1024
G. Appendino et al. / Phytochemistry 51 (1999) 1021±1026
tometer. HR-MS were taken on
a
MAT 95ST
6.67 (PABA-BB'), 4.82 (br s, H-3), 4.49 (br t, J=6 Hz,
H-7), 4.27 (d, J=11 Hz, H-29a), 3.96 (d, J=11 Hz, H-
29b), 1.20 (s, H-26), 1.15, 1.13, 1.01 (s, H-23+H-
25+H-28+H-30), 0.97 (s, H-27).
1
Finnigan MAT apparatus (70 eV, EI mode). H- and
13C NMR spectra were taken on Bruker AM 400 (400
and 100 MHz, respectively) and a Bruker DRX (500
and 125 MHz, respectively) instruments. H- and 13C-
1
NMR chemical shifts refer to CHCl3 at 7.26 ppm and
CDCl3 at 77.0 ppm, respectively. Silica gel 60 (70±230
mesh, Merck) was used for open-column chromatog-
raphy. A Waters Microporasil column (0.8 Â 30 cm)
was used for HPLC, with detection by a Waters dier-
ential refractometer 340.
3.4. 3-O-p-Aminobenzoyl-29-O-benzoylmulti¯ora-
7,9(11)-diene-3a,29-diol (2a)
Powder (Et2O), m.p. 204±2068C; [a]D25 130 (CHCl3,
EtOH
max
c 0.90); UV l
n
nm (log e): 290 (4.06), 240 (sh); IR
cm 1: 3476, 3380, 1707, 1630, 1603, 1370, 1314,
KBr
max
1277, 1171, 1117; CI-MS (isobutane) m/z (rel. int.):
664 [C44H57NO4+H]+ [M+H]+ (100). HR-EIMS:
663.4287 [M]+ (1.5) (calculated for C44H57NO4:
663.4288).
3.1. Plant material
Seeds of pumpkin, zucchini and cucumber were pur-
chased from Sementi Dotto, Mortigliano, UD, Italy.
The seeds of bualo gourd were obtained from fruits
collected by AM around Fenix (Arizona, USA) in
February 1998. Voucher specimens of all these seeds
are kept at the Istituto Sperimentale per la Nutrizione
delle Piante, Torino.
3.5. Acetylation of 1a
To a soln. of 1a (111 mg, 0.163 mmol) in pyridine
(1.5 ml), an excess Ac2O was added (1.5 ml). The reac-
tion was stirred at room temp. for 24 h and then
worked up by dilution with water and extraction with
EtOAc. After washing with dil. HCl, NaHCO3 and
brine, the organic phase was dried (Na2SO4) and evap-
orated. The residue was puri®ed by CC (ca. 5 ml silica
3.2. Isolation of the constituents
Isolation from the seeds of zucchini as representative
(Varieta nano verde di Milano): dried and powdered
seeds (500 g) were extracted with acetone at room
temp. (1 Â 2 l; 2 Â 1 l). Removal of the solvent left a
reddish oily residue (210 g) which was partitioned
between hexane (1.0 l) and acetonitrile (1.0 l). After 16
h the two phases were separated and the lower aceto-
nitrile phase was further washed with hexane (2 Â 100
ml). Evaporation of the acetonitrile phase left a red-
dish residue (4.7 g) which was separated by column
chromatography (60 ml silica gel, elution with mixtures
of hexane±EtOAc). Elution with hexane±EtOAc 9:1
gave 2a (65 mg after crystallisation from ether) as a
white powder; elution with hexane±EtOAc 8:2 gave
342 mg 1a as a green oil. Further puri®cation was
achieved by HPLC (microporasil column, hexane±
EtOAc 8:2 as eluant) to give 210 mg of a crystalline
powder. Attempts to remove the green colour from
crude 1a by C18 RP silica gel gave mainly the dehy-
drated product 2a.
gel, hexane±EtOAc 7:3 as eluant) to give 65 mg 1b as
liquid ®lm
a colourless powder, m.p. 161±1628C; IR n
max
cm 1: 3374, 1707, 1599, 1526, 1370, 1279, 1173, 1113,
713; CI-MS (isobutane) m/z (rel. int.): 447 765
[C48H63NO7+H]+ [M+H]+ (80).
3.6. Attempted silylation of 1a
To a soln. of 1a (70 mg, 0.103 mmol) in DMF (1.0
ml), imidazole (26 mg, 0.309 mmol, 3 mol. equiv.) and
triethylsilyl chloride (65 ml, 58 mg, 3 mol. equiv.) were
added. After stirring at room temp. overnight, the
reaction was worked up by slowly pouring into a
slurry of celite (ca. 1 g) in water (ca. 5 ml). The slurry
was then ®ltered, and the cake washed with water to
remove DMF and then with EtOAc to recover the
product. After washing with brine and drying, the ®l-
trate was evaporated and the residue puri®ed by CC
(ca. 5 g silica gel, hexane±EtOAc 7:3) to give 30 mg 2a
as a colourless powder.
3.3. 3-O-p-Aminobenzoyl-29-O-benzoylmulti¯ora-8-ene-
3a,7b,29-triol (1a)
3.7. Degradation of 1b in CDCl3
Powder (Et2O), m.p. 158±1608C; [a ]D25 52 (pyridine,
A sample of 1b (ca. 10 mg) was dissolved in CDCl3
1
EtOH
KBr
c 0.90); UV l
nm (log e): 289 (4.06); IR n
and its H NMR spectrum was taken at regular inter-
max
max
cm 1: 3465, 3376, 1701, 1688, 1636, 1603, 1516, 1367,
1277, 1169, 1109, 714; CI-MS (isobutane) m/z (rel.
int.): 682 [C44H59NO5+H]+ [M+H]+ (100); HR-
EIMS: 663.4290 [M-H2O]+ (2) (calculated for
vals. After 24 h, complete conversion to a ca. 10:1
mixture of 2b and 2c was observed. When the sample
was exposed to the air, formation of 2d was observed
from 2b. For the spectroscopic data of 2b and 2d, see
Table 1Table 2. Diagnostical 1H-NMR signals
(CDCl3) for 2c: d 4.84 (br s, H-3), 5.57 (br d, J=5 Hz,
1
C44H57NO4: 663.4288); H NMR (CDCl3): d 8.04 (Bz-
AA'), 7.79 (PABA-AA'), 7.61 (Bz-C), 7.53 (Bz-BB'),