G.D.A. Martin et al. / Phytochemistry 65 (2004) 701–710
709
3
.10. Stemod-12-ene (13)
was purified on silica gel. Elution with 50–70% ethyl
acetate in petrol afforded 2a,7b,13(S)-trihydroxy-
stemodane (17) (76 mg) which crystallised from metha-
Stemod-12-ene (13) was prepared from stemod-12-en-
-one (9) (2.0 g, 6.99 mmol) in accordance with the
ꢁ ꢁ
nol as cubes, mp 240–241 C; [ꢁ] +22.6 (MeOH, c
D
27
2
ꢁ
ꢁ
modified Wolff Kishner conditions (Huang-Minlon,
949; Manchand et al., 1973). Stemod-12-ene (13) (0.9
g) crystallised from chloroform as an amorphous solid,
0.44); lit. mp 241–245 C, [ꢁ] ꢀ2.9 (c 1.35, py) (Badria
D
1
and Hufford, 1991).
Elution with ethyl acetate yielded 2a,3b,13(S),16a-
tetrahydroxystemodane (18) (38 mg) which crystallised
ꢁ
27
D
ꢁ
mp 43–45 C; [ꢁ] +33.6 (c 5.77, CHCl ); lit. mp 52–
5
1
3
ꢁ ꢁ
3 C; [ꢁ] +36.6 (CHCl , c 1.00) (Manchand et al.,
D 3
from methanol as prisms, mp 239–240 C; [ꢁ] ꢀ17.9ꢁ
ꢁ
27
D
(MeOH, c 0.47); FTIR: ꢂmax cm 3404 (OH); ESMS
ꢀ
1
1
ꢀ1
973); FTIR: ꢂmax cm 1678 (>C¼O), 1447, 1378; H
NMR (200 MHz, CDCl ): ꢀ 0.87 (3H, s, H-19), 0.89
3
(EI) m/z (rel. int.): 361.2347 [M+Na]+ (100)
1
(
5
3H, s, H-20), 0.95 (3H, s, H-18), 1.64 (3H, s, H-17),
.00 (1H, bs, H-12).
[(C H O +Na) requires 361.2354]; H NMR (200
2
0
34
2
MHz, CD OD): ꢀ 0.87 (3H, s, H-19), 0.97 (3H, s, H-20),
3
1
.03 (3H, s, H-18), 1.14 (3H, s, H-17), 3.07 (1H, br d,
J=10.0 Hz, H-3), 3.58 (1H, dt, J=10.3, 4.3 Hz, H-2),
.32 (1H, s, H-16).
3
.11. Methyl betulinate (15)
4
To a solution of potassium hydroxide (1.0 g, 17.82
mmol) dissolved in ethanol (16.5 ml) and water (5 ml) at
3.14. Incubation of stemodinone (8)
ꢁ
6
5
C was slowly added a solution of N-methyl-N-
nitroso-p-toluenesulfonamide (diazald) (6.9 g, 32.20
mmol) dissolved in diethyl ether (52 ml). The ethereal
diazomethane distillate was added to a solution of
betulinic acid (14) (1.0 g, 2.19 mmol) in dichloro-
methane (170 ml) at 0 C (ice bath). The organic solu-
tion was removed in vacuo to afford methyl betulinate
Stemodinone (8) (1 g) was fed to the growing fungus.
The mycelial (0.57 g) and broth (0.54 g) extracts were
combined and chromatographed. Elution with 40%
ethyl acetate in petrol provided 6a,13(S)-dihydroxy-
stemodan-2-one (19) (14.3 mg) which crystallised from
ꢁ
ꢁ
27
ꢁ
acetone as needles, mp 191–193 C; [ꢁ]D +55.6
(CHCl , c 4.5); FTIR ꢂ
ꢀ
1
(15) (1 g) which crystallised from dichloromethane as
needles, mp 220–221 C; [ꢁ] +10.3 (CH Cl , c 1.55);
cm
max
3388 (OH), 1696
+
3
ꢁ
27
ꢁ
(C¼C); HRMS (EI): m/z (rel. int.): 320.2351 [M] (41)
D
lit. mp 221–223 C; [ꢁ] +5 (Monaco and Previtera,
2
2
ꢁ
ꢁ
+
[C H O requires 320.2351], 302.2246 [M–H O] (30),
20 32
D
3
2
ꢀ
1
1
1
984); FTIR: ꢂmax cm 3568 (OH), 3370 (OH), 1726
233.1905 (100); H NMR (200 MHz, CDCl ): ꢀ 0.99
3
(
NMR (200 MHz, CDCl ): ꢀ 0.75 (3H, s, H-24), 0.82
>C¼O), 1635 (C¼C), 1452, 1378, 1154, 1046; 1H
(3H, s, H-20), 1.15 (3H, s, H-17), 1.20 (3H, s, H-19),
1.31 (3H, s, H-18), 3.86 (1H, dt, J=11.0, 4.4 Hz, H-6).
3
(
3H, s, H-25), 0.91 (3H, s, H-23), 0.96 (6H, s, H-26,
H-27), 1.68 (3H, s, H-30), 2.99 (1H, m, w/2=14.9 Hz,
H-19), 3.19 (1H, dd, J=9.5, 5.5 Hz, H-3), 3.67 (3H, s,
3.15. Incubation of 2ꢃ,13(S)-dihydroxystemodane (10)
w
H-31), 4.60 (1H, m, / =4.7 Hz, H -29), 4.74 (1H, d,
2
Incubation of 2b,13(S)-dihydroxystemodane (10) (1 g)
with the fungus gave mycelial (0.89 g) and broth (0.34 g)
extracts after work up. Thin layer chromatography
(TLC) showed that five metabolites were formed. The
combined extract (1.23 g) was purified on silica gel.
Elution in 10% acetone in dichloromethane gave
2b,7b,13(S)-trihydroxystemodane (20) (53 mg) as a
a
J=1.6 Hz, H -29).
b
3
.12. Betulin (16)
To a solution of the methyl betulinate (15) (0.70 g,
.489 mmol) in tetrahydrofuran (35.0 ml) was added
1
2
7
ꢁ
ꢀ1
lithium aluminium hydride (0.23 g, 6.06 mmol).
The reaction mixture was refluxed for 3 h and ethyl
acetate (35 ml) was added. Water (100 ml) was added
and the mixture was extracted with ethyl acetate (3 Â 35
ml). The organic solution was dried, filtered and
removed in vacuo to afford betulin (16) (0.65 g) which
gum, [ꢁ]D +24.6 (MeOH, c 3.6); FTIR ꢂ
cm 3380
max
+
(OH); HRMS (EI) m/z (rel. int.): 321.2657 [M-1] (8.6)
[C H O requires 322.2508], 289.2530 [M–CH –
2
0
34
3
3
+
1
H O] (22.2), 250.2190 (19.8), 233.2173 (100); H NMR
2
(200 MHz, CDCl ): ꢀ 0.93 (3H, s, H-18), 1.11 (3H, s, H-
3
19), 1.14 (3H, s, H-17), 1.19 (3H, s, H-20), 3.34 (1H, ddd,
J=14.6, 9.3, 5.3 Hz, H-7), 4.08 (1H, q, J=3.8 Hz, H-2).
Purification of the other metabolites proved imprac-
tical.
ꢁ
crystallised from acetone as needles, mp 245–246 C;
2
7
ꢁ ꢁ
ꢁ] +17.9 (Me CO, c 0.64); lit. mp 236–238 C; [ꢁ]
D
2 D
+
[
ꢁ
24 (c 0.075, py) (Tinto et al., 1992).
3
.13. Incubation of stemodin (2)
3.16. Incubation of stemod-12-en-2-one (9)
Stemodin (2) (1.0 g) was incubated with R. oryzae.
The fermentation was harvested to provide mycelial
0.36 g) and broth (0.38 g) extracts. The broth extract
Stemod-12-en-2-one (9) (500 mg) was fed to R. ory-
zae. Workup yielded mycelial (0.73 g) and broth (0.44 g)
extracts which were pooled and purified on silica gel.
(