Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz. Avda. República Saharaui, s/n, 11510 Puerto Real (Cádiz), Spain. *firstname.lastname@example.org
Allelochemicals are an important potential source for new herbicides and agrochemicals since they offer new modes of action, more specific interactions with weeds, and potentially less environmental damage. Sesquiterpene lactones constitute a wide group of compounds with several biological activities, including allelopathic.
The natural occurring sesquiterpene lactones dehydrocostuslactone and cynaropicrin has been modified in three different ways: preparation of 11,13-oxetanelactones, addition of a second Michael acceptor and reduction of the α-methylene-γ-lactone. Their biological activities were tested in two different levels: wheat coleoptile bioassay and phytotoxicity using Lactuca sativa, Lolium rigidum, and Echinocloa crus-galli as target species. Parallel assays of pigment (chlorophyll A, chlorophyll B, and carotens) contents in treated hypocotyls were performed.
This study suggest that guaianolides may be good candidates for the development of new natural product-based herbicides. In this way, small modifications can induce conformational and reactivity changes that modulate or change the bioactivity.
Twenty three guaianolides have been prepared and bioassayed on Lactuca sativa, Lolium rigidum and Echinocloa crus-galli. Inhibitory activities for these compounds have been found.
Guaianolides, phytotoxicity, oxetane, Echinochloa crus-galli, Lactuca sativa, Lolium rigidum
Allelochemicals are important potential source for new herbicides and agrochemicals, since they offer new modes of action, more specific interactions with weeds, and potentially less environmental damage (Vyvyan 2002, Duke 1986a, Duke 1986b, Macías 1995, Duke et al. 1997, Rice 1995, Waller and Yamasaki 1996).
Sesquiterpene lactones constitute a wide group of compounds with several biological activities, including allelopathic (Picman and Picman 1984, Macias et al. 1993, Macías et al. 1996).They present a wide range of functional groups. Among them, five natural oxetane lactones, clementein, clementein B, clementein C, subexpinnatin B, and subexpinnatin C, were isolated from the Spanish endemic knapweed Centaurea clementei Boiss (Massanet et al. 1983, Collado et al. 1986) and the Canary Islands endemism Centaurea canariensis Brouss var. subexpinnata Burchd,(Collado et al. 1985). Some of them have been obtained later by hemi-synthesis methods (Macías et al. 1993). It is known that compounds containing oxetane ring have been reported to display a wide range of biological activities and this structural feature is regarded to be essential for their bioactivity (Wang et al. 2000, Hosono et al. 1994).
In this way, we have achieved the synthesis of four new oxetanelactones using the natural product dehydrocostuslactone as starting material. The semisynthesis also yielded other 6 derivatives, which present different chemical features and stereochemistry and have been obtained for first time in this study.
Dehydrocostus lactone (1) was obtained from crude costus resin oil (Saussurea lappa) by previous column chromatography (CC) separation and then purified by crystallization from hexane/ethyl acetate mixtures. Compounds 2-12 were obtained using previously reported methodology (Collado et al. 1987, Macías et al. 1990, Macías et al. 1992a, Macías et al. 1992b). Compounds 13, 15 and 23 were isolated from artichoke (Cynara scolymus) (Bernhard et al. 1979, Bernhard 1982) and 14, 16-21 were prepared from 13 using different synthetic procedures (Macías et al. 1999, Macías et al. 2000).
Coleoptiles were obtained from 3 days-old wheat seedlings sown on 15 cm diameter Petri dishes fitted with Whatman #1 filter paper and grown at 24°C in the dark. The etiolated seedlings were removed from the dishes and selected for size uniformity under a green safelight. The selected etiolated seedlings were placed in a Van der Wij guillotine, and the apical 2 mm were cut off and discarded. The next 4 mm portions were selected for bioassay and kept in an aqueous nutritive buffer for 1 h before being used to synchronize the growth.
Products were purified (+99%) by HPLC previous to the bioassay and tested at 1000 µM to 10 µM in a buffered nutritive aqueous solution (citric acid-sodium hydrogenphosphate buffer, pH 5.6; 2% sucrose). Mother solutions of pure compounds were prepared in DMSO and diluted to the proper concentration with the buffer to a 0.5% v/v DMSO final maximum concentration. Following dilutions were prepared maintaining the same buffer and DMSO concentrations. Bioassays were performed in 10 mL test tubes as follows: five coleoptiles were placed per tube containing 2 mL of test solution each; three replicates were prepared for each test solution and the experiments were run in duplicate. Test tubes were placed in a roller tube apparatus and rotated at 6 rpm for 24 h at 22°C in the dark. Increments of coleoptile elongation were measured by digitalization of their photographic images and data were statistically analyzed.
Phytotoxicity tests were performed in 24 well plates. Each treatment consisted of two replicates and two controls. A 10 mM stock solution of each compound was prepared in DMSO and diluted with water to a final concentration of 100 µM DHZ in 1% DMSO aq. solution. Control treatments received 1 % DMSO without the test compound. Seeds were grown on moistened Whatman #1 filter paper. Five seeds per plate well were used for lettuce (Lactuca sativa var. Romana), barnyardgrass (Echinochloa crus-galli) and rigid ryegrass (Lolium rigidum). Plates were incubated at 25 ± 2°C under fluorescent lights maintaining a 16 h photoperiod at 400 µmol m–2 s–1 PAR. Germination and growth rates, as well as chlorophyll and carotenoid contents were measured on 7-d-old plants for all species. Chlorophyll was extracted from cotyledons each treatment, two replicates each, in 3.5 ml dimethyl sulfoxide (Hiscox and Israelstam 1979), and concentrations determined spectrophotometrically according to Arnon (Arnon 1949).
The naturally occurring sesquiterpene lactones dehydrocostuslactone and cynaropicrin have been modified in three different ways:
- Preparation of 11,13-oxetanelactones
- Addition of a second Michael acceptor
- Reduction of the α-methylene-γ-lactone.
Together with the modified molecules, the natural compound grosshemin (23) was also included. So Twenty-three molecules with different stereochemistry (seven of them natural products: 1, 2, 13, 15, 16, 18 and 23) were tested. This includes compounds with α-methylene-α-lactone, two different Michael acceptors, γ-hydroxylactones, 1,4-dicarbonyl compounds and oxetanelactones (Figure 1).
The biological activity was tested in two different ways: wheat coleoptile bioassay and phytotoxicity using lettuce (L. sativa var. Romana), barnyardgrass (E. crus-galli) and rigid ryegrass (L. rigidum) as target species. Parallel assays of pigment (chlorophyll A, chlorophyll B and carotens) content in treated were performed.
The results of the coleoptile bioassay showed that those compounds with logP lower than 2 were almost inactive, being the most active ones those with alkylable moieties as oxetane ring (9, 10, 11, and 12) or α,β-unsaturated carbonyl groups (1, 13, 14, 15, 16, and 23).
The phytotoxicity observed was species dependent. Thus, L. sativa showed high resistance to this kind of compounds, and only oxetane lactones showed moderate activity. Oxetanelactones (9, 10, 11, and 12) derived from dehydrocostuslactone (1) showed high activity levels on L. rigidum with EC50 values about ten times lower than the commercial herbicide Logran. Compounds with no alkylating moieties showed different profiles of activity, affecting the pigment contents. This could indicate a different mode of action depending on the nature of the functional groups present in the molecule. The compounds that most affect on the growth of E. crus-galli were the oxetanelactones (9, 10, 11 and 12), cynaropicrin (14), and the 1,4-dicarbonyl derivative 3.
Figure 1. Guaianolides tested.
Guaianolides seems to be good candidates for the development of new natural product based herbicides. In this way, small modifications can induce high conformational and reactivity changes that modulate or change the bioactivity.
This research has been supported by the Ministerio de Educación y Ciencia, Spain (MEC; Project No. AGL2004-08357-C04-04).
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