2002 Journal of Natural Products, 2008, Vol. 71, No. 12
Tao et al.
0071102) has been deposited in the Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming, People’s Republic of China.
Extraction and Isolation. Air-dried and ground entire plants (2.2
kg) were extracted with 95% EtOH (10 L × 3) at room temperature.
After removal of the solvents under reduced pressure, the obtained
residual material (303.4 g) was distributed between EtOAc and water,
and the EtOAc-soluble portion (100.6 g) was subjected to vacuum-
column chromatography over silica gel eluted with Me2CO-petroleum
ether in a gradient manner. Two light yellow resinous fractions, A (2.2
g) and B (1.8 g), were collected from the elution of Me2CO-petroleum
ether, 1:9 to 1:8 and 1:5 fractions, respectively. Fractions A and B
were separated over Sephadex LH-20 eluted with Me2CO. Subfraction
A1 (2.0 g) showed a spot by TLC on silica gel eluted with
CHCl3-CH3OH (10:1) at Rf 0.50 and gave four main peaks for
compounds 3 (tR 14.9 min), 1 (tR 17.4 min), 5 (tR 20.3 min), and 7 (tR
24.1 min) by HPLC analysis (YMC-Pack ODS-A, 4.6 × 250 mm, 5
µm; H2O-CH3OH (2.5:97.5), 1 mL/min; 30 °C; 275 nm). In turn,
subfraction B1 (1.8 g) showed a spot at Rf 0.45 on TLC and gave four
main peaks for compounds 4 (tR 13.7 min), 2 (tR 16.1 min), 6 (tR 18.8
min), and 8 (tR 22.4 min), under the same HPLC conditions. Purification
of these compounds was performed initially by semipreparative HPLC
(YMC-Pack ODS-A, 10 × 250 mm, 5 µm) eluted with H2O-CH3OH
(2.5:97.5) at a flow of 4 mL/min at 30 °C, respectively. Each subfraction
contained two main peaks for compounds 1 and 2, 3 and 4, 5 and 6,
and 7 and 8, respectively. Finally, purification was carried out by
semipreparative HPLC eluted with an anhydrous mixture of
CH3OH-CH3CN (7:3) at a flow rate of 2.8 mL/min at 30 °C by heart
cutting and independent reinjection. The pure compounds 3 (15 mg, tR
18.6 min), 1 (58 mg, tR 21.1 min), 5 (50 mg, tR 24.0 min), and 7 (38
mg, tR 27.9 min) from A1, and 4 (19 mg, tR 17.4 min), 2 (15 mg, tR
19.9 min), 6 (30 mg, tR 22.7 min), and 8 (45 mg, tR 26.2 min) from B1
were obtained, respectively.
Pescaprein XVI (7): amorphous, white powder; [R]20D -20 (c 0.14,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.1), 279 (4.0) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1382.0 [M + H]+;
positive ESIMS/MS m/z 1382.0,1235.9, 835.6, 689.5, 507.3, 361.2;
positive HRESIMS m/z 1381.7867 ([M + H]+) (calcd for C72H117O25,
1381.7884).
Pescaprein XVII (8): amorphous, white powder; [R]20D -6 (c 0.17,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (3.9), 280 (3.9) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1382.0 [M + H]+;
positive ESIMS/MS m/z 1382.0, 1235.9, 835.6, 689.5, 507.3, 361.2;
positive HRESIMS m/z 1381.7858 ([M + H]+) (calcd for C72H117O25,
1381.7884).
Intramolecular trans-Cinnamoyl Migration under Water. Com-
pound 1 (1 mg) was stirred in a solution of MeOH-H2O (95:5, 1 mL)
for 30 min at room temperature and evaporated to dryness in vacuo.
The residue was identified as a mixture of compounds 1 and 2 by HPLC
analysis. Compound 2 also gave a mixture of 1 and 2 when subjected
to the same procedure. Compounds 3 and 4, 5 and 6, and 7 and 8 were
treated as above, and similar results were obtained.
Transesterification of the Resin Glycoside Mixture of Fractions
A1 and B1 Containing 1-8. A solution of NaOMe (5 mg) in
anhydrous MeOH (1 mL) was added dropwise to a solution of fraction
A1 (15 mg) and B1 (15 mg) in anhydrous MeOH (4 mL) at room
temperature and then refluxed for 2 h. The reaction mixture was adjusted
to pH 7.0 with positive ion-exchange resin and filtered. The filtrate
was concentrated and subjected to separation over Sephadex LH-20
eluted with MeOH-CHCl3 (1:1) to discard the byproduct in the
reaction. Subfractions were collected and evaporated in vacuo. The
residue obtained (18 mg) was identified as simonic acid B methyl ester
(9) by 2D NMR data analysis and comparison of its mass spectrum,
1H and 13C NMR data, and physical properties (mp 112-114 °C; [R]20
D
-81 (c 0.76, MeOH)) with the literature values.15
The air-dried and ground entire plant (5 g each) was extracted twice
with 80 mL of anhydrous CHCl3 and petroleum ether (dried over
anhydrous Na2SO4) to afford 250 mg and 180 mg extracts, respectively.
The MeOH-soluble portion was applied to HPLC analysis (YMC-Pack
ODS-A, 4.6 × 250 mm, 5 µm; H2O-CH3OH (2.5:97.5), 1 mL/min;
30 °C; 275 nm).
Saponification of the Resin Glycoside Mixture of Fractions A1
and B1 Containing 1-8. A solution of fractions A1 (80 mg) and B1
(80 mg) in 5% KOH-H2O (5 mL) was refluxed for 2 h. The reaction
mixture was acidified to pH 4.0 with 4 N HCl and extracted with CH2Cl2
(10 mL × 2). The water layer was adjusted to pH 7.0 with 1 N NaOH
and evaporated to dryness in vacuo. The residue was subjected to
Sephadex LH-20 eluted with MeOH to desalt, and 100 mg of simonic
acid B (10) was obtained as a colorless oil. It afforded key fragments
at m/z 1003.4 [M + H]+, 1025.4 [M + Na]+, 857.4 [M + H - 146.0]+,
711.3 [857.4 - 146.1]+, 565.3 [711.3 - 146.0]+, 419.2 [565.3 -
146.1]+, and 273.2 [419.2 - 146.0]+ in the positive ESIMS. Its NMR
data were assigned by comparison with those of simonic acid B methyl
ester (9). The organic layer was washed with H2O, dried over anhydrous
Na2SO4, concentrated in vacuo, and directly analyzed by GC-MS (30
m × 0.32 mm × 0.25 µm HP-5 MS column: He, 1 mL/min; 40 °C, 2
min, 40-250 °C, ∆ 15 °C/min, 250 °C, 10 min): 2-methylpropanoic
acid (tR 3.38 min): m/z 88 [M]+ (12), 73 (47), 60 (3), 55 (7), 45 (14),
43 (100), 41 (48); 2-methylbutanoic acid (tR 4.87 min): m/z 102 [M]+
(1), 87 (25), 74 (100), 73 (15), 57 (47), 45 (11), 41 (35); n-decanoic
acid (tR 10.57 min): m/z 172 [M]+ (7), 143 (10), 129 (58), 115 (14),
101 (8), 87 (17), 73 (100), 60 (98), 57 (42), 55 (40), 43 (40), 41 (42);
Pescaprein X (1): amorphous, white powder; [R]20 -17 (c 0.14,
D
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.2), 279 (3.9) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1354.1 ([M + H]+);
positive ESIMS/MS m/z 1354.1, 1208.0, 807.6, 661.5, 507.3, 361.2;
positive HRESIMS m/z 1353.7589 ([M + H]+) (calcd for C70H113O25,
1353.7571).
Pescaprein XI (2): amorphous, white powder; [R]20D -12 (c 0.15,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.5), 279 (3.8) nm;1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1354.1 ([M+H]+);
positive ESIMS/MS m/z 1354.1, 1208.0, 807.6, 661.5, 507.3, 361.2;
positive HRESIMS m/z 1353.7563 ([M+H]+) (calcd for C70H113O25,
1353.7571).
Pescaprein XII (3). amorphous white powder; [R]20D -15 (c 0.13,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.7), 279 (3.9) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1339.9 [M + H]+;
positive ESIMS/MS m/z 1339.9, 1193.8, 793.5, 647.4, 493.3, 347.2;
+
trans-cinnamic acid (tR 11.18 min): m/z 148 [M] (74), 147 (100),
positive HRESIMS m/z 1361.7253 ([M
+
Na]+) (calcd for
131 (21), 120 (5) 103 (45), 102 (23), 91(21), 77 (34), 74 (7), 63 (5),
51 (23), 50 (9), 45 (6); and n-dodecanoic acid (tR 12.27 min): m/z 200
[M]+ (12), 183 (2), 171 (12), 157 (32), 143 (11), 129 (42), 115 (18),
101 (13), 85 (30), 73 (100), 60 (82), 57 (42), 55 (46), 43 (48), 41 (44).
By the same procedure, pure compound 1 gave 2-methylbutanoic
acid [tR 5.09 min; m/z 102 [M]+ (1), 87 (25), 74 (100), 73 (14), 57
(48), 45 (12), 41 (35)], n-decanoic acid [tR 10.62 min; m/z 172 [M]+
(6), 143 (11), 129 (60), 115 (14), 101 (8), 87 (20), 83 (13), 73 (100),
71 (38), 60 (98), 57 (40), 55 (39), 43 (38), 41 (40)], and trans-cinnamic
acid [tR 11.27 min; m/z 148 [M]+ (68), 147 (100), 131 (19), 103 (44),
102 (21), 91 (20), 77 (32), 74 (8), 51 (21), 50 (8), 45 (3)].
Preparation of (R)-1-Phenylethyl-(S)-2-methylbutanoate. Trace
DMAP (1.5 mg) was added to the solution of the above carboxylic
acids (a quarter from saponification) in CH2Cl2 (2 mL) and cooled to
0 °C. Then (R)-1-phenylethanol (7.0 µL) and DCC (15.0 mg) were
added. The reaction mixture was stirred at 0 °C for 10 min and room
temperature for 12 h and then filtered. The standard (R)-1-phenylethyl-
(S)-2-methylbutanoate was also prepared from (R)-1-phenylethanol and
(S)-2-methylbutanoic acid by the same process. The reaction product
was analyzed by GC-MS (30 m × 0.32 mm × 0.25 µm HP-5 MS
C69H110O25Na, 1361.7234).
Pescaprein XIII (4): amorphous, white powder; [R]20D -18 (c 0.14,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.1), 279 (3.8) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1339.9 [M + H]+;
positive ESIMS/MS m/z 1339.9, 1193.8, 793.5, 647.4, 493.3, 347.2;
positive HRESIMS m/z 1361.7238 ([M
+
Na]+) (calcd for
C69H110O25Na, 1361.7234).
Pescaprein XIV (5): amorphous, white powder; [R]20D -26 (c 0.14,
MeOH); UV (MeOH) λmax (log ꢀ) 200 (4.3), 279 (4.2) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1368.0 [M + H]+;
positive ESIMS/MS m/z 1368.0, 1222.0, 821.6, 675.5, 493.3, 347.2;
positive HRESIMS m/z 1367.7706 ([M + H]+) (calcd for C71H115O25,
1367.7727).
Pescaprein XV (6): amorphous, white powder; [R]20D -13 (c 0.13,
MeOH); UV (MeOH) λmax (log ꢀ) 200 (4.0), 280 (3.9) nm; 1H and 13
C
NMR, see Tables 1 and 2; positive ESIMS m/z 1368.0 [M + H]+;
positive ESIMS/MS m/z 1368.0, 1222.0, 821.6, 675.5, 493.3, 347.2;
positive HRESIMS m/z 1367.7783 ([M + H]+) (calcd for C71H115O25,
1367.7727).