Total synthesis of agosterol A: An MDR-modulator from a marine sponge

Article abstract of DOI:10.1002/1521-3765(20010618)7:12<2663::AID-CHEM26630>3.0.CO;2-U

The first total synthesis of agosterol A, a modulator or multidrug resistance (MDR) mediated by P-gp and MRP1, and isolated from a marine sponge, was achieved from ergosterol by utilizing a regioselective epoxy-cleavage reaction and regioselective dehydro

Full text of DOI:10.1002/1521-3765(20010618)7:12<2663::AID-CHEM26630>3.0.CO;2-U

FULL PAPER
Total Synthesis of Agosterol A: an MDR-Modulator from a Marine Sponge
Nobutoshi Murakami, Masanori Sugimoto, Mari Morita, and Motomasa Kobayashi*[a]
Abstract: The first total synthesis of agosterol A, a modulator of multidrug resistance
Keywords: drug research
drug resistance ´ MRP1 ´ steroids
´ total synthesis
´ multi-
(MDR) mediated by P-gp and MRP1, and isolated from a marine sponge, was
achieved from ergosterol by utilizing a regioselective epoxy-cleavage reaction and
regioselective dehydroxylation as the key reactions.
hydroxyl functions. In the first instance, an oxidative cleavage
of the double bond between C22 and C23 in 2, after protection
of the diene portion, would afford a precursory aldehyde,
which is further submitted to nucleophilic substitution by a
Grignard reagent to give an undesired 22(S)-alcohol (vi)
under the direction of Cramꢀs rule. Thereafter, the 22(S)-
hydroxyl group would be inverted to a 22(R)-hydroxyl group
by the Mitsunobu reaction. The 11a-hydroxyl group would be
built up by a regioselective, reductive epoxy-cleavage of the
9a,11a-epoxide (iv) prepared from the 5,7,9(11)-triene (v),
which is obtained by dehydrogenation from the homoannular
diene. After protection of the homoannular diene of iii,
Introduction
The development of multidrug resistance (MDR), which is
observed as a response to chemotherapy, is a very serious
problem in the treatment of cancer.[1] The major documented
mechanism by which tumor cells acquire this MDR pheno-
type is the increased expression of an ATP-dependent
membrane glycoprotein, which serves as an efflux pump for
antitumor agents.[2] Besides a representative and well-charac-
terized glycoprotein called P-glycoprotein (P-gp),[2] an in-
creased expression of a multidrug-resistance-associated pro-
tein (MRP1)[3] has been found in many non-P-gp mediated
MDR cells. Since there have been few MDR-modulating
substances mediated by MRP1, we have been searching for
new modulators from extracts of marine invertebrates on the
basis of bioassay. Recently, we characterized a potent
modulator of MDR mediated by P-gp and MRP1 named
agosterol A, which was isolated from a marine sponge:
Spongia sp., and investigated the structure ± activity relation-
ship by the use of naturally occurring congeners and synthetic
analogues.[4, 5] Limited availability from natural sources com-
pelled us to explore a synthetic protocol of agosterol A. As a
result of preliminary study, we recently achieved the synthesis
of a 4-deacetoxyl analogue of agosterol A.[6] In this report, we
wish to present the first total synthesis of agosterol A from
ergosterol.
ˆ
regioselective introduction of a C3 C4 double bond, followed
by selective dihydroxylation would afford 3b,4b-dihydroxyl
functions. Continuing on from this, a 6b-hydroxyl group
would be introduced by regio- and stereo-selective hydro-
boration from the less-hindered a-side. Finally, differen-
tiated removal of the protecting group and acetylation would
lead to agosterol A (1). This strategy was executed as
follows:
After protection of the hydroxyl group in ergosterol (2) as a
methoxymethyl (MOM) ether, masking of the 5,7-diene
portion with 4-phenyl-1,2,4-triazoline-3,5-dione,[7] a typical
protecting group of cisoid dienes, provided a 6,22-diene
derivative. However, further selective ozonolysis of the
ˆ
C22 C23 double bond proved to be unsuccessful. Therefore,
the 5,7-diene portion of the MOM ether of 2 was protected
with 1,4-dihydrophthalazine-1,4-dione,[8] prepared from the
corresponding tetrahydro precursor and Pb(OAc)4 in situ, to
give compound 3 in 82% yield from 2 (Scheme 2). In contrast,
ozonolysis of 3 in the presence of pyridine cleaved the
Results and Discussion
Scheme 1 delineates our retrosynthetic analysis for agosterol
A (1) from commercially available ergosterol (2); this
includes the sequential, stereoselective introduction of four
ˆ
C22 C23 double bond selectively to give an aldehyde in 90%
yield. The aldehyde was further subjected to a Grignard
reaction by using 3-methylbutylmagnesium bromide to fur-
nish an undesired 22(S)-alcohol 4 under the direction of
Cramꢀs rule. Inversion of the 22-hydroxyl group in 4 by the
conventional Mitsunobu reaction[9] with triphenylphosphine,
diethyl azodicarboxylate (DEAD), and 4-nitrobenzoic acid
gave a 22(R)-benzoate in poor yield (20%). As a result of
[a] Prof. M. Kobayashi, Dr. N. Murakami, M. Sugimoto, M. Morita
Graduate School of Pharmaceutical Sciences, Osaka University
1-6 Yamada-oka, Suita, Osaka 565-0871 (Japan)
Fax : (‡81)6-6879-8219
E-mail: kobayasi@phs.osaka-u.ac.jp
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M. Kobayashi et al.
FULL PAPER
Scheme 1. Retrosynthetic steps for the synthesis of agosterol A from ergosterol.
Scheme 2. Reagents and conditions: a) MOMCl, iPr2NEt, CH2Cl2, 96% from 8; b) phthalhydrazide, Pb(OAc)4, CH2Cl2-AcOH, two steps 82%; c) O3,
CH2Cl2-Py, then, Me2S; d) 3-methylbutylmagnesium bromide, THF, two steps 90%; e) TMAD, PMe3, pOCH3BzOH, THF, 66%; f) LiAlH4, THF, 89%;
g) (S)-( )-MTPA[(R)-(‡)-MTPA], EDCI ´ HCl, DMAP, CH2Cl2, 5a: 82%, 5b: 76%; h) TBSOTf, 2,6-lutidine, DMF-CH2Cl2, 91%; i) Hg(OAc)2, EtOH/
CHCl3/AcOH, 9: 80%, 10: 85%; j) 4-phenyl-1,2,4-triazoline-3,5-dione, CH2Cl2, 89% from 9, 94% from 10; k) mCPBA, CHCl3, 11: 71%, 12: 64%;
l) LiAlH4, THF, 48% from 11.
examining several reaction conditions, a combination of
N,N,N',N'-tetramethylazodicarboxamide (TMAD), trimethyl-
phosphine, and 4-methoxybenzoic acid[10] provided the de-
sired 22(R)-benzoate in 66% yield. On the other hand,
CsOAc treatment of the chloromethanesulfonate[11] of 4 in the
presence of [18]crown-6 furnished the acetate with 22(R)
configuration in 56% yield. Reductive cleavage of both the
4-methoxybenzoyl group and the protecting group of the
diene portion occurred spontaneously, and the configuration
at the C22 hydroxyl group in 5 was confirmed by the modified
Mosher method.[12] Namely, the proton signals due to the
23-H2, 24-H2, 26-H3, and 27-H3 of the (R)-a-methoxy-a-
trifluoromethylphenylacetic acid (MTPA) ester (5b) ap-
peared at higher field than those of the (S)-MTPA ester
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Synthesis of Agosterol A
2663±2670
(5a), whereas the signals ascribable to the protons 20-H, 21-
H3, and 18-H3, settled in the left-side region of the 22-
hydroxyl group in 5b, were observed at lower field, as can be
seen in Figure 1.
alcohol, in which BH3-LiBH4,[14] AlH3,[15] potassium triphen-
ylborohydride-Ph3B,[16] and Cp2TiCl2-Mn-1,4-cyclohexa-
diene[17] were used, did not afford any satisfactory outcome.
We also attempted the use of representative metal-hydride
reagents such as Super-Hydrideꢂ, l-Selectrideꢂ, and Red-Alꢂ
for this conversion. Among them, only Red-Al gave the
expected 11a-alcohol 14 in poor yield (16%).
This finding directed us to examine the cooperation of a
Lewis acid with LiAlH4. Three of the four Lewis acids tested
(the exception being TiCl4) afforded the 11a-alcohol 14
(Table 1, Scheme 3). In particular, Et2AlCl enhanced the
epoxy ring cleavage along with removal of the triazolidine
group to furnish the desired 11a-alcohol 14 as compared with
the reaction conditions in the absence of a Lewis acid (run 5).
Intensive investigation of the reaction conditions revealed
that the treatment of 12 with Et2AlCl under reflux prior to
addition of LiAlH4 improved the yield of 14, bringing it up to
90% (run 7). Replacement of LiAlH4 by diisobutylaluminum
hydride (DIBAL-H) or Red-Al resulted in a slight decrease in
the yields of 14 (DIBAL-H: 80%; Red-Al: 52%). Hence,
Figure 1. Distribution of Dd(ˆ dS dR) values (ppm, 500 MHz).
After protection of the 22-hydroxyl group in 5 with tert-
butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) and
[13]
2,6-lutidine, dehydrogenation with Hg(OAc)2
afforded a
triene 9 in 73% yield over two steps. Protection of the
homoannular 5,7-diene of 9 by using 4-phenyl-1,2,4-triazoline-
3,5-dione, and subsequent epoxidation with meta-chloroper-
benzoic acid in CHCl3 predominantly afforded a 9a,11a-
epoxide 11 in 71% yield. The stereochemistry on the epoxy
moiety in 11 was established by NOE observation between
the 11b-H, 19-H, and 18-H. Reductive cleavage of the epoxy
ring and deprotection of the triazoline group in 11 by LiAlH4
gave the desired 11a-alcohol 13 in unsatisfactory yield (48%).
To overcome this undesirable outcome, we carefully
examined the reaction conditions utilizing 12 as a model
substrate. The epoxide 12 was prepared from 7,8-didehydro-
cholesterol (8) through the same three-step transformation,
after protection of the 3-hydroxyl group in 8 as an MOM
ether. The reported procedures to lead from the epoxide to an
Table 1. Investigation on regioselective reductive epoxy cleavage of 12.
Run
Lewis Acid
Hydride
Pretreatment
Yield
1
2
3
4
5
6
7
8
±
LiAlH4
LiAlH4
LiAlH4
LiAlH4
LiAlH4
LiAlH4
LiAlH4
LiAlH4 ‡ Et2AlCl
±
46%
35%
18%
0%
58%
70%
90%
10%
BF3 ´ Et2O
Al(OiPr)3
TiCl4
Et2AlCl
Et2AlCl
Et2AlCl
6 h
6 h
6 h
1 min
2 h
6 h
±
Each reaction was carried out in THF under reflux. After pre-treatment by
2 mol equiv. of Lewis acid, 10 mol equiv. of hydride reagent was added.
Scheme 3. Reagents and conditions: a) LiAlH4, Et2AlCl, THF, 84% from 11, 90% from 12; b) TBSOTf, 2,6-lutidine, toluene, 95% from 13; c) Fe(CO)5,
1-(4-methoxyphenyl)-4-phenyl-1-azabuta-(E, E)-1,3-diene, PhCH3, 94%; d) MgBr2-Et2O, Me2S, CH2Cl2, 73%; e) TsCl, Py, quant.; f) DBN, PhH, quant.;
g) OsO4, Py, then aq. NaHSO3, 80%; h) TESOTf, DMAP, Py, quant.; i) Me3NO, PhH, 95%; j) BH3Me2S, THF, then, H2O2, aq. NaOH, 62%; k) TBAF, THF,
90%; l) Ac2O-Py, 91%; m) HF-Py, THF, 84%.
Chem. Eur. J. 2001, 7, No. 12
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M. Kobayashi et al.
FULL PAPER
chelation between the epoxy oxygen and the aluminum atom
might participate in this reductive epoxy-cleavage reaction.
The stereochemistry at C9 in 14 was confirmed to be R by the
coupling constant (J ˆ 9.3 Hz) between 9-H and 11-H. This
stereoselectivity indicates that the 11a-alcohol 14 will be
given through protonation from the less hindered a-side to
the C9 carbocation intermediate.
product[4] isolated from the marine sponge in comparison of
1
the spectroscopic features such as H NMR, 13C NMR, IR,
MS, and optical rotation data.
Conclusion
We have achieved the first total synthesis of agosterol A (1)
from ergosterol (2), a readily available, naturally occurring
sterol, in 3.5% total yield through 23 steps. The present
synthetic protocol includes a regioselective, reductive epoxy-
cleavage reaction and regioselective dehydroxylation as the
key reactions.
Application of this conversion condition to 11 furnished 13
in nearly the same yield (84%) as the preparation for 14,
without losing the TBS group.[18] After protection of the 11-
hydroxyl group in 13 as a TBS ether, the 5,7-diene portion was
masked by using pentacarbonyl iron and 1-(4-methoxyphen-
yl)-4-phenyl-1-azabuta-(E,E)-1,3-diene[19] to give a tricarbon-
yl iron complex in 90% yield for two steps. In contrast,
replacement of the protecting group by phthalazine and
triazoline afforded no desired 3-ene compounds because of
the lability of both under basic conditions. The MOM group in
the complex was removed by TMSBr[20] treatment to give an
alcohol 15 in unsatisfactory yield (50%). In contrast, treat-
ment with MgBr2-etherate[21] and Me2S afforded 15 in 73%
yield without deprotection of the tricarbonyl iron moiety.
Next, quantitative treatment of 15 with para-toluenesulfonyl
chloride in pyridine provided the corresponding tosylate,
which was further subjected to an elimination reaction in 2,6-
lutidine solution to selectively give an undesired 2-ene
derivative. On the other hand, treatment of the tosylate with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 2,6-lutidine in
toluene gave a 3-ene 16 and a 2-ene derivative in a ratio of 2:1
quantitatively. Detailed examination of the reaction condi-
tions disclosed that the treatment of the tosylate with 1,5-
diazabicyclo[4.3.0]non-5-ene (DBN) in benzene under reflux
furnished only the desired 16 in excellent yield.
Dihydroxylation of 16 with osmium tetraoxide proceeded
stereoselectively from the b-side free from interruption by the
tricarbonyl iron function to give a cis-3b,4b-diol 17 in 80%
yield. The 1H NMR spectrum of 17 exhibited two oxymethine
signals due to the 3-H (brd, J ˆ 11.1 Hz) and the 4-H (brs),
indicating construction of the desired 3(S), 4(R)-configura-
tions. The newly generated hydroxyl groups in 17 were
protected by triethylsilyl trifluoromethanesulfonate (TE-
SOTf) and 4-dimethylaminopyridine (DMAP) in pyridine to
give a di-TES ether. Subsequent cleavage of the tricarbonyl
iron residue by using trimethylamine N-oxide[22] afforded a
diene 18 (95% for two steps). Hydroboration of 18 by use of a
borane dimethyl sulfide complex, followed by H2O2 treatment
proceeded with moderate stereo- and regio-selectivity (62%)
to give a 6a-alcohol. Deprotection of the TES groups by using
tetrabutylammonium fluoride (TBAF)[23] provided a triol 19
in 90% yield. The stereochemistry on the C5 and C6 carbons
in 19 was established to be a by the physico-chemical feature;
three characteristic signals assignable to the oxymethine
protons (d ˆ 3.56, m, H3; d ˆ 4.29, brs, H4; d ˆ 4.32, brd,
J ˆ 9.5 Hz, H6) were observed in the 1H NMR spectrum, and
the chemical shifts and coupling constants resembled those of
trideacetylagosterol A.[5] On acetylation with Ac2O in pyr-
idine, the triol 19 was converted to a triacetate, which was
further subjected to HF-pyridine treatment[24] to furnish
agosterol A (1) in 76% yield for two steps. The synthesized
agosterol A was shown to be superimposable with the natural
Experimental Section
General: The following instruments were used to obtain physical data: a
Jasco DIP-370 digital polarimeter for specific rotations, a JASCO FT/IR-
5300 infrared spectrometer for IR spectra, a JEOLJMS SX-102 mass
spectrometer for FAB-MS,
a JEOLJNM LA-500 (500 MHz), a
JEOLJNM-AL300 (300 MHz) spectrometer for 1H NMR spectra
(1H NMR: CDCl3 solution with tetramethylsilane (TMS) as internal
standard unless otherwise specified), and a Yanaco CHNCORDER(MT-5)
for elemental analysis. All new compounds bearing the 5,7-conjugated
diene portion were characterized by high-resolution FAB-MS because of
the lability in light; HPLC was performed by using an Hitachi L-6000 pump
equipped with an Hitachi L-4000H UV detector. Silica gel (Merck 60 ± 230
mesh) and precoated thin-layer chromatography plates (Merck, Kiesel gel,
60F254) were used for column chromatography and TLC, respectively. Spots
on TLC plates were detected by spraying Ce(SO4)2/H2SO4 [Ce(SO4)2 ´
nH2O (10 g) in 6.3% aqueous H2SO4 (1.0 L)] or acidic para-anisaldehyde
solution [para-anisaldehyde (25 mL), c-H2SO4 (25 mL), AcOH (5 mL),
EtOH (425 mL)] with subsequent heating.
Conversion from ergosterol (2) to compound 3: iPr2NEt (20 mL,
115 mmol) was added to a solution of ergosterol (15.2 g, 38.3 mmol) in
CH2Cl2 (190 mL) at 08C, then the whole mixture was stirred for 5 min.
Methoxymethyl (MOM) chloride (7.2 mL, 96 mmol) was added to the
reaction mixture at 08C, then the whole mixture was stirred at room
temperature in the dark for a further 36 h. The reaction mixture was poured
into saturated aqueous NH4Cl, then the whole mixture was extracted with
EtOAc. The EtOAc extract was successively washed with 5% aqueous HCl
and saturated aqueous NaCl, then dried over MgSO4. Removal of the
solvent from the EtOAc extract under reduced pressure gave a MOM ether
(19.4 g). A solution of Pb(OAc)4 (28.4 g, 64 mmol) in CH2Cl2 (420 mL) and
glacial AcOH (5.0 mL) was slowly added to a solution of the MOM ether
(18.8 g) and phthalhydrazide (27.9 g, 172 mmol) in CH2Cl2 (630 mL),
keeping the temperature between 58C and 08C. Then the whole mixture
was stirred at 08C for 1 h. The reaction was quenched with Al2O3 (76 g,
745 mmol) and stirred for 30 min. After filtration, the filtrate was
successively washed with H2O, saturated aqueous NaHCO3, and saturated
aqueous NaCl, then dried over MgSO4. Removal of the solvent from the
CH2Cl2 layer under reduced pressure gave a crude product, which was
purified by column chromatography (SiO2 240 g, n-hexane/EtOAc 4:1) to
furnish compound 3 as a yellow amorphous solid. Yield: 18.2 g, 82%;
1
[a]2D3
ˆ
126.88 (c ˆ 1.89 in CHCl3); H NMR (500 MHz, CDCl3) d ˆ 8.13
(m, 2H; PhH), 7.32 (m, 2H; PhH), 6.65, 6.27 (both d, J ˆ 7.9 Hz, 1H; H6,7),
5.22 (dd, J ˆ 15.2, 7.9 Hz, 1H; H22 or 23), 5.14 (dd, J ˆ 15.2, 7.3 Hz, 1H;
H22 or 23), 4.72, 4.63 (ABq, J ˆ 6.7 Hz, 2H; 3-OCH2OCH3), 3.54 (m, 1H;
H3), 3.34 (s, 3H; 3-OCH2OCH3), 1.02 (s, 3H; H19), 1.02, 0.90 (both d, J ˆ
6.7 Hz, 3H; H21,28), 0.84 (s, 3H; H18), 0.83, 0.81 (both d, J ˆ 6.7 Hz, 3H;
H26,27); IR nÄmax (KBr): 2955, 1653, 1310, 1044 cm 1; FAB-MS m/z: 601
‡
[M‡H] ; FAB-HRMS m/z: calcd for C38H53N2O4: 601.4005, found:
601.3994; elemental analysis calcd (%) for C38H52N2O4 (600.8): C 75.96,
H 8.72, N 4.66; found C 76.31, H 8.63, N 4.88.
Ozonolysis of compound 3 followed by Grignard reaction giving compound
4: Ozone gas was bubbled through a solution of compound 3 (15.5 g,
25.8 mmol) in dry CH2Cl2 (1.5 L) and pyridine (44 mL, 541 mmol) at
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Synthesis of Agosterol A
2663±2670
608C for 3 h. The excess O3 was removed by bubbling with Ar, then the
whole mixture was treated with Me2S (9.4 mL, 129 mmol) at 608C for
30 min. Removal of the solvent from the reaction mixture under reduced
An (R)-MTPA ester (5b), a colorless amorphous solid, was prepared from
5 (4.7 mg, 0.0106 mmol) in the same manner. Yield: 5.1 mg, 76%; [a]D20
ˆ
1
14.58 (c ˆ 0.19 in CHCl3); H NMR (500 MHz, CDCl3) d ˆ 7.54 (m, 2H;
(OCH3)PhCF3), 7.39 (m, 3H; (OCH3)PhCF3), 5.58 (dd, J ˆ 5.5, 1.9 Hz, 1H;
H6), 5.41 (dt, J ˆ 5.5, 2.5 Hz, 1H; H7), 5.10 (brd, J ˆ 10.2 Hz, 1H; H22),
4.72, 4.70 (ABq, J ˆ 6.6 Hz, 2H; 3-OCH2OCH3), 3.58 (s, 3H;
(OCH3)PhCF3), 3.53 (m, 1H; H3), 3.38 (s, 3H; 3-OCH2OCH3), 1.90 (m,
1H; H20), 1.43 (m, 4H; H23,24), 0.97 (d, J ˆ 6.6 Hz, 3H; H21), 0.95 (s, 3H;
H19), 0.80 (d, J ˆ 6.4 Hz, 3H; H27), 0.75 (d, J ˆ 6.4 Hz, 3H; H26), 0.64 (s,
3H; H18),; IR nÄmax (KBr) ˆ 2949, 1736, 1454, 1269, 1167 cm 1; FAB-MS
pressure gave
a crude aldehyde (18.3 g). Mg (22.7 g, 0.95 mol) was
activated by heating with a burner then cooled, and THF (630 mL) was
added. 3-Methylbutylbromide (78.6 mL, 0.63 mol) was slowly added to this
suspension, and the mixture was stirred for 1 h to give a solution of
3-methylbutylmagnesium bromide (1.0m in THF). Some of this solution
(153 mL, 153 mmol) was added to a solution of the crude aldehyde (18.3 g)
in THF (220 mL) at 788C, then the whole mixture was stirred for 1 h. The
reaction mixture was quenched with saturated aqueous NH4Cl and
extracted with EtOAc. The EtOAc extract was successively washed with
saturated aqueous Na2S2O3 and saturated aqueous NaCl, then dried over
MgSO4. Removal of the solvent from the EtOAc extract under reduced
pressure gave a crude product, which was purified by column chromatog-
raphy (SiO2 200 g, PhH/acetone 10:1) to furnish compound 4 as a yellow
‡
m/z: 683 [M‡Na] ; FAB-HRMS m/z: calcd for C39H55F3O5Na: 683.3899,
found: 683.3901.
Conversion from compound 5 to compound 9: 2,6-Lutidine (2.4 mL,
20.4 mmol), then TBSOTf (4.2 mL, 18.4 mmol) were added to a solution of
compound 5 (4.55 g, 10.2 mmol) in DMF/CH2Cl2 (2:1, 93 mL) at room
temperature and the whole mixture was stirred for 1 h in the dark. The
reaction mixture was poured into saturated aqueous NH4Cl, then the whole
mixture was extracted three times with EtOAc/PhCH3 (1:1). The organic
layer was washed with saturated aqueous NaCl, then dried over MgSO4.
Removal of the solvent from the organic layer under reduced pressure gave
a crude product, which was purified by column chromatography (SiO2
100 g, n-hexane/EtOAc 30:1) to furnish compound 6 (5.2 g, 91%). A
amorphous solid. Yield: 12.1 g, 90%; [a]D23
ˆ
114.68 (c ˆ 0.61 in CHCl3);
1H NMR (500 MHz, CDCl3) d ˆ 8.14 (m, 2H; PhH), 7.64 (m, 2H; PhH),
6.66, 6.27 (both d, J ˆ 7.9 Hz, 1H; H6,7), 4.72, 4.63 (ABq, J ˆ 6.7 Hz, 2H;
3-OCH2OCH3), 3.63 (m, 1H; H22), 3.54 (m, 1H; H3), 3.35 (s, 3H;
3-OCH2OCH3), 1.03 (s, 3H; H19), 0.91 (d, J ˆ 6.7 Hz, 3H; H21), 0.89 (d,
J ˆ 6.7 Hz, 6H; H26, 27), 0.81 (s, 3H; H18); IR nÄmax (KBr) ˆ 3482, 2951,
1651, 1312 cm 1; FAB-MS m/z: 605 [M‡H] ; FAB-HRMS m/z: calcd for
‡
solution of compound
6 (5.2 g, 9.25 mmol) in EtOH/AcOH/CHCl3
C37H53N2O5: 605.3954, found: 605.3959; elemental analysis calcd (%) for
C37H52N2O5 (604.8): C 73.48, H 8.67, N 4.63; found C 73.73, H 8.73, N 4.81.
(100:0.056:70, 263 mL) was treated with Hg(OAc)2 (14.7 g, 46.3 mmol) at
room temperature for 17 h in the dark. After filtration through a SiO2
column (25 g) with EtOAc/CHCl3 (1:1) as eluant, removal of the solvent
from the filtrate under reduced pressure gave a crude product, a solution of
which in CHCl3 was passed through a Florisilꢂ column to give a triene 9
(5.1 g). Since 9 was partly degraded in the SiO2 column chromatography,
the next transformation was carried out without further purification, except
for separating the sample for characterization. An aliquot of the crude
product (21 mg) was purified by SiO2 column chromatography (SiO2 2 g, n-
hexane/EtOAc 20:1) to furnish a triene 9 as a colorless amorphous solid.
Yield: 17 mg, 80%; [a]D22 ˆ ‡152.98 (c ˆ 1.49 in CHCl3); 1H NMR
(500 MHz, CDCl3) d ˆ 5.68, 5.50, 5.40 (all brd, J ˆ ca. 6 Hz, 1H;
H6,7,11), 4.69 (brs, 2H; 3-OCH2OCH3), 3.90 (brd, J ˆ 8.7 Hz, 1H; H22),
3.49 (m, 1H; H3), 3.37 (s, 3H; 3-OCH2OCH3), 1.25 (s, 3H; H19), 0.87 (m,
18H; 22-OSi(CH3)2C(CH3)3, H21,26,27), 0.56 (s, 3H; H18), 0.03 (s, 6H; 22-
OSi(CH3)2C(CH3)3); IR nÄmax (KBr) ˆ 2951, 1464, 1254, 1044 cm 1; FAB-MS
Inversion of hydroxyl group in compound 4 followed by LiAlH4 reduction:
PMe3 (4.5 mL, 43 mmol) was added to a solution of compound 4 (11.9 g,
19.6 mmol) and para-anisic acid (6.6 g, 43 mmol) in THF (56 mL), then
TMAD (7.4 g, 43 mmol) was added to the reaction mixture at 08C and this
was stirred for 10 min. The whole mixture was stirred at room temperature
for 20 min, then warmed to 508C and stirred at this temperature for 1.5 h.
Removal of the solvent from the reaction mixture under reduced pressure
gave a crude product, which was purified by column chromatography (SiO2
150 g, n-hexane/EtOAc 3:1) to furnish a para-anisyl ester (9.6 g, 66%). A
solution of the para-anisyl ester (9.5g, 12.9 mmol) in THF (193 mL) was
treated with LiAlH4 (4.9 g, 129 mmol) under reflux for 2 h in the dark. The
reaction mixture was slowly quenched with 0.5n aqueous potassium
sodium tartrate at 08C, then the whole mixture was extracted with EtOAc.
The EtOAc extract was washed with saturated aqueous NaCl, then dried
over MgSO4. Removal of the solvent from the EtOAc extract under
reduced pressure gave a crude product, which was purified by column
chromatography (SiO2 120 g, n-hexane/EtOAc 6:1) to furnish compound 5
‡
m/z: 579 [M‡Na] ; FAB-HRMS m/z: calcd for C35H60O3SiNa: 579.4210,
found: 579.4194.
Conversion from 7,8-didehydrocholesterol (8) to compound 10: The same
treatment of compound 8 (2.9 g, 7.8 mmol) as in the preparation for the
MOM ether of compound 2 gave a crude product, which was purified by
column chromatography (SiO2 70 g, n-hexane/EtOAc 30:1) to furnish 7
(3.07 g, 96%). The MOM ether 7 (1.65 g, 3.86 mmol) was transformed to a
triene 10 (1.4 g) in the same manner as for the preparation for compound 9.
Because of the same property as compound 9, the triene 10 was also
converted without further purification. An aliquot of the crude product
(20 mg) was purified by column chromatography (SiO2 2 g, n-hexane/
EtOAc 20:1) to furnish compound 10 as a colorless amorphous solid. Yield:
16 mg, 85%; [a]2D3 ˆ ‡162.58 (c ˆ 4.59 in CHCl3); 1H NMR (500 MHz,
CDCl3) d ˆ 5.67, 5.50, 5.40 (all brd, J ˆ ca. 6 Hz, 1H; H6,7,11), 4.68 (brs,
2H; 3-OCH2OCH3), 3.49 (m, 1H; H3), 3.37 (s, 3H; 3-OCH2OCH3), 1.24 (s,
3H; H19), 0.92 (d, J ˆ 6.1 Hz, 3H; H21), 0.87, 0.86 (both d, J ˆ 6.7 Hz, 3H;
H26,27), 0.56 (s, 3H; H18); IR nÄmax (KBr) ˆ 2949, 1466, 1148, 1105,
as a colorless amorphous solid. Yield: 5.1 g, 89%; [a]D23
ˆ
46.28 (c ˆ 1.13
1
in CHCl3); H NMR (500 MHz, CDCl3) d ˆ 5.56 (dd, J ˆ 5.5, 1.9 Hz, 1H;
H6), 5.38 (dt, J ˆ 5.5, 2.6 Hz, 1H; H7), 4.72, 4.70 (ABq, J ˆ 6.7 Hz, 2H;
3-OCH2OCH3), 3.63 (m, 1H; H22), 3.49 (m, 1H; H3), 3.38 (s, 3H;
3-OCH2OCH3), 0.95 (d, J ˆ 7.1 Hz, 3H; H21), 0.94 (s, 3H; H19), 0.91, 0.90
(both d, J ˆ 6.4 Hz, 3H; H26,27), 0.64 (s, 3H; H18); IR nÄmax (KBr) ˆ 3482,
1
‡
2946, 2876, 1462, 1044 cm ; FAB-MS m/z: 467 [M‡Na] ; FAB-HRMS
m/z: calcd for C29H48O3Na: 467.3502, found: 467.3503.
Preparation of (S)- and (R)-MTPA esters of compound 5: DMAP (2.7 mg,
0.022 mmol), EDCI ´ HCl (6.3 mg, 0.033 mmol) and (S)-MTPA (6.6 mg,
0.028 mmol) were successively added to a solution of compound 5 (5.0 mg,
0.011 mmol) in CH2Cl2 (1.0 mL), then the whole mixture was stirred at
room temperature for 9 h in the dark. The reaction mixture was poured into
saturated aqueous NH4Cl, then the whole mixture was extracted with
EtOAc. The EtOAc extract was washed with saturated aqueous NaCl, then
dried over MgSO4. Removal of the solvent from the EtOAc extract under
reduced pressure gave a crude product, which was purified by column
chromatography (SiO2 2 g, n-hexane/EtOAc 10:1) to furnish an (S)-MTPA
1
‡
1044 cm ; FAB-MS m/z: 449 [M‡Na] ; FAB-HRMS m/z: calcd for
C29H46O2Na: 449.3395, found: 449.3385.
Conversion from compound 9 to compound 11: A solution of compound 9
(4.8 g) in CH2Cl2 (121 mL) was treated with 4-phenyl-1,2,4-triazoline-3,5-
dione (1.53 g, 8.72 mmol) at room temperature for 1 h. Removal of the
solvent from the reaction mixture under reduced pressure gave a crude
product, which was purified by column chromatography (SiO2 100 g, n-
hexane/EtOAc 5:1) to furnish a Diels ± Alder adduct (4.8 g, 71% from
compound 6). A solution of the Diels ± Alder adduct of compound 9 (4.6 g,
6.34 mmol) in CHCl3 (127 mL) was treated with 3-chloro peroxybenzoic
acid (mCPBA) (10.9 g, 63.4 mmol) at room temperature for 17 h. The
reaction mixture was poured into 20% aqueous K2CO3, then the whole
mixture was extracted with EtOAc. The EtOAc extract was successively
washed with saturated aqueous NaHCO3 and saturated aqueous Na2S2O3/
saturated aqueous NaCl (1:1), then dried over MgSO4. Removal of the
ester as a colorless amorphous solid. Yield: 5.8 mg, 82%; [a]D23
ˆ
67.38
(c ˆ 0.44 in CHCl3); 1H NMR (500 MHz, CDCl3) d ˆ 7.54 (m, 2H;
(OCH3)PhCF3), 7.39 (m, 3H; (OCH3)PhCF3), 5.58 (dd, J ˆ 5.5, 1.9 Hz,
1H; H6), 5.40 (dt, J ˆ 5.5, 2.5 Hz, 1H; H7), 5.08 (brd, J ˆ 10.5 Hz, 1H;
H22), 4.72, 4.70 (ABq, J ˆ 6.6 Hz, 2H; 3-OCH2OCH3), 3.56 (s, 3H;
(OCH3)PhCF3), 3.52 (m, 1H; H3), 3.38 (s, 3H; 3-OCH2OCH3), 1.83 (m,
1H; H20), 1.48 (m, 4H; H23, 24), 0.95 (s, 3H; H19), 0.87, 0.85 (both d, J ˆ
6.6 Hz, 3H; H26,27), 0.75 (d, J ˆ 6.6 Hz, 3H; H21), 0.62 (s, 3H; H18); IR
1
nÄmax (KBr) ˆ 2949, 1742, 1454, 1269, 1169 cm
; FAB-MS m/z: 683
‡
[M‡Na] ; FAB-HRMS m/z: calcd for C39H55F3O5Na: 683.3900, found:
683.3889.
Chem. Eur. J. 2001, 7, No. 12
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0712-2667 $ 17.50+.50/0
2667

M. Kobayashi et al.
FULL PAPER
solvent from the EtOAc extract under reduced pressure gave a crude
product, which was purified by column chromatography (SiO2 120 g, n-
hexane/EtOAc 2:1) to furnish a mixture of compound 11 and an 9(11)-en-
6,7-epoxide isomer in a ratio of 3:1 (4.5 g, 95%). An aliquot of this mixture
(20 mg) was purified by HPLC [column: COSMOSIL 5C18-AR-II (20 mm
ù  250 mm), mobile phase: MeOH/H2O 99:1, detection: UV (l ˆ 230 nm),
flow rate: 5.0 mLmin 1] to furnish compound 11 (14 mg) and the 9(11)-en-
6,7-epoxide isomer (4.7 mg) both as colorless amorphous solids.
was refluxed for 6 h in the dark. The reaction mixture was treated with
LiAlH4 (1.5 g, 40 mmol) under reflux for 1 h in the dark. Work-up in the
same manner as for the preparation of compound 5 above gave a crude
product, which was purified by column chromatography (SiO2 50 g, n-
hexane/EtOAc 4:1) to furnish compound 13 as a colorless amorphous solid.
Yield: 1.5 g, 84%; [a]D21
ˆ
20.18 (c ˆ 0.16 in CHCl3); 1H NMR (500 MHz,
CDCl3) d ˆ 5.54 (dd, J ˆ 5.5, 1.9 Hz, 1H; H6), 5.38 (dt, J ˆ 5.5, 2.6 Hz, 1H;
H7), 4.72, 4.70 (ABq, J ˆ 6.7 Hz, 2H; 3-OCH2OCH3), 4.21 (m, 1H; H11),
3.55 (m, 2H; H3,22), 3.38 (s, 3H; 3-OCH2OCH3), 2.03 (brd, J ˆ 8.5 Hz, 1H;
H9), 1.11 (s, 3H; H19), 0.95 (d, J ˆ 6.7 Hz, 3H; H21), 0.88 (s, 9H; 22-
OSi(CH3)2C(CH3)3), 0.87 (d, J ˆ 6.4 Hz, 6H; H26,27), 0.61 (s, 3H; H18),
0.03, 0.02 (both s, 3H; 22-OSi(CH3)2C(CH3)3); IR nÄmax (KBr) ˆ 3401, 2948,
Compound 11: [a]D22
ˆ
58.38 (c ˆ 1.22 in CHCl3); 1H NMR (500 MHz,
CDCl3) d ˆ 7.42 (m, 4H; PhH), 7.31 (m, 1H; PhH), 6.53, 6.30 (both d, J ˆ
8.5 Hz, 1H; H6,7), 4.80, 4.72 (ABq, J ˆ 6.6 Hz, 2H; 3-OCH2OCH3), 4.27
(m, 1H; H3), 3.57 (dt, J ˆ 8.2, 3.6 Hz, 1H; H22), 3.38 (s, 3H;
3-OCH2OCH3), 3.25 (brd, J ˆ 5.6 Hz, 1H; H11), 1.09 (s, 3H; H19), 0.90
(d, J ˆ 6.6 Hz, 3H; H21), 0.88 (s, 9H; 22-OSi(CH3)2C(CH3)3), 0.86, 0.85
‡
1464, 1042 cm 1; FAB-MS m/z: 597 [M‡Na] ; FAB-HRMS m/z: calcd for
C35H62O4SiNa: 597.4315, found: 597.4321.
Conversion from compound 12 to compound 14: The same treatment of
compound 12 (542 mg, 0.877 mmol) as in the preparation for compound 13
was carried out to furnish compound 14 (351 mg, 90%) through chromato-
(both d, J ˆ 6.6 Hz, 3H; H26,27), 0.75 (s, 3H; H18), 0.03, 0.02 (both s, 3H;
1
22-OSi(CH3)2C(CH3)3); IR nÄmax (KBr) ˆ 2953, 1759, 1711, 1399, 1046 cm
;
‡
FAB-MS m/z: 770 [M‡Na] ; FAB-HRMS m/z: calcd for C43H65N3O6SiNa:
770.4540, found: 770.4513; elemental analysis calcd (%) for C43H65N3O6Si
(748.1): C 69.04, H 8.76, N 5.62; found C 68.74, H 8.73, N 5.29.
graphic purification (SiO2 20 g, n-hexane/EtOAc 6:1) as
a colorless
1
amorphous solid: [a]D21
ˆ
44.68 (c ˆ 0.34 in CHCl3); H NMR (500 MHz,
CDCl3) d ˆ 5.54 (dd, J ˆ 5.5, 1.9 Hz, 1H; H6), 5.37 (dt, J ˆ 5.5, 2.6 Hz, 1H;
H7), 4.71, 4.69 (ABq, J ˆ 6.9 Hz, 2H; 3-OCH2OCH3), 4.21 (ddd, J ˆ 10.8,
9.3, 5.5 Hz, 1H; H11), 3.54 (m, 1H; H3), 3.38 (s, 3H; 3-OCH2OCH3), 2.02
(d, J ˆ 9.3 Hz, 1H; H9), 1.11 (s, 3H; H19), 0.96 (d, J ˆ 6.5 Hz, 3H; H21),
0.87, 0.86 (both d, J ˆ 6.7 Hz, 3H; H26,27), 0.62 (s, 3H; H18); IR nÄmax
9(11)-En-6,7-epoxide: [a]D23
ˆ
19.88 (c ˆ 0.64 in CHCl3); 1H NMR
(500 MHz, CDCl3) d ˆ 7.44 (m, 4H; PhH), 7.34 (m, 1H; PhH), 5.78 (brd,
J ˆ 5.5 Hz, 1H; H11), 4.79, 4.68 (ABq, J ˆ 6.6 Hz, 2H; 3-OCH2OCH3), 4.03
(m, 1H; H3), 3.59 (m, 1H; H22), 3.37 (s, 3H; 3-OCH2OCH3), 3.62, 3.35
(both d, J ˆ 4.6 Hz, 1H; H6,7), 1.36 (s, 3H; H19), 0.91 (d, J ˆ 6.8 Hz, 3H;
H21), 0.89 (s, 9H; 22-OSi(CH3)2C(CH3)3), 0.86, 0.85 (both d, J ˆ 6.4 Hz,
3H; H26,27), 0.81 (s, 3H; H18), 0.03 (s, 6H; 22-OSi(CH3)2C(CH3)3); IR
(KBr) ˆ 3405, 2955, 1466, 1107, 1042 cm 1; FAB-MS m/z: 467 [M‡Na] ;
‡
FAB-HRMS m/z: calcd for C29H48O3Na: 467.3501, found: 467.3530.
nÄmax (KBr) ˆ 2951, 1765, 1713, 1399, 1044 cm 1; FAB-MS m/z: 748 [M‡H] ;
‡
Conversion from compound 13 to compound 15: 2,6-Lutidine (1.07 mL,
9.2 mmol), then TBSOTf (1.75 mL, 7.64 mmol) were added to a solution of
compound 13 (1.36 g, 3.06 mmol) in PhCH3 (99 mL) at room temperature
and the whole mixture was stirred for 1 h in the dark. Work-up in the same
manner as for the preparation of the TBS ether of compound 5a gave a
crude product, which was purified by column chromatography (SiO2 50 g,
n-hexane/EtOAc 30:1) to furnish an TBS ether as a colorless amorphous
FAB-HRMS m/z: calcd for C43H66N3O6Si: 748.4721, found: 748.4724;
elemental analysis calcd (%) for C43H65N3O6Si (748.1): C 69.04, H 8.76, N
5.62; found C 69.19, H 8.81, N 5.72.
Conversion from compound 10 to compound 12: The same treatment of
compound 10 (1.36 g, 3.18 mmol) as in the preparation for compound 11
gave a crude product, which was purified by column chromatography (SiO2
36 g, n-hexane/EtOAc 7:2) to furnish a Diels ± Alder adduct of compound
10 (1.8 g, 94%). Epoxidation of the 22-deoxy congener was conducted in
the same manner as for the preparation of compound 11 to furnish
compound 12 and an 6,7-epoxide isomer in a ratio of 4:1 (1.3 g, 80%)
through chromatographic purification (SiO2 40 g, n-hexane/EtOAc 5:2).
An aliquot of this mixture (21 mg) was purified by HPLC [column:
COSMOSIL 5C18-AR-II (20 mm ù Â 250 mm), mobile phase: MeOH/H2O
99:1, detection: UV (l ˆ 230 nm), flow rate: 5.0 mLmin 1] to furnish
compound 12 (15 mg) and the 9(11)-en-6,7-epoxide isomer (3.8 mg) as
colorless amorphous solids.
solid. Yield: 2.0 g, 95%; [a]D22
ˆ
15.78 (c ˆ 0.10 in CHCl3); 1H NMR
(500 MHz, CDCl3) d ˆ 5.50 (dd, J ˆ 5.5, 1.9 Hz, 1H; H6), 5.33 (dt, J ˆ 5.5,
2.6 Hz, 1H; H7), 4.71, 4.69 (ABq, J ˆ 6.7 Hz, 2H; 3-OCH2OCH3), 4.27 (td,
J ˆ 10.7, 5.4 Hz, 1H; H11), 3.55 (m, 2H; H3, 22), 3.38 (s, 3H;
3-OCH2OCH3), 2.14 (d, J ˆ 10.7 Hz, 1H; H9) 1.08 (s, 3H; H19), 0.94 (d,
J ˆ 6.7 Hz, 3H; H21), 0.89 (s, 18H; 11, 22-OSi(CH3)2C(CH3)3), 0.87 (d, J ˆ
6.4 Hz, 6H; H26,27), 0.59 (s, 3H; H18), 0.10, 0.09 (both s, 3H; 11,22-
OSi(CH3)2C(CH3)3), 0.03 (s, 6H); IR nÄmax (KBr) ˆ 2953, 1466, 1256,
‡
1047 cm 1; FAB-MS m/z: 711 [M‡Na] ; FAB-HRMS m/z: calcd for
C41H76O4Si2Na: 711.5180, found: 711.5170.
1-(4-Methoxyphenyl)-4-phenyl-1-azabuta-(E,E)-1,3-diene
(190 mg,
Compound 12: [a]D21
ˆ
91.98 (c ˆ 1.07 in CHCl3); 1H NMR (500 MHz,
0.8 mmol) and Fe(CO)5 (18 mL, 138 mmol) were added to a solution of
the TBS ether (1.9 g, 2.76 mmol) in PhCH3 (18 mL), and the whole mixture
was refluxed for 27 h in the dark. Removal of the solvent from the reaction
mixture under reduced pressure gave a crude product, which was purified
by column chromatography (SiO2 50 g, n-hexane/acetone 30:1) to furnish a
tricarbonyl iron complex (2.1 g, 94%). MgBr2 ´ Et2O (2.3 g, 9.0 mmol) was
gradually added to a solution of this complex (1.24 g, 1.5 mmol) in CH2Cl2
(15 mL) and Me2S (3.3 mL, 45 mmol) at room temperature, then the whole
mixture was stirred for 4 h in the dark. Work-up in the same manner as for
the preparation of the TBS ether of compound 5a gave a crude product,
which was purified by column chromatography (SiO2 50 g, n-hexane/
EtOAc 5:1) to furnish compound 15 as a yellow amorphous solid. Yield:
CDCl3) d ˆ 7.42 (m, 4H; PhH), 7.31 (m, 1H; PhH), 6.54, 6.29 (both d, J ˆ
8.4 Hz, 1H; H6,7), 4.81, 4.72 (ABq, J ˆ 6.8 Hz, 2H; 3-OCH2OCH3), 4.27
(m, 1H; H3), 3.38 (s, 3H; 3-OCH2OCH3), 3.25 (brd, J ˆ 5.5 Hz, 1H; H11),
1.09 (s, 3H; H19), 0.92 (d, J ˆ 6.5 Hz, 3H; H21), 0.86, 0.85 (both d, J ˆ
6.7 Hz, 3H; H26,27), 0.75 (s, 3H; H18); IR nÄmax (KBr): 2953, 1757, 1707,
1399, 1046 cm 1; FAB-MS m/z: 618 [M‡H] ; FAB-HRMS m/z: calcd for
‡
C37H52N3O5: 618.3907, found: 618.3922; elemental analysis calcd (%) for
C37H51N3O5 (617.8): C 71.93, H 8.32, N 6.80; found C 72.13, H 8.62, N 6.85.
9(11)-Ene-6,7-epoxide: [a]D20
ˆ
26.28 (c ˆ 0.36 in CHCl3); 1H NMR
(500 MHz, CDCl3) d ˆ 7.43 (m, 4H; PhH), 7.33 (m, 1H; PhH), 5.77 (dd,
J ˆ 6.7, 1.6 Hz, 1H; H11), 4.80, 4.68 (ABq, J ˆ 6.8 Hz, 2H; 3-OCH2OCH3),
4.08 (m, 1H; H3), 3.63, 3.35 (both d, J ˆ 4.8 Hz, 1H; H6,7), 3.37 (s, 3H;
3-OCH2OCH3), 1.35 (s, 3H; H19), 0.93 (d, J ˆ 6.5 Hz, 3H; H21), 0.86 (d,
J ˆ 6.8 Hz, 6H; H26,27), 0.82 (s, 3H; H18); IR nÄmax (KBr) ˆ 2930, 1763,
860 mg, 73%; [a]2D3
ˆ
21.68 (c ˆ 0.13 in CHCl3); 1H NMR (500 MHz,
CDCl3) d ˆ 5.22, 4.92 (both d, J ˆ 4.4 Hz, 1H; H6,7), 3.68 (m, 1H; H3), 3.53
(m, 2H; H11,22), 1.09 (s, 3H; H19), 0.90 (d, J ˆ 7.0 Hz, 3H; H21), 0.88 (m,
15H; 11 or 22-OSi(CH3)2C(CH3)3, H26,27), 0.85 (s, 9H; 11 or 22-
OSi(CH3)2C(CH3)3), 0.69 (s, 3H; H18), 0.08 (s, 3H), 0.02 (s, 6H), 0.01 (s,
3H) [11,22-OSi(CH3)2C(CH3)3]; IR nÄmax (KBr) ˆ 3414, 2957, 2033, 1962,
1711, 1412, 1044 cm 1; FAB-MS m/z: 618 [M‡H] ; FAB-HRMS m/z: calcd
‡
for C37H52N3O5: 618.3907, found: 618.3906; elemental analysis calcd (%) for
C37H51N3O5 (617.8): C 71.93, H 8.32, N 6.80; found C 72.09, H 8.43, N 6.93.
1470, 1254, 1063 cm 1; FAB-MS m/z: 807 [M‡Na] ; FAB-HRMS m/z:
‡
Reductive cleavage of the epoxide 11: A solution of the epoxide 11 (21 mg,
0.028 mmol) in THF (1.8 mL) was treated with LiAlH4 (10.6 mg,
0.28 mmol) under reflux for 15 h in the dark. Work-up in the same manner
as for the preparation of compound 5 gave a crude product, which was
purified by column chromatography (SiO2 2 g, n-hexane/EtOAc 4:1) to
furnish compound 13 (6.0 mg, 48%).
calcd for C42H72FeO6Si2Na: 807.4115, found: 807.4088.
Conversion from compound 15 to compound 16: TsCl (2.07 g, 10.9 mmol)
was added to a solution of compound 15 (850 mg, 1.08 mmol) in pyridine
(21.7 mL), then the whole mixture was stirred at room temperature for 12h
in the dark. The reaction mixture was poured into 5% aqueous HCl, then
the whole mixture was extracted with EtOAc. The EtOAc extract was
washed with saturated aqueous NaCl, then dried over MgSO4. Removal of
the solvent from the EtOAc extract under reduced pressure gave a crude
Reductive cleavage of the epoxide 11 by using Et2AlCl: A solution of
Et2AlCl (1.0m in n-hexane; 8.0 mL, 80.0 mmol) was added to a solution of
the epoxide 11 (3.0 g, 4.0 mmol) in THF (251 mL), then the whole mixture
2668
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0712-2668 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 12

Synthesis of Agosterol A
2663±2670
product, which was purified by column chromatography (SiO2 50 g, n-
hexane/EtOAc 15:1) to furnish a tosylate (1.1 g, quant.). A solution of the
tosylate (884 mg, 0.94 mmol) in PhH (56 mL) was heated under reflux with
DBN (11.7 mL, 94 mmol) for 26 h in the dark. Work-up in the same manner
as for the preparation of the TBS ether of compound 5a gave a crude
product, which was purified by column chromatography (SiO2 30 g, 100%
n-hexane) to furnish compound 16 as a yellow amorphous solid. Yield:
EtOAc. The EtOAc extract was washed with saturated aqueous NaCl, then
dried over MgSO4. Removal of the solvent from the EtOAc extract under
reduced pressure gave a crude product, which was purified by column
chromatography (SiO2 2 g, n-hexane/EtOAc 1:3) to furnish compound 19
as a colorless amorphous solid. Yield: 7.4 mg, 90%; [a]2D2 ˆ ‡15.08 (c ˆ 0.18
in CHCl3); 1H NMR (500 MHz, CDCl3) d ˆ 5.33 (brs, 1H; H7), 4.32 (brd,
J ˆ 9.5 Hz, 1H; H6), 4.29 (brs, 1H; H4), 3.99 (td, J ˆ 10.9, 5.2 Hz, 1H;
H11), 3.56 (m, 2H; H3, 22), 1.14 (s, 3H; H19), 0.91 (d, J ˆ 6.6 Hz, 3H; H21),
0.89, 0.88 (both s, 9H; 11 or 22-OSi(CH3)2C(CH3)3), 0.87 (d, J ˆ 6.6 Hz, 6H;
H26,27), 0.53 (s, 3H; H18), 0.09, 0.08 (both s, 3H; 11, 22-
OSi(CH3)2C(CH3)3), 0.02 (s, 6H); IR nÄmax (KBr) ˆ 3409, 2957, 1472, 1254,
722 mg, quant.; [a]D23
ˆ
152.38 (c ˆ 0.34 in CHCl3); 1H NMR (500 MHz,
CDCl3) d ˆ 5.59 (brs, 2H; H3,4), 5.19, 4.99 (both d, J ˆ 4.3 Hz, 1H; H6,7),
3.62 (td, J ˆ 10.5, 4.9 Hz, 1H; H11), 3.54 (m, 1H; H22), 0.97 (s, 3H; H19),
0.92 (d, J ˆ 6.9 Hz, 3H; H21), 0.88 (m, 24H; 11,22-OSi(CH3)2C(CH3)3,
H26,27), 0.72 (s, 3H; H18), 0.07, 0.02 (both s, 6H; 11,22-
OSi(CH3)2C(CH3)3); IR nÄmax (KBr) ˆ 2959, 2033, 1960, 1468, 1256,
‡
1063, 835 cm 1; FAB-MS m/z: 701 [M‡Na] ; FAB-HRMS m/z: calcd for
C39H74O5Si2Na: 701.4973, found: 701.4948.
‡
1059 cm 1; FAB-MS m/z: 789 [M‡Na] ; FAB-HRMS m/z: calcd for
Conversion from compound 19 to agosterol A (1): A solution of compound
19 (5.6 mg, 0.0082 mmol) in pyridine (1.6 mL) was treated with Ac2O
(0.4 mL) at 508C for 24 h. Work-up in the same manner as for the
preparation of compound 5a gave a crude product, which was purified by
column chromatography (SiO2 2 g, n-hexane/EtOAc 3:1) to furnish a
triacetate (6.0 mg, 91%). A solution of the triacetate (4.0 mg, 0.005 mmol)
in THF (1.5 mL) was treated with 70% HF-pyridine (0.3 mL) at room
temperature for 24 h. The reaction mixture was poured into saturated
aqueous NaHCO3, then the whole mixture was extracted with EtOAc. The
EtOAc extract was successively washed with saturated aqueous NaCl,
saturated aqueous NaHCO3, and saturated aqueous NaCl, then dried over
MgSO4. Removal of the solvent from the EtOAc extract under reduced
pressure gave a crude product, which was purified by column chromatog-
raphy (SiO2 2 g, n-hexane/EtOAc 1:1) to furnish agosterol A (1) (2.4 mg,
84%). The synthesized agosterol A was identical with the authentic sample
as shown by 1H NMR, 13C NMR, IR, MS, and optical rotation.
C42H70FeO5Si2Na: 789.4009, found: 789.3998.
Dihydroxylation of compound 16 with OsO4: A solution of compound 16
(617 mg, 0.805 mmol) in pyridine (8.6 mL) was treated with OsO4 (1.0m in
Py, 1.17 mL, 1.17 mmol) at room temperature for 5.5 h in the dark. The
reaction mixture was successively treated with H2O (34 mL) and NaHSO3
(11.7 g, 97.5 mmol), then the whole mixture was stirred at room temper-
ature for 24 h in the dark. The mixture was extracted with EtOAc, then the
EtOAc extract was washed with saturated aqueous NaCl and dried over
MgSO4. Removal of the solvent from the EtOAc extract under reduced
pressure gave a crude product, which was purified by column chromatog-
raphy (SiO2 15 g, n-hexane/EtOAc 3:1) to furnish compound 17 as a yellow
amorphous solid. Yield: 493 mg, 80%; [a]D23
ˆ
35.28 (c ˆ 0.79 in CHCl3);
1H NMR (500 MHz, CDCl3) d ˆ 5.41, 5.30 (both d, J ˆ 4.5 Hz, 1H; H6,7),
3.84 (brd, J ˆ 11.1 Hz, 1H; H3), 3.80 (brs, 1H; H4), 3.52 (m, 2H; H11,22),
1.23 (s, 3H; H19), 0.90 (d, J ˆ 6.8 Hz, 3H; H21), 0.89 (d, J ˆ 7.2 Hz, 3H;
H26, 27), 0.87 (d, J ˆ 7.2 Hz, 3H), 0.88 (s, 9H), 0.85 (s, 9H; [11, 22-
OSi(CH3)2C(CH3)3]), 0.68 (s, 3H; H18), 0.04 (s, 3H), 0.02 (s, 6H), 0.01 (s,
3H) [11,22-OSi(CH3)2C(CH3)3]; IR nÄmax (KBr) ˆ 3405, 2953, 2035, 1964,
Acknowledgements
1466, 1256, 1061 cm 1; FAB-MS m/z: 823 [M‡Na] ; FAB-HRMS m/z:
‡
calcd for C42H72FeO7Si2Na: 823.4064, found: 823.4060.
The authors are grateful to the Naito Foundation, the Houansha
Foundation, and the Ministry of Education, Science, Sports, and Culture
of Japan for financial support.
Conversion from compound 17 to compound 18: DMAP (6.0 mg,
0.049 mmol) was added to
a solution of compound 17 (42.7 mg,
0.053 mmol) in pyridine (1.0 mL), then the reaction mixture was treated
with TESOTf (89 mL, 0.39 mmol) at room temperature for 1 h in the dark.
Work-up in the same manner as for the preparation of compound 5a gave a
crude product, which was purified by column chromatography (SiO2 2 g, n-
hexane/EtOAc 100:1) to furnish a di-TES ether (56.0 mg, quant.). A
solution of the di-TES ether (55.0 mg, 0.052 mmol) in PhH (5.2 mL) was
treated with Me3NO (78 mg, 1.04 mmol) at room temperature for 28 h in
the dark. Work-up in the same manner as for the preparation of compound
5a gave compound 18 as a colorless amorphous solid. Yield: 44 mg, 95%;
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[a]2D2
ˆ
76.18 (c ˆ 1.46 in CHCl3); 1H NMR (500 MHz, CDCl3) d ˆ 5.66 (d,
J ˆ 5.6 Hz, 1H; H6), 5.40 (dt, J ˆ 5.6, 2.8 Hz, 1H; H7), 4.28 (m, 1H; H11),
4.10 (brs, 1H; H4), 3.57 (brd, J ˆ 8.4 Hz, 1H; H22), 3.51 (brd, J ˆ 11.4 Hz,
1H; H3), 1.20 (s, 3H; H19), 0.95 (3,4-OSi(CH2CH3)3, 21H; H21), 0.90, 0.89
(both s, 9H; 11 or 22-OSi(CH3)2C(CH3)3), 0.88 (d, J ˆ 6.6 Hz, 6H; H26,27),
0.60 (s, 15H; 3,4-OSi(CH2CH3)3, H18), 0.10, 0.09 (both s, 3H; 11 or 22-
OSi(CH3)2C(CH3)3), 0.03 (s, 6H); IR nÄmax (KBr) ˆ 2953, 1462, 1254, 1090,
1
‡
835 cm
;
FAB-MS m/z: 911 [M‡Na] ; FAB-HRMS m/z: calcd for
C51H100O4Si4Na: 911.6597, found: 911.6587.
Hydroboration followed by deprotection of TES groups giving compound
19: A solution of BH3 ´ SMe2 (2.0m, 0.048 mmol) in THF, (27 mL) was added
to a solution of compound 18 (21.0 mg, 0.024 mmol) in THF (0.8 mL) at
08C, then the whole mixture was stirred at room temperature for 2 h in the
dark. The reaction mixture was treated with MeOH (50 mL) at 08C, then
1.0 n aqueous NaOH (66 mL) and 30% aqueous H2O2 (17 mL) were
successively added to the reaction mixture at 08C. After being stirred at
room temperature for 1 h, the whole mixture was poured into saturated
aqueous NaCl, and was then extracted with EtOAc. The EtOAc extract was
washed with saturated aqueous Na2S2O3/saturated aqueous NaCl (1:1),
then dried over MgSO4. Removal of the solvent from the EtOAc extract
under reduced pressure gave a crude product, which was purified by
column chromatography (SiO2 2 g, n-hexane/EtOAc 40:1) to furnish an 6a-
alcohol (13.4 mg, 62%). A solution of the 6a-alcohol (11.0 mg, 0.012 mmol)
in THF (1.84 mL) was treated with TBAF (1.0m in THF, 120 mL,
0.12 mmol) at 208C for 24 h. The reaction mixture was poured into
saturated aqueous NaCl, then the whole mixture was extracted with
Chem. Eur. J. 2001, 7, No. 12
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0947-6539/01/0712-2669 $ 17.50+.50/0
2669

M. Kobayashi et al.
FULL PAPER
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column chromatography. Since isolation of the resulting 11a-alcohol
13 was very easy, this conversion was executed as a mixture in the
synthesis of agosterol A (1).
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Received: January 11, 2001 [F2998]
2670
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0947-6539/01/0712-2670 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 12