C-3
stereogenic centers in (3R)-10 and (3S)-11 adopt similar
orientations in space (see Figure 5A).
Figure 5. Superimposition of the six-membered rings in the energy-
minimized structures. A. (3R)-10 with (3S)-11; B. (3R)-10 and (3S)-11 with
IP3 (4, in blue); C. (3R)-10 and (3S)-11 with adenophostinA (1, in blue); D.
Alternative orientation of (3R)-10 and (3S)-11 with IP3 (4, in blue).
The relative location of P-3 in spirophostins (3R)-10 and
(3S)-11 with respect to the corresponding phosphates P-1 and
P-2' in IP3 and adenophostinA, respectively, was determined
by comparison of the superimpositions of the respective
energy-minimized conformers (Figure 5B and 5C). Perusal of
Table 5 and Table 6 indicates that the distance between the
P-3 in (3R)-10 and the P-1 or P-2' is slightly smaller than for
the P-3 in (3S)-11. In this respect, it is of interest to note that a
more distinct positioning of the P-3 does not occur owing to
the high rotational energy barrier (>2 kcalmolÀ1) of torsion
Conclusion
The conformationally restricted adenophostinA analogues
(3R)-10 and (3S)-11 were successfully synthesized and eval-
uated for their biological activity. Molecular modeling showed
À
Table 6. P P distances in most stable conformations of spirophostins (3R)-10 and (3S)-11.
Stereo-Conf.No.
DE [kcalmolÀ1
]
Puckering of five-membered ring
P P distances [Š]
Distance between 3-P and 2'-P[a] [Š]
À
3 ± 8
3 ± 9
8 ± 9
Model B
Model C
R-1
R-7
R-17
R-44
R-68
R-106
0.0
0.4
0.9
1.7
2.0
2.6
C5-exo
10.2
10.3
10.3
10.3
9.8
9.5
9.5
9.3
9.0
8.0
8.9
4.1
4.1
4.1
4.1
3.9
4.1
Mean:
4.1
4.1
4.1
4.1
4.1
3.9
1.8
1.6
1.2
0.8
1.8
0.7
1.3
2.0
3.4
2.5
1.7
1.6
1.4
2.1
3.3
3.5
3.8
4.3
5.7
4.5
C5-exo/O1-endo
O1-endo/C5-exo
O1-endo
C2-exo-C3-endo
O1-endo/C2-exo
10.3
S-1
S-7
S-9
S-12
S-26
S-123
0.0
0.3
0.5
0.7
1.2
3.0
O1-endo/C2-exo
C5-exo/O1-endo
O1-endo
O1-endo-C2-exo
C2-exo/O1-endo
C2-exo
10.2
9.5
10.0
10.3
10.3
10.4
9.5
9.6
9.7
9.6
9.5
9.6
3.2
2.3
2.8
3.4
3.6
4.0
Mean:
[3]
[a] Distance between 3-P of spirophostin and 2'-P of adenophostin (Model B & C)
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when the glucose rings of both molecules were superimposed.
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Spirophostins
2696±2704
127.4, 126.8 (CH arom), 98.4 (2 Â Cq BDA), 76.8, 75.5, 68.5, 62.1 (C-2, C-3,
C-4, C-5), 73.2, 72.4 (2 Â CH2 Bn), 47.0 (2 Â CH3 OMe), 16.8 (2 Â CH3
BDA).
that the conformation of both spirophostins are quite similar
which could explain the small difference in their IP3R-binding
and Ca2‡-releasing properties. Moreover, the orientation of
P-3 in spirophostins (3R)-10 and (3S)-11 prevents optimal
interaction with the receptor, thus explaining the reduced
biological activities in comparison with adenophostinA (1)
and IP3 (4). The latter observation, together with other
reports,[3, 6, 7] suggests that the activity of IP3 cannot be
exceeded with conformationally restricted adenophostinA
analogues lacking the inextricable interplay between the P-2'
and the adenine moiety.
(2'S,3'S)-5,9-Di-O-benzyl-6,7-di-O-(2',3'-dimethoxybutane-2',3'-diyl)-1,2,3-
trideoxy-d-gluconon-1-en-4-ulopyranose (14): A solution of allylmagnesi-
um bromide in THF (1.0m, 4.55 mL) was added dropwise to a cooled
(À788C) solution of compound 13 (2.1 g, 4.5 mmol) in THF (45 mL) under
a N2 atmosphere. After stirring for 30 min the mixture was quenched with
aqueous NH4Cl (10%), extracted with EtOAc, and washed with H2O. The
organic layer was dried (MgSO4) and concentrated. Purification by column
chromatography (Et2O/light petroleum, 1:2, v/v) afforded allyl ketosugar
14 as a colorless oil. a/b ꢀ9:1. Yield 2.0 g, (3.9 mmol, 86%). Rf ˆ 0.82
(EtOAc/light petroleum, 3:1, v/v); 1H NMR (CDCl3, 300 MHz, HH-
COSY): d ˆ 7.38 ± 7.23 (m, 10H, H arom), 5.92 ± 5.78 (m, 1H, H-2), 5.15 (dd,
1H, H-1a, J1a,1b ˆ 2.1 Hz, J1a,2 ˆ 10.2 Hz), 5.08 (dd, 1H, H-1b, J1b,2
ˆ
17.2 Hz), 4.88 (AB, 2H, CH2 Bn, J ˆ À11.2 Hz), 4.59 (AB, 2H, CH2 Bn,
J ˆÀ 12.2 Hz), 4.19 (t, 1H, H-6, J5,6 ˆ J6,7 ˆ 9.8 Hz), 4.08 (ddd, 1H, H-8,
J7,8 ˆ 10.2 Hz, J8,9a ˆ 4.3 Hz, J8,9b ˆ 2.0 Hz), 3.76 (t, 1H, H-7), 3.73 (ABX,
2H, H-9, J9a,9b ˆ À11.3 Hz), 3.54 (d, 1H, H-5, J5,6 ˆ 9.7 Hz), 3.31, 3.22 (2  s,
Experimental Section
General methods and materials: CH2Cl2 and toluene were dried by
distillation from P2O5 (5 gLÀ1) and stored over molecular sieves 4 Š
(Acros). Et3N was refluxed for 2 h in the presence of CaH2 (5 gLÀ1) and
subsequently distilled. CH3CN (Rathburn), CHCl3, 1,4-dioxane, propan-2-
ol, DMF, DMSO (Baker), acetone, and THF (Acros), p. a. grade, were
stored over molecular sieves 4 Š. MeOH (HPLC-grade, Rathburn) was
stored over molecular sieves 3 Š. Acetic acid and acetic anhydride (p. a.,
Baker) were used as received. Butane-2,3-dione, camphorsulfonic acid,
trifluoromethanesulfonic acid, sodium hydride and 1H-tetrazole (Acros),
Dowex 50WX4, tert-butyl hydroperoxide (80% in di-tert-butyl peroxide),
(R)-(À)- and (S)-(‡)-2-methoxy-2-(trifluoromethyl)phenylacetic acid
chloride (Fluka), allylmagnesium bromide (1.0m in THF), N-iodosuccin-
imide and 10% Pd/C (Aldrich), benzyl bromide, and trifluoroacetic acid
(Merck) were used as received. Di-O-benzyl-(N,N-diisopropyl) phosphor-
amidite (21) was prepared as described.[17]
6H, 2  CH3 OMe), 2.91 (s, 1H, OH), 2.47 (dd, 2H, H-3, J2,3 ˆ 7.2 Hz, J1b,3
ˆ
0.8 Hz), 1.35, 1.30 (2  s, 6 H, 2  CH3 BDA); 13C{1H} NMR (CDCl3): d ˆ
138.0 (2 Â Cq Ph), 132.2 (C-2), 127.8 ± 126.9 (CH arom), 118.8 (C-1), 99.0
(2 Â Cq BDA), 97.8 (C-4), 77.5, 71.4, 69.7, 65.8 (C-5, C-6, C-7, C-8), 74.4, 72.8
(2 Â CH2 Bn), 67.8 (C-9), 47.5, 47.4 (2 Â CH3 OMe), 42.8 (C-3), 17.5, 17.3
‡
‡
‡
(2  CH3 BDA); MS (ES): 532 [M‡NH4] , 537 [M‡Na] , 553 [M‡K] ;
C29H38O8: calcd C 67.69, H 7.44; found: C 67.48, H 7.43%.
(3R) and (3S) (5S,7R,8R,9S,10R,2'S,3'S)-10-Benzyloxy-7-benzyloxymeth-
yl-8,9-di-O-(2',3'-dimethoxy-2',3'-dioxybutane-2',3'-diyl)-3-hydroxy-1,6-di-
oxaspiro[4.5]decane; (3R)-19a and (3S)-20a: To a solution of compound 14
(0.89 g, 1.7 mmol) in acetone/H2O (20 mL, 4:1, v/v) was added N-morpho-
line oxide (0.50 g, 3.4 mmol), followed by a catalytic amount of K2OsO4 ´
2H2O (HAZARDOUS!, 31 mg, 0.08 mmol). After the mixture had been
stirred for 1 h, TLC analysis revealed complete conversion of starting
material into a lower running product. The mixture was stirred over 1 h in
the presence of Na2SO3 (3 g) and extracted with Et2O. The organic layer
was washed with brine, H2O, and dried over MgSO4. Removal of the
solvents yielded an inseparable mixture of triols (2S)-15‡(2R)-16 (ratio
1:1) in quantitative yield that was used without purification in the next
reaction. Rf ˆ 0.33 (EtOAc/light petroleum, 3:1, v/v); MS (ES): 571
All experiments were performed under anhydrous conditions at room
temperature unless stated otherwise. Reactions were monitored by TLC
analysis conducted at Schleicher and Schüll DC Fertigfolien
(F 1500LS254). Compounds were visualized by UV light and by spraying
with 20% sulfuric acid in EtOH or ammonium molybdate (25 gLÀ1) and
ceric ammonium sulfate (10 gLÀ1) in 10% aqueous H2SO4, followed by
charring at 1408C. Column chromatography was performed on silica gel60,
0.063 ± 0.200 mm (Baker) or for crucial separations on silica gel60, 0.040 ±
0.063 mm (Merck). 1H NMR, 13C NMR, and 31P NMR spectra were
recorded with a JEOL JNM-FX-200 (200/50.1/80.7 MHz), a Bruker WM-
300 (300/75.1/121.0 MHz) or a Bruker DMX-600 spectrometer (600/150/
242.1 MHz). All spectra were recorded at 200/50.1/80.7 MHz, respectively,
unless otherwise stated. 1H and 13C chemical shifts are given in ppm (d)
relative to tetramethylsilane as internal standard and 31P chemical shifts
relative to 85% H3PO4 as external standard. Elemental analyses were
performed with a Perkin ± Elmer Series II Analyzer 2400. Mass spectra
were recorded on a Finnigan MAT TSQ-70 or a PE-SCIEX API 165 mass
spectrometer equipped with an Electrospray Interface (ES). Optical
rotations were measured at 589 nm with an automatic Propol polarimeter.
‡
‡
[MNa] , 587 [M‡K] . The crude mixture of triols (2S)-15‡(2R)-16
(0.34 g, 0.62 mmol) was treated according to entries 3 and 4 in Table 1.
TLC analysis (light petroleum/EtOAc, 1:3, v/v) indicated the conversion of
starting material into higher running products. In cases when CSA or TfOH
was used the reaction mixture was neutralized with Et3N prior to
concentration under reduced pressure. Separation of the isomers was
achieved by column chromatography (MeOH/EtOAc/toluene, 2.5:25:90,
v/v/v; silica gel 0.040 ± 0.063 mm) to give the two isomers in near equal
amounts. The 3R and 3S isomers were separable as mixtures of a/b
anomers. (3R)-19a: Yield 0.14 g (0.27 mmol, 44%); Rf ˆ 0.61 (toluene/
EtOAc/MeOH, 180:50:5, v/v/v); 1H NMR (600 MHz, H ± H COSY,
CDCl3): d ˆ 7.45 ± 7.24 (m, 10H, H arom), 4.87 (AB, 2H, CH2 Bn, J ˆ
À10.7 Hz), 4.56 (AB, 2H, CH2 Bn, J ˆ À12.2 Hz), 4.23 (t, 1H, H-9, J8,9
J9,10 ˆ 9.9 Hz), 4.17 (ddd, 1H, H-3, J2a,3 ˆ 2.1 Hz, J3,4a ˆ 5.1 Hz, JOH,3
ˆ
ˆ
(2'S,3'S)-2,6-Di-O-benzyl-3,4-di-O-(2',3'-dimethoxybutane-2',3'-diyl)-d-
glucono-1,5-lactone (13): To a vigorously stirred solution of ethyl (2'S,3'S)-
2,6-di-O-benzyl-3,4-di-O-(2',3'-dimethoxybutane-2',3'-diyl)-1-thio-b-d-glu-
copyranoside (12)[10] (2.9 g, 5.6 mmol) in CH2Cl2/H2O (35 mL, 10:1, v/v)
was added a solution of N-iodosuccinimide (7.5 mmol) and TfOH (50 mL,
0.56 mmol) in CH2Cl2/THF (40:1, v/v) in a dropwise manner. After 2 h the
reaction mixture was washed with aqueous Na2S2O3 (20%), aqueous
NaHCO3 (10%), brine, and H2O. The organic layer was dried (MgSO4) and
concentrated in vacuo. The residue was subjected to column chromatog-
raphy (Et2O/light petroleum, 1:2, v/v) to afford the glucopyranose
derivative. Yield 2.3 g, (4.9 mmol, 88%). Rf 0.34 (Et2O/light petroleum,
2:1, v/v). The product (2.3 g, 4.88 mmol) was coevaporated with toluene
(3 Â 10 mL) and stirred in a mixture of DMSO (18 mL) and acetic
anhydride (9 mL). After 2 h at 708C, when TLC analysis indicated the
complete conversion into a higher-running product, the reaction mixture
was concentrated and evaporated with toluene (3 Â 10 mL). The crude
product 13, susceptible to hydrate formation, was used immediately in the
next reaction without purification. Rf ˆ 0.92 (Et2O/light petroleum, 2:1,
v/v); 13C{1H} NMR (CDCl3): d ˆ 168.2 (C-1), 137.1, 137.0 (2  Cq Ph), 127.5,
11.6 Hz), 4.00 (ddd, 1H, H-7, J7,11b ˆ 1.9 Hz, J7,11a ˆ 4.2 Hz, J7,8 ˆ 10.3 Hz),
4
3.85 (dd, 1H, H-2a, J2a,2b ˆ À9.5 Hz, J2a,4b ˆ 1.4 Hz), 3.82 (dd, 1H, H-2b,
J2b,3 ˆ 2.2 Hz), 3.80 (t, 1H, H-8), 3.66 (ABX, 2H, H-11, J11a,11b ˆ À11.2 Hz),
3.58 (d, 1H, H-10), 3.52 (d, 1H, OH), 3.34, 3.21 (2 Â s, 6H, OMe BDA), 2.23
(dd, 1H, H-4a, J4a,4b ˆ À14.5 Hz), 2.01 (d, 1H, H-4b), 1.36, 1.29 (2  s, 6 H,
CH3 BDA); 13C{1H} NMR (CDCl3): d ˆ 138.1, 136.9 (2  Cq Ph), 128.9 ±
126.8 (CH arom), 107.2 (C-5), 99.4, 99.3 (2 Â Cq BDA), 77.8 (C-3), 75.8, 75.4
(2 Â CH2 Bn), 73.4 (C-2), 72.0, 71.9, 71.1, 66.0 (C-7, C-8, C-9, C-10), 68.0 (C-
11), 48.0 (2  CH3 OMe), 46.1 (C-4), 17.8, 17.6 (2  CH3 BDA); [a]2D0 ‡123.2
‡
‡
(c ˆ 1.0 CHCl3); MS (ES): 548 [M‡NH4] , 553 [M‡Na] ; C29H38O9: calcd
C 65.64, H 7.22; found: C 65.38, H 7.19.
(3S)-20a: 0.14 g (0.26 mmol, 42%); Rf ˆ 0.55 (toluene/EtOAc/MeOH,
1
180:50:5, v/v/v); H NMR (600 MHz, H ± H COSY, CDCl3): d ˆ 7.36 ± 7.25
(m, 10H, H arom), 4.82 (AB, 2H, CH2 Bn, J ˆ À11.6 Hz), 4.53 (AB, 2H,
CH2 Bn, J ˆ À12.2 Hz), 4.26 (m, 1H, H-3), 4.21 (t, 1H, H-9, J9,10 ˆ J8,9
ˆ
9.8 Hz), 4.16 (dd, 1H, H-2a, J2a,2b ˆ À9.9 Hz, J2a,3 ˆ 4.7 Hz), 4.07 (m, 1H,
H-7), 3.95 (d, 1H, H-2b), 3.68 ± 3.56 (m, 4H, H-8, H-11, H-10), 3.31, 3.21
(2  s, 6 H, 2  OMe BDA), 3.20 (d, 1H, OH, JOH,3 ˆ 11.0 Hz), 2.16 (dd, 1H,
Chem. Eur. J. 2000, 6, No. 15
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0615-2701 $ 17.50+.50/0
2701

J. H. van Boom et al.
FULL PAPER
H-4a, J4a,4b ˆ À13.4 Hz, J3,4a ˆ 5.7 Hz), 1.86 (d, 1H, H-4b), 1.36, 1.29 (2  s,
6H, CH3 BDA); 13C{1H} NMR (CDCl3): d ˆ 138.4, 137.4 (2  Cq Ph),
128.6 ± 127.6 (CH arom), 109.5 (C-5), 99.7 (2 Â Cq BDA), 79.2 (C-3), 75.4,
75.1, 75.0 (C-2, 2 Â CH2 Bn) 72.1, 71.9, 70.9, 66.2 (C-7, C-8, C-9, C-10), 66.2
(C-11), 48.1, 48.0 (2 Â CH3 OMe), 42.0 (C-4), 17.9, 17.7 (2 Â CH3 BDA);
1H, H-2a, J2a,2b ˆ À10.3 Hz, J2a,3 ˆ 6.1 Hz), 4.18 (t, 1H, H-9, J8,9 ˆ J9,10 ˆ
9.8 Hz), 3.86 (dd, 1H, H-2b, J2b,3 ˆ 3.1 Hz), 3.85 (t, 1H, H-8, J7,8 ˆ 9.9 Hz),
3.81 (ddd, 1H, H-7, J7,11a ˆ 1.8 Hz, J7,11b ˆ 3.0 Hz), 3.66 (ABX, 2H, H-11,
J10a,10b ˆ À13.6 Hz), 3.57 (d, 1H, H-10), 3.51 (s, 3H, OMe MTPA ester),
3.31, 3.21 (2  s, 6 H, 2  OMe BDA), 2.41 (dd, 1H, H-4a, J4a,4b ˆ À14.4 Hz,
J3,4a ˆ 7.8 Hz), 2.03 (dd, 1H, H-4b, J3,4b ˆ 1.3 Hz), 1.35, 1.28 (2  s, 6 H, 2 Â
‡
‡
[a]2D0 ‡106.8 (c ˆ 1.0 CHCl3); MS (ES): 548 [M‡NH4] , 553 [M‡Na] ;
‡
C29H38O9: calcd C 65.64, H 7.22; found: C 65.44, H 7.20.
CH3 BDA); MS (ES): 769 [M‡Na] .
S-Mosher ester derivative of (3S,5R,7R,8R,9S,10R,2'S,3'S)-10-benzyloxy-
7-benzyloxymethyl-8,9-di-O-(2',3'-dimethoxy-2',3'-dioxybutane-2',3'-diyl)-
3-hydroxy-1,6-dioxaspiro[4.5]decane: 1H NMR (600 MHz, H ± H COSY,
CDCl3): d ˆ 7.50 ± 7.21 (m, 15H, H arom), 5.36 (m, 1H, H-3), 4.88 (AB, 2H,
CH2 Bn, J ˆ À11.9 Hz), 4.46 (AB, 2H, CH2 Bn, J ˆ À12.2 Hz), 4.37 (dd,
(1R,2''S,3''S)-1,5-Di-O-benzyl-2,3-di-O-(2'',3''-dimethoxybutane-2'',3''-di-
yl)-1-(2'-furyl)-d-lyxitol (21): Spiroketalization of triols (2S)-15‡(2R)-16
(0.25 g, 0.46 mmol) according to the conditions listed in entry 2 of Table 1
gave, in addition to the spiroketals (3R)-19a and (3S)-20a, furan derivative
21 as a higher running product according to TLC analysis. Yield 36%
(85 mg, 0.17 mmol); Rf ˆ 0.82 (toluene/EtOAc/MeOH, 180:50:5, v/v/v);
1H NMR (200 MHz, CDCl3): d ˆ 7.41 ± 7.16 (m, 11H, H-5', H arom), 6.50 (d,
1H, H-2', J2',3' ˆ 3.4 Hz), 6.37 (dd, 1H, H-4', J3',4' ˆ 1.8 Hz), 4.91 (d, 1H, H-1,
J1,2 ˆ 2.9 Hz), 4.53 (AB, 2H, CH2 Bn, J ˆ À12.4 Hz), 4.49 (s, 2H, CH2 Bn),
1H, H-2a, J2a,2b ˆ À10.3 Hz, J2a,3 ˆ 6.0 Hz), 4.19 (t, 1H, H-9, J8,9 ˆ J9,10
ˆ
9.6 Hz), 3.93 (dd, 1H, H-2b, J2b,3 ˆ 3.7 Hz), 3.86 (m, 2H, H-8, H-7), 3.68
(ABX, 2H, H-11, J11a,11b ˆ À11.5 Hz, J7,11a ˆ 2.9 Hz, J7,11b ˆ 4.2 Hz), 3.56 (d,
1H, H-10), 3.46 (s, 3H, OMe MTPA ester), 3.32, 3.21 (2 Â s, 6 H, 2 Â OMe
BDA), 2.40 (dd, 1H, H-4a, J4a,4b ˆ À14.4 Hz, J3,4a ˆ 8.0 Hz), 1.99 (d, 1H,
4.18 (dd, 1H, H-3, J3,4 ˆ 5.7 Hz, J2,3 ˆ 9.6 Hz), 3.82 (dd, 1H, H-2, J1,2
ˆ
‡
H-4b), 1.35, 1.29 (2  s, 6 H, 2  CH3 BDA); MS (ES): 769 [M‡Na] .
2.9 Hz), 3.74 ± 3.55 (m, 3H, H-4, H-5), 3.23 (s, 3H, CH3 OMe), 3.21 (brs,
1H, OH), 2.91 (s, 3H, CH3 OMe), 1.28, 1.22 (2 Â s, 6 H, 2 Â CH3 BDA);
13C{1H} NMR (CDCl3): d ˆ 152.2 (C-2'), 141.5 (C-5'), 138.1, 137.9 (2  Cq
Ph), 128.6 ± 127.5 (CH arom), 110.4, 109.1 (C-3', C-4'), 99.0, 98.3 (2 Â Cq
BDA), 72.8, 71.7, 70.0, 68.3 (C-1, C-2, C-3, C-4), 73.1, 70.7, 70.2 (2 Â CH2 Bn,
C-5), 47.8, 47.5 (2 Â CH3 OMe), 17.4, 16.9 (2 Â CH3 BDA); MS (ES): 535
(3R,5S,7R,8R,9S,10R)-10-Benzyloxy-7-benzyloxymethyl-3,8,9-trihydroxy-
1,6-dioxaspiro[4.5]decane [(3R)-17a]: The a/b mixture of spiroketal (3R)-
19 obtained in entry 3, Table 1 (0.19 g, 0.36 mmol) was dissolved in aqueous
trifluoroacetic acid (95%, 8 mL) and was stirred for 2 h. The reaction
mixture was concentrated in vacuo and coevaporated with toluene (2 Â
5 mL). The oily residue was purified by column chromatography (light
petroleum/EtOAc, 1:1 ! 0:1, v/v) to give (3R)-17a as a white foam. Yield
73% (0.11 g, 0.26 mmol). 1H NMR (600 MHz, H ± H COSY, CDCl3): d ˆ
7.44 ± 7.25 (m, 10H, H arom), 4.90 (AB, 2H, CH2 Bn, J ˆ À11.0 Hz), 4.56
‡
‡
[M‡Na] , 551 [M‡K] ; C29H36O8: calcd C 67.95, H 7.08; found: C 68.08, H
7.06.
Preparation of the Mosher ester derivatives of (3R)-19a and (3S)-20a: The
BDA-protected spiroketal 19a or 20a (3.3 mg, 6.2 mmol) and (N,N-
dimethyl)aminopyridine (0.2 mg, 1.6 mmol) were coevaporated with freshly
distilled (CaH2) pyridine (2 Â 1 mL). The residue was dissolved in a mixture
of 1,2-dichloroethane and pyridine (3 mL, 2:1, v/v) which was concentrated
to a volume of 0.5 mL. The solution was stirred with molecular sieves (3 Š)
under an Ar atmosphere and subsequently (R)-(À)- or (S)-(‡)-2-methoxy-
2-(trifluoromethyl)phenylacetic acid chloride (2.9 mL, 16 mmol) was added
by syringe. After 3 h the reaction mixture was filtered over a layer of silica
gel (0.6 Â 6.0 cm) which was eluted with 1,2-dichloroethane (10 mL). The
solution was washed with H2O (5 mL), 2% aqueous NaHCO3 (5 mL), H2O
(5 mL), dried over MgSO4 and concentrated. Traces of organic solvents
were removed by coevaporation with carbon tetrachloride. The products
obtained in this way were of enough purity for analysis by NMR
spectroscopy. Note that the use of (R)-acid results in the formation of the
(S)-ester. See Scheme 2 for a summary of the spectroscopic analysis of the
Mosher esters.
(AB, 2H, CH2 Bn, J ˆ À12.2 Hz), 4.20 (m, 1H, H-3), 4.03 (t, 1H, H-9, J8,9
ˆ
J9,10 ˆ 9.3 Hz), 3.85 (dd, 1H, H-2a, J2a,2b ˆ À9.4 Hz, 4J2a,4b ˆ 1.4 Hz), 3.83 (m,
1H, H-7), 3.80 (dd, 1H, H-2b, J2b,3 ˆ 2.5 Hz), 3.64 (ABX, 2H, H-11, J7,11a
ˆ
4.3 Hz, J7,11b ˆ 4.2 Hz, J11a,11b ˆ À10.5 Hz), 3.57 (t, 1H, H-8, J7,8 ˆ 9.6 Hz),
3.40 (d, 1H, H-10), 3.38 (brs, 3H, OH), 2.16 (dd, 1H, H-4a, J4a,4b
ˆ
À14.5 Hz, J3,4a ˆ 5.4 Hz), 1.95 (dd, 1H, H-4b); 13C{1H} NMR (CDCl3):
d ˆ 136.8 (2  Cq Ph), 129.2 ± 127.7 (CH arom), 107.1 (C-5), 79.5, 75.6, 72.5,
71.8, 71.5 (C-3, C-7, C-8, C-9, C-10), 75.7, 75.5 (2 Â CH2 Bn), 73.6 (C-2), 70.0
‡
(C-11), 45.5 (C-4); MS (ES): 439 [M‡Na] ; [a]2D0: ‡8.8 (c ˆ 0.5 CHCl3);
C23H28O7: calcd C 66.33, H 6.78; found: C 66.39, H 6.77.
(3S,5S,7R,8R,9S,10R)-10-Benzyloxy-7-benzyloxymethyl-3,8,9-trihydroxy-
1,6-dioxaspiro[4.5]decane [(3S)-18a]: The a/b mixture of spiroketal (3S)-
20 obtained in entry 3, Table 1 (99 mg, 0.19 mmol) was converted into (3S)-
18a as described for the preparation of (3R)-17a. Yield 69% (55 mg,
1
0.13 mmol); H NMR (600 MHz, H ± H COSY, CDCl3): d ˆ 7.42 ± 7.23 (m,
R-Mosher ester derivative of (3R,5S,7R,8R,9S,10R,2'S,3'S)-10-benzyloxy-
7-benzyloxymethyl-8,9-di-O-(2',3'-dimethoxy-2',3'-dioxybutane-2',3'-diyl)-
3-hydroxy-1,6-dioxaspiro[4.5]decane: 1H NMR (600 MHz, H ± H COSY,
CDCl3): d ˆ 7.59 ± 7.21 (m, 15H, H arom), 5.50 (m, 1H, H-3), 4.76 (AB, 2H,
CH2 Bn, J ˆ À11.5 Hz), 4.55 (AB, 2H, CH2 Bn, J ˆ À12.1 Hz), 4.19 (t, 1H,
10H, H arom), 4.77 (AB, 2H, CH2 Bn, J ˆ À11.6 Hz), 4.53 (AB, 2H, CH2
Bn, J ˆ À12.1 Hz), 4.28 (m, 1H, H-3), 4.13 (dd, 1H, H-2a, J2a,2b ˆ À9.9 Hz,
J2a,3 ˆ 4.6 Hz), 3.96 (d, 1H, H-2b), 3.94 (t, 1H, H-9, J8,9 ˆ J9,10 ˆ 9.2 Hz), 3.88
(m, 1H, H-7), 3.60 (ABX, 2H, H-11, J7,11a ˆ 3.2 Hz, J7,11b ˆ 6.6 Hz, J11a,11b
ˆ
À10.3 Hz), 3.44 (t, 1H, H-8, J7,8 ˆ 9.2 Hz), 3.42 (d, 1H, H-10), 3.10 (brs, 3H,
OH), 2.13 (dd, 1H, H-4a, J4a,4b ˆ À13.4 Hz, J3,4a ˆ 5.7 Hz), 1.87 (d, 1H,
H-4b); 13C{1H} NMR (CDCl3): d ˆ 137.7, 137.6 (2  Cq Ph), 128.5 ± 127.6
(CH arom), 107.7 (C-5), 78.8, 75.2, 71.8, 71.3, 70.5 (C-3, C-7, C-8, C-9, C-10),
77.9, 75.2, 73.4, 69.9 (2 Â CH2 Bn, C-2, C-11), 41.5 (C-4); MS (ES): 439
H-9, J9,10 ˆ J8,9 ˆ 9.8 Hz), 4.15 (dd, 1H, H-2a, J2a,2b ˆ À10.7 Hz, J2a,3
5.9 Hz), 4.05 (dd, 1H, H-2b, J2b,3 ˆ 2.2 Hz), 3.84 (ddd, 1H, H-7, J7,8
ˆ
ˆ
10.0 Hz, J7,11a ˆ 2.0 Hz, J9,11b ˆ 4.5 Hz), 3.74 (t, 1H, H-8), 3.69 ± 3.57 (m,
3H, H-11, H-10), 3.48 (s, 3H, OMe MTPA ester), 3.29, 3.19 (2 Â s, 6 H, 2 Â
OMe BDA), 2.30 (dd, 1H, H-4a, J4a,4b ˆ À13.9 Hz, J3,4a ˆ 8.0 Hz), 2.12 (dd,
1H, H-4b, J3,4b ˆ 4.5 Hz), 1.34, 1.28 (2  s, 6 H, 2  CH3 BDA); MS (ES): 769
‡
[M‡Na] ; [a]2D0: ‡21.2 (c ˆ 0.5 CHCl3); C23H28O7: calcd C 66.33, H 6.78;
found: C 66.27, H 6.76.
‡
[M‡Na] .
(3R,5S,7R,8R,9S,10R)-10-Benzyloxy-7-benzyloxymethyl-3,8,9-trihydroxy-
1,6-dioxaspiro[4.5]decane 3,8,9-tris-(di-O-benzyl)phosphate (22): A mix-
ture of compound (3R)-17a (85 mg, 0.20 mmol) and dibenzyloxy-(N,N-
diisopropylamino) phosphine[17] (21, 0.27 mL, 0.82 mmol) was dried by
coevaporation with 1,4-dioxane (2 Â 5 mL) and dissolved in 1,2-dichloro-
ethane (6 mL). A solution of 1H-tetrazole (72 mg, 1.0 mmol) in acetonitrile
(3.0 mL) was added under a N2 atmosphere. After 30 min TLC analysis
(light petroleum/Et2O, 1:1, v/v) showed complete conversion of starting
material into a higher running product (Rf ˆ 0.76). The reaction mixture
was cooled (08C), tert-butyl hydroperoxide (0.47 mL, 80% in di-tert-butyl
peroxide) was added and stirring was continued for 30 min. TLC analysis
revealed complete disappearance of the phosphite triester intermediate
into a lower running product. The reaction mixture was diluted with
EtOAc, washed with H2O, and dried (MgSO4), and concentrated in vacuo.
Compound 22 was obtained as a colorless oil after purification by column
chromatography (light petroleum/EtOAc, 3:1 ! 0:1, v/v). Yield 60%
(0.15 g, 0.12 mmol); Rf 0.53 (EtOAc/light petroleum, 3:2, v/v); 1H NMR
(CDCl3, 300 MHz, H ± H COSY): d ˆ 7.42 ± 7.03 (m, 40H, H arom), 5.11 ±
S-Mosher ester derivative of (3R,5S,7R,8R,9S,10R,2'S,3'S)-10-benzyloxy-
7-benzyloxymethyl-8,9-di-O-(2',3'-dimethoxy-2',3'-dioxybutane-2',3'-diyl)-
3-hydroxy-1,6-dioxaspiro[4.5]decane: 1H NMR (600 MHz, H ± H COSY,
CDCl3): d ˆ 7.49 ± 7.24 (m, 15H, H arom), 5.52 (m, 1H, H-3), 4.82 (AB, 2H,
CH2 Bn, J ˆ À11.6 Hz), 4.55 (AB, 2H, CH2 Bn, J ˆ À12.3 Hz), 4.19 (t, 1H,
H-9, J8,9 ˆ J9,10 ˆ 9.8 Hz), 4.14 (dd, 1H, H-2a, J2a,2b ˆ À10.6 Hz, J2a,3
6.2 Hz), 3.95 (dd, 1H, H-2b, J2b,3 ˆ 2.3 Hz), 3.84 (ddd, 1H, H-7, J7,8
ˆ
ˆ
10.2 Hz, J7,11a ˆ 1.9 Hz, J7,11b ˆ 4.3 Hz), 3.75 (t, 1H, H-8), 3.64 (ABX, 2H,
H-11, J11a,11b ˆ À11.0 Hz), 3.58 (d, 1H, H-10), 3.42 (s, 3H, OMe MTPA
ester), 3.29, 3.20 (2 Â s, 6 H, 2 Â OMe BDA), 2.36 (dd, 1H, H-4a, J4a,4b
ˆ
À13.8 Hz, J3,4a ˆ 8.0 Hz), 2.19 (dd, 1H, H-4b, J3,4b ˆ 4.9 Hz), 1.34, 1.26 (2 Â
‡
s, 6 H, 2  CH3 BDA); MS (ES): 769 [M‡Na] .
R-Mosher ester derivative of (3S,5S,7R,8R,9S,10R,2'S,3'S)-10-benzyloxy-
7-benzyloxymethyl-8,9-di-O-(2',3'-dimethoxy-2',3'-dioxybutane-2',3'-diyl)-
3-hydroxy-1,6-dioxaspiro[4.5]decane: 1H NMR (600 MHz, H ± H COSY,
CDCl3): d ˆ 7.51 ± 7.23 (m, 15H, H arom), 5.39 (m, 1H, H-3), 4.83 (AB, 2H,
CH2 Bn, J ˆ À11.5 Hz), 4.43 (AB, 2H, CH2 Bn, J ˆ À12.1 Hz), 4.34 (dd,
2702
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Chem. Eur. J. 2000, 6, No. 15

Spirophostins
2696±2704
3
incubated with a constant amount of H-IP3 (approx. 9000 d.p.m. (disinte-
grations per minute) per assay; stock: 21 CimmolÀ1; NEN) and adrenal
cortex membranes; incubations were stopped after 30 min at 48C by rapid
vacuum filtration.[18] Nonspecific binding was defined in the presence of
10mm IP3. Each displacement isotherm (see Figure 2) was used to obtain an
estimate of the IC50 value (see Table 2) using GraphPad Prism and are
given as Àlog IC50 values (Æs.e. mean).
4.85 (m, 14H, 6  CH2 Bn, H-3, H-9), 4.71 (AB, 2H, CH2 Bn, J ˆ
À11.5 Hz), 4.63 (q, 1H, H-8, J8,9 ˆ J7.8 ˆ 3J8,p ˆ 9.6 Hz), 4.41 (AB, 2H, CH2
3
Bn, J ˆ À12.1 Hz), 4.05 (dd, 1H, H-2a, J2a,2b ˆ À 10.2 Hz, J2a,4 ˆ 1.6 Hz),
3.91 ± 3.82 (m, 2H, H-2b, H-7), 3.71 (ABX, 2H, H-11, J7,11a ˆ 4.4 Hz, J7,11b
ˆ
1.8 Hz, J11a,11b ˆ À11.0 Hz), 3.57 (d, 1H, H-10, J9,10 ˆ 9.7 Hz), 2.17 (m, 2H,
H-4); 13C{1H} NMR (CDCl3): d ˆ 138.1, 137.5, 136.1 ± 135.4 (Cq Bn),
133.0 ± 127.5 (CH arom), 107.3 (C-5), 79.6, 77.9, 76.6, 74.6, 70.5 (C-3, C-7,
C-8, C-9, C-10), 75.0, 73.0, 72.4 (2 Â CH2 Bn, C-2), 69.8 ± 69.3 (CH2 Bn),
68.1 (C-11), 41.3 (C-4); 31P NMR (CDCl3): d ˆ À0.67, À1.15, À1.57; MS
45Ca2‡-Release experiments: Assays were performed using SH-SY5Y
human neuroblastoma cells (passage 20 ± 30) essentially as described
previously[23] with certain modifications. Confluent monolayers of SH-
SY5Y cells were washed and harvested using 10mm HEPES, 0.9% NaCl,
0.02% EDTA, pH 7.4 and recovered by centrifugation (400 Â g, 3 min).
Cells were resuspended in an intracellular-like buffer (ICB: 20mm HEPES,
‡
‡
(ES): 1198 [M‡H] , 1220 [M‡Na] ; [a]D20
:
‡17.8 (c ˆ 1.0 CHCl3);
C65H67O16P3: calcd C 65.21, H 5.64; found: C 65.25, H 5.63.
(3S,5S,7R,8R,9S,10R)-10-Benzyloxy-7-benzyloxymethyl-3,8,9-trihydroxy-
1,6-dioxaspiro[4.5]decane 3,8,9-tris(di-O,O-benzyl)phosphate (23): Spiro-
ketal (3S)-18a (55 mg, 0.13 mmol) was phosphorylated (as described for
the preparation of 22) to give 23. Yield 65% (0.10 g, 85 mmol). Rf 0.64
(EtOAc/light petroleum, 3:2, v/v); 1H NMR (CDCl3): d ˆ 7.41 ± 7.11 (m,
40H, H arom), 5.07 ± 4.82 (m, 14H, 6 Â CH2 Bn, H-3, H-9), 4.63 (q, 1H,
H-8, J8,9 ˆ J7.8 ˆ 3J8,p ˆ 9.6 Hz), 4.45 (AB, 2H, CH2 Bn, J ˆ À11.9 Hz), 4.36
(AB, 2H, CH2 Bn, J ˆ À12.4 Hz), 4.09 (dd, 1H, H-2a, J2a,2b ˆ À9.9 Hz,
J2a,3 ˆ 4.4 Hz), 4.00 (m, 2H, H-7), 3.90 (d, 1H, H-2b), 3.72 (ABX, 2H, H-11,
13mm KCl, 2.5mm MgCl2, 2mm ATP, 20 mm CaCl2, Ph 7.1; the free [Ca2‡
]
was buffered to 100 ± 150nm by addition of EGTA) and centrifuged
(400 Â g, 3 min). This latter step was repeated and the final cell pellet was
gently resuspended in ICB supplemented with an ATP regenerating system
(10mm phosphocreatine, 10 UmLÀ1 creatine phosphokinase) and perme-
abilization was achieved by addition of 50 mgmLÀ1 b-escin. After 2 min
1 mCimLÀ1 45Ca2‡ (1000 CimmolÀ1, Amersham, Little Chalfont, UK) was
added and the permeabilized cell suspension was added to ICB containing
different concentrations of adenophostinA (1), IP3 (4), spirophostin (3R)-
10 or (3S)-11. Incubations were continued for 2 min (IP3, 30 s) and samples
were then centrifuged (13,000 Â 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 (13000 Â 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 scintillation cocktail (Packard Bioscience BV,
Groningen, the Netherlands). Samples were stored overnight in the dark
before scintillation counting. The total releaseable 45Ca2‡-pool was defined
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 percentage of the total
ionomycin-releaseable pool) using GraphPad Prism.
J7,11a ˆ 4.3 Hz, J7,11b ˆ 1.9 Hz, J11a,11b ˆ À11.4 Hz), 3.50 (d, 1H, H-10, J9,10
ˆ
9.8 Hz), 2.05 (m, 2H, H-4); 13C{1H} NMR (CDCl3): d ˆ 138.2, 137.6, 135.7
(Cq Bn), 129.1 ± 127.0 (CH arom), 106.9 (C-5), 79.9, 79.8, 76.8, 74.1, 69.9
(C-3, C-7, C-8, C-9, C-10), 75.2, 72.8, 70.7 (2 Â CH2 Bn, C-2), 69.9 ± 69.4
(CH2 Bn), 67.9 (C-11), 40.0 (C-4); 31P NMR (CDCl3): d ˆ À0.55 (P-3),
‡
À1.56 (P-8, P-9); MS (ES): 1220 [M‡Na] ; [a]2D0: 11.4 (c ˆ 1.0 CHCl3);
C65H67O16P3: calcd C 65.21, H 5.64; found: C 65.15, H 5.58.
(3R,5S,7R,8R,9S,10R)-7-Hydroxymethyl-3,8,9,10-tetrahydroxy-1,6-dioxa-
spiro[4.5]decane 3,8,9-trisphosphate; spirophostin (3R)-10: Compound 22
(0.15 g, 0.12 mmol) was dissolved in a mixture of 1,4-dioxane, propan-2-ol
and H2O (15 mL, 4:2:1, v/v/v) containing NaOAc (0.12 g, 1.4 mmol). The
clear solution was degassed and 10% Pd/C (75 mg) was added under an
atmosphere of N2. The mixture was stirred under H2 (1 atm) for 16 h and
the catalyst was removed by filtration. The filtrate was concentrated under
reduced pressure and the crude product was purified by gel-filtration over a
Fractogel HW-40 column (elution: 0.15m triethyl ammonium bicarbonate
buffer). Concentration and coevaporation (MeOH/H2O, 4 :1, v/v, 3 Â 5 mL)
of the appropriate fractions, followed by lyophilization gave trisphosphate
Molecular modeling: All calculations were run on IRIS workstations
according to Hotoda et al.[3] The stable conformers (Table 6), within
3.0 kcalmolÀ1 from the lowest-energy conformers R-1 and S-1, were
selected from a total of 434 and 363 stable conformations found for the R
and S isomer, respectively. After determination of the distance between the
P-3 of (3R)-10 or (3S)-11 and the P-2' in superimpositions with two models
of adenophostinA (models B and C in ref.[3]), it was established that the
spirophostins matched better with model B. This model, which also
corresponded better with the solution structure of adenophostinA assigned
by NMR spectroscopy, was therefore used in Tables 4 and 5 in the
Discussion.
‡
(3R)-10 in pure form. The product was converted into the Na -form by ion-
‡
exchange with Dowex 50Wx4 (Na -form) followed by lyophilization. Yield
87% (64 mg, 0.11 mmol). 1H NMR (D2O, 600 MHz, H ± H-COSY): d ˆ
4.87 (m, 1H, H-3), 4.28 (q, 1H, H-9, J8,9 ˆ J7,8 ˆ 3J9,P ˆ 9.0 Hz), 4.05 (ABX,
2H, H-2, J2a,3 ˆ 4.9 Hz, J2b,3 ˆ 1.9 Hz, J2a,2b ˆ À10.1 Hz), 3.96 (q, 1H, H-8,
3J8,P ˆ 9.7 Hz), 3.84 (dd, 1H, H-11a, J11a,7 ˆ 4.3 Hz, J11a,11b ˆ À13.5 Hz), 3.66
(m, 3H, H-11b, H-7, H-10), 2.35 (d, 2H, H-4, J3,4 ˆ 5.5 Hz); 13C{1H} NMR
(D2O): d ˆ 108.8 (C-5), 79.4 (C-3, J3,P ˆ 3.1 Hz), 74.8, 73.0, 72.0 (C-7, C-8,
C-9, C-10, J8,P ˆ 5.0 Hz, J9,P ˆ 5.3 Hz), 74.3 (C-2, 3J2,P ˆ 4.6 Hz), 60.7 (C-11),
42.9 (C-4, 3J4,P ˆ 5.0 Hz); 31P{1H} NMR (D2O, 242 MHz, P ± H-COSY): d ˆ
2.83 (P-8), 2.35 (P-9), 1.68 (P-3); MS (ES): 475 [M À H]À, 497 [M À
Acknowledgement
‡
‡
2H‡Na ]À, 519 [M À 3H‡2Na ]À; [a]D20: ‡23.8 (c ˆ 0.4 H2O); HRMS
We thank Rajendra Mistry (Department of Cell Physiology & Pharmacol-
ogy, 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 Hans vanderElst and Nico Meeuwe-
noord for recording mass spectra. This research has been financially
supported by the Council for Chemical Sciences of the Netherlands
Organization for Scientific Research (CW-NWO).
(ES) calcd C9H18O16P3 (M À H)À 474.9807; found 474.9821.
(3S,5S,7R,8R,9S,10R)-7-Hydroxymethyl-3,8,9,10-tetrahydroxy-1,6-dioxa-
spiro[4.5]decane 3,8,9-trisphosphate; spirophostin (3S)-11: Deprotection
of 23 (0.10 g, 85 mmol) and purification of the resulting trisphosphate was
accomplished as described for the synthesis of spirophostin (3R)-10 to
‡
afford the Na -salt of (3S)-11. Yield 57% (30 mg, 60 mmol). 1H NMR (D2O,
600 MHz, H ± H-COSY, 78C): d ˆ 4.65 (m, 1H, H-3), 4.16 (m, 2H, H-9,
H-2a), 3.88 (m, 2H, H-8, H-2b), 3.71 (ABX, 2H, H-11a, J7,11a ˆ 4.0 Hz,
J7,11b ˆ 1.7 Hz, J11a,11b ˆ À13.2 Hz), 3.66 (t, 1H, H-7, J7,8 ˆ 9.8 Hz), 3.53 (d,
[1] a) M. J. Takahashi, T. Kagasaki, T. Hosoya, S. Takahashi, J. Antibiot.
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1H, H-10, J9,10 ˆ 9.4 Hz), 2.48 (dd, 1H, H-4a, J4a,4b ˆ À14.6 Hz, J3,4a
ˆ
8.1 Hz), 1.99 (dd, 1H, H-4b, J3,4b ˆ 2.1 Hz); 13C{1H} NMR (D2O): d ˆ
108.6 (C-5), 79.3 (C-3, J3,P ˆ 5.8 Hz), 73.9, 73.2, 73.0, 72.7 (C-7, C-8, C-9,
C-10, J8,P ˆ 5.1 Hz, J9,P ˆ 4.6 Hz), 75.3 (C-2, 3J2,P ˆ 4.9 Hz), 61.2 (C-11), 41.7
3
(C-4, J4,P ˆ 4.7 Hz); 31P{1H} NMR (D2O, 242 MHz, P-H-COSY): d ˆ 3.45
‡
(P-3), 2.92 (P-8), 2.76 (P-9); MS (ES): 475 [M À H]À, 497 [M À 2H‡Na ]À,
‡
519 [M À 3H‡2Na ]À; [a]D20: ‡14.8 (c ˆ 0.1 H2O); HRMS (ES) calcd
C9H18O16P3 (M À H)À 474.9807; found 474.9816.
3H-IP3 Displacement binding experiments: A P2 fraction of bovine adrenal
cortex was prepared as described previously.[21] Increasing concentrations
of adenophostinA,[22] IP3, and the spirophostins (3R)-10 and (3S)-11 were
Chem. Eur. J. 2000, 6, No. 15
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0615-2703 $ 17.50+.50/0
2703

J. H. van Boom et al.
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Received: December 29, 1999 [F2218]
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