Sterols from Phycomyces blakesleeanus
J ournal of Natural Products, 1998, Vol. 61, No. 12 1495
(
3:1) (12 mL) was treated with p-toluenesulfonic acid (18 mg)
BSTFA and analyzed by GC-MS. TRR′ (1.090) and the mass
spectrum of the TMS derivative of 3 coincided with those
obtained from fungal neoergosterol.
19-Nor er gosta -5,7,9-tr ien -3â-ol (2). The catalyst (10%
Pd/C, 40 mg) was added to a solution of 3 (167 mg) in EtOH
and anhydrous MgSO (370 mg), and the solution was stirred
at 70 °C for 1 h. The reaction mixture was then filtered,
diluted with ether (50 mL), and washed with a saturated
solution of NaHCO
layer was dried over anhydrous Na
4
3
(25 mL × 3) and then brine. The organic
2
SO and filtered, and the
4
(35 mL). The mixture was stirred for 2 days under H
2
at 1
solvent was removed to give a residue which was flash
chromatographed (hexane, t-BuOMe 85:15) obtaining 375 mg
atm. The suspension was filtered, and the solvent was
2
5
1
removed, obtaining 135 mg of 2: [R]
D
-2° (c 1.0, CHCl
3
); H
-
1
1
of 25: oil; IR (film) νmax 1735, 1696, 1672 cm
CDCl , 300 MHz) δ 6.88 (1H, d, J ) 7.9 Hz, H-6), 6.83 (1H, d,
J ) 7.9 Hz, H-7), 6.77 (1H, dd, J ) 15.8, 9.0 Hz, H-22), 6.11
1H, d, J ) 15.8 Hz, H-23), 5.14 (1H, dddd, J ) 9.7, 7.1, 4.8,
.2 Hz, H-3), 3.08 (1H, dd, J ) 16.3, 4.8 Hz, H-4R), 2.86 (1H,
dd, J ) 16.3, 7.1 Hz, H-4â), 2.84 (1H, heptuplet, J ) 6.9 Hz,
H-25), 2.05 (3H, s, CH CO), 1.18 (3H, d, J ) 6.6 Hz, H-21),
.12 (6H, d, J ) 6.9 Hz, H-26, H-27), 0.63 (3H, s, H-18);
;
H NMR
NMR (CDCl , 300 MHz) δ 6.90 (1H, d, J ) 8.0 Hz, H-6), 6.85
3
(
3
(1H, d, J ) 8.0 Hz, H-7), 4.11 (1H, dddd, J ) 9.5, 8.0, 4.5, 3.3
Hz, H-3), 3.06 (1H, dd, J ) 16.0, 4.5 Hz, H-4R), 2.76 (1H, dd,
J ) 16.0, 8.0 Hz, H-4â), 1.00 (3H, d, J ) 6.3 Hz, H-21), 0. 87
(3H, d, J ) 6.8 Hz, H-28), 0.799 (3H, d, J ) 6.8 Hz, H-26),
(
3
1
3
0.797 (3H, d, J ) 6.8 Hz, H-27), 0.56 (3H, s, H-18); C NMR
+
3
(Table 2); EIMS, as TMS-ether, m/z 454 [M] (7), 439 (5), 364
1
3
1
C
(100), 349 (3), 237 (35), 210 (33), 195 (49); HREIMS m/z
+
NMR (Table 2); EIMS m/z 362 [M - AcOH] (100), 237 (2),
21 (9), 210 (11), 195 (20), 181 (13), 141 (14).
9-Nor -24-oxoer gosta -5,7,9-tr ien -3â-yl Aceta te (26). A
mixture of 25 (300 mg), Bu SnH (240 mg, 97% pure), and
382.3247 (calcd for C27H42O, 382.3236). Cholesterol (0.5 mg)
2
was added to 2 mg of 2. The mixture was derivatized with
BSTFA and analyzed by GC-MS. TRR′ (1.190) and the mass
spectrum of TMS derivative of 2 coincided with those obtained
from phycomysterol B.
Rea ction of Er goster yl Aceta te w ith Dieth yl Azod i-
ca r boxyla te: Acetic anhydride (5 mL) was added to a solution
of ergosterol (12, 500 mg) in 5 mL of pyridine. The reaction
mixture was stirred at room-temperature overnight. Then it
was poured onto crushed ice and extracted with ether. The
1
3
azaisobutyronitrile (AIBN, 2 mg) in 1 mL of toluene was stirred
under Ar for 6 h at 80 °C. The reaction mixture was cooled,
the solvent removed, and the residue purified by flash chro-
matography (hexane, t-BuOMe 85:15) yielding 26 (210 mg).
1
Oil; H NMR (CDCl
3
, 300 MHz) δ 6.88 (1H, d, J ) 7.9 Hz,
H-6), 6.84 (1H, d, J ) 7.9 Hz, H-7), 5.13 (1H, dddd, J ) 9.0,
7
2
1
.8, 4.9, 3.2 Hz, H-3), 3.08 (1H, dd, J ) 16.3, 4.9 Hz, H-4R),
.85 (1H, dd, J ) 16.3, 7.8 Hz, H-4â), 2.05 (3H, s, CH CO),
.01 (6H, d, J ) 6.9 Hz, H-26, H-27), 0.99 (3H, d, J ) 6.4 Hz,
organic layer was washed with saturated solutions of KHSO
4
and NaHCO and brine and dried over anhydrous Na SO ,
4
3
3
2
yielding 360 mg of ergosteryl acetate. Diethyl azodicarboxylate
(0.15 mL) was added to a solution of ergosteryl acetate (360
mg) in sodium-dried benzene (5 mL), and the solution was
refluxed under argon for 8 h. Removal of the solvent gave a
mixture of compounds 27 (93 mg), 28 (44 mg), and 29 (72 mg),
which were isolated using semipreparative TLC (hexane,
t-BuOMe 7:3). Spectroscopic data for compounds 27 and 28
1
3
H-21), 0.58 (3H, s, H-18); C NMR (Table 2); EIMS m/z 364
+
[
1
(
M-AcOH] (100), 278 (7), 237 (7), 236 (4), 235 (9), 210 (19),
95 (24), 183 (14), 141 (7), 71 (3); HRFABMS m/z 447.2878
calcd for C28 Na, 447.2875).
9-Nor er gosta -5,7,9,24(28)-tetr a en -3â-ol (1). t-BuOK
0.5 mL of a solution 1 M in t-BuOH) was added to a
40 3
H O
1
(
3
0
suspension of methyltriphenylphosphonium bromide (175 mg)
in anhydrous toluene (1.25 mL) and heated at 70 °C for 40
min. A solution of 26 (50 mg) in toluene (1 mL) was then
added, and the mixture stirred at 70 °C for 3 h. The reaction
mixture was allowed to cool and was flash chromatographed
were in agreement with those previously reported.
Com-
, 300 MHz) δ 6.50 (1H, d, J ) 8.3
pound 29: 1H NMR (CDCl
3
Hz, H-7 or H-6), 6.11 (1H, d, J ) 8.3 Hz, H-6 or H-7), 5.20
(1H, m, H-3), 5.20 (1H, dd, J ) 15.3, 8.0 Hz, H-23), 5.12 (1H,
dd, J ) 15.3, 8.0 Hz, H-22), 2.01 (3H, s, CH
3
CO), 1.22 (3H, t,
CH CH ),
2
5
with hexane: t-BuOMe (60:40), yielding 20 mg of 1: oil; [R]
6.5 (c 0.4, CHCl ); IR (film) νmax 3339, 3081, 2958, 2870, 1639,
462, 1233, 1051, 887, 810 cm ; H NMR (CDCl
δ 6.90 (1H, d, J ) 7.8 Hz, H-6), 6.85 (1H, d, J ) 7.8 Hz, H-7),
.74 (1H, br s, H-28a), 4.68 (1H, d, J ) 1.4 Hz, H-28b), 4.11
1H, dddd, J ) 10.3, 8.0, 4.2, 3.1 Hz, H-3), 3.06 (1H, dd, J )
5.5, 4.2 Hz, H-4R), 2.76 (1H, dd, J ) 15.5, 8.0 Hz, H-4â), 1.05
3H, d, J ) 6.8 Hz, H-26), 1.04 (3H, d, J ) 6.8 Hz, H-27), 1.03
D
J ) 7.1 Hz, CO CH CH ), 1.13 (3H, t, J ) 7.1 Hz, CO
2
2
3
2
2
3
+
1
3
0.97 (3H, d, J ) 6.6 Hz, H-21), 0.92 (3H, s, H-19), 0.89 (3H, d,
-
1 1
3
, 400 MHz)
J ) 6.8 Hz, H-28), 0.82 (3H, d, J ) 6.6 Hz, H-27), 0.801 (3H,
1
3
3
d, J ) 6.5 Hz, H-26), 0.799 (3H, s, H-18); C NMR (CDCl ,
4
(
1
3 2 2
100 MHz) δ 170.3 (s, CH CO), 159.9 (s, CO Et), 155.9 (s, CO -
Et), 140.8 (d, C-7 or C-6), 135.5 (d, C-22), 132.1 (d, C-23), 128.8
(d, C-6 or C-7), 70.3 (d, C-3), 66.3 (s, C-8 or C-5), 65.1 (s, C-5
or C-8), 62.0 (t, CO CH CH ), 61.2 (t, CO CH CH ), 57.0 (d,
2 2 3 2 2 3
C-17), 50.8 (d, C-14), 50.0 (d, C-9), 43.9 (s, C-13), 42.8 (d, C-24),
40.5 (s, C-10), 39.9 (d, C-20), 39.5 (t, C-12), 34.8 (t, C-1), 33.2
(d, C-25), 32.0 (t, C-2), 28.4 (t, C-16), 26.8 (t, C-4), 24.8 (t, C-15),
(
(
2
1
3
3H, d, J ) 6.0 Hz, H-21), 0.59 (3H, s, H-18); C NMR (Table
); EIMS, as TMS-ether, m/z 452 [M]+ (17), 437 (10), 362
100), 347 (11), 278 (59), 237 (45), 235 (59), 210 (81), 195 (99);
HREIMS m/z 380.3072 (calcd for C27 40O, 380.3079). Cho-
(
H
21.5 (q, CH
3
CO), 21.2 (t, C-11), 20.8 (q, C-21), 20.0 (q, C-27),
lesterol (0.5 mg) was added to 2 mg of 1. The mixture was
derivatized with BSTFA and analyzed by GC-MS. TRR′ (1.175)
and the mass spectrum of TMS derivative of 1 coincided with
those obtained from phycomysterol A.
19.8 (q, C-26), 19.0 (q, C-19), 17.6 (q, C-28), 14.7 (q, CO -
2
1
13
CH
2
CH
3
), 14.4 (q, CO
2
CH
2
CH
3
), 13.4 (q, C-18); H and
C
NMR data were assigned on the basis of 2D NMR experi-
1
1
ments: H/ H homonuclear correlation (COSY), as well as
1
1
9-Nor er gosta -5,7,9,22-tetr a en -3â-ol (3). The acetate 22
direct (HETCOR) and long-range (COLOC) heteronuclear H/
1
3
(
400 mg) was dissolved in 25 mL of 1 N KOH/MeOH. The
C correlations; HRFABMS m/z 635.4023 (calcd for C36
Na, 635.4036).
56 2 6
H N O -
solution was refluxed for 30 min, and then the EtOH was
removed and 50 mL of water was added. The mixture was
extracted with ether (40 mL × 3), and the organic layers
Syn th esis of Er gosta -5,8,22-tr ien -3â-ol (15). This sterol
was obtained from compound 27 by the procedure previously
described.30 H NMR data, mp, and [R]
1
25
washed together with brine and dried over anhydrous Na
2
-
D
were in agreement
with those previously reported;3 C NMR (Table 2); HREIMS
0 13
SO
4
.
When the solvent was eliminated, 341 mg of 3 was
2
5
obtained. Colorless needles; mp 150-152 °C; [R]
CHCl
MHz) δ 6.90 (1H, d, J ) 6.8 Hz, H-6), 6.84 (1H, d, J ) 6.8 Hz,
H-7), 5.24 (2H, m, H-22, H-23), 4.11 (1H, dddd, J ) 9.6, 8.1,
D
-7° (c 0.5,
m/z 396.3392 (calcd for C28H44O, 396.3392). Cholesterol (0.25
-
1
1
3
); IR (film) νmax 3338, 970 cm ; H NMR (CDCl , 300
3
mg) was added to 1 mg of 15. The mixture was derivatized
with BSTFA and analyzed by GC-MS. The chromatogram
showed two peaks: the major peak had TRR′ (1.070) and MS
identical to those for TMS ether of fungal lichesterol, whereas
TRR′ (1.090) and MS of the minor peak (17% abundance relative
to the major peak) coincided with those obtained from TMS-
ether of neoergosterol.
Syn th esis of Er gosta -5,8(14),22-tr ien -3â-ol (30). The
intermediate 28 (44 mg) was dissolved in ethylamine (2 mL),
treated with lithium (35 mg), and stirred at -20 °C for 1 h. A
4 mL amount of water was then added, and the suspension
4
.9, 3.2, H-3), 3.06 (1H, dd, J ) 16.0, 4.9 Hz, H-4R), 2.76 (1H,
dd, J ) 16.0, 8.1 Hz, H-4â), 1.10 (3H, d, J ) 6.6 Hz, H-21), 0.
9
0
4 (3H, d, J ) 6.8 Hz, H-28), 0.86 (3H, d, J ) 6.8 Hz, H-26),
.84 (3H, d, J ) 6.8 Hz, H-27), 0.61 (3H, s, H-18); C NMR
1
3
+
(
(
Table 2); EIMS, as TMS-ether, m/z 452 [M] (18), 437 (7), 362
100), 237 (64), 235 (20), 210 (34), 195 (49); HREIMS m/z
3
80.3076 (calcd for C27
H40O, 380.3079). Cholesterol (0.5 mg)
was added to 2 mg of 3. The mixture was derivatized with