Synthesis and anti-tumor activity of new steroidal nuclear analogues of aragusterol A

Article abstract of DOI:10.1248/cpb.51.640

3α,7α-Dihydroxy-5-epiaragusterol A (3) was synthesized from bile acid (cholic acid) as a new steroidal nuclear analogue of antitumor marine steroid aragusterol A. 7α-Hydroxyaragusterol A (4) was also derived from xestokerol B. The in vitro anti-proliferat

Full text of DOI:10.1248/cpb.51.640

640
Chem. Pharm. Bull. 51(6) 640—645 (2003)
Vol. 51, No. 6
Synthesis and Anti-tumor Activity of New Steroidal Nuclear Analogues of
Aragusterol A
*
Hidemichi MITOME, Masakazu SHINOHARA, Hiroaki MIYAOKA, and Yasuji YAMADA
School of Pharmacy, Tokyo University of Pharmacy and Life Science; 1432–1 Horinouchi, Hachioji, Tokyo 192–0392,
Japan. Received January 15, 2003; accepted April 4, 2003; published online April 8, 2003
3a,7a-Dihydroxy-5-epiaragusterol A (3) was synthesized from bile acid (cholic acid) as a new steroidal nu-
clear analogue of antitumor marine steroid aragusterol A. 7a-Hydroxyaragusterol A (4) was also derived from
xestokerol B. The in vitro anti-proliferative activity of each of these analogues toward KB cells as well as in vivo
anti-tumor activity of 5-epiaragusterol A (2) previously synthesized by the authors and 3 were assessed.
Key words synthesis; anti-tumor; steroid; analogue; aragusterol A
Aragusterol A (1) having a structurally unique marine A-containing marine sponge were carried out so as to obtain
steroid with a 12b-hydroxy group and rare 26,27-cyclo struc- new steroidal nuclear analogues of aragusterol A. The in
ture, was isolated from an Okinawan marine sponge of the vitro anti-proliferative activity of 3 and 4 toward KB cells
genus Xestospongia.1—6) This compound has been found to and in vivo antitumor activity of 2 and 3 were assessed and
strongly inhibit KB cell proliferation when present at IC50 are discussed in the following.
0.042 mg/ml and express potent anti-tumor activity toward
L1210 leukemia in mice (T/C 220% at 1.6 mg/kg).1) A com- Results and Discussion
parison of the activity of this compound with that of other
New steroidal nuclear analogues of aragusterol A were
aragusterols and their derivatives, indicated the configura- synthesized in this study by a method based on previously
tions of C-12, C-22 hydroxy and C-20 epoxy groups to be obtained results.8,9) Prior to these syntheses, the method for
significantly essential for expression of the anti-tumor activ- obtaining side-chain segment was improved. (S)-(Ϫ)-Cit-
ity of aragusterol A. A study on the underlying mechanism ronellol was transformed to ester 5 by protection of the hy-
for inhibition of cellular proliferation has disclosed aragus- droxy group as tert-butyldiphenylsilyl (TBDPS) ether, oxida-
terol A to arrest human non-small-cell lung-cancer (NSCLC) tive degradation of the double bond, oxidation of resulting
cells in the late G1 phase of the cell cycle by inhibiting pRb aldehyde and esterification of the carboxyl group (Chart 1).
phosphorylation.7) The synthesis of this compound was pre- Ester 5 was converted to allylic alcohol 6 as follows: (1)
viously carried out via the coupling of a steroidal nuclear treatment with lithium diisopropylamide (LDA) and then
segment obtained from (ϩ)-hecogenin with the side-chain diphenyldisulfide to afford phenylsulfide, (2) m-chloroper-
segment produced in an enantioselective manner.8) The large benzoic acid (mCPBA) oxidation of the sulfide to sulfoxide,
scale synthesis of aragusterol A has proven difficult owing to (3) introduction of a carbon–carbon double bond by thermal
the scarcity of (ϩ)-hecogenin in nature. The synthesis of 5- elimination and (4) diisobutylaluminum hydride (DIBAH)
epiaragusterol A (2) was thus previously carried out from reduction of the ester. Diastereoselective cyclopropanation of
bile acid (deoxycholic acid) abundantly present in nature and allylic alcohol 6 was done with Charette’s dioxaborolane lig-
the compound was noted to express strong anti-proliferative and 710—13) to give alcohol 8 as the sole product. Bromide 9
activity at IC50 0.041 mg/ml toward KB cells in vitro, the ex- was obtained from alcohol 8 by conversion of the alcohol
tent being comparable to that of aragusterol A.9) In the pre- into bromide, reduction of bromide by LiAlH4, deprotection
sent study, the synthesis of 3a,7a-dihydroxy-5-epiaragus- of TBDPS ether and coversion of the corresponding alcohol
terol A (3) from cholic acid and that of 7a-hydroxyaragus- into bromide. By this method, side-chain segment could be
terol A (4) from xestokerol B4,5) isolated from an aragusterol produced on a large scale with greater efficiency than was
Chart 1
* To whom correspondence should be addressed. e-mail: yamaday@ps.toyaku.ac.jp
© 2003 Pharmaceutical Society of Japan

June 2003
641
Chart 2
Chart 4
iPr2NMgBr.22) Finally, epoxidation of allylic alcohol 16 with
mCPBA gave 3a,7a-dihydroxy-5-epiaragusterol A (3) as a
single product.23)
Treatment of xestokerol B with p-toluenesulfonic acid
(TsOH) in MeOH provided 20(22)E-olefin 17 along with
the 20(22)Z-isomer in a 4 : 1 ratio (Chart 4).19) Epoxidation
t
of olefin 17 was conducted with BuOOH in the presence
of VO(acac)2, giving (20R,22R)-epoxide 18 along with
(20S,22S)-isomer in a 25 : 1 ratio.21) Epoxide 18 was con-
verted to allylic alcohol 19 by treatment with iPr2NMgBr. Al-
lylic alcohol 19 was made to undergo epoxidation23) and on
deprotection of the ketal, 7a-hydroxyaragusterol A (4) was
obtained as the sole product.
3a,7a-Dihydroxy-5-epiaragusterol A (3) and 7a-hydroxy-
aragusterol A (4) were found to inhibit KB cellular growth
when present at IC50 1.241 and 0.048 mg/ml in vitro, respec-
tively. 5-Epiaragusterol A (2) and 3a,7a-dihydroxy-5-epiara-
gusterol A (3) expressed anti-tumor activity toward L1210
leukemia in mice (T/C 152% at 7.25 mg/kg and 205% at
Chart 3
possible previously.8)
From ketone 10, obtained by degradation of the side-chain 25 mg/kg, respectively). In vitro, the lesser activity of 3a,7a-
from cholic acid,14,15) diol 11 was produced via the following dihydroxy-5-epiaragusterol A (3) and greater effective dose
steps: (1) protection of the ketone as a ketal, (2) selective requirement of 5-epiaragusterol A (2) and 3 compared to ara-
methanolysis of acetate at C-3 by K2CO3 in MeOH, (3) pro- gusterol A (1) may possibly owe to differences in the physi-
tection of the hydroxy group as methoxymethyl (MOM) cal properties of these compounds. In vivo assessment of 7a-
ether and (4) deprotection of acetates by K2CO3 in MeOH hydroxyaragusterol A (4) activity has not been possible
under reflux (Chart 2). Diol 11 thus obtained was treated owing to the its scarcity of this compound.
with pyridinium chlorochromate (PCC) to give diketone in
89% yield. Treatment of this diketone with L-Selectride® af- Conclusion
forded diol 12 having the 12b-hydroxy group along with the
The authors carried out the synthesis of 3a,7a-dihydroxy-
12a-isomer 11 in a 5 : 2 ratio.16) Deprotection of the ketal 5-epiaragusterol A (3) from cholic acid and 7a-hydroxyara-
followed by protection of the hydroxy groups as MOM ether gusterol A (4) from xestokerol B as new steroidal nuclear
gave nuclear segment 13.
analogues of aragusterol A. 3a,7a-Dihydroxy-5-epiaragus-
Reaction of nuclear segment 13 with alkyllithium, ob- terol A (3) and previously synthesized 5-epiaragusterol A (2)
tained from bromide 9 with lithium naphthalenide,17,18) fol- were clearly shown to express in vitro anti-proliferative and
lowed by treatment with conc. HCl gave 20(22)E-olefin 14 antitumor activity. It would thus appear that the steroidal nu-
and the 20(22)Z-isomer in a 12 : 1 ratio (Chart 3).19) Stereose- cleus functions as determinant only of the physical properties
lective epoxidation of olefin 14 was carried out by its treat- of the analogues of aragusterol A, not its biological activity.
t
ment with BuOOH in the presence of vanadium (III) acety- Modification of the steroidal nucleus of aragusterol A may
lacetonate (VO(acac)2)20) to afford (20R,22R)-epoxide 15 possibly prove useful for synthesizing analogues of aragus-
along with (20S,22S)-isomer in a 13 : 1 ratio.21) Allylic alco- terol A possessing greater medical potential.
hol 16 was derived from epoxide 15 by treatment with

642
Vol. 51, No. 6
Experimental
Na2HPO4 (32.0 g, 223 mmol). The cold (Ϫ40 °C) suspension was carefully
treated with a solution of mCPBA (66.0 g, 268 mmol) in CHCl3 (600 ml).
The reaction mixture was allowed to warm slowly to room temperature,
stirred for 2 h at this temperature, filtered through celite, and then concen-
trated under reduced pressure. The residue was dissolved in Et2O (600 ml)
and washed successively with water and saturated NaCl solution, dried over
MgSO4 and concentrated under reduced pressure. Resulting crude sulfoxide
was used for subsequent reaction without further purification.
The solution of the above crude sulfoxide in toluene (500 ml) was refluxed
for 2 h and concentrated under reduced pressure. The residue was chro-
matographed on a silica gel column with hexane–Et2O (9 : 1) to give a,b-un-
saturated ester (78.0 g, 3 steps 85% yield) as a colorless oil: [a]D30 Ϫ16.0°
(cϭ1.4, CHCl3). ESI-MS m/z: 419 (MϩϩNa). HR-ESI-MS m/z: 419.2058
(Calcd for C24H32O3NaSi: 419.2018). IR (neat) cmϪ1: 3072, 1729, 1659. 1H-
NMR (400 MHz, CDCl3) d: 1.03 (3H, d, Jϭ6.8 Hz), 1.04 (9H, s), 1.60 (2H,
m), 2.59 (1H, m), 3.64 (1H, d, Jϭ6.1 Hz), 3.67 (1H, d, Jϭ6.3 Hz), 3.73 (3H,
s), 5.78 (1H, dd, Jϭ15.7, 1.1 Hz), 6.87 (1H, dd, Jϭ15.7, 7.8 Hz), 7.40 (6H,
m), 7.65 (4H, m).
Melting points were measured on Yazawa BY-2 micro melting point appa-
ratus without correction. Optical rotation was measured with a JASCO DIP-
360 automatic polarimeter. Infrared (IR) spectra were recorded with a
Perkin-Elmer FT-IR 1710 or a JASCO FT/IR-620 spectrometer. 1H- and 13C-
NMR spectra were recorded with a Varian Gemini-300, a Bruker AM-400 or
DPX-400. Chemical shifts were expressed on a d (ppm) scale with tetram-
ethylsilane (TMS) as the internal standard (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad). Electron ionization mass spectra (EI-MS),
electrospray ionization mass spectra (ESI-MS), high-resolution electron ion-
ization mass spectra (HR-EI-MS) and high-resolution electrospray ioniza-
tion mass spectra (HR-ESI-MS) were obtained with a Hitachi M-80, a
Thermo Quest TSQ 700, a VG Auto Spec E or a Micromass LCT spectrom-
eter.
Methyl (S)-6-(tert-Butyldiphenylsiloxy)-4-methylhexanoate (5) To a
solution of (S)-(Ϫ)-citronellol (5.00 g, 32.0 mmol, Ͼ99% ee) in 1,2-
dichloroethane (32 ml) were added triethylamine (11.0 ml, 77.0 mmol), 4-di-
methylaminopyridine (DMAP, 390 mg, 3.20 mmol) and TBDPSCl (10.0 ml,
38.0 mmol). The mixture was stirred at room temperature for 8 h. The reac-
tion mixture was diluted with Et2O (200 ml) and washed successively with
water and saturated NaCl solution. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was chromatographed
on a silica gel column with hexane–Et2O (9 : 1) to give TBDPS ether (12.6 g,
99% yield) as a colorless oil: [a]D26 ϩ2.7° (cϭ4.1, CHCl3). EI-MS m/z: 394
(Mϩ). HR-EI-MS m/z: 394.2692 (Calcd for C26H38OSi: 394.2692). IR (neat)
To a cold (Ϫ78 °C) solution of the above a,b-unsaturated ester (1.20 g,
2.90 mmol) in CH2Cl2 (10 ml) was added DIBAH (7.0 ml, 6.40 mmol, 0.93 M
in hexane). The reaction mixture was stirred for 30 min, diluted with Et2O
(300 ml), treated with saturated NaCl solution (10 ml) and then stirred vigor-
ously for further 30 min. The organic layer was dried over MgSO4 and con-
centrated under reduced pressure. The residue was chromatographed on a
silica gel column with hexane–Et2O (2 : 1) to give allylic alcohol 6 (1.00 g,
97% yield) as a colorless oil: [a]D30 Ϫ7.8° (cϭ0.8, CHCl3). ESI-MS m/z: 391
(MϩϩNa). HR-ESI-MS m/z: 391.2057 (Calcd for C23H32O2NaSi: 391.2069).
1
cmϪ1: 2930. H-NMR (300 MHz, CDCl3) d: 0.86 (3H, d, Jϭ6.5 Hz), 1.08
(9H, s), 1.17 (1H, m), 1.36 (2H, m), 1.62 (3H, s), 1.63 (2H, m), 1.71 (3H, s),
1.99 (2H, m), 3.71 (2H, m), 5.11 (1H, m), 7.42 (6H, m), 7.71 (4H, m).
To a solution of the above TBDPS ether (24.0 g, 60.8 mmol) in
CHCl3–MeOH (1 : 1, 600 ml) was added NaHCO3 (16.0 g, 182 mmol). The
mixture was stirred at Ϫ15 °C with introducing ozone until pale blue color
persisted. Excess ozone was removed by a flow of argon. The reaction mix-
ture was treated with Me2S (14.0 ml, 182 mmol), allowed to worm slowly to
room temperature over 2 h. The reaction mixture was stirred for 8 h at room
temperature and concentrated under reduced pressure. The residue was di-
luted with Et2O (100 ml) and washed successively with water and saturated
NaCl solution. The organic layer was dried over MgSO4 and concentrated
under reduced pressure to give crude aldehyde. The crude product was used
for subsequent reaction without further purification.
1
IR (neat) cmϪ1: 3327, 2930. H-NMR (400 MHz, CDCl3) d: 0.97 (3H, d,
Jϭ6.8 Hz), 1.04 (9H, s), 1.34 (1H, m), 1.56 (2H, m), 2.37 (1H, m), 3.65
(2H, t, Jϭ10.5 Hz), 4.03 (2H, d, Jϭ4.6 Hz), 5.53 (2H, m), 7.43 (6H, m),
7.66 (4H, m).
{2-[(R)-4-(tert-Butyldiphenylsiloxy)butyl]-(1R,2S)-cyclopropyl}-
methanol (8) To the cold solution (0 °C) of Et2Zn (8.40 ml, 8.40 mmol,
1.0 M in hexane) in CH2Cl2 (30 ml) was carefully added CH2I2 (1.40 ml,
16.7 mmol) with vigorous stirring.24) The mixture was stirred at 0 °C for
30 min, and a preformed solution of dioxaborolane 7 (25.0 ml, 2.50 mmol,
0.1 M in CH2Cl2) and allylic alcohol 6 (770 mg, 2.10 mmol) was added via
cannula. The resulting mixture was stirred at room temperature for 3 h, di-
luted with Et2O (200 ml), washed successively with saturated NH4Cl solu-
tion, water and saturated NaCl solution. The organic layer was dried over
MgSO4 and concentrated under reduced pressure. The residue was chro-
matographed on a silica gel column with hexane–Et2O (3 : 2) to give alcohol
8 (795 mg, 99% yield) as a colorless oil: [a]D26 Ϫ8.0° (cϭ1.5, CHCl3). ESI-
MS m/z: 405 (MϩϩNa). HR-ESI-MS m/z: 405.2229 (Calcd for
C24H34O2NaSi: 405.2226). IR (neat) cmϪ1: 3354, 2998. 1H-NMR (400 MHz,
CDCl3) d: 0.31 (2H, m), 0.40 (1H, m), 0.88 (1H, m), 0.91 (3H, d,
Jϭ6.3 Hz), 0.99 (1H, m), 1.04 (9H, s), 1.52 (1H, dq, Jϭ13.4, 6.7 Hz), 1.66
(1H, dq, Jϭ13.4, 6.7 Hz), 3.53 (2H, m), 3.72 (1H, dd, Jϭ10.3, 6.7 Hz), 7.40
(6H, m), 7.68 (4H, m).
(R)-1-Bromo-3-[(1R,2R)-2-methylcyclopropyl]butane (9) To a solu-
tion of alcohol 8 (607 mg, 1.60 mmol) in CH2Cl2 (5.0 ml) were added Ph3P
(500 mg, 1.90 mmol) and CBr4 (630 mg, 1.90 mmol). The mixture was
stirred at room temperature for 10 min, diluted with Et2O (50 ml), filtered
through silica gel, and concentrated under reduced pressure. Resulting crude
bromide was used for subsequent reaction without further purification.
To a cold (0 °C) solution of the above crude bromide in Et2O–THF (2 : 1,
5.10 ml) was carefully added LiAlH4 (90.0 mg, 2.40 mmol). The mixture was
stirred at room temperature for 1 h, diluted with Et2O (50 ml), treated with
saturated NaCl solution (2.0 ml) and then stirred vigorously for further
30 min. The organic layer was dried over MgSO4 and concentrated under re-
duced pressure. Resulting crude TBDPS ether was used for subsequent reac-
tion without further purification.
To a solution of the above crude aldehyde and 2-methyl-2-butene
(30.0 ml, 269 mmol) in tert-butanol (240 ml) were added dropwise aqueous
(60 ml) solution of NaClO2 (19.0 g, 209 mmol) and NaH2PO4 (9.30 g,
60.0 mmol) with vigorous stirring. The mixture was stirred at room tempera-
ture for 2 h. The reaction mixture was diluted with Et2O (600 ml) and
washed successively with 30% NaH2PO4 solution and saturated NaCl solu-
tion. The organic layer was dried over MgSO4 and concentrated under re-
duced pressure to give crude carboxylic acid. The crude product was used
for subsequent reaction without further purification.
To a solution of the above crude carboxylic acid in CH3CN (300 ml) were
added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 36.0 ml, 240 mmol) and
CH3I (7.50 ml, 120 mmol). The mixture was stirred at room temperature for
8 h. The reaction mixture was diluted with Et2O (150 ml) and washed suc-
cessively with water and saturated NaCl solution. The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column with hexane–Et2O (9 : 1) to
give ester 5 (16.7 g, 3 steps 69% yield) as a colorless oil: [a]D26 ϩ1.1°
(cϭ3.0, CHCl3). EI-MS m/z: 383 (MϩϪCH3). HR-EI-MS m/z: 383.2018
(Calcd for C23H31O3Si: 383.2043). IR (neat) cmϪ1: 2931, 1741. 1H-NMR
(300 MHz, CDCl3) d: 0.84 (3H, d, Jϭ6.4 Hz), 1.04 (9H, s), 1.36 (1H, m),
1.46 (1H, m), 1.62 (2H, m), 2.29 (2H, m), 3.66 (3H, s), 3.70 (2H, m), 7.40
(6H, m), 7.67 (4H, m).
(R,E)-6-(tert-Butyldiphenylsiloxy)-4-methylhex-2-en-1-ol (6) To
a
cold (Ϫ78 °C) solution of diisopropylamine (45.0 ml, 328 mmol) in tetrahy-
drofuran (THF, 300 ml) was added nBuLi (181 ml, 280 mmol, 1.55 M in
hexane). The mixture was stirred for 1 h, treated with a solution of ester 5
(93.0 g, 233 mmol) in THF (400 ml), and stirred for further 1 h. The mixture
was transferred to a solution of diphenyldisulfide (62.0 g, 284 mmol) in THF
(300 ml) via cannula. The reaction mixture was stirred at room temperature
for 30 min, diluted with Et2O (1500 ml) and washed successively with water
and saturated NaCl solution. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was chromatographed on
a silica gel column with hexane–Et2O (9 : 1) to give crude sulfide. The crude
product was used for subsequent reaction without further purification.
To a solution of the above crude sulfide in CHCl3 (1200 ml) was added
To a solution of the above crude TBDPS ether in THF (5.0 ml) was added
nBu4NF (2.40 ml, 2.40 mmol, 1.0 M in THF). The mixture was stirred at
room temperature for 1 h, diluted with Et2O (50 ml), washed successively
with water and saturated NaCl solution. The organic layer was dried over
MgSO4 and concentrated under reduced pressure. The residue was chro-
matographed on a silica gel column with pentane–Et2O (1 : 1) to give alcohol
(180 mg, 3 steps 88% yield) as a colorless oil: [a]D29 Ϫ36.6° (cϭ1.3, CHCl3).
EI-MS m/z: 110 (MϩϪH2O). HR-EI-MS m/z: 110.1084 (Calcd for C8H14:
110.1096). IR (neat) cmϪ1: 3388, 2994. 1H-NMR (300 MHz, CDCl3) d: 0.16
(3H, m), 0.46 (1H, m), 0.85 (1H, m), 0.93 (3H, d, Jϭ6.3 Hz), 1.00 (3H, d,
Jϭ5.8 Hz), 1.63 (1H, dq, Jϭ13.8, 6.9 Hz), 1.65 (1H, dq, Jϭ13.8, 6.9 Hz),
3.72 (2H, t, Jϭ6.9 Hz).

June 2003
643
HR-EI-MS m/z: 419.2431 (Calcd for C24H35O6: 419.2434). IR (KBr) cmϪ1
:
To a solution of the above alcohol (2.60 g, 20.1 mmol) in CH2Cl2 (100 ml)
were added Ph3P (7.90 g, 30.2 mmol) and N-bromosuccinimide (NBS,
5.40 g, 30.2 mmol). The mixture was stirred at room temperature for 10 min,
diluted with Et2O (500 ml), filtered through silica gel, and concentrated
under reduced pressure. The residue was distilled (50 °C, 3 mmHg) to give
bromide 9 (2.60 g, 13.5 mmol, 67% yield) as a colorless oil: [a]D23 Ϫ26.4°
(cϭ0.9, CHCl3). EI-MS m/z: 190 (Mϩ). HR-EI-MS m/z: 190.0355 (Calcd
1
2937, 1709. H-NMR (300 MHz, CDCl3) d: 1.10 (3H, s), 1.27 (3H, s), 1.27
(3H, s), 1.90 (1H, m), 1.99 (1H, dd, Jϭ13.0, 2.1 Hz), 2.06 (1H, dd, Jϭ12.5,
5.2 Hz), 2.24 (1H, m), 2.70 (1H, t, Jϭ12.9 Hz), 2.70 (1H, dd, Jϭ10.2,
9.4 Hz), 2.77 (1H, br t, Jϭ11.7 Hz), 2.87 (1H, dd, Jϭ12.9, 6.0 Hz), 3.31 (3H,
s), 3.46 (1H, m), 3.82 (1H, q, Jϭ7.0 Hz), 3.84 (1H, q, Jϭ7.4 Hz), 3.93 (1H,
q, Jϭ7.4 Hz), 3.97 (1H, q, Jϭ7.0 Hz), 4.60 (2H, br s). 13C-NMR (75 MHz,
CDCl3) d: 13.0, 22.1, 22.5, 24.5, 24.7, 27.0, 34.0, 34.5, 35.9, 37.9, 45.1,
45.4, 48.3, 48.4, 51.5, 55.2, 56.5, 62.9, 65.4, 75.4, 94.6, 111.1, 209.2, 211.6.
To a cold (0 °C) solution of the above 7,12-diketone (100 mg, 0.23 mmol)
in THF (2.0 ml) was added dropwise L-Selectride® (0.7 ml, 0.7 mmol, 1.0 M
in THF). The mixture was stirred at room temperature for 2 h, treated with
1 N NaOH solution and 30% H2O2 solution, and stirred further 30 min. The
mixture was diluted with Et2O (20 ml), washed successively with saturated
NH4Cl solution, water and saturated NaCl solution, dried over MgSO4 and
concentrated under reduced pressure. The residue was chromatographed on
a silica gel column with EtOAc to give 7a,12b-diol 12 (53.2 mg, 52% yield)
along with 12a isomer 11 (22.2 mg, 22% yield): 7a,12b-diol 12: colorless
amorphous. [a]D26 ϩ14.3° (cϭ1.8, CHCl3). EI-MS m/z: 423 (MϩϪCH3).
1
for C8H15Br: 190.0357). IR (neat) cmϪ1: 2875. H-NMR (400 MHz, CDCl3)
d: 0.13 (2H, d, Jϭ4.7 Hz), 0.20 (1H, d, Jϭ8.6 Hz), 0.57 (1H, m), 0.95 (3H,
s), 1.02 (3H, d, Jϭ6.0 Hz), 1.90 (2H, m), 3.51 (2H, t, Jϭ7.5 Hz).
7a,12a-Dihydroxy-3a-[(methoxymethyl)oxy]-5b-pregnan-20-one Eth-
ylene Ketal (11) To a solution of 3a,7a,12a-triacetoxy-5b-pregnan-20-
one8) (10, 1.00 g, 2.20 mmol) in benzene (25 ml) were added ethylene glycol
(250 ml, 4.30 mmol) and TsOH (20 mg). The mixture was stirred for 2 h
under reflux to remove the H2O produced in the reaction (Dean-Stark trap).
After addition of pyridine (100 ml) at room temperature, the reaction mixture
was concentrated under reduced pressure. The residue was chromatographed
on a silica gel column with hexane–EtOAc (1 : 1) to give triacetate (802 mg,
73% yield) as a colorless amorphous: [a]D24 ϩ86.7° (cϭ0.6, CHCl3). EI-MS
m/z: 505 (MϩϪCH3). HR-EI-MS m/z: 505.2816 (Calcd for C28H41O8:
HR-EI-MS m/z: 423.2730 (Calcd for C24H39O6: 423.2747). IR (KBr) cmϪ1
:
1
505.2801). IR (KBr) cmϪ1: 2942, 1735, 1249. H-NMR (300 MHz, CDCl3)
3495, 3435, 2937. 1H-NMR (300 MHz, CDCl3) d: 0.75 (3H, s), 0.90 (3H, s),
1.31 (3H, s), 2.18 (1H, br t, Jϭ11.1 Hz), 3.35 (3H, s), 3.36 (1H, m), 3.86
(1H, m), 4.01 (5H, m), 4.67 (2H, m). 13C-NMR (75 MHz, CDCl3) d: 7.8,
22.6, 23.1, 23.4, 25.4, 27.6, 28.0, 31.7, 35.0, 35.2, 35.2, 36.6, 37.6, 41.2,
48.1, 48.8, 55.1, 57.5, 63.3, 63.7, 68.0, 76.7, 77.9, 94.3, 111.5.
d: 0.81 (3H, s), 0.90 (3H, s), 1.19 (3H, s), 2.03 (3H, s), 2.07 (3H, s), 2.11
(3H, s), 2.27 (1H, br t, Jϭ9.9 Hz), 3.87 (4H, m), 4.56 (1H, m), 4.90 (1H, m),
5.08 (1H, m). 13C-NMR (75 MHz, CDCl3) d: 13.2, 21.4, 21.5, 21.6, 22.3,
22.4, 22.5, 22.6, 24.0, 25.2, 26.9, 29.0, 31.2, 34.4, 34.6, 37.2, 40.9, 43.2,
44.6, 49.7, 63.3, 64.8, 70.7, 74.1, 74.9, 111.2, 170.3, 170.5, 170.6.
To a solution of the above triacetate (100 mg, 0.19 mmol) in MeOH
(2.0 ml) was added K2CO3 (30.0 mg, 0.21 mmol). The mixture was stirred at
room temperature for 3 h and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column with EtOAc to give 3a-
alcohol (85.0 mg, 95% yield) as a colorless amorphous: [a]D23 ϩ73.8°
(cϭ0.4, CHCl3). EI-MS m/z: 463 (MϩϪCH3). HR-EI-MS m/z: 463.2682
(Calcd for C26H39O7: 463.2696). IR (KBr) cmϪ1: 3413, 2931, 1733, 1245.
1H-NMR (300 MHz, CDCl3) d: 0.80 (3H, s), 0.89 (3H, s), 1.15 (3H, s), 2.06
(3H, s), 2.10 (3H, s), 2.30 (1H, br t, Jϭ10.1 Hz), 3.48 (1H, m), 3.86 (4H, m),
4.89 (1H, m), 5.07 (1H, m). 13C-NMR (75 MHz, CDCl3) d: 13.2, 21.5, 21.6,
22.3, 22.4, 22.5, 24.0, 25.2, 29.0, 30.4, 31.3, 34.3, 34.8, 37.2, 38.6, 41.0,
43.2, 44.6, 49.6, 63.2, 64.7, 70.8, 71.6, 74.9, 111.2, 170.6, 170.7.
3 ,7 ,12 -Tri[(methoxymethyl)oxy]-5 -pregnan-20-one (13) A solu-
tion of 7a,12b-diol 12 (1.20 g, 2.70 mmol) in 80% acetic acid was stirred at
room temperature for 2 h, and then concentrated under reduced pressure. Re-
sulting crude 7,12-dihydroxyketone was used for subsequent reaction with-
out further purification.
a
a
b
b
To a solution of the above 7,12-dihydroxyketone in 1,2-dichloroethane
i
(15 ml) were added Pr2NEt (3.80 ml, 21.6 mmol) and MOMCl (1.20 ml,
16.2 mmol). The mixture was stirred for 16 h at room temperature. The reac-
tion mixture was diluted with Et2O (100 ml), and washed successively with
water and saturated NaCl solution. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was chromatographed
on a silica gel column with hexane–Et2O (1 : 2) to give ketone 13 (900 mg, 2
steps 69% yield) as a colorless viscous oil. [a]D27ϩ42.5° (cϭ1.35, CHCl3).
EI-MS m/z: 482 (Mϩ). HR-EI-MS m/z: 451.3047 (Calcd for C26H43O6:
451.3060). IR (KBr) cmϪ1: 2938, 1707. 1H-NMR (300 MHz, CDCl3) d: 0.72
(3H, s), 0.91 (3H, s), 0.99 (1H, dt, Jϭ14.1, 2.9 Hz), 1.91 (1H, dt, Jϭ13.0,
4.8 Hz), 2.20 (3H, s), 2.73 (1H, br t, Jϭ8.7 Hz), 3.30 (3H, s), 3.34 (3H, s),
3.35 (3H, s), 3.35 (1H, m), 3.42 (1H, dd, Jϭ10.9, 4.6 Hz), 3.65 (1H, m),
4.68 (6H, m). 13C-NMR (75 MHz, CDCl3) d: 8.8, 22.6, 24.0, 26.0, 27.0,
27.8, 30.7, 32.2, 32.3, 35.3, 35.4, 36.2, 38.5, 41.4, 49.3, 50.2, 55.1, 55.6,
56.1, 61.6, 74.3, 77.0, 86.0, 94.6, 95.4, 96.0, 211.3.
To a solution of the above alcohol (3.80 g, 8.00 mmol) in CH2Cl2 (40 ml)
were added iPr2NEt (2.30 ml, 16.1 mmol) and MOMCl (0.92 ml, 12.1 mmol).
The mixture was stirred for 3 h at room temperature, diluted with Et2O
(200 ml), washed successively with water and saturated NaCl solution, dried
over MgSO4 and concentrated under reduced pressure. The residue was
chromatographed on a silica gel column with hexane–Et2O (1 : 1) to give
7a,12a-diacetate (4.20 mg, Ͼ99% yield) as a colorless amorphous: [a]D27
ϩ65.1° (cϭ0.7, CHCl3). EI-MS m/z: 507 (MϩϪCH3). HR-EI-MS m/z:
507.2944 (Calcd for C28H43O8: 507.2958). IR (KBr) cmϪ1: 2934, 1734,
(20E,24R,25R,26R)-26,27-Cyclo-3 ,7 ,12 -trihydroxy-24,26-di-
a
a
b
1
1249. H-NMR (300 MHz, CDCl3) d: 0.80 (3H, s), 0.88 (3H, s), 1.17 (3H,
b
methyl-5 -cholest-20(22)-ene (14) To a solution of naphthalene (1.50 g,
11.7 mmol) in THF (20 ml) was added Li (70.0 mg, 10.0 mmol). The mixture
was stirred at room temperature to become a homogeneous solution. Bro-
mide 9 (1.00 g, 5.00 mmol) was added to the mixture at Ϫ78 °C. After stir-
ring for 30 min, a solution of ketone 13 (800 mg, 1.66 mmol) in THF
(9.00 ml) was added dropwise to the mixture. The reaction mixture was
stirred for 30 min at Ϫ78 °C and then allowed to warm slowly to room tem-
perature. Reaction mixture was diluted with Et2O (150 ml), and washed suc-
cessively with saturated NH4Cl solution, water and saturated NaCl solution.
The organic layer was dried over MgSO4 and concentrated under reduced
pressure. Resulting crude product was used for subsequent reaction without
further purification.
s), 2.05 (3H, s), 2.07 (3H, s), 2.30 (1H, br t, Jϭ9.9 Hz), 3.34 (3H, s), 3.37
(1H, m), 3.86 (4H, m), 4.65 (2H, s), 4.89 (1H, m), 5.07 (1H, m). 13C-NMR
(75 MHz, CDCl3) d: 13.2, 21.4, 21.5, 22.2, 22.4, 22.5, 22.6, 24.0, 25.2, 27.9,
29.0, 31.4, 34.5, 34.9, 36.0, 37.2, 41.2, 43.2, 44.5, 49.6, 55.1, 63.2, 64.7,
70.8, 74.9, 94.7, 111.2, 170.5, 170.7.
To a solution of the above diacetate (4.10 g, 7.90 mmol) in MeOH (80 ml)
was added K2CO3 (5.50 g, 39.5 mmol). The mixture was stirred under reflux
for 3 d and concentrated under reduced pressure. The residue was dissolved
in Et2O (500 ml), washed successively with water and saturated NaCl solu-
tion, dried over MgSO4 and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column with EtOAc to give
7a,12a-diol 11 (2.50 g, 73% yield) as a colorless amorphous: [a]D31 ϩ33.6°
(cϭ0.3, CHCl3). EI-MS m/z: 423 (MϩϪCH3). HR-EI-MS m/z: 423.2743
(Calcd for C24H39O6: 423.2747). IR (KBr) cmϪ1: 3473, 2935. 1H-NMR
(400 MHz, CDCl3) d: 0.77 (3H, s), 0.87 (3H, s), 1.27 (3H, s), 2.32 (1H, br t,
Jϭ10.8 Hz), 3.34 (3H, s), 3.35 (1H, m), 3.82 (1H, m), 3.92 (5H, m), 4.65
(2H, m). 13C-NMR (100 MHz, CDCl3) d: 13.9, 22.7, 22.8, 23.0, 23.9, 27.4,
27.6, 27.8, 34.7, 35.1, 35.4, 36.7, 39.2, 41.8, 42.1, 46.3, 49.8, 55.2, 63.8,
64.3, 68.4, 72.4, 77.5, 94.4, 112.2.
7a,12b-Dihydroxy-3a-[(methoxymethyl)oxy]-5b-pregnan-20-one Eth-
ylene Ketal (12) To a solution of 7a,12a-diol 11 in 1,2-dicholoroethane
(100 ml) were added molecular sieves (4A, powder, 6.50 g) and PCC (6.50 g,
17.1 mmol). The mixture was stirred at room temperature for 4 h, diluted
with Et2O (200 ml), filtered through silica gel, and concentrated under re-
duced pressure to afford 7,12-diketone (2.20 g, 89% yield) as a colorless
amorphous: [a]D31 ϩ35.9° (cϭ0.3, CHCl3). EI-MS m/z: 419 (MϩϪCH3).
To a solution of the above crude product in MeOH (8.0 ml) was added
TsOH (8.0 mg). The mixture was stirred for 12 h under reflux. The reaction
mixture was added pyridine (0.5 ml), and then concentrated under reduced
pressure. The residue was chromatographed on a silica gel column with
Et2O to give 20(22)E-olefin 14 (450 mg, 2 steps 60% yield) and 20(22)Z-iso-
mer (35.0 mg, 2 steps 5% yield).
20(22)E-Olefin 14: Colorless amorphous. [a]D28Ϫ36.6° (cϭ0.1, CHCl3).
EI-MS m/z: 444 (Mϩ). HR-EI-MS m/z: 444.3592 (Calcd for C29H48O3:
444.3604). IR (KBr) cmϪ1: 3424, 2951. 1H-NMR (400 MHz, CDCl3) d: 0.11
(1H, m), 0.18 (2H, m), 0.47 (1H, m), 0.75 (3H, s), 0.91 (3H, s), 0.92 (3H, d,
Jϭ6.6 Hz), 0.98 (3H, d, Jϭ6.0 Hz), 1.70 (3H, br s), 2.37 (1H, t, Jϭ9.9 Hz),
3.46 (1H, m), 3.65 (1H, dd, Jϭ11.3, 5.0 Hz), 3.88 (1H, m), 5.56 (1H, br t,
Jϭ7.2 Hz). 13C-NMR (100 MHz, CDCl3) d: 8.6, 11.8, 12.7, 15.3, 19.0, 19.8,
22.6, 23.7, 25.2, 26.9, 28.2, 30.6, 32.0, 34.8, 35.1, 35.3, 35.9, 38.2, 39.0,
39.8, 41.2, 47.7, 49.1, 60.8, 68.2, 71.8, 80.0, 127.6, 139.3.

644
(20R,22R,24R,25R,26R)-26,27-Cyclo-20,22-epoxy-24,26-dimethyl-5b-
Vol. 51, No. 6
20(22)Z-Isomer: Colorless amorphous. [a]D26Ϫ20.0° (cϭ0.2, CHCl3). EI-
MS m/z: 488 (Mϩ). HR-EI-MS m/z: 488.3860 (Calcd for C31H52O4:
cholestane-3a,7a,12b-triol (15) To a solution of 20(22)E-olefin 14
(435 mg, 0.98 mmol) in benzene (5 ml) were added VO(acac)2 (3.00 mg,
0.01 mmol) and tert-butylhydroperoxide25) (TBHP, 0.50 ml, 1.47 mmol, 3.0 M
in CH2Cl2). The mixture was stirred for 3 d at room temperature. To the mix-
ture was added Me2S (0.5 ml) and stirred for 1 h. The reaction mixture was
diluted with Et2O (40 ml), and washed successively with water and saturated
NaCl solution. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The residue was chromatographed on a silica gel
column with EtOAc to give (20R,22R)-epoxide 15 (293 mg, 65% yield) and
(20S,22S)-isomer (22.0 mg, 5% yield).
488.3866). IR (KBr) cmϪ1: 3447. H-NMR (300 MHz, CDCl3) d: 0.11 (1H,
1
m), 0.20 (2H, m), 0.49 (1H, m), 0.77 (3H, s), 0.80 (3H, s), 0.87 (3H, d,
Jϭ6.4 Hz), 1.00 (3H, d, Jϭ6.0 Hz), 1.78 (3H, br s), 3.13 (3H, s), 3.18 (3H,
s), 3.66 (1H, dd, Jϭ11.0, 4.9 Hz), 3.86 (1H, m), 5.42 (1H, m). 13C-NMR
(75 MHz, CDCl3) d: 9.0, 10.5, 11.7, 12.7, 19.0, 19.2, 22.2, 23.6, 24.7, 26.8,
28.3, 28.4, 34.7, 34.9, 35.1, 35.9, 36.4, 36.6, 38.4, 39.2, 44.6, 47.5, 47.5,
48.0, 49.5, 50.4, 67.6, 79.5, 100.2, 128.5, 138.6.
(20R,22R,24R,25R,26R)-26,27-Cyclo-20,22-epoxy-7a,12b-dihydroxy-
24,26-dimethy-l5b-cholestan-3-one Dimethyl Ketal (18) To a solution of
20(22)E-olefin 17 (286 mg, 0.59 mmol) in benzene (6.0 ml) were added
VO(acac)2 (2.00 mg, 5.90 mmol) and TBHP (0.25 ml, 0.74 mmol, 3.0 M in
CH2Cl2). The mixture was stirred for 2 h at room temperature. The reaction
mixture was treated with Me2S (0.5 ml), and stirred for 1 h. The mixture was
diluted with Et2O (40 ml), washed successively with water and saturated
NaCl solution. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The residue was chromatographed on a silica gel
column with hexane–Et2O (1 : 2) to give (20R,22R)-epoxide 18 (220 mg,
75% yield) and (20S,22S)-isomer (14.0 mg, 0.5% yield).
(20R,22R)-Epoxide 15: Colorless amorphous. [a]D26Ϫ10.8° (cϭ1.1,
CHCl3). EI-MS m/z: 460 (Mϩ). HR-EI-MS m/z: 460.3566 (Calcd for
C29H48O4: 460.3553). IR (CHCl3) cmϪ1: 3412, 2951. 1H-NMR (300 MHz,
CDCl3) d: 0.14 (1H, m), 0.22 (2H, m), 0.53 (1H, m), 0.70 (3H, s), 0.88 (3H,
s), 1.00 (3H, d, Jϭ6.5 Hz), 1.02 (3H, d, Jϭ6.0 Hz), 1.30 (3H, s), 3.32 (1H,
dd, Jϭ11.2, 4.6 Hz), 3.40 (1H, t, Jϭ6.1 Hz), 3.43 (1H, m), 3.85 (1H, m).
13C-NMR (75 MHz, CDCl3) d: 9.0, 12.0, 12.8, 19.1, 20.0, 20.1, 22.6, 22.8,
26.3, 26.9, 28.2, 30.7, 31.9, 35.0, 35.1, 35.3, 36.1, 36.8, 37.7, 39.7, 41.2,
48.0, 49.1, 57.3, 61.7, 64.1, 68.0, 71.8, 76.9.
(20R,22R)-Epoxide 18: Colorless amorphous. [a]D26Ϫ4.4° (cϭ0.3,
CHCl3). EI-MS m/z: 504 (Mϩ). HR-EI-MS m/z: 504.3801 (Calcd for
(22R,24R,25R,26R)-26,27-Cyclo-24,26-dimethyl-5b-cholest-20-ene-
i
3a,7a,12b,22-tetraol (16) To Pr2NH (0.91 ml, 6.50 mmol) in flask was
C31H52O5: 504.3815). IR (CHCl3) cmϪ1: 3399. H-NMR (400 MHz, CDCl3)
1
added dropwise MeMgBr (6.0 ml, 0.93 M in THF) at room temperature.
After stirring for 1 h, resulting solution was added to (20R,22R)-epoxide 15
(250 mg, 0.54 mmol) in another flask, and stirred for 3 d at room tempera-
ture. The reaction mixture was diluted with Et2O (20 ml), and washed suc-
cessively with saturated NH4Cl solution, water and saturated NaCl solution.
The organic layer was dried over MgSO4 and concentrated under reduced
pressure. The residue was chromatographed on a silica gel column with
EtOAc to give allylic alcohol 16 (173 mg, 70% yield) as a colorless amor-
phous: [a]D26Ϫ1.0° (cϭ1.1, CHCl3). EI-MS m/z: 460 (Mϩ). HR-EI-MS m/z:
460.3533 (Calcd for C29H48O4: 460.3553). IR (CHCl3) cmϪ1: 3382, 2927,
1639. 1H-NMR (300 MHz, CDCl3) d: 0.09 (1H, m), 0.17 (2H, m), 0.51 (1H,
m), 0.75 (3H, s), 0.90 (3H, d, Jϭ6.4 Hz), 0.94 (3H, s), 0.99 (3H, d,
Jϭ6.0 Hz), 3.44 (1H, m), 3.64 (1H, dd, Jϭ11.6, 4.3 Hz), 3.88 (1H, m), 4.31
(1H, dd, Jϭ8.3, 5.8 Hz), 4.91 (1H, s), 5.08 (1H, s). 13C-NMR (75 MHz,
CDCl3) d: 8.0, 11.6, 12.9, 19.3, 19.6, 22.6, 23.1, 27.5, 29.9, 30.5, 31.5, 32.7,
34.3, 34.6, 34.8, 35.4, 39.0, 39.1, 41.3, 44.4, 48.0, 48.6, 50.6, 67.9, 71.5,
76.1, 79.0, 112.8, 151.2.
d: 0.14 (1H, m), 0.22 (2H, m), 0.53 (1H, m), 0.69 (3H, s), 0.77 (3H, s), 0.97
(3H, d, Jϭ6.6 Hz), 1.01 (3H, d, Jϭ6.0 Hz), 1.29 (3H, s), 3.11 (3H, s), 3.17
(3H, s), 3.23 (1H, dd, Jϭ10.9, 4.2 Hz), 3.81 (1H, m). 13C-NMR (100 MHz,
CDCl3) d: 9.2, 10.6, 12.9, 19.2, 20.2, 20.2, 23.0, 26.5, 27.1, 28.5, 28.8, 34.8,
35.0, 35.2, 35.3, 36.0, 36.3, 36.8, 36.9, 38.1, 44.6, 47.6, 47.6, 48.2, 49.2,
57.6, 61.8, 64.1, 67.6, 77.5, 100.4.
(20S,22S)-Isomer: Colorless amorphous. [a]D26Ϫ12.8° (cϭ0.4, CHCl3).
EI-MS m/z: 504 (Mϩ). HR-EI-MS m/z: 504.3813 (Calcd for C31H52O5:
504.3815). IR (CHCl3) cmϪ1: 3427. 1H-NMR (400 MHz, CDCl3) d: 0.11
(1H, m), 0.22 (2H, m), 0.53 (1H, m), 0.81 (3H, s), 0.85 (3H, s), 0.99 (3H, d,
Jϭ6.6 Hz), 1.00 (3H, d, Jϭ6.1 Hz), 2.85 (1H, t, Jϭ7.0 Hz), 3.13 (3H, s),
3.18 (3H, s), 3.27 (1H, dd, Jϭ10.8, 5.6 Hz), 3.88 (1H, m). 13C-NMR
(100 MHz, CDCl3) d: 9.3, 10.5, 11.9, 12.6, 15.7, 19.1, 20.0, 23.8, 24.7, 26.7,
26.8, 27.0, 28.4, 28.7, 34.7, 35.0, 35.1, 35.8, 36.0, 36.7, 38.5, 44.6, 47.6,
48.0, 52.3, 59.6, 61.0, 67.8, 78.0, 100.3.
(22R,24R,25R,26R)-26,27-Cyclo-7a,12b,22-trihydroxy-24,26-di-
methyl-5b-cholest-20-en-3-one Dimethyl Ketal (19) To Pr2NH (0.73 ml,
i
3a,7a-Dihydroxy-5-epiaragusterol A (3) To a cold (0 °C) solution of
allylic alcohol 16 (150 mg, 0.33 mmol) in CH2Cl2 (1.50 ml) were added
Na2HPO4 (140 mg, 0.98 mmol) and mCPBA (160 mg, 0.65 mmol). After stir-
ing for 1 h, the mixture was treated with Me2S (0.5 ml), and stirred for fur-
ther 1 h. The mixture was diluted with Et2O (15 ml), washed successively
with water, saturated NaHCO3 solution, water again and saturated NaCl so-
lution. The organic layer was dried over MgSO4 and concentrated under re-
duced pressure. The residue was chromatographed on a silica gel column
with EtOAc to give 3a,7a-dihydroxy-5-epiaragusterol A (3, 139 mg, 88%
yield) as a colorless powder: mp 66—67 °C. [a]D26Ϫ1.0° (cϭ1.1, CHCl3).
EI-MS m/z: 476 (Mϩ). HR-EI-MS m/z: 476.3495 (Calcd for C29H48O5:
5.20 mmol) in flask was added dropwise MeMgBr (4.7 ml, 0.93 M in THF).
The mixture was stirred at room temperature for 1 h. The mixture was added
to a solution of (20R,22R)-epoxide 18 (219 mg, 0.43 mmol) in THF (2.0 ml),
and stirred for 16 h at room temperature. The reaction mixture was diluted
with Et2O, and washed successively with saturated NH4Cl solution, water
and saturated NaCl solution. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was chromatographed on
a silica gel column with Et2O to give allylic alcohol 19 (205 mg, 94% yield)
as a colorless amorphous: [a]D26Ϫ21.3° (cϭ0.4, CHCl3). EI-MS m/z: 504
(Mϩ). HR-EI-MS m/z: 504.3810 (Calcd for C31H52O5: 504.3815). IR
(CHCl3) cmϪ1: 3344. 1H-NMR (300 MHz, CDCl3) d: 0.09 (1H, m), 0.17
(2H, m), 0.51 (1H, m), 0.71 (3H, s), 0.79 (3H, s), 0.91 (3H, d, Jϭ6.6 Hz),
0.99 (3H, d, Jϭ5.9 Hz), 3.11 (3H, s), 3.16 (3H, s), 3.58 (1H, dd, Jϭ10.9,
4.2 Hz), 4.28 (1H, dd, Jϭ13.4, 5.5 Hz), 4.89 (1H, s), 5.06 (1H, s). 13C-NMR
(75 MHz, CDCl3) d: 7.9, 10.4, 11.8, 12.8, 19.1, 19.8, 23.3, 27.5, 28.2, 29.8,
31.8, 34.5, 34.7, 34.8, 35.0, 35.7, 36.3, 39.1, 44.5, 45.0, 47.4, 47.5, 47.7,
48.7, 49.7, 67.3, 76.2, 78.1, 100.3, 112.9, 151.4.
1
476.3502). IR (CHCl3) cmϪ1: 3416, 2932. H-NMR (300 MHz, CDCl3) d:
0.16 (1H, m), 0.23 (2H, m), 0.48 (1H, m), 0.68 (3H, s), 0.90 (3H, s), 0.92
(3H, br s), 1.01 (3H, d, Jϭ6.0 Hz), 2.34 (1H, br t, Jϭ9.6 Hz), 2.79 (1H, d,
Jϭ3.8 Hz), 3.04 (1H, d, Jϭ3.8 Hz), 3.35 (2H, m), 3.57 (1H, dd, Jϭ11.1,
4.5 Hz), 3.84 (1H, m). 13C-NMR (75 MHz, CDCl3) d: 7.7, 12.4, 12.5, 18.8,
19.2, 22.7, 23.3, 27.4, 27.6, 28.8, 31.0, 31.9, 34.8, 35.0, 35.3, 35.4, 37.7,
39.9, 41.3, 46.5, 48.7, 48.9, 51.2, 66.0, 67.6, 71.8, 74.0, 77.4.
(20E,24R,25R,26R)-26,27-Cyclo-7a,12b-dihydroxy-24,26-dimethyl-
7a-Hydroxyaragusterol A (4) To a cold (0 °C) solution of allylic alco-
hol 19 (204 mg, 0.40 mmol) in CH2Cl2 (4.0 ml) were added Na2HPO4
(172 mg, 1.21 mmol) and mCPBA (200 mg, 0.81 mmol). The mixture was
stirred at 0 °C for 4 h, treated with Me2S (0.2 ml), and stirred for further 1 h.
The mixture was diluted with Et2O, and washed successively with water, sat-
urated NaHCO3 solution, water again and saturated NaCl solution. The or-
ganic layer was dried over MgSO4 and concentrated under reduced pressure.
The residue was chromatographed on a silica gel column with Et2O to give
7a-hydroxyaragusterol A dimethyl ketal (165 mg, 78% yield) as a colorless
amorphous: [a]D26ϩ5.0° (cϭ0.2, CHCl3). EI-MS m/z: 520 (Mϩ). HR-EI-MS
m/z: 520.3779 (Calcd for C31H52O6: 520.3764). IR (CHCl3) cmϪ1: 3403. 1H-
NMR (300 MHz, CDCl3) d: 0.15 (1H, m), 0.24 (2H, m), 0.48 (1H, m), 0.69
(3H, s), 0.78 (3H, s), 0.93 (3H, s), 1.01 (3H, d, Jϭ6.0 Hz), 2.17 (1H, dd,
Jϭ10.7, 2.0 Hz), 2.94 (1H, d, Jϭ4.8 Hz), 3.07 (1H, d, Jϭ4.8 Hz), 3.13 (3H,
s), 3.18 (3H, s), 3.36 (1H, dd, Jϭ10.9, 4.2 Hz), 3.49 (1H, d, Jϭ8.7 Hz), 3.84
(1H, m). 13C-NMR (75 MHz, CDCl3) d: 8.1, 10.4, 12.4, 12.4, 18.8, 19.1,
23.1, 26.7, 27.6, 28.2, 28.9, 29.7, 34.6, 34.9, 35.1, 35.8, 36.6, 37.8, 40.1,
5b-cholest-20(22)-en-3-one Dimethyl Ketal (17) To
a solution of
xestokerol B4,5) (500 mg, 1.09 mmol) in MeOH (20 ml) was added TsOH
(20.0 mg). The mixture was stirred for 18 h under reflux. The reaction mix-
ture was added pyridine (1.0 ml), and concentrated under reduced pressure.
The residue was chromatographed on a silica gel column with hexane–Et2O
(2 : 1) to give 20(22)E-olefin 17 (286 mg, 54% yield) and 20(22)Z-isomer
(71.5 mg, 13% yield).
20(22)E-Olefin 17: Colorless amorphous. [a]D26Ϫ16.5° (cϭ0.2, CHCl3).
EI-MS m/z: 488 (Mϩ). HR-EI-MS m/z: 488.3865 (Calcd for C31H52O4:
1
488.3866). IR (KBr) cmϪ1: 3446. H-NMR (300 MHz, CDCl3) d: 0.09 (1H,
m), 0.16 (2H, m), 0.46 (1H, m), 0.74 (1H, m), 0.80 (3H, s), 0.91 (3H, d,
Jϭ6.6 Hz), 0.97 (3H, d, Jϭ6.0 Hz), 1.69 (3H, br s), 3.13 (3H, s), 3.18 (3H,
s), 3.58 (1H, dd, Jϭ10.4, 4.5 Hz), 3.84 (1H, m), 5.55 (1H, t, Jϭ6.6 Hz). 13C-
NMR (75 MHz, CDCl3) d: 8.6, 10.5, 11.7, 12.7, 15.3, 19.0, 19.7, 23.7, 25.2,
26.9, 28.3, 28.5, 34.6, 34.9, 35.0, 35.8, 35.9, 36.4, 38.4, 39.0, 44.6, 47.5,
47.5, 47.7, 49.0, 60.8, 67.5, 79.8, 100.2, 127.5, 139.2.

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645
44.3, 47.5, 47.5, 48.2, 48.6, 48.8, 50.4, 65.6, 67.5, 71.8, 77.1, 100.2.
819 (2000).
To the above ketal (144 mg, 0.28 mmol), pyridinium p-toluenesulfonate
(PPTS, 0.1% in acetone) was added. The mixture was stirred for 4 h at room
temperature. The reaction mixture was added pyridine (0.1 ml), and concen-
trated under reduced pressure to give crude product. The solid was recrystal-
lized from EtOAc to provide 7a-hydroxyaragusterol A (4, 84.0 mg, 64%
8) Mitome H., Miyaoka H., Nakano M., Yamada Y., Tetrahedron Lett.,
36, 8231—8234 (1995).
9) Mitome H., Miyaoka H., Takahashi H., Yamada Y., Bioorg. Med.
Chem. Lett., 7, 691—692 (1997).
10) Charette A. B., Juteau H., J. Am. Chem. Soc., 116, 2651—2652 (1994).
yield) as a colorless amorphous: [a]D26ϩ18.0° (cϭ0.2, CHCl3). EI-MS m/z: 11) Charette A. B., C&EN, 73, February 6, 2 (1995).
474 (Mϩ). HR-EI-MS m/z: 474.3344 (Calcd for C29H46O5: 474.3345). IR 12) Charette A. B., Prescott S., Brochu C., J. Org. Chem., 60, 1081—1083
1
(CHCl3) cmϪ1: 3359, 1712. H-NMR (400 MHz, CDCl3) d: 0.16 (1H, m),
(1995).
0.25 (2H, m), 0.50 (1H, m), 0.73 (3H, s), 0.95 (3H, s), 1.00 (3H, s), 1.02 13) Charette A. B., Juteau H., Lebel H., Molinaro C., J. Am. Chem. Soc.,
(3H, d, Jϭ6.0 Hz), 2.95 (1H, d, Jϭ4.0 Hz), 3.07 (1H, d, Jϭ4.0 Hz), 3.39 120, 11943—11952 (1998).
(1H, dd, Jϭ10.0, 6.9 Hz), 3.49 (1H, d, Jϭ8.9 Hz), 3.88 (1H, m). 13C-NMR 14) Barton D. H. R., Wozniak J., Zard S. Z., Tetrahedron, 45, 3741—3754
(100 MHz, CDCl3) d: 8.0, 10.3, 12.4, 12.4, 18.8, 19.1, 23.1, 26.7, 27.6, 29.2, (1989).
34.8, 35.6, 36.9, 37.8, 38.0, 38.0, 38.9, 40.1, 43.8, 44.0, 48.1, 48.7, 48.7, 15) Meystre C., Frey H., Wettstein A., Miescher K., Helv. Chim. Acta, 27,
50.5, 65.7, 67.0, 72.9, 77.0, 211.3.
1815—1824 (1944).
16) In this reaction, 7b-isomer was not obtained.
Acknowledgments The authors thank Taisho Pharmaceutical Co., Ltd. 17) Screttas C. G., J. Chem. Soc., Chem. Commun., 1972, 752—753
for measurements of biological activity. This work was supported in part by
(1972).
a Grant-in-Aid for Scientific Research from the Ministry of Education, Cul- 18) Freeman P. K., Hutchinson L. L., J. Org. Chem., 45, 1924—1930
ture, Sports, Science and Technology of Japan.
(1980).
19) E-Configuration of the olefin in 14 and 17 was revealed by the
References and Notes
15.3 ppm (C-21) signal in the 13C-NMR spectrum.
1) Iguchi K., Fujita M., Nagaoka H., Mitome H., Yamada Y., Tetrahedron 20) Sharpless K. B., Michaelson R. C., J. Am. Chem. Soc., 95, 6136—
Lett., 34, 6277—6280 (1993). 6137 (1973).
2) Shimura H., Iguchi K., Yamada Y., Nakaike S., Yamagishi T., Ma- 21) Diastereoselectivity in the epoxidation of 14 and 17 can be explained
tsumoto K., Yokoo C., Experientia, 50, 134—136 (1994).
3) Iguchi K., Shimura H., Taira S., Yokoo C., Matsumoto K., Yamada Y.,
J. Org. Chem., 59, 7499—7502 (1994).
4) Miyaoka H., Shinohara M., Shimomura M., Mitome H., Yano A.,
Iguchi K., Yamada Y., Tetrahedron, 53, 5403—5412 (1997).
5) Kobayashi J., Ishida K., Naitoh K., Shigemori H., Mikami Y., Sasaki
T., J. Nat. Prod., 56, 1350—1355 (1993).
6) Kobayashi M., Chen Y.-J., Higuchi K., Aoki S., Kitagawa I., Chem.
Pharm. Bull., 44, 1840—1842 (1996).
7) Fukuoka K., Yamagishi T., Ichihara T., Nakaike S., Iguchi K., Yamada
Y., Fukumoto H., Yoneda T., Samata K., Ikeya H., Nanaumi K., Hi-
rayama N., Narita N., Saijyo N., Nishio K., Int. J. Cancer, 88, 810—
based on the transition state in which vanadium metal is coordinated
with hydroxy group at C-12. See ref. 8.
22) Fukuyama T., Akasaka K., Karanewsky D. S., Wang C.-L. J., Schmidt
G., Kishi Y., J. Am. Chem. Soc., 101, 262—263 (1979).
23) The stereoselectivity of epoxidation with mCPBA is explained by pref-
erential attack of the reagent from the less hindered side. See ref. 8.
24) On a larger scale, this procedure will sometimes lead to violent explo-
sions. See ref. 12 and 13.
25) 3.0 M tert-Butylhydroperoxide solution in CH2Cl2 was prepared by
using Sharpless’s procedure. See: Gao Y., Hanson R. M., Klunder J.
M., Ko S. Y., Masamune H., Sharpless K. B., J. Am. Chem. Soc., 109,
5765—5780 (1987).