Structure elucidation and phytotoxicity of C13 nor-isoprenoids from Cestrum parqui

Article abstract of DOI:10.1016/j.phytochem.2003.11.018

Twelve C₁₃ nor-isoprenoids have been isolated from the leaves of Cestrum parqui (Solanaceae). The structure (2R,6R,9R)-2,9-dihydroxy-4- megastigmen-3-one has been assigned to the new compound. All the structures have been determined by spectros

Full text of DOI:10.1016/j.phytochem.2003.11.018

Phytochemistry 65 (2004) 497–505
www.elsevier.com/locate/phytochem
Structure elucidation and phytotoxicity of C13 nor-isoprenoids from
Cestrum parqui
Brigida D’Abroscaa, Marina DellaGrecab, Antonio Fiorentinoa,*, Pietro Monacoa,
Palma Orianoa, Fabio Temussib
a
`
Dipartimento di Scienze della Vita, Seconda Universita di Napoli, via Vivaldi 43, I-81100 Caserta, Italy
`
Dipartimento di Chimica Organica e Biochimica, Universita Federico II, via Cynthia 4, I-80126 Napoli, Italy
b
Received 9 May 2003; received in revised form 3 July 2003
Abstract
Twelve C13 nor-isoprenoids have been isolated from the leaves of Cestrum parqui (Solanaceae). The structure (2R,6R,9R)-2,9-
dihydroxy-4-megastigmen-3-one has been assigned to the new compound. All the structures have been determined by spectroscopic
means and chemical correlations. The compounds showed phytotoxic effect on the germination and growth of Lactuca sativa L.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Cestrum parqui; Solanaceae; Spectroscopic analysis; Phytotoxicity; C13-Nor-isoprenoids
1. Introduction
chloride in a separator funnel. A combination of
various chromatographic separations of the organic
extract, afforded the C13 nor-isoprenoids 1–13 (Fig. 1).
The new compound 1 was identified as (2R,6R,9R)
2,9-dihydroxy-4-megastigmen-3-one. Its EI mass spec-
trum showed the molecular ion at m/z 226, which,
together with the elemental analysis was in good agree-
ment with the molecular formula of a bisnorsesqui-
terpene C13H22O3.
In a study of the potential allelopathic effects of
spontaneous plants present in Italy, we recently repor-
ted that some metabolites isolated from Sambucus nigra
inhibited the germination and growth of some mono-
and dicotiledones (D’Abrosca et al., 2001).
Continuing the phytochemical study of common
weeds from the Mediterranean area, we investigated
Cestrum parqui L’Herrit.1788 (Solanaceae).
1
In the H NMR spectrum (Table 1) were present two
This plant, commonly named green cestrum, is a per-
ennial shrub indigenous to South America, naturalised
and now widely distributed in the Mediterranean area as
one of the major weeds. It grows in dense masses, crowd-
ing out other species and it is noted for its extreme toxicity
to farm animals (McLennan and Kelly, 1984). Pearce et
al. (1992) revealed the presence of ent-kaurene glyco-
sides, named parquin and carboxyparquin, from the
alcoholic extract of C. parqui.
methyls at ꢀ 0.88 and 1.23 as singlets, two methyl
doublets at ꢀ 1.22 and 2.02, five aliphatic protons as two
multiplets ranging from 1.27 to 2.05 ppm, two methine
protons geminal to hydroxyls as a multiplet at ꢀ 3.78
and a singlet at ꢀ 4.18, and an olefinic proton as singlet
at ꢀ 5.90. The 13C NMR spectrum (Table 1) showed 13
carbon signals, identified, by a DEPT experiment, as
four methyls, two methylenes, three aliphatic methines,
two olefinic carbons, one of them tetrasubstituted, a
quaternary carbon and a carbonyl carbon. All the
carbons were correlated to the corresponding protons
on the basis of a HMQC experiment.
2. Results and discussion
The COSY experiment showed a correlation series
beginning with the carbinol methine at ꢀ 3.78, assigned
to H-9, which was coupled with the doublet methyl at
d 1.22 assigned to H-10, and with the H-8 methylene
signals at ꢀ 1.58. These latter protons were correlated to
the H-7 protons at ꢀ 1.55 and 1.27, which were coupled
with the methine at ꢀ 2.05 attributed at the H-6. The
Leaves of C. parqui were infused in aq. 10% MeOH
for 48 h. The aq. soln. was extracted with methylene
* Corresponding author. Tel.: +39-0823-274576; fax: +39-0823-
274571.
E-mail address: antonio.fiorentino@unina2.it (A. Fiorentino).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2003.11.018

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B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
1
signal at ꢀ 4.18, in the H NMR spectrum indicated the
presence of a further hydroxyl group in the molecule. It
was positioned at the C-2 on the basis of heterocorela-
tion observed in the HMBC experiment, that showed
the correlations of the H-2 proton with the carbons C-1,
C-3, C-11, C-12, C-4, and between the carbinol carbon
at ꢀ 76.0 with the protons H-4, H-11 and H-12.
Vilburnum dilatatum (Machida and Kikuchi, 1996).
The CD spectrum showed a positive Cotton effect,
Áꢁ239.5 nm +20.9, confirming an R configuration at C-6
(Harada and Nakanishi, 1982). The R configuration at
C-3 carbon was confirmed by Mosher’s method.
Compound 4, (6R,7E,9R)-9-hydroxy-4,7-megastig-
madien-3-one, was identified as the aglycone of a glu-
coside isolated from Rubus idaeus (Pabst et al., 1992).
The R configuration at the C-9 carbon was attributed
by Mosher’s method, and the R configuration at the C-6
carbon was confirmed by the CD spectrum, which
showed a positive Cotton effect, Áꢁ251.7 nm +193.6.
Compound 5 has been identified as (3R,6R,7E,9R)-
3,9-dihydroxy-4,7-megastigmadiene, already obtained
from the Greek tobacco (Behr et al., 1978). The con-
figurations at the C-3 and C-9 carbons have been
attributed by Mosher’s method. The positive Cotton
effect in the CD spectrum (Áꢁ231.9 nm +77.1) indicated
a R configuration for the C-6 as the correlated
compounds 3 and 4.
Compound 6 was identified as (3S,7E,9R)-3,9-di-
hydroxy-5,7-megastigmadiene. It was previously isolated
as glucoside from Bunias orientalis leaves (Dietz and
Winterhalter, 1996). The spectroscopic data were in
agreement with those of the compound described by
Perez et al. (1996), and the configurations at the C-3 and
C-9 carbons were established by Mosher’s method.
The absolute configurations at the carbinol carbons
have been established as follows: compound 1 was con-
verted into the diasteromeric MTPA diesters, and the
Mosher’s method (Dale and Mosher, 1973) was applied
for the configuration of the C-9 carbon. Comparison of
the chemical shifts of the signals due to protons H-8 and
H-10 in both the R and the S derivatives and the calcu-
lation of the corresponding differences, expressed as
ÁdRꢀS, were in agreement with a R configuration for C-
9. For the C-2 carbon a modified Mosher method was
utilised (Ohtani et al., 1991). The positive and the
negative ÁdRꢀS values for the H-4 and the H-11 and H-
12 protons were found, respectively, on the right and
the left sides of the MTPA plane (Fig. 2) indicating an R
configuration for C-2 carbon, too.
Compound 2 was identified as (6R,9R) 9-hydroxy-4-
megastigmen-3-one. Its spectral data were in agreement
with those isolated from Greek tobacco (Aasen et al.,
1974). The MTPA esters of 2 indicated an R configur-
ation at the C-9 carbon. The configuration R at the C-6
carbon was established by MnO2 oxidation to the
corresponding diketone, which had the same rotation as
(6R)-4-megastigmen-3,9-dione (Aasen et al., 1974).
Compound 3 showed NMR spectra identical with
those reported for (3R,6R,7E)-3-hydroxy-4,7-mega-
stigmadien-9-one, a C-13 nor-terpene isolated from
Table 1
NMR data for compound 1a
Position
ꢀC
DEPT
ꢀH
HMBC (C!H)
1
2
3
4
5
6
7
42.0
76.0
C
CH
C
—
2, 6, 11, 12
4, 6, 11, 12
2, 4
4.18 s
198.8
122.4
166.1
52.9
—
CH
C
5.90 s
2, 6, 13
4, 7, 13
—
2.05 m
1.55 m
CH
CH2
2, 4, 8, 11, 12
8, 9
24.7
1.27 m
1.58 m
3.78 m
8
38.8
67.6
23.8
23.8
21.2
24.3
CH2
CH
6, 7, 9, 10
7, 8, 10
9, 8
9
10
11
12
13
CH3
CH3
CH3
CH3
1.22 d (6.0)
0.88 s
1.23 s
2, 6
2, 6
2.02 d (1.2)
4, 6
a
Values were recorded at 500 MHz for 1H and 125 MHz for 13C in
CDCl3 with J values in Hz in parentheses.
Fig. 1. C13 nor-isoprenoids 1–13.
Fig. 2. MTPA plane of diester 1.

B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
499
Compound 7 was identified as (3S,5R,6S,7E)-5,6-
epoxy-3-hydroxy-7-megastigmen-9-one. The NMR data
were in good agreement with annuionone D, isolated
from sunflower leaves (Macıas et al., 1999). A comparison
of the optical rotation of this compound with the lit-
erature data (Broom et al., 1992) suggested
the enantiomeric structure for 7. According with the
assigned structure, oxidation of 5 with MnO2 gave 13
and the following treatment with MCPBA led to
compound 7 as the main product.
Also compound 8 was characterised by an oxirane
ring at the 5,6 carbons, but the spectral data indicated
the presence of a hydroxyl group at the C-9 position.
Like compound 6, it had already been isolated as 3-O-
glucoside from Pistia stratiotes (DellaGreca et al.,
1995).
Fig. 3. Effect of C13-norisoprenoids on germination (A), root length (B) and shoot lenght (C) of Lactuca sativa L. Value presented as percentage
differences from control and are not significantly different with P>0.05 for Student’s t-test. (a) P<0.01; (b) 0.01<P<0.05.

500
B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
ꢁ
Compound 9, was identified as the (3S,5R,6R,7E,9R)-
heated for 5 min at 110 C. Prep. TLC was performed
on Merck Kieselgel 60 F254 plates, with 0.5 or 1 mm film
thickness. Flash column chromatography (FCC) was
performed on Merck Kieselgel 60 (230–400 mesh) at
medium pressure. Column chromatography (CC) was
performed on Merck Kieselgel 60 (70–240 mesh) or on
Sephadex LH-201 (Pharmacia).
3,5,6,9-tetrahydroxy-7-megastigmene. Like the previous
compound, it was isolated as 3-O-glucosides from the
aquatic plant Pistia stratiotes (DellaGreca et al., 1996).
Compound 10 had spectral data identical to the 4-
oxo-b-ionol obtained from the biotransformation of b-
ionone by Cunninghamella blakesleeana (Hartman et al.,
1988).
Compounds 11 and 12 were identified as geometric
isomers at the Á6 double bond. They were obtained by
chemical synthesis (Ito et al., 1997) and now is the first
time that they have been reported as natural products.
The MTPA esters of both the nor-terpenes indicated an
S configuration for the C-9 carbon.
In order to evaluate their potential phytotoxicity, all
the compounds were tested on the seeds of Lactuca
sativa L. (Macias et al., 2000), with exception of 10
because of an insufficiency samples. The results are
reported in Fig. 3. Aq. solns. of nor-terpenes, ranging
between 10ꢀ4 and 10ꢀ7 M, were tested on germination,
root and shoot length of the lettuce. With the exception
of the C-13 nor-terpene 3, the compounds had no effect
on germination, but they had a moderate inhibitory
profile on root and shoot growth.
3.2. Plant material
Plants of Cestrum parqui were collected in Sant’Agata
de’ Goti, near Caserta (Italy), and identified by Dr
Assunta Esposito of the Second University of Naples. A
voucher specimen (CE125) has been deposited at the
Herbarium of the Dipartimento di Scienze della Vita of
Second University of Naples.
3.3. Extraction and isolation
Fresh leaves of C. parqui (30 kg) were frozen at
ꢀ80 ꢁC, powdered and extracted with MeOH–H2O (1:9)
ꢁ
for 48 h at 10 C. The aq. soln. obtained was extracted
in a separator funnel using methylene chloride. The
organic layer was dried with Na2SO4 and concentrated
under vacuum yielding 8 g of residual material.
3. Experimental
3.3.1. Organic extract fractionation
The CH2Cl2 extract was chromatographed on silica
gel, with CHCl3 and EtOAc solns, to give four fractions
A–D.
3.1. General experiment procedures
1
NMR spectra were recorded at 500 MHz for H and
125 MHz for 13C on a Varian 500 spectrometer Fourier
transform NMR, with 0.05 M solns. in CDCl3 or
Fraction A, eluted with CHCl3–EtOAc (19:1) was
rechromatographed by FCC on SiO2, to give two frac-
tions: the first was purified on HPLC using an RP-18
preparative column, and eluting with MeOH–MeCN–
H2O (2:1:2), to have 10 (2 mg); the second fraction was
purified on prep HPLC using an RP-18 preparative
column, and eluting with MeOH–MeCN–H2O (3:2:5),
to have 3 (5 mg).
Fraction B, eluted with CHCl3–EtOAc (9:1), was
rechromatographed on Sephadex LH-20 eluting with
hexane–CHCl3–MeOH (3:1:1), to produce two frac-
tions: the first was purified on HPLC using an RP-18
preparative column, and eluting with MeOH–MeCN–
H2O (2:1:2) to give pure 1 (15 mg) and 2 (21 mg); the
second fraction was chromatographed on C-18 reverse
phase silica with MeOH–MeCN–H2O (3:2:5) and then
purified on SiO2-HPLC, eluting with CHCl3–iso-PrOH
(49:1) to give pure isomers 5 (12 mg) and 6 (15 mg).
Fraction C, eluted with CHCl3–EtOAc (4:1) was
rechromatographed on C-18 reverse phase silica with
MeOH–MeCN–H2O soln. to have three fractions. The
first, obtained with MeOH–MeCN–H2O (1:1:3), was
chromatographed on prep. TLC eluting with toluene–
Me2CO (3:2) to give 7 (7 mg); the second fraction,
obtained with MeOH–MeCN–H2O (1:1:2), was purified
on SiO2-HPLC, eluting with CHCl3–Me2CO (9:1) to
ꢁ
CD3OD at 37 C. Proton-detected heteronuclear corre-
lations were measured using HMQC (optimised for
1JHC=145 Hz) and HMBC (optimised for 1JHC=7 Hz).
Optical rotations were measured on a Perkin-Elmer 343
polarimeter. CD spectra were obtained in CHCl3 solns.
on a Jasco J-715 Spectrophotometer Polarimeter. IR
spectra were determined in CHCl3 solns. on a FT-IR
Perkin-Elmer 1740 spectrometer. UV spectra were
obtained on
a Perkin-Elmer Lambda 7 spectro-
photometer in CHCl3 or EtOH solns. Electronic impact
mass spectra (EI-MS) were obtained with a HP 6890
spectrometer equipped with a MS 5973 N detector. The
HPLC apparatus consisted of a pump (Shimadzu LC-
10AD), a refractive index detector (Shimadzu RID-
10A) and a Shimadzu Chromatopac C-R6A recorder.
Preparative HPLC was performed using RP-8 (Luna 10
mm, 250ꢂ10 mm i.d., Phenomenex), RP-18 (Luna 10 mm,
250ꢂ10 mm i.d., Phenomenex) or SiO2 (Maxsil 10 silica,
10 mm, 250ꢂ10 mm i.d., Phenomenex), columns.
Analytical TLC was performed on Merk Kieselgel 60
F254 or RP-18 F254 plates with 0.2 mm layer thickness.
Spots were visualized by UV light or by spraying with
H2SO4–AcOH–H2O (1:20:4). The plates were then

B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
501
give pure 4 (12 mg) and 11 (5 mg) besides 2; the third
fraction, obtained with MeOH–MeCN–H2O (1:1:1),
was purified on HPLC, using an NH2 preparative
column and eluting with hexane–Me2CO (9:1) to give
pure 12 (6 mg) besides 2 and 4.
Fraction D, eluted with CHCl3–EtOAc (3:1), was
rechromatographed on SiO2 by FCC. The fraction
obtained by eluting with CHCl3–MeOH (19:1) was first
chromatographed on C-8 silica with MeOH–MeCN–
H2O (3:2:1), and then purified by prep. TLC with tolu-
ene–Me2CO (3:2) to give pure 8 (15 mg) and a mixture
that was purified by RP-8 HPLC eluting with MeOH–
MeCN–H2O (3:2:5) to give pure 9 (4 mg).
3.3.2. Compound characterization
(2R,6R,9R)-2,9-Dihydroxy-4-megastigmen-3-one (1).
Colourless oil; [ꢂ]2D5 +102.7ꢁ (CH2Cl2, c 0.56); UV
(CHCl3) lmax (log ꢁ): 245 (3.94); 1H NMR and 13C
NMR: see Table 1; EI-MS: m/z 226 [M]+, 208 [M–
H2O]+; elemental analysis: found: C, 69.01; H, 9.83.
C13H22O3 requires: C, 68.99; H, 9.80%.
(6R,9R)-9-Hydroxy-4-megastigmen-3-one (2). Col-
ourless oil; [ꢂ]D25 +83.6ꢁ (CH2Cl2, c 0.61); UV (CHCl3)
lmax (log ꢁ): 243 (3.48); 1H NMR: see Table 2; 13C
NMR: see Table 3; EI-MS: m/z 210 [M]+, 192
[MꢀH2O]+; elemental analysis: found: C, 74.41; H,
10.13. Calc. for C13H22O2: C, 74.24; H, 10.54%.
(3R,6R,7E)-3-Hydroxy-4,7-megastigmadien-9-one (3).
Colourless oil; [ꢂ]2D5 +37.1ꢁ (CH2Cl2, c 0.21); CD
(CHCl3) +20.9 (239.5 nm); UV (CHCl3) lmax (log ꢁ):
207 (3.95); 1H NMR: see Table 2; 13C NMR: see
Table 3; EI-MS: m/z 208 [M]+, 190 [MꢀH2O]+; ele-
mental analysis: found: C, 74.58; H, 9.73. Calc. for
C13H20O2: C, 74.96; H, 9.68%.
(6R,7E,9R)-9-Hydroxy-4,7-megastigmadien-3-one (4).
Colourless oil; [ꢂ]2D5 +292.0ꢁ (CH2Cl2, c 0.42); CD
(CHCl3) +193.6 (251.7 nm); UV (CHCl3) lmax (log ꢁ):
245 (4.06); 1H NMR: see Table 2; 13C NMR: see
Table 3; EI-MS: m/z 208 [M]+, 190 [MꢀH2O]+; ele-
mental analysis: found: C, 74.72; H, 9.65. C13H20O2
requires: C, 74.96; H, 9.68%.
(3R,6R,7E,9R)-3,9-Dihydroxy-4,7-megastigmadiene
(5). Colourless oil; [ꢂ]2D5 +25.9ꢁ (CH2Cl2, c 0.53); CD
(CHCl3) +77.1 (231.9 nm); UV (CHCl3) lmax (log ꢁ):
207 (3.93); 1H NMR: see Table 2; 13C NMR: see
Table 3; EI-MS: m/z 210 [M]+, 192 [MꢀH2O]+; ele-
mental analysis: found: C, 74.31; H, 10.68. Calc. for
C13H22O2: C, 74.24; H, 10.54%.
(3S,7E,9R)-3,9-Dihydroxy-5,7-megastigmadiene (6).
Colourless oil; [ꢂ]2D5 ꢀ97.9ꢁ (CH2Cl2, c 0.48); UV
1
(CHCl3) lmax (log ꢁ): 243 (3.63); H NMR: see Table 2;
13C NMR: see Table 3; EI-MS: m/z 210 [M]+, 192
[MꢀH2O]+; elemental analysis: found: C, 74.31; H,
10.68. C13H22O2 requires: C, 74.24; H, 10.54%.
(3S,5R,6S,7E)-5,6-Epoxy-3-hydroxy-7-megastigmen-9-
one (7). Colourless oil; [ꢂ]2D5 ꢀ43.7ꢁ (CH2Cl2, c 0.39);

502
B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
UV (CHCl3) lmax (log ꢁ): 243 (3.76); 1H NMR: see
Table 2; 13C NMR: see Table 3; EI-MS: m/z 224 [M]+,
206 [MꢀH2O]+; elemental analysis: found: C, 70.08; H,
9.04. Calc. for C13H20O3: C, 69.61; H, 8.99%.
perature, EtOAc (5 ml) and H2O (5 ml) were added to
the reaction mixture. The organic layer, separated by
centrifugation at 4000 rpm for 10 min, gave a crude
extract which was purified by prep. TLC eluting with
1
(3S,5R,6S,7E,9R)-5,6-Epoxy-3,9-dihydroxy-7-mega-
stigmene (8). Colourless oil; [ꢂ]2D5 ꢀ53.9ꢁ (CH2Cl2, c
hexane–EtOAc (4:1). The (S)-MTPA diester had the H
NMR spectral data (500 MHz, CDCl3): ꢀ 1.00 (3H, s,
H-11), 1.05 (3H, s, H-12), 1.39 (3H, d, J=6.0 Hz, H-10),
1.85 (3H, s, H-13), 2.00 (1H, m, H-6a), 3.59 (6H, s,
OMe), 5.12 (1H, m, H-9), 5.42 (1H, s, H-2a), 5.83 (1H,
s, H-4), 7.38 (3H, m), 7.44 (3H, m), 7.52 (2H, m), 7.66 (2H,
m). The (R)-MTPA diester was prepared, from (S)-(+)-
MTPA chloride, using the same procedure. 1H NMR (500
MHz, CDCl3): ꢀ 0.90 (3H, s, H-11), 0.94 (3H, s, H-12),
1.32 (3H, d, J=6.0 Hz, H-10), 1.94 (3H, d, J=1.0 Hz,
H-13), 1.97 (1H, m, H-6a), 3.51 (3H, s, OMe), 3.67 (3H,
s, OMe), 5.11 (1H, m, H-9), 5.56 (1H, s, H-2a), 5.90
(1H, s, H-4), 7.41 (6H, m), 7.51 (2H, m), 7.78 (2H, m).
1
0.47); UV (CHCl3) lmax (log ꢁ): 245 (3.95); H NMR:
see Table 2; 13C NMR: see Table 3; EI-MS: m/z 226
[M]+, 208 [MꢀH2O]+; elemental analysis: found: C,
68.84; H, 9.74. C13H22O3 requires: C, 68.99; H, 9.80%.
(3S,5R,6R,7E,9R) - 3,5,6,9 - Tetrahydroxy - 7 - mega-
stigmene (9). Colourless oil; [ꢂ]2D5 33.8ꢁ (MeOH, c 0.53);
UV (EtOH) lmax (log ꢁ): 207 (3.95); 1H NMR: see
Table 2; 13C NMR: see Table 3; EI-MS: m/z 244 [M]+,
226 [MꢀH2O]+; elemental analysis: found: C, 63.98; H,
9.87. C13H24O4 requires: C, 63.91; H, 9.90%.
(7E,9ꢃ)-9-Hydroxy-5,7-megastigmadien-4-one (10).
Colourless oil; [ꢂ]2D5 +101.1ꢁ (CH2Cl2, c 0.39); UV
1
(CHCl3) lmax (log ꢁ): 264 (4.03); H NMR: see Table 2;
3.3.4. Preparation of (S) and (R)-MTPA esters of 2
(R)-(ꢀ)-MTPA chloride (2.5 ml, 13 mmol) was added
to a soln. of pure 2 (1.5 mg, 7.1 mmol) in dry pyridine
(25 ml). After 6 h under magnetic stirring at room
temperature, EtOAc (5 ml) and H2O (5 ml) were added
to the reaction mixture. The organic layer, separated by
centrifugation at 4000 rpm for 10 min, gave a crude
extract which was purified by prep. TLC eluting with
13C NMR: see Table 3; EI-MS: m/z 208 [M]+, 190
[MꢀH2O]+; elemental analysis: found: C, 74.85; H,
9.71. Calc. for C13H20O2: C, 74.96; H, 9.68%.
(6E,9S)-9-Hydroxy-4,6-megastigmadien-3-one (11).
Colourless oil; [ꢂ]2D5 +4.7ꢁ (CH2Cl2, c 0.42); UV
1
(CHCl3) lmax (log ꢁ): 285 (4.68); H NMR: see Table 2;
13C NMR: see Table 3; EI-MS: m/z 208 [M]+, 190
[MꢀH2O]+; elemental analysis: found: C, 74.85; H,
9.87. Calc. for C13H20O2: C, 74.96; H, 9.68%.
1
hexane–EtOAc (4:1). The (S)-MTPA ester had the H
NMR spectral data (500 MHz, CDCl3): ꢀ 0.91 (3H, s,
H-12), 0.96 (3H, s, H-11), 1.35 (3H, d, J=6.5 Hz, H-10),
1.86 (3H, d, J=1.5 Hz, H-13), 1.95 (1H, d, J=17.5 Hz,
H-2a), 2.17 (1H, d, J=17.5 Hz, H-2b), 3.59 (3H, s,
OMe), 5.09 (1H, m, H-9), 5.76 (1H, s, H-4), 7.40 (3H,
m), 7.53 (2H, m). The (R)-MTPA ester was prepared
from (S)-(+)-MTPA chloride, using the same proce-
dure. 1H NMR (500 MHz, CDCl3): ꢀ 0.99 (3H, s, H-12),
1.00 (3H, s, H-11), 1.29 (3H, d, J=6.0 Hz, H-10), 1.93
(3H, s, H-13), 2.01 (1H, d, J=17.0 Hz, H-2a), 2.27 (1H,
d, J=17.0 Hz, H-2b), 3.50 (3H, s, OMe), 5.08 (1H, m,
H-9), 5.82 (1H, s, H-4), 7.41 (3H, m), 7.55 (2H, m).
(6Z,9S)-9-Hydroxy-4,6-megastigmadien-3-one (12).
Colourless oil; [ꢂ]2D5 +28.5ꢁ (CH2Cl2, c 0.37); UV
1
(CHCl3) lmax (log ꢁ): 287 (4.65); H NMR: see Table 2;
13C NMR: see Table 3; EI-MS: m/z 208 [M]+, 190
[MꢀH2O]+; elemental analysis: found: C, 74.59; H,
9.54. Calc. for C13H20O2: C, 74.96; H, 9.68%.
3.3.3. Preparation of (S) and (R)-MTPA diesters of 1
(R)-(ꢀ)-MTPA chloride (5 ml, 26 mmol) was added to
a soln. of pure 1 (1.5 mg, 6.6 mmol) in dry pyridine (50
mL). After 6 h under magnetic stirring at room tem-
Table 3
13C NMR data for compounds 2–12a
2
3
4
5
6
7
8
9
10
11
12
1
36.2
47.1
199.6
125.1
165.8
51.1
26.2
38.6
68.0
24.6
27.2
28.8
23.7
33.9
44.0
36.0
47.4
33.4
44.4
36.8
48.2
35.3
40.8
64.2
47.0
67.5
69.7
142.6
132.8
197.6
28.5
29.6
25.2
20.0
34.9
40.8
64.2
47.0
66.3
69.5
124.9
137.8
68.2
23.6
29.5
24.7
19.8
36.4
42.1
65.0
48.5
68.5
71.6
139.6
126.4
69.1
24.3
30.5
25.4
20.6
35.4
34.3
38.1
53.6
40.9
53.0
2
3
65.6
199.0
125.7
161.7
55.3
65.8
65.0
42.2
37.2
198.9
130.9
143.1
154.8
125.4
39.1
199.1
129.0
144.6
155.9
126.6
39.6
4
126.0
135.4
54.3
124.6
137.4
54.0
192.0
160.1
140.3
125.3
140.3
68.9
5
125.8
136.2
126.6
138.5
69.3
6
7
147.0
133.5
199.0
27.0
126.6
138.5
68.2
129.1
137.6
68.7
8
9
67.9
23.4
68.2
23.5
10
11
12
13
23.5
27.0
23.6
29.4
23.6
30.0
23.5
28.6
29.2
24.7
28.9
29.0
28.0
28.1
27.8
23.4
24.0
22.6
28.4
21.2
27.3
19.9
22.8
22.3
24.8
a
Values were recorded at 125 MHz in CDCl3.

B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
503
3.3.5. Oxidation of 2
1. The (S)-MTPA diester had the 1H NMR spectral
data (500 MHz, CDCl3): ꢀ 0.82 (3H, s, H-12), 0.88 (3H,
s, H-11), 1.42 (3H, d, J=6.0 Hz, H-10), 1.51 (1H, dd,
J=4.0 and 14.1 Hz, H-2a), 1.58 (3H, s, H-13), 1.84 (1H,
dd, J=6.0 and 14.1 Hz, H-2b), 2.25 (1H, d, J=8.0 Hz,
H-6a), 3.55 (3H, s, OMe), 3.56 (3H, s, OMe), 5.44 (1H,
dd, J=6.0 and 15.5 Hz, H-8), 5.47 (1H, dd, J=8.0 and
15.5 Hz, H-7), 5.49 (1H, m, H-3), 5.52 (1H, brs, H-4),
5.57 (1H, m, H-9), 7.40 (6H, m), 7.52 (4H, m). The (R)-
Active MnO2 (60 mg, 0.69 mmol) was added to a soln.
of 2 (6 mg, 28 mmol) in CHCl3 (0.8 ml). The reaction
mixture was kept under magnetic stirring at room
temperature. After 6 h, the reaction was filtered on SiO2
with CHCl3–Me2CO (19:1), and the residue was purified
on prep TLC eluting with CHCl3–Me2CO (19:1) to
1
obtain pure (6R)-4-megastigmen-3,9-dione (2 mg): H
NMR spectral data (500 MHz, CDCl3): ꢀ 1.02 (3H, s,
H-12), 1.07 (3H, s, H-11), 2.00 (3H, d, J =1.0, H-13),
2.05 (1H, d, J=17.5 Hz, H-2ax), 2.16 (3H, s, H-10), 2.37
(1H d, J=17.5 Hz, H-2eq), 5.83 (1H, s, H-4).
1
MTPA ester had the H NMR spectral data (500 MHz,
CDCl3): ꢀ 0.81 (3H, s, H-12), 0.83 (3H, s, H-11), 1.36
(3H, d, J=6.5 Hz, H-10), 1.44 (1H, dd, J=4.0 and 14.5
Hz, H-2a), 1.64 (3H, s, H-13), 1.82 (1H, dd, J=6.5 and
14.5 Hz, H-2b), 2.28 (1H, d, J=8.5 Hz, H-6a), 3.57 (6H,
s, OMe), 5.49 (1H, dd, J=6.5 and 15.0 Hz, H-8), 5.50
(1H, m, H-3), 5.52 (1H, dd, J=8.5 and 15.0 Hz, H-7),
5.55 (1H, m, H-9), 5.56 (1H, s, H-4), 7.40 (3H, m), 7.43
(3H, m), 7.52 (2H, m), 7.63 (3H, m).
3.3.6. Preparation of (S) and (R)-MTPA esters of 3
The (S) and (R)-MTPA esters of 3 were prepared
using the same procedure described for the compound
1
2. The (S)-MTPA ester had the H NMR spectral data
(500 MHz, CDCl3): ꢀ 0.92 (3H, s, H-12), 0.96 (3H, s, H-
11), 1.62 (1H, dd, J=4.0 and 14.3 Hz, H-2a), 1.65 (3H,
s, H-13), 1.94 (1H, dd, J=6.0 and 14.3 Hz, H-2b), 2.26
(3H, s, H-10), 2.46 (1H, d, J=10,0 Hz, H-6), 3.55 (3H,
s, OMe), 5.57 (1H, m, H-3), 5.60 (1H, brs, H-4), 6.08
(1H, d, J=15.8 Hz, H-8), 6.53 (1H, dd, J=10.0 and 15.8
Hz, H-7), 7.41 (3H, m), 7.54 (2H, m). The (R)-MTPA
ester had the 1H NMR spectral data (500 MHz,
CDCl3): ꢀ 0.88 (3H, s, H-12), 0.88 (3H, s, H-11), 1.49
(1H, dd, J=3.8 and 14.5 Hz, H-2a), 1.68 (3H, s, H-13),
1.87 (1H, dd, J=6.0 and 14.5 Hz, H-2b), 2.27 (3H, s, H-
10), 2.46 (1H, d, J=10,0 Hz, H-6), 3.56 (3H, s, OMe),
5.57 (1H, m, H-3), 5.65 (1H, brs, H-4), 6.08 (1H, d,
J=15.8 Hz, H-8), 6.52 (1H, dd, J=10.0 and 15.8 Hz, H-
7), 7.40 (3H, m), 7.53 (2H, m).
3.3.9. Preparation of (S) and (R)-MTPA diesters of 6
The (S) and (R)-MTPA diesters of 6 were prepared
using the same procedure described for the compound
1. The (S)-MTPA diester had the 1H NMR spectral
data (500 MHz, CDCl3): ꢀ 1.01 (3H, s, H-11), 1.10 (3H,
s, H-12), 1.47 (3H, d, J=6.1 Hz, H-10), 1.59 (1H, dd,
J=4.0 and 14.1 Hz, H-2ax), 1.82 (1H, dd, J=6.1 and
14.1 Hz, H-eq), 1.70 (3H, s, H-13), 2.23 (1H, dd, J=16.6,
10.2 Hz, H-4ax), 2.48 (1H, dd, J=16.6, 5.5 Hz, H-4eq),
5.31 (1H, m, H-3), 5.35 (1H, dd, J=6.0 and 15.5 Hz, H-
8), 3.55 (6H, s, OMe), 5.62 (1H, m, H-9), 6.10 (1H, dd,
J=8.0 and 15.5 Hz, H-7), 7.39 (6H, m), 7.52 (4H, m).
1
The (R)-MTPA diester had the H NMR spectral data
(500 MHz, CDCl3): ꢀ 1.04 (3H, s, H-11), 1.11 (3H, s, H-
12), 1.39 (3H, d, J=6.1 Hz, H-10), 1.66 (1H, m, H-2ax),
1.88 (1H, m, H-2eq), 1.67 (3H, s, H-13), 2.12 (1H, dd,
J=16.4, 9.9 Hz, H-4ax), 2.43 (1H, dd, J=16.4, 5.6 Hz,
H-4eq), 5.30 (1H, m, H-3), 5.39 (1H, dd, J=6.0 and 15.5
Hz, H-8), 3.55 (6H, s, OMe), 5.59 (1H, m, H-9), 6.15 (1H,
dd, J=8.0 and 15.5 Hz, H-7), 7.39 (6H, m), 7.50 (4H, m).
3.3.7. Preparation of (S) and (R)-MTPA esters of 4
The (S) and (R)-MTPA esters of 4 were prepared
using the same procedure described for the compound
1
2. The (S)-MTPA ester had the H NMR spectral data
(500 MHz, CDCl3): ꢀ 0.90 (3H, s, H-12), 1.00 (3H, s, H-
11), 1.43 (3H, d, J=6.0 Hz, H-10), 1.83 (3H, s, H-13),
2.05 (1H, d, J=16.5 Hz, H-2a), 2.23 (1H, d, J=16.5 Hz,
H-2b), 2.48 (1H, d, J=8.0 Hz, H-6a), 3.56 (3H, s,
OMe), 5.55 (1H, dd, J=6.0 and 15.5 Hz, H-8), 5.57 (1H,
m, H-9), 5.60 (1H, dd, J=8.0 and 15.5 Hz, H-7), 5.89
(1H, s, H-4), 7.39 (3H, m), 7.53 (2H, m). The (R)-MTPA
ester had the 1H NMR spectral data (500 MHz,
CDCl3): ꢀ 0.93 (3H, s, H-12), 1.02 (3H, s, H-11), 1.38
(3H, d, J=6.0 Hz, H-10), 1.86 (3H, s, H-13), 2.09 (1H,
d, J=17.0 Hz, H-2a), 2.29 (1H, d, J=17.0 Hz, H-2b),
2.53 (1H, d, J=9.0 Hz, H-6a), 3.52 (3H, s, OMe), 5.58
(1H, m, H-9), 5.65 (1H, dd, J=6.5 and 15.5 Hz, H-8),
5.72 (1H, dd, J=9.0 and 15.5 Hz, H-7), 5.91 (1H, s, H-
4), 7.40 (3H, m), 7.54 (2H, m).
3.3.10. Preparation of compound 7 from 6
Oxidation of 6. Active MnO2 (25 mg, 0.29 mmol) was
added to a soln. of 6 (5 mg, 24 mmol) in CHCl3 (0.5 ml).
The reaction mixture was kept under magnetic stirring
at room temperature. After 2 h, the reaction was filtered
on SiO2 with CHCl3–iso-PrOH (24:1) and the residue
was purified on prep TLC eluting with CHCl3–iso-
PrOH (24:1) to obtain pure (3R,7E)-3-hydroxy-5,7-
megastigmadien-9-one (2 mg). The oxidation product
13 had the 1H NMR spectral data (500 MHz, CDCl3): ꢀ
1.13 (3H, s, H-12), 1.14 (3H, s, H-11), 1.50 (1H, t,
J=12,2 Hz, H-2ax), 1.79 (3H, s, H-13), 1.80 (1H, m, H-
2eq), 2.10 (1H, dd, J=10.1 and 16.5 Hz, H-4ax), 2.32
(3H, s, H-10), 2.46 (1H, dd, J=8.0 and 16.5 Hz, H-4eq),
4.04 (1H, m H-3), 6.13 (1H, d, J=16.5 Hz, H-8), 7.22
(1H, d, J=16.5 Hz, H-7).
3.3.8. Preparation of (S) and (R)-MTPA diesters of 5
The (S) and (R)-MTPA esters of 5 were prepared
using the same procedure described for the compound

504
B. D’Abrosca et al. / Phytochemistry 65 (2004) 497–505
Epoxidation of 13. 3-Chloroperbenzoic acid (4.2 mg,
13), 2.19 (2H, s, H-2), 2.66 (2H, m, H-8), 3.52 (3H, s,
OMe), 5.28 (1H, m, H-9), 5.62 (1H, t, J=6.9 Hz, H-7),
5.92 (1H, s, H-4), 7.39 (6H, m), 7.52 (4H, m). The (R)-
MTPA diester had the 1H NMR spectral data (500
MHz, CDCl3): ꢀ 1.06 (3H, s, H-11), 1.09 (3H, s, H-12),
1.40 (3H, d, J=6.0 Hz, H-10), 2.16 (2H, s, H-2), 2.17
(3H, s, H-13), 2.62 (2H, m, H-8), 3.55 (3H, s, OMe),
5.26 (1H, m, H-9), 5.53 (1H, t, J=6.9 Hz, H-7), 5.90
(1H, s, H-4), 7.40 (6H, m), 7.52 (4H, m).
31.4 mmol) was added to a soln. of 13 (2 mg, 9.5 mmol)
in CHCl3 (0.5 ml). The reaction mixture was kept under
magnetic stirring at room temperature. After 4 h 5%
NaHCO3 was added and the mixture was extracted with
CH2Cl2. The organic layer was washed with 10%
Na2SO3, 5% Na2SO3 and, finally with H2O until neu-
trality. The residue was chromatographed on prep. TLC
[toluene–Me2CO (3:1)] to give compound 7, as the main
product along with a small amount of the isomeric
(3R,5S,6R,7E)-5,6-epoxy-3-hydroxy-7-megastigmen-9-one.
3.4. Bioassays
3.3.11. Preparation of (S) and (R)-MTPA esters of 8
The (S) and (R)-MTPA esters of 8 were prepared
using the same procedure described for the compound
1. The (S)-MTPA diester had the 1H NMR spectral
data (500 MHz, CDCl3): ꢀ 0.92 (3H, s, H-12), 0.95 (3H,
s, H-11), 1.14 (3H, s, H-13), 1.28 (1H, dd, J=9.0 and
13.5 Hz, H-2ax), 1.42 (1H, d, J=6.0 Hz, H-10), 1.68
(1H, dd, J=6.0 and 13.5 Hz, H-2eq), 1.86 (1H, dd, J=7.5
and 14.5 Hz, H-4ax), 2.46 (1H, dd, J=5.5 and 14.5 Hz, H-
4eq), 3.54 (3H, s, OMe), 3.56 (3H, s, OMe), 5.16 (1H, m,
H-3), 5.62 (1H, m, H-9), 5.65 (1H, dd, J=7.0 and 14.5
Hz, H-8), 5.89 (1H, d, J=14.5 Hz, H-7), 7.40 (6H, m),
Seeds of Lactuca sativa L. (cv Parella) collected dur-
ing 2001, were obtained from Blumen1 (Milan, Italy).
All undersized or damaged seeds were discarded and the
assay seeds were selected for uniformity.
Bioassays used Petri dishes (90 mm diameter) with
one sheet of Whatman Nꢁ 1 filter paper as support. In
four replicate experiments, germination and growth
were conducted in aq. solns. at controlled pH. Test
solns. (10ꢀ4 M) were prepared using MES (2-[N-mor-
pholino]ethanesulfonic acid, 10 mM, pH 6) and the rest
(10ꢀ5–10ꢀ7 M) were obtained by dilution. Parallel con-
trols were performed. After adding 25 seeds and 5 ml
test solns, Petri dishes were sealed with Parafilm1 to
ensure closed-system models. Seeds were placed in a
1
7.51 (4H, m). The (R)-MTPA ester had the H NMR
spectral data (500 MHz, CDCl3): ꢀ 0.95 (3H, s, H-12),
1.04 (3H, s, H-11), 1.10 (3H, s, H-13), 1.36 (1H, d, J=6.0
Hz, H-10), 1.40 (1H, dd, J=9.0 and 13.5 Hz, H-2ax), 1.74
(1H, dd, J=2.5 and 13.5 Hz, H-2eq), 1.77 (1H, dd, J=7.0
and 14.5 Hz, H-4ax), 2.40 (1H, dd, J=5.0 and 14.5 Hz, H-
4eq), 3.53 (6H, s, OMe), 5.18 (1H, m, H-3), 5.60 (1H, m,
H-9), 5.71 (1H, dd, J=7.0 and 15.5 Hz, H-8), 5.99 (1H,
d, J=15.5 Hz, H-7), 7.40 (6H, m), 7.51 (4H, m).
ꢁ
growth chamber KBW Binder 240 at 25 C in the dark.
Germination percentage was determined daily for five
days (no more germination occurred after this time).
ꢁ
After growth, plants were frozen at ꢀ20 C to avoid
subsequent growth until the measurement process.
Data are reported as percentage differences from
control in the graphics and tables. Thus, zero represents
the control; positive values represent the stimulation of
the parameter studied and negative values represent
inhibition.
3.3.12. Preparation of (R) and (S)-MTPA esters of 11
The (R) and (S)-MTPA esters of 11 were prepared
using the same procedure described for the compound
2. The (S)-MTPA diester had the 1H NMR spectral
data (500 MHz, CDCl3): ꢀ 1.25 (6H, s, H-11 and H-12),
1.36 (3H, d, J=6.0 Hz, H-10), 2.03 (3H, s, H-13), 2.25
(2H, s, H-2), 2.79 (2H, m, H-8), 3.55 (3H, s, OMe), 5.30
(1H, m, H-9), 5.94 (1H, t, J=6.9 Hz, H-7), 5.93 (1H, s,
H-4), 7.40 (6H, m), 7.52 (4H, m). The (R)-MTPA diester
3.5. Statistical treatment
The statistical significance of differences between
groups was determined by a Student’s t-test, calculating
mean values for every parameter (germination average,
shoot and root elongation) and their population var-
iance within a Petri dish. The level of significance was
set at P<0.05.
1
had the H NMR spectral data (500 MHz, CDCl3): ꢀ
1.25 (3H, s, H-11), 1.26 (3H, s, H-12), 1.44 (3H, d,
J=6.0 Hz, H-10), 2.23 (2H, s, H-2), 2.02 (3H, s, H-13),
2.75 (2H, m, H-8), 3.52 (3H, s, OMe), 5.32 (1H, m, H-9),
5.84 (1H, t, J=6.9 Hz, H-7), 5.89 (1H, s, H-4), 7.39 (6H,
m), 7.52 (4H, m).
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