Stereoselective syntheses of diastereomers of antitumor natural product pericosine A from (-)-quinic acid

Article abstract of DOI:10.1055/s-2007-990798

The stereoselective syntheses of diastereomers of antitumor natural pericosine A from (-)-quinic acid were achieved. One of the diastereomers, methyl (3R,4S,5S,6R)-6-chloro-3,4,5-trihydroxycyclohex-1-enecarboxylate, possesses the reported structure of per

Full text of DOI:10.1055/s-2007-990798

PAPER
3219
Stereoselective Syntheses of Diastereomers of Antitumor Natural Product
Pericosine A from (–)-Quinic Acid
Stereoselective
Sy
o
ntheses of
D
s
h
rs of
A
ntitum
i
or Na
h
tural
P
roduct
i
P
eric
o
d
sine
A
e Usami,* Yasuko Ueda
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Fax +81(726)901005; E-mail: usami@gly.oups.ac.jp
Received 18 May 2007; revised 26 June 2007
This paper is dedicated to Professor Hiroshi Shimizu on the occasion of his retirement from Gifu Pharmaceutical University.
COOMe
2
COOMe
COOMe
Abstract: The stereoselective syntheses of diastereomers of antitu-
mor natural pericosine A from (–)-quinic acid were achieved. One
of the diastereomers, methyl (3R,4S,5S,6R)-6-chloro-3,4,5-trihy-
droxycyclohex-1-enecarboxylate, possesses the reported structure
of pericosine A. The reported relative configuration of natural peri-
cosine A was shown to be incorrect.
Cl
Cl
MeO
HO
6
5
3
4
HO
HO
OH
OH
OH
OH
OH
OH
3
1
2
Key words: stereoselective synthesis, marine natural product, per-
icosine A, diastereomer
Figure 1
cytotoxicity against P388 lymphocytic leukemia cells
(ED50 0.12 and 4.0 mg/mL, respectively).
The search for bioactive marine natural products has been
pursued intensively over the past two decades.1,2 A new
trend has emerged that involves the study of the metabo-
lites of microorganisms from marine sources.3,4 In the
course of such studies, unique multifunctionalized C7-cy-
clohexenoids, designated as pericosines A and B, were
isolated in 1977 as metabolites of Periconia byssoides
OUPS-N133 originally separated from the sea hare, Aply-
sia kurodai.5 The reported structures 1 and 2 are presented
in Figure 1. These compounds showed significant in vitro
Pericosines have the structural character of carbasugars,
and have been reported to exhibit antiviral, antifungal, and
antitumor activities.6 We are interested in the syntheses of
natural pericosines and related compounds.7,8
In 1998, Donohoe and co-workers achieved the total syn-
thesis of 2 and established its absolute configuration.9 In
2005, Ruano and co-workers reported their synthetic ef-
forts to produce 2 using asymmetric Diels–Alder reaction,
but their total synthesis could not be completed.10
COOMe
Cl
HO
OH
COOMe
COOMe
O
HO
Cl
OH
Cl
1
O
O
HO
COOMe
O
OH
Cl
5
6
HO
OH
OH
3
O
O
O
HO
HO
COOH
OH
HO
HO
Cl
Cl
O
O
O
3
HO
HO
O
O
OH
OTBDMS
OTBDMS
OTBDMS
7
8
9
(–)-quinic acid (4)
Scheme 1 Retrosynthetic strategy for 1 and 3 from (–)-quinic acid
SYNTHESIS 2007, No. 20, pp 3219–3225
x
x.
x
x
.
2
0
0
7
Advanced online publication: 21.09.2007
DOI: 10.1055/s-2007-990798; Art ID: F09207SS
© Georg Thieme Verlag Stuttgart · New York

3220
Y. Usami, Y. Ueda
PAPER
O
In spite of the significant in vivo antitumor activity of per-
icosine A against murine P388 leukemia cells (the T/C
value was estimated to be 122%),5 there has been no re-
port of its absolute configuration. Thus, we have attempt-
ed the stereoselective total synthesis of 1 in order to
elucidate the absolute configuration of pericosine A as a
promising seed compound for cancer drug candidates. We
have already stated that 1 is an incorrect structure for per-
icosine A in a preliminary report.11 We describe herein the
full details of the stereoselective syntheses of diastereo-
mers 1 and 3 of pericosine A from (–)-quinic acid (4),12,13
O
HO
5
HO
b, c, d
e
a
X
4
O
O
3
R1O
4
O
OR2
OTBDMS
9: X = H
8: X = Cl
10: R1 = H, R2 = H
11a: R1 = TBDMS, R2 = H
11b: R1 = H, R2 = TBDMS
O
O
COOMe
OH
HO
Cl
1
HO
HO
together with the results of H and 2D NMR analysis of
the intermediates and the products. Improvements of
chemical yields were also examined.
Cl
Cl
f, g
h
6
O
O
R1O
O
HO
Our synthetic strategy is summarized in Scheme 1. Both
synthetic target molecules 1 and 3 possessing a different
configuration at C3 could be prepared from common in-
termediate enone 5 by independent stereoselective reduc-
tion of the C3 ketone in 5. Enone 5 was prepared by
dehydration of b-hydroxy ketone 6, which was obtained
through the lactone ring opening of diol 7. Diol 7 was ob-
tained by stereoselective reduction of a-chloro ketone 8.
a-Chloro ketone 8 was prepared by the stereoselective
chlorination of known ketone 9 derived from commercial-
ly available 4.
OR2
12: R1 = R2 = H
13: R1 = R2 = isopropylidene
O
OTBDMS
7
14
i
j
COOMe
HO
6
Cl
OH
HO
OH
15
COOMe
HO
COOMe
k, l, m
n, o
Cl
Cl
The synthesis of 1 was achieved as shown in Scheme 2.
Using a literature procedure,14 4 was converted into lac-
tone 10, which was silylated to give a mixture of 4-silyl
ether 11a and 5-silyl ether 11b. These were separated and
the pure tert-butyldimethylsilyl ether 11b was oxidized
with Dess–Martin periodinane to give known lactone 9 in
95% yield. The trimethylsilyl enol ether derived from 9
was chlorinated with N-chlorosuccinimide in N,N-di-
methylformamide with excellent selectivity to give the
desired a-chloro ketone 8 in 45% yield in two steps.
3
3
R1O
O
O
R1O
OR2
OR2
6: R1 = R2 = isopropylidene
16: R1 = R2 = H
17: R1 = R2 = TMS
18: R1 = R2 = TMS
5: R1 = R2 = H
COOMe
Cl
COOMe
Cl
p
q
The stereochemistry at C6 was deduced from the low-
field shift (d = 3.29) of H2a in the 1H NMR spectrum of 8
compared with that of 9 (d = 2.86). This stereoselectivity
was the same as for bromination or fluorination described
in the literature14,15 showing that the bromo ketone as-
sumed a chair conformation with 6a-axial-bromo atom,
causing the low-field shift of H2aax. Long-range cou-
plings H6/H2eq (J = 2.5 Hz), H4/H2eq (J = 1.1 Hz), and
H4/H6 (J = 1.4 Hz) in 8 also supported its chair conforma-
tion (Scheme 3). The proximity of the chlorinating agent
to the silyl enol ether from the opposite side of the hin-
dered lactone bridge was surmised to cause this stereose-
lectivity.
O
OH
HO
OH
O
OH
1
19
Scheme 2 Reagents and conditions: (a) Ref. 14; (b) Dess–Martin
periodinane, CH2Cl2 (95%); (c) TMSOTf, Et3N, toluene, reflux; (d)
NCS, DMF (33% in 2 steps from 9); (e) NaBH4, MeOH (68%), (57%
in 3 steps from 9); (f) Bu4NF, THF (67%); (g) 2,2-dimethoxypropane,
PPTS, acetone, reflux (30%); (h) NaOMe, MeOH (90%); (i) TFA,
MeOH, 60 °C (38%); (j) 2,2-dimethoxypropane, cat. TsOH, CH2Cl2
(65%); (k) Dess–Martin periodinane, CH2Cl2 (78%); (l) TFA, MeOH
(70%); (m) TMSCl, Et3N (71%); (n) Martin’s sulfurane dehydrating
agent, CH2Cl2 (65%); (o) TFA, MeOH (quant.); (p) Bu4NBH(OAc)3,
AcOH–AcCN (64%); (q) 2,2-dimethoxypropane, cat. TsOH, acetone
(72%).
The following reduction of 8 with sodium borohydride
gave single diol 7 in 68% yield with the desired stereo-
chemistry that was proved completely, as described later.
The chemical yield of 7 was improved by sequential reac- tronegative C4–O (aax) and C6–Cl (aax) should stabilize
tions from 45% in two steps to an overall yield of 57% in the Felkin–Anh transition state for the b-side hydride at-
three steps from ketone 9. The stereoselectivity observed tack to C5 carbonyl (Scheme 3), despite the presence of a
in this reduction was explained with the Felkin–Anh mod- lactone bridge in the molecule. An alternative plausible
el.16,17 In this case, the lactone bridge should play a role in explanation was steric repulsion effected by neighboring
fixing the conformation of the cyclohexane ring. Elec- bulky substituents at C4 or C6.
Synthesis 2007, No. 20, 3219–3225 © Thieme Stuttgart · New York

PAPER
Stereoselective Syntheses of Diastereomers of Antitumor Natural Product Pericosine A
3221
H–
NOEs
O
O
H
H
H
H
O
O
O
6
H
4
4
H
4
H
3
6
OH
H
OH
H
H
Cl
HO
HO
2
3
2b
H
H
COOMe
H
HO
H
H
H
Cl
Cl
OH
H
OH
OTBDMS
OTBDMS
8
7
15
Scheme 3
Diol 7 was treated with tetrabutylammonium fluoride to ture in 64% yield. Proof of the stereochemistry will be de-
afford triol 12 in 67% yield, which was then transformed scribed later.
into acetonide 13 in 30% yield. The lactone ring of 13 was
opened with sodium methoxide to afford diol 14 in 90%
yield.
However, the NMR data of 1 and acetonide 19 did not
agree with those of natural pericosine A and those of its
acetonide described in the literature.5 Furthermore, the
Secondary alcohol 14 was oxidized with Dess–Martin isopropylidene bridge in 19 derived from 1 was located
periodinane18 to give hydroxy ketone 6 in 78% yield. The between C4 and C5, whereas that of the acetonide of per-
NOESY cross peaks H4/H2b, H6/H2b, and H4/H6 were icosine A was between C3 and C4.5 This was supported by
observed in 6.
NOESY cross peaks between one of two methyl groups
and H4 or H5 of 19. These NOESY cross peaks in 19 and
the NOESY cross peaks H4/H6 in 18 described above also
denied the possibility of epimerization at C4 and C6 dur-
ing the conversion from 17 into 18.
Because of the low chemical yields of these steps, an al-
ternative route was examined. Diol 7 was converted into
tetraol 15 in 38% yield with trifluoroacetic acid in metha-
nol under reflux with recovery of dechlorinated methyl
quinate in 30% yield. Compound 15 was confirmed to
have the chair conformation based on analyses of the cou-
pling constants of 1H NMR and NOESY cross peaks H4/
H2b, H6/H2b, and H4/H6 (Scheme 3). This experiment
proved the stereochemistry at C6 in 8 or C5 in 7. Treat-
ment of 15 with 2,2-dimethoxypropane and catalytic 4-
toluenesulfonic acid in dichloromethane under reflux
gave 14 in 65% yield.
COOMe
COOMe
Cl
Cl
a, b
c
18
R1O
OH
HO
O
OR2
O
20: R1 = R2 = TMS
3: R1 = R2 = H
21
Scheme 4 Reagents and conditions: (a) NaBH4, MeOH; (b) TFA,
CH2Cl2 (quant. in 2 steps); (c) 2,2-dimethoxypropane, cat. TsOH
(55%).
However, 6 was not dehydrated with various kinds of de-
hydrating agents. Then, the isopropylidene moiety of 6
was removed by reacting with trifluoroacetic acid to give
triol 16 in 70% yield, which was then protected as the
bis(trimethylsilyl ether) to give reactive hydroxy ketone
17 in 71% yield. The HMBC cross peak H2a/C6 in 16
confirmed the assignment of the NMR signals. Observing
NOESY cross peaks between H4 and H6 in 6, 16, and 17
implied that no epimerization had occurred during the
preparation of these compounds.
The stereochemistry at C3 in 1 was proved by the follow-
ing experiments. Common intermediate 18 was reduced
with sodium borohydride to afford allyl alcohol 20 as a
single product. Then, 20 was deprotected to afford our
second target diastereomer 3 quantitatively in two steps
(Scheme 4). Product 3 was transformed into 3,4-O-ace-
tonide 21 in 55% isolated yield. NOESY cross peaks be-
tween one of two methyl groups of the isopropylidene
moiety and H3 or H4 implied that the newly generated hy-
droxy group at C3 and the neighboring hydroxy group at
C4 had the cis configuration in 21. Furthermore a NOESY
cross peak between H3 and H5 in 3 implied that H3 and
H5 had the cis configuration. Therefore, the stereochem-
istry of synthesized 3 was confirmed (Scheme 4). The
spectral data of 3 were different from those of 1 or natural
pericosine A. Based on these experiments, synthesized 1
must have an a-hydroxy group at C3. All the experimental
results described above guaranteed correctness of the ste-
reostructure of synthesized 1.
Dehydration of 17 to give a,b-unsaturated ketone 18 was
carried out with Martin’s sulfurane dehydrating agent
(bis[a,a-bis(trifluoromethyl)benzyloxy]diphenylsulfur)19
in 65% yield. Dehydration with thionyl chloride and tri-
ethylamine afforded 18 in 30% yield together with the
deprotected dihydroxy a,b-unsaturated ketone 5 in 16%
yield. The HMBC cross peaks H6/C1, H5/C1, H2/C1, H4/
C3, and H5/C3 in 18 led the assignment of the NMR sig-
nals. The cross peak between H4 and H6 in the NOESY
spectra of 18 implied that H4 and H6 had the cis configu-
ration.
Enone 18 was treated with trifluoroacetic acid and metha-
nol to give 5 quantitatively, and 5 was reduced with tet-
rabutylammonium triacetoxyborohydride20 in a stereo-
selective manner to give 15 having the desired stereostruc-
Through this study, we have shown that 1 was the incor-
rect stereostructure of pericosine A.
Synthesis 2007, No. 20, 3219–3225 © Thieme Stuttgart · New York

3222
Y. Usami, Y. Ueda
PAPER
MS (CI): m/z = 321 [M + H]+.
HRMS: m/z [M – t-Bu]+ calcd for C9H12O5SiCl: 263.0142; found:
In conclusion, the stereoselective syntheses of diastereo-
mers 1 and 3 of pericosine A from (–)-quinic acid were
achieved. Detailed NMR analysis of the intermediates or 263.0129.
the products confirmed that the stereostructures of 1 and 3
were methyl (3R,4S,5S,6R)- and (3S,4S,5S,6R)-6-chloro-
3,4,5-trihydroxycyclohex-1-enecarboxylate, respectively.
This study confirmed that the structure of antitumor natu-
ral pericosine A was not 1 or its diastereomer 3. The re-
sults of this study have motivated us to conduct the total
synthesis of real pericosine A assigned to (3S,4S,5S,6S)-
6-chloro-3,4,5-trihydroxycyclohex-1-enecarboxylate as
our next work.21,22
Anal. calcd for C13H21ClO5Si·0.25 H2O: C, 47.96; H, 6.73; found;
C, 47.89; H, 6.74.
(1S,3R,4R,5S,6S)-4-(tert-Butyldimethylsiloxy)-6-chloro-1,5-di-
hydroxycyclohexane-1,3-carbolactone (7)
Method A: A soln of chloro ketone 8 (32.1 mg, 0.10 mmol) in
MeOH (1 mL) was added dropwise to a suspension of NaBH4 (3.8
mg, 0.10 mmol) in MeOH (2 mL) at –15 °C. The mixture was stirred
for 30 min, quenched with H2O, and extracted with EtOAc. The or-
ganic layer was washed with brine, dried (MgSO4), filtered, and
concentrated under reduced pressure to give crude diol that was pu-
rified by chromatography (2% MeOH–CH2Cl2) to afford 7 (22 mg,
68%).
IR spectra were obtained with a Perkin Elmer FT-IR spectrometer
1720X. EIMS was determined with a Hitachi 4000H mass spec-
trometer. NMR spectra were recorded at 27 °C on Varian Unity In-
ova-500, Gemini-2000, and Mercury-300 spectrometers in CDCl3
with TMS as internal reference; for comparative purposes, the as-
signments are always given according to the numbering given in
Scheme 2. Melting points were determined on a Yanagimoto mi-
cromelting point apparatus and are uncorrected. Liquid column
chromatography was conducted over silica gel (Nacalai Tesque, sil-
ica gel 60, mesh 70–230 or 230–400). Analytical TLC was per-
formed on precoated Merck aluminum sheets (DC-Alufolien
Kieselgel 60 F254), and compounds were detected by spraying an
EtOH soln of phosphomolybdic acid followed by heating. Anhyd
THF was distilled over sodium benzophenone ketyl under argon at-
mosphere.
Method B: Crude chloro ketone 9, which was obtained by the same
procedure as that described above on the same scale (1.0 mmol of
starting material 11b), was dissolved in THF (3 mL) and the mix-
ture was added to a suspension of NaBH4 (37.8 mg, 1 mmol) in
MeOH (3 mL) at –15 °C. The mixture was treated in the same man-
ner to afford 7 (184.0 mg, 57% from starting ketone) as colorless
crystals; mp 128–131 °C (CH2Cl2).
[a]D26 –44.5 (c 0.45, MeOH).
IR (KBr): 3437 (OH), 1807 (C=O) cm–1.
1H NMR (CDCl3): d = 0.11 (s, 3 H, SiMe), 0.16 (s, 3 H, SiMe), 0.94
(s, 9 H, t-Bu), 2.24 (ddd, J = 12.4, 6.0, 1.4 Hz, 1 H, H2b), 2.75 (d,
J = 12.4 Hz, 1 H, 5-OH), 3.10 (d, J = 12.4 Hz, 1 H, H2a), 3.94 (ddd,
J = 12.4, 5.2, 4.4 Hz, 1 H, H5), 4.12 (dd, J = 4.9, 4.4 Hz, 1 H, H4),
4.35 (dd, J = 5.2, 1.4 Hz, 1 H, H6), 4.67 (dd, J = 6.0, 4.9 Hz, 1 H,
H3).
(1S,3R,4S)-4-(tert-Butyldimethylsiloxy)-1-hydroxy-5-oxocyclo-
hexane-1,3-carbolactone (9)
To a suspension of Dess–Martin periodinane (3.74 g, 8.82 mmol) in
CH2Cl2 (10 mL) was added 11b (1.27 g, 4.41 mmol) in CH2Cl2 (2
mL) at r.t. After stirring for 2 h, the mixture was diluted with t-
BuOMe and treated with aq Na2S2O3 and aq NaHCO3. The organic
layer was washed with brine, dried (MgSO4), and filtered, and the
solvent was removed under reduced pressure to give a crude residue
that was recrystallized to afford 9 (1.20 g, 95%).14
13C NMR (CDCl3): d = –4.9 (q), –4.6 (q), 17.9 (s), 25.6 (3 q), 32.0
(t), 65.7 (d), 66.7 (d), 67.3 (d),75.5 (s), 76.0 (d), 174.3 (s).
HRSIMS: m/z [M + H]+ calcd for C13H24ClO5Si: 323.1080; found:
323.1084.
(1S,3R,4R,5S,6S)-6-Chloro-1,4,5-trihydroxycyclohexane-1,3-
carbolactone (12)
To a soln of 7 (257 mg, 0.80 mmol) in THF (3 mL) was added 1.0
M Bu4NF in THF (0.8 mL, 0.80 mmol,) under argon. The mixture
was stirred at r.t. for 3 h, and TsOH·H2O (152 mg, 0.80 mmol) was
added. The mixture was stirred for a further 30 min and concentrat-
ed directly under reduced pressure to give a residue that was puri-
fied by column chromatography (silica gel, gradient, EtOAc–
hexane, 1:1 to EtOAc) to afford 12 (111.1 mg, 67%) as colorless
crystals; mp 111–115 °C (EtOAc–hexane).
[a]D26 –57.5 (c 0.58, MeOH).
IR (KBr): 3403 (OH), 1785 (C=O) cm–1.
1H NMR (CD3OD): d = 2.14 (ddd, J = 12.1, 6.0, 2.2 Hz, 1 H, H2b),
3.03 (d, J = 12.1 Hz, 1 H, H2a), 3.86 (br t, J = 4.7 Hz, 1 H, H4), 4.04
(t, J = 4.7 Hz, 1 H, H5), 4.29 (ddd, J = 4.7, 2.2, 1.1 Hz, 1 H, H6),
4.71 (dd, J = 6.0, 4.7 Hz, 1 H, H3).
(1S,3R,4S,6R)-4-tert-(Butyldimethylsiloxy)-6-chloro-1-hy-
droxy-5-oxocyclohexane-1,3-carbolactone (8)
To a toluene soln (6 mL) of ketone 9 (284 mg, 1.00 mmol) were
added successively Et3N (578 mL, 4.00 mmol) and TMSOTf (609
mL, 3.4 mmol) and the resulting soln was refluxed for 8 h with stir-
ring under argon. The mixture was diluted with hexane mixed with
aq NaHCO3 and extracted. The organic layer was washed with
brine, dried (MgSO4), filtered, and concentrated under reduced
pressure to give the crude silyl enol ether. To the soln of silyl enol
ether in DMF (5 mL) was added NCS (133 mg, 1.00 mmol) and the
mixture was stirred at r.t. for 50 h under argon. The resulting mix-
ture was extracted with n-hexane, dried (MgSO4), filtered, and con-
centrated under reduced pressure to give the crude chloro ketone
that was purified by recrystallization to afford 8 (107 mg, 33%) as
colorless crystals (Et2O); mp 67–70 °C.
13C NMR (CD3OD): d = 32.8 (t), 67.0 (d), 67.2 (d), 67.6 (d), 76.7
[a]D26 –11.0 (c 1.7, MeOH).
IR (KBr): 3521 (OH), 1820 (C=O), 1739 (C=O) cm–1.
1H NMR (CDCl3): d = 0.13 (s, 3 H, SiMe), 0.16 (s, 3 H, SiMe), 0.90
(s, 9 H, t-Bu), 2.51 (dddd, J = 12.6, 6.3, 2.5, 1.1 Hz, 1 H, H2b), 3.29
(d, J = 12.6 Hz, 1 H, H2a), 4.13 (ddd, J = 4.1, 1.4, 1.1 Hz, 1 H, H4),
4.17 (dd, J = 2.5, 1.4 Hz, 1 H, H6), 4.74 (dd, J = 6.3, 4.1 Hz, 1 H,
H3).
(s), 77.2 (d), 177.1 (s).
HRMS (CI): m/z [M + H]+ calcd for C7H10ClO5: 209.0216; found:
209.0217.
(1S,3R,4R,5S,6S)-6-Chloro-1-hydroxy-4,5-(isopropylidene-
dioxy)cyclohexane-1,3-carbolactone (13)
A mixture of 12 (73.8 mg, 0.36 mmol), PPTS (8.9 mg, 0.04 mmol),
and 2,2-dimethoxypropane (1 mL) in acetone (5 mL) was refluxed
for 2 h, cooled to r.t., and concentrated to give a crude residue that
13C NMR (CDCl3): d = –5.2 (q), –5.0 (q), 18.1 (s), 25.5 (3 q), 31.4
(t), 62.1 (d), 71.1 (d), 74.6 (d), 74.9 (d), 173.6 (s), 197.7 (s).
Synthesis 2007, No. 20, 3219–3225 © Thieme Stuttgart · New York

PAPER
Stereoselective Syntheses of Diastereomers of Antitumor Natural Product Pericosine A
3223
was purified by column chromatography (silica gel, EtOAc–n-
hexane, 1:1) to afford 13 (26.0 mg, 30%) as colorless crystals
(CH2Cl2); mp 72–75 °C.
[a]D26 +4.4 (c 0.68, MeOH).
Methyl (1S,2S,3S,4S,5R)-2-Chloro-1,5-dihydroxy-3,4-(isopro-
pylidenedioxy)cyclohexanecarboxylate (14) from 15
2,2-Dimethoxypropane (1 mL, excess) and a catalytic amount of
TsOH·H2O (2.0 mg) were added to a CH2Cl2 soln (5 mL) of 15 (22.0
mg, 0.09 mmol). The mixture was stirred under reflux with 3A mo-
lecular sieves for 2 h. After removing the volatile components under
reduced pressure, CH2Cl2 was added to the residue. The supernatant
that was separated from the residue was concentrated under reduced
pressure to give almost pure 14 (16.8 mg, 65%).
IR (KBr): 3437 (OH), 1768 (C=O) cm–1.
1H NMR (CDCl3): d = 1.41 (s, 3 H, acetonide-Me), 1.67 (s, 3 H, ac-
etonide-Me), 2.36 (dd, J = 12.1, 5.2 Hz, 1 H, H2b), 2.95 (d, J = 12.1
Hz, 1 H, H2a), 4.39 (m, 1 H), 4.48 (m, 2 H), 4.99 (m, 1 H).
13C NMR (CDCl3): d = 25.4 (q), 25.5 (q), 31.9 (t), 63.3 (d), 72.0 (d),
Methyl (1S,4S,5R,6S)-6-Chloro-1-hydroxy-4,5-(isopropylidene-
dioxy)-3-oxocyclohexanecarboxylate (6)
72.5 (d), 74.4 (s), 75.1 (s), 113.1 (s), 174.1 (s).
MS (CI): m/z = 249 [M + H]+.
HRMS: m/z [M – CH3]+ calcd for C9H10ClO5: 233.0216; found:
To a suspension of Dess–Martin periodinane (579 mg, 1.35 mmol)
in CH2Cl2 (5 mL) was added 14 (190 mg, 0.68 mmol) in CH2Cl2 (2
mL) at r.t. After stirring for 2 h, the mixture was diluted with t-
BuOMe and treated with aq Na2S2O3 and aq NaHCO3. The organic
layer was washed with brine, dried (MgSO4), and filtered, and the
solvent was removed under reduced pressure to give a crude residue
that was purified by column chromatography (silica gel, 1%
MeOH–CH2Cl2) to afford 6 (146.2 mg, 78%) as colorless crystals
(Et2O–hexane); mp 115–118 °C.
[a]D26 +33.9 (c 0.45, MeOH).
IR (liquid film): 3275 (OH), 1760 (C=O), 1744 (C=O) cm–1.
1H NMR (CDCl3): d = 1.44 (s, 3 H, acetonide-Me), 1.50 (s, 3 H,
acetonide-Me), 2.85 (d, J = 14.4 Hz, 1 H, H2a), 3.00 (d, J = 14.4
Hz, 1 H, H2b), 3.89 (s, 3 H, COOMe), 4.52 (d, J = 5.5 Hz, 1 H, H4),
4.81 (dd, J = 5.5, 3.9 Hz, 1 H, H5), 4.91 (d, J = 3.9 Hz, 1 H, H6).
233.0221.
Methyl (1S,3R,4S,5S,6S)-6-Chloro-1,3-dihydroxy-4,5-(isopro-
pylidenedioxy)cyclohexanecarboxylate (14)
To a soln of 13 (24.0 mg, 0.10 mmol) in MeOH (1 mL) was added
NaOMe (7.0 mg, 0.13 mmol). The mixture was stirred at r.t. for 1 h,
after which a drop of AcOH was added and stirring was continued
for a further 15 min. The mixture was concentrated directly to give
a crude residue that was purified by column chromatography (silica
gel, 3% MeOH–CH2Cl2) to afford 14 (24.4 mg, 90%) as a colorless
oil.
[a]D26 +9.5 (c 1.71, MeOH).
IR (liquid film): 3474 (OH), 1742 (C=O) cm–1.
1H NMR (CDCl3): d = 1.41 (s, 3 H, acetonide-Me), 1.59 (s, 3 H, ac-
etonide-Me), 1.94 (dd, J = 14.0, 10.7 Hz, 1 H, H2b), 2.24 (dd,
J = 14.0, 4.4 Hz, 1 H, H2a), 2.55 (br s, 1 H OH), 3.58 (s, 1 H, OH),
3.86 (s, 3 H, COOMe), 4.06 (dd, J = 6.9, 5.2 Hz, 1 H, H4), 4.17
(ddd, J = 10.7, 6.9, 4.4 Hz, 1 H, H3), 4.53 (dd, J = 5.2, 4.1 Hz, 1 H,
H5), 4.60 (d, J = 4.1 Hz, 1 H, H6).
13C NMR (CDCl3): d = 26.0 (q), 27.0 (q), 47.4 (t), 53.8 (q), 57. 8 (d),
78.5 (d), 78.8 (d), 79.3 (s), 111.9 (s), 171.0 (s), 201.4 (s).
MS (EI): m/z = 263 [M – CH3]+.
HRMS: m/z [M – CH3]+ calcd for C10H12ClO6: 263.0321; found:
263.0326.
13C NMR (CDCl3): d = 25.8 (q), 28.2 (q), 38.6 (t), 53.7 (q), 58.8 (d),
Methyl (1S,4S,5R,6S)-6-Chloro-1,4,5-trihydroxy-3-oxocyclo-
hexanecarboxylate (16)
67.1 (d), 76.67 (d), 76.71 (s), 80.4 (d), 110.3 (s), 173.2 (s).
MS (CI): m/z = 281 [M + H]+.
HRMS: m/z [M – CH3]+ calcd for C10H14ClO6: 265.0478; found:
Compound 6 (16.1 mg, 0.058 mmol) was dissolved in MeOH (3
mL) and TFA (1 mL, excess) was added with stirring at r.t. After 1
h, the solvent was removed from the mixture under reduced pres-
sure to afford a residue that was purified by column chromatogra-
phy (silica gel, 5% MeOH–CH2Cl2) to afford 16 (10.1 mg, 70%) as
a colorless oil.
[a]D24 +16.7 (c 0.36, MeOH).
IR (liquid film): 3470 (OH), 1736 (C=O) cm–1.
1H NMR (CDCl3): d = 2.91 (d, J = 14.1 Hz, 1 H, H2b), 3.07 (dd,
J = 14.1, 0.9 Hz, 1 H, H2a), 3.92 (s, 3 H, COOMe), 4.42 (dd,
J = 3.8, 0.9 Hz, 1 H, H4), 4.56 (dd, J = 3.8, 2.7 Hz, 1 H, H5), 4.80
(d, J = 2.7 Hz, 1 H, H6).
265.0483.
Methyl (1S,3R,4S,5S,6S)-6-Chloro-1,3,4,5-tetrahydroxycyclo-
hexanecarboxylate (15)
Compound 7 (476 mg, 1.47 mmol) was dissolved in MeOH (4 mL)
and TFA (2 mL, excess) and the reaction flask was heated overnight
at 60 °C. After cooling, the solvent was removed under reduced
pressure to give a residue that was purified by column chromatog-
raphy (silica gel, 2% then 5% then 10% MeOH–CH2Cl2 gradient) to
afford pure 15 (129 mg, 38%) as colorless crystals; mp 157–160 °C;
together with methyl quinate (87.6 mg, 30%).
13C NMR (CDCl3): d = 47.4 (t), 54.1 (q), 59.1 (d), 76.8 (d), 77.3 (d),
[a]D26 –20.9 (c 0.49, MeOH).
IR (liquid film): 3441 (OH), 1748 (C=O) cm–1.
1H NMR (CD3OD): d = 1.81 (dd, J = 13.5, 11.7 Hz, 1 H, H2b), 2.19
(dd, J = 13.5, 5.0 Hz, 1 H, H2a), 3.45 (dd, J = 9.6, 3.0 Hz, 1 H, H4),
3.78 (s, 3 H, COOMe), 3.98 (ddd, J = 11.7, 9.6, 5.0 Hz, 1 H, H3),
4.10 (dd, J = 3.0, 2.8 Hz, 1 H, H5), 4.49 (d, J = 2.8 Hz, 1 H, H6).
13C NMR (CD3OD): d = 42.4 (t), 53.4 (q), 62.1 (d), 66.7 (d), 76. 7
(d), 76.7 (2 d), 80.6 (s), 173.8 (s).
HRMS: m/z [M + H]+ calcd for C8H14ClO6: 241.0478; found:
241.0482.
81.0 (s), 170.7 (s), 203.0 (s).
HRMS: m/z [M – H2O]+ calcd for C8H9ClO5: 220.0138; found:
220.0145.
Methyl (1S,4S,5R,6S)-6-Chloro-1-hydroxy-4,5-bis(trimethyl-
siloxy)-3-oxocyclohexanecarboxylate (17)
To soln of 16 (6.2 mg, 0.026 mmol) in CH2Cl2 (2 mL) were added
Et3N (14.5 mL, 0.10 mmol) and TMSCl (9.9 mL, 0.09 mmol,). The
mixture was stirred overnight at r.t., treated with sat. aq NH4Cl, and
extracted with CH2Cl2. The organic layer was dried (MgSO4) and
filtered, and the solvent was removed under reduced pressure to
give a crude residue that was purified by column chromatography
(silica gel, 1% MeOH–CH2Cl2) to afford 17 (7.1 mg, 71%) as col-
orless crystals (CH3Cl); mp <40 °C.
Anal. Calcd for C8H13O6Cl: C, 39.30; H, 5.45; found: C, 39.42; H,
5.46.
[a]D22 +23.4 (c 0.57, MeOH).
Synthesis 2007, No. 20, 3219–3225 © Thieme Stuttgart · New York

3224
Y. Usami, Y. Ueda
PAPER
IR (KBr): 3497 (OH), 1737 (C=O), 1732 (C=O) cm–1.
was treated with 1.0 M sodium potassium tartrate and sat. aq
NaHCO3, and extracted with EtOAc. The organic layer was dried
(MgSO4) and filtered, and the solvent was removed under reduced
pressure to give a crude residue that was purified by column chro-
matography (silica gel, 5% MeOH–CH2Cl2) to afford 1 (3.7 mg,
64%) as an oil.
[a]D30 –71.1 (c 0.09, EtOH).
IR (liquid film): 3383 (OH), 1721 (C=O), 1652 (C=C) cm–1.
1H NMR (CDCl3): d = 0.16 (s, 9 H, 4-OTMS), 0.18 (s, 9 H, 5-
OTMS), 2.89 (d, J = 14.9 Hz, 1 H, H2b), 2.97 (dd, J = 14.9, 0.9 Hz,
1 H, H2a), 3.85 (s, 3 H, COOMe), 4.38 (dd, J = 3.0, 0.9 Hz, 1 H,
H4), 4.50 (dd, J = 3.0, 2.1 Hz, 1 H, H5), 4.78 (d, J = 2.1 Hz, 1 H,
H6), 4.89 (s, 1 H, OH).
13C NMR (CDCl3): d = 0.05 (q), 0.1 (q), 49.2 (t), 53.4 (q), 59.7 (d),
77.8 (d), 81.1 (s), 81.2 (d), 171.1 (s), 201. 3 (s).
HRMS: m/z [M]+ calcd for C14H27ClO6Si2: 382.1021; found:
382.1027.
1H NMR (acetone-d6): d = 3.72 (dd, J = 5.9, 2.3 Hz, 1 H, H4), 3.76
(s, 3 H, COOMe), 4.16 (dd, J = 4.1, 2.3 Hz, 1 H, H5), 4.60 (m, 1 H,
H3), 5.07 (dt, J = 4.1, 1.4 Hz, 1 H, H6), 6.73 (d, J = 3.0, 1.4 Hz, 1
H, H2).
13C NMR (acetone-d6): d = 52.2 (q), 58.0 (d), 69.6 (d), 70.6 (d), 75.
3 (d), 130.9 (d), 141.6 (s), 166.4 (s).
Methyl (4R,5S,6R)-6-Chloro-4,5-bis(trimethylsiloxy)-3-oxo-
cyclohex-1-enecarboxylate (18)
Method A: To a CH2Cl2 soln (5 mL) of 17 (79.5 mg, 0.21 mmol)
were added Me3N (228 mL, 2.10 mmol) and SOCl2 (153 mL, 2.10
mmol) at –10 °C. After stirring at 0 °C for 3 h, the mixture was treat-
ed with sat. aq NaHCO3 and extracted with CH2Cl2. The organic
layer was dried (MgSO4) and filtered, and the solvent was removed
under reduced pressure to give a crude residue that was purified by
column chromatography (silica gel, 1% MeOH–CH2Cl2) to afford
18 (22.6 mg, 30%) and desilylated 16 (7.3 mg, 16%).
HRMS: m/z [M]+ calcd for C8H1235ClO5: 223.0372; found:
223.0360.
Methyl (3R,4S,5S,6R)-6-Chloro-3-hydroxy-4,5-(isopropy-
lidenedioxy)cyclohex-1-enecarboxylate (19)
To a soln of 1 (2.0 mg, 0.009 mmol) in acetone (0.5 mL) were added
2,2-dimethoxypropane (0.5 mL, excess) and a catalytic amount of
TsOH·H2O. After stirring at r.t. for 3 h, volatile components were
removed under reduced pressure to give a crude residue that was pu-
rified by column chromatography (silica gel, 2% MeOH–CH2Cl2)
to afford 19 (1.7 mg, 72%) as an oil.
Method B: To a CH2Cl2 soln (1 mL) of 17 (7.9 mg, 0.021 mmol) was
added 1 M Martin’s sulfurane dehydrating agent in CH2Cl2 (0.93
mL, 0.93 mmol) at 0 °C, and the mixture was stirred for 3 h. The
mixture was treated in the same manner as method A to afford 18
(4.9 mg, 65%) as colorless crystals (CH2Cl2); mp 88–90 °C.
[a]D22 +34.4 (c 0.87, MeOH).
IR (KBr): 1732 (C=O), 1711 (C=O), 1508 (C=C) cm–1.
1H NMR (CDCl3): d = 0.12 (s, 9 H, OTMS), 0.17 (s, 9 H, OTMS),
3.86 (s, 3 H, COOMe), 4.27 (d, J = 2.3 Hz, 1 H, H4), 4.37 (dd,
J = 3.4, 2.3 Hz, 1 H, H5), 5.12 (dd, J = 3.4, 2.3 Hz, 1 H, H6), 6.53
(d, J = 2.3 Hz, 1 H, H2).
13C NMR (CDCl3): d = 0.15 (q), 0.3 (q), 52.7 (q), 56.5 (d), 76.5 (d),
77.9 (d), 130.6 (d), 143.7 (s), 165.5 (s), 195.8 (s).
IR (liquid film): 3446 (OH), 1718 (C=O), 1646 (C=C) cm–1.
1H NMR (CDCl3): d = 1.41 (s, 3 H, acetonide-Me), 1.62 (s, 3 H, ac-
etonide-Me), 3.82 (s, 3 H, COOMe), 4.31 (ddd, J = 8.7, 5.7, 0.7 Hz,
1 H, H4), 4.36 (dd, J = 8.7, 4.8 Hz, 1 H, H5), 4.98 (dd, J = 5.7, 2.1
Hz, 1 H, H3), 5.22 (dd, J = 4.8, 0.7 Hz, 1 H, H6), 7.14 (ddd, J = 2.1,
1.1, 0.7 Hz, 1 H, H2).
HRMS: m/z [M + H]+ calcd for C11H1635ClO5: 263.0685; found:
263.0695.
Methyl (3S,4S,5S,6R)-6-Chloro-3,4,5-trihydroxycyclohex-
1-enecarboxylate (3)
HRMS: m/z [M]+ calcd for C14H2535ClO5Si2: 364.0927; found:
364.0930.
To a suspension of NaBH4 (0.74 mg, 0.02 mmol) in MeOH (1.5 mL)
was added dropwise 18 (7.7 mg, 0.02 mmol) in MeOH (0.5 mL) at
–10 °C. After stirring for 30 min, the mixture was treated with sat.
aq NH4Cl and extracted with CH2Cl2. The organic layer was dried
(MgSO4) and filtered, and the solvent was removed under reduced
pressure to give almost pure 20 (7.3 mg) as an oil.
1H NMR (CDCl3): d = 0.17 [s, 9 H, Si(CH3)3], 0.18 [s, 9 H,
Si(CH3)3], 3.79 (s, 3 H, COOMe), 4.77 (br d, J = 4.2 Hz, 1 H, H6),
6.81 (br d, J = 2.6 Hz, 1 H, H2).
Methyl (4R,5S,6R)-6-Chloro-4,5-dihydroxy-3-oxocyclohex-
1-enecarboxylate (5)
Compound 18 (27.3 mg, 0.075 mmol) was dissolved in MeOH (1
mL) and TFA (0.5 mL, excess) was added with stirring at r.t. After
15 min, the solvent was removed from the mixture under reduced
pressure to afford a residue that was purified by column chromatog-
raphy (silica gel, 2% MeOH–CH2Cl2) to afford 5 (16.7 mg, 100%)
as colorless crystals (CH2Cl2); mp 97–100 °C.
[a]D22 undetermined (almost 0).
IR (KBr): 3501 (OH), 1725 (C=O), 1708 (C=O), 1625 (C=C) cm–1.
1H NMR (CDCl3): d = 3.90 (s, 3 H, COOMe), 4.37 (d, J = 2.5 Hz,
1 H, H4), 4.60 (dd, J = 3.4, 2.5 Hz, 1 H, H5), 5.27 (dd, J = 3.4, 2.3
Hz, 1 H, H6), 6.72 (d, J = 2.3 Hz, 1 H, H2).
13C NMR (CDCl3): d = 53.0 (q), 56.3 (d), 73.2 (d), 76.0 (d), 129.6
(d), 144.7 (s), 164.8 (s), 196.4 (s).
MS (CI): m/z = 221 [M + H]+.
HRMS: m/z [M – H2O]+ calcd for C8H735ClO4: 202.0030; found:
Without purification, 20 was dissolved in MeOH (1 mL) and TFA
(0.5 mL). After stirring at r.t. for 30 min, the solvent was removed
under reduced pressure to afford a residue that was purified by col-
umn chromatography (silica gel, 5% MeOH–CH2Cl2) to afford 3
(4.8 mg, 100% in 2 steps) as an oil.
[a]D22 +58.3 (c 0.01, EtOH).
IR (liquid film): 3373 (OH), 1726 (C=O), 1653 (C=C) cm–1.
1H NMR (acetone-d6): d = 3.78 (s, 3 H, COOMe), 3.89 (ddd,
J = 8.7, 5.0, 2.3 Hz, 1 H, H5), 4.10 (m, 1 H OH), 4.16 (d, J = 9.2 Hz,
1 H, OH), 4.22 (m, 1 H, H3), 4.28 (d, J = 8.7 Hz, 1 H, 5-OH), 4.36
(m, 1 H, H4), 5.03 (br d, J = 5.0 Hz, 1 H, H6), 6.81 (br d, J = 2.5 Hz,
1 H, H2).
13C NMR (acetone-d6): d = 52.3 (q), 56.5 (d), 68.8 (d), 67.0 (d), 71.6
(d), 132.0 (d), 142.7 (s), 168.0 (s).
202.0031.
Methyl (3R,4S,5S,6R)-6-Chloro-3,4,5-trihydroxycyclohex-
1-enecarboxylate (1)
To a soln of Me4NBH(OAc)3 (52.8 mg, 0.20 mmol,) in AcOH–
MeCN (1:1, 1 mL) was added dropwise 5 (5.8 mg, 0.026 mmol) in
MeCN (0.5 mL) at –20 °C. After stirring for 30 min, the mixture
HRMS: m/z [M]+ calcd for C8H1235ClO5: 223.0373; found:
223.0379.
Synthesis 2007, No. 20, 3219–3225 © Thieme Stuttgart · New York

PAPER
Stereoselective Syntheses of Diastereomers of Antitumor Natural Product Pericosine A
3225
Methyl (3R,4R,5S,6R)-6-Chloro-5-hydroxy-3,4-(isopropy-
lidenedioxy)cyclohex-1-enecarboxylate (21)
(5) Numata, A.; Iritani, M.; Yamada, T.; Minoura, K.;
Matsumura, E.; Yamori, T.; Tsuruo, T. Tetrahedron Lett.
1997, 38, 8215.
(6) For reviews, see (a) Suami, T. Pure Appl. Chem. 1987, 59,
1509. (b) Suami, T.; Ogawa, S. Adv. Carbohydr. Chem.
Biochem. 1990, 48, 21. (c) Suami, T. Top. Curr. Chem.
1990, 154, 257. (d) Berecibar, A.; Grandjean, C.;
Sinwardena, A. Chem. Rev. 1999, 99, 779.
To a soln of 3 (29.8 mg, 0.134 mmol) in acetone (1 mL) were added
2,2-dimethoxypropane (1 mL, excess) and a catalytic amount of
TsOH·H2O. After stirring at r.t. for 15 min, volatile components
were removed under reduced pressure to give a crude residue that
was purified by column chromatography (silica gel, 1% MeOH–
CH2Cl2) to afford 21 (19.3 mg, 55%) as an oil.
(7) Usami, Y.; Numata, A. Chem. Pharm. Bull. 2004, 52, 1125.
(8) (a) Usami, Y.; Hatsuno, C.; Yamamoto, H.; Tanabe, M.;
Numata, A. Chem. Pharm. Bull. 2004, 52, 1130. (b) Usami,
Y.; Hatsuno, C.; Yamamoto, H.; Tanabe, M.; Numata, A.
Chem. Pharm. Bull. 2005, 53, 721; errata.
(9) Donohoe, T. J.; Blades, K.; Helliwell, M.; Waring, M. J.;
Newcombe, N. J. Tetrahedron Lett. 1998, 39, 8755.
(10) Garcia Ruano, J.; Lopez-Cantarero, J.; Alemparte, C.
Phosphorus, Sulfur Silicon Relat. Elem. 2005, 180, 1493.
(11) Usami, Y.; Ueda, Y. Chem. Lett. 2005, 34, 1062.
(12) Barco, A.; Benetti, S.; Risi, C. D.; Marchetti, P.; Pollini, G.
P.; Zanirato, V. Tetrahedron: Asymmetry 1997, 8, 3515.
(13) Banwell, M. G.; Hungerford, N. L.; Jolliffe, K. A. Org. Lett.
2004, 6, 2737.
IR (liquid film): 3431 (OH), 1718 (C=O), 1646 (C=C) cm–1.
1H NMR (CDCl3): d = 1.39 (s, 3 H, acetonide-Me), 1.42 (s, 3 H,
acetonide-Me), 3.02 (d, J = 11.9 Hz, 1 H, 5-OH), 3.84 (s, 3 H,
COOMe), 3.96 (dddd, J = 11.9, 4.9, 3.1, 1.1 Hz, 1 H, H5), 4.56
(ddd, J = 5.9, 3.1, 1.1 Hz, 1 H, H4), 4.77 (ddd, J = 5.9, 3.5, 0.9 Hz,
1 H, H3), 5.10 (t, J = 4.9, 0.9 Hz, 1 H, H6), 6.92 (dd, J = 3.5, 0.9 Hz,
1 H, H2).
HRMS: m/z [M + H]+ calcd for C11H1635ClO5: 263.0685; found:
263.0693.
Acknowledgment
We are grateful to Dr. K. Minoura, Ms. M. Fujitake, and Ms. S.
Okabe of this University for NMR measurements, MS measure-
ments, and elemental analyses, respectively. This work was suppor-
ted in part by a Grant-in-Aid for ‘High-Tech Research Center’
Project for Private Universities: matching fund subsidy from
MEXT (Ministry of Education, Culture, Sports, Science and Tech-
nology), 2002–2006, Japan.
(14) Manthey, M. K.; Gonzalez-Bello, C.; Abell, C. J. Chem.
Soc., Perkin Trans. 1 1997, 625.
(15) Gonzalez-Bello, C.; Manthey, M. K.; Harris, J. H.; Hawkins,
A. R.; Coggins, J. R.; Abell, C. J. Org. Chem. 1998, 63,
1591.
(16) Cherest, M.; Felkin, H. Tetrahedron Lett. 1968, 9, 2205.
(17) Anh, N. T. Top. Stereochem. 1980, 88, 145.
(18) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
(19) Alhalt, R. J.; Martin, J. C. J. Am. Chem. Soc. 1972, 94, 5003.
(20) Evans, D. A.; Chapman, K. T. Tetrahedron Lett. 1986, 27,
5939.
(21) Usami, Y.; Horibe, Y.; Takaoka, I.; Ichikawa, H.; Arimoto,
M. Synlett 2006, 1598.
(22) Usami, Y.; Takaoka, I.; Ichikawa, H.; Horibe, Y.;
Tomiyama, S.; Ohtsuka, M.; Imanishi, Y.; Arimoto, M. J.
Org. Chem. 2007, 72, 6127.
References
(1) Yotsu, M.; Yamazaki, T.; Meguro, Y.; Endo, A.; Murata,
M.; Naoki, H.; Yasumoto, T. Toxicon 1987, 25, 225.
(2) Moore, R. E.; Helfrich, P.; Patterson, G. M. L. Oceanus
1989, 25, 54.
(3) Newman, D. J.; Cragg, G. M. Curr. Drug Targets 2006, 7,
279.
(4) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666.
Synthesis 2007, No. 20, 3219–3225 © Thieme Stuttgart · New York