1
202
Vol. 52, No. 10
13
Table 1.
C-NMR Data of Myrrhanol A (1) and Myrrhanone A (2)
pressure provided the 50% aqueous methanolic extract (105 g, 5.68%). The
aqueous methanolic extract (96.8 g) was partitioned in an EtOAc–H O (1 : 1,
2
1
2
v/v) mixture. Removal of the solvent from the EtOAc- and H O-soluble frac-
tions under reduced pressure yielded an EtOAc-soluble fraction (34.0 g,
2
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
37.9
27.0
78.6
38.8
55.0
20.2
44.3
73.9
61.1
38.8
25.5
31.3
125.1
135.0
39.6
26.5
124.5
134.6
39.3
26.1
125.7
134.7
28.1
15.4
15.5
23.7
16.2
16.0
13.7
68.6
38.3
33.9
217.0
47.4
55.1
21.3
43.7
73.6
60.3
38.5
25.8
31.2
124.8
135.1
39.6
26.5
124.4
134.6
39.3
26.1
125.7
134.6
26.3
21.3
14.8
23.5
16.2
16.0
13.7
68.6
2.00%) and H O-soluble fraction (62.8 g, 3.68%), respectively.
Normal-phase silica gel column chromatography [BW-200 (Fuji Silysia
Chemical, Ltd., 1 kg), n-hexane–EtOAc (3 : 1Æ1 : 1, v/v)ÆCHCl –MeOH
(10 : 1, v/v)ÆMeOH] of the EtOAc-soluble extract (29.0 g) gave nine frac-
tions [Fr. 1 (1.51 g), 2 (1.13 g), 3 (5.22 g), 4 (4.50 g), 5 (3.60 g), 6 (3.20 g), 7
(4.80 g), 8 (3.80 g), 9 (1.24 g)]. Fraction 2 (1.10 g) was purified by
2
3
reversed-phase silica gel column chromatography [30 g, MeOH–H O
2
(80 : 20Æ90 : 10, v/v)ÆMeOH] and HPLC [MeOH–H O (90 : 10, v/v)] to
2
C-9
give (8R)-3b,8-dihydroxypolypoda-13E,17E,21-triene (5, 118 mg, 0.0084%)
and (8R)-3-oxo-8-hydroxypolypoda-13E,17E,21-triene (6, 52 mg, 0.0037%).
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
Fraction 4 (1.00 g) was purified by HPLC [MeOH–H O (90 : 10, v/v)] to
2
give myrrhanone A (2, 84 mg, 0.026%). Fraction 5 (3.60 g) was separated by
reversed-phase silica gel column chromatography [100 g, MeOH–H O
2
(70 : 30Æ80 : 20, v/v)ÆMeOH] and HPLC [MeOH–H O (85 : 15, v/v)] to
2
give myrrhanol A (1, 523 mg, 0.036%). Fraction 6 (3.20 g) was separated by
reversed-phase silica gel column chromatography [100 g, MeOH–H O
2
(90 : 10, v/v)ÆMeOH] and HPLC [MeOH–H O (75 : 25, v/v)] to give
2
myrrhanol B (3, 36 mg, 0.0025%), myrrhanone B (4, 187 mg, 0.013%), and
(ꢁ)-diayangambin (7, 10 mg, 0.0007%). The known compounds (3—7)
1
were identified by comparison of their physical data ([a] , IR, H-NMR,
D
1
3
8)
C-NMR) with those of authentic samples.
2
7
Myrrhanol A (1): Colorless oil, [a]D ꢁ12.2° (cꢀ1.00, MeOH). High-res-
olution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa) : 483.3814.
ꢁ
3
0
52
3
ꢂ1
1
Found: 483.3830. IR (KBr): 3432, 2936, 1670, 1461, 1387, 1087 cm . H-
NMR (500 MHz, CDCl ) d: 0.75, 0.79, 0.98, 1.13, 1.65 (3H each, all s, 24,
3
25, 23, 26, 29-H ), 0.88 (1H, dd, Jꢀ2.1, 9.5 Hz, 5-H), 1.02 (1H, dd, Jꢀ3.9,
3
3.9 Hz, 9-H), 1.13, 1.70 (1H each, both m, 1-H ), 1.27, 1.45 (1H each, both
2
m, 11-H ), 1.32, 1.65 (1H each, both m, 6-H ), 1.32, 1.87 (1H each, both m,
2
2
7-H ), 1.60 (6H, s, 27, 28-H ), 2.00 (4H, m, 15, 19-H ), 2.08 (4H, m, 12, 16-
2
3
2
H ), 2.12 (2H, m, 20-H ), 3.21 (1H, dd, Jꢀ4.9, 11.6 Hz, 3-H), 3.96 (2H, s,
2
2
3
0-H ), 5.12 (1H, dd, Jꢀ5.8, 6.7 Hz, 17-H), 5.16, 5.38 (1H each, both dd-
2
1
3
1
25 MHz, CDCl3.
like, 13, 21-H). C-NMR (125 MHz, CDCl ) d : given in Table 1. Positive-
3 C
ꢁ
ion FAB-MS: m/z 483 (MꢁNa) .
Myrrhanone A (2): Colorless oil, [a] ꢁ11.9° (cꢀ1.00, MeOH). High-
2
8
D
ꢁ
nary carbon bearing an oxygen function (d 73.6, C-8) and resolution positive-ion FAB-MS: Calcd for
C
H
O
Na (MꢁNa) :
C
30
50
3
4
1
81.3658. Found: 481.3669. IR (KBr): 3453, 2930, 1709, 1650, 1456, 1385,
080 cm . H-NMR (500 MHz, CDCl ) d: 0.95, 1.02, 1.09, 1.19, 1.65 (3H
carbonyl carbon (d 217.0, C-3), which were found to be su-
perimposable on those of 1, except for the signal due to the
carbonyl group. In the HMBC experiment of 2, long-range
C
ꢂ1 1
3
each, all s, 25, 24, 23, 26, 29-H ), 1.13 (1H, dd, Jꢀ4.0, 4.0 Hz, 9-H), 1.30,
3
1
.52 (1H each, both m, 11-H ), 1.37, 1.60 (1H each, both m, 6-H ), 1.46
2
2
correlations were observed between the 23,24-methyl pro- (1H, m, 5-H), 1.46, 1.90 (1H each, both m, 7-H ), 1.52, 1.90 (1H each, both
2
tons and 3-carbonyl carbon (Fig. 1). In addition, reduction of m, 1-H
), 1.60 (6H, s, 27, 28-H
), 2.00 (4H, m, 15, 19-H ), 2.10 (6H, m, 12,
3 2
2
1
5
6, 20-H ), 2.40, 2.60 (1H each, both m, 2-H ), 3.96 (2H, s, 30-H ), 5.12,
2
with sodium borohydride (NaBH ) yielded 1, so that the
2
2
2
4
13
.16, 5.39 (1H each, all dd-like, 17, 13, 21-H). C-NMR (125 MHz, CDCl )
3
absolute stereostructure of myrrhanone A was determined
to be (5R,8R,9R,10S)-3-oxo-8,30-dihydroxypolypoda-13E,
ꢁ
d : given in Table 1. Positive-ion FAB-MS: m/z 481 (MꢁNa) .
C
Preparation of the (R)-MTPA Ester (1a) and the (S)-MTPA Ester (1b)
from Myrrhanol A (1) A solution of 1 (2.0 mg) in CH Cl (1.0 ml) was
29)
1
7E,21E-triene (2).
2
2
treated with (R)-MTPA (10.0 mg) in the presence of EDC·HCl (10.0 mg)
Experimental
and 4-DMAP (5.0 mg), and the mixture was heated under reflux for 2 h. The
The following instruments were used to obtain physical data: specific ro- reaction mixture was poured into ice-water, and the whole was extracted
tations, Horiba SEPA-300 digital polarimeter (lꢀ5 cm); UV spectra, Shi- with EtOAc. The EtOAc extract was successively washed with 5% aqueous
madzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrome- HCl, aqueous saturated NaHCO , and brine, then dried over MgSO and fil-
3
4
ter; EI-MS and high-resolution MS, JEOL JMS-GCMATE mass spectrome- tered. Removal of the solvent from the filtrate under reduced pressure fur-
ter; FAB-MS and high-resolution MS, JEOL JMS-SX 102A mass spectrom- nished a residue, which was purified by a normal-phase silica gel column
1
13
eter; H-NMR spectra, JNM-LA500 (500 MHz) spectrometer; C-NMR [1.0 g, n-hexane–EtOAc (4 : 1)] to give 1a (1.1 mg, 38%) and 1 (0.5 mg).
spectra, JNM-LA500 (125 MHz) spectrometer with tetramethylsilane as an Through a similar procedure, 1b (1.0 mg, 37%) and 1 (0.6 mg) were pre-
internal standard; and HPLC detector, Shimadzu RID-6A refractive index pared from 1 (2.0 mg) by the use of (S)-MTPA (10.0 mg), EDC·HCl
detector.
The following experimental conditions were used for chromatography: or-
dinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji
Silysia Chemical, Ltd., 150—350 mesh); reverse-phase silica gel column
(10.0 mg), and 4-DMAP (5.0 mg).
1a: Colorless oil. H-NMR (500 MHz, CDCl ) d: 0.78, 0.82, 0.83, 1.13,
1.59 (3H each, all s, 24, 25, 23, 26, 29-H ), 1.61 (6H, s, 27, 28-H ), 1.25,
1
3
3
3
1.79 (1H each, both m, 1-H ), 1.79, 1.88 (1H each, both m, 2-H ), 3.56 (6H,
2
2
chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., s, OMeꢃ2), 4.66, 4.71 (2H, ABq, Jꢀ13.1 Hz, 30-H ), 4.73 (1H, dd-like, 3-
2
1
0
00—200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck, H), 5.11, 5.16, 5.51 (1H each, both dd-like, 17, 13, 21-H), 7.39—7.55 (10H,
ꢁ
.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (re- m, Ph-H). Positive-ion FAB-MS: m/z 915 (MꢁNa) .
1
verse phase); reverse-phase HPTLC, precoated TLC plates with Silica gel
RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by spraying
with 1% Ce(SO ) –10% aqueous H SO , followed by heating.
1b: Colorless oil. H-NMR (500 MHz, CDCl ) d: 0.79, 0.80, 0.91, 1.13,
3
1.59 (3H each, all s, 25, 24, 23, 26, 29-H ), 1.24, 1.65 (1H each, both m, 1-
H ), 1.61 (6H, s, 27, 28-H ), 1.65, 1.80 (1H each, both m, 2-H ), 3.56 (6H, s,
3
4
2
2
4
2
3
2
Extraction and Isolation The resin of B. mukul [1.85 kg, collected in
OMeꢃ2), 4.46, 4.71 (2H, ABq, Jꢀ13.1 Hz, 30-H ), 4.73 (1H, dd-like, 3-H),
2
Rajastan, India and purchased from Sharangdhar Pharmaceuticals PTV, Ltd.
(
5.11, 5.16, 5.51 (1H each, both dd-like, 17, 13, 21-H), 7.38—7.51 (10H, m,
Pune, India)] was finely minced and extracted three times with 50% aque- Ph-H). Positive-ion FAB-MS: m/z 915 (MꢁNa) .
ꢁ
ous methanol under reflux for 3 h. Evaporation of the solvent under reduced
NaBH Reduction of Myrrhanone A (2) A solution of 2 (10.0 mg) was
4