Synthetic racemate and enantiomers of cytosporone E, a metabolite of an endophytic fungus, show indistinguishably weak antimicrobial activity

Article abstract of DOI:10.1271/bbb.67.2584

The racemate and the enantiomers of cytosporone E [3-heptyl-4,5,6- trihydroxyphthalide (1)], a metabolite of the endophytic fungus, CR200 (Cytospora sp.), were synthesized. The key steps were (i) Sharpless asymmetric dihydroxylation of an alkene (8) and (

Full text of DOI:10.1271/bbb.67.2584

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Synthetic Racemate and Enantiomers of Cytosporone
E, a Metabolite of an Endophytic Fungus, Show
Indistinguishably Weak Antimicrobial Activity
Tomoya OHZEKIa & Kenji MORIb
a Department of Chemistry, Graduate School of Science, Science University of
TokyoKagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan
b Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, RIKEN
Research Center for Allergy and Immunology, c/o Seikagaku CorporationTateno 3-1253,
Higashiyamato City, Tokyo 207-0021, Japan
Published online: 22 May 2014.
To cite this article: Tomoya OHZEKI & Kenji MORI (2003) Synthetic Racemate and Enantiomers of Cytosporone E, a
Metabolite of an Endophytic Fungus, Show Indistinguishably Weak Antimicrobial Activity, Bioscience, Biotechnology, and
Biochemistry, 67:12, 2584-2590, DOI: 10.1271/bbb.67.2584
To link to this article: http://dx.doi.org/10.1271/bbb.67.2584
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Biosci. Biotechnol. Biochem., 67 (12), 2584–2590, 2003
Synthetic Racemate and Enantiomers of Cytosporone E, a Metabolite of an
*
Endophytic Fungus, Show Indistinguishably Weak Antimicrobial Activity
1
2,
†
Tomoya OHZEKI and Kenji MORI
1Department of Chemistry, Graduate School of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan
2Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, RIKEN Research Center for Allergy
and Immunology, c o Seikagaku Corporation, Tateno 3-1253, Higashiyamato City, Tokyo 207-0021, Japan
W
Received June 19, 2003; Accepted July 28, 2003
The racemate and the enantiomers of cytosporone E
[3-heptyl-4,5,6-trihydroxyphthalide (1)], a metabolite of
the endophytic fungus, CR200 (Cytospora sp.), were
synthesized. The key steps were (i) Sharpless asymmetric
dihydroxylation of an alkene (8) and (ii) HPLC separa-
tion of the enantiomers of tert-butyldimethylsilyl ether
(12) on a chiral stationary phase. The racemate and
enantiomers of cytosporone E showed only weak
antimicrobial activity with no diŠerence among them.
Key words: antimicrobial compound; asymmetric
dihydroxylation; enantiomeric separa-
tion; endophytic fungus; phthalide
In 2000, Clardy and his co-workers isolated
Scheme 1. Synthesis of (
Reagents: (a) (C2H5)2NH, THF (95
(CH3)2 N(CH2)2 N(CH3)2, THF. ii) CH3(CH2)6CHO. iii) KOH,
CH3OH, H2O. iv) 2 HCl (71 ). (c) BBr3, CH2Cl2 (79 ).
±
)-Cytosporone E (1).
cytosporones A-E as new octaketide metabolites of
the endophytic fungus, CR200 (Cytospora sp.).1)
Two of them, cytosporone D and E (1), were report-
ed to show strong antimicrobial activity, while the
others were biologically inactive.1) Their structures
were clariˆed mainly by an NMR analysis. In the case
of cytosporone C (see Scheme 1), its structure was
resolved by an X-ray crystallographic analysis.
According to Clardy et al., the space group in which
it crystallized required cytosporone C to be isolated
as a racemic mixture.1) Although the speciˆc rotation
values of cytosporones were not reported, it could be
speculated that cytosporones in general might have
been isolated as racemates. This speculation aroused
our curiosity to scrutinize whether there would be
any diŠerence between the enantiomers of cyto-
sporone E (1) with regard to their antimicrobial ac-
tivity. It is well established that the enantiomers of a
bioregulator usually diŠer in their biological activi-
ties.2,3) We therefore undertook the syntheses of
the racemate and enantiomers of cytosporone E
[3-heptyl-4,5,6-trihydroxyphthalide (1)]. This paper
describes the synthesis and biological evaluation of
z
). (b) i) sec-BuLi,
M
z
z
±
(
)-1, (R)-1 and (S)-1 which revealed their anti-
microbial activities to be weak and almost the same.
Results and Discussion
±
Our synthesis of ( )-cytosporone E (1) is summa-
rized in Scheme 1. In analogy with our previous
synthesis of bioactive and naturally occurring
phthalides,4,5) the Snieckus's ortho lithiation
methodology6,7) was employed as the key step.
Accordingly,
trimethoxybenzoyl chloride (2) was treated with
diethylamine to give -diethylamide 3. Lithiation
of 3 with sec-butyllithium in the presence of
-tetramethylethylenediamine (TMEDA)
commercially
available
3,4,5-
N, N
N, N, N?, N?
was followed by the addition of octanal. The result-
ing crude mixture was saponiˆed with potassium
hydroxide and then acidiˆed with dilute hydrochloric
*
Synthetic Microbial Chemistry, Part 41. For Part 40, see ref. 5.
To whom correspondence should be addressed.
†

Synthesis of Cytosporone E
2585
±
±
acid to give lactone ( )-4. Treatment of ( )-4 with
±
boron tribromide aŠorded ( )-1, mp 162–1659C,
whose H- and 13C-NMR data were in good agree-
1
ment with those reported for cytosporone E.1) The
overall yield of (
To synthesize the enantiomers of 1, we ˆrst expect-
ed that our methods for the synthesis of ( )- and ( )-
±
)-1 was 53 based on 2 (3 steps).
z
R
S
3-butyl-7-hydroxyphthalide would work perfectly:5)
(i) Miyaura-Suzuki coupling (Scheme 2, 7
ª
8),8) (ii)
ª
9)9) and
Sharpless asymmetric dihydroxylation (8
(iii) Barton radical deoxygenation.10) Our expectation
concerning the high enantioselectivity of the asym-
metric dihydroxylation turned out to be too optimis-
tic, and we ˆnally had to use the less sophisticated
method of the enantiomeric separation of 12 by
preparative HPLC.
Considering the ease of its later removal, the
benzyl group was selected as the protective group for
the phenolic hydroxy groups of 1, and commercially
available methyl 3,4,5-trihydroxybenzoate (5) was
benzylated to give known 6.11) Bromination of
6
with
N-bromosuccinimide (NBS) aŠorded 7,
which was subjected to Miyaura-Suzuki coupling
with (E
)-1-octeneboronic acid12) in the presence of
tetrakis(triphenylphosphine)palladium and potassi-
um carbonate to give oleˆn 8. Asymmetric dihydro-
9) proceeded sluggishly
}
xylation of 8 with AD-mix-
b
to furnish enantiomerically impure (3
e.e. as determined by an HPLC analysis on Daicel
Chiralcel} OG) in a 54
yield after a week at 4 C.
R,1?R)-9 (45
z
z
9
This unfavorable result was in contrast to our previ-
ous observation that asymmetric dihydroxylation
of methyl (
E
)-2-benzyloxy-6-(1
?
-pentenyl)benzoate
gave the desired product of
À
99 e.e. The
z
neighboring benzyloxy group in 8 must have inter-
fered and caused deterioration in the enantioselec-
tivity of the reaction.
Scheme 2. Synthesis of (
Reagemts: (a) BnBr, K2CO3, DMF (96
(96 ). (c) )-CH3(CH2)5CH CHB(OH)2, Pd(PPh3)4,
K2CO3, C6H6 EtOH (5:1) (71 of 8 and 19 of recovered 7).
(d) AD-mix- H2O (1:1) [54 of
}, CH3SO2NH2, tert-BuOH
(3 ,1 )-9 and 46 of recovered 8]. (e) 1,1-thiocarbonyldii-
midazole, CH2Cl2 (quant.). (f) ( -Bu)3SnH, AIBN, toluene
(90 ). (g) H2, Pd(OH)2-C, EtOAc (92 ). (h) TBSCl, imida-
zole, DMF (98 ). (i) i) HPLC separation. ii) 4 HCl EtOH
(1:5) [40 of ( )-1 and 58 of recovered ( )-12]. ( j) AD-mix-
}, CH3SO2NH2, tert-BuOH
H2O (1:1) [39
R
)- and (
S
)-Cytosporone E (1).
Although resulting enantiomerically impure
z
). (b) NBS, DMF
(3R,1?R)-9 was an oil, there was a possibility that
one of the intermediates or even ˆnal product (S)-1
z
(
W
E
＝
z
z
W
b
z
could be su‹ciently crystalline to enable us its puriˆ-
cation by recrystallization. We therefore continued
the synthesis. In order to deoxygenate the hydroxy
R
?
R
z
n
z
z
group at C-1
9, it was converted to thiocarbonyl derivative
(3 ,1 ,1 )-10
)-10. Barton deoxygenation10) of (3
gave tribenzylated ( )-cytosporone E (11) which was
debenzylated with the Pearlman catalyst under
hydrogen to give ( )-cytosporone E (1) with low
? of enantiomerically impure (3R,1?R)-
z
S
M
W
z
z
S
z
a
W
of (3
S
,1?S
)-9 and
R
?R
R ?R
59
z
of recovered 8].
S
S
enantiomeric purity. Unfortunately, none of these
compounds was acceptably crystalline, and we had to
search for other possibilities to improve the enantio-
Chiralcel} OD to give pure (
Deprotection of the TBS groups of (
conventional conditions with tetra(
um ‰uoride, however, caused partial racemization of
)-1. Fortunately, treatment of ( )-12 with dilute
ethanoic hydrochloric acid at room temperature
caused no appreciable racemization to give ( )-1,
yield. The
deprotection conditions were so mild that starting
S
)-12 (98.4
z
e.e.).
S
)-12 under
n
-butyl)ammoni-
meric purity of (
Upon examining the HPLC behavior of (
its tribenzyl derivative ( )-11 and other derivatives
of ( )-1, we found that the enantiomers of tris-
tert-butyldimethlysilyl(TBS)-protected derivative
12 could be separated by preparative HPLC on
S)-1.
S
)-1,
(
S
S
S
S
S
24
－
90.7 (c 1.00, acetone), in a 40z
[a
]
D

2586
T. OHZEKI and K. MORI
)-1, and Itraconazole}
±
Table 1. Antimicrobial Activities of ( )-Cytosporone E (1), (
R
)-1, (
S
Minimum Concentration for Growth Inhibition (
Sample
mgWml)
Microorganism
(
)-1
(
R
)-1
(S)-1
Itraconazole}
±
Candida albicans ATCC24433
Candida albicans SANK51486
Candida albicans TIMM3164
Candida albicans ATCC64550
Candida parapsilosis ATCC90018
Candida glabrata ATCC90030
Candida krusei ATCC6258
Candida tropicalis ATCC750
Cryptococcus neoformans TIMM1855
Aspergillus fumigatus ATCC26430
Aspergillus fumigatus SANK10569
Aspergillus ‰avas SANK18497
À
À
8
8
8
8
8
4
8
8
8
8
8
8
À
À
8
8
8
8
8
4
8
8
8
8
8
8
À
À
8
8
8
8
8
4
8
8
8
8
8
8
0.125
0.031
0.25
1
0.125
1
0.25
0.5
0.25
0.25
0.25
0.5
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
(
S
)-12 was recovered in a 58
was proved to be of 98.4
)-12 which could be precisely analyzed by HPLC.
z
yield. Resulting (
S
)-1
NMR spectra were recorded at 500 MHz with a Jeol
z
e.e. by its conversion to
JNM-LA500 spectrometer. The peaks for TMS (at
d
＝
＝
(
S
0.00) and acetone-
d
5 (at
d
2.04) were used for the
For the synthesis of (
R
)-cytosporone E (1), asym-
internal standards. 13C-NMR data were recorded at
126 MHz with a Jeol JNM-LA500 spectrometer. The
metric dihydroxylation of 8 was conducted with AD-
}
＝ ＝
77.0) and acetone-d6 [at d
mix-
a
to give (3
S
,1
?
S
)-9 of only 36
z
e.e. This was
peaks for CDCl3 (at
d
converted to (
R
)-12 of low e.e. and separated by
)-cytosporone E (1),
1.00, acetone), whose enantiomeric
99 e.e. as estimated by an HPLC
analysis of corresponding tris-TBS derivative ( )-12.
With all three of the desired samples ( )-1, ( )-1
and ( )-1 in hand, the antimicrobial activities of
29.8 (CD3)] were used for the internal standards.
Optical rotation was measured with a Jasco P-1010
polarimeter, and EI-MS data were recorded with a
Jeol JMS-AX505HA spectrometer. Column chro-
matography was carried out with Merck Kieselgel 60
Art 1.07734, and TLC analyses were performed with
Merck 60F-254 silica gel plates.
preparative HPLC to give (
R
25
＋
[
a
]
91.3 (
c
D
purity was
À
z
R
±
R
S
these samples were examined at Sankyo Co. by
employing twelve diŠerent microorganisms, includ-
N, N-Diethyl-3,4,5-trimethoxybenzamide (3). Un-
ing four strains of Candida albicans
C. glabrata C. krusei C. tropicalis
neoformans, two strains of Aspergillus fumigatus
,
C. parapsilosis
,
der an argon atmosphere, a solution of 3,4,5-
trimethoxybenzoyl chloride (2.31 g, 10.0 mmol) in
THF (20 ml) was stirred and cooled with an ice-water
bath, treated with diethylamine (4.14 ml, 40.0
mmol), and then further stirred for 30 min at room
temperature. This mixture was concentrated in
vacuo, and the residue was diluted with EtOAc. The
solution was successively washed with water and
brine, and dried with MgSO4, the solvent being
removed in vacuo to give a colorless solid. The solid
,
,
,
Cryptococcus
,
and Asp. ‰avas. The results are shown in Table 1.
All three samples showed the same degree of anti-
microbial activity, which was far weaker than that of
the practical antimicrobial agent, itraconazole}
(Janssen).
±
In conclusion, ( )-, (R)-, and (S)-cytosporone E
(1) were synthesized. Although cytosporone E has
been claimed to show strong antimicrobial activity,1)
our synthetic samples were only weakly active as
antimicrobial agents. The stereochemistry at C-3 of 1
had no particular eŠect on the bioactivity of 1, and
no diŠerence was apparent with regard to the
antimicrobial activity of the enantiomers of 1. In this
particular case, therefore, bioactivity did not depend
on the absolute conˆguration of the bioactive
molecule.
was recrystallized from hexane-EtOAc to give 3
1
(2.54 g, 95
z
＝
), mp 48.0–49.0
9
＝
C. IR nmax (KBr) cm－
:
1630 (s, C O), 1585 (s, C C), 1505 (w), 1470 (s),
1410 (s), 1235 (s), 1125 (s), 1005 (s). NMR dH
(500 MHz, CDCl3): 1.18 (3 H, br., NCH2C
(3 H, br., NCH2C
H, br., NC
H
3), 1.23
2), 3.53 (2
2), 3.86 (3 H, s, 4-OMe), 3.87 (6 H, s,
H3), 3.30 (2 H, br., NCH
H
3-OMe, 5-OMe), 6.60 (2 H, s, 2-H, 6-H). NMR dC
(126 MHz, CDCl3): 12.8 (q), 14.2 (q), 39.2 (t), 43.2
(t), 56.1 (q), 60.7 (q), 103.5 (d), 132.5 (s), 138.6 (s),
153.1 (s), 170.8 (s). Anal. Found: C, 62.95; H, 7.90;
Experimental
N, 5.12
N, 5.24
z. Calcd. for C14H21NO4: C, 62.90; H, 7.92;
Melting point (mp) data were measured with a
z
.
Yanaco MP-S3 instrument. IR spectra were meas-
1
ured with a Jasco FT IR-410 spectrometer, and H-
W

Synthesis of Cytosporone E
2587
±
±
4
(
)-1-Heptyl-3,4,5-trimethoxyphthalide [( )-
].
l,
br. s, OH), 8.33 (1 H, br. s, OH), 9.09 (1 H, br. s,
Under an argon atmosphere, a ‰ask was charged with
OH). NMR dC (126 MHz, acetone-d6): 14.3, 23.3,
－
THF (50 ml) and cooled to 78
9
C. TMEDA (990
m
25.4, 29.9, 30.0, 32.5, 33.8, 80.0, 102.9, 118.0, 130.3,
7.00 mmol) followed by sec-butyllithium in cyclo-
139.5, 140.6, 147.6, 170.9. HREIMS m z (M＋):
W
hexane-hexane (1.0
M
, 7.00 ml, 7.00 mmol) were then
calcd. for C15H20O5, 280.1311; found, 280.1303.
added. A solution of 3 (1.34 g, 5.01 mmol) in THF
(5 ml) was next added dropwise to the ‰ask. This
Methyl 2-Bromo-3,4,5-tribenzyloxybenzoate (
7).
solution was stirred for 1 h, before octanal (760
m
l,
A solution of -bromosuccinimide (196 mg, 1.10
N
－
7.70 mmol) was added to the solution at 78
9
C, and
mmol) in DMF (4.0 ml) was added dropwise to a
solution of 6 (455 mg, 1.00 mmol) in DMF (1.0 ml),
and the mixture was stirred for 16 h at room tempera-
ture. The mixture was diluted with water, and
extracted with EtOAc. The organic layer was succes-
sively washed with water and brine, dried with
MgSO4, and concentrated in vacuo to give crude 7 as
a brown solid. This crude product was recrystallized
from hexane-EtOAc to give 7 as needles (513 mg,
the cooling bath was removed after 5 min. The solu-
tion was stirred for 3 h, and water was added. This
mixture was concentrated in vacuo, and the residue
was diluted with EtOAc. The solution was successive-
ly washed with water and brine, dried with MgSO4,
and concentrated under reduced pressure. A solution
of KOH (3.92 g, 70 mmol) in methanol (20 ml) and
water (2 ml) was added to the residue, and the
mixture was stirred for 16 h. The reaction mixture
was washed with hexane. The aqueous layer was
96
z
＝
), mp 92.0–93.0
9
＝
C. IR
n
max (KBr) cm－1: 1720 (s,
O), 1585 (w, C C), 1560 (w), 1370 (s), 1335 (s),
1100 (s), 740 (s), 700 (s). NMR dH (500 MHz,
C
acidiˆed with 4
M
HCl aq., and extracted with
EtOAc. The organic layer was successively washed
with water and brine, dried with MgSO4, and the
solvent was removed in vacuo. The residue was chro-
matographed on silica gel (40 g, eluted with hexane-
CDCl3): 3.92 (3 H, s, OMe), 5.03 (2 H, s, PhC
5.09 (2 H, s, PhC 2), 5.10 (2 H, s, PhC
H
H
2),
2),
H
7.26–7.51 (16 H, m, 6-H, aromatic). NMR dC
(126 MHz, CDCl3): 52.5, 71.3, 75.4, 75.7, 110.3,
112.1, 127.6, 127.7, 128.2, 128.25, 128.31, 128.4,
128.58, 128.61, 128.7, 136.0, 136.6, 136.7, 145.8,
150.9, 151.7, 166.4. Anal. Found: C, 65.67; H,
EtOAc) to give (
±
)-4 (1.15 g, 71
z
) as a colorless oil.
1
＝
＝
IR
n
max (ˆlm) cm－ : 1765 (s, C O), 1615 (w, C C),
1480 (s), 1345 (s), 1255 (w), 1105 (s). NMR dH
(500 MHz, CDCl3): 0.87 (3 H, t, 6.9 Hz, 7 -H),
1.22–1.24 (10 H, m, 2 , 3 , 4 , 5 -H), 1.66–1.73
(1 H, m, 1 -H), 2.16–2.22 (1 H, m, 1 -H), 3.92 (3 H,
s, OMe), 3.94 (3 H, s, OMe), 3.98 (3 H, s, OMe),
J
＝
?
z z
4.82 . Calcd. for C29H25BrO5: C, 65.30; H, 4.72 .
?
?
?
?, 6?
?
?
(E )-Methyl 2-(1-Octenyl)-3,4,5-tribenzyloxyben-
zoate ( ). To a stirred solution of 7 (24.0 g, 45.0
mmol) in benzene (300 ml) and ethanol (50 ml) were
added ( )-1-octeneboronic acid (9.36 g, 60.0 mmol),
8
＝
7.8, 2.9 Hz, 3-H), 7.25 (1 H, s,
5.46 (1 H, dd,
J
7-H). NMR C (126 MHz, CDCl3): 13.9 (q), 22.4 (t),
d
E
24.5 (t), 28.9 (t), 29.1 (t), 31.5 (t), 33.3 (t), 56.2 (q),
60.6 (q), 60.9 (q), 80.0 (d), 102.4 (d), 121.2 (s), 135.2
(s), 146.6 (s), 147.4 (s), 155.4 (s), 170.3 (s). Anal.
Pd(PPh3)4 (1.04 g, 0.900 mmol), and K2CO3 (37.3 g,
270 mmol). The mixture was re‰uxed for 14 h under
an Ar atmosphere. The mixture was then diluted with
Found: C, 67.12; H, 8.38
67.06; H, 8.13
z. Calcd. for C18H26O5: C,
EtOAc and successively washed with 1
M
NaOH aq.,
z
.
water and brine. The organic layer was dried with
MgSO4, and the solvent was removed in vacuo. The
residue was chromatographed on silica gel (100 g,
(
±
)-Cytosporone E [(
±
)-
1
]. A solution of boron
, 5.31 ml, 5.31
tribromide in dichloromethane (1.0
M
eluted with hexane-EtOAc) to give 8 (18.6 g, 71
a colorless oil and starting material 7 (4.57 g, 19
8: IR nmax (ˆlm) cm－1: 1720 (s, C
O), 1590 (m, C
C), 1560 (w), 1330 (s), 1110 (s), 735 (s), 695 (s). NMR
z
z
) as
mmol) was added dropwise to a stirred solution of
).
phthalide 4 (380 mg, 1.18 mmol) in dichloromethane
＝
＝
(5 ml), stirring being continued for 15 min at 09C.
The reaction mixture was quenched with water and
diluted with EtOAc. The organic layer was succes-
sively washed with water and brine, dried with
MgSO4, and concentrated in vacuo. The residue was
chromatographed on silica gel (20 g, eluted with
dH (500 MHz, CDCl3): 0.88 (3 H, t,
J
＝
6.9 Hz,
-H), 2.18 (1 H,
-H), 3.84 (3 H, s, OMe),
2), 5.09 (2 H, s, PhC 2), 5.10
16.2, 6.9 Hz,
16.2, 1.5, 1.5 Hz, 1 -H),
8?
-H), 1.25–1.43 (8 H, m, 4
?
, 5
?
, 6
?
, 7
?
＝
14.5, 7.2, 1.4 Hz, 3?
ddd,
J
4.92 (2 H, s, PhC
H
H
＝
H2), 6.03 (1 H, dt, J
(2 H, s, PhC
±
＝
CHCl3–CH3OH) to give ( )-1 (262 mg, 79
z
) as an
2?-H), 6.60 (1 H, ddd,
J
?
amorphous brown solid, mp 162–165
9
C. IR nmax
7.19–7.44 (16 H, m, 6-H, aromatic). NMR dC
(126 MHz, CDCl3): 14.1, 22.6, 29.0, 29.2, 31.8, 33.8,
52.1, 71.0, 75.0, 75.5, 110.9, 122.9, 125.8, 127.6,
127.7, 127.9, 128.0, 128.1, 128.25, 128.28, 128.4,
128.5, 128.6, 136.4, 137.2, 137.3, 145.0, 150.9,
(CHCl3) cm－ : 3360 (br. m, O-H), 1740 (s, C O),
1620 (m), 1515 (m), 1500 (m), 1380 (s), 1340 (s), 1310
(s), 1290 (s), 1065 (m), 870 (m). NMR dH (500 MHz,
1
＝
＝
, 6?
acetone-
1.40 (10 H, m, 2
m, 1 -H), 2.21–2.27 (1 H, m, 1
7.3, 3.1 Hz, 3-H), 6.81 (1 H, s, 7-H), 8.24 (1 H,
d
6): 0.86 (3 H, t,
J
?
6.9 Hz, 7
-H), 1.69–1.74 (1 H,
-H), 5.42 (1 H, dd,
?-H), 1.27–
?
, 3 , 4 , 5
?
?
151.2, 168.6. Anal. Found: C, 78.74; H, 7.42
Calcd. for C37H40O5: C, 78.69; H, 7.14
z
.
?
?
z.
J
＝

2588
T. OHZEKI and K. MORI
(3R,1
phthalide [(3R,1
(31.1 g) and MeSO2NH2 (2.11 g, 22.2 mmol) in tert
?
R)-4,5,6-Tribenzyloxy-3-(1
?
-hydroxybutyl)-
22.0 min [67.8
(3 ,1
)-9]. Its IR, H- and 13C-NMR spectra were
identical with those of (3 ,1 )-9. Anal. Found: C,
76.29; H, 7.03 . Calcd. for C35H38O6: C, 76.30; H,
6.76
z, (3S,1?S)-9], 72.3 min [32.2z,
1
?R)-9
]. A solution of AD-mix-b}
R ?R
-
R ?R
BuOH (150 ml) and water (150 ml) was stirred for 1 h
z
at 0
9
C. Alkene 8 (4.18 g, 7.40 mmol) was added, and
z
.
･
the mixture was stirred for 7 d at 49C. Na2SO3 7H2O
(40 g, 160 mmol) was then added, and the mixture
was stirred for 1 h at room temperature. This mixture
was diluted with EtOAc, successively washed with
water and brine, and dried with MgSO4. The solvent
was removed in vacuo. The residue was chro-
matographed on silica gel (150 g; hexane-EtOAc) to
(3R,1
carbonyloxy)phthalide [(3R,1
atmosphere, 1,1-thiocarbonyldiimidazole (90
ty, 103 mg, 0.521 mmol) was added to a solution of
(3 ,1 )-9 (197 mg, 0.347 mmol) and DMAP (4.3
?
R)-4,5,6-Tribenzyloxy-3-(1
R)-10]. Under an Ar
puri-
?-imidazolylthio-
?
z
R
?R
mg, 0.035 mmol) in CH2Cl2 (10 ml). After stirring
for 18 h at room temperature, the solvent was
removed in vacuo. The residue was chromatographed
on silica gel (5 g, eluted with hexane-EtOAc) to give
give (3
R
,1
?
R
)-9 (2.26 g, 54
z
) and starting material
8 (1.91 g, 46
z
). Desired and crude (3
R,1?R)-9 was
obtained as an amorphous solid, mp 112–115
9
C,
25
＋
[
a
]
16.2 (
c
0.60, CHCl3). The enantiomeric purity
enantiomerically impure (3
quant.) as a colorless oil. IR
max (ˆlm) cm－1: 1765 (s,
O), 1615 (w, C C), 1535 (w), 1390 (s), 1330 (s),
1285 (s), 1100 (s). NMR dH (500 MHz, CDCl3): 0.90
(3 H, t, 6.9 Hz, 7 -H), 1.29–1.37 (8 H, m, 3 , 4
, 6 -H), 5.01 (1 H, d,
R,1?R)-10 (250 mg,
D
of resulting (3
R
,1?R
)-9 was found to be 45.4
z
e.e.
n
＝
C
＝
by an HPLC analysis. HPLC [column, Daicel
Chiralcel} OG (0.46 cm 25 cm); eluent, hexane 2-
×
W
propanol
10 C; detection, UV (254 nm)]
(3 ,1 )-9], 72.3 min [72.7
＝
3 2; ‰ow rate, 0.5 ml min; temperature,
W
J
＝
?
?
?,
W
＝
J
9
t
R: 22.0 min [27.3
z
,
5
?
?
-H), 1.90–1.99 (2 H, m, 2
?
＝
S
?S
z
, (3
R
,1
?
R
)-9]. IR nmax
1.2 Hz, 3-H), 5.06 (2 H, dd,
J
18.6, 11.3 Hz,
2),
2), 6.04 (1 H,
-H), 6.91 (1 H, d,
0.6 Hz, aromatic), 7.17–7.40 (7-H, m, 17 H,
(KBr) cm－ : 3415 (br. m, O-H), 1730 (s, C O), 1610
PhC
5.18 (2 H, dd,
ddd, 7.9, 6.5, 1.2 Hz, 1
H
2), 5.07 (2 H, dd,
J
18.6, 11.3 Hz, PhCH
1
＝
＝
＝
＝
＝
J 27.8, 10.9 Hz, PhC
(w, C C), 1500 (w, C C), 1455 (s), 1105 (s), 765
H
(m), 750 (m), 700 (m). NMR dH (500 MHz, CDCl3):
J
＝
?
＝
＝
J
0.89 (3 H, t,
, 5 , 6
OH), 4.05 (1 H, bs, 1
3-H), 5.12–5.21 (6 H, m, PhC
J
6.9 Hz, 7
-H), 1.37–1.51 (2 H, m 2
-H), 4.95 (1 H, d,
2), 7.25–7.47 (16 H,
?
-H), 1.27–1.30 (8 H, m, 3
-H), 1.71 (1 H, bs,
1.8 Hz,
?,
4?
?
?
?
aromatic), 8.01 (1 H, s, aromatic). NMR dC (126
MHz, CDCl3): 14.0 (q), 22.4 (t), 25.0 (t), 28.8 (t),
30.2 (t), 31.5 (t), 71.3 (t), 75.4 (t), 75.6 (t), 77.9 (d),
80.6 (d), 104.4 (d), 117.3 (d), 121.3 (s), 127.6 (d),
128.29 (d), 128.34 (d), 128.4 (d), 128.6 (d), 128.7 (d),
130.8 (d), 132.0 (s), 135.6 (s), 136.2 (s), 136.6 (s),
?
J
＝
H
m, 7-H, aromatic). NMR dC (126 MHz, CDCl3):
14.1, 22.5, 25.7, 29.0, 31.7, 33.7, 70.7, 71.4, 75.7,
75.8, 81.9, 104.8, 122.0, 127.7, 128.37, 128.41,
128.7, 128.78, 128.82, 133.9, 135.8, 136.3, 136.5,
146.4, 155.0, 170.2. Anal. Found: C, 76.35; H,
136.9 (s), 146.3 (s) 146.7 (s), 155.5 (s), 169.4 (s),
＋
＋
182.8 (s). HR-FABMS m z ([M H] ): calcd. for
W
7.02
z
. Calcd. for C36H38 O6: C, 76.30; H, 6.76
z
.
C40H40N2O6S, 677.2685; found, 677.2683.
(3S,1
?
S)-4,5,6-Tribenzyloxy-3-(1
?
-hydroxybutyl)-
(3S,1
carbonyloxy)phthalide [(3S,1
manner to that just described, (3
0.229 mmol) was converted into 152 mg (quant.) of
enantiomerically impure (3 ,1
)-10. Its IR, 1H- and
13C-NMR spectra were identical with those of
?
S)-4,5,6-Tribenzyloxy-3-(1
S)-10]. In a similar
,1 )-9 (130 mg,
?-imidazolylthio-
}
phthalide [(3S,1
?S)-
9
]. A solution of AD-mix-
a
?
(29.4 g) and MeSO2NH2 (2.00 g, 21.0 mmol) in tert
BuOH (150 ml) and water (150 ml) was stirred for 1 h
at 0 C. Alkene 8 (3.95 g, 7.00 mmol) was added to
the mixture which was then stirred for 7 d at 4 C,
before Na2SO3 7H2O (40 g, 160 mmol) was added,
-
S ?S
9
S ?S
9
･
(3R,1?R)-10. HR-FABMS m z ([M
＋
H]＋): calcd.
W
the mixture ˆnally being stirred for 1 h at room tem-
perature. This mixture was diluted with EtOAc and
successively washed with water and brine, before
being dried with MgSO4. The solvent was removed in
vacuo. The resulting residue was chromatographed
on silica gel (150 g, eluted with hexane-EtOAc) to
give crude (3
material 8 (2.35 g, 59
9 was obtained as an amorphous solid, mp 112–
for C40H40N2O6S, 677.2685; found, 677.2682.
(S)-4,5,6-Tribenzyloxy-3-heptylphthalide [(S)-11].
Under an Ar atmosphere, AIBN (5.7 mg, 0.035
mmol) was added to a solution of (3
R
,1
?
R
)-10 (250
l,
mg, 0.347 mmol) and tributyltin hydride (468
m
S
,1?S
)-9 (15.6 g, 39
z
) and starting
1.74 mmol) in toluene (10 ml), and the mixture was
re‰uxed for 15 min. It was then diluted with EtOAc
and successively washed with water and brine. The
organic layer was dried with MgSO4 and concentrat-
ed in vacuo. The residue was chromatographed twice
z
). Desired and crude (3
S,1?S)-
23
115
9
C, [a] 11.3 (c 0.99, CHCl3). The enantiomer-
－
D
ic purity of resulting (3
S,1?S)-9 was found to be
×
on silica gel (10 g 2, eluted with hexane-EtOAc) to
35.6
z
e.e. by an HPLC analysis. HPLC [column,
Daicel Chiralcel} OG (0.46 cm 25 cm); eluent,
give (
1.5109. IR
C), 1500 (w, C
S
)-11 (172 mg, 90
z
) as a colorless oil, nD24
×
1
＝
＝
hexane 2-propanol 3 2; ‰ow rate, 0.5 ml min;
n
max (ˆlm) cm－ : 1765 (s, C O), 1615 (w,
W
W
W
temperature, 10
9
C; detection, UV (254 nm)] tR
:
C
＝
＝
C), 1465 (s), 1340 (s), 1100 (s),

Synthesis of Cytosporone E
2589
740 (m), 700 (m). NMR dH (500 MHz, CDCl3):
rate, 0.5 ml min; temperature, 10
9
z
C; detection, UV
R: 12.2 min [0.8 , ( )-12], 13.4
)-12]; ( )-12 R: 12.5 min [99.8
, (R)-12]. (S
)-12: oil, n2D4
W
＝
0.87 (3 H, t,
m, 2 ,3 ,4 ,5
J
6.8 Hz, 7?-H), 1.23–1.39 (10 H,
(254 nm)], (
min [99.2
S
)-12
t
R
?
?
?
?
,6
?
-H), 1.56–1.67 (1 H, m, 1
?
＝
-H),
z
, (
S
R
t
z
,
2.09–2.14 (1 H, m, 1
?
-H), 5.00 (1 H, dd,
J
8.0,
14.6, 11.0 Hz,
2),
(
S
)-12], 14.1 min [0.4
z
24
3.0 Hz, 3-H), 5.14 (4 H, dd,
J
＝
1.4939, [
a
]
D
－
38.4 (
c
0.992, hexane). IR nmax (ˆlm)
PhCH
2), 5.15 (2 H, dd,
J
14.9, 10.6 Hz, PhC
H
cm－ : 1770 (s, C O), 1610 (w, C C), 1585 (w),
1
＝
＝
＝
7.27–7.47 (16 H, m, 7-H, aromatic). NMR dC
(126 MHz, CDCl3): 14.1, 22.6, 24.6, 29.0, 29.1, 31.7,
33.1, 71.3, 75.4, 75.6, 80.3, 104.4, 121.5, 127.6,
128.30, 128.32, 128.37, 128.44, 128.5, 128.58,
128.63, 128.64, 135.9, 136.5, 136.59, 136.63, 146.5,
146.8, 154.7, 170.4. Anal. Found: C, 78.44; H,
1455 (s), 1355 (s), 1255 (s), 1090 (s), 935 (m), 905 (m),
870 (w), 840 (s), 785 (s). NMR
d
H (500 MHz, CDCl3):
0.01 (3 H, s, SiMe), 0.10 (3 H, s, SiMe), 0.15 (3 H,
s, SiMe), 0.16 (3 H, s, SiMe), 0.24 (3 H, s, SiMe),
－
＝
6.9 Hz, 7?-H),
0.30 (3 H, s, SiMe), 0.85 (3 H, t,
0.91 (9 H, s, tert-Bu), 0.95 (9 H, s, tert-Bu), 0.99
(9 H, s, Bu), 1.22–1.29 (10 H, m, 2 , 3 , 4 , 5
-H), 1.61–1.70 (1 H, m, 1 -H), 2.15–2.21 (1 H, m,
-H), 5.38 (1 H, dd, 7.5, 2.9 Hz, 3-H), 7.02 (1 H,
s, 7-H). NMR dC (126 MHz, CDCl3): 4.4, 4.2,
J
6.90
z
. Calcd. for C36H38O5: C, 78.52; H, 6.96
z
.
t
?
?
?
?,
6
?
1?
?
(R)-4,5,6-Tribenzyloxy-3-heptylphthalide [(R)-11].
In a similar manner to that just described, (3 ,1 )-
10 (152 mg, 0.229 mmol) was converted into 106 mg
J
＝
－
－
S
?S
－
－
－
－
4.15, 3.6, 3.13, 3.06, 14.0, 18.3, 18.7, 18.8,
1
(84
NMR spectra were identical with those of (
Anal. Found: C, 78.58; H, 6.88 . Calcd. for
C36H38O5: C, 78.52; H, 6.96
z
) of (
R
)-11, n2D4 1.5123. Its IR, H- and 13C-
22.6, 24.5, 26.01, 26.04, 26.2, 29.1, 29.3, 31.6, 32.8,
80.4, 110.5, 118.8, 135.7, 142.5, 145.1, 150.6,
S
)-10.
z
170.8. HR-FABMS m z ([M
＋
H]＋): calcd. for
W
z
.
C33H62O5Si3, 623.3983; found, 623.3990. (
R
)-12: oil,
24
1
n2D4 1.4939, [
a
]
39.6 (c 1.01, hexane). Its IR, H-
＋
D
Debenzylation of Enantiomerically Impure (S)-11
and (R)-11 Pearlman's catalyst [20 Pd(OH)2 on
carbon; wet, 218 mg] was added to a solution of
)-11 (172 mg, 0.312 mmol) in EtOAc (5 ml), and
and 13C-NMR spectra were identical with those of
＋
＋
.
z
(
S
)-12. HR-FABMS m z ([M H] ): calcd. for
W
C33H62O5Si3, 623.3983; found, 623.3990.
(
S
the suspension was stirred under H2 for 40 min. The
mixture was then ˆltered through Celite, and the
solvent was removed in vacuo to give enantiomerical-
(S)-Cytosporone E [(S)-
1
]. Puriˆed (
S)-12 (98.4z
e.e., 136 mg, 0.218 mmol) in 4
M
HCl-ethanol (1:5,
30 ml) was stirred for 4 d at room temperature. The
mixture was extracted with EtOAc. The organic layer
was successively washed with water and brine, dried
with MgSO4, and concentrated in vacuo. The residue
was chromatographed on silica gel (5 g, eluted with
z
ly impure (S)-1 (80.0 mg, 92 ) as a brown solid.
This solid was employed in the next step without
further puriˆcation. In a similar manner to that just
described, (
ed into 50.7 mg (94
NMR spectra were identical with those of (
R)-11 (106 mg, 0.192 mmol) was convert-
1
z
) of (
R
)-1. Its IR, H- and 13C-
)-1.
hexane-EtOAc) to give (S)-1 (24.4 mg, 40z) and
±
recovered (
S
)-12 (79.0 mg, 58
). Mp. 169–173
z
9
C.
25
1
－
±
[a
]
90.7 0.1 (
c
1.00, acetone). Its IR, H- and
D
13
±
C-NMR spectra were identical with those of ( )-1.
Puriˆcation of Tris-TBS Derivatives (S)- and
(R)-12 by HPLC. A solution of ( )-enriched 1
(35.6 e.e., 50.7 mg, 0.181 mmol) and imidazole
S
HR-EIMS m z (M＋): calcd. for C H O , 280.1311;
W
15
20
5
z
found, 280.1314.
(111 mg, 1.63 mmol) in DMF (3 ml) was stirred and
cooled with an ice-water bath. TBSCl (246 mg,
1.63 mmol) was added to the solution, and the
mixture was stirred for 14 h at room temperature. It
was then diluted with EtOAc, and the solution was
successively washed with water and brine. The organ-
ic layer was dried with MgSO4 and concentrated in
vacuo. The resulting residue was chromatographed
on silica gel (10 g, eluted with hexane-EtOAc) to give
(R)-Cytosporone E [(R)-
1
]. In a similar manner to
)-12 (99.4 e.e.,
that just described, puriˆed (
R
z
174 mg, 0.279 mmol) was converted into 32.6 mg
(42 ) of ( )-1 and recovered ( )-12 (45.4 mg,
56
z
R
R
24
z
). Mp 168–172
9
C, [
a
]
91.3 0.1 (c 1.00,
＋ ±
D
acetone). Its IR, 1H- and 13C-NMR spectra were
identical with those of ( )-1. HR-EIMS m z (M＋):
±
W
calcd. for C15H20O5, 280.1311; found, 280.1314.
(
S
)-enriched 12 (111 mg, 98
z). This compound 12
was puriˆed by HPLC [column, Daicel Chiralcel
Acknowledgment
OD} (2 cm 25 cm); eluent, hexane 2-propanol
×
＝
W
500 1; ‰ow rate, 16 ml min; temperature, 20
9
C;
We thank Dr. T. Hasegawa (Sankyo Co. Ltd.) for
W
W
detection, UV (254 nm)]
t
R: 12.5 min [(
R
)-12], 13.4
his help in carrying out the bioassay of ( )-, (R)-
±
min [(
)-12 and (
98.4 e.e. and 99.4
S
)-12]. The enantiomeric purities of resulting
and (S)-1 at Institute of Science and Technology,
(
S
R
)-12 were respectively found to be
Sankyo Co. Ltd. Our thanks are due to Prof. T.
Saito (Science University of Tokyo) for his en-
couragement.
z
z
e.e. by an HPLC analysis.
HPLC [column, Daicel Chiralcel} OD (0.46 cm
×
25 cm); eluent, hexane 2-propanol
＝
500 1; ‰ow
W
W

2590
T. OHZEKI and K. MORI
(1998).
7) Snieckus, V., Directed ortho metalation. Tertiary
amide and -carbamate directors in synthetic strate-
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