Abstract

Media summary

Key Words

Introduction

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.

Methods

Compounds preparation

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).

Coleoptile bioassay

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 bioassay

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).

Result and Discussion

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.

Conclusion

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.

Acknowlegements

This research has been supported by the Ministerio de Educación y Ciencia, Spain (MEC; Project No. AGL2004-08357-C04-04).

References

Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Journal of Plant Physiology. 24, 1-15.

Bernhard HO (1982) Quantitative determination of bitter sesquiterpenes from Cynara scolymus L. (artichoke) and Cynara cardunculus L. (Kardone) (Compositae). Pharmaceutica Acta Helvetiae 57, 179-180

Bernhard HO, Thiele K and Pretsch E (1979) Cynaratriol, a new guajanolide from the thistle Cynara cardunculus L. and the artichoke C. scolymus L. (Compositae). Helvetica Chimica Acta 62, 1288-1297.

Collado IG, Macías FA, Massanet GM and Rodríguez-Luis F (1985) Guainolides from Centaurea canariensis. Phytochemistry, 24, 2107-2109.

Collado IG, Macías FA, Massanet GM and Rodríguez-Luis F (1986) Structure, chemistry and stereochemistry of clementeins, sesquiterpene lactones from Centaurea clementei. Tetrahedron, 42, 3611-22.

Collado IG, Macías FA, Massanet GM, Molinillo JMG and Rodriguez-Luis F (1987) Chemical Transformation of Deacylsubexpinnatin into the Natural Oxetane Lactone Subexpinnatin C. The Journal of Organic Chemistry. 52, 3323-3326.

Duke SO (1986a) In ‘The Science of Allelopathy’ (Ed. Putnam AR and Tang CS) pp. 287–304 (Wiley, New York)

Duke SO (1986b) Naturally occurring chemical compounds as herbicides. Reviews of Weed Science. 2, 15–44.

Duke SO, Dayan FE, Hernandez A, Duke MV and Abbas, H. K. (1997) Natural products as leads for new herbicide modes of action. Brighton Crop Protection Conference-Weeds, Vol. 2, 579–586.

Hiscox JD and Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany. 57, 1332-4.

Hosono F, Nishiyama S, Yamamura S, Izawa T and Kato K (1994) Synthesis and antiviral evaluation of [(2'S,3'S)-bis(hydroxymethyl)azetidin-1-yl]pyrimidine nucleosides: analogs of oxetanocin-A. Bioorganic & Medicinal Chemistry Letters. 4, 2083-6.

Macías FA (1995) In (Ed.Inderjit KMM Dakshini and Einhellig FA) ACS Symposium Series, 582 pp. 310–329 (ACS, Washington)

Macias FA, Galindo JCG, Castellano D and Velasco RF (2000) Sesquiterpene Lactones with Potential Use as Natural Herbicide Models. 2. Guaianolides. Journal of Agricultural and Food Chemistry. 48, 5288-5296.

Macias FA, Galindo JCG, Molinillo JMG, Castellano D, Velasco RF and Chinchilla D (1999) Developing new herbicide models from allelochemicals. Pesticide Science. 55, 633-675.

Macias FA, Molinillo JMG and Massanet GM (1992a) First Synthesis of Two Naturraly Occurring Oxetane Lactones: Clementein and Clementein B. Tetrahedron. 49, 2499-2508.

Macias FA, Molinillo JMG, Collado IG, Massanet GM and Rodriguez-Luis F (1990) An efficient and mild entry to 1,4-dicarbonyl compounds via photochemical addition of acyl radical to electron-deficient olefins. Tetrahedron Letters. 31, 3063-3066.

Macias FA, Molinillo JMG, Massanet GM and Rodriguez-Luis F (1992b) Study of Photochemical Addition of Acyl Radical to Electron-Deficient Olefins. Tetrahedron. 48, 3345-3352.

Macías FA, Torres A, Molinillo JMG, Varela RM and Castellano D (1996). Phytochemistry 43, 1205–1215.

Macías FA, Varela RM, Torres A and Molinillo JMG (1993) Potential allelopathic guaianolides from cultivar sunflower leaves, var. SH-222. Phytochemistry 34, 669–673.

Massanet GM, Collado IG, Macias FA, Bohlmann F and Jakupovic J (1983) Tetrahedron Letters 24, 1641-2.

Picman J and Picman AK (1984) Autotoxicity in Parthenium hysterophorus and its possible role in control of germination. Biochemical Systematic Ecology 12, 287–292.

Rice EL. (1995) Biological Control of Weeds and Plant Diseases (University of Oklahoma Press, Norman)

Vyvyan JR. (2002) Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron 58, 1631–1646.

Waller GR and Yamasaki K (1996) Advances in Experimental Medicine and Biology Vol. 405 (Plenum, New York)

Wang M, Cornett B, Nettles J, Liotta DC and Snyder JP (2000) The oxetane ring in taxol. Journal of Organic Chemistry. 65, 1059-1068.