348 J ournal of Natural Products, 2003, Vol. 66, No. 3
Reino et al.
acetone was removed by blowing nitrogen over the solution,
and the product was recovered in EtOAc. The EtOAc extract
was washed (×2) with aqueous NaHCO3 and brine. The solvent
was evaporated under reduced pressure, and the crude product
was subjected to column chromatography to afford 23 (10.1
C-8), 51.9 (d, C-1), 52.5 (t, C-7), 60.2 (d, C-5), 64.1 (t, C-10),
73.1 (d, C-4), 75.3 (t, C-15), 84.3 (s, C-9).
Com p ou n d 26. A 1 M aqueous solution of K2CO3 (5 mL)
was added to a solution of 25 (50 mg, 0.169 mmol) in methanol.
The mixture was stirred and refluxed for 3 h, the methanol
was removed, and the product was extracted with EtOAc (×4).
The residue obtained after evaporation of the solvent was
purified by normal-phase HPLC to yield 264 (15 mg, 0.059
mmol, 35%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ
0.89 (3H, d, J 11-2 ) 6.5 Hz, H-11), 1.10 (1H, q, J 3â-3R ) J 3â-2
) J 3â-4 ) 12.3 Hz, H-3â), 1.21 (1H, d, J 7R-7â ) 12.4 Hz, H-7R),
1.23 (3H, s, H-14), 1.32 (3H, s, H*-12), 1.29 (3H, s, H*-13),
1.47 (1H, ddd, J 1-2 ) 11.5 Hz, J 1-10 ) 6.7 Hz, J 1-10′ ) 4.5 Hz,
H-1), 1.51 (1H, d, J 5-4 ) 9.9 Hz, H-5), 1.71 (1H, m, J 2-1 ) 11.5
Hz, J 2-11 ) 6.5 Hz, J 2-3R ) 3.0 Hz, J 2-3â ) 12.3 Hz, H-2), 1.90
(1H, ddd, J 3R-3â ) 12.3 Hz, J 3R-2 ) 3.0 Hz, J 3R-4 ) 4.7 Hz,
H-3R), 1.91 (1H, d, J 7â-7R ) 12.4 Hz, H-7â), 3.40 (1H, d, J 15′-15
) 10.2 Hz, H-15′), 3.89 (1H, d, J 15-15′ ) 10.2 Hz, H-15), 3.95
(1H, ddd, J 4-3R ) 4.7 Hz, J 4-3â ) 12.3 Hz, J 4-5 ) 9.9 Hz, H-4),
3.67 (1H, dd, J 10′-10 ) 12.0 Hz, J 10′-1 ) 4.5 Hz, H-10′), 3.97
(1H, dd, J 10-10′ ) 12.0 Hz, J 10-1 ) 6.7 Hz, H-10) (*interchange-
able); 13C NMR (CDCl3, 50 MHz) δ 20.3 (q, C-11), 26.3 (q, C-14),
27.4 (q, C-13), 30.6 (d, C-2), 36.7 (q, C-12), 39.5 (s, C-6), 44.4
(t, C-3), 46.0 (s, C-8), 52.0 (d, C-1), 52.4 (t, C-7), 64.0 (t, C-10),
65.1 (d, C-5), 70.2 (d, C-4), 75.1 (t, C-15), 84.3 (s, C-9).
Botr yoloic Acid (28). Lactone 27 (14 mg, 0.045 mmol) in
methanol was stirred with a 1 N aqueous solution of K2CO3
(240 µL) for 12 h at room temperature. The solution was
diluted with H2O, acidified with cold 2 N HCl, and extracted
with EtOAc (30 mL, ×3). The solvent was evaporated and the
residue subjected to column chromatography (eluted with
hexane/EtOAc, 4:6, with drops of acetic acid) to afford 28 (10
25
mg, 0.029 mmol, 30%) as a white solid: mp 93-95 °C; [R]D
+8° (c 0.65, CHCl3); IR (dry film) νmax 3506, 1737, 1243, 1211,
1066, 1017 cm-1 1H NMR (CDCl3, 400 MHz) δ 0.87 (3H, s,
;
J 11-2 ) 6.4 Hz, H-11), 1.03 (1H, d, J 7R-7â ) 12.8 Hz, H-7R),
1.17 (3H, s, H-12), 1.20 (3H, s, H-13), 1.23 (3H, s, H-14), 1.34
(3H, s, H*-17), 1.40 (1H, q, J 3â-3R ) 12.1 Hz, H-3â), 1.41 (3H,
s, H*-18), 1.48 (1H, m, H-2), 1.72 (1H, d, J 7â-7R ) 12.8 Hz,
H-7â), 1.83 (1H, q, J 1-2 ) 6.6 Hz, H-1), 1.99 (1H, d, H-5), 2.00
(3H, s, CH3COO- superimposed on H-5), 2.02 (1H, m, H-3R
superimposed on H-5 and CH3COO-), 2.96 (1H, d, J 15′-15 ) 13.6
Hz, H-15′), 3.74 (1H, dd, J 10′-10 ) 10.6 Hz, J 10′-1 ) 6.6 Hz,
H-10′), 3.82 (1H, dd, J 10-10′ ) 10.6 Hz, J 10-1 ) 6.3 Hz, H-10),
3.83 (1H, d, J 15-15′ ) 13.6 Hz, H-15), 4.22 (1H, s, -OH), 5.02
(1H, dt, J 4-3R ) 4.8 Hz, J 4-3â ) J 4-5 ) 11.4 Hz, H-4),
(*interchangeable); 13C NMR (CDCl3, 50 MHz) δ 20.4 (q, C-11),
21.5 (q, CH3COO-), 21.9 (q, C-14), 24.5 (q, C*-18), 25.0 (q,
C*-17), 27.8 (q, C-13), 30.0 (d, C-2), 36.0 (q, C-12), 36.3 (s,
C-6), 40.0 (t, C-3), 51.7 (s, C-8), 52.2 (d, C-1), 55.1 (t, C-7), 62.4
(t, C-10), 63.1 (d, C-5), 66.6 (t, C-15), 73.9 (d, C-4), 88.0 (s, C-9),
101.1 (s, C-16) (*interchangeable); EIMS m/z 296 [M-
(CH3)2CO]+ (0.9), 281 [M-(CH3)2CO-CH3]+ (1), 221 [M-
(CH3)2CO-CH3-AcOH]+ (31), 140 (85), 109 (59), 96 (100).
Botr y-5(9)-en ed iol (24). The acetonide 23 (12 mg, 0.034
mmol) was dissolved in dry pyridine (2 mL) and then treated
with SOCl2 (100 µL) for 1 h at room temperature. The mixture
was diluted with H2O and extracted with EtOAc. The organic
phase was washed with an aqueous solution of CuSO4 and
brine, dried, and concentrated to dryness. The residue was
purified by means of normal-phase HPLC with hexane/EtOAc
(7:3) as solvent to afford 24 (5 mg, 0.017 mmol, 50%) as an
amorphous solid: [R]D26 -34° (c 0.14, CHCl3); IR (dry film) νmax
3414, 1717, 1254, 1216 cm-1; 1H NMR (CDCl3, 400 MHz) δ 1.05
(3H, d, J 11-2 ) 7.2 Hz, H-11), 1.08 (6H, s, H*-13, H*-14),
1.14 (3H, s, H*-12), 1.47 (1H, d, J 7R-7â ) 13.2 Hz, H-7R), 1.61
(1H, m, J 3R-3â ) 15.2 Hz, J 3R-4 ) 1.6 Hz, H-3R), 1.92 (1H, d,
25
mg, 0.030 mmol, 67%) as a white solid: mp 203-205 °C; [R]D
+7° (c 1.07, CHCl3); IR (dry film) νmax 3390, 1728, 1470, 1246,
1025, 953 cm-1; H NMR (CD3COCD3, 400 MHz) δ 0.97 (3H,
1
d, J 11-2 ) 6.2 Hz, H-11), 1.06 (1H, ddd, J 3â-3R ) 12.4 Hz, J 3â-4
) 11.3 Hz, H-3â), 1.08 (3H, s, H-14 superimposed on H-3â),
1.09 (3H, s, H-13), 1.18 (1H, d, J 7R-7â ) 12.8 Hz, H-7R), 1.29
(3H, s, H-12), 1.92 (1H, d, J 5-4 ) 11.3 Hz, H-5), 2.00 (1H, m,
J 2-1 ) 12.3 Hz, J 2-11 ) 6.2 Hz, J 2-3R ) 3.2 Hz, H-2), 2.03 (3H,
s, CH3COO-), 2.10 (1H, m, J 3R-3â ) 12.4 Hz, J 3R-4 ) 4.8 Hz,
J
7â-7R ) 13.2 Hz, H-7â), 1.99 (3H, s, CH3COO-), 2.12 (1H, m,
J 3â-3R ) 15.2 Hz, J 3â-4 ) 5.5 Hz, H-3â), 2.17 (1H, m, J 1-10
)
H-3R), 2.50 (1H, d, J 1-2 ) 12.3 Hz, H-1), 2.58 (1H, d, J 7â-7R )
J 1-10′ ) 3.5 Hz, H-1), 2.25 (1H, m, J 2-11 ) 7.2 Hz, J 2-3â ) 10.1
12.8 Hz, H-7â), 3.33 (1H, d, J 15′-15 ) 11.5 Hz, H-15′), 3.92 (1H,
d, J 15-15′ ) 11.5 Hz, H-15), 5.10 (1H, dt, J 4-3R ) 4.8 Hz, J 4-3â
) J 4-5 ) 11.3 Hz, H-4); 13C NMR (CD3COCD3, 100 MHz) δ
21.1 (q, C-11), 21.4 (q, CH3COO-), 22.0 (q, C-14), 28.3 (q, C-13),
30.2 (d, C-2), 36.8 (s, C-6), 36.4 (q, C-12), 39.5 (t, C-3), 50.5
(s, C-8), 53.4 (t, C-7), 61.6 (d, C-1), 65.0 (d, C-5), 67.5 (t,
C-15), 73.4 (d, C-4), 88.6 (s, C-9), 174.8 (s, C-10), 170.1 (s,
CH3COO-); EIMS m/z 295 [M - H2O - CH3]+ (0.2), 267 [M -
HCOOH - CH3]+ (0.1), 253 [M - AcOH - CH3]+ (0.4), 252 [M
- HCOOH - 2CH3]+ (2), 235 [M - HCOOH - CH2OH]+ (14),
232 [M - HCOOH - CH2OH-H2O]+ (6), 177 (47), 96 (100).
Cells a n d Cu ltu r e Con d ition s. Human breast ductal
carcinoma (Hs578T) and adenocarcinoma (MDA-MB-231),
fibrosarcoma (HT-1080), glioblastoma (U87-MG), and embry-
onic fibroblasts (IMR-90) were obtained from the American
Type Culture Collection (Rockville, MD). Human umbilical
vein endothelial cells (HUVEC) were isolated from newborns.14
Cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) (Life Technologies, Paisley, Scotland) containing 10%
(v/v) fetal calf serum (FCS) (BioWhittaker, Walkersville, MD),
1% glutamin, 10 µg/mL insulin (Hs578T), and 1% (v/v)
vitamins (HT-1080 cells) (the latter three from Life Technolo-
gies). U87-MG cells were grown in Minimum Essential Me-
dium (MEM) with 10% FCS, 1% sodium pyruvate, and 1%
nonessential amino acids (the latter two from Life Technolo-
gies). MDA-MB-231 cells were grown in Leibovitz’s L-15
medium (BioWhittaker) with 10% FCS. Primary cells HUVEC
were grown in DMEM containing medium 199 (Life Technolo-
gies) with 20% FCS, 10 mM HEPES, 2.5 µg/mL fungizone (the
latter two from Life Technologies), 100 µg/mL heparin, and
100 µg/mL endothelial cell growth supplement (ECGS) (the
latter two from Sigma, St. Louis, MO). All cell types were
cultured at 37 °C with 100 IU/mL penicillin and 0.1 mg/mL
Hz, H-2), 3.39 (1H, dd, J 10-10′ ) 11.0 Hz, J 10-1 ) 3.5 Hz, H#-
10), 3.42 (1H, d, J 15′-15 ) 10.6 Hz, H-15′), 3.56 (1H, d, J 15-15′
)
10.6 Hz, H-15), 3.77 (1H, dd, J 10′-10 ) 11.0 Hz, J 10′-1 ) 3.5 Hz,
H#-10′), 5.42 (1H, dd, J 4-3R ) 1.6 Hz, J 4-3â ) 5.5 Hz, H-4)
(*,#interchangeable); 13C NMR (CDCl3, 50 MHz) δ 20.3 (q,
C-11), 21.5 (q, CH3COO-), 24.6 (q, C-12), 27.0 (d, C-2), 29.3
(q, C-14), 30.6 (q, C-13), 31.6 (t, C-3), 43.4 (d, C-1), 43.9 (s,
C-6), 50.4 (s, C-8), 51.1 (t, C-7), 64.1 (t, C-10), 64.4 (d, C-4),
69.6 (t, C-15), 125.0 (s, C*-9), 143.6 (s, C*-5) (*interchangeable);
EIMS m/z 265 [M - CH2OH]+ (15), 236 [M - AcOH]+ (5), 223
(100), 205 [M - CH2OH - AcOH]+ (74).
Com p ou n d 25. A solution of 22 (33 mg, 0.105 mmol) in
EtOAc (5 mL) was treated with oxalic acid for 48 h, as
described above for botrydial (1). The mixture was neutralized
and extracted with EtOAc. The crude product obtained after
evaporation of the solvent was purified by means of HPLC with
hexane/EtOAc (13:7) to give 254 (10.5 mg, 0.035 mmol, 34%)
as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.88 (3H, d,
J 11-2 ) 6.3 Hz, H-11), 1.05 (1H, q, J 3â-3R ) J 3â-2 ) J 3â-4
)
12.0 Hz, H-3â), 1.10 (3H, s, H-14), 1.23 (1H, d, J 7R-7â ) 12.1
Hz, H-7R), 1.28 (3H, s, H-12), 1.26 (3H, s, H-13), 1.52 (1H, ddd,
J 1-10â ) 6.8 Hz, J 1-10R ) 4.9 Hz, J 1-2 ) 11.9 Hz, H-1), 1.76
(1H, m, J 2-11 ) 6.3 Hz, J 2-3â ) 12.0 Hz, J 2-1 ) 11.9 Hz, H-2),
1.82 (1H, d, J 5-4 ) 12.0 Hz, H-5), 1.92 (1H, d, J 7â-7R ) 12.1
Hz, H-7â), 2.02 (3H, s, CH3COO-), 2.03 (1H, m, J 3R-3â ) 12.0
Hz, J 3R-4 ) 4.6 Hz, H-3R), 3.42 (1H, d, J 15R-15â ) 10.2 Hz,
H-15R), 3.67 (1H, dd, J 10R-10â ) 12.1 Hz, J 10R-1 ) 4.9 Hz,
H-10R), 3.90 (1H, d, J 15â-15R ) 10.2 Hz, H-15â), 3.96 (1H, dd,
J
10â-10R ) 12.1 Hz, J 10â-1 ) 6.8 Hz, H-10â), 5.06 (1H, dt, J 4-3â
) J 4-5 ) 12.0 Hz, J 4-3R ) 4.6 Hz, H-4); 13C NMR (CDCl3, 50
MHz) δ 20.2 (q, C-11), 21.5 (q, CH3COO-), 26.5 (q, C-13), 27.2
(q, C-14), 30.4 (d, C-2), 36.2 (q, C-12), 39.6 (t, C-3), 45.9 (s,