Sesquiterpene lactones with potential use as natural herbicide models. 2. Guaianolides

Article abstract of DOI:10.1021/jf0005364

A structure-activity study to evaluate the effect of 17 guaianolide sesquiterpene lactones (in a range of 100-0.001 μM) on the growth and germination of several mono- and dicotyledon target species is accomplished. Results are compared with those obtained in the same bioassay with an internal standard, the commercial herbicide Logran, to validate the results with a known active formulation and to compare the results with a commercial product to test their potential use as natural herbicide models. Specific conditions for the selective mono- or polyhydroxylation of guaianolides using the SeO₂/tert-butyl hydroperoxide system are presented and discussed. The high regio- and stereo-selectivities of the reaction are explained through the specific structural requirements of the bulky first adduct formed during the ene reaction. These compounds appear to have deeper effects on the growth of either monocots or dicots than the previously tested germacranolides. Otherwise, the lactone group seems to be necessary for the activity, though it does not necessarily need to be unsaturated. However, the presence of a second and easily accessible unsaturated carbonyl system greatly enhances the inhibitory activity. Lipophilicity and the stereochemistry of the possible anchoring sites are also crucial factors for the activity. Finally, the levels of growth inhibition obtained with some compounds on dicots or monocots are totally comparable to those of Logran and allow proposing them as lead compounds.

Full text of DOI:10.1021/jf0005364

5288
J. Agric. Food Chem. 2000, 48, 5288−5296
Sesqu iter p en e La cton es w ith P oten tia l Use a s Na tu r a l Her bicid e
Mod els. 2. Gu a ia n olid es†
Francisco A. Mac´ıas,* J uan C. G. Galindo, Diego Castellano, and Rau´l F. Velasco
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias, Universidad de Ca´diz, c/ Repu´blica Saharaui
s/n, Apdo. 40, 11510-Puerto Real, Ca´diz, Spain
A structure-activity study to evaluate the effect of 17 guaianolide sesquiterpene lactones (in a range
of 100-0.001 µM) on the growth and germination of several mono- and dicotyledon target species
is accomplished. Results are compared with those obtained in the same bioassay with an internal
standard, the commercial herbicide Logran, to validate the results with a known active formulation
and to compare the results with a commercial product to test their potential use as natural herbicide
models. Specific conditions for the selective mono- or polyhydroxylation of guaianolides using the
SeO2/tert-butyl hydroperoxide system are presented and discussed. The high regio- and stereo-
selectivities of the reaction are explained through the specific structural requirements of the bulky
first adduct formed during the ene reaction. These compounds appear to have deeper effects on the
growth of either monocots or dicots than the previously tested germacranolides. Otherwise, the
lactone group seems to be necessary for the activity, though it does not necessarily need to be
unsaturated. However, the presence of a second and easily accessible unsaturated carbonyl system
greatly enhances the inhibitory activity. Lipophilicity and the stereochemistry of the possible
anchoring sites are also crucial factors for the activity. Finally, the levels of growth inhibition obtained
with some compounds on dicots or monocots are totally comparable to those of Logran and allow
proposing them as lead compounds.
Keyw or d s: Guaianolide; dehydrocostuslactone; isozaluzanin C; dehydrozaluzanin C; Logran; allylic
oxidation; phytotoxicity; allelopathy; herbicide; Lactuca sativa L.; Lycopersicon esculentum L.;
Lepidium sativum L.; Allium cepa L.; Triticum aestivum L.
INTRODUCTION
obtained from natural sources. Compounds have been
tested in our phytotoxic bioassay system (Mac´ıas et al.,
2000b) using monocot and dicot species as targets and
the commercial herbicide Logran as internal standard.
Water solubility is one of the main objections made
to the use of these compounds and their possible
mechanism of action. However, a certain degree of
lipophilicity is needed to cross the membranes, and
depending on their target site, amphipathic compounds
are desirable. Thus, a methodology to introduce in
consecutive steps different hydroxyl groups in selected
positions has been developed using the soft oxidant
selenium dioxide/tert-butylhydroperoxide or the hexa-
methylphosphoric triamide (HMPA)/Na2CO3 system,
and their effect on the phytotoxicity is discussed.
Since the mode of action of these compounds is yet
unknown, no attempts to establish any further com-
parison between the mode of action of Logran and the
SLs are made. The study has been focused on germina-
tion and root and shoot elongation effects. They have
been accepted as indirect measures of the effects caused
by the chemicals on their target sites.
Guaianolides and the closely structurally related
pseudoguaianolides are an especially bioactive group of
sesquiterpene lactones (SLs). They have been reported
to have several activities such as cytotoxic (Yuuya et
al., 1999), antifungal (Wedge et al., 2000), larvaecidal
(Neves et al., 1999), antiplasmodial (Fournet et al.,
1993), and phytotoxic (Galindo et al., 1999; Mac´ıas et
al., 2000a). SLs have been reported as allelopathic
agents in many plants of the family Compositae (Mac´ıas
et al., 1993; Pandey, 1994), being the guaianolide- and
pseudoguaianolide-like compounds most frequently
tested.
In continuation of our systematic study on SLs as
potential lead herbicides (Mac´ıas et al., 1999), herein
we present the results of a structure-activity relation-
ship (SAR) study accomplished with 17 SLs bearing the
guaianolide backbone. Most of them have been synthe-
sized from the readily available dehydrocostuslactone
(1) (compounds 2-18) (Mac´ıas et al., 1992, 2000b,c;
Wedge et al., 2000) (Figure 1), while 8R-hydroxyachillin
(19) and 8R-acetoxyachillin (20) (Figure 1) have been
MATERIALS AND METHODS
* To whom correspondence should be addressed. E-mail:
famacias@uca.es. Phone: +34-956-016370. Fax: +34-956-
016288, +34-956-016193.
Dehydrocostuslactone (1) was obtained from crude costus
resin oil (Saussurea lappa) by previous column chromatogra-
phy (CC) separation and then purified by crystallization from
hexane/ethyl acetate mixtures. 8R-Hydroxyachillin (19) and
8R-acetoxyachillin (20) were obtained from Artemisia lanata
Wild extract by CC. All the structures of the natural com-
pounds were confirmed by comparison of their spectroscopic
† Natural Products as Allelochemicals. 13. Part 12: Macı´as,
F. A.; Simonet, A. M.; Pacheco, P. C.; Barrero, A. F.; Cabrera,
E.; J ime´nez-Gonza´lez, D. Natural and synthetic podolactones
with potential use as natural herbicide models. J . Agric. Food
Chem. 2000, 48, 3003-3007.
10.1021/jf0005364 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/06/2000

Guaianolides as Natural Herbicide Models
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5289
[M]+ (1), 228 [M - H2O]+ (3), 218 [M - CO]+ (6); 1H NMR, see
Table 2; 13C NMR, see Table 3; HREIMS (M+) found 246.1250,
C15H18O3 requires 246.1256.
Compound 13. Colorless crystals; IR (neat, KBr) νmax 3450
(OH), 1746 (carbonyl group) cm-1; EIMS m/z (rel intens) 264
1
[M]+ (2), 246 [M - H2O]+ (5), 228 [M - 2H2O]+ (4); H NMR,
see Table 2; 13C NMR, see Table 3; HREIMS (M+) found
264.1370, C15H20O4 requires 264.1362.
Compound 14. Colorless crystals; IR (neat, KBr) νmax 3428
(OH), 1752 (carbonyl group) cm-1; EIMS m/z (rel intens) 280
1
[M]+ (1), 262 [M - H2O]+ (7), 244 [M - 2H2O]+ (4); H NMR,
see Table 2; 13C NMR, see Table 3; HREIMS (M+) found
280.1319, C15H20O5 requires 280.1311.
Dih yd r oxyla tion of SLs 1 a n d 11. Compound 1 (240 mg,
1.04 mmol, 1 equiv) was dissolved in CHCl3 (10 mL, 0.1 M)
and strongly stirred at room temperature. Then, SeO2 (27 mg,
0.24 mmol, 1 equiv) and TBHP (1 mL, 4 equiv) were added.
The reaction was stopped 2 h later by filtering through silica
gel and the solvent evaporated under vacuum. CC of the crude
product of the reaction yielded 2 (38 mg, 15%) and 5 (164 mg,
60%). Treatment of 11 (200 mg) under the same conditions
yielded 13 (38 mg, 18%), 14 (109 mg, 51%), and trace amounts
of 15.
Compound 15. Colorless crystals; IR (neat, KBr) νmax 4332
(OH), 1745 (carbonyl group) cm-1; EIMS m/z (rel intens) 264
1
[M]+ (3), 246 [M - H2O]+ (5), 228 [M - 2H2O]+ (8); H NMR,
see Table 2; 13C NMR, see Table 3; HREIMS (M+) found
264.1359, C15H20O4 requires 264.1362.
Hyd r oxyla tion of 5. Compound 5 (100 mg, 0.38 mmol, 1
equiv) was dissolved in CHCl3 (3.8 mL, 0.1 M) and strongly
stirred at room temperature. Then, SeO2 (34 mg, 0.31 mmol,
2 equiv) and TBHP (35 mL, 1 equiv) were added. After 12 h,
the reaction was stopped as usual, yielding 6 (30 mg, 28%) as
the unique product (Mac´ıas et al., 1992).
Hyd r oxyla tion of 1 u n d er Reflu x. Compound 1 (2.17
mmol, 1 equiv) was dissolved in CHCl3 (0.1 M solution) with
SeO2 (120 mg, 1.08 mmol, 1 equiv) and TBHP (2 mL, 1 equiv).
The reaction mixture was refluxed over 90 min and then
stopped as usual, yielding 5 (42%), 6 (16%), and 7 (10%).
Compound 7. Colorless crystals; IR (neat, KBr) νmax 3280
(OH), 1756 (carbonyl group) cm-1; EIMS m/z (rel intens) 278
[M]+ (1), 260 [M - H2O]+ (3), 242 [M - 2H2O]+ (5), 224 [M -
3H2O]+ (4); 1H NMR, see Table 2; 13C NMR, see Table 3;
HREIMS M+, found 278.1162, C15H18O5 requires 278.1154.
Hyd r oxyla tion of 1 w ith HMP A/Na 2CO3. Compound 1
(150 mg, 0.65 mmol, 1 equiv) was dissolved in HMPA (15 mL),
and then, an aqueous solution of Na2CO3 (20% (w/v)) was
added dropwise until the mixture remained unclear. The
reaction mixture was strongly stirred and heated at 90-95
°C over 7 days. Workup: The reaction mixture was extracted
with AcOEt (5×), and the combined organic phases were
washed consecutively with aqueous HCl (1 N) (3×), aqueous
Na2CO3 (20% (w/v)) (3×), and brine (3×). The organic phase
was dried over anhydrous sodium sulfate, the solvent evapo-
rated under vacuum, and the crude product of the reaction
purified by CC (hexane/ethyl acetate mixture), yielding 10 (72
mg, 30%), 11 (60 mg, 21%), and 12 (11 mg, 6%), with 45 mg
(30%) of starting material 1 remaining.
F igu r e 1. Guaianolides tested.
data (IR, EIMS, 1H and 13C NMR) with those in the literature.
Logran (terbutryn (59.4%) + triasulfuron (0.6%)) was supplied
by Novartis.
Gen er a l Exp er im en ta l P r oced u r es. 1H NMR and 13C
NMR spectra were recorded at 399.952 and 100.577 MHz,
respectively, on a Varian UNITY-400 spectrometer using
CDCl3 as solvent. The resonances of residual chloroform at
1
δH 7.25 ppm in the H spectra and of CDCl3 at δC 77.00 ppm
in the 13C spectra were used as internal references. Mass
spectra were obtained by using a VG 1250 or a Kratos MS-
80-RFA instrument at 70 eV. The infrared (IR) spectra were
recorded on a Bio-Rad FTS-7. Column chromatography was
performed on silica gel (35-75 mesh), and TLC analysis was
carried out using aluminum-packed precoated silica gel plates.
For HPLC, LiChrosorb silica 60 was used in the normal-phase
mode using differential refractometer (RI) and UV detectors,
with a Hitachi L-6020A HPLC instrument. All solvents were
spectral grade or distilled from glass prior to use.
Sod iu m Bor oh yd r yd e (Na BH4) Red u ction s. A 2 mL
methanolic solution with 15 mg each of compounds 5 and 6
was kept in a Dewar glass at 0 °C. While the solution was
stirring, NaBH4 (1.4 equiv) was added during the first 5 min
of reaction. After 1 h, the reaction was stopped by addition of
2 mL of distilled water. Extraction with AcOEt and HPLC
purification (hexane/ethyl acetate mixture) yielded dihydro
derivatives 17 and 18 (average 90%).
Mon oh yd r oxyla tion of SLs 1 a n d 11. Compound 1 (500
mg, 2.17 mmol, 1 equiv) was dissolved in CHCl3 (75 mL, 0.03
M solution) and strongly stirred at room temperature with
selenium dioxide (SeO2, 60 mg, 0.54 mmol, 0.5 equiv). Then,
tert-butyl hydroperoxide (TBHP; 2 mL, 2 equiv) was added
dropwise at a rate of 0.2 mL/min and allowed to react for 10
min. Filtering the reaction mixture through silica gel stopped
the reaction, and the solvent was then evaporated under
vacuum. The crude product of the reaction was purified by CC
using a hexane/ethyl acetate mixture as eluent, yielding 2 (294
mg, 55%), 4 (150 mg, 28%), 5 (74 mg, 13%), and trace amounts
of 3 (Mac´ıas et al., 1992). Treatment of 11 under the same
conditions yielded 13 (335 mg, 63%) and 14 (68 mg, 12%).
Compound 17. White powder; IR (neat, KBr) νmax 3220 (OH),
1756 (carbonyl group) cm-1; EIMS m/z (rel intens) 264 [M]+
1
(2), 248 [M - H2O]+ (5); H NMR, see Table 2; 13C NMR, see
Table 3; HREIMS (M+) found 264.1355, C15H20O4 requires
264.1362.
Compound 18. White powder; IR (neat, KBr) νmax 3430 (OH),
1756 (carbonyl group) cm-1; EIMS m/z (rel intens) 280 [M]+
(2), 262 [M - H2O]+ (5), 244 [M - 2H2O]+ (4), 226 [M - 3H2O]+
Compound 4. Colorless crystals; IR (neat, KBr) νmax 3401
(OH), 1754 (carbonyl group) cm-1; EIMS m/z (rel intens) 246

5290 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Mac´ıas et al.
Ta ble 1. Resu m e of th e Bioa ctivity Da ta of Com p ou n d s 1-9 a n d 11-20a
lettuce
R
cress
R
tomato
R
wheat
R
onion
G
S
G
S
G
S
G
S
G
R
S
1
2
3
4
5
6
7
8
9
11
12
13
14
16
17
18
19
20
L
-
0
0
+
+
(+)
(-)
+
-, +
)
(-)
(+)
)
0
)
)
0
-
0
0
0
0
0
)
0
0
)
0
0
)
0
)
0
0
0
0
), +
)
)
-
0
)
(+)
+
)
(-)
-
0
0
0
+
0
0
0
0
-
-
(+)
), ++
), ++
)
(+)
(-)
)
0
0
)
0
)
0
0
0
(-)
0
-
-
0
0
0
0
0
0
)
0
0
0
0
0
0
0
0
0
0
0
)
0
-
)
0
0
)
0
)
0
0
0
0
0
0
0
0
)
0
0
+
0
(-)
0
(+)
0
0
0
0
)
0
-, (+)
(+)
0
(+)
0
0
0
0
-
(+)
-
(+)
+
)
(-)
-
0
0
0
(+)
-
0
0
(-)
-
0
0
-, ++
-, ++
), ++
0
0
(-)
)
0
0
)
0
)
0
0
0
0
0
0
0
0
(-)
(+)
+
-
+
(+)
)
0
), ++
(+)
0
0, (+)
0
0
(+)
)
0
0
(+)
0
0
+
0
0
(+)
0
)
(+)
(+)
0
(-)
-
0
0
-
0
)
)
+
(-)
0
0
0
0
0
0
0
)
++
)
0
-
)
0
0
(+)
0
)
++
)
0
(+)
-
0
0
(+)
0
+
-
0
0
(+)
++
++
(+)
+
++
0
(-)
)
+
+
0
(+)
++
0
(-)
)
0
0
(-)
(-)
)
0
0
0
)
0
)
0
-
0
)
0
)
-
-
0
)
)
a
Key: 0, no activity; (+), (-), stimulatory or inhibitory values below 20%; +, -, stimulatory or inhibitory values between 20% and
40%; ++, ), stimulatory or inhibitory values higher than 40%. G ) germination, R ) root length, and S ) hypocotyl length. Values of
Logran activity presented here correct the previous ones in Mac´ıas et al. (1999).
Ta ble 2. 1H NMR Da ta for Com p ou n d s 4, 7, 13-15, 17, 18, a n d 21 (400 MHz in CDCl3, Sign a l of th e Resid u a l CHCl3
Cen ter ed a t δ 7.25 p p m )a
4
7
13
14
15
17
18
21
1
2R
2â
3R
3â
5
2.69 dd
1.82 dd
2.19 dd
2.57 dd
2.77 dd
3.25
2.08 m
2.08 m
-
4.63
-
3.97 d
3.48
2.25
1.53 ddd
3.01 m
2.15 ddd
1.83 ddd
-
4.63
3.06 m
3.88 dd
2.37
2.10 m
1.27 dddd
2.45 ddd
1.99 ddd
2.33 ddd
2.79 dd
2.10 m
2.10 m
2.70 m
1.82 dddd
2.20
2.54 ddd
2.70 m
-
4.23
2.69
1.40
2.17 m
2.17 m
2.70 m
2.42 m
2.81 dd
2.25 m
2.17 ddd
-
2.84 m
2.52 dd
2.03 dd
-
-
-
4.60 br dd
4.70 br s
4.57 dddd
-
-
4.05 d
-
4.09
-
2.76
3.72 dd
2.41
6
7
6.18 dd
3.26 ddddd
2.19 ddd
1.45 m
4.05 d
2.39 m
2.00
2.44 m
1.32 dddd
2.10 m
2.43 m
2.37 m
2.73
-
3.84
3.73
4.87
2.36 dddd
2.05 m
1.33 m
2.12
2.53 ddd
2.26 dq
8R
8â
9R
9â
11â
12
13a
13b
14
14′
15
15′
1.33 m
1.32
2.08
2.54
2.23
-
1.28
2.24 m
2.48 m
-
-
5.52 d
6.24 d
4.92 s
4.82 s
5.41 dd
5.10 dd
4.46 dd
-
-
5.51
6.15
5.02
4.76
-
-
-
1.25 d
5.71 br d
5.04
5.26
4.81 dd
4.72
5.20 m
5.00 m
3.96 dd
3.68 dd
4.86
3.80 dd
3.94 dd
4.81 br s
4.91 br s
5.34 s
4.93
4.72
5.61 d
5.43 d
4.91
4.75
5.59
5.40
4.68
5.35 dd
5.29 dd
4.75
5.53 d
5.34 d
5.61 d
5.40 d
5.08 s
a
Multiplicities are not repeated if identical with those of the preceding column. J (Hz): (4) 1,2R ) 2R,3R ) 2R,2â ) 5.1; 1,2â ) 7.9;
2R,3â ) 9.2; 2R,2â ) 13.2; 3R,3â ) 11.3; 6,7 ) 8.8; 7,13a ) 3.2; 7,13b ) 3.6; 7,8R ) 4.7; 8R,9R ) 2.2; 8R,9â ) 8.3; 8R,8â ) 10.6; 15,3R )
15,3â ) 2.4; 15′,3R ) 15′,3â ) 2.1; (7) 1,2R ) 4.3; 1,2â ) 8.5; 2R,2â ) 14.1; 2R,3â ) 2â,3â ) 7.6; 3â,15 ) 1.8; 3â,15′ ) 1.6; 6,7 ) 8.8; 7,8R
) 3.2; 8R,8â ) 10.1; 8R,9â ) 8â,9â ) 6.6; 7,13a = 3.1; 7,13b ) 3.4; (13) 1,2R ) 3.8; 1,2â ) 7.3; 1,5 ) 1.7; 2R,2â ) 13.2; 2R,3â ) 6.8; 2â,3â
) 6.9; 5,6 ) 6,7 ) 9.3; 8R,8â ) 12.6; 8R,9R ) 8â,9R ) 4.1; 8R,9â ) 12.3; 8â,9R ) 11.8; 7,8â ) 8â,9â ) 4.6; 9R,9â ) 12.9; 11,13a ) 3.3;
11,13b ) 4.4; 13a,13b ) 11.8; 15,1 ) 15,3â ) 1.9; 15′,1 ) 15′,3â ) 1.8; (14) 1,2R ) 3.6; 1,2â ) 7.2; 2R,3â ) 2â,3â ) 7.6; 3â,15 ) 2.2;
3â,15′) 1.8; 6,7 ) 9.7; 7,11 ) 8.8; 11,13a ) 11,13b ) 4.1; 13a,13b ) 11.6; (15) 1,2R ) 2R,3R ) 4.0; 2R,2â ) 13.2; 2R,3â ) 4.9; 6,7 ) 9.7;
7,8R ) 5.8; 8R,9R ) 11.3; 8R,8â ) 14.9; 9R,9â ) 16.3; 11,13a ) 6.1; 11,13b ) 4.3; 13a,13b ) 11.4; 15,2R ) 15,2â ) 2.3; 15′,2R ) 15′,2â )
2.4; (17) 1,2R ) 3.3; 1,2â ) 8.8; 2R,2â ) 15.4; 2â,3â ) 6.8; 6,7 ) 9.6; 7,8R ) 2.4; 7,8â ) 11.5; 7,11 ) 11.5; 9R,9â ) 11.0; 8R,9â ) 8â,9â )
5.6; 11,13 ) 3.5; 15,3â ) 1.8; 15′,3â ) 1.5; (18) 2R,2â ) 15.4; 2â,3â ) 6.8; 6,7 ) 9.6; 7,11 ) 11.2; 9R,9â ) 13.2; 8R,9â ) 8â,9â ) 6.4; 11,13
) 3.5; 15,3â ) 1.6; 15′,3â ) 1.4; (21) 5,6 ) 6,7 ) 9.2; 7,13a ) 2.7; 7,13b ) 3.1; 12,13a ) 1.8; 12,13b ) 1.3; 1,14′ ) 9,14′ ) 1.4.
(7); 1H NMR, see Table 2; 13C NMR, see Table 3; HREIMS
(M+) found 280.1308, C15H20O5 requires 280.1311.
Table 3; HREIMS (M+) found 232.1455, C15H20O2 requires
232.1463.
Diisobu tyla lu m in u m Hyd r id e (DIBALH) Red u ction . A
dry toluene solution of 1 (100 mg, 0.43 mol, 1 equiv) was stirred
with a DIBALH toluene solution (1.1 equiv) in a Dewar glass
(-70 °C, N2 atmosphere, 1.5 h). Workup: MeOH and distilled
water were added consecutively; the gelatinous precipitate was
homogenized and extracted with AcOEt. All organic phases
were dried over anhydrous Na2SO4, and the solvent was
evaporated under vacuum to yield 21 (62 mg, 61%).
Molecu la r Mod elin g. Minimum-energy conformations and
molecular properties were obtained using MMX and PM3
calculations (PCModel version 6.0, Serena Software, Bloom-
ington, IN; MOPAC version 6.00). Conformers were obtained
using the randomize command in PCModel, and the local
minimum energy structures obtained were used for further
semiempirical minimization with MOPAC using the PM3
method. For semiempirical calculations the parameters PRE-
CISE, GEOM-OK, and T ) 86400 were used. Theoretical ∆Hf
values produced with MOPAC allowed us to discriminate and
to obtain the minimum-energy conformer in each case.
Compound 21. Colorless oil; IR (neat, KBr) νmax 3220 (OH),
1756 (carbonyl group) cm-1; EIMS m/z (rel intens) 232 [M]+
(4), 214 [M - H2O]+ (18); 1H NMR, see Table 2; 13C NMR, see

Guaianolides as Natural Herbicide Models
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5291
Ta ble 3. 13C NMR Da ta for Com p ou n d s 4, 13-15, 17, 18,
a n d 21 (100 MHz in CDCl3, Sign a l of CDCl3 Cen ter ed a t δ
77.00 p p m )a
4
7
13
14
15
17
18
21
1
2
3
4
5
6
7
8
9
55.4 d 45.5
28.2 t 38.9
31.2 t 73.9 d 74.4
49.6
38.0
58.4
30.7
71.6
55.0
28.2
52.7
38.6
80.1 s 48.8 d
44.8
30.5
31.5 t 74.9 d 71.5
32.1 t
154.2 s 150.1 154.4 152.5 154.5 157.0 155.5 151.9
81.2 s 79.1
85.8 d 86.0
40.2 d 35.6
31.5 t 37.6
49.3 d 83.6 s 81.3
80.3 s 82.7
48.2 d
83.0
52.5
32.7
32.3
85.9
43.6
32.4
83.0
44.4
32.2
86.4
38.4
32.7
37.9
85.8
41.6
32.9
38.9
83.5
39.9
31.4
38.5
37.0 t 73.3 d 39.7 t 46.5
10 139.5 s 139.2 149.1 150.3 148.3 147.8 147.7 149.9
11 148.2 s 155.7 43.1 d 49.6 49.0 44.2 43.8 153.5
12 176.9 s 170.8 177.4 177.2 177.3 178.4 177.3 97.4 d
13 121.0 t 120.7 58.6 59.2 60.1
13.2 q 14.1 108.2b
t
14 110.7 t 113.2 t 112.4 116.4 113.2 113.5 115.7 110.9
15 113.7 t 115.0 112.4 114.7 110.3 114.0 117.9 108.1b
a
Multiplicities are not repeated if identical with those of the
b
preceding column. Values may be interchanged.
Ger m in a tion a n d Seed lin g Gr ow th Bioa ssa ys. Seeds
of Lactuca sativa L. cv. Roman, Lycopersicon esculentum L.
cv. Tres Cantos, Lepidium sativum L. cv. Comu´n, Allium cepa
L. cv. Valenciana, and Triticum aestivum L. cv. Cortex were
obtained from FITOÄ , S.L. (Barcelona, Spain). All undersized
or damaged seeds were discarded, and the assay seeds were
selected for uniformity. Bioassays were carried out in 9 cm L
plastic Petri dishes, using Whatman No. 1 filter paper as
support.
Bioa ssa y Meth od ology. The general procedure was as
follows: L. sativum, 25 seeds per dish, 5 mL of test solution,
4 days in the dark, 25 °C, and four replicates of each
concentration; L. sativa and L. esculentum, 25 seeds per dish,
5 mL of test solution, 5 days in the dark, 25 °C, and four
replicates of each concentration; A. cepa, 25 seeds per dish, 5
mL of test solution, 7 days in the dark, 25 °C, and four
replicates of each concentration; T. aestivum, 10 seeds per dish,
5 mL of test solution, 5 days in the dark, 25 °C, and 10
replicates of each concentration (Mac´ıas et al., 2000a).
F igu r e 2. (a, top) 0.03 M in CHCl3; 1/SeO2/TBHP (1:0.5:2);
room temperature; 30 min; (b, bottom) 0.1 M in CHCl3; 1/SeO2/
TBHP (1:1:4); 2 h.
Test solutions (10-3 or 10-4 M) were prepared using MES
(2-[N-morpholino]ethanesulfonic acid) (10 mM), and the rest
were obtained by dilution. Parallel controls were performed.
All pH values were adjusted to 6.0 before bioassay with MES.
All products were purified prior to the bioassay using HPLC
equipped with a refractive index detector. The minimum
degree of purity was 99% as extracted from the chromato-
grams.
Data are presented as percentage differences from control
in graphics (Figures 5 and 6). Thus, zero represents the control;
positive values represent stimulation of the studied parameter,
and negative values represent inhibition.
Sta tistica l Tr ea tm en t. Germination and root and shoot
length values were tested by Welch’s test (Zar, 1984), the
differences between test solutions and controls being signifi-
cant with P e 0.01.
F igu r e 3. Selected NOE effects on the minimum-energy
conformer of compound 7 obtained by using PM3 calculations.
RESULTS
Interestingly, while hydroxylation at room tempera-
ture of 5 led to the 1,3,5-trihydroxyl derivative 6 as a
unique product, treatment under reflux of 1 led to the
mixture of 5, 6, and the 1,3,9-trihydroxyl derivative 7.
The position and stereochemistry of the hydroxyl group
as C-9R was unambiguously established through 1H-
1H COSY and NOE experiments (Figure 3). The mini-
mum-energy conformation (MEC) that explained all the
NOE effects observed was fully coincident with that
obtained using the PM3 calculation. The trihydroxylated
compounds 6 and 7 were obtained in similar yields. No
trace of compound 7 could be detected when 5 was
treated at room temperature.
Selective mono- and dihydroxylations are obtained
depending upon the reaction conditions (Figure 2).
Compounds 2, 3, and 5 spectral data (MS, IR, H and
1
13C NMR) were fully consistent with those in the
literature. Moreover, X-ray analysis of compounds 2 and
5 confirmed the structures and the stereochemistry
proposed for the hydroxyl groups. For the newly re-
ported compound 4, the IR and the EIMS data con-
firmed the incorporation of a hydroxyl group, whereas
the position and stereochemistry of the new group was
1
established as C-5R through the analysis of its H NMR
spectrum and comparison with data obtained for 5.

5292 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Mac´ıas et al.
L. sa tivu m L. (F igu r e 5). Main inhibitory effects on
germination are observed for 2 (1 mM), 5 (1 mM and
100 µM), and 12 (10-100 µM). Moderate effects are
obtained for compounds 9 and 11, the rest remaining
inactive.
Important inhibitory effects on radicle and hypocotyl
growth with good dose-response curves are obtained
for compounds 2, 5, 11, 12, and 14, turning 2, 4, and 5
to stimulatory with the dilution. The acetonide deriva-
tive 9 acts as a strong growth promoter.
L. escu len tu m L. (F igu r e 5). Tomato is usually the
less sensitive dicot plant species, and this is well
correlated with the results obtained in germination,
where no strong inhibitory or promoting activities are
obtained.
Similar results are obtained in radicle and hypocotyl
growth, where only compounds 2, 5, and 11 (1 mM)
present important inhibitions on root and shoot elonga-
tion.
T. a estivu m L. (F igu r e 6). The effects observed are
less intense than with the dicots assayed. Germination
is poorly affected with main inhibitory effects for
compounds 2 (1 mM), 11 (0.1 and 1 mM), and 20 (0.1
and 1 mM). Compounds 2, 3, 5, 11, and 17 inhibit
radicle and hypocotyl growth, while compounds 7 and
16 enhance growth.
A. cepa L. (F igu r e 6). Onion is a more sensitive
species, and the growth is deeply inhibited by 2, 5, and
11 (1 mM) and compound 4 (0.1 mM). Germination is
not affected by any of the compounds.
F igu r e 4. Selected comparison of minimum-energy conform-
ers obtained by using PM3 calculations.
In view of these results, the order of reactivity seems
to be C-3 > C-5 > C-1 > C-9. This order does not seem
to change when the starting material is compound 11,
lacking the hydroxymethylene moiety in the lactone
ring.
Mono- and dihydroxyl derivatives at the C-11 and
C-13 positions were obtained by treatment with aqueous
Na2CO3/HMPA according to the previously published
methods (Mac´ıas et al., 2000c). New compounds 13-15
were obtained from compound 2 using the same meth-
odology as described above and their structures and
stereochemistry unequivocally established through com-
parison of their spectral data.
Bioassay results are presented in Figures 5 and 6.
Table 1 presents a resume of the main trends shown
by every compound tested on each target species. Data
corresponding to phytotoxic activities shown by com-
pound 8 have already been reported (Mac´ıas et al.,
2000b), but they have been introduced in Table 1 by
means of making suitable comparisons.
DISCUSSION
The proposed mechanism of the allylic oxidation with
SeO2/TBHP proceeds through an ene reaction of the
t-ButOO-SeO2H adduct and the double bond of the
lactone followed by a [2,3]-sigmatropic rearrangement
(Haruna and Ito, 1981). Among the three possible sites
of reaction, the exo-methylene group in the lactone ring
is electron-deficient and thus a low reactive position.
MMX calculations show that the adduct of the ene
reaction at the C-10-C-14 positions is of higher energy
than that resulting from the attack at C-4-C-15. This
would explain the preferred order of reactivity C-3-C-5
> C-1 > C-9. Parametrization of the selenium atom in
PCModel was not possible, and a silicon atom was
selected as a suitable and reasonable approach to
calculate and to compare the energy of the possible
conformers.
Moreover, among all possible conformers at C-14,
those with the selenium atom lying down or aligned
with the plane of the molecule and the tert-butyl
hydroperoxide moiety coming out from the molecule are
of lower energy. In these cases, the hydroxyl groups of
the selenium atom have to approach the double bond
from the R-face to give the [2,3]-sigmatropic rearrange-
ment (Figure 4). There is also a slight difference of
energy between the two adducts with the double bond
at C-3-C-4 and C-4-C-5 that favors the former. This
is also in agreement with the higher reactivity obtained
at the C-3 position. This situation is reproduced with
the two possible C-14 adducts that lead to the R-hy-
droxyl derivatives at C-1 and C-9, thus explaining the
high regioselectivity obtained in the reaction, where the
bulky tert-butyl hydroperoxide moiety is the driving
force.
L. sa tiva L. (F igu r e 5). Main inhibitory effects on
the germination can be observed for compounds 14 and
18, both of them lacking the exomethylene system in
the lactone ring, and compound 8. However, the effects
observed for the rest of the compounds are moderate or
active, even though some of them have been tested at 1
mM.
The most important effects are those observed on the
growth (radicle and hypocotyl). In most of the com-
pounds, there is a general trend to stimulate growth.
The results obtained are especially high in compounds
13, 14, and 18 in the range between 0.001 and 10 µM.
Only compound 11 shows an important inhibitory effect
on radicle growth, while 5 shows a good dose-response
curve for both parameters, although the values of
activity are moderate. All of these data are in good
accordance with previous results obtained for the root-
promoting activity of some guaianolides and some other
sesquiterpenes (Kalsi et al., 1981, 1983).
The preferred R-approximation and disposition of
the adduct in compounds 1, 2, and 5 can be explained

Guaianolides as Natural Herbicide Models
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5293
F igu r e 5. Germination and growth effects of selected compounds on lettuce, cress, and tomato. Values are expressed as percentage
differences from the control. L ) Logran.
through the chairlike conformation of the seven-
membered ring, which forces the C-4-C-15 double bond
to bend to the same face of the lactone ring. Thus, any
bulky group, such as the TBHP-SeO2 adduct, has to
approach from the less hindered R-face.
Bioa ssa y Discu ssion . From the results shown in
Table 1 and Figures 5 and 6, four general observations
can be made.
First, not all the compounds tested present activity,
even though many of them present an exo-methylene
moiety in the lactone ring, which has been usually
claimed to be responsible for the activity observed.
Moreover, compound 11, which is one of the most active,
does not present this group. The most active compounds
are 2, 5, and 11, followed by 4, 9, and 14.
belongs to monocotyledons or dicotyledons, or even on
the species within the same family. This was already
reported in an earlier paper for trans,trans-germacrano-
lides (Mac´ıas et al., 1999). Guaianolides, when active
on monocots, are growth inhibitors, but never show an
enhancing effect at low concentrations. Germination,
except in two cases, remains untouched. On the other
hand, active guaianolides are inhibitory to the germina-
tion of dicots, although their main effects remain on
growth. They are usually inhibitory at high concentra-
tions (0.1-1.0 mM) and turn to stimulatory at low
concentrations (10-0.01 µM).
Third, tomato is again the less sensitive plant
species, which is in good accordance with previous
results (Mac´ıas et al., 1999, 2000). However, intense
growth inhibition is obtained with some compounds
at 1 mM.
Second, the different mode of action shown by these
compounds depends on whether the target species

5294 J. Agric. Food Chem., Vol. 48, No. 11, 2000
Mac´ıas et al.
F igu r e 6. Germination and growth effects of selected compounds on wheat and onion. Values are expressed as percentage
differences from the control. L ) Logran.
Finally, comparison of the values and profiles of
activity shown by these lactones and those of the
herbicide Logran reveals that they are as effective
or more effective than the commercial product on di-
cotyledons, but in many cases with opposite profiles of
activities. They show similar values of activity on
monocotyledons.
Since a wide range of concentrations has been chosen,
a dose-response curve should be expected if no other
factors such as solubility are going to affect the bio-
activity. Previously (Mac´ıas et al., 1999) it has been
proposed that SLs probably do not act as phytohormones
and an allelopathic or/and antifeedant defensive role is
most reasonable (Picman, 1986; Castillo et al., 1998).
From this point of view, molecules can fall into one of
two classifications: (a) Inhibitors. If the range of
concentrations is correctly chosen, bioactivity usually
exhibits a dose-response format; compounds tend to act
as inhibitors at the highest concentrations, activity
decreasing with dilution. In many cases, the activity
may turn into stimulatory (phytohormone-like activity)
as the test solution is diluted. (b) The test compound is
not a powerful inhibitor, or the range of concentrations
chosen falls out of the “inhibitory” zone. Then, low or
no activity at the higher concentrations should be
observed (no inhibition), and enhancing activities are
obtained as the test solutions are more diluted. Once
the maximum of stimulatory activity has been reached,
the activity decreases again, tending to zero.
It is striking that the guaianolides tested may fill up
each of the two categories depending on whether the
target species is a monocot or a dicot: growth of the
monocot species is never stimulated, while most dicot
growth is enhanced at low concentrations. This could
be indicative of different mechanisms of binding to the
targets, or even of different targets.

Guaianolides as Natural Herbicide Models
J. Agric. Food Chem., Vol. 48, No. 11, 2000 5295
Ta ble 4. Selected Th eor etica l Molecu la r P r op er ties
Obta in ed by Usin g P M3 Ca lcu la tion s for Som e
Com p ou n d s Cor r ela ted w ith Th eir Biologica l Activitya
conformational effects on pseudorigid systems are of
much less importance than on flexible rings such as
germacranolides (Mac´ıas et al., 1992; Velasco, 2000).
preferred
conformation
dipole
moment, D
activity
(dicots)
activity
(monocots)
Moreover, the activities of some compounds without
the R-methylene-γ-lactone group (10-20) show very
good levels of activity on dicots and monocots for some
of them. Actually, compound 11 presents as good of
levels of growth inhibition as compounds 1, 2, 5, and 8;
compounds 12 and 14 also present good levels of growth
inhibition on dicots, while they are inactive on monocots.
However, when another hydroxyl group is added (com-
pounds 13 and 15), no inhibition is again observed.
Finally, the lactone-ring-lacking compound 16 presents
no activity on any of the target plants. With all of these
data in mind, it can be advanced that, even though the
lactone ring seems to be necessary for the activity, the
presence of a conjugated exocyclic double bond does not
necessarily mean growth inhibition. Moreover, when
active, modes of action can differ depending on whether
the lactone ring is saturated or unsaturated, or even
on the presence of other groups (e.g., hydroxyl groups)
in the lactone ring.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
17
chair
chair
chair
boat
boat
boat
chair
twist chair
boat
4.432
4.937
4.929
3.628
2.565
4.131
1.896
5.188
4.434
9.875
4.140
9.849
2.666
2.982
2.426
-, +
)
(-), +
-, +
-
)
)
(+)
)
-
boat
chair
chair
boat
boat
boat
-
-
(+)
-, +
a
Key: empty cells, activity below 20% or no activity; -, +,
stimulatory or inhibitory activities between 20% and 40%; ), ++,
stimulatory or inhibitory activities higher than 40%.
Activity may depend on the following factors: the
presence or not of certain functionalizations, preferred
conformation, water solubility, and lipophilicity, among
others.
It was previously reported that some 11,13-dihydro-
germacranolides without the double bond in the lactone
ring presented good levels of inhibitory activity (Mac´ıas
et al., 1999). Nevertheless, in this collection, compounds
17 and 18 present growth-enhancing activity, while
their unsaturated precursors (compounds 5 and 6) are
inhibitors or weak growth stimulators, respectively.
Regarding the influence of different functional groups,
the effect of one or two R,â-unsaturated carbonyl
systems (cyclopentenones 8, 19, and 20, compounds 1-7
and 9, or lactol 21), the presence of hydroxyl groups in
different positions, and the introduction of bulky groups
(9) are discussed. Compounds 1-9, 19, and 21 fill the
first requirement. However, not all of them show growth
inhibitory activity. It has been demonstrated that
unsatured γ-lactones and cyclopentanone carbonyl groups
react with sulfhydryl groups of important biomolecules
such as glutathione (GSH) (Galindo et al., 1999; Schmidt,
1997), the last one being up to 10 times more reactive
that the lactone group (Schmidt, 1997).
In a recent report, two different modes of action have
been proposed for 8: one which proceeds through the
cyclopentenone group and causes plasma membrane
leakage, and one which has been attributed to the
lactone ring (Galindo et al., 1999). The main difference
among cyclopentenones 8, 19, and 20 is that the first
one presents an exo-methylene group in the cyclopen-
tane ring, while the methyl group hinders the double
bond in compounds 19 and 20, which also lack the
exocyclic double bond at the lactone ring. Thus, a steric
factor in the cyclopentenone ring could be proposed to
explain the lack of activity of 19 and 20.
In view of the results presented here, other factors
should be considered to explain the lack of inhibition
shown by compounds 3, 4, 6, 7, and 9. All of them
present the R-methylene-γ-lactone ring, the reactivity
of this group to GSH in compound 2 also being demon-
strated (Galindo et al., 1999). There are two extreme
conformational possibilities for the double ring system
in these compounds: chairlike and boatlike conforma-
tions (Figure 4). Molecular mechanics calculations using
PM3 methods (MOPAC, 1990) lead to a chairlike
minimum-energy conformer for compounds 1-3 and 7,
a boatlike conformer for compounds 4-6 and 9, and a
twisted conformation for compound 8. The boat confor-
mation induces a certain difficulty of access to the
double bond by the upper (â) face. However, no correla-
tion between the preferred conformation and the activity
could be established (Table 4). As can be observed,
Which other factors could be involved to explain the
order of bioactivity? It has been proposed that a certain
degree of lipophilicity is necessary to cross the cell
membranes (Reynolds, 1987) or to concentrate inside
them and thus to exert a biological effect. On the other
hand, and depending on the threshold level of activity
in each compound, water solubility is also needed: too
polar compounds would not be allowed to cross the
membranes and extremely low water soluble compounds
would not be soluble enough. In this study, compound
1, which represents the simplest molecule of this col-
lection, presents a good degree of activity on cress and
onion growth. The inhibition is enhanced when an
R-hydroxyl group is introduced (2 and, in a lower
extension, 4). The introduction of a â-hydroxyl group
(3) results in no activity. The activity of the dihydrox-
ylated derivative 5 is similar to that of 2. However, a
third hydroxyl group dramatically results in no activity
(6 and 7). Then, two possible hypotheses can be ad-
vanced: (a) One is to postulate that a third hydroxyl
group introduces too much charge in the molecule, thus
making crossing the membranes more difficult. (b)
Hydroxyl groups need a specific stereochemistry to fit
the requirements for binding in the appropriate orienta-
tion, thus allowing a better approach of the active part
of the molecule to the reactive center of the target; the
best orientations are obtained with 3R- and 3R,5R-
dihydroxyl derivatives, while 1R- and 9R-hydroxyl de-
rivatives would place the molecule in the wrong orien-
tation. Obviously, either hypothesis is in need of further
substantiation.
Finally, some conclusions can be obtained from these
results in terms of inhibitory activity.
(a) The lactone group is necessary for the activity, but
it does not seem to be necessary for it to be unsaturated.
(b) The introduction of a second and easily accessible
R,â-unsaturated carbonyl system results in an enhanced
activity.

5296 J. Agric. Food Chem., Vol. 48, No. 11, 2000
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Mac´ıas, F. A.; Galindo, J . C. G.; Molinillo, J . M.; Castellano,
D. Dehydrozaluzanin C, a potent plant growth regulator
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(c) The introduction of an increasing number of
hydroxyl groups leads to the loss of activity when
polarity reaches a value that makes their transport
through the membranes difficult. Until this limit, the
activity is enhanced.
(d) The hydroxyl groups need a specific orientation
and placement in the molecule to provoke the appropri-
ate conformational changes that correctly orient the
active part toward the reactive center of the target.
(e) The levels of activity of some of these compounds
are similar to those of Logran on monocotyledons and
dicotyledons. Thus, compounds such as dehydrocostus-
lactone (1), isozaluzanin C (2), 5R-hydroxyisozaluzanin
C (5), dehydrozaluzanin C (8), and its 11â,13-dihydro-
13-hydroxydehydrocostus lactone (11) can be proposed
as lead compounds for the development of new models
of herbicides.
(f) Main stimulatory effects are observed for the
growth of the dicots cress and lettuce (in decreasing
order of activity) at low concentrations. Such effects are
especially notorious for compounds 13, 14, and 18
(lettuce) and 2, 4, 5, and 9 (cress).
(g) It is striking the lack of important growth-
promoting activities on monocots, even though signifi-
cant levels of growth inhibition are obtained. The
differences between dose-response curves in monocots
and dicots could be due to different mechanisms of
binding to the targets.
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Picman, A. K. Biological activities of sesquiterpene lactones.
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Schmidt, T. J . Helenanolide-type sesquiterpene lactones-III.
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ABBREVIATIONS
DCM, dichloromethane; GSH, glutathione; HOMO,
highest occupied molecular orbital; LUMO, lowest un-
occupied molecular orbital; SAR, structure-activity
relationship; TBHP, tert-butyl hydroperoxide.
ACKNOWLEDGMENT
We thank FITOÄ , S.A. and Novartis for providing seeds
and herbicides, respectively.
Velasco, R. F. Chemical transformations in germacranolides,
leads for natural herbicides. M.S. Dissertation, University
of Ca´diz, Spain, 2000.
Wedge, D.; Galindo, J . C. G.; Mac´ıas, F. A. Fungicidal activity
of several natural and synthetic sesquiterpene lactones.
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modulators: Synthesis and biological activities of dehydro-
costuslactone, mokko lactone, eremanthin, and their de-
rivatives. J . Nat. Prod. 1999, 62, 22-30.
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