isolation, synthesis and structure-activity relationships ...10021/fulltext01.pdf · biological...

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Akademisk avhStockholm framdoktorsexamen Åkroken, MittuBiological Chem

Isolation, Synthesis and ture-Activity Relationships of dants against the Pine Weevil,

Hylobius abietis

Carina Eriksson Doctoral Thesis Sundsvall 2006

andling som med tillstånd av Kungliga Tekniska Högskolan ilägges till offentlig granskning för avläggande av filosofie

i organisk kemi, fredagen den 28:e april 2006, kl 14:00 i sal O102niversitetet, Sundsvall. Fakultetsopponent: Professor John Pickett,istry Division, Rothamsted Research, Harpenden, England.

Cover picture: The pine weevil, Hylobius abietis L., (Coleoptera: Curculionidae). Photo: Rune Axelsson © Carina Eriksson Chemistry, Department of Natural Sciences, Mid Sweden University, SE-851 70 Sundsvall, SWEDEN. Organic Chemistry, Department of Chemistry, Royal Institute of Technology (KTH), SE-100 44 Stockholm, SWEDEN. ISBN 91-7178-301-6 ISRN KTH/IOK/FR--06/101--SE ISSN 1100-7974 TRITA-IOK Forskningsrapport 2006:101 Universitetsservice US AB, Stockholm

Abstract

Isolation, Synthesis and Structure-Activity Relationships of Antifeedants against the Pine Weevil, Hylobius abietis Carina Eriksson, Department of Natural Sciences, Mid Sweden University, SE-851 70 Sundsvall Sweden and KTH Chemistry, Organic Chemistry, SE-100 44 Stockholm, Sweden © Carina Eriksson, 2006, Doctoral Thesis, ISBN: 91-7178-301-6 The large pine weevil, Hylobius abietis L., is a major insect pest on conifer seedlings in northern Europe. Due to its feeding newly planted trees get girdled, resulting in high seedling mortality (up to 80%). As a consequence great financial losses to the forest industry occur. Today the seedlings are protected with the pyrethroid insecticide cypermethrin. This insecticide is toxic to aquatic organisms and is, from 2010, prohibited for use in Sweden by the Swedish Chemicals Inspectorate. An alternative to insecticides is to protect the seedlings with antifeedants, compounds that, either through taste or smell or both, deter the weevils from feeding. This thesis describes the search for and the synthesis of such antifeedant compounds.

Bark extracts of several woody species, known to be non-palatable to the weevil, were prepared and found to display antifeedant activity against H. abietis. The major chemical constituents of the extracts were tested for antifeedant activity. Antifeedants such as eugenol, 2-phenylethanol and benzylalcohol, but also feeding stimulants such as β-sitosterol and linoleic acid, were identified. An extract of linden bark, Tilia cordata, was shown to contain nonanoic acid, a highly active antifeedant. Other aliphatic carboxylic acids were also found to display high antifeedant activities against the weevil, both in laboratory and in field tests.

The enantiomers of dihydropinidine, a piperidine alkaloid present in several conifer species, were prepared by dimethylzinc mediated allylation of 2-methyltetrahydropyridine-N-oxide. When tested in micro feeding assays, no difference in antifeedant activity was found for the enantiomers. In a field test high antifeedant activity, comparable with that of the presently used insecticide cypermethrin, was found for (±)-dihydropindine. Other naturally occurring piperidine alkaloids were synthesised and also found to display high antifeedant activities in laboratory tests.

Structure-activity relationships were evaluated for methoxy substituted benzaldehydes, benzoic acids and cinnamic aldehydes, -acids, -esters and -alcohols. While the carboxylic acids were inactive or even feeding stimulants, the aldehydes were the most active antifeedants.

Keywords: Pine weevil, Hylobius abietis, semiochemicals, antifeedant, feeding deterrent, feeding stimulant, bark extract, carboxylic acid, piperidine alkaloids, structure-activity, cinnamic aldehyde, benzaldehyde.

III

Abbreviations

n-Bu n-butyl ∆X reflux Dibal-H diisobutylaluminium hydride dr diastereomeric ratio ee enantiomeric excess Et ethyl GC gas chromatography L. Linné LC liquid chromatography Me methyl MS mass spectrometry NOE Nuclear Overhauser Effect NOESY Nuclear Overhauser Enhancement Spectroscopy n-Pr n-propyl R generalised structure unit rt room temperature THF tetrahydrofuran TLC thin layer chromatography

IV

List of Publications

I. Antifeedants against Hylobius abietis pine weevils: An active compound in extract of bark of Tilia cordata linden Månsson, P. E.; Eriksson, C.; Sjödin, K. J. Chem. Ecol. 2005, 31, 989-1001

II. Nonanoic acid, other long-chain carboxylic acids and related compounds as antifeedants in Hylobius abietis pine weevils Månsson, P. E.; Schlyter, F.; Eriksson, C.; Sjödin, K. Entomol. Exp. Appl. submitted

III. Antifeedants and feeding stimulants in bark extracts of ten woody non-host species of the pine weevil, Hylobius abietis Eriksson, C.; Månsson, P. E.; Sjödin, K.; Schlyter, F. Manuscript

IV. Synthesis of (+)- and (−)-dihydropinidine by diastereoselective dimethylzinc promoted allylation of 2-methyltetrahydropyridine-N-oxide with an allylboronic ester Eriksson, C.; Sjödin, K.; Schlyter, F.; Högberg, H.-E. Tetrahedron: Asymmetry submitted

V. Benzaldehyde, cinnamic aldehyde and related compounds as antifeedants against the pine weevil, Hylobius abietis: A study of structure-activity relationships Eriksson, C.; Schlyter, F.; Sjödin, K.; Högberg, H.-E. Manuscript

Appendix: Synthesis of (±)-pinidine (cis-2-methyl-6-[(1E)-propenyl]piperidine) and (±)-“Z-pinidine” (cis-2-methyl-6-[(1Z)-propenyl]piperidine) from racemic Betti base [1-(aminophenylmethyl)-2-naphthol]. Eriksson, C.

Paper I was reprinted with kind permission from Springer Science and Buisness Media.

VI

Contribution report

The author’s contributions to the papers behind this thesis:

I: Experimental work, except for biological testing, shared writing of the article.

II: Performed experimental work, except for biological testing,

shared writing of the article.

III: Experimental work, except for the biological testing, shared writing of the article.

IV: Performed planning, experimental work and writing of the

article, supervised by Prof. H.-E. Högberg and Dr. K. Sjödin. V: Performed all experimental work except for the biological

testing. The multivariate data analysis was performed with help from Dr. K. Sjödin. Major contribution to the writing of the article.

Appendix: Performed planning and writing of the appendix. The experimental work was performed with some help from Rebecka From, undergraduate student supervised by the author.

VII

Contents

1. Introduction.....................................................................................................1 1.1 Semiochemicals ..........................................................................................1

1.1.1 Allelochemicals in insect pest control ..................................................2 1.1.2 Insect antifeedants................................................................................4

1.2 The pine weevil, Hylobius abietis ...............................................................5 1.3 Semiochemicals and stereochemistry..........................................................6 1.3 This thesis ...................................................................................................8

2. Biological test methods ...................................................................................9 2.1 Laboratory tests...........................................................................................9 2.2 Field tests ..................................................................................................10

3. Antifeedants against the pine weevil from non-host woody speciesI-III ..... 11 3.1 Identification of an antifeedant in bark from linden, Tilia cordata ...........12 3.2 Antifeedant activity of aliphatic carboxylic acids and related compounds13

3.2.1 Laboratory tests..................................................................................14 3.2.2 Field tests ...........................................................................................17

3.3 Antifeedant activity of chemical constituents in ten non-host woody species .............................................................................................................18

3.3.1 Bark extraction and antifeedant activity of prepared extracts ............18 3.3.2 Chemical content of non-host bark extracts and antifeedant activity of identified compounds..................................................................................19 3.3.3 Fractionation of horse chestnut bark extract ......................................24

4. Synthesis and antifeedant activity of piperidine alkaloidsIV, Appendix ..........27 4.1 Synthesis of dihydropinidine.....................................................................28

4.1.1 Dimethylzinc mediated allylation of 2-methyltetrahydropyridine-N-oxide............................................................................................................28 4.1.2 Synthesis of (±)-dihydropinidine for field tests..................................30

4.2 Synthesis of pinidine.................................................................................31 4.3 Synthesis of epidihydropinidine................................................................33 4.4 Antifeedant activity of piperidine alkaloids in laboratory- and field tests.34

5. Structure-activity relationships of cinnamic aldehyde, benzaldehyde and related compounds as antifeedants against the pine weevilV.........................37

5.1 Synthesis of α-alkylcinnamic aldehydes ...................................................38 5.2 Test compounds.........................................................................................40 5.3 Multivariate data analysis and structure-activity relationships .................40

6. Conclusions and future work .......................................................................46 Acknowledgements............................................................................................47 References ..........................................................................................................48

IX

1. Introduction Chemical ecology is a relatively young interdisciplinary science. It aims to investigate and explain the role that naturally produced chemicals have for the relationships and interactions within and between species in the ecosystem. The isolation and identification of such biologically active natural products and their preparation by synthesis for structure verification and biological studies are essential parts of chemical ecology. The compounds are often required in enantiomerically pure forms and as a consequence organic chemistry is one of the key disciplines in this area. A close cooperation between biologists and chemists is necessary for successful progress in this field. This is particularly important when it comes to the development of insect pest management techniques that utilise biological methods including the use of synthetically prepared naturally occuring compounds. 1.1 Semiochemicals

The compounds used by organisms for communication within and between species are called semiochemicals (Nordlund, 1981). Semiochemicals can be divided into two groups, pheromones and allelochemicals (Figure 1.1). Pheromones are compounds, emitted by an organism to the environment, which elicit a reaction in another organism of the same species (Nordlund, 1981). Pheromones are divided further into subgroups such as alarm pheromones, sex pheromones and aggregation pheromones.

Semiochemicals

PheromonesIntraspecificinteractions

AllelochemicalsInterspecific interactions

AllomonesEmitter

favourable

SynomonesEmitter and receiver

favourable

KairomonesReceiver

favourable

ApneumonesEmitted by

nonliving material

Figure 1.1. The classification system of semiochemicals.

Allelochemicals are defined as compounds that are responsible for chemical mediated interactions between individuals of different species. The allelochemicals are divided into four subgroups: allomones, kairomones, synomones and apneumones. Allomones are compounds that benefit the emitter but not the receiver. A well-known example of allelopathy is that of the walnut (Juglans spp.) which produces a naphthalene glucoside in the leaves and roots.

1

The compound is hydrolyzed by microorganisms in the soil and oxidised to give juglone (1, Scheme 1.1), a compound that prevents germination of many other species in the vicinity of the tree (Kaufman et al., 1999).

O

HOOH

OHO

OHHO

HO Enzymatic hydrolysis

H2OOH

OHOH

Nonenzymatic oxidation

O

OOH

Hydrojuglone-β-D-glucopyranoside

1

Scheme 1.1. Hydrolysis of hydrojuglone-β-D-glucopyranoside to juglone (1) (Duroux et al., 1998).

Kairomones are beneficial to the receiver; an example is Anopheles gambiae sensu stricto, a malaria mosquito that uses human volatiles such as lactic acid, ammonia and several carboxylic acids for host orientation (Smallegange et al., 2005). Synomones induce a response in the receiver that is favourable to both the emitter and receiver; floral scents that attract pollinators of various types can be regarded as synomones (Nordlund, 1981). Apneumones are allelochemicals emitted from non-living material.

1.1.1 Allelochemicals in insect pest control Among the approximately one million insect species in the world, 10 000 have been regarded as harmful, causing up to 14% of all crop losses (Dev and Koul, 1997). Therefore, efficient control methods for insect pests are necessary in modern production of food and fibre. For a naturally occurring compound to be successfully exploited for this purpose, several aspects have to be considered.

The compound or its metabolites should not be toxic to humans or other organisms and it must be readily available at a low cost, either by extraction from natural material or by synthesis. A highly active compound that can be used in low concentrations is preferable. Under the varying and often demanding conditions met in field, the compound, in a suitable formulation, must be easily applicable, stable and long-lasting.

There are over 2000 plant species possessing insecticidal activity (Dev and Koul, 1997). The most important example of a commercial natural insecticide used in crop protection is the pyrethrum extract derived from the daisy-like

2

flower Tanacetum or Chrysanthemum cinerariifolium (Casida, 1980). The extract consists of a mixture six esters (Figure 1.2) derived from either (+)-trans-chysanthemic acid (2, serie I) or (+)-trans-pyrethric acid (3, serie II) (Dev and Koul, 1997).

H

HO

O

O

R

H

HOH

O

MeOOC

H

HOH

O

2 3

MeOOC

H

HO

O

O

R

Serie I Pyrethrin I R= CH=CH2Cinerin I R= MeJasmolin I R= C2H5

Serie IIPyrethrin II R= CH=CH2Cinerin II R= MeJasmolin II R= C2H5

Figure 1.2. Pyrethrins from Tanacetum or Chrysanthemum cineariifolium.

The structures of these so called pyrethrins have served as models or “leads” for extensive structure-activity studies during the development of synthetic pyrethroids with high insecticidal activity and prolonged persistence (Matsuo and Miyamoto, 1997) such as permethrin (4) and cypermethrin (5) (Figure 1.3).

OO

O

Cl

Cl OO

O

Cl

ClCN

4 5

Figure 1.3. Synthetic pyrethroids developed from the pyrethrins.

3

Another example of a widely used natural product is the triterpenoid, azadirachtin (6, Figure 1.4), which is a constituent in the seeds of the Indian neem tree, Azadirachta indica (Isman et al. 1997). This compound acts as an insect growth regulator and may also serve as a feeding deterrent for some insects. It is used to control, among other insects: aphids, beetles, weevils, caterpillars and moths. Its complex structure prevents large scale synthesis but despite this, the development of sophisticated extraction methods from its natural source makes azadirachtin viable for use in pest insect control (Immaraju, 1998).

OO

OH

H

O

H

O

HO OH

OMeO

O

O

OH

OAcO

OMe

H

H

6

Figure 1.4. The complex structure of azadirachtin.

1.1.2 Insect antifeedants The definition of the term insect antifeedant varies widely (Månsson, 2005). Some authors prefer a restrictive definition where volatile compounds are excluded while others include toxic compounds with postingestive effects. Using a broad definition, insect antifeedants are compounds that temporarily or permanently reduce or prevent insects from feeding. When the antifeedant is a naturally occurring compound it can be regarded as an allelochemical belonging to the subgroup allomones. Active synthetic compounds, not yet found in nature, can also be regarded as antifeedants.

Antifeedants differ from insecticides by their indirect, rather than direct, action. Thus an antifeedant should not kill the insect. However, death can be caused by starvation due to inhibited feeding (Munakata, 1975). Consequently the feeding inhibition can be a result of the compounds acting either on gustatory or olfactory receptors in the insect. Moreover, antifeedant compounds may vary in volatility. A repellent is a volatile compound that prevents feeding by repelling the insect prior to contact with the food source. Thus, a repellent can be active at a longer distance from the food source. A compound that prevents or reduces feeding after the insect has already tasted the plant material is termed a deterrent (Norris, 1999). Therefore deterrents are often non volatile compounds.

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1.2 The pine weevil, Hylobius abietis

The large pine weevil, Hylobius abietis L. (Coleoptera: Curculionidae), is a major insect pest on conifers, such as Pinus sylvestris L. (Scots pine) and Picea abies (L.) H. Karst (Norway spruce) in northern Europe (Långström and Day, 2004). The weevils are attracted to host monoterpenes such as α-pinene (Nordlander et al. 1986), 3-carene and α-terpineol (Selander, 1976). Ethanol, which is released from injured or dying conifers, also attracts the weevils (Lindelöw et al., 1993). Ethanol has a synergistic effect on the attraction of both larvae and adult weevils to α-pinene, host-turpentine and host material (Tilles et al., 1986a; Lindelöw et al., 1993; Nordenhem and Nordlander, 1994). The attractiveness of ethanol varies depending on the stage of the weevils in their life cycle and on the time of the season (Nordenhem and Eidmann, 1991). Ethanol in combination with α-pinene signals suitable breeding sites. Thus, reproductive weevils are significantly attracted whereas pre-reproductive weevils are not. Limonene, however, which is also a component in Scots pine and Norway spruce, has been found to inhibit the attraction to α-pinene and reduces the attraction to the combination of α-pinene and ethanol as well as to host material (Nordlander, 1990, 1991). Verbenone is a feeding deterrent, probably because this compound is a signal of food deterioration to the weevils (Lindgren et al., 1996).

Host volatiles released from the damaged wood on clear-cuttings attracts flying weevils which immigrate to the area (Solbreck and Gyldberg, 1979, Solbreck, 1980). This results in high population densities at the sites of clear-cutting. Incoming weevils will feed mainly in the crowns of the trees surrounding the clear cut (Örlander et al., 2000). However, this feeding causes little or no harm to fully grown trees. By olfactory orientation towards host volatiles diffusing through the soil (Nordlander et al., 1986), arriving females locate and oviposit in the roots of fresh stumps from the recently felled coniferous trees. The following year, newly emerged weevils encounter the recently planted saplings upon which they start feeding. The weevils often girdle the stem of the young seedlings. As a consequence the seedlings die, resulting in considerable economic losses for the forest industry. Thus, the method of reforestation presently used, clear cutting without stump removal and replanting within two years, provides ideal conditions for the pine weevil (Örlander et al., 1997).

Hitherto no pheromone has been found for H. abietis (Tilles et al., 1986b; Nordlander et al., 1986) except for a short-range mating stimulant from the body of female weevils (Tilles et al., 1988). However, the active compound remains to be identified. An aggregation pheromone has been suggested (Selander, 1978) although the long-range attraction of weevils is probably caused by the increased amount of host terpenes and ethanol released when weevils are feeding on host material (Tilles et al., 1986b; Zagatti et al., 1997).

In the attempts to control the damage caused by the pine weevil, several approaches have been used, with varying success. Since no long-range pheromone has been found for H. abietis, damage control by mass-trapping and

5

mating disruption techniques are not viable (Schlyter, 2004). Thus, protection by insecticides has been widely used. For example, the pyrethroid, permethrin (4, Figure 1.3), has been employed for efficient protection of the young conifer saplings against pine weevils. This insecticide, however, has been found to cause allergic reactions in forestry workers (Kolmodin-Hedman, et al. 1995) and is highly toxic to water dwelling organisms (McLeese et al., 1980). Therefore, since 2003, the National Chemicals Inspectorate prohibits permethrin for use in plant protection in Sweden. It is nowadays replaced by cypermethrin (5, Figure 1.3), another pyrethroid, which has been found to be even more toxic to aquatic organisms than permethrin (McLeese et al., 1980) and will be prohibited from 2010. Antifeedant compounds (section 1.1.2) are an alternative to toxic insecticides (Salom, et al., 1994; Klepzig and Schlyter, 1999; Bratt et al., 2001; Thacker et al., 2003; Schlyter, 2004). Other alternatives for the protection of young trees have been explored also, such as planting under shelterwood trees (von Sydow and Örlander, 1994), mounding and delay of planting (Örlander and Nilsson, 1999) and the use of physical barriers as shelters around the seedlings (Hagner and Jonsson, 1995).

1.3 Semiochemicals and stereochemistry

Stereochemistry deals with chemistry in three dimensions and is of great importance in organic chemistry. Stereoisomers are compounds that have the same molecular formula but differ in configuration or sometimes in conformation, i.e the arrangement of atoms in space differs. Stereoisomers are divided into enantiomers and diastereomers.

Stereoisomers

H

H

HH

trans -2-butenecis -2-butene

Enantiomers Diastereomers

O OMirror plane

(R)-Carvone (S)-Carvone(7a) (7b)

H H

Figure 1.5. The classification of stereoisomers and examples thereof.

A pair of enantiomers are mirror images of each other but can not be superposed, i.e. they can not be put on top of each other so that every part of the molecule coincides; they are chiral (Greek; cheir = hand). Many objects in nature are chiral; an example is our left and right hands. The right hand is the mirror image

6

of the left hand but they can not be superposed so that every part of the hands coincides. Diastereomers are stereoisomeric molecules that are not mirror images to each other.

Even though most physical properties, such as the melting point, solubility and density are the same (unless examined in a chiral environment) enantiomers can display different biological properties. An example is that of the monoterpene carvone whose enantiomers (Figure 1.5) have different odours. The R-enantiomer (7a) has the scent of spearmint and the other, the S-enantiomer (7b), has the scent of caraway. Carvone is a potent antifeedant against the pine weevil, H. abietis, and it was shown that the S-enantiomer was slightly more active than the R-enantiomer (Schlyter et al., 2004a).

Another, more drastic effect of chirality, connected to chemical ecology, is when a trace of the “wrong” stereoisomer of a pheromone completely inhibits the action of the pheromone (Mori, 1997). However, there are also examples where both enantiomers are required for bioactivity (Mori, 1997). Thus, when a compound is to be used for protection against insect pests it is useful and, in some cases, even necessary to know which stereoisomer is associated with the desired activity and whether other possible stereoisomers are inactive or not. Even though the desired compound is naturally occurring, the amount that can be obtained from its natural source is often not sufficient for further biological studies. Instead, enantioselective synthesis of the compound in large quantities is often required.

Thus, synthesis of enantiopure or enantiomerically enriched compounds is of great importance in the field of chemical ecology. The organic chemist involved in chemical ecology will often use already known and reliable synthetic methods and consequently, is not always working in the frontline inventing new methods of synthesis. Nevertheless the synthesis of semiochemicals is an important and necessary contribution to such interdisciplinary research and can be regarded as a demonstration of the usefulness and scale-up potential of already developed reactions.

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1.3 This thesis

In order to find environmentally friendly alternatives to insecticides for control of the pine weevil, a collaboration project was started in 1996, involving chemists and biologists: ”Protection of conifer seedlings against Hylobius abietis pine weevils by allomones”, a project within the research programme: “Pheromones and Kairomones for Insect Control” sponsored by MISTRA1. This thesis describes the progresses of this project. The work has been concentrated on finding compounds, predominantly naturally occurring ones, with antifeedant activity against the pine weevil.

An optimal antifeedant should deter the pine weevil, without killing it, from feeding on conifer saplings, while directing its feeding to other available food sources of no economic interest to the forest industry.

The strategies used in the search for such active compounds are:

1. Extraction of non-host plant species upon which the weevils avoid feeding and subsequent testing and fractionation of the extracts in order to identify the active compounds.

2. Screening of a large number of compounds, naturally occurring or synthetic, for antifeedant activity in order to build a library of active compounds.

3. Structure-activity studies of lead compounds, i.e. compounds with high antifeedant activity, from the resulting library.

A number of aspects, such as volatility, availability and toxicity, have to be considered for a compound to be used in the field. Compounds of low volatility in a suitable formulation have to be used in order to achieve a long-lasting effect in the field and methods for large-scale synthesis of active compounds have to be developed.

1 Foundation for Strategic Environmental Research

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2. Biological test methods 2.1 Laboratory tests

Three types of laboratory tests were used to determine the antifeedant activity of chemical compounds against H. abietis. A minimum limit of ~95% chemical purity was set for the compounds to be tested. The first test, the micro feeding assay, in which an antifeedant index [AFI(m)] (Klepzig and Schlyter, 1999) was determined, was compulsory for all compounds. This assay (Schlyter et al., 2004b; I; II) is a choice test in which two TLC-plates; one treated with the test compound (10% w/w in an appropriate solvent) and one control plate (treated with solvent only) were used. A sucrose solution was added to both plates to stimulate feeding after which the two plates were presented to a weevil in a Petri dish. The AFI(m) was calculated, based on the area eaten by the weevil on each plate respectively, according to equation 1:

Equation 1 AFI=Area fed on control − Area fed on treatment

Area fed on control + Area fed on treatment

Thus an AFI of +1 means total inhibition of feeding by the treatment, an AFI near 0 means no effect and negative AFI values are achieved for compounds that stimulate feeding.

When the AFI(m) of bark extracts was determined the area of the bark was determined before the extraction in order to apply a similar amount/area of the compounds on the TLC plate as present in the bark. The volume of the resulting extract was adjusted to 10 µl × N (were N is the number of TLC plates, each of an area of 25 mm2, corresponding to the total area of the extracted bark and 10 µl is the volume of the extract applied on each TLC plate) (I).

For compounds with high AFI(m)-values, the ED50(m), i.e. the dose required to achieve an AFI value of 0.5, was determined from dose-response tests (Klepzig and Schlyter, 1999, I; II). A total of 50-100 mg of the test compounds was required for the micro feeding assays and the dose-response tests.

The compounds with the highest activity, i.e. the ones with ED50(m) values within the range of a few percent, were tested in the dose-response no-choice twig assay (Klepzig and Schlyter, 1999; I; II) which requires ~2 g of the compound that is to be tested. In the twig assay the compounds were tested on the natural host of the weevils. The activity of the compounds has to be high in order to withstand the attractiveness of host volatiles that evaporate from the twigs once the weevil has bitten and tasted the bark. An AFI(t) was calculated according to equation 1 based on the area eaten on treated pine twigs versus untreated control twigs. As in the micro feeding assay an ED50(t) was calculated from dose-response twig tests (Klepzig and Schlyter, 1999). For several compounds with high activity in the micro feeding assay and the subsequent dose-response test, it was often found that the activity was considerably lower in the more demanding no-choice twig test.

9

2.2 Field tests

The most active compounds, as determined by the twig assay, were tested in the field. The test compound in a suitable formulation was painted on the lower part (~10 cm) of the stem on conifer saplings. A variety of formulations have been tested. Some compounds were simply dissolved in molten paraffin wax (m.p.= 52-54 °C) and applied on the saplings. For compounds not soluble in this wax other types of formulations were considered (II; section 4.4). An optimal formulation will protect the compounds from the harsh conditions often met in the field and will confer a slow release of the compounds resulting in long-lasting protection of the trees. A field test often requires at least 10 g of a test compound.

The planting in the field and the damage evaluation were performed at intervals ranging from weeks to months after planting and were made impartially by persons not involved in the research project. As in the micro feeding assays and the twig tests an AFI was calculated, according to equation 1, based on the area eaten on untreated control plants versus treated plants.

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3. Antifeedants against the pine weevil from non-host woody speciesI-III

In spite of the severe damages caused by the pine weevil on newly planted coniferous trees at reforestation areas (section 1.2), the weevil appears to be a polyphagous insect, feeding on non-conifer species as well (Scott and King, 1974; Löf, 2000). The feeding preferences of H. abietis have been extensively studied (Månsson and Schlyter, 2004). There are plant species that the weevils will not feed upon even when no choice is given and some species of which the weevils remove only small amounts of the bark and phloem (Table 3.1). Several known insecticidal and antifeedant compounds emanate from natural sources. Thus, it is reasonable to hypothesise that the non-feeding behaviour of the weevils, when presented to these woody species is a result of chemical compounds with antifeedant activity present in those plants. Table 3.1. Examples of woody species, not palatable to H. abietis (Månsson and Schlyter, 2004).

Species Latin name Linden Tilia cordata Mill. Guelder rose Viburnum opulus L. Spindle tree Evonymus europaeus L. Alder Alnus glutinosa (L.) Gaertner Walnut Juglans regia L. Beech Fagus sylvatica L. Horse chestnut Aesculus hippocastanum L. Yew Taxus baccata L. Holly Ilex aquifolium L. Aspen Populus tremula L. Lilac Syringa vulgaris L.

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3.1 Identification of an antifeedant in bark from linden, Tilia cordata

One of the species, rejected as a food source by H. abietis, is linden (T. cordata) (Månsson and Schlyter, 2004). In order to identify possible antifeedants in the bark from this species, extracts of the bark were prepared and tested for antifeedant activity as described in section 2.1 (I). Various methods for the preparation and extraction of the bark were evaluated (Table 3.2) and the resulting extracts were tested for antifeedant activity in micro feeding assays. Out of the five linden extracts only three (C, D and E) displayed antifeedant activity while one extract stimulated feeding (A, AFI(m) <0). Table 3.2. Methods for linden bark extraction and antifeedant activity of resulting extracts.

Extract Bark treatment Extraction method Solvent Antifeedant activity

A Ground frozen

High pressure

Soxhlet extraction*

CO2 (l) Negative

(significant attraction)

B Freeze dried

Solvent Soxhlet#

MeOH/CH2Cl2 1:9

No

C Fresh Solvent Soxhlet Hexane→

CH2Cl2→MeOHPositive,

weak

D Ground frozen Solvent Soxhlet MeOH/CH2Cl2

1:9 Positive

E Levigated fresh in

blender with MeOH

Solvent Soxhlet

MeOH Positive

*Bøwadt and Hawthorne, 1995; I, #Furniss et al., 1989

To be able to isolate and identify the compound/compounds responsible for the antifeedant activity in some of the bark extracts the classical method of fractionation followed by biological testing of the resulting fractions was used. Thus, one of the active extracts obtained from linden bark, extract D (Table 3.2), was fractionated by LC. The resulting fractions were tested in the micro feeding assay, in the same way as the total extract (I). Whereas some fractions were attractive to the weevils, most of the fractions were inactive (Figure 3.1). Two fractions did, however, display a high antifeedant activity (no 12 and 13).

12

10911108912128711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h 1.5

1.0

0.5

0.0

-0.5

-1.0

9

Merged fractions # 1-17

Micro feeding assay, merged fractions

1091110891212711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h 1.5

1.0

0.5

0.0

-0.5

-1.089



1091110891212711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h

1.0

0.5

0.0

-0.5

-1.089



1091110891212711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h 1.5

1.0

0.5

0.0

-0.5

-1.089



1091110891212711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h 1.5

1.0

0.5

0.0

-0.5

-1.089



1091110891212711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h 1.5

1.0

0.5

0.0

-0.5

-1.089



1091110891212711101112101211N =171615141312111087654321

95%

CI A

ntife

edan

tInd

ex 4

h

1.0

0.5

0.0

-0.5

-1.089



Figure 3.1. Antifeedant index of fractions of T. cordata bark extract D.

The two active fractions were analysed by GC and one major compound (~ 84%, relative area) was found in fraction no 13. The same compound was also present in fraction no 12 together with some minor unidentified constituents. This compound was identified, by GC-MS and NMR (comparison of spectras with those of an authentic sample), as nonanoic acid. Moreover, high antifeedant activity (AFI(m)= 0.99 ± 0.01, N = 9) was found when commercially available nonanoic acid was tested against H. abietis in a micro feeding assay.

3.2 Antifeedant activity of aliphatic carboxylic acids and related compounds

There are several reports of insecticidal, antifeedant and deterrent activities of different carboxylic acids against a variety of insects. For example some fatty acids are feeding deterrents to the boll weevil, Anthonomus grandis (Bird et al., 1987), and some are feeding deterrents against drywood termites, Incisitermes minor (Scheffran and Rust, 1983). Nonanoic acid, which exhibited strong antifeedant activity against the pine weevil, occurs in almost all plant- and animal species. It is also present at low levels in the food we eat and it is easily degraded in the environment (U.S. Environmental Protection Agency, 2006). Hence, this acid could be regarded as a relatively safe candidate for use as an antifeedant against H. abietis. However, a carboxylic acid with a higher boiling point than that of nonanoic acid would have a longer persistence under field conditions and would therefore be more suitable for use in the field. Based on these facts an investigation of the antifeedant activities of aliphatic carboxylic acids against H. abietis was initiated.

13

3.2.1 Laboratory tests Unbranched, aliphatic carboxylic acids, in the range of C6-C13, and some methyl branched aliphatic carboxylic acids were tested in micro feeding assays (Figures 3.2 and 3.3) (II).

1816171541271926N =

C13C12C11C10C9C8C7C6

Ant

ifeed

ant I

ndex

±95

% C

.I. 1.5

1.0

0.5

0.0

−0.5

Carboxylic acids

Figure 3.2. Antifeedant index of unbranched carboxylic acids (C6-C13) in micro feeding assays.

All unbranched acids with a chain length of 6-10 carbons were more active than the ones with longer chains (Figure 3.2). A similar tendency was observed for the methyl branched acids with maximum activity obtained for 2-methyldecanoic acid (Figure 3.3).

14

Antif

eeda

nt In

dex

±95%

C.I.

1713171417N =

1.5

1.0

0.5

0.0

−0.5

Methyl branched carboxylic acids2MC122MC112MC102MC92MC8

Figure 3.3. Antifeedant index of branched carboxylic acids in micro feeding assays: 2-methyloctanoic acid (2MC8), 2-methylnonanoic acid (2MC9), 2-methyldecanoic acid (2MC10), 2-methylundecanoic acid (2MC11) and 2-methyldodecanoic acid (2MC12).

The activity of the carboxylic acids seemed to be related to the volatility of the compounds, with a decreased activity for acids with higher boiling points. A similar result was obtained when fatty acids were tested for settling and larviposition deterrent activity against the green peach aphid, Myzus persicae (Greenway et al., 1978). The acids of shorter chain-lengths (C8-C13) were deterrents while those with a chain length >C16 were stimulants. In contrast, the deterrent activity of carboxylic acids against western drywood termites, I. minor, was lower for shorter saturated acids than for longer unsaturated ones (Scheffrahn and Rust, 1983). Interestingly, decanoic acid has been found to deter feeding of the boll weevil, A. grandis, while nonanoic acid which exhibits a high antifeedant activity against the pine weevil, is a feeding stimulant to A. grandis (Bird et al., 1987).

While the antifeedant activity against the pine weevil decreased for unbranched acids with more than 10 carbons, 2-methyldecanoic acid, consisting of 11 carbons was the most active of the branched acids. The reported boiling point of 2-methyldecanoic acid (~350 °C, Berglund et al., 1993) is considerably higher than the one for decanoic acid (~270 °C, Aldrich catalogue, 2005-2006). Thus the addition of a methyl group provided an efficient way to decrease the volatility without any decrease in the antifeedant activity. Several of the unbranched carboxylic acids were also tested in the twig assays. In these tests nonanoic acid was the most active carboxylic acid (II).

15

When the sugar solution, which is added to the TLC plates as a feeding stimulant, was acidified with HCl to a pH of ~4.5, i.e. the pH achieved when mixing nonanoic acid with a sugar solution, no antifeedant activity was found. Thus the antifeedant effect of the carboxylic acids was not the result of the acidity of the carboxylic acid.

In order to investigate the effect of the functional group, some compounds related to nonanoic acid, such as 1-nonanol, nonanal, sodium nonanoate and nonanoic acid anhydride were tested in micro feeding- and twig assays (Table 3.3). Whereas sodium nonanoate was not active in the micro feeding assay, a high AFI(m)-value was obtained for 1-nonanol. The ED50(m) of 1-nonanol, however, was not as low as that of nonanoic acid (II). Moreover, when the alcohol was tested on twigs the activity was lower than of nonanoic acid. When tested in the more demanding twig test several compounds displayed a lower activity. Once the weevil has tasted the bark, only compounds with exceptional antifeedant activity counteracts the attractiveness of the host volatiles released from the twig (section 2.1).

When the aldehyde, nonanal, was tested in the micro feeding assay, it exhibited an even lower antifeedant activity than that of the corresponding alcohol, 1-nonanol. When tested in the micro feeding assay, nonanoic anhydride displayed a high activity which was lost in the twig test. This result might be explained by the fact that when tested in the micro feeding assay, the anhydride is hydrolysed by the added sugar solution, forming the active nonanoic acid. In the twig test however, no sugar solution is added. Thus, the compound is not hydrolysed to the same extent. Apparently to achieve the highest activity for aliphatic compounds of this type, a carboxylic acid moiety is required.

Table 3.3. Antifeedant activity of derivatives of nonanoic acid in laboratory tests.

Compound AFI(m) 10%

ED50(m) (%)

AFI(t) 10%

ED50(t) (%)

1-Nonanol

≈ 1 >1 >0 >1

Nonanal

>0 0

Nonanoic anhydride

≈1 0

Sodium nonanoate 0

16

3.2.2 Field tests During the summer of 2003, octanoic-, nonanoic- and decanoic acids formulated in paraffin were tested in the field (II). The formulation was painted on the lower part of the stem of Norway spruce seedlings. An increase in activity with increasing chain length was found, with decanoic acid being the most active acid (Figure 3.4).

40804080N =C10 in Wax

C9 in Wax

C8 in Wax

Wax Blank

1.0

0.5

0.0

Antif

eeda

nt In

dex

±95%

C.I.

(3 m

onth

s)

Figure 3.4. Antifeedant index of octanoic- (C8), nonanoic- (C9) and decanoic acid (C10), formulated in paraffin, in the first field test 2003 (Asa Forest Experimental Station,

Lammhult, Småland).

In a second field test that started in August 2003 the overall weevil damage was low. However, similar to the first test, decanoic acid was the most active acid. The release rates of the acids, formulated in paraffin, from treated saplings were determined and, as expected, showed slower evaporation of the less volatile decanoic acid than that of octanoic- and nonanoic acid (II). Thus, the higher activity of decanoic acid in the field test, compared to octanoic- and nonanoic acid, could be explained by the longer persistence of this compound.

Unexpectedly, in both field tests, several of the treated plants died from something other than a weevil attack. This was assumed to be caused by phytotoxicity of the test compounds as no control plants suffered from this type of damage. The phytotoxicity increased with the chain length of the applied acid. Phytotoxicity of carboxylic acids has been observed earlier on a number of plants treated with C6-C14 carboxylic acids with C9-C11 being the most herbicidal. The carboxylic acids are believed to cause damage to the cell membrane system, resulting in electrolytic leakage and subsequent death of the plants (Fukuda et al., 2004).

17

In a third field test that started in June 2004 it was demonstrated that the phytotoxic side effect could partially be circumvented. An extra protective layer of the paraffin based formulation, without the active carboxylic acid, was applied onto the stem before the application of the formulation containing the active ingredient, i.e. nonanoic acid (II). In this way the damage, due to phytotoxicity of nonanoic acid was reduced from 75% to 25%. This clearly demonstrates the importance of a proper formulation strategy.

3.3 Antifeedant activity of chemical constituents in ten non-host woody species

The finding that linden bark contains the highly active antifeedant nonanoic acid resulted in the plausible assumption that the other ten woody species (Table 3.1), known to be rejected by H. abietis as a food source (Månsson and Schlyter, 2004), also contain such active compounds. Thus, extracts of these species were prepared and tested for antifeedant activity (III).

Aliphatic carboxylic acids have been found in several of the extracted non-host species. Apart from in the flowers (Vidal and Richard, 1986) and bark from linden (I), the highly active pine weevil antifeedant, nonanoic acid, has also been found in horse chestnut seeds and peels (Buchbauer et al., 1994a). Octanoic and decanoic acids have been found in a hydrolysed extract of needles from yew (Erdemoglu et al., 2003). Other carboxylic acids in the range C6-C10 have been found in walnut leaves (Nahrstedt et al., 1981; Buttery et al., 2000) and in beech wood (Guillén and Ibargoitia, 1996).

Our hypothesis was that the activity of the extracts might be explained by the presence of these acids or by the presence of other, already known antifeedant compounds, such as carvone and carvacrol (Klepzig and Schlyter, 1999; Schlyter et al., 2004a).

3.3.1 Bark extraction and antifeedant activity of prepared extracts Bark from guelder rose, spindle tree, alder, walnut, beech, horse chestnut, yew, holly, aspen and lilac (Table 3.1) were prepared according to procedure D (Table 3.2). However, the extraction method differed; the bark from each species was first extracted with pentane after which the solvent was changed to methanol. In this way a separation of non-polar and polar compounds in the bark was obtained.

The resulting 20 extracts (10 pentane- and 10 methanol extracts) were tested in micro feeding assays. The extracts of aspen, holly, yew and horse chestnut showed antifeedant activity (Figure 3.5). None of the pentane extracts showed any antifeedant activity and some, i.e. those of horse chestnut, beech, lilac and guelder rose, even stimulated feeding. The fact that some of the bark extracts displayed antifeedant activity supported the hypothesis that compounds with antifeedant activity were actually present in the corresponding bark.

18

9157712141718149 814112818167169N =

Viburnum

Syringa

Populus

Ilex

Taxus

Fagus

Juglans

Aesculus

Alnus

Euonymus

Anti-

feed

ant I

ndex

±95

% C

.I. 1.50

1.00

.50

0.00

−.50

−1.00

Figure 3.5. Antifeedant index of pentane (|- -○- -|) and methanol (|—■—|) bark extracts of pine weevil non-hosts, spindle tree (Evonymus), alder (Alnus), horse chestnut (Aesculus),

walnut (Juglans), beech (Fagus), yew (Taxus), holly (Ilex), aspen (Populus) lilac (Syringa) and Guelder rose (Viburnum).

3.3.2 Chemical content of non-host bark extracts and antifeedant activity of identified compounds The 20 extracts (section 3.3.1) were analysed by GC-MS and repeatedly occurring major compounds were identified by comparing their retention times and MS-spectra with reference samples. The identified compounds were tested for antifeedant activity (Table 3.5, following 3 pages).

19

Table 3.5. Antifeedant activity in micro feeding assays of compounds identified in pentane and methanol bark extracts of non-host woody species.

Compound

10% AFI(m) ±SE (N)

Gue

lder

rose

Spin

dle

tree

Ald

er

Wal

nut

Bee

ch

Hor

se c

hest

nut

Yew

Hol

ly

Asp

en

Lila

c

Hexanoic acid O

OH

0.98*±0.01 (26)α

P P

Nonanoic acid O

OH

0.94*±0.05 (41)α,β

P

Carvacrol OH

1.00*±0.00 (26)γ

M

Eugenol

OH

OMe

1.00*±0.00

(6) M P/M M M

2-Phenylethanol OH

1.00*±0.01 (19)

P P

Carvone O

0.83*±0.06 (49)γ

P P

P) Identified by GC-MS in pentane extract, M) Identified by GC-MS in methanol extract, M) Methanol extract with significant antifeedant activity, P) Pentane extract with significant feeding stimulating activity, *) AFI significantly different from 0 by 95% confidence interval for the mean of N observations, α) II, β) I, γ) Schlyter et al., 2004a, δ) Klepzig and Schlyter, 1999 (enantiomers not analysed).

20

Table 3.5. Continued.

Compound

10% AFI(m) ±SE (N)

Gue

lder

rose

Spin

dle

tree

Ald

er

Wal

nut

Bee

ch

Hor

se c

hest

nut

Yew

Hol

ly

Asp

en

Lila

c

Benzylalcohol

OH

0.78*±0.22

(8) M P P/M

Hexadecanoic acid

COOH13

0.12 ±0.12 (19)

P/M M P/M P/M P/M P/M P P P/M M

Limonene

<0.05± >0.13

(19)δ P M M

Vanillin CHO

OHOMe

0.01 ±0.25 (14)

P/M P P P P

Benzoic acid COOH

0.01 ±0.16

(9) M P P/M P/M P/M P P/M M

Benzaldehyde CHO

−0.01±0.18 (10)

P/M M M P/M P/M M


21

Table 3.5. Continued.

Compound

10% AFI(m) ±SE (N)

Gue

lder

rose

Spin

dle

tree

Ald

er

Wal

nut

Bee

ch

Hor

se c

hest

nut

Yew

Hol

ly

Asp

en

Lila

c

Cinnamic acid COOH

−0.08±0.21

(8) P/M P/M

β-Sitosterol

HOH H

−0.16±0.13 (19)

P/M M P/M P/M P/M P/M P/M P P/M

2-Furaldehyde

OO

H

−0.21±0.16 (17)

M M M P/M M M M M

Linolenic acid

COOH6

−0.22±0.11 (20)

P

Linoleic acid

COOH64

−0.29*±0.12 (20)

M M P/M P/M P/M P/M P/M P P/M M

5-(Hydroxymethyl)-2-furaldehyde

OO

HHO

−0.32*±0.13 (18)

M M P/M M


22

Surprisingly, the only carboxylic acids detected in the range C6-C10 were nonanoic acid and hexanoic acid. Both were found in pentane extracts that did not show any antifeedant activity (Table 3.5). In accordance with the decreased activity found for C11-C13 carboxylic acids (Figure 3.2), hexadecanoic acid identified in most of the extracts did not exhibit any antifeedant activity. Two other carboxylic acids, linoleic- and linolenic acid, were feeding stimulants that were found both in feeding stimulating extracts and extracts with antifeedant activity. Linoleic acid has previously been found to be a feeding deterrent to the boll weevil, A. grandis (Bird et al., 1987).

The known pine weevil antifeedant, carvone (Schlyter et al., 2004a) was only found in the feeding stimulating pentane extracts of beech and holly. The antifeedant active methanol extracts of horse chestnut, yew and aspen all contained one or more of the active antifeedants carvacrol, eugenol, and benzylalcohol. The highly active antifeedant 2-phenylethanol was found only in feeding stimulating extracts.

Two aldehydes, 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde were identified in several extracts (Table 3.5). In contrast to the high antifeedant activity found for some aromatic aldehydes (see section 5.2), these heteroaromatic aldehydes exhibited weak feeding stimulating activity. One of the aldehydes, 5-(hydroxymethyl)-2-furaldehyde, has been reported earlier as a constituent of the essential oil and head space extract of horse chestnut flowers (Buchbauer et al., 1994b). However, both aldehydes might be artefacts formed by carbohydrate dehydration (Kallury et al., 1986; Antal Jr et al., 1990), either during the extraction procedure or in the injector of the GC.

Another compound with feeding stimulating activity, β-sitosterol was found in almost all extracts. Sitosterol has earlier been found to display some antifeedant activity against the cotton boll weevil, A. grandis (Miles et al., 1987) but has also been found to stimulate feeding of other weevil species (Coleoptera: Curculionidae) (Doss et al., 1982; Shanks and Doss, 1987). The feeding stimulating effect of sitosterol was, for some of the weevils, synergistically enhanced by adding sucrose (Shanks and Doss, 1987).

Thus all extracts were complex mixtures of inactive, feeding stimulating and feeding deterrent compounds. Active antifeedants were found in feeding stimulating extracts and vice versa, i.e. feeding stimulants were found in extracts with feeding deterrent properties. However, when comparing the chromatograms of antifeedant active versus feeding stimulating extracts, the amount of the feeding stimulating compounds β-sitosterol and linoleic acid was less in the antifeedant active extracts than in the feeding stimulating extracts. Thus, whether an extract showed antifeedant activity or feeding stimulating properties seemed to be dependent on the efficiency of the pentane extraction in removing these feeding stimulants. Of course there is also a concentration dependence connected to the antifeedant activity. Consequently several active compounds can be present in an extract in concentrations that are too low to induce any response of the weevils.

23

3.3.3 Fractionation of horse chestnut bark extract The successful fractionation of an active extract from linden (section 3.1), inspired us to a closer examination of the other extracts described above (section 3.3.2). The methanol extract of horse chestnut, A. hippocastanum, was chosen for fractionation (Figure 3.5) (III). A first fractionation of this extract by reverse phase LC resulted in one active fraction which, according to GC-MS, contained several compounds. This fraction was further fractionated by straight phase LC which resulted in three fractions exhibiting some antifeedant activity, i.e. M1:3:1, M1:3:8 and M1:3:9 (Figure 3.6).

6678875910N =

M1:3:9M1:3:8M1:3:7M1:3:6M1:3:5M1:3:4M1:3:3M1:3:2M1:3:1

Anti-

feed

ant I

ndex

±SE

1.00

.50

0.00

−.50

−1.00

Figure 3.6. Antifeedant index of subfractions (M1:3:1-M1:3:9) of fraction no 3 from the first methanol extract of horse chestnut.

Two compounds found both in fraction M:1:3:8 and M:1:3:9 were tentatively identified by GC-MS as aliphatic esters of hexanoic acid. The MS-spectra and retention times were compared with those of reference samples of some esters of hexanoic acid (tetradecyl-, tridecyl-, dodecyl- and hexyl hexanoate). However, none of them were identical with the compounds in the fractions. When tested in a micro feeding assay, high antifeedant activity was found for hexyl hexanoate (AFI(m)= 0.87) and some hexenyl- and branched butyl esters of hexanoic acid (III). However, the activity was lower for the less volatile tetradecyl hexanoate (AFI(m)= 0.32). Further investigation of the MS-spectra of fraction M1:3:8 indicated the presence of some unidentified branched alcohols. Due to the limited material left in the active fractions, no further characterisation was possible.

24

Thus, a second batch of horse chestnut bark was extracted, this time using an extraction sequence starting with pentane, followed by dichloromethane and finally methanol. Again the methanol extract was the only extract that displayed antifeedant activity. After LC fractionation of this extract, three fractions were found to deter feeding (M2:12-13, M2:14-15 and M2:16-21) and one fraction stimulated feeding (M2:01) (Figure 3.7).

7889499N =

5 x 5 mm TLC plates, 5µL solute, 5 µL 1M Sucrose

M2:22-34M2:16-21M2:14-15 M2:12-13M2:03-11M2:02M2:01

Ant

i-fee

dant

Inde

x ±

SE

1.00

.50

0.00

−.50

−1.00

Figure 3.7. Antifeedant index of fractions obtained from the second methanol extract.

25

Surprisingly, the feeding stimulant 5-(hydroxymethyl)-2-furfural was found in the fractions exhibiting antifeedant activity. However, the major compounds common for the antifeedant active fractions were tentatively identified as branched aliphatic alcohols. Their MS-spectra and retention times were compared with the ones of some available alcohols of that type, i.e. 2-butyl-1-octanol, 2-hexyl-1-decanol and 2-ethyl-1-hexanol. Among those, the latter has been found in walnut peels (Buchbauer et al., 1993). Although similarities in the MS-spectra were found, the retention times of the reference alcohols differed from those of the compounds in the fractions. Two of these reference alcohols, however, exhibited antifeedant activity when tested in micro feeding assays (Table 3.4). The feeding stimulant, β-sitosterol was found in M2:01, explaining its feeding stimulating properties.

Thus, we were not able, as we had done for linden, to isolate one single active compound responsible for the observed antifeedant activity of the fractions from horse chestnut bark. The fact that some of the tested alcohols and hexanoate esters were active and that these types of compounds were present in several active fractions, indicated that branched alcohols and hexanoate esters, together with other identified antifeedants, were responsible for the antifeedant activity of horse chestnut bark. However, there are probably additional, not yet identified, antifeedants present in the bark of this species.

Table 3.4. Antifeedant index of branched chained alcohols.

Compound AFI(m) 10% ±SE (N)

2-Ethyl-1-hexanol OH

0.93 ±0.08 (27)

2-Butyl-1-octanol OH

0.86 ±0.10 (27)

2-Hexyl-1-decanol OH

0.17 ±0.09 (29)

26

4. Synthesis and antifeedant activity of piperidine alkaloidsIV, Appendix The first alkaloids isolated from conifer species were α-pipecoline [(+)-8, Figure 4.1] and an alkaloid given the trivial name pinidine (Tallent et al., 1955). The structure and absolute configuration of pinidine was determined as 2R-methyl-6R-(2E-propenyl)-piperidine [(−)-9, Figure 4.1] (Tallent and Hornig, 1956; Hill et al., 1965). Since then several other piperidine alkaloids have been isolated from Pinus and Picea species (Stermitz and Miller, 1990; Schneider and Stermitz, 1990; Schneider et al., 1991; Tawara et al., 1993, 1995; Stermitz et al., 1994; Todd et al., 1995; Gerson and Kelsey, 2002) (Figure 4.1).

HN

HN

O

(+)-12

HN

(−)-9

HN

10

HN

(−)-13

OH

(+)-11

HN

(+)-8

Figure 4.1. Examples of piperidine alkaloids found in conifer species; α-pipecoline [(+)-8], (−)-pinidine [(−)-9], dihydropinidine (10), (+)-euphococcinine [(+)-11], epidihydropinidine

[(+)-12] and (−)-pinidinol [(−)-13].

In general spruce species contain both cis- and trans-piperidine alkaloids, while the pines seem to contain cis-piperidine alkaloids only (Tawara et al. 1993, Stermitz et al., 1994). Several of the alkaloids common for pine and spruce species have also been found in insects. For example, the Mexican bean beetle, Epilachna varivestis, produces dihydropinidine (10) (Attygalle et al., 1993, absolute configuration not reported) and euphococcinine [(+)-11]. Euphococcinine probably serves as an allelochemical that keeps predators at a distance (Eisner et al., 1986).

In a preliminary study the hydrochloride salt of (±)-dihydropinidine (10) showed high antifeedant activity against the pine weevil (Schlyter et al., unpublished results). Thus, this compound might be suitable for use in the protection of young conifer plants against feeding of the pine weevil. The compound has been found in needles from Picea pungens, (Todd et al., 1995) and in needles and bark from Picea sitchensis (Gerson and Kelsey, 2002). However, the absolute configuration of the compound in these species has not been reported.

27

Enantiomers and diastereomers of a semiochemical can display antagonistic biological effects (Mori, 1997). It was therefore of interest to test the enantiomers of dihydropindine for antifeedant activity separately. Accordingly, synthesis of the two enantiomers of dihydropinidine [(+)-10 and (−)-10] was necessary.

In spite of the apparently simple structures of the piperidine alkaloids, the challenge that lies in the asymmetric synthesis of these and other 2,6-disubstituted piperidine alkaloids is demonstrated by the numerous methods developed for their preparation (Bailey et al., 1998; Husson and Royer, 1999; Groaning and Meyers, 2000, Laschat and Dickner, 2000).

4.1 Synthesis of dihydropinidine

4.1.1 Dimethylzinc mediated allylation of 2-methyltetrahydropyridine-N-oxideSince it has been reported that when vinylboronic esters are added to certain nitrones in the presence of dimethylzinc N-allylic hydroxylamines are formed (Pandya et al., 2003), it was decided to test that method for the preparation of the enantiomers of dihydropinidine, (+)-10 and (−)-10 (IV). As the synthetic sequence proceeds via hydrogenation of (−)-9 and (+)-9, respectively, these compounds would also be available to test for antifeedant activity against H. abietis (Scheme 4.1).

N+O-

B O

O

+

NOH

NOH

+

14 15

HN

E-cis-16 E-trans-16

(−)-9

HN

(+)-10

H2, Pd/C

Zn, HOAc

Me2Zn

Scheme 4.1. Planned synthesis of (−)-pinidine [(−)-9] and (+)-dihydropinidine [(+)-10].

28

The required nitrones, (R)-2-methyltetrahydropyridine-N-oxide (14) and its enantiomer (S)-2-methyltetrahydropyridine-N-oxide (ent-14) were prepared by a modification of an earlier reported procedure (Chackalamannil and Wang, 1997; IV) (Scheme 4.2). Starting from d- and l-alanine respectively, N-Cbz-alanine methyl esters (17 and ent-17) were prepared in two steps (Wipf and Heimgartner, 1988; Kim et al., 1985). The nitrone 14 was used immediately in the subsequent vinylboration reaction (Scheme 4.1).

O

O

P+Ph3 Br-

NH2

O

O

NHOCOPh

O

O

NHOH

O

O N+O-

NHCOOCH2Ph

COOMe

17

14

a) 19

b)

NHCOOCH2Ph

CHO

18

NHCOOCH2Phd)c) e)

f)

O

O

1

23

4

20

Scheme 4.2. Synthesis of nitrone (14). a) Dibal-H (2.0 equiv), CH2Cl2, −78 °C, ~96%. b) 1. 19 (1.38 equiv), THF, KOt-Bu, 2. addition of 18, rt, ~51%. c) H2, Pd/C, EtOH/EtOAc,

~99%. d) Ph(CO)2O2 (2.0 equiv), CH2Cl2, NaHCO3/NaOH buffer (pH 10.5), ~56%. e) LiOH (2 M aq.), THF/MeOH, ~95%. f) HCl (2 M aq.), 15 min.

Unfortunately, when the vinylic boronic ester, 1-propenyl pinacolyl boronate (15), was added to the nitrone in the presence of dimethylzinc, according to the procedure described by Pandya et al. (2005) (Scheme 4.1), only a low conversion of the nitrone was observed. Several attempts to convert the nitrone to the corresponding hydroxylamines E-cis-16 and E-trans-16 were performed, using various types of solvents, varying the amount and type of dialkylzinc as well as varying the temperature. Moreover, the order of addition of the reagents was altered. In case an alkyl/vinyl exchange on zinc was a prerequisite for the reaction to proceed, the dialkylzinc reagent was mixed with the boronic ester before addition of the nitrone. Still, no satisfactory conversion of the nitrone could be achieved.

29

However, changing the vinylic boronic ester 15 to allylboronic ester 21 resulted in full conversion of the nitrone 14, yielding the hydroxylamines cis-22 and trans-22 (dr: 79/21), which could be separated by chromatography on silica (Scheme 4.3). The desired isomer cis-22 was obtained in ~30% yield calculated from hydroxylamine 20. Similarly addition of allylboronic ester 21 to the nitrone ent-14 gave the corresponding hydroxylamines ent-cis-22 and ent-trans-22. Palladium catalysed hydrogenation of cis-22 and ent-cis-22, gave (+)- and (−)-dihydropinidine [(+)-10 and (−)-10] respectively, which were isolated as their hydrochloride salts. The enantiomeric purity of respective enantiomer was >97% ee as determined by GC-analysis of the trifluoroacetamides of (+)- and (−)-10. The optical rotations of the enantiomers were in agreement with earlier reported values (IV). When tested in micro feeding assays both enantiomers showed the same, high antifeedant activity against H. abietis (see section 4.4).

N+O-

B O

O

+

NOH

NOH

+

14 21

HN

cis-22 trans-22

H2, Pd/C

Me2Zn (2.0 equiv)

CH2Cl2, −78 oC

(+)-10

Scheme 4.3. Synthesis of (+)-dihydropinidine [(+)-10].

4.1.2 Synthesis of (±)-dihydropinidine for field tests In field tests several grams of the compound to be tested is needed. Since the hydrochloride salts of the enantiomers of dihydropinidine (+)-10 and (−)-10, both showed high antifeedant activity in laboratory tests (see section 4.4), a racemate of the compound could be used. Thus, racemic dihydropinidine (10) was synthesised by a successful scale-up of the method developed by Wang et al. (2005) (Scheme 4.4).

The required Betti base (23) was prepared according to Szatmári et al. (2003). Following the procedures described by Wang et al. (2005), the Betti base was reacted with pentane-1,5-dial and benzotriazole (Bt) to give 24, which after methylmagnesium chloride addition furnished 25. Reaction of this with propylmagnesium bromide diastereoselectively gave the cis-dihydropinidine derivative 26. Palladium catalysed hydrogenation of 26, in the presence of CH2Cl2, which has been reported to be hydrodechlorinated during the hydrogenation (Wang et al, 2005), produced the hydrochloride salt, 10-HCl obtained in a total yield of ~42% calculated from Betti base (23). When tested in field high activity was found for (±)-dihydropinidine hydrochloride 10-HCl (see section 4.4).

30

O

N

HN

*HCl

b)a)

d)

Bt

OH

NH2

23 24

O

N

25

N

26

OHc)

7a

10-HCl

Benzotriazole = Bt

+

11

NH

NN

Scheme 4.4. Synthesis of (±)-dihydropinidine hydrochloride (10-HCl) for field tests (Wang et al., 2005). a) OHC(CH2)3CHO, CH2Cl2, 0 °C, pH 8-9, 20 min, 97.2%. b) MeMgCl, THF,

0 °C, 2h, 98.7%. c) n-PrMgBr, Et2O, −10 °C, 10 min, 73.9%. d) 10% Pd/C, H2, MeOH/CH2Cl2, rt, 58.7%.

4.2 Synthesis of pinidine

Dimethylzinc mediated addition of the vinylic boronic ester 15 to the nitrone 14 resulted in poor conversion of the nitrone (Scheme 4.1, section 4.1.1; IV). Thus, the method chosen for the synthesis of the enantiomers of dihydropinidine 10 did not give access to the pinidine enantiomers (−)-9 and (+)-9. However, it was observed during the development of the method for the preparation of (+)-10 and (−)-10 that when the nitrone was reacted with either 1-propenylmagnesium bromide or di-1-propenylzinc full conversion was achieved (Scheme 4.5). Unfortunately, the products were a virtually equimolar mixture of the hydroxylamines cis-16 and trans-16. Moreover, the alkenyl side chain was a mixture of E- and Z-isomers. While separation of the cis- and trans-isomers was easily achieved by chromatography on silica, the separation of the E- and Z-isomers, i.e. separation of E-cis-16 from Z-cis-16 and E-trans-16 from Z-trans-16, was not successful.

N+O-

14

NOH

E-cis -16

NOH

E-trans-16

+

NOH

Z-cis -16 Z-trans-16

+

MgBr

or

Zn

NOH

Scheme 4.5. Hydroxylamine isomers obtained from reaction of nitrone 14 with 1-propenylmagnesium bromide or di-1-propenylzinc.

31

The results described in the preceding paragraph warranted the exploration of an alternative route to racemic pinidine [(±)-9]. Thus, based on a few modifications of the Betti base procedure used to prepare racemic dihydropinidine [(±)-10] for field tests, a new route was explored (Scheme 4.6; Appendix). Compound 24 was alkylated at C11 using 1-propenylmagnesium bromide in THF, without any dialkylation at C7a. The products were an almost equimolar mixture of E- and Z-isomers, which were separated by chromatography on silica. Based on the known configuration of compound 24 (Xu et al., 2004) and the assumption that alkylation at C11 occurred in an SN2 manner (Wang et al., 2005), the configurations of the products were tentatively assigned to be those of E-27 and Z-27 (Scheme 4.6).

N

OH

cis-28major

O

N

Bt

O

N

O

N24

E-27

Z-27

11

7a

b) +

N

OH

trans-28minora)

Scheme 4.6. Synthesis towards racemic pinidine [(±)-9]. a) 1-propenylMgBr, THF, 0 °C. b) MeMgBr in Et2O, CH2Cl2, 0 °C.

Methylation at C7a of the E-isomer E-27 gave cis-28 as the major isomer (Scheme 4.6). In contrast to the high diastereoselectivity achieved when 25 was alkylated with n-propylmagnesium bromide (Scheme 4.4; Wang et al., 2005), a minor amount of trans-28 was detected by NMR. Without separation of cis- and trans-28, the naphthalene moiety was removed using α-chloroethyl chloroformate (Olofson et al., 1984) following an earlier described procedure (Karlsson and Högberg, 2002) (Scheme 4.7).

HN

a) N

cis-29

O O

Cl

b)N

O O

Cl

+cis-28

+

trans-28(±)-9trans-29

Scheme 4.7. Synthesis of (±)-9. a) ClCOOCH(Cl)CH3, 1,8-bis(dimethylamino)-naphthalene, 1,2-dichloroethane, ∆x. b) Chromatography on silica gel.

32

After work-up the diastereomeric carbamate cis-29 was detected by GC-MS, together with traces of the trans-diastereomers trans-29. After chromatography on silica the free amine, i.e. (±)-9 was isolated and converted to the hydrochloride salt which was isolated in ~38% yield as calculated from E-27. The trans-isomer of (±)-9 was detected in a minor number of the fractions after chromatography but was not isolated. Apparently the carbamates cis-29 and trans-29 decomposed during the chromatography. Starting from Z-27, cis-2-methyl-6-[1Z-propenyl]piperidine hydrochloride (30, Figure 4.2) was prepared following the same procedure and isolated in ~70% yield, as calculated from Z-27.

H*HClN

30

Figure 4.2. “(±)-Z-pinidine” hydrochloride (cis-2-methyl-6-[1Z-propenyl]piperidine hydrochloride) (30).

The racemic Betti base is easily resolved into its pure enantiomers (Cardellicchio et al., 1998) therefore both enantiomers of either pinidine 9 or Z-pinidine 30 will potentially be accessible via the new Betti base route (Schemes 4.6 and 4.7).

4.3 Synthesis of epidihydropinidine

It was discovered that when di-2-propenylzinc was added to the nitrone (14), either with or without boronic ester 21, the diastereoselectivity was reversed in favour of trans-22 (Scheme 4.8; cf Scheme 4.3).

ZnN+O-

14

NOH

cis -22minor

NOH

trans-22major

+ +CH2Cl2 /Et2O

Scheme 4.8. Addition of di-2-propenylzinc to nitrone 14.

33

However, when trans-22 was subjected to palladium catalysed hydrogenation a mixture of (+)-dihydropinidine [(+)-10] and (+)-epidihydropinidine [(+)-12] was obtained. Due to the limited amount of substance left, attempts to separate these compounds by fractional crystallisation of the hydrochlorides failed. Double bond migration during palladium catalysed hydrogenation of alkenes is a common phenomenon that has been observed for a variety of alkenes (Huntsman et al., 1963; Smith et al., 1973). Thus, the observed epimerisation is probably due to the migration of the double bond forming the enamine 31 (Scheme 4.9).

NAlkene migration

trans-22

OHNOH

Hydrogenation

HN

31 (+)-10

Scheme 4.9. Double bond migration leading to inversion of configuration during palladium catalysed hydrogenation of trans-22.

When 31 is hydrogenated the thermodynamically favoured product, i.e. the cis-isomer (+)-10, is probably formed in excess. An alternative explanation may be that since the reduction of the hydroxylamine functionality is slower than the hydrogenation of the alkene, palladium can coordinate to the oxygen of the hydroxylamine 31 and thereby influence the diastereoselectivity during the hydrogenation. Presumably this double bond migration also occurred during the hydrogenation of the enantiomers of cis-22. However, hydrogenation of 31 in that case, led to the desired isomer. Due to these problems racemic epidihydropinidine [(±)-12] was prepared according to an earlier described procedure (Bubnov et al., 1998) (Scheme 4.10).

N 3B1. MeLi2.

3. MeOH4. H2O, OH−

HN Pd/C, EtOH

HN

(±)-12

Scheme 4.10. Synthesis of epidihydropinidine [(±)-12] according to Bubnov et al. (1998).

4.4 Antifeedant activity of piperidine alkaloids in laboratory- and field tests

Whether the piperidine alkaloids found in spruce and pine serve as protective allelochemicals against the feeding of herbivores or not remain to be proven (Gerson and Kelsey, 2002). However, an alkaloid mixture containing (−)-pinidinol [(−)-13] and (+)-epidihydropinidine [(+)-12] was found to have antifeedant activity against the Eastern spruce budworm, Choristoneura fumiferana (Schneider et al., 1991). Euphococcinine [(+)-11] was found to be an active deterrent against ants and spiders in laboratory tests (Eisner et al., 1986).

34

High antifeedant activity against H. abietis was found for all the tested piperidine alkaloids (Table 4.1). The fact that these non volatile salts were active indicates that their action as feeding deterrents is as a gustatory rather than an olfactory repellent. Table 4.1. Antifeedant activity of piperidine alkaloids in laboratory micro feeding assays and no-choice twig tests (Schlyter et al., unpublished results).

Compound

AFI(m) 10% ±SE (N)

ED50(m) (%)

AFI(t) 10% ±SE (N)

ED50(t) (%)

(±)-dihydropinidine

hydrochloride (10-HCl)

0.91 ±0.04 (22)

0.99 ±0.63

(4)

0.88 ±0.02 (10)

1.41 ±0.63

(2)

(±)-epidihydropinidine

hydrochloride [(±)-12-HCl]

0.95 ±0.03 (17)

1.39 ±0.73

(3)

0.74 ±0.02 (10)

(−)-dihydropinidine

hydrochloride [(−)-10-HCl]

0.90 ±0.09 (15)

1.02 ±0.19

(3)

(+)-dihydropinidine

hydrochloride [(+)-10-HCl]

0.96 ±0.03 (26)

1.00 ±0.42

(4)

(±)-pinidine

hydrochloride [(±)-9-HCl]

0.75 ±0.09 (26)

2.41 ±1.04

(3)

(±)-Cis-2-methyl-6-[(1Z)-

propenyl]piperidine hydrochloride (30)

0.91 ±0.04 (26)

9.20 ±7.96

(3)

Because no difference in activity was found for the enantiomers of dihydropinidine [(+)-10-HCl and (−)-10-HCl], a racemate (10-HCl) was tested in the field during the summer of 2005 (Shtykova et al., unpublished results). Due to the insolubility of the hydrochloride in wax, the compound was formulated in a water-based polymer formulation chosen to give a slow release of the compound. Evaluation of weevil damage was first performed one month after planting. A high activity (AFI= 0.91 ± 0.06) identical to that of the standard insecticide treatment, i.e. the pyrethroid cypermethrin (5, Figure 1.3) (AFI= 0.91 ± 0.06), was observed.

35

At the second damage evaluation performed two months after planting, a lower antifeedant activity (AFI= 0.86 ± 0.07) was observed. However, the activity was still comparable with that of cypermethrin (AFI= 0.89 ± 0.05). Thus, the hydrochloride salt of (±)-dihydropinidine (10-HCl) is a highly active, non-volatile antifeedant against H. abietis.

Some drawbacks of using these types of alkaloids have, however, been reported. An alkaloid mixture from P. ponderosa, containing some of the alkaloids shown in Figure 4.1, was toxic and teratogenic to frog embryos (Tawara et al., 1993). Some other structurally related alkaloids, coniine (32) and γ-coniceine (33) (Figure 4.3), are also known to be toxic to animals (López et al., 1999). If dihydropinidine is indeed toxic, a formulation that inhibits leakage of the water soluble compound to the surroundings may be used2. Otherwise the compound is not suitable for the use as an antifeedant against H. abietis in field.

HN N

32 33

Figure 4.3. Toxic piperidine alkaloids, coniine (32) and γ-coniceine (33).

2 A research project [“Formulation of semiochemicals for plant protection against forest pest insects”, sponsored by The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas)] regarding formulation of antifeedants was recently started.

36

5. Structure-activity relationships of cinnamic aldehyde, benzaldehyde and related compounds as antifeedants against the pine weevilV

Quantitative structure-activity relationship (QSAR) studies employ methods to link the biological activity of a chemical compound to the molecular properties/structure of the compound. QSAR is frequently utilised in the pharmaceutical industry in order to obtain a compound with optimal biological activity when new drugs are developed from lead compounds. The molecular properties and structure of a lead compound are systematically varied and the changes in biological activity are observed. Multivariate data analysis is often used to evaluate the results.

In the early days of this project a number of compounds, selected to represent a diversity of structures, were screened for antifeedant activity against H. abietis (Schlyter et al., unpublished results). Thus, a library of active antifeedants was created. However, since the purpose was to find antifeedant compounds which could be used in large scale field trials, the availability of the compounds had to be considered and toxic compounds or compounds with other adverse effects were excluded.

Some compounds that showed high antifeedant activity in the initial tests were o-methoxybenzaldehyde (34) and p-methoxy-α-methyl-trans-cinnamic aldehyde (35) (Figure 5.1). The structures of these compounds were systematically modified by synthesis. Mono-, di- and trimethoxy analogues of the α-methylcinnamic aldehyde 35 were prepared from the corresponding commercially available benzaldehydes. The alkyl chain in the α-position was varied in length and the aldehyde functional group was converted to alcohol, carboxylic acid, and esters of different types. Several of the modifications gave compounds with decreased volatility, which is one of the desired qualities of an optimal antifeedant for use in the field. The cinnamic aldehydes so prepared and a number of similarly substituted benzaldehydes were tested for antifeedant activity in micro feeding assays and no-choice twig tests. Multivariate data analysis was used to establish structure-activity relationships for these compounds.

H

O

OMe

H

O

MeO

34 35

Figure 5.1. o-methoxybenzaldehyde (34) and p-methoxy-α-methyl-trans-cinnamic aldehyde (35).

37

5.1 Synthesis of α-alkylcinnamic aldehydes

The syntheses of the majority of the cinnamic aldehydes were straight forward, employing a simple aldol condensation between the appropriate aromatic aldehyde and an aliphatic aldehyde of varying chain length (Scheme 5.1).

H

O

(MeO)n

n = 0-2

+ H

O

(MeO)n

RH

ONaOH or KOH

EtOH/H2O

R = H, Me, Et, n-Pr, n-Bu, C6H13

R

Scheme 5.1. Synthesis of cinnamic aldehydes via aldol condensation.

Although the synthetic strategy was versatile and very simple, some problems were encountered. When trimethoxy substituted benzaldehydes were used as starting materials, the extent of conversion was low. This is probably a consequence of the electron releasing nature of the methoxy substituents. The methoxy groups donate electrons which results in an electron rich aromatic ring and hence an electron rich carbonyl carbon with low susceptibility to nucleophilic attack. Moreover, for some aldehydes, the methoxy substituents cause increased steric hindrance around the carbonyl group, which further decreases the reactivity. In order to circumvent these problems another strategy was employed for the synthesis of the trimethoxy compounds (Scheme 5.2).

H

O

(MeO)3

+ POEt

EtOO

CO2Et a)

b)

(MeO)3

OEt

O

(MeO)3

OHc)

(MeO)3

H

O

Scheme 5.2. a) Synthesis of trimethoxy substituted α-methylcinnamic aldehydes. a) LiBr, Et3N, THF, rt. b) LiAlH4, THF, rt. c) MnO2, CH2Cl2, rt. Up to 65% over three steps.

Thus, these compounds were synthesised by the Horner-Wadsworth-Emmons (HWE) modification (Boutagy and Thomas, 1974) of the Wittig reaction, which employs a phosphonate ester as the olefinating agent. This method frequently leads to olefins having an E-configuration (Wadsworth et al., 1965). Usually, relatively strong bases, such as n-butyl lithium and potassium tert-butoxide, are used to achieve the desired metal enolate of the phosphonate ester. However, in

38

the presence of magnesium or lithium halides the HWE reaction can proceed using a weaker base, such as triethylamine (Rathke and Novak, 1985). This also turned out to be case when these α,β-unsaturated esters depicted in scheme 5.2 were prepared. One attempt to reduce the ester directly to aldehyde was performed using diisobutylaluminium hydride. However, since only alcohol was obtained no further attempts to optimise this reaction was performed. Instead, subsequent reduction of the resulting esters using LiAlH4 followed by MnO2 oxidation of the alcohols gave the desired α,β-unsaturated aldehydes with overall yields of up to ~65%.

The expected E-configuration of the alkene moiety of the synthesised compounds was confirmed by the 1H NMR coupling constants between the olefin protons (for aldehydes with no branching in α-position) or by NOESY experiments observing the NOE contribution between the aldehyde proton and the olefinic β-proton. This NOE would not have been observed for compounds with Z-configuration (Figure 5.2).

(MeO)n

RO

HH

NOE

R = Me, Et, n-Pr, n-Bu, C6H13

n = 0-3

(MeO)n

RH NOE

O H

E Z

Figure 5.2. NOE effects depending on the configuration of the alkene moiety.

Several of the α,β-unsaturated aldehydes prepared in this work are also naturally occurring. All of them are derivatives of cinnamic aldehyde (36), a well known constituent of several plant species (Huang and Sheu, 2000; He et al., 2005; Lee et al., 1999). For example, the leaf oil from cinnamon, Cinnamomum zeylanicum, has been found to contain α-hexylcinnamic aldehyde (37, Figure 5.3) (Raina et al., 2001). In addition, an extract of flowers from Agastache mexicana subsp. mexicana has been found to contain 3-methoxycinnamic aldehyde (38, Figure 5.3) (Estrada-Reyes et al., 2004). In a parallel work we identified cinnamic acid (39, Figure 5.3) in bark extracts of yew and aspen, plant species known to be rejected by H. abietis as a food source (III).

OH

O

H

OH

O

3937 38

H

O

36

MeO

Figure 5.3. Examples of naturally occurring cinnamic aldehydes.

39

5.2 Test compounds

A total of 64 compounds (V) were tested for antifeedant activity against H. abietis in micro feeding assays and no-choice twig tests (section 2.1). Included in these compounds were 12 carboxylic acids, 5 cinnamic esters, 3 alcohols, 15 benzaldehydes, 28 cinnamic aldehydes and 1 dihydro cinnamic aldehyde (Figure 5.4). Normally the microassay dose-response is not determined for compounds with an AFI(m) <0.5 (section 2.1). However, for the structure-activity studies it was necessary to determine the ED50(m) of some of the compounds with low AFI(m) values. Similarly the twig tests are usually only performed for the compounds with the highest AFI(m)-values and ED50(m)-values in the range of a few percent while in this case it was also necessary to test some compounds of lower activity.

(MeO)n

R

(MeO)n

O

HH

O

(MeO)

OH

(MeO)n

R

(MeO)

O

OHOH

O

(MeO)n

R1OR2

O

R = H, Men= 0-2

R1 = H, MeR2 = Me, Et

n= 0-2

R = H, Me, Et, n-Pr, n-Bu, C6H13n= 0-3

n= 0-3

Figure 5.4. Examples of structural variation of compounds included in the QSAR-studies.

5.3 Multivariate data analysis and structure-activity relationships

The data obtained from the microassays and the twig tests were subjected to multivariate data analysis3 using projection to latent structures by means of partial least squares (PLS) analysis.

When tested both in the micro feeding assays and on twigs, all the cinnamic- and benzoic acids were either inactive or even acted as feeding stimulants (AFI(m)= −1.00 to 0, AFI(t)= −0.47 to 0.38) (V). Similarily, Watkins et al. (1999) found that cinnamic aldehyde was a more active repellent against pigeons (Columba livia) than cinnamic acid. The activity of these compounds is in contrast to the high antifeedant activity against H. abietis found for nonanoic

3 Umetrics, SIMCA-P 10.0

40

acid and some other aliphatic carboxylic acids but not for nonanal (section 3, I, II). When included in the PLS-models, the feeding stimulating properties of the carboxylic acids made them strongly separated from the other compound classes tested. Thus, they were not included in further data analysis. A PLS-analysis of a model [Y-variable= AFI(m)] that included all tested compounds, except the carboxylic acids, showed that high antifeedant activity, as determined by the micro feeding assay, was connected to cinnamic aldehydes lacking methoxy groups or containing only one methoxy group (Figure 5.5). Compounds containing three methoxy groups were less active. The alcohols and cinnamic esters were found close to zero on the p1-axis (Figure 5.5) which corresponds to the intermediate antifeedant activity observed for these compounds (V).

Figure 5.5. PLS-analysis based on AFI(m), including all tested compounds except for carboxylic acids, [Q2(0.0754) > limit(0.05)]. a) Score plot, 1) AFI >0.90, 2) AFI= 0.50-0.90, 3) AFI <0.50; b) Loading plot, UA= cinnamic aldehyde, BA= benzaldehyde, E= cinnamic ester, OH= cinnamyl alcohol, 0M= no methoxy substitution, 1M= 1 methoxy group, 2M= 2 methoxy groups, 3M= 3 methoxy groups, M2-M6= position of methoxy

group.

41

A PLS-analysis [Y-variable= AFI(m)] including only the cinnamic aldehydes again showed that the highest activity was connected to compounds containing a maximum of one methoxy group (Figure 5.6). For the majority of the cinnamic aldehydes an increased substitution with two or three methoxy groups reduced the activity. This corresponds well with the low ED50(m)-values (0.001% to 1.5%, V) obtained for cinnamic aldehydes with less than three methoxy groups (n= 0-2, Figure 5.4). Whether an alkyl substituent in the α-position was present or not was not shown to influence the activity (Figure 5.6; V). Disappointingly, when tested on twigs, the activities of the cinnamic aldehydes were profoundly reduced. Only cinnamic aldehyde (36, Figure 5.3) retained its activity on twigs. Thus, the strategy to decrease the volatility of cinnamic aldehyde by the addition of methoxy groups or alkyl chains in α-postion, while maintaining a high activity, failed. As mentioned earlier (section 2.1), when tested in the more demanding twig test, decreased activity compared with that in the micro feeding assay, has been observed for several compounds.

42

Figure 5.6. PLS-analysis based on AFI(m), including cinnamic aldehydes, Q2(0.343) > limit(0.05). a) Score plot, 1) AFI >0.90, 2) AFI= 0.50-0.90, 3) AFI <0.50; b) Loading plot, Ak= α-alkyl chain, 0Ak= no α-alkyl chain, 0M= no methoxy substitution, 1M= 1 methoxy

group, 2M= 2 methoxy groups, 3M= 3 methoxy groups, M2-M6= position of methoxy group.

43

No difference in activity was observed between the dihydro cinnamic aldehyde, 3-(2-methoxyphenyl)-2-methyl-propanal (40, Figure 5.7), and the corresponding unsaturated aldehyde 41 (Figure 5.7). Previously, a similar result was obtained when cinnamic aldehyde and its corresponding “saturated” derivative, 3-phenylpropanal, were tested for ovipositional-deterrent activity against the onion fly, Delia antiqua (Cowles et al., 1990).

H

OOMe

H

OOMe

40 41

Figure 5.7. 3-(2-methoxyphenyl)-2-methyl-propanal (40) and 2-methoxy-α-methyl-trans-cinnamic aldehyde (41).

A model including only benzaldehydes [Y-variable= AFI(m)] showed that benzaldehydes containing one or two methoxy groups, preferentially in the m-position, are more active than those with three methoxy groups (Figure 5.8). In general low ED50(m)-values (0.64% to 1.82%; V) were obtained for benzaldehydes with less than three methoxy groups. Benzaldehyde itself was not active (AFI(m)= −0.10). Three benzaldehydes were tested on twigs. They were mono-methoxy substituted and were all active. Thus, for the benzaldehydes, introduction of one methoxy group is a possible way to decrease the volatility, while retaining antifeedant activity even in twig tests.

44

Figure 5.8. PLS-analysis based on AFI(m), including benzaldehydes, Q2(0.276) > limit(0.05). a) Score plot, 1) AFI >0.90, 2) AFI= 0.50-0.90, 3) AFI <0.50; b) Loading plot,

0M= no methoxy substitution, 1M= 1 methoxy group, 2M= 2 methoxy groups, 3M= 3 methoxy groups, M2-M6= position of methoxy group.

45

6. Conclusions and future work Several new antifeedants against H. abietis, including single compounds and classes of compounds, have been identified. By examining the chemical content of bark extracts prepared from non-host species of the pine weevil, we found the highly active antifeedant, nonanoic acid, to be at least in part responsible for the reluctance of the weevils to feed on linden, T. cordata. Apart from nonanoic acid, some other naturally occurring aliphatic carboxylic acids were shown to exhibit high antifeedant activities in both laboratory and field tests.

The identification of active compounds in other non-host species, excluding linden was not as straight forward. However, even though the extracts contained a complicated mixture of antifeedants and feeding stimulants that were not easily separated by chromatography, some active compounds were identified. Additional laboratory and field tests of these compounds are needed. Further investigation of extracts with antifeedant activity might lead to the identification of additional antifeedant compounds.

When screening a number of compounds for antifeedant activity the piperidine alkaloid dihydropinidine [(±)-10] was found to be a highly active antifeedant against H. abietis. A method for the synthesis of the enantiomers of 10, involving dimethylzinc mediated addition of allylboronic ester 21 to the nitrone 14 as a key step, was developed. When (±)-10 was tested in the field high antifeedant activity comparable with that of the insecticide cypermethrin was observed.

Among the active compounds found in the screening procedure, benzaldehydes and cinnamic aldehydes were choosen for structure-activity studies. It was found that introduction of one or two methoxy groups on benzaldehyde (itself inactive) considerably enhanced the antifeedant activity. Although several cinnamic aldehydes were active in the micro feeding assays, only cinnamic aldehyde itself (36) retained a high activity in the twig tests. Thus, evaluation of the activities of cinnamic aldehydes and benzaldehydes in the field would be of great interest.

A proper formulation of the active compound is required to secure the slow release necessary for long term protection of the seedlings. Moreover, preliminary tests showed that a proper formulation technique could reduce damages on conifer seedlings caused by phytotoxic aliphatic carboxylic acids. Even though several potent antifeedants have been identified, the development of efficient formulations techniques is essential for further progress in this area. When developing a formulation there are several requirements that must be considered. The formulation must be easy to apply and should prevent leakage of the active compound to the surroundings. A slow degradation of the formulation into compounds that are environmentally safe is preferred.

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Acknowledgements Bakom varje färdig doktor står många (förvånade?) personer. Jag vill tacka följande personer som under denna tid, på ett eller annat sätt, stått bakom mig och hjälpt mig att förverkliga denna avhandling:

Professor Hans-Erik Högberg och Dr. Kristina Sjödin för idéer, handledning, stöd och lärorika diskussioner.

Professor Christina Moberg i egenskap av huvudhandledare på KTH och för värdefulla kommentarer på manuskriptet till avhandlingen.

Docent Fredrik Schlyter och Dr. Per Månsson för gott samarbete och terapi i det ljuva Asa.

Dr. Tessa Pocock for valuable comments on manuscripts and on the thesis.

Alla nuvarande och tidigare medlemmar i organkemi gruppen: Docent Erik Hedenström, Palle, Fredrik, Jimmy, Jonas, Helen, Marica, Olle, Ove, Ba-Vu, Micke, Staffan och Linda. Ett speciellt tack till Dan och Sunil som har tagit sig tid att läsa delar av avhandlingen. Anna, tack för rese-sällskap. Mona och Jessica för allt roligt vi har haft både på jobbet och på fritiden.

Övrig nuvarande och tidigare personal på NAT (gamla NTM och KEP). Ett speciellt Tack till Håkan och Torborg, vad vore det för ordning på lab. utan Er? Siw, Viktoria, Ingrid, Johnny och Jenny för Er hjälp med allt möjligt och omöjligt.

Studenter som under årens lopp har kämpat på lab. med diverse startmaterial jag har behövt. Särskilt mycket hjälp har jag fått av Rebecka From.

Anders för att du har härdat ut under den här tiden.

Farmor, vad jag än har för problem blir jag alltid glad i din närhet.

Casper för alla skogspromenader som fått mig att tänka på annat.

Maria, du är världens bästa syster. Jag är så glad att jag har dig och jag är glad att du har Jonas, en så’n trevlig sambo.

Mamma och Pappa, även om ”Cia kan sälv” hade hon aldrig klarat sig utan Er. Ni är bäst!

Financial support was kindly provided by MISTRA (Foundation for Strategic Environmental Research) and the Aulin-Erdtman Foundation.

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